A Comparative Study of the Pharmacokinetics of Clozapine N-Oxide and Clozapine N-Oxide Hydrochloride Salt in Rhesus Macaques.

Translating chemogenetic techniques from nonhuman primates to potential clinical applications has been complicated in part due to in vivo conversion of the chemogenetic actuator, clozapine N-oxide (CNO), to its pharmacologically active parent compound, clozapine, a ligand with known side effects, including five boxed warnings from the Food and Drug Administration. Additionally, the limited solubility of CNO requires high concentrations of potentially toxic detergents such as dimethylsulfoxide (DMSO). To address these concerns, pharmacokinetic profiling of commercially available CNO in DMSO (CNO-DMSO, 10% v/v DMSO in saline) and a water-soluble salt preparation (CNO-HCl, saline) was conducted in rhesus macaques. A time course of blood plasma and cerebrospinal fluid (CSF) concentrations of CNO and clozapine was conducted (30-240 minutes post-administration) following a range of doses (3-10 mg/kg, i.m. and/or i.v.) of CNO-DMSO or CNO-HCl. CNO-HCl resulted in 6- to 7-fold higher plasma concentrations of CNO compared to CNO-DMSO, and relatively less clozapine (3%-5% clozapine/CNO in the CNO-DMSO group and 0.5%-1.5% clozapine/CNO in the CNO-HCl group). Both groups had large between-subjects variability, pointing to the necessity of performing individual CNO pharmacokinetic studies prior to further experimentation. The ratio of CNO measured in the CSF was between 2% and 6% of that measured in the plasma and did not differ across drug preparation, indicating that CSF concentrations may be approximated from plasma samples. In conclusion, CNO-HCl demonstrated improved bioavailability compared with CNO-DMSO with less conversion to clozapine. Further investigation is needed to determine if brain concentrations of clozapine following CNO-HCl administration are pharmacologically active at off-target monoaminergic receptor systems in the primate brain.

Discriminative Stimulus Effects and Metabolism of Ethanol in Rhesus Monkeys.

BACKGROUND: Animal models are an essential feature of drug and pharmacotherapy development for treating alcohol use disorders (AUDs). The rhesus macaque is a robust animal model for many aspects of AUDs particularly in exploiting individual differences in oral self-administration of ethanol (EtOH), endocrine orchestration of stress response, and menstrual cycle characteristics. However, the clearance rates of EtOH have not been reported in this species, and the GABAA and N-methyl-D-aspartate (NMDA) receptor involvement in EtOH's discriminative stimulus effects has not been fully characterized. METHODS: EtOH clearance rates following 2 doses of EtOH on separate days (0.5 and 1.0 g/kg, i.g.) were determined in 8 young adult male rhesus macaques. The EtOH was given by nasogastric gavage, and repeated blood samples were taken over 5 hours without sedation. Next, all subjects were trained on a 2-choice 1.0 g/kg EtOH (i.g.) versus water discrimination with a 60-minutes pretreatment period to capture peak blood EtOH concentration (BEC). Substitution testing was conducted with GABAA ligands pentobarbital (i.g. and i.m.) and midazolam (i.g.), as well as NMDA antagonist MK-801 (i.m.). RESULTS: Peak BECs were 34 and 87 mg/dl for 0.5 and 1.0 g/kg doses, respectively, and occurred at 66 and 87 minutes following gavage. All GABAA and NMDA ligands tested resulted in responding on the EtOH-appropriate lever with the potency ranking of MK-801 (ED50 : 0.017 mg/kg) > midazolam (ED50 : 1.6 mg/kg) > pentobarbital (ED50 : 3.7 mg/kg) > EtOH (ED50 : 700 mg/kg, or 0.7 g/kg) in these subjects. CONCLUSIONS: These results suggest that the compound discriminative stimulus effects of EtOH are highly consistent across species, providing further support for the rhesus macaque as strong model for pharmacotherapy development for AUD.

Intra-VTA deltorphin, but not DPDPE, induces place preference in ethanol-drinking rats: distinct DOR-1 and DOR-2 mechanisms control ethanol consumption and reward.

