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.

Keywords: Alcohol abuse; Astrocyte activation; Binge drinking; Cytokine; Hippocampus; Neuroimmune.

Fig 1.

Representative photomicrographs at 100X of IL-1β within the hippocampus of mice exposed to water (a), 3 sucrose DID cycles (b), 1 ethanol DID cycle (c), and 3 ethanol DID cycles (d) are shown. The inset of panel d shows a cell expressing IL-1β at 400X.Our analyses indicated that 3 cycles of ethanol resulted in an increase in IL-1β immunoreactivity within the DG (h) andCA1 (i) but not the CA2/3(j). Moreover, no statically significant differences were observed between water and sucrose drinking group in any subregion of the hippocampus (e-g). The scale bar =10 μm (c). Each photomicrograph has traces outlining the hippocampal fissure. All data are presented as mean ± SEM. *p < 0.05 compared to water group.

Fig 2.

Representative photomicrographs of IL-6 within the hippocampus of mice exposed to water (a), 3 sucrose DID cycles (b), 1 ethanol DID cycle (c), and 3 ethanol DID cycles (d) are shown. The inset of panel c shows a cell expressing IL-6 at 400X. Analyses indicated that neither ethanol (h-j) nor sucrose (e-g) had any effect on IL-6 expression within the regions of the hippocampus compared with the water control. The scale bar =10 μm (c). Each photomicrograph has traces outlining the hippocampal fissure.

Fig 3.

Representative photomicrographs of IL-10 within the hippocampus of mice exposed to water (a), 3 sucrose DID cycles (b), 1 ethanol DID cycle (c), and 3 ethanol DID cycles (d) are shown. The inset of panel b shows a cluster of cells expressing IL-10 at 400X. Analyses indicated that 3 cycles of ethanol reduced IL-10 within the CA1 (i), but this effect of ethanol was not present in either the DG (h) or the CA2/3(j). Moreover, no statically significant differences were observed between water and sucrose drinking group in any subregion of the hippocampus (e-g). The scale bar =10 μm (c). Each photomicrograph has traces outlining the hippocampal fissure. All data are presented as mean ± SEM. *p < 0.05 compared to water group.

Fig 4.

Representative photomicrographs of IL-4 within the hippocampus of mice exposed to water (a), 3 sucrose DID cycles (b), 1 ethanol DID cycle (c), and 3 ethanol DID cycles (d) are shown. The inset of panel a shows a cell expressing IL-4 at 400X. Analyses indicated that neither ethanol (h-j) nor sucrose (e-g) had any effect on IL-4 expression within the regions of the hippocampus. The scale bar =10 μm (c). Each photomicrograph has traces outlining the hippocampal fissure.

Fig 5.

Representative photomicrographs of astrocytes within the dentate gyrus of mice exposed to water (a), 3 sucrose DID cycles (b), 1 ethanol DID cycle (c), and 3 ethanol DID cycles (d) indicate an effect of ethanol on GFAP immunoreactivity. The inset of panel d shows GFAP+ cells at 400X. Our analyses showed that 3 cycles of ethanol resulted in an increase in GFAP immunoreactivity within the DG (h) andCA1 (i) but not the CA2/3(j). Moreover, no statically significant differences were observed between water and sucrose drinking group in any subregion of the hippocampus (e-g). The scale bar =10 μm (c). Each photomicrograph has traces outlining the hippocampal fissure. All data are presented as mean ± SEM. *p < 0.05 compared to water group.

Fig 6.

One cycle of DID exposure caused increased GFAP mRNA expression in the hippocampus in the ethanol group compared with the water control. This effect was only seen during alcohol intoxication and did not persist days after abstinence. All data are presented as mean ± SEM. *p < 0.05 compared to water group.