Presynaptic CRF1 Receptors Mediate the Ethanol Enhancement of GABAergic Transmission in the Mouse Central Amygdala

Corticotropin-releasing factor (CRF) is a 41-amino-acid neuropeptide involved in stress responses initiated from several brain areas, including the amygdala formation. Research shows a strong relationship between stress, brain CRF, and excessive alcohol consumption. Behavioral studies suggest that the central amygdala (CeA) is significantly involved in alcohol reward and dependence. We recently reported that the ethanol augmentation of GABAergic synaptic transmission in rat CeA involves CRF1 receptors, because both CRF and ethanol significantly enhanced the amplitude of evoked GABAergic inhibitory postsynaptic currents (IPSCs) in CeA neurons from wild-type (WT) and CRF2 knockout (KO) mice, but not in neurons of CRF1 KO mice. The present study extends these findings using selective CRF receptor ligands, gene KO models, and miniature IPSC (mIPSC) analysis to assess further a presynaptic role for the CRF receptors in mediating ethanol effects in the CeA. In whole-cell patch recordings of pharmacologically isolated GABAAergic IPSCs from slices of mouse CeA, both CRF and ethanol augmented evoked IPSCs in a concentration-dependent manner, with low EC50s. A CRF1 (but not CRF2) KO construct and the CRF1-selective nonpeptide antagonist NIH-3 (LWH-63) blocked the augmenting effect of both CRF and ethanol on evoked IPSCs. Furthermore, the new selective CRF1 agonist stressin1, but not the CRF2 agonist urocortin 3, also increased evoked IPSC amplitudes. Both CRF and ethanol decreased paired-pulse facilitation (PPF) of evoked IPSCs and significantly enhanced the frequency, but not the amplitude, of spontaneous miniature GABAergic mIPSCs in CeA neurons of WT mice, suggesting a presynaptic site of action. The PPF effect of ethanol was abolished in CeA neurons of CRF1 KO mice. The CRF1 antagonist NIH-3 blocked the CRF- and ethanol-induced enhancement of mIPSC frequency in CeA neurons. These data indicate that presynaptic CRF1 receptors play a critical role in permitting or mediating ethanol enhancement of GABAergic synaptic transmission in CeA, via increased vesicular GABA release, and thus may be a rational target for the treatment of alcohol abuse and alcoholism

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

μ-Opioid Receptors Selectively Regulate Basal Inhibitory Transmission in the Central Amygdala: Lack of Ethanol Interactions

Endogenous opioid systems are implicated in the actions of ethanol. For example, μ-opioid receptor (MOR) knockout (KO) mice self-administer less alcohol than the genetically intact counterpart wild-type (WT) mice (Roberts et al., 2000). MOR KO mice also exhibit less anxiety-like behavior than WT mice (Filliol et al., 2000). To investigate the neurobiological mechanisms underlying these behaviors, we examined the effect of ethanol in brain slices from MOR KO and WT mice using sharp-electrode and whole-cell patch recording techniques. We focused our study in the central nucleus of the amygdala (CeA) because it is implicated in alcohol drinking behavior and stress behavior. We found that the amplitudes of evoked inhibitory postsynaptic currents (IPSCs) or inhibitory postsynaptic potentials (IPSPs) were significantly greater in MOR KO mice than WT mice. In addition, the baseline frequencies of spontaneous and miniature GABAA receptor-mediated inhibitory postsynaptic currents were significantly greater in CeA neurons from MOR KO than WT mice. However, ethanol enhancements of evoked IPSP and IPSC amplitudes and the frequency of miniature IPSCs were comparable between WT and MOR KO mice. Baseline spontaneous and miniature excitatory postsynaptic currents (EPSCs) and ethanol effects on EPSCs were not significantly different between MOR KO and WT mice. Based on knowledge of CeA circuitry and projections, we hypothesize that the role of MOR- and GABA receptor-mediated mechanisms in CeA underlying reinforcing effects of ethanol operate independently, possibly through pathway-specific responses within CeA