45th meeting of the International Narcotics Research ConferenceMontreal, Quebec, Canada
July 13‐18, 2014

Program and abstracts

Committees/Acknowledgments
Program Committee

Executive Committee

Louis Gendron (Canada, Chair)
Catherine Marie Cahill (USA, Co‐chair)John Traynor (USA, President/Secretary)
Lakshmi Devi (USA, Past President)
Lawrence Toll (USA, Treasurer/Vice
President)

Michael Bruchas (USA)Ivy Carroll (USA)Charles Chavkin (USA)
Lakshmi Devi (USA)
Susan Ingram (USA)Jose Moron Concepcion (USA)
Minoru Narita (Japan)
Amynah Pradhan (USA)
Lawrence Toll (USA, Treasurer)
Tuan Trang (Canada)
John Traynor (USA, INRC President)

Elena Bagley (Australia)
Stephen Husbands (UK)Thomas Koch (Germany)
Dominique Massotte (France)
Jay McLaughlin (USA)Jose Moron‐Concepcion (USA)
Chaim Pick (Israël)Tom Prisinzano (USA)Ichiro Sora (Japan)

Acknowledgments
This conference would have not been possible without the strong support of public
institutions and sponsors.
‐ Alkermes, Inc. (USA)‐ ASPET (The Neuropharmacology division)‐ Centre de Recherche du CHUS (Canada)‐ Charles River (USA)‐ Denator AB (Sweden)‐ Faculté de médecine et des sciences de la santé
Université de Sherbrooke (Canada)‐ Hotchkiss Brain Institute
University of Calgary (Canada)‐ Institut de pharmacologie de Sherbrooke
Université de Sherbrooke (Canada)‐ Institute of Neuroscience, Mental Health and Addiction (INMHA)
Canadian Institute for Health Research (Canada)‐ National Institute on Drug Addiction (NIDA)‐ Quebec Pain Research Network
Fonds de la Recherche du Québec – Santé (Canada)‐ Reckitt Benckiser Pharmaceuticals (Canada)‐ Stoelting Co. (USA)‐ Tourisme Montréal (Canada)‐ Trevena Inc. (USA)Cover
Images from: Tourisme Montréal, Robert Burch, Michel Caty, Victor Lamich Diaz,, Benoit Cécile

Thank you to our Sponsors
Quebec Level Sponsors

The Neuropharmacology Division of ASPET has contributed towards
the ongoing training of future generations of neuropharmacologists

Montreal Level Sponsors

Other Sponsors

Granting Agencies

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Floor Plan
(first floor)

Program
Sunday July 13th

15:00‐20:00
18:00‐20:00

Registration
Welcome reception (Le Portage)

Monday July 14th
8:00‐15:00
6:30‐8:00

Registration
Breakfast provided (Fontaine A)

7:50‐8:00

Welcoming Remarks (Louis Gendron and John Traynor)

Plenary lecture #1 (Westmount)
8:00‐8:45
Roger Sunahara (University of Michigan)“Structural and functional characterization of the receptor‐G protein cooperativity”

Symposium #1 (Westmount)
RECEPTOR CRYSTAL STRUCTURE AND NOVEL OPIOID CHEMISTRY
Chair: Peter Schiller (Institut de Recherche Clinique de Montréal)
Discussion leader: Ivy Carroll (Research Triangle Institute)
8:45‐9:10
Gustavo Fenalti (Scripps Research Institute)“Molecular Control of Opioid Receptor Signaling”

9:10‐9:35

Nurulain Zaveri (Astraea Therapeutics)“Which way is up? Rethinking Nociceptin Ligand Design and SAR From Nociceptin Opioid
Receptor Crystal Structure and Active‐State Homology Model comparisons”

9:35‐10:00

Philip Portoghese (University of Minnesota)“MOR heteromers as targets for treatment of inflammatory and neuropathic pain”

10:00‐10:20

Nutritional break

10:20‐10:45

Stephen Husbands (University of Bath)“Relapse prevention: targeting multiple receptors”

10:45‐10:55

Hot Topics‐D. Tourwé“Synthesis and biological evaluation of a compact, conformationally constrained bifunctional
opioid agonist‐neurokinin‐1 antagonist”

10:55‐11:05‐K.E. Livingston

11:05‐11:15‐P.D. Mosier“Probing Allosteric Modulation of the Mu Opioid Receptor”“Analogs of salvinorin A bearing sulfur‐containing substituents at position C2 are high‐affinity kappa‐opioid receptor partial agonists with a potentially novel binding mode”

11:15‐11:25‐A.A. Harland“Synthesis, Optimization, and Evaluation of Mixed‐Efficacy ‐Opioid Receptor Agonists/ ‐Opioid Receptor Antagonists: A Balancing Act”

11:25‐11:35‐R.D. Howells“Novel small molecule triazoles modeled from naltrindole potently inhibit the proliferationof human multiple myeloma cells”

11:35‐12:00

Discussion period

12:00‐13:00

Lunch provided (Fontaine A)

Symposium #2 (Westmount)
REVISITING THE THERAPEUTIC POTENTIAL OF DELTA OPIOID RECEPTOR LIGANDS
Chair: Junzo Kamei (Hoshi University)
Discussion leader: Emily Jutkiewicz (University of Michigan)
13:00‐13:25
Gregory Scherrer (Stanford University School of Medicine)“Delta Opioid Receptor Function in Skin: Controlling Pain Where it Starts”

13:25‐13:50

Jon Violin (Trevena Inc.)“Biased ligands at mu and delta opioid receptors: targeting selective signalling to develop
improved therapeutics”

13:50‐14:15

Manojkumar Puthenveedu (Carnegie Mellon University)“Increasing delta opioid receptor bioavailability”

14:15‐14:40

Amynah Pradhan (University of Illinois at Chicago)“The Potential of Delta Opioid Receptor Agonists for the Treatment of Migraine”

14:40‐15:00

Nutritional break

15:00‐15:10

Hot Topics‐M. Bigliardi‐Qi

15:10‐15:20‐A. Saitoh

15:20‐15:30‐A.B. Tudashki“Delta Opioid receptors: Important Role In wound healing”“The DOR2 antagonist naltriben abolishes KNT‐127‐induced anxiolytic‐like effects in rats”“Ligand ability to evoke Gi activation and to promote internalization of DOR can predict
response duration over a short period”

15:30‐16:00

Discussion period

16:00‐18:00

Poster session #1 (odd numbers; Fontaine B‐C)Tuesday July 15th
8:00‐15:00
6:30‐8:00

Registration
Breakfast provided (Fontaine A)

Founder’s lecture (Westmount)
8:00‐9:00
Brigitte Kieffer (McGill University) andChristopher Evans (University of California Los Angeles)“Cloning: twice in one day”
Symposium #3 (Westmount)
CHANGES IN REWARD CIRCUITRY IN CHRONIC PAIN.
Chair: Howard Fields (University of California San Francisco)Discussion leader: Catherine M. Cahill (University of California Irvine)
9:00‐9:25
Marco L. Loggia (Harvard Medical School)“Disrupted brain circuitry for pain‐related reward/punishment in fibromyalgia”

9:25‐9:50

Lucia Hipolito (Columbia University)“Chronic pain increases opioid self‐administration through an accumbal dopaminergic
mechanism”

9:50‐10:10

Nutritional break

10:10‐10:35

Anna Taylor (University of California Irvine)

10:35‐11:00

Zhizhong Pan (University of Texas)“Neuropathic Pain Modulates Dopaminergic Circuitry: Role for Microglial Activation”“Pain facilitates response to opioid reward via a shared epigenetic pathway”

11:00‐11:10

Hot Topics‐S.D. Comer“Abuse liability of oxycodone and morphine in buprenorphine‐maintained participants withor without chronic pain”

11:10‐11:20‐A. Bendiksen“The Use of Opioids in the Treatment of Young People with Chronic Non‐Malignant Pain at a
Multidisciplinary Pain Clinic in Denmark”

11:20‐11:30‐I. Gomes“Analysis of Morphine Regulated Striatal Synaptic Networks using Proteomics and Network
Analysis”

11:30‐12:00

Discussion period

12:00‐13:00
12:00‐13:00

Lunch provided (Fontaine A)
Lunch (Executive committee) (Salon Fundy)

Symposium #4 (Westmount)
DISSECTING THE FUNCTIONS OF NOCICEPTIN/ORPHANIN FQ RECEPTORS
Chair: Michael Bruchas (Washington University)
Discussion leader: Larry Toll (Torrey Pines Institute)13:00‐13:25

Raül Andero Galí (Emory University)“Amygdala‐dependent fear is regulated by opioid receptor‐like 1 in mice and humans with
PTSD”

13:25‐13:50

Kelly Standifer (University of Oklahoma College of Pharmacy)

13:50‐14:15

Thomas Jhou (Medical University of South Carolina)

14:15‐14:40

Robert Innis (National Institutes of Mental Health)“Neuroinflammatory actions of N/OFQ following traumatic stress”“Nociceptin/Orphanin‐FQ in the rostromedial tegmental nucleus”“Imaging of nociceptin/orphanin FQ peptide (NOP) receptors in human brain and whole‐body using a novel positron emission tomography radioligand, [11C]NOP‐1A”

14:40‐15:00

Nutritional break

15:00‐15:10

Hot Topics‐A. Ozawa“EGFP‐NOP Mice: Location in Spinal Cord and DRG”

15:10‐15:20‐S.M. Spangler

15:20‐15:30‐Y. Zhang“Dissecting Nociceptin Receptor Modulation of Reward”“Effect of the nociception/orphanin FQ peptide receptor antagonist JTC‐801 on cytokine
expression following exposure to the single prolonged stress model of PTSD”

15:30‐16:00

Discussion period

16:00‐18:00

Poster session #2 (even numbers; Fontaine B‐C)
Wednesday July 16th

8:00‐12:00
6:30‐8:00

Registration
Breakfast provided (Fontaine A)

Plenary lecture #2 (Westmount)
8:00‐8:45
Antonello Bonci (NIDA intramural program director)“Optogenetic approaches to study synaptic plasticity and substance abuse”
Symposium #5 (Westmount)
DYSPHORIA AND AVERSION IN ADDICTION AND CHRONIC PAIN
Chair: Chris Evans (University of California Los Angeles)
Discussion leader: Charles Chavkin (University of Washington)
8:45‐9:10
Julie Kauer (Brown University)“Acute stress activates kappa opioid receptors and triggers persistent synaptic changes inthe VTA”

9:10‐9:35

Joel Schlosburg (Scripps Research Institute, San Diego)“Dynorphin‐kappa opioid receptor activation regulates the dynamics of drug‐taking in
the progression towards addiction”

9:35‐10:00

Brendan Walker (Washington State University)“Quiescent kappa‐opioid receptors in the medial prefrontal cortex: the rollercoaster ofcognitive control”

10:00‐10:20

Nutritional break

10:20‐10:30

Hot Topics‐A. Riegel

10:30‐10:40‐L.G. Rosen“The Functional Rewiring of Cortical Synapses in a Translational Model of Neuropathic Pain”“Opiate exposure state controls a molecular switch in opiate reward memory formation in
the basolateral amygdala‐prefrontal cortical pathway”

10:40‐10:50‐A. Lesniak

10:50‐11:00‐S. Schattauer“Opioid receptor expression in mice with persistent, long‐term neuropathic pain”

11:10‐11:20“Ligand‐directed signaling at kappa opioid receptors: differential mechanisms of JNKMAPK activation by U50,488 and norBNI”‐G.R. Matyas“Development of a Vaccine against Heroin”

11:20‐11:45

Discussion period

12:00‐13:30

Data Blitz (lunch boxes provided)
Thursday July 17th

8:00‐15:00
6:30‐8:00

Registration
Breakfast provided (Fontaine A)

Plenary lecture #3 (Westmount)
8 :00‐8:45
Yves De Koninck (Université Laval)“Expanding your toolbox; new approaches to probe the brain with light”

Symposium #6 (Westmount)(Sponsored by the CIHR’s Institute of Neuroscience, Mental Health and Addiction)
WORKSHOP ON NEW TECHNOLOGIES IN NEUROSCIENCE AND DRUG

8:45‐9:10

ADDICTION
Chair: Fred Nyberg (Uppsala University)
Discussion leader: Jose Moron‐Concepcion (Columbia University)
Maxime Descoteaux (Université de Sherbrooke)

9:10‐9:35

Philippe Seguela (McGill University)

9:35‐10:00

Sotiris Masmanidis (University of California Los Angeles)“Diffusion MRI: imaging the wiring of the brain”"Optogenetic control of pain pathways in vivo"“Large‐scale electrophysiology and virtual reality for reward circuit research”

10:00‐10:20

Nutritional break

10:20‐10:30

Hot Topics‐K. Skold“Post‐sampling changes in opioid peptide levels and the effect of tissue stabilization”

10:30‐10:40‐A.K. Fakira“NR2B‐mediated changes in hippocampal spine morphology following morphine conditioned
place preference”

10:40‐10:50‐M.J. Schmidt“Spatiotemporal Control of Mu‐Opioid Signaling and Behavior”

10:50‐11:15

Discussion period

Symposium #7 (Westmount)
DISSECTING OPIOID NEURAL CIRCUITS
11:15‐12:00
YOUNG INVESTIGATOR AWARD SYMPOSIUM
Chair: John Traynor (University of Michigan)
Discussion leader: Elyssa Margolis (University of California San Francisco)Michael Bruchas (Washington University)“Spatiotemporal Control of Opioid Systems in Reward and Aversion”

12:00‐13:00

Lunch provided (Fontaine A)

13:00‐13:25

Thomas Kash (University of North Carolina)

13:25‐13:50

Matthew Banghart (Harvard University)“Dynorphin controls the gain of an amygdalar anxiety circuit““A photochemical approach to peptidergic neuromodulation”

13:50‐14:00

Hot Topics‐E.N. Bobeck“Endosomal Endothelin Converting Enzyme‐2: A regulator of opioid receptor trafficking”

14:00‐14:10‐K.J. Tonsfeldt“Extrasynaptic GABAA receptors in the periaqueductal gray are involved in descending pain
modulation and chronic inflammatory pain‐induced plasticity”

14:10‐14:20‐L. Kotecki“GIRK channel activation in midbrain GABA neurons is not required for opioid‐induced
locomotor stimulation”

14:20‐14:45

Discussion period

14:45‐15:00

Nutritional breakSymposium #8 (Westmount)
GENETIC AND EPIGENETIC REGULATION IN ADDICTION AND CHRONIC PAIN
Chair: Laura Stone (McGill University)
Discussion leader: Mary Jeanne Kreek (The Rockefeller University)
15:00‐15:25
Luda Diatchenko (McGill University)“Expansion of mu‐opioid receptor gene locus: new functional variants”

15:25‐15:50

Pamela Kennedy (University of California Los Angeles)“Chromatin Cross‐Talk in the Nucleus Accumbens: Implications for Cocaine‐Induced
Behavioral and Molecular Adaptations”

15:50‐16:15

Marcello Wood (University of California Irvine)“The role of HDAC3 in acquisition and extinction of drug‐seeking behavior”

16:15‐16:25

Hot Topics‐O. Levran“Dopaminergic system gene polymorphisms and heroin addition: further support for a
protective effect of casein kinase 1e (CSNK1E) variants”

16:25‐16:35‐J.S. Wieskopf“Broad spectrum analgesic effcacy of IBNtxA is mediated by exon 11‐associated splice variantsof the mu‐opioid receptor gene”

16:35‐16:55

Discussion period

16:55‐17:45

Business meeting (Westmount)

Shuttles to the Banquet dinner leave between 18:15 and 18:30 from the Hilton
19:00‐lateBanquet Dinner
Friday July 18th
End of the Conference and Departure

INRC 2014 Awardees
Dr Brigitte Kieffer

Founder’s lecture

B. L. Kieffer is Professor at McGill Univerity and at the Université de Strasbourg
France. Her team uses mouse genetic approaches to tackle the role of opioid
receptors in brain physiology and disorders, and to search for novel genes involvedin psychiatric disorders. She has developed and shared exquisite genetic tools
worldwide, and has developed innovative research lines with strong impact inneuroscience and biomedical research. Her work has important implications forthe development of treatment of pain, drug abuse and emotional disorders. She ispart of the Center for Opioid Receptors and Drugs of Abuse or CSORDA.
Pr. Kieffer is recipient of the Jules Martin (French Academy of Science, 2001) andthe Lounsbery (French and US Academies of Science, 2004) Awards, and has
become an EMBO Member in 2009. In 2012 she received the Lamonica Award of
Neurology (French Academy of Science) and was nominated Chevalier de la Légion
d’honneur. In December 2013 she was elected as a member of the French Academyof Sciences. She started as the Scientific Director of the Douglas Hospital ResearchCentre, affiliated to McGill University in January 2014.

Dr Christopher Evans
Christopher Evans received his Ph.D. from Imperial College London, conducting his
thesis research on endorphins and enkephalins, at the Medical Research Council
Institute in Mill Hill. After a postdoctoral fellowship at Stanford University, hejoined the UCLA faculty in the Department of Psychiatry and Biobehavioral Science.
His research accomplishments have included identification of a number of novel
endogenous opioid peptides and the cloning of the first opioid receptor.

Young Investigator Award

Dr. Evans is currently Director of the UCLA Brain Research Institute and the Stefan
Hatos Professor directing the Shirley and Stefan Hatos Center for
Neurophamacology in the UCLA Semel Institute. He is also director of a NIH‐funded
center – The Center for Opioid Receptors and Drugs of Abuse or CSORDA with thebroad aim of understanding the action of opioid drugs such as morphine andheroin at the molecular, cellular and behavioral levels.

Dr Michael Bruchas
Michael Bruchas’s research training is in GPCR pharmacology and neuroscience.
His graduate work focused on adrenergic receptors while his post‐doctoral work
was in the department of pharmacology, at the University of Washington, Seattle,
in Charles Chavkin’s laboratory. Here he studied opioid receptor biased signaling
in behavior using mouse genetics and behavioral approaches. His laboratory at
Washington University (St. Louis) investigates interactions between stress, GPCRs,
neuropeptides, and neural circuits in affective behavior.
Recent efforts by his group have focused on developing tools for wireless
optogenetics and optical GPCRs for examining signaling pathways and behavior invivo. Using a variety of genetic, pharmacological, engineering, and optogenetic
approaches, he will discuss recent efforts by his team to dissect the role of opioid
peptides and receptor signalling in models of affective behavior.

Dr Brigitte Kieffer, Founder’s Award, INRC 2014

When a scientist receives an honour it is usually for a single major contribution toadvancing the field, but for Brigitte Kieffer I would identify three major contributions to
opioid science which have been world‐leading and have had a very significant impact
on both INRC and on the wider scientific community. Firstly, her cloning of the delta
opioid receptor (published in PNAS), secondly the development and characterisation of
the mu opioid receptor knockout mouse (published in Nature) and thirdly thedevelopment and characterisation of the first conditional opioid receptor knockout
mouse, with a fascinating behavioural phenotype that will be published shortly. In
between there have been many other significant publications, including really elegant
work on the role of opioids in emotional responses (in Nature Genetics) and in maternal
attachment (in Science).
Brigitte first came to an INRC meeting in 1990 at Noordwijkerhout, and at that point several laboratories had spent nearly adecade trying to clone the first opioid receptor. In the 1980’s I listened to a lot of talks on the cloning of the opioid receptor
that really had nothing to say! I felt sorry for a string of PhD students who had to write a thesis with precious little to show
for their heroic efforts. So when Brigitte Kieffer and Chris Evans independently succeeded in cloning the first opioid receptor
in 1992 these were landmark publications. Chris published in Science and Brigitte published in PNAS, though I know sheoriginally submitted the manuscript to Nature. The editors foolishly said “it was just the cloning of another G‐protein coupled
receptor” and rejected the manuscript. This paper is now cited over 850 times and I am sure the editors of Nature are kicking
themselves. Nature did not make the same mistake twice and when Brigitte submitted her phenotypic characterisation of
the first opioid receptor knockout mouse in December 1996 the referee and editors reports were glowing. The citation
count for this paper is now approaching four figures. She presented this seminal work at the INRC meeting in Long Beachearlier that year; a clearcut deletion of the mu opioid receptor and a remarkable loss of key behaviours that this receptor isinvolved in. I recall there was stunned silence in the room, as INRCers listened to this major field advance. Avram Goldstein
who was standing next to me remarked “this is almost too good to be true”. But it was one of those set of experiments that
we would all love to be involved with; I was privileged to be one of the collaborators on this work. Sometimes, experiments
don’t work out as you had planned. Brigitte had set out to make a delta opioid receptor knockout mouse, and succeeded
first in making a mu knockout – the kappa gene knockout came next (again a first) and the delta knockout last. Sometimes
the direction of science is rather serendipitous.
There is a third landmark discovery to come and I suspect in her Founders lecture she might talk about her work in developing
a conditional deletion of an opioid receptor to enable us to identify the role of these receptors in specific regions in thebrain. This work and the previous development of knockout mice, have taken many years and stoic perseverance to cometo fruition. It reflects Brigitte’s critical attention to detail, a characteristic I have seen when writing both grant applications
and research publications with her. She has very many successful collaborations in both Europe and worldwide, and inlooking at the author lists of her papers you might think she collaborates with everyone. This is not true – she chooses her
collaborators very carefully. I remember her telling me when she cloned the delta opioid receptor that she gave the clones
to everyone who asked; but far too many of those that asked did not do anything useful with them! So when the opioid
knockouts were developed she took a much more cautious approach, and it has delivered over 200 high quality publications.
Brigitte is a person who is in love with her science and her success has been recognised by four national prizes from both
French and US academies of science. She was appointed as Director of the world‐leading IGBMC in Strasbourg in 2012 wheremost of her opioid research has been carried out, but she always said that she must not allow leadership and management
to compromise the important science she still had to do. So this year she moved to be scientific director of the Douglas
Institute at McGill University in Canada – there is more to come, investigating opioid involvement in emotion.
For me, it has been an honour to collaborate with Brigitte for 20 years. There has been some great science and greatfriendship. In the early 1980’s you would have found Hans Kosterlitz (one of the founders of INRC) in the bar late at night at
INRC meetings, working hard and playing hard. From 1992, you would find Chris Evans and Brigitte Kieffer (and me!) there
too. As I said, great science and great friendship; it is what INRC is all about.
Professor Ian Kitchen
University of Surrey, UK
April 2014

Dr Christopher Evans, Founder’s Lecture Award, INRC 2014
When I was asked by the organizers of this year INRC to write a short text toacknowledge Chris Evans’ contribution to our society, I was wondering where
has the time gone. It seems like yesterday that Chris and I first met, at the1981 Kyoto meeting, when INRC was at its first golden age. It was at the time
when endogenous opioid peptides were being identified and the society
experienced a large influx of scientists whose research focuses deviated fromthe traditional approaches used by the anatomists, biochemists, medicalchemists, pharmacologists and physiologists that populated the society atthat time. Chris is one of those scientists who joined INRC during that timeand his scientific footprints since then have been tremendous. His thesis
training was as a peptide chemist with Derek Smyth at MRC England. Christhen went to Stanford for a postdoctoral position with Jack Barchas where, inclose collaboration with Eckard Weber, he isolated several new opioid
peptides, including metorphamide, and developed antibody probes forstudying the different processing products of proenkephalin, prodynorphin and proopiomelanocortin.
Everyone in the society knows that Chris was instrumental in the cloning of the opioid receptors. His initial report on the
successful cloning of ‐opioid receptor by expression cloning in 1992 has been cited >1000 times. Together with Brigitte
Kieffer, who independently reported the cloning of ‐opioid receptor at the same time, they launched INRC into its second
golden age. Subsequent successful attempts to clone other opioid receptors and the Orphanin F/Q receptor are based on
the two cDNA sequences reported by Chris and Brigitte. But few know that before his successful attempt, Chris was involvedin a failed attempt to clone the receptor by expression cloning. I was one member on the team that failed. To his credit andpersistency, an ingenious method of synthesizing 125I‐labeled peptide for high affinity binding and autoradiography toidentify clones, and a library generated in collaboration with Rob Edwards that eliminated secondary mRNA structure thus
enabling complete reverse transcription of the receptor mRNA, Chris and his talented technician Duane Keith, weresuccessful while many of us failed in cloning the receptor. For this singular achievement, Chris’ contribution to the society is
unparalleled.
In addition to the cloning of the receptor, another significant contribution that alters the course of INRC is Chris’ work on
biased agonism. Together with Mark von Zastrow, based on my initial observations that morphine is a partial agonist in the
‐opioid receptor and will not internalize the receptor, Chris demonstrated unequivocally that biased agonism exists in the
opioid receptor systems. Chris’ initial reports on the differential regulation of receptor trafficking laid the foundation forbiased agonism, probable mechanisms for differential opiate tolerance and addiction, and probable drug development that
one day might eliminate the many side‐effects of opiate analgesics, the holy grail of opioid receptor research.
Chris’ contribution to the society is not limited to his scientific accomplishments. Chris has been an active participant inyearly INRC meetings, has served on the Executive Committee, and local organizing committees. But I think the other major
impact that Chris has on INRC is his mantra for collaborative research and out of the box ideas. I am sure many of us havebenefited from Chris’ generosity in providing reagents and clones. Our studies on the opioid genes transcription andgeneration of the µ‐opioid receptor knockout mice would not be possible if not for his initial willingness to share the yet tobe reported ‐opioid receptor cDNA clone. Sometimes, I still have problems comprehending his ideas, seemingly come out
from the right field, to paraphrase an American baseball idiom. A case and point is his continued enthusiasm and efforts on
the phylogenetic studies of the opioid receptors and peptides. I guess one of these days, another high impact study will
come out of Chris’ group that will change the course of how we think about the biological functions of opioids.
What I have attempted does not detail all Chris’ contributions to INRC. What I have summarized is Chris’ contributions thatchange the course of INRC, contributions that rank with those of INRC founders, such as Avram Goldstein, Eric Simon andHans Kosterlitz. For these contributions, Chris definitely deserves the honor of being one of this year Founder’s Lecture
speakers. I am honored to have the opportunity to write this tribute and called Chris as my friend and collaborator.
Professor Ping‐Yee Law
University of Minnesota, USA
May 2014

TABLE OF CONTENTS
Plenary lectures
Roger K. Sunahara
Allosteric modulation of the orthosteric binding site by G proteins: mechanism of high affinity agonist
binding

12

Antonello Bonci, MD
Optogenetic approaches to understanding synaptic plasticity and substance use disorders.

13

Yves De Koninck
Expanding your toolbox; new approaches to probe the brain with light

14

Founder’s lecture
Brigitte Kieffer and Christorpher Evans
Cloning: Twice in One Day

15

Young Investigator Award
Michael R. Bruchas
Spatiotemporal Control of Opioid Systems in Reward and Aversion

16

Symposium #1
Gustavo Fenalti
Molecular Control of δ-Opioid Receptor Signaling

17

NURULAIN T. ZAVERI, Ph.D.
Which way is up? Rethinking Nociceptin Ligand Design and SAR from Nociceptin Receptor Crystal
Structure and Active-State Homology Model comparisons18

Philip S. Portoghese
MMG22 selectively targets a putative MOR-mGluR5 heteromer for treatment of chronic inflammatory
pain

19

Stephen Husbands
Potential Relapse Prevention Agents Targetting Multiple Opioid Receptors

20

Symposium #2
Gregory Scherrer
Delta Opioid Receptor Function in Skin: Controlling Pain Where it Starts

21

Jonathan Violin
Biased ligands at mu and delta opioid receptors: targeting selective signalling to develop improved
therapeutics

22

Manojkumar A. Puthenveedu
Increasing delta opioid receptor bioavailability

23

Amynah A Pradhan
Delta Opioid Receptor Agonists for the Treatment of Migraine

24

Symposium #3
Marco L. Loggia
Disrupted Brain Circuitry for Pain-Related Reward/Punishment in Fibromyalgia

125Lucia Hipolito
Chronic pain increases opioid self-administration through an accumbal dopaminergic mechanism

26

Anna Taylor
Neuropathic Pain Modulates Dopaminergic Circuitry: Role for Microglial Activation

27

Zhizhong Pan
Pain facilitates response to opioid reward via a shared epigenetic pathway

28

Symposium #4
Raül Andero Galı́
Amygdala-dependent fear is regulated by Oprl1 in mice and humans with PTSD

29

Kelly Standifer
NEUROINFLAMMATORY ACTIONS OF N/OFQ FOLLOWING TRAUMATIC STRESS

30

Thomas Jhou
Nociceptin/Orphanin-FQ and opioid mechanisms involving the rostromedial tegmental nucleus

31

Robert Innis
Imaging of nociceptin/orphanin FQ peptide (NOP) receptors in human brain and whole-body using anovel positron emission tomography radioligand, [11C]NOP-1A

32

Symposium #5
Julie A. Kauer
Blocking kappa opioid receptors, even after stress, rescues LTPGABA and prevents reinstatement ofcocaine seeking

33

Joel E Schlosburg
Dynorphin-kappa opioid receptor activation regulates the dynamics of drug-taking in the progression
towards addiction

34

Brendan M. Walker
Quiescent kappa-opioid receptors in the medial prefrontal cortex: the rollercoaster of cognitive control

35

Symposium #6
Maxime Descoteaux
Diffusion MRI : imaging the wiring of the brain

36

Philippe SéguélaOptogenetic control of pain pathways in vivo

37

Sotiris Masmanidis
Large-scale electrophysiology and virtual reality for reward circuit research38

Symposium #7
Thomas Kash
Kappa Opioid Receptor signaling controls the gain of an amygdalar anxiety circuit

39

Matthew Banghart
A photochemical approach to peptidergic neuromodulation

40

Symposium #8

2

Luda Diatchenko
Mu-opioid receptor gene locus: new functional variants

41

Pamela Kennedy
Chromatin Cross-Talk in the Nucleus Accumbens: Implications for Cocaine-Induced Behavioral andMolecular Adaptations

42

Marcelo A. Wood
The role of HDAC3 in acquisition and extinction of drug-seeking behavior

43

POSTERS
1.

A. Samoshkin
Structural and functional interaction between 6TM MOR isoform and β2-adrenoreceptors

45

2.

Aaron M. Bender
Synthesis and evaluation of mixed efficacy mu opioid receptor (MOR), delta opioid receptor (DOR)
ligands45

3.

Achla Gupta
The fungal collybolides as a new class of sesquiterpenes that selectively interact with the kappa opioid
receptor

46

4.

Adie Wilson-Poe
Chronic Morphine Treatment Alters Cannabinoid Modulation of GABAergic Synaptic Transmission in
the Periaqueductal Grey

46

5.

Akihiko Ozawa
EGFP-NOP Mice: Location in Spinal Cord and DRG

47

6.

Akiyoshi Saitoh
The DOR2 antagonist naltriben abolishes KNT-127-induced anxiolytic-like effects in rats.

47

7.

Amanda K. Fakira
NR2B-mediated changes in hippocampal spine morphology following morphine conditioned place preference48

8.

Andrea Bedini
Prolonged activation of Toll-like receptor 4 (TLR4) down-regulates NOPr expression in human glioblastoma cells, hampering nociceptin ability to counteract TLR4-mediated elevation of [Ca2+]i and induction of NF-kB

48

9.

Andrzej W. Lipkowski
Neuroprotective effects of biphalin in a mouse model of mild traumatic brain injury (mTBI)

49

10.

Anette Bendiksen
The Use of 0pioids in the Treatment of Young People with Chronic Non-Malignant Pain at a Multidisciplinary Pain Clinic in Denmark.

49

11.

Anna Lesniak
Opioid receptor expression in mice with persistent, long-term neuropathic pain

50

12.

Arthur Riegel
The Functional Rewiring of Cortical Synapses in a Translational Model of Neuropathic Pain

50

13.

Aubrie A. Harland
Synthesis, Optimization, and Evaluation of Mixed-Efficacy µ-Opioid Receptor Agonists/ δ-Opioid Receptor Antagonists: A Balancing Act

51

3

14.

Bernard P. Roques
DENKIs (Dual Enkephalins Inhibitors), a pharmacological and therapeutic breakthrough

51

15.

Bigliardi, PL
Delta Opioid receptors: Important Role In wound healing

52

16.

Brady Atwood
Effects of alcohol on mu opioid receptor-mediated plasticity at dorsal striatal inputs

52

17.

Brenda M. Ray
Naltrexone, like Nalmefene, is a Kappa opioid receptor partial agonist

53

18.

Brian Cox
Effects of morphine on GABAergic plasticity on dopamine neurons of the VTA: roles of PKA, AKAP,
calcineurin, and GABA-A receptor trafficking

53

19.

Brian Reed
Trypanosoma brucei as Novel Vaccine Platform for Oxycodone

54

20.

C. Mollereau
Mass spectrometry analysis of agonist-induced MOP receptor phosphorylation in the mouse brain.

54

21.

CJ Schier
The activity of HIV-1 Tat and morphine on Toll-like receptor 4 signaling Implications for mechanism ofactions contributing to HIV-related neuroinflammation

55

22.

Caroline C. Churchill
Adeno-associated virus mediated gene transfer of arginine decarboxylase reduces morphine analgesic
tolerance.

55

23.

Christine Neumann
The Delta-Opioid Receptor Affects Epidermal Homeostasis via ERK-Dependent Inhibition of Transcription Factor POU2F3

56

24.

Christoph Stein
Effect of Peripheral Opioid Receptor Blockade on Opioid Analgesic Demand: A Randomized Clinical
Trial

56

25.

Christopher R. McCurdy
SYNTHESIS AND EVALUATION OF DUAL-ACTIVITY OPIOID-NPFF LIGANDS FOR RECEPTOR
AFFINITY, ANTINOCICEPTION AND TOLERANCE LIABILITIES.

57

26.

Courtney Donica
Platelet-Derived Growth Factor Receptor-beta Antagonism Restores Morphine Analgesic Efficacy
Against Neuropathic Pain

57

27.

Cristina D Peterson
Intrathecal delivery of a bivalent ligand targeting a mu opioid Receptor mGLuR5 Receptor heteromer
reduces nerve-injury hyperalgesia

58

28.

Cullen L. Schmid
Novel ligands at the mu opioid receptor that display bias toward G protein coupling over βarrestin2
recruitment

58

29.

Dana Selley
Cannabinoid CB1 Receptor Induction of ΛFosB Mediates Mu Opioid Receptor Cross-Sensitization in
the Nucleus Accumbens

59

4

30.

Derek Simon
Calmodulin-dependent Kinase II Protein is Increased in the Hippocampus of Oxycodone Selfadministered Adult C57Bl/6 Mice.

59

31.

Devon Collins
Oxycodone causes greater locomotor activation in female compared to male C57BL/6J mice

60

32.

Dirk Tourwe
SYNTHESIS AND BIOLOGICAL EVALUATION OF A COMPACT, CONFORMATIONALLY CONSTRAINED BIFUNCTIONAL OPIOID AGONIST - NEUROKININ-1 ANTAGONIST

60

33.

Dominique Massotte
CO-LOCALIZATION OF MU AND DELTA OPIOID RECEPTORS IN THE NERVOUS SYSTEM USINGDOUBLE FLUORESCENT KNOCK-IN MICE

61

34.

Dr. Andrea Bedini
Ligand-directed signaling at mu opioid receptors: differential mechanisms of MAPK activation by morphine and fentanyl

61

35.

Dr. Selena Schattauer
Nalfurafine is a KOR agonist that produces analgesia, pruritus, inhibition of luteinizing hormone release,and ERK1/2 MAPK activation, but has low potency for p38 MAPK activation

62

36.

Dr. Selena Schattauer
Ligand-directed signaling at kappa opioid receptors: differential mechanisms of JNK MAPK activation
by U50,488 and norBNI

62

37.

Eriko Nakata
OPIOID DELTA RECEPTOR-MEDIATED β-ARRESTIN SIGNALING MODULATES CONVULSIVE
EFFECTS

63

38.

Erin Bobeck
Endosomal Endothelin Converting Enzyme-2: A regulator of opioid receptor trafficking

63

39.

Francia Fang
Increased Emergency Department Visits Caused by Tramadol Abuse are Associated with Opioid-RelatedOver-Dose Death in the U.S.

64

40.

Fraser McIntosh
Tramadol is an effective analgesic in an acute and chronic rat pain model

64

41.

Gary R. Matyas
Development of a Vaccine against Heroin

65

42.

Georgina Thompson
Comprehensive analysis of factors influencing quantification of biased signalling at the mu-opioid receptor

65

43.

Gina F. Marrone
Supraspinal endomorphin analgesia requires exon 11 variants of the mu opioid receptor (MOR-1)

66

44.

H. Machelska
Pain inhibition by leukocytic opioid receptors

66

45.

Hamid I Akbarali
The role of beta-arrestin2 in the development of morphine tolerance in the enteric nervous system

67

5

46.

Hanieh Bagheri Tudashki
Ligand ability to evoke Gαi activation and to promote internalization of DOR can predict response
duration over a short period.

67

47.

Heather Leduc-Pessah
Involvement of Microglial P2X7 Receptors in the Development of Morphine Analgesic Tolerance

68

48.

Heather Leduc-Pessah
Toll-like receptor 4 mutant and null mice retain morphine-induced tolerance, hyperalgesia, and physicaldependence

68

49.

Helen Sanderson
Agonist-induced phosphorylation of Thr370 and Ser375 in the COOH-terminus of the mu-opioid receptor

69

50.

Helmut Schmidhammer
Synthesis and Biological Characterization of Tritium-Labelled HS665, a Highly Potent and Selective
Agonist at the κ Opioid Receptor

69

51.

Hiroshi Ueda
Anti-opioid NMDA receptor theory underlying the lack of morphine analgesia in fibromyalgia-like model
mice

70

52.

Hélène Beaudry
Cyclin-depedent kinase 5 regulates Mu and Delta opioid receptor activities

70

53.

Iness Charfi
Molecular determinants of delta-opioid receptor recycling

71

54.

Ivone Gomes
Analysis of Morphine Regulated Striatal Synaptic Networks using Proteomics and Network Analysis

71

55.

JP McLaughlin
Evaluating the optimal ratio of mixed MOR/KOR agonism to prevent liabilities of use and cocaineconditioned place preference.

72

56.

Jacob Mahoney
Mechanism for the allosteric effects of G proteins on orthosteric ligand binding to GPCRs

72

57.

Jane Aldrich
Characterization of the Antinociceptive and Kappa Opioid Receptor Antagonist Activity of Analogs ofthe Macrocyclic Tetrapeptide CJ-15,208

73

58.

Jeffrey Wieskopf
Broad spectrum analgesic efficacy of IBNtxA is mediated by exon 11-associated splice variants of the
mu-opioid receptor gene

73

59.

Jenny Morgenweck
Functionally selective kappa opioid receptor agonist efficacy in pruritis

74

60.

Jessica Anand
DEVELOPMENT OF PERIPHERALLY AVAILABLE MIXED EFFICACY MOR/DOR LIGANDS THATDISPLAY REDUCED DEVELOPMENT OF TOLERANCE

74

61.

Jin XuExploring pharmacological functions of mu opioid receptor carboxyl termini in gene targeted mousemodels

75

6

62.

Joel Schlosburg
Optimization of an active vaccine against heroin: immunotherapeutic approaches towards long-term
blockade of heroin activity

75

63.

John M. Streicher
Identification of Novel Signaling Regulators of the Mu Opioid Receptor

76

64.

Jordan G. McCall
Wireless devices for optogenetic and pharmacological dissection of neural circuits in behavior

76

65.

K. A. Hymel
Stress-induced increases in depression-like and cocaine-seeking behaviors are reversed by disruption ofmemories during reconsolidation

77

66.

Karen J Tonsfeldt
Extrasynaptic GABAA receptors in the periaqueductal gray are involved in descending pain modulationand chronic inflammatory pain–induced plasticity.

77

67.

Karim Nagi
STRATIFICATION OF OPIOID ANALGESICS ACCORDING TO BIAS FOOTPRINTS AT MU- (MOR)
AND DELTA-OPIOID RECEPTORS (DORs).

78

68.

Karl Sköld
Post-sampling changes in opioid peptide levels and the effect of tissue stabilization

78

69.

Kathryn Livingston
Probing Allosteric Modulation of the Mu Opioid Receptor

79

70.

Kazuo Nakamoto
The involvement of brain GPR40 signaling in regulation of the descending pain inhibitory system

79

71.

Khaled Abdallah
Mapping delta opioid receptors in the rat central nervous system using an irreversible biotinylated ligand

80

72.

Laura G. Rosen
Opiate exposure state controls a molecular switch in opiate reward memory formation in the basolateral
amygdala-prefrontal cortical pathway

80

73.

Lee-Yuan Liu-Chen
Functional selectivity at the kappa opioid receptor (KOPR): from in vitro to in vivo

81

74.

Lydia Kotecki
GIRK channel activation in midbrain GABA neurons is not required for opioid-induced locomotor stimulation

81

75.

Magdalena I. Bujalska-Zadrożny
Influence of disulfiram on the development of tolerance on analgesic action of morphine

82

76.

Magdalena I. Bujalska-Zadrożny
Effect of new form of magnesium salt on analgesic activity of tramadol

82

77.

Mariana Spetea
Anti-inflammatory and Antinociceptive Action of 6β-Tryptophan Substituted 14-O-methyloxymorphone
in Mouse Models of Inflammatory Bowel Diseases

83

7

78.

Mark R. Nilges
Endomorphin analog analgesics lack reinforcement qualities and are promising candidate medications
for the treatment of opioid addiction

83

79.

Martin J. Schmidt and Madison A. Baird
Spatiotemporal Control of Mu-Opioid Signaling and Behavior

84

80.

Matthew Hearing
Repeated Morphine Promotes Bidirectional and Cell-Type Specific Adaptations in AMPAR plasticity in
Medium Spiny Neurons of the Accumbens Core and Shell

84

81.

Matthew Randesi
Variants of stress-related genes and their role in heroin self-exposure, addiction, and response to treatment

85

82.

Meritxell Canals
Delineating the dynamics of µ Opioid Receptor signalling and regulation

85

83.

Minghua-Hua Li
Postnatal changes in excitability and GABAergic transmission in rat rostral ventromedial medulla (RVM)
neurons

86

84.

Nicole Burma
Spinal P2X7R receptors are critically involved in the development of morphine physical dependence

86

85.

Orna Levran
Dopaminergic system genes polymorphisms and heroin addiction: further support for a protective effectof casein kinase 1 (CSNK1E) variants

87

86.

Osamu Nakagawasai
Angiotensin II type 1 receptor-mediated increase in spinal p38 MAPK phosphorylation leads to the
induction of nociceptive behavior in mice

87

87.

P. D. Mosier
Analogs of salvinorin A bearing sulfur-containing substituents at position C2 are high-affinity kappaopioid receptor partial agonists with a potentially novel binding mode

88

88.

Philippe Bourassa
Surface plasmon resonance as a label-free approach to monitor the mu opioid receptor-mediated signaling.

88

89.

Pierre-Eric Lutz
Opioid signaling in early-life adversity: a translational study

89

90.

Reagan L. Pennock
Differential effector coupling does not underlie resistance or susceptibility to desensitization of presynaptic mu opioid and GABAB receptors

89

91.

Ream Al-Hasani
Bimodal role for nucleus accumbens dynorphinergic neurons in aversion and reward

90

92.

Richard D. Howells
Novel small molecule triazoles modeled from naltrindole potently inhibit the proliferation of humanmultiple myeloma cells.

90

93.

Rob Hill
Tolerance to morphine respiratory depression: reversal by ethanol

91

8

94.

Ruihua Chen
Differential Modulation of the Signaling of µ–Opioid Receptor Agonists by the Positive Allosteric Modulator, BMS-986121

91

95.

Sandra D. Comer
Abuse liability of oxycodone and morphine in buprenorphine-maintained participants with or without
chronic pain

92

96.

Sarah L Withey
Reversal by Tamoxifen of tolerance to morphine-induced respiratory depression.

92

97.

Seikwan Oh
SYNTHESIS OF 3,4,5-TRIMETHOXYCINNAMIC ACID DERIVATIVES AND THEIR ANTINARCOTIC EFFECT

93

98.

Shay Q. Neufeld
Opioid modulation of inhibitory synapses in striatal patch compartments

93

99.

Shogo Tokuyama
Involvement of ERM proteins in the Development of Morphine Analgesic Tolerance through Pglycoprotein at the Blood–Brain Barrier

94

100.

Shuanglin Hao
Interleukin 4 mediated by HSV vector attenuates morphine tolerance and physical withdrawal in rats

94

101.

Sima Ajami
Substance Dependence Treatment Data Set; According to the Comparative Study of Substance Dependence Treatment Information System

95

102.

Sima Ajami
The Advantages and Barriers to Implement Substance Dependence Treatment Information System(SDTIS)

95

103.

Skylar Spangler
Dissecting Nociceptin Receptor Modulation of Reward

96

104.

St-Louis E., Parent J.-L., Gendron L.
Analysis of the second intracellular loop of DOP: A role in the membrane targeting of DOP

96

105.

Stephanie Puig
Platelet-derived growth factor circuitry underlying opioid tolerance

97

106.

Steven D. Chang
Quantitative Analysis of Functional Selectivity at the Nociceptin Opioid Receptor

97

107.

Susruta Majumdar
Utilizing the Ugi multi-component reaction to synthesize opioids

98

108.

