Extracellular matrix abnormalities in the hippocampus of subjects with substance use disorder.

Contextual triggers are significant factors contributing to relapse in substance use disorders (SUD). Emerging evidence points to a critical role of extracellular matrix (ECM) molecules as mediators of reward memories. Chondroitin sulfate proteoglycans (CSPGs) are a subset of ECM molecules that form perineuronal nets (PNN) around inhibitory neurons. PNNs restrict synaptic connections and help maintain synapses. Rodent models suggest that modulation of PNNs may strengthen contextual reward memories in SUD. However, there is currently a lack of information regarding PNNs in the hippocampus of people with SUD as well as how comorbidity with major depressive disorder (MDD) may affect PNNs. We used postmortem hippocampal tissues from cohorts of human and nonhuman primates with or without chronic alcohol use to test the hypothesis that PNNs are increased in subjects with SUD. We used histochemical labeling and quantitative microscopy to examine PNNs, and qRT-PCR to examine gene expression for ECM molecules, synaptic markers and related markers. We identified increased densities of PNNs and CSPG-labeled glial cells in SUD, coinciding with decreased expression of the ECM protease matrix metalloproteinase 9 (Mmp9), and increased expression for the excitatory synaptic marker vesicle associated membrane protein 2 (Vamp2). Similar increases in PNNs were observed in monkeys with chronic alcohol self-administration. Subjects with MDD displayed changes opposite to SUD, and subjects with SUD and comorbid MDD had minimal changes in any of the outcome measures examined. Our findings demonstrate that PNNs are increased in SUD, possibly contributing to stabilizing contextual reward memories as suggested by preclinical studies. Our results also point to a previously unsuspected role for CSPG expression in glial cells in SUD. Evidence for increased hippocampal PNNs in SUD suggests that targeting PNNs to weaken contextual reward memories is a promising therapeutic approach for SUD, however comorbidity with MDD is a significant consideration.

Extracellular Matrix Abnormalities in the Hippocampus of Subjects with Substance Use Disorder.

Contextual triggers are significant factors contributing to relapse in substance use disorders (SUD). Emerging evidence points to a critical role of extracellular matrix (ECM) molecules as mediators of reward memories. Chondroitin sulfate proteoglycans (CSPGs) are a subset of ECM molecules that form perineuronal nets (PNN) around inhibitory neurons. PNNs restrict synaptic connections and help maintain synapses. Rodent models suggest that modulation of PNNs may strengthen contextual reward memories in SUD. However, there is currently a lack of information regarding PNNs in the hippocampus of people with SUD as well as how comorbidity with major depressive disorder (MDD) may affect PNNs. We used postmortem hippocampal tissues from cohorts of human and nonhuman primates with or without chronic alcohol use to test the hypothesis that PNNs are increased in subjects with SUD. We used histochemical labeling and quantitative microscopy to examine PNNs, and qRT-PCR to examine gene expression for ECM molecules, synaptic markers and related markers. We identified increased densities of PNNs and CSPG-labeled glial cells in SUD, coinciding with decreased expression of the ECM protease matrix metalloproteinase 9 (Mmp9), and increased expression for the excitatory synaptic marker vesicle associated membrane protein 2 (Vamp2). Similar increases in PNNs were observed in monkeys with chronic alcohol self-administration. Subjects with MDD displayed changes opposite to SUD, and subjects with SUD and comorbid MDD had minimal changes in any of the outcome measures examined. Our findings demonstrate that PNNs are increased in SUD, possibly contributing to stabilizing contextual reward memories as suggested by preclinical studies. Our results also point to a previously unsuspected role for CSPG expression in glial cells in SUD. Evidence for increased hippocampal PNNs in SUD suggests that targeting PNNs to weaken contextual reward memories is a promising therapeutic approach for SUD, however comorbidity with MDD is a significant consideration.

Quantification of observable behaviors following oral administration of oxycodone and nalfurafine in male rhesus monkeys.

