Timing and Efficacy of Ca²⁺ Channel Activation in Hippocampal Mossy Fiber Boutons

MFB Ca²⁺ channels have steep voltage dependence and rapid activation kinetics

To determine the proportion of Ca²⁺channels opened by the fast presynaptic AP waveform, we analyzed the gating properties of the presynaptic Ca²⁺channels in MFBs (Figs. 2-4). Figure2A shows whole-cell voltage-clamp recordings of pharmacologically isolated Ca²⁺ currents evoked by 20 msec voltage steps from −70 mV to +60 mV, and Figure2B illustrates the corresponding current–voltage (I–V) relationship. The Ca²⁺ currents were activated above −40 mV and reached a maximal current amplitude of 98 ± 11 pA at 0 mV (n = 16). At more positive potentials the inward currents decreased, resulting from a reduction in the driving force (Augustine et al., 1985; Borst and Sakmann, 1998).

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Fig. 2.

Presynaptic voltage-gated Ca²⁺currents in MFBs. A, Voltage-clamp recording of Ca²⁺ currents evoked by 20 msec voltage pulses from a holding potential of −80 mV to potentials of −70 to 60 mV in an MFB. B, The maximal current amplitude during the pulses is plotted against pulse potential (n = 16) and fitted with Equation 2 (continuous line; see Materials and Methods). C, To obtain the steady-state activation curve, tail current integrals were plotted against the amplitude of the preceding pulse (5 msec duration). Data were normalized to the maximal value in each experiment, averaged across experiments, and then normalized to the maximal average value (n = 6). The activation curve was fitted with a Boltzmann function (continuous line). norm., Normalized.

To obtain the steady-state activation curve, we plotted the integral of the tail currents against the voltage of the preceding pulse (Fig.2C). Because the tail currents were measured at the same potential, the integral gives a precise measure of the number of channels open at the end of the preceding pulse. The steady-state activation data were adequately fitted by a Boltzmann function with a slope factor of 7.1 mV and a midpoint potential of −3.9 mV. We then fitted the bell-shaped I–V relation in Figure2B with the product of a Boltzmann function representing channel activation and a modified Goldman–Hodgkin–Katz equation describing the driving force for ion flow (Sala, 1991). The parameters of the Boltzmann function were constrained to the previously determined values. From the fitted curve, the extrapolated reversal potential was determined to be 75.0 mV (Fig. 2B). In conclusion, presynaptic Ca²⁺ channels in MFBs show a high activation threshold and a steep voltage dependence.

We next examined the activation and deactivation kinetics of presynaptic Ca²⁺ channels. The time course of activation could be well fitted by a monoexponential function with delayed onset relative to the onset of the pulse (Fig.3A,B). The activation time constant τ and the delay are plotted in Figure3C as a function of voltage. The activation τ was fast and voltage dependent, ranging from τ = 842 ± 137 μsec at −10 mV to 201 ± 35 μsec at 50 mV (n = 6). The voltage dependence could be fitted by Equation 3 with coefficientsk₁ = 1.12 msec⁻¹ andk₂ = 0.14 msec⁻¹ as well as slope factors V₁ = 31.5 mV andV₂ = 8.6 mV. In contrast, the delay was independent of voltage, with a mean value of ∼200 μsec. Linear regression revealed that the slope of the fitted line was not significantly different from 0 (p > 0.4).

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Fig. 3.

MFB Ca²⁺ currents show fast voltage-dependent activation kinetics. A, Ca²⁺ currents evoked by rectangular voltage pulses from −30 to 50 mV were analyzed. The part indicated by thehorizontal bar is shown in B at an expanded time scale. B, The activation of the Ca²⁺ currents could be well fitted by a monoexponential function with a short delay relative to the onset of the pulse (thick superimposed lines). C, The activation time constant τ (○) and the delay (■) are plotted against voltage (n = 6). The voltage dependence of the activation τ was fitted with Equation 3 (continuous line). The voltage dependence of the delay was fitted by linear regression (dashed line).