Tomoe Kanbara
THE CONTRIBUTION OF Gi/o PROTEIN TO OPIOID ANTINOCICEPTION IN AN OXALIPLATININDUCED NEUROPATHY RAT MODEL

98

109.

Vadim Yuferov
Association of the OPRK1 intronic SNP rs6473797 with prolactin response to dynorphin administrationin normal male volunteers

99

9

110.

Vinod Tiwari
Subcutaneous injection of DALDA inhibits ongoing neuropathic pain and persistent inflammatory pain
in rats

99

111.

Wataru Nemoto
Antagonistic effect of angiotensin (1-7) on angiotensin II-induced nociceptive behavior in mice

100

112.

Yan ZhouInvolvement of kappa opioid receptors in chronic escalation alcohol drinking in mice

100

113.

Yanan Zhang
Tetrahydroisoquinoline-based orexin 1 receptor antagonists: structure-activity relationships at the 1benzyl position

101

114.

Yi-Ting Chiu
Deciphering agonist- and species-dependent KOPR phosphorylation using LC-MS/MS and phosphospecific KOPR antibodies

101

115.

Yingxian Pan
A heroin addiction severity-associated intronic single nucleotide polymorphism modulates alternative
pre-mRNA splicing of mu opioid receptor gene, OPRM1, as a splicing modifier via hnRNPH interactions

102

116.

Yong Zhang
EFFECT OF THE NOCICEPTIN/ORPHANIN FQ PEPTIDE RECEPTOR ANTAGONIST JTC-801
ON CYTOKINE EXPRESSION FOLLOWING EXPOSURE TO THE SINGLE PROLONGED STRESSMODEL OF PTSD

102

117.

ZG. LuRescue of IBNtxA analgesia, but not morphine, in a double E1/E11 MOR-1 knockout mouse modelusing lentiviral-mediated gene delivery of the 6 transmembrane (6TM) domain variant mMOR-1G

103

118.

Zafiroula Georgoussi
RGS4 and RGS2 proteins, new modulators of the κ-opioid receptor signaling

103

10

ORAL PRESENTATIONS

11

Allosteric modulation of the orthosteric binding site by G proteins:mechanism of high affinity agonist binding
Roger K. Sunahara
Department of Pharmacology, University of Michigan Medical School
Recent advances in the structural biology of G protein-coupled receptors (GPCRs) have unraveled many
intricacies of ligand binding. Our recent efforts to elucidate the crystal structure of the β2-adrenergic
receptor (β2AR), in a complex with the stimulatory G protein, Gs, have provided insight into how GPCR
activation leads to GDP release, the principle step that precedes GTP binding and G protein activation.
The structural data also reveal a possible mechanism through which G proteins in turn allostericallymodulate hormone binding. These and more recent high resolution structural data using β2AR-selective
camelid antibodies (nanobodies) suggest that occupation of the binding site for the G protein C-terminus
stabilizes high affinity agonist binding. The conformation changes results in the formation of a cap over
the orthosteric site, located at the extracellular face of the receptor, slowing ligand dissociation. Here we
provide functional evidence to suggest that this ‘closed’ conformation garners high affinity agonist-binding
properties through slowing the dissociation rates of ligands bound at the orthosteric site. Moreover, coupling of a nucleotide-free G protein or nanobody to a ligand-free, or empty receptor, can also stabilize
the ‘closed’ conformation and physically prevent a ligand’s access to the hormone binding site. Taken
together these structural changes and the slower observed ligand dissociation rates account for the Gprotein-dependent high affinity binding properties of agonists.

12

Optogenetic approaches to understanding synaptic plasticity and substanceuse disorders.
Antonello Bonci, MD
National Institute on Drug Abuse
The ventral tegmental area (VTA), nucleus accumbens (NAC) and prefrontal cortex (PFC) are all part ofthe limbic system and play a fundamental role in motivation, reward- and drug-dependent behaviors. A
few years ago, my laboratory has shown that drugs of abuse such as cocaine can produce long-term synaptic plasticity and that the duration of such plasticity is dependent upon the modality of drug or rewardadministration. By applying a multidisciplinary approach that includes electrophysiology, optogenetics
and behavioral procedures, my laboratory has produced a series of studies aimed at defining the pathways
that control and modulate reward and drug-dependent behaviors. During my presentation, I will present
the latest data on the cellular mechanisms and pathways that underlie reward substance use disorders.

13Expanding your toolbox; new approaches to probe the brain with light
Yves De Koninck
Université Laval and Institut universitaire en santé mentale de Québec, Canada
The future of neuroscience lies in our ability to assess, in a context sensitive manner, how each componentof the enormously complex CNS integrates, processes and transfers neurochemical information. Althoughcolossal advances in understanding cell signalling events have been made using ex vivo preparations, thehighly reactive and plastic CNS imposes in vivo studies to assess their relevance to normal function andpathology. Thus, the true enabling discovery technologies will be those that bridge single cell molecularsignalling studies with whole animal physiological and behavioural assessments.
The recent advent of photoactivatable proteins to generate novel sensors and actuators open new arraysof possibilities on this front. Yet, harnessing their full potential remains limited by properties of light suchas diffraction, absorption and scattering which restrict resolution and depth of observation/intervention.
Thus, our ability to probe and control cellular and molecular events across the length and time scales(from subcellular compartments to neuronal networks; from milliseconds to days) in vivo hinges on thedevelopment of novel techniques to deliver light and measure events with extreme sensitivity and precision.
I will describe recent techniques developed to undertake these challenges. At one end of the spectrum,
fluorescence fluctuation approaches to conduct quantitative analysis of molecular interactions in situ at
previously unachievable resolutions. At the other end of the spectrum, optical approaches to enable single
cell signalling and electrophysiology studies in deep brain structures. I will also describe means to detecting direct molecular constituents, such as lipids, without any label or contrast agent, which is critical fortranslation to human studies. I will finally discuss challenges that remain to develop methods to probe
and control neuronal activity non-invasively during behavior.
This work was funded by the Canadian Institutes of Health Research, the Pfizer-Fonds Recherche Québec– Santé Innovation Fund and the Natural Science and Engineering Research Council of Canada.

14

Cloning: Twice in One Day
Brigitte Kieffer and Christorpher Evans
Douglas Research Centre, McGill University, Canada; UCLA, USA
Cloning of opioid receptors was a significant and frustrating blockade for the progress of INRC researchfrom the mid 80’s till the final cloning of the delta opioid receptor in 1992. There were many approaches
attempted; some based on receptor purification, others on subtractive cloning or G-protein coupled receptor homology cloning. However, expression cloning won the day with two groups, one in Strasbourg,
led by Brigitte Kieffer, and one in Los Angeles, led by Chris Evans, successfully cloning the delta opioid
receptor from NG108-15 cells using very similar strategies and over the same time period. Papers were
submitted in the summer of 1992 and the delta opioid receptor sequence known by all by the winter of 1992.
As a result of cloning the delta opioid receptor, sequences of the kappa and mu opioid receptors werequickly cloned or discovered by homology. A fourth receptor, the Opioid Receptor-Like (ORL) Receptor
was also discovered by several homology cloners and a new GPCR system revealed. The cloning of the
opioid receptor family opened many new exciting possibilities for opioid research. These included; theidentification of the selectivity and structure activities of the opioid receptor family, the ability to tag
and follow receptor trafficking within live cells both in vitro and in vivo, and the ability to knockout andknock-in opioid receptors to determine receptor participation in innate and drug-modified behaviors. The
cloning clearly pushed the opioid field to another era of discovery but was just one piece of the puzzle.
The Holy Grail remains as to whether separating analgesic from addictive mu opiates can be achieved, andligand-biased signaling at these receptors may hold promise. Current questions in the field also include
evaluation of the true therapeutic potential for delta agonists or kappa antagonists in the treatment ofmood disorders. Finally, the complexity of endogenous opioid physiology, including receptor signaling andwithin-system interactions throughout the broad opioidergic neural networks, will surely maintain thisfascinating system at the center of neuroscience research.

15

Spatiotemporal Control of Opioid Systems in Reward and Aversion
Ream Al-Hasani, Jordan G. McCall, Gunchul Shin, Sung-Il Park, Jenny M. Wong, Omar S. Mabrouk,
Nicole A. Capik, Gavin Schmitz, Blessan Sebastian, Daniel Y. Hong, Michael J. Krashes, Bradford B.
Lowell, Thomas L. Kash, Robert T. Kennedy, John Rogers, Michael R. Bruchas
Washington University (RAH, JGM, GS, BS, DYH, MRB), University of Michigan (JMW, OSM), University of North Carolina (NAC, TK), University of Illinois (GS,SP, JR), Harvard University (BL, MK)
Stress responses are largely controlled by specific neurotransmitters and their receptors in the central
nervous system. Many of these signals are conveyed through activation of both neuropeptide (i.e. CRF
and Opioid) and monoamine (norepinephrine, dopamine, serotonin) receptor systems. These receptors areseven transmembrane spanning G-protein coupled receptors (GPCR) and they can stimulate a variety ofsignaling cascades following neurotransmitter/neuropeptide release. Neuropeptide and monoamine circuits
are engaged by stress, and elicit a complex array of behavioral responses relevant to anxiety, addiction,and depression. Two neuropeptide systems of major interest in stress responsivity include dynorphin
opioid peptides and CRF. These systems and circuits have classically been studied using pharmacologicalapproaches, in vivo and in vitro electrophysiology and biochemical methods. Here we will describe recent
advances in optogenetic technology including development and implementation of cellular scale wireless
optogenetic devices for in vivo behavioral measures. In addition, we report divergence of behavioral responses using optical control of discrete brain region subnucleai containing dynorphin expressing neurons.
We find that optical control of this neuropeptide system in select regions results in differences in reward
and aversion behavior. Finally, we will also discuss recent advances in controlling various monoamine andpeptide GPCR signaling pathways with optogenetic strategies and how these technologies reveal novel
insights into neuromodulator function in stress-induced affective behavior.

16

Molecular Control of δ-Opioid Receptor Signaling
Gustavo Fenalti, Patrick M. Giguere, Vsevolod Katritch, Xi-Ping Huang, Aaron A. Thompson, Vadim
Cherezov, Bryan L. Roth, Raymond C. Stevens
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550
North Torrey Pines Road, La Jolla, CA 92037, USA; National Institute of Mental Health Psychoactive
Drug Screening Program / Department of Pharmacology and Division of Chemical Biology and Medicinal
Chemistry, University of North Carolina Chapel Hill Medical School, Chapel Hill, NC 27599, USA
Opioids represent widely prescribed and abused medications, although their signal transduction mechanisms are not well understood. Here we present the 1.8Å high-resolution crystal structure of the humanδ-opioid receptor (δ-OR), revealing the presence and fundamental role of a sodium ion mediating allostericcontrol of receptor functional selectivity and constitutive activity. The distinctive δ-OR sodium ion sitearchitecture is centrally located in a polar interaction network in the 7-transmembrane bundle core, withthe sodium ion stabilizing a reduced agonist affinity state, and thereby modulating signal transduction.
Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue
Asn131 to Ala or Val augments constitutive arrestin-ergic signaling. Mutation of Asp95Ala, Asn310Ala,
and Asn314Ala transform classical δ-opioid antagonists like naltrindole into potent β-arrestin-biased agonists. The data establish the molecular basis for allosteric sodium ion control in opioid signaling, revealingthat sodium-coordinating residues act as “efficacy-switches” at a prototypic G protein-coupled receptor.

17

Which way is up? Rethinking Nociceptin Ligand Design and SAR from
Nociceptin Receptor Crystal Structure and Active-State Homology Modelcomparisons
NURULAIN T. ZAVERI, Ph.D.
Astraea Therapeutics
The antagonist-bound crystal structure of the nociceptin opioid receptor (NOP) was recently reported
along with those of the other opioid receptors bound to opioid antagonists. Crystal structures of theactive-state opioid receptor GPCRs bound to opioid agonists still remain to be determined. We recently
reported the first homology model of the ‘active-state’ of the nociceptin receptor (Daga and Zaveri, 2012).
Using this NOP ‘active-state’ homology model for docking NOP agonists and for molecular dynamics simulations was used to understand NOP ligand structure-activity relationships and agonist-induced receptor
activation. However, comparing binding poses of NOP antagonists in the NOP crystal structure to thebinding pose of NOP agonists revealed some interesting differences, which may be important to consider
during lead optimization of NOP receptor ligands. These insights have assisted our structure-based drug
design efforts for novel nociceptin ligand discovery. Grant Support: R01DA014026 and R01DA027811.

18

MMG22 selectively targets a putative MOR-mGluR5 heteromer fortreatment of chronic inflammatory pain
P.S. Portoghese, E. Akgun, M.M. Lunzer, M.I. Javed, A.J. Beitz, B.A. Smeester
Medicinal Chemistry, College of Pharmacy; Veterinary and Biomedical Sciences, University of Minnesota
The management of chronic pain with clinically employed analgesics is problematic due to developmentof tolerance and other adverse effects. As an approach to this problem, a bivalent ligand (MMG22) containing both mu agonist and mGluR5 antagonist pharmacophores recently was reported (PNAS USA,
2013:110, 11595) to produce potent antinociception via a putative MOR-mGluR5 heteromer in mice with
LPS-induced acute inflammatory pain . Here we present studies of MMG22 in a chronic inflammatory pain
model in mice implanted with fibrosarcoma cells. As intrathecal MMG22 exhibited 250,000-times greater
potency than a mixture of mu opioid agonist (M19) and mGluR5 antagonist (MG20) monovalent ligands,
the data are consistent with interaction of MMG22 with the protomers of a putative MOR-mGluR5 heteromer. Since a 572-fold potency increase of MMG22 observed over a period of 21 days after implantationof fibrosarcoma cells in mice mirrored the time-course increase of hyperalgesia, we investigated the possibility that activated NMDAR might be inhibited via a MMG22-occupied heteromer. After inhibition of
NMDAR-mediated hyperalgesia with MK801, the potency of MMG22 was reduced over a thousand-fold
and was similar to that of control mice and TLR4 KO mice. These results suggest that the NMDAR is
inhibited through physical association with a MMG22-occupied MOR-mGluR5 heteromer via allostericinteraction with the mGluR5 protomer. Presumably, the potent antinociception of MMG22 is due to a
combination of NMDAR inhibition and activation of the MOR protomer. There was no significant difference in the ED50 of morphine between control and LPS-pretreated mice in the presence or absence ofMK801, supporting the crucial role of inhibited mGluR5 protomer in blocking activated NMDAR. Given
the high potency, long duration (<24 hrs) of action, and absence of tolerance, MMG22 has potential asan analgesic for chronic inflammatory pain.
This project was supported by NIH grant DA030316.

19

Potential Relapse Prevention Agents Targetting Multiple Opioid Receptors
Stephen Husbands
University of BathCurrent treatments for drug addiction are only marginally effective; 70% of treated addicts relapse within
the first year following treatment, while the majority of drug addicts take more than one addictive drug
at any one time (poly-drug abuse).
Buprenorphine and mu opioid (MOP) receptor antagonists have been combined and shown, in preclinicaland early clinical studies, to be effective as a treatment of cocaine, alcohol and opiate abuse (particularly
relapse prevention) and also against major depressive disorder. The objective of this project has been to
mimic these combinations by developing single chemical entities that have potent kappa and delta opioid(KOP, DOP) receptor antagonist and measurable nociceptin/ORL-1 (NOP) receptor partial agonist activity coupled with substantially lower efficacy at MOP receptors than displayed by buprenorphine. Leadcompounds have been identified within the C7-methyl orvinol series. One such compound, BU10119, hasbeen confirmed as an opioid antagonist in vivo, exhibiting no antinociceptive activity in the acetic acidwrithing assay, while in the warm water tail withdrawal (50oC) assay BU10119 (10mg/kg sc) completely
suppressed the antinociceptive activity of the KOP receptor agonist EKC, and was not surmountable up
to 10mg/kg EKC (approx 100-times the ED50 dose of EKC) indicating very potent KOP antagonism.
Antagonism lasted for 24h, indicating an extended, but not overly long, duration of action. In the acetic
acid writhing assay BU10119 (1 mg/kg, sc) completely prevented the effect of 1 mg/kg morphine (sc),confirming MOP antagonism. In an assay for hyperalgesia, which responds to very low opioid agonist activity and NOP agonism there was a significant effect of BU10119 that was blocked by the NOP antagonist
J113397 but not blocked by the MOP antagonist naltrexone, confirming NOP receptor agonism in vivo.
Importantly, this compound which has MOP, KOP and DOP antagonist activity and NOP partial agonist
activity is effective at blocking reinstatement of cocaine seeking in rats induced by the pharmacologicalstressor yohimbine; a model of stress-induced relapse to cocaine.
Supported by NIDA Grant DA07315

20Delta Opioid Receptor Function in Skin: Controlling Pain Where it Starts
Gregory Scherrer
Stanford University
The delta opioid receptor (DOR) has long been considered a promising target for the treatment of chronic
pain. Specifically, numerous studies indicate that DOR agonists can reduce tactile hypersensitivity inrodent models of neuropathic or inflammatory pain. However, how and where DOR operates along neuralcircuits to control pain has remained unclear. Here I will provide evidence that the skin is a major locus of
DOR-mediated modulation of nociception. Early during embryonic development discrete populations oflarge-diameter somatosensory neurons of the dorsal root ganglia (DRG) start to express DOR. These neurons, in which DOR expression is maintained during adulthood, will give rise to myelinated mechanosensory
fibers that innervate the glabrous and hairy skin. Additionally DOR is expressed by cutaneous unmyelinated mechanonociceptors that co-express the G protein-coupled receptor MrgprD. Recordings using an
ex vivo somatosensory system preparation demonstrated that DOR-expressing DRG neurons respond tomechanical stimulation of the skin. In these mechanosensory neurons, DOR is present at the plasma membrane and trafficked along both central and peripheral axons. DOR-positive peripheral axons terminate
either as free nerve endings that meander through the epidermis to innervate the stratum granulosum, as
seen for MrgprD-positive fibers, or innervate differentiated structures in the skin, including Merkel celltouch domes, hair follicles or Meissner corpuscles, to form touch-detecting organs. I will also discuss thefunction of DOR in these peripheral terminals and how DOR regulates neuronal excitability in skin.
Financial support: National Institute on Drug Abuse grant DA031777.

21

Biased ligands at mu and delta opioid receptors: targeting selective
signalling to develop improved therapeutics
Jonathan Violin
Trevena Inc.
On-target adverse effects have limited the clinical utility of drugs targeting opioid receptors. ”Biased
ligands” stabilize subsets of receptor conformations compared to classical agonists and antagonists, andmay offer the possibility of dissecting beneficial and adverse pharmacology at the same receptor.
TRV130 is a G protein-biased ligand at the mu opioid receptor: it stimulates G protein coupling withpotency and efficacy similar to strong opioid analgesics, but stimulates markedly reduced coupling tobeta-arrestins. This profile translates to an improved therapeutic index in preclinical models of pain andopioid-related adverse events. Early clinical trials of TRV130 suggest that this profile may deliver enhanced analgesia with improved safety and tolerability compared to morphine. These results support theconcept that biased ligands can deliver differentiated pharmacology at opioid receptors.
Similarly, emerging data suggests that biased ligands targeting the delta opioid receptor may be able
to reduce the on-target seizure liability that has hindered drug discovery targeting selective delta opioid
agonists. Preclinical studies of beta-arrestin knockout mice and G protein-biased ligands suggests that
analgesic, anti-depressant, and anti-Parkinsonian benefits of delta opioid receptor activation can be engaged without engaging seizure liability.
Further discovery and characterization of biased ligands may uncover new opportunities to deliver differentiated therapeutics with improved efficacy, safety and tolerability.
Conflict of interest: Dr. Violin is an employee of Trevena Inc, a pharmaceutical company developing
some of the compounds that will be described in this presentation.

22

Increasing delta opioid receptor bioavailability
Manojkumar A. Puthenveedu
Dept of Biological Sciences, Carnegie Mellon University
The delta opioid receptor (DOR) is an attractive candidate as an alternate target for pain management.
However, DOR agonists are not very effective in vivo, although they are efficient at initiating signaling invitro. One potential explanation for this lower efficacy is that DOR is predominantly retained in intracellular compartments in neurons, therefore not being available at the cell surface to bind ligands. This
leads to the simple hypothesis that release of this retention will increase surface delivery of DOR, and,
therefore, higher efficacy of DOR agonists. We have developed a tractable model using PC12 cells andprimary sensory neuronal cell cultures to test this hypothesis at a molecular level. I will present our workon the molecular mechanisms and functional relevance of the retention and regulated surface delivery of
DOR. This work is supported by internal funds from CMU.

23

Delta Opioid Receptor Agonists for the Treatment of Migraine
Amynah A Pradhan
Department of Psychiatry, University of Illinois at Chicago
Migraine is an extraordinarily common brain disorder for which therapeutic options continue to be limited. The aim of this study was to use new and established preclinical mouse models to examine theeffects of delta opioid receptor agonists on basic mechanisms of migraine. The nitric oxide donor, nitroglycerin, is a consistent migraine trigger in humans, and was shown to evoke both acute mechanicaland thermal hyperalgesia in mice. Chronic intermittent administration of nitroglycerin resulted in a progressive and sustained hyperalgesia, as well as conditioned place aversion. Several delta opioid receptor
agonists, including SNC80, significantly reduced nitroglycerin-evoked hyperalgesia. SNC80 also abolishednitroglycerin-induced conditioned place aversion, suggesting that delta opioid receptor activation may also
alleviate the negative emotional state associated with migraine. In addition, we found that SNC80 significantly attenuated cortical spreading depression, a model of migraine aura that is considered useful in
predicting the efficacy of migraine preventive therapies. We have also used conditional knockout mice todetermine the different roles of central vs. peripheral delta opioid receptors in mediating the migrainerelieving effects of delta agonists. Together, our data demonstrates that delta opioid receptor agonists
modulate multiple basic mechanisms of migraine, and indicate that delta opioid receptors are a promisingtherapeutic target for this disorder.
This research was supported by NIH-NIDA Grants DA05010 and DA031243, the Shirley and Stefan Hatos
Research Foundation, and the Department of Psychiatry UIC.
A.Pradhan has a contract with Trevena.

24

Disrupted Brain Circuitry for Pain-Related Reward/Punishment in
Fibromyalgia
Marco L. Loggia
Harvard Medical School, Charlestown, MA
While patients suffering from fibromyalgia (FM) are known to exhibit hyperalgesia, the central mechanisms contributing to this altered pain processing are not fully understood. In this study we investigate
potential dysregulation of the neural circuitry underlying cognitive and hedonic aspects of the subjective
experience of pain such as anticipation of pain and of pain relief.
FMRI was performed on 31 FM patients and 14 controls while they received cuff pressure pain stimuli on
their leg, calibrated to elicit a pain rating of 50/100. During the scan, subjects also received visual cues
informing them of impending pain onset (pain anticipation) and pain offset (relief anticipation).
Patients exhibited less robust activations during both anticipation of pain and anticipation of relief withinregions commonly thought to be involved in sensory, affective, cognitive and pain-modulatory processes.
In healthy controls, direct searches and region-of-interest analyses in the ventral tegmental area (VTA)
revealed a pattern of activity compatible with the encoding of punishment: activation during pain anticipation and pain stimulation, but deactivation during relief anticipation. In FM patients, however, VTA
activity during pain and anticipation (of both pain and relief) periods was dramatically reduced or abolished.
FM patients exhibit disrupted brain responses to reward/punishment. The VTA is a source for rewardlinked dopaminergic/GABAergic neurotransmission in the brain and our observations are compatible with
reports of altered dopaminergic/GABAergic neurotransmission in FM. Reduced reward/punishment signaling in FM may relate to the augmented central processing of pain and reduced efficacy of opioid
treatments in these patients.

25

Chronic pain increases opioid self-administration through an accumbal
dopaminergic mechanism
Lucia Hipolito1 , Elaine Zhong1 , Jose Gonzalez-Romero1 , Laszlo Virag1 , Robert Whittington1 , Sandra D.
Comer2 , Susan M. Carlton3 , Michael R. Bruchas4 , Jose A. Morón 1(1) Department of Anesthesiology, Columbia University, 630 West 168th St., P&S Box 46, New York,NY 10032 USA. (2) Department of Psychiatry, Division on Substance Abuse, New York State Psychiatric Institute, College of Physicians and Surgeons of Columbia University, New York, NY 10032 USA.
(3) Department of Neuroscience & Cell Biology. University of Texas Medical Branch Galveston, Texas77555 USA. (4) Departments of Anesthesiology, Department of Anatomy and Neurobiology, Washington
University Pain Center, Washington University School of Medicine, Box 8054 St. Louis, MO 63110 USA.
Prescription opioid use is at an all-time high, and abuse of this class of medications has skyrocketedin the past two decades. Here we investigated the relationship between chronic pain and opioid selfadministration. We found a striking rightward shift in the dose response curve for heroin self-administrationin the presence of chronic pain with a concomitant alteration of dopamine release in the nucleus accumbens. Together, these results identify a previously unknown connection between chronic pain and loss of
dopaminergic signaling in opioid self-administration that induces opioid intake at very high doses, which
may act to promote opioid dose escalation and perhaps abuse.
This work was supported by NIH, NIDA grants DA027460 (JAM), DA035144 (MRB), and by the National
Center for Research Resources and the National Center for Advancing Translational Sciences, National
Institutes of Health, through Grant Number UL1 RR024156.

26

Neuropathic Pain Modulates Dopaminergic Circuitry: Role for Microglial
Activation
Anna M.W.Taylor, Annie Castonguay, Atefeh Ghogha, Niall P. Murphy, Yves Dekoninck, Christopher J.
Evans, Catherine M. Cahill
UC Irvine, UC Los Angeles, Laval University
The use of opioids for the treatment of non-cancer chronic pain is controversial because of suboptimal
analgesia and risk of iatrogenic addiction. The mesolimbic dopamine system, consisting of dopaminergic
cell bodies in the ventral tegmental area (VTA) projecting to the ventral striatum, is of particular interestin pain research because of its overlapping function in the negative affective state of pain, opioid reward,
and analgesia. In this study, we examine how chronic pain alters signaling in the mesolimbic dopamine
system and the effect this has on the reinforcing and analgesic properties of opioids.
A neuropathic pain condition was modeled in C57bl/6 male mice via a loose ligation of the left sciatic
nerve with a polyethylene cuff (chronic constriction injury; CCI). Ventral striatal dopamine release wasmeasured via microdialysis. After behavioral testing, brains were isolated and prepared for molecular andchemical analysis.
Systemic morphine two weeks after nerve injury failed to stimulate dopamine release in the ventral striatum. Nerve-injury also led to a disruption in the chloride (Cl-) gradient in GABAergic neurons in the
VTA, caused by a decrease in K+/Cl- co-transporter function. This disrupted Cl- gradient in GABA
neurons led to an increased inhibition on dopaminergic neurons as measured by lowered cFOS activationin dopamine cell bodies stimulated by systemic morphine in nerve-injured animals. GABAergic KCC2
function could be restored by interfering with BDNF signaling or by chronic treatment with the microglial
inhibitor, minocycline. This treatment also recovered morphine-stimulated dopamine release in the ventral
striatum in nerve-injured animals.
These results point to a global dysregulation in mesolimbic reward processing in chronic pain states that
may undermine the analgesic and hedonic attributes of opioids. Interfering with BDNF signaling or blocking microglial activation may represent a novel therapeutic target to ameliorate the analgesic effectivenessof opioids in chronic pain states.
This work is supported by NIH, CIHR and The UCLA Hatos Center. The authors declare no conflict ofinterest.

27

Pain facilitates response to opioid reward via a shared epigenetic pathway
Zhi Zhang, Wenjuan Tao, Yuan-Yuan Hou, Wei Wang, Paul J. Kenny, Zhizhong Z. Pan
University of Texas MD Anderson Cancer Center
In an animal model of chronic pain, we show that pain facilitates the acquisition of behaviors of opioid reward. The pain effect is mediated by pain- and opioid-mediated upregulation of the transcription regulator
MeCP2 in the central nucleus of the amygdala (CeA) that integrates emotional responses. The downstream
effects of MeCP2 upregulation include repression of the histone dimethyltransferase G9a and activation of
BDNF function. These findings suggest a shared epigenetic regulation pathway of MeCP2–G9a–BDNF in
CeA regulation of emotional responses to pain and opioid reward.
Supported by NIH National Institute on Drug Abuse grants DA023069 and DA027541. The authors
declare no financial conflict of interest.

28

Amygdala-dependent fear is regulated by Oprl1 in mice and humans with
PTSD
Raül Andero Galı́
Emory University
The amygdala-dependent molecular mechanisms driving the onset and persistence of posttraumatic stressdisorder (PTSD) are poorly understood. Recent observational studies have suggested that opioid analgesia
in the aftermath of trauma may decrease the development of PTSD. Using a mouse model of dysregulatedfear, we found altered expression within the amygdala of the Oprl1 gene (opioid receptor-like 1), which
encodes the amygdala nociceptin (NOP)/orphanin FQ receptor (NOP-R). Systemic and central amygdala
infusion of SR-8993, a new highly selective NOP-R agonist, impaired fear memory consolidation. In humans, a single-nucleotide polymorphism (SNP) within OPRL1 is associated with a self-reported historyof childhood trauma and PTSD symptoms (n = 1847) after a traumatic event. This SNP is also associated with physiological startle measures of fear discrimination and magnetic resonance imaging analysisof amygdala-insula functional connectivity. Together, these data suggest that Oprl1 is associated with
amygdala function, fear processing, and PTSD symptoms. Further, our data suggest that activation of
the Oprl1/NOP receptor may interfere with fear memory consolidation, with implications for preventionof PTSD after a traumatic event.

29

NEUROINFLAMMATORY ACTIONS OF N/OFQ FOLLOWINGTRAUMATIC STRESS
Kelly M. Standifer, Courtney L. Donica, Cindy D. Simpson-Durand and Yong Zhang
Department Pharmaceutical Sciences, College of Pharmacy; Oklahoma Center for Neuroscience and Deptof Cell Biology, College of Medicine; University of Oklahoma HSC, OKC, OK, USA
Post-traumatic stress disorder (PTSD) frequently shares co-morbidity with chronic pain and negatively
impacts the course of treatment for both disorders. We recently reported that persistent mechanical allodynia and thermal hyperalgesia were produced in an animal model of PTSD, single prolonged stress(SPS), in the absence of additional physical injury. The appearance of allodynia was preceded by elevated
TNFα in serum, and followed by elevated nociceptin/orphanin FQ (N/OFQ) in the CSF at day 9 and invarious brain regions associated with pain and emotional processing between days 9-28. In addition, levelsof the pro-inflammatory cytokine, IL-6, or its mRNA were elevated in spinal cord and amygdala during thesame time period. The neuropeptide, N/OFQ, has immunomodulatory actions in addition to modulating
nociceptive sensitivity, anxiety and many other responses. When rats received daily injections of the NOP
receptor antagonist JTC-801 on Day 7 of SPS, JTC-801 treatment reversed allodynia and hyperalgesia,
and returned N/OFQ and IL-6 levels back to baseline by day 21.
To better understand how N/OFQ modulates cytokine expression, cytokine mRNA levels in U937 human monocyte cells were assessed by quantitative PCR (qPCR). N/OFQ-induced up-regulation of IL-6
mRNA was blocked by NOP receptor antagonists and inhibitors of NFκB, p38 and Akt signaling. It was
not blocked by inhibitors of PKC or ERK MAP kinase. This suggests potential mechanisms by which
N/OFQ increases IL-6. Our findings suggest that N/OFQ may maintain chronic pain states by stimulating IL-6 up-regulation during chronic stress and that this up-regulation may contribute to allodynia andhyperalgesia in the PTSD model.
Animal protocol was approved by the OUHSC IACUC committee & U.S. Army Research and MaterielCommand ACURO office, and complied with the Animal Welfare Act and adhered to the principles
described in the Guide for Care and Use of Laboratory Animals. This study was supported by the Department of the Army DMRDP W81XWH-11-2-0077 and the Oklahoma Center for the Advancement of
Science and Technology. No conflict exists.

30

Nociceptin/Orphanin-FQ and opioid mechanisms involving the rostromedial
tegmental nucleus
Thomas Jhou
Medical University of South Carolina
The rostromedial tegmental nucleus (RMTg) was identified in 2009 as a midbrain GABAergic region that
encodes negative reward prediction errors, projects intensely to midbrain dopamine neurons, and receives
major inputs from the lateral habenula, frontal cortex, and other regions involved in motivated behavior. Subsequent studies showed the RMTg to be a critical modulator of aversive behavior, and of somephysiological and motivational effects of mu opioids. We now report that RMTg neurons in both rats
and transgenic mice express mRNA for prepronociceptin (PNOC), the precursor to the peptide nociceptin(also called orphanin FQ), as well as nocicstatin. The predicted function of PNOC in these neurons issomewhat different from its apparent roles in other brain regions, adding to an already diverse view of
PNOC roles in motivated behavior.
This work is supported by 1R03DA034431 and 1R01DA037327. No financial conflicts of interest are
declared.

31

Imaging of nociceptin/orphanin FQ peptide (NOP) receptors in humanbrain and whole-body using a novel positron emission tomography
radioligand, [11C]NOP-1A
T. G. Lohith1 , S. S. Zoghbi1 , C. L. Morse1 , N. A. Goebl2 , J.T. Tauscher2 , V.W. Pike1 , R. B. Innis1 , M.
Fujita1
1 Molecular Imaging Branch, NIMH, Bethesda, MD, USA, 2 Eli Lilly & Co., Lilly Research Laboratories,
Indianapolis, IN, USA
Introduction: Nociceptin/orphanin FQ peptide (NOP) receptor is a new member of opioid receptor that
may be linked to pathophysiology of pain and other neuropsychiatric disorders. To image NOP receptor
in vivo, we developed a new positron emission tomography (PET) ligand, [11C]NOP-1A, that has highaffinity (Ki = 0.15 nM) at NOP and high binding (50 - 70% specific) in monkey brain. Next, we assessed
the ability of [11C]NOP-1A to image and quantify NOP receptors for the first time in human brain andestimate its radiation safety profile in humans.
Methods: After i.v. injection of [11C]NOP-1A, two hour dynamic PET scans were performed in healthy
subjects for brain (n = 7), brain test and retest in same subject (n = 11), and whole-body imaging (n= 9). During brain scans, arterial blood was sampled serially to measure [11C]NOP-1A concentrations inplasma. From brain scans, total distribution volume (VT; a measure of receptor density) was determinedby kinetic modeling. Whole-body scans provided radioactivity biodistribution and radiation exposure estimates (effective dose).
Results: [11C]NOP-1A showed high radioactivity uptake ( 5–7 peak SUV) in brain. VT values (mL/cm3)
were high, ranged from 10.1 in temporal cortex to 5.6 in cerebellum, well identified ( 1.1% SE), and stable
after 70 min scan time indicating minimal entry of radiometabolites in brain. The retest variability andreliability of VT were moderately good averaging 12% and 0.46 for large brain regions. In whole-body,the radioactivity distributed in NOP receptor expressing peripheral organs - such as heart, pancreas andspleen. The average effective dose was 4.3 µSv/MBq, which is safe and similar to other 11C-labeled radioligands.
Conclusion: [11C]NOP-1A is an excellent PET ligand to study changes in NOP receptor density or receptor occupancy by novel drugs in living subjects.
Disclosures: Study was supported by NIMH IRP and by CRADA with Eli Lilly & Co. Authors declare no conflicts of interest.

32

Blocking kappa opioid receptors, even after stress, rescues LTPGABA andprevents reinstatement of cocaine seeking
Abigail M. Polter, Rachel A. Bishop, Lisa A. Briand, R. Christopher Pierce, Julie A. Kauer
1Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence
RI, 02908 2Department of Psychiatry Perelman School of Medicine at the University of Pennsylvania
Philadelphia, PA 19104
Stress can be an important precipitating factor that drives escalation or relapse of drug-seeking behavior,and dopaminergic neurons in the ventral tegmental area (VTA) are an important site of convergence ofdrugs and stress. We previously identified a long-term potentiation of GABAergic synapses onto theseneurons (LTPGABA). Multiple drugs of abuse and stress block or inhibit LTPGABA (Nugent et al, Nature, 2007; Niehaus et al, Eur J Neurosci 2010), suggesting that this common mechanism may play a rolein addiction and stress-related diseases. Our recent work shows that the block of LTPGABA by stressrequires glucocorticoid (GR, Niehaus et al 2010) and kappa opioid receptors (KOR, Graziane et al, Neuron, 2013), however, it remains unknown how long this lasts. As previously shown, LTP of GABAergic
synapses onto dopamine neurons in VTA slices can be triggered by application of a nitric oxide donor(part of the LTP signaling cascade) (IPSC amplitude, 155±28% of baseline values). After a five-minutecold water swim stress, LTPGABA is blocked for at least five days and returns to normal levels by ten
days. Thus, stress causes long-lasting, but not permanent, alterations in plasticity of VTA GABAergic
synapses. Remarkably, administration of the KOR antagonist nor-BNI 2 hours (131±7% of baseline) or 24hours (138±15% of baseline) after stress also rescues LTPGABA and preliminary data suggest rescue evenat 4 days after stress. In parallel, post-stress administration of nor-BNI (2 hours after stress) also prevents
reinstatement of cocaine self-administration. Our results show that a single exposure to acute stress cancause days-long changes in the reward circuitry mediated by KORs rescued by a KOR antagonist. No
conflicts of interest. This research was supported by NIH grants DA011289 (JAK), MH019118 (NMG
and AMP),AA007459 (AMP), DA033372 (LAB), DA15214 (RCP), DA22339 (RCP), DA33641 (RCP),
DA18678 (RCP) and a NARSAD Young Investigator Award (AMP).

33

Dynorphin-kappa opioid receptor activation regulates the dynamics ofdrug-taking in the progression towards addiction
Schlosburg JE, Vendruscolo LF, Park PE, Cohen A, Schmeichel BE, Whitfield TW, Crawford EF, & Koob
GF
1) Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La Jolla, CA;
2) Skaggs School of Pharmacy & Pharmaceutical Sciences, UCSD, La Jolla, CA
In an effort to understand the neurobiology, and potential therapeutic targets, involved in drug addiction,
identifying underlying differences between non-dependent use and escalating drug dependence is critical.
Increasing efforts are focusing on the negative reinforcing properties of drug withdrawal and activation ofbrain stress/dysphoric systems, which oppose the positive reinforcing/euphoric effects of drugs of abuse.
Key amongst these systems is the dynorphin-kappa opioid receptor system, which has established roles indecreasing dopamine response to drugs of abuse, as well as stress-induced drug relapse.
Using an extended-access model of drug self-administration (6-23 hr/session), we have demonstrated thata single systemic injection of the long-acting kappa opioid antagonist, nor-BNI, has powerful effectivenessin suppressing the progressive escalation and increased motivation for drug infusions typical in the transition to dependent drug-taking. Though to varying degrees, nor-BNI significantly blunts the increased
drug-seeking in self-administration of heroin, methamphetamine, cocaine, and nicotine. This inhibitionof drug escalation is paralleled by concurrent decreases in progressive ratio breakpoints for drug infusion,indicating that nor-BNI prevents enhancement of drug reinforcement efficacy. Evidence also suggests that
increasing drug intake produces increased prodynorphin content in key brain regions involved in reward(e.g. nucleus accumbens) and stress (e.g. central nucleus of the amygdala), which may guide points ofintervention for preventing continued progression of the addiction cycle. However, the specific patternof prodynorphin dysregulation appears to be drug-specific. Using intracranial infusions of nor-BNI, or
virally-mediated prodynorphin shRNA knockdown, we can replicate systemic effects into these same identified regions. Together, the evidence suggests dynorphin-kappa receptor overactivation is a key mediatorin excessive drug-seeking and taking.
Research supported by DA004043. Training for JES & TWT provided by AA007456 and DA033726,
respectively. The authors declare no conflicts of interest with this research.

34

Quiescent kappa-opioid receptors in the medial prefrontal cortex: therollercoaster of cognitive control
Brendan M. Walker
Washington State University
Understanding the neurobiological basis of impulsive-like behavior is critically important for the treatmentof numerous neuropsychiatric disorders. Impulsivity declines with age, but is a hallmark symptom of numerous neuropsychiatric disorders, including addictive disorders such as alcohol dependence. Utilizing a
combination of neurobehavioral techniques and agonist-stimulated GTPγS assays, we demonstrate that
central kappa-opioid receptor (KOR) stimulation induces an impulsive phenotype in rodents with greater
effects in younger animals than those that are older. Additional analyses identified that within the medial prefrontal cortex (mPFC), KORs show increased function in younger compared to older Wistar rats.
Furthermore, mPFC KOR function declined with the maturity to the extent of becoming ‘silent’ in older
rats. However, following chronic alcohol exposure, KOR function in the mPFC increased significantly
and may account for the cognitive deficits commonly observed in alcoholics. In support of this hypothesis, site-specific administration of a KOR agonist into the mPFC was shown to induce working memory
deficits. The present data highlight the role of the mPFC KORs as an endogenous impulse/cognitive control mechanism and are could be highly relevant in the clinical management of impulse/cognitive control
deficits commonly observed in many neuropsychiatric disorders. Disclosures: Support for this research
was provided in part by R01AA020394-01 from the National Institute on Alcohol Abuse and Alcoholism
and the WSU Alcohol and Drug Abuse Research Program according to the State of Washington Initiative
No. 171. BMW is a consultant for H. Lundbeck A/S, Denmark.

35

Diffusion MRI : imaging the wiring of the brain
Maxime Descoteaux
Sherbrooke Connectivity Imaging Lab (SCIL), Computer Science, Université de Sherbrooke
In this talk, diffusion MRI (dMRI) will be introduced, from its basics to its many applications for humanbrain mapping. We will see how one can go from raw diffusion data to full brain white matter structuralconnectivity. This computerized process is called tractography. In particular, we will see how diffusion
tractography is currently at the heart of connectomics studies and how it can be used for research andclinical applications. Connectomics is at the heart of modern neurosciences, neuroimaging and neuroinformatics.

36

Optogenetic control of pain pathways in vivo
Philippe SéguélaMontreal Neurological Institute- McGill University
Nociceptors are specialized primary sensory neurons activated by noxious stimuli. Their selective activation
and/or silencing can control pain perception. Using a conditional genetic strategy, we generated transgenic
rodent models in which pain is either optically evoked by activation of excitatory channelrhodopsin-2 innociceptors or inhibited by silencing nociceptor activity using hyperpolarizing opsins. Our approach consists of expressing the proton pump archeorhodopsin (ArchT) fused to EGFP in the Nav1.8+ nociceptors,
using the Nav1.8-Cre driver line. Cellular distribution of the ArchT-EGFP actuator was assessed in fluorescence in DRG, trigeminal ganglia, sciatic nerve, glabrous skin and dorsal horn of the spinal cord, andshowed a selective expression of ArchT in nociceptive somata and distal processes. Strong labeling both inspinal laminae I and II and in glabrous skin demonstrated an efficient trafficking of ArchT from cell soma
to central terminals and peripheral targets. Electrophysiological recordings confirmed significant outward
photocurrents and hyperpolarizations in ArchT-expressing cultured DRG neurons in response to yellow
light (589 nm) stimulation. These light-evoked inhibitions were sufficiently large to block electricallyas well as chemically-induced action potentials. Analgesic effects of transdermal illumination with yellow light were observed in freely moving transgenic mice. A complete behavioral analysis is currently
in progress as sensory phenotyping is conducted under several acute and chronic pain conditions. Our
results validate the development of constitutive transgenic models in which the function of geneticallydefined neuronal populations in somatosensory pathways can be optically and non-invasively controlledwith high spatiotemporal resolution. This technology allows the selective interrogation of candidate cellular targets in search of genetic or pharmacological treatment of chronic pain conditions resistant to opioids.

37

Large-scale electrophysiology and virtual reality for reward circuit research
Sotiris Masmanidis
University of California, Los Angeles
I will introduce a recently developed technology, nanofabricated silicon-based probes, for recording largescale neural activity across multiple brain areas. This technology routinely allows simultaneous recordingof over one hundred neurons from the basal ganglia and related brain areas. Additionally, I will discuss thedevelopment of a virtual conditioned place preference assay relying on olfactory cues, and some preliminary results in combining these techniques to understand the network-level mechanisms of reward-basedlearning and addiction.

