Phoenix, AZ

46th meeting of the International Narcotics Research Conference inconjunction with the 77th annual College on Problems of Drug
Dependence
Phoenix, Arizona, USA
June 15-19, 2015
Official Program with Poster Abstracts

Committees and Acknowledgements
Executive Committee
Fred Nyberg (Sweden, President)
John Traynor (USA, Past-President)
Lawrence Toll (USA, Treasurer)
Craig W. Stevens (USA, Information Officer)
Tom Prisinzano (USA)Jose Moron-Concepcion (USA)Catherine Cahill (USA)Thomas Koch (Germany)
Dominique Massotte (France)
Christoph Stein (Germany)
Ichiro Sora (Japan)
Chaim Pick (Israel)
Paul Bigliardi (Singapore)
Program Committee
Todd W. Vanderah (USA, Chair)
Jose Moron-Concepcion (USA, Co-Chair)
Christopher Bailey (UK)Louis Gendron (Canada)
Robert Gereau, IV (USA)Graeme Henderson (UK)
Susan Ingram (USA)
Tally Largent-Milnes (USA)Jay McLaughlin (USA)Michael Morgan (USA)Fred Nyberg (Sweden, President)
John Traynor (USA)
Christopher Vaughn (Australia)
George Wilcox (USA)

Acknowledgments
This meeting is supported by the following:

Front Cover Photos by Philippe Coursodon Photography, Tucson, Arizona; http://www.phcphotography.com/
Back Cover Photo from http://visitbath.co.uk/

2

INRC- Arizona Biltmore Hotel and Meeting Floor Plans

PosterSessions

INRC
Sessions

End ofConference
BanquetWelcome
Reception

3

INRC Schedule at a glance
Time/Date
7:00

Monday6.15.15

8:00
8:30

Tuesday
6.16.15
Registration
8:00-9:00
CPDD/INRC
Plenary 1 Nora Volkow
9:00-9:15
Break

Wednesday
6.17.15
Registration

8:00-9:45 INRC
Plenary: John
Williams &related Hot
topics

9:00
9:15 -11:30
Session 1 Addiction to
Prescription
Opioids (Chair:Fred Nyberg,
INRC
President)

9:30
10:00
10:30
11:00
11:30
12:00

11:30-1:30
Lunch +
Poster session(INRC, Evens)

12:30
13:00

13:30
14:00
14:30
15:00
15:30
16:00
Registration
begins
16:30
17:00
17:30
18:00
18:30
19:00
20:00

Welcomereception

1:30-2:45
Founders
Lecture Gavril
Pasternak
2:45-3:00
Break
3:00 - 5:00
Session 2 Advances in
Therapeutic
Design(Chairs: Jay
Mc Laughlin
and Jane
Aldrich)9:45-10:00
BreakThursday
6.18.15
Registration
8:00- 9:45
Session 5 Vaccines toprevent Drug
Abuse (Chairs:
Mary Jeanne
Kreek andChristopher
Evans)
9:45-10:00
Break

10:00- 12:00
Session 3 - Pain
and Reward(Chair: Frank
Porreca)

10:00-12:00
CPDD/INRC
Plenary 3 Cannabinoids(Chair: Todd
Vanderah)

12:00-2:00
Lunch + Poster
session (INRC,
Odds)

12:00-1:00
Lunch + Data
Blitz (Chair:
Susan Ingram)
1:00-2:00 Early
Career EventMock Study
Section(Optional)

2:00-4:00
CPDD/INRC
Plenary 2 Opioid Abuse(Chair: Sandra
Comer)

Friday6.19.15
Registration
8:00-9:45 Session
6 - Opioid
Tolerance andHyperalgesia(Chair: Michael
Morgan)9:45-10:00 Break
10:00-12:00
Session 7 - NonCanonical Actionsof Opioids (Chair:
Michael Morgan)

Lunch

1:00-2:00 Young
Investigator
Plenary (Gregory
Scherrer)

2:00-3:30 Young
Investigator
Session (Chair:
Gregory Scherrer)

4:00-4:15
Break
4:15-5:15
Session 4 Optogenetics
and pain (Chair:
Robert Gereau)

Free afternoon

Saturday
6.20.15

3:30-4:00 Break
4:00-5:00
Business meeting

Closing Banquet

DepartConferenceINRC2015 Awardees
Founders’ Award – Gavril Pasternak Dr. Gavril Pasternak holds the Anne Burnett Tandy Chair in
Neurology at Memorial Sloan-Kettering Cancer Center and is a Laboratory Head in the Molecular
Pharmacology and Chemistry Program within the Sloan-Kettering Institute. After receiving his M.D.
and Ph.D. degrees from the Johns Hopkins University he completed his clinical training in
Neurology at Johns Hopkins Hospital and then joined the faculty at Memorial Sloan-Kettering in
1979. He is a Fellow of the American Academy of Neurology and a Fellow of the American
Neurological Association. His research has focused on opioid receptors and their mechanisms ofaction, with over 400 publications. He has served on the Editorial Boards of numerous scientificjournals. He is a recipient of a Senior Scientist Award and a MERIT Award from the National
Institute on Drug Abuse and has served on their Board of Scientific Counselors. He is a member of
the Johns Hopkins Society of Scholars and has been awarded the Millenium Prize from the Norwegian University of
Science and Technology, the John J. Bonica and the Raymond W. Houde Awards from the Eastern Pain Association, theJulius Axelrod Award of the American Society of Pharmacology and Experimental Therapeutics, the S. Weir MitchellAward from the American Academy of Neurology. He has been honored with the 2014 William Potter Lecture at ThomasthJefferson University, the 6 Donald W. Benson Lectureship on Pain Medicine at the Johns Hopkins School of Medicine,st
the 1 Annual Machaskee Memorial Lectureship at the Cleveland Clinic and the Stitzel Lecture at West Virginia School of
Medicine.
Comments about Dr. Pasternak from the Selection Committee:
Dr. Pasternak is recognized for his major contributions to the differential roles of opiate receptor subtypes in relieving painwith diminished side effects. Over the years his research has focused upon the characterization of opiate
receptors. Using both ligand binding and molecular biological techniques, Dr. Pasternak has uncovered several novel
receptors derived by alternative splicing of the mu opiate receptor gene. His discoveries have significantly increased ourunderstanding of how opiates act and have led to novel, potent analgesics with reduced side effects. Also, in his recent
research Dr. Pasternak has discovered some new selective opioid drugs that are much more potent than morphine withdiminished adverse effects with less potential for causing physical dependence. Dr. Pasternak has been a frequentattendant at the INRC meetings throughout his career and together with his students and collaborators provided
important contributions to our organization.
Young Investigator Award – Grégory Scherrer Dr. Grégory Scherrer received his PhD in
Molecular and Cellular Biology from Strasbourg University in 2005 under the supervision of Dr.
Brigitte Kieffer. In 2006 he joined Dr. Allan Basbaum’s laboratory at UCSF for his postdoctoral
training. From 2009-20012 he continued as a postdoctoral fellow with Dr. Amy MacDermott at
Columbia University in NYC. In 2012 he started his own research laboratory at Stanford University
as a faculty member of the Neurosciences Institute and the Departments of Anesthesiology andMolecular and Cellular Physiology. His laboratory combines a variety of experimental approaches
including molecular and cellular biology, neuroanatomy, electrophysiology, opto-/pharmacogenetics and behavior in mouse to resolve the functional organization of pain neural circuits innormal conditions and during injury- or disease-induced chronic pain, and how opioids modulate neuronal function to
produce analgesia and detrimental side effects. A major goal of the Scherrer laboratory is to use novel insights into
opioids’ mechanisms of action develop more efficacious and safer therapeutics to treat patients suffering from pain.
Comments about Dr. Scherer from the Selection Committee: While working on his thesis, conducted under thesupervision of Dr. Brigitte Kieffer, Dr. Gregory Scherrer studied the role of delta opioid receptor (DOR) in pain control,
emotional response and cognitive process utilizing advanced techniques, including molecular biology and transgenic
animals. Upon obtaining his PhD, he then moved to San Francisco to work with Professor Alan Basbaum at UCSF to
conduct studies on the function and anatomical localization of DOR in pain processing pathways. During a post-doctoral
period with Dr. Amy MacDermott, he focused on the organization of neuronal circuits that are regulated by opioids in the
spinal cord. Upon finishing his work at Columbia he was recruited by Standford University as an Assistant Professor in the
Department of Anesthesiology where, Greg continues his research on cellular and molecular mechanisms of pain and its
control by opioids. Since the beginning of his career, Dr Scherrer’s research has been original and important, resulted inseveral publications in high impact journals such as PNAS, PLoS One, Pain and Cell, and led to the awarding of acompetitive Pathway to Independence (K99/R00) Award from NIDA to examine the analgesic actions of opioid peptides inchronic pain. Over the years Dr. Scherrer has been a regular attendee at INRC meetings and has contributed excellent
science related to our organization.

5

Full Program

Chris McCurdy (University of Mississippi)
Sigma receptor antagonists in peripheral pain: Potential
for improved diagnosis and pharmacotherapy withsignificantly reduced CNS liabilities

Monday, June 15
15:00-20:00 Registration

Andy Coop (University of Maryland)
UMB425: A unique opioid analgesic with lower tolerance

18:00-20:00 Welcome Reception*Aztec Ballroom and patio

Jessica Anand (University of Michigan)
Development of bifunctional MOR agonist/DOR
antagonist peptides: Successes and failures

Tuesday, June 16
8:00-9:00 INRC/CPDD Plenary 1 Nora Volkow
Director, National Institute on Drug Abuse (NIDA)*Arizona Biltmore Ballroom

Jane Aldrich (University of Florida)
Analogs of the novel macrocyclic tetrapeptide CJ-15,208
exhibit diverse opioid activity profiles

9:00 – 9:15 Break
9:15-10:15 Session 1 “Addiction to Prescription
Opioids”, Chair: Fred Nyberg, Uppsala University
Thomas Clausen (University of Oslo)
Prescription
opioid-abuse
andScandinavian countries

dependence

16:00 – 16:30 Hot Topics
Todd Hillhouse (University of Michigan)
Enhancement of opioid-mediated antinociception by the
mu-opioid receptor positive allosteric modulator, BMS986122 **poster #31

in

Gabriele Fischer (Medical University of Vienna)
Addiction and diversion of prescription opioids in Europe

Achla Gupta (Mt. Sinai Hospital)
Collybollides are highly selective and potent agonists ofkappa opioid receptors **poster #27

Pauline Voon (St. Paul's Hospital, Vancouver, CAN)
Nonmedical prescription opioid use in North America

James Zadina (Tulane University)An
endomorphin
analogprovidingprolonged
antinociception with substantial reduction of multiple
adverse side effects relative to morphine **poster #103

10:15 – 11:00 Hot Topics
Naoko Kuzumaki (Hoshi University, Tokyo)
Chronic pain and addiction **poster #38

16:30 - 17:00 Discussion
Wednesday, June 17

Rachel Enga (Virginia Commonwealth University)
Precipitated oxycodone withdrawal reduces the startle
reflex and bodyweight in C57BL/6J and control mice, but
not in HIV-1 Tat-expressing mice **poster #18
Darrow Khosh-Chashm (University of Texas - Houston)
Impact of benzodiazepine use on retention andcompliance, relapse, and safety in buprenorphinemaintained patients: A Literature Review **poster #34
11:00-11:30 Discussion
11:30-13:30 Lunch & Even numbered posters (withCPDD) *Frank Lloyd Wright Ballroom

8:00 - 08:45 INRC Plenary 1 - John Williams (Vollum
Institute)
What is all the confusion about opioid actions on
dopamine neurons?
8:45 – 9:10 Elyssa Margolis (UCSF)
Opioid actions in the VTA are diverse and depend on
projection target
9:10 – 9:35 MOP in the VTA, plenary follow-up
Adrianne R. Wilson-Poe (Columbia University)
Inflammatory pain dysregulates opioid function in themesolimbic pathway

13:30-14:45 Founders’ Lecture
Gavril Pasternak (Memorial Sloan-Kettering Cancer
Center)40 years on drugs: It’s all in the telling of the tailMichael Bruchas (Washington University-St. Louis)
Spatiotemporal control of opioid signaling and behavior

14:45-15:00 Break

9:45 - 10:00 Break

15:00-16:00 Session 2 “Advances in Therapeutic
Design”, Chairs: Jay McLaughlin, Torrey Pines Institutefor Molecular Studies; Jane Aldrich, University of Florida

10:00 - 11:45 Session 3 “Pain and Reward”, Chair:Frank Porreca, University of Arizona

9:35 – 9:45 Discussion

Howard Fields (UCSF)

6

How the nucleus accumbens enables the approach or
avoid decision
Frank Porreca (University of Arizona)
Endogenous opioids and reward of pain relief
Catherine Cahill (UC-Irvine)
Neuropathic pain modulates dopaminergic circuitry: A
role for microglial activation
Siri Leknes (Oslo University Hospital)
Endogenous opioids in human pain and reward
11:45 - 12:00 Discussion
12:00 - 14:00 Lunch & Odd-numbered posters (withCPDD) *Frank Lloyd Wright Ballroom
14:00 - 16:00 INRC/CPDD Plenary 2 “Novel Strategies
for Reducing Opioid Abuse”, Chair: Sandra Comer,
Columbia University *Arizona Biltmore Ballroom
Kathy Cunningham (University of Texas Medical Branch,
Galveston)
Prospects for serotonin therapeutics in addictive
disordersMarco Pravetoni (University of Minnesota)
Development of vaccines for opioid abuse
Jermaine Jones (Columbia University)
Assessing the ability of glial inhibitors to alter the abuseliability of opioids using laboratory models in humans

8:00 - 9:00 Session 5 “Vaccines to Prevent Drug
Abuse”, Chairs: Mary Jeanne Kreek, The Rockefeller
University; Christopher Evans, UCLA
Brian Reed (The Rockefeller University)
Application of a novel small molecule vaccine platform tooxycodone immunization
Gary Matyas (Walter Reed Army Institute of Research)
Development and optimization of a heroin vaccine
Paul Bremer (Scripps Research Institute)
Design of haptens and conjugate vaccines for heroin
addiction
Joel Schlosburg (Scripps Research Institute)
Selectivity, capacity, durability: putting a heroin vaccine
through its paces and clearing the major hurdles towards
clinical viability
9:00 – 9:30 Hot Topics
Rashmi Jalah (Walter Reed Army Institute of Research)
Development of a combination Heroin-HIV vaccine**poster #46
Oscar Torres (Walter Reed Army Institute of Research)
Comparison of equilibrium dialysis (ED) combined withultra-high performance liquid chromatography/tandem
mass spectrometry (UPLC/MS/MS) and competitive
ELISA for the determination of dissociation constants of
6-acetylmorphine (6AM) and morphine to polyclonal
antibodies **poster #88

Theodore Cicero (Washington University, St. Louis)
Epidemiology on the impact of opioid abuse-deterrent
medications on opioid abuse in the community

Lee W. Hutson (University of North Carolina-Chapel Hill)
Interleukin-1 signaling in the basolateral amygdala
mediatesheroin-conditioned
immunosuppression**poster #35

16:00 - 16:15 Break

9:30 – 9:45 Discussion

16:15 - 17:00 Session 4 “Optogenetics in Pain”,
Chair: Robert W. Gereau IV, Wash U.-St. Louis
Robert W. Gereau IV (Washington University-St. Louis)
Fully-implantable wireless optoelectronic systems forinterrogation of spinal and peripheral pain circuitry

9:45 – 10:00 Break

Anthony Pickering (University of Bristol)
Opto- and chemo-genetic activation of brainstem POMC
neurones reveals opioid-mediated analgesic andcardiorespiratory roles
Zhizhong Z. Pan (The University of Texas MD Anderson)
Dissecting the brain's pain circuits
17:00 - 17:15 Discussion
Thursday, June 18
8:00-10:00 INRC posters on display*Frank Lloyd Wright Ballroom

10:00-12:00 INRC/CPDD Plenary 3 “Cannabinoid
Mechanisms and Interventions”, Chair: Todd W.
Vanderah, University of Arizona*Arizona Biltmore Ballroom
Alexandros Makriyannis (Northeastern University)
Therapeutic interventions through the modulation of
CB1/CB2 cannabinoid receptor function
Aron Lichtman (Virginia Commonwealth University)
Inhibition of the 2-AG biosynthetic enzyme diacylglycerol
lipase beta: Antinociception through a cannabinoid
receptor independent pathway
Todd W. Vanderah (University of Arizona)
Synergistic actions of opioid/cannabinoid agents fordiscrete pain etiologies

7Andrea Hohmann (Indiana University)The
therapeuticpromiseofcannabinoid analgesics

12:00 - 13:00 Data Blitz + Lunch Chair: Susan L.
Ingram, Oregon Health and Science University

11:00 – 11:30 Hot Topics
John Streicher (University of New England)
Heat shock protein 90 regulates mu opioid receptor
signaling in vitro and in mouse periaqueductal grey**poster #84

13:00 - 14:00 Optional Early Career Event - Mock
Study Section Chair: José Moron-Concepción,
Columbia University

Jin Xu (Memorial Sloan-Kettering Cancer Center)
Truncated six transmembrane mu opioid receptors
mediate opioid analgesia **poster #48

nonpsychoactive

11:30 - 12:00 Discussion
Friday, June 19
12:00 -13:00 Lunch
8:00 - 9:15 Session 6 “Opioid Tolerance andHyperalgesia- Real Consequences, Treatment,
Mechanisms”, Chair: Michael M. Morgan, Washington
State University-Vancouver
Michael M. Morgan (Washington State UniversityVancouver)Why are there so many mechanisms of opioid
tolerance?
Bradley K. Taylor (University of Kentucky)
Endogenous opioid dependence
Lori Eidson (Georgia State University)
Role of glia in opioid tolerance
Jürgen Sandkühler (Medical University of Vienna)
CNS
mechanisms
leadingtoopioid-induced
Hyperalgesia
Dorit Pud (University of Haifa)
Opioids and abnormal pain perception in human

13:00 - 14:00 Young Investigator Plenary
Gregory Scherrer (Stanford University)
Functional organization of the opioid system in pain
neural circuits
14:00 - 14:45 Session 8 Young Investigator (Chair:
Gregory Scherrer)
Sarah Ross (University of Pittsburgh)
Dynorphin is released from spinal B5-I interneurons andfunctions as an inhibitory neuromodulator of itch
Steeve Bourane (The Salk Institute for BiologicalStudies)
Identification of functional populations of interneurons in
dorsal spinal cord
14:45 - 15:15 Hot Topics
Edita Navratilova (University of Arizona)
Distinguishing brain circuits mediating pain relieving andaddictive effects of systemic morphine **poster #73

9:15 – 9:45 Discussion
9:45 – 10:00 Break
10:00 - 11:00 Session 7 “Non-canonical Actions of
Opioids”, Chair: Michael M. Morgan, Washington State
University-Vancouver
Eamonn Kelly (University of Bristol)
GRKs, arrestins and bias at the mu opioid receptor

Gina Marrone (Memorial Sloan-Kettering Cancer Center)
U50,488H requires truncated 6 transmembrane variantsof the mu opioid receptor for analgesia but not side
effects **poster #39
Erica Levitt (Oregon Health and Science University)
Mu opioid receptors hyperpolarize respiratory-controlling
Kölliker-Fuse neurons **poster #60
15:15 - 15:30 Discussion

Lakshmi Devi (Mt. Sinai Hospital)
Regulation of opioid receptor function by post-endocytic
peptide processing
Michael Lacagnina (Duke University)
Neuroimmune signaling and opioids: impact of earlylife experience on glial function and addiction-related
behaviors
Wendy Walwyn (UCLA)
A role for constitutively active mu opioid receptors in therecovery from chronic pain

15:30 – 15:45 Break
15:45-17:00 Business Meeting (INRC President, Fred
Nyberg)*Special Recognition Award, Rao Rapaka (NIDA)
17:00– 18:30 Break
18:30 - end Closing Banquet *Gold Room
Saturday, June 20: Depart Conference8

Talk Abstracts (Main Sessions only)“Hot Topics” abstracts can be found in poster abstracts
Tuesday June 16th

INRC/CPDD Plenary 1
1. INRC/CPDD Plenary 1 Nora Volkow Director, National Institute on Drug Abuse (NIDA)
INRC Session 1
2. The involvement of prescription opioid-abuse and dependence in Scandinavian countries
Thomas Clausen (University of Oslo)
The Scandinavian countries share many similarities cultural and political characteristics, all providing health careprimarily through publicly funded universal health care systems. Scandinavian countries share a common feature of welldeveloped national health registries, including prescription databases. , Set against this relatively similar and comparable
context, the presentation will highlight and compare prevalence of opioid use, estimates of abuse and dependence fromthe Scandinavian region, and specifically address the involvement of prescription opioids in overdose deaths. The accessto comparative data offer the opportunity for exploring further some observed differences.
3. Addiction and diversion of prescription opioids in Europe
Gabriele Fischer (Medical University of Vienna)
In Europe, the overall numbers of new heroin clients in treatment are declining from a peak of N = 59,000 in 2007 to N
= 31,000 in 2012. However, the misuse of opioids other than heroin is an increasing concern in the EU, with more than
10% of first-time opioid clients entering specialised treatment misusing opioids other than heroin (including methadone,
buprenorphine and fentanyl). In addition, prescription opioid misuse and addiction among chronic pain patients is an
emerging public health issue, which may be associated with long-term opioid pharmacotherapy for these serious medical
conditions. European studies report that up to 14% of patients, who receive treatment with opioid analgesics for pain
relief, develop an opioid dependence.
4. Nonmedical prescription opioid use in North America
Pauline Voon (St. Paul's Hospital, Vancouver, Canada)
The rates of prescription opioid use in North America are among the highest in the world, and have been paralleled bya rise in opioid-related morbidity and mortality. However, many monitoring surveys currently fail to differentiate between
prescription opioid use for the purpose of euphoria versus pain or withdrawal management. This presentation will reporton recent findings and avenues for intervention and research in order to mitigate the individual, social and structuralproblems related to undertreated pain and prescription opioid use.
5. Founders’ Lecture - 40 years on drugs: It’s all in the telling of the tailGavril Pasternak (Memorial Sloan-Kettering Cancer Center)
Opioids are unique in that our extensive clinical experience with them predated the identification of their receptor
targets. Over the past 40 years, our understanding of opioid action at the molecular level has evolved to provide insights
into the extraordinary complexity of opioids and their targets implied by decades of clinical observation

INRC Session 2
6. Sigma receptor antagonists in peripheral pain: Potential for improved diagnosis and pharmacotherapy withsignificantly reduced CNS liabilities
Chris McCurdy (University of Mississippi)
Peripheral nerve injury, as a consequence of trauma, surgery, inflammation, or other causes, is a major medical
problem. This type of injury is often associated with chronic pain. About 100 Million people suffer from chronic pain in the
United States. Diagnosis and treatment are still considered as unmet medical needs. Current clinical imaging methods
used to evaluate chronic pain are centered on anatomic alterations, which do not necessarily reflect the origin of chronic
pain. A potential biomarker associated with nerve injury and neuroinflammation is the sigma-1 receptor (S1R). In
addition, S1Rs appear to play an active role in pain modulation, both peripherally and centrally. We recently identified ahighly selective S1R antagonist that was transformed into a PET probe candidate and demonstrated high specificity and9selectivity for imaging S1Rs in mice, rats, and monkeys. We have utilized this probe in a rat model of nerve injury via
PET/MRI. The results have helped promote the clinical use of the agent in identifying peripheral pain generators inpatients suffering from neuropathic pain. Furthermore, we have investigated the cold compound and similar derivatives
as potential pharmacotherapies for neuropathic pain in mouse models of nerve injury. These compounds have equipotent
or superior analgesic efficacy to the clinically utilized gabapentin. The compounds have also been examined for liabilities
in locomotor, rotorod, conditioned place preference and in some cases, self-administration assays. The results indicate
the analgesic effects produced by S1Rs antagonists are not associated with these potential liabilities. These results
confirm the ability of S1Rs to serve as potential diagnostic and analgesic agents for neuropathic pain without CNS
liabilities. Funding provided by NIDA (DA023205), NIGMS (GM104932), the Center for Biomedical Imaging at Stanford
University, and the State of Florida, Executive Office of the Governor’s Office of Tourism, Trade, and Economic
Development.
7. UMB425: A unique opioid analgesic with lower tolerance
Andy Coop (University of Maryland)
Opioids remain the primary medication for the treatment of chronic pain, but their use is significantly hindered throughthe development of tolerance and dependence. Previous studies have shown that the co-administration of the mu agonist
morphine together with a delta antagonist attenuates the development of tolerance and dependence in rodents.
Strengthened by studies with delta knock-out animals, and peptide ligands with a dual profile of mu agonism/deltaantagonism, UMB425 was designed as a novel non-peptide ligand possessing the same dual profile. In mouseantinociceptive assays, UMB425 was shown to possess similar potency to morphine, but showed significantly lesstolerance development when an ED90 dose was administrated twice a day for 5 days. UMB425 thus has the potential to
be developed into a new clinical analgesic with important advantages over current opioid analgesics.
8. Development of bifunctional MOR agonist/DOR antagonist peptides: Successes and failures
Jessica Anand, Emily Jutkiewitz, and Henry I Mosberg (University of Michigan)
Historically, medicinal chemists and pharmacologists have sought to develop highly selective ligands for specifictherapeutic targets. The prevailing thought was that the more selective a ligand was for its desired target, the fewer
adverse events occur. However recently, there has been a growing consensus that the simultaneous modulation ofmultiple targets often generates a more desirable drug profile. This is of particular interest in the field of opioid analgesics,
where it has been reported that the co-administration of a mu opioid receptor (MOR) agonist with a delta opioid receptor(DOR) antagonist produces MOR mediated analgesia, but with reduced tolerance and dependence liabilities, featuresthat limit the clinical use of MOR agonist opioid analgesics. Early work in our lab focused on generating selective ligands
for MOR or DOR as potential therapeutics. More recently, we have reexamined past “failed” selective ligands as starting
points for developing bifunctional MOR agonist/DOR antagonist compounds. Using SAR and computational modeling wehave developed and reported a peripherally bioavailable, constrained peptide, VRP26 that displays MOR agonist/DOR
antagonist features in vitro. More interestingly, VRP26 produces antinociception in vivo, with reduced development oftolerance and dependence. We have further explored the rewarding effects of VRP26 in rodents. In parallel, we haveattempted to translate the key binding elements of VRP26 to a more easily synthesized linear peptide scaffold.
9. Analogs of the novel macrocyclic tetrapeptide CJ-15,208 exhibit diverse opioid activity profiles
Jane Aldrich (University of Florida)
The macrocyclic peptide CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) exhibits mixed opioid agonist/kappa opioid receptor(KOR) antagonist activity in vivo, while its D-Trp isomer exhibits predominantly KOR antagonism. Both compounds areactive after oral administration and are promising lead compounds for potential development as treatments for pain anddrug abuse. We are exploring structure-activity relationships for both opioid agonist and KOR antagonist activities. We
expected that conservative amino acid substitutions in these peptides would be well tolerated by opioid receptors and thatthe resulting analogs would exhibit similar opioid activity profiles to the parent peptides with potentially greater potency invivo. The analogs exhibited diverse opioid activity profiles. The analogs generally retained opioid receptor affinities,
determined in radioligand binding assays, similar to or higher than the parent peptides. As with the parent peptide, theoanalogs of CJ-15,208 exhibited antinociception in the mouse 55 C warm water tail withdrawal assay in vivo, but with awide range (80-fold) of antinociceptive potencies and varying opioid receptor involvement. Of particular interest was ananalog that produced antinociception mediated primarily by mu opioid receptors, while exhibiting antagonism at delta
opioid receptors. The analogs of [D-Trp]CJ-15,208 generally exhibited minimal opioid agonist activity, similar to the parent
peptide. KOR antagonist activity proved sensitive to structural modification, with most of the analogs exhibiting loss of
KOR antagonism. Thus minor structural changes had substantial impacts on the opioid activity observed in vivo andresulted in analogs with promising activity profiles for potential development. Research supported by NIDA grants R01
DA023924 and DA032928.

10

th

Wednesday, June 17

INRC Plenary session
10. What is all the confusion about opioid actions on dopamine neurons?
John Williams (Oregon Health and Science University)
This presentation will focus on the action of opioids on dopamine neurons in vivo and in brain slices. The
heterogeneity of dopamine neurons distinguished by projection area, direct opioid sensitivity and the sensitivity of afferent
inputs to opioids will be presented. Finally there will be a comment on the requirements necessary to obtain reliable andreproducible results using electrophysiological methods.
11. Opioid actions in the VTA are diverse and depend on projection target
Elyssa Margolis (UCSF)
MOP receptor activation in the VTA is required for systemic opiate reward, however recent discoveries suggest thatclassical local circuit concepts proposed to explain how this happens need to be revised. We recently reported a MOP
receptor induced direct excitatory effect on a subset of VTA neurons, and we report here that these include dopaminergic
neurons that project to the infralimbic, but not the prelimbic, cortex. As we previously reported that VTA dopamine
neurons that project to prefrontal cortex are inhibited by KOP receptor activation, these data raise the possibility that the
VTA projection to infralimbic cortex conveys a valence difference between the aversive KOP and appetitive MOP signals
from the VTA.
12. Inflammatory pain dysregulates opioid function in the mesolimbic pathway
Adrianne R. Wilson-Poe and Jose Moron-Concepcion (Columbia University)
Chronic pain triggers a myriad of compensatory processes in the central nervous system. Much of our understandingof these adaptations comes from studies in the spinal cord and other regions that are classically involved in paintransmission and modulation. However, recent work in our laboratory indicates that inflammatory pain can also influence
the function and behavioral output of the mesolimbic pathway. We utilized the complete Freund’s adjuvant (CFA) model to
induce persistent inflammatory pain. Microdialysis, self-administration, and in vitro electrophysiology were used to quantify
pain-induced changes in opioid function in the ventral tegmental area (VTA) and nucleus accumbens (NAc). Inflammatory
pain was associated with diminished dopamine release in the NAc, as triggered by microinjection of the mu-opioid
receptor (MOR) agonist DAMGO directly into the VTA. These results were complemented by in vitro whole-cell patch
clamp experiments, which further demonstrated pain-induced MOR desensitization in the VTA in response to DAMGO.
The pain-induced decrease in MOR-dependent accumbal DA release was also associated with a higher propensity to selfadminister large doses (200 μg/kg) of heroin. These data indicate that inflammatory pain induces changes in the MOR
system in mesolimbic regions which are typically associated with the rewarding effects of opioids. Thus, pain-induced
alterations in opioid function in this pathway may contribute to enhanced opioid abuse liability in chronic pain patients.
Support from the National Institute on Drug Abuse (DA034464, DA027460). None of the authors has a conflict of interest
related to this research.
13. Spatiotemporal control of opioid signaling and behavior
Michael Bruchas (Washington University-St. Louis)
Optogenetics is now a widely accepted tool for spatiotemporal manipulation of neuronal activity. However, a majorityof optogenetic approaches use binary on/off control schemes. Here we extend the optogenetic toolset by developing aneuromodulatory approach using a rationale-based design to generate a Gi- coupled, optically-sensitive, mu-opioid-likereceptor, we term opto-MOR. We demonstrate that opto-MOR engages canonical mu-opioid signaling through inhibition of
adenylyl cyclase, activation of MAPK and G protein-gated inward rectifying potassium (GIRK) channels, and internalizes
with similar kinetics as the mu-opioid receptor. To assess in vivo utility we expressed a Cre-dependent viral opto-MOR in
RMTg/VTA GABAergic neurons, which led to a real-time place preference. In contrast, expression of opto-MOR in
GABAergic neurons of the ventral pallidum hedonic cold spot, led to real-time place aversion. This tool has generalizable
application for spatiotemporal control of opioid signaling and, furthermore, can be used broadly for mimicking endogenous
neuronal inhibition pathways.
INRC Session 3
14. How the nucleus accumbens enables the approach or avoid decision
Howard Fields (UCSF)
Howard Fields will review the evidence that the Nucleus Accumbens is not simply part of the reward circuit but cangenerate both approach and avoidance behaviors. Opioids acting in the Nucleus Accumbens can shift decision making topromote approach and suppress responses to pain.

1115. Endogenous opioids and reward of pain relief
Frank Porreca (University of Arizona)Frank Porreca will present evidence that relief of ongoing pain by non-opioid treatments requires release ofendogenous opioids in the rostral anterior cingulate cortex.

