Differential effects of intra-ventral tegmental area ghrelin and glucagon-like peptide-1 on the stimulatory action of D-amphetamine and cocaine-induced ethanol intake in male Sprague Dawley rats.

In order to further elucidate the role of mesolimbic peptides in the expression of ethanol reward, the present study investigated the effects of ghrelin and glucagon-like peptide-1 (GLP-1) on ethanol intake, in addition to ethanol intake stimulated by systemic d-amphetamine or cocaine treatment. While a number of studies suggest that ghrelin plays an important role in mesolimbic reward, emerging data now indicate that GLP-1 receptor mechanisms inhibit reward signaling, possibly by directly or indirectly inhibiting ghrelinergic activity within the mesolimbic system. In the present study all rats were initially habituated to a 6% ethanol solution. We then demonstrated that intraperitoneal injections of d-amphetamine and cocaine increased ethanol intake compared to the vehicle condition. In subsequent testing we examined the effects of ventral tegmental area (VTA) ghrelin or vehicle paired with a fixed dose of d-amphetamine or vehicle. In separate rats we then investigated the impact of the GLP-1 agonist exendin-4 (Ex-4), injected into the VTA, on ethanol intake alone, or when Ex-4 was co-administered with d-amphetamine or cocaine. Our results indicated that VTA ghrelin significantly increased ethanol intake, and most importantly, potentiated the effect of d-amphetamine and cocaine on ethanol consumption. Conversely, VTA Ex-4 inhibited ethanol intake and antagonized the stimulatory effect of d-amphetamine and cocaine on ethanol consumption. In a final study we further demonstrated that VTA Ex-4 treatment significantly inhibited the combined stimulatory effects of ghrelin paired with d-amphetamine or ghrelin paired with cocaine. Overall our findings are consistent with a critical role for both ghrelin and GLP-1 receptor mechanisms in mesolimbic ethanol reward circuitry. Moreover, our results further suggest that ghrelin and GLP-1 modulate the stimulatory effect of psychostimulants on ethanol intake.

Adjuvant use of melatonin for pain management in dysmenorrhea - a randomized double-blinded, placebo-controlled trial.

PURPOSE: Dysmenorrhea is a common, recurring, painful condition with a global prevalence of 71%. The treatment regime for dysmenorrhea includes hormonal therapies and NSAID, both of which are associated with side effects. A dose of 10 mg melatonin daily has previously been shown to reduce the level of pelvic pain in women with endometriosis. We chose to investigate how this regime, administered during the week of menstruation, would affect women with dysmenorrhea but without any signs of endometriosis, as adjuvant analgesic treatment. METHODS: Forty participants with severe dysmenorrhea were randomized to either melatonin or placebo, 20 in each group. Our primary outcome was pain measured with numeric rating scale (NRS); a difference of at least 1.3 units between the groups was considered clinically significant. Secondary outcomes were use of analgesics, as well as absenteeism and amount of bleeding. Mixed model was used for statistical analysis. RESULTS: Eighteen participants completed the study in the placebo group and 19 in the melatonin group. Mean NRS in the placebo group was 2.45 and 3.18 in the melatonin group, which proved to be statistically, although not clinically significant. CONCLUSION: This randomized, double-blinded, placebo-controlled trial could not show that 10 mg of melatonin given orally at bedtime during the menstrual week had better analgesic effect on dysmenorrhea as compared with placebo. However, no adverse effects were observed. CLINICAL TRIALS: NCT03782740 registered on 17 December 2018.

Comparing behavior following binge ethanol in adolescent and adult DBA/2 J mice.

