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INTRODUCTION |
Calcium influx plays an important role in regulating activity in neural circuits. Calcium current can underlie rhythmic oscillations in membrane potential (Soltesz et al. 1991
), the triggering of transmitter release (Mulkey and Zucker 1993
), and the regulation of a number of ion currents (Sah 1995
; Wisgirda and Dryer 1994
; Yamoah et al. 1994
). Different functions such as these are often mediated by different calcium channel types that are pharmacologically and biophysically distinguishable (Elliott et al. 1995
; Gonzales-Burgos et al. 1995
). In addition, calcium channels can mediate changes in neural circuitry by participating in events such as neuromodulation or activity-dependent changes in excitability (Del Mar et al. 1994
; Hong and Lnenicka 1993
; Zengel et al. 1993
) .
In the crustacean stomatogastric ganglion (STG), calcium influx also plays an important regulatory role. Calcium ions are necessary for a number of functions important to pattern generation, such as membrane potential oscillation (Harris-Warrick and Flamm 1986
, 1987
; Hermann and Wadepuhl 1987
), synaptic transmission (Graubard et al. 1983
), neuromodulation (Zhang and Harris-Warrick 1995
), and activity-dependent changes of intrinsic membrane properties (Turrigiano et al. 1994
). Although it has been hypothesized that different types of calcium channel may subserve some of these different functions (Hartline and Graubard 1992
), different types of calcium channel have not yet been well characterized in this system (but see Turrigiano et al. 1995
).
A generally important correlate of the function of calcium channel types is their location. Calcium channel subtypes are often segregated in anatomically and functionally distinct regions of neurons (Llinás et al. 1992
; Westenbroek et al. 1992
), or are closely colocalized with other proteins that are anatomically segregated, such as synaptotagmin or calcium-dependent potassium channels (Leveque et al. 1992
; Roberts et al. 1990
).
Single STG neurons are complex in structure, with anatomically distinct regions showing differences in function. The peripheral cell bodies give rise to a single primary neurite that branches profusely in a central neuropil. It is between the distal fine neurites that synaptic connections between STG neurons are found (Baldwin and Graubard 1995
; Kilman and Marder 1996
; King 1976
). The primary neurite also gives rise to one or more axons that synapse onto target muscles. These functional differences between regions make STG neurons good candidates for segregation of different types of calcium channel to different regions. Indeed, depolarization-evoked calcium changes do show regional variation when imaged with calcium-sensitive dyes (Graubard and Ross 1985
; Ross and Graubard 1989
) consistent with the idea that the channels underlying the calcium changes also show regional variation. It has also been hypothesized that low- and high-threshold channels must be differentially distributed (Hartline and Graubard 1992
) because the threshold for synaptic transmission is approximately
60 mV (Graubard et al. 1983
), whereas the threshold for calcium current measured from the soma appears to be higher (Golowasch and Marder 1992
; Graubard and Hartline 1991
; Zhang and Harris-Warrick 1995
). However, no pharmacological separation of calcium channel types has been accomplished in stomatogastric neurons.
The goal of this study was to search for pharmacological agents that would allow separation of calcium channel types. We took advantage of the geometry of STG neurons to study two isolated preparations: unidentified cell bodies and identified neuromuscular junctions. These two preparations were necessary to expand the number of differentially distributed calcium channel types studied, because space clamp of inward currents is difficult to achieve in stomatogastric neurons (Hartline and Graubard 1992
). Using a range of selective calcium channel blockers on these two preparations, we have distinguished at least two calcium channel types that are distinct in pharmacology, function, and location.
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METHODS |
Animals
Male red rock crabs (Cancer productus) were collected south of Marina Beach in Edmonds, Washington. The animals were maintained for a period of up to 1 mo in aerated seawater tanks at 10-13°C until use.
Neuron culture
Crabs were anesthetized before surgery by chilling on ice for
30 min. Cells were cultured using modifications of existing protocols (Graf and Cooke 1990
; Krenz et al. 1990
; Panchin et al. 1993
; Turrigiano and Marder 1993
). Modifications included the use of 2 mg/ml of pronase for enzymatic digestion of the connective tissue between neurons before culturing and the use of a sharp glass micropipette to dissect off small groups of cell bodies. The addition of antibiotic/antimycotic to the cell culture medium was sufficient to prevent contamination by bacteria and fungus.
