NMDA receptor contribution to the climbing fiber response in the adult mouse Purkinje cell

Abstract

Among integrative neurons displaying long-term synaptic plasticity, adult Purkinje cells seemed to be an exception by lacking functional NMDA receptors (NMDA-Rs). Although numerous anatomical studies have shown both NR1 and NR2 NMDA-R subunits in adult Purkinje cells, patch-clamp studies failed to detect any NMDA currents. Using more recent pharmacological and immunodetection tools, we demonstrate here that Purkinje cells from adult mice respond to exogenous NMDA application and that postsynaptic NMDA-Rs carry part of the climbing fiber-mediated EPSC (CF-EPSC), with undetectable contribution from presynaptic or polysynaptic NMDA currents. We also detect NR2-A/B subunits in adult Purkinje cells by immunohistochemistry. The NMDA-mediated CF-EPSC is barely detectable before 3 weeks postnatal. From the end of the third week, the number of cells displaying the NMDA-mediated CF-EPSC rapidly increases. Soon, this EPSC becomes detectable in all the Purkinje cells but is still very small. Its amplitude continues to increase until 12 weeks after birth. In mature Purkinje cells, we show that the NMDA-Rs contribute to the depolarizing plateau of complex spikes and increase their number of spikelets. Together, these observations demonstrate that mature Purkinje cells express functional NMDA receptors that become detectable in CF-EPSCs at approximately 21 d after birth and control the complex spike waveform.

Figure 1.

NMDA currents recorded in adult (>8 weeks) mouse Purkinje cells and their contribution to the climbing fiber EPSC. A, Bath application of 50 μm NMDA performed in the presence of NBQX (10 μm), bicuculline (20 μm), and TTX (1 μm). B, Inward currents induced by ionophoretic applications of NMDA (left) are completely blocked by 50 μm d-APV (right). Recordings made in the presence of NBQX, bicuculline, and TTX in the bath (same concentrations as in A). C, PF-EPSCs are blocked by NBQX. Averaged PF-EPSCs from one cell in control conditions (left) and in the presence of 10 μm NBQX (right). D, CF-EPSCs display an NMDA-mediated component. Averaged CF-EPSCs recorded in control conditions (bicuculline only, left), after addition of 10 μm NBQX (middle), and in the presence of NBQX plus 50 mm d-APV (right). Inset, Superimposed scaled control-mediated (black) and NMDA-mediated (gray) CF-EPSCs.

Figure 2.

Most of the NBQX-resistant CF-EPSC is carried by NMDA-Rs in the absence of Mg²⁺. A, d-APV (50 μm) reversibly blocks the NBQX-resistant CF-EPSC. A1, Averaged CF-EPSCs recorded in one cell before, during, and after the application of d-APV (as indicated on the traces). A2, Same cell as A1. Amplitude of the CF-EPSCs plotted over time; note the washout of d-APV. B, Effect of Mg²⁺ on the NBQX-resistant CF-EPSC. Superimposed averaged NBQX-resistant CF-EPSCs recorded before and after addition of 1 mm external Mg²⁺ (as indicated on the traces) are shown. Additional application of 50 μm d-APV does not further block the response. C, Blocking glutamate transporters with 100 μm of dl-TBOA potentiates the NMDA-mediated CF-EPSC. C1, Averaged NBQX-resistant CF-EPSCs (trace 1, black) are potentiated by TBOA (trace 2, dark gray). These potentiated responses are blocked by the final addition of 50 μm d-APV (trace 3, light gray) showing that they are carried by NMDA-Rs. C2, Mean ± SEM amplitude (n = 5 cells) of the NMDA-mediated CF-EPSCs in control (NBQX at 10 μm), after addition of TBOA (100 μm), and after further addition of d-APV (50 μm). Numbers correspond to C1. D, Blocking group 1 mGluRs with AIDA (100 μm) has no effect on the NBQX-resistant CF-EPSC. D1, Averaged NBQX-resistant CF-EPSCs in control (NBQX alone) and during bath application of AIDA (NBQX + AIDA). Sweeps are merged for comparison (right). D2, Amplitude of the NBQX-resistant CF-EPSC plotted over time before and during the application of AIDA (as indicated).

Figure 3.

