Alcohol use disorder (AUD) is highly prevalent and one of the leading causes of disability in the US and around the world. There are some molecular biomarkers of heavy alcohol use and liver damage which can suggest AUD, but these are lacking in sensitivity and specificity. AUD treatment involves psychosocial interventions and medications for managing alcohol withdrawal, assisting in abstinence and reduced drinking (naltrexone, acamprosate, disulfiram, and some off-label medications), and treating comorbid psychiatric conditions (e.g., depression and anxiety). It has been suggested that various patient groups within the heterogeneous AUD population would respond more favorably to specific treatment approaches. For example, there is some evidence that so-called reward-drinkers respond better to naltrexone than acamprosate. However, there are currently no objective molecular markers to separate patients into optimal treatment groups or any markers of treatment response. Objective molecular biomarkers could aid in AUD diagnosis and patient stratification, which could personalize treatment and improve outcomes through more targeted interventions. Biomarkers of treatment response could also improve AUD management and treatment development. Systems biology considers complex diseases and emergent behaviors as the outcome of interactions and crosstalk between biomolecular networks. A systems approach that uses transcriptomic (or other -omic data, e.g., methylome, proteome, metabolome) can capture genetic and environmental factors associated with AUD and potentially provide sensitive, specific, and objective biomarkers to guide patient stratification, prognosis of treatment response or relapse, and predict optimal treatments. This Review describes and highlights state-of-the-art research on employing transcriptomic data and artificial intelligence (AI) methods to serve as molecular biomarkers with the goal of improving the clinical management of AUD. Considerations about future directions are also discussed.

Keywords: RNA seq; alcohol dependence; alcoholism; biomarker; microarray; transcriptome.

Apremilast is a phosphodiesterase (PDE) type 4 inhibitor that is nonselective at subtypes PDE4A-D. It modulates ethanol and GABAergic responses via protein kinase A (PKA) phosphorylation of specific GABA_A receptor subunits and has opposite effects on ethanol-induced ataxia in wild-type and GABA_A β3-S408/409A knock-in mice. We hypothesized that these different effects are due to preferential actions at different PDE4 subtypes. To test this hypothesis, we compared effects of selective PDE4 inhibitors on responses to ethanol and GABAergic drugs in male and female C57BL/6J mice. The PDE4B inhibitor A33 accelerated recovery from ataxia induced by ethanol and diazepam but did not alter ataxia induced by propofol. The PDE4D inhibitor D159687 accelerated recovery from diazepam-induced ataxia but prolonged recovery from ethanol- and propofol-induced ataxia. A33 shortened, while D159687 prolonged, the sedative-hypnotic effects of ethanol. Both drugs shortened diazepam's sedative-hypnotic effects. The modulatory effects of A33 and D159687 were completely prevented by the PKA inhibitor H89. Only D159687 prevented development of acute functional tolerance to ethanol-induced ataxia. D159687 transiently reduced two-bottle choice drinking in male and female mice that had consumed ethanol for 3 weeks and transiently reduced two-bottle choice, every-other-day drinking in male mice. A33 did not alter ethanol drinking in either procedure. Neither drug altered binge-like ethanol consumption or blood ethanol clearance. Thus, D159687 produced behavioral effects similar to apremilast, although it produced a more transient and smaller reduction in drinking. These results indicate that PDE4D inhibition contributes to apremilast's ability to reduce ethanol drinking, whereas PDE4B inhibition is not involved.

In this study we investigated the effects of a neonatal handling protocol that mimics the handling of sham control pups in protocols of neonatal exposure to brain insults on dendritic arborization and glycosaminoglycan (GAG) levels in the developing brain. GAGs are long, unbranched polysaccharides, consisting of repeating disaccharide units that can be modified by sulfation at specific sites and are involved in modulating neuronal plasticity during brain development. In this study, male and female Sprague-Dawley rats underwent neonatal handling daily between post-natal day (PD)4 and PD9, with brains analyzed on PD9. Neuronal morphology and morphometric analysis of the apical and basal dendritic trees of CA1 hippocampal pyramidal neurons were carried out by Golgi-Cox staining followed by neuron tracing and analysis with the software Neurolucida. Chondroitin sulfate (CS)-, Hyaluronic Acid (HA)-, and Heparan Sulfate (HS)-GAG disaccharide levels were quantified in the hippocampus by Liquid Chromatography/Mass Spectrometry analyses. We found sex by neonatal handling interactions on several parameters of CA1 pyramidal neuron morphology and in the levels of HS-GAGs, with females, but not males, showing an increase in both dendritic arborization and HS-GAG levels. We also observed increased expression of glucocorticoid receptor gene Nr3c1 in the hippocampus of both males and females following neonatal handling suggesting that both sexes experienced a similar stress during the handling procedure. This is the first study to show sex differences in two parameters of brain plasticity, CA1 neuron morphology and HS-GAG levels, following handling stress in neonatal rats.

