Two-bottle Choice Paradigm Research Articles

Genotypic Differences in the Effects of Menthol on Nicotine Intake and Preference in Mice

Menthol has been shown to exacerbate elements of nicotine addiction in humans and rodents; however, the mechanisms mediating its effects are not fully understood. This study examined the impact of genetic factors in menthol’s effects on oral nicotine consumption by comparing two inbred mouse strains with differing sensitivities to nicotine. C57BL/6J (B6J) mice are nicotine-preferring, while DBA/2J (D2J) mice are not. While the effects of menthol on oral nicotine consumption have been highlighted in B6J mice, it is unknown if they extend to the D2J strain as well. Consequently, adolescent (PND 21) and adult (PND 63), male and female D2J mice were subjected to the nicotine two-bottle choice (2BC) paradigm with orally and systemically administered menthol. Then, we evaluated its impact on nicotine pharmacological responses in conditioned reward and nociception after systemic administration and, lastly, investigated the potential involvement of the TAAR1 gene and α7 nAChRs in menthol’s effects. Menthol failed to enhance oral nicotine consumption in adult and adolescent female and male D2J mice. Moreover, this lack in effect was not due to nicotine concentration, oral aversion to menthol, or basal preference for nicotine. Menthol also failed to augment nicotine reward or enhance nicotine-induced antinociception in D2J mice, demonstrating that genetic background plays a significant role in sensitivity to menthol’s effects on nicotine. Furthermore, TAAR1 or α7 nAChRs did not seem to mediate menthol’s differential effects in D2J mice. These findings support the existence of genotype-specific mechanisms that may contribute to the variable effects of menthol in different populations.

Maternal Separation Alters Ethanol Drinking and Reversal Learning Processes in Adolescent Rats: The Impact of Sex and Glycine Transporter Type 1 (GlyT1) Inhibitor.

Adverse early life experiences are associated with an enhanced risk for mental and physical health problems, including substance abuse. Despite clinical evidence, the mechanisms underlying these relationships are not fully understood. Maternal separation (MS) is a commonly used animal model of early neglect. The aim of the current study is to determine whether the N-methyl-D-aspartate receptor (NMDAR)/glycine sites are involved in vulnerability to alcohol consumption (two-bottle choice paradigm) and reversal learning deficits (Barnes maze task) in adolescent rats subjected to the MS procedure and whether these effects are sex dependent. By using ELISA, we evaluated MS-induced changes in the NMDAR subunits (GluN1, GluN2A, GluN2B) expression, especially in the glycine-binding subunit, GluN1, in the prefrontal cortex (PFC) and ventral striatum (vSTR) of male/female rats. Next, we investigated whether Org 24598, a glycine transporter 1 (GlyT1) inhibitor, was able to modify ethanol drinking in adolescent and adult male/female rats with prior MS experience and reversal learning in the Barnes maze task. Our findings revealed that adolescent MS female rats consumed more alcohol which may be associated with a substantial increase in GluN1 subunit of NMDAR in the PFC and vSTR. Org 24598 decreased ethanol intake in both sexes with a more pronounced decrease in ethanol consumption in adolescent female rats. Furthermore, MS showed deficits in reversal learning in both sexes. Org 24598 ameliorated reversal learning deficits, and this effect was reversed by the NMDAR/glycine site inhibitor, L-701,324. Collectively, our results suggest that NMDAR/glycine sites might be targeted in the treatment of alcohol abuse in adolescents with early MS, especially females.

