RESUMEN

The infralimbic cortex (IL) is part of the medial prefrontal cortex (mPFC), exerting top-down control over structures that are critically involved in the development of alcohol use disorder (AUD). Activity of the IL is tightly controlled by γ-aminobutyric acid (GABA) transmission, which is susceptible to chronic alcohol exposure and withdrawal. This inhibitory control is regulated by various neuromodulators, including 5-hydroxytryptamine (5-HT; serotonin). We used chronic intermittent ethanol vapor inhalation exposure, a model of AUD, in male Sprague-Dawley rats to induce alcohol dependence (Dep) followed by protracted withdrawal (WD; 2 weeks) and performed ex vivo electrophysiology using whole-cell patch clamp to study GABAergic transmission in layer V of IL pyramidal neurons. We found that WD increased frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs), whereas miniature IPSCs (mIPSCs; recorded in the presence of tetrodotoxin) were unaffected by either Dep or WD. The application of 5-HT (50 µM) increased sIPSC frequencies and amplitudes in naive and Dep rats but reduced sIPSC frequencies in WD rats. Additionally, 5-HT2A receptor antagonist M100907 and 5-HT2C receptor antagonist SB242084 reduced basal GABA release in all groups to a similar extent. The blockage of either 5-HT2A or 5-HT2C receptors in WD rats restored the impaired response to 5-HT, which then resembled responses in naive rats. Our findings expand our understanding of synaptic inhibition in the IL in AUD, indicating that antagonism of 5-HT2A and 5-HT2C receptors may restore GABAergic control over IL pyramidal neurons. SIGNIFICANCE STATEMENT: Impairment in the serotonergic modulation of GABAergic inhibition in the medial prefrontal cortex contributes to alcohol use disorder (AUD). We used a well-established rat model of AUD and ex vivo whole-cell patch-clamp electrophysiology to characterize the serotonin modulation of GABAergic transmission in layer V infralimbic (IL) pyramidal neurons in ethanol-naive, ethanol-dependent (Dep), and ethanol-withdrawn (WD) male rats. We found increased basal inhibition following WD from chronic alcohol and altered serotonin modulation. Exogenous serotonin enhanced GABAergic transmission in naive and Dep rats but reduced it in WD rats. 5-HT2A and 5-HT2C receptor blockage in WD rats restored the typical serotonin-mediated enhancement of GABAergic inhibition. Our findings expand our understanding of synaptic inhibition in the infralimbic neurons in AUD.

Asunto(s)

Alcoholismo , Etanol , Potenciales Postsinápticos Inhibidores , Corteza Prefrontal , Ratas Sprague-Dawley , Serotonina , Síndrome de Abstinencia a Sustancias , Transmisión Sináptica , Ácido gamma-Aminobutírico , Animales , Masculino , Serotonina/metabolismo , Ratas , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Alcoholismo/metabolismo , Alcoholismo/fisiopatología , Etanol/farmacología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Síndrome de Abstinencia a Sustancias/metabolismo , Síndrome de Abstinencia a Sustancias/fisiopatología , Ácido gamma-Aminobutírico/metabolismo , Neuronas GABAérgicas/efectos de los fármacos , Neuronas GABAérgicas/metabolismo , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo

RESUMEN

Chronic ethanol dependence and withdrawal activate corticotropin releasing factor (CRF)-containing GABAergic neurons in the medial prefrontal cortex (mPFC), which tightly regulate glutamatergic pyramidal neurons. Using male CRF1:GFP reporter mice, we recently reported that CRF1-expressing (mPFCCRF1+) neurons predominantly comprise mPFC prelimbic layer 2/3 pyramidal neurons, undergo profound adaptations following chronic ethanol exposure, and regulate anxiety and conditioned rewarding effects of ethanol. To explore the effects of acute and chronic ethanol exposure on glutamate transmission, the impact of chronic alcohol on spine density and morphology, as well as persistent changes in dendritic-related gene expression, we employed whole-cell patch-clamp electrophysiology, diOlistic labeling for dendritic spine analysis, and dendritic gene expression analysis to further characterize mPFCCRF1+ and mPFCCRF1- prelimbic layer 2/3 pyramidal neurons. We found increased glutamate release in mPFCCRF1+ neurons with ethanol dependence, which recovered following withdrawal. In contrast, we did not observe significant changes in glutamate transmission in neighboring mPFCCRF1- neurons. Acute application of 44 mM ethanol significantly reduced glutamate release onto mPFCCRF1+ neurons, which was observed across all treatment groups. However, this sensitivity to acute ethanol was only evident in mPFCCRF1- neurons during withdrawal. In line with alterations in glutamate transmission, we observed a decrease in total spine density in mPFCCRF1+ neurons during dependence, which recovered following withdrawal, while again no changes were observed in mPFCCRF- neurons. Given the observed decreases in mPFCCRF1+ stubby spines during withdrawal, we then identified persistent changes at the dendritic gene expression level in mPFCCRF1+ neurons following withdrawal that may underlie these structural adaptations. Together, these findings highlight the varying responses of mPFCCRF1+ and mPFCCRF1- cell-types to acute and chronic ethanol exposure, as well as withdrawal, revealing specific functional, morphological, and molecular adaptations that may underlie vulnerability to ethanol and the lasting effects of ethanol dependence.

RESUMEN

Post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) are often comorbid. Few treatments exist to reduce comorbid PTSD/AUD. Elucidating the mechanisms underlying their comorbidity could reveal new avenues for therapy. Here, we employed a model of comorbid PTSD/AUD, in which rats were subjected to a stressful shock in a familiar context followed by alcohol drinking. We then examined fear overgeneralization and irritability in these rats. Familiar context stress elevated drinking, increased fear overgeneralization, increased alcohol-related aggressive signs, and elevated peripheral stress hormones. We then examined transcripts of stress- and fear-relevant genes in the central amygdala (CeA), a locus that regulates stress-mediated alcohol drinking. Compared with unstressed rats, stressed rats exhibited increases in CeA transcripts for Crh and Fkbp5 and decreases in transcripts for Bdnf and Il18. Levels of Nr3c1 mRNA, which encodes the glucocorticoid receptor, increased in stressed males but decreased in stressed females. Transcripts of Il18 binding protein (Il18bp), Glp-1r, and genes associated with calcitonin gene-related peptide signaling (Calca, Ramp1, Crlr-1, and Iapp) were unaltered. Crh, but not Crhr1, mRNA was increased by stress; thus, we tested whether inhibiting CeA neurons that express corticotropin-releasing factor (CRF) suppress PTSD/AUD-like behaviors. We used Crh-Cre rats that had received a Cre-dependent vector encoding hM4D(Gi), an inhibitory Designer Receptors Exclusively Activated by Designer Drugs. Chemogenetic inhibition of CeA CRF neurons reduced alcohol intake but not fear overgeneralization or irritability-like behaviors. Our findings suggest that CeA CRF modulates PTSD/AUD comorbidity, and inhibiting CRF neural activity is primarily associated with reducing alcohol drinking but not trauma-related behaviors that are associated with PTSD/AUD.

