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 calcium imaging in cerebellar astrocytes of mice which have undergone a cerebellar craniotomy. Multicell bolus loading (MCBL) is performed using the synthetic 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.