AAV2/5 does not cause more Ca²⁺ signals as measured by Fluo-4AM. The data reported in the Results show that many more Ca²⁺ signals were measured with GECIs than with Fluo-4AM. The data strongly indicate that this is because GECIs sample entire astrocytes. However, to make sure that AAV2/5-mediated expression of a fluorescent protein did not alter the number of Ca²⁺ signals, we made AAV2/5 for the red fluorescent protein tdTomato and imaged Ca²⁺ signals from tdTomato-expressing cells using Fluo-4 (A; GCaMP3 cannot be used in conjunction with Fluo-4 to address this issue because the fluorophores have spectral overlap). The Ca²⁺ signals measured from cells expressing tdTomato were indiscernible from those measured using Fluo-4AM in control uninjected mice (B). These data provide strong evidence that AAV2/5-mediated expression of GECIs and tdTomato does not alter astrocyte Ca²⁺ signaling.