The Journal of Neurophysiology Vol. 80 No. 5 November 1998, pp. 2268-2273
Copyright ©1998 by the American Physiological Society

L-Type Ca²⁺ Channels Mediate the Slow Ca²⁺-Dependent Afterhyperpolarization Current in Rat CA3 Pyramidal Cells In Vitro

Mitsuo Tanabe, Beat H. Gähwiler, and Urs Gerber

Brain Research Institute, University of Zurich, CH-8057 Zurich, Switzerland

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Tanabe, Mitsuo, Beat H. Gähwiler, and Urs Gerber. L-type Ca²⁺ channels mediate the slow Ca²⁺-dependent afterhyperpolarization current in rat CA3 pyramidal cells in vitro. J. Neurophysiol. 80: 2268-2273, 1998. Single-electrode voltage-clamp recordings were obtained from CA3 pyramidal cells in rat hippocampal organotypic slice cultures, and the slow Ca²⁺-dependent K⁺ current or afterhyperpolarization current (I_AHP) was elicited with brief depolarizing voltage jumps. The slow I_AHP was suppressed by the selective L-type Ca²⁺ channel antagonists isradipine (2 µM) or nifedipine (10 µM). In contrast, neither -conotoxin MVIIA (1 µM) nor -agatoxin IVA (200 nM), N-type and P/Q-type Ca²⁺ channel antagonists, respectively, attenuated this slow outward current. The slow I_AHP was significantly reduced by thapsigargin (10 µM), a Ca²⁺ ATPase inhibitor that depletes intracellular Ca²⁺ stores, and by ryanodine (10-100 µM), which blocks Ca²⁺-induced Ca²⁺ release from intracellular compartments. At this concentration thapsigargin did not modify high-threshold Ca²⁺ current, which was, however, blocked by isradipine. Thus, in hippocampal CA3 pyramidal cells, Ca²⁺ influx through L-type Ca²⁺ channels is necessary to trigger the slow I_AHP. Furthermore, intracellular Ca²⁺-activated Ca²⁺ stores represent a critical component in the transduction pathway leading to the generation of the slow I_AHP.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The slow I_AHP, a Ca²⁺-dependent K⁺ current, modulates the firing pattern of many classes of neurons by inducing accommodation of action potential discharge. The physiological importance of this current is reflected in the multitude of neurotransmitter systems and second messenger pathways that evolved for its regulation (Nicoll 1988). In hippocampal pyramidal cells the slow I_AHP plays a prominent role in determining neuronal activity patterns and is sensitive to regulation by the cholinergic, glutamatergic, and diverse aminergic afferents impinging on these cells. Earlier studies to characterize the slow I_AHP in hippocampal pyramidal cells have shown that this K⁺ current depends on influx of extracellular Ca²⁺ (Gustafson and Wigström 1981; Hablitz 1981; Hotson and Prince 1980; Schwartzkroin and Stafstrom 1980), is relatively insensitive to voltage and tetraethylammonium chloride (TEA) (Lancaster and Adams 1986), is not blocked by apamin, as opposed to the medium and slow I_AHPs in most other cell types (Lancaster and Nicoll 1987; Storm 1989), and underlies accommodation of cell firing (Madison and Nicoll 1984). However, although a rise in intracellular Ca²⁺ clearly triggers the slow I_AHP, the channels mediating this Ca²⁺ influx in hippocampal pyramidal cells were not yet identified. In various other types of neurons the Ca²⁺ channels underlying the induction of the apamin-insensitive slow I_AHP were recently described. For example, in rat sensorimotor cortical pyramidal neurons and in cholinergic nucleus basalis neurons, mainly N- and P-type Ca²⁺ channels are responsible for initiating this current (Pineda et al. 1996; Williams et al. 1997).

