Neuroinflammatory responses and blood-brain barrier injury in chronic alcohol exposure: role of purinergic P2 × 7 Receptor signaling.

Alcohol consumption leads to neuroinflammation and blood‒brain barrier (BBB) damage, resulting in neurological impairment. We previously demonstrated that ethanol-induced disruption of barrier function in human brain endothelial cells was associated with mitochondrial injury, increased ATP and extracellular vesicle (EV) release, and purinergic receptor P2 × 7R activation. Therefore, we aimed to evaluate the effect of P2 × 7R blockade on peripheral and neuro-inflammation in ethanol-exposed mice. In a chronic intermittent ethanol (CIE)-exposed mouse model, P2 × 7R was inhibited by two different methods: Brilliant Blue G (BBG) or gene knockout. We assessed blood ethanol concentration (BEC), brain microvessel gene expression by using RT2 PCR array, plasma P2 × 7R and P-gp, serum ATP, EV-ATP, number of EVs, and EV mtDNA copy numbers. An RT2 PCR array of brain microvessels revealed significant upregulation of proinflammatory genes involved in apoptosis, vasodilation, and platelet activation in CIE-exposed wild-type animals, which were decreased 15-50-fold in BBG-treated-CIE-exposed animals. Plasma P-gp levels and serum P2 × 7R shedding were significantly increased in CIE-exposed animals. Pharmacological or genetic suppression of P2 × 7R decreased receptor shedding to levels equivalent to those in control group. The increase in EV number and EV-ATP content in the CIE-exposed mice was significantly reduced by P2 × 7R inhibition. CIE mice showed augmented EV-mtDNA copy numbers which were reduced in EVs after P2 × 7R inhibition or receptor knockout. These observations suggested that P2 × 7R signaling plays a critical role in ethanol-induced brain injury. Increased extracellular ATP, EV-ATP, EV numbers, and EV-mtDNA copy numbers highlight a new mechanism of brain injury during alcohol exposure via P2 × 7R and biomarkers of such damage. In this study, for the first time, we report the in vivo involvement of P2 × 7R signaling in CIE-induced brain injury.

Embryonic alcohol exposure alters cholinergic neurotransmission and memory in adult zebrafish.

Alcohol is the most consumed addictive substance worldwide that elicits multiple health problems. Consumption of alcoholic beverages by pregnant women is of great concern because pre-natal exposure can trigger fetal alcohol spectrum disorder (FASD). This disorder can significantly change the embryo's normal development, mainly by affecting the central nervous system (CNS), leading to neurobehavioral consequences that persist until adulthood. Among the harmful effects of FASD, the most reported consequences are cognitive and behavioral impairments. Alcohol interferes with multiple pathways in the brain, affecting memory by impairing neurotransmitter systems, increasing the rate of oxidative stress, or even activating neuroinflammation. Here, we aimed to evaluate the deleterious effects of alcohol on the cholinergic signaling and memory in a FASD zebrafish model, using inhibitory avoidance and novel object recognition tests. Four months after the embryonic exposure to ethanol, the behavioral tests indicated that ethanol impairs memory. While both ethanol concentrations tested (0.5 % and 1 %) disrupted memory acquisition in the inhibitory avoidance test, 1 % ethanol impaired memory in the object recognition test. Regarding the cholinergic system, 0.5 % ethanol decreased ChAT and AChE activities, but the relative gene expression did not change. Overall, we demonstrated that FASD model in zebrafish impairs memory in adult individuals, corroborating the memory impairment associated with embryonic exposure to ethanol. In addition, the cholinergic system was also affected, possibly showing a relation with the cognitive impairment observed.

Effects of voluntary chronic intermittent access to ethanol on the behavioral performance in adult C57BL/6 J mice.