BACKGROUND: Recent preclinical studies have investigated the atypical kappa-opioid receptor (KOR) agonist, nalfurafine, as a co-formulary with mu-opioid receptor (MOR) agonists as a potential deterrent for misuse. However, no study has investigated effects of nalfurafine combined with a MOR agonist using an oral route of administration. The objective of the current study was to measure behavioral effects of orally administered oxycodone and nalfurafine, alone and combined, in rhesus monkeys using a quantitative behavioral observation procedure. METHODS: Adult male rhesus monkeys (N=5) were orally administered vehicle, oxycodone (0.56-1.8mg/kg), nalfurafine (0.001-0.0056mg/kg), or mixtures (1.0mg/kg oxycodone/0.001-0.0056mg/kg nalfurafine) in a Jell-O vehicle at multiple timepoints (10-320min). Species-typical and drug-induced behaviors were recorded by observers blinded to conditions. RESULTS: Oxycodone alone significantly increased scratch and face-rub behaviors without affecting other behaviors. Nalfurafine decreased baseline levels of scratch without affecting other behaviors, and oxycodone-nalfurafine combinations resulted in reduced oxycodone-induced scratching at a dose (0.001mg/kg) that did not produce sedation-like effects. Oxycodone combined with larger nalfurafine doses (0.0032-0.0056mg/kg) also reduced oxycodone induced scratch that were accompanied with sedation-like effects (i.e., increased lip droop). CONCLUSIONS: Nalfurafine was orally active in rhesus monkeys, and it reduced oxycodone-induced pruritus at a dose that did not produce sedation-like effects that are commonly observed with prototypical KOR agonists. Combinations of low doses of nalfurafine with MOR agonists such as oxycodone may be well-tolerated by humans who are prescribed MOR agonists for the treatment of pain.

Pharmaco-EEG analysis of ligands varying in selectivity for α1 subunit-containing GABAA receptors during the active phase in rats.

RATIONALE: Benzodiazepines are known to evoke changes in cortical electrophysiological activity that can be correlated with action at distinct γ-aminobutyric acid type A (GABAA) receptor subtypes. OBJECTIVES: We used electroencephalography (EEG) paired with electromyography (EMG) to evaluate the role of α1 subunit-containing GABAA receptors (α1GABAARs) in benzodiazepine-induced sedation and changes in EEG band frequencies during the active phase of the light/dark cycle. METHODS: Male Sprague-Dawley rats (N = 4/drug) were surgically instrumented with EEG/EMG electrodes. The rats were injected i.p. with zolpidem, an α1GABAAR-preferring compound, or L-838,417, which has selective efficacy for α2/3/5 subunit-containing GABAARs (i.e., α1GABAAR-sparing compound), in comparison with the non-selective benzodiazepine, triazolam. RESULTS: All ligands evaluated induced changes in sleep-wake states during the active phase consistent with an increase in slow-wave sleep (SWS). The degree of SWS increase appeared to be related to the magnitude of delta power band changes induced by the ligands, with the strongest effects engendered by the α1GABAAR-preferring drug zolpidem and the weakest effects by the α1GABAAR-sparing compound, L-838,417. Consistent with other research, a selective increase in beta band power was observed with L-838,417, which may be associated with α2GABAAR-mediated anxiolysis. CONCLUSIONS: Overall, these findings support the establishment of pharmaco-EEG "signatures" for identifying subtype-selective GABAA modulators in vivo.

Behavioral effects of triazolam and pregnanolone combinations: reinforcing and sedative-motor effects in female rhesus monkeys.

Introduction: Benzodiazepines (BZs) are prescribed as anxiolytics, but their use is limited by side effects including abuse liability and daytime drowsiness. Neuroactive steroids are compounds that, like BZs, modulate the effects of GABA at the GABAA receptor. In a previous study, combinations of the BZ triazolam and neuroactive steroid pregnanolone produced supra-additive (i.e., greater than expected effects based on the drugs alone) anxiolytic effects but infra-additive (i.e., lower than expected effects based on the drugs alone) reinforcing effects in male rhesus monkeys, suggestive of an improved therapeutic window. Methods: Female rhesus monkeys (n=4) self-administered triazolam, pregnanolone, and triazolam-pregnanolone combinations intravenously under a progressive-ratio schedule. In order to assess characteristic sedative-motor effects of BZ-neuroactive steroid combinations, female rhesus monkeys (n=4) were administered triazolam, pregnanolone, and triazolam-pregnanolone combinations. Trained observers, blinded to condition, scored the occurrence of species-typical and drug-induced behaviors. Results: In contrast to our previous study with males, triazolam-pregnanolone combinations had primarily supra-additive reinforcing effects in three monkeys but infra-additive reinforcing effects in one monkey. Scores for deep sedation (i.e., defined as atypical loose-limbed posture, eyes closed, does not respond to external stimuli) and observable ataxia (any slip, trip, fall, or loss of balance) were significantly increased by both triazolam and pregnanolone. When combined, triazolam-pregnanolone combinations had supra-additive effects for inducing deep sedation, whereas observable ataxia was attenuated, likely due to the occurrence of robust sedative effects. Discussion: These results suggest that significant sex differences exist in self-administration of BZ-neuroactive steroid combinations, with females likely to show enhanced sensitivity to reinforcing effects compared with males. Moreover, supra-additive sedative effects occurred for females, demonstrating a higher likelihood of this adverse effect when these drug classes are combined.

