NR2B- and NR2D-containing synaptic NMDA receptors in developing rat substantia nigra pars compacta dopaminergic neurones

Abstract

NMDA receptors are present at glutamatergic synapses throughout the brain, and are important for the development and plasticity of neural circuits. Their subunit composition is developmentally regulated. We have investigated the developmental profile of functional synaptic NMDA receptor subunits in dopaminergic neurones of the substantia nigra pars compacta (SNc). In SNc dopaminergic neurones from rats aged postnatal day (P)7, ifenprodil inhibited NMDA-EPSCs with an estimated IC(50) of 0.36 microm and a maximum inhibition of 73.5 +/- 2.7% (10 microm), consistent with a substantial population of NR1/NR2B-containing diheteromeric receptors. UBP141, a novel NR2D-preferring antagonist, inhibited NMDA-EPSCs with an estimated IC(50) of 6.2 microm. During postnatal development, the maximum inhibitory effect of 10 microm ifenprodil significantly decreased. However, NMDA-EPSCs were not inhibited by Zn(2+) (200 nM) or potentiated by the Zn(2+) chelator TPEN (1 microm), and the effect of UBP141 did not increase during development, indicating that NR2B subunits are not replaced with diheteromeric NR2A or NR2D subunits. The time course of the decay of NMDA-EPSCs was not significantly changed in ifenprodil at any age tested. Together, these data suggest that diheteromeric NR1/NR2A or NR1/NR2D receptors do not account for the ifenprodil-resistant component of the NMDA-EPSC. We propose that NR1/NR2B/NR2D triheteromers form a significant fraction of synaptic NMDA receptors during postnatal development. This is the first report of data suggesting NR2D-containing triheteromeric NMDA receptors at a brain synapse.

Figure 1. NMDA-EPSCs throughout early postnatal development

A, example recording of EPSCs at +40 mV from a SNc dopaminergic neurone in a slice from a rat aged P7. Traces are the average of 10 EPSCs. ‘Total’ EPSC (black trace) was recorded in picrotoxin (50 μm) and glycine (10 μm), ‘AMPA’-EPSC (dark grey trace) was recorded in the presence of 50 μm d-AP5, and ‘NMDA’-EPSC was obtained by subtracting the AMPA-EPSC from the Total EPSC. B, bar graph showing the ratio of the AMPA-EPSC to the NMDA-EPSC (obtained by measuring the responses shown in A at the three developmental stages tested. There was no significant difference during the first 3 weeks of postnatal development (ANOVA P > 0.05; ‘n’ in parentheses). C, current–voltage relationship for NMDA-EPSCs recorded in 1.3 mm extracellular Mg²⁺ and normalized to the response at +40 mV (rat aged P7). D, bar graph showing the ratio of the NMDA-EPSC recorded at −40 mV to that recorded at +40 mV in slices from rats aged P7, P14 and P21. Data for P14 slices are shown at 1.3 mm and 0.1 mm extracellular Mg²⁺. ANOVA revealed a significant difference (P < 0.05), and the post hoc tests revealed a significant difference only between 0.1 and 1.3 mm Mg²⁺ at P14 (*P < 0.05, Bonferroni's multiple comparison post hoc test; ‘n’ in parentheses).

Figure 2. Developmental change in functional NR2B receptors

A, example recordings of NMDA-EPSC amplitude (pA) over time in a slice from a P7 rat (open circles) and a slice from a P21 rat (grey circles). Ifenprodil (10 μm) was added after a 10 min stable baseline was recorded, d-AP5 (50 μm) was added at the end of each experiment. B, combined data of NMDA-EPSCs over time showing the effect of ifenprodil (10 μm) at the three developmental stages tested (open circles, P7, n = 6; black circles, P14, n = 17; grey circles, P21, n = 7). In all three age groups ifenprodil caused a significant inhibition. C, bar graph comparing the mean inhibition (%) induced by 10 μm ifenprodil at P7 (measured 20–30 min post-drug), P14 and P21 (both measured 10–20 min post-drug). Significant difference detected with ANOVA; *P < 0.01, Bonferroni's multiple comparison post hoc test (‘n’ in parentheses). D, inhibition curves showing the effect of increasing concentrations of ifenprodil on NMDA-EPSCs at the three developmental stages. The fit of the data (see Methods) was used to obtain the IC₅₀ values reported in the text. Numbers of slices at each concentration and age are as follows: P7 (open circles) 0.1 μm, n = 4; 1 μm, n = 5; 10 μm, n = 6; P14 (black circles) 0.1 μm, n = 6; 1 μm, n = 5; 3 μm, n = 6; 10 μm, n = 17; P21 (grey circles) 0.1 μm, n = 4; 1 μm, n = 5; 3 μm, n = 4; 10 μm, n = 7.

