Functional NR2B- and NR2D-containing NMDA receptor channels in rat substantia nigra dopaminergic neurones

Abstract

NMDA receptors regulate burst firing of dopaminergic neurones in the substantia nigra pars compacta (SNc) and may contribute to excitotoxic cell death in Parkinson's disease (PD). In order to investigate the subunit composition of functional NMDA receptors in identified rat SNc dopaminergic neurones, we have analysed the properties of individual NMDA receptor channels in outside-out patches. NMDA (100 nm) activated channels corresponding to four chord conductances of 18, 30, 41 and 54 pS. Direct transitions were observed between all conductance levels. Between 18 pS and 41 pS conductance levels, direct transitions were asymmetric, consistent with the presence of NR2D-containing NMDA receptors. Channel activity in response to 100 nm or 200 microm NMDA was not affected by zinc or TPEN (N,N,N',N'-tetrakis-[2-pyridylmethyl]-ethylenediamine), indicating that SNc dopaminergic neurones do not contain functional NR2A subunits. The effect of the NR2B antagonist ifenprodil was complex: 1 microm ifenprodil reduced open probability, while 10 microm reduced channel open time but had no effect on open probability of channels activated by 100 nm NMDA. When the concentration of NMDA was increased to 200 microm, ifenprodil (10 microm) produced the expected reduction in open probability. These results indicate that NR2B subunits are present in SNc dopaminergic neurones. Taken together, these findings indicate that NR2D and NR2B subunits form functional NMDA receptor channels in SNc dopaminergic neurones, and suggest that they may form a triheteromeric NMDA receptor composed of NR1/NR2B/NR2D subunits.

Figure 1. Large and small-conductance NMDA receptor channels in SNc dopaminergic neurones

A, example currents recorded from outside-out patches from SNc dopaminergic neurones at a membrane potential of −60 mV in the presence of a, 100 nm NMDA and 10 μm glycine showing four different current amplitudes corresponding to the four conductance levels; b, 10 μm NMDA and 10 μm glycine; and c, 200 μm NMDA and 10 μm glycine. B, stability plot of channel amplitudes for one patch throughout the duration of the recording. C, amplitude distribution for the patch illustrated in B, fitted with the sum of four Gaussian components. The standard deviations were constrained to be the same (0.205 pA) for each component. The mean amplitude and relative area of each component are shown on the histogram, and correspond to chord conductances of 16.7 pS, 30.5 pS, 39.8 pS and 51.2 pS. The amplitude distribution contains current amplitudes for all openings that were longer than two filter rise-times.

Figure 2. NMDA receptors in SNc dopaminergic neurones contain NR2D subunits

A, examples of direct transitions between 18 pS and 41 pS and 41 pS and 54 pS, with percentage occurrence observed in this patch indicated for each transition. Transitions were identified using A_crit values of 1.45 pA, 2.12 pA and 2.90 pA, calculated from the fit to the amplitude distribution shown in Fig. 1C. B, plot of channel amplitudes before and after direct transitions from the same patch illustrated in A. Each point on the graph represents a single direct transition. The density of points illustrates that direct transitions between 41 pS and 54 pS occur with equal frequency, while transitions between 18 pS and 41 pS are asymmetric, occurring more frequently from 41 pS to 18 pS than from 18 pS to 41 pS. C, Three-dimensional plot of the same data shown in B.

Figure 3. Large-conductance NMDA receptor channels in SNc dopaminergic neurones are unaffected by TPEN

A, stability plots of P_open, mean open time and mean shut time for single-channel currents in the presence of a, 100 nm NMDA and 10 μm glycine and b, in the presence of NMDA, glycine and 1 μm TPEN show that TPEN did not affect the NMDA channel kinetics. Bar graphs summarizing data (mean ±s.e.m.) from four patches showing that TPEN had no significant effect on P_open (B), opening frequency (C) or open (D) and shut (E) times.

Figure 4. Large-conductance NMDA receptor channels in SNc dopaminergic neurones are unaffected by zinc

A, example currents recorded in the presence of a, 200 μm NMDA and 1 μm TPEN; and b, 200 μm NMDA and 200 nm zinc, illustrating that zinc has no effect on channel activity. B, bar graph (mean ±s.e.m.) summarizes the lack of effect of zinc on channel activity in response to a low (10 μm, 4 patches) or saturating (200 μm, 6 patches) concentration of NMDA.

Figure 5. Large-conductance NMDA receptor channels in SNc dopaminergic neurones contain NR2B subunits

A, example currents recorded a, in the presence of 100 nm NMDA; b, in the presence of 100 nm NMDA and 10 μm ifenprodil; c, in the presence of 200 μm NMDA; and d, in the presence of 200 μm NMDA and 10 μm ifenprodil, showing that ifenprodil reduces channel activity in response to a higher but not a lower concentration of NMDA. B, stability plots of P_open, mean open time and mean shut time for single-channel currents in 100 nm NMDA and in the presence of 100 nm NMDA and 10 μm ifenprodil showing that, in this patch, in addition to decreasing channel open time, ifenprodil also decreased P_open and channel shut time.

Figure 6. Ifenprodil reduces NMDA receptor channel activity in response to a high agonist concentration in SNc dopaminergic neurones

A, amplitude distributions in the presence of a, 10 μm NMDA; and b, 10 μm NMDA and 10 μm ifenprodil, fitted with the sum of four Gaussian components. The standard deviations were constrained to be the same (0.141 pA and 0.189 pA, respectively) for each component. Ifenprodil did not change the mean amplitude and relative area of each component. B, all-point amplitude distributions in the presence of a, 200 μm NMDA; and b, 200 μm NMDA and 10 μm ifenprodil, showing that ifenprodil reduces the channel activity.

Figure 7. Analysis of direct transitions between channel conductance levels

Examples of direct transitions between conductance levels, as indicated above each trace. Broken lines indicate the boundaries of each conductance level, determined by the A_crit values calculated from the Gaussian components in the amplitude distributions for each patch. There were a significant number of transitions (Wilcoxon rank sum test, P < 0.01) for all 12 possible ways that four different current levels can be connected in pairs (see Table 1).