Alternative reinforcers enhance the effects of opioid antagonists, but not agonists, on oxycodone choice self-administration in nonhuman primates.

Clinical reports suggest that the most effective strategies for managing opioid use disorder comprise a comprehensive treatment program of both pharmacological and non-pharmacological approaches. However, the conditions under which these combinations are most effective are not well characterized. This study examined whether the presence of an alternative reinforcer could alter the efficacy of FDA-approved opioid antagonist or agonist medications, as well as the non-opioid flumazenil, in decreasing oxycodone choice self-administration in nonhuman primates. Adult squirrel monkeys (n=7; 4 females) responded under concurrent second-order FR3(FR5:S);TO45s schedules of reinforcement for intravenous oxycodone (0.1mg/kg) or saline on one lever and 30% sweetened condensed milk or water on the other. Doses of naltrexone (0.00032-1.0mg/kg), nalbuphine (0.32-10mg/kg), buprenorphine (0.0032-0.032mg/kg), methadone (0.32-1.0mg/kg), or flumazenil (1-3.2mg/kg) were administered intramuscularly prior to oxycodone self-administration sessions that occurred with either milk or water as the alternative. Naltrexone, a m-opioid receptor antagonist, was >30-fold more potent when milk was available compared to water and abolished oxycodone intake (injections/session) while concomitantly increasing milk deliveries at the highest dose tested. Pretreatment with the low efficacy m-agonist nalbuphine was most effective in the presence of milk compared to water, decreasing oxycodone preference to <50% of control values. The higher efficacy m-agonists, methadone and buprenorphine, and the benzodiazepine antagonist flumazenil, did not appreciably alter the reinforcing potency of oxycodone under either condition. These results suggest that antagonist medications used in combination with alternative reinforcers may be an effective strategy to curtail opioid abuse-related behaviors. Significance Statement Clinical treatment programs for opioid use disorder utilize a combination of pharmacological and non-pharmacological approaches. However, the conditions under which these combinations are most effective have not been fully characterized. This study examined whether the effectiveness of m-opioid medications to decrease oxycodone self-administration is altered in the presence of an alternative reinforcer. The results suggest that alternative reinforcers enhance the effects of antagonist or low efficacy partial agonists suggesting they may be a more effective strategy to curtail opioid use.

Reinforcing and Stimulant-Like Effects of Methamphetamine Isomers in Rhesus Macaques.

Monoamine releasers such as d-methamphetamine (d-MA) can reduce cocaine use in laboratory studies and have been forwarded for the management of cocaine use disorder (CUD). However, the proven abuse liability of d-MA has limited enthusiasm for clinical use. The levorotatory isomer of MA, l-MA, appears to have lesser stimulant effects, possibly due to its preferential norepinephrine-releasing properties compared with dopamine. The present study evaluated the abuse potential of l-MA by comparing its reinforcing effects with known stimulant drugs of abuse in nonhuman primates. Adult rhesus macaques (N = 4) responded for intravenous injections of cocaine, d-MA, methcathinone (MCAT), or l-MA under a fixed-ratio (FR) schedule of reinforcement; reinforcing effectiveness was evaluated using behavioral economic demand procedures. In a separate cohort (N = 9), daily activity and food-reinforced responding were assessed during 100 days of treatment with daily dosages of l-MA (2.3 mg/kg per day, i.v.) or d-MA (0.74 mg/kg per day, i.v.) previously shown to decrease cocaine self-administration. Results show that all drugs maintained self-administration, with peak injections reaching ∼100 inj per session for cocaine, MCAT, and d-MA and ∼50 inj per session for l-MA . In demand studies, self-administration of each drug gradually decreased as FR size increased. The exponential model of demand indicated that the reinforcing effectiveness of l-MA was significantly less than the other drugs studied. Chronic l-MA treatment did not appreciably alter daily activity and only transiently suppressed food-reinforced responding. These data, coupled with previous findings that l-MA effectively reduces stimulant self-administration, suggest that l-MA, or other norepinephrine-preferring releasers, may serve as agonist medication for CUD with lesser abuse liability than common psychostimulants. SIGNIFICANCE STATEMENT: Development of pharmacotherapies for cocaine use disorder remains a formidable challenge. Agonist-based therapies show promise, but enthusiasm is tempered by the abuse liability of previously proposed medications. This study evaluated the abuse liability and chronic treatment effects of methamphetamine's levorotatory isomer (l-MA). l-MA demonstrated lower abuse liability compared with commonly abused stimulants and produced few untoward effects. In the context of recent studies demonstrating that l-MA attenuates stimulant self-administration, these findings support l-MA's potential as a pharmacotherapy for stimulant addiction.

