Electrical Properties of a Cockroach Motor Neuron Soma Depend on Different Characteristics of Individual Ca Components

Janette D. Mills and Robert M. Pitman

School of Biological and Medical Sciences, Gatty Marine Laboratory, University of St. Andrews, Fife KY16 8LB, United Kingdom

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Mills, Janette D. and Robert M. Pitman. Electrical properties of a cockroach motor neuron soma depend on different characteristics of individual Ca components. J. Neurophysiol. 78: 2455-2466, 1997. The "fast" coxal depressor motor neuron (Df) of the cockroach is among the most extensively studied of insect neurons. It has been shown that the cell body of this neuron can exhibit active electrical properties, which may change over time or with chemical modulation. To further understand these electrical events and their modulation, inward currents in Df have been characterized under conditions in which outward currents have been suppressed. The inward current activated at potentials positive to -60 mV and peaked between -10 and 0 mV when measured in barium saline and between 0 and +10 mV when measured in calcium saline. The inward current was insensitive to Ni2+ (100 µM) but reduced by verapamil (50 µM) and abolished by Cd2+ (1 mM). Two components of ICa were identified by their sensitivity to either 50 µM nifedipine or micromolar Cd2+. The nifedipine-sensitive component activated positive to -60 mV and peaked between -10 and 0 mV, whereas the Cd2+-sensitive component activated positive to -40 mV and peaked between +10 and +20 mV. Immediately after dissection, depolarization of Df evoked plateau potentials, whereas 1-4 h after dissection, depolarization evoked action potentials. The plateau potentials were insensitive to 100 µM Cd2+ but blocked by 50 µM nifedipine, whereas the spikes required a combination of nifedipine (50 µM) and Cd2+ (100 µM) for complete suppression, indicating that only one component of ICa contributes to the plateau potential, whereas both components contribute to action potentials. Currents measured in calcium saline decayed faster than currents measured in barium saline. The inactivation characteristics were investigated with the use of double-pulse voltage-clamp experiments. ICa showed a greater degree of inactivation and slower recovery from inactivation than did IBa. Current decay and the extent of inactivation were reduced after injection of the calcium-chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). This suggests that the calcium current of this neuron displays calcium-dependent inactivation. An additional mechanism, most probably voltage-dependent inactivation, also occurs because IBa, even in neurons injected with BAPTA, displayed some inactivation. The inactivation characteristics may be important in determining activity displayed by Df. Indirect evidence suggests that intracellular calcium is high immediately after dissection. At this time, the calcium current may therefore be reduced due to calcium-dependent inactivation. This may, at least partly, explain why the cell does not spike shortly after dissection.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Voltage-dependent calcium currents are important in regulating a wide range of cellular activities, such as synaptic transmission, muscle contraction, and hormone secretion as well as contributing to the inward current of active electrical events like action potentials and plateau potentials (reviewed by Bean 1989; Hess 1990). In vertebrates, calcium channels have been characterized on the basis of their electrophysiological and pharmacological properties as either low-threshold T type or high-threshold L, N, and P type (reviewed by Bean 1989; Hess 1990) and more recently as Q and R type (Dunlap et al. 1995; Olivera et al. 1994). In insects, voltage-dependent calcium currents have been recorded in Drosophila (Byerly and Leung 1988; Saito and Wu 1991), Manduca (Hayashi and Levine 1992), Carausius (Ashcroft and Stanfield 1982), Schistocerca (Laurent et al. 1993; Pearson et al. 1993), Locusta (Bickmeyer et al. 1994a,b), and Periplaneta (Christensen et al. 1988; David and Pitman 1995a; Grolleau and Lapied 1996; Wicher and Penzlin 1997). High- and low-threshold component subtypes have also been identified in cockroach dorsal unpaired median (DUM) neurons (Grolleau and Lapied 1996; Wicher and Penzlin 1997). However, these calcium currents have properties that do not correspond to vertebrate channels in terms of their pharmacology, activation threshold, or kinetics (Bickmeyer et al. 1994a; Pearson et al. 1993; Wicher and Penzlin 1997).

The soma of the cockroach fast coxal depressor motor neuron, Df, may generate either action potentials or plateau potentials on depolarization, each of which are calcium dependent (Hancox and Pitman 1991, 1992); immediately after dissection, depolarization evokes plateau potentials, which are relatively low-amplitude, long-duration events, whereas 1-4 h after dissection, depolarization evokes all-or-none action potentials (Hancox and Pitman 1992). The soma of this neuron does not normally appear to possess tetrodotoxin (TTX)-sensitive sodium channels (Pitman 1979), but does exhibit calcium currents (ICa) (David and Pitman 1995a). In this paper we have investigated the inactivation properties and pharmacology of ICa to establish the role of different components of ICa in determining the type of electrical activity recorded from this neuron at different times. We have shown that ICa consists of nifedipine-sensitive and cadmium-sensitive components; the nifedipine-sensitive current underlies the plateau potential, whereas both components underlie the action potential. The inactivation characteristics of calcium currents also may be important in determining electrical activity in Df.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

All experiments were performed on the metathoracic "fast" coxal depressor motor neuron, Df (Pearson and Iles 1970), of adult male cockroaches (Periplaneta americana). Animals were decapitated, the mesothoracic and metathoracic ganglia and the first three abdominal ganglia were dissected out, then the metathoracic ganglion was desheathed for electrophysiological recording (see Pitman 1975). Experiments were performed in circulating oxygenated saline containing (in mM) 214 NaCl, 3.1 KCl, 9 CaCl2, and 10 N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer, pH 7.2. For experiments using a barium saline, CaCl2 was replaced with equimolar BaCl2. Nifedipine (Sigma) was first dissolved in ethanol (100%), and then the stock solution (10-3 M) was prepared by carefully adding saline while stirring and gently heating. Appropriate concentrations of ethanol were applied to the preparation as controls. Twenty- or 200-µl aliquots of nifedipine and verapamil (Sigma) were added to a side compartment of the chamber (total volume, 2 ml), where the oxygenation system mixed and diluted these agents before they reached the preparation. Concentrations are expressed as final values attained after agents had mixed in the experimental chamber. The calcium-chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; Sigma) was iontophoretically injected into neurons by 300-ms current pulses sufficient to hyperpolarize the membrane by 40 mV (4-10 nA). These pulses were delivered at 0.1 Hz for up to 20 min. In such experiments, electrodes were filled with solution containing 100 mM BAPTA and 100 mM KCl. Experiments were carried out at room temperature (20-23°C).

