Maturation of EPSCs and Intrinsic Membrane Properties Enhances Precision at a Cerebellar Synapse

Spontaneous EPSCs in immature and mature GCs are quantal events

To quantify the developmental change in synaptic conductance waveform between immature and mature MF–GC synapses, we made recordings at physiological temperature (∼37°C) in acute slices of cerebellum from mice in two age ranges. Immature GCs were examined between P7 and P9 (8.2 ± 0.1; n = 72 cells; “P8”), and mature GCs were examined between P34 and P52 (39.1 ± 0.3; n = 96; “P39”).

In the presence of the selective GABA_AR antagonist 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl) pyridazinium bromide (10 μm; SR-95531) and the NMDAR antagonists AP5 (20 μm) and 7-CK (20 μm), spontaneous synaptic currents were detected that were fully blocked by the non-NMDAR antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (5 μm) or the selective AMPAR antagonist GYKI 53655 (100 μm; data not shown). These AMPAR-mediated EPSCs occurred with a similar frequency at both ages and had similar mean peak amplitudes (Table 1). Consistent with all events being quantal in nature, blockade of action potentials with tetrodotoxin (TTX; 0.5 μm) affected neither the amplitude nor the frequency of the currents (Fig. 1A–C). The c.v. of the peak amplitudes was 43.5 ± 2.0% at P8 and 40.5 ± 3.1% at P39. In contrast with miniature EPSCs in GCs from mature rats (Wall and Usowicz, 1998; Wall et al., 2002), all amplitude histograms, although skewed toward larger amplitudes, lacked multiple discrete peaks (Fig. 1B,D,E). Moreover, we found no evidence of inflections in their rising-phase large events, and there was no positive correlation between their amplitude and their rise time (Fig. 1D,E) (cf. Wall and Usowicz, 1998) or their width (data not shown). Thus, at both immature and mature synapses, spontaneous EPSCs exhibit properties of individual quantal events, with a high amplitude variance.

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Figure 1.

Properties of spontaneous EPSCs in mature and immature GCs. A, Continuous records showing spontaneous EPSCs recorded from a'P39′ GC (P35) in the absence and presence of TTX (V_m = -70 mV). B, Amplitude distributions of events from a'P8′ GC (P7) recorded in the absence and presence of TTX. Superimposed lines show cumulative amplitude histograms (p > 0.05; Kolmogorov–Smirnov test). C, Interevent interval histograms from the same cell as B, showing the exponential distribution of interevent intervals and the lack of effect of TTX. Inset, Summary of results from both age groups. Dotted line indicates cell shown in C (Δf denotes mean change in frequency across all cells). D, Superimposed spontaneous EPSCs from a P7 GC (V_m = -70 mV). Right-hand panel shows a plot of peak amplitude against 10–90% rise time for the same cell, with superimposed distributions. E, Corresponding data for a P36 GC. In both D and E there is no correlation between peak and 10–90% rise time (p > 0.05; Spearmann rank order correlation). The solid lines show the fitted linear regressions, and the dotted lines show the 95% confidence limits.

AMPAR-mediated quantal EPSCs become faster during development

The decay of the AMPAR-mediated quantal EPSCs was best described by a double exponential (Fig. 2A), with parameters at P8 broadly similar to those reported for spontaneous EPSCs recorded from GCs of immature rats (Silver et al., 1996; Wall et al., 2002). In mature GCs, the rise and decay of the EPSCs were significantly faster (Table 1). The reduction in rise time did not simply reflect altered RC filtering by the electrode–cell circuit, because the mean corner frequency was not different at the two ages (1.35 ± 0.07 kHz at P8 and 1.47 ± 0.12 kHz at P39; p > 0.05). The speeding of the decay was caused by a reduction in τ_fast with no significant change in its relative amplitude or in the value of τ_slow (Table 1, Fig. 2B). Overall, these kinetic changes resulted in an approximate halving of the charge transfer during the AMPAR–quantal EPSC (Fig. 2C).

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Figure 2.

Speeding of the AMPAR-mediated quantal EPSC. A, Representative averaged AMPAR–quantal EPSCs at -70 mV, from 'P8′ and 'P39′ GCs (P8 and P36). At both ages, the decay phase was best fitted by a double exponential function (solid line). The individual components of the fit are shown as dotted lines. B, For each age group the mean decay time constants (τ_fast and τ_slow) are plotted against their relative contributions to the EPSC amplitudes. Error bars indicate SEM. At P8, τ_fast = 0.66 ± 0.05 msec (88.9 ± 1.8%) and τ_slow = 6.91 ± 1.71 msec; at P39,τ_fast = 0.59 ± 0.04 msec (91.4 ± 1.3%) andτ_slow = 4.38 ± 1.02 msec. Asterisk indicates significant difference between τ_fast values (Mann–Whitney U test). C, Histogram of spontaneous EPSC 10–90% rise, τ_decay, and quantal charge in both age groups. Asterisks indicate significant difference between groups (rise andτ_decay, t test; quantal charge, Mann–Whitney U test).