BACKGROUND: Chronic alcohol drinking increases the risk of alcohol use disorders, causing various neurological disorders. However, the impact of different ethanol levels on a spectrum of behaviors during chronic drinking remains unclear. In this study, we established an intermittent access to ethanol in a two-bottle choice (IA2BC) procedure to explore the dose-dependent effects of ethanol on the behavioral performance of C57BL/6 J mice. METHODS: Adult male C57BL/6 J mice were provided voluntary access to different ethanol concentrations (0 %, 5 %, 10 %, and 20 % ethanol) under a 12-week IA2BC paradigm. A battery of behavioral tests was administered to assess alterations in pain threshold, anxiety-like behaviors, locomotor activity, motor coordination, and cognition. Ethanol consumption and preference were monitored during each session. Moreover, the liver, heart, and lung tissues were examined using pathological microscopy. RESULTS: The average (standard deviation) ethanol consumption of mice under the IA2BC paradigm increased dose-dependently to 5.1 (0.2), 8.7 (0.7), and 15.9 (0.8) g/kg/24 h with 5 %, 10 %, and 20 % ethanol, respectively. However, there is no significant difference in ethanol preference among all the ethanol groups. Chronic ethanol drinking caused hyperalgesia, cognitive impairment, and motor incoordination, but caused no changes in body temperature, locomotor activity, or anxiety-like behaviors. Minor histopathological alterations in the liver were detected; however, no major abnormal pathology was observed in the heart or lungs. CONCLUSION: These findings clarify the link between ethanol dosage and behavioral changes in mice over a 12-week IA2BC paradigm, thereby bridging the knowledge gap regarding the effects of chronic ethanol drinking on neurological disorders.

Identification of Phosphodiesterase-7A (PDE7A) as a Novel Target for Reducing Ethanol Consumption in Mice.

BACKGROUND: Ethanol elicits a rapid stimulatory effect and a subsequent, prolonged sedative response, which are potential predictors of EtOH consumption by decreasing adenosine signaling; this phenomenon also reflects the obvious sex difference. cAMP (cyclic Adenosine Monophosphate)-PKA (Protein Kinase A) signaling pathway modulation can influence the stimulatory and sedative effects induced by EtOH in mice. This study's objective is to clarify the role of phosphodiesterase (PDE) in mediating the observed sex differences in EtOH responsiveness between male and female animals. METHODS: EtOH was administered i.p. for 7 days to identify the changes in PDE isoforms in response to EtOH treatment. Additionally, EtOH consumption and preference of male and female C57BL/6J mice were assessed using the drinking-in-the-dark and 2-bottle choice tests. Further, pharmacological inhibition of PDE7A heterozygote knockout mice was performed to investigate its effects on EtOH-induced stimulation and sedation in both male and female mice. Finally, Western blotting analysis was performed to evaluate the alterations in cAMP-PKA/Epac2 pathways. RESULTS: EtOH administration resulted in an immediate upregulation in PDE7A expression in female mice, indicating a strong association between PDE7A and EtOH stimulation. Through the pharmacological inhibition of PDE7A KD mice, we have demonstrated for the first time, to our knowledge, that PDE7A selectively attenuates EtOH responsiveness and consumption exclusively in female mice, whichmay be associated with the cAMP-PKA/Epac2 pathway and downstream phosphorylation of CREB and ERK1/2. CONCLUSIONS: Inhibition or knockdown of PDE7A attenuates EtOH responsivenessand consumption exclusively in female mice, which is associated with alterations in the cAMP-PKA/Epac2 signaling pathways, thereby highlighting its potential as a novel therapeutic target for alcohol use disorder.

Chronic ethanol exposure produces long-lasting, subregion-specific physiological adaptations in RMTg-projecting mPFC neurons.

