Increased number of cryptosporidiosis cases with travel history to Croatia might be related to swimming pools, Germany, 2023.

In August and September 2023, an unusually high number of cryptosporidiosis cases identified by routine German surveillance had travelled to Croatia (n = 23). Nine cases had stayed in the same camping resort and seven further cases had stayed at other camping sites within 15 km. Based on our standardised questionnaires, the most likely source of infection was swimming pools (93%). Further environmental investigations on site might reveal potential common sources of contamination that could be targeted by control measures.

Pairing food and drink: A physiological model of blood ethanol levels for a variety of drinking behaviors.

We present a blood ethanol concentration compartment model which utilizes an animal's ethanol intake, food intake, and weight to predict the animal's blood ethanol concentration at any given time. By incorporating the food digestion process into the model we can predict blood ethanol concentration levels over time for a variety of drinking and eating scenarios. The model is calibrated and validated using data from cohorts of male monkeys, and is able to capture blood ethanol concentration kinetics of the monkeys from a variety of drinking behavior classifications.

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.

Synaptic effects of IL-1ß and CRF in the central amygdala after protracted alcohol abstinence in male rhesus macaques.

A major barrier to remission from an alcohol use disorder (AUD) is the continued risk of relapse during abstinence. Assessing the neuroadaptations after chronic alcohol and repeated abstinence is important to identify mechanisms that may contribute to relapse. In this study, we used a rhesus macaque model of long-term alcohol use and repeated abstinence, providing a platform to extend mechanistic findings from rodents to primates. The central amygdala (CeA) displays elevated GABA release following chronic alcohol in rodents and in abstinent male macaques, highlighting this neuroadaptation as a conserved mechanism that may underlie excessive alcohol consumption. Here, we determined circulating interleukin-1ß (IL-1ß) levels, CeA transcriptomic changes, and the effects of IL-1ß and corticotropin releasing factor (CRF) signaling on CeA GABA transmission in male controls and abstinent drinkers. While no significant differences in peripheral IL-1ß or the CeA transcriptome were observed, pathway analysis identified several canonical immune-related pathways. We addressed this potential dysregulation of CeA immune signaling in abstient drinkers with an electrophysiological approach. We found that IL-1ß decreased CeA GABA release in controls while abstinent drinkers were less sensitive to IL-1ß's effects, suggesting adaptations in the neuromodulatory role of IL-1ß. In contrast, CRF enhanced CeA GABA release similarly in controls and abstinent drinkers, consistent with rodent studies. Notably, CeA CRF expression was inversely correlated with intoxication, suggesting that CRF levels during abstinence may predict future intoxication. Together, our findings highlight conserved and divergent actions of chronic alcohol on neuroimmune and stress signaling on CeA GABA transmission across rodents and macaques.

Effect of chronic ethanol consumption in rhesus macaques on the nucleus accumbens core transcriptome.

The nucleus accumbens core (NAcc) has been repeatedly demonstrated to be a key component of the circuitry associated with excessive ethanol consumption. Previous studies have illustrated that in a nonhuman primate (NHP) model of chronic ethanol consumption, there is significant epigenetic remodeling of the NAcc. In the current study, RNA-Seq was used to examine genome-wide gene expression in eight each of control, low/binge (LD*), and high/very high (HD*) rhesus macaque drinkers. Using an FDR < 0.05, zero genes were significantly differentially expressed (DE) between LD* and controls, six genes between HD* and LD*, and 734 genes between HD* and controls. Focusing on HD* versus control DE genes, the upregulated genes (N = 366) were enriched in genes with annotations associated with signal recognition particle (SRP)-dependent co-translational protein targeting to membrane (FDR < 3 × 10-59 ), structural constituent of ribosome (FDR < 3 × 10-47 ), and ribosomal subunit (FDR < 5 × 10-48 ). Downregulated genes (N = 363) were enriched in annotations associated with behavior (FDR < 2 × 10-4 ), membrane organization (FDR < 1 × 10-4 ), inorganic cation transmembrane transporter activity (FDR < 2 × 10-3 ), synapse part (FDR < 4 × 10-10 ), glutamatergic synapse (FDR < 1 × 10-6 ), and GABAergic synapse (FDR < 6 × 10-4 ). Ingenuity Pathway Analysis (IPA) revealed that EIF2 signaling and mTOR pathways were significantly upregulated in HD* animals (FDR < 3 × 10-33 and <2 × 10-16 , respectively). Overall, the data supported our working hypothesis; excessive consumption would be associated with transcriptional differences in GABA/glutamate-related genes.

