Brain Functional Connectivity Mapping of Behavioral Flexibility in Rhesus Monkeys.

The predisposition to engage in autonomous habitual behaviors has been associated with behavioral disorders, such as obsessive-compulsive disorder and addiction. Attentional set-shifting tasks (ASSTs), which incorporate changes governing the association of discriminative stimuli with contingent reinforcement, are commonly used to measure underlying processes of cognitive/behavioral flexibility. The purpose of this study was to identify primate brain networks that mediate trait-like deficits in ASST performance using resting-state fMRI. A self-pacing ASST was administered to three cohorts of rhesus monkeys (total n = 35, 18 female). Increased performance over 30 consecutive sessions segregated the monkeys into two populations, termed High Performers (HP, n = 17) and Low Performers (LP, n = 17), with one anomaly. Compared with LPs, HPs had higher rates of improving performance over sessions and completed the 8 sets/sessions with fewer errors. LP monkeys, on the other hand, spent most of each session in the first set and often did not acquire the first reversal. A whole-brain independent components analysis of resting-state fMRI under isoflurane identified four strong networks. Of these, a dual regression analysis revealed that a designated "executive control network," differed between HPs and LPs. Specific areas of connectivity in the rhesus executive control network, including frontal cortices (ventrolateral, ventromedial, and orbital) and the dorsal striatum (caudate, putamen) correlated with perseverative errors and response latency. Overall, the results identify trait-like characteristics of behavioral flexibility that are associated with correlated brain activity involving specific nuclei of frontostriatal networks.SIGNIFICANCE STATEMENT Resting state functional connectivity MRI in rhesus monkeys identified specific nuclei in frontostriatal circuitry that were associated with population differences in perseverative and impulsive aspects of cognitive flexibility.

Profiling of extracellular vesicle-bound miRNA to identify candidate biomarkers of chronic alcohol drinking in nonhuman primates.

BACKGROUND: Long-term alcohol drinking is associated with numerous health complications including susceptibility to infection, cancer, and organ damage. However, due to the complex nature of human drinking behavior, it has been challenging to identify reliable biomarkers of alcohol drinking behavior prior to signs of overt organ damage. Recently, extracellular vesicle-bound microRNAs (EV-miRNAs) have been found to be consistent biomarkers of conditions that include cancer and liver disease. METHODS: In this study, we profiled the plasma EV-miRNA content by miRNA-Seq from 80 nonhuman primates after 12 months of voluntary alcohol drinking. RESULTS: We identified a list of up- and downregulated EV-miRNA candidate biomarkers of heavy drinking and those positively correlated with ethanol dose. We overexpressed these candidate miRNAs in control primary peripheral immune cells to assess their potential functional mechanisms. We found that overexpression of miR-155, miR-154, miR-34c, miR-450a, and miR-204 led to increased production of the inflammatory cytokines TNFα or IL-6 in peripheral blood mononuclear cells after stimulation. CONCLUSION: This exploratory study identified several EV-miRNAs that could serve as biomarkers of long-term alcohol drinking and provide a mechanism to explain alcohol-induced peripheral inflammation.

Behavioral Flexibility in Alcohol-Drinking Monkeys: The Morning After.

