Climbing-fibre activation of NMDA receptors in Purkinje cells of adult mice

Abstract

Among principal neurons, adult Purkinje cells have long been considered unusual in lacking functional NMDA receptors. This view has emerged largely from studies on rats, where NMDA receptors are expressed in Purkinje cells of newborn animals, but are lost after 2 weeks. By contrast, immunolabelling data have shown that Purkinje cells from adult mice express multiple NMDA receptor subunits, suggesting a possible species difference. To investigate the presence of functional NMDA receptors in Purkinje cells of mice, and to explore the contribution of different receptor subunits, we made whole-cell and single-channel patch-clamp recordings from Purkinje cells of wild-type and NR2D-/- mice of different ages. Here we report that multiple NMDA receptor subtypes are indeed expressed in Purkinje cells of young and adult mice; in the adult, both NR2A- and NR2B-containing subtypes are present. Furthermore, we show that NMDA receptor-mediated EPSCs can be evoked by climbing fibre stimulation, and appear to be mediated mainly by NR2A-containing receptors.

Figure 1

Low- and high-conductance NMDARs in PCs from young mice A, continuous recording from wild-type and NR2D−/− patches, in the presence of 10 μm NMDA and 5 μm glycine (P6; −60 mV). Single-channel currents are absent from the NR2D−/− patch. B–D, properties of NMDARs in wild-type mice. B, single-channel currents recorded at various membrane potentials and C, the corresponding current–voltage relationship (same patch as A). Slope conductances for this patch are indicated on the I–V plot, and indicated as dotted lines on the traces. The mean slope conductances from 3 patches were 42.5 ± 1.0 and 21.3 ± 0.3 pS. D, typical of NR2D-containing NMDARs, direct transitions from the main- to the subconductance level occurred more frequently than vice versa, as shown by the ‘asymmetry plot’, where i^th represents the first and (i + 1)^th represents the second subconductance level for each opening (see also Methods). E and F, high-conductance NMDARs in patches from wild-type and NR2D−/− P6 mice. E, example trace, and its all-point histogram, showing both high- and low-conductance openings (wild-type). F, NMDAR openings recorded from an NR2D−/− patch (slope conductance 50 and 38 pS) and their block by 2 mm extracellular Mg²⁺ at negative potentials.

Figure 2

Functional NMDARs in PCs from adult wild-type mice A, representative whole-cell response to bath applied NMDA (50 μm glycine, −60 mV) and its subsequent block by d-AP5. Right: the bar graph shows amplitudes of currents evoked by different concentrations of NMDA, and block by d-AP5 in pooled data from 7 experiments (concentrations in μm). B, superimposed single-channel currents recorded in the presence of 100 μm NMDA (50 μm glycine; −60 mV) in an outside-out patch from a P67 mouse. Lower trace shows block in the presence of Mg²⁺ (4 mm). C, NMDA-channel currents recorded at various membrane potentials in a patch from a P74 mouse (100 μm NMDA plus 50 μm glycine; dotted lines indicate the slope conductance for this patch). Right panel shows I–V plot of pooled data from 7 experiments (all mice older than P61; mean slope conductance 54 pS).

Figure 3

Zinc sensitivity of extrasynaptic NMDARs in PCs from adult mice A, single-channel currents activated by 100 μm NMDA (50 μm glycine) before and after the application of 5 or 200 nm Zn²⁺ (P74; −60 mV) in an outside-out patch exhibiting ‘high’ zinc sensitivity. Bar plot shows pooled data from 11 experiments (all mice older than P70) and illustrates the reduction of NMDAR-mediated charge transfer (Q(t)) in the presence of Zn²⁺ (symbols indicate values from individual patches; the number of patches is indicated in brackets). Note that, on average, the sensitivity of NMDARs to Zn²⁺ in these patches is typical of recombinant NR2A-containing NMDARs (as indicated by the dotted lines; see Rachline et al. 2005). B, single-channel currents in a patch exhibiting ‘low’ zinc sensitivity (P77; −60 mV). Bar plots illustrate pooled data from 3 experiments. Dotted lines indicate the level of Zn²⁺ inhibition expected for recombinant NR2B-containing NMDARs (see Rachline et al. 2005).

Figure 4

Ifenprodil sensitivity of extrasynaptic NMDARs in PCs from adult mice A, superimposed NMDAR channel openings in an outside-out patch from an adult PC exposed to 100 μm NMDA (50 μm glycine) before and after the application of 5 μm ifenprodil (P63; −60 mV). In this patch, ifenprodil reduced channel activity. B, bar plot of pooled data from 10 experiments showing that, on average, ifenprodil reduced the NMDAR charge transfer (Q(t)) to 63.7 ± 0.5% of control. Note that some patches (right hand bar) were unaffected by ifenprodil. Symbols indicate values from individual patches. Note that the scatter of the data was likely to be due to the presence of more than one NMDAR in each patch. Dotted lines indicate the level of inhibition expected for recombinant NR2A-, NR2A–NR2B- or NR2B-containing NMDAR, in the presence of 3 μm ifenprodil (see Hatton & Paoletti, 2005).

Figure 5

NMDAR activation during CF-EPSC in PCs from adult mice A, CF-EPSCs evoked in 1 μm NBQX (left). After full block of remaining AMPARs by an additional 20 μm NBQX (middle), CF-EPSCs were greatly reduced by d-AP5. The d-AP5-sensitive current is shown on the right. As illustrated in the inset (scaled currents), this NMDAR-mediated component rose more slowly than the pure AMPAR-mediated component of the EPSC (NBQX-sensitive current). In this cell the 10–90% rise time was 6.2 ms (versus 2.4 ms for the NBQX-sensitive current) and the 37% decay was 27 ms; mean 37% decay 29 ± 4 ms (n = 7). The d-AP5-sensitive current was obtained by subtracting the average CF-EPSC recorded in the presence of 50–100 μm d-AP5 from the average CF-EPSC recorded in the presence of 20 μm NBQX. The NBQX-sensitive current was obtained by subtracting the average CF-EPSC recorded in the presence of 20 μm NBQX from the average CF-EPSC recorded in the presence of 1 μm NBQX. B, CF-EPSCs from a different PC, recorded initially in the presence of 1 μm NBQX. Addition of a further 20 μm NBQX left a residual component that was unaffected by 5 μm ifenprodil but eliminated by d-AP5. Right: same traces on an expanded time scale. Stimulation artefacts were blanked. Bottom right: plot of pooled data from 3 experiments showing the lack of effect of ifenprodil on the peak and the area (charge) of NMDAR-mediated CF-EPSCs (see text for details). Symbols connected by continuous lines show the mean values and the vertical error bars indicate the s.e.m.