Differential regulation of G protein-coupled receptor-associated proteins in the caudate and the putamen of cynomolgus macaques following chronic ethanol drinking.

The dorsal striatum is composed of the caudate nucleus and the putamen in human and non-human primates. These two regions receive different cortical projections and are functionally distinct. The caudate is involved in the control of goal-directed behaviors, while the putamen is implicated in habit learning and formation. Previous reports indicate that ethanol differentially influences neurotransmission in these two regions. Because neurotransmitters primarily signal through G protein-coupled receptors (GPCRs) to modulate neuronal activity, the present study aimed to determine whether ethanol had a region-dependent impact on the expression of proteins that are involved in the trafficking and function of GPCRs, including G protein subunits and their effectors, protein kinases, and elements of the cytoskeleton. Western blotting was performed to examine protein levels in the caudate and the putamen of male cynomolgus macaques that self-administered ethanol for 1 year under free access conditions, along with control animals that self-administered an isocaloric sweetened solution under identical operant conditions. Among the 18 proteins studied, we found that the levels of one protein (PKCß) were increased, and 13 proteins (Gαi1/3, Gαi2, Gαo, Gß1γ, PKCα, PKCε, CaMKII, GSK3ß, ß-actin, cofilin, α-tubulin, and tubulin polymerization promoting protein) were reduced in the caudate of alcohol-drinking macaques. However, ethanol did not alter the expression of any proteins examined in the putamen. These observations underscore the unique vulnerability of the caudate nucleus to changes in protein expression induced by chronic ethanol exposure. Whether these alterations are associated with ethanol-induced dysregulation of GPCR function and neurotransmission warrants future investigation.

Chronic alcohol consumption alters sex-dependent BNST neuron function in rhesus macaques.

Repeated alcohol drinking contributes to a number of neuropsychiatric diseases, including alcohol use disorder and co-expressed anxiety and mood disorders. Women are more susceptible to the development and expression of these diseases with the same history of alcohol exposure as men, suggesting they may be more sensitive to alcohol-induced plasticity in limbic brain regions controlling alcohol drinking, stress responsivity, and reward processing, among other behaviors. Using a translational model of alcohol drinking in rhesus monkeys, we examined sex differences in the basal function and plasticity of neurons in the bed nucleus of the stria terminalis (BNST), a brain region in the extended amygdala shown to be a hub circuit node dysregulated in individuals with anxiety and alcohol use disorder. We performed slice electrophysiology recordings from BNST neurons in male and female monkeys following daily "open access" (22 h/day) to 4% ethanol and water for more than one year or control conditions. We found that BNST neurons from control females had reduced overall current density, hyperpolarization-activated depolarizing current (Ih), and inward rectification, as well as higher membrane resistance and greater synaptic glutamatergic release and excitatory drive, than those from control males, suggesting that female BNST neurons are more basally excited than those from males. Chronic alcohol drinking produced a shift in these measures in both sexes, decreasing current density, Ih, and inward rectification and increasing synaptic excitation. In addition, network activity-dependent synaptic inhibition was basally higher in BNST neurons of males than females, and alcohol exposure increased this in both sexes, a putative homeostatic mechanism to counter hyperexcitability. Altogether, these results suggest that the rhesus BNST is more basally excited in females than males and chronic alcohol drinking produces an overall increase in excitability and synaptic excitation. These results shed light on the mechanisms contributing to the female-biased susceptibility to neuropsychiatric diseases including co-expressed anxiety and alcohol use disorder.

Chronic alcohol consumption alters sex-dependent BNST neuron function in rhesus macaques.

Repeated alcohol drinking contributes to a number of neuropsychiatric diseases, including alcohol use disorder and co-expressed anxiety and mood disorders. Women are more susceptible to the development and expression of these diseases with the same history of alcohol exposure as men, suggesting they may be more sensitive to alcohol-induced plasticity in limbic brain regions controlling alcohol drinking, stress responsivity, and reward processing, among other behaviors. Using a translational model of alcohol drinking in rhesus monkeys, we examined sex differences in the basal function and plasticity of neurons in the bed nucleus of the stria terminalis (BNST), a brain region in the extended amygdala shown to be a hub circuit node dysregulated in individuals with anxiety and alcohol use disorder. We performed slice electrophysiology recordings from BNST neurons in male and female monkeys following daily "open access" (22 hr/day) to 4% ethanol and water for more than one year or control conditions. We found that BNST neurons from control females had reduced overall current density, hyperpolarization-activated depolarizing current (Ih), and inward rectification, as well as higher membrane resistance and greater synaptic glutamatergic release and excitatory drive, than those from control males, suggesting that female BNST neurons are more basally excited than those from males. Chronic alcohol drinking produced a shift in these measures in both sexes, decreasing current density, Ih, and inward rectification and increasing synaptic excitation. In addition, network activity-dependent synaptic inhibition was basally higher in BNST neurons of males than females, and alcohol exposure increased this in both sexes, a putative homeostatic mechanism to counter hyperexcitability. Altogether, these results suggest that the rhesus BNST is more basally excited in females than males and chronic alcohol drinking produces an overall increase in excitability and synaptic excitation. These results shed light on the mechanisms contributing to the female-biased susceptibility to neuropsychiatric diseases including co-expressed anxiety and alcohol use disorder.