Because the amount of Ca²⁺ inflow depends on how long the channels remain open during the presynaptic AP, we further analyzed the deactivation kinetics after a voltage step from 0 mV to negative potentials (−10 mV to −60 mV) (Fig.4A). As shown in Figure4B, the deactivation could be adequately fitted by a monoexponential function. The deactivation τ was fast, ranging from 813 ± 90 μsec at −10 mV to 148 ± 11 μsec at −60 mV (n = 6). The voltage dependence could be described with an exponential function with an e-fold change per 15.0 mV (Fig. 4C). Thus both activation and deactivation time constants were fast and highly voltage dependent.

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Fig. 4.

MFB Ca²⁺ currents show fast voltage-dependent deactivation kinetics. A, A voltage pulse to 0 mV was used to activate the Ca²⁺channels. The deactivation time course was analyzed after the membrane potential was stepped back to different potentials. The part indicated by the horizontal bar is shown in B at an expanded time scale. B, The deactivation time course could be well fitted with a monoexponential function (thick superimposed lines). C, The deactivation time constant τ is plotted against voltage (n = 6; same MFBs as in Fig. 3). The voltage dependence of the deactivation τ was fitted by a monoexponential function (continuous curve).

Presynaptic Ca²⁺ channels are effectively opened by MFB APs

To study the presynaptic Ca²⁺ inflow during natural stimuli, we used previously recorded AP waveforms as voltage-clamp commands (Scroggs and Fox, 1992) (Fig.5). A propagated AP evoked a large and brief Ca²⁺ inflow starting at the peak of the AP (Fig. 5A). During the repolarization phase the current rapidly increased, reaching a peak amplitude of 187 ± 36 pA (n = 9). This current was substantially larger (177 ± 17%) than the maximum peak current amplitude during rectangular voltage pulses to 0 mV in the same MFBs (102 ± 16 pA;p < 0.01). The half-duration of the AP-evoked Ca²⁺ current was very short (540 ± 12 μsec; n = 9), most probably because of the fast deactivation of the channels at negative potentials. Similar results were obtained with a locally evoked AP waveform (Fig. 5C,dashed line). The relative peak amplitude and half-duration were 175 ± 10% and 590 ± 19 μsec (n = 18), very similar to the respective values for the propagated AP. If data with both waveforms were pooled, a peak amplitude of 171 ± 20 pA (corresponding to a relative amplitude of 173 ± 9%), a half-duration of 579 ± 15 μsec, and a total charge of 119 ± 13 fC were obtained (n = 24). This corresponds to an inflow of ∼370,000 Ca²⁺ ions per single AP.

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Fig. 5.

Rapid and effective activation of Ca²⁺ currents during AP waveforms. A, An AP waveform applied as a voltage-clamp command (top trace) instead of a rectangular voltage pulse evoked a transient Ca²⁺ inward current in an MFB (bottom trace). The orthodromically propagated AP was previously recorded from a different MFB. B, For a 5 msec prepulse period the command voltage was digitally set to +40 mV, which evoked a slightly larger Ca²⁺ peak current (continuous line) as compared with control (dashed line). C, An AP recorded from an MFB (dashed line) and a much slower somatic AP waveform (continuous line) evoked Ca²⁺currents with different amplitudes and time courses (bottom traces). Both somatic and MFB AP were evoked by direct current injection. D, The amplitude and half-duration of currents evoked by different AP waveforms are shown. The peak current was normalized to the maximal current amplitude of theI–V relationship (at 0 mV) in the same boutons (corresponding to 100%). The bar graphsummarizes data from MFB AP experiments (n = 24), prepulse experiments (2–5 msec; n = 9), and soma AP experiments (n = 5); in all cases the waveforms were applied to MFBs.

