Innate Immune Signaling in Microglia Regulates Inhibitory Circuit Maturation and Behavioral Outcomes

Abstract

Decades of studies have illustrated the long-term impacts of perinatal inflammation on brain development. As the brain-resident macrophage, microglia are uniquely positioned to detect and respond to immune challenges. Microglia also perform functions early in life that are critical to the health and function of developing neurons. A common neuronal subtype impacted by early life challenges are GABAergic parvalbumin+ interneurons (PVIs). PVIs have a unique fast-spiking quality, leaving them very sensitive to perturbations. The deposition of specialized extracellular matrix structures, called perineuronal nets (PNNs), helps usher PVIs into their mature functions, protects them from damage, and limits their synaptic plasticity. PNNs have also been shown to be impacted by early life experiences, but little is known about the mechanisms that regulate their developmental remodeling. Although developmental interactions between microglia and excitatory neurons have been well characterized, less is known about the interactions between microglia, interneurons, and the extracellular matrix. We hypothesized that in the context of early life inflammation, ablation of microglial pro-inflammatory signaling would be protective for developing PVIs. We used a mouse line in which toll-like receptor adaptor protein MyD88 is removed from microglia in the brain, blunting pro-inflammatory cytokine release. Male and female mice with and without microglial-MyD88 were injected with saline or lipopolysaccharide (LPS) at postnatal day 4, and brains were collected across development and in adulthood. Adult male mice without microglial-MyD88 had significantly more PVIs across the dorsal hippocampus, as well as increased PNN colocalization, PVI synaptic protein, and increased inhibitory signaling by electrophysiology. This led to male-specific adult behavior deficits related to hippocampal-dependent discrimination. To better understand the role of microglial-MyD88 in PVI development, we quantified male microglial phagocytic capacity at P12 and found that MyD88-deficient microglia were more phagocytic than controls, and preferentially engulf inhibitory synaptic material (VGAT+) over excitatory synaptic material (VGlut2+). Sequencing of isolated microglia acutely after saline injection revealed that loss of MyD88 alone led to many transcriptional changes in both male and female microglia, in particular genes related to extracellular matrix remodeling in males. We identified that a neuronally-derived cytokine, interleukin (IL)-33, which has been previously shown to modulate microglial interactions with the extracellular matrix in adulthood, is developmentally regulated in the hippocampus. At the peak of the expression of this cytokine and its receptors on microglia, brain injection of IL-33 leads to changes in microglial ramification and increased engulfment of aggrecan, a key extracellular matrix component. We found that MyD88 deficient microglia are not responsive to acute IL-33 stimulation but gene expression or morphology. Indeed, male MyD88 deficient microglia engulf less aggrecan at P15 than control microglia with intact MyD88, identifying the likely mechanism through which MyD88 loss in microglia impacts inhibitory system development in the hippocampus. Together, these results show baseline microglia innate immune signaling plays a role in regulating the development of PVIs in the dorsal hippocampus in a sex-specific manner. This work provides insight into the indirect extracellular mechanisms of early life inflammation on susceptible developing GABAergic neurons.