Chronic ethanol consumption exacerbates future stress‐enhanced fear learning, an effect mediated by dorsal hippocampal astrocytes

Abstract

Background: Alcohol use disorder (AUD) and post-traumatic stress disorder (PTSD) are highly comorbid, yet there is a lack of preclinical research investigating how prior ethanol (EtOH) dependence influences the development of a PTSD-like phenotype.Furthermore, the neuroimmune system has been implicated in the development of both AUD and PTSD, but the extent of glial involvement in this context remains unclear. A rodent model was developed to address this gap in the literature. Methods:We used a 15-day exposure to the 5% w/v EtOH low-fat Lieber-DeCarli liquid diet in combination with the stress-enhanced fear learning (SEFL) paradigm to investigate the effects of chronic EtOH consumption on the development of a PTSDlike phenotype. Next, we used a reverse transcription quantitative real-time polymerase chain reaction to quantify mRNA expression of glial cell markers GFAP (astrocytes) and CD68 (microglia) following severe footshock stress in EtOH-withdrawn rats.Finally, we tested the functional contribution of dorsal hippocampal (DH) astrocytes in the development of SEFL in EtOH-dependent rats using astrocyte-specific G i designer receptors exclusively activated by designer drugs (G i -DREADD).Results: Results demonstrate that chronic EtOH consumption and withdrawal exacerbate future SEFL. Additionally, we found significantly increased GFAP mRNA expression in the dorsal and ventral hippocampus and amygdalar complex following the severe stressor in EtOH-withdrawn animals. Finally, the stimulation of the astroglial G i -DREADD during EtOH withdrawal prevented the EtOH-induced enhancement of SEFL. Conclusions: Collectively, results indicate that prior EtOH dependence and withdrawal combined with a severe stressor potentiate future enhanced fear learning. Furthermore, DH astrocytes significantly contribute to this change in behavior. Overall, these studies provide insight into the comorbidity of AUD and PTSD and the potential neurobiological mechanisms behind increased susceptibility to a PTSD-like phenotype in individuals with AUD.

Sex-dependent factors of alcohol and neuroimmune mechanisms

“…Furthermore, an increase in GFAP immunostaining and morphological alterations of astrocytes were observed in the HPC in both males and females after 10 days of oral alcohol administration by gavage ( Kane et al, 2014 ). Similarly, using a low-fat Lieber-DeCarli liquid diet (5% w/v alcohol) combined with fear-conditioned stress increased GFAP mRNA expression in the HPC and amygdala in male rats ( Barkell et al, 2022 ). Astrocytic glutamate transporter (GLAST) knockout mice exhibited decreased alcohol consumption and preference in a two-bottle free-choice paradigm in both sexes ( Karlsson et al, 2012 ).…”

Evidence for an alcohol‐related “harm paradox” in individuals with internalizing disorders: Test and replication in two independent community samples

“…The “congenital” pathway to shared neurobiology (see above) is consistent with the notion that the presence of either INTD or AUD marks a neurobiological status conducive to the development of the other. Additionally, there are theoretical (e.g., Koob and Le Moal, 2008), clinical (e.g., Schuckit & Hesselbrock, 1994), and empirical (e.g., Barkell et al, 2022) observations suggesting that the neurobiological consequences of chronic alcohol use (a “scar”) can serve to increase the risk of developing INTD symptoms and possibly syndromes. While an INTD‐related harm paradox effect associated with AUD is implied by our hypothesis and important if true, studying this effect is beyond the scope of the present work.…”

“…The "congenital" pathway to shared neurobiology (see above) is consistent with the notion that the presence of either INTD or AUD marks a neurobiological status conducive to the development of the other. Additionally, there are theoretical (e.g., Koob andLe Moal, 2008), clinical (e.g., Schuckit &Hesselbrock, 1994), and empirical (e.g., Barkell et al, 2022)…”

Inhibiting CRF Projections from the Central Amygdala to Lateral Hypothalamus and Amygdala Deletion of CRF Alters Binge-Like Ethanol Drinking in a Sex-Dependent Manner

“…In order to confirm genetic deletion of CRF and CRF1R, brain collection, cryosectioning and FISH was performed as previously described 35 . RNA extraction, cDNA synthesis, and RT-qPCR were performed as previously described 36,37 . Please refer to extended methods on supplemental materials for full methodology.…”

“…Tissue punches were collected in bead homogenizing tubes containing 500 mL of TriReagent (Molecular Research Center, Cincinnati, OH). RNA extraction, cDNA synthesis, and RT-qPCR were performed as previously described (Barkell et al, 2022;Paniccia et al, 2018). Briefly, RNA from each sample was extracted and purified using the Qiagen RNEasy Tissue Mini Kit for RNA purification (Qiagen, Hilden, Germany).…”