Synaptic Activation of Ca2+ Action Potentials in Immature Rat Cerebellar Granule Cells In Situ

Egidio D'angelo,giovannade Filippi, Paola Rossi, and Vanni Taglietti

Istituto di Fisiologia Generale, I-27100 Pavia, Italy

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

D'Angelo, Egidio, Giovanna De Filippi, Paola Rossi, and Vanni Taglietti. Synaptic activation of Ca2+ action potentials in immature rat cerebellar granule cells in situ. J. Neurophysiol. 78: 1631-1642, 1997. Although numerous Ca2+ channels have been identified in cerebellar granule cells, their role in regulating excitability remained unclear. We therefore investigated the excitable response in granule cells using whole cell patch-clamp recordings in acute rat cerebellar slices throughout the time of development (P4-P21, n = 183), with the aim of identifying the role of Ca2+ channels and their activation mechanism. After depolarizing current injection, 46% of granule cells showed Ca2+ action potentials, whereas repetitive Na+ spikes were observed in an increasing proportion of granule cells from P4 to P21. Because Ca2+ action potentials were no longer observed after P21, they characterized an immature granule cell functional stage. Ca2+ action potentials consisted of an intermediate-threshold spike (ITS) activating at -60/-50 mV and sensitive to voltage inactivation and of a high-threshold spike (HTS), activating at above -30 mV and resistant to voltage inactivation. Both ITS and HTS comprised transient and protracted Ca2+ channel-dependent depolarizations. The Ca2+ action potentials could be activated synaptically by excitatory postsynaptic potentials, which were significantly slower and had a proportionately greater N-methyl-D-aspartate (NMDA) receptor-mediated component than those recorded in cells with fast repetitive Na+ spikes. The NMDA receptor current, by providing a sustained and regenerative current injection, was critical for activating the ITS, which was not self-regenerative. Moreover, NMDA receptors determined temporal summation of impulses during repetitive mossy fiber transmission, raising membrane potential into the range required for generating protracted Ca2+ channel-dependent depolarizations. The nature of Ca2+ action potentials was considered further using selective ion channel blockers. N-, L-, and P-type Ca2+ channels generated protracted depolarizations, whereas the ITS and HTS transient phase was generated by putative R-type channels (RITS and RHTS, respectively). RHTS channels had a higher activation threshold and were more resistant to voltage inactivation than RITS channels. At a mature stage, most of the Ca2+-dependent effects depended on the N-type current, which promoted spike repolarization and regulated the Na+-dependent discharge frequency. These observations relate Ca2+ channel types with specific neuronal excitable properties and developmental states in situ. Synaptic NMDA receptor-dependent activation of Ca2+ action potentials provides a sophisticated mechanism for Ca2+ signaling, which might be involved in granule cell development and plasticity.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The granule cells of mature rat cerebellum generate a repetitive spike discharge, regulating information transfer along the mossy fiber pathway (D'Angelo et al. 1995). However, the excitable response at early developmental stages may have different properties and involve different channels than at a mature stage. Immature neurons can generate Ca2+ action potentials (Baccaglini and Spitzer 1977; McCleskey 1994; Spitzer 1991), involving specific Ca2+ channel currents (McCobb et al. 1989; Yaari et al. 1987). Specific Ca2+ channel expression indeed has been suggested at an immature stage of granule cell development (Rossi et al. 1994).

The Ca2+ channels of granule cells have been characterized extensively in cell culture, revealing high-voltage activated (HVA) Ca2+ channels of the L, N, and P type. In addition to these, other channels also may be expressed, namely the Q- and R-type channels (Amico et al. 1995; Bossu et al. 1994; Ellinor et al. 1993; Forti et al. 1994; Pearson et al. 1995; Randall and Tsien 1995; Sather et al. 1993; Tottene et al. 1996; Zhang et al. 1993). These channels have different gating and kinetic properties and coexist in granule cells. Their role in regulating granule cell excitability remained elusive, however, because no correlation with functional states in situ had been established. By using current-clamp patch-clamp recordings in cerebellar slices, we found that a large proportion of granule cells of the developing cerebellum generated Ca2+ action potentials with complex time course and voltage dependence due to the opening of L-, P-, N-, and putative R-type channels. At a mature stage, the Ca2+ action potentials disappeared, and N-type Ca2+ channels participated in regulating Na+-dependent spike discharge.

Granule cells are activated synaptically by mossy fibers (Eccles et al. 1967) involving glutamate receptors of the N-methyl-D-aspartate (NMDA) type, which, at an early developmental stage, account for most of the synaptic current (D'Angelo et al. 1993). We found that synaptic activation of the NMDA receptors, by providing a protracted and voltage-dependent current injection, was critical for sustaining Ca2+ action potential generation.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Cerebellar slices (250 µM thick) were obtained from 4- to 21-day-old rats (Wistar strain, day of birth = P1) as reported previously (D'Angelo et al. 1993, 1995). The rats were anesthetized with halothane (Aldrich) before being killed by decapitation. Krebs solution for slice cutting and recovery contained (in mM) 120 NaCl, 2 KCl, 1.2 MgSO4, 26 NaHCO3, 1.2 KH2PO4, 2CaCl2, and 11 glucose. This solution was equilibrated with 95% O2-5% CO2 (pH 7.4). Slices were maintained at room temperature before being transferred to the recording chamber (1.5 ml) mounted on the stage of an upright microscope (Zeiss Standard-16). The preparations were superfused at a rate of 5-10 ml/min with a Krebs solution to which 10 µM glycine and 10 µM bicuculline were added, and maintained at 30°C with a feed-back Peltier device (HCC-100A, Dagan, Minneapolis, MN).

Whole cell current-clamp recordings

The experimental technique was substantially similar to that used in our previous paper (D'Angelo et al. 1995). Granule cells in lobules IV-IX were recorded in the whole cell patch-clamp configuration (Edwards et al. 1989) using the "blind-patch" approach. Recordings were performed with an Axopatch-1D or an Axopatch 200-A (fast current-clamp mode) amplifier. The data were sampled with a TL-1 DMA Interface (sampling time = 250 µs for current-clamp recordings, 10 µs for voltage-clamp recordings) and analyzed with pClamp software (Axon Instruments, Foster City, CA). Mossy fiber stimulation was performed with a bipolar tungsten electrode (Clark Instruments, Pangbourne, UK) via a stimulus isolation unit. The stimulating electrode was placed over the mossy fiber bundle, and stimuli were applied at the frequency of 0.1 Hz or in 500-ms trains of 5, 10, or 50 Hz. In some experiments, a second stimulating electrode was placed in proximity of the Purkinje cell layer to test granule cell antidromic excitation.