16. Neuropathic pain modulates dopaminergic circuitry: role for microglial activation
Catherine Cahill (UC-Irvine)
Catherine Cahill will present recent evidence that peripheral nerve injury causes microglial activation in the VTA. This
activation leads to disruption in reward behavior and dopamine release in the nucleus accumbens via changes in
GABAergic neuronal activity.
17. Endogenous opioids in human pain and reward
Siri Leknes (Oslo University Hospital)
Siri Leknes will discuss the role of the opioid system for pain and reward. Recent evidence from pharmacologicalstudies suggests extensive parallels between human and rodent findings not only for pain relief, but also for reward 'liking'and 'wanting' behaviours.
INRC/CPDD Plenary 2 - Novel Strategies for Reducing Opioid Abuse
18. Prospects for serotonin therapeutics in addictive disorders
Kathy Cunningham (University of Texas Medical Branch, Galveston)
19. Development of vaccines for opioid abuse
Marco Pravetoni (University of Minnesota)
20. Assessing the ability of glial inhibitors to alter the abuse liability of opioids using laboratory models inhumansJermaine Jones (Columbia University)
21. Epidemiology on the impact of opioid abuse-deterrent medications on opioid abuse in the community
Theodore Cicero (Washington University, St. Louis)
INRC Session 4
22. Fully-implantable wireless optoelectronic systems for interrogation of spinal and peripheral pain circuitry
Robert W. Gereau IV (Washington University-St. Louis)
The use of optogenetics has previously required remote light sources and fiber optic delivery schemes that impose
significant physical constraints on natural behaviors, and thus limit utility in typical animal behavioral studies. In this talk,we discuss technologies we have developed that combine soft, compliant neural interfaces with fully-implantable,
stretchable wireless power and control systems to achieve optogenetic modulation of nearly any region of the nervous
system including the spinal cord and peripheral nerves, in freely behaving animals. The utility of these devices in the
study of pain will be demonstrated.

23. Opto- and chemo-genetic activation of brainstem POMC neurones reveals opioid-mediated analgesic andcardiorespiratory roles
Anthony Pickering (University of Bristol)

Beta-endorphin plays a key role in the mediation of stress-induced analgesia and is synthesised by POMC neuronesin the brain. These POMC neurones located in two discrete clusters in the arcuate nucleus of the hypothalamus and also
in the nucleus of the solitary tract (NTS) in the medulla. Using opto- and chemo-genetic targeting approaches we showthat the NTS population of several hundred neurones exerts potent, opioid mediated effects on somatic nociception andcardiorespiratory control in vivo. These findings identify the NTS POMC neurones as being cross-modality command-likeneurones and provide a proof of concept demonstration that a chemogenetic strategy can be used to generate asynthetic analgesic by targeting a defined cell population.
1224. Dissecting the brain's pain circuits
Zhizhong Z. Pan (The University of Texas MD Anderson)

13Thursday June 18th
Session 5
25. Application of a novel small molecule vaccine platform to oxycodone immunization
Brian Reed (The Rockefeller University)
Vaccines against drugs of abuse could potentially yield circulating antibodies which protect against the CND-mediatedeffects of exposure to the relevant drug, including rewarding effects, thus preventing progression to addiction. To beeffective, such vaccines need to elicit a significant level of high affinity antibody, which have selectivity for the molecule ofinterest, and which circulate for substantial time periods. Small molecule vaccine platforms to date largely consist of
conjugating a hapten closely related to the molecule of interest on a carrier protein which is injected one or more times
with an immune stimulating adjuvant. We have begun investigations of alternative small molecule platforms to determine,in comparison with conventional carrier proteins. We have synthesized Trypanasoma brucei brucei coated withoxycodone-conjugated surface coat proteins using selective enzymatic coupling (“Sortagging” using sortase A). The
approach makes use of the ability of the natural pathogen, T. brucei, to stimulate a vigorous immune response.
26. Development and optimization of a heroin vaccine
Gary Matyas (Walter Reed Army Institute of Research)
Development of a heroin vaccine requires that the vaccine induce antibodies that react with heroin, 6-acetylmorphine(6AM) and morphine. The 3-hydroxyl, 6-hydroxyl or the bridgehead nitrogen of heroin/morphine can be attached to carrier
proteins and used as hapten immunogens. Depending on the attachment site, different faces of the heroin/morphinemolecule are presented to the immune system. The immunogenicity and efficacy of chemically stable haptens containing
linkers at the 3- (6-AmHap) and 6- (MorHap) positions and the bridgehead nitrogen (DiAmHap) conjugated to tetanus
toxoid and adjuvanted with liposomes containing monophosphoryl lipid A were investigated. The haptens induced hightiter antibodies in mice. Following subcutaneous challenge with heroin (1 mg/kg), 6-AmHap and MorHap immunizedanimals had low %MPE in a tail-flick antinociception assay. DiAmHap immunized animals had a 59% MPE. Variation of
the MorHap density on tetanus toxoid indicated that the highest hapten density immunogens induced the highest efficacy
from heroin challenge. The Kd of the antibodies to 6AM and morphine was measured by equilibrium dialysis withquantitation by UPLC-MS/MS. DiAmHap-induced antibodies had a Kd of >0.1 µM for 6AM and did not bind morphine.
MorHap and 6-AmHap-induced antibodies had a Kd of <25 nM for 6AM and morphine, suggesting that the efficacy of
MorHap and 6-AmHap was due to the induction of high affinity antibodies. This work was supported through a
Cooperative Agreement Award (W81XWH-07-2-067) between the Henry M. Jackson Foundation and the U.S. Army
Medical Research and Materiel Command and a NIDA Avant Garde award (1DP1DA034787-01). The views expressed inthis article are those of the authors and do not necessarily reflect the official policy of the Department of the Army,
Department of Defense, or NIH, or the U.S. Government.
27. Design of haptens and conjugate vaccines for heroin addiction
Paul Bremer (Scripps Research Institute)
Heroin is a highly addictive opioid that incurs a huge cost to society. In addition to its negative contribution to criminal
activity, productivity loss and medical care, heroin can facilitate the spread of blood-borne pathogens such as HIV and
HCV via intravenous administration. Furthermore, heroin addiction leads to abuse of prescription opioid analgesics suchas OxyContin (oxycodone) and Vicodin (hydrocodone). Abuse of heroin and other opioids has reached epidemic levels in
the past decade, precipitating a rise in overdose deaths. In pursuing therapies to curb the rise of opioid addiction, we havedeveloped a heroin conjugate vaccine. In order to maximize efficacy, we have rigorously optimized each component of thevaccine i.e. heroin hapten structure, conjugation chemistry, carrier protein, adjuvant and dosing. After 3 immunizations,
our refined vaccine elicits unprecedented levels of ‘immunoantagonistic’ protection from heroin; it shifts the heroin EC 50
curves by ~25 times in mouse antinociceptive testing. The protective effects of the vaccine last over three months without
boosters and can be maintained indefinitely with boosters. Furthermore, the heroin vaccine is formulated with components
already approved for human use, showing ample promise for future clinical trials. This work is supported by the National
Institute on Drug Abuse (R01DA026625, F31DA037709 and K99DA037344). The authors declare no conflict of interest
related to this research.
28. Selectivity, capacity, durability: putting a heroin vaccine through its paces and clearing the major hurdles
towards clinical viability
Joel Schlosburg (Scripps Research Institute)Despite the fact that demand for treatment of opiate dependence has more than doubling in the United States in thepast decade, few effective therapeutic options result in long-term abstinence. In the 1970s, researchers demonstrated
proof-of-concept that administering a drug-like structure attached to an immunogenic protein can produce antibodies
which effectively bind opiates in the bloodstream. However, the limitation of being easily surmounted by additional drug
administration left the immunoantagonistic promise of a heroin vaccine unfulfilled for decades to follow. After recent
development of new formulations, screening for acute antagonism in basic antinociceptive tests identified a potentially14effective vaccine. This vaccine was examined in an increasingly complex series of behavioral heroin screens, including:acquisition of self-administration, conditioned place preference, drug-primed reinstatement, and suppression of dependent
drug escalation. Capacity of the lead formulation was estimated at 5-6 mg/kg using heroin cumulative dosing in the hot
plate test, greater than the average daily intake of our dependent heroin self-administering rats. Following extensive
optimization procedures focused on comparing formulations, based on quantitative capacity to block heroin in vivo, thefinal formulation now shifts the heroin dose-response curves over 25-fold versus unvaccinated animals. The blockade ofheroin activity can be maintained by reboosting at least 8 months, and is now being further tested in anticipation of atransition to clinical trials. This heroin vaccine may represent an effective therapeutic for heroin addiction, and the
behavioral procedures followed may demonstrate the procedures needed to identify an effective, clinically-ready drug
vaccine. Research Supported by R01DA026625, K99DA037344, and the Pearson Center for Alcoholism and Addiction
Research. The authors declare no conflicts of interest for this research.
INRC/CPDD Plenary 3 “Cannabinoid Mechanisms and Interventions”
29. Therapeutic interventions through the modulation of CB1/CB2 cannabinoid receptor function
Alexandros Makriyannis (Northeastern University)
The endocannabinoid system is composed of two GPCRs CB1 and CB2. It also encompasses two families ofendogenous ligands represented by anandamide and 2-arachidonoyl glycerol (2AG) as well as the enzymes involved intheir metabolism. A number of these protein targets are being explored as potential drug targets. I shall be discussing
some recent efforts in my laboratory to identify new drug leads as well as methods we developed to carry out the drug
discovery effort. (Supported by grants from NIDA)
30. Inhibition of the 2-AG biosynthetic enzyme diacylglycerol lipase beta: Antinociception through a cannabinoid
receptor independent pathway
Aron H. Lichtman, Department of Pharmacology and Toxicology, Virginia Commonwealth University
This presentation will provide an overview of the consequences of pharmacological inhibition or genetic deletion of
diacylglycerol lipase (DAGL)-β in the mouse LPS model of inflammatory pain. Intraplantar LPS evoked mechanicalallodynia that was associated with increased expression of DAGL-β that co-localized with increased tumor necrosis factorα, and prostaglandins in paws. DAGL-β (-/-) mice or wild type mice treated with the DAGL-β inhibitor KT109 displayed
reductions LPS-induced allodynia. The antinociceptive effects of KT109 did not undergo tolerance following repeatedadministration and did not elicit discernable side effects (e.g., motor deficits, anxiogenic behavior, or gastric ulcers).
Further work showed that KT109 reversed LPS-induced allodynia through a local site of action, and also reversed
allodynia in mouse neuropathic pain models. These findings suggest that local inhibition of DAGL-β at the site of
inflammation represents a novel avenue to treat pathological pain, with no apparent untoward side effects.
31. Synergistic actions of opioid/cannabinoid agents for discrete pain etiologies
Todd W. Vanderah (University of Arizona)
Neuropathic pain is a complex pain disorder that is difficult to manage. Current therapeutic options not only lackefficacy, but are also associated with adverse events. Therapeutics such as Vicodin® and Percocet® that combine
opioids with NSAIDs for a synergistic analgesic effect are limited by their association with hepatotoxicity, GI irritation,
constipation and the potential for abuse. Cannabinoid 2 receptors (CB2) are not known to possess psychotropic,
hepatotoxic or GI effects and have been shown to be active against inflammatory pain. My presentation will focus onwhether synergistic analgesic activity can be achieved by combining CB2 agonists with mu opioid agonist in models of
inflammation and neuropathic pain. In addition, I will present whether CB2 agonists are effective at significantly reducing
opioid-induced unwanted side effects and their ability to modify metastatic bone cancer.
32. The Therapeutic Promise of Nonpsychoactive Cannabinoid Analgesics
Andrea G. Hohmann, Department of Psychological and Brain Sciences and Gill Center for Biomolecular Science, Indiana
University
Direct activation of cannabinoid CB1 receptors produces desirable therapeutic properties but also unwanted(psychoactive) side effects that limit clinical use. CB1 receptors are densely expressed in the central nervous system
whereas CB2 receptors are expressed primarily in immune cells. The discovery of an allosteric binding site on thecannabinoid CB1 receptor− a site distinct from the classical (orthosteric) binding site − has fostered drug discovery efforts
aimed at developing positive allosteric modulators (PAMs) of CB1 signaling that lack cannabimimetic effects associatedwith direct CB1 agonists. CB2 receptors, which are upregulated in response to inflammation and injury, also represent atherapeutic target. Therapeutic efficacy of CB2 agonists, classical CB1/CB2 mixed agonists and CB1 PAMs were
evaluated in a mouse model of toxic neuropathy produced by the chemotherapeutic agent paclitaxel. We evaluated antiallodynic efficacy, possible tolerance, and cannabimimetic effects (i.e., hypothermia, catalepsy, and CB 1-dependent
withdrawal signs) using CB1 knockout (CB1KO), CB2 knockout (CB2KO), and wildtype (WT) mice. Chronic treatment with

15

THC produced tolerance to both cardinal signs of CB1 intoxication and therapeutic efficacy. Challenge with a CB1
antagonist also produced robust physical withdrawal. By contrast, CB2 agonists and CB1 PAMs suppressedchemotherapy-induced neuropathic pain without producing tolerance, CB1-dependent withdrawal, or unwanted CB1mediated side effects. Thus, CB2 agonists and CB1 PAMs offer therapeutic potential to produce a more circumscribedand beneficial spectrum of biological effects compared with direct activation of CB1 receptors. Supported by DA037673,
DA009158, and DA035068

Friday, June 19th
Session 6
33. Why are there so many mechanisms of opioid tolerance?
Michael M. Morgan, Washington State University- Vancouver
Tolerance to the antinociceptive effect of opioids develops rapidly with repeated administration. Over 50 distinct
neural mechanisms for opioid tolerance have been described. Some of this variability is caused by the fact that there aredifferent types of tolerance (e.g., associative and non-associative). In addition, distinct mechanisms for tolerance areengaged in different parts of the nervous system. For example, NMDA receptors contribute to tolerance produced byspinal administration of opioids, but not following administration into the periaqueductal gray (PAG). Distinct mechanismsof tolerance also are engaged depending on the model of tolerance (e.g., desensitization), whether the development or
expression of tolerance is examined, and the opioid administered. However, problems with experimental design also
contribute to the wide range of mechanisms proposed to underlie opioid tolerance. These problems include failing torecognize the difference between correlation and causation, designing experiments to distinguish changes that occur as aresult of tolerance as opposed to repeated testing, and the necessity of determining whether tolerance can be alteredindependent of antinociception. In order to identify specific mechanisms of tolerance, a well-designed experiment must
focus on specific neurons (e.g., PAG neurons), a specific type of tolerance (e.g., development of non-associative
tolerance), and a specific opioid (e.g., morphine). Control groups are needed to avoid confounds such as changes thatare secondary to repeated testing or alterations in antinociception. There are no conflicts of interest associated with thiswork. Funded by NIH grant DA015498
34. Endogenous opioid dependence
Bradley K. Taylor (University of Kentucky)
Mu opioid receptor constitutive activity (MORCA) keeps chronic pain in remission for months after the induction of
inflammation with complete Freund’s adjuvant in mice. Spinal analgesic dependence on MOR CA was manifested withsigns of cellular withdrawal upon spinal administration of inverse agonists, including MOR-G-protein uncoupling, cAMP2+
overshoot, and neuronal ERK phosphorylation and [Ca ]i mobilization. Supraspinal MORCA dependence was revealed
upon systemic or central administration of naltrexone with altered gene expression (Fos), somatomotor behaviors (pawflutters, teeth chatters, wet-dog shakes), escape behaviors (jumping, rearing, hyperlocomotion), and motivation to seek
pain relief (the negative reinforcing capacity of intrathecal lidocaine in a conditional place preference paradigm).
35. Role of glia in opioid tolerance
1
2
2
2
1
Lori N. Eidson , Kiyoshi Inoue , Larry J. Young , Malu G. Tansey & Anne Z. Murphy
1
2
Neuroscience Institute, Georgia State University, Psychiatry, Emory University; Atlanta, GA
Opioid-based narcotics remain an integral part of clinical pain management with morphine being amongst the mosteffective drugs. However, the acute and long-term analgesic efficacy of morphine is limited by the ensuingneuroinflammatory response. The midbrain periaqueductal gray (PAG) is an essential neural substrate for opioidmediated analgesia, and we have previously reported that chronic morphine administration activates ventrolateral PAG(vlPAG) glia via the innate immune receptor toll-like receptor 4 (TLR4), leading morphine tolerance. TLR4 activation
results in a robust release of cytokines, including tumor necrosis factor (TNF), and TNF signaling increases neuronal
excitability by modulating glutamate homeostasis and signaling via neuronal TNF receptor 1 (TNFR1). Our overarching
hypothesis is that morphine administration increases PAG neural excitability in a TNF-dependent manner to decrease thehyperpolarizing effects of morphine and facilitate opioid tolerance. Using anti-TNF gene therapy and an anti-TNF biologic
that crosses the BBB, we demonstrate that sequestration of PAG soluble TNF (solTNF) abolished tolerance to systemicmorphine and naïve tolerance to morphine induced by intra-vlPAG injections of the TLR4 agonist LPS. Morphine
tolerance was accompanied by a significant increase in vlPAG TLR4 and proinflammatory cytokine (IL-1β) mRNA anddecrease in vlPAG astrocytic glutamate transporter mRNA (GLT-1 and GLAST) that was attenuated or eliminated bysequestration of solTNF. These results support our working hypothesis and indicate that morphine binds to TLR4 within
the vlPAG, leading to the release of solTNF. Our results further suggest that solTNF mediates morphine tolerance in the
PAG via neuronal TNFR1 signaling and augmentation of glutamate homeostasis.
36. CNS mechanisms leading to opioid-induced Hyperalgesia

16Jürgen Sandkühler (Medical University of Vienna)
37. Opioids and abnormal pain perception in humans
Dorit Pud, PhD, University of Haifa, Haifa, Israel
Opioids are the cornerstone therapy for the treatment of moderate to severe acute and chronic pain. Yet, while somepatients achieve proper analgesia with opioid treatment, others – with similar diagnoses – do not benefit or evenexperience negative effects from the same treatment. Thus, if based solely on pain diagnoses, the decision of whether or
not to initiate opioid treatment is often counterproductive. Therefore, exploring the response to opioid treatment is of asignificant clinical importance as well as identifying patients who are likely to benefit from opioid treatment. Clinicalevidence suggests that opioid usage per-se can alter pain sensitivity. This has been consistently demonstrated in opioid
addicts, who show paradoxical increased (opioid induced hyperalgesia=OIH) responsiveness to various painful stimuli. I
my presentation I will review the alterations in pain perception in opioid addicts and in patients receiving opioids for thetreatment of chronic pain and become more sensitive to pain. Better understanding of these issues can potentially
optimize opioid analgesic effectiveness and mitigate opioid-related risks.
Session 7
38. GRKs, arrestins and bias at the mu opioid receptor
1211Eamonn Kelly , Eduard Sabido Aguade , Graeme Henderson and Alexandra Cooke
1
2
School of Physiology and Pharmacology, University of Bristol, Bristol UK; Proteomics Unit, Centre de RegulacióGenòmica (CRG), Barcelona, Spain
The cellular response to a mu opioid receptor (MOPr) agonist is usually defined in terms of a single output, such as
inhibition of cyclic AMP signal, but it is likely that the global cellular response will involve many intracellular signaling
pathways. In terms of bias, it is increasingly important that we are able to monitor many different outputs from the receptor
rather than just the basic G protein versus arrestin signals, which could miss important forms of bias. Here we have useda SILAC (stable isotope labeling with amino acids in cell culture) approach with mass spectrometry-based
phosphoproteomics to begin to address this issue, monitoring the signaling induced by a 10 min treatment of HEK293
cells stably expressing MOPr with morphine or endomorphin-2 (each 30 µM). Preliminary results from thephosphoproteomics and bioinformatics analysis indicate that MOPr activation in HEK293 cells 1) leads to regulation ofmany signaling pathways, some of which are not normally associated with MOPr, and 2) whilst morphine andendomorphin-2 regulate the same pathways in some instances, in others they regulate different pathways, or regulatethem in opposite directions. These results suggest that quantitative phosphoproteomics offers an important means tostudy bias. Supported by the Prime-XS European Consortium
39. Regulation of opioid receptor function by post-endocytic peptide processing
Lakshmi Devi (Mt. Sinai Hospital)
Opioid receptor function is modulated by post-activation events such as receptor endocytosis, recycling and/or
degradation. While it is generally thought that the peptide ligand gets co-endocytosed with the receptor, relatively few
studies have investigated the role of the endocytosed peptide and peptide processing enzymes in regulating receptor
function. In this study, we focused on endocytic endothelin-converting enzyme 2 (ECE2), a member of the neprilysin
family of metallopeptidases that exhibits an acidic pH optimum, localizes to an intracellular compartment and selectively
processes some, but not all, opioid peptides at acidic pH. We examined the role of ECE2 in endocytic processing of opioid
peptides and its effect on opioid receptor function by using selective ECE2 inhibitors. We find that the highly selective
ECE2 inhibitor, S136492, and reagents that increase the pH of the acidic compartment impair receptor recycling byprotecting the endocytosed peptide from degradation. This, in turn, leads to a substantial decrease in surface receptor
signaling. We also find that ECE2 inhibition modulates antinociception mediated only by opioid peptides that are ECE2
substrates. Finally, we find that ECE-2 inhibition modulates stress-induced opioid analgesia. These results suggest that
ECE2, by selectively processing endogenous opioid peptides in the endocytic compartment, plays a role in modulating
opioid receptor activity in vitro and in vivo. This work was supported in part by NIH grants DA008863 and NS026880 (toL.A.D.)
40. Neuroimmune signaling and opioids: impact of early-life experience on glial function and addiction-related
behaviors
Michael Lacagnina (Duke University)
Identifying the biological and environmental factors that influence risk or resilience to opioid abuse is an issue ofsignificant clinical importance. While remarkable progress has been made in characterizing the neurobiologicalconsequences of acute and repeated opioid exposure, considerably less is known about the contribution that glial cells
may play in the molecular and behavioral outcomes associated with drug abuse. Here I will discuss several lines ofevidence suggesting that opioids activate glia and alter expression of neuroimmune signaling pathways in the rat central

17nervous system, as well as the potential mechanisms whereby early-life conditions can potently influence later-life glial
activation and subsequent behavioral responding to opioids. During an acute morphine challenge there is a rapid increasein chemokine gene transcription within the nucleus accumbens (NAc) that can be blocked with the nonspecific glial
attenuator ibudilast. Importantly, a neonatal handling procedure that promotes enriched maternal care also attenuates themorphine-induced chemokine response and prevents reinstatement of morphine conditioned place preference.
Furthermore, neonatal handling reduces self-administration of remifentanil with no appreciable effect on operant
responding for food or sucrose. The enduring effect of neonatal handling may be explained by epigenetic mechanisms
altering expression of the anti-inflammatory cytokine interleukin-10 (IL-10) exclusively within microglia. Collectively, theseresults support the concept that neural-glial interactions in the NAc are involved in the establishment of addiction-related
behaviors, and early-life environmental conditions that affect this signaling may substantially influence the reinforcing
properties of opioids. Research supported by NIH/NIDA grant DA034185. No conflicts of interest are declared.
41. A role for constitutively active mu opioids receptors in the recovery from chronic pain
Wendy Walwyn,Ph.D., Dept. of Psychiatry, UCLA, Los Angeles, CA, USA
G-Protein Coupled Receptors (GPCRs) are recognized as being able to activate downstream signaling cascades in
the absence of an agonist. However, such constitutive signaling is seldom observed in an endogenous, unperturbed
system. We have previously shown that mu opioid receptors expressed in dorsal root ganglia neurons are no exception
and do not signal constitutively in the basal state. However, deleting β-arrestin 2 or inhibiting Src increases such signaling,possibly by revealing a caveolin-dependent recycling pathway. We have recently found that chronic pain increases
constitutive inhibition of calcium channels by mu opioid receptors in these primary afferent neurons. Such constitutive
activity is partially responsible for the return to a normal analgesic state following chronic inflammatory pain. However,there are key differences between this adaptation to chronic pain and the β-arrestin 2 and Src-mediated constitutive
activity previously observed. The pathways involved and the consequences of this adaptation to chronic pain will be
discussed.
42. Young Investigator Plenary Functional organization of the opioid system in pain neural circuits
Gregory Scherrer (Stanford University)
Young Investigator Session
43. Dynorphin is released from spinal B5-I interneurons and functions as an inhibitory neuromodulator of itch
Sarah Ross (University of Pittsburgh)
Mu opioids are known to relieve pain. The effect if of kappa opioids on somatosensation, however, is less clear. Our
previous work revealed that B5-I spinal interneurons are a population of inhibitory neurons that function to inhibit itch.
Here we show that B5-I interneurons release the kappa opioid dynorphin. Our data indicate that spinal kappa opioids
bidirectionally modulate itch sensitivity. Thus, dynorphin acts as a neuromodulator to inhibit itch in the dorsal horn of the
spinal cord, raising the possibility that kappa opioids may be a broadly effective therapy for pathological itch. Finally, weshow that B5-I neurons are innervated by menthol-, capsaicin-, and mustard oil-responsive sensory neurons and are
required for the inhibition of itch by menthol. These findings suggest that dynorphin may be involved in the inhibition of itchby chemical counterstimuli. This research was supported by NIH grants R01 AR063772 and R21 AR064445 to S.E.R. andby grants to A.J.T. from the MRC (MR/L003430/1) and BBSRC (BB/J001082). Part of this work was supported by a grantfrom the Rita Allen Foundation to S.E.R, a Whitehall Foundation Research Grant to S.E.R., and the Wellcome Trust to
A.J.T. None of the authors has a conflict of interest related to this research.
44. Identification of functional populations of interneurons in dorsal spinal cord
Steeve Bourane (The Salk Institute for Biological Studies)
The somatosensory system plays an essential role in protecting the body from noxious insults and the fine tuning of
motor activity that ensures the flexible and coordinated limb movements animals need for locomotion, posture andvolitional movements such as reaching and grasping. The dorsal spinal cord is the primary center for integrating andprocessing a number of these somatosensory modalities including heat, cold, pain, itch and touch. Incoming
somatosensory information is processed by complex neural circuits comprised of different excitatory and inhibitory
interneuron cell types (INs) that then relay this information to several brain areas via projection neurons. Changes to thedorsal horn circuitry have been implicated in the development and maintenance of somatosensory disorders like chronic
pain and allodynia, which are major healthcare problems with few effective treatments. In the present study, we havefunctionally characterized different population of excitatory and inhibitory INs in the dorsal spinal cord and have identified
discrete roles for them in processing and transmitting noxious and innocuous touch modalities. We find that the RORα INs
are important for innocuous touch, while Sst excitatory neurons respond to, and relay, noxious mechanical stimuli.
Supported by the Catharina Foundation, the NIH grants NS080586, NS086372. There is no author conflict of interest
related to this research.

18

Poster Abstracts“Evens”- Tuesday presentation; “Odds”- Wednesday presentation
1. A peripherally available mixed efficacy MOR/DOR ligand that displays reduced tolerance, dependence, andabuse liability
Jessica P. Anand1, Henry I. Mosberg2, and Emily M. Jutkiewicz1
1Department of Pharmacology and 2Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI, 48109,
USA
It has been demonstrated that the co-administration of a mu opioid receptor (MOR) agonist with a delta opioid receptor(DOR) antagonist produces analgesia with limited development of undesirable neurobiological adaptations and reduced
abuse liability. We have previously reported a peptide, VRP26, which displays MOR agonism and DOR antagonism in asingle molecule. VRP26 contains a C-terminal β-glucoserine to improve blood-brain barrier penetration and therefore
displays antinociception in vivo after peripheral administration. In this study we examined the ability of VRP26 to produce
tolerance to its antinociceptive effects or physical dependence after chronic administration as well as its ability to produce
conditioned place preference as a measure of reward. Unlike fentanyl, seven day continuous administration of VRP26
produced no rightward shift in the antinociceptive dose response curve and fewer signs of withdrawal than fentanyl.
Further, antinociceptive doses of VRP26, unlike fentanyl, do not produce conditioned place preference in mice.
Additionally, VRP26 does not produce increased locomotor activity, while fentanyl does, suggesting that VRP26 andfentanyl may have differential effects on the dopaminergic system. VRP26 demonstrates proof of concept that mixed
efficacy opioid ligands may be better alternatives to traditional opioid analgesics for chronic pain management, producing
pain relief with limited tolerance development and abuse liability.
2. Signal biased agonists of delta opioid receptors for alcohol withdrawal-induced hyperalgesia
Doungkamol Alongkronrusmee and Richard M. van Rijn
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA
Chronic pain and hyperalgesia are major health issue for which adequate treatment options are still lacking.
Withdrawal from long-term alcohol consumption can induce hyperalgesia in both humans and rodents. Yet, the molecularmechanisms underlying this phenomenon are not well understood, which stifles development of new therapeutics. We
have previously shown that delta opioid receptors (DORs) are upregulated during prolonged alcohol exposure. Moreover,alcohol withdrawal-induced hyperalgesia is exacerbated in DOR knockout mice, suggesting that DORs are promisingtargets for treatment of chronic pain induced by alcohol withdrawal. We therefore proposed to study how DORs modulated
hyperalgesia during chronic alcohol withdrawal. We also investigated the contribution of β-arrestin in DOR-mediatedanalgesia using signal biased DOR agonists. For alcohol withdrawal-induced hyperalgesia, mice were exposed to alcohol
either voluntarily or by oral gavage for three weeks followed by a period of abstinence during which the mice exhibited
hyperalgesia. Mechanical hyperalgesia and analgesia were measured using von Frey filaments. We have found that DOR
agonists are able to reduce alcohol withdrawal-induced hyperalgesia and those oral gavaged mice show robust andpersistent hyperalgesia. This study will contribute to our understanding of the role of DORs in hyperalgesia and further
assist in rational design of DOR agonists for potential therapeutic use in chronic pain conditions. Support by the Ralph W.
and Grace M. Showalter Research Trust and the NIH Pathway to Independent Award (K99/R00). None of the authors has
a conflict of interest related to this research.
3. Measurement of phasic dopamine signals in the rat nucleus accumbens core and shell in response to noxious
stimuli
Christopher Atcherley1,2, Jennifer Xie2, Levi Lazarus3, Michael L. Heien3, Frank Porreca1,2,4
1. Department of Research, Mayo Clinic, Scottsdale, AZ, 85259; 2. Department of Pharmacology, Arizona Health Sciences
Center, University of Arizona, Tucson, AZ, 85724; 3. Department of Chemistry and Biochemistry, University of Arizona,
Tucson, AZ 85721; 4. Department of Anesthesiology, Arizona Health Sciences Center, University of Arizona, Tucson, AZ,
85724
Pain serves a critical function in survival and impacts all aspects of well-being. The aversiveness of pain demands a
behavioral response that is associated with dopamine signaling in the nucleus accumbens (NAc). Mesolimbic
dopaminergic pathways have been implicated in action selection. Both pain, and pain relief, have been shown to produce
dopamine release in the nucleus accumbens, however the exact mechanism is not yet understood. In this study, we usedfast scan cyclic voltammetry to capture and temporally correlate phasic dopamine signals in the NAc core (NAcc) and NAc
shell (NAcs) in response to a noxious thermal stimulus. Male, S.D. rats (320 - 360 g) were anesthetized with isoflurane(0.75 – 1.0 %). The level of anesthesia was regulated to elicit a tail-flick response with a latency of 3.8 – 5.2 sec.
Following anesthesia, biocompatible carbon-fiber microelectrodes were implanted into the NAcc or NAcs for fast-scancyclic voltammetry (FSCV) enabling real-time measurements of dopamine (DA). A triangle waveform (-0.4 V to 1.3 V at
400 V/sec) was applied to the electrode every 100 ms and the resultant voltammogram recorded. The dopaminergic signal
was extracted from the voltammogram by principal component regression, a mathematical tool to identify and quantify
endogenous DA changes based on wave-shape. Electrode placement was optimized by electrically evoking DA release19with a stimulating electrode placed in the median forebrain bundle (24 pulse, 4 ms per pulse, 300 µA). Placement of the
microelectrode was verified with histology by locating the electrode tip, which was marked with an electrical lesion (600µA). The noxious thermal stimulus produced NAc DA efflux in both the NAcc and NAcs that was time-locked to the tailflick. The temporal relationship to pain onset and pain offset varied in these two regions. These methods provide anapproach to assess how NAc dopaminergic signaling may contribute to pain-motivated behaviors.
4. Dorsal striatal mu opioid receptors inhibit cholinergic interneuron-driven dopamine release
Brady K. Atwood, Yolanda Mateo, David Lovinger
Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health,Rockville, MD, USA
Dopaminergic input to the dorsal striatum from the substantia nigra is important for many forms of striatal plasticity andstriatum-mediated behaviors. The regulation of dopamine release in this brain region is complex and involves multiple
neurotransmitters. The role of the mu opioid receptor (MOR) in regulating dopamine release from these inputs is unclear.
Some studies show that MOR activation within the dorsal striatum decreases dopamine release, which is curious
considering the absence of MOR on dopaminergic inputs. However, MORs found on cholinergic interneurons (CINs) may
allow for regulation of dopamine release independent of direct effects on dopaminergic terminals. CINs make up less than
5% of striatal neurons, yet have extensively arborized axons that broadly influence neurotransmission. Acetylcholine
released by these interneurons drives local striatal dopamine release by activating nAChRs on dopaminergic terminals.
Recent evidence demonstrates that activation of MORs on CINs inhibits the activity of these neurons. We tested the
hypothesis that MOR-mediated reduction of CIN activity is responsible for the effect of MOR agonists on dopaminergictransmission. We utilized fast-scan cyclic voltammetry to determine the effects of opioid ligands on dopamine release instriatal brain slices. The MOR agonist DAMGO inhibited dopamine release elicited by both electrical stimulation andoptogenetic activation of CINs. These data demonstrate that MORs are important regulators of CIN-driven dopamine
release. We are currently exploring the effects of genetic manipulations of MOR expression on CIN-driven dopamine
release. No conflict of interest. Work is supported by the Division of Intramural Clinical & Biological Research of NIAAA.
5. Synaptic adaptations in CA1 area of the mouse hippocampus following conditioned place preference tomorphine
Chris P. Bailey, Peter T. Rigby
Department of Pharmacy and Pharmacology, University of Bath, UK
Drug addiction has a strong learning and memory component, thought to contribute to relapse (Portugal GS et al(2014) J Neurosci 34:527-38). Our aim was to examine memory-related synaptic adaptations in the mouse hippocampus
following contingent and non-contingent administration of morphine. Mice were trained to exhibit morphine-induced
conditioned place preference (‘morphine CPP’): 1x i.p. injection per day for 4 days (alternating between 10 mg/kg morphineand saline). Immediately following CPP testing, mice were killed and ventral hippocampal slices prepared forelectrophysiological recording (Nicholls RE et al (2006) Proc Natl Acad Sci USA 103:6380-5. Separate groups of animals(‘non-contingent morphine’) received identical injections but were confined to home cage. There were 2 control groups:‘non-contingent saline’ and ‘saline CPP’. Using extracellular field recordings, stimulus-induced LTP was reduced in bothmorphine groups (‘morphine CPP’ and ‘non-contingent morphine’) vs saline controls. But, exposure to the CPP
environment (‘morphine CPP’ or ‘saline CPP’) increased both the magnitude and variance of LTP compared with noncontingent controls. Using whole-cell patch-clamp recordings, the AMPA/NMDA ratio was increased in the ‘non-contingent
morphine’ group. The ‘morphine CPP’ group showed a non-significant trend to increased AMPA/NMDA ratio, but a highly
significant increase in the variance. These data suggest that in vivo morphine administration induces LTP in the ventral
hippocampus, but that conditioning induces more complex, possibly cell-specific, changes. Ongoing work is attempting to
uncover these cell-specific effects of morphine conditioning on synaptic plasticity. Funding: BBSRC studentship; no conflictof interest
6. Mu-opioid receptor modulation of thalamo-striatal afferents.
William T. Birdsong, Barbara J. Hunnicutt, Tainyi Mao, John T. Williams
Vollum Institute, Oregon Health & Science University, Portland, OR, USA
Opioid dependent inhibition of glutamate inputs to the striatum/Nucleus Accumbens using electrical stimulation is small
and variable. This variability may result from the large number of different afferent inputs. The medial thalamus is known tosend broad glutamatergic projections to both the striatum and Accumbens. Medial thalamus also expresses a high densityof mu-opioid receptors, MOPr. Therefore, it was hypothesized that specific thalamo-striatal projections would be moresensitive to modulation by opioid receptor agonists than other glutamatergic afferents in the striatum— cortico-striatal, forexample. The current project uses whole cell voltage clamp recordings in mouse striatum coupled with viral mediatedexpression of channel rhodopsin in either thalamus or cortex. Optical stimulation of thalamo-striatal and cortico-striatal
afferents as well as bulk electrical stimulation of striatal inputs were used to compare the opioid sensitivity of thalamic andcortical glutamatergic inputs onto striatal medium spiny neurons. The results suggest that while cortico-striatal and bulk
glutamate inputs are relatively insensitive to inhibition by MOPr activation, thalamo-striatal projections are highly sensitive20

to inhibition by MOPr agonists. Thus, opioid treatment may alter the balance of glutamatergic input from thalamus andcortex to the striatum. This work was supported by the National Institutes of Health National Institute on Drug Abuse[DA08163].

