Ca2+ Channels Involved in the Generation of the Slow Afterhyperpolarization in Cultured Rat Hippocampal Pyramidal Neurons

M. Shah and D. G. Haylett

Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Shah, M. and D. G. Haylett. Ca2+ Channels Involved in the Generation of the Slow Afterhyperpolarization in Cultured Rat Hippocampal Pyramidal Neurons. J. Neurophysiol. 83: 2554-2561, 2000. The advantages of using isolated cells have led us to develop short-term cultures of hippocampal pyramidal cells, which retain many of the properties of cells in acute preparations and in particular the ability to generate afterhyperpolarizations after a train of action potentials. Using perforated-patch recordings, both medium and slow afterhyperpolarization currents (mIAHP and sIAHP, respectively) could be obtained from pyramidal cells that were cultured for 8-15 days. The sIAHP demonstrated the kinetics and pharmacologic characteristics reported for pyramidal cells in slices. In addition to confirming the insensitivity to 100 nM apamin and 1 mM TEA, we have shown that the sIAHP is also insensitive to 100 nM charybdotoxin but is inhibited by 100 µM D-tubocurarine. Concentrations of nifedipine (10 µM) and nimodipine (3 µM) that maximally inhibit L-type calcium channels reduced the sIAHP by 30 and 50%, respectively. However, higher concentrations of nimodipine (10 µM) abolished the sIAHP, which can be partially explained by an effect on action potentials. Both nifedipine and nimodipine at maximal concentrations were found to reduce the HVA calcium current in freshly dissociated neurons to the same extent. The N-type calcium channel inhibitor, omega -conotoxin GVIA (100 nM), irreversibly inhibited the sIAHP by 37%. Together, omega -conotoxin (100 nM) and nifedipine (10 µM) inhibited the sIAHP by 70%. 10 µM ryanodine also reduced the sIAHP by 30%, suggesting a role for calcium-induced calcium release. It is concluded that activation of the sIAHP in cultured hippocampal pyramidal cells is mediated by a rise in intracellular calcium involving multiple pathways and not just entry via L-type calcium channels.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In hippocampal pyramidal cells, a train of action potentials is followed by a multicomponent afterhyperpolarization (AHP), comprising a fast AHP (fAHP), a medium AHP (mAHP), and a slow AHP (sAHP) (Storm 1987, 1989; see Sah 1996 for a review). The fAHP occurs immediately after an action potential, lasts 1-10 ms, and is primarily a result of activation of the large conductance calcium-activated potassium ion channels (BK channels) (Sah 1996) that are involved in the repolarization of action potentials (Lancaster and Nicoll 1987; Yoshida et al. 1991). The mAHP has a fast onset (<10 ms), can last up to several hundred milliseconds, and has a decay time constant of ~39 ms. It appears to result from the activation of a number of different ion channels (Alger et al. 1994; Storm 1987, 1989), including an apamin-sensitive small conductance calcium-activated potassium ion channel (SK channel) (Stocker et al. 1999). In contrast the sAHP has slow kinetics and is most easily detected after a train of action potentials (for review see Storm 1990). The current underlying the sAHP (sIAHP) shows a distinct rising phase, peaks between 400-700 ms after a train of action potentials, and decays with a time constant of ~1.5 s at 30°C (Lancaster and Adams 1986). The sAHP is insensitive to the bee venom toxin apamin (100 nM), 1 mM tetraethyammonium (TEA), and 1 mM 4-aminopyridine (4AP) (Lancaster and Nicoll 1987; see Storm 1990). It has been shown that the potassium ion channels giving rise to the sAHP are calcium-activated (Lancaster and Adams 1986) and have an estimated conductance of 10 pS (Valiante et al. 1998). Taken together, these findings suggest that apamin-insensitive SK channels underlie the sAHP. Of the three subtypes of cloned SK channels (SK1-SK3) (Kohler et al. 1996), only SK1 channels (expressed in Xenopus oocytes) are insensitive to apamin and thus may potentially underlie the sIAHP (Ishii et al. 1997; Kohler et al. 1996; Vergara et al. 1998). No specific blocker of the sAHP or SK1 channels has yet been identified to allow a test of this possibility. The sAHP can however be inhibited by neurotransmitters such as acetylcholine, noradrenaline, and histamine and potentiated by adenosine (see Storm 1990).

Studies of the source of calcium required to activate the sAHP in hippocampal cells have provided conflicting results. In hippocampal pyramidal slices, the sAHP was only partially reduced by the L-type calcium channel inhibitor nimodipine (Moyer et al. 1992; Rascol et al. 1991). This contrasts with findings in pyramidal cells in organotypic slice cultures where the sAHP was almost completely blocked by the L-type channel inhibitor isradipine (Tanabe et al. 1998). These studies suggest that the source of calcium for the sAHP may differ between hippocampal preparations. Similarly, there is evidence both for (Tanabe et al. 1998; Torres et al. 1996) and against (Lancaster and Zucker 1994; Zhang et al. 1995) a role for calcium-induced calcium release (CICR) from intracellular stores.