Patch pipettes were pulled from borosilicate glass capillaries (Hingelberg, Malsfeld, Germany) and had 8-12 MOmega resistance before a seal was formed with a filling solution containing (in mM) 126 K-gluconate, 4 NaCl, 1 MgSO4, 0.02 CaCl2, 0.1 bis-(o-aminophenoxy)-N,N,N',N'tetraacetic acid (BAPTA), 15 glucose, 3 ATP, 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH was adjusted to 7.2 with KOH). This solution buffered intracellular Ca2+ at 100 nM, similar to the resting Ca2+ concentration measured in granule cells (Marchetti et al. 1995). After a giga-seal was formed (seal resistance was usually >20 GOmega ), the electrode capacitance was canceled carefully before rupturing the patch to allow for the electronic compensation of pipette charging during subsequent current-clamp recordings (D'Angelo et al. 1995). Once in the whole cell configuration, the current transients elicited by 10-mV hyperpolarizing pulses from the holding potential of -70 mV in voltage-clamp mode showed a mono-exponential relaxation (time constant = 81 ± 27 µs, n = 40), and were used to estimate series resistance (21.1 ± 8.7 MOmega , n = 40) and input resistance and capacitance. Depending on the high-input-to-series resistance ratio, bridge balancing in current-clamp recordings proved of little effect and was not routinely used (D'Angelo et al. 1995). Membrane potential was measured relative to an agar-bridge reference electrode. Reported membrane potential values have been adjusted off-line for liquid-junction potentials (usually <= 5 mV).

In the current-clamp mode, the granule cell input resistance (Rm) was monitored repeatedly by measuring the steady state membrane potential change generated by hyperpolarizing current pulses from -70 to -80 mV. Experiments in which Rm changed by more than ±10% during recordings were rejected.

Experimental tracings were analyzed using pClamp software. HW denotes duration of excitatory postsynaptic potentials (EPSPs) or spikes at half-amplitude. Data are reported as means ± SD, and statistical comparisons were done using Student's t-test (NS, not significant).

Drugs

In the present experiments, control and test solutions [including channel blockers or ethylene glycol-bis(beta -aminoethylether)-N,N,N',N'-tetracetic acid (EGTA)] were applied locally through a multibarrel pipette. Perfusion of control solution was commenced before seal formation and was maintained until switching to test solutions.

Glycine, bicuculline, tetrodotoxin (TTX), tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP), nifedipine, and amiloride were obtained from SIGMA. The conotoxin omega -CTX GVIA was obtained from Peninsula Laboratories (Belmont, CA), the conotoxin omega -CTX MVIIC from Bachem (Bubendorf, Switzerland), the agatoxin omega -Aga IVA from Peptide International (Louisville, KY), apamine from Alomone (Tel-Aviv, Israel), and BAPTA tetrapotassium salt from Molecular Probes (Eugene, OR). Stock solutions were prepared for all drugs and stored frozen at -20°C. Nifedipine was dissolved in ethanol (final concentration 0.05%), amiloride in dimethyl sulfoxide (final concentration 0.1%), and omega -CTX MVIIC in 0.1% TEA (final concentration 20 µM), and all the other drugs in water. The drugs were dilutedto their final concentration in the appropriate Krebs solutionbefore use.

The action of organic channel blockers on the Ca2+ action potentials was tested in preliminary experiments. The action of 10 µM nifedipine occurred in 1.3 ± 0.6 min (n = 7) and was readily reversible on wash. The action of omega -CTX GVIA occurred in 2-4 min and was almost irreversible after a 30-min wash (n = 12). A threefold increase in either nifedipine or omega -CTX GVIA concentration did not increase their inhibitory effect. The action of omega -Aga IVA was maximum after 4.5 ± 1.4 min at 3 nM (n = 7) and 3.8 ± 2 min at 30-300 nM (n = 8). Thereafter, omega -Aga IVA block was irreversible during a 15-min wash (n = 12). The observed action times, the reversibility (or irreversibility) in drug action, and the apparent saturation of the effects at the concentrations used in the present experiments are consistent with observations reported in experiments in cell culture (e.g., Amico et al. 1995; Pearson et al. 1995; Randall and Tsien 1995). Any residual Ca2+ action potentials could be blocked with 1 mM Ni2+ at the end of the experiments.

The glutamate receptor antagonists D-2-amino-5-phosphonovaleric acid (APV), 7-chlorokinurenic acid (7-Cl-kyn), and 6cyano-7-nitroquinoxaline-2,3-dione (CNQX) were obtained from Tocris Cookson (Bristol, UK) and prepared as reported previously (D'Angelo et al. 1993, 1995). No noticeable difference in the synaptic response was noted whether or not 10 µM glycine was present in the extracellular solutions (n = 4), indicating that ambient glycine in the slice was probably enough to saturate the glycine binding site on the NMDA receptor (Thomson 1989; cf. D'Angelo et al. 1995).

