Properties of Voltage-Activated Ca2+ Currents in Acutely Isolated Human Hippocampal Granule Cells

H. Beck1, R. Steffens1, U. Heinemann2, and C. E. Elger1

1 Department of Epileptology, University of Bonn Medical Center, D-53105 Bonn, Germany; and 2 Department of Neurophysiology, Institute of Physiology, Charité Berlin, D-10000 Berlin, Germany

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Beck, H., R. Steffens, U. Heinemann, and C. E. Elger. Properties of voltage-activated Ca2+ currents in acutely isolated human hippocampal granule cells. J. Neurophysiol. 77: 1526-1537, 1997. Properties of Ba2+ currents through voltage-dependent Ca2+ channels (IBa) were investigated in 61 dentate granule cells acutely isolated from the resected hippocampus of nine patients with therapy-refractory temporal lobe epilepsy (TLE). Currents with a high threshold of activation (HVA) peaked at 0 mV, and showed some time-dependent inactivation and a voltage of half-maximal steady-state inactivation (V1/2inact) of -16.4 mV. Application of saturating doses of omega -conotoxin (omega -CgTx) GVIA or nifedipine distinguished characteristic N-type (38%) and L-type (62% of HVA currents) Ca2+ currents. Combined application of both agents blocked HVA currents by >95%. In a 10-mo-old child but not in adult patients, an omega -agatoxin IVA (omega -AgaTx IVA)-sensitive but omega -CgTx MVIIC-insensitive, noninactivating component of HVA currents (~24%) was present that most probably corresponds to a P-type current. A T-type Ca2+ current could be separated from HVA components on the basis of its steady-state voltage-dependent inactivation(V1/2inact = -71.0 mV). The T-type Ca2+ current isolated by subtraction peaked at more negative potentials (-10 mV), showed a significantly more rapid time-dependent inactivation, and could be selectively blocked by low concentrations of Ni2+. It was insensitive to nifedipine and omega -CgTx GVIA. We conclude that L-, N-, and T-type currents are present in adult human dentate granule cells and an additional P-type current is present in neurons from a 10-mo-old patient. These data may provide a basis for comparison with animal models of epilepsy and for the elucidation of mechanisms of action of drugs intended for use in human disease.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The electrophysiological analysis of hippocampal tissue from patients undergoing temporal lobe resections for therapy-refractory temporal lobe epilepsy (TLE) in vitro presents a unique opportunity to study the intrinsic and synaptic properties of neurons in the human (Schwartzkroin 1993). Voltage-dependent Ca2+ channels can influence the electrical activity of neurons by contributing to the propagation of postsynaptic depolarizing input (Amitai et al. 1993; Reuveni et al. 1993) or by activating repolarizing Ca2+-dependent K+ channels (Lancaster and Nicoll 1987; Lancaster et al. 1991). Furthermore, influx of Ca2+ is important for a variety of cellular events including vesicular exocytosis, long-term potentiation, gene transcription, or induction of apoptosis. For these reasons, alterations of voltage-dependent Ca2+ channels in chronic disease are of considerable interest. Indeed, alterations of Ca2+ currents have been reported after electrical kindling in rats in CA1 pyramidal neurons and in dentate granule cells (Köhr and Mody 1991; Vreugdenhil and Wadman 1994).

In voltage-clamp experiments, Ca2+ conductances with a high threshold of activation (HVA Ca2+ currents) can be separated from those with a low threshold of activation (LVA or T-type channels; Hess 1990; Swandulla et al. 1991). HVA currents can be further differentiated with the use of specific antagonists such as dihydropyridines or the Conus geographus toxin omega -conotoxin (omega -CgTx) GVIA, which block L- and N-type Ca2+ currents, respectively (for review see Bean 1989). The funnel web spider toxin omega -agatoxin (omega -AgaTx IVA) blocks a noninactivating (P type; Brown et al. 1994) and an inactivating (Q type) Ca2+ current. Additional HVA components may be insensitive to all of these agents (R-type currents; Randall and Tsien 1995; Sather et al. 1993). Single-channel analyses of Ca2+ conductances present on the granule cell soma have shown that multiple Ca2+ channel subtypes coexist in these neurons (Blaxter et al. 1989; Fisher et al. 1990; Johnston et al. 1992). In addition, a careful pharmacological analysis of whole cell Ca2+ currents in dissociated guinea pig granule cells has identified N-, L-, and putative P-type Ca2+ currents in addition to a T-type current (Eliot and Johnston 1994). The purpose of the present study was to provide a characterization of the pharmacological and kinetic properties of calcium currents in isolated human dentate granule cells to compare these properties with those previously described in the kindling model of epilepsy and in adult control animals (Eliot and Johnston 1994; Köhr and Mody 1991) as well as in human neocortical neurons (Sayer et al. 1993).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Patient data

Surgical specimens from eight patients with pharmaco-resistant TLE were obtained for electrophysiological analysis (average age at surgery 33.0 ± 5.1 yr, mean ± SE). In addition, the hippocampus of a 10-mo-old child who underwent a hemispherectomy for therapy of therapy-refractory seizures caused by hemimegalencephaly was obtained. The mean duration of the TLE in the adult patients was 24.9 ± 8.2 (SE) yr and the mean age at the onset of seizures was 8.3 ± 4.2 yr. All adult patients suffered from complex partial seizures, with additional simple partial seizures in five patients and additional secondary generalized seizures in seven patients. The 10-mo-old child suffered from frequent Blitz-Nick-Salaam seizures. The seizure frequency per month over the 3 months preceding presurgical evaluation ranged from 2 to 10 complex partial seizures per month. None of the patients had known episodes of status epilepticus. In all adult patients, the hippocampus was shown to be intimately involved in the generation of temporal lobe seizures by noninvasive and invasive diagnostic procedures as described elsewhere (Engel et al. 1992). The surgical removal of the hippocampus was clinically indicated in every case to achieve seizure control. A lesionectomy with amygdalohippocampectomy (n = 2) and a selective amygdalohippocampectomy (n = 6) were performed. The 10-mo-old child underwent functional hemispherectomy. All patients were under a full antiepileptic drug regimen at the time of operation. Six patients showed a histopathological diagnosis of solitary Ammon's horn sclerosis with severe neuronal loss in the CA1, CA3, and CA4 subfield and relative sparing of CA2 (Margerison and Corsellis 1966). Two patients showed dysembryoblastic tumors not involving the hippocampus proper without any evidence for Ammon's horn sclerosis. Informed consent was obtained from all patients for additional histopathological and electrophysiological evaluation. In the case of the 10-mo-old child, consent was obtained from the parents. All procedures were approved by the ethics committee of the University of Bonn Medical Center and conform to standards set by the Declaration of Helsinki (1989).

Preparation of acutely isolated dentate granule cells

Isolated hippocampal granule cells were prepared similarly to methods described by Mody et al. (1989) and Sayer et al. (1993). Human hippocampal specimens were placed in ice-cold artificial cerebrospinal fluid containing (in mM) 125 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 10 glucose, and 26 NaHCO3, pH 7.4, 95% CO2-5% O2, immediately after surgical removal. A 4- to 5-mm-thick coronal segment of the corpus of the hippocampus was prepared with a razor blade and the tissue block was transferred to the stage of a vibratome (Campden Instruments, Longborough, UK). Coronal slices (400 µm) were prepared and alternate slices were transferred to an interface chamber for slice recording and a storage chamber with warmed artificial cerebrospinal fluid (95% CO2-5% O2) for preparation of acutely isolated neurons. In the storage chamber, isolation of viable neurons was possible up to 10 h after the preparation of vibratome slices. After an equilibration period of 60 min, the first section was transferred to a conical polystyrene tube with 5 ml of incubation medium containing (in mM) 125 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 piperazine-N,N-bis-2-ethanesulfonic acid (PIPES), and 25 glucose, 35°C, pH 7.4, 100% O2. Pronase (protease type XIV, Sigma) (2-3 mg/ml) was added to the oxygenated medium. After incubation for 25 min the slice was washed in ice-cold trituration medium containing (in mM) 126 NaCl, 2 KCl, 1 CaCl2, 2 MgCl2, 26 PIPES, 10 glucose, and 1.25 NaHPO4, pH 7.4, 100% O2. The dentate gyrus was dissected under a binocular microscope (Zeiss, Oberkochem, Germany) and triturated in 2 ml of ice-cold trituration solution with fire-polished glass pipettes. The cell suspension was then placed in a petri dish for subsequent patch-clamp recordings. At least two subsequent washes with extracellular recording solution (see below) were performed before whole cell recording was started. Isolated cells showed a round or ovoid small soma with a single process. This appearance is reminiscent of granule cell morphology in situ. Another type of neuron occurring in low numbers in the acutely isolated preparation showed a multipolar morphology with several processes emanating from the soma. Only neurons with a granule-cell-like morphology were included in the present study. The isolated cells were superfused with an extracellular solution containing 140 mM tetraethylammonium chloride (TEA), 5 mM 4-aminopyridine, 5 mM BaCl2, 10 mM glucose, 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), and 1 µM tetrodotoxin (chemicals obtained from SIGMA). The osmolarity was adjusted to 283 mosM with sucrose.

Patch-clamp whole cell recording

Patch pipettes were fabricated from borosilicate glass capillaries (1.5 mm OD, 1 mm ID; Science Products, Hofheim, Germany) on a Narishige P83 puller (Narishige, Tokyo, Japan). Pipettes usually had a resistance of 2-3 Momega . The pipettes were filled with an intracellular solution containing (in mM) 80 cesium methanesulfonate, 20 TEA, 1 CaCl2, 5 MgCl2, 11 ethyleneglykole-bis-(2-aminoethyl)-tetraacetic acid (EGTA), 10 HEPES, 10 adenosine-5'-triphosphate, and 0.5 guanosine-5'-triphosphate, osmolarityadjusted to 275-280 mosM with sucrose. Tight-seal whole cell recordings were obtained at room temperature (21-24°C) according to Hamill et al. (1981). Membrane currents were recorded with the use of a patch-clamp amplifier (EPC9, HEKA Elektronik, Lambrecht/Pfalz, Germany) and collected on-line with the TIDA for Windows acquisition and analysis program (HEKA Elektronik, Lambrecht/Pfalz, Germany). Command voltages were adjusted for -5- to -6-mV liquid junction potentials measured according to Neher (1992). The membrane capacitance was measured with the use of the EPC9 capacitance cancellation according to Sigworth et al. (1995). This estimate can yield results slightly better than those obtained with sine wave techniques (Gillis 1994; Sigworth et al. 1995). The mean membrane capacitance was 11.0 ± 2.7 pF. The input resistance of the examined neurons was determined by short hyperpolarizing voltage steps from a -50-mV holding potential and was >1 GOmega in most neurons with the recording solution (see above). The series resistance estimated by exponential fitting of the capacitance artifacts during brief hyperpolarizing voltage commands from -50 to -60 mV conformed well to the values obtained with the EPC9 capacitance cancellation paradigm (Sigworth et al. 1995; average 10.3 ± 3.7 MOmega for values obtained with the EPC9). Series resistance compensation was employed to improve the voltage-clamp control (30-40%). The maximal residual voltage error estimated from the product of series resistance and the maximal current Imax did not exceed 5.5 mV. All results were expressed as means ± SE.

Drug application

Drugs were applied with a four-barreled superfusion pipette placed at a distance of 30-50 µm from the cell body. The superfusion rate was adjusted by hydrostatic pressure. In some experiments, 30 µM CdCl2 or 1 or 5 µM NiCl2 were applied. omega -CgTx GVIA, omega -CgTx MVIIC, and omega -AgaTx IVA were prepared as a stock solution of 1 mM, 1 mM, and 400 µM, respectively, in deoxygenated water, divided into usable-sized aliquots, and stored at 20°C. Stock solution at 2 µl was added per 1 ml of extracellular medium for an end concentration of 2 µM omega -CgTx GVIA or MVIIC and per 0.5 µl/ml extracellular medium for an end concentration of 200 nM omega -AgaTx IVA. omega -CgTx GVIA and omega -CgTx MVIIC were obtained from RBI (Natick, MA). omega -AgaTx IVA was a generous gift from Pfeiser Central Research Division (Groton, CT). Toxins were applied in extracellular solution containing 500 µg/ml of bovine cytochrome C (Sigma) in most cases. Nifedipine was solubilized in dimethylsulfoxide at 10 mM (0.025%), stored in the dark, and diluted in the bath solution at 10 µM immediately before use. The pH was adjusted to 7.4 in all extracellular and intracellular solutions before recording.

Data analysis

Comparison of pharmacological effects on different current components was carried out with the aid of an analysis of variance. Because this test assumes normally distributed samples, a nonparametric test was carried out in addition (Mann-Whitney U-Wilcoxon Rank test). All statistical tests were carried out with the programme SPSS, version 6.1.2. (SPSS, Munich, Germany). All results were expressed as means ± SE.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Recordings from 61 human neurons were obtained between 5 and 90 min after isolation from hippocampal coronal slices of the resected hippocampus of eight patients with therapy-refractory TLE. Only measurements in which tight-seal whole cell recordings could be obtained for >20 min were included in the study; in most neurons, stable recordings could be obtained for between 30 and 80 min. Between 3 and 11 neurons were studied from each individual patient.

Properties of IBa in human dentate granule cells

When voltage steps to 0 mV were applied from a holding potential of -50 mV, slowly activating and inactivating Ba2+ inward currents could be observed. The voltage steps were separated by 10-s intervals to avoid current inactivation. The currents could be blocked completely by >30 µM Cd2+ (n = 4, not shown) and by 66.5% after addition of 10 µM Cd2+ to the recording medium in two neurons. The maximal amplitudes of IBa increased within the 1st min after the whole cell configuration was established, with a slow subsequent rundown. Amplitudes of IBa were reduced by 20.4 ± 12.9% after 27.5 ± 4.7 min of whole cell recording. Nevertheless, the kinetic parameters of IBa were all evaluated 5-10 min after patching onto the cell to minimize artifacts due to rundown. Fig. 1A shows a current family of inward Ba2+ currents elicited by depolarization to the various potentials indicated, showing a threshold of activation at -30-mV command potential. Currents evoked from holding potentials of -100 and -50 mV, respectively, are superimposed (Fig. 1A, left). When depolarizations were carried out from a potential of -100 mV, the amplitude of IBa was augmented in all cells under study. The average amplitude of IBa normalized for the cell capacitance was 144.3 ± 24.3 pA/pF when elicited from -100 mV (command voltage 0 mV). When currents elicited from -50 mV were subtracted from those elicited from -100 mV, the resulting traces seemed to show a more rapid inactivation during the 50-ms command pulse (Fig. 1A, right). At a holding potential of -50 mV (open circle ) as well as -100 mV (Fig. 1B, bullet ), there was no indication of a Ba2+ current with a low threshold of activation. The peak currents showed a bell-shaped dependence on the command pulse potential with a threshold at about -30 mV and a maximum at depolarizations to 0 mV (averages from 12 neurons; Fig. 1B). However, the current-voltage dependence of the current component obtained by subtraction (Fig. 1A, right) showed peak amplitudes at more negative potentials (about -10 mV, Fig. 1C).


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FIG. 1. Ba2+ currents IBa in human dentate granule cells. A, left: currents were evoked with 50-ms command pulses to the various potentials indicated on the left margin. Traces were obtained after a 2-s prepulse to -100 mV or directly from the holding potential of -50 mV (superimposed). When depolarizations were carried out from a potential of -100 mV, the amplitude of Ba2+ currents was augmented in all cells under study. A, right: difference current resulting from the subtraction of traces obtained from -50 mV from those obtained from -100 mV at the various command potentials indicated on the left margin. Note the different calibration bars. B: current-voltage relations of IBa (averages from 12 neurons) obtained from -50 mV (open circle ) and -100 mV (bullet ). Peak current values were measured, normalized to the maximal current obtained from -100 mV, and plotted vs. the command voltage. C: voltage dependence of the current component obtained by subtraction as in A, right (-50-mV from -100-mV holding potential; averages from 12 neurons). The resulting values were normalized to the maximal amplitude of the subtracted component and plotted vs. the command pulse voltage.

Steady-state inactivation of IBa

Because the differing amplitudes of IBa when elicited from -100 and -50 mV may reflect properties of steady-state voltage-dependent inactivation, the steady-state inactivation of IBa was investigated by varying 1-s prepulses between -100 and +40 mV and subsequently evoking IBa with a command pulse to 0 mV (see Fig. 2A, inset). Indeed, a gradual reduction of current amplitudes could be observed when the conditioning prepulses were varied in a range from -100 to -50 mV. Figure 2B shows the corresponding current-voltage relations. Because a component of IBa could not be inactivated by prepulses as positive as +40 mV, we subtracted current traces evoked after prepulses to +40 mV from those evoked at the various other potentials (see Fig. 2B, inset). These current values were then normalized to Imax and averaged and plotted as a function of the membrane potential. The data points were then fitted by a Boltzmann equation of the form
<IT>I</IT>/<IT>I</IT><SUB>max</SUB>= [1 + exp(<IT>V</IT><SUB>1/2</SUB><IT>− V</IT>)/<IT>K</IT>)]<SUP>−1</SUP> (1)
where I is the voltage-dependent current amplitude, V is the voltage-clamp potential, V1/2 is the voltage at which half-maximal values of IBa occur, and K is the slope factor. The data points could not be fitted well by a single Boltzmann equation as described in rat dentate granule cells with identical stimulation paradigms (Köhr and Mody 1991). Nevertheless, the best Boltzmann fit with a single exponential is shown superimposed on the data points (Fig. 2B,- - -), yielding values of voltage of half-maximal steady-state inactivation (V1/2inact) of -20.9 ± 1.3 mV (K = 11.0 ± 1.1). In contrast, the data points could be approximated very well with the sum of two Boltzmann functions of the above forms, yielding two voltages of half-maximal activation of -16.4 ± 0.9 mV (K = 7.9 ± 0.6; Imax1 = 0.80 ± 0.04) and -71.0 ± 4.8 mV (K = 12.1 ± 6.1; Imax2 = 0.23 ± 0.07). Best fits of the sum of two Boltzmann functions with these parameters are shown superimposed on the data points (Fig. 2B, ------). Whereas the more positive value of V1/2inact is in good agreement with values obtained for an HVA IBa (Mody et al. 1989), the current inactivating at more negative potentials may correspond to the difference currents showing rapid time-dependent inactivation in Fig. 1. These properties suggest that this conductance corresponds to a T-type current with similar properties of inactivation that has been described in guinea pig dentate granule cells (Eliot and Johnston 1994). By subtracting ideal current values---obtained from substituting fitted parameters for the HVA ICa---into the above Boltzmann equation from the total amplitude of IBa, we attempted to isolate the inactivation properties of the T-type ICa. The resulting values were normalized to the maximal amplitude and plotted versus the prepulse voltage (Fig. 2C, open circle ). A Boltzmann function is superimposed on the data points. For comparison, the ideal values for the HVA ICa used for subtractive isolation of the T-type current are shown (Fig. 2C, bullet ).


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FIG. 2. Steady-state voltage-dependent inactivation of ICa. A: steady-state inactivation of ICa was investigated by varying 1-s prepulses between -100 and +40 mV and subsequently evoking ICa with a 50-ms command pulse to 0 mV (see inset). B: current traces evoked after prepulses to +40 mV were subtracted from those evoked at the various other potentials (see inset). These values were normalized to maximum current (Imax), averaged, and plotted as a function of the membrane potential. The data points were fitted by a single Boltzmann equation (Eq. 1), shown superimposed on the data points(- - -) yielding values of voltage of half-maximal steady-state inactivation (V1/2inact) of -20.9 ± 1.3 mV (K = 11.0 ± 1.1), or by the sum of 2 Boltzmann functions of the above forms, yielding 2 voltages of half-maximal activation of -16.4 ± 0.9 mV (K = 7.9 ± 0.6; Imax1 = 0.80 ± 0.04) and -71.0 ± 4.8 mV (K = 12.1 ± 6.1; Imax2 = 0.23 ± 0.07; ------). C: isolation of the T-type current by subtracting ideal current values obtained from substituting fitted parameters for the high threshold of activation (HVA) ICa into the above Boltzmann equation (bullet ) from the total amplitude of ICa. Resulting values were normalized, plotted vs. the prepulse voltage (open circle ), and fitted with a Boltzmann function.

Time-dependent inactivation and activation of IBa

Because the T-type current seemed to inactivate more rapidly than the HVA Ca2+ currents, we attempted to characterize the properties of time-dependent inactivation of these current components. The time-dependent inactivation of the HVA ICa was determined from a holding potential of -50 mV. During 1-s command pulses, a slow time-dependent inactivation of the HVA ICa became apparent (Fig. 3A). The time-dependent decay of IBa during 1-s command pulses could be fitted adequately by a monoexponential equation of the form
<IT>I</IT><SUB>(t)</SUB><IT>= A</IT><SUB>0</SUB><IT>+ A</IT><SUB>1</SUB>* exp(−<IT>t</IT>/τ) (2)
where A0 is constant and A1 is the amplitude of IBa showing time-dependent decay with the time constant tau . Figure 3A shows the fitted curves obtained for the various command potentials superimposed on the decay of the HVA IBa. The decay time constants tau inact were voltage dependent, showing a steady decrease with more depolarizing command potentials (Fig. 3C). The T-type current was again isolated by subtracting current traces obtained from -50-mV holding potential from those obtained from -100 mV (Fig. 3B). These current traces showed a more rapid time-dependent inactivation during 1-s command pulses that could also be fitted by a single exponential (Fig. 3B). The decay time constant of the T-type current was also somewhat voltage dependent, showing a U-shaped dependence on the command pulse potential.


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FIG. 3. Properties of time-dependent inactivation of Ca2+ currents (ICa). A: time-dependent inactivation of the HVA Ca2+ currents was determined during 1-s depolarizations from a holding potential of -50 mV to the various command potentials indicated on the right margin. The time-dependent decay of ICa during 1-s command pulses could be fitted with a monoexponential equation (see Eq. 2). Fitted curves obtained for the various command potentials are superimposed on the current decay. B: time-dependent decay of the T-type current isolated by subtraction (see inset). C: voltage dependence of the decay time constants tau inact of the HVA (bullet ) and T-type components (black-square).

The time-dependent activation described by fitting a monoexponential equation to the rising phase of IBa was also dependent on the membrane potential and was faster with more depolarizing voltage steps (not shown). The time-dependent activation of the T-type current obtained by subtraction was not significantly different from that seen on depolarization from -50 mV. None of the kinetic parameters determined above were significantly different when properties of neurons isolated from the hippocampus of the 10-mo-old patient were compared with those from adult hippocampus. After the kinetic characterization of the two current components, we attempted a further discrimination of the different Ba2+ current components by pharmacological means.

Effects of nifedipine on HVA currents

Dihydropyridines have been shown to be a specific probe for L-type channels that do not seem to affect other Ca2+ channel subtypes (Bean 1989; Mintz et al. 1992; Nowycky et al. 1985). When depolarizing command pulses were delivered from a holding potential of -50 mV to inactivate most of the T-type current, nifedipine blocked 62.1 ± 10.9% of IBa with command pulses to 0 mV (Fig. 4A, n = 7). The difference currents obtained by subtraction of currents elicited in the presence of nifedipine from those in control solution showed a slow increase of current amplitude during the 50-ms command pulse (Fig. 4A, bottom). Comparison of the blocking effects by nifedipine at the end of the command pulse and 10 ms after the onset of the command pulse showed that a higher proportion of current could be blocked at the end of the command pulse (P < 0.05, 2-tailed Student's t-test, not shown). Because this behavior points to a voltage dependence of the blocking effects of dihydropyridines on L-type channels that has been previously described in various preparations (Eliot and Johnston 1994), we examined the effects of 1-s conditioning prepulses to various potentials from -100 to -20 mV on the fraction of IBa blocked by nifedipine. This fraction was normalized to the maximal blocking effect observed and plotted versus the prepulse voltage (Fig. 4B). This experiment showed that higher proportions of IBa could be blocked when depolarizing conditioning prepulses to -20 mV were applied before the test pulse (Fig. 4B). This may reflect either the contribution of the nifedipine-insensitive T-type current with a V1/2inact in a hyperpolarized potential range or a voltage-dependent action of nifedipine as suggested above (Fig. 4A). No shifts in the current-voltage dependence could be observed under 10 µM nifedipine (Fig. 4C); however, voltage ramps were not applied to exclude small shifts in current-voltage dependence as described in guinea pig dentate granule cells (Eliot and Johnston 1994). The voltage dependence of the nifedipine-sensitive difference current obtained as in Fig. 4A also peaked at 0 mV (Fig. 4D). The blocking action of nifedipine seemed not to be due to a shift in steady-state inactivation because the inactivation curves could be fitted with a sum of two Boltzmann functions as in Fig. 2 with no significant difference in both values of V1/2inact (not shown).


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FIG. 4. Effects of nifedipine. A, top: example for the blocking effect of 10 µM nifedipine. Currents were elicited by 50-ms command pulses to 0 mV from a holding potential of -50 mV. A, bottom: difference current obtained by subtracting the above traces in the presence of 10 µM nifedipine from control traces. B: to further assess the voltage dependence of the nifedipine blocking effects, 1-s conditioning prepulses were applied from -100 to -20 mV before Ca2+ currents were elicited with a 50-ms command pulse to 0 mV. The fraction of peak Ca2+ current during the command pulse blocked by nifedipine after the various 1-s conditioning prepulses was determined and then normalized to the maximal blocking effect observed. C: activation properties of HVA Ca2+ currents obtained by application of 50-ms command pulses to the various potentials from -50 to +50 mV. D: voltage dependence of the nifedipine-sensitive difference current obtained by subtracting traces in the presence of nifedipine from those in control solution at the various command potentials.

Effects of omega -CgTx GVIA, omega -CgTx MVIIC, and omega -AgaTx IVA on HVA currents

To further differentiate the different types of HVA currents in human granule cells, we applied toxins specific for different types of neuronal Ca2+ channels. Application of saturating concentrations of omega -CgTx GVIA (2 µM; Bean 1989; Fox et al. 1987a,b; Randall and Tsien 1995) resulted in block of 37.8 ± 17.6% of HVA IBa under control conditions at a command pulse potential of 0 mV (Fig. 5A; n = 9 in adult neurons). Application of 10 µM omega -CgTx GVIA did not result in additional block (n = 4, not shown). The omega -CgTx GVIA-sensitive difference current (Fig. 5A, bottom) seemed to show some time-dependent inactivation during 50-ms command pulses. When longer command pulses of 1 s were used, the time-dependent inactivation of this component became more apparent. This behavior was clearly distinct from that observed for nifedipine (Fig. 4A). The effects of omega -CgTx GVIA (10 µM) were clearly additive to those of nifedipine. Combined application of both agents blocked >95% of HVA currents when the T-type current was inactivated by a holding potential of -50 mV. The block by 2 µM omega -CgTx GVIA was to a small degree reversible in some neurons (Fig. 5, B and C). Current-voltage relations could be obtained for three neurons in control solution and in the presence of omega -CgTx GVIA and did not suggest shifts in the voltage-dependent activation behavior (Fig. 5C). In these experiments, the average blocking effect of 2 µM omega -CgTx GVIA was 27 ± 4%. As observed for nifedipine, the voltage dependence of the omega -CgTx GVIA-sensitive difference current obtained as in Fig. 5A peaked at 0 mV, indicating similar activation properties of these components (Fig. 5D).


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FIG. 5. Effects of omega -conotoxin (omega -CgTx) GVIA and omega -agatoxin IVA (omega -AgaTx IVA). A, top: 2 µM omega -CgTx GVIA resulted in block of 37.8 ± 17.6% of HVA ICa under control conditions. Traces were elicited with 50-ms command pulses to 0 mV from a holding potential of -50 mV (n = 9). A, bottom: difference current obtained by subtracting the above traces in the presence of 2 µM omega -CgTx GVIA from preceding traces in control solution with command pulses of 50 ms and durations of 1 s. B: time course of the block by omega -CgTx GVIA in juvenile (bottom) and adult (top) hippocampus. Currents were elicited with 50-ms pulses from -50-mV holding potential to 0 mV every 10 s. Horizontal bars: duration of toxin application. C: activation properties of HVA Ca2+ currents obtained by application of 50-ms command pulses to the various potentials from -50 to +50 mV. D: voltage dependence of the omega -CgTx GVIA-sensitive difference current obtained by subtracting traces in the presence of omega -CgTx GVIA from those in control solution at the various command potentials. E, top): effects of omega -AgaTx IVA in neurons derived from the hippocampus of a 10-mo-old child. E, bottom: difference current obtained by subtracting the above traces in the presence of 200 nM omega -AgaTx GVIA from preceding traces in control solution. F, top: time course of representative experiments in a neuron showing effects of omega -AgaTx GVIA (cell 2) and a neuron lacking an effect (cell 1). F, bottom: insensitivity of Ca2+ currents in immature granule cells to omega -CgTx MVIIC.

Effects of omega -AgaTx IVA

omega -AgaTx IVA showed blocking effects (23.7 ± 8.0%; Fig. 5E) in 3 of 12 neurons. These effects were partly reversible (Fig. 5F). The difference current obtained by subtracting currents in the presence of omega -AgaTx IVA from immediately preceding current traces in control solution yielded current traces that did not inactivate with time (Fig. 5E, bottom). In these neurons, additional application of omega -CgTx GVIA yielded block of another 25.1 ± 6% of HVA IBa, a somewhat lower proportion than could be blocked by omega -CgTx GVIA alone in adult neurons. All three neurons showing omega -AgaTx IVA effects were derived from the hippocampus of a 10-mo-old child who underwent a hemispherectomy (3 of 9 neurons, Fig. 5F, top, cell 2). The remaining neurons from this patient were not omega -AgaTx IVA sensitive (n = 6; Fig. 5F, top, cell 1). An omega -AgaTx IVA-sensitive component was not observed in neurons isolated from adult hippocampus (n = 3; not shown). Because omega -CgTx MVIIC may be useful in differentiating P-type from Q-type channels in immature dentate granule cells, we applied omega -CgTx MVIIC in concentrations of 2 µM. In the tested neurons from immature hippocampus, omega -CgTx MVIIC did not show significant blocking effects (n = 6, Fig. 5F, bottom).

Effects of Ni2+ on T-type and HVA Ba2+ currents

Because low concentrations of Ni2+ have been shown to have a preferential blocking effect on T-type currents and to block conductances resistant to the dihydropyridines omega -CgTx GVIA and omega -AgaTx IVA (Eliot and Johnston 1994), we applied Ni2+ in concentrations of 1 and 5 µM. HVA Ba2+ currents were elicited from a holding potential of -50 mV (Fig. 6A, top). T-type currents were isolated as described above by subtracting traces elicited from -50 mV from those elicited from -100 mV (Fig. 6A, bottom). The effects of Ni2+ were somewhat variable. Figure 6A shows an individual neuron in which 1 µM Ni2+ blocked the T-type component completely while the HVA currents were relatively unaffected. Such a complete block of a T-type conductance was obtained in three of nine neurons. Superfusion of 5 µM Ni2+ resulted in a complete blockade of T-type Ba2+ currents in the remainder of the neurons. However, in this concentration a substantial additional blocking effect on HVA IBa became apparent in all neurons (Fig. 6A, top). In those cells showing a block of the T-type current with 1 µM Ni2+, this effect was reflected in the steady-state inactivation curve (Fig. 6B, same cell as Fig. 6A). Although the sum of two Boltzmann functions had to be be employed to adequately fit the voltage-dependent inactivation in control solution (black-square), the data points could be fitted well with only one Boltzmann equation after application of 1 µM (bullet ) or 5 µM Ni2+ (Fig. 6B, open circle ). Figure 6C shows the average of all investigated neurons (n = 9). When the Ni2+-sensitive difference current was isolated by subtracting traces in the presence of 1 µM Ni2+ from preceding traces in control solution, the current-voltage relation of the difference current could be shown to peak at about -10 mV (Fig. 6D). Note the similarities to the current-voltage relation of the T-type component depicted in Fig. 1.


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FIG. 6. Effects of Ni2+. A, top: effects of 1 and 5 µM Ni2+ on HVA currents elicited from -50 mV. A, bottom: effects of 1 and 5 µM Ni2+ on the T-type component isolated by subtraction (see inset). A neuron is depicted in which the selective block of the T-type component by 1 µM Ni2+ was particularly prominent, whereas the HVA currents were relatively unaffected. B: steady-state inactivation in the neuron depicted in A. With 1 and 5 µM Ni2+, the steady-state inactivation could be fitted well with only 1 Boltzmann equation that is superimposed on the data points. Legend shown for B and C. C: steady-state inactivation averaged for all investigated neurons for 1 and 5 µM Ni2+. D: activation properties of the Ni2+-sensitive difference current.

Pharmacological properties of the T-type current

In contrast to the sensitivity of the T-type current to Ni2+, we expected this current component to be insensitive to omega -CgTx GVIA and to dihydropyridines. Indeed, T-type currents isolated by subtraction were not sensitive to 10 µM nifedipine or 2 µM omega -CgTx GVIA (Fig. 7, A-C; data from different cells). Figure 7A summarizes the blocking effects observed with the different blocking agents on the T-type current in comparison with the effects on HVA Ba2+ currents. We quantified the amplitudes of these two components of IBa during application of a blocking agent and normalized these values to the amplitudes in control solution. The results confirm that 1 µM Ni2+ preferentially blocks the T-type ICa (P < 0.05, analysis of variance), whereas 10 µM nifedipine and 2 µM omega -CgTx GVIA preferentially block the HVA IBa without affecting the T-type current (Fig. 7A; P < 0.05, analysis of variance). These results could be confirmed by a Mann-Whitney U-Wilcoxon Rank test. The small average depression observed for T-type currents on application of nifedipine or omega -CgTx GVIA may reflect contamination by HVA Ca2+ current components.


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FIG. 7. Pharmacological properties of the T-type current component compared with the HVA Ca2+ currents. A: amplitudes of both components under superfusion of a blocking agent were quantified and normalized to the amplitudes in control solution. Asterisks: significance of <0.05 (analysis of variance and Mann-Whitney U-Wilcoxon Rank test). B and C: individual traces showing the insensitivity of the T-type component isolated by subtraction to omega -CgTx GVIA and nifedipine (data from different neurons).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The electrophysiological properties of human (Isokawa et al. 1991, 1993; Williamson et al. 1993) and rat dentate granule cells (see Fricke and Prince 1984; Stanton et al. 1989) have been investigated in a number of studies employing intracellular recording with sharp microelectrodes. In this configuration, the analysis of voltage-dependent ion currents underlying the firing behavior of neurons is severely limited by the electrotonic properties of the intact processes. Therefore we have analyzed the properties of Ba2+ currents through voltage-dependent Ca2+ currents in acutely isolated human dentate granule cells from patients with therapy-refractory TLE. To reduce Ca2+-dependent inactivation of ICa as far as possible, we employed Ba2+ as a charge carrier and dialyzed the neurons with an intracellular solution containing 10 mM EGTA. In this recording configuration, we were able to discriminate multiple components of Ca2+ currents.

Kinetic properties of an HVA ICa

One component showed characteristic properties of HVA Ca2+ currents, namely, a high threshold of activation around -30 mV with maximal current on depolarization to 0 mV, a half-maximal inactivation around -16 mV, and a slow time-dependent inactivation under condition where intracellular Ca2+ is strongly buffered. These kinetic properties of the HVA ICa correspond well to those described in isolated rat dentate granule cells (Köhr and Mody 1991) and human cortical neurons (Sayer et al. 1993). Because considerable overlap in kinetic characteristics has been described for HVA Ca2+ currents, the contribution of different subtypes of Ca2+ currents required specific pharmacological tools. Because human material is relatively scarce, we decided to apply the different antagonists in concentrations known to be saturating from animal experiments without investigating the specificity and dose-response characteristics in detail.

Dihydropyridine-sensitive HVA Ca2+ current

In recent years, a number of specific pharmacological agents acting on subtypes of HVA Ca2+ currents has been described. Dihydropyridines block L-type Ca2+ channels but have been shown not to affect T-, N-, or P-type Ca2+ currents (Bean 1989; Mintz et al. 1992; Nowycky et al. 1985). Therefore we used nifedipine in concentrations of 10 µM as a probe to test for the presence of L-type Ca2+ channels in human dentate granule cells. Nifedipine blocked the largest component of whole cell Ca2+ current (62%) with depolarizations from -50 mV. Similar blocking effects (68%) for this holding potential were obtained in guinea pig dentate granule cells with saturating concentrations of nimodipine (Eliot and Johnston 1994). The dependence of the magnitude of block on the holding potential can be attributed to two effects. First, the T-type channel present in this preparation undergoes steady-state inactivation in a more negative potential range, leading to a higher proportion of L-type current in the control current. Second, the voltage dependence of the nifedipine effect itself may contribute to this phenomenon, as has been described for L-type currents in other preparations (Bean 1984; Eliot and Johnston 1994). That blocking effects of nifedipine increase on depolarization is also suggested by the increased effects at the end compared with the current 10 ms after onset of the command pulse. Data on the expression levels of different L-type channel subunits in the human hippocampus are not presently available, but high levels of L-type channels have been detected in the rat dentate gyrus on the protein level as well as on the mRNA level (Ahlijanian et al. 1990; Chin et al. 1992). Therefore the finding that a large proportion of the whole cell Ca2+ current in human dentate granule cells is dihydropyridine sensitive is not particularly surprising.

omega -CgTx GVIA-sensitive HVA Ca2+ current

omega -CgTx GVIA, a peptide component from the toxin of the marine snail C. geographus, has been well characterized as a specific blocker of N-type Ca2+ channels (Hess 1990; Sher and Clementi 1991). In human dentate granule cells, omega -CgTx GVIA blocked a slowly inactivating component that compares well with the kinetics of the N-type current in guinea pig dentate gyrus granule cells (Eliot and Johnston 1994). The magnitude of block (38%) was somewhat larger in our hands compared with ~20% in the guinea pig. The additive effects of omega -CgTx GVIA and nifedipine with combined application of both antagonists blocking >95% of Ca2+ currents suggests that HVA currents in adult human dentate granule cells are mainly carried by L-type and N-type conductances. This does not preclude the presence of distinct Ca2+ conductances on distal dendrites that cannot be detected in acutely isolated cells (Blaxter et al. 1989).

omega -AgaTx IVA-sensitive HVA Ca2+ current in immature hippocampus

The funnel web spider toxin omega -AgaTx IVA (Mintz et al. 1992) was used to characterize a further component of Ca2+ currents. omega -AgaTx IVA blocks P-type channels with a high degree of specificity with a half-maximal block in the nanomolar range in Purkinje cells (Randall and Tsien 1995), amygdala neurons (Foehring and Scroggs 1994), and other central and peripheral neurons (Brown et al. 1994; Mintz et al. 1992). At higher concentrations in the range of hundreds of nanomolars, omega -AgaTx IVA blocks an additional high-threshold current mediated by Q-type channels (Randall and Tsien 1995; Randall et al. 1996). Therefore the concentration of 200 nM omega -AgaTx IVA should block both the Q-type and the P-type channel if present in these neurons. omega -AgaTx IVA inhibited a small proportion of ICa in part of the neurons derived from immature hippocampus. The rapid onset of block and noninactivating decay kinetics as well as the insensitivity to omega -CgTx MVIIC suggests that the blocking effects of omega -AgaTx IVA indicate the presence of a P-type Ca2+ channel (Eliot and Johnston 1994; Mintz et al. 1992; Randall and Tsien 1995; Randall et al. 1993; Sather et al. 1993; Zhang et al. 1993). These data compare well with the presence of a putative P-type current in dentate granule cells obtained from guinea pig pups aged 8-20 days (Eliot and Johnston 1994) and to the absence of P-type Ca2+ channels in the adult rat dentate gyrus (Hillman et al. 1991).

Properties of the T-type ICa

The T-type Ca2+ current component could be discriminated by its distinct steady-state inactivation properties with a voltage of half-maximal inactivation around -70 mV. Several lines of evidence indicate that this component can be classified as T-type Ca2+ current. First, the steady-state inactivation in such a hyperpolarized range compares well with T-type channels in other preparations, where V1/2inact is around -80 mV (O'Dell and Alger 1991; Swandulla et al. 1991). The properties of steady-state inactivation permitted analysis of this current in relative isolation by subtracting currents elicited from -50-mV from those elicited from -100-mV holding potential. Second, the T-type current isolated by subtraction showed a more rapid time-dependent decay than the HVA Ca2+ currents (Bean 1989; Fedulova et al. 1985; Hess 1990; Swandulla et al. 1991) and a small shift of the peak amplitudes in the current-voltage relation in a hyperpolarizing direction as expected for T-type Ca2+ currents. Third, the T-type current component could be discriminated pharmacologically, showing significantly higher sensitivity to low concentrations of Ni2+ (1 µM) than the HVA ICa. Conversely, the T-type current showed almost no sensitivity toward nifedipine and omega -CgTx GVIA, in contrast to the HVA ICa. The component described in human dentate granule cells is very similar to a current component in guinea pig dentate granule cells showing strong voltage-dependent inactivation between -60 and -80 mV, rapid inactivation during depolarizing voltage commands, insensitivity to toxins, and high sensitivity to low concentrations of Ni2+ (Eliot and Johnston 1994). On the other hand, the extremely high sensitivity toward Ni2+ in human dentate granule cells suggests that the Ni2+-sensitive current may be partly due to expression of a channel similar to doe-1 or rbE-II. These channel proteins conduct currents that are very sensitive to Ni2+, and show peak amplitudes in a depolarized potential range (Ellinor et al. 1993; Soong et al. 1993; Zhang et al. 1993). The small relative amplitude of the T-type current in our experiments compares well with the small proportion of T-type channels observed in single-channel recordings (Fisher et al. 1990) and in whole cell recordings in guinea pig dentate granule cells (Eliot and Johnston 1994). In previous analyses of the HVA ICa in rat dentate granule cells with the use of Ca2+ as a charge carrier with strongly reduced extrinsic Ca2+ buffers, such a component was not observed (Köhr and Mody 1991).

Changes related to epilepsy

Up-regulation of Ca2+ currents has been reported in the kindling model of epilepsy in CA1 pyramidal neurons of the rat (Vreugdenhil and Wadman 1994), thus making voltage-dependent Ca2+ currents an interesting candidate for alterations in human TLE. In addition, loss of intracellular buffering capacity for Ca2+ reportedly leads to significant increase in Ca2+-dependent inactivation of voltage-dependent Ca2+ currents (Köhr and Mody 1991). In TLE patients, the discrimination of epilepsy-related changes from species differences is difficult because of the lack of control tissue. T-, N-, and L- type Ca2+ current components in immature control guinea pig (Eliot and Johnston 1994) are present in roughly the same proportions as in our study, with rather similar kinetic characteristics, suggesting that these currents may not be altered during chronic epileptogenesis. On the other hand, on comparison, the current density measured in human dentate granule cells was 144.3 ± 24.3 pA/pF, whereas the current density measured with an identical configuration in rat dentate granule cells was 91.3 ± 30.2 pA/pF (n = 10; P < 0.005, unpublished data). Although these data may point to a diffuse up-regulation of Ca2+ currents without shifts in the contribution of different subtypes in human TLE, these findings must nevertheless be treated with caution, because it is possible that Ca2+ currents in both normal and epileptic human tissue differ in the same way from normal rat Ca2+ currents.

In summary, we describe the coexistence of N-, L-, T-, and P-type Ca2+ currents in human hippocampal granule cells. The properties and relative proportions of these currents seem to be similar to those observed in rodent dentate granule cells. Whether the Ca2+ currents described here are similar to those in normal human beings cannot be decided unequivocally. Nevertheless, the data presented here may provide a basis for comparison with properties of Ca2+ currents in animal models of epilepsy and for the elucidation of mechanisms of action of drugs intended for use in human disease.

    ACKNOWLEDGEMENTS

  We thank Pfeiser Central Research Division, Groton, for the generous gift of omega -AgaTx IVA. In addition, we thank Dr. C. Steinhäuser and Prof. Urban for enlightening discussions, and Prof. Schramm, Prof. Zentner, and Dr. van Roost for providing neurosurgical specimens.

  This research was supported by a grant from the Ministry of Science and Education, Northrhine-Westfalia, University of Bonn Center Grant BONFOR 111/2, the Sonderforschungsbereich SFB 400 of the Deutsche Forschungsgemeinschaft, and the Stiftung Peter.

    FOOTNOTES

  Address for reprint requests: H. Beck, Dept. of Epileptology, University of Bonn Medical Center, Sigmund-Freud Str. 25, D-53105 Bonn, Germany.

  Received 12 August 1996; accepted in final form 2 December 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

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