Developmental Ethanol Exposure Impacts Purkinje Cells but Not Microglia in the Young Adult Cerebellum.

Fetal alcohol spectrum disorders (FASD) caused by developmental ethanol exposure lead to cerebellar impairments, including motor problems, decreased cerebellar weight, and cell death. Alterations in the sole output of the cerebellar cortex, Purkinje cells, and central nervous system immune cells, microglia, have been reported in animal models of FASD. To determine how developmental ethanol exposure affects adult cerebellar microglia and Purkinje cells, we used a human third-trimester binge exposure model in which mice received ethanol or saline from postnatal (P) days 4-9. In adolescence, cerebellar cranial windows were implanted and mice were aged to young adulthood for examination of microglia and Purkinje cells in vivo with two-photon imaging or in fixed tissue. Ethanol had no effect on microglia density, morphology, dynamics, or injury response. However, Purkinje cell linear frequency was reduced by ethanol. Microglia-Purkinje cell interactions in the Purkinje Cell Layer were altered in females compared to males. Overall, developmental ethanol exposure had few effects on cerebellar microglia in young adulthood and Purkinje cells appeared to be more susceptible to its effects.

Developmental ethanol exposure has minimal impact on cerebellar microglial dynamics, morphology, and interactions with Purkinje cells during adolescence.

Introduction: Fetal alcohol spectrum disorders (FASD) are the most common cause of non-heritable, preventable mental disability, occurring in almost 5% of births in the United States. FASD lead to physical, behavioral, and cognitive impairments, including deficits related to the cerebellum. There is no known cure for FASD and their mechanisms remain poorly understood. To better understand these mechanisms, we examined the cerebellum on a cellular level by studying microglia, the principal immune cells of the central nervous system, and Purkinje cells, the sole output of the cerebellum. Both cell types have been shown to be affected in models of FASD, with increased cell death, immune activation of microglia, and altered firing in Purkinje cells. While ethanol administered in adulthood can acutely depress the dynamics of the microglial process arbor, it is unknown how developmental ethanol exposure impacts microglia dynamics and their interactions with Purkinje cells in the long term. Methods: To address this question, we used a mouse model of human 3rd trimester exposure, whereby L7cre/Ai9+/-/Cx3cr1G/+ mice (with fluorescently labeled microglia and Purkinje cells) of both sexes were subcutaneously treated with a binge-level dose of ethanol (5.0 g/kg/day) or saline from postnatal days 4-9. Cranial windows were implanted in adolescent mice above the cerebellum to examine the long-term effects of developmental ethanol exposure on cerebellar microglia and Purkinje cell interactions using in vivo two-photon imaging. Results: We found that cerebellar microglia dynamics and morphology were not affected after developmental ethanol exposure. Microglia dynamics were also largely unaltered with respect to how they interact with Purkinje cells, although subtle changes in these interactions were observed in females in the molecular layer of the cerebellum. Discussion: This work suggests that there are limited in vivo long-term effects of ethanol exposure on microglia morphology, dynamics, and neuronal interactions, so other avenues of research may be important in elucidating the mechanisms of FASD.

Microglia and astrocytes show limited, acute alterations in morphology and protein expression following a single developmental alcohol exposure.

Fetal alcohol spectrum disorders (FASD) are the most common cause of nonheritable, preventable mental disability and are characterized by cognitive, behavioral, and physical impairments. FASD occurs in almost 5% of births in the United States, but despite this prevalence there is no known cure, largely because the biological mechanisms that translate alcohol exposure to neuropathology are not well understood. While the effects of early ethanol exposure on neuronal survival and circuitry have received more attention, glia, the cells most closely tied to initiating and propagating inflammatory events, could be an important target for alcohol in the developing brain. Inflammation is known to alter developmental trajectories, but it has recently been shown that even small changes in both astrocytes and microglia in the absence of full-blown inflammatory signaling can alter brain function long-term. Here, we studied the acute response of astrocytes and microglia to a single exposure to ethanol in development across sexes in a mouse model of human third trimester exposure, in order to understand how these cells may transition from their normal developmental path to a different program that leads to FASD neuropathology. We found that although a single ethanol exposure delivered subcutaneously on postnatal day 4 did not cause large changes in microglial morphology or the expression of AldH1L1 and GFAP in the cortex and hippocampus, subtle effects were observed. These findings suggest that even a single, early ethanol exposure can induce mild acute alterations in glia that could contribute to developmental deficits.