Program Book

INRC

International Narcotics Research Conference
July 9-14, 2017 | Chicago IL USA
Hyatt Centric Magnificent MileINRC Executive Committee
Fred Nyberg (Sweden, President)
John Traynor (USA, Past-President)
Larry Toll (USA, Treasurer)
Craig W. Stevens (USA, Information Officer)
Cathy Cahill (USA)Amynah Pradhan (USA)Jim Zadina (USA)
Chris Bailey (UK)
Lucia Hipolito (Spain)
Christoph Stein (Germany)
Paul Bigliardi (Singapore)
Meritxell Canals (Australia)
Kazutaka Ikeda (Japan)

Organizing Committee
Amynah Pradhan (USA)Ream Al-Hasani (USA)Michael Bruchas (USA)Cathy Cahill (USA)Louis Gendron (Canada)
Susan Ingram (USA)
Emily Jutkiewicz (USA)
Nicolas Massaly (USA)Michael Morgan (USA)Jose Moron-Concepcion (USA)
Manojkumar Puthenveedu (USA)
Tuan Trang (Canada)
John Traynor (USA)Richard van Rijn (USA)Adie Wilson-Poe (USA)Contents
Pages
Exhibitor & Sponsor Information……………………………… 2-3
Location Map…………………………………………………… 4
INRC 2017 Awardees………………………………………… . 5-6
Meeting Schedule……………………………………………… 7-13
List of Posters……………………………………………………14-23
Abstracts………………………………………………………… 24-103

Photographic Policy
Please note that photographs taken at this meeting may be used on our website, social
networking sites, and in other publications. If you do not wish to have your image used
for this purpose, please speak to a member of the INRC Executive Committee or email
apradhan@psych.uic.edu.

1Thank You to our Generous Sponsors!

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INRC 2017 AWARDEESFounders’ Award – Professor John Traynor
Dr. John Traynor is Professor of Pharmacology and Associate
Chair for Research at the University of Michigan Medical School.
He holds the Edward F Domino Research Professorship in
Pharmacology and is Principal Investigator of a NIDA-fundedtraining grant on the biology of drug abuse and holder of a NIDA
Merit Award. Also, Dr. Traynor chairs the Neuropharmacology
Division of the American Society for Pharmacology andExperimental Therapeutics and is a standing member of
Neurobiology of Motivated Behavior Study Section of CSR(Center for Scientific Review) of the National Institutes of Health.
He has served as Director of the University of Michigan
Substance Abuse Research Centre for a four years term and wasa member of the Medical School’s Biomedical Research Council
from 2010-2016. Dr. Traynor was a permanent member of The National Institute on Drug Abuse(NIDA) Training and Career Development Committee and has served on the editorial boards ofJournal of Neurochemistry and the Journal of Medicinal Chemistry. He is currently on the editorial
board of the British Journal of Pharmacology.
Dr. Traynor earned degrees in Pharmacy (B.S.) and Medicinal Chemistry (Ph.D.), both from the
University of Aston in the United Kingdom. His post-doctoral training at the University of Gottingen
in Germany concentrated on Biochemical Pharmacology. For more than nearly three decades,
Dr. Traynor’s research has focused on opioids and includes areas such as structure-activity
relationships, signaling and behavior. He has trained a large number of PhD students andpostdoctoral fellows as well as many Masters and Undergraduate students.
Dr. Traynor’s laboratory employs a wide range of techniques from molecular biology to behavioral
pharmacology. Strong analgesic drugs such as morphine are extremely important both for thetreatment of pain and for the medical and social consequences of opiate (heroin) addiction. The
laboratory uses in vitro and in vivo models to characterize the mechanism of action of these drugs.
In recent years, Dr. Traynor’s research has focused on alternative ways to better harness theanalgesic effects of endogenous opioid peptides by targeting a specific family of intracellular
proteins (RGS proteins) that act as negative regulators of a variety of neurotransmitters, and/or
developing allosteric compounds that modulate opioid receptors in unique ways. The long-term
aim of these studies is to identify novel targets and medications to treat pain, drug dependence
and depression.
Dr. Traynor is also widely appraised for his role in organizing symposia in national andinternational conferences. He has been a frequent attendant at the INRC meetings throughout his
career and together with his students and collaborators provided outstanding contributions to ourorganization. He was President of the International Narcotics Research Conference (INRC, 20102014) and continues to serve on the Executive Committee of INRC as the immediate pastpresident. Over the years he has really been an active attendant at the INRC meetings andparticipated in discussion of actual topics in a way that reveal an outstanding competence andexperience in questions related to the field of opioids. Unambiguously, Dr. Traynor belongs to thecore of successful and prominent researchers that has been essential for development andmaintenance of INRC as an organization promoting high-quality science in the area of opioid
research.
5Young Investigators Award – Dr. Aashish Manglik
Dr. Aashish Manglik is currently a Stanford Distinguished Fellow
and Instructor of Molecular and Cellular Physiology at Stanford
University School of Medicine. He has an independent position inwhich he has his own laboratory and funding, including an Early
Independence Award from the NIH Director.
Dr. Manglik has been recognized for his work on G protein-coupled
receptor signaling and in in silico drug discovery. His work has
elucidated the structural basis of opioid receptor function, thereby
yielding insight into the mechanism of action of fundamental andwidely used analgesics like morphine and codeine. Using thesestructures, he has pioneered the development of novel opioid
analgesics, identified by in silico methods, which potentially possess
reduced dose-limiting side effects. Dr. Manglik’s work has been published in more than twenty
peer-reviewed articles, and he was a key contributor to the invention of the core technologies at
a company he co-founded called Ab Initio Biotherapeutics.
Dr. Manglik received his B.A. in Biology and Chemistry at Washington University in St. Louis,
where he was awarded the Spector Prize for his outstanding academic and researchachievements. At Stanford, he was awarded an American Heart Association Pre-doctoral
Fellowship in 2012. In his research there, Dr. Manglik used biophysical techniques such as X-ray
crystallography and NMR spectroscopy. Obtaining purified opioid receptors and subsequent
crystal structures is a tremendous achievement and requires the very highest abilities andperseverance. These results also rely on the development of new tools to stabilize thesechallenging proteins. Several of his papers and patents describe the development and design ofsingle domain antibodies (nanobodies) for different conformational states of the mu-opioid
receptor. Dr. Manglik trained as an MD as well as a PhD, and his medical training allows him tocover a broad picture generated by the uncovering of these structures. For example, his mostrecent achievement, and work that he presented at INRC 2016, has been published in Nature. It
describes the structure-based discovery of PZM21, an opioid with reduced side-effects in rodent
models. This was a mammoth effort across the laboratories of prominent and well-reputed
scientists and demonstrates not only Dr. Manglik’s ability to interact and collaborate with top
researchers but also his breadth of knowledge.
Dr. Manglik has presented many of his findings at meetings and universities in the US but alsoabroad, e.g. at the above mentioned INRC meeting in Bath (UK) in 2016. At the meeting in Bath,he gave a highly impressive talk on his work on the mu-opioid receptor structure and the discovery
of PZM21. His engagement at this meeting attracted many INRC members and his creative
attitude will certainly bring an excitement to INRC and encourage other young scientists working
in related areas to become regular attendees.

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Schedule for INRC 2017
Sunday July 9, 2017
3:00-5:30 PM - Registration
5:30-6:00 PM – Pre-welcome reception for trainees
6:00-8:00 PM - Welcome Reception

Monday July 10, 2017
7:00-8:30 AM - Breakfast
8:15-8:30 AM - Welcome Address
Plenary Lecture
8:30-9:30 AM - Mark von Zastrow – University of California San Francisco, CA, USA - Cell
biology of opioid drug action
Opioid Receptor Trafficking & Signaling
Chair: Aki Ozawa - Torrey Pines Institute, FL, USA
Discussion Leader: Louis Gendron – Université de Sherbrooke, QC, Canada
9:30-9:55 AM – Manoj Puthenveedu – Carnegie Mellon University, PA, USA- Endocytic control
of functional selectivity of opioid receptors
9:55-10:20 AM – Lee-Yuan Liu-Chen – Temple University, PA, USA– Characterization of amutant mouse line expressing a fusion protein of kappa opioid receptor and tdTomato
10:20-10:35 AM – Hot Topic 1 – Dominique Massotte - Institut des Neurosciences Cellulaires etIntegratives, Strasbourg, France - Trafficking & signaling of endogenous mu-delta heteromers.
10:35-10:55 AM - Coffee Break
10:55-11:20 AM - Nathaniel Jeske – University of Texas Health Science Center at San Antonio,
TX, USA – Peripheral Opioid Receptor Regulation
11:20-11:45 AM - Meritxell Canals – Monash University, Melbourne, Australia - GPCR signaling
platforms for pain and analgesia
11:45-12:00 PM – Hot Topic 2 - Marta Filizola - Icahn School of Medicine at Mount Sinai, NY,
USA - Structural and Dynamic Elements of µ-Opioid Receptor Functional Selectivity

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12:00-12:15 PM – Hot Topic 3 – William Birdsong – Oregon Health and Sciences University,
OR, USA - Opioid modulation of synaptic transmission in affective pain circuitry
12:15-12:30 PM – Discussion
12:30-1:30 PM – Lunch
Kappa opioid and nociceptin peptides in reward and aversion circuits.
Chair: Ream Al-Hasani - St. Louis College of Pharmacy and Washington University, MO, USA
Discussion Leader: Larry Toll – Torrey Pines Institute, FL, USA
1:30-1:55 PM - Ream Al-Hasani - St. Louis College of Pharmacy and Washington University,
MO, USA - Circuit dynamics of in vivo dynorphin release in the nucleus accumbens shell
1:55-2:20 PM - Hugo Tejeda - Bonci Lab, NIDA-IRP, MD, USA - Kappa-opioid receptor control
of nucleus accumbens synaptic integration differentially gates D1 and D2 MSN activity
2:20-2:45 PM - Olivier George – Scripps Research Institute, CA, USA - Upregulation ofdynorphin in the central nucleus of the amygdala mediates the negative emotional states of
nicotine withdrawal but not escalation of nicotine intake.
2:45-3:05 PM – Coffee Break
3:05-3:30 PM - Andrew Hardaway - Kash Lab, University of North Carolina, NC, USA, - Central
amygdala Prepronociceptin-expressing neurons mediate palatable food consumption & reward
3:30-3:55 PM - Kyle Parker - Bruchas Lab Washington University, MO, USA - Midbrain
nociceptin neurons modulate reward behaviors
3:55-4:10 PM – Hot Topic 1 - Anushree N. Karkhanis - Wake Forest School of Medicine, NC,
USA - Adolescent social isolation increases kappa opioid receptor function in the nucleus
accumbens and basolateral amygdala of rats.
4:10-4:25 PM – Hot Topic 2 - Kabirullah Lutfy – Western University, CA, USA - Male but not
female mice lacking nociceptin exhibit higher anxiety and enhanced reward following exposure
to the elevated plus maze test.
4:25-4:40 PM – Discussion
4:40-5:10 PM – Data blitz Session 1
5:10-7:00 PM – Poster Session A

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Tuesday July 11, 2017
7:00-8:30 AM - Breakfast
Plenary Lecture
8:30-9:30 AM - Michael Salter – Hospital for Sick Children & University of Toronto, Toronto,
Canada - Sex, Pain and Microglia
Targeting opioid receptors to treat alcohol use disorders
Chair: Meredith Robins – Purdue University, IN, USA
Discussion Leader: Kabirullah Lutfy – Western University, CA, USA
9:30-9:55 AM – Richard van Rijn – Purdue University, IN, USA - The potential of delta opioid
receptors as novel target for alcohol use disorders
9:55-10:20 AM – Subhash Pandey - University of Illinois at Chicago & Jesse Brown VA, IL, USA- A role of epigenetically regulated dynorphin/kappa-opioid receptor and neuropeptide Y system
in alcohol tolerance
10:20-10:35 AM – Hot Topic 1- Braulio Munoz – Indiana University School of Medicine Ethanol disrupts synaptic specific mu opioid receptor-mediated long term depression in dorsal
striatum
10:35-10:55 AM - Coffee Break
10:55-11:20 AM - Annika Thorsell – Linkoping University, Linkoping, Sweden – The OPRM1
A118G SNP in drug reinforcement and treatment response to naltrexone: Evidence from ahumanized mouse model.
11:20-11:45 AM - Linda Rorick-Kehn – Lilly Laboratories, Eli Lilly and Company, IN,USA - The
novel nociceptin receptor antagonist LY2940094 reduces ethanol-seeking and selfadministration in animal models and significantly reduces heavy drinking in alcohol-dependent
subjects.
11:45-12:00 PM – Hot Topic 2 – Lucia Hipolito - University of Valencia, Valencia, Spain –Inflammatory pain alters the vulnerability to alcohol relapse without impacting the intensity of therelapse in rats.
12:00-12:15 PM – Hot Topic 3 – Moriya Yuki - Tohoku University, Sendai, Japan - The effects ofchronic stress on alcohol consumption in μ-opioid receptor knockout mice
12:15-12:30 PM – Discussion
12:30-1:30 PM – Lunch

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Opioids and Pain
Chair: Nicolas Massaly – Washington University, MO, USA
Discussion Leader: Cathy Cahill – University of California Irvine, CA, USA
1:30-1:55 PM – Jim Wang - University of Illinois at Chicago, IL, USA - Protein kinasemechanisms in opioid-induced hyperalgesia
1:55-2:20 PM – Greg Scherrer – Stanford University, CA, USA - Circuits and synaptic
mechanisms for pain control by endogenous and exogenous opioids
2:20-2:35 PM – Hot Topic 1 - Shiwei Liu – University of California Irvine, CA, USA - Kappa
opioid receptor up-regulation contributes to mood and reward dysregulation in neuropathic pain.
2:45-3:05 PM – Coffee Break
3:05-3:30 PM – Hiroshi Ueda – Nagasaki University, Nagasaki, Japan - Therapeutic recovery
from diminished morphine analgesia in chronic pain state
3:30-3:55 PM – Vanna Zachariou, Icahn School of Medicine at Mount Sinai, NY, USA Targeting RGSz1 to optimize the actions of opioid analgesics.
3:55-4:10 PM – Hot Topic 2 – Moe Watanabe - Hoshi University, Tokyo, Japan Characterization of the endogenous small-sized peptides released in the nucleus accumbens
related to pain modulation
4:10-4:25 PM – Hot Topic 3 – Gregory Corder – Stanford University, CA, USA - A neuralnetwork for abstracting nociceptive information into an aversive pain perception
4:25-4:40 PM – Discussion
4:40-5:10 PM – Data blitz Session 2
5:10-7:00 PM – Poster Session B

Wednesday July 12, 2017
7:00-8:30 AM - Breakfast
Founders’ Lecture
8:30-9:30 AM – John Traynor – University of Michigan, MI, USA - Opioid Research: pastachievements – future challenges

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Emerging Techniques & Hot Topics
Chair: Amanda Fakira - Icahn School of Medicine at Mount Sinai, NY, USA
Discussion Leader: Michael Bruchas – Washington University, MO, USA
9:30-9:45 AM – Brigitte Kieffer – McGill University/Douglas Institute, Montreal, Canada –Probing whole-brain connectivity in live mice: receptors and drugs
9:45-10:00 AM – Elena Romanova – University of Illinois Urbana-Champaign, IL, USA –Unbiased characterization of intercellular signaling peptides using mass spectrometry
10:00-10:15 AM – Emma Childs – University of Illinois at Chicago, IL, USA – Human laboratory
paradigms to study conditioned cues
10:15-10:30 AM – Michael Morgan – Washington State University, WA, USA - Preclinical
Assessment of opioid analgesia, side effects, and withdrawal
10:30-10:50 AM - Coffee Break
10:50-11:05 AM - Damien Jullie - University of California San Francisco, CA, USA - Probing
axonal trafficking with live cell imaging of pHluorin tagged Mu Opioid Receptor
11:05-11:20 AM - Hot Topic 1 – Louis Gendron – Université de Sherbrooke, QC, Canada Investigating delta opioid receptors in a new conditional knockin mouse
11:20-11:35 AM – Hot Topic 2 – Aliza Ehrlich – McGill University, Douglas Research Center,
QC, Canada - Distinct MOR agonist profiles revealed by visualizing Mu opioid receptor
trafficking with MOR-Venus mice
11:35-11:50 AM – Hot Topic 3 – Emily Jutkiewicz - University of Michigan, MI, USA - Allostericactivation of delta opioid receptors in vivo: promising behavioral profile
11:50-12:05 PM – Hot Topic 4 – Sidney Williams – Washington University, MO, USA Development of a Virtual Reality Paradigm for in vivo Hippocampal Imaging During Morphine
Conditioned Place Preference
12:05-12:30 PM – Discussion
12:30-1:30 PM - Lunch
1:30 PM – Free afternoon and breakout session on Antibodies & Mutant Mice (Christopher
Evans - University of California Los Angeles USA, and Brigitte Kieffer - McGill University Douglas Institute, Canada)

11Thursday July 13, 2017
7:00-8:30 AM - Breakfast
Plenary Lecture
8:30-9:30 AM - Plenary Lecture – Catherine Bushnell, National Center for Complementary andIntegrative Health, NIH, Bethesda, MD, USA - Alterations of the opioid system in chronic pain
Opioid Epidemic
Chair: Kyle Windisch - Rockefeller University, NY, USA
Discussion Leader: Jose Moron-Concepcion – Washington University, MO, USA
9:30-9:55 AM – Theodore J Cicero – Washington University, MO, USA - History of Prescription
Opioid abuse and the Recent Transition to Heroin
9:55-10:20 AM – Sandra Comer – Columbia University and the New York State PsychiatricInstitute, NY, USA – Pharmacological Therapies for Opioid Use Disorder: Implications ofFentanyl Availability on Treatment Effectiveness
10:20-10:35 AM – Hot Topic 1 – Barbara Belli - Orexigen Therapeutics, CA, USA - OREX-1019;
a small molecule with potential for addiction maintenance treatment and relapse prevention
10:35-10:55 - Coffee Break
10:55-11:20 AM - Andrew Charles – University of California Los Angeles, CA, USA –Prescription opioids and migraine: the basic and clinical science of a major public health issue
11:20-11:45 AM Tuan Trang – Hotchkiss Brain Institute, University of Calgary, Calgary, Canada- Blocking microglial pannexin-1 channels alleviates opioid withdrawal,
11:45-12:00 PM – Hot Topic 2 - Laura Moye – University of Illinois at Chicago - Identifying therole of peripheral delta opioid receptors in chronic migraine
12:00-12:15 PM – Hot Topic 3 – Seven Tomek – Arizona State University, AZ, USA - Effects of
Heroin on Prosocial Behavior in Rats.
12:15-12:30 PM – Discussion
12:30-1:30 PM – Lunch
Young Investigator Symposium – Ligand Directed Signaling at Opioid Receptors
1:30-2:15 PM – Aashish Manglik - Stanford University, CA, USA - Structural dynamics in G
protein-coupled receptor signaling12

2:15-2:40 PM - Susruta Majumdar – Memorial Sloan Kettering Cancer Center, NY, USA Kratom derived natural products as leads to design analgesics with mu opioid G-biased
agonism and delta opioid antagonism
2:40-2:55 PM – Hot Topic 1 - Mariana Spetea - University of Innsbruck, Innsbruck, Austria Ligand-receptor and structure-function relationship studies on differently substituted
diphenethylamines interacting with the κ-opioid receptor
2:55-3:15 PM – Coffee Break
3:15-3:35 PM – Jonathan Violin – Trevena Inc, PA, USA - Discovery and development ofoliceridine, a G protein-biased ligand targeting the mu-opioid receptor
3:35-3:50 PM – Hot Topic 2 - Kylie B. McPherson - Oregon Health & Science University, OR,
USA – Agonist Functional Selectivity determined by RGS proteins.
3:50-4:05 PM – Hot Topic 3 – Indrajeet Sharma – University of Oklahoma, OK, USA –Collybolide, a novel biased agonist to study kappa-opioid receptor pharmacology.
4:05-4:20 PM – Discussion
4:20-5:00 PM – INRC Business Meeting
5:00 PM – Conference Close
6:00-7:00 PM – Closing Reception - Odyssey Cruise Boarding
7:00-9:30 PM – Cruise Banquet and Dancing

Friday July 14, 2017
Departure13

Poster Presentations
1 - Political Priority for Tobacco Control among Adolescents in Local Governments
Areas, Osun State, Nigeria
Ademola Adelekan
2 - Effectiveness of Provider Initiative Approach for Smoking Cessation among Pregnant
Women in Osun State, Nigeria
Ademola Adelekan
3 - Ring Substitution in the Macrocyclic Opioid Peptide CJ-15,208 Alters the Opioid
Activity Profile in vivo
J. V. Aldrich
4 - Identification of a novel interaction site of the mu-delta opioid receptor heterodimers
Doungkamol Alongkronrusmee
5 - Low Abuse Liability of the Endomorphin Analog ZH853 is Supported by Reduced
Psychomotor Activation and Withdrawal.
Ariel Amgott-Kwan
6 - Morphine influences cell proliferation, migration and apoptosis of tumor cell lines
endogenously expressing mu opioid receptors
Andrea Bedini
7 - OREX-1019; a small molecule with potential for addiction maintenance treatment andrelapse prevention
Barbara Belli PhD
8 - OREX-1038; a potent analgesic molecule with potential for reduced abuse liability
Barbara Belli PhD
9 - Rewarding effects of opioidergic projections from the ventral pallidum to substantia
nigra
Julio Bernardi
10 - Opioid receptors modulate skin ageing and melanocytic disorders
Bigliardi PL1
11 - μ-Opioid receptor trafficking in human keratinocytes
Bigliardi-Qi M*
12 - Opioid modulation of synaptic transmission in affective pain circuitry
William Birdsong
13 - Antidepressant activity of the buprenorphine analog BU10119
Caroline A. Browne

1414 - Peripherally restricted opioid combination therapy synergizes in multiple pain states
Daniel J. Bruce
15 - Characterization of opioid receptor subtypes and biased signaling as factors formodulation of alcohol use in mice.
Robert J. Cassell
16 - Nucleus accumbens mu-opioid receptors are recruited and necessary for theenhancement of motivated behaviors
Daniel C Castro
17 - Characterization of Sigma 1 Receptor (S1R) antagonist CM-304 and a related analog,
AZ-66: Evaluating novel therapeutics for allodynia and induced pain
Thomas J. Cirino
18 - Oxycodone-induced conditioned place preference and locomotor activity in male andfemale Oprm1 A112G mice
Devon Collins
19 - A neural network for abstracting nociceptive information into an aversive pain
perception
Gregory Corder
20 - On Target effects of Mu opioid receptor activation on brain activity and connectivity
identified by mouse fMRI
Emmanuel Darcq
21 - Differential regulation of delta opioid receptor-mediated behaviors by arrestin2 inmice
Isaac J. Dripps
22 - Distinct MOR agonist profiles revealed by visualizing Mu opioid receptor trafficking
with MOR-Venus mice
Aliza T. Ehrlich
23 - Percutaneous Electrical Nerve Stimulation (Electro-Acupuncture) elevates Agmatine
in Spinal Cords of Nerve-Injured Rats.
Samuel J. Erb
24 - Evaluation of opioid-nociceptin bifunctional ligands by biochemical,
pharmacological and structural modeling tools
Anna I. Erdei
25 - How did the Opioid System Evolve?
Chris Evans
26 - The role of striatal mu opioid receptor populations in reward-related behaviors.
Christopher Evans15

27 - The role of the deorphanized receptor, GPR83, in stress and reward.
Amanda K. Fakira
28 - Assessment of ZH853, a novel endomorphin analog, versus morphine in acute andlong term dosing for chronic pain.
Amy Feehan
29 - Structural Insights into Opioid Peptidomimetics with Bifunctional Mu-Opioid
Agonism and Delta-Opioid Antagonism
Thomas Fernández
30 - Structural and Dynamic Elements of µ-Opioid Receptor Functional Selectivity
Marta Filizola
31 - Characterizing the biochemical and synaptic properties of CNIH3, a novel component
in individual risk for opioid dependence
Hannah Frye
32 - Mechanistic insights into opioid-induced chemoresistance in breast cancer cells:
DOR stimulation increases P-glycoprotein abundance by exploiting EphA2/c-src
signaling
Daniela A. Fux
33 - Investigating delta opioid receptors in a new conditional knockin mouse
Louis Gendron
34 - Oxycodone conditioned place preference alters hippocampal mossy fiber leuenkephalin levels in a sex-dependent manner
Jason D. Gray
35 - Delta-opioid receptor (DOR) antagonism/inverse agonism reduces the developmentof mu-opioid receptor (MOR) tolerance
Nicholas Griggs
36 - Selective Inhibition of M5 Muscarinic Acetylcholine Receptors Attenuates
Remifentanil Self-Administration without Blocking Morphine-induced Analgesia in Rats
Barak W. Gunter
37 - Functional consequences of OPRM1 A118G (MOR N40D) on humanneurotransmission
Apoorva Halikere
38 - Crucial role of the κ-opioid receptor system in tumor angiogenesis
Yusuke Hamada
39 - CaMKIIα controls the biogenesis of let-7 microRNAs in opioid tolerance
Ying He16

40 - The G-protein biased opioid agonist PZM21 does indeed depress respiration
Rob Hill
41 – Inflammatory pain alters the vulnerability to alcohol relapse without impacting theintensity of the relapse in rats.
Lucia Hipolito
42 - Simultaneous administration of a chemokine receptor antagonist with morphineenhances the analgesic effect of morphine on incisional pain in rats
S. Inan
43 - Dermatolgical manifestations in substance abuse and how the community responses
in developing country
Shambhu D Joshi
44 - Opioid receptors reveal a parallel cycle of regulated presynaptic membranetrafficking
Damien Jullie
45 - Probe Dependence of High and Low Affinity Agonist Binding to the Delta Opioid
Receptor is Allosterically Regulated by BMS-986187 and Na+ Ions
Evan Schramm
46 - Allosteric Agonism at the Delta Opioid Receptor: Potential Mechanism for Functional
Selectivity
Matthew Stanczyk
47 - Allosteric activation of delta opioid receptors in vivo: promising behavioral profile
Emily Jutkiewicz
48 - Analysis of medication overuse headache after repeated morphine or THC
administration using home cage wheel running
Ram Kandasamy
49 - Effect of opioids on the cellular responses to anti-cancer drugs
Leena Karhinen
50 - Adolescent social isolation increases kappa opioid receptor function in the nucleus
accumbens and basolateral amygdala of rats.
Anushree N. Karkhanis
51 - Norbuprenorphine pharmacokinetics and pharmacodynamics: First in man
evaluation
Evan D. Kharasch17

52 - Serum and urine concentrations of morphine and morphine metabolites in patientswith advanced cancer receiving continuous intravenous morphine: an observational
study
Soo Jin Kim
53 - Evaluation of the dual kappa and delta opioid receptor agonist MP1104 in rat modelsof cocaine self-administration, analgesia and behavioural side-effects
Bronwyn Kivell
54 - Regulation of morphine sensitivity and adenylate cyclase sensitization by carboxylterminus of V1b vasopressin receptors
Taka-aki Koshimizu
55 - Generation and characterization analysis of human iPS cell derived-sensory neurons
as a useful research tool for pain and opioid systems
Naoko Kuzumaki
56 - Factors associated with ketamine use in pancreatic cancer patient in a single
hospice center
Kyung min Kwon
57 - Sex differences in opioid signaling
Emily Leff
58 - Genetic deletion of mu opioid receptors from Kölliker-Fuse neurons reducesmorphine-induced respiratory depression
Erica Levitt
59 - Cannabinoid CB2 receptor plasticity in rostral ventromedial medulla in inflammatory
pain
Ming-Hua Li*
60 - The CB2 cannabinoid receptor agonist LY2828360 suppresses chemotherapyinduced neuropathic pain with sustained efficacy and attenuates morphine tolerance anddependence
Xiaoyan Lin
61 - Kappa opioid receptor up-regulation contributes to mood and reward dysregulationin neuropathic pain
Shiwei Liu
62 - The role of endogenous dynorphins in amphetamine sensitization and depressionlike behaviors in mice
Kabirullah Lutfy
63 - AT-328, a selective NOP agonist, reduces the rewarding action of ethanol andcocaineKabirullah Lutfy
18

64 - The role of mu opioid receptors in hyperlocomotion and sensitization induced byamphetamine
Kabirullah Lutfy
65 - Male but not female mice lacking nociceptin exhibit higher anxiety and enhanced
reward following exposure to the elevated plus maze test
Kabirullah Lutfy
66 - Activation of δOR alleviates κOR-mediated place aversion: MP1104, a dual κOR-δOR
agonist is an analgesic and anti-addiction agent with attenuated side effects
Susruta Majumdar
67 - Dissecting dopamine pathways altered by pain-induced dysfunction in opioid
signaling
Tamara Markovic
68 - Pain Recruits Accumbal Kappa Opioid System And Alters Opioid Consumption.
Nicolas Massaly
69 - Design and Synthesis of Collybolide Probes for Kappa-Opioid Receptor
Nicholas P. Massaro
70 - Trafficking and Signaling of Endogenous Mu-Delta Heteromers
Dominique Massotte
71 - Measurement of Heroin Antibody Binding Affinity Using Microscale Thermophoresis
Gary Matyas
72 - Neuroinflammatory effects of HIV-1 Tat protein correlate with elevated brain
dopamine levels and increased morphine consumption and conditioned place preference(CPP), and are prevented by indomethacin treatment
Jay McLaughlin
73 - Agonist functional selectivity determined by RGS proteins
Kylie B. McPherson
74 - Single-particle tracking as an effector-independent readout of mu opioid receptor
activity after agonist exposure
Marissa Metz
75 - The delta opioid receptor as an emerging therapy for mTBI-induced headaches
Laura Moye
76 - Identifying the role of peripheral delta opioid receptors in chronic migraine
Laura Moye19

77 - Ethanol disrupts synaptic specific mu opioid receptor-mediated long term
depression in dorsal striatum
Braulio Munoz
78 - Free fatty acid receptor hypothalamic GPR40/FFAR1 regulate β-endorphin release via
prohormone convertase 2 protein expression
Kazuo Nakamoto
79 - Opiate Use Disorder in Pregnancy in Crawford County, Ohio: A Case Study
Madeline L. Novack
80 - The effects of commonly used opioids in cell cultures
Erik Nylander
81 - Metformin acting through the mammalian target of rapamycin complex 1 (mTORC1)
attenuates morphine efficacy in a mouse model of neuropathic pain.
Ilona Obara
82 - Development of a Selective Antagonist for the Mu-Delta Heterodimer
Keith M. Olson
83 - Nociceptin receptor trafficking in midbrain dopamine neurons
Patrick R O'Neill
84 - Spinal pain regulation of the N/OFQ-NOP receptor system in a spinal nerve injury
mouse modelAkihiko Ozawa
85 - A novel non-canonical role of RGS4 in δ-opioid receptor mediated neuronaloutgrowth and differentiation
Paschalina Pallaki
86 - Genetic dissociation of morphine analgesia from hyperalgesia in mice
Gavril Pasternak
87 - Analgesic and Anti-Inflammatory Effects of Novel Kappa Opioid Receptor Agonist
16-Ethynyl Salvinorin A
Kelly Paton
88 - Morphine-dosing affects development of antinociceptive tolerance
Alok Kumar Paul89 - Role of mu opioid receptor A112G SNP in the mesolimbic neurocircuitry
Dina Popova
90 - Imatinib prevents morphine tolerance without inducing MOR endocytosis.
Puig S.

20

91 - Serotonergic and adrenergic gene variants and vulnerability to non-dependent
opioid use and heroin dependence in a Dutch population
Matthew Randesi
92 - Conditioned place aversion to an “extremely” G-protein biased kappa opioid
receptor agonist in C57BL6 mice
Brian Reed
93 - Critical role of Gi-protein signaling in the dorsal striatum for the reduction ofvoluntary alcohol consumption in C57Bl/6 mice
Meridith Robins
94 - Convergent and Divergent Behavioral Changes Caused by Different Patterns ofMorphine Exposure in Mice
Patrick E. Rothwell
95 - Anxiolytic-like and antidepressant-like effects of a novel delta opioid receptor
agonist NC-2800 in rats
Akiyoshi Saitoh
96 - Ligand-receptor and structure-function relationship studies on differently
substituted diphenethylamines interacting with the κ-opioid receptor
Helmut Schmidhammer
97 - Carboxyl-terminal multisite phosphorylation regulates µ-opioid receptor
desensitization and tolerance
Stefan Schulz
98 - Beyond cAMP assays: GIRK channels as ultra-sensitive sensors for thecharacterization of novel opioids using the voltage-sensitive dye FMP
Stefan Schulz
99 - COLLYBOLIDE, A NOVEL BIASED AGONIST TO STUDY KAPPA-OPIOID RECEPTOR
PHARMACOLOGY
Indrajeet Sharma
100 - Opioid-Elicited Erk1/2 Phosphorylation, Elk-1 and CREB Activation, and Lipid Raft
Localization in HEK 293 Cells Coexpressing μ-Opioid and Nociceptin Receptors
Cynthia Wei-Sheng Lee
101 - Effect of Magnetic Acupuncture Attachment on Neigun(acupoint P6) on Autonomic
Nervous System
Hyun Hwa Shin
102 - TRV0109101, a G protein-biased agonist of the µ-opioid receptor, does not promote
opioid-induced mechanical allodynia following chronic administration
Edward R. Siuda
21

103 - Dissecting Mechanisms of Nociceptin Opioid Peptide Receptor Regulation
Skylar Spangler
104 - Injectable peptide-based hydrogels as efficient matrices for the controlled-deliveryof opioid analgesics
Mariana Spetea
105 - Studies with Nociceptin/Orphanin FQ Peptide (NOP) Receptor knockout rats reveal
NOP receptor-dependent sex differences in nociceptive sensitivity, anxiety-like behaviors
and serum N/OFQ and CORT levels in response to traumatic stress
Kelly M. Standifer
106 - A pH-sensitive opioid analgesic designed by modeling of pathological receptorligand interactions
Christoph Stein
107 - Hsp90 and PEBP Promote Opioid Anti-Nociception Through the Regulation of Mu
Receptor Signal Transduction Cascades
John M. Streicher
108 - Modulation of endogenous opioid-mediated antinociception by regulator of G
protein signaling (RGS) proteins
Sherrica Tai
109 - Label-free mass spectrometry based quantitation of peptides related to chronic
migraine and opioid-induced hyperalgesia (OIH) in mice
Krishna D. B. Anapindi
110 - Overlapping mechanisms between opioid induced hyperalgesia and chronic
migraine
Alycia Tipton
111 - Influence of tramadol in global cerebral ischemia-induced mechanical allodynia
Shogo Tokuyama
112 - In Vitro And In Vivo Profile Of PPL-101 And PPL-103: Mixed Opioid Partial Agonist
Analgesics With Low Abuse Potential
Lawrence Toll
113 - Effects of Heroin on Prosocial Behavior in Rats
Seven Tomek
114 - Characterization of the endogenous small-sized peptides released in the nucleus
accumbens related to pain modulation
Moe Watanabe

22115 - Automated detection of Mu Opiod Receptor recycling reveals distinct
subpopulations of exocytic events
Zara Weinberg
116 - Development of a Virtual Reality Paradigm for in vivo Hippocampal Imaging During
Morphine Conditioned Place Preference
Sidney Williams
117 - Activation, Desensitization and Trafficking of total phosphodeficient MORs in the
MOR-knockout rat
John T Williams
118 - Inflammatory Pain Changes The Expression and Function Of CB1 Receptors in The
Periaqueductal Gray
Adrianne R. Wilson-Poe
119 - Naltrexone and Nalmefene Attenuate Cocaine Place Preference but Differentially
Effect Cocaine Locomotor Sensitization
KA Windisch120 - A Novel assay for measuring human μ-opioid receptor’s functional activity
Jinhua Wu
121 – Potential of circulating mu-opioid receptor and toll-like receptor-4 activation inmorphine-administered mice.
Nan XIE
122 - Alternatively spliced mu opioid receptor C termini impact the diverse actions ofmorphine
Jin Xu123 - Design and synthesis a novel κ opioid receptor agonists isolating both sedation
and aversion.
Masahiro Yata
124 - The effects of chronic stress on alcohol consumption in μ-opioid receptor knockout
mice
Moriya Yuki
125 - Commonly used opioids induce neurotoxic effects in neuronal cell cultures
Sofia Zelleroth23

Speaker Abstracts
Plenary 1 - An expanding view of functional selectivity in GPCR-directed drug action
Mark von Zastrow; UCSF
The concept of functional selectivity in GPCR-directed drug action has its early roots in the cellbiological study of opioid receptors and, although highly controversial when first proposed, is now
generally accepted to the point of motivating modern drug discovery campaigns. The biochemical
and cellular basis of functional selectivity, however, remains enigmatic. An early hypothesis thatis now widely recognized, and presently called biased agonism, posits that functional selectivity
among ligands is determined through differential coupling of ligand-occupied receptors toheterotrimeric G proteins relative to β-arrestins. I will briefly discuss the biochemical genesis ofsuch agonist bias at opioid receptors, focusing on ligand discrimination mediated by receptor
multi-phosphorylation. I will then discuss emerging evidence suggesting that there exists a
discrete and additional source of functional selectivity among opioid receptor-directed ligands,
determined by differences in the subcellular location at which receptors are activated. Such'spatial bias' is particularly striking in neurons and has interesting implications because it reveals
cellular effects produced by alkaloid drugs such as morphine that are not produced by peptide
agonists. I will then generalize the concept of spatial bias to other GPCRs, focusing on
mechanistic insight into spatial drug discrimination gained through study of adrenergic
receptors. Finally, I will discuss ongoing efforts to more incisively elucidate functional selectivity
using proximity labeling and quantitative proteomic methodologies.

