Native subtypes of NMDA receptors with distinct developmental and anatomical distribution are present in the brain. Knowledge of their function and pharmacology greatly helps in understanding the involvement of NMDA receptors in physiological and pathological processes in the central nervous system. Multiple patch-clamping methods were carried out in my experiments. First, the single-channel properties of native NMDA receptors were studied in outside-out patches excised from neuronal cell bodies in substantia nigra pars compacta (SNc) and subthalamic nucleus (STN) from 7-day old rats. The steady state channel activations produced by NMDA (100 nM or 200 uM) and glycine (10 uM) were studied at -60 mV. The results showed that both large and small conductance channels are present on STN and SNc neurons. These large conductance NMDA channels from SNc and STN display a high ifenprodil sensitivity, suggesting NR2B-containing NMDA receptors are present on SNc and STN neurons. In addition, direct transition analysis suggests that the small conductance channels may be due to NR2D-containing NMDA receptors. Second, whole-cell patch-clamp recording of bath application-induced NMDA receptor-mediated currents (Inmda) in dopaminergic neurons of SNc in brain slices from P7 rats was used to characterize the ifenprodil inhibition and voltage-dependence of Mg2+ block of NMDA receptors. The NMDA-induced whole-cell currents were evoked by NMDA (10 uM or 200 uM) and glycine (10 pM). There are two main findings from the whole-cell experiments. (1) The NMDA-induced whole-cell currents display a high ifenprodil sensitivity, suggesting the presence of NR2B-containing NMDA receptors on P7 rat SNc neurons. (2) The combined application of Mg2+ and ifenprodil reduced the voltage-dependent Mg2+ block, which is consistent with recombinant NR2D-containing NMDA receptors that have a lower affinity for Mg2+. This suggests that NR2D-containing NMDA receptors are present in P7 rat SNc. Third, in order to evaluate the deactivation kinetics of extrasynaptic NMDARs, outside-out patches containing multiple channels were obtained from P7 rat dopaminergic neurons of SNc and stimulated with a brief synaptic-like (l-4ms) pulse of ImM glutamate. The results suggested that the triheteromeric NR1-NR2B-NR2D rather than diheteromeric NR1-NR2D NMDA receptors are present on extrasynaptic sites of SNc neurons. In addition, in order to investigate voltage-dependent Mg2+ block and memantine block, several channel block models were developed to evaluate multiple Mg2+ effects on native NMDA receptors and competition between Mg2+ and memantine for block of NMDAR channels.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:498636 |
| Date | January 2008 |
| Creators | Huang, Zhuo |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1443978/ |
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