Discriminative-Stimulus Effects of Synthetic Cathinones in Squirrel Monkeys.

BACKGROUND: Synthetic cathinones display overlapping behavioral effects with psychostimulants (e.g., methamphetamine [MA]) and/or entactogens (e.g., 3,4-methylenedioxymethaphetamine [MDMA])-presumably reflecting their dopaminergic and/or serotonergic activity. The discriminative stimulus effects of MDMA thought to be mediated by such activity have been well characterized in rodents but have not been fully examined in nonhuman primates. METHODS: The present studies were conducted to systematically evaluate the discriminative stimulus effects of 5 abused synthetic cathinones (methylenedioxypyrovalerone [MDPV], α-pyrrolidinovalerophenone [α-PVP], methcathinone [MCAT], mephedrone, and methylone) in adult male squirrel monkeys trained to distinguish intramuscular injections of MA (0.1 mg/kg; n = 4) or MDMA (0.6 mg/kg; n = 4) from vehicle. RESULTS: Each training drug produced dose-dependent effects and, at the highest dose, full substitution. MDMA produced predominantly vehicle-like responding in the MA-trained group, whereas the highest dose of MA (0.56 mg/kg) produced partial substitution (approximately 90% appropriate lever responding in one-half of the subjects) in the MDMA-trained group. MDPV, α-PVP, and MCAT produced full substitution in MA-trained subjects, but, at the same or higher doses, only substituted for MDMA in one-half of the subjects, consistent with primarily dopaminergically mediated interoceptive effects. In contrast, mephedrone and methylone fully substituted in MDMA-trained subjects but failed to fully substitute for the training drug in MA-trained subjects, suggesting a primary role for serotonergic actions in their interoceptive effects. CONCLUSIONS: These findings suggest that differences in the interoceptive effects of synthetic cathinones in nonhuman primates reflect differing compositions of monoaminergic actions that also may mediate their subjective effects in humans.

Vigilance demand and the effects of stimulant drugs in a five-choice reaction-time procedure in mice.

Stimulant drugs used for treating attention-deficit hyperactivity disorder (ADHD) increase signal-detection accuracy in five-choice serial reaction-time procedures. These increases may result from drug-induced increases in control exerted by the stimuli that prompt responses, which was assessed in the present study. Mice were trained with food reinforcement to nose poke into one of five holes after its illumination (signal), and effects of methylphenidate, d-amphetamine, and pentobarbital were assessed. Subsequently, the time from trial onset to signal was changed from fixed to variable for one group of subjects. A 'warning' stimulus (change in ambient lighting) preceding the signal was added for a second group. Effects of the drugs were reassessed. Dose-related increases in accuracy of signal detection (nose pokes in hole where a signal was displayed) were obtained with methylphenidate and d-amphetamine, but not with pentobarbital. When the presignal time was variable, increases in signal detection were not obtained with either stimulant. When a warning stimulus preceded the signal, the increases in accuracy were similar to those obtained without the warning stimulus. Hence, a procedure that increased vigilance demand (using a variable prestimulus period) eliminated the effects of drugs useful in treating ADHD, whereas a procedure that decreased vigilance demand (adding the warning light) had no appreciable effects on the response to stimulant drugs. Taken together, the present results suggest that the five-choice serial reaction-time has predictive validity for selecting drugs effective for treating ADHD, although effects can depend critically on the stimulus conditions used and the vigilance required by the procedure.

Interactions between nicotine and drugs of abuse: a review of preclinical findings.

Polysubstance abuse is common among substance-use disorder patients, and nicotine is one of the most commonly co-used substances. Epidemiological and clinical laboratory studies suggest that nicotine, when combined with other drugs of abuse, increases intake of one or both substances. This review focuses on the preclinical literature regarding nicotine's interaction with alcohol, stimulants (i.e., cocaine, amphetamines), opioids (i.e., morphine, heroin), and Δ9-tetrahydrocannabinol (THC). The current understanding of how these various classes of abused drugs may interact with nicotine on behavioral, physiological, and pharmacological indices that may be important in maintaining co-use of one or both substances in human populations are highlighted. Suggestions as to future areas of research and gaps in knowledge are offered.

Preclinical efficacy of N-substituted benztropine analogs as antagonists of methamphetamine self-administration in rats.