Role of β‐Arrestin2 as a Regulator of Fluid Homeostasis and Blood Pressure

Low‐renin hypertension is a common subset of hypertensive patients, which is associated with salt sensitivity. Deoxycorticosterone (DOCA)‐salt hypertension, a prototypical model of low‐renin hypertension, requires activation of the angiotensin type‐1 receptor (AT1R) in specific brain areas. The canonical G‐protein‐mediated signaling of the AT1R is known to induce pressor responses while the non‐canonical β‐Arrestin mediated signaling is thought to counterbalance the detrimental effects of canonical signaling. We hypothesized that β‐Arrestin activation contributes to fluid homeostasis and blood pressure (BP) regulation in DOCA‐salt hypertension. Global β‐Arrestin1 (Arrb1) and β‐Arrestin2 (Arrb2) knockout (KO) mice were employed to evaluate drinking behavior and BP. Age‐ and sex‐matched C57BL/6J mice served as controls. Mice were subjected to the two‐bottle choice paradigm, in which the animals were presented with two bottles, one containing water and one containing varying concentrations of NaCl (0.15, 0.30, and 0.45 mol/L). At baseline, Arrb2‐KO mice exhibited a significant increase in saline intake when presented with 0.15M and 0.30M but not 0.45M (WT0.15M=2.4±0.8 and WT0.30M 1.2±0.4 vs Arrb2‐KO0.15M=4±1.7 and Arrb2‐KO0.30M=2.3±1.1 mL/day; p0.15M=0.23 and p0.30M<0.05; n=19 and 24, respectively). This resulted in a saline preference, which means mice preferred saline over water by more than 50% by volume. Under DOCA, Arrb2‐KO, but not Arrb1‐KO, exhibited a significant increase in saline intake when presented with 0.15M and 0.30M, but not 0.45M saline (WT0.15M=10.9±3.6 and WT0.30M 3.8±2 vs Arrb2‐KO0.15M=15.2±6.3 and Arrb2‐KO0.30M=6±2 mL/day; p<0.05; n=19 and 24, respectively) when compared with wildtype. In addition, Arrb2‐KO mice showed a significant increase in fluid intake when presented to 0.15M, 0.30M, and 0.45M saline. Telemetric BP was measured under three conditions: 1) baseline without saline in drinking water 2) baseline with a choice of saline and water, and 3) administration with DOCA with a choice of saline and water. Arrb2‐KO did not exhibit significant differences in BP in absence of DOCA infusion when given water alone or a choice for saline. However, Arrb2‐KO exhibited an increase in the pressor response to DOCA‐salt (WT=152.2±11.9 vs Arrb2‐KO=163.7±4.2 mmHg; p=0.18; n=6). Surprisingly, no alterations in water intake, saline intake, total fluid intake, saline preference, or BP was observed in Arrb1 KO at any given saline concentration under either baseline or DOCA condition. These findings suggest that in low renin hypertension, Arrb2, but not Arrb1, might counterbalance the detrimental canonical signaling of pathological G protein‐coupled receptors including AT1R.

Attenuation of oleoylethanolamide-induced reduction of alcohol consumption in adult rats exposed intermittently to alcohol during adolescence

Oleoylethanolamide (OEA) is an endogenous N-acylethanolamine that reduces both food and alcohol intake through the activation of peripheral sensory nerves in the gut. These effects are opposite to those of anandamide, a main endogenous cannabinoid type 1 receptor (CB1R) agonist. The present study aims to characterize the impact of intermittent and voluntary alcohol intoxications (using the two-bottle choice paradigm) during adolescence on inhibitory actions of OEA and the CB1R antagonist/inverse agonist SR141716A on voluntary alcohol intake in adulthood. In the present study we show that both OEA (5 mg/kg) and SR141716A (3 mg/kg) reduce alcohol drinking in adult rats using a two-bottle choice paradigm. These effects lasted for 24 h and were not additive when both compounds were co-administered. However, when OEA and SR141716A were administered to adult rats with a history of intermittent alcohol exposure during adolescence (from postnatal day 31 to 55), the effects of OEA were attenuated. Moreover, the co-administration of OEA and SR141716A was not as effective as the administration of SR141716A alone. These data suggest that adolescent exposure to alcohol alters the inhibitory actions of OEA on alcohol drinking, which results in the loss of a protective mechanism that might account for the long-term effects of alcohol exposure in the adolescence. The implications for the vulnerability to alcohol addiction is discussed.

Early-Life Sleep Deprivation Enhanced Alcohol Consumption in Adolescent Rats.

Evidence in the literature suggests that sleep deprivation during early-life developmental stages, by impacting important processes such as the reward circuit maturation, may increase the vulnerability for alcohol and substance use. The mechanisms involved are not fully understood. In this study, utilizing our previously established model, we examined the impact of early-life sleep deprivation on alcohol consumption in adolescent rats. Male Sprague Dawley rats served as either the control (CON) or sleep-deprived (SD) group. Sleep deprivation was induced using a Pinnacle automated sleep deprivation apparatus. The SD group of rats was sleep deprived for 6–8 h/day for 14 days from postnatal day (PND)19 to PND32. At PND33, anxiety- and depression-like behaviors were assessed in rats using elevated plus maze and sucrose splash test, respectively. At PND39, alcohol consumption was assessed in rats for five consecutive days using the two-bottle choice paradigm, water versus 5% ethanol. SD rats exhibited significant anxiety- and depression-like behaviors as compared to CON rats. Interestingly, SD rats consumed a larger volume of alcohol when compared to CON rats, which was significantly higher at day 5 (mean of alcohol consumption (ml) ± SD; CON = 6.67 ± 3.42; SD = 19.00 ± 6.05, p = 0.0126). SD rats also showed high preference for alcohol over water, which was significantly higher at day 5 (mean of alcohol preference (%) ± SD; CON = 26.85 ± 14.97; SD = 57.69 ± 5.61, p = 0.014). Our data suggest that early-life sleep deprivation enhanced alcohol consumption in adolescent rats.