NMDA receptors do not contribute to quantal EPSCs in mature GCs

To investigate the contribution of NMDARs to quantal EPSCs, we made recordings in the presence or absence of NMDAR antagonists. To determine the activation of NMDARs under physiological conditions, we included 1 mm Mg²⁺ in the external solution and recorded currents at two holding potentials: the first near rest (-60 and -80 mV for P8 and P39, respectively) and the second near action potential threshold (-25 and -35 mV, respectively) (see Fig. 4), where we would expect partial relief of Mg²⁺ block. In P8 GCs, blockade of NMDARs accelerated the decay of quantal EPSCs at both membrane potentials (Fig. 3A,C). The decay time (τ_decay) was reduced from 3.0 ± 0.5 to 1.6 ± 0.2 msec at -60 mV, and from 7.1 ± 1.4 to 1.9 ± 0.4 msec at -25 mV (n = 5). Subtracting the current recorded in AP5 from that recorded in control conditions revealed the NMDAR-mediated component. At -25 mV, the amplitude of this component was 25.2% of the dual-component quantal EPSC (Table 1). As expected, given the voltage-dependent blocking action of Mg²⁺, the contribution of NMDARs to the peak of the quantal EPSCs was reduced at -60 mV to 9.5% (Fig. 3A). In marked contrast, blockade of NMDARs in P39 GCs had no effect on the decay of quantal EPSCs, either at rest or near action potential threshold (Fig. 3B,C). The absence of an NMDAR-mediated component to the quantal current was confirmed in additional recordings, made under conditions designed to maximize the likelihood of detecting any NMDAR activation (+40 mV, with added glycine increased from 3 to 10 μm; data not shown; n = 5). These results indicate that in adult GCs, NMDARs are not activated during quantal events.

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Figure 3.

Developmental change in NMDAR contribution to quantal EPSCs. A, Scaled population average EPSCs recorded from P8 GCs (n = 5). Recordings were made at two potentials: resting potential (-60 mV) and action potential threshold (-25 mV) (Fig. 4). B, Population average EPSCs from P39 GCs at the corresponding potentials (n = 9 at -80 mV and n = 7 at -35 mV). Calibration applies to both A and B. The term +AP5 denotes addition of both AP5 and 7-CK. C, Histogram showing the effect of NMDAR blockade on theτ_decay of the EPSCs. Note the significant effect in immature GCs (^*p < 0.05) and the lack of effect in mature GCs (note also the 10-fold change in scale on the ordinate).

GC intrinsic membrane properties change during development

Changes in the kinetics of the quantal EPSC occur against a background of developmental changes in GC intrinsic membrane properties, and it is the interplay of these changes that will determine the developmental changes in the EPSP. Although previous studies (D'Angelo et al., 1994; Brickley et al., 1996, 2001) have identified several developmental changes in GC properties, these have generally been examined over a restricted age range and only at room temperature. To characterize intrinsic membrane properties at physiological temperature, we examined the response of immature and mature GCs to constant current injection (Fig. 4A). P8 GCs had an input capacitance (C_in) of 3.9 ± 0.1 pF (n = 59), an input conductance (G_in) of 0.73 ± 0.1 nS (n = 28), and a resting membrane potential (V_m) of -58.8 ± 1.5 mV (n = 51). P39 GCs were smaller (C_in 3.0 ± 0.1 pF; n = 96), had a higher input conductance (G_in 1.48 ± 0.10 nS; n = 37), and were more hyperpolarized (V_m - 78.6 ± 0.5 mV; n = 84; all p < 0.05). In agreement with previous studies, mature GCs had action potentials that were brief, and all cells supported high-frequency firing in response to sustained depolarization (D'Angelo et al., 1995; Brickley et al., 1996), whereas P8 GCs fired broad action potentials that tended to accommodate at high stimulus intensities (data not shown). The subthreshold current–voltage relationship of P8 GCs was linear, whereas P39 GCs exhibited both inward and outward rectification (Fig. 4B,C). The spike threshold potential also decreased during development, by ∼8.5 mV (p < 0.05; Mann–Whitney U test) (Fig. 4D). The emergence of the mature phenotype was reflected in a nearly three-fold increase in the current required to reach spike threshold (p < 0.05) (Fig. 4E).

Somatic conductance injection effectively mimics a dendritic synaptic input

To determine how the observed developmental changes in GCs interact to influence the EPSP and EPSP-spike coupling, we used the technique of dynamic current clamp (Robinson and Kawai, 1993). Using a conductance command waveform to evoke EPSPs, rather than recording spontaneous events, obviated the need for pharmacological intervention (which may alter intrinsic membrane properties) (Brickley et al., 1996; Hausser and Clark, 1997) and allowed manipulation of the conductance waveform in a single cell.

