5/11 Microglial MyD88 in Mouse Models of Excessive Alcohol Intake

Abstract
Repeated alcohol exposure can be a potent neuroinflammatory stimulus. Alcohol activates microglia within the
brain via toll-like-receptors (TLRs); this activation leads to production of inflammatory mediators which contribute
to dependence and abuse 4,5. Fast spiking parvalbumin positive (PV+) GABAergic interneurons (PVIs) are critical
for normal brain function. PVIs are often surrounded by specialized extracellular matrix molecules called
perineuronal nets (PNNs) which protect and buffer them from inflammation and oxidative stress. Notably,
increased PNNs are implicated in alcohol use disorder (AUD), which may be due to a "locking in" of critical
neuroplasticity mechanisms that underlie habitual behavior such as drug seeking in the face of adverse
consequences 2. Our preliminary data show that mice with microglial-specific ablation of the critical TLR adaptor
molecule myeloid differentiation primary response 88 [MyD88] (Cx3cr1-CreBT-MyD88f/f [hereafter called "Cre+
mice"]) exhibit decreased proinflammatory responses (e.g. IL-1, TNF) to a lipopolysaccharide (LPS) injection
compared to Cre- controls, as expected. Surprisingly, however, in response to LPS, male (but not female) Cre+
mice exhibit increased numbers of PVIs and PV/PNN interactions within the frontal cortex. Cre+ male mice further
exhibit markedly increased alcohol intake in an acute drinking-in-the dark (DID) protocol compared to controls,
consistent with findings from previous INIA-Neuroimmune projects using MyD88-/- (knockout) mice and chronic
alcohol paradigms 6. Taken together, these data suggest microglia critically regulate PVI number and/or
PNN envelopment in response to inflammatory signals via MyD88 signaling, and that increased PVI/PNN
interactions in the absence of MyD88 may underlie increased drinking in Cre+ mice. The overall goal of
this proposal is to test this hypothesis. Aim 1 will determine if microglial-MyD88 regulates the number of PVIs
and/or PNN interactions directly in response to chronic alcohol by assessing the phagocytosis of PVI-specific
inhibitory synapses and PNNs by microglia. Aim 2 will determine if microglial-MyD88 regulates the number of
PVIs and/or PNN interactions indirectly in response to chronic alcohol by assessing the "secretome" of microglia
and the production of PNN components by astrocytes and PVIs. Aim 3 will determine the role of PVIs and/or
PNN interactions in excessive drinking behavior in mice with and without microglial MyD88 by assessing the
impact of (1) degrading PNNs and (2) chemogenetic inhibition vs. activation of microglia on excessive drinking.
This is an innovative new project designed to complement existing strengths of several other INIA-Neuroimmune
investigators. If completed, these data would be the first to show a direct role for microglia in PVI/PNN
development and their role in excessive drinking, which could lead to novel treatment options.

Grant Number: 5U01AA029969-05
NIH Institute/Center: NIH
Principal Investigator: Staci Bilbo