Modeling cue-exposure therapy for alcohol use disorder in rhesus monkeys: Effects of putative cognitive enhancers.

BACKGROUND: Cue-exposure therapy (CET) is an effective approach for anxiety-related disorders, but its effectiveness for substance use disorders is less clear. One potential means of improving CET outcomes is to include a cognitive-enhancing pharmacotherapy. This study evaluated d-cycloserine (DCS) and RY-023, putative cognitive enhancers targeting glutamate and GABA systems, respectively, in a monkey model of CET for alcohol use disorder. METHODS: Male rhesus monkeys (n = 4) underwent multiple cycles of the CET procedure. During baseline (Phase 1), monkeys self-administered an ethanol solution under a fixed-ratio schedule and limited access conditions such that every 5th response in a 3-h session resulted in 30-s access to a drinking spout and a change in ethanol-paired cue lights from white to red. Behavior then was extinguished (Phase 2) by omitting the ethanol solution yet retaining the ethanol-paired stimulus lights. Monkeys also received injections of vehicle, DCS (3 mg/kg), a partial agonist at the glycine modulatory site on glutamatergic NMDA receptors, or the α5GABAA receptor-selective inverse agonist RY-023 (0.03 or 0.3 mg/kg). Once responding declined, monkeys underwent a cue reactivity test (Phase 3), and then returned to self-administration the following day to assess reacquisition (Phase 4). RESULTS: Through multiple cycles, self-administration remained stable. Compared to vehicle, DCS facilitated extinction of ethanol seeking (Phase 2) and delayed reacquisition of ethanol self-administration (Phase 4). In contrast, RY-023 facilitated extinction (Phase 2) and reduced cue reactivity (Phase 3). CONCLUSIONS: Adjunctive pharmacotherapy can improve CET outcomes, but the choice of pharmacotherapy should be dependent on the outcome of interest.

Daily, limited access to methamphetamine self-administration during pregnancy leads to increased methamphetamine sensitivity in adult offspring.

Methamphetamine use by women, even throughout pregnancy, is common. But there is limited knowledge about the effects in prenatally methamphetamine-exposed children. This study investigated how prenatal methamphetamine exposure in rats, via maternal i.v. self-administration, affected the sensitivity of adult offspring to methamphetamine in comparison with controls. The offspring were generated from dams either self-administering methamphetamine daily under limited-access conditions prior to and throughout pregnancy, or their respective saline-yoked control dams. Spontaneous and methamphetamine-induced locomotor activity was assessed in male and female offspring of both exposure groups after a range of methamphetamine doses. In a separate group of offspring, acquisition of i.v. methamphetamine self-administration, responding under fixed and progressive ratio schedules of methamphetamine reinforcement, and reinstatement of extinguished drug-seeking behavior were assessed. Methamphetamine dose-dependently increased locomotor activity in both exposure groups. However, methamphetamine-exposed males showed significantly enhanced locomotor activity compared with controls at 1 mg/kg, and methamphetamine-exposed females showed significantly enhanced locomotor activity compared with controls at 3.2 mg/kg. Methamphetamine-exposed offspring of both sexes acquired methamphetamine self-administration faster and showed overall higher levels of methamphetamine-induced reinstatement compared with controls. Taken together, these results indicate that prenatal methamphetamine exposure to relatively low levels alters methamphetamine sensitivity in male and female adult offspring.

The G-protein biased kappa opioid agonists, triazole 1.1 and nalfurafine, produce non-uniform behavioral effects in male rhesus monkeys.