Session 1: Endocytic control of functional selectivity of opioid receptors
Zara Y. Weinberg, Tiffany Phan, Manojkumar Puthenveedu PhD; Carnegie Mellon University,
Pittsburgh, PA 15213
Functional selectivity at the µ opioid receptor (µR) has been a major focus for drug discovery in
the recent past. Efforts to modify functional selectivity has focused on developing new orthostericor allosteric modulators that target µR itself. We test the hypothesis that functional selectivity of
any given agonist can be independently modified by trafficking events that regulate spatial
localization of µR. We identify specific sequence elements on µR that determine the dynamics ofµR residence in specialized endocytic domains, along with arrestin. Different ligands can leveragesequence interactions on µR to regulate receptor lifetimes in spatially separated membrane
domains, and, therefore, regulate the magnitude of arrestin signaling. Independently manipulating
endocytic dynamics was sufficient to modulate arrestin signaling without changing G protein
mediated signals. Our results show that functional selectivity in the opioid system can be
independently tuned by changing the spatial localization of receptors.

24

Session 1: mTOR pathway is involved in KOR agonist-induced aversion
Jeffrey J. Liu2, Kelly M. DiMattio1, Chongguang Chen1, Yi-Ting Chiu1, Taylor Gentile1, John
Muschamp1, Alan Cowan1, Matthias Mann2, Lee-Yuan Liu-Chen1
1
Center for Substance Abuse Research & Department of Pharmacology, Temple University,
School of Medicine, Philadelphia, PA 19140, USA, 2Department of Proteomics and Signal
Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
The KOR agonist nalfurafine is used in Japan for treatment of uremic pruritus in hemodialysis
patients; significantly, at the therapeutic doses, dysphoria was not observed as an adverse
reaction. We took a “bedside to bench” approach to investigate possible mechanisms underlying
KOR-mediated aversion. Nalfurafine produced conditioned place aversion (CPA) in mice at higher
dose than the doses causing analgesia and anti-scratch, whereas the opposite was true for MOMSalB and U50,488H, two structurally distinct KOR agonists. At ~A50 in the anti-scratch test,
U50,488H, but not nalfurafine, significantly increased the baseline threshold in intracranial selfstimulation. Thus, our findings on nalfurafine mimicked clinical observations. In mouse brains, atthe lowest doses producing maximal anti-scratch effects, U50,488H and MOM-SalB promoted
robust KOR phosphorylation at T363 and S369, but nalfurafine did not. Shotgun
phosphoproteomic analyses provide an unbiased view of changes in downstream
phosphorylation events. In mouse striatum, more than 390 phosphosites were different between
U50,488H and nalfurafine treatment. In addition, the two agonists activated distinct downstream
pathways in different brain regions. For example, proteins in the mTOR signaling pathway were
phosphorylated by U50,488H, but not by nalfurafine, in striatum and cortex; however, nodifferential phosphorylation of mTOR pathway was found in hippocampus. Inhibition of the mTOR
pathway by rapamycin abolished U50,488H-induced CPA, without affecting analgesic and antiscratch effects. These results indicate that the mTOR pathway is involved in U50,488H-induced
CPA and lack of CPA by nalfurafine may be related to its inability to cause KOR phosphorylationand to activate the mTOR pathway.

Session 1: Peripheral Opioid Receptor Regulation
Nathaniel A. Jeske PhD; University of Texas Health Science Center of San Antonio
Opioid receptors expressed in peripheral sensory neurons have the ability to serve as asignificant analgesic target in multiple pain phenotypes. Furthermore, targeting peripheral opioid
receptors significantly reduce addiction potential and avoid other serious, negative side effects
associated with central nervous system opioid receptor activation. However, opioid receptors
expressed in nociceptors exist in an analgesically desensitized state. New research from ourgroup has uncovered the mechanism behind this reduced responsiveness, and has identified atarget for pharmaceutical consideration that would increase analgesic efficacy of opioids in the
periphery. This avenue for treatment would reduce systemic dosing requirements to alleviate
pain, thereby addressing treatment concerns associated with the current and ongoing opioid
epidemic.

25

Session 1 - GPCR signaling platforms for pain and analgesia
Meritxell Canals PhD; Monash University, Australia
The realization that G protein-coupled receptors (GPCRs) can continue to signal once internalized
has triggered major revision of the classical view of GPCRs as proteins predominantly signalingfrom the plasma membrane. In addition, it has become apparent that the localization of a GPCR
within the plasma membrane can also dictate its signaling outcomes. It has therefore become
important to understand how both receptor and signal compartmentalization dictate cellular andphysiological responses. I will present work that illustrates these concepts in the context of the
mu-opioid receptor (MOPr) and the neurokinin 1 receptor (NK1R), two receptors that are key inthe (patho)physiology of pain and analgesia. First, using a range of biophysical approaches(including FRET and BRET biosensors) we have shown how the localization of the MOPr within
the plasma membrane once activated by different ligands dictates distinct spatiotemporalsignaling profiles. Second, combining FRET and BRET with ex vivo and in vivo experiments, wehave recently shown that the NK1R generates signals from endosomes that underlie pain andreveal a critical role for endosomal signaling of GPCRs in complex pathophysiological processes.
Taken together, our results illustrate the importance of understanding molecular mechanisms thatgovern the formation and location of GPCR signaling platforms in order to provide new venues
for the generation of safer and more effective analgesics.
Session 2: Circuit dynamics of in vivo dynorphin release in the nucleus accumbens shell
Ream Al-Hasani1,2,3, Jenny-Marie T. Wong6, Jordan G. McCall1,2,3, Omar S. Mabrouk6, Gavin
Schmitz1,2, Dionnet L. Bhatti1, Kirsten Porter-Stransky4, Brandon Aragona4, Robert T. Kennedy4,
Michael Bruchas1,3; 1Washington University, St. Louis, MO; 2St. Louis College of Pharmacy;
3
Washington University School of Medicine; 4University of Michigan, Ann Arbor, Michigan
We recently used an optogenetic approach to demonstrate that stimulation of dynorphinergic cells
in the ventral nucleus accumbens shell (vNAcSh) elicits robust aversive behavior andphotostimulation of dorsal NAcSh dynorphin (dNAcSh) cells induces a place preference and ispositively reinforcing. Both of which appear to be dependent on kappa opioid receptor (KOR)
activation. To follow these recently published findings, we are investigating how KOR is able tomediate these opposing behaviors in two distinct regions of the NAcSh. We are using an optomicrodialysis approach which combines optogenetics with microdialysis for use in awake, freely
moving mice. This system allows quantification of neuropeptide release while directly modulatingcell-type specific neuronal firing in the NAcSh. We have identified that the amount of dynorphin
and met-enkephalin released during optogenetic stimulation is equal in the dNAc and vNAc.
Interestingly, release of leu-enkephalin and dopamine is only detectable following
photostimulation in the dNAc release. To further understand the circuitry driving the opposing
unique behaviors and distinct neuropeptide release profiles, we are mapping the projections toand from discrete regions with the dyn-reporter mouse (dyn-CretdTomato) and using tracing
approaches (Rabies, canine adenovirus and cholera-toxin B). Thus far we have identified
projections from the lateral septum, dorsal and ventral tegmental area (VTA). Together theseexperiments will help us understand how these distinct populations of dynorphin neurons in the
NAcSh are engaged, altered, and recruited in stress and reward-related behaviors. Supported by
NIDA K99/R00 DA038725 (RA), NIDA R01 DA037152 (MRB) and R01 DA037152 EUREKA NIDA (MRB) .

26

Session 2: Kappa-opioid receptor control of nucleus accumbens synaptic integrationdifferentially gates D1 and D2 MSN activity
Hugo Tejeda; National Institute on Drug Abuse
Endogenous dynorphin signaling via the kappa-opioid receptor (KOR) in the nucleus accumbens(NAcc) powerfully mediates negative affective states and stress reactivity. Excitatory inputs fromthe hippocampus and amygdala play a fundamental role in shaping the activity of both NAcc D1
and D2 MSNs, which encode positive and negative motivational valences, respectively. However,a circuit-based mechanism by which KOR modulation of excitation-inhibition balance modifies D1
and D2 MSN activity is lacking. Here, we provide a comprehensive synaptic framework whereinpresynaptic KOR inhibition decreases excitatory drive of D1 MSN activity by the amygdala, but
not hippocampus. Conversely, presynaptic inhibition by KORs of inhibitory synapses on D2 MSNs
enhances integration of excitatory drive by the amygdala and hippocampus. In conclusion, we
describe a circuit-based mechanism showing differential gating of afferent control of D1 and D2
MSN activity by KORs in a pathway specific manner.

Session 2: Upregulation of dynorphin in the central nucleus of the amygdala mediates
the negative emotional states of nicotine withdrawal but not escalation of nicotine intake.
Olivier George PhD; The Scripps Research InstituteAbstinence from nicotine often results in emergence of a negative emotional state that predict
relapse and escalation of nicotine intake in humans and rats. Prodynorphin and activation of
kappa receptors have been shown to produce withdrawal-like symptoms, suggesting that
activation of the dynorphin-kappa system may be responsible for the emergence of a negative
emotional state leading to escalation of nicotine intake. However, the causal role of activation of
the dynorphin-kappa system on measures of negative emotional states and nicotine intake independent animals remains to be demonstrated. To test this hypothesis we tested the effect ofviral vector downregulation of prodynorphin in the central nucleus of the amygdala on withdrawal
induced-pain, conditioned place aversion, escalation of nicotine intake and stress-induced
reinstatement. Immunohistochemical analysis showed that prodynorphin’s content was increasedin the CeA in nicotine dependent rats during abstinence, but not in non-dependent
rats. Downregulation of CeA prodynorphin using AAV-shPdyn did not affect nicotine escalation,
but significantly decreased withdrawal-induced hyperalgesia, aversion to withdrawal and stressinduced reinstatement using the pharmacological stressor Yohimbine (1.25mg/kg). These results
demonstrate that while activation of kappa receptors mediates both the negative emotional stateof withdrawal and the increased motivation for nicotine after abstinence, increased prodynorphin
levels in the CeA only mediates the negative emotional state of nicotine withdrawal. This reportprovides preclinical evidence for the efficacy of kappa antagonists in reducing the motivational
effects of nicotine withdrawal, and identify that upregulation of prodynorphin in the CeA is
responsible for the emergence of the negative emotional state of nicotine withdrawal.

27

Session 2: Central amygdala Prepronociceptin-expressing neurons mediate palatable
food consumption and reward
J. Andrew Hardaway1, Christopher M. Mazzone1, Dipanwita Pati1, Michelle Kim1, Jennifer
Jensen1, Jeffrey F. Diberto1, Ami Shiddapur1, Jonathan A. Sugam1, Michael P. Saddoris1, Greg
Tipton1, Zoe McElligott1, Michael R. Bruchas2, Garret D. Stuber1, Thomas C. Jhou1, Cynthia M.
Bulik1, Thomas L. Kash1
1
UNC Chapel Hill, 2Washington University
Food palatability is one of many sensory factors that drive food consumption, and the drive tofeed for its rewarding properties is a key contributor to obesity and binge eating. We haveidentified a population of cells expressing prepronociceptin, the gene encoding the opioid-likeneuropeptide nociceptin, in the central amygdala (PnocCeA) that are activated by palatable foodconsumption. Ablation or chemogenetic inhibition of these cells reduces palatable foodconsumption. Further, ablation of PnocCeA cells reduces the increase bodyweight and adiposity
accompanied by continuous high fat diet access. PnocCeA neurons project to the vBNST, PBN,
and NTS, and activation of axons in the PBN and NTS produces reward behavior. These datasuggest that the PnocCeA hindbrain network is critical for promoting the reinforcing and rewarding
properties of palatable food. In my current work, I am using slice electrophysiology and in vivo
Ca2+ imaging to measure the recruitment of PnocCeA cells during palatable food consumption andthe impact of nociceptin receptor signaling on synaptic physiology.

Session 2: Midbrain nociceptin neurons modulate reward behaviors
Kyle Parker PhD; Washington University School of Medicine in St. Louis
Nociceptin/Orphanin FQ Opioid Receptor (NOPR) and its endogenous ligand, nociceptin (NOFQ)
are widely distributed throughout the brain and have been demonstrated to have a role inmediating pain, stress, anxiety, feeding, and reward behaviors. In particular, NOPR stimulation
has been shown to inhibit mesolimbic dopamine transmission and reduce reward-related
behaviors. However, the endogenous sources of nociceptin within these reward circuits are notcompletely understood. Using a novel transgenic prepronociceptin (PNOC)-Cre mouse andretrograde tracing techniques, we examined a population of nociceptin neurons located within theparanigral and paraintrafascicular nuclei of the ventral tegmental area (VTA). We found that thesenociceptin neurons have monosynaptic projections onto dopamine cells and ablation of thesecells enhances sucrose seeking under fixed and progressive ratio operant tasks. In addition, we
found that chemogenetic stimulation of these nociceptin neurons reduces operant responding
under the same progressive ratio task and can drive a conditioned place aversion (CPA). It wasalso found that optogenetic stimulation of these VTA-projecting cells during a real-time place
Preference task drive aversion-like behaviors as well as reduce operant behavior performance.
These findings show a previously unknown population of nociceptin-containing neurons that are
positioned to tonically suppress reward motivation via dopamine cell inhibition. Understanding
how this discrete population may be critical to the regulation of normal reward seeking behaviors
could provide insight into behaviors dysregulated during motivational states such as addiction.

28

Plenary 2: Sex, Pain and Microglia
Michael W. Salter MD PhD; Hospital for Sick Children & University of Toronto, Toronto, Canada
Neuron-microglial interactions are increasingly recognized as being key for physiological andpathological processes in the CNS. Microglia are found to play a causal role in neuropathic pain
behaviours resulting from peripheral nerve injury, and a core neuron-microglia-neuron signaling
pathway has been elucidated. Within the dorsal horn, microglia suppress neuronal inhibition by acascade involving activation of microglial P2X4 receptors causing the release of brain derived
neurotrophic factor (BDNF). BDNF acts on trkB receptors leading to a rise in intracellular Clconcentration in dorsal horn nociceptive output neurons, transforming the response properties ofthese neurons. In addition to suppressing inhibition, peripheral nerve injury causes activitydependent potentiation at dorsal horn glutamatergic synapses which enhances nociceptive
transmission. BDNF mediates the enhancement of synaptic NMDAR responses throughactivation of TrkB and the Src-family kinase, Fyn. We have discovered that microglia-to-neuron
signaling is not only critical for pain hypersensitivity after nerve injury but also for the paradoxical
hyperalgesic effect of morphine and other opioids. This core signaling pathway has been
extensively characterized, in studies using male mice. We have recently discovered that infemale mice, however, pain hypersensitivity depends upon the adaptive immune system, likely
upon T cells. Despite this profound difference in cellular mechanisms, pain hypersensitivity infemale mice is as robust as that in male mice. Taking into consideration sex differences in the
spinal immune-neuronal signaling has important implications ranging from diagnostics, totherapeutics, to prevention of chronic pain. Supported by CIHR, Krembil Fdn, CRC, Anne and Max
Tanenbaum Chairs, and Northbridge Chair.

Session 3: The potential of δ-opioid receptors as novel target for alcohol use disorders
Richard M. van Rijn1,2,3, Terrance Chiang1, Meridith T. Robins1,3, Robert J Cassell1,2,3, Mohamed
El-Sayed1,3, Mark S. Cushman1,3; 1Purdue University; 2Purdue Institute for Drug Discovery,
3
Purdue Institute for Integrative Neuroscience
Alcohol use disorder (AUD) is defined as a chronic, relapsing brain disease and as such carriesa large socioeconomic burden; it is estimated that 17 million adults in the United States sufferfrom AUD. The delta opioid receptors (DORs) are an exciting new target for treatment of AUD asactivation of DORs can not only reduce alcohol intake, but can also reduce anxiety anddepression, which are often co-morbid with AUD. We have previously shown that depending on
the agonist DOR activation can either decrease or increase alcohol use in mice. We have recently
identified that the ability of DOR agonist to modulate alcohol consumption in mice is tightly
correlated with a DOR agonist’s ability to recruit beta-arrestin2, such that weak beta-arrestin
recruiters decrease intake, but strong beta-arrestin recruiters increase alcohol intake. Here we
report on our ongoing efforts to pharmacologically characterize DORs for their role in modulating
alcohol-related behavior using an approach that runs the gamut from medicinal chemistry, tomolecular and cellular pharmacology ending with preclinical testing in mouse models of alcoholuse. Specifically, we will provide evidence that implicates the dorsal striatum as important brain
region for the DOR-mediated effects and that biased signal transduction strongly impacts the
directional response of alcohol use. We will also describe our efforts to synthesize novel DORselective, G-protein biased agonists. Finally we will discuss the impact of interspecies differenceson drug development of signal-biased DOR agonists.
29

Session 3: A role of epigenetically regulated dynorphin/K-opioid receptor and
Neuropeptide Y system in alcohol tolerance
Subhash C Pandey PhD, Martina Palmisano, Harish Krishnan, Huaibo Zhang
University of Illinois at Chicago & Jesse Brown VA Medical Center Chicago, IL
Alcohol tolerance has been associated with promoting alcohol intake. Evidence suggests that thedynorphin (DYN) /kappa opioid (KOP) receptor and neuropeptide Y (NPY) along with brainderived neurotrophic factor (BDNF) contributes to the negative reinforcing effects of alcohol. Here,
we investigated the role of epigenetic regulation of DYN/KOP, NPY and BDNF systems in rapid
ethanol tolerance (RET) to anxiolysis using an animal model. Adult male Sprague-Dawley rats
were treated with ethanol (1g/kg intraperitoneal) or n-saline once or twice (24hr apart). Single but
not two injections of ethanol exert anxiolysis. Ethanol increases PDYN mRNA levels in the
amygdala (AMY) that remain increased after the second injection. Acute ethanol increases NPY
and BDNF but not KOP mRNA levels. An increase in KOP receptor expression, but no change in
NPY and BDNF expression was observed during RET. The changes in PDYN and KOP receptor
gene expression do not seem to be mediated by H3K9/K14 acetylation or H3K9 dimethylation,
but may be related to higher occupancy of trimethylated H3K27 and H3K4 at promoter regions.
However, changes in NPY and BDNF expression may be regulated by H3K9/14 acetylation during
RET. Interestingly, nor-BNI (KOP antagonist) and HDAC inhibitor treatment pretreatment or
BDNF infusion into AMY prevented RET. Nor-BNI treatment increased NPY mRNA levels in the
AMY of tolerant rats. Together, these data suggest that epigenetic regulation of DYN/KOP system
and its interaction with NPY/BDNF in the amygdala could be involved in RET. Supported by NIHNIAAA grants and by the VA Senior Research Career Scientist award to SCP

Session 3: The OPRM1 A118G SNP in drug reinforcement and reward: modulation oftreatment response to naltrexone
Annika Thorsell PhD; Linköpings Universitet, Linköping, Sweden
Therapeutic responses to naltrexone may be moderated by variation at the mu-opioid receptor
gene locus (OPRM1: Oprm-A118G). This however remains controversial since human results
vary. We have generated “humanized” mice carrying the respective human OPRM1 A118G
alleles (i.e.118AA and 118GG). Here, we examine the role of the OPRM1 A118G variation in
animal models of drug reinforcement and reward, as well as in treatment response to opioid
antagonist treatment (naltrexone and/or nalmefene). For reward, alcohol-induced dopaminerelease in the nucleus accumbens was measured, as well as brain stimulation reward thresholds
in the right medial forebrain bundle in the lateral hypothalamus. Reward-related behavior foralcohol and opiates was measured using the CPP model. Alcohol intake was measured using
both operant and non-operant paradigms. Alcohol lowered brain stimulation reward thresholds in
118GG, but not 118AA, mice, and this was blocked by naltrexone. In addition, brain microdialysis
showed greater peak dopamine-response to alcohol in 118GG than in 118AA mice. Furthermore,
the A118G SNP modulated reward-related behavior in the CPP-model. In the home cage, 118GG
showed increased alcohol intake and naltrexone selectively suppressed alcohol intake in 118GG.
Similarly, both naltrexone and nalmefene were more effective in suppressing operant alcohol selfadministration in 118GG mice. Therefore, OPRM1 A118G variation is a genetic determinant of
dopamine responses to alcohol, a mechanism by which it likely modulates alcohol reward,
and robustly moderates effects of opioid antagonism on alcohol reward and consumption. These30

findings strongly support a personalized medicine approach to alcoholism treatment that takes
into account OPRM1 genotype.
Session 3: The novel nociceptin receptor antagonist LY2940094 reduces ethanol-seeking
and self-administration in animal models and significantly reduces heavy drinking inalcohol-dependent subjects
Linda Rorick-Kehn PhD; Lilly Research Laboratories, Indianapolis, IN
Nociceptin is a 17 amino acid peptide that produces physiological effects through activation of the
opioid-receptor-like (ORL1, or NOP) receptor. The NOP receptor is localized in the mesolimbic
reward pathway and has been suggested to play a role in feeding, mood, stress, andaddiction. We discovered a novel NOP receptor antagonist (LY2940094) and found it to havehigh binding affinity (Ki = 0.11 nM), antagonist potency (Kb = 0.17 nM) and selectivity for NOP
receptors. LY2940094 is orally bioavailable, CNS penetrant and potently occupies NOP
receptors in vivo. Herein, we characterize LY2940094 in several rodent models of ethanol
consumption. After Phase 1 safety studies, we conducted an 8-week proof-of-concept doubleblind, placebo-controlled clinical study to evaluate the efficacy of LY2940094 in reducing alcohol
consumption in heavy drinking subjects with alcohol dependence. In preclinical experiments,
LY2940094 dose-dependently reduced homecage ethanol self-administration in rats, without
affecting food/ water intake or locomotor activity. LY2940094 attenuated progressive ratio
operant responding and breakpoints for ethanol in rats, as well as stress-induced reinstatementof ethanol-seeking. Furthermore, LY2940094 blocked ethanol-stimulated dopamine release in
response to ethanol challenge (1.1 g/kg, IP). In the clinical study, LY2940094 increased
abstinence rates in alcohol-dependent subjects and reduced both heavy drinking days andplasma GGT levels. Our findings demonstrate for the first time that blockade of NOP receptors
attenuates ethanol self-administration and ethanol-motivated behaviors in rodents and may help
alcohol-dependent individuals initiate and maintain abstinence from alcohol drinking. Results
suggest that LY2940094 may have potential therapeutic utility in treating alcohol addiction.

Session 4: Protein kinase mechanisms in opioid-induced hyperalgesia
Zaijie Jim Wang; University of Illinois at Chicago
In addition to the development of drug addiction and tolerance, chronic treatment with opioiddrug(s) also produces paradoxical opioid-induced hyperalgesia (OIH) in humans. OIH can also
be established in preclinical models where opioid-induced hypersensitivity to cold (cold allodynia),
heat (heat hyperalgsia), and mechanical stimuli (mechanical allodynia or hyperalgesia) can bedemonstrated. Intriguingly, animals with OIH did not show condition place preference in an
attempt to determine spontaneous pain. I will present evidence for potential roles of protein kinase
in OIH especially those of CaMKII-alpha and protein kinase C isoforms. Elucidating the protein
kinase-mediated cellular signaling pathways may help to understand the mechanisms of OIH andpotentially offer new targets for interventions.

31Session 4: Circuits and synaptic mechanisms for pain control by endogenous andexogenous opioids
Gregory Scherrer PhD; Stanford University, Stanford, CA, USA
Opioids represent the mainstay treatment for the management of severe pain; however, themechanisms underlying their analgesic properties remain unclear. Elucidating the functionalorganization of the opioid system in pain neural circuits is urgently needed to develop innovative
analgesic therapies with limited side effects. We use mouse genetics, neuroanatomy,
electrophysiology, electrophysiology, opto/ chemogenetics and in vivo calcium imaging to localize
opioid peptides and receptors along pain neural circuits and determine how opioids modulate thedifferent components of pain experience. We found that mu opioid receptors (MORs) expressedby primary afferent nociceptors initiate analgesic tolerance and OIH development. Deletion of
MORs specifically in TRPV1+ nociceptors eliminated morphine tolerance, hyperalgesia, andpronociceptive synaptic long-term potentiation, without altering antinociception. Furthermore, coadministration of methylnaltrexone bromide, a peripherally restricted MOR antagonist, is sufficient
to abrogate morphine tolerance and in perioperative and chronic pain models. We also identified
the spinal neurons expressing MOR, delta opioid receptors (DORs), and both receptors. In thedorsal horn, MORs and DORs are present in largely separate neural populations, with limited coexpression in subclasses of excitatory lamina II interneurons and NK1R+ lamina I projection
neurons of the anterolateral tract. Conditional knockout experiments revealed that DOR agonists
act on DORs expressed by somatostatin+ dorsal horn interneurons to reduce mechanical pain,
but can also non-selectively activate spinal MORs to diminish heat pain. Unexpectedly, we reportthat the majority of spinal neurons co-expressing DOR and MOR correspond to ventral horn V1
inhibitory interneurons that regulate the activity of motoneurons.

Session 4: Therapeutic recovery from diminished morphine analgesia in chronic pain
state
Hiroshi Ueda1, Hiroyuki Neyama; Nagasaki University Graduate School of Biomedical Sciences
1-14, Bunkyo-machi, Nagasaki 852-8521, Japan
We have often experienced the cases of less sensitivity or resistance of various types of pain to
opioid treatments in clinic. In relation to such clinical evidence, our group has studied thediminished morphine analgesia and underlying mechanisms in various experimental models in
mice, such as 1) morphine analgesic tolerance against acute pain following repeated treatments
through an anti-opioid NMDA receptor mechanism (J Neurosci, 23:6529-6536, 2003), 2)
decreased peripheral morphine analgesic activity in a model of neuropathic pain following partial
sciatic nerve ligation/pSNL through an epigenetic gene silencing of MOPr (J Neurosci, 30: 48054814, 2010)) and 3) loss of central morphine analgesia in models of experimental fibromyalgialike pain model following intermittent cold or psychological (empathy) stress in mice (Neurosci
Lett, 472: 184-187, 2010; Neurobiol Pain, in press). In the present study we will introduce somesuccessful examples to recover the diminished morphine analgesia in models of pSNL-typeneuropathic pain and intermittent stress-induced fibromyalgia-like pain, by using novel inhibitorsof interaction between repressor and co-repressor or NMDA receptor antagonists.

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Session 4: Targeting RGSz1 complexes to optimize the actions of opioid analgesics
Sevasti Gaspari1, Immanuel Purushothaman1, Valeria Cogliani1, Rachael L. Neve1, Elliot Ross1,
Li Shen1, Venetia Zachariou1
1
Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, 10029
Regulators of G protein signalling z1 (RGSz1) is a member of the RGS family of proteins, present
in several networks expressing mu opioid receptors (MOPR). Using genetic mouse models for
global or brain region-targeted manipulations of RGSz1 expression we demonstrate thatprevention of RGSz1 action increases the analgesic efficacy of MOPR agonists in male andfemale groups of mice. Importantly, prevention of RGSz1 action delays the development ofmorphine tolerance, but decreases sensitivity to the rewarding and locomotor activating effects ofmorphine. We used information from next generation RNA Sequencing (RNA-Seq) analysis andapplied a number of molecular and biochemical studies to understand the detailed signaltransduction mechanisms by which RGSz1/Gaz complexes affect functional responses of MOPR
in addiction and analgesia networks. Our studies reveal that RGS complexes promote thedevelopment of morphine tolerance by controlling the availability of Gaz protein to effector
molecules, including components of the Wnt/beta-catenin pathway. Interventions in RGSz1controlled signal transduction may be used to optimize the analgesic actions of opioids anddecrease the risk of dependence or addiction.

Founder’s Lecture: Opioid Research: Past achievements and future challenges
John Traynor PhD; Department of Pharmacology, University of Michigan, Ann Arbor, Michigan
Opiates and opioids have long fascinated scientists by the complexity of their chemistry, cellbiology and physiology. It is easy to understand how the Founders of INRC come to introduce
this conference as a forum to meet and discuss their latest research.
A reoccurring theme of the Founders lecture has been “How did we get here and where are wegoing next”. In this spirit I will review previous research that has inspired my work, concentratingon the receptors, their ligands and signaling pathways. I will discuss findings on alternative
approaches to traditional opioid therapies aimed to increase the activity of the endogenous opioid
peptides. This includes enhancement of the activity of the receptors with allosteric modulators or
promoting signaling downstream of the receptors with regulator of G protein signaling proteins.
However, in spite of all the outstanding science that has been carried out to understand the control
of endogenous opioid systems, how opioid ligands activate receptors, the intricacies of signaling
pathways and the mapping of brain circuits modulated by opioids, many challenges still remain if
we are to understand the full beauty of the opioid systems.

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Session 5: Probing whole-brain connectivity in live mice: receptors and drugs
Brigitte L Kieffer; McGill University, Douglas Research CenterIn humans, resting state magnetic resonance neuroimaging (rsfMRI) has opened the era ofconnectome/imaging genetics, in order to elucidate how genetic factors affect brain organizationand connectivity in healthy individuals and disease. Yet the causal impact of a single gene on
anatomical, functional and effective connectivity remains largely unknown, and animal researchis best suited to this goal. We tested whether rsfMRI in living animals would reveal connectivity
alterations upon targeted inactivation of a single gene, the mu opioid receptor (MOR) gene (usedhere as a proof-of-principle). MOR is broadly distributed throughout the nervous system andmediates the remarkably potent analgesic and addictive properties of opiates like morphine.
Hypothesis-free analysis of rsfMRI data revealed pronounced modifications of functional
connectivity (FC) in MOR knockout mice, indicating for the first time that loss of one geneproduces alterations at whole-brain level, which are detectable non-invasively in live animals.
Further, fine-grained rsfMRI mapping captured a unique MOR gene-to-network signature andrevealed strongest perturbations in connectional patterns of pain/aversion-related nodes,
consistent with current knowledge of MOR function. We also found specific FC alterations in
conditional mice lacking MOR in the striatum, as well as a very distinct signature in mice lacking
another G protein coupled receptor, and all the latter findings strongly correlate to known
behavioral phenotypes of these mice. This pioneering approach, which is fully translatable tohuman research, opens an entirely new view on functional implications of gene activities onwhole-brain networks and how these ultimately drive behavior.
Session 5: Unbiased characterization of intercellular signaling peptides using mass
spectrometry. Elena V Romanova PhD; University of Illinois, Urbana-Champaign
Often times there are indications of peptides involvement in the response to drug exposure, but(neuro)peptide identity may not be known. Mass spectrometry measurements referred to as
peptidomics allow comprehensive, targeted or exploratory analyses of unknown, endogenous
signaling peptides. The advantage of peptidomics is its ability to evaluate and characterize final
gene products at the location of action, which provides information most other “omics”
technologies cannot. Dynamic peptide level changes potentially associated with illicit drug use,pain or therapeutic response to narcotics can be evaluated. At the NIDA UIUC Neuroproteomics
Center, we apply exploratory MS-based approaches to understand peptide changes in various
brain areas relevant to effects of illicit drugs, stimulants, and pain medication. For example, using
a rodent model of cocaine addiction, we showed that distinct peptide profiles in the mPFC
statistically correlate with individuals that exhibit reduced sensitivity to the behavioral effects ofacute cocaine administration. With AMPH, we found that chronic exposure leads to significant
peptide dynamics in the brain’s reward circuitry, thus suggesting that in addition to the mesolimbic
dopamine system concomitant chemical changes in other pathways and in neuronal organization
may play a part in the overall effect of chronic AMPH exposure. In the most recent work, we usedMS-based quantitation of peptide levels in mouse models of migraine and opioid-induced
hyperalgesia to probe the role of neuropeptides in pain phenomena. The combination of
peptidomics measurements with behavioral assessment of pharmacological intervention provides
unique avenues for peptide discovery, functional analysis and, ideally, intervention and treatment.

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Session 5: Human laboratory paradigms to study conditioned cues
Emma Childs PhD; University of Illinois at Chicago
Learned associations between a drug and the cues surrounding drug experiences are considered
critically important in the development of addiction. These associations, formed through theprocesses of drug conditioning, are robust and long-lasting, inducing cravings and relapse longafter the user is drug-free. However, few clinical studies have examined the conditioning
processes and little is known about how associations are formed and how they influence behavior
including drug use. Recently, we have established a human laboratory model of de novo drug
conditioning, based on the Conditioned Place Preference (CPP) paradigm used in rodents. Using
this model, we have shown that humans, like animals, come to prefer and spend more time in anenvironment paired with a rewarding drug. Moreover, individuals exhibit altered behavior, drug
effects and drug taking in the drug-paired environment. Together the findings demonstrate that
we can use this model to better understand how conditioned associations control behavior. Thisknowledge will help us to design novel approaches to counteract their powerful influences.

Session 5: Preclinical Assessment of opioid analgesia, side effects, and withdrawal
Michael Morgan PhD; Washington State University, WA, USA
A revolution in the preclinical assessment of nociception from pain-evoked to pain-depressed
behaviors has occurred over the past decade. Pain-evoked tests such as the hot plate and vonFrey tests require a response to a noxious stimulus. Such tests are useful for assessing
nociception, but are inadequate for assessment of analgesic effects because of potential motor
confounds (e.g., drugs can block responding on the hot plate test because of sedation orparalysis). In contrast, pain-depressed behavioral tests assess reductions in ongoing behaviors
caused by pain. These tests are particularly useful for assessment of analgesia because
analgesic and motor effects are opposite (e.g., analgesia is defined as a restoration of behavior,
whereas a sedative drug reduces behavior). This presentation will describe a series of studies
showing that home cage wheel running is a particularly useful pain-depressed behavior for the
preclinical assessment of analgesic efficacy. Because home cage wheel running assesses normal
daily activity, it is sensitive to a wide range of disruptive stimuli. Data will be presented showing
depression of wheel running caused by pain (hindpaw inflammation, migraine), drug side effects(morphine, THC), and spontaneous morphine withdrawal. These findings indicate that home cage
wheel running is an objective and clinically relevant method to screen treatments for analgesia,side effects, and withdrawal profile.

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Session 5: Probing axonal trafficking with live cell imaging of pHluorin tagged Mu Opioid
Receptor
Damien Jullie; University of California San Francisco
Much is known about the synaptic vesicle cycle and its local modulation by presynaptic GPCRs,
but little is known about how GPCRs traffic at terminals. The super ecliptic pHluorin (SEP) is a pH
sensitive variant of the GFP which is not fluorescent in an acidic environment like the lumen of
intracellular compartments. Using a combination of live cell imaging and local perfusion, pHluorin
is a useful tool to probe the trafficking of surface receptors. Using such approaches to study the
trafficking of the Mu Opioid Receptor (MOR) in axons, we found that receptors can undergo ligand
dependent endocytosis and that internalization is essentially happening within presynaptic
terminals. After internalization, MOR containing endosomes support local recycling of thereceptors back to the axonal surface, unravelling that another endocytic cycle operates withinpresynaptic terminals.

Plenary 3: Alterations of the opioid system in chronic pain
Catherine Bushnell; National Center for Complementary and Integrative Health, NIH, Bethesda,
MD, USA
There is growing evidence that opioid systems may be disrupted in chronic pain. We examined infibromyalgia patients and healthy controls the consequences of blocking mu-opioid receptors
using naloxone on the pleasantness and intensity of skin brushing. At baseline, the healthy
subjects preferred slow brushing over fast, and rated fast brushing as more intense. The pain
patients showed a blunted distinction between slow and fast brushing for both pleasantness andintensity. Naloxone increased the perceived pleasantness of skin brushing for healthy subjects,
but not for pain patients. In contrast, naloxone decreased touch intensity ratings only for patients.
These findings revealed that opioids are involved in touch pleasantness, but this involvement isaltered in chronic pain.
We also investigated, using a rodent model, the effect of chronic pain on opioid receptor
availability. Studies have shown altered opioid receptor availability in pain patients, but it is notknown if this is caused by the pain or related to other factors. Thus, we conducted a controlled 3month longitudinal study, comparing nerve injured rats (spared nerve injury; SNI) to matched
controls. Using [18F]-diprenorphine to label unbound opioid receptors and examining their densitywith PET, we confirmed in rodents the finding from pain patients of reduced opioid receptor
availability in multiple brain areas, including the insula and caudate-putamen. These findings
provide new evidence that chronic pain alters the brain opioid system, which might explain thelimited effectiveness of opioid therapy in chronic pain patients.

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Session 6: History of Prescription Opioid abuse and the Recent Transition to Heroin
Theodore Cicero; Washington University in St. Louis, School of Medicine
Two events late in the last century and early 2000s led to a surge in the use and abuse ofprescription opioid drugs: First, a precipitous increase in opioid prescribing rates due to arenewed and perhaps ill-informed focus on treating pain; and, second, the introduction and heavily
promoted extended release oxycodone (i.e., OxyContin) which led to diversion of unprecedented
amounts of pure oxycodone unadulterated with acetaminophen/NSAIDs. Over the past 15-20
years, admission to treatment programs for opioid use disorder, opioid overdose fatalities andopioid-related emergency room visits have reached epidemic levels. Recently, a number of steps
have been taken to reduce the diversion and abuse of these drugs, including the development ofabuse-deterrent formulations and efforts to promote better utilization of these products fortherapeutic purposes (e.g., closure of “pill mill”, prescription monitoring programs to deter “doctor
shopping”). Many of these efforts appear to have been successful, but there was a major
unanticipated outcome. Faced with a reduced supply of prescription opioids in the drug-seeking
culture, and the increased cost of those that were available, many users transitioned to heroin
which became a much cheaper and far more accessible alternative. Thus, while the prescription
opioid abuse problem shows signs of diminishing in its impact, a resurgence in the abuse of heroin
has occurred with all of the complications of such use – overdose deaths and transmission ofblood-borne pathogens (e.g., HIV and Hepatitis C).