7. Protein kinases C and GRK2/3 are involved in U50,488H-promoted KOPR phosphorylation
Yi-Ting Chiu, Chongguang Chen, Anika Mann*, Stefan Schulz*, Lee-Yuan Liu-Chen
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
Agonist-promoted KOPR phosphorylation plays a key role in signaling and receptor regulation. Here we characterizedmolecular mechanisms of U50,488H-induced KOPR phosphorylation. Neuro2a cells stably transfected with FLAGmKOPR-6His were treated with vehicle or U50,488H and solubilized and receptor was partially purified with Ni-NTA
agarose and resolved by SDS-PAGE followed by immunoblotting with specific antibodies recognizing KOPR
phosphorylated at S356/T357, T363 and S369. U50,488H (0.1µM) caused phosphorylation of KOPR at S356/T357, T363
and S369 very quickly, reaching peaks at 1 min. The effect of U50,488H (2 min) was dose-dependent with the EC50
values of 0.1µM, 0.48µM and 0.24µM for phosphorylation at S356/T357, T363 and S369, respectively. The pan-PKC
inhibitor GF109203X (4µM) inhibited U50,488-induced phosphorylation at S356/T357> T363>> S369. The selective PKC α
and β1 inhibitor Go6976 (100nM) inhibited U50,488-induced phosphorylation at S356/T357 and S369, but not at T363. The
PKC activator phorbol 12-myristate 13-acetate (PMA, 0.1µM) caused dose-dependent phosphorylation of S356/T357,
S369 and T363 with the EC50 values of 6.6nM, 7.5nM and 0.2nM, respectively. PMA produced similar phosphorylationlevel at S369 as U50,488H, but reached the peak (at 15 min) later than U50,488H. PMA promoted lower maximal response
at pT363, but higher maximal effect at pS356/T357, than U50,488H. siRNA knockdown of GRK2, GRK3 or GRK2+GRK3
reduced U50,488h-promoted phosphorylation at S356/T357, T363 and S369. These results indicate phosphorylation of
KOPR at different residues is differentially regulated by GRKs and PKC. We will further examine which isoforms of PKC
are involved and if GRK5/6 participate in U50,488H-induced KOPR phosphorylation. (This work was supported by NIH
grants DA017302 and DA036802. The authors have no conflicts of interest.)
8. Differential phosphorylation of the KOPR by U50,488H and etorphine: quantitation with SILAC
Chongguang Chen and Lee-Yuan Liu-Chen
Center for Substance Abuse Research, Dept. of Pharmacology, Temple University School of Med., Philadelphia, PA 19140
U50,488H promoted robust KOPR internalization, whereas etorphine caused low or no internalization. Here we used
SILAC (stable isotope labeling by amino acids in cell culture) to quantitatively compare U50,488H- and etorphine-induced
KOPR phosphorylation. Neuro2A mouse neuroblastoma cells stably transfected with the mouse KOPR epitope tagged withFLAG at the N-terminus and 6 x His at the C-terminus (FmK6H) were grown in the presence of Arg0, Arg6 or Arg10 for atleast 10 passages to label proteins to 99%. Cells were treated with vehicle (C, Arg0), etorphine (E, Arg6) or U50,488H (U,Arg10) for 30 min and the FmK6H was purified with Nickel-NTA column followed by anti-FLAG affinity chromatography.
The FmK6H was resolved as a broad band of Mr 53kDa by SDS-PAGE, in-gel digested with endoproteinase Glu-C andanalyzed with LC-MS/MS. A group of mixed single-, double- and triple-phosphopeptides were identified and quantified,revealing distinct imprints of the two different ligands on KOPR phosphorylation. The two ligands promoted single-site
phosphorylation primarily at T363 and S369 (U/E=2) whereas S356/T357 had basal phosphorylation and U50,488H
induced only slight increases. Both agonists also induced two- and three-site phosphorylation at S356-T363, T357-S369,
T363-S369, S356-T357-S369 with ratios of U/E=2, 3.4, 3.3, 5.6, respectively. Therefore, U50,488H produced higher levelsof single-, double- or triple-phosphorylated form of the KOPR and U50,488H differs more markedly from etorphine inhigher-order KOPR phosphorylation. In addition, because all phosphorylation occurs within the fragment
RQSTNRVRNTVQDPASMRD, the unphosphorylated fragment was used to calculate the % of total KOPR being
phosphorylated. About 60% and 30% of the receptors are phosphorylated following U50 and etorphine treatment,
respectively. (This work was supported by NIH grants DA017302 and DA036802. The authors have no conflicts ofinterest).
9. Optogenetic control of the spinal cord in behaving mice.
Amelia J. Christensen1, Shrivats M. Iyer2, Saurabh Vyas2, Amaury Francois3,4, Sam Vesuna2, Charu Ramakrishnan2,
Karl Deisseroth2,6,7, Gregory Scherrer3,4,5, Scott L. Delp2,8.
Departments of 1Electrical Engineering, 2Bioengineering, 3Anesthesiology, Perioperative and Pain Medicine, 4Molecular
and Cellular Physiology, 5Stanford Neurosciences Institute, 6Psychiatry and Behavioral Sciences, 7Howard Hughes
Medical Institute, 8Mechanical Engineering, Stanford University, Stanford, Ca.
Spinal cord circuits perform extensive computation, process sensory and motor information, and play a critical role inthe development of pathology. However, progress in understanding the function and role of these neurons using
optogenetic techniques has been stymied by a lack of experimental tools for light delivery to the spinal cord. Here, wedeveloped methods to enable optogenetic interrogation of spinal cord circuits using off-the-shelf components,

21

demonstrating modulation of dorsal horn neurons in freely moving animals. We use these techniques to show that
activation of somatostatin interneurons is sufficient to induce pain, and investigate the pathway through which this signal isrelayed to the brain. This work eliminates roadblocks in spinal cord neuroscience, facilitating a wide range of studies fromother groups. This study was supported by the U.S. National Institutes of Health (NINDS R01-NS080954), and the Stanford
Bio-X NeuroVentures program. AJC was supported by a Texas Instruments Stanford Graduate fellowship. None of theauthors has a conflict of interest related to this work.
10. Effects of sex and genotype on corticosterone levels in a mouse model of OPRM1 (A118G) polymorphism
Devon Collins, Mary Jeanne Kreek, Yong Zhang
The Laboratory of the Biology of Addictive Disease, The Rockefeller University, New York, NY, USA
The A118G single nucleotide polymorphism (SNP) of the human mu-opioid receptor gene (OPRM1) is widely studied
due to its association with multiple pain and addiction phenotypes in humans. To elucidate the functional consequences ofthis SNP, mice bearing an equivalent nucleotide substitution (A112G) in the mouse opioid receptor gene were generated(Julie Blendy, UPenn). 112G mice show pain-, stress-, and addiction-related phenotypes similar to humans bearing the118G allele. Previous work from our group shows that the 118G allele affects physiological, agonist-, and antagonistmediated changes in cortisol levels. To our knowledge, there are no studies of the effects of the 112G allele on
corticosterone levels in mice. Thus, we determined baseline serum levels of corticosterone in stress-minimized, adult maleand female mice homozygous for either the 112A (AA) or 112G (GG) allele using a commercially available
radioimmunoassay. Corticosterone was higher in female mice compared to male mice (females 137.75 ± 19.06 ng/mL,
males 54.45 ± 4.45 ng/mL; p < 0.05). Neither males (AA 50.80 ± 7.07 ng/mL, GG 57.05 ± 5.98 ng/mL; p > 0.05) nor
females (AA 104.46 ± 18.44 ng/mL, GG 165.50 ± 16.84 ng/mL; p > 0.05) showed a significant effect of genotype. There
was a trend toward higher corticosterone in GG mice (overall AA 77.63 ± 13.04 ng/mL, overall GG 111.28 ± 11.47 ng/mL; p= 0.068). This work was supported by NIH 1R01DA029147 (YZ), the Adelson Medical Research Foundation (MJK), andthe David Rockefeller Graduate Program (DC). The authors declare no conflict of interest.
11. Receptor dissociation kinetics determines opioid receptor signalling bias
MacDonald J Christie1, Setareh Sianati1, Anna Wang1, Meritxell Canals2, Zoltan Decan3, Paul F Alewood3, RobertCapon3
1Discipline of Pharmacology University of Sydney, Sydney, NSW, 2Monash Institute of Pharmaceutical Sciences, Monash
University, Vic, 3Institute for Molecular Bioscience, University of Queensland, QLD, Australia.
Recently there has been much interest in the possibility that biased signalling at the MOPr may be effective in skewing
the analgesic versus side-effect profiles of MOPr agonists. We have used a single cell type to examine the signalling
efficacy of a series of opioids with varying efficacy for G-protein signalling versus endocytosis, including a novel peptidic
agonist with extreme G-protein bias. We determined activation of G-protein signalling (GIRK activation), Ser375
phosphorylation (immunohistochemistry), β-arrestin-2 binding (BRET) and endocytosis (immunohistochemistry) underconditions, where possible, that association and dissociation kinetics could be determined. We and others have previously
established that all regulatory steps of MOPr are reversible at the cell surface. Our major findings are that the dissociation
kinetics of GIRK signalling, Ser375 phosphorylation, and β-arrestin-2 binding are highly correlated for agonists at each stepof the regulatory process, with correlation coefficients of approximately 0.9. This demonstrates that reversal of eachregulatory step is ligand-dissociation rate dependent rather than pathway (e.g. phosphatase kinetics) limited. Importantly,
when we account for initial GIRK signalling efficacy, dissociation rate strongly predicts efficacy for endocytosis, with rapidly
dissociating agonists displaying the greatest GIRK versus arresting bias. This suggests that development of high G-protein
efficacy agonists with rapid dissociation rates can be systematically developed to limit adverse effects of opioids.
12. Lipophilicity Efficiency is a new paradigm for balancing receptor affinity and in vivo antinociceptive efficacy of
opioid peptides
Rossella De Marco1, Santi Spampinato2, Andrea Bedini1, Luca Gentilucci1
1Department of Chemistry “G. Ciamician”, University of Bologna, via Selmi 2, 40126 Bologna, Italy; 2Department ofPharmacy and Biotechnology, University of Bologna, via Irnerio 48, 40126 Bologna, Italy
The lipophilicity efficiency indices LLE and LELP have been recently proposed to support balanced optimization ofpotency and ADMET profile. Ligand lipophilicity efficiency LLE represents the activity of a ligand without the contribution ofits lipophilicity. LLE consents high potency while maintaining moderate lipophilicity. Ligand efficiency-dependent lipophilicity
LELP has been recently proposed to combine lipophilicity, molecular size, and potency into one composite descriptor. In
this respect, we compare for the first time the lipophilic efficiency indices to affinity and in vivo efficacy in the field of opioid
compounds. We analyzed of a mini-library of lipophilic opioid peptides, carrying a Trp equipped with methyl, nitro, andhalo-substituents at various positions of the indole ring. Substitution had a strong impact on experimental MOR affinities.
We also observed that the different groups at Trp influenced stability and lipophilicity. Peptides with 5-nitro and 7-Br-2-Me
Trp showed moderately faster analgesia as compared to the unsubstituted compounds in mouse tail flick assay. The
antinociceptive efficacy profiles nicely correlated to the calculated LLE and LELP, suggesting that lipophilicity efficiency
indices might represent a new and useful predictive tool for the design of centrally or peripherally acting analgesic

22compounds. Support by Fondazione Umberto Veronesi
13. The evolution of amygdala neuronal ensembles encoding neuropathic pain states
Gregory Corder1,2,6, Biafra Ahanonu3,6, Benjamin Grewe4,6, Mark Schnitzer3,4,5,6, and Grégory Scherrer1,2,6
1Dept. of Anesthesiology, Perioperative and Pain Medicine, 2Dept. of Molecular and Cellular Physiology, Dept. of
Neurosurgery, 3Dept. of Biology; 4Dept. of Applied Physics; 5HHMI, 6Stanford Neurosciences Institute, Stanford
University, Palo Alto, CA, USA
Pain is a multidimensional experience comprising sensory and emotional modules. The basolateral amygdala (BLA),
classically involved in fear and anxiety, assigns an emotional valence, such as unpleasantness, to highly salient
environmental stimuli. To understand how the BLA encodes the experience of aversive stimuli and how this activity
changes during the development of chronic neuropathic pain we followed BLA ensemble dynamics for multiple weeks after
the induction of a peripheral nerve injury. To visualize the BLA network activity in awake, freely behaving mice we
implanted a small microendoscope that allowed us to image large populations of BLA projection neurons expressing thecalcium indicator GCaMP6m. We monitored the evolution of neural activity patterns in the BLA in response to innocuousand noxious stimuli, before and for weeks after a peripheral nerve injury that induced neuropathic pain. Prior to nerve
injury, noxious, but not innocuous, stimuli evoked time-locked Ca2+ spikes in a subpopulation of BLA neurons. Strikingly,
after the establishment of neuropathic pain the neuronal ensemble responses evoked by prior innocuous stimulitransformed such that the network representation was indistinguishable from activity patterns evoked by frankly noxious
stimuli. Our results point to a possible neural substrate for allodynia and show for the first time how neural ensemble
coding may shape the perception of pain unpleasantness. We are presently examining the functional importance of this
BLA network representation to several behavioral features of chronic pain. Studies supported by NIDA DA031777 (GS),
T32-DA035165 (GC), NSF DGE-114747 (BA) and DARPA (MS). No conflicts of interest.
14. Nalfurafine shows lower propensity to cause aversion in mice and produces lower KOPR phosphorylation thanU50,488H and MOM-Sal B
Kelly M. DiMattio, Chongguang Chen, Yi-Ting Chiu, Alan Cowan & Lee-Yuan Liu-Chen
Center for Substance Abuse Research & Department of Pharmacology, Temple University School of Medicine,Philadelphia, PA 19140, USA
KOPR agonists are potentially useful as antipruiritic agents, analgesics and water diuretics; however, theirdevelopment has been limited by dysphoria. Nalfurafine, U50,488H and methoxymethyl salvinorin B (MOM-SalB) are
structurally distinct selective KOPR agonists. Nalfurafine is used in Japan for treatment of uremic pruiritus in hemodialysis
patients; however, surprisingly, at the therapeutic doses, nalfurafine does not produce dysphoria. Here we examined thethree compounds for inhibition of compound 48/80-induced scratching, antinociceptive effect in the formalin test andaversive effect in conditioned place aversion (CPA) in male CD-1 mice to determine if there were dose differences among
the three tests for each compound. The three compounds produced antinociception and anti-scratching effects in dosedependent fashion with antinociception A50 values of 5.8 μg/kg (nalfurafine), 17 µg/kg (MOM-SalB) and 0.58 mg/kg(U50,488H) and the corresponding A50 values for antiscratching effect of 8.0 μg/kg, 70.2 µg/kg and 2.07 mg/kg,
respectively. Significant aversion was seen at all doses of MOM-Sal B (10-300 µg/kg) and U50,488 (0.25-10 mg/kg),
whereas nalfurafine only produced significant aversion at the 20 μg/kg. Thus, U50,488H and MOM-SalB produced CPA atlower doses than those required for antinociceptive and anti-scratching effects, whereas the reverse was true fornalfurafine. In neuro2A mouse neuroblastoma cells expressing the mouse KOPR (mKOPR), the three compounds were fullagonists in promoting [35S]GTPγS binding. All three compounds induced mKOPR phosphorylation at S356/T357, T363
and S369, but nalfurafine caused T363 phosphorylation to a much lower extent. These results indicate that nalfurafine actson the KOPR in a different manner from U50,488H and MOM-SalB, which may account for the unique in vivo
pharmacological profile. In addition, our mouse models may be useful for screening for KOPR agonists with lower
propensity to cause dysphoria. Supported by NIH grants (DA036802 and DA013429). The authors have no conflicts ofinterest to disclose.
15. Effect of a novel fentanyl derivate on mu-opioid receptor-induced G-protein activation
Giovanna Del Vecchio1, Olga Scharkoi2, Markus Weber2, Christoph Stein1.
1Klinik für Anaesthesiologie und operative Intensivmedizin, Charité Universitätsmedizin Berlin, Campus Benjamin Franklin,
Hindenburgdamm 30, Berlin 12203, Germany; 2Abteilung Numerische Analysis und Modellierung, Zuse-Institut Berlin,
Takustr. 7, Berlin, 14195, Germany
The use of mu-opioid receptor (MOR) agonists in the treatment of pain is greatly limited by their side effects. Therefore,
new drugs with a disease-specific action precluding sedation, addiction, respiratory depression and other adverse effects
are needed. Opioids activate the G-protein coupled receptor (GPCR) MOR, leading to conformational rearrangements ofinhibitory Gα and Gβγ subunits, their activation and targeting of intracellular mediators. Despite the progresses made inunderstanding GPCR activation, the diversity of signaling by different MOR ligands and in different tissue milieus requires
further characterization. Fluorescence Resonance Energy Transfer (FRET) provides novel methodological impulses to thestudy of dynamic protein-protein interactions in live cells. Here, we used FRET to investigate MOR-induced G-protein

23

activation upon treatment with Fentanyl and Fluor-Fentanyl. The latter was derived by computer-aided drug design toselectively activate peripheral MOR during extracellular acidosis, a hallmark of inflammation following tissue injury. Gprotein subunits and MOR were expressed in HEK293 cells and FRET between fluorescent Gαi1-mTq and Venus-Gγ2 wasmeasured over time. Fentanyl induced a marked decrease in FRET, likely due to rearrangement/dissociation of G-protein
complexes, which was blocked by co-treatment with naloxone. Fluor-fentanyl selectively activated G-proteins at low
extracellular pH, while under physiological conditions (pH 7.4), its effects were indistinguishable from vehicle-treated cells.
These data suggest that our novel fentanyl derivate activates MOR and G-proteins preferably at low pH. Supported by
BMBF-VIP0272/03V0364. Conflicts of interest: Patent applications WO 2013026787 A1; WO 2013102681 A1.
16. Differential regulation of delta opioid receptor-mediated behaviors by RGS proteins in mice
Isaac J. Dripps1, Richard R. Neubig2, and Emily M. Jutkiewicz1
1Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI; 2Department of Pharmacology,
Michigan State University, East Lansing, MI
The delta opioid receptor (DOPr) is a member of the opioid receptor family of G protein-coupled receptors (GPCRs).
Activation of DOPr induces antinociception and antidepressant-like effects in animal models without the constipation,
respiratory depression, and abuse liability associated with mu opioid receptor agonists such as morphine. In addition, some
DOPr agonists cause convulsions, hindering their development as therapeutics in humans. However, the intracellularsignaling pathways that generate these behaviors have yet to be determined. Regulator of G protein signaling (RGS)
proteins act as negative modulators of GPCR signaling and may differentially regulate DOPr-mediated behaviors. To
investigate this, we compared behaviors induced by the DOPr agonist SNC80 in Gαo RGS-insensitive (RGSi) knock-in and
RGS4 knockout mice. SNC80-induced peripherally-mediated antinociception in the acetic acid stretch assay wasenhanced in both the Gαo RGSi knock-in and RGS4 knockout animals. Loss of RGS4 also potentiated SNC80-induced
centrally-mediated antihyperalgesia in a nitroglycerin-induced thermal hyperalgesia assay. In the tail suspension test,
RGS4 knockout mice showed no significant difference in SNC80-induced antidepressant-like effects. Furthermore, there
was no change in the dose of SNC80 needed to induce a convulsion in RGS4 knockout mice. Taken together, these data
demonstrate that RGS4 negatively modulates some, but not all DOPr-mediated behaviors. Because RGS proteinsmodulate DOPr-mediated G protein signaling, these data are consistent with G protein independent signaling mechanisms
generating DOPr-mediated antidepressant-like effects and convulsions. Future studies will focus on verifying which DOPrmediated behaviors signal through G protein independent mechanisms.
17. Enhanced enkephalin tone in the striatum facilitates locomotion following deletion of D2 receptors in striatal
medium spiny neurons
Lauren K. Dobbs, Mariah Blegen, Veronica A. Alvarez
National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, USA
The endogenous opioid peptide enkephalin is expressed in medium spiny neurons (MSNs) of the striatum and nucleus
accumbens (NAc) where, through activation of opioid receptors, enkephalin is thought to mediate morphine reward. The
level of enkephalin mRNA in the striatum is regulated by the activity of dopamine D2 receptors (D2R) but it is unclear which
population of D2R-expressing neurons exerts this control and whether changes in expression level have any functionalsignificance on striatal behaviors, such as opioid-induced locomotor activity and reward. We found that a transgenic
mouse with selective deletion of D2Rs from D2R-containing MSNs (MSN-Drd2-KO) show an 85% increase in enkephalin
mRNA levels in the striatum compared to littermate controls. The effect of met-enkephalin on GABAA-mediated inhibitory
post-synaptic currents (IPSCs) was measured in D1R-containing MSNs triggered by optogenetic stimulation of D2-MSN
axon collaterals in slices containing the NAc. Met-enkephalin attenuated optogenetic-evoked IPSC amplitude equally in
MSN-Drd2-KOs and controls, suggesting that opioid receptor levels are functionally equivalent. However, when
enkephalin degradation was inhibited optogenetic-evoked IPSC amplitude and spontaneous IPSC frequency wasdecreased in MSN-Drd2-KOs, but not controls, strongly suggesting that these mice have enhanced enkephalin tone.
Furthermore, systemic administration of the opioid antagonist naloxone induced a locomotor response and a statedependent place aversion only in MSN-Drd2-KOs; however, both genotypes showed a locomotor response to oxycodone
and acquired morphine conditioned place preference. These data indicate that selective deletion of D2Rs from D2Rcontaining MSNs enhances striatal enkephalin tone to facilitate locomotion without affecting opioid conditioned reward. The
authors have no conflict of interest to declare.
18. Precipitated oxycodone withdrawal reduces the startle reflex and bodyweight in C57BL/6J and control mice,but not in HIV-1 Tat-expressing mice
Rachel M. Enga1, Edward G. Hawkins1, Kristen M. Lee1, Pamela E. Knapp2, Kurt F. Hauser1 and Patrick M. Beardsley1,3
1Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond VA
2Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond VA
3Center for Biomarker Research and Precision Medicine, Virginia Commonwealth University, Richmond VA
Abuse of prescription opiates, such as oxycodone, has increased in recent decades. HIV-infected individuals have an
enhanced risk of opiate abuse. There is evidence that opiates worsen the cellular and behavioral toxicity symptomatic of24

HIV-associated neurocognitive disorders (HAND) presumably through their interaction with the neurotoxic HIV-1 Tat
protein. Previously, in agreement with clinical findings in HAND-diagnosed patients, we found significant deficits in
prepulse inhibition of the startle reflex (PPI), a measure of sensorimotor gating, in transgenic mice that centrally express
HIV-1 Tat. We subsequently hypothesized that even greater PPI deficits would occur in Tat-expressing vs Tat-null mice
during oxycodone dependence. Physical dependence upon oxycodone was induced by administering a 10-day escalating
regimen (9-33 mg/kg, b.i.d., s.c.) in Tat(+), Tat(-), and C57BL/6J mice. Startle/PPI was tested in a 25-min session underchronic and precipitated withdrawal conditions. Chronic oxycodone administration did not significantly affect startle or PPI
in any group. Precipitating withdrawal with naloxone (1 mg/kg, s.c.) significantly (p<0.05) reduced the startle reflex andbody weights in C57BL/6J and Tat(-) mice, but surprisingly not in Tat(+) mice. Also surprisingly, PPI was unaffected in allgroups. Contrary to our hypothesis, these results suggest that Tat-expressing mice may be more resistant to opiate
withdrawal effects. This research was supported by NIDA training grant T32DA007027 and NIH grant R01 DA018633.
None of the authors have a conflict of interest.
19. Characterization of the diimidazo-diazepine TPI-2065-14, a mixed KOR and DOR agonist with peripherallyrestricted activity and minimal liabilities of use.
Shainnel O. Eans,1,2 Michelle L. Ganno,1 Elisa Mizrachi,1 Richard A. Houghten,1 Colette T. Dooley,1 Jay P.
McLaughlin,1,2 Adel Nefzi1
1Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL; 2Univ. of Florida, Gainesville, FL
Agonists with mixed selectivity for kappa and delta opioid receptors could increase analgesic potency while reducing
adverse effects. Moreover, we hypothesized that peptidomimetic ligands might display reduced blood brain barrier (BBB)
permeability, resulting in safer peripherally-selective opioid analgesics. Accordingly, the 2065-series of fuseddiazaheterocyclics was synthesized from reduced tripeptides. In vitro screening with radioligand competition binding
assays demonstrated variable opioid receptor affinity across the series, with the diimidazo-diazepine TPI-2065-14
displaying nanomolar affinity for KOR and DOR. Central (i.c.v.) or intraperitoneal (i.p.) administration of 2065-14 produced
dose-dependent, opioid-receptor mediated antinociception in the mouse 55oC warm-water tail-withdrawal and acetic-acidwrithing assays. The potency ratio of 2065-14 in the two assays was 32.6 times higher than morphine, and antinociception
was blocked by pretreatment with naloxone methiodide only when administered on the same side of the BBB, suggesting
possible peripherally-restricted activity. Confirming this, although readily detected in blood, only trace amounts of 2065-14
were detected in brain by LC-MS/MS following peripheral administration, which supports our earlier hypothesis.
Administration of 2065-14 did not alter gastrointestinal transit or produce seizures at any antinociceptive therapeutic dosetested, and did not demonstrate acute antinociceptive tolerance. Even when centrally administered (i.c.v.), maximal
antinociceptive doses of 2065-14 did not produce locomotor effects in the rotorod assay or cause conditioned-place
preference or aversion. Together, these data support that mixed-selectivity opioid agonists may increase analgesic potency
with minimal liabilities and that diimidazo-diazepine ligands may possess innate peripheral selectivity, with 2065-14 offering
early therapeutic potential as a novel analgesic with fewer clinical liabilities. This work was funded by the Florida Drug
Discovery Acceleration Program by the State of Florida and by R01-DA31370 (to RAH).
20. Morphine conditioned place preference induces structural plasticity of dendritic spines in the hippocampus.
Amanda K. Fakira1, Alexandra Berman1, Omid Cohensedgh1, Kaylee Wedderburn-Pugh1, David Sulzer2 and Jose A.
Morón Concepcion1
1Department of Anesthesiology, College of Physicians and Surgeons, Columbia University Medical Center, New York;2Departments of Neurology, Psychiatry, and Pharmacology, Columbia University Medical Center, New York, New York
Opiate abuse is emerging problem in the US with 2.1 million people abusing opiate prescription drugs. Relapse occurs
in 80% of abstinent abusers indicating a need to develop methods to prevent cravings that drive relapse. Our previous
studies demonstrate that the NMDA-NR2B subunit in the hippocampus (HPC) is necessary for opiate induced behaviors.
Dendritic spine morphology, controlled by actin polymerization, is regulated by NR2B-mediated Ca2+ influx and activationof the Rho signaling cascade. We have found that morphine conditioned place preference (CPP) induces a decrease inspines on CA1 pyramidal cells in the hippocampus mediated by a decrease in thin type spines. Additionally, we havefound that morphine CPP increases RhoA expression in synaptosomal fractions while overall expression in unalteredfollowing morphine CPP. Suggesting that morphine CPP alters RhoA trafficking in the hippocampus. Future studies will
investigate whether the morphine CPP induced effects on thin spines and RhoA trafficking are mediated by increases in
NR2B function. Studies thus far demonstrate that morphine CPP induces plasticity in the hippocampus, a brain area that
sends projections to the medial prefrontal cortex (mPFC). The mPFC is also involved in the acquisition and retrieval of
drug associated memories. Ongoing studies are investigating the role of vHPC-mPFC projections in reward, and inparticular opiate induced behaviors. Our preliminary studies demonstrate that optogenetic activation of the vHPC can
induce a CPP and future studies will determine the role of hippocampal projections to the mPFC in the development of
CPP and their role in opiate craving. This work was supported by NIH grant R01 DA027460 to JMC. Authors have noconflict of interest.