BACKGROUND: While there is a growing body of evidence that the delta opioid receptor (DOR) modulates ethanol (EtOH) consumption, development of DOR-based medications is limited in part because there are 2 pharmacologically distinct DOR subtypes (DOR-1 and DOR-2) that can have opposing actions on behavior. METHODS: We studied the behavioral influence of the DOR-1-selective agonist [D-Pen(2) ,D-Pen(5) ]-Enkephalin (DPDPE) and the DOR-2-selective agonist deltorphin microinjected into the ventral tegmental area (VTA) on EtOH consumption and conditioned place preference (CPP) and the physiological effects of these 2 DOR agonists on GABAergic synaptic transmission in VTA-containing brain slices from Lewis rats. RESULTS: Neither deltorphin nor DPDPE induced a significant place preference in EtOH-naïve Lewis rats. However, deltorphin (but not DPDPE) induced a significant CPP in EtOH-drinking rats. In contrast to the previous finding that intra-VTA DOR-1 activity inhibits EtOH consumption and that this inhibition correlates with a DPDPE-induced inhibition of GABA release, here we found no effect of DOR-2 activity on EtOH consumption nor was there a correlation between level of drinking and deltorphin-induced change in GABAergic synaptic transmission. CONCLUSIONS: These data indicate that the therapeutic potential of DOR agonists for alcohol abuse is through a selective action at the DOR-1 form of the receptor.

Dopamine, corticostriatal connectivity, and intertemporal choice.

Value-based decisions optimize behavioral outcomes. Because delayed rewards are discounted, an increased tendency to choose smaller, immediate rewards can lead to suboptimal choice. Steep discounting of delayed rewards (impulsivity) characterizes subjects with frontal lobe damage and behavioral disorders including substance abuse. Correspondingly, animal studies and indirect evidence in humans suggest that lower dopamine in the frontal cortex contributes to steeper discounting by impairing corticostriatal function. To test this hypothesis directly, we performed a randomized, double-blind, counterbalanced, placebo-controlled study in which we administered the brain penetrant catechol-O-methyltransferase inhibitor tolcapone or placebo to healthy subjects performing a delay discounting task. Tolcapone significantly increased choice of delayed monetary rewards, and this tolcapone-induced increase covaried with increased BOLD activity in the left ventral putamen and anterior insula. Tolcapone also changed corticostriatal connectivity: specifically, by inducing a decrease in the coherence between ventral putamen and pregenual cingulate cortex. These results indicate that raising cortical dopamine levels attenuates impulsive choice by changing corticostriatal function.

Characterization of DREADD receptor expression and function in rhesus macaques trained to discriminate ethanol.

Circuit manipulation has been a staple technique in neuroscience to identify how the brain functions to control complex behaviors. Chemogenetics, including designer receptors exclusively activated by designer drugs (DREADDs), have proven to be a powerful tool for the reversible modulation of discrete brain circuitry without the need for implantable devices, thereby making them especially useful in awake and unrestrained animals. This study used a DREADD approach to query the role of the nucleus accumbens (NAc) in mediating the interoceptive effects of 1.0 g/kg ethanol (i.g.) in rhesus monkeys (n = 7) using a drug discrimination procedure. After training, stereotaxic surgery was performed to introduce an AAV carrying the human muscarinic 4 receptor DREADD (hM4Di) bilaterally into the NAc. The hypothesis was that decreasing the output of the NAc by activation of hM4Di with the DREADD actuator, clozapine-n-oxide (CNO), would potentiate the discriminative stimulus effect of ethanol (i.e., a leftward shift the ethanol dose discrimination curve). The results showed individual variability shifts of the ethanol dose-response determination under DREADD activation. Characterization of the expression and function of hM4Di with MRI, immunohistochemical, and electrophysiological techniques found the selectivity of NAc transduction was proportional to behavioral effect. Specifically, the proportion of hM4Di expression restricted to the NAc was associated with the potency of the discriminative stimulus effects of ethanol. Together, these experiments highlight the NAc in mediating the interoceptive effects of ethanol, provide a framework for validation of chemogenetic tools in primates, and underscore the importance of robust within-subjects examination of DREADD expression for interpretation of behavioral findings.