Session 6: Pharmacological Therapies for Opioid Use Disorder: Implications of Fentanyl
Availability on Treatment Effectiveness
Sandra D Comer; Columbia University and NYSPI
Misuse and abuse of opioids is currently a major public health concern. Efforts to address thisproblem include prescription drug monitoring programs, increased education for prescribers,
development of abuse deterrent formulations of opioids, and expansion of access to medicationassisted treatment. Although misuse of prescription opioids appears to be reaching a plateau or
declining slightly, heroin use is increasing and the number of overdose deaths attributable toprescription opioids and heroin is continuing to rise. A particularly disturbing new trend is theincrease in illicit fentanyl and structurally related analogs that are now being sold on the streets.
Fentanyl is either added to or sold as heroin or made to look like prescription drugs such as
OxyContin or Xanax. Therefore, in many cases, users are unaware that they are injecting,insufflating, or ingesting fentanyl. Because fentanyl is 50-100x more potent than heroin, the riskof opioid overdose is increased. In addition, its margin of safety is narrow with regard to analgesic
effects versus respiratory depression and it has high efficacy at mu opioid receptors. During thispresentation, both preclinical and clinical research on fentanyl and structurally similar agonists
will be shown in order to highlight the concerns about the risks of fentanyl abuse compared toother mu opioid agonists and how its pharmacology may make it more difficult to treat with the
medications that are currently available for managing opioid use disorder.

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Session 6: Prescription opioids and migraine: the basic and clinical science of a major
public health issue
Andrew Charles; Headache Research and Training Program, UCLA, CA, USA
Migraine is one of the most prevalent of all disorders worldwide, and represents a common pathto overuse of prescription opioids. The most commonly prescribed opioids are primarily mu
receptor agonists. Despite the fact that the majority of patients report that they are not particularly
effective in relieving migraine headache, these medications are nonetheless widely prescribed for
migraine in the United States. There is now substantial evidence that use of currently available
opioids for migraine is associated with worsening of the disorder, including greater frequency andseverity of attacks, and reduced responsiveness to migraine-specific therapies. Underlying
mechanisms for worsening of migraine by mu opioid agonists may be similar to those that cause
opioid-induced hyperalgesia. Overuse of a prescription opioids by migraine patients is asignificant public health issue. Potential approaches to dealing with this issue for migraine as well
as for other pain disorders will be discussed.

Session 6: Blocking Pannexin-1 Channels on Microglia Alleviates Opioid Withdrawal
Tuan Trang; University of Calgary
The mechanisms involved in opiate withdrawal are poorly understood, and the limited clinicalstrategies for treating withdrawal are ineffective. Here, we identified the pannexin-1 (Panx1)channel as a novel therapeutic target for treating morphine withdrawal. We discovered thatmorphine treatment induces synaptic plasticity in spinal lamina I/II neurons, which manifests as
long-term synaptic facilitation upon naloxone-precipitated morphine withdrawal. This synaptic
facilitation is critically gated by activation of Panx1 channels expressed on
microglia. Pharmacologically blocking Panx1, or genetically ablating this channel specificallyfrom microglia, blocked spinal synaptic facilitation and alleviated the behavioural sequelae ofmorphine withdrawal. Targeting Panx1 represents a potential novel therapeutic approach fortreating the symptoms of opiate withdrawal.
Young Investigator Award: Structural dynamics in G protein-coupled receptor signaling
Aashish Manglik MD PhD; Stanford University, Stanford, CA, USA
The recent determination of X-ray crystal structures of the four major opioid receptor subtypes
has revealed key facets of opioid ligand recognition. These inactive-state structures, combinedwith a structure of the active µOR, have provided a high-resolution molecular description of opioid
action. Using these structural insights, we have embarked on new computational discovery
campaigns to identify novel opioid analgesics. An early success of uncovering new chemistry has
been the discovery of a Gi biased opioid agonist, called PZM21, with unique signaling properties
and in vivo efficacy. The remarkable efficacy of PZM21 and other novel biased opioids highlights
the relevance of G protein-coupled receptor (GPCR) structural plasticity in opioid signaling andphysiology. I will describe the approaches and tools that my newly-launched independent
laboratory will employ to dissect the biophysical basis of opioid receptor signaling complexity.
These approaches may ultimately enable rationally designed control of opioid receptor signaling
and may portend exciting new ways to manipulate the vast physiology coordinated by the broader
GPCR family.
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Session 7: Kratom derived natural products as leads to design analgesics with mu opioid
G-biased agonism and delta opioid antagonism
Susruta Majumdar; Memorial Sloan Kettering Cancer Center, NY, USA
Mitragyna speciosa or kratom has been used by Southeast Asian people for hundreds of years
both as an analgesic and an opioid antidote. Mitragynine and 7-hydroxy mitragynine are two
kratom alkaloids that have been studied in detail and are known to act as opioid receptor
modulators. The pharmacology of a related semi-synthetic derivative, mitragynine pseudoindoxyl(MP) has not been well characterized. We have shown that MP is a G-protein biased mu opioid
agonist and delta antagonist. It is a potent analgesic in mice, which exhibited no reward,
attenuated respiratory depression and constipation when compared with morphine. In addition,
MP showed slower development of tolerance and minimal physical dependence. The design,synthesis, and pharmacological evaluation of novel analgesics based on this template will be
discussed.

Session 7: Discovery and development of oliceridine, a G protein-biased ligand targetingthe mu-opioid receptor
Jonathan Violin PhD; Trevena Inc
Opioids are powerful analgesics and are widely employed for the management of moderate tosevere acute pain, but cause many adverse effects, including gastrointestinal dysfunction, abuseand dependence liability, and potentially fatal respiratory depression. In the hospital and similar
settings, injectable opioids remain necessary for many patients, but opioid-related adverse events(ORAEs) increase hospital costs and can threaten patient safety. Morphine pharmacology in βarrestin2 knockout mice suggested that a ligand that promotes coupling of the μ-opioid receptor(MOR) to G proteins, but not β-arrestins, might result in higher analgesic efficacy, lessgastrointestinal dysfunction, and less respiratory depression than conventional opioids. Using cellbased assays and iterative medicinal chemistry, we sought and discovered oliceridine (TRV130)– a G protein biased MOR ligand that largely avoids the β-arrestin pathway. Oliceridine
demonstrated a wider therapeutic window than morphine in rodents, showing less respiratory andgastrointestinal dysfunction at equianalgesic doses. Intravenous oliceridine has been compared
to intravenous morphine in Phase 1, Phase 2, and Phase 3 clinical trials. Evidence from thesestudies, using multiple measures of efficacy, safety, and tolerability, suggest that oliceridine may
offer patients who require intravenous opioids the pain relief associated with conventional opioids
with potential for less post-operative nausea and vomiting, and reduced risk of opioid-induced
respiratory depression. This presentation will review the discovery and development ofoliceridine, with an emphasis on recent Phase 3 results. Oliceridine is an investigational agent
not approved by the FDA.

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Poster Abstracts40

1 - Political Priority for Tobacco Control among Adolescents in Local Governments
Areas, Osun State, Nigeria
Ademola Adelekan; University of Ibadan
Supervisory Counsellors for Health (SCH) have great responsibility to play for Tobacco Control(TC) as political head of health administration in Local Government (LG). This study therefore
designed to determine the political priority for TC among adolescents in LG areas in Osun State,
Nigeria. The study was a descriptive qualitative study. All consenting 27 out of 30 SCH in Osun
State were interviewed using in-depth interview guide. Interviews took place at the participant’s
office at the time convenient for them. Data was recorded with tape recorder and analysis wasdone using thematic method. Majority of the respondents were aware that cigarette smoking
among adolescents is high in Nigeria but did not know the significant of this extent. All the
respondents were aware of TC policy in Nigeria but only few had ever read through this policy.
Almost all the respondents were not aware of any international policy on Tobacco control. Many
reported knowing at least five adolescents in their community who smoke cigarette and had agood knowledge of health consequences of tobacco smoking. Only few reported to have TC unit
in their LG and among these, almost all reported that no specific programme in place for educating
young people. Local Government SCH awareness of tobacco magnitude and political priority for
its control among adolescents was low. Tobacco education programme should be intensified forpolitical leaders to develop adequate measures on this issue and making necessary funds
available.

2 - Effectiveness of Provider Initiative Approach for Smoking Cessation among Pregnant
Women in Osun State, Nigeria
Ademola Adelekan; University of Ibadan
Women who smoke during pregnancy need assistance in quitting, and obstetric health careproviders are in a unique position to help them. This study therefore determined the effectivenessof Provider Initiated Approach (PIA) for smoking cessation among pregnant women receiving
Antenatal Care (ANC) services in selected primary health care facilities in Osun State, Nigeria. A
total of 150 frontline health workers who are ANC providers were trained on PIA to help theirpatients quit smoking. They were equipped with smoking knowledge and skills on how to integratesmoking cessation with ANC services. Smoking cessation messages were discussed with women
who reported smoking through micro-teaching, IEC materials and as well as client provider
interaction. A total of 22,845 pregnant women were screened for smoking from March-September,2014 and data were analysed using descriptive statistics and t-test. A total of 1426 (6.2%)
respondents’ were identified to be currently smoking with mean age of 27.5 ± 10.1 years. Other
reported substances currently using among respondents were alcohol (98.6%) and marijuana(1.6%). A total of 12.9%, 56.9% and 32.7% reported to cease smoking complete within one, twoand three months of this intervention respectively. Reasons adduced for smoking included social
integration (76.4%), peer pressure (56.3%) and to reduce stress (45.8%). The prevalence use ofalcohol and marijuana also decreased to 12.9% and 0.0% respectively. A significant reduction insmoking associated with this intervention was observed. More health care providers should betrained on using this approach.

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3 - Ring Substitution in the Macrocyclic Opioid Peptide CJ-15,208 Alters the Opioid
Activity Profile in vivo
J. V. Aldrich1, C. Reedy2, H. M. Stacy1, J. M. Medina1, S. O. Eans1, T. J. Cirino1, T. F. Murray3, J.
P. McLaughlin1; 1University of Florida, 2University of Kansas, 3Creighton University
The macrocyclic peptide CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) exhibits mixed agonist/kappa opioid
receptor (KOR) antagonist activity and prevents stress-induced reinstatement of drug-seeking behavior
after oral administration. As part of our exploration of the structure-activity relationships (SAR) of this
peptide, we incorporated different substitutions in the aromatic residues. Analogs were examined for opioid
receptor affinity in radioligand binding assays and for both opioid agonist and antagonist activity in vivo after
intracerebroventricular (i.c.v.) administration using the mouse 55 oC warm-water tail-withdrawal assay.
Selected analogs were further evaluated for their ability to prevent stress- and cocaine-induced
reinstatement of extinguished cocaine-conditioned place preference (CPP) responses. Incorporation of a
substitution on one of the phenyl rings of CJ-15,208 was generally well tolerated by KOR, with variable
effects on the affinity for mu opioid receptors (MOR). All of the analogs exhibited antinociception in vivo at
a dose of 30 nmol i.c.v. The most potent of these analogs produced MOR- and KOR-mediatedantinociception with an ED50 (and 95% C.I.) value of 5.95 (3.91-8.77) nmol i.c.v. and subsequent KOR
antagonism. The identity of the substituent affected antagonist activity, with fluorination of either
phenylalanine residue abolishing KOR antagonism in vivo. One analog exhibited antagonism of MOR anddelta opioid receptors as well as KOR and prevented stress-, but not cocaine-induced, reinstatement of
extinguished cocaine-CPP. In addition to advancing our understanding of their SAR, these results further
support exploring the potential application of such macrocyclic peptides for the treatment of drug abuse.
This research supported by NIDA grant R01 DA023924.

4 - Identification of a novel interaction site of the mu-delta opioid receptor heterodimers
Doungkamol Alongkronrusmee, Hamed T. Ghomi, Shiqi Tang, Markus A. Lill, Richard M. vanRijn; Purdue University, West Lafayette, IN, USA
Mu opioid receptor (MOR) agonists remain highly effective for the treatment of pain, and would represent
ideal analgesics if their side effects could be prevented. Evidence suggests that MORs can interact withdelta opioid receptors (DORs) to form MOR-DOR heterodimers which may contribute to certain adverse
effects of mu opioids. Our goal is to develop drug-like compounds that disrupt the heterodimers to lessen
these side effects. Yet, a lack of tools to investigate the role of MOR-DOR heterodimers in preclinical andclinical models is stifling our ability to target the heterodimers with drugs. To aid in the development of suchtools, it is important to understand the MOR-DOR interaction by determining which amino acids in theheterodimer interface play major roles in the MOR-DOR formation. We constructed DOR mutants to probe
for destabilization of the MOR-DOR heterodimers. We used a multipronged approach including calcium
signaling assay, bimolecular fluorescence complementation and co-immunoprecipitation to investigate theimpact of mutations on the stability and function of the heterodimers. We confirmed that amino acids 242,
243 and 244 located in the intracellular loop, as previously reported by others, were crucial in stabilizing
the heterodimers. Intriguingly, we identified novel amino acids 208, 209 and 288 to be necessary for theheterodimer formation. These amino acids are located on the extracellular regions, thereby providing apromising new target for the heterodimer disruption. These findings will move us closer to our goal todevelop small-molecule drugs that can be used with opioids to prevent their side effects.

42

5 - Low Abuse Liability of the Endomorphin Analog ZH853 is Supported by Reduced
Psychomotor Activation and Withdrawal.
Ariel Amgott-Kwan1, Terrence Hunter1, Amy Feehan1, James Zadina1,21
Neuroscience Program, Brain Institute, Tulane University, 2SE Louisiana Veterans HCS, New
Orleans, LA, Depts. of Medicine and Pharmacology, Tulane University School of Medicine
Opioids are highly effective analgesics, but their use is limited by adverse side effects. We previously
demonstrated that an endomorphin analog (ZH853), a selective agonist of the mu-opioid receptor, provides
potent analgesia with reduced or absent side effects, including reduced reward, respiratory depression,
motor impairment, tolerance, hyperalgesia, and glial activation as compared to morphine (1). Here we
further characterize the potential of this analog for reduced abuse liability. The progression from casual
drug use to addiction is thought to be initially motivated by positive reinforcing properties of the drug,
mediated by activation of mesolimbic dopamine signaling. Later, continued drug use is driven by negatively
reinforcing effects, including physical dependence (2). Our previous study showed that ZH853 failed toinduce conditioned place preference or self-administration, indicating reduced reward (1). Increased
locomotion in mice is an indicator of dopamine release, which in turn is associated with reward (3). Acutesubcutaneous administration of morphine, but not equi-antinociceptive doses of ZH853, induced locomotor
activation in CF1 mice, indicating differential dopaminergic activation. A negatively reinforcing property ofabused drugs, withdrawal, was also assessed. Repeated exposure to ZH853 failed to produce naloxoneprecipitated jumping or weight loss, significant signs of physical dependence. These data suggest ZH853
may provide effective long-term treatment with low risk of abuse compared to currently-used opioid
medications.
(1) Zadina, JE et al. (2016). Neuropharmacology (105): 215-227.
(2) Koob, GF, & Volkow, ND. (2016). The Lancet (8): 760-773.
(3) Wise, RA, & Bozarth, MA (1987). Psychological Review (94):469 – 492.

6 - Morphine influences cell proliferation, migration and apoptosis of tumor cell lines
endogenously expressing mu opioid receptors
Andrea Bedini1, Monica Baiula1, Santi Spampinato1
1
Department of Pharmacy and Biotechnology (FaBiT) – University of Bologna – Italy
Opioids are widely used to treat acute, chronic, post-operative cancer pain. Mu opioid receptor (MOR) is
expressed in several human malignancies but opioid-mediated effects on uncontrolled cancer cell growth,invasion of normal tissues, resistance to apoptosis and tumor recurrence aren’t still fully understood. We
aimed at characterizing any morphine-mediated effect on cell proliferation, migration, apoptosis of different
tumor cell lines expressing MOR. MOR mRNA and protein levels were preliminarily measured in each cellline; [3H]thymidine incorporation, wound-healing and annexin assays were employed to evaluate cellproliferation, migration and apoptosis, respectively. HeLa cervix carcinoma cells displayed the highest MOR
mRNA levels, followed by DAOY medulloblastoma, U87-MG astrocytoma, HT-29 colon-carcinoma. MOR
protein expression, evaluated by receptor binding assay, did not mirror that of mRNA, as DAOY cells
displayed the highest expression, followed by HeLa, U87-MG, HT-29 cells. Morphine tripled HeLa and HT29 cell proliferation after 24h and 48h, respectively; HeLa migration after 18h, and only moderately
increased apoptosis in both cell lines after 12h; these effects were sensitive to the MOR antagonist betafunaltrexamine.Morphine increased U87-MG viability, migration and proliferation with peaks at 12h, 18h,
48h, respectively; beta-funaltrexamine and naloxone significantly reduced U87-MG and HeLa basal
migration, possibly unmasking a contribution of endogenous opioids to cell migration.DAOY cells express
the highest levels of MOR; however, morphine did not influence cell proliferation, migration or apoptosis,
nor antagonized forskolin-induced cAMP accumulation. Sequencing MOR in DAOY cells and investigations
on other medulloblastoma cell lines are currently ongoing and will be presented at the conference.

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7 - OREX-1019; a small molecule with potential for addiction maintenance treatment andrelapse prevention
Barbara Belli PhD1, Charles P. France PhD2,3, David R. Maguire PhD2, Lisa R. Gerak PhD2, Monica Bennett
PhD4, Gerta Cami-Kobeci PhD5, Stephen M. Husbands PhD5, Peter Flynn PhD1
1Orexigen Therapeutics, Inc., La Jolla, CA, 2Dept of Pharmacology, University of Texas Health Sciences
Center at San Antonio, TX, 3Dept of Psychiatry, University of Texas Health Science Center at San
Antonio, TX, 4Genea Biocells, San Diego, CA, 5Dept of Pharmacy and Pharmacology, University of Bath,
Bath BA2 7AY
In the US, over 16 million people have used prescription opioids or heroin for non-medical purposes,
resulting in death rate of nearly 9 per 100,000 population in 2016. OREX-1019 is a novel small molecule indevelopment for addiction maintenance. Binding assays across the human opioid receptor family revealed
an affinity for all receptors less than 10 nM; similar receptor binding affinities were obtained forbuprenorphine. Agonist activity of compounds was evaluated by measuring the enhancement of [ 35S]
GTPgS binding and antagonism was evaluated by measuring inhibition of control agonist-induced [35S]
GTPgS binding. Agonist/antagonist activity (% effect) values for OREX-1019 were: mu (MOP) 21/69; kappa(KOP) 27/81; delta (DOP) 49/65; and nociception/orphaninFQ peptide (NOP) 24/34. Agonist/antagonist
values for buprenorphine were: MOP 79/39; KOP 32/93, DOP 25/125 and NOP 0/100, respectively. OREX1019 was tested in nonhuman primates (NHP) for its ability to maintain self-administration responding (i.v.)
and its ability to block self-administration of remifentanil. Up to a dose of 1.0 mg/kg, OREX-1019 failed tomaintain self-administration responding that was greater than vehicle. At doses of 0.1 or 0.3 mg/kg s.c.,
OREX-1019 reduced the average number of daily infusions of remifentanil (0.32 µg/kg/infusion) from 2528 to 5-15. Collectively these results suggest that OREX-1019 might be effective for managing opioid
addiction and have less abuse liability compared with currently available medications.

8 - OREX-1038; a potent analgesic molecule with potential for reduced abuse liability
Barbara Belli PhD1, Charles P. France PhD2,3, David R. Maguire PhD2, Lisa R. Gerak PhD2, Monica Bennett
PhD4, Gerta Cami-Kobeci PhD5, Stephen M. Husbands PhD5, Peter Flynn PhD1
1Orexigen Therapeutics, Inc., La Jolla, CA, 2Dept Pharmacology, University of Texas Health Science
Center at San Antonio, TX, 3Dept Psychiatry University of Texas Health Science Center at San Antonio,
TX, 4Genea Biocells, San Diego, CA, 5Dept Pharmacy and Pharmacology, University of Bath, BathOREX-1038 is a novel opioid-based small molecule in development for acute and chronic pain. Binding
assays across the opioid receptor family revealed potency at the human mu-opioid receptor (MOP) and thenociception/orphaninFQ peptide (NOP) receptor with pIC50 values of 10.04 and 8.3 nM, respectively.
Agonist and antagonist activity was determined and OREX-1038 was demonstrated to have mixedagonist/antagonist activity at the human mu-opioid receptor (MOP, 50/46% effect) and agonist activity at
the nociception/orphaninFQ peptide (NOP) receptor (40/0% effect). Following subcutaneous (s.c.)administration, OREX-1038 (20 mg/kg) exhibited potent antinociceptive effects in the Brennan rat model ofincisional pain comparable to morphine (5 mg/kg s.c.). The duration of the analgesic effect was longer, 24
hr versus 6 hr for morphine. OREX-1038 did not affect motor coordination in the rat rotarod assay and did
not reduce GI transit time. In the non-human primate (NHP) warm water tail withdrawal assay OREX-1038(0.03 mg/kg s.c.) treatment increased tail withdrawal latency, between 40-60% of the maximum possible
effect 2-3 hours after administration and in some experiments maintained the antinociceptive effect for up
to 24 hrs. In a NHP self-administration study, OREX-1038 maintained responding that was greater thansaline but less than remifentanil with some monkeys receiving 20 or more infusions of 1.0 µg/kg/infusion(30 times the maximally effective dose for antinociception). Self-administration of OREX-1038 decreased
over consecutive sessions. These data suggest that OREX-1038 may be a potent, novel analgesic withsignificantly reduced potential for abuse.

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9 - Rewarding effects of opioidergic projections from the ventral pallidum to substantia
nigra
Julio Bernardi, Ludwig Trillo, Dionnet L. Bhatti, Yu Jin Shin, Jose Moron-Concepcion, Michael R.
Bruchas; Washington University in Saint Louis
Opioid addiction has become an epidemic, but the role of endogenous opioid circuits is still poorly
understood. The ventral pallidum (VP) is one the strongest projections to the midbrain dopamine neurons
and expresses enkephalin at high levels. We hypothesized that stimulation of VP enkephalin projections tothe substantia nigra pars compacta (SNc) would be rewarding and mediated by enkephalin. We used PenkIRES-Cre mice for optogenetic, chemogenetic and fiber photometry interrogations of this circuit.
Optogenetic stimulation of VP enkephalin terminals in the SNc was reinforcing in real time place preference(RTTP) and operant intracranial self-stimulation paradigms. Chemogenetic inhibition of these neurons wasaversive in a conditioned place aversion paradigm, demonstrating that the enkephalinergic VP-SNc circuit
bi-directionally modulates reinforcement behaviors. To demonstrate that the evoked rewarding behaviors
is due to signaling at the level of the SNc rather than antidromic activation of VP projections to other regions,
we performed lidocaine microinfusions in the VP and photostimulation of VP terminals in the SNc, which
failed to abolish the self-stimulation or real time preference behavior. To understand the dynamic circuit
properties of the VP-SNc enkephalin neurons during reward-seeking behaviors, we measured GCAMP6s
calcium transients of VP terminals in the SNc through fiber photometry in a Pavlovian paradigm with twocues, one that predicted sucrose reward availability and one that didn’t. We found that VP-SNc differentially
encoded both reward-predicting and nonreward-predicting cues with less than 200 ms latency. Together,
our results implicate the enkephalin VP-SNc circuit as an important bidirectional modulator of reward andaversion.

10 - Opioid receptors modulate skin ageing and melanocytic disorders
Bigliardi PL11,3, Neumann C1, Heng Gu2, Bigliardi-Qi M1
1IMB/A*STAR, 8A Biomedical Grove, #06-06, Singapore, 2Chinese Academy of Medical Science, Instituteof Dermatology, Nanjing, PR China, 3NUHS, UMC, Singapore
Typical signs of skin ageing are changes in skin thickness, pigmentation and delayed wound healing. On
cellular level ageing affects differentiation, proliferation, migration of various cell types including
keratinocyts, melanocytes and fibroblasts. Previously we have shown that the d- opioid receptor system(DOPr) affects differentiation and migration capabilities of the epithelial keratinocytes resulting in woundhealing delay and significant alterations of the important skin differentiation upon DOPr expression levelchanges.
Further investigation of opioid receptor system in skin ageing, a multidecade study was performed on
Chinese population comparing sun-exposed and unexposed skin. Small excisional skin biopsies were takenfrom 30 healthy Chinese women at different age from the anterior surface of the upper arm (sun-protected)
and the posterior surface of the forearm (sun-exposed). Various parameters such as epidermal thickness,pigmentation and m- (MOPr) and d- (DOPr) expression in epidermis were evaluated in these different age
group. Interestingly, the expression of DOPr (but not MOPr) decreased significantly with age. This was
independent from photoageing and sign for biological ageing in skin. In addition the reduction of epidermal
DOPr expression correlated significantly with an increase of epidermal melanin content with age.
Consequently, we performed in-vitro experiments using monolayer 2D cultures of human pigmented cells
and observe a significant modulation of melanin production in these cultures by various opioid ligands.
Similar results were obtained by using 3D organotypic co-culture models.
This has implications on benign skin pigmentation in aged skin and other dyspigmentations in malignant
melanotic cells with potential clinical applications.

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11 - μ-Opioid receptor trafficking in human keratinocytes
Bigliardi-Qi M1, Neumann C2, Bhimsen R1,3, Ramasami S1, and Bi1
1
Institute of Medical Biology, A*STAR, Singapore; National University of Singapore, YLL Schoolof Medicine; National University Hospital, UMC, Singapore; 3ICES, A*STAR, Singapore;2
University of Basel, Switzerland
Opioids receptors are mostly studied for their function in the CNS, but less known for their
peripheral effects. Our group discovered opioid receptors in skin and the epidermis of the skin,which shares the same ectoderm origin as the nervous system, is most affected by the regulatory
function of the opioid receptors. This receptor system is not only involved in modulating peripheral
sensation, but also in skin homeostasis, wound healing and skin ageing.
Topical treatment with the opioid antagonist naltrexone in chronic pruritus relieves itching by
modifying the localization of the epidermal µ-opioid receptor (µ-OR), but the precise molecularmechanisms of µ-OR modulation are unclear. Here, we evaluated the real-time behavior of µOR–ligand complexes in the presence of OR agonist and antagonists. We conjugated the μ-OR
ligand endomorphine-1 (EM) to the fluorescent dye to directly visualize OR dynamics in cultured
keratinocytes. By live-cell imaging we identified fast trafficking of the endogenous fluorophore
conjugated µ-OR complex. Furthermore, acute and chronic treatment of keratinocytes withnaltrexone plus EM increased fluorophore conjugated µ-OR complex binding to the µ-OR. Overall,competition for the μ-OR with OR agonist (EM), or antagonists elicited canonical and noncanonical effects on basal and differentiated keratinocytes. Our approach has helped uncover thecomplexity of skin-specific μ-OR–ligand dynamics and the mechanistic consequences ofantagonists and agonists on the µ-OR. Such studies will facilitate the development of novel
treatment strategies for peripheral sensory disorders and could be used for predicting
interindividual differences in OR behaviour and trafficking in the CNS.

12 - Opioid modulation of synaptic transmission in affective pain circuitry
Bart Jongbloets, Tianyi Mao, William Birdsong
Oregon HealthAffective/ motivational pain processing involves the medial thalamus, anterior cingulate cortex(ACC) and striatum. Opioid analgesics are known to modulate this circuitry and relieve thenegative emotional effects of pain. However, the identity of the opioid receptors, their cellularlocations, and their physiological effects on this affective circuitry remain uncharacterized. In thepresent study, synaptic connections between the three brain regions; thalamus, striatum, andACC, were characterized based on the effect of opioid agonists to modulate glutamatergictransmission between these regions. Using optogenetic and electrophysiological approaches, it
was determined that single striatal projection neurons in the dorsomedial striatum received direct
input from both thalamic and cortical afferents. It was found that opioid receptors differentiallymodulated thalamic and cortical glutamate release in striatum. Additionally, µ and ∂-opioid
receptors were found to play distinct roles in modulating thalamo-cortical and intracortical synaptictransmission in the ACC. These data identify opioid receptor subtype-dependent inhibition of
specific pathways within the thalmao-cortico-stiatal circuitry, providing insights into how opioid
analgesics modulate affective pain at the synaptic and circuit level.

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13 - Antidepressant activity of the buprenorphine analog BU10119
Caroline A. Browne1,2, Shivon A. Robinson1, Edgardo Falcon1, Stephen M. Husbands3, Irwin
Lucki1,2
1
University of Pennsylvania, 2Uniformed Services University of the Health Sciences, 3Universityof Bath
Buprenorphine, a potent kappa opioid receptor (KOR) antagonist and mu opioid receptor (MOR) partial
agonist, rapidly and effectively alleviates symptoms in treatment-resistant major depressed patients.
Previously, our laboratory demonstrated that low-dose buprenorphine reversed behavioral deficits induced
in two rodent models of depression, chronic mild stress and chronic social defeat. Moreover, we
determined that at 24 h KOR antagonism mediates buprenorphine’s antidepressant effects, whereas
blockade of MORs facilitates buprenorphine’s anxiolytic action. Development of buprenorphine as atreatment modality for depression may be complicated by its MOR agonist activity and consequently its
potential abuse liability in a vulnerable patient population. Here we evaluated the behavioral effects of anovel buprenorphine analog, BU10119, which was modified to reduce efficacy at MORs while retaining thebeneficial profile at KORs. BU10119 was screened in male C57BL/6J mice using the forced swimming test
(FST) at 1 and 24 h post administration and in novelty induced hypophagia (NIH) 24 h post treatment andcompared with buprenorphine, the selective KOR antagonist CERC-501 and the selective MOR antagonist
cyprodime. BU10119 was active both at 1 and 24 h post admisntration, producing a similar inverted Ushape dose reponse curve to that of buprenorphine, reducing immobility in the FST and latency scores in
the NIH, in the absence of MOR agonist induced hyperactivity. Cyprodime was only effective at 1 h. CERC501 reduced immobility at both timepoints, but reduced latency scores only at the highest dose at 24 h.
Overall, these data show that BU10119 retains the beneficial behavioral profile of buprenorphine.

14 - Peripherally restricted opioid combination therapy synergizes in multiple pain states
Daniel J. Bruce, Cristina Peterson, Kelley F. Kitto, Catherine Harding-Rose, Kevin Wickman,
Carolyn Fairbanks, George Wilcox
University of Minnesota
The adverse effects of opioids are largely mediated by mu-opioid receptors (MORs) in the CNS. However,
opioid receptors also mediate analgesia on the peripheral terminals of primary sensory afferents. Despite
decades of research targeting peripheral MORs, no peripherally directed opioid has provided effective pain
control without the risk for addiction and diversion. Because recordings from spinal cord slices had
demonstrated presynaptic synergy between the MOR agonist loperamide (Lo) and the DOR agonist
oxymorphindole (OMI) in the central terminals of nociceptors in spinal cord, we tested for anti-hyperalgesic
synergy in the periphery. Using models of inflammatory, nerve injury, post-operative, or tumor-induced pain
and intraplantar, systemic, or topical application showed that when combined in a 1:1 dose ratio OMI-Lo
produces a robust, 50- to 100-fold anti-hyperalgesic synergy. This synergy was blocked by the peripherally
restricted opioid antagonist, naloxone methiodide, reinforcing the peripheral localization of the effect. The
synergistic interaction is also completely ablated when G protein-coupled, inwardly rectifying potassiumchannels (GIRKs) are blocked or knocked out. Although repeated dosing with supra-therapeutic doses
induces tolerance, therapeutic doses do not. Initial self-administration studies also indicate a significantly
reduced abuse liability compared to prescription opioids such as oxycodone. We conclude that MOR
agonists significantly synergize with DOR agonists at peripheral sites of action, providing strong evidencein support of peripherally restricted combination opioid therapy. The systemic and topical efficacy of the
combination, along with loperamide’s extremely low abuse liability, suggests that this combination might betherapeutically useful to control multiple pain modalities in the clinic.

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15 - Characterization of opioid receptor subtypes and biased signalling as factors formodulation of alcohol use in mice.
Robert J. Cassell1,2,3, Meridith T. Robins1,3, Kendall L. Mores1, Rajendra Uprety4, Susruta
Majumdar4, Richard M. van Rijn1,2,3
1
Purdue University, Department of Medicinal Chemistry and Molecular Pharmacology, 2Purdue
Institute for Drug Discovery, 3Purdue Institute for Integrative Neuroscience, 4Memorial Sloan
Kettering Cancer Center
The non-subtype selective opioid receptor antagonist naltrexone is one of the few FDA-approved drug
treatments for alcohol use disorder. However, opioid receptor subtypes may each uniquely modulate
alcohol use, exemplified by opposing alcohol consumption patterns in mu (MOR) and delta (DOR) opioid
receptor knockout mice. Additionally, we have recently shown that biased signalling at DORs is correlatedwith alcohol intake (Chiang et al., 2016). We hypothesize that Gi-protein signalling is important for DOR
selective agonists to reduce alcohol intake, but that other pharmacological mechanisms may determine
how non-DOR selective agonists control alcohol use in mice. To test our hypothesis we characterized DOR
and MOR agonists with different degrees of subtype selectivity and signalling bias in cellular assays andfor their ability to modulate alcohol intake in C57BL/6 mice. We find that G-protein biased mixed agoniststhat activate DOR and MOR are capable of reducing alcohol drinking behavior in mice within volitional
alcohol self-administration assays and that their effects are only partly abolished in DOR knockout mice.
Interestingly the G-protein biased mixed MOR/KOR agonist TRV130 has no effect on alcohol use. Currently
our data is suggestive of at least two distinct mechanisms by which opioid receptor agonists are able tomodulate drinking behavior in rodents. We have synthesized DOR-selective G-protein biased agonists that
could help further elucidate the mechanism by which DORs can reduce alcohol intake. The goal is to identify
specific pharmacological properties of opioid receptor agonists that can be utilized to develop new drugs
for the treatment of AUD.

16 - Nucleus accumbens mu-opioid receptors are recruited and necessary for theenhancement of motivated behaviors
Daniel C Castro, Anthony Guglin, Tochi Onyeador, Michael Bruchas
Washington University, St. Louis
Mu opioid receptors (MORs) are involved in motivation for natural and drug rewards. One site for MOR
action is nucleus accumbens medial shell (mNAc). MOR stimulation in this mesocorticolimbic hub enhances
appetitive behaviors such as food intake and social interactions. By contrast, disruption of MOR signaling
has yielded ambiguous results. Here, we sought to determine when mu opioid receptors are recruited tomediate motivated behaviors by disrupting MOR signaling during baseline or enhanced motivated states(i.e., ad libitum versus food deprived, social versus isolated housing). To disrupt MOR signaling, we used
constitutive MOR-/- knockout (KO), conditional knockout (Oprm1fl/fl), and pharmacological approaches. KOs
showed normal baseline behaviors compared to wildtype controls, but failed to show enhanced motivation
after food deprivation or social isolation. Targeted viral deletion of MORs in mNAc of Oprm1fl/fl mice showeda similar pattern, suggesting that MORs are recruited to enhance, not generate motivated behaviors. Next,
to determine whether MORs are dynamically recruited, we microinjected CTAP (MOR antagonist) into
mNAc. MOR blockade only reduced motivated behaviors when animals were in heightened motivatedstates. Finally, to pinpoint which neuronal populations are important in mNAc for MOR-mediatedenhancements, we crossed Oprm1fl/fl mice with dynorphin- or enkephalin-cre mice to delete MORs fromthose neuronal populations. Results suggested that MORs act primarily via D1/dynorphin neurons, but not
D2/enkephalin neurons. Future work will test MOR sufficiency to enhance behaviors by either selectively
rescuing MORs in knockout mice or optically stimulating novel photosensitive MOR chimeras. This work
was supported by grants T32NS007205 and R01DA042499.

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17 - Characterization of Sigma 1 Receptor (S1R) antagonist CM-304 and a related analog,
AZ-66: Evaluating novel therapeutics for allodynia and induced pain
Thomas J. Cirino1, Shainnel O. Eans1, Jessica M. Medina1, Marco Mottinelli2, Sebastiano Intagliata2,
Christopher McCurdy2, Jay P. McLaughlin1
1Department of Pharmacodynamics, University of Florida, Gainesville, FL, 2Department of Medicinal
Chemistry, University of Florida, Gainesville, FL
Sigma-1 receptors (S1R) are associated with activity-induced spinal sensitization and pain, particularly withrespect to nerve injury. The purpose of this study was to investigate the in vivo analgesic and anti-allodynic
activity of two novel sigma receptor antagonists, the S1R selective CM-304 and a related analog, the nonselective S1R/Sigma-2 receptor(S2R) antagonist AZ-66, in mouse assays of thermal, chemical, induced
inflammatory pain, as well as the chronic nerve constriction injury (CCI) model of neuropathic pain. Bothcompounds produced time- and dose-dependent analgesia and anti-allodynic effects in each respective
pain model. Interestingly, while less potent than morphine in a 55oC warm-water tail-withdrawal assay, bothCM-304 and AZ-66 produced antinociception in the writhing test (0.48 (0.09-1.82) and 2.31 (1.02-4.81)
mg/kg, i.p., respectively), equivalent to morphine (1.75 (0.31-7.55) mg/kg, i.p.). Likewise, pretreatment (1030 mg/kg, i.p.) with either sigma-receptor antagonist dose-dependently produced antinociception in theformalin paw assay. While the selective S1R antagonist CM-304 dose-dependently (10-45 mg/kg, i.p.)
reduced allodynia in the CCI neuropathic pain model equivalent to the control analgesic gabapentin (50mg/kg, i.p.), AZ-66 demonstrated a much longer duration of action. Place conditioning with either sigmareceptor antagonist with doses up to 45 mg/kg, i.p. produced neither rewarding nor aversive effects, andneither compound produced hyperlocomotion as observed with morphine (10 mg/kg, i.p.). Although eachcompound exhibited modest respiratory depression at high doses (30 mg/kg, i.p.), this seemed likely due
to modest sedative effects as observed in the rotorod assay. Overall, this data demonstrates sigma
receptor-selective antagonists produce antinociception with fewer liabilities.

