Adolescent ethanol drinking promotes hyperalgesia, neuroinflammation and serotonergic deficits in mice that persist into adulthood

Abstract

Adolescent alcohol use can permanently alter brain function and lead to poor health outcomes in adulthood. Emerging evidence suggests that alcohol use can predispose individuals to pain disorders or exacerbate existing pain conditions, but the underlying neural mechanisms are currently unknown. Here we report that mice exposed to adolescent intermittent access to ethanol (AIE) exhibit increased pain sensitivity and depressive-like behaviors that persist for several weeks after alcohol cessation and are accompanied by elevated CD68 expression in microglia and reduced numbers of serotonin (5-HT)-expressing neurons in the dorsal raphe nucleus (DRN). 5-HT expression was also reduced in the thalamus, anterior cingulate cortex (ACC) and amygdala as well as the lumbar dorsal horn of the spinal cord. We further demonstrate that chronic minocycline administration after AIE alleviated hyperalgesia and social deficits, while chemogenetic activation of microglia in the DRN of ethanol-naïve mice reproduced the effects of AIE on pain and social behavior. Chemogenetic activation of microglia also reduced tryptophan hydroxylase 2 (Tph2) expression and was negatively correlated with the number of 5-HT-immunoreactive cells in the DRN. Taken together, these results indicate that microglial activation in the DRN may be a primary driver of pain, negative affect, and 5-HT depletion after AIE.

Keywords: Adolescence; Depression; Dorsal raphe; Ethanol; Microglia; Pain; Serotonin.

Fig. 1.

Hyperalgesia and social deficits in adult mice after adolescent alcohol exposure. (A) Experimental timeline for adolescent alcohol exposure and behavioral testing. (B) Average ethanol intake (g/kg/day) and (C) preference (%) over the 4-week alcohol drinking paradigm. (D-E) Time spent in the open arms and distance traveled in the elevated plus maze (EPM). (F-G) Time spent in the center and distance traveled in an open field. (H) Thermal pain threshold in the Hargreaves test and (I-J) mechanical pain thresholds in the Von Frey test. (K) Time spent in social interaction in the 3-chambered social interaction test. (L-M) Time spent immobile and frequency of inactive bouts in the forced swim test. *p < 0.05, **p < 0.01, ***p < 0.001. Figure 1A was created with BioRender.

Fig. 2.

5-HT depletion and microglial activation in the DRN following AIE. (A) Representative confocal images of Tph2, 5-HT, CD68 and 5-HT-CD68 overlay in the DRN and (B) MRN of water control and alcohol drinking mice. (C) Brain atlas schematic illustrating the approximate positions of the DRN (blue) and MRN (magenta) included in this analysis. (D) Histograms of total cell counts in the DRN and MRN for (D) Tph2-IR neurons, (E) 5-HT-IR neurons, and (F) density of CD68-IR microglia. (G) Correlation between CD68 optical density and 5-HT-IR cells in the DRN. Scale bar = 100 μm. *p < 0.05.

Fig. 3.

Loss of 5-HT input and microglial activation in pain processing regions of the brain and spinal cord. Representative confocal images of 5-HT and CD68 from water control and alcohol drinking mice and approximate brain atlas coordinates for the (A) medullary raphe, (B) posterior complex of the thalamus (Po), (C) hypothalamus (Hyt), (D) anterior cingulate cortex (ACC), (E) amygdala (Amy), and (F) dorsal horn (DH) of the spinal cord. (G) Histogram of total 5-HT-IR neurons in the medullary raphe. Histograms of (H) 5-HT-IR area and (I) 5-HT optical density, (J) CD68-IR cell density, and (K) CD68 optical density in the Po, hyt, ACC, Amy and DH. Scale bar = 50 μm *p < 0.05.

Fig. 4.

Minocycline alleviates pain and depressive-like behaviors after AIE. (A) Experimental time of alcohol exposure, minocycline administration and behavioral tests. (B) Average minocycline dose over the exposure period. (C-D) Mechanical pain thresholds in the Von Frey and (E) Hargreaves tests showing that minocycline alleviates hyperalgesia after AIE. (F) Time spent in social interaction in the 3-chambered social interaction test indicating that minocycline can mitigate depressivelike behaviors after AIE. (G) Representative confocal images of CD68-IR microglia in vehicle and minocycline treated water control and AIE mice. (H-I) Immunohistochemical analysis of CD68-IR microglia in the DRN following AIE and chronic minocycline administration. Scale bar = 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 4A was created with BioRender.

Fig. 5.

Chemogenetic activation of microglia in the DRN induces hyperalgesia and depressive-like behaviors in ethanol-naïve mice. (A) Experimental timeline for surgeries, injections and behavioral experiments in Cx3cr1-cre::hM3Dq^DRN mice. (B) Schematic of stereotaxic injections of AAV into the DRN. (C) Specificity of DREADD expression in microglia and transduction efficiency of AAV-mediated hM3Dq expression in DRN microglia. (D-G) Representative confocal images of Tph2, Cx3cr1-eGFP, hM3Dq-mCherry and overlay in the DRN. (H-I) Mechanical pain thresholds in the Von Frey test, (J) thermal pain thresholds in the Hargreaves test and (K) social investigation time in the 3-chambered social interaction test in Cx3cr1-cre::hM3Dq^DRN relative to Cx3cr1-cre::mCherry^DRN mice following CNO injection. Scale bar = 50 μm (15 μm for inset). *p < 0.05, ****p < 0.0001.

Fig. 6.

Chemogenetic activation of microglia induces CD68 and inhibits Tph2 expression in the DRN. (A) Experimental timeline of injections and perfusions for histology in Cx3cr1-cre × hM3Dq mice. (B) Representative confocal images of Cx3cr1-GFP, HA, and overlay in the DRN. (C) Specificity of DREADD expression in microglia and % of microglia transduced with hM3Dq in the DRN. (D) Histogram of GFP and CD68 expression, (E) Cell density of 5-HT-fos double positive neurons, (F) Tph2 and 5-HT optical density and (G) neuronal density in the DRN of Cx3cr1-cre × hM3Dq and control (Cx3cr1-cre) mice following CNO injection. Correlation analysis between CD68 OD and (H) Tph2 optical density and (I) 5-HT neuronal density. Representative confocal images of (J)Tph2, (K) 5-HT, (L) CD68 and (M) GFP in control and DREADD-expressing mice after CNO. Scale bar = 50 μm in panels B, L-M, and 100 μm in panels J-K. *p < 0.05, **p < 0.01.