The adolescent brain undergoes maturation in areas critically involved in reward, addiction, and memory. Adolescents consume alcohol more than any other drug, typically in a binge-like manner. While adults also binge on alcohol, the adolescent brain is more susceptible to ethanol-related damages due to its ongoing development, which may result in persistent behavioral and physical changes, including differences in myelination in the frontal cortex. Sex also impacts ethanol metabolism and addiction progression, suggesting females are more sensitive than males. This study addressed memory, sociability, ethanol sensitivity, and myelin gene expression changes due to binge ethanol, sex, and age. DBA/2 J males and females were exposed to intermittent binge ethanol (4 g/kg, i.g.) from postnatal day (PND) 29-42 or as adults from PND 64-77. Age groups were tested for behaviors at the early phase (24 h - 7 days) and late phase (starting 3 weeks) after the last dose. Adult prefrontal cortex was collected at both phases. Adolescent ethanol impaired late phase memory while adult ethanol showed no impairment. Meanwhile, adolescent males showed early phase tolerance to ethanol-induced locomotor activation, while adult females showed tolerance at both phases. Adult-treated mice displayed reductions in social interaction. Adult ethanol decreased Mal expression, a gene involved in myelin integrity, at the early phase. No differences in myelin gene expression were observed at the late phase. Thus, adolescent binge ethanol more severely impacts memory and myelin gene expression compared to adult exposure, while adult mice display ethanol-induced reductions in social interaction and tolerance to ethanol's locomotor activation.

The interactions of alcohol and cocaine regulate the expression of genes involved in the GABAergic, glutamatergic and endocannabinoid systems of male and female rats.

Although the pharmacological and behavioural interactions between cocaine and alcohol are well established, less is known about how polyconsumption of these drugs affects the neurotransmitter systems involved in their psychoactive effects and in particular, in the process of addiction. Here, rats of both sexes at two stages of development were studied under a chronic regime of intravenous cocaine and/or alcohol administration. Brain samples from the medial prefrontal cortex, nucleus accumbens, hippocampus and amygdala were extracted to analyse the mRNA expression of genes encoding subunits of the GABA, NMDA and AMPA receptors, as well as the expression of the CB1 receptor, and that of enzymes related to the biosynthesis and degradation of endocannabinoids. Moreover, two synaptic scaffold proteins related to GABA and NMDA receptors, gephyrin and PSD-95, were quantified in Western blots. Significant interactions between cocaine and alcohol were common, affecting the GABAergic and endocannabinoid systems in the medial prefrontal cortex and amygdala of young adults, whereas such interactions were evident in the glutamatergic and endocannabinoid systems in adults, as well as a more pronounced sex effect. Significant interactions between these drugs affecting the scaffold proteins were evident in the medial prefrontal cortex and nucleus accumbens of young adults, and in the nucleus accumbens and amygdala of adults, but not in the hippocampus. These results highlight the importance of considering the interactions between cocaine and alcohol on neurotransmitter systems in the context of polyconsumption, specifically when treating problems of abuse of these two substances.

Independent of differences in taste, B6N mice consume less alcohol than genetically similar B6J mice, and exhibit opposite polarity modulation of tonic GABAAR currents by alcohol.

Genetic differences in cerebellar sensitivity to alcohol (EtOH) influence EtOH consumption phenotype in animal models and contribute to risk for developing an alcohol use disorder in humans. We previously determined that EtOH enhances cerebellar granule cell (GC) tonic GABAAR currents in low EtOH consuming rodent genotypes, but suppresses it in high EtOH consuming rodent genotypes. Moreover, pharmacologically counteracting EtOH suppression of GC tonic GABAAR currents reduces EtOH consumption in high alcohol consuming C57BL/6J (B6J) mice, suggesting a causative role. In the low EtOH consuming rodent models tested to date, EtOH enhancement of GC tonic GABAAR currents is mediated by inhibition of neuronal nitric oxide synthase (nNOS) which drives increased vesicular GABA release onto GCs and a consequent enhancement of tonic GABAAR currents. Consequently, genetic variation in nNOS expression across rodent genotypes is a key determinant of whether EtOH enhances or suppresses tonic GABAAR currents, and thus EtOH consumption. We used behavioral, electrophysiological, and immunocytochemical techniques to further explore the relationship between EtOH consumption and GC GABAAR current responses in C57BL/6N (B6N) mice. B6N mice consume significantly less EtOH and achieve significantly lower blood EtOH concentrations than B6J mice, an outcome not mediated by differences in taste. In voltage-clamped GCs, EtOH enhanced the GC tonic current in B6N mice but suppressed it in B6J mice. Immunohistochemical and electrophysiological studies revealed significantly higher nNOS expression and function in the GC layer of B6N mice compared to B6Js. Collectively, our data demonstrate that despite being genetically similar, B6N mice consume significantly less EtOH than B6J mice, a behavioral difference paralleled by increased cerebellar nNOS expression and opposite EtOH action on GC tonic GABAAR currents in each genotype.

