Functional heterogeneity of NMDA receptors in rat substantia nigra pars compacta and reticulata neurones

Abstract

The nigra substantia nigra pars compacta (SNc) and substantia pars reticulata (SNr) form two major basal ganglia components with different functional roles. SNc dopaminergic (DA) neurones are vulnerable to cell death in Parkinson's disease, and NMDA receptor activation is a potential contributing mechanism. We have investigated the sensitivity of whole-cell and synaptic NMDA responses to intracellular ATP and GTP application in the SNc and SNr from rats on postnatal day (P) 7 and P28. Both NMDA current density (pA/pF) and desensitization to prolonged or repeated NMDA application were greater in the SNr than in the SNc. When ATP levels were not supplemented, responses to prolonged NMDA administration desensitized in P7 SNc DA neurones but not at P28. At P28, SNr neurones desensitized more than SNc neurones, with or without added ATP. Responses to brief NMDA applications and synaptic NMDA currents were not sensitive to inclusion of ATP in the pipette solution. To investigate these differences between the SNc and SNr, NR2 subunit-selective antagonists were tested. NMDA currents were inhibited by ifenprodil (10 microM) and UBP141 (4 microM), but not by Zn(2+) (100 nm), in both the SNr and SNc, suggesting that SNc and SNr neurones express similar receptor subunits; NR2B and NR2D, but not NR2A. The different NMDA response properties in the SNc and SNr may be caused by differences in receptor modulation and/or trafficking. The vulnerability of SNc DA neurones to cell death is not correlated with NMDA current density or receptor subtypes, but could in part be related to inadequate NMDA receptor desensitization.

Fig. 1

Whole-cell I_h and rapid NMDA responses in SNc neurones. (A) Sample I_h response of a neurone in the SNc region of a brain slice from a P28 rat. Cells were voltage-clamped at −60 mV, and the membrane potential was stepped to −120 mV for 1.8 s in order to activate the I_h current. Superimposed (grey dashed line) is the fit of a single exponential that was used to determine the I_h amplitude and time constant. (B) Sample response of a P28 SNc neurone to rapid application of NMDA (200 μm for 15 s). In order to allow rapid access of the agonist, neurones were ‘cleaned’, as originally described by Edwards et al. (1989), before the whole-cell recording configuration was obtained.

Fig. 2

NMDAR current density in SNc and SNr neurones. NMDA (50 μm) and glycine (10 μm) were applied for 500 s to midbrain slices, and current responses were recorded from SNc and SNr neurones. (A) Sample traces recorded from P7 SNc and SNr neurones (both with intracellular ATP supplementation), illustrating that current density is greater in SNr neurones. (B) Concentration–response curves for NMDA-activated currents (expressed per unit of capacitance, pA/pF) for SNc (black) and SNr (grey) neurones at P7 (left) and P28 (right). Solid lines show fits to the Hill–Langmuir equation, assuming that the two cell types have the same [A]₅₀ for NMDA [[A]₅₀ value of 25 μm, and Hill coefficient of 1.22 at both P7 and P28 (not shown)]. The ratios of the predicted maxima were 1.5 at P7 and 1.8 at P28. Experiments were performed with ATP in the pipette solution. (C) SNr neurones had significantly greater current density than SNc neurones at P7 and P28, as indicated by *P < 0.05 (Bonferroni multiple comparison post hoc tests following two-way anova). (D) Examples of synaptic currents in P28 SNc neurones ± ATP. The AMPA receptor component of the current was recorded in the presence of 50 μm D-AP5. The AMPA/NMDA current ratio was not significantly different with or without ATP (unpaired t-test).

Fig. 3

NMDAR desensitization to prolonged agonist application: amplitude of responses in SNc neurones (A) and SNr neurones (B) to NMDA (500-s application) at P7 (i) or P28 (ii). Open symbols represent recordings made without ATP supplementation, and filled symbols represent recordings made with added intracellular ATP. In paired t-tests, there was a significant difference in the NMDA current amplitude at 300 s as compared with peak current in P7 SNc neurones with no added ATP (open triangles), P7 SNr neurones with no added ATP (open diamonds), and P28 SNr neurones with added ATP (filled diamonds). *P < 0.05 (paired t-test). (C) The change in NMDA current at 30 s as compared with NMDA current at 300 s was used to estimate NMDAR desensitization during prolonged agonist application. NMDA responses in P28 SNc neurones (Cii) did not significantly desensitize. At P28, SNr neurones desensitized more than SNc neurones (Cii). There was a significant decrease in desensitization between P7 and P28 (no ATP) in SNc neurones. In SNr neurones, there was a specific increase in desensitization between P7 and P28 when ATP was added (anova with Bonferroni multiple comparison post hoc tests; *P < 0.05).

Fig. 4

NMDAR desensitization to consecutive NMDA applications. (A) Sample recording of the response to paired applications of NMDA (200 μm, 60 s indicated by bar, 5 min between each application) recorded from SNr neurones at P7. (B) Bar graphs show the ratio of the second and the first response to NMDA. (C) Sample recording of the response to brief (15 s), rapid application of NMDA recorded from an SNc neurone at P28. (D) Adding ATP did not significantly change the N2/N1 ratios of responses to rapid application. Ages and numbers of cells are indicated below and on the bars, respectively. ***P < 0.001. (E) Slow (NMDAR-mediated) EPSCs recorded from P28 SNc neurones show little evidence of desensitization with repeated activation at 0.2 Hz, with or without added ATP. (F) NMDA EPSC amplitude after 150 s of repetitive stimulation, as a percentage of the initial amplitude; there is no significant effect of adding ATP (unpaired t-test).

Fig. 5

Pharmacology of NMDARs in SNc and SNr neurones. Sample traces in response to paired applications of NMDA (200 μm, 60 s indicated by bar, 5 min between each application) recorded from SNc neurones at P28, with N2 recorded in the presence of (A) 10 μm ifrenprodil or (B) 4 μm UBP141. ATP was added for all recordings. Bar graphs show the percentage inhibition of NMDA currents (corrected for control N2/N1 as described in Materials and methods). Ages and numbers of cells are shown below and on bars, respectively. There was no significant difference in the mean inhibition with ifenprodil or with UBP141 between the different groups (anova).