25

21. The kappa opioid receptor agonist 16-bromosalvinorin A reduces cocaine seeking in rats
Amy W. Ewald1, John H. Miller1, Thomas E. Prisinzano2, and Bronwyn M. Kivell1
1Centre for Biodiscovery, Victoria University of Wellington, Wellington, NEW ZEALAND; 2Medicinal Chemistry, The
University of Kansas, Lawrence, KS, USA
Kappa opioid receptor (KOPr) agonists have known anti-cocaine effects but produce various side effects that limit
therapeutic use. The aim of the current study was to determine if the novel salvinorin A analog 16-bromosalvinorin A(brSalA), could attenuate reinstatement of cocaine seeking preclinically with reduced side effects compared to traditional
KOPr agonists. Male Sprague-Dawley rats trained to self-administer cocaine in daily 2 hour sessions were subjected toextinction followed by administration of vehicle or brSalA (0.3 mg/kg, 1 mg/kg; n=6) and drug-prime reinstatement testing(20 mg/kg cocaine, i.p). Sedative effects were evaluated by determining locomotor activity in rats for 60 min following
brSalA (1 mg/kg, i.p, n=6). To evaluate the effects of brSalA on natural reward pathways, drug naïve rats were trained toself-administer sucrose pellets and effects on responding after brSalA administration (1 mg/kg, i.p, n=7) determined.
Depressive effects were assessed by subjecting rats to forced swimming conditions for 5 min following an injection of brSal
A (1 mg/kg, i.p, n=8-9). BrSalA significantly decreased cocaine seeking but did not reduce locomotor activity and sucrose
intake or increase immobility times in rats. This highlights a specific effect of brSal A on drug seeking not due to sedation or
modulation of natural reward, and the minimal side effects of brSalA at the effective dose. These findings provide
information for ongoing work in developing eﬀective anti-addiction pharmacotherapies. Support by Neurological Foundationof New Zealand. AWE was supported by a Victoria University Doctoral Scholarship. The authors have no conflicts ofinterest to declare.
22. Impact of linker/address region in the discovery of potent multifunctional ligands with μ/δ opioid
agonist/neurokinin 1 (NK1) Antagonist Activities
Aswini Kumar Giri1, Christopher R. Apostol1, Peg Davis2, David Rankin2, Gabriella Molnar2, Todd W. Vanderah2, Frank
Porreca2, Victor J. Hruby1
1Departments of Chemistry and Biochemistry, University of Arizona, 1306 E. University Blvd., Tucson, Arizona 85721;
2Department of Pharmacology, University of Arizona, 1501 N. Campbell Ave., Tucson, AZ 85724
Multifunctional ligands with µ/δ opioid agonist/NK1 antagonist activities were designed and synthesized with theconcept of overlapping and adjacent pharmacophores. Conformationally flexible, as well as rigid, amino acids were used
as linker/address region to examine the role played by it in binding selectivity between µ and δ opioid receptors. Influenceof structural changes in opioid pharmacophores in the biological profile at MOR, DOR, and NK1R were studied. The
structure-activity relationships (SARs) study revealed that the N-terminus modification, which was expected to haveinfluence on the biological profiles at µ/δ opioid receptors, has appreciable impact on NK1 receptor binding affinities andactivities. Use of rigid amino acid at the address region enhanced the MOR binding selectivity over DOR. Functional
assays showed that the ligand AKG113 (H-Tyr-D-Ala-Gly-NMePhe-Gly-Pro-Leu-Trp-NH-Bn(3ʹ,5ʹ-(CF3)2)) containing aflexible linker (i.e. Gly) has two times more agonist activity at DOR compared to that at MOR. This study led to thediscovery of a potent ligand AKG177 (H-Tyr-D-Ala-Gly-NMePhe-4-Apac-Pro-Leu-Trp-NH-Bn(3ʹ,5ʹ-(CF3)2)) having a
relatively rigid linker (i.e. 4-Apac) with balanced agonist activity at MOR and DOR while maintaining high antagonist activity
at NK1 receptor.
23. Temporally coordinated presynaptic inhibition by enkephalin and GABA gates primary afferent input in the
spinal cord dorsal horn
Amaury Francois1, Elizabeth I Sypek1, Adam Hantman2, Grégory Scherrer1
1Department of Anesthesiology, Perioperative and Pain Medicine, Department of Molecular and Cellular Physiology,
Department of Neurosurgery, Stanford Neurosciences Institute, Stanford University, Palo Alto, CA, USA; 2Janelia Farm
Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Enkephalins are endogenous peptidergic agonists that can bind to both delta and mu opioid receptors (DOR and MOR)
to regulate neurotransmission. Here we combined mouse genetics, neuroanatomy, optogenetics and slice
electrophysiology to resolve the mechanism of action of enkephalins in the spinal cord. We generated knockin mice inwhich the Cre recombinase is expressed by enkephalinergic neurons (Penk-Cre mice) to identify and manipulate theseneurons. In situ hybridization experiments confirmed Cre activity in neurons expressing enkephalins. We first resolved theidentity of spinal enkephalinergic neurons in laminae I-III and found that they represent a heterogeneous population of
GABAergic and glutamatergic interneurons. Furthermore, enkephalins were concentrated in axons and presynaptic buttonsof these Penk-Cre+ interneurons, which formed axo-axonic synapses with primary afferents. We then expressed channel
rhodopsin-2 in Penk-Cre+ neurons to elucidate how release of enkephalins at these synapses modulates
neurotransmission between somatosensory and spinal neurons. Strikingly, we found that light-evoked depolarization of
enkephalinergic neurons caused a long-lasting depression in glutamate release that followed GABA-mediated presynaptic
inhibition. This enkephalinergic presynaptic inhibition is highly selective and topographically organized as it requires thepresence of MOR on terminals of afferents projecting to lamina I-IIo and of DOR on afferents projecting to laminae IIi-III.
Altogether our results uncover a mechanism by which the coordinate action of GABA and enkephalins provide immediate
but sustained presynaptic inhibition to gate primary afferent input in the spinal cord dorsal horn. Support by NIH/NIDA

26

(DA031777, G.S.) and Stanford Dean’s Fellowship (A.F.) None of the authors have conflicts of interest.
24. GPR171, the receptor for hypothalamic neuropeptide BigLEN, is involved in the regulation of stress andreward-related behaviors
Ivone Gomes1, Erin N. Bobeck1, Wakako Fujita1,2, and Lakshmi A. Devi1
1Department of Pharmacology and System Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY, USA,
2Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki City, Japan.
Neuropeptidomic studies show that proSAAS derived peptides are abundantly present in the hypothalamus andenriched in agouti related peptide (AgRP) neurons of the arcuate nucleus. We recently identified BigLEN, a proSAAS
derived peptide, as an endogenous agonist of GPR171. Lentiviral shRNA-mediated knockdown of GPR171 in the
hypothalamus indicates receptor involvement in modulation of food intake and metabolism. In order to explore the role of
GPR171 in vivo non-peptidic ligands, both agonists and antagonists, are needed. Therefore, we generated a homology
model of GPR171 based on the crystal structure of a phylogenetically related receptor, and virtually screened an in-housesmall-molecule library. This led to the identification of MS0015203 as an agonist and MS0021570 as an antagonist.
Biochemical characterization demonstrated that these compounds bind to and exert their effects selectively via GPR171.
Administration of MS0015203 increases receptor activation in the paraventricular nucleus and food intake; this is not seenin mice with knockdown of GPR171 in the ventral hypothalamus. Interestingly the antagonist, MS0021570, blocks theincreases in food intake seen following selective activation of AgRP neurons that would lead to release of BigLEN.
Immunohistochemical studies show that, in addition to hypothalamus, GPR171 is expressed in the basolateral amygdala.
We find that GPR171 knockdown in this brain region not only prevents the acquisition of fear memories but also ofmorphine conditioned place preference. Together, these results show an involvement of GPR171 in stress and rewardrelated behaviors. This work was supported in part by NIH grants DA008863 and NS026880 (to L.A.D.) and E.N.B. is
supported by NIDA training grant (DA007135).
25. Casein kinase- 1 epsilon deletion enhances opioid reward and is associated with increased striatal Oprm1 andNpas4 expression
Lisa R. Goldberg1,2, Neema Yazdani1-3, Stacey L. Kirkpatrick1, Olga Lacki1, W. Evan Johnson4, and Camron D.
Bryant1
1Laboratory of Addiction Genetics, Department of Pharmacology and Experimental Therapeutics and Psychiatry, Boston
University School of Medicine; 2Biomolecular Pharmacology Training Program, Department of Pharmacology andExperimental Therapeutics; 3Transformative Training Program in Addiction Science, Department of Pharmacology andExperimental Therapeutics, Boston University School of Medicine; 4 Department of Medicine, Division of Computational
Biomedicine, Boston University School of Medicine
Genetic and pharmacological studies indicate that Casein kinase-1 epsilon (Csnk1e) contributes to psychostimulant,
opioid, and ethanol behaviors. Pharmacological inhibition of CSNK1E enhanced psychomotor sensitivity to the selective
mu-opioid receptor agonist fentanyl, indicating a negative regulatory role in drug behavior. Here, we tested the hypothesisthat Csnk1e negatively regulates fentanyl reward using the conditioned place preference assay (CPP) in Csnk1e knockout(KO) and wild-type (WT) mice. KOs showed a leftward shift in the inverted u-shaped curve for opioid reward versus WTs,
exhibiting enhanced reward at lower doses (0.05 mg/kg) and decreased reward at higher doses (0.2 mg/kg). No significant
differences were observed in fentanyl analgesia in the 52.5°C hot plate assay (0-0.4 mg/kg) or naloxone conditioned place
aversion (4 mg/kg), suggesting a neural mechanism selective for dopaminergic reward circuitry. To generate novel
hypotheses regarding the molecular mechanisms that mediate enhanced opioid reward in Csnk1e KOs, we usedtranscriptome analysis via mRNA sequencing of striatum from naïve KO and WT mice to identify the transcription factor
Npas4 as the top hit (2.2-fold increase in expression; p=4.96 x10-136), supporting Npas4 transcript covariance withmorphine reward (Piechota et al., 2010). Importantly, expression of Oprm1 (mu-opioid receptor) was higher in KOs
compared to WTs (1.7-fold increase; p=2.14x10-4). The activity of one of the top upstream regulators identified by
Ingenuity Pathway Analysis, STAT3, is negatively regulated by CSNK1E and Stat3 has previously been shown to regulate
Oprm1 expression. We conclude that Csnk1e deletion enhances opioid reward, possibly via a STAT3-mediated increase in
mu-opioid receptor expression. FUNDING SOURCES: R00DA029635 (NIDA; C.D.B.), T32GM008541, HG005692(W.E.J.), Transformative Training Program in Addiction Science (Burroughs Wellcome 9550300872). None of the authors
has a conflict of interest related to this research.
26. Development of stable dynorphinA analogues at the bradykinin 2 receptor for the treatment of neuropathic
painSara M. Hall1, Yeon Sun Lee1, Cyf Nadine Ramos Colón1, Frank Porreca2, Josephine Lai2, Victor J. Hruby1
Departments of 1Biochemistry and Chemistry, 2Pharmacology, University of Arizona, Tucson, Arizona, U.S.A.
Current treatments for neuropathic pain involve opioids, non-steroidal anti-inflammatory drugs (NSAIDS), andanticonvulsants. Many of these treatments are highly efficacious for acute pain but are not very effective in neuropathic
pain and have serious side effects caused by long-term administration. Treatment for this disease is difficult withconventional methods, partly because the mechanism of this disease is not well known. One target for neuropathic pain27

treatment may be the blockade of Dynorphin A (Dyn A). Experimental evidence has shown that des-tyrosine fragments ofdynorphin A can interact with bradykinin 2 receptors (B2R) to activate calcium influx through a non-opioid mechanism. This
neuronal excitatory effect is proposed to underlie the pronociceptive actions of spinal Dyn A. Therefore, the development of
B2R antagonists can be used to block Dyn A which can lead to novel therapeutics for pain management. We previously
discovered a minimum pharmacophore for the B2R that was found to reverse thermal hyperalgesia and mechanicalhypersensitivity when administered intrathecally in nerve-injured rats (spinal nerve ligation). Based on its structure, thispharmacophore is susceptible to degradation by peptidases. In an effort to improve the peptide stability, modified Dyn A
analogs were designed. These modifications showed improved stability and gave us further insight into the interaction of
Dyn A analogs with the B2R. This research was supported by grants from the National Institute on Drug Abuse of the NIH(P01DA006284 and R01DA013449). SMH was supported by a Marshall Dissertation Fellowship. None of the authors havea conflict of interest.
27. Collybollides are highly selective and potent agonists of kappa opioid receptors
Achla Gupta1, Erin N. Bobeck1, Ivone Gomes1, Adrien Cave3, Heidi E. Hamm3, Joseph Parello3, andLakshmi A. Devi1,2
1Department of Pharmacology and System Therapeutics, 2Department of Neuroscience and Friedman Brain Institute,Icahn School of Medicine at Mount Sinai, New York, NY, USA; 3Department of Pharmacology, Vanderbilt University
School of Medicine, Nashville, TN, USA
Ligands selective for the κ opioid receptor (κOR), both agonists and antagonists, have a potential to be developed astherapeutics in the treatment of psychiatric and neurological disorders as well as obesity. Salvinorin A, a non-nitrogenous
diterpene derived from the hallucinogenic mint Salvia divinorum, is a highly selective and potent κOR agonist. The
structure of Salvinorin A has a furyl-δ-lactone motif; this motif is also present in collybolides, sesquiterpenes extracted fromthe fungus Collybia maculata. We therefore examined whether collybolide and its sesquiterpene diastereoisomer, 9-epicollybolide function as opioid receptor ligands. Competition binding assays as well as functional and trafficking assays
carried out with human embryonic kidney cells expressing either μ, δ or κ opioid receptors show that collybolide, and to alesser extent 9-epi-collybolide, is a highly selective κ opioid receptor agonist. Interestingly, a hemisynthetic epimer of
collybolide at the level of its furan group, 7-epi-collybolide, exhibits inverse agonist activity. Taken together our results
demonstrate that collybolides represent novel κOR agonists that could be developed as therapeutics in the treatment ofdrug addiction, mood disorders or pain. This work was supported in part by NIH grants DA008863 and NS026880 (toLAD.) and E.N.B. is supported by NIDA training grant (DA007135).
28. AMPA receptor positive allosteric modulators (PAMs) attenuate opioid tolerance, dependence, and opioid
induced-hyperalgesia
Xiaoyu Hu1, Rui Wang1, Xuebi Tian1, Jia Zhou3, Zaijie Jim Wang1
1Department of Biopharmaceutical Sciences and Cancer Center, University of Illinois, Chicago, IL, USA; 3Department ofPharmacology and Toxicology and Center for Addiction Research, University of Texas Medical Branch, Galveston, TX,
USA
Development of opioid tolerance, dependence, and paradoxical hyperalgesia hinders the use of opioids for thetreatment of chronic pain. It is reported that antagonists of a-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA)
receptors attenuate morphine tolerance and dependence. In this study, we found that positive allosteric modulators (PAMs)of AMPA receptors can also attenuate acute morphine antinociceptive tolerance and physical dependence. In mice treatedwith morphine (100mg/kg s.c.), acute morphine tolerance and dependence developed in 4-6 h. Treatment with aniracetam,an AMPA PAM, was able to prevent the development of morphine antinociceptive tolerance and physical dependence(86.1 ± 4.9% v.s. 16.7 ± 5.1% MPE, p<0.001 compared with morphine tolerance group). Aniracetam also partially reversed
the established acute morphine tolerance and dependence in mice (57.1 ± 12.6% MPE, p<0.01). In addition, Aniracetam
was effective in blocking established opioid-induced hyperalgesia (0.62 ± 0.08g vs. 0.03 ± 0.02g; p<0.001 for mechanicalallodynia and 10.36 ± 0.82s v.s. 4.95 ± 0.34s; p<0.001 for thermal hyperalgesia, compared with chronic morphine group).
These data suggest that positive allosteric modulators of AMPA receptors can attenuate morphine tolerance, dependence,and opioid-induced hyperalgesia.
29. Presence of spontaneous sensory hypersensitivity in mice lacking OPRM1
Ying He, Rui Wang, Jonathan Nazari, Zaijie Jim Wang
Department of Biopharmaceutical Sciences and Cancer Center, University of Illinois, Chicago, IL USA
The μ opioid receptor (MOR) is necessary to mediate the analgesic and addictive effects of opioids. Taking advantageof the MOR knockout mouse model, this study aimed to examine the functional involvement of MOR in the endogenous
pain modulation circuits, by characterizing spontaneous and evoked pain behaviors in mice lacking the functional µ opioid
receptor gene (OPRM1). We found MOR-null mice did not produce significant changes to the locomotor coordination in therotarod test. Both the wild-type and MOR-null mice exhibited normal activities and movements in open field exploration
test. MOR-null mice displayed nociceptive responses to innocuous mechanical and noxious thermal stimuli,
indistinguishable from those in the wild-type littermate mice, indicting the absence of evoked sensory hypersensitivity in28MOR-null mice. We previously validated a method of employing negative reinforcement to study spontaneous pain in miceand applied the conditioned place preference (CPP) paradigm in the present study to investigate spontaneous pain in
MOR-null mice. After a single trial conditioning, (clonidine: 1 µg in 5 µL of saline, i.t.), MOR-null mice spent significantly
more time in clonidine-paired chambers than in saline-paired chambers. In contrast, clonidine did not elicit CPP in the wildtype control mice. These data suggest the presence of stimulus-independent spontaneous pain in the MOR-null mice,
supporting a physiological role of the endogenous opioid system in tonic pain suppression. None of the authors has aconflict of interest related to this research.

30. The involvement of free fatty acid receptor GPR40/FFA1 signaling on the development of chronic pain
Kazuo Nakamoto1, Takashi Nishinaka1, Aizawa Fuka1, Mitsumasa Mankura2 , Yutaka Koyama3, Fumiyo Kasuya4 andShogo Tokuyama1
1Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kobe Gakuin University; 2Faculty of Food Culture,
Kurashiki Sakuyo University; 3Laboratory of Pharmacology, Faculty of Pharmacy, Osaka Ohtani University; 4Biochemical
Toxicology Laboratory, Faculty of Pharmaceutical Sciences, Kobe Gakuin University
We have demonstrated that the activation of the G protein-coupled receptor 40/free fatty acid receptor 1(GPR40/FFA1) signaling pathway may play an important role in the regulation of the descending pain control system. Here,
we examined the involvement of supraspinal GPR40/FFA1 signaling in the development of chronic pain. We used acomplete Freund's adjuvant (CFA)-induced inflammatory chronic pain and postoperative pain mouse model. Mechanicalallodynia and thermal hyperalgesia were evaluated using von Frey filaments and plantar test, respectively. Long-lasting
hyperplasia of paw, persistent mechanical allodynia and thermal hyperalgesia were elicited in CFA-treated mice. The
intracerebroventricular (i.c.v.) injection of docosahexaenoic acid (DHA) (50 µg) and GW9508 (1.0 µg), a GPR40/FFA1
agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection.
These effects were inhibited by the i.c.v. pretreatment with GW1100 (10 µg), a GPR40/FFA1 antagonist. Furthermore, The
repeated GW1100 treated mice significantly increased phosphorylated ERK (p-ERK) in the spinal cord after low thresholdtouch stimulation, and exacerbated postoperative pain-induced mechanical allodynia compared to vehicle treated mice.
Our findings suggest that the activation of the supraspinal GPR40/FFA1 signaling might provide valuable information
regarding a novel therapeutic approach for pain control.
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.
31. Enhancement of opioid-mediated antinociception by the mu-opioid receptor positive allosteric modulator,
BMS-986122.
Todd Hillhouse, James Hallahan, Kathryn Livingston, Claire Meurice, Neil Burford, Andrew Alt and John Traynor
Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA and Bristol-Myers Squibb Company,
Wallingford, CT, USA
We recently described positive allosteric modulators (PAMs) of the mu-opioid receptor. Such compounds bind to a siteon the mu-receptor that is distinct from the orthosteric site for endogenous opioid peptides and opioid drugs. Mu-receptor
PAMs could be analgesics themselves and/or enhance the action of traditional opioid agonists and thereby reduce thelevel of opioid drug required to afford pain relief. In cells expressing the mu-receptor, BMS-986122 alone did not activate
GTPgamma(35)S binding or recruit beta-arrestin. However, BMS-986122 enhanced the binding affinity and/or potency of
mu-opioid receptor orthosteric agonists in a probe-dependent manner with the most robust increase in affinity being seen
with R(-)-methadone; BMS-986122 also showed a robust increase in the potency of R(-)-methadone to stimulate
GTPgamma(35)S binding in mouse brain homogenates. Consequently, we sought to examine the effect of BMS-986122 invivo. Using the hot-plate assay we observed that BMS-986122 given intracerebroventricularly (i.c.v.) caused a substantial
increase in the antinociceptive activity of R(-)-methadone given i.c.v. or intraperitoneally. At higher doses BMS-986122
alone (i.c.v.) afforded a short-lived antinociception that was completely prevented by pretreatment of the mice withnaloxone or the selective mu-antagonist beta-FNA. In addition, BMS-986122 promoted swim-stress induced
antinociception. These studies are the first to demonstrate that mu-opioid receptor PAMs enhance the antinociceptive
activity of opioid drugs and suggest the PAMs may also increase endogenous opioid peptide activity. Supported by the
National Institute on Drug Abuse of the National Institutes of Health (T32 DA07268). The authors report no conflict ofinterest.
32. Exploring the contribution of the anterior cingulate cortex in opioid-sensitive descending pain modulatory
circuitry
Lusine Gomtsian1, Edita Navratilova1, Alec Okun1, Chaoling Qu1, Xu Yue1, Dong Lu1, Janice Oyarzo2, Dagoberto
Robles1 and Frank Porreca1
1Department of Medical Pharmacology, University of Arizona, Tucson, AZ, USA; 2Mayo Clinic, Scottsdale, AZ, USA
Neuropathic pain is a complex, multidimensional phenomenon consisting of sensory and affective components.
Opioids such as morphine are effective for moderate to severe pain, but have many harmful side effects. There is a29

growing, urgent medical and societal need to elucidate the mechanisms and opioid-sensitive circuits by which pain andpain relief is modulated. Cortical regions may contribute to descending pathways modulating pain through projections to
the peri-aqueductal gray (PAG) and the rostral ventromedial medulla (RVM). Here, we explore the effect of morphine
injection at different regions of the ACC and its effect on tactile hypersensitivity in a rodent model of neuropathic pain.
Adult, male Sprague-Dawley rats received nerve injury by ligation of the L5/L6 spinal nerves (SNL injury). Evoked tactile
hypersensitivity was evaluated following ACC and RVM morphine injections. Rats with SNL developed tactile
hypersensitivity that was not reversed by morphine injection into various region of the ACC. In contrast, morphine injectionin the RVM showed increased evoked thresholds in SNL animals, as demonstrated previously. We have shown that sites
in the rACC can modulate the affective components of neuropathic pain without altering the sensory component. The
overarching goal of this research is to understand the nature of the neuroanatomical contributions of the ACC and its rolein descending pain modulatory pathways. Further studies exploring other sites within the ACC may ultimately lead to abetter understanding of opioid-sensitive circuitry. Supported by DA034975. The authors have no conflict of interest.
33. Accelerating basic research and drug discovery (from hundreds of compounds to trillions!)
Richard Houghten, Torrey Pines Institute for Molecular Studies
Over the past 25 years, Torrey Pines Institute has generated very large mixture-based peptide combinatorial libraries(10s of thousands to millions, billions and even trillions of compounds) that are systematically arranged allowing for thescreening of these libraries in virtually any assay that is capable of low to medium throughput. Essentially, these libraries
allow for high throughput screening of millions of compounds using an exponentially lower number of samples required tobe screened. The Torrey Pines platform technology of highly diverse libraries and deconvolution strategies, have beenutilized in dozens of in vitro assays to produce highly potent and specific individual compounds. More recently, theselibraries have been screened directly in disease-relevant animal models with the hypothesis that this approach will yield
more “advanced” therapeutic candidates, decreasing both the time and costs inherent in the drug discovery process. Our
libraries are now being used in a robust program that blankets the State of Florida in a program known as the “Florida Drug
Discovery Acceleration Program”. Examples of the successes we have had in pain modulation will be described in detail.
34. Impact of benzodiazepine use on retention and compliance, relapse, and safety in buprenorphine-maintained
patients: A Literature Review.
Darrow Khosh-Chashm M.D. Department of Psychiatry & Behavioral Sciences, University of Texas Health Science Center– Houston, Houston, TX USA
Benzodiazepine use is prevalent among buprenorphine-maintained patients. Our objective was to assess the impact ofbenzodiazepine use on compliance, retention, resolution, and safety in patients being treated with buprenorphine for opioid
use disorders. We searched Ovid MEDLINE PubMed, OneSearch, and Science Direct. We did not use any date or
language restrictions in the electronic searches for trials. We included all studies pertaining to buprenorphine withconcurrent benzodiazepine use published between January 2002 and March 2015. The primary outcomes werecompliance with treatment and relapse to illicit opioid use. Our secondary outcome was safety. We included 7 trials in thisreview; 4 trials were conducted in the USA, 1 in Norway, 1 in Finland, and 1 in England. Patients actively using
benzodiazepines relapsed more often although one study contradicted these findings. There was a statistically significant
increase in patients who tested positive for benzodiazepines and non-compliance with treatment. For issues of safety, we
found that lethal overdose would be unexpected to occur in the context of controlled oral co-administration of therapeutic
dosages of benzodiazepines and buprenorphine. Our findings for buprenorphine and concurrent IV benzodiazepine use
was consistent with previous literature with a more than three-fold increased odds of accidental injury compared to those
without a benzodiazepine prescription, with the greatest odds of injury among females. Benzodiazepine use was also
associated with slowed sensory processing and psychomotor impairment. Our review confirms that benzodiazepine use
among buprenorphine-maintained patients is associated with a greater risk of relapse, poor compliance, and safetyconcerns. There was no conflict of interest related to this research.
35. Interleukin-1 signaling in the basolateral amygdala mediates heroin-conditioned immunosuppression
Lee. W. Hutson, Christina L. Lebonville, & Donald T. Lysle
Department of Psychology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Studies in our laboratory have demonstrated that the immunosuppressive properties of heroin can be conditioned to a
specific environmental context (CS), resulting in a previously neutral stimulus eliciting immunosuppressive effects.
Moreover, we have shown that the nucleus accumbens shell (NAcS) and basolateral amygdala (BLA) are important neuralsubstrates that mediate this effect. Recently, we began investigating the mechanisms that mediate heroin-conditioned
immunosuppression within these regions. Cytokines have been traditionally thought of as peripheral immune molecules,
but, in fact, they also serve as neuromodulators in the brain. For example, studies suggest that central interleukin-1 (IL-1)
has an important modulatory role in associative learning. The present study investigated the role of IL-1 signaling within the
BLA and NAcS in the expression of heroin-conditioned immunosuppression. Rats received five, 60-minute conditioning
sessions, during which heroin was administered prior to placement into the CS. On test day, rats received microinfusionsof interleukin-1 receptor antagonist (IL-1Ra) or saline into the BLA or NAcS 30 minutes prior to CS re-exposure. Following

30

re-exposure, rats received systemic lipopolysaccharide treatment to induce an immune response. Our findings
demonstrate that bilateral administration of IL-1Ra into the BLA, but not the NAcS, attenuated nitrate/nitrite levels in bloodplasma and expression of inducible nitric oxide synthase in spleen tissue following re-exposure to the CS, indicating IL-1
signaling in the BLA, but not the NAcS, plays an important role in heroin-conditioned immunosuppression. Supported by
the National Institute on Drug Abuse (DA034721). None of the authors have a conflict of interest related to this research.
36. Blockade of dopamine D3 receptor by YQA14 attenuates methamphetamine-induced behavioral sensitizationand conditioned place preference in mice
Rui Song1, Li Sun1, 2, Ying Chen1, Ri-Fang Yang1, Ning Wu1*, Rui-Bin Su1, Jin Li1*
1Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, China; 2Departments of Anesthesiology, General
Hospital of Beijing Military Command, Beijing, 100700, China
Drug addiction is characterized by high rate of relapses to drug use after abstinence. The neural mechanisms
underlying high vulnerability to relapse are incompletely understood. We have recently reported that blockade of dopamine(DA) D3 receptors by YQA14 attenuates cocaine reward and relapse to drug-seeking. In the present study, we investigatedwhether YQA14 similarly inhibits methamphetamine (METH)-induced locomotor sensitization and conditioned place
preference (CPP) in mice. Here we report that chronic administration or a single injection of YQA14 (6.25, 12.5 and 25
mg/kg, i.p., 20 min prior to METH) significantly inhibited the acquisition and expression of METH-induced locomotor
sensitization. In contrast, chronic administration of YQA14 did not alter the acquisition, while a single injection of YQA14
significantly attenuated the expression of METH-induced CPP. In addition, chronic administration of YQA14 also facilitatedextinction and decreased the reactivation of METH-induced CPP. These findings suggest that brain D3 receptors are
critically involved in METH’s rewarding and psychomotor-stimulating effects. Thus, YQA14 deserves further studies as apotential medication for METH addiction. Support by Natural Science Foundation of China (Grant No.81102425), Project of
National Science and Technology Support Program in China (2012BAI01B07), Beijing Nova Program xx2014A014, Keyproject of Natural Science Foundation of Beijing (7131010) and the National Basic Research Program of China (Grant No.
2009CB522008). None of the authors has a conflict of interest related to this research.
37. An opioid agonist/NK1 antagonist inhibits nociception in an animal model of neuropathic pain while lacking
positive reinforcement.
Alexander J. Sandweiss1, Josh Stark1, Tally M. Largent-Milnes1, and Todd W. Vanderah1
1Medical Pharmacology, University of Arizona, Tucson, AZ
Chronic pain affects approximately 100 million Americans. Opioids are the mainstay therapy for the treatment ofchronic pain. While physicians and patients alike are apprehensive about using opioids due to their side effects including
respiratory depression and addiction, 259 million opioid prescriptions were written in 2012. Although opioids are the best
available analgesics, they increase both positive and negative reinforcement, ultimately leading to addiction. The pronociceptive neurotransmitter substance p can potentiate positive reinforcement, suggesting that neurokinin-1 receptors(NK1R) located on dopaminergic neurons originating in the ventral tegmental area (VTA) of the midbrain and projecting to
the nucleus accumbens (NAc) may play a role in opioid reward. Here, we show that a multivalent pharmacophore, TY032,
acting as both an opioid receptor agonist and NK1R antagonist inhibits preclinical neuropathic pain without increasing
extracellular dopamine in the nucleus accumbens. As tested by an infrared (IR) thermal nociception assay and von Freyfilament probing, intrathecal administration of TY032 dose dependently attenuates thermal hypersensitivity and mechanicalallodynia for 3 hours post injection. TY032 microinjection into the VTA did not increase extracellular dopamine release inNAc dialysate. These data indicate dual targeting of the dopamine reward circuitry may be an effective therapy with limited
abuse liability warranting further investigation into how substance p signaling affects opioid induced reward in the VTA.
Research supported by University of Arizona departmental funds. No conflict of interest exists.
38. Chronic pain modulates the molecular function of the opioid-reward network
Naoko Kuzumaki1 , Michiko Narita1 and Minoru Narita1,2
1 Dept. of Pharmacol., Hoshi Univ. Sch. of Pharm. and Pharmaceut. Sci., Tokyo, Japan
2 Life Science Tokyo Advanced Research Center (L-StaR), Tokyo, Japan
Pain is a multidimensional experience with both sensory-discriminative and motivational-affective components. The
mesolimbic dopaminergic system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens (NAc),
is a crucial mediator of the reinforcing effects of μ-agonists. We previously demonstrated that the release of dopamine in
the NAc after morphine treatment was markedly suppressed by sciatic nerve ligation. miRNAs are small, noncoding RNA
molecules that direct the post-transcriptional suppression of gene expression and play an important role in regulating
synaptic plasticity. In the present study, we found that sciatic nerve ligation induced a drastic decrease in the expression of
miR200b and miR429 in NAc neurons. Under the tonic activation or suppression of VTA-NAc dopamine neurons by an
optogenetic technique, the expression of miR200b and miR429 in the NAc was altered. Along with these changes inmiRNAs, the expression of DNA methyltransferase 3a (DNMT3a), which is one of the predicted targets of miR200b/429,
was significantly increased in the NAc after sciatic nerve ligation. To identify the methylated gene that is associated withthis increase in DNMT3a, MBD2-seq was performed in the NAc after sciatic nerve ligation. As a result, we found some31

candidate genes that were decreased and highly methylated. They included genes for intracellular enzymes that are
related to the cAMP signaling pathway. These findings suggest that chronic nociceptive stimuli induce an epigenetic/posttranscriptional modification along with an attenuation of the “opioid-associated motivation/valuation circuitry”.
39. U50,488H requires truncated 6 transmembrane variants of the mu opioid receptor for analgesia but not side
effects
Gina F. Marrone, Steven G. Grinnell, Grace C. Rossi, Valerie LeRouzic, Zhigang Lu, Susruta Majumdar, Ying-Xian Pan,
Gavril W. Pasternak
Molecular Pharmacology and Chemistry Program, Department of Neurology, Memorial Sloan Kettering Cancer Center,
1275 York Avenue, New York, NY 10065 (USA).
The mu opioid receptor (MOR) contains two independent promoters associated with exon 1 and exon 11. Most exon
11 variants are truncated 6 transmembrane (TM) receptors, lacking the first TM domain encoded by exon 1. We previously
reported that certain mu analgesics and the novel opioid IBNtxA require exon 11 variants for analgesia. Here, we reportthat the kappa opioid receptor selective analgesic U50,488H also requires exon 11 variants for analgesia, but not otheractions. In contrast to wildtype controls, U50,488H failed to elicit significant antinociceptive responses in mice lacking exon
11 variants (E11 KO) following systemic, supraspinal, and spinal administration. Restoring expression of the 6TM variantmMOR-1G via lentivirus injection either spinally (i.t.) or supraspinally (i.c.v.) rescued U50,488H analgesia, confirming a roleof this 6TM variant in its analgesia. Aversion and hypolocomotion are prototypical side effects of kappa agonists. Unlike
analgesia, the conditioned place aversion and suppression of locomotor activity produced by U50,488H in wildtype mice
were unchanged in E11 KO mice. Together, our findings demonstrate that U50,488H analgesia is dependent upon atruncated 6TM mu receptor variant and that this analgesia is separable from prototypical KOR side effects in an exon 11dependent manner.
40. Analysis of changes in the expression of opioid- and dopamine-related genes in Parkinson’s disease specificiPS cells derived-dopaminergic neuron
Yukari Suda1, Naoko Kuzumaki1,2, Michiko Narita1, Daigo ikegami1, Katsuhide Igarashi3, Makoto Suematsu4, Nobutaka
Hattori5, Hideyuki Okano2,3, and Minoru Narita1,3
1Dept. Pharmacol., Hoshi Univ. Sch. Pharm., Tokyo, Japan; 2Dept. Physiol., Keio Univ. Sch. Med., Tokyo, Japan; 3Life
Science Tokyo Advanced Research Center (L-StaR), Tokyo, Japan; 4Dept. Biochem., Keio Univ. Sch. Med., Tokyo, Japan;
5Dept. Neurol., Juntendo Univ. Grad. Sch. Med., Tokyo, Japan
iPS cells (iPSCs) are pluripotent cells which give rise to all cells in the organism. A potential solution is to utilize
reprogramming technology to generate disease-specific iPSCs. iPSC derived from patients with Parkinson’s disease canthen be differentiated to neurons for disease modelling. In the present study, we generated iPSCs from two Parkinson’s
patients and two control subjects. All of the clones differentiated into neurons included tyrosine hydroxylase-positive
neurons. Under the present condition, we performed the comprehensive gene expression analysis of dopaminergic
neurons derived from control iPSCs or Parkinson’s disease specific-iPSCs. We found significant differences in theexpression of several dopamine-related genes between control and patients. Among those, the expression level of COMT
was dramatically increased in Parkinson’s disease specific iPSC-derived dopaminergic neurons. Unlike dopamine-related
genes, expression levels of opioid-related genes, such as μ-opioid receptor (OPRM1), δ-opioid receptor (OPRD1), κ-opioid
receptor (OPRK1), proopiomelanocortin (POMC), prodynorphin (PDYN) and proenkephalin (PENK) were not changed in
Parkinson’s disease specific iPSC-derived dopaminergic neurons. Subsequently, we profiled DNA methylation in
Parkinson’s disease specific iPSCs and control iPSC-derived dopaminergic neurons using the Illumina Infinium
HumanMethylation 450 BeadChips. We found differences in pattern of DNA methylation at the transcriptional start sites
with CpG island of dopamine-, but not opioid-, related genes in Parkinson’s disease specific iPSC-derived dopaminergic
neurons. These findings suggest that change in the expression of dopamine-related genes with epigenetic modification in
Parkinson’s disease specific-iPSC-derived dopaminergic neurons could lead to neuronal dysfunction in Parkinson’s
disease.
41. Crucial role of Κ-opioidergic system for tumor angiogenesis
Yusuke Hamada1, Kohei Yamamizu2,3, Michiko Narita1, Naoko Kuzumaki1, Mitsuaki Yamazaki4, Hiroshi Nagase5, Jun K.
Yamashita2,3, Minoru Narita1,61Dept. Pharamcol., Hoshi Univ. Sch. Pharm. Pharmaceut. Sci., Tokyo, Japan; 2Department of Cell Growth andDifferentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan; 3Laboratory of Stem
Cell Differentiation, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; 4Department of Anesthesiology, Toyama University School of Medicine, Toyama, Japan; 5Department of Medicinal
Chemistry, International Institute for Integrative Sleep Medicine (IIIS), University of Tsukuba, Tsukuba, Japan; 6Life
Science Tokyo Advanced Research Center (L-StaR), Tokyo, Japan
Opioids are effective analgesics for the management of moderate to severe cancer pain. However, little is known about
the role of opioid system in tumor angiogenesis. The tumor angiogenesis is required for tumor progression with the highly
expression of several activators such as VEGF. In the previous study, we demonstrated that the Κ opioid receptor agonist