Quantification of Ca2+ channel blockage

To quantify the effect of Ca2+ channel blockers, we estimated the ionic current generating ITS, iITS. This could be done considering that the current injected through the microelectrode (iinj) divides into a capacitive (iC) and an ionic (iion) component and that iion comprises a leakage current (iL) as well as the voltage-sensitive current (iITS) giving rise to the action potential (Jack et al. 1975). It follows the equation
<IT>i</IT><SUB>ITS</SUB><IT>= i</IT><SUB>inj</SUB>− (<IT>i</IT><SUB>C</SUB><IT>+ i</IT><SUB>L</SUB>) (1)
where iC = Cm dV/dt and iL = V/Rm could be measured directly from voltage tracings (Cm and Rm values were obtained as explained above, and V indicates membrane potential), and iinj was known. Because the ITS rising phase was slow, the relative contribution of iL was taken into account in our measurements, although this term often is neglected (cf. Jack et al. 1975; McCormick et al. 1992). In seven cells recorded under pharmacological Na+ and K+ current blockage (1 µM TTX, 20 mM TEA, 4 mM 4-AP)---the cells had Cm = 5.7 ± 1.4 pF and Rm = 3.9 ± 0.3 GOmega and were maintained at a holding potential of -79.7 ± 3.5 mV---the maximum inward current involved in ITS generation was imax = -2.9 ± 0.4 pA/pF at -26 ± 0.6 mV. imax was consistent with Ca2+ current measurements previously performed in immature granule cells in situ (Rossi et al. 1994). It should be noted that imax depended on the effectiveness of the regenerative process and could not be used for measuring fractional channel block (which is assessed conveniently in voltage-clamp experiments). Equation 1 also was used to estimate the current sustaining the protracted ITS component (ipr) by considering that, following the ITS ballistic phase, iC is zero and ITS current is simply iITS = iinj - iL.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Patch-clamp recordings were performed on 183 neurons in the internal granular layer of rat cerebellar slices from P4 to P21. The neurons did not display spontaneous firing either during seal formation or after having established the whole cell recording configuration and had the low membrane capacitance and high-input resistance typical of granule cells (Table 1) (D'Angelo et al. 1995). When recorded from near rest (-70 mV), the granule cells showed two basic firing patterns on depolarizing current injection. Forty-six percent of granule cells showed characteristic action potentials comprising components with different threshold and time course (Fig. 1A). The principal component consisted of a broad action potential (HW > 30 ms) activating at a threshold between -60 and -50 mV, which is intermediate between low- and high-threshold spikes in thalamic (Jahnsen and Llinas 1984) and inferior olivary neurons (Llinas and Yarom 1981). By analogy, this granule cell action potential has been termed intermediate-threshold spike, or ITS. When stimulus intensity was sufficiently high, ITS could prime a high-threshold spike at above -30 mV (HTS: cf. the two uppermost tracings in Fig. 1A). Because these excitable responses were not usually recorded beyond P21, they were related to an immature stage of granule cell development. Other granule cells fired fast spikes (HW < 2 ms) repeatedly at high-frequency but did not show any ITS (Fig. 1B), like the cells recorded at a mature developmental stage (D'Angelo et al. 1995). Electrophysiologically mature granule cells were observed occasionally as early as P5 and became more frequent thereafter (4% at P4-P9, 22% at P10-P21). Some granule cells (24%) showed firing patterns including both the ITS/HTS and fast repetitive action potentials, whereas other granule cells (18%) did not show any excitable response (not shown).

 
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TABLE 1. Passive membrane properties of granule cells


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FIG. 1. Diverse excitable properties in cerebellar granule cells. A and B: voltage response to DC injection from holding potential of -70 mV in 2 different granule cells recorded at P15. Granule cell in A generates a broad nonrepetitive action potential at a relatively low threshold (ITS) and a more spiky depolarization at high threshold (HTS). Granule cell in B generates a fast repetitive spike dicharge. Note also the marked inward rectification in the subthreshold voltage response in B, but not in A. In this and following figures, the current-clamp protocol is shown at the bottom. C: subthreshold voltage-current relationships of the cells in A and B, documenting the different intensity of inward rectification. Membrane voltage values were measured at 600 ms, where the tracings attained a steady level.

The subthreshold membrane charging of granule cells was smooth with no humps or afterpotentials. Granule cells with ITS/HTS had an almost linear subthreshold voltage response, whereas granule cells with a fast repetitive firing showed marked inward rectification (Fig. 1, A and B), as demonstrated in the voltage-current plots in Fig. 1C. Consequently, Rm was larger in granule cells with ITS/HTS than with fast repetitive firing over most of the subthreshold membrane potential range, although Rm values were similar close to the threshold for action potential activation (Table 1). Cm was also larger in granule cells with ITS/HTS than with fast repetitive firing, probably reflecting morphological rearrangement during development (Altman 1972). The larger Rm and Cm accounted for passive membrane transients being slower in granule cells with ITS/HTS than with fast repetitive firing (Fig. 1, A and B, Table 1).

The ionic nature of granule cell action potentials was investigated using specific ion channel blockers. The Na+ channel blocker TTX (1 µM) blocked the fast repetitive action potentials (Fig. 2B, n = 9). TTX-resistant action potentials (ITS and HTS) were blocked by 1 mM Ni2+ (Fig. 2A, n = 10), indicating that they had been generated by Ca2+ channel currents. Therefore ITS and HTS were Ca2+ action potentials, whereas Na+ action potentials sustained the fast repetitive firing. It should be noted that TTX caused appreciable modifications in HTS in 6 of 10 granule cells that did not show repetitive firing (cf. Fig. 2A) and that Ca2+ and repetitive Na+ action potentials coexisted in another 5 granule cells (not shown).


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FIG. 2. Na+- and Ca2+-dependent action potentials. Sequential application of 1 µm tetrodotoxin (TTX) and 1 mM Ni2+ (+1 µm TTX) in a cell generating ITS and HTS (A) and in a cell generating fast repetitive spikes (B). ···, approximate activation thresholds for ITS (-55 mV) and HTS (-20 mV). Note that ITS and HTS were blocked by Ni2+, whereas fast action potentials were blocked by TTX. Same scale and experimental sequence in A and B (P16).

In the presence of 1 µM TTX, local perfusion of a solution containing the Ca2+ chelator 5 mM EGTA and no added Ca2+ abolished both ITS and HTS (n = 6). In addition to bearing out the Ca2+-dependence of both ITS and HTS, this ruled out any noticeable contribution of persistent TTX-resistant Na+ currents (Llinas 1989).

Synaptic activation of Ca2+ action potentials

In 15 of 17 granule cells generating Ca2+ action potentials, EPSPs and characteristic EPSP/action potential complexes (comprising ITS and HTS) were observed after mossy fiber stimulation (Fig. 3A). EPSPs were activated with a delay of 1-1.5 ms, compatible with monosynaptic granule cell activation. Similarly as in granule cells with fast repetitive Na+ spikes (Fig. 3B), transition between neighboring levels in the EPSP and EPSP/Ca2+ action potential complex occurred with small changes in the stimulus intensity, suggesting that an increasing number of mossy fiber synapses was recruited. If the response to the minimal stimulus intensity is assumed to correspond to unitary synaptic events (see D'Angelo et al. 1995; Silver et al. 1996), the unitary EPSP should have an amplitude of 8.8 ± 2.4 mV from the holding membrane potential of -71.4 ± 2.3 mV (n = 6). Higher levels of synaptic activation then would correspond to the recruitment of up to four mossy fiber synapses, in good agreement with the morphological evidence that granule cells make an average of four synapses with as much as different mossy fibers (Eccles et al. 1967). Accordingly, 2.6 ± 1.1 (n = 8) mossy fiber synapses had to be activated synchronously to generate ITS. Despite using stimulus intensities higher than those effectively activating mossy fibers, stimulation close to the Purkinje cell layer failed to elicit any antidromic spikes (n = 11), suggesting that the ascending axon in granule cells generating Ca2+ action potentials was inefficient in propagating excitation.


