INRC

Program

INTERNATIONAL NARCOTICS RESEARCH CONFERENCE

June 12-16, 2018
San Diego, California USA

Contents
Pages
Executive Committee and Meeting Organizers…………………………………2
Exhibitor and Sponsor Information……………………………………………….3
INRC 2018 Awardees……………………………………………………………...4-5
Meeting Schedule…………………………………………………………………..6-11
Abstract List………………………………………………………………………...12-19
Symposium Abstracts……………………………………………………………...20-30
Poster Abstracts……………………………………………………………………31-78

INRC Officers
Fred Nyberg (President, Sweden)
John Traynor (Past-President, USA)
Larry Toll (Treasurer, USA)
Craig Stevens (Information Officer, USA)

INRC Executive Committee
USA
Amynah Pradhan (USA)Jim Zadina (USA)Ream Al-Hasani (USA)EuropeChris Bailey (UK)
Lucia Hipolito (Spain)
Stefan Schulz (Germany)
Rest of World
Kazutaka Ikeda (Japan)
Meritxell Canals (Australia)
Shogo Tokuyama (Japan)

INRC 2018 Organizing Committee
Larry Toll
Cathy Cahill
Chris Evans
Nurulain Zaveri
Sandra Comer
Rita Valentino
Fred Nyberg
Lee-Yuan Liu-Chen
Elyssa Margolis
Tuan Trang
2

Thank you to our generous SPONSORS!

3

INRC 2018 AWARDEESFounders’ Lecture – Professor Macdonald Christie
Dr. Macdonald (Mac) Christie is Professor of Pharmacology,
University of Sydney School of Medical Sciences. Mac is aneuropharmacologist, an internationally renowned electrophysiologist and expert in ion channel and synaptic physiology andpharmacology and the leading basic opioid pharmacologist inAustralia. He leads a research team that studies cellular andmolecular mechanisms in opioid receptor signaling in pain
pathways, the biological basis of adaptations that produce chronic
pain and drug dependence and is Chief Investigator on a NHMRC
Program grant involving preclinical development of novel pain
therapeutics. Professor Christie is internationally renowned for his
studies on ion channel function and neural network plasticity innervous system disorders such as chronic pain and drug
dependence. His research is fundamentally important given the increasing use of opioids tomanage moderate to severe chronic pain is complicated by adverse effects such as
tolerance, opioid dependence and in some cases, addiction. Mac is a past Director (Basic
Research) and Professor at the Pain Management Research Institute, Royal North Shore
Hospital, and Director of Pain and Addiction Research at the Brain and Mind ResearchInstitute, University of Sydney. He is a member of the Board of Directors of the Woolcock
Institute of Medical Research and of the Sydney Cancer Institute and member of the NSW
Tissue Resource Centre Management Committee.
Professor Christie’s research has heralded numerous ‘firsts’. He contributed to the firstcloning of the D2 dopamine receptor (Nature) and achieved the first expression of a
mammalian voltage-gated potassium channel (Science), and first functional demonstrationof its heteropolymerisation (Neuron). His later research uncovered the mechanisms ofplasticity of G-protein receptor coupling in neurons that are transforming thinking regarding
opioid tolerance and ion channel plasticity in chronic pain states. His group discovered thatµ and δ opioid receptors in nerve cells share common G-Protein receptor couplingmechanisms and provided the first evidence of direct coupling of µ-receptors to GIRK
channels and of coupling of the opioid-related receptor ORL1, to GIRK channels in brain as
well as in cannabinoid signaling. He discovered a novel signalling mechanism for G-Protein
Coupled Receptors (GPCR) in synapses in brain (Nature). This provided a basis for seminal
findings on plasticity of signaling in opioid withdrawal published in Neuron (2005) andrecently in Nature Neuroscience (2011).
Mac received a BSc (Hons, First Class) from the School of Biological Sciences, Flinders
University of South Australia in 1978 and Ph.D. From the Department of Pharmacology at
The University of Sydney in 1983. Mac had postdoctoral fellowships at the University of
Melbourne and MIT and was a Senior Research Associate at the Vollum Institute, Oregon
Health Sciences University, where he produced his seminal work on opiate receptors with
Dr. Alan North. He returned to the University of Sydney in 1990 and has been there eversince.
4

Young Investigator Award – Dr. Tuan
Trang
Dr. Tuan Trang is Associate Professor of Physiology andPharmacology at the University of Calgary. Tuan’s research
focuses on discovering the fundamental molecules andprocesses involved in chronic pain and enhancing the utilityof opioid drugs in treating pain conditions. A strong focus of
his research is the role of microglia, the immune cells in thecentral nervous system, and the complex interplay between
microglia and neurons in chronic pain and opioid analgesia.
Understanding the key molecules and processes that underlie chronic pain and that
contribute to the unwanted side effects of opioid use is a major step towards improving
current therapies and identifying novel targets for creating entirely new, more effective
strategies for treating pain.
Dr. Trang graduated with first-class academic distinction from his undergraduate (1999) andgraduate studies (2005) at Queen’s University, one of the top Canadian academic
institutions. He then completed a postdoctoral fellowship in the laboratory of Dr. Michael
Salter, a world leading pain researcher at Toronto’s SickKids Hospital. Tuan’s productivity
as a trainee was exemplary. From his postdoc alone he published 8 peer-reviewed articles,
including first authored articles in the Journal of Neuroscience, Nature Neuroscience, andNature Medicine. This adds to 8 papers published as a graduate student, 7 of which as firstauthor in excellent journals such as Pain and British Journal of Pharmacology. He has
published a total of 29 peer-reviewed articles and has a rising h-index of 18. Tuan’s highlevel of achievement is evidence by prestigious scholarships and awards at each and every
stage of his training.
Since arriving in Calgary, Tuan has been remarkably successful in obtaining competitive
grants, including grants from NSERC and CIHR. Not only did he land a CIHR grant on his
first attempt, but also his grant was the top ranked grant on the Pharmacology and
Toxicology committee. Adding further to his success, Tuan has received numerous
prestigious awards, including a CIHR New Investigator Award, and a Rita Allen Foundation
Award in Pain, which is given to outstanding new investigators in Canada and the UnitedStates for distinguished achievement in pain research.
Tuan has built a vibrant and young lab with an upward trajectory. Most impressively, herecently published a senior authored paper in Nature Medicine – this is an outstandingachievement at any career stage, but it is especially remarkable for a young investigator
only four and half years into a faculty position. He also has published a paper in the Journalof Neuroscience, and currently has manuscripts under review at the Journal of Clinical
Investigation, and Pain. Tuan has therefore established a highly productive independent
research program that is taking the opioid and pain research field in exciting new directions.

5

Schedule for INRC 2018
Tuesday June 12, 2018
2:00-5:00 PM -- Registration
5:30-7:00 PM -- Welcome Reception Indigo Terrace
Wednesday June 13, 2018
8:00 AM -- INRC/CPDD Plenary Lecture
Nora Volkow, NIDA

Indigo BCFG

9:15 AM-12:00 PM -- Symposium 1, INRC/CPDD
Fentanyl
Chairs: Sandra Comer and Cathy Cahill

Indigo BCFG

Speakers:
F. Ivy Carroll, RTI International, USA -Synthesis of fentanyl: A little goes a long way
James Woods, University of Michigan, USA -Behavioral pharmacology of fentanyl: Preclinical models
Alexander Walley, Boston University School of Medicine, USA -Illicitly made fentanyl overdose: Prevention and management in real-world settings
Kim Janda, The Scripps Research Institute, USA -Opioid use disorders: Vaccination as a therapeutic strategy
Sandra Comer, Columbia University, USA -Fentanyl: How did we get here and what do we do about it?
INRC Hot Topics:
Rob Hill, University of Bristol, UK -Fentanyl Depression of Respiration in Mice
Neil Varshneya, Virginia Commonwealth University, USA -Antinociceptive and locomotor effects of Fentanyl-related substances in mice
12:00-1:30 PM -- Lunch
and CPDD posters

Aqua Salon EF
Sapphire ABC/EFG

1:30-4:00 PM -- Symposium 2, INRC/CPDD
Opioid Co-Morbidity
6

Indigo BCFG

Chairs: Rita Valentino and Chris Evans
Speakers:
Tom Kosten, Baylor College of Medicine, USA -Buprenorphine: Opioid dependence and depression
Brigitte Kieffer, McGill University, Canada -Opioid receptors and diverse contributions to mood regulation
Charles Chavkin, University of Washington, USA-Kappa/dynorphin systems and anxiety statesLouisa Degenhardt, National Drug and Alcohol Research Centre, Australia, -Co-occurrence of mental health and opioid use disorders
Rita Valentino, National Institute on Drug Abuse, Discussant -Comorbidity of opioid addiction and other psychiatric disorders
INRC Hot Topics:
Sarah Palumbo -- Clinical Characteristics of Opioid Use Disorder
Gregory Corder -- Dynamic encoding of aversive pain perception within hierarchical neuralensembles
Keith Olsen -- The non-selective opioid diprenorphine produces delta-opioid receptormediated rapid antidepressant-like effects in mice
4:15-4:30 PM -- Data Blitz

Indigo BCFG

4:30-7 PM -- INRC Posters
Sapphire DHLP
Please attend your poster on Wednesday if you have an odd numbered abstract and on
Thursday if you have an even numbered abstract.

Thursday June 14, 2018
9:00-10:00 AM -- INRC/CPDD Plenary Lecture
George Koob, NIAAA

Indigo BCFG

10:00 AM-12:30 PM -- Symposium 3, INRC/CPDD
Opioid Global Recommendations
Chairs: Gabriele Fischer and Fred Nyberg

Indigo BCFG

Speakers:
Gabriele Fischer, Medizinische Universitat Wien, Austria -7

Guidelines for the psychosocially assisted pharmacological treatment of opioid
dependence within EU
Thomas Clausen, The Norwegian Institute of Public Health, Norway -A Nordick Network promoting optimal service provision in addiction treatment in theNordick region
Pauline Voon, BC Centre on Substance Use, Canada -Opioid use disorder clinical guidelines: A Canadian model
Gavin Bart, University of Minnesota, MN -Methadone treatment guidelines development for States in Southeast Asia
Fred Nyberg, Uppsala University, Sweden -Opioid treatment programs – Aspects to deal with in the future perspective

INRC Hot Topics:
Caio Sarmento, University of Kansas, USA –The trends of opioid prescription patterns among patients with Fibromyalgia
Insiya Attarwala, University of Pittsburgh, USA –Computational modeling and analysis of Oxycodone suggest high-risk drug-druginteractions with commonly co-administered benzodiazepines
12:30 PM-2:00 PM
and CPDD posters

Aqua Salon EF
Sapphire ABC/EFG

2:00-4:30 PM -- Symposium 4, INRC/CPDD
New Targets for Drug Abuse Medications
Chairs: Tom Prisanzano and Nurulain Zaveri

Indigo BCFG

Speakers:
Nicholas Cosford, NCI-Designated Cancer Center, USA -Progress towards the development of novel therapeutics for drug dependence
Mei-Chuan (Holden) Ko, Wake Forest University, USA -NOP receptor-targeted agonists as non-addictive analgesics and potential anti-opioid
treatment in primates
Scott Runyon, RTI International, USA -Dissecting the neuropeptide S system and its role in substance abuse
Huburt Yin, University of Colorado, USA -Small molecule TLR modulators targeting neurological diseases
Corey Hopkins, University of Nebraska, USA -8

The synthesis, optimization and biological characterization of a novel series of D4
antagonists as a potential new treatment for drug abuse
INRC Hot Topics:
Aliza Ehrlich, McGill University, Canada –Preserved functional selectivity of MOR ligands in HEK293 cells and MOR-Venus DRG
neurons reveals the physiological reality of biased signaling.
Giordano de Guglielmo, The Scripps Research Institute, USA –Nociceptin/orphanin FQ in the central nucleus of the amygdala selectively reduces
oxycodone self-administration in rats with high addiction-like behaviors
4:45-5:00 PM -- Data Blitz

Indigo BCFG

5:00-7:30 PM -- INRC Posters
Sapphire DHLP
Please attend your poster on Wednesday if you have an odd numbered abstract and on
Thursday if you have an even numbered abstract.

Friday June 15, 2018
8:30-9:30 AM -- INRC Founders Lecture
Macdonald Christie, University of Sidney

Aqua Salon DEF

9:30 AM-12:15 PM -- Symposium 5, INRC
Kappa, Novel Uses
Chair: Lee-Yuan Liu-Chen

Aqua Salon DEF

Speakers:
Jeff Liu, Max Planck Institute of Biochemistry, Germany -In vivo Brain KOR Signaling Elucidated by Phosphoproteomics
Tao Che, University of North Carolina School of Medicine, USA -Structure-guided ligand design of kappa opioid receptor
William Carlezon, Harvard Medical School, USA -Sleep architecture regulated by selective alterations in the activity of dopamine D1
receptor-expressing cells within the nucleus accumbens
Frances Leslie, University of California, Irvine, USA -Age- and sex-dependent interaction of kappa opioid receptors with nicotine and alcohol
Hot Topics:
Hannah Marie Harris, University of Florida, USA -Evaluation of two peripherally-restricted kappa-opiod receptor agaonists for safer
analgesia without CNS liabilities.
Qianhan Ma, University of Bath, UK -9

Sex differences in kappa opioid receptor activated brain networks implicated in the
response to stress and drugs of abuse
12:15 PM – Lunch --- Box lunches
Free Afternoon or
Research Society on Alcoholism, Satellite Symposium. Aqua Salon DEF
Saturday June 16, 2018
8:30-9:30 AM --INRC Plenary Lecture
Howard Fields, UCSF

Aqua Salon DEF

9:30 AM-12:15 PM -- Symposium 6, INRC
Opioid Circuitry
Chair, Elyssa Margolis

Aqua Salon DEF

Speakers:
Mary Heinricher, Oregon Health and Sciences University, USA -Linking pain transmission to pain modulation
Tomohisa Mori, Hoshi University, Japan -Opioid Circuits: Pain and Reward
Elyssa Margolis -- UC San Francisco, CA, USA
Mu opioid receptor activation inhibits hypothalamic inputs to the lateral habenula andrelieves tonic pain
Dean Kirson -- Oregon Health and Science University, USA
Oxycodone self-administration dysregulates central amygdala kappa opioid receptor
systemHot Topics:
Will Birdsong, Oregon Health and Science University, USA -Mu- and delta-opioid modulation of striatal glutamate release
Donita Robinson, University of North Carolina, USA -Local mu-opioid receptor antagonism blunts evoked phasic dopamine release in rat
nucleus accumbens
Kylie McPherson, Oregon Health and Science University, USA -Diverse cell types within the vlPAG exhibit unique adaptations to membrane firingproperties after inflammation
12:15-1:15 PM – Lunch

Aqua Salon foyer

1:15-2:00 PM -- Young Investigator Lecture
10

Aqua Salon DEF

Tuan Trang, University of Calgary
Targeting the immune system to alleviate opioid side effects
2:00-4:00 PM -- Young Investigator Symposium
Chair: Tuan Trang

Aqua Salon DEF

Speakers:
Nicolas Massaly, Washington University in St.Louis, USA -Pain-induced Negative Affect is Mediated via Recruitment of the Nucleus Accumbens
Kappa Opioid System
Michael Morgan, Washington State University, USA -Pain Enhances Spontaneous Opioid Withdrawal in the Rat
Tuomas Lilius, University of Helsinki, Finland -Ketamine in the treatment of opioid tolerance: does the choice of opioid play a role?
Brad Taylor, University of Pittsburgh, USA -Endogenous kappa opioid receptor inhibition of persistent pain
Hot Topic:
Catherine Thomas, University of Calgary, Canada
The effects of opioid sensitization on incentive sensitization and dopamine
neurotransmission
4:00-5:00 PM -- Business Meeting

Aqua Salon DEF

7:00 PM -- Banquet: at Hotel

Aqua Salon DEF

11

Abstracts list
Please attend your poster on Wednesday if you have an odd numbered abstract and on
Thursday if you have an even numbered abstract.

1 - The effect of mu-opioid receptor activation on synaptic transmission within theorbital frontal cortex
Brittany Ambrose
2 - Fentanyl Reexamined
Jessica Anand
3 - Labeling endogenous opioid receptors in living neurons via ligand-directedchemistrySeksiri Arttamangkul
4 - Investigating the anti-addiction and chemotherapy induced neuropathic pain
effects of MP1104, a novel mixed acting kappa, delta opioid receptor agonist inrodents
Diana Atigari
5 - Computational modeling and analysis of Oxycodone metabolism suggest highrisk drug-drug interactions with commonly co-administered benzodiazepines
Insiya Y. Attarwala
6 - Differential G-Protein Expression Alters Pharmacological Properties of Kappa
Opioid Receptor
Miriam E. Barnett
7 - Pharmacological Properties of Buprenorphine and Samidorphan Alone and in
Combination Being Developed as an Adjunctive Treatment for Major Depressive
DisorderJean M. Bidlack
8 - mu- and delta-opioid modulation of striatal glutamate release
William Birdsong
9 - The neuropeptide system, BigLEN-GPR171, interacts with the opioid system torelieve pain and decrease tolerance
Erin Bobeck
10 - Morphine-induced condition place preference activates alpha-CaMKII but does
not require its phosphorylation at Thr286.
Fernando Boix12

11 - The active heroin metabolite 6-acetylmorphine has reinforcing properties andcan sustain self-administration in the rat
Fernando Boix
12 - Cell type specific loss of striatal Gαolf signaling regulates morphine-induced
behaviors in mice
Gloria Brunori
13 - Investigating agonist-dependent changes in the membrane organization of the
μ-opioid receptor using fluorescence correlation spectroscopy
Meritxell Canals
14 - Risk factors attributing to severity of pressure ulcers in adult palliative carepatients with cancer.
Minkyung Cho
15 - Neuroadaptations at nicotinic acetylcholine receptors and behavioral responses
to nicotine in neuropathic rats
Andrea Cippitelli
16 - Dynamic encoding of aversive pain perception within hierarchical neuralensembles
Gregory Corder
17 - Nociceptin/orphanin FQ in the central nucleus of the amygdala selectively
reduces oxycodone self-administration in rats with high addiction-like behaviors
Giordano de Guglielmo
18 - Role of mu-opioid receptors in indirect-pathway striatal neurons in cocaine
rewardLauren K. Dobbs
19 - Synthesis of FGF21, a Hormone Which Modulates the Dopamine Reward
Pathway, Is Increased by GSK3 and HDAC Inhibitors
Louben Dorval
20 - G-protein biased kappa opioid receptor agonist attenuates cocaine selfadministration in male mice
Alexandra Dunn
21 - Identifying kappa opioid receptor-protein interactions following activation by
unbiased and G-protein biased ligands
Amelia Dunn
22 - Preserved functional selectivity of MOR ligands in HEK293 cells and MORVenus DRG neurons reveals the physiological reality of biased signaling
Aliza T. Ehrlich13

23 – Nociception-evoked impulsivity in rats and its reversal with morphine
Nidia Espinoza
24 - Endomorphin analog treatment expedites recovery from chronic inflammatory
pain and protects against latent sensitization
Amy Feehan
25 - Intrinsic G-protein efficacy, not ß-arrestin 2 dependent signaling, confers
reduced opioid-induced respiratory depression
Alexander Gillis
26 - Exploring rapid desensitization of mu-opioid receptors induced by G proteinbiased agonists
Sam Groom
27 - Preclinical Characterization of Mitragyna Speciosa Alkaloids for the Treatmentof Alcohol Use Disorder
Anna Gutridge
28 - Evaluation of two peripherally-restricted kappa-opioid receptor agonists forsafer analgesia without CNS liabilities
Hannah Harris
29 - Specific PKC isoform mediates ongoing pain in a mouse model of migraine
YING HE
30 - Mechanisms of presynaptic suppression of GABA release by opioid receptors
in the hippocampus
X. Jenny He
31 - Fentanyl Depression of Respiration in Mice
Rob Hill
32 - Co-administration of chemokine receptor antagonists with opioids: Potentiationof analgesic effects on incisional pain in rats
Saadet Inan
33 - Multidisciplinary Approach in the Optimization of a Highly Selective Sigma-1
Receptor Antagonist
Sebastiano Intagliata
34 - Evaluation of mixed efficacy opioid ligands in drug discrimination assays
Emily Jutkiewicz
35 - An enzymatic approach reverses nicotine dependence, decreases compulsivelike intake and prevents relapse in rats
Marsida Kallupi

1436 - Pharmacological effects of modifying the N-substituent of hydromorphone
Sophia Kaska
37 - A selective bivalent antagonist for the mu-delta opioid receptor heterodimer
potentiates oxymorphone anti-nociception while sharply reducing morphinewithdrawal
Attila Keresztes
38 - DPP4 inhibitors, Ile-Pro-Ile (IPI) and vildagliptin produce diverse
antihyperalgesic effects following intrathecal administration in inflammatory pain
models in rats
Kornél Király
39 - Evaluation of the analgesic and side-effects of G-protein biased mu-opioid
receptor Salvinorin A analogues kurkinorin and kurkinol
Bronwyn Kivell
40 - Enhanced opioid analgesia and loss of tolerance but exacerbated side effects inmice expressing G-protein biased, phosphorylation-defic
Andrea Kliewer
41 - The complex roles of µ-opioid receptor phosphorylation and ß-arrestins inmediating opioid analgesia with fewer side effects
Andrea Kliewer
42 - Contribution of β-arrestin and ERK signaling in the anxiolytic-like effects of theδ-opioid receptor (δOR) agonist SNC80
Mee Jung Ko
43 - Interruption of continuous morphine administration in mice negatively impacts
brain and behavior
Emilia Lefevre
44 - Ketamine in the treatment of opioid tolerance: does the opioid have a role?
Tuomas O. Lilius
45 - Roles of Protein Kinase C in Kappa Opioid Receptor-Mediated Effects in vivo:
Pharmacological and Phosphoproteomic Approaches
Jeffrey J. Liu
46 - Correlation of KOR phosphorylation in mouse brains with sedation, motor
incoordination and aversion induced by agonists
Lee-Yuan Liu-Chen
47 - Identification of local GPCR protein interaction networks using proteomics andproximity labeling
Braden T. Lobingier15

48 - Sex differences in kappa opioid receptor activated brain networks implicated in
the response to stress and drugs of abuse
Qianhan Ma
49 - Dissecting Agonist-induced Nociceptin/Orphanin FQ Receptor Phosphorylationin vitro and in vivo
Anika Mann
50 - A Stable Heroin Analog Vaccine Formulation that Induces Long Duration
Antibody Titers that Block the Antinociceptive Effects of Heroin andHydromorphone
Gary Matyas
51 - Morphine and HIV-1 Tat-induced calcium flux in hippocampal neurons isdependent on L-type calcium channels and NMDAR signaling
Virginia D. McLane
52 - Diverse cell types within the vlPAG exhibit unique adaptations to membrane
firing properties after inflammation.
Kylie McPherson
53 - Mobility of the mu opioid receptor as an effector independent assay foralterations in signaling state
Marissa Metz
54 - Multi-site phosphorylation is required for sustained interaction with GRKs andarrestins in mediating rapid mu-opioid receptor desensitization.
Elke Miess55 - Chronic Pain Enhances Spontaneous Fentanyl Withdrawal in Rats
Michael M. Morgan
56 - Exposure of early life stress reduces the expression of μ-opioid receptor in the
PAG of adult mice brain and morphine analgesia
Kazuo Nakamoto
57 - Inside the Net: morphine-induced conditioned place preference and effects on
perineuronal nets in the ventral tegmental area
Toni A.E. Nigro
58 - Cannabinoid Type-1 Receptors can Mediate the Opioid-Sparing Effects of Delta9-tetrahydrocannabinol in Nonhuman PrimatesMark R. Nilges
59 - Growth hormone and Insulin-like growth factor-1 promotes the recovery ofneurons after methadone exposure
Erik Nylander16

60 - The non-selective opioid diprenorphine produces delta-opioid receptormediated rapid antidepressant-like effects in mice
Keith M Olson
61 - Inhibition of alpha7 nicotinic receptors in heroin-primed reinstatement ofconditioned place preference
Josephine C Palandri
62 - Clinical Characteristics of Opioid Use Disorder
Sarah A. Palumbo
63 - Development of vaccines to treat heroin and prescription opioid abuse suitablefor clinical trials
Marco Pravetoni
64 - In vivo EEG signatures during chronic fentanyl exposure, spontaneous
withdrawal, and protracted abstinenceCorey Puryear
65 - Differential Activation and Cellular Localization of Protein Kinases in the
Periaqueductal Gray Following Morphine Treatment
Akila Ram
66 - Sex differences in plasticity and stress-related genes in the rat following
oxycodone conditioned place preference
Matthew Randesi
67 - The effects of remifentanil dose on the acquisition and persistence of
responding for drug-paired cues.
Stephen H. Robertson
68 - Local mu-opioid receptor antagonism blunts evoked phasic dopamine releasein rat nucleus accumbens
Donita L. Robinson
69 - The Trends of Opioid Prescription Patterns Among Patients with Fibromyalgia
Caio Sarmento
70 - HA-epitope tag knockin mice demonstrate lack of µ-opioid receptor splicevariants in mouse brain
Stefan Schulz
71 - The association of long-term opium use with overall and cause-specificmortality: Results from a long-term, prospective population-based study
Ramin Shakeri
72 - Predictive factors for quality of life and impact of physical activity in Koreanbreast cancer survivors
Hyun Hwa Shin17

73 - A genetically encoded biosensor reveals location bias of opioid drug action
Miriam Stoeber
74 - The effects of opioid sensitization on incentive sensitization and dopamine
neurotransmission
Catherine Thomas
75 - Modulation of opiate reward and intake, as well as antinociceptive effects by the
OPRM1 A118G SNP.
Annika Thorsell
76 - Interaction between spinal high-mobility group box-1 and glial cells in global
cerebral ischemia-induced mechanical allodynia
Shogo Tokuyama
77 - Identifying a role for gonadotropin-releasing hormone and kisspeptin neurons
in the development of opioid-induced hypogonadism
Karen J. Tonsfeldt
78 - Further evidence for the utility of central μ-opioids for cancer patients.
Kazuhiro Torigoe
79 - Antinociceptive and Locomotor Effects of Fentanyl-Related Substances in Mice
Neil B. Varshneya
80 - AT-312 (1-(1-((cis)-4-isopropyl cyclohexyl) piperidine-4-yl)-1H-indol-2-yl)
methanol) a novel therapeutic compound for opioid relapse in an adolescent rat
model
Dolores Vazquez Sanroman
81 - Physical association between μ opioid and dopamine D1 receptors：
Implications for modulation of locomotor sensitization in dopamine-independent
mannerYu-Jun Wang
82- AMPA Receptor Positive Allosteric Modulators Attenuate Opioid Tolerance and
Dependence
Zaijie Jim Wang
83 - Characterization of JVA 4001, a novel mixed opioid agonist/ kappa opioid
receptor antagonist that attenuates drug and stress-induced reinstatement of
extinguished morphine-conditioned place preference
Lisa Wilson
84 - Comparative Characterization of Operant Oxycodone Self-Administration in
Male BALB/c and C57Bl/6 Mice
Kyle Windisch18

85 - Generation of conditional Oprm1 knockout rat models using Easi-CRISPR withlong ssDNA donors
Jin Xu86 - The roles of alternatively spliced mu opioid receptor intracellular C-termini
encoded by exon 7 on fentanyl actions
Jin Xu87 - The Relation between Serum Uric acid and Spirometric Values and the Risk of
Respiratory Disease
Jiwon Yoon
88 - Increasing G-protein signalling bias for µ-opioid receptor agonists enhances
protein kinase C dependent desensitization.
Arsalan Yousuf
89 - Profile of a Novel bifunctional NOP/MOP receptor agonist for Opioid Use
Disorder and Non-addicting Analgesia: Efficacy and lack of opioid side effects innonhuman primates
Nurulain Zaveri
90 - Structurally different Anabolic Androgenic Steroids induce neurotoxic effects inprimary cortical cell cultures
Sofia Zelleroth
91 - Morphine induced alteration in gut microbiome contributes to analgesic
tolerance by modulating the Gut-Immune-CNS Axis
Li Zhang
92 - Diarylurea-based allosteric modulators of the cannabinoid CB1 receptor:
Structure-activity relationship studies on the pyrrolidinylpyridinyl group
Yanan Zhang

19Symposium Abstracts
Joint CPDD/INRC Symposia
Wednesday June 13
Symposium 1 -- Fentanyl Abuse: From Medicinal Chemistry to ClinicalManagement
Chairs: Sandra Comer and Catherine Cahill
Opioid use disorder (OUD) and its associated morbidity and mortality currently are at epidemic levelsin the U.S. Beginning in the early 1990's, the increased incidence of OUD was mostly attributed toabuse of prescribed opioids, but an increase in heroin use has been observed in recent years.
Despite concentrated efforts to address the problem, the rate of opioid overdose deaths is surging
nationwide. This surge is due in part to the introduction of illicitly made fentanyl and its analogs.
Despite the fact that fentanyl has been used in clinical settings for decades, its abuse liability, toxicity,and responsiveness to treatment medications are not well characterized. In the current symposium,
Dr. Ivy Carroll will describe the ease of synthesizing fentanyl and its analogs relative to that of
obtaining natural opioids and their analogs, as well as the relative in vitro and in vivo properties offentanyl. Dr. James Woods will describe the behavioral pharmacology of fentanyl in preclinicalmodels, as well as the ability of opioid antagonists and partial agonists to
reduce its effects. Dr. Alex Walley will describe the observed course of fentanyl-related overdoses
and the utility of naloxone to reverse them in real-world settings. Dr. Kim Janda will describe afentanyl vaccine as a potential novel therapeutic approach to treating fentanyl abuse. Dr. Sandra
Comer will serve as the Discussant. This symposium should provide a broad overview of thechemistry and pharmacology of fentanyl, as well as the clinical characteristics of fentanyl overdoseand opportunities for treatment

Symposium 2 -- Comorbidity of Opioid Addiction and Other Psychiatric
Disorders
Chairs: Rita Valentino and Christopher Evans
Psychiatric disorders, including post-traumatic stress disorder and depression, often accompany
opioid use disorder (OUD). Chronic pain syndromes are also comorbid with anxiety and depressive
disorders and individuals in pain are more likely to be using opioids, creating a direct pathway to
OUD in comorbid patients. The symposia will merge both clinical and basic research perspectives
on the role of different opioid systems, namely mu, kappa and delta, in both contributing to, andrelieving mood-related disorders. Dr. Kosten will first review clinical data on depression and opiate
dependence, highlighting results from treatment with the kappa antagonist/partial mu agonist
buprenorphine. Dr. Kieffer will then review basic research on the role of different opioid receptors inmood, and present her more recent work on the contribution of mu-receptors in selective cell-types
to aversive behaviors triggered during opiate withdrawal. Dr. Chavkin will discuss how stress-induced
activation of the dynorphin-kappa opioid receptor system can produce dysphoric and anxiety-likestates that increase the rewarding valence of addictive drugs, promote drug seeking behaviors, andtrigger relapse of drug-seeking in abstinent animals. Finally, Dr. Louisa Degenhardt will discuss theclinical implications of co-morbidity in cohorts she has studied in Australia. Questions in the symposia
will address the salience of opioid drugs to individuals with mental disorders and/or pain and theissue of self-medication combined with distress during withdrawal states. Insights into contributionsof different opioid receptors to mediating or alleviating symptoms exhibited in various comorbid
mental states with OUD could guide future treatment strategies.