Df somata were penetrated by two thin-walled, fiber-filled borosilicate glass microelectrodes (Clark Electromedical, Pangbourne, UK). Microelectrodes for current-clamp recording contained 2 M potassium acetate and had resistances of 12-20 MOmega . For voltage-clamp recordings, the microelectrodes were filled with 2 M cesium chloride; the voltage electrode (for monitoring membrane potential) had a resistance between 8 and 15 MOmega , whereas the current electrode (used to apply current) had a resistance between 5 and 10 MOmega . These electrodes enabled voltage control of the soma to within ±0.2 mV as determined by a second independent voltage electrode. Outward potassium currents were blocked by applying 50 mM tetraethylammonium chloride (TEA+, Sigma) to the saline, and by leakage of cesium ions into the cell from the microelectrodes. This leakage of cesium ions was facilitated by applying a train of positive pulses. Current was monitored using a laboratory-built "virtual earth" amplifier circuit connected to the reference electrode in the experimental chamber. Data from current-clamp experiments were recorded on tape using a DTR 1204 digital tape recorder (Biological Science Instruments) and displayed on a Gould 1604 oscilloscope. A CED 1401 Plus computer interface (Cambridge Electronic Design) and associated software were used for generating voltage command pulses, recording digitized data, and for off-line analysis. This system provided a sampling frequency of 5-18 kHz, which was optimized automatically by the software, according to the parameters used (i.e., lower acquisition frequencies would operate during long sample periods). Where required, leakage correction was applied off-line using CED patch- and voltage-clamp analysis software. Hardcopy data were down-loaded from tape or computer using a Gould Colorwriter 6120 plotter or a Hewlett-Packard Laserjet 4P printer. All statistical data are presented as means ± SE.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Current isolation

Inward currents were measured with the use of a two-electrode voltage-clamp technique under conditions that minimized contamination by outward potassium currents (see METHODS). When the Df soma was depolarized from a holding potential of -70 mV, which is near the normal resting membrane potential recorded in these experiments (mean resting membrane potential: -70 ± 0.7 mV, n = 107), to progressively more positive membrane potentials, inward currents were elicited in saline containing either 9 mM calcium chloride or in saline in which equimolar barium chloride had replaced the calcium chloride (Fig. 1A). Typical current-voltage (I-V) relationships are shown in Fig. 1B, which were taken from the same cell first bathed in calcium saline and then in barium saline; the barium current (IBa) peaked between -10 and 0 mV and the calcium current (ICa) between 0 and +10 mV. A difference in calcium and barium I-V relationships has been observed in other preparations and is generally considered to be due to more effective surface charge screening by calcium ions, which would shift the gating characteristics of channels to more positive membrane potentials (Byerly and Hagiwara 1982). The currents reversed at potentials less positive than the predicted calcium equilibrium potential; the I-V relationship for IKCa indicates that ECa is more positive than +120 mV (David and Pitman 1995a; Thomas 1984). A similar discrepancy between the observed and predicted reversal potential of currents through calcium channels has been seen in other preparations, where it has been attributed to outward movement, through the calcium channel, of intracellular monovalent cations such as K+ (or Cs+ in Cs+-loaded neurons) (Brezina et al. 1994; Eckert and Chad 1984; Lee and Tsien 1982). Although, potassium currents were minimized by external TEA+ (50 mM) and intracellular cesium, activation of unblocked potassium currents could also contaminate the inward current and cause a negative shift in reversal potential. It is unlikely that chloride currents would significantly contaminate inward currents recorded from this neuron, because lowering the external chloride concentration had no discernible effect on the I-V relationship (data not shown). TTX had no effect on electrical activity measured under current-clamp (Hancox and Pitman 1992), nor did it block inward currents (not illustrated), indicating that voltage-dependent TTX-sensitive Na channels do not make a significant contribution to electrical events recorded from this preparation. Although the mean peak current amplitude in barium saline (measured at -10 mV) was 230 ± 27 nA (SE, n = 13 neurons) and the mean peak calcium current amplitude (measured at +10 mV) was 186 ± 23 nA (n = 14 neurons), this difference is not significant (P = 0.22), suggesting that the permeability for the two ions is similar.


View larger version (16K):
[in this window]
[in a new window]
 


View larger version (15K):
[in this window]
[in a new window]
 
FIG. 1. Calcium and barium currents recorded from motor neuron, Df. A: ICa and IBa elicited by stepping from a holding potential of -70 mV to the command potentials indicated. Note the faster decay of ICa compared with IBa. B: current-voltage (I-V) relationship for peak inward ICa and IBa measured in the same cell first in calcium saline, then after changing to a barium saline. The potential was held at -70 mV, and jumped for 50 ms to potentials between -50 and +70 mV.

Evidence indicating that calcium currents may be subdivided into components with different thresholds is provided by experiments in which the holding potential was varied. In most preparations (12 of 15), inward currents elicited by stepping from a holding potential more negative than the resting potential were larger than those seen from -70 mV (no change in current amplitude was recorded in the other 3 cases). Figure 2 shows recordings from a neuron in which currents elicited from a holding potential of -100 mV were larger than those obtained from -70 mV. As the holding potential was taken more positive than -70 mV, there was a progressive decrease in the amplitude of inward currents until, at a holding potential of -20 mV, no current was elicited on depolarization. As the holding potential became more depolarized, the peak of the I-V relationship shifted in a depolarizing direction (n = 4). At a holding potential of -100 mV, the currents activated by stepping to potentials positive to -60 mV and peaked at -10 mV, whereas using a holding potential of -40 mV, currents activated positive to -30 mV and peaked at 0 mV (Fig. 2). Because the inward current displays inactivation even at the resting membrane potential, this suggests that it may have a low-threshold component.


