High Alcohol-drinking Rats Research Articles

Gut Microbiota-Derived Extracellular Vesicles Influence Alcohol Intake Preferences in Rats.

Growing preclinical and clinical evidence suggests a link between gut microbiota dysbiosis and problematic alcohol consumption. Extracellular vesicles (EVs) are key mediators involved in bacteria-to-host communication. However, their potential role in mediating addictive behaviour remains unexplored. This study investigates the role of gut microbiota-derived bacterial extracellular vesicles (bEVs) in driving high alcohol consumption. bEVs were isolated from the gut microbiota of a high alcohol-drinking rat strain (UChB rats), either ethanol-naïve or following chronic alcohol consumption and administered intraperitoneally or orally to alcohol-rejecting male and female Wistar rats. Both types of UChB-derived bEVs increased Wistar's voluntary alcohol consumption (three bottle choice test) up to 10-fold (p<0.0001), indicating that bEVs are able and sufficient to transmit drinking behaviour across different rat strains. Molecular analysis revealed that bEVs administration did not induce systemic or brain inflammation in the recipient animals, suggesting that the increased alcohol intake triggered by UChB-derived bEVs operates through an inflammation-independent mechanism. Furthermore, we demonstrate that the vagus nerve mediates the bEV-induced increase in alcohol consumption, as bilateral vagotomy completely abolished the high drinking behaviour induced by both intraperitoneally injected and orally administered bEVs. Thus, this study identifies bEVs as a novel mechanism underlying gut microbiota-induced high alcohol intake in a vagus nerve-dependent manner.

Evaluating sex and line differences in successive negative contrast and ethanol consumption using alcohol preferring and high alcohol drinking rats.

The loss of a job or relationship are a couple of examples of unexpected reward loss. Life events, such as these can induce negative emotional reactions (e.g., anxiety and stress), which have been associated with increased alcohol consumption and in turn, an increased risk of developing an alcohol use disorder (AUD). The present study analyzed consummatory successive negative contrast (SNC) for the first time in alcohol preferring (P) and high alcohol drinking (HAD) rats that have been selectively bred to consume high amounts of ethanol. Following reward loss, animals were given free access to ethanol to determine whether consumption would increase as a possible indication of any negative emotional reaction. Male and female P and HAD rats were split into shifted and unshifted groups receiving either 32% or 4% sucrose for 5 min across 10 preshift days. Subsequently, all animals received 4% sucrose for four postshift days, across which, animals were given access to 20% ethanol for 30 min after access to 4% sucrose. Male and female P rats demonstrated a longer contrast effect than HAD rats, indicated by a longer recovery time following the downshift in reward. Conversely, HAD males did not demonstrate a contrast effect following this downshift in reward unlike their female counterparts. Surprisingly, P rats who experienced a loss of reward consumed significantly less ethanol than animals who did not. Lastly, individual measure of contrast size, or shift ratio, was significantly associated with greater ethanol consumption in HAD males only, who did not display a contrast effect. These data indicate different reactivity to SNC between these two lines and sexes, suggesting different genetic and sex-related mechanisms underlying sensitivity to an unexpected loss of reward and ethanol consumption following this loss.

Sex differences in cognitive performance and alcohol consumption in High Alcohol-Drinking (HAD-1) rats

Sex differences in cognitive performance and alcohol consumption in High Alcohol-Drinking (HAD-1) rats

Alcohol Drinking and Blood Alcohol Concentration Revisited.