38

Kappa Opioid Receptor signaling controls the gain of an amygdalar anxiety
circuitThomas Kash, Nicole Crowley
University of North Carolina Chapel Hill
A large body of behavioral evidence indicates that activation of kappa opioid receptors (KOR) increases
anxiety-like behavior. However, to date the circuit that mediates KOR induced anxiety has yet to be
identified. Using a combination of electrophysiological and optogenetic techniques we found that activation of KOR inhibits glutamate release in the bed nucleus of the stria terminalis (BNST) and prevents
the anxiolytic phenotype seen with activation of basolateral amygdala (BLA) to BNST projections. In
addition, optically-evoked dynorphin-induced heterosynaptic plasticity of glutamate inputs in the BNST
is demonstrated. These results demonstrate a local dynorphin-dependent, neuropeptidergic mechanism ofinhibiting an anxiolytic pathway, providing a discrete therapeutic target for anxiety treatment

39

A photochemical approach to peptidergic neuromodulation
Matthew R. Banghart1 , Shay Q. Neufeld1 , John T. Williams2 , Bernardo L. Sabatini1
1Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School, Boston, MA,
USA 2Vollum Institute, Oregon Health-Sciences University, Portland, OR, USA
The mammalian striatum is rich in the opioid neuropeptides enkephalin and dynorphin, as well as thetachykinin Substance P. Despite their abundance, little is known about their role in modulating striatal
activity. The inability to deliver these relatively large and hydrophobic molecules with precision in brain
tissue has limited quantitation of their actions and hindered studies into the kinetics of peptidergic transmission. The use of photoactivatable or ‘caged’ signaling molecules is widespread in neuroscience due tothe spatiotemporal precision afforded by light. To facilitate our studies into peptidergic transmission in
the striatum, we have developed caged opioid, tachykinin and cholecystokinin neuropeptides and a caged
analog of the opioid antagonist naloxone. Photochemical release of opioid peptides during electrophysiological experiments from striatal brain slices revealed two temporal domains of opioid signaling that are
specific to distinct targets within the striatal microcircuit. Furthermore, we have successfully integrated
optogenetic tools with our photochemical reagents using dual-color one-photon excitation to study peptidergic modulation of genetically identified synapses in the striatum. These tools should be generally
useful to the opioid research community.
This work was supported by the Helen Hay Whitney Foundation (MRB), the Charles A. King Trust/Bankof America Co-Trustee (MRB), the National Institute on Drug Abuse (K99DA034648 to MRB; RO1DA08163
to JTW), the Canadian Institute of Health Research (SQN), the National Institute of Mental Health(RO1MH085418 & R01MH100568 to BLS), and the Howard Hughes Medical Institute (BLS).

40

Mu-opioid receptor gene locus: new functional variants
L. Diatchenko1 , S.A. Shabalina5 , A.A. Samoshkin1 , C.T. Viet2 , M. Convertino3 , W. Maixner4 , N.V.
Dokholyan3 , B. Schmidt2 .
(1) Alan Edwards Centre for Research on Pain, McGill University, Montreal, Quebec, Canada; (2) Oraland Maxillofacial Surgery, NYU, New York, NY, USA; (3) Computational and Experimental Biophysics,
UNC, Chapel Hill, NC, USA; (4) Center for Neurosensory Disorders, UNC, Chapel Hill, NC, USA; (5)NCBI, NIH, Bethesda, MD, USA
Opioids are the most widely used analgesics to treat moderate to severe pain. However, the analgesic
efficacy of opioids is compromised by wide range of optimal dose response and side-effects such as tolerance, dependence and hyperalgesia (OIH). The primary target for clinically used opioids is the µ-opioid
receptor (MOR). Genetic variability within human OPRM1 gene locus, coding for MOR receptor, hasbeen proposed to explain variation in responses to opioids, with the primary focus on the non-synonymouschange A118G. Through the association study design we recently identified new functional genetic variant within OPRM1 gene locus, SNP rs563649, that led to the cloning and characterization of new human
MOR isoform MOR-1K. MOR-1K isoform codes for 6 transmembrane (TM) MOR variant, instead of 7TMstructure that is characteristic for GPCR, and shows substantial functional differences in comparison withmajor isoform.
Supported by NIH/NIDA grant # 5R41DA032293-02;
Conflict of interest: Shareholder and Consultant for Algynomics Inc

41

Chromatin Cross-Talk in the Nucleus Accumbens: Implications for
Cocaine-Induced Behavioral and Molecular Adaptations
Pamela J Kennedy (1) , Jian Feng (2) , AJ Robison (3) , I Maze (4) , Ana Badimon (2) , Ezekiel Mouzon (2) ,Dipesh Chaudhury (5) , Diane Damez-Werno (2) , Stephen J Haggarty (6) , Ming-Hu Han (5) , Rhonda BasselDuby (7) , Eric Olson (7) & Eric Nestler (2) .
(1) Department of Psychology, University of California, Los Angeles, Los Angeles, California, USA. (2)
Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount
Sinai, New York, New York, USA. (3) Department of Physiology, Michigan State University, East Lansing, Michigan, USA. (4) Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University,
New York, New York, USA. (5) Department of Pharmacology and Systems Therapeutics, Friedman Brain
Institute, Mount Sinai School of Medicine, New York, New York, USA. (6) Center for Human Genetic
Research, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston,
Massachusetts, USA. (7) Department of Molecular Biology, University of Texas Southwestern Medical
Center, Dallas, Texas, USA.
Repeated exposure to cocaine results in increased histone acetylation and decreased histone methylationin the nucleus accumbens (NAc), both of which promote drug-induced alterations in gene expression.
Histone deacetylases (HDACs) are a family of enzymes that regulate the acetylation of histone tails. We
show that local knockdown of HDAC1 from the NAc of floxed HDAC1 mice blocks cocaine-induced behavioral plasticity. We further show that pharmacological inhibition of class I HDACs in the NAc increases
global levels of histone acetylation (H3K9ac and H3K14ac), but also induces repressive histone methylation (H3K9me2), and blocks cocaine-induced molecular and behavioral adaptations. Repeated exposure
to cocaine increased expression of many GABAA receptor subunit genes in the NAc and increased thefrequency of inhibitory post-synaptic currents on medium spiny neurons. These effects of cocaine were
blocked by HDAC inhibition which was correlated with a prominent increase in repressive histone methylation at the promoters of GABAA receptor subunit genes. Our findings suggest a novel mechanism bywhich prolonged and selective HDAC inhibition can alter behavioral and molecular adaptations to cocaine.
Kennedy: Canadian Institutes for Health Research (ref 90086)
Eric Nestler: NIDA and NIH

42

The role of HDAC3 in acquisition and extinction of drug-seeking behavior
Melissa Malvaez, George Rogge, and Marcelo A. Wood
University of California Irvine
Repeated use of drugs of abuse causes persistent alterations in gene expression responsible for the longterm behavioral and structural changes in central reward pathways. Recently, it has been suggested that
epigenetic mechanisms are responsible, in part, for these drug-induced changes in gene expression. Epigenetic regulation of gene expression may provide transient and potentially stable conditions, which in turnmay ultimately participate in the molecular mechanisms required for neuronal changes subserving longlasting changes in drug-seeking behavior. Our research is focused on understanding the role of chromatin
modifying enzymes in the acquisition and extinction of context-drug associated memory formation. In
particular, we examine how the histone acetyltransferase CREB-binding protein (CBP) and the histone
deacetylase 3 (HDAC3) are pivotally involved in regulating histone acetylation required for transcription
underlying context-cocaine associated memory formation using the conditioned place preference (CPP)
paradigm. One exciting result of this research is that HDAC inhibition after establishing a CPP significantly facilitates extinction of drug-seeking behavior in a manner that is refractive to reinstatement.
Thus, understanding chromatin modifying mechanisms that establish and maintain drug-dependent plasticity changes may lead to a better understanding of substance abuse disorders as well as novel approaches
for treatment.
This research is currently supported by NIDA.

43

POSTER PRESENTATIONS

44

No. 1 - Structural and functional interaction
between 6TM MOR isoform andβ2-adrenoreceptors

No. 2 - Synthesis and evaluation of mixed
efficacy mu opioid receptor (MOR), delta
opioid receptor (DOR) ligands

A. Samoshkin1 , C.T. Viet2 , M. Convertino3 , W. Maixner4 ,N.V. Dokholyan3 , B. Schmidt2 , L. Diatchenko1

Aaron M. Bender1,2 , Aubrie A. Harland1,2 , Mary J.
Clark3 , Emily M. Jutkiewicz3 , John R. Traynor3 , Henry I.
Mosberg1,2

1Alan Edwards Centre for Research on Pain, McGill University,
Montreal, Quebec, Canada; 2Bluestone Center for Clinic Research,
New York University, New York, New York, USA; 3Departmentof Biochemistry and Biophysics, University of North Carolina at
Chapel, Chapel Hill, NC, USA; 4Center for Pain Research andInnovation, School of Dentistry, University of North Carolina at
Chapel Hill, Chapel Hill, NC, USA

Opioids are the most widely used analgesics to treat moderate to severe pain. However, the analgesic efficacy of
opioids is compromised by side-effects such as tolerance,
dependence and hyperalgesia (OIH). The primary target
for opioids is the 7 transmembrane (7TM) µ-opioid receptor (MOR). 6TM, an alternatively spliced isoform of
MOR, lacks the first TM and extracellular domains andhas been associated with high pain sensitivity and poor
response to morphine (Shabalina et al., 2009). Two independent groups recently showed that, 6TM is not localized
at the cell membrane, instead is retained in the intracellularcompartments (Gris et al., 2010; Majumdar et al., 2011).
Current literature hypothesizes that 6TM localization andfunctional activities could be strongly linked to a chaperone or a specific GPCR partner. It was also suggested the
β2-adrenoreceptor (AR) could be such co-factor (Liang etal., 2007; Hudson et al., 2010). To investigate this important possible link between 6TM MOR and AR we screened
three AR subtypes (β1-, β2- and β3-ARs) for their ability
to traffic 6TM to the cell surface. We expressed 6TM alone
or in combination with three AR subtypes and we screened
for their ability to alter 6TM localization in human cells.
We observed that, when expressed alone, 6TM was mainlypresent in the intracellular compartments and colocalizedwith marker for ER but not with the marker for plasma
membrane. When coexpressed with β2-ARs, 6TM wastranslocated from ER compartments to the plasma membrane. However, upon coexpression with β1- or β3- AR,
6TM retained its intracellular localization, suggesting thatβ2 could be the only subtype of AR responsible for 6TM
alterations. To further investigate the mechanism of action,
we demonstrate that coexpression of 6TM with β2-ARs in
neuronal Be(2)C cells increases the percentage of cells withpositive Ca2+ response upon treatment by a selective ligand for 6TM - 10µM IBNtxA (Majumdar et al., 2011),
compared to the cells transfected with 6TM alone.Our datastrongly suggest that β2-AR could be a structural and functional partner of 6TM MOR.

1.
Department of Medicinal Chemistry, College of Pharmacy,
University of Michigan, Ann Arbor, Michigan 48109, United States
2.Interdepartmental Program in Medicinal Chemistry, University ofMichigan, Ann Arbor, Michigan 48109, United States 3. Departmentof Pharmacology, University of Michigan, Ann Arbor, Michigan 48109

Although opioid analgesics represent the gold standard forthe treatment of acute and chronic pain, their usage isoften accompanied by undesirable side effects such as thedevelopment of dependence and tolerance. A considerable
amount of research has thus been done to find a potent
analgesic that does not display these negative attributes.
In general, clinically used opioids such as morphineevoke both the desired and undesired effects throughactivation of the mu opioid receptor (MOR). Numerous
reports have indicated that the undesired MOR-relatedside effects may be ameliorated by concomitant ligand
interaction with the delta opioid receptor (DOR). Ourcurrent research is focused on the development of small
molecule opioid ligands that display a mixed MOR/DOR
efficacy profile. Here, we describe a diverse series ofstructural modifications to opioid peptidomimetic 1, which
employs a tetrahydroquinoline (THQ) scaffold and ismodeled on a series of cyclic tetrapeptide opioid agonists.
Compound 1 is currently the only small molecule MOR
agonist/DOR antagonist reported to cross the blood brain
barrier, and is approximately equipotent to morphine in
the mouse warm water tail withdrawal assay following
intraperitoneal administration. Structural modifications
focused on increasing the metabolic half life of compound
1 will be presented, based on the results of metabolite
identification studies.
We also describe a series of 4-substituted piperidine andpiperazine compounds based on THQ 2, a compound thatshows balanced, low nanomolar binding affinity for MOR
and DOR. We have shown that by changing the lengthand flexibility profile of the side chain in this position,binding affinity is improved at both receptors by asignificant degree. Furthermore, several of the compounds
in this series also display good efficacy at MOR, while
simultaneously displaying DOR antagonism.

45

No. 3 - The fungal collybolides as a new classof sesquiterpenes that selectively interact withthe kappa opioid receptor
Achla Gupta1 , Ivone Gomes1 , Adrien Cavé3 , Heidi E.
Hamm3 , Joseph Parello3 , and Lakshmi A. Devi1,2
1Department of Pharmacology and Systems Therapeutics, 2Department of Neuroscience and Friedman Brain Institute, Icahn Schoolof Medicine at Mount Sinai, New York, NY, USA; 3Departmentof Pharmacology, Vanderbilt University School of Medicine,
Nashville,TN ,USANo. 4 - Chronic Morphine Treatment Alters
Cannabinoid Modulation of GABAergic
Synaptic Transmission in the Periaqueductal
Grey
Wilson-Poe, A.R., & Vaughan. C.W.
University of Sydney, Faculty of Medicine, Kolling Institute ofMedical Research

Selective kappa opioid receptor agonists and antagonists
have potential as therapeutics for the treatment of
psychiatric and neurological disorders, as well as obesity.
Most kappa receptor ligands are nitrogenous compoundswith tertiary amine function. An exception to this issalvinorin A, a non-nitrogenous diterpene extracted fromthe hallucinogenic Mexican mint Salvia divinorum thatexhibits potent and highly selective agonistic activity at
kappa opioid receptors. The structure of salvinorin A has
a furyl-δ-lactone motif; this motif is also present in collybolides, sesquiterpenes extracted from the fungus Collybia
maculata. We therefore examined whether collybolide
and its sesquiterpene diastereoisomer, 9-epi-collybolide
function as opioid receptor ligands. Competition binding
assays as well as functional GTPγS binding and cAMPassays carried out with human embryonic kidney cells
expressing either mu, delta or kappa opioid receptors showthat both compounds are highly selective kappa opioid
receptor agonists. Interestingly, collybolides display abiphasic binding and signaling profile with a transitionat very low ligand concentration (EC50 >10-12 M) thatis reported for the first time and likely originates from a
receptor population functionally different from the major
population associated with the transition at higher ligand
concentration. Interestingly, 7-epi-collybolide, a hemisynthetic epimer at the level of the furan group, also exhibitsa partial agonistic activity at higher concentrations. Taken
together, these studies show that collybolides represent a
novel class of selective kappa opioid receptor agonists that
could be developed as therapeutics in the treatment ofdrug addiction, mood disorders or pain.

Cannabinoids and opioids produce antinociception bymodulating GABAergic synaptic transmission in adescending analgesic pathway from the midbrain periaqueductal grey (PAG). While chronic opioid treatment
produces opioid tolerance, it has recently been shown toenhance cannabinoid-induced antinociception within the
PAG. This study examined the effect of chronic opioid
treatment on opioid and cannabinoid modulation of
GABAergic synaptic transmission in the PAG. Midbrain
PAG slices were prepared from untreated animals, andanimals that had undergone chronic morphine (4x 5
mg/kg), or saline pre-treatment over 2 days. Whole cellvoltage-clamp recordings were made from neurons within
the ventrolateral PAG. In slices from untreated animals,
the pan-cannabinoid receptor agonist WIN55212 andthe opioid agonist DAMGO reduced the amplitude ofelectrically evoked GABAa receptor-mediated inhibitory
postsynaptic currents (IPSCs) in PAG neurons, with
EC50s of 30 and 100 nM, respectively. The inhibitionof evoked IPSCs produced by WIN55212 (30 nM) andDAMGO (100 nM) was similar in PAG neurons frommorphine and saline treated animals. However, wheneIPSCs were recorded in the presence of the CB1 antagonist AM251, DAMGO-mediated inhibition of the current
was significantly greater in the morphine-treated neurons.
Finally, AM251 increased the frequency of spontaneous
miniature IPSCs in PAG neurons from chronic morphine,
but not saline treated animals. These results indicate thatwhile the potency of cannabinoid agonist-induced inhibition of GABAergic synaptic transmission is unaffectedby chronic morphine treatment, there is an enhancementof endocannabinoid-mediated tonic inhibition.
Thus,
endocannabinoid modulation of synaptic transmission
should be an important consideration in the developmentof novel opioid analgesic therapies.

This work was supported by NIH grants DA008863,
DA019521, and NS026880 to L.A.D.

Research was supported by the National Institute on
Drug Abuse of the National Institutes of Health underAward Number F32DA034464.

46No. 5 - EGFP-NOP Mice: Location in Spinal
Cord and DRG
Akihiko Ozawa1 , Jinhua Wu1 , Daniela Mercatelli1 , Gloria
Brunori1 , Sarah Low2 , Grégory Scherrer2 , , Brigitte
Kieffer3 ; Lawrence Toll1
1 Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL,
USA, 2 Stanford University School of Medicine, Stanford, CA USA,
3 Douglas Institute, McGill University, Montreal, Canada

NOP receptors and N/OFQ are found throughout thebrain, in the spinal cord, dorsal root ganglia, and a variety of peripheral tissues. To better characterize the localization of the receptor, immunohistochemical experiments
were conducted on mice in which the NOP receptor is replaced by an active NOP-eGFP receptor C-terminal fusion
protein. After perfusion and fixation, sections from dorsal
root ganglia and spinal cord were stained with anti-GFP
antibodies, as well as with antibodies to NF200, CGRP,
PKCγ, and with the isolectin IB4. In the DRG, NOPeGFP receptors are expressed on large, medium, and relatively small NF200+ (myelinated) cells that are mostly
CGRP-, suggesting they are not the peptidergic Aδ nociceptors. This suggests that the NOP-eGFP receptors
might be on myelinated low-threshold mechanosensory neurons, including down-hair afferents. Down-hair afferents are
the most sensitive mechano-sensitive somatosensory neurons and contribute to touch perception. Down-hair afferents have myelinated axons and relatively small diameter
cell bodies, and are NF200+. These cells contain neither
mu nor delta receptors. They project to the ventral border of inner lamina II of the spinal cord where PKCγ+
spinal neurons are located. There are also a number ofsmall cells that are NOP-eGFP+, NF200-, CGRP+: these
are likely the typical peptidergic nociceptive C-fibers, all ofwhich co-express mu receptors. In the spinal cord, NOPeGFP receptors are highly expressed in laminae I-III, colocalizing with PKCγ (ventral border of lamina II inner) as
well as with IB4 (dorsal border of lamina II inner), but appear to be expressed ventrally throughout the spinal cord.
In most of the cells visualized, whether they be from thespinal cord, DRG, or brain, NOP receptors appear to be located throughout the cell, rather than being localize to theplasma membrane. In summary, our immunohistochemical characterization reveals information regarding the cells
types containing NOP receptors in DRG and the laminar
location in the spinal cord, information that will be usefulin understanding the mechanism by which NOP receptors
alter a nociceptive response.
This work was funded by NIH

No. 6 - The DOR2 antagonist naltriben
abolishes KNT-127-induced anxiolytic-like
effects in rats.
Akiyoshi Saitoh (1) , Azusa Sugiyama (1,2) , Jun-Ichiro Oka(2), Hiroshi Nagase (3) , Mitsuhiko Yamada (1)(1) Dept. of Neuropsychopharmacology, NIMH, NCNP, Tokyo,
Japan, (2) Lab. Pharmacol, Fac. Pharm. Sci., Tokyo Univ. Sci.,
Chiba, Japan, (3), Dept. Med. Chem, IIIS, WPI, Tsukuba Univ.,
Ibaraki, 305-8577, JapanThe DOR2 antagonist naltriben abolishes KNT-127induced anxiolytic-like effects in rats.
Akiyoshi Saitoh (1), Azusa Sugiyama (1,2), Jun-Ichiro Oka(2),Hiroshi Nagase (3), Mitsuhiko Yamada (1)(1) Dept. of Neuropsychopharmacology, NIMH, NCNP,
Tokyo, Japan, (2) Lab. Pharmacol, Fac. Pharm. Sci.,
Tokyo Univ. Sci., Chiba, Japan, (3) Dept. Med. Chem,
IIIS, WPI, Tsukuba Univ., Ibaraki, Japan
Objective: We previously reported that the delta opioid receptor (DOP) agonists KNT-127 produce potent
anxiolytic-like effects in animal models. Here, we reporton the roles of DOR subtypes (DOR1 and DOR2) play inmediating KNT-127-induced anxiolytic-like effects.
Methods: Male Wistar rats were used for behavioral
experiments. The anxiolytic-like effect was evaluated using
the elevated-plus maze test. As reference, the antinociceptive effects were evaluated using the formalin test.
KNT-127 was administered 30 min before the test session.
The DOR1-selective antagonist 7-benzylidenenaltrexone(BNTX) and the DOR2-selective antagonist naltriben(NTB) were administered 30 min before KNT-127 treatment.
Results: Pretreatment with NTB completely abolishedKNT-127-induced anxiolytic-like effects in rats. By contrast, BNTX produced no effect at a dose that completely
blocked the antinociceptive effects of KNT-127.
Conclusion: We clearly demonstrated that the DOR2selective antagonist, but not the DOR1-selective antagonist, abolishes the anxiolytic-like effects of the DOR
agonist KNT-127, suggesting different roles of these DOR
subtypes in anxiety. We propose that DOR2-selective
agonists would be good candidates for future developmentof anxiolytic drugs.
Financial Disclosures: The authors report no biomedical
financial interests or potential conflicts of interest. This
research was supported by research grants from an
Intramural Research Grant (24-2) for Neurological andPsychiatric Disorders of NCNP.

47

No. 7 - NR2B-mediated changes inhippocampal spine morphology following
morphine conditioned place preference

No. 8 - Prolonged activation of Toll-likereceptor 4 (TLR4) down-regulates NOPr
expression in human glioblastoma cells,
hampering nociceptin ability to counteract
TLR4-mediated elevation of [Ca2+]i andinduction of NF-kB

A.K. Fakira1 , G.S. Portugal1 , R. Al-Hasani2,3 , S.A.
Golden5 , S.J. Russo5 , M.R. Bruchas2,3 ,4 , D. Sulzer6 andJ.A. Morón Concepcion1 .
1Department of Anesthesiology, College of Physicians and Surgeons,
Columbia University Medical Center, New York 2Departmentof Anesthesiology, Division of Basic Research, 3Department of
Anatomy–Neurobiology, and 4Washington University Pain Center,
Washington University School of Medicine, St. Louis, Missouri
5Fishberg Department of Neuroscience and Friedman Brain Institute,
Graduate School of Biomedical Sciences at the Ichan School of
Medicine at Mount Sinai, New York, New York 6Departments of
Neurology, Psychiatry, and Pharmacology, Columbia University
Medical Center, New York, New York,The hippocampus plays a critical role in the developmentof opiate preference and cravings triggered by environmental cues. The non-medical use of prescription drugs occurs
in 13% of young adults and 1.7% of the overall population.
Moreover, relapse to opiate addiction occurs at alarming
rates which signifies the need for improved therapeutics.
To understand the mechanisms that control the formation
and retrieval of drug associated memories we investigate
how morphine exposure in a novel context modifies hippocampal function. Following morphine conditioned place
preference (CPP), levels of the NR2B NMDA receptor subunit are increased in post-synaptic density fractions of the
hippocampus which persists after extinction of morphine
CPP. This may be important for cue dependent relapse as
hippocampal infusion of the NR2B antagonist ifenprodil
blocks morphine induced reinstatement. Furthermore, hippocampal LTP is impaired after morphine CPP and extinction, indicating that long lasting changes in synaptic
function occur after repeated morphine exposure. To investigate this, we are examining dendritic spines on CA1
pyramidal neurons of which there are three main varieties;
thin, stubby and mushroom. The formation and retraction of these spines is controlled by Rho family GTPases
by regulation of actin polymerization. Preliminary datasuggests that the density of thin spines is decreased following morphine CPP. Interestingly, it is known that increased
RhoA activity can decrease the number of thin spines andthat RhoA regulators p250GAP and kalirin-7 both form
direct interactions with the NR2B subunit. Future studies using a combination of 2-photon and confocal imaging
will determine how NR2B-RhoA signaling is involved inalterations of spine morphology after morphine CPP. Altogether, our data highlight new mechanisms underlying
context-dependent drug seeking behavior and may lead todevelopment new therapeutic approaches to preventing relapse.
NIH grant R01 DA025036

Andrea Bedini, Monica Baiula, Marco Caprini, Francesco
Formaggio, Jacopo Baldi and Santi Spampinato
Department of Pharmacy and Biotechnology (FaBiT) – University of
Bologna – Italy

INTRODUCTION: The bidirectional interaction between
neurons and neighboring glia contributes to the onset andmaintenance of chronic pain. Toll-like receptor 4 (TLR4) isa key glial activator inducing the release of cytokines andchemokines. The endogenous peptide nociceptin/orphanin
FQ (N/OFQ) and its receptor (NOPr) are involved in paintransmission and inflammatory response. Interestingly,
NOPr is expressed in glia and may be activated by N/OFQ
released by other neuronal or immune cells, possibly
playing a role in the glial adaptive responses to thealtered activity of nociceptive neurons in chronic pain.
We aimed to investigate any cross-talk between TLR4and NOPr-mediated intracellular signaling in a model ofhuman glia exposed to E. coli lipopolysaccharide (LPS), a
TLR4 agonist.
MEHTODS: Experiments were carried out in U87-MG
glioblastoma cells after acute (0-24h) or prolonged (72h)exposure to LPS (0.05–10ng/ml) and/or N/OFQ (1µM).
Interleukin-1β (IL-1β) mRNA levels were detected by
qPCR; LPS-induced elevation of [Ca2+ ]i and transcriptional activation of NF-kB reporter gene were detectedby calcium imaging and dual reporter gene assays,
respectively. LPS-induced changes of NOPr transcripts
and density of NOPr binding sites were investigated by
qPCR and receptor binding assays.
RESULTS: An acute activation of TLR4 by LPS induces
a significant increase of IL1-β mRNA that is blocked by
the simultaneous exposure to N/OFQ, which is also able
to counteract LPS-induced increase of [Ca2+ ]i and NF-kB
activation. The selective NOPr antagonist [Nphe(1)]Nociceptin (1-13)-NH2 prevents the inhibitory effect of
N/OFQ. Interestingly, a prolonged activation of TLR4
by LPS significantly down-regulates NOPr expression
through transcriptional processes requiring the activationof p38MAPK. In these conditions, N/OFQ-mediatedinhibition of LPS-induced increase of [Ca2+ ]i and NF-kB
activation is absent. These findings suggest a relevant role
played by N/OFQ/NOPr system, balancing glial activation
after an acute exposure to LPS; this action is lost after
a prolonged exposure to LPS that mimics activation of
TLR4 observed in chronic pain.
Funded by FARB2012, RFO2012, RFO2013

48

No. 9 - Neuroprotective effects of biphalin ina mouse model of mild traumatic brain injury(mTBI)
Anna Lesniak1 , Mariusz Sacharczuk2 , Chaim G Pick3 ,Andrzej W. Lipkowski1
1Mossakowski Medical Research Centre, Polish Academy of Sciences,
Warsaw, PL 02106, 2Institute of Genetics and Animal Breeding,
Polish Academy of Sciences, Jastrzebiec, PL 05552, 3Tel Aviv
University, Tel Aviv, Israel,

Traumatic brain injury (TBIs) concussion is often a resultof traffic accidents, contact sports as well as battlefield or
terrorist explosions. TBI is classified based on severity. A
mild form of traumatic brain injury (mTBI), sometimes
called mild head injury – MHI, often results in the postconcussion syndrome (PCS). Unfortunately, PCS is usually
underestimated, because the immediate physical symptoms
decrease rapidly and conventional neuroimaging studies ofthe brains of most mTBI victims often do not express any
radiological evidence of brain lesions. However, cognitive
impairments persist for weeks, months or even years
after the incident. A mouse weight drop model mirrors
well the mTBI-induced long-lasting learning and memory
impairments observed in humans [1].
Recent results
indicate that opioids, especially biphalin show promisinganti-neurodegenerative properties [2,3]. Therefore, we
decided to assess if an immediate post-injury injectionof biphalin provided any benefits in mTBI behavioral
impairments. After a systemic administration of biphalin
we observed an improvement in spatial and recognition
memory in the Morris Water Maze and Novel Object
Recognition tests 7 and 30 days post-trauma. Our new
data suggest that opioid receptor activation may provide
neuroprotection of post-traumatic neurodegeneration
processes. Further investigations will be carried out inthe development of optimal post-accidental therapeutictime-window for efficacious treatment of mTBI.
Acknowledgements: This work was supported by thePolish National Science Center (NCN), grant no.
2011/03/N/NZ4/02021 for Anna Lesniak. The technicalsupport by Zdzislawa Kowalska is highly acknowledged.
References:
1. Zohar O, Rubovitch V, Milman A, Schreiber S, Pick
CG (2011), Acta Neurobiol Exp 71: 36-45
2. Kawalec M, Kowalczyk JE, Beresewicz M, Lipkowski
AW, Zablocka B (2011), Neurochem Res 36: 2091-2095,
3. Yang L, Wang H, Shah K, Karamyan VP, Abbruscato
TJ (2011). Brain Res. 1383:307–316

No. 10 - The Use of 0pioids in the Treatment
of Young People with Chronic Non-Malignant
Pain at a Multidisciplinary Pain Clinic in
Denmark.
Anette Bendiksen, M.D.
The Multidisciplinary Pain Clinic, Friklinikken, Give, Denmark.

Background: In Denmark the general opinion among doctors is that as far as possible opioids should be avoided in
the treatment of young people with chronic non-malignant
pain.
Aim: To study the use of opioids in young patients from
17-28 years of age with chronic non-malignant pain treatedby trained pain specialists in a multidisciplinary pain clinic
in Denmark with a total of 3000 referrals a year.
Method: A retrospective systematic review of the recordsof all 60 young patients (45 female and 15 male) treated
medically in the clinic during 12 months from 2013-2014.
Results: 52% were in opioid therapy when referred to ourclinic. At the end of their therapy 65% of all patients wereon opioid treatment, 64% of the women and 66% of themen. In the clinic they were all placed on antidepressantsand anticonvulsants before opioids were started. The
opioids used when well treated were methadone in 51% of
the patients with a mean dose of 18 mg a day (4,5-40mg),
buprenorphine in 10% with a mean dose of 0,36 mg a day(0,12-0,6 mg), slow release morphine in 8% with a meandose of 18 mg a day (15-20mg), slow release tramadolein 26% with a mean dose of 225 mg a day (50-400 mg)
and tapentadol in 5% with a mean dose of 350 mg a day(300-400mg).
Conclusion: Despite the view on opioid usage in young
people with chronic non-malignant pain and the attempt
to avoid addictive drugs, this study shows that opioids
are necessary analgesics in young patients too in orderto achieve a satisfactory pain relief. Relatively low doses
and only long-acting opioids were used. There was nodifference between female and male patients with regard
to opioid use. Methadone was the preferred opioid because
it was well tolerated by most, and because of low level oftolerance development.

49

No. 11 - Opioid receptor expression in micewith persistent, long-term neuropathic pain
Jonsson A., Gronbladh A., Lesniak A., Hallberg M.,
Nyberg F.
Division of Biological Research on Drug Dependence, Department ofPharmaceutical Biosciences, Uppsala University, Uppsala, Sweden

Introduction
Chronic pain arising from peripheral nerve lesions serves achallenge due to its multifactorial character and consequent
resistance to treatment. Despite extensive research there is
still not enough knowledge about this phenomenon in the
aspect of developing effective pain-relief. There is muchcontroversy around the use of opioids in neuropathic pain.
Numerous studies bring forward hypotheses explaining
reduced effectiveness of opioid analgesia under neuropathic
pain conditions. Among them are the alterations in opioid
receptor expression, but studies employing different modelsof neuropathy demonstrate variable results. A decrease or
no change in opioid receptor expression was reported, whileothers claim that this is a dynamic process changing overtime. Therefore, our aim was to study the expression of 3
types of opioid receptors in a mouse model of long-lastingneuropathic pain.
Methods
To study opioid receptor expression we utilized a sparednerve injury (SNI) mouse model. Male, NMRI mice weresacrificed after 40 days after nerve ligation or sham surgery
and the dorsal and ventral portions of the lumbar andsacral parts of the spinal cord were removed. The relative
abundances of opioid receptor mRNAs was quantified in
qPCR analysis using specific primer sets.
Results
Transcripts of all 3 opioid receptors were detected in thedorsal horn of the spinal cord ipsilateral to the ligation.
But their amount did not differ as compared to the shamor control mice. There was also no alteration in theexpression level of either of the receptors.
Conclusion
Lack of alterations in opioid receptor expression in the
spinal cord dorsal horn in long-term neuropathy is thefirst step towards a notion that opioids may be adequate
for clinically meaningful analgesia in patients with a long
history of neuropathic pain. However, opioid receptor
functionality and interaction with other neurotransmitter
systems demands further investigation to support thisclaim.
Acknowledgements
This work was supported by the Berzelii Technology Centre for Neurodiagnostics and Swedish Medical ResearchCouncil (Grant 9459).

No. 12 - The Functional Rewiring of Cortical
Synapses in a Translational Model of
Neuropathic Pain
B. Harlan (1) , H. Hughes (1) , W. Buchta (1) , R. Wang (2) ,T. Shippenberg (2) , and A.C. Riegel (1)(1) Department of Neurosciences, Medical University of South
Carolina (MUSC), Charleston SC 29425; (2) Integrative Neuroscience
Branch, National Institute on Drug Abuse (NIDA), Baltimore, MD
21224

Neuropathic pain is a progressive neurological disease
and often refractory to therapeutic intervention. The
negative affect and emotion associated with chronic
pain suggests a rewiring of higher cognitive functions
and a dysfunctional cortex. To investigate the cellularmechanisms underlying this behavioral state, we used thespared nerve injury (SNI) model of neuropathic pain inwild-type and transgenic rats to assess structural andneuronal plasticity in the prelimbic cortex. This strategy
enabled us to link a persistent (<40d) SNI-induced
regional increase in C-Fos activation and increased densityof long/thin dendritic spine filaments ballistically loadedwith fluorescent DiI, with an overactive release of calcium
from the endoplasmic reticulum (ER) and neuronal
hyperexcitability. The increased firing measured in brainslices from SNI animals was not attenuated by blockadeof NMDA/AMPA receptors or activation of GABA-A/B
receptors, indicating an adaptation beyond synaptic
inputs. Instead, the hyperexcitability was linked to a
failure of intrinsic-inhibition mediated by KCNQ (Kv7)
ion channels, a loss of the slow after-hyperpolarization(AHP) and an elimination of inhibitory accommodation(spike-frequency adaptation)—all of which could be restored by blockade of PKA, manipulation of calcium storesor stabilization of KCNQ channels with retigabine. These
SNI-induced functional adaptations demonstrated an
unexpected specificity to CaMKII-positive, deep cortical(5/6) layer neurons in the prelimbic cortex contralateral to
the SNI.
Likewise, the SNI-induced structural changes in spine
density and filament length suggestive of new synaptic
connections in prelimbic layer 5/6 neurons were notobserved in pyramidal neurons in the visual cortex. Taken
together these findings demonstrate (1) the complexity of
the neuroadaptations during neuropathic pain, and (2)that the enduring hyperexcitability in the cortex during
this state often attributed to NMDA-dependent LTP may
actually result from an ER calcium-dependent loss of
intrinsic inhibition.
This work was supported by NIDA/NIH (DA033342& DA028811). No conflicts of interest.

50

No. 13 - Synthesis, Optimization, andEvaluation of Mixed-Efficacy µ-Opioid
Receptor Agonists/ δ-Opioid Receptor
Antagonists: A Balancing Act

No. 14 - DENKIs (Dual Enkephalins
Inhibitors), a pharmacological andtherapeutic breakthroughBernard P. Roques

2

1

2

Aubrie A. Harland , Larisa Yeomans , Aaron M. Bender ,Mary J. Clark3 , Jessica Anand1,3 , Qin Wang3 , Ira
Pogozheva1 , John R. Traynor3 , Henry I. Mosberg1,2
1 Department of Medicinal Chemistry, College of Pharmacy,
University of Michigan, Ann Arbor, Michigan 48109, United States
2 Interdepartmental Program in Medicinal Chemistry, University ofMichigan, Ann Arbor, Michigan 48109, United States 3 Departmentof Pharmacology, Medical School, University of Michigan, Ann
Arbor, Michigan 48109, United States

Several studies have shown that selective δ-opioid receptor (DOR) antagonism in the presence of a µ-opioid receptor (MOR) agonist or administration of a MOR agonist
in DOR knockout mice greatly reduces the developmentof MOR-mediated morphine tolerance and dependence.13 Unfortunately, multi-drug regimens and administrationof “drug cocktails” are often impractical and difficult toimplement in clinical practice due to undesired side effects and decreased patient compliance. With these limitations in mind, our lab is interested in creating and synthesizing a bi-functional ligand, or series of bi-functional
ligands, with a mixed-efficacy profile that display MOR
agonism/DOR antagonism. Although our original opioid
peptidomimetic series displayed the desired profile and validated our tetrahydroquinoline (THQ) scaffold as being asuitable and bioavailable template, the compounds in thisoriginal series displayed 10-130-fold preference for MOR
over DOR, and many of the compounds still exhibited
considerable affinity and efficacy at the κ-opioid receptor(KOR).4 In a subsequent generation of compounds, we have
explored two different modifications to our original scaffold in attempt to balance the affinity at MOR and DOR
while reducing KOR affinity and efficacy. One modification
included replacement of a THQ scaffold with a tetrahydronaphthalene (THN) scaffold. However, this change not
only unbalanced MOR and DOR affinities by preferentially
increasing affinity at MOR, it also disrupted the efficacy
profiles at all three receptors. Additionally, we modified
the original scaffold through N-acetylation of the nitrogenin the THQ core. This modification not only improved
DOR affinity, but also reduced KOR affinity without significantly altering efficacy profiles, thereby creating a bifunctional ligand with a cleaner and more balanced MOR
agonist/DOR antagonist profile.

Université Paris Descartes, INSERM U1022, 4 avenue del’Observatoire, 75006 Paris ; Pharmaleads, 11 rue Watt, 75013 Paris,
FranceEnkephalins are endogenous peptide-ligands of opioid
receptors with affinities for the mu (MOR) and delta(DOR) receptors in the range of morphine [1]. They are
released by intense stimuli such as pain but their effects
are transient (≤ min) due to their rapid degradationby two metallopeptidases, NEP and APN. As early as
1984, we have developed inhibitors named DENKIs (Dual
ENKephalinase Inhibitors) increasing concentration andhalf-life of ENKs, specifically in the painful area, hence
inducing a “physiological” analgesia. The most advanced
DENKIs are PL37 and PL265 both in clinical trials.
DENKI-induced analgesia is first new concept emerging in
the clinical treatment of pain since decades [1]. DENKIs
have two main indications : i) via i.v., severe acute or
chronic pain due to surgery, traumatisms or cancer; ii)
p.o., severe neuropathic pain, such as direct nerve suffering
due to chemical (antitumor drugs), metabolic (diabetes),
infectious (shingles) or mechanical (accidental section
during surgery) lesions.
Both types of pain are unsatisfactorily treated. Morphine(MO) remains the current treatment, but its side-effects
restrain its use. As DENKIs are devoid of MO side effects
P37 can be substituted, combined or alternated with
MO. Furthermore, the analgesia induced by protected
ENKs is amplified by their stronger effects than MO on
the emotional status of the suffering patient by DORs
recruitment in the whole limbic system of reward. This isfar from negligible when treating severe chronic pain.
Current medications for neuropathic pain have beendeveloped for other indications: depression for duloxetine,
seizures for pregabalin but are effective in less than half ofpatients, with total relief in only 30% of responders.
Opiates are sometimes prescribed in neuropathic pain,
but two issues limit their use: i) an inhibitory effect on
the spino-bulbo-spinal descending pathway, reducing theiranalgesic effects [1]; ii) need of high doses generating
numerous and inacceptable side-effects (tolerance andconstipation). As shown, none of these inconvenients wereobserved with ENKs. Finally, a six-fold synergy has been
demonstrated when PL37 and pregabalin are combined.
[1] B.P. ROQUES, M.C. FOURNIE-ZALUSKI and
M. WURM, Inhibiting the breakdown of endogenous
opioids and cannabinoids to alleviate pain. Nature Rev.
Drug Discovery, 2012, 11, 292-311.

51

No. 15 - Delta Opioid receptors: ImportantRole In wound healing

No. 16 - Effects of alcohol on mu opioid
receptor-mediated plasticity at dorsal striatal
inputsBigliardi-Qi M., 1 Neumann C., 1 , Bigliardi PL. 1,2
Brady K. Atwood & David M. Lovinger
1Institute of Medical Biology, Agency for Science, Technology &Research (A*STAR) 8A Biomedical Grove, 06-06 (Immunos), Singapore 138648 2Department of Internal Medicine, School of Medicine,National University Hospital, 5 Lower Kent Ridge Road,Singapore

In addition to its analgesic functions, the peripheral opioid
receptor system affects skin homeostasis through effects
on cell differentiation, proliferation, apoptosis, migration
and adhesion. Our group has shown that delta-opioid
receptor (DOR) knockout mice reveal as phenotype an
atrophic epidermis and over-expression of keratin 10. In
addition, the DOR knockout mice have delayed woundhealing in a burn wound model and have on the woundmargin epidermal hypertrophy and over-expression ofdermal MMP-2 and Collagen IV. This suggests that DOR
effects keratinocyte migration and/or proliferation during
skin homeostasis.
Recent results from our group reveal that not only
keratinocytes, but also human fibroblasts are under control
of delta opioid receptors and its ligands. We used an
in-vitro wound scratch model using human immortalized
keratinocytes (N/TERTS) and primary human fibroblasts
and we could observe that the DOR ligands were much
more stimulating migration than MOR ligands and that
there exists a complicated agonist/antagonist relationship.
New results on mice by using topically applied opioid
ligands show a significant improvement of wound healing
in a full thickness, punch biopsy model. In addition,
for the past a few years, it has been our major task to
ask what is the molecular consequence of delta opioid
receptor activation in keratinocytes and how could that
influence skin differentiation, homeostasis and finallywound healing. Our discovery of a transcription factor exclusively expressed in epidermis and acts as a bi-functionaltranscription factor activating and repression of keratin
expression is involved in this important signaling pathway.
no conflict of interests to declare

National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda,
MD, USAThe dorsal striatum is one brain area in which the opioid
system is strongly impacted by alcohol use. Alcohol use
produces changes in mu opioid receptor (MOR) levels
and functionality in dorsal striatum. Alcohol also alters
the levels of enkephalins, the endogenous MOR ligands
in the striatum. It is likely that these changes will alter
MOR-mediated signaling. It is unknown however, what
effect alcohol has on MOR-mediated synaptic plasticity
in this brain region. We previously showed that MOR
mediates long-term depression (mOP-LTD) of excitatorytransmission in dorsal striatum and that this plasticity
was sensitive to in vivo oxycodone exposure. mOP-LTD
is mutually occlusive with endocannabinoid-LTD and
endocannabinoid-LTD is impaired by both oxycodone andethanol exposure. We therefore hypothesized that alcohol
would impair mOP-LTD at dorsal striatal inputs similar
to oxycodone.
To determine the effects of in vivo oxycodone andethanol exposure, we used single I.P. injections of oxycodone and a chronic intermittent ethanol exposure(CIE) paradigm using vapor chambers in C57BL/6J mice.
Whole-cell patch clamp electrophysiology was used toprobe striatal inputs in mouse brain slices for changes to
MOR-mediated synaptic plasticity induced by exposure tooxycodone or alcohol.
DAMGO induces mOP-LTD of excitatory transmission in dorsal striatal brain slices. A single in vivo
exposure to oxycodone disrupted mOP-LTD for multiple
days following exposure. We also found that, similar
to oxycodone, 2 weeks of CIE disrupted the ability of
DAMGO to induce mOP-LTD in the dorsal striatum ofmouse brain slices. Therefore two distinct drugs of abusecause the same disruption to MOR-mediated plasticity inthe dorsal striatum. We are currently using an optogenetic
approach to determine whether these effects are broad or
restricted to specific striatal inputs.
No conflict of interest.
Work is supported by theDivision of Intramural Clinical & Biological Research of
NIAAA.

52

No. 17 - Naltrexone, like Nalmefene, is a
Kappa opioid receptor partial agonist

No. 18 - Effects of morphine on GABAergic
plasticity on dopamine neurons of the VTA:
roles of PKA, AKAP, calcineurin, and
GABA-A receptor trafficking

Ray, B.M., Ducat, E., Altschuler, J., Rahm, S., Reed, B.,
Kreek, MJ
The Laboratory of the Biology of Addictive Diseases, The Rockefeller
University, New York, USA

In prior published work from this Laboratory, nalmefene(10 mg) and naloxone (10 mg), both selective mu opioid
receptor antagonists, were administered intravenously (IV)to healthy human subjects on separate days. In males,
nalmefene caused a modest rise in prolactin levels, whereas
naloxone did not, demonstrating the kappa opioid receptor
partial agonist properties of nalmefene, as documented
in appropriate binding studies. We have now comparednalmefene to naltrexone administered IV to evaluate
kappa opioid receptor agonist activity of naltrexone. In
total, 13 healthy subjects participated: 5 males and 8
females. Males and females were analyzed separately, sincefemales have higher and more variable basal prolactin
levels than males. In males, naltrexone or nalmefene (10mg, IV) were given on separate study days. The serum
prolactin levels area under the curve (AUC; from 0-120
minutes post-antagonist administration) was similar forboth drugs. The AUC following naltrexone administration
was elevated compared to the AUC for the baselineday (Student’s T-test; p>0.005). The AUC following
nalmefene administration was similarly elevated compared
to baseline (Student’s T-test, p>0.01). In females, there
was an apparent elevation in serum prolactin levels after
naltrexone, as well as after nalmefene administrationcompared to baseline. However, due to the wide variability
both at baseline and after antagonist administration,
the apparent changes were not significant. These data
demonstrate for the first time that naltrexone is a kappa
opioid receptor partial agonist in humans.
Support: Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.
The authors declare no conflict of interest.