Atypical dopamine-uptake inhibitors have low abuse potential and may serve as leads for development of cocaine-abuse treatments. Among them, the benztropine (BZT) derivatives, N-butyl (JHW007), N-allyl (AHN2-005), and N-methyl (AHN1-055) analogs of 3α-[bis(4'-fluorophenyl)methoxy]-tropane dose-dependently decreased cocaine self-administration without effects on food-maintained responding. Our study examined selectivity by assessing their effects on self-administration of other drugs. As with cocaine, each BZT analog (1.0-10.0 mg/kg i.p.) dose-dependently decreased maximal self-administration of d-methamphetamine (0.01-0.32 mg/kg/infusion) but was inactive against heroin (1.0-32.0 µg/kg/infusion) and ketamine (0.032-1.0 mg/kg/infusion) self-administration. Further, standard dopamine indirect-agonists [WIN35,428 ((-)-3ß-(4-fluorophenyl)-tropan-2-ß-carboxylic acid methyl ester tartrate), d-amphetamine (0.1-1.0 mg/kg i.p., each)] dose-dependently left-shifted self-administration dose-effect curves for d-methamphetamine, heroin, and ketamine. Noncompetitive NMDA-glutamate receptor/channel antagonists [(+)-MK-801 (0.01-0.1 mg/kg i.p.), memantine (1.0-10.0 mg/kg i.p.)] also left-shifted dose-effect curves for d-methamphetamine and ketamine (but not heroin) self-administration. The µ-agonists [dl-methadone and morphine (1.0-10.0 mg/kg i.p., each)] dose-dependently decreased maximal self-administration of µ-agonists (heroin, remifentanil) but not d-methamphetamine or ketamine self-administration. The µ-agonist-induced decreases were similar to the effects of BZT analogs on stimulant self-administration and effects of food prefeeding on responding maintained by food reinforcement. Radioligand-binding and behavioral studies suggested that inhibition of dopamine transporters and σ receptors were critical for blocking stimulant self-administration by BZT-analogs. Thus, the present results suggest that the effects of BZT analogs on stimulant self-administration are similar to effects of µ-agonists on µ-agonist self-administration and food prefeeding on food-reinforced responding, which implicates behavioral mechanisms for these effects and further supports development of atypical dopamine uptake inhibitors as medications for stimulant abuse.

A Multimodal Preclinical Assessment of MDMA in Female and Male Rats: Prohedonic, Cognition Disruptive, and Prosocial Effects.

Background: Frontline antidepressants such as selective serotonin reuptake inhibitors (SSRIs) leave many patients with unmet treatment needs. Moreover, even when SSRIs reduce depressive symptoms, anhedonia, the loss of pleasure to previously rewarding activities, often remains unabated. This state of affairs is disheartening and calls for the development of medications to more directly treat anhedonia. The atypical psychedelic 3,4-methylenedioxymethamphetamine (MDMA) might have promise as a prohedonic medication given its efficacious applications for treatment-resistant post-traumatic stress disorder and comorbid depression. However, in addition to its prosocial effects as an entactogen, MDMA is also associated with neurotoxic cognitive deficits. The present studies were designed to examine the relative potency of MDMA in female and male rats across three distinct behavioral domains to assist in defining a preclinical profile of MDMA as a candidate prohedonic therapeutic. Methods: First, signal detection metrics of reward responsivity were examined using the touchscreen probabilistic reward task (PRT), a reverse-translated assay used to objectively quantify anhedonic phenotypes in humans. Second, to probe potential cognitive deficits, touchscreen-based assays of psychomotor vigilance and delayed matching-to-position were used to examine attentional processes and short-term spatial memory, respectively. Finally, MDMA's entactogenic effects were studied via pairwise assessments of social interaction facilitated by machine-learning analyses. Results: Findings show (1) dose-dependent increases in reward responsivity as quantified by the PRT, (2) dose-dependent deficits in attention and short-term memory, and (3) dose-dependent increases in aspects of prosocial interaction in male but not female subjects. Neither the desirable (prohedonic) nor undesirable (cognition disruptive) effects of MDMA persisted beyond 24 h. Conclusions: The present results characterize MDMA as a promising prohedonic treatment, notwithstanding some liability for short-lived cognitive impairment following acute administration.

Effects of chronic naltrexone treatment on relapse-related behavior and neural responses to fentanyl in awake nonhuman primates.