Keywords: Dendritic arborization; Glycosaminoglycans (GAGs); Neonatal handling; Sex differences.

Excessive alcohol use disrupts neuroimmune signaling across various cell types, including neurons, microglia, and astrocytes. The present review focuses on recent, albeit limited, evidence of sex differences in biological factors that mediate neuroimmune responses to alcohol and underlying neuroimmune systems that may influence alcohol drinking behaviors. Females are more vulnerable than males to the neurotoxic and negative consequences of chronic alcohol drinking, reflected by elevations of pro-inflammatory cytokines and inflammatory mediators. Differences in cytokine, microglial, astrocytic, genomic, and transcriptomic evidence suggest females are more reactive than males to neuroinflammatory changes after chronic alcohol exposure. The growing body of evidence supports that innate immune factors modulate synaptic transmission, providing a mechanistic framework to examine sex differences across neurocircuitry. Targeting neuroimmune signaling may be a viable strategy for treating AUD, but more research is needed to understand sex-specific differences in alcohol drinking and neuroimmune mechanisms.

Compulsive alcohol drinking is a key symptom of alcohol use disorder (AUD) that is particularly resistant to treatment. An understanding of the biological factors that underly compulsive drinking will allow for the development of new therapeutic targets for AUD. One animal model of compulsive alcohol drinking involves the addition of bitter-tasting quinine to an ethanol solution and measuring the willingness of the animal to consume ethanol despite the aversive taste. Previous studies have demonstrated that this type of aversion-resistant drinking is modulated in the insular cortex of male mice by specialized condensed extracellular matrix known as perineuronal nets (PNNs), which form a lattice-like structure around parvalbumin-expressing neurons in the cortex. Several laboratories have shown that female mice exhibit higher levels of aversion-resistant ethanol intake but the role of PNNs in females in this behavior has not been examined. Here we compared PNNs in the insula of male and female mice and determined if disrupting PNNs in female mice would alter aversion-resistant ethanol intake. PNNs were visualized in the insula by fluorescent labeling with Wisteria floribunda agglutinin (WFA) and disrupted in the insula by microinjecting chondroitinase ABC, an enzyme that digests the chondroitin sulfate glycosaminoglycan component of PNNs. Mice were tested for aversion-resistant ethanol consumption by the addition of sequentially increasing concentrations of quinine to the ethanol in a two-bottle choice drinking in the dark procedure. PNN staining intensity was higher in the insula of female compared to male mice, suggesting that PNNs in females might contribute to elevated aversion-resistant drinking. However, disruption of PNNs had limited effect on aversion-resistant drinking in females. In addition, activation of the insula during aversion-resistant drinking, as measured by c-fos immunohistochemistry, was lower in female mice than in males. Taken together, these results suggest that neural mechanisms underlying aversion-resistant ethanol consumption differ in males and females.

Astrocytes release numerous factors known to contribute to the process of synaptogenesis, yet knowledge about the signals that control their release is limited. We hypothesized that neuron-derived signals stimulate astrocytes, which respond by signaling back to neurons through the modulation of astrocyte-released synaptogenic factors. Here we investigate the effect of cholinergic stimulation of astrocytes on synaptogenesis in co-cultured neurons. Using a culture system where primary rat astrocytes and primary rat neurons are first grown separately allowed us to independently manipulate astrocyte cholinergic signaling. Subsequent co-culture of pre-stimulated astrocytes with naïve neurons enabled us to assess how prior stimulation of astrocyte acetylcholine receptors uniquely modulates neuronal synapse formation. Pre-treatment of astrocytes with the acetylcholine receptor agonist carbachol increased the expression of synaptic proteins, the number of pre- and postsynaptic puncta, and the number of functional synapses in hippocampal neurons after 24 hours in co-culture. Astrocyte secretion of the synaptogenic protein thrombospondin-1 increased after cholinergic stimulation and the inhibition of the target receptor for thrombospondins prevented the observed increase in neuronal synaptic structures. Thus, we identified a novel mechanism of neuron-astrocyte-neuron communication, i.e. , neuronal release of acetylcholine stimulates astrocytes to release synaptogenic proteins leading to increased synaptogenesis in neurons. This study provides new insights into the role of neurotransmitter receptors in developing astrocytes and into our understanding of the modulation of astrocyte-induced synaptogenesis.