The Impact of Sex, Circadian Disruption, and the ClockΔ19/Δ19 Genotype on Alcohol Drinking in Mice.

Shift work is associated with increased alcohol drinking, more so in males than females, and is thought to be a coping mechanism for disrupted sleep cycles. However, little is presently known about the causal influence of circadian rhythm disruptions on sex differences in alcohol consumption. In this study, we disrupted circadian rhythms in female and male mice using both environmental (i.e., shifting diurnal cycles) and genetic (i.e., ClockΔ19/Δ19 mutation) manipulations, and measured changes in alcohol consumption and preference using a two-bottle choice paradigm. Alcohol consumption and preference, as well as food and water consumption, total caloric intake, and weight were assessed in adult female and male ClockΔ19/Δ19 mutant mice or wild-type (WT) litter-mates, housed under a 12-hour:12-hour light:dark (L:D) cycle or a shortened 10-hour:10-hour L:D cycle. Female WT mice (under both light cycles) increased their alcohol consumption and preference over time, a pattern not observed in male WT mice. Compared to WT mice, ClockΔ19/Δ19 mice displayed increased alcohol consumption and preference. Sex differences were not apparent in ClockΔ19/Δ19 mice, with or without shifting diurnal cycles. In conclusion, sex differences in alcohol consumption patterns are evident and increase with prolonged access to alcohol. Disrupting circadian rhythms by mutating the Clock gene greatly increases alcohol consumption and abolishes sex differences present in WT animals.

Sucrose intake and preference by Wistar Han rats are not influenced by sex or food/water deprivation

Anhedonia is the decreased ability to experience pleasure from rewarding or enjoyable activities, a core symptom of depression. The sucrose preference test (SPT), based on a two-bottle choice paradigm, is a widely used behavioural paradigm for the evaluation of anhedonia in rodents. Up to now, different protocols have been reported regarding water/food deprivation and duration of exposure to the water/sucrose solutions. In this work, by comparing six of the most used SPT protocols regarding sucrose preference and total intake, in both male and female Wistar Han rats, we showed (i) food/water deprivation does not significantly impact sucrose intake and preference; (ii) increasing the duration of the test is associated with an increased sucrose preference and (iii) no sex-specific differences in the basal sucrose preference of Wistar Han rats. Our results call for standardization of protocols and suggest a protocol without food/water deprivation and a 12-hour duration (lights out) as more efficacious in the measurement of anhedonia in rodents. This protocol not only reduces the confounding factors of drinking patterns and the stress-inducing food/water deprivation but also is not sensitive to sex-specific differences in the total intake of liquid in Wistar Han rats.

Influence of Age and Genetic Background on Ethanol Intake and Behavioral Response Following Ethanol Consumption and During Abstinence in a Model of Alcohol Abuse.

Genetic background and age at first exposure have been identified as critical variables that contribute to individual vulnerability to drug addiction. Evidence shows that genetic factors may account for 40–70% of the variance in liability to addiction. Alcohol consumption by young people, especially in the form of binge-drinking, is becoming an alarming phenomenon predictive of future problems with drinking. Thus, the literature indicates the need to better understand the influence of age and genetic background on the development of alcohol dependence. To this aim, the inbred rat strains Lewis (LEW, addiction prone) and Fischer 344 (F344, addiction resistant) were used as a model of genetic vulnerability to addiction and compared with the outbred strain Sprague-Dawley (SD) in a two-bottle choice paradigm as a model of alcohol abuse. During a 9-week period, adolescent and adult male rats of the three strains were intermittently exposed to ethanol (20%) and water during three 24-h sessions/week. Adult and adolescent SD and LEW rats escalated their alcohol intake over time reaching at stable levels, while F344 rats did not escalate their intake, regardless of age at drinking onset. Among adolescents, only F344 rats consumed a higher total amount of ethanol than adults, although only SD and LEW rats escalated their intake. Adult LEW rats, albeit having a lower ethanol consumption as compared to SD rats but greater than F344, showed a more compulsive intake, consuming higher amounts of ethanol during the first hour of exposure, reaching a higher degree of ethanol preference when start drinking as adolescents. Behavioral analysis during the first hour of ethanol consumption revealed significant strain differences, among which noticeable the lack of sedative effect in the LEW strain, at variance with F344 and SD strains, and highest indices of withdrawal (most notable jumping) in LEW rats during the first hour of abstinence days. The present results underscore the importance of individual genetic background and early onset of alcohol use in the progression toward abuse and development of alcohol addiction.