It is widely assumed that the amount of alcohol in the blood reflects the amount of alcohol consumed. However, several factors in addition to amount of alcohol consumed can influence blood alcohol concentration (BAC). This study examines the effect of alcohol dose, concentration, and volume on BAC in rats with a high-alcohol-drinking (HAD) phenotype. Study 1 examined the relationship between the amount of alcohol consumed and BAC. Alcohol-naïve, male, HAD rats (N=7) were given access to alcohol for 2h/d for 9 consecutive days with food and water adlibitum. Alcohol intake and BAC were measured at 30, 60, and 90minutes after onset of access. Study 2 examined the effects of altering alcohol dose, concentration, and volume on BAC (as measured by area under the curve). Alcohol-naïve, male, HAD rats (N=39) were infused, via an intragastric cannulus, with 1.16, 2.44, or 3.38g alcohol/kg body weight (BW), produced by varying alcohol volume while holding concentration constant or by holding volume constant while varying concentration. Other rats were infused with 10, 15, or 20% v/v alcohol solutions while holding dose constant. BAC was more strongly correlated with the ratio of alcohol intake (g/kg BW) to total fluid intake (mls) (R=0.85 to 0.97, p<0.05 to p<0.001) than it was with the amount of alcohol consumed (g/kg BW) (R=0.70 to 0.81, p<0.05). No effect of alcohol dose was seen during the first hour following the onset of an alcohol infusion regardless of whether dose was achieved by altering alcohol volume or concentration. After 1hour, higher alcohol doses were predictive of greater BACs. The fact that a 3-fold difference in alcohol dose did not result in significant differences in BACs during the first 30minutes after ingestion of alcohol has potentially important implications for interpretation of studies that measure alcohol-sensitive end points during this time.

Effect of voluntary alcohol consumption on Maoa expression in the mesocorticolimbic brain of adult male rats previously exposed to prolonged maternal separation.

Discordant associations between monoamine oxidase A (MAOA) genotype and high alcohol drinking have been reported in human and non-human primates. Environmental influences likely moderate genetic susceptibility. The biological basis for this interplay remains elusive, and inconsistencies call for translational studies in which conditions can be controlled and brain tissue is accessible. The present study investigated whether early life stress and subsequent adult episodic alcohol consumption affect Maoa expression in stress- and reward-related brain regions in the rat. Outbred Wistar rats were exposed to rearing conditions associated with stress (prolonged maternal separation) or no stress during early life, and given free choice between alcohol and/or water in adulthood. Transcript levels of Maoa were assessed in the ventral tegmental area, nucleus accumbens (NAc), medial prefrontal cortex, cingulate cortex, amygdala and dorsal striatum (DS). Blood was collected to assess corticosterone levels. After alcohol consumption, lower blood corticosterone and Maoa expression in the NAc and DS were found in rats exposed to early life stress compared with control rats. An interaction between early life stress and voluntary alcohol intake was found in the NAc. Alcohol intake before death correlated negatively with Maoa expression in DS in high alcohol-drinking rats exposed to early life stress. Maoa expression is sensitive to adulthood voluntary alcohol consumption in the presence of early life stress in outbred rats. These findings add knowledge of the molecular basis of the previously reported associations between early life stress, MAOA and susceptibility to alcohol misuse.

Daily patterns of ethanol drinking in adolescent and adult, male and female, high alcohol drinking (HAD) replicate lines of rats

Daily patterns of ethanol drinking in adolescent and adult, male and female, high alcohol drinking (HAD) replicate lines of rats

Binge Drinking: In Search of its Molecular Target via the GABAA Receptor

Binge drinking, frequently referred to clinically as problem or hazardous drinking, is a pattern of excessive alcohol intake characterized by blood alcohol levels ≥0.08 g% within a 2-h period. Here, we show that overexpression of α1 subunits of the GABAA receptor contributes to binge drinking, and further document that this involvement is related to the neuroanatomical localization of α1 receptor subunits. Using a herpes simplex virus amplicon vector to deliver small interference RNA (siRNA), we showed that siRNA specific for the α1 subunit (pHSVsiLA1) caused profound, long-term, and selective reduction of gene expression, receptor density, and binge drinking in high-alcohol drinking rats when delivered into the ventral pallidum (VP). Scrambled siRNA (pHSVsiNC) delivered similarly into the VP failed to alter gene expression, receptor density, or binge drinking. Silencing of the α1 gene in the VP, however, failed to alter binge sucrose or water intake. These results, along with our prior research, provide compelling evidence that the α1-containing GABAA receptor subunits are critical in the regulation of binge-like patterns of excessive drinking. Collectively, these data may be useful in the development of gene-based and novel pharmacological approaches for the treatment of excessive drinking.