Background and purpose: Chronic pain is considered a key factor contributing to alcohol use disorder (AUD). The mechanisms responsible for chronic pain associated with chronic alcohol consumption are unknown. We evaluated the development of chronic pain in a mouse model of alcohol dependence and investigate the role of neuroinflammation.

Experimental approach: The chronic-intermittent ethanol two-bottle choice CIE-2BC paradigm generates three groups: alcohol-dependent with escalating alcohol intake, nondependent (moderate drinking) and alcohol-naïve control male and female mice. We measured mechanical allodynia during withdrawal and after the last voluntary drinking. Immunoblotting was used to evaluate the protein levels of IBA-1, CSFR, IL-6, p38 and ERK2/1 in spinal cord tissue of dependent and non-dependent animals.

Key results: We found significant escalation of drinking in the dependent group in male and female compared with the non-dependent group. The dependent group developed mechanical allodynia during 72 h of withdrawal, which was completely reversed after voluntary drinking. We observed an increased pain hypersensitivity compared with the naïve in 50% of non-dependent group. Increased IBA-1 and CSFR expression was observed in spinal cord tissue of both hypersensitivity-abstinence related and neuropathy-alcohol mice, and increased IL-6 expression and ERK1/2 activation in mice with hypersensitivity-related to abstinence, but not in mice with alcohol-evoked neuropathic pain.

Conclusions and implications: The CIE-2BC model induces two distinct pain conditions specific to the type of ethanol exposure: abstinence-related hypersensitivity in dependent mice and alcohol-evoked neuropathic pain in about a half of the non-dependent mice.

We previously discovered using transcriptomics that rats undergoing withdrawal after chronic ethanol exposure had increased expression of several genes encoding RNA splicing factors in the hippocampus. Here, we examined RNA splicing in the rat hippocampus during withdrawal from chronic ethanol exposure and in postmortem hippocampus of human subjects diagnosed with alcohol use disorder (AUD). We found that expression of the gene encoding the splicing factor, poly r(C) binding protein 1 (PCBP1), was elevated in the hippocampus of rats during withdrawal after chronic ethanol exposure and AUD subjects. We next analyzed the rat RNA-Seq data for differentially expressed (DE) exon junctions. One gene, Hapln2, had increased usage of a novel 3' splice site in exon 4 during withdrawal. This splice site was conserved in human HAPLN2 and was used more frequently in the hippocampus of AUD compared to control subjects. To establish a functional role for PCBP1 in HAPLN2 splicing, we performed RNA immunoprecipitation (RIP) with a PCBP1 antibody in rat and human hippocampus, which showed enriched PCBP1 association near the HAPLN2 exon 4 3' splice site in the hippocampus of rats during ethanol withdrawal and AUD subjects. Our results indicate a conserved role for the splicing factor PCBP1 in aberrant splicing of HAPLN2 after chronic ethanol exposure. As the HAPLN2 gene encodes an extracellular matrix protein involved in nerve conduction velocity, use of this alternative splice site is predicted to result in loss of protein function that could negatively impact hippocampal function in AUD.