Beyond Genes: Inclusion of Alternative Splicing and Alternative Polyadenylation to Assess the Genetic Architecture of Predisposition to Voluntary Alcohol Consumption in Brain of the HXB/BXH Recombinant Inbred Rat Panel

Post transcriptional modifications of RNA are powerful mechanisms by which eukaryotes expand their genetic diversity. For instance, researchers estimate that most transcripts in humans undergo alternative splicing and alternative polyadenylation. These splicing events produce distinct RNA molecules, which in turn yield distinct protein isoforms and/or influence RNA stability, translation, nuclear export, and RNA/protein cellular localization. Due to their pervasiveness and impact, we hypothesized that alternative splicing and alternative polyadenylation in brain can contribute to a predisposition for voluntary alcohol consumption. Using the HXB/BXH recombinant inbred rat panel (a subset of the Hybrid Rat Diversity Panel), we generated over one terabyte of brain RNA sequencing data (total RNA) and identified novel splice variants (via StringTie) and alternative polyadenylation sites (via aptardi) to determine the transcriptional landscape in the brains of these animals. After establishing an analysis pipeline to ascertain high quality transcripts, we quantitated transcripts and integrated genotype data to identify candidate transcript coexpression networks and individual candidate transcripts associated with predisposition to voluntary alcohol consumption in the two-bottle choice paradigm. For genes that were previously associated with this trait (e.g., Lrap, Ift81, and P2rx4) (Saba et al., Febs. J., 282, 3556–3578, Saba et al., Genes. Brain. Behav., 20, e12698), we were able to distinguish between transcript variants to provide further information about the specific isoforms related to the trait. We also identified additional candidate transcripts associated with the trait of voluntary alcohol consumption (i.e., isoforms of Mapkapk5, Aldh1a7, and Map3k7). Consistent with our previous work, our results indicate that transcripts and networks related to inflammation and the immune system in brain can be linked to voluntary alcohol consumption. Overall, we have established a pipeline for including the quantitation of alternative splicing and alternative polyadenylation variants in the transcriptome in the analysis of the relationship between the transcriptome and complex traits.

Role of 5-HT2A receptors in the effects of ayahuasca on ethanol self-administration using a two-bottle choice paradigm in male mice.

Ayahuasca has been proposed as a potential treatment of alcohol (ethanol) use disorder (AUD). The serotonin 5-HT2A receptor agonist N,N-dimethyltryptamine (DMT) is the main psychoactive component of ayahuasca, suggesting that its therapeutic effects may be mediated by 5-HT2A receptors. The aim of the present study was to investigate the effects of ayahuasca on the expression of ethanol self-administration using a two-bottle choice procedure and the role of 5-HT2A receptors in those effects. Male mice had intermittent access to ethanol (10% v/v) in a two-bottle choice procedure for 30days. Animals were then submitted to 3 treatment phases, each followed by ethanol re-exposure tests. During the treatment phase, every 3days, animals received i.p. injections of either vehicle or the 5-HT2A receptor antagonist M100907 (M100, 1mg/kg) followed by an i.g. (gavage) administration of vehicle or ayahuasca (100mg/kg) and were exposed to the self-administration apparatus with no ethanol availability. During re-exposure tests, animals were submitted to the same conditions as during acquisition, with no treatments prior to those sessions. Treatment with ayahuasca blocked the expression of ethanol self-administration, decreasing ethanol intake and preference during re-exposure tests. Pretreatment with M100 blocked the effects of ayahuasca on ethanol drinking without significantly attenuating ethanol self-administration. Treatment with ayahuasca during alcohol abstinence blocked the expression of alcohol self-administration in mice, and 5-HT2A receptor activation is critical for those effects to emerge. Our findings support a potential for ayahuasca and other 5-HT2A receptor agonists as adjunctive pharmacotherapies for the treatment of AUD.

Behavioral characterization of withdrawal following chronic voluntary ethanol consumption via intermittent two-bottle choice points to different susceptibility categories.