Ethanol consumption in non-human primates alters plasma markers of bone turnover but not tibia architecture.

Ethanol consumption is associated with positive, negative, and neutral effects on the skeletal system. Our previous work using a nonhuman primate model of voluntary ethanol consumption showed that chronic ethanol use has an impact on skeletal attributes, most notably on biochemical markers of bone turnover. However, these studies were limited by small sample sizes and resulting lack of statistical power. Here, we applied a machine learning framework to integrate data from 155 monkeys (100 ethanol and 55 controls) to identify the bone features associated with chronic ethanol use. Specifically, we analyzed the influence of ethanol consumption on biomarkers of bone turnover and cancellous and cortical bone architecture in tibia. We hypothesized that chronic ethanol use for 6 months to 2.5 years would result in measurable changes to cancellous features and the biochemical markers compared to control animals. We observed a decrease in bone turnover in monkeys exposed to ethanol; however, we did not find that ethanol consumption resulted in measurable changes in bone architecture.

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.

Synchrony between midbrain gene transcription and dopamine terminal regulation is modulated by chronic alcohol drinking.

Alcohol use disorder is marked by disrupted behavioral and emotional states which persist into abstinence. The enduring synaptic alterations that remain despite the absence of alcohol are of interest for interventions to prevent relapse. Here, 28 male rhesus macaques underwent over 20 months of alcohol drinking interspersed with three 30-day forced abstinence periods. After the last abstinence period, we paired direct sub-second dopamine monitoring via ex vivo voltammetry in nucleus accumbens slices with RNA-sequencing of the ventral tegmental area. We found persistent augmentation of dopamine transporter function, kappa opioid receptor sensitivity, and dynorphin release - all inhibitory regulators which act to decrease extracellular dopamine. Surprisingly, though transcript expression was not altered, the relationship between gene expression and functional readouts of these encoded proteins was highly dynamic and altered by drinking history. These results outline the long-lasting synaptic impact of alcohol use and suggest that assessment of transcript-function relationships is critical for the rational design of precision therapeutics.

Effects of repeated alcohol abstinence on within-subject prefrontal cortical gene expression in rhesus macaques.

Male rhesus monkeys (n = 24) had a biopsy of prefrontal cortical area 46 prior to chronic ethanol self-administration (n = 17) or caloric control (n = 7). Fourteen months of daily self-administration (water vs. 4% alcohol, 22 h access/day termed "open-access") was followed by two cycles of prolonged abstinence (5 weeks) each followed by 3 months of open-access alcohol and a final abstinence followed by necropsy. At necropsy, a biopsy of Area 46, contralateral to the original biopsy, was obtained. Gene expression data (RNA-Seq) were collected comparing biopsy/necropsy samples. Monkeys were categorized by drinking status during the final post-abstinent drinking phase as light (LD), binge (BD), heavy (HD) and very heavy (VHD drinkers). Comparing pre-ethanol to post-abstinent biopsies, four animals that converted from HD to VHD status had significant ontology enrichments in downregulated genes (necropsy minus biopsy n = 286) that included immune response (FDR < 9 × 10-7) and plasma membrane changes (FDR < 1 × 10-7). Genes in the immune response category included IL16 and 18, CCR1, B2M, TLR3, 6 and 7, SP2 and CX3CR1. Upregulated genes (N = 388) were particularly enriched in genes associated with the negative regulation of MAP kinase activity (FDR < 3 × 10-5), including DUSP 1, 4, 5, 6 and 18, SPRY 2, 3, and 4, SPRED2, BMP4 and RGS2. Overall, these data illustrate the power of the NHP model and the within-subject design of genomic changes due to alcohol and suggest new targets for treating severe escalated drinking following repeated alcohol abstinence attempts.

Six months of voluntary alcohol consumption in male cynomolgus macaques reduces intracortical bone porosity without altering mineralization or mechanical properties.