Causal role for the primate superior colliculus in the computation of evidence for perceptual decisions.

Trained monkeys performed a two-choice perceptual decision-making task in which they reported the perceived orientation of a dynamic Glass pattern, before and after unilateral, reversible, inactivation of a brainstem area-the superior colliculus (SC)-involved in preparing eye movements. We found that unilateral SC inactivation produced significant decision biases and changes in reaction times consistent with a causal role for the primate SC in perceptual decision-making. Fitting signal detection theory and sequential sampling models to the data showed that SC inactivation produced a decrease in the relative evidence for contralateral decisions, as if adding a constant offset to a time-varying evidence signal for the ipsilateral choice. The results provide causal evidence for an embodied cognition model of perceptual decision-making and provide compelling evidence that the SC of primates (a brainstem structure) plays a causal role in how evidence is computed for decisions-a process usually attributed to the forebrain.

Cross-Species Translational Findings in the Discriminative Stimulus Effects of Ethanol.

The progress on understanding the pharmacological basis of ethanol's discriminative stimulus effects has been substantial, but appears to have plateaued in the past decade. Further, the cross-species translational efforts are clear in laboratory animals, but have been minimal in human subject studies. Research findings clearly demonstrate that ethanol produces a compound stimulus with primary activity through GABA and glutamate receptor systems, particularly ionotropic receptors, with additional contribution from serotonergic mechanisms. Further progress should capitalize on chemogenetic and optogenetic techniques in laboratory animals to identify the neural circuitry involved in mediating the discriminative stimulus effects of ethanol. These infrahuman studies can be guided by in vivo imaging of human brain circuitry mediating ethanol's subjective effects. Ultimately, identifying receptors systems, as well as where they are located within brain circuitry, will transform the use of drug discrimination procedures to help identify possible treatment or prevention strategies for alcohol use disorder.

Effect of repeated abstinence on chronic ethanol self-administration in the rhesus monkey.

RATIONALE: Abstinence-based approaches to treating alcohol use disorder (AUD) are highly prevalent, but abstinence from chronic drinking may exacerbate subsequent levels of alcohol intake in relapse. OBJECTIVE: Use a non-human primate model that encompasses a range of chronic voluntary ethanol drinking to isolate biological responses to repeated cycles of imposed abstinence as a function of baseline voluntary alcohol drinking levels. METHODS: Over a 26-month protocol, young adult male rhesus macaques were first induced to drink alcohol and then given continuous access to 4% (w/v) ethanol (n = 8) or water (n = 4) for approximately 14 months, followed by three 28- to 35-day abstinence phases, with 3 months of ethanol access in between. Ethanol intake and blood ethanol concentration (BEC) were the primary dependent variables. Observational signs of physical dependence and circulating ACTH and cortisol were monitored. RESULTS: Prior to abstinence, stable, categorical, individual differences in voluntary ethanol intake under chronic access conditions were found. Following abstinence, categorical "non-heavy" drinking subjects increased drinking transiently (increased between 0.7 and 1.4 g/kg/day in first month after abstinence) but returned to baseline after 3 months. Categorical "heavy" drinkers, however, maintained drinking 1.0-2.6 g/kg above baseline for over 3 months following abstinence. Signs of physical dependence were rare, although huddling and social withdrawal increased in ethanol and control subjects. The most prominent effect on hormonal measures was heightened cortisol during abstinence that increased to a greater extent in ethanol subjects. CONCLUSION: Involuntary abstinence increases drinking in the absence of overt physical withdrawal symptoms, and heavy drinkers are more robustly affected compared to non-heavy drinkers.

Social setting, social rank and HPA axis response in cynomolgus monkeys.