Alcohol is among the most commonly abused drugs worldwide. Cessation of chronic alcohol consumption can result in the appearance of withdrawal symptoms that commonly promote relapse in individuals with alcohol use disorder (AUD). Thus, preclinical models of voluntary alcohol consumption, in which animals manifest spontaneous signs of withdrawal after alcohol cessation, can be useful for studying AUD and its treatment. The intermittent two-bottle choice paradigm (I2BC) has been used extensively to examine alcohol intake in rodents. However, previous studies have reported conflicting observations regarding its potential to result in the spontaneous manifestation of withdrawal upon alcohol cessation. We employed a battery of behavioral tests to examine the emergence of affective and physical signs of withdrawal in female and male mice exposed to alcohol in the I2BC for 10weeks. Specifically, mice of both sexes undergoing 24-h withdrawal from the I2BC were tested for physical signs of withdrawal, anxiety-like behavior in the open field arena (OFA) and elevated plus maze (EPM), and anxiety/compulsive-like behavior in the marble burying test (MBT). The main outcomes from these tests were combined into a behavioral severity score to describe the overall behavioral phenotype. Both female and male mice undergoing withdrawal from the I2BC displayed elevated physical signs of withdrawal and anxiety-associated behavior in the EPM and MBT. Analysis of the overall behavioral severity score revealed more severe phenotypes in female and male mice undergoing withdrawal from the I2BC than controls. Additionally, stratification of the mice based on severity scores demonstrated a differential distribution of severities between the exposure groups. We confirmed that a significant fraction of mice chronically exposed to alcohol in the I2BC display spontaneous withdrawal. In addition, we showed that computing a severity score from a combination of behavioral metrics can be useful in preclinical research to model evaluation tools used in patients with AUD.

Whey-Adapted versus Natural Cow's Milk Formulation: Distinctive Feeding Responses and Post-Ingestive c-Fos Expression in Laboratory Mice.

The natural 20:80 whey:casein ratio in cow’s milk (CM) for adults and infants is adjusted to reflect the 60:40 ratio of human milk, but the feeding and metabolic consequences of this adjustment have been understudied. In adult human subjects, the 60:40 CM differently affects glucose metabolism and hormone release than the 20:80 CM. In laboratory animals, whey-adapted goat’s milk is consumed in larger quantities. It is unknown whether whey enhancement of CM would have similar consequences on appetite and whether it would affect feeding-relevant brain regulatory mechanisms. In this set of studies utilizing laboratory mice, we found that the 60:40 CM was consumed more avidly than the 20:80 control formulation by animals motivated to eat by energy deprivation and by palatability (in the absence of hunger) and that this hyperphagia stemmed from prolongation of the meal. Furthermore, in two-bottle choice paradigms, whey-adapted CM was preferred against the natural 20:80 milk. The intake of the whey-adapted CM induced neuronal activation (assessed through analysis of c-Fos expression in neurons) in brain sites promoting satiation, but importantly, this activation was less pronounced than after ingestion of the natural 20:80 whey:casein CM. Activation of hypothalamic neurons synthesizing anorexigenic neuropeptide oxytocin (OT) was also less robust after the 60:40 CM intake than after the 20:80 CM. Pharmacological blockade of the OT receptor in mice led to an increase in the consumption only of the 20:80 CM, thus, of the milk that induced greater activation of OT neurons. We conclude that the whey-adapted CM is overconsumed compared to the natural 20:80 CM and that this overconsumption is associated with weakened responsiveness of central networks involved in satiety signalling, including OT.

Sex differences in behavioral, cognitive and voluntary ethanol-intake effects in Dexamethasone-induced depression-like state in Wistar rat

The stress response is attached to psychosomatic and psychiatric disorders. Therefore, it is important to comprehend the underlying mechanisms influencing this relationship. Moreover, men and women respond differently to stress–both psychologically and biologically. These differences should be studied to have an enhanced understanding of the gender difference. However, researches shedding light on sex dimorphism implication have historically been insufficient. Based on observations that advocate the inclusion of sex as a biological variable in stress response, the present study was designed to explore sex differences in (i) depressive-like, (ii) anxiety-like behaviors, (iii) cognitive-like performances, and (iv) voluntary ethanol intake (VEI) in Wistar rat submitted to dexamethasone (DEX)-stress simulation. Rats were administered daily with DEX (1.5 mg/kg, s.c., 21 days) or vehicle. Behavior, cognitive, and VEI states of rats were evaluated in the following paradigms: forced swimming test (FST); saccharin preference test (SPT); open field test (OFT); elevated plus-maze test (EPMT); novelty suppressed feeding test (NSFT); spatial learning and memory in Morris water maze test (MWMT); VEI in two-bottle choice paradigm. DEX-treated rats showed a set of depression-like behaviors: increased time of immobility; reduced preference for saccharin consumption; increased anxiety-like behavior; cognitive impairments; and enhanced VEI. Sexual dimorphism was recorded in this study. Females were more impaired in FST, SPT, EPMT, NSFT, and VEI. Results demonstrate that DEX-treatment induced a behavioral alterations related to anxiety-depressive-like state with learning and memory impairments; confirm the facilitatory role of glucocorticoids on VEI and reveal sexual dimorphism in stress response.