For many neurons, the location of a synaptic conductance within the dendritic tree profoundly influences the amplitude and time course of the resulting somatic EPSP (Rall, 1967; Reyes, 2001). In such neurons, injection of a conductance at the soma will not mimic the somatic EPSP that would result from dendritic input. GCs receive MF excitatory input at the distal end of their dendrites (Hamori and Somogyi, 1983). Although these dendrites are short, we wished to determine whether GCs at all ages behaved as single electrical compartments (Silver et al., 1992) or whether the developmental changes in their morphology (Ramon y Cajal, 1995) could also impact on the properties of the somatic EPSP. Figure 5A shows confocal images of representative immature and mature GCs. P8 GCs had a soma diameter of 8.5–10.5 μm (9.5 ± 0.5 μm; n = 5) with 10–20 neurites (13.4 ± 1.8) and a single axon extending into the molecular layer. P39 GCs had a smaller soma (6.3–8.9 μm; 7.4 ± 0.5 μm; n = 5; p < 0.05) and only four to five dendrites (4.2 ± 0.2). These dendrites often terminated in claw-like structures, formed by dendritic digits. These claws wrap around the MF terminal to generate the cerebellar glomerulus characteristic of mature MF–GC synapses (Hamori and Somogyi, 1983).

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Figure 5.

Effect of GC morphology on EPSP properties. A, Z-projections of confocal images from neurobiotin-filled P8 and P39 GCs (P9 and P38) in sagittal cerebellar slices. For the P8 GC, note the numerous dendrites and the fine axon extending into the molecular layer. For the P39 GC, note the four dendrites, each of which terminates in dendritic digits. Scale bars, 10 μm. ML, Molecular layer; PL, Purkinje cell layer; IGL, internal granule cell layer. B, Shape plots of the two GCs reconstructed in NEURON. C, Plots showing the placement of the conductance injection in each simulation. In each case arrowhead 1 indicates the soma (gray). Calibration applies to B and C. D, Somatic EPSPs recorded in response to excitation at somatic and dendritic locations, as indicated. Arrowheads (sequentially numbered according to decreasing peak amplitude; 1–5 for P9 and 1–3 for P39) indicate EPSPs originating from the corresponding sites shown in C.

We reconstructed the GCs shown in Figure 5A, incorporated them into passive simulations within NEURON, and injected into each cell conductance waveforms based on the filter-corrected population average P8 and P39 AMPAR–quantal EPSCs (see Materials and Methods) (Fig. 5C). In the P8 cell, somatic EPSPs evoked from dendritic terminals, when compared with the EPSP induced by somatic conductance injection, were filtered only modestly (Fig. 5D) (amplitude -9.5%, rise + 15.5%, half-width + 2.5%; mean of the most proximal and distal dendritic injections). For the P39 cell, the corresponding changes were -4.5, +6.0, and +1.0%. These simulations suggest that both immature and mature GCs are indeed electrically compact and approximate single electrical compartments. Accordingly, conductance injection at the soma can effectively mimic a dendritic synaptic input.

AMPAR–EPSC kinetics and membrane properties differentially shape the quantal EPSP

For conductance stimulation of real neurons we used waveforms based on the population average quantal EPSCs obtained at P8 and P39 (Fig. 6A) (see Materials and Methods). We use the terms “g_syn8” to denote the dual-component P8 waveform, “g_syn8A” to denote the AMPAR-only P8 waveform, and “g_syn39” to denote the waveform from P39 GCs.

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Figure 6.

AMPAR–EPSPs evoked by conductance injection. A, Representative responses of 'P8′ and 'P39′ GCs (P7 and P37) to injection of synaptic conductance waveforms (g_syn8A and g_syn39). For these cells, the P8 EPSP had a peak amplitude of 4.0 mV, a 10–90% rise-time of 0.99 msec, and half-width of 13.2 msec; the P39 EPSP had a peak amplitude of 2.6 mV, a 10–90% rise of 0.43 msec, and half-width of 4.3 msec. B, Mean responses (±SEM, dotted lines) of P8 (n = 8) and P39 GCs (n = 15) to g_syn8A or g_syn39 at -80 mV. The right-hand panel shows a histogram of EPSP peak amplitude, 10–90% rise-time, and half-width at the two ages. P39 EPSPs were significantly smaller and faster then P8 EPSPs. C, Mean responses of P39 GCs to injection of g_syn 8A and g_syn 39(both n = 8). Changing g_syn affects only the amplitude and the rise of the EPSP. D, Mean responses of P8 and P39 GCs to g_syn 39 (both n = 8). The different intrinsic properties of the two GCs affect only the rise and decay of the EPSP.