In this study we have sought to study the role of L- and N-type calcium channels and CICR in the activation of the sAHP. To improve drug access to neurons, we decided to use isolated pyramidal cells. Previous studies have reported difficulties in recording the sAHP in cultured hippocampal pyramidal cells (Alger et al. 1994) but we describe an isolated cell preparation that makes this possible. A potential criticism of using cultured cells is that the isolation procedure and culture conditions may lead to changes in calcium and potassium channel distributions and densities and possibly to changes in channel regulation. It was thus important to explore the basic properties and the pharmacology of the sIAHP in these cultured cells. Using this preparation, we have been able to study the effects on the sIAHP of the L-type calcium ion channel inhibitors, nifedipine and nimodipine, the N-type calcium channel inhibitor, omega -conotoxin GVIA, and ryanodine, an inhibitor of CICR. Some of this work has been published previously in abstract form (Shah and Haylett 1999).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hippocampal cell culture method

Four-day-old Sprague-Dawley rats were decapitated and the whole brain was removed and placed in cold (4°C) Gey's Balanced Salt Solution (GBSS) supplemented with 0.6% wt/vol glucose and 8 mM MgCl2 as described by Allen et al. (1993). The brain was hemisected and 500-µm-thick coronal slices were obtained using a MacIlwain tissue chopper. The CA1 and CA3 regions were dissected out. These regions were incubated in Ca2+- and Mg2+-free Hanks' Balanced Salt Solution (HBSS) containing 0.125% wt/vol trypsin and 1 mM HEPES buffer (Sigma, pH 7.3) at 37°C for 1 h. After the 1 h enzyme treatment, the slices were repeatedly washed with Ca2+- and Mg2+-free HBSS supplemented with 8 mM MgCl2, 1 mg/ml bovine serum albumin, and 10% heat-inactivated fetal calf serum (FCS). Trituration was carried out in this solution with three fire-polished sterilized Pasteur pipettes of decreasing bore diameters (1-0.2 mm) to release individual pyramidal cells. The cell suspension was centrifuged at 27 × g and the cells resuspended in neurobasal medium supplemented with 2% B27 serum free supplement, 0.02 mg × ml-1 gentamicin, 0.5% wt/vol L-glutamine, and 10% FCS. 200 µl of the cell suspension was plated in 35-mm tissue culture dishes (Nunc) coated with poly-D-lysine (molecular weight > 300,000). The cells were maintained in culture in incubators continuously gassed with 95% O2-5% CO2 at 37°C. After 24 h in culture the cells were re-fed with the culture medium without FCS and retained in this medium until use.

Measurement of the sIAHP

The pyramidal cell cultures were used between 8 and 15 days after preparation. All studies were made at a temperature of ~28°C. The cell cultures were superfused in the culture dishes at 5 ml × min-1 with a bathing solution composed of (in mM) 130 NaCl, 3 KCl, 2.5 CaCl2, 1.2 MgCl2, 5 HEPES free acid, 10 glucose, and 26 NaHCO3 (pH maintained at 7.2 by continuously gassing with 95% O2-5% CO2). DNQX (6,7-dinitroquinoxaline-2,3-dione; 5 µM) was added to block excitation by endogenously released glutamate. Patch electrodes with resistances of 4-10 MOmega were pulled from thin borosilicate glass (Clark Electromedical Instruments; GC15OTF-15). The tips of the pipettes were coated with Sylgard and fire polished. To obtain perforated patches, the electrodes were filled with a solution composed of 126 mM KMeSO4, 14 mM KCl, 10 mM HEPES, 3 mM MgCl2, 2 mM Na2ATP, 0.3 mM Na2GTP, and 1.2 mg × ml-1 amphotericin B (pH adjusted to 7.25 with 1 M KOH).

Cells with a soma of 12-18 µm diameter and with at least one thick process were assumed to be pyramidal (Fig. 1B). The sIAHP was recorded under hybrid clamp conditions using an Axoclamp 2A amplifier (Axon Instruments). A train of 13 action potentials was evoked by passing 5-ms current pulses (at a frequency of 76.5 Hz) under discontinuous current-clamp conditions and the cell was then voltage-clamped at approximately -50 mV to record the sIAHP (Axoclamp sampling rate 1.5-5kHz). The pulse protocol was provided by a Master-8 pulse generator (Intracel). The sIAHP was evoked every 10 s. The current signals were filtered using the Axoclamp 2A low-pass filter at 0.3 kHz. For action potential recording, the voltage signals were filtered at 3 kHz. Signals were recorded on a chart recorder and an oscilloscope. Signals were also digitized at 48 kHz (VR-10 digital data recorder; Instrutech) and recorded continuously on a video recorder. sIAHPs were also stored on a computer using pClamp6 (Axon Instruments) for later analysis. The first 2 s of the sIAHP were acquired at a sampling frequency of 2 kHz and the remainder at a sampling frequency of 0.5 kHz. Action potentials were acquired separately at a sampling frequency of 20 kHz.