Naltrexone, an opioid antagonist that blocks the reinforcing properties of opioid agonists, is often prescribed to preclude relapse to opioid use disorder (OUD) following detoxification. However, few laboratory studies have directly investigated the ability of naltrexone to alter relapse-inducing effects of opioid agonists, including their priming strength in reinstatement studies and their impact in brain regions known to be involved in drug-induced reinforcement in MRI studies. Here we directly address this issue by investigating the effects of continuous exposure to naltrexone on 1) fentanyl-induced reinstatement of drug-seeking behavior, 2) fentanyl-induced patterns of blood oxygenation level dependent (BOLD) activation in the nucleus accumbens (NAcc), and 3) fentanyl-induced changes in NAcc functional connectivity (FC) in awake non-human primates that are engaged in ongoing opioid self-administration studies. We found that naltrexone antagonizes the priming strength of fentanyl as shown by a rightward shift in its reinstatement dose-effect curve and that naltrexone surmountably antagonizes the BOLD response induced by fentanyl. However, while naltrexone also countered fentanyl's effects on NAcc FC, the effects were not surmounted by a higher dose of fentanyl. Together, these data suggest that, in contrast to naltrexone's modulation of fentanyl's effects on behavior and BOLD responses, their interactive effects on FC between multiple brain regions do not reflect their receptor-mediated activity. Additionally, we demonstrated opposing effects in the absence and presence of naltrexone on NAcc FC at baseline (i.e., in the absence of any fentanyl prime) suggesting that naltrexone alters FC at baseline, even though naltrexone appears behaviorally silent in the absence of an agonist prime. Together these data provide additional insight into ways in which naltrexone interacts with opioid agonists, both behaviorally and in the brain. Further understanding the effects of opioid agonists on patterns of FC could help elucidate our understanding of the neural processes that contribute to the initiation of and relapse to opioid-seeking behavior in OUD.

Prefrontal-Limbic Circuitry Is Associated With Reward Sensitivity in Nonhuman Primates.

BACKGROUND: Abnormal reward sensitivity is a risk factor for psychiatric disorders, including eating disorders such as overeating and binge-eating disorder, but the brain structural mechanisms that underlie it are not completely understood. Here, we sought to investigate the relationship between multimodal whole-brain structural features and reward sensitivity in nonhuman primates. METHODS: Reward sensitivity was evaluated through behavioral economic analysis in which monkeys (adult rhesus macaques; 7 female, 5 male) responded for sweetened condensed milk (10%, 30%, 56%), Gatorade, or water using an operant procedure in which the response requirement increased incrementally across sessions (i.e., fixed ratio 1, 3, 10). Animals were divided into high (n = 6) or low (n = 6) reward sensitivity groups based on essential value for 30% milk. Multimodal magnetic resonance imaging was used to measure gray matter volume and white matter microstructure. Brain structural features were compared between groups, and their correlations with reward sensitivity for various stimuli was investigated. RESULTS: Animals in the high sensitivity group had greater dorsolateral prefrontal cortex, centromedial amygdaloid complex, and middle cingulate cortex volumes than animals in the low sensitivity group. Furthermore, compared with monkeys in the low sensitivity group, high sensitivity monkeys had lower fractional anisotropy in the left dorsal cingulate bundle connecting the centromedial amygdaloid complex and middle cingulate cortex to the dorsolateral prefrontal cortex, and in the left superior longitudinal fasciculus 1 connecting the middle cingulate cortex to the dorsolateral prefrontal cortex. CONCLUSIONS: These results suggest that neuroanatomical variation in prefrontal-limbic circuitry is associated with reward sensitivity. These brain structural features may serve as predictive biomarkers for vulnerability to food-based and other reward-related disorders.

Resting-State Networks of Awake Adolescent and Adult Squirrel Monkeys Using Ultra-High Field (9.4†T) Functional Magnetic Resonance Imaging.

Resting-state networks (RSNs) are increasingly forwarded as candidate biomarkers for neuropsychiatric disorders. Such biomarkers may provide objective measures for evaluating novel therapeutic interventions in nonhuman primates often used in translational neuroimaging research. This study aimed to characterize the RSNs of awake squirrel monkeys and compare the characteristics of those networks in adolescent and adult subjects. Twenty-seven squirrel monkeys [n = 12 adolescents (6 male/6 female) ∼2.5 years and n = 15 adults (7 male/8 female) ∼9.5 years] were gradually acclimated to awake scanning procedures; whole-brain fMRI images were acquired with a 9.4 T scanner. Group-level independent component analysis (ICA; 30 ICs) with dual regression was used to detect and compare RSNs. Twenty ICs corresponding to physiologically meaningful networks representing a range of neural functions, including motor, sensory, reward, and cognitive processes, were identified in both adolescent and adult monkeys. The reproducibility of these RSNs was evaluated across several ICA model orders. Adults showed a trend for greater connectivity compared with adolescent subjects in two of the networks of interest: (1) in the right occipital region with the OFC network and (2) in the left temporal cortex, bilateral occipital cortex, and cerebellum with the posterior cingulate network. However, when age was entered into the above model, this trend for significance was lost. These results demonstrate that squirrel monkey RSNs are stable and consistent with RSNs previously identified in humans, rodents, and other nonhuman primate species. These data also identify several networks in adolescence that are conserved and others that may change into adulthood.