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FIG. 3. Synaptic activation of action potentials. A and B: excitatory postsynaptic potential (EPSP) and action potentials in the same cells as in Fig. 1 after mossy fiber stimulation (triangle ) at increasing stimulus intensities. Granule cell in A shows failures (<4.3 V), EPSPs (4.3 V, 4.9 V), and EPSPs surmounted by an ITS (6.5 V) or ITS/HTS complex (10 V). Granule cell in B shows failures (<5.5 V), EPSPs (5.5 V, 7.5 V), and an EPSP with a fast action potential superimposed (12 V). C: EPSPs (average of 12 tracings) are shown in control (contr) and after a sequential block of the N-methyl-D-aspartate [NMDA; 100 µM D-2-amino-5-phosphonovaleric acid (APV) + 50 µM 7-chlorokinurenic acid (7-Kyn)] and non-NMDA component (10 µM 6-cyano-7-nitroquinoxaline-2,3-dione). The NMDA EPSP (obtained by subtracting from control the tracings measured after application of APV + 7-Cl-Kyn) and non-NMDA EPSPs are shown (right). - - -, passive membrane discharge has been superimposed on both the NMDA and non-NMDA EPSP. Holding potential -70 mV. D: EPSPs (average of 4 tracings) were elicited from different holding potentials in control (contr) and after block of the NMDA component (APV + 7-Cl-Kyn). NMDA EPSPs obtained by subtraction are shown (right). Note that EPSPs in C and D remained subthreshold for ITS activation.

EPSPs in granule cells generating Ca2+ action potentials comprised a NMDA and a non-NMDA receptor-mediated component. The NMDA component was slower than passive membrane transients, whereas the non-NMDA component and passive transients decayed at a similar rate (Fig. 3C). EPSPs showed a marked voltage dependence, increasing in size and slowing down the more the membrane was depolarized (Fig. 3D), as expected from voltage-dependent increase and slowing down of the NMDA current during membrane depolarization (D'Angelo et al. 1994b, 1995). It should be noted that the EPSPs were slower in granule cells generating Ca2+ than Na+ action potentials (Fig. 3, A and B, Table 2), probably due to a larger membrane time constant and a greater contribution of the NMDA relative to non-NMDA component (D'Angelo et al. 1993).

 
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TABLE 2. Properties of granule cell EPSPs

Requirements for Ca2+ action potential generation

ITS activation showed a marked dependence on the intensity of injected current pulses. Moreover, a brief current injection failed to prime ITS activation, whereas ITS could be activated as the pulse duration was increased (Fig. 4A). This observation indicates that ITS is not self-regenerative and that ITS generation requires a protracted supporting current, which must be provided by synaptic channels during mossy fiber transmission. Blocking the NMDA receptors precluded ITS activation (Fig. 4B). The residual depolarization decayed close to passive membrane transients as expected from a non-NMDA EPSP. The higher efficiency of NMDA than non-NMDA receptors in generating ITS apparently was related to the slow regenerative inward current produced by NMDA receptors during depolarization (D'Angelo et al. 1994b) (see Fig. 3D). Repetitive stimulation at frequencies between 5 and 50 Hz generated ITS and HTS followed by a protracted depolarization (Fig. 4C). At these frequencies, a doublet of EPSPs resulted in a saturation of the response, which was maintained by any subsequent input. Blocking NMDA receptors revealed that non-NMDA EPSPs were too short and small to determine effective temporal summation and sustain Ca2+ channel-dependent responses. Results similar to those shown in Fig. 4 have been obtained in six other granule cells to which glutamate receptor antagonists have been applied, revealing a primary role for the NMDA receptors in driving Ca2+ action potential generation.


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FIG. 4. Requirements for Ca2+ action potential activation. A: ITS showed graded activation with protracted current pulses of increasing intensity (left). ITS could not be activated as current pulse was shortened (middle). Short current pulses proved ineffective despite their intensity was higher than that used in protracted current pulses (right). B: ITS and HTS were activated during low-frequency mossy fiber stimulation (0.1 Hz). After NMDA receptor blockage with APV + 7-Cl-Kyn, the ITS/HTS complex could no longer be elicited, and EPSP decay only slightly deviated from passive membrane discharge (···). Note that the non-NMDA EPSP had nearly the same amplitude as control EPSP and crossed ITS threshold. D: during high-frequency mossy fiber stimulation (12 Hz), temporal summation sustained an intense protracted depolarization. After APV + 7-Cl-Kyn application, a dramatic reduction in temporal summation precluded any protracted depolarization, uncovering non-NMDA EPSP depression along the trains. Same cell (P14), holding potential (-80 mV), and scale in A-C.

Voltage-inactivation in ITS and HTS

In addition to having distinct activation thresholds, ITS and HTS showed distinct voltage inactivation. ITS could be activated from low holding potentials (usually less than -60 mV, Fig. 5A, bottom) but was inactivated at potentials higher than -50 mV (Fig. 5A, top). This mechanism resulted in rebound ITS activation on return to a membrane potential around -50 mV after a period of hyperpolarization at around -80 mV, as illustrated in Fig. 5A and observed in another eight granule cells. Conversely, HTS still could be activated from -50 mV. Therefore ITS had much more pronounced voltage inactivation than HTS. Differential ITS and HTS voltage inactivation also could be evidenced during synaptic transmission. ITS, but not HTS synaptic activation, failed when the cell was held at around -50 mV for 5 s (Fig. 5B). The voltage dependence of ITS inactivation occurred in the range of membrane potentials between -90 and -50 mV, as observed using different conditioning membrane potentials (n = 7, Fig. 5C, left). Paired stimulation experiments demonstrated that inactivation developed during the ITS, causing refractoriness in ITS reactivation. After a conditioning ITS at -80 mV, refractoriness was removed in ~500 ms (n = 3; Fig. 5C, right).


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FIG. 5. Inactivation in ITS and HTS. A: a granule cell was stimulated by depolarizing and hyperpolarizing current steps from the holding membrane potential of -50 mV. A HTS followed by an irregular plateau was elicited beyond -30 mV, whereas ITS was elicited on rebound depolarization at -55 mV. B: an ITS and ITS/HTS complex were activated by mossy fiber stimulation from the membrane potential of -80 mV. Using same stimulus intensity, HTS, but not ITS, could be activated after 5 s conditioning at -52 mV. C: stimulation with a depolarizing current step of constant intensity after 5 s conditioning at different potentials evidenced voltage-dependent ITS inactivation (left; note that ITS generated from the lowest holding potential grew enough to activate an HTS). Stimulation at different time intervals after a conditioning ITS evidenced ITS time-dependent deinactivation (right). Same scale in A-C, cells recorded at P12, P13, and P16, respectively.