20

Thursday June 14

Symposium 3 -- International Opioid Guidelines
Chairs: Fred Nyberg and Gabriele Fischer
Over the past decade, the prescribing of opioids has increased dramatically in Western countries
and in particular in North America. In parallel, increases in opioid addiction, overdose, and associateddeaths have been seen. A similar situation occurs in Europe. According to the EMCDDA assessmentof the drug-induced deaths, next to Estonia, Sweden is the country with the most drug-induced
deaths per million population in Europe. From 2006 to 2014 a 40% increase in drug-related deaths
has been recorded in Sweden, mainly due to opioids. This increase has been seen in both sexes.
Moreover, it should be noted that a great part of the increase is seen in opioid deaths combined withbenzodiazepines. Although opioid addiction is effectively treated using a multidisciplinary approach
including agonist opioid treatment and psychosocial intervention there are some difficulties. Misuseand diversion of pain medicines, like fentanyl and buprenorphine comprise a significant problem in
Sweden, but also in other Nordic countries with some of the highest frequencies in Europe. These
inconveniences are associated with poor treatment compliance and increases in risk of blood-borne
infections, crime, and mortality. To address this problem, changes in medicines used in treatment in
Finland and Iceland have already been implemented and considerations are under way in Norway
and Sweden. At this symposium, international well-known speakers will highlight current
perspectives on Guidelines for opioid treatment as well as opioid overdoses and related deaths in
Nordic countries but also in other countries within EU, North America and in Southeast Asia.

Symposium 4 -- New Targets for Drug Abuse Therapy
Chairs: Thomas Prisinzano and Nurulain Zaveri
This symposium will highlight new and emerging therapeutic approaches for the treatment of drug
abuse. Among the topics covered will be receptor modulators, neuropeptide S receptor antagonists,
toll-like receptor modulators, and dopamine receptor antagonists.

21

INRC Symposia
Friday June 15
Symposium 5,
Kappa, Novel Uses
Chair, Lee-Yuan Liu-Chen
In vivo Brain KOR Signaling Elucidated by Phosphoproteomics
Jeffrey J. Liu1, Yi-Ting Chiu3, Luca Zangrandi2, Chongguang Chen3, Sean J. Humphrey1,
Kirti Sharma1, Mariana Spetea4, Christoph Schwarzer2, Lee-Yuan Liu-Chen3, Matthias
Mann 1,5*
1Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry,
Martinsried 82152, Germany 2Department of Pharmacology, Medical University of Innsbruck, 6020
Innsbruck, Austria 3Center for Substance Abuse Research and Department of Pharmacology,
Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USA 4Department ofPharmaceutical Chemistry, Institute of Pharmacy and Center for Molecular Biosciences Innsbruck(CMBI), University of Innsbruck, 6020 Innsbruck, Austria 5Novo Nordisk Foundation Center forProtein Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, 2200,
Denmark
A systems view of G protein-coupled receptor (GPCR) signaling in its native environment is the key
in development of GPCR therapeutics with fewer side effects. Using the kappa-opioid receptor (KOR)
as a model, we employed high-throughput phosphoproteomics to investigate signaling induced bystructurally diverse agonists in five mouse brain regions. We employed the classic KOR agonist
U50,488H as the reference and comparatively studied downstream phosphorylation changes
induced by 6’GNTI and newly reported compounds in one study, and Nalfurafine in another. Fromthese two different phosphoproteomic studies, we observed strong regional specificity of KOR
signaling, due to differences in protein-protein interaction networks, neuronal contacts and thedifferent tissues in neuronal circuitries. Agonists with distinct signaling profiles elicited differential
dynamic phosphorylation of synaptic proteins, linking GPCR signaling to the modulation of brain
functions. The large-scale de-phosphorylation of synaptic proteins in striatum after 5 min agonist
stimulation was partially blocked by Protein Phosphotase 2A (PP2A) inhibitors, underscoring the
involvement of PP2A in KOR mediated synaptic functions. Pathway analysis in both
phosphoproteomic studies revealed enrichment of mTOR signaling by agonists associated withaversion. Consequently, mTOR inhibition during KOR activation abolished aversion, while
preserving beneficial antinociceptive, anti-scratch and anticonvulsant effects. Our results establish
high-throughput phosphoproteomics as a general strategy to investigate GPCR in vivo signaling,enabling prediction and modulation of behavioral outcomes.

22Structure-guided ligand design of kappa opioid receptor

Tao Che1, Daniel Wacker1, Susruta Majumdar2, Magdalena Korczynska3, EIs Pardon4,
Jan Steyaert4, Ivy F. Carroll5, Brian K. Shoichet3, Bryan L. Roth1
1
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill,
NC, USA; 2Molecular Pharmacology Program and Department of Neurology, Memorial
Sloan Kettering Cancer Center, New York, NY, USA; 3Department of Pharmaceutical
Chemistry, University of California, San Francisco, CA, USA; 4VIB Center for Structural
Biology, VIB, Brussels, Belgium; 5Research Triangle Institute, Research Triangle Park, NC,
USA
The kappa-opioid receptor (KOR) mediates the actions of psychotomimetics and proposed
non-addictive analgesics. A complete understanding of KOR activation is necessary to
elucidate the pharmacological properties of this important drug target. While there exist
several orthosteric KOR ligands with diverse chemotypes, the allosteric mechanism of KOR
still remains poorly understood due to the lack of such modulators. Here we present two
distinct crystal structures of the KOR in complex with either a positive or negative allostericnanobody. Comparing these multi-state structures reveals: 1) a mechanism for opioid
receptor activation and ligand selectivity; 2) key residues that explain the pharmacology,function, and biased signaling of the KOR; 3) new avenues for opiate-based therapy anddesign of allosteric modulators.

Sleep architecture regulated by selective alterations in the activity of
dopamine D1 receptor-expressing cells within the nucleus accumbens
Kenneth McCullough, Galen Missig, William A. Carlezon Jr.
Department of Psychiatry, Basic Neuroscience Division, McLean Hospital, Harvard Medical School,
Belmont MA, USA.
Stress plays a critical role in the neurobiology of mood and anxiety disorders. We recently showedthat chronic social defeat stress in mice causes persistent alterations in sleep architecture. Olderwork shows that stress activates the transcription factor CREB within the nucleus accumbens (NAc),
and that non-selective elevations in NAc CREB function produce depressive-like effects whereas
disruptions in CREB function produce antidepressant- and anxiolytic-like effects. Elevated NAc
CREB function is associated with increases in expression of dynorphin (an endogenous agonist at
kappa-opioid receptors) and neuronal excitability. However, the ways in which dopamine D1- andD2-expressing medium spiny neurons (MSNs) within the NAc contribute to the persistent effects ofstress is unclear. Here we examined how selective manipulation of D1 MSNs, which co-express
dynorphin, affects sleep architecture using a wireless EEG system that enables continuous datacollection in freely-moving mice. We used viral vectors to express excitatory or inhibitory DREADDs
in the NAc of mice expressing cre-recombinase in D1 MSNs (GENSAT FK-150). Chronic activationof D1-MSNs (via hM3Dq) produced decreases in paradoxical sleep (PS) without perturbing slow
wave sleep (SWS) or wakefulness (W) times. Conversely, chronic inhibition of D1-MSNs (via hM4Di)
produced increases in PS without affecting SWS or W. These effects persisted following a 5-day
DREADD ligand washout, suggesting that even transient activation or inhibition of this neuronal
population produces long-lasting effects on sleep. Our findings indicate that alterations in thefunction of NAc MSNs resembling those caused by stress are sufficient to produce complex effects
on sleep and wakefulness.
Support: MH063266 (to WAC)

23

Age- and sex-dependent interaction of kappa opioid receptors with
nicotine and alcohol
Frances M. Leslie*, Armando Lárraga, & Sarah J. Cross
Department of Pharmacology, University of California, Irvine CA, USA
Concurrent use of nicotine and alcohol represents a major public health concern, and use of bothsubstances typically begins during adolescence. We have studied the role of the kappa opioid
receptor (KOR) in initial acquisition of intravenous drug self-administration in adolescent and adultrats, and have found both drug- and sex-differences. Blockade of KOR by pretreatment with
norbinaltorphimine (norBNI) enhances initial self-administration of ethanol (EtOH) in adolescentmales but not adults, and in adult females but not adolescents. In contrast, self-administration of
nicotine + EtOH is enhanced by norBNI pretreatment in adult males but not adolescents. KOR
blockade does not significantly affect combined drug intake in females, or nicotine self-administrationin either sex. These data suggest that KOR activation may differentially block drug reinforcement inan age- and sex-dependent manner. To test the hypothesis that KOR activity is both age- and sexdependent, we used an autoradiographic method to assess U69,593-stimulated GTPγS binding inspecific brain regions. KOR activity in drug naïve animals was found to differ across age and sex,
with adolescent males having greater KOR activity in the ventral tegmental area and median raphecompared to adult males, whereas adult females showed higher KOR activity in the basolateral
amygdala than adolescent females. Overall, these findings suggest that KORs undergo sex- andregion-dependent functional maturation during adolescence that may underlie observed differencesin the reinforcing effects of nicotine and alcohol.

Saturday June 16
Symposium 6,
Opioid Circuity
Chair, Elyssa Margolis
Linking pain transmission to pain modulation
Mary Heinricher
Oregon Health and Sciences University, Portland OR, USA
Descending modulatory pathways are a primary site of action of centrally acting analgesic drugs,
including opioids and cannabinoids. The principal output node mediating descending control is the
rostral ventromedial medulla (RVM). A significant focus of research in the last several decades has
been on defining the outputs from the RVM, showing that bidirectional control from this region ismediated by two physiologically defined cell classes, “ON-cells” and “OFF-cells,” that respectively
facilitate and inhibit dorsal horn nociceptive transmission under different conditions. However,ascending transmission pathways are intimately intertwined with this descending control system, andRVM pain-modulating neurons also receive noxious input: ON-cells are activated by noxious
stimulation, giving a “burst” of activity, whilst OFF-cell firing is suppressed, producing a “pause” inany ongoing activity. We showed recently that the lateral parabrachial complex is a major relay ofacute noxious information to the RVM. PB is a nociceptive and autonomic processing and relay
center that, at least in rodent, is the target of the bulk of supraspinal projections from the superficial
dorsal horn. We will discuss recent evidence that PB projects directly to RVM to modulate the activityof identified pain-modulating neurons in the region, including how that circuit switches from acontralateral to ipsilateral dominance in persistent pain states. We will also consider recent evidence
for plasticity in cannabinoid regulation of this circuit.

24

Opioid Circuits: Pain and Reward
Mori T, Watanabe M, Hamada Y, Narita M, Kuzumaki N, Navratilova E, Porreca F, Narita
M
Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical
Sciences, Tokyo, Japan, Department of Pharmacology, Arizona Health Sciences Center,
University of Arizona, Tucson, Arizona
Opioid research has been mostly focused on drug dependence and pain control. Pain is a subjective
experience and could be modified by affective and motivational factors such as attention, distraction,
mood and/or pleasure. Pain itself can negatively alter pleasure or motivation, indicating that there iscross-talk between pain and motivation. The mesolimbic dopaminergic system integrates positive
and negative motivational signals and determines the appropriate behavioral response. Therefore,
we hypothesized that activation of mesolimbic dopaminergic neurons by systemic administration ofmorphine could be altered by pain. Interestingly, we found that morphine-induced reward could be
suppressed under pain conditions accompanied by inhibition of the mesolimbic dopaminergic
neurons, suggesting that pain alters activation of dopaminergic system and suppresses theacquisition of rewarding effect of morphine. On the other hand, optogenetic or chemogenetic
activation of mesolimbic dopamine neurons using, respectively, light sensitive opsins or designer
receptors exclusively activated by designer drugs (DREADDs) reduced hyperalgesia in animalmodels of chronic pain. To further investigate the involvement of mesolimbic dopaminergic neurons
in morphine-induced analgesia, we generated transgenic mice expressing enhanced natronomonas
pharaonis halorhodopsin (eNpHR-EYFP) under the control of the c-fos promoter using a tamoxifen
inducible Cre-loxP system (c-Fos-eNpHR mice), allowing us to label morphine-activated cells with
eNpHR-EYFP. Optical suppression of morphine-activated (c-fos positive) dopamine neurons in theventral tegmental area (VTA), which expressed eNpHR-EYFP, significantly inhibited morphineinduced analgesia. We next generated transgenic mice expressing Gq-coupled human muscarinic
M3 DREADD (hM3Dq), which can be specifically targeted by clozapine N-oxide (CNO) to induce
neuronal activation, or channelrhodopsin-2 (ChR2), under the control of the c-fos promoter using atamoxifen inducible Cre-loxP system. Reactivation of morphine-activated neurons in the VTA
through the DREADD system had no effect on pain thresholds under normal conditions, whereas
optical reactivation of morphine-activated neurons in the VTA alleviated thermal hyperalgesia inanimal models of neuropathic and cancer pain. These findings provide further evidence thatmorphine-activated VTA-dopamine neurons may, at least partially, contribute to morphine-induced
analgesia and modulate pain transmission.

25

Mu opioid receptor activation inhibits hypothalamic inputs to the lateralhabenula and relieves tonic pain
Maggie W. Waung1, Joseph R. Driscoll1, Elyssa B. Margolis1;
1
Department of Neurology, University of California, San Francisco, CA, USA 94143
The lateral habenula (LHb) is a small, phylogenetically conserved, diencephalic nucleus implicatedin depression, drug abuse, and the processing of aversive or disappointing information. Composedof physiologically homogeneous glutamatergic neurons, the LHb receives inputs from and projects
to many components of the limbic and basal ganglia circuits. LHb neurons are activated by acuteand persistent pain, raising the possibility that our previously reported inhibition of LHb neurons andtheir glutamatergic inputs by mu opioid receptor (MOR) activation (Margolis and Fields, 2016)
decreases the aversive LHb output signal. We have found that in rats with neuropathic pain,
microinjecting the MOR agonist DAMGO directly into the LHb reversed the mechanical sensitivity
induced by the injury. Rats also develop a place preference for the context paired with intra-LHb
DAMGO, but only when experiencing chronic pain, not following sham injury, suggesting thepreference is driven by pain relief, rather than positive reinforcement. By optogenetically isolating
different inputs to the LHb, we found that MOR inhibition of glutamate release is strongest in LHb
terminals arising from neurons in the lateral preoptic area (LPOA) of the hypothalamus. Barker etal. (2017) recently reported that optogenetic stimulation of this glutamatergic projection from LPOA
to the LHb is aversive in naïve animals, consistent with the possibility that MOR inhibition of thiscircuit can cause relief by decreasing activity in this aversive input. Together, these observations
implicate the LHb and its hypothalamic inputs as novel targets for inhibiting the aversive componentof tonic pain.

26

Oxycodone self-administration dysregulates central amygdala kappa
opioid receptor system
Dean Kirson
The Scripps Research Institute, La Jolla, CA, USA
Non-medical opioid abuse is a significant global problem, with an estimated 2 million people
in the US having a prescription opioid related abuse disorder and prescription opioid related
overdose deaths drastically increasing over the last two decades. Despite the growing
impact of prescription opioids on public health, few studies have investigated synaptic
changes resulting from abuse of oxycodone, one of the most commonly prescribed
medications. The negative affective state associated with chronic exposure to drugs ofabuse is thought to be mediated in part by dynorphin/kappa opioid receptor (KOR) signaling.
Increased KOR activity is aversive and KOR antagonists are currently being investigated as
potential treatments for stress-induced drug-seeking behaviors and withdrawal-relatedanhedonia. The CeA is involved in the processing of internal and external emotional stimuli
related to anxiety and stress and thus is a critical mediator of escalated drug intake. The
CeA is a primarily GABAergic nucleus that also contains numerous stress-related
neuropeptides, and dynorphin/KOR inhibition of GABAergic transmission becomesdysregulated in the CeA following escalated intake of other drugs of abuse, suggesting thatalteration of CeA KOR-mediated signaling may be involved in the escalation of drug intake.
We investigated the effects of the KOR agonist (-)-U-50488 hydrochloride (U-50488) and
KOR antagonist nor-binaltorphamine dihydrochloride (norBNI) on locally evoked and action
potential independent spontaneous (mIPSC) GABAergic signaling in the central amygdalaof rats that self-administered oxycodone on short and long access schedules. We find that
12 hour oxycodone access on a 5 day a week schedule selectively reverses the effects of
norBNI from increasing mIPSC frequency (increasing GABA release) to decreasing it(decreasing GABA release). This effect has been seen with other drugs of abuse and isusually accompanied by a reversal in the effects of U-50488, which we do not find here.
Rats that had 12 hour access on a 7 day a week schedule, decreasing abstinent time, orwere pretreated with norBNI, to antagonize KOR signaling, had decreased dysregulation
similar to rats that received shorter 1 hour oxycodone access periods. Thus, we find thatchronic oxycodone intake dysregulates CeA KOR system uniquely from other drugs ofabuse and dysphoric signaling from intermittent long withdrawal periods further exacerbates
this dysregulation.

27

Young Investigator Award
Targeting the immune system to alleviate opioid side effects
Young Investigator Lecture -- Tuan Trang,
University of Calgary, Calgary, Alberta, Canada
Microglia are immune cells in the central nervous system that are key targets of opioid action.
Repeated opioid treatment drives microglial activation, which critically underlies a variety ofside effects associated with opioid use. How opioids modulate microglial function is poorly
understood. Here, we identified an essential transcription factor expressed in microglia thatis modulated by opioid treatment. Pharmacological and genetic manipulation of thistranscription factor alters the microglial response to opioid treatment and impacts thedevelopment of opioid tolerance, dependence, and hyperalgesia. Collectively, our results
identify a transcription factor that critically modulates microglial function and response toopioid treatment.

Symposium 7
Young Investgator Symposium
Pain-induced Negative Affect is Mediated via Recruitment of the Nucleus
Accumbens Kappa Opioid System.
N. Massaly
Washington University in St.Louis, St.Louis, MO, USA
Prolonged negative affect significantly impacts quality of life for patients suffering from pain.
These maladaptive emotional states can lead to severe depression, suicide, involuntary
opioid overdose, and related neuropsychiatric comorbidities. The nucleus accumbens (NAc)shell, which integrates both the aversive and rewarding valence of stimuli, exhibits allostaticchanges in the presence of pain. In discrete regions of this structure, activation of the kappa
opioid receptor (KOR), either by dynorphin, its endogenous agonist, or pharmacologicalligands, acutely decreases the reinforcing properties of rewards and induces dysphoria andaversive behaviors. Using a wide range of complementary techniques including
pharmacology, optogenetics, chemogenetics, physiology, biochemistry and in vivo positronemission tomography (PET) imaging, our current findings demonstrate that in vivo
recruitment of NAc shell dynorphin neurons, acting through KOR, is both necessary andsufficient to drive pain-induced negative affect.
Furthermore, we reveal that the presence of inflammatory pain impacts patterns ofconsumption of fentanyl using an intra-venous self-administration paradigm. Thoseparticular patterns, where rats in pain display bursts of consumption interrupted by periodsof “rest”, could lead to the occurrence of respiratory depression and subsequent involuntary
overdose. Taken together, our results provide evidence that adaptations in the kappa opioid
system within the NAc shell represent a functional target for therapeutic intervention in painthat could circumvent affective disorders and may prevent life threatening episodes.

28Pain Enhances Spontaneous Opioid Withdrawal in the Rat
Michael Morgan
Washington State University Vancouver
An increase in the number of people taking opioids for pain has led to an increase in thenumber of people dependent on opioids. The withdrawal symptoms that occur upon
termination of opioid use can cause patients to transition to opioid abuse. Animal studies
provide a unique opportunity to systemically examine the factors (sex, type of opioid, chronicpain) that may contribute to opioid withdrawal. Although spontaneous opioid withdrawal isdifficult to measure in animals, depression of home cage wheel running provides anobjective and continuous measure of both the magnitude and duration of opioid withdrawal.
Our data show that the duration of spontaneous opioid withdrawal is greatly enhanced inrats with hindpaw inflammation. This enhancement is evident in male and female rats
following withdrawal from morphine or fentanyl. These enhanced withdrawal symptoms
suggest that pain patients may be especially susceptible to opioid abuse as a means to
avoid opioid withdrawal.

Ketamine in the treatment of opioid tolerance: does the choice of opioid
play a role?
Tuomas Lilius, MD, PhD
Pharmacology, Clinical Pharmacology, HUSLAB University of Helsinki, Helsinki, Finland
The effects of N-methyl-D-aspartic acid (NMDA) receptor antagonists in enhancing opioid
analgesia and attenuating opioid tolerance have been of great interest both in the laboratory
and the clinic. Preclinical studies have mostly focused on the pharmacodynamic interactions
between ketamine and morphine, whereas only scarce information about co-administrationof ketamine and the widely used oxycodone exists. Pharmacokinetic interactions between
ketamine and opioids have been only little studied. Further, the role of ketamine metabolites
in opioid tolerance remains unclear.
Our recent studies in rats show that co-administration of low-dose ketamine or norketamine
effectively attenuated morphine tolerance, whereas tolerance to oxycodone was lessaffected. This difference may be partly attributable to a pharmacokinetic interaction between
morphine and ketamine leading to vastly increased concentrations of morphine, ketamine,and norketamine in the central nervous system. Co-administration of oxycodone andketamine did not show changes in drug concentrations. Norketamine, the main active
metabolite of ketamine, should be further studied in the treatment of opioid tolerance, as it
attenuated morphine tolerance with minor adverse effects. In contrast, (2S,6S;2R,6R)hydroxynorketamine, a major secondary metabolite with no NMDA receptor antagonist
properties, did not have tolerance-attenuating effects.
In addition to pharmacodynamic differences, pharmacokinetic interactions between
ketamine and different opioids may explain the wide variation in treatment response andadverse effects observed in clinical ketamine-opioid interaction studies. These results
also warrant studies of possible pharmacokinetic ketamine-opioid interactions in humans.

29

Endogenous kappa opioid receptor inhibition of persistent pain
Bradley Taylor
University of Pittsburgh, Department of Anesthesiology
Injury triggers a sustained silent form of neuronal plasticity in spinal signaling (latentsensitization, LS) that engenders vulnerability to chronic pain. Intrathecal pharmacology
studies using LY2456302 or nor-binaltorphimine revealed that LS in mice can be kept inremission by spinal kappa opioid receptors (KOR). This endogenous analgesic action lasts
for over one year, attenuates pronociceptive cAMP receptor signaling, and is sexdependent. Strategies that promote endogenous spinal KOR analgesia may prevent thetransition from acute to chronic pain.

30

POSTER ABSTRACTS
1 - The effect of mu-opioid receptor activation on synaptic transmission within the orbital
frontal cortex
Brittany Ambrose1, Ben Lau2, Stephanie Borgland2
1
Cumming School of Medicine, University of Calgary, 2Department of Physiology andPharmacology, University of Calgary
The orbital frontal cortex (OFC) plays a critical role in estimating the likelihood of specific outcomesto guide future responses. Mu-opioid receptor (MOP) stimulation within selective regions of the OFC
has been shown to enhance feeding and hedonic responses to sucrose. Furthermore, the selective
MOP agonist DAMGO activated Fos in the rostral, but not caudal OFC. MOP receptors are
expressed on parvalbumin positive basket cells in other parts of the prefrontal cortex. In other regionsof the prefrontal cortex, mu-opioid agonists act pre-synaptically suppress GABAergic synaptictransmission. However, their precise cellular mechanism of action across the different sub-regionsof the OFC remains unknown. Due to the functional heterogeneity and differences in inputs, it isimportant to characterize possible differences in mu-opioid actions between these sub-regions.
Thus, we investigated the cellular actions of mu-opioids within the medial and lateral OFC.
Immunohistochemistry revealed the presence of the endogenous opioid, β-endorphin, throughout
both the medial and lateral OFC of adult male C57BL/6 mice. Using patch clamp electrophysiology,
we show that DAMGO produced a significant suppression of evoked GABAergic inhibitory post
synaptic currents (IPSCs) onto layer 2/3 pyramidal neurons within the medial, but not lateral OFC.
This effect was inhibited by pre-application of the MOP antagonist, CTAP. CTAP application after
DAMGO-mediated inhibition of IPSCs did not washout the effect, indicating that DAMGO-induces along-term depression of medial OFC inhibitory synapses. Our results suggest that mu-opioids arepresent in the OFC and MOP inhibition of inhibitory synaptic transmission in regionally selective in
the OFC.
2 - Fentanyl Reexamined
Jessica Anand1, John Traynor2
1
Univeristy of Michigan, 2University of Michigan, Ann Arbor, USA
The number of drug seizures and overdose deaths involving fentanyl has risen steeply since 2016
and as a result emergency core scheduling for fentanyl analogs was put into place in late 2017. In
light of the role fentanyl and its analogs play in the current opioid epidemic we chose to reexamine
fentanyl and characterize its function in vitro and in vivo. Fentanyl is commonly known as a highly
potent and efficacious mu opioid receptor (MOR) agonist with significant abuse potential. However,
we have observed that fentanyl appears to have a delta opioid receptor (DOR) agonist component
in vivo. Morphine and fentanyl both display dose dependent antinociception in the mouse warm water
tail withdrawal assays, as expected for prototypical MOR agonists. Pretreatment with naltrindole (3.2
mg/kp ip), a DOR antagonist, did not shift the dose response curve for morphine; surprisingly
naltrindole at this same dose produced a significant right-ward shift in the dose response curve forfentanyl. We decided to further explore this apparent DOR agonist activity of fentanyl in vitro and invivo. Binding affinities and efficacy were determined at MOR, DOR, the kappa opioid receptor (KOR),
and the nociception receptor (NOP) in vitro. The effects of MOR and DOR selective antagonists were
examined in vitro to determine what effect, if any, receptor cross-talk has on the overall agonist
activity of fentanyl. The contribution of DOR to the in vivo antinociceptive effects of fentanyl was
examined using both antagonists and knock out mice. Funded by DA 033397.