Stimulation of immature GCs with g_syn8A at -80 mV generated quantal EPSPs that had a peak amplitude of 4.1 ± 0.2 mV (n = 15), a rise-time of 0.9 ± 0.1 msec, and a half-width of 11.3 ± 1.4 msec (Fig. 6A, left, B). In mature GCs, the EPSPs evoked by g_syn39 were smaller and faster, with a peak amplitude of 2.7 ± 0.1 mV (-34%; n = 8), a 10–90% rise-time of 0.55 ± 0.04 msec (-45%), and a half-width of 5.6 ± 0.5 msec (-50%; all p < 0.05 vs P8) (Fig. 6A, right, B). In the latter case, these parameters were not significantly different from those of spontaneous EPSPs recorded without blockade of GABA_A receptors (n = 16; data not shown), confirming the validity of the conductance stimulation approach.

To quantify the respective roles of AMPAR–quantal EPSC kinetics and membrane properties in shaping the EPSP, we next compared the effects of the two conductance waveforms injected into cells of the same age, as well as the effects of injecting a common waveform into cells of different ages (an approach used previously at glycinergic synapses) (Singer et al., 1998). As shown in Figure 6C, for P39 GCs, stimulation using g_syn39 produced EPSPs that had a half-width similar to those evoked with g_syn8A (-6%; n = 8; p > 0.05), but they were 32% smaller and rose 29% faster (p < 0.05). Qualitatively similar results were obtained with P8 GCs, either at their resting potential (-60 mV) or when held at -80 mV (n = 14; data not shown). Conversely, when a common command waveform (g_syn39) was used to stimulate P8 and P39 GCs (Fig. 6D), EPSPs in the mature cells (n = 8) did not differ in amplitude from those in the immature cells (n = 14; +3%; p > 0.05) but they had a faster rise (-23%) and decay (-47%; both p < 0.05). Comparable results were obtained with g_syn8A (data not shown). Overall, these data illustrate how the size and shape of quantal EPSPs in GCs change with age because of alterations in both EPSC kinetics and intrinsic membrane properties. Developmental changes in the AMPAR–quantal EPSC waveform influence the peak and rise of the EPSP but not its half-width. On the other hand, the change in intrinsic membrane properties between P8 and P39 influences the rise and decay of the EPSP but not its amplitude.

Loss of NMDAR-mediated component speeds the quantal EPSP in mature GCs

We next examined the significance of the developmental loss of the NMDAR-mediated component of the quantal EPSC. When immature GCs were stimulated at their resting potential (-62.4 ± 0.8 mV) with the dual-component quantal EPSC waveform (g_syn8), the EPSP was larger (4.5 ± 0.6 mV; +35%; n = 9) and slower (rise-time 2.0 ± 0.6 msec; +107%; half-width 22.8 ± 3.0 msec; +86%; all p < 0.05) than that evoked by the AMPAR conductance alone (g_syn8A) (Fig. 7A). The developmental loss of the NMDAR-mediated component from mature synapses acts in combination with the speeding of the AMPAR conductance to cause a further reduction in amplitude and duration of the EPSP. When measured at their respective resting membrane potentials, P39 EPSPs (Fig. 6A, right panel) were smaller than P8 EPSPs resulting from dual-component currents (-39%) (Fig. 7A, right panel), with a faster rise time (-74%) and decay (half-width -75%; all p < 0.05). This difference could be replicated in a simplified single-compartment passive simulation (see Materials and Methods), in which we compared the EPSPs at a common potential of -60 mV (Fig. 7B). The model also allowed us to quantify (in a manner analogous to that shown in Fig. 6, C and D) the respective roles of the change in membrane properties and the overall change in synaptic conductance. The results were qualitatively similar to those shown in Figure 6, with the exception that consideration of the dual-component waveform shows that the acceleration in the EPSC reduces not only the peak and rise of the EPSP but also its half-width (Fig. 7C,D). Overall, the developmental loss of the NMDAR-mediated component of the quantal EPSC at P39 acts in concert with the speeding AMPAR-mediated component and the change in intrinsic membrane properties to reduce the amplitude and speed the kinetics of the quantal EPSP.

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Figure 7.

Effect of NMDARs on EPSPs evoked by conductance injection. A, Representative responses of P8 GCs (P8) to injection of synaptic conductance waveforms (g_syn8A and g_syn8) at -60 mV. The g_syn8A-induced EPSP had a peak amplitude of 4.6 mV, a 10–90% rise time of 2.34 msec, and half-width of 21.9 msec. The g_syn8-induced EPSP had a peak amplitude of 3.2 mV, a 10–90% rise time of 0.82 msec, and half-width of 8.2 msec. B, EPSPs from single-compartment passive NEURON models of P8 and P39 GCs, evoked using g_syn8 or g_syn39. Model parameters were chosen to match properties of P8 GCs [diameter 12.7 μm to give C_in = 4.0 pF; specific membrane conductance (G_m) 1.56e ^-4 S/cm ² to give G_in = 0.52 nS] and P39 GCs (diameter 10.3 μm to give C_in = 3.0 pF; G_m 4.42e ^-4 S/cm ² to give G_in = 1.47 nS). The right-hand panel shows a histogram of EPSP peak amplitude, 10–90% rise time, and half-width at the two ages. P39 EPSPs were significantly smaller and faster than P8 EPSPs. For comparison, the dashed lines in the P8 (open bars) indicate the results obtained with the AMPAR-mediated component alone. C, Responses from modeled P39 GCs to injection of g_syn8 and g_syn39. Changing g_syn affects the amplitude, the rise, and the decay of the EPSP. D, Responses of modeled P8 and P39 GCs to g_syn8_. The different intrinsic properties of the two GCs affect only the rise and decay of the EPSP. Voltage and time calibrations in A also apply to B–D.