Kappa-opioid receptor (KOR) agonists have been studied as potential treatments for pain, pruritus, and substance-use disorders, but prototypical KOR agonists produce side-effects like dysphoria and sedation. Atypical KOR agonists that exhibit G-protein biased signaling at the KOR have been reported to produce therapeutic-like effects with fewer or reduced side effects relative to prototypical KOR agonists. In the current report, behavioral profiles were determined using a behavioral scoring system that was modified to quantify drug-induced behaviors in nonhuman primates (NHPs). Profiles were determined for a prototypical and two biased KOR agonists, alone and combined with the mu-opioid receptor (MOR) agonist, oxycodone. Five adult male rhesus monkeys implanted with intravenous catheters were administered a range of doses of the KOR agonist, U50-488H (0.01-0.1 mg/kg) and the biased KOR agonists, nalfurafine (0.0001-0.001 mg/kg) and triazole 1.1 (0.32-1.0 mg/kg), alone and combined with the MOR agonist, oxycodone (0.01-0.32 mg/kg). In addition, the largest triazole 1.1 dose tested (1.0 mg/kg) was administered in time-course determinations (0-56 min), alone and combined with oxycodone (0.1 mg/kg). U50-488H and nalfurafine produced sedative-like and motor-impairing effects. Triazole 1.1 had a milder side-effect profile, in some instances producing sedative-like effects but to a lesser degree compared with the other KOR agonists, particularly for lip droop and rest/sleep posture. All KOR agonists reduced oxycodone-induced scratch, but nalfurafine produced behavior-disrupting and sedative-like effects when combined with oxycodone that were not observed with triazole 1.1. The duration of triazole 1.1's behavioral effects was relatively short, dissipating entirely by 56 min. Our results suggest that KOR agonists with comparable pharmacology to triazole 1.1 may be useful therapeutics with reduced side-effect profiles, and the mechanisms conferring these benefits may be attributed to factors other than G-protein bias.

Influence of Pair-housing on Sleep Parameters Evaluated with Actigraphy in Female Rhesus Monkeys.

Rhesus monkeys are naturally social animals, and behavioral management strategies have focused on promoting pairhousing in laboratory settings as an alternative to individual or group housing. In humans, co-sleeping can have a major impact on bed partners' sleep, raising the possibility that pair-housing also may influence sleep parameters in monkeys. In the present study, we investigated if pair-housing would impact home-cage partner's sleep in female rhesus monkeys, and if nighttime separation using socialization panels would alter this pattern. Sleep parameters of 10 experimentally naïve adult female rhesus monkeys (5 pairs) were evaluated for 7 consecutive days using actigraphy monitors attached to primate collars. Paired animals then were separated by socialization panels during the night, and sleep-associated measures were evaluated for 7 consecutive days. The data showed that sleep efficiency was significantly lower when monkeys were pairhoused as compared with when they were separated. On the nights when subjects were pair-housed, a positive correlation was detected for sleep measures (both sleep latency and efficiency) of both members of a pair (R2's = 0.16-0.5), suggesting that pair-housing influences sleep quality. On nights when subjects were separated, no correlations were observed for sleep measures between members of the pairs (R2's = 0.004-0.01), suggesting that when separated, the home-cage partner's sleep no longer influenced the partner's sleep. Our results indicate that pair-housing has a strong impact on the home-cage partner's sleep, and that this pattern can be prevented by nighttime separation using socialization panels. Studies evaluating sleep in pair-housed monkeys should consider the effects that the partner's sleep may have on the subject's sleep. Sleep is a biologic phenomenon and experimental outcome that affects physical and behavioral health and altered sleep due to pair-housing may affect a range of research outcomes.

Tolerance and dependence following chronic alprazolam treatment in rhesus monkeys: Role of GABAA receptor subtypes.

BACKGROUND: To assess GABAA receptor subtypes involved in benzodiazepine tolerance and dependence, we evaluated the ability of subtype-selective and non-selective ligands to substitute for (i.e., produce "cross-tolerance") or precipitate withdrawal during chronic alprazolam treatment. METHODS: Four female rhesus monkeys (Macaca mulatta) were implanted with chronic intravenous catheters and administered alprazolam (1.0 mg/kg every 4 h). Following 14+ days of chronic alprazolam, acute administration of selected doses of non-selective and subtype-selective ligands were substituted for, or administered with, alprazolam, followed by quantitative behavioral observations. The ligands included alprazolam and midazolam (positive modulators, non-selective), zolpidem (positive modulator, preferential affinity for α1-containing GABAA receptors), HZ-166 (positive modulator, preferential efficacy at α2- and α3-containing GABAA receptors), and ßCCT (antagonist, preferential affinity for α1-containing GABAA receptors). RESULTS: Acutely, alprazolam and midazolam both induced observable ataxia along with a mild form of sedation referred to as "rest/sleep posture" at a lower dose (0.1 mg/kg, i.v.), whereas at a higher dose (1.0 mg/kg, i.v.), induced deep sedation and observable ataxia. With chronic alprazolam treatment, observable ataxia and deep sedation were reduced significantly, whereas rest/sleep posture was unchanged or emerged. Zolpidem showed a similar pattern of effects, whereas no behaviors engendered by HZ-166 were changed by chronic alprazolam. Administration of ßCCT, but not HZ-166, resulted in significant withdrawal signs. CONCLUSIONS: These results are consistent with a role for α1-containing GABAA receptor subtypes in tolerance and dependence observed with chronic alprazolam, although other receptors may be involved in the withdrawal syndrome.