Action of K+ channel blockers on ITS and HTS

To assess the role of repolarizing currents in the Ca2+ action potentials, we used the broad spectrum K+ channel blocker, TEA. Application of 20 mM TEA could modify ITS (Fig. 6A), although this effect was observed inconstantly and was statistically not significant (8 ± 13% imax change; n = 9; NS). Therefore the ITS time course should be determined largely by Ca2+ channel and passive membrane properties. Conversely the application of TEA as low as 1 mM increased HTS, revealing that HTS was limited severely by repolarizing K+ conductances. A marked HTS broadening was observed as the TEA concentration was increased <= 20 mM (Fig. 6B). The differential action of TEA on ITS and HTS could be explained if we consider that effective activation of TEA-sensitive K+ currents occurs at around -30 mV (Bardoni and Belluzzi 1993; Gabbiani et al. 1994), thus not significantly affecting the ITS time course. At more than -30 mV, K+ currents rapidly increase, limiting the development of HTS. The comparison of ITS and HTS after TEA application also shows that ITS had faster inactivation than the HTS current.


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FIG. 6. Action of K+ channel blockers on ITS and HTS (all solutions contained 1 µM TTX). A: ITS could be enhanced by 20 mM tetraethylammonium chloride (TEA) application, showing a faster rising phase, slower decay phase, and increased peak amplitude. However, these changes were observed only in 4 of 9 cells and were statistically not significant. B: HTS became higher and broader at increasing TEA concentrations (1 and 20 mM). C and D: Ca2+ action potentials were not affected by 2 mM 4-aminopyridine or by 500 nM apamine. Same scale in A-D, cells recorded at P12, P11, and P13, respectively.

Among TEA-insensitive K+ currents, the transient IA current and Ca2+-dependent K+ currents (see Bardoni and Belluzzi 1993) may be activated during the action potentials. Application of the IA blocker 4-AP (2 mM; Fig. 6C) had, however, no remarkable effect on ITS (-6 ± 7% imax change, n = 4, NS). The peptidic toxin apamine (500 nM; Fig. 6D) was also ineffective (+2 ± 6% imax change, n = 5, NS), ruling out the involvement of TEA-insensitive Ca2+-dependent K+ channels in action potential repolarization.

Pharmacological identification of ITS components

Recent findings demonstrate the coexistence of multiple HVA Ca2+ current components in cerebellar granule cells in culture (Amico et al. 1995; Pearson et al. 1995; Randall and Tsien 1995). Indeed, ITS was inhibited by 10 µM nifedipine (n = 14), by the Agelenopsys Aperta peptidic toxin 3-30 nM omega -Aga IVA (n = 13), and by the Conus Geographus peptidic toxin 5 µM omega -CTX GVIA (n = 15), which are known to block L, P, and N channels, respectively (Fig. 7, A and B). All these Ca2+ channel antagonists reduced both a transient and a protracted ITS component, as shown below. ITS was not affected at all by 500 µM amiloride (-2 ± 3% imax change, n = 6, NS; Fig. 4C) as expected from previous studies showing that low-voltage activated channels are not expressed in granule cells (see, for instance, Randall and Tsien 1995; Rossi et al. 1994).


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FIG. 7. Action of organic Ca2+ channel blockers on ITS (all perfused solutions contained 1 µM TTX and 20 mM TEA). A: ITS was reduced by a 3-min application of 5 µM omega -CTX GVIA. B: omega -CTX GVIA resistant ITS was reduced by sequential applications of 10 µM nifedipine, and 10 µM nifedipine + 300 nM omega -Aga IVA (all solutions contained 5 µM omega -CTX GVIA). Tracings are shown after a steady drug effect was observed (5-10 min). A spiky ITS shape in these experiments might be favoured by blockage of repolarizing K+ currents and of Ca2+ currents with slow kinetics and by using rather negative holding potentials (see also Fig. 8). Although membrane potential could rise at around -20 mV, a full HTS activation was never observed, indicating a marginal contribution of HTS channels. C: a 15-min perfusion with 500 µM amiloride did not affect ITS. D: coperfusion of 5 µM omega -CTX GVIA, 10 µM nifedipine, and 30 nM omega -Aga IVA did not block ITS, which then was reduced severely by 5 µM omega -CTX MVIIC. Action of omega -CTX MVIIC was reversible after 3 min wash (bottom, - - -). Results in this figure provided evidence for the coexistence of N-, L-, P-, and putative R-type channels (RITS) in granule cells. Preparations were obtained at P12-P15, and 60-min preincubation with 5 µM omega -CTX GVIA was used in B and D.

The sequential application of 5 µM omega -CTX GVIA, 10 µM nifedipine, and 300 nM omega -Aga IVA showed N-, L-, and P-type channel coexpression in granule cells (Fig. 7B). In eight experiments, imax changed by -14 ± 11%, -37 ± 14%, -52 ± 21%, respectively, and ipr by -51 ± 18%,-73 ± 16%, -89 ± 23%, respectively (n = 8; P < 0.05 for each measurement). In these experiments, a possible action of omega -Aga IVA on L- and N-type channels (Pearson et al. 1995) was prevented by nifedipine and omega -CTX GVIA. A Q-type channel might coexist with a P-type channel and be blocked by omega -Aga IVA (Randall and Tsien 1995). This possibility was investigated by using an omega -Aga IVA at a concentration that should discriminate P- against Q-type channel block (30 nM) and the Conus Magus peptidic toxin omega -CTX MVIIC (Randall and Tsien 1995; Tottene et al. 1996). The rationale of these experiments was that, if a fraction of putative Q-type channels is left unblocked by 30 nM omega -Aga IVA, it then should be blocked irreversibly by omega -CTX MVIIC (Sather et al. 1993). The application of 5 µM omega -CTX MVIIC in the presence of 5 µM omega -CTX GVIA, 10 µM nifedipine, and 30 nM omega -Aga IVA inhibited ITS (-74 ± 13% imax change, n = 8, P < 0.01; Fig. 7D). This effect was, however, almost completely reversible after5 min perfusion of a solution without omega -CTX MVIIC(-12 ± 5% imax change, n = 8, NS; Fig. 7D, bottom), indicating that the major omega -CTX MVIIC-sensitive ITS component has different binding properties than the Q-type channel reported previously (Sather et al. 1993).