Drugs were applied by switching to a superfusion fluid containing the drug using a multiway tap. The flow of the superfusion solution was directed onto the patched cell.

Measurement of calcium ion currents

Because cultured hippocampal cells have extensive dendritic processes, it is difficult to voltage-clamp them adequately. Therefore calcium ion currents were studied using acutely dissociated cells under whole cell conditions. The pyramidal cells were isolated and plated as described above. Recordings were made from cells between 3.5 and 8 h after isolation.

Pyramidal cells could again be identified by their morphology (Fig. 1A). The cells were superfused with a solution composed of the following (in mM): 115 NaCl, 2 KCl, 2 CaCl2, 0.5 MgCl2, 11 glucose, and 10 HEPES (pH adjusted to 7.4 with 1 M NaOH). TEA (25 mM), TTX (0.3 µM), and DNQX (5 µM) were added to block potassium currents, sodium currents, and AMPA and kainate receptor activation, respectively. Patch-clamp recordings were made with a List EPC-7 amplifier using 7-10 MOmega pipettes filled with solution composed of the following (in mM): 135 CsCl, 0.5 CaCl2, 2 MgCl2, 10 HEPES, 3 EGTA, 2 Na2ATP, and 0.3 Na2GTP (pH adjusted to 7.3 with NaOH). The calcium ion current was generated using a procedure similar to that described by Deak et al. (1998). The cells were voltage clamped at -80 mV. Using a ramp protocol, the cells were depolarized from -100 to +40 mV at a speed of 1400 mV × s-1 every 10 s. The leak current was determined using Cd2+ (200 µM) to block calcium currents. Because the maximum voltage error was calculated to be <5 mV, series resistance compensation was not carried out. The signals were filtered at a frequency of 1 kHz (8 pole bessel filter) and digitized at a sampling frequency of 3.33 kHz using pClamp6 software (Axon Instruments).

Data analysis

Data were analyzed using pClamp6 software. The current traces shown in the figures are the average of three records. The average of the amplitude of three successive records of the sIAHP in the presence of the drug was expressed as a percentage of the amplitude of the average of six successive records before application of the drug. The effects of drugs on action potential duration were measured at a potential of -20 mV. The effects of calcium channel blockers on the time course of the sIAHP were measured by fitting the sIAHP empirically to the equation
<IT>y</IT><IT>=</IT><IT>y</IT><SUB><IT>0</IT></SUB><IT>+</IT><IT>A</IT><SUB><IT>1</IT></SUB><IT>e</IT><SUP><IT>−</IT><IT>t</IT><IT>/&tgr;<SUB>1</SUB></IT></SUP><IT>+</IT><IT>A</IT><SUB><IT>2</IT></SUB><IT>e</IT><SUP><IT>−</IT><IT>t</IT><IT>/&tgr;<SUB>2</SUB></IT></SUP><IT>+</IT><IT>A</IT><SUB><IT>3</IT></SUB><IT>e</IT><SUP><IT>−</IT><IT>t</IT><IT>/&tgr;<SUB>3</SUB></IT></SUP>
which provided a satisfactory fit of the decaying mIAHP, the growth and decay phases of the sIAHP, and allowed for the holding current at -50 mV. The time to peak was determined from the fitted line. All results are expressed as mean ± SE. Statistical analysis was carried out using the Student's t-test (paired or unpaired as appropriate).

When analyzing the effects of drugs on the calcium ion current, the average peak current of the last two records in the presence of the drug (2-min bath application) was expressed as a percentage of the average peak current of two successive records obtained both before application of the drug and after its washout. All records were leak subtracted.

Materials

All solutions and chemicals were obtained from Sigma except for the following: GBSS, Ca2+, Mg2+-free HBSS, neurobasal medium, B27 serum-free supplement, gentamicin, and FCS were obtained from Gibco; bovine serum albumin was obtained from ICN Pharmaceuticals; KMeSO4 was purchased from Pfaltz and Bauer; and omega -conotoxin GVIA was obtained from Peptide Institute and Alamone Laboratories.

Stock solutions of most drugs were made in water and stored at -20°C until required. Stock solutions of nifedipine, nimodipine, and ryanodine were made in dimethyl sulfoxide (DMSO) and stored at -20°C. These were diluted to the appropriate concentrations in the external bathing solution. The maximum concentration of DMSO applied (0.1%) had no effect on the sIAHP. Suitable precautions were taken to ensure that nifedipine, nimodipine, and ryanodine were protected from exposure to light.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Both freshly dissociated and cultured hippocampal pyramidal cells were identified by their morphology (Fig. 1). The soma had a width of 12-18 µM and at least one apical dendrite emerging from it. The cultured pyramidal cells were easily visualized as glial cell growth was minimized by the use of the B27 serum free supplement (Brewer et al. 1993). Recordings were made only from neurons that had resting membrane potentials of at least -50 mV. Conventional whole cell recording was observed to cause significant rundown of the sIAHP, which quite often disappeared within 1-2 min of establishing whole cell conditions. This was much less marked when perforated patches were tested. Stable recordings of the sIAHP could be obtained for up to 60 min.



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Fig. 1. Morphology of pyramidal cells in short and long term culture. A: dissociated hippocampal pyramidal cell ~3 h after isolation. Cell can be clearly identified from its morphology. Cells of this type were used to record calcium ion currents. B: hippocampal pyramidal cell in culture for 8 days. sIAHPs were routinely recorded from cells of this appearance. Horizontal bar on each picture represents 20 µm.

General properties of the sIAHP

Approximately 50-60% of the cells exhibited a sIAHP with an amplitude >20pA. The sAHP recorded under current-clamp conditions in these cells (Fig. 2A) had similar kinetics to the sIAHP recorded using the hybrid clamp (Fig. 2B). In some cells exhibiting a sIAHP, a mIAHP was also observed (Fig. 3A). In a few cells (~20%), a mIAHP was observed without any sIAHP. The amplitude of the sIAHP increased at more positive potentials (Fig. 2B) as expected for a selective change in potassium permeability. To record the sIAHP, cells were routinely held at -50 mV. The amplitude of the sAHP increased with the number of action potentials (Fig. 2, C and D) as has been reported in slices (Lancaster and Adams 1986). However, the time-to-peak and the decay time constant of the sIAHP did not change with the action potential number. The amplitude of the sIAHP was near maximal with a train of 13 or more action potentials (Fig. 2D). Based on this observation, a train of 13 action potentials was used to generate the sIAHP in subsequent experiments.



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Fig. 2. Dependence of sIAHP on holding potential and number of action potentials. A: sAHP after a train of 13 action potentials recorded under current clamp conditions at resting membrane potential of cell (-69 mV). B: sIAHP recorded from the same cell under hybrid clamp conditions at a holding potential of (i) -50 mV and (ii) -65 mV. Extra holding current at -50 mV has been offset to superimpose traces. C: sIAHPs activated using (i) 12, (ii) 10, (iii) 8, and (iv) 6 action potentials. D: dependence of sIAHP on number of action potentials in a train. Amplitude of the sIAHP has been expressed as a percentage of amplitude after 13 action potentials. Values plotted are means of the number of observations shown in brackets. Where n > 2, SE is also shown.



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Fig. 3. Pharmacological characterization of sIAHP. Effects on sIAHP of 2-min bath applications of 100 nM apamin (A), 200 µM cadmium (B), 1 mM TEA (C), 100 µM tubocurarine (D), and 100 nM charybdotoxin (E). All control traces were recorded just before drugs were applied and are shown superimposed on traces in the presence of drug just before washout. Calibration bars shown in B apply for A-E. F: effects of 100 nM charybdotoxin on last action potential from train of action potentials in the same cell as E. Control action potential is indicated by dotted line.

The effect of drugs on the sIAHP was investigated in cells exhibiting a sIAHP >50pA. In these cells, the mean amplitude of the sIAHP was 173 ± 15 (SE) pA (n = 78). The sIAHP peaked ~600 ms after the last action potential and had a decay time constant of 1.30 ± 0.06 s (n = 32), which is similar to the value reported for the sIAHP (1.5 s) in hippocampal slices (see Storm 1990).

Pharmacological characterization of the sIAHP

The sIAHP could be abolished by a 2-min bath application of 200 µM Cd2+ (n = 4; Fig. 3B) and is therefore dependent on calcium entry. The sIAHP was observed to be insensitive to 100 nM apamin (inhibition -8.9 ± 7.6%, n = 6; Fig. 3A) and 1 mM TEA (2.0 ± 6.4%, n = 6; Fig. 3C) as previously reported (Lancaster and Adams 1986; Lancaster and Nicoll 1987). Although TEA appeared to have no effect on the sIAHP, the width of the action potentials increased from 1.27 ± 0.18 to 2.03 ± 0.46 ms (P = 0.057). Apamin did not have any effect on the action potentials (P > 0.05). In cells that had both a mIAHP and a sIAHP, 2-min bath applications of either 100 nM apamin (Fig. 3A) or 200 µM Cd2+ abolished the mAHP suggesting that apamin-sensitive SK channels mediate the mAHP in these cultured neurons (see also Stocker et al. 1999). The effects of Cd2+ and TEA were reversible within 5 min of washout. Although there was little change in the amplitude of the sIAHP for 15 min after washout, the effect of apamin on the mIAHP was irreversible within this time period.

Apamin-sensitive SK channels are sensitive to the plant alkaloid, D-tubocurarine (Cook and Haylett 1985; Kohler et al. 1996) and AHPs in various neurons can also be blocked by tubocurarine (IC50 < 100 µM) (Bourque and Brown 1987; Dun et al. 1986). In chromaffin cells, which also express apamin-sensitive SK channels, the IC50 for tubocurarine is 20 µM (Park 1994). In cultured hippocampal pyramidal cells, tubocurarine at a concentration of 50 µM had little effect on the sIAHP (an increase in amplitude of 15.4 ± 18.5%, n = 4). Increasing the concentration to 100 µM reduced the amplitude of the sIAHP by 25.9 ± 8.3% (n = 6; Fig. 3D), which was completely reversed within a 5-min washout period. The shape of the sIAHP was unaffected by tubocurarine. It was observed that both 50 and 100 µM tubocurarine completely abolished the mIAHP in cells that had both the mIAHP and the sIAHP, providing further evidence that apamin-sensitive SK channels underlie the mAHP in these cultured neurons (Stocker et al. 1999).

Charybdotoxin is a potent blocker of both large conductance (BK) and intermediate conductance (IK) calcium-activated potassium ion channels (McManus 1991). Because magnocellular neurons of the rat supraoptic nucleus have been demonstrated to have a charybdotoxin-sensitive sAHP (Greffrath et al. 1998), we decided to test the effects of charybdotoxin on the sIAHP in cultured hippocampal pyramidal cells. A 2-min application of charybdotoxin (100 nM) significantly increased the amplitude of the sIAHP by 21.7 ± 5.4% (n = 7; P < 0.01; Fig. 3E) and also significantly (P < 0.01) increased the action potential width from 1.30 ± 0.27 ms (under control conditions) to 1.56 ± 0.29 ms (Fig. 3F). This broadening of the action potentials can be explained by block of BK channels which are involved in action potential repolarization in hippocampal pyramidal cells (Lancaster and Nicoll 1987; Yoshida et al. 1991). The increase in amplitude of the sIAHP may be a result of increased influx of calcium through voltage-gated calcium channels.

The sIAHP was sensitive to neurotransmitters, as previously reported (see Storm 1990). Muscarine (3 µM; n = 4) and noradrenaline (1 µM; n = 3) abolished the sIAHP (Fig. 4, A and B). The effects of both reversed in <5 min. Both agonists also caused a reduction of the outward holding current (Fig. 4C), an effect which has also been demonstrated in previous studies (see Storm 1990). Neither agonist modified the action potentials.



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Fig. 4. Muscarine and noradrenaline suppress sIAHP. A and B: effects of 2-min bath applications of 1 µM noradrenaline and 3 µM muscarine respectively. sIAHP traces in absence and presence of the 2 agents have been superimposed. C: effect of muscarine on resting outward current in the same cell used in B. To illustrate effects of muscarine on sIAHP and resting outward current more clearly, action potentials are not shown. Note that although muscarine and noradrenaline have abolished the sIAHP, the mIAHP (as indicated in B and C) has remained largely unaffected.

Calcium channel inhibitors

The L-type calcium channel inhibitor, nifedipine (1 µM), inhibited the sIAHP amplitude by 29.1 ± 4.0% (n = 10). This effect was maximal within 2 min of bath application and was also reversible within 5 min of washout. Increasing the concentration of nifedipine to 10 µM did not inhibit the sIAHP any further (28.3 ± 5.1%, n = 14; Fig. 5A), suggesting that L-type channels had been maximally inhibited at both concentrations. Nifedipine had no effect on the time-to-peak or decay time constant of the sIAHP (control value for time-to-peak = 0.85 ± 0.09 s and tau 3 = 0.68 ± 0.06 s; in the presence of nifedipine time-to-peak = 0.83 ± 0.10 s and tau 3 = 0.66 ± 0.05 s; n = 14; P > 0.05; Fig. 5A). The possibility that the degree of block by nifedipine depends on the number of action potentials was also tested. Nifedipine produced almost the same degree of block when applied to cells in which the sIAHP was induced using either 8 or 20 action potentials [27.2 ± 3.3%, (n = 3) and 29.4 ± 4.5%, (n = 5) respectively]. Therefore in this test the effect of nifedipine on the sIAHP is independent of the number of action potentials.