Modulation of relapse-like drinking in male Sprague-Dawley rats by ligands targeting the α5GABAA receptor.

Preclinical evidence suggests a key role for GABAA receptors containing the α5 subunit (i.e., α5GABAA receptors) in the abuse-related effects of alcohol, including the reinforcing and discriminative stimulus effects, as well as cue-induced alcohol-seeking behavior. However, the contribution of this GABAA receptor subtype to relapse-like drinking behavior remains unknown. The present study evaluated the capacity of ligands targeting α5GABAA receptors to modulate the alcohol deprivation effect (ADE), a model of relapse-like drinking. Groups of Sprague-Dawley rats underwent repeated cycles of long-term access to alcohol solutions (5%, 10%, 20% v/v) and water in the home cage followed by water only deprivation periods. Upon evidence that the ADE could be reliably expressed across cycles, drug treatment was initiated. One group received the α5GABAA receptor-preferring agonist QH-ii-066 and the other group received the α5GABAA receptor-selective inverse agonist L-655,708. At the end of ADE testing, rats underwent testing in the elevated zero maze under vehicle or L-655,708 treatment for assessment of anxiety-like behavior. The ADE was reliably expressed across repeated cycles of alcohol access/deprivation in a subset of rats. Low doses of QH-ii-066 enhanced expression of the ADE; whereas, L-655,708 dose-dependently inhibited expression of the ADE. L-655,708 did not engender anxiogenic effects in the elevated zero maze under the conditions evaluated. These findings suggest a key role for α5GABAA receptor mechanisms in relapse-like drinking. Moreover, they suggest that α5GABAA receptors may represent a novel pharmacological target for the development of medications to prevent or reduce alcohol relapse.

8-Substituted Triazolobenzodiazepines: In Vitro and In Vivo Pharmacology in Relation to Structural Docking at the α1 Subunit-Containing GABAA Receptor.

In order to develop improved anxiolytic drugs, 8-substituted analogs of triazolam were synthesized in an effort to discover compounds with selectivity for α2/α3 subunit-containing GABAA subtypes. Two compounds in this series, XLi-JY-DMH (6-(2-chlorophenyl)-8-ethynyl-1-methyl-4H-benzo [f][1,2,4]triazolo[4,3-a][1,4]diazepine) and SH-TRI-108 [(E)-8-ethynyl-1-methyl-6-(pyridin-2-yl)-4H-benzo [f][1,2,4]triazolo[4,3-a][1,4]diazepine], were evaluated for in vitro and in vivo properties associated with GABAA subtype-selective ligands. In radioligand binding assays conducted in transfected HEK cells containing rat αXß3γ2 subtypes (X = 1,2,3,5), no evidence of selectivity was obtained, although differences in potency relative to triazolam were observed overall (triazolam > XLi-JY-DMH > SH-TRI-108). In studies with rat αXß3γ2 subtypes (X = 1,2,3,5) using patch-clamp electrophysiology, no differences in maximal potentiation of GABA-mediated Cl- current was obtained across subtypes for any compound. However, SH-TRI-108 demonstrated a 25-fold difference in functional potency between α1ß3γ2 vs. α2ß3γ2 subtypes. We evaluated the extent to which this potency difference translated into behavioral pharmacological differences in monkeys. In a rhesus monkey conflict model of anxiolytic-like effects, triazolam, XLi-JY-DMH, and SH-TR-108 increased rates of responding attenuated by shock (anti-conflict effect) but also attenuated non-suppressed responding. In a squirrel monkey observation procedure, both analogs engendered a profile of sedative-motor effects similar to that reported previously for triazolam. In molecular docking studies, we found that the interactions of the 8-ethynyl triazolobenzodiazepines with the C-loop of the α1GABAA site was stronger than that of imidazodiazepines XHe-II-053 and HZ-166, which may account for the non-sedating yet anxiolytic profile of these latter compounds when evaluated in previous studies.