Transient receptor potential ankyrin 1 channel modulates the abuse-related mechanisms of methamphetamine through interaction with dopamine transporter.

BACKGROUND AND PURPOSE: Methamphetamine (METH) use disorder has risen dramatically over the past decade, and there are currently no FDA-approved medications due, in part, to gaps in our understanding of the pharmacological mechanisms related to METH action in the brain. EXPERIMENTAL APPROACH: Here, we investigated whether transient receptor potential ankyrin 1 (TRPA1) mediates each of several METH abuse-related behaviours in rodents: self-administration, drug-primed reinstatement, acquisition of conditioned place preference, and hyperlocomotion. Additionally, METH-induced molecular (i.e., neurotransmitter and protein) changes in the brain were compared between wild-type and TRPA1 knock-out mice. Finally, the relationship between TRPA1 and the dopamine transporter was investigated through immunoprecipitation and dopamine reuptake assays. KEY RESULTS: TRPA1 antagonism blunted METH self-administration and drug-primed reinstatement of METH-seeking behaviour. Further, development of METH-induced conditioned place preference and hyperlocomotion were inhibited by TRPA1 antagonist treatment, effects that were not observed in TRPA1 knock-out mice. Similarly, molecular studies revealed METH-induced increases in dopamine levels and expression of dopamine system-related proteins in wild-type, but not in TRPA1 knock-out mice. Furthermore, pharmacological blockade of TRPA1 receptors reduced the interaction between TRPA1 and the dopamine transporter, thereby increasing dopamine reuptake activity by the transporter. CONCLUSION AND IMPLICATIONS: This study demonstrates that TRPA1 is involved in the abuse-related behavioural effects of METH, potentially through its modulatory role in METH-induced activation of dopaminergic neurotransmission. Taken together, these data suggest that TRPA1 may be a novel therapeutic target for treating METH use disorder.

Single nuclei transcriptomics in human and non-human primate striatum in opioid use disorder.

In brain, the striatum is a heterogenous region involved in reward and goal-directed behaviors. Striatal dysfunction is linked to psychiatric disorders, including opioid use disorder (OUD). Striatal subregions are divided based on neuroanatomy, each with unique roles in OUD. In OUD, the dorsal striatum is involved in altered reward processing, formation of habits, and development of negative affect during withdrawal. Using single nuclei RNA-sequencing, we identified both canonical (e.g., dopamine receptor subtype) and less abundant cell populations (e.g., interneurons) in human dorsal striatum. Pathways related to neurodegeneration, interferon response, and DNA damage were significantly enriched in striatal neurons of individuals with OUD. DNA damage markers were also elevated in striatal neurons of opioid-exposed rhesus macaques. Sex-specific molecular differences in glial cell subtypes associated with chronic stress were found in OUD, particularly female individuals. Together, we describe different cell types in human dorsal striatum and identify cell type-specific alterations in OUD.

Effects of methcathinone and 3-Cl-methcathinone (PAL-434) in cocaine discrimination or self-administration in rhesus monkeys.

Monoamine releasers with varying selectivity for dopamine (DA)/norepinephrine and serotonin (5-HT) release are potential treatment medications for cocaine abuse. Although DA-selective monoamine releasers effectively reduce cocaine abuse, their clinical usefulness is limited by abuse liability. It is hypothesized that increasing 5-HT neurotransmission may reduce the abuse-related effects of DA releasers, but the optimal DA:5-HT release ratio remains to be determined. This study in rhesus monkeys compared the effects of two compounds with differing potency for 5-HT release. Methcathinone and 3-Cl-methcathinone (PAL-434) have equal potency for DA release, but PAL-434 has 10-fold higher potency for 5-HT release. In drug discrimination studies, monkeys were trained to discriminate cocaine (0.4 mg/kg i.m.) from saline in a two-key, food-reinforced procedure. In drug self-administration studies, a separate group of monkeys was trained to respond for cocaine [0.01 mg/kg/injection (inj)] and food (1 g pellets) under a second order schedule of reinforcement [FR2(VR16:S)]. When responding was stable, methcathinone (0.1­0.56 mg/kg.h i.v.) or PAL-434 (0.32­1.8 mg/kg.h i.v.) was administered chronically (one injection every 20 min for 23 h/d) for 7­10 d. In discrimination studies, both compounds dose-dependently increased cocaine-like responding but with different potencies (cocaine=methcathinone >PAL-434). Chronic treatment with methcathinone or PAL-434 dose-dependently and selectively reduced cocaine self-administration. PAL-434 was about 4-fold and methcathinone about 1.6-fold more potent at decreasing cocaine- over food-maintained responding. These data suggest that compounds with moderate selectivity for DA vs. 5-HT release (8­15-fold) may be effective for the treatment of cocaine dependence.