Phosphatidylethanol in whole blood of rhesus monkeys correlates with ethanol consumption.

BACKGROUND: Phosphatidylethanol (PEth) homologs are ethanol metabolites used to identify and monitor alcohol drinking in humans. In this study, we measured levels of the 2 most abundant homologs, PEth 16:0/18:1 and PEth 16:0/18:2, in whole blood samples from rhesus macaque monkeys that drank ethanol daily ad libitum to assess the relationship between PEth levels and recent ethanol exposure in this animal model. METHODS: Blood samples were obtained from The Monkey Alcohol Tissue Research Resource. The monkeys were first induced to consume 4% (w/v) ethanol in water from a panel attached to their home cage. Then, monkeys were allowed to drink ethanol and water ad libitum 22 h daily for 12 months and the daily amount of ethanol each monkey consumed was measured. Whole, uncoagulated blood was collected from each animal at the end of the entire experimental procedure. PEth 16:0/18:1 and PEth 16:0/18:2 levels were analyzed by HPLC with tandem mass spectrometry, and the ethanol consumed during the preceding 14 days was measured. Combined PEth was the sum of the concentrations of both homologs. RESULTS: Our results show that (1) PEth accumulates in the blood of rhesus monkeys after ethanol consumption; (2) PEth homolog levels were correlated with the daily average ethanol intake during the 14-day period immediately preceding blood collection; (3) the application of established human PEth 16:0/18:1 cutoff concentrations indicative of light social or no ethanol consumption (<20 ng/ml), moderate ethanol consumption (≥ 20 and < 200 ng/ml) and heavy ethanol consumption (≥ 200 ng/ml) predicted significantly different ethanol intake in these animals. PEth homologs were not detected in ethanol-naïve controls. CONCLUSIONS: This study confirms that PEth is a sensitive biomarker for ethanol consumption in rhesus macaque monkeys. This nonhuman primate model may prove useful in evaluating sources of variability previously shown to exist between ethanol consumption and PEth homolog levels among humans.

Effects of graded increases in ethanol consumption on biochemical markers of bone turnover in young adult male cynomolgus macaques.

Chronic heavy alcohol use is often associated with reduced bone mineral density and altered bone turnover. However, the dose response effects of ethanol on bone turnover have not been established. This study examined the effects of graded increases of ethanol consumption on biochemical markers of bone turnover in young adult male cynomolgus macaques (Macaca fascicularis). For this study, 6.6-year-old (95% CI: 6.5, 6.7) male macaques were subjected to three 30-day sessions of increased ethanol intake over a 90-day interval. During the first 30 days, the monkeys drank a predetermined volume of ethanol corresponding to 0.5 g/kg/day, followed by 1.0 g/kg/day and 1.5 g/kg/day. Osteocalcin, a marker of bone formation, and carboxyterminal cross-linking telopeptide of type 1 collagen (CTX), a marker of resorption, were measured during each 30-day session. In addition, the ratio of osteocalcin to CTX was determined as a surrogate measure of global turnover balance. Mean osteocalcin decreased by 2.6 ng/mL (1.8, 3.5) for each one-half unit (0.5 g/kg/day) increase in dose (p < 0.001). Mean CTX decreased by 0.13 ng/mL (0.06, 0.20) for each one-half unit increase in dose (p < 0.001). Furthermore, there was an inverse relationship between dose and the ratio of osteocalcin to CTX, such that the mean ratio decreased by 0.9 (0.3, 1.5) for each one-half unit increase in dose (p = 0.01). In summary, male cynomolgus macaques had decreased blood osteocalcin and CTX, and osteocalcin to CTX ratio during the 90-day interval of graded increases in ethanol consumption, indicative of reduced bone turnover and negative turnover balance, respectively. These findings suggest that over the range ingested, ethanol resulted in a linear decrease in bone turnover. Furthermore, the negative bone turnover balance observed is consistent with reported effects of chronic alcohol intake on the skeleton.