RESUMEN

Protein kinase C epsilon (PKCε) regulates behavioural responses to ethanol and plays a role in anxiety-like behaviour, but knowledge is limited on downstream substrates of PKCε that contribute to these behaviours. We recently identified brain-specific serine/threonine-protein kinase 1 (BRSK1) as a substrate of PKCε. Here, we test the hypothesis that BRSK1 mediates responses to ethanol and anxiety-like behaviours that are also PKCε dependent. We used in vitro kinase assays to further validate BRSK1 as a substrate of PKCε and used Brsk1-/- mice to assess the role of BRSK1 in ethanol- and anxiety-related behaviours and in physiological responses to ethanol. We found that BRSK1 is phosphorylated by PKCε at a residue identified in a chemical genetic screen of PKCε substrates in mouse brain. Like Prkce-/- mice, male and female Brsk1-/- mice were more sensitive than wild-type to the acute sedative-hypnotic effect of alcohol. Unlike Prkce-/- mice, Brsk1-/- mice responded like wild-type to ataxic doses of ethanol. Although in Prkce-/- mice ethanol consumption and reward are reduced in both sexes, they were reduced only in female Brsk1-/- mice. Ex vivo slice electrophysiology revealed that ethanol-induced facilitation of GABA release in the central amygdala was absent in male Brsk1-/- mice similar to findings in male Prkce-/- mice. Collectively, these results indicate that BRSK1 is a target of PKCε that mediates some PKCε-dependent responses to ethanol in a sex-specific manner and plays a role distinct from PKCε in anxiety-like behaviour.

Asunto(s)

Etanol , Proteína Quinasa C-epsilon , Animales , Femenino , Masculino , Ratones , Ansiedad , Encéfalo/metabolismo , Etanol/farmacología , Ratones Endogámicos C57BL , Fenotipo , Proteína Quinasa C-epsilon/genética , Proteína Quinasa C-epsilon/metabolismo , Serina , Treonina/genética

RESUMEN

Large conductance potassium (BK) channels are among the most sensitive molecular targets of ethanol and genetic variations in the channel-forming α subunit have been nominally associated with alcohol use disorders. However, whether the action of ethanol at BK α influences the motivation to drink alcohol remains to be determined. To address this question, we first tested the effect of systemically administered BK channel modulators on voluntary alcohol consumption in C57BL/6J males. Penitrem A (blocker) exerted dose-dependent effects on moderate alcohol intake, while paxilline (blocker) and BMS-204352 (opener) were ineffective. Because pharmacological manipulations are inherently limited by non-specific effects, we then sought to investigate the behavioral relevance of ethanol's direct interaction with BK α by introducing in the mouse genome a point mutation known to render BK channels insensitive to ethanol while preserving their physiological function. The BK α K361N substitution prevented ethanol from reducing spike threshold in medial habenula neurons. However, it did not alter acute responses to ethanol in vivo, including ataxia, sedation, hypothermia, analgesia, and conditioned place preference. Furthermore, the mutation did not have reproducible effects on alcohol consumption in limited, continuous, or intermittent access home cage two-bottle choice paradigms conducted in both males and females. Notably, in contrast to previous observations made in mice missing BK channel auxiliary ß subunits, the BK α K361N substitution had no significant impact on ethanol intake escalation induced by chronic intermittent alcohol vapor inhalation. It also did not affect the metabolic and locomotor consequences of chronic alcohol exposure. Altogether, these data suggest that the direct interaction of ethanol with BK α does not mediate the alcohol-related phenotypes examined here in mice.

Asunto(s)

Consumo de Bebidas Alcohólicas , Etanol , Ratones Endogámicos C57BL , Animales , Etanol/farmacología , Masculino , Ratones , Consumo de Bebidas Alcohólicas/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Femenino

RESUMEN

Impairments in the function of the hypothalamic-pituitary-adrenal (HPA) axis and enhanced glucocorticoid receptor (GR) activity in the central amygdala (CeA) are critical mechanisms in the pathogenesis of alcohol use disorder (AUD). The GR antagonist mifepristone attenuates craving in AUD patients, alcohol consumption in AUD models, and decreases CeA γ-aminobutyric acid (GABA) transmission in alcohol-dependent rats. Previous studies suggest elevated GR activity in the CeA of male alcohol-preferring Marchigian-Sardinian (msP) rats, but its contribution to heightened CeA GABA transmission driving their characteristic post-dependent phenotype is largely unknown. We determined Nr3c1 (the gene encoding GR) gene transcription in the CeA in male and female msP and Wistar rats using in situ hybridization and studied acute effects of mifepristone (10 µM) and its interaction with ethanol (44 mM) on pharmacologically isolated spontaneous inhibitory postsynaptic currents (sIPSCs) and electrically evoked inhibitory postsynaptic potentials (eIPSPs) in the CeA using ex vivo slice electrophysiology. Female rats of both genotypes expressed more CeA GRs than males, suggesting a sexually dimorphic GR regulation of CeA activity. Mifepristone reduced sIPSC frequencies (GABA release) and eIPSP amplitudes in msP rats of both sexes, but not in their Wistar counterparts; however, it did not prevent acute ethanol-induced increase in CeA GABA transmission in male rats. In msP rats, GR regulates CeA GABAergic signaling under basal conditions, indicative of intrinsically active GR. Thus, enhanced GR function in the CeA represents a key mechanism contributing to maladaptive behaviors associated with AUD.