31

3 - Labeling endogenous opioid receptors in living neurons via ligand-directed chemistry
Arttamangkul S1, Placzek A1, Birdsong W1, Farrens D1, Williams JT1
1
Oregon Health Science University
Locating endogenous opioid receptors in brain tissues has been difficult even when antibodies areavailable. Due to large molecular weight and size, the penetration of antibody is often limited andthin slices from fixed tissues are necessary. To visualize the endogenous receptors in thicker living
slices, we took a chemical approach where a small antagonist, naltrexamine is used to direct Alexa
594 dye to reside in proximity and form covalent bond with nucleophilic side chain of an amino acidon extracellular face of the receptor. The key functional group acyl imidazole reported to successfullylabel AMPA and GABA receptors is placed in the linker arm of the compound Nal-AI-594 and thus
allows naltrexamine to be free from the binding pocket while leaving the dye on the receptor. With
this strategy, the receptors can be visualized and electrophysiologically recorded. In this study, NalAI-594 was synthesized and used to label endogenous mu opioid receptors of mouse and rat locus
coeruleus neurons. It also identified mu opioid receptors in other areas of the brain such as midbrain,habenula and parabrachial nucleus. Detailed characterization for specificity and labeling efficiency
will be presented.
4 - Investigating the anti-addiction and chemotherapy induced neuropathic pain effects of
MP1104, a novel mixed acting kappa, delta opioid receptor agonist in rodents
Diana Atigari1, Rajendra Uprety2, Susruta Majumdar3, Bronwyn Kivell1
1
School of Biological Sciences, Centre for Biodiscovery, Victoria University of Wellington, New
Zealand, 2Department of Neurology, Memorial Sloan Kettering Cancer Centre, New York, USA,
3
Center of Clinical Pharmacology, St. Louis College of Pharmacy and Washington University
School of Medicine, St. Louis, MO, USA
Introduction: Development of effective non-addictive pain therapies are urgently needed. Kappaopioid receptor (KOPr) activation reduces drug-seeking behaviour in preclinical models of drug use
and also attenuates pain and inflammation. In this study, we utilised a chemotherapy-induced
neuropathic pain (CINP) model to evaluate the effects of both acute and chronic treatment of
MP1104, a mixed opioid agonist, on mechanical and cold allodynia. We also investigated the anticocaine effects of MP1104 in rats trained to self-administer cocaine.
Methods: To induce neuropathic pain, male C57BL/6J mice (n=60) were administered paclitaxel, achemotherapy drug to give a cumulative dose of 16 mg/kg. Mechanical and cold allodynia measured
using von Frey filaments and by placing a drop of acetone to hind paw and recording reaction time
duration. For acute treatment, on day 15 mice were administered cumulative doses of MP1104 (ormorphine), mechanical & cold allodynia evaluated. Chronic daily administration of MP1104 or
morphine from day 16 to 38. Male Sprague-Dawley rats (n=20) were used to investigate the anticocaine effects of MP1104 in cocaine self-administration models.
Results: MP1104 dose-dependently reduced mechanical (p<0.0001) and cold allodynia (p<0.0001)
to pre-paclitaxel levels. Non-linear regression analysis revealed MP1104 was more potent thanmorphine at reducing mechanical allodynia (EC50 = 0.45 mg/kg) and cold allodynia (EC50 =0.47
mg/kg).In rats trained to self-administer cocaine, MP1104 reduced cocaine prime reinstatement ofdrug-seeking behaviour at doses of 0.3 & 1mg/kg (p<0.001), caused significant downward shift(p<0.0001) in cocaine self-administration dose-response curve (two-way ANOVA with repeatedmeasures).
Conclusion: MP1104 has potent effects in reducing both mechanical and cold allodynia in CINP
following both acute and chronic treatment in mice. Moreover, MP1104 has no rewarding properties,
thus making it suitable for developing better pain medications.

32

5 - Computational modeling and analysis of Oxycodone metabolism suggest high-risk drugdrug interactions with commonly co-administered benzodiazepines
Insiya Y. Attarwala1, Zhaojia Zhang1,2, Xiaoqing Tan1,2, Lirong Wang1,2, Xiang-Qun Xie1,2,3
1
NIH National Center of Excellence for Computational Drug Abuse Research, 2Department ofPharmaceutical Sciences and Computational Chemical Genomics Screening Center, School of
Pharmacy, 3Department of Computational Biology and Structural Biology, School of Medicine,
University of Pittsburgh
In the past decade, prescription opioids have risen to be the leading cause of drug overdoses in the
United States. Oxycodone, indicated for the treatment of moderate to severe pain, is a commonlyused opioid with an unfortunately high abuse potential and risk of death due to overdose. Adding tothis crisis are drug-drug interactions (DDIs) that occur when patients are simultaneously taking
additional medications. Analysis of FDA Adverse Event Reporting Systems for oxycodone showsthat most fatal outcomes involve concomitant use of other drugs, of which nearly half result from coadministration with benzodiazepines, commonly prescribed sedative-anxiolytics. This study
examined the interaction between oxycodone and Diazepam, as a result of their shared Phase I
metabolism by the CYP450 3A4 enzyme. The benzodiazepine diazepam is also a competitive
inhibitor of CYP3A4. We hypothesized that concomitant administration of diazepam may affect the
metabolism of oxycodone and may lead to increased or prolonged adverse effects of oxycodone.
Using pharmacokinetic (PK) data of oxycodone at different clinical doses, we obtained a PK
simulation of oxycodone using a one-compartment model with first-order dosing and linear
elimination. We then simulated the oxycodone concentration-time profile in the presence ofdiazepam. Our simulation showed that in the presence of diazepam, levels of oxycodone and active
metabolite Oxymorphone (from CYP2D6) accumulate. Our quantitative simulations with clinicaldoses show potential DDI and overdose risk thresholds for concomitant oxycodone diazepam use.
Such information raises awareness for healthcare professionals prescribing opioids with othermedications in the current climate of the opioid crisis.
6 - Differential G-Protein Expression Alters Pharmacological Properties of Kappa Opioid
Receptor
Miriam E. Barnett1, Brian I. Knapp1, Jean M. Bidlack1
1
Department of Pharmacology and Physiology, University of Rochester
In the canonical model, the kappa opioid receptor (KOR) signals through the Gαi and Gαo proteins.
However, the involvement of other G proteins to downstream signaling events has not been
rigorously characterized. The milieu of G proteins within a particular cell type may greatly influence
the pharmacological properties of opioid ligands. Characterization of G-protein alpha subunit
expression at both the mRNA and protein level in HEK 293 and CHO cells revealed that each line
had distinct expression profiles. Notably, HEK 293 cells expressed the pertussis-insensitive Gαz anddid not express Gαo, while CHO cells expressed Gαo and not Gαz. Changes in cyclic AMP levels
were measured in HEK 293 and CHO cells stably expressing the human KOR after administrationof classical kappa-selective agonists and antagonists. Like U50488, a Kappa-selective agonist,
antagonists inhibited cyclic AMP levels in HEK 293 cells, but not in the CHO cells. G-protein specifictoxins and overexpression systems were used to selectively measure signaling through specific G
proteins in each cell line. The individual G proteins had different effects on the pharmacologicalproperties of opioid ligands resulting in functionally biased KOR signaling. (Supported by NIH
T32GM068411 and the Margo Cleveland Fund)33

7 - Pharmacological Properties of Buprenorphine and Samidorphan Alone and in
Combination Being Developed as an Adjunctive Treatment for Major Depressive Disorder
Jean M. Bidlack1, Brian I. Knapp1, Daniel R. Deaver2, Margarita Plotnikava2, Derrick Arnelle2, Angela
M. Quinn2, May Fern Toh2, Sokhom S. Pin2, Mark N. Namchuk2
1
University of Rochester, 2Alkermes
A combination of buprenorphine and samidorphan at a 1:1 dose ratio (mg/mg), is being investigatedas an adjunctive treatment for major depressive disorder (BUP/SAM, ALKS 5461). This study
characterized the receptor and [35S]GTPγS binding properties of buprenorphine and samidorphan
alone and in combination. [3H]Buprenorphine had a slow dissociation from the μ opioid receptor(MOR). In the [35S]GTPγS binding assay, buprenorphine was a partial agonist at all three opioid
receptors. Samidorphan was an antagonist at the MOR and a partial agonist at the κ and δ opioid
receptors (KOR and DOR, respectively). A 1:5 molar ratio of buprenorphine:samidorphan, similar to
steady-state plasma levels in humans, showed that samidorphan attenuated buprenorphine’s
efficacy without affecting its potency at the MOR. At the KOR, the same ratio did not change
buprenorphine’s efficacy. Similar results were obtained when MOR and KOR signaling, mediated by
Gαi proteins, was evaluated in a BRET assay. When the MOR signaled through Gαo and Gαz
proteins, buprenorphine had Emax values between 87 and 92% and samidorphan had Emax values
between 27 and 33%. At the KOR, buprenorphine and samidorphan were partial agonists when the
KOR signaled through Gαi and Gαo and full agonists signaling through Gαz proteins. Buprenorphine
produced some β-arrestin recruitment mediated by the MOR, while a 1:3 molar ratio ofbuprenorphine:samidorphan produced no β-arrestin recruitment. The collective data show thatsamidorphan attenuates the in vitro efficacy of buprenorphine at the MOR but, did not alterbuprenorphine’s activity at the KOR.
8 - Mu- and Delta-opioid Modulation of Striatal Glutamate Release
William Birdsong1, Bart Jongbloets1, Kim Engeln1, Dong Wang2, Gregory Scherrer2, Tiany Mao1
1
Oregon Health & Science University, Portland, USA, 2Stanford University, Stanford, USA
Affective/ motivational pain processing involves the medial thalamus, anterior cingulate cortex (ACC)and striatum. Opioid analgesics are known to modulate this circuitry and relieve the negative
emotional effects of pain. However, the identity of the opioid receptors, their cellular locations, andtheir physiological effects on this affective circuitry remain uncharacterized. In the present study,
synaptic connections between the three brain regions; thalamus, striatum, and ACC, werecharacterized based on the effect of opioid agonists to modulate glutamatergic transmission 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 andcortical afferents. It was found that opioid receptors differentially modulated thalamic and corticalglutamate release in striatum. Additionally, µ and ∂-opioid receptors were found to play distinct roles
in modulating thalamo-cortical and intracortical synaptic transmission in the ACC. Imaging revealed
that ∂-opioid receptors are expressed on nearly all parvalbumin positive interneurons in the ACC.
These data identify opioid receptor subtype-dependent inhibition of specific pathways within thethalmao-cortico-stiatal circuitry, whereby µ-opioids inhibited striatal glutamate release while ∂-opioid
activation facilitated release, providing insights into how opioid analgesics may modulate affective
pain at the synaptic and circuit level.

34

9 - The neuropeptide system, BigLEN-GPR171, interacts with the opioid system to relieve
pain and decrease tolerance
Leela Afrose1, Ivone Gomes2, Lakshmi Devi2, Erin Bobeck1
1
Utah State University, 2Icahn School of Medicine at Mount Sinai
Recently BigLEN was identified (a ProSAAS derived peptide) as the endogenous peptide for theorphan receptor GPR171. BigLEN and GPR171 are both highly expressed in the descending pain
modulatory pathways, including the periaqueductal gray (PAG). In addition, we find that GPR171 is
co-localized with mu opioid receptors (MOPr) in the ventrolateral PAG in a portion of vGLut2 andGAD67 positive neurons. Given the expression pattern of GPR171, we hypothesized that GPR171
contributes to antinociception and tolerance. To test this, mice were injected with a GPR171
antagonist (MS21570; 5 mg/kg, i.p.) followed by the tail flick test which produced an increase inantinociception compared to controls. However, this compound is not as efficacious as morphineand pretreatment with MS21570 10 min prior to morphine (10 mg/kg, s.c.) did not alter morphineinduced antinociception. To assess the role of GPR171 in morphine tolerance, mice were injectedwith morphine twice daily for 5 days. A subset of mice were pretreated with MS21570 10 min prior
to each morphine injection. Those mice pretreated with MS21570 showed a decrease in thedevelopment of morphine tolerance. To understand how these systems may be working together,
GPR171 was knocked down in Neuro2A cells which led to a decrease in MOPr-mediated GTPγSsignaling by DAMGO, but had no effect on GTPγS signaling by the delta opioid receptor agonist,
deltorphin. Taken together, this data shows that GPR171 and MOPr interact with one another and
GPR171 antagonist may be a novel target to treat pain or opioid tolerance.
10 - Morphine-induced condition place preference activates alpha-CaMKII but does not
require its phosphorylation at Thr286.
Jannike M. Andersen1, Siri H. Opdal2, Christian P. Müller3, Fernando Boix1
1
Section for Drug Abuse Research, Department of Forensic Sciences, Oslo University Hospital, Oslo,
Norway, 2Section for Pediatric Forensic Medicine, Department of Forensic Sciences, Oslo University
Hospital, Oslo, Norway, 3Department of Psychiatry and Psychotherapy, University Hospital,
Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany
Background: Conditioned Place Preference (CPP) is an association learning procedure mediatedby synaptic changes in striatal neurons. Activation of Calcium/calmodulin-dependent protein kinase
II (CaMKII), by phosphorylation at Thr286, has been shown to be important for synaptic plasticity
processes to occur. In this study, we examined the contribution of alpha-CaMKII (αCaMKII) and its
autophosphorylation at Thr286 (p-αCaMKII) on CPP induced by morphine. Methods: The effects of
10 or 30 µmol/kg morphine on αCaMKII, p-αCaMKII, and β-actin levels in dorsal and ventral striatum
and hippocampus were examined in mice after acute administration, morphine-induced CPP, and asubchronic treatment mirroring the drug administrations during CPP. The acquisition of CPP was
also tested in autophosphorylation deficient αCaMKIIT286A mice (Giese, Science 279, 1998) using
the same morphine doses. Results: After morphine-induced CPP, the levels of αCaMKII and βactin, but not p-αCaMKII, were significantly increased in the three brain areas. These protein levels
were also increased after the acute morphine administration, but only in striatum. No changes were
found after the subchronic morphine treatment. Besides, no differences were observed between
αCaMKIIT286A and wild type mice in the expression of CPP induced by morphine. Conclusions:
Morphine-induced CPP is accompanied by increases in the levels of β-actin and overall αCaMKII instriatum, but does not require phosphorylation of αCaMKII at Thr286. This is in contrast to cocaineinduced CPP (Easton, Transl Psychiatry 4, 2014). These changes were also observed in
hippocampus after morphine-induced CPP, but not after acute morphine, indicating that this area isrecruited during CPP acquisition.

35

11 - The active heroin metabolite 6-acetylmorphine has reinforcing properties and cansustain self-administration in the rat
Riccardo Avvisati1, Jannike M. Andersen2, Vigdis Vindenes2, Inger Lise Bogen2, Jørg Mørland3, Aldo
Badiani1, Fernando Boix2
1
Sussex Addiction Research and Intervention Centre (SARIC), University of Sussex, Brighton, UK,2
Section for Drug Abuse Research, Department of Forensic Sciences, Oslo University Hospital,
Oslo, Norway, 3Norwegian Institute of Public Health, Oslo, Norway
Introduction: 6-Acetylmorphine (6-AM) is the main active metabolite found immediately after heroin
administration. However, 6-AM’s reinforcing properties, which could be central for heroin’s high drug
abuse potential, have been sparsely examined. The present study examines the acquisition,
extinction, relapse, and retraining of self-administration of 6-AM in rats compared with equimolar
doses of heroin. Methods: Sprague-Dawley rats were submitted to an intravenous selfadministration procedure with 6-AM or heroin encompassing: 1. acquisition (five FR1 sessions andtwo FR2 sessions) using 0.135 µmol/kg (44.3 μg/kg 6-AM, 50 μg/kg heroin) per infusion; 2. extinction(five sessions); 3. relapse (one session) using a single 0.068 µmol/kg (22.15 μg/kg 6-AM, 25 μg/kg
heroin) priming infusion; and 4. retraining (one session) using 0.068 or 0.135 µmol/kg. Results:
Whereas the rate of self-administration was similar for 6-AM and heroin during FR1, it wassignificantly higher for 6-AM during FR2. No differences were observed during extinction. Primingwith 0.068 µmol/kg reinstated lever presses for both drugs during the first hour of the relapse session.
Retraining with the dose used in acquisition (0.135 µmol/kg) restored lever presses for both drugs at
a level similar to the last acquisition session. Retraining with half the dose (0.068 µmol/kg)
significantly increased self-administration in animals receiving 6-AM, but not in animals receiving
heroin. Conclusions: Intravenous 6-AM shows similar reinforcing properties as heroin, supporting
its proposed involvement in the rewarding and abuse properties of its parent drug. However, someobserved disparities also indicate the existence of some differences in their rewarding qualities after
systemic administration.
12 - Cell type specific loss of striatal Gαolf signaling regulates morphine-induced behaviors
in mice
Gloria Brunori1, Anna Stauffer1, Kathleen Sheridan2, Janet Robishaw1
1
Department of Biomedical Science, College of Medicine, Florida Atlantic University, Boca Raton,
FL, USA, 2Weis Center for Research, Geisinger Clinic, Danville, PA, USA
G protein signaling pathways are essential for the translation of neurotransmitter activity into
behavior. Within the striatum, the Gαolf isoform mediates stimulation of adenylyl cyclase (AC)upon activation of dopamine D1 receptors (D1R) and adenosine A2AR. The Gγ7 protein is alsoenriched in the striatum, where it orders the assembly and sets the functional level of the striatalspecific Gαolfβ2γ7 heterotrimer. Activation of opioid receptors and inhibition of AC in the striatum are
key mechanisms for psychomotor and rewarding effects of morphine. Preliminary data obtainedin Gng7 gene (encoding Gγ7) global knock-out mice indicated that Golf signaling is required tomediate the locomotor-enhancing effects of morphine. To dissect the role of Golf signaling pathway
in the main subpopulations of striatal medium spiny neurons, we generated new mouse models inwhich Gng7 gene is deleted specifically in D1R- or D2R (also expressing A2AR)-expressing neurons.
Analysis of striatal membranes showed reduced levels of Gαolf protein and decreased levels of
forskolin-stimulated AC activity in both strains. AC activity was also reduced following stimulation of
D1R and A2AR in D1R- and D2R-Gng7 deleted mice, respectively. Notably, mice lacking the Gγ7
subunit in D1R-positive neurons showed reduced sensitivity to the acute locomotor-enhancing effects
of morphine, although they developed normal behavioral sensitization and conditioned place
preference in response to chronic morphine. In contrast, loss of Gγ7 subunit in D2R-positive neurons
produced increased locomotor response to acute morphine. Collectively, these findings provide
novel evidence regarding specific contributions of the striatal-specific Golf signaling to opioidmediated behaviors.

36

13 - Investigating agonist-dependent changes in the membrane organization of the μ-opioid
receptor using fluorescence correlation spectroscopy
Arisbel Batista-Gondin1,2, Michelle L Halls2, Stephen J Briddon1, Meritxell Canals1,2
1
Centre of Membrane Proteins and Receptors (COMPARE), and School of Life Sciences,
University of Nottingham, UK, 22 Drug Discovery Biology, Monash Institute of Pharmaceutical
Sciences, Monash University, Australia
Differential regulation of the MOR by morphine and other agonists has been linked to opioid tolerance
and dependence. At a cellular level, MOR regulation occurs via multiple mechanisms including
receptor desensitization, internalization, recycling and plasma membrane redistribution. Here wehave used advanced imaging techniques (Fluorescence Correlation Spectroscopy, FCS;
Fluorescence Recovery After Photobleaching, FRAP) to monitor agonist-dependent changes in
MOR organization within living cells.
SNAP-hMOR was stably expressed in HEK293 cells in the basal state and upon stimulation with theantagonist naloxone, the low internalizing agonist morphine and the high internalizing agonist
DAMGO. FCS measurements were taken on the upper cell membrane and diffusion co-efficients(D), receptor number (N) and molecular brightness (e) were determined using autocorrelation andphoton counting histogram analysis as previously descirbed1. Immobile MOR fraction and macro-D
were determined using FRAP on the lower cell membrane.
DAMGO, but not morphine, caused a time- and concentration-dependent MOR clustering on theupper cell membrane, as indicated by a decreased D, reduced receptor number and an increased
proportion of very bright receptor species. These effects were prevented by pre-incubation withnaloxone. Similarly addition of DAMGO, but not morphine, caused a reduction of the proportion ofmobile receptors. Interestingly, these changes are independent of MOR internalization, but
dependent on GRK2/3-mediated phosphorylation. The role of Gβγ and PKC was also investigated.
Overall, these results demonstrate agonist-specific changes in the micro-organization of MOR at theplasma membrane, probably occurring prior to internalization. They provide insights into anothermolecular event differentially regulated by distinct opioids.
14 - Risk factors attributing to severity of pressure ulcers in adult palliative care patients withcancer.
Minkyung Cho1, Jiwon Yoon1, Hwanseok Choi1
1
Seoul St. Mary's Hospital, Catholic Medical Center, Korea
Background Pressure ulcers are one of the most common medical problems in palliative carepatients with cancers. A number of studies about predictors of formation of pressure ulcers havebeen conducted but there have been few studies about risk factors affecting severity of pressure
ulcers. We analyzed factors associated with the severity of pressure ulcers in patients with cancers
hospitalized in a palliative care ward setting. Methods This study was retrospective analysis of 186
patients with pressure ulcers with a mean age (±standard deviation) of 70.22±13.48 years (range:
22-96 years, median 73.0 years; 50.5% women), who were admitted in Seoul St. Mary’s hospital
palliative care ward in 2015. Results Patients were hospitalized for mean 20.69±17.10 days (1-91
days, median 21.5days). 159 patients (85.5%) had low grade (stage 1-2) and 27 patients (14.5%)
had high grade (stage 3-6) pressure ulcers. Chi-square test was used to examine the differences ofgrade according to categorical variables and independent sample t-test was used for continuous
variables; PPS (Palliative Performance Scale) and Braden scale appeared to have significantly
associated to grade of pressure ulcers. Two factors assessed at admission appear to predict theseverity of pressure ulcers in the multivariate logistic regression model; PPS (odds ratio [OR]=0.600(95% CI=0.390-0.925,) P=0.021), Braden scale (OR=0.783 (95% CI=0.655-0.935), P=0.007).
Conclusion PPS and Braden scale may contribute to severity of pressure ulcers in patients withcancers in palliative care ward.

37

15 - Neuroadaptations at nicotinic acetylcholine receptors and behavioral responses tonicotine in neuropathic rats
Andrea Cippitelli1,2, Gloria Brunori1,2, Jennifer Schoch1,2, Akihiko Ozawa1,2, Lawrence Toll1,21
Biomedical Science Department, Charles E. Schmidt College of Medicine, Florida Atlantic
University, Boca Raton, Florida, USA, 2Torrey Pines Institute for Molecular Studies, Department of
Neuropharmacology, Port St Lucie, Florida, USA
Tobacco addiction and chronic pain represent highly prevalent and comorbid conditions thatengender considerable burdens upon individuals and systems. This complex relationship is poorly
understood at both molecular and behavioral levels. Here we describe experiments aimed atunderstanding whether a chronic pain state induces neuroadaptations at nicotinic acetylcholine
receptors (nAChRs) that lead to increased vulnerability to nicotine addiction or to the developmentof coping strategies to relieve pain symptoms. We found dramatic downregulation in the mRNA
expression levels of α3, α4, α5, α7, β2 and β4 nAChR subunits in dorsal root ganglia and a timedependent downregulation of discrete subunits in the cingulate cortex and the amygdala of rats
subjected to ligation of the L5 spinal nerve, a procedure that models neuropathic pain, as compared
to sham-operated rats. In contrast, expression of the nAChR subunits examined in brain regions
important for rewarding processes was comparable in these rat groups. Spinal nerve ligated (SNL)and sham-operated rats showed no changes in patterns of acquisition, motivation for nicotine taking,extinction and cue-induced relapse into nicotine seeking, although the ligated group failed toreinstate lever pressing when relapse was induced by nicotine priming. Nicotine-experienced SNL
rats did not show anxiogenic-like activity following administration of a nicotine dose that produced
anxiety in control animals. SNL and sham rats were equally sensitive to nicotine-induced
antinociception; however, nicotine produced a potent and long-lasting antiallodynic effect in SNL
rats. Altogether, these results contribute to explain epidemiological studies demonstrating increased
cigarette consumption in chronic pain individuals.
16 - Dynamic encoding of aversive pain perception within hierarchical neural ensembles
Gregory Corder1, Biafra Ahanonu1, Benjamin Grewe1, Mark Schnitzer1, Gregory Scherrer2
1
Stanford University, Palo Alto, USA, 2Stanford University, Stanford, USA
An unpleasant percept dominates the affective dimension of pain, which provides a motivational
drive to initiate protective behaviors that limit exposure to noxious stimuli. While detailed mechanisms
underlying the sensory detection and spinal processing of nociception have been uncovered, it
remains unclear how brain circuits transform this emotionally inert information into an affective pain
perception. Injury-induced plasticity within affective circuits, such as the basolateral amygdala (BLA),
may lead to a miscoding of sensory information concomitant with the emergence of chronic pain. To
identify the principles of nociceptive information coding in the BLA, we used a head-mounted
miniature microscope to monitor the calcium activity dynamics of individual BLA neurons in freely
behaving mice presented with a diverse set of painful and innocuous stimuli. We tracked thelongitudinal dynamics of BLA neural coding across 9,777 cells before and after the development ofneuropathic allodynia from a peripheral nerve injury. We found that prior to nerve injury,
multidimensional and population vector analysis of Ca2+ transients revealed that a unique nociceptive
neural ensemble in the BLA, distinct from positive valence ensembles, encodes a diverse array of
painful stimuli. Silencing of this ensemble alleviated pain affective-motivational behaviors without
altering the detection of noxious stimuli, withdrawal reflexes, anxiety, or reward. After theestablishment of neuropathic pain, the ensemble representations of prior innocuous and noxious
stimuli became more similar. Collectively, our results identify a neural representation of nociception
in the amygdala that is necessary for the instantiation of the negative affective qualities of acute andchronic pain.

38

17 - Nociceptin/orphanin FQ in the central nucleus of the amygdala selectively reduces
oxycodone self-administration in rats with high addiction-like behaviors
de Guglielmo G1, Kallupi M1, Maturin L1, Schweitzer P1, George O1
1
Department of Neuroscience, The Scripps Research Institute, La Jolla, CA, USA
Over the past 15 years, oxycodone consumption has increased nearly 500%, and opioid-related
overdose deaths have quadrupled. Recent work found that the dysregulation of thenociceptin/orphanin FQ (N/OFQ) receptor, may be important after chronic exposure to opioids. We
investigated the role of N/OFQ on oxycodone addiction in heterogeneous stock (HS) rats, a uniqueoutbred strain with high genetic variability that mimics genetic diversity in humans. We usedoxycodone self-administration (150 μg/kg/inj, 12 h/session) combined with progressive-ratio
responding and withdrawal-induced hyperalgesia. We measured robust individual differences andestablished an Addiction Index as a comprehensive evaluation of addiction-like behaviors for eachindividual, identifying rats with high (HA) and low (LA) addiction-like behavior. We found that HA rats,
compared with LA rats, exhibited a significant increase in spontaneous g-aminobutyric acid (GABA)
ergic transmission, reflected by isolated spontaneous inhibitory postsynaptic currents (sIPSCs), in
the central nucleus of the amygdala (CeA). Superfusion of the CeA slice with N/OFQ (500 nM)
decreased spontaneous GABA sIPSCs more in HA rats, suggesting that low levels of N/OFQ in the
CeA may be responsible for high GABA release and high oxycodone intake. Finally, intra-CeA
N/OFQ infusions (1 μg/site) significantly reduced oxycodone intake in HA rats, but not in LA ratssuggesting that the downregulation of N/OFQ levels in the CeA may be responsible for thehyperGABAergic tone in the CeA of HA rats. The development and repurposing of small moleculesthat target the N/OFQ system may have therapeutic efficacy in the treatment of opioid use disorder,
particularly in heavy users.
18 - Role of mu-opioid receptors in indirect-pathway striatal neurons in cocaine reward
Lauren K. Dobbs1, Carolyn Bowering1, Aya Matsui1, Veronica A. Al1
1
National Institute on Alcohol Abuse and Alcoholism, NIH
Cocaine-induced enhancement of dopamine levels within the striatum is important for cocaine’s
rewarding and stimulating effects. Interestingly, though, accumulating evidence from constitutive
knockout models and pharmacological manipulations indicates that striatal opioid peptides andreceptors also contribute to cocaine seeking and taking. Within the striatum, the mu-opioid receptor(MOR) is expressed in both the indirect- and direct-pathway medium spiny neurons (iMSNs anddMSNs, respectively), and thus the specific population of MORs contributing to cocaine reward
remains unknown. Here, we generated mice with a selective deletion of MORs from iMSNs (iMSNMORKO) and tested cocaine-mediated reward and locomotion. iMSN-MORKO mice had a 48%
reduction of MOR mRNA in the striatum, with no change in expression in the periaqueductal gray.
iMSN-MORKO mice were slower to acquire a cocaine conditioned place preference, suggesting
MORs expressed in iMSNs facilitate conditioned cocaine reward. We next used slice
electrophysiology to probe the functional role of MORs in iMSNs on striatal synaptic transmission.
Application of met-enkephalin or the MOR-agonist DAMGO inhibited spontaneously-evoked GABAAmediated inhibitory post-synaptic currents (IPSCs) when measured in dMSNs, as well as IPSCs
evoked by optogenetic stimulation of iMSN axon collaterals. These findings suggest that activationof MORs in iMSNs has an effect similar to dopamine D2 receptor activation in iMSNs on restraining
collateral GABA transmission. Preliminary data suggest iMSN-MORKO mice are basally hyperactive
and have an enhanced cocaine locomotor response. Future experiments will follow-up on thisfinding and also determine whether MORs in iMSNs are functionally lost in iMSN-MORKO mice usingslice electrophysiology.