PRECLINICAL STUDY: Effects of concurrent access to multiple ethanol concentrations and repeated deprivations on alcohol intake of high‐alcohol‐drinking (HAD) rats

High-alcohol-drinking rats, given access to 10% ethanol, expressed an alcohol deprivation effect (ADE) only after multiple deprivations. In alcohol-preferring (P) rats, concurrent access to multiple ethanol concentrations combined with repeated cycles of EtOH access and deprivation produced excessive ethanol drinking. The current study was undertaken to examine the effects of repeated alcohol deprivations with concurrent access to multiple concentrations of ethanol on ethanol intake of HAD replicate lines of rats. HAD-1 and HAD-2 rats received access to 10, 20 and 30% (v/v) ethanol for 6 weeks. Rats from each replicate line were assigned to: (1) a non-deprived group; (2) a group initially deprived of ethanol for 2 weeks; or (3) a group initially deprived for 8 weeks. Following the restoration of the ethanol solutions, cycle of 2 weeks of ethanol exposure and 2 weeks of alcohol deprivation was repeated three times for a total of four deprivations. Following the initial ethanol deprivation period, deprived groups significantly increased ethanol intakes during the initial 24-hour re-exposure period. Multiple deprivations increased ethanol intakes, shifted preference to higher ethanol concentrations and prolonged the duration of the elevated ethanol intakes for up to 5 days. In addition, repeated deprivations increased ethanol intake in the first 2-hour re-exposure period as high as 5-7 g/kg (which are equivalent to amounts consumed in 24 hours by HAD rats), and produced blood ethanol levels in excess of 150 mg%. The results indicate that HAD rats exhibit 'loss-of-control' of alcohol drinking with repeated deprivations when multiple ethanol concentrations are available.

Rats bred for high alcohol drinking are more sensitive to delayed and probabilistic outcomes

Alcoholics and heavy drinkers score higher on measures of impulsivity than nonalcoholics and light drinkers. This may be because of factors that predate drug exposure (e.g. genetics). This study examined the role of genetics by comparing impulsivity measures in ethanol-naive rats selectively bred based on their high [high alcohol drinking (HAD)] or low [low alcohol drinking (LAD)] consumption of ethanol. Replicates 1 and 2 of the HAD and LAD rats, developed by the University of Indiana Alcohol Research Center, completed two different discounting tasks. Delay discounting examines sensitivity to rewards that are delayed in time and is commonly used to assess 'choice' impulsivity. Probability discounting examines sensitivity to the uncertain delivery of rewards and has been used to assess risk taking and risk assessment. High alcohol drinking rats discounted delayed and probabilistic rewards more steeply than LAD rats. Discount rates associated with probabilistic and delayed rewards were weakly correlated, while bias was strongly correlated with discount rate in both delay and probability discounting. The results suggest that selective breeding for high alcohol consumption selects for animals that are more sensitive to delayed and probabilistic outcomes. Sensitivity to delayed or probabilistic outcomes may be predictive of future drinking in genetically predisposed individuals.

Effects of short deprivation and re-exposure intervals on the ethanol drinking behavior of selectively bred high alcohol-consuming rats

Effects of short deprivation and re-exposure intervals on the ethanol drinking behavior of selectively bred high alcohol-consuming rats

Effects of multiple alcohol deprivations on operant ethanol self-administration by high-alcohol-drinking replicate rat lines

Effects of multiple alcohol deprivations on operant ethanol self-administration by high-alcohol-drinking replicate rat lines

Dopamine and serotonin content in select brain regions of weanling and adult alcohol drinking rat lines

Dopamine and serotonin content in select brain regions of weanling and adult alcohol drinking rat lines

Effects of Long‐Term Episodic Access to Ethanol on the Expression of an Alcohol Deprivation Effect in Low Alcohol–Consuming Rats