View larger version (20K):
[in this window]
[in a new window]
 
FIG. 2. Effect of holding potential on the I-V relationship of IBa. The membrane potential was held at the potentials indicated on each graph and then stepped to command potentials between -60 and +20 mV. Insert: current traces obtained by stepping from the holding potentials indicated to a command potential of -10 mV.

Pharmacology of inward currents

Calcium channels have been characterized according to their sensitivity to a number of drugs and toxins and by their voltage threshold. Although a number of compounds, such as verapamil and its analogue D-600 block calcium channels but do not discriminate well between different channel classes, other antagonists exhibit selectivity. Divalent cations can show some discrimination between different subclasses of calcium channels; low-threshold T-type channels are preferentially inhibited by micromolar concentrations of nickel (Ni2+), whereas high-threshold channels are preferentially blocked by micromolar concentrations of cadmium ions (Cd2+) (Fox et al. 1987). In cockroach DUM cells, low-threshold Ni-sensitive and Ni-insensitive calcium currents have been identified (Grolleau and Lapied 1996).

As in other preparations, the inward calcium and barium currents in Df were sensitive to verapamil, which, at a concentration of 50 µM, produced up to 90% (85 ± 4%, n = 3) block of inward current (Fig. 3). A subsequent addition of 1 mM cadmium chloride blocked the remaining current (Fig. 3). However, calcium and barium currents were insensitive to Ni2+ at 100 µM (Fig. 4; n = 5), suggesting that there is no current similar to the Ni2+-sensitive low-threshold "T" type of vertebrates or the low-threshold Ni-sensitive current identified in cockroach DUM cells (Grolleau and Lapied 1996; Wicher and Penzlin 1997). Both ICa and IBa were sensitive to Cd2+; a partial block was produced by 10 µM Cd2+ (peak inward current inhibited by 28 ± 1%, n = 4) and complete block by 0.5-1 mM Cd2+ (n = 15, Fig. 4). Low concentrations of Cd2+ (<100 µM) preferentially blocked inward currents that activated at potentials positive to -40 mV (Fig. 4). In the presence of Cd2+, the peak of the I-V relationship was shifted by 10 mV in a hyperpolarizing direction (n = 4; arrows, Fig. 4). This suggests that micromolar Cd2+ preferentially blocks calcium currents that are activated at relatively positive potentials and supports the view that calcium currents in Df have more than one component as reported previously (David and Pitman 1995a). However, at higher concentrations of Cd2+, this selectivity was lost, and currents activated at lower potentials were also suppressed (Fig. 4).


View larger version (25K):
[in this window]
[in a new window]
 
FIG. 3. Effect of verapamil and Cd2+ on ICa. I-V relationships for peak inward ICa constructed from a holding potential of -70 mV to command potentials between -80 and +30 mV in control (bullet ), 50 µM verapamil (black-square), and after a subsequent application of 1 mM Cd2+ in the continued presence of verapamil (black-triangle). Insert: current traces elicited by stepping to 0 mV.


View larger version (24K):
[in this window]
[in a new window]
 
FIG. 4. Effect of Ni2+ and Cd2+ ions on the I-V relationship for IBa. Holding potential was -80 mV. There was little difference in the I-V relationship measured in control (bullet ) and 100 µM Ni2+ (black-diamond ). Low concentrations of Cd2+ (10-50 µM) preferentially blocked inward current elicited by stepping to potentials positive -40 mV. In the presence of Cd2+, the peak inward current was shifted by 10 mV in a negative direction (see arrows). At higher concentrations of Cd2+, the selectivity was lost; inward current activated at more negative potentials was also blocked. At concentrations >500 µM Cd2+, all inward current was inhibited.

Components of inward current show differential sensitivities to nifedipine and micromolar cadmium

Mammalian L-type channels are blocked by dihydropyridines, such as nifedipine applied in nanomolar concentrations (Bean 1984; Fox et al. 1987). In Df somata, nifedipine inhibited ICa at concentrations above 20 µM; 100 µM blocked 47 ± 7% (n = 6) of peak ICa that was activated by stepping from -70 to 0 mV (Fig. 5A). A subsequent application of 10 µM Cd2+ further inhibited the current and 1 mM Cd2+ completely blocked inward current (Fig. 5A). Comparison of the effects of 50 µM nifedipine and 10 µM Cd2+ on the I-V relationship of ICa indicates that the two drugs target currents activated at different potentials; the Cd2+-insensitive current that activated negative to -40 mV (see Fig. 4) was inhibited by nifedipine (Fig. 5B). Nifedipine, however, was less effective at blocking currents that activated by stepping to test potentials more positive than zero, which were inhibited by subsequent application of Cd2+ (still in the presence of nifedipine; Fig. 5, B and C). After block of ICa by nifedipine, the peak of the I-V relationship shifted in a depolarizing direction (Fig. 5B). The percentage block of total ICa by 50 µM nifedipine or by 10 µM Cd2+ at different membrane potentials is compared in Fig. 5C. This figure shows that the block by nifedipine decreases, whereas the block by Cd2+ increases as the membrane is stepped from a holding potential of -80 mV to progressively more positive potentials.