32

could act as the novel endogenous angiogenesis inhibitor in vascular development. In the present study, we investigatedwhether Κ opioid receptor system could inhibit tumor angiogenesis in the process of tumor growth. We confirmed thattreatment with Κ opioid receptor agonists, U50,488H and nalfurafine (TRK-820), significantly inhibited human umbilical vein
endothelial cell (HUVEC) migration and tube formation by suppressing VEGFR2 expression. In contrast, treatment with a µ
opioid receptor agonist DAMGO or a δ opioid receptor agonist SNC80 failed to prevent angiogenesis in HUVECs.
Furthermore, lewis lung carcinoma or B16 melanoma grafted in Κ opioid receptor knockout mice and prodynorphin (PDYN)
knockout mice increased proliferation and remarkably enhanced tumor angiogenesis. Repeated intraperitoneal injection of
nalfurafine (0.1–10 mg/kg, b.i.d.) significantly inhibited tumor growth by suppressing tumor angiogenesis. These findings
indicate that Κ opioidergic system may be involved in tumor angiogenesis. This knowledge could lead to a novel strategy
for cancer therapy.
42. Endogenous opioid peptides and functional selectivity at the κ-opioid receptor
Keith Olson1, Justin Lavigne2, John Streicher2, Frank Porreca3, Victor J. Hruby1
1Department of Chemistry and Biochemistry, University of Arizona; 2 Department of Pharmacology, University of New
England; 3 Department of Pharmacology, University of Arizona
The opioidergic system – composed of the µ,δ, Κ and NOP receptor subtypes and over 20 neuropeptides – modulates
numerous animal behaviors including pain, analgesia, dysphoria, addiction and anxiety. Over the past 30+ years,
pharmacologists discovered that 1) endogenous neuropeptides display low receptor selectivity between receptor subtypes,
and 2) exogenous ligands can preferentially activate certain signaling pathways over others at the same receptor – aphenomenon defined as functional selectivity. However, the unique function of the numerous endogenous opioid peptides
at a given receptor remains unclear. These studies investigate whether endogenous opioid peptides display functional
selectivity. Our data demonstrate enkephalins and dynorphins differentially regulate two ubiquitous signaling modules – βarrestin and G-protein – at the ΚOR. Dynorphin A and Dynorphin B swap potency rank orders for β-arrestin2 recruitment
and GTPγS signaling, indicating two distinct signaling platforms are formed. Based on previously explored regulatory rolesfor β-arrestin and G-protein, further investigation probed whether receptor regulation was differentially regulated by the
dynorphins and enkephalins. Dynorphin A and enkephalin treatment but not Dynorphin B treatment simulated JNK –aknown regulatory kinase that can be scaffolded by β-arrestin. Furthermore, internalization studies showed additionalchanges in potency – where Dynorphin B stimulated more robust internalization than Dynorphin A. Thus, these in vitro
assays show dynorphins and enkephalins differentially regulate signals at the ΚOR receptor. Observing distinct signaling
and regulatory of Dynorphin A, Dynorphin B and the enkephalins in vivo is the major future goal of this work.
43. Changes in VTA-MOR function associated with opioid reward by neuropathic pain and chronic treatment withethanol
Masahiro Shibasaki1, Naoko Kuzumaki1, Michiko Narita1, Katsuhide Igarashi2, Tomohisa mori1, Tsutomu Suzuki3, Minoru
Narita1,2
1Dept. of Pharmacol., Hoshi Univ. Sch. of Pharm. and Pharmaceut. Sci., Tokyo, Japan; 2Life Science Tokyo Advanced
Research Center (L-StaR), Tokyo, Japan; 3Addiction Research laboratory, Hoshi Univ. Sch. of Pharm. and Pharmaceut.
Sci., Tokyo, Japan
Morphine causes psychological dependence via activation of μ-opioid receptors (MOR) in the ventral tegmental area
(VTA). However, μ-opioid receptor function is able to be changed under many of pathological conditions. In the present
study, we investigated a possible change of VTA-MOR function and opioid reward in neuropathic pain and ethanoldependent models. As a result, the morphine-induced place preference was significantly suppressed by sciatic nerve
ligation, whereas it was enhanced by chronic treatment with ethanol. Using [35S] GTPgS binding assay, MOR-mediatedG-protein activation in the VTA was significantly suppressed by sciatic nerve injury, while it was enhanced by chronic
treatment with ethanol. These results suggest that alteration of VTA-MOR function by several pathological conditions may
modulate the development of morphine dependence.
Support by part by Grants-in-Aid for Scientific Research from the Ministry of Health, Labor and Welfare, and the Ministry of
Education, Culture, Sports, Science and Technology of Japan. None of the authors has a conflict of interest related to thisresearch.
44. Nalmefene: G-protein biased partial agonist of the kappa opioid receptor
Joshua Hillman, Amelia Dunn, Eduardo Butelman, Brian Reed, and Mary Jeanne Kreek
Laboratory of the Biology of Addictive Diseases, Rockefeller University, New York, NY, USA
In vitro pharmacological studies of G-protein coupled receptor (GPCR) ligands have largely focused on G-protein
mediated signaling endpoints (e.g. cyclic AMP levels). Recent indications that GPCR signaling can also proceed via nonG-protein mediated pathways, particularly β-arrestin mediated pathways, have advanced our understanding of ligand
mediated signaling. Reexamination of the molecular pharmacology of receptor ligands in light of this advance is warranted.
We previously demonstrated that nalmefene exhibits partial agonism of the kappa opioid receptor (KOP-r), utilizing
prolactin as a biomarker of kappa agonist activity in vivo and GTP-gamma-S binding stimulation in vitro (Bart et al., 2005,

33

Neuropsychopharm., 30:2254-62), while also exhibiting antagonism of the mu (MOP-r) opioid receptors. Nalmefene has
recently been approved for use in the European Union for treatment of alcoholism; both the antagonism of MOP-r and thepartial agonism of KOP-r may contribute to its efficacy. We have compared the effects of nalmefene on KOP-r signaling via
downstream G-protein mediated signaling (GTP-gamma-S binding) and arrestin recruitment. Consistent with our prior
results, nalmefene exhibited partial agonism of stimulation of GTP-gamma-S binding. In contrast, investigation of liganddirected beta-arrestin recruitment to KOP-r demonstrated that nalmefene functions as an antagonist, with minimal betaarrestin recruitment, and full blockade of U69,593 arrestin signaling. Understanding the functional consequences of thedifferential signaling properties of nalmefene via KOP-r in relation to its in vivo effects will require further exploration.
The authors have no conflicts of interest. These studies were supported by the Robertson Development Fund and theMiriam and Sheldon Adelson Medical Research Foundation.

45. Involvement of the mesolimbic dopaminergic pathway in morphine-induced analgesia
Moe Watanabe1, Akira Yamashita2, Michiko Narita1, Yusuke Hamada1, Daigo Ikegami1, Naoko Kuzumaki1, Akihiro
Yamanaka2, Minoru Narita1, 3
1Dept. Pharmacol., Hoshi Univ. Sch. of Pharm. and Pharmaceut. Sci., Tokyo, Japan; 2Dept. Neurosci. II, RIEM, Nagoya
Univ. Aichi, Japan; 3Life Science Tokyo Advanced research center (L-StaR), Tokyo, Japan
Morphine is frequently used for the treatment of cancer pain and moderate to severe non-cancer pain. It has been well
documented that morphine analgesia is produced by activating descending pain modulatory system in brain stem anddecreasing release of pain transmitter in the spinal dorsal horn. On the other hand, morphine induces rewarding behavior
through the activation of the ventral tegmental area (VTA) dopamine neurons. VTA is the origin of the dopaminergic cellbodies of the mesolimbic dopamine pathway and is widely implicated in the reward, motivation and pleasure. VTA
dopamine neuron projects to limbic structures, most prominently the nucleus accumbens (N.Acc.). The recent studies haveshown that the reduction of neuronal activity in the N.Acc. under the neuropathic pain contributes to pain transmission. In
this study, we investigated the role of the mesolimbic dopaminergic system in morphine-induced analgesia using by
optogenetics and designer receptors exclusively activated by designer drugs (DREADD) system. We generated thetransgenic mice expressing enhanced natronomonas pharaonis halorhodopsin (eNpHR) or Gi-coupled human muscarinic
M4 DREADD (hM4Di) protein under the control of the tyrosine hydroxylase promoter. The eNpHR is activated by yellow
light illumination and leading to neuronal inhibition. The hM4Di is activated by designer drug, clozapine N-oxide (CNO) andleading to neuronal inhibition. Optical suppression of VTA dopamine neurons significantly inhibited morphine-induced
analgesia. As well as the result of optogenetics, CNO-induced suppression of VTA dopamine neurons also inhibitedmorphine-induced analgesia. These findings suggest that mesolimbic dopaminergic pathway may, at least contribute tomorphine-induced analgesia.
46. Development of a combination Heroin-HIV vaccine
Rashmi Jalah1,2, Oscar B. Torres1,2, Fuying Li3,4, Joshua F. G. Antoline3,4, Zoltan Beck1,2, Arthur E. Jacobson3,4,
Kenner C. Rice3,4, Carl R. Alving1, Gary R. Matyas1
1US Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring MD; 2U.S. Military HIV
Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD; 3Drug Design
and Synthesis Section, National Institute on Drug Abuse and 4National Institute on Alcohol Abuse and Alcoholism, National
Institutes of Health, Bethesda, MD, USA
There is a high prevalence of HIV-1 infection in injection drug users. We are developing a combination heroin-HIV
vaccine to address this global health problem. The heroin component of the vaccine was developed by conjugating achemically stable heroin/morphine hapten (MorHap) to tetanus toxoid (TT). To demonstrate the feasibility of a combination
heroin-HIV vaccine, the TT-MorHap conjugate was mixed with a recombinant HIV envelope antigen, CN54 gp140.
Alternatively, MorHap was directly conjugated to gp140 at varying hapten densities of 20-60 MorHap/gp140 molecule. Mice
were immunized with 3 doses every 3 weeks using formulations containing these vaccines mixed with liposomes
containing monophosphoryl lipid A as an adjuvant. The magnitude and durability of serum antibodies and inhibition ofheroin-induced antinociception after subcutaneous heroin (1 mg/kg) challenge was assayed. The combination vaccines
elicited high titer and durable antibodies to MorHap (~1 mg/mL) comparable to that elicited by TT-MorHap alone.
Antibodies to HIV Env for the TT-MorHap+gp140 mixed vaccine and gp140-MorHap conjugate with low MorHap density of
20 were comparable to that obtained by gp140 alone (~3 mg/mL). The Env antibodies decreased for gp140-MorHap
conjugates with higher MorHap densities. All mice immunized with the dual vaccines showed inhibition of heroin-induced
antinociception with a % maximal potential effect (%MPE) ranging from 15-40, while MorHap-TT immunized animals had a%MPE of 8. The most promising formulations for a combination heroin-HIV vaccine were TT-MorHap mixed withunconjugated gp140 or gp140-MorHap with low MorHap density. Both induced high antibody titers to gp140 and protected
mice from heroin challenge.
47. Polymorphisms in the mu opioid receptor (OPRM1) and monoamine genes associate with pain in sickle celldisease34

Ellie H. Jhun1, Yingwei Yao2, Ying He1, Robert E. Molokie1, 3, 4, A. Kyle Mack5, Diana J. Wilkie2, Zaijie Jim Wang1
1Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago (UIC), IL; 2Departmentof Biobehavioral Health Science, College of Nursing, UIC; 3Division of Hematology/Oncology, College of Medicine, UIC,
60612; 4Comprehensive Sickle Cell Center, University of Illinois Hospital and Health Sciences System, Chicago, IL.
5Northwestern University-Feinberg School of Medicine, Division of Pediatric Hematology/Oncology/Stem Cell
Transplantation, Children’s Memorial Hospital, Chicago, IL, USA.
Pain is pervasive throughout a sickle cell disease (SCD) patient’s life and effective pain management is suboptimal.
SCD pain is heterogeneous and identifying the mechanisms that underlie this variation may aid in new therapeuticadvances and management. As opioid analgesics are the mainstay in adult SCD pain therapy, candidate polymorphisms
in the OPRM1 and monoamine genes have been collected for our study. Adult and pediatric sickle cell patients wererecruited during routine outpatient clinic visits where blood and buccal swab samples were collected for DNA extraction
and genotyping. Association studies are performed for candidate gene polymorphisms by using phenotype data gatheredfrom PAINReportIt®, a pain assessment tool. Composite pain index (CPI) score is a marker for baseline pain andrepresents the multidimensional pain experience. Utilization is defined as admissions to the emergency department and/or
the acute care center resulting from a sickle cell pain crisis and is a surrogate marker for acute pain in SCD. We foundassociations of acute and chronic SCD pain with SNPs in OPRM1 and monoamine genes including dopamine receptor D3(DRD3), adrenergic receptor B2 (ADRB2) and catechol-O-methyltransferase (COMT). Rs1799971 (Asn40Asp) G allele isassociated with an increase in utilization rate (p=0.003) in an additive negative binomial regression analysis including
covariates (ethnicity, CPI, sickle cell genotype, age, sex). Polymorphisms in the mu opioid receptor and monoamine
neurotransmitter system genes may help to elucidate the variability seen in pain and opioid therapy in SCD and aid in thedevelopment of precision medicine.
48. Truncated six transmembrane mu opioid receptors mediate opioid analgesia
Jin Xu1, Zhigang Lu1, Grace C. Rossi2, Susruta Majumdar1, Gavril W. Pasternak1, Ying-Xian Pan1
1 Department of Neurology and the Molecular Pharmacology and Chemistry 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.
Potent opioid analgesics lacking side-effects may be possible by targeting truncated six transmembrane (TM) mu
receptors generated from alternative splicing of the mu opioid receptor (Oprm1) gene. IBNtxA (3-iodobenzoyl-6βnaltrexamide) is a potent analgesic against thermal, inflammatory and neuropathic pain and fails to produce the typical side
effects associated with traditional opiates. IBNtxA analgesia is lost in an exon 11 knockout mouse lacking a set of 6TM
truncated mu receptors, suggesting it acts through the 6TM truncated mu receptors. In the current study we demonstratethat truncated 6TM splice variants, including mMOR-1G, mMOR-1M and mMOR-1N, can rescue IBNtxA analgesia in acomplete mu opioid receptor knockout mouse lacking all Oprm1 splice variants due to disruptions of both exons 1 and 11.
No mu opioids were active in these mice. A lentivirus containing the 6TM variant mMOR-1G or mMOR-1M or mMOR-1N
administered intrathecally restored IBNtxA, but not morphine, analgesia, confirming that the truncated 6TM variants areboth necessary and sufficient for IBNtxA analgesia. RT-PCR and immunohistochemistry confirmed expression of thelentivirus in the spinal cord. The lentivirus expressing mMOR-1G also restored 125I-IBNtxA binding to the 6TM-associatedtargets. Interestingly, U50,488H analgesia was also lost in our complete MOR-1 KO mouse, and rescued by the 6TMvariants, suggesting involvement of the 6TM variants in U50,488H actions. Furthermore, we demonstrated thatbuprenorphine analgesia was mediated through both truncated 6TM and full-length 7TM mu receptors by the lentivirus
studies in both complete MOR-1 KO and exon 11 KO mice. (Supported by DA13997 & DA029244 (Y.-X.P) and DA02615& DA07242 (G.W.P) from the National Institute on Drug Abuse; and a core grant CA08748 from the National Cancer
Institute to MSKCC)
49. Involvement of AMPA receptors in the antidepressant-like effects of the delta opioid receptor agonist SNC80
Aaron M Chadderdon and Emily M Jutkiewicz
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI
Delta-opioid receptor agonists produce rapid antidepressant-like effects in a number of animal models. However, themechanisms underlying these behavioral effects are unknown. The present study investigated the role of glutamatergic
AMPA receptors in the antidepressant-like effects of the delta opioid agonist SNC80. The AMPA receptor antagonist 2,3dihycdroxy-6-nitro-7-sulfoamoylbenzo(f)quinoxaline (NBQX) (0, 10, 18 mg/kg i.p.) was administered 15 min prior to SNC80(s.c.). SNC80-induced antidepressant-like effects were evaluated 60 min later in the forced swim test in rats. In addition,
locomotor activity immediately following SNC80 administration was measured with and without NBQX pretreatment.
SNC80 significantly decreased immobility in the forced swim test, indicating an antidepressant-like effect. These rapid
antidepressant-like effects of SNC80 were attenuated by NBQX in a dose-dependent manner. In addition, SNC80stimulated locomotor activity was partially blocked by NBQX. These findings suggest that downstream AMPA receptor
activation may play an important role in the antidepressant-like effects of SNC80 as well as other behavioral effects. Thispotential mechanism of action is consistent with mechanisms thought to be important in the fast-acting antidepressanteffects of ketamine. Future studies will probe other mediators involved in this pathway, such as changes in glutamate

35

receptor ratios and mTOR signaling. The authors have no conflicts of interest and this work was supported by start-up
funds to EM Jutkiewicz.
50. Novel mixed-action mu-opioid receptor (MOR) agonist/delta-opioid receptor (DOR) antagonist that preventsstress-induced reinstatement of extinguished cocaine-seeking behavior
Kristen A. Hymel1,2, Shainnel O. Eans,1,2 Michelle L. Ganno,1 Elisa Mizrachi,1 Nicolette Ross,1,3 Sanjeewa N.
Senadheera,3 Jane V. Aldrich,2,3 Jay P. McLaughlin1,2
1Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL; 2Univ. of Florida, Gainesville, FL; 3Univ. of Kansas,
Lawrence, KS
We hypothesized that a mixed-action MOR agonist/DOR antagonist would produce analgesia with reduced liabilities,
and prevent reinstatement of extinguished drug-seeking behavior. Accordingly, we examined two mixed opioid
agonist/antagonists, (-)pentazocine and a novel cyclic peptide 1, in C57BL/6J and MOR -/- mice for opioid efficacy andselectivity in the 55oC warm-water tail-withdrawal assay, and determined each compounds’ effects on the reinstatement ofcocaine-seeking behavior in a conditioned place preference (CPP) assay. (-)Pentazocine and 1 demonstrated full efficacy
in the tail-withdrawal assay, with ED50 (and 95% CI) values of 3.3 (2.8-3.9) and 20.2 (15.1-25.8) nmol, i.c.v., respectively.
Compound 1 also produced significant antinociception after oral administration (18.0 (12.7-28.8) mg/kg, p.o.). Whereas ()pentazocine antinociception was mediated by all three opioid receptors, the antinociception produced by 1 was primarily
MOR mediated. Pretreatment (>2 h, i.c.v.) with either 1 or (-)pentazocine selectively antagonized DOR antinociception.
Repeated treatment with 1 or (-)pentazocine did not result in significant acute opioid antinociceptive tolerance. Acutepretreatment with 1 (30 or 100 nmol, i.c.v.) produced conditioned place aversion, whereas higher doses of (-)pentazocine
produced CPP in C57BL/6J (but not MOR -/-) mice. However, after pretreatment (>2 h), both compounds dosedependently prevented stress-induced reinstatement in C57BL/6J mice. These data suggest the therapeutic value of DOR
antagonists may expand beyond the treatment of alcoholism. Moreover, with its distinct opioid activity profile, the cyclic
peptide 1 is a promising therapeutic for development as both an analgesic with reduced liabilities and as a maintenancemedication to reduce relapse to cocaine-seeking behavior. Funding provided by NIDA (DA018832 and DA032928) and theState of Florida, Executive Office of the Governor’s Office of Tourism, Trade, and Economic Development.
51. Assessment of morphine antinociception in male and female rats on a novel pain-suppressed wheel-runningassay
Ram Kandasamy1, Jonas J. Calsbeek2, Michael M. Morgan1,2
1Graduate Program in Neuroscience, Washington State University, Pullman, WA, USA; 2Department of Psychology,
Washington State University Vancouver, Vancouver, WA, USA
Although nociception in animals is typically assessed with an acute noxious stimulus, such pain-evoked tests do not
mimic the disruptive effects of pain on daily life functions. Our objective was to use suppression of wheel-running in rats asa model of pain-suppressed behavior in humans. Although a number of other pain-suppressed tests have been developed,
suppression of wheel-running is expected to be a useful model because wheel-running is a natural rodent behavior,assessment takes place in the rat’s home cage (a low-stress environment), and measurements can be assessed 24 hours
a day (which reflects the human pain experience). Importantly, there have been no studies examining females in painsuppressed tests, despite the greater prevalence of chronic pain in women. Thus, we studied the effect of peripheral
inflammation induced by intraplantar administration of Complete Freund’s Adjuvant (CFA) on voluntary wheel-running inmale and female rats. Both male and female rats reach stable levels of running within 4 days, although female rats ran
significantly more than male rats. Administration of CFA into the right hindpaw suppressed wheel-running in both sexes,
but running recovered faster in male compared to female rats. Administration of morphine (0.32-10 mg/kg, s.c.) had both
sedative and antinociceptive effects that varied depending on the dose, time post-injection, and sex of the rat. These data
indicate that suppression of wheel-running may be a clinically relevant method to assess nociception. Support provided by
NIH/NIDA DA027625. Authors report no conflict of interest.
52. Development of a high-sensitivity method for the detection of human exposure to fentanyl and six fentanyl
analogs in urine and plasma using liquid chromatography mass spectrometry
Pearl Kaplan1, Rebecca L. Shaner2, Elizabeth I. Hamelin2, and Rudolph C. Johnson2
1ORISE Fellow, Centers for Disease Control and Prevention, 4770 Buford Highway, Atlanta, GA 30341
2Emergency Response Branch, Centers for Disease Control and Prevention, 4770 Buford Highway, Atlanta, GA 30341
Increasing reports of fentanyl abuse highlight the need for sensitive and specific test methods to detect human
exposure to this class of potent analgesics. During pain management sensitive and rapid test methods are required toaccurately measure fentanyls, and in cases of overdose and poisoning highly specific tests are needed to determine theexact fentanyl analog of exposure. A highly-sensitive method for the analysis of fentanyl, six analogs, and five metabolites,in both human urine and plasma was developed and validated using liquid chromatography tandem mass spectrometry
and a single sample preparation approach. The high-sensitivity nature of this method is well suited to quantitate
concentrations of fentanyls relating to both clinical and abuse scenarios. The limits of detection of target analytes in thevalidated method in urine and plasma range from 0.011 to 0.0038 ng/mL. Simulated patient and overdose samples were36

analyzed to evaluate the performance of the method. This high-sensitivity method uses a single sample preparation
technique for urine and plasma and is able to distinguish between multiple fentanyl compounds, which is required inpoisoning cases when an initial positive test warrants confirmatory analysis to determine the exact fentanyl analog of
exposure. Research was supported by the Centers for Disease Control and Prevention and the Oak Ridge Institute for
Science and Education. None of the authors has a conflict of interest related to this research.
53. Structural insights into function and pharmacology of the δ-Opioid receptor ligands
Vsevolod Katritch1, Gustavo Fenalti3,*, Peter W. Schiller4, Bryan L. Roth5,6, Vadim Cherezov2 and Raymond C.
Stevens1,2
Departments of 1Biological Sciences and 2Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA
90089, USA; 3Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla,
CA 92037, USA; 4Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, Montreal,
Quebec, Canada H2W 1R7; 5National Institute of Mental Health Psychoactive Drug Screening Program, and 6Departmentof Pharmacology, University of North Carolina Chapel Hill Medical School, Chapel Hill, NC, USA; *Current address:
Celgene Corporation, San Diego, CA 92121 USA
The breakthroughs in crystallography of G protein-coupled receptors have led to 3D structure determination for all four
Opioid receptors (ORs). Among them, the structure of δ-opioid receptor in complex with naltrindole has been solved at
highest resolution (1.8 Å), revealing not only atomic details of ligand interactions, but also description of tightly boundwaters and the conserved sodium binding site at the center of 7TM (seven-transmembrane) helical bundle. Most recently,
serial femtosecond crystallography technology also helped to determine the structure of human δ-OR bound to the bifunctional δ-OR antagonist and µ-OR agonist tetrapeptide DIPP-NH2. Ligands with such bi-functional µ-OR and δ-OR
profiles are considered as potential therapeutic alternatives to alkaloid opiate analgesics with diminished side effects.
Further computational analysis of these δ-OR complexes and other ORs helps to reveal structural basis for subtype
selectivity, allosteric modulation, functional and pharmacological profiles of these receptors, providing a path to structurebased discovery of improved analgesics with reduced side effects. These studies are supported by the National Institute on
Drug Abuse of the NIH grants DA038858 (V.K.), DA035764 (V.C., V.K., B.L.R. and R.C.S.) and DA004443 (P.W.S.),
National Institute of General Medical Sciences grants U54 GM094618 (R.C.S., V.C., V.K.) and R01 GM108635 (V.C.), andthe US National Institute of Mental Health Psychoactive Drug Screening Program (B.L.R.). None of the authors has aconflict of interest related to this research.
54. Preclinical development of Salvinorin A analogues for the treatment of drug-addiction
Bronwyn M. Kivell 1, Amy WM. Ewald1, David Young1, and Thomas E. Prisinzano2
1School of Biological Sciences, Centre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand.
2Department of Medicinal Chemistry, University of Kansas, Kansas, USA.
There are currently no effective FDA approved treatments for psychostimulant abuse. Although drugs targeting the
dopamine system have some anti-addictive properties, they also have unwanted side effects that limit their therapeuticuse. Activation of the kappa opioid receptor (KOPr), which regulates the function of the dopamine system, may hold the
key to designing better therapeutic compounds to treat addiction. Utilising the novel neoclerodane diterpene structure of
Salvinorin A, we have identified two novel, long acting, selective KOPr agonists, 16-bromo-Salvinorin A and mesylSalvinorin B. Male rats trained to self-administer cocaine were subjected to extinction followed by acute administration
KOPr agonist and drug-prime reinstatement testing performed. Bromo-Salvinorin A (1 mg/kg), and mesyl-Salvinorin B (0.3mg/kg) significantly attenuated cocaine-seeking. We also show these compounds have reduced side effects compared to
traditional KOPr agonists and do not cause sedation in locomotor tests, conditioned place aversion (mesyl-Salvinorin B) or
depression in the forced swim test (bromo-Salvinorin A) or anxiety in the light/dark test and elevated plus maze (bromoSalvinorin A, mesyl-Salvinorin B). Characterisation of these new ligands is expected to lead to new medications to treat
drug dependence, as well as, new molecular tools for chemical biology. By linking analogue design with anti-addiction
effects we will be able to generate better tolerated anti-addiction compounds. Support by the Neurological Foundation of
New Zealand (BMK). AE received support from Victoria University of Wellington Postgraduate Scholarship. None of theauthors has a conflict of interest related to this research.
55. Loss of morphine reward and dependence in mice lacking G protein–coupled receptor kinase 5
Andrea Kliewer1, Laura Glück1, Anastasia Loktev1, Lionel Moulédous2, Catherine Mollereau2, Ping-Yee Law3, and Stefan
Schulz1
1Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany;
2Institut de Pharmacologie et de Biologie Structurale, CNRS/Université de Toulouse, Toulouse Cedex, France;
3Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota.
The clinical benefits of opioid drugs are counteracted by the development of tolerance and addiction. We provide invivo evidence for the involvement of G protein–coupled receptor kinases (GRKs) in opioid dependence in addition to theirroles in agonist selective mu-opioid receptor (MOR) phosphorylation. In vivo MOR phosphorylation was examined byimmunoprecipitation and nanoflow liquid chromatography–tandem mass spectrometry analysis. Using the hot-plate and37

conditioned place preference test, we investigated opioid-related antinociception and reward effects in mice lacking GRK3
or GRK5. Etonitazene and fentanyl stimulated the in vivo phosphorylation of multiple carboxyl-terminal phosphate acceptor
sites, including threonine 370, serine 375, and threonine 379, which was predominantly mediated by GRK3. By contrast,
morphine promoted a selective phosphorylation of serine 375 that was predominantly mediated by GRK5. In contrast to
GRK3 knockout mice, GRK5 knockout mice exhibited reduced antinociceptive responses after morphine administrationand developed morphine tolerance similar to wild-type mice but fewer signs of physical dependence. Also, morphine wasineffective in inducing conditioned place preference in GRK5 knockout mice, whereas cocaine conditioned place
preference was retained. However, the reward properties of morphine were evident in knock-in mice expressing aphosphorylation-deficient S375A mutation of the MOR. These findings show for the first time that MOR phosphorylation isregulated by agonist-selective recruitment of distinct GRK isoforms that influence different opioid-related behaviors.
Modulation of GRK5 function could serve as a new approach for preventing addiction to opioids, while maintaining theanalgesic properties of opioid drugs at an effective level. Support by Deutsche Forschungsgemeinschaft Grant Nos.
SCHU924/11-2 and SCHU924/15-1 and National Institute on Drug Abuse Grant No. 1R01DA031442-01A1. None of theauthors has a conflict of interest related to this research.