RATIONALE: Hypothalamic-pituitary-adrenal (HPA) axis activity under different social settings in non-human primates is understudied. OBJECTIVE: The aim of this study is to evaluate the response of pituitary-adrenal hormones (adrenocorticotropic hormone (ACTH) and cortisol) to pharmacological challenges of the HPA axis in male cynomolgus macaques under different social settings. METHODS: Male cynomolgus macaques (Macaca fascicularis, n = 11) were individually (A) and socially housed (B) in alternation, over consecutive months, in an ABA design. During each experimental phase, plasma ACTH and cortisol were measured in response to low- and mild-intensity psychological stressors and following administration of saline, naloxone, ovine-corticotropin-releasing factor (oCRF), and dexamethasone. RESULTS: These data demonstrate that cortisol measured under low stress conditions is sensitive to social rank (dominance hierarchy) and distinguishes dominant from non-dominant animals during both individual and social settings. Administration of naloxone resulted in elevated circulating ACTH and cortisol, while oCRF only increased circulating cortisol. During social housing, the cortisol response to naloxone and oCRF was increased, whereas dexamethasone suppression of ACTH and cortisol remained consistent across all social settings. CONCLUSIONS: Circulating ACTH and cortisol are differentially sensitive to changes in social settings in non-human primates. Cortisol response increased during social housing and could be stimulated by both naloxone and oCRF, whereas ACTH response was generally not influenced by social setting or oCRF but was increased by naloxone. These data show differential adrenal and pituitary response to changes in social settings and a small, but consistent, effect of social dominance.

Ranking Cognitive Flexibility in a Group Setting of Rhesus Monkeys with a Set-Shifting Procedure.

Attentional set-shifting ability is an executive function underling cognitive flexibility in humans and animals. In humans, this function is typically observed during a single experimental session where dimensions of playing cards are used to measure flexibility in the face of changing rules for reinforcement (i.e., the Wisconsin Card Sorting Test (WCST)). In laboratory animals, particularly non-human primates, variants of the WCST involve extensive training and testing on a series of dimensional discriminations, usually in social isolation. In the present study, a novel experimental approach was used to assess attentional set-shifting simultaneously in 12 rhesus monkeys. Specifically, monkeys living in individual cages but in the same room were trained at the same time each day in a set-shifting task in the same housing environment. As opposed to the previous studies, each daily session began with a simple single-dimension discrimination regardless of the animal's performance on the previous session. A total of eight increasingly difficult, discriminations (sets) were possible in each daily 45 min session. Correct responses were reinforced under a second-order schedule of flavored food pellet delivery, and criteria for completing a set was 12 correct trials out of a running total of 15 trials. Monkeys progressed through the sets at their own pace and abilities. The results demonstrate that all 12 monkeys acquired the simple discrimination (the first set), but individual differences in the ability to progress through all eight sets were apparent. A performance index (PI) that encompassed progression through the sets, errors and session duration was calculated and used to rank each monkey's performance in relation to each other. Overall, this version of a set-shifting task results in an efficient assessment of reliable differences in cognitive flexibility in a group of monkeys.

Nicotinic receptors in non-human primates: Analysis of genetic and functional conservation with humans.

Nicotinic acetylcholine receptors (nAChRs) are highly conserved between humans and non-human primates. Conservation exists at the level of genomic structure, protein structure and epigenetics. Overall homology of nAChRs at the protein level is 98% in macaques versus 89% in mice, which is highly relevant for evaluating subtype-specific ligands that have different affinities in humans versus rodents. In addition to conservation at the protein level, there is high conservation of genomic structure in terms of intron and exon size and placement of CpG sites that play a key role in epigenetic regulation. Analysis of single nucleotide polymorphisms (SNPs) shows that while the majority of SNPs are not conserved between humans and macaques, some functional polymorphisms are. Most significantly, cynomolgus monkeys express a similar α5 nAChR Asp398Asn polymorphism to the human α5 Asp398Asn polymorphism that has been linked to greater nicotine addiction and smoking related disease. Monkeys can be trained to readily self-administer nicotine, and in an initial study we have demonstrated that cynomolgus monkeys bearing the α5 D398N polymorphism show a reduced behavioral sensitivity to oral nicotine and tend to consume it in a different pattern when compared to wild-type monkeys. Thus the combination of highly homologous nAChR, higher cortical functions and capacity for complex training makes non-human primates a unique model to study in vivo functions of nicotinic receptors. In particular, primate studies on nicotine addiction and evaluation of therapies to prevent or overcome nicotine addiction are likely to be highly predictive of treatment outcomes in humans. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.