39

19 - Synthesis of FGF21, a Hormone Which Modulates the Dopamine Reward Pathway, Is
Increased by GSK3 and HDAC Inhibitors
Louben Dorval1, Brian I. Knapp2, Jean M. Bidlack3
1
Department of Pharmacology and Physiology, University of Rochester School of Medicine andDentistry, 2University of Rochester, 3University of Rochester
Fibroblast growth factor 21 (FGF21), a regulator of glucose and lipid metabolism, acts as a global
starvation signal to modulate energy expenditure and suppress growth. Expressed in liver, brown
adipose tissue, glia, and neurons, FGF21 crosses the blood-brain barrier and acts centrally to induce
the sympathetic nervous system. Previous studies have shown that FGF21 decreases alcohol andsweet preference in mice. Furthermore, the effects of FGF21 on taste preference correlated with adecrease in dopamine levels in the nucleus accumbens, a primary brain region in the reward
pathway. Therefore, FGF21 may also modulate the effects of morphine and other opioids in the
reward pathway. This current study explored the effects of histone deacetylase (HDAC) andglycogen synthase kinase3 (GSK-3) on FGF21 mRNA expression levels. C6 glioma cells weretreated with HDAC inhibitors, trichostatin A (TSA), vorinostat (SAHA), and valproate (VPA) and GSK3 inhibitors, LiCl, 1-azakenpaullone (1-aza), SB-216763, and TWS119. FGF21 mRNA was
upregulated (10-70 fold) by HDAC inhibitors. GSK-3 inhibitors also upregulated FGF21 mRNA
expression. Notably, the specific GSK-3binhibitors TWS119 and 1-aza induced a higher increase (520 fold) in FGF21 mRNA expression compared to the nonselective GSK-3(a-b) inhibitors (1.7-3 fold).
The increase in FGF21 mRNA levels by the HDAC and GSK inhibitors was both time- andconcentration- dependent. These results demonstrate that GSK-3 and HDAC are targets in theregulation of FGF21 expression, which will be key in understanding the different effects of FGF21 in
the brain. (Supported by NIH grant DA044766)
20 - G-protein biased kappa opioid receptor agonist attenuates cocaine self-administrationin male mice
Alexandra Dunn1, Jose Erazo1, Amelia Dunn1, Kyle Windisch1, Brian Reed1, Mary Jeanne Kreek1
1
The Rockefeller University, New York, USA
INTRODUCTION: Biased kappa opioid receptor (KOR) agonism, in which compounds selectively
activate specific intracellular signaling pathways (e.g. G-protein but not β-arrestin mediatedpathways), has emerged as a potentially viable therapeutic approach for the treatment of cocaine
addiction. However, the specific effects of biased KOR agonists have not been fully elucidated invivo. We investigated the effects of a recently characterized G-protein biased KOR agonist, N-butylN-phenylethyl-N-3-hydroxyphenylethyl-amine (BPHA; Dunn et al. 2018), on cocaine selfadministration in mice.
METHODS: Adult male C57BL6 mice (n=25) were initially trained to respond for 0.5mg/kg/infusion
cocaine during daily 2-hour FR1 intravenous self-administration sessions. Following acquisition,
mice were pretreated with 30mg/kg BPHA on two consecutive sessions, followed by 45mg/kg BPHA
for a single session. On a subsequent day, mice received 3mg/kg LY2444296, a short-acting KOR
antagonist, prior to 45mg/kg BPHA pretreatment of cocaine self-administration.
RESULTS AND DISCUSSION: 30mg/kg BPHA pretreatment did not significantly decrease cocaine
intake, whereas pretreatment with 45mg/kg BPHA significantly decreased cocaine selfadministration (49 +/- 11%) compared to vehicle (p<0.05). Attenuation of cocaine intake by 45mg/kg
BPHA was not blocked by pretreatment with a single dose of LY2444296 (3mg/kg), indicating thatthe BPHA-induced reduction in cocaine intake may not be KOR-mediated. Further studies areneeded to elucidate the mechanism(s) of action and involvement of the KOR system in BPHAinduced attenuation of cocaine self-administration.
These studies were supported by the Robertson Therapeutic Discovery Fund (BR, MJK) and the Dr.
Miriam and Sheldon G. Adelson Research Foundation (MJK).

40

21 - Identifying kappa opioid receptor-protein interactions following activation by unbiased
and G-protein biased ligands
Wenzhu Zhang1, Amelia Dunn1, Brian Reed1, Brian Chait1, Mary Jeanne Kreek1,2
1Rockefeller University, New York, USA, 2Rockefeller University, New York, USA
Biased agonists selectively activate certain signaling pathways downstream of their receptor. They
are being developed for several drug targets including the kappa opioid receptor (KOR), a potential
target for addiction. Biased ligands differentially effect signaling by inducing distinct conformationalchanges in the receptor. It is poorly understood how these conformations lead to differential
signaling. Distinct receptor conformations possibly lead to specific receptor-protein interactions,driving signaling differences. In this study, changes in KOR-protein interactions were identified
following drug treatmenT and compared across treatment groups. HEK293 cells expressing taggedKOR were treated with U69,593, MCBPHA (N-methylcyclobutyl-N-phenylethyl-N-3hydroxyphenylethyl-amine) or BPHA (N-butyl- N-phenylethyl-N-3-hydroxyphenylethyl-amine).
MCBPHA and BPHA are recently described biased KOR agonists that selectively activate G-protein
over arrestin signaling when compared to U69,593 (Dunn, et. al. 2018, IJNP, in press).
Immunoprecipitation of the receptor with stable-isotope labeled amino acids in cell culture (SILAC)
followed by mass spectrometry was used to identify changes in receptor-protein interactions after
drug treatment. Significant correlations were found between treatment by the two biased agonists,
BPHA and MCBPHA. There was no correlation between the changes in receptor-protein interactions
caused by U69,593 and either biased agonist. This study demonstrates that changes in receptorprotein interactions can be identified by immunoprecipitation followed by mass spectrometry, andsuggests that changes in receptor-protein interactions may be correlated to extent of observed ligand
bias. Candidate receptor-protein interactions identified will be further characterized for their role in
KOR signaling. Supported by the Robertson Therapeutic Development Fund and the Dr. Miriam and
Sheldon G. Adelson Medical Research Foundation.
22 - Preserved functional selectivity of MOR ligands in HEK293 cells and MOR-Venus DRG
neurons reveals the physiological reality of biased signaling
Aliza T. Ehrlich1,2, Meriem Semache3, Dillon Dafonte1, Leonie Runtz1, Christine Colley1,4, Amina Mezni1,4,
Christian LeGouill3, Florence Gross3, Viktoriya Lukasheva3, Emmanuel Darcq1, Michel Bouvier3, Brigitte
L. Kieffer1,2
1
Department of Psychiatry, McGill University, Douglas Hospital Research Center, Montréal, QC, Canada,2Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch-Graffenstaden, France,
3
Department of Biochemistry, Université de Montréal, Institute for Research in Immunology and Cancer,
Université de Montréal, Montréal, QC, Canada, 4Department of Biochemical Sciences, Faculty of Healthand Medical Sciences, University of Surrey, Guildford, Surrey, United Kingdom
Over-production and prescription of opioids has caused a major crisis of opioid abuse, addiction andoverdose with a global burden most strongly impacting North America. Biased agonists for Mu Opioid
Receptors (MORs) are emerging drugs that are proposed to produce the desired G protein-effect of
pain relief without unwanted βarrestin2-effects such as euphoria or respiratory depression. In thisstudy, in vitro and ex vivo assays were assessed by employing 10 preclinical (Met-Enkephalin,
DAMGO, Endomorphin-1, TRV130, PZM21) and clinical MOR agonists, (Fentanyl, Loperamide,
Oxycodone, Morphine, Buprenorphine) to examine which drugs possess biased properties andshould be promoted in the clinic and which assays are useful for the development of safer opioids.
We employed transfected MORs in HEK-293 cells and used resonance energy transfer (RET)
biosensors to established signaling signatures for 10 compounds. To examine endogenous MORs,
we generated and fully characterized new knock-in mice expressing physiological levels of functional
MORs. Agonist-induced MOR-Venus internalization and endosome co-localization were examinedin MOR-Venus neurons and correlated well with over-expressed MORs in HEK-293 cell profiling,
despite significant differences in levels of receptors and effectors. In both systems, the partial MOR
agonist, buprenorphine’s profile was uniquely identifiable. This study demonstrates, thatobservations in transfected systems are indicative of native MOR function and buprenorphine may
be a better alternative for naïve opioid, chronic pain patients.

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23 - Nociception-evoked impulsivity in rats and its reversal with morphine
Nidia Espinoza1, Javier Ibias1, Arbi Nazarian1
1
Western University of Health Sciences
According to the U.S. National Institute on Drug Abuse, about 21-29% of patients who are prescribed
opioids for chronic pain misuse them and 8-12% develop an opioid use disorder. Opioid use
disorders and addiction to opioids is believed to be significantly impacted by changes in impaired
decision making, and impulsivity. The goal of this project was to establish a nociception-evokedmodel of impulsivity in rats; to determine if morphine reduces impulsivity; and examine whether
impulsivity and/or morphine effects are sexually dimorphic. To measure impulsive choice, animals
were trained in a delay discounting task for food pellets using a delay schedule of 0, 8, 16, and 32
seconds. After training was complete and baseline measurements were established, rats received
intraplantar Complete Freund’s Adjuvant (CFA). Animals were then subjected to the delay
discounting task again under two treatment groups, saline and morphine for two weeks. The results
showed that CFA treatment increased delayed discounting (i.e. more impulsive) and this effect was
marginally more robust in female than male rats. Moreover, morphine treatment decreased
nociception-evoked delayed discounting. The effects of morphine were gradually lost as thetreatment continued during the two weeks testing period, suggestive of the development of morphine
tolerance. The present findings demonstrate nociception-evoked impulsivity in rats that is sensitive
to opioids and highlights the need for continued examination of impulsivity as a potential measure of
the affective component of pain, as well as a factor in examining opioid abuse in cases of chronic
pain conditions.
24 - Endomorphin analog treatment expedites recovery from chronic inflammatory pain andprotects against latent sensitization
Amy Feehan1, James E Zadina1,2,3,4
1
Tulane Brain Institute, 2Department of Medicine Tulane University, 3Department of Pharmacology
Tulane University, 4SE Louisiana Veterans HCS, New Orleans, LA
Opioids are unrivaled for relief of moderate to severe pain, but in addition to well-known side effects,
including abuse potential, opioids have also been shown to paradoxically cause pain sensitization
over time, thus limiting their use. Morphine, for example, worsens and prolongs mechanical allodynia
and thermal hyperalgesia after Complete Freund’s Adjuvant (CFA) injection into the hind paw. ZH853
is a novel endomorphin analog that has reduced side effects, including abuse liability, and superior
analgesia compared to morphine. For preclinical development, we wanted to test whether chronic
drug administration, before or after inflammatory pain, would induce sensitization of pain, as
morphine does. In the current study, adult male Sprague-Dawley rats were implanted with intrathecal
catheters and administered drug via osmotic minipump for 5 days. Inflammation was induced by CFA
injection to a hind paw and sensitivity was measured with von Frey and Hargreaves testing. Studies
have also shown gait impairments in this paradigm, and we used the CatWalk XT to determine
whether morphine and/or ZH853 made these impairments worse. When all animals had recovered
to baseline, naltrexone was used to probe whether latent sensitization had developed. We found thatmorphine prolonged and intensified hypersensitivity in both paradigms, while ZH853 reduced thetotal time and intensity of hypersensitivity, and protected against CFA-induced latent sensitization
versus vehicle and morphine treatment. This study suggests that, unlike currently used opioids,
ZH853 can treat chronic inflammatory pain effectively without causing further sensitization of thepain system.

42

25 - Intrinsic G-protein efficacy, not ß-arrestin 2 dependent signaling, confers reduced
opioid-induced respiratory depression
Alexander Gillis1, Arsalan Yousuf1, Preeti Manandhar2, Setareh Sianati1, Andrea Kliewer3, Stefan
Schulz3, MacDonald J Christie1
1
University of Sydney, 2Macquarie University, 3Friedrich Schiller University, Jena, Germany
ß-arrestin 2 (ß-arr2) dependent signaling via the µ-opioid receptor (MOPr) has been proposed tocontribute to opioid analgesic side effects. We studied opioid-induced respiratory depression using
whole-body plethysmography and antinociception in a hotplate assay. ß-arr2 knockout (KO) andphosphorylation deficient, arrestin non-recruiting MOPr knockin mice (11S/T-A), were used to study
the contribution of ß-arr2 dependent mechanisms to opioid-induced respiratory depression.
Morphine severely depressed respiration in wild-type, ß-arr2 KO and 11S/T-A mice, with nosignificant genotype effect on potency or maximal effect. G-protein biased agonists that do notstrongly induce MOPr-ß-arr2 interactions have been developed and appear to have favourable
therapeutic profiles in preclinical and early clinical trials. In vitro studies of G-protein activation
using GIRK channels with receptor inactivation by partial irreversible antagonism showed that Gprotein biased agonists PZM21 and TRV130 have lower intrinsic efficacy than morphine. In vivo,
these compounds induced similar respiratory depression to morphine but with lower maximal
effect. The very low efficacy agonist buprenorphine has been characterised as having a low ceiling
effect in respiratory depression, leading to the hypothesis that the favourable profiles of PZM21 andTRV130 are due to their low efficacy. Heterozygous MOPr K.O. (+/-) animals have reduced receptor
expression and are a reasonable genotypic model of low efficacy agonists activating a fraction ofreceptors. In MOPr +/- animals morphine produces full analgesia, but only partially depresses
respiratory frequency compared to wild-type. Low intrinsic G-protein efficacy, rather than ß-arrestin
2 dependent signaling, may explain reduced respiratory depression induced by apparently G-protein
biased agonists.
26 - Exploring rapid desensitization of mu-opioid receptors induced by G protein-biased
agonists
Sam Groom1, Alex Conibear2, Alex Disney1, Stephen Husbands1, Yangmei Li3, Lawrence Toll4,
Graeme Henderson2, Eamonn Kelly2, Chris Bailey1
1
University of Bath, Bath, UK, 2University of Bristol, Bristol, UK, 3Torrey Pines Institute for MolecularStudies, Port St. Lucie, USA, 4Florida Atlantic University, Boca Raton, USA
G-protein-biased agonists of mu-opioid receptors (MOPrs) have the potential to induce less receptor
desensitization and tolerance than traditional opioids. The aim of this study was to investigate MOPr
desensitisation induced by 2 potential G-protein-biased MOPr agonists: PZM21 (Manglik et al.,(2016) Nature 537:185-190) and ‘Compound 1’ (Tyr-c[D-Lys-Phe-Tyr-Gly]) (Li et al., (2016) J Med
Chem 59:1239-1245). In HEK293 cells, agonist-induced phosphorylation of Ser375 (a GRK
substrate) on MOPr after agonist exposure was measured using Western blotting. Whole-cellvoltage-clamp recordings were taken from rat locus coeruleus neurons. MOPrs couple to GIRK
channels, with the amplitude of observed K+ currents providing a measure of MOPr activation.
Opioids (30µM) were perfused for 10 minutes, with reductions in evoked-current amplitude tracked
as a measure of receptor desensitisation. Morphine and DAMGO both induced Ser375
phosphorylation. In contrast, PZM21 and Compound 1 induced negligible Ser375 phosphorylation,suggesting they are G-protein-biased. The amplitude of K+ currents evoked by morphine, PZM21
and Compound 1 were broadly similar and significantly less than those evoked by Met-Enkephalin.
This indicates that, like morphine, PZM21 and Compound 1 are partial MOPr agonists. While PZM21
induced less MOPr desensitization than morphine, Compound 1 induced robust MOPr
desensitization of greater magnitude than morphine. These data indicate that although G proteinbiased agonists at MOPrs induce minimal Ser375 phosphorylation and promote little arrestin
recruitment, their abilities to induce rapid MOPr desensitization can be vastly different. Subsequent
experiments are ongoing to investigate the intracellular mechanisms involved, and the in vivo
consequences.

43

27 - Preclinical Characterization of Mitragyna Speciosa Alkaloids for the Treatment of
Alcohol Use Disorder
Anna Gutridge1, Meridith Robins1, Rajendra Uprety2, Robert J. Cassell1, Gavril Pasternak2, Susruta
Majumdar2,3, Richard van Rijn1
1
Purdue University, Medicinal Chemistry and Molecular Pharmacology and Purdue Institute ofIntegrative Neuroscience, West Lafayette, IN, USA, 2Memorial Sloan Kettering Cancer Center,
Molecular Pharmacology and Chemistry Program, and Neurology, New York, NY, USA., 3Centerfor Clinical Pharmacology, Washington University School of Medicine and St. Louis College of
Pharmacy, St. Louis, MO, USA
Mitragyna speciosa (kratom) is an alkaloid containing plant gaining traction in opioid-dependent
individuals to mitigate opioid withdrawal effects. Several alkaloids present in kratom target opioid
receptors, but interact in a manner which does not recruit β-arrestin. Mu opioid receptor (µOR) drugs
biased towards G-proteins over β-arrestin show advantages including reduced respiratory
depression and dependence liability at equianalgesic doses to morphine. We have previously
demonstrated delta opioid receptor (δOR) agonists with minimal β-arrestin2 recruitment decrease
voluntary alcohol consumption in C57BL/6 mice; therefore, we hypothesized kratom-derived
alkaloids will decrease alcohol consumption but not display robust conditioned place preference(CPP).
We characterized kratom and the kratom-derived alkaloids mitragynine, 7hydroxymitragynine, speciogynine and paynantheine for G-protein signaling and β-arrestin2
recruitment in vitro at δORs and µORs. Behavioral effects of alkaloid administration were
investigated in a limited-access, 2-bottle choice 10% alcohol paradigm, locomotor test and acute
CPP to assess potential reward. Kratom alkaloids showed G-protein signaling (inhibition of cAMP
production), but undetectable β-arrestin2 recruitment at µOR and δOR. Kratom, mitragynine,
paynantheine and 7-OH-mitragynine dose-dependently reduced alcohol intake in mice. Reduction of
alcohol intake by 7-OH-mitragynine was attenuated in δOR-knockout mice. Mitragynine, kratom and7-OH-mitragynine show attenuated place preference at therapeutic doses, compared to morphine.
Our results indicate kratom alkaloids most likely reduce alcohol use by targeting δORs in addition to
in a G-protein biased manner. We propose kratom alkaloids may be a useful chemical template todevelop novel pharmacological interventions for the treatment of alcohol use disorder.

44

28 - Evaluation of two peripherally-restricted kappa-opioid receptor agonists for safer
analgesia without CNS liabilities
Hannah Harris1, Shyam Kamble1, Shainnel O. Eans1, Jessica M. Medina1,2, Michelle L. Ganno2, MarcelloA. Giulianotti2, Richard A. Houghten2, Bonnie A. Avery1, Jay McLaughlin1
1
University of Florida, Gainesville, USA, 2Torrey Pines Institute for Molecular Studies
Clinically-used mu opioid receptor (MOR) agonists produce robust analgesia, but central nervous
system (CNS) action also results in liabilities such as sedation, respiratory depression and abuse.
Peripherally-located kappa opioid receptors (KOR) are thought to mediate significant analgesia voidof many side effects produced by centrally acting opioids. Two potential candidates previously
identified from a library of 6,250,000 tetrapeptides, ffir-NH2 and ff(nle)r-NH2, demonstrated affinity(IC50 = ~2 nM) and selectivity for the KOR (Dooley et al., 1998). This study characterized ffir-NH2 andff(nle)r-NH2 in mice after central (i.c.v.), intraperitoneal (i.p.) or oral (p.o.) administration for dosedependent, peripherally-restricted opioid-receptor mediated antinociception and potential
liabilities. In the 55°C warm-water tail-withdrawal test, each tetrapeptide produced robust
antinociception after all routes of administration, with a response after i.p. administration that was
9.9- and 21.0-times more potent than morphine, respectively (ffir-NH2 ED50=0.53 (0.08-2.50) mg/kg;
ff(nle)r-NH2 ED50=0.25 (0.05-1.08) mg/kg). Pretreatment with opioid antagonists and testing in KOR
KO mice demonstrated KOR selectivity, and naloxone methiodide pretreatment suggested activity
that was peripherally-restricted. Supporting this, pharmacokinetic analysis following i.v.
administration of either tetrapeptide demonstrated plasma levels consistent with the duration ofantinociception, but no detected tetrapeptide in brain tissue. The tetrapeptides further demonstrated
dose-dependent antinociception and antiallodynia in mouse models of inflammatory and neuropathic
pain. High peripheral doses of ffir-NH2, but not ff(nle)r-NH2, increased urine production, motor
impairment and respiratory depression, but neither tetrapeptide demonstrated place conditioning.
Together, these data show that ffir-NH2 and ff(nle)r-NH2 produce rapid, potent, peripherally-restricted
antinociception with fewer side effects.
29 - Specific PKC isoform mediates ongoing pain in a mouse model of migraine
YING HE1, Zaijie Jim Wang2
1
University of Illinois at Chicago, Chicago, USA, 2University of Illinois at Chicago, Chicago, USA
Migraine is one of the most common neurological disorders characterized by recurrent attacks oftypically throbbing and unilateral headache, affecting up to 20 % of the population worldwide. Despite
the high prevalence and severity of this primary headache disorder, limited progress has been madein understanding and treating migraine. By characterizing and validating a mouse migraine model,
this study aimed to investigate the functional contribution of PKC isoforms in migraine. Systemic
administration of NO donor, nitroglycerin (NTG) induced significant and prolonged mechanicalhypersensitivity in female mice. We found the presence of migraine-like ongoing pain in mice after
chronic intermittent treatment of NTG. The peptide antagonist of calcitonin gene related peptide(CGRP), αCGRP (8-37) effectively blocked ongoing pain and elicited pain relief-induced CPP in
NTG-treated mice. Prominent activation of specific PKC isoform was observed in the superficial
laminae of the spinal cord dorsal horn in chronic NTG-treated mice. Functional inhibition of the PKC
isoform significantly attenuated ongoing spontaneous pain in chronic NTG-treated mice.
Furthermore, we recapitulated the NTG-triggered migraine model in PKC isoform-null mice. In
response to repeated administration of NTG, ongoing spontaneous pain was not developed in micelacking the specific PKC isoform. This study is the first to identify the presence of ongoing pain inmice treated with nitroglycerin, a known human migraine trigger that closely resembles the common
manifestation of spontaneous migraine attacks in humans. These findings demonstrated a criticalregulatory role of spinal PKC in migraine pathophysiology, which may offer new pharmacologicaltargets for anti-migraine treatment.

45

30 - Mechanisms of presynaptic suppression of GABA release by opioid receptors in the
hippocampus
X. Jenny He1, Matthew Banghart1
1
University of California San Diego
Opioid receptors are prominent in interneurons of the hippocampus where they modulate GABAergic
inhibition onto pyramidal cells. Mu opioid receptor (MOR) activation is known to suppress GABA
release from parvalbumin (PV) basket cells in the CA1 region, which positions opioids to affect spiketiming, oscillations, and synchronization of neuronal populations in the hippocampus. Although thedelta opioid receptor (DOR) is also prominent in the hippocampus, its functional role in distinct
interneuron classes has not been established, nor have the molecular mechanisms that underlie
MOR and DOR-mediated suppression of synaptic transmission. Using a combination ofelectrophysiology and optogenetics, we discovered that both MOR and DOR strongly inhibit GABA
release from PV+ basket cells in a mutually-occlusive manner. Although both receptors appeared toutilize a presynaptic mechanism, they were differentially prone to desensitization. We further
characterized the molecular mechanisms of opioid receptor-mediated suppression using two-photon
calcium imaging and pharmacology and found evidence for modulation of processes downstream ofvoltage-gated calcium channel activation. Because the small size of presynaptic terminals in central
neurons makes them largely inaccessible to pharmacological manipulations with membraneimpermeant peptide probes, we also explored a genetic approach for identifying the moleculareffectors downstream of receptor activation. Our results suggest a previously unappreciated role for
DOR in modulating perisomatic inhibition in the hippocampus and reveal the molecular mechanisms
by which both MOR and DOR regulate GABA release from PV basket cells in CA1.
31 - Fentanyl Depression of Respiration in MiceR.Hill 1, W.L.Dewey 2, E.Kelly 1, G.Henderson 1
1
University of Bristol, 2Virginia Commonwealth University
In 2016 more opioid overdose deaths in the USA involved fentanyl-based opioids than heroin orprescription opioids such as oxycodone. It has been suggested that fentanyl is resistant to naloxone
reversal. This investigation examined fentanyl depression of respiration, reversal by naloxone andacute tolerance to fentanyl.
Experiments were conducted in male mice (CD-1). Respiration was measured by whole bodyplethysmography in mice breathing either 95% air/5% CO2 or 100% air. All drugs were administeredintraperitoneally.
Fentanyl (0.05-1.35 mg/kg) dose dependently depressed respiration (20-75%). Morphine (1-10mg/kg) also dose dependently depressed respiration (15-40%). Acute administration of morphine(10 mg/kg) or fentanyl (0.15 mg/kg) produced equivalent levels of respiratory depression (~ 40%
decrease in minute volume). Administration of naloxone (0.3 and 1 mg/kg) fully reversed morphine
depression of respiration, but not that induced by fentanyl. Administration of 3 mg/kg naloxone fully
reversed both morphine and fentanyl induced depression of respiration.
Administration of two doses of fentanyl (0.15 mg/kg) 2 hrs apart resulted in significantly lessdepression of respiration by the second dose of fentanyl compared to the first. Pre-treatment with
the GRK2/3 inhibitor compound 101 (10 mg/kg) or the PKC inhibitor calphostin C (45 mg/kg) did notprevent this tolerance. Also, compound 101 did not decrease acute fentanyl depression of respirationsuggesting that it did not involve GRK2/3 and arrestin binding.
These data indicate that fentanyl is more resistant to naloxone antagonism compared to a classicalopioid agonist such as morphine.

