Glutamatergic transmission in the central nucleus of the amygdala is selectively altered in Marchigian Sardinian alcohol-preferring rats: Alcohol and CRF effects

The CRF system of the central nucleus of the amygdala (CeA) is important for the processing of anxiety, stress, and effects of acute and chronic ethanol. We previously reported that ethanol decreases evoked glutamate transmission in the CeA of Sprague Dawley rats and that ethanol dependence alters glutamate release in the CeA. Here, we examined the effects of ethanol, CRF and a CRF1 receptor antagonist on spontaneous and evoked glutamatergic transmission in CeA neurons from Wistar and Marchigian Sardinian Preferring (msP) rats, a rodent line genetically selected for excessive alcohol drinking and characterized by heightened activity of the CRF1 system. Basal spontaneous and evoked glutamate transmission in CeA neurons from msP rats was increased compared to Wistar rats. Ethanol had divergent effects, either increasing or decreasing spontaneous glutamate release in the CeA of Wistar rats. This bidirectional effect was retained in msP rats, but the magnitude of the ethanol-induced increase in glutamate release was significantly smaller. The inhibitory effect of ethanol on evoked glutamatergic transmission was similar in both strains. CRF also either increased or decreased spontaneous glutamate release in CeA neurons of Wistar rats, however, in msP rats CRF only increased glutamate release. The inhibitory effect of CRF on evoked glutamatergic transmission was also lost in neurons from msP rats. A CRF1 antagonist produced only minor effects on spontaneous glutamate transmission, which were consistent across strains, and no effects on evoked glutamate transmission. These results demonstrate that the genetically altered CRF system of msP rats results in alterations in spontaneous and stimulated glutamate signaling in the CeA that may contribute to both the anxiety and drinking behavioral phenotypes

Genetic and Pharmacologic Manipulation of TLR4 Has Minimal Impact on Ethanol Consumption in Rodents

Toll-like receptor 4 (TLR4) is a critical component of innate immune signaling and has been implicated in alcohol responses in preclinical and clinical models. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium tested the hypothesis that TLR4 mediates excessive ethanol drinking using the following models: (1) Tlr4 knock-out (KO) rats, (2) selective knockdown of Tlr4 mRNA in mouse nucleus accumbens (NAc), and (3) injection of the TLR4 antagonist (+)-naloxone in mice. Lipopolysaccharide (LPS) decreased food/water intake and body weight in ethanol-naive and ethanol-trained wild-type (WT), but not Tlr4 KO rats. There were no consistent genotypic differences in two-bottle choice chronic ethanol intake or operant self-administration in rats before or after dependence. In mice, (+)-naloxone did not decrease drinking-in-the-dark and only modestly inhibited dependence-driven consumption at the highest dose. Tlr4 knockdown in mouse NAc did not decrease drinking in the two-bottle choice continuous or intermittent access tests. However, the latency to ethanol-induced loss of righting reflex increased and the duration decreased in KO versus WT rats. In rat central amygdala neurons, deletion of Tlr4 altered GABAA receptor function, but not GABA release. Although there were no genotype differences in acute ethanol effects before or after chronic intermittent ethanol exposure, genotype differences were observed after LPS exposure. Using different species and sexes, different methods to inhibit TLR4 signaling, and different ethanol consumption tests, our comprehensive studies indicate that TLR4 may play a role in ethanol-induced sedation and GABAA receptor function, but does not regulate excessive drinking directly and would not be an effective therapeutic target., SIGNIFICANCE STATEMENT Toll-like receptor 4 (TLR4) is a key mediator of innate immune signaling and has been implicated in alcohol responses in animal models and human alcoholics. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium participated in the first comprehensive study across multiple laboratories to test the hypothesis that TLR4 regulates excessive alcohol consumption in different species and different models of chronic, dependence-driven, and binge-like drinking. Although TLR4 was not a critical determinant of excessive drinking, it was important in the acute sedative effects of alcohol. Current research efforts are directed at determining which neuroimmune pathways mediate excessive alcohol drinking and these findings will help to prioritize relevant pathways and potential therapeutic targets

Alcohol Dependence Disrupts Amygdalar L-Type Voltage-Gated Calcium Channel Mechanisms