BACKGROUND: Heavy alcohol drinking has aspects of inflexible behavior. This study addressed the consequences of chronic alcohol drinking on cognitive and sensory-motor domains of behavioral flexibility in rhesus monkeys. METHODS: Behavioral flexibility was assessed in 12 monkeys (n = 9, ethanol [EtOH] drinkers) with a set-shifting visual discrimination procedure before alcohol self-administration and while maintaining consumption of 1.5 g/kg/d EtOH. Task performance was assessed in the morning after ~18 hours of drinking 1.5 g/kg, and 1 hour before the next day's drinking session began. The first 10 set-shifting sessions had the original (preethanol) test parameters and were used to determine retention of preethanol performance. Then, an effect of sensory-motor challenge (60% reduction in the size of the discriminative stimuli) on performance was assessed during 10 additional sessions. RESULTS: There were no average group-dependent differences in the performance between control and EtOH groups at the preethanol time-point. The daily consumption of 1.5 g/kg/d produced binge alcohol intakes in 7 of 9 monkeys (blood EtOH concentration [BEC ≥ 80 mg/dl]). Chronic daily intakes of 1.5 g/kg had no effect on retention of the task in the sober state. However, when challenged with a reduction in the size of the stimuli, daily 1.5 g/kg EtOH resulted in a decrement in performance due to an increase in the number of errors. CONCLUSIONS: Rhesus monkeys consuming 1.5 g/kg alcohol daily perform equally as could as control monkeys in retention of a well-learned cognitive task. However, this pattern of daily alcohol intake robustly decreased the ability to flexibly adjust behavior when confronted with novel changes to perceptual stimuli.

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.

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.

Chronic alcohol self-administration in monkeys shows long-term quantity/frequency categorical stability.

BACKGROUND: The current criteria for alcohol use disorders (AUDs) do not include consumption (quantity/frequency) measures of alcohol intake, in part due to the difficulty of these measures in humans. Animal models of ethanol (EtOH) self-administration have been fundamental in advancing our understanding of the neurobiological basis of AUD and can address quantity/frequency measures with accurate measurements over prolonged periods of time. The nonhuman primate model of voluntary oral alcohol self-administration has documented both binge drinking and drinking to dependence and can be used to test the stability of consumption measures over time. METHODS: Here, an extensive set of alcohol intakes (g/kg/d) was analyzed from a large multi-cohort population of Rhesus (Macaca mulatta) monkeys (n = 31). Daily EtOH intake was uniformly distributed over chronic (12 months) access for all animals. RESULTS: Underlying this distribution of intakes were subpopulations of monkeys that exhibited distinctive clustering of drinking patterns, allowing us to categorically define very heavy drinking (VHD), heavy drinking (HD), binge drinking (BD), and low drinking (LD). These categories were stable across the 12 months assessed by the protocol, but exhibited fluctuations when examined at shorter intervals. CONCLUSIONS: The establishment of persistent drinking categories based on quantity/frequency suggests that consumption variables can be used to track long-term changes in behavioral, molecular, or physiochemical mechanisms related to our understanding of diagnosis, prevention, intervention, and treatment efficacies.

Monkey alcohol tissue research resource: banking tissues for alcohol research.

BACKGROUND: An estimated 18 million adults in the United States meet the clinical criteria for diagnosis of alcohol abuse or alcoholism, a disorder ranked as the third leading cause of preventable death. In addition to brain pathology, heavy alcohol consumption is comorbid with damage to major organs including heart, lungs, liver, pancreas, and kidneys. Much of what is known about risk for and consequences of heavy consumption derive from rodent or retrospective human studies. The neurobiological effects of chronic intake in rodent studies may not easily translate to humans due to key differences in brain structure and organization between species, including a lack of higher-order cognitive functions, and differences in underlying prefrontal cortical neural structures that characterize the primate brain. Further, rodents do not voluntarily consume large quantities of ethanol (EtOH) and they metabolize it more rapidly than primates. METHODS: The basis of the Monkey Alcohol Tissue Research Resource (MATRR) is that nonhuman primates, specifically monkeys, show a range of drinking excessive amounts of alcohol (>3.0 g/kg or a 12 drink equivalent per day) over long periods of time (12 to 30 months) with concomitant pathological changes in endocrine, hepatic, and central nervous system (CNS) processes. The patterns and range of alcohol intake that monkeys voluntarily consume parallel what is observed in humans with alcohol use disorders and the longitudinal experimental design spans stages of drinking from the EtOH-naïve state to early exposure through chronic abuse. Age- and sex-matched control animals self-administer an isocaloric solution under identical operant procedures. RESULTS: The MATRR is a unique postmortem tissue bank that provides CNS and peripheral tissues, and associated bioinformatics from monkeys that self-administer EtOH using a standardized experimental paradigm to the broader alcohol research community. CONCLUSIONS: This resource provides a translational platform from which we can better understand the disease processes associated with alcoholism.