46

32 - Co-administration of chemokine receptor antagonists with opioids: Potentiation ofanalgesic effects on incisional pain in rats
Saadet Inan1, Mia N Watson1, Menahem Doura1, Christopher S Tallarida1, Xiaohong Chen1, Joseph
J Meissler1, Ellen B Geller1, Scott M Rawls1, Alan Cowan1, Toby K Eisenstein1, Martin W Adler1
1Center for Substance Abuse Research, Lewis Katz School of Medicine at Temple University,
Philadelphia, PA 19140
Our laboratories have shown that there is cross-desensitization between opioid and chemokine
receptors and that chemokines can inhibit the analgesic activity of opioids. Here, we investigatedwhether co-administration of chemokine receptor antagonists (CRAs) with opioids would enhance
the analgesic effect of opioids on incisional pain in rats. We chose morphine and oxycodone, which
are the most commonly prescribed opioids for pain. Male Sprague-Dawley rats (200-230 g)underwent incisional surgery on the left hind paw. Pain responses were evaluated measuring
mechanical allodynia at various times post-surgery from 15 to 360 minutes. Morphine, oxycodone,
maraviroc (a CCR5 antagonist), AMD3100 (a CXCR4 antagonist), or their vehicles were injecteds.c., alone or in combination, at 25 minutes post-surgery. We found that a) morphine and oxycodone
significantly reversed mechanical allodynia in a time- and dose-dependent manner; b) neither
maraviroc nor AMD3100 had an effect by themselves; c) addition of AMD3100 significantly shifted
both morphine and oxycodone dose-responses to the left (1.8- and 2.0-fold, respectively); d) additionof maraviroc shifted the dose-response curves of morphine (2.4-fold) and oxycodone (1.5-fold) tothe left; and e) addition of the two CRAs with morphine further shifted the dose-response curve tothe left (3.3-fold). These results indicate that the combination of CRAs with opioids permits use oflower doses of the opioids to achieve an analgesic effect equal to that obtained with opioids alone.
Using opioids in lower doses has the potential to reduce unwanted side effects and the developmentof tolerance and dependence. DoD grant W81XWH-15-1-0252; NIDA P30 DA013429
33 - Multidisciplinary Approach in the Optimization of a Highly Selective Sigma-1 Receptor
Antagonist
Sebastiano Intagliata1, Theresa A. Kopajtic2, Jonathan L. Katz2, Michelle L. Ganno3, Abhisheak
Sharma4, Bonnie A. Avery4, Shainnel O. Eans3,5, Jay P. McLaughlin5, Christopher R. McCurdy1
1
Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610,
USA, 2Psychobiology Section, Intramural Research Program, Department of Health and Human
Services, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA,
3
Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987, USA, 4Department ofPharmaceutics, College of Pharmacy, University of Florida, Gainesville, FL 32610, USA, 5Department
Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, FL 32610, USA
Chronic pain is one of the leading causes of adult disability in the United States, and it is estimatedthat about 20 million individuals suffer from some form of peripheral neuropathy. Chronic pain
conditions represent a major health-care cost, due to both the medical expenses and the lost workdays. Current treatments possess notable liabilities such as tolerance, respiratory depression, andaddiction in relation to opioids; or sedation and dizziness in the case of antiepileptic drugs. Thus,
novel analgesics which have fewer side effects than current pain treatments are still needed. The
sigma-1 receptor (S1R) has been recently proposed as novel target, due to the ability of the S1R
antagonists to modulate the pain effectively with no liabilities. Our research group has developed
CM304 (FTC146) the most potent and selective S1R ligand to date for this receptor, which hasentered into clinical trials as a positron emission tomography and magnetic resonance imaging(PET/MRI) diagnostic agent for peripheral nerve injury. Despite CM304 demonstrating antialloydinic
activity in rodents, it possess a short half-life and poor oral bioavailability, rendering it unsuitable fortherapeutic development. Herein, we present a multidisciplinary approach for the lead-optimizationof a highly selective S1R antagonist. The methods include: synthesis and structure activityrelationship (SAR) investigation, in vitro/in vivo pharmacology evaluation, and in vitro/in vivo
pharmacokinetic studies of novel CM304 analogs. Synthetic pathways of new compounds along withpreliminary binding data, and in vitro metabolic stability are reported being reported.

47

34 - Evaluation of mixed efficacy opioid ligands in drug discrimination assays
Emily Jutkiewicz1, Ashleigh L Matthews1, Henry I Mosberg1
1
University of Michigan, Ann Arbor, USA
Mixed efficacy opioid ligands have been developed as alternatives to mu opioid receptor agonists
for producing analgesia with potentially fewer side effects, such as tolerance, physical dependence,
and abuse liability. To determine if mixed efficacy ligands produce similar interoceptive effects toreceptor-selective opioid agonists, the current study evaluated the effects of mixed efficacy opioid
ligands in rats trained to discriminate either a selective mu- or delta-opioid receptor agonist. Drugdiscrimination assays are useful for categorizing common drug stimulus effects, predicting potential
abuse liability, and obtaining quantitative pharmacological analysis of drug effects in vivo. For theseexperiments, rats were trained to discriminate either 3 mg/kg morphine or SNC80 from saline on a
fixed ratio 10 schedule of reinforcement in a food-reinforced, drug discrimination paradigm. In rats
trained to discriminate morphine, hydrocodone and buprenorphine fully generalize to thediscriminative stimulus effects of morphine. In rats trained to discriminate SNC80, the SNC80
analogs AZD2327 and DPI287 fully generalized to the discriminative stimulus effects of SNC80. The
peptidomimetic MOR agonist/DOR antagonist AAH8 produces antinociception in rats but fails togeneralize to either the discriminative stimulus effects of morphine or SNC80. Similarly, the MOR
agonist/DOR agonist MMP2200 does not generalize to the discriminative stimulus effects ofmorphine but fully generalizes to the discriminative stimulus effects of SNC80. These data suggestthat incorporating high affinity, DOR activity with a MOR agonist may alter the discriminative stimulus
effects of MOR agonists. Supported by DA041565 and DA003910.

35 - An enzymatic approach reverses nicotine dependence, decreases compulsive-likeintake and prevents relapse in rats
Marsida Kallupi1,2, Song Xue2, Bin Zhou2, Kim Janda2, Olivier george1,2
1
The Scripps Research Institute, 2The Scripps Research Institute, La Jolla, USA
Tobacco use disorder is the leading cause of disease and preventable death worldwide, but current
medications that are based on pharmacodynamics have low efficacy. Novel pharmacokinetic
approaches to prevent nicotine from reaching the brain have been tested using vaccines, but such
efforts have failed because antibody affinity and concentration are not sufficient to completely
prevent nicotine from reaching the brain. In the present study, we provide preclinical evidence of theefficacy of an enzymatic approach to reverse nicotine dependence, reduce compulsive-like nicotine
intake, and prevent relapse in rats with a history of nicotine dependence. Chronic administration of
NicA2-J1, an engineered nicotine-degrading enzyme that was originally isolated from P. putida S16,
completely prevented nicotine from reaching the brain and reversed somatic signs of withdrawal,
hyperalgesia, and irritability-like behavior in nicotine-dependent rats with a history of escalation of
nicotine self-administration. NicA2-J1 also decreased compulsive-like nicotine intake, reflected byresponding despite the adverse consequences of contingent footshocks, and prevented nicotineand stress (yohimbine)-induced relapse. These results demonstrate the efficacy of enzymatictherapy in treating nicotine addiction in advanced animal models and provide a firm grounding forthe development of biological therapies for smoking cessation in humans.

48

36 - Pharmacological effects of modifying the N-substituent of hydromorphone
Sophia Kaska1, Rachel S. Crowley1, Thomas C. Irvin2, Meining Wang2, Arthur E. Jacobson2, Thomas
E. Prisinzano1, Kenner C. Rice2
1
Department of Medicinal Chemistry, School of Pharmacy, The University of Kansas, Lawrence,KS, 2Drug Design and Synthesis Section, Molecular Targets and Medications Discovery Branch,
IRP, National Institute on Drug Abuse and the National Institute on Alcohol Abuse and Alcoholism,
NIH, Department of HHS, Bethesda, MD, United States
Hydromorphone (Dilaudid) is a potent and effective opioid analgesic for the treatment of pain. Whileopioid medications are effective analgesics for the treatment of pain through G-proteins, they present
a number of undesired side effects, such as tolerance, dependence, and respiratory
depression. These side-effects have been said to be mediated by beta-arrestin
recruitment. Importantly, structural modification of hydromorphone has demonstrated therelationship of its substituents and the effect on biological activity. For instance, when the N-methyl
substituent is replaced with an N-phenethyl substituent in some opioids, their activity can betransformed from agonist to antagonist. In this collaboration, a number of N-derivatives of opioids
have been generated to explore their agonist and antagonist activity, as well as to evaluate betaarrestin recruitment. The N-phenethyl derivative of hydromorphone is relatively inert, but when apara-nitro group is added, the agonist activity is transformed. Interestingly, other N-substituents havealso converted weak mu agonists into more potent agonists. Furthermore, to evaluate theintracellular signaling mechanisms that lead to the negative side effects of opioid medications, betaarrestin recruitment was investigated. Two of these compounds demonstrate some recruitment ofbeta-arrestin, while further evaluation of other compounds for bias towards this signaling pathway isin progress. Overall, these data suggest that the N-substituent of an opioid is important for dictating
the agonist function and activity of a molecule and that elucidation of these structure-activity
relationships will assist in the development of effective opioid analgesics with reduced side effects.
37 - A selective bivalent antagonist for the mu-delta opioid receptor heterodimer potentiates
oxymorphone anti-nociception while sharply reducing morphine withdrawal
Attila Keresztes1, Paul Nguyen1, Keith Olson1,2, Victor Hruby2, John M. Streicher1
1
Department of Pharmacology, College of Medicine, University of Arizona, Tucson AZ USA,2
Department of Chemistry and Biochemistry, College of Science, University of Arizona, Tucson AZ
USA
Experimental observations over decades have shown that the mu (MOR) and delta (DOR) opioid
receptor systems interact. One hypothesis for this interaction is that the MOR and DOR
heterodimerize to form a distinct signaling unit (MDOR). However, few MDOR-selective tools exist,
and no MDOR-selective antagonists, creating controversy as to the role or even existence of the
MDOR in vivo. We thus created a novel series of bivalent antagonists by linking MOR and DOR
antagonist pharmacophores by a variable-length spacer. We found a lead compound D24M with
0.85 nM potency and ≥89 fold selectivity at the MDOR, which was selective for the MDOR in CD-1
mice up to intracerebroventricular (icv) doses of 10 nmol. We further tested D24M in mouse models
of opioid anti-nociception and dependence. We found that 1 nmol of icv D24M strongly potentiatedoxymorphone anti-nociception in the tail flick (AUC increase of 52.3%), paw incision (increase of628%), and chemotherapy-induced peripheral neuropathy pain models (CIPN; increase of 249.6%).
Importantly, the MOR antagonist CTAP and the DOR antagonist naltrindole could not replicate thisincrease, suggesting an MDOR mechanism. We further found that 1 nmol of icv D24M given 5
minutes prior to naloxone precipitation of withdrawal strongly blocked jumping behavior in acute andchronic models of morphine dependence, while D24M alone produced no withdrawal behavior.
These results suggest that the MDOR has a role in blocking anti-nociception and promoting
withdrawal. Our results also suggest that MDOR antagonists could be novel therapeutics to enhance
opioid anti-nociception and treat opioid withdrawal.

49

38 - DPP4 inhibitors, Ile-Pro-Ile (IPI) and vildagliptin produce diverse antihyperalgesic
effects following intrathecal administration in inflammatory pain models in rats
Kornél Király1, Mihály Balogh1, Bence Varga1, Dávid Karádi1, Mahmoud Al-Khrasani1, Zita Puskár2,
Márk Kozsurek2, Pál Riba1
1
Department of Pharmacology and Pharmacotherapy, Semmelweis University, 2Department of
Anatomy, Histology and Embryology, Semmelweis University
We have recently reported the ineffectiveness of Ile-Pro-Ile and vildagliptin following intrathecal
administration in tail-flick test. They produced robust opioid-mediated antihyperalgesic action incarrageenan-induced inflammatory pain and a limited non-opioid analgesia in neuropathic pain.
Further analysis suggested a different mechanisms, Ile-Pro-Ile acts exclusively through μ receptors
while vildagliptin has a mixed action with δ receptor dominancy. The present study was aimed toassess their actions on CFA-induced monoarthritis model and formalin test.
Methods: male Wistar rats (160-220g) were used. CFA-induced hyperalgesia was measured by
Randall-Selitto test and dynamic plantar aesthesiometer (DPA) on the 4th day. In formalin test pain
behaviors were counted from 0 to 60 min.
The test compounds restored the prehyperalgesic threshold evoked by CFA when Randall-Selitto
but not DPA test was applied. Ile-Pro-Ile showed pure μ-mediated effect while vildagliptin had mixed,
δ-dominated action. In formalin test only Ile-Pro-Ile was able to significantly reduce pain reactions of
the 2nd phase but not the 1st phase. This effect was abolished by μ selective or nonselective opioid
antagonists and significantly, though partially, by a selective δ antagonist. Our results are inagreement with our previously published work proving that the two DPP4 inhibitors could activate
different analgesic pathways. In both pathways endogenous opioid peptides are involved.
Endomorphins or β-endorphin could be involved in the action of Ile-Pro-Ile whereas enkephalins
could mediate that of vildagliptin. The Ile-Pro-Ile activated analgesic pathway might play a role incontrolling pain transmission in formalin test. The molecular background of these diverse effects
need further elaboration.
39 - Evaluation of the analgesic and side-effects of G-protein biased mu-opioid receptor
Salvinorin A analogues kurkinorin and kurkinol
Bronwyn Kivell1, Amy Alder1, Rachel Crowley2, Thomas Prisinzano2
1
Victoria University of Wellington, Wellington, New Zealand, 2Department of Medicinal Chemistry,
University of Kansas, Lawrence, KS, USA
Development of G-protein biased mu-opioid receptor agonists with reduced β-arrestin signaling arehighly sought after for the development of safer pain medications with reduced abuse liability,
tolerance, dependence, constipation and respiratory depression. Here we evaluate the analgesic
and side-effects of novel G-protein biased mu-opioid agonists kurkinorin and kurkinol based on the
structural scaffold of salvinorin A. Analgesic effects were evaluated in C57BL/6 mice using the 50°C
hot-water tail withdrawal assay using cumulative administration of kurkinorin or kurkinol andanalgesic effects were compared to morphine. Kurkinorin showed equal potency to morphine (ED50
6.43 ± 0.27 (mg/kg)), whereas kurkinol was more potent (ED50 values of 2.35 ± 0.25; p<0.001).
Tolerance was determined following daily administration of morphine (10 mg/kg), kurkinorin (10mg/kg) or kurkinol (5 mg/kg) and dose-response tail-withdrawal effects evaluated on Day 9. Unlike
morphine, which showed significant tolerance from days 5-9 with an ED50 on day 9 of 16.35 ± 0.75(mg/kg), kurkinorin showed reduced tolerance and kurkinol showed no significant tolerance effects(ED50 values on day 9 of (7.9 and 3.60 (mg/kg))) respectively. Effects on motor co-ordination werealso evaluated using the accelerating rotarod, and latency to fall recorded. Kurkinorin and kurkinol
showed a shorter duration of motor impairment effects compared to morphine (p<0.001). Morphinesignificantly inhibited gastrointestinal transit via transit of charcoal meal (p<0.0001), whereas
kurkinorin shows no change. The G-protein biased mu-opioid agonists kurkinorin and kurkinol havepotent analgesic effects and reduced side-effects and support the development of biased agonists
for development of analgesics with reduced side-effects.

50

40 - Enhanced opioid analgesia and loss of tolerance but exacerbated side effects in miceexpressing G-protein biased, phosphorylation-defic
Andrea Kliewer1, Frank Schmiedel1, Setareh Sianati2, Alexis Bailey3, Jordan T Bateman4, Erica S
Levitt4, John T Williams5, MacDonald J Christie2, Stefan Schulz1
1
Friedrich Schiller University, Jena, Germany, 22Discipline of Pharmacology, School of Medical
Sciences, University of Sydney, NSW 2006, Australia., 3Institute of Medical and Biomedical
Education, St George's University of London, London SW17 ORE, UK, 4Department ofPharmacology and Therapeutics, University of Florida, Gainesville, FL 32608, 5The Vollum
Institute, Oregon Health and Science University, 3181S.W. Sam Jackson Pk. Rd., Portland, OR,
97239, USA
Opioid analgesics are powerful pain relievers, however, over time pain control is diminished due todevelopment of analgesic tolerance and life threatening side effects limit utility. Until now the initialmolecular mechanisms causing tolerance remain unresolved. In vitro evidence indicates that rapidµ-opioid receptor desensitization and ß-arrestin interaction is controlled by hierarchicalphosphorylation of multiple intracellular serine (S) and threonine (T) residues. To assess thecontribution of phosphorylation to µ-receptor signaling in vivo, we created three lines of knockin micewith a series of carboxyl-terminal S/T to alanine mutations that are increasingly unable to recruit
regulatory arrestin proteins. Here we show that desensitization is inhibited in locus coeruleus
neurons from mice with phosphorylation-deficient µ-opioid receptors. Consistent with this, acutefentanyl- and morphine-induced analgesia are both strongly enhanced. Upon chronic administration
analgesic tolerance to fentanyl and morphine is either abrogated or greatly diminished, while signsof physical dependence persist. Unexpectedly, opioid side effects including constipation andrespiratory depression are unchanged or exacerbated, indicating that arrestin recruitment does notcontribute to severity of these side effects. Our findings identify carboxyl-terminal multi-site
phosphorylation as the key step that drives acute µ-opioid receptor desensitization and long-term
tolerance. They also predict that G protein-biased opioid agonists will produce more effective
analgesia and reduced tolerance but also severe adverse effects.

41 - The complex roles of µ-opioid receptor phosphorylation and ß-arrestins in mediating
opioid analgesia with fewer side effects
Andrea Kliewer1, Frank Schmiedel1, Stefan Schulz1
1
Friedrich Schiller University, Jena, Germany
The efficiency of µ-opioid receptor signaling is regulated by coordinated receptor phosphorylationand subsequent interaction with ß-arrestin-1 and ß-arrestin-2. We have recently reported thattolerance to fentanyl and morphine is either abrogated or greatly diminished in total phosphorylationmutant µ-opioid receptor knockin (TPD) mice. This study was designed to examine whether residual
morphine tolerance in TPD mice can be abolished by genetic deletion of ß-arrestin-2. A head-tohead comparison of the two mouse lines revealed that ßarr2-KO mice exhibit only a very modest
analgesic phenotype compared to TPD mice. Unlike TPD mice, the analgesic efficacy of fentanyl ormorphine was not enhanced or prolonged in ßarr2-KO mice. Moreover, analgesic tolerance as aresult of chronic administration of fentanyl or morphine in ßarr2-KO mice was indistinguishable fromwild-type mice. Combination of phosphorylation- and ß-arrestin-2-deficiency did not result in any
additional inhibition of morphine tolerance. Notably, opioid side effects such as respiratory
depression and constipation induced by subanalgesic doses of fentanyl or morphine were reducedin ßarr2-KO mice but exacerbated in TPD mice. Thus, these findings do not support role of a ßarrestin-2 in regulating µ-opioid receptor responsiveness in vivo. We conclude that a rigorous reexamination of the physiological functions of ß-arrestin proteins will require novel animal models inthat µ-opioid receptor neurons are devoid of both ß-arrestin-1 and ß-arrestin-2.

51

42 - Contribution of β-arrestin and ERK signaling in the anxiolytic-like effects of the δ-opioid
receptor (δOR) agonist SNC80
Mee Jung Ko1,3,4, Terrance Chiang1, Arbaaz M. Mukadam1, Grace E. Mulia1, Marcus M. Weera2,3,4,Julia A. Chester2,3,4, Richard M. van Rijn1,3,4
1

Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University,
West Lafayette, Indiana 47907, 2Department of Psychological Sciences, College of Health and Human
Sciences, Purdue University, West Lafayette, Indiana 47907, 3Interdisciplinary Life Science PhD Program(PULSe), Purdue University, West Lafayette, Indiana 47907, 4Purdue Institute for Integrative Neuroscience,
Purdue University, West Lafayette, Indiana 47907

Opioids have been considered as a therapeutic approach for anxiety disorders. Although it has beensuggested that β-arrestin proteins may contribute to the side effect profile of many opioids, the roleof β-arrestin in δ-opioid receptor (δOR)-mediated anxiolysis has not been fully understood. Previous
studies have identified that the selective δOR agonist, SNC80, which strongly recruits β-arrestin, has
an anxiolytic-like profile in rodents. Here, we further characterized the distinctive role of β-arrestinbiased δOR signaling in anxiety-like behavior and conditioned fear responses. In wild-type mice,systemic administration of SNC80 (s.c. 20 mg/kg) demonstrated anxiolytic-like effects in the elevatedplus maze (EPM) and the dark-light transition box, whereas these effects were abolished in global
β-arrestin-2 knockout mice. We also discovered that SNC80 caused strong extracellular signalregulated kinase1/2 (ERK1/2) phosphorylation in CHO cells (overexpressing δOR and β-arrestin-2)
as well as in the amygdala and the striatum of wild-type mice. The anxiolytic-like effects of SNC80
were blocked by the MEK (MAPK/ERK kinase, an upstream kinase of ERK1/2) inhibitor, SL327. We
further expanded our study by exploring conditioned fear, and found that SNC80 also decreased
conditioned fear-potentiated startle (FPS) responses in wild-type mice. These effects, however, were
not impacted by a global knockout of β-arrestin-2. It is possible that the lack of β-arrestin 2
requirements for δOR-mediated FPS responses may be indicative of low β-arrestin-2 expression in
the brain regions responsible for the conditioned fear responses. Taken together, we suggest that βarrestin-2-biased δOR signaling modulates anxiety-like behavior through the downstream ERK1/2
phosphorylation.

52

43 - Interruption of continuous morphine administration in mice negatively impacts brain
and behavior
Emilia Lefevre1, Marc Pisansky1, Lauren Bystrom1, David Leipold1, Thomas Kono1, Federico
Buffaldi1, Marco Pravetoni1, Patrick Rothwell1
1
University of Minnesota, Minneapolis, USA
Clinical use of opiate-based analgesics can lead to negative consequences like dependence andaddiction. We propose that maladaptive changes in brain function occur when otherwise continuous
opioid exposure is interrupted by short periods of withdrawal. To test this hypothesis, male andfemale mice were implanted with osmotic minipumps to continuously deliver morphine (63mg/kg/day), and administered twice-daily naloxone injections (10 mg/kg) to interrupt opioid receptor
stimulation. Continuous morphine exposure produced tolerance of psychomotor activation. In
contrast, interruption of morphine exposure with naloxone caused psychomotor sensitization, an
addiction-related behavior related to adaptations in the nucleus accumbens (NAc). We next
performed RNA-seq analysis of NAc tissue, to analyze differential gene expression after continuous
or interrupted morphine exposure. After controlling for false discovery rate, we found no genes thatwere significantly regulated by continuous morphine exposure alone, whereas interrupted morphine
exposure significantly regulated 1,328 genes. Genes that were differentially regulated by continuous
and interrupted morphine exposure included GABA-A receptor and ion channel subunits, prompting
further analysis of cellular properties using ex-vivo slice physiology. We recorded from NAc medium
spiny neurons (MSNs) identified by expression of the D1 or D2 dopamine receptor. Intrinsic
excitability was significantly, and selectively, increased in D2 MSNs after interrupted morphine.
Continuous morphine exposure increased the amplitude of spontaneous inhibitory postsynaptic
currents (sIPSCs) onto D1 MSNs, whereas interrupted morphine exposure tended to increase sIPSC
amplitude onto D2 MSNs. This study illustrates dramatic and divergent adaptations in cellularfunction that occur with different patterns of opiate administration.
44 - Ketamine in the treatment of opioid tolerance: does the opioid have a role?
Tuomas O. Lilius1,2,3, Emilia Kangas1, Hanna Viisanen1, Viljami Jokinen1, Mikko Niemi2,3, Pekka V.
Rauhala1, Eija A. Kalso1,4
1
Pharmacology, University of Helsinki, Helsinki, Finland, 2Clinical Pharmacology, University of
Helsinki, Helsinki, Finland, 3HUSLAB, Helsinki University Hospital, Helsinki, Finland, 4Departmentof Anaesthesiology, Intensive Care Medicine, and Pain Medicine, University of Helsinki andHelsinki University Hospital, Helsinki, Finland
The effects of N-methyl-D-aspartic acid (NMDA) receptor antagonists in enhancing opioid
analgesia and attenuating opioid tolerance have been of great interest both in the laboratory andthe clinic. Preclinical studies have mostly focused on the pharmacodynamic interactions between
ketamine and morphine, whereas only scarce information about co-administration of ketamine andthe widely used oxycodone exists. Pharmacokinetic interactions between ketamine and opioids
have been only little studied. Further, the role of ketamine metabolites in opioid tolerance remains
unclear. Our recent studies show that co-administration of low-dose ketamine or norketamine
effectively attenuated morphine tolerance in rats, whereas tolerance to oxycodone was lessaffected. This difference may be partly attributable to a pharmacokinetic interaction between
morphine and ketamine leading to vastly increased concentrations of morphine, ketamine, andnorketamine in the central nervous system. Co-administration of oxycodone and ketamine did notshow changes in drug concentrations. Norketamine, the main active metabolite of ketamine,should be further studied in the treatment of opioid tolerance, as it attenuated morphine tolerancewith minor adverse effects. In contrast, (2S,6S;2R,6R)-hydroxynorketamine, a major secondary
metabolite with no NMDA receptor antagonist properties, did not have tolerance-attenuating
effects. In addition to pharmacodynamic differences, pharmacokinetic interactions between
ketamine and different opioids may explain the wide variation in treatment response and adverse
effects observed in clinical ketamine-opioid interaction studies. These results also warrant studies
of possible pharmacokinetic ketamine-opioid interactions in humans.

53

45 - Roles of Protein Kinase C in Kappa Opioid Receptor-Mediated Effects in vivo:
Pharmacological and Phosphoproteomic Approaches
Jeffrey J. Liu1, Yi-Ting Chiu2, Chongguang Chen2, Matthias Mann1, Lee-Yuan Liu-Chen2
1
Max Planck Institute of Biochemistry, Martinsried, Germany, 2Temple University Lewis Katz
School of Medicine, Philadelphia, PA, USA
Kappa opioid receptor (KOR) agonists possess the capacity to become anti-pruritic drugs as well as
the next generation of analgesics, but are limited by their dysphoric, psychotomimetic andhallucinogenic properties. Recent evidence demonstrated that the KOR-mediated adverse effects
are reduced in some class of KOR agonists, suggesting that distinct downstream biochemical
pathways, such as protein kinase C (PKC), may be responsible for different behavioral outcomes.
Here, we showed that PKC inhibition preserved the beneficial antinociceptive and antipruritic effects
induced by the KOR agonist U50,488H, but attenuated the adverse condition placed aversion (CPA),
sedation and motor incoordination. We also found that PKC inhibition diminished phosphorylation of
KOR at S369. Using a large-scale shotgun mass spectrometry-based phosphoproteomics, weinvestigated the downstream phosphorylation changes induced by U50,488H with and without PKC
inhibition, in a temporal- and brain region-specific fashion. At five-minute, we observed PKCdependent modulation of GRKs and Wnt Signaling pathways, whereas at thirty-minute, we observed
PKC-dependent modulation of calcium signaling, stress signaling, cytoskeleton changes and mTOR
signaling. We previously showed that the mTOR pathway was involved in KOR agonist-promoted
CPA. Altogether, we investigated behavioral effects and biochemical signaling pathway changes asa result of PKC inhibition during KOR signaling. We also further demonstrated that adverse
behavioral effects from KOR activation can be separated through downstream effector inhibition.
(supported by NIH grants R01 DA041359 and P30 DA013429)
46 - Correlation of KOR phosphorylation in mouse brains with sedation, motor
incoordination and aversion induced by agonists
Chongguang Chen1, Peng Huang1, Jeffrey Liu2, Yi-Ting Chiu1, John Logue1, Brigitte Kieffer3,
Matthias Mann2, Lee-Yuan Liu-Chen1
1
Temple University Lewis Katz School of Medicine, Philadelphia, PA, USA, 2Max Planck Institute of
Biochemistry, Martinsried, Germany, 3Douglas Mental Health Institute, Department of Psychiatry,
McGill University, Montreal, Quebec, Canada
Selective KOR agonists are promising analgesics and antipruritic agents. However, clinicalapplications of KOR agonists are limited by side effects, including dysphoria, sedation, motor
incoordination and psychotomimetic effects in humans, except for nalfurafine in most patients.
Nalfurafine has been used in Japan for treatment of pruritus in hemodialysis patients. We havepreviously found that in male CD-1 mice, nalfurafine produced analgesic and anti-scratch effects
dose-dependently, like U50,488H. However, at doses effective for analgesic and anti-scratch effects,
U50,488H, but not nalfurafine, caused aversion, anhedonia, sedation and motor incoordination. In
this study, we demonstrated that U50,488H, but not nalfurafine, promoted KOR phosphorylation at
T363 and S369 in mouse brains as detected by immunoblotting with phospho-KOR specificantibodies. In addition, U50,488H, but not nalfurafine, caused KOR internalization in the brain of amutant mouse line expressing a fusion protein of KOR conjugated at the C-terminus with tdTomato(KtdT). Furthermore, in phosphoproteomic studies, U50,488H, but not nalfurafine, promoted
phosphorylation of GRK5/6 at S484/T485 in the striatum and cortex, which was shown to enhance
GRK5 activities. Taken together, these data reveal a correlation between agonist-promoted KOR
phosphorylation and behaviors considered to be detrimental side effects, such as sedation, aversion
and motor incoordination. These results also suggest that agonist-induced KOR phosphorylation inmouse brains may be used to screen for KOR agonists devoid of these side effects. (Supported by
NIH grants R01 DA041359 and P30 DA013429)

54

47 - Identification of local GPCR protein interaction networks using proteomics andproximity labeling
Braden T. Lobingier1, Ruth Huttenhain2, Brian Shoichet3, Nevan J. Krogan2, Mark von Zastrow1,2
1
Department of Psychiatry, University of California San Francisco, 2Department of Cellular andMolecular Pharmacology, University of California San Francisco, 3Pharmaceutical Chemistry,
University of California San Francisco, 4Pharmaceutical Chemistry
Cellular activity is inherently organized in space and in time. Here we describe a method to capture
information about these dimensions and define the protein networks underlying rapidly evolving
cellular processes. The approach builds on proximity labeling, combined with quantitative
proteomics and spatial references, to obtain temporally resolved ‘snapshots’ of the environment
around a protein of interest with sub-organelle spatial resolution. We apply this method with labeling
periods of 30 seconds to interrogate the local protein networks engaged by G protein-coupled
receptors upon ligand-induced stimulation. We demonstrate that the methodology provides aquantitative readout of receptor engagement with previously known protein partners, and that itallows discovery of new network components. We now apply this approach to examine the protein
interaction networks engaged by the mu-type opioid receptor when stimulated by full, partial, and G
protein biased agonists.

