In vivo two-photon calcium imaging provides the opportunity to monitor activity in multiple components of neural circuitry at once. Here we report the use of bulk-loading of fluorescent calcium indicators to record from axons, dendrites, and neuronal cell bodies in cerebellar cortex in vivo. In cerebellar folium crus IIa of anesthetized rats, we imaged the labeled molecular layer and identified all major cellular structures: Purkinje cells, interneurons, parallel fibers, and Bergmann glia. Using extracellular stimuli we evoked calcium transients corresponding to parallel fiber beam activity. This beam activity triggered prolonged calcium transients in interneurons, consistent with in vitro evidence for synaptic activation of N-methyl-D-aspartate receptors via glutamate spillover. We also observed spontaneous calcium transients in Purkinje cell dendrites that were identified as climbing-fiber-evoked calcium spikes by their size, time course, and sensitivity to AMPA receptor antagonist. Two-photon calcium imaging of bulk-loaded cerebellar cortex is thus well suited to optically monitor synaptic processing in the intact cerebellum.
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