Mifepristone Decreases Chronic Voluntary Ethanol Consumption in Rhesus Macaques.

The efficacy of short-term treatment with mifepristone (MIFE), a high-affinity, nonselective glucocorticoid receptor antagonist, to reduce ethanol drinking was tested in a rhesus macaque model. Stable individual daily ethanol intakes were established, ranging from 1.6 to 4.0 g/kg per day (n = 9 monkeys). After establishment of chronic ethanol intake, a MIFE dosing regimen that modeled a study of rodent drinking and human alcohol craving was evaluated. Three doses of MIFE (17, 30, and 56 mg/kg per day) were each administered for four consecutive days. Both 30 and 56 mg/kg decreased ethanol intake compared with baseline drinking levels without a change in water intake. The dose of 56 mg/kg per day of MIFE produced the largest reduction in ethanol self-administration, with the average intake at 57% of baseline intakes. Cortisol was elevated during MIFE dosing, and a mediation analysis revealed that the effect on ethanol drinking was fully mediated through cortisol. During a forced abstinence phase, access to 1.5 g/kg ethanol resulted in relapse in all drinkers and was not altered by treatment with 56 mg/kg MIFE. Overall, these results show that during active drinking MIFE is efficacious in reducing heavy alcohol intake in a monkey model, an effect that was related to MIFE-induced increase in cortisol. However, MIFE treatment did not eliminate ethanol drinking. Further, cessation of MIFE treatment resulted in a rapid return to baseline intakes, and MIFE was not effective in preventing a relapse during early abstinence. SIGNIFICANCE STATEMENT: Mifepristone reliably decreases average daily ethanol self-administration in a nonhuman primate model. This effect was mediated by cortisol, was most effective during open-access conditions, and did not prevent or reduce relapse drinking.

Chronic Voluntary Ethanol Drinking in Cynomolgus Macaques Elicits Gene Expression Changes in Prefrontal Cortical Area 46.

BACKGROUND: Genome-wide profiling to examine brain transcriptional features associated with excessive ethanol (EtOH) consumption has been applied to a variety of species including rodents, nonhuman primates (NHPs), and humans. However, these data were obtained from cross-sectional samples which are particularly vulnerable to individual variation when obtained from small outbred populations typical of human and NHP studies. In the current study, a novel within-subject design was used to examine the effects of voluntary EtOH consumption on prefrontal cortex (PFC) gene expression in a NHP model. METHODS: Two cohorts of cynomolgus macaques (n = 23) underwent a schedule-induced polydipsia procedure to establish EtOH self-administration followed by 6 months of daily open access to EtOH (4% w/v) and water. Individual daily EtOH intakes ranged from an average of 0.7 to 3.7 g/kg/d. Dorsal lateral PFC area 46 (A46) brain biopsies were collected in EtOH-naïve and control monkeys; contralateral A46 biopsies were collected from the same monkeys following the 6 months of fluid consumption. Gene expression changes were assessed using RNA-Seq paired analysis, which allowed for correction of individual baseline differences in gene expression. RESULTS: A total of 675 genes were significantly down-regulated following EtOH consumption; these were functionally enriched for immune response, cell adhesion, plasma membrane, and extracellular matrix. A total of 567 genes that were up-regulated following EtOH consumption were enriched in microRNA target sites and included target sites associated with Toll-like receptor pathways. The differentially expressed genes were also significantly enriched in transcription factor binding sites. CONCLUSIONS: The data presented here are the first to use a longitudinal biopsy strategy to examine how chronic EtOH consumption affects gene expression in the primate PFC. Prominent effects were seen in both cell adhesion and neuroimmune pathways; the latter contained both pro- and antiinflammatory genes. The data also indicate that changes in miRNAs and transcription factors may be important epigenetic regulators of EtOH consumption.

Voluntary Chronic Heavy Alcohol Consumption in Male Rhesus Macaques Suppresses Cancellous Bone Formation and Increases Bone Marrow Adiposity.

