Today, more Americans die each year because of drug overdoses than are killed in motor vehicle accidents. In fact, in 2015, more than 33,000 individuals died due to an overdose of heroin or prescription opioids. Sadly, 40-60 % of patients on current opioid addiction treatment medications relapse. Studies have shown that the addiction/abuse liability of opioids are abolished in mu opioid receptor (MOR) knock-out mice; this indicates that the addiction and abuse liability of opioids are mainly mediated through MOR. Utilizing the “message-address concept”, the our laboratory reported a novel non-peptide, reversible MOR selective ligand 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α (isoquinoline-3-carboxamido)morphinan (NAQ). Molecular modeling and mutagenesis studies revealed that the selectivity of NAQ for MOR is because of the π-π stacking of the isoquinoline ring of NAQ with W318. Therefore, other heterocyclic ring systems were explored to obtain a diverse library of compounds with similar or different molecular interactions and pharmacologic characteristics as NAQ. The newly designed compounds were indole analogs of 6α/β-naltrexamine. The compounds were synthesized and the affinity and selectivity for MOR determined using the radioligand binding assay while the functional activity at MOR was determined using the [35S]GTPγS binding assay. The indole analog of 6α-naltrexamine substituted at position 7 (compound 6) was found to be very potent and had the lowest efficacy in the [35S]GTPγS functional assay while the indole analog of 6β-naltrexamine substituted at position 2 (compound 10) was identified as a MOR agonist and had the greatest efficacy. In vivo studies were conducted using the warm-water immersion assay to find whether the synthesized compounds had antinociceptive effects and/or blocked the antinociceptive effects of morphine. Not surprisingly, compound 10 was identified as an opioid agonist while compound 6 almost completely blocked morphine’s antinociceptive effects. The opioid antagonist effect of compound 6 was found to be dose dependent with an AD50 of 2.39 mg/kg (0.46-12.47). An opioid withdrawal assay was conducted on compound 6 using morphine-pelleted mice. Compound 6 produced significantly less withdrawal symptoms at 50 mg/kg than naltrexone at 1 mg/kg. Therefore, compound 6 has the potential to be used in opioid addiction and withdrawal treatment.

Cocaine is interrelated with the opioid system on many levels, especially via the mu opioid receptor (MOR). Also, cocaine has been involved in modulating nitric oxide (NO) actions within the cell. The effect of cocaine was first assessed on the MOR, and then on transcription by the use of 1 µg/ mL actinomycin D inhibitor. Several signaling pathways that cocaine may exert its action in modulating the MOR up-regulation in protein expression were also explored. Two dosage regimens were used in cocaine treatment, single continuous treatment (SCT), and repeated intermittent treatment (RIT). Different pathway inhibitors were used on PC12 cells, as follows: the PLC-PKC inhibitors 5 µM U-73122 and 10 µM BIS-1 used to investigate the involvement of the PKC signaling pathways in MOR expression levels, the evaluation of MAPK pathway by the use of 50 µM U0126 inhibitor, and the 10 µM LY94002 inhibitor was used to investigate the PI3K/Akt pathway. Moreover, the effect of NO on these signaling pathways was investigated by the use of 20 mM nonselective L-NAME inhibitor and qualitatively by DAF-2 florescence. Western blot analysis indicated that cocaine up-regulated MOR protein expression. Also, RIT cocaine treatment increased MOR protein levels via transcription. All three signaling pathways, MAPK, Akt and PKC modulated cocaine-induced increase of MOR following SCT cocaine treatment (post-transcriptional). Both MAPK and Akt have been found to modulate the cocaine-induced transcription of MOR via the two dosage regimens of cocaine, SCT and RIT. Also, inhibition of both PLC and PKC did not prevent cocaine-induced increase in MOR transcription, according to RIT of cocaine. Furthermore, Akt and PKC appeared to modulate cocaine-induced NO