NMDA receptor subunits GluRε1, GluRε3 and GluRζ1 are enriched at the mossy fibre–granule cell synapse in the adult mouse cerebellum


: Professor Masahiko Watanabe, as above.
E-mail: watamasa@med.hokudai.ac.jp


Cerebellar N-methyl-d-aspartate (NMDA) receptors are concentrated in the granular layer and are involved in motor coordination and the induction of long-term potentiation at mossy fibre–granule cell synapses. In the present study, we used immunohistochemistry to examine the distribution of NMDA receptor subunits in the adult mouse cerebellum. We found that appropriate pepsin pretreatment of sections greatly enhanced the sensitivity and specificity of immunohistochemical detection. As a result, intense immunolabelling for GluRε1 (NR2A), GluRε3 (NR2C), and GluRζ1 (NR1) all appeared in synaptic glomeruli of the granular layer. Double immunofluorescence showed that these subunits were colocalized in individual synaptic glomeruli. Within the glomerulus, NMDA receptor subunits were located between centrally-located huge mossy fibre terminals and peripherally-located tiny Golgi axon terminals. By immunoelectron microscopy, all three subunits were detected at the postsynaptic junction in granule cell dendrites, forming synapses with mossy fibre terminals. Consistent with the known functional localization, GluRε1, GluRε3, and GluRζ1 are, thus, anatomically concentrated at the mossy fibre–granule cell synapse. By contrast, immunohistochemical signals were very low in Purkinje cell somata and dendrites in the molecular layer. The lack of GluRζ1 immunolabelling in Purkinje cells was unexpected because the cells express GluRζ1 mRNA at high levels and high levels of GluRζ1 protein in the molecular layer were revealed by immunoblot. As Purkinje cells are exceptionally lacking GluRε expression, the discrepant result may provide in vivo evidence suggesting the importance of accompanying GluRε subunits in synaptic localization of GluRζ1.