56. Chronic junk food consumption induces aberrant cue-induced food-seeking in rats.
Alisa R. Kosheleff, Jingwen Zhou, Jennifer Hsueh, Andrew Le, Kevin Quizon, Sean B. Ostlund, Nigel T. Maidment, Niall P.
MurphyThe pervasiveness of highly-palatable, energy-dense foods is considered a leading cause of obesity, which is asignificant public health concern. While the idea that food is addictive is under debate, overeating shares many
characteristics with drug addiction, such as compulsive pursuit in the face of dire health consequences. Cues associatedwith palatable foods (e.g., images, odors) can trigger food-seeking, even when sated, which could lead to decreased
inhibitory control over food intake. Here, we explored if a junk food diet (i.e., “cafeteria diet;” CD) impacts the ability ofanimals to respond appropriately to food-paired cues in a Pavlovian-to-instrumental transfer test. Male rats were firsttrained to press a lever to receive a food reward, subsequently they learned to associate free delivery of the reward with anauditory cue. Rats were then exposed to either normal chow (Control rats) or chow and CD for either 2 hrs (Binge rats) or
24 hrs (All-Day rats) per day, for 1, 3 or 6 weeks. After this, all rats were switched to 2 hrs-per-day chow access forapproximately 1 week prior to testing. At test, rats were sated for 1 hour on chow, and presented with a food-paired (CS+)
and neutral (CSo) cue, and lever presses and food cup entries recorded. Control rats increased lever pressing and foodcup entries at the onset of the CS+, but not the CSo, as expected. Binge rats increased lever pressing to both the CS+ and
the CSo, though food cup entries increased only during the CS+. All-Day rats showed markedly reduced lever pressing andfood cup entries to both cues. These results suggest that chronic junk food consumption induces atypical responding toenvironmental stimuli predictive of food rewards, and that different dietary access produces unique behavioral
abnormalities. Specifically, Binge rats generalize the excitatory properties of reward-paired cues to other, neutral cues,
while All-Day rats appear insensitive to the motivational properties of either cue. Together these studies emphasize thatjunk food diets induce aberrant food-seeking in response to environmental cues, and this action may contribute tomaladaptive feeding behavior leading to obesity.
57. Tachykinin-opioid chimeric peptide in acute pain treatment in Wistar rats
Piotr Kosson1, Anna Kosson 1, Aleksandra Misicka1,2, Andrzej W. Lipkowski1
1Mossakowski Medical Research Centre Polish Academy of Science, Warsaw, Poland; 2Faculty of Chemistry, Biologicaland Chemical Research Centre, University of Warsaw, Warsaw, Poland
Morphine and its derivatives are the most widely used narcotic analgesics for relieving severe acute pain.
Unfortunately, the use of opioids in treating is limited due to significant side effects e.g. analgesic tolerance, addiction,
respiratory depression. Numerous endogenous components that participate in the formation, transmission, modulation andperception of pain signals offer new strategies for the development of new analgesic. Substance P (SP) is one of this, theundecapeptide Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 is widely distributed throughout the central nervous
system and is highly expressed on areas that are critical for the regulation of pain influx, affective behavior and stress. For
activation of the receptor neurokinin-1 (NK-1) the C-terminal sequence of SP is essential. However, not all the effects of SP
are mediated through the C-terminal fragment. N-terminal fragment, such SP(1-7) induces antinociception anddesensitization of several SP-induced behaviors. SP(1-7) has been suggested to modulate the expression of opiate
tolerance and withdrawal behaviors in rodents. In the present study, we have synthesized the chimeric compoundcontaining two fragments N- and C-terminal functional domains of the endogenous opioid and the tachykinin SP,
respectively. In our study we have shown that the relative balance of activity between opioid and tachykinin
pharmacophores can produce different effects (pro-nociceptive, antinociceptive, or neutral). The presentation will focus on
the way from an idea through synthesis to the application of the new tachykinin-opioid chimeric compound (AWL 3106) inacute pain treatment. Acknowledgments: This work was supported by the European Union Grant NormoLife3858. Biological activity of peptides with both, opioid agonist and tachykinin antagonist components
Jolanta Dyniewicz1, Piotr Kosson1, Aleksandra Misicka1,2, Andrzej W. Lipkowski1
1Mossakowski Medical Research Centre Polish Academy of Science, Warsaw, Poland
1,2Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland
Bi- or multifunctional drugs may have much improved potency due to synergic effect or may produce fewer side effects
than compounds acting at a single target. Substance P plays an important role in pain signals generation and transmission
from periphery to CNS. In contrast, opioids suppress pain signals mainly through suppression of substance P release.
These mechanisms of neurophysiological actions were fundamentals for invention of hybrid compounds that act as both
antagonists of substance P to reduce postsynaptic activation of NK1 receptors as well as opioid agonists to activatepresynaptic opioid receptors that result in decrease substance P release. We developed a new chimeric analgesics inwhich opioid pharmacophore is covalently hybridized with tachykinin pharmacophore that positively modulate effects of the
opioid part. Synergistic enhancement of opioid analgesia and/or decrease of unwanted side-effects should result from such
hybridization. Therefore, to the list of already synthesized and characterized compounds presented in the literature weelaborated new series of compounds that combine peptide opioid pharmacophore (biphalin) with 3,5 bis-trifluoromethylbenzyl derivative, responsible for antagonist at NK1 receptor. Opioid and tachykinin pharmacophores are connected by few
kinds of linkers which are derivative of hydrazine. We will present pharmacological and analgesic properties of new
bifunctional peptides, which exhibit affinity to opioid (µ and δ) and NK1 receptors. Acknowledgement: This work was
supported by the Polish National Science Center (NCN) DEC-2011/03/N/ST5/04725.
59. Amphipathic non-opioid dynorphin a analogs to inhibit neuroexcitatory effects at central bradykinin receptors
Yeon Sun Lee1, Sara M. Hall1, Cyf Ramos-Colon1, Michael Remesic1, David Rankin2, Todd W. Vanderah2, Frank
Porreca2, Josephine Lai2, Victor J. Hruby1
1Department of Chemistry and Biochemistry; 2Department of Pharmacology, The University of Arizona, Tucson, AZ
85721, USA
Nerve injury and inflammation cause up-regulation of dynorphin A (Dyn A), an endogenous opioid ligand, in the spinal
cord resulting in hyperalgesia via the interaction with bradykinin receptors (BRs).1 This is a non-opioid neuroexcitatory
effect that cannot be blocked by opioid antagonists. Our systematic structure-activity relationships study on Dyn A
identified a lead ligand, LYS1044: [des-Arg7]-Dyn A-(4-11), along with the key structural feature (i.e. amphipathicity) for theBRs.2 Our lead ligand showed antihyperalgesic effect in nerve injured animals and inhibited non-opioid Dyn A-induced
neuroexcitatory effects in naïve animals. The inhibitory effects may be localized to the CNS and thus there would be little
cardiovascular effects in the periphery. In an effort to improve the metabolic stability of our lead ligand with retained
biological activities, various modifications were performed and as the results, we were able to develop very stable Dyn A
analogues that possess high therapeutic potential for pathological pain states via a novel mechanism of BRs in the CNS.
60. Mu opioid receptors hyperpolarize respiratory-controlling Kölliker-Fuse neurons
Erica S Levitt1, Ana P Abdala2, Julian FR Paton2, John M Bissonnette3 and John T Williams1
1Vollum Institute, Oregon Health & Science University, Portland, OR, USA; 2School of Physiology and Pharmacology,
University of Bristol, Bristol, UK; 3Department of Obstetrics and Gynecology, Oregon Health & Science University,
Portland, OR, USA
Respiratory depression is the primary cause of death from opioid overdose. Opioid-induced respiratory effects include
aspiration and difficulty swallowing, suggesting impairment of the upper airways. The pontine Kölliker-Fuse (KF) controls
upper airway patency and regulates respiration, in particular the inspiratory/expiratory phase transition. Given theimportance of the KF in coordinating respiratory pattern, the mechanisms of mu opioid receptor activation in this nucleus
were investigated at the systems and cellular level. Injection of the mu opioid agonist DAMGO into the KF of anesthetizedvagi-intact rats resulted in a decrease in respiratory drive. DAMGO applied directly into the KF of the in situ arteriallyperfused working heart-brainstem preparation of rat resulted in robust apneusis. Both of these effects were rapidly
reversed by the opioid antagonist naloxone. In brain slice preparations, activation of mu opioid receptors on KF neurons
hyperpolarized a distinct population (61 %) of neurons. In voltage-clamp recordings the outward current produced by theopioid agonist [Met5]enkephalin (ME) was concentration-dependent, reversed at the potassium equilibrium potential andwas blocked by BaCl2, characteristics of a G protein-coupled inwardly rectifying potassium (GIRK) conductance. The
clinically used drug morphine produced an outward current in KF neurons with similar potency to morphine-mediatedcurrents in locus coeruleus brain slice preparations. Thus, the population of KF neurons that are hyperpolarized by mu
opioid agonists are likely mediators of the opioid-induced loss of post-inspiration and induction of apneusis. Supported by
DA08163 (JTW) and DA38069 (ESL). The authors have no conflict of interest to declare.

39

61. GABAergic transmission and reduced modulation by endocannabinoids in adult rat rostral ventromedial
medulla (RVM) neurons following persistent inflammation
Ming-Hua Li, Katherine L. Suchland, and Susan L. Ingram
Department of Neurological Surgery, Oregon Health & Science University, Portland, OR USA 97239
Chronic pain is a major health issue that affects ~ 20% of the population worldwide and costs >$500 billion a year.
RVM neuron activity is altered during persistent inflammation induced by complete Freund’s adjuvant (CFA). Using wholecell patch-clamp recordings from adult rat RVM slices, we find that GABA release in the RVM is significantly increased inrats pretreated with CFA. Endocannabinoids normally inhibit presynaptic GABA release in adult RVM so that superfusion of
the cannabinoid receptor 1 (CB1) selective inhibitor rimonabant (SR141716, 3 μM) increases the frequency of GABAergic
miniature inhibitory postsynaptic currents (mIPSCs) by 88 ± 31%. Interestingly, there is a reduction in endocannabinoid
inhibition of GABA release following CFA treatment. SR141716 increased GABA mIPSC frequency by only 13 ± 9%.
Further, we find that the inhibition of GABA release by the CB receptor agonist Win55,212-2 is reduced in CFA-treated (11± 6%) compared with naïve rats (40 ± 5%). In contrast to cannabinoid modulation of presynaptic GABA release, the muopioid receptor agonist DAMGO (1 µM)-mediated inhibition of GABAergic mIPSC frequency was not different in naïve (52 ±6%) compared to CFA-treated (52 ± 10%) RVM neurons. Increased GABA release in the RVM reduces activation ofdescending pain inhibitory fibers resulting in increased nociceptive transmission from primary afferents. The loss of
endocannabinoid modulation in the RVM may be a key mechanism in the hyperalgesia produced by persistent
inflammation. Supported by NIH (DA027625 and DA035316 SLI) and American Heart Association (13SDG14590005,
MHL). There are no conflicts of interest.
62. The neuroprotective effects of Memantine on methamphetamine-induced cognitive deficits
Liu Yao1, Jiao Dongliang1, Liu Jinggen2, Zhao Min1
1Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2State Key
Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
Abuse of amphetamine-type stimulants, especially methamphetamine (METH), has become a global public healthproblem. Methamphetamine can not only lead to addiction, it can also cause severe neurotoxicity, which is closely
associated with cognitive deficits. The present study was undertaken to understanding the mechanisms of METH-induced
cognitive dysfunction, and determining whether memantine, a non-competitive antagonist of the NMDA receptor, canprevent the METH-induced cognitive deficits induced by METH. By using an object recognition animal model to test thenon-spatial cognitive memory in mice, we found that administration of METH (4 mg/kg ×4, i.p., 2 h interval) abolished longterm memory of mice in recognition task, without affecting short-term memory at 1 week after treatment. We also foundthat METH administration induced neuron apoptosis in the medial prefrontal cortex (mPFC) at 72 h after injection,
manifested as a reduced expression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2). More importantly, METHinduced memory impairment was prevented by pretreatment with memantine (5 mg/kg, p.o.). The present study suggested
that METH-induced cognitive deficits might be related to METH-induced apoptosis in mPFC, and memantine prevented
METH-induced cognitive impairment through an anti-apoptotic mechanism. The effects of memantine on preventing neuron
apoptosis in the mPFC need to be further investigated. Support by the Chinese National Nature Science Foundation(81271468, 81130020), The Ministry of Education (20120073110089), Shanghai city health and family planning committee(2014ZYJB0002), China Postdoctoral Science Foundation (2014M561482). None of the authors has a conflict of interest
related to this research.
63. Role of PDGF receptor and its ligands in nociception in Drosophila
Roger Lopez-Bellido1, Seol Hee Im2, Juyeon Jo2, Patrick Huang2, Michael J Galko2, Howard B Gutstein1
1Department of Anesthesiology, The University of Texas MD Anderson Cancer Center; 2Department of Genetics, The
University of Texas MD Anderson Cancer Center
Recent studies suggest that platelet-derived growth factor receptor-β (PDGFR-β)-mediated signaling plays a key rolein morphine tolerance and resistance to morphine analgesia in neuropathic pain. However, the exact molecular mechanismof the PDGFR downstream signaling in regulating pain is unknown. We took advantage of Drosophila, a highly tractable
genetic model of nociception to investigate whether the PDGFR-like receptor (Pvr) and its PDGF-like ligands (Pvf1, Pvf2,
and Pvf3) regulate nociception. To determine if PDGF regulation of nociception is conserved we assayed thermal andmechanical baseline nociceptive responses in Pvr gain-of-function and Pvr and Pvf loss-of-function conditions. Tissuespecific Pvr hyperactivation and overexpression in neurons required for aversive responses to noxious thermal andmechanical stimuli showed that Pvr can induce thermal and mechanical allodynia and hyperalgesia. Larvae homozygous
for hypomorphic loss of function alleles and transheterozygous for hypomorphic/null alleles of Pvr were hyposensitive tonoxious thermal and mechanical stimuli. Interestingly, neuron-specific RNAi targeting of Pvr led to defects only inmechanical hyperalgesia. Finally, we observed that hypomorphic loss of function alleles of Pvf2, and Pvf3 showed ahyposensitive phenotype to both thermal and mechanical stimuli. These results indicate that PDGFR and its ligands are
key mediators of different nociceptive responses in Drosophila and suggest that PDGFR and its ligands have evolutionarily
related functions between flies and mammals. They also reveal that the ligands and receptor may be acting in different

40

cells types to differentially mediate mechanical and thermal nociceptive responses in Drosophila. NIH/NIDA grant
R01DA36680, H.B.G. The authors declare no conflict of interest.
64. Role of G protein-coupled receptor kinases 2 and 3 in mu-opioid receptor desensitization and internalization.
Janet D. Lowe1, Helen S. Sanderson1, Alexandra E. Cooke1, Mehrnoosh Ostovar2, Elena Tsisanova1, Sarah L. Withey1,
Charles Chavkin3, Stephen M. Husbands2, Eamonn Kelly1, Graeme Henderson1, and Chris P. Bailey2
1School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom; 2Department of Pharmacy andPharmacology, University of Bath, Bath, United Kingdom; 3Department of Pharmacology, University of Washington Schoolof Medicine, Seattle, WA, USA
There is ongoing debate about the role of G protein receptor kinases (GRKs) in agonist-induced desensitization of the
mu-opioid receptor (MOPr) in brain neurons (see Williams et al. 2013, Pharmacol Rev 15;223-254). In the present study
we have used a novel, membrane permeable, small molecule inhibitor of GRK2 and GRK3, Takeda Compound 101(Cmpd101), to study the involvement of GRK2/3 in acute agonist-induced MOPr desensitization. We observed that
Cmpd101 inhibits the desensitization of the G protein activated potassium current (GIRK) evoked by receptor- saturating
concentrations of Met Enkephalin, DAMGO, endomorphin-2 and morphine in rat and mouse locus coeruleus (LC) neurons.
In LC neurons from GRK3 knockout mice Met Enkaphalin-induced desensitization was unaffected implying a role for GRK2
in MOPr desensitization. Quantitative analysis of the loss of MOPr function following acute agonist exposure revealed that
Cmpd101 partially reversed MOPr desensitization. Inhibition of ERK, PKC or JNK did not inhibit the Cmpd101-insensitive
component of desensitization. In HEK 293 cells Cmpd101 produced almost complete inhibition of DAMGO-induced MOPr
phosphorylation at Ser375, arrestin translocation and MOPr internalization. Our data demonstrate a role for GRK2 (andpotentially also GRK3) in agonist-induced MOPr desensitization in the LC, but leave open the possibility that another, as
yet unidentified, mechanism of desensitization also exists. Support from the MRC [MR/J013269/1], BBSRC[BB/J003506/1],
Wellcome Trust Value in People award and USPHS-NIDA grant RO1DA030074. None of the authors has a conflict ofinterest related to this research.
65. Agonist-induced nociception/orphanin FQ peptide receptor phosphorylation revealed by phosphosite-specificantibodies
Anika Mann and Stefan Schulz
Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany
The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor is the most recently discovered and least characterized
member in the opioid receptor family (MOR, KOR and DOR). NOP receptors are widely distributed across tissues andmodulate several physiological processes by its endogenous ligand nociceptin The NOP receptor is a potential target forthe development of ligands with therapeutic use in several pathophysiological states (e.g. chronic and neuropathic pain).
Consequently, there is increasing interest in understanding the molecular regulation of NOP receptor. Recently, we
generated phosphosite-specific antibodies directed against pS351 and pT362/pS363 and a phosphorylation-independent
antibody. First results show that nociception, Ro64-6198, SCH221510 and MCOPPB induce a robust phosphorylation at
both S351 and T362/S363, which can be blocked by the selective antagonist J113397. Buprenorphine andnorbuprenophine failed to induce a phosphorylation at these sites. In the presence of nociceptin, S351 phosphorylation
occurred at a faster rate than phosphorylation of T362/S363 indicating that S351 is the primary site of agonist-dependent
phosphorylation. After activation of PKC by phorbol 12-myristate 13-acetate only S351 but not T362/S363 phosphorylation
is increased indicating that S351 can also undergo heterologous PKC-mediated phosphorylation. Using NOPR-GFP knockin mice, we detected phosphorylation at S351 and T362/S363 in vivo after application of AT202. Together, these data
provide new and quantitativ insight into the molecular regulation of NOP receptors in vivo and in vitro. None oft he authors
has a conflict of interest releated to this research.
66. Heterologous regulation of agonist-independent μ-opioid receptor phosphorylation by protein kinase C.
Susann Illing*, Anika Mann* and Stefan Schulz
Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany;*These authors contributed equally
Homologous agonist-induced phosphorylation of μ-opioid receptor (MOR) is initiated at the carboxyl-terminal S375,
followed by phosphorylation of T370, T376 and T379. In HEK293 cells, this sequential and hierarchical multi-site
phosphorylation is specifically mediated by G-protein coupled receptor kinases 2 and 3. In the present study, we provide
evidence for a selective and dose-dependent phosphorylation of T370 after activation of PKC by phorbol esters. We used a
combination of phospho site-specific antibodies, kinase inhibitors and siRNA knockdown screening to identify kinases thatmediate agonist-independent phosphorylation of MOR. In addition phospho site-specific antibodies were also used to study
constitutive phosphorylation at S363 of MORs in mouse brain. Activation of PKC by phorbol esters or heterologous
activation of substance P receptors co-expressed with MORs in the same cell induced a selective and dose-dependent
phosphorylation of T370 that specifically requires the PKCα isoform. Inhibition of PKC activity did not compromise
homologous agonist-driven T370 phosphorylation. In addition, S363 was constitutively phosphorylated in both cells andmouse brain. Constitutive S363 phosphorylation required ongoing PKC activity. When basal PKC activity was decreased,

41

S363 was also a substrate for homologous agonist-stimulated phosphorylation. Our results have disclosed novel
mechanisms of heterologous regulation of MOR phosphorylation by PKC. These findings represent a useful starting point
for definitive experiments elucidating the exact contribution of PKC-driven MOR phosphorylation to diminished MOR
responsiveness in morphine tolerance and pathological pain. This work was supported by the Deutsche
Forschungsgemeinschaft [SCH924/11-2 and SCH924/15-1]. None of the authors has a conflict of interest related to thisresearch.
67. Pain-induced decrease in motivation is mediated by kappa opioid receptors in the nucleus accumbens
Massaly, N.1, Hipólito, L.1, Sirohi, S.2, Walker, M.2, Bruchas, M.3 & Morón-Concepcion, J.A.1
1Dept. of Anesthesiology, Columbia University Medical Center, New York, NY. 2Dept. of Psychology, Washington State
University, Pullman, WA. 3Dept. of Anesthesiology, Washington University, Saint Louis, MO
During chronic inflammatory pain, the nucleus accumbens (NAc) undergoes long term changes affecting mood andrewarding properties of reinforcers. The opioid system is at least partially involved in these modifications and could explain
the modification of reinforcing effects of natural rewards and drugs of abuse. Kappa opioid receptor (KOR) activation in the
NAc leads to dysphoria and other aversive effects. These receptors also have a role in chronic pain and adaptations in
KOR system could underlie pain-induced changes in motivation. We assessed this hypothesis by microinjecting
norbinaltorphimine (NorBNI) directly into the NAc shell. Motivation to self-administer food was tested 48 hours after
injection of complete Freund’s adjuvant (CFA), using a progressive ratio schedule of reinforcement. We then measured
KOR and prodynorphin expression together with KOR function, using GTPgammaS and neuronal activation, using cfos
marker. Our data reveal an increase in both KOR expression and dynorphin A-stimulated GTPgammaS binding in the NAc
Shell 48 hours after CFA administration. Furthermore we showed that blockade of these receptors reverses the paininduced decrease in motivation for food reward. These results, together with the role of KORs in mediating the negative
effects of long access and chronic exposure to alcohol and drugs of abuse, further support the hypothesis of kappa opioid
system involvement in the suppression of reward/reinforcement circuitry during both addiction and pain processes. This
study is supported by the NIH grants DA027460 and AA020394. None of the authors has a conflict of interest related tothis research.
68. Neuropeptide FF (NPFF)-induced hyperalgesia and acute opioid antinociceptive tolerance is prevented by VBJ192, a dual-activity mu-opioid receptor (MOR) agonist-NPFF receptor antagonist
Jay P. McLaughlin1,2, Shainnel O. Eans,1,2 Jessica M. Medina,1,2 Michelle L. Ganno,1 V. Blair Journigan,3 Stephen J.
Cutler,3 Christopher R. McCurdy3
1Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL; 2Univ. of Florida, Gainesville, FL; 3Univ. of Mississippi,
University, MS
Tolerance limits the analgesic value of MOR agonists. NPFF mediates hyperalgesia and opioid-induced tolerance
through the activation of NPFF1 and 2 receptors. We hypothesized that VBJ-192, a dual-activity MOR agonist/NPFF
receptor antagonist in vitro, would produce antinociception with reduced tolerance. Repeated i.c.v. administration of two
MOR agonists, morphine and DAMGO, was first characterized in a model of acute antinociceptive tolerance using themouse 55oC warm-water tail-withdrawal assay. Whereas morphine demonstrated significant acute antinociceptive
tolerance with a 9.6-fold rightward shift in potency upon a second administration 8 h later, the 2.5-fold rightward shiftobserved with DAMGO was not significant. NPPF-receptor mediation of opioid tolerance was confirmed in tests where
initial co-administration of NPFF (10 nmol) with DAMGO (1.7 nmol) significantly increased the rightward shift in DAMGO
dose-response 9.7-fold. Administration of NPFF alone was sufficient to significantly shift rightward (7.6-fold) theantinociception of DAMGO administered 8 h later. Conversely, initial co-administration of the NPFF-receptor antagonist
RF9 (1 nmol) with morphine (3 nmol) prevented the rightward shift of antinociceptive potency produced by a second
administration of morphine. Although VBJ-192 produced antinociception lasting up to 90 min (with an ED50 value of 6.88
(4.72-9.47) nmol, i.c.v.), it also dose-dependently prevented NPFF-induced hyperalgesia in a 48oC warm-water tailwithdrawal assay for an additional hour. Unlike morphine, VBJ-192 did not reduce respiration, and repeated administration
did not demonstrate significant acute antinociceptive tolerance. Together, these results confirm the mediating effect ofNPFF on opioid tolerance, and suggest the potential of dual-action opioid-NPFF ligands as analgesics with fewer liabilities.
Funding provided by NIDA (DA034777) and the State of Florida, Executive Office of the Governor’s Office of Tourism,
Trade, and Economic Development.
69. Agonist-selective multi-site phosphorylation of the µ-oipoid receptor regulates ß-arrestin recruitment
Elke Miess1 and Stefan Schulz1
1Department of Pharmacology and Toxicology, Jena University Hospital - Friedrich Schiller University Jena, Germany
Opioid drugs are the most potent analgesics, which are used in the clinic; however, by activating the μ-opioid receptor(MOR) they also produce several adverse side effects including constipation, antinociceptive tolerance, and physicaldependence. There is substantial evidence suggesting that G protein-coupled receptor kinases (GRKs) and β-arrestins
play key roles in regulating MOR signaling and responsiveness. Following phosphorylation by GRKs, β-arrestins bind tophosphorylated MORs, which prevents further interactions between the receptor and G proteins even in the continued

42presence of agonist resulting in diminished G protein-mediated signaling. We have previously shown that agonist-induced
phosphorylation of MOR occurs at a conserved 10-residue sequence, 370TREHPSTANT379, in the carboxyl-terminal
cytoplasmic tail. Morphine induces a selective phosphorylation of serine375 (S375) in the middle of this sequence that is
predominantly catalyzed by G protein-coupled receptor kinase 5 (GRK5). By contrast, high-efficacy opioids not only induce
phosphorylation of S375 but also drive higher-order phosphorylation on the flanking residues threonine370 (T370),
threonine376 (T376), and threonine379 (T379) in a hierarchical phosphorylation cascade that specifically requires GRK2/3
isoforms. To investigate this mechanism further, we have adapted a β-galactosidase complementation assay for βarrestin1 and β-arrestin2. Using this assay, we were able to show that activation of MOR by high-efficacy agonists such as
DAMGO results in recruitment of both β-arrestin1 and β-arrestin2, whereas activation by low-efficacy agonists such asmorphine results only in detectable recruitment of β-arrestin2 but not β-arrestin1. The morphine-induced β-arrestin
recruitment was strongly enhanced by overexpression of GRK2 or GRK3. Conversely, siRNA knock down of GRK2 or
GRK3 strongly inhibits DAMGO-induced β-arrestin recruitment. Mutation of S375 to alanine strongly inhibited β-arrestin
recruitment. However, mutation of all 11 carboxyl-terminal serine and threonine residues of MOR was required tocompletely abolish interaction with β-arrestin1 and β-arrestin2. None of the authors has a conflict of interest related to thisresearch.
70. Small-molecule NOP agonists reduce alcohol reward in mouse models of alcoholism
Nurulain T. Zaveri,1 Paul Marquez,2 Michael E. Meyer,1 Abdul Hamid,2 and Kabirullah Lufty2
1Astraea Therapeutics, Mountain View, CA, USA; 2Department of Pharmaceutical Sciences, College of Pharmacy,
Western University of Health Sciences, Pomona, CA, USA
Studies in alcohol-preferring rats suggest that dysregulation of the nociceptin opioid receptor NOP and its ligand
N/OFQ are a pathophysiological consequence of excessive alcohol intake. Further, administration of N/OFQ blocks the
rewarding effects of alcohol in the conditioned place preference (CPP) paradigm in mice, as well as reinstatement of
extinguished alcohol CPP, suggesting that NOP agonists may be a promising approach for treating alcohol addiction. We
report the efficacy of small-molecule NOP agonists in reducing ethanol-induced CPP in mice. Mice tested for baseline
place preference were subjected to three conditioning trials, during which they were treated subcutaneously with vehicle ora NOP agonist, followed by ethanol (2 g/kg) or saline and confined to the vehicle-paired or drug-paired chamber for 15 min.
In the afternoon of each day, mice received the alternative treatment and confined to the opposite chamber for 15 min. On
day 5, mice were tested for place preference. Among the compounds tested, selective NOP agonist AT-312 dosedependently reduced ethanol-induced CPP. AT-312 alone had no motivational effects at the dose at which it blocked
ethanol CPP. The inhibitory effect of the agonists on alcohol reward was completely abolished in mice lacking NOP.
Together, these data suggest that AT-312 reduced alcohol reward via the NOP receptor and was devoid of any
motivational effects. Studies are ongoing to assess effect of NOP agonists in the two-bottle-choice paradigm, which is
widely used as a model of alcohol-taking behaviors and reinforcement. Authors declare no conflicts of interest. Support:
Contract HHSN275201300005C.
71. NPYFa, dualsteric chimeric peptide of met-enkephalin and NPFF, prevents opioid induced tolerance
Annu Mudgal1, 2, Santosh Pasha1, 2*
1Academy of Scientific and Innovative Research (AcSIR), CSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB)
Campus, New Delhi, India; 2Peptide Synthesis Laboratory, CSIR-IGIB, New Delhi, India
Methionine-enkephalin-Arg-Phe (MERF) is a known endogenous amphiactive analgesic peptide. Neuropeptide FF(NPFF) is reported for long lasting analgesia, role in opioid modulation and tolerance development. Based on these reports
a dualsteric chimeric peptide NPYFa (YGGFMKKKPQRFamide) was designed, having Met-enkephalin (opioid) and PQRF
sequence of NPFF at C-terminal which can target both opioids and NPFF receptors. The aim of the present study was todetermine opioid induced analgesia upon acute treatment and its tolerance development upon chronic exposure. NPYFa
demonstrated early onset, dose dependent and prolonged anti-nociception. Antagonists (μ, κ and δ receptor) pretreatment
studies alone or together and with NPFF receptors antagonist demonstrated κ-opioid receptors mediated anti-nociception.
RF9, NPFF receptor antagonist exhibited additive effect to NPYFa acute analgesia, suggesting participation of NPFF
receptors. In addition both Eu-GTP-γS binding assay and FACS analysis further corroborated the observed acuteanalgesia showing significant binding with KOR and NPFF2 receptors suggesting its multiple binding nature. Further
chronic (6 days) treatment effect of NPYFa showed up-regulation of protein expression of these receptors suggesting notolerance development to the NPYFa acute analgesia. Thus, NPYFa demonstrated potent, long lasting anti-nociception
without tolerance development. Hence NPYFa may prove to be a potent analgesic probe with less tolerance development.
72. Morphine analgesic potency is higher in the affective versus the sensory component of formalin-evoked pain
in male and female rats
Lisa Harton, Janell R. Richardson, Arbi Nazarian
Department of Pharmaceutical Sciences, Western University of Health Sciences, Pomona, CA, USA.
Sensation of pain is a multifaceted process that can be divided into sensory (somatic/discriminative) and affective(emotional) components. These components play unique and important roles in the pain experience. Therefore, we

43performed a side by side examination of the analgesic effects of morphine in sensory and affective components of theformalin pain model in male and female rats.
The sensory component of formalin-evoked pain was measured byassessing paw flinches across a 60 min testing session; whereas the affective component was measured using theconditioned place aversion (CPA) paradigm. Our preliminary findings suggest that male and female rats exhibited similarlevels of formalin-evoked flinching behavior and CPA. Morphine treatment reduced the formalin flinching behavior and the
CPA in a dose dependent manner. However, the formalin-evoked CPA was blocked with lower doses of morphine thanthose blocking the flinching response. These findings suggest a greater potency of morphine in blocking the affective
versus the sensory component of pain in rats. Moreover, formalin-evoked c-Fos expression was measured in the
superficial dorsal horn of the spinal cord. Systemic morphine treatment did not reduce the formalin-evoked c-Fos
expression. These findings describe a critical difference in morphine’s ability to reduce sensory and affective pain andhighlight the importance of considering the affective component of pain when examining various pain types and analgesics.
Supported by funds provided by the Western University of Health Sciences. The authors declare no conflict of interest.
73. Distinguishing brain circuits mediating pain relieving and addictive effects of systemic morphine
Edita Navratilova, Diana Meske, Kozo Morimura, Jennifer Xie, Chaoling Qu, Xue Yue, Janice Oyarzo, Michael Ossipov andFrank Porreca
Department of Pharmacology, University of Arizona, Tucson, AZ 85724, USA
Relief of ongoing pain is a natural reward that activates the mesolimbic dopamine (DA) reward pathway. Opiates suchas morphine can relieve ongoing pain by reducing sensory nociceptive transmission, however, opiates act primarily by
alleviating the affective features of pain (i.e., unpleasantness). Opiates also directly activate mesolimbic pathways, aneffect associated with their addictive properties. Using a rat model of neuropathic pain we assessed motivated behaviors(conditioned place preference; CPP) and performed neurochemical analyses (in vivo microdialysis) to show thatmorphine’s effects on modulation of pain affect (i.e., the anti-aversive effects) occur at doses that do not alter evokedsensory responses. Additionally, the anti-aversive doses of morphine did not activate reward circuits in uninjured animals,suggesting that the anti-aversive and addictive effects are mediated by different mechanisms. We hypothesized, that ininjured animals, morphine acts by engaging opioid receptors in the anterior cingulate cortex (ACC), an area implicated inpain aversiveness, to alleviate the affective features of pain. Microinjection of morphine in the ACC elicited CPP and NAc
DA release in neuropathic rats without affecting evoked hypersensitivity and without activating reward circuits in uninjured
states. Moreover, blockade of opioid receptors in the ACC abolished the anti-aversive effects of intravenous morphine
while the rewarding effects were not dependent on the ACC. Thus, the anti-aversive and rewarding effects of morphine canbe dissociated both pharmacologically and anatomically. Supported by a grant from the National Institutes on Drug Abuse(DA034975). The authors have no conflict of interest.
74. Methadone-induced neuronal injury: a possible NMDA receptor-mediated mechanism
Erik Nylander, Alfhild Grönbladh, Shanti Diwakarla, and Mathias Hallberg
Dept. of Pharmaceutical Biosciences, Uppsala University, Uppsala, SWEDEN
Methadone is a long-lasting drug used as a therapy against opiate dependency and is a commonly used pain reliever
for neuropathic pain and cancer. Despite its efficacy, little is known about the damaging effects caused by methadone in
the brain. Methadone is known for its affinity to the endogenous opioid receptors, such as the µ-opioid receptor, however, it
has also been suggested to act as a antagonist at the N-methyl-D-aspartate receptor (NMDA-R). The NMDA-R is a key
receptor throughout the central nervous system, and activation is considered an underlying mechanism for several cellular
phenomena, such as synaptic plasticity, long-term potentiation, and neuronal survival. In this study, we assessed theneurotoxic effects of the opioid receptor agonists methadone and morphine, and the NMDA-R antagonist MK-801 on
mature primary cortical cultures using a cell viability assay. We found that both methadone and MK-801 were neurotoxic tocells in a dose-dependent manner. Morphine also induced cell death, but only at high concentrations. Notably, the opioid
receptor antagonist naloxone (10 µM) was unable to inhibit the effects of methadone, suggesting that neurotoxicity may be
mediated by the NMDA-R. These results led us to examine the potential neuroprotective effects of the endogenous peptide
growth hormone (GH), which has been reported to improve cognitive function in patients chronically exposed tomethadone. Our preliminary data suggests that GH may act as a neuroprotectant in cortical cells exposed to methadone.
Support: Swedish Research Council & Kjell and Märta Beijer Foundation. The authors declare no conflict of interest.
75. A potent mu-opioid agonist that is inactive in tests of abuse liability
Nilges MR1, Zhang X4, Zadina JE1,2,3,4
1Neuroscience Program, 2Departments of Medicine and 3Pharmacology, Tulane University, Tulane University School of
Medicine, 4 SE Louisiana Veterans HCS, New Orleans , LA
For many years a large proportion of novel opioid discovery focused on developing safer analgesics by modifying thestructure of opium derivatives. The failure of this approach is clear: Opioid addiction and overdoses are higher than ever,and chronic pain continues to be poorly treated. Endomorphins (EM) are endogenous ligands for the pain modulating muopioid receptor. EM analogs were developed with the goal of providing analgesic effects comparable to morphine with
reduced side effects. In reward assays, EM analogs were not self-administered (SA), did not produce conditioned place