48 - Sex differences in kappa opioid receptor activated brain networks implicated in the
response to stress and drugs of abuse
Qianhan Ma1, Sue Wonnacott1, Sarah J. Bailey1, Chris P. Bailey1
1
Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, UK
Introduction: Kappa opioid receptors (KOPrs) are involved in both stress responses and addictionrelated behaviour (Bruchas et al (2010) Brain Research 1314:44-55). It has been shown that the roleof KOPrs in motivated behaviour is sex dependent (Russell et al (2014) Biol Psychiatry 76:213-222).
This study aimed to investigate the influence of sex on brain regions that are activated by stress andcompare the effect of in vivo stressors and KOPrs activation.
Methods: Adult (8-13wks) male and female C57BL/6 c-Fos-GFP transgenic mice, in which
expression of green fluorescent protein (GFP) is driven by the activation of c-Fos (Barth et al (2004)
J Neurosci 24:6466-75). Mice were administered either KOPr agonist (U50,488; 20mg/kg) or aphysical stressor – forced swim stress (FSS). Following transcardial perfusion fixation, brain sections
were taken and immunolabelled to assess cFos and cFos-driven GFP expression, to indicate brain
regions activated by the in vivo treatments.
Results: In male mice, both cFos and cFos-driven GFP expression in the nucleus accumbens(NAcc) and prelimbic prefrontal cortex (PFC) were increased by both U50,488 and FSS. In contrast,
U50,488 did not increase cFos or cFos-driven GFP expression in either NAcc or PFC in female mice,
FSS increased expression in NAcc not PFC. The effects of U50,488, but not FSS, were blocked by
pre-administration of the KOPr antagonist norBNI (10mg/kg).
Conclusion: Together these data suggest that sex differences in brain regions activated by KOPr
agonists and physical stressors, potentially reflecting the sex differences seen in the behaviouraleffects of KOPrs.

55

49 - Dissecting Agonist-induced Nociceptin/Orphanin FQ Receptor Phosphorylation in vitro
and in vivo
Anika Mann1, Thomas Guenther1, Lionel Moulédous2, Carine Froment3, Patrick R. O´Neill4, Gloria
Brunori5, Nurulain T. Zaveri6, Michael R. Bruchas4, Brigitte L. Kieffer7, Lawrence Toll5, Stefan Schulz1
1Institute of Pharmacology and Toxicology, Jena Universtiy Hospital, Friedrich Schiller University Jena,
Drackendorfer Str. 1, 07747, Jena, Germany, 2Research Center on AnimalCognition, Center for
Integrative Biology, Toulouse University, CNRS, UPS, France, 3Institut de Pharmacologie et de Biologie
StructurateUniversité de Toulouse, CNRS, UPS, France, 4Departments ofAnesthesiology,
Neuroscience, andPsychiatry, Washington University School ofMedicine, St. Louis, Missouri 63110,
5
Torrey Pines Institute forMolecular Studies, Port St. Lucie, FL, USA, 6Astraea Therapeutics, LLC,
Mountain View, California, 7Douglas Research Center, Department ofPsychiatry, FacultyofMedicine,
McGill University, Canada
The nociceptin/orphanin FQ opioid peptide (NOP) receptor is the fourth member of the opioid
receptor family and a promising target for the development of novel analgesics. The detailed
molecular events underlying its agonist-dependent regulation remain incompletely understood. Here,
we generated a series of phosphosite-specific antibodies and examined agonist-induced NOP
receptor phosphorylation. We found that agonist-induced phosphorylation occurs primarily at four
carboxyl-terminal serine and threonine residues in human NOP receptor, namely S346, S351, T362
and S363. Agonist-induced NOP receptor phosphorylation proceeds with a temporal hierarchy, inwhich S346 appears to be the primary site of phosphorylation. In NOP-eGFP mice, NOP receptor
multisite phosphorylation and internalization occurred in a dose-dependent and agonist-selective
manner that could be blocked by specific antagonists. A comparison of chemically distinct NOP
receptor agonists revealed dissociation between G protein signaling and receptor phosphorylation.
We also show that G protein-coupled receptor kinases 2 and 3 (GRK2/3) cooperate during agonistinduced phosphorylation, which in turn facilitates NOP receptor desensitization and internalization.
Together, we showed for the first time agonist-selective NOP receptor phosphorylation, in vitro as
well as in vivo.
50 - A Stable Heroin Analog Vaccine Formulation that Induces Long Duration Antibody
Titers that Block the Antinociceptive Effects of Heroin and Hydromorphone
Gary Matyas1, Oscar Torres1,2, Agnieszka Sulima3,4, Zoltan Beck1,2, Arthur E. Jacobson3,4, Kenner
C. Rice3,41Walter Reed Army Institute of Research, 2Henry M. Jackson Foundation for the Advancement ofMilitary Medicine, 3National Institute on Drug Abuse, 4National Institute on Alcohol Abuse andAlcoholism, National Institutes of Health
We have recently described a new heroin hapten, 6-AmHap, when conjugated to tetanus toxoid (TT),mixed with Army Liposome Formulation (ALF) as an adjuvant, and used to immunize mice protectedboth mice and rats from both subcutaneous and intravenous heroin challenge (Sulima et al., J. Med
Chem 2018). The vaccine induced antibodies bound to heroin, its degradation products and crossreacted with other opioids. In separate studies, the duration of the antibody response has beenmonitored in both mice and rats. Although the experiments are still ongoing, the antibody titers to 6AmHap have remained high for 6 months following the last immunization. Challenge of the animals
with heroin indicates that vaccine-induced efficacy is still maintained in tail immersion and hot plate
assays. Based on the strong cross-reactivity of the antibodies to hydromorphone from competition
ELISA, mice were immunized with the 6-AmHap-TT conjugate mixed with ALF and aluminumhydroxide adjuvants. The animals were challenged with heroin or hydromorphone at 1 mg/kg by the
subcutaneous route every 20 min. Control animals reached 100% MPE in a tail-flick assay after only
1 dose of heroin or hydromorphone. Immunized animals challenged with heroin required 2-3 doses
to reach 100% MPE, while those challenged with hydromorphone required 3-4 doses. The average
EC50 of the immunized animals was 1.7 ± 0.27 and 1.0 ± 0.15 mg/kg for heroin and hydromorphone,respectively. Based on this data, the 6-AmHap-based heroin vaccine also is expected to haveefficacy against hydrocodone and other opioids.

56

51 - Morphine and HIV-1 Tat-induced calcium flux in hippocampal neurons is dependent on
L-type calcium channels and NMDAR signaling
Virginia D. McLane1, Pamela E. Knapp1,2,3, Kurt F. Hauser1,2,3
1
Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA,
USA, 2Department of Anatomy & Neurobiology, Virginia Commonwealth University, Richmond, VA,
USA, 3Institute for Drug & Alcohol Studies, Virginia Commonwealth University, Richmond, VA, USA
Opiates exacerbate HIV-induced neuropathogenesis within vulnerable regions of the central nervous
system, such as the hippocampus. Prolonged (72 h) exposure to neurotoxic HIV-1 proteintransactivator of transcription (Tat, 100 nM) reduced primary murine hippocampal neuron survival in
vitro, particularly in GAD67+, GABAergic cells (p<0.001). Acute Tat exposure increased neuronal
intracellular calcium concentration ([Ca2+]i) by 193.9 ± 53.9% compared to controls, which was
exacerbated by morphine (312.0 ± 31.5%) and reversed by GABAA agonist muscimol (100 nM, threeway interaction, p<0.001). [Ca2+]i returned to baseline within 20 min in Tat-exposed cells (114.4 ±
30.3%); however, GABAA antagonist bicuculline (20 µM) prolonged Tat-induced elevation of [Ca2+]iin the presence of morphine (208.0 ± 22.8%, p<0.001). To identify key sources of Tat-induced
calcium influx, potential extracellular [NMDAR (MK-801), AMPAR (CNQX), L-type Ca2+ channels(nimodipine, isradipine)] and intracellular [RyR1,3 (dantrolene), σ1R (BD-1063)] calcium sources were
blocked pharmacologically. Tat-induced [Ca2+]i was significantly reduced by nimodipine (10 µM,
p<0.001) and MK-801 (20 µM, p<0.001), but not BD-1063 (1 µM, p=0.381). Morphine-induced
calcium influx was reduced by isradipine (5 µM, p=0.009) and BD-1063 (p=0.038). Bicuculline andmorphine interactive effects within Tat-exposed cells were reduced by CNQX (500 nM, p<0.001).
Overall, these data indicate morphine, bicuculline, and HIV-1 Tat elevate [Ca2+]i within hippocampal
neurons through both independent (i.e., NMDAR, RyR, σ1) and shared (i.e., Cav1.2) calcium sources.
Thus, GABAA signaling appears to ameliorate aspects of Tat-induced overexcitability. The potential
loss of vulnerable GABAergic interneurons within the hippocampus may exacerbate opiate- and HIVinduced neuropathology. Support: NIH R01 DA018633.
52 - Diverse cell types within the vlPAG exhibit unique adaptations to membrane firingproperties after inflammation.
Kylie McPherson1, Katherine Suchland1, Susan Ingram1,2
1
Oregon Health & Science University, 2Oregon Health & Science University, Portland, USA
The periaqueductal grey (PAG) is an important integration site within the descending pain modulatory
pathway that receives diverse inputs. The vlPAG is a highly heterogeneous region with diverse cell
types that have yet to be characterized fully in terms of their response to noxious stimuli and opioids.
Using whole-cell patch-clamp recordings of membrane firing properties in naïve rats we defined 5
distinct cell-types within the vlPAG: onset-spiking, fast-spiking, transiently fast-spiking, randomspiking, and dopamine neurons. Animals that experienced inflammation (2 h), induced by complete
Freund’s Adjuvant (CFA) injected into the hindpaw, have neurons that exhibit hyper-excitablecharacteristics including increased firing frequency and altered firing patterns. Fos expression isrobustly increased 2 h after CFA, with a significant reduction 6 d after CFA that remains significantly
greater than the naïve animals—suggesting altered levels of neuronal activity after CFA that change
between early and persistent inflammation. Studies are examining whether inflammation-induced
Fos expression labels sensitized vlPAG neurons at acute (2 and 24 hours) and after persistent
inflammation (6 days) using Fos-GFP expression in Long-Evans rats. Additionally, the studies use
retrograde fluorescent CTb labeling from the rostral ventromedial medulla (RVM) to examine intrinsic
properties of vlPAG neurons that project to the RVM. The effects of opioids on the intrinsic membrane
properties of these cell types will also be examined.

57

53 - Mobility of the mu opioid receptor as an effector independent assay for alterations insignaling state
Marissa Metz1,2, Reagan Pennock1, Shane Hentges1,2, Diego Krapf3,4
1
Department of Biomedical Sciences, Colorado State University, 2Molecular, Cellular, and Integrative
Neurosciences Program, Colorado State University, 3Electrical and Computer Engineering, Colorado
State University, 4School of Biomedical Engineering, Colorado State University, 5Colorado State
University
Assays for opioid receptor activity typically exploit the signaling of a relevant effector, such as
potassium channel currents or cyclic AMP generation. However, these assays do not directly sample
events occurring at the receptor itself, potentially masking important processes that happen
upstream of effector activation. One way to better understand opioid receptor activity is to directly
observe receptor mobility using single particle tracking. Flag-tagged mu opioid receptors weretracked using spinning disk and TIRF microscopy in response to application of DAMGO. In earlysignaling stages as desensitization commenced but before internalization begins, receptors
exhibited an increase in overall mobility with a decrease in mobile state dwell times withincompartmentalized membrane areas. However, in late signaling stages when internalization begins,
Flag-MORs exhibited decreases in overall mobility with increases in dwell times of remaining mobile
receptors. Further analysis revealed that free portions of mobile states were in part a result of gprotein activation. Association with clathrin accounted for a small portion of both mobile and immobile
tracks, with a larger proportion of immobile receptors colocalized with clathrin when they were treatedwith DAMGO for 10 minutes. Understanding of which mobility states correspond to different signalingstates will allow for better, more direct assays of receptor signaling, and may pose an interesting,effector-free avenue for assaying drugs with different efficacy profiles for opioid signaling pathways.
54 - Multi-site phosphorylation is required for sustained interaction with GRKs and arrestinsin mediating rapid mu-opioid receptor desensitization.
Elke Miess1, Arisbel B Gondin2, Arsalan Yousuf3, Ralph Steinborn1, Nadja Mösslein4, Yunshi Yang4,
Martin Göldner4, Julia G. Ruland4, Moritz Bünemann4, Cornelius Krasel4, MacDonald J. Christie3,
Michelle L Halls2, Stefan Schulz1, Meritxell Canals2
1
Department of Pharmacology and Toxicology, Jena University Hospital - Friedrich Schiller University
Jena, Germany, 2Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences,
Monash University, Melbourne, Australia, 3Discipline of Pharmacology, University of Sydney, Sydney,
Australia, 4Department of Pharmacology and Toxicology, Philipps-University Marburg, Germany
G protein-coupled receptor kinases and β-arrestins are key regulators of μ-opioid receptor (MOP)signaling and trafficking. We have previously shown that high-efficacy opioids such as DAMGO
stimulate a GRK2/3-mediated multisite phosphorylation of conserved C-terminal tail Ser and Thr
residues, which facilitate internalization of the receptor. In contrast, morphine-induced
phosphorylation is limited to Ser375 and it is not sufficient to drive substantial receptor internalization.
Here, we report how specific multisite phosphorylation controls the dynamics of GRK and β-arrestininteractions with MOP and show how such phosphorylation mediates receptor desensitization. We
show that the kinetics of GRK2/3 recruitment to a DAMGO-activated MOP are faster than the kineticsof β-arrestin recruitment. β-arrestin recruitment requires GRK2 activity and MOP phosphorylation,
but surprisingly, GRK recruitment is also dependent on the integrity of the phosphorylation sites in
the C-terminus. Translocation of both regulatory proteins and their stable interaction with MOP aredependent on phosphorylation of Ser and Thr residues within the 375STANT379 motif of the C-tail. Our
results also suggest that other residues outside this motif participate in the initial and transient
recruitment of GRK and β-arrestins. Finally, using complementary patch clamp approaches, we showthat high efficacy agonist desensitization of MOP has two components; a sustained component,
which requires GRK2-mediated phosphorylation and a potential soluble factor, and a rapid
component likely mediated by GRK2 but independent of receptor phosphorylation. Elucidating thesecomplex receptor-effector interactions represents an important step towards a mechanistic
understanding of MOP desensitization that leads to the development of tolerance and dependence.

58

55 - Chronic Pain Enhances Spontaneous Fentanyl Withdrawal in Rats
Andrea T. Lee1, Tammy N. Hilgendorf1, Ram Kandasamy1, Michael M. Morgan1
1
Washington State University, 2WA
Overdose deaths caused by fentanyl and its analogs have increased dramatically in recent years.
Despite over 20,000 deaths caused by fentanyl overdose in the US in 2016, almost all animal
research focuses on morphine dependence. Although morphine and fentanyl produce
antinociception and dependence by binding to the mu-opioid receptor, the distinct signalingmechanisms engaged and short half-life of fentanyl may induce more severe withdrawal symptoms.
This hypothesis was tested by examining the magnitude and duration of spontaneous fentanyl
withdrawal in rats with and without inflammatory pain. Dependence was induced by continuous
subcutaneous fentanyl infusion (1 mg/kg/day) for 3 days. CFA was injected into the hindpaw to
induce inflammation while rats were anesthetized for implantation of the fentanyl pump. Withdrawal
was assessed by measuring depression of home cage wheel running as described previously(Kandasamy et al., 2017). Withdrawal was evident as a 50% reduction in wheel running during the
24 hours following removal of the fentanyl pump in pain-free rats. Running had recovered to nearbaseline levels of running by Day 2. In contrast, a near complete inhibition of wheel running occurredfollowing removal of the fentanyl pump in rats with hindpaw inflammation. Although there was a
gradual increase in wheel running across days, wheel running was significantly depressed
throughout the 6 days of withdrawal assessment. These data indicate that chronic pain significantly
enhances spontaneous fentanyl withdrawal. Such pronounced and prolonged withdrawal likely
contributes to the continued use and abuse of fentanyl and fentanyl analogs.

56 - Exposure of early life stress reduces the expression of μ-opioid receptor in the PAG ofadult mice brain and morphine analgesia
Kazuo Nakamoto1, Asami Imanishi1, Natsuki Kyoya1, Masataka Kasuga1, Shogo Tokuyama1
1
Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kobe Gakuin University
Early life stress relates to the pathogenesis of psychiatric disorders and chronic pain in adult patients.
However, information about the mechanisms for chronic pain which induced by early life stress
exposure is limited. In this study, to elucidate the mechanism underlying early life stress-induced
increase of pain sensitivity, we investigated the changes of each opioid receptors mRNA expression
in some brain area of mice which subjected to maternal separation combined with social isolation(MSSI) as an early life stress. Furthermore, we tested the analgesic effect of morphine on thermal
stimulation in tail flick test. In the periaqueductal gray (PAG) area, a region that is implicated in the
opioid control of nociception, μ-, δ-and κ-opioid receptor (MOR, DOR and KOR) mRNA expression
were significantly decreased in MSSI model mice compared to control mice. In the amygdala, KOR
mRNA is significantly increased in MSSI model mice, but not control mice. In the medial prefrontal
cortex and rostral ventral medulla area, MOR mRNA showed tendency to decrease in MSSI model
mice. However, there are no significant difference between control and MSSI model mice in otherarea of the brain. In the tail flick test, MSSI model mice showed significantly decrease of morphine
induced analgesia compared to control mice. On the other hand, morphine induced hyper-locomotion
did not change between two groups. Finally, we conclude that MSSI induced decrease of MOR
mRNA expression in the PAG area, suggesting that these phenomenon could be induced theincrease of pain sensitivity in MSSI model mice.

5957 - Inside the Net: morphine-induced conditioned place preference and effects on
perineuronal nets in the ventral tegmental area
Toni A.E. Nigro1,2, Dolores Beatriz Vazquez Sanroman1
1
Oklahoma State University Center for Health Sciences, 2Oklahoma State University College of
Osteopathic Medicine
The ventral tegmental area (VTA) is the origin of the brain dopamine-rewarding pathway implicatedin morphine reinforcing properties. VTA dopaminergic neurons activity is modulated by VTA-GABA
neurons. Indeed, GABAergic neurons are important substrates for morphine effects and aresurrounded by perineuronal nets (PNNs). PNNs are aggregations that form net-like structures
surrounding GABAergic interneurons and are important for plasticity during critical periods, such asadolescent neurodevelopment, and during drug-associated learning and memory. The present
experiments examined whether morphine conditioned place preference (CPP) in adolescent rats
lead to changes in the PNNs and neuronal activity within the VTA. Twenty-four Sprague Dawley
adolescent rats were used. In the present set of experiments, we explored the acquisition, extinction,and reinstatement of a morphine-induced CPP using an ‘unbiased’ apparatus for all stages, rats
were given 2-daily 20-minutes sessions for a total of eight pairings for each phase. Rats received 5
mg/kg or saline during conditioning sessions. Further, 8-extintion sessions (drug-free). Followed bya drug-primed (2.5 mg/kg) or saline session. Morphine significantly increased the percentage timeon the drug-paired floor [One way ANOVA: F (1,22)=4.65, p< 0.05; mor-sal 60%; sal-sal 50%].
Morphine-prime induced reinstatement was effective only for 40% of the animals. No sex differenceswere found regarding morphine preference score [X2(1) = 0.85, p=0.68]. At the cellular level,morphine preference decreased PNN expression [X2(1) = 7.91, p<0.01]. Our results showed thatmorphine inducing PNN-dependent plasticity in the brain reward system of adolescent rats.
58 - Cannabinoid Type-1 Receptors can Mediate the Opioid-Sparing Effects of Delta-9tetrahydrocannabinol in Nonhuman PrimatesMark R. Nilges1, Zachary Bondy1, Joseph A. Grace1, Peter J. Winsauer1,2
1Louisiana State University Health Sciences Center, 2Alcohol and Drug Abuse Center of Excellence
Combinations of opioids and cannabinoids can produce synergistic antinociceptive effects. These“opioid-sparing” effects could potentially reduce some of the adverse effects produced by opioids. In
this experiment, thermal antinociception was assessed using a warm-water tail-withdrawal
procedure in nonhuman primates (rhesus macaques). The acute antinociceptive effects ofcumulative doses of heroin were studied in the presence or absence of the cannabinoid receptor
ligands delta-9-tetrahydrocannabinol (Δ9-THC), rimonabant, cannabidiol (CBD), or cannabinol(CBN). We also studied the effects of a serotonin-noreprinephrine reuptake inhibitor, amitriptyline,
as a positive control. When CBN or CBD was administered prior to cumulative doses of heroin, no
shift was evident in the dose-effect curve compared to heroin alone. However, a combination of Δ9THC (1 mg/kg) and heroin shifted the ED50 for heroin 3.4-fold leftward compared to heroin alone. A
selective cannabinoid type-1 receptor (CB1R) antagonist, rimonabant, was then tested to determine
if the enhanced antinociceptive effects produced by Δ9-THC could be antagonized. When rimonabant(0.32 – 1 mg/kg) alone was administered prior to heroin, the dose-effect curve was shifted rightward;
however, this shift was not evident 15 minutes after the cumulative heroin injections. When thesedoses of rimonabant preceeded Δ9-THC, the leftward shift of the Δ9-THC-heroin dose-effect curve
was dose-dependently antagonized. This is consistent with the notion that Δ9-THC can produce
opioid-sparing effects in a CB1R-dependent manner, and rimonabant can produce
antagonist/inverse agonist effects in nonhuman primates.

60

59 - Growth hormone and Insulin-like growth factor-1 promotes the recovery of neurons
after methadone exposure
Erik Nylander1, Sofia Zelleroth1, Fred Nyberg1, Alfhild Grönbladh1, Mathias Hallberg1
1
Uppsala University, Sweden
Background:
Accumulating data suggest that long-term use of methadone may be linked to reduced cognitive
function. These effects are associated with increased neuronal cell death and reduced neurogenesis.
Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) are suggested to act pro-cognitive andneuroprotective in the CNS and may counteract some of these effects. The aim for present study
was to examine if GH/IGF-1 acts protective or increases the recovery of the mitochondrial function
or membrane integrity in methadone-treated cells.
Methods:
Primary cortical cells from rats were harvested and grown for seven days in vitro. Cells were theneither a) exposed to a repeated methadone treatment for three days with or without GH/IGF-1 or b)
exposed to methadone for 24h, washed, and later exposed to GH/IGF-1 for 48h.
The mitochondrial function, as assessed in the tetrazolium-bromide (MTT) assay, and membrane
integrity, as assessed in the lactate dehydrogenase (LDH) assay, were evaluated at the end of theexperiments.
Results:
Growth hormone, but not IGF-1, significantly decreased LDH-release when co-treated withmethadone but no effects was seen in the MTT-assay. Furthermore, both GH and IGF-1 significantly
reduced LDH release and increased the mitochondrial function after cells had been treated for 24hwith methadone.
Conclusion:
Growth hormone protects cells when co-treated with methadone for three days by stabilizing themembrane integrity. No protective effects were seen using IGF-1. However, both GH and IGF-1
improved the recovery of the mitochondrial function and the membrane integrity after acute 24hmethadone treatment.
60 - The non-selective opioid diprenorphine produces delta-opioid receptor-mediated rapid
antidepressant-like effects in mice
Keith M Olson1, Todd M. Hillhouse1, James E Hallahan1, Evan Schramm1, John Traynor1
1
University of Michigan, Ann Arbor
Major depressive disorder (MDD) is the most common mood disorder worldwide with a lifetime
prevalence of ~15%. However, current FDA approved treatments are limited by a multi-week delayed
onset of action and minimal efficacy in some patients. Preclinical evidence indicates delta-opioid
receptor (DOR) agonists and kappa-opioid receptor (KOR) antagonists may produce rapid onset
antidepressant effects and provide a novel depression target in treatment resistant patients [3H]diprenorphine saturation binding experiments in membranes expressing the mu-opioid receptor(MOR); (KDm= 0.31 +/- 0.04); DOR (KDd= 1.1 nM +/- 0.16); and KOR (KOR KDk= 0.36 +/- 0.09)
revealed non-selective affinity, as expected. In vitro[35S]GTPgS assays in the same cell lines
revealed diprenorphine is a DOR and KOR partial agonist (EC50d= 4.1 nM +/- 2.0, EMAXd= 40 % +/5.0; EC50k= 0.71 nM +/- 0.21, EMAXk= 41 % +/- 5.3); it also has potent MOR antagonist against
DAMGO (KB= 0.18 +/- 0.05). In vivodiprenorphine produced anti-depressive-like effects in the tailsuspension test and the novelty-induced hypophagia test that were blocked by the DOR-selective
antagonist naltrindole. While classical DOR agonists, such as SNC80, produce seizure activity,
diprenorphine did not produce seizures and blocked SNC80 mediated seizures indicating that higher
DOR efficacy is needed to produce seizures than anti-depressant-like effects. Alternatively, the
DOR/KOR partial agonist and MOR antagonist profile of diprenorphine may mitigate against
seizures. Future directions include the development of diprenorphine analogues to determine if
modulating DOR/KOR agonist activity improves diprenorphine anti-depressant-like efficacy.

6161 - Inhibition of alpha7 nicotinic receptors in heroin-primed reinstatement of conditioned
place preference
Josephine C Palandri1,2, Sharon Smith3, David J Heal3, Susan Wonnacott2, Chris Bailey1
1
Department of Pharmacy & Pharmacology, University of Bath, Bath BA2 7AY, UK, 2Department of
Biology & Biochemistry, University of Bath, Bath BA2 7AY, UK, 3RenaSci, Nottingham, UK
Drug addicts form associative memories in the context of drug use, which can trigger cravings andprompt relapse (Kauer JA & Malenka RC (2007) Nat Rev Neurosci 8:844-858). We and others haveshown that methyllycaconitine (MLA), an α7-nicotinic acetylcholine receptor (α7nAChR) antagonist
attenuates the reinstatement of morphine-primed conditioned place preference (CPP) in rodents(Feng B et al (2011) Behav Brain Res 220:100-105; Wright VL et al (2018) Addict Biol in press). The
aim of this study is to investigate the effects of MLA on CPP with a more clinically relevant opiate,
heroin. All rats (male, Wistar, 6-7 weeks) underwent heroin CPP (1 injection daily over 4 days,
alternating between saline and heroin (1mg/kg) (Cordery SF et al. (2014) Addict Biol 19:575-86). To
test the effects of MLA on heroin CPP acquisition, rats were given MLA (4mg/kg) 20 minutes prior toeach conditioning injection. To test the effects on reinstatement, animals underwent CPP extinction
and received either saline or MLA (4mg/kg) 20 minutes prior to heroin-priming (1mg/kg). Although
MLA pre-treatment had no effect on the acquisition of heroin CPP, a single MLA dose prereinstatement completely prevented heroin-primed reinstatement. These data show that MLA does
not affect heroin reward per se, but can selectively affect heroin-related memories, suggesting a rolefor α7nAChRs in opiate relapse. The ability to pharmacologically distinguish between the different
stages of addiction is exciting for potential therapies targeted at opiate relapse. Analysis of the effectof MLA on intravenous self-administration of heroin is ongoing.
62 - Clinical Characteristics of Opioid Use Disorder
Sarah A. Palumbo1, Vanessa Troiani2, Kayleigh M. Adamson2, Sarah Robishaw2, Sarathbabu
Krishnamurthy2, Raghu Metpally2, Laney Jones2, Sarah Pendergrass2, Joseph Chronowski2,
Christopher Lam2, Richard C. Crist3, Glenn A. Doyle3, Thomas Ferraro4, Mingyao Li3, Wade H.
Berrettini3, Janet Robishaw1
1
Florida Atlantic University, Boca Raton, FL, 2Geisinger, Danville, PA, 3University of Pennsylvania,
Philadelphia, PA, 4Cooper Medical School of Rowan University, Camden, NJ
The Opioid Epidemic affects 100 million people in the United States with 115 people per day dying
from an opioid overdose. The US Burden of Disease Collaborators lists Pennsylvania as 4th in thenation for drug abuse deaths at 708 per 100,000 in 2016; and, CDC data reports Pennsylvania as
showing the largest increase in Emergency Department visits for opioid overdoses from July 2016
to September 2017. The goal of this NIH-funded study is to investigate clinical and genetic predictorsof Opioid Use Disorder (OUD) among chronic pain patients served by the Geisinger health system
in Pennsylvania.
Using electronic health record data, we identified two groups: 1) MUA, participants with a medication
use agreement for opioids; and 2) TMUA, participants with a terminated MUA resulting from ruleviolations indicative of OUD. Notably, the TMUA group showed a higher prevalence of International
Classification of Diseases (ICD) pain codes for lumbar/sacral region (p = 10-13), musculoskeletal
system (p = 10-9), and multiple systems (p = 10-11). In addition, the TMUA group exhibited a higher
frequency of ICD10 codes for polysubstance use disorders, including nicotine (p = 10-67), alcohol (p= 10-21), and other substances (p = 10-78). Finally, the TMUA group revealed a higher incidence of
ICD10 codes for mental health disorders, including anxiety (p = 10-18) and major depression (p = 1012). Having identified one group of stable users and another of presumptive OUD patients, we willconduct genome-wide association studies to identify a polygenic risk score for development of OUD.