18 - Oxycodone-induced conditioned place preference and locomotor activity in male andfemale Oprm1 A112G mice
Devon Collins1, Yong Zhang1, Mary Jeanne Kreek2
1The Rockefeller University, 2Rockefeller University
A common single nucleotide polymorphism (SNP) in the human mu-opioid receptor (MOR) gene (OPRM1),
A118G, has been demonstrated to alter mu-opioid receptor function in healthy humans, and is associatedwith opioid addiction. Mice bearing an equivalent SNP, A112G, in the mouse MOR gene were generated,characterized, and studied (Mague et al., 2009). Male and female mice homozygous for the 112G allele
show genotype-dependent reductions in morphine-stimulated locomotor activity and genotype-dependent,
sex-specific reductions in morphine-induced conditioned place preference (Mague et al., 2009). GG miceshow enhanced heroin self-administration (Zhang et al., 2015). Female mice bearing one or two copies of
the 112G allele show reduced buprenorphine-stimulated locomotor activity, blunted buprenorphine-induced
analgesia, diminished sensitivity to buprenorphine-mediated reduction of novelty-induced hypophagia, andsimilar depressive-like behavior in the forced-swim test (Browne et al., 2017). We have previously
characterized conditioned place behavior and locomotor responses to oxycodone in male and female wildtype mice, but we have not yet investigated these effects in A112G mice. We found that oxycodone (1mg/kg) produced a similar conditioned place preference in AA and GG mice regardless of sex. Oxycodone(1 mg/kg) produced a significant increase in locomotor activity during conditioning sessions in A112 males
only; there was no such increase in GG males or females regardless of genotype. These data suggest thatdifferences in opioid-mediated behavior in GG mice may be drug- and sex-specific.
This work was supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation.

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19 - A neural network for abstracting nociceptive information into an aversive pain
perception
Gregory Corder, Biafra Ahanonu, Benjamin F. Grewe, Mark J. Schnitzer, Grégory Scherrer
Stanford University
Pain is an unpleasant experience that drives motivated behaviors to limit exposure to noxious
stimuli. However, how activity in affective neural networks generates the aversive quality of pain
remains unexplored. Here, we utilized recurrent in vivo calcium imaging and manipulation ofactivity in nociceptive ensembles to determine the neural basis of pain aversion. We identified adiscrete ensemble in the basolateral amygdala, distinctive from salience and positive valence
networks, in which painful stimuli are innately represented through combinatorial coding. Silencingof this ensemble selectively alleviated pain affective-motivational behaviors, without affecting
detection of noxious stimuli, withdrawal reflexes, anxiety, or reward. Furthermore, innocuous
stimuli engage this nociceptive ensemble in neuropathic pain state, to facilitate the pathologicaldysfunctions characterizing chronic pain. Collectively, these results identify the neuralrepresentation of nociception in the amygdala, necessary for the instantiation of the innate
negative qualities of acute and chronic pain experiences.
20 - On Target effects of Mu opioid receptor activation on brain activity and connectivity
identified by mouse fMRI
Emmanuel Darcq1,2, Md Taufiq Nasseef1,2, Praveen Kulkarni3, Brigitte Kieffer1,2
1
McGill, 2Douglas Hospital Research Centre, 3Center for Translational Neuro-Imaging,Northeastern University, Boston, MA, United States
Success in developing effective drugs has been limited. Although drug-induced molecular
processes are well understood at the cellular level, and behavioral effects have beencharacterized in animal models, the overall impact of target activation and inhibition on neuralnetwork connectivity remains largely unknown. Our goal is to develop a new platform to identify
and screen novel drugs, using functional Magnetic Resonance Imaging (fMRI) in living mice. Here
we present a proof-of-principle study based on Mu opioid Receptor (MOR) responses tomorphine. MORs are expressed in brain areas belonging to pain and addiction and, using geneknockout (KO) in mice, our laboratory demonstrated that MOR mediates both the remarkably
analgesic and addictive properties of morphine. MOR is a prime target in the development ofnovel potent analgesics, possibly devoid of adverse effects, and also new treatments for
substance use and mood disorders.
The approach measures morphine-induced modifications of Blood-oxygen-level-dependent
signal in the brain of live WT and MOR KO animals. Images are acquired using 7 Tesla Scannerand functional data are preprocessed using standard pipeline. Activation maps are thenestablished, functional connectivity and information flow are characterized. Comparing WT and
MOR KO data will allow subtracting off-target effects, and establish the on-target signature of
MOR-mediated activation of networks. Altogether, the characterization of activation andconnectivity effects induced by the prototypic opiate drug provides a reference dataset to further
test other mu opiates used in the clinic and under development, and constitutes a unique platform
to characterize drugs effects on the functional-connectome.

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21 - Differential regulation of delta opioid receptor-mediated behaviors by arrestin2 inmice
Isaac J. Dripps, Emily M. Jutkiewicz
University of Michigan
The delta opioid receptor (DOR) has been shown to produce multiple active conformations that differentiallyengage downstream signaling effectors, including both G protein- and arrestin-mediated pathways.
However, the in vivo consequences of this phenomenon, known as functional selectivity or biased agonism,
is not readily understood. To investigate the potential in vivo consequences of acute arrestin-mediatedsignaling at DOR, we evaluated the behavioral effects of the DOR agonist SNC80 in arrestin2 and arrestin3
knockout mice. Antihyperalgesia was evaluated using a nitroglycerin-induced thermal hyperalgesia assay.
Antidepressant-like effects were measured using the forced swim test. Mice were also observed
continuously for convulsive activity following SNC80 administration. In both arrestin2 and arrestin3
knockout mice, the acute antihyperalgesic and antidepressant-like effects of SNC80 were not significantly
altered. There was also no change in SNC80-induced convulsions in arrestin3 knockout mice. In arrestin2
knockout mice, the potency of SNC80 to produce convulsions was enhanced, as demonstrated by aleftward shift in the dose response curve. Furthermore, these mice often exhibited multiple convulsions in
response to a single dose of SNC80. Loss of arrestin2 also slowed the development of tolerance to the
convulsive effects of SNC80 following repeated daily treatments of SNC80. Arrestin2 knockout mice did not
convulse following treatment with KNT-127, a DOR agonist purported to not produce convulsions. Taken
together, these data suggest that arrestin2 is an important negative regulator of SNC80-stimulated
convulsions. Future studies will further evaluate the in vivo consequences of functional selectivity at DOR.

22 - Distinct MOR agonist profiles revealed by visualizing Mu opioid receptor trafficking
with MOR-Venus mice
Aliza T. Ehrlich1, Meriem Semanche2, Dillon Dafonte1, Léonie Runtz1, Florence Gross1,2, Christine
Colley1, Emmanuel Darcq1, Michel Bouvier2, Brigitte L. Kieffer1
1
McGill University, Douglas Research Center, 2Universite De Montreal, IRIC
G protein-coupled receptors (GPCRs) are cell membrane receptors which bind to stimuli likeneurotransmitters or hormones, to activate cell signaling responses that govern cellular changes which inturn influence behavior and physiology. Thus, GPCRs are highly exploitable targets in biomedicine. The
mu opioid receptor (MOR) is activated by morphine, a widely prescribed powerful pain reliever encumberedby aversive aspects including constipation, respiratory depression, tolerance and addictive euphoria. The“Holy Grail” pain reliever therefore looks like morphine in respect to pain relief but lacks aversive effects(Kieffer, 2016). Two distinct signaling pathways may distinguish these behaviors: the former occurring via
G proteins while the latter due to β-arrestin recruitment (Bohn et al., 1999; Bohn et al., 2000). Here, we
generated a functional fluorescent MOR knock-in mouse, MOR-Venus, in a manner previously described(Scherrer et al., 2006; Erbs et al., 2015) and found morphine induced analgesic responses in hotplate andtail-immersion tests were comparable to wild-type. Next, we mapped the receptor in the mouse brain andprepared primary neurons and DRGs from these mice to examine MOR agonist-induced trafficking toscreen compounds based on the redistribution of the receptor. We tested mu agonist, DAMGO, and foundinternalization of MOR in primary neurons. We are now testing several other compounds to demonstrate
how these analyses reveal a unique method to evaluate MOR agonist-induced MOR trafficking profiles
which may be useful to predict the clinical behavior of an agonist.

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23 - Percutaneous Electrical Nerve Stimulation (Electro-Acupuncture) elevates Agmatine in Spinal
Cords of Nerve-Injured Rats.
Samuel J. Erb1,2, Kelley F. Kitto2, Kathleen A. Sluka4, Carolyn A. Fairbanks1,2,3
1Dept Pharmaceutics, 2Dept Neuroscience, 3Dept. Pharmacology, University of Minnesota-Twin Cities,
Minneapolis, MN, 4Dept. Physical Therapy and Rehabilitation Science, University of Iowa, Iowa City, IA
Decarboxylated L-arginine (Agmatine) prevents or reduces the development of opioid tolerance and selfadministration, as well as manifestations of neuropathic pain in rodents. Since Agmatine is an endogenous
moiety, we hypothesized that Agmatine released in response to percutaneous electrical nerve stimulation(electro-acupuncture) may contribute to alleviation of neuropathic pain in rodents. We report here that
Agmatine is elevated in spinal microdialysate collected from nerve-injured rats during and following electroacupuncture stimulation. Male Sprague Dawley rats were nerve-injured and allowed to recover for one
week before implantation of transverse spinal cord microdialysis fibers (AN69 Hospal). Dialysate was
perfused at a flow rate of 5 µL/min and collected in 15-minute bins. Baseline samples were collected for 1
hour before and during a 30-minute electro-acupuncture stimulation (4 Hz, Zsusanli acupoint), which took
place under isoflurane anesthesia. Control subjects were anesthetized, but did not receive
stimulation. Samples were collected for 3 hours following cessation of stimulation and anesthesia. Rats
were then assessed via von Frey stimulation for anti-allodynic effects of the treatment. Samples were
derivatized and assessed for Agmatine content via HPLC fluorescence detection. Spinal Agmatine waselevated in microdialysate collected from electro-acupuncture stimulated subjects, but not anesthetized
controls. von Frey thresholds were elevated in electro-acupuncture stimulated, but not anesthetized
controls, at 3 hours post-stimulation (following conclusion of microdialysate sample collection).
Percutaneous electrical nerve stimulation (electro-acupuncture) mediates release of endogenous
Agmatine, which may contribute to alleviation of neuropathic pain responses in rats.
24 - Evaluation of opioid-nociceptin bifunctional ligands by biochemical, pharmacological andstructural modeling tools
Anna I. Erdei1, Adina Borbély2, Anna Magyar2, Nóra Taricska3, András Perczel3,4, Ottó Zsíros5, Győző Garab5, Edina
Szűcs1, Ferenc Ötvös1, Ferenc Zádor1, Mihály Balogh6, Mahmoud Al-Khrasani6, Sándor Benyhe1
1Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, H-6726 Szeged, Hungary,
2MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd University,
Budapest, 3Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University,
Budapest, 4MTA-ELTE Protein Modelling Research Group, Institute of Chemistry, Hungarian Academy of Sciences,
Eötvös Loránd University, Budapest, 5Institute of Plant Biology, Biological Research Center, Hungarian Academy of
Sciences, Szeged, Hungary, 6Dept Pharmacology and Pharmacotherapy, Semmelweis University, Budapest
Our novel synthesized opioid-nociceptin hybrid peptides H-YGGFGGGRYYRIK-NH2, H-YGGFRYYRIK-NH2 and AcRYYRIKGGGYGGFL-OH were designed to target opioid and NOP receptors as bifunctional ligands. Two of thesechimeric molecules are composed of the minimum opioid tetrapeptide structure YGGF targeting the opioid receptors,
and a NOP receptor specific synthetic sequence Ac-RYYRIK-NH2 isolated from combinatorial chemical libraries. The
pharmacophores were connected directly or with a triglycine spacer. These chimeric pharmacophores were studied
with circular dichroism spectroscopy, molecular dynamics calculations, radioligand competition, ligand-stimulated[35S]GTPgS binding assays, and electrically stimulated mouse vas deferens (MVD) bioassays, to study their solution
structures, receptor binding affinities, potencies and efficacies, respectively. In circular dichroism spectroscopy, more
a-helical periodicity was present in the structure of H-YGGFRYYRIK-NH2, while H-YGGFGGGRYYRIK-NH2 showed atendency of having either unordered or b-sheet structures, and Ac-RYYRIKGGGYGGFL-OH had the most b-sheet
structures. In molecular dynamics calculations, the amount of turns in the peptides was proportional to the activity. In
contrast, the presence of helices showed a reverse effect, as the most helical H-YGGFRYYRIK-NH2 proved to beinactive on NOPr. In radioligand competition receptor binding assays H-YGGFGGGRYYRIK-NH2 showed apreference for NOPr and KOPr only, while H-YGGFRYYRIK-NH2 and Ac-RYYRIKGGGYGGFL-OH displayed DOPraffinity as well. In [35S]GTPgS binding assays, H-YGGFRYYRIK-NH2 and H-YGGFGGGRYYRIK-NH2 exhibited highly
efficacious agonist actions. In MVD bioassay Ac-RYYRIKGGGYGGFL-OH was the most potent compound.

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25 - How did the Opioid System Evolve?
Chris Evans, Alden Huang, Anna Taylor
UCLA
Endogenous opioid systems (enkephalin, endorphin, dynorphin and nociceptin/orphanin FQ) comprise atleast four genetically homologous precursor-proteins and four genetically homologous G-protein coupled
receptors in every vertebrate species thus far examined. However, little is known about how opioid systems
might have evolved from invertebrates. We recently leveraged de-novo transcriptome and low-coveragewhole-genome assembly in the Pacific hagfish to identify and characterize the first full-length coding
sequence for a functional opioid receptor in a cyclostome. Additionally, we defined two novel endogenous
opioid precursors in this species that predict several novel opioid peptides (submitted for publication).
Bioinformatic analysis shows no closely related opioid receptor genes in invertebrates with regard either tothe genomic organization or to conserved opioid receptor- specific sequences that are common in allvertebrates. Furthermore, no proteins analogous to vertebrate opioid precursors could be identified ingenomic searches despite previous claims of protein or RNA-derived sequences in several invertebrate
species. The presence of an orthologous receptor and opioid precursors in hagfish indicates that afunctional opioid system arose from a common ancestor of cyclostomes and gnathostomes some 550 Mya,
earlier than all previous authenticated accounts, and probably in the common ancestor to all extant
vertebrates. The premise that opioid systems evolved from invertebrate systems concerned with
antimicrobial defense systems is a likely scenario and we speculate that the high concentrations of opioid
precursors in tissues such as the testes, gut, and activated immune cells are key remnants of thisevolutionary role.

26 - The role of striatal mu opioid receptor populations in reward-related behaviors.
Wendy Walwyn1, Nathanial Valerde1, Joshua Hakimian1, Ani Minasyan1, Ralph Albert1, Carlos
Torres1, Nicole Romaneschi1, Camille Johnston1, Anna Taylor1, Amie Severino1, Brigitte Kieffer2,
Christopher Evans1
1
Department of Psychiatry and Bioebehavioral Sciences, David Geffen School of Medicine,
UCLA, 2Faculty of Medicine, Department of Biochemistry, McGill University
Mu opioid receptors are densely expressed in the different neuronal populations of the striatum yet the roleof these populations in regulating reward behaviors is relatively unknown. So as to examine how thesereceptor populations modulate reward, we bred a genetically engineered mouse in which exons 2 and 3 ofthe mu opioid receptor gene are flanked by LoxP cassettes 1 with different cre driver lines2. We first verified
the mu receptor deletion from D1 and D2 neurons. We then assessed the initial hyperlocomotor response
and subsequent sensitization of this response to oxycodone or morphine. Mice lacking mu receptors on
D1 neurons showed an initial response to these opioids but no sensitization thereafter, whereas thoselacking mu receptors on D2 neurons showed reduced exploratory behavior and an enhanced sensitization
to repeated opioids. We also generated a profile of contingent opioid self-administration, assessing
establishment of this behavior under a short-access FR1 schedule over 9 days. This was followed by 3days of an extinction profile of behavior in which all cues except the drug were presented in a contingent
manner under an FR1 schedule. There was no effect of the deletion during the initial establishment phasebut mice lacking mu receptors on D1 and D2 neurons showed a different profile of extinction behaviors.
These findings demonstrate the divergent roles of striatal mu receptor populations in different behavioral
paradigms of reward.
1.Weibel, R. et al. PLoS One 8, e74706, doi:10.1371/journal.pone.0074706 (2013).2. Gong, S. et al. . J
Neurosci 27, 9817-9823, doi:10.1523/JNEUROSCI.2707-07.2007 (2007).

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27 - The role of the deorphanized receptor, GPR83, in stress and reward.
Amanda K. Fakira1,2, Lakshmi Devi1,2
1
Icahn School of Medicine at Mount Sinai, 2Department of Pharmacological Sciences
Anxiety and drug dependence are prevalent disorders that often occur in a comorbid state
therefore therapeutics that target common mechanisms are highly desirable. The recently
deorphanized G-protein coupled receptor, GPR83, is highly expressed in the nucleus accumbens,
medial prefrontal cortex and central nucleus of the amygdala therefore GPR83 is a potential
regulator of reward and anxiety related behaviors. Preliminary studies demonstrate that morphine
conditioning induces an increase in GPR83 expression in the nucleus accumbens and that
GPR83 downregulation attenuates preference for morphine. Analysis of genes that regulate thedopamine and opiate system in GPR83 knockout mice reveal potential increases in expression
for the dopamine transporter, delta opioid and kappa opioid receptors in the nucleus accumbens.
GPR83 has also been shown to be regulated by glucocorticoids indicating that this receptor
system is involved the stress and anxiety. Our preliminary data show that global GPR83 deletion
reduces anxiety-related behaviors, especially in female mice. Overall, these data suggest thatblocking GPR83 function is a potential therapeutic target for anxiety and substance abuse
disorders however more details about the GPR83 system need to be understood. Future studies
are to determine in which brain regions GPR83 is necessary and sufficient for anxiety and reward
behaviors and whether GPR83 is a critical regulator of stress induced drug relapse.

28 - Assessment of ZH853, a novel endomorphin analog, versus morphine in acute andlong term dosing for chronic pain.
Amy Feehan1, Ariel Amgott-Kwan1, Xing Zhang2, James Zadina1,2,3
1
Neuroscience Program, Brain Institute, Tulane University, 2Departments of Medicine andPharmacology, Tulane University School of Medicine, 3SE Louisiana Veterans HCS, New
Orleans, LA
Chronic pain affects 100 million Americans, many of whom are inadequately treated. Whileopioids are effective against many types of chronic pain, significant side effects limit patient andphysician willingness to use them for these conditions. We have developed a mu-opioid receptor
selective endomorphin (EM) analog (ZH853) with reduced or absent side effects of traditionallyprescribed opioids including: abuse liability, respiratory depression, motor impairment, tolerance,and glial activation (1). In the current study, we evaluated ZH853 and morphine in chronic pain
states after both acute and chronic dosing.
Acutely, both intravenous andintrathecal administration of ZH853 produced a long lasting reversal of hypersensitivity.
Additionally, in ongoing experiments, we are evaluating whether chronic, moderate doses of
ZH853 prolong pain like morphine does (2). Classical pain tests (e.g. von Frey) are used todetermine pain sensitivity, while functional recovery is assessed with a multitude of gaitparameters from the CatWalk XT system. Using these tests, we will determine whether functional
and pain recovery correlate, and whether ZH853 or morphine alter the time-course of recovery.
(1) Zadina, JE et al. (2016). Neuropharmacology (105): 215-227.
(2) Grace, PM et al. (2016). Proc Natl Acad Sci (113): E3441-50.

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29 - Structural Insights into Opioid Peptidomimetics with Bifunctional Mu-Opioid
Agonism and Delta-Opioid Antagonism
Thomas Fernández1, Nicholas Griggs1, Anthony Natase2, Jessica Anand1, Henry Mosberg2, John
Traynor1, Emily Jutkiewicz1
1
Departments of Pharmacology, Medical School, 2Medicinal Chemistry, College of Pharmacy
Opioid analgesics exert both their beneficial and unwanted effects by activation of the mu-opioid receptor(MOPR). Furthermore, repeated administration of opioid analgesics and subsequent chronic MOPR
activation promotes opioid tolerance and physical dependence. It has been proposed that activation of
MOPR with concurrent antagonism of the delta-opioid receptor (DOPR) can ameliorate the development of
opioid tolerance and dependence. Here we report on compounds containing a tetrahydroquinoline core
(THQ) that are bifunctional in that they activate MOPR and inhibit DOPR. Compounds were evaluated by
two in vitro assays in membrane preparations of cloned cell lines expressing either MOPR, DOPR, or
kappa-opioid receptors (KOPR): 1) [35S]-GTPγS binding assays to measure maximal G protein activation;
full agonists at each opioid receptor (DAMGO, DPDPE, and U69,593, respectively) were used as standards.
Maximum binding and potency (EC50) values were determined from concentration-effect curves. 2)
Competition binding assays using [3H]-diprenorphine to determine affinity (Ki) values. Modification of the
THQ core led to changes in G protein activation and binding affinity at MOPR and DOPR. In addition,
sulfone side chain substitutions revealed greater receptor activation at MOPR and KOPR while leaving
DOPR inactive. The observed structure-activity relationships will guide further chemical modification anddrug development of analgesic opioid compounds devoid of tolerance and dependence liabilities.
Supported by DA-03910 and the PREP program at the University of Michigan.

30 - Structural and Dynamic Elements of µ-Opioid Receptor Functional Selectivity
Abhijeet Kapoor1, Gerard Martinez-Rosell2, Davide Provasi1, Gianni de Fabritiis2, Marta Filizola1
1
Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York,NY, USA, 2Computational Biophysics Laboratory (GRIB-IMIM), Universitat Pompeu Fabra,
Barcelona Biomedical Research Park (PRBB), Barcelona, Spain
The wealth of structural information on opioid receptors that has appeared in the literature over the past
five years offers a superb opportunity to study these systems at an unprecedented level of molecular detailand to provide novel testable hypotheses about the molecular determinants responsible for opioid receptor
function. Since static information from X-ray crystallography is not sufficient to provide a comprehensive,mechanistic understanding of the diverse biological functions mediated by the µ-opioid receptor (MOR) and
induced by ligands with different efficacy profiles, we complement this information with dynamic
representations using state-of-the-art computational methods. Although the dynamics of MOR could also
be studied experimentally using various spectroscopic and imaging techniques, these do not provide thenecessary level of atomistic detail that one would need to identify the molecular determinants that are
directly responsible for the receptor’s functional selectivity. Molecular dynamics (MD) simulations are aparticularly valuable tool to deduce these details for sparsely-populated conformational states of thereceptor that are impossible or difficult to retrieve experimentally, especially when these computations are
conducted alongside experiments. Coupled with state-of-the-art algorithms for statistical analyses of MD
simulation data, these simulations provide a very powerful approach to identify new and potentiallydruggable metastable states that have not been (and may never be) observed by crystallography, as wellas the slowest and therefore most important conformational degrees of freedom underlying opioid receptor
functional selectivity.

55

31 - Characterizing the biochemical and synaptic properties of CNIH3, a novel component
in individual risk for opioid dependence
Hannah Frye, Christopher Trousdale, Elliot Nelson, Joseph Dougherty, Jose Moron-Concepcion
Washington University in St. LouisIn the last decade, the CDC reported a drastic increase in opioid use across socioeconomic status, agegroup, and sex. In a recent genome-wide association study (GWAS) by Nelson et al., the presence of single
nucleotide polymorphisms (SNPs) in cornichon family AMPAR auxiliary protein 3 (CNIH3) was the strongest
correlating factor for protection against addiction despite drug misuse (Mol Psychiatry, 2016). Previous
studies have either focused exclusively on its homolog CNIH2, or used dual CNIH2/3 knock-out mousemodels. However, CNIH2 had no significant impact on opioid dependence in the GWAS. Although more
information on the unique function of neuronal CNIH3 is needed, CNIH3 is suspected to aid in the traffickingof AMPA receptors (AMPARs) to the post-synaptic density (PSD). CNIH3 is highly expressed in the
hippocampus, a key area for AMPAR mediated opioid-associated memory and learning. This study aims toestablish the molecular-level foundation for CNIH3 activity in the hippocampus using a CNIH3 knock-out
mouse model as a prelude to future opioid studies. Biochemically, we found that CNIH3 plays a critical rolein maintaining both GluA1 and GluA2 AMPAR subunits at the PSD. After measuring the levels of PSD
synaptic scaffolding proteins, synaptic spines, and PSD morphology, we found that CNIH3 may also
regulate the synaptic properties of hippocampal neurons. This study sets the stage for future investigations
into the biochemical, physiological, and behavioral relevance of hippocampal CNIH3 in opioid-induced
plasticity and conditioned behavior.

32 - Mechanistic insights into opioid-induced chemoresistance in breast cancer cells:
DOR stimulation increases P-glycoprotein abundance by exploiting EphA2/c-src
signaling
Alexandra Schoos, Vanessa Knab, Ralph Gradisch, Daniela A. Fux
University of Veterinary Medicine Vienna
Opioids were recently shown to promote the development of drug resistance in breast cancer cells towards
cytotoxic chemotherapeutics. The signaling mechanisms of the phenomenon, however, have not been
identified so far. As the development of drug resistance is often related to an up-regulation of the drug efflux
transporter P-glycoprotein (P-gp), we investigated here whether and how opioids may modulate P-gp
abundance in triple negative breast cancer cells. For the purpose, delta-opioid receptor (DOR) expressing
4T1 and MDA-MB-231 breast cancer cells were incubated with [D-Ala2, D-Leu5]Enkephalin (DADLE) andtested for P-gp abundance by Western blot analysis. In both cell lines, DADLE treatment resulted in asignificant, time- and DOR-dependent increase of P-gp abundance. The increase of P-gp abundance wasassociated with a reduced activation of caspase-3 by the P-gp substrate Doxorubicin. The finding implies
that DOR stimulation increases the expression of functionally active P-gp and protects breast cancer cells
from Doxorubicin-induced apoptosis. Further analysis revealed that DADLE exposure leads to an activationof the Ephrin receptor tyrosine kinases EphA2 in 4T1 and MDA-MB-231 cells. Inhibition of EphA2 activity
by Dasatinib diminished DADLE-mediated P-gp up-regulation. In addition, P-gp regulation was alsoabolished by an inhibition of the EphA2 down-stream substrate c-src kinase. Together the findings let us
suggest that stimulation of DORs in breast cancer cells may trigger Doxorubicin resistance by increasing
P-gp abundance via stimulating EphA2/c-src kinase signaling pathway.

56

33 - Investigating delta opioid receptors in a new conditional knockin mouse
Khaled Abdallah1, Jade Degrandmaison1, Véronique Blais1, Kelly Bradbury3, Francis Bergeron1,
Kimberly Fontes3, Christine Lavoie1, Catherine Cahill2, Jim Boulter4, Jean-Luc Parent1, Louis
Gendron1
1
Université de Sherbrooke, 2University of California Irvine, 3Bishop's University, 4University ofCalifornia in Los Angeles
Delta opioid receptors (DOPrs) represent a promising target for the treatment of chronic pain and emotional
disorders. In order to further investigate the role of this receptor, we have generated a geneticallyengineered mouse in which a Flag epitope has been introduced between the first and second amino acidsof DOPr, and a floxed translational stop cassette introduced upstream of the initiation codon. In the absenceof recombination, these mice (STOP::Flag-DOPr) behave as DOPr knockout mice. By breeding
STOP::Flag-DOPr mice with Zp3-Cre mice, we generated a mouse line expressing a Flag-tagged DOPr inplace of the endogenous receptor (Flag-DOPr knockin). In the present study, we thoroughly characterizedthese mice using pharmacological and behavioral approaches. We found that the Flagged receptor isexpressed at similar levels and in the same areas as its endogenous counterpart and that DOPr agonists
produce similar behavioral effects than in wildtype animals. Most importantly, we succeeded in
immunoprecipitating Flag-DOPr endogenously expressed in the brain of these mice. Not only the FlagDOPr knockin mice are a useful complementary tool to existing genetic mouse models (ex. DOPr-KO andcKO-, DOPr-eGFP knockin), they also represent a unique opportunity to better understand the role and theregulation of DOPr, in vivo, in specific brain regions or in a subpopulation of neurons.
This work was supported by the Quebec Pain Research Network (QPRN), the Canadian Institutes for
Health Research (MOP-123399), and the Fonds de Recherche Québec-Santé.

34 - Oxycodone conditioned place preference alters hippocampal mossy fiber leuenkephalin levels in a sex-dependent manner
Jason D. Gray1, Natalina Contoreggi2, Farah Bshesh2, James Ryan2, Yan Zhou1, Konrad T. Ben1,
Joshua F. Kogan1, Bruce S. McEwen1, Mary Jeanne Kreek1, Teresa A. Milner2
1
The Rockefeller University, 2Feil Family Brain and Mind Research Institute, Weill Cornell
Medical College
In the rat hippocampus, females with elevated estrogen states (proestrus) compared to males contain twice
the levels of leu-enkephalin (LENK)-immunoreactivity (ir) in mossy fibers and three times as many delta
opioid receptor (DOR)-labeled CA3 dendritic spines contacted by mossy fibers. We examined the effects
of repeated exposure to the opioid agonist oxycodone (3mg/kg, I.P.) during a 10-day conditioned place
preference (CPP) task on LENK mossy fiber levels and in the subcellular distribution of DORs in CA3
dendritic spines contacted by mossy fibers of female and male rats. All female rats were in proestrus/estrus
as assessed by vaginal smear cytology. Hippocampal sections were processed for quantitative lightmicroscopic localization of LENK-ir using immunoperoxidase and electron microscopic localization of DOR
using silver-intensified gold (SIG). In females only, oxycodone CPP resulted in decreased LENK-ir levels
in two subregions of the mossy fiber pathway: stratum lucidum of CA2/CA3a and the central hilus of thedentate gyrus. In both females and males, oxycodone CPP did not alter the number of DOR SIG particles
in CA3 pyramidal cell dendritic spines contacted by mossy fibers. These results suggest that in females
oxycodone CPP results in the selective depletion of LENK in mossy fibers that overlap with pyramidal cell
dendrites known to be enriched in phosphorylated DORs and with GABAergic hilar interneurons known tocontain DORs. Moreover, they also suggest that following oxycodone-CPP DORs remain positioned in
CA3 neuron dendritic spines where they could promote learning processes in females.
Supported by: NIH-DA08259 (TAM, MJK, BSM)

57

35 - Delta-opioid receptor (DOR) antagonism/inverse agonism reduces the developmentof mu-opioid receptor (MOR) tolerance
Nicholas Griggs, Henry Mosberg, John Traynor
University of Michigan
Opioid analgesics are prescribed to combat moderate to severe pain yet there are many problems
associated with the use of these medications, including constipation, respiratory depression, and physicaldependence. Previous studies have reported that elimination of the delta-opioid receptor (DOR) or coadministration of naltrindole, a DOR antagonist, with the mu-opioid receptor (MOR) agonist morphine
reduces the development of analgesic tolerance and also lessens dependence. As such we and others
have designed and synthesized opioid ligands with a MOR agonist-DOR antagonist profile that show
antinociception in rodent models but with reduced tolerance liability. To probe the mechanism behind theaction of these so called “mixed efficacy” compounds we have employed SH-SY5Y neuroblastoma cells,
which endogenously express both human opioid receptor types. MOR tolerance at the level of the Gprotein, determined as a decrease in GTPγ35S binding, was induced by overnight incubation with highconcentrations of the selective MOR agonist DAMGO. Tolerance was observed as a loss of the maximal
effect and potency of DAMGO and morphine. Co-treatment of the cells with the DOR antagonist naltrindoleor the DOR inverse agonist ICI-174864 at DOR-selective concentrations reduced the development oftolerance. These findings provide support for the hypothesis that DOR inhibition is advantageous for
improved MOR agonist activity and that at least one contributing mechanism involves receptors in a single
cell. Overall, these data support the development of “mixed-efficacy” MOR agonist-DOR antagonist
compounds. Funded by DA-03910 and NIGMS-GM007767.

36 - Selective Inhibition of M5 Muscarinic Acetylcholine Receptors Attenuates
Remifentanil Self-Administration without Blocking Morphine-induced Analgesia in Rats
Barak W. Gunter1,3, Robert W. Gould1,3, Michael P. Miller1,3, Michael Bubser1,3, Craig W.
Lindsley1,2,3, Carrie K. Jones1,31
Department of Pharmacology, Vanderbilt University, 2Department of Chemistry, Vanderbilt
University, 3Vanderbilt Center for Neuroscience Drug Discovery
Opioid use disorder (OUD) has reached epidemic proportions in the United States; yet current treatment
options remain limited and are associated with abuse potential and other adverse side effects.
Accumulating evidence suggests that selective inhibition of the M 5 muscarinic acetylcholine receptor(mAChR) subtype may provide a novel approach for the treatment of OUD. Recently, we reported thesuccessful discovery and optimization of a novel series of highly selective M 5 negative allosteric modulators(NAMs), represented by the M5 NAM tool compound ML375. Since our previous studies have demonstrated
that ML375 can reduce cocaine self-administration in rodents, we set out to determine if ML375 could also
attenuate opioid self-administration in rats without blocking opioid-induced analgesia.
In the present study, male Sprague-Dawley rats were trained to self-administer remifentanil under a fixedratio schedule of reinforcement. The ability of ML375 to attenuate the reinforcing effects of remifentanil at
each unit dose was then determined. The effects of ML375 were also determined when rats responded
under a progressive ratio (PR) schedule for remifentanil alone and in combination with cocaine. Finally, thepotential analgesic effects of ML375 alone and in combination with morphine were assessed using a hot
plate apparatus.
ML375 reduced remifentanil self-administration under the FR 10 schedule and under the PR schedule.
Moreover, preliminary data indicate that ML375 has no effect on morphine-induced analgesia. Taken
together, these results suggest that selective negative allosteric modulation of the M 5 receptor may
represent a promising novel pharmacotherapy for the treatment of OUD.

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37 - Functional consequences of OPRM1 A118G (MOR N40D) on humanneurotransmission
Apoorva Halikere1, Jennifer C. Moore2, Mavis Swerdel3, Jay Tischfield2, Ronald P. Hart3, Zhiping P. Pang1;
1Child Health Institute of New Jersey, Robert Wood Johnson Medical School, Rutgers University,
2RUCDR/Infinite Biologics, The Human Genetics Institute of New Jersey, Department of Genetics, Rutgers
University, 3The Human Genetics Institute of New Jersey, Department of Cell Biology and Neuroscience,
Rutgers University
Functional genetic variation at the mu opioid receptor (MOR), particularly rs1799971 (ORPM1 A118G, MOR
N40D), has been linked to addiction and other reward-related behaviors. To investigate the functional
consequences of this SNP in a human neuronal context, we derived human induced neuronal (iN) cells
from induced pluripotent stem (iPS) cell lines generated from human subjects carrying either homozygous
N40 or D40 alleles. Our data obtained from induced human neuronal cells exposed to DAMGO reveal two
new fundamental insights into the functional consequences of this SNP: 1) D40 MOR exhibits stronger
inhibition of synaptic release, suggesting enhanced acute signaling and 2) D40 MOR iN cells exhibit
defective signaling after chronic (7 days) DAMGO exposure compared to N40 MOR iNs, suggesting a
possible impaired receptor re-sensitization is associated with the D40 MORs. Additionally, in order torigorously control for background genetic variation among human subjects that may confound diseasephenotypes, we used CRISPR/Cas9 mediated gene targeting to create two isogenic pairs of human stem
cell lines from which we recapitulated differences in DAMGO sensitivity observed in subject derived
neurons, providing strong additional evidence to support our new data. Our study provides important insight
into how functional genetic variation in OPRM1 may modify synaptic transmission in a human neuronalcontext.

38 - Crucial role of the κ-opioid receptor system in tumor angiogenesis
Yusuke Hamada1, Kohei Yamamizu2,3, Michiko Narita1, Naoko Kuzumaki1, Hiroshi Nagase4, Jun K
Yamashita2,3, Minoru Narita1,5; 1Dept Pharmacology, Hoshi University, School of Pharmacy andPharmaceutical Sciences, Tokyo, 2Dept Cell Growth and Differentiation, Center for iPS Cell Research andApplication (CiRA), Kyoto University, Kyoto, 3Laboratory of Stem Cell Differentiation, Stem Cell ResearchCenter, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 4Dept Medicinal Chemistry,
International Institute for Integrative Sleep Medicine (IIIS), University of Tsukuba, Tsukuba, 5Life Science
Tokyo Advanced Research Center (L-StaR), Hoshi Univ. Sch. Pharm. Pharmaceut. Sci., Tokyo, Japan
Opioids are effective analgesics for the management of moderate to severe cancer pain. However, little is
known about the role of opioid systems in tumor angiogenesis. The tumor angiogenesis is required fortumor progression with the highly expression of several activators such as vascular endothelial growth
factor (VEGF). In the previous study, we demonstrated that the κ opioid receptor (KOR) ligand could act asa novel endogenous angiogenesis inhibitor in vascular development. In the present study, we investigatedwhether KOR system could inhibit tumor angiogenesis in the process of tumor growth. We confirmed thattreatment with KOR agonists, U50,488H and nalfurafine (TRK-820), significantly inhibited human umbilicalvein 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 toprevent angiogenesis in HUVECs. Furthermore, Lewis lung carcinoma or B16 melanoma grafted in KOR
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. Finally, treatment with nalfurafine enhanced
the survival advantage induced by treatment with gemcitabine in pancreatic cancer-bearing mice. Thesefindings indicate that KOR system could act as an inhibitory modulator of tumor angiogenesis via
suppressing VEGF signaling. This knowledge could lead to a novel strategy for cancer therapy.