To estimate the relative open probability (P_open) of the Ca²⁺ channels during the AP, we added a prepulse to +40 mV to the AP waveform (Fig.5B,D) (Borst and Sakmann, 1998). According to the steady-state activation curve (Fig. 2C), this pulse should activate the Ca²⁺channels with a maximal P_open. The peak amplitude of the evoked Ca²⁺currents, however, was only slightly larger as compared with control (124 ± 3% of control; p < 0.01;n = 9). This indicates that the presynaptic Ca²⁺ channels are opened very effectively by the AP waveform and that the peakP_open with a natural stimulus reaches ∼80% of the maximal value.

To further elucidate the relation between AP waveform and Ca²⁺ channel activation, we compared the Ca²⁺ current evoked by a fast MFB AP with that evoked by a slow somatic AP waveform (Fig.5C,D). The somatic AP evoked a Ca²⁺ current with a half-duration of 1232 ± 51 μsec, significantly larger than that evoked by the brief AP (p < 0.01). However, the peak amplitude was reduced (84.6 ± 1.1% of control; p< 0.01; n = 5). This decrease in peak current amplitude appears to be fundamentally different from the increase in current during AP broadening reported previously for both somatic (Scroggs and Fox, 1992; Wheeler et al., 1996) and presynaptic Ca²⁺ channels (Augustine, 1990; Sabatini and Regehr, 1997). In conclusion, the data suggest that MFB Ca²⁺ channels are effectively opened by the presynaptic APs. To understand the complex dynamic process of activation and deactivation during AP waveforms, we developed a computational model of Ca²⁺ channel gating.

Ca²⁺ channel gating in MFBs can be described by a five-state kinetic model

The pore-forming α₁ subunit of voltage-gated Ca²⁺ channels consists of four domains, each having a positively charged S4 segment, which is believed to be involved in the voltage-dependent gating of the channels (Hille, 2001). Therefore, a model with four voltage-dependent transitions between four closed states and one open state was used (Fig. 6A). The microscopic rate constants α_i and β_i were assumed to be exponentially dependent on voltage (Eq. 4) (Fig. 6B). For a given parameter set, the occupancy of the open state during rectangular voltage pulses was simulated, and the resulting traces were fitted with exponential functions, similar to the experimental data. The parameters for the rate constants α_i and β_i were changed until the best approximation of the experimental values for activation τ, delay, deactivation τ, and steady-state activation were achieved. Figure 6, C andD, shows a comparison of the experimental data with the predictions of the final model (continuous lines). In the final set of rate constants, the last transition shows the steepest voltage dependence (V_i = 49, 42, 55, and 27 mV), suggesting cooperativity in channel gating (Fig.6B) (see also Materials and Methods).

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Fig. 6.

A kinetic model of Ca²⁺ channel gating in MFBs. A, A serial model with four closed states and one open state was developed (top panel). The occupancy of the open state was simulated (bottom panel), and the resulting traces were fitted with a monoexponential function with delayed onset, in the same way as the experimental data. I_max is the current corresponding to an open probability of 1. Pulse from −80 to 0 mV. B, The microscopic rate constants for the transitions α_i(V) and β_i(V) as calculated according to Equation 4, using the best-fit parameters given in Materials and Methods. Note the different steepness of the voltage dependence of the rates. C, Comparison of measured activation τ (filled circles), deactivation τ (open circles), and delay (squares) with the values predicted by the model (continuous lines).D, Comparison of the measured steady-state activation (filled circles) with the steady-state activation curve of the model (continuous line).