62

63 - Development of vaccines to treat heroin and prescription opioid abuse suitable forclinical trials
Baruffaldi F1, Raleigh M1, Peterson S1, Laudenbach M1, Winston S2, Runyon S3, Carroll FI3, Pentel
PR1,4, Pravetoni M1,41
Minneapolis Medical Research Foundation, MN, 2Winston Biopharmaceutical Consulting, CO,3
RTI International, NC, 4University of Minnesota Department of Medicine, MN
Vaccines for the treatment of opioid use disorders and prevention of fatal overdoses have shownpromising pre-clinical efficacy. However, readying vaccine candidates for human testing presents
unique quality and regulatory challenges. This study first focused on optimization andcharacterization of a conjugate vaccine targeting oxycodone, and then applied this technology toformulate analogous heroin and fentanyl vaccines. To this end, several batches of an oxycodone
hapten conjugated to GMP-grade keyhole limpet hemocyanin (OXY-KLH) carrier protein adsorbedon alum adjuvant were produced to test the effect of coupling reaction temperature, time, buffer
composition, and protein:alum ratio on vaccine immunogenicity and efficacy in rats. Small conjugates
could be evaluated by gel electrophoresis and size exclusion chromatography but were notimmunogenic while larger conjugates were immunogenic but their size could only be measured bydynamic light scattering (DLS). OXY-KLH purification using tangential flow filtration (TFF, scalable)
was compared to ultrafiltration (non-scalable) and both elicited equivalent oxycodone-specific serum
IgG antibody titers and blockage of brain oxycodone compared to control. Following TFF, levels ofresiduals were negligible. OXY-KLH formulated in alum was stable over 6 months at 4o-37oC as
confirmed by immunogenicity and reduced brain oxycodone levels by 66 – 73% compared to control
in rats. An analogous heroin/morphine vaccine (M-KLH) was formulated under conditions similar tothe oxycodone vaccine. An analogous fentanyl vaccine (F-KLH) showed efficacy in blocking fentanyl
distribution to the brain in mice and rats. These data suggest that KLH-based opioid vaccines
adsorbed on alum adjuvant are suitable for pharmaceutical manufacturing.
64 - In vivo EEG signatures during chronic fentanyl exposure, spontaneous withdrawal, andprotracted abstinenceCorey Puryear1, Angelica Patino1, Connie Sanchez-Morillo1
1
Alkermes, Waltham, USA
Preclinical studies in rodent models of opioid addiction are commonly used to characterize long-term
effects on brain function. However, understanding the effects opioid agonists on cortical circuit
activity may better elucidate the functional impact of opioids. Chronic opioid exposure andwithdrawal has been shown to produce dynamic changes in oscillatory activity in brain areas central
to the pathophysiology of drug addiction. Therefore, it is important to understand whether these
dynamic changes can be observed in skull-level EEG recordings, which hold greater translational
potential to clinical EEG studies. As such, we characterized the effects of chronic fentanyl exposure,withdrawal and abstinence on cortical EEG patterns.
Fentanyl-treated rats exhibited significant physical dependence, as evidenced by transient weightloss and somatic phenotypes during withdrawal. Cortical EEG dynamics displayed distinct
oscillatory signatures that varied according to the phase of fentanyl exposure orwithdrawal. Fentanyl exposure caused a transient increase in delta (1-4Hz) and alpha (8-12Hz)
power, and a transient decrease in gamma (30-80Hz) power. In contrast, spontaneous withdrawal
was characterized by acute, transient increases in multiple frequency bands. Interestingly, alphapower remained increased above baseline levels during the subsequent three weeks of abstinence.
These results demonstrate the profound impact of fentanyl on cortical circuit function. Normalizationof opioid-induced changes in oscillatory dynamics, and subsequent rebound during withdrawal
suggests the engagement of allostatic mechanisms that may compensate for the persistentactivation of mu-opioid receptors. The persistent elevation of alpha power during protracted
abstinence suggests that chronic fentanyl exposure has a long-term impact on cortical circuitry.

63

65 - Differential Activation and Cellular Localization of Protein Kinases in the Periaqueductal
Gray Following Morphine Treatment
Akila Ram1, Erin N. Bobeck1
1
Department of Biology, Utah State University
Morphine is one of the oldest, and one of the most commonly prescribed painkillers for acute andchronic pain despite detrimental side effects after long-term use. Morphine functions as an agonistof the m-opioid receptor (MOPr), which is an inhibitory G-protein coupled receptor. In this study, we
examine the molecular consequences of chronic morphine treatment in the mouse ventrolateralperiaqueductal gray (vlPAG) which is a key brain region known to be involved in the development oftolerance. The purpose of this research was to investigate activation of different kinases within thevlPAG following repeated morphine treatment. Wild-type mice were administered 9 boluses ofmorphine (10 mg/kg, s.c.) or saline over 5 days, following which the brains were harvested. We
performed fluorescence immunohistochemistry to map the localization and phosphorylation of
extracellular signal-regulated kinase (ERK), protein kinase-C (PKC), and protein kinase-A (PKA).
We observed significantly greater activated ERK in the vlPAG of morphine-treated animals. We also
note that phosphorylated PKC tends to localize to the plasma membrane following morphine
treatment. Further, we observed that phosphorylated PKA tends to localize to the nucleus and that
there is a significant reduction in nuclear punctae of pPKA following morphine treatment. In addition,
we observed putative differential activation patterns of all three proteins along the rostral-caudal axis.
Taken together, this study demonstrates a differential activation and localization of ERK, PKC and
PKA, and opens avenues to explore the role of chronic morphine treatment on G-protein or β-arrestinsignaling and kinase nuclear transport.
66 - Sex differences in plasticity and stress-related genes in the rat following oxycodone
conditioned place preference
Matthew Randesi1, Yan Zhou1,2, Jason Gray3, Batsheva Reich4, Bruce S. McEwen3, Teresa Milner4,
Kreek 1
1
The Rockefeller University, New York, USA, 2Rockefeller University, 3The Rockefeller University,
New York, USA, 4Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York,NY
Oxycodone abuse is a pressing public health issue. This study was conducted to determine if there
are sex differences in gene expression in the hippocampus of rats following oxycodone conditioned
place preference. Rat brains were harvested and the medial (dentate gyrus/CA1) and lateral(CA2/CA3) dorsal hippocampus were isolated. Following total RNA isolation, cDNA was preparedfor gene expression analysis using a RT2 Profiler PCR expression array (Qiagen, Inc.). This custom
designed qPCR expression array contained a total of 25 genes; including those genes coding for
opioid peptides and receptors, as well as candidate genes involved in synaptic plasticity, including
those upregulated following oxycodone self-administration in mice, and genes involved in stressresponses. The gene expression levels of male and female oxycodone exposed rats were comparedto male and female saline controls.
Gene expression in the medial hippocampus saw twice as many significant changes in gene
expression than in the lateral hippocampus. Within the dentate gyrus/CA1 region, there were nearly
twice as many sex (male/female) differences found than there were condition (saline/oxycodone)
differences. We found a significant Sex x condition interaction for the expression levels of the genethat codes for protein kinase B (Akt1) in the medial region, with oxycodone exposed females having
significantly greater expression that both control females as well as oxycodone males(P=0.0497 and P=0.0032, respectively). In conclusion, we found modest sex- and regionaldifferences in expression of opioid, stress response, plasticity and kinase/signaling related genes in
the rat hippocampus following oxycodone exposure.

6467 - The effects of remifentanil dose on the acquisition and persistence of responding for
drug-paired cues.
Stephen H. Robertson1, Emily M. Jutkiewicz1
1
University of Michigan
Drug-paired cues take on reinforcing properties that can promote drug seeking and taking. Previous
research has shown that, following response-independent infusions of remifentanil paired with a cue,rats learned to make a novel response for presentations of a drug-paired cue to a greater extent thancontrol rats. In the current experiment, we first established a cue as a conditioned reinforcer bypairing i.v. infusions of remifentanil (1.0, 3.2, or 10.0 ug/kg/infusion) with the cue. Twenty infusionsof remifentanil were paired with 20 cue presentations that were delivered according to a variable
time 3-min schedule each day for 5 days. The control group received the same number of drug
infusions and cue presentations, but they were not explicitly paired. For the next 28 sessions, rats
were allowed to freely respond on a nose poke manipulandum for presentations of the drug-pairedcue, which were delivered according to a random ratio 2 schedule. We found that responding to
produce drug-paired cues was elevated in the experimental group relative to the control group. The
number of responses emitted was dose-dependent such that following 3.2 and 10 ug/kg/infusion, but
not 1 ug/kg/infusion, animals in the experimental group responded more than controls. For animals
conditioned with 3.2 ug/kg/infusion, responding persisted for 24 days; whereas responding following
conditioning with 10 ug/kg/infusion persisted for 7 days. These findings demonstrate that drugpaired cues sustain drug seeking behavior, suggesting that Pavlovian conditioning processes
contribute to prolonged drug use.
68 - Local mu-opioid receptor antagonism blunts evoked phasic dopamine release in rat
nucleus accumbens
Alexander Gómez-A1, Tatiana A. Shnitko1, Haley Barefoot1, Eleanor L. Brightbill1, Leslie A.
Sombers2, Saleem M. Nicola3, Donita L. Robinson1
1
University of North Carolina - Chapel Hill, 2North Carolina State University, Raleigh, NC, 3Albert
Einstein College of Medicine, Bronx, New YorkThe present study addressed the hypothesis that mu-opioid receptors (MORs) within the nucleus
accumbens (NAc) regulate dopamine release. Specifically, we infused the MOR antagonist CTAP
(D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) into the NAc core while evoked dopamine release
was measured by fast-scan cyclic voltammetry. We report that CTAP dose-dependently inhibited
electrically-evoked dopamine release, with full blockade achieved with the 8 µg dose. In contrast,
evoked dopamine release increased after nomifensine infusion and was unchanged after vehicle
infusion. These findings demonstrate profound local control of dopamine release by MORs within
the NAc core, which have implications for regulation of reward processing.

6569 - The Trends of Opioid Prescription Patterns Among Patients with Fibromyalgia
CVM Sarmento1, M Maz1, T Pfeifer1, W Liu1
1
The University of Kansas Medical Center
Although opioid medications may be effective for the management of acute pain, several studies
have been reporting a lack of effectiveness of opioid treatment in chronic pain conditions such as
fibromyalgia (FM) and Rheumatoid Arthritis (RA), yet previous studies speculated that opioidprescriptions for patients with FM have been increasing in the past years. Aim: This study aimed to
identify patterns of prescribing opioid medications for patients with FM in the past eight years at the
University of Kansas Medical Center. Methods: Fibromyalgia, chronic pain, narcotics, opioids,
medication, and drugs were used as search terms on the Healthcare Enterprise Repository for
Ontological Narration (HERON) database to identify patients at our institution with a diagnosis of FM
who had received opioid prescriptions from January 1st, 2010 to December 31st, 2017. The collected
data were analyzed descriptively. In addition, a Chi-square test for trend was used to analyze apossible linear relationship between the proportions over time Results: Although opioid medications
were prescribed more frequently in 2010 (40%) and 2011 (42%), the number of opioid prescriptions
have decreased since 2012 and reached the lowest number in 2016 (27%). The Chi-square test fortrend shows that from 2010 to 2017 the prescriptions of opioids had a statistically significant
decrease, p < 0.0001. Conclusion: Contrary to the previous literature and the hypothesis that opioids
had been increasingly prescribed for FM patients in the past decade, the findings of this analysis
indicated that the frequency of opioid prescriptions has decreased since 2012.

70 - HA-epitope tag knockin mice demonstrate lack of µ-opioid receptor splice variants inmouse brain
Sebastian Fritzwanker1, Andrea Kliewer1, Stefan Schulz1
1
Friedrich Schiller University, Jena, Germany
GPCRs are notoriously difficult to detect in native tissues. In an effort to resolve this problem, we
have developed a novel mouse model with insertion of HA-epitope tag sequence fused to the aminoterminus of the µ-opioid receptor. Here, we provide proof-of-principle that this approach provides a
powerful means for highly efficient immunodetection of low abundant GPCR targets in their tissuesof origin. We show that the HA-tag facilitates both super-resolution imaging of endogenous receptors
and quantitative mass spectrometry of biochemically isolated receptors. Mass spectrometricanalyses confirmed post-translational modifications and protein-protein-interactions of MOP
previously observed in cultured cells, most notably agonist-selective phosphorylation of carboxylterminal serine and threonine residues and activity-dependent interaction with Gai1 and Gao proteins.
Mass spectrometry also unequivocally identified the carboxyl-terminal 387LENLEAETAPLP398 motif,
which is part of the canonical sequence. At least 14 isoforms have been proposed to arise fromalternative splicing of the MOP carboxyl-terminus such that this sequence is specifically replaced byvarious unrelated sequences while the rest of the receptor protein remains unchanged.
Unexpectedly, our mass spectrometric analysis failed to detect any of the alternative sequences.
The 387LENLEAETAPLP398 motif is the epitope of the rabbit monoclonal antibody UMB-3. The use of
UMB-3 for multiple successive rounds of immunodepletion revealed that mouse brain homogenates
contain >98% of HA-tagged MOP which also contain the UMB-3 epitope. We conclude that nearly
all MOP receptors in mouse brain contain the canonical amino acid sequence and that alternative
spliced variants may represent heteronuclear mRNA that is either not or poorly translated.

66

71 - The association of long-term opium use with overall and cause-specific mortality:
Results from a long-term, prospective population-based study
Ramin Shakeri1, Farin Kamangar2, Hossein Poustchi3, Akram Pourshams3, Shahin Merat3, Arash
Etemadi4, Siavosh Nasseri Moghaddam1, Masoud Sotoudeh5, Masoud Khoshnia6, Abdolsamad
Gharavi6, Amir Ali Sohrabpour5, Farhad Islami7, Atieh Rahmati5, Christian C Abnet4, Sanford M
Dawsey4, Nicholas E Day8, Paul D Pharoah8, Paolo Boffetta9, Paul Brennan10, Neal D Freedman4,
Reza Malekzadeh5
1Digestive Oncology Research Center, Digestive Disease Research Institute, Shariati Hospital,
Tehran University of Medical Sciences, Tehran, Iran, 2Department of Biology, School of Computer,
Mathematical, and Natural Sciences, Morgan State University, Baltimore, Maryland, USA, 3Liverand Pancreaticobiliary Research Center, Digestive Disease Research Institute, Tehran Universityof Medical Sciences, Tehran, Iran, 4Metabolic Epidemiology Branch, Division of Cancer
Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, Maryland, USA, 5Digestive
Disease Research Center, Digestive Disease Research Institute, Tehran University of Medical
Sciences, Tehran, Iran, 6Golestan Research Center of Gastroenterology and Hepatology, Golestan
University of Medical Sciences, Gorgan, Iran, 7Surveillance and Health Services Research,
American Cancer Society, Atlanta, Georgia, USA, 8Departments of Oncology and Public Healthand Primary Care, University of Cambridge, Cambridge, UK, 9The Tisch Cancer Institute, Ichan
School of Medicine at Mount Sinai, New York, USA, 10Genetic Epidemiology Group, International
Agency for Research on Cancer, Lyon, France
Background: Over 35 million people use opioids for medicinal or recreational purposes. Opioid
overdose is well known to cause death. However, there is a dearth of data on the long-term healtheffects of opioids, including opium itself. Therefore, we compared overall and cause-specific mortalityof long-term opium users to non-users.
Methods: Detailed and validated data on opium use were obtained at baseline from 50,045
participants of the Golestan Cohort Study. Data were also collected on other covariates, including
sex, age, tobacco and alcohol use, education, and income.
Results: At baseline, 17% (n=8,487) of the study participants reported ever using opium, of whom
89.8% were current users. During 500,718 person-years of follow-up, 6,466 of the participants died.
Ever use of opium was associated with higher risk of all-cause mortality, with an adjusted HR (95%
CI) of 1.68 (1.58-1.79). It was also significantly associated with higher risk of death from ischemic
heart disease (1.92; 95% CI 1.66-2.22), cerebrovascular disease (1.41; 1.17-1.71), cancers of theesophagus (1.47; 1.07-2.02) and stomach (1.44; 1.04-1.98), COPD (4.38; 2.95-6.51), asthma (2.96;
1.39-6.30), liver disease (2.83; 1.72-4.63), and infectious diseases (1.72; 1.22-2.42). The
associations persisted after stringent sensitivity analyses. Overall, 40% of deaths among opium
users, and 10% of all deaths in this population were attributable to opium use.
Conclusions: Together, these results strongly suggest that chronic opium use causes numerous
chronic diseases, including cardiovascular disease and several forms of cancer. Future studies inother populations using these and other forms of opioids are urgently needed.

67

72 - Predictive factors for quality of life and impact of physical activity in Korean breast
cancer survivors
Hyun Hwa Shin1, Chul-Min Kim1, Yoon Jee Oh1
1
The Catholic University of Korea, Seoul, Republic of Korea
Background Improving long term life quality after cancer management is a growing concern forbreast cancer survivors. We aimed to investigate the associated factors and the impact of regularphysical activity on life quality in breast cancer survivors.
Methods Using the data from 2007 – 2012 Korea National Health and Nutrition Examination Survey,
155 subjects with a history of breast cancer were included. Life quality was assessed by EQ-5D
index, and EQ-5D index level above mean value was regarded as high life quality. Association
between various physical activities and life quality was analyzed.
Results Among inclusion, 104 (67.1%) had high life quality (EQ-5D index ≥0.87) and 51 (32.9%)
had low life quality (EQ-5D index <0.87). Of those, 13.7%, 16.4%, and 60.2% were regularly doing
high, moderate, and light intensity physical activity. Young age, current economic activity, no
bereaved spouse, favorable subjective health status, absence of arthritis or myocardial infarction or
angina, and the presence of walking and stretching exercise were significantly associated with highlife quality. Presence of high or moderate intensity exercise did not affect life quality. In multivariate
analysis, subjective health status, arthritis, walking, and stretching exercise were independently
associated with high life quality.
Conclusion Light intensity exercise including walking and stretching is associated with high life
quality in breast cancer survivors. Comprehensive lifestyle management considering subjective
health status, musculoskeletal disease, and encouraging light intensity exercise are important tomaintain favorable life quality.

73 - A genetically encoded biosensor reveals location bias of opioid drug action
Miriam Stoeber2, Damien Jullie2, Braden Lobingier2, Jan Steyaert3, Peter W. Schiller4, Aashish
Manglik2, Mark von Zastrow2
1
UCSF, Department of Psychiatry, 2University of California San Francisco, San Francisco, USA,
3
Structural Biology Brussels, Vrije Universiteit Brussel, Belgium, 4Clinical Research Institute ofMontreal
Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but
distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental questionis how drugs differ from peptides in their actions on target neurons. Here we show that drugs differ
in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor
that directly detects ligand-induced activation of ORs and uncover a real-time map of thespatiotemporal organization of OR activation in living neurons. Peptide agonists produce acharacteristic activation pattern initiated in the plasma membrane and propagating to endosomes
after receptor internalization. Drugs produce a different activation pattern by additionally driving OR
activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic
arbor. These results establish an approach to probe the cellular basis of neuromodulation and revealthat drugs distort the spatiotemporal landscape of neuronal OR activation.

68

74 - The effects of opioid sensitization on incentive sensitization and dopamine
neurotransmission
Catherine Thomas1, Cassandra Klune1, Alicia Zumbusch1, Una Mrdja1, Stephanie Borgland1,2,
Vedran Lovic1,2,3
1
University of Calgary, 2Hotchkiss Brain Institute, 3ACHRI
Through repeated pairings with a drug, drug-associated stimuli or cues (e.g., needles, syringes, pillbottles) can become imbued with incentive (motivational) value. This incentive property empowers
drug cues to instigate and intensify drug seeking, consumption, and relapse. Repeated drug
exposure produces enduring changes that can intensify the physiological and behavioural effects offurther drug exposure (sensitization). All drugs of abuse potentiate dopamine (DA)neurotransmission in the nucleus accumbens (NAcc), a key node in the brain’s reward circuitry
thought to be necessary for the attribution of motivational value to drugs and drug-cues. The effects
of opioid sensitization on attribution of incentive value to an opioid-cue and cue-evoked DA
neurotransmission remain unknown. We found that an opioid cue (compound light-tone) gained
incentive value after a single Pavlovian cue-drug pairing session. This effect was potentiated by a
10-day opioid sensitization regimen (i.e., escalating doses of remifentanil in the absence of any
discrete cues). Furthermore, opioid sensitization leads to significantly greater cue-evoked DA
neurotransmission, compared to non-sensitized controls. The relationship between opioid preexposure, incentive value attribution and cue- and drug-evoked DA neurotransmission may offer aunique opportunity to predict the ability of drug cues to spur on drug seeking, consumption, andrelapse.
75 - Modulation of opiate reward and intake, as well as antinociceptive effects by the
OPRM1 A118G SNP.
A. Thorsell1, L. Holm1, M. Heilig1
1
CSAN/IKE, Linköping University, Sweden
Both the therapeutic response to mu-opioid receptor (MOR) antagonists, as well as sensitivity to therewarding effects of MOR-agonists may be moderated by a variation at the mu-opioid receptor gene
locus (OPRM1: Oprm-A118G). We have previously generated and described a “humanized” mouse
carrying the respective human OPRM1 A118G alleles (i.e.118AA and 118GG). Here, we examine
the role of the OPRM1 A118G variation in reward sensitivity and opiate intake, using animal models
of drug reinforcement and reward. Reward-related behavior for opiates (including morphine,
oxycodone) was measured using the Conditioned Place Preference (CPP) model. Drug intake wasmeasured using an oral limited access model as well as a continuous access paradigm. Pain
sensitivity was evaluated using the tail-flick and hot plate tests, and anxiety-related behavior during
acute withdrawal following opiate-administration was measured on the elevated plus-maze (EPM).
The OPRM1-A118G’s as well as C57Bl6/J(Rj) were used in the experiments.
The OPRM1 118AA carriers displayed a blunted anxiogenic behavioral response following acutewithdrawal from opiates compared to the 118GG’s. Additionally, the 118GG’s overall displayed more
pronounced CPP, and furthermore at doses not inducing CPP in the C57Bl6/J for morphine.
Oxycodone conditioning generated strong CPP in all animals used. A higher consumption of oral
morphine was seen in the 118GG’s compared to the 118AAs. Pain responses were also differentiallymodulated by morphine, but not by oxycodone.
In conclusion, for morphine and oxycodone, the OPRM1 A118G modulates reward sensitivity in amanner not easily related to the antinociceptive effects of the respective compounds.

69

76 - Interaction between spinal high-mobility group box-1 and glial cells in global cerebral
ischemia-induced mechanical allodynia
Shogo Tokuyama1, Wataru Matsuura1, Shinichi Harada1, Keyue Liu2, Masahiro Nishibori2
1
Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kobe Gakuin University,2
Department of Pharmacology, Graduate School of Medicine, Dentistry and Pharmacological
Sciences, Okayama University
Central post-stroke pain (CPSP) is one of the complications of cerebral ischemia and neuropathic
pain. We have previously shown that spinal high-mobility group box-1 (HMGB1) plays an importantrole in the induction of CPSP. It has been reported that HMGB1 exacerbates inflammation and pain
condition through its receptor as toll-like receptor 4 (TLR4) or receptor for advanced glycation endproducts (RAGE). Furthermore, the glial cells also involve in pain exacerbation. In this study, weinvestigated whether the interaction between spinal glial cells and HMGB1 signaling is involved in
the induction of CPSP. 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 Freytest. Spinal HMGB1, microglia and astrocyte expression were clearly increased on day 3 after BCAO.
Although HMGB1 colocalized with NeuN (marker of neurons), but not with Iba1 (marker of microglia)
and GFAP (marker of astrocyte) on day 3 after BCAO. Intrathecal (i.t.) injection of
lipopolysaccharides from Rhodobacter sphaeroides (LPS-RS, a TLR4 antagonist) and lowmolecular-weight heparin (LMWH, a RAGE antagonist) significantly suppressed mechanicalallodynia on day 3 after BCAO. In addition, BCAO-induced increase of spinal microglia and astrocyte
were suppressed by i.t. injection of anti-HMGB1 monoclonal antibody and LPS-RS administration,
but not LMWH administration. These results suggest that the interaction between spinal glial cells
and HMGB1/TLR4 may be involved in the induction of CPSP. On the other hand, HMGB1/RAGE
signaling may relate to other mechanisms instead of spinal glial cells activation for CPSP.

77 - Identifying a role for gonadotropin-releasing hormone and kisspeptin neurons in thedevelopment of opioid-induced hypogonadism
Karen J. Tonsfeldt1, Laura J. Cui1, Pamela L Mellon1
1
University of California San Diego
One understudied aspect of opioid use is the development of opioid-induced hypogonadism, which
occurs in the vast majority of opioid users regardless of sex, route of administration, or duration ofuse. Hypogonadism is characterized by low levels of estrogen and testosterone. The hypothalamuspituitary-gonad axis consists of Kisspeptin (Kiss1) neurons that stimulate gonadotropin-releasing
hormone (GnRH) neurons. Pulses of GnRH stimulate pulses of luteinizing hormone (LH) and folliclestimulating hormone (FSH) from the pituitary to act on the gonads to promote the production of
estrogen and testosterone. We are exploring the actions of opioids and mu-opioid receptor signalingin Kiss1 and GnRH neurons. We have identified the mu opioid receptor (MOR) in the immortalized
kisspeptin neurons, KT-aRs, and GnRH neurons, GT1-7s, using qPCR and dermorphin-A594
binding. We have also found that morphine or DAMGO treatment decreases Kiss1 and GnRH
transcriptional activity. GT1-7 cells transfected a GnRH-luc showed reduced GnRH transcription
following morphine treatment. KT-aR cells treated with DAMGO show a marked decrease in Kiss1
mRNA using qPCR. These studies suggest a direct effect of MOR on Kiss1 and GnRH transcription,
which may contribute to the development of opioid-induced hypogonadism. Responsiveness of
GnRH neurons to kisspeptin can be measured by the increase in serum LH following exogenous
kisspeptin challenge. The increase in LH following kisspeptin administration was abolished in acutely
morphine-treated mice, indicating interference of GnRH neuron responsiveness by morphine.
Overall, our preliminary findings are beginning to establish the roles of the kisspeptin and GnRH
neurons in the etiology of opioid-induced hypogonadism.