In all these experiments, omega -CTX GVIA, nifedipine, and omega -Aga IVA were used at saturating concentrations, and their action was allowed to progress until a maximum effect was attained (see METHODS). Nevertheless, a considerable ITS component remained unblocked (48% of control imax, 11% of control ipr, n = 8). This non-L, non-N, non-P ITS component, which was inhibited partially and reversibly by omega -CTX MVIIC, thus should reflect activation of R-type Ca2+ channels (Ellinor et al. 1993; Pietrobon et al. 1996; Randall and Tsien 1995; Tottene et al. 1996; Zhang et al. 1993), and it henceforth will be defined as RITS.

Differential roles of Ca2+ channels in ITS generation

The protracted depolarizations in ITS were reduced after 5 µM omega -CTX GVIA, 10 µM nifedipine, and 300 nM omega -Aga IVA coapplication, so that RITS (which could be blocked reversibly by 5 µM omega -CTX MVIIC) appeared narrower than control ITS (Fig. 8A). Exponential fitting showed that RITS decay conformed to passive membrane discharge (Fig. 8A). Therefore the putative RITS current should inactivate more rapidly than N-, L-, and P-type currents, as well as of passive membrane discharge (time constant <= 32.2 ± 9.6 ms, n = 5), and largely accounted for the transient componentin ITS.


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FIG. 8. Differential roles of Ca2+ channels in ITS generation. After control recordings, the RITS component was isolated by blocking N, L, and P channels with 5 µM omega -CTX GVIA + 10 µM nifedipine + 300 nM omega -Aga IVA (all the perfused solutions contained 1 µM TTX and 20 mM TEA). A; RITS component (reversibly inhibited by 5 µM omega -CTX MVIIC) was narrower than control ITS and decayed after an exponential time course (···) with a time constant of 38 ms. An exponential curve with the same time constant was fitted to passive membrane potential charging (- - -). B: using a relatively small injected current, no ITS could be observed after L-, N-, and P-channel block. However, the RITS component (reversibly inhibited by 5 µM omega -CTX MVIIC, inset) could be recovered by increasing injected current (from 4 to 5 pA, right). An exponential curve with time constant of 36 ms fits both ITS decay (···) and the passive membrane potential charging (- - -). C: recordings from different conditioning membrane potentials show greater voltage-inactivation in the transient (RITS) than protracted (L, N, P) ITS component. Cells were recorded at P12-P15. It should be noted that, although control ITS in A and C grew more than -20 mV, no full HTS activation was observed.

The role of L-, N-, and P-type currents was investigated further in experiments in which coapplication of 5 µM omega -CTX GVIA, 10 µM nifedipine, and 300 nM omega -Aga IVA almost fully prevented ITS generation (-96 ± 16% imax change, P < 0.01, n = 7; Fig. 8B). Subsequently, the RITS component (which decayed conforming to passive membrane discharge and was reduced reversibly by 5 µMomega -CTX MVIIC) could be restored by increasing the injected current intensity (-42 ± 18% imax change P < 0.01, n = 7). These observations suggested that L-, N-, and P-type currents boosted regenerative activation in the transient ITS component.

Figure 8C shows differential voltage inactivation in the ITS components. In control solution, changing the holding membrane potential from -85 to -65 mV caused a greater inactivation in the transient ITS component than in the subsequent protracted depolarization (-54 ± 12% imax vs. -14 ± 9% ipr change, n = 5, P < 0.01). This difference was even larger when ITS elicited from -85 mV were compared with ITS elicited from -58 mV (-97 ± 6% imax vs. -23 ± 8% ipr change, n = 5, P < 0.01). After perfusing a solution containing 5 µM omega -CTX GVIA, 10 µM nifedipine, and 300 nM omega -Aga IVA, the transient ITS component still showed voltage-dependent inactivation. The transient RITS was therefore more voltage inactivated than protracted L-, P-, and N-type currents.

HTS pharmacology

The pharmacology of HTS was investigated from a membrane potential of around -50 mV in the presence of 20 mM TEA. The application of 5 µM omega -CTX GVIA markedly and irreversibly inhibited HTS (Fig. 9, n = 15). The residual action potential could activate repeatedly, suggesting that the residual Ca2+ current is incompletely voltage inactivated at potentials as high as -30 mV. A subsequent application of 10 µM nifedipine, 300 nM omega -Aga IVA, and 5 µM omega -CTX MVIIC reduced the efficiency of depolarizing pulses (Fig. 6, n = 5), as expected from the action of these drugs on protracted depolarizations (see previous section). Neither of these drugs, however, could suppress the residual action potentials, which were blocked finally by 1 mM Ni2+ (n = 12). The constant observation of a conspicuous HTS component insensitive to omega -CTX GVIA, nifedipine, omega -Aga IVA, and omega -CTX MVIIC suggested activation of putative R-type channels different from those involved in ITS, which we shall define as RHTS.


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FIG. 9. HTS pharmacology. In this and similar experiments, membrane potential was maintained at around -50 mV for 5 s to inactivate the transient ITS component before activating ITS (inset). Experimental tracings illustrate sequential application of 5 µM omega -CTX GVIA, 10 µM nifedipine + 300 nM omega -Aga IVA + 5 µM omega -CTX MVIIC, and 1 mM Ni2+ in solutions containing 1 µM TTX and 20 mM TEA. A conspicuous HTS remained after coapplication of the organic Ca2+ channel blockers. Full HTS block was obtained with Ni2+ perfusion (note that no regenerative membrane depolarizations were observed despite the current intensity was increased). Cell recorded at P13.

Ca2+ channel activation in Na+ spikes

Activation of Ca2+ channels in granule cells with fast repetitive Na+ spikes was investigated at P19-P21, when the probability of recording cells with a mature firing pattern was high. As noted in Fig. 2B, these granule cells did not show any ITS or HTS after Na+ spike blockage by 1 µM TTX. However, a HTS was revealed on applying 20 mM TEA. This HTS was inhibited strongly by 5 µM omega -CTX GVIA (Fig. 10A, n = 9), and subsequent application of 10 µM nifedipine, 300 nM omega -Aga IVA, and 5 µM omega -CTX MVIIC had no noticeable effect.