Enhancement of cue-induced reinstatement of alcohol seeking by acute total sleep restriction in male Wistar rats.

Clinical studies suggest that sleep impairment is a barrier to successful treatment in alcohol use disorder (AUD) patients, with sleep disruption associated with relapse to alcohol taking. To date, no preclinical study has evaluated the relationship between impaired sleep and alcohol relapse. In the present study, we used a self-administration model to investigate the effects of sleep restriction on reinstatement induced by alcohol-paired environmental cues. Using a sucrose fading protocol, male Wistar rats (N = 8) were trained to self-administer alcohol under a fixed-ratio 2 schedule of alcohol delivery such that completion of every second response resulted in the delivery of the alcohol solution and activation of the alcohol-paired cue light. Once self-administration was stable, behavior was extinguished by omitting delivery of the alcohol solution and the alcohol-paired cues. When responding reached low, stable levels, alcohol seeking was induced by re-presentation of the alcohol-paired cues but with no alcohol solution available for self-administration. To evaluate the effects of sleep restriction on cue-induced alcohol seeking, reinstatement tests were conducted after 6-h of total (slow wave + rapid eye movement [REM]) sleep restriction using the gentle handling method or after 6-h of REM sleep-only restriction using the flower pot method. Relevant control conditions also were evaluated. The results showed that acute restriction of total sleep, but not REM sleep primarily, significantly augmented cue-induced reinstatement of alcohol seeking. This increase was specific to total sleep restriction conditions and cannot be attributed to differences in alcohol intake, responding, or days to extinction. Our findings imply that acute slow wave sleep restriction is necessary and/or sufficient for the enhancement of cue-induced alcohol seeking and, further, suggest that decreased slow wave sleep in AUD patients places individuals at a unique risk for relapse.

Tolerance and dependence following chronic alprazolam treatment: quantitative observation studies in female rhesus monkeys.

RATIONALE: In order to understand mechanisms underlying tolerance and dependence following chronic benzodiazepine treatments, quantitative and reproducible behavioral models of these phenomena are required. OBJECTIVES: This research evaluated the ability of chronic treatment with a commonly prescribed benzodiazepine, alprazolam, to induce tolerance to sedative effects and physical dependence using a novel set of behavioral measurements in rhesus monkeys. METHODS: Four female rhesus monkeys (Macaca mulatta) were implanted with chronic intravenous catheters and administered i.v. alprazolam (1.0 mg/kg every 4 h, 38 days total). Quantitative observation measures were obtained during the 38 days of treatment. Acute administration of the benzodiazepine receptor antagonist flumazenil (0.1, 0.3 mg/kg, i.v.) was given to assess precipitated withdrawal. On day 39, saline was substituted for alprazolam and withdrawal signs were assessed for 7 days. RESULTS: Maximal sedation ("deep sedation") was evident on day 1 but was not significantly different from baseline levels by day 4 and was absent for the remainder of the 38 days of treatment. A milder form of sedation, "rest/sleep posture," emerged by day 3 and did not decline over 38 days. Cessation of alprazolam treatment resulted in significant withdrawal signs (nose rub, vomit, procumbent posture, tremor/jerk, rigid posture) that dissipated by day 3. These signs also were observed with flumazenil (0.3 mg/kg). CONCLUSIONS: Chronic alprazolam treatment resulted in rapid tolerance to some behaviors (e.g., deep sedation) but no tolerance to others (e.g., rest/sleep posture). Physical dependence was observed via both spontaneous and precipitated withdrawal. Based on previous research, these phenomena may reflect differential plasticity at GABAA receptor subtypes.

Evaluation of the anti-conflict, reinforcing, and sedative effects of YT-III-31, a ligand functionally selective for α3 subunit-containing GABAA receptors.