Brian M. Cox, Matthieu Dacher, Shawn Gouty, Jayaraj
Kodangattil & Fereshteh S. Nugent
Department of Pharmacology,
Bethesda MD 20814 USA

Uniformed

Services

University,

Morphine treatment modulates the synaptic strength of
GABAergic synapses onto dopamine (DA) neurons of the
VTA by modulating both long-term potentiation (LTP)
and long-term depression (LTD). Morphine inhibits a formof LTP mediated by NO and cGMP at these GABA
synapses (Nugent et al, Nature 446:1086, 2007). A single
exposure to morphine also blocks a recently discovered formof long-term depression (LTD-GABA) in DA neurons of
VTA (Dacher & Nugent, Neuropharmacol. 61:1166, 2011).
In LTD-GABA, the postsynaptic A-kinase-anchoring scaffolding protein 79/150 (AKAP79/150) signaling complex
selectively regulates inhibitory synaptic transmission bytethering protein kinase A (PKA) and calcineurin (CaN) to
GABAergic synapses (Dacher et al, J. Neurosci. 33:2650,
2013). Using immunofluorescence and whole-cell patch
clamp recording in rat midbrain slices, we report that postsynaptic DA D2 receptor activation in the VTA induces
LTD of GABAergic synapses on DA neurons through CaN
activation accompanied by PKA inhibition in a process requiring AKAP79/150 as a bridging signaling molecule. Activation of CaN through AKAP induces a clathrin-mediatedloss of GABA-A receptors in the synapse and a reductionin the magnitude of the IPSP, a process that is inhibited byprior morphine exposure. In contrast, disruption of AKAPmediated anchoring of PKA does not affect glutamatergic synapses onto DA neurons, suggesting that the PKAAKAP-CaN complex is uniquely situated at GABAA receptor synapses where it regulate plasticity associated withGABAA receptor trafficking. Opiate–induced disruptionof GABAergic LTP and LTD mediated through these recently elucidated signaling pathways in the VTA has thepotential to persistently alter the output of individual DA
neurons and of the VTA as a whole. These actions may
contribute to the reinforcing and/or addictive properties of
opiates.[Supported by the Whitehall Fndn and NARSAD(to FN) and USU].

53

No. 19 - Trypanosoma brucei as Novel
Vaccine Platform for Oxycodone
Brian Reed, Jason Pinger, Pete Stavrapoulos, Eduardo
Butelman, Mary Jeanne Kreek, Nina Papavasiliou
Laboratory of the Biology of Addictive Diseases; Laboratory of
Lymphocyte Biology - The Rockefeller University

Vaccination against abused drugs holds promise as ameans to prevent drug addiction, both as an aide toprevent recidivism in drug addicted persons in recovery, as
well as to prevent progression from those in early stagesof the drug addiction trajectory to prevent developmentof addiction. Prior attempts to develop such vaccines
demonstrated that immune system production of specificantibodies to drugs of abuse can suppress the effectsof these drugs, but indicate a need for better vaccine
platforms with more sustained production of higher levelsof antibodies, and with reduced individual variability.
Trypanasoma brucei brucei, a nonvirulent (in humans)
strain related to virulent T. brucei strains responsible
for African sleeping sickness, has properties of immune
stimulation suggesting it may serve as an improved
vaccine platform compared to conventional approaches,e.g. non-specific hapten conjugation to proteins such as
keyhole limpet hemocyanin, co-administered with adjuvant. Oxycodone was conjugated via the 6-ketone-position
using amino-oxy-propanethiol linkage to a peptide which
was then enzymatically coupled to the N-terminus of thesingle uniform coat protein of T.brucei, with up to 100
million uniformly presented oxycodone molecules present
on each T.brucei organism. In our initial studies in mice,
we detected oxycodone specific antibodies following 2
successive injections of 1.0x106 T. Brucei with conjugatedoxycodone. In parallel studies, we also detected antibodies
specific for a different small molecule, nitrophenol, following inoculation with T. brucei coupled to nitrophenol.
This seminal proof-of-concept study will serve as the basisof further studies of the immune response to T.brucei as asmall molecule vaccine platform.
Supported by: NIH-NIDA-CEBRA grant R21-DA036365(P.S., E.B.), NIH-NCATS-CTSA UL1-TR000043 Pilot
Award (B.R., J.P.), Dr. Miriam and Sheldon G. Adelson
Medical Research Foundation (M.J.K.) The authors haveno conflicts of interest.

No. 20 - Mass spectrometry analysis ofagonist-induced MOP receptor
phosphorylation in the mouse brain.
C. Mollereau1 , C. Froment1 , O. Schilz1 , L. Moulédous1 , S.
Schulz2 .
1 Institut de Pharmacologie et Biologie Structurale CNRS/Universitéde Toulouse, 205 route de Narbone, 31077 Toulouse, France 2
Institute of Pharmacology and Toxicology, Jena University Hospital,
Friedrich Schiller University Jena, 1 Drackendorfer strasse, D607747
Jena, Germany

Phosphorylation of the mu-opioid (MOP) receptor isthought to be one of the initial steps leading to thedevelopment of analgesic tolerance. Many in vitro data
have shown that variation in the efficacy of several opioid
drugs in terms of desensitization, arrestin recruitement andinternalization is correlated to differential MOP receptor
phosphorylation. High-efficacy agonists such as DAMGO
induce higher levels of MOP receptor phosphorylation andinternalization than low-efficacy agonists such as morphine.
The relative importance of particular residues has been
approached by site-directed mutagenesis and developmentof phosphosite-specific antibodies and, more recently, bymass spectrometry analyses which is the only methodable to demonstrate the presence of multiphosphorylation
on a single receptor. Phosphorylation of MOP receptor
occurs primarily at S363 and within a cluster of serine andthreonine residues (T370, and 375STANT379) within thecytoplasmic tail of the receptor. Depending on the drug,
multiple phosphorylation sites have been identified andshown to be sequentially and differentially phosphorylatedby diverse GRK. Most of these data have been obtainedin cellular models and it is now essential to study the
post-translational modifications of MOP receptors in vivo
in order to understand the molecular basis for the diversityof acute and long-term opioid effects.
Here, we describe for the first time by using Nanoflow
Liquid Chromatography-tandem Mass Spectrometry(NanoLC-MS/MS), the analysis of endogenous MOP
receptor phosphorylation from the brain of mice administered with different opioids. The agonist-induced
phosphorylated residues, including multi-phosphorylated
clusters on a single receptor, were identified and theirrelative abundance quantified by a label-free method.
The data contribute to demonstrate drug-selective andhierarchical multiphosphorylation of MOP receptor invivo. A part of this work is reported in Glück et al. (BiolPsy, in press).
Financial support : Region Midi-Pyrénées and European
funds (FEDER)

54

No. 21 - The activity of HIV-1 Tat andmorphine on Toll-like receptor 4 signaling
Implications for mechanism of actions
contributing to HIV-related
neuroinflammation
CJ Schier1 , N El-Hage1 , EM Podhaizer1 , M Rodriguez2 ,PE Knapp1 , KF Hauser1
1Virginia Commonwealth University, Richmond, VA 23298, 2Universidad Central del Caribe School of Medicine, Bayamon, PR 00960

Opiate drug use by HIV-1 positive individuals is reported
to exacerbate HIV-associated neuroinflammation. In addition to µ-opioid receptors, opiate drugs reportedly interact
with the Toll-like receptor 4 complex (TLR4) in models ofchronic pain and neuroinflammation. Likewise, TLR4 is a
plausible target for HIV-1 virotoxins such as Tat; therefore, we aimed to clarify the role of morphine and Tat in
TLR4 activation using acute HIV–morphine in vitro models. We initially employed a human, HEK cell line engineered to stably express CD14 and MD2 either with TLR4
[TLR4(+) cells] or without TLR4 [control, TLR4(-) cells],
and containing a NF-κB-inducible reporter that quantifies
TLR4 activation. We found that an acute, 16-h exposure
to HIV-1 Tat1-86 (100 nM) increased TLR4 reporter activity; however, at the time and conditions assessed, exposure to morphine (500 nM–5 µM) did not activate the
TLR4 reporter. Using an ELISA, we noted that a 24-h exposure to Tat, but not morphine, increased CCL2 releasein TLR4(+), but not TLR4(-) cells. LPS-EK and LPSRS were used as positive and negative controls to assure
TLR4 specificity. We sought to confirm these results using acutely treated, primary, mixed-glial cultures isolated
from wild-type and TLR4-null mice. Wild-type, but not
TLR4-null mixed glia, showed increased release of CCL2
and CCL5 after 12 h of stimulation with TLR4 agonist LPSEK, Tat, or combined Tat and morphine, but not morphine
alone. Model validation using TLR2 (peptidoglycan) andTLR3 (Poly IC) agonists assessing the capacity of TLR4null glia and macrophages to activate NF-κB independently
of TLR4 are planned. Overall, our results suggest that
acute exposure to Tat, but not morphine, activates TLR4
and chemokine release. Thus, TLR4 expression plays alarge role in the acute, Tat-induced chemokine response.
Further experiments are required to explore more chronic
HIV and opiate exposures, in which prolonged elevations
in TLR4 levels may lead to fundamentally different interactions with morphine compared to acute models. Support:
NIH T32 DA007027, K02 DA027374, and R01 DA034231.
The authors declare no conflicts of interest.

No. 22 - Adeno-associated virus mediatedgene transfer of arginine decarboxylase
reduces morphine analgesic tolerance.
Caroline C. Churchill1 , Daniel Schuster2 , Lalitha Belur3 ,Stephen Schnell2 , Maureen Riedl2 , Cristina Peterson1 ,,Kelley F. Kitto1 , R. Scott McIvor3 , Lucy Vulchanova4 ,Carolyn A. Fairbanks1, 2 ,5,6
Departments of 1Experimental and Clinical Pharmacology, 2Neuroscience, 3Genetics Cell Biology and Development, 4Molecular
Veterinary Biosciences 5Pharmaceutics, 6Pharmacology, Universityof Minnesota, Minneapolis, MN 55455 (USA).

Background: The enzyme arginine decarboxylase (ADC),
decarboxylates arginine to produce agmatine. Agmatine
has been shown to prevent the development of opioid
analgesic tolerance when given by many routes of administration and in multiple models of tolerance. Since
agmatine is produced endogenously, the opportunity arises
to develop a method for enhancing expression of ADC andpotentially the therapeutic effects of agmatine, locally andconstiutively. We have recently characterized a method fordelivery gene therapeutics to the central nervous systemusing simple intrathecal injection of adeno-associated virus(AAV) vectors by direct lumbar puncture in conscious
mice. We report here the effects of gene transfer of ADC
in a model of opioid induced tolerance.
Methods: ICR-CD1 male mice were pre-treated witheither AAV5-ADC vector or saline eight weeks prior to
testing. Thermal sensory responses were determined using
the warm water tail flick immersion test (52.5°C). Mice
were give seven subcutaneous (s.c.) injections of either
morphine (3 mg/kg (4 times) and 5 mg/kg (3 times)) or
saline over the course of 3 days. On day four, tail flick test
was performed and cumulative dose-response curves to s.c.
morphine (1, 3, 10, 20 mg/kg) were constructed in eachof the four pre-treatment groups (Saline-Saline, SalineMorphine, AAV5-ADC-saline, AAV5-ADC-morphine.)
Relative potency (Tallarida 1987) was calculated for eachdose-response curve and the development of morphine
tolerance assessed in each of the two primary treatmentgroups.
Results: A significant rightward shift in the morphine dose-response curve was observed in control
subjects, as expected. However, in subjects pre-treatedwith AAV5-ADC, morphine tolerance did not develop.
Conclusion: We conclude that the observed AAV5ADC prevention in morphine tolerance is likely due
to production of the decarboxylase product of ADC,
agmatine, which has been shown repeatedly to prevent
opioid tolerance.

55

No. 23 - The Delta-Opioid Receptor Affects
Epidermal Homeostasis via ERK-Dependent
Inhibition of Transcription Factor POU2F3

No. 24 - Effect of Peripheral Opioid Receptor
Blockade on Opioid Analgesic Demand: A
Randomized Clinical Trial

Neumann, C. 1 , Bigliardi-Qi, M. 1 , Widmann, C. 2 ,Bigliardi, P.L. 1, 3

Christina A. Jagla, Peter Martus, Christoph Stein
Freie Universitaet Berlin - Charité

1 Experimental Dermatology Laboratory, Institute of Medical
Biology, A*STAR, 138648 Singapore; 2 Department of Physiology,
University of Lausanne, 1005 Lausanne, Switzerland; 3 NUH/NUS,
National University/Hospital of Singapore, 119228 Singapore

The expression of several neurohormones, neurotransmitters, and neuropeptides, including pro-opiomelanocortin
derivatives, such as β-endorphin and [Met5]-Enkephalin,
as well as expression of the corresponding receptors has
been shown in skin. In view of the constant environmental
assaults that the skin must endure, the presence of thisneuroendocrine system has been suggested to be importantfor cutaneous homeostasis and during wound healing.
However, there is currently a lack of understanding of themolecular mechanisms by which neuropeptide signalling
can modulate keratinocyte function.
Here, we show by Western blot that DOPr signalling
specifically activates the ERK 1/2 MAPK pathway inhuman keratinocyte cell lines.
Quantitative imaging
suggested a reduced proliferation rate of DOPr activatedkeratinocytes in culture. Furthermore, DOPr activation
markedly delays induction of keratin intermediate filament
Keratin 10 (KRT 10) and KRT 1 during in vitro differentiation, observed both by quantitative real-time PCR andWestern blot, and abolished the induction of KRT 10 inan organotypic skin model. This is accompanied by deregulation of involucrin, loricrin, and filaggrin, illustrated bya markedly reduced induction of their expression upon
initiation of differentiation in vitro. Additionally, POU2F3
was identified as a transcription factor mediating the
DOPr-induced regulation of keratinocyte differentiation
related genes.
It was revealed that DOPr-mediated
ERK-dependent downregulation of this factor affects key
aspects of keratinocyte function.
Complementing previous studies in DOPr-deficient mice,these data suggest that DOPr activation in human keratinocytes significantly influences epidermal morphogenesis
and homeostasis, and might have a major impact during
the wound healing process.

Peripherally mediated opioid analgesia is a promising
approach to pain treatment that avoids the deleterious side
effects of centrally acting opioids and nonsteroidal analgesics. To gauge the therapeutic potential of this strategy,
we assessed the proportion of morphine analgesia mediatedoutside the central nervous system. We hypothesizedthat the selective blockade of peripheral opioid receptors
by methylnaltrexone (MNX) will increase the patients’demand for morphine to achieve satisfactory postoperative pain relief. In a double-blind, placebo-controlled,sequential two-center trial, 50 patients undergoing knee
replacement surgery were randomized to receive MNX (0.9mg/kg s.c.) (hospital I: n = 14; hospital II: n = 11) orsaline (hospital I: n = 13; hospital II: n = 12) at the end of
surgery. Primary outcome was the cumulative amount ofi.v. morphine administered over 8 hours postoperatively.
Secondary outcomes were pain scores at rest and during
movement, vital signs and adverse side effects. After
MNX, demands for morphine (hospital I: 35.31 ± 12.99 mg
vs. 25.51 ± 7.92 mg, P=0.03; hospital II: 35.42 ± 11.73
mg vs. 24.8 ± 7.84 mg, P = 0.02; pooled data: P > 0.001;
means ± SD) were strongly (by about 40 %) increased.
Secondary outcomes were similar in all groups (P <0.05). Thus, a significant proportion of analgesia producedby systemically administered morphine is mediated byperipheral opioid receptors. Drugs that selectively activate
such receptors should have the potential to produce
powerful clinical pain relief.
The study was funded by International Anesthesia
Research Society, Helmholtz Gemeinschaft, and European
Commission (FP7-602891-2).

The authors have declared that no competing interests exist.
These studies were supported by funding from Swiss
National Science Foundation (SNSF) grant 31003A-116811
to M. Bigliardi-Qi and by the Singapore Biomedical
Research Council of the Singapore Agency for Science,
Technology and Research (A*STAR).

56

No. 25 - SYNTHESIS AND EVALUATIONOF DUAL-ACTIVITY OPIOID-NPFF
LIGANDS FOR RECEPTOR AFFINITY,
ANTINOCICEPTION AND TOLERANCE
LIABILITIES.

No. 26 - Platelet-Derived Growth Factor
Receptor-beta Antagonism Restores
Morphine Analgesic Efficacy Against
Neuropathic Pain
Donica, CL, Cui, Y, Shi, S, and Gutstein, HB

CR McCurdy1 , VB Journigan1 , C Mesangeau1 , CN
Kapanda1 , SJ Cutler1 , SO Eans2 , ML Ganno2 , JP
McLaughlin2
1 Dept of Biomolecular Sciences, The University of Mississippi,
University, MS 38677, USA 2 Torrey Pines Institute for MolecularStudies, Port St. Lucie, FL 34987, USA

Neuropeptide FF (NPFF) receptor antagonists prevent
morphine-mediated antinociceptive tolerance. Dual-acting
compounds with opioid agonist and NPFF antagonist may
produce antinociception without tolerance.
Methods: In an effort to produce selective, non-peptide
NPFF probes, we identified compounds with dual affinity
at NPFF and opioid receptors. The affinity and activityof these compounds for opioid and NPFF receptors wasdetermined with radioligand competition binding assays
as well as functional assays. Three compounds wereselected for in vivo investigation in the mouse warm-water
tail-withdrawal test, as well as an initial screen for acuteantinociceptive tolerance. Antinociceptive activity andopioid selectivity of these compounds was determined after
intracerebroventricular (icv) administration.
Results: A series of compounds were generated andexamined in receptor binding and functional assays.
Structure-activity relationships were identified in the seriesof compounds, conveying increased affinity in the nM
range for opioid receptors and the uM range for NPFF
receptors with opioid agonism and NPFF antagonism
in functional assays. Compounds 1, 2 and 3 produced
equipotent antinociception lasting at least 50 min, with
ED50 (and 95%CI) values of 6.9(4.7-9.5), 5.0(4.2-5.8) and2.7(2.1-3.7) nmol, icv, respectively that was antagonized by
pretreatment with MOR or KOR antagonists. Moreover,although repeat treatment with morphine shifted theED50 value 9.6-fold rightward, the 1.6-fold rightward shift
caused by compound 1 did not produce significant acuteantinociceptive tolerance.
Conclusions: Our investigations of structure activity
relationships yielded ligands that retained the receptor
affinity and activity of the parent compounds. Compounds
selected from the in vitro screening demonstrated full
antinociception, with one compound showing an absence ofacute antinociceptive tolerance. These results suggest thedevelopment of dual-action opioid-NPFF ligands may offer
therapeutic promise as analgesics with fewer liabilities ofuse.

Department of Anesthesiology, Department of Biochemistry andMolecular Biology, The University of Texas – MD Anderson Cancer
Center, Houston, Texas

Background: Chronic, intractable pain is a problem of
pandemic proportions. Pain caused by nerve injuries(neuropathic pain) is extremely difficult to treat. For
centuries, opiates such as morphine have been the first-line
treatment for severe chronic pain. However, opiates areoften ineffective against neuropathic pain, leaving few
options for suffering patients. In these studies, we determined whether PDGFR-beta inhibition could improve theeffectiveness of morphine for neuropathic pain treatment.
Results and Findings: Spinal nerve ligation was performed in male Sprague-Dawley rats. The clinically used
PDGFR antagonist imatinib did not relieve mechanicalpain in a nerve injury model as determined by Von Frey
assay. Surprisingly, combining imatinib with a previously
ineffective dose of morphine led to complete pain relief.
Scavenging released platelet-derived growth factor B(PDGF-B) also markedly augmented the analgesic effectof morphine. Tolerance to the analgesic effect of morphine
did not develop when the PDGFR was inhibited or
PDGF-B was scavenged.
Conclusions:
These findings suggest the novel hypothesis that PDGF-B released by injured nerves renders
animals resistant to morphine, implying that PDGFR-beta
inhibition could potentially eliminate the tremendous
suffering caused by neuropathic pain.
These studies were funded by CPRIT grant awarded
to HG and PhRMA Foundation Fellowship to CD.

Funding: Supported by funds from the University ofMississippi and the State of Florida
COI: none to declare57

No. 27 - Intrathecal delivery of a bivalent
ligand targeting a mu opioid Receptor
mGLuR5 Receptor heteromer reduces
nerve-injury hyperalgesia
Cristina D. Peterson, Kelley F. Kitto, Eyup Akgün, Mary
Lunzer, Phil S. Portoghese, Carolyn A. Fairbanks
University of Minnesota

Background: Reports on the presence of MOR andmGluR5 in the spinal cord, and the existence of MORmGluR5 heteromer in HEK293 cells coexpressed with
MOR and mGluR5 has raised the likelihood that thisputative heteromer may be present in the CNS. Such aheteromer could be involved in the inflammatory process,given the known functional interaction of mGluR5 with
the NMDA receptor. Thus, a bivalent ligand (MMG22)
that contains both mGluR5 antagonist and mu agonist
pharmacophores should antagonize the mGluR5 protomer and activate the MOR protomer of the putative
MOR-mGluR5 heteromer. This in fact proved to be thecase, in that intrathecal MMG22 has been reported to
produce exceptional antinociception without tolerance inmouse models of inflammatory pain (Akgün, E et al. 2013).
Methods: ICR-CD1 males mice were used. Sensory
thresholds were determined using an electronic vonFrey algesiometer. Mice were subjected to spared nerve
injury (Decostered and Woolf, 2001). At multiple days
post-injury, tactile hypersensitivity levels were measured todetermine magnitude of hyperalgesia. MMG22, morphine,
MPEP, or the combination of morphine + MPEP wereadministered intrathecally according to the method ofHylden and Wilcox (1980).
Sensory thresholds were
evaluated post-injection at 5, 12, 20, 60, 90, and 120
minutes post-injection. Data were analyzed by ANOVA
and dose-response curves constructed by the method of
Tallarida (1997).
Results: MMG22 (1, 100, 1000 pmol) was effective
in reversing nerve-injury induced tactile hypersensitivity
at 5 and 12 minutes post injection. Higher doses (1-10nmol) were effective for a duration equivalent to that ofeffective doses of morphine, MPEP or the combination.
Conclusion: We conclude that MMG22 demonstrates
anti-hyperalgesic efficacy in a mouse model of neuropathic
pain. The potency of MMG22 appears to be greater thanthat of either morphine or MPEP given individually or in
combination.

No. 28 - Novel ligands at the mu opioid
receptor that display bias toward G protein
coupling over βarrestin2 recruitment
Cullen L. Schmid (1) , Nicolette C. Ross (2) , Zhizhou Yue, Jenny Morgenweck (1) , Michael D. Cameron (1,3) ,Thomas D. Bannister (2) and Laura M. Bohn(1)(2)(1) The Departments of Molecular Therapeutics and Neuroscience;(2) Translational Research Institute—Medicinal Chemistry; (3)Translational Research Institute—Drug Metabolism and Pharmacokinetics, The Scripps Research Institute, Jupiter, Florida 33458

Introduction:
Advances in the understanding of functionally selective
ligands, or drugs that differentially and preferentially
activate or inhibit signaling pathways downstream ofreceptor binding, have led to drug development efforts
specifically targeting certain pathways in the hope ofenhancing the beneficial aspects of receptor activation
while avoiding negative effects in vivo. While opioids,
which mediate their effects through activation of the mu
opioid receptor (MOR), are the gold standard for the treatment of moderate to severe pain, their therapeutic use islimited by their significant adverse side effects. Previously
we have demonstrated in mice that the absence of thescaffolding and regulatory protein βarrestin2 (βarr2-KOmice) results in enhanced and prolonged antinociception
to morphine without the development of antinociceptive
tolerance, respiratory suppression and constipation.
Based on these findings, we hypothesize that functionallyselective compounds which activate MOR without inducing interactions with βarrestin2 would recapitulate theeffects of morphine in the βarr2-KO mice and would serve
as more effective analgesics with reduced side effect profiles.
Methods:
Herein, we utilize cell lines to evaluate novel ligands
for their ability to activate MOR-mediated signalingcascades and induce βarrestin2 interactions with thereceptor. Pharmacokinetic and antinociceptive profilesof lead compounds were then determined in C57Bl/6J mice.
Results:
Through this screening process, we have identified
compounds that display bias towards the activationof MOR-mediated signaling cascades and away fromβarrestin2 recruitment. These compounds are also brain
penetrant and induce potent antinociception.
Conclusions:
These functionally selective MOR agonists may serveas useful tools in vivo for evaluating the contribution ofβarrestin2 interactions with the receptor to MOR-mediatedphysiologies and may aid drug discovery efforts for thedevelopment of more effective opioid analgesics.
Funding from NIDA: DA033073

58

No. 29 - Cannabinoid CB1 Receptor
Induction of ΛFosB Mediates Mu Opioid
Receptor Cross-Sensitization in the Nucleus
Accumbens

No. 30 - Calmodulin-dependent Kinase II
Protein is Increased in the Hippocampus of
Oxycodone Self-administered Adult C57Bl/6
Mice.

Dana E. Selley1 , Matthew F. Lazenka1 , Aaron Tomarchio1 ,Eric J. Nestler2 and Laura J. Sim-Selley1

Derek P. Simon, PhD, Yong Zhang, PhD, and Mary
Jeanne Kreek, MD

1Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA; 2Department of Neuroscience,Friedman Brain Institute, Mt. Sinai School of Medicine, New York,NYThe Laboratory of the Biology of Addictive Diseases, the Rockefeller
University, New York, NY 10065

Introduction. Repeated administration of abused drugs,
such as opiates and Λ9-tetrahydrocannabinol (THC), themajor psychoactive constituent in marijuana, induces theAP1 transcription factor ΛFosB in the striatum. Repeatedcannabinoid agonist treatment also cross-sensitizes rodents
to the reinforcing and motor stimulant effects of mu opioid
receptor (MOR) agonists. We reported that inducible
transgenic expression of ΛFosB in D1/dynorphin positive
medium spiny striatal neurons enhanced MOR signaling in
the nucleus accumbens (NAc) and enhanced the rewarding
effects of morphine without affecting MOR expression.
Moreover, repeated THC administration induced ΛFosB in
the NAc, but inducible transgenic expression of ΛFosB did
not enhance cannabinoid CB1 receptor (CB1R) signaling
in this region.
Methods. Mice were injected twice daily for one weekwith 10 mg/kg THC or a ramping dose paradigm of
10, 30 and 60 mg/kg THC, with the dose increasing
every other day. ΛFosB induction was determined byimmunoblot, and ΛFosB co-localization was determinedby dual immunohistochemistry. G-protein activation wasdetermined by agonist-stimulated [35S]GTPγS binding,either in membranes isolated from dissected brain regions
or by [35S]GTPγS autoradiography.
Results. Repeated THC administration induced ΛFosB
in the NAc and dorsal striatum. ΛFosB was co-localizedwith dynorphin, suggesting induction in D1/dynorphin
positive medium spiny neurons. Similarly to transgenic
over-expression of ΛFosB, repeated THC administration
enhanced MOR-mediated G-protein activation in theNAc, whereas CB1R-mediated G-protein activity was notincreased. This effect was limited to NAc because neither
THC nor inducible transgenic over-expression of ΛFosB
altered MOR-mediated G-protein activation in the dorsal
striatum. Importantly, inducible transgenic expression of
ΛcJun, a dominant negative inhibitor of AP1-mediatedtranscription, blocked THC-induced enhancement of MOR
activity in NAc.
Conclusion. These results suggest that cross-sensitizationof MOR-mediated G-protein activation by repeated THC
is mediated by induction of ΛFosB.
Supported by DA014277, DA008227, DA030404 andDA030227 from NIDA.

Prescription opioid (PO) abuse is reaching epidemic levels amongst all socio-economic groups in the United States.
PO such as oxycodone are powerfully addictive, incur ahigh risk of fatal overdose, and lead to use of even moredangerous opioids. Progression to addiction involves learning to associate drug use with drug reward, thus the formation of drug-associated memories is an essential componentof addictive disease. The hippocampus is acknowledged
as the brain region critical for the formation/storage ofmemories. Memory consolidation in hippocampal neurons
involves augmentation of glutamatergic synaptic strength
through repeated activation of Calmodulin-dependent kinase II (CamKII). However, the mechanisms underlying drug-associated reward learning are still unclear for
oxycodone addiction. To examine molecular correlates
in the hippocampus with oxycodone-reward learning, we
conducted an oxycodone self-administration experiment.
Adult C57Bl/6 male mice were implanted with an intravenous catheter and allowed to self-administer oxycodone
2 hours/day for 14 days (0.25mg/kg/nose poke). Salineyoked animals were used as control. Mice were then sacrificed and the entire hippocampus was dissected and protein lysate analyzed by Western Blot. We found a statistically significant increase in the amount of CamKII protein in the hippocampus of oxycodone-self administeredmice compared to controls, though no difference in phosphorylated CamKII, or the memory-related factors NMDAR1 and Egr1, was observed. These data suggest thatan increase of CamKII-dependent drug-associated memory
formation may have occurred in the oxycodone-self administered animals and that CamKII may represent a potential therapeutic target for the disruption of drug-associatedreward learning. This work was supported by the Kopf
Family Postdoctoral Fellowship (DPS), NIH R01DA029147(YZ) and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (MJK). No conflicts of interest to
declare.

59

No. 32 - SYNTHESIS AND BIOLOGICALEVALUATION OF A COMPACT,CONFORMATIONALLY CONSTRAINEDBIFUNCTIONAL OPIOID AGONIST NEUROKININ-1 ANTAGONISTNo. 31 - Oxycodone causes greater locomotor
activation in female compared to male
C57BL/6J mice
D Collins, Y Zhang, S Rahm, B Reed, MJ Kreek
The Laboratory of the Biology of Addictive Disease, The Rockefeller
University, New York, NY

Nonmedical use of the oxycodone is a major publichealth problem. Males and females may present different
behavioral consequences related to its use. Unfortunately,
many rodent studies of oxycodone’s behavioral effects are
conducted in mixed groups or males only and subsequently
generalized to females. Here, we compare locomotor
activity data in drug-naı̈ve vs. oxycodone-exposed maleand female mice.
Adult male and female C57BL/6J mice (10 weeksold) were randomly assigned to one of two groups (5mice/sex/group), drug-naı̈ve and oxycodone-treated, were
injected (IP) 3x/day (every 6hr; 1st injection at 0800) for
14 days. Naı̈ve mice received only saline at each injection.
Oxycodone-treated mice received oxycodone (10 mg/kg)
at each injection. Each day, immediately following the
1st injection, locomotor activity, defined as total distancetraveled, was measured in a standard photobeam enclosure
during a 2-hr session.
Three-way ANOVA, sex x drug x session, with repeated measures on the last factor, revealed a main
effect of sex on locomotor activity in both oxycodoneand saline-treated mice [F (1,16) = 29.06, p > 0.0001]
as well as a main effect of drug [F(1,16) =189.09, p> 0.00001] with no significant interaction of sex anddrug. Student-Neuman-Keuls post-hoc tests revealed asignificant increase in locomotor activity in oxycodoneversus saline-injected animals in both sexes [p > 0.0005].
Taken together, these results suggest that sex-based
differences produce unique oxycodone-induced locomotorstimulating effects in male and female C57BL/6J mice.
This work was supported by NIH 1R01DA029147(YZ), the Adelson Medical Research Foundation (BR& MJK), and the David Rockefeller Graduate Program(DTC). The authors declare no conflict of interest.

Dirk Tourwé1 , Karel Guillemyn,1 Patrycia Kleczkowska,4Olivier Van der Poorten,1 Isabelle Van den Eynde,1 Attila
Keresztes,2 Eva Varga,2 Josephine Lai,2 Frank Porreca,2
Nga N. Chung,3 Carole Lemieux,3 Joanna Mika,5 Wioletta
Makuch,5 Barbara Przewlocka,5 Jozef Vanden Broeck,6Andrzej W. Lipkowski,4 Peter W. Schiller,3 and Steven
Ballet1 *1 Laboratory of Organic Chemistry, Departments of Chemistry
and Bioengineering Sciences, Vrije Universiteit Brussel, Brussels,
Belgium, 2 Department of Pharmacology, University of Arizona,
Tucson AZ, U.S.A., 3 Department of Chemical Biology and
Peptide Research, Clinical Research Institute, Montreal, Canada, 4
Neuropeptide Laboratory, Medical Research Centre, Polish Academyof Sciences, Warsaw, Poland, 5 Department of Pain Pharmacology,
Institute of Pharmacology, Polish Academy of Sciences, Kraków,
Poland, 6 Animal Physiology and Neurobiology Department,
Katholieke Universiteit Leuven, Leuven, Belgium.

The co-administration of an opioid agonist combined with
NK1 receptor (NK1R) antagonists, as separate compounds,
or as one hybrid compound, led to an enhanced antinociceptive potency, with suppressed tolerance effects upon prolonged administration.[1][2]We reported the compact peptidomimetic Dmt-D-Arg-AbaGly-(3,5-(CF3)2)NMe-benzyl, (SBCHM01), possessing amixed opioid agonist-NK1R antagonist profile (MOR Ki:
0.4 nM, DOR Ki: 10.4 nM, NK1R pA2: 7.8), with potent antinociceptive properties in the mouse tail flick assay when given iv, indicating that it was able to cross the
BBB.[3] The compound however induced tolerance. Control over the relative potency of a dual ligand with overlapping pharmacophores represents a major challenge. We
have made modifications in SBCHM01 in order to influence opioid potency and NK1R antagonism, which identified Dmt-D-Arg-Aba-beAla-NMe-benzyl (KGCHM02), aligand with improved opioid potency (MOR Ki: 0.08 nM,
DOR Ki: 0.3 nM), and slightly reduced NK1R antagonism(pA2: 6.44). In the mouse tail flick assay, after iv administration, KGCHM02 was less potent than SBCHM01,
indicating that its BBB permeability is reduced. The hybrids were injected it and compared to their opioid parts
lacking the NK1R part in a neuropathic pain model(CCI).
The hybrids, but not the parent opioid parts decreased allodynia in the von Frey test, KGCHM02 being more potent
than SBCHM01. Also in the cold plate test, the hybrids
decreased hyperalgesia in contrast to the opioid parts, witha potency stronger than that of morphine.
[1] Misterek et al; Life Sci.54,393(1994)[2] Largent-Milnes etal.;Br. J. Pharmacol.161,986(2010)[3]Ballet J.Med. Chem.
54,2467(2011)60

No. 33 - CO-LOCALIZATION OF MU AND
DELTA OPIOID RECEPTORS IN THE
NERVOUS SYSTEM USING DOUBLEFLUORESCENT KNOCK-IN MICE
Eric Erbs*, Lauren Faget*, Gregory Scherrer°, Audrey
Matifas*, Laurent Vasseur*, Pierre Veinante#, Brigitte L.
Kieffer*, Dominique Massotte#
INCI UPR 3212, CNRS, Strasbourg, France,* IGBMC UMR7104,
Illkirch, France, °Stanford University, CA USA

We have generated double knock-in mice expressing delta
opioid receptors in fusion with the green fluorescent protein
and mu receptors in fusion with a red fluorescent protein.
We have used these animals to map the two receptors in thebrain, spinal cord and dorsal root ganglia. Special attention was given to the profile of mu-delta co-expression at thecellular level. Our data indicate that neurons co-expressing
the two receptors are particularly abundant within regions
involved in nociception. This suggests potential physiological relevance and prompts to investigate in vivo the functional role of mu-delta heteromers to assess their potential
as a new therapeutic target

No. 34 - Ligand-directed signaling at mu
opioid receptors: differential mechanisms of
MAPK activation by morphine and fentanyl
Andrea Bedini, Jamie Rose Kuhar, Erica Melief, Charles
Chavkin
Department of Pharmacology, University of Washington, Seattle(USA) Department of Pharmacy and Biotechnology, University of
Bologna (Italy)

INTRODUCTION: The ability of opioid ligands toselectively activate particular signaling pathways overothers represents an intriguing opportunity in opioid
pharmacology to develop more effective drugs. However,in order to exploit ligand-directed signaling at mu opioid
receptors (MOR), a deeper investigation of the different
signaling modalities elicited by opioid ligands is necessary.
The aim of this research, therefore, has been to characterize
the differential processes of JNK and ERK1/2 MAPK
activation by two prototypical MOR agonists, morphineand fentanyl.
METHODS: Wild type, JNK1-/-, JNK2-/-, JNK3-/-, orGRK3-/- male C57/Bl6 mice were administered saline,
morphine (10mg/kg, s.c), or fentanyl (0.3mg/kg, s.c.)30-60 minutes prior to harvesting spinal cords. Spinal cord
cell lysates were analyzed for phospho-JNK immunoreactivity. To further characterize mechanisms of JNK
activation, additional experiments were carried out in
MOR expressing HEK293 cells. Cells were pretreated withsiRNA against arrestin-2 or arrestin-3, or inhibitors of Src
family kinases or PKC, and then treated with morphine or
fentanyl (10µM, 30 min). Cell lysates were analyzed forphospho-JNK and phospho-ERK1/2 immunoreactivity.
RESULTS: Both morphine and fentanyl selectively
increase pJNK2-ir in vivo. Morphine activation of JNK
was GRK3-independent, whereas fentanyl activation of
JNK was GRK3-dependent.
In vitro, morphine andfentanyl both activated ERK1/2 in a Src-dependent but
arrestin-independent manner. Morphine activation of JNK
was PKC- and Src-dependent, but arrestin-independent,
whereas fentanyl-mediated activation of JNK was arrestin
2- and Src-dependent, but PKC-independent.
These
distinct modalities in JNK activation may represent
the molecular basis of the differences observed between
morphine vs.
fentanyl-induced tolerance, which are
JNK-dependent and JNK-independent, respectively.
Work supported by DA030074, DA07278, DA020570.

61

No. 35 - Nalfurafine is a KOR agonist that
produces analgesia, pruritus, inhibition ofluteinizing hormone release, and ERK1/2
MAPK activation, but has low potency for
p38 MAPK activation

No. 36 - Ligand-directed signaling at kappa
opioid receptors: differential mechanisms of
JNK MAPK activation by U50,488 andnorBNI
Selena Schattauer, Andrea Bedini, Charles Chavkin

Selena Schattauer, Jamie Rose Kuhar, Allisa Song, Ashley
Angell, Robert Steiner, Charles Chavkin
Department of Pharmacology and Department of Physiology andBiophysics, University of Washington, Seattle (USA)Previous studies have shown the aversive propertiesof KOR activation to be GRK3/p38 MAPK-mediated,indicating that KOR agonists that selectively activate
G-protein signaling without activating p38 may havetherapeutic potential as non-dysphoric analgesics. Clinical
trials investigating the efficacy of the KOR agonist
nalfurafine in treating pruritus in dialysis patients did notreport dysphoric side effects, indicating nalfurafine may
have an attractive pharmacology for clinical use. This
research aimed to characterize nalfurafine activation of
ERK1/2 and p38 MAPK, as well as the in vivo effects ofnalfurafine in analgesia, pruritus, and luteinizing hormone(LH) release.
HEK293 cells expressing human or rat KOR were treatedwith 10pm-10µM nalfurafine or 1µM U50,488 prior to
analysis of phospho-ERK1/2 (5 min) or phospho-p38(30 min) immunoreactivity to measure the efficacy andpotency of nalfurafine for activation of these MAPKs.
Analgesia was measured in male C57/BL6 mice using
the warm water tail withdrawal assay before and 30 min
after U50,488 (1-30mg/kg, i.p.) or nalfurafine (1-150µg/kgi.p.) To measure pruritus, male wild type, MOR-/-, or
KOR-/- C57/Bl6 mice were administered saline or 50µg/kg
nalfurafine 20 minutes prior to 5’GNTI (50µg/kg, s.c.)and scratches over 30 minutes were counted. To measure
LH, blood was collected prior to or 20 minutes following
administration of saline or 1-50µg/kg nalfurafine in female
C57/Bl6 mice 13 days post-ovariectomy.
Nalfurafine was
250 fold more potent for ERK1/2
activation as compared to p38 activation in hKOR
expressing HEK293 cells, and 20 fold more potent for
ERK1/2 activation as compared to p38 activation in rKOR
expressing HEK293 cells. U50,488 (15 and 30mg/kg) andnalfurafine (50 and 150µg/kg) produced a norBNI-sensitive
analgesic response in the warm water tail withdrawal
assay.
5’GNTI increased scratching in a KOR- andMOR-independent manner and nalfurafine pretreatment
resulted in a KOR-dependent and MOR-independent
reduction in scratching. U50,488 and nalfurafine both
reduced LH levels in ovariectomized mice.

Department of Pharmacology, University of Washington, Seattle(USA) Department of Pharmacy and Biotechnology, University of
Bologna (Italy)

Exploiting ligand-directed signaling at kappa opioid receptors (KOR) represents an opportunity for the developmentof safer and more effective analgesic drugs, but further
investigation of signaling modalities is needed. It has
previously been shown that both U50,488 and norBNI canboth stimulate KOR-dependent phosphorylation of JNKMAPK in vivo and in vitro; however norBNI activation of
JNK results in receptor desensitization, whereas U50,488
activation of JNK does not. Therefore, the aim of thisresearch is to characterize the process of JNK activationby these ligands to better understand the differential
effects on desensitization. HEK293 or wild type mouseembryonic fibroblast (MEF) cells expressing KOR-GFP
were treated with norBNI or U50,488 (10µM), and celllysates were analyzed for phospho-JNK immunoreactivity.
U50,488 resulted in biphasic JNK activation in HEK293
cells, with peaks at 15 and 60 min, whereas norBNI
resulted in monophasic JNK activation peaking at 60
min. In contrast, U50,488 caused JNK activation in MEF
cells peaking at 15min whereas norBNI was ineffective.
To further characterize the signaling mechanisms, KOR
expressing HEK293 cells were treated with norBNI or
U50,488 following pretreatment with either Src family
kinase inhibitors, Rho family GTPase inhibitors, or siRNAagainst arrestin-2 or arrestin-3. JNK activation by U50,488
had an early, arrestin-independent component (15min) anda slower arrestin-3 dependent component (60min). Thisis similar to the previously reported biphasic activationof ERK by U50,488. The monophasic norBNI activationof JNK was significantly reduced by both arrestin-2 andarrestin-3 siRNA. Inhibition of Rac1, RhoA, or Src prevented JNK activation by norBNI at 60min and U50,488
at 15 and60 min. U50,488 and norBNI were both able toincrease KOR/arrestin interactions, as assessed by BRET
analysis using venus tagged arrestins and luciferase tagged
KOR. These differences in signaling cascades mediating
JNK activation by norBNI and U50,488 may account forthe differences in JNK-mediation desensitization.
Work supported by DA030074, DA07278, DA020570.

Work supported by DA030074, DA07278, DA020570.

62

No. 37 - OPIOID DELTA
RECEPTOR-MEDIATED β-ARRESTIN
SIGNALING MODULATES CONVULSIVE
EFFECTS
Eriko Nakata (1) , Junichi Sakai (1) , Takashi Kanda (1) ,Yoshikazu Watanabe (1, 2) , Daisuke Saito (1) , Toshihiro
Takahashi (1) , Takashi Iwai (2) , Shigeto Hirayama (2) ,Hideaki Fujii (2) , Tomio Yamakawa (1) and Hiroshi Nagase(2, 3)(1) Discovery Research Laboratories, Nippon Chemiphar Co. Ltd.,
Saitama, Japan, (2) School of Pharmacy, Kitasato University, Tokyo,
Japan and (3) International Institute for Integrative Sleep Medicine,
University of Tsukuba, Ibaraki, Japan

Opioid δ receptor agonists are potential therapeutic agentsfor pain and various indications. However, convulsive
effects through the δ receptor activation were reported in
preclinical studies. Although successful separation between
convulsions and the desired pharmacological effects has
been reported for some δ agonists, its mechanism has not
yet been explored.
We designed and synthesized the characteristic δ receptor
agonists with a novel fundamental oxazatricyclodecane
structure, and identified their unique pharmacological
profile. With regard to four δ receptor agonists, which
had a slightly different structure and showed similar G
protein-mediated agonistic activities in cAMP assay using
the δ receptor-expressing CHO cells, we analyzed their δreceptor mediated β-arrestin2 signaling activities in vitro
and convulsive effects in mice. All compounds showed
analgesic activities in acetic acid-induced writhing test
and two compounds induced the convulsion. Interestingly,
two δ agonists without the convulsion appeared to be aconsequence reduced β-arrestin2 recruitment and followedinternalization activities, suggested that the δ receptorinduced convulsive effects are mediated by β-arrestin2
signaling.
These results indicated that δ agonists preferred G proteinmediated signaling to β-arrestin2-mediated signaling could
be useful therapeutic agents.
This study was supported by the Japan Science andTechnology Agency (JST) grants.

