cerebellar circuit activation through the mossy fiber-parallel fiber pathway using high-resolution VSD imaging in acute cerebellar slices

It is not yet fully clear how mossy fiber inputs activate the granular layer retransmitting signals through the ascending axon and parallel fibers to Purkinje cells. We have characterized the spatio-temporal properties of cerebellar circuit activation in response to mossy fiber stimulation by using Voltage-Sensitive Dye (VSD) imaging in sagittal and coronal rat cerebellar slices (P20-P25). Fluorescent signals generated by Di4-ANNEPS were sampled at 1 KHz with a Micam-Ultima camera (SciMedia). The granular layer was activated in spots of about 30 micrometer diameter showing distinct intensities of response with delays of 2.1±0.15 ms (n=15). Then activation propagated into the molecular and Purkinje cell layers with an additional 3.6±1.1 ms delay (n=10). Simultaneous patch-clamp recordings from granule cells and Purkinje cells showed a direct correlation between intracellular depolarization and VSD signal. In sagittal slices, mossy fiber stimulation activated the overlaying area supporting vertical transmission, while in coronal slices activation also propagated laterally demonstrating spread of excitation along the parallel fibers. Transmission through the granular layer and to the molecular layer was more pronounced using high-frequency bursts rather than single isolated stimuli (+85.5%, n=8, from 0.1 Hz to 500 Hz), was markedly reduced by NMDA receptor blockers (e.g. -17.9%, n=4, at 200 Hz) and was enhanced by GABA-A receptor blockers (e.g. +36.9 %; n=4 at 200 Hz). VSD recordings reveal therefore (1) that the granular layer activates in spots depending on NMDA and GABA-A receptors, (2) that signals are transmitted toward the molecular layer depending on the frequency of the mossy fiber input and (3) that Purkinje cell excitation through the ascending granule cell axon coexist with that due to parallel fiber transmission. These observations provide the basis for a detailed investigation of spatio-temporal signal processing in the cerebellar circuit.