NMDARs contribute to evoked EPSCs in mature GCs

To examine how the changes in quantal properties are reflected in action potential-evoked events, we used minimal extracellular stimulation in the GC layer to activate single MF inputs (Silver et al., 1996). We recorded the evoked currents (evoked EPSCs) in standard 2 Ca/1 Mg external solution in the presence and absence of NMDAR antagonists (AP5 and 7-CK; 20 μm), and to reveal the full extent of any NMDAR activation, we held the cells at +40 mV.

In agreement with our previous data from rat GCs at room temperature (Cathala et al., 2000), MF stimulation consistently activated NMDARs in both immature and mature GCs (Fig. 8A,B, left). The presence of an NMDAR-mediated current in the mature evoked EPSCs, albeit reduced in relative amplitude (NMDA/AMPA ratio, 0.84 ± 0.19 at P8 vs 0.20 ± 0.05 at P39; p < 0.05), contrasts markedly with the absence of this component in the quantal EPSCs (Fig. 3). This disparity reflects a genuine difference between quantal and evoked events: in all P39 GCs, quantal events recorded before or during periods of MF stimulation were mediated solely by AMPARs (n = 4; data not shown).

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Figure 8.

Action potential-evoked EPSCs and simulated EPSPs accelerate with age. A, Population average EPSCs recorded from P8 and P39 GCs at +40 mV (n = 5 and 4). The NMDAR-mediated component was determined by subtraction of the current (AMPA) recorded in AP5. B, Scaled multi-exponential fits of the evoked EPSCs shown in A. C, Conductance waveforms with the NMDAR-mediated component scaled for the appropriate resting potential. Right panels show simulated EPSPs from single-compartment passive NEURON models (details as in Fig. 7). D, Scaled conductance waveforms and corresponding dual-component EPSPs on expanded time scales.

In both P8 and P39 GCs, the amplitudes of the evoked EPSCs were ∼5- to 10-fold those of the corresponding quantal EPSCs, indicating the release of multiple vesicles in response to each MF action potential, which increases the glutamate concentration beyond the synapse (DiGregorio et al., 2002). The fact that in mature GCs NMDAR activation was seen only under this condition suggests that the receptors are located outside the synapse. By contrast, in immature GCs, the presence in the quantal EPSC of an NMDAR-mediated component with a submillisecond rise time suggests that NMDARs are colocalized with AMPARs within the postsynaptic density at this age.

The action potential-evoked EPSP accelerates with age

Similar to the quantal EPSC, the AMPAR-mediated component of the evoked EPSC became faster with age. The 10–90% rise time decreased from 0.29 ± 0.04 msec at P8 to 0.18 ± 0.01 msec at P39 (n = 5 and 4; p < 0.05), and the major component of the current decay (τ_fast) was accelerated from 1.11 ± 0.09 msec at P8 to 0.77 ± 0.05 msec at P39 (p < 0.05), with no change in relative amplitude (A_fast 83.1 ± 2.2% at P8 and 81.5 ± 1.6% at P39) (Fig. 8 A). The developmental speeding of the evoked EPSC decay (τ_fast P39/τ_fast P8 ratio, 0.69) was comparable with that seen for the AMPAR-mediated quantal EPSC (0.65). This is consistent with the proposition (Wall et al., 2002) that an increased synchrony of transmitter release is unlikely to account for the overall speeding of the current. In fact, convolution of a Gaussian latency distribution (Silver et al., 1996) with the quantal waveform suggests that when the quantal decay is significantly slower than the width of the latency distribution, changes in latency affect the rise time but not the decay of the evoked EPSC, which is determined primarily by the time course of the quantal event (cf. Diamond and Jahr, 1995). Thus, in GCs the developmental speeding of the evoked EPSC decay is governed by the speeding of the underlying quantal events.

