Astrocytes exist throughout the CNS and affect neural circuits and behavior through intracellular Ca²⁺ signaling. Studying the function(s) of astrocyte Ca²⁺ signaling has proven difficult because of the paucity of tools to achieve selective attenuation. Based on recent studies, we generated and used male and female knock-in mice for Cre-dependent expression of mCherry-tagged hPMCA2w/b to attenuate astrocyte Ca²⁺ signaling in genetically defined cells in vivo (CalEx^flox mice for Calcium Extrusion). We characterized CalEx^flox mice following local AAV-Cre microinjections into the striatum and found reduced astrocyte Ca²⁺ signaling (∼90%) accompanied with repetitive self-grooming behavior. We also crossed CalEx^flox mice to astrocyte-specific Aldh1l1-Cre/ERT2 mice to achieve inducible global CNS-wide Ca²⁺ signaling attenuation. Within 6 d of induction in the bigenic mice, we observed significantly altered ambulation in the open field, disrupted motor coordination and gait, and premature lethality. Furthermore, with histologic, imaging, and transcriptomic analyses, we identified cellular and molecular alterations in the cerebellum following mCherry-tagged hPMCA2w/b expression. Our data show that expression of mCherry-tagged hPMCA2w/b with CalEx^flox mice throughout the CNS resulted in substantial attenuation of astrocyte Ca²⁺ signaling and significant behavioral alterations in adult mice. We interpreted these findings candidly in relation to the ability of CalEx to attenuate astrocyte Ca²⁺ signaling, with regards to additional mechanistic interpretations of the data, and their relation to past studies that reduced astrocyte Ca²⁺ signaling throughout the CNS. The data and resources provide complementary ways to interrogate the function(s) of astrocytes in multiple experimental scenarios.SIGNIFICANCE STATEMENT Astrocytes represent a significant fraction of all brain cells and tile the entire central nervous system. Unlike neurons, astrocytes lack propagated electrical signals. Instead, astrocytes are proposed to use diverse and dynamic intracellular Ca²⁺ signals to communicate with other cells. An open question concerns if and how astrocyte Ca²⁺ signaling regulates behavior in adult mice. We approached this problem by generating a new transgenic mouse line to achieve inducible astrocyte Ca²⁺ signaling attenuation in vivo We report our data with this mouse line and we interpret the findings candidly in relation to past studies and within the framework of different mechanistic interpretations.

Astrocytes and neurons extensively interact in the brain. Identifying astrocyte and neuron proteomes is essential for elucidating the protein networks that dictate their respective contributions to physiology and disease. Here we used cell-specific and subcompartment-specific proximity-dependent biotinylation¹ to study the proteomes of striatal astrocytes and neurons in vivo. We evaluated cytosolic and plasma membrane compartments for astrocytes and neurons to discover how these cells differ at the protein level in their signalling machinery. We also assessed subcellular compartments of astrocytes, including end feet and fine processes, to reveal their subproteomes and the molecular basis of essential astrocyte signalling and homeostatic functions. Notably, SAPAP3 (encoded by Dlgap3), which is associated with obsessive-compulsive disorder (OCD) and repetitive behaviours^2-8, was detected at high levels in striatal astrocytes and was enriched within specific astrocyte subcompartments where it regulated actin cytoskeleton organization. Furthermore, genetic rescue experiments combined with behavioural analyses and molecular assessments in a mouse model of OCD⁴ lacking SAPAP3 revealed distinct contributions of astrocytic and neuronal SAPAP3 to repetitive and anxiety-related OCD-like phenotypes. Our data define how astrocytes and neurons differ at the protein level and in their major signalling pathways. Moreover, they reveal how astrocyte subproteomes vary between physiological subcompartments and how both astrocyte and neuronal SAPAP3 mechanisms contribute to OCD phenotypes in mice. Our data indicate that therapeutic strategies that target both astrocytes and neurons may be useful to explore in OCD and potentially other brain disorders.