Post-traumatic stress disorder (PTSD) leads to enhanced alcohol drinking and development of alcohol use disorder (AUD). Identifying shared neural mechanisms might help discover new therapies for PTSD/AUD. Here, we employed a rat model of comorbid PTSD/AUD to evaluate compounds that inhibit FK506-binding protein 51 (FKBP5), a co-chaperone modulator of glucocorticoid receptors implicated in stress-related disorders. Male and female rats received a familiar avoidance-based shock stress followed by voluntary alcohol drinking. We then assessed trauma-related behaviors through sleep bout cycles, hyperarousal, fear overgeneralization, and irritability. To evaluate the role of stress and alcohol history on the sensitivity to FKBP5 inhibitors, in two separate studies, we administered two FKBP5 inhibitors, benztropine (Study 1) or SAFit2 (Study 2). FKBP5 inhibitors were administered on the last alcohol drinking session and prior to each trauma-related behavioral assessment. We also measured plasma corticosterone to assess the actions of FKBP5 inhibitors after familiar shock stress and alcohol drinking. Benztropine reduced alcohol preference in stressed males and females, while aggressive bouts were reduced in benztropine-treated stressed females. During hyperarousal, benztropine reduced several startle response outcomes across stressed males and females. Corticosterone was reduced in benztropine-treated stressed males. The selective FKBP5 inhibitor, SAFit2, reduced alcohol drinking in stressed males but not females, with no differences in irritability. Importantly, SAFit2 decreased fear overgeneralization in stressed males and females. SAFit2 also reduced corticosterone across stressed males and females. Neither FKBP5 inhibitor changed sleep bout structure. These findings indicate that FKBP5 inhibitors modulate stress-related alcohol drinking and partially modulate trauma-related behaviors. This work supports the hypothesis that targeting FKBP5 may alleviate PTSD/AUD comorbidity.

The neuroimmune system of the brain, which is comprised primarily of astrocytes and microglia, regulates a variety of homeostatic mechanisms that underlie normal brain function. Numerous conditions, including alcohol consumption, can disrupt this regulatory process by altering brain levels of neuroimmune factors. Alcohol and neuroimmune factors, such as proinflammatory cytokines IL-6 and TNF-alpha, act at similar targets in the brain, including excitatory and inhibitory synaptic transmission. Thus, alcohol-induced production of IL-6 and/or TNF-alpha could be important contributing factors to the effects of alcohol on the brain. Recent studies indicate that IL-6 plays a role in alcohol drinking and the effects of alcohol on the brain activity following the cessation of alcohol consumption (post-alcohol period), however information on these topics is limited. Here we used homozygous and heterozygous female and male transgenic mice with increased astrocyte expression of IL-6 to examined further the interactions between alcohol and IL-6 with respect to voluntary alcohol drinking, brain activity during the post-alcohol period, IL-6 signal transduction, and expression of synaptic proteins. Wildtype littermates (WT) served as controls. The transgenic mice model brain neuroimmune status with respect to IL-6 in subjects with a history of persistent alcohol use. Results showed a genotype dependent reduction in voluntary alcohol consumption in the Drinking in the Dark protocol and in frequency-dependent relationships between brain activity in EEG recordings during the post-alcohol period and alcohol consumption. IL-6, TNF-alpha, IL-6 signal transduction partners pSTAT3 and c/EBP beta, and synaptic proteins were shown to play a role in these genotypic effects.

Background and purpose: The endocannabinoid (eCB) system plays an important homeostatic role in the regulation of stress circuits and has emerged as a therapeutic target to treat stress disorders and alcohol use disorder (AUD). Extensive research has elucidated a role for the eCB anandamide (AEA), but less is known about 2-arachidonoylglycerol (2-AG) mediated signalling.

Experimental approach: We pharmacologically enhanced eCB signalling by inhibiting the 2-AG metabolizing enzyme, monoacylglycerol lipase (MAGL), in male and female Marchigian Sardinian alcohol-preferring (msP) rats, a model of innate alcohol preference and stress hypersensitivity, and in control Wistar rats. We tested the acute effect of the selective MAGL inhibitor MJN110 in alleviating symptoms of alcohol drinking, anxiety, irritability and fear.

Key results: A single systemic administration of MJN110 increased 2-AG levels in the central amygdala, prelimbic and infralimbic cortex but did not acutely alter alcohol drinking. MAGL inhibition reduced aggressive behaviours in female msPs, and increased defensive behaviours in male msPs, during the irritability test. Moreover, in the novelty-induced hypophagia test, MJN110 selectively enhanced palatable food consumption in females, mitigating stress-induced food suppression. Lastly, msP rats showed increased conditioned fear behaviour compared with Wistar rats, and MJN110 reduced context-associated conditioned fear responses, but not cue-probed fear expression, in male msPs.

Conclusions and implications: Acute inhibition of MAGL attenuated some stress-related responses in msP rats but not voluntary alcohol drinking. Our results provide new insights into the sex dimorphism documented in stress-induced responses. Sex-specific eCB-based approaches should be considered in the clinical development of therapeutics.