RESUMEN

Alcohol use disorder (AUD) and anxiety disorders are frequently comorbid and share dysregulated neuroimmune-related pathways. Here, we used our established rat model of comorbid post-traumatic stress disorder (PTSD)/AUD to characterize the interleukin 18 (IL-18) system in the central amygdala (CeA). Male and female rats underwent novel (NOV) and familiar (FAM) shock stress, or no stress (unstressed controls; CTL) followed by voluntary alcohol drinking and PTSD-related behaviors, then all received renewed alcohol access prior to the experiments. In situ hybridization revealed that the number of CeA positive cells for Il18 mRNA increased, while for Il18bp decreased in both male and female FAM stressed rats versus CTL. No changes were observed in Il18r1 expression across groups. Ex vivo electrophysiology showed that IL-18 reduced GABAA-mediated miniature inhibitory postsynaptic currents (mIPSCs) frequencies in CTL, suggesting reduced CeA GABA release, regardless of sex. Notably, this presynaptic effect of IL-18 was lost in both NOV and FAM males, while it persisted in NOV and FAM females. IL-18 decreased mIPSC amplitude in CTL female rats, suggesting postsynaptic effects. Overall, our results suggest that stress in rats with alcohol access impacts CeA IL-18-system expression and, in sex-related fashion, IL-18's modulatory function at GABA synapses.

Asunto(s)

Alcoholismo , Núcleo Amigdalino Central , Trastornos por Estrés Postraumático , Ratas , Masculino , Femenino , Animales , Alcoholismo/complicaciones , Núcleo Amigdalino Central/metabolismo , Interleucina-18/metabolismo , Etanol/farmacología , Consumo de Bebidas Alcohólicas , Ácido gamma-Aminobutírico/metabolismo

RESUMEN

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.

RESUMEN

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.

Asunto(s)

Neuralgia , Enfermedades Neuroinflamatorias , Enfermedades Neuroinflamatorias/inducido químicamente , Enfermedades Neuroinflamatorias/metabolismo , Etanol/toxicidad , Modelos Animales de Enfermedad , Neuralgia/inducido químicamente , Neuralgia/metabolismo , Dolor Crónico/inducido químicamente , Dolor Crónico/metabolismo , Ratones Endogámicos C57BL , Masculino , Femenino , Animales , Ratones , Conducta Animal

RESUMEN

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.

Asunto(s)

Alcoholismo , Núcleo Amigdalino Central , Animales , Núcleo Amigdalino Central/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Etanol/farmacología , Femenino , Humanos , Masculino , Ratas , Receptores de Hormona Liberadora de Corticotropina/genética , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica , Ácido gamma-Aminobutírico/farmacología