Effect of enterally administered sleep-promoting medication on the intravenous sedative dose and its safety and cost profile in mechanically ventilated patients: A retrospective cohort study.

BACKGROUND: The clinical effect of enteral administration of sleep-promoting medication (SPM) in mechanically ventilated patients remains unclear. This study aimed to investigate the relationship between enteral SPM administration and the intravenous sedative dose and examine the safety and cost of enteral SPM administration. METHODS: This single-center retrospective cohort study was conducted in a Japanese tertiary hospital intensive care unit (ICU). The exposure was enteral SPM administration during mechanical ventilation. The outcome was the average daily propofol dose per body weight administered as a continuous sedative during mechanical ventilation. Patients were divided into three groups based on the timing of SPM administration at ICU admission: "administration within 48 hours (early administration [EA])," "administration after 48 hours (late administration [LA])," and "no administration (NA)." We used multiple linear regression models. RESULTS: Of 123 included patients, 37, 50, and 36 patients were assigned to the EA, LA, and NA groups, respectively. The average daily propofol dose per body weight was significantly lower in the EA group than in the LA and NA groups (ß -5.13 [95% confidence interval (CI) -8.93 to -1.33] and ß -4.51 [95% CI -8.59 to -0.43], respectively). Regarding safety, enteral SPM administration did not increase adverse events, including self-extubation. The total cost of neuroactive drugs tended to be lower in the EA group than in the LA and NA groups. CONCLUSIONS: Early enteral SPM administration reduced the average daily propofol dose per body weight without increasing adverse events.

Persistence of cerebellar ataxia during chronic ethanol exposure is associated with epigenetic up-regulation of Fmr1 gene expression in rat cerebellum.

BACKGROUND: Alcohol intoxication produces ataxia by affecting the cerebellum, which coordinates movements. Fragile X mental retardation (FMR) protein is a complex regulator of RNA and synaptic plasticity implicated in fragile X-associated tremor/ataxia syndrome, which features ataxia and increased Fmr1 mRNA expression resulting from epigenetic dysregulation of FMRP. We recently demonstrated that acute ethanol-induced ataxia is associated with increased cerebellar Fmr1 gene expression via histone modifications in rats, but it is unknown whether similar behavioral and molecular changes occur following chronic ethanol exposure. Here, we investigated the effects of chronic ethanol exposure on ataxia and epigenetically regulated changes in Fmr1 expression in the cerebellum. METHODS: Male adult Sprague-Dawley rats were trained on the accelerating rotarod and then fed with chronic ethanol or a control Lieber-DeCarli diet while undergoing periodic behavioral testing for ataxia during ethanol exposure and withdrawal. Cerebellar tissues were analyzed for expression of the Fmr1 gene and its targets using a real-time quantitative polymerase chain reaction assay. The epigenetic regulation of Fmr1 was also investigated using a chromatin immunoprecipitation assay. RESULTS: Ataxic behavior measured by the accelerating rotarod behavioral test developed during chronic ethanol treatment and persisted at both the 8-h and 24-h withdrawal time points compared to control diet-fed rats. In addition, chronic ethanol treatment resulted in up-regulated expression of Fmr1 mRNA and increased activating epigenetic marks H3K27 acetylation and H3K4 trimethylation at 2 sites within the Fmr1 promoter. Finally, measurement of the expression of relevant FMRP mRNA targets in the cerebellum showed that chronic ethanol up-regulated cAMP response element binding (CREB) Creb1, Psd95, Grm5, and Grin2b mRNA expression without altering Grin2a, Eaa1, or histone acetyltransferases CREB binding protein (Cbp) or p300 mRNA transcripts. CONCLUSIONS: These results suggest that epigenetic regulation of Fmr1 and subsequent FMRP regulation of target mRNA transcripts constitute neuroadaptations in the cerebellum that may underlie the persistence of ataxic behavior during chronic ethanol exposure and withdrawal.

Adolescent exposure to delta-9-tetrahydrocannabinol and ethanol heightens sensitivity to fear stimuli.