BACKGROUND: Chronic heavy alcohol consumption is an established risk factor for bone fracture, but comorbidities associated with alcohol intake may contribute to increased fracture rates in alcohol abusers. To address the specific effects of alcohol on bone, we used a nonhuman primate model and evaluated voluntary alcohol consumption on: (i) global markers of bone turnover in blood and (ii) cancellous bone mass, density, microarchitecture, turnover, and microdamage in lumbar vertebra. METHODS: Following a 4-month induction period, 6-year-old male rhesus macaques (Macaca mulatta, n = 13) voluntarily self-administered water or ethanol (EtOH; 4% w/v) for 22 h/d, 7 d/wk, for a total of 12 months. Control animals (n = 9) consumed an isocaloric maltose-dextrin solution. Tetracycline hydrochloride was administered orally 17 and 3 days prior to sacrifice to label mineralizing bone surfaces. Global skeletal response to EtOH was evaluated by measuring plasma osteocalcin and carboxyterminal collagen cross-links (CTX). Local response was evaluated in lumbar vertebra using dual-energy X-ray absorptiometry, microcomputed tomography, static and dynamic histomorphometry, and histological assessment of microdamage. RESULTS: Monkeys in the EtOH group consumed an average of 2.8 ± 0.2 (mean ± SE) g/kg/d of EtOH (30 ± 2% of total calories), resulting in an average blood EtOH concentration of 88.3 ± 8.8 mg/dl 7 hours after the session onset. Plasma CTX and osteocalcin tended to be lower in EtOH-consuming monkeys compared to controls. Significant differences in bone mineral density in lumbar vertebrae 1 to 4 were not detected with treatment. However, cancellous bone volume fraction (in cores biopsied from the central region of the third vertebral body) was lower in EtOH-consuming monkeys compared to controls. Furthermore, EtOH-consuming monkeys had lower osteoblast perimeter and mineralizing perimeter, no significant difference in osteoclast perimeter, and higher bone marrow adiposity than controls. No significant differences between groups were detected in microcrack density (2nd lumbar vertebra). CONCLUSIONS: Voluntary chronic heavy EtOH consumption reduces cancellous bone formation in lumbar vertebra by decreasing osteoblast-lined bone perimeter, a response associated with an increase in bone marrow adiposity.

Taar1 gene variants have a causal role in methamphetamine intake and response and interact with Oprm1.

We identified a locus on mouse chromosome 10 that accounts for 60% of the genetic variance in methamphetamine intake in mice selectively bred for high versus low methamphetamine consumption. We nominated the trace amine-associated receptor 1 gene, Taar1, as the strongest candidate and identified regulation of the mu-opioid receptor 1 gene, Oprm1, as another contributor. This study exploited CRISPR-Cas9 to test the causal role of Taar1 in methamphetamine intake and a genetically-associated thermal response to methamphetamine. The methamphetamine-related traits were rescued, converting them to levels found in methamphetamine-avoiding animals. We used a family of recombinant inbred mouse strains for interval mapping and to examine independent and epistatic effects of Taar1 and Oprm1. Both methamphetamine intake and the thermal response mapped to Taar1 and the independent effect of Taar1 was dependent on genotype at Oprm1. Our findings encourage investigation of the contribution of Taar1 and Oprm1 variants to human methamphetamine addiction.

Dose-dependent effects of chronic alcohol drinking on peripheral immune responses.

It is well established that chronic heavy alcohol drinking (CHD) results in significant organ damage, increased susceptibility to infections, and poor outcomes following injury. In contrast, chronic moderate drinking (CMD) has been associated with improved cardiovascular health and immunity. These differential outcomes have been linked to alterations in both innate and adaptive branches of the immune system; however, the mechanisms remain poorly understood. To address this question, we determined the impact of chronic drinking on the transcriptional and functional responses of peripheral blood mononuclear cells (PBMC) collected from male rhesus macaques classified as CMD or CHD after 12 months of voluntary ethanol self-administration. Our analysis suggests that chronic alcohol drinking, regardless of dose alters resting transcriptomes of PBMC, with the largest impact seen in innate immune cells. These transcriptional changes are partially explained by alterations in microRNA profiles. Additionally, chronic alcohol drinking is associated with a dose dependent heightened inflammatory profiled at resting and following LPS stimulation. Moreover, we observed a dose-dependent shift in the kinetics of transcriptional responses to LPS. These findings may explain the dichotomy in clinical and immunological outcomes observed with moderate versus heavy alcohol drinking.

