Injection of Recombinant Adenovirus for Delivery of Genetically Encoded Calcium Indicators into Astrocytes of the Cerebellar Cortex

The cerebellar cortex contains two astrocyte types: the Bergmann glia of the molecular layer and the velate protoplasmic astrocytes of the granule cell layer. In vivo, these cell types generate both subcellular calcium transients and trans-glial calcium waves. This protocol outlines a method for in vivo calcium imaging in cerebellar astrocytes, using the injection of a replication-incompetent recombinant adenovirus for gene transfer of the fluorescent calcium indicator protein (FCIP) G-CaMP2. The adenovirus contains a cytomegalovirus (CMV) immediate-early (IE) promoter which confines expression of G-CaMP2 to astrocytes. Expression is sufficiently high to allow calcium signals to be recorded in Bergmann glial processes as well as the processes and somata of velate protoplasmic astrocytes. To obtain structural information, G-CaMP2 fused with the brighter chromophore DsRed allows threedimensional (3D) reconstruction of cells. G-CaMP2 expression lasts for at least 3 wk, enabling long-term functional imaging in both anesthetized and awake animals.