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FIG. 10. Ca2+ channel activation in Na+ spikes. A: after having blocked Na+-dependent firing with 1 µM TTX, a HTS was revealed by 20 mM TEA application. This HTS was inhibited strongly by 5 µM omega -CTX GVIA. A set of tracings is shown from a hyperpolarized holding potential to demonstrate the absence of ITS. - - -, obtained after a subsequent application of 10 µM nifedipine + 300 nM omega -Aga IVA + 5 µM omega -CTX MVIIC. B: spike afterhyperpolarization was reduced by 5 µM omega -CTX GVIA. No greater inhibition was obtained by subsequent 1 mM Ni2+ application (- - -). C: application of 5 µM omega -CTX GVIA increased the firing frequency. Cells recorded at P19-P20.

The role of Ca2+ currents in the fast Na+-dependent spiking discharge was tested by application of 5 µM omega -CTX GVIA. omega -CTX GVIA caused a reduction in spike afterhyperpolarization (AHP; n = 5; Fig. 10B). As expected from the experiments on HTS inhibition shown in Fig. 10A, no noticeable enhancement of omega -CTX GVIA action was observed following a further application of 1 mM Ni2+. The N-type channel enhancement of AHP probably depended on the activation of Ca2+-dependent repolarizing currents (Gabbiani et al. 1994). In association with AHP reduction, omega -CTX GVIA increased spike frequency (n = 5; Fig. 10C), indicating an important role for the N-type channels in controlling granule cell coding properties during repetitive discharge.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

We have investigated the excitable and synaptic properties of granule cells in the internal granular layer of developing rat cerebellum using patch-clamp whole cell recordings in acute slice preparations (Edwards et al. 1989). During the first three postnatal weeks, when migration and major developmental changes take place (Altman 1972), nearly 50% of granule cells generated Ca2+ action potentials. The Ca2+ action potentials depended on L-, N-, P-, and putative R-type Ca2+ channel activation, concurring to generate depolarizations with different threshold, kinetics, and voltage sensitivity. These properties were no longer observed after the third postnatal week, when granule cells show fast repetitive Na+-dependent firing (D'Angelo et al. 1995), and Ca2+-dependent regulation of excitability depends prominently on N-type channels. Therefore, consistent with changes in Ca2+ currents (Rossi et al. 1994), the Ca2+ action potentials appeared as a property of granule cells at immature developmental stage. The Ca2+ action potentials could be activated synaptically by mossy fiber stimulation, revealing a critical role for NMDA receptors in Ca2+ action potential generation.

Synaptic activation of Ca2+ action potentials

Ca2+ action potentials consisted of a spike termed intermediate threshold (ITS), because it activates at potentials (-60/-50 mV) laying between those typical of low-threshold spike (LTS) and HTS (Jahnsen and Llinas 1984; Llinas and Yarom 1981; McCormick et al. 1992), and of a HTS, activating at more than -30 mV. Both ITS and HTS comprised transient and protracted Ca2+ channel-dependent depolarizations. ITS was less affected by repolarizing K+ currents, and ITS transient phase showed more marked voltage- and time-dependent inactivation than HTS In inferior olivary (Llinas and Yarom 1981) and thalamic relay neurons (Jahnsen and Llinas 1984), LTS is somatic, whereas HTS originates from remote dendritic regions. Dendritic channel activation (Spruston et al. 1995) may generate local current flows, causing an apparent increase in spike threshold and inducing repetitive spike activation. However, the granule cell is electrotonically compact and behaves like a lumped somato-dendritic compartment (D'Angelo et al. 1993, 1995; Silver et al. 1992, 1996), so that Ca2+ channel location is unlike to remarkably influence the generation of Ca2+ action potentials.

The granule cells did not generate spontaneous membrane potential oscillations. Oscillatory activity was not observed in the cell-attached or perforated-patch configuration (E. D'Angelo, G. De Filippi, P. Rossi, and V. Taglietti, unpublished observation), ruling out the possibility that autorythmicity was disrupted by cytoplasmic changes caused by the pipette solution. The natural stimulus for Ca2+ action potential activation probably is provided by mossy fiber activity. Synaptic activation of Ca2+ action potentials depended on NMDA receptors, although non-NMDA receptors contributed to synaptic depolarization. The requirement for NMDA receptor activation was related to the slow kinetics and regenerative behavior of the NMDA current, which increases and lasts longer the more the membrane is depolarized (D'Angelo et al. 1994b). Thus the NMDA receptors provided the protracted inward current required to sustain the transient phase of ITS, which was not self-regenerative. During repetitive mossy fiber activity, NMDA receptors sustained temporal summation of impulses at a frequency as low as 5 Hz, raising membrane potential into the range required for generating protracted Ca2+ channel-dependent depolarizations.

An important determinant of Ca2+ action potential generation was the granule cell membrane potential. On the one hand, ITS was inactivated strongly by membrane depolarization, and ITS repriming required several hundred milliseconds at a rather negative membrane potential (typically lower than -65 mV). On the other hand, HTS still could be activated from membrane potentials causing ITS inactivation (see Fig. 5). Another critical factor was the pattern of mossy fiber stimulation. Our present as well as previous results (D'Angelo et al. 1995) suggest that a small number (usually 1-4) of mossy fibers could be recruited by increasing the stimulus intensity (see Fig. 4). Accordingly, ITS generation should require synchronous activity in at least two mossy fibers, and the recruitment of additional mossy fibers should activate the whole ITS/HTS complex. During repetitive mossy fiber stimulation, the response saturated, rendering the granule cell refractory to any subsequent input. Response saturation might be prevented by the repolarizing action of Golgi cell inhibitory postsynaptic potentials (Brickley et al. 1996; E. D'Angelo, P. Rossi, Armano, and V. Taglietti, unpublished observations), allowing ITS recovery from inactivation and subsequent rebound ITS activation (cf. Fig. 5A). Therefore inactivating properties may play an important role in relating ITS to inhibitory activity in Golgi cell fibers (Eccles et al. 1967; Shimono et al. 1976).