NMDA-mediated CF-EPSCs are postsynaptic. A, Internal MK801 blocks the NBQX-resistant CF-EPSC. Left, Averaged total CF-EPSCs recorded in one cell with standard K-based internal solution in the presence of 200 nm NBQX to reduce voltage-clamp escape and after addition of 10 μm NBQX. Right, Another cell recorded in the same conditions but with 3 mm MK801 added to the intracellular medium. Note that the infusion of MK801 in the Purkinje cell illustrated in the right blocks the NBQX-resistant CF-EPSC. B, Left, NBQX-resistant CF-EPSCs recorded from one cell at different holding potentials (indicated on the left) to establish their I–V curve. Top traces, In the presence of external Mg²⁺; bottom traces, in nominally Mg²⁺-free BBS. Right, Corresponding I–V curve established with the mean ± SEM peak amplitude of three successive EPSCs recorded at a given potential, in the presence (filled circles; standard BBS) or in the absence of external Mg²⁺ (open circles; Mg-free).

Figure 4.

Distribution of NR2-A/B immunofluorescence on sagittal slices observed with confocal imaging. A1, Single NR2-A/B immunolabeling showing the soma and primary dendrites of a Purkinje cell. A2, Same area observed in differential interference contrast microscopy (DIC). Scale bar, 20 μm. ML, Molecular layer; IGL, internal granular layer. Note the presence of presumably NR2-A at the level of glomeruli in the IGL. B, Successive 1-μm-spaced confocal sections showing NR2-A/B (red), calbindin (CaBP, blue), as well as VGluT2 (green) labeling. Scale bar, 40 μm. C, Three-dimensional reconstruction of a stack of 30 successive 1-μm-spaced confocal sections displaying the NR2-A/B immunolabeling alone (left) and NR2-A/B (red) plus CaBP (blue) plus VGluT2 (green) merged (right). Same zone as in B. Note that the merged 3D stack reveals that distal dendrites of Purkinje cell in the upper third of the molecular layer displays less intense immunoreactivity for NR2-A/B. D, High-power image (4×) of CaBP-positive (blue) dendrites showing NR2-A/B (red) and VGluT2 (green). Scale bar, 10 μm.

Figure 5.

Development of the NMDA-mediated CF-EPSCs and their blockade by CNQX. A, Amplitude of the NBQX-resistant CF-EPSC over time, in Mg²⁺-free external medium, in two different Purkinje cells from P17 and P26 mice. Addition of 50 μm d-APV in the bath reversibly blocks the NBQX-resistant CF-EPSC at P26, whereas it has no effect at P17. B, Proportion of Purkinje cells displaying a detectable NMDA component, observed at different postnatal ages. The total number of cells (n) recorded at each age is indicated above the bars. C, Percentage of blockade of the NBQX-resistant CF-EPSCs induced by d-APV (50 μm) over the development. Ages are indicated at the bottom of the graph. The total number of cells recorded at each age is indicated above the bars. D, Mean ± SEM peak amplitude of the NBQX-resistant CF-EPSCs in control (10 μm NBQX alone) and after addition of three different concentrations of CNQX (10, 20, or 50 μm as indicated). On the right, further application of 200 μm glycine tends to reverse the blockade by CNQX. Final addition of d-APV blocks the response reduced by CNQX and partially reversed by glycine, showing that it is carried by NMDA-Rs.

Figure 6.

Blocking the complex spike with NBQX reveals an NMDA-EPSP. Whole-cell current-clamp recording of a Purkinje cell from a mature animal (6 months old) in the absence of external Mg²⁺. Complex spike illustrated before (control) and after addition of NBQX (10 μm). NBQX suppresses spikes and spikelets of the complex spike and reveals an NBQX-resistant CF-EPSP (top middle trace) that is completely inhibited by 50 μm d-APV (top right trace). Note the kinetics of the complex spike and of NMDA-EPSP superimposed below.

Figure 7.

NMDA-R contribution to the complex spike in standard (1 mm Mg²⁺) BBS. Whole-cell recordings from mature Purkinje cells in the presence of external Mg²⁺ (1 mm) at room temperature. A1, Complex spikes recorded in bicuculline only (black trace) and in bicuculline plus d-APV (50 μm, gray trace). In this example, the ADP was measured at 70 ms after the stimulation onset. Note that d-APV reduces both the fast depolarization plateau (top) and the ADP. A2, Same cell as in A1. Amplitude of the ADP (bars) and resting potential (squares) plotted over time. B, Same type of recording in another mature Purkinje cell. B1, Complex spikes before (1), during (2), and after (3) the application of d-APV (50 μm). In this cell, d-APV reduced the plateau amplitude (arrows) and shifted the appearance of the spikelets (as in traces merged in bottom). B2, Same cell as B1. Delay of spikes and spikelets of the complex spike plotted over time. Note the occurrence of a third spikelet in some trials during the washout of d-APV (asterisk on the right trace of B1). This third spikelet occurred in the time window used to estimate the plateau amplitude, therefore causing the presence of peaks in the plot of amplitude of the plateau over time.