70

78 - Further evidence for the utility of central μ-opioids for cancer patients.
Kazuhiro Torigoe1, Yusuke Hamada2,3, Michiko Narita2, Yoshiaki Machida1, Tomohisa Mori2, Naoko
Kuzumaki2, Minoru Narita2,3
1Div. Practical Edu., Hoshi Univ., Tokyo, Japan, 2Dept. Pharmacol., Hoshi Univ., Tokyo, Japan, 3LStaR, Hoshi Univ., Tokyo, Japan
In a clinical setting, μ-opioid agonists are widely used for pain relief of cancer patients. Generally, itis thought that the peripheral μ-opioid receptor is involved in the occurrence of side effects such as
constipation. Although centralμ-opioid receptors are thought to be important for the development ofanalgesic effects, a little is known about the effects of peripheral and central μ-opioids on tumor
growth. Here we investigated the effect of μ-opioid receptor stimulation on tumor growth in the
peripheral or central region by activating the peripheral μ-opioid receptor or artificially activating the
hypothalamic μ-opioidergic neurons. Lewis lung carcinoma cells were transplanted subcutaneously
into the right hip of the mouse, and tumor-bearing animal were prepared.Quantitative analysis oftumor volume was performed in saline-treated, loperamide, a peripheral μ-opioid receptor agonist,(10 mg/kg/day, b.i.d, i.p.) - treated or methylnaltrexone (MNTX), a peripheral μ-opioid receptor
antagonist, (10 mg/ kg/day) - treated mice at 4,7,11 and 14 days after tumortransplantation. Treatment of loperamide resulted in tumor growth. On the other hand, treatment
ofMNTX inhibited tumor growth. Furthermore, the activation of pro-opiomelanocortinneurons in
the arcuate nucleus significantly suppressed tumor growth via the activation of cell-mediatedimmunity. These findings suggest that the stimulation of central μ-opioid receptors may enhance
antitumor immunity via cell-mediated immunity and the use of μ-opioids that selectively activate
central μ-opioid receptors may produce not only more effective analgesic effect but also antitumor
effect.
79 - Antinociceptive and Locomotor Effects of Fentanyl-Related Substances in Mice
Neil B. Varshneya1, D. Matthew Walentiny1, Lea T. Moisa1, Teneille D. Walker2, Luli R. Akinfiresoye2,
Patrick M. Beardsley1
1
Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA,
USA, 2Diversion Control Division, Drug Enforcement Administration, United States Department ofJustice, Springfield, VA, USA
In 2016, drug overdose was the leading cause of death for Americans under the age of 52 andresulted in more than 42,000 opioid-related fatalities. A five-fold increase in deaths involving fentanyl
or fentanyl analogs between 2013 and 2016 contributed to this problem. In the present study,
fentanyl-related substances were tested in adult male Swiss Webster mice for their effects on
locomotion and antinociception and compared to those of fentanyl and morphine. In locomotor
activity tests, fentanyl (1 and 10 mg/kg), morphine (100 and 180 mg/kg), crotonyl fentanyl (10 mg/kg),
and para-fluorobutyrylfentanyl (10 and 100 mg/kg) produced significant (p ≤ 0.05) dose-dependent
increases in locomotion. However, valeryl fentanyl was without effects on locomotion up to 100
mg/kg. In warm-water tail-withdrawal tests, fentanyl (0.1 and 1 mg/kg), morphine (10 and 32 mg/kg),
crotonyl fentanyl (0.01-10 mg/kg), para-fluorobutyrylfentanyl (1-10 mg/kg), and valeryl fentanyl (10and 32 mg/kg) produced significant (p ≤ 0.05) dose-dependent increases in antinociception withincreasing ED50s (CI) for doing so of fentanyl [0.08 (0.04-0.16)] > crotonyl fentanyl [0.23 mg/kg(0.18-0.29)] > para-fluorobutyrylfentanyl [0.91 mg/kg (0.46-1.58)] > valeryl fentanyl [6.43 mg/kg(3.91-10.46)] > morphine [7.82 mg/kg (5.42-11.02)]. Naltrexone pretreatment (1 mg/kg) increased
antinociceptive ED50s several fold with decreasing magnitude of valeryl fentanyl (11.90x) > parafluorobutyrylfentanyl (10.87x) > crotonyl fentanyl (6.27x) > fentanyl (3.95x) > morphine (1.48x). Ourdata show that within some fentanyl-related substances doses eliciting hyperlocomotion andantinociception are separable, and suggest that their antinociceptive effects can be even more
susceptible to antagonism than those of morphine.

71

80 - AT-312 (1-(1-((cis)-4-isopropyl cyclohexyl) piperidine-4-yl)-1H-indol-2-yl) methanol) anovel therapeutic compound for opioid relapse in an adolescent rat model
Vazquez Sanroman D1, Bardo M.T.2, Nurulain Zaveri3
1
Oklahoma State University, 2University of Kentucky, 3Astraea Therapeutics
Nociceptin opioid peptide (NOP) receptor activation translates into inhibition of opioid-mediatedreward. The ventral tegmental area (VTA) is not only the origin of the brain dopamine reward
pathway, but also an important substrate for NOP receptor effects. Indeed, VTA-GABAergic neurons
are surrounded by perineuronal nets (PNNs), which consist of extracellular matrix elements
necessary for the modulation of nociception and synaptic plasticity involved in drug addiction. To
construct a model of opioid relapse in adolescent rats, we evaluated the effect of the NOP receptor
full agonist AT-312 on the acquisition, extinction and reinstatement of morphine-induced conditioned
place preference (CPP) in adolescent female and male (PND34) Sprague Dawley rats. In Experiment
1, we examined the effects of AT-312 on morphine (5 mg/kg) CPP. In Experiment 2, we analyzed
the effect of AT-312 on extinction. After conditioning, rats underwent daily AT-312 (3 mg/kg, s.c.)administration and extinction sessions. In Experiment 3, we tested the effect of AT-312 on thereinstating effects of a priming dose of morphine (2.5 mg/kg). Our results showed that systemic
administration of AT-312 in adolescent rats blocked morphine-induced CPP [F (1, 5) = 25.36, p <.05], facilitated morphine-conditioned extinction [F (1, 5) = 25.36, p < .05], and remarkably reduced
drug-primed reinstatement [X2(1) = 10.53, p<0.05]. At the cellular level, AT 312 also decreased
PNN expression [X2(1) = 7.91, p<0.01]. Our results showed that the NOP agonist AT-312 alteredmorphine reward and reinstatement, while also inducing PNN-dependent plasticity in the brain
reward system of adolescent rats.
81 - Physical association between μ opioid and dopamine D1 receptors：Implications formodulation of locomotor sensitization in dopamine-independent manner
Yi-Min Tao1, Chuan Yu1, Yu-Jun Wang1, Jing-Gen Liu1
1
Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of
Science, Shanghai 201203, China;
Introduction: Exposed to opiates induces locomotor sensitization in rodents, which may be related
to the development of compulsive drug-seeking behavior. Numerous studies have demonstrated that
locomotor sensitization can occur in a dopamine transmission-independent manner, however, theunderlying mechanisms are unclear.
Methods: The coimmunoprecipitation, BRET and cross-antagonism assays were used todemonstrating the existence of receptor heterodimers. The function of heterodimers was evaluatedby behavioral studies of locomotor sensitization.
Results/Conclusion: We demonstrated that dopamine D1 receptor (D1R) antagonist SCH23390
antagonized the signaling originated by stimulation of μ opioid receptor (μOR) with agonists intransfected cells expressing two receptors and in wild type but not D1R KO mouse striatal tissues,suggesting that D1R antagonist SCH23390 was able to modify μOR function via receptor
heteromers, since the ability of an antagonist of one of the receptors to inhibit signals originated bystimulation of the partner receptor was a biochemical characteristic of receptor heteromers. The
existence of D1R/μOR heterodimers was further supported by biochemical and biophysical assays.
Moreover, in vivo we demonstrated that, on condition that dopamine release was absent (e.g.
destruction of the dopaminergic projection from the ventral tegmental area to the striatum),
SCH23390 still significantly inhibited μ agonist-induced behavioral responses in rats. Additionally,
we demonstrated that D1R or μOR KO mice, which were unable to form D1R/μOR heterodimer,
failed to express locomotor sensitization to morphine. These results suggest that D1R/μOR
heterodimers may be involved in dopamine independent expression of locomotor sensitization to
opiates.

72

82 - AMPA Receptor Positive Allosteric Modulators Attenuate Opioid Tolerance and
Dependence
Xiaoyu Hu1, Xuebi Tian1, Xiao Guo1, Ying He1, Haijun Chen2, Jia Zhou2, Zaijie Jim Wang1
1
University of Illinois, 2University of Texas Medical Branch, Galveston, TX
Development of opioid tolerance and dependence hinders the use of opioids for the treatment ofchronic pain. In searching for the mechanism and potential intervention for opioid tolerance anddependence, we studied the action of two positive allosteric modulators of the α-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid receptor (AMPAR PAMs). In mice treated with morphine (100mg/kg, s.c.), acute morphine tolerance and dependence developed in 4-6 h. Treatment withaniracetam, a well-established AMPAR PAM, was able to completely prevent and reverse thedevelopment of acute antinociceptive tolerance to morphine. Partial, but significant, effects ofaniracetam on acute morphine induced-physical dependence were also observed. Moreover,aniracetam significantly reversed the established morphine tolerance and dependence in a chronicmodel of morphine tolerance and dependence produced by intermittent morphine (10 mg/kg, s.c. for5d). In addition, HJC0122, a new AMPAR PAM was found to have similar effects as aniracetam butwith a higher potency. These previously undisclosed actions of AMPAR PAMs are intriguing andmay shed lights on understanding the APMA signaling pathway in opioid addiction.
Supported by R01DA041809, P30DA28821, R01DA038446.
No conflict of interest.

83 - Characterization of JVA 4001, a novel mixed opioid agonist/ kappa opioid receptor
antagonist that attenuates drug and stress-induced reinstatement of extinguishedmorphine-conditioned place preference
Lisa Wilson1, Ariana Brice1, Shainnel O. Eans1, Grant Simpson1, Mike Ferrecane1, Jane V. Aldrich1,
Jay McLaughlin1, Heather Stacy1
1
University of Florida, Gainesville, USA
Mixed opioid agonist/kappa opioid receptor (KOR) antagonists such as the macrocyclic tetrapeptide
CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp) show promise as treatments for both pain and drug abuse.
Accordingly, we hypothesized that a mixed mu/kappa opioid receptor agonist/KOR antagonist analog
of CJ-15,208, JVA 4001, would produce potent antinociception while also preventing both drug- andstress-induced reinstatement of extinguished cocaine and morphine conditioned place preference(CPP). Characterized in the mouse 55oC warm-water tail withdrawal assay, JVA 4001 produced
short-acting but potent antinociception after both central (ED50 and 95% C.I. value = 0.11(0.05-0.21)
nmol, i.c.v.) and peripheral (i.p. and p.o.) administration mediated by MOR and (to a lesser extent)
KOR agonism. Furthermore, JVA 4001 demonstrated potent KOR-selective antagonism, preventing
U50,488-induced antinociception for up to 4 h after administration through central (1 pmol, i.c.v.) or
peripheral (1 mg/kg, i.p. or 10 mg/kg, p.o.) routes. Although JVA 4001 demonstrated
hyperlocomotion and respiratory depression consistent with MOR agonism, these effects were lessthan those produced by traditional MOR agonists, and JVA 4001 did not produce the CPP
demonstrated by morphine. In contrast, JVA 4001 dose-dependently blocked both stress- and druginduced reinstatement of extinguished place-preference responses of cocaine- and morphine-CPP
mice in a time-dependent manner. Overall, although additional pharmacokinetic studies are needed,
the promising antinociception with reduced liabilities and the ability to prevent both drug- and stressinduced reinstatement of multiple abused substances highlight JVA 4001 as an excellent leadcandidate for the treatment of pain and drug abuse.

73

84 - Comparative Characterization of Operant Oxycodone Self-Administration in MaleBALB/c and C57Bl/6 Mice
Kyle Windisch1, Brian Reed1, Amelia Dunn1, Alexandra Dunn1, Marco Pravetoni2, Mary Jeanne
Kreek11
Rockefeller University, New York, USA, 2Minneapolis Medical Research Foundation, andDepartment of Medicine and Pharmacology, University of Minnesota, Minneapolis, MN, USA
INTRODUCTION: The BALB/c mouse strain is commonly used in the development of addiction
vaccines due in part to the strain’s robust immunological response. However, use of BALB/c micefor the development of opioid vaccines is challenged by the limited behavioral outputs demonstrated
with these mice. BALB/c mice have been shown to acquire heroin, but not morphine, intravenous
self-administration (IVSA) but only for very low unit doses. Here we investigated BALB/c oxycodone
IVSA. METHODS: For acquisition, separate groups of male BALB/cAnNCrl had eight daily 1-hr
oxycodone IVSA sessions (56, 112, or 250 ug/kg/inf), followed subsequently by dose response
sessions (28, 56, 112, 250 and 500 ug/kg/inf; 2 days/dose). For maintenance, separate groups of
BALB/cAnNCrl and C57Bl/6NCrl mice had 14 daily 2-hr oxycodone sessions (28 or 250 ug/kg/inf).
RESULTS: All mice met criteria for acquisition by the final acquisition session. A standard inverted
U dose response was observed with no significant difference between groups. BALB/c mice had
lower initial responding for 250 ug/kg/inf oxycodone (day 1) compared to C57Bl/6 mice; however, asimilar pattern of escalated intake was observed between the BALB/c and C57Bl/6 mice.
CONCLUSION: This is the first report of BALB/c mice stably self-administering an opioid with unit
doses commensurate to those used with other mouse strains. Further research is needed todetermine if this is unique to oxycodone or the BALB/cAnNCrl substrain.
Support: Gary R. Helman Postdoctoral Research Fellowship (KAW), Miriam and Sheldon Adelson
Medical Research Foundation (MJK), and NIH-NIDA R01 DA041730 (MP).
85 - Generation of conditional Oprm1 knockout rat models using Easi-CRISPR with long
ssDNA donors
Jin Xu1, Valerie P. Le Rouzic1, Rolen Quadros2, Gavril W. Pasternak1, Miyasaka Yoshiki3, Tomoji
Mashimo*3, Thomas L. Saunders*4, Channabasavaiah B. Gurumurthy*2, Yingxian Pan*1
1
Memorial Sloan-Kettering Cancer Center, New York, USA, 2Mouse Genome Engineering Core
Facility, University of Nebraska Medical Center, Omaha, NE 68198 USA, 3Institute of Experimental
Animal Sciences, Graduate School of Medicine, Osaka University, Osaka 565-0871 Japan,
4
Transgenic Animal Core, University of Michigan, Ann Arbor, MI 48109 USA
A single-copy mu opioid receptor (OPRM1) gene generates a vast array of mu opioid receptor splicevariants through extensive alternative pre-mRNA splicing that is conserved from mouse to rat tohuman. Two distinct promoters associated with either exon 1 (E1) or exon 11 (E11) control theexpression of these splice variants. E1-associated variants mainly encode full-length carboxyl (C-)
terminal, 7-transmembrane (7-TM) domain receptors, whereas most E11-associated variants aretruncated, 6-TM domain receptors. Using gene targeted mouse models, we have demonstrated thefunctional relevance and difference of the E1-associated and E11-associated variants. While mousemodels are valuable, rats have many advantages both in behavioral modeling and in vivo
manipulation. In the present study, we have generated two conditional Oprm1 knockout (KO) models
in Sprague Dawley rats using a newly developed Easi-CRISPR with long ssDNA donors approach.
The first rat model is the conditional E11 KO model in which E11 codon and adjacent intron
sequences were flanked by two loxPs. Cre-mediated E11 deletion was confirmed through breedinga transgenic CAG-Cre rat. The second rat model is the conditional E1 KO model with a similar
strategy. Currently, these rat models are being bred and used for initial pharmacological studies.
These conditional Oprm1 KO rat models will provide valuable resources for the research community,
and would expand our ability explore mu opioid pharmacology with approaches and techniques not
feasible in mice. *Correspondent authors. Supported by grants from NIH (DA029244, DA06241,
DA042888 and CA08748)

7486 - The roles of alternatively spliced mu opioid receptor intracellular C-termini encoded byexon 7 on fentanyl actions
Jin Xu1, Valerie P. Le Rouzic1, Zhigang Lu2, Gavril Pasternak1,3, Yingxian Pan4
1
Memorial Sloan-Kettering Cancer Center, New York, USA, 2Nanjing University of Chinese
Medicine, Nanjing, China, 3Memorial Sloan-Kettering Cancer Center, New York, USA, 4Memorial
Sloan-Kettering Cancer Center, New York, USA
The mu opioid receptor gene (OPRM1) undergoes extensive alternative pre-mRNA splicing,
generating multiple splice variants that are conserved from rodents to humans. One type of the
splice variants is full-length 7-transmembrane (TM) C-terminal variants that are identical except forthe sequences at the tip of the intracellular C-terminal tail. Increasing evidence supports thepharmacological importance of these 7TM C-terminal variants. Particularly, exon 7-associated Cterminal truncation in C57BL/6J mice (mE7M-B6) diminished morphine tolerance and reward without
altering physical dependence, like those seen in a β-arrestin2 KO mouse. Together with the resultsfrom cell-based studies, it suggests a physical and functional interaction of E7-associated C-terminal
tails with β-arrestin2 that contributes to morphine desensitization and tolerance. In the present study,
we further investigate the roles of E7-associated C-terminal tails on fentanyl actions in mE7M-B6
mice. In mE7M-B6 mice in which a stop codon was inserted at the beginning of E7 and all E7associated C-termini were abolished, fentanyl analgesia determined by dose-response curve in aradiant heat tail-flick assay was not affected. However, fentanyl-induced tolerance was significantly
reduced, while fentanyl-induced physical dependence determined by naloxone-precipitatedwithdrawal was not changed. These data further support the functional relevance of E7-associatedsplice variants in opioid pharmacology.
Supported in part by grants from NIH (DA029244, DA06241, DA042888 and CA08748), the Peter F.
McManus Charitable Trust, Mr. William H. Goodwin and Mrs. Alice Goodwin and the CommonwealthFoundation for Cancer Research.
87 - The Relation between Serum Uric acid and Spirometric Values and the Risk of
Respiratory Disease
Jiwon Yoon1, Minkyung Cho1, Chulmin Kim1
1
Seoul St. Mary's Hospital, Catholic Medical Center, Korea
Background : Serum uric acid (UA), the final product of purine degradation, has been shown to be
increased in the tissue hypoxic state. Several studies have been reported that patients with chronic
obstructive pulmonary disease (COPD) have high serum levels of UA. However, the relationshipbetween UA levels and spirometric values has not been investigated.
Methods: The subjects of this study were 3,037 individuals aged 19 years and or older who
underwent pulmonary function tests (PFT), using the data from the Korea National Health andNutritional Survey(KNHANES) conducted in 2016. Chi-square test was used to evaluate baselinecharacteristics. Logistric regression and linear regression analysis were used to evaluate
associations between UA and spirometric values
Results: Elevated levels of UA were significantly increased in males (90.5%) than in females (9.5%).
Percent predicted forced vital capacity (FVC %), forced expiratory volume in 1 s (FEV1 %) andFEV1/FVC were inversely correlated with UA levels. Linear regression analysis showed that FVC %and FEV1 % were predictive for UA levels, independently of sex, age, smoking, drinking. Logistic
regression analysis indicated that Elevated levels of UA were significantly associated with risk of
COPD and Obstructive pulmonary diseases, after adjustment for sex, age, smoking, drinking,physical activity (odds ratio, 1. 480; 95% confidence interval, 1.004-2.182)
Conclusion: FVC % and FEV1 % were significantly associated with serum uric acid. Elevated levels
of UA were increased with risk of respiratory disease in a general population of Korea
Key words: uric acid, spirometry, COPD

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88 - Increasing G-protein signalling bias for µ-opioid receptor agonists enhances protein
kinase C dependent desensitization.
Arsalan Yousuf1, Setareh Sianati1, Marco Avena1, MacDonald J. Christie1
1
University of Sydney, Australiaµ-Receptor agonists have the ability to differentially activate distinct signalling pathways such as Gprotein signalling versus β-arrestin-2-dependent regulation, a phenomenon termed biased signalling.
Biased signalling has been suggested to influence side effect profile, as well as tolerance anddependence on opioids (Schmid et al., 2017). We and others have established that morphine, which
weakly recruits β-arrestin-2, induces desensitization of the µ-receptor via a protein kinase C (PKC)
dependent mechanism but peptides such as met-enkalphalin that strongly recruit β-arrestin-2
desensitise via G-protein receptor kinase/β-arrestin-2 dependent mechanisms (Yousuf et al., 2015).
To determine the role of G-protein versus arrestin bias in PKC-dependent regulation ofdesensitization we examined a spectrum of biased opioids. We first established the G-protein versus
arrestin bias of the series using perforated patch clamp recordings of GIRK channel conductance(G-protein signalling) and β-arrestin-2 recruitment to the receptor (BRET), as well as endocytosis(immunohistochemically) in AtT20 cells stably expressing µ-receptors. Acute desensitization wasthen measured in the presence and absence of the potent PKC blocker, calphostin–C, to resolve theproportion of desensitization mediated by PKC. Our results showed that endomorphin-2, the moststrongly arrestin biased opioid in the series, showed the smallest effect of calphostin-C (7 ± 3 %reduction of desensitization) with a progressive increase in PKC-dependence for met-enkephalin,morphine, to bilorphin, a novel, strongly G-protein biased peptidic agonist, which showed strong
dependence on PKC (80 ± 4 % inhibition by calphostin-C). In conclusion, our results show that
increasing bias towards G-protein versus arrestin signalling increases PKC-mediated receptor
desensitization.
89 - Profile of a Novel bifunctional NOP/MOP receptor agonist for Opioid Use Disorder andNon-addicting Analgesia: Efficacy and lack of opioid side effects in nonhuman primates
Nurulain Zaveri1, Mei-Chuan Ko3, Huiping Ding3, Norikazu Kiguchi3,4, Dennis Yasuda1, Pankaj R.
Daga1, Willma E. Polgar1, James J. Lu1, Paul W. Czoty3, Shiroh Kishioka4
1
Astraea Therapeutics, 2Wake Forest University, 3Department of Physiology and Pharmacology,
Wake Forest School of Medicine, 4Department of Pharmacology, Wakayama Medical University
The opioid crisis, declared as a public health emergency, is in essence a double epidemic of opioid
addiction and chronic pain. Over 25 million people have daily pain and over 2 million are addicted to
opioids. Since opioids provide the most effective pain relief, efforts to treat pain with opioids have
fueled the opioid use disorders characterized by physical dependence and craving, potentiallyleading to serious consequences such as abuse and respiratory depression. New pharmacologicalapproaches that provide alternatives for safe, effective analgesia and address opioid use disorders
are urgently needed, but are, as yet, unavailable to patients. Agonists for the nociceptin opioid
receptor (NOP) have been known to modulate the pharmacology of mu opioid (MOP) agonists,
particularly antinociception and opioid-induced reward. We have hypothesized that bifunctional
NOP/MOP agonists may have an attractive profile of potent analgesic effects and lack opioid
rewarding effects. Here we report such a profile for a novel NOP/MOP bifunctional agonist AT-121,
discovered using structure-based drug design and medicinal chemistry optimization. AT-121
possesses partial agonist activity at the NOP and MOP receptor in a single molecule. Detailed
characterization in nonhuman primates shows that AT-121 has “dual” pharmacological efficacy suchthat it suppresses oxycodone’s reinforcing effects and also shows potent analgesia (with lower ED50
than morphine). AT-121 lacks the side effects of opioids, such as respiratory depression, abusepotential, opioid-induced hyperalgesia, physical dependence and tolerance. Bifunctional NOP/MOP
agonists with an appropriate balance of NOP and MOP agonist efficacy may provide an innovative
solution for the opioid crisis.

76

90 - Structurally different Anabolic Androgenic Steroids induce neurotoxic effects inprimary cortical cell cultures
Sofia Zelleroth1, Erik Nylander1, Fred Nyberg1, Alfhild Grönbladh1, Mathias Hallberg1
1
Uppsala University, Uppsala, Sweden
Anabolic Androgenic steroids (AASs) are suggested as a gateway to opioid abuse and dependenceof other drugs of abuse. The AASs are used to enhance physical appearance and performance insports, which have become a growing problem among non-athletes. Many of the individuals abusing
AAS also use opioids and several heroin-abusers have a history of previous AAS misuse. This raises
the question, how does AASs affect the brain resulting in increased sensitivity to othernarcotics? The aim was to investigate the toxic effects of structurally different AASs to gain more
knowledge about how the brain is affected by AAS abuse.
The toxicity of testosterone, nandrolone, stanozolol and trenbolone was studied in primary corticalcell cultures. The cells were exposed to increasing concentrations of the AASs for 24 hours or 3
days. The cells were co-treated with the androgen receptor (AR) antagonist, flutamide, to determine
if the effects were mediated by the AR. Thereafter, cellular toxicity was determined by measuringmitochondrial activity, lactate dehydrogenase release and caspase activity.
All AASs studied induced toxic effects in primary cortical cells, in a time-dependent and dosedependent manner. Testosterone, nandrolone and trenbolone caused their toxic effects by inductionof apoptosis. In contrast, stanozolol seemed to cause necrosis. Flutamide were able to abolish or
diminish the AAS-induced effects.
In conclusion, supraphysiological concentrations of the AASs studied induce neurotoxic effects, but
to different extent and possibly by different mechanisms. However, the main effects seem to be
mediated by activation of the classical AR-pathway.
91 - Morphine induced alteration in gut microbiome contributes to analgesic tolerance bymodulating the Gut-Immune-CNS Axis
Li Zhang1,2, Jingjing Meng2, Ramakrishnan Sundaram2, Sabita Roy1,2
1
University of Minnesota, 2University of Miami
Opioids are the most prescribed analgesic agent for pain relieve clinically. However its clinical use
is limited by co-morbidities such as tolerance and dependence. We have previously shown thatchronic morphine treatment results in significant sustained inflammation as a consequence of gut
barrier disruption and bacterial translocation that is mediated by increased gut epithelial expressionof TLR2 and TLR4 proteins. A significant body of literature shows that chronic inflammation iscorrelated with morphine analgesic tolerance. Our recent study demonstrates that morphine
treatment results in significant gut microbial disruption, a significant expansion of gram-positive
pathogenic bacteria. The purpose of this study was to investigate the role of gut microbiome in thedevelopment of morphine induced analgesic tolerance. We show that morphine treatment of germ
free mice and pan antibiotic treated mice results in significantly attenuated morphine induced antinociceptive tolerance, bacterial translocation, TLR2 and TLR4 expression, and reduced systemic
inflammation. Moreover, morphine induced analgesic tolerance was significantly decreased in in
TLR2KO and TLR4KO mice. Notable treatment of mice with the probiotics, VSL#3, which consistedof eight beneficial bacteria, reduced morphine induced analgesic tolerance, TLR2 and TLR4
expression and chronic inflammation by restoring gut homeostasis. In conclusion, we demonstrated
that morphine-induced dysbiosis modulated analgesic tolerance through gut-immune-CNS axis.
Maintaining gut homeostasis is a therapeutic target for morphine tolerance, which extends theeffectiveness of opioids in clinical pain management.

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92 - Diarylurea-based allosteric modulators of the cannabinoid CB1 receptor: Structureactivity relationship studies on the pyrrolidinylpyridinyl group
Thuy Nguyen1, Thomas F. Gamage1, Ann M. Decker1, Nadezhda German1, Charlotte E. Farquhar1,
Tiffany L. Langston1, Jenny Wiley1, Brian F. Thomas1, Terry Kenakin2, Yanan Zhang1
1
Research Triangle Institute, 2University of North Carolina Chapel Hill
Allosteric modulators have attracted significant interest in the modulation of the CB1 signaling fortherapeutic benefits while avoiding the adverse effects associated with orthosteric ligands. In ourprevious studies on the CB1 receptor allosteric modulator PSNCBAM-1 we have reported that the
pyrrolidinyl ring is not required for CB1 modulation and the pyridinyl moiety could be replaced by asimple phenyl or substituted phenyl rings. Here we extended the structure-activity relationships ofthese diarylureas by introducing heterocyclic groups at the place of the pyridinyl ring of PSNCBAM1. These novel compounds showed good potencies and binding affinities in calcium mobilization,
GTP-γ-S binding and radioligand binding assays. Most compounds possessed low nanomolar IC50
values at CB1 receptor without any significant activities at the CB2 receptor. These modulators
reduced the Emax of the orthosteric CB1 receptor agonist CP55940, as expected with negative
allosteric modulators (NAMs). Interestingly, this new series of compounds displayed signaling
pathway bias and variations in cooperativities with the orthosteric agonist CP55,940 and antagonist
SR141716A. These results suggest that the CB1 signaling could be fine-tuned based on theorthosteric ligands and signaling pathways.
Support: NIH grant DA040693 (YZ).

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Notes79

2019
50th Anniversary

See you in New York!
July 7-11, 2019
Grand Hyatt, New York, NY USA