Network-level encoding of local neurotransmitters in cortical astrocytes

Astrocytes, the most abundant non-neuronal cell type in the mammalian brain, are crucial circuit components that respond to and modulate neuronal activity via calcium (Ca2+) signaling. Astrocyte Ca2+ activity is highly heterogeneous and occurs across multiple spatiotemporal scales—from fast, subcellular activity to slow, synchronized activity across connected astrocyte networks—to influence many processes. However, the inputs that drive astrocyte network dynamics remain unclear. Here we used ex vivo and in vivo two-photon astrocyte imaging while mimicking neuronal neurotransmitter inputs at multiple spatiotemporal scales. We find that brief, subcellular inputs of GABA and glutamate lead to widespread, long-lasting astrocyte Ca2+ responses beyond an individual stimulated cell. Further, we find that a key subset of Ca2+ activity—propagative activity—differentiates astrocyte network responses to these two major neurotransmitters, and may influence responses to future inputs. Together, our ..., Animals
Experiments were carried out using young adult mice, in accordance with protocols approved by the University of California, San Francisco Institutional Animal Care and Use Committee (IACUC). Animals were housed in a 12:12 light-dark cycle with food and water provided ad libitum. Animal housing rooms were kept at 68–74 degrees Fahrenheit and 30–70% humidity. Male and female mice were used whenever available. Transgenic mice used in this study were Cx43fl/fl mice56 from the Bhattacharya Lab (UCSF, USA) and EAAT2-tdT mice57 from the Yang Lab (Tufts University, USA). For in vivo imaging, all experiments were performed at the same time each day. 
Surgical procedures
For viral expression for ex vivo experiments, neonatal Swiss Webster or C57Bl/6 (P0–3) mice were anesthetized on ice for 3 min before injecting viral vectors (AAV5.GfaABC1D.GCaMP6f.SV40 [Addgene, 52925-AAV5], AAV9.hGfap.pinkFlamindo, pENN.AAV9.Gfap.iGluSnFr.WPRE.SV40 [Addgene, 98930-AAV9], or AAV5.GFAP(0.7).RFP.T2A.iCre [..., , # Network-level encoding of local neurotransmitters in cortical astrocytes

[https://doi.org/10.5061/dryad.83bk3jb0j](https://doi.org/10.5061/dryad.83bk3jb0j)

## Description of the data and file structure

**Datasets**

**AQuA Cyto-GCaMP6f receptor agonist bath application (Fig. 1 and associated extended data figures)**

16 slices, 4 mice. 400µm thick, acute, coronal V1 slices were prepared from mice injected with GfaABC1D-cyto-GCaMP6f. Simultaneous 2P imaging of astrocyte Ca2+ activity and bath application of receptor agonists in the presence of TTX (1 µM), to block neuronal action potentials. Two recordings (10 min, 853 frames each at 1.42Hz) were acquired for each slice, GABA and glutamate receptor agonists were applied sequentially (Baclofen and t-ACPD, respectively), separated by an inter-imaging interval of >20 min, including an ACSF washout period of > 10min. Four concentrations of agonists were tested (5–100 µM), and the order of agonists was alternated between concentrat...