Chronic ethanol exposure produces neuroadaptations in the medial prefrontal cortex (mPFC) that are thought to facilitate maladaptive behaviors that interfere with recovery from alcohol use disorder. Despite evidence that different cortico-subcortical projections play distinct roles in behavior, few studies have examined the physiological effects of chronic ethanol at the circuit level. The rostromedial tegmental nucleus (RMTg) is functionally altered by chronic ethanol exposure. Our recent work identified dense input from the mPFC to the RMTg, yet the effects of chronic ethanol exposure on this circuitry is unknown. In the current study, we examined physiological changes after chronic ethanol exposure in prelimbic (PL) and infralimbic (IL) mPFC neurons projecting to the RMTg. Adult male Long-Evans rats were injected with fluorescent retrobeads into the RMTg and rendered dependent using a 14-day chronic intermittent ethanol (CIE) vapor exposure paradigm. Whole-cell patch-clamp electrophysiological recordings were performed in fluorescently-labeled (RMTg-projecting) and -unlabeled (projection-undefined) layer 5 pyramidal neurons 7-10 days following ethanol exposure. CIE exposure significantly increased intrinsic excitability as well as spontaneous excitatory and inhibitory postsynaptic currents (sE/IPSCs) in RMTg-projecting IL neurons. In contrast, no lasting changes in excitability were observed in RMTg-projecting PL neurons, although a CIE-induced reduction in excitability was observed in projection-undefined PL neurons. CIE exposure also increased the frequency of sEPSCs in RMTg-projecting PL neurons. These data uncover novel subregion- and circuit-specific neuroadaptations in the mPFC following chronic ethanol exposure and reveal that the IL mPFC-RMTg projection is uniquely vulnerable to long-lasting effects of chronic ethanol exposure. This article is part of the Special Issue on "PFC circuit function in psychiatric disease and relevant models".

Patterns of neuronal activation following ethanol-induced social facilitation and social inhibition in adolescent cFos-LacZ male and female rats.

Alcohol-associated social facilitation together with attenuated sensitivity to adverse alcohol effects play a substantial role in adolescent alcohol use and misuse, with adolescent females being more susceptible to adverse consequences of binge drinking than adolescent males. Adolescent rodents also demonstrate individual and sex differences in sensitivity to ethanol-induced social facilitation and social inhibition, therefore the current study was designed to identify neuronal activation patterns associated with ethanol-induced social facilitation and ethanol-induced social inhibition in male and female adolescent cFos-LacZ rats. Experimental subjects were given social interaction tests on postnatal day (P) 34, 36, and 38 after an acute challenge with 0, 0.5 and 0.75 g/kg ethanol, respectively, and ß-galactosidase (ß-gal) expression was assessed in brain tissue of subjects socially facilitated and socially inhibited by 0.75 g/kg ethanol. In females, positive correlations were evident between overall social activity and neuronal activation of seven out of 13 ROIs, including the prefrontal cortex and nucleus accumbens, with negative correlations evident in males. Assessments of neuronal activation patterns revealed drastic sex differences between ethanol responding phenotypes. In socially inhibited males, strong correlations were evident among almost all ROIs (90 %), with markedly fewer correlations among ROIs (38 %) seen in socially facilitated males. In contrast, interconnectivity in females inhibited by ethanol was only 10 % compared to nearly 60 % in facilitated subjects. However, hub analyses revealed convergence of brain regions in males and females, with the nucleus accumbens being a hub region in socially inhibited subjects. Taken together, these findings demonstrate individual and sex-related differences in responsiveness to acute ethanol in adolescent rats, with sex differences more evident in socially inhibited by ethanol adolescents than their socially facilitated counterparts.

Effects of MC-100093 on Ethanol Drinking and the Expression of Astrocytic Glutamate Transporters in the Mesocorticolimbic Brain Regions of Male and Female Alcohol-Preferring Rats.

Chronic ethanol consumption increased extracellular glutamate concentrations in several reward brain regions. Glutamate homeostasis is regulated in majority by astrocytic glutamate transporter 1 (GLT-1) as well as the interactive role of cystine/glutamate antiporter (xCT). In this study, we aimed to determine the attenuating effects of a novel beta-lactam MC-100093, lacking the antibacterial properties, on ethanol consumption and GLT-1 and xCT expression in the subregions of nucleus accumbens (NAc core and NAc shell) and medial prefrontal cortex (Infralimbic, mPFC-IL and Prelimbic, mPFC-PL) in male and female alcohol-preferring (P) rats. Female and male rats were exposed to free access to ethanol (15% v/v) and (30% v/v) and water for five weeks, and on Week 6, rats were administered 100 mg/kg (i.p) of MC-100093 or saline for five days. MC-100093 reduced ethanol consumption in both male and female P rats from Day 1-5. Additionally, MC-100093 upregulated GLT-1 and xCT expression in the mPFC and NAc subregions as compared to ethanol-saline groups in female and male rats. Chronic ethanol intake reduced GLT-1 and xCT expression in the IL and PL in female and male rats, except there was no reduction in GLT-1 expression in the mPFC-PL in female rats. Although, MC-100093 upregulated GLT-1 and xCT expression in the subregions of NAc, we did not observe any reduction in GLT-1 and xCT expression with chronic ethanol intake in female rats. These findings strongly suggest that MC-100093 treatment effectively reduced ethanol intake and upregulated GLT-1 and xCT expression in the mPFC and NAc subregions in male and female P rats.