View larger version (28K):
[in this window]
[in a new window]
 
FIG. 5. Effect of nifedipine and Cd2+ on ICa. A: time course of inhibition of ICa; the cell was held at -70 mV and stepped to 0 mV once every 15 s. The current was partially blocked by nifedipine at the concentrations indicated. A further inhibition was seen after a subsequent application of 10 µM Cd2+, and complete block after perfusing with 1 mM Cd2+. B: effect of 50 µM nifedipine and 10 µM Cd2+ on the I-V relationship of ICa (holding potential -80 mV). Nifedipine was applied first, followed by Cd2+ (still in the presence of nifedipine). C: mean percentage block of ICa obtained for peak inward currents elicited by stepping the membrane potential from a holding potential of -80 mV to command potentials between -40 and +20 mV by either nifedipine (50 µM; n = 5) or Cd2+ (10 µM; n = 6). D: effects of 50 µM nifedipine and 10 µM Cd2+ on the I-V relationship of ICa at a holding potential of -40 mV. Values obtained for the same cell as in B. E: mean percentage block of ICa obtained for peak inward currents elicited from a holding potential of -40 mV to command potentials between -30 and +20 mV by either nifedipine (50 µM; n = 5) or Cd2+ (10 µM; n = 6).

L-type calcium currents display less voltage-dependent inactivation than other subtypes; for example in chick neurons, L-type current can be isolated by the use of a holding potential of -40 mV, which inactivates T and N type (Fox et al. 1987). However, in Df, currents activated from a holding potential of -40 mV were less sensitive to nifedipine than currents activated from -80 mV (Fig. 5D). With the use of a holding potential of -40 mV, the percentage block produced by nifedipine or cadmium did not vary with command potential (Fig. 5E), indicating that the current activated from this potential consists primarily of the cadmium-sensitive component. The fact that, at this holding potential, nifedipine produces some block of the inward current suggests that it may exert some small effect on the Cd2+-sensitive current component. After application of nifedipine, the I-V relationship, constructed from a holding potential of -40 mV, did not show a shift in a depolarizing direction (Fig. 5D) such as that observed when the holding potential was -80 mV (Fig. 5B). This would be expected if nifedipine-sensitive current has undergone steady-state inactivation at a holding potential of -40 mV. The nifedipine-sensitive current in Df therefore displays steady-state inactivation at more negative potentials than a typical L-subtype channel, which does not display substantial inactivation at a holding potential of -40 mV.

Nifedipine- and Cd2+-sensitive currents were obtained by subtracting the current that was blocked by either 50 µM nifedipine or 10 µM Cd2+ from those measured under control conditions (Fig. 6A). Nifedipine-sensitive currents showed faster inactivation than the Cd2+-sensitive currents (Fig. 6A and Table 1). Although nifedipine-sensitive currents appeared to activate slightly more rapidly than Cd2+-sensitive currents, the difference was not statistically significant. Mean I-V curves for the nifedipine- and Cd2+-sensitive currents have been constructed (Fig. 6B). The nifedipine-sensitive current activated at potentials positive to -60 mV and peaked between -20 and 0 mV (Fig. 6Bi), whereas the Cd2+-sensitive current activated positive to -50 mV and peaked at potentials between +10 and +20 mV (Fig. 6Bii).


View larger version (27K):
[in this window]
[in a new window]
 
FIG. 6. Nifedipine and Cd2+-sensitive calcium currents. Difference currents were obtained by subtracting currents obtained in the presence of the channel blocker from corresponding control currents. Ai: current traces of nifedipine-sensitive currents elicited by stepping the membrane potential from a holding potential of -80 mV to command potentials between -40 and 0 mV in 10-mV increments. Aii: current traces of Cd2+-sensitive currents elicited by stepping the membrane potential from a holding potential of -80 mV to potentials between -30 and +10 mV in 10-mV increments (different Df neuron to Ai). Bi: mean I-V relationship of calcium currents sensitive to 50 µM nifedipine (n = 8). Bii: mean I-V relationship of calcium currents sensitive to 10 µM Cd2+ (n = 9).

 
View this table:
[in this window] [in a new window]
 
TABLE 1. Time constant of decay of nifedipine- and cadmium-sensitive currents

Thus, to summarize, the differential effects of Cd2+ and nifedipine indicate that there are two components to the inward current in the soma of Df: one component that activates at potentials positive to -60 mV, displays rapid activation and inactivation, and is sensitive to nifedipine, but insensitive to micromolar Cd2+; and a second component that activates at potentials positive to -50 mV, is sensitive to micromolar Cd2+, and displays slower kinetics than the nifedipine-sensitive component.

Effect of cadmium and nifedipine on electrical activity under current clamp

Upon depolarization under current clamp, Df somata can generate plateau potentials shortly after dissection and isolation of the nerve cord, but usually produce action potentials several hours later (Hancox and Pitman 1992, 1993). Plateau potentials have a threshold between -60 and -40 mV (mean: -50.9 ± 0.7, n = 64) and reach a mean membrane potential of -37.2 ± 1.1 mV, (n = 68) (unpublished observations). It might be expected from the I-V relationships for ICa, that plateau potentials would be insensitive to micromolar concentrations of Cd2+, which does not block ICa over this range of potentials, but could be blocked by nifedipine, which does block ICa at these potentials. The effects of micromolar Cd2+ and nifedipine on electrical activity in Df measured under current-clamp conditions are shown in Fig. 7. As predicted, the plateau potential is insensitive to Cd2+ at concentrations up to 100 µM (Fig. 7Aii) but is depressed by 50 µM nifedipine (Fig. 7Aiii, n = 5). Cadmium (100 µM), however, only partially suppressed the action potentials (Fig. 7Bii), and complete block of the action potential was only attained in the presence of both nifedipine and Cd2+ (Fig. 7Biii, n = 6). This is not surprising since both currents are activated in the range of membrane potentials encountered during an action potential (-50.5 ± 1 mV up to -21 ± 2.5 mV, n = 14). These observations suggest that a nifedipine-sensitive current underlies the plateau potential, whereas both nifedipine- and Cd2+-sensitive currents underlie the action potentials.