Antisense-induced downregulation of major circadian genes modulates the expression of histone deacetylase-2 (HDAC-2) and CREB-binding protein (CBP) in the medial shell region of nucleus accumbens of mice exposed to chronic excessive alcohol consumption.

Circadian genes in the medial accumbal shell (mNAcSh) region regulate binge alcohol consumption. Here, we investigated if antisense-induced knockdown of major circadian genes (Per1, Per2, and NPAS2) in the mNAcSh of mice exposed to intermittent access two-bottle choice (IA2BC) paradigm modulates the expression of histone deacetylase-2 (HDAC-2) and CREB-binding protein (CBP), key epigenetic modifiers associated with withdrawal-associated behaviors such as anxiety. Adult male C57BL/6J mice (N=28), surgically implanted with bilateral guide cannulas above the mNAcSh, were chronically (4weeks) exposed to alcohol (20% v/v) or saccharin (0.03%) via IA2BC paradigm. In the fourth week, a mixture of antisense (AS-ODNs; N=14/group) or nonsense (NS-ODNs; N=14/group) oligodeoxynucleotides against circadian genes were bilaterally infused into the mNAcSh. Subsequently, alcohol/saccharin consumption and preference were measured followed by euthanization of animals and verification of microinjection sites by visual inspection and the expression of HDAC-2 and CBP by using RT-PCR along with the verification of antisense-induced downregulation of circadian genes in the mNAcSh. As compared with NS-ODNs, AS-ODNs infusion significantly attenuated the alcohol-induced increase in HDAC-2 and reduction in CBP expression in the mNAcSh along with a significant reduction in alcohol consumption and preference. No significant effect was observed on either saccharin consumption or preference. Our results suggest that circadian genes in the mNAcSh may have a causal to play in mediating epigenetic changes observed after chronic alcohol consumption.

Ethanol interaction with α3β4 nicotinic acetylcholine receptors in neurons of the laterodorsal tegmentum.

Nicotinic acetylcholine receptors (nAChRs) play a key role in the rewarding effects of ethanol (EtOH), and while several nAChR subtypes have been implicated, attention has recently shifted to a role for the α3β4 nAChR. The laterodorsal tegmental nucleus (LDTg), a brainstem cholinergic nucleus that sends excitatory projections to the ventral tegmental area, is an Integral part of the brain reward pathway. Here we investigate a potential role for LDTg α3β4 nAChRs in EtOH self-administration and reward. Sprague-Dawley rats were given ad libitum access to a 20% EtOH solution, as part of a two-bottle choice paradigm. Approximately 1week after removal of EtOH access, we measured LDTg α3β4 nAChR current responses to focal application of acetylcholine (ACh), using whole-cell patch clamp electrophysiology recordings in acute brain slices. In addition, we used whole-cell electrophysiology to assess the acute effects of EtOH on the sensitivity of LDTg α3β4 nAChRs. Focal application of ACh onto LDTg neurons resulted in large α3β4 nAChR-mediated inward currents, the magnitude of which showed a positive correlation with levels of EtOH self-administration. In addition, using brain slices taken from EtOH-naïve rats, bath application of EtOH resulted in a moderate potentiation of LDTg α3β4 nAChR sensitivity. Using a rat model, increased α3β4 nAChR function was associated with greater EtOH self-administration, with α3β4 nAChR function also acutely potentiated by EtOH. Assuming that similar findings apply to humans, the α3β4 nAChR could be a therapeutic target in the treatment of EtOH use disorder.

Antagonism of GluK1-containing kainate receptors reduces ethanol consumption by modulating ethanol reward and withdrawal

Alcohol use disorder (AUD) is a neuropsychiatric condition affecting millions of people worldwide. Topiramate (TPM) is an antiepileptic drug that has been shown to reduce ethanol drinking in humans. However, TPM is associated with a variety of adverse effects due to its interaction with many receptor systems and intracellular pathways. GluK1-containing kainate receptors (GluK1*KARs) are non-selectively inhibited by TPM, and genetic association studies suggest that this receptor system could be targeted to reduce drinking in AUD patients. We examined the efficacy of LY466195, a selective inhibitor of GluK1*KAR, in reducing ethanol consumption in the intermittent two-bottle choice paradigm in mice. The effect of LY466195 on various ethanol-related phenotypes was investigated by quantification of alcohol intake, physical signs of withdrawal, conditioned place preference (CPP) and in vivo microdialysis in the nucleus accumbens. Selective GluK1*KAR inhibition reduced ethanol intake and preference in a dose-dependent manner. LY466195 treatment attenuated the physical manifestations of ethanol withdrawal and influenced the rewarding properties of ethanol. Interestingly, LY466195 injection also normalized changes in dopamine levels in response to acute ethanol in ethanol-dependent mice, but had no effect in ethanol-naïve mice, suggesting ethanol state-dependent effects. The data point to GluK1*KARs as an attractive pharmacological target for the treatment of AUD.