production while MAPK did not. NO seemed to be involved with the PKC and Akt pathways in up-regulating MOR in RIT of cocaine directly by the Akt pathway, and indirectly by the PKC pathway. On the other hand, NO and MAPK modulated the MOR up-regulation expression simultaneously, but in an individual/parallel manner. Furthermore, signaling pathway activation levels were tested using L-NAME which concluded that NO modulated cocaine-induced increase in total Akt protein levels, but did not appear to have an effect on phosphorylated MAPK activation levels. In conclusion, different treatment regimens of cocaine activate different pathways; SCT of cocaine activated all three signaling pathways, however, RIT of cocaine activated only the MAPK and Akt pathways. / Saudi Bureau in Canada

abstract: Intermittent social defeat stress produces vulnerability to drugs of abuse, a phenomena known as cross-sensitization, which is proceeded by a corresponding upregulation of ventral tegmental area (VTA) mu-opioid receptors (MORs). Since VTA MORs are implicated in the expression of psychostimulant sensitization, they may also mediate social stress-induced vulnerability to drugs of abuse. Social stress and drugs of abuse increase mesolimbic brain-derived neurotrophic factor (BDNF) signaling with its receptor, tropomyosin-related kinase B (TrkB). These studies examined whether VTA MOR signaling is important for the behavioral and cellular consequences of social stress. First, the function of VTA MORs in the behavioral consequences of intermittent social defeat stress was investigated. Lentivirus-mediated knockdown of VTA MORs prevented social stress-induced cross-sensitization, as well as stress-induced social avoidance and weight gain deficits. Next it was examined whether VTA MOR expression is critical for stress-induced alterations in the mesocorticolimbic circuit. At the time cross-sensitization was known to occur, lentivirus-mediated knockdown of VTA MORs prevented stress-induced increases in VTA BDNF and its receptor, TrkB in the nucleus accumbens (NAc), and attenuated NAc expression of delta FosB. There was no effect of either stress or virus on BDNF expression in the prefrontal cortex. Since social stress-induced upregulation of VTA MORs is necessary for consequences of social stress, next activity dependent changes in AKT, a downstream target of MOR stimulation associated with sensitization to psychostimulant drugs, were investigated. Using fluorescent immunohistochemical double labeling for the active form of AKT (pAKT) and markers of either GABA or dopamine neurons in the VTA, it was determined that social stress significantly increased the expression of pAKT in GABA, but not dopamine neurons, and that this effect was dependent on VTA MOR expression. Moreover, intra-VTA inhibition of pAKT during stress prevented stress-induced weight gain deficits, while acute inhibition of VTA pAKT blocked the expression of cross-sensitization in subjects that had previously exhibited sensitized locomotor activity. Together these results suggest that social stress upregulates MORs on VTA GABA neurons, resulting in AKT phosphorylation, and that increased VTA MOR-pAKT signaling may represent a novel therapeutic target for the intervention of substance abuse disorders. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2015

Most clinical opioids produce analgesia through the Mu Opioid Receptor (MOR) providing the only effective treatment for chronic pain patients. These studies explore three pre-clinical strategies to improve MOR analgesia and minimize side effects: 1) compounds that target G-protein Coupled Receptors (GPCRs) heterodimers, such as heterodimerization between the Delta Opioid Receptor (DOR) and MOR (MDOR); 2) multi-functional compounds that target multiple receptor systems for synergistic effects, such as a MOR agonist and a the serotonin reuptake transporter (SERT) inhibitor; or 3) biased agonists that preferentially activate one signaling pathway associated with analgesia over another associated with side effects at the same receptor. First, several indirect lines of evidence indicate the MOR-DOR heterodimer (MDOR) can regulate MOR opioid tolerance and withdrawal. However, studying MDOR remains difficult because no selective MDOR antagonists are available. To address this need, we created a novel series of bivalent MDOR antagonists by connecting a low affinity MOR antagonist (H-Tyr-Pro-Phe-D1Nal-NH2) to a moderate affinity DOR (H- Tyr-Tic-OH) antagonist with variable length polyamide spacers (15-41 atoms). In vitro radioligand binding and [35S]-GTPγS coupling assays in MOR, DOR, and MDOR expressing cell lines show bivalent ligands produce a clear length dependence in MDOR but not MOR or DOR cell lines. The lead compound – D24M with a 24-atom spacer – displayed high potency (IC50MDOR = 0.84 nM) with 91-fold selectivity for MDOR:DOR and 1,000-fold MDOR:MOR selectivity. Second, clinicians have long appreciated subtle but distinct differences in analgesia and side effects of MOR opioids. A variety of non-MOR targets including DOR, Kappa Opioid Receptor (KOR), the Cannabinoid Receptor-1 (CB1), the Sigma-1 Receptor (σ1R), the Dopamine- (DAT), Serotonin- (SERT) and Norepinephrine- Reuptake Transporters (NET) induce analgesia and/or modulate MOR mediated side effects. To determine if different opioid profiles arise from non-MOR interactions, we evaluated the binding and function of nine clinical analgesics at the nine aforementioned targets revealing several clinical opioids contain previously unidentified affinity’s or activity’s. Hydrocodone displayed low affinity at the MOR (KI = 1800 nM) and only ~2 fold less affinity at the σ1R (KI = 4000 nM). Second buprenorphine promoted monoamine influx at DAT, SERT and NET with EC50 > 1,000 nM. These novel interactions suggest the nuanced differences of clinical opioids may arise from previously unappreciated off-target effects. Future studies will assess whether these in vitro results predict hydrocodone and buprenorphine activity in vivo. Finally, the unique function of the numerous endogenous opioid peptides at a given receptor remains unclear. How endogenous ligands interact with ORs produces obvious drug design consequences. These studies show two endogenous Dynorphin analogues – Dynorphin A and Dynorphin B – differentially regulate two ubiquitous signaling modules – βarrestin2 and Gαi/o– at the DOR. Dynorphin A and Dynorphin B swap potency rank orders for β-arrestin2 recruitment and [35S]-GTPγS signaling, indicating two distinct signaling platforms are formed. Dynorphin A but not Dynorphin B treatment simulated AC super activation, while Dynoprhin B internalized DOR better than Dynorphin A. These in vitro assays suggest endogenous Dynorphin analogues differentially regulate signals at the DOR in vitro. Future work includes further characterizing signaling differences in vitro and testing these changes in vivo.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22366号 / 医博第4607号 / 新制||医||1043(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 岩田 想, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Release of endomorphin (ENDO)-2 like substances from the dorsal horn of the isolated rat spinal cord was measured by the immobilized-antibody microprobe technique. Spinal cords were removed from anesthetized 4-6 week old rats and superfused with oxygenated Krebs solution at room temperature. Glass microprobes coated with ENDO-2 antibodies were inserted into the dorsal horn of the lumbar spinal cord 1.5 mm lateral to the midline to a depth 2.5 mm below the dorsal surface of the cord. Each probe remained in situ for 10 min periods before, during and after electrical stimulation applied to the dorsal root entry zone of the same spinal segment. There was no detectable basal release of immunoreactive endomorphin-2 like substance (irENDO) from the dorsal horns during the pre-stimulation, nor following the stimulation period. A significant release of irENDO was measured during the electrical stimulation. These results provide the first evidence of a irEndo release that is correlated spatially with the dorsal horn laminae I and II where ENDO-2-immunoreactive fibers are concentrated in the dorsal horn in response to electrical activation of primary afferent fibers.

