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

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Abstract
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. The NR2B subunit-selective NMDAR antagonist ifenprodil (10 μM) reduced whole-cell NMDA-evoked currents by ≈80 %. The NMDA-evoked currents were unaffected by the Zn2+ chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN; 1 μM) suggesting the absence of NMDARs containing NR2A subunits. Outside-out patches from Golgi cells exhibited a population of ‘high-conductance’ 50 pS NMDAR openings. These were inhibited by ifenprodil, with an IC50 of 19 nM. 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. The NMDAR-mediated component of EPSCs, evoked by parallel fibre stimulation or occurring spontaneously, was not affected by 1 μM TPEN. However, it was reduced (by ≈60 %) in the presence of 10 μM 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. 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.