Abstract 43: Non-Canonical AT1R/b-Arrestin2 Activation In Brain As A Regulator Of Fluid Homeostasis And Blood Pressure

The brain renin angiotensin system (RAS) is known for its role in cardiovascular and metabolic regulation. Angiotensin II (Ang II) is the major active product of the RAS, exerting most of its physiological effects through the angiotensin type-1 receptor (AT 1 R). Canonical or G-protein-mediated signaling of the AT 1 R within the brain has long been known to induce a dipsogenic and pressor response upon Ang II stimulation. Non-canonical or β-Arrestin mediated signaling is thought to counterbalance the detrimental effects of canonical signaling. However, the non-canonical AT 1 R/β-Arrestin pathway within the brain is understudied. Therefore, it is hypothesized that β-Arrestin activation contributes to fluid homeostasis and blood pressure (BP) regulation. Global β-Arrestin1 ( Arrb 1) and β-Arrestin2 ( Arrb 2) knockout (KO) mice were employed to evaluate drinking behavior and BP with and without deoxycorticosterone acetate (DOCA). Age- and sex-matched C57BL/6J mice served as controls. Mice were subjected to the two-bottle choice paradigm, in which the animals were presented with two bottles, one containing water and one containing 0.15M saline. In the absence of DOCA, mice lacking β-Arrestin2 had increased saline intake when compared to β-Arrestin1-KO and wildtype (WT=2.2±0.2 and Arrb 1-KO=2±0.4 vs Arrb 2-KO=5±0.7 mL/day; p<0.001; n=13, 11 and 9, respectively). This resulted in a saline preference, which means mice preferred saline over water by more than 50% by volume. In the presence of DOCA, mice lacking β-Arrestin2 had increased saline intake when compared to β-Arrestin1-KO and wildtype (WT=10.6±1.2 and Arrb 1-KO=6.5±0.8 vs Arrb 2-KO=16.6±2 mL/day; p<0.001; n=13, 11 and 9, respectively). However, these mice did not develop a saline preference. Preliminarily, β-Arrestin2-KO mice exhibited higher BP when compared to WT at baseline (WT=108±5 vs Arrb 2-KO=124±6 mmHg; n=2), which was exacerbated in response to DOCA (WT=122±6 vs Arrb 2-KO=140±5 mmHg; n=2). These findings suggest that β-Arrestin2 might counterbalance effects of canonical activation of the AT 1 R through G proteins. Overall, β-Arrestin2 appears to protect against cardiovascular diseases since the genetic ablation of β-Arrestin2 resulted in an increase in saline intake and exacerbated BP.

Expression of the hippocampal PTCH during early abstinence is associated with drinking patterns in a rat model of voluntary alcohol intake.

Perinatal alcohol exposure induces fetal alcohol syndrome partially through Sonic Hedgehog (SHH) impairment; however, the relationship between SHH signaling cascade and alcohol drinking pattern in adulthood remains obscure. We studied the expression of SHH and components of respective signaling cascade [PTCH receptor (Patched), SMO co-receptor (Smoothened) and downstream transcriptional factor Glioma-associated oncogene (GLI)] during early abstinence in brain regions of rats demonstrating different drinking patterns in intermittent access two-bottle choice paradigm (IA2BC). Male Wistar rats were subjected to twenty 24-h sessions of free access to two-bottle choice (water or 20% ethanol) with 24-h withdrawal periods (water only). Control animals had access to water only. Quantitative PCR and western blotting were used to assess transcript and protein levels in the brain, respectively. During the course of the IA2BC, one part of animals demonstrated gradual escalation from low to high alcohol intake and preference of alcohol over water (group I), while the other one consumed alcohol at stable high level (group II) (Peregud et al., 2021). Three days after the last drinking session, PTCH mRNA elevated in the hippocampus of group I rats as compared to the control group. However, SHH, SMO and GLI mRNA levels in the hippocampus did not change. The protein content of PTCH in the hippocampus of group I rats was higher as compared to both control and group II. PTCH elevation is a known marker of SHH cascade activity. Thus, activated hippocampal SHH signaling cascade is a hallmark of rats demonstrating gradual escalation of alcohol intake in the IA2BC procedure.