L-type voltage-gated calcium channels (LTCCs) are implicated in several psychiatric disorders that are comorbid with alcoholism and involve amygdala dysfunction. Within the amygdala, the central nucleus (CeA) is critical in acute alcohol's reinforcing actions, and its dysregulation in human alcoholics drives their negative emotional state and motivation to drink. Here we investigated the specific role of CeA LTCCs in the effects of acute alcohol at the molecular, cellular physiology, and behavioral levels, and their potential neuroadaptation in alcohol-dependent rats. Alcohol increases CeA activity (neuronal firing rates and GABA release) in naive rats by engaging LTCCs, and intra-CeA LTCC blockade reduces alcohol intake in nondependent rats. Alcohol dependence reduces CeA LTCC membrane abundance and disrupts this LTCC-based mechanism; instead, corticotropin-releasing factor type 1 receptors (CRF1s) mediate alcohol's effects on CeA activity and drive the escalated alcohol intake of alcohol-dependent rats. Collectively, our data indicate that alcohol dependence functionally alters the molecular mechanisms underlying the CeA's response to alcohol (from LTCC- to CRF1-driven). This mechanistic switch contributes to and reflects the prominent role of the CeA in the negative emotional state that drives excessive drinking.SIGNIFICANCE STATEMENT The central amygdala (CeA) plays a critical role in the development of alcohol dependence. As a result, much preclinical alcohol research aims to identify relevant CeA neuroadaptions that promote the transition to dependence. Here we report that acute alcohol increases CeA neuronal activity in naive rats by engaging L-type calcium channels (LTCCs) and that intra-CeA LTCC blockade reduces alcohol intake in nondependent rats. Alcohol dependence disrupts this LTCC-based mechanism; instead, corticotropin-releasing factor type 1 receptors (CRF1s) mediate alcohol's effects on CeA activity and drive the escalated alcohol intake of alcohol-dependent rats. This switch reflects the important role of the CeA in the pathophysiology of alcohol dependence and represents a new potential avenue for therapeutic intervention during the transition period

Alcohol Dependence Disrupts Amygdalar L-Type Voltage-Gated Calcium Channel Mechanisms

L-type voltage-gated calcium channels (LTCCs) are implicated in several psychiatric disorders that are comorbid with alcoholism and involve amygdala dysfunction. Within the amygdala, the central nucleus (CeA) is critical in acute alcohol's reinforcing actions, and its dysregulation in human alcoholics drives their negative emotional state and motivation to drink. Here we investigated the specific role of CeA LTCCs in the effects of acute alcohol at the molecular, cellular physiology, and behavioral levels, and their potential neuroadaptation in alcohol-dependent rats. Alcohol increases CeA activity (neuronal firing rates and GABA release) in naive rats by engaging LTCCs, and intra-CeA LTCC blockade reduces alcohol intake in nondependent rats. Alcohol dependence reduces CeA LTCC membrane abundance and disrupts this LTCC-based mechanism; instead, corticotropin-releasing factor type 1 receptors (CRF1s) mediate alcohol's effects on CeA activity and drive the escalated alcohol intake of alcohol-dependent rats. Collectively, our data indicate that alcohol dependence functionally alters the molecular mechanisms underlying the CeA's response to alcohol (from LTCC- to CRF1-driven). This mechanistic switch contributes to and reflects the prominent role of the CeA in the negative emotional state that drives excessive drinking.SIGNIFICANCE STATEMENT The central amygdala (CeA) plays a critical role in the development of alcohol dependence. As a result, much preclinical alcohol research aims to identify relevant CeA neuroadaptions that promote the transition to dependence. Here we report that acute alcohol increases CeA neuronal activity in naive rats by engaging L-type calcium channels (LTCCs) and that intra-CeA LTCC blockade reduces alcohol intake in nondependent rats. Alcohol dependence disrupts this LTCC-based mechanism; instead, corticotropin-releasing factor type 1 receptors (CRF1s) mediate alcohol's effects on CeA activity and drive the escalated alcohol intake of alcohol-dependent rats. This switch reflects the important role of the CeA in the pathophysiology of alcohol dependence and represents a new potential avenue for therapeutic intervention during the transition period

Nociceptin/Orphanin FQ Decreases Glutamate Transmission and Blocks Ethanol-Induced Effects in the Central Amygdala of Naive and Ethanol-Dependent Rats

The central nucleus of the amygdala (CeA) mediates several addiction-related processes and nociceptin/orphanin FQ (nociceptin) regulates ethanol intake and anxiety-like behaviors. Glutamatergic synapses, in the CeA and throughout the brain, are very sensitive to ethanol and contribute to alcohol reinforcement, tolerance, and dependence. Previously, we reported that in the rat CeA, acute and chronic ethanol exposures significantly decrease glutamate transmission by both pre- and postsynaptic actions. In this study, using electrophysiological techniques in an in vitro CeA slice preparation, we investigated the effects of nociceptin on glutamatergic transmission and its interaction with acute ethanol in naive and ethanol-dependent rats. We found that nociceptin (100-1000 nM) diminished basal-evoked compound glutamatergic receptor-mediated excitatory postsynaptic potentials (EPSPs) and spontaneous and miniature EPSCs (s/mEPSCs) by mainly decreasing glutamate release in the CeA of naive rats. Notably, nociceptin blocked the inhibition induced by acute ethanol (44 mM) and ethanol blocked the nociceptin-induced inhibition of evoked EPSPs in CeA neurons of naive rats. In neurons from chronic ethanol-treated (ethanol-dependent) rats, the nociceptin-induced inhibition of evoked EPSP amplitude was not significantly different from that in naive rats. Application of [Nphe1]Nociceptin(1-13)NH2, a nociceptin receptor (NOP) antagonist, revealed tonic inhibitory activity of NOP on evoked CeA glutamatergic transmission only in ethanol-dependent rats. The antagonist also blocked nociceptin-induced decreases in glutamatergic responses, but did not affect ethanol-induced decreases in evoked EPSP amplitude. Taken together, these studies implicate a potential role for the nociceptin system in regulating glutamatergic transmission and a complex interaction with ethanol at CeA glutamatergic synapses