Social setting interacts with hyper dopamine to boost the stimulant effect of ethanol.

Alcohol consumption occurring in a social or solitary setting often yields different behavioural responses in human subjects. For example, social drinking is associated with positive effects while solitary drinking is linked to negative effects. However, the neurobiological mechanism by which the social environment during alcohol intake impacts on behavioural responses remains poorly understood. We investigated whether distinct social environments affect behavioural responses to ethanol and the role of the dopamine system in this phenomenon in the fruit fly Drosophila melanogaster. The wild-type Canton-S (CS) flies showed higher locomotor response when exposed to ethanol in a group setting than a solitary setting, and there was no difference in females and males. Dopamine signalling is crucial for the locomotor stimulating effect of ethanol. When subjected to ethanol exposure alone, the dopamine transport mutant flies fumin (fmn) with hyper dopamine displayed the locomotor response similar to CS. When subjected to ethanol in a group setting, however, the fmn's response to the locomotor stimulating effect was substantially augmented compared with CS, indicating synergistic interaction of dopamine signalling and social setting. To identify the dopamine signalling pathway important for the social effect, we examined the flies defective in individual dopamine receptors and found that the D1 receptor dDA1/Dop1R1 is the major receptor mediating the social effect. Taken together, this study underscores the influence of social context on the neural and behavioural responses to ethanol.

Chronic Alcohol Exposure Alters the Levels and Assembly of the Actin Cytoskeleton and Microtubules in the Adult Mouse Hippocampus.

BACKGROUND: Alcohol abuse, a prevalent global health issue, is associated with the onset of cognitive impairment and neurodegeneration. Actin filaments (F-actin) and microtubules (MTs) polymerized from monomeric globular actin (G-actin) and tubulin form the structural basis of the neuronal cytoskeleton. Precise regulation of the assembly and disassembly of these cytoskeletal proteins, and their dynamic balance, play a pivotal role in regulating neuronal morphology and function. Nevertheless, the effect of prolonged alcohol exposure on cytoskeleton dynamics is not fully understood. This study investigates the chronic effects of alcohol on cognitive ability, neuronal morphology and cytoskeleton dynamics in the mouse hippocampus. METHODS: Mice were provided ad libitum access to 5% (v/v) alcohol in drinking water and were intragastrically administered 30% (v/v, 6.0 g/kg/day) alcohol for six weeks during adulthood. Cognitive functions were then evaluated using the Y maze, novel object recognition and Morris water maze tests. Hippocampal histomorphology was assessed through hematoxylin-eosin (HE) and Nissl staining. The polymerized and depolymerized states of actin cytoskeleton and microtubules were separated using two commercial assay kits and quantified by Western blot analysis. RESULTS: Mice chronically exposed to alcohol exhibited significant deficits in spatial and recognition memory as evidenced by behavioral tests. Histological analysis revealed notable hippocampal damage and neuronal loss. Decreased ratios of F-actin/G-actin and MT/tubulin, along with reduced levels of polymerized F-actin and MTs, were found in the hippocampus of alcohol-treated mice. CONCLUSIONS: Our findings suggest that chronic alcohol consumption disrupted the assembly of the actin cytoskeleton and MTs in the hippocampus, potentially contributing to the cognitive deficits and pathological injury induced by chronic alcohol intoxication.

Differential regulation of G protein-coupled receptor-associated proteins in the caudate and the putamen of cynomolgus macaques following chronic ethanol drinking.