On the basis of the delayed activation and prolonged decay of the slow I_AHP, a number of mechanisms that might explain the slow action of Ca²⁺ was proposed. These include a redistribution of cytoplasmic Ca²⁺ by simple diffusion (Lancaster and Zucker 1994; Zhang et al. 1995), Ca²⁺-induced Ca²⁺ release from intracellular Ca²⁺ stores (Sah and McLachlan 1991), and Ca²⁺-induced activation of an intracellular signal transduction cascade (Lasser-Ross et al. 1997; Schwindt et al. 1992). Currently, however, the actual mechanism involved remains unclear.

The goal of this investigation was to establish which Ca²⁺ channels are involved in the generation of the slow I_AHP in hippocampal CA3 pyramidal cells and to clarify whether Ca²⁺-induced Ca²⁺ release contributes to this current.

	METHODS

Abstract Introduction Methods Results Discussion References

Experiments were performed with organotypic hippocampal slice cultures. Hippocampi were removed aseptically from 6-day-old Wistar rats that were killed by cervical dislocation. Tissue slices of 400 µm thickness were prepared and cultured by means of the roller tube technique as described previously (Gähwiler 1981).

Electrophysiological recordings

After 15-30 days in vitro, the cultures were transferred to a recording chamber mounted onto the stage of an inverted microscope (Axiovert 35 M, Zeiss, Jena, Germany) and superfused with an external solution (at 32°C, pH 7.4) containing (in mM) 148.9 Na⁺, 2.7 K⁺, 146.2 Cl, 2.8 Ca²⁺, 0.5 Mg²⁺, 11.6 HCO₃, 0.4 H₂PO₄, and 5.6 D-glucose. Single-electrode voltage-clamp recordings were made from CA3 pyramidal cells (Axoclamp 2 amplifier, Axon Instruments, Foster City, CA) with microelectrodes filled with 2 M potassium methylsulfate (KMeSO₄) and tip resistances of 50-80 M. The switching frequency ranged between 1.5 and 2 kHz, which provided sufficient resolution to analyze the slow I_AHP signals, and headstage output was monitored continuously to ensure adequate settling time between samples. Input resistance was assessed with 500-ms hyperpolarizing voltage commands of 10 mV.

The slow I_AHP was induced with brief depolarizing voltage jumps (from a holding potential of between 55 and 60 mV to 0 mV for 50-100 ms, 0.04 Hz) in the presence of tetrodotoxin (TTX, 0.5 µM). When Ca²⁺ currents were elicited, 20-mV depolarizing voltage jumps were applied from a holding potential of 40 mV for 0.5-1 s (0.05 Hz) in the presence of TTX (0.5 µM) and TEA (10 mM) with microelectrodes filled with 2 M cesium chloride (CsCl). Excitatory postsynaptic potentials (EPSPs) were evoked in current-clamp mode at a membrane potential of approximately 80 mV by electrically stimulating the mossy fibers (0.05 Hz) in the presence of the N-methyl-D-aspartate (NMDA) receptor antagonist 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), the -aminobutyric acid type A (GABA_A) receptor antagonist bicuculline, the GABA_B receptor antagonist CGP 52 432 (10 µM each), and a nonsaturating concentration of the non-NMDA receptor antagonist and 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX, 10 µM).

Numerical data in the text are expressed as means ± SE. Student's t-test was used to compare means when appropriate. P < 0.05 was considered significant.

Drugs and chemicals

()-Bicuculline methochloride, thapsigargin, and ryanodine were purchased from Sigma (St. Louis, MO), CNQX was from Tocris Neuramin (Bristol, UK), TTX was from Sankyo (Tokyo), -agatoxin IVA was from Latoxan (Rosans, France) or from the Peptide Institute (Osaka, Japan), and synthetic -conotoxin MVIIA was from Latoxan. Isradipine, CPP, and CGP 52 432 were kindly donated by Novartis (Basel). Drugs were directly applied via the superfusion solution. Peptide toxins used to block Ca²⁺ channels were bath-applied in the presence of cytochrome C (0.1 mg/ml) to reduce nonspecific binding.

	RESULTS

Abstract Introduction Methods Results Discussion References

Hippocampal pyramidal cells express voltage-gated Ca²⁺ channels belonging to the L-, N-, P-, and Q-types (Ahlijanian et al. 1990; Westenbroek et al. 1990, 1992, 1995). Essentially the same pattern of Ca²⁺-channel distribution was described in CA3 pyramidal cells in hippocampal slice cultures (Elliott et al. 1995). In a first series of experiments, specific Ca²⁺-channel antagonists were tested to determine the channel types associated with the induction of I_AHP.

L-type Ca²⁺-channel blockers suppress slow I_AHP

Isradipine, a dihydropyridine (DHP) that selectively blocks L-type Ca²⁺ channels (Hof et al. 1984), significantly reduced the slow I_AHP in CA3 pyramidal cells. Figure 1A illustrates the typical time course of I_AHP inhibition in response to isradipine (2 µM) in a representative cell. In seven cells, isradipine superfused for 7 min depressed the slow I_AHP from 247.3 ± 48.6 pA to 45.8 ± 13.1 pA (a decrease of 82.3 ± 4.2%, P < 0.01). Attempted washout of the drug for >30 min did not lead to recovery. Representative traces of the slow I_AHP are depicted in Fig. 1B. An alternative DHP, nifedipine (10 µM for 5-10 min), similarly suppressed the slow I_AHP from 309.1 ± 37.4 pA to 178.9 ± 22.8 pA (a decrease of 42.1 ± 4.7%, n = 7, P < 0.01, not shown). The weaker action of nifedipine probably reflects the lower affinity of this compound for L-channels versus isradipine (Cognard et al. 1986; Ohya and Sperelakis 1990).

View larger version (14K): [in this window] [in a new window]   FIG. 1. L-type Ca2+ channels mediate the slow IAHP in hippocampal CA3 cells. The cell was clamped close to 55 mV and the slow IAHP was elicited with brief depolarizing voltage jumps to 0 mV (50-100 ms, 0.04 Hz) in the presence of tetrodotoxin (TTX). Isradipine (2 µM), a specific L-type Ca2+ channel antagonist, suppresses the slow IAHP. A: time course of the inhibition of slow IAHP. Each point represents the mean amplitude and SE of 4 consecutive slow IAHPs. B: examples of individual traces recorded at the times indicated on the graph.

N- and P/Q-type Ca²⁺-channel blockers do not reduce slow I_ahp

In contrast to the potent inhibition by L-type Ca²⁺-channels blockers, the specific N-type Ca²⁺-channel antagonist -conotoxin MVIIA (CmTx) (Olivera et al. 1987) did not reduce the slow I_AHP. In fact, superfusion of CmTx (1 µM for 5 min) slightly, but significantly, increased the amplitude of slow I_AHP by 11.4% (from 326.6 ± 19.4 pA to 363.9 ± 23.8 pA, n = 5, P < 0.05, Fig. 2A). This observation, which may be explained by improved membrane space clamping as a result of closure of N-type Ca²⁺ channels, was not further investigated. To confirm that CmTx was indeed blocking Ca²⁺ channels, we examined its effects on pharmacologically isolated EPSPs evoked in CA3 cells by stimulating mossy fiber afferents with an electrode positioned in the dentate hilus area. As we previously reported for CA3 pyramidal cells in this preperation (Poncer et al. 1997), EPSPs were rapidly reduced in the presence of CmTx (by 55.0 ± 4.5%, n = 5, Fig. 2B).

View larger version (20K): [in this window] [in a new window]   FIG. 2. N- and P/Q-type Ca2+ channels do not contribute to the slow IAHP. A: -conotoxin MVIIA (1 µM), a specific N-type Ca2+ channel antagonist does not reduce the amplitude of the slow IAHP. Each point represents the mean amplitude and SE of 4 consecutive slow IAHPs in a typical cell. Average current records at the times indicated on the graph are shown superimposed for A and C. C: slow IAHP was not reduced by -agatoxin IVA (200 nM, the P/Q-type Ca2+-channel antagonist). B and D: in contrast, excitatory postsynaptic potentials were suppressed by -conotoxin MVIIA (1 µM) or -agatoxin IVA (200 nM). Each sample record represents the average of 4 consecutive EPSPs.

Experiments were then performed with -agatoxin IVA (Aga IVA) at 200 nM, a concentration that blocks both P- and Q-type Ca²⁺ channels (Olivera et al. 1994; Zhang et al. 1993). Aga IVA had no effect on the slow I_AHP (a 2.7% increase P > 0.05, n = 4, Fig. 2C) but reduced evoked EPSPs (by 53.1 ± 7.7%, n = 3, Fig. 2D).

Thus Ca²⁺ influx via N- or P/Q-type Ca²⁺ channels does not mediate the slow I_AHP in hippocampal CA3 pyramidal cells.

Intracellular Ca²⁺ stores contribute to the generation of slow I_AHP

A characteristic feature of the slow I_AHP is its prolonged time course lasting several seconds and the slow onset and slow decay times. This suggests that Ca²⁺ influx does not directly activate the K⁺ channels conducting the I_AHP. It is reasonable to assume that Ca²⁺ release from intracellular stores might contribute to the slow time course of this response in CA3 cells.

We examined this possibility by depleting intracellular Ca²⁺ stores with thapsigargin, a Ca²⁺ ATPase inhibitor that prevents Ca²⁺ uptake into intracellular compartments. In six of seven cells, bath-application of thapsigargin (10 µM for 28-30 min) reduced the slow I_AHP from 225.7 ± 34.3 pA to 83.9 ± 21.1 pA (P < 0.05). In one remaining cell, the slow I_AHP was not affected by thapsigargin. The time course of the response to thapsigargin and representative traces from a typical cell are illustrated in Fig. 3A.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Ca2+ release from intracellular Ca2+ stores contributes to the generation of slow IAHP. A: bath-application of thapsigargin (10 µM) decreases the amplitude of slow IAHP. Each point represents the mean amplitude of 4 consecutive slow IAHPs in a typical cell. B: bath-application of ryanodine (10 µM) results in a similar attenuation of slow IAHP, although this effect is only partially reversible.

Two functionally separate releasable pools of intracellular Ca²⁺ can be distinguished in neurons based on the ligands Ca²⁺- or inositol 1,4,5-trisphosphate (IP₃), which mediate the respective liberation of the sequestered Ca²⁺ through channels spanning the membranes of the endoplasmic reticulum (Henzi and MacDermott 1992). Hippocampal CA3 pyramidal cells primarily express the Ca²⁺-activated Ca²⁺-release channels that are sensitive to ryanodine (Pauda et al. 1991; Sharp et al. 1993). Furthermore, a recent study in cerebellar neurons demonstrated that these ryanodine receptors can functionally couple with L-type Ca²⁺ channels (Chavis et al. 1996). We therefore tested whether Ca²⁺-activated Ca²⁺ release contributes to the generation of the slow I_AHP induced by Ca²⁺ influx through L-type Ca²⁺ channels. Superfusion of ryanodine (10-100 µM for 4-20 min) to inhibit Ca²⁺-activated Ca²⁺ release reduced the slow I_AHP from 219.7 ± 40.9 pA to 100.3 ± 15.7 pA (n = 8, P < 0.05). This action was only partially reversible after 30 min of wash. The time course of the ryanodine effect and representative traces from one cell are shown in Fig. 3B. This result indicates that the Ca²⁺ signal mediated by L channels causes the release of intracellular Ca²⁺ stores that contribute to the activation of the K⁺ conductance underlying the slow I_AHP.

It has been shown that thapsigargin, in addition to depleting intracellular Ca²⁺ pools, can also inhibit L-type Ca²⁺ current (Buryi et al. 1995; Nelson et al. 1994). Thus the observed inhibition of slow I_AHP in response to thapsigargin may result from a direct inhibition of L-type Ca²⁺ channels rather than the emptying of Ca²⁺ stores. Under our experimental conditions, however, this did not appear to be the case. Cells were clamped at 40 mV and stepped to 20 mV to elicit Ca²⁺ current with a strong L-type component (Gähwiler and Brown 1987). Thapsigargin (10 µM for 25 min) did not significantly decrease Ca²⁺ current (a reduction of 16.6 ± 3.5%, P > 0.05, n = 3, Fig. 4A). On the other hand, isradipine (2 µM for 7 min) reduced Ca²⁺ current by 80.1 ± 7.0% (n = 5, P < 0.05, Fig. 4B).

View larger version (8K): [in this window] [in a new window]   FIG. 4. Distinct actions of thapsigargin and isradipine on Ca2+ currents. Suppression of the slow IAHP by thapsigargin is not due to its effect on the Ca2+ current. Cells were clamped at 40 mV, and Ca2+ currents were elicited by voltage steps to 20 mV (for 0.5-1 s, 0.05 Hz) in the presence of TTX (0.5 µM) and tetraethylammonium chloride (10 mM) with micloelectrodes filled with 2 M CsCl. A: thapsigargin (10 µM) produced no significant change in the Ca2+ current. B: isradipine (2 µM) suppressed Ca2+ currents. Each sample trace represents the average of 4 consecutive Ca2+ currents.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The principal finding of this study is that Ca²⁺ influx through L-type Ca²⁺ channels initiates the generation of the slow I_AHP in hippocampal CA3 pyramidal cells. Furthermore, it appears that Ca²⁺ release from intracellular stores is required to induce the slow I_AHP. This suggests that L-type Ca²⁺ channels can functionally couple to Ca²⁺-activated Ca²⁺ stores, which may account in part for the prolonged time course of the slow I_AHP.

Ca²⁺ influx through L-type Ca²⁺ channels triggers the slow I_AHP

Our results obtained with DHP Ca²⁺-channel antagonists are consistent with an involvement of L-type Ca²⁺ channels in the generation of the slow I_AHP in the hippocampus. A number of previous studies demonstrating the colocalization of L-type Ca²⁺ channels with the K⁺ channels thought to underlie the slow I_AHP in hippocampal pyramidal cells provide support for this conclusion.

L-type Ca²⁺ channels are expressed mainly in the cell body and proximal dendrites of CA3 pyramidal cells (Ahlijanian et al. 1990; Westenbroek et al. 1990), and their activation results in a pronounced somatic Ca²⁺ transient (Elliott et al. 1995). Such Ca²⁺ transients are closely associated in time with the induction of slow I_AHP (Knöpfel and Gähwiler 1992; Lasser-Ross et al. 1997). In contrast, N and P/Q channels in these cells are expressed primarily in axon terminals where they mediate neurotransmitter release. Although there is some expression of these channels on the dendrites, they do not contribute significantly to Ca²⁺ transients induced by stimulating the cell body (Elliott et al. 1995).

The channels that conduct the slow I_AHP in hippocampal pyramidal cells were identified as Ca²⁺-dependent small conductance potassium channels referred to as SK channels, with an estimated conductance of 2-5 pS (Sah and Isaacson 1995). Similarly to the L-type Ca²⁺ channels, these SK channels appear to be concentrated on the proximal dendrites (Sah and Bekkers 1996). Moreover, cell-attached, patch-clamp recordings in hippocampal pyramidal cells have shown that L-type Ca²⁺ channels colocalize exclusively with SK channels, whereas patches with N-type Ca²⁺ channels are associated with large conductance Ca²⁺-dependent K⁺ channels (Tavalin and Marrion 1997).

Further evidence that activation of SK channels does indeed underlie the slow I_AHP was recently obtained with the cloning of a family of these K⁺ channels. One of these, the human SK1 channel (hSK1) with a single channel conductance of 9.9 pS displays all the properties associated with the slow I_AHP characterized in hippocampal pyramidal cells, including apamin insensitivity and voltage independence (Köhler et al. 1996).

It was reported that DHP antagonists also inhibit certain K⁺ currents. For example, DHPs rapidly inactivated Shaker K⁺ channels expressed in Xenopus oocytes (Avdonin et al. 1997). In cerebellar granule cells, DHPs were found to block one-third of the large conductance Ca²⁺-dependent K⁺ channels (Fagni et al. 1994). In contrast, SK channels do not appear to be sensitive to DHPs. In both cortical pyramidal neurons (Foehring and Waters 1995) as well as cholinergic nucleus basalis neurons (Williams et al. 1997), DHPs had no effect on the apamin-insensitive slow I_AHP, which is mediated primarily by Ca²⁺ influx through N-type channels in these cells.

A Ca²⁺-induced Ca²⁺ release mechanism is involved in generating slow I_AHP

Releasable intracellular Ca²⁺ stores in brain cells perform important functions, not only in Ca²⁺ homeostasis but also in the modulation of diverse neuronal responses. Neurons express two main types of intracellular Ca²⁺ channels, which are activated, respectively, by IP₃ or by Ca²⁺ itself (Henzi and MacDermott 1992). In CA3 pyramidal cells Ca²⁺ release is mediated mainly by Ca²⁺ binding to ryanodine receptors, whereas in CA1 pyramidal cells Ca²⁺ stores are regulated by IP₃ receptors (Pauda et al. 1991; Sharp et al. 1993). In keeping with this expression pattern, our data indicate that ryanodine-sensitive Ca²⁺ stores are involved in the activation of the slow I_AHP in CA3 pyramidal cells. Similarly, Ca²⁺-activated Ca²⁺ stores were found to be necessary for the induction of an apamin-insensitive slow AHP in vagal motor neurons (Sah and McLachlan 1991) and in locus ceruleus neurons (Osmanovic and Shefner 1993). In these studies, however, the voltage-gated Ca²⁺ channels mediating Ca²⁺ influx were not characterized.

A close functional association between L-type Ca²⁺ channels and ryanodine-sensitive Ca²⁺ stores was demonstrated in a variety of cell types. The best-studied example is the coupling between L-type channels and ryanodine receptors that constitutes the key element of the excitation-contraction coupling machinery in skeletal muscle (Ashcroft 1991). Functional coupling between L-type Ca²⁺ channels and ryanodine receptors was also described in neurons, where this may play a role in regulating electrical properties and in synaptic plasticity (Chavis et al. 1996). On the basis of our results, we hypothesize that in CA3 pyramidal cells Ca²⁺-activated Ca²⁺ stores may represent one component in an intracellular protein scaffold involved in the delivery of Ca²⁺ from the L-type channel to the appropriate binding sites on the SK channel. This arrangement may limit the diffusion of incoming Ca²⁺, thereby enhancing the specificity of this response. Furthermore, Ca²⁺ release from intracellular stores may serve to amplify the primary Ca²⁺ signal.

	ACKNOWLEDGEMENTS

  We thank L. Heeb, R. Kägi, H. Kasper, L. Rietschin, and R. Schöb for excellent technical assistance. This research was supported by Sankyo Ltd., the Prof. Dr. Max Cloëtta Foundation, and the Swiss National Science Foundation (31-45547.95).

	FOOTNOTES

  Address for reprint requests: U. Gerber, Brain Research Institute, University of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland.

  Received 8 May 1998; accepted in final form 14 July 1998.

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