Projection-dependent heterogeneity of cerebellar granule cell calcium responses

Cerebellar granule cells (GCs) relay mossy fiber (MF) inputs to Purkinje cell dendrites via their axons, the parallel fibers (PFs), which are individually located at a given sublayer of the molecular layer (ML). Although a certain degree of heterogeneity among GCs has been recently reported, variability of GC responses to MF inputs has never been associated with their most notable structural variability, location of their projecting PFs in the ML. Here, we utilize an adeno-associated virus (AAV)-mediated labeling technique that enables us to categorize GCs according to the location of their PFs, and compare the Ca²⁺ responses to MF stimulations between three groups of GCs, consisting of either GCs having PFs at the deep (D-GCs), middle (M-GCs), or superficial (S-GCs) sublayer. Our structural analysis revealed that there was no correlation between position of GC soma in the GC layer and location of its PF in the ML, confirming that our AAV-mediated labeling was important to test the projection-dependent variability of the Ca²⁺ responses in GCs. We then found that the Ca²⁺ responses of D-GCs differed from those of M-GCs. Pharmacological experiments implied that the different Ca²⁺ responses were mainly attributable to varied distributions of GABA_A receptors (GABA_ARs) at the synaptic and extrasynaptic regions of GC dendrites. In addition to GABA_AR distributions, amounts of extrasynaptic NMDA receptors appear to be also varied, because Ca²⁺ responses were different between D-GCs and M-GCs when glutamate spillover was enhanced. Whereas the Ca²⁺ responses of S-GCs were mostly equivalent to those of D-GCs and M-GCs, the blockade of GABA uptake resulted in larger Ca²⁺ responses in S-GCs compared with D-GCs and M-GCs, implying existence of mechanisms leading to more excitability in S-GCs with increased GABA release. Thus, this study reveals MF stimulation-mediated non-uniform Ca²⁺ responses in the cerebellar GCs associated with the location of their PFs in the ML, and raises a possibility that combination of inherent functional variability of GCs and their specific axonal projection contributes to the information processing through the GCs.