Functional topology of the mossy fibre-granule cell-Purkinje cell system revealed by imaging of intrinsic fluorescence in mouse cerebellum

We report an activity-induced green fluorescence signal observed when mouse cerebellar slices were illuminated with blue light and parallel fibre-Purkinje cell synapses were activated. The optical signal consisted of an initial increase in fluorescence that peaked within 1-2 s after the onset of stimulation, followed by a long lasting (40 s) transient decrease in fluorescence. Single or tetanic electrical stimuli applied to the molecular layer elicited 'beam-shaped' fluorescence changes along the trajectory of parallel fibres. These signals reported activation of Purkinje cells as they were depressed by antagonists of ionotropic and metabotropic glutamate receptors at Purkinje cells and correlated with Purkinje cell spiking activity. Optical responses induced by direct pharmacological activation of glutamate receptors were reduced by a calcium-free extracellular medium, consistent with the hypothesis that they reflect metabolic activity due to an increased intracellular calcium load associated with neuronal activation. We used these intrinsic fluorescence signals to address the question of whether granule cells excite Purkinje cells only locally via the ascending branches of their axons, or more widespread along the parallel fibre trajectory. White matter stimulation of the mossy fibres also elicited a beam-like fluorescence change along the trajectory of parallel fibres. Simultaneous imaging and extracellular recording demonstrated the association between the beam-like fluorescence signal and Purkinje cell spiking. This non-invasive imaging technique supports the notion that parallel fibre activity, evoked either locally or through the mossy fibre-granule cell pathway, can activate postsynaptic Purkinje cells along more than 3 mm of the parallel fibre trajectory.