Corticosterone Levels and Glucocorticoid Receptor Gene Expression in High Drinking in the Dark Mice and Their Heterogeneous Stock (HS/NPT) Founder Line.

The High Drinking in the Dark (HDID-1) line of mice has been selectively bred for achieving high blood alcohol levels (BALs) in the Drinking in the Dark task, a model of binge-like drinking. Recently, we determined that glucocorticoid receptor (GR) antagonism with either mifepristone or CORT113176 (a selective GR antagonist) reduced binge-like ethanol intake in the HDID-1 mice, but not in their founder line, HS/NPT. Here, we examined whether the selection process may have altered glucocorticoid functioning by measuring (1) plasma corticosterone levels and (2) expression of the genes encoding GR (Nr3c1) and two of its chaperone proteins FKBP51 and FKBP52 (Fkbp5 and Fkbp4) in the brains (nucleus accumbens, NAc) of HDID-1 and HS/NPT mice. We observed no genotype differences in baseline circulating corticosterone levels. However, HDID-1 mice exhibited a greater stimulated peak corticosterone response to an IP injection (of either ethanol or saline) relative to their founder line. We further observed reduced basal expression of Fkbp4 and Nr3c1 in the NAc of HDID-1 mice relative to HS/NPT mice. Finally, HDID-1 mice exhibited reduced Fkbp5 expression in the NAc relative to HS/NPT mice following an injection of 2 g/kg ethanol. Together, these data suggest that selective breeding for high BALs may have altered stress signaling in the HDID-1 mice, which may contribute to the observed selective efficacy of GR antagonism in reducing binge-like ethanol intake in HDID-1, but not HS/NPT mice. These data have important implications for the role that stress signaling plays in the genetic risk for binge drinking.

Regulation of alcohol drinking by ventral striatum and extended amygdala circuitry.

Alcohol use disorder is a complex psychiatric disorder that can be modeled in rodents using a number of drinking paradigms. Drinking-in-the-dark (DID) is widely used to model the binge/intoxication stage of addiction, and chronic intermittent ethanol vapor procedures (CIE) are used to induce dependence and model withdrawal/negative affect induced escalation of drinking. We discuss experiments showing the ventral striatum (vStr) and extended amygdala (EA) are engaged in response to ethanol in rodents through c-Fos/Fos immunoreactivity studies. We also discuss experiments in rodents that span a wide variety of techniques where the function of vStr and EA structures are changed following DID or CIE, and the role of neurotransmitter and neuropeptide systems studies in these ethanol-related outcomes. We note where signaling systems converge across regions and paradigms and where there are still gaps in the literature. Dynorphin/κ-opioid receptor (KOR) signaling, as well as corticotropin releasing factor (CRF)/CRF receptor signaling were found to be important regulators of drinking behaviors across brain regions and drinking paradigms. Future research will require that females and a variety of rodent strains are used in preclinical experiments in order to strengthen the generalizability of findings and improve the likelihood of success for testing potential therapeutics in human laboratory studies.

Central nucleus of the amygdala projections onto the nucleus accumbens core regulate binge-like alcohol drinking in a CRF-dependent manner.

RATIONALE: The nucleus accumbens (NAc) is important for regulating a number of behaviors, including alcohol and substance use. We previously found that chemogenetically manipulating neuronal activity in the NAc core regulates binge-like drinking in mice. The central amygdala (CeA) is also an important regulator of alcohol drinking, and projects to the NAc core. We tested whether neuronal projections from the CeA to the NAc core, or neuropeptides released by the CeA in the NAc core, could regulate binge drinking. METHODS: For experiment 1, mice were administered AAV2 Cre-GFP into the NAc core and a Cre-inducible DREADD [AAV2 DIO- hM3Dq, -hM4Di, or -mCherry control] into the CeA. We tested the effects of altering CeA to NAc core activity on binge-like ethanol intake (via "Drinking in the Dark", DID). For experiment 2, we bilaterally microinfused corticotropin releasing factor (CRF), neuropeptide Y (NPY), or somatostatin (SST) into the NAc core prior to DID. For experiment 3, we tested whether intra-NAc CRF antagonism prevented reductions in drinking induced by CNO/hM3Dq stimulation of CeA->NAc projections. RESULTS: Chemogenetically increasing activity in neurons projecting from the CeA to NAc core decreased binge-like ethanol drinking (p < 0.01). Intra-NAc core CRF mimicked chemogenetic stimulation of this pathway (p < 0.05). Binge-like drinking was unaffected by the doses of NPY and SST tested. Lastly, we found that intra-NAc CRF antagonism prevented reductions in drinking induced by chemogenetic stimulation of CeA->NAc projections. These findings demonstrate that neurons projecting from the CeA to NAc core that release CRF are capable of regulating binge-like drinking in mice.

Effects of chemogenetic manipulation of the nucleus accumbens core in male C57BL/6J mice.

Binge drinking is a widespread public health concern with limited effective treatment options. To better select pharmaceutical targets, it is imperative to expand our knowledge of the underlying neural mechanisms involved in binge drinking. Our previous experiments in C57BL/6J female mice found that increasing activity in the nucleus accumbens (NAc) core using excitatory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) reduced binge-like drinking. These results differed from what has been found in males; however, it is unclear whether differences in experimental procedures or sex underlie these discrepancies. We matched the conditions used in our female study and asked whether bidirectional manipulation of NAc core activity has different effects on binge-like drinking in males. Male C57BL/6J mice were stereotaxically injected with AAV2 hSyn-HA hM3Dq (excitatory), -hM4Di (inhibitory), or -eGFP bilaterally into the NAc core. We tested the effects of altering NAc activity on binge-like ethanol intake using Drinking in the Dark (DID). During the first week, mice were pre-treated with vehicle to establish baseline ethanol intake. In week 2, mice were treated with 1 mg/kg CNO prior to DID to determine the effects of DREADD-induced changes in NAc core activity on ethanol intake. Decreasing activity via CNO/hM4Di significantly decreased binge-like drinking in male mice relative to eGFP and hM4Di groups. We also measured intake of sucrose, quinine, and water after CNO treatment and found that increasing NAc core activity via CNO/hM3Dq increased quinine intake, and increased water intake over time. We did not observe significant differences in the GFP or hM4Di groups. This work suggests there exist apparent sex-related differences in NAc core contributions to binge-like alcohol drinking, thus demonstrating the need for inclusion of both sexes in future work.