View larger version (17K):
[in this window]
[in a new window]
 
FIG. 7. Effect of Cd2+ and nifedipine on electrical activity recorded under current-clamp conditions. A: a plateau potential elicited by depolarization (Ai) is not blocked by 100 µM Cd2+ (Aii) but is blocked by 50 µM nifedipine (Aiii). B: time-dependent spikes require a combination of micromolar Cd2+ and nifedipine for complete suppression. The action potentials (Bi) are partially suppressed by 100 µM Cd2+ (Bii) and completely blocked by a further addition of 50 µM nifedipine (Biii).

Inactivation of currents

One possible reason why the soma of Df is unable to spike immediately after dissection could be that the intracellular concentration of calcium in the neuron is elevated at this time. This proposal is supported by the observation that intracellular injection of calcium chelators enables the cell to spike (Pitman 1979), whereas agents such as caffeine that increase intracellular calcium cause a spiking cell to revert to plateaux (personal observation). A high intracellular calcium concentration could prevent spiking first by enhancing the calcium-dependent potassium current and, second by depressing the calcium current itself if it displays calcium-dependent inactivation. Although we already have evidence to support the first mechanism (unpublished observations), experiments were performed to establish whether the inactivation characteristics of the calcium current could contribute to the transition in the electrical properties of Df. Evidence has already been presented indicating that some steady-state inactivation of Ca currents occurs at potentials close to the resting potential of the neuron (Figs. 2 and 5).

In the experiments presented here, no attempt was made to dissect the current into its nifedipine and Cd2+ components because both contribute to the inward current of spikes. Inactivation of ICa and IBa was evident as a decay in the inward current during depolarizing steps (Figs. 1 and 8). The time course of decay (tau ) for ICa was faster than that for IBa. Using depolarizations <100 ms duration, both calcium and barium currents showed decays that could be best fitted by a single exponential (Fig. 8A). For ICa, tau  was 52 ± 6.5 ms (n = 9) at -30 mV, was fastest between -20 and -10 mV (31 ± 2 ms, n = 19) and then increased with further depolarization (Fig. 8B). IBa decayed with a time constant approximately double that of ICa; at -40 mV, tau  for IBa was 105 ± 8 ms (n = 6), was fastest at -20 mV (67 ± 2 ms, n = 12), and increased again at more positive membrane potentials (Fig. 8B). The faster inactivation of ICa compared with IBa suggests that ICa shows calcium-dependent inactivation. Further evidence to support this is that intracellular injection of the calcium-chelator, BAPTA, into the soma of Df slows current decay (Fig. 8C, n = 3). For ICa measured during longer duration depolarizing pulses (500 ms) in the absence of BAPTA, a double exponential was required to accurately fit the current decay; after injection of BAPTA, a single exponential was sufficient (Fig. 8C). Decay of IBa measured during long depolarizing pulses could normally be fitted with a single exponential. This suggests that the faster component of decay of ICa was due to a calcium-dependent inactivation process.


View larger version (15K):
[in this window]
[in a new window]
 
FIG. 8. Time constant of decay of ICa and IBa. A: peak inward currents induced by stepping from -80 to -10 mV (IBa) or +10 mV (ICa) for 50 ms can be fitted (dashed line) between the vertical lines with a single exponential (using the equation, A + Be-t/tau where tau  is the time constant of decay). B: time constant of decay with respect to command potential for IBa and ICa (mean ± SE, n values between 6 and 11). Ci: peak inward current for ICa elicited by stepping from -80 to +10 mV for 500 ms can be best fitted between the vertical lines by a double exponential shown as a dashed line (using the equation, A + Be-t/tau 1 + C e-t/tau 2 where tau 1 and tau 2 are the time constants of decay). Cii: after injection of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) into the soma, decay is best fitted by a single exponential.

The extent of inactivation can be determined with the use of a double pulse protocol. During the first pulse (prepulse), inactivation of the current developed. Test pulses to a potential sufficient to evoke maximum inward current (+10 mV in calcium saline and -10 mV in barium saline) showed the fraction of channels not inactivated during the preceding prepulse. An interpulse interval of 10 ms was chosen during which the membrane potential was returned to -70 mV. Typical current records are shown in Fig. 9A for ICa and IBa. When the test pulse was not preceded by a prepulse (traces 1), a large inward current could be recorded during the test pulse. A prepulse that activated a large inward current produced a great deal of inactivation as can be seen by the small amplitude current induced by the test pulse (traces 2). Application of prepulses sufficiently positive to produce a smaller inward current resulted in less inactivation, and a larger current was induced by the subsequent test pulse (Fig. 9A, traces 3). Prepulses of 200 ms were normally sufficient to produce maximal inactivation of calcium currents, whereas 400-ms pulses were necessary to have a similar effect on barium currents (not illustrated). Therefore 500-ms duration was normally used in the experiments to ensure that maximal inactivation would occur, while avoiding irreversible rundown of inward currents, which frequently occurred when prepulses of >= 1 s were used. Inactivation curves for IBa, ICa in both uninjected and in BAPTA-filled neurons were constructed by normalizing the current flowing during the test pulse (I) to its size in the absence of a prepulse. The mean inactivation curves for ICa (n = 10) and IBa (n = 12) and ICa in BAPTA-filled neurons (n = 4) showed steep inactivation at membrane potentials more positive than -60 mV for IBa and -50 mV for ICa in uninjected neurons and in BAPTA-filled neurons (Fig 9B). ICa in both uninjected and in BAPTA-filled neurons showed maximum inactivation between 0 and +20 mV, whereas inactivation of IBa peaked between -20 and 0 mV (Fig. 9B). The potential at which half inactivation occurred was at -39 ± 4 mV (n = 16) for IBa, at -28 ± 4 mV (n = 13) for ICa, and at -24 ± 5 mV (n = 4) for ICa in BAPTA-filled neurons (Fig. 9B). Maximum inactivation was greater for ICa (93 ± 9%, n = 15), than for IBa (70 ± 4%, n = 10) or for ICa in BAPTA-filled neurons (61 ± 3%, n = 4). The difference between the inactivation curves obtained for ICa in uninjected and BAPTA-injected neurons gives an indication of the extent of calcium-dependent inactivation.