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.

Long-term drinking stability in the open-access self-administration monkey model.

The Non-Human Primate (NHP) model for the study of Alcohol Use Disorders (AUD) as developed in our laboratories is critical to our understanding of the pathophysiology of voluntary, chronic, ethanol consumption. Previous work in this model established categories of ethanol consumption that parallel reported categories of human consumption across a spectrum spanning low drinking, binge drinking, heavy drinking, and very heavy drinking, albeit at generally higher daily intakes across categories than documented in people. Original categories assigned to ethanol consumption patterns were established using a limited cohort of rhesus macaques. This study revisits the validity of categorical drinking using an additional 28 monkeys. In addition to finding categorical representations consistent with the original 2014 report, our findings demonstrate that drinking categories remain stable across the observed 12 months of nearly consistent access to ethanol (22 h/day), termed "open access". Animals occupying the two ends of the spectrum, "low" and "very heavy" drinkers, exhibit the largest stability. The findings also indicate a slight escalatory drift over time, with very heavy drinking animals experiencing fatigue near the end of open access.

Impact of putamen inhibition by DREADDs on schedule-induced drinking in rhesus monkeys.

The putamen is a nucleus within the sensory-motor striatal network that is involved in automatic, habitual actions. Schedule-induced polydipsia (SIP) is highly automated behavior, reliably occurring under intermediate interval schedules of reinforcement. The effect of putamen inhibition in mediating SIP of water and ethanol (4% w/v) under a Fixed Time 5-min (FT-5 min) schedule for food delivery was tested in 12 rhesus monkeys (6 male, 6 female). Water and ethanol SIP sessions ended after set volumes were consumed. Baseline patterns of SIP intake differed between water and ethanol SIP in volume but not in pattern of drinking. Activation of the designer receptor exclusively activated by designer drug (DREADD: hM4Di) with deschloroclozapine (DCZ; 300 µg/kg, i.m.) administered 30 min prior to the onset of the SIP session, for four consecutive sessions. DCZ administration increased the postpellet drink volume and reduced the time to drink both water and ethanol. Although the effect of DCZ treatment was similar for increasing SIP with either water or ethanol, post-DCZ return to baseline SIP rates of differed, perhaps highlighting the effect of a state dependency with ethanol SIP. Overall, the study shows that targeting the putamen with the inhibitory DREADD produces a reversible, reproducible and reliable increase in adjunctive drinking.

Dose-response effects of alcohol on biochemical markers of bone turnover in non-human primates: Effects of species, sex and age of onset of drinking.

PURPOSE: Alcohol consumption suppressed bone turnover in male non-human primates; however, it is unclear the extent to which this effect depends upon biological variables. Using archived plasma samples, we investigated whether sex, age of onset of alcohol intake, and species influence the effects of graded increases in alcohol consumption on bone turnover markers. METHODS: 91 male and female macaques (rhesus and cynomolgus), ranging in age from 4 years (adolescent) to 10 years (adult) were required to increase their consumption of ethanol in 30-day increments: 0 g/kg/day, followed by 0.5 g/kg/day, 1.0 g/kg/day, and, finally, 1.5 g/kg/day. Plasma osteocalcin (formation), plasma CTX (resorption) and osteocalcin to CTX ratio (turnover balance) were measured during these intervals to assess the dose-response effects of alcohol. RESULTS: We detected no relationship between dose and osteocalcin when all monkeys were combined, but there was a significant effect of sex (lower levels in females) and interactions between alcohol dose and sex (osteocalcin levels increased with dose in rhesus females). In contrast, we detected a negative linear dose-response relationship for ethanol and CTX. We did not detect a relationship between dose and osteocalcin to CTX ratio overall, but there was a significant positive relationship detected in females (no change in males). Increased age predicted lower biomarker levels for both osteocalcin and CTX. Species was a significant predictor for osteocalcin and the osteocalcin to CTX ratio in these models. CONCLUSION: These findings indicate that age, sex, and species influence bone turnover and support the concept that factors beyond quantity of alcohol affect skeletal response to alcohol consumption.

