Imaging Calcium Waves in Cerebellar Bergmann Glia

Abstract

This protocol describes methods for recording synaptically evoked Ca²⁺ waves from individual Bergmann glia (BG) in slices of cerebellar cortex. Unlike protoplasmic, star-shaped astrocytes, whose thin processes pose a serious challenge to stable Ca²⁺ measurements, BG are large radial cells, with several main processes that run over distances of several hundred micrometers toward the pia and ensheathe thousands of parallel fiber (PF) synapses. Stimulation of PF synapses with brief bursts can trigger long-lasting Ca²⁺ responses in BG processes, which can be reliably recorded using a cooled charge-coupled device (CCD) camera. This protocol was developed to enable measurements of Ca²⁺ waves in individual BG loaded with a high-affinity Ca²⁺ indicator such as Fura-2 for up to 2 h. Because BG recorded in slices rarely display spontaneous (i.e., tetrodotoxin [TTX]-sensitive) or intrinsic Ca²⁺ transients, Ca²⁺ waves can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Fluorescence measurements obtained using CCD technology offer a straightforward means of characterizing the mechanisms and potential functional consequences of widespread and long-lasting, store-mediated Ca²⁺ increases in astrocytes.