No. 38 - Endosomal Endothelin Converting
Enzyme-2: A regulator of opioid receptor
trafficking
Erin N. Bobeck1 , Achla Gupta1 , Wakako Fujita1 , Ivone
Gomes1 , and Lakshmi A. Devi1,2
1Department of Pharmacology and Therapeutics; 2The Friedman
Brain institute Icahn Medical School at Mount Sinai; New York, NY

Trafficking of opioid receptors following activation has
been studied extensively, however very few studies haveevaluated the fate of the internalized neuropeptide.
It is generally assumed that the internalized peptide
is hydrolyzed by endopeptidases; virtually nothing is
known about specific enzyme(s) involved in this process.
Previously we have shown endothelin-converting enzyme-2(ECE-2), a metallopeptidase, to be involved in opioid
peptide processing in vitro and opioid actions in vivo.
In this study, we examined the involvement of ECE-2 inmodulating µ and δ opioid receptor trafficking and its
impact on receptor function. In cells stably expressing
ECE-2 and µ or δ receptors, opioid peptide treatment
leads to an increase in colocalization of µ or δ receptors
with ECE-2 within the endosomal compartment. Following
removal of the peptide, there is a decrease in colocalization
with ECE-2, and this is prevented by ECE-2 inhibition.
We also find that ECE-2 inhibition leads to substantial
decrease in receptor recycling and signaling without significantly affecting receptor internalization. Interestingly wefind that ECE-2 inhibition affects recycling of receptors
internalized in response to only those agonists that aresubstrates of ECE-2. Similar results were obtained withdorsal root ganglion cells and primary cortical neurons that
endogenously express the receptors and ECE-2. To assess
the role of ECE-2 in vivo, we examined the effect of ECE-2
inhibition on intrathecal DAMGO or fentanyl-mediatedantinociception.
We find that the co-administrationof the ECE-2 inhibitor prolongs DAMGO-mediatedantinociception (as compared to controls) but not the
fentanyl-mediated antinociception. Finally, we find thatadministration of the ECE-2 inhibitor significantly affects
stress-induced analgesia in wild-type mice, but not inmice lacking δ receptors suggesting opioid receptors andECE-2 in this process. Taken together these data reveal anovel role of ECE-2 in modulating the opioid system bypostendocytic processing of opioid peptides.
Supported by NIH grants, DA019521 and NS026880 (toLAD); ENB was supported by the NIDA Postdoctoral
Training Program DA007135.

63

No. 39 - Increased Emergency Department
Visits Caused by Tramadol Abuse are
Associated with Opioid-Related Over-Dose
Death in the U.S.
Francia Fang (1) , Jing Wu (2)(1) Chapel Hill High School, Chapel Hill, NC 27516. USA (2) University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. USA

Introduction: Tramadol is an uncontrolled narcotic opioidin the U.S. and its increased abuse or misuse poses serious
health risks and adverse outcomes. To better understanda geographic pattern associated to its adverse healtheffects, a comprehensive analysis of numbers of emergency
department (ED) visits caused by Tramadol abuse and its
correlation with opioid over-dose death were conducted.
Methods: The cohort data (2004 - 2010) was collected from three major public drug abuse surveillancesystems including (1) drug abuse-related ED visits fromDrug Abuse Warning Network (DAWN); numbers ofindividual deaths due to tramadol abuse from (2) The
Centers for Disease Control and Prevention (CDC)
Morbidity and Mortality Weekly Report and (3) The
Annual Medical Examiners Report of Florida Departmentof Law Enforcement (FDLE).
Results: The results from DAWN ED visits demonstrated a substantial increase in the number of Tramadol
abuse among the middle-aged group and that womencomprise 58% of the ED visits. A correlation study ofthe numbers of ED visit and prescribed opioid over-dose
death from CDC and FDLE showed that the increased ED
visits involved in Tramadol are positively correlated with
the increase in overdose death rates in the US (r2= 0.741;
r = 0.861) nationally as well as in the state of Florida(r2= 0.978; r = 0.989). The analytic result is statisticallysignificant (p > 0.01).
Conclusions: The study results showed that increased
abuse of Tramadol positively correlated with increased
deaths due to opioid over-dose and a further legal andregulatory implementation may be required to reduce thenon-control access of Tramadol and subsequent public risk
due to Tramadol abuse.

No. 40 - Tramadol is an effective analgesic in
an acute and chronic rat pain model
Fraser McIntosh, Harunor Rashid, Kari L Riggs, Kimberley H Chappell
Charles River, Elanco

BACKGROUND:
The analgesic tramadol is a widely used drug for a variety of chronic pain conditions including osteoarthritis (OA)
patients who have failed to respond to non-steroidal antiinflammatory drugs (NSAIDS).
OBJECTIVE:
The aim of these studies was to assess whether tramadol
would be efficacious in a rat model of OA pain which exhibits both NSAID sensitive and insensitive phases of joint
pain.
METHODS:
Rat Formalin: Animals(n=8 per group) were administeredeither vehicle, tramadol (20-80mg/kg PO)or duloxetine before injection of formalin (30uL) into the left footpad. Nocifensive behaviours were captured by video camera andanalysed later offline. Phase 1 (0-5mins) and Phase 2(2035mins) responses were quantified for all groups.
Rat Mono-iodoacetate(MIA): Joint pain was induced byinjection of MIA (25uL) into the left knee of each animal (n=8 per group). Dynamic weight bearing (DWB) responses were quantified using the BioSebTM system. Baseline responses were analysed either on Day 1 or Day 21 andanimals were randomized to treatment groups. Tramadol
was administered (60mg/kg PO) and DWB responses were
measured at 0.5,2,4 and 48hours. For each animal, data
was quantified as weight borne on the left limb (injured) right limb (uninjured).
RESULTS:
Treatment with tramadol resulted in a significant dose dependent reduction in both Phase 1 and Phase 2 responses
in the rat formalin model. Based on these data, 60mg/kg(oral, BID) was evaluated in the rat MIA model. In theearly NSAID sensitive phase, tramadol demonstrated significant efficacy (40-60%) at all timepoints following both
single and BID dosing. In the late NSAID insensitive phaseof the model, tramadol was similarly effective (35-55%).
CONCLUSIONS:
Tramadol is an effective analgesic demonstrating efficacy inboth NSAID sensitive and insensitive phases of a chronic
joint pain model. The rat MIA model employing a quantitative, non-subjective DWB endpoint, is a useful system
to study OA joint pain.

64No. 41 - Development of a Vaccine against
HeroinGary R. Matyas1 , Oscar Torres1,4 , Rashmi Jalah1,4 ,Kenner C. Rice2,3 , Fuying Li2,3 , Joshua F.G. Antoline2,3 ,Kejun Cheng2,3 , Malliga R. Iyer3 , Arthur E. Jacobson2,3 ,Zoltan Beck1,4 , Carl R. Alving1
1Laboratory of Adjuvant and Antigen Research, US Military HIV
Research Program, Walter Reed Army Institute of Research, 503
Robert Grant Avenue, Silver Spring, MD 20910, USA

One novel approach to the treatment of drug abuse isto develop vaccines that prevent the pharmacologicaleffects of the drug. Although the vaccines do not reduce
the physical dependence of the drug in addicts, vaccines
will prevent the drug-induced euphoria and may also be
important in preventing relapse and drug overdose. The
mechanism of protection of vaccines is to induce antibodies
that bind to the drug, sequester it in the blood and prevent
it from the crossing the blood-brain barrier. However,the development of a vaccine to heroin has a numberof challenges. Heroin is too small to be immunogenic
when injected and therefore, surrogate heroin haptens
must be coupled to a carrier to induce antibody responses
to heroin. Both high titer and high affinity antibodies
that have a long duration are required. Heroin vaccine
development is particularly difficult because heroin is
rapidly metabolized to 6-acetylmorphine and morphine
after injection. Consequently, a vaccine for heroin must
induce antibodies that bind not only to heroin, but also its
metabolites. Two heroin haptens, DiAmHap and MorHap,
were tested as antigens for a candidate heroin vaccine.
MorHap is a morphine analog containing the functionalgroup used for coupling at the 6 hydroxyl position.
DiAmHap is a heroin analog in which amide groups aresubstituted for the acetyl groups at the 3 and 6 hydroxyl
positions and the coupling group is at the bridge nitrogenat position 17. MorHap and DiAmHap were coupled totetanus toxoid as the carrier and mixed with liposomes
containing monophosphoryl lipid A as an adjuvant. Mice
were immunized and boosted twice at 3 week intervals.
Nine weeks after the primary immunization, sera wereassayed for antibodies to the hapten and the animals were
tested by a hot-plate nociception assay. Antibody titers
were very high with endpoint titers of 3 and 2.5 million
for MorHap and DiAmHap immunized mice, respectively.
Both groups of immunized animals exhibited reduced
antinociceptive effects of heroin after the injection ofheroin. MorHap immunization induced antibodies thatreacted with 6-acetylmorphine and morphine, whereas
immunization with DiAmHap induced antibodies thatreacted with heroin, 6-acetylmorphine and morphine. This
study indicates that stable heroin haptens can be used in
vaccine formulations and that resulting antibodies block
the pharmacological effects of heroin.

No. 42 - Comprehensive analysis of factors
influencing quantification of biased signallingat the mu-opioid receptor
Georgina Thompson, Arthur Christopoulos and Meritxell
CanalsMonash Institute of Pharmacological Sciences, Drug Discovery
Biology Laboratory. Parkville, Victoria, Australia

The dynamic structure of GPCRs allows different ligands
to stabilise the receptor into unique active conformations,
resulting in differential activation cell signalling pathways
and, eventually, in distinct physiological outcomes. Thisphenomenon known as “signalling bias” or “functional selectivity” can be exploited to design new opioids that selectively activate signalling pathways that lead to analgesia
whilst minimising side effects that are elicited by activation of other signalling pathways. However, bias is not an
absolute quality, it is a dynamic and multidimensional phenomenon; it is always relative to a reference ligand, and it
is dependent on both cellular protein complement as wellas the spatiotemporal properties of the different signalling
endpoints.
By quantifying signalling bias at the µ opioid receptor(MOR) across multiple different signalling pathways in two
different cell lines, we show how the cellular backgroundinfluences the direction of biased agonism. In addition, we
have examined the impact of signalling kinetics on the detection of bias. This work has shown that the time points
selected for measuring activation of different signalling effectors can significantly alter the bias observed across different pathways.
Altogether this demonstrates the changeable nature of ligand bias, which presents a significant challenge when attempting to predict biased signalling in vivo. However thesestudies have also identified ligands that maintain uniquesignalling bias profiles in different cell lines which may be
predictive of distinct physiological outcomes. A greater understanding about biased signalling in native tissue and invivo, will enable links between distinct signalling bias profiles and specific physiological responses to be established,providing essential new information for the design opioids
with effective analgesia and less side effects.

65No. 43 - Supraspinal endomorphin analgesia
requires exon 11 variants of the mu opioid
receptor (MOR-1)

No. 44 - Pain inhibition by leukocytic opioid
receptors
M.Ö. Celik, D. Labuz, H. Machelska

Marrone G.F. 1, 2 , Grinnell S.G. 1, 2 , Pan Y.X. 1 , Pintar
J. 4 , Majumdar, S. 1 , Pasternak G.W. 1, 2 ,3
1Mol. Pharmacol. and Chem., Mem. Sloan-Kettering Cancer Ctr.,
New York, NY; 2Dept. of Neurosci., 3Dept. of Pharmacol., Weill
Cornell Med. Col., New York, NY, 4Dept. of Neurosci. and Cell
Bio., Robert Wood Johnson Medical School, Piscataway, NJ

Multiple mu opioid receptors (MOR-1) have been confirmedwith the cloning of several mu splice variants. These variants are generated by distinct promoters associated withexon 1 and exon 11. Previously, our lab observed thatthe potency of morphine-6β-glucuronide, heroin, and fentanyl analgesia was reduced in mice lacking truncated 6transmembrane (6TM) exon 11 variants of MOR-1. In
contrast, morphine analgesia was intact. We now present
evidence that the mu selective peptides endomorphin-1
and endomorphin-2 also require exon 11 variants for theiranalgesic actions supraspinally. An endomorphin-2 analog, DAPP (dmt-DAla-Phe-Phe-NH2), produces analgesia
via exon 11 variants of MOR-1 as well. In vitro, approximately 50% of [125I]DAPP binding was lost in the brainsof exon 11 knockout mice, whereas no specific binding was
detected in mice lacking both MOR-1 exon 1 and exon 11
variants. Consistent with binding in mouse brain, we observed increased binding in CHO cells coexpressing MOR-1
and exon 11 6TM variant MOR-1G compared to CHO cells
expressing MOR-1 alone. No binding was observed in cells
expressing MOR1G alone. Together, our results indicatethat 6TM exon 11 variants are involved in endomorphin
analgesia and binding.

Department of Anesthesiology, Charité – Universitätsmedizin Berlin,
Campus Benjamin Franklin, Berlin, Germany

Introduction: Traditionally, opioids produce analgesia byacting at opioid receptors on sensory neurons and blocking
the release of excitatory neurotransmitters. Althoughopioid receptors are also expressed by leukocytes, theirsignificance to pain transmission has not been addressed.
Here we tested the hypothesis that activation of opioid
receptors in leukocytes releases opioid peptide Metenkephalin (ENK), which activates peripheral neuronal
receptors, to ameliorate neuropathic pain.
Methods: As a model of neuropathy we used a chronic
constriction injury (CCI) of the sciatic nerve in mice.
Mechanical sensitivity was evaluated with von Freyfilaments. Immune cells were isolated from damaged
nerves, and ENK extracellular levels were measured byradioimmunoassay.
Results: In vivo, exogenous µ-, δ- and κ-opioid receptor
agonists injected at the CCI site infiltrated by opioid
peptide-containing leukocytes produced analgesia, which
was attenuated by ENK inactivation or leukocyte depletion. The decreased analgesia was restored by re-injectionof wild type, but not of µ-, δ- or κ-receptor lacking
leukocytes.
In vitro, the exogenous opioids secreted
ENK from leukocytes. This release was abolished by
the respective opioid receptor antagonists, by blocking
Gαi or Gβγ subunits, by removing intracellular, but not
extracellular calcium, and was attenuated by inhibitors ofphospholipase C or inositol 1,4,5-trisphosphate receptors.
Conclusion: Our findings indicate that activation of
Gαi-coupled opioid receptors in immune cells leads tothe intracellular calcium-regulated secretion of ENK, andconsequently decreases neuropathy-induced mechanicalsensitivity. The leukocytic opioid receptor-induced opioid
secretion represents a previously unknown mechanistic
basis for peripheral pain relief.
There is no conflict of interest.
Supported by theDeutsche Forschungsgemeinschaft grant.

66

No. 45 - The role of beta-arrestin2 in thedevelopment of morphine tolerance in theenteric nervous system

No. 46 - Ligand ability to evoke Gαi activationand to promote internalization of DOR can
predict response duration over a short period.

Hamid I Akbarali, Tricia H. Smith, Dipanjana D. De,
Hercules Maguma and William L. Dewey

Hanieh Bagheri Tudashki, Derek N. Robertson, Peter W.
Schiller and Graciela Pineyro

Department of Pharmacology and Toxicology, Virginia Commonwealth University

Centre de recherche, Sainte-Justine Hospital, Montreal; Departmentof Pharmacology, Faculty of Medicine, University of Montreal;
Laboratory of Chemical Biology and Peptide Research, ClinicalResearch Institute of Montreal.

Clinical and anecdotal data suggest that tolerance to opioids does not develop to constipation upon long-term treatment. This is supported by studies demonstrating lack oftolerance development in the isolated mouse colon upon repeated morphine exposure. However, tolerance develops toupper gastrointestinal motility and in the isolated ileum. In
the ileum, but not the colon, tolerance is associated witha decrease in β-arrestin2 (BARR2). We have previously
shown that tolerance to morphine occurs in the colon fromBARR2 knock-out (KO) mice. We have now examined a)
localization of BARR2 in the myenteric plexus, b) development of tolerance in a single adult mouse enteric neuron
from the BARR2 KO mice, and c) changes in the expression of the µ-OR in the ileum and colon following expressionof morphine antinociceptive tolerance. Immunohistochemistry was performed on whole-mount longitudinal muscle
myenteric plexus from mouse ileum. Enteric neurons wereisolated, cultured and cell excitability examined by wholecell patch clamp recordings. The mRNA expression forµ-OR was determined by qPCR. BARR2 was specifically
localized in the cell bodies of neurons in the myenteric ganglia co-expressing ChAT, but not Substance P or nNOS.
Isolated neurons from the ileum of wild-type mice exposed
to morphine (10 µM) overnight or from morphine-pelleted(5 days) mice, responded to naloxone (1 µM) with an increased number of evoked action potentials and a decreased
rheobase, indicative of enhanced excitability upon precipitated withdrawal. Neurons from the colon of wild-type miceremained unchanged. However, neurons from the colon of
BARR2 KO mice when treated with morphine overnight, responded with enhanced excitability to naloxone treatment. In morphine-pelleted mice, the expression of OPRM1 was significantly reduced compared to controls in theileum but not the colon. The expression of OPRM-1 wasreduced in the colon of BARR2 KO mice. The decrease inileum of wild-type and colon of BARR2 KO mice was due
to reduced mRNA stability. These studies suggest that therole of BARR2 in the enteric neurons is to maintain theexpression of µ-OR. Supported by NIH DA024009.

Opioids are the most powerful analgesics but their prolonged use can cause the development of analgesic tolerance. The tolerance may be associated with the durationof response to cAMP One approach to assess the production of cAMP in response to activation of adenylate cyclaseis through the use of radiolabeled precursors. This method
requires long periods of activation of the receptor to havea detectable level of cAMP production. During this period, we cannot distinguish the actual response regulatedby the delta opioid receptor (DOR). For this study, we useda biosensor based on BRET technology that provides measurements of cAMP levels as a function of real time. The
aim of our study was to determine whether there is a correlation between the internalization profile of DOR whenstimulated by different ligands, with respect to the duration of signaling in the short-term (≤15 min) and long term
(120 min). During the 15 min of stimulation, inhibition ofcAMP production by DOR activation is characterized byan increase in the inhibition of cAMP, and after 6-7 minutes, a reduction in inhibition of production cAMP. Thisevolution of the duration of cAMP inhibition is biphasic
and is explained in part by the efficiency of ligands to promote Gαi activation and by the profile of internalizationfor each of the different ligands used in this study. For
example, deltorphin II which displayed high efficiency topromote Gαi activation and internalization shows a more
pronounced decline in cAMP response. Unlike morphine
which displayed low efficiency to promote Gαi activation,
and a poor sequestration, displaying a minimal response
decay of inhibition of cAMP. Blocking internalization of
DOR by dynasore reduced response decay for internalizing agonists (e.g.: deltorphin II). However, ligand ability
to promote internalization of DOR does not explain the kinetic profile of a persistent inhibition of cAMP over a longer
period of 120 min. In conclusion this data indicates thata ligands ability to evoke G protein activation or promoteendocytosis were predictive of response duration over short
but not sustained periods of cAMP inhibition.

67

No. 47 - Involvement of Microglial P2X7
Receptors in the Development of Morphine
Analgesic Tolerance

No. 48 - Toll-like receptor 4 mutant and nullmice retain morphine-induced tolerance,
hyperalgesia, and physical dependence

Heather Leduc-Pessah 1 , Nicholas Weilinger 2 , Churmy
Fan 1 , Roger Thompson 2 , Tuan Trang 1

Heather Leduc-Pessah, Theresa Alexandra Mattioli,
Graham Skelhorne-Gross, Chris J.B. Nicol, Brian Milne,
Tuan Trang, Catherine M. Cahill

Departments of Comparative Biology & Experimental Medicine,and Physiology & Pharmacology 1, Hotchkiss Brain Institute 2,
University of Calgary, Calgary, Alberta, Canada

Introduction: Morphine is indispensable in the treatmentof acute and chronic pain. However, its use is limited by
the development of analgesic tolerance, such that higher
and more frequent doses are required to achieve the samelevel of pain control. Growing evidence suggests that microglia are causally involved in the development of opioid
tolerance. In the present study we examined the importance of ATP gated P2X7 receptors (P2X7R) expressed on
spinal microglia in the development of morphine tolerance.
Methods: Sprague Dawley rats were administered morphine (15mg/kg; IP) once daily over 7 days. Morphine
anti-nociception was assessed using thermal tail flick andmechanical paw pressure tests. Cultured BV2 microglial
cells were treated with 5 day morphine(1µM). Changes in
P2X7R protein expression were assessed by Western blot,
whereas modulation of P2X7R function was determined bywhole cell patch clamp recordings and calcium imaging.
Results: We found that repeated morphine administration
caused a progressive decline in morphine anti-nociception
and a loss in morphine analgesic potency, the two key features of morphine analgesic tolerance. The developmentof morphine tolerance correlated with an increase in spinal
dorsal horn microglial P2X7R protein expression. Pharmacologically blocking P2X7Rs with the selective antagonist A740003 attenuated the development of tolerance
but did not reverse established tolerance. In BV2 microglial cells, repeated morphine treatment increased total P2X7R protein expression, an effect recapitulated by
the mu-opioid receptor agonist DAMGO, and suppressedby the mu-receptor antagonist, CTAP. In addition to increased P2X7R expression, we found that repeated morphine markedly potentiated P2X7R-mediated calcium responses and inward current in BV2 microglia.
Conclusion: Taken together, our findings suggest that microglial P2X7Rs are causally involved in the development,
but not expression, of morphine analgesic tolerance. We
also discovered that microglial P2X7R expression and function are critically modulated by activity of mu-opioid receptors.
Acknowledgements: Supported by CIHR, NSERC and CFI
Authors report no conflict of interest

University of Calgary, Queen’s University and University of California
IrvineIntroduction: The innate immune system modulates
opioid-induced effects within the central nervous systemand one target that has received considerable attention is
the toll-like receptor 4 (TLR4). Here, we examined thecontribution of TLR4 in the development of morphine
tolerance, hyperalgesia, and physical dependence in
two inbred mouse strains: C3H/HeJ mice which havea dominant negative point mutation in the TLR4 generendering the receptor non-functional, and B10ScNJ mice
which are TLR4 null mutants.
Methods:
Opioid physical dependence was established using a 5-day escalating morphine dosing paradigm
where mice received intraperitoneal injections of systemicmorphine at 8 h intervals. On day 5 mice were challenged
with a single injection of the opioid receptor antagonist,
naloxone (2 mg/kg), to precipitate morphine withdrawal
and signs of withdrawal were scored. Morphine analgesic
tolerance and hyperalgesia were assessed in mice administered morphine (10mg/kg; i.p) once daily for 5 days or
escalating morphine twice daily for 7 days, respectively.
Anti-nociception was assessed using thermal tail flick
and mechanical paw pressure tests. Gene expression was
analyzed using quantitative real-time PCR and microglial
activation and cfos immunoreactivity were assessed using
immunohistochemical labelling of fixed spinal cord sections.
Results: We found that neither acute antinociceptive
response to a single dose of morphine, nor the developmentof analgesic tolerance to repeated morphine treatment,
was affected by TLR4 genotype. Likewise, opioid induced
hyperalgesia and opioid physical dependence were notaltered in TLR4 mutant or null mice. We also examined
the behavioural consequence of two stereoisomers ofnaloxone: (-) naloxone, an opioid receptor antagonist,and (+) naloxone, a purported antagonist of TLR4.
Both stereoisomers of naloxone suppressed opioid induced
hyperalgesia in wild-type control, TLR4 mutant, andTLR4 null mice.
Conclusion: Collectively, our data suggest that TLR4
is not required for opioid-induced analgesic tolerance,
hyperalgesia, or physical dependence.
Funding: CIHR (TT, CMC) and NSERC (TT, TAM)

68

No. 49 - Agonist-induced phosphorylation of
Thr370 and Ser375 in the COOH-terminus of
the mu-opioid receptor

No. 50 - Synthesis and Biological
Characterization of Tritium-Labelled HS665,
a Highly Potent and Selective Agonist at theκ Opioid Receptor

H.S.Sanderson, G.Henderson and E.Kelly
University of Bristol

Agonist-induced activation of the mu-opioid receptor(MOPr) leads to phosphorylation of multiple residues in
the COOH-terminus of the receptor (1,2,3). The role ofthese phosphorylation events in MOPr function is stilllargely unknown, however phosphorylation of Ser375 isthought to be mediated by GRKs (1,4) and leads toarrestin association and MOPr internalization (2). In
the present study we used phospho-specific antibodies
to study the agonist-induced phosphorylation of Thr370
and Ser375. HA-tagged MOPr was stably expressed in
HEK293 cells and following agonist addition, the receptor
was immuno-precipitated from cells (1,3) and subject toWestern blotting.
Under receptor saturating concentrations, DAMGO,
Endomorphin-2 and methadone all induced robust phosphorylation of Thr370 and Ser375 within 10 min. In
contrast, morphine induced lower levels of phosphorylation
at both theseresidues.
The role of PKC in phosphorylation of
Thr370 and Ser375 was studied using the PKC inhibitor
GF109203X (10 µM, added 30 min before agonist). Pretreatment with GF109203X abolished morphine-induced
phosphorylation of Thr370 but did not affect morphineinduced phosphorylation of Ser375. Pre-treatment with
GF109203X also reduced
DAMGO- and Endomorphin-2-induced phosphorylation of
Thr370, but did not affect the ability of these agonists to
induce phosphorylation of Ser375.
In summary, agonist-induced activation of MOPr
leads to phosphorylation of Thr370 and Ser375 in the
COOH-terminus of the receptor, with morphine inducing
less phosphorylation than other agonists such as DAMGO
and Endomorphin-2. Furthermore, whereas inhibitionof PKC blocked morphine-induced phosphorylation of
Thr370, it did not affect morphine-induced phosphorylation of Ser375. This indicates that PKC activity plays a
key role (either directly or indirectly) in morphine-induced
phosphorylation of Thr370.
(1) Chen Y-J et al, J Neurochem 124:189, 2013
(2) Lau EK et al, Sci Signal 4:ra52, 2011
(3) Just S et al, Mol Pharmacol 83:633, 2013
(4) Doll C et al, Br J Pharmacol 167:1259, 2012

Helmut Schmidhammer1 , Elena Guerrieri1 , Jayapal Reddy
Mallareddy2 , Geza Toth2 , Mariana Spetea1
1Department of Pharmaceutical Chemistry, Institute of Pharmacy,
University of Innsbruck, Innsbruck, Austria; 2Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences,
Szeged, Hungary

Introduction: Activation on the κ opioid (KOP) receptor
leads to significant analgesia, while it is not involved in theunwanted side effects of the µ opioid (MOP) receptor. Besides the analgesic activity, KOP agonists have also shown
other beneficial effects such as antipruritic, anti-arthritic,
anti-inflammatory, and neuroprotective effects. On theother hand, inhibition of the KOP receptor may be usefulfor the treatment of depression, anxiety, stress and drug
addiction. Several N-substituted diphenethylamine analogues have been synthesized and biologically characterized
for opioid receptor activities. The N-cyclobutylmethyl
substituted analogue (HS665) was identified as a novel
highly selective KOP agonist with potent antinociceptive
action mediated through KOP receptor mechanisms [1].
Methods: Bromination of HS665 with N-bromosuccinimide
in dichloromethane afforded the dibrominated analogue, which was tritium labelled with tritium gas over
PdO/BaSO4 catalyst to yield [3H]HS665 with a specificactivity of 30.65 Ci/mmol. Kinetic studies and opioid
binding characteristics of [3H]HS665 were determinedusing radioligand binding assays.
Results: In Chinese hamaster ovary (CHO) cells expressing
the human KOP receptors (CHO-hKOP), binding of the
new tritiated opioid ligand was demonstrated to be rapid,
specific and saturable. [3H]HS665 specifically labelled
a single class of opioid binding sites with an affinity inthe subnanomolar range, together with an extremely low
nonspecific binding (>10%). Furthermore, the abilityof a number of type-selective opioid ligands to displace
its binding from CHO-hKOP cells was assessed. The
competition binding experiments proved the KOP receptor
specificity of this novel radioligand.
Conclusion: Altogether, these properties clearly define theprospective of the tritium-labelled HS665 to become an
important and useful research tool for investigating andunderstanding KOP receptor pharmacology.
[1] M. Spetea, I. P. Berzetei-Gurske, E. Guerrieri and H.
Schmidhammer (2012). J. Med. Chem. 55, 10302-10306.

69

No. 51 - Anti-opioid NMDA receptor theory
underlying the lack of morphine analgesia infibromyalgia-like model mice
Hiroshi Ueda, Yosuke Matsushita, Takehiro Mukae
Department of Pharmacology and Therapeutic Innovation, Nagasaki
University Graduate School of Biomedical Sciences

We have developed an experimental fibromyalgia model inmice, which had been given intermittent cold stress (MolPain, 2008). As seen in fibromyalgia patients, these miceshow long-lasting hyperalgesia and allodynia for manyweeks. Female-dominant gender difference is also observed.
From the point of therapeutic view, such abnormal pain
behaviors could be effectively inhibited by gabapentinoids
and many anti-depressants (Mol Pain 2011). Among all,no morphine analgesia is observed (Neurosci Lett, 2010).
Based on the assumption that excess stress may drive
endogenous opioid systems, we speculated that opioid
tolerance might be caused by the actions of endogenous
opioids. In addition, we have previously demonstrated
that NMDA receptor subunit NR2A plays a crucial rolein the development of morphine analgesic tolerance (Jof Neurosci, 2003). In the present study we will discuss
the possible roles of NMDA receptor system in such“endogenous opioid tolerance” by use of specific TG miceand therapeutic drugs.

No. 52 - Cyclin-depedent kinase 5 regulates
Mu and Delta opioid receptor activities
Hélène Beaudry, Andrée-Ann Mercier-Blais, Caroline
Delaygue, Christine Lavoie, Jean-Luc Parent, Witold
Neugebauer and Louis Gendron
Institut de pharmacologie de Sherbrooke, Faculté de médecine et dessciences de la santé, Université de Sherbrooke, Québec, Canada

Introduction: We have shown that chronic morphine and
CFA-induced inflammation increase the density of cell surface delta opioid receptors (DOPs) in rodent spinal cordand dorsal root ganglia neurons. Among other mechanisms,
receptor phosphorylation represents an important step inthe regulation of receptor trafficking and functions. Interestingly, the phosphorylation of DOP on Thr161 by Cyclindependent kinase 5 (Cdk5) was shown to be involved inreceptor trafficking. The present study therefore aimed todetermine the role of Cdk5 in regulating the functions of
DOP in inflamed- and morphine-treated rats. Since mu(MOP) and DOP have been shown to be coregulated, we
also sought to determine if Cdk5-mediated regulation of
DOP also affects MOP.
Methods: The role of Cdk5 on the regulation of opioid receptors in inflamed- and morphine-treated rats was studied using roscovitine as a selective kinase inhibitor andC11-DOPi2, a cell-penetrant peptide mimicking endogenous DOP’s second intracellular loop.
Results: We found that roscovitine dose-dependently (1-30µg, i.t.) reduced the antinoniceptive effect of deltorphine(Dlt II) in morphine-treated rats. Similarly roscovitine induced a robust decrease in Dlt II-induced antihyperalgesia
in the CFA inflammation model. Administration of the
C11-DOPi2 mimicking peptide also produced a robust decrease in Dlt II-mediated antinociceptive and antihyperalgesic effects. Interestingly, although MOP does not contain the putative phosphorylation motif for Cdk5, we observed that roscovitine significantly increased the DAMGOinduced antinociception and antihyperalgesia. Moreover,blockade of DOP phosphorylation with DOPi2 significantly
increased MOP antinociceptive and antihyperalgesic effects.
Conclusion: Together, our results demonstrate that Cdk5 isa key player of DOP regulation in inflamed- and morphinetreated rats. Moreover, our results indicate that the regulation of DOP by Cdk5 is sufficient to modulate MOP
functions by an indirect process. Further studies are required to understand the precise mechanisms of action of
Cdk5 on MOP and DOP. Supported by CIHR, FRQS and
NSERC. Authors declare no financial disclosure.

70

No. 53 - Molecular determinants ofdelta-opioid receptor recycling

No. 54 - Analysis of Morphine Regulated
Striatal Synaptic Networks using Proteomics
and Network Analysis

Iness Charfi, Graciela Pineyro
Sainte-Justine research center, University of Montreal

Opioids are the most potent analgesics for the treatment
of severe pain. Delta opioid receptor (DOR) agonists induce their analgesic actions with fewer side effects thanthose of mu receptor, making them a target of interest fordevelopment of novel analgesics. However, they induce tolerance to analgesia. Recent hypotheses suggest that distinct profile of tolerance of different opioid analgesics could
result from the stabilization of different ligand-specific conformations of the receptor, each having different trafficking profiles. Here, we compared different DOR ligands toassess whether they display different post-endocytic trafficking (recycling) properties. The experiments were donein HEK293 cells and in cortical neurons transfected with
DORs. Our results indicate that in HEK293 cells, DORs
internalized by DPDPE colocalize with the mannose-6phosphate receptor (M6PR),a marker for late endosomes(LE) and trans-golgi network (TGN). Inhibition of Rab9
and TIP47 (two proteins involved in the transport between
LE and TGN) affects DOR recycling. Furthermore, the kinase PKD (which is known to intervene in trafficking fromTGN to the membrane), the Rab7 (which enables retromermediated transport from sorting endosome (SE) to TGN)
and Rab11 (which mobilizes recycling cargo from perinuclear compartment to the membrane) were also involved inthis process. We can conclude that in HEK293 cells, theDOR may recycle from the SE directly or via the LE and/or
the TGN. Then we studied DOR recycling in neurons. Heretoo, DORs colocalized with M6PR and the recycling wasdependant on Rab9 and TIP47. We can conclude to datethat in neurons, translocation from LE to TGN constitutes
a route for internalized DORs to reach the membrane. Our
outlook is to study these mechanisms for different DOR
agonists and to correlate these properties with the potential of drugs to induce tolerance. These results will enable
the development of ligands with a longer analgesic activity.
(CIHR, NSERC)

Ivone Gomes1 , Steven D. Stockton Jr. 1,2 , Tong Liu3 ,
Chandrakala Moraje1 , Lucia Hipolito-Cubedo4 , Avi
Ma’ayan1 ,, Jose Morón-Concepcion4 , Hong Li3 andLakshmi A. Devi1,2
1Department of Pharmacology and Systems Therapeutics, 2Department of Neuroscience and Friedman Brain Institute, Mount Sinai
School of Medicine, New York, New York, 3Center for Advanced
Proteomic Research and Department of Biochemistry and Molecular
Biology, New Jersey Medical School Cancer Center, UMDNJ,
Newark, NJ, 4 Department of Anesthesiology, Columbia University
Medical Center, New York, NY

The clinical use of morphine in the treatment of chronic
pain is limited by the development of tolerance, dependence and addiction. These undesired effects are thought
to be due to alterations in synaptic transmission andneuroplasticity within the reward circuitry, particularlyin the striatum. In this study, using a combination ofsubcellular fractionation, quantitative proteomics andsystems biology approaches, we examined changes instriatal postsynaptic density fractions following chronicmorphine administration. Differential isotopic labeling andmass spectrometry analysis of fractions from saline andmorphine treated animals identified over 2,600 proteins ofwhich 38 were differentially altered (25 upregulated and 13
downregulated) in response to morphine. Several of thesedifferentially regulated proteins were validated by Western
blot analysis. Next, using graph theory-inspired network
analysis we identified a morphine-regulated network where
upregulated and downregulated proteins were connectedby 40 significant intermediates. Follow up studies witha few of the interesting intermediates in the network(such as caspase-3, receptor-interacting serine/threonine
protein kinase 3 and E3-ubiquitin ligase neural precursor
cell expressed developmentally down-regulated protein 4(NEDD4)) revealed that they were localized to the postsynpase and upregulated in response to chronic morphine
administration. Finally, since many of the proteins in
the morphine-regulated network predicted alterations inproteasomal degradation, we hypothesized that the global
ubiquitination state of the postsynaptic density proteins
were substantially altered by chronic morphine treatment.
Direct examination revealed a significant increase in thelevel of ubiquitination of postsynpatic density proteins inmorphine treated animals as compared to saline treatedanimals.
Taken together, these findings suggest an
emerging role for posttranslational modification of proteinssuch as ubiquitination in opiate tolerance, dependence andaddiction.
This work was supported by NIH grants DA008863,
DA019521, and NS026880 to L.A.D.

71

No. 55 - Evaluating the optimal ratio of mixed
MOR/KOR agonism to prevent liabilities ofuse and cocaine-conditioned place preference.

No. 56 - Mechanism for the allosteric effectsof G proteins on orthosteric ligand binding to
GPCRs

JP McLaughlin1 , SO Eans1 , ML Ganno1 , NC Ross1 , SN
Senadheera2 , JV Aldrich2

Brian DeVree1 , Giselle Velez-Ruiz1 , Jacob Mahoney1 ,Soren Rasmussen2 , Adam Kuszak1 , Jan Steyaert3 , Els
Pardon3 , Brian Kobilka4 , and Roger Sunahara1

1:Torrey pines Institute for Molecular Studies, Port St Lucie, FL
34987, USA; 2:Dept of Medicinal Chemistry, The University of
Kansas, Lawrence, KS 66045, USA

Mixed activity mu opioid receptor (MOR) and kappa
opioid receptor (KOR) agonists may prevent the acute rewarding effects of abused drugs. We used two macrocyclic
tetrapeptide mixed opioid agonists with differing ratiosof MOR and KOR agonism to evaluate the balanced of
mixed MOR/KOR agonism that would minimize liabilities
while preserving the therapeutic ability to suppress the
rewarding effects of cocaine.
Methods: The ratio of MOR to KOR agonist activities possessed by the two peptides CJ-15,208 (Ross et al.,2012) and [D-Ala1,D-Trp4]CJ-15,208 (Aldrich et al., 2014)
were determined in the 55oC warm-water tail-withdrawal
test, using wild-type C57BL/6J, MOR knockout (MOR
KO) and KOR knockout (KOR KO) mice. Sedation andrespiratory depression were evaluated in the rotorod assay
and CLAM system. Conditioned place preference (CPP)
testing directly evaluated the rewarding effect of thepeptides and their ability to prevent cocaine CPP.
Results:
CJ-15,208 and [D-Ala1,D-Trp4]CJ-15,208
produced equipotent antinociception in wild-type mice,with ED50 (and 95%CI) values of 1.74(0.62-4.82) and3.03(2.16-4.59) nmol, icv, respectively. CJ-15,208 showed
an equal mix of MOR and KOR agonism, whereas [DAla1,D-Trp4]CJ-15,208 antinociception was predominantly
KOR mediated. Neither compound produced sedation
at doses up to 100 nmol icv. CJ-15,208 produced initialconditioned place aversion (CPA) at a 3 nmol icv dose andprevented cocaine-CPP, but at higher doses demonstrated
MOR-mediated CPP. In contrast, [D-Ala1,D-Trp4]CJ15,208 (3 nmol icv) was without effect in the conditioned
place preference assay, but demonstrated CPA at higher
doses that also prevented cocaine-CPP. Both of theseeffects were absent in KOR KO mice.
Conclusions: The addition of MOR agonism to KOR
agonism alleviates some liabilities attributed to KORselective agonists, yet retains the ability to counteract the
rewarding effects of cocaine. However, an even ratio of
MOR/KOR agonist activity proved detrimental at higher
doses, suggesting an upper limit to the desired amount of
MOR agonism.

1University of Michigan, 2University of Copenhagen, 3Vrije Universiteit Brussel, 4Stanford University

G protein-coupled receptors (GPCRs) are allostericmachines that translate extracellular ligand binding into
intracellular signaling via transition between distinct
conformational subsets. Agonist-mediated stabilizationof an active GPCR conformation, and thus promotionof G protein interaction, is a fundamental tenet of
GPCR biology. In addition, G protein-mediated allostericenhancement of agonist affinity has been observed in many
GPCRs. However, the intricacies of this high-affinity stateremain poorly understood, and the mechanism by which G
proteins allosterically regulate orthosteric ligand binding
is unclear. Based on structural data, we hypothesizedthat transition to an active receptor conformation leads
to constriction of the orthosteric binding site around its
ligand, thus slowing dissociation and enhancing affinity.
We used equilibrium and kinetic radioligand binding
assays to characterize the effects of G protein and G
protein-mimetic nanobodies on the ligand-binding properties of multiple GPCRs, and also investigated the roles ofindividual residues within β2-adrenergic receptor (β2AR).
When bound to nucleotide-free Gs heterotrimer, β2AR
displayed a Bmax decrease which was restored by GDP or
GTPγS. [3H]DHAP association was slowed when β2AR
was bound to nucleotide-free Gs heterotrimer, and GDP
reversed this effect in a concentration-dependent manner.
The Gs mimetic nanobody Nb80 slowed both [3H]DHAP
association and dissociation. However, this effect was
not antagonist-specific, as Nb80 also inhibited associationof the agonist [3H]Formoterol.
Together, these datasuggest that transit into or out of the β2AR orthostericsite is restricted due to a “closed” orthosteric site in
the active β2AR conformation (bound either to Gs or
Nb80). Furthermore, it appears that high-affinity agonist
binding is only supported when receptor is bound to
nucleotide-free G protein. Interestingly, the M2 muscarinic
acetylcholine receptor and mu opioid receptor displayed
ligand-binding phenomena similar to those we observedfor β2AR, suggesting that this behavior may extend to
GPCRs beyond β2AR and the amine receptor family.
This work was supported by AHA 13PRE17110027.

Funding:
NIDA grant R01 DA032928 and funds
from the State of Florida.
Conflicts of interest: none.

72

No. 57 - Characterization of theAntinociceptive and Kappa Opioid Receptor
Antagonist Activity of Analogs of theMacrocyclic Tetrapeptide CJ-15,208
J.V. Aldrich (1) , S.N. Senadheera (1) , T.F. Murray (2) ,M.L. Ganno (3) , S.O. Eans (3) , E. Mizrachi (3) , and J.P.
McLaughlin (3)1) Department of Medicinal Chemistry, The University of Kansas,
Lawrence, KS 66045, USA; 2) Creighton University School of
Medicine, Omaha, NE 68178, USA; 3) Torrey Pines Institute for
Molecular Studies, Port St. Lucie, FL 34987, USA

The macrocyclic peptide CJ-15,208 (cyclo[Phe-D-Pro-PheTrp], Saito et al., J. Antibiot. 2002, 55, 847) exhibits mixed
opioid agonist/kappa opioid receptor (KOR) antagonist
activity in vivo (Ross et al., Br. J. Pharmacol. 2012, 165,
1097) and is a promising lead compound for developing
novel therapeutics for the treatment of pain and drug
abuse. We synthesized analogs of this lead peptide to
explore the influence of the aromatic residues on theanalogs’ opioid activity profiles.
Methods. The analogs were synthesized by a combination of solid phase synthesis with cyclization in solution
under optimized reaction conditions. The peptides were
evaluated in radioligand binding assays for mu, kappaand delta opioid receptor affinities in vitro, and in themouse 55 oC warm water tail withdrawal assay in vivo for
antinociception and opioid antagonist activity.
Results. The analogs generally exhibited potent antinociception in vivo comparable to CJ-15,208 following both
intracerebroventricular and oral administration in spite of
reductions in opioid receptor affinities in vitro; multiple
opioid receptors contributed to the antinociception ofeach analog. In contrast to the parent peptides, only
one of the analogs exhibited KOR antagonism in vivo.
Interestingly, when screened in an acute tolerance model,
the antinociceptive tolerance observed varied markedly
among the peptides, from no significant display of tolerance
to a rightward shift in the dose-response curve equal to or
greater than that exhibited by morphine.

No. 58 - Broad spectrum analgesic efficacy ofIBNtxA is mediated by exon 11-associatedsplice variants of the mu-opioid receptor geneJeffrey S. Wieskopf1 , Ying-Xian Pan2 , Jaclyn Marcovitz1 ,Alexander H. Tuttle1 , Susruta Majumdar2 , Gavril W.
Pasternak2 , and Jeffrey S. Mogil1
1Dept. of Psychology and Alan Edwards Centre for Research on Pain,
McGill University, Montreal, QC H3A 1B1 CANADA; 2Departmentof Neurology and Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065

Mu-opioids remain vastly important for the treatment ofpain, and would represent ideal analgesics if their analgesic
effects could be separated from their many side effects. A
recently synthesized compound, iodobenzoylnaltrexamide(IBNtxA), acting at 6-transmembrane (6-TM) splicevariants of the mu-opioid receptor gene, was shown to havepotent analgesic actions against acute, thermal pain accompanied by a vastly improved side-effect profile comparedto 7 TM-acting drugs such as morphine. Whether suchanalgesia can be seen in longer lasting and non-thermal
algesiometric assays is not known. The current study
demonstrates potent and efficacious IBNtxA inhibitionof a wide variety of assays, including inflammatory andneuropathic hypersensitivity and spontaneous pain. We
further demonstrate the dependence of such analgesia on
6-TM mu-opioid receptor variants using isobolographic
analysis and the testing of Oprm1 (the mu-opioid receptor
gene) exon 11 null mutant mice. Finally, the effect of nerve
damage (spared nerve injury) and inflammatory injury(complete Freund’s adjuvant) on expression of mu-opioid
receptor variant genes in pain-relevant central nervous
system loci was examined, revealing a complex pattern ofchanges. These findings are supportive of the potential
value of 6-TM-acting drugs as novel analgesics.
The Louise and Alan Edwards Foundation (J.S.M.)
and the National Institute on Drug Abuse of the NIH(DA02615, DA06241, DA07242, G.W.P.).
JSW wassupported by a Vanier Canada Graduate Scholarship fromthe Canadian Institutes for Health Research. None of
the authors has a conflict of interest related to this research.