The alcohol-preferring (P) and -nonpreferring (NP) and high alcohol-drinking (HAD) and low alcohol-drinking (LAD) rats have been selectively bred for divergent preference for ethanol over water. In addition, both P and HAD rats display an alcohol deprivation effect (ADE). This study was undertaken to test whether the NP, LAD-1, and LAD-2 lines of rats could display an ADE as well. Adult female NP, LAD-1, and LAD-2 rats were given concurrent access to multiple concentrations of ethanol [5, 10, 15% (v/v)] and water in an ADE paradigm involving an initial 6 weeks of 24-hr access to ethanol, followed by four cycles of 2 weeks of deprivation from and 2 weeks of re-exposure to ethanol (5, 10, and 15%). A control group had continuous access to the ethanol concentrations (5, 10, and 15%) and water through the end of the fourth re-exposure period. For NP rats, a preference for the highest ethanol concentration (15%) was evident by the end of the fifth week of access (approximately 60% of total ethanol fluid intake). Contrarily, LAD rats did not display a marked preference for any one concentration of ethanol. All three lines displayed an ADE after repeated cycles of re-exposure to ethanol, with the general ranking of intake being LAD-1 > NP > LAD-2 (e.g., for the first day of reinstatement of the third re-exposure cycle, intakes were 6.5, 2.9, and 2.4 g/kg/day compared with baseline values of 3.1, 2.0, and 1.3 g/kg/day for each line, respectively). By the 13th week, rats from all three lines, with a ranking of LAD-1 > NP > LAD-2, were drinking more ethanol (3.3, 2.2, and 2.0 g/kg/day, respectively) compared with their consumption during the first week of access (approximately 1.1 g/kg/day for all three lines). These data indicate that access to multiple concentrations of ethanol and exposure to multiple deprivation cycles can partially overcome a genetic predisposition of NP, LAD-1, and LAD-2 rats for low alcohol consumption. In addition, the findings suggest that genetic control of low alcohol consumption in rats is not associated with the inability to display an ADE.

Innate differences in neuropeptide Y (NPY) mRNA expression in discrete brain regions between alcohol-preferring (P) and -nonpreferring (NP) rats: a significantly low level of NPY mRNA in dentate gyrus of the hippocampus and absence of NPY mRNA in the medial habenular nucleus of P rats

Innate differences in neuropeptide Y (NPY) mRNA expression in discrete brain regions between alcohol-preferring (P) and -nonpreferring (NP) rats: a significantly low level of NPY mRNA in dentate gyrus of the hippocampus and absence of NPY mRNA in the medial habenular nucleus of P rats

Corticotropin-releasing factor gene expression is down-regulated in the central nucleus of the amygdala of alcohol-preferring rats which exhibit high anxiety: a comparison between rat lines selectively bred for high and low alcohol preference

Corticotropin-releasing factor gene expression is down-regulated in the central nucleus of the amygdala of alcohol-preferring rats which exhibit high anxiety: a comparison between rat lines selectively bred for high and low alcohol preference

High-alcohol-drinking rats exhibit persistent freezing responses to discrete cues following Pavlovian fear conditioning

High-alcohol-drinking rats exhibit persistent freezing responses to discrete cues following Pavlovian fear conditioning

Effects of Neuropeptide Y on Sucrose and Ethanol Intake and on Anxiety‐Like Behavior in High Alcohol Drinking (HAD) and Low Alcohol Drinking (LAD) Rats

Background: In a previous study, neuropeptide Y (NPY) administered into the lateral ventricles decreased ethanol intake in alcohol‐preferring (P) rats but not in alcohol‐nonpreferring (NP) or unselected Wistar rats. The purpose of the present investigation is to extend these findings in selectively‐bred high‐alcohol‐drinking (HAD)1 and low‐alcohol‐drinking (LAD)1 rats by examining the effects of intracerebroventricularly administered NPY on the elevated plus maze test of anxiety and on ethanol and sucrose intake.Methods: Female HAD1 and LAD1 rats were surgically implanted with cannula into the lateral ventricle. Following recovery, a test of anxiety was conducted in which the rats (n= 12–13/group) received either artificial cerebrospinal fluid (aCSF) or NPY (10 μg) 10 min prior to a 5‐min test on an elevated plus maze. Following anxiety testing, 11 HAD and 11 LAD rats were trained to self‐administer ethanol (8% w/v), and 5 HAD and 8 LAD rats were trained to self‐administer sucrose (2.5%) during daily 2‐hr sessions. A within‐subject design was used in which the rats were pretreated once a week with aCSF, 5 μg NPY, or 10 μg NPY prior to the drinking sessions.Results: HAD and LAD rats treated with aCSF did not differ in time spent in open arms of the plus maze. NPY increased time spent on the open arms to similar degrees in both rat lines. HAD rats consumed more ethanol and sucrose than LAD rats. NPY increased sucrose intake in both rat lines. However, the same doses of NPY reduced ethanol intake in HAD but not in LAD rats.Conclusion: The plus maze results indicated that selective breeding for high and low alcohol preference in the HAD1 and LAD1 rats, respectively, did not yield differences in anxiety‐like behavior and in response to the anxiolytic effects of NPY. The increases in sucrose intake were consistent with the known orexigenic effects of NPY. The decreased ethanol intake following NPY administration in HAD rats was similar to previous observations with P rats and is consistent with the hypothesis that ethanol intake and NPY activity may be inversely related.