59

39 - CaMKIIα controls the biogenesis of let-7 microRNAs in opioid tolerance
Ying He, Zaijie Jim Wang
Department of Biopharmaceutical Sciences and Cancer Center, University of Illinois, Chicago,
IL USA
Emerging evidence suggests that microRNAs (miRNAs) – mediated cellular adaptations are critical for drug
addiction. We previously reported that let-7 family miRNAs contribute to the development of opioid tolerance
by targeting the µ opioid receptor. Chronic morphine treatment induced a marked increase of let-7
expression, which functionally correlated with the development of opioid tolerance. The aim of this study
was to understand the mechanisms how let-7 is regulated by opioids. We first determined the transcription
status of let-7 and found that the expression of primary let-7 (pri-let-7) remained unchanged in SH-SY5Y
cells that were treated with morphine (1 μM, for 48 h). In agreement with the in vitro observation, chronicmorphine tolerance (one 75 mg morphine pellet/mouse, s.c.) did not alter the level of pri-let-7 in mousebrain front cortex region. These findings suggested that the robust elevation of let-7 occurred at the posttranscriptional level. In the presence of KN93, inhibitor of Ca 2+ /calmodulin-dependent protein kinase II(CaMKII), chronic morphine treatment failed to generate let-7 up-regulation in SH-SY5Y cells. We further
determined whether inactivation of CaMKIIα by T286A point mutation would affect let-7 expression andopioid tolerance. Indeed, antinociceptive tolerance was absent in CaMKIIαT286A mutant mice. Meanwhile,the level of let-7 in CaMKIIαT286A mutant mice was much lower than that in wild-type mice, and was resistant
to chronic morphine stimulation. Taken together, these data suggested that the activity of CaMKIIα wasessential for the biogenesis of let-7 in opioid tolerance.

40 - The G-protein Biased Opioid Agonist PZM21 Does Indeed Depress Respiration
Rob Hill1, Alex Disney2, Alex Conibear1, Katy Sutcliffe1, Steve Husbands2, Chris Bailey2, Eamonn
Kelly1, Graeme Henderson1
1
University of Bristol, 2University of BathPZM21 is a novel opioid agonist reported to produce G-protein biased signalling at the MOPr. PZM21 wasalso reported to have decreased side effects, including a lack of significant respiratory depression [1]. In
the current investigation we sought to replicate those results.
Bioluminescence resonance energy transfer (BRET) was used to investigate the relative activation of Gprotein signalling or arrestin-3 recruitment by PZM21. Respiration was measured by whole bodyplethysmography in male mice (CD-1) breathing either 95% air/5% CO2 or 100% air.
PZM21 (0.01-30 µM) showed little to no recruitment of arrestin-3 in the BRET assay compared to significant
activation of the G-protein signalling pathway (EC50=0.27 µM). For G-protein activation PZM21 had higher
efficacy than morphine but lower efficacy than DAMGO. PZM21 thus displayed significant bias for G-protein
activation over arrestin recruitment at MOPr.
Acute administration of morphine (10 mg/kg i.p.) rapidly depressed respiration in both 100% air and 95%
air/5% CO2. Saline did not decrease respiration under either condition. Contrary to the previous report [1]acute administration of an equi-analgesic dose [1] of PZM21 (40 mg/kg i.p.) produced significant respiratory
depression under both conditions that was not significantly different from that observed following morphine.
This resulted from a decrease in respiratory rate, not tidal volume. PZM21 induced respiratory depression
was completely inhibited by naloxone (1 mg/kg).
These data demonstrate that PZM21 is indeed a G-protein biased MOPr agonist. However, despite thisbiased signalling profile, PZM21 retains the undesired side effect of respiratory depression.

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41 - Inflammatory pain alters the vulnerabilitiy to alcohol relapse without impacting theintensity of the relapse in rats.
Yolanda Campos, Lucía Martí-Prats, Luis Granero, Ana Polache, Teodoro Zornoza, Lucia Hipolito
University of Valencia
Clinical research has highlighted the relationship between pain and drug use disorders, especially opiatesand ethanol. Moreover, a recent clinical study showed that the correct management of pain in patients witha previous history of alcohol use disorder decreases the risk of relapsing, revealing that pain may increase
the vulnerability to alcohol relapse. Recent data demonstrated that, in rats, inflammatory pain desensitizes
the mu opioid receptors located in the mesocorticolimbic dopamine system and consequently increases
heroin voluntary intake at high doses. It is well known that alcohol reinforcing effects and alcohol relapse
are also mediated through the activation of mu opioid receptors in the mesolimbic pathway. However, therole of inflammatory pain in the consumption or relapse into ethanol has not been explored yet. In this study
we evaluated the effect of inflammatory pain on the alcohol deprivation effect (ADE) in long-term ethanolexperienced rats using a non-operant paradigm. To this end, rats were exposed to four free ethanol intake
periods followed by four forced abstinence periods. The complete Freund's adjuvant was used to induce
pain during the fourth period of imposed ethanol deprivation. In the group of rats presenting ADE in all
periods of reintroduction, the presence of pain did not induce changes in the ADE. Noteworthy in the ratsthat never showed ADE, the development of an inflammatory pain condition increased the amount of ratsthat spontaneously presented ADE phenomenon. Our results suggest that pain may affect the vulnerability
to relapse without altering the intensity of the ADE phenomenon.

42 - Simultaneous administration of a chemokine receptor antagonist with morphineenhances the analgesic effect of morphine on incisional pain in rats
S. Inan1,2, X. Chen1,2, A. Cowan1,2, J.J. Meissler1,2, E.B. Geller1,2, C. Tallarida1,2, S. Rawls1,2, T.K.
Eisenstein1,2, M.W. Adler1,2
1Center for Substance Abuse Research, 2Temple University Lewis Katz Medical School
Cross-desensitization between opioid and chemokine receptors is well established. It has also been shownthat chemokines are involved in pain modulation. We investigated if simultaneous administration of achemokine receptor antagonist with morphine would enhance the analgesic effect of morphine on incisional
pain in rats. Dose-response curves for morphine (1-10 mg/kg) and maraviroc (0.5-5 mg/kg), a CCR5
antagonist, alone were established using male rats. Animals underwent incisional surgery on the left hindpaw. Pain responses were evaluated using von Frey filaments at various times post-surgery 15 to 360
minutes and 24 to 96 hours. Morphine, maraviroc, or their vehicles were injected s.c. at 25 minutes postsurgery. While morphine significantly reduced pain in a time- and dose-dependent manner, maraviroc had
no effect. Mean % reversal of mechanical allodynia was established at 60 min post-surgery with 5 (56 ±14%) and 10 (86 ± 5%) mg/kg of morphine. Another group was given both maraviroc (2.5 mg/kg) andmorphine (5 mg/kg) at different s.c. sites (t = 25). Mean % reversal of mechanical allodynia at 60 min was
significantly increased (100 ± 0%). The data show that combining morphine with maraviroc significantly
enhanced the analgesic effect of morphine. This result indicates that the same analgesic effect of morphine
alone can be achieved with lower doses of morphine when combined with maraviroc, which could haveclinical treatment implications for some types of pain. Immune changes induced by the incision and the
drugs were measured.
Supported by Grant (W81XWH-15-1-0252) from the DoD and U.S. Army.

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43 - Dermatolgical manifestations in substance abuse and how the community responses
in developing country
Shambhu D Joshi1, Bhatta A2
1
Far western community Hospital, 2Vardhaman mahabir medical college
Introduction: It has been estimated that there are around 60000 drug abusers in Nepal. Some ofthe herbal by products are used as a cultural, medicinal and traditional exchange from generation
to generation such as marijuana, alcohol, cannabis. Later on, other products such as
pharmaceutical drugs either by import or for export in this globalization era.
Methods: It was a study 2015-2016, of patients who came for treatment with dermatologic relateddisorders in the clinical dermatology practice. 163 cases which were taken in-depth interview andcounseling.
Results/Conclusion:: Most of the cases are adolescent group (16-35) , M:F;60:3. 65% of the cases
have complained of dryness of mouth and skin. 12% having erythema and swelling of Injectingsite and 4% having an infection for which they had taken broad spectrum antibiotics. Keloid,
abscess formation and post Inflammatory-pigmentation also seen in 6% of the cases.
Staphylococcus aureus , streptococcal species, and E.coli found in the culture and sensitivity test.
Those who are taking marijuana regularly 45% had Common skin manifestations include
premature aging such as periorbital darkening, hair loss and graying of hair.
30% of the cases those were chronic alcoholic have had features of CNS depressant, impairing
motor function and coordination, decision-making and judgment, and memory.
In a resource poor setting areas, dermatologist plays a vital role for the treatment, managementwith a multidisciplinary team. Early diagnosis, treatment, and rehabilitation are important toimprove the quality of life of people in developing country

44 - Opioid receptors reveal a parallel cycle of regulated presynaptic membranetrafficking
Damien Jullie, M Stoeber, JB Sibarita, Mark von Zastrow
UCSF
Much is known about how presynaptic GPCRs regulate the synaptic vesicle (SV) cycle, but little
is known about GPCRs trafficking at the presynapse. We show that mu opioid neuropeptide
receptors (MORs), GPCRs that mediate inhibition of SV cycling, localize diffusely along the axonal
plasma membrane when not activated and rapidly concentrate at presynaptic terminals in
response to ligand-induced activation. This dynamic accumulation process occurs through ligandinduced endocytosis of MORs into a population of endosomes that accumulate in boutons
separately from SV membranes and are specifically marked by retromer. These endosomes, inturn, locally source vesicular insertion of receptors into the presynaptic membrane. The GPCR
cycle is mechanistically distinct from the SV cycle because it is stimulated, rather than inhibited,by ligand-induced activation of MORs and operates in a fully calcium-independent manner. Thesefindings reveal a discrete cycle of presynaptic membrane trafficking that dynamically accumulates
neuromodulator receptors at the presynapse.

6245 - Probe Dependence of High and Low Affinity Agonist Binding to the Delta Opioid
Receptor is Allosterically Regulated by BMS-986187 and Na+ Ions
Evan Schramm1, Kathryn Livingston1,2, John Traynor1
1
University of Michigan, 2University of California-San Francisco
BMS-986187 is a positive allosteric modulator (PAM) for the delta opioid receptor (DOPr) thatenhances the binding affinity and/or the efficacy of orthosteric agonists. The probe dependence
of BMS-986187, or the differential cooperativity between orthosteric ligand types and BMS986187, was examined by measuring the effect of the PAM on DOPr binding affinity of a varietyof ligands. Competition binding was performed using the non-selective ligand 3H-diprenorphinein CHO-hDOPr membrane fractions in the presence of 100mM NaCl and 10uM GTPγS to promote
inactive (R) receptor states. The results demonstrated that BMS-986187 increased agonist
affinity. The affinities of the endogenous opioid peptides β-endorphin, dynorphin A (1-17),
dynorphin B, leu-enkephalin, and met-enkephalin were enhanced by approximately 140, 50, 11,30, and 40-fold respectively. The small molecule agonist SNC80 shifted 14-fold whereas the shift
for both TAN67 and BW373U86 was 3-fold. These values were compared to the binding affinitiesof the ligands in the absence of NaCl and GTPγS, representing a high affinity (R*) form of thereceptor. The increase in affinity in the presence of BMS-986187 closely correlated with binding
affinities obtained in the absence of NaCl and GTPγS. BMS-986187 likely modulates the DOPrin part by destabilizing the Na+ binding site to promote agonist binding and receptor activation.
This work was supported by R01 DA033396 and T32GM008597.

46 - Allosteric Agonism at the Delta Opioid Receptor: Potential Mechanism for Functional
Selectivity
Matthew Stanczyk, John Traynor
University of Michigan
The delta opioid receptor (DOPr) has gained attention in recent years as a pharmacological target.
DOPr agonists have been shown to promote analgesia and relieve depression. Additionally,
evidence suggests that targeting DOPr results in less respiratory depression and abuse liability
than the commonly targeted mu-opioid receptor. Unfortunately, the potential use of DOPr ligands
is limited by their rapid development of tolerance and induction of convulsions. Evidence suggeststhat biased ligands may be capable of activation of downstream effectors associated with thepositive effects while mitigating the negative ones. BMS 986187 is an allosteric modulator of the
DOPr with apparent agonist properties, thus acting as an “Ago-PAM”. Since Ago-PAMs act at asite other than the orthosteric site it can be hypothesized that they may show unique signaling
properties. This study set out to validate BMS 986187 as an Ago-PAM and to determine thedownstream signaling complement associated with its activation of DOPr in the absence oforthosteric ligand. We confirmed that BMS 986187 is an Ago-PAM at the DOPr. Additionally, BMS
986187 shows biased agonism favoring G protein activation over β-arrestin relevant pathways
including receptor phosphorylation and internalization. This is the first evidence of an opioid AgoPAM showing bias and suggests that targeting the DOPr through this allosteric site may be aunique way to potentially mitigate side effects. Funded by DA 033397 and 5T32GM007767-38.

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47 - Allosteric activation of delta opioid receptors in vivo: promising behavioral profile
Emily Jutkiewicz1, Isaac Dripps2
1
University of Michigan, 2University of Michigan
Positive allosteric modulators (PAMs) bind to receptor sites distinct from the orthosteric binding site, canmodify the affinity and activity of orthosteric ligands, and may have activity on their own. It has been
proposed that PAMs could have distinct advantages as compared with orthosteric agonists, such asincreased receptor selectivity, improved margins of safety, and selective augmentation of endogenous
tone. Activation of delta-opioid receptors (DORs) at the orthosteric binding site produces analgesic andantidepressant effects in animal models; however, prototypical DOR agonists, such as SNC80, also
produce adverse convulsive effects. Therefore, this study aimed to evaluate the in vivo effects of the DOR
PAM BMS-986187, alone and in combination with endogenous and exogenous agonists, to determine its
profile of activity. We evaluated potential pain-relieving effects in the acetic acid-induced stretch assay andin the nitroglycerin-induced hyperalgesia assay in mice. We measured antidepressant-like effects in themouse forced swim and tail suspension tests and convulsions by continuous observation. Alone, BMS986187 produced only antidepressant-like effects that were attenuated by the DOR antagonist naltrindole
and absent in the DOR knockout mice. BMS-986187, but not the orthosteric agonist SNC80, enhanced theantidepressant-like effects of the enkephalinase inhibitor RB101. BMS-986187 increased the potency
and/or efficacy of the DOR agonists SNC80 and KNT127 to produce antihyperalgesia and antidepressantlike effects but did not alter their convulsive effects. These findings suggest that the DOR PAM has apromising profile of behavioral effects, likely by enhancing endogenous tone to alter mood without
seizurogenic activity.

48 - Analysis of medication overuse headache after repeated morphine or THC
administration using home cage wheel running
Ram Kandasamy, Cole T. Dawson, Tammy N. Hilgendorf, Michael M. Morgan
Washington State University Vancouver
Medication overuse headache (MOH) is characterized by more frequent and intense headaches upon
repeated use of anti-migraine treatments. Thus, emerging anti-migraine treatments must be tested for MOH
in preclinical studies. We have shown that activation of TRPA1 receptors on dural afferents via
microinjection of allyl isothiocyanate (AITC) mimics migraine-like pain as indicated by depression of home
cage wheel running. Although opioids can reduce migraine pain, they are not recommended because of
the potential for MOH. The objective of this study was to assess morphine-induced MOH in rats anddetermine whether MOH also occurs with repeated administration of ∆9-tetrahydrocannabinol (THC). Rats
had access to a running wheel in their home cage 23 hrs a day for one week. Baseline running wasassessed on the last day. Rats were injected intraperitoneally twice-daily for 2.5 days with either THC (1.0mg/kg), morphine (3.2 mg/kg), or vehicle. Migraine-like pain was induced 24 hrs following the last injection
via dural microinjection of AITC (10%; 10 μL). Immediately after, rats received either THC (0.1, 0.32 mg/kg),
morphine (0.32, 1.0 mg/kg), or vehicle intraperitoneally. Dural AITC depressed running for 3 hours.
Administration of 0.32 mg/kg THC prevented AITC-induced depression of wheel running in THC- andvehicle-pretreated animals. Administration of 1.0 mg/kg of morphine prevented depressed wheel runningfor one hour in saline-pretreated animals, but was ineffective in morphine-pretreated animals. AITCdepressed wheel running was prolonged in morphine-pretreated animals compared to THC- and vehiclepretreated animals. These data indicate that repeated morphine, but not repeated THC, administration
elicits MOH

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49 - Effect of opioids on the cellular responses to anti-cancer drugs
Leena Karhinen1, Tuomas Lilius1,3, Krister Wennerberg2, Eija Kalso1,3
1
University of Helsinki, 2Institute for Molecular Medicine Finland, 3Helsinki University Hospital
Cancer patients routinely receive opioids during surgery and for the management of severe cancer-related
pain. In preclinical studies, morphine has been found to drive breast cancer progression (1-3). In patient
cohorts, polymorphism of the mu-opioid receptor (MOR)-coding gene OPRM1 has been proposed to belinked with the onset and prognosis of breast cancer (4,5) and oesophageal squamous cell carcinoma (6).
Specifically, the A118G polymorphism may correlate with the better survival of breast cancer patients (5,our unpublished results). Morphine has even been suggested to promote stemness and chemoresistancein breast cancer cells (7). Chemotherapy is the main line of treatment in the most severe breast cancer
cases that lack targeted treatments. Thus, the role of opioids in chemoresistance might play a role in their
prognosis.
In this study, we address the effects of different commonly used opioid MOR-agonist analgesics and one
opioid antagonist on breast cancer cells in vitro, in combination with cytostatics. We study whether
morphine, oxycodone or fentanyl affect proliferation, migration and invasion of breast cancer cells in vitro,
and whether opioids have a role in chemoresistence. Our first results suggest that morphine may enhanceproliferation of cells. Currently, we are studying the combinations with chemotherapeutics.
1. Nguyen J .. Br.J.Anaesth. 113 Suppl 1:i4-13, 2014
2. Gupta K .. Cancer Res. 62:4491-8, 2002
3. Farooqui M .. Br.J.Cancer. 97:1523-31, 2007
4. Cieslinska A .. Tumour Biol. 36:4655-60, 2015
5. Bortsov AV .. Anesthesiology.116:896-902, 2012
6. Wang S.. Int J.Clin.Oncol. 18:666-9, 2013
7. Niu DG .. Oncotarget. 6:3963-76, 2015

50 - Adolescent social isolation increases kappa opioid receptor function in the nucleus
accumbens and basolateral amygdala of rats.
Anushree N. Karkhanis, Jeffrey L. Weiner, Sara R. Jones
Department of Physiology and Pharmacology, Wake Forest School of Medicine
Adverse social experiences during adolescence increase the risk of developing alcohol use disorder during
adulthood in humans. Similarly, rats exposed to adolescent social isolation show greater ethanol intake inadulthood compared to group housed controls. Acute stress elevates dynorphin levels, a kappa opioid
receptor (KOR) ligand, which regulates dopamine. Activation of KORs inhibits dopamine release in the NAc
and the BLA. The NAc and BLA are interconnected and play integral roles in the neurobiology of stress,
anxiety, and reward-seeking behavior. In order to investigate early-life stress-induced changes in KORs,
we housed the rats individually (social isolation, aSI; 1 rat/cage) or in groups (group housed, aGH; 4
rats/cage) for six weeks (PD 28-74). Baseline levels of dopamine were significantly lower in the NAc and
BLA of aSI compared to aGH rats. KOR inhibition increased baseline dopamine levels in both NAc andBLA. The aSI rats showed increased dopamine responses to ethanol (2 g/kg) in both NAc (200% of
baseline) and BLA (280% of baseline). Ethanol augmented dopamine responses in the NAc of aSI rats pretreated with norBNI, but attenuated responses in the BLA. The inhibitory effects of U50,488 on dopamine
release were enhanced in the NAc of aSI rats suggesting that chronic stress increases KOR function. It is
possible that KOR differences may explain in part the effects of ethanol on behaviors related to specificbrain regions, for example, augmentation of DA in the NAc results in increased reinforcement, whereas
augmentation in DA in the BLA may contribute to decreased anxiety.

6551 - Norbuprenorphine pharmacokinetics and pharmacodynamics: First in man evaluation
Evan D. Kharasch1,2, Alicia M. Flaker1, Kristi Kraus1, Jane Blood1
1Washington University in St Louis, 2St Louis College of Pharmacy(Bup) is a partial µ agonist, δ and κ antagonist, and nociceptin receptor agonist, with complex
pharmacology. It is extensively metabolized, primarily to norbuprenorphine (norBup); both Bup and norBup
undergo glucuronidation. Metabolite exposure can exceed that of Bup. NorBup, Bup-glucuronide andnorBup-glucuronide are pharmacologically active. Mechanisms of Bup pharmacology in humans remain
undefined. We conducted a series of first-in-man studies to evaluate norBup clinical pharmacology. The
first was a dose-escalation study (0.005-1 mg) with venous plasma sampling. The second evaluated a 1 hr
infusion of 0.3 mg, with arterial and venous sampling, and measurement of pupil diameter, respiratory rate,
end-expired CO2, and response to thermal stimulus. NorBup was well-tolerated, without adverse effects.
Plasma norBup-glucuronide exceeded norBup concentrations. NorBup and norBup-glucuronide plasma
AUCs, and urine excretion, were proportional to dose (r>0.9) throughout the entire dose range. NorBupglucuronide was formation-rate limited. NorBup (0.3 mg) caused mild miosis (maximum pupil diameterchange 1.3 ± 0.8 mm), minimal respiratory depression (respiratory rate decreased from 16 ± 1 to 14 ± 3
and end-expired CO2 increased from 39 ± 2 to 41 ± 2 mmHg), and was slightly anti-analgesic (maximum
tolerated temperature decreased from 49 ± 1 to 48 ± 1 °C and NRS pain ratings to a preset temperature
increased to 113 ± 13% of baseline). Drug effects were negligible after 24 hr. Effect (miosis) vsconcentration showed hysteresis. NorBup is pharmacologically active in man, shows both µ agonist and κ
antagonist properties and may contribute to the pharmacologic effects of parent Bup.
52 - Serum and urine concentrations of morphine and morphine metabolites in patients withadvanced cancer receiving continuous intravenous morphine: an observational study
Soo Jin Kim1, Yong Joo Lee2, Sang Yeon Suh3, Junghan Song4, Pal Klepstad5, Sanghee Shiny Lee6, Ah
Ram Seo6, Hong Yup Ahn6, Myung Ah Lee7, Chul Min Kim1
1DeptFamily Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea,
Seoul, 2Dept Palliative Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic Universityof Korea, Seoul, 3Dept Medicine, Dongguk University School of Medicine, Seoul, 4Dept Labaratory
Medicine, Seoul National University Bundang Hospital, College of Medicine, Seoul, 5Dept
Anaesthesiology and Intensive Care Medicine, St Olvas University Hospital, Trondheim, Norway, 6Dept
Statistics, Dongguk University, Seoul, 7Division of Oncology, Dept Internal Medicine, Seoul St. Mary's
Hospital, College of Medicine, Catholic University of Korea, Seoul, South Korea
The feasibility and clinical implication of drug monitoring of morphine, morphine-6-glucuronide (M6G) andmorphine-3-glucuronide (M3G) need further investigation. This study aimed to determine what predicts
serum concentrations of morphine in cancer patients receiving continuously intravenous morphine, therelationships between serum concentration of morphine/its metabolites and urinary concentrations, and therelation between morphine concentrations and with clinical outcomes. We collected serum and urine
samples from 24 patients with advanced cancer undergoing continuously intravenous morphine therapy.
Serum samples were obtained at day one. Spot urine samples were collected once daily on threeconsecutive days. Pain and adverse drug events were assessed using the Korean version of MD Anderson
Symptom Inventory. A total of 96 samples (72 urine and 24 serum samples) were collected. Median doseof morphine was 82.0mg/24hours. In a multivariate analysis, total daily morphine dose was the mostsignificant predictors of both serum and urine concentration of morphine. Morphine, M6G, and M3G inserum and urine were statistical significantly correlated (correlation coefficient = 0.81, 0.44, 0.56; p values< 0.01, 0.03, 0.01, respectively). There was no significant correlation between clinical symptoms andmorphine/metabolite concentrations. Spot urine concentrations of morphine and its metabolites were highly
correlated to those of serum. Total dose of daily morphine was related to both serum and urine
concentration of morphine and its metabolites.

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53 - Evaluation of the dual kappa and delta opioid receptor agonist MP1104 in rat modelsof cocaine self-administration, analgesia and behavioural side-effects
Diana Atigari1, Rajendra Uprety2, Gavril Pasternak2, Susruta Majumdar2, Bronwyn Kivell1
1
Victoria University of Wellington, 2Dept Neurology, Memorial Sloan Kettering Cancer Center
Activation of kappa-opioid receptors (KOPr) have demonstrated anti-cocaine effects and analgesic
properties and have therapeutic potential as non-addictive pain medications and anti-addiction
pharmacotherapies. However, side-effects limit their clinical development. We hypothesise that mixedagonists activating both delta-opioid receptors (DOPr) and KOPr, such as MP1104, will have desired
analgesic and anti-cocaine effects with reduced side-effects. To evaluate this, male Sprague Dawley rats
were administered MP1104 (0.3 and 0.6 mg/kg/i.p.) and analgesic effects evaluated in the tail-withdrawal
assay. MP1104 showed analgesia at 1 h, with peak effects at 3 h and a duration of 8 h. To evaluate theanti-cocaine effects of MP1104 rats were trained to self-administer cocaine (0.5 mg/kg/iv), followed byextinction and then subject to cocaine-prime reinstatement-tests. We show that MP1104 dose-dependently
decreased reinstatement (0.3 and 1 mg/kg/i.p.) (p<0.01 & p<0.001 respectively) in a KOPr dependent, but
not DOPr dependent manner. In cocaine dose-response experiments, acute administration of MP1103 (0.3
(p<0.01) & 0.6 mg/kg (p<0.0001)) caused a downwards shift in the cocaine dose-response curve,
demonstrating potent anti-cocaine effects. Evaluation of side-effects, including aversion in Conditioned
Place Aversion tests, depression, in the Forced Swim Test and anxiety in the Elevated Plus Maze showedno significant effects compared to vehicle treated controls following MP1104 at 0.3 or 0.6 mg/kg doses.
However, MP1104 at 1 mg/kg (p<0.01) but not 0.3 or 0.6 mg/kg doses showed sedative effects inspontaneous locomotor tests. MP1104 shows potent analgesic and anti-cocaine effects with few sideeffects to the selective KOPr agonist U50,488.

54 - Regulation of morphine sensitivity and adenylate cyclase sensitization by carboxylterminus of V1b vasopressin receptors
Taka-aki Koshimizu1, Kenji Honda2, Yukio Takano2
1
Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University,2
Dept Physiology and Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University
Chronic morphine exposure accelerates adenylate cyclase (AC) signaling and reduces the analgesic
efficacy, a condition known as opioid tolerance. The mechanism of how non-opioid neurotransmittersmodulate AC responsiveness during chronic morphine exposure has not been fully clarified. In tail-flick andhot plate tests, we show that morphine sensitivity was increased in mice lacking vasopressin V1b receptors(V1bRs), but not in mice lacking V1a receptors. Analgesic tolerance to morphine developed with significant
delay in V1bR-deficient mice and in mice administered a V1bR antagonist into lateral ventricle. Morespecifically, administrations of a V1bR antagonist to rostral ventromedial medulla (RVM), where V1bRs andμ-type opioid receptors (MORs) were co-expressed, delayed tolerance development. In HEK cell model,
the carboxyl-terminus of the V1bR was constitutively phosphorylated and associated with β-arrestin 2.
Complex formation between constitutive V1b-β-arrestin 2 and MOR was detected in bioluminescenceresonance energy transfer experiments. V1bR-mediated association between β-arrestin 2 and MOR wasnecessary for vasopressin-mediated accelerations of ERK phosphorylation and AC sensitization. Inhibitorof ERK signaling to the RVM alleviated morphine tolerance. Genome editing and deletion of the V1bR
carboxyl-terminus, which was necessary for β-arrestin 2 binding, increased morphine analgesia in miceand suppressed morphine-induced AC sensitization in the RVM. These findings indicated that inhibition of
V1bR that is functionally associated with MOR provides a novel approach to enhance morphine analgesia
without accelerating analgesic tolerance.

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55 - Generation and characterization analysis of human iPS cell derived-sensory neurons
as a useful research tool for pain and opioid systems
Naoko Kuzumaki1, Chizuru Iwasawa1, Yuya Igeta1, Yukari Suda1, Michiko Narita1, Minoru Narita1,21
Dept. Pharmacol., Hoshi Univ., Tokyo, Japan, 2L-StaR, Hoshi Univ., Tokyo, Japan
Chronic pain is a major challenge to clinical practice and basic science. The lack of access tosensory neuron has limited our understanding of its development and the physiology of pain inhumans. Induced pluripotent stem cell (iPSC) technology allows generation of cells from patients,
and can recapitulate disease pathology. Such cells can be used in place of human tissue or animalmodels for disease modeling, drug screening, and even cell replacement therapies. In the present
study, we generated sensory neuron-derived from human iPS cells following a small molecule
differentiation protocol, which results in the differentiation of human iPS to sensory neurons of a
nociceptor phenotype. This protocol involves dual SMAD inhibition, which efficiently induces
neuro-ectoderm formation from human embryonic stem cells. This is followed by inhibition of
GSK-3, γ-secretase, and vascular endothelial growth factor receptor/fibroblast growth factor
receptor, which enables fate specification toward a sensory phenotype followed by maturation of
the neurons with growth factors. Under these conditions, we found the cells expressing markers
of sensory neurons such as TAC1 (substance P), SLC17A6 (VGLUT2), NTRK1 (trkA) and TRPV1
at 15 days after differentiation. Furthermore, the μ-opioid receptor expression was clearly foundin sensory neurons derived from human iPS cells with the progress of cell differentiation. Thesefindings suggest that human iPS cells derived sensory neuron is useful tool for development andfunctional analysis of sensory neuron and opioid system. In near future, we intend to apply thistechnique to pain and opioid research.
56 - Factors associated with ketamine use in pancreatic cancer patient in a single
hospice center
Kyung min Kwon, Young joo Lee, Chang jin Choi
St. Mary's hospital, South Korea
Up to 90% of pancreatic cancer patient suffer from neuropathic pain. In palliative care setting,pain control in pancreatic cancer patient is one of the major goals. Ketamine is a NMDA receptor
antagonist effective in neuropathic pain. Also there have been studies about opioid sparing effectof ketamine. This study was held in palliative care unit among pancreatic cancer patients to findout the factors related to ketamine use and the opioid sparing effect.
Medical records of pancreatic cancer patients admitted to St.Mary’s hospital palliative care unit
from 2013.1 to 2014.12 were reviewed. Patients were divided in to 2 categories according toketamine use. Also opioid use before and after ketamine use was compared in ketamine group.
Compared to non ketamine use group, patients in ketamine group required higher dose of opioid.
Total opioid dose, daily opioid dose, number of daily rescue medication, daily average rescue
dose were statistically significantly higher in ketamine group. Opioid requirement was increased
after ketamine administration.
In this study, ketamine group required more opioid. Ketamine is frequently considered in patientswith severe pain, requiring high amount of opioid. Also ketamine did not have a opioid sparing
effect. Future studies about palliative use of ketamine in larger number of patients are required.

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57 - Sex differences in opioid signaling
Emily Leff, Seksiri Arttamangkul, John Williams
Oregon Health Sciences University
To further understand opioid desensitization and its effect on the clinical population, it is important
to investigate the presence of sex-based differences in our rodent models. We focused on thedensely packed, homogenous population of Locus Coeruleus (LC) neurons. These neurons
express mu-opioid receptors that are coupled to G-coupled inwardly rectifying potassiumchannels (GIRKs) and have been previously shown to undergo opioid desensitization. Using
whole-cell voltage-clamp recordings from LC neurons in acute brain slices from opioid naïve
Sprague-Dawley rats, concentration response curves to morphine and [Met5] enkephalin (ME)were constructed in slices from male and female rats. The morphine and ME currents werenormalized to the current induced by a saturating concentration of the α2-adrenergic agonist UK.
Interestingly, while there was no significant difference in the ME concentration response curves,the morphine concentration response obtained in slices from female rats had a larger maximum
current induced by morphine than experiments in slices from male rats. Intracellular recordings
were used to elucidate sex differences in ME desensitization. There was no significant difference
between males and females in the degree of desensitization during ME application at a saturating
concentration, but females showed a reduction in the recovery from desensitization. The potential
differences in tolerance induced by chronic morphine treatment will be investigated.

58 - Genetic deletion of mu opioid receptors from Kölliker-Fuse neurons reducesmorphine-induced respiratory depression
John Bissonnette1, Brigitte Kieffer2, Erica Levitt3
1
Oregon Health & Science University, 2Douglas Hospital Research Centre, 3University of Florida
Activation of mu opioid receptors in the pontine Kölliker-Fuse (KF) causes a reduction in
respiratory rate in anesthetized rats. We have previously shown that mu opioid agonists
hyperpolarize a population of KF neurons (Levitt et al., J Physiol, 2015). The goal of theseexperiments was to determine the contribution of the opioid-sensitive KF neurons to the reduction
in respiratory rate caused by systemic administration of the opioid agonist morphine. Targeted
deletion of mu opioid receptors was accomplished by viral expression of Cre recombinase in the
KF of mice with floxed mu opioid receptors. Experiments were performed 4-5 weeks later. In
whole-cell recordings from brain slices, infected KF neurons were no longer hyperpolarized by
opioid agonist, confirming deletion of mu opioid receptors. Head-out plethysmography was used
to measure respiratory rate. Morphine (10 mg/kg, ip) caused a significantly smaller reduction in
respiratory rate in mice with bilateral deletion of mu opioid receptors from KF, compared touninjected or GFP injected controls. The time-course for the effect of morphine was also delayedin mice lacking mu receptors in KF neurons. Thus, opioid-sensitive KF neurons contribute to, but
are not entirely responsible for, systemic morphine-induced respiratory depression.

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59 - Cannabinoid CB2 receptor plasticity in rostral ventromedial medulla in inflammatory
pain
Ming-Hua Li, Katherine L Suchland, Susan L Ingram
Dept. of Neurological Surgery, Oregon Health & Science University, Portland OR 97239
Treatment of chronic pain is still challenging. Emerging data indicate that drugs that target endocannabinoid
systems produce analgesia with fewer side effects than opioids. The analgesic activity of cannabinoids is
mediated by activation of CB receptors (CB1R and CB2R). However, the benefits of CB1R analgesia arelimited by its psychotropic effects and the development of tolerance. We have recently demonstrated thatthe function and expression of CB1R was reduced in the rostral ventromedial medulla (RVM) in persistent
inflammation induced by complete Freund’s adjuvant (CFA). Using whole-cell patch-clamp recordings fromadult RVM slices, the effects of CB2 agonists on GABAergic neurotransmission were studied. CB2 agonist
inhibition of GABAergic miniature IPSC (mIPSC) frequency is increased in RVM neurons from CFA-treatedrats. CB2 antagonists increased mIPSC frequency only in CFA-treated RVM neurons indicating an
endocannabinoid tone in inflamed rats. Systemic administration of a CB2 agonist (AM1241, 3mg/kg, i.p.)
reverses mechanical hypersensitivity in CFA-treated rats. Finally, we find that administration of minocycline,
a microglia inhibitor, blocks the development of CB2 plasticity in the RVM during inflammation. Our data
provide that CB2 receptor function emerges in the RVM in persistent inflammation and that selective CB2
receptor agonists may be useful for treatment of persistent inflammatory pain.

60 - The CB2 cannabinoid receptor agonist LY2828360 suppresses chemotherapyinduced neuropathic pain with sustained efficacy and attenuates morphine tolerance anddependence
Xiaoyan Lin1,2, Megan Huibregtse1,2, Ken Mackie1,2,3,4, Andrea G. Hohmann1,2,3,4
1
Program in Neuroscience, Indiana University, 2Psychological and Brain Sciences, Inidana
University, 3Interdisciplinary Biochemistry Program, Indiana University, 4Gill Center for
Biomolecular Science, Indiana University
Cannabinoid CB2 receptors represent an attractive target for pharmacotherapies relative to CB1 receptors
due to the lack of psychotropic effects following activation of this receptor. The CB2 agonist LY2828360
lacked both toxicity and efficacy in a clinical trial for osteoarthritis. However, whether LY2828360 shows
efficacy in neuropathic pain models and/or enhances efficacy of narcotic analgesics has not been reported.
We evaluated anti-allodynic efficacy of chronic treatments with LY2828360 and the opioid analgesic
morphine in paclitaxel-treated wild-type (WT) mice and CB2 knockout (CB2KO) mice. The impact of
LY2828360 treatment on naloxone-precipitated morphine withdrawal was also assessed. LY2828360
suppressed paclitaxel-induced allodynia without producing tolerance in WT mice. Anti-allodynic efficacy of
LY2828360 was absent in CB2KO mice. Morphine tolerance developed in CB2KO mice but not WT micewith a history of LY2828360 treatment. LY2828360-induced anti-allodynic efficacy was preserved in WT
mice previously rendered tolerant to morphine but absent in morphine-tolerant CB2KO mice.
Coadministration of LY2828350 with morphine blocked morphine tolerance in WT but not CB2KO mice.
WT mice that received coadministration of LY2828360 and morphine also exhibited a trend towards lower
numbers of naloxone-precipitated jumps compared to CB2KO mice. In conclusion, the CB2 agonist
LY2828360 attenuates chemotherapy-induced neuropathic pain without producing tolerance, and may
prolong effective analgesia while reducing opioid dependence. Our studies suggest that the CB2 agonist
LY2828360 may be useful as a first line treatment in chemotherapy-induced neuropathic pain and may behighly efficacious in neuropathic pain states that are refractive to opioid analgesics.

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61 - Kappa opioid receptor up-regulation contributes to mood and reward dysregulationin neuropathic pain
Shiwei Liu1, Sarah Pickens1, Anna Taylor2, Lihua Xue3, Piper Williams5, Juli Wu5, F. Ivy Carroll4,
Pat Levitt5, Frances Leslie1, Catherine Cahill1
1
University of California, Irvine, 2University of California, Los Angeles, 3Queens University,
4
Research Triangle Institute, 5University of Southern California
Many patients with chronic pain also present with mood disorders such as anxiety or depression, which
negatively impact their quality of life. The kappa opioid receptor (KOR) is known to regulate mood,
motivation and reward pathways in the brain by modulating dopamine neurotransmission. Our study aimed
to determine whether KOR activity is increased in chronic neuropathic (NP) pain and whether antagonismof this receptor alleviates the tonic aversive component of pain and restores reward.
We induced NP pain in adult male C57/BL6 mice and Long Evans rats via polyethylene cuff implantation
around their left hind limb sciatic nerve. We discovered that KOR signaling was sensitized in NP pain mice
where pain animals exhibited exacerbated conditioned place aversion to KOR agonist U50488. Using qRTPCR and [35S]-GTPγS autoradiography, we also found that NP pain mice exhibited significant increases in
KOR gene expression and protein activation in the nucleus accumbens (NAc) and ventral tegmental area
(VTA). Furthermore, fluorescent in situ hybridization of the VTA confirmed the majority of this KOR upregulation occurred within dopaminergic neurons. NP pain led to a blunting of reward because intra-VTA
injection of MOR agonist DAMGO failed to produce a conditioned place preference (CPP), an effect that
could be reversed with pretreatment of KOR-selective antagonist JDTic. The mechanism of reward
disruption was determined using microdialysis to be KOR-mediated inhibition of dopamine release at theNAc. These results highlight the impact of KOR activation on NP pain, which has negative consequences
on mood and reward.