Cannabis use disorder (CUD) has doubled in prevalence over the past decade as a nation-wide trend toward legalization allows for increased drug accessibility. As a result, marijuana has become the most commonly used illicit drug in the United States particularly among the adolescent population. This is especially concerning since there is greater risk for the harmful side effects of drug use during this developmental period due to ongoing brain maturation. Increasing evidence indicates that CUD often occurs along with other debilitating conditions including both alcohol use disorder (AUD) and anxiety disorders such post-traumatic stress disorder (PTSD). Additionally, exposure to cannabis, alcohol, and stress can induce alterations in glutamate regulation and homeostasis in the prefrontal cortex (PFC) that may lead to impairments in neuronal functioning and cognition. Therefore, in order to study the relationship between drug exposure and the development of PTSD, these studies utilized rodent models to determine the impact of adolescent exposure to delta-9-tetrahydrocannabinol (THC) and ethanol on responses to fear stimuli during fear conditioning and used calcium imaging to measure glutamate activity in the prelimbic cortex during this behavioral paradigm. The results from these experiments indicate that adolescent exposure to THC and ethanol leads to enhanced sensitivity to fear stimuli both behaviorally and neuronally. Additionally, these effects were attenuated when animals were treated with the glutamatergic modulator N-acetylcysteine (NAC). In summary, these studies support the hypothesis that adolescent exposure to THC and ethanol leads to alterations in fear stimuli processing through glutamatergic reliant modifications in PFC signaling.

Binge ethanol drinking associated with sex-dependent plasticity of neurons in the insula that project to the bed nucleus of the stria terminalis.

Modifications in brain regions that govern reward-seeking are thought to contribute to persistent behaviors that are heavily associated with alcohol-use disorder (AUD) including binge ethanol drinking. The bed nucleus of the stria terminalis (BNST) is a critical node linked to both alcohol consumption and the onset, maintenance and progression of adaptive anxiety and stress-related disorders. Differences in anatomy, connectivity and receptor subpopulations, make the BNST a sexually dimorphic region. Previous work indicates that the ventral BNST (vBNST) receives input from the insular cortex (IC), a brain region involved in processing the body's internal state. This IC-vBNST projection has also been implicated in emotional and reward-seeking processes. Therefore, we examined the functional properties of vBNST-projecting, IC neurons in male and female mice that have undergone short-term ethanol exposure and abstinence using a voluntary Drinking in the Dark paradigm (DID) paired with whole-cell slice electrophysiology. First we show that IC neurons projected predominantly to the vBNST. Next, our data show that short-term ethanol exposure and abstinence enhanced excitatory synaptic strength onto vBNST-projecting, IC neurons in both sexes. However, we observed diametrically opposing modifications in excitability across sexes. In particular, short-term ethanol exposure resulted in increased intrinsic excitability of vBNST-projecting, IC neurons in females but not in males. Furthermore, in females, abstinence decreased the excitability of these same neurons. Taken together these findings show that short-term ethanol exposure, as well as the abstinence cause sex-related adaptations in BNST-projecting, IC neurons.

Lateral hypothalamus-projecting noradrenergic locus coeruleus pathway modulates binge-like ethanol drinking in male and female TH-ires-cre mice.

A growing body of literature implicates noradrenergic (NE) signaling in the modulation of ethanol consumption. However, relatively few studies have detailed specific brain pathways that mediate NE-associated binge-like ethanol consumption. To begin to fill this gap in the literature, male and female C57BL6/J and TH-ires-cre mice underwent pharmacological and chemogenetic testing, respectively, in combination with "drinking in the dark" procedures to model binge-like consumption of ethanol or sucrose solutions. First, we showed that intraperitoneal administration of the NE reuptake inhibitor, reboxetine, blunted binge-like ethanol intake in C57BL6/J mice. Chemogenetic activation of locus coeruleus (LC) tyrosine hydroxylase (TH)-expressing neurons blunted binge-like ethanol intake regardless of sex. Chemogenetic activation of LC projections to the lateral hypothalamus (LH), a region implicated in ethanol consumption, blunted binge-like ethanol drinking without altering sucrose intake in ethanol-experienced or ethanol-naïve mice. In C57BL/6 J mice, LH-targeted microinfusion of an α1-adrenergic receptor (AR) agonist blunted binge-like ethanol intake across both sexes, while LH infusion of a ß-AR agonist blunted binge-like ethanol intake in females exclusively. Finally, in mice with high baseline ethanol intake both an α1- AR agonist and an α-2 AR antagonist blunted binge-like ethanol intake. The present results provide novel evidence that increased NE tone in a circuit arising from the LC and projecting to the LH reduces binge-like ethanol drinking in mice, and may represent a novel approach to treating binge or heavy drinking prior to the development of dependence. This article is part of the special Issue on "Neurocircuitry Modulating Drug and Alcohol Abuse".