To investigate how the different components of the P8 and P39 evoked EPSCs influence the time course of the corresponding EPSPs, we injected conductance waveforms derived from multi-exponential fits to the population average AMPAR- and NMDAR-mediated currents into single-compartment passive models of GCs. An expanded view of the fitted currents at +40 mV is shown in Figure 8B. With respect to the AMPAR-mediated component, the NMDAR-mediated component exhibited a slow rise (10–90% rise, 2.1 ± 0.3 msec at P8 and 2.3 ± 0.6 msec at P39) and a delayed onset, which increased with age (0.58 msec at P8 and 0.77 msec at P39). To produce NMDAR conductance waveforms appropriate for the respective resting membrane potentials of the P8 and P39 cells, we scaled the waveforms measured at +40 mV, according to the expected voltage-dependent Mg²⁺ block (see Materials and Methods) (Fig. 8C, compare also B, D).

At resting membrane potential, the NMDAR-mediated conductance slowed the rise of the P8 EPSP (+31%) and increased both its peak (+15%) and its half-width (+50%) (Fig. 8C). In contrast, at P39 the EPSP was dominated by the AMPAR-mediated conductance (Fig. 8C), and the NMDAR conductance had little affect on the rise, peak, or width of the EPSP (+1.5, +0.4, and + 12%, respectively). The same qualitative difference was apparent when the EPSPs were compared at a common potential (-80 mV). Overall, the EPSP produced by the P39 conductance had a faster rise and shorter duration than the EPSP produced by the P8 conductance (10–90% rise, 0.7 vs 1.9 msec; half-width, 6.9 vs 16.2 msec) (Fig. 8D). The developmental change in the evoked EPSPs (rise -65%; half-width -58%) parallels that seen for the quantal EPSPs (-69% and -77%), despite the presence of spillover after multiquantal release (DiGregorio et al., 2002).

The speeding of the somatic EPSP is predicted to play a critical role in the efficacy of spike generation, influencing the relationship between MF input and GC output (Gabbiani et al., 1994; Maex and Schutter, 1998) as well as the precision and reliability of spike timing (Fricker and Miles, 2000; Harsch and Robinson, 2000; Futai et al., 2001; Carter and Regehr, 2002).

Determinants of EPSP–spike coupling

To compare EPSP–spike coupling in immature and mature GCs, we injected a variable number of quanta (constructed from integer multiples of g_syn8, g_syn8A, and g_syn39) into P8 or P39 cells at their resting potentials to initiate a spike with a probability of ∼0.5 (0.59 ± 0.04, 0.52 ± 0.05, and 0.57 ± 0.05, respectively; n = 8–9; p > 0.05). We considered this approach preferable to the stimulation of MFs, because it allowed us to examine the effect on spiking of different postsynaptic parameters, independently of stochastic variation in the release process. Because we were interested primarily in the effects caused by the speeding of the early phase of the EPSP, we adopted the simplified approach of using variable numbers of synchronous quantal events.

At both ages, conductance injection evoked only a single spike. In immature GCs, the spikes were triggered with a latency of 4.6 ± 1.1 msec and a temporal jitter (c.v. of latency) of 19.7 ± 4.2% (n = 9). In mature GCs, both spike latency and jitter were reduced (1.9 ± 0.1 msec and 10.5 ± 0.8%; both p < 0.05; n = 9) (Fig. 9A). The efficacy of EPSP–spike coupling depends not only on the amplitude and shape of the EPSP, but also on the intrinsic spike generation mechanism of the cell. To investigate the importance of membrane properties to the development changes in spike latency and jitter, we stimulated P8 and P39 GCs with a common command waveform (g_syn8A). Spikes were initiated with comparable latencies (2.7 ± 0.2 vs 2.4 ± 0.1 msec; p > 0.05), but the jitter was significantly reduced in P39 GCs (17.3 ± 3 vs 11.5 ± 1.2; p < 0.05; n = 8 and 6) (Fig. 9B). We also compared the effect of different waveforms (g_syn8A and g_syn39) in the same cell (P39). With g_syn8A, spikes were initiated with a longer latency than with g_syn39 (2.4 ± 0.1 vs 2.0 ± 0.1 msec; p < 0.05), although the jitter was unaffected (11.5 ± 1.2 vs 11.3 ± 0.8%; p > 0.05; n = 6) (Fig. 9B). Comparison of Figure 9, B and C, shows that the developmental speeding of the AMPAR–EPSC significantly reduces the latency of EPSP–spike coupling, whereas the temporal jitter is set by the intrinsic membrane properties. Together, these results indicate that the speeding of the quantal EPSC and the

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Figure 9.

