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.

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 (mPFC^CRF1+) neurons comprise a distinct population that exhibits neuroadaptations following withdrawal from chronic ethanol underlying AUD-related behavior. We found that mPFC^CRF1+ neurons comprise a glutamatergic population with distinct electrophysiological properties and regulate anxiety and conditioned rewarding effects of ethanol. Notably, mPFC^CRF1+ 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 mPFC^CRF1+ 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 mPFC^CRF1+ neurons is sufficient to decrease glutamate transmission, heighten anxiety, and abolish ethanol reinforcement, providing mechanistic insight into the observed mPFC^CRF1+ synaptic adaptations in withdrawal that drive these behavioral phenotypes. Together, these findings highlight mPFC^CRF1+ 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.

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.

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 naive, 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.

Purinergic P2X receptors (P2X) are ATP-gated ion channels widely expressed in the CNS. While the direct contribution of P2X to synaptic transmission is uncertain, P2X reportedly affect N-methyl-D-aspartate receptor (NMDAR) activity, which has given rise to competing theories on the role of P2X in the modulation of synapses. However, P2X have also been shown to participate in receptor cross-talk: an interaction where one receptor (e.g., P2X2) directly influences the activity of another (e.g., nicotinic, 5-HT3 or GABA receptors). In this study, we tested for interactions between P2X2 or P2X4 and NMDARs. Using two-electrode voltage-clamp electrophysiology experiments in Xenopus laevis oocytes, we demonstrate that both P2X2 and P2X4 interact with NMDARs in an inhibited manner. When investigating the molecular domains responsible for this phenomenon, we found that the P2X2 c-terminus (CT) could interfere with both P2X2 and P2X4 interactions with NMDARs. We also report that 11 distal CT residues on the P2X4 facilitate the P2X4–NMDAR interaction, and that a peptide consisting of these P2X4 CT residues (11C) can disrupt the interaction between NMDARs and P2X2 or P2X4. Collectively, these results provide new evidence for the modulatory nature of P2X2 and P2X4, suggesting they might play a more nuanced role in the CNS.

Significance: The 4 intravenous anesthetics currently in clinical use are associated with undesirable side effects such as lower blood pressure. These agents are poorly tolerated in young and elderly patients, limiting their use and increasing their risks. Additionally, etomidate, an anesthetic that achieves stable cardiovascular conditions, causes significant adrenal suppression by inhibiting steroid biosynthesis. Even though the exact mechanisms of anesthesia remain unknown, a significant component appears to be mediated by γ-aminobutyric acid type A receptors (GABA _A Rs). By targeting an anesthetic binding site in these receptors, we are designing anesthetics with reduced side effects. A unique agent based on this design is now shown to be potent and selective for GABA _A -slow receptors and produces anesthesia with minimal hemodynamic side effects.