Chronic Low-Dose Alcohol Consumption Attenuates Post-Ischemic Inflammation via PPARγ in Mice.

Ischemic stroke is one of the leading causes of death and permanent disability in adults. Recently, we found that light alcohol consumption (LAC) suppresses post-ischemic inflammatory response, which plays an important role in ischemic brain damage. Our goal was to determine the role of peroxisome proliferator-activated receptor-gamma (PPARγ) in the anti-inflammatory effect of LAC against transient focal cerebral ischemia. In in vivo study, male C57BL/6J wild type (WT) and endothelial-specific conditional PPARγ knockout mice were gavage fed with 0.7 g/kg/day ethanol or volume-matched water daily for 8 weeks. From the 7th week, 3 mg/kg/day GW9662 (a selective PPARγ antagonist) was intraperitoneally given for two weeks. Cerebral ischemia/reperfusion (I/R) injury and expression of manganese superoxide dismutase (MnSOD) and adhesion molecules, neutrophil infiltration, and microglial activation in the cerebral cortex before and following a 90 min unilateral middle cerebral artery occlusion (MCAO)/24 h reperfusion were evaluated. In in vitro study, the impact of chronic alcohol exposure on expression of PPARγ and MnSOD in C57BL/6J mouse brain microvascular endothelial cells (MBMVECs) was measured. PPARγ and MnSOD were significantly upregulated in the cerebral cortex of ethanol-fed WT mice and low-concentration ethanol-exposed C57BL/6J MBMVECs. GW9662 significantly inhibited alcohol-induced upregulation of MnSOD. Eight-week ethanol feeding significantly reduced cerebral I/R injury and alleviated the post-ischemic inflammatory response (upregulation of intercellular adhesion molecule-1 (ICAM-1) and E-selectin, microglial activation, and neutrophil infiltration). Treatment with GW9662 and endothelial-specific conditional knockout of PPARγ did not alter cerebral I/R injury and the inflammatory response in the control mice but abolish the neuroprotective effect in ethanol-fed mice. In addition, GW9662 and endothelial-specific conditional knockout of PPARγ diminished the inhibitory effect of LAC on the post-ischemic expression of adhesion molecules and neutrophil infiltration. Our findings suggest that LAC may protect against cerebral I/R injury by suppressing the post-ischemic inflammation via activation of PPARγ.

Adjuvant use of melatonin for relieving symptoms of painful diabetic neuropathy: results of a randomized, double-blinded, controlled trial.

PURPOSE: The trial aimed to investigate the effectiveness of exogenous melatonin as an adjuvant to pregabalin for relief of pain in patients suffering from painful diabetic neuropathy (PDN). PATIENTS AND METHODS: This randomized, double-blind, placebo-controlled trial was carried out between October 2019 and December 2020 in an outpatient specialty clinic in Iran. One-hundred-three type 2 diabetic patients suffering from PDN were randomized into either the melatonin group (n = 52) or the placebo group (n = 51). Besides pregabalin at a dose of 150 mg per day, patients started with melatonin or an identical placebo, at a dose of 3 mg/day at bedtime for 1 week, which was augmented to 6 mg/day for further 7 weeks. The primary outcomes were changes in mean NRS (numerical rating scale) pain score from baseline to endpoint and responder rate (patients with a reduction of 50% and higher in average pain score compared with baseline). Secondary endpoints were changes in mean NRS pain-related sleep-interference score, overall improvement evaluated by Patient and Clinical Global Impressions of Change (PGIC, CGIC), and impact of the intervention on patient's Health-related quality of life (QOL). All analyses were conducted on an Intention-to-Treat (ITT) analysis data set. RESULTS: At the study endpoint, treatment with melatonin resulted in a considerably higher reduction in the mean NRS pain score in comparison with placebo (4.2 ± 1.83 vs. 2.9 ± 1.56; P-value < 0.001). In terms of treatment responders, a greater proportion of melatonin-treated patients satisfied the responder criterion than placebo-treated patients (63.5% vs. 43.1%). Melatonin also reduced pain-related sleep interference scores more than did placebo (3.38 ± 1.49 vs. 2.25 ± 1.26; P-value < 0.001). Further, at the endpoint, more improvement was also seen in terms of PGIC, CGIC, and Health-related QOL in patients treated with melatonin than placebo. Melatonin was also well tolerated. CONCLUSION: The present results showed that melatonin as an adjunct therapy to pregabalin might be helpful for use in patients with PDN. However, confirmation of these results requires further studies.