RESUMEN

Prefrontal circuits are thought to underlie aberrant emotion contributing to relapse in abstinence; however, the discrete cell-types and mechanisms remain largely unknown. Corticotropin-releasing factor and its cognate type-1 receptor, a prominent brain stress system, is implicated in anxiety and alcohol use disorder (AUD). Here, we tested the hypothesis that medial prefrontal cortex CRF1-expressing (mPFCCRF1+) neurons comprise a distinct population that exhibits neuroadaptations following withdrawal from chronic ethanol underlying AUD-related behavior. We found that mPFCCRF1+ neurons comprise a glutamatergic population with distinct electrophysiological properties and regulate anxiety and conditioned rewarding effects of ethanol. Notably, mPFCCRF1+ neurons undergo unique neuroadaptations compared to neighboring neurons including a remarkable decrease in excitability and glutamatergic signaling selectively in withdrawal, which is driven in part by the basolateral amygdala. To gain mechanistic insight into these electrophysiological adaptations, we sequenced the transcriptome of mPFCCRF1+ neurons and found that withdrawal leads to an increase in colony-stimulating factor 1 (CSF1) in this population. We found that selective overexpression of CSF1 in mPFCCRF1+ neurons is sufficient to decrease glutamate transmission, heighten anxiety, and abolish ethanol reinforcement, providing mechanistic insight into the observed mPFCCRF1+ synaptic adaptations in withdrawal that drive these behavioral phenotypes. Together, these findings highlight mPFCCRF1+ neurons as a critical site of enduring adaptations that may contribute to the persistent vulnerability to ethanol misuse in abstinence, and CSF1 as a novel target for therapeutic intervention for withdrawal-related negative affect.

Asunto(s)

Alcoholismo , Síndrome de Abstinencia a Sustancias , Humanos , Receptores de Hormona Liberadora de Corticotropina/genética , Etanol/farmacología , Alcoholismo/genética , Hormona Liberadora de Corticotropina , Neuronas , Ansiedad

RESUMEN

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 α1B, ß1, and ß2 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, ß 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 α1B 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.

Asunto(s)

Alcoholismo , Núcleo Amigdalino Central , Consumo de Bebidas Alcohólicas , Animales , Núcleo Amigdalino Central/metabolismo , Etanol/farmacología , Humanos , Masculino , Norepinefrina , ARN Mensajero , Ratas , Receptores Adrenérgicos/metabolismo

RESUMEN

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.

Asunto(s)

Alcoholismo/fisiopatología , Amígdala del Cerebelo/efectos de los fármacos , Neuronas GABAérgicas/efectos de los fármacos , Antagonistas de Hormonas/farmacología , Mifepristona/farmacología , Receptores de Glucocorticoides/antagonistas & inhibidores , Sinapsis/efectos de los fármacos , Amígdala del Cerebelo/fisiopatología , Animales , Neuronas GABAérgicas/fisiología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Sinapsis/fisiología

RESUMEN

A major barrier to remission from an alcohol use disorder (AUD) is the continued risk of relapse during abstinence. Assessing the neuroadaptations after chronic alcohol and repeated abstinence is important to identify mechanisms that may contribute to relapse. In this study, we used a rhesus macaque model of long-term alcohol use and repeated abstinence, providing a platform to extend mechanistic findings from rodents to primates. The central amygdala (CeA) displays elevated GABA release following chronic alcohol in rodents and in abstinent male macaques, highlighting this neuroadaptation as a conserved mechanism that may underlie excessive alcohol consumption. Here, we determined circulating interleukin-1ß (IL-1ß) levels, CeA transcriptomic changes, and the effects of IL-1ß and corticotropin releasing factor (CRF) signaling on CeA GABA transmission in male controls and abstinent drinkers. While no significant differences in peripheral IL-1ß or the CeA transcriptome were observed, pathway analysis identified several canonical immune-related pathways. We addressed this potential dysregulation of CeA immune signaling in abstient drinkers with an electrophysiological approach. We found that IL-1ß decreased CeA GABA release in controls while abstinent drinkers were less sensitive to IL-1ß's effects, suggesting adaptations in the neuromodulatory role of IL-1ß. In contrast, CRF enhanced CeA GABA release similarly in controls and abstinent drinkers, consistent with rodent studies. Notably, CeA CRF expression was inversely correlated with intoxication, suggesting that CRF levels during abstinence may predict future intoxication. Together, our findings highlight conserved and divergent actions of chronic alcohol on neuroimmune and stress signaling on CeA GABA transmission across rodents and macaques.

