Project Summary / Abstract Astrocytes are pervasive throughout the CNS and are the most abundant non-neuronal cell type. They are an essential component of neural circuits, and increasing evidence demonstrates they are specialized for specific brain regions. For example, proteomic, transcriptomic, and electrophysiology experiments show that hippocampal and striatal astrocytes are distinct. However, it is unknown if astrocytes are heterogeneous within brain regions such as the striatum where neuronal subtypes are largely uniform. Thus, the dorsal lateral striatum (DLS) and the nucleus accumbens (NAc) receive different afferents, extend different efferents, and encode different behaviors, and yet there is no basic understanding of astrocyte heterogeneity within striatal areas. My pilot data demonstrate that NAc astrocytes have different morphologies compared to DLS astrocytes, and single- cell sequencing molecularly identified 5 astrocyte subtypes in the striatum. In Aim 1, I will extend these findings and use spatial transcriptomics and RNAscope to identify the location of each striatal astrocyte subtype. This unbiased approach will determine if astrocyte subtypes are localized to subregions of the striatum. Ventral striatal astrocytes are also functionally distinct from dorsal striatal astrocytes, as NAc astrocytes have a significantly stronger intracellular Ca2+ signaling response to dopamine than do dorsal astrocytes. Dopamine (DA) in the NAc is critical for reward-learning such as Pavlovian conditioning. In Aim 2, I will therefore determine if the distinct NAc Ca2+ responses are functionally relevant to DA-encoded Pavlovian stimulus-reward learning. Together, this project proposes a comprehensive investigation of striatal astrocyte heterogeneity (Aim 1 – Priority Area #1), which is essential for forming mechanistic hypotheses and determining causality of NAc astrocyte Ca2+ signaling in DA-encoded reward learning (Aim 2 – Priority Area #4).

Project Narrative The aim of this proposal is to rigorously explore striatal astrocyte heterogeneity and identify functional roles of NAc astrocyte Ca2+ in reward learning. I will map molecularly-identified astrocyte subtypes on to regions of the striatum using spatial transcriptomics and RNAscope (BRAIN initiative Goal 1 – Discovery Diversity). In addition, I will use precise astrocyte-targeted interventional tools to attenuate or stimulate astrocyte intracellular calcium signaling to explore the role of NAc astrocyte Ca2+ signaling in reward learning (BRAIN initiative Goal 4 – Demonstrate Causality).