Preferential Loading of Bergmann Glia with Synthetic Acetoxymethyl Calcium Dyes

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 calciumimagingincerebellarastrocytesofmicewhichhaveundergonea cerebellarcraniotomy. Multicell bolusloading (MCBL) is performed using thesynthetic calcium indicators Fluo-5F AM and Fluo-4 AM. In the cerebellum, a degree of cell-type specificity can be achieved by varying the depth of injection. This protocol describes a loading procedure following craniotomy which allows preferential labeling of Bergmann glia.