NMDA receptors are present at glutamatergic synapses throughout the brain, and are important for the development and plasticity of neural circuits. Their subunit composition is developmentally regulated. We have investigated the developmental profile of functional synaptic NMDA receptor subunits in dopaminergic neurones of the substantia nigra pars compacta (SNc). In SNc dopaminergic neurones from rats aged postnatal day (P)7, ifenprodil inhibited NMDA‐EPSCs with an estimated IC₅₀of 0.36 μmand a maximum inhibition of 73.5 ± 2.7% (10 μm), consistent with a substantial population of NR1/NR2B‐containing diheteromeric receptors. UBP141, a novel NR2D‐preferring antagonist, inhibited NMDA‐EPSCs with an estimated IC₅₀of 6.2 μm. During postnatal development, the maximum inhibitory effect of 10 μmifenprodil significantly decreased. However, NMDA‐EPSCs were not inhibited by Zn²⁺(200 nm) or potentiated by the Zn²⁺chelator TPEN (1 μm), and the effect of UBP141 did not increase during development, indicating that NR2B subunits are not replaced with diheteromeric NR2A or NR2D subunits. The time course of the decay of NMDA‐EPSCs was not significantly changed in ifenprodil at any age tested. Together, these data suggest that diheteromeric NR1/NR2A or NR1/NR2D receptors do not account for the ifenprodil‐resistant component of the NMDA‐EPSC. We propose that NR1/NR2B/NR2D triheteromers form a significant fraction of synaptic NMDA receptors during postnatal development. This is the first report of data suggesting NR2D‐containing triheteromeric NMDA receptors at a brain synapse.

N‐Methyl‐d‐aspartate glutamate receptors (NMDARs) contribute to neural development, plasticity and survival, but they are also linked with neurodegeneration. NMDARs at synapses are activated by coincident glutamate release and depolarization. NMDARs distal to synapses can sometimes be recruited by ‘spill‐over’ of glutamate during high‐frequency synaptic stimulation or when glutamate uptake is compromised, and this influences the shape of NMDAR‐mediated postsynaptic responses. In substantia nigra dopamine neurons, activation of NMDARs beyond the synapse during different frequencies of presynaptic stimulation has not been explored, even though excitatory afferents from the subthalamic nucleus show a range of firing frequencies, and these frequencies change in human and experimental Parkinson's disease. This study reports that high‐frequency stimulation (80 Hz/200 ms) evoked NMDAR‐excitatory postsynaptic currents (EPSCs) that were larger and longer lasting than those evoked by single stimuli at low frequency (0.1 Hz). MK‐801, which irreversibly blocked NMDAR‐EPSCs activated during 0.1‐Hz stimulation, left a proportion of NMDAR‐EPSCs that could be activated by 80‐Hz stimulation and that may represent activity of NMDARs distal to synapses. TBOA, which blocks glutamate transporters, significantly increased NMDAR‐EPSCs in response to 80‐Hz stimulation, particularly when metabotropic glutamate receptors (mGluRs) were also blocked, indicating that recruitment of NMDARs distal to synapses is regulated by glutamate transporters and mGluRs. These regulatory mechanisms may be essential in the substantia nigra for restricting glutamate diffusion from synaptic sites and keeping NMDAR‐EPSCs in dopamine neurons relatively small and fast. Failure of glutamate transporters may contribute to the declining health of dopamine neurons during pathological conditions.

Key points: Calcium ion influx throughN‐methyl‐d‐aspartate receptors (NMDARs) may contribute to substantia nigra pars compacta (SNc) dopaminergic neurone dysfunction in Parkinson's disease. Responses of NMDARs in dopaminergic neurones showed use‐dependent run‐down that was not readily reversible and was partly dependent on Ca²⁺influx and partly dependent on clathrin‐mediated endocytosis. Thus, we report regulation of NMDARs in SNc dopaminergic neurones by intracellular Ca²⁺at both synaptic and extrasynaptic sites and provide evidence for activity‐dependent changes in receptor trafficking. N‐Methyl‐d‐aspartate receptors (NMDARs) are Ca²⁺‐permeable glutamate receptors that play a critical role in synaptic plasticity and promoting cell survival. However, overactive NMDARs can trigger cell death signalling pathways and have been implicated in substantia nigra pars compacta (SNc) pathology in Parkinson's disease. Calcium ion influx through NMDARs recruits Ca²⁺‐dependent proteins that can regulate NMDAR activity. The surface density of NMDARs can also be regulated dynamically in response to receptor activity via Ca²⁺‐independent mechanisms. We have investigated the activity‐dependent regulation of NMDARs in SNc dopaminergic neurones. Repeated whole‐cell agonist applications resulted in a decline in the amplitude of NMDAR currents (current run‐down) that was use dependent and not readily reversible. Run‐down was reduced by increasing intracellular Ca²⁺buffering or by reducing Ca²⁺influx but did not appear to be mediated by the same regulatory proteins that cause Ca²⁺‐dependent run‐down in hippocampal neurones. The NMDAR current run‐down may be mediated in part by a Ca²⁺‐independent mechanism, because intracellular dialysis with a dynamin‐inhibitory peptide reduced run‐down, suggesting a role for clathrin‐mediated endocytosis in the regulation of the surface density of receptors. Synaptic NMDARs were also subject to current run‐down during repeated low‐frequency synaptic stimulation in a Ca²⁺‐dependent but dynamin‐independent manner. Thus, we report, for the first time, regulation of NMDARs in SNc dopaminergic neurones by changes in intracellular Ca²⁺at both synaptic and extrasynaptic sites and provide evidence for activity‐dependent changes in receptor trafficking. These mechanisms may contribute to intracellular Ca²⁺homeostasis in dopaminergic neurones by limiting Ca²⁺influx through the NMDAR.