Keywords: 2-AG; alcohol use disorder; endocannabinoid system; monoacylglycerol lipase; sex differences; stress.

Stress increases alcohol consumption in dependent animals and contributes to the development of alcohol use disorder. The nucleus of the solitary tract (NTS) is a critical brainstem region for integrating and relaying central and peripheral signals to regulate stress responses, but it is not known if it plays a role in alcohol dependence- or in stress-induced escalations in alcohol drinking in dependent mice. Here, we used RNA-sequencing and bioinformatics analyses to study molecular adaptations in the NTS of C57BL/6J male mice that underwent an ethanol drinking procedure that uses exposure to chronic intermittent ethanol (CIE) vapor, forced swim stress (FSS), or both conditions (CIE + FSS). Transcriptome profiling was performed at three different times after the last vapor cycle (0-hr, 72-hr, and 168-hr) to identify changes in gene expression associated with different stages of ethanol intoxication and withdrawal. In the CIE and CIE + FSS groups at 0-hr, there was upregulation of genes enriched for cellular response to type I interferon (IFN) and type I IFN- and cytokine-mediated signaling pathways, while the FSS group showed upregulation of neuronal genes. IFN signaling was the top gene network positively correlated with ethanol consumption levels in the CIE and CIE + FSS groups. Results from different analyses (differential gene expression, weighted gene coexpression network analysis, and rank-rank hypergeometric overlap) indicated that activation of type I IFN signaling would be expected to increase ethanol consumption. The CIE and CIE + FSS groups also shared an immune signature in the NTS as has been demonstrated in other brain regions after chronic ethanol exposure. A temporal-based clustering analysis revealed a unique expression pattern in the CIE + FSS group that suggests the interaction of these two stressors produces adaptations in synaptic and glial functions that may drive stress-induced drinking.

The collaborative cross (CC) founder strains include five classical inbred laboratory strains [129S1/SvlmJ (S129), A/J (AJ), C57BL/6J (B6), NOD/ShiLtJ (NOD), and NZO/HILtJ (NZO)] and three wild-derived strains [CAST/EiJ (CAST), PWK/PhJ (PWK), and WSB/EiJ (WSB)]. These strains encompass 89% of the genetic diversity available in Mus musculus and ∼10–20 times more genetic diversity than found in Homo sapiens. For more than 60 years the B6 strain has been widely used as a genetic model for high ethanol preference and consumption. However, another of the CC founder strains, PWK, has been identified as a high ethanol preference/high consumption strain. The current study determined how the transcriptomes of the B6 and PWK strains differed from the 6 low preference CC strains across 3 nodes of the brain addiction circuit. RNA-Seq data were collected from the central nucleus of the amygdala (CeA), the nucleus accumbens core (NAcc) and the prelimbic cortex (PrL). Differential expression (DE) analysis was performed in each of these brain regions for all 28 possible pairwise comparisons of the CC founder strains. Unique genes for each strain were identified by selecting for genes that differed significantly [false discovery rate (FDR) < 0.05] from all other strains in the same direction. B6 was identified as the most distinct classical inbred laboratory strain, having the highest number of total differently expressed genes (DEGs) and DEGs with high log fold change, and unique genes compared to other CC strains. Less than 50 unique DEGs were identified in common between B6 and PWK within all three brain regions, indicating the strains potentially represent two distinct genetic signatures for risk for high ethanol-preference. 338 DEGs were found to be commonly different between B6, PWK and the average expression of the remaining CC strains within all three regions. The commonly different up-expressed genes were significantly enriched (FDR < 0.001) among genes associated with neuroimmune function. These data compliment findings showing that neuroimmune signaling is key to understanding alcohol use disorder (AUD) and support use of these 8 strains and the highly heterogeneous mouse populations derived from them to identify alcohol-related brain mechanisms and treatment targets.