The dorsal striatum is composed of the caudate nucleus and the putamen in human and non-human primates. These two regions receive different cortical projections and are functionally distinct. The caudate is involved in the control of goal-directed behaviors, while the putamen is implicated in habit learning and formation. Previous reports indicate that ethanol differentially influences neurotransmission in these two regions. Because neurotransmitters primarily signal through G protein-coupled receptors (GPCRs) to modulate neuronal activity, the present study aimed to determine whether ethanol had a region-dependent impact on the expression of proteins that are involved in the trafficking and function of GPCRs, including G protein subunits and their effectors, protein kinases, and elements of the cytoskeleton. Western blotting was performed to examine protein levels in the caudate and the putamen of male cynomolgus macaques that self-administered ethanol for 1 year under free access conditions, along with control animals that self-administered an isocaloric sweetened solution under identical operant conditions. Among the 18 proteins studied, we found that the levels of one protein (PKCß) were increased, and 13 proteins (Gαi1/3, Gαi2, Gαo, Gß1γ, PKCα, PKCε, CaMKII, GSK3ß, ß-actin, cofilin, α-tubulin, and tubulin polymerization promoting protein) were reduced in the caudate of alcohol-drinking macaques. However, ethanol did not alter the expression of any proteins examined in the putamen. These observations underscore the unique vulnerability of the caudate nucleus to changes in protein expression induced by chronic ethanol exposure. Whether these alterations are associated with ethanol-induced dysregulation of GPCR function and neurotransmission warrants future investigation.

Acute ethanol disrupts conditioned inhibition in the male rat.

RATIONALE: Alcohol can disrupt conditioned sexual inhibition (CSI) established by first-order conditioning in male rats. CSI can also be induced using second-order conditioning, during which male rats are trained to associate a neutral odor with a nonreceptive female. As a result, when given access to two receptive females (one scented and one unscented) during a copulatory preference test, they display CSI toward the scented female. OBJECTIVE: The present study examined the effect of low-to-moderate doses of alcohol on CSI and brain activation following exposure to alcohol and the olfactory cue alone. METHODS: Sexually-naïve Long-Evans rats received alternate conditioning sessions with unscented receptive or scented (almond extract) non-receptive females. Following the conditioning phase, males were injected with saline, alcohol 0.5 g/kg or 1 g/kg, 45 min before a copulatory test with two receptive females, with one bearing the olfactory cue. Fos activation was later assessed, following exposure to alcohol and the olfactory cue alone, in several brain regions involved in the expression and regulation of male sexual behavior. RESULTS: While males in the saline group displayed sexual avoidance towards the scented female, those injected with alcohol before the copulatory test, regardless of the dose, copulated indiscriminately with both females. Subsequent exposure to alcohol and the olfactory cue alone induced different Fos expression between groups in several brain regions. CONCLUSIONS: Low to moderate doses of alcohol disrupt conditioned sexual inhibition in male rats and induce a differential pattern of neural activation, particularly in regions involved in the expression and regulation of sexual behavior.

Environmental enrichment enhances ethanol preference over social reward in male swiss mice: Involvement of oxytocin-dopamine interactions.

The impact of environmental enrichment (EE) on natural rewards, including social and appetitive rewards, was investigated in male Swiss mice. EE, known for providing animals with various stimuli, was assessed for its effects on conditioned place preference (CPP) associated with ethanol and social stimuli. We previously demonstrated that EE increased the levels of the prosocial neuropeptide oxytocin (OT) in the hypothalamus and enhanced ethanol rewarding effects via an oxytocinergic mechanism. This study also investigated the impact of EE on social dominance and motivation for rewards, measured OT-mediated phospholipase C (PLC) activity in striatal membranes, and assessed OT expression in the hypothalamus. The role of dopamine in motivating rewards was considered, along with the interaction between OT and D1 receptors (DR) in the nucleus accumbens (NAc). Results showed that EE mice exhibited a preference for ethanol reward over social reward, a pattern replicated by the OT analogue Carbetocin. EE mice demonstrated increased social dominance and reduced motivation for appetitive taste stimuli. Higher OT mRNA levels in the hypothalamus were followed by diminished OT receptor (OTR) signaling activity in the striatum of EE mice. Additionally, EE mice displayed elevated D1R expression, which was attenuated by the OTR antagonist (L-368-889). The findings underscore the reinforcing effect of EE on ethanol and social rewards through an oxytocinergic mechanism. Nonetheless, they suggest that mechanisms other than the prosocial effect of EE may contribute to the ethanol pro-rewarding effect of EE and Carbetocin. They also point towards an OT-dopamine interaction potentially underlying some of these effects.