We then simulated the Ca²⁺ inflow during APs, using the established model of Ca²⁺channel gating and the driving force from theI–V relation fitted with Equation 2 (Fig.7). If the brief MFB AP was applied as a voltage command, the simulated Ca²⁺current had a peak amplitude of 161 pA and a half-duration of 596 μsec. The peak amplitude corresponded to 176% of the current amplitude during a rectangular voltage pulse to 0 mV, very similar to the experimental data (relative current amplitude 173%; half-duration 579 μsec) (Fig. 5). As shown in Figure 7A, the channels started to open during the rising phase of the AP and reached an open probability of 51% at the peak of the AP. The maximal open probability occurred during the early repolarization phase at a potential of 15 mV (P_open = 90%). The peak Ca²⁺ inward current occurred slightly later, at a potential of −20 mV (P_open = 68%). Subsequently, the channels closed very rapidly and deactivated before complete repolarization to the resting potential. Consistent with the effective opening of the Ca²⁺ channels during the MFB AP, the peak current amplitude was only slightly increased by a 5 msec prepulse to 40 mV (Fig. 7B).

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Fig. 7.

High open probability of the model Ca²⁺ channels during AP waveforms. A, A propagated MFB AP was used as a voltage command to simulate the activation of the model channels. The calculated open probability (gray line) was multiplied by the driving force for Ca²⁺ using Equation 2 to simulate the Ca²⁺ current. B, A 5 msec prepulse leads to a slight increase of the Ca²⁺ current (continuous line) as compared with control (dashed line). C, Comparison of the MFB AP (dashed line) with a slower somatic AP (continuous line). Note that the peak of the Ca²⁺ current occurs at different potentials, as indicated. D, Comparison of the relative peak current amplitude and the half-duration during an MFB AP, the prepulse simulation, and the soma AP application (filled bars). For the relative peak current, 100% corresponds to the simulated current amplitude during a rectangular voltage pulse to 0 mV.

To examine the mechanisms underlying the reduction in peak amplitude of the Ca²⁺ current during the slower somatic AP (Fig. 5C), we used simulations to compare Ca²⁺ channel activation by fast and slow APs. Similar to the experimental data, the simulated Ca²⁺ current during a broad somatic AP had a prolonged time course and a reduced amplitude (Fig. 7C). The simulation shows that the reduction in peak amplitude is caused by a reduction in driving force at this point (−10 vs−20 mV). By contrast, the open probability of the channels at the peak of the Ca²⁺ current was very similar. Figure7D shows a comparison of the relative peak amplitude and half-duration of simulated Ca²⁺ currents evoked by different waveforms. The simulated values for the MFB AP were in close agreement with experimentally determined values (Fig. 5). The results of experiments and modeling converge toward the conclusion that a brief AP waveform leads to efficient and rapid activation of the Ca²⁺ channels, resulting in a high peak open probability.

Open probability of presynaptic Ca²⁺ channels is highly sensitive to changes in AP waveform

The shape of the presynaptic AP in MFBs is not constant but regulated in an activity-dependent manner (Geiger and Jonas, 2000). Thus we systematically examined the effects of modified AP waveforms, scaled in amplitude and shape in comparison with the original MFB AP (Fig. 8). Figure 8Ashows the effect of amplitude scaling on the evoked Ca²⁺ inflow, and Figure8B illustrates plots of peak amplitude and charge as a function of AP amplitude. The simulations show that the control AP waveform (vertical lines) is a very effective stimulus, producing a presynaptic Ca²⁺ signal with near-maximal peak amplitude and total charge (89 and 98% of the maximal value, respectively). By contrast, a reduction of the AP amplitude by Na⁺-channel inactivation during high-frequency activity or steady-state depolarization, for example by presynaptic kainate receptors, will substantially reduce the presynaptic Ca²⁺ inflow (Kamiya and Ozawa, 2000; Schmitz et al., 2000).

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Fig. 8.

Presynaptic Ca²⁺ inflow is highly sensitive to changes in AP amplitude and shape.A, The amplitude of the measured MFB AP was scaled from 10 to 120% in 10% steps and used to activate the Ca²⁺ channel model. B, The resulting peak amplitude and the total charge were plotted against the AP amplitude. The amplitude of the measured AP is indicated by thedashed vertical lines. C, The decay phase of the AP waveform was scaled by digitally shrinking or expanding the timescale of the trace after the peak from 50 to 500% in 50% steps.D, The resulting peak amplitude and the total charge were plotted against the AP half-duration. The half-duration of the measured AP is indicated by the dashed vertical lines. In B the data points were connected by straight lines, and the curves in D are fits with an exponential function plus a constant (top graph) or linear function (bottom graph).

