Central amygdala corticotropin-releasing factor neurons promote hyponeophagia but do not control alcohol drinking in mice
Max Kreifeldt, Melissa A Herman, Harpreet Sidhu, Agbonlahor Okhuarobo, Giovana C Macedo,
Roxana Shahryari, Pauravi J Gandhi, Marisa Roberto, Candice Contet
Corticotropin-releasing factor (CRF) signaling in the central nucleus of the amygdala (CeA) plays a critical role in rodent models of excessive alcohol drinking. However, the source of CRF acting in the CeA during alcohol withdrawal remains to be identified. In the present study, we hypothesized that CeA CRF interneurons may represent a behaviorally relevant source of CRF to the CeA increasing motivation for alcohol via negative reinforcement. We first observed that Crh mRNA expression in the anterior part of the mouse CeA correlates positively with alcohol intake in C57BL/6J males with a history of chronic binge drinking followed by abstinence and increases upon exposure to chronic intermittent ethanol (CIE) vapor inhalation. We then found that chemogenetic activation of CeA CRF neurons in Crh-IRES-Cre mouse brain slices increases gamma-aminobutyric acid (GABA) release in the medial CeA, in part via CRF1 receptor activation. While chemogenetic stimulation exacerbated novelty-induced feeding suppression (NSF) in alcohol-naïve mice, thereby mimicking the effect of withdrawal from CIE, it had no effect on voluntary alcohol consumption, following either acute or chronic manipulation. Furthermore, chemogenetic inhibition of CeA CRF neurons did not affect alcohol consumption or NSF in chronic alcohol drinkers exposed to air or CIE. Altogether, these findings indicate that CeA CRF neurons produce local release of GABA and CRF and promote hyponeophagia in naïve mice, but do not drive alcohol intake escalation or negative affect in CIE-withdrawn mice. The latter result contrasts with previous findings in rats and demonstrates species specificity of CRF circuit engagement in alcohol dependence.
Figure 4. Chemogenetic stimulation of CeA CRF neurons does not affect alcohol drinking.
A. Crh-IRES-Cre male mice were injected with a Cre-dependent hM3Dq-encoding vector in the anterior CeA and were tested for voluntary ethanol intake under different experimental conditions. B. Experimental timeline. Each box represents one week, the color code indicates the experimental procedure conducted during that week. Two-bottle choice (2BC) drinking sessions were conducted Mon-Fri, starting at the beginning of the dark phase and lasting 2 h (blue boxes). Mice were given ten baselining sessions prior to testing the acute effect of CNO (0, 1, 5 and 10 mg/kg, i.p., 30-min pretreatment) according to a within-subject Latin-square design over four consecutive days (red arrows; data shown in panel C). An additional 2BC session without pretreatment was conducted and mice were then split in two groups exhibiting equivalent baseline ethanol intake, which were repeatedly injected with either CNO (5 mg/kg) or vehicle. Weeks of CNO or vehicle administration (once per day, Tue-Fri; red boxes) were alternated with weeks of 2BC drinking sessions (Mon-Fri, as described above; blue boxes; data shown in panel D) for a total of 3 rounds. Mice were then given a 3-week ethanol deprivation period (white boxes), after which 2BC sessions were resumed for a week (blue box; data shown in panel E). Next, the mice were exposed to four cycles of chronic intermittent ethanol exposure (16-h ethanol vapor inhalation followed by 8-h air inhalation, Mon-Fri; yellow box). The mice were then returned to their home cages and 2BC sessions resumed four days later (Tue-Fri; blue box; data shown in panel F). On the third session (Thu), CNO (5 mg/kg) or vehicle was administered 30 min prior to the session (red arrow). C-F. Ethanol intake is expressed in g ethanol per kg body weight in 2-h session. Data are shown as mean ± s.e.m. Number of mice per group is shown in the legend of each graph.