Project Summary An emerging model of obesity pathogenesis posits a central role for the inflammatory activation of hypothalamic microglia localized to the arcuate nucleus (ARC, a key brain area for the control of food intake and body weight) in the pathogenesis of diet-induced obesity (DIO), a finding observed in mammalian species ranging from rodents to humans. While microglia activation in the ARC is a known determinant of weight gain in high fat diet (HFD)-fed mice, it paradoxically improves glucose tolerance in DIO, but the mechanisms underlying both of these metabolic effects remain unclear. Herein, we report the novel finding that in mice, DIO induces loss of specialized extracellular matrix (ECM) structures known as perineuronal nets (PNNs) in the same brain area where reactive gliosis occurs. PNNs can powerfully influence the activity of neurons that they enmesh, and in the ARC, a large proportion of AgRP and a subset of POMC neurons are among those enmeshed by PNNs. These neurons are central regulators of energy homeostasis, and their altered function in DIO is strongly implicated in obesity pathogenesis and glucose regulation. Importantly, ablating or silencing microglia reduces Npy and AgRP levels and increases POMC neuron excitability, suggesting a link between microglial activation and ARC neuronal function that promotes weight gain. Furthermore, DIO is associated with loss of hypothalamic PNNs in proportion to the degree of microglial activation, with PNN stability enhanced by interventions that ablate or limit the inflammatory capacity of microglia. Finally, removal of hypothalamic PNNs experimentally causes gliosis, hyperphagia and rapid weight gain with preserved glucose tolerance in rodents, providing strong evidence that microglia and PNNs are linked in a critical mechanism that underlies obesity pathogenesis. Here, we investigate the inter-related hypotheses that obesity-associated microglial activation induces loss of PNN enmeshment of ARC neurons, thereby altering AgRP and Pomc neuron function in ways that promote excess fat accumulation but maintain glucose tolerance. Proposed studies will first quantify the role played by microglia to regulate ARC PNN turnover. We will then 1) determine whether the effect of DIO to induce loss of ARC PNNs depends on microglial activation, 2) identify the roles of both microglial activation and PNN loss in obesity-associated dysfunction of AgRP and POMC neurons, and 3) determine the bidirectional contributions linking ARC PNN loss and microglial activation to DIO susceptibility.