44

preference (CPP), and did not elicit locomotor sensitization (LS). Morphine promoted reward behaviors in all of theseparadigms and produced dopamine neuron soma size reductions in the ventral tegmental area from these rats, whileanalog 4 did not. We further screened EM analogs in a drug discrimination (DD) model to test the substitution effects of EM
analogs for morphine. Rats were trained to associate injections of morphine with one lever for food reward, while saline
cued another lever for the food. During test sessions, rats dose-dependently responded on the drug-paired lever when preinjected with EM analogs, indicating that rats perceive the analogs as being more similar to morphine than vehicle, eventhough the analogs were not self-administered. Morphine, but not analogs, impaired lever responding for food. Results
indicate that EM analogs could substitute for morphine with less abuse potential. This suggests a potential therapeutic rolefor EM analogs in the treatment of pain and opioid addiction. Funded by VA, DOD, and ONR.
76. Characterization of the NOP receptor distribution using NOP-eGFP mice
Akihiko Ozawa1, Gloria Brunori1, Daniela Mercatelli1, Jinhua Wu1, Melissa Williams1, Sarah Low2, Grégory Scherrer2,
Brigitte L. Kieffer3, and 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
The NOP-N/OFQ system regulates a wide range of biological actions including pain. To better characterize thereceptor localization in the brain, spinal cord and DRG, immunohistochemical experiments are conducted on a knock-in
mouse that expresses NOP receptor C-terminal fused with eGFP. The current study demonstrates that NOP-eGFP is
widely presented throughout the brain structures including the regions related to pain, as well as coexpressed with mu
receptors in specific subpopulation of DRG and brain regions. In DRG, NOP-eGFP receptors are expressed on a numberof myelinated neurons indicating that the receptors are mostly expressed in A fibers. Of which, there are a small number of
medium myelinated-neurons that are mostly CGRP-, sugges
implying the possibility that the receptors might be on myelinated low-threshold mechanosensory neurons. The receptors
are also expressed on small unmyelinated-neurons that are likely either the typical peptidergic (CGRP+/mu+) or
nonpeptidergic (IB4+) C-nociceptors, which are essential to heat or mechanical pain modalities. Additionally, strong
immunoreactivity of NOP-eGFP receptors is also found in specific spinal laminae I-III, the regions important for the
regulation of pain, itch and touch. Altogether our immunohistochemical characterization provides us with information
regarding the cell types containing NOP receptors in DRG and the lamina location in the spinal cord as well as the brain
regions related to pain. It will be useful information for understanding the mechanism by which NOP receptors alter a
nociceptive response. This work was funded by NIH grant (DA023281, L.T.; DA031777, G.S.).
77. Exploration of possible effects of chronic neuropathic pain on sucrose reward in rats
Alec Okun 1,2, Bethany Remeniuk1 Janice Oyarzo 1, David Mc Kinzie2 Jeffrey D. Kennedy 2, and Frank Porreca 1
1 Department of Pharmacology, University of Arizona, Tucson, AZ 85724, USA; 2 Neuroscience Discovery, Eli Lilly &Company, Indianapolis, USA
Considerable co-morbidity is observed in patients with chronic pain and major depressive disorder (MDD). Anhedonia
is a hallmark of depression and one of the principle diagnostic criteria for MDD. We employed a sucrose preference assay
to determine whether chronic neuropathic pain influences reward in the rat. Protocols were approved by IACUC prior toperforming experiments. Spinal nerve ligation (SNL) or sham surgery was performed on groups of 8-10 male Harlan SD
rats by tightly ligating L5/L6 spinal nerves. Rats were given a choice of 1% sucrose or water solutions. The preference forsucrose and the total volume of fluid consumed were recorded and assessed by two-way ANOVA (p<0.05). Preference forsucrose was evaluated on days 2, 7, 14 and 21. No differences in sucrose preference or total volume consumed wereobserved between sham-operated and nerve-injured animals. When evaluated up to day 21 after SNL, there was noevidence of changes in rewarding qualities of sucrose. Improved mechanistic understanding of the possible effects of pain
on reward may facilitate novel treatments for comorbid chronic pain and depression. Acknowledgments/Disclosures: Eli
Lilly & Company funded these studies and currently employs AO and, DM. Conflicts of interest: The authors have noconflicts of interest to declare.
78. A bivalent ligand (MCC22) potently inhibits inflammatory and neuropathic pain via
putative MOR-CCR5 heteromers in mouse spinal cord
Philip S. Portoghese,1 Mary M. Lunzer,1 Michael D. Powers,1 Muhammad I, Javed,1 Yuk K. Sham,2 Giuseppe Cataldo,3Donald A. Simone,5 Eyup Akgun1
1Department of Medicinal Chemistry, 2Center for Drug Design, School of Dentistry,3 University of Minnesota, Minneapolis,
MN, USA
Conventional opioid analgesics are not highly effective for treatment of pain associated with various chronic diseases
due to tolerance and other adverse effects. Given that crosstalk between opioid and chemokine receptors reduces thepotency of opioids, and reports on the existence of MOR-CCR5 heteromers in cultured cells, has led us to consider a novel
therapeutic target for a more effective treatment of inflammatory and neuropathic pain. In this regard, we have designed aseries of bivalent ligands (MCC series) whose members are comprised of MOR agonist and CCR5 antagonist
pharmacophores tethered by variable length spacers. In LPS and CFA pretreated mice, the bivalent ligand with a 22-atom

45

spacer (MCC22) was exceptionally potent in the tail-flick and von Frey assays. MCC22 (i.t.) has an ED50=0.015
pmol/mouse and exhibits >700-fold enhancement of antinociception over the 20-atom spacer homolog, MCC20, and is
3500x more potent than a mixture of monovalent mu agonist and CCR5 antagonist. Molecular simulation studies of MCC22
bound to MOR-CCR5 heteromer with a TM5,6 interface is consistent with the bridging of its protomers. The 7500-foldgreater i.t. vs i.c.v. potency of MCC22 in inflamed mice, suggests MOR-CCR5 heteromers reside in the spinal cord.
Evaluation of MCC22 in a cisplatin-induced neuropathy mouse model revealed it to be effective at a 0.1 fmol i.t. dose. This
research was supported by National Institute on Drug Abuse grant R01DA030316. None of the authors have conflict ofinterest related to this research.
79. Enkephalin and endorphin-based glycosides as analgesics
Evan M. Jones1, Lajos Z. Szabò1, Mark Lefever1, Yingxue Li1, Bobbi Anglin1, Nicholas Laude1, Denise Giuvelis2, Brian I.
Knapp3, Michael Heien1, Jean M. Bidlack3, Edward J. Bilsky2 and Robin Polt1
1Dept of Chemistry & Biochemistry, BIO5, University of Arizona, Tucson, AZ, 85721, USA; 2Dept of Biomedical Sciences,
University of New England, 11 Hill Beach Rd, Biddeford, ME, 04005 USA; 3Dept of Pharmacology and Physiology,
University of Rochester School of Medicine & Dentistry, 601 Elmwood Avenue, Rochester, NY, 14642, USA
Glycopeptides related to β-endorphin penetrate the blood-brain barrier (BBB) of mice to produce antinociception.
Short glycopeptides (5–7 residues) produce mu agonism, delta agonism, or synergistic mu + delta agonism. By linking
helical amphipathic “addresses” to these opioid “messages” it is possible to enhance their effects in vivo.† The molecularweights (MW) of the glycopeptides do not appear to affect BBB penetration rates, at least in the range of MW’s examinedso far, 550—3,500 Daltons. Mouse tail-flick assays and behavioral studies suggest that the mixed mu-delta agonism
produces a powerful synergism to enhance anti-nociceptive effects while reducing sedative effects and locomotor
stimulation. Use of separation-free MSN analysis was used to study the stability of glycopeptides in rat serum. We also
used a nano LC-MSN system with chip-based electrospray ionization (ESI chip) to lower the detection limits for theglycopeptides. A modified on-line preservation system minimizes degradation, enabling quantifiable limits (~100 pM) of
both endogenous peptides and exogenous glycopeptide drugs from the brain of a conscious unsedated mouse. We thankthe National Institute of Neurological Disease and Stroke (NINDS) and the Michael J. Fox Foundation for the support ofthese studies. There are no conflicts of interest.
80. Chronic neuropathic pain and the resulting loss of intrinsic inhibition in the prefrontal cortex causes enduring
structural, electrophysiological and behavioral adaptations
Benjamin Harlan1, Hannah Hughes1, Bethany Pavlinchak, William Buchta1, Reggie Wang2, Toni Shippenberg2, andArthur Riegel1
1Department of Neuroscience, Medical University of South Carolina (MUSC), Charleston SC 29425; 2Integrative
Neuroscience Branch, National Institute on Drug Abuse (NIDA), Baltimore, MD 21224
The negative affect and emotion associated with chronic neuropathic pain suggests a rewiring of higher cognitive
functions and a dysfunctional cortex. To investigate the cellular mechanisms underlying this behavioral state, we used thespared nerve injury (SNI) model of neuropathic pain in wild-type and transgenic rats to assess structural and neuronalplasticity in the prelimbic (PL) cortex. SNI treatment resulted in neuronal adaptations indicative of an enduringhyperexcitability including: 1) slowly evolving but persistent (>40d) regional C-Fos activation, 2) increased density oflong/thin dendritic spine filaments as shown with ballistically loaded fluorescent DiI, 3) overactive release of calcium fromthe endoplasmic reticulum (ER) and 4) elevated basal firing rates (>300%). These adaptations occurred in CaMKII-positive,
deep cortical (5/6) layer PL neurons contralateral to the lesion. The hyperexcitability was unaltered by antagonists for
NMDA/AMPA receptors, but involved a failure of intrinsic-inhibition, mediated by the slow after-hyperpolarization (AHP).
This resulted in loss of inhibitory accommodation (spike-frequency adaptation) — which could be restored by blockade of
PKA, manipulation of calcium stores or stabilization of KCNQ channels with retigabine. In SNI animals, microinfusion ofretigabine or baclofen/muscimol into the PL attenuated the mechanical allodynia to sham-levels. These findings
demonstrate that the enduring cortical hyperexcitability induced by neuropathic pain (often attributed to NMDA-dependent
LTP) may actually result from an ER calcium-dependent loss of intrinsic inhibition that measurably impacts pain-related
behaviors. This work was supported by NIDA/NIH (DA033342 & DA028811). None of the authors has a conflict of interest
related to this research.
81. Cell-autonomous regulation of mu-opioid receptor recycling and resensitization by substance p.
Shanna L. Bowman1, Amanda L. Soohoo1, Daniel J. Shiwarski1, Stefan Schulz2, Amynah A. Pradhan3, Manojkumar A.
Puthenveedu1.
1Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA; 2Institute of Pharmacology
and Toxicology, Friedrich Schiller University Jena, Drackendorfer Straße 1, Jena, Germany; 3Department of Psychiatry,
University of Illinois, Chicago, 1601 West Taylor Street, Chicago, IL 60612, USA.
Opioid analgesics are known to modulate the pain pathway, but whether and how pain can modulate neuronalsensitivity to opioids is less clear despite early reports of paradoxical antinociceptive effects of inflammatory neuropeptides.
Here, we show that substance P (SP), a neuropeptide associated with inflammatory pain, reprograms opioid receptor

46

recycling and signaling and sensitizes peripheral neurons to opioid analgesics. SP, through activation of the neurokinin 1(NK1R) receptor, increases the post-endocytic recycling of the mu-opioid receptor (MOR) in trigeminal ganglion (TG)neurons in response to endogenous opioids and fentanyl, but nor morphine. SP-mediated protein kinase C (PKC)
activation is both required and sufficient for increasing recycling of exogenous and endogenous MOR in TG neurons. The
target of this cross-regulation is MOR itself, as mutation of either of two PKC phosphorylation sites on MOR abolishes the
SP-induced increase in recycling and resensitization. Further, SP abolishes acute tolerance to fentanyl-induced, but notmorphine-induced, antinociception in mice. Our results define a physiological pathway that cross-regulates opioid receptor
recycling via direct modification of MOR and suggest a mode of homeostatic interaction between the pain and analgesic
systems. S.L.B. was supported by an NIH T32 grant NS007433, A.A.P. was supported by NIH DA031243, and M.A.P. was
supported by NIH DA024698 and DA036086. None of the authors has a conflict of interest related to this research.
82. Collybolide, a novel scaffold to study kappa-opioid receptor pharmacology
Nicholas P. Massaro1, Jean-Louis Banères2, Brice Kauffmann3, Jean Dessolin3, Michel Laguerre3, Markus W. Voehler4,
Alicia Jornet5, Karren Hyde5, Adrien Cavé5, Heidi E. Hamm5, Joseph Parello4, Achla Gupta6,7, Ivone Gomes6,7,Lakshmi A. Devi6,7 and Indrajeet Sharma1
1Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA;
2Institut des Biomolécules Max Mousseron (IBMM), Equipe de Pharmacologie Cellulaire, UMR 5247 CNRS-Universités
Montpellier I & II, Montpellier, France; 3Institut Européen de Chimie et de Biologie IECB, UMR 5248 CBMN, Pessac,
France ; 4Department of Chemistry, 5Department of Pharmacology, Vanderbilt, Nashville, TN, USA; 6Department ofPharmacology and System Therapeutics, 7Department of Neuroscience and Friedman Brain Institute, Icahn School of
Medicine at Mount Sinai, New York, NY, USA.
Collybolide, recently discovered to be a potent and selective kappa opioid receptor (KOR) agonist, has become aprospective pharmacological target due to a furyl-δ-lactone core structure similar to salvinorin A, the first non-nitrogenous
ligand with a unique mode of binding to KOR. Understanding how collybolide achieves its remarkable selectivity for human
KOR (hKOR) is an essential step to study kappa opioid pharmacology by establishing the SAR of collybolide to KOR.
Through our multidisciplinary collaboration, we are developing a diverse library of collybolide analogues using twocomplementary approaches, i.e. semi-synthesis and diversity-oriented total synthesis. In the semi-synthetic approach, weare pursuing a rational structural diversification of the natural collybolides. Diversity-oriented total synthesis of collybolide iscurrently underway starting from sugars, an inherently rich source of chirality provided by Mother Nature. The absoluteconfiguration of two natural collybolides (from the fungus Collybia maculata) and their conformational plasticity were
determined by X-ray crystallography and dynamic NMR, respectively. Additionally, hKOR has been obtained as arecombinant protein stabilized in amphiphilic media and will be used for planned SAR studies in vitro. A theoreticalsimulation of human KOR (hKOR) in complex with collybolide suggests that the sesquiterpene interacts with hKORselective Tyr residues. Altogether, these studies will help in establishing the SAR of collybolide at KOR for discoveringnovel therapeutic agents to treat pain and substance abuse. Support by OU Start-up (to I.S.) and NIH grants DA008863
and NS026880 (to L.A.D.). None of the authors has a conflict of interest related to this research.
83. Interaction between methylphenidate, methadone and different antidepressant drugs in mice, and possible
clinical implications
Shaul Schreiber1, Miaad Bader2, Vardit Rubovitch2, and Chaim G. Pick2,3
1Department of Psychiatry, Tel Aviv Sourasky Medical Center & Tel Aviv University Sackler Faculty of Medicine, Tel Aviv,
Israel; 2Department of Anatomy, and Anthropology, Sackler Faculty of Medicine, and 3Sagol School of Neuroscience, Tel ‫‫‬‫‫‬Aviv University, Tel Aviv, Israel
Methylphenidate (MPH), a psychostimulant used for the treatment of attention deficit hyperactivity disorder (ADHD), is
widely used by patients treated with antidepressants and / or methadone maintenance treatment. Preclinical studies in rats
showed that MPH has an analgesic effect when given alone or in combination with morphine. In the present experiments
we studied the interaction of acute doses of MPH with sub threshold doses of methadone and different antidepressant
medications. Next, we studied the interaction of increasing doses of MPH with chronic methadone using implanted minipumps. For these goals we performed the hot plate assay on mice. The addition of a sub threshold dose of venlafaxine,
desipramine and clomipramine to MPH produced significant augmentation of MPH antinociceptive effect with each
medication (p<0.05). No interactions were found when sub threshold doses of escitalopram and methadone were added toacute doses of MPH. On the other hand, addition of increasing doses of MPH to chronic methadone given for 2 weeks
using osmotic mini pumps induced augmentation of the antinociceptive effect of chronic methadone exclusively at highdose of MPH. These findings may implicate the need of an excessive attention to the administration of MPH given to MMT
patients. The findings of the no interaction between MPH and escitalopram may hint to the possibly safe co-administrationof methylphenidate and SSRIs to depressed ADHD patients. Further studies are needed before these possible clinicalimplications can be validated. Supported by (i) The Dr. Miriam and Sheldon G. Adelson Center for the Biology of Addictive
Diseases. (ii) The Ari and Regine Aprijaskis Fund, at Tel-Aviv University. None of the authors has a conflict of interest
related to this research.

47

84. Heat shock protein 90 regulates mu opioid receptor signaling in vitro and in mouse periaqueductal grey
Sarah McCarthy, Nate Mullen, Courtney Brann, James Cormier, Katie Edwards, Edward Bilsky, and John M. Streicher
Department of Biomedical Sciences, College of Osteopathic Medicine, University of New England,
Biddeford ME USA
Recent efforts to develop opioids with fewer side effects have focused on signalling proteins responsible for different
aspects of the response, and targeting these regulators with functionally selective drugs and other strategies. To find new
targets for this approach, we mined proteomic databases to identify unknown candidates for mu opioid receptor (MOR)
regulators. One such candidate was Heat shock protein 90 (Hsp90), a ubiquitous, highly expressed, signalling regulator,
which is altered in brain by chronic opioid treatment. We treated MOR expressing CHO, HEK, U2OS, and SH-SY5Y
neuroblastoma cells with 17-AAG, a selective Hsp90 inhibitor, followed by the MOR agonist DAMGO. We found changes in
evoked signalling and protein expression that varied by cell type. These changes included strongly decreased βarrestin2,
MOR, and Akt expression, alterations in ERK signalling baseline and evoked DAMGO response, and increased Hsp70
expression. We continued these studies in vivo by intracerebroventricular injection of 17-AAG into mouse brain, followedby vehicle or DAMGO. We found an increased ERK signalling baseline and abolished DAMGO induction in theperiaqueductal grey, along with increased Hsp70 expression. Furthermore we found that 17-AAG treatment did not alter
the acute anti-nociceptive response to DAMGO in the tail-flick test. Studies are in progress to test the effect of 17-AAG
treatment on chronic opioid induced tolerance and dependence, which we expect to be decreased. These findings
demonstrate that Hsp90 has a strong regulatory role in MOR signalling in vitro and in vivo, and suggest the possibility of
Hsp90 inhibitor co-therapy to improve opioid therapeutic index.
85. Implication of COPB1 in intracellular retention of the delta opioid receptor
Etienne St-Louis1,4, Jean-Luc Parent2,3,4 and Louis Gendron1,3,4
1Départements de pharmacologie-physiologie et de 2Rhumatologie, Faculté de médecine et des sciences de la santé,
3Institut de pharmacologie de Sherbrooke, 4Centre de recherche clinique du CHUS, Sherbrooke, QC, CANADA.
As opposed to most G protein-coupled receptors, the delta opioid receptor (DOPr) is poorly addressed at the plasma
membrane. Indeed, under normal conditions, only a small proportion of DOPr can escape the endoplasmic reticulum (ER)and reach the plasma membrane. Most recently, we have described an important role for T161 phosphorylation by cdk5 inmediating morphine and inflammation-induced up-regulation of DOPr. In this study, we sought to better describe themolecular mechanisms involved in the regulation of DOPr trafficking. We investigated the role of the putative COPB1
binding motifs located in the intracellular loops (ICL) of DOPr. In particular, we identified a COPB1 binding motif within theconsensus cdk5 phosphorylation motif in the ICL2 of the DOPr. Using confocal microscopy, co-immunoprecipitation and
GST-pulldown assays we found COPB1 to colocalize and interact with the COPB1 binding motifs in ICL2 and ICL3 of
DOPr. Most interestingly, the replacement of T161 by phosphomimetic residues decreased the ability of COPB1 to bind the
ICL2 of DOPr. We finally studied the impact of mutations altering the COPB1 binding motifs on the surface expression of
Flag-DOPr in HEK cells using an ELISA assay and found a major role for the two di-lysine binding sites in the retention of
DOPr into the ER. Together, our results suggest that COPB1 is involved in the intracellular retention of DOPr and that thephosphorylation of T161 can interfere with COPB1 binding to ICL2 promoting the escape of DOPr from the ER, increasing
its expression at the plasma membrane. This work was supported by the Canadian Institute of Health Research grant#MOP-123399.
86. Peripheral mu-opioid receptor activation attenuates ongoing neuropathic and inflammatory pain in rats
Vinod Tiwaria, Fei Yanga, Shao-Qiu Hea, Ronen Shechtera, Chen Zhangb, Bin Shua,c, Tong Zhangd, VineetaTiwaria, Yun
Wangb, Xinzhong Donge,f, Yun Guana, Srinivasa N. Rajaa
aDepartment of Anesthesiology and Critical Care Medicine, Johns Hopkins University, School of Medicine, Baltimore,
Maryland, 21205; bDepartment of Anesthesiology, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020,
China; cDepartment of Anesthesiology, Tongji Hospital, Tongji Medical College, Wuhan, 430030, China; dDepartment of
Neurology, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, China; eThe Solomon H. Snyder
Department of Neuroscience, Center for Sensory Biology, Johns Hopkins University, School of Medicine, Baltimore,
Maryland, 21205; fHoward Hughes Medical Institute, Johns Hopkins University, School of Medicine, Baltimore, Maryland,
21205.
Ongoing pain, a common clinical presentation in patients suffering from tissue or nerve injury, is difficult to assess in
pre-clinical studies. Using a novel assay, conditioned place preference (CPP) that relies on reward from pain relief, weinvestigated if DALDA, a hydrophilic mu-opioid receptor (MOR) agonist, can prevent spontaneous pain in rats withneuropathic (SNL) and inflammatory insults. SNL rats spent significantly more time in DALDA (10 mg/kg s.c.) pairedchamber during post-conditioning, compared to pre-conditioning. DALDA did not induced CPP in naive rats, suggestingthat DALDA’s effects in SNL rats is likely due to the reward from pain relief. Intraperitoneal pretreatment with theperipherally restricted MOR-preferring antagonist, methylnaltrexone (5 mg/kg), blocked DALDA-induced CPP in SNL rats,suggesting a peripheral mechanism for DALDA’s effects on ongoing pain. Systemic treatment with resiniferatoxin (0.1mg/kg i.p) blocked DALDA-induced CPP in injured rats, suggesting an inhibition of TRPV1-expressing neurons is important
to DALDA-induced CPP. DALDA pretreatment also inhibited ongoing pain behavior at 15-60 min (phase II) after48

intraplantar formalin injection (50 µL, 1%) and attenuated the subsequent development of mechanical hyperalgesia. In
neurophysiological studies, spontaneous activity of WDR neurons in SNL rats was significantly decreased at 20-30 min
after DALDA, but not vehicle, treatment. Pre-treatment with methylnaltrexone blocked the inhibitory effect of DALDA on
WDR neuronal activity. Our findings suggest that DALDA alleviates ongoing neuropathic and inflammatory pain in rats byactivating peripheral MORs, especially those expressed in TRPV1-positive neurons. Further studies are required todelineate the underlying cellular and molecular mechanisms. Conflict of interest: There is no conflict of interest.
Grant/Other Support: NIH Grant NS70814, NS26363.

87. Galectin-3 is upregulated in primary afferent neurons and satellite glial cells following peripheral nerve injury,
but not in spinal neurons or microglia
Elizabeth I. Sypek1, Vivianne L. Tawfik1, Grégory Scherrer1
1Department of Anesthesiology, Perioperative and Pain Medicine, Department of Molecular and Cellular Physiology,
Department of Neurosurgery, Stanford Neurosciences Institute, Stanford University, Palo Alto, CA, USA.
Galectins are a class of carbohydrate binding protein that bind beta-galactoside sugars and have been implicated incell adhesion, communication, and immune responses. Galectin-3 was shown to be upregulated in microglia following
stroke and has thus been used as a marker for microglia activation. Here we wanted to determine the contribution of
microglial gal-3 to neuropathic pain. We performed unilateral sciatic nerve axotomy and, strikingly, failed to observe
galectin-3 in activated spinal microglia. Rather, we observed a significant increase in galectin-3-immunoreactivity in thecentral terminals of IB4+ and CGRP+ nociceptors in laminae I-IIo of the dorsal horn. Corresponding galectin-3 upregulation
was observed in sensory neuron cell bodies in lumbar dorsal root ganglia following injury. We observed no galectin-3 inspinal cord neurons, astrocytes, or microglia before or after sciatic nerve injury. However, following direct injury to thespinal cord galectin-3 is expressed by a subset of microglia and invading macrophages. We also found an upregulation of
galectin-3 in dorsal root ganglion satellite glial cells associated with somata of IB4+ nociceptors. We are currently
investigating the mechanism of action of galectin-3 in nociceptors and SCGs to test the hypothesis that galectin-3 might bea signaling molecule that regulates nociception or regeneration following nerve injury. Regardless of galectin-3 mechanismof action, our results reveal that either distinct microglial populations, or distinct gene expression programs in these cells,
are engaged in spinal microglia following peripheral and central injuries. Support by NIH/NIDA (DA031777, G.S.), FAER
Research Fellowship (V.L.T) and NDSEG Fellowship (E.S.).
88. Comparison of equilibrium dialysis (ED) combined with ultra-high performance liquid chromatography/tandem
mass spectrometry (UPLC/MS/MS) and competitive ELISA for the determination of dissociation constants of 6acetylmorphine (6AM) and morphine to polyclonal antibodies
Oscar B. Torres1,4, Rashmi Jalah1,4, Fuying Li2,3, Joshua F. G. Antoline2,3, Arthur E. Jacobson2,3, Carl R. Alving1,
Kenner C. Rice2,3, and Gary R. Matyas1
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; 2Drug Design and Synthesis Section, Molecular
Targets and Medications Discovery Branch, National Institute on Drug Abuse, National Institutes of Health, Department of
Health and Human Services, 9800 Medical Drive, Bethesda, MD 20892 USA; 3National Institute on Alcohol Abuse andAlcoholism, National Institutes of Health, 9800 Medical Drive, Bethesda, MD 20892 USA; 4U.S. Military HIV Research
Program, Henry M. Jackson Foundation (HJF) for the Advancement of Military Medicine, 6720A Rockledge Drive,
Bethesda, MD 20817 USA
Vaccines to drugs of abuse function by producing antibodies that prevent the drug from crossing the blood-brain
barrier. In vivo, heroin rapidly is metabolized into 6AM and morphine. Thus, a heroin vaccine must induce high titer andhigh affinity antibodies to heroin, 6AM and morphine. ED and competitive ELISA were performed on sera from mice
immunized with DiAmHap, 6-ProxyHap and MorHap haptens tetanus toxoid (TT) conjugates. The concentrations of thedrugs were quantified using ED-UPLC/MS/MS and dissociation constant (Kd) was calculated by plotting [Bound Drug] vs[Free Drug]. The Kd was calculated by competitive ELISA using the opiates as inhibitors and hapten-BSA conjugates ascoating agents. Immunization of mice with 6-PrOxyHap-TT and MorHap-TT conjugates induced antibodies that have highaffinities for both 6AM and morphine (Kd< 25 nM), while DiAmHap-TT conjugates induced antibodies that do not bindmorphine and had low affinities for 6AM (Kd> 100 nM) as measured by ED-UPLC/MS/MS. In contrast, competitive ELISA
yielded apparent Kd that were in the M range. Due to the chemical instability of heroin, the Kd for heroin cannot be
determined by either of the methods. We described a method that utilized ED-UPLC MS/MS that provided a more accurate
apparent dissociation constants (Kd ~ nM) than competitive ELISA (Kd ~ high M) for the measurement of antibodies to
opiates. This work was supported through a Cooperative Agreement Award (W81XWH-07-2-067) between the HJF and the
U.S. Army Medical Research and Materiel Command and an Avant Garde award from NIDA (NIH grant no.
1DP1DA034787-01).
89. The NOP Receptor Antagonist SB612111, but not the Agonist AT-202 Blocks Ethanol Drinking in Mice49

Gloria Brunori, Andrea Cippitelli, Michelle Gorman, Nurulain Zaveri, Lawrence Toll
Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987, USA
The NOP receptors is found throughout the brain, spinal cord, and dorsal root ganglia and is involved in many CNS
pathways including those for pain and reward. It has been demonstrated repeatedly that both N/OFQ, the endogenous
ligand for NOP, and small molecule agonists can block conditioned place preference (CPP) of many abused drugs, andcan reduce extracellular dopamine levels in the nucleus accumbens. Results with self-administration studies have beenless conclusive. N/OFQ does not block heroin self-administration in rats, and small molecule agonists have had equivocal
results in blocking self-administration of alcohol in rats and opiates in monkeys. When exploring alcohol consumption inmice using the one-bottle and the two-bottle choice drinking in the dark (DID) paradigms we found the NOP agonist AT-202
to have no effect on alcohol consumption at non-sedative doses. Conversely, the NOP receptor antagonist SB612111 wasable to attenuate alcohol consumption in both types of experiments. At 30 mg/kg SB612111 reduced alcohol consumption
by 30% when 20% alcohol was present for 4 h in the one-bottle DID. SB6121211 was more effective, reducing alcohol
consumption by 50%, when mice were exposed to both 15% ethanol and water for 4h. This surprising result suggests that
there is a difference between the reward inducing actions of the CPP and self-administration tests, and that the NOP
system influences these two different drug abuse paradigms differently. The mechanism by which NOP antagonists
attenuate alcohol drinking behavior is current unclear. Supported by NIDA grant 1R01DA023281-01S1.
90. Structure-based approaches to designing bifunctional NOP-mu opioid receptor ligands from NOP-selective
scaffolds
Dennis Yasuda, Edward Tuan, Pankaj R. Daga, and Nurulain T. Zaveri1
Astraea Therapeutics, 320 Logue Avenue, Mountain View, CA, USA
Nociceptin/OrphaninFQ (N/OFQ), the endogenous peptide ligand for the NOP receptor, has been shown to decrease
opioid-induced reward and tolerance development. The antinociceptive activity of selective NOP agonists, however, isdependent on the route of administration and the pain modality. We have previously hypothesized that NOP-mu opioid
bifunctional agonists, possessing mu opioid-based antinociceptive efficacies and reduced tolerance/rewarding properties
due to NOP agonist efficacy, may have desirable profiles as ‘non-addicting analgesics’. We reported the design of such
NOP-mu bifunctional agonists from the NOP-selective dihydroindolin-2-one class of NOP agonists. Ro 64-6198, the wellstudied selective NOP agonist from the 1,3,8-triazaspirodecanone class of NOP agonists, only shows antinociceptive
activity in models of acute thermal pain and inflammatory pain when administered systemically. Here, we report the designof bifunctional NOP-mu agonists from this NOP-selective 1,3,8-triazaspirodecanone scaffold using NOP and mu opioid
receptor structure-based approaches. We designed rational structural modifications of Ro 64-6198 that increased its
binding affinity at the mu opioid receptor without adversely affecting its NOP affinity. The resulting NOP/mu ligands
maintained NOP full agonist efficacy and had varying levels of mu agonist efficacy to afford several new bifunctional NOPmu agonists. These drug design approaches for obtaining the desired bifunctional profiles, guided by receptor structural
information as well as the detailed pharmacological characterization of binding affinities and functional efficacies, will bepresented. Authors declare no conflicts of interest. This work was supported by NIDA grant R01DA027811 (NZ).
91. Synaptic plasticity in the medial prefrontal cortex: role of 7 nicotinic acetylcholine receptors
Matt Udakis1, Sue Wonnacott1, Huib Mansvelder2 and Chris Bailey1
1University of Bath, Bath, UK; 2Vrije University, Amsterdam, Netherlands
The medial prefrontal cortex (mPFC) is a key brain region implicated in drug-related associative learning (1). 7
nicotinic acetylcholine receptors (nAChR) have a modulatory role in the mPFC (2) and 7 nAChR antagonism can inhibit
reinstatement to opioid drug seeking (3). We have explored the mechanisms by which 7 nAChRs modulate excitation in
the mPFC as a pre-requisite to investigating their influence on drug-associated learning.
We measured spontaneous EPSCs and IPSCs in brain slices from drug naïve mice, in response to 7 nAChR positive
allosteric modulator (PNU-120596), selective agonist (PNU-282987) and antagonists (MLA). Functional somatic 7nAChRs on inhibitory interneurons and presynaptic 7 nAChRs on glutamatergic terminals were discriminated. This
enables 7nAChRs to play a complex dynamic role in both excitation and inhibition in the mPFC, with endogenous tonic
acetylcholine preferentially enhancing excitatory transmission via 7 nAChRs. Using field recordings to study stimulusinduced plasticity in mPFC slices, both inhibition and activation of 7 nAChRs reduced stimulus-induced LTP, whilst MLA
enhanced LTD. Optogenetic strategies are currently being applied to define the glutamatergic input pathways bearing 7
nAChRs. These studies will pave the way for an analysis of the roles of 7 nAChRs in the mPFC in drug-related learning.
Funded by an MRC studentship and bursaries from Bath Alumni, British Pharmacological Society and Guarantors of Brain
to MU. We declare no conflict of interest.
92. Mitragyna alkaloids revisited: pharmacology of mitragynine and related natural products
András Váradi, Gina F. Marrone, Travis C. Palmer, Amanda Hunkele, Valerie Le Rouzic, Gavril W. Pasternak, Ying-Xian
Pan, Jay McLaughlin and Susruta Majumdar
Molecular Pharmacology and Chemistry Program, Department of Neurology, Memorial Sloan Kettering Cancer Center,50