Oxycodone self-administration and reinstatement in male and female squirrel monkeys: Effects of alternative reinforcer availability.

The use of non-drug alternative reinforcers has long been utilized as a component of therapeutic interventions for the management of substance use disorder; however, the conditions under which alternative reinforcers are most effective are not well characterized. This study evaluated the impact of varying the magnitude of an alternative reinforcer on oxycodone self-administration and reinstatement in male and female squirrel monkeys. Subjects (n=4/sex) were trained under concurrent second-order schedules of reinforcement for intravenous oxycodone (0.001-0.1mg/kg/inj) on one lever, and sweetened condensed milk (5, 10, 20, 30% in water) on another. Oxycodone-primed reinstatement was evaluated by administering 0.32mg/kg oxycodone prior to sessions in which saline was available on the drug-paired lever. During oxycodone self-administration sessions, milk availability decreased oxycodone self-administration and preference in a concentration-dependent manner; low milk concentrations were more effective at decreasing oxycodone’s reinforcing potency in males. During reinstatement tests, milk significantly attenuated oxycodone-primed responding in both males and females; low milk concentrations were more effective at decreasing the priming effects of oxycodone in females. That alternative reinforcers differentially impacted self-administration and reinstatement in a sex-dependent manner suggests that treatment strategies that utilize alternative reinforcers may be more effective in males or females depending on when they are implemented.

Resting state networks of awake adolescent and adult squirrel monkeys using ultra-high field (9.4T) functional magnetic resonance imaging.

Resting state networks (RSNs) are increasingly forwarded as candidate biomarkers for neuropsychiatric disorders. Such biomarkers may provide objective measures for evaluating novel therapeutic interventions in nonhuman primates often used in translational neuroimaging research. This study aimed to characterize the RSNs of awake squirrel monkeys and compare the characteristics of those networks in adolescent and adult subjects. Twenty-seven squirrel monkeys ( n =12 adolescents [6 male/6 female] ∼2.5 years and n =15 adults [7 male/8 female] ∼9.5 years) were gradually acclimated to awake scanning procedures; whole-brain fMRI images were acquired with a 9.4 Tesla scanner. Group level independent component (IC) analysis (30 ICs) with dual regression was used to detect and compare RSNs. Twenty ICs corresponding to physiologically meaningful networks representing a range of neural functions, including motor, sensory, reward (e.g., basal ganglia), and cognitive processes were identified in both adolescent and adult monkeys. Significant age-related differences between the adult and adolescent subjects (adult > adolescent) were found in two networks of interest: (1) the right upper occipital region with an OFC IC and (2) the left temporal cortex, bilateral visual areas, and cerebellum with the cingulate IC. These results demonstrate that squirrel monkey RSNs are stable and consistent with RSNs previously identified in humans, rodents, and other nonhuman primate species. These data also identify several networks in adolescence that are conserved and others that may change into adulthood. Significance Statement: Functional magnetic resonance imaging procedures have revealed important information about how the brain is modified by experimental manipulations, disease states, and aging throughout the lifespan. Preclinical neuroimaging, especially in nonhuman primates, has become a frequently used means to answer targeted questions related to brain resting-state functional connectivity. The present study characterized resting state networks (RSNs) in adult and adolescent squirrel monkeys; twenty RSNs corresponding to networks representing a range of neural functions were identified. The RSNs identified here can be utilized in future studies examining the effects of experimental manipulations on brain connectivity in squirrel monkeys. These data also may be useful for comparative analysis with other primate species to provide an evolutionary perspective for understanding brain function and organization.

Estimating and mitigating the effects of systemic low frequency oscillations (sLFO) on resting state networks in awake non-human primates using time lag dependent methodology.

