PROJECT SUMMARY Perineuronal nets (PNNs) are condensed extracellular matrix around a subset of neurons. PNNs can provide physical protection and ion buffering for neurons and regulate their synaptic plasticity and intracellular signaling. Recent evidence indicates that PNNs enmesh GABA neurons in the arcuate nucleus of the hypothalamus (ARH) to regulate energy and glucose homeostasis. However, little is known about the potential metabolic function of PNNs in other brain regions that are also implicated in metabolic health. We observed abundant PNNs in the posterodorsal medial amygdala (MePD) and found that PNN-enmeshed neurons in the MePD are mostly GABA neurons. Digestion of PNNs drastically reduces excitability and synaptic activity of these GABAMePD neurons. Importantly, we showed that chronic disruption of MePD PNNs leads to hyperphagia and modest weight/fat gain in chow-fed mice. On the other hand, chronic increases in MePD PNNs result in lower body weight/fat in chow-fed mice. These results suggest that MePD PNNs and PNNs-enmeshed GABAMePD neurons are required to prevent overeating and obesity. We will follow up these findings to establish the function of MePD PNNs (Aim 1) and GABAMePD (Aim 2) neurons in energy and glucose homeostasis. Meanwhile, we will delineate the mechanisms by which PNNs regulate excitability and synaptic activity of GABAMePD neurons. 17β-estradiol (E2) can act on estrogen receptor-α (ERα) to regulate energy and glucose balance in both females and males. Previous reports showed that ERα in many brain regions mediates the metabolic effects of E2 only in female mice, but not male mice. Our pilot observations indicate that E2 can increase PNNs in MePD in both male and female mice. Importantly, loss of ERα in the medial amygdala (containing the MePD) causes obesity not only in female mice, but also in male mice, suggesting the potential metabolic role of MePD in male metabolic health. In Aim 3, we will follow up these findings to test whether E2 induces PNNs to facilitate GABA neurons in the MePD to prevent obesity and metabolic dysregulation in both male and female mice. Together, we will reveal a new metabolic regulation mechanism: E2-ERα-PNNs-GABAMePD-energy/glucose homeostasis. Our studies will advance our understanding about the estrogen biology in both female and male metabolism and the metabolic role of PNNs in a new brain region MePD. Finally, we will identify a unique anorexigenic GABA population, which is different from previous reported orexigenic GABA neural populations in many other brain regions.