Maternal circulating miRNAs that predict infant FASD outcomes influence placental maturation.

Prenatal alcohol exposure (PAE), like other pregnancy complications, can result in placental insufficiency and fetal growth restriction, although the linking causal mechanisms are unclear. We previously identified 11 gestationally elevated maternal circulating miRNAs (HEamiRNAs) that predicted infant growth deficits following PAE. Here, we investigated whether these HEamiRNAs contribute to the pathology of PAE, by inhibiting trophoblast epithelial-mesenchymal transition (EMT), a pathway critical for placental development. We now report for the first time that PAE inhibits expression of placental pro-EMT pathway members in both rodents and primates, and that HEamiRNAs collectively, but not individually, mediate placental EMT inhibition. HEamiRNAs collectively, but not individually, also inhibited cell proliferation and the EMT pathway in cultured trophoblasts, while inducing cell stress, and following trophoblast syncytialization, aberrant endocrine maturation. Moreover, a single intravascular administration of the pooled murine-expressed HEamiRNAs, to pregnant mice, decreased placental and fetal growth and inhibited the expression of pro-EMT transcripts in the placenta. Our data suggest that HEamiRNAs collectively interfere with placental development, contributing to the pathology of PAE, and perhaps also, to other causes of fetal growth restriction.

Detecting Neurodevelopmental Effects of Early-Gestation Ethanol Exposure: A Nonhuman Primate Model of Ethanol Drinking During Pregnancy.

BACKGROUND: Gestational ethanol (EtOH) exposure is associated with multiple developmental abnormalities, collectively termed fetal alcohol spectrum disorder (FASD). While the majority of women abstain from EtOH following knowledge of pregnancy, one contributing factor to the high FASD prevalence is that pregnancy is not detected until 4 to 6 weeks. Thus, EtOH consumption continues during the initial stages of fetal development. METHODS: An experimental protocol is described in which rhesus macaques self-administer 1.5 g/kg/d EtOH (or isocaloric maltose dextrin) prior to pregnancy and through the first 60 days of a 168-day gestation term. Menstrual cycles were monitored, including measurements of circulating estradiol and progesterone levels. The latency to consume 1.5 g/kg EtOH and blood EtOH concentration (BEC) was measured. RESULTS: Twenty-eight fetuses (14 EtOH and 14 controls) were generated in this study. EtOH did not affect menstrual cycles or the probability of successful breeding. No EtOH-induced gross adverse effects on pregnancy were observed. Individual variability in latency to complete drinking translated into variability in BEC, measured 90 minutes following session start. Drinking latencies in controls and EtOH drinkers were longer in the second gestational month than in the first. All pregnancies reached the planned experimental time point of G85, G110, or G135, when in utero MRIs were performed, fetuses were delivered by caesarean section, and brains were evaluated with ex vivo procedures, including slice electrophysiology. Fetal tissues have been deposited to the Monkey Alcohol Tissue Research Resource. CONCLUSIONS: This FASD model takes advantage of the similarities between humans and rhesus macaques in gestational length relative to brain development, as well as similarities in EtOH self-administration and metabolism. The daily 1.5 g/kg dose of EtOH through the first trimester does not influence pregnancy success rates. However, pregnancy influences drinking behavior during the second month of pregnancy. Future publications using this model will describe the effect of early-gestation EtOH exposure on anatomical and functional brain development at subsequent gestational ages.

Distinct Roles for Two Chromosome 1 Loci in Ethanol Withdrawal, Consumption, and Conditioned Place Preference.

