The use of non-human primates (NHPs) in studies of volitional, oral self-administration of alcohol can help address the complex interplay between stress and excessive alcohol consumption. There are ...aspects to brain, endocrine and behavior of NHPs, particularly macaques, that provide a critical translational link towards understanding the risks and consequences of alcohol use disorders (AUDs) in humans. These include wide individual differences in escalating daily alcohol intake, accurate measures of hypothalamic-pituitary-adrenal (HPA) axis hormonal interactions, neuroanatomical specificity of synaptic adaptations to chronic alcohol, genetic similarities to humans, and the ability to conduct in vivo brain imaging. When placed in a framework that alcohol addiction is a sequence of dysregulations in motivational circuitry associated with severity of AUD, the NHP can provide within-subject information on both risks for and consequences of repeatedly drinking to intoxication. Notably, long-term adaptations in neurocircuitry that mediate behavioral reinforcement, stress responses and executive functions are possible with NHPs. We review here the substantial progress made using NHPs to address the complex relationship between alcohol and stress as risk factors and consequences of daily drinking to intoxication. This review also highlights areas where future studies of brain and HPA axis adaptations are needed to better understand the mechanisms involved in stress leading to excessive alcohol consumption.
This article is part of the Special Issue entitled “Alcoholism”.
•The use of NHPs in volitional alcohol self-administration studies with the interaction of stress to exacerbate drinking.•The NHP provides within-subject information on both risks for and consequences of repeatedly drinking to intoxication.•This review highlights areas where future studies of brain and HPA axis are needed to understand the mechanisms.
•A microbial community (MC) degrading ibuprofen was selected for sludge bioaugmentation.•Microbial acclimatization after ibuprofen (IBP) toxic shock was experimentally estimated.•Transient change in ...IBP removal rate along the acclimatization period was quantified.•Bioaugmented sludge diminished the acclimatization period after a toxic shock.•Original performance of a wastewater treatment mini-plant is not recovered after an IBP shock.
The determination of the peak volumetric removal rate of ibuprofen in batch culture was used to select a microbial community utilizable for augmentation. For a continuous removal process, this rate, particularly the peak value of the volumetric removal rate is an essential factor reflecting the catabolic behavior (metabolic quotient qS) and the physiological state of a microbial community which depends on environmental conditions. Thus, if stable environmental conditions are maintained in a homogeneous continuous regime, a high metabolic quotient and volumetric removal rate could be maintained for the selected microbial community.
Using equations derived from the material balance of ibuprofen in a wastewater treatment mini-plant (WTMP) operating in a homogeneous condition, the beginning and end of an acclimatization period after an IBP toxic shock were estimated by comparison of the experimental values of IBP accumulated to its theoretical accumulation. Using the experimental values of IBP accumulated within the WTMP, the actual transient change in the volumetric removal rate along the acclimatization period was calculated from another derived equation. This procedure allows to measure the change in the volumetric removal rate, showing the kinetic response of the system and the acclimatization time of a microbial consortium after a toxic shock.
Chronic heavy drinking (CHD) of alcohol is a known risk factor for increased susceptibility to bacterial and viral infection as well as impaired wound healing. Evidence suggests that these defects ...are mediated by a dysregulated inflammatory response originating from myeloid cells, notably monocytes and macrophages, but the mechanisms remain poorly understood. Our ability to study CHD is impacted by the complexities of human drinking patterns and behavior as well as comorbidities and confounding risk factors for patients with alcohol use disorders. To overcome these challenges, we utilized a translational rhesus macaque model of voluntary ethanol self-administration that closely recapitulates human drinking patterns and chronicity. In this study, we examined the effects of CHD on blood monocytes in control and CHD female macaques after 12 months of daily ethanol consumption. While monocytes from CHD female macaques generated a hyper-inflammatory response to
LPS stimulation, their response to
was dampened. In depth scRNA-Seq analysis of purified monocytes revealed significant shifts in classical monocyte subsets with accumulation of cells expressing markers of hypoxia (
) and inflammation (NFkB signaling pathway) in CHD macaques. The increased presence of monocyte subsets skewed towards inflammatory phenotypes was complemented by epigenetic analysis, which revealed higher accessibility of promoter regions that regulate genes involved in cytokine signaling pathways. Collectively, data presented in this manuscript demonstrate that CHD shifts classical monocyte subset composition and primes the monocytes towards a more hyper-inflammatory response to LPS, but compromised pathogen response.
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 (
= 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.
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.
Rationale
Hypofunction of striatal dopamine neurotransmission, or hypodopaminergia, is a consequence of excessive ethanol use and is hypothesized to be a critical component of alcoholism, driving ...alcohol intake in an attempt to restore dopamine levels; however, the neurochemical mechanisms involved in these dopaminergic deficiencies are not fully understood.
Objective
Here we examined the specific dopaminergic adaptations that produce hypodopaminergia and contribute to alcohol use disorders using direct, sub-second measurements of dopamine signaling in nonhuman primates following chronic ethanol self-administration.
Methods
Female rhesus macaques completed 1 year of daily (22 h/day) ethanol self-administration. Subsequently, fast-scan cyclic voltammetry was used in nucleus accumbens core brain slices to determine alterations in dopamine terminal function, including release and uptake kinetics, and sensitivity to quinpirole (D2/D3 dopamine receptor agonist) and U50,488 (kappa opioid receptor agonist) induced inhibition of dopamine release.