CB1 receptor neutral antagonist treatment epigenetically increases neuropeptide Y expression and decreases alcohol drinking

Alcohol consumption is mediated by several important neuromodulatory systems, including the endocannabinoid and neuropeptide Y (NPY) systems in the limbic brain circuitry. However, molecular mechanisms through which cannabinoid-1 (CB1) receptors regulate alcohol consumption are still unclear. Here, we investigated the role of the CB1 receptor-mediated downstream regulation of NPY via epigenetic mechanisms in the amygdala. Alcohol drinking behavior was measured in adult male C57BL/6J mice treated with a CB1 receptor neutral antagonist AM4113 using a two-bottle choice paradigm while anxiety-like behavior was assessed in the light-dark box (LDB) test. The CB1 receptor-mediated changes in the protein levels of phosphorylated cAMP-responsive element binding protein (pCREB), CREB binding protein (CBP), H3K9ac, H3K14ac and NPY, and the mRNA levels of Creb1, Cbp, and Npy were measured in amygdaloid brain structures. Npy-specific changes in the levels of acetylated histone (H3K9/14ac) and CBP in the amygdala were also measured. We found that the pharmacological blockade of CB1 receptors with AM4113 reduced alcohol consumption and, in an ethanol-naïve cohort, reduced anxiety-like behavior in the LDB test. Treatment with AM4113 also increased the mRNA levels of Creb1 and Cbp in the amygdala as well as the protein levels of pCREB, CBP, H3K9ac and H3K14ac in the central and medial nucleus of amygdala, but not in the basolateral amygdala. Additionally, AM4113 treatment increased occupancy of CBP and H3K9/14ac at the Npy gene promoter, leading to an increase in both mRNA and protein levels of NPY in the amygdala. These novel findings suggest that CB1 receptor-mediated CREB signaling plays an important role in the modulation of NPY function through an epigenetic mechanism and further support the potential use of CB1 receptor neutral antagonists for the treatment of alcohol use disorder.

Assessing Gut‐Brain Interactions on Alcohol Consumption: The Role of Fecal Microbiota Transplantation

Microbial representation in the gastrointestinal tract plays an important role in general health and illness. It is known that direct communication pathways exist between the gut and the brain, allowing for reciprocal influence via the gut-brain axis (GBA). The GBA can also be influenced on either side via interactions with the hypothalamic-pituitary-adrenal (HPA) axis and the immune system. Importantly, alcohol consumption has been correlated with changes in the HPA axis and can alter the levels of pro- and anti-inflammatory cytokines, though, the exact alterations are dependent upon the levels of alcohol administered. Additionally, research shows that alcohol consumption is correlated with dysbiosis of the gut microbiome, a condition that may be corrected via fecal microbiota transplantation (FMT). Therefore, this study was designed to assess the interconnected neurobiological impacts of FMT following a moderate alcohol consumption paradigm (drinking-in-the-dark; DID) on corticotropin releasing factor (CRF) and microglial reactivity. Adult male, Sprague-Dawley rats were exposed to alcohol following a repeated DID procedure in order to escalate voluntary consumption. After three rounds of DID, a proton pump inhibitor was provided in a suspension of Vanilla Ensure Plus for voluntary consumption prior to FMT. This was given in order to reduce gastric acid secretion and promote a successful FMT. FMT was administered via oral gavage using fecal samples obtained from either self-donations or those from healthy, non-experimental donor rats. Following FMT, the animals were exposed to a two-bottle choice paradigm with water and a 10% ethanol solution for 14 days to measure alcohol craving. Afterwards, brain tissue was collected, sliced, and stained using immunohistochemical techniques for either CRF as a marker of HPA activation or Iba1 as a marker of microglial reactivity indicative of immune activation. Initial results from a densitometric analysis of CRF expression in the paraventricular nucleus of the hypothalamus (PVN) failed to reveal any significant effects of FMT or alcohol consumption on this aspect of HPA axis activity. Additional quantification of CRF in the central nucleus of the amygdala also failed to demonstrate alterations to CRF levels following treatment. Quantification of Iba1 reactivity in the multiple brain regions and evaluation of the efficacy of FMT is ongoing. Although these initial results were non-significant, the complexity of the potential mechanisms underlying a role for gut health in contributing to excessive alcohol consumption warrants further investigation.