Developmental changes in EPSP–spike coupling. A, Representative examples of spikes induced in 'P8′ and 'P39′ GCs (P9 and P35) from their resting potentials, by the injection of multiple quanta. In each cell, by varying the number of quanta injected, the spiking probability was set close to 0.5 and was not significantly different between each test group. In the examples shown, spiking probability was 0.56 at P8 and 0.68 at P39. The right-hand panels summarize the spike latency and mean spike jitter (n = 9 at P8 and P39). The cumulative distributions of latency contain pooled data from all cells (an equal number of events from each cell) and are significantly different (p < 0.05; Kolmogorov–Smirnov test). In the histogram of spike jitter (Latency c.v.), the vertical error bars indicate SEM; ^*p < 0.05. B, Representative examples of spikes induced by g_syn8A injected into P8 and P39 GCs at their resting potentials. The right-hand panel shows a histogram of mean latency and jitter (n = 8 for P8 and 6 for P39). Error bars indicate SEM; ^*p < 0.05. The relatively hyperpolarized resting potential of the P8 cell shown reflects the spread of the data, and at this age there was no correlation between resting potential and spike latency (p = 0.52; Spearman rank order correlation) or spike jitter (p = 0.43). C, Representative examples of spikes induced by g_syn8A and g_syn39 injected into a single P39 cell. The right-hand panel shows a histogram of mean latency and jitter (n = 6).

Relevance of developmental changes in quantal EPSC properties

Quantal events are the fundamental building blocks for synaptic transmission at central synapses, and their basic properties are key determinants of information transfer. We found that at the MF–GC synapse the mean amplitude of quantal EPSCs did not change, but their kinetics became markedly faster, attributable to both a loss of the NMDAR-mediated component and a speeding of the AMPAR-mediated component. Because the duration of vesicular release after MF stimulation is brief relative to the quantal decay (Silver et al., 1996; DiGregorio et al., 2002; Wall et al., 2002), the properties of evoked EPSCs mostly reflect those of underlying quantal events, and the developmental speeding of their initial decay mirrors the speeding of the AMPAR-mediated component of the quantal events. Although spillover of glutamate onto AMPARs at neighboring MF synapses within a glomerulus (DiGregorio et al., 2002) and onto extrasynaptic NMDARs (see below) can influence the waveform of the evoked EPSC at mature synapses, such spillover may contribute significantly only at low frequencies of MF firing, when release probability is high. Thus the kinetics of the quantal EPSC are likely to remain a major determinant of the postsynaptic conductance waveform for physiologically relevant high-frequency inputs (van Kan et al., 1993), when release probability decreases and spillover becomes minimal (DiGregorio et al., 2002). Indeed, any activity-dependent reduction in release probability, by preferentially reducing the spillover components, should allow the quantal parameters to determine the EPSP shape, thus preserving the temporal precision of spike generation.

Impact of developmental changes in AMPAR–EPSCs

The kinetics of the synaptic conductance have been shown to be important in shaping local EPSPs in thin dendrites (Rall, 1967; Jack et al., 1975; Hausser and Clark, 1997) and somatic EPSPs when synapses are located electrotonically close to the soma (Geiger et al., 1997; Trussell, 1997). In these cases the EPSP decay is governed primarily by the decay of the synaptic conductance; hence AMPAR–EPSC kinetics play a key role in determining the window for temporal summation of EPSPs. This contrasts with the situation in GCs, where the duration of the somatic EPSP is unaffected by developmental speeding in the AMPAR-mediated component of the EPSC. Instead, the speeding in AMPAR–EPSC kinetics affects specifically the early component of the EPSP, reducing its rise time and magnitude. These changes result in an increase of ∼50% in the number of synchronous quanta being required to reach spike threshold (our unpublished observation) and a reduction in the latency of EPSP–spike coupling.

Significance of developmental loss of synaptic NMDARs

In agreement with previous studies (Silver et al., 1992), our results show that quantal EPSCs at immature MF–GC synapses are mediated by both AMPARs and NMDARs. Although an unimpeded diffusion of glutamate could allow activation of high-affinity NMDARs located outside the synapse (Chen and Diamond, 2002), the submillisecond rise time of both current components suggests that the receptors are colocalized within the postsynaptic density. In mature GCs, NMDARs were activated after minimal stimulation of individual mossy fibers, but quantal EPSCs were mediated solely by AMPARs. This result strongly suggests that at these synapses functional AMPARs and NMDARs are not colocalized. In support of this finding, recent immunohistochemical observations from adult MF–GC synapses show that AMPAR subunits are located exclusively within postsynaptic densities (DiGregorio et al., 2002), whereas the majority of intraglomerular NMDAR subunits are located outside, or at the periphery of the synaptic specialization (Yamada et al., 2001), with a preponderance of labeling at intraglomerular attachment plaques (Petralia et al., 2002). Because the competitive inhibition of glutamate uptake does not increase AMPAR-mediated cross-talk between adjacent synapses within the glomerulus (DiGregorio et al., 2002), it is unlikely that differences in uptake underlie the developmental changes in NMDAR activation that we observe. Alterations in the glutamate content of vesicles, synaptic geometry, NMDAR affinity, or NMDAR gating could conceivably contribute (Jonas and Spruston, 1994; Conti and Weinberg, 1999), but the most likely explanation for our findings is that a redistribution of NMDARs occurs, such that AMPARs and NMDARs are spatially segregated in mature GCs. Thus, during development there is a switch from a situation in which a single quantum of transmitter activates both AMPARs and NMDARs to a situation in which the release of multiple vesicles is required to activate NMDARs. Because NMDARs would operate only during glutamate spillover, the level of presynaptic activity will determine their activation in mature GCs. Our data from mature GCs are comparable with recent observations showing that minimally evoked EPSCs in cerebellar stellate cells (Clark and Cull-Candy, 2002) and miniature EPSCs in retinal ganglion cells (Chen and Diamond, 2002) are mediated solely by AMPARs. However, in these cases the degree of glutamate spillover required to recruit extrasynaptic NMDARs differs. In stellate cells, high-frequency stimulation or activation of multiple presynaptic fibers is needed (Carter and Regehr, 2000; Clark and Cull-Candy, 2002), whereas in ganglion cells blockade of glutamate transport enables NMDAR activation during the largest miniature EPSCs (Chen and Diamond, 2002).

