The density of AMPA receptors activated by a transmitter quantum at the climbing fibre-Purkinje cell synapse in immature rats

Abstract

We aimed to estimate the number of AMPA receptors (AMPARs) bound by the quantal transmitter packet, their single-channel conductance and their density in the postsynaptic membrane at cerebellar Purkinje cell synapses. The synaptic and extrasynaptic AMPARs were examined in Purkinje cells in 2- to 4-day-old rats, when they receive synaptic inputs solely from climbing fibres (CFs). Evoked CF EPSCs and whole-cell AMPA currents displayed roughly linear current-voltage relationships, consistent with the presence of GluR2 subunits in synaptic and extrasynaptic AMPARs. The mean quantal size, estimated from the miniature EPSCs (MEPSCs), was approximately 300 pS. Peak-scaled non-stationary fluctuation analysis of spontaneous EPSCs and MEPSCs gave a weighted-mean synaptic channel conductance of approximately 5 pS (approximately 7 pS when corrected for filtering). By applying non-stationary fluctuation analysis to extrasynaptic currents activated by brief glutamate pulses (5 mM), we also obtained a small single-channel conductance estimate for extrasynaptic AMPARs (approximately 11 pS). This approach allowed us to obtain a maximum open probability (Po,max) value for the extrasynaptic receptors (Po,max = 0.72). Directly resolved extrasynaptic channel openings in the continued presence of glutamate exhibited clear multiple-conductance levels. The mean area of the postsynaptic density (PSD) of these synapses was 0.074 microm2, measured by reconstructing electron-microscopic (EM) serial sections. Postembedding immunogold labelling by anti-GluR2/3 antibody revealed that AMPARs are localised in PSDs. From these data and by simulating error factors, we estimate that at least 66 AMPARs are bound by a quantal transmitter packet at CF-Purkinje cell synapses, and the receptors are packed at a minimum density of approximately 900 microm-2 in the postsynaptic membrane.

Figure 1. Identification and morphology of immature Purkinje cells in thin slices

A, IR-DIC view of Purkinje cells in a cerebellar slice (300 μm thick) of a P4 rat. EGL, external germinal layer. B, confocal image of another P4 Purkinje cell loaded with the fluorescent dye calcein (500 μm) through a whole-cell patch pipette. Overlay of 35 images (taken for every 0.5 μm Z-axis). Calibration bars, 10 μm.

Figure 10. Density of AMPARs at CF-Purkinje cell synapse

A, an example of complete serial ultrathin sections (70 nm thickness) of a P3 CF-Purkinje cell synapse. Arrows indicate edges of the PSD. B, immunogold labelling by anti-GluR2/3 antibody was localised within PSDs of CF-Purkinje cell synapses. C, histograms of the PSD area (top) and of the quantal size (bottom). Pooled data of 1112 MEPSCs recorded at 33–34 °C from four cells, and of 122 randomly sampled PSDs, are shown.

Figure 2. Pharmacological identification and similarity in Ca²⁺ permeability of synaptic and extrasynaptic AMPARs in immature Purkinje cells

A, CF-evoked EPSCs recorded from a P3 Purkinje cell. Averaged traces at stimulation frequency of 0.1 Hz, before and 1 min after NBQX (5 μm) application are superimposed. B, whole-cell current response to bath-applied glutamate (1 mm) in another P3 Purkinje cell. NBQX blocked 88 % of the glutamate-induced steady-state current. C, averaged CF EPSCs in the same cell as in A at V_h=+40 and −40 mV. Stimulus artifact was subtracted using records obtained at V_rev= 0 mV. Stimulation frequency, 0.1 Hz. Intracellular solution contained 50 μm spermine. D, current-voltage relationship of AMPA (10 μm)-induced steady-state current obtained by applying voltage-ramp pulses. P4 Purkinje cell. Bathing solution contained TTX (1 μm), bicuculline (10 μm), strychnine (1 μm), AP-5 (100 μm), cyclothiazide (30 μm) and CPCCOEt (100 μm). Averaged currents of 10 trials in the absence of AMPA were subtracted from that in the presence of AMPA. V_rev, +2.6 mV. Calibration for the inset, 1 nA and 0.1 s.

Figure 3. Climbing fibre MEPSCs in an immature Purkinje cell

A, examples of MEPSCs recorded from a P3 Purkinje cell in the presence of 5 mm Ca²⁺ and 0.3 μm TTX. V_h, −70 mV. B, amplitude histogram of 636 MEPSCs recorded from the cell in A. Noise histogram is scaled for clarity. CV of MEPSC amplitude was calculated after subtracting the noise variance. C, averaged waveform of MEPSCs fitted with a double-exponential function (superimposed continuous line). Dotted lines indicate fast and slow components.