Asunto(s)

Abstinencia de Alcohol , Núcleo Amigdalino Central , Hormona Liberadora de Corticotropina , Interleucina-1beta , Transmisión Sináptica , Animales , Núcleo Amigdalino Central/fisiopatología , Hormona Liberadora de Corticotropina/metabolismo , Potenciales Postsinápticos Inhibidores , Interleucina-1beta/metabolismo , Macaca mulatta , Masculino , Ácido gamma-Aminobutírico/metabolismo

RESUMEN

Alcohol use disorder (AUD) and affective disorders are frequently comorbid and share underlying mechanisms that could be targets for comprehensive treatment. Post-traumatic stress disorder (PTSD) has high comorbidity with AUD, but comprehensive models of this overlap are nascent. We recently characterized a model of comorbid AUD and PTSD-like symptoms, wherein stressed rats receive an inhibitory avoidance (IA)-related footshock on two occasions followed by two-bottle choice (2BC) voluntary alcohol drinking. Stressed rats received the second footshock in a familiar (FAM, same IA box as the first footshock) or novel context (NOV, single-chambered apparatus); the FAM paradigm more effectively increased alcohol drinking in males and the NOV paradigm in females. During abstinence, stressed males displayed avoidance-like PTSD symptoms, and females showed hyperarousal-like PTSD symptoms. Rats in the model had altered spontaneous action potential-independent GABAergic transmission in the central amygdala (CeA), a brain region key in alcohol dependence and stress-related signaling. However, PTSD sufferers may have alcohol experience prior to their trauma. Here, we therefore modified our AUD/PTSD comorbidity model to provide 3 weeks of intermittent extended alcohol access before footshock and then studied the effects of NOV and FAM stress on drinking and PTSD phenotypes. NOV stress suppressed the escalation of alcohol intake and preference seen in male controls, but no stress effects were seen on drinking in females. Additionally, NOV males had decreased action potential-independent presynaptic GABA release and delayed postsynaptic GABAA receptor kinetics in the CeA compared to control and FAM males. Despite these changes to alcohol intake and CeA GABA signaling, stressed rats showed broadly similar anxiogenic-like behaviors to our previous comorbid model, suggesting decoupling of the PTSD symptoms from the AUD vulnerability for some of these animals. The collective results show the importance of alcohol history and trauma context in vulnerability to comorbid AUD/PTSD-like symptoms.

Asunto(s)

Alcoholismo , Trastornos por Estrés Postraumático , Consumo de Bebidas Alcohólicas/psicología , Alcoholismo/terapia , Animales , Comorbilidad , Femenino , Masculino , Fenotipo , Ratas , Trastornos por Estrés Postraumático/psicología

RESUMEN

AIMS: Alcohol use disorder (AUD) is linked to hyperactivity of brain stress systems, leading to withdrawal states which drive relapse. AUD differs among the sexes, as men are more likely to have AUD than women, but women progress from casual use to binge and heavy alcohol use more quickly and are more likely to relapse into repetitive episodes of heavy drinking. In alcohol dependence animal models of AUD, the central amygdala (CeA) functions as a hub of stress and anxiety processing and gamma-Aminobutyric acid (GABA)ergic signaling within the CeA is involved in dependence-induced increases in alcohol consumption. We have shown dysregulation of CeA GABAergic synaptic signaling in alcohol dependence animal models, but previous studies have exclusively used males. METHODS: Here, we used whole-cell patch clamp electrophysiology to examine basal CeA GABAergic spontaneous inhibitory postsynaptic currents (sIPSC) and the effects of acute alcohol in both naïve and alcohol dependent rats of both sexes. RESULTS: We found that sIPSC kinetics differ between females and males, as well as between naïve and alcohol-dependent animals, with naïve females having the fastest current kinetics. Additionally, we find differences in baseline current kinetics across estrous cycle stages. In contrast to the increase in sIPSC frequency routinely found in males, acute alcohol (11-88 mM) had no effect on sIPSCs in naïve females, however the highest concentration of alcohol increased sIPSC frequency in dependent females. CONCLUSION: These results provide important insight into sex differences in CeA neuronal function and dysregulation with alcohol dependence and highlight the need for sex-specific considerations in the development of effective AUD treatment.