Cell body recordings
Electrophysiological recordings were made from cells with a compact, spherical morphology after 3-5 days in culture (Fig. 1A). Recordings were usually made at room temperature (~22°C). In several experiments, recordings were also made at both 15 and 22°C. In these cases, temperature did not change the maintained amplitude of the calcium current. Two-electrode voltage-clamp recordings using an Axoclamp 2A (Axon Instruments; Foster City, CA) were accomplished as in Graubard and Hartline (1991)
, with the exception that the current injection electrode was not shielded. Electrode filling solutions contained
1 M chloride; cultured somata had greatly lowered resistances with higher chloride concentrations. Current amplitudes were measured with the use of the Axoclamp 2A (internal circuit) and pClamp software. Input resistance was measured from the current-voltage relationship to small hyperpolarizing voltage steps. Membrane capacitance was measured with sawtooth voltage commands as in Moody and Bosma (1985)
. Leak current, measured from voltage steps of 10-30 mV negative to resting potential, which ranged from
25 to
60, was subtracted during analysis.

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| FIG. 1.
Diagrams illustrative of the preparations used in this study. A: photograph of an unidentified stomatogastric neuron in cell culture above a sample series of voltage commands and the resulting calcium currents. B: neuromuscular preparation stretches of nerve containing stomatogastric axons were left attached to their target muscle. Action potentials were stimulated in the presynaptic axon with brief current pulses delivered to the nerve, and postsynaptic potentials (PSPs) were recorded intracellularly from muscle fibers.
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Both inward and outward currents were measured; outward currents were measured to determine whether there was a calcium-dependent component. Inward and outward currents were recorded under different conditions. For recording outward currents, intracellular microelectrodes were filled with 1 M KCl and 0.5 M K2SO4, and the extracellular bath contained normal crab saline.To reveal the smaller inward currents, an electrode filling solution of 1 M tetraethyl ammonium chloride (TEACl) and 1 or 5 µM 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) and a high-calcium bath saline containing TEA were used. Sodium currents were blocked with tetrodotoxin (TTX). The current injection electrode was 10 M
, and the voltage recording electrode was 20 M
when filled with 1 M TEACl. Currents were examined in both barium and calcium salines during the experiments on cultured cell bodies; similar results were obtained for organic blockers in both salines.
Experimental treatment commenced when inward current had stabilized, usually 10-15 min after ionophoretic injection of TEA and BAPTA. Toxins were added directly to the recording chamber. Dose-response measurements represent values for currents after toxin addition that were stable for
5 min.
Neuromuscular junction
Neuromuscular transmission served as an assay for presynaptic calcium entry. Neuromuscular preparations were isolated by cutting sections of nerve leading to the muscle, so that the connections of the axons to the cell bodies were severed. A suction electrode attached to the cut end of the motor nerve leading to either the intact cv2 (glutamatergic) or the gm1b (cholinergic) muscle was used to evoke axonal action potentials (Fig. 1B). The inferior cardiac (IC) neuron innervates the cv2 muscle, whereas gastric mill (GM) neurons innervate the gm1b muscle (Govind and Lingle 1987
). Synaptic potentials at these muscles are exclusively excitatory; inhibitory inputs have not been found (Govind and Lingle 1987
). Isolated 1-ms voltage pulses at just above action-potential threshold were fed into the bipolar suction electrode to stimulate the axons in the nerve. In addition, a bipolar pin electrode made of stainless steel and insulated with a mixture of heavy mineral oil and petroleum jelly was sometimes placed along the nerve containing the IC axon between the suction electrode and the muscle (Fig. 1B), allowing extracellular action potentials in the IC axon to be recorded (A-M Systems differential amplifier model 1700). Postsynaptic potentials (PSPs) were recorded with a 3 M KCl intracellular microelectrode (10 M
resistance) in a muscle fiber. Potentials were measured in the current-clamp configuration of the Axoclamp 2A and stored on tape for later analysis.
The extracellular medium was normal crab saline, and toxins were added directly to the recording chamber. Recordings were made at 15°C or at room temperature, 22°C. No difference in the effects of toxins was observed with different temperatures. Controls for any increase or decrease of PSP amplitude during the experiment (both of these occurred) consisted of a series of measurements taken over a time roughly equivalent to the expected time course of blocker effect (30 min to 1 h).
