Identification of subunits contributing to synaptic and extrasynaptic NMDA receptors 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 were 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 currents were unaffected by the Zn(2+) chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN; 1 mu M) suggesting the absence of NMDARs containing NR2A subunits. 3. Outside-out patches from Golgi cells exhibited a population of 'high-conductance' 50 pS NMDAR openings. These were inhibited by ifenprodil, with an IC(50) 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 pS, 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 stimulation or occurring spontaneously, was not affected by 1 mu M TPEN. However, it was reduced (by similar to 60 %) in the presence of 10 mu M ifenprodil, to leave a residual NMDAR-mediated current that exhibited fast decay kinetics. This is, therefore, unlikely to have arisen from receptors composed of NR1/NR2D subunits. 6. We conclude that in cerebellar Golgi cells, the high- and low-conductance NMDAR channels arise from NR2B- and NR2D-containing receptors, respectively. We found no evidence for NR2A-containing receptors in these cells. While NR2B-containing receptors are present in both the synaptic and extrasynaptic membrane, our results indicate that NR1/NR2D receptors do not contribute to the EPSC and appear to be restricted to the extrasynaptic membrane.