View larger version (13K):
[in this window]
[in a new window]
 
FIG. 9. Inactivation characteristics of ICa and IBa. A: example of typical current traces elicited during a double pulse experiment for ICa (Ai) and IBa (Aii). The test pulse evoked maximum inward current (to +10 mV for ICa and -10 mV for IBa). When the test pulse was not preceded by a prepulse (1), a large current could be recorded during the test pulse. When maximum inward current was elicited during the prepulse (2), little inward current was elicited during the test pulse. A prepulse to a more positive potential (+40 mV) at which net inward current was smaller (3), resulted in less inactivation as can be seen by the slightly larger currents induced by the subsequent test pulse (3). B: mean inactivation curves for calcium currents (n = 10), barium currents (n = 12), and calcium currents in neurons injected with BAPTA (n = 4), constructed by normalizing the current flowing during a test pulse to its size in the absence of a prepulse.

At more positive membrane potentials there was a decrease in inactivation as a function of depolarization, resulting in U-shaped inactivation curves (Fig. 9B). The underlying mechanism is not clear. In principle, this could be produced by facilitation; in some other preparations, prepulses to relatively positive membrane potentials can enhance the calcium current, which is a process known as facilitation (review by Dolphin 1996). In bovine chromaffin cells, an L-type conductance is normally quiescent but can be activated by a large predepolarization (to +120 mV) or repetitive depolarization to physiological potentials (Artalejo et al. 1990, 1991), which involves voltage-dependent phosphorylation (Artalejo et al. 1992). A similar enhancement of calcium current has been seen for cardiac L-type calcium channels, where the normal gating mode is changed to a mode characterized by long openings and high open probability (Pietrobon and Hess 1990). If facilitation was occurring as well as inactivation at positive potentials in Df, then this could explain why the inactivation curves were U shaped. To determine whether facilitation was occurring in Df, shorter duration prepulses to positive potentials were used because these should favor facilitation over inactivation. Prepulse potentials up to +120 mV of duration between 5 and 50 ms did not produce any facilitation of current generated by subsequent test pulses for either ICa or IBa in uninjected or BAPTA-injected cells (n = 7; Fig. 10). The U-shaped inactivation curves are therefore unlikely to be the result from facilitation of the current at positive potentials. The most likely reason for the U-shaped inactivation curve is a reduction in the extent of inactivation at positive membrane potentials produced by 1) influx of calcium or of other cations or 2) by some undescribed inactivation component.


View larger version (9K):
[in this window]
[in a new window]
 
FIG. 10. ICa does not display facilitation at positive membrane potentials. A prepulse to +100 mV for 5 ms does not increase the amplitude of current induced by a subsequent test pulse to +10 mV compared with current induced in the absence of a prepulse.

Recovery from inactivation and cumulative inactivation

Recovery from inactivation was investigated by increasing the interval between the prepulse and test pulse. Increasing the interval between the prepulse and test pulse from 10 to 200 ms in barium saline decreased the inactivation measured at -10 mV by 31 ± 5% (n = 3; Fig. 11A). In calcium saline, <5% recovery from inactivation was seen by increasing the interpulse interval from 10 to 200 ms (n = 3, Fig. 11B). This suggests that recovery from inactivation is faster for IBa than for ICa, probably because removal of intracellular calcium (either by buffering or by pumping into stores or into the extracellular space) is slower than recovery from voltage-dependent inactivation.


View larger version (25K):
[in this window]
[in a new window]
 
FIG. 11. Recovery from inactivation. A: increasing the interpulse interval from 10 ms to the values indicated (between 50 and 200 ms) decreases the extent of inactivation of IBa, showing that recovery from inactivation can occur within this time scale. B: no recovery from inactivation was observed for ICa using the same protocol as in A. C: IBa displays cumulative inactivation measured by stepping repeatedly from -80 mV to a test potential of -10 mV at the intervals indicated (between 1 and 10 s). Current is normalized relative to that measured during the 1st pulse. Complete recovery from cumulative inactivation required an interpulse interval of 10 s. D: cumulative inactivation for ICa using test potentials to +10 mV shows similar characteristics to that measured for IBa.

The effect of cumulative inactivation was determined by giving a train of test pulses (50 ms duration) to a potential that evoked maximal inward current (-10 mV in barium saline, n = 3, and +10 mV in calcium saline, n = 3). The amount of inactivation was measured by normalizing the current during successive test pulses to that measured during the first pulse. The interval between the pulses was varied between 1 and 15 s to estimate the time required to recover from inactivation. For both IBa and ICa, cumulative inactivation of the current was seen with each successive pulse (Fig. 11, C and D). This was most apparent when the interpulse interval was <3 s; at an interpulse interval of 1 s, less inactivation was produced for IBa (Fig. 11C) than for ICa (Fig. 11D). However, with longer interpulse intervals, there was little difference in the extent of inactivation between ICa and IBa. Although little inactivation occurred when the interpulse interval was >5 s, an interval of 10-15 s was normally required for recovery to occur completely.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

This paper describes the characteristics of calcium currents in the soma of the cockroach Df motor neuron and their importance in determining the type of electrical activity exhibited by this cell.