Conclusions.
Changes in the macrocyclic tetrapeptide structure were well tolerated with retention ofantinociceptive activity, while the KOR antagonism wasvery sensitive to changes in the aromatic residues. The
identification of compounds with potent antinociceptionthat exhibit minimal tolerance in the initial screening
is a promising development in the search for potential
analgesics with improved liability profiles.
Research supported by NIDA grant R01 DA018832.

73

No. 60 - DEVELOPMENT OFPERIPHERALLY AVAILABLE MIXEDEFFICACY MOR/DOR LIGANDS THATDISPLAY REDUCED DEVELOPMENT OF
TOLERANCE

No. 59 - Functionally selective kappa opioid
receptor agonist efficacy in pruritis
J. Morgenweck
L.M.Bohn (1)(1), K. Frankowski(2), J. Aubé(2),(1) Department of Molecular Therapeutics and Neurosciences, The
Scripps Research Institute, Jupiter, FL 33458, (2) Department ofMedicinal Chemistry, The University of Kansas, Lawrence, KS 66045

Introduction: The only clinically utilized KOR agonist
on the market is the partial agonist Nalfurafine which isused in the treatment of pruitis or itch. Conversely, KOR
antagonists induce acute pruritis emphasizing that KOR
may be a very good target for the treatment of pruitis.
The mechanism through which KOR modulates this
response remains unclear. In this study we behaviorally
investigate the role of KOR and the GPCR regulatory
protein, βarrestin2, in the itch response in mice.
Methods: Itch is evaluated by using an establishedmouse model of acute pruritis wherein a single subcutaneous (s.c.) injection of an itch-inducing irritant (either
KOR antagonist or chloroquine phosphate) is used toinduce robust scratching bouts that are quantified over a
60 minute observation period.
Results: Two KOR antgonists, 5’GNTI and norBNI,
induce itch in C57BL/6J mice in a dose-dependent manner
which is decreased (but not eliminated) in KOR KO
mice supporting the involvement of the KOR system inpruritis. This response can be dose-dependently reducedwith pretreatment of the conventional KOR agonist,
U50,488H, as previously shown. A functionally selective
KOR agonist that preferentially promotes G protein
signaling over βarrestin2 recruitment is also efficacious in
suppressing the itch response. Like U50,488H, the biased
KOR agonist is also efficacious in preventing chloroquine
phosphate-induced itch. To further test the βarrestin2
involvement in this response, itch was also evaluated βarr2
WT and KO mice.
Conclusions:
These observations suggest that agonist efficacy at KOR
for treating pruitis may be due to its utilization of G
protein signaling pathways. Further studies are needed todetermine the mechanistic underpinnings of KOR functionin the development and treatment of pruitis.

Jessica P. Anand (1) , Vanessa R. Porter-Barrus (1) , Emily
M. Jutkiewicz (2) , John R. Traynor (2) *, and Henry I.
Mosberg (1)(1)(1) Department of Medicinal Chemistry, and (2) Departmentof Pharmacology, University of Michigan, Ann Arbor, MI, 48109, USA

It has been shown that the co-administration of a mu opioid receptor (MOR) agonist with a delta opioid receptor(DOR) antagonist displays MOR mediated analgesia, butalso produces reduced negative side effects notably the development of tolerance and dependence, features that limit
the clinical use of opioid analgesics. Administering drug
cocktails has considerable disadvantages related to potential diverse pharmacokinetic properties. Therefore we have
explored the development of multifunctional ligands that
display both MOR agonism and DOR antagonism in a single molecule. We have generated a series of constrained
mixed efficacy peptides using our previously published homology models of the opioid receptors to guide ligand design. These peptides displayed low nM affinity for MOR
and DOR in vitro with selectivity over the kappa opioid
receptor. This series of ligands also displayed MOR efficacy similar to morphine and reduced DOR activation,with some compounds showing a complete lack of stimulation at DOR. As low bioavailability is typical of peptides,
we installed a C-terminal sugar moiety, in which the side
chain hydroxyl of Ser is covalently O-linked to a glucose or
lactose as a means of improving blood-brain barrier penetration. Both glycosylated and unglycosylated derivatives
were assessed in vivo for antinociceptive properties. Those
ligands that produced antinociception were further evaluated for the development of tolerance and dependence. Ligands in this series displayed a reduced development of acute
tolerance, as desired. This demonstrates that although peptides are traditionally considered to be poor drug scaffolds,
they can be viable tools for exploring therapeutic peptide
analgesics as well as potentially developing molecules with
reduced dependence liability and extended therapeutic use.
This work was funded by NIH grants DA003910 (H.I.M.)
MH083754 (J.R.T), DA007267 (V. P.-B.), DA007281 and
GM007767 (J.P.A.)

Support: NIH/NIDA: R01DA031927 (LMB and JA)
Disclosure:74

No. 61 - Exploring pharmacological functionsof mu opioid receptor carboxyl termini ingene targeted mouse models
J. Xu1 , V. Le Rouzic1 , ZG. Lu1 , M. Xu1 , T. Brown1 , W.
Hoefer2 , G.C. Rossi2 , G.W. Pasternak1 , Y.-X. Pan1
1 Department of Neurology the Molecular Pharmacology andChemistry Program, Memorial Sloan-Kettering Cancer Center, New
York, NY 10065, U.S.A. ;2Department of Psychology, CW Post
College, Long Island University, Brookville, NY 11568, U.S.A.

Mu opioid receptor (OPRM1) gene undergoes extensive alternative pre-mRNA splicing, creating an array of splicevariants or isoforms that are conserved from rodent to human. These splice variants have been categorized into threemajor types based upon receptor structure, full-length carboxyl (C-) terminal variants with 7-transmembrane (TM)domains, truncated variants containing 6-TM domains andtruncated variants with single TM. Structurally, all the Cterminal variants shared the entire receptor structure predicted from exons 1/2/3 with the original MOR-1, exceptthat they have different intracellular C-terminal tails. The
functional significance of these C-terminal variants has beensuggested by differences in region-specific expression, mu
agonist-induced G protein coupling, phosphorylation, internalization and post-endocytic sorting. However, little has
been known for their in vivo function. In the present study,
we generate three gene targeted mouse models: 1) mE3M
in which all C-terminal tails are eliminated by introducing
a stop codon in the end of exon 3; 2) mE4M and 3) mE7M,
in which C-terminal tails encoded by exon 4 or exon 7 are
disrupted by inserting a stop codon in the beginning of exon
4 or exon 7, respectively. All three mouse models displayed
normal analgesic responses toward various mu opioids, buteach mouse model exhibited unique profiles in terms of
mu opioid-induced tolerance, physical dependence and rewarding. For example, mE7M homozygous mice showed
resistance to morphine tolerance, contrasting to mE4M homozygous mice that were vulnerable to morphine tolerance.
Eliminating C-terminal tails encoded by exon 7 had no effect on morphine-6β-glucuronide (M6G) dependence. However, disrupting C-terminal tails encoded by exon 4 significantly reduced M6G dependence. Our data highlight thefunctional importance of the C-terminal tails in mediating
the actions of various mu opioids, and suggest distinct rolesof individual C-terminal tails in the complex mu opioid actions. (Supported by DA13997 & DA029244 (Y.-X.P) andDA02615 & DA07242 (G.W.P) from the National Instituteon Drug Abuse; CA08748 (Core grant, MSKCC) from the
National Cancer Institute)

No. 62 - Optimization of an active vaccine
against heroin: immunotherapeutic
approaches towards long-term blockade ofheroin activity
Schlosburg JE1,2 , Bremer PT2 , Stowe GN2 , Vendruscolo
LF1 , Lockner JW2 ,Lively JM2 , Koob GF1 , & Janda KD2
1Committee on the Neurobiology of Addictive Disorders; 2Departments of Chemistry, Immunology, and Microbial Science, The Scripps
Research Institute, La Jolla, CA 92037

Heroin continues to be the most statistically likely drug
to result in addiction among users, with few effective
therapeutic options that result in long-term abstinence.
While opioid antagonists can be administered in slowrelease formulations, these treatments often preclude othercommon treatment options, have potential for side-effects,
and require moderate medical monitoring.
We havepreviously demonstrated a novel heroin vaccine hapten
design that is capable of preventing heroin antinociception, conditioned place preference, reinstatement, andre-escalation of self-administration with high specificity
without targeting opioid receptor activity. This vaccine
attached heroin to keyhole limpet hemocycanin (KLH),
via a thiol linker attached at the nitrogen bridgehead,allowing the metabolically labile portion of heroin toremain exposed. When delivered in alum adjuvant, theresult was generally high titer levels of antibodies that hadbinding specificity for heroin (4±1 µM) and especially highaffinity its primary metabolite, 6-acetylmorphine (35±1nM).
Using serum antibody titer ELISA measurements andquantitative screening methods of heroin-induced hot
plate and tail immersion responses (54°C) in mice, wehave engaged in a multi-step optimization of the original
vaccine. The process systematically examined all thecomponents of the heroin vaccine formulation: route ofadministration, protein immunogen, chemical linkers,
and adjuvants. We have carried the top performing
variations forward through each successive test, withthe final result being an optimized vaccine formulationthat appears to have roughly 5-10 times the capacity inpreventing heroin psychoactivity of the original vaccine,
shifting heroin dose-response curves over 20-fold versus
unvaccinated animals. The blockade of heroin activity iseffective for at least several months, and may represent
an effective adjunct therapeutic option for heroin addiction.
Research Supported by DA026625 and the Pearson
Center for Alcoholism and Addiction Research. The
authors declare no conflicts of interest for this research.

75

No. 63 - Identification of Novel Signaling
Regulators of the Mu Opioid Receptor
Justin LaVigne, Katie Edwards, Kathlene Pangilinan,
Kyle Hong, Nate Mullen and John M. Streicher
University of New England, Department of Biomedical Sciences,
Biddeford, ME USA

Chronic pain is a serious health concern with high costs
and a significant impact on patient quality of life. A
multi-decade drug discovery effort targeted at finding
new pain therapeutics has met with limited success.
Recent work has demonstrated that specific signalingcascades (i.e. βarrestin2) downstream of the mu opioid
receptor (MOR) mediate specific behavioral effects, andthat functionally selective drugs that specifically activate
the desired cascades can have an enhanced therapeuticindex. However, very few regulatory proteins which canbe targeted in this manner have been described. In orderto facilitate the identification of such targets, we haveapplied two methods of unbiased screening to find novelsignaling regulators of the MOR. For one, we have createda MOR-CHO cell line, and used co-immunoprecipitation of
drug activated receptor complexes followed by proteomic
analysis in an attempt to find novel associated proteins.
For the other, we will transfect a MOR-HEK cell line witha pooled shRNA library, covering 15,000 human genes.
Populations of cells with increased or decreased signaling
will be isolated by flow cytometry, and the candidate genes
which regulate MOR induced signaling will be identified
by next-gen sequencing. From these data, we will identify
a list of potential signaling regulators of the MOR, andwill further test the effect of candidate knockdown on
MOR induced signaling. Using this approach, we haveinitial confirmation that a novel scaffold protein previously
unassociated with the MOR regulates MOR induced
ERK activation without altering receptor expression or
activation. We have also begun to test other candidate
regulators with a similar approach. Overall, we hope
to use this data to further define the MOR signaling
complex, and to identify new targets and strategies for
drug discovery to treat chronic pain.
Conflicts: None
Funding:
COBRE Pilot Project Grant (underP20GM103643)

No. 64 - Wireless devices for optogenetic andpharmacological dissection of neural circuits
in behavior
Jordan G. McCall, Gunchul Shin, Sung il Park, Jae-Woong
Jeong, Daniel V. Harburg, Ream Al-Hasani, Edward R.
Siuda, Martin J. Schmidt, Daniel S. Brenner, Robert W.
Gereau, IV, John A. Rogers, and Michael R. Bruchas(1) Division of Biological and Biomedical Sciences (DBBS), Neurosciences Program, (2) Dept. of Anesthesiology, (3) Anatomy and
Neurobiology, (4) Pain Center, Washington University, St. Louis,
MO, USA, (5) Dept. of Materials Science and Engineering, (6)Chemistry, (7) Mechanical Science and Engineering, (8) Electricaland Computer Engineering, (9) Beckman Institute for Advanced
Science and Technology (10) Frederick Seitz Materials Research Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL, USA

The rise of optogenetics provides a unique opportunity toadvance both materials engineering and neuroscience. We
report a series of novel devices for studying and perturbing
intact neural systems through optogenetics, microfluidic
pharmacology, and electrophysiology. Unlike optogenetic
approaches that rely on rigid, glass fiber optics coupled toexternal light sources, these novel devices utilize flexible
substrates to carry microscale, inorganic light emitting
diodes (µ-ILEDs) and multimodal sensors into the brain.
As with previous versions of these devices, wireless control
is possible using radiofrequency (RF) power scavenging.
Increasing the carrier frequency of this RF signal allows
for miniaturization of these harvesters. We demonstratethat these devices can be accurately implanted into deepbrain structures while mounted completely subcutaneously under the scalp. Because of their architecture,light from these devices can be delivered at different
angles allowing complete dorsal-ventral targeting of deepbrain structures. We demonstrate the utility of these
independently-controlled µ-ILEDs in isolating anatomical
subregions of dynorphinergic neurons. In addition to
optogenetic control, we also present devices for wirelessdelivery of conventional and UV-caged opioid agonists andantagonists. These wireless microfluidic devices introduce
a second wireless scheme based on battery-powered microprocessors for combined optogenetic and pharmacologicalmanipulation of neural circuits. Here we demonstrate theability to wirelessly deliver the µ-opioid receptor agonist,
DAMGO, in a homecage environment. Finally, acute invivo electrophysiological study of opioid circuits is possible
with completely dissolvable recording and stimulating
electrodes. These devices allow for chronic implantation ofa transient electrode that is fully resorbed by brain tissue
over time. In sum, we present a diverse toolset of devices
capable of achieving previously untenable control ofneuropeptide and monoamine neural circuits. Supported
by NIDA R01DA037152 (MRB), NIH Common Fund
NINDS R01NS081707 (MRB, RWG, JAR), and NIMH
F31MH101956 (JGM).

76

No. 65 - Stress-induced increases in
depression-like and cocaine-seeking behaviors
are reversed by disruption of memories duringreconsolidation

No. 66 - Extrasynaptic GABAA receptors in
the periaqueductal gray are involved indescending pain modulation and chronic
inflammatory pain–induced plasticity.

K. A. Hymela, S. O. Eansa, K. Sitchenkob, S. M. Gomes
a,b, A. L. Lukowskya, J. M. Medinaa, E. I. Sypekb, A. N.
Careyb,c and J. P. McLaughlina,bKaren J. Tonsfeldt, Katherine L. Suchland, Minghua Li &Susan L. Ingram.

aTorrey Pines Institute of Molecular Studies, Port St. Lucie, Florida
34987, USA; bDepartment of Psychology, Northeastern University,
Boston, Massachusetts 02115, USA; cDepartment of Psychology,
Simmons College, Boston, Massachusetts 02115, USA

Maladaptive behavioral responses characteristic of posttraumatic stress disorder (PTSD) are notably resistant
to treatment after establishment. We hypothesized that
disrupting memory reconsolidation during stress reexposure may reverse previously established stress-induced
increases in depression-like and cocaine-seeking behaviors.
C57BL/6Jmice were subjected to repeated social defeat
stress (SDS), and examined for time spent immobile
in a subsequent forced swim test (FST). Additional
SDS-exposed mice were place conditioned with cocaine,and tested for conditioned place preference (CPP). To
determine if memory disruption could reverse PTSD-likesequelae, mice were administered vehicle or drugs provento disrupt memory formation during reconsolidation(propranolol (1 or 10 mg/kg, i.p.) or cycloheximide (2.2 or
4.4 mg/kg, i.p.)) both 1 h prior and 2 h after re-exposure
to a single additional trial of SDS. Mice were then again
tested for FST or CPP the next day. SDS-exposed micedisplayed increases in socially defeated postures across SDS
trials and subsequently demonstrated significant increases
in time spent immobile in the FST or in the cocaine-pairedchamber compared to controls. Vehicle-treatment followedby additional SDS-exposure did not alter these behaviors,
but propranolol- or cycloheximide-treatment reversed
each of the potentiated responses in a dose-dependent
manner. Overall, these results demonstrate that while
repeated exposure to a social defeat stressor subsequently
increased depression-like and cocaine-seeking behaviors,
pharmaceutical disruption of traumatic memories madelabile by re-exposure to SDS during reconsolidation may
have therapeutic value in the treatment of established
PTSD-related behaviors.
Source of Funding: This research was supported bya Northeastern University Provost’s RSDF award to JPM
and funds from the State of Florida, Executive Office of
the Governor’s Department of Economic Opportunity.

Dept. of Neurological Surgery, Oregon Health & Science University,
Portland, OR

Plasticity changes induced by chronic inflammatory pain
have been described in many areas of the descending modulatory pathway, but remain relatively unstudied in theperiaqueductal gray (PAG). Opioid inhibition of GABAergic synaptic activity in the PAG activates a descending
antinociceptive pathway. The extrasynaptic, tonic chloride current produced by delta-subunit containing GABAA
receptors has been identified in the PAG, but its role inchronic inflammatory pain has not been explored. Extrasynaptic and synaptic GABAA currents were measured inmale and female rats treated with Complete Freud’s Adjuvant (CFA) or untreated (naı̈ve). Electrophysiologicalrecordings were made from slices containing the PAG sixdays after CFA injection. Basal GABA mIPSC frequency
was increased in CFA (2.2 ± 0.5 Hz, n=12 cells) comparedto saline pretreated rats (1.1 ± 0.2 Hz, n=8 cells). The
amplitude of the GABA mIPSC was significantly increasedin CFA treated rats (45 ± 4 pA, n=10), compared to naı̈ve
rats (32 ± 4 pA, n=11). Superfusion with the GABAA antagonist bicuculline (BIC) reduced the holding current by
18 ± 4 pA (n = 10) in naive male rats. The GABAA antagonist picrotoxin (300 µM) changed the holding current by 7
± 2 pA (n=6) and this effect was enhanced by CFA treatment in female rats (16 ± 6 pA, n=3). When measured
using qRT-PCR, CFA-treated rats had significantly moreGABAA-delta subunit mRNA (2.8 ± 0.4 (n=4) comparedto naı̈ve rats (1.1 ± 0.4 relative units, n=3).These studies
indicate that the extrasynaptic GABAA current mediatedby receptors containing the delta subunit may be an important site of plasticity during chronic inflammatory pain.
We used the delta subunit agonist THIP to examine how
activation of the tonic current alters pain behavior. On its
own, THIP (100 ng) microinjected into the vlPAG had noeffect on hot plate latency but significantly attenuated morphine (5 µg)-induced antinociception. In addition, DS2, a
new allosteric agonist of the delta subunit, shifts the morphine dose-response curve of morphine to the right. These
data support a role of the extrasynaptic GABAA receptor
in pain modulation in vivo.
Support: AG023477 (KJT) and DA027625 (SLI)

Conflict of Interest: none declaredskdfhagvbujagvk;

77

No. 67 - STRATIFICATION OF OPIOIDANALGESICS ACCORDING TO BIAS
FOOTPRINTS AT MU- (MOR) AND
DELTA-OPIOID RECEPTORS (DORs).
Karim Nagi (1,2) , Johanie Charbonneau (1,2) , Victoria
Lukashova (1) , Christian Le Gouill (1) , Michel Bouvier (1)and Graciela Pineyro (1,2)(1) University of Montreal and (2) Ste-Justine Hospital ResearchCenter, Montreal, Quebec, Canada.

Opioid analgesics are elective in the treatment of severe
pain but their use is restricted by severe side effects.
Signaling bias has been proposed as a viable means for
improving this situation but we still ignore the whether
signaling preferences are indeed predictive of desired andunwanted actions of opioid analgesics. To start addressingthese issues HEK293 cells were transfected with different
BRET-based biosensors to compare clinically available
opioids and selective DOR agonists in their efficiency
to induce: activation of different G proteins (Gαi/o/z),
stimulation of analgesic effectors (Kir3.1/3.2 channels),
inhibition of cAMP production and recruitment of βarr2
in presence or absence of GRK2, GRK5 and GRK6. Bias
was calculated from operational transduction coefficients(log(Tau/KA)) using Met-enkephalin as the standard(Met-ENK). Responses elicited by rat DORs indicatedthat deltorphin II defined a group on its own being the only
ligand with no bias between Kir3 and cyclase responses.
For the rest of ligands Kir3 log(Tau/KA) ratios were atleast one order of magnitude greater than those observed
for cyclase inhibition.
Among them βarr2 responses
allowed to define three additional groups: a) drugs thatfailed to elicit any βarr2 response (buprenorphine andTIPP); b) drugs whose log(Tau/KA) ratios for different
βarr2 readouts were not different from those corresponding
to cyclase responses (morphine, oxycodone and DPDPE)
and c) drugs whose efficiency to inhibit cAMP production
was at least one order of magnitude lower than that
displayed for βarr2 recruitment (loperamide, AR-M100390
and SNC-80). Although with different efficiencies, allligands activated Gαi1/αo/αz proteins, displaying no bias
among these responses. Comparing results obtained with
rat DORs to human DORs and to rat MORs indicatedthat bias profiles were reasonably maintained among DORsof different species but varied among receptor subtypes. If
these signaling profiles are predictive of in vivo outcomes,
they should allow to segregate ligands into groups with
distinct potential for analgesia and/or side effects.
Funding: This work was supported by Pfizer (M.B
and G.P).

No. 68 - Post-sampling changes in opioid
peptide levels and the effect of tissue
stabilization
Jenny Gustafsson1 , Lova Segerström1 , Karl Sköld2 , Mats
Borén2 , Ingrid Nylander1
1Department of Pharmaceutical Biosciences, Uppsala University,
Uppsala, Sweden 2Research and Development, Denator AB, Uppsala,SwedenAim of Investigation: Immediately after sacrifice ofexperimental animals or tissue excision during sampling,
enzymes present in tissues may start to degrade proteinsand peptides, and thus give rise to analytical results basedon post-mortem conditions that are not representative tothe in vivo conditions. The present project evaluates theuse of conductive heat transfer for sample stabilization(enzymatic inactivation) in a routine radioimmunoassay
protocol for measurement of opioid peptides in rat brain.
Methods:
The Stabilizor T1 instrument (Denator
AB, Gothenburg, Sweden) was used for stabilization.
Different post-mortem treatments were tested on stabilized
and non-stabilized tissue and the effect was evaluated on
the levels of opioid peptides in rat hypothalamus (HT),striatum (Str), and cingulate cortex (Cg). Dynorphin A(Dyn A), Dynorphin B (Dyn B), Leu-enkephalin-Arg6(Leu-Arg), and Met-enkephalin-Arg6Phe7 (MEAP) was
analysed using radioimmuneassay (RIA).
Results: Levels of Dyn A, Dyn B, and MEAP in
HT were higher in stabilized tissue than in non-stabilized.
Leu-Arg, a degradation product from the dynorphins,
showed an opposite pattern. Stabilization before freezing
resulted in higher Dyn A, Dyn B, and MEAP compared tosamples which were stabilized in a frozen state.
Conclusions: This work proves that samples enzymatically stabilized using the Stabilizor T1 instrument
can be analyzed using standard RIA protocols and keep
the sample integrity. All of the peptide levels were stable
two hours post-stabilization in room temperature, proving
the instrument’s application in the clinic.
However,
to fully evaluate the results obtained in this project,complimentary mass spectroscopy analyzes of the different
post-mortem treatment groups are necessary to determine
from which peptide, or peptides, the measured immunoreactivity is derived from.
Acknowledgments:
Funding was provided from theSwedish Medical Research Council and ERAB

78

No. 69 - Probing Allosteric Modulation of the
Mu Opioid Receptor

No. 70 - The involvement of brain GPR40
signaling in regulation of the descending pain
inhibitory system

Livingston KE, Traynor JR
University of Michigan; Department of Pharmacology

The mu opioid receptor (MOPr), particularly the orthosteric site, is the major target for all clinically used opioid drugs. We have begun to investigate positive allostericmodulation of MOPr as a novel avenue for managing pain
and have recently characterized the first positive allostericmodulator (PAM) of MOPr, BMS-986122 (BMS). The objective of the current study was to investigate the probe
selectivity, and possible mechanism of action of BMS fora variety of orthosteric MOPr ligands by radioligand binding assays and by activation of G protein using the [35S]GTPγS assay. In membranes from C6 glioma cells expressing MOPr, BMS alone did not displace orthosteric ligands
from MOPr, nor did it alter basal [35S]-GTPγS binding.
As a MOPr PAM, BMS increased the binding affinity ofseveral clinically used opioid drugs, including methadone
and loperamide, as well as endogenous peptides including
methionine-enkephalin and β-endorphin. In addition, BMS
enhanced the potency of these ligands to cause activationof G protein with allosteric cooperatively (α) values ranging from 6 to 18. In contrast, binding affinity and potency
to stimulate [35S]-GTPγS binding of the opioids morphineand congeners as well as fentanyl was unaffected by BMS.
For these compounds, there was an enhancement of maximal G protein stimulation. Sodium ions are known to stabilize inactive states of G protein-coupled receptors, including MOPr, thereby reducing agonist affinity. To test the
hypothesis that BMS promotes active-like states of MOPr,
we compared the sensitivity of MOPr agonists to BMS with
their sensitivity to Na+ ions and found an excellent correlation. Moreover, the potency of Na+ ions to reduce
DAMGO binding was markedly attenuated in the presenceof BMS. Overall, these findings show that BMS exhibits
distinct probe dependence for MOPr agonists and that thismay be related to an ability of BMS to interfere with thebinding of Na+ ions to the receptor. Supported by T32
DA007267.

Kazuo Nakamoto (1) , Naoya Sato (1) , Takashi Nishinaka(1), Aizawa Fuka (1) , Mitsumasa Mankura (2) , Yutaka
Koyama (3) , Fumiyo Kasuya (4) and Shogo Tokuyama (1)(1) Department of Clinical Pharmacy, School of Pharmaceutical
Sciences, Kobe Gakuin University, (2) Bizen Kasei Chemical Co.
Ltd., (3) Laboratory of Pharmacology, Faculty of Pharmacy, Osaka
Ohtani University, (4) Biochemical Toxicology Laboratory, Facultyof Pharmaceutical Sciences, Kobe Gakuin University

Omega-3 polyunsaturated fatty acids (PUFAs) such as
docosahexaenoic acid (DHA) have an antinociceptive
effect against pain caused by inflammation and neuropathy; however, the underlying mechanisms remain
poorly understood. Previously, we demonstrated that
DHA-induced antinociception, effectuated by increased
β-endorphin release, may be mediated by GPR40, along-chain fatty acid receptor. Here, we examine theinvolvement of GPR40 signaling in the supraspinal areain the regulation of the descending pain inhibitory systemcomprising serotonergic and noradrenergic neurons.
Immunohistochemistry revealed that GPR40 protein was
colocalized with NeuN (a neuron marker) in the medulla
oblongata, but not with GFAP (an astrocyte marker).
In addition, GPR40 protein colocalized with tryptophanhydroxylase (TPH, a serotonergic neuron marker) anddopamine beta hydroxylase (DBH, a noradrenergic neuron
marker). A single intracerebroventricular (i.c.v.) injectionof GW9508, a GPR40 agonist, significantly increased thenumber of c-Fos-positive cells in the locus coeruleus andraphe magnus of the medulla oblongata compared with
the 0.2%DMSO-treated group. In addition, the number ofneurons double-labeled for c-Fos and TPH and DBH wereincreased in the mice treated with GW9508, comparedwith the 0.2%DMSO-treated group. The pre treatmentof 6-hydroxy dopamine (6-OHDA), a neuron toxic, or
p-chrolophenylalanine (PCPA) significantly inhibited
GW9508-induced aninociception. Interestingly, the i.c.v.
injection of GW9508 significanly increased noradrenaline
and serotonine level in the spinal cord. Furthermore, thei.c.v. injection of GW1100, a GPR40 antagonist, significantly increased formalin-induced pain-related behavior in
the late phase, but not in the early phase, compared with
the 0.2%DMSO-treated group. In conclusion, our findings
suggest that the GPR40 signaling pathway may play animportant role in the regulation of the descending pain
control system.
This study was supported by a Grant-in-Aid for Scientific
Research (C) (24592364) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.

79No. 71 - Mapping delta opioid receptors in
the rat central nervous system using an
irreversible biotinylated ligand
Khaled Abdallah (1) , Jane V. Aldrich (5) , Xin Wang (5) ,Jean-Luc Parent (2,3 ,4) & Louis Gendron (1,3 ,4)(1) Département de Physiologie et Biophysique, (2) Service deRhumatologie, Département de Médecine, Faculté de Médecine etdes sciences de la santé, (3) Institut de Pharmacologie de Sherbrooke,
Université de Sherbrooke and (4) The Centre de recherche duCHUS, Sherbrooke, Québec J1H5N4, Canada, and (5) Departmentof Medicinal Chemistry, The University of Kansas, Lawrence, Kansas
66045, USA.

Over the last decades, growing evidence revealed thatdelta opioid receptor (DOP) agonists may be of interestfor the treatment of chronic pain, emotional disorders,and neurodegenerative diseases. The expression patternof DOP in the central nervous system (CNS), however,remains a matter of controversy. In fact, the selectivityof most commercially available DOP antibodies has beenchallenged. To address this issue, we used an antibody-free
approach to map DOP in the brain and the spinal cordof rats. We combined an irreversible affinity label (AL)derivative of the DOP antagonist TIPP with a biotin
derivative (DSB) to take advantage of the high affinity ofbiotin derivatives to bind avidin. Brain and spinal cord
slices were incubated with 100 nM of TIPP-AL-DSB andthen the labeled receptors detected using an avidin-biotinperoxidase reaction followed by 3,3’diaminobenzidine(DAB) staining. This pharmacological labeling of DOP
revealed a wide distribution of the receptor throughout
the CNS with high staining in the olfactory bulb, cortex,
striatum, hypothalamus, thalamus, some pontine nuclei,
and brainstem as well as in the spinal cord. At thecellular level DOP labeling appeared to be relatively
homogenous within the neuronal cell bodies and dendrites
sharing different morphological features in size and shape.
Topographical analysis indicated a selective laminarorganization of DOP mainly in cerebral cortex and spinal
cord. While DOP staining in the somatosensory cortex
was mostly observed in pyramidal cells of layer V, it waslargely distributed throughout the gray matter of the
spinal cord (mostly in laminae I-V of the dorsal horn andin some motoneurons of the ventral horn). Notably, thepattern of DOP expression observed with TIPP-AL-DSB
appears to be very similar to previous anatomical studies(binding and in situ hybridization studies). In conclusion,
TIPP-AL-DSB is a useful tool to study DOP and could
potentially be used as an alternative to antibodies.
Financial support: Supported by CIHR grant MOP273137
No conflict of interest to declareNo. 72 - Opiate exposure state controls amolecular switch in opiate reward memory
formation in the basolateral
amygdala-prefrontal cortical pathway
Laura G. Rosen, Danika Lyons, Walter J. Rushlow, Steven
R. Laviolette
Department of Anatomy and Cell Biology, Department of Psychiatry,
Schulich School of Medicine and Dentistry, The University of Western
Ontario

The potent rewarding effects of opiate class drugs facilitate
the formation of strong associative memories linked tothe drug experience that play a key role in triggering
relapse. These opiate reward memories are encoded andstored in the basolateral amygdala (BLA) and medial
prefrontal cortex (mPFC) along a temporal gradient.
Interestingly, intra-BLA processing of opiate-related
reward memories is mediated by dopamine D1R and D2R
signaling as a function of opiate exposure state, where D1R
receptors are required for acute opiate memory formationin the previously drug-naı̈ve state, but D2R signaling isnecessary for memory formation during opiate dependence
and withdrawal. Linkages between D1R and ERK 1/2
and between D2R and CaMKIIα suggest these signaling
molecules may underlie the state-dependent opiate memory
formation “switch”. Using an unbiased place conditioning
procedure paired with targeted microinfusions, we showthat associative memories are processed in the BLA
via an ERK-dependent mechanism in the naı̈ve state,
but via a CaMKIIα-dependent mechanism following thetransition to dependence and withdrawal. Interestingly,
intra-mPFC memory acquisition requires CaMKIIαsignaling in the drug-naı̈ve state, but does not appear torequire ERK1/2 in either opiate exposure state. Western
blotting analyses demonstrated a further dissociation inthese signaling molecules across the BLA-mPFC pathway.
Protein analysis revealed a reduction in both ERK1/2
and CaMKIIα expression in BLA tissue, but an increasein both CaMKIIα and ERK1/2 in the mPFC. Together,
these results demonstrate a functional interaction between
the BLA and mPFC during the processing and storageof opiate-related associative memories, during the switchfrom the non-dependent, to dependent/withdrawn opiate
exposure states.
Funding sources: CIHR grant to SRL

80

No. 73 - Functional selectivity at the kappa
opioid receptor (KOPR): from in vitro to invivo

No. 74 - GIRK channel activation in midbrain
GABA neurons is not required for
opioid-induced locomotor stimulation

DiMattio KM, Cowan A, Tallarida RJ, Liu-Chen L-Y

Lydia Kotecki1 , Matthew C. Hearing2 , Ezequiel Marron
Fernandez de Velasco1 , Nicole Victoria1 , Zhilian Xia1 ,Marco Pravetoni1 , C. David Weaver3 , Kevin Wickman1

Temple University School of Medicine, Center for Substance Abuse
Research and Department of Pharmacology, Philadelphia, PA, USA

It has been previously shown that analgesia at the
KOPR is G protein mediated and dysphoria is β-arrestin
mediated. There have been several recent reports ofligand bias at the KOPR. However, there have been nocomprehensive reports of the degree of bias for multiple
available KOPR agonists at the human and mouse KOPRs.
Here we examined the ability of over 20 KOPR agonists toactivate G proteins and to internalize the receptor in vitro.
We used clonal mouse neuroblastoma (N2a) cells stablytransfected with the human or mouse KOPR (hKOPR andmKOPR, respectively) for our in vitro studies. We usedagonist-induced [35S]GTPγS binding to membranes as ameasure of G protein activation and KOPR internalization
as a readout of β-arrestin recruitment employing on-cellwestern technique on live cells. The method of Ehlert
and colleagues was used to quantify the degree of ligand
bias. Several ligands were identified to be biased for
either the G protein or β-arrestin pathway, with several
species differences. We chose a G biased compound,
MOM-Salvinorin B, an arrestin biased ligand, U50,488H,
and a balanced ligand, nalfurafine (TRK820) identified invitro at the mKOPR to perform in vivo behavioral studies
in CD1 mice. Three different behavioral tests were used:(1) inhibition of compound 48/80-induced scratching, (2)the formalin test, and (3) conditioned place aversion. We
hypothesized that there would be separation between thedose-response relationship for analgesia and dysphoria in
the arrestin biased and G protein biased ligands. At all
doses of the three compounds that produced analgesia
and anti-scratching effects, significant aversion was seen
with U50,488 and MOM-Salvinorin B, whereas nalfurafine
only produced significant aversion at the highest dose of
20 µg/kg. Nalfurafine was the only compound studied tohave a separation between the ED50 values for antinociception and anti-scratching vs. aversion. Thus, there aredifferences in ligand bias between in vitro and in vivo, thereasons for which will be discussed.
This work was supported by NIH grants T32 DA007237(K.M.D.) and R01 DA017302 (L.Y.L-C). The authors
declare no conflicts of interest.

1Department of Pharmacology, University of Minnesota, Minneapolis, MN 2Department of Neuroscience, University of Minnesota,
Minneapolis, MN 3Department of Pharmacology, Vanderbilt Instituteof Chemical Biology, Vanderbilt University Nashville, TN

The ventral tegmental area (VTA) and interconnected
brain regions, including the nucleus accumbens and medial
prefrontal cortex comprise, in part, the mesocorticolimbic
dopamine (DA) system. The mesocorticolimbic DA system
mediates the reward-related behavioral effects of drugs ofabuse, including opioids. Opioids are believed to indirectly
stimulate dopamine (DA) release from direct inhibition of
GABAergic neurons located in the VTA and/or rostromedial tegmental nucleus (RMTg). Innervation of VTA DA
neurons by VTA GABA neurons is evident, however, thespecific mechanism whereby these local GABA neurons disinhibit VTA DA neurons remains unclear. Reward-related
effects of opioids, including locomotor stimulation, require
activation of mu opioid receptors (MOR). MOR activation
modulates downstream effectors that decrease neuronal excitability, including G protein-gated inwardly-rectifying K+(GIRK/Kir3) channels. GIRK channels are localized to
midbrain GABA neurons and have long been thought to underlie the direct inhibitory effect of opioids on VTA GABA
neurons, and consequently, the disinhibition of VTA DA
neurons and opioid-induced reward-related behavior. Here,
we show that selective genetic and pharmacologic manipulations of GIRK channel activity in the VTA or RMTg
do not disrupt the locomotor stimulatory effects of opioids. The prototypical GIRK channel is a heterotetramer
formed by GIRK1 and GIRK2 subunits, however, VTA DA
neurons do not express Girk1. Leveraging this knowledge,
we microinfused a novel GIRK channel agonist, ML297,
which selectively activates GIRK1-containing heteromers.
This approach permitted selective inhibition of midbrain
GABAergic, but not DAergic neurons. Neither intra-VTA
or -RMTg infusion of ML297 was sufficient to promote locomotor stimulation. In contrast to our hypothesis, thesedata suggest that GIRK channel activation is not required
for reward-related behavior induced by opioids.
Supported by DA034696, DA011806, DA007097

81No. 75 - Influence of disulfiram on thedevelopment of tolerance on analgesic actionof morphine

No. 76 - Effect of new form of magnesium salt
on analgesic activity of tramadol
Magdalena I. Bujalska-Zadrożny, Anna E. de CordéAnna E. de Cordé, Magdalena I. Bujalska-Zadrożny
Medical University of Warsaw, Poland
Medical University of Warsaw, Poland

INTRODUCTION
The use of opioids in the treatment of chronic pain islimited by a rapid development of opioid tolerance.
It is know that dopamine participates in the developmentof tolerance on analgesic activity of opioids.
Since
disulfiram and its metabolites inhibit the activity of
dopamine-B-hydrolase leading to increase of dopaminelevel, we hypothesize that disulfiram may delay opioid
tolerance.
METHODS
The studies were performed on male Wistar rats. Twotypes of nociceptive stimuli, mechanical (Randall-Selittotest) and thermal (Tail-flick test and Plantar test) wereused.
RESULTS
Disulfiram administered alone on 21 Connecticut days
slightly increased the threshold for mechanical (RandallSelitto test) and thermal stimuli (tail-flick and plantar
tests) in dose-dependent manner.
Morphine administered alone on 21 consecutive days
gradually increased the threshold for mechanical andthermal stimuli. The antinociception reached its maximum
on day 6 in Randall-Selitto test and tail-flick test or on
day 4 in plantar test, then following repeated treatment
slowly decreased and achieved baseline values on day 14(Randall-Selitto test), on day 13 (plantar test) and on day
21 (tail-flick test).
Premedication with disulfiram (12.5 mg/kg p.o.) prevented
the development of tolerance on analgesic activity of morphine (25 mg/kg p.o.) for consecutive 21 days. Similar
but stronger effect was observed after premedication withdisulfiram at doses 25 or 50 mg/kg p.o. before morphine.
CONCLUSIONSDisulfiram delays the development of tolerance on analgesic
action of morphine in acute pain model after mechanicaland thermal stimuli.
Research subject implemented with CePT infrastructure financed by the EU – the European Regional
Development Fund within the Operational Program“Innovative economy” for 2007-2013.

INTRODUCTION
As previously reported, magnesium sulphate administeredparenterally significantly increased an opioid analgesia
in different kinds of pain. It seems that the combined
administration of opioid and magnesium may offer in the
future very valuable therapeutic possibilities, moreoverthe concomitant administration of those compounds orally
would hypothetically allow for the use of this combinationin outpatient conditions.
Since the typical form of magnesium salts are weakly
and slowly absorbed from the gastrointestinal tract, we
decided to investigate how magnesium salt in new andbetter absorbed form administered orally modify theopioid analgesia, in comparison with conventional form ofmagnesium salt.
METHODS
The studies were performed on male Wistar rats. Twotypes of nociceptive stimuli, mechanical (Randall-Selittotest) and thermal (Tail-flick test), were used.
RESULTS
Conventional and new forms of magnesium salts at adose of 15 mg of magnesium ions/kg did not modify thenociceptive threshold after mechanical and thermal stimuli.
Tramadol administered at a low dose of 125 mg/kg had
only a little antinociceptive effect.
A pre-treatment with a new form of magnesium saltenhanced the analgesic activity of orally administeredtramadol significantly faster and more effectively incomparison to the conventional form of magnesium salt.
CONCLUSIONS
A new form of magnesium salt enhances analgesic effect oftramadol. This observation may be relevant clinically.
Research subject implemented with CePT infrastructure financed by the EU – the European Regional
Development Fund within the Operational Program“Innovative economy” for 2007-2013.
This work was supported by the National Centre for
Research and Development of Poland.

82No. 78 - Endomorphin analog analgesics lack
reinforcement qualities and are promising
candidate medications for the treatment of
opioid addiction

No. 77 - Anti-inflammatory andAntinociceptive Action of 6β-Tryptophan
Substituted 14-O-methyloxymorphone inMouse Models of Inflammatory Bowel
Diseases

Mark R. Nilges, Chloe Cable, James E. Zadina
Mariana Spetea1 , Tanila Ben Haddou1 , Marta Sobczak2 ,Jakub Fichna2 , Helmut Schmidhammer1
1Department of Pharmaceutical Chemistry, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, Universityof Innsbruck, Innsbruck, Austria; 2Department of Biomolecular
Chemistry, Faculty of Medicine, Medical University of Lodz, Lodz, Po

Introduction: Inflammatory bowel diseases (IBD) constitute a large group of severe and chronic gastrointestinal(GI) disorders characterized by intestinal inflammation,
mucosal damage, and severe pain that is still inadequately
managed. µ Opioid (MOP) receptors are distributed
throughout the central and peripheral nervous systems andvarious non-neuronal tissues. Several lines of evidence showthat MOP receptors are involved in the pathophysiology
of inflammatory GI disorders, directing toward the clinicalrelevance of targeting MOP receptors for the treatmentof IBD. Herein, we describe the anti-inflammatory andantinociceptive actions of 6β-tryptophan substituted
14-O-methyloxymorphone (HS1333), a novel peripherally
acting MOP agonist, in mouse models of IBD.
Methods: Opioid receptor activities were determinedusing radioligand binding and [35S]GTPγS functional
assays.
A fluorescence cell-based assay was used tomonitor the nuclear factor-κB (NF-κB) activity upon
stimulation with tumor necrosis factor-α (TNF-α) andlipopolysaccharide (LPS). Anti-inflammatory effects wereassessed in trinitrobenzene sulfonic acid (TNBS)- anddextran sodium sulphate (DSS)-induced colitis in mice.
Antinociceptive action of HS1333 was evaluated in mousemodels of abdominal pain after.
Results: HS1333 displays high MOP binding affinity and isa potent and full agonist toward the MOP receptor. It significantly inhibits, in a concentration-dependent manner,
NF-κB activation in TNF- and LPS-stimulated humanmonocytic THP-1 Blue cells. In mice, the MOP receptor
agonist (0.1 and 1 mg/kg, s.c.) twice daily attenuates
TNBS- and DSS-induced colitis. Dose-dependent andsignificant antinociceptive effects were produced by s.c.
HS1333 in acetic acid-induced abdominal stretching andmustard oil-induced acute colitis in mice.
Conclusion: Novel MOP agonists acting in the peripherywith combined immunosuppressive and antinociceptive
properties may provide a new approach for the treatmentof IBD. (Supported by the Austrian Science Fund (FWF):
P21350, Tyrolean Research Fund (TWF): UNI-0404/949
and S&T Cooperation Fund Austria-Poland 2013-2014:
PL13/2013.