Ethanol effects on local cerebral glucose utilization in high-alcohol-drinking and low-alcohol-drinking rats

Ethanol effects on local cerebral glucose utilization in high-alcohol-drinking and low-alcohol-drinking rats

Quantitative Autoradiography of Mu‐Opioid Receptors in the CNS of High–Alcohol‐Drinking (HAD) and Low–Alcohol‐Drinking (LAD) Rats

The binding of [3H]DAMGO to mu-opioid sites was measured in the CNS of selectively bred high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) rats to test the hypothesis that high alcohol preference is associated with higher densities of mu-opioid receptors. Adult, alcohol-naïve male HAD and LAD rats from replicate line 1 were decapitated and their brains frozen in isopentane. Brain sections were incubated with 5 nM [3H]DAMGO, and nonspecific binding was determined in the presence of unlabeled DAMGO. Films were exposed for 60 days, then analyzed using quantitative autoradiography. The densities of [3H]DAMGO binding sites were measured within subregions of neocortex, limbic system, basal ganglia, diencephalon, and brainstem. LAD rats had significantly higher [3H]DAMGO binding (10-30%) than HAD rats within the anterior dorsal hippocampus (CA2), posterior hippocampus (dorsal CA1, and ventral CA1, CA3, and dentate gyrus), thalamus (medial dorsal, lateral, medial dorsal, central, ventral lateral, ventral medial, and ventral medial geniculate nuclei), habenula, and amygdala. No significant interline differences were found in the prefrontal, cingulate, frontal, parietal, temporal, occipital or entorhinal cortices, olfactory tubercle, nucleus accumbens, lateral septum, ventral tegmental area, hypothalamus, caudate-putamen, substantia nigra, claustrum, central gray, or superior colliculus. The present findings with the HAD and LAD lines do not support the hypothesis that high alcohol preference is associated with higher densities of CNS mu-opioid receptors. Instead, the present results, in combination with previously published findings, suggest that the mu-opioid system may play a complex role in regulating high-alcohol-drinking behavior.

A Characterization of Approach and Avoidance Learning in High-Alcohol-Drinking (HAD) and Low-Alcohol-Drinking (LAD) Rats

This study was undertaken as one of a series of experiments designed to examine basic behavioral characteristics present in rats bred specifically for alcohol preference. The basic premise for these experiments has been the idea that alcohol-preferring and -nonpreferring rats may differ in basic activation and inhibition control mechanisms that govern behavior and that different lines of alcohol-preferring rats may demonstrate differential deficits in behavioral activation and behavioral inhibition tendencies. In the present experiment, conditioned approach and avoidance behaviors were studied in alcohol-naïve high-alcohol-drinking (HAD), low-alcohol-drinking (LAD), and N/NIH rats to evaluate behavioral activation in this line of rats. High alcohol drinking (HAD1), low alcohol drinking (LAD1), and N/Nih stock rats were trained to press a response bar during a tone signal to avoid a mild foot shock or receive a food reward. In addition, HAD2 and LAD2 rats, independently-bred replicate lines of the HAD1/LAD1 rats, were trained on the avoidance task. Although the HAD1 rats easily learned the appetitive version of the bar-pressing task, they did not learn the avoidance response. The LAD1 and N/Nih rats learned both the approach and the avoidance tasks normally. Similar to HAD1 rats, the HAD2 rats did not learn the avoidance response whereas LAD2 rats showed significant avoidance performance levels. The present data demonstrated that both HAD1 and HAD2 rats had a rather specific behavioral activation deficit: although they easily learned to press a bar to receive food reinforcement, they did not learn to press the bar to avoid a foot shock. We speculate that this failure to learn the avoidance response may be related to heightened anxiety in the HAD rats and that this excessive anxiety may lead to the development of high levels of alcohol consumption in these selectively bred rats.