Figure 4. Stability analysis of CF EPSCs

Analysis of spontaneous EPSCs recorded from a P3 Purkinje cell. V_h, −70 mV. External Ca²⁺, 5 mm. A, stability plots of amplitude (top) and weighted decay time (middle), and plot of 10–90 % rise time against the amplitude (bottom) of 153 consecutive EPSCs. Straight lines indicate linear regression. The lack of correlation between plotted parameters was confirmed by Spearman's rank test. B, the amplitudes and decay times of EPSCs were not correlated. C, EPSCs were averaged separately according to peak amplitude grouped into three ranges (top). The three separate averages were normalized and superimposed, to illustrate the lack of correlation between decay time course and amplitude (bottom).

Figure 5. Peak-scaled non-stationary fluctuation analysis of CF EPSCs

Procedures of PS-NSFA, applied to 153 spontaneous EPSCs analysed in Fig. 3. A, averaged waveform (red trace) scaled at the peak of an individual EPSC (black trace). Dotted lines indicate binning to 30 fractions. B, subtraction of the peak-scaled average from the individual EPSC shown in A. Arrows indicate the range in which the subtraction was applied. For clarity, data are represented as points. The sum-squared difference was calculated for each bin. The procedure was repeated for each EPSC and cumulated averages are plotted in C. C, the straight red line indicates the fit of the initial one-third of the plot to the theoretical equation (see Methods). The dotted line indicates the baseline variance (2.6 pA²). The weighted mean synaptic channel conductance was 4.6 pS. D, PS-NSFA on randomly re-sampled events was repeated 100 times. Means and s.d.s of the 100 current-variance plots are shown. The individual plot was fitted as in C. E, estimates of the channel conductance obtained by fitting bootstrapped data displayed a normal distribution (red curve). F, relationship of the bootstrap data CV versus the number of events, on simulated current (line-connected symbols) and the real EPSCs (green, pooled data in 5 and 2 mm Ca²⁺). The bootstrap data CV were also affected by the binning and fitting conditions.

Figure 7. Effects of filtering on the quantal size and single-channel conductance estimated from PS-NSFA

A, example of a simulated EPSC, in the absence (black) and presence (red) of an RC filter (0.72 kHz) cascaded with a Gaussian filter (2 kHz). The single-channel conductance was 5 pS, the number of channels was 100, and the driving force was −100 mV. The glutamate pulse was set to rise instantaneously to the peak concentration (3.1 mm) and decay as a double-exponential waveform (Clements, 1996), as shown in B. Inset, expansion of the indicated part (dotted line) of currents. B, averaged currents of simulated EPSCs (1000 events averaged in each condition). C, current-variance plots of simulated EPSCs obtained by PS-NSFA. Each plot is derived from 1000 events simulated as in A and B. The initial one-third of the plot was fitted to the theoretical equation (Methods).

Figure 6. Skew in the current-variance plot by PS-NSFA is expected from the activation kinetics of the Purkinje cell AMPAR model

Using the kinetic scheme of AMPARs in Purkinje cells (Häusser & Roth, 1997), EPSCs were simulated using four different concentrations of glutamate (0.3 ms, step pulse), as indicated. The single-channel conductance was set at 5 pS, the number of channels was set at 100, and the driving force was −100 mV. A, averages of simulated currents. Upper trace indicates the glutamate pulses. B, current-variance relationships by PS-NSFA (continuous lines) were obtained for an individual set of simulated EPSCs (1000 events each) at indicated peak P_o values. Open circles represent current-variance plots obtained by Sigworth-type NSFA, imposed on the Sigworth-type theoretical relationships (dotted curves). C, current-variance relationships of 4000 pooled events by PS-NSFA.

Figure 8. Non-stationary fluctuation analysis of AMPAR-mediated currents in outside-out patches activated by rapid application of glutamate

Data derived from the same patch excised from P4 Purkinje cell. A and D, current-response activated by short (1 ms) or long (100 ms) pulses of glutamate (5 mm). Averaged response and an individual response are superimposed. Top traces indicate the timing of solution exchange measured as changes in liquid junction currents. B and E, stability of peak amplitude of glutamate-activated currents was confirmed by Spearman's rank test. Red lines, linear regression. C and F, open circles are current-variance plots by Sigworth-type NSFA applied to the glutamate-activated currents of extrasynaptic non-NMDARs. The blue dotted lines indicate the fit to the theoretical Sigworth equation (Methods). The red lines in C and F show the current-variance relationship by PS-NSFA. Channel open probability at the peak of the current was 0.57 for the short pulse.

Figure 9. Multiple single-channel conductances of somatic AMPARs in Purkinje cell patches

A and C, examples of single-channel openings in the continued presence of agonist, recorded from outside-out patches of P3 Purkinje cells. V_h, −100 mV. External Ca²⁺, 5 mm. Baseline (closed) level is indicated as C. B and D, all-point amplitude histograms of single AMPA channel currents. The baseline noise has been subtracted from histograms after fitting Gaussian distributions to the noise components.