Similarly, we modified the half-duration of the AP waveform by slowing the time course of repolarization in comparison with the MFB AP (Fig.8C,D). The peak amplitude was exponentially dependent on the half-duration (Fig. 8D, top graph). In contrast, the total charge could be fitted by linear regression (Fig. 8D, bottom graph), yielding a slope of 73 fC/msec. Thus the model predicts a total Ca²⁺ inflow of ∼77–121 fC for the experimental half-durations ranging from 540 to 1140 μsec. Furthermore, it explains the previous observation that dynamic AP broadening leads to a decrease in peak amplitude but an increase in total charge of presynaptic Ca²⁺ inflow (Geiger and Jonas, 2000).

In conclusion, the simulations show that the natural AP is a highly effective stimulus for the activation of the presynaptic Ca²⁺ channels. Additionally, the Ca²⁺ signal can be regulated via the half-duration of the presynaptic AP waveform.

Ca²⁺ channel gating is optimal for large Ca²⁺ inflow

Similarly, we examined the effects of alterations in channel gating on the amplitude and charge of Ca²⁺inflow (Fig. 9). Figure 9Ashows the effects of slower gating, generated by multiplication of all rates with constant factors in the range 1–0.1, and Figure9B shows the effects of acceleration of gating, using factors in the range 1–10. In all simulations, the natural MFB AP waveform was used as a stimulus. Plots of peak amplitude and total charge against the scaling factor indicate that gating kinetics is a critical determinant of the total Ca²⁺inflow (Fig. 9C,D). Surprisingly, both slowing down and speeding up the rate constants reduced the Ca²⁺ peak current and the integral. The peak amplitude and total charge produced by the native channels (scaling factor of 1) were 99 and 97% of the maximal values, which were reached with factors slightly below 1. Furthermore, both slowing down and speeding up of the rates lead to prolonged time course of Ca²⁺ inflow. In particular, the acceleration of the rates by a factor of >2 leads to a biphasic Ca²⁺ current, increasing the total time interval during which Ca²⁺ inflow occurs (Fig. 9B). In conclusion, these simulations unequivocally show that the gating of presynaptic Ca²⁺channels in MFBs is optimal for producing a transient Ca²⁺ inflow with maximal amplitude and minimal duration. Whether this conclusion also holds true at physiological temperatures remains to be established. However, because the Q₁₀ values for AP duration (∼2.2) (Geiger and Jonas, 2000; this paper) and Ca²⁺channel activation kinetics are similar (∼2.5) (Swandulla and Armstrong, 1988), this appears to be very likely.

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Fig. 9.

Presynaptic Ca²⁺ inflow is highly sensitive to changes in gating kinetics. A, All rate constants in the kinetic model were multiplied by a constant factor (1–0.1 in steps of 0.1) to selectively slow channel gating without affecting steady-state activation. Simulated Ca²⁺ currents are shown superimposed.B, The gating was accelerated by multiplying rate constants with a constant factor (1–10 in steps of 1).C, D, Peak amplitude (C) and charge (D) of the simulated current, plotted against the scaling factor on a logarithmic scale. The properties of the original Ca²⁺ channel are indicated by the vertical dashed lines. Curves inC and D represent cubic spline interpolations.

