The basolateral amygdala (BLA) controls numerous behaviors, like anxiety and reward seeking, via the activity of glutamatergic principal neurons. These BLA neurons receive excitatory inputs primarily via two major anatomical pathways - the external capsule (EC), which contains afferents from lateral cortical structures, and the stria terminalis (ST), containing synapses from more midline brain structures. Chronic intermittent ethanol (CIE) exposure/withdrawal produces distinct alterations in these pathways. Specifically, 10 days of CIE (via vapor inhalation) increases presynaptic function at ST synapses and postsynaptic function at EC synapses. Given that 10-day CIE/withdrawal also increases anxiety-like behavior, we sought to examine the development of these alterations at these inputs using an exposure time-course in both male and female rats. Specifically, using 3, 7, and 10 days CIE exposure, we found that all three durations increase anxiety-like behavior in the elevated plus maze. At BLA synapses, increased presynaptic function at ST inputs required shorter exposure durations relative to post-synaptic alterations at EC inputs in both sexes. But, synaptic alterations in females required longer ethanol exposures compared to males. These data suggest that presynaptic alteration at ST-BLA afferents is an early neuroadaptation during repeated ethanol exposures. And, the similar patterns of presynaptic-then-postsynaptic facilitation across the sexes suggest the former may be required for the latter. These cooperative interactions may contribute to the increased anxiety-like behavior that is observed following CIE-induced withdrawal and may provide novel therapeutic targets to reverse withdrawal-induced anxiety.

The current experiment examined the effects of 10 days of chronic intermittent ethanol (CIE) exposure on anxiety-like behavior and home cage ethanol intake using a 20% intermittent access (M, W, F) paradigm in male and female Long-Evans rats. Withdrawal from alcohol dependence contributes to relapse in humans and increases in anxiety-like behavior and voluntary ethanol consumption in preclinical models. Our laboratory has shown that 10 days of CIE exposure produces both behavioral and neurophysiological alterations associated with withdrawal in male rats; however, we have yet to examine the effects of this exposure regime on ethanol intake in females. During baseline, females consumed more ethanol than males but, unlike males, did not show escalations in intake. Rats were then exposed to CIE and were again given intermittent access to 20% ethanol. CIE males increased their intake compared to baseline, whereas air-exposed males did not. Ethanol intake in females was unaffected by CIE exposure. Notably, both sexes expressed significantly elevated withdrawal-associated anxiety-like behavior in the plus maze. Finally, rats were injected with the cannabinoid CB1 receptor antagonist, SR141716A (0, 1, 3, 10 mg/kg, i.p.) which reduced ethanol intake in both sexes. However, females appear to be more sensitive to lower doses of this CB1 receptor antagonist. Our results show that females consume more ethanol than males; however, they did not escalate their intake using the intermittent access paradigm. Unlike males, CIE exposure had no effect on drinking in females. It is possible that females may be less sensitive than males to ethanol-induced increases in drinking after a short CIE exposure. Lastly, our results demonstrate that males and females may have different pharmacological sensitivities to CB1 receptor blockade on ethanol intake, at least under the current conditions.

Negative affect promotes dysregulated alcohol consumption in non-dependent and alcohol-dependent animals, and cues associated with negative affective states induce withdrawal-like symptoms in rats. This study was designed to test the hypotheses that: (1) the kappaopioid receptor (KOR) system mediates phenotypes related to alcohol withdrawal and withdrawal-like negative affective states and (2) cues associated with negative affective states would result in dysregulated alcohol consumption when subsequently presented alone. To accomplish these goals, intracerebroventricular infusion of the KOR antagonist nor-binaltorphimine (nor-BNI) was assessed for the ability to attenuate the increase in 22-kHz ultrasonic vocalizations (USVs) associated with alcohol withdrawal and KOR activation in adult male wistar rats. Furthermore, cues associated with a KOR agonist-induced negative affective state were assessed for the ability to dysregulate alcohol consumption and the efficacy of intracerebroventricular KOR antagonism to reduce such dysregulation was evaluated. KOR antagonism blocked the increased number of 22-kHz USVs observed during acute alcohol withdrawal and a KOR agonist (U50,488) resulted in a nor-BNI reversible increase in 22-kHz USVs (mimicking an alcohol-dependent state). Additionally, cues associated with negative affective states resulted in escalated alcohol self-administration, an effect that was nor-BNI sensitive. Taken together, this study implicates negative affective states induced by both alcohol withdrawal and conditioned stimuli as being produced, in part, by activity of the DYN/KOR system.

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. Significance StatementAccumulating evidence suggests that the medial prefrontal cortex (mPFC) and its projections to the basolateral amygdala (BLA) bidirectionally modulate fear-related behaviors. Since the neuronal circuits for fear and anxiety are thought to overlap, we sought to examine the role of dorsal and ventral subdivisions of the mPFC and their inputs to the BLA in regulating anxiety. Specifically, we focused on alcohol withdrawalinduced anxiety-like behavior, which is a commonly reported cause of relapse in humans with alcoholism. In our study, we used optogenetics and chemogenetics to demonstrate, for the first time, that withdrawal from chronic ethanol exposure strengthens dorsal mPFC (dmPFC) synapses, but weakens ventral mPFC synapses in the BLA and that inhibiting glutamate release from dmPFC terminals in the BLA reduces anxiety-like behavior. The authors declare no competing financial interests. Author contributions: M.M.M. and B.A.M. designed research; M.M.M., B.C.P., A.M.C., and N.J.A. performed research; M.M.M., B.C.P., N.J.A., and B.A.M. analyzed data; M.M.M. and B.A.M. wrote the paper.

Cocaine is a commonly abused central nervous system stimulant that enhances dopamine (DA) neurotransmission through its ability to block dopamine transporters (DATs). Recent evidence suggests there may be an interaction between DATs and D2/D3 autoreceptors that modulates cocaine's effects. The purpose of this study was to explore how D2/D3 autoreceptors modulate the ability of cocaine to inhibit DA uptake through DATs on presynaptic DA terminals. Using fast-scan cyclic voltammetry in brain slices containing the nucleus accumbens core from male and female C57BL/6J mice, we first sought to examine the effects of global autoreceptor blockade using the nonselective D2/D3 autoreceptor antagonist, raclopride. We found that the ability of cocaine to inhibit DA uptake was increased by raclopride and that this effect was consistent across sexes. Further, using D2 (L-741,626) or D3 (SB-277011-A) autoreceptor selective antagonists, we discovered that blockade of D3, but not D2, autoreceptors was responsible for the increased cocaine potency. Alterations in cocaine potency were attributable to alterations in uptake inhibition, rather than cocaine effects on vesicular DA release, suggesting that these results may be a product of a functional D3/DAT interaction apart from the canonical inhibitory actions of D3 autoreceptors on DA release. Additionally, application of D2 (sumanirole) and D3 (PD 128907) autoreceptor specific agonists had inverse effects, whereby D2 autoreceptor activation decreased cocaine potency and D3 autoreceptor activation had no effect. Together, these data show that dopamine autoreceptors dynamically regulate cocaine potency at the DAT, which is important for understanding cocaine's rewarding and addictive properties.