Microglia Toll-like Receptor 4 Drives Sex Differences in Ethanol-Mediated Pain Sensitization, Comorbid Anxiety-, & Depressive-like Behaviors

Lifestyle factors perpetuate pain states and, in this study, we asked if alcohol modulates comorbid behavioral pathologies such as anxiety and depression. Casual alcohol consumption, such as “wine-mom culture”, is not widely studied for its role in contributing to alcohol-induced behavioral deficits. We recently reported sex differences in pain sensitization in mice in a short-term, low-dose ethanol paradigm. We found microglia in female mice had greater activation than in male mice. Toll-like receptor-4 has been implicated in alcohol-induced behavioral deficits. We therefore utilized a novel genetic model of TLR4 knockout mice that allows for re-expression of TLR4 on microglia only to assess its role in driving pain comorbid behaviors. Mice were baselined for pain and affective behaviors then administered a 5% (v/v) ethanol diet. At day 19 mice were injected with a subthreshold dose of PGE2 and tested for pain and affective behaviors. Our investigation revealed that TLR4 only on microglia is enough to drive pain sensitization and comorbid depressive-like behavior in females only and anxiety-like behaviors in both sexes. To correlate behavioral responses with microglia activation, morphological changes and c-Fos counts were assessed in mPFC, BLA, and CA1. Mice with whole-body knockout of TLR4 showed no behavioral or morphological differences in any condition. Mice with TLR4 reactivation showed increased microglial activation and higher c-Fos counts in the mPFC and CA1 for females, and in the BLA for males. This work reveals the direct role of microglial TLR4 in driving sexual dimorphisms in pain comorbid behaviors in non-pathological drinkers. Funding: R35GM147094, R21DK130015, UT Systems STAR Award.