| The identity of NR2 subunits is critical in determining the functional properties of NMDA receptors. In order to explore the subunit composition of synaptic NMDA receptors in developing cerebellum granule cells, we studied the action potential evoked autaptic NMDA receptor mediated excitatory postsynaptic currents (autaptic NMDA-EPSCs) using solitary cerebellar neurons cultured in microislands from wild-type (+/+), NR2A subunit knockout (NR2A-/-) and NR2C knockout (NR2C-/-) mice. The peak amplitude of autaptic NMDA-EPSCs significantly increased for all genotypes from days in vitro 8 (DIV8) to DIV13. Compared to +/+ group, the autaptic NMDA-EPSCs were always smaller in NR2A-/- groups; while in NR2C-/- groups, they were similar at DIV8 and became significantly larger at DI V 13. The decay time constants of autaptic NMDAEPSCs were alike in +/+ and NR2C-/- groups, which became slightly but significantly faster with development; in contrast, the autaptic NMDA EPSCs recorded in NR2A-/groups were always slower compared to the other two genotypes and didn't exhibit any changes with age in vitro. Investigation on quantal parameters indicated that quantal contents increased in all genotypes from DIV8 to DIV13, which was largely due to the increase in the number of release sites. Compared to +/+ group at DIV13, the quantal size of autaptic NMDA-EPSCs was smaller in NR2A-/- group and larger in NR2C-/- group. Measured with MK-801, roughly 50% of surface NMDA receptors were consistently present at synaptic sites in +/+ group with age in vitro. The ratio of peak amplitude of maximal autaptic NMDA-EPSCs to whole cell current indicated that a larger portion of surface NMDA receptors located at synaptic sites in NR2A-/- and NR2C-/- groups at DIV13. The NR2B-selective antagonists Conantokin G and CP101,606 differed in their actions on autaptic NMDA-EPSCs, suggesting the presence of both heterodimeric NR1/NR2B and heterotrimenc NR1/NR2A/NR2B receptors. The significantly differences in peak amplitude and charge transfer of autaptic NMDA-EPSCs between +/+ and NR2C-/- groups strongly suggested that NR2C containing NMDA receptors contributed to synaptic NMDA receptors as well, which was further supported by the difference in Mg 2+ sensitivity of synaptic NMDA receptors between these two genotypes. |