Resurgence of alcohol seeking following abstinence induced by punishment in male and female rats.

To better approximate the human condition, animal models of relapse to drug and alcohol seeking have increasingly employed negative consequences to generate abstinence. Here we report the first demonstration of relapse to punishment-suppressed alcohol seeking induced by loss of non-drug reward (i.e., resurgence). We also report the first examination of potential sex differences in any form of relapse to alcohol seeking following suppression by punishment. Male and female rats first pressed a lever for 20 % oral alcohol. Next, lever pressing for one group continued to produce alcohol, but also produced occasional footshock. For another group, lever pressing similarly produced alcohol and occasional footshock, and a nose-poke response produced alternative non-drug reward (i.e., food). Males showed similar suppression of alcohol seeking by punishment alone and punishment + alternative non-drug reward, whereas females showed less suppression by punishment alone. Finally, when alternative reinforcement and punishment were suspended, resurgence occurred for both sexes in the group that previously had access to non-drug reward. Exposure to and then removal of punishment alone did not produce relapse for males, but it did for females. These results suggest that loss of alternative non-drug reward can generate relapse to alcohol seeking following abstinence induced by negative consequences. Future research should further examine the role of potential sex differences in sensitivity to punishment and how such differences may contribute to relapse more broadly.

Adult and adolescent C57BL/6J and DBA/2J mice are differentially susceptible to fear learning deficits after acute ethanol or MK-801 treatment.

Ethanol and other drugs of abuse disrupt learning and memory processes, creating problems associated with drug use and addiction. Understanding individual factors that determine susceptibility to drug-induced cognitive deficits, such as genetic background, age, and sex, is important for prevention and treatment. Comparison of adolescent and adult mice of both sexes across inbred mouse strains can reveal age, sex, and genetic contributions to phenotypes. We treated adolescent and adult, male and female, C57BL/6J and DBA/2J inbred mice with ethanol (1 g/kg or 1.5 g/kg) or MK-801 (0.05 mg/kg or 0.1 mg/kg), an NMDA receptor antagonist, prior to fear conditioning training. Contextual and cued fear retention were tested one day and eight or nine days after training. After ethanol exposure, adult C57BL/6J mice experienced greater deficits in contextual learning than adult DBA/2J mice. C57BL/6 J adolescents were less susceptible to ethanol-induced contextual learning disruptions than C57BL/6J adults, and adolescent males of both strains exhibited greater ethanol-induced contextual learning deficits than adolescent females. After MK-801 exposure, adolescent C57BL/6J mice experienced more severe contextual learning deficits than adolescent DBA/2J mice. Both ethanol and MK-801 had greater effects on contextual learning than cued learning. Collectively, we demonstrate that genetic background contributes to contextual and cued learning outcomes after ethanol or MK-801 exposure. Further, we report age-dependent drug sensitivities that are strain-, sex-, and drug-specific, suggesting that age, sex, and genetic background interact to determine contextual and cued learning impairments after ethanol or MK-801 exposure.

High ethanol preference and dissociated memory are co-occurring phenotypes associated with hippocampal GABAAR-δ receptor levels.