Asunto(s)

Alcoholismo/fisiopatología , Núcleo Amigdalino Central/efectos de los fármacos , Ácido gamma-Aminobutírico/efectos de los fármacos , Animales , Etanol/farmacología , Femenino , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

Alcohol elicits a neuroimmune response in the brain contributing to the development and maintenance of alcohol use disorder (AUD). While pro-inflammatory mediators initiate and drive the neuroimmune response, anti-inflammatory mediators provide an important homeostatic mechanism to limit inflammation and prevent pathological damage. However, our understanding of the role of anti-inflammatory signaling on neuronal physiology in critical addiction-related brain regions and pathological alcohol-dependence induced behaviors is limited, precluding our ability to identify promising therapeutic targets. Here, we hypothesized that chronic alcohol exposure compromises anti-inflammatory signaling in the central amygdala, a brain region implicated in anxiety and addiction, consequently perpetuating a pro-inflammatory state driving aberrant neuronal activity underlying pathological behaviors. We found that alcohol dependence alters the global brain immune landscape increasing IL-10 producing microglia and T-regulatory cells but decreasing local amygdala IL-10 levels. Amygdala IL-10 overexpression decreases anxiety-like behaviors, suggesting its local role in regulating amygdala-mediated behaviors. Mechanistically, amygdala IL-10 signaling through PI3K and p38 MAPK modulates GABA transmission directly at presynaptic terminals and indirectly through alterations in spontaneous firing. Alcohol dependence-induces neuroadaptations in IL-10 signaling leading to an overall IL-10-induced decrease in GABA transmission, which normalizes dependence-induced elevated amygdala GABA transmission. Notably, amygdala IL-10 overexpression abolishes escalation of alcohol intake, a diagnostic criterion of AUD, in dependent mice. This highlights the importance of amygdala IL-10 signaling in modulating neuronal activity and underlying anxiety-like behavior and aberrant alcohol intake, providing a new framework for therapeutic intervention.

Asunto(s)

Alcoholismo , Consumo de Bebidas Alcohólicas , Animales , Ansiedad , Núcleo Amigdalino Central , Etanol , Interleucina-10 , Ratones , Ácido gamma-Aminobutírico

RESUMEN

Alcohol use disorder (AUD) and anxiety disorders are frequently comorbid and share mechanisms that could be therapeutic targets. To facilitate mechanistic studies, we adapted an inhibitory avoidance-based "2-hit" rat model of posttraumatic stress disorder (PTSD) and identified predictors and biomarkers of comorbid alcohol (ethanol)/PTSD-like symptoms in these animals. Stressed Wistar rats received a single footshock on two occasions. The first footshock occurred when rats crossed into the dark chamber of a shuttle box. Forty-eight hours later, rats received the second footshock in a familiar (FAM) or novel (NOV) context. Rats then received 4 weeks of two-bottle choice (2BC) ethanol access. During subsequent abstinence, PTSD-like behavior responses, GABAergic synaptic transmission in the central amygdala (CeA), and circulating cytokine levels were measured. FAM and NOV stress more effectively increased 2BC drinking in males and females, respectively. Stressed male rats, especially drinking-vulnerable individuals (≥0.8 g/kg average 2-h ethanol intake with >50% ethanol preference), showed higher fear overgeneralization in novel contexts, increased GABAergic transmission in the CeA, and a profile of increased G-CSF, GM-CSF, IL-13, IL-6, IL-17a, leptin, and IL-4 that discriminated between stress context (NOV > FAM > Control). However, drinking-resilient males showed the highest G-CSF, IL-13, and leptin levels. Stressed females showed increased acoustic startle and decreased sleep maintenance, indicative of hyperarousal, with increased CeA GABAergic transmission in NOV females. This paradigm promotes key features of PTSD, including hyperarousal, fear generalization, avoidance, and sleep disturbance, with comorbid ethanol intake, in a sex-specific fashion that approximates clinical comorbidities better than existing models, and identifies increased CeA GABAergic signaling and a distinct pro-hematopoietic, proinflammatory, and pro-atopic cytokine profile that may aid in treatment.