Chronic ethanol exposure affects the glutamatergic system in several brain reward regions such as the nucleus accumbens (NAc). Our laboratory has shown that chronic exposure to ethanol reduced the expression of glutamate transporter 1 (GLT-1) and cystine/glutamate exchanger transporter (xCT) and, as a result, increased extracellular glutamate concentrations in the NAc of alcohol preferring (P) rats. Moreover, previous study from our laboratory reported that chronic ethanol intake altered the expression of certain metabotropic glutamate receptors in the brain. In addition to central effects, chronic ethanol consumption induced liver injury, which is associated with steatohepatitis. In the present study, we investigated the effects of chronic ethanol consumption in the brain and liver. Male P rats had access to free choice of ethanol and water bottles for five weeks. Chronic ethanol consumption reduced GLT-1 and xCT expression in the NAc shell but not in the NAc core. Furthermore, chronic ethanol consumption increased fat droplet content as well as peroxisome proliferator-activated receptor alpha (PPAR-α) and GLT-1 expression in the liver. Importantly, treatment with the novel beta-lactam compound, MC-100093, reduced ethanol drinking behavior and normalized the levels of GLT-1 and xCT expression in the NAc shell as well as normalized GLT-1 and PPAR-α expression in the liver. In addition, MC100093 attenuated ethanol-induced increases in fat droplet content in the liver. These findings suggest that MC-100093 might be a potential lead compound to attenuate ethanol-induced dysfunction in glutamatergic system and liver injury. Significance Statement This study identified a novel beta-lactam, MC100093, that has upregulatory effects in GLT-1. MC-100093, reduced ethanol drinking behavior and normalized levels of GLT-1 and xCT expression in the NAc shell as well as normalizing GLT-1 and PPAR-α expression in the liver. In addition, MC100093 attenuated ethanol-induced increases in fat droplet content in the liver.

Aberrant glucocorticoid signaling via glucocorticoid receptors (GR) plays a critical role in alcohol use disorder (AUD). Acute alcohol withdrawal and protracted abstinence in dependent rats are associated with increased GR signaling and changes in GR-mediated transcriptional activity in the rat central nucleus of the amygdala (CeA). The GR antagonist mifepristone decreases alcohol consumption in dependent rats during acute withdrawal and protracted abstinence. Regulation of CeA synaptic activity by GR is currently unknown. Here, we utilized mifepristone and the selective GR antagonist CORT118335 (both at 10 μM) as pharmacological tools to dissect the role of GR on GABA transmission in male, adult Sprague-Dawley rats using slice electrophysiology. We subjected rats to chronic intermittent alcohol vapor exposure for 5-7 weeks to induce alcohol dependence. A subset of dependent rats subsequently underwent protracted alcohol withdrawal for 2 weeks, and air-exposed rats served as controls. Mifepristone reduced the frequency of pharmacologically-isolated spontaneous inhibitory postsynaptic currents (sIPSC) in the CeA (medial subdivision) without affecting postsynaptic measures in all groups, suggesting decreased GABA release with the largest effect in dependent rats. CORT118335 did not significantly alter GABA transmission in naïve, but decreased sIPSC frequency in dependent rats. Similarly, mifepristone decreased amplitudes of evoked inhibitory postsynaptic potentials only in dependent rats and during protracted withdrawal. Collectively, our study provides insight into regulation of CeA GABAergic synapses by GR. Chronic ethanol enhances the efficiency of mifepristone and CORT118335, thus highlighting the potential of drugs targeting GR as a promising pharmacological avenue for the treatment of AUD.

Alcohol use disorder (AUD) is a chronically relapsing disease characterized by loss of control in seeking and consuming alcohol (ethanol) driven by the recruitment of brain stress systems. However, AUD differs among the sexes: men are more likely to develop AUD, but women progress from casual to binge drinking and heavy alcohol use more quickly. The central amygdala (CeA) is a hub of stress and anxiety, with corticotropin-releasing factor (CRF)-CRF1 receptor and Gamma-Aminobutyric Acid (GABA)-ergic signaling dysregulation occurring in alcohol-dependent male rodents. However, we recently showed that GABAergic synapses in female rats are less sensitive to the acute effects of ethanol. Here, we used patch-clamp electrophysiology to examine the effects of alcohol dependence on the CRF modulation of rat CeA GABAergic transmission of both sexes. We found that GABAergic synapses of naïve female rats were unresponsive to CRF application compared to males, although alcohol dependence induced a similar CRF responsivity in both sexes. In situ hybridization revealed that females had fewer CeA neurons containing mRNA for the CRF1 receptor (Crhr1) than males, but in dependence, the percentage of Crhr1-expressing neurons in females increased, unlike in males. Overall, our data provide evidence for sexually dimorphic CeA CRF system effects on GABAergic synapses in dependence.