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.

Ethanol alters the relationship between IGF-1 and bone turnover in male macaques.

Insulin-like growth factor 1 (IGF-1) influences bone turnover. Transient decreases in IGF-I levels and/or bioavailability may contribute to the detrimental effects of alcohol on bone. The goals of this non-human primate study were to i) evaluate the 20-h response of bone turnover markers to ethanol consumption and ii) assess how ethanol consumption influences the relationship between IGF-1 and these markers. Osteocalcin (bone formation), carboxyterminal cross-linking telopeptide of type 1 collagen (CTX, bone resorption), IGF-1, and IGF binding protein 1 (IGFBP-1) were measured in plasma from male rhesus macaques (N = 10, 8.4 ± 0.3 years) obtained at 12:00, 16:00, and 06:00 h during two phases: pre-ethanol (alcohol-naïve) and ethanol access. During the ethanol access phase, monkeys consumed 1.5 g/kg/day ethanol (4% w/v) beginning at 10:00 h. Osteocalcin and CTX were lower, and the ratio of osteocalcin to CTX was higher at each time point during ethanol access compared to the pre-ethanol phase. Pre-ethanol marker levels did not vary across time points, but markers varied during ethanol access. IGF-1 levels, but not IGFBP-1 levels, varied during the pre-ethanol phase. In contrast, IGF-1 levels were stable during ethanol access but IGFBP-1 levels varied. There were positive relationships between IGF-1 and turnover markers during the pre-ethanol phase, but not during ethanol access. In conclusion, chronic ethanol consumption reduces levels of bone turnover markers and blocks the normal positive relationship between IGF-1 and turnover markers and alters the normal relationship between IGF-1 and IGFBP-1. These findings support the hypothesis that chronic alcohol consumption leads to growth hormone/IGF-1 resistance.

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.

Low cognitive flexibility as a risk for heavy alcohol drinking in non-human primates.

Chronic alcohol abuse is frequently considered a habitual or inflexible behavior; however, measures of pre-existing cognitive flexibility prior to initiation of alcohol use are usually not available. This study used rhesus monkeys and an attentional set-shifting task to investigate whether pre-existing cognitive flexibility would predict increased risk for heavy alcohol drinking. As previously reported, monkeys were given 30 daily set-shifting sessions prior to alcohol access. These sessions consisted of the same sequence of eight unique visual discriminations (sets) of two objects that varied on two dimensions (shapes and colors). The ratio of errors per trials, session duration, and maximum set reached were primary dependent variables from each session and were used to compose a session performance index (PI) that ranged from a low performance PI of 31 to an optimal performance PI of 247. Here, animals underwent an alcohol induction period followed by 22 weeks of daily (22-h) self-administration sessions with free access to water and alcohol. Based on average daily alcohol intake during 22 weeks of 22-h/day access, the monkeys were categorized as non-heavy (mean = 2.0 ± 0.3 g/kg/day; n = 3) and heavy (mean = 3.3 ± 0.5 g/kg/day; n = 6) drinkers. The two groups diverged in performance on the set-shifting task across the 30 pre-alcohol sessions, and at the end of the pre-alcohol testing, the group average PI was 216 ± 27 and 137 ± 71 for the future non-heavy and heavy drinkers, respectively. The data show that low cognitive flexibility assessed with a set-shifting procedure was predictive of future classification as a heavy alcohol drinker. The data highlight individual differences in both cognitive flexibility and in alcohol self-administration in this population of rhesus monkeys.

Time for a Drink? A Mathematical Model of Non-human Primate Alcohol Consumption.

We simulate a non-human primate's alcohol drinking pattern in order to better understand temporal patterning of alcoholic drinks that can lead to the excessive intakes associated with alcohol use disorder. A stochastic mathematical model of alcohol consumption pattern is developed, where model parameters are calibrated to an individual monkey's drinking history. The model predicts a time series that simulates a monkey's alcohol intake in time, and we analyze this drinking pattern to understand the variations in day and night drinking, the lengths of drinks (intake in 5 or more consecutive secs), and lengths of bouts (1 or more drinks per 5 min occasion). This time series can predict a lifetime categorical drinking level (light, binge, heavy, or very heavy), thus correlating an individual monkey's parameters with distinct long term drinking classifications.

Drinking typography established by scheduled induction predicts chronic heavy drinking in a monkey model of ethanol self-administration.

BACKGROUND: We have developed an animal model of alcohol self-administration that initially employs schedule-induced polydipsia (SIP) to establish reliable ethanol consumption under open access (22 h/d) conditions with food and water concurrently available. SIP is an adjunctive behavior that is generated by constraining access to an important commodity (e.g., flavored food). The induction schedule and ethanol polydipsia generated under these conditions affords the opportunity to investigate the development of drinking typologies that lead to chronic, excessive alcohol consumption. METHODS: Adult male cynomolgus monkeys (Macaca fascicularis) were induced to drink water and 4% (w/v in water) ethanol by a Fixed-Time 300 seconds (FT-300 seconds) schedule of banana-flavored pellet delivery. The FT-300 seconds schedule was in effect for 120 consecutive sessions, with daily induction doses increasing from 0.0 to 0.5 g/kg to 1.0 g/kg to 1.5 g/kg every 30 days. Following induction, the monkeys were allowed concurrent access to 4% (w/v) ethanol and water for 22 h/day for 12 months. RESULTS: Drinking typographies during the induction of drinking 1.5 g/kg ethanol emerged that were highly predictive of the daily ethanol intake over the next 12 months. Specifically, the frequency in which monkeys ingested 1.5 g/kg ethanol without a 5-minute lapse in drinking (defined as a bout of drinking) during induction strongly predicted (correlation 0.91) subsequent ethanol intake over the next 12 months of open access to ethanol. Blood ethanol during induction were highly correlated with intake and with drinking typography and ranged from 100 to 160 mg% when the monkeys drank their 1.5 g/kg dose in a single bout. Forty percent of the population became heavy drinkers (mean daily intakes >3.0 g/kg for 12 months) characterized by frequent "spree" drinking (intakes >4.0 g/kg/d). CONCLUSION: This model of ethanol self-administration identifies early alcohol drinking typographies (gulping the equivalent of 6 drinks) that evolve into chronic heavy alcohol consumption in primates (drinking the equivalent of 16 to 20 drinks per day). The model may aid in identifying biological risks for establishing harmful alcohol drinking.

Prodrug thiamine analogs as inhibitors of the enzyme transketolase.

Transketolase, a key enzyme in the pentose phosphate pathway, has been suggested as a target for inhibition in the treatment of cancer. Compound 5a ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of the transketolase cofactor thiamine, is a potent transketolase inhibitor but suffers from poor pharmacokinetics due to high clearance and C(max) linked toxicity. An efficient way of improving the pharmacokinetic profile of 5a is to prepare oxidized prodrugs which are slowly reduced in vivo yielding longer, sustained blood levels of the drug. The synthesis of such prodrugs and their evaluation in rodent models is reported.

Non-charged thiamine analogs as inhibitors of enzyme transketolase.

Inhibition of the thiamine-utilizing enzyme transketolase (TK) has been linked with diminished tumor cell proliferation. Most thiamine antagonists have a permanent positive charge on the B-ring, and it has been suggested that this charge is required for diphosphorylation by thiamine pyrophosphokinase (TPPK) and binding to TK. We sought to make neutral thiazolium replacements that would be substrates for TPPK, while not necessarily needing thiamine transporters (ThTr1 and ThTr2) for cell penetration. The synthesis, SAR, and structure-based rationale for highly potent non-thiazolium TK antagonists are presented.