62 - The role of endogenous dynorphins in amphetamine sensitization and depressionlike behaviors in mice
Abdul Hamid, Patrick Chan, Kabirullah Lutfy
College of Pharmacy, Western University of Health Scieces
Endogenous dynorphin has been implicated in the action of amphetamine as well as in depression-likebehaviors in rodents. In the present study, using male and female mice lacking the preprodynorphin(ppDYN) gene and thier wild-type littermates/controls, we determined the role of endogenous dynorphin in
amphetamine-induced hyperlocomotion and sensitization as well as in depression-like behaviors in naive
as well as following short-term amphetamine withdrawal. Our results showed that amphetamine increased
locomotor activity in both male and female mice, the magnitude of which was greater in wild-type comparedto ppDYN knockout mice. Sensitization developed to the action of amphetamine in male mice but there wasno difference between mice of the two genotypes. On the other hand, sensitization develped in female micelacking the ppDYN gene but not in their wild-type controls. The immobility time was not different between
wild-type and knockout mice of either sex, showing that male and female mice of the two genotypes
exhibited comparable depression-like behaviors. Similarly, the magnitude of depression-like behaviors
following amphetamine cessation was not different between mice lacking the ppDYN gene and their wildtype controls. On the other hand, male mice lacking the ppDYN gene exposed to the FST and then tested
for locomotor activity exhibited sensitized response to an amphetamine challenge dose compared to theirrespective non-stressed naive mice. In contrast, amphetamine-induced hyperlocomotion was comparable
between wild-type mice exposed to the FST and their non-stressed controls. Taken together, these results
suggest that dynorphin does not play any functional role in depression-like behaviors in mice.

7163 - AT-328, a selective NOP agonist, reduces the rewarding action of ethanol andcocaineNurulain Zaveri1, Paul Marquez2, Blair V. Journigan1, Abdul Hamid2, Kabirullah Lutfy2
1
Astraea Therapeutics, 2College of Pharmacy, Western University of Health Sciences
The NOP receptor has been characterized as a potential target to reduce the rewarding action of addictive
drugs. In the present study, we characterized the selectivity and affinity of a novel small-molecule NOP
agonist AT-328 toward NOP compared to classical opioid receptors, and tested its effects on the rewarding
actions of ethanol and cocaine using the conditioned place preference (CPP) paradigm. Mice lacking the
NOP receptor and their wild-type littermates/controls were tested for baseline place preference on day 1,
in which each mouse was placed in the neutral chamber of the CPP apparatus and allowed to freely explore
the chambers for 15 min. The amount of time that mice spent in each chamber was recorded. On days 24, mice received twice daily conditioning with the NOP ligand (3 mg/kg) or its vehicle 5 min prior to ethanol(2 g/kg) or cocaine (15 mg/kg) or vehicle followed by saline in a counterbalance manner. On day 5, animals
were tested for preference toward the conditioning chambers, as described for day 1. Our results showedthat ethanol and cocaine each induced a significant CPP response in both wild-type and knockout
mice; however, the magnitude of this response was not different between mice of the two genotypes. AT328 blocked the development of CPP induced by either drug in wild-type but not knockout mice, suggestingthat the novel NOP agonist reduced the rewarding action of ethanol and cocaine via the NOP receptor.
Thus, NOP agonists may represent potential pharmacotherapy to treat drug addiction.

64 - The role of mu opioid receptors in hyperlocomotion and sensitization induced byamphetamine
Abdul Hamid, Paul Marquez, Kabirullah Lutfy
College of Pharmacy, Western University of Health Sciences
The endogenous opioid system has been implicated in hyperlocomotion and sensitization induced by anumber of addictive drugs including amphetamine. For example, naltrexone the non-selective opioid
receptor antagonist, has been shown to attenuate the motor stimulatory action of amphetamine. In thepresent study, we determined the role of mu opioid receptor in amphetamine-induced hyperlocomotion andsensitization in female mice. Mice lacking the mu opioid receptor and their wild-type littermates/controls
were habituated to the locomotor activity chambers for one hour, injected with amphetamine (1 or 3 mg/kg)
and motor activity was recorded for another hour. Mice in the high-dose amphetamine-treated groups were
given the same treatment for 3 consecutive days and then tested for sensitization following a challengedose of amphetamine (1 mg/kg). We also tested these mice for amphetamine-induced reward in theconditioned place preference (CPP) paradigm. Mice were tested for baseline place preference on day 1,received conditioning with saline/amphetamine (3 mg/kg) or amphetamine/saline in a counterbalancemanner on days 2 and 3, and then tested for CPP on day 4. On each test day, the amount of time that micespent in each CPP chamber was recorded for 15 min. Our results showed that the motor stimulatory actionof amphetamine was not altered in mice lacking the mu opioid receptor compared to their wild-type controls.
However, amphetamine-induced locomotor sensitization was attenuated in knockout mice. Likewise, thissingle-conditioning paradigm induced CPP in wild-type but not knockout mice, suggest that the mu opioid
receptor may be involved in amphetamine-induced sensitization.

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65 - Male but not female mice lacking nociceptin exhibit higher anxiety and enhanced
reward following exposure to the elevated plus maze test
Sidney Perez2, Paul Marquez1, Osman Farhad1, Abdul Hamid1, Kabirullah Lutfy1
1
College of Pharmacy, Western University of Health Sciences, 2Cal Poly Pomona
We determined the role of endogenous nociceptin in anxiety-like behaviors and assessed if asubsequent test for ethanol-induced conditioned place preference (CPP) would be altered inthese compared to non-exposed mice. Male and female mice lacking N/OFQ and wild-type mice
were tested for anxiety-like behaviors in the elevated plus maze (EPM) test on days 1 and 2 andin the presence of ethanol (0.5 mg/kg) on day 5 followed by a test for CPP a month later. In the
EPM test, mice were placed in the center of the maze facing one of the open arms and the amountof time that mice spent on each arm was recorded. For the CPP experiments, naive mice (nonexposed) as well as mice exposed to the EPM test (exposed group) were tested for baseline
preference on day 1, received twice-daily conditioning on days 2-4 with saline/ethanol (2 g/kg) ina counterbalance manner, and then tested for CPP on day 5. Our results showed that male micelacking N/OFQ expressed greater anxiety-like behaviors compared to their wild-type controls.
However, this response was not observed in female mice. The rewarding action of ethanol wasenhanced in exposed male but not female mice lacking N/OFQ compared to wild-type mice. On
the other hand, there was no significant difference in ethanol-induced CPP in non-exposed maleor exposed female mice of the two genotypes. Together, these results suggest that lack of N/OFQ
increases anxiety-like behaviors in males as well as leads to enhanced reward in exposed malemice.

66 - Activation of δOR alleviates κOR-mediated place aversion: MP1104, a dual κOR-δOR
agonist is an analgesic and anti-addiction agent with attenuated side effects
Susruta Majumdar1, Rajendra Uprety1, Steven G Grinnell2, Andras Varadi1, Gina Marrone1, Gavril
W Pasternak1, Jonathan Javitch2, S O Eans3, M L Eanno3, M L Ganno3, Jay P McLaughlin3
1
Memorial Sloan Kettering Cancer Center, 2Columbia University, 3University of Florida
Kappa receptor agonists are known to be agents with analgesic and anti-addictive properties
whose therapeutic benefit is limited by dysphoria and sedation. MP1104, a morphinan derivative
and an analog of IBNtxA is a picomolar binder at δOR and κOR. In functional assays, it is a fullagonist at both δOR and κOR while displaying arrestin bias at these receptors. In vivo, it is apotent analgesic in mice and exhibits its actions through kappa and delta receptors. MP1104 also
shows no convulsions at doses 30-fold higher than its analgesic ED50. Surprisingly, in spite of itsvery high affinity for kappa receptors it shows no conditioned place preference or aversion(CPP/CPA) at doses 3-fold higher than its analgesic ED50. Closer evaluation of this compound in
the place aversion assays in the presence of delta antagonists shows place aversion. We now
report that dual activation of kappa and delta receptors leads to a separation of analgesia andcocaine blockade from aversion. Our preliminary findings suggest that activation of delta
receptors alleviates kappa-mediated place aversion and could be an effective strategy tocounteract kappa-mediated adverse effects.

7367 - Dissecting dopamine pathways altered by pain-induced dysfunction in opioid
signaling
Tamara Markovic1, Nicolas Massaly1, Lucia Hipolito2, Jose Morón-Concepcion1
1
Washington University in Saint Louis, 2University of Valencia
Pain is a complex phenomenon composed of sensory and emotional-affective components. In addition tosensory disturbances, patients experiencing pain report the presence of other negative symptoms such as
dysphoria, anhedonia, and depression. These pain-induced psychiatric disorders are commonly linked to adecrease in dopamine (DA) levels within the mesolimbic reward pathway (VTA-NAc). Recent studies fromour lab demonstrated that opioid-evoked DA release in the nucleus accumbens (NAc) is attenuated in thepresence of pain. This suppression of DA transmission in a condition of pain is likely due to downregulationof mu opioid receptor function in the VTA GABAergic neurons. Moreover, changes in DA transmission
within the mesolimbic pathway are associated with impaired motivated responses to natural and drug
rewards. Thus, we hypothesize that restoring DA levels by stimulating DA neurons in the ventral tegmental
area (VTA) is sufficient to prevent motivational deficits induced by pain. Here, we utilized a chemogenetic
approach to determine the role of DA transmission in the pain-induced attenuation of motivation. We
demonstrate that activation of DA neurons in the VTA is sufficient to prevent pain-induced decrease inmotivation. Furthermore, using an intersectional approach, we assessed whether this effect is mediated bya subset of VTA neurons projecting to the NAc or prefrontal cortex (PFC). Our results indicate that theoverall activation of VTA DA neurons (but not solely VTA-NAc or VTA-PFC) is sufficient to prevent
motivational deficits during inflammatory pain. Our findings provide mechanistic evidence for the
involvement of DA pathways in pain-induced negative emotional states.

68 - Pain Recruits Accumbal Kappa Opioid System And Alters Opioid Consumption.
Nicolas Massaly1,2, Adrianne Wilson-Poe1,2, Lucia Hipolito4, Tamara Markovic1,2, Michael R.
Bruchas1,2,3, Jose A. Morón1,2,3
1
Dept. of Anesthesiology, Pain Center, 2Washington University in St. Louis, School of Medicine,3
Dept. of Neurosciences, 4University of Valencia
The quality of life for patients suffering from chronic pain is impacted by co-morbidities such as prolonged
negative affective states. The presence of these severe emotional disturbances might underlie the misuseof drugs of abuse reported in 25% of pain patients, and lead to involuntary overdose. Thus, uncovering themechanisms responsible for pain-induced negative affect represents an important step to tackle the opioid
epidemic. In the mesolimbic pathway, the nucleus accumbens (NAc) shell plays a central role in the
integration of aversive and rewarding valence of stimuli. Prior work has revealed that the dynorphin-Kappa
Opioid Receptor (KOR) system decreases reinforcing properties of rewards and induces dysphoria andaversive behaviors. The abundance of dynorphin neurons and KORs in the NAc shell led us to hypothesize
a role for this system in pain-induced negative affect.
In this study we demonstrate that inflammatory pain, induced by Complete Freund’s Adjuvant (CFA)
injection in the hindpaw, enhances dynorphin A content and dynorphin neurons excitability in the NAc Shell.
A PET scan imaging revealed that CFA-induced inflammation decreases radiotracer binding potential, an
indirect measurement of KORs binding pockets occupancy. Lastly, we silenced dynorphin neurons, using
a chemo-genetic approach, in inflamed versus controls rats to demonstrate the role of dynorphin release in
the pain-induced misuse of fentanyl self-administration.
Our results confirm the recruitment of dynorphin-KORs system in the NAc Shell to drive pain-induced
negative affect. This work provides a novel path for future studies aiming to prevent the pain-induced opioid
misuse and involuntary overdoses.

7469 - Design and Synthesis of Collybolide Probes for Kappa-Opioid Receptor
Nicholas P. Massaro1, Adrien Cavé2, Heidi E. Hamm22, Joseph Parello2, Vsevolod Katritch3,
Lakshmi Devi4, Indrajeet Sharma1
1
University of Oklahoma, 2Vanderbilt University, 3University of Southern California, 4Icahn
School of Medicine at Mount Sinai
Studies suggest that selective kappa-opioid receptor (KOR) agonists biased towards G-protein signaling
could be novel therapeutics to treat itch and pain with reduced side effects. Deciphering the structuralrequirements essential for KOR selectivity through novel chemical probes is the first step in developing
selective- and biased-KOR ligands. In this vein, we have identified collybolide, a non-nitrogenous
sesquiterpene natural product extracted from the mushroom Collybia maculata as a highly selective KOR
agonist having in vivo analgesic and antipruritic activity. Collybolide has a furyl-δ-lactone core similar to thatof salvinorin A, however differs from the latter in exhibiting biased agonistic activity. Collybolide shows noconditioned place aversion unlike classical kappa agonists at doses at which it blocks non-histaminemediated itch, suggesting the possibility of separating kappa mediated liabilities from its ability to block itchwith this novel natural product based template. However, there are liabilities associated with use of
collybolide as a drug candidate such as aqueous solubility, efficacy, and metabolic stability. These liabilities
can be overcome by rational structural diversification of collybolide, as supported by the preliminary studies.
The goal is to comprehensively examine the SAR of collybolide, which will yield a set of selective- andbiased-KOR ligands with the potential of becoming lead drug candidates for itch and pain with reduced side
effects. Further, high-throughput screening at the NIMH-PDSP against 50 CNS receptors could find newhits, which could be optimized further to explore new therapeutic opportunities.

70 - Trafficking and Signaling of Endogenous Mu-Delta Heteromers
Lyes Derouiche, Florian Pierre, Dominique Massotte
Institut des Neurosciences Cellulaires et Integratives, CNRS UPR3212, Strasbourg, France
Heteromerization of Mu-Delta opioid receptors was postulated more than a decade ago using heterologous
expression systems. Accordingly, signaling and trafficking properties of Mu-Delta heteromers have been
extensively studied in transfected cells. However, little is known about the functional impact of endogenous
Mu-Delta heteromerization and, until recently, evidence for in vivo Mu-Delta physical proximity remained
limited. Using double knock-in mice co-expressing functional fluorescent Mu and Delta receptors, wepreviously revealed strong Mu-Delta neuronal co-localization in pathways associated with nociception andopiate withdrawal and showed close physical proximity of the two receptors, which strongly supports theexistence of Mu-Delta heteromers in the brain (Erbs et al. 2015 Brain Structure Function). Using primaryneuronal cultures from double fluorescent knock-in mice (Delta-eGFP/Mu-mcherry), we have now
examined whether Mu-Delta heteromerization induces bias in pharmacologically-induced receptor
trafficking and/or signaling compared to neurons only expressing one receptor type. Pharmacologicalstimulation included the use of selective mu or delta agonist alone or in combination with an antagonist forthe other receptor, administration of the Mu-Delta agonist CYM51010 or stimulation by endogenous opioid
peptides. Modifications in receptor trafficking resulting from Mu-Delta co-expression were identified byimmunocytochemistry in primary neuronal cultures. In parallel, Erk1/2 phosphorylation analyzed byWestern blot to address G protein and beta-arrestin signaling also revealed Mu-Delta specificities upon
stimulation with the different agonists.

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71 - Measurement of Heroin Antibody Binding Affinity Using Microscale Thermophoresis
Gary Matyas1, Oscar B. Torrres1,2, Alexander Duval1,2, Agnieszka Sulima3, Joshua F. G. Antoline3, Arthur
E. Jacobson3, Kenner C. Rice3, Carl R. Alving1
1Walter Reed Army Institute of Research, 2Henry M. Jackson Foundation for the Advancement of Military
Medicine, 3National Institute on Drug Abuse, National Institutes of HealthAn effective heroin vaccine needs to induce high affinity antibodies that bind heroin and its metabolites. We
developed a microscale thermophoresis (MST)-based method that accurately measures affinity of serum
antibodies to heroin and other structurally related drugs. MST exploits the movement of molecules in
microscopic temperature gradients to measure binding interactions. The thermophoresis is monitored bymeasuring the changes in a fluorescence signal. The technique measures the competition of afluorescently-tagged hapten tracer with opioids. This MST assay can be used with small molecule/protein
interactions in serum. The fluorescent tracer (MorHap-Cy5) was used as a probe in MST measurements.
The binding affinities (Ki) of a monoclonal antibody to morphine (Ab1060) and hapten-induced polyclonal
antibodies (6-AmHap antibodies) to heroin and other opioids were calculated by comparing the antibody K dagainst tracer to the IC50 of the drug competitor against an antibody:tracer complex. The Ki of ab1060
measured by MST was consistent with the reported Kd value of 2 nM. The 6-AmHap antibodies had highaffinities to heroin and its metabolites (6-acetylmorphine, morphine, morphine-3-β-glucuronide, morphine6-β-glucuronide and normorphine). The TT-6-AmHap vaccine induced antibodies that cross-reacted withheroin and other opioids. The Kd of 6-AmHap antibodies were consistent with the facial recognition
hypothesis, which holds that antibodies are generated to the face of the hapten opposite the carrier
attachment site. The antibodies cross-reacted with the same face on other opioids. This study demonstrates
that MST is a valuable technique for the measurement of the Ki of opioid with proteins.
72 - Neuroinflammatory effects of HIV-1 Tat protein correlate with elevated brain dopamine levels
and increased morphine consumption and conditioned place preference (CPP), and are prevented
by indomethacin treatment
Jay McLaughlin1, Thomas Cirino1, Shainnel Eans1, Heather Stacy1, Jae Kim2, Dionyssios Mintzopoulos2,
Matthew Strauss3, Luyi Zhou3, Wei Lun Sun3, Pamela M. Quizon3, Jun Zhu3, Marc J. Kaufman2
1Dept. Pharmacodynamics, Univ. of Florida, Gainesville, FL 32610, 2McLean Imaging Center, McLean
Hospital, Harvard Medical School, Belmont, MA 02478, 3Dept. Drug Discovery and Biomedical Sciences,
Univ. of South Carolina, Columbia SC 29208
The HIV-1 Transactivator of Transcription (Tat) protein activates brain microglia and increases brain
temperature, both markers of neuroinflammation. Exposure to Tat protein also increases the rewarding
effects of cocaine and ethanol. We hypothesized that mice exposed to Tat protein would demonstrateelevated dopamine levels concordant with the potentiation of morphine-CPP and increased morphine
consumption in a two bottle choice (TBC) assay. Western blot analysis confirmed the expression of Tat
protein in GT-tg bigenic (iTat) mice, in which brain-selective Tat expression is induced by activation of a
doxycycline (Dox) promotor for 7 days. HPLC analysis of iTat mouse brains demonstrated increased
dopamine content in prefrontal cortex and Nucleus accumbens by 92% and 37%, respectively. Consistent
with these neuroadaptive changes in the dopamine system, a 7-day Tat exposure significantly potentiatedmorphine-CPP 3-fold over saline-treated littermates. The magnitude of potentiation depended on the
degree of Tat exposure. iTat mice treated 7d with Dox significantly increased voluntary consumption ofmorphine in the TBC, effects that lasted up to a week after Tat induction. Confirming the contribution ofneuroinflammation to these effects, daily pretreatment with the anti-inflammatory indomethacin (10mg/kg/d, i.p.) during the 7-d induction prevented Tat-induced changes in morphine-CPP and TBC. Overall,these data suggest that expression of HIV-1 Tat protein potentiated the rewarding effects of morphinethrough the elevation of neuroinflammation and dopamine in reward systems, suggesting a biological basis
by which HIV infection may increase the vulnerability to opioid abuse. Supported by R01-MH085607 and
R01-DA039044 (JPM and MK) and R21-DA041932 (Zhu).

7673 - Agonist functional selectivity determined by rgs proteins
Kylie B. McPherson1, Ming-hua Li1, Katherine L. Suchland1, Janet D. Lowe1, John R. Traynor2,
Susan L. Ingram1
1
Dept. of Neurological Surgery, Oregon Health & Science University, Portland, OR, 2Departmentof Pharmacology, University of Michigan
RGS proteins are GTPase accelerating proteins that accelerate the hydrolysis of Galpha-GTP at GPCRs.
Using a transgenic knock-in mouse that expresses Galphao proteins insensitive to RGS proteins, we showthat GPCR coupling to effectors in the periaqueductal gray (PAG) is altered by the mutation. We comparedmu opioid receptor (MOR), nociception/orphanin FQ and GABAB receptor agonist inhibition of evoked
GABA release and activation of postsynaptic GIRK channels. We also confirmed the role of Gao proteinsin the different receptor-mediated signaling pathways using myristoylated-peptide inhibitors of Gao and Gai.
MOR agonists met-enkephalin and morphine induced greater inhibition of presynaptic GABA release in theknock-in mice. Maximal DAMGO and fentanyl inhibition of mIPSCs were not altered suggesting that low
efficacy agonists are more affected by RGS activity. MOR agonist activation of GIRK currents was reduced
for DAMGO and fentanyl but not for met-enkephalin. The GABAB agonist baclofen produced comparable
inhibition of GABA release in both genotypes but GIRK currents were significantly reduced in the knock-in
mice. Nociceptin-mediated inhibition of GABA release was not altered in the knock-in mice over a range ofconcentrations (300 nM - 3 µM) and activation of GIRK currents was not reduced. Thus, not all Galphao/icoupled GPCRs are modulated by the mutation reducing RGS interactions with Galphao.

74 - Single-particle tracking as an effector-independent readout of mu opioid receptor
activity after agonist exposure
Marissa Metz, Reagan Pennock, Shane Hentges
Colorado State University
Mu opioid receptors (MORs) mediate analgesia and reward by changing excitability and output fromneurons expressing them. However, the cellular effects of MOR agonism often diminish with continued
agonist exposure as the receptors undergo desensitization, a process that likely contributes to analgesic
tolerance. Most current approaches to observe opioid receptor function, such as electrophysiology or
fluorescent cyclic AMP indicators, only resolve signaling consequences based on the output of entire MOR
populations within a cell. These assays cannot identify the physical properties of individual receptors in
different signaling states and how differentially signaling receptors contribute to an overall cellular response
to agonist. Here we established single-particle tracking as a method to measure agonist-induced changes
in individual receptors’ mobilities, which could reflect unique signaling states between receptors. Single
particle tracking in AtT-20 cells expressing Flag-tagged MORs (FLAG-MORs) revealed immobile andmobile populations of receptors under basal conditions. When these cells were treated with DAMGO for 10
minutes, a higher fraction of receptors were immobile. Other agonists with varying pharmacological profiles,
such as morphine, were also tested due to known differences in internalization, desensitization, andcovalent modifications they induce. These results demonstrate heterogeneity of MOR mobility under basal
conditions that is shifted, but not eliminated, after activation with an agonist. Further experiments
simultaneously tracking known MOR interacting partners could identify physical receptor properties that
correspond to specific mobilities, and tracking in neurons from transgenic FLAG-MOR mice would allow
endogenous pharmacological dynamics to be better understood both within entire cells and distinct cellularcompartments.

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75 - The delta opioid receptor as an emerging therapy for mTBI-induced headaches
Laura Moye, ML Novack, HR Krishnan, AF Tipton, SC Pandey, AA Pradhan
University of Illinois at Chicago
Post-traumatic headache (PTH) is a migraine-like headache disorder secondary to mild traumatic brain
injury (mTBI). Up to 90% of mTBI patients develop PTH, which often progresses as a chronic condition.
Many PTH patients are highly dependent on anti-migraine medications, as there are no PTH-specificpharmacotherapies. Delta opioid receptor (DOR) agonists may be an emerging therapy for these mTBIinduced headaches because they are uniquely anti-nociceptive in chronic pain and migraine-associatedstates. DOR agonists, which have anxiolytic and antidepressant effects, may also combat post-traumaticstress disorder which is often co-morbid with PTH. In this study, we characterized a novel mouse model of
PTH, and determined the effectiveness of DOR as a therapeutic target. The PTH model combines theclosed-head weight-drop method and the nitroglycerin (NTG) chronic migraine model. To induce mTBI, a30-gram weight was dropped on the intact crania of anesthetized C57Bl6/J adult male mice. After recovery,
mice were chronically treated with saline, a subthreshold dose (0.1 mg/kg), or a high dose of NTG (10mg/kg) over 5 test days. Basal and post-treatment mechanical thresholds were assessed using von Freyhair stimulation. Only the mTBI group developed a progressive and sustained basal hypersensitivity to thesubthreshold dose of NTG. Additionally, sumatriptan and topiramate, commonly used migraine therapies,
were partially effective in reversing PTH-associated pain. SNC80, a hallmark DOR agonist, reversed acute
hyperalgesia, and had a protective effect on basal hyperalgesia. Overall, mTBI increases sensitivity todeveloping migraine-associated pain, and DOR activation may be effective in alleviating PTH-associatedpain.

76 - Identifying the role of peripheral delta opioid receptors in chronic migraine
Laura Moye, AF Tipton, ML Novack, AA Pradhan
University of Illinois at Chicago
Chronic migraine is a debilitating disorder with limited therapeutic options. We have previously shown thatdelta opioid receptor (DOR) agonists are effective in models of migraine-associated pain, aura, andnegative affect. The aim of this study was to further characterize the effectiveness of DOR agonists in othermodels of headache associated pain, and to determine the specific role of peripheral DORs in migraine.
We initially tested the effect of SNC80 in a model of medication overuse headache (MOH) and opioid
induced hyperalgesia (OIH). We also tested conditional knockout mice, in which DORs were deleted inNav1.8-expressing ganglia, within the nitroglycerin (NTG) migraine model; and we started a preliminary
characterization in trigeminal ganglia of DOR relative to the pro-migraine neuropeptide, CGRP. C57BL6
mice were treated chronically with sumatriptan or morphine which produced significant mechanicalsensitivity – a hyperalgesia that was inhibited by SNC80. DORNav1.8 and floxed counterparts were treatedwith vehicle or NTG (10 mg/kg, ip) every other day for 9 days. SNC80 inhibited migraine-associated pain
in floxed controls, an effect that was partially blunted in DOR Nav1.8 cKOs. Furthermore, we also observedthat in DOR-eGFP mice there was a small amount of co-expression between DOR-eGFP and CGRP in
trigeminal ganglia of naïve/pain-free animals. Future studies will examine the effect of NTG on DOR-eGFP
and CRGP expression. Overall, our results show that DOR activation can inhibit other types of pain that
contribute to migraine, and that peripheral/trigeminal ganglia are an important site of action for the antimigraine effects of DOR agonists.

78

77 - Ethanol disrupts synaptic specific mu opioid receptor-mediated long term
depression in dorsal striatum
Braulio Munoz, Brandon Fritz, Brady Atwood
Indiana University School of Medicine
Background: One of the current treatments for alcohol use disorders (AUDs) is the use of the
opioid receptor antagonist naltrexone, indicating that the μ opioid receptor (MOPr) has a role in
AUD. MOPr activation in dorsal striatum produces long-term synaptic depression (LTD) of specificstriatal synapses. Interestingly, the dorsal striatum (DS) is a brain region involved in action
selection, control of movement and motivation, and habit development all of which are affected in
AUD. Therefore, it’s possible that MOPr function in DS can be related to regulation of synaptic
plasticity in AUDs.
Results: We found that MOPr activation by DAMGO (0.3 μM) produces static LTD of striatal
glutamatergic transmission on to medium spiny neurons in mouse brain slices. Using
optogenetics to probe specific dorsal striatal inputs, we found that MOR activation at thalamic
glutamatergic inputs produces short term depression (STD), but MOR activation at corticalglutamatergic input produces a static LTD. We determined that a single in vivo exposure to 2.0
g/kg (i.p.) of ethanol (EtOH) disrupts corticostriatal-mediated LTD. Similarly, experiments using amouse model of binge drinking, drinking-in-the-dark, show that LTD was disrupted at 72 hrs after
the last exposure of binge drinking. In addition, we determined that the MOR LTD at specificcortical afferents was disrupted by in vivo EtOH exposure.
Conclusion: The study shows that MOPr activation leads to specific neuroplastic changes at
cortical inputs to the DS and plays an important role in alcohol’s effects on synaptic
regulation possibly regulates dorsal striatal-dependent habitual alcohol abuse.

78 - Free fatty acid receptor hypothalamic GPR40/FFAR1 regulate β-endorphin release via
prohormone convertase 2 protein expression
Kazuo Nakamoto1, Aizawa Fuka1, Shogo Tokuyama1
1
Kobe Gakuin University
Previous studies have shown that the administration of docosahexaenoic acid (DHA) or GW9508,
a GPR40/FFAR1 (free fatty acid receptor) agonist, facilitates β-endorphin release in the arcuate
nucleus of the hypothalamus in mice. However, the mechanisms mediating β-endorphin releaseinduced by GPR40/FFAR1 agonists remain unknown. In this study, we focused on the changes
in expression of hypothalamic prohormone convertase (PC) 2, which is a calcium-dependent
subtilisin-related proteolytic enzyme. The intracerebroventricular injection of DHA or GW9508
significantly increased PC2 protein expression in the hypothalamus. This increase in PC2
expression was inhibited by pretreatment with GW1100, a GPR40/FFAR1 antagonist.
Furthermore, PC2 protein expression gradually increased over time after complete Freund’s
adjuvant. These increase in PC2 expression were inhibited by pretreatment with GW1100.
However, GW1100 by itself had no effect on PC2 levels. Taken together, our findings suggestthat activation of the hypothalamic GPR40/FFAR1 signaling pathway may regulate β-endorphin
release via PC2, and regulate the endogenous pain control system.
This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.

7979 - Opiate Use Disorder in Pregnancy in Crawford County, Ohio: A Case Study
Madeline L. Novack
Crawford County Health Partners
Opiate Use Disorder (OUD), characterized by opiate addiction and misuse, has increased in the
U.S. in recent years. This rise in OUD has affected pregnant women and their infants. An
increasing number of infants are diagnosed with neonatal abstinence syndrome (NAS), requiring
treatment for withdrawal from opiates postpartum. Furthermore, heavy opiate use during
pregnancy is associated with lower birthweight, smaller head circumference, and lower testscores later in a child’s life. This case study utilizes a meta-analysis of 26 relevant researchstudies, birth records from Crawford County’s largest maternity ward, and interviews with
Crawford County healthcare officials and outside OUD experts. Additionally, this study includes
anecdotes from a local woman who became pregnant during treatment for her heroin addiction.
According to Crawford County’s birth records, the prevalence of opiate use in pregnancy in 2015
was 6.7%; opiates were the most used drug during pregnancy, after marijuana. Opiate-positive
infants accounted for 5% of all births in that year. The effect of increasing opiate use during
pregnancy is apparent: Crawford County’s NAS rate is the highest in any surrounding county, with12.1 per 1,000 live births. Furthermore, half of the infants transferred from Crawford County tosurrounding neonatal intensive care units in 2015 were due to complications of NAS. The data
indicate that although all local healthcare officials acknowledge the opioid epidemic, biases
regarding treatment strategy have resulted in medical inaction. Unbiased approaches to treating
OUD in pregnancy will prevent further harm to addicted mothers and their infants.

80 - The effects of commonly used opioids in cell cultures
Erik Nylander, Sofia Zelleroth, Alfhild Grönbladh, Leo Leoson, Esra Flaieh, Fred Nyberg, Mathias
Hallberg
Uppsala University
Introduction. Accumulating evidence suggest that long-term opioid consumption are associatedwith cognitive dysfunctions such as reduced memory and learning. The non-medical use of
opioids as well as the number of opioid prescriptions is increasing, even in young adults. The
observed cognitive decline can be a cause of increased neuronal cell death, reduced
neurogenesis, and morphological changes in the brain. Taken together, these toxic effects
associated with opioid consumption must be further evaluated. The aim for present study was
therefore to investigate the neurotoxic effects of commonly used opioids using neuronal cell
cultures. This study is part of a larger project were the toxic effects of the opioids methadone,
morphine, ketobemidone, fentanyl, oxycodone, hydromorphone, buprenorphine, as well as theopioid antagonist naloxone are evaluated.
Methods. Cells were exposed to opioids for 4, 24 or 72h and the neurotoxic effects were
measured using a battery of cell viability assays.
Results. There was a varying degree of toxicity between the different opioids and time points. At
4h exposure, toxicity occurred with some opioids while at 24h or longer a more pronounce toxicity
was observed with most of the opioids used.
Conclusion. This study reveals that commonly used opioids may induce toxic effects in neuronalcell cultures.
80

81 - Metformin acting through the mammalian target of rapamycin complex 1 (mTORC1)
attenuates morphine efficacy in a mouse model of neuropathic pain.
Amal Alsubayiel1, JoãoPaulo Ferreira Bertacchi1, Sam Eldabe1,2, I 1, Ilona Obara1
1
Durham University, School of Medicine, Pharmacy and Health, Stockton on Tees, UK, 2TheJames Cook University Hospital, Middlesbrough, UK
There is a pressing need for the identification of new strategies to improve efficacy of opioid-based
treatments. Here, by extending upon studies on the mammalian target of rapamycin complex 1 (mTORC1)
in chronic pain, for the first time we explored the effect of relatively safe and widely used anti-diabetic drug
metformin that inhibits mTORC1 through activation of the 5' adenosine monophosphate-activated protein
kinase (AMPK) on the modulation of morphine analgesic efficacy in neuropathic mice. The originality of ourstudy showed that chronic metformin administration (200 mg/kg i.p., 24 h before each morphine) blocked
the development and maintenance of morphine tolerance (40 mg/kg, i.p., twice daily for up to 10
consecutive days) in adult male C57BL/6J mice (n=6-8) subjected to spared nerve injury model (SNI) andassessed by the tail-flick, von Frey and acetone tests. Also, a single metformin dose injected in morphine
tolerant SNI mice fully restored the analgesic effect of morphine. In addition, when metformin was injectedin combination with morphine (3, 10, 20 mg/kg, i.p.) in SNI mice it potentiated dose-dependently analgesic
effect of morphine. The involvement of mTORC1 in these behavioural effects was confirmed by our parallel
studies using the direct mTORC1 inhibitor CCI-779 (25 mg/kg, i.p.), and by western blotting andimmunohistochemistry showing inhibition of mTORC1 downstream targets P-p70 S6 kinase and P-S6
ribosomal protein in the dorsal spinal cord after metformin treatment. Together, our results define the roleof mTORC1 in opioid analgesia and thus support a novel approach for the improvement of opioid therapy.

82 - Development of a Selective Antagonist for the Mu-Delta Heterodimer
Keith M. Olson1, Victor J. Hruby1, John M. Streicher2
1
Department of Chemistry and Biochemistry, University of Arizona, Tucson, Az. USA,2
Department of Pharmacology, University of Arizona, Tucson AZ USA
Mu and delta opioid receptor (MOR, DOR) agonists induce anti-nociception, tolerance, and withdrawal invivo; however, selective MOR and DOR ligands modulate the other receptor’s activity. For example,
morphine mediated side effects – including tolerance and withdrawal – are attenuated by DOR selective
antagonists. Whether these synergies arise from the formation of MOR and DOR heterodimers (MDOR)
remains unclear. Current tools using immunochemical, genetic, or chemical methods show MDOR
upregulation after chronic morphine treatment in mice, in addition to potential roles in tolerance,
dependence, and drug seeking behavior. However, directly assessing MDOR-mediated behaviors isextremely difficult because no selective MDOR antagonists are available. To address this need, we createda novel series of bivalent MDOR antagonists by connecting low affinity MOR (H-Tyr-Pro-Phe-D1Nal-NH2)
and moderate affinity DOR (H-Tyr-Tic-OH) pharmacophores with variable length polyamide spacers (1541 atoms). We tested the in vitro selectivity of this series using radioligand binding and [35S]-GTPγS
coupling in MOR, DOR, and MDOR expressing cell lines. In vitro screening of MDOR antagonism andaffinity show a clear length dependence in MDOR but not MOR or DOR cell lines. The lead compound –D24M with a 24-atom spacer – displayed high potency (IC50MDOR = 0.84 nM) with 91-fold selectivity for
MDOR:DOR and >1:1,000 MDOR:MOR selectivity. Preliminary tail-flick anti-nociceptive assays indicate
D24M blocks CYM51010 mediated anti-nociception – a compound previously reported to mediate
antinociception through MDOR. After completing in vivo characterization, this first-in-class MDOR
antagonist will be used to probe how MDOR influences opioid behaviors including withdrawal and tolerance.

8183 - Nociceptin receptor trafficking in midbrain dopamine neurons
Patrick R O'Neill, Michael Bruchas
Washington University, St. Louis
Agonists that activate the nociceptin/orphanin FQ receptor (NOPR) have been found to inhibit the
rewarding effects of commonly abused drugs including cocaine, morphine, amphetamine, andalcohol in the conditioned place preference (CPP) paradigm in rodents. The Bruchas Lab has
obtained recent data demonstrating that the site of NOPR action for inhibiting natural reward
seeking (sucrose), as well as cocaine CPP is through its action in dopaminergic (DA) neurons of
the ventral tegmental area (VTA). However, little is known about NOPR signaling, or its regulation
by receptor trafficking, in these neurons. Here we used primary cultures of ventral midbrainneurons from NOPR-YFP knock-in mice to examine NOPR trafficking at (or very near)endogenous receptor expression levels. We also examined NOPR trafficking selectively in DA
neurons in primary cultures using a Cre-dependent AAV construct to express NOPR-YFP only in
DA neurons. Using live cell imaging, we found that NOPR is primarily localized to the plasma
membrane, and that activation by nociceptin causes pronounced receptor internalization within
10 minutes. This system will be used to identify molecular mechanisms controlling NOPR
trafficking in midbrain DA neurons, and to determine if synthetic NOPR ligands can differentiallycontrol its trafficking. This work is funded by the NIH through NIDA award K01-DA042219.