Chronic alcohol exposure disrupts CB₁ regulation of GABAergic transmission in the rat basolateral amygdala

The basolateral nucleus of the amygdala (BLA) is critical to the pathophysiology of anxiety-driven alcohol drinking and relapse. The endogenous cannabinoid/type 1 cannabinoid receptor (eCB/CB(1)) system curbs BLA-driven anxiety and stress responses via a retrograde negative feedback system that inhibits neurotransmitter release, and BLA CB(1) activation reduces GABA release and drives anxiogenesis. Additionally, decreased amygdala CB(1) is observed in abstinent alcoholic patients and ethanol withdrawn rats. Here we investigated the potential disruption of eCB/CB(1) signaling on GABAergic transmission in BLA pyramidal neurons of rats exposed to 2–3 weeks intermittent ethanol. In the naïve rat BLA, the CB(1) agonist WIN 55,212-2 (WIN) decreased GABA release and this effect was prevented by the CB(1) antagonist AM251. AM251 alone increased GABA release via a mechanism requiring postsynaptic calcium-dependent activity. This retrograde tonic eCB/CB(1) signaling was diminished in chronic ethanol exposed rats, suggesting a functional impairment of the eCB/CB(1) system. In contrast, acute ethanol increased GABAergic transmission similarly in naïve and chronic ethanol exposed rats, via both pre- and postsynaptic mechanisms. Notably, CB(1) activation impaired ethanol’s facilitation of GABAergic transmission across both groups, but the AM251- and ethanol-induced facilitation of GABA release were additive, suggesting independent presynaptic sites of action. Collectively, the present findings highlight a critical CB(1) influence on BLA GABAergic transmission that is dysregulated by chronic ethanol exposure and thus, may contribute to the alcohol dependent state

Chronic ethanol exposure decreases CB₁ receptor function at GABAergic synapses in the rat central amygdala

The endogenous cannabinoids (eCBs) influence the acute response to ethanol and the development of tolerance, dependence and relapse. Chronic alcohol exposure alters eCB levels and type 1 cannabinoid receptor (CB(1)) expression and function in brain regions associated with addiction. CB(1) inhibits GABA release, and GABAergic dysregulation in the central nucleus of the amygdala (CeA) is critical in the transition to alcohol dependence. We investigated possible disruptions in CB(1) signaling of rat CeA GABAergic transmission following intermittent ethanol exposure. In the CeA of alcohol-naïve rats, CB(1) agonist WIN 55,212-2 (WIN) decreased the frequency of spontaneous and miniature GABA(A) receptor-mediated inhibitory postsynaptic currents (s/mIPSCs). This effect was prevented by CB(1) antagonism, but not type 2 cannabinoid receptor (CB(2)) antagonism. After 2–3 weeks of intermittent ethanol exposure, these WIN inhibitory effects were attenuated, suggesting ethanol-induced impairments in CB(1) function. The CB(1) antagonist AM251 revealed a tonic eCB/CB(1) control of GABAergic transmission in the alcohol-naïve CeA that was occluded by calcium chelation in the postsynaptic cell. Chronic ethanol exposure abolished this tonic CB(1) influence on mIPSC, but not sIPSC, frequency. Finally, acute ethanol increased CeA GABA release in both naïve and ethanol exposed rats. Although CB(1) activation prevented this effect, the AM251- and ethanol-induced GABA release were additive, ruling out a direct participation of CB(1) signaling in the ethanol effect. Collectively, these observations demonstrate an important CB(1) influence on CeA GABAergic transmission and indicate that the CeA is particularly sensitive to alcohol-induced disruptions of CB(1) signaling

Interleukin-1 Interacts with Ethanol Effects on GABAergic Transmission in the Mouse Central Amygdala