Rapid gating of presynaptic Ca²⁺ channels

The gating of presynaptic Ca²⁺channels could be adequately described by a serial gating model with four closed states and one open state. In contrast, neither alternative models with two or three closed states nor Hodgkin–Huxley models with an integer number of independent gating particles provided a sufficient description of the data (J. Bischofberger, unpublished data). In the models with two or three closed states, the delay of activation was smaller than that observed experimentally. In Hodgkin–Huxley models, deactivation is faster than activation at the same potential, with a ratio that approaches the number of gating particles (Kay and Wong, 1987; Zidanic and Fuchs, 1995; Mennerick and Matthews, 1998). Thus the functional properties of presynaptic Ca²⁺channels in MFBs are inconsistent with the assumption of independently moving gating particles.

Our model suggests that the voltage dependence differs among the transition rates. This has to be interpreted as a different movement of the S4 segments, the putative voltage sensors, because the number of positive charges per S4 segment is comparable among domains (approximately five) (Soong et al., 1993). For the first three transitions, the steepness factors range from 42 to 55 mV, which would correspond to an S4 segment movement by 9–12% along the total electric field. In contrast, for the final transition leading to the open state, the steepness factor is 27 mV, which would correspond to an S4 segment movement by 19%. Interdomain differences in voltage-dependent movement of S4 segments were experimentally demonstrated for voltage-dependent Na⁺channels (Cha et al., 1999). An analogous biophysical approach will reveal whether similar interdomain differences exist in voltage-gated Ca²⁺ channels, as our results would suggest.

Efficiency and timing of Ca²⁺ channel opening during single APs

The activation kinetics of the presynaptic Ca²⁺ channels in MFBs appear to be much faster than the activation of Ca²⁺channels in invertebrate presynaptic terminals (Llinás et al., 1981; Wright et al., 1996). For the squid giant synapse it was suggested that the relative open probability of Ca²⁺ channels during a single AP was ∼10% (Pumplin et al., 1981; Augustine, 1990), markedly less than the value estimated for the MFB (∼90%). The gating is also faster than that described for somatodendritic high-voltage-activated Ca²⁺ channels in CA1 pyramidal neurons (Kay and Wong, 1987). This may be correlated to the slower time course of somatodendritic voltage signals, e.g., synaptic potentials or backpropagating APs (Magee and Johnston, 1995; Normann et al., 2000).

The activation kinetics of the Ca²⁺channels in MFBs, however, is similar to that reported for presynaptic Ca²⁺ channels in giant vertebrate synaptic terminals (Zidanic and Fuchs, 1995; Borst and Sakmann, 1998; Mennerick and Matthews, 1998). In the calyx of Held, single APs evoke a Ca²⁺ current with a relative peak open probability of ∼70% and an average half-duration of ∼400 μsec (24°C) (Borst and Sakmann, 1998), comparable with the values reported here. Thus, although the two types of synapses differ in morphological properties, subunit composition of postsynaptic receptors, and functional role in the neuronal circuit, the efficacy and timing of presynaptic Ca²⁺ inflow appear to be similar (Von Gersdorff and Borst, 2002).

In the MFB, the Ca²⁺ channels open very early during the rising phase of the AP with a relativeP_open of ∼50% at the peak of the AP; however, no substantial current can be detected at this point, because of the small driving force. The Ca²⁺ inflow is restricted to the repolarization phase of the AP (Figs. 5A, 7A). In contrast, for parallel fiber synapses in the cerebellum, it was reported that rapid Ca²⁺ channel gating leads to substantial Ca²⁺ current during the upstroke and peak of the presynaptic AP (Sabatini and Regehr, 1996,1997). This situation appears to be very different from that in the MFB because the experimentally determined I–Vrelationship for the Ca²⁺ currents in the MFB is obviously inconsistent with a large driving force at the peak of the AP (at ∼45 mV) (Fig. 2B). If these discrepancies are not caused by technical difficulties in deriving the voltage and Ca²⁺ current time course from fluorescence signals (Sabatini and Regehr, 1996, 1997), they may suggest a synapse-specific difference in presynaptic ion flow.

How many Ca²⁺ ions are necessary for transmitter release?