BACKGROUND: In recent years, pharmacological strategies have implicated α3 subunit-containing GABAA (α3GABAA) receptor subtypes in the anxiety-reducing effects of benzodiazepines, whereas transgenic mouse approaches have implicated α2 or α5 subunit-containing GABAA receptors. AIMS: We investigated the role of α3GABAA subtypes in benzodiazepine-induced behaviors by evaluating the anti-conflict, reinforcing, and sedative-motor effects of the novel compound YT-III-31, which has functional selectivity for α3GABAA receptors. METHODS: Female and male rhesus monkeys were trained under a conflict procedure (n = 3), and a progressive-ratio schedule of reinforcement with midazolam as the training drug (n = 4). Sedative-like behavior was assessed using a quantitative behavioral observation procedure (n = 4). A range of doses of YT-III-31 was administered in all tests using the i.v. route of administration. RESULTS: In the conflict procedure, increasing doses of YT-III-31 resulted only in dose-dependent attenuation of non-suppressed responding. In the progressive-ratio model of self-administration, YT-III-31 maintained average injections/session above vehicle levels at 0.1 and 0.18 mg/kg/injection. In quantitative observation procedures, YT-III-31 engendered mild sedative effects ("rest/sleep posture"), and deep sedation at the highest dose tested (5.6 mg/kg, i.v.), along with a suppression of tactile/oral exploration and increased observable ataxia. In contrast to other benzodiazepine-like ligands, YT-III-31 uniquely engendered a biphasic dose-response function for locomotion and suppressed self-groom. CONCLUSIONS: The finding that YT-III-31 lacked anti-conflict properties is in accordance with transgenic mouse research indicating no role for α3GABAA subtypes in benzodiazepine-mediated anxiety reduction. Instead, our results raise the possibility of a role for α3GABAA receptors in the abuse potential and sedative effects of benzodiazepine-type drugs.

Changes in the elimination and resurgence of alcohol-maintained behavior in rats and the effects of naltrexone.

Resurgence may be a mechanism of relapse in alcohol use disorder patients upon discharge from treatment as part of an abuse-treatment-relapse cycle. Adjunctive pharmacotherapies may be a means to facilitate behavioral treatments and block resurgence. Experiments were conducted using a model of alcohol self-administration to assess the repeatability of the elimination and resurgence of alcohol-maintained behavior and the effects of naltrexone. Experiments had three phases. In Phase 1, behavior was maintained by oral alcohol under a fixed-ratio schedule. In Phase 2, behavior was extinguished via condensed milk delivery under a differential-reinforcement-of-other-behavior (DRO) schedule. In Phase 3, the DRO schedule was eliminated. In Experiment 1, this 3-phase cycle was replicated 4 times. Across replications, response rates and dose of alcohol consumed did not differ in Phase 1, alcohol-maintained behavior was eliminated more rapidly in Phase 2, and the resurgence effect was generally stable in Phase 3. In Experiment 2, naltrexone was administered in Phase 2, Phase 3, or both Phases 2 and 3, to separate groups of rats. Naltrexone facilitated the elimination of alcohol-maintained behavior in Phase 2 and, the resurgence of alcohol-maintained behavior was reduced only for those rats that received naltrexone in both phases. Together, these experiments demonstrate that the resurgence of alcohol-maintained behavior is replicable within-subjects and, further, resurgence of alcohol-maintained behavior may be a useful model to evaluate pharmacological interventions to facilitate behavioral treatments and reduce the likelihood of relapse. Results with naltrexone support the use of medication-assisted therapy approaches to reduce relapse risk in patients. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

GABAA Receptor Subtypes and the Abuse-Related Effects of Ethanol in Rhesus Monkeys: Experiments with Selective Positive Allosteric Modulators.

BACKGROUND: Previous studies have investigated α1GABAA and α5GABAA receptor mechanisms in the behavioral effects of ethanol (EtOH) in monkeys. However, genetic studies in humans and preclinical studies with mutant mice suggest a role for α2GABAA and/or α3GABAA receptors in the effects of EtOH. The development of novel positive allosteric modulators (PAMs) with functional selectivity (i.e., selective efficacy) at α2GABAA and α3GABAA receptors allows for probing of these subtypes in preclinical models of the discriminative stimulus and reinforcing effects of EtOH in rhesus macaques. METHODS: In discrimination studies, subjects were trained to discriminate EtOH (2 g/kg, intragastrically) from water under a fixed-ratio (FR) schedule of food delivery. In oral self-administration studies, subjects were trained to self-administer EtOH (2% w/v) or sucrose (0.3 to 1% w/v) under an FR schedule of solution availability. RESULTS: In discrimination studies, functionally selective PAMs at α2GABAA and α3GABAA (HZ-166) or α3GABAA (YT-III-31) receptors substituted fully (maximum percentage of EtOH-lever responding ≥80%) for the discriminative stimulus effects of EtOH without altering response rates. Full substitution for EtOH also was engendered by a nonselective PAM (triazolam), an α5GABAA -preferring PAM (QH-ii-066) and a PAM at α2GABAA , α3GABAA , and α5GABAA receptors (L-838417). A partial (MRK-696) or an α1GABAA -preferring (zolpidem) PAM only engendered partial substitution (i.e., ~50 to 60% EtOH-lever responding). In self-administration studies, pretreatments with the functionally selective PAMs at α2GABAA and α3GABAA (XHe-II-053 and HZ-166) or α3GABAA (YT-III-31 and YT-III-271) receptors increased EtOH, but not sucrose, drinking at doses that had few, or no, observable sedative-motor effects. CONCLUSIONS: Our results confirm prior findings regarding the respective roles of α1GABAA and α5GABAA receptors in the discriminative stimulus effects of EtOH and, further, suggest a key facilitatory role for α3GABAA and potentially α2GABAA receptors in several abuse-related effects of EtOH in monkeys. Moreover, they reveal a potential role for these latter subtypes in EtOH's sedative effects.