We previously identified a region on chromosome 1 that harbor quantitative trait loci (QTLs) with large effects on alcohol withdrawal risk using both chronic and acute models in mice. Here, using newly created and existing QTL interval-specific congenic (ISC) models, we report the first evidence that this region harbors two distinct alcohol withdrawal QTLs (Alcw11and Alcw12), which underlie 13% and 3-6%, respectively, of the genetic variance in alcohol withdrawal severity measured using the handling-induced convulsion. Our results also precisely localize Alcw11 and Alcw12 to discreet chromosome regions (syntenic with human 1q23.1-23.3) that encompass a limited number of genes with validated genotype-dependent transcript expression and/or non-synonymous sequence variation that may underlie QTL phenotypic effects. ISC analyses also implicate Alcw11and Alcw12 in withdrawal-induced anxiety-like behavior, representing the first evidence for their broader roles in alcohol withdrawal beyond convulsions; but detect no evidence for Alcw12 involvement in ethanol conditioned place preference (CPP) or consumption. Our data point to high-quality candidates for Alcw12, including genes involved in mitochondrial respiration, spatial buffering, and neural plasticity, and to Kcnj9 as a high-quality candidate for Alcw11. Our studies are the first to show, using two null mutant models on different genetic backgrounds, that Kcnj9-/- mice demonstrate significantly less severe alcohol withdrawal than wildtype littermates using acute and repeated exposure paradigms. We also demonstrate that Kcnj9-/- voluntarily consume significantly more alcohol (20%, two-bottle choice) than wildtype littermates. Taken together with evidence implicating Kcnj9 in ethanol CPP, our results support a broad role for this locus in ethanol reward and withdrawal phenotypes. In summary, our results demonstrate two distinct chromosome 1 QTLs that significantly affect risk for ethanol withdrawal, and point to their distinct unique roles in alcohol reward phenotypes.

Cross-species molecular dissection across alcohol behavioral domains.

This review summarizes the proceedings of a symposium presented at the "Alcoholism and Stress: A Framework for Future Treatment Strategies" conference held in Volterra, Italy on May 9-12, 2017. Psychiatric diseases, including alcohol-use disorders (AUDs), are influenced through complex interactions of genes, neurobiological pathways, and environmental influences. A better understanding of the common neurobiological mechanisms underlying an AUD necessitates an integrative approach, involving a systematic assessment of diverse species and phenotype measures. As part of the World Congress on Stress and Alcoholism, this symposium provided a detailed account of current strategies to identify mechanisms underlying the development and progression of AUDs. Dr. Sean Farris discussed the integration and organization of transcriptome and postmortem human brain data to identify brain regional- and cell type-specific differences related to excessive alcohol consumption that are conserved across species. Dr. Brien Riley presented the results of a genome-wide association study of DSM-IV alcohol dependence; although replication of genetic associations with alcohol phenotypes in humans remains challenging, model organism studies show that COL6A3, KLF12, and RYR3 affect behavioral responses to ethanol, and provide substantial evidence for their role in human alcohol-related traits. Dr. Rob Williams expanded upon the systematic characterization of extensive genetic-genomic resources for quantifying and clarifying phenotypes across species that are relevant to precision medicine in human disease. The symposium concluded with Dr. Robert Hitzemann's description of transcriptome studies in a mouse model selectively bred for high alcohol ("binge-like") consumption and a non-human primate model of long-term alcohol consumption. Together, the different components of this session provided an overview of systems-based approaches that are pioneering the experimental prioritization and validation of novel genes and gene networks linked with a range of behavioral phenotypes associated with stress and AUDs.

On the relationships in rhesus macaques between chronic ethanol consumption and the brain transcriptome.

This is the first description of the relationship between chronic ethanol self-administration and the brain transcriptome in a non-human primate (rhesus macaque). Thirty-one male animals self-administered ethanol on a daily basis for over 12 months. Gene transcription was quantified with RNA-Seq in the central nucleus of the amygdala (CeA) and cortical Area 32. We constructed coexpression and cosplicing networks, and we identified areas of preservation and areas of differentiation between regions and network types. Correlations between intake and transcription included largely distinct gene sets and annotation categories across brain regions and between expression and splicing; positive and negative correlations were also associated with distinct annotation groups. Membrane, synaptic and splicing annotation categories were over-represented in the modules (gene clusters) enriched in positive correlations (CeA); our cosplicing analysis further identified the genes affected only at the exon inclusion level. In the CeA coexpression network, we identified Rab6b, Cdk18 and Igsf21 among the intake-correlated hubs, while in the Area 32, we identified a distinct hub set that included Ppp3r1 and Myeov2. Overall, the data illustrate that excessive ethanol self-administration is associated with broad expression and splicing mechanisms that involve membrane and synapse genes.