A m-opioid receptor (OPRM1) single-nucleotide-polymorphism, found in both humans and rhesus macaques mediates the mother-infant attachment bond. Because mothers treat their sons and daughters differently, it is somewhat surprising that the role of infant sex has not been assessed in the context of a maternal-OPRM1-genotype-by-infant-sex interaction. The present study investigates the effect of maternal-OPRM1-genotype and infant sex on mother-infant behaviors. Over the first six months of offspring life, mother-infant behavioral data assessing attachment quality was collected twice weekly from a large number of rhesus monkey mother-infant pairs (N=161 dyads; n=64 female infants, n=97 male infants). Mothers were genotyped for OPRM1 variation. Factor analysis of the observed behaviors showed two factors: Attachment (maternal-infant cradling, rejections, and infant approaches and leaves), and Maternal Restraints (mother restrains infant, preventing exploration). Further analyses showed a two-way, maternal-genotype-by-infant-sex interaction for both factors. For Attachment, mothers with the CC genotype cradled and restrained (Maternal Restraints) their female infants more and rejected them less, when compared to female infants of CG mothers. Perhaps as a consequence, female infants of CC genotype mothers approached and left their mothers less often, when compared to female infants of CG mothers, likely an indication that female infants from mothers with CG genotype play a greater role in maintaining the mother-infant bond than do female infants from CC genotype mothers. This finding may also indicate a more secure attachment in infants from CC genotype mothers. Unlike female infants, on average, the mother-infant relationship of dyads with a male infant was largely undifferentiated by maternal genotype. These findings suggest that, in contrast to female infants from CG mothers, CC mothers and their female infants appear to have a closer mother-infant relationship which may portend close life-long bonds, as mothers and female offspring remain together throughout life. Male offspring appear to have a more aloof mother-infant bond regardless of OPRM1-genotype. The results of this study indicate that maternal-OPRM1 variation mediates mother-infant attachment behaviors for female infants and has less effect for male infants. This suggests that offspring sex should be included in studies investigating the effect of maternal-OPRM1 genotype on the mother-infant attachment relationship.

No description available.

Animal models have proven to be useful tools for modeling human neurochemical and behavioral responses to drugs of abuse, including cocaine. Cocaine is a psychomotor stimulant that facilitates monoaminergic neurotransmission by binding to transporters and inhibiting the reuptake of dopamine, serotonin and norepinephrine into presynaptic neurons. Many of the behavioral effects of cocaine, including its locomotor-activating and reinforcing properties, have been attributed to the ability of cocaine to enhance dopaminergic activity. In addition to its direct effects on monoamine neurotransmitters, cocaine impacts other neurotransmitter systems including the endogenous opioid system. The effects of selectively antagonizing mu opioid receptors on cocaine-induced behaviors were evaluated during this research. This research also evaluated the effect of selectively antagonizing dopamine D1 or D2 receptors on cocaine-induced mu opioid receptor occupancy by endogenous opioid ligands. This research furthered our understanding of how the endogenous opioid and dopaminergic systems interact to mediate cocaine-induced behaviors. Although data support the role of mu opioid receptors in modulating cocaine-mediated locomotion and reward, the location of the mu opioid receptors involved has not been established. An evaluation of the effects of a selective mu opioid receptor antagonist administered directly into specific brain regions on cocaine-induced behaviors is important for understanding how the endogenous opioid and dopaminergic systems interact to mediate cocaine-induced behaviors. The studies outlined herein sought to determine the contribution of mu opioid receptors in specific regions of the mesocorticolimbic system to the rewarding and locomotor-activating effects of cocaine in the rat. In addition, to further understand the role of mu opioid receptors in cocaine reward, neuronal activation was studied via cFos activation following the expression of cocaine-induced place preference. Results of the research outlined herein demonstrate the importance of mu opioid receptors in cocaine-induced reward and activity, and demonstrate the anatomical selectivity of mu receptors within the nucleus accumbens, VTA and caudate putamen in this regard. These data suggest that cocaine causes the release of endogenous opioid peptides and that these peptides contribute to the rewarding and