84 - Spinal pain regulation of the N/OFQ-NOP receptor system in a spinal nerve injury
mouse modelAkihiko Ozawa1, Gloria Brunori1, Andrea Cippitelli1, Nicolas Toll1, Jennifer Schoch1, Brigitte L.
Kieffer2, Lawrence Toll1
1
Torrey Pines Institute for Molecular Studies, 2Douglas Research Center, McGill University
The endogenous opioid system regulates pain sensitivity and is targeted by both endogenousand exogenous opioid reagents in order to manage pathological pain. The NOP receptor system
plays a significant role in the regulation of pain, and interestingly functions differently in the spinal
cord and supraspinal tissues. Yet, the mechanism by which NOP receptor agonists regulate paintransmission is still understudied due to lack of appropriate tools to interpret the development of
NOP receptor-based analgesics. Here we examined changes in the peripheral and spinal NOP
receptor distribution mediated by spinal nerve ligation (SNL) using NOP-eGFP mice, as well as
the antihyperalgesic and antiallodynic effects of intrathecally (i.t) administrated N/OFQ in SNL
mice. The immunoreactivity of NOP-eGFP receptors in the spinal superficial lamina that is
generally responsible for noxious heat stimulation was significantly decreased in SNL mice. In
contrast, immunoreactivity was unchanged in the ventral border of lamina II inner, a region
essential for injury-induced allodynia. NOP-eGFP expression was also significantly decreased ina large number of primary afferents in the L4 DRG of SNL mice, especially in small DRG neurons.
However, SNL mice showed increased sensitivity, compared to sham animals, to the effects of i.t
administered N/OFQ with respect to mechanical as well as thermal stimuli. These results suggest
the possibility that the spinal NOP receptor system directly down-regulates the neurons intrinsic
to the spinal cord but not the excitability of DRG neurons to attenuate injury-induced hyperalgesia
under certain chronic pain conditions.

82

85 - A novel non-canonical role of RGS4 in δ-opioid receptor mediated neuronaloutgrowth and differentiation
Paschalina Pallaki1, Eirini-Maria Georganta1, Ioannis Serafimidis2, Maria-Pagona Papakonstantinou1,
Vassilis Papanikolaou1, Sofia Koutloglou1, Elsa Papadimitriou3, Adamantia Agalou1, Aggeliki Tserga1,
Alexandra Simeonof1, Dimitra Thomaidou3, Maria Gaitanou3, Zafiroula Georgoussi1
1Laboratory of Cellular Signalling and Molecular Pharmacology, Institute of Biosciences and Applications,
National Centre for Scientific Research «Demokritos», 15310 Athens, Greece, 2Laboratory ofDevelopmental Biology, Biomedical Research Foundation of the Academy of Athens, 11527 Athens,
Greece, 3Laboratory of Cellular and Molecular Neurobiology, Hellenic Pasteur Institute, 11521 Athens,
GreeceThe Regulator of G-protein signaling 4 (RGS4) has been found to be a negative regulator of μ, δ and κopioid receptor signaling that confers selectivity for G protein coupling to these receptors by directlyinteracting with them (Georgoussi et al., 2006, 2012; Leontiadis et al. 2009; Papakonstantinou et al., 2015).
It was also shown that δ-opioid receptor (δ-OR) forms a multicomponent signaling complex, consisting of
Gi/Go proteins and the Signal Transducer and Activator of Transcription STAT5B, that leads to neurite
outgrowth and neuronal differentiation via a phosphoryalted-STAT5B-Gαi/o pathway (Georganta et al.,2010, 2013). Knowing that RGS4 is a multitask protein we questioned whether RGS4 could be implicatedin neuronal differentiation and neurite outgrowth upon δ-OR activation. We demonstrate that RGS4 directlyinteracts with STAT5B in vitro and in living cells independently of δ-OR presence. This interaction involves
the N-terminal portion of RGS4 and the DNA-binding-SH3 domain of STAT5B. Expression of RGS4 in
HEK293 cells expressing δ-OR and/or erythropoietin receptor results in inhibition of DSLET anderythropoietin-dependent STAT5B phosphorylation and transcriptional activation. Measurements of
DSLET-dependent neurite outgrowth of neuroblastoma cells is also blocked by RGS4, whereas primarycortical cultures of RGS4−/− mice exhibit enhanced neuronal sprouting after δ-OR activation. Finally, neuralprogenitor cells from RGS4−/− mice exhibit enhanced proliferation with concomitant increases of the mRNAlevels of the Bcl2 and Bcl-xl STAT5B target genes. These observations suggest that RGS4 plays asignificant regulatory role in opioid dependent neuronal differentiation and neurite outgrowth via a “noncanonical” signaling pathway involving STAT5B-directed responses.

86 - Genetic dissociation of morphine analgesia from hyperalgesia in mice
Gina Marrone1,2, Valerie Le Rouzic1, Andras Varadi1, Jin Xu1, Anjalie Rajadhyaksha2, Susruta
Majumdar1, Yingxian Pan1, Gavril Pasternak1,2
1
Department of Neurology, Memorial Sloan Kettering Cancer Center, 2Department of Neurology
and Neuroscience, Weill Cornell Medical College, 3Memorial Sloan-Kettering Cancer Center,
4
Memorial Sloan Kettering Cancer Center
Morphine is the prototypic mu opioid, producing its analgesic actions through traditional 7 transmembrane
domain (7TM) G-protein coupled receptors generated by the mu opioid receptor gene (Oprm1). However,the Oprm1 gene undergoes extensive alternative splicing to yield three structurally distinct sets of splicevariants. In addition to the full length 7TM receptors, it produces a set of truncated variants comprised ofonly 6 transmembrane domains (6TM). This study explored the relative contributions of 7TM and 6TMvariants in a range of morphine actions. Loss of the 6TM variants in an exon 11 knockout (E11 KO) mouse
did not affect morphine analgesia, reward or respiratory depression. However, E11 KO mice lacking 6TMvariants failed to show morphine-induced hyperalgesia, developed tolerance more slowly than wildtype
mice and did not display hyperlocomotion. Together, our findings confirm the established role of 7TM mu
receptor variants in morphine analgesia, reward and respiratory depression, but reveal an unexpected
obligatory role for 6TM variants in morphine-induced hyperalgesia and a modulatory role in morphine
tolerance and dependence.

8387 - Analgesic and Anti-Inflammatory Effects of Novel Kappa Opioid Receptor Agonist
16-Ethynyl Salvinorin A
Kelly Paton1, Samuel Williamson2, Thomas Prisinzano2, Bronwyn Kivell1
1
School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand,
2
Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS
Current pain medications are highly addictive. As an alternative, kappa opioid receptor (KOR) agonists
have proven analgesic effects without rewarding properties. 16-ethynyl Salvinorin A (Ethynyl SalA) is apotent analogue of Salvinorin A (SalA) and has been shown to attenuate cocaine-prime induced drugseeking behaviour in rats without causing sedative, anxiogenic, aversive or pro-depressive side effects.
Here, we investigated the ability of Ethynyl SalA to modulate pain behaviours in preclinical models of
nociceptive, inflammatory and neuropathic pain. The analgesic effects were evaluated in C57BL/6 miceusing the 2% intradermal formalin and warm-water tail-withdrawal assays. Ethynyl SalA (2 mg/kg i.p.)
showed a significant analgesic effect in the tail-withdrawal assay and a longer duration of action in than
SalA. At 2 mg/kg (i.p.) Ethynyl SalA significantly reduced phase one nociceptive (p<0.0001) and phase two
inflammatory (p<0.0001) pain in the formalin assay and reduced the accompanying paw oedema(p=0.0011), which was reversed with the KOR antagonist nor-binaltorphimine (10 mg/kg). The paclitaxelinduced neuropathic pain model was used to assess the cumulative dose response effects of Ethynyl SalA,
morphine and traditional KOR agonist U50,488, on mechanical and cold allodynia. Paclitaxel-induced
neuropathy was evaluated at baseline and every second consecutive day, with dose-response effects
evaluated on day 15. Non-linear regression analysis revealed that Ethynyl SalA was more potent atreversing paclitaxel-induced mechanical and cold allodynia than either SalA, U50,488 or morphine. Thesefindings demonstrate that Ethynyl SalA significantly reduces nociceptive, inflammatory and neuropathic
pain without the risk of abuse.

88 - Morphine-dosing affects development of antinociceptive tolerance
Alok Kumar Paul1, Nuri Gueven1, Nikolas Dietis1,2
1
Division of Pharmacy, School of Medicine, University of Tasmania, Australia, 2Medical School,
University of Cyprus, Cyprus
Clinical development of antinociceptive tolerance after repeated administration morphine limits its chronic
use. Despite growing knowledge about the molecular mechanisms of morphine tolerance, we know little
about the influence of dosage regimen in its development. We hypothesized that the morphine dose, as
well as the increments of dose after tolerance, contribute to tolerance development and can affect total
antinociception levels. Four groups of male Sprague Dawley rats received different daily doses ofintermittent subcutaneous morphine for 14 days. After the development of antinociceptive tolerance,
different increments of morphine doses were administered until tolerance redeveloped (Group A:
2.5(b.i.d)→5→10 mg/kg/day, Group B: 5(b.i.d.)→10 mg/kg/day, Group C: 5(b.i.d.)→15 mg/kg/day andGroup D: 10(b.i.d.)→20 mg/kg/day). Antinociceptive responses were measured daily by tail-flick and hotplate assays pre-treatment and at various post-treatment time-points. Total antinociception was calculated
as AUC over the duration of treatment. Animals treated with smaller morphine starting-doses developed
antinociceptive tolerance faster than those started on higher doses. Higher starting-doses and higher doseincrements after tolerance development resulted in more sustained antinociception and delayed theredevelopment of tolerance. Hyperalgesia was absent from all groups, shown by the insignificant variation
in basal antinociception levels. Our observations were replicated by both assays and were therefore notassay-specific. These results suggest that morphine dosing regimen in rats significantly influence the
manifestation of antinociceptive tolerance and the total antinociception produced. Our results also highlightthe need for standardized and validated testing protocols to compare different studies as indispensable totranslate pre-clinical results into the clinic.

84

89 - Role of mu opioid receptor A112G SNP in the mesolimbic neurocircuitry
Dina Popova1, Julie A. Blendy2, Zhiping Pang1
1
Child Health Institute, Rutgers University, 2Perelman School of Medicine, University ofPennsylvania
Mu opioid receptors (MORs) are highly enriched in the reward circuitry and can be activated byendogenous ligands as well as drugs of abuse. Individuals with the A118G single nucleotide
polymorphism (SNP) in OPRM1 exhibit increased risk for drug addiction and require higher
morphine doses to achieve adequate analgesia. Genetically engineered mice carrying A112G
SNPs (homologous to human A118G) display less total MOR mRNA and protein expression andreduced analgesic response to morphine. In this study, we are investigating how A112G in MOR
affects opioid regulation of synaptic transmission in the ventral tegmental (VTA). Previously, it has
been proposed that opioids excite dopamine (DA) neurons in the VTA through inhibition of local
interneurons; however, recent reports show significant diversity in DA neurons response to MOR.
We addressed role of A118G polymorphism in MOR considering VTA heterogeneity, focusing on
the mesolimbic pathway. Using slice electrophysiology, we found that both inhibitory andexcitatory inputs to VTA DA neurons projecting to the nucleus accumbens medial shell (mAcb sh)were highly inhibited by application of MOR agonist DAMGO, leading to a moderate increase of
DA neurons firing. Interestingly, mice carrying minor allele (i.e. GG112) exhibited no change insynaptic transmission compared to their littermate major allele controls (i.e. AA112), but hadoverall lower DAMGO sensitivity. Additionally, no significant differences in DA neurons firing were
found between genotypes. Our current focus is to study the underlying molecular and cellularmechanisms of the A118G SNP which contribute to altered sensitivity of VTA DA neurons to
opioids.
90 - Imatinib prevents morphine tolerance without inducing MOR endocytosis.
Puig S.1, White P.1, Szott S.1, Gutstein H.B.1
1
Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, PA
Analgesic tolerance limits the use of opioids for the treatment of chronic pain. It was previously
hypothesized that sustained signaling by the mu opioid receptor (MOR) could cause morphine
tolerance, which could be prevented by promoting MOR endocytosis. However, some studies
have challenged this theory. We have shown that Imatinib, by inhibiting PDGFR-β (plateletderived growth factor receptor-beta), selectively prevents morphine tolerance. In this study, we
determine whether inhibition of morphine analgesic tolerance with Imatinib involves endocytosisof the MOR. Rats were injected i.t. with either vehicle, 0.6 nM morphine, 10 µg imatinib or
morphine + imatinib. Analgesia was measured daily using tail flick latency (TFL). After either one
or five days of treatment, spinal cords were extracted and processed for immunohistochemistrywith anti-NeuN (Millipore) and anti-MOR (Abcam) antibodies. Images of the substantia gelatinosa(SG) were analyzed using confocal microscopy. The average number of internalized vesicles
containing MOR in neurons was determined utilizing an automated and unbiased softwarealgorithm (Imaris, Bitplane). Co-administration of Imatinib with morphine prevented tolerance
without altering morphine’s analgesic effect. Interestingly, the levels of MOR internalization in SG
neurons were not different between groups. Our results show that imatinib did not prevent
tolerance by promoting MOR endocytosis, challenging the hypothesis that the lack of MORinternalization could be the cause of morphine tolerance.
8591 - Serotonergic and adrenergic gene variants and vulnerability to non-dependent
opioid use and heroin dependence in a Dutch population
Matthew Randesi1, Wim van den Brink2,3, Orna Levran1, Vadim Yuferov1, Peter Blanken2,4, Jan
M. van Ree2,5, Jurg Ott1, Mary Jeanne Kreek1
1
Rockefeller University, 2Central Committee on the Treatment of Heroin Addicts (CCBH),
3
University of Amsterdam, 4Parnassia Addiction Research Centre, 5Utrecht University
Drug addiction is a chronic relapsing brain disease caused by a combination of genetic, epigenetic,
environmental and drug-induced factors. The aim of this case-control association study was to determine
if genetic variants in the serotonergic and adrenergic pathways are associated with non-dependent opioid
use and heroin dependence in a Dutch Caucasian cohort. Subjects were ascertained in the Netherlands,
consisting of the following groups: not opioid dependent (NOD), previous opioid users [n=162]; opioid
dependent (OD) patients in methadone maintenance treatment [n=143] or heroin-assisted treatment[n=135]; and healthy controls with no history of heroin use (HC) [n=153]. A total of 152 variants in 19 genes
were genotyped using a modified Illumina GoldenGate platform. After filtering for quality, HWE and MAF,
126 SNPs were used in case-control analyses to establish the role of the serotonergic and adrenergic genevariants in (a) non-dependent opioid use, and (b) heroin dependence. The following groups were compared:
HC vs. OD; [HC+NOD] vs. OD and HC vs. NOD. In the comparison of HC vs. OD, 4 SNPs in ADRA1A andone SNP each in SLC6A2, SLC18A2, S100A10 and HTR3B were found significant (p<0.05). In thecomparison of [HC+NOD] vs. OD, 3 SNPs in both SLC18A2 and ADRA1A and 1 SNP in SLC6A2 andHTR3B were found significant (p<0.05). Further studies are warranted to confirm and elucidate the potential
roles of these variants in the vulnerability to illicit drug use and drug addiction.

92 - Conditioned place aversion to an “extremely” G-protein biased kappa opioid
receptor agonist in C57BL6 mice
Brian Reed, Amelia D. Dunn, Alexandra M. Dunn, Eduardo R Butelman, Mary Jeanne Kreek
Rockefeller University
Studies of derivatives of N-phenylethyl-N-3-hydroxyphenylethyl-amine have indicated that selectsubstituents confer preferential binding and GTP-gamma-S stimulation at the kappa opioid receptor (KOR),
compared to other opioid receptors or dopamine receptors. We have demonstrated that different Nsubstituents results in differential G-protein versus arrestin mediated KOR signaling bias. We havedetermined that N-n-butyl-N-phenylethyl-N-3-hydroxyphenylethyl-amine (BPHA) is “extremely” biased
towards G-protein mediated KOR signaling, with full agonism in KOR-mediated [35S]GTPγS stimulation,with no stimulation and full antagonism of KOR coupling to β-arrestin 2 . HPHA showed no agonism in mu
or delta opioid receptor (MOR, DOR) signaling, although binding inhibition was observed. BPHA showed
29-fold selectivity for KOR versus MOR binding affinity, and 335-fold selectivity for KOR versus DOR
binding. In vivo in C57BL6 mice, BPHA resulted in increased serum prolactin levels 30 minutes afteradministration (10-30 mg/kg) , which was blocked by prior administration of KOR antagonist
LY2444296. Conversely, 30 mg/kg BPHA had no effect on rotarod incoordination, in contrast to theunbiased agonist U50,488, supporting the hypothesis that beta-arrestin mediated signaling is required for
KOR mediated motor incoordination. In a conditioned-place-preference/aversion model in mice, with 4
conditioning sessions, equivalent levels of aversive conditioning were expressed following BPHA as
following the unbiased agonist U50,488. This occurred at a dose of BPHA which had no effect on morphineanalgesia. Overall, our findings suggest that KOR-mediated aversive behavior is not dependent on KORβ-arrestin 2 mediated signaling.
Supported by the Robertson Therapeutic Discover Fund and the Miriam and Sheldon G Adelson Medical
Research Foundation.

86

93 - Critical role of Gi-protein signaling in the dorsal striatum for the reduction of voluntary
alcohol consumption in C57Bl/6 mice
Meridith Robins1,2, Terrance Chiang1,2, Doungkamol Alongkronrusmee1,2, MeeJung Ko1,2, Richard van
Rijn1,2
1Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette IN,
USA, 2Purdue Institute of Integrative Neuroscience, Purdue University, West Lafayette IN, USA
Despite the large burden of alcohol abuse at the individual and society level, pharmacological treatment
options are limited. Recently, we discovered that systemic administration of Gi-protein biased delta-opioid
receptors (DORs) agonists decreased alcohol consumption in a volitional alcohol use mouse model, while
β-arrestin-2 biased agonists increased alcohol consumption. We hypothesized that striatal DORs are
crucial for modulation of alcohol intake. To test our hypothesis, mice were trained to consume 10% alcohol
in a two-bottle, limited-access, drinking-in-the-dark paradigm. We then activated endogenous Gi-protein
coupled DORs in the dorsal striatum of cannulated mice by drug microinfusion. Through this approach, we
recapitulated our systemic findings in the dorsal striatum: infusion of Gi-protein biased DOR agonist TAN67 decreased voluntary alcohol consumption (p < 0.02), whereas the β-arrestin-2 biased DOR agonist
SNC80 increased alcohol intake in the mice (p < 0.01). Studies were repeated in β-arrestin-2 knockout mice
where SNC80 decreased alcohol consumption (p < 0.005) resulting from DOR activation only through Gisignaling. To study the general role of striatal Gi-signaling on alcohol use, we then utilized a chemogenetic
approach to locally express designer muscarinic M4 receptor (hM4Di, DREADD) in neurons of the dorsal
striatum using adeno-associated virus. Administration of the DREADD-specific agonist clozapine-N-oxidesignificantly decreased alcohol consumption in mice expressing the DREADD but not those expressing
control GFP. Thus, using both pharmacologic and chemogenetic approaches, we demonstrated that
selective Gi-signaling in the dorsal striatum is sufficient to decrease alcohol intake in mice. In contrast, βarrestin-2 signaling in the dorsal striatum may increase alcohol intake.
94 - Convergent and Divergent Behavioral Changes Caused by Different Patterns of Morphine
Exposure in Mice
Kerry J. Trotter1, Federico Buffaldi2, Marco Pravetoni2, Patrick E. Rothwell1
1Department of Neuroscience, University of Minnesota, 2Minneapolis Medical Research Foundation and
Departments of Medicine and Pharmacology, University of Minnesota
Many studies have documented the behavioral changes that develop during chronic opiate exposure inrodents. While analgesic tolerance is widely reported, other behavioral effects (like psychomotor activationand conditioned place preference) can be either enhanced or reduced by prior opiate exposure. Thisvariability is often related to the pattern of chronic opiate administration, with intermittent exposure causing
sensitization and continuous exposure causing tolerance. However, few studies have conducted wellcontrolled comparisons between different patterns of opiate exposure. In an initial series of experiments,
we assessed morphine-induced analgesia and psychomotor activation across a range of doses, andcompared the effects of intermittent exposure (daily injections) with continuous exposure (viasubcutaneously implanted pumps). Both patterns of morphine exposure caused analgesic tolerance, butintermittent exposure caused long-lasting sensitization of psychomotor activation, whereas continuous
exposure caused transient psychomotor tolerance. These behavioral differences were apparent after
controlling for a variety of factors, including total drug dose, peak analgesic effect, and serum drug level.
To determine if these divergent behavioral changes were related to the continuity of morphine exposure,
we implanted osmotic minipumps to continuously deliver morphine, and administered daily naloxone
injections to interrupt opioid receptor stimulation. This manipulation prevented the initial development of
psychomotor tolerance; we are currently evaluating long-term changes in morphine sensitivity. Thesestudies provide a framework for ongoing investigation of the neurobiological consequences of different
patterns of opiate exposure. Our results may inform the way opiates are used clinically to treat pain, andsuggest new strategies to reduce opiate abuse.

8795 - Anxiolytic-like and antidepressant-like effects of a novel delta opioid receptor
agonist NC-2800 in rats
Akiyoshi Saitoh1, Eriko Nakata2, Satoshi Nakashima1, Masatoshi Ukezono1, Leo Gotoh1, Masaaki
Hirose2, Junichi Sakai2, Hideaki Fujii3, Mitsuhiko Yamada1, Hiroshi Nagase4, Tomio Yamakawa2
1
Dept. of Neuropsychopharmacology. NIMH, NCNP, Tokyo, Japan, 2Discovery Research Lab.,
Nippon Chemiphar Co. Ltd., Saitama, Japan, 3Lab. Med. Chem., School Pharm. Kitasato Univ.,
Tokyo Japan, 4WPI-IIIS, Univ. of Tsukuba, Ibaraki Japan
Delta opioid receptor (DOR) agonists have been proposed to be attractive targets for the development of
the novel anxiolytic and/or antidepressant. Recently, we succeeded in synthesizing a novel small moleculecompound, named NC-2800, containing the morphinan structure. NC-2800 showed DOR agonistic activity
in functional cAMP assays using recombinant cells stably expressing opioid mu, delta or kappa receptor,
respectively. In this study, we investigated the anxiolytic-like / antidepressant-like effects of NC-2800 in
rats. In the elevated plus maze test, which is a screening model for anxiolytics, the single treatment with
NC-2800 (1-10 mg/kg, p.o.) significantly increased the time spent on the open arm. In the forced swimming
test, which is a screening model for antidepressants, the single treatment with NC-2800 (0.3-10 mg/kg,
p.o.) significantly decreased the immobility score and increased the swimming score. In the olfactorybulbectomized rat, which is an animal model of depression, NC-2800 (1 mg/kg/day, s.c. for 14 days)
significantly decreased the total score of hyperemotional responses over the entire period for 14 days fromday one. In conclusion, it is suggested that NC-2800 is a novel candidate for anxiolytic and/or
antidepressant. This study was supported by the Japan Agency for Medical Research and Development(AMED) grant.

96 - Ligand-receptor and structure-function relationship studies on differently
substituted diphenethylamines interacting with the κ-opioid receptor
Helmut Schmidhammer1, Filippo Erli1, Elena Guerrieri1, Aquilino Lantero1, Tanila Ben Haddou1,
Shainnel O. Eans2,3, Michelle L. Ganno2, Jay P. McLaughlin2,3, Mariana Spetea1
1
University of Innsbruck, 2Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL, USA,
3
University of Florida, Gainesville, FL, USA
The κ-opioid receptor (KOR) has an important role in pain modulation and other behavioural responses.
Although KOR activation does not produce dependence, euphoria or leads to respiratory suppression, it
induces dysphoria, sedation and psychotomimesis. Accumulated evidence indicates that KOR-mediatedantinociception results from G protein-mediated signalling events, while alternative signalling pathways (i.e.
β-arrestin2) promotes adverse effects. The concept of biased agonism at the KOR has gained significanceto drug discovery, with G protein-biased KOR agonists emerging as prospective analgesics with an
improved benefit/risk profile. We have designed and pharmacologically investigated a series of structurallydistinct KOR ligands from the class of diphenethylamines. Binding studies showed that these compounds
are KOR selective with several analogs demonstrating picomolar affinity for the human KOR. They activate
G proteins with high potency as full or partial agonists, while several ligands also display much lower
potencies and efficacies in promoting β-arrestin2 recruitment. They induce marked antinociception in themouse writhing test. Two diphenethylamines HS665 (agonist) and HS666 (partial agonist) were also shown
to produce potent centrally KOR-mediated antinociception in the tail-withdrawal assay, associated with
reduced sedation and also an absence of conditioned place aversion with HS666. In summary, HS666
displays pharmacological characteristics of a KOR analgesic with diminished aversive liability correlatingwith its low efficacy for β-arrestin2 recruitment. These results offer valuable structural and functional insights
into the design and/or discovery of drugs targeting the KOR with improved pharmacological profiles for thetreatment of pain.

88

97 - Carboxyl-terminal multisite phosphorylation regulates µ-opioid receptor
desensitization and tolerance
Stefan Schulz
University Hospital Jena, Germany
Phosphorylation of G protein-coupled receptors (GPCRs,) by GPCR kinases (GRKs) plays anessential role in the regulation of receptor function by promoting interactions of the receptors withinteracting proteins. For the mu-opioid receptor (MOR), agonist-induced phosphorylation 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 thissequence that is predominantly catalyzed by 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 hierarchicalphosphorylation cascade that specifically requires GRK2/3 isoforms. In vivo GRK3 facilitates
MOR desensitization, whereas GRK5 seems to be required for opioid dependence. Thesefindings suggest that agonist-selective recruitment of distinct GRKs can influence different opioidrelated behaviors. To further explore the physiological consequences of MOR phosphorylation,
we have now generated and characterized a series of different phosphorylation-deficient mice.
98 - Beyond cAMP assays: GIRK channels as ultra-sensitive sensors for thecharacterization of novel opioids using the voltage-sensitive dye FMP
Stefan Schulz
Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University
Jena, Germany
All members of the opioid receptor family activate G protein-coupled inwardly rectifying potassiumchannels (GIRK). Therefore these potassium channels are important effectors of opioid-mediatedanalgesia. Using GIRK channels as an assay read-out for receptor activation reflects therefore
on a naturalistic event of opioid-mediated analgesia. Here, we report on a FLIPR assay system
based on the proprietary membrane potential bisoxonole dye FMP that uses HEK293 cells as
expression system for both GIRK channel subunits and opioid receptors. As expected, theGIRK1/2 channel selective activator ML297 (VU0456810) induces a decrease of fluorescencesignal intensity which is indicative for GIRK channel-mediated membrane hyperpolarization onlyin the presence of both the GIRK1 and GIRK2 subunits. After co-expression of the GIRK2 subunit
and the NOP or the µ receptor we detected a rapid agonist-induced decrease of fluorescencesignal intensity induced by Nociceptin/Orphanin FQ or DAMGO respectively. The detected signals
were both Pertussis-Toxin (PTX) and Tertiapin-Q (TPN-Q) dependent, yielding EC50 values in
the nanomolar range. We tested a panel of selective NOP/µ receptor ligands and multi-opioid
receptor agonist. Our findings are in accordance with those made in AtT-20 cells which natively
express prototypical neuronal GIRK1/2 channels. We assume that this easy-to-use assay format
can be potentially applied 1) to determine the βγ-subunit fraction of G protein signaling, 2) as a
general HTS-compatible screening assay for the identification of new opioid ligands, 3) for theidentification of GPCR-interacting proteins (GIP) such as GRKs, 4) to identify functional GPCRGPCR interaction and many other potential applications.

89

99 - Collybolide, a novel biased agonist to study kappa-opioid receptor pharmacology
Indrajeet Sharma1, Nicholas P. Massaro1, Adrien Cavé2, Heidi E. Hamm2, Joseph Parello2,
Vsevolod Katritch3, Lakshmi Devi4
1
University of Oklahoma, 2Vanderbilt University, 3University of Southern California, 4Icahn
School of Medicine at Mount Sinai
Kappa-opioid receptor (KOR) agonists are promising alternative as therapeutics to mu-opiateswith a low risk of dependence and abuse. The use of KOR agonists as analgesics has been
limited because of dose dependent neuropsychiatric effects including sedation and dysphoria.
Studies with KOR agonists biased to G-protein–mediated signaling (RB64 and triazole 1.1)
suggest the possibility of development of novel therapeutics with reduced side effects. In this vein,
we have identified collybolide, a non-nitrogenous sesquiterpene natural product extracted fromthe mushroom Collybia maculata as a highly selective and biased KOR agonist with in vivo
analgesic and antipruritic activity (Gupta et. al. PNAS 2016, 113, 6041–6046). Collybolide has afuryl-δ-lactone core similar to that of salvinorin A, however differs from the latter in exhibiting
biased agonistic activity. Collybolide shows no conditioned place aversion unlike classical kappa
agonists at doses at which it blocks non-histamine-mediated itch, suggesting the possibility ofseparating kappa mediated liabilities from its ability to block itch with this novel natural productbased template. However, there are liabilities associated with use of collybolide as a drugcandidate such as aqueous solubility, efficacy, and metabolic stability. These liabilities can beovercome by rational structural diversification of the collybolide template. Design, docking, SAR
studies, and pharmacological characterization of collybolide analogues will be discussed. Our
hypothesis is to comprehensively examine the SAR of collybolide, which will yield a set of
selective- and biased-KOR ligands with the potential of becoming lead drug candidates for itchand pain with reduced side effects.

100 - Opioid-Elicited Erk1/2 Phosphorylation, Elk-1 and CREB Activation, and Lipid Raft
Localization in HEK 293 Cells Coexpressing μ-Opioid and Nociceptin Receptors
Cynthia Wei-Sheng Lee1, Tzu-Yu Lin1, Ing-Kang Ho1,2
1
Center for Drug Abuse and Addiction, China Medical University Hospital, China Medical
University, Taichung, Taiwan, 2Graduate Institute of Clinical Medical Science, China Medical
University, Taichung, Taiwan
We aimed to explore the responses of Erk1/2 (p44/42 MAPK), transcription factors Elk-1 and
CREB, and lipid rafts elicited by opioids through coexpressed μ-opioid (MOP) andnociceptin/orphanin FQ (NOP) receptors in human embryonic kidney (HEK) 293 cells.
Phosphorylation of Erk1/2 was assessed by immunoblotting, Elk-1 and CREB activation was
revealed by reporter assays, and receptor colocalization on lipid rafts was detected byimmunocytochemistry in HEK 293 cells expressing MOP, NOP, and MOP+NOP receptors after
treatment with DAMGO, nociceptin, morphine, methadone, or buprenorphine. We found that
coexpressing NOP with MOP compromised the potency of methadone on Erk1/2 activation in
MOP-expressing cells. Nociceptin, but not morphine, induced Elk-1 and CREB in MOP+NOPexpressing cells. These results showed that opioids could modulate human Erk1/2, localizationof opioid receptors on the lipid rafts, and transcription factors Elk-1 and CREB, in MOP+NOPexpressing cells at the cytosolic, plasma membrane and nuclear levels.
90

101 - Effect of Magnetic Acupuncture Attachment on Neigun(acupoint P6) on Autonomic
Nervous System
Hyun Hwa Shin1, Joon Seoung Noh2, Whan Seok choi3
1Department of Family Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic Universityof Korea, 2Department of Family Medicine, Suwon St. Vincent's Hospital, College of Medicine, The
Catholic University of Korea, 3Department of Family Medicine, Seoul St. Mary's Hospital, College ofMedicine, The Catholic University of Korea
Neigun(acupoint P6) is known to be good acupoint for treating nausea and vomiting occur after surgery andchemotherapy. In addition, it has been reported to stabilize the sympathetic nervous system. The purposeof this study was to investigate the effect of non-invasive magnetic acupuncture attachment on theautonomic nervous system by measuring HRV(Heart Rate Variability). 20 healthy volunteers were recruited
through advertisement. Magnetic acupuncture was attached to either Neigun(acupoint P6) or Zu San
Li(acupoint ST36) by the randomization table, and HRV was measured before its attachment and 20
minutes after its attachment. One week later, we repeated the procedure at the same time on different
acupoint. We measured HRV using SA-2000(Medicare, Korea). Of the 20 subjects who completed the trial,
5 were male and 15 were female, and the mean age of each was 26 and 27. There was no significant
difference in HRV indices before attaching the magnetic acupuncture. However, the Neigun(acupoint P6)
stimulation showed significant increase in the percent change of SDNN(The Standard deviation of Normal
to Normal intervals) when stimulated by magnetic acupuncture(P=0.018) compared to Zu San Li(acupoint
ST36) stimulation, but, the percent change of RMSSD(Square root of the mean of the sum of the square ofdifferences between adjacent NN intervals), which mainly reflects parasympathetic activity, was not
significant(P=0.069). The result of this study suggest that attaching the magnetic acupuncture on
Neigun(acupoint P6) has a positive effect on autonomous function.

102 - TRV0109101, a G protein-biased agonist of the µ-opioid receptor, does not promote
opioid-induced mechanical allodynia following chronic administration
Michael Koblish, Richard Carr III, Edward R. Siuda, David H. Rominger, William GowenMacDonald, Conrad L. Cowan, Aimee L. Crombie, Jonathan D. Violin, Michael W. Lark
Trevena, Inc.
Prescription opioids remain essential for the management of acute moderate to severe pain. However,chronic use leads to adverse events including tolerance and opioid-induced hyperalgesia (OIH). OIH is aprocess of nociceptive sensitization in which individuals paradoxically experience exaggerated nociceptive
responses, instead of pain reduction, when chronically administered opioids. Although the characteristics
of OIH have been described in patient populations with high opioid usage, the molecular mechanisms of
OIH remain elusive. Here, we describe the pharmacological and pharmacokinetic properties of a G proteinbiased µ opioid receptor (MOR) agonist, TRV0109101, a close analog of oliceridine (TRV130). Likeoliceridine, TRV0109101 is a potent and selective agonist of MOR-mediated G protein-signaling with
reduced β-arrestin2 recruitment compared to morphine. In a model of OIH, continuous infusion of traditional,
unbiased opioids in mice promoted significant and sustained hyper-responsiveness to innocuous
mechanical stimuli, while continuous infusion of TRV0109101 did not induce mechanical allodynia. Further,
after a single dose, TRV0109101 promoted acute reversal of established morphine-induced mechanicalallodynia. In subsequent thermal antinociceptive tests, TRV0109101 displayed similar analgesic properties
to other conventional opioids with comparable tolerance following repeated dosing or continuous infusion,suggesting the development of OIH is mechanistically distinct from tolerance. Congruent with
TRV0109101’s pharmacological profile and efficacy, the development of OIH, but not tolerance, is likely
dependent on contributions from β-arrestin as oxycodone-induced OIH was absent in β-arrestin1 and βarrestin2 KO mice. We posit that the use of a MOR G-protein biased agonist may provide robust analgesia
with reduced risk of OIH.

91

103 - Dissecting Mechanisms of Nociceptin Opioid Peptide Receptor Regulation
Skylar Spangler, Patrick O'Neill, Michael Bruchas
Washington University in Saint LouisThe nociceptin opioid peptide receptor (NOPR) is highly expressed in dopaminergic (DA) neurons of theventral tegmental area (VTA). It is well known that these VTA-DA neurons are associated with rewardrelated behaviors. Not only does NOPR agonism inhibit the rewarding effects of virtually all drugs of abuse,
but nociceptin has been shown to inhibit VTA-DA firing in slice and dramatically decrease accumbal
dopamine release. Currently, the properties of NOPR regulation and inhibition of VTA-DA neuronalensembles has yet to be elucidated. We examined NOPR regulation in heterologous cells using cAMPmeasures and beta-arrestin recruitment (BRET) assays and in acute VTA slices, with endogenous NOPR,
using two-photon (2P) imaging. Our preliminary data suggest that NOPR still recruits arrestin3 despite
mutated c-terminal phosphorylation sites, but multiple of these sites may be involved in acutedesensitization of NOPR. We also demonstrate our ability to use 2P imaging to observe spontaneous andevoked calcium transients in VTA-DA neurons. This method was used to assess the dynamics of NOPR
signaling on neuronal activity, including receptor desensitization. We also used real-time 2P-imaging tovisualize trafficking of the receptor in brain tissue of a novel NOPR-eYFP mouse line, generated by our lab.
We couple these imaging approaches to simultaneously study receptor internalization and desensitizationin both heterologous cells and DA neurons. These data provide the groundwork for studies probing the
NOPR regulation and signaling in live neuronal populations, which will provide insight on endogenous opioid
regulation in neuronal networks and the cellular mechanisms that underline NOPR-mediated behaviors.