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.

Identifying Future Drinkers: Behavioral Analysis of Monkeys Initiating Drinking to Intoxication is Predictive of Future Drinking Classification.

BACKGROUND: The Monkey Alcohol Tissue Research Resource (MATRR) is a repository and analytics platform for detailed data derived from well-documented nonhuman primate (NHP) alcohol self-administration studies. This macaque model has demonstrated categorical drinking norms reflective of human drinking populations, resulting in consumption pattern classifications of very heavy drinking (VHD), heavy drinking (HD), binge drinking (BD), and low drinking (LD) individuals. Here, we expand on previous findings that suggest ethanol drinking patterns during initial drinking to intoxication can reliably predict future drinking category assignment. METHODS: The classification strategy uses a machine-learning approach to examine an extensive set of daily drinking attributes during 90 sessions of induction across 7 cohorts of 5 to 8 monkeys for a total of 50 animals. A Random Forest classifier is employed to accurately predict categorical drinking after 12 months of self-administration. RESULTS: Predictive outcome accuracy is approximately 78% when classes are aggregated into 2 groups, "LD and BD" and "HD and VHD." A subsequent 2-step classification model distinguishes individual LD and BD categories with 90% accuracy and between HD and VHD categories with 95% accuracy. Average 4-category classification accuracy is 74%, and provides putative distinguishing behavioral characteristics between groupings. CONCLUSIONS: We demonstrate that data derived from the induction phase of this ethanol self-administration protocol have significant predictive power for future ethanol consumption patterns. Importantly, numerous predictive factors are longitudinal, measuring the change of drinking patterns through 3 stages of induction. Factors during induction that predict future heavy drinkers include being younger at the time of first intoxication and developing a shorter latency to first ethanol drink. Overall, this analysis identifies predictive characteristics in future very heavy drinkers that optimize intoxication, such as having increasingly fewer bouts with more drinks. This analysis also identifies characteristic avoidance of intoxicating topographies in future low drinkers, such as increasing number of bouts and waiting longer before the first ethanol drink.

Genetic Polymorphisms Affect Mouse and Human Trace Amine-Associated Receptor 1 Function.

Methamphetamine (MA) and neurotransmitter precursors and metabolites such as tyramine, octopamine, and ß-phenethylamine stimulate the G protein-coupled trace amine-associated receptor 1 (TAAR1). TAAR1 has been implicated in human conditions including obesity, schizophrenia, depression, fibromyalgia, migraine, and addiction. Additionally TAAR1 is expressed on lymphocytes and astrocytes involved in inflammation and response to infection. In brain, TAAR1 stimulation reduces synaptic dopamine availability and alters glutamatergic function. TAAR1 is also expressed at low levels in heart, and may regulate cardiovascular tone. Taar1 knockout mice orally self-administer more MA than wild type and are insensitive to its aversive effects. DBA/2J (D2) mice express a non-synonymous single nucleotide polymorphism (SNP) in Taar1 that does not respond to MA, and D2 mice are predisposed to high MA intake, compared to C57BL/6 (B6) mice. Here we demonstrate that endogenous agonists stimulate the recombinant B6 mouse TAAR1, but do not activate the D2 mouse receptor. Progeny of the B6XD2 (BxD) family of recombinant inbred (RI) strains have been used to characterize the genetic etiology of diseases, but contrary to expectations, BXDs derived 30-40 years ago express only the functional B6 Taar1 allele whereas some more recently derived BXD RI strains express the D2 allele. Data indicate that the D2 mutation arose subsequent to derivation of the original RIs. Finally, we demonstrate that SNPs in human TAAR1 alter its function, resulting in expressed, but functional, sub-functional and non-functional receptors. Our findings are important for identifying a predisposition to human diseases, as well as for developing personalized treatment options.