In immature GCs, because the NMDAR-mediated conductance is slower than the membrane time constant, this component affects not only the early part of the quantal EPSP but also its width. Loss of this NMDAR-mediated component in older animals clearly leads to smaller and faster quantal EPSPs, but it is also likely to modify the voltage dependence of transmission as well as the magnitude and spatial distribution of any glutamate receptor-mediated postsynaptic calcium entry. Together, these changes would be expected to alter the precision of spike generation (Harsch and Robinson, 2000; Futai et al., 2001; Maccaferri and Dingledine, 2002) and influence the expression of synaptic plasticity (D'Angelo et al., 1999). However, further studies will be required to determine the mechanism of any NMDAR redistribution (Carroll and Zukin, 2002; Losi et al., 2003; Wenthold et al., 2003) and its impact on MF–GC transmission under physiological conditions, when it is likely that multiple quanta are released in response to complex patterns of presynaptic activity.

The impact of developmental changes in intrinsic membrane properties

Synaptic integration depends on the duration of the EPSPs, because this determines the time window within which they summate to reach spike threshold. This is important because fast EPSPs allow neurons to behave as coincidence detectors, whereas neurons with slow EPSPs may behave as temporal integrators (Geiger et al., 1997; Trussell, 1997; Taschenberger and von Gersdorff, 2000). In mature GCs the decay of the quantal EPSP is governed primarily by the membrane time constant, and the increase in membrane conductance during development reduces the window for temporal summation. Accordingly, to attain spike threshold, synaptic events in mature GCs would have to occur at shorter intervals, increasing the requirement for coincident input. Several processes are thought to contribute to the increase in membrane conductance seen during GC development. These include a significant increase in both tonic GABA_AR-mediated (Brickley et al., 1996, 2001; Hamann et al., 2002) and potassium-dependent conductances (D'Angelo et al., 1994; Millar et al., 2000; Brickley et al., 2001). Both of these may be dynamically regulated (Brickley et al., 1996; Millar et al., 2000), and both will increase the temporal precision of spike generation, consistent with predictions from GC models (Gabbiani et al., 1994; Maex and Schutter, 1998).

The developmental speeding of the EPSP decay, by reducing the duration of the EPSP plateau, should also increase temporal precision of EPSP–spike coupling in mature GCs (Fricker and Miles, 2000; Galarreta and Hestrin, 2001). Indeed, we have shown that the developmental change in intrinsic membrane properties reduced the temporal jitter of spike initiation. Whether the greater temporal jitter observed in immature GCs reflects only the slow EPSP decay, or is also affected by the immature state and lower number of sodium channels (D'Angelo et al., 1994), remains to be determined. Indeed, fewer sodium channels could mean that the timing of spike initiation will be more susceptible to the stochastic gating of the channels (Schneidman et al., 1998). In either case, any reduction in spike latency and variability would allow for a more accurate temporal coding of mossy fiber inputs in adult GCs. Moreover, with development, the hyperpolarization of the resting membrane potential and the increased input conductance will dampen GC excitability and should result not only in a reduction of the window in which temporal summation can occur but also in an increase of the absolute number of quanta required to reach spike threshold, compared with the situation in immature GCs.

Functional consequences for mature GCs

As indicated above, the altered passive properties of mature GCs, brought about in part by the increased tonic component of GABA-mediated inhibition (Brickley et al., 1996), will act in concert with the developmental changes in the EPSC waveform to increase the likelihood that GCs fire only in response to closely timed MF inputs. In vivo, the firing of individual MFs is modulated over a wide range and may reach frequencies of several hundred Hertz (van Kan et al., 1993). Given that the conductance attributable to tonic GABA_AR activation increases with Golgi cell activity (Brickley et al., 1996) and should thus reflect the frequency of MF input, the developmental changes that we have described are likely to favor the sparse coding of MF input by GCs, which is thought to be necessary for coherent motor control (Marr, 1969; Tyrrell and Willshaw, 1992).