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Fig. 5. Inhibitory effects of nifedipine and omega -conotoxin. A: record of sIAHP in presence of a supramaximal concentration of nifedipine (10 µM) applied for 2 min. B: effects of a supramaximal concentration of omega -conotoxin GVIA (100 nM) applied alone for 20 min and together with nifedipine (10 µM). Traces in absence and presence of drugs have been superimposed. Traces in A and B were obtained from the same cell. Control trace in B was obtained after recovery from effect of nifedipine shown in A. Effect of omega -conotoxin was irreversible. C: for cell shown in B, amplitude of sIAHP before, during, and after application of omega -conotoxin GVIA alone and together with nifedipine has been plotted against time. Location of records shown in B are indicated in C as (i), (ii), and (iii). D and E: effects of 10 µM nifedipine and 100 nM omega -conotoxin respectively on last action potential in train of 13 action potentials. Dotted lines represent control traces. Calibration bars shown in B and E also apply to A and D, respectively. Change in membrane potential from D to E occurred during time gap of 10 min between washout of nifedipine and application of omega -conotoxin. It is not a consequence of application of omega -conotoxin.

A 20-min bath application of omega -conotoxin GVIA (100 nM), which is a selective blocker of N-type calcium channels (Tsien et al. 1988), irreversibly reduced the sIAHP by 35.8 ± 3.4% (n = 8; Fig. 5B), suggesting calcium entry via N-type calcium ion channels also plays a role in the activation of the sIAHP. Because of the irreversibility of the effects of omega -conotoxin, it was essential to use only those cells that demonstrated stable sIAHPs for at least 5 min before drug application (as shown in Fig. 5C). It should also be noted that after washout of omega -conotoxin, the sIAHPs remained at their reduced amplitude for at least 15 min and occasionally up to 30 min (see Fig. 5C). Application of omega -conotoxin had no effects on the time-to-peak or decay time constant of the sIAHP (control value for time-to-peak = 0.82 ± 0.13 s, control tau 3 = 1.15 ± 0.12 s; after 20-min application of omega -conotoxin time-to-peak = 0.83 ± 0.09 s and tau 3 = 1.15 ± 0.18 s; n = 8; P > 0.05; Fig. 5B). The IC50 for omega -conotoxin block of N-type channels has been given as 60 pM (Wagner et al. 1988). Application of nifedipine (10 µM) and omega -conotoxin (100 nM) together reduced the sIAHP by 69.6 ± 2.6% (n = 4; Fig. 5B). Neither nifedipine nor omega -conotoxin had any effects on the duration of the action potentials (Fig. 5, D and E). With coapplication of nifedipine and omega -conotoxin, the action potential width increased from 0.98 ± 0.10 ms (under control conditions) to 1.70 ± 0.31 ms (P = 0.056).

The effects of a second L-type calcium channel antagonist, nimodipine, were also studied. Nimodipine at concentrations up to 3 µM only caused a partial inhibition of the sIAHP (Fig. 6, A and B). The maximal effects of nimodipine occurred within 2 min of bath application and reversed within 5 min of washout. The block with 3 µM nimodipine was not significantly different from that with 1 µM (P > 0.05). The IC50 for inhibition of L-type channels by nimodipine is ~50 nM in neurons (Marchetti et al. 1995) so that it is likely that almost maximal inhibition of L-type channels was achieved by nimodipine at the concentrations used in this study (0.3-10 µM; see also results of direct measurements of calcium currents). There was no effect of nimodipine (at either 1 or 3 µM) on the time-to-peak or decay time constant of the sIAHP (control value for time-to-peak = 0.51 ± 0.01 s and tau 3 = 0.67 ± 0.15 s; in the presence of nimodipine time-to-peak = 0.54 ± 0.03 s and tau 3 = 0.64 ± 0.11 s; n = 9; P > 0.05; Fig. 6B), nor was there any change in the action potential width.



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Fig. 6. Effects of nimodipine on sIAHP. A: histogram to show effects of different concentrations of nimodipine on amplitude of sIAHP. Means and the SE have been plotted and number of observations are shown in brackets. B and C: illustrations of effects of 3 and 10 µM nimodipine on sIAHP in 2 different cells. Tails of currents have been superimposed. Calibration bars in C also apply to B.

Increasing the nimodipine concentration to 10 µM resulted in complete abolition of the sIAHP (Fig. 6C) within 2 min of application. This can at least partially be explained by the ability of 10 µM nimodipine to reduce the action potential amplitude during a train of 13 action potentials in a use-dependent fashion (Fig. 7). The threshold of firing of the action potentials was also raised (see Fig. 7, B and D) and the last current pulse during the train of 13 often failed to trigger an action potential (Fig. 7B). The use-dependent effect on the action potentials reversed during the 10-s intervals between trains. These findings suggest that the complete block of the sIAHP by nimodipine at high concentrations cannot be solely attributed to block of L-type calcium ion channels.



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Fig. 7. Effects of 10 µM nimodipine on action potentials. A: train of action potentials before application of 10 µM nimodipine. B: in the same cell a train of 13 action potentials in presence of 10 µM nimodipine applied 2 min beforehand. Calibration bar in B applies also to A. C and D: first and second action potentials in train of 13 action potentials in absence and presence of 10 µM nimodipine. Action potentials have been superimposed. Dotted lines represent control action potentials. Calibration bars in D also apply to C. Ordinates represent cell membrane potential (mp).

The effects of nimodipine on the sIAHP as well as the action potentials were reversible within 5 min at all concentrations.

The effects of inhibitors on calcium currents

The effects of both nifedipine and nimodipine on the calcium current were examined directly in freshly dissociated cells. Using the protocol described in METHODS to evoke the calcium current, a low-voltage-activated (LVA) and a high-voltage-activated (HVA) calcium current were observed in all cells. It should be noted that rundown of the calcium current was quite common and therefore the inhibition of drugs was calculated as a percentage of the average of control and recovery currents. Data were only collected from cells in which the calcium current recovered to within 70% of the control after washout.

Nifedipine (10 µM), nimodipine (3 µM), and nimodipine (10 µM) reduced the HVA calcium current by 18.8 ± 3.5% (n = 7), 20.8 ± 4.3% (n = 4), and 23 ± 1.7% (n = 5), respectively (Fig. 8). There were no significant differences between these values (P > 0.05). These results are consistent with those published in previous studies (Deak et al. 1998; Potier and Rovira 1999) and it can be concluded that at these concentrations, both nifedipine and nimodipine cause a complete inhibition of the current carried by L-type channels in these cells.



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Fig. 8. Effects of nifedipine and nimodipine on calcium currents. Records in absence and presence of 10 µM nifedipine (A), 3 µM nimodipine (B), and 10 µM nimodipine (C). Calcium currents were evoked from a holding potential of -80 mV using a voltage ramp (D). Bathing solution contained 1 µM tetrodotoxin and 25 mM TEA to inhibit sodium and potassium ion channels, respectively. Patch pipette filling solution contained CsCl to provide additional block of potassium currents. In each record, leak current was determined in presence of 200 µM cadmium, which completely blocked HVA current but only part of LVA current. Records are from 3 different cells. Calibration bar shown in A applies to B and C as well.

Nimodipine (3 µM) also caused a small reduction (Fig. 8B) of the LVA calcium ion current, with a greater effect at 10 µM (Fig. 8C). Nimodipine (10 µM) has previously been observed to inhibit an LVA current component (Avery and Johnston 1996). In this study the sIAHP was recorded at a holding potential of -50 mV. At this potential, the LVA current is expected to be inactivated and is therefore unlikely to contribute to the generation of the sIAHP.

Effect of ryanodine

Ryanodine was applied at a maximal concentration of 10 µM (Tanabe et al. 1998). A steady block of 29.4 ± 6.1% (n = 5; Fig. 9) of the sIAHP was achieved at ~20 min. Because of the slow onset of action, particular care was taken to select cells that showed very little rundown of the sIAHP (as shown in Fig. 9B). These findings are consistent for a role of CICR in the activation of the sIAHP in cultured neurons. There were no obvious effects of ryanodine on the duration or amplitude of the action potential, confirming previous studies (Sandler and Barbara 1999).



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Fig. 9. Effects of ryanodine on sIAHP. A: record of sIAHP in absence (i) and presence (ii) of a maximal concentration of ryanodine. Ryanodine was applied for 20 min. Traces have been superimposed. B: amplitude of sIAHP before, during, and after application of ryanodine has been plotted against time. Location of records shown in A are indicated in B as (i) and (ii).


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Using perforated patches to reduce rundown, we have demonstrated that a sIAHP can be detected in 50-60% of cultured rat hippocampal pyramidal cells. The sIAHP peaks ~600 ms after a train of action potentials and has an average decay time constant of ~1.3 s. The sIAHP was abolished by application of Cd2+, a blocker of voltage-gated calcium ion channels, indicating that the sIAHP is activated by a rise in intracellular calcium. As reported for hippocampal slices, the sIAHP was insensitive to 100 nM apamin (Lancaster and Nicoll 1987) and 1 mM TEA (Lancaster and Adams 1986) but was suppressed by application of 1 µM noradrenaline and 3 µM muscarine. The time course and pharmacologic characteristics of the sIAHP in the cultured pyramidal cells appear to be similar to those reported for pyramidal cells in slices (see Storm 1990). The amplitude of the sIAHP was significantly increased in the presence of 100 nM charybdotoxin, which is probably a consequence of the widening of action potentials (Fig. 3F). The sIAHP was also discovered to be relatively insensitive to D-tubocurarine. In view of the insensitivity of the sIAHP to apamin and D-tubocurarine, of the SK channels so far identified, only the SK1 channel pharmacology closely matches the pharmacology of the sIAHP. Thus as suggested by Vergara et al. (1998), it is possible that SK1 underlies the sIAHP.

