Characterization of the Hippocampal Neuroimmune Response to Binge-Like Ethanol Consumption in the Drinking in the Dark Model

Abstract

Objectives: Alcohol dependence leads to dysregulation of the neuroimmune system, but the effects of excessive alcohol consumption on key players of the neuroimmune response after episodic binge drinking in nondependence has not been readily assessed. These studies seek to determine how the neuroimmune system within the hippocampus responds to binge-like consumption prior to dependence or evidence of brain damage. Methods: C57BL/6J mice underwent the drinking in the dark (DID) paradigm to recapitulate binge consumption. Immunohistochemical techniques were employed to determine the effects of ethanol on cytokine and astrocyte responses within the hippocampus. Astrocyte activation was also assessed using qRT-PCR. Results: Our results indicated that binge-like ethanol consumption resulted in a 3.6-fold increase in the proinflammatory cytokine interleukin (IL)-1β immunoreactivity in various regions of the hippocampus. The opposite effect was seen in the anti-inflammatory cytokine IL-10. Binge-like consumption resulted in a 67% decrease in IL-10 immunoreactivity but had no effect on IL-4 or IL-6 compared with the water-drinking control group. Moreover, astrocyte activation occurred following ethanol exposure as GFAP immunoreactivity was increased over 120% in mice that experienced 3 cycles of ethanol binges. PCR analyses indicated that the mRNA increased by almost 4-fold after one cycle of DID, but this effect did not persist in abstinence. Conclusions: Altogether, these findings suggest that binge-like ethanol drinking prior to dependence causes dysregulation to the neuroimmune system. This altered neuroimmune state may have an impact on behavior but could also result in a heightened neuroimmune response that is exacerbated from further ethanol exposure or other immune-modulating events.

Chemogenetic manipulation of astrocytic signaling in the basolateral amygdala reduces binge‐like alcohol consumption in male mice

“…In fact, this is the first report to indicate that non-dependent binge drinking increases GFAP immunoreactivity in the amygdala following the DID model (Figure 1). Our previous report found a similar response in the hippocampus with increased GFAP density after multiple cycles of DID ethanol exposure (Grifasi, McIntosh, et al, 2019). This finding agrees with the work of others who have shown that binge consumption in the DID model is associated with gene network expression changes that include astrocytic genes that regulate the neuroimmune response and glutamate synthesis (Mulligan et al, 2011).…”

“…The few studies on astrocytes and non-dependent alcohol consumption that are published focus on the effects that alcohol has on the astrocytes (Grifasi, McIntosh, et al, 2019;Mulligan et al, 2011), but this study also examined the effects of altered astrocyte function on alcohol consumption. The major findings of our study are as follows:…”

“…Fluorescent immunohistochemical procedures were used to colabel DREADD and GFAP, but anti-GFAP was conjugated to 3,3′-Diaminobenzidine (DAB) for astrogliosis measures. Immunohistochemical procedures were similar to our previous publications (Grifasi, Evans, et al, 2019;Grifasi, McIntosh, et al, 2019;. For astrogliosis studies, mice were split into groups undergoing one cycle (water n = 5, sucrose n = 7; ethanol n = 7) or three cycles (water n = 5; sucrose n = 6; ethanol n = 8) of DID.…”

Repetitive binge-like consumption based on the Drinking-in-the-Dark model alters the microglial population in the mouse hippocampus

“…Immunoreactivity was measured with the open-source software Qupath 0.2.3 (University of Edinburg, Edinburg, UK) using experimenter-determined optical density thresholds [ 46 ]. Immunoreactivity is expressed as percent area (immunoreactive positive area/total area of ROI) [ 47 ]. Microglia were also assessed using profile cell counts of Iba-1 and are expressed as Iba-1+ cells/section [ 18 ].…”

“…Quantitative polymerase chain reaction (qRT-PCR) was performed similarly to our previous report [ 47 ]. Briefly, un-anesthetized mice were euthanized by rapid decapitation, and the hippocampus and amygdala were microdis-sected out and snap-frozen.…”