Figure 3. NR2A-containing receptors are absent during early postnatal development

A, example recording of NMDA-EPSC amplitude (pA) over time in a slice from a P14 rat. Zn²⁺ (200 nm) was added after a 10 min stable baseline, d-AP5 (50 μm) was added at the end of the experiment. Inset shows NMDA-EPSCs in control, Zn²⁺ and d-AP5. B, combined data of NMDA-EPSCs over time showing the lack of effect of TPEN (1 μm; grey circles, n = 8) and Zn²⁺ (200 nm; black circles, n = 6) in slices from rats aged P13–P22. C, combined data of NMDA-EPSCs over time showing the effect of NVP-AAM077 (10 nm; open circles, n = 9; grey circles, 30 nm, n = 10) in slices from rats aged P13–P22. Inset shows NMDA-EPSCs in control and 30 nm NVP-AAM077. D, bar graph summarizes the lack of effect of TPEN and Zn²⁺ alone or Zn²⁺ following a baseline in TPEN, and the modest effects of NVP-AAM077, suggestive of an effect on NR2B-containing receptors.

Figure 4. NR2D receptors are present throughout early postnatal development

A, combined data of NMDA-EPSCs over time showing the effect of UBP141 (3 μm) at the three developmental stages tested (open circles, P7, n = 6; black circles, P14, n = 5; grey circles, P21, n = 4). In all three age groups UBP141 caused a significant inhibition. B, inhibition curves showing the effect of increasing concentrations of UBP141 on NMDA-EPSCs at the three developmental stages. The fit of the data was used to obtain the IC₅₀ values reported in the text. Numbers of slices at each concentration and age are as follows: P7 (open circles) 1 μm, n = 6; 3 μm, n = 6; 10 μm, n = 4; P14 (black circles) 1 μm, n = 5; 3 μm, n = 5; 10 μm, n = 4; P21 (grey circles) 1 μm, n = 5; 3 μm, n = 4; 10 μm, n = 4.

Figure 5. NR2B and NR2D subunits may form triheteromeric NMDA receptors

A, example recording of NMDA-EPSCs at +40 mV from a SNc dopaminergic neurone in a slice from a rat aged P14. Traces are the average of 20 NMDA-EPSCs. ‘Control’ EPSC (black trace) was recorded in picrotoxin, glycine and DNQX (10 μm), ‘Ifenprodil’ EPSC (grey trace; Ifen) was recorded in the presence of 10 μm ifenprodil, and the two traces have been scaled for comparison of the decay. B, bar graph showing the time constant of the decay (weighted from a two-component exponential fit of the control NMDA-EPSC at P7, P14 and P21 and of the NMDA-EPSC component remaining in the presence of 10 μm ifenprodil at each age. ANOVA revealed a significant difference (P < 0.05); post hoc tests revealed a significant difference between control τ_W at P7 and P21 (*P < 0.05, Bonferroni's multiple comparison post hoc test; ‘n’ in parentheses). C, current–voltage relationship for NMDA-EPSCs recorded in 1.3 mm extracellular Mg²⁺ (normalized to the response at +40 mV) in control conditions (n = 7) and in the presence of 10 μm ifenprodil (n = 4) in slices from P14 rats. D, bar graph showing the ratio of the NMDA-EPSC recorded at −40 mV to that recorded at +40 mV in control and ifenprodil.