Results
Ethanol drinking greatly increased uptake rates, which were positively correlated with lifetime ethanol intake. Furthermore, the sensitivity of dopamine D2/D3 autoreceptors and kappa opioid receptors, which both act as negative regulators of presynaptic dopamine release, was moderately and robustly enhanced in ethanol drinkers.
Conclusions
Greater uptake rates and sensitivity to D2-type autoreceptor and kappa opioid receptor agonists could converge to drive a hypodopaminergic state, characterized by reduced basal dopamine and an inability to mount appropriate dopaminergic responses to salient stimuli. Together, we outline the specific alterations to dopamine signaling that may drive ethanol-induced hypofunction of the dopamine system and suggest that the dopamine and dynorphin/kappa opioid receptor systems may be efficacious pharmacotherapeutic targets in the treatment of alcohol use disorders.
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.
This novel study shows that PEth, a direct biomarker of ethanol intake, is present in the blood of Rhesus monkeys after consuming ethanol ad libitum; and that the levels of the two most abundant PEth homologs, PEth 16:0/18:1 and PEth 16:0/18:2, are detectable as a function of daily ethanol consumed. This pre‐clinical model provides a greater experimental control over ethanol dosing, which could help understand and interpret variability found in PEth in human blood samples.
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.
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.
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.
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.
•Age, sex, and species influenced markers of bone turnover in non-human primates.•Ethanol consumption resulted in a dose-dependent reduction in CTX.•Ethanol consumption resulted in increased osteocalcin in rhesus females.
Rationale
Hypothalamic-pituitary-adrenal (HPA) axis activity under different social settings in non-human primates is understudied.
Objective
The aim of this study is to evaluate the response of ...pituitary-adrenal hormones (adrenocorticotropic hormone (ACTH) and cortisol) to pharmacological challenges of the HPA axis in male cynomolgus macaques under different social settings.
Methods
Male cynomolgus macaques (
Macaca fascicularis
,
n
= 11) were individually (A) and socially housed (B) in alternation, over consecutive months, in an ABA design. During each experimental phase, plasma ACTH and cortisol were measured in response to low- and mild-intensity psychological stressors and following administration of saline, naloxone, ovine-corticotropin-releasing factor (oCRF), and dexamethasone.
Results
These data demonstrate that cortisol measured under low stress conditions is sensitive to social rank (dominance hierarchy) and distinguishes dominant from non-dominant animals during both individual and social settings. Administration of naloxone resulted in elevated circulating ACTH and cortisol, while oCRF only increased circulating cortisol. During social housing, the cortisol response to naloxone and oCRF was increased, whereas dexamethasone suppression of ACTH and cortisol remained consistent across all social settings.
Conclusions
Circulating ACTH and cortisol are differentially sensitive to changes in social settings in non-human primates. Cortisol response increased during social housing and could be stimulated by both naloxone and oCRF, whereas ACTH response was generally not influenced by social setting or oCRF but was increased by naloxone. These data show differential adrenal and pituitary response to changes in social settings and a small, but consistent, effect of social dominance.
Background
Recent work with long‐term ethanol (EtOH) self‐administration in nonhuman primate models has revealed a complex array of behavioral and physiological effects that closely mimic human ...alcohol abuse. Detailed neurophysiological analysis in these models suggests a myriad of pre‐ and postsynaptic neurobiological effects that may contribute to the behavioral manifestations of long‐term EtOH drinking. The molecular mechanisms regulating presynaptic effects of this chronic EtOH exposure are largely unknown. To this end, we analyzed the effects of long‐term EtOH self‐administration on the levels of presynaptic SNARE complex proteins in Macaca mulatta basolateral amygdala, a brain region known to regulate both aversive and reward‐seeking behaviors.
Methods
Basolateral amygdala samples from control and EtOH‐drinking male and female monkeys were processed. Total basolateral amygdala protein was analyzed by Western blotting using antibodies directed against both core SNARE and SNARE‐associated proteins. We also performed correlational analyses between protein expression levels and a number of EtOH drinking parameters, including lifetime grams of EtOH consumed, preference, and blood alcohol concentration.
Results
Significant interactions or main effects of sex/drinking were seen for a number of SNARE core and SNARE‐associated proteins. Across the range of EtOH‐drinking phenotypes, SNAP25 and Munc13‐1 proteins levels were significantly different between males and females, and Munc13‐2 levels were significantly lower in animals with a history of EtOH drinking. A separate analysis of very heavy‐drinking individuals revealed significant decreases in Rab3c (females) and complexin 2 (males).
Conclusions
Protein expression analysis of basolateral amygdala total protein from controls and animals following long‐term EtOH self‐administration suggests a number of alterations in core SNARE or SNARE‐associated components that could dramatically alter presynaptic function. A number of proteins or multiprotein components were also correlated with EtOH drinking behavior, which suggest a potentially heritable role for presynaptic SNARE proteins.
Functional impact of sex and long‐term drinking by Macaca mullata on presynaptic vesicle priming and release proteins from lateral amygdala samples examined. Parentheses indicate significant main effects, either sex or ethanol drinking, sound in the western analysis. Asterisks denote proteins significantly correlated with either total voluntary ethanol consumption or average blood alcohol concentration.
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