Neuroscience Program, Departments of Medicine and Pharmacology,
Tulane University

Endomorphins (EM) have the highest selectivity of any endogenous peptide for the pain modulating mu-opioid receptor (MOR). We have synthesized several metabolicallystable, blood-brain barrier permeable EM analogs that haveequal or greater analgesic potency than morphine, the current gold standard MOR agonist. Morphine and otherexogenously derived opioid analgesics produce detrimental side effects such as respiratory depression, tolerance,
glial cell activation, and have a high potential for abuse.
We have shown that EM analogs do not induce respiratory depression, produced substantially less tolerance thanmorphine, and do not induce markers of microglia or astrocyte activation. In abuse liability assays, intravenous(i.v.) morphine produced a robust and dose-dependent conditioned place preference effect, but the analogs did not.
In self-administration models, lever pressings for morphine
progressively escalated as lever pressing requirements increased or the available mg/kg dose decreased, suggestinghigh abuse liability. By contrast, rats with access to i.v.
EM analog infusions lever pressed no more than controls
with access to i.v. vehicle, suggesting the analogs lack reinforcing qualities. In this study we trained rats to discriminate i.v. infusions of morphine from vehicle in a 2-lever foodchoice drug-discrimination (DD) task to assess the substitution potential of the analogs for morphine. Methadone fully
substitutes for morphine, but suppresses motor/appetitive
responses for food in the DD model. A compound whichlacks self-administration but substitutes for morphine without motor/appetitive impairment has potential as a pharmacotherapy for opioid addiction. We found that analogs 2
and 4 fully substituted for morphine, but did not impair
motor/appetitive responding. This suggests the analogs
may provide a better therapeutic window than methadone
since motor impairment is a major limitation of methadone
maintenance therapy. These data indicate EM analogs havea better safety profile than morphine, lack reinforcement
qualities, provide potent analgesia, and are novel candidates for opioid addiction pharmacotherapy.
Support: VA, DOD, ONR

83

No. 79 - Spatiotemporal Control of
Mu-Opioid Signaling and Behavior
Martin J. Schmidt1 *, Madison A. Baird1 *, Edward Siuda1 ,Will Planer1 , Vijay Samineni1,3 , Bryan Copits1,3 , Skylar
Spangler1,4 , Robert W. Gereau IV1 -4 , and Michael R.
Bruchas1 -4
1.Department of Anesthesiology, Washington University in St.
Louis 2.Department of Neurobiology, Washington University in
St.
Louis 3.Washington University Pain Center 4.Program in
Neuroscience/DBBS, *Co-presenters

Tools for isolating microcircuitry and kinetics of opioid action with discrete spatiotemporal control in freely moving
animals are not yet available. We created optically sensitive mu opioid receptors (oMORs) for using optogenetics
to drive mu opioid-like signaling in in vivo at the level ofmicrocircuitry with cell-type specificity. Here, we present
the initial characterization of these receptors in vitro andin vivo. oMOR chimeric receptor constructs were designed
to incorporate the extracellular loops of rat rhodopsin with
the intracellular components of rat mu opioid receptors.
First, we compared the pharmacology of oMOR vs. wildtype MOR in inhibiting cAMP, activating MAPKs, andinternalizing in response to photo-stimulation. Sustained
photo-stimulation of oMOR caused suppression of forskolininduced cAMP, activated pERK MAPK and caused receptor internalization with kinetics that are consistent with endogenous MOR activation. For in vivo cell-type specificity,oMORs were cloned and packaged into a cre-dependent
adenovirus (AAV5-EF1a-oMOR-WPRE) and injected into
VGAT-IRES-Cre (GABA-selective) mice in brain structures relevant for mu opioid-driven behaviors such as conditioned place preference and analgesia. As expected, thevirus was expressed in the VTA and RMTg of VGATIRES-Cre mice. Whole cell slice physiology recordings fromthese mice showed that photo-stimulation of oMOR caused
a robust outward GIRK current, in a similar manner to
DAMGO at wild-type MOR. Furthermore, 10mW 473nm
laser light delivered through fiber optic ferrules caused significant real time place preference. Here we show that
oMORs are light sensitive Gi-coupled GPCRs that signal
and traffic similar manner as compared to MOR and are sufficient to promote reward-like behavior in mice. This tool
will enable spatiotemporal dissection of neural pathways,
behaviors associated with mu opioid receptor activation,and could generally be used to examine effects of inhibitory
G-protein activation in neural circuits. Authors have nocompeting financial interests. Supported by R01DA037152
to MRB, and HHMI to MAB.

No. 80 - Repeated Morphine Promotes
Bidirectional and Cell-Type Specific
Adaptations in AMPAR plasticity in Medium
Spiny Neurons of the Accumbens Core and
Shell
Matthew Hearing, Jakub Jedynak, Rachel Fischer, andMark Thomas
Department of Neuroscience, University of Minnesota

Introduction: In animal models of addiction, plasticity ofglutamatergic synapses in the nucleus accumbens (NAc)exerts powerful control over drug-seeking behavior. However, little is known about whether, how or when experience with drugs may trigger synaptic plasticity in this key
nucleus. Here we identify adaptations in glutamatergic signaling within the NAc following withdrawal from repeatedmorphine exposure.
Methods: Whole-cell voltage-clamp recordings were performed in medium spiny neurons (MSNs) in sagittal brainslices containing the NAc core or shell regions from adult
C57BL/J6 and Drd1a-tdTomato mice. Mice received 5
daily injections of morphine (10 mg/kg, i.p.) or saline,
followed by 10-14 d of withdrawal, at which point animals
were left undisturbed (no challenge) or injected with saline
or morphine 24 h before electrophysiological study (challenge).
Results: Initial studies in NAc shell MSNs of wild-typemice showed a significant increase in the ratio of AMPAR:NMDAR currents, a reduction in paired-pulse ratios,
and an increase in miniature excitatory postsynaptic current (mEPSC) amplitude, but not frequency in morphinetreated mice following withdrawal (no challenge). In contrast, preliminary data from the core indicate that withdrawal from morphine does not significantly alter any ofthese parameters. Studies using Drd1a-tdTomato miceshow that within the shell, AMPAR/NMDAR ratios, aswell as mEPSC amplitude and frequency are increased in
D1- but not in non-D1-MSNs (i.e., D2-MSNs), while a significant reduction in frequency was found in D2-MSNs of
the core. Whether AMPAR:NMDAR ratios are altered in
the core is currently being explored. In drug-challenged
mice, data indicate that a single re-exposure to morphineduring withdrawal becomes a potent stimulus for synaptic
depression and reverses the initially observed potentiation
within the shell. These effects, and the role of depotentiation in the expression of sensitization are currently being
explored further.
Conclusion: Our results indicate that in vivo morphine exerts dynamic bidirectional control over excitatory synaptic
strength in NAc that displays neuronal and anatomical selectivity.

84No. 81 - Variants of stress-related genes andtheir role in heroin self-exposure, addiction,and response to treatment

No. 82 - Delineating the dynamics of µ Opioid
Receptor signalling and regulation
Meritxell Canals and Michelle Halls

M. Randesi(1) , Orna Levran(1) , V. Yuferov(1) , Joel Correa
da Rosa(2) , P. Blanken(3) , W. van den Brink(3) , J.M. vanRee(3) , M.J. Kreek(1)(1) The Laboratory of the Biology of Addictive Diseases, The
Rockefeller University, New York, NY, USA; (2) Center for Clinicaland Translational Science, The Rockefeller University, New York,NY, USA; (3) Central Committee on the Treatment of Heroin
Addicts (CCBH), Utrecht, the Netherlands

Here we report part of a larger study designed to identify
genetic variants that are associated with susceptibility
to develop heroin addiction and response to treatment
thereof. The goal was to determine if specific variants (120)of stress-related genes (26) contribute to heroin addiction
in a unique cohort of 597 Dutch Caucasian subjects, consisting of four groups: 1) subjects with no history of druguse (n=153); 2) subjects self-exposed to illicit opiates atleast 5 times but not addicted (n=163); 3) former heroindependent subjects successfully methadone-maintained(n=143); and 4) treatment-resistant heroin-dependent
subjects, offered heroin-assisted methadone treatment(n=138). Genotyping was performed using a custom
GoldenGate array (Illumina). Initially, a one-way ANOVA
was performed to test for differences in genotype frequency among the four groups of subjects for each single
nucleotide polymorphism (SNP), separately. Four SNPs
from three genes were identified as nominally significant(p>0.05): arginine vasopressin receptor 1B (AVPR1B)
SNP rs33933482; galanin (GAL) SNP rs3136541; and in the
glucocorticoid receptor gene (NR3C1) SNPs rs10482672
and rs17339455. Analysis comparing the control group to
those subjects self-exposed without addiction, found two
SNPs in the neuropeptide Y gene (NPY), rs2234759 andrs6536721, as nominally significant. When the methadone
maintained subjects were compared to the group that
required augmentation with heroin, four SNPs in fourgenes were nominally significant: corticotropin releasing
hormone receptor 1 (CRHR1) SNP rs4792887; glycine
receptor, alpha 1 (GLRA1) SNP rs2964608; NPY SNP
rs16148; and NR3C1 SNP rs10482672. Further analyses
are in progress to determine if there are differences between the combined groups 1+2 and combined groups 3+4.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical
Sciences, Monash University

Receptor signal compartmentalisation (the restriction of
second messengers in space and time) provides a mechanism
whereby receptors can direct the assembly of focused “platforms” that facilitate second messenger production, the organisation and scaffolding of effectors, and co-ordinationof regulatory events. Decades of research have focused,without success, on the differential regulation and internalisation of µOR by morphine compared to DAMGO, inan effort to design more effective analgesics. To date, there
are no studies examining compartmentalised signalling ofµOR in live cells, no information on µOR spatiotemporal signalling profiles, and no knowledge of whether thisis regulated by receptor trafficking. Whether µOR resides
within biochemically-defined lipid-rich plasma membrane(PM) regions is controversial, and the lack of a unifying
view is likely due to the invasive nature of methods used
to isolate these domains. We now show that the regulationof µOR trafficking and signal compartmentalisation are interdependent. We have used BRET to measure µOR endocytic trafficking and have correlated this with signallingmeasured from bulk compartments in single cells using
FRET biosensors. µOR initially resides within a unique
PM microdomain. Morphine stimulation of µOR initiatesa PM-localised Gαi/o-Gβγ-PKC activation. PKC phosphorylation of the receptor causes sequestration of µOR
and a sustained signalling profile: sustained membranePKC phosphorylation, and sustained cytosolic ERK phosphorylation. In contrast, DAMGO does not activate PKC
allowing µOR translocation within the PM and a transient signalling profile: transient Gαi/o-mediated cytosolic
ERK phosphorylation, βArr recruitment, receptor internalisation, and transient βArr-mediated nuclear ERK phosphorylation. Our results suggest that µOR-mediated signalling is highly compartmentalised and ligand-dependentand that distinct µOR membrane localisations lead to diverse spatiotemporal signalling.

Supported by pilot funds from NIDA [MJK], CCBH
ZonMw 310000465, and The Dr. Miriam & Sheldon G.
Adelson Medical Research Foundation.
The authors report no conflicts of interest.

85No. 83 - Postnatal changes in excitability and
GABAergic transmission in rat rostral
ventromedial medulla (RVM) neurons

No. 84 - Spinal P2X7R receptors are criticallyinvolved in the development of morphine
physical dependence

Ming-Hua Li, Katherine L. Suchland, and Susan L. Ingram

Nicole Burma & Tuan Trang

Oregon Health & Science University

Departments of Comparative Biology & Experimental Medicine, andPhysiology & Pharmacology, Hotchkiss Brain Institute, University ofCalgary, Calgary, Alberta, Canada

Neurons in the rostral ventromedial medulla (RVM) play
critical and complex roles in pain modulation. Recent
studies have shown that electrical stimulation of the RVM
produces pain facilitation in young animals (postnatal day(PN) > 21) but biphasic pain facilitation and inhibitionin adults, and that opioids applied in this region havedifferent effects at different ages. The cellular mechanisms
underlying these changes in the influence of the RVM on
pain behaviors are not known. This is in part because
whole-cell patch-clamp studies in RVM to date havebeen in young (PN > 16) animals and the fact thatthe organization and abundance of myelinated fibers
in this region make it a challenging area for whole-cellpatch-clamp recording in adults. Neurotransmission inseveral systems, including GABA and glutamate neurotransmission, undergo developmental changes that mature
by PN 21-30. Thus, we focused on optimizing whole-cellpatch-clamp recordings for RVM neurons in animals older
than PN day 30 and compared the results to animals 10- 21 days PN. Our results demonstrate that there aresubstantial differences in neuronal excitability, GABAergic
neurotransmission and opioid action in RVM neurons
between these time periods. Differences in these properties
of RVM neurons may underlie the developmental changes
in descending control of pain from the RVM to the spinal
cord.

These studies were supported by the grants from
NIH (DA027625, S.L.I.), and America Heart Association(13SDG14590005, MH.L.).

Introduction: Opioids, such as morphine, are among themost powerful and widely prescribed analgesics for managing pain. However, repeated opioid use can lead to physical dependence, a crippling condition that manifests as awithdrawal syndrome upon drug cessation. The spinal dorsal horn is a primary site of action for opioids, and cellular changes in this region are causally implicated in opioid
physical dependence. In the present study, we examined
the importance of spinal ATP-gated P2X7 purinoceptors(P2X7R) in the development of morphine physical dependence.
Methods: Opioid physical dependence was established using a 5-day escalating morphine dosing paradigm. Adult
male Sprague Dawley rats were given twice daily intraperitoneal injections of morphine at 8 hour intervals (day 1:
10 mg/kg; day 2: 20 mg/kg; day 3: 30 mg/kg; day 4: 40
mg/kg). To examine the contribution of spinal P2X7Rs
in the development of morphine physical dependence, intrathecal injections of the P2X7R antagonist brilliant blue
G (BBG) or A740003 were co-administered with morphine.
On day 5, rats received a morning injection of 45 mg/kg
and 2 hours later challenged with a single injection of the
opioid receptor antagonist, naloxone (2 mg/kg), to precipitate morphine withdrawal.
Results: We found that naloxone challenge precipitated arobust withdrawal syndrome in morphine treated animals.
The severity of morphine withdrawal correlated with increased spinal expression of ionized calcium-binding adaptor molecule (iba-1), a cellular marker of microglial activation, and the upregulation of c-Fos, a marker of neuronal
activation. Intrathecal administration of the P2X7R antagonist, BBG or A740003, significantly attenuated the behavioural signs of withdrawal. Blocking spinal P2X7R also
prevented the naloxone-induced increase in iba-1 and c-Fos
expression.
Conclusions: Blocking P2X7Rs during repeated morphine
treatment decreases withdrawal symptoms and reduces activation of spinal microglia. Collectively, our findings reveal
a novel and critical role of spinal P2X7Rs in the development of morphine dependence.
[Supported by grants from CIHR, NSERC, and CFI]
Authors declare no conflicts of interest.

86No. 85 - Dopaminergic system genes
polymorphisms and heroin addiction: further
support for a protective effect of casein kinase1 (CSNK1E) variants

No. 86 - Angiotensin II type 1
receptor-mediated increase in spinal p38
MAPK phosphorylation leads to the
induction of nociceptive behavior in mice

*Levran, O.1 , Peles, E.2,3 , Randesi, M.1 , Correa da Rosa,
J.4 , Rotrosen, J.5 , Adelson, M.1,2 ,6 , Kreek, M.J.1

Osamu Nakagawasai1 , Wataru Nemoto1 , Yoshiki Ogata1 ,Fukie Yaoita1 , Takeshi Tadano2 , Koichi Tan-No1

1The Laboratory of the Biology of Addictive Diseases, The Rockefeller University, New York, NY, USA, 2Dr. Miriam and Sheldon G.
Adelson Clinic for Drug Abuse Treatment and Research, Tel Aviv
Elias Sourasky Medical Center, Tel Aviv, Israel, 3Sackler Facultyof Medicine, Tel Aviv University, Tel Aviv, 4Center for Clinicaland Translational Science, The Rockefeller University, 5VA New
York Harbor Healthcare System and NYU School of Medicine, New
York, 6Dr. Miriam and Sheldon G. Adelson Clinic for Drug Abuse
Treatment and Research, Las Vegas, NV, USA

1 Department of Pharmacology, Tohoku Pharmaceutical University,
Japan; 2 Laboratory of Environmental and Health Sciences,
College of Medical Pharmaceutical and Health Sciences, Kanazawa
University, Japan.

The dopaminergic reward pathways have been implicatedin the etiology of drug addictions. We have previously
reported a tentative association of CSNK1E (casein kinase1) SNPs and haplotype with heroin addiction in subjectsof European ancestry.
The aims of this association study were to determine if genetic variants in genes encoding proteins of the
dopaminergic pathway account for the heritable factors insusceptibility to heroin addiction, as well as to replicate
our previous findings in a larger cohort. The study was
performed on 828 unrelated former severe heroin addicts
treated at methadone maintenance treatment programs in
NYC, Las Vegas and Israel, as well as 233 healthy controls.
The study was limited to subjects of predominantly
European ancestry, verified by STRUCTURE analysis of
155 AIMs. A total of 118 SNPs in 13 genes were genotyped
using the Illumina GoldenGate platform. After filteringfor quality, HWE, MAF and complete LD, 82 SNPs wereanalyzed for association by logistic regression using the
PLINK program.
Nominally significant associations were observed at
12 SNPs located in the genes CSNK1E, DRD2/ANKK1,
DRD3,and DBH (P>0.05). The association of the intronic
CSNK1E SNP rs1534891 (a protective effect of the minor
allele) remained significant after correction for multiple
testing. This study supports our previous report of association of this SNP and a related haplotype with protection
from heroin addiction, in subjects of European descent.
CSNK1E encodes a serine/threonine-selective enzyme
isoform that interacts with several signaling pathways
including circadian rhythms and DARPP-32 regulation of
the dopaminergic pathway. Csnk1e has been implicated innegative regulation of sensitivity to stimulants and opioids,
in rodents. CSNK1E may be a target in the treatment ofdrug addiction.

It has been demonstrated that angiotensin II (Ang II) participates in either the inhibition or the facilitation of nociceptive transmission depending on the brain area. However, the role of spinal Ang II in nociceptive transmission
remains unclear. Therefore, in order to elucidate the roleof Ang II in nociceptive transmission in the spinal cord,
we examined the effect of intrathecal (i.t.) administrationof Ang II into mice. I.t. administration of Ang II produced a behavioral response in mice mainly consisting ofbiting and/or licking of the hindpaw and the tail along withslight hindlimb scratching directed toward the flank. The
behavior induced by Ang II was dose-dependently inhibited by intraperitoneal injection of morphine, suggestingthat the behavioral response is related to nociception. The
nociceptive behavior was also inhibited dose-dependently
by i.t. co-administration of losartan, an Ang II type 1(AT1) receptor antagonist, and SB203580, a p38 MAPK
inhibitor. However, the Ang II type 2 (AT2) receptor antagonist PD123319, the upstream inhibitor of ERK1/2 phosphorylation U0126, and the JNK inhibitor SP600125 had
no effect on Ang II-induced nociceptive behavior. Western blot analysis showed that the i.t. injection of Ang II
induced phosphorylation of p38 MAPK in the lumbar dorsal spinal cord, which was inhibited by losartan, without
affecting ERK1/2 and JNK. Furthermore, we found that
AT1 receptor expression was relatively high expressed in
the lumbar superficial dorsal horn. Our data show thati.t. administration of Ang II induces nociceptive behavior accompanied by the activation of p38 MAPK signaling
mediated through AT1 receptors. This observation indicates that Ang II may act as a neurotransmitter and/or
neuromodulator in the spinal transmission of nociceptive
information.

Supported by the Dr. Miriam and Sheldon G. Adelson
Medical Research Foundation and 2UL1 RR024143-06(JC).
The authors have no conflict of interest.

87No. 87 - Analogs of salvinorin A bearing
sulfur-containing substituents at position C2
are high-affinity kappa-opioid receptor partial
agonists with a potentially novel binding mode
P. D. Mosier (1) , P. R. Polepally (2) , E. Vardy (3) , J. K.
Zjawiony (2) , B. L. Roth (3)(1) Dept. of Med. Chem., Virginia Commonwealth Univ., Richmond,
Virginia, 23298, USA, (2) Dept. of Pharmacognosy, Univ. ofMississippi, University, Mississippi, 38677, USA, (3) Dept.
ofPharmacol., Univ. of North Carolina, Chapel Hill, North Carolina,
27599, USA

The mechanism by which the highly affine and non-basic
agonist salvinorin A and its analogs interact with andmodulate the function of the kappa-opioid receptor (KOP)
is not only intriguing from a receptor–ligand interaction
standpoint but also highly relevant to the potential
treatment of several disease states, including but notlimited to those associated with visceral pain (e.g. irritablebowel disease; IBS). Here we report the affinity, potency
and putative interaction modes of 19 salvinorin A analogs
with either an S-alkyl or S-aryl thiocarbonate or dual
ester/S-aryl- or ester/S-alkyl-thioether substituent at theC2-position at the human KOP receptor. Affinity data
for the compounds at the wild-type KOP receptor were
determined using a competitive binding assay with thearylacetamide agonist [3H]U69,593 as the radioligand.
Nine of the compounds were shown to have substantial
binding affinity (Ki 4–177 nM). Notably, positional isomers
were found to have greatly varying Ki values. Functional
data for these compounds at the wild-type KOP receptor
was determined using a cellular cAMP response assay
and showed that each of the tested compounds havepartial agonist activity (EC50 = 35–1850 nM) relative tosalvinorin A. Experimentally-guided automated docking
routines were used to derive putative binding modes forthe partial agonists using the crystal structure of thehuman KOP receptor bound to the antagonist JDTic(PDB ID = 4DJH). Interestingly, a “reverse” binding
mode not previously reported was identified in which the
2-substituent interacts with transmembrane (TM) helix 2,TM3 and extracellular loop 2 (EL2), while the diterpenoid
salvinorin scaffold interacts with TM3, TM6 and TM7.
These results suggest that the uncharged nature of theligands combined with the spacious and largely symmetricorthosteric binding pocket allow salvinorin A analogs toeffectively bind and stabilize active conformations of the
KOP receptor through alternative binding modes.

No. 88 - Surface plasmon resonance as alabel-free approach to monitor the mu opioid
receptor-mediated signaling.
P. Bourassa1 , H. Bagheri Tudashki4 , G. Pineyro5 , M.
Grandbois2,3 and L. Gendron1,3
1. Département de physiologie et biophysique, 2. Départementde pharmacologie, 3. Institut de pharmacologie, Université de
Sherbrooke, Québec, Canada. 4. Département de pharmacologie, 5.
Département de psychiatrie, Université de Montréal, Québec, Canada

Nowadays, most analgesics used for the treatment ofmoderate to severe pain conditions target the mu opioid
receptor (MOP). Interestingly, all MOP agonists do
not activate the numerous receptor-mediated signalingcascades to the same level. In this study, we used Surface
Plasmon Resonance (SPR) spectroscopy as a label-free
approach to investigate the contribution of G protein
coupling, ERK1/2 activation and receptor internalization
to the SPR signals generated by various ligands in living
HEK cells expressing the human Flag-MOP.
In the present study, we found that SPR signals triggeredby DAMGO and morphine were similar. However, weobserved that they were differently affected by various
pharmacological inhibitors. Indeed, U0126, dynasore andcholera toxin (CTX) all affected the amplitude of thesignal induced by DAMGO and morphine. CTX also
affected the maximal slope of morphine SPR response.
However, it had no effect on the agonist-induced ERK
phosphorylation. The SPR signals induced by DAMGO
and morphine were completely abolished by pertussis toxin(PTX) pretreatment, indicating an important role for Gαi.
We also evaluated four MOP antagonists (CTAP, CTOP,
naloxone and naltrexone). We found that CTOP, naloxone
naltrexone and CTAP, which intriguingly is the lone ligandof our study to induce a rapid decrease in reflectance, allgenerated PTX-sensitive SPR signals. Our BRET analysis
further revealed that MOP antagonists can trigger conformational changes within the Gαiβγ heteromer. Together,
our results suggest that CTOP, naloxone and naltrexone
are partial agonists while CTAP is likely an inverse agonist.
We demonstrated that SPR spectroscopy is a sensitive approach allowing for the monitoring of aggregate
cellular responses occurring as a result of receptor activation. With this cell-based, label-free approach we showthat it is possible to detect responses for ligands for which
no responses are normally detected in formal biochemical
assays.

Support: NIH-NIMH MH-R01DA017204 (BLR).
Conflict of interest: authors have no conflict of interest to declare.
Funding: Supported by NSERC-CIHR Collaborative
Health Research Project grant #RPCS-385882-2010 (M.Gand L. G) and by NSERC grant #311997 (G. P)

88

No. 89 - Opioid signaling in early-life
adversity: a translational study
Pierre-Eric Lutz, Dominique Filliol, Sabine Dhir, Emmanuel Darcq, Michael Meaney, Brigitte Kieffer, Gustavo
TureckiDouglas Hospital Research Centre, McGill University, Montréal,CanadaIntroduction. Childhood maltreatment (CM) represents an
extreme form of emotional pain that affects children of allages, race, economic, and cultural backgrounds. There isa strong relationship between CM and mental health outcomes. Accordingly, sexual and physical abuse, as well as
profound emotional neglect, are among the strongest predictors of psychiatric pathology, such as depression and suicide.
Hypothesis. We hypothesize that CM disrupts opioid
signaling in brain structures processing emotional pain,
thereby increasing the risk of depression and suicide
throughout life. We also propose that epigenetic adaptations in the opioid system genes may account for such longterm effects.
Methods. Our project combines the study of human postmortem tissues and a robust and validated rat model. We
focus our analyses on three brain regions critically regulating emotional pain: the anterior insula, the anterior cingulate cortex and the dorso-medial thalamus.
In human, we measure the expression (quantitative PCR)
and G-protein coupling ([35S]-GTPγS) of opioid receptors,
as a function of past histories of severe CM, depression andsuicide. In rat, we quantify the expression of the opioid
system as a function of naturally occurring variations inmaternal care. Our group of investigators has provided important evidence that low levels of maternal care in thismodel are relevant to the study of neurobiological adaptations to early-life adversity, which may be conserved acrossspecies.
Results. Our results indicate that a history of severe CM
associates with a significant decrease in kappa opioid receptor mRNA levels in the human anterior insula. Surprisingly,
CM did not modify the maximal coupling efficiency of the
kappa opioid receptor (Emax) in this brain region.
Conclusion. This is the first study reporting how CM may
impact the opioid system in the human brain. As a next
step, we are currently investigating potential epigenetic
mechanisms accounting for maladaptive opioid signaling associated with CM, focusing on DNA methylation.
Funding sources:
Canadian Institute for Health Research
Fondation Fyssen (PEL) and Fondation BettencourtShueller (PEL)

No. 90 - Differential effector coupling does
not underlie resistance or susceptibility todesensitization of presynaptic mu opioid and
GABAB receptors
Reagan L. Pennock and Shane T. Hentges
Colorado State University, Department of Biomedical Sciences

Mu opioid receptor (MOR)-mediated inhibition of GABA
release onto proopiomelanocortin (POMC) neurons in the
hypothalamus resists acute desensitization. In contrast,
acute desensitization of GABAB receptor (GABABR)mediated inhibition of GABA release is observed at roughly
one quarter of POMC neurons. It is currently unclear
whether this differential susceptibility to desensitization isdue to differing physical properties of presynaptic MORs
and GABABRs, or due to differential effector coupling.
In the present study, whole-cell voltage clamp recordings
were made in POMC neurons. The ability of MOR and
GABABR agonists to suppress inhibitory postsynaptic potentials (IPSCs) was measured in the presence of pharmacological blockers that target known effectors of MORs and
GABABRs. Neither DAMGO- nor baclofen-induced inhibition of IPSCs was occluded when voltage-dependent K+
channels were blocked or when a Ca2+ -free external recording solution was used. DAMGO and baclofen also induced
robust inhibition of IPSCs induced by ionomycin-mediated
Ca2+ influx into terminals presynaptic to POMC neurons.
Together, these data suggest that MOR- and GABABRmediated inhibition of GABA release onto POMC neurons does not require the activation of voltage-dependent
K+ channels or the inhibition of calcium influx. However,when recordings were made in an external solution containing Sr2+ instead of Ca2+ , Sr2+-evoked IPSCs were robustly inhibited by both DAMGO and baclofen. This result
suggests that MOR- and GABABR-mediated inhibition of
GABA release can occur through the inhibition of voltagedependent Ca2+ channels (VDCC). Together with the observation that presynaptic inhibition of GABA release by
MORs and GABABRs is not occluded by unregulated calcium influx due to ionomycin, it appears that inhibition of
release from both types of receptors can be mediated by either the inhibition of Ca2+ channels or by direct inhibitionof vesicular release. Altogether, the results demonstratethat presynaptic MORs and GABABRs are coupled similarly, and that differential receptor-effector coupling is unlikely to explain differential desensitization of presynaptic
receptors. NIH grant R01DA032562

89No. 91 - Bimodal role for nucleus accumbens
dynorphinergic neurons in aversion andrewardNo. 92 - Novel small molecule triazoles
modeled from naltrindole potently inhibit the
proliferation of human multiple myeloma cells.

Ream Al-Hasani*, Jordan G. McCall*, Jenny M. Wong,
Omar S. Mabrouk, Nicole A. Capik, Gavin Schmitz,
Blessan Sebastian, Daniel Y. Hong, Michael J. Krashes,
Bradford B. Lowell, Thomas L. Kash, Robert T. Kennedy,
Michael R. Bruchas

R.D. Howells1 , T.C. Yoon1 , A.M. Meillier1 , Y. Peng2 , andW.J. Welsh2

Washington University in St. LouisMultiple myeloma (MM) is an incurable malignancy derived from plasma cells that proliferates in the bone marrow. In 2014, the American Cancer Society estimated that
24,050 people will be diagnosed with MM, and 11,090 will
die of the disease. Current therapies have improved the
5-year survival estimate to 40%, however, there is an obvious need for new, effective treatment options. We reported
that naltrindole (Nti) inhibited human MM cell proliferation in vitro and in a murine xenograft model in vivo, byinteraction with a non-opioid receptor target (Mundra etal., 2012). Molecular modeling of the Nti pharmacophore
led to the synthesis of a series of trisubstituted triazoles,
and in the present study, we have discovered a lead compound, MM-900, that is 50-fold more potent than Nti ininhibiting the proliferation of MM cells. For cell proliferation assays, human U266 cells are plated in 12-well dishes at
a density of 100,000 cells/ml in RPMI 1640 medium containing 10% FBS. U266 cells were incubated for 72 h at
37o C in a humidified atmosphere containing 5% CO2 in
the presence and absence of Nti or MM-triazoles at concentrations ranging from 5-50 µM. Following incubation, cells
were gently dispersed and aliquots were analyzed in duplicate in a Vi-Cell instrument to quantify the number of
viable cells/ml and the overall percent viability. In these assays, Nti inhibits MM cell proliferation with an EC50 of 20µM. Approximately 20 MM-triazoles have been screened,
and the majority displayed EC50‘s in the 10-80 µM range,
however, 3 compounds inhibited MM cell growth at significantly lower concentrations. Thus, MM-900, MM-902 andMM-902S reduced U266 cell proliferation with EC50‘s of400, 490 and 520 nM, respectively, reflecting an increasein potency relative to Nti of 40-50-fold. We have shownthat Nti significantly inhibits human MM tumor growth in
SCID mice with daily dosing of 15 mg/kg, ip, therefore,
the most active MM-triazoles that are obtained will also be
tested in vivo in the mouse/human xenograft model. It is
anticipated that this approach will yield novel therapeutic
compounds to be used alone or in combination with current
drugs used to treat MM.

The dynorphin/kappa opioid system is implicated in stressand vulnerability to drug abuse. It is thought that stresscauses dynorphin release activating kappa-opioid receptors(KOR) within dopaminergic and serotonergic nuclei andtheir ventral striatal targets. Consequently, much attention
has focused on these systems in the modulation of KORmediated responses. Despite our current knowledge of central dynorphinergic cell body populations, a clear description of the axonal projections of these neurons is unknown.
To address this we crossed the Cre-dependent tdTomato(Ai9) reporter mouse to a mouse expressing Cre recombinase under the same promoter as dynorphin (Dyn-Cre)so only dynorphinergic cells express tdTomato, allowing
complete visualization of dynorphinergic circuitry throughout the brain. We show robust dynorphin expression incell bodies throughout the brainstem and forebrain. We
were also able to use these mice in conjunction with viral
retrograde approaches to isolate and identify NAc dynorphinergic projections throughout the brain. Dynorphinergic neurons within the accumbens are particularly implicated in stress and drug abuse. Therefore we investigated whether specific modulation of dynorphinergic neuronal firing in the NAc is sufficient to induce aversive behaviors. We virally targeted channelrhodopsin-2 to striatal dynorphinergic neurons and optogenetically activatedneuronal populations in both the dorsal and ventral NAc
shell. Activation of dorsal NAc shell induces a place preference and is positively reinforcing in an FR1 operant task
paradigm while activation of ventral NAc shell drives conditioned and real-time aversive behavior. This photoactivation of dynorphinergic neurons in the ventral NAc also
increased dynorphin release, as measured using microdialysis and mass spectroscopy. Understanding the mechanisms by which the dynorphin/kappa opioid system regulates negative affective behaviors will provide valuable insight into potential treatments for drug abuse and depression. Work supported by NIDA R01DA033396 (M.R.B,
R.A), NIH Common Fund, NINDS R01NS081707 (M.R.B),
NIMH F31MH101956 (J.G.M). Authors declare no conflictof interest.

Department of Biochemistry and Molecular Biology, Rutgers-NJ
Medical School1, Newark, NJ; Department of Pharmacology,
Rutgers-RWJ Medical School, Piscataway, NJ2.

90

No. 93 - Tolerance to morphine respiratory
depression: reversal by ethanol

No. 94 - Differential Modulation of the
Signaling of µ–Opioid Receptor Agonists by
the Positive Allosteric Modulator,
BMS-986121

R Hill, E Kelly, G Henderson
University of Bristol

Ethanol has been reported to reverse both morphine
cellular tolerance in rat locus coeruleus neurones (1) andmorphine antinociceptive tolerance in vivo in mice (2).
The present investigation examined the effect of a low doseof ethanol on tolerance to morphine respiratory depression
in mice.
Respiration (minute volume) was measured by wholebody plethysmography in male mice (CD-1) breathing 95%
air/5% CO2. To measure changes in respiration, the data
for each mouse was normalised to pre-drug baseline andthe area under the curve (AUC) calculated in arbitrary
units for a 30 min post-drug period. All data are given as
mean ± standard error.
Morphine (10 mg/kg ip; n = 8) rapidly depressed
respiration. In comparison to saline injected animals the
AUC for morphine was -11.0 ± 1.3. Ethanol (0.3 g/kg
ip; n = 6) caused no significant depression of respirationcompared to saline (n=8) with AUC = -1.4 ± 0.6 Vs -0.7
± 0.6 respectively.
Tolerance to morphine was induced by subcutaneous
implantation of a 75mg morphine pellet. After 6 days ofpellet implantation respiration had returned to pre pellet
implantation levels (minute volume pre pellet 154 ± 5
ml/min, n = 12; 6 days after pellet implantation 149 ± 4
ml/min, n = 12).
In morphine pelleted mice, challenge with morphine(10 mg/kg ip; n = 6) on day 6 caused significantly
less depression of respiration than in placebo pellet
implanted mice (AUC = -3.3 ± 0.6 for morphine treatedVs -10.4 ± 0.6 for placebo, P>0.001). When morphinepelleted mice (n = 6) were injected simultaneously withmorphine (10 mg/kg) and ethanol (0.3 g/kg) there
was a significant depression of respiration compared tomorphine alone (AUC = -8.3 ± 0.8 Vs -3.3 ± 0.6, P>0.001).
These data provide evidence that ethanol reverses
tolerance to morphine respiratory depression as previously
observed with both cellular and antinociception tolerance(1, 2).

Shweta Bhargava, Ruihua Chen, Gregg Crumley, Jianxi
Liu, Yeung-Wai Lock, Wendy S. Miller, Aniket Patel,
Thomas M. Stormann, Marla L. Watt, and Donald J. Kyle
Discovery Research, Purdue Pharma L.P., Cranbury, New Jersey,
United States, 08512

The search for novel opioids with strong analgesic properties and reduced side effect profiles has been ongoingfor decades. Efforts have focused on compounds acting
at the orthosteric, “opiate” site on the µ opioid receptor(MOR). Recently, BMS-986121 was described as a positive
allosteric modulator (PAM) acting on the MOR. Understanding how such a PAM molecule modulates signaling ofµ agonists via G protein and β-arrestin2 (β-Arr2) pathways
may aid the discovery of novel, analgesic µ PAMs with reduced side effect profiles. We examined the effects of BMS986121 on MOR activation of G protein and β-Arr2 signaling by a panel of opioid agonists. BMS-986121 effects onagonist-stimulated MOR internalization and MOR/β-Arr2
co-localization were also examined. Methods: Agoniststimulated GTPγS binding was used to measure G protein
signaling. Signaling events in the β-Arr2 pathway werestudied with: a β-Arr2 recruitment assay (DiscoveRx); fluorescent SnapTag-MOR and β-Arr2-ZsGreen proteins withhigh content imaging analysis on a GE INCell 6000 device were used to measure MOR internalization and colocalization with β-Arr2. Results: DAMGO was a full agonist for GTPγS binding and β-Arr2 recruitment with nobiased signaling observed; it also exhibited full efficacy for
MOR internalization, with internalized MOR and β-Arr2
highly co-localized. BMS-986121 increased DAMGO potency for all parameters with no effect on efficacy, leaving the unbiased signaling profile intact. Morphine wasa G protein-biased, partial agonist, with modest internalization of MOR and minimal subsequent co-localization
with β-Arr2. BMS-986121 increased the potency and efficacy of morphine for both G protein and β-Arr2 signaling. The PAM enhanced morphine potency and efficacy
for MOR internalization as well as co-localization with βArr2. Buprenorphine was a weak, partial agonist for G
protein signaling with negligible recruitment of β-Arr2 giving a G protein-biased profile. Buprenorphine produced
no MOR internalization or MOR/β-Arr2 co-localization.
BMS-986121 enhanced buprenorphine efficacy for various
parameters, with little impact on potency.

1.Llorente et al, Mol Pharmacol 84:252, 2013
2.Hull LC et al, J Pharmacol Exp Ther 345:512,201391

No. 95 - Abuse liability of oxycodone andmorphine in buprenorphine-maintained
participants with or without chronic pain

No. 96 - Reversal by Tamoxifen of tolerance
to morphine-induced respiratory depression.
Sarah L Withey, Eamonn P Kelly, Graeme Henderson

Comer S.D., Metz V., Vosburg S.K., Mogali S., Manubay
J.M., Jones J.D., Vogelman J., Sullivan M.A.
New York State Psychiatric Institute and Columbia University, New
York, NY 10032

Abuse of prescribed opioids is currently a serious publichealth concern in the U.S. The problem is complicated by
the fact that it is difficult to differentiate between opioid abusers who do not need these medications for pain
and patients who may be misusing opioids (e.g., taking
larger doses than prescribed) because they are experiencing inadequate pain relief. The purpose of our study is
to examine the subjective and reinforcing effects of morphine (MOR) and oxycodone (OXY) in participants meeting DSM-V criteria for opioid use disorders who either do or
do not have chronic pain. All participants live in the hospital and are maintained on sublingual buprenorphine (BUP;
4 mg BID) throughout the 4-week study. Following a 1week BUP stabilization period, participants receive MOR(360 mg), OXY (120 mg), or placebo (PBO) orally during
separate laboratory sessions. The drugs are administeredunder single-blind conditions and labeled Drug A, B, or C.
Participants then have the opportunity to choose to selfadminister Drug A, B, or C during 5 separate choice sessions (1 choice opportunity per day). Preliminary analysesof the data were conducted for 7 participants with chronic
pain (6M,1F; average pain score (0-10): 4.3±1.2) and 9
without pain (8M,1F; average pain score: 1.3±0.6). Ratings of “High” and “Good effects” significantly increased
after administration of MOR in those with pain and after
OXY in those without pain compared to PBO, but drug
“Liking” only increased in those without pain and only after OXY. Furthermore, the percentage of drug choices did
not differ in patients with pain (37±11%, 37±15%, 26±13%
for PBO, MOR, and OXY, respectively), but choice ofOXY (58±12%) was greater than PBO (24±9%) and MOR(16±11%) in those without pain. The reasons cited for
drug choice also differed between the groups. The majorityof participants without pain chose OXY and MOR in orderto “Get high,” whereas those with pain chose MOR most often to “Enhance the study medication (BUP)” and OXY to“Get high.” These data suggest that opioid-seeking behaviors and the reasons for them differ between opioid users
with and without chronic pain. Supported by DA16759(SDC).

University of Bristol

Opioid analgesics are the leading class of prescription
drugs that cause unintentional overdose deaths throughrespiratory depression. In single neurones and in studies ofantinociception, tolerance to morphine is reversed by both
inhibitors of protein kinase C (PKC) and ethanol (Hullet al., 2013; Llorente et al., 2013). The investigation of
PKC activity in respiration experiments in freely moving
animals is limited by the lack of PKC inhibitors that crossthe blood-brain barrier. Here we investigate the effectsof Tamoxifen, a drug known to inhibit PKC (O’Brian etal., 1985), on tolerance to morphine-induced respiratory
depression.
To induce tolerance mice were implanted with a 75
mg morphine pellet (sc) for 6 days. On day 6, respiration
was recorded using individual plethysmograph chambers.
To assess the level of tolerance mice were given a challengedose of morphine (10 mg/kg ip). Changes in minutevolume were recorded for 30 minutes post challenge andanalysed as a percentage of pre-challenge baseline.
Morphine treated animals demonstrated significant
tolerance to the respiratory depressant effects of morphine,when compared to naı̈ve controls. On morphine challenge,
respiration was 92.8 ± 3.5 % and 60.7 ± 4.3 % of pre-druglevels respectively, p>0.001. Tamoxifen (0.6 mg/kg ip),
injected 30 minutes before morphine, significantly reversed
tolerance to respiratory depression in morphine treatedanimals (on morphine challenge respiration was 59.3 ± 5.3% of pre-drug levels p>0.001). Tamoxifen alone had noeffect on respiration in morphine pelleted or naı̈ve animals.
We have demonstrated that tolerance to respiratory
depression by morphine can be reversed by the PKC
inhibitor, Tamoxifen. Previously we have reported thatethanol reverses tolerance to both the antinociceptive andrespiratory depressant effects of morphine. This could
suggest that ethanol exerts its effects by inhibition of PKC.
References
Hull LC et al., (2013), J Pharmacol Exp Ther. 345(3):5129
Llorente J et al., (2013), Mol Pharmacol. 84(2):252-60
O’Brian CA, et al., (1985), Cancer Res. 45; 2462
Research funded by the BBSRC.

92

No. 97 - SYNTHESIS OF
3,4,5-TRIMETHOXYCINNAMIC ACIDDERIVATIVES AND THEIRANTI-NARCOTIC EFFECT
K. Yun, J. Kim, Y. Lee, J. Kim, S. Oh
Dept. of Neuroscience and TIDRC, School of Medicine, Ewha
Womans Univ., Seoul, Korea

The purpose of this study is to characterize the putative anti-narcotic effects of 3,4,5-trimethoxycinnamic acid
(TMCA) constituent of Polygala tenuifolia. A simple synthesis and biological evaluation of 3,4,5-trimethoxyphenyl
acrylamides 1a-f as novel antinarcotic agents is described.
TMCA derivatives were administered 30 min prior to theinjection of morphine in C57BL/6 mouse for 7 days. It was
found that compounds 1d-f exhibited good inhibitory effects on the morphine withdrawal syndrome in mice. Interestingly, TMCA derivatives showed specific binding affinity
on serotonin receptors, especially on the 5-HT1A receptor.
The cells were treated with 3,4,5-trimethoxyphenyl acrylamides 1a-f (10 uM) for 30 min, pERK expression was elevated by tested compounds in cortical neuronal cells. The
elevation of pERK expression by 3,4,5-trimethoxyphenyl
acrylamides treatment were suppressed by WAY 100635 (1uM), 5-HT1A receptor-specific antagonist. Compounds 1a
and 1c demonstrated significant action to pERK expression and good response to WAY 100635 in cultured cortical neurons among those analogues. These results suggest
simple synthesis of 3,4,5-trimethoxyphenyl acrylamides andkey fragments are useful for the development of antinarcotic
agents.

No. 98 - Opioid modulation of inhibitory
synapses in striatal patch compartments
Matthew R. Banghart, Shay Q. Neufeld, Bernardo L.
Sabatini
Howard Hughes Medical Institute, Department of Neurobiology,
Harvard Medical School, Boston, MA, USA

Opioid peptides and their receptors are prominent in thedorsal striatum, a brain region critical for the generationof purposeful movements and goal-directed behavior.
Mu-opioid receptors (MORs) are highly enriched instriatal patch compartments, distinct limbic microcircuits
embedded within the predominately sensorimotor dorsal
striatum.
Previous work has revealed patch specificsuppression of inhibition by the MOR agonist DAMGO,
however it is not known which pre- and post-synaptic neurons within patches are targeted by DAMGO. In addition,
the action of opioid peptides, which can activate multiple
opioid receptors, is not understood. Using a combinationof transgenic mice, optogenetics, and photoactivatable
peptides, we identified the inhibitory synapses suppressedby opioid peptides, investigated the underlying receptors,
and defined the kinetics of modulation.
We validated a transgenic mouse line that allows simultaneous observation of patches and post-synaptic cell
identity. We confirmed that enkephalin (enk) – the only
endogenous ligand for MORs in striatum – suppresses local
inhibition onto both direct and indirect pathway striatal
projection neurons selectively in patches. To determine
the synaptic targets of enk we conditionally expressed
ChR2 in specific cell types and found that particularsubsets of inhibitory synapses were suppressed by enk.
Investigation of the underlying receptors revealed that
MOR is not the only relevant opioid receptor. Finally, weprobed the time course of opioid peptide modulation with
caged enk, allowing instantaneous delivery of saturating
peptide concentrations. Surprisingly, maximal synaptic
suppression required the presence of peptide for several
minutes. These results delineate how opioid release instriatum could shift the balance of limbic-associatedstriatal output by suppressing a subset of inhibitory
synapses in patches.
This work was supported by the Charles A. King Trust(MRB), National Institute on Drug Abuse (K99DA034648
to MRB), Canadian Institute of Health Research (SQN),
the National Institute of Mental Health (RO1MH085418& R01MH100568 to BLS), and Howard Hughes Medical
Institute (BLS).