Aim: Resting-state fMRI (rs-fMRI) is often used to infer regional brain interactions from the degree of temporal correlation between spontaneous low-frequency fluctuations, thought to reflect local changes in the BOLD signal due to neuronal activity. One complication in the analysis and interpretation of rs-fMRI data is the existence of non-neuronal low frequency physiological noise (systemic low frequency oscillations; sLFOs) which occurs within the same low frequency band as the signal used to compute functional connectivity. Here, we demonstrate the use of a time lag mapping technique to estimate and mitigate the effects of the sLFO signal on resting state functional connectivity of awake squirrel monkeys. Methods: Twelve squirrel monkeys (6 male/6 female) were acclimated to awake scanning procedures; whole-brain fMRI images were acquired with a 9.4 Tesla scanner. Rs-fMRI data was preprocessed using an in-house pipeline and sLFOs were detected using a seed regressor generated by averaging BOLD signal across all voxels in the brain, which was then refined recursively within a time window of -16-12 s. The refined regressor was then used to estimate the voxel-wise sLFOs; these regressors were subsequently included in the general linear model to remove these moving hemodynamic components from the rs-fMRI data using general linear model filtering. Group level independent component analysis (ICA) with dual regression was used to detect resting-state networks and compare networks before and after sLFO denoising. Results: Results show sLFOs constitute ~64% of the low frequency fMRI signal in squirrel monkey gray matter; they arrive earlier in regions in proximity to the middle cerebral arteries (e.g., somatosensory cortex) and later in regions close to draining vessels (e.g., cerebellum). Dual regression results showed that the physiological noise was significantly reduced after removing sLFOs and the extent of reduction was determined by the brain region contained in the resting-state network. Conclusion: These results highlight the need to estimate and remove sLFOs from fMRI data before further analysis.

A new class of 5-HT2A /5-HT2C receptor inverse agonists: Synthesis, molecular modeling, in vitro and in vivo pharmacology of novel 2-aminotetralins.

BACKGROUND AND PURPOSE: The 5-HT receptor subtypes 5-HT2A and 5-HT2C are important neurotherapeutic targets, though, obtaining selectivity over 5-HT2B and H1 receptors is challenging. Here, we delineated molecular determinants of selective binding to 5-HT2A and 5-HT2C receptors for novel 4-phenyl-2-dimethylaminotetralins (4-PATs). EXPERIMENTAL APPROACH: We synthesized 42 novel 4-PATs with halogen or aryl moieties at the C(4)-phenyl meta-position. Affinity, function, molecular modeling and 5-HT2A receptor mutagenesis studies were performed to understand structure-activity relationships at 5-HT2 -type and H1 receptors. Lead 4-PAT-type 5-HT2A /5-HT2C receptor inverse agonists were compared with pimavanserin, a selective 5-HT2A /5-HT2C receptor inverse agonist approved to treat Parkinson's disease-related psychosis, in the mouse head twitch response and locomotor activity assays, models relevant to antipsychotic drug development. KEY RESULTS: Most 4-PAT diastereomers in the (2S,4R)-configuration bound non-selectively to 5-HT2A , 5-HT2C and H1 receptors, with >100-fold selectivity over 5-HT2B receptors, whereas diastereomers in the (2R,4R)-configuration bound preferentially to 5-HT2A over 5-HT2C receptors and had >100-fold selectivity over 5-HT2B and H1 receptors. Results suggest that G2385.42 and V2355.39 in 5-HT2A receptors (conserved in 5-HT2C receptors) are important for high affinity binding, whereas interactions with T1945.42 and W1584.56 determine H1 receptor affinity. The 4-PAT analog (2S,4R)-4-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, (2S,4R)-2k, a potent and selective 5-HT2A /5-HT2C receptor inverse agonist, had activity like pimavanserin in the mouse head twitch response assay but was distinct in not suppressing locomotor activity. CONCLUSIONS AND IMPLICATIONS: The novel 4-PAT chemotype can yield selective 5-HT2A /5-HT2C receptor inverse agonists for antipsychotic drug development by optimizing ligand-receptor interactions in transmembrane domain 5. Chirality can be exploited to attain selectivity over H1 receptors, which may circumvent sedative effects.

Fentanyl-induced changes in brain activity in awake nonhuman primates at 9.4 Tesla.

Functional magnetic resonance imaging (fMRI) has been used to study the influence of opioids on neural circuitry implicated in opioid use disorder, such as the cortico-striatal-thalamo-cortical (CSTC) circuit. Given the increase in fentanyl-related deaths, this study was conducted to characterize the effects of fentanyl on patterns of brain activation in awake nonhuman primates. Four squirrel monkeys were acclimated to awake scanning procedures conducted at 9.4 Tesla. Subsequently, test sessions were conducted in which a dose of fentanyl that reliably maintains intravenous (IV) self-administration behavior in monkeys, 1 µg/kg, was administered and the effects on patterns of brain activity were assessed using: (1) a pharmacological regressor to elucidate fentanyl-induced patterns of neural activity, and (2) seed-based approaches targeting bilateral anterior cingulate, thalamus, or nucleus accumbens (NAc) to determine alterations in CSTC functional connectivity. Results showed a functional inhibition of BOLD signal in brain regions that mediate behavioral effects of opioid agonists, such as cingulate cortex, striatum and midbrain. Functional connectivity between each of the seed regions and areas involved in motoric, sensory and cognition-related behavior generally decreased. In contrast, NAc functional connectivity with other striatal regions increased. These results indicate that fentanyl produces changes within CSTC circuitry that may reflect key features of opioid use disorder (e.g. persistent drug-taking/seeking) and thereby contribute to long-term disruptions in behavior and addiction. They also indicate that fMRI in alert nonhuman primates can detect drug-induced changes in neural circuits and, in turn, may be useful for investigating the effectiveness of medications to reverse drug-induced dysregulation.

