PROJECT SUMMARY Astrocytes have been shown to play an important role in synaptic transmission. While there is emerging research about the effects of neurotransmitters on astrocytes, there is little known about how other signaling molecules such as hormones impact astrocyte signaling. One important hormone that is well studied with neuronal signaling is estradiol (E2). This has important relevance in motivated behaviors such as drug addiction where astrocyte signaling and estrogen signaling affect drug seeking behavior. However, it is unknown how estrogen specifically affects astrocyte signaling. This proposal will determine how estrogen affects astrocyte signaling and its subsequent regulation of tripartite synaptic transmission. For this proposal, I will focus on astrocytes in the nucleus accumbens (NAc) as this brain region is critical for drug seeking behaviors and displays structural and functional sex differences. I hypothesize that E2 will increase intracellular calcium in astrocytes by binding to estrogen receptors on astrocytes. This receptor binding will lead to gliotransmitter release which will in turn increase excitatory synaptic transmission. To this test hypothesis, I will use two photon microscopy to examine calcium activity in astrocytes. I will first establish that E2 increases calcium activity in NAc astrocytes (Aim 1a). I will then determine the location and ER subtype as well as cell type in which ER is expressed (Aim 1b). For Aim 2, I will test the effects of E2 on excitatory synaptic transmission. First, I will determine where E2 stimulates gliotransmitter release by measuring slow inward currents, a biological assay of astrocytic glutamate release (Aim 2a). In addition, I will monitor excitatory post synaptic currents (EPSCs) and determine the gliotransmitter responsible for changes in EPSCs (Aim 2b). This proposal will fill the knowledge gap of how estrogen affects astrocyte signaling which could lead to further understanding of sex differences in NAc related behaviors. This proposal will also allow me to master many research techniques such as two photon calcium imaging as well as electrophysiology, which will promote my career goals to become an independent academic neuroscience researcher.