93

No. 99 - Involvement of ERM proteins in theDevelopment of Morphine Analgesic
Tolerance through P-glycoprotein at the
Blood–Brain Barrier
(1)

(1,2)

Shogo Tokuyama , Takuro Kobori, Shinichi Harada(1), Hideo Takahashi (2) and Kazuo Nakamoto (1)(1) Department of Clinical Pharmacy, School of Pharmaceutical
Sciences, Kobe Gakuin University, (2) Department of Pharmacology,
Kinki University, Faculty of Medicine.

Introduction: Many of opioid analgesics such as morphine
are recognized as substrate for P-glycoprotein (P-gp), a
drug efflux transporter. Therefore, P-gp has considerable
impact on pharmacokinetics and pharmacological effects
of opioid analgesics. Cumulative evidence proposes thatchronic treatment with morphine increases P-gp expression
in the brain capillary endothelial cells (BCECs), an integral
component of blood brain barrier (BBB), leading to thedevelopment of morphine analgesic tolerance. However,the detailed mechanism remains unexplained.
Ezrin/radixin/moesin (ERM) are scaffold proteins regulating the plasma membrane localization of some drugtransporters such as P-gp in peripheral tissues, althougha few reports focus its role in the central nervous system
as well. In this study, we investigated the involvement of
ERM in the development of morphine analgesic tolerance
through altered P-gp expression in BCECs.
Methods: Male ddY mice aged 4 weeks were given morphine (50 mg/kg/day) subcutaneously for 5 days and its
analgesic effect against thermal stimuli was evaluated with
tail-flick test every day during experimental period. Each
protein expression in the membrane fraction of BCECs wasdetermined using western blotting. The protein-protein
interactions between P-gp and each ERM proteins, ortheir immunohistological localization were analyzed bymeans of immunoprecipitation or immunofluorescenceassay, respectively.
Results & Conclusion: Repeated treatment with morphinedecreased its analgesic effect and increased P-gp protein
expression in BCECs. Under the same condition, thelevels of total ERM proteins were similar between saline
and morphine group. The protein expression of moesin
was significantly increased whereas that of ezrin was
significantly decreased and no change was observed inthat of radixin. Furthermore, immunoprecipitation or
immunofluorescence studies revealed the interaction
between moesin and P-gp along with co-localization.
In conclusion, an increase in moesin accounts, at leastin part, for the increased expression of P-gp in BCECs,
leading to the development of morphine analgesic tolerance.

No. 100 - Interleukin 4 mediated by HSV
vector attenuates morphine tolerance andphysical withdrawal in rats
Xuexing Zheng, Shue Liu, Wenwen Zheng, Roy C. Levitt,
Keith A. Candiotti, David A. Lubarsky & Shuanglin Hao
Department of Anesthesiology, University of Miami Miller School of
Medicine, Miami, FL33136

Introduction:
Morphine is one of the most effective and potent analgesics
currently available. Their utility in the management ofpain is well recognized, however, extending the applicationof opioids to the management of chronic pain has met withconsiderable resistance. This resistance is due in part toconcerns related to the development of tolerance, incorrect
drug usage, and addiction. Recent evidence suggests
chronic morphine may induce the release of proinflammatory cytokines which are involved in morphine tolerance.
Methods:
In the morphine tolerance model, chronic morphine wasadministrated intraperitoneally for 7 days.
Thermallatency was measured using hotplate test. HSV vector
expressing IL-4 or control vector was injected into thehindpaws.
Neurochemical factors in the spinal cord
and/or DRG were examined using western blots and/or
immunohistochemistry. In the physical withdrawal model,
rats were treated with escalated morphine for 5 days
followed by naloxone precipitation. HSV vector expressing
IL-4 or control vector was injected into the periaqueductal
gray (PAG) 1 week before morphine. Physical withdrawal
symptoms were observed.
Results:
Subcutaneous inoculation of these vectors into hindpaws
delayed the development of chronic morphine tolerance inrats. IL-4 overexpression mediated by the HSV vectors
reduced TNFα and the phosphorylation of p38 in morphine
tolerance in the DRG and the spinal dorsal horn in chronicmorphine
Furthermore, we found that HSV-mediated IL-4 overexpression in the PAG reduced the morphine withdrawal.
Conclusion:
These results support the concept that proinflammatory
factors may play an important role in the pathogenesis ofmorphine tolerance induced by chronic morphine. Thesestudies indicate that the nonreplicative HSV-derived
vectors expressing immunomodulatory molecules mightprove a novel approach (gene therapy) to morphine
tolerance/physical withdrawal.
The study was supported by the NIH DA20078 (S.H.),
DA026734 (S.H.), and DA034749 (S.H.). The authors
declare that there are no competing interests in the work.

94No. 101 - Substance Dependence Treatment
Data Set; According to the Comparative
Study of Substance Dependence Treatment
Information System

No. 102 - The Advantages and Barriers to
Implement Substance Dependence Treatment
Information System (SDTIS)
Sima Ajami, Zahra Mellat-Karkevandi

1

Sima Ajami , Zahra Mellat-Karkevandi

2

Isfahan University of Medical Sciences
Isfahan University of Medical Sciences

Addiction is a phenomenon that causes structural changes
in different systems of society. Studies show for planningof addiction prevention and treatment, it is necessary tocreate an information management system. In addiction
treatment, without access to identification and causal information, distribution of personal trusteeships and treatment equipment won’t be possible. In order to plan for
addicts’ treatment, information has to be collected by interviewing and examining patient and regarding substanceusing history and social activity. This information helps
clinicians to specify treatment plan for detoxification andcontinuing supportive treatment. The aim of this study wasto establish substance dependence treatment data sets according to a comparative study on America, Australia, andEngland Minimum Data Set. In this study, which was anapplied one and from comparative-exploratory study, data
sets of substance dependence treatment information systems in America, Australia, and England were compared.
Sample countries were chosen based on related available information on the internet and also development of thesecountries in health information management. Information
source included library sources, electronic sources and counseling with specialists inside and outside of country. Findings analysis was done in theory-comparative method andby using comparative table. In America admission data set
and discharge data set, in Australia established-level items
and episode-level items, and in England adults and young
people, were titles of main groups in data set of substancedependence treatment. Presence of a standard data set in
substance dependence treatment field is base of establishinga treatment information system and so base of this disease
prevention and treatment. Thus, according to lack of thisdata set in Iran, we hope that data sets studied in thisresearch can be a good guidance to establish a Minimum
Data Set (MDS) for substance dependence treatment.
Key words: Data Set; Minimum Data Set; Information System; Substance Dependence; Substance Dependence Treatment
Conflict of interest: Nil

Addiction is a phenomenon that causes structural changes
in different systems of society. Studies show for planningof addiction prevention and treatment, it is necessary tocreate an information management system. In addiction
treatment, without access to identification and causal information, distribution of personal trusteeships and treatment equipment won’t be possible. The aim of information system is informing others, thus information systemgives proper information to users based on their information need. The information system is a set of organizedcomponents which is responsible for collecting, storing anddistributing of information. Substance dependence information systems refer to systems which collect, analyse andreport data related to substance dependence information.
The aim of this study was to identify advantages and barriers to implement Substance Dependence Treatment Information System (SDTIS). We employed the following keywords and their combinations in different areas of articles. In this study, 22 of collected articles and reports
were selected based on their relevancy. The most important ones are recording sufficient, complete and accurate
information and easy and timely access to them; supporting
clinical decision making; supporting audits of clinical practice; helping prevention, control and treatment planning;monitoring and enhancing the quality of care; evaluatingrisk factors; being effectively used for research purposes;
helping creation a network among treatment centers andadministrative centers and allowing longitudinal tracking
of client health-related outcomes by having linkages withother healthcare information systems. Also, it may be necessary to modify the assessment to transfer information
when patients move between levels of care. In general, it
is a useful and credible tool in measuring improvements inhealthcare and one of vital prerequisites for creating andusing an Electronic Health Record (EHR) in every country.
Developing formal protocols to examine data quality andconsistency shortly after admission can help us to solve thelast problem. Key words: Registry, Management Information System, Substance Dependence95No. 103 - Dissecting Nociceptin Receptor
Modulation of Reward
Skylar M. Spangler 1,2 ,3,4 ; Elisha E. Peterson 2,4 ; Ream
Al-Hasani 2,3 ,4 ; William J Planer 2,3 ,4 ; Garret D. Stuber
5,6 8, ; Thomas Kash 7,8 ; Thomas Jhou 9,10 ; Michael R.
Bruchas 1,2 ,3,4
1) Division of Biology and Biomedical Sciences (Neuroscience) 2)
Dept of Anesthesiology 3) Anatomy and Neurobiology 4) Washington
University Pain Center Washington University School of Medicine,Saint Louis, MO, USA 5) Dept of Psychiatry 6) Dept o

Nociceptin/Orphanin FQ Opioid Receptor (NOPR) and its
endogenous ligand, nociceptin (NOFQ) have been shown toaffect the rewarding properties of drugs of abuse. BothNOPR and N/OFQ are highly expressed in the Ventral Tegmental Area (VTA), widely regarded as a criticalanatomical region for both drug and natural reward. The
circuitry of the NOPR/NOFQ receptor system in the context of reward, however, remains unknown. The aim of
this study was to dissect the role of NOPR and NOFQ inreward modulation. First, we pharmacologically targeted
NOPR receptors with potent and selective NOPR agonist,
SCH 221510. Consistent with prior studies on NOPR agonism, we demonstrated that SCH221510 significantly attenuates drug-seeking behavior in a cocaine conditioned place
preference (CPP) model. Then, to determine whether theeffect of SCH221510 was specific to NOPR expressed in
dopaminergic VTA (DA-VTA) neurons, we targeted a credependent NOPR virus into the VTA of NOPR knockout
crossed with Tyrosine hydroxylase-Cre mice. Our preliminary data suggests activation of NOPR specifically within
the dopaminergic neurons of the VTA is sufficient to attenuate drug seeking behavior in a CPP model. To determine
the source of endogenous nociceptin to the VTA that modulates reward behavior, we generated a novel Cre mouseline, Noci-IRES-Cre, in which the promoter for the NOFQ
gene drives Cre recombinase expression. We then created areporter mouse by crossing a Noci-Cre mouse with a Credependent tdTomato (Ai9) reporter mouse. In this reportermouse, tdTomato is expressed in NOFQ positive neurons
and allows us to examine both cell bodies and projection
sites. We show NOFQ is expressed in the expected cellbodies of brain loci involved in reward and affective behavior, including the Rostromedial Tegmental nucleus (RMTg)
and the VTA, which inhibit DA-VTA neurons, and also
the Central Amygdala (CeA). We optogenetically targetedthese nociceptin containing cells and evaluated their effects
on reward and aversion behavior. These data provide thegroundwork for NOPR containing neural circuits that regulate reward and aversive-like behavior.

No. 104 - Analysis of the second intracellularloop of DOP: A role in the membranetargeting of DOP
Etienne St-Louis, Jean-Luc Parent, Louis Gendron
Université de Sherbrooke

We have previously shown that activation of the mu opioid
receptor and inflammation increase the plasma membrane
density of delta opioid receptors (DOPs) in neurons.
Interestingly, this effect is accompanied by an increased
analgesic efficacy of DOP agonists. In a recent study, we
found that inhibiting cyclin-dependent kinase 5 (cdk5)
impairs the regulation of DOP triggered by morphineand inflammation. The regulation of DOP by cdk5 islikely mediated by the phosphorylation of T161 localized
within the second intracellular loop (ICL2) of DOP. The
exact mechanisms involved in the cdk5-mediated DOP
regulation are however unknown.
We have identified a putative COPB1 binding site (KxK)near to the cdk5 consensus phosphorylation motif in the
ICL2 of DOP. Since COPB1 is involved in the retrograde
transport of proteins from the Golgi to the endoplasmic
reticulum (ER), we hypothesized that the cdk5-mediatedphosphorylation of T161 attenuates the ability of COPB1
to bind the 164KAK putative ER retention motif, thus
promoting the export of DOP to the cell surface. To study
the role of this putative retention motif in the regulation of
DOP, we generated a mutation in the ICL2. Using ELISA,
we found that the K166A mutant has a higher level of cellsurface expression compared to the wildtype Flag-DOP.
The K166A mutation also induced a mobility shift of
Flag-DOP on SDS-PAGE compatible with a variation in
the glycosylation levels. Finally, our preliminary results
suggest that COPB1 can be co-immunoprecipitated withwildtype Flag-DOP. A reduction in the association of
COPB1 with the K166A mutant remains to be determined.
Our results suggest that the KxK motif localized in the
ICL2 of DOP is part of the cellular mechanisms involvedin the retention of DOP. Mutation of this motif possibly
decreases the ability of COPB1 to associate and retain
DOP in the ER. Because this motif is adjacent to theputative cdk5 phosphorylation site, we now propose that
cdk5 regulates DOP by promoting the phosphorylationof T161, which interferes with the association of COPB1
with DOP.
Financial support: Supported by CIHR grant MOP273137
No conflict of interest to declare96No. 105 - Platelet-derived growth factor
circuitry underlying opioid tolerance
Stephanie Puig, Howard B. Gutstein
University of Texas MD Anderson Cancer Center. Department ofAnesthesiology and PeriOp. Medicine.

For centuries, opioids have been the gold standard fortreating severe pain. However, with repeated dosing, theanalgesic effect of opioids decreases. The mechanisms
underlying opioid tolerance remain poorly understood. We
recently made the groundbreaking discoveries that inhibition of the platelet-derived growth factor receptor beta
(PDGFR-β) signaling prevented morphine tolerance fromoccurring and completely reversed established tolerance.
Based on these findings, we hypothesize that 1) PDGFR-βsignaling is a general mediator of opioid tolerance; 2)
PDGFR-β expressing cells in the pain processing pathways
form neural circuits underlying tolerance; and 3) These
circuits are modified by opioid exposure. Using immunohistofluorescence and confocal microscopic imaging, wehave defined the specific types of cells expressing the
PDGFR-β and the platelet-derived growth factor-BB(PDGF-BB) in the dorsal root ganglia (DRG) and thesubstantia gelatinosa (SG) under basal conditions. These
proteins were characterized in various cell subtypes of the
SG and the DRG. Experiments are underway investigating
the co-localization of PDGFR-β and the MOR underbasal conditions. We are also determining whether the
distribution and co-localization of these markers is alteredby chronic administration of various opioids. These studies
will provide unique insights into the neural substrates
underlying opioid tolerance.
The authors declare no conflict of interest.
Tis research is funded by a CPRIT grant to H.B. Gutstein.

No. 106 - Quantitative Analysis of Functional
Selectivity at the Nociceptin Opioid Receptor
Steven D. Chang, Skylar Spangler, Wayne Mascarella,
Vsevolod Gurevich, F. Ivy Carroll, Michael R. Bruchas
Washington University in St. Louis, RTI International, Vanderbilt
University

The Nociceptin/Orphanin FQ Opioid Receptor (NOPR)
is the most recently discovered and least characterized Gprotein coupled receptor in the opioid receptor family. The
actions of NOPR and its endogenous ligand, nociceptin,
have been thought to play a role in neuromodulation in thecontexts of pain, opioid tolerance and addiction, as wellas the modulation of stress and anxiety behaviors. It isthought that the NOP receptor, like other opioid receptors and GPCRs, may bind functionally selective ligandsthat can bias signal transduction, resulting in diverse behavioral effects. The potential for functional selectivity at
the NOPR has yet to be explored. To quantitatively study
the signal transduction profiles of NOPR ligands in-depth,
we used real-time live-cell cAMP and Bioluminescence Resonance Energy Transfer (BRET) assays to study the ligandinduced Gα-protein activation and arrestin 2/3 recruitment
at NOPR. Additionally, we employed high-resolution confocal microscopy to identify differences ligand-induced receptor internalization. We screened multiple NOPR-selective
ligands, as well as novel, custom-designed small molecules
aimed at elucidating structural significance in signal transduction. We have found that the NOPR, like other opioid
receptors exhibits different signaling profiles, depending on
the ligand type. Furthermore, we show that minute changes
in ligand structure can illicit opposing signaling profiles
ranging from agonism to inverse agonism. Additionally,
we propose plausible ligand-receptor docking configurations
that may help to elucidate the relationship between conformation and signal transduction at the NOP receptor.
Finally, we show that ligands are capable of eliciting different degrees of receptor internalization. Together, thesedata provide new and quantitative insight into the structural relationship between ligands and signal transduction
at the NOP receptor, which will ultimately facilitate additional studies examining NOPR ligand bias in behavior.
Supported by: NIH R00 DA025182 and T32 DA007261.

97No. 107 - Utilizing the Ugi multi-component
reaction to synthesize opioids

No. 108 - THE CONTRIBUTION OF Gi/o
PROTEIN TO OPIOID
ANTINOCICEPTION IN ANOXALIPLATIN-INDUCED NEUROPATHYRAT MODEL

András Váradi, Joan J Subrath, Valerie LeRouzic, Gavril
W. Pasternak, Gina Marrone, and Susruta Majumdar
Department of Neurology, Memorial Sloan-Kettering Cancer Center,
NY, NY 10065

In multicomponent reactions (MCRs), two or more
starting materials react simultaneously to yield a single
product. High structural diversity can be achieved in just
one synthetic step, reaction yields are generally high andthe purification of products is simple. Thus, MCRs are
suitable for the quick and convenient synthesis of drug-likelibraries.
Our approach utilizes the isocyanide-based four-component
Ugi reaction to obtain synthetic opioid scaffolds ina one-pot, single step fashion. Reaction between Nphenethylpiperidone (ketone), aniline (amine), propionicacid (carboxylic acid) and isocyanides yielded a small
library of bis-amide analogs of carfentanyl. Our leadcompound showed high affinity for mu (MOR) and delta(DOR) opioid receptors with poor affinity for kappa(KOR) receptors in radioligand binding assays. It had
moderate analgesic potency in vivo but showed a reasonable side-effect profile over morphine.
This MCR approach has the promise to deliver morepotent and efficacious small-molecule opioids with anadvantageous side-effect profile and potential clinicalapplicability.
This work was supported by research grant from the
National Institute on Drug Abuse (DA034106) to SM and(DA06241) to GWP.

Tomoe Kanbara (1, 2) , Atsushi Nakamura (1, 2) Masahiro
Shibasaki (2) , Tomohisa Mori (2) , Tsutomu Suzuki (2) ,Minoru Hasegawa (1) , Gaku Sakaguchi (1) , Toshiyuki
Kanemasa (1)(1) Pain & Neurology, Medicinal Research Laboratories, SHIONOGI
Co., Ltd., Osaka, Japan; (2) Department of Toxicology, Hoshi
University School of Pharmacy and Pharmaceutical Sciences, Tokyo,
Japan

Purpose: Oxaliplatin is a chemotherapeutic agent thatinduces chronic refractory neuropathy.
To determine
whether opioids are effective for this type of neuropathy,
we investigated the efficacies of morphine, oxycodone,
and fentanyl, and the mechanisms underlying opioid
antinociception, in oxaliplatin-induced neuropathy in rats.
Method: We assessed the antinociceptive effects of opioids
in rats with oxaliplatin-induced neuropathy using the vonFrey test. To investigate the µ-opioid receptor functionof each opioid in the oxaliplatin model, a [35S]-GTPγSbinding assay was performed.
Result: Rats exhibited significant mechanical allodynia
following 2 weeks of chronic oxaliplatin administration.
Within the range of doses that did not induce sedation, morphine (3 mg/kg, subcutaneously [s.c.]) andoxycodone (0.3–0.56 mg/kg, s.c.) completely reversed
oxaliplatin-induced mechanical allodynia, whereas fentanyl(0.0056–0.03 mg/kg, s.c.) showed partial antinociception.
In the oxaliplatin model, the antinociceptive effects of the
optimal doses of morphine and oxycodone were completely
inhibited by pertussis toxin (PTX; 0.5 µg/rat, i.c.v.), a
Gi/o protein inhibitor, while the partial effect of fentanyl
was not affected by PTX. In the [35S]-GTPγS binding
assay, activation of µ-opioid receptor by fentanyl, but notby morphine or oxycodone, in the mediodorsal thalamusof oxaliplatin-treated rats was significantly reduced.
Conclusion: Morphine and oxycodone showed robust
antinociceptive effects via PTX-sensitive Gi/o protein
activation in oxaliplatin-induced neuropathy. The partial
antinociception of fentanyl in the oxaliplatin model might
result in part from the loss of that protein activation.
The degree of Gi/o protein activation might be related
to the potency of antinociceptive effect of opioids on
oxaliplatin-induced neuropathy.
Conflict of interest
None of the authors have any conflicts of interest to
disclose relating to this submission.

98No. 109 - Association of the OPRK1 intronic
SNP rs6473797 with prolactin response todynorphin administration in normal malevolunteers
Yuferov V, Levran O, Ducat E, Randesi M, Ho A, Kreek
MJ
Laboratory of the Biology of Addictive Diseases, The Rockefeller
University, New York, USA

Pituitary prolactin (PRL) secretion is under inhibitory hypothalamic tuberoinfundibular dopaminergic control in humans. Extra pituitary brain prolactin has anxiolytic andanti-stress properties through activation of PRL receptors.
The kappa opioid receptor (OPRK1) and its ligands dynorphin peptides (Dyn) modulate the activity of the mesolimbic, nigrostriatal and hypothalamic dopaminergic neurons.
This study evaluated the effect of an OPRK1 variant, which
we have previously shown to be associated with heroin
addiction, on prolactin response to dynorphin administration in healthy males. Method. Twenty four healthy
male volunteers participated in the study conducted in the
Rockefeller University Hospital. On each testing day, subjects received Dyn1-13 i.v.. at doses of 0, 120 and 500µg/kg. Serum prolactin levels were determined at sequential time points from 0 to 120 min by immunoradiometricassay. The change in prolactin response of each dynorphin
dose from the saline (delta area under the curve, AUC)
was calculated for each subject. OPRK1 intronic SNP
rs6473797 was genotyped using Illumina GoldenGate array. A two-way ANOVA, Genotype by Condition (Low
Dose, High Dose), with repeated measures on the second factor, was used to examine delta AUCs. All subjects gave consent for genetic studies. Results. There
was no significant main effect of Genotype, F(2,21)=2.41,
P=0.11. There was a significant main effect of Condition,
F(1,21)=36.50, p>0.00001, and a significant Genotype X
Condition interaction, F(2,21)=3.94, p>0.05. While there
was no difference among genotypes at the low dose, individuals with the GG genotype had significantly lower response to the higher dynorphin dose than individuals with
AA and AG (p>0.05). Conclusion. The results suggestthat the OPRK1 intron 2 variant alters prolactin levels in
response to Dyn1-13 administration. This alteration may
be due to lower expression of the receptor and therefore lessinhibition of dopaminergic tone in the hypothalamus. Support: NIH NIDA-P60-05130 (MJK), NIH-NCRR CTSA,UL1-TR000043, Dr. Miriam and G. Sheldon Adelson Medical Research Foundation. The authors declare no conflictof interest.

No. 110 - Subcutaneous injection of DALDA
inhibits ongoing neuropathic pain andpersistent inflammatory pain in rats
Vinod Tiwari, Shao-Qiu He, Fei Yang, Qian Xu, Ronen
Shechter, Alene Carteret, Yun Guan, Srinivasa N. Raja
Department of Anesthesiology and Critical Care Medicine, Johns
Hopkins University, School of Medicine, Baltimore, MD, USA

Introduction & Methodology: Patients with tissue or
nerve injury commonly suffer from ongoing and/or
paroxysmal spontaneous pain. Although ongoing painis the most common and bothersome clinical complaint,
most preclinical animal studies rely on reflex response to
evoked sensory hypersensitivities which does not always
correlate with analgesic efficacy in humans. Here, we usedthe conditioned place preference (CPP) to examine if
subcutaneous injection of dermorphin [D-Arg2,Lys4](1-4)
amide (DALDA), a mu-opioid receptor (MOR) agonist,
can reduce ongoing pain associated with nerve injury andtissue inflammation
Results: Compared to the preconditioning, rats with L5
spinal nerve ligation (SNL) surgery spent significantly
more time in the DALDA (5, 10 mg/kg s.c., n=8-12/dose)paired chamber during post-conditioning. Importantly,
neither dose of DALDA (5 and 10 mg/kg, s.c.,) induced
CPP in naive rats, suggesting that DALDA-induced CPP
in SNL rats is likely due to the reward from pain relief,
but not due to drug penetration into central nervous
system (CNS) which will directly activate CNS reward
circuitry, an action known to CNS-penetrating mu-opioids(e.g., morphine). Intraperitoneal (i.p.) pretreatment withmethylnaltrexone (5 mg/kg), a peripherally restricted
MOR-preferring antagonist, at 10 min before DALDA injection blocked DALDA-induced CPP in SNL rats, further
suggesting a peripheral mechanism for DALDA-induced
relief of ongoing pain. Gabapentin (60 mg/kg, i.p), which
we used as a positive control, also induced CPP in SNL
rats, but not in naive rats. In a rat model of persistent
inflammatory pain induced by intraplantar injection offormalin (50 µL, 1%), DALDA pretreatment (10 mg/kgs.c,) significantly inhibited spontaneous pain behavior at
15-60 min (phase II) after formalin injection, as comparedto saline pretreatment.
Conclusions: Current findings suggest that subcutaneous
injection of DALDA may alleviate ongoing pain associatedwith neuropathic and inflammatory insults in rats, andthe drug action may be preferentially by targeting MORs
in the peripheral nervous system. Further studies are
required to examine the underlying neurophysiologicalmechanisms.

Grant/Other Support: NIH Grant NS70814, NS26363.

99

No. 111 - Antagonistic effect of angiotensin(1-7) on angiotensin II-induced nociceptive
behavior in mice

No. 112 - Involvement of kappa opioid
receptors in chronic escalation alcohol
drinking in mice

Wataru Nemoto1 , Yoshiki Ogata1 , Osamu Nakagawasai1 ,Fukie Yaoita1 , Takeshi Tadano2 , Koichi Tan-No1

Yan Zhou, F. Ivy Carroll, Mary Jeanne Kreek
Rockefeller University, NY; Research Triangle Institute, NC

1Department of Pharmacology, Tohoku Pharmaceutical University,
4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan; 2Laboratory of Environmental and Health Sciences, College of Medical
Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa

We have recently demonstrated that intrathecal (i.t.) administration of angiotensin II (Ang II) induces nociceptive
behavior in mice accompanied by a phosphorylation of p38
MAPK mediated through Ang II type 1 (AT1) receptors(Nemoto et al., Mol. Pain 2013;9:38). The N-terminal fragment of Ang II, Ang (1-7), plays a pivotal role in counterbalancing many of the well-established actions induced by
Ang II. However, the role of Ang (1-7) in spinal nociceptive transmission remains unclear. Therefore, we examined
whether i.t. administration of Ang (1-7) can inhibit theAng II-induced nociceptive behavior in mice. The nociceptive behavior induced by Ang II was dose-dependently
inhibited by the co-administration of Ang (1-7) (0.03-0.3pmol), whereas Ang (1-7) (0.3-30 pmol) alone did not elicit
nociceptive behavior. The inhibitory effect of Ang (1-7)
was reversed by the co-administration of A779, a Mas receptor antagonist, at a dose of 0.1 pmol, suggesting that the
inhibitory effect of Ang (1-7) was mediated through Mas receptors. Western blot analysis showed that the increase inspinal p38 MAPK phosphorylation following the i.t. administration of Ang II was also inhibited by Ang (1-7), and
the Ang (1-7) induced-inhibition was prevented by A779.
Furthermore, we found that spinal Mas receptors are colocalized with AT1 receptors in the superficial dorsal horn.
Our data show that the i.t. administration of Ang (1-7)
attenuates an Ang II-induced nociceptive behavior and isaccompanied by the inhibition of p38 MAPK phosphorylation mediated through Mas receptors. This observation
indicates that Ang (1-7) may modulate nociceptive transmission in the spinal cord.

Alcohol consumption affects multiple neurobiological systems including the dynorphin/kappa opioid receptor (KOPr) system. Specifically, activation of the KOP-r system has
been implicated in the negative reinforcement aspects of alcohol addiction. Though KOP-r antagonists are found to
reduce alcohol consumption in alcohol-dependent rats, it isunclear whether KOP-r activation is involved in high alcohol drinking in mice. Therefore, the present study was undertaken to determine if pharmacological blockade of KOPr could alter alcohol drinking (consumption & preference at
7.5, 15 and 30% concentrations) in male adult C57BL/6J
mice, using two protocols: 1) 5-day drinking-in-the-dark(DID) with 1 recording/day (4-h); and 2) 3-week chronic
escalation drinking (CED) (2-bottle choice & each otherday) with 3 recordings/day (4, 8 & 24-h). Selective KOPr antagonist JDTic (3, 10 or 20 mg/kg, ip) administration
was given 1 day before the test after mice established drinking behavior. Saccharin DID drinking (0.2%) with JDTic
were tested as control. The alcohol (2g/kg) induced conditioned place preference (CPP) was tested with JDTic at
20mg/kg. Mice exposed to the DID for 5 days at each of
3 alcohol concentrations established stable drinking behavior, with 4-6 g/kg/4-h alcohol consumption. JDTic had noeffect on alcohol or saccharin DID drinking. With the CED
protocol, mice exposed to 15% alcohol vs. water gradually developed high alcohol consumption from 12g/kg/day
at the start to 20g/kg/day after 3 weeks, with increased
preference ratio [PR] from 0.4-0.6 to 0.7-0.8. In contrast
to the DID, after 3-week CED drinking, JDTic at 10 &20mg/kg (but not 3mg/kg) decreased alcohol drinking at
all 3 time points. The total daily consumption was reduced
to 15g/kg after 20mg/kg JDTic, with no effects on the
PR. The alcohol CPP expression was unaltered by JDTic
at 20mg/kg. The results show that pharmacological blockade of KOP-r reduced the CED, but not the DID binge,
drinking or CPP rewarding expression, providing new evidence for the involvement of the KOP-r system in alcohol
addiction. Support: Dr. Miriam & Sheldon G. Adelson
Med Res Foundation, DA09045. No conflict of interest.

100No. 113 - Tetrahydroisoquinoline-based orexin
1 receptor antagonists: structure-activity
relationships at the 1-benzyl position
Yanan Zhang, David Perrey, Nadezhda German, Ann
Decker, Brian P. Gilmour, and Brian F. Thomas

No. 114 - Deciphering agonist- andspecies-dependent KOPR phosphorylation
using LC-MS/MS and phosphospecific KOPR
antibodies
Yi-Ting Chiu, Chongguang Chen, Anika Mann*, Stefan
Schulz*, Lee-Yuan Liu-Chen

Research Triangle Institute

Blockade of the OX1 receptor has been suggested to be a
promising strategy for the treatment of drug addiction. Unfortunately, currently available orexin antagonists are either
mixed OX1/OX2 antagonists designed to treat sleep disorders or OX1 selective antagonists with undesirable pharmacological properties. To address the pressing need forpotent and selective OX1 antagonist, we have developed
orexin antagonists based on a tetrahydroisoquinoline scaffold, the core structure of both the dual OX1/OX2 receptor antagonist ACT-078573 and the OX2 selective antagonist TCS-OX2-29. Recently, we reported the synthesis andin vitro and in vivo evaluation of a series of substituted
tetrahydroisoquinolines, one of which showed excellent invitro OX1 potency and selectivity and attenuated cocaineinduced conditioned place preference in rats (J. Med Chem.,
2013, p6901). We herein report our efforts in further exploring the structure-activity relationships focusing on the
1-benzyl position of the tetrahydroisoquinoline. Structuralfeatures important for OX1 potency and selectivity havebeen identified including the requirement for aromatic functionality at the 1-position. Combined structural modifications from several positions have been conducted which
resulted in compounds having low nanomolar potency at
the OX1 receptor and excellent selectivity over the OX2
receptor. These finding will help expedite the development of potent and selective OX1 antagonists as potential
medications for the treatment of OX1-mediated disorders
such as drug addiction. This work was supported by research grants from the National Institute on Drug Abuse(DA032837 and DA026582).

Center for Substance Abuse Research and Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA
19140 *Institute of Pharmacology and Toxicology, Jena University
Hospital, Friedrich-Schiller-University, Jena, Germany 07747

We found that etorphine caused lower phosphorylationand internalization of the KOPR than U50,488H. Here
we examined the sites of phosphorylation by the two
agonists. The human and mouse KOPRs were tagged at
N-terminus with FLAG and at C-terminus with 6xHis(FhK6H and FmK6H, respectively) and stably expressedin Neuro2A mouse neuroblastoma cells.
Cells weretreated with vehicle or 10 µM U50,488H for 30 min.
FhK6H and FmK6H were affinity-purified with Ni-NTA
agarose followed by anti-FLAG M2 antibody-agarose,
resolved with SDS–PAGE and stained. The FhK6H or
FmK6H protein, a diffused band of 52kDa, was excisedand in-gel micro-digestion was performed with Glu-C.
LC-MS/MS analysis revealed that U50,488H promoted
hKOPR phosphorylation at S356, T357, S358 and T363
with T363 having the most hits. U50,488H enhanced
mKOPR phosphorylation at S356, T357, T363 and S369
with S369 having the most hits. Peptides containing 1, 2
and 3 phosphates were detected. Rabbit antiserum was
generated against KLH conjugates of four phosphopeptides
containing pS356/pT357, pS358, pT363, and pS369.
Antiserum was tested for phosphopeptide specificity by
dot-blot and those with high specificity were purified.
Cells were treated with saline, U50,488H (10 µM) or
etorphine (1 µM) for 30 min, solubilized and receptor waspartially purified with a Ni-NTA agarose. For FhK6H andFmK6H, purified phosphopeptide antibodies all detecteddiffuse bands of 52kDa. Staining intensity of hKORP
with anti-pS358 and anti-pS363 was in the order of U50 <etorphine < control, whereas that with anti-pS356/pT357
was U50 < etorphine control. The U50,488H effect was
blocked by naloxone. Staining intensity of mKOPR with
anti-pS356/pT357 was U50< etorphine control; with antiT363 was U50<< etorphine < control; with anti-S369 was
U50<etorphine << control. These results indicate that
KOPR phosphorylation is agonist- and species-dependent.
We will further investigate the dose-response relationshipand time courses of the two agonists in phosphorylating
different residues to disclose possible differences in site andhierarchy of phosphorylation in both KOPRs.
This work was supported by NIH grant DA17302. The
authors have no conflicts of interest.

101No. 115 - A heroin addiction
severity-associated intronic single nucleotide
polymorphism modulates alternative
pre-mRNA splicing of mu opioid receptor
gene, OPRM1, as a splicing modifier via
hnRNPH interactions

No. 116 - EFFECT OF THE
NOCICEPTIN/ORPHANIN FQ PEPTIDERECEPTOR ANTAGONIST JTC-801 ON
CYTOKINE EXPRESSION FOLLOWINGEXPOSURE TO THE SINGLEPROLONGED STRESS MODEL OF PTSD

J. Xu1 , Z.G. Lu1 , M. Xu1 , L. Pan1 , Y. Deng1 , X. Xie2 , H.
Liu2 , S. Ding3 , Y.L. Hurd4 , G.W. Pasternak1 , R.J. Klein5 ,L. Cartegni6 , W. Zhou2 and Y.-X. Pan1

Yong Zhang (1) , Cindy D. Simpson-Durand (1) and Kelly
M. Standifer (1, 2)

1Dept.
of Neurology and the Molecular Pharmacology andChemistry Program, Memorial Sloan-Kettering Cancer Center, New
York, NY 10065, USA 2Lab. of Behavioral Neuroscience, Ningbo
Addiction Research and Treatment Center, School of Medicine,
Ningbo University, Ningbo, Zhejiang, P.R. China 3Lab. of Clinical
Biochemistry, Ningbo No. 2 Hospital, Zhejiang, P.R. China 4Depts.
of Psychiatry and Neuroscience, Icahn School of Medicine at Mount
Sinai, New York, NY 10029, USA 5Dept. of Medicine, Memorial
Sloan-Kettering Cancer Center, New York, NY 10065, USA 6Susan
Lehman Cullman Lab for Cancer Research, Ernest Mario School of
Pharmacy Rutgers, The State University of New Jersey, Piscataway,
NJ 08854, USA

Single nucleotide polymorphisms (SNP) in the OPRM1
gene have been implicated to associate with vulnerabilityof dependence on opioids such as heroin. The functionalsignificance of several SNPs has been demonstrated in influencing opioid binding, G protein coupling, and receptor
expression, as well as promoter activity. Yet, little has been
known for the role of SNPs on OPRM1 alternative splicing. The present studies identify association of an intronic
SNP (rs9479757) with the heroin addiction severity among
Chinese male heroin abusers, and describe its roles as an
OPRM1 alternative splicing modifier. Although no significant difference between heroin abuser (n=332) and normal
control (n=190) groups, this SNP were significantly associated with the heroin addiction severity (Mild. Moderate
and Severe groups) defined by criteria including route, duration and daily dose of heroin intake among 332 heroinabusers. Haplotype-based analysis together with additional
22 SNPs further supported strong association of rs9479757
with the heroin addiction severity, suggesting that this SNP
is a candidate marker for predicting degree of heroin addiction in Chinese male heroin abusers. In vitro EMSA andUV-crosslinking studies indicated that hnRNPH is the major binding partner for the G-containing SNP site, whereas
the A to G transition significantly lowers affinity toward hnRNPH. Minigene studies indicate that the A to G transition
facilitates exon 2 skipping, leading to altered expressionof the OPRM1 splice variants, which was consistent withthe data from human postmortem brains, suggesting that
the G-containing SNP site functions as an intronic splicing
enhancer. The roles of this SNP in modulating OPRM1
alternative splicing through hnRNPH was further demonstrated by using siRNA, antisense morpholino oligos, andRNA affinity purification-coupled LC-MS/MS approaches
in human neuroblastoma Be(2) cells. Our studies suggest afunctional link between a SNP-containing splicing modifier
and the heroin addiction severity through the underlying
mechanisms involving hnRNPH.

(1) Department Pharmaceutical Sciences, College of Pharmacy, (2)Oklahoma Center for Neuroscience and Dept of Cell Biology, Collegeof Medicine, University of Oklahoma HSC, OKC, OK, USA

Post-traumatic stress disorder (PTSD) is frequently associated with increased inflammatory cell activity and higher
levels of circulating cytokines in plasma. Cytokine profiles
in animal models of PTSD have not been reported to date.
We previously reported that single-prolonged stress (SPS),
an established animal model for PTSD, induces long-lastingmechanical and thermal allodynia. Further, JTC-801, an
N/OFQ peptide (NOP) receptor antagonist, reversed SPSinduced pain- and anxiety-like behavior, as well as elevated N/OFQ levels and NOP receptor expression in brain.
To test our hypothesis that neuroinflammation is a pivotal component of the mechanism underlying pain and/or
PTSD, we examined the effect of JTC-801 on cytokine protein and gene expression in brain after SPS. Male SpragueDawley rats were subjected to sham or SPS, followed byonce daily injection of JTC-801 (6 mg/kg, i.p.) or vehicle
from day 7 until 21 days after initiation of SPS. Cytokine
mRNA levels were measured using real-time quantitative
PCR. JTC-801 alleviated SPS-induced mechanical allodynia and reversed thermal threshold to baseline levels. JTC801 reversed increased IL-2 and IL-6 mRNA in amygdala
after SPS. In hippocampus, there was a significant interaction effect between SPS and JTC-801 on IL-6 mRNA levels. SPS increased IL-1α and IL-2 mRNA in periaqueductal
grey (PAG), and TNF-α mRNA in hippocampus, but thiswas not reversed by JTC-801. When cytokine proteins were
examined using Rat Cytokine 10-Plex Panel, there was asignificant interaction effect between SPS and JTC-801 on
IL-6 level in spinal cord. Higher IL-4, IL-10, IL-12, GMCSF and IFN-γ levels were noted in hippocampus after
SPS, which were not reversed by JTC-801. These results
suggest that inflammatory cytokine expression in brain was
differentially affected by SPS, and only partially mediatedthrough the NOP receptor; expression of neuroinflammatory mediators may contribute to the maintenance of PTSD
symptoms. Animal protocol was approved by the OUHSCIACUC committee and U.S. Army Research and MaterielCommand ACURO office. This study was supported by the
Department of the Army DMRDP W81XWH-11-2-0077.
No conflict exists.

102

No. 117 - Rescue of IBNtxA analgesia, but
not morphine, in a double E1/E11 MOR-1
knockout mouse model using
lentiviral-mediated gene delivery of the 6transmembrane (6TM) domain variantmMOR-1G

No. 118 - RGS4 and RGS2 proteins, newmodulators of the κ-opioid receptor signaling
Maria-Pagona Papakonstantinou and Zafiroula Georgoussi
Laboratory of Cellular Signalling and Molecular Pharmacology,
Institute of Biosciences and Applications, National Centre for
Scientific Research Demokritos, Athens, Greece

ZG. Lu1 , J. Xu1 , G.C. Rossi2 , S. Majumdar1 , G.W.
Pasternak1 , Y.-X. Pan1
1 Department of Neurology and the Molecular Pharmacology andChemistry Program, Memorial Sloan-Kettering Cancer Center, New
York, NY 10065, U.S.A. 2Department of Psychology, Long Island
University, Post Campus, Brookville, NY 11568, U.S.A.

Extensive alternative pre-mRNA splicing of the mu
opioid receptor (OPRM1) gene creates multiple variants
or isoforms that are conserved from rodent to human.
Expression of these variants is controlled by two distinct
promoters associated with exon 1 (E1) and exon 11(E11). Almost all the full-length (7TM) splice variants
and the single TM variants are under the control of
the E1 promoter whereas the truncated 6TM variants
are transcribed through the E11 promoter. The E11
knockout (KO) mouse has provided important functional
information on the 6TM variants. Loss of the E11 variants
eliminates
IBNtxA
(3-iodobenzoyl-6β-naltrexamide)
analgesia while morphine analgesia was intact. IBNtxA
is a new potent opiate analgesic lacking the traditional
side effects associated with classical opiates, including
respiratory depression, constipation, physical dependence
and reinforcing behavior. In contrast, morphine analgesia
is lost in an E1 knockout mouse while IBNtxA analgesia
remained intact. Recently, we developed a new KO mousemodel in which both E1-associated and E11-associatedvariants are disrupted (E1/11 double KO), leading theloss of all MOR-1 splice variants. No mu opioids show
analgesic activity In this E1/11 double KO mouse,
including morphine, M6G and IBNtxA. In the current
study we used a lentiviral-mediated gene delivery of the
6TM variant mMOR-1G to determine if we could rescue(i.e. restore) IBNtxA analgesia in the E1/11 double KO
mouse. Intrathecal (i.t.) or intracerebroventricular (i.c.v.)
administration of lentiviral particles expressing mMOR-1G
in E1/11 double KO mice restored IBNtxA analgesia, but
not morphine analgesia. These studies confirm a role forE11 variants in IBNtxA analgesia and illustrates the utilityof Lentiviral-mediated gene delivery in studying in vivo thefunction of the OPRM1 splice variants and as a potential
therapeutic strategy to pain management. (Supported by
DA13997 & DA029244 (Y.-X.P) and DA02615 & DA07242(G.W.P) from the National Institute on Drug Abuse; anda core grant CA08748 from the National Cancer Instituteto MSKCC.

Previous studies have shown that RGS4 associates with the
C-termini of µ- and δ-opioid receptors in living cells andplays a key role in Gi/Go protein coupling selectivity andsignaling of these receptors (Georgoussi et al., 2006; Leontiadis et al., 2009). To deduce whether similar partners
are involved in κ-opioid receptor (κ-OR)signaling and define the ability of members of B/R4-RGS family to interact with this receptor we performed pulldown experiments
using GST fusion peptides encompassing specific regionsof the κ-OR. These experiments indicated that RGS2 andRGS4 interact within a specific amino acid stretch of the
carboxyl terminus of κ-OR. Co-immunoprecipitation studies indicated that RGS2 and RGS4 associate with κ-OR
constitutively and upon receptor activation and confer selectivity for coupling with a specific subset of G proteins.
Functional assays have shown that both members of B/R4RGS family attenuate κ-OR mediated inhibition of adenylyl
cyclase and ERK1,2 phosphorylation displaying a differential amplitude in their functional effect. Collectively, ourresults demonstrate that RGS2 and RGS4 are new interacting partners and negative modulators of κ-OR signaling.This work was supported by the EU grant “Normolife”(LSHC-037733) and the European COST Action CM1207(GLISTEN).

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June 15th – 19th 2015