Asunto(s)

Alcoholismo , Citocinas/sangre , Neuronas GABAérgicas/fisiología , Factores Sexuales , Trastornos por Estrés Postraumático , Transmisión Sináptica , Consumo de Bebidas Alcohólicas , Amígdala del Cerebelo , Animales , Femenino , Masculino , Ratas , Ratas Wistar

RESUMEN

Excessive serotonin (5-HT) signaling plays a critical role in the etiology of alcohol use disorder. The central nucleus of the amygdala (CeA) is a key player in alcohol-dependence associated behaviors. The CeA receives dense innervation from the dorsal raphe nucleus, the major source of 5-HT, and expresses 5-HT receptor subtypes (e.g., 5-HT2C and 5-HT1A) critically linked to alcohol use disorder. Notably, the role of 5-HT regulating rat CeA activity in alcohol dependence is poorly investigated. Here, we examined neuroadaptations of CeA 5-HT signaling in adult, male Sprague Dawley rats using an established model of alcohol dependence (chronic intermittent alcohol vapor exposure), ex vivo slice electrophysiology and ISH. 5-HT increased frequency of sIPSCs without affecting postsynaptic measures, suggesting increased CeA GABA release in naive rats. In dependent rats, this 5-HT-induced increase of GABA release was attenuated, suggesting blunted CeA 5-HT sensitivity, which partially recovered in protracted withdrawal (2 weeks). 5-HT increased vesicular GABA release in naive and dependent rats but had split effects (increase and decrease) after protracted withdrawal indicative of neuroadaptations of presynaptic 5-HT receptors. Accordingly, 5-HT abolished spontaneous neuronal firing in naive and dependent rats but had bidirectional effects in withdrawn. Alcohol dependence and protracted withdrawal did not alter either 5-HT1A-mediated decrease of CeA GABA release or Htr1a expression but disrupted 5-HT2C-signaling without affecting Htr2c expression. Collectively, our study provides detailed insights into modulation of CeA activity by the 5-HT system and unravels the vulnerability of the CeA 5-HT system to chronic alcohol and protracted withdrawal.SIGNIFICANCE STATEMENT Elevated GABA signaling in the central nucleus of the amygdala (CeA) underlies key behaviors associated with alcohol dependence. The CeA is reciprocally connected with the dorsal raphe nucleus, the main source of serotonin (5-HT) in the mammalian brain, and excessive 5-HT signaling is critically implicated in the etiology of alcohol use disorder. Our study, using a well-established rat model of alcohol dependence, ex vivo electrophysiology and ISH, provides mechanistic insights into how both chronic alcohol exposure and protracted withdrawal dysregulate 5-HT signaling in the CeA. Thus, our study further expands our understanding of CeA cellular mechanisms involved in the pathophysiology of alcohol dependence and withdrawal.

Asunto(s)

Alcoholismo/metabolismo , Núcleo Amigdalino Central/metabolismo , Serotonina/metabolismo , Síndrome de Abstinencia a Sustancias/metabolismo , Transmisión Sináptica , Ácido gamma-Aminobutírico/metabolismo , Potenciales de Acción , Alcoholismo/fisiopatología , Animales , Núcleo Amigdalino Central/fisiología , Potenciales Postsinápticos Inhibidores , Masculino , Ratas , Ratas Sprague-Dawley , Síndrome de Abstinencia a Sustancias/fisiopatología