To activate muscle receptors directly, a solution of 1 mM acetylcholine chloride in normal crab saline, made up on the day of use, was pressure ejected directly onto the gm1b muscle through glass pipettes with tip diameter of 50-100 µm. Pipettes were placed close to the intracellular recording site and positioned to give the largest response. Pressure was regulated with a Picospritzer II (General Valve).
Salines and chemicals
The recipes for salines were modified from Graubard and Hartline (1991)
for lobster saline and Graubard and Ross (1985)
for crab saline. Culture medium consisted of equal amounts L-15 medium and 1.5-1.75× saline, with 1% antibiotic/antimycotic and 6 g/l D-glucose, filtered through a 0.2-µm pore diameter cellulose acetate filter. The pH of all salines was adjusted to 7.42 with 1 M NaOH.
Chemicals were obtained from the following companies: Eastman (TEACl); Atlanta Biologicals (fetal bovine serum); Calbiochem (collagenase/dispase, pronase); RBI (nicardipine). All other purchased chemicals were obtained from Sigma.
Ion channel blockers
Calcium channels were blocked with divalent cations (CdCl2, MnCl2, and CoCl2) and organic blockers. Three classes of organic calcium channel blockers were used to pharmacologically characterize stomatogastric calcium channels, including the following. 1) The dihydropyridines, nifedipine and nicardipine. Nifedipine was stored dry in the refrigerator, and nicardipine was stored dry at room temperature. They were dissolved just before use in 95% ethyl alcohol (EtOH) and diluted down in saline to a stock solution. The final concentration of EtOH in the saline bathing the cells was
1%. At this concentration, EtOH alone did not affect the amplitudes of ion currents or of PSPs. Dihydropyridines are light sensitive, therefore the microscope light source and nearby room lights were turned off during dihydropyridine addition. 2)
-Agatoxin was stored in a 0.5 mg/ml stock solution in distilled water at
70°C. Immediately before use, it was diluted in saline containing 1 mg/ml of lysozyme to occupy nonspecific binding sites. Lysozyme did not affect calcium current at this concentration. 3)
-Conotoxin GVIA was diluted with distilled water to 100 µMand stored at
70°C. It was thawed immediately before use.
TTX was diluted in saline immediately before use from a stock solution of 100 µM in distilled water kept at
20°C. Four-aminopyridine (4-AP) and 2,3-diaminopyridine were dissolved in saline and adjusted to pH 7.42 no more than 2-3 h before use. Group values are expressed as means ± SE.
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RESULTS |
Cultured cell bodies
SOMA CULTURE.
We used cell bodies in culture to measure calcium current directly because in intact stomatogastric neurons, good space clamp is difficult to achieve (Graubard and Hartline 1991
; Zhang and Harris-Warrick 1995). In addition, cultured cell bodies were synaptically isolated from other neurons, and drugs could be expected to have more rapid and uniform effects than in the stomatogastric neuropil. Stomatogastric somata were isolated from intact stomatogastric neurons with <20 µm of primary neurite to make them more electrically compact. They showed little obvious outgrowth over 3-4 days in culture (Fig. 1A). Approximately 15-30% of the somata from any given ganglion survived the pulling process. Mortality during cell culture was widely variable. Neurons were not identified before being cultured, because the manipulations needed to do so further decreased survival rate. Somata varied greatly in size; after 2-3 days in culture, capacitances ranged from 0.2 to 0.7 nF, and input resistances ranged from 50 to 200 M
in normal saline. The cells also expressed a range of active properties such as hyperpolarization after a depolarizing current, postinhibitory rebound, and the tendency for voltage to oscillate in high calcium saline containing TEACl. Somata also sometimes oscillated spontaneously or fired broad action potentials that could be blocked by 0.2-0.5 µM TTX. These active properties, along with a stable leak current, were indicative of healthy neurons. After
4 days in culture, an increasing leak current together with an increasingly spherical and vacuolated appearance was usually correlated with a decline in active properties over this time period; such neurons were excluded from study.

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| FIG. 2.
