Alterations in Microglial function moderate the development of maladaptive drinking behaviors following early life stress and are exacerbated by ethanol consumption

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PROJECT SUMMARY Currently 14.5 million people suffer from alcohol use disorder (AUD) in the United States. Identifying early risk factors that increase the likelihood of developing AUD could help elucidate potential models for preventative care and mitigate the impact AUD has on society. One highly studied risk factor associated with developing AUD is early life stress (ELS). The mechanisms that facilitate ELS's impact on the development of AUD is poorly understood. One consistent finding in ELS literature shows a role of microglia in the development of a host of neurological disorders including AUD. Over the past several years, new evidence has been uncovered identifying a role of microglia in the development and maintenance of AUD. To expand this field of work and discover novel targets for treatment, my project seeks to implement a model of early life stress: periadolescent social stress (PSS) and a model to promote voluntary binge-like consumption of ethanol: drinking in the dark. Using these models, I will identify how microglia respond to these factors in isolation and when combined. Over the past two years, I have run experiments to optimize the PSS paradigm and drinking in the dark protocol. I have performed immunohistochemical staining and run qPCR to determine how microglia respond to these stimuli. I have found that combined PSS and binge ethanol consumption decrease microglia number, increase microglial reactivity, and alter microglia morphology in the ventral hippocampus. In addition, markers related to homeostasis are reduced following PSS and binge ethanol consumption. While these results are interesting, evidence suggest that microglia are present heterogeneously throughout the brain and serve a variety of functions. To better characterize the effects of early life stress and ethanol consumption on microglia function, my proposal aims to identify key regions that demonstrate alterations in microglia count, reactivity, and phenotype; and identify how these changes relate to changes in microglial RNA. To determine these key regions, I will use light sheet microscopy. This approach will provide us an opportunity to create an unbiased 3D reconstruction of the whole brain with single microglia resolution. I will use this technique to identify how microglia are distributed throughout the brain and phenotypically altered due to these environmental exposures. Focusing on the regions identified with light sheet microscopy, I will perform targeted purification of polysomal mRNA sequencing (TRAP-seq). Using a CX3CR1CreER transgenic mouse line that primarily labels for microglia in GFP, I will sort microglia from other cell types and identify changes in microglial RNA expression due to PSS and ethanol exposure. Following each of these main experiments, I will determine whether microglia inhibition during PSS will prevent changes in binge-like ethanol consumption and microglial adaptations. This work will mark a significant advancement in the field and will call for the consideration of microglia function as a target for treatment of ELS and prevention of AUD.