Chronic Intermittent Ethanol Exposure Dysregulates Nucleus Basalis Magnocellularis Afferents in the Basolateral Amygdala.

Nucleus basalis magnocellularis (NBM) cholinergic projections to the basolateral amygdala (BLA) regulate the acquisition and consolidation of fear-like and anxiety-like behaviors. However, it is unclear whether the alterations in the NBM-BLA circuit promote negative affect during ethanol withdrawal (WD). Therefore, we performed ex vivo whole-cell patch-clamp electrophysiology in both the NBM and the BLA of male Sprague Dawley rats following 10 d of chronic intermittent ethanol (CIE) exposure and 24 h of WD. We found that CIE exposure and withdrawal enhanced the neuronal excitability of NBM putative "cholinergic" neurons. We subsequently used optogenetics to directly manipulate NBM terminal activity within the BLA and measure cholinergic modulation of glutamatergic afferents and BLA pyramidal neurons. Our findings indicate that CIE and withdrawal upregulate NBM cholinergic facilitation of glutamate release via activation of presynaptic nicotinic acetylcholine receptors (AChRs). Ethanol withdrawal-induced increases in NBM terminal activity also enhance BLA pyramidal neuron firing. Collectively, our results provide a novel characterization of the NBM-BLA circuit and suggest that CIE-dependent modifications to NBM afferents enhance BLA pyramidal neuron activity during ethanol withdrawal.

Chronic Ethanol Exposure Potentiates Cholinergic Neurotransmission in the Basolateral Amygdala.

Chronic intermittent ethanol (CIE) exposure dysregulates glutamatergic and GABAergic neurotransmission, facilitating basolateral amygdala (BLA) pyramidal neuron hyperexcitability and the expression of anxiety during withdrawal. It is unknown whether ethanol-induced alterations in nucleus basalis magnocellularis (NBM) cholinergic projections to the BLA mediate anxiety-related behaviors through direct modulation of GABA and glutamate afferents. Following 10 days of CIE exposure and 24 h of withdrawal, we recorded GABAergic and glutamatergic synaptic responses in BLA pyramidal neurons with electrophysiology, assessed total protein expression of cholinergic markers, and quantified acetylcholine and choline concentrations using a colorimetric assay. We measured α7 nicotinic acetylcholine receptor (nAChR) dependent modulation of presynaptic function at distinct inputs in AIR- and CIE-exposed BLA coronal slices as a functional read-out of cholinergic neurotransmission. CIE/withdrawal upregulates the endogenous activity of α7 nAChRs, facilitating release at both GABAergic' local' interneuron and glutamatergic synaptic responses to stria terminalis (ST) stimulation, with no effect at GABAergic lateral paracapsular cells (LPCs). CIE caused a three-fold increase in BLA acetylcholine concentration, with no changes in α7 nAChR or cholinergic marker expression. These data illustrate that α7 nAChR-dependent changes in presynaptic function serve as a proxy for CIE-dependent alterations in synaptic acetylcholine levels. Thus, cholinergic projections appear to mediate CIE-induced alterations at GABA/glutamate inputs.

Withdrawal from chronic ethanol exposure increases postsynaptic glutamate function of insular cortex projections to the rat basolateral amygdala.

A key feature of alcohol use disorder (AUD) is negative affect during withdrawal, which often contributes to relapse and is thought to be caused by altered brain function, especially in circuits that are important mediators of emotional behaviors. Both the agranular insular cortex (AIC) and the basolateral amygdala (BLA) regulate emotions and are sensitive to ethanol-induced changes in synaptic plasticity. The AIC and BLA are reciprocally connected; and the effects of chronic ethanol exposure on this circuit have yet to be explored. Here, we use a combination of optogenetics and electrophysiology to examine the pre- and postsynaptic changes that occur to AIC-BLA synapses following withdrawal from 7- or 10-days of chronic intermittent ethanol (CIE) exposure. While CIE/withdrawal did not alter presynaptic glutamate release probability from AIC inputs, withdrawal from 10, but not 7, days of CIE increased AMPA receptor-mediated postsynaptic function at these synapses. Additionally, NMDA receptor-mediated currents evoked by electrical stimulation of the external capsule, which contains AIC afferents, were also increased during withdrawal. Notably, a single subanesthetic dose of ketamine administered at the onset of withdrawal prevented the withdrawal-induced increases in both AMPAR and NMDAR postsynaptic function. Ketamine also prevented the withdrawal-induced increases in anxiety-like behavior measured using the elevated zero maze. Together, these findings suggest that chronic ethanol exposure increases postsynaptic function within the AIC-BLA circuit and that ketamine can prevent ethanol withdrawal-induced alterations in synaptic plasticity and negative affect.

Chronic Ethanol Differentially Modulates Glutamate Release from Dorsal and Ventral Prefrontal Cortical Inputs onto Rat Basolateral Amygdala Principal Neurons.

The medial prefrontal cortex (mPFC) and the basolateral amygdala (BLA) have strong reciprocal connectivity. Projections from the BLA to the mPFC can drive innate, anxiety-related behaviors, but it is unclear whether reciprocal projections from the mPFC to BLA have similar roles. Here, we use optogenetics and chemogenetics to characterize the neurophysiological and behavioral alterations produced by chronic ethanol exposure and withdrawal on dorsal mPFC (dmPFC) and ventral mPFC (vmPFC) medial prefrontal cortical terminals in the BLA. We exposed adult male Sprague Dawley rats to chronic intermittent ethanol (CIE) using vapor chambers, measured anxiety-like behavior on the elevated zero maze, and used electrophysiology to record glutamatergic and GABAergic responses in BLA principal neurons. We found that withdrawal from a 7 d CIE exposure produced opposing effects at dmPFC (increased glutamate release) and vmPFC (decreased glutamate release) terminals in the BLA. Chemogenetic inhibition of dmPFC terminals in the BLA attenuated the increased anxiety-like behavior we observed during withdrawal. These data demonstrate that chronic ethanol exposure and withdrawal strengthen the synaptic connections between the dmPFC and BLA but weakens the vmPFC-BLA pathway. Moreover, facilitation of the dmPFC-BLA pathway during withdrawal contributes to anxiety-like behavior. Given the opposing roles of dmPFC-BLA and vmPFC-BLA pathways in fear conditioning, our results suggest that chronic ethanol exposure simultaneously facilitates circuits involved in the acquisition of and diminishes circuits involved with the extinction of withdrawal-related aversive behaviors.