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Title: NMDA receptor kinetics and synaptic integration in cortical pyramidal neurons
Author: Vargas-Caballero, Mariana
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2004
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Understanding the synaptic function of N-methyl-D-aspartate receptors (NMDARs) and their contribution to membrane excitability requires a better comprehension of their kinetics in physiological conditions. Most previous studies of NMDAR gating have focussed on responses to jumps in agonist or blocker concentrations, or on single-channel gating at equilibrium. This thesis exposes the kinetics of NMDARs in nucleated patch and whole-cell recordings of rat cortical pyramidal neurons during nonstationary conditions of voltage and agonist concentration. It was found that the timing of voltage-dependent removal of Mg2+ block of NMDARs can determine their nonlinear contribution to excitability. At room temperature, the observed kinetics of unblock showed a very fast component (t < 1 ms), together with a slower component (t = 10-40ms). The amplitude and time constant of the slow component both increased with depolarisation, and it accounted for half of the current for steps from -70 to +40 mV. Block was effectively instantaneous for voltage steps from +40 to -70 mV, but had major slow components for steps from +40 to -40 mV, where up to 40% of current blocks with time constants between 3 and 6 ms. Additionally, the voltage-dependence of deactivation kinetics were studied in nucleated patches during fast and sustained perfusion with agonist. Currents at +40 mV had a time-constant of decay two-fold larger than those at -40 mV. The voltage dependence observed was, however, much less pronounced than suggested previously (Konnerth et al, 1990). This discrepancy may be explained by the improved space clamp conditions and calcium buffering achieved with nucleated patches as compared to whole cell recordings. Depolarisations from rest at different time intervals during the decay phase recovered a current of similar amplitude to that recorded during sustained depolarisation. The observations on block/unblock during voltage steps and voltage dependence of decay strongly suggest a Mg2+ -trapping block mechanism for the NMDAR. However, a trapping block kinetic scheme with identical rate constants for the receptor bound and unbound to Mg2+ (Sovolebsky and Yelshansky, 2000) does not predict the slow unblock and fast tail currents observed after a depolarising voltage step. Instead, the slow unblock was found to be consistent with faster closing kinetics for the channel when it is bound to Mg2+, which was termed an asymmetric trapping block (ATB) model. During nonstationary voltage conditions, the observed time-dependence of block and unblock results in a very different pattern of activation from the instantaneous I-V relation that has been assumed for the NMDAR in previous modelling studies. Voltage-clamp of nucleated patches with action potential waveforms, at both room temperature and at 33°C, during stationary NMDAR activation, showed that the rising phase of single Na+ action potentials unblocks far less NMDAR current than expected from the stationary voltage-dependence, while a large current is uncovered during the upstroke of slow Ca2+ action potentials. The repolarisation of fast Na+ action potentials uncovers an NMDAR tail current, much bigger than the predicted steady-state level of current. Thus, retarding the boosting effect of depolarisation and resisting the repolarisation, and therefore prolonging, dendritic Ca2+ spikes. The functional consequences of slow unblock were studied in a simple model of pyramidal cell excitability.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral