The Journal of Neurophysiology Vol. 78 No. 5 November 1997, pp. 2269-2279
Copyright ©1997 by the American Physiological Society

Properties of Calcium Spikes Revealed During GABA_A Receptor Antagonism in Hippocampal CA1 Neurons From Guinea Pigs

Masami Miura¹, Masatomo Yoshioka¹, Hiroyoshi Miyakawa², Hiroshi Kato¹, and Ken-Ichi Ito¹

¹ Department of Physiology, Yamagata University School of Medicine, Yamagata 990-23; and ² Laboratory of Life Science, School of Life Science, Tokyo College of Pharmacy, Hachioji 192-03, Japan

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Miura, Masami, Masatomo Yoshioka, Hiroyoshi Miyakawa, Hiroshi Kato, and Ken-Ichi Ito. Properties of calcium spikes revealed during GABA_A receptor antagonism in hippocampal CA1 neurons from guinea pigs. J. Neurophysiol. 78: 2269-2279, 1997. Intracellular electrical responses and changes in intracellular calcium concentration ([Ca²⁺]_i) in response to activation of synaptic inputs and to DC injections were recorded simultaneously from CA1 pyramidal neurons (n = 42) in guinea pig hippocampal slices. In the presence of the -aminobutyric acid-A (GABA_A) receptor antagonists, bicuculline (25 µM) and picrotoxin (10 µM), broad (>20 ms) all-or-none spikes were induced by activation of synaptic inputs (20 pulses, 30 Hz) and were accompanied by a simultaneous rapid and large rise in [Ca²⁺]_i (34 of 34 cells). By contrast, direct depolarizing current (0.7 nA, 1 s) induced spikes having short duration, during which time the spike firing pattern was observed not to be significantly affected. When Na⁺ channels were blocked by QX-314 applied intracellularly through the recording microelectrode in the presence of GABA_A receptor antagonists, broad spikes were frequently generated by activation of synaptic inputs (32 of 33 cells). These broad spikes were blocked by Cd²⁺ (200 µM) or in Ca²⁺-free medium (6 of 6 cells) but were resistant to either tetrodotoxin (TTX; 1 µM; 6 of 6 cells) or QX-314, whereas short-duration spikes were blocked by both TTX andQX-314. Based on these findings we conclude that broad and short-duration spikes are Ca²⁺ and Na⁺ spikes, respectively. To investigate the properties of the Ca²⁺ spikes, antagonists of a voltage-operated Ca²⁺ channel were applied to the evoked responses. Nifedipine (30 µM), a L-type Ca²⁺ channel blocker, suppressed the generation of Ca²⁺ spikes, whereas Ni²⁺ (100 µM), theT- and R-type Ca²⁺ channel blocker, and -agatoxin-IVA (-Aga-IVA, 60 nM), a P-type Ca²⁺ channel blocker, had little effect on the generation of Ca²⁺ spikes. Nifedipine suppressed the rise in [Ca²⁺]_i induced by synaptic inputs up to 26% of the control in the soma and 18-32% in the dendrites (n = 5), respectively, whereas Ni²⁺ suppressed the rise by 12-27% (n = 5) in both soma and dendrites. -Aga-IVA showed little effect (less than a 10% change; n = 7). These results suggest that the GABA_A inhibitory system tonically suppresses dendritic Ca²⁺ spikes, and the L-type Ca²⁺ channel plays a major role in the generation of Ca²⁺ spikes and in Ca²⁺ influx.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Intracellular Ca²⁺ ions play an important role in neuronal functions through Ca²⁺-dependent activation of enzymes. Therefore, to understand basic process involved in such functions, it is essential to be able to measure intracellular Ca²⁺ concentration ([Ca²⁺]_i) and its dynamic changes subsequent to neural activation. Because the rise in [Ca²⁺]_i occurs through various pathways, such as voltage-operated Ca²⁺ channels (VOCCs) in the plasma membrane (Christie et al. 1995; Miyakawa et al. 1992; Regehr and Tank 1992) or IP₃ receptors located on the endoplasmic reticulum in the cytoplasm (Berridge 1993; Shirasaki et al. 1994), it is also important to examine which types of VOCCs are involved in the elevation of [Ca²⁺]_i, as well as in regional changes in [Ca²⁺]_i within a neuron.

A fluorometric method using a fluorescent Ca²⁺ indicator, fura-2, is now widely accepted for the measurement of [Ca²⁺]_i in single neurons (Jaffe et al. 1992; Lasser-Ross et al. 1991). The long-lasting rise in [Ca²⁺]_i evoked by synaptic inputs in hippocampal CA1 neurons has been shown to depend primarily on the generation of Na⁺ spikes (Miyakawa et al. 1992; Regehr and Tank 1992). Alford et al. (1993) further demonstrated that D,L-2-amino-5-phosphonopropionic acid (AP5), an antagonist that selectively blocks N-methyl-D-aspartate (NMDA) glutamate receptors, inhibits the increase in [Ca²⁺]_i observed in the absence of Na⁺ spikes. On the basis of these findings, it was concluded that the elevation of [Ca²⁺]_i is mediated by VOCCs and NMDA receptor-coupled channels.

However, there is as yet no definite evidence that the large elevation of [Ca²⁺]_i corresponds to Ca²⁺ spikes observed in CA1 neurons, even though the cells have been shown electrophysiologically to be able to generate Ca²⁺ spikes (Fujita and Sakata 1962; Schwartzkroin and Slawsky 1977; Wong et al. 1979). It may be that the lack of such evidence stems from the difficulty in observing Ca²⁺ spikes under normal recording conditions. If reproducible Ca²⁺ spikes are generated under certain experimental conditions, the role of VOCCs in the production of Ca²⁺ spikes could be examined readily by simultaneous recording of electrical events and [Ca²⁺]_i by fluorometric methods.

The activation of -aminobutyric acid-A (GABA_A) receptors reduces Ca²⁺ influx into CA1 neurons induced by application of high K⁺, therefore Ca²⁺ entry through VOCCs may be regulated by a GABA_A receptor-coupled mechanism (Wadman and Connor 1992). In the present study, we first tested for the participation of GABA_A receptors in the generation of Ca²⁺ spikes. Because we could induce broad and all-or-none spikes under conditions of block of GABA_A receptors, and these spikes had been shown to be Ca²⁺ spikes based on their properties, we further examined the extent of the contribution of each type of VOCC to the rise in [Ca²⁺]_i during Ca²⁺ spikes.

	METHODS

Abstract Introduction Methods Results Discussion References

Slices (500 µM) were obtained from the hippocampus of guinea pigs (220-350 g) using a Rotor Slicer (Dosaka EM, DTY-1000), and were incubated at least 1 h in an artificial cerebrospinal fluid (aCSF) consisting of (in mM) 124 NaCl, 5.0 KCl, 2.5 CaCl₂, 2.0 MgCl₂, 22 NaHCO₃, 1.25 NaH₂PO₄, and 10 glucose, the latter of which was well aerated with a gas mixture of 95% O₂-5% CO₂. The temperature of the medium was maintained at 30-31°C (Fujii et al. 1991; Miyakawa and Kato 1986).

As shown in Fig. 1A, a stimulating electrode (Stim.) consisting of two cashew-coated tungsten wires was placed in the stratum radiatum of the CA1/CA2 region to synaptically activate CA1 pyramidal neurons through Schaffer collateral/commissural pathways (SC). A recording glass micropipette (Rec.) was used for impalement of the soma of a CA1 pyramidal neuron. The tip of the recording micropipette was filled with fura-2 (6 mM), a fluorescent Ca²⁺ dye, and then back-filled with K-acetate (3 M). Inmost experiments, 25-50 mM N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314, Research Biochemicals, Natick, MA), a Na⁺ channel blocker (Connors and Prince 1982; Hille 1977; Strichartz 1973), was dissolved in the fura-2 solution and was used to fill the tip of the microelectrode (80-120 M). The QX-314 in the tip of the microelectrode was loaded into the cell by applying depolarizing pulses of 0.2 nA for 0.2 s at 1 Hz for 5 min.

View larger version (37K): [in this window] [in a new window]   FIG. 1. Experimental methods for recording electrical responses and the change in [Ca2+]i. A: schematic view of a hippocampal slice. Stimulating electrode (Stim.) was placed in stratum radiatum of CA1 region (CA1) to provide synaptic stimulation to CA1 pyramidal neurons through Schaffer collaterals (SC). Recording electrode (Rec.), containing calcium-sensitive fluorescent dye, fura-2, was placed in stratum pyramidale (p) and was used for impalement of the soma of a CA1 pyramidal neuron. B: diagram of experimental system. Hippocampal slice (S) was placed in the chamber on the stage of an upright microscope and perfused with the medium by a constant flow pump. Intracellular responses to synaptic stimulation were recorded by microelectrode. These were amplified, analyzed, and stored by computer. To examine the change in [Ca2+]i in the impaled neuron, excitation light (380 nm) was produced by a Xenon lamp, and the emission light was detected with a cooled charge coupled device (CCD) camera attached to the top of a microscope. Data were also analyzed and stored by computer. C: an example of representative data. Left panel: a fine-grain picture of a stained pyramidal neuron observed microscopically that was displayed on the computer screen. The 4 boxes indicate the regions where the changes in [Ca2+]i were recorded: basal dendrite (BD), soma (Soma), proximal apical dendrite (PAD), and distal apical dendrite (DAD). Bottom right panel: intrasomatic electrical responses to DC injection are illustrated. Top right panel: %change in fluorescence (F/F) in 4 regions corresponding to each region shown in the left panel. Similar plots are used for subsequent figures to express the change in parameters using the identical protocol.

Intracellular potentials were recorded every 30 s, amplified(Axoclamp 2B, Axon Instrument, Foster City, CA), converted from A/D (PC-9800, NEC, Tokyo), and stored on a microcomputer. The resting membrane potential (V_m) of neurons analyzed in this experiment was more negative than 50 mV; the mean value of V_m was 67 mV (n = 56), and the membrane resistance (R_m) ranged between 38 and 58 M; the mean value of R_m was 45.3 M (n = 50). Changes in [Ca²⁺]_i were also recorded, analyzed, and stored every 5-10 min by computer as explained in the following section. Two types of stimuli, trans-synaptic and DC stimulation, were used one after the other at intervals of 30 s. The former includes a train of pulses that consists of 20 pulses at 30 Hz with a pulse duration of 0.2 ms and a pulse intensity of 0.1-0.3 mA applied through the stimulating electrode. As for the latter, a depolarizing current of 0.7 nA for 1 s was injected through the microelectrode in the cell.

As shown in Fig. 1B, one of the incubated slices (S) was placed in a fixed location in the chamber on the stage of an upright microscope (Optiphoto, Nikon) and was perfused with the aCSF by the aid of a constant-flow pump. After impaling a CA1 pyramidal neuron with a microelectrode, fura-2 was loaded iontophoretically for 10-20 min with a steady hyperpolarizing current of0.2-0.4 nA so as to fill the cell as far into the fine dendrites as possible (Jaffe et al. 1992; Miyakawa et al. 1992). Most impaled neurons were located 50-100 µm from the surface of the slice. The shape of the loaded neuron was displayed on a computer screen through a water immersion lens (×25, 0.8 NA) and a cooled charge coupled device (CCD) camera (CC200, Photometrics). After confirming the staining of the cell with fura-2 including the fine branches of dendrites, the hyperpolarizing current was slowly removed so as not to trigger spontaneous action potentials. Changes in [Ca²⁺]_i were measured by a cooled CCD camera detecting the emission of fura-2 fluorescence following excitation at a wavelength of 380 nm by a Xenon lamp. The CCD camera was operated in sequential transfer mode (Lasser-Ross et al. 1991). The frame interval was 20 ms. The impaled and stained neuron was exposed to ultraviolet light only during the measurement of [Ca²⁺]_i to prevent photic damage of neurons.

The [Ca²⁺]_i measurements were performed in four regions that were designated on the screen by the aid of the computer (Fig. 1C, rectangles at left): 1) the basal dendrites ~50 µm from the soma (BD), 2) the soma (Soma), 3) the proximal apical dendrites ~100 µm from the soma (PAD), and 4) the distal apical dendrites ~200 µm from the soma (DAD). The correction for background fluorescence was performed by subtraction of the background levels, obtained by taking the mean of the values of two areas unloaded with fura-2 (around 20 × 20 µm²) in each frame, away from the original fluorescence image. This is necessary because the intensity of the background fluorescence gradually increased during the acquisition of data over the time course of a recording session that typically lasted 2-3 h. The change in [Ca²⁺]_i was expressed as the %ratio of F₃₈₀/F₃₈₀, where F₃₈₀ was the intensity of fluorescence to 380 nm excitation and F₃₈₀ was the change from F₃₈₀ during stimulation (Fig. 1C, right).

AP5 (50 µM, Tocris Neuramin, Bristol, UK), an NMDA receptor antagonist, was used in all experiments and was continuously perfused throughout each experiment to minimize the Ca²⁺ influx through NMDA receptors. To block GABA_A receptors, ()-bicuculline methiodide (BMI, 25 µM, Research Biochemicals, Natick, MA) and picrotoxin (PTX, 10 µM, Extrasynthese, Genay, France) were simultaneously administered. Four kinds of VOCC blockers were applied via the perfusion aCSF: nifedipine (30 µM, Sigma, St. Louis, MO) for L-type channels, -Agatoxin-IVA (-Aga-IVA, 60 nM, Peptide Institute, Osaka, Japan) for P-type channels, NiCl₂ (100 µM) for T- and R-type channels, and CdSO₄ (200 µM) as a general blocker of all types of VOCC channels. The perfusion rate was set at 2-3 ml/min, so that the solution in the recording chamber was replaced within 5 min.

	RESULTS

Abstract Introduction Methods Results Discussion References

To investigate the properties of Ca²⁺ spikes and the rise in [Ca²⁺]_i, three experimental strategies were adopted by the administration of three kinds of drugs. First, GABA_A receptor antagonists, BMI and PTX, were added to the aCSF to eliminate the influence of the inhibitory system that limits the transient increase in [Ca²⁺]_i (Callaway et al. 1995; Wadman and Connor 1992). Second, the intracellular Na⁺ channel blocker, QX-314, was added to the fura-2 containing solution and was filled into the micropipette so that the transient increase in [Ca²⁺]_i triggered by Na⁺ spikes would be blocked (Jaffe et al. 1992; Miyakawa et al. 1992). Third, specific blockers of VOCCs were added to the aCSF to evaluate the contribution of each type of VOCC to the [Ca²⁺]_i elevation related to Ca²⁺ spikes. One of the VOCC blockers was added to the perfusion aCSF, which contained BMI and PTX as well as AP5 (50 µM). The pathways for Ca²⁺ influx were mainly limited to VOCCs, because Ca²⁺ influx triggered by Na⁺ spikes and through NMDA receptor-coupled channels was blocked by QX-314 and AP5, respectively.

Forty-two cells were investigated in total, and typical responses are shown in Fig. 2. Simultaneously recorded responses showing the changes in [Ca²⁺]_i (top 4 traces) and electrical potentials (bottom trace in each panel) obtained from two different neurons are depicted, in which intracellular recordings were carried out from a neuron shown in top panels (A-D) without the injection of QX-314, and from the other neuron (E-H) in which QX-314 was injected. In both neurons, responses to synaptic input and to direct depolarizing current injection are shown on the right and left, respectively, each of which consists of responses before (Control), and after blocking GABA_A receptors by application of both BMI and PTX.

View larger version (24K): [in this window] [in a new window]   FIG. 2. Examples of responses from 2 neurons (top panels in A-D, and bottom panels in E-H) for changes in [Ca2+]i (top 4 traces in each panel) and electrical responses (bottom trace) to synaptic inputs (left panel of A, B, E, and F, indicated with a bar in the bottom of each panel) and to depolarizing current injection (C, D, G, and H, indicated with a bar at the bottom). The neuron for A-D has 68 to 70 mV of Vm and 55 M of Rm. The other neuron for E-H has 68 to 73 mV of Vm and 38 M of Rm. Four traces show an increase in F/F in the regions corresponding to basal dendrites (BD; dotted line in narrow space), soma (Soma; line), proximal apical dendrites (PAD; broken line), and distal apical dendrites (DAD; dotted line in wide space). For the neuron represented by the data of the top panels, a fura-2-loaded microelectrode was used, whereas for the neuron represented by the data of bottom panels, QX-314 was further added to the microelectrode to block Na+ channels. After recording the responses of A and E, and C and G, bicuculline (BMI) and picrotoxin (PTX) were added to the perfusate. Thereafter, responses B and F, and D and H were recorded. For each neuron, a pair of recordings (e.g., A and C) were made almost at the same time, within a few minutes. A: electrical and [Ca2+]i responses evoked by synaptic stimulation of 20 pulses at 30 Hz. B: responses after the application of BMI and PTX to the same stimulation as in A. E: in the presence of QX-314, electrical and [Ca2+]i responses induced by the synaptic stimulation. F: furthermore addition of BMI and PTX, electrical and [Ca2+]i responses. C, D, G, and H: responses to the depolarizing current injection. Calibration bars: 500 ms refer to all F/F traces, and 50 mV and 500 ms refer to all electrical traces.

As shown in Fig. 2, A and C, the neuron showed a rise in [Ca²⁺]_i in accompaniment to fast and short-duration spikes in response to synaptic inputs (A) and to DC injection (C). These results are in good agreement with those of previous studies by Jaffe et al. (1992) and Miyakawa et al. (1992), who showed that the rise in [Ca²⁺]_i corresponds to Na⁺ spikes in the soma and dendrites.

In contrast, the neuron loaded with QX-314 failed frequently to generate short-duration spikes to both synaptic and direct stimulation (Fig. 2, E and G). Instead, synaptic inputs evoked only one short-duration spike with a small change in [Ca²⁺]_i (Fig. 2E), and a depolarizing current injection elicited a broad spike with a steep rise in [Ca²⁺]_i (Fig. 2G; n = 34).

After recording these responses, antagonists of GABA_A receptors, BMI (25 µM) and PTX (10 µM), were administered. In the case of the neuron shown at the top of the figure, synaptic inputs enhanced the rise in [Ca²⁺]_i in all regions and produced a long-lasting large depolarization on which broad spikes appeared together with short-duration spikes; the number of short-duration spikes was suppressed (Fig. 2B). In the case of the bottom neuron injected with QX-314, synaptic input produced an additional two broad spikes and prominent changes in [Ca²⁺]_i (Fig. 2F; 32 of 33 cells) in the presence of BMI and PTX, whereas depolarizing current injection caused no significant change in both [Ca²⁺]_i or in the response of the broad spike as shown in Fig. 2H (compare H with G). These data suggest that the inhibitory synaptic input sensitive to BMI and PTX usually suppresses the appearance of broad spikes and suppresses [Ca²⁺]_i elevation elicited by synaptic activation in CA1 pyramidal neurons, even though these cells appear to have an intrinsic Ca²⁺ spike.

Figure 3 illustrates representative recordings from two different neurons (top panels of A and B, and bottom panels of C and D) during administration of BMI, PTX, QX-314, and AP5, showing a train of broad spikes with a short-duration spike at the beginning, and a stepwise increase in [Ca²⁺]_i in response to a depolarizing current injection (Fig. 3, A and C). Tetrodotoxin (TTX; 1.0 µM; n = 6) completely eliminated the short-duration spike but caused no significant change in the broad spikes or in the sharp, stepwise rise in [Ca²⁺]_i, as shown in Fig. 3B. By contrast, Cd²⁺ (200 µM), a nonspecific blocker of VOCCs, or Ca²⁺-free medium eliminated broad spikes (n = 6), leaving a short-duration spike at the beginning and abolished the stepwise rise in [Ca²⁺]_i seen in Fig. 3C (Fig. 3D). When synaptic inputs were activated at threshold levels (0.13 mA, 20 pulses at 30 Hz), the broad spike was observed to be all-or-none in nature: a short-duration spike followed by a broad spike and the rise in [Ca²⁺]_i was induced (Fig. 4A). On other occasions in the same cell, when the same intensity of stimulation produced only a short-duration spike, there was no corresponding rise in [Ca²⁺]_i observed (Fig. 4B). Thus, in the following text, we refer to the short-duration and the broad spike as Na⁺ and Ca²⁺ spikes, respectively.

View larger version (16K): [in this window] [in a new window]   FIG. 3. A: [Ca2+]i elevation and broad spikes in precise temporal correspondence induced by injection of depolarizing current in the presence of BMI, PTX, and QX-314. Vm = 67 mV, Rm = 41 M. B: elevation of [Ca2+]i and broad spikes were resistant to TTX, whereas the Na+ spike appearing at the beginning of the response was blocked. C and D: in a different neuron from A and B, the effect of Cd2+ on both elevation of [Ca2+]i and broad spikes.Vm = 65 mV, Rm = 53 M.

View larger version (11K): [in this window] [in a new window]   FIG. 4. Responses of [Ca2+]i and broad spikes to synaptic stimulation at threshold intensity, recorded in the same neuron. A: both short-duration and broad spikes. B: only short-duration spike. Vm = 70 mV, Rm = 45 M.

The relationship between the generation of Na⁺ or Ca²⁺ spikes and the rise in [Ca²⁺]_i was examined by expanding the time scale of both types of recording. As is shown in Fig. 2, E and F, a Na⁺ spike was associated with a small rise in [Ca²⁺]_i, and two Ca²⁺ spikes with a steep and large rise in [Ca²⁺]_i. The two Ca²⁺ spikes in F correspond to the first two peaks appearing in the rising phase of the F/F traces within 20 ms; a "nick" on the rising phase in the [Ca²⁺]_i traces corresponds to the time between the two Ca²⁺ spikes. It is clearly seen in Fig. 3, A-C, that the stepwise rise in [Ca²⁺]_i corresponds to the occurrence of Ca²⁺ spikes. These findings indicate that the increase in [Ca²⁺]_i during the Ca²⁺ spike is much greater than that associated with the Na⁺ spike (compare Fig. 2, E with F, and Fig. 4, A with B).

Because Ca²⁺ spikes were readily induced by activation of synaptic inputs or by depolarizing current injections during the block of GABA_A receptors and of Na⁺ spikes, we examined the effects of selective inhibitors of various types of VOCCs involved in Ca²⁺ spikes during the above experimental conditions (Fig. 2, D and H)

Data representative of the effects of blockers on the responses are shown in Figs. 5-7, in which the responses to synaptic input or to DC stimulation are depicted in the top and bottom panels, respectively. Both panels show the response before (control, left) and after (right) application of each VOCC blocker. Figure 8 summarizes the suppressant effects of each VOCC blocker on transient [Ca²⁺]_i in response to activation of synaptic input (A) and of depolarizing current injection (B). The effects of the blocker were expressed as the %change of control of the magnitude of the first peak of [Ca²⁺]_i accompanied by the first Ca²⁺ spike, so as to avoid measuring errors related to the different numbers of spikes or different intervals of spikes. If a stimulation produced multiple Ca²⁺ spikes, the first [Ca²⁺]_i peak was judged by the nick indicated by the arrow in the trace of the maximal response in four regions of Figs. 5-7. After impalement with the micropipette containing QX-314 to block the Na⁺ spike, iontophoretic ejection was performed in the neuron, and BMI and PTX were perfused in the aCSF throughout this experiment. None of the calcium channel blockers at the concentrations tested altered excitatory postsynaptic potentials elicited by synaptic activation, which was subthreshold for elicitation of an action potential, suggesting that these blockers could not significantly affect synaptic transmission.

View larger version (18K): [in this window] [in a new window]   FIG. 5. Effects of nifedipine on Ca2+ spikes and on the rise in [Ca2+]i evoked by synaptic stimulation (A and B) and by depolarizing current injection (C and D).Vm = 66 to 73 mV, Rm = 38 M.

View larger version (24K): [in this window] [in a new window]   FIG. 8. Summary of the suppressant effects of voltage-operated Ca2+ channel (VOCC) blockers on [Ca2+]i evoked by synaptic stimulation (A), and by depolarizing current injection (B). The mean percent for the suppression of F/F with SE (vertical small bars) was plotted against the distance of the interested regions from the soma segmented by 50 µm. The negative numbers of the abscissa refer to the basal dendrites and positive to the apical ones. The suppressant effects of each blocker were expressed by the %change from control of the magnitude of the early peak of F/F accompanied by Ca2+ spikes. *Significant difference (P < 0.05) of suppressant effects between control and each region and between the soma.

Nifedipine (30 µM), a specific blocker of L-type channels, suppressed the number of Ca²⁺ spikes and inhibited the [Ca²⁺]_i elevation evoked by synaptic stimulation in the soma by 26.2 ± 4.9% (mean ± SE, n = 5) and in the dendrites by 18-32% (Figs. 5B and 8A). Nifedipine decreased [Ca²⁺]_i in the distal dendrites significantly less than in the soma (Fig. 8A). For the response to DC injection, nifedipine decreased the number of Ca²⁺ spikes (Fig. 5D) in association with a suppression of [Ca²⁺]_i elevation by 21-34% (n = 5). The magnitude of the inhibition induced by nifedipine in the soma did not differ significantly from the other neuronal regions analyzed (Fig. 8).

Ni²⁺ (100 µM), an antagonist of T- and R-type channels, did not decrease the number of Ca²⁺ spikes (Fig. 6, B and D). However, Ni²⁺ suppressed the synaptically induced [Ca²⁺]_i elevation in the soma by 27.2 ± 6.4% (n = 5) and in the apical dendrites by 12-21% (Figs. 6B and 8A), but the extent of this inhibition in the distal dendrites (>100 µm) was not significant. Ni²⁺ suppressed the [Ca²⁺]_i elevation evoked by DC injection in the soma by 6.3% ± 7.7% (n = 5), and in the dendrites by 2-9% (Figs. 6D and 8B).

View larger version (18K): [in this window] [in a new window]   FIG. 6. Effects of Ni2+ on Ca2+ spikes and on the rise in [Ca2+]i evoked by synaptic stimulation (A and B) and by depolarizing current injection (C and D). Vm = 60 mV, Rm = 40 M.

-Aga-IVA (60 nM), a specific antagonist of P-type channels, suppressed the synaptically evoked [Ca²⁺]_i elevation in the soma by 5.4 ± 6.7% (n = 7) and showed little effect in the dendrites (Fig. 7, B and D; Fig. 8, A and B). When T-, R-, L-, and P-type antagonists were applied simultaneously, Ca²⁺ transients and broad spikes still remained (data not shown).

View larger version (19K): [in this window] [in a new window]   FIG. 7. Effects of -Aga-IVA on Ca2+ spikes and on the rise in [Ca2+]i evoked by synaptic stimulation (A and B) and by depolarizing current injection (C and D). Vm = 65 to 70 mV, Rm = 40 M.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Generation of Ca²⁺ spikes and changes in [Ca²⁺]_i

In the present study, two types of spikes were identified with respect to their durations. The short-duration spike was sensitive to both TTX and QX-314. In contrast, the broad spike was resistant to TTX, but sensitive to Cd²⁺, a nonspecific VOCC antagonist. Both spikes appeared to have all-or-none properties. Although the short-duration spike was accompanied by only a small increase in [Ca²⁺]_i, the broad spike was associated with a large increase in [Ca²⁺]_i that corresponded precisely in the times of their occurrences. On the basis of these findings, we conclude that the former is a Na⁺ spike and the latter is a Ca²⁺ spike. This conclusion is supported further by the evidence that 1) QX-314 had no effect on both broad spikes and on the elevation of [Ca²⁺]_i (Fig. 2, G and H) and 2) nifedipine and Ni²⁺, VOCC blockers, partially but significantly suppressed the rise of [Ca²⁺]_i associated with broad spikes (Figs. 5, 6, and 8). Although the application of QX-314 failed to eliminate the first Na⁺ spike appearing at the beginning of the response (Figs. 2-7), this could be due to the fact that QX-314 is an open Na⁺ channel blocker (Connors and Prince 1982; Hille 1977; Ragsdale et al. 1994; Strichartz 1973). Until recently, QX-314 was believed to be a specific Na⁺ channel blocker (Conners and Prince 1982; Hille 1977; Strichartz 1973). Andreasen and Hablitz (1993), however, have reported other effects of QX-314 and other derivatives of lidocaine, in that they block outward K⁺ currents, resulting in tonic depolarization. This effect of K⁺ channel block could also be helpful in producing Ca²⁺ spikes. These results support the view that hippocampal CA1 neurons have the ability to generate Ca²⁺ spikes, which is in good agreement with previous findings from electrophysiological studies (Benardo et al. 1982; Fujita and Sakata 1962; Schwartzkroin and Slawsky 1977; Wong et al. 1979).

Effects of block of GABA_A receptors on Ca²⁺ spike generation

In the hippocampal CA1 region, there are at least three types of inhibitory interneuron, and they innervate CA1 pyramidal neurons through both feed-forward and feedback pathways. The inhibitory inputs act on pyramidal neurons through GABA_A receptors. (Buhl et al. 1994; Lacaille and Schwartzkroin 1988a,b).

Miles et al. (1996) reported that inhibitory inputs block both Ca²⁺ spikes and the rise in [Ca²⁺]_i, and that bicuculline, a GABA_A receptor antagonist, eliminates these inhibitory effects. The block of GABA_A receptors in CA1 neurons facilitated Ca²⁺ spikes and enhanced [Ca²⁺]_i elevation, indicating that inhibitory interneurons are usually operative in suppressing the generation of the Ca²⁺ spike.

Although we used the [Ca²⁺]_i imaging technique employed in this study, we have not been able to determine the precise region of the neuron where the Ca²⁺ spike is generated. This is partly due to the relatively slow time resolution of [Ca²⁺]_i imaging (20 ms) and also due to the way the GABA_A receptor antagonists were administered via superfusion causing effects to be distributed across the entire surface of the neuronal membrane.

GABA_A receptor antagonists had no significant effect on the generation of Na⁺ spikes in response to DC injection (Fig. 2, C and D), but this observation could be due to the stimulating paradigm. In this condition, the involvement of the inhibitory system was minor, because the inhibitory action at the impaled cell probably operated only through a recurrent pathway.

Contribution of VOCC subtypes to the generation of Ca²⁺ spikes and the rise in [Ca²⁺]_i

To evaluate the contributions of each subtype of VOCC to Ca²⁺ spikes and [Ca²⁺]_i elevation, nifedipine, Ni²⁺, and -Aga-IVA were administered so as to block L-, T-, R-, and P-type VOCCs, respectively. In all these experiments, the pathways for Ca²⁺ influx through NMDA receptor/channels and the activation of Na⁺ spikes had been blocked by AP5 and QX-314, respectively, in addition to BMI and PTX. Thus the possible routes for Ca²⁺ influx could be, for the most part, restricted to VOCCs.

The L-type antagonist, nifedipine, suppressed the number of Ca²⁺ spikes. L-type VOCCs are considered to be involved in the generation of Ca²⁺ spikes, especially in repetitive Ca²⁺ firing due to the slow inactivating property of L-type VOCCs and the subsequent increase of Ca²⁺-dependent K⁺ conductances. The properties of L-type VOCCs and Ca²⁺ spikes have been reported in inferior olivary neurons and neocortical neurons. In the former type, high-threshold Ca²⁺ spikes lack refractoriness (Llinás and Yarom 1981). In the latter, a Ca²⁺ plateau (>200 ms) has been reported in the presence of TTX and tetraethylammonium (TEA) (Yuste et al. 1994). It is likely that nifedipine would block L-type VOCC and low-threshold VOCC, which is involved to maintain the resting level of [Ca²⁺]_i because nimodipine (similar to nifedipine) blocks low-threshold Ca²⁺ entry at resting membrane potential in CA1 hippocampal neurons (Magee et al. 1996). Our observations that the block of L-type VOCCs by nifedipine suppresses the number of Ca²⁺ spikes indicate that the L-type VOCC also exists in hippocampal pyramidal neurons.

Ni²⁺ (100 µM), had no noticeable effect on Ca²⁺ spikes in terms of their numbers, but it suppressed the rise in [Ca²⁺]_i. Because T-type VOCC has been shown to be rapidly inactivating, the block of T-type VOCCs might be expected to decrease [Ca²⁺]_i mediated by the first Ca²⁺ spike with no further modulation thereafter of Ca²⁺ spikes.

The lesser effectiveness of -Aga-IVA on Ca²⁺ spikes and [Ca²⁺]_i could be due to the reduced distribution of this type of VOCC in hippocampal neurons (Magee and Johnston 1995; Mintz et al. 1992). Despite the fact that -Aga-IVA is a large protein, it is likely that in the present study, this molecule penetrated the slices because previous studies employing the same superfusion method of -Aga-IVA administration (60 nM) to examine its effects on -burst stimulation induced LTP showed a block of synaptic plasticity using identical concentrations and procedures (Ito et al. 1995) as followed in the present study. Furthermore, even when nifedipine, Ni²⁺, and -Aga-IVA were added simultaneously in the aCSF, Ca²⁺ spikes still occurred. This observation indicates that other VOCCs exist in CA1 pyramidal neurons and contribute to the residual Ca²⁺ spikes (Fisher et al. 1990; Mills et al. 1994).

Nifedipine and Ni²⁺ affect the rise in [Ca²⁺]_i differently depending on both the region studied and the type of stimulation employed (Fig. 8, A and B). Christie et al. (1995) and Magee and Johnston (1995) have reported that L-type VOCCs were mainly located in somatic and proximal apical dendritic regions, whereas Ni²⁺-sensitive channels (of the T- and R-type) were relatively more distributed in the distal dendrites. Although the distribution of the L-type channel in our study is consistent with these reports, it is not so for the Ni²⁺-sensitive channel. This discrepancy might be caused by different types of spikes being responsible for the induced Ca²⁺ influx: we used Ca²⁺ spikes for the investigation of the VOCCs contribution to Ca²⁺ influx; however, the other studies employed Na⁺ spike-induced Ca²⁺ influx. The suppressant effect on [Ca²⁺]_i in response to activated synaptic input was pronounced at the soma, whereas the effect on [Ca²⁺]_i in response to DC injection was relatively more pronounced in distal dendritic regions. An explanation for these findings is difficult, but one possibility to account for the results is that there is a propagation of Ca²⁺ spikes from the site where they are generated, to a neighboring region, i.e., the propagation of the Ca²⁺ spike would occur from the dendrite to the soma in the case of synaptic inputs, and from the soma to the dendrites in the case of DC injection. If the propagation were blocked by a VOCC blocker, [Ca²⁺]_i would become smaller, and thus the pronounced suppressant effect would be exerted in the region where the Ca²⁺ spike is blocked. However, another possibility cannot be excluded and that is that there is a different distribution of VOCC subtypes along the surface of the membrane of the neuron.

In summary, the present study indicates first that hippocampal CA1 neurons can generate Ca²⁺ spikes, which may originate both from the dendrite and the soma; second, that a GABA_A inhibitory system normally exerts a suppressant effect on Ca²⁺ spikes in the dendrites; and third, that theL-type of VOCC is involved in the generation of Ca²⁺ spikes, whereas the contribution of T-, R-, and P-types of VOCC may be involved relatively less. Thus, in addition to the known roles of the dendrites and somata in the electrophysiological integration and modulation of signal flow through Na⁺ and K⁺ channel mechanisms, the increase in [Ca²⁺]_i caused by Ca²⁺ spikes may play important roles contributing to neuronal function, particularly with reference to such processes as synaptic plasticity, various biochemical responses, morphological changes, and in gene expression (Ghosh and Greenberg 1995).

	ACKNOWLEDGEMENTS

  We thank Dr. T. P. Hicks for correcting the English in the manuscript and for critical comments.

  This study was supported by a grant from the Naito Foundation.

	FOOTNOTES

  Address for reprint requests: K.-I. Ito, Dept. of Physiology, School of Medicine, Yamagata University, 2-2-2 Iida Nishi, Yamagata 990-23, Japan.

  Received 13 August 1996; accepted in final form 15 July 1997.

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