Calcium channels in vertebrate neurons

In vertebrates, calcium channels have been classified on the basis of their pharmacological and electrophysiological properties as either high-threshold or low-threshold (Nowycky et al. 1985; Tsien et al. 1988; reviews by Bean 1989; Hess 1990; Olivera et al. 1994). The T-type channel is an example of a low-threshold calcium channel; in dorsal root ganglion cells, it activates positive to -70 mV, reaching a maximum between -40 and -20 mV, shows rapid transient channel kinetics, steady-state inactivation even for small depolarizations, greater sensitivity to Ni2+ than to Cd2+, and has similar permeabilities to calcium and barium (Fox et al. 1987). As yet, no specific blocking ligand has been found for the T-type channel. The high-threshold group of calcium channels shows greater permeability to barium than calcium and is insensitive to Ni2+ but blocked by micromolar Cd2+. This group can be further subdivided into L, N, P, Q, or R type. The L type is dihyropyridine sensitive, activates positive to -20 mV, and is slowly inactivating. The N-type channel is blocked by omega -conotoxin GVIA and shows inactivation kinetics intermediate between L- and T-type channels. Other subtypes of high-threshold calcium channels (P and Q type) have been identified by their sensitivity to omega -agatoxin IVA and omega -conotoxin MVIIC (Mintz et al. 1992; Olivera et al. 1994; Uchitel and Protti 1994). Another class of high-threshold calcium channel, R type, which has been described recently, is insensitive to dihydropyridines, omega -conotoxins, and omega -agatoxin IVA (Dunlap et al. 1995; Zhang et al. 1993).

Calcium channels identified in cockroach neurons

The calcium current in the soma of Df consists of at least two different components: one that activates positive to -60 mV, peaks between -10 and 0 mV, and is preferentially blocked by nifedipine, and another component that activates positive to -50 mV, peaks between +10 and +20 mV, and is inhibited by 10 µM Cd2+. It is possible that more than one conductance may underlie each component, especially the Cd2+-sensitive component, because Cd2+ has been shown to block a number of high-threshold calcium channels in other preparations. The calcium current in Df is less sensitive to nifedipine, and displays steady-state inactivation at more negative membrane potentials than a typical vertebrate L-type current. Nanomolar concentrations of nifedipine inhibit vertebrate L-type current, although at high concentrations, selectivity may be lost; at 100 µM it may blockT-type channels (Cohen and McCarthy 1987; Cohen et al. 1992; Van Skiver et al. 1989). It is possible therefore that, in these experiments, more than one class of calcium channel may have been blocked. This is a general problem that can occur when vertebrate channel pharmacology is applied to invertebrate preparations. The nifedipine-sensitive current in Df, like the T type, is activated by small depolarizations and shows relatively fast inactivation kinetics; however, it is not blocked by Ni2+ ions and so cannot be considered to be a typical T-type channel. Therefore it appears to show characteristics intermediate between the high-threshold L type and the low-threshold T type. The threshold of activation and Ni insensitivity of the nifedipine-sensitive current are similar to the low-threshold current found in the cockroach DUM cells (Grolleau and Lapied 1996). That current displays little inactivation at normal [Ca2+]i, but almost completely inactivates when [Ca2+]i is elevated to 0.1 mM (Grolleau and Lapied 1996). It is possible that this low-threshold current recorded from DUM neurons is similar to the LVA current reported here, if the [Ca2+]i in Df is intermediate between the two concentrations used by Grolleau and Lapied (1996).

The current that is blocked by micromolar Cd2+ shows properties of a typical high-threshold channel in its activation threshold and inactivation kinetics. However, as yet we have not further characterized this conductance. Because no block has been observed by omega -conotoxin GVIA (Pitman, personal observation), it cannot be classified as a typical N-type channel. It may therefore belong to the P/Q/R class or even a unique subclass.

Calcium currents in other insects

The calcium currents measured in Df somata show some similarities to currents measured in other insect neurons. In locust medial neurosecretory cells of the pars intercerebralis, a partial sensitivity to dihydropyridines but no block byomega -conotoxin GVIA has been seen (Bickmeyer et al. 1994a). Dihydropyridines also had only a small effect on the calcium current in Drosophila neurons (Byerly and Leung 1988). Verapamil and low concentrations of Cd2+ inhibit a sustained component of inward current in locust neurons (Pearson et al. 1993). The calcium current measured in Kenyon cells of the honeybee are partially suppressed by nifedipine and verapamil and completely blocked by 50 µM Cd2+ (Schäfer et al. 1994). A high-voltage-activated calcium channel in locust medial neurosecretory cells has been shown to be suppressed by the P-type channel blocker, omega -agatoxin IVA (Bickmeyer et al. 1994b). It is possible that the current in Df may be similar to this current, although the effects of this toxin on Df have not been studied. Steady-state Ca channel inactivation has been demonstrated for neurons of a number of insect species (locust: Bickmeyer et al. 1994b; Pearson et al. 1993; honeybee Kenyon cells: Schäfer et al. 1994; Manduca: Hayashi and Levine 1992; Carausius: Ashcroft and Stanfield 1982).

Inactivation of the calcium current is both voltage and calcium dependent

Calcium current inactivation in Df depends both on membrane potential and on internal calcium concentration. Calcium-dependent inactivation is evident because currents decay faster and steady-state inactivation is greater for ICa than for IBa. Calcium currents also decay more slowly and display less steady-state inactivation after injection of the calcium buffer, BAPTA. Calcium-dependent inactivation may be an important feedback process, preventing [Ca2+]i rising too high (Eckert and Chad 1984). Because both barium and calcium currents measured after injection of BAPTA still display steady-state inactivation, it appears that there is an additional mechanism of inactivation, which is most probably voltage dependent. The steady-state inactivation curves for both calcium and barium currents are U shaped, because of a decline in inactivation that occurs at more positive membrane potentials. This characteristic has been observed for barium currents in other preparations (Brehm and Eckert 1978; Nakayama and Brading 1993; Tillotson 1979). This observation has led to the suggestion that, because the extent of inactivation reflects the magnitude of inward current (as measured from the I-V relationship), then barium ions are capable of producing inactivation. Alternatively, intracellular barium ions may exert their effect by displacing calcium from intracellular stores as has been demonstrated in Helix neurons, (Meech and Thomas 1980). One problem in interpreting the experiments reported here is that it is extremely difficult to completely remove all external calcium because the surface membrane of Df is highly invaginated (Lane et al. 1982; Smith and Treherne 1965). In the current study, some nerve cords were dissected and washed in barium saline to remove external calcium, and BAPTA was injected to chelate intracellular calcium. Despite this, it is still possible that, if the calcium-binding site responsible for inactivation is located very close to the inner mouth of the channel pore, even BAPTA may be unable to completely abolish calcium-dependent inactivation (Zong and Hofmann 1996). However, it has been shown that calcium and voltage-dependent inactivation may coexist in some preparations; including heart (Argibay et al. 1988; Hadley and Hume 1987; Hadley and Lederer 1991; Kass and Sanguinetti 1984; Lee et al. 1985), molluscan neurons (Brown et al. 1981; Gutnick et al. 1989), and sensory neurons (Akaike et al. 1988). In frog cardiocytes, the rapidly falling phase of the steady-state inactivation curve is thought to be largely a voltage-dependent process, and calcium-dependent inactivation is more prominent at more positive membrane potentials (Argibay et al. 1988). Inactivation in Df appears to be similar, because caffeine, which increases intracellular calcium levels, has no effect on the steady-state inactivation curve between -70 and -10 mV, but increases the extent of inactivation at more positive membrane potentials (unpublished observations). Zong and Hofmann (1996) have shown thatan alpha 1c subunit of the L-type channel expressed in CHOand HEK 293 cells is mainly responsible for calcium-dependent inactivation and that coexpression with the auxiliary subunits accelerated the voltage-dependent inactivation of the channel.

Calcium current decay in many other preparations has been fitted with a double exponential function. In Df, fitting was normally carried out on currents activated by pulses of <100 ms and required only a single exponential, whereas currents induced by longer duration pulses required a double exponential function. Similar observations have been reported in Helix neurons (Brown et al. 1981). The fast component of decay has normally been assigned to a calcium-dependent process and the slow component assigned to a voltage-dependent process (Hadley and Hume 1987; Kass and Sanguinetti 1984; Lee et al. 1985). For calcium currents in Df, the faster component of decay appears to be calcium-dependent, because, after iontophoresis of BAPTA, this component of decay is abolished, leaving a slower presumably voltage-dependent component of decay.

Both ICa and IBa show cumulative inactivation with interpulse intervals <10 s, this being greater in calcium saline for an interpulse interval of 1 s. At interpulse intervals >1 s, there is little difference between inactivation measured in calcium or barium saline. An interpulse interval between 10 and 15 s is required for complete recovery from inactivation. This may be important functionally in limiting spiking frequency. Using a standard double-pulse protocol, some recovery from inactivation could be demonstrated for IBa by increasing the interpulse interval from 10 to 100 ms. However, this was not observed for ICa, suggesting that recovery from calcium-mediated inactivation has not occurred in this time scale. This may reflect the time required to lower intracellular calcium by sequestration into intracellular stores or extrusion through the surface membrane. Alternatively, it has been suggested that calcium-dependent inactivation may be the result of dephosphorylation of the calcium channel by the calcium-sensitive phosphatase, calcineurin (Klee et al. 1979, 1983), which enhances the rate of calcium-dependent inactivation when added to the intracellular perfusate of a Helix neuron (Chad and Eckert 1986). Recovery from inactivation may then depend on the rate of rephosphorylation or be related to reduction of the phosphatase activity.

Differential pharmacological block of electrical activity

The soma of Df can display both calcium-dependent action potentials and calcium-dependent plateau potentials (Hancox and Pitman 1992). Plateau potentials can be recorded immediately after dissection of the CNS, whereas action potentials can normally only be observed at periods >2 to 4 h after isolation. The plateau potential can be blocked by nifedipine but not by micromolar Cd2+, whereas the action potentials are only blocked by a combination of both drugs, suggesting that the nifedipine-sensitive current underlies the plateau potential and that both the nifedipine-sensitive and Cd2+-sensitive currents contribute to the spikes.

Voltage-dependent steady-state inactivation may be important in influencing the ability of the cell to plateau, because the nifedipine-sensitive current that underlies the plateau potentials appears to be inactivated at a holding potential of -40 mV. Although the steady-state inactivation properties of the nifedipine- and cadmium-sensitive currents were not examined in detail, it seems likely that the nifedipine-sensitive current does not show substantial calcium-dependent inactivation, because this conductance underlies the plateau potential, and agents that increase intracellular calcium levels such as caffeine do not block plateau potentials nor do they affect the inward current that activates negative to -40 mV except at very high concentrations (unpublished observations). However, caffeine does block spikes and depresses currents at more positive potentials, suggesting that the Cd2+-sensitive conductance may display calcium-dependent inactivation. Muscarinic agonists, which have been shown to increase intracellular calcium levels (David and Pitman 1996), suppress inward calcium currents, presumably through calcium-dependent inactivation (David and Pitman 1995b) and can convert a neuron that responds to depolarization by spiking to one that can only produce plateau potentials (unpublished observations). Calcium-dependent inactivation of the inward current may also partly explain why the cell displays plateau potentials but is unable to spike immediately after dissection when it is thought that intracellular calcium levels are high. A high calcium-dependent potassium conductance at this time may also depress the ability of the cell to spike (unpublished observations). In cells that are already displaying plateau potentials, their duration can be influenced by outward conductances (unpublished observations). The results shown here also demonstrate that decay of the calcium current could also contribute toward plateau termination since substantial decay of ICa occurs within the time course of plateau potentials (100 ms to 1 s). The properties of the inward calcium currents in the soma of Df, can therefore explain the different types of electrical activity exhibited by this neuron; whether the neuron generates action potentials or plateau potentials will depend on the relative magnitude of the different Ca channels in the neuron. This will be determined by the intracellular calcium concentration, which itself may be under the influence of neurotransmitters or neuromodulators.

    ACKNOWLEDGEMENTS

  We thank the Biotechnology and Biological Sciences Research Council for supporting this work.

    FOOTNOTES

  Address reprint requests to R. M. Pitman.

  Received 4 February 1997; accepted in final form 7 July 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

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