Multiple Ca2+ channels in ITS and HTS

The analysis of ITS and HTS inhibition by nifedipine, omega -CTX GVIA, omega -Aga IVA, and omega -CTX MVIIC suggested a selective involvement of L-, N-, P-, and putative R-type Ca2+ channels. Dissection of Ca2+ currents in culture also has suggested the expression of Q-type channels (Randall and Tsien 1995), an alpha 1A subunit-related channel that has been expressed previously in Xenopus oocytes (Sather et al. 1993). A distinctive property of the Q channel is the irreversible high-affinity block by omega -CTX MVIIC. In ITS, however, the omega -CTX MVIIC block was readily reversible and may affect a fraction of the RITS current. It should be noted that a putative R-type current has been reported in granule cells in culture; this current was blocked partially and reversibly by omega -CTX MVIIC (Ellinor et al. 1993). Ellinor's et al. (1993) R-type current showed activation threshold, voltage-dependent inactivation, and decay kinetics compatible with RITS and with the transient non-L, non-N, non-P current component specific to immature granule cells (Rossi et al. 1994). RITS may be composite itself, including the G2 and G3 channels that have been identified recently in granule cells in culture (Pietrobon et al. 1996; Tottene et al. 1996). The insensitivity to amiloride differentiates RITS from T-type channels expressed in other immature neurons (McCobb et al. 1989; Yaari et al. 1987), although similarities of R-type and T-type channel functional properties have been reported (Carbone et al. 1996). Not only an ITS but also an HTS component may reflect activation of R-type Ca2+ channels. RHTS showed poor voltage inactivation and high apparent activation threshold (more than -30 mV), therefore differing from RITS. RHTS also differed from Q-type channels, which are inactivated almost completely at -50 mV and are sensitive to omega -Aga IVA and omega -CTX MVIIC (see Carbone et al. 1996).

The multiple Ca2+ channels provided Ca2+ action potentials with complex regulatory properties. The N-, L-, and P-type channels generated protracted depolarizations, whereas the RITS and RHTS channels generated transient depolarizations with different activation thresholds. The activation mechanism of ITS was particularly interesting. RITS channels had more pronounced voltage-dependent inactivation than the other channels, allowing selective modulation of ITS generation depending on the cell membrane potential. Moreover, N-, L-, and P-type channel currents enhanced regenerative RITS-channel activation, reinforcing a cooperative mechanism initiated by the NMDA channel current at the synapse. In addition to having different gating and kinetics properties, the multiple Ca2+ channels provide targets for chemical modulation (Amico et al. 1995; Haws et al. 1993) suitable for changing the efficiency of ITS and HTS generation.

Development of granule cell electroresponsiveness

The N-type channels play a relevant role in granule cell migration (Komuro and Rakic 1993) and are maintained thereafter. A role for L-, P-, and putative R-type Ca2+ channels becomes apparent in postmigratory cells with the generation of Ca2+ action potentials (Ca2+ action potentials are not usually observed in premigratory cells) (D'Angelo and Rossi, unpublished observation). Ca2+ action potentials then are observed throughout the first three postnatal weeks, probably reflecting continuous transformation of neurons migrating from the external to the internal granular layer.

The disappearance of Ca2+ action potentials after P21 (D'Angelo et al. 1995), together with the observation of Na+ action potentials at the beginning of the migration time, suggest that granule cell firing properties develop quickly (1-2 days) from a Ca2+-dependent to a Na+-dependent mode, as demonstrated in other neurons (Baccaglini and Spitzer 1977; Spitzer 1991). The development from Ca2+- to Na+-dependent action potentials involved multiple concerted changes in membrane currents, including the disappearance of L-, P-, and putative R-type Ca2+ currents, and an increase in Na+ and K+ currents (D'Angelo et al. 1994a). Overlap of different phases of channel expression may explain the firing patterns comprising both the Ca2+ and Na+ action potentials. At a mature stage of development, Ca2+ channels contributed to regulating Na+-dependent firing, probably through a Ca2+-dependent activation of K+ currents (Gabbiani et al. 1994). Most of the Ca2+-dependent effects depended on the N-type current (Rossi et al. 1994). We cannot rule out the possibility that a small proportion of non-N channels is also present (Westenbroek et al. 1995), although their functional role remains to be established. Other types of Ca2+ channels are located at the parallel fiber synapses, controlling neurotransmitter release (Mintz et al. 1995).

Development of mossy fiber transmission

Simultaneous to action potential changes, major changes occurred in mossy fiber activation of granule cells. Granule cells generating Ca2+ action potentials showed slower EPSPs than those generating Na+ action potentials. The slower EPSP time course probably was related to a greater membrane time constant, depending both on the absence of inward rectification and a greater membrane capacitance. Moreover, contribution of a slow NMDA relative to a fast non-NMDA receptor-mediated component was greater in granule cells generating Ca2+ than Na+ action potentials, probably reflecting the developmental changes in synaptic currents reported previously (D'Angelo et al. 1993). Changes in NMDA current kinetics accompanying developmental substitution in NMDA receptor subunits (Ebralidize et al. 1996; Takahashi et al. 1996; Vallano et al. 1996) also may contribute to the observed differences in synaptic depolarization, although a correlation between expression of synaptic and membrane channels remains to be demonstrated.

Conclusions

Synaptic transmission at the mossy fiber synapse activated Ca2+ action potentials in granule cells at an immature developmental stage. This excitable response required NMDA receptor activation and involved the opening of L-, N-, P-, and putative R-type Ca2+ channels. The cooperative interaction of NMDA and Ca2+ channels in regulating granule cell synaptic excitation may be extended by considering that NMDA and Ca2+ channels are the two major pathways for Ca2+ influx through the neuronal membrane. Synaptic activation of Ca2+ action potentials, by amplifying NMDA receptor-mediated Ca2+ influx, may generate a network-dependent signal suitable for influencing granule cell development and plasticity (Garthtwaite 1994; Spitzer 1991). At a mature stage, most of the Ca2+ channel-dependent effects were due to N-type channels, which promoted Na+ spike repolarization regulating granule cell spike-frequency coding. An increasing proportion of functionally mature granule cells helped explaining the improvement of mossy fiber efficiency to relay information to Purkinje cells observed in vivo during the first three postnatal weeks (Puro and Woodward 1977; Shimono et al. 1976).

    ACKNOWLEDGEMENTS

  We thank Dr. M. Toselli and D. Pietrobon for discussion of an early version of the manuscript. This work was supported by grants of the Ministero dell' Università e della Ricerca Scientifica e Tecnologica, Consiglio Nazionale delle Ricerche, and Istituto Nazionale Fisica della Materia. P. Rossi was partially supported by Telethon Grant E0.464.

    FOOTNOTES

  Address for reprint requests: E. D'Angelo, Istituto di Fisiologia Generale, Via Forlanini 6, I-27100 Pavia, Italy.

  Received 13 September 1996; accepted in final form 3 June 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society