Alcohol use disorder (AUD) frequently co-occurs with dissociative disorders and disorders with dissociative symptoms, suggesting a common neurobiological basis. It has been proposed that facilitated information processing under the influence of alcohol, resulting in the formation of dissociated memories, might be an important factor controlling alcohol use. Access to such memories is facilitated under the effect of alcohol, thus further reinforcing alcohol use. To interrogate possible mechanisms associated with these phenotypes, we used a mouse model of dissociative amnesia, combined with a high-alcohol preferring (HAP) model of AUD. Dissociated memory was induced by activation of hippocampal extrasynaptic GABA type A receptor delta subunits (GABAAR-δ), which control tonic inhibition and to which ethanol binds with high affinity. Increased ethanol preference was associated with increased propensity to form dissociated memories dependent on GABAAR-δ in the dorsal hippocampus (DH). Furthermore, the DH level of GABAAR-δ protein, but not mRNA, was increased in HAP mice, and was inversely correlated to the level of miR-365-3p, suggesting an miRNA-mediated post-transcriptional mechanism contributing to elevated GABAAR-δ. The observed changes of DH GABAAR-δ were associated with a severe reduction of excitatory projections stemming from GABAAR-δ-containing pyramidal neurons in the subiculum and terminating in the mammillary body. These results suggest that both molecular and circuit dysfunction involving hippocampal GABAAR-δ receptors might contribute to the co-occurrence of ethanol preference and dissociated information processing.

Altered excitatory transmission in striatal neurons after chronic ethanol consumption in selectively bred crossed high alcohol-preferring mice.

Genetic predisposition to heavy drinking is a risk factor for alcohol misuse. We used selectively bred crossed high alcohol-preferring (cHAP) mice to study sex differences in alcohol drinking and its effect on glutamatergic activity in dorsolateral (DLS) and dorsomedial (DMS) striatum. We performed whole-cell patch-clamp recording in neurons from male and female cHAP mice with 5-week alcohol drinking history and alcohol-naïve controls. In DMS, alcohol-naïve males' neurons displayed lower cell capacitance and higher membrane resistance than females' neurons, both effects reversed by drinking. Conversely, in DLS neurons, drinking history increased capacitance only in males and changed membrane resistance only in females. Altered biophysical membrane properties were accompanied by disrupted glutamatergic transmission. Drinking history increased spontaneous excitatory postsynaptic current (sEPSC) amplitude in DMS and frequency in DLS female neurons, compared to alcohol-naïve females, without effect in males. Acute ethanol differentially impacted DMS and DLS neurons by sex and drinking history. In DMS, acute alcohol significantly increased sEPSC frequency only in neurons from alcohol-naïve females, an effect that disappeared after drinking history. In DLS, acute alcohol had opposing effects in males and females based on drinking history. Estrous cycle also impacted DMS and DLS neurons differently: sEPSC amplitudes were higher in DMS cells from drinking history than alcohol-naïve females, whereas estrous cycle, not drinking history, modified DLS firing rate. Our data show sex differences in cHAP ethanol consumption and neurophysiology, suggesting differential dysregulation of glutamatergic drive onto DMS and DLS after chronic ethanol consumption.

Broad and dynamic neurochemical alterations in the brain of alcoholic rats.

Ethanol is the active ingredient in alcoholic beverages. As ethanol consumption increases from zero to very high, it is still unknown which metabolites are present at different times and which are essential to normal functioning. In this article, we used an intermittent-access 20% ethanol drinking paradigm to make Wistar male rats voluntarily drink large amounts of ethanol for 10, 20, 30, and 50 days, respectively. A hydrogen-1 nuclear magnetic resonance approach was used to investigate the time-dependent neurochemical metabolites spectra in the hippocampus, striatum, nucleus accumbens and prefrontal cortex. Multivariate pattern recognition techniques were used to analyze the hydrogen-1 nuclear magnetic resonance spectra data. Metabolic profiling was obtained, differentiating the ethanol-treated and control rats. The ethanol-affected metabolites disrupted processes associated with neurotransmitters, oxidative stress, energy metabolism and amino acids. Together, our findings demonstrate broad, dynamic, and time-dependent endogenous metabolic alterations in rats treated with ethanol.

Combined nicotine and ethanol age-dependently alter neural and behavioral responses in male rats.