104 - Injectable peptide-based hydrogels as efficient matrices for the controlled-deliveryof opioid analgesics
Mariana Spetea1, Charlotte Martin2, Helmut Schmidhammer1, Steven Ballet2
1
University of Innsbruck, Innsbruck, Austria, 2Vrije Universiteit Brussel, Brussels, Belgium
Central goals in chronic pain control aim to provide analgesia of adequate efficacy and duration. Frequent
drug administration is commonly required to achieve sustained analgesia. To overcome drawbacks related
to repeated opioid administration during the treatment of chronic pain, several controlled-drug delivery
systems have been designed. Injectable peptide-based hydrogels represent suitable and attractive
controlled-drug delivery matrices, with advantages including protection of the drug against enzymatic
degradation by encapsulation in the hydrogel network, while maintaining the therapeutic plasma drugconcentration over a long period of time via diffusion from the hydrogel and by degradation of the hydrogel
network. Consequently, lower dosage and frequency of administration are possible, and result in an
improvement of the drug efficacy, while reducing the risk of side effects. This work reports on the design
and investigations of a family of short amphipathic peptide-based hydrogels as controlled-drug delivery
systems for opioids. Based on the lead sequence H-FEFQFK-NH2, distinct peptide hydrogelators weretargeted including β3-homo and D-amino acids. Such hydrogelator sequences show increased proteolytic
stability, a feature hypothesized to prolong the in vivo duration of action. The resulting peptide-basedhydrogels form thixotropic injectable hydrogels under physiological conditions. Subcutaneous injection ofmorphine and 14-methoxymetopon (a µ-opioid agonist 500-fold more potent than morphine with an
improved side effect profile), co-formulated with different hydrogelators produced prolonged antinociceptive
effects up to 96 h in the mouse tail-flick test. These results establish the potential of these peptide-basedhydrogels as efficient systems for the controlled-delivery of opioids.

92

105 - Studies with Nociceptin/Orphanin FQ Peptide (NOP) Receptor knockout rats reveal
NOP receptor-dependent sex differences in nociceptive sensitivity, anxiety-like behaviors
and serum N/OFQ and CORT levels in response to traumatic stress
Kelly M. Standifer1,2, Cindy Durand1, Ian Schalo1, Yong Zhang1
1
University of Oklahoma Health Sciences C, 2Oklahoma Center for Neuroscience
Nociceptin/Orphanin FQ (N/OFQ) modulates pain transmission, learning/memory, stress andanxiety via activation of the N/OFQ peptide (NOP, OPRL1) receptor. We previously reported thatsingle-prolonged stress (SPS), an established animal model of PTSD, produces allodynia andanxiety-like behaviors, elevates N/OFQ and decreases circulating corticosterone in maleSprague-Dawley rats that was reversed by NOP receptor antagonist treatment following SPS.
The objective of this study was to further examine the role of the N/OFQ-NOP receptor systemwith male AND female Wistar Han wildtype (WT) and NOP receptor knockout (OPRL1-/-) rats
randomly divided into control or SPS groups by gender. In male and female WT rats, paw
withdrawal thresholds (PWT) to von Frey and paw withdrawal latencies (PWL) to radiant heatstimuli, respectively, decreased within 7 days after SPS, lasting through day 28. Baseline
sensitivity did not differ between genotypes. However, while male OPRL1-/- rats were protectedfrom SPS-induced nociceptive hypersensitivity, female OPRL1-/- rats exhibited exacerbated
nociceptive hypersensitivity. Male OPRL1-/- rats had a lower anxiety index (AI) than WT, and it
was not increased by SPS. In contrast, SPS increased AI in WT and OPRL1-/- female rats. SPS
increased N/OFQ and decreased circulating corticosterone levels in male WT, but not in maleOPRL1-/- rats. SPS did not alter serum N/OFQ or CORT levels in either WT or OPRL1-/- female
rats. Male OPRL1-/- rat results are consistent with previous findings with the NOP receptor
antagonist, and indicate that endogenous N/OFQ-NOP receptor signaling plays an important, but
distinct, role in males and females following traumatic stress.

106 - A pH-sensitive opioid analgesic designed by modeling of pathological receptorligand interactions
Viola Spahn1, Giovanna Del Vecchio1, Dominika Labuz1, Antonio Rodriguez-Gaztelumendi1,
Nicolas Massaly1, Julia Temp1, Vedat Durmaz2, Peggy Sabri2, Marco Reidelbach2, Halina
Machelska1, Marcus Weber2, Christoph Stein1
1
Freie Universitaet Berlin - Charité, 2Computational Molecular Design, Zuse-Institut Berlin
Indiscriminate activation of opioid receptors provides pain relief but also severe central andintestinal side effects. We hypothesized that exploiting pathological (rather than physiological)
conformation dynamics of opioid receptor-ligand interactions might yield ligands without adverse
actions. By computer simulations at low pH, a hallmark of injured tissue, we designed an agonist
that, due to its low pKa, selectively activates peripheral mu-opioid receptors at the source of pain
generation. Unlike the conventional opioid fentanyl, this agonist showed pH-sensitive binding, Gprotein subunit dissociation, and cAMP inhibition in vitro. It produced injury-restricted analgesia
in rats with different types of inflammatory pain without exhibiting respiratory depression, sedation,
constipation or addiction potential.

93107 - Hsp90 and PEBP Promote Opioid Anti-Nociception Through the Regulation of Mu
Receptor Signal Transduction Cascades
Wei Lei1, Justin LaVigne1, David Duron1, Katie Edwards1, John M. Streicher1
1
Department of Pharmacology, College of Medicine, University of Arizona, Tucson AZ USA
Recent advances in understanding the signal transduction cascades of the mu opioid receptor (MOR) havesuggested new strategies for opioid drug discovery to minimize side effects – such as βarrestin2 biased
agonists. One limitation to this approach is that the signaling complex of the MOR is not well understood,
including our mechanistic understanding of how signal transduction molecules transduce changes inbehavior. To address this limitation, we sought to identify new signaling regulators of the MOR, and to
determine their role in MOR-mediated anti-nociception. Through the intracerebroventricular administrationof selective small molecule antagonists in mice, we found that the chaperone protein Heat shock protein
90 (Hsp90) and the GRK2/Raf1 sequestering protein phosphatidylethanolamine-binding protein (PEBP)
both strongly promote opioid anti-nociception. Hsp90 inhibition completely blocked morphine antinociception in HIV neuropathic pain and strongly blocked in post-surgical pain, while only slightly impacting
tail-flick anti-nociception. PEBP inhibition by contrast strongly blocked tail-flick anti-nociception. Seeking
mechanisms for these changes, we found that Hsp90 inhibition blocked ERK MAPK activation in the brain,
and this signaling change could account for all the impact of Hsp90 on opioid anti-nociception. In
comparison, we found that PEBP regulates the potency/efficacy of βarrestin2 recruitment in vitro,suggesting a role in regulating arrestin recruitment in the brain with subsequent effects on tail-flick antinociception. Through these studies, we have thus identified novel MOR signaling regulators with strongroles in promoting anti-nociception, and begun to illuminate their molecular mechanisms. Future work could
utilize this information for the creation of novel drug development strategies.

108 - Modulation of endogenous opioid-mediated antinociception by regulator of G
protein signaling (RGS) proteins
Sherrica Tai1, Nicholas Senese1, Susan Ingram2, John Traynor1
1
University of Michigan, 2Oregon Health & Science University
RGS proteins are negative modulators of G protein signaling downstream of G protein-coupled receptors,
including the mu-opioid receptor (MOPR) and serve to switch-off signaling. We have shown that Gαo is the
predominant Gα protein for mediating MOPR agonist-mediated antinociception in mice. Consequently,
129SvEv mice expressing Gαo that is insensitive to RGS protein activity (RGSi-Gαo) exhibit an increased
antinociceptive response to MOPR agonists in the hot-plate test. In contrast, there was a paradoxicalinhibition, or no change, of opioid antinociception in the tail-withdrawal test. Here we show, using the hotplate test as a measure of heat nociception, that endogenous opioid peptide signaling is also enhanced inthese RGSi-Gαo mice. The enkephalinase inhibitor RB-101, which increases the half-life of endogenous
opioid peptides, administered intracerebroventricular to wild type mice produced antinociception that was
reversed by an opioid antagonist, confirming a role for endogenous opioid peptides. This antinociceptive
effect was enhanced in RGSi-Gαo mice. A brief swim stress triggered antinociception that was greater in
RGSi-Gαo mice compared to their wild-type littermates. This response was also reversed by opioid
antagonists. The results show the opioid peptide system is more active in mice expressing RGSi-Gαo andthat inhibition of RGS proteins may provide a therapeutic target. On the other hand, the role of RGS proteinsin negatively modulating MOPR signaling is not reflected as consistent inhibition of behavioral responses
across models suggesting RGS proteins may be affecting other systems and/or acting on different
intracellular substrates. Supported by DA035316 and DA035316-03S1.

94

109 - Label-free mass spectrometry based quantitation of peptides related to chronic migraine andopioid-induced hyperalgesia (OIH) in mice
Krishna D. B. Anapindi1, Ning Yang1, Elena V. Romanova1, Stanislav S. Rubakhin1, Alycia Tipton2, Amynah
Pradhan2, Jonathan V. Sweedler1
1University of Illinois at Urbana Champaign, 2University of Illinois at Chicago
Migraine is a pain disorder characterized by recurrent headaches that debilitate around 40 million people
annually in the United States. The mechanism that leads to this chronic pain state is currently unknown.
Opioid drugs are commonly prescribed medications to treat migraine. However, persistent use of opioids
can lead to more severe and increased frequency of migraine. This paradoxical situation is likely related to
the known effects of opioid use- opioid-induced hyperalgesia (OIH) - where patients administered with
opioids becomes more sensitive to a pain stimulus. Considering that opioids can push episodic migraine
into a chronic condition indicates that OIH may share overlapping mechanisms with chronic migraine.
Though the same pain signaling pathways and nervous system morphological structures are involved indevelopment and sustaining of both conditions, precise molecular mechanisms driving them are still not
well understood. Neuropeptides, which act as cell-to-cell signaling molecules between different regions of
the nervous system are believed to play an important modulatory role in controlling these disorders. To
identify the peptide complement in the brain regions associated with these disorders, we performed a mass
spectrometry based de novo sequencing of peptides followed by their label-free quantitation using the open
source platform SKYLINE. 10 peptides with significantly changed signals were identified from 6 different
prohormones, including pro-CGRP, pro-opiomelanocortin and pro-Enkephalin prohormones. 3 of these 6
prohormones are common to both the conditions. The peptide and prohormone biomarker candidates
identified in this study may help elucidate the mechanistic processes leading to OIH and migraine.
110 - Overlapping mechanisms between opioid induced hyperalgesia and chronic migraine
Alycia Tipton1, Elena V. Romanova2,3, Stanislav S. Rubakhin2,3, Krishna D. B. Anapindi2,3, Ning Yang2,
Jonathan V. Sweedler2,3, Amynah A. Pradhan11Dept Psychiatry, University of Illinois at Chicago, 2Dept Chemistry, University of Illinois at UrbanaChampaign, 3Beckman Institute for Advanced Science and Technology, University of Illinois at UrbanaChampaign
Opioids are commonly used to treat migraine, but can ultimately result in refractory headache and migraine
chronification. This exacerbated pain is likely related to opioid induced hyperalgesia (OIH), a phenomenon
in which opioid treatment results in a paradoxical increase in pain. It is currently unknown how mu opioid
receptor agonists negatively regulate migraine. The aim of this study was to identify overlapping
mechanisms between OIH and chronic migraine. Initially, we determined if morphine could promote chronic
migraine-associated pain. We used the known human migraine trigger, nitroglycerin (NTG), which we’veshown previously produces a migraine-associated hyperalgesia in mice. Mice were treated daily for 11 days
with morphine or vehicle. Following two days of pre-treatment mice were injected with a sub-threshold doseof NTG or vehicle every second day for 9 days. Morphine and NTG treated animals developed a severe
chronic hyperalgesia, which was greater than either treatment alone. Further, changes in neuropeptide
expression are hallmarks of both chronic migraine and OIH. An additional aim of this study was to performa peptidomic screen to determine changes in neuropeptides following chronic NTG vs. OIH treatment. Mice
were chronically administered a maximal dose of NTG to induce chronic migraine-associated pain; or
escalating doses of morphine to induce OIH, along with corresponding vehicle controls. Tissue frommultiple pain and reward-processing regions were collected for quantitative mass spectrometry and multiple
significantly changed peptides were detected, including a pro-pain/migraine CGRP-related peptide. Our
results indicate that opioids may synergistically promote a chronic migraine state.

95

111 - Influence of tramadol in global cerebral ischemia-induced mechanical allodynia
Shogo Tokuyama, Wataru Matsuura, Shinichi Harada
Kobe Gakuin University
Central post-stroke pain (CPSP) is one of the complications of cerebral ischemia and neuropathic pain
syndrome. Although CPSP is a serious condition, details pertaining to underlying mechanisms are not wellestablished, making current standard treatments only partially effective. In this study, we assessed theeffects of tramadol, an analgesic drug mediated by opioid receptors, using a mouse model of global cerebral
ischemia. Male ddY mice were subjected to 30 min of bilateral carotid artery occlusion (BCAO). The
development of hind paw mechanical hyperalgesia was measured using the von Frey test. Mechanicalallodynia was significantly and dose-dependently suppressed by intraperitoneal (i.p.) tramadol (10 or 20
mg/kg). These effects which peaked at 10 min and continued for at least 60 min were inhibited by naloxone(a nonselective opioid receptor antagonist, 1 mg/kg, i.p.). Tramadol antinociception was significantly
negated by beta-funaltrexamine (a selective μ-opioid receptor antagonist, 20 mg/kg, i.p.) but not naltrindole(a selective δ-opioid receptor antagonist, 5 mg/kg, i.p.) or nor-binaltorphimine (a selective κ-opioid receptor
antagonist, 10 mg/kg, i.p.) after 5 min, by b-funaltrexamine and nor-binaltorphimine but not naltrindole after
10 min, and by all selective opioid receptor antagonists at 15 and 30 min after tramadol treatment. These
results suggested that antinociception induced by tramadol through various opioid receptors was timedependent. Furthermore, it is possible that the opioid receptors involved in tramadol-induced
antinociception change over time with metabolism of this drug. Support by Grants-in-Aid and by special
coordination funds from Grants-in-Aid for Scientific Research (C) (16K10988)

112 - In Vitro And In Vivo Profile Of PPL-101 And PPL-103: Mixed Opioid Partial Agonist
Analgesics With Low Abuse Potential
Lawrence Toll1, Andrea Cippitelli1, Nicholas Toll1, John A. Lawson3, William Crossman3, Taline
Khroyan2, Willma E. Polgar2
1
Torrey Pines Institute for Molecular Stu, 2SRI International, 3Phoenix PharmaLabs
Opiates are the most effective and widely used treatments for acute and chronic pain. However, problems
associated with morphine and other standard opioid analgesics severely limit their effectiveness in theclinic. PPL-101 and PPL-103, derived from morphine and morphinan ring systems contain a chiral Nsubstituent, which confers a combination of high binding affinities and partial agonist activities at mu, delta,
and kappa opioid receptors, leading to unique in vivo pharmacology compared to other conventional
opioids. Acute antinociceptive and reward acquisition of PPL-101 and PPL-103 were assessed in miceusing the tail flick assay and CPP paradigm, respectively. The reinforcing effects of these compounds wereassessed in rats using the self-administration paradigm. In mice, PPL-101 and PPL-103 produced
antinociception reaching maximal effects equivalent to morphine at approximately one third and one-tenthof morphine’s dose, respectively. PPL-101-induced antinociception was attenuated following pretreatment
with the kappa antagonist JDTic, but not the mu opioid antagonist beta-FNA. In mice, PPL-101 and PPL103 produced dose-dependent decreases in activity, similar to other kappa agonists, however, they did not
produce conditioned place aversion, and in fact elicited a trend towards CPP. In rats, neither PPL-101 nor
PPL-103 were self-administered when substituted for morphine. Collectively, these results indicate that
mixed opioid receptor partial agonists can produce potent antinociceptive activity with a lack of dysphoria
in mice and without being self-administered in rats. Compounds with this profile could be superior
analgesics with greatly reduced addiction liability and fewer side-effects compared to traditional opiates.

96113 - Effects of Heroin on Prosocial Behavior in Rats
Seven Tomek, Gabriela Stegmann, M. Foster Olive
Arizona State University
Previous research indicates that rats are capable of prosocial or empathetic behavior. Under normal
conditions, when a “free” rat is placed in the vicinity of rat trapped in a restraint, the rat will release or “save”
the other rat from confinement. Human empathy research shows a correlation between heroin use andlower levels of empathy. This study was aimed to determine the effects of heroin on prosocial behavior inrats. For two weeks, rescuer rats were given the opportunity to save their cage-mate, and the occurrence
and latency to free the confined rat was recorded. After the saving behavior was established rats wererandomly selected into either the sucrose (control) or heroin (treatment) group in which they were allowed
to self-administer heroin (0.06 mg/kg/inf, i.v.) or sucrose pellets (orally) for 10-14 days. Next, rats wereretested for saving behavior once daily for 3 days, during which they had a choice between freeing thetrapped rat or continuing to self-administer their respective reinforcer. Results indicated that sucrose rats
continued to save their cage-mate instead of self-administering sucrose, whereas heroin rats chose to selfadminister heroin and not save their cage-mate. Thus, rats with a history of heroin self-administration show
deficits in prosocial behavior, consistent with specific diagnostic criteria for opiate use disorder. Since
activity in the insula is known to be correlated with both drug craving and important abilities needed forsocial interaction, future studies are planned to attempt to restore prosocial behavior following heroin intake
using chemogenetic approaches.

114 - Characterization of the endogenous small-sized peptides released in the nucleus
accumbens related to pain modulation
Moe Watanabe1, Naohiko Uchiyama1, Yuki Sugiura2, Michiko Narita1, Takashige Kondo1,
Yoshitaka Iwayama1, Hidetsugu Ogata1, Naoko Kuzumaki1, Minoru Narita1,31
Dept. Pharmacol., Hoshi Univ., Tokyo, Japan, 2Dept. Biochem., Keio Univ. Sch. Med., Tokyo,
Japan, 3L-StaR, Hoshi Univ., Tokyo, Japan
A growing body of evidences indicates the alteration of excitability in the nucleus accumbens (N.Acc) under
the chronic pain. They suggest that the neuronal activity via chemical signaling in the N.Acc plays a role forpain modulation. However, there is little knowledge about what kind of small molecules are involved in thisevent. In this study, we investigated the role of the small molecules released from the N.Acc induced bypain or administration of analgesics. We generated mice expressing channerhodopsin-2 in the nociceptive
neurons with the injection of AAV6-hSyn-ChR2(ET/TC)-EGFP. Two weeks after AAV injection, we collected
the dialysate in the N.Acc after the activation of the nociceptive neurons by optical stimulation. We alsocollected the dialysate in the N.Acc. after the systemic administration of morphine. Then we performed
comparative metabolome analysis by fourier transform mass spectrometry (FT-MS). In the present study,
89 metabolites were identified in the N.Acc. Among those, N-acetylaspartylglutamate (NAAG) and
acetoacetate were decreased after the activation of the nociceptive neurons, whereas both were increased
after the systemic administration of morphine in the N.Acc. As theoretically considered, direct injection of
the NAAG into the N.Acc significantly attenuated the nociceptive stimuli induced by the activation of thenociceptive neurons by optical stimulation. These results suggest that NAAG released in the N.Acc. could
be, at least in part, associated with pain modulation.

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115 - Automated detection of Mu Opiod Receptor recycling reveals distinct
subpopulations of exocytic events
Zara Weinberg, Tiffany Phan, Manojkumar Puthenveedu
Carnegie Mellon University
GPCR internalization and recycling-dependent resensitization is an integral part of how cells
regulate their responsiveness to intercellular signaling. Receptor recycling has previously beenassessed either through gross measures of cell surface receptor density using antibody labelingor through monitoring of individual exocytic events using TIRF microscopy. We have used
quantitative image analysis of single exocytic events of the Mu Opioid Receptor, coupled with amachine learning algorithm, to identify distinct subpopulations of these exocytic events that can
provide insight into the multiple regulated and constitutive recycling pathways present in cells.
This insight into recycling can help elucidate the regulation of receptor recycling in basal
conditions, as well as provide opportunities for modifying specific recycling pathways to change
cellular responsiveness.

116 - Development of a Virtual Reality Paradigm for in vivo Hippocampal Imaging During
Morphine Conditioned Place Preference
Sidney Williams, Moises W. Arriaga, Edward B. Han, Jose Moron-Concepcion
Washington University, Saint LouisLong term associations between a specific environment and drugs of abuse, such as opioids, cantrigger craving and relapse in people with previous history of drug misuse. These maladaptive
memories are partly formed by structural and functional changes in the dorsal hippocampus.
Using ex vivo imaging approaches, our lab has shown that morphine conditioned place preference(mor CPP) development decreases the number of dendritic spines on hippocampal CA1 neurons
through NR2B-containing NMDA receptors. Yet the timing and dynamics of these events and theirpotential relationship to the association between morphine reward and context are unknown. To
observe neural networks in real time as mor CPP and reinstatement take place, we have designed
a virtual reality conditioned place preference (VR-CPP) paradigm paired with two-photon imaging.
The three chamber VR-CPP apparatus contains a neutral middle chamber and two conditioning
chambers containing distinct visual cues. Mice are head fixed in the VR environment and allowed
to freely run on a Styrofoam ball suspended by air pressure. Movement of the ball is tracked by acomputer mouse, converted to forward and yaw velocities by custom written software in LabView,
and then fed to a virtual reality engine written in Matlab, which updates the visual scene permitting
the animal to navigate through the VR environment. The mor CPP protocol (Portugal et al., 2014)is then executed in the VR environment. When combined with two-photon in vivo imaging, thisnovel behavioral paradigm allows unprecedented spatio-temporal resolution in following structuraland activity changes that accompany mor CPP.

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117 - Activation, Desensitization and Trafficking of total phosphodeficient MORs in the
MOR-knockout rat
John T Williams, Seksiri Arttamangkul, Daniel Heinz, James R Bunzow
Oregon Health Sciences University
Desensitization and trafficking of receptors is thought to be dependent on the phosphorylation ofmultiple sites on the C-terminus of MORs. Both processes were examined in locus coeruleus (LC)neurons in brain slice preparations. Wild type and mutant MORs were virally expressed in a MOR
knockout rat. Both receptors had an N-terminal GFP tag such that trafficking of receptors inducedby the application of agonists could be examined. All phosphorylation sites on the C-terminus
were mutated to alanine in a construct termed total phosphodeficient (TPD) MOR. Whole cellrecordings and imaging experiments were carried out in preparations expressing either wild typeor the TPD MOR. Activation of both receptors induced outward currents. Application of asaturating concentration of [Met]5enkephalin (30 µM) resulted in a peak and decline in current
over a period of 10 min in experiments from wild type receptors. In experiments with the TPD
MOR the decline in the current was significantly reduced, indicating a dramatic reduction indesensitization. Imaging of membrane-associated receptors was carried out using an anti-GFP
nanobody conjugated with Alexa594. The wild type receptors internalized within 10 min following
the application of ME, however there was no internalization of the TPD MORs. The results showthat acutely TPD MORs function similar to wild type but that desensitization and trafficking is
dramatically disrupted.
118 - Inflammatory Pain Changes The Expression and Function Of CB1 Receptors in The
Periaqueductal Gray
Adrianne R. Wilson-Poe, Chris Trousdale, and Jose Morón
Washington University in Saint LouisThe periaqueductal gray (PAG) mediates the antinociceptive properties of analgesics, including
opioids and cannabinoids. Administration of either opioids or cannabinoids into the PAG induces
antinociception, and the presumed mechanism of analgesia is the disinhibition of GABAergic
neurons. However, most studies characterizing the antinociceptive properties of cannabinoids in
the PAG have been conducted in naive animals. Few studies have reported on the role of CB1
receptors in the PAG during conditions which would prompt the administration of analgesics;
namely, in chronic pain. In this study, we used the CFA model to characterize CB1 receptor
expression and function during persistent inflammatory pain. Using cellular fractionation andwestern blot analysis, we demonstrate that 48 hours after CFA injection, there is a significant
upregulation in the expression of synaptic CB1 receptors. To assess whether this protein
upregulation induces a functional change, we measured the anti-hyperalgesic action of WIN
55,212-2, and used whole cell patch clamp electrophysiology to identify pain-induced alterationsin the PAG. By studying the potency of WIN during conditions in which analgesics would normallybe administered, we have unveiled a novel compensatory change in the descending pain pathway
during persistent pain. Opioids are still the most commonly prescribed analgesics for chronic pain.
Because there are neuroanatomical and functional interactions between PAG CB1 and mu-opioid
systems, these pain-induced changes provide critical insight into the analgesic efficacy of thesedrugs during persistent pain, and may aid the development of novel pharmaco-therapies.

99119 - Naltrexone and Nalmefene Attenuate Cocaine Place Preference but Differentially
Effect Cocaine Locomotor Sensitization
KA Windisch, B Reed, A Dunn, MJ Kreek
The Rockefeller University
INTRODUCTION/METHODS: Previously we showed that nalmefene (NMF) exhibits partial agonism at
kappa opioid receptors (KOPr), while also exhibiting antagonism at mu opioid receptors, using in vitro
GTPγS binding stimulation and in vivo prolactin release as an biomarker of kappa agonist activity in humans(Bart et al., 2005, Neuropsychopharmacol, 30:2254-62). Here we examine the effect of low (1 mg/kg) andhigh (10 mg/kg) dose naltrexone (NTX) and NMF on cocaine place preference and locomotor sensitization.
RESULTS: Both doses of NTX and high dose NMF significantly reduced cocaine place preference.
Conversely, a significant place avoidance was observed for high dose NTX and both NMF doses.
Interestingly, blockade of cocaine induced locomotor sensitization was observed with both NTX doses andlow, but not high, dose NMF. CONCLUSIONS: Overall, NTX and NMF block the hedonic effects of cocaine
and induce a negative/aversive subjective state when administered alone. However, unlikely high dose
NTX, a significant locomotor sensitization to cocaine was observed with high dose NMF pretreatment (asmeasured by crossovers during CPP conditioning) suggesting potential pathway or receptor specificdifferences between NTX and NMF in vivo. This is an important step in understanding the potential
mechanism(s) of action of NTX and NMF for the development of more efficacious pharmacologicaltreatments of substance use disorders. Support: the Gary R. Helman Postdoctoral Research Fellowship(KAW), the Robertson Development Fund (EB, BR, MJK), and the Miriam and Sheldon Adelson Medical
Research Foundation (MJK).

120 - A Novel assay for measuring human μ-opioid receptor’s functional activity
Jinhua Wu1,2, Akihiko Ozawa1, Jennifer Schoch1, Lawrence R Toll1,21Torrey Pines Institute for Molecular Studies, 2Assuage Pharmaceuticals, Inc
Traditionally GTPgS binding and adenylyl cyclase (AC) activity inhibition are often used to measure µopioid receptor (MOR) activation. We developed a novel assay in which intact live cells can be used todevelop new ligands (agonist or antagonist) for human mu-opioid receptor. Chinese hamster ovary cells
were stably transfected with human MOR, and μ-opioid receptor’s expression was confirmed byimmunohistochemistry and binding affinity assay. CHO cells expressing mu were seeded in 96-wellplates, loaded with membrane potential-sensitive fluorescent dye for 60 minutes, and then treated with orwithout mu agonists. CHO-MOR cells hyperpolarized upon the treatment of its own agonist, which isprobably mediated by G protein-coupled inwardly-rectifying potassium channel or other certain potassiumchannel. Membrane potential change induced by CHO-MOR activation can be detected only throughmembrane potential-sensitive fluorescent potassium dye but not through calcium dye. The resulting
fluorescence changes could be recorded in real time. The μ-opioid receptor agonist DAMGO has an
EC50 of ~3.28nM in mu cells, and 100nM DAMGO-induced μ-opioid receptor activation can be inhibitedby μ-opioid receptor antagonist naloxone in a dose-dependent manner, with an IC50 of is ~40nM. This
novel functional assay provides a simple, quick and safe method for real-time measurement of mureceptor activation or inhibition through their action on native CHO cells expressing human MOR. Thismethod can greatly facilitate the drug discovery for opioid receptors.

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121 - POTENTIAL OF CIRCULATING MU-OPIOID RECEPTOR AND TOLL-LIKE RECEPTOR-4
ACTIVATION IN MORPHINE-ADMINISTERED MICE
Nan XIE1, Fabio Pereira GOMES1, Vandana DEORA1, Kye GREGORY2, Tharindu VITHANAGE2, Zeyad D
NASSAR1, Peter CABOT1, David STURGESS2, Paul Nicholas SHAW 1, Marie-Odile PARAT1
1Dept School of Pharmacy, 2Dept Mater Research Institute, The University of Queensland, Australia
In this study, we set up new cell-based assays to quantify circulating receptor activation potential andunveiled the ability of morphine and its metabolites present in biological samples to activate the µ-opioid
receptor (MOR) and toll-like receptor-4 (TLR-4). Alphascreen cyclic AMP (cAMP) assay and MOR
overexpressing HEK293 cells were employed to quantify the MOR activation. HEK-Blue hTLR4 were
utilized to measure TLR-4 activation. Both assays were standardised using morphine, its MOR-active
metabolite morphine-6 glucuronide (M6G) and its MOR-inactive, but TLR4-active metabolite morphine-3
glucuronide (M3G) in the presence/absence of plasma. Specificity was verified using opioid and TLR-4
antagonists or inhibitors. These methods were then employed to measure the receptors activation potential
in the circulation of morphine-treated mice (1 or 10 mg/kg every 12 h for 3 days). Plasma spiked withmorphine or M6G could activate MOR, whereas M3G, which lack MOR activation potential, led to moderate
but consistent TLR-4 activation. Plasma from morphine-treated mice showed MOR activation potential,
reversed by MOR antagonists. Morphine administration led to TLR4 activation at time points where M3G ismeasured in plasma, but also to TLR4-independent NF-κB activation via elevation of circulating cytokines
such as TNFα at time points where M3G is not detected. Our study has established new methods toevaluate the effect of perioperatively administered opioids on the biology of cancer cells and otherprominent tumour-associated cells such as macrophages. Furthermore, the study suggests a novel nonopioid-mediated role for morphine administered perioperatively to cancer surgery patients.
122 - Alternatively spliced mu opioid receptor C termini impact the diverse actions of morphine
Jin Xu1, Zhigang Lu2, Ankita Narayan1, valerie P. Le rouzic1, Mingming Xu1, Amanda Hunkele1, Taylor G.
Brown1, William F. Hoefer3, Grace C. Rossi3, Richard C. Rice4, Arlene Martinez-Rivera4, Anjali M.
Rajadhyaksha4, Luca Cartegni5, Daniel L. Bassoni6, Gavril W. Pasternak1, Ying-Xian Pan1
1Memorial Sloan-Kettering Cancer Center, 2Key Laboratory of Acupucture and Medicine Research of
Ministry of Education, and First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing,
China, 3Dept Psychology, Long Island University, Brockville, New York, 4Division of Pediatric Neurology,
Dept Pediatrics, Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, 5Dept
Chemical Biology, Emest Mario School of Pharmacy, Rutgers, The State University of New Jersey,
Piscataway, New Jersey, 6DiscoverX Corporation, Fremont, California
Extensive 3′ alternative splicing of the mu opioid receptor gene OPRM1 creates multiple C-terminal splicevariants. However, their behavioral relevance remains unknown. The present study generated 3 mutant
mouse models with truncated C termini in 2 different mouse strains, C57BL/6J (B6) and 129/SvEv (129).
One mouse truncated all C termini downstream of Oprm1 exon 3 (mE3M mice), while the other two
selectively truncated C-terminal tails encoded by either exon 4 (mE4M mice) or exon 7 (mE7M mice).
Studies of these mice revealed divergent roles for the C termini in morphine-induced behaviors, highlighting
the importance of C-terminal variants in complex morphine actions. In mE7M-B6 mice, the exon 7–
associated truncation diminished morphine tolerance and reward without altering physical dependence,
whereas the exon 4–associated truncation in mE4M-B6 mice facilitated morphine tolerance and reduced
morphine dependence without affecting morphine reward. mE7M-B6 mutant mice lost morphine-induced
receptor desensitization in the brain stem and hypothalamus, consistent with exon 7 involvement inmorphine tolerance. In cell-based studies, exon 7–associated variants shifted the bias of several mu opioids
toward β-arrestin 2 over G protein activation compared with the exon 4–associated variant, suggesting aninteraction of exon 7–associated C-terminal tails with β-arrestin 2 in morphine-induced desensitization andtolerance. Together, the differential effects of C-terminal truncation illustrate the pharmacologicalimportance of OPRM1 3′ alternative splicing.

101123 - Design and synthesis a novel κ opioid receptor agonists isolating both sedation
and aversion.
Masahiro Yata1, Naoshi Yamamoto2, Yasuyuki Nagumo2, Hiroshi Nagase1,2
1Graduate School of Pure and Applied Sciences, University of Tsukuba, 2International Institute forIntegrative Sleep Medicine (WPI-IIIS), University of Tsukuba
Highly selective κ opioid receptor (KOR) agonists were expected to be potent analgesic drugs withoutmorphine-like side effects derived from μ opioid receptor. Nalfurafine is the only KOR agonist without both
addiction and aversion in the efficacious dose, which was launched in Japan in 2009 as an antipruritic agent
for kidney dialysis and chronic liver disease patients.1 However, nalfurafine could not be applied to use for
analgesic because of the sedative effect derived from the KOR. Accordingly, we have attempted to designand synthesize nalfurafine derivatives without the sedative effect. The detailed conformational analysis andstructure activity relationship studies of nalfurafine led us to the hypothesis that the orientation of the 6amide side chain would significantly influence the affinity for the KOR and the side effects derived from
KOR, like aversive and sedative effects.2-3 On the basis of this hypothesis, we designed and synthesized
the 4,5-epoxy morphinan derivatives with a bicyclo[2.2.2]octene skeleton whose 6-amide side chainoriented to the upper side of the C-ring. The final obtained derivative YNT-1612 showed the higher agonist
activity for the KOR than that of nalfurafine. Furthermore, YNT-1612 showed no sedative and aversive
effects even at much higher dose than that of nalfurafine. We will report the design, synthesis, and thepharmacological effects of YNT-1612 derivatives.
1. Nagase, H. Jpn. J. Pharm. Palliat. Care Sci. 2010, 3, 115.
2. Fujii, H. et al. Bioorg. Med. Chem. Lett. 2010, 20, 121.
3. Watanabe, Y. et al. Bioorg. Med. Chem. Lett. 2014, 24, 4980.

124 - The effects of chronic stress on alcohol consumption in μ-opioid receptor knockout
mice
Moriya Yuki1,2,3, Yoshiyuki Kasahara2,3, F. Scott Hall4,5, George R. Uhl4,6, Kazutaka Ikeda1, Ichiro Sora2,7
1Tokyo Metropolitan Institute of Medical Science, 2Department of Biological Psychiatry, Tohoku University
Graduate School of Medicine, Japan, 3Department of Disaster Psychiatry, International Research Instituteof Disaster Science (IRIDeS), Tohoku University, 4Molecular Neurobiology Branch, Intramural Research
Program, National Institute on Drug Abuse, USA, 5Department of Pharmacology and Experimental
Therapeutics, University of Toledo, USA, 6Research Service, New Mexico VA Healthcare System,
Albuquerque, NM, USA, 7Department of Psychiatry, Kobe University Graduate School of Medicine, Japan
Adverse life experiences are associated with an increased risk of developing alcohol use disorders (AUD).
Female drinking levels and drinking problems are increasing and more closely approximating those seenamong men. One factor that has not been adequately examined in previous studies of the genetic
contributions to AUD is sex, which is a substantial shortcoming in the field given that there are significant
influences of sex on alcohol consumption patterns and alcoholism in humans and in animal models. Genetic
factors, such as allelic variation in opioid receptor system genes, have a substantial influence on alcohol
consumption, but only a limited set of such genetic influences on behavioral activity associated with forced
drinking have been examined. In our previous study, disturbances of mu-opioid receptors influenced theeffects of isolation-rearing on ethanol consumption in a sex-dependent manner. The preset study was
based on the hypothesis that the effects of restrain stress on ethanol consumption, would be also influenced
by both sex and the functioning of mu-opioid receptor systems. The effects of restrain stress on ethanol
intake were assessed using a two-bottle home-cage consumption (8% v/v ethanol vs. water) paradigm inmale and female wild-type and mu-opioid gene knockout mice. Restraint stress modestly increased ethanol
consumption in female mu-opioid knockout mice but not in female and male wild-type mice. The study
shows that disturbances of mu-opioid receptors influences the behavioral consequences of ethanol
consumption following stress in a sex-dependent manner.

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125 - Commonly used opioids induce neurotoxic effects in neuronal cell cultures
Sofia Zelleroth1, Erik Nylander1, Esra Flaieh1, Leo Leoson1, Alfhild Grönbladh1, Mathias Hallberg1,
Fred Nyberg1
1
Uppsala University, 2Uppsala University
Non-medical use of opioids is a global problem affecting health, social and economic welfare. The
frequency of prescription drug abuse is increasing and the consequences of this abuse aredevastating. In addition to unintentional overdose death there are several opioid-related disorders.
Chronic non-medical use of opioids has been proposed to cause cognitive dysfunction by
inhibiting neurogenesis and inducing neuronal apoptosis. Furthermore, as many of the opioid
abusers are young adults with still undergoing development of the central nervous system it is ofimportance that these neurotoxic events are further evaluated.
The study is part of a larger project were the toxic effects of the opioids methadone, morphine,ketobemidone, fentanyl, oxycodone, buprenorphine, as well as the opioid antagonist naloxone
are evaluated. The purpose of the present study is to examine the neurotoxic effects of thecommonly used opioids in neuronal cell cultures.
The toxic effects of commonly used opioids were studied on neuronal cell cultures. The cells were
given the opioid as a single dose for 4h and 24h, or as repeated treatment during 72h. Thereafter,
the opioid induced toxicity was analysed by measuring mitochondrial activity and cell cytotoxicity.
The results indicate that common prescription opioids induce neurotoxic effects to different extent.
Primarily, differences in neurotoxicity depend on treatment time and dose.
The present study suggests that opioids induce neurotoxic effects on neuronal cell cultures. The
result further highlights the problems associated with opioids and can provide further insight on
how to counteract these problems.

103See you in San Diego!
June 12-16, 2018
Bayfront Hilton | San Diego CA USA

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