Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC286
Use of burst analysis and agonist concentration jumps to investigate the properties of di- and tri-heteromeric NMDA receptors in dopaminergic neurons of neonatal rat substantia nigra
Q. Zhao1, A. Gibb1
1. Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom.
While many NMDA receptors are diheteromers composed of two glycine-binding (GluN1) and two glutamate binding (GluN2) subunits, there is evidence for triheteromeric GluN1/GluN2B/GluN2D receptors in the midbrain (Dunah et al., 1998; Jones & Gibb, 2005) and cerebellum (Brickley et al., 2003). Single channel amplitude distributions and ifenprodil inhibition has shown the presence of high conductance GluN2B subunit containing receptors (Jones & Gibb, 2005) while low conductance openings with asymmetrical transition frequencies between high and low conductances suggest GluN2D subunits are present. Here we further utilise ifenprodil block as a tool in steady-state recordings and in agonist concentration jump experiments to test for triheteromeric GluN1/GluN2B/GluN2D receptors in substantia nigra dopaminergic neurons in 300 μm thick midbrain slices from 7 day old rats. In order to examine the properties of single receptor activations, outside-out patch recordings (n=5) were made in very low concentrations (20nM) of NMDA plus 10μM glycine. Burst length distributions showed that 1μM ifenprodil produced a briefer intermediate burst component (1.43±0.09ms) compared with control (2.56±0.26ms), but no significant change in overall mean burst length (control, 6.67±1.1ms; ifenprodil, 6.63±1.17ms; P=0.9751) or in the slowest component of the burst length distribution (control, 20.44±1.82ms; ifenprodil, 23.56±3.97ms; P=0.3353) that is likely to dominate the response to an agonist concentration jump (Wyllie et al., 1998). Analysis of the total open time per burst showed a significant decrease in mean open time per burst (control, 2.26±0.38ms; ifenprodil, 1.45±0.21ms; P =0.0228). The time course of the macroscopic response to a brief 4 ms pulse of 1mM NMDA could be best fitted with three exponential components with mean rise-time of 5.49±0.15ms and decay of 59.40±0.41ms (relative amplitude, 10.06±0.01pA) and 1016.01±16.91 ms (relative amplitude, 0.21±0.01pA) (n=5). 1μM ifenprodil inhibited the peak current by 60.4±9.3% (n=5) and decreased the slow time constant to 309.32±10.93ms (relative amplitude, 0.43±0.02pA), but neither the time constant describing the rising (6.03±0.24ms) nor the fast decay time constant (56.48±0.39ms) changed. These results suggest NMDA receptor activation in substantia nigra dopaminergic neurons produces bursts of channel openings, which combined with the first latencies to activation, generate the familiar slowly rising and decaying macroscopic NMDA response. The co-localization of kinetically distinct GluN2B and GluN2D in a single triheteromeric GluN1/GluN2B/GluN2D receptor may account for the effect of ifenprodil on bursts of openings and on the macroscopic NMDA current decay.
Where applicable, experiments conform with Society ethical requirements