Application of 10 µM ryanodine reduced the sIAHP in cultured neurons by ~30% suggesting that CICR plays a role in the activation of the sIAHP. This finding is in agreement with the results of Tanabe et al. (1998), who observed ~55% inhibition with 10-100 µM ryanodine, but differs from those of Torres et al. (1996) and Zhang et al. (1995), neither of whom detected any effect of ryanodine at a concentration of 20 µM. However, Torres et al. (1996) and Tanabe et al. (1998) observed a reduction of the sIAHP with thapsigargin, an indirect inhibitor of CICR.

This study also provides further information on the role of L- and N-type calcium channels in generation of the sIAHP. Nifedipine, at both 1 µM and 10 µM, inhibited the sIAHP by only 30%. Another L-type calcium channel blocker, nimodipine, at concentrations that specifically inhibit calcium channels, also reduced the sIAHP by <50%. It should be noted that in the acute cell preparation both nifedipine and nimodipine inhibited the HVA calcium current to similar extents (Fig. 8) suggesting both drugs inhibit L-type calcium channels maximally at the concentrations used. This suggests that because L-type channels were maximally inhibited by these concentrations, L-type calcium channels are not exclusively concerned in the activation of the sIAHP in cultured hippocampal pyramidal cells. This conclusion is in keeping with studies employing hippocampal slices (Moyer et al. 1992; Rascol et al. 1990) in which inhibition of L-type calcium channels produced only partial inhibition of the sAHP. In organotypic slice cultures, however, the L-type calcium channel blocker, isradipine, produced almost complete inhibition of the sIAHP, suggesting a dominant role for L-type calcium channels (Tanabe et al. 1998). Therefore the source of calcium for the generation of the sAHP may differ in different preparations.

At a high concentration (10 µM), nimodipine was found to abolish the sIAHP, despite blocking HVA calcium channels to the same extent as 10 µM nifedipine and 3 µM nimodipine (Fig. 8). One factor contributing to the complete block of the sIAHP is likely to be the inhibitory effect on action potentials (Fig. 7) resulting in a reduction in calcium entry into the neurons. Nimodipine, at this concentration, may also be inhibiting the potassium channel underlying the sIAHP. Dihydropyridines have been found to inhibit potassium channels in various other preparations (for example Ellory et al. 1992; Mlinar and Enyeart 1994). Thus in this study, nimodipine appears to have multiple actions that are not shared by nifedipine.

Although N-type channel inhibitors have been shown to have no effect on the sAHP in rat hippocampal slices (Rascol et al. 1990) or in rat hippocampal organotypic slice cultures (Tanabe et al. 1998), in our cultured cells omega -conotoxin GVIA (100 nM) irreversibly inhibited the sIAHP by 35% (Fig. 5B). This suggests that N-type calcium channels also have a role in activation of the sIAHP. This observation coupled with the finding that L-type calcium channel blockers only partially reduced the sIAHP in these cells supports the conclusion that calcium entry via L-type calcium channels is not solely responsible for the activation of the sIAHP. Application of nifedipine and omega -conotoxin together reduced the sIAHP by 70 ± 3% (Fig. 5B), significantly <100%. Because cadmium completely abolished the sIAHP, the indication is that calcium entry via other types of HVA calcium channels may also play a role in activation of the sIAHP.

It should be noted that in rat neocortical pyramidal cells (Pineda et al. 1998) and in guinea-pig nucleus basalis neurons (Williams et al. 1997), an apamin-insensitive sIAHP is activated predominantly by calcium entry via N-type calcium ion channels. Thus it is clear that the types of calcium channels involved in the activation of the apamin-insensitive sIAHP vary between neurons and may depend on the particular calcium channels present in the vicinity of the potassium channels responsible for the sIAHP.

In conclusion, we have found that the sIAHP can be successfully recorded from cultured hippocampal pyramidal cells and that activation by a train of action potentials, the time course and inhibition by muscarine and noradrenaline are very similar to the findings in hippocampal slices (Sah 1996; Storm 1990). Thus cultured neurons provide a useful alternative to hippocampal slices for the further study of hippocampal physiology and pharmacology. In addition, in cultured rat hippocampal pyramidal cells, calcium entry via both the L- and N- type calcium ion channels contributes to the activation of the sIAHP. Taken together with published work, it is clear that the coupling of the potassium channel underlying the sAHP to calcium sources is not well established and requires further studies.


    ACKNOWLEDGMENTS

We are grateful to Prof. D. H. Jenkinson and Drs. T.G.J. Allen and D.C.H. Benton for helpful discussions and Dr. S. J. Marsh for help with the photography of the cells.

This work was supported by the Wellcome Trust. M. Shah is a Medical Research Council scholar.


    FOOTNOTES

Address for reprint requests: D. G. Haylett, Dept. of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 September 1999; accepted in final form 11 January 2000.


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