Mixing energy drinks and alcohol during adolescence impairs brain function: A study of rat hippocampal plasticity.

In the last decades, the consumption of energy drinks has risen dramatically, especially among young people, adolescents and athletes, driven by the constant search for ergogenic effects, such as the increase in physical and cognitive performance. In parallel, mixed consumption of energy drinks and ethanol, under a binge drinking modality, under a binge drinking modality, has similarly grown among adolescents. However, little is known whether the combined consumption of these drinks, during adolescence, may have long-term effects on central function, raising the question of the risks of this habit on brain maturation. Our study was designed to evaluate, by behavioral, electrophysiological and molecular approaches, the long-term effects on hippocampal plasticity of ethanol (EtOH), energy drinks (EDs), or alcohol mixed with energy drinks (AMED) in a rat model of binge-like drinking adolescent administration. The results show that AMED binge-like administration produces adaptive hippocampal changes at the molecular level, associated with electrophysiological and behavioral alterations, which develop during the adolescence and are still detectable in adult animals. Overall, the study indicates that binge-like drinking AMED adolescent exposure represents a habit that may affect permanently hippocampal plasticity.

Serotonin transporter knockdown relieves depression-like behavior and ethanol-induced CPP in mice after chronic social defeat stress.

Patients with stress-triggered major depression disorders (MDD) can often seek comfort or temporary relief through alcohol consumption, as they may turn to it as a means of self-medication or coping with overwhelming emotions. The use of alcohol as a coping mechanism for stressful events can escalate, fostering a cycle where the temporary relief it provides from depression can deepen into alcohol dependence, exacerbating both conditions. Although, the specific mechanisms involved in stress-triggered alcohol dependence and MDD comorbidities are not well understood, a large body of literature suggests that the serotonin transporter (SERT) plays a critical role in these abnormalities. To further investigate this hypothesis, we used a lentiviral-mediated knockdown approach to examine the role of hippocampal SERT knockdown in social defeat stress-elicited depression like behavior and ethanol-induced place preference (CPP). The results showed that social defeat stress-pro depressant effects were reversed following SERT knockdown demonstrated by increased sucrose preference, shorter latency to feed in the novelty suppressed feeding test, and decreased immobility time in the tail suspension and forced swim tests. Moreover, and most importantly, social stress-induced ethanol-CPP acquisition and reinstatement were significantly reduced following hippocampal SERT knockdown using short hairpin RNA shRNA-expressing lentiviral vectors. Finally, we confirmed that SERT hippocampal mRNA expression correlated with measures of depression- and ethanol-related behaviors by Pearson's correlation analysis. Taken together, our data suggest that hippocampal serotoninergic system is involved in social stress-triggered mood disorders as well as in the acquisition and retrieval of ethanol contextual memory and that blockade of this transporter can decrease ethanol rewarding properties.

Potentiation of the depressant effect of alcohol by flunitrazepam in rats: an electrocorticographic, respiratory and electrocardiographic study.

Alcohol, a widely commercialized psychotropic drug, and the benzodiazepine Flunitrazepam, an anxiolytic widely prescribed for patients with anxiety and insomnia problems, are well known drugs and both act on the central nervous system. The misuse and the association of these two drugs are public health concerns in several countries and could cause momentary, long-lasting and even lethal neurophysiological problems due to the potentiation of their adverse effects in synergy. The present study observed the result of the association of these drugs on electrophysiological responses in the brain, heart, and respiratory rate in Wistar rats. 8 experimental groups were determined: control, one alcohol group (20% at a dose of 1 ml/100 g VO), three Flunitrazepam groups (doses 0.1; 0.2 and 0.3 mg/kg) and three alcohol-Flunitrazepam groups (20% at a dose of 1 ml/100 g VO of alcohol, combined with 0.1; 0.2 and 0.3 mg/kg of Flunitrazepam, respectively). The results showed that there was a more pronounced reduction in alpha and theta wave power in the alcohol-Flunitrazepam groups, a decrease in the power of beta oscillations and greater sedation. There was a progressive decrease in respiratory rate linked to the increase of Flunitrazepam dose in the alcohol-Flunitrazepam associated administration. It was observed alteration in heart rate and Q-T interval in high doses of Flunitrazepam. Therefore, we conclude that the association alcohol-Flunitrazepam presented deepening of depressant synergistic effects according to the increase in the dose of the benzodiazepine, and this could cause alterations in low frequency brain oscillations, breathing, and hemodynamics of the patient.

A Role for GABAA Receptor ß3 Subunits in Mediating Harmaline Tremor Suppression by Alcohol: Implications for Essential Tremor Therapy.

Background: Essential tremor patients may find that low alcohol amounts suppress tremor. A candidate mechanism is modulation of α6ß3δ extra-synaptic GABAA receptors, that in vitro respond to non-intoxicating alcohol levels. We previously found that low-dose alcohol reduces harmaline tremor in wild-type mice, but not in littermates lacking δ or α6 subunits. Here we addressed whether low-dose alcohol requires the ß3 subunit for tremor suppression. Methods: We tested whether low-dose alcohol suppresses tremor in cre-negative mice with intact ß3 exon 3 flanked by loxP, and in littermates in which this region was excised by cre expressed under the α6 subunit promotor. Tremor in the harmaline model was measured as a percentage of motion power in the tremor bandwidth divided by overall motion power. Results: Alcohol, 0.500 and 0.575 g/kg, reduced harmaline tremor compared to vehicle-treated controls in floxed ß3 cre- mice, but had no effect on tremor in floxed ß3 cre+ littermates that have ß3 knocked out. This was not due to potential interference of α6 expression by the insertion of the cre gene into the α6 gene since non-floxed ß3 cre+ and cre- littermates exhibited similar tremor suppression by alcohol. Discussion: As α6ß3δ GABAA receptors are sensitive to low-dose alcohol, and cerebellar granule cells express ß3 and are the predominant brain site for α6 and δ expression together, our overall findings suggest alcohol acts to suppress tremor by modulating α6ß3δ GABAA receptors on these cells. Novel drugs that target this receptor may potentially be effective and well-tolerated for essential tremor. Highlights: We previously found with the harmaline essential tremor model that GABAA receptors containing α6 and δ subunits mediate tremor suppression by alcohol. We now show that ß3 subunits in α6-expressing cells, likely cerebellar granule cells, are also required, indicating that alcohol suppresses tremor by modulating α6ß3δ extra-synaptic GABAA receptors.

Neutral sphingomyelinase controls acute and chronic alcohol effects on brain activity.

Alcohol consumption is a widespread phenomenon throughout the world. However, how recreational alcohol use evolves into alcohol use disorder (AUD) remains poorly understood. The Smpd3 gene and its coded protein neutral sphingomyelinase (NSM) are associated with alcohol consumption in humans and alcohol-related behaviors in mice, suggesting a potential role in this transition. Using multiparametric magnetic resonance imaging, we characterized the role of NSM in acute and chronic effects of alcohol on brain anatomy and function in female mice. Chronic voluntary alcohol consumption (16 vol% for at least 6 days) affected brain anatomy in WT mice, reducing regional structure volume predominantly in cortical regions. Attenuated NSM activity prevented these anatomical changes. Functional MRI linked these anatomical adaptations to functional changes: Chronic alcohol consumption in mice significantly modulated resting state functional connectivity (RS FC) in response to an acute ethanol challenge (i.p. bolus of 2 g kg-1) in heterozygous NSM knockout (Fro), but not in WT mice. Acute ethanol administration in alcohol-naïve WT mice significantly decreased RS FC in cortical and brainstem regions, a key finding that was amplified in Fro mice. Regarding direct pharmacological effects, acute ethanol administration increased the regional cerebral blood volume (rCBV) in many brain areas. Here, chronic alcohol consumption otherwise attenuated the acute rCBV response in WT mice but enhanced it in Fro mice. Altogether, these findings suggest a differential role for NSM in acute and chronic functional brain responses to alcohol. Therefore, targeting NSM may be useful in the prevention or treatment of AUD.