Chronic heavy alcohol consumption is a risk factor for low trauma bone fracture. Using a non-human primate model of voluntary alcohol consumption, we investigated the effects of 6 months of ethanol intake on cortical bone in cynomolgus macaques (Macaca fascicularis). Young adult (6.4 ± 0.1 years old, mean ± SE) male cynomolgus macaques (n = 17) were subjected to a 4-month graded ethanol induction period, followed by voluntary self-administration of water or ethanol (4 % w/v) for 22 h/d, 7 d/wk. for 6 months. Control animals (n = 6) consumed an isocaloric maltose-dextrin solution. Tibial response was evaluated using densitometry, microcomputed tomography, histomorphometry, biomechanical testing, and Raman spectroscopy. Global bone response was evaluated using biochemical markers of bone turnover. Monkeys in the ethanol group consumed an average of 2.3 ± 0.2 g/kg/d ethanol resulting in a blood ethanol concentration of 90 ± 12 mg/dl in longitudinal samples taken 7 h after the daily session began. Ethanol consumption had no effect on tibia length, mass, density, mechanical properties, or mineralization (p > 0.642). However, compared to controls, ethanol intake resulted in a dose-dependent reduction in intracortical bone porosity (Spearman rank correlation = -0.770; p < 0.0001) and compared to baseline, a strong tendency (p = 0.058) for lower plasma CTX, a biochemical marker of global bone resorption. These findings are important because suppressed cortical bone remodeling can result in a decrease in bone quality. In conclusion, intracortical bone porosity was reduced to subnormal values 6 months following initiation of voluntary ethanol consumption but other measures of tibia architecture, mineralization, or mechanics were not altered.

Prenatal delta-9-tetrahydrocannabinol exposure alters fetal neurodevelopment in rhesus macaques.

Prenatal cannabis use is associated with adverse offspring neurodevelopmental outcomes, however the underlying mechanisms are relatively unknown. We sought to determine the impact of chronic delta-9-tetrahydrocannabinol (THC) exposure on fetal neurodevelopment in a rhesus macaque model using advanced imaging combined with molecular and tissue studies. Animals were divided into two groups, control (n = 5) and THC-exposed (n = 5), which received a daily THC edible pre-conception and throughout pregnancy. Fetal T2-weighted MRI was performed at gestational days 85 (G85), G110, G135 and G155 to assess volumetric brain development. At G155, animals underwent cesarean delivery with collection of fetal cerebrospinal fluid (CSF) for microRNA (miRNA) studies and fetal tissue for histologic analysis. THC exposure was associated with significant age by sex interactions in brain growth, and differences in fetal brain histology suggestive of brain dysregulation. Two extracellular vesicle associated-miRNAs were identified in THC-exposed fetal CSF; pathway analysis suggests that these miRNAs are associated with dysregulated axonal guidance and netrin signaling. This data is indicative of subtle molecular changes consistent with the observed histological data, suggesting a potential role for fetal miRNA regulation by THC. Further studies are needed to determine whether these adverse findings correlate with long-term offspring neurodevelopmental health.

Recurrent activity within microcircuits of macaque dorsolateral prefrontal cortex tracks cognitive flexibility.

Human and non-human primate data clearly implicate the dorsolateral prefrontal cortex (dlPFC) as critical for advanced cognitive functions 1,2 . It is thought that intracortical synaptic architectures within dlPFC are the integral neurobiological substrate that gives rise to these processes, including working memory, inferential reasoning, and decision-making 3-7 . In the prevailing model, each cortical column makes up one fundamental processing unit composed of dense intrinsic connectivity, conceptualized as the 'canonical' cortical microcircuit 3,8 . Each cortical microcircuit receives sensory and cognitive information from a variety of sources which are represented by sustained activity within the microcircuit, referred to as persistent or recurrent activity 4,9 . Via recurrent connections within the microcircuit, activity can propagate for a variable length of time, thereby allowing temporary storage and computations to occur locally before ultimately passing a transformed representation to a downstream output 4,5,10 . Competing theories regarding how microcircuit activity is coordinated have proven difficult to reconcile in vivo where intercortical and intracortical computations cannot be fully dissociated 5,9,11,12 . Here, we interrogated the intrinsic features of isolated microcircuit networks using high-density calcium imaging of macaque dlPFC ex vivo . We found that spontaneous activity is intrinsically maintained by microcircuit architecture, persisting at a high rate in the absence of extrinsic connections. Further, using perisulcal stimulation to evoke persistent activity in deep layers, we found that activity propagates through stochastically assembled intracortical networks, creating predictable population-level events from largely non-overlapping ensembles. Microcircuit excitability covaried with individual cognitive performance, thus anchoring heuristic models of abstract cortical functions within quantifiable constraints imposed by the underlying synaptic architecture.