Effects of cannabinoid exposure on short-term memory and medial orbitofrontal cortex function and chemistry in adolescent female rhesus macaques.

Aim: There is increasing concern that cannabinoid exposure during adolescence may disturb brain maturation and produce long-term cognitive deficits. However, studies in human subjects have provided limited evidence for such causality. The present study utilized behavioral and neuroimaging endpoints in female non-human primates to examine the effects of acute and chronic exposure during adolescence to the cannabinoid receptor full agonist, AM2389, on cognitive processing and brain function and chemistry. Materials and methods: Adolescent female rhesus macaques were trained on a titrating-delay matching-to-sample (TDMTS) touchscreen task that assays working memory. TDMTS performance was assessed before and during chronic exposure to AM2389, following antagonist (rimonabant) administration, and after discontinuation of the chronic regimen. Resting-state fMRI connectivity and magnetic resonance spectroscopy data were acquired prior to drug treatment, during chronic exposure, and following its discontinuation. Voxels were placed in the medial orbitofrontal cortex (mOFC), a region involved in memory processing that undergoes maturation during adolescence. Results: TDMTS performance was dose-dependently disrupted by acute AM2389; however, chronic treatment resulted in tolerance to these effects. TDMTS performance also was disrupted by discontinuation of the chronic regimen but surprisingly, not by rimonabant administration during chronic AM2389 treatment. mOFC N-acetylaspartate/creatine ratio decreased after acute and chronic administration but returned to baseline values following discontinuation of chronic treatment. Finally, intra-network functional connectivity (mOFC) increased during the chronic regimen and returned to baseline values following its discontinuation. Conclusion: Neural effects of a cannabinergic drug may persist during chronic exposure, notwithstanding the development of tolerance to behavioral effects. However, such effects dissipate upon discontinuation, reflecting the restorative capacity of affected brain processes.

Decreases in cocaine self-administration with dual inhibition of the dopamine transporter and σ receptors.

Sigma receptor (σR) antagonists attenuate many behavioral effects of cocaine but typically not its reinforcing effects in self-administration procedures. However, the σR antagonist rimcazole and its N-propylphenyl analogs, [3-(cis-3,5-dimethyl-4-[3-phenylpropyl]-1-piperazinyl)-propyl]diphenylamine hydrochloride (SH 3-24) and 9-[3-(cis-3,5-dimethyl-4-[3-phenylpropyl]-1-piperazinyl)-propyl]carbazole hydrobromide (SH 3-28), dose-dependently decreased the maximal rates of cocaine self-administration without affecting comparable responding maintained by food reinforcement. In contrast, a variety of σR antagonists [N-phenethylpiperidine oxalate (AC927), N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine dihydrobromide (BD 1008), N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino) ethylamine dihydrobromide (BD 1047), N-[2-(3,4-dichlorophenyl) ethyl]-4-methylpiperazine dihydrochloride (BD 1063), and N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100)] had no effect on cocaine self-administration across the range of doses that decreased rates of food-maintained responding. Rimcazole analogs differed from selective σR antagonists in their dual affinities for σRs and the dopamine transporter (DAT) assessed with radioligand binding. Selective DAT inhibitors and σR antagonists were studied alone and in combination on cocaine self-administration to determine whether actions at both σRs and the DAT were sufficient to reproduce the effects of rimcazole analogs. Typical DAT inhibitors [2ß-carbomethoxy-3ß-(4-fluorophenyl)tropane (WIN 35,428), methylphenidate, and nomifensine] dose-dependently shifted the cocaine dose-effect curve leftward. Combinations of DAT inhibitor and σR antagonist doses that were behaviorally inactive alone decreased cocaine self-administration without effects on food-maintained responding. In addition, whereas the DAT inhibitors were self-administered at rates similar to those of cocaine, neither rimcazole analogs nor typical σR antagonists (NE-100 and AC927) maintained responding above control levels across a wide range of doses. These findings suggest that the unique effects of rimcazole analogs are due to dual actions at the DAT and σRs and that a combined target approach may have utility in development of medical treatments for cocaine abuse.