Alcohol-induced changes in the gut microbiome and metabolome of rhesus macaques.

RATIONALE: Increasing evidence has demonstrated that changes in the gut microbiome, including those associated with dietary influences, are associated with alterations in many physiological processes. Alcohol consumption is common across human cultures and is likely to have a major effect on the gut microbiome, but there remains a paucity of information on its effects in primates. OBJECTIVES: The effects of chronic alcohol consumption on the primate gut microbiome and metabolome were studied in rhesus macaques that were freely drinking alcohol. The objectives of the study were to determine what changes occurred in the gut microbiome following long-term exposure to alcohol and if these changes were reversible following a period of abstinence. METHODS: Animals consuming alcohol were compared to age-matched controls without access to alcohol and were studied before and after a period of abstinence. Fecal samples from rhesus macaques were used for 16S rRNA sequencing to profile the gut microbiome and for metabolomic profiling using mass spectrometry. RESULTS: Alcohol consumption resulted in a loss of alpha-diversity in rhesus macaques, though this was partially ameliorated by a period of abstinence. Higher levels of Firmicutes were observed in alcohol-drinking animals at the expense of a number of other microbial taxa, again normalizing in part with a period of abstinence. Metabolomic changes were primarily associated with differences in glycolysis when animals were consuming alcohol and differences in fatty acids when alcohol-drinking animals became abstinent. CONCLUSIONS: The consumption of alcohol has specific effects on the microbiome and metabolome of rhesus macaques independent of secondary influences. Many of these changes are reversed by a relatively short period of abstinence.

α5GABAA subunit-containing receptors and sweetened alcohol cue-induced reinstatement and active sweetened alcohol self-administration in male rats.

RATIONALE: GABAA receptors containing the α5 subunit (i.e., α5GABAA receptors) appear to be critically involved in the reinforcing and subjective effects of alcohol. Their role in alcohol relapse remains unknown. OBJECTIVES: Pharmacological approaches were used to probe the role of α5GABAA receptors in alcohol seeking induced by re-exposure to a sweetened alcohol-paired cue, as well as in alcohol + sucrose vs. sucrose self-administration. METHODS: For reinstatement studies, rats were trained to self-administer alcohol under a fixed-ratio schedule in which responding was maintained by alcohol + sucrose deliveries and an alcohol-paired stimulus. Sweetened alcohol seeking was extinguished by eliminating solution deliveries and the sweetened alcohol-paired stimulus. During reinstatement tests, animals received pretreatments of an α5GABAA inverse agonist (L-655,708) or an agonist (QH-ii-066) prior to sessions in which presentation of the sweetened alcohol-paired stimulus was restored, but no solution was delivered. For self-administration studies, rats were trained to self-administer alcohol + sucrose or sucrose under a fixed-ratio schedule. Once stable, animals received pretreatments of QH-ii-066, L-655,708, the inverse agonist RY-023, or naltrexone. RESULTS: L-655,708 attenuated reinstatement of sweetened alcohol seeking by alcohol + sucrose-paired cues; whereas sweetened alcohol-seeking behavior was augmented by QH-ii-066, albeit at different doses in different rats. Both L-655,708 and RY-023 selectively reduced alcohol + sucrose vs. sucrose self-administration. In contrast, naltrexone reduced both alcohol + sucrose and sucrose self-administration; whereas QH-ii-066 enhanced sucrose self-administration only. CONCLUSIONS: α5GABAA receptors play a key role in the modulation of sweetened alcohol cue-induced reinstatement, as well as in alcohol + sucrose but not sucrose self-administration. Inverse agonist activity at α5GABAA receptors may offer a novel strategy for both the reduction of problematic drinking and the prevention of relapse.