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

Abstract

The medial prefrontal cortex (mPFC) and the basolateral amygdala (BLA) have strong reciprocal connectivity. Projections from the BLA to the mPFC can bidirectionally modulate anxiety-related behaviors but it is unclear if the same is true for mPFC to BLA projections. Our laboratory is specifically interested in withdrawal-related anxiety-like behavior and the underlying synaptic plasticity. Here, we use optogenetics and chemogenetics to characterize the neurophysiological and behavioral alterations produced by chronic ethanol exposure and withdrawal on dorsal mPFC/prelimbic (dmPFC/PL) and ventral mPFC (vmPFC/IL) 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 (EZM), and used electrophysiology to record glutamatergic and GABAergic responses in BLA principal neurons. We found that 24-hour withdrawal following a 7-day CIE exposure significantly increased the glutamate release probability from PL/dmPFC terminals, but significantly decreases the glutamate release probability from IL/vmPFC terminals. Chemogenetic inhibition of PL/dmPFC terminals in the BLA attenuated the increased withdrawal-dependent, anxiety-like behavior. These data demonstrate that chronic ethanol exposure and withdrawal strengthens the PL/dmPFC -BLA pathway but weakens the IL/vmPFC -BLA pathway. Moreover, we provide novel evidence that the PL/dmPFC -BLA pathway can modulate anxiety-like behavior. These findings suggest that mPFC-BLA circuits known to regulate the acquisition of aversive behaviors are up-regulated by chronic ethanol while those involved with the extinction of these behaviors are down-regulated. Significance StatementAccumulating evidence suggests that the medial prefrontal cortex and its projections to the basolateral amygdala bidirectionally modulate fear-related behaviors. Since the neuronal circuits for fear and anxiety are thought to overlap, we sought to examine the role of prelimbic and infralimbic subdivisions of the medial prefrontal cortex and their inputs to the basolateral amygdala in regulating anxiety. Specifically, we focused on alcohol withdrawal-induced anxietylike behavior, which is a commonly reported cause of relapse in human alcoholics. In our study, we used optogenetics and chemogenetics to demonstrate, for the first time, that withdrawal from chronic ethanol exposure strengthens prelimbic synapses, but weakens infralimbic synapses in the basolateral amygdala and that inhibiting glutamate release from prelimbic terminal in the basolateral amygdala reduces anxiety-like behavior.

Downregulation of Synaptotagmin 1 in the Prelimbic Cortex Drives Alcohol-Associated Behaviors in Rats

“…Specifically, changes in regulation of BLA excitability have been associated with behavioral consequences that include increased anxiety, dysregulation of emotional responsiveness, and stressinduced relapse to drug use, behaviors commonly observed in patients with addictive disorders (37,40,41). In preclinical studies, hyperexcitability of the BLA has been observed after chronic intermittent alcohol exposure (42). Collectively, these data suggest that a history of alcohol dependence may modulate neuronal activity of the BLA through Syt1 modulation and that this mechanism contributes to behavioral consequences of dependence.…”

Sex- and Subtype-Specific Adaptations in Excitatory Signaling Onto Deep-Layer Prelimbic Cortical Pyramidal Neurons After Chronic Alcohol Exposure

“…As shown in Figure 3A, we observe a significant shift from facilitation towards RPR depression after alcohol exposure in Type-A PCs of male rats that is not observed in females (Figure 3B) [ [11,34], we hypothesized that reducing extracellular calcium would reduce release probability, observed as a shift towards RPR facilitation, to a greater degree in alcohol-exposed PCs. Figures 3C-E demonstrate that reducing extracellular calcium elicits a significant enhancement in RPR facilitation in both male and female Type-A cells [males: F (1,127) CalciumMod=93.4, p<0.0001: females: F (1,175) CalciumMod=81.26, p<0.0001] to equivalent levels between water/ethanol-exposed groups.…”

“…The vast interconnectivity of the PFC permits this region to influence a wide variety of behaviors. As such, altering prelimbic PFC signaling elicits a number of cognitive impairments including deficits in working memory [23,35] and the expression of anxiety-like behaviors [11,12,34,36]. On this basis, we therefore sought to determine whether the observed subtypespecific changes in deep-layer PCs after chronic alcohol exposure were associated with specific changes in behavior.…”

“…Indeed, others report that increased PFC signaling into the adjacent basolateral amygdala (BLA) during acute withdrawal is necessary and sufficient to elicit such behaviors [34], though again it is unclear from which PC subclass these terminals emanate. Considered together, it seems reasonable to hypothesize that hyperactivity specifically within Type-A cells diminishes the specificity of PFC top-down control over the expression of fear/anxiety-like behaviors, contributing to negative affect states characteristic of ethanol dependence.…”

Leveraging Neural Networks in Preclinical Alcohol Research

“…Specific neuronal ensembles critical for alcohol dependent drinking within the CEA were identified by the chemogenetic silencing of activated neurons using Daun02 [ 91 ]. Inhibition of dorsal mPFC projections to the BLA via chemogenetics reduced withdrawal symptoms associated with abstinence for alcohol dependence [ 132 ]. With chemogenetic methods, receptors are targeted via the injection of a ligand, resulting in a less specific time of effect.…”