We have examined the changes in the pharmacological properties and decay kinetics of NMDAR-mediated EPSCs during development of cerebellar Golgi cells. NMDARs are heteromeric assemblies containing NR1- and at least one type of NR2-subunit (NR2A-D). In situ hybridisation studies of the cerebellum have indicated that mRNA expression of the four NR2-subunits differs between cell types and is developmentally regulated (Watanabe et al. 1994). The deactivation kinetics and pharmacological properties of NMDAR-mediated currents are dependent on the type of NR2-subunit contributing to the assembly (see Cull-Candy et al. 2001). For example, acceleration of the NMDAR-EPSC that occurs during development at various central synapses is thought to reflect an NR2B to NR2A switch.

We examined spontaneous EPSCs in Golgi cells under whole-cell patch-clamp in thin slices from humanely killed animals (Misra et al. 2000). Currents were recorded at -80 mV in Mg²⁺-free solution containing GABA- and glycine receptor blockers. EPSCs evoked by parallel fibre stimulation were also examined. At postnatal day 7 (P7), the mean decay time constant of spontaneous NMDAR-EPSCs was 72.0 ± 5.4 ms (mean ± S.E.M., n = 11). By P15 this was significantly reduced, with a time constant of 46.0 ± 6.0 ms (n = 22; P < 0.01, Student’s unpaired t test). This speeding up of the decay kinetics suggests an increased contribution of the NR2A subunit at P15. We further investigated the possible developmental change in the contribution of NR2B and NR2A to the NMDAR-EPSC by using NR2 subunit-selective drugs. Ifenprodil selectively inhibits NR1/NR2B receptors, and the Zn²⁺ chelator N,N,Nâ,Nâ-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) is known to potentiate currents through NR1/NR2A receptors. TPEN (1 mM) had little effect at P7, but significantly augmented EPSCs by P15 (38 ± 12 % of control, n = 7; P < 0.01, paired t test). On the other hand, inhibition by 10 mM ifenprodil was significantly reduced from 71 ± 3 % at P7 (n = 11) to 42 ± 8 % by P15 (n = 7; P < 0.01, unpaired t test). We found a clear correlation between the faster decay time of NMDAR-EPSCs and the reduction in ifenprodil block observed during development. Similar developmental changes in decay kinetics and pharmacology were observed with parallel fibre-evoked EPSCs. It is of note that by P35 spontaneous NMDAR-EPSCs were reduced in amplitude to 35 % of that at P7.

In conclusion, our data on the pharmacological and kinetic properties of NMDAR-EPSCs indicate a developmentally regulated reduction in the contribution of NR2B- and an increasing role for the NR2A-subunit at Golgi cell synapses.

This work is supported by The Wellcome Trust.

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