1275 York Avenue, New York, NY 10065 (USA).
Mitragynine, an indole alkaloid isolated from the Southeast Asian rubiaceous plant Mitragyna speciosa, is one of the
few examples of opioid natural product small molecules that are structurally unrelated to morphine. Mitragyna-based herbal
products (often marketed as Kratom) are legally available in the US. Anecdotal reports indicate they are usedrecreationally, as pain relievers, and to prevent opioid withdrawal. Mitragynine, as well as its naturally occurring congeners
such as 7-hydroxymitragynine and mitragynine pseudoindoxyl, have received attention in the past decades because they
produce antinociceptive effects in rodents. . However, the published pharmacological data on this alkaloid family is very
diverse, making the comparison of results difficult. Our goal was to pharmacologically characterize mitragynine, related
natural products, and several semi-synthetic analogs. Mitragynine, 7-hydroxymitragynine, mitragynine pseudoindoxyl, andtheir analogs were evaluated in cloned opioid receptor transfected cell lines in in vitro radioligand binding assays,[35S]GTPγS functional assays, and β-arrestin biased agonism assays. All compounds were also evaluated in vivo in tailflick analgesia assays in the absence and presence of antagonists selective for several opioid receptor subtypes. Ourpresentation will discuss the implications of the results in opioid drug discovery and possibilities to diversify this template todevelop novel analgesics. Supported by DA034106 to SM from NIDA.
93. Synthesis and pharmacology of a dual kappa-delta opioid receptor agonist which blocks cocaine conditioned
place preference
András Váradia, Gina F. Marronea, Daniel Afonina, Joan J. Subratha, Valerie Le Rouzica, Amanda Hunkelea, Gavril W.
Pasternaka, Jay P. McLaughlinb, Susruta Majumdara
aMolecular Pharmacology and Chemistry Program, Department of Neurology, Memorial Sloan Kettering Cancer Center,
1275 York Avenue, New York, NY 10065 (USA); bDepartment of Pharmacodynamics, University of Florida, 1345 Center
Drive, Gainesville, FL 32610, USA
Opioids can be used to treat opioid, alcohol, and potentially cocaine abuse disorders. Analgesics with selectivity for aparticular opioid receptor family have prototypical side effects including abuse liability (MOR), dysphoria (KOR), andseizures (DOR). An important goal of opioid drug development is to eliminate these unwanted effects. We have recently
discovered a new class of opioid analgesics targeting 6-transmembrane splice variants of MOR (6TM/E11) with highaffinity. The lead compound in this class, 3-iodobenzoyl-β-naltrexamine (IBNtxA) displays potent antinociception without
side-effects associated with activation of traditional opioid receptors. During our SAR studies on IBNtxA, we discovered an
opioid ligand (MP1104) with picomolar binding affinity for all opioid receptors. In functional assays in vitro, it was 574 and
107-fold more potent than prototypic KOR and DOR agonists, respectively. In vivo, MP1104 was 18-fold more potent thanmorphine. We used both selective opioid antagonists and knockout mice to determine the relative contributions of opioid
receptor families to MP1104 analgesia. MP1104 appears to produce analgesia exclusively through KOR and DOR
receptors. MP1104 did not precipitate seizures at doses 45-fold higher than the analgesic ED50. When evaluated in
conditioned place preference/aversion assays, MP1104 showed no rewarding or aversive effects at a dose three times
higher than its analgesic ED50, but attenuated cocaine-conditioned place preference. There are presently no FDA
approved drugs to treat cocaine addiction. MP1104 may represent an attractive strategy to treat pain as well as cocaine
dependence without the characteristic side effects seen with opioid analgesics. Supported by DA034106 to SM from NIDA
94. Beta-arrestin1 preferentially mediates analgesic tolerance to SNC80 in a ligand-biased manner
Vicente-Sanchez A.1, Tipton A.F.1, Akbari H.1, Segura L.1, Pradhan A.A.1
1Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, USA.
Ligand directed signaling via the delta opioid receptor (DOPR) has important implications given the potential
therapeutic uses of delta agonists in the treatment of chronic pain and emotional disorders. We had previously shown that
repeated injection of the high-internalizing delta agonist (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3methoxybenzyl]-N,N-diethylbenzamide or SNC80, produced acute behavioral desensitization while the low-internalizing
delta agonist N,N-diethyl-4-(phenyl-piperidin-4-ylidenemethyl)-benzamide or ARM390 did not. Since β-arrestins are wellknown to regulate G protein-coupled receptors signaling and trafficking, we therefore investigated the behavioral
significance of ligand-specific interactions between β-arrestin 1 and the DOPR. In the Complete Freund’s Adjuvant (CFA)
tail model of inflammatory pain, mice lacking β-arrestin 1 showed enhanced and longer lasting pain-relieving effects of
SNC80, and decreased behavioral tolerance following repeat exposure to the agonist. In contrast, ARM390 produced
similar analgesic effects and no tolerance in both WT and KO animals. Following chronic treatment, the absence of βarrestin 1 attenuated the extent of tolerance to SNC80, but not to ARM390. Furthermore, chronic treatment with SNC80
abolished delta-agonist induced GTPγS binding in WT brain membranes, whereas DOPR-G protein coupling remained
intact in KO mice. Overall, these results indicate that DOPR agonists interact with β-arrestins in a ligand-biased manner,and that the high-internalizing agonist SNC80 preferentially recruits β-arrestin 1. This work was supported by the NIH Grant
DA031243, the Shirley and Stefan Hatos Research Foundation, and the Dept. of Psychiatry at the University of Illinois at
Chicago. None of the authors has a conflict of interest related to this research.

51

95. Cocaine self-administration induces changes in synaptic transmission and plasticity in ventral hippocampus
Madhusudhanan M. Keralapurath1,2, Sherri Briggs1,3 and John J. Wagner1,2,3
1Department of Physiology & Pharmacology, 2Interdisciplinary Toxicology Program, 3Neuroscience Program, University ofGeorgia, Athens, USA
Allowing rats extended access to cocaine self-administration is thought to recapitulate key aspects of cocaine
addiction. Understanding the mechanisms that underlie drug-induced neuroadaptations that persist in the brain after
protracted abstinence is crucial for developing therapeutic interventions. Whole-cell voltage-clamp and extracellular
recording technique were used to assess changes in neurotransmission and long-term potentiation (LTP) in the CA1 region
using a ventral hippocampal slice preparation. Rats allowed to self-administer cocaine daily, including “long access” (6hr)
sessions, exhibited an increase in the AMPA/NMDA current ratio and enhanced excitatory transmission following 3-5
weeks of abstinence. Inhibitory transmission was also significantly decreased in long access animals and the AMPA/NMDA
ratio measured in the absence of GABAergic blockers was greatly enhanced. We also observed a significant reduction of
LTP magnitude. Conclusions: These findings suggest the presence of synergistic effects of enhanced AMPA anddiminished GABA neurotransmission under physiological conditions in the CA1 region of cocaine-taking animals,
supporting the conclusion that persisting enhancement of AMPA-mediated transmission and concomitant inhibition of
GABA-mediated transmission promoted a chronic state of potentiation that partially occluded further LTP. As a result, thisincreased output from the ventral hippocampus to other limbic areas such as the nucleus accumbens would be expected tocontribute to sensitization-like responses mediating the escalation of drug intake and promote the reinstatement of drugseeking behaviors. Supported by the National Institute on Drug Abuse, NIH (DA016302 to J.J.W.). None of the authors has
a conflict of interest related to this research.
96. Biphasic effects of Κ opioid receptor agonist U50488H in anxiety-related behaviors
Yu-Jun Wang, Ai Hang, Jing-Gen Liu
Department of pharmacology, State key laboratory of drug research, Shanghai Institute of Materia Medica, Chinese
Academy of Science, Shanghai 201203, China
Accumulative evidences indicated that Κ opioid receptor (KOR)/dynorphin system is involved in regulating states of
emotion and motivation. There is a general consensus that activation of KORs produced negative affective states, such as
dysphoria in human and depressive-like or anxiety-like effects in rodents. However, a number of studies demonstrated thatadministration of KOR agonists produced anxiolytic effects in rodents. The molecular mechanism underlying the biphasic
effects of KORs in anxiety-related behavior was unknown. In the present study, we tested the effects of different doses of Κ
agonist U50488H on anxiety-related behavior in mice. We found that systemically administration of low dose of U50488H(0.125 mg/kg, s.c.) produced anxiogenic effects in the elevated plus maze test, whereas higher dose of U50488H (2.5mg/kg, s.c.) produced anxiolytic effects. The low dose-dependent effect of U50488H was completely blocked by
pretreatment with Κantagonist norBNI, whilst higher dose-dependent effect was partially attenuated by norBNI. pERK wasused as neuronal activity marker to study the neurobiological substrates responsible for the biphasic effects of U50488H.
We found that U50488H anxiogenic effect was paralleled with robust increased pERK activity in the nucleus accumbens,
whereas U50488H anxiolytic effect was paralleled with robust increased pERK activity in the hypothalamus and septum.
Thus, the present study firstly provided the neurobiological substrates to explain the biphasic effect of KORs in anxietyrelated behaviors. Support by grant 81401107 from National Natural Science Foundation of China. None of the authors has
a conflict of interest related to this research.
97. Identification and connectivity of spinal neurons that express delta opioid receptors
Dong Wang1, Vivianne L. Tawfik1, Sarah A Low1, Brigitte L. Kieffer2, Allan I. Basbaum3, Grégory Scherrer1
1Department of Anesthesiology, Perioperative and Pain Medicine & Department of Molecular and Cellular Physiology &Department of Neurosurgery, Stanford Neurosciences Institute, Stanford University, Palo Alto, CA, USA; 2Depatment of
Psychiatry, Douglas Institute, McGill University, Montreal, QC, CANADA; 3Departments of Anatomy, University ofCalifornia San Francisco, San Francisco, CA, USA
Here we used DOR-GFP reporter mice as well as in situ hybridization and electrophysiological recordings in wildtype
mice to identify spinal neurons that express the delta opioid receptor (DOR). In the dorsal horn, DOR+ neurons areconcentrated in lamina II inner. Coimmunolabeling revealed that these DOR+ neurons co-express calbindin, somatostatin
and PKCgamma, identifying them as excitatory interneurons known to process mechanical stimuli. Consistent with thisidea, mechanical stimulation of the mouse hindpaw induced c-Fos expression in DOR+ neurons. Whole-cell patch clamp
recordings in slices showed that DOR+ neurons in lamina II inner displayed either single, delayed or gap action potential
firing patterns, consistent with their excitatory nature. Dorsal root stimulation experiments indicated that these DOR+
neurons receive Adelta or Abeta afferent inputs. Furthermore, DOR agonists activate GIRK channels, reducing excitability
to myelinated low-threshold mechanoreceptor inputs. We also report striking dorso-ventral segregation in the identity of
DOR+ neurons, and DOR presence in motor control circuits. Thus the majority of DOR+ neurons located in lamina III andmore ventral coexpress markers of inhibitory neurons, including NOS+ interneurons in lamina III, parvalbumin+ neurons inlamina IV-V, and engrailed 1+ V1 interneurons in the ventral horn. Our results suggest that beside their action on primary52

afferent mechanoreceptors, DOR agonists could also decrease injury-induced mechanical hypersensitivity by apostsynaptic action on lamina II excitatory interneurons. Support by NIH grants (DA031777, G.S; T32GM089626, D.W.), a
FAER fellowship (V.L.T.) and a HHMI fellowship (S.A.L.). None of the authors has a conflict of interest related to thisresearch.
98. The relationship between reward perception and analgesic effects of heroin in rats
Charlotte Wincott, Brian Reed, and Mary Jeanne Kreek
Laboratory of the Biology of Addictive Diseases; The Rockefeller University, NY, NY
Individual differences in sensitivity to pain and reward likely reflect a combination of genetic, developmental, andenvironmental factors. In this study, we investigated potential correlates of heroin reward perception in conditioned place
preference (CPP) in Sprague-Dawley rats. In the same animals, we also conducted a hot-plate experiment to investigate
potential correlations of the analgesic and reward effects of heroin. For CPP, animals were exposed to heroin (1 mg/kg) orsaline during conditioning. On test day, total time spent in the unconditioned chamber was subtracted from total time spentin the conditioned chamber. For hot-plate, response latency was measured after heroin (1mg/kg) or saline. In CPP, a ttest revealed a significant difference between the saline (n=10) and heroin (n=12) groups [t(18.65)=2.285, p=0.0342], withthe heroin group spending greater time in the heroin-paired chamber. Substantial individual variability was observed within
the heroin group: the upper quartile (n=3) showed high CPP (mean=411.4+29.20 s) and the lower quartile (n=3) exhibited
no CPP (mean=-62.53+52.58 s). Heroin resulted in significantly increased latency in the hot-plate assay compared withsaline, indicating an analgesic effect. Additionally, a positive correlation was observed in the heroin group between %maximum response in hot-plate (at 15 minutes) and increased time spent in the heroin-paired chamber [r(9)=0.66,
p=0.026, n=11]. Our findings suggest a relationship between heroin-induced reward and pain perception. Authors have noconflicts of interest. This work was supported by a David Novick Postdoctoral Fellowship (CW) and the Dr. Miriam and
Sheldon G. Adelson Medical Research Foundation (MJK).
99. The role of the hippocampal and medial prefrontal cortex α7 nicotinic acetylcholine receptors in morphine
primed reinstatement to conditioned place preference
Victoria L. Wright1, Christopher P. Bailey2, David J. Heal3, Polymnia Georgiou5, Alexis Bailey5 and Susan Wonnacott1
1Dept. of Biology and Biochemistry, University of Bath, UK 2Department of Pharmacy and Pharmacology, University ofBath, UK 3Renasci, Nottingham, UK; 5University of Surrey, Guildford, UK.
Nicotinic acetylcholine receptors (nAChRs) play a key role in addiction-related behaviours (Rahman et al (2015) Front
Neurosci 8:426). We have shown that the α7 nAChR antagonist methyllycaconitine (MLA) can selectively inhibit
reinstatement to morphine-CPP (Wright et al., companion abstract). The aim of this study was to investigate themechanisms of this effect. Stimulus-induced LTP in the medial prefrontal cortex (mPFC) was attenuated by the presence of
α-bungarotoxin, a neurotoxin known to antagonise the α7 nAChR. Using autoradiography we examined potential changes
in AMPA and NMDA receptor binding in mice that had undergone MLA treatment at morphine reinstatement. There wereno significant changes in NMDA receptor binding (using [3H]MK801) but significant changes were observed in [3H]AMPA
binding in the hippocampus, but not in other brain regions examined (including mPFC, nucleus accumbens, amygdala,
VTA). These findings suggest potential roles for the hippocampus and the mPFC in the α7 effects. Behaviouralexperiments using intracranial infusions of MLA into hippocampus or mPFC are ongoing. Supported by funding fromBBSRC and a CASE studentship with RenaSci, Nottingham, UK. None of the authors has a conflict of interest related tothis research.
100. α7 nicotinic receptor antagonism selectively reduces reinstatement to morphine-conditioned place
preference
Susan Wonnacott1, Christopher P. Bailey2 , David J. Heal3 , and Victoria L. Wright1
1Dept. of Biology and Biochemistry, University of Bath, UK 2Department of Pharmacy and Pharmacology, University ofBath, UK 3Renasci, Nottingham, UK
Maintaining long-term abstinence and preventing relapse after re-exposure to drug-associated cues is the main
challenge for treating drug addiction. Nicotinic acetylcholine receptors (nAChR) have been implicated in responses todrugs of abuse other than nicotine (Rahman et al (2015) Front Neurosci 8:426). The aim of this work was to characterise
the role of α7 nAChRs in morphine reward learning using conditioned place preference (CPP). The α7 nAChR antagonist
methyllycaconitine (MLA) was used to determine if these receptors contribute to specific stages of drug-paired learning,
namely acquisition, reconsolidation or reinstatement of morphine-CPP. In 7-8week old C57BL/6J mice MLA (4mg/kg, s.c),given 20min prior to a conditioning dose of morphine (10mg/kg, i.p) or post-test trial, had no effect on the acquisition,
reconsolidation or expression of morphine-CPP. However, when given 20min prior to a priming dose of morphine (5mg/kg,i.p), MLA (4mg/kg, s.c) significantly inhibited drug-induced reinstatement. These results suggest that α7 nAChR may
selectively control reinstatement to morphine-CPP. Supported by funding from BBSRC CASE studentship with RenaSci,
Nottingham, UK. None of the authors has a conflict of interest related to this research.
101. Evidence for the involvement of hippocampal α7 nicotinic acetylcholine receptors in morphine primed

53

reinstatement to conditioned place preference
Victoria L. Wright1, Christopher P. Bailey2, David J. Heal3, Polymnia Georgiou5, Alexis Bailey5 and Susan Wonnacott1
1Dept. of Biology and Biochemistry, University of Bath, UK 2Department of Pharmacy and Pharmacology, University ofBath, UK 3Renasci, Nottingham, UK; 5University of Surrey, Guildford, UK.
Nicotinic acetylcholine receptors (nAChRs) play a key role in addiction-related behaviours (Rahman et al (2015) Front
Neurosci 8:426). We have shown that the α7 nAChR antagonist methyllycaconitine (MLA) can selectively inhibit
reinstatement to morphine-CPP (Wonnacott et al., companion abstract). The aim of this study was to investigate themechanisms of this effect. Using autoradiography we examined potential changes in AMPA and NMDA receptor binding inmice treated with either saline of MLA at morphine reinstatement. There were no significant changes in NMDA receptor
binding (using [3H]MK801) but morphine reinstatement significantly increased [3H]AMPA binding in the hippocampus, but
not in other brain regions examined (including mPFC, nucleus accumbens, amygdala, VTA). The selective increase in the
hippocampus was partially antagonized by MLA, linking α7 nAChR activation to glutamatergic synaptic plasticity in the
hippocampus. Morphine reinstatement following intracranial infusions of saline or MLA directly into the hippocampus andmPFC are ongoing. Supported by funding from BBSRC CASE studentship with RenaSci, Nottingham, UK. None of theauthors has a conflict of interest related to this research.
102. Identification and expression of a truncated OPRK1 splice variant with the novel termination codon in humanpost-mortem brain
Yuferov V1, Randesi M1, Reed B1, Morgello S2, Kreek MJ1
1Laboratory of the Biology of Addictive Diseases, The Rockefeller University, New York, NY; 2Dept Neurology,
Neuroscience and Pathology, Mount Sinai Medical center, New York, NY
A novel splice variant of the human kappa-opioid receptor (OPRK1) was identified by RT-PCR amplification andsequence analysis of a human whole brain cDNA library (Clontech). The new OPRK1 splice variant (hOPRK1tm1) ischaracterized by a deletion of 88 bp at 5´-end of exon 3. The deletion in this region produces a premature translation
termination codon. Translation of this variant would be predicted to result in the protein of 90 amino acids in length,
retaining the extracellular N-terminal region, the first transmembrane domain (TM1) and five amino acids of the first
intracellular domain. To compare the level of expression of the hOPRK1tm1with that of full-length OPRK1 in human postmortem brain tissues we have performed a quantitative RT- PCR assay in two brain regions, the nucleus accumbens andanterior cingulate in ten HIV-negative subjects, obtained from the Manhattan HIV Brain Bank. The hOPRK1tm1specific
PCR primer was designed to span the junction site of the canonical exon 2 and the new truncated exon 3. We have founda substantial hOPRK1tm1expression levels in the nucleus accumbens and anterior cingulate, although lower than the fulllength OPRK1 (p<0.001, two-tailed t-test). Further studies are needed to elucidate potential functions of the truncatedsplice variant of OPRK1 in humans. Support: Dr. Miriam and G. Sheldon Adelson Medical Research Foundation (MJK),
NIMH-U24-MH100931 (SM). The authors declare no conflict of interest.
103. An endomorphin analog providing prolonged antinociception with substantial reduction of multiple adverse
side effects relative to morphine
James E. Zadina1,2,3,4, Mark R. Nilges 4, Jenny Morgenweck 2, Jennifer Jernberg1,4, Xing Zhang 1,2, and Melita B.
Fasold1,2.
1SE Louisiana Veterans HCS, New Orleans, LA and Depts of 2Medicine; 3Pharmacol.; 4 Neurosci. Program, Tulane Univ.
Sch. of Med., New Orleans, LA, USA
Opioids acting at the mu opioid receptor (MOR) are the most effective analgesics, but multiple adverse side effects ofavailable MOR agonists limit their use. Endomorphins (EMs) are potent and selective agonists at MOR. We synthesized
several metabolically stable EM analogs and selected four for in-depth analysis of antinociceptive vs. side-effect profiles in
the rat. The analogs showed high affinity, selectivity, and efficacy at MOR relative to delta and kappa opioid receptors.
Morphine induced significant conditioned place preference and self-administration, while the analogs did not, consistent
with reduced abuse potential. At equi-antinociceptive doses, respiratory depression was significantly reduced for all
analogs; analogs 1 and 4 did not induce respiratory depression at doses producing significantly longer antinociception thanmorphine. Motor impairment on a rotorod was less, and antinociception longer, after analogs 2 and 4 than after morphine.
Morphine induced cognitive impairment in the Morris water maze while several analogs did not. Tolerance was determinedwith cumulative dosing before and after 7-day intrathecal administration by osmotic minipump. Analogs were initially 30fold more potent than morphine and only produced a 13-fold shift in the dose-response curve vs a 38-fold shift bymorphine, reflecting significantly less tolerance. Morphine produced significant glial activation (increased GFAP, Iba1 andpp38 staining), while the analogs did not. In summary, our novel EM analogs, especially analog 4, show a highly favorable
profile relative to morphine as potent analgesics with reduced 1) reward/abuse liability, 2) respiratory depression, 3) motor
impairment, 4) cognitive impairment 5) tolerance and 6) glial activation. Funded by the VA, DOD, and ONR.
104. Small-molecule NOP agonists reduce alcohol reward in mouse models of alcoholism
Nurulain T. Zaveri,1 Paul Marquez,2 Michael E. Meyer,1 Abdul Hamid,2 and Kabirullah Lufty2
1Astraea Therapeutics, Mountain View, CA, USA; 2Department of Pharmaceutical Sciences, College of Pharmacy,

54

Western University of Health Sciences, Pomona, CA, USA
Studies in alcohol-preferring rats suggest that dysregulation of the nociceptin opioid receptor NOP and its ligand
N/OFQ are a pathophysiological consequence of excessive alcohol intake. Further, administration of N/OFQ blocks the
rewarding effects of alcohol in the conditioned place preference (CPP) paradigm in mice, as well as reinstatement of
extinguished alcohol CPP, suggesting that NOP agonists may be a promising approach for treating alcohol addiction. We
report the efficacy of small-molecule NOP agonists in reducing ethanol-induced CPP in mice. Mice tested for baseline
place preference were subjected to three conditioning trials, during which they were treated subcutaneously with vehicle ora NOP agonist, followed by ethanol (2 g/kg) or saline and confined to the vehicle-paired or drug-paired chamber for 15 min.
In the afternoon of each day, mice received the alternative treatment and confined to the opposite chamber for 15 min. On
day 5, mice were tested for place preference. Among the compounds tested, selective NOP agonist AT-312 dosedependently reduced ethanol-induced CPP. AT-312 alone had no motivational effects at the dose at which it blocked
ethanol CPP. The inhibitory effect of the agonists on alcohol reward was completely abolished in mice lacking NOP.
Together, these data suggest that AT-312 reduced alcohol reward via the NOP receptor and was devoid of any
motivational effects. Studies are ongoing to assess effect of NOP agonists in the two-bottle-choice paradigm, which is
widely used as a model of alcohol-taking behaviors and reinforcement. Authors declare no conflicts of interest. Support:
Contract HHSN275201300005C.
105. The role of mu opioid receptors in D1 and D2 neurons in opioid reward-related behaviors
1
1
1
2
3
1
1
David Pena , Ani Minasyan , Ralph Albert , J. David Jentsch , Brigitte Kieffer , Christopher J. Evans and Wendy Walwyn
123
Dept of Psychiatry and Biobehavioral Sciences, and Dept of Psychology UCLA, CA, USA Dept of Psychiatry, Faculty of
Medicine, McGill University, Montreal, Canada
The abuse of prescriptions opioids and fatalities from opioid overdose has increased exponentially over the last
decade. Yet our understanding of the molecules involved and underlying neural circuitry mediating opioid reward islimited. We have begun to examine the role of specific mu opioid receptor populations in neurons that also express
dopamine receptor 1 (D1) or dopamine receptor 2 (D2), important striatal intermediates of the mesolimbic reward
pathway. Using conditional mu opioid (mu) receptor mice generated by Kieffer and colleagues (PMC3771900) bred with
D1 or D2 Cre recombinase mice, mu receptors were deleted from D1 or D2 neurons. Opioid reward-related behaviors;
opioid-induced hyperlocomotion or opioid self-administration, were then assessed. We found that mice lacking mu
receptors in D1 neurons showed a reduction in both the locomotor effect and sensitization profile of morphine (10mg/kg),
and oxycodone (10mg/kg), yet the locomotor profile of cocaine (20mg/kg) remained unchanged. Conversely, mice lacking
mu receptors in D2 neurons showed enhanced sensitization to oxycodone (10mg/kg). Intravenous self-administration of
oxycodone (0.25mg/kg/inf 2h access) revealed several deficits of the self-administration profile; a reduction in the numberof infusions earned and of goal-directed opioid seeking in mice lacking mu receptors in D1 neurons. Collectively, these
initial data show that mu receptors expressed in D1 or D2 neurons play specific roles in opioid-reward related behaviors.
These data also suggest that opioid reward results from an interplay of mu receptor signaling in D1 and D2 neurons.
Supported by NIDA (DA005010: JDJ, BK, CJE, WW and 5T32DA024635-07: DP) and the Hatos Foundation.
106. Coupled activation of primary sensory neurons contributes to chronic pain
Yu Shin Kim1, Kyoungsook Park1, Saijilafu2, Liang Han1, Zhe Li1, Catherine Gong1, LeAnne Young1, Shaoqiu He3,
Fengguan Zhou2, Yun Guan3, Michael J. Caterina1,4 and Xinzhong Dong1,5
1Department of Neuroscience, Center of Sensory Biology, the Johns Hopkins University School of Medicine, 725 N. Wolfe
Street, Baltimore, MD 21205 USA; 2Department of Orthopedic Surgery, the Johns Hopkins University School of Medicine,
725 N. Wolfe Street, Baltimore, MD 21205 USA; 3Department of Anesthesiology, the Johns Hopkins University School of
Medicine, 725 N. Wolfe Street, Baltimore, MD 21205 USA; 4Department of Biological Chemistry, the Johns Hopkins
University School of Medicine, Baltimore, MD 21205, USA; 5Howard Hughes Medical Institute, the Johns Hopkins
University School of Medicine, Baltimore, MD 21205, USA
Primary sensory neurons in the dorsal root ganglia (DRG) play an essential role in initiating pain by detecting painfulstimuli in the periphery and sending signals to the spinal cord via their axons. Pathological conditions such as
inflammation and nerve injury can sensitize DRG neurons, causing heightened pain sensitivity and often leading tochronic pain. Although the mechanisms of hypersensitivity of individual neuron have been extensively studied, how DRG
neurons function at a population level under physiological and pathological conditions is unclear due to the lack of proper2+
tools. By specifically expressing a genetically-encoded Ca indicator in almost all dorsal root ganglion (DRG) andtrigeminal ganglion (TG) neurons in Pirt-GCaMP mice. Using this technique, we developed an in vivo live imaging
technique which allowed us to simultaneously monitor the activation of >1,700 neurons/DRG in response to mechanicalstimulation applied to the receptive field in live mice. Using this powerful technique, we revealed a striking neuronal
coupling phenomenon that is adjacent neurons tend to activate together in mice with inflammation or nerve injury, whichrarely happens in naïve animals. The coupled activation occurs among various sizes of neurons including small-diameter
nociceptors and large-diameter low-threshold mechanoreceptors. The transferring of non-membrane permeable dye
between coupled-activating neurons suggests that the coupled activation is likely due to non-diffusible cell-to-cellcommunication. Combining pharmacological and genetic approaches with the imaging technique, we found the coupling55

phenomenon is mediated by the upregulation of gap junction in satellite glial cells surrounding DRG neurons after injury.
Blocking gap junction significantly attenuates neuronal coupling in the DRG and mechanical hypersensitivity. Therefore,
neuronal coupling represents a new form of neuronal plasticity in the DRG and by “hijacking” neighboring neurons throughgap junction it contributes to pain hypersensitivity. Finally, this study creates a new way of characterizing the physiologicalproperties and functions for developing new pain- or other modality-specific drug target for a treatment with few side
effects.
107. Comparative effects of a semi-synthetic salvinorin A analog, 16-Br-salvinorin A in a translational
neuroendocrine biomarker assay, and on sedation scores
Eduardo R. Butelman1, Andrew P. Riley2, Thomas E. Prisinzano2 and Mary Jeanne Kreek1
1Laboratory on the Biology of Addictive Diseases, The Rockefeller University, and 2Dept. of Medicinal Chemistry,
University of Kansas School of Pharmacy, Lawrence KS
The neoclerodane salvinorin A, from the plant Salvia divinorum, is a potent high efficacy KOP-r agonist, and has
several “on-target” undesirable effects, including sedation, aversion and hallucinogenesis. KOP-r ligands may have usefulpharmacotherapeutic properties in modulating mood diseases and addictive diseases. Synthetic efforts are examining
whether novel salvinorin A analogs can show differential potency in causing KOP-r agonist effects in vivo, as compared tothe aforementioned undesirable effects. Recent studies with the novel analog 16-Br-salvinorin A (Riley et al., 2014; J.
Med. Chem. 57:10464-10475) showed that this compound retained high potency KOP-r agonist effects in vitro; it was alsoable to decrease reinstatement of cocaine self-administration in rats, in the absence of locomotor sedative-like effects. We
examined the effects of 16-Br-salvinorin A in a translational neuroendocrine biomarker assay of KOP-r agonist effects(prolactin release), in male non-human primates (n=3), while also monitoring its ability to produce sedative-like effects
common in reference KOP-r agonists. 16-Br-salvinorin A (0.0032-0.1 mg/kg, i.v.) was equieffective to salvinorin A (0.0010.032 mg/kg), but was approximately 3-fold less potent in the biomarker assay. However, 16-Br-salvinorin A caused little
or no sedation, as scored with an observational rating scale over this dose range (non-blinded observer rating). Thesestudies indicate that novel salvinorin A analogs may retain KOP-r agonist effects, but with a potentially lower burden ofundesirable “on-target” effects. We gratefully acknowledge funding primarily by NIH-NIDA grant DA 018151. None of theauthors has a conflict of interest related to this research.

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We look forward to seeing you next year in

Bath, England for INRC 2016,
June 10-14
Check http://www.inrcworld.org/2016/2016mtg.htm for
updates61