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

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. At many central excitatory synapses, the transmitter L-glutamate activates a mixture of NMDARs and non-NMDARs. The NMDARs are unique among mammalian synaptic receptors in displaying a voltage-dependent block by Mg2+ ions, and requiring the binding of both the transmitter and the co-agonist, glycine, for their activation (reviewed by Johnson & Ascher, 1996). The functional properties of NMDARs are influenced by a wide variety of endogenous modulators including Zn2+ ions, protons and polyamines (see Dingledine et al. 1999). Several functionally distinct subtypes of NMDARs are present in central neurons and these differ in their responses to glutamate, glycine and modulators (reviewed by Feldmeyer & Cull-Candy, 1996). Thus, the type of NMDAR present at a particular synapse has clear functional implications for transmission. Molecular techniques have identified three families of NMDAR subunits. The NR1 family exists in a variety of splice variants, and is widespread throughout the central nervous system (CNS). The NR2 family comprises four members, NR2A, -2B, -2C and -2D, which are differentially distributed. More recently, the NR3 subunit has been identified, which has a relatively restricted distribution (see McBain & Mayer, 1994; Dingledine et al. 1999). Our knowledge of the relationship between NMDAR properties and their subunit composition has been greatly advanced by studies of native receptors, whose subunit complement has been inferred from in situ hybridization, immunocytochemistry or single cell PCR (Farrant et al. 1994; Momiyama et al. 1996; Paoletti et al. 1997; Plant et al. 1997; Stocca & Vicini, 1998) and by experiments on recombinant receptors (see for example, Stern et al. 1992; Monyer et al. 1994; Vicini et al. 1998; Wyllie et al. 1998). Two functionally distinct classes of native NMDAR channels have been identified in the CNS: ‘high-conductance’ NMDAR channels, with conductance levels of 40 and 50 pS, and ‘low-conductance’ channels with levels of ∼18 and ∼38 pS (see Momiyama et al. 1996, and references therein). Studies of both recombinant and native receptors have ascribed the high-conductance events to receptors formed from NR1/NR2A or NR1/NR2B subunits (Stern et al. 1992; Farrant et al. 1994; Brimecombe et al. 1997). The low-conductance class of receptor channel is associated with receptor assemblies containing NR1/NR2C (Stern et al. 1992; Farrant et al. 1994; Takahashi et al. 1996) or NR1/NR2D subunits (Momiyama et al. 1996; Wyllie et al. 1996). We have been particularly interested in the low-conductance NR1/NR2D channels since they exhibit a number of unusual properties, including a low sensitivity to voltage-dependent block by extracellular Mg2+ (see Monyer et al. 1994; Momiyama et al. 1996) and unusually slow deactivation rate, following a brief pulse of glutamate (Monyer et al. 1994; Vicini et al. 1998; Wyllie et al. 1998). Thus, if present at the synapse, such receptors might be expected to produce currents, and Ca2+ entry, lasting several seconds. In situ hybridization data suggest that the NR2D-containing NMDARs may be widespread in the cerebellum, being the only NR2 subunit mRNA detected in young Purkinje cells, stellate cells and Golgi cells of the rat (Watanabe et al. 1994; Akazawa et al. 1994). Previous work on immature Purkinje cells has demonstrated that while these cells express a homogeneous population of receptors with properties characteristic of NR1/NR2D assemblies, the receptors appear not to be activated synaptically (Momiyama et al. 1996). Golgi cells are GABAergic interneurons, classically thought to contribute to the filtering of mossy fibre sensory input during its relay via granule cells to Purkinje cells (see Gabianni et al. 1994 for references). The importance of Golgi cells to cerebellar function is underlined by the fact that their selective ablation causes acute disruption of motor co-ordination (Watanabe et al. 1998). Recently, it has been suggested that the feedback excitation of Golgi cells via granule cell axons (parallel fibres) acts to synchronise activity of both cell types (Maex & DeSchutter, 1998; Vos et al. 1999). Although parallel fibre input to Golgi cells activates both AMPA- and NMDA-type glutamate receptors (Dieudonne, 1998), the subunit composition of the latter and their importance to parallel fibre efficacy remain unclear. In the present study we have examined the biophysical and pharmacological properties of synaptic and extrasynaptic NMDARs in cerebellar Golgi cells. Contrary to expectations based on in situ hybridization data, our results indicate that Golgi cells express at least two types of NMDAR channel, only one of which appears to be expressed at the synapse.