Use of alcohol (EtOH) and nicotine (Nic) typically begins during adolescence. Smoking and drinking often occur together and lead to a higher consumption of alcohol. Although we have shown that Nic+EtOH is reinforcing in self-administration tests in adolescent male rats, whether Nic+EtOH affects other behaviors or neuronal activity in an age-dependent manner is unknown. To address this, adolescent and adult male rats were given intravenous injections of Nic (30 µg/kg)+EtOH (4 mg/kg) and evaluated for locomotor and anxiety-like behaviors. Regional neuronal activity, assessed by cFos mRNA expression, was measured and used to evaluate functional connectivity in limbic regions associated with anxiety and motivation. Nic+EtOH increased locomotor activity and was anxiolytic in adolescents, but not adults. The posterior ventral tegmental area (pVTA), a critical regulator of drug reward, was selectively activated by Nic+EtOH in adults, while activity in its target region, the NAc-shell, was decreased. Drug-induced alterations in functional connectivity were more extensive in adults than adolescents and may act to inhibit behavioral responses to Nic+EtOH that are seen in adolescence. Overall, our findings suggest that brief, low-dose exposure to Nic+EtOH produces marked, age-dependent changes in brain and behavior and that there may be an ongoing maturation of the pVTA during adolescence that allows increased sensitivity to Nic+EtOH's reinforcing, hyperlocomotor, and anxiolytic effects. Furthermore, this work provides a potential mechanism for high rates of co-use of nicotine and alcohol by teenagers: this drug combination is anxiolytic and recruits functional networks that are unique from protective, inhibitory networks recruited in the mature and adult brain.

Sex-dependent and ontogenetic effects of low dose ethanol on social behavioral deficits induced by mouse maternal separation.

Early life stress can induce lifelong emotional and social behavioral deficits that may in some cases be alleviated by drugs or alcohol. A model for early life stress, rodent maternal separation, recapitulates these behavioral sequelae, which are not limited to potentiated anxiety-like behavior, attenuated social motivation, and altered reward-seeking. Here we employed mouse maternal separation with early weaning (MSEW), consisting of pup-dam separation lasting 4-8 hours on postnatal days (PD) 2-16, with early weaning on PD 17. Prior MSEW studies have limited subjects by age or sex, so we more comprehensively investigated MSEW effects in both sexes, during adolescence and adulthood. We found universal effects of MSEW to include lifelong enhancement of anxiety-like and despair behavior, as well as deficits in social motivation. We also observed some sex-dependent effects of MSEW, namely that female MSEW mice exhibited social habituation to a greater degree than their male counterparts. Low dose ethanol administration had no major effects on the social behavior of non-stressed mice. But interestingly, MSEW-induced social habituation was counteracted by low dose ethanol in adolescent female mice, and potentiated in adolescent male mice. These effects were absent in adult animals, suggesting that ethanol may exert differential effects on the developing brain in such a manner to produce age-, sex-, and stress-dependent effects upon social behavior. Together, results indicate that MSEW reliably produces long-lasting impairments in emotional and social behaviors in both sexes and across the lifespan, but may exert more salient social behavioral effects on female animals.

Heritability of ethanol consumption and pharmacokinetics in a genetically diverse panel of collaborative cross mouse strains and their inbred founders.

BACKGROUND: Interindividual variation in voluntary ethanol consumption and ethanol response is partially influenced by genetic variation. Discovery of the genes and allelic variants that affect these phenotypes may clarify the etiology and pathophysiology of problematic alcohol use, including alcohol use disorder. Genetically diverse mouse populations, which demonstrate heritable variation in ethanol consumption, can be utilized to discover the genes and gene networks that influence this trait. The Collaborative Cross (CC) recombinant inbred strains, Diversity Outbred (DO) population and their 8 founder strains are complementary mouse resources that capture substantial genetic diversity and can demonstrate expansive phenotypic variation in heritable traits. These populations may be utilized to discover candidate genes and gene networks that moderate ethanol consumption and other ethanol-related traits. METHODS: We characterized ethanol consumption, preference, and pharmacokinetics in the 8 founder strains and 10 CC strains in 12-hour drinking sessions during the dark phase of the circadian cycle. RESULTS: Ethanol consumption was substantially heritable, both early in ethanol access and over a chronic intermittent access schedule. Ethanol pharmacokinetics were also heritable; however, no association between strain-level ethanol consumption and pharmacokinetics was detected. The PWK/PhJ strain was the highest drinking strain, with consumption substantially exceeding that of the C57BL/6J strain, which is commonly used as a model of "high" or "binge" drinking. Notably, we found strong evidence that sex moderated genetic effects on voluntary ethanol drinking. CONCLUSIONS: Collectively, this research serves as a foundation for expanded genetic study of ethanol consumption in the CC/DO and related populations. Moreover, we identified reference strains with extreme consumption phenotypes that effectively represent polygenic models of excessive ethanol use.