Quantification of the density of cooperative neighboring synapses required to evoke endocannabinoid signaling

•A genetically encoded calcium indicator is used to image firing axons.•Parallel fiber input density in acute cerebellar slices is derived from this imaging.•The endocannabinoid-mediated plasticity correlates with the determined input density.•This plasticity requires activated synapses to be less than 1.5 μm apart.

The spatial pattern of synapse activation may impact on synaptic plasticity. This applies to the synaptically-evoked endocannabinoid-mediated short-term depression at the parallel fiber (PF) to Purkinje cell synapse, the occurrence of which requires close proximity between the activated synapses. Here, we determine quantitatively this required proximity, helped by the geometrical organization of the cerebellar molecular layer. Transgenic mice expressing a calcium indicator selectively in granule cells enabled the imaging of action potential-evoked presynaptic calcium rise in isolated, single PFs. This measurement was used to derive the number of PFs activated within a beam of PFs stimulated in the molecular layer, from which the density of activated PFs (input density) was calculated. This density was on average 2.8 μm−2 in sagittal slices and twice more in transverse slices. The synaptically-evoked endocannabinoid-mediated suppression of excitation (SSE) evoked by ten stimuli at 200 Hz was determined from the monitoring of either postsynaptic responses or presynaptic calcium rise. The SSE was significantly larger when recorded in transverse slices, where the input density is larger. The exponential description of the SSE plotted as a function of the input density suggests that the SSE is half reduced when the input density decreases from 6 to 2 μm−2. We conclude that, although all PFs are truncated in an acute sagittal slice, half of them remain respondent to stimulation, and activated synapses need to be closer than 1.5 μm to synergize in endocannabinoid signaling.