Background: Individuals with alcohol use disorder (AUD) have a heightened risk of contracting HIV infection. The effects of these two diseases and their comorbidity on brain structure have been well described, but their effects on brain function have never been investigated at the scale of whole-brain connectomes.

Methods: In contrast with prior studies that restricted analyses to specific brain networks or examined relatively small groups of participants, our analyses are based on whole-brain functional connectomes of 292 participants.

Results: Relative to participants without AUD, the functional connectivity between the anterior cingulate cortex and orbitofrontal cortex was lower for participants with AUD. Compared with participants without AUD+HIV comorbidity, the functional connectivity between the anterior cingulate cortex and hippocampus was lower for the AUD+HIV participants. Compromised connectivity between these pairs was significantly correlated with greater total lifetime alcohol consumption; the effects of total lifetime alcohol consumption on executive functioning were significantly mediated by the functional connectivity between the pairs.

Conclusions: Taken together, our results suggest that the functional connectivity of the anterior cingulate cortex is disrupted in individuals with AUD alone and AUD with HIV infection comorbidity. Moreover, the affected connections are associated with deficits in executive functioning, including heightened impulsiveness.

Background: Alcohol use disorder (AUD) is a leading preventable cause of death. The central amygdala (CeA) is a hub for stress and AUD, while dysfunction of the noradrenaline stress system is implicated in AUD relapse.

Methods: Here, we investigated whether alcohol (ethanol) dependence and protracted withdrawal alter noradrenergic regulation of the amygdala in rodents and humans. Male adult rats were housed under control conditions, subjected to chronic intermittent ethanol vapor exposure to induce dependence, or withdrawn from chronic intermittent ethanol vapor exposure for 2 weeks, and ex vivo electrophysiology, biochemistry (catecholamine quantification by high-performance liquid chromatography), in situ hybridization, and behavioral brain-site specific pharmacology studies were performed. We also used real-time quantitative polymerase chain reaction to assess gene expression of α_1B, β₁, and β₂ adrenergic receptors in human postmortem brain tissue from men diagnosed with AUD and matched control subjects.

Results: We found that α₁ receptors potentiate CeA GABAergic (gamma-aminobutyric acidergic) transmission and drive moderate alcohol intake in control rats. In dependent rats, β receptors disinhibit a subpopulation of CeA neurons, contributing to their excessive drinking. Withdrawal produces CeA functional recovery with no change in local noradrenaline tissue concentrations, although there are some long-lasting differences in the cellular patterns of adrenergic receptor messenger RNA expression. In addition, postmortem brain analyses reveal increased α_1B receptor messenger RNA in the amygdala of humans with AUD.

Conclusions: CeA adrenergic receptors are key neural substrates of AUD. Identification of these novel mechanisms that drive alcohol drinking, particularly during the alcohol-dependent state, supports ongoing new medication development for AUD.

We previously showed that apremilast, an FDA-approved PDE4 inhibitor, selectively alters behavioral responses to ethanol and certain GABAergic drugs in a PKA-dependent manner in C57BL6/J mice. Here, we investigated if PKA phosphorylation of β3 GABAA receptor subunits is involved in apremilast regulation of ethanol, propofol, or diazepam responses. Apremilast prolonged rotarod ataxia and loss of the righting reflex by ethanol and propofol in wild-type mice, but not in β3-S408A/S409A knock-in mice. In contrast, apremilast hastened recovery from the ataxic and sedative effects of diazepam in both genotypes. These findings suggest that apremilast modulation of ethanol and propofol behaviors in wild-type mice is mediated by β3 subunit phosphorylation, whereas its actions on diazepam responses involve a different mechanism. The PKA inhibitor H-89 prevented apremilast modulation of ethanol-induced ataxia. Apremilast sensitized wild-type males to ethanol-induced ataxia and decreased acute functional tolerance (AFT) in females but had no effect in β3-S408A/S409A mice of either sex. These results could not be attributed to genotype differences in blood ethanol clearance. There were also no baseline genotype differences in ethanol consumption and preference in two different voluntary drinking procedures. However, the ability of apremilast to reduce ethanol consumption was diminished in β3-S408A/S409A mice. Our results provide strong evidence that PKA-dependent phosphorylation of β3 GABAA receptor subunits is an important mechanism by which apremilast increases acute sensitivity to alcohol, decreases AFT, and decreases ethanol drinking.