Identification of subunits contributing to synaptic and extrasynaptic NMDAreceptors in Golgi cells of the rat cerebellum

1. To investigate the properties of N-methyl-D-aspartate receptors (NMDARs) in cerebellar Golgi cells, patch-clamp recordings were made in cerebellar slices from postnatal day 14 (P14) rats. To verify cell identity, cells wer e filled with Neurobiotin and examined using confocal microscopy. 2. The NR2B subunit-selective NMDAR antagonist ifenprodil (10 mu M) reduced whole-cell NMDA-evoked currents by similar to 80 %. The NMDA-evoked curren ts were unaffected by the Zn2+ chelator N,N,N',N'-tetrakis-(2-pyridylmethyl )-ethylenediamine (TPEN; 1 mu M) suggesting the absence of NMDARs containin g NR2A subunits. 3. Outside-out patches from Golgi cells exhibited a population of 'high-con ductance' 50 pS NMDAR openings. These were inhibited by ifenprodil, with an IC50 of 19 nM. 4. Patches from these cells also contained 'low-conductance' NMDAR channels , with features characteristic of NR2D subunit-containing receptors. These exhibited a main conductance of 39 pS, with a sub-conductance level of 19 p S, with clear asymmetry of transitions between the two levels. As expected of NR2D-containing receptors, these events were not affected by ifenprodil. 5. The NMDAR-mediated component of EPSCs, evoked by parallel fibre stimulat ion or occurring spontaneously, was not affected by 1 mu M TPEN. However, i t was reduced (by similar to 60 %) in the presence of 10 mu M ifenprodil, t o leave a residual NMDAR-mediated current that exhibited fast decay kinetic s. This is, therefore, unlikely to have arisen from receptors composed of N R1/NR2D subunits. 6. We conclude that in cerebellar Golgi cells, the high- and low-conductanc e NMDAR channels arise from NR2B- and NR2D-containing receptors, respective ly. We found no evidence for NR2A-containing receptors in these cells. Whil e NR2B-containing receptors are present in both the synaptic and extrasynap tic membrane, our results indicate that NR1/NR2D receptors do not contribut e to the EPSC and appear to be restricted to the extrasynaptic membrane.