Neuroinflammation is hypothesized to enhance alcohol consumption and contribute to the development of alcoholism. GABAergic transmission in the central amygdala (CeA) plays an important role in the transition to alcohol dependence. Therefore, we studied the effects of interleukin-1β (IL-1β) a proinflammatory cytokine mediating ethanol-induced neuroinflammation, and its interaction with ethanol on CeA GABAegic transmission in B6129SF2/J mice. We also assessed ethanol intake in B6129SF2/J mice. Intake with unlimited (24 hours) ethanol access was 9.2-12.7 g/kg (3-15% ethanol), while limited (2 hours) access produced an intake of 4.1±0.5 g/kg (15% ethanol). In our electrophysiology experiments, we found that recombinant IL-1β (50 and 100 ng/ml) significantly decreased the amplitude of evoked inhibitory postsynaptic potentials (eIPSPs), with no significant effects on paired-pulse facilitation (PPF). IL-1beta (50 ng/ml) had dual effects on spontaneous miniature inhibitory postsynaptic currents (mIPSCs): increasing mIPSC frequencies in most CeA neurons, but decreasing both mIPSC frequencies and amplitudes in a few cells. The IL-1β receptor antagonist (IL-1ra; 100 ng/ml) also had dual effects on mIPSCs and prevented the actions of IL-1β on mIPSC frequencies. These results suggest that IL-1β can alter CeA GABAergic transmission at pre- and postsynaptic sites. Ethanol (44 mM) significantly increased eIPSP amplitudes, decreased PPFs, and increased mIPSC frequencies. IL-1β did not alter ethanol’s enhancement of the eIPSP amplitude, but, in IL-1β-responsive neurons, the ethanol effects on mIPSC frequencies were lost. Overall, our data suggest that the IL-1 system is involved in basal GABAergic transmission and that IL-1β interacts with the ethanol-induced facilitation of CeA GABAergic transmission

Role of MyD88 in IL-1β and Ethanol Modulation of GABAergic Transmission in the Central Amygdala

Myeloid differentiation primary response protein (MyD88) is a critical neuroimmune adaptor protein in TLR (Toll-like receptor) and IL-1R (Interleukin-1 receptor) signaling complexes. These two pro-inflammatory families play an important role in the neurobiology of alcohol use disorder, specifically MyD88 regulates ethanol drinking, ethanol-induced sedation, and ethanol-induced deficits in motor coordination. In this study, we examined the role of MyD88 in mediating the effects of IL-1β and ethanol on GABAergic transmission in the central amygdala (CeA) of male mice using whole-cell patch-clamp recordings in combination with pharmacological (AS-1, a mimetic that prevents MyD88 recruitment by IL-1R) and genetic (Myd88 knockout mice) approaches. We demonstrate through both approaches that IL-1β and ethanol’s modulatory effects at CeA GABA synapses are not dependent on MyD88. Myd88 knockout potentiated IL-1β’s actions in reducing postsynaptic GABAA receptor function. Pharmacological inhibition of MyD88 modulates IL-1β’s action at CeA GABA synapses similar to Myd88 knockout mice. Additionally, ethanol-induced CeA GABA release was greater in Myd88 knockout mice compared to wildtype controls. Thus, MyD88 is not essential to IL-1β or ethanol regulation of CeA GABA synapses but plays a role in modulating the magnitude of their effects, which may be a potential mechanism by which it regulates ethanol-related behaviors

Cessation of fluoxetine treatment increases alcohol seeking during relapse and dysregulates endocannabinoid and glutamatergic signaling in the central amygdala.

Administration of selective serotonin reuptake inhibitors (SSRIs), typically used as antidepressants, induces long-lasting behavioral changes associated with alcohol use disorder (AUD). However, the contribution of SSRI (fluoxetine)-induced alterations in neurobiological processes underlying alcohol relapse such as endocannabinoid and glutamate signaling in the central amygdala (CeA) remains largely unknown. We utilized an integrative approach to study the effects of repeated fluoxetine administration during abstinence on ethanol drinking. Gene expression and biochemical and electrophysiological studies explored the hypothesis that dysregulation in glutamatergic and endocannabinoid mechanisms in the CeA underlie the susceptibility to alcohol relapse. Cessation of daily treatment with fluoxetine (10 mg/kg) during abstinence resulted in a marked increase in ethanol seeking during re-exposure periods. The increase in ethanol self-administration was associated with (a) reductions in levels of the endocannabinoids N-arachidonoylethanolomine and 2-arachidonoylglycerol in the CeA, (b) increased amygdalar gene expression of cannabinoid type-1 receptor (CB1), N-acyl phosphatidylethanolamine phospholipase D (Nape-pld), fatty acid amid hydrolase (Faah), (c) decreased amygdalar gene expression of ionotropic AMPA (GluA2 and GluA4) and metabotropic (mGlu3) glutamate receptors, and (d) increased glutamatergic receptor function. Overall, our data suggest that the administration of the antidepressant fluoxetine during abstinence dysregulates endocannabinoid signaling and glutamatergic receptor function in the amygdala, facts that likely facilitate alcohol drinking behavior during relapse