Kappa-opioid receptor stimulation in the nucleus accumbens shell and ethanol drinking: Differential effects by rostro-caudal location and level of drinking.

Although the kappa-opioid receptor (KOR) and its endogenous ligand, dynorphin, are believed to be involved in ethanol drinking, evidence on the direction of their effects has been mixed. The nucleus accumbens (NAc) shell densely expresses KORs, but previous studies have not found KOR activation to influence ethanol drinking. Using microinjections into the NAc shell of male and female Long-Evans rats that drank under the intermittent-access procedure, we found that the KOR agonist, U50,488, had no effect on ethanol drinking when injected into the middle NAc shell, but that it promoted intake in males and high-drinking females in the caudal NAc shell and high-drinking females in the rostral shell, and decreased intake in males and low-drinking females in the rostral shell. Conversely, injection of the KOR antagonist, nor-binaltorphimine, stimulated ethanol drinking in low-drinking females when injected into the rostral NAc shell and decreased drinking in high-drinking females when injected into the caudal NAc shell. These effects of KOR activity were substance-specific, as U50,488 did not affect sucrose intake. Using quantitative real-time PCR, we found that baseline gene expression of the KOR was higher in the rostral compared to caudal NAc shell, but that this was upregulated in the rostral shell with a history of ethanol drinking. Our findings have important clinical implications, demonstrating that KOR stimulation in the NAc shell can affect ethanol drinking, but that this depends on NAc subregion, subject sex, and ethanol intake level, and suggesting that this may be due to differences in KOR expression.

Adolescent intermittent ethanol use in male rats do not change cerebellar cell numbers but initiate astroglial reaction.

Alcohol consumption during adolescence causes negative structural changes in the cerebellum and can lead to cognitive and motor skill disorders. Unfortunately, the age at which individuals begin drinking alcohol has decreased in recent years, which has drawn attention to the effects of alcohol on neurological changes during preadolescence. In this study, we investigated the effects of adolescent intermittent ethanol (AIE) exposure on the cellular composition of the cerebellum in male rats, particularly when alcohol consumption begins early. The male rats received eight doses of intermittent intraperitoneal injection of 25% (v/v) ethanol (3 g/kg) or saline from postnatal days (PND) 25 to PND 38. In rats, 28-42 days old corresponds to 10-18 years old in humans. Two hours after the last injection, the cells, neurons, and non-neuronal cells in the cerebellum were immunocytochemically labeled and the total numbers of related cells were calculated using the Isotropic Fractionator method. We found that AIE exposure does not change the cell numbers of the cerebellum in the short term, but it does activate astrocytes in the white matter of the cerebellum. These findings suggest that alcohol use during adolescence impairs the innate immune system and negatively affects brain plasticity.

Effects of social housing conditions on ethanol-induced behavioral sensitization in Swiss mice.

RATIONALE: In previous animal model studies, it was shown that drug sensitization is dependent upon physical environmental conditions. However, the effects of social housing conditions on drug sensitization is much less known. OBJECTIVE: The aim of the present study was to investigate the effects of social conditions, through the size of housing groups, on ethanol stimulant effects and ethanol-induced behavioral sensitization in mice. MATERIALS AND METHODS: Male and female Swiss mice were housed in groups of different sizes (isolated mice, two mice per cage, four mice per cage and eight mice per cage) during a six-week period. A standard paradigm of ethanol-induced locomotor sensitization was then started with one daily injection of 2.5 g/kg ethanol for 8 consecutive days. RESULTS: The results show that social housing conditions affect the acute stimulant effects of ethanol. The highest stimulant effects were observed in socially isolated mice and then gradually decreased as the size of the group increased. Although the rate of ethanol sensitization did not differ between groups, the ultimate sensitized levels of ethanol-induced stimulant effects were significantly reduced in mice housed in groups of eight. CONCLUSIONS: These results are consistent with the idea that higher levels of acute and sensitized ethanol stimulant effects are observed in mice housed in stressful housing conditions, such as social isolation.