locomotor-stimulating effects of cocaine. Further, these data also suggest that opioid peptides are released in the nucleus accumbens shell during the expression of cocaine place preferences and that mu opioid receptors in this region are critical for the manifestation of this behavior. Although data demonstrate that extracellular levels of endogenous opioid peptides are increased following cocaine administration, the time- and dose-dependent occupancy of mu opioid receptors within specific brain regions had not been established in previous studies. The present research sought to determine the time- and dose-dependent occupancy of mu opioid receptors, measured indirectly by displacement of 3H-DAMGO binding, within specific brain regions. 3H-DAMGO binding was measured by in vitro autoradiography. In addition, the contribution of dopamine D1 and D2 receptors in cocaine-induced 3H-DAMGO displacement was evaluated. Results demonstrate that cocaine administration caused a dose- and time-dependent displacement of 3H-DAMGO binding to mu opioid receptors within the nucleus accumbens core and shell. This displacement was attenuated by pretreatment with a selective D2 dopamine receptor antagonist, demonstrating that cocaine, acting via D2 dopamine receptors, can cause the release of an endogenous opioid peptide that binds to mu opioid receptors within the nucleus accumbens core and shell. Previous studies have demonstrated that chronic administration of non-selective mu opioid receptor antagonists has profound effects on mu opioid receptor density and signaling. The research presented herein sought to determine whether chronic treatment with the selective mu opioid receptor antagonist, CTAP, would increase mu opioid receptor density and agonist-stimulated G-protein activation. In addition, this research sought to determine whether chronic CTAP administration would sensitize animals to the locomotor stimulating effects of cocaine. Results outlined herein demonstrate that chronic CTAP treatment sensitized animals to the locomotor effects of cocaine and that this sensitization occurred in conjunction with an increase in mu opioid receptor density within the nucleus accumbens core and shell. / Pharmacology

Chemokines, immune proteins that induce chemotaxis and adhesion, and their G-protein coupled receptors distribute throughout the central nervous system (CNS), regulate neuronal patterning, and mediate neuropathology. These chemo-attractant molecules may provide a neuro-immune "link" by regulating CNS systems. The purpose of this study was to investigate the interactions of specific chemokines, stromal cell-derived factor (SDF)-1a/CXCL12, and fractalkine/CX3CL1, and their receptors, CXCR4 and CX3CR1, with the serotonin (5-hydroxytryptamine; 5-HT) and opioid systems using anatomical and electrophysiological techniques in the rat brain. In the serotonin dense midbrain raphe nuclei (RN), SDF-1a, CXCR4, fractalkine and CX3CR1 co-localize over 70% with 5-HT neurons. CX3CR1 also localizes to microglia in the RN and hippocampus. Functionally, SDF-1a (10 nM) increases spontaneous inhibitory postsynaptic current (sIPSC) frequency and evoked IPSC (eIPSC) amplitude, while decreasing paired-pulse ratio (PPR) selectively in 5-HT neurons, thus stimulating presynaptic GABA release at these neurons. Alternatively, fractalkine (10 nM) increases sIPSC and eIPSC amplitude without changing PPR selectively in 5-HT neurons, thereby elevating the postsynaptic GABA receptor number or sensitivity. These results are dose-dependent and receptor-mediated. Chemokine interactions with serotonin, a neurotransmitter regulating mood, may lead to therapies for depression comorbid with immune diseases. Additional immunohistochemical analysis in the brain shows CXCR4 and CX3CR1 neuronal co-localization with the mu-opioid receptor (MOR) in the hippocampus, cingulate cortex, periaqueductal grey (PAG), nucleus accumbens, ventral tegmental area, globus pallidus, but not in the striatum or habenular nuclei, suggesting region specific receptor interactions. Electrophysiological recordings following morphine, SDF-1?? or fractalkine in vitro treatment reveal morphine (10 ?M)-mediated hyperpolarization of the membrane potential and reduction of the input resistance of PAG neurons, however, SDF-1??and fractalkine at 10 nM do not impact either parameter. In combination, SDF-1? inhibits morphine's actions in all PAG neurons tested, and fractalkine blocks morphine-mediated changes in 60% of PAG neurons examined. Thus, CXCR4 as well as CX3CR1, although less consistently, both appear to desensitize MOR at the neuronal level. Chemokine-opioid receptor interactions may mediate novel mechanisms to treat neuro-inflammatory pain and opiate abuse. The combined anatomical and electrophysiological results support chemokines as neuromodulatory proteins that may provide communication between the nervous and immune systems. / Anatomy