The present results provide quantitative estimates of fundamental parameters of the early presynaptic events that are relevant for both efficacy and timing of synaptic transmission. We found that the average peak amplitude of the presynaptic Ca²⁺current evoked by a natural AP is 170 pA. With a single-channel current of 0.2 pA at physiological external Ca²⁺concentrations (Gollasch et al., 1992), this corresponds to 850 Ca²⁺ channels open at the peak in a presynaptic terminal. The total Ca²⁺inflow evoked by a single AP was 120 fC, corresponding to ∼370,000 Ca²⁺ ions per bouton.

MFBs have multiple release sites (up to 37 in an incompletely reconstructed large MFB) (Chicurel and Harris, 1992). Thus the measured presynaptic Ca²⁺ current would approximately correspond to 23 Ca²⁺channels open at the peak at a single release site, producing an inflow of ∼10,000 Ca²⁺ ions per site. Although these are only rough estimates, the results are consistent with previous suggestions at boutons of neocortical pyramidal cells (∼40 channels per bouton, ∼5500 Ca²⁺ ions) (Koester and Sakmann, 2000) and the calyx of Held (∼20 channels per site, ∼13000 Ca²⁺ ions) (Borst and Sakmann, 1996; Sätzler et al., 2002). Furthermore, they are consistent with the cooperativity between different types of Ca²⁺ channels in triggering transmitter release at the mossy fiber–CA3 synapse (Castillo et al., 1994) and other cortical glutamatergic synapses (Luebke et al., 1993; Wheeler et al., 1994; Dunlap et al., 1995). Although we cannot exclude a subpopulation of release sites endowed with a single Ca²⁺ channel (Stanley, 1997), our results suggest that most of the sites are controlled by multiple channels. This may increase the reliability of excitation–secretion coupling in central glutamatergic synapses.

Functional significance for synaptic transmission at MFB–CA3 pyramidal neuron synapse

The quantitative properties of the presynaptic Ca²⁺ inflow are also critical for the timing of transmitter release. A large Ca²⁺ signal may help to minimize synaptic delay. Transmitter release requires the binding of multiple Ca²⁺ ions to the Ca²⁺ sensor (Augustine, 2001). As shown for the calyx of Held, the sequential binding of multiple Ca²⁺ ions introduces a significant delay between the Ca²⁺ inflow and the transmitter release (Schneggenburger and Neher, 2000). This delay was strongly dependent on the Ca²⁺concentration. Thus, a large Ca²⁺ signal will provide a rapid onset of neurotransmitter release. Additionally, the fast deactivation of the presynaptic Ca²⁺ channels may be important for the termination of exocytosis. The rapid Ca²⁺signaling will be a critical determinant of the precise timing of transmitter release at the MFB to CA3–pyramidal cell synapse, where the half-width of the first latency distribution is ∼500 μsec (Jonas et al., 1993). Similar rules may apply for synapses formed by the mossy fiber system onto other types of target cells, e.g., at the granule cell to basket cell synapse, formed by mossy fiber collaterals on inhibitory interneurons (Geiger et al., 1997).

The precise timing during single APs may be relevant for the temporal encoding of information in the hippocampus (Lisman, 1999). It was shown that granule cells and pyramidal cells fire APs in a specific temporal relationship to the theta rhythm when the animal passes through the place field of the cell (Jung and McNaughton, 1993; O'Keefe and Recce, 1993). When the animal is in the center of the place field of a given neuron, this neuron fires APs earlier in the theta cycle than neurons with adjacent place fields (Skaggs and McNaughton, 1996; Skaggs et al., 1996). For temporal coding with high fidelity, neurons with the same place field should fire exactly at the same time (Lisman, 1999). Because the mossy fiber tract is the major excitatory pathway that connects the associative network in the dentate gyrus/hilus with that in the CA3 region, the precise timing of presynaptic Ca²⁺ inflow and transmitter release might be important for the correct temporal sequence of place cell firing and, more generally, for the temporal coding of episodic memory.