Voltage dependence of activation ( ) and inactivation ( ) of calcium current. Normalized steady-state inactivation was measured at pulses to +10 mV from a varying holding potential. Activation is normalized current during variable-amplitude voltage steps from a holding potential of 70 mV. and , peak current; and , current at the end of the pulse. Data shown were taken from one experiment for inactivation and a different experiment for activation.
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| FIG. 3.
Sensitivity of somatic calcium current to organic blockers. Current-voltage relationships are shown with insets of raw current traces during single voltage pulses. Current was measured at the end of the voltage pulses. Two dihydropyridines, nicardipine (A) and nifedipine (B) block calcium current. -Agatoxin IVA also blocks calcium current (C), but -Conotoxin GVIA does not (D). These records were not corrected for contamination by outward current. Command voltages were to +10 mV for the raw current traces seen in B-D and to +20 mV for A.
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SOMATIC CALCIUM CURRENT.
Most recordings were done at 2-3 days after plating in culture, to maximize both calcium current amplitude and cell health. In two-electrode voltage clamp, calcium current was only apparent when other currents were blocked (Fig. 1A). Outward currents were blocked with a combination of up to 100 mM TEA substituted for Na in the bathing saline and ionophoretic injection of TEA through recording electrodes. Inward current activated by hyperpolarizing pulses ("sag" current) was also present and at increasing hyperpolarization caused the current-voltage relationship to deviate from linearity. Hyperpolarization-activated inward current was sometimes blocked by the substitution of up to 20 mM Cs for Na in the bath saline (Golowasch and Marder 1992
; Kiehn and Harris-Warrick 1992
) and was never included in the calculation of leak current. Sodium current was blocked by addition of TTX. Contamination by outward currents still existed, however, especially by a transient A-like current. In other crustacean neurons, the presence of contaminating A-current obscures inward current, especially from relatively negative holding potentials, but can be blocked by 4-AP (Tazaki and Cooke 1990
). In cultured C. productus neurons, 4-AP was toxic, inducing a large and increasing leak current, and neither2,3-diaminopyridine nor TEA effectively blocked A current.
We took four different approaches to reduce the effect of outward current on our measurements. First, we often measured calcium current from a holding potential of
30 mV to reduce A-current. This would have inactivated outward A-current, but it also would have inactivated low-threshold inward current, so in some experiments holding potential was more negative, from
80 to
100 mV. Second, unless otherwise stated, measurements of calcium current amplitude were taken late in the voltage step, when the large transient component of A-current was minimal. Such late measuring times would have made transient calcium current correspondingly difficult to detect. Third, barium was sometimes substituted for calcium in the bath saline. Barium increased the amplitude of inward current, making it easier to observe. Barium also decreased the amplitude of outward current in normal saline, but did not do so in the calcium-recording protocol, which included internal and external TEA and internal BAPTA. Fourth, calcium current was measured by subtracting current measured with calcium channel blockers present from control current. When this subtraction was done, the current-voltage relationship peaked at 14 ± 3 mV (mean ± SE, n = 6). The apparent reversal potential was +52 ± 1 mV (n = 6).
NO EVIDENCE FOR MULTIPLE CALCIUM CURRENTS IN CULTURED SOMATA.
We examined both the voltage dependence and pharmacological sensitivities of total calcium current in cultured cell bodies to determine whether it could be divided into subtypes.
Voltage dependence. Total calcium current of cultured cell bodies was high-threshold, independent of holding potential. Calcium current began to activate at voltages more positive than
40 mV. No transient low-threshold calcium current was seen, even for steps from negative holding potentials of
90 to
100 mV. However, a small and rapid low-threshold calcium current could have been present, but obscured by remaining outward current contamination. Current evoked by a single voltage step often had both a transient peak and a sustained component (Fig. 2, left inset). The ratio of peak to sustained current varied, with the sustained component falling to as little as 39% of the peak value during a 200-ms voltage step. The time constant of this decay ranged from 15 to 150 ms. The decay was present even under conditions minimizing A-current and in barium salines, and so was probably not due to outward current contamination.
High-voltage-activated calcium currents can vary in their kinetics and voltage sensitivities (Randall and Tsien 1995
). To test whether different current components could be distinguished on this basis, we compared the voltage dependence of the total peak versus the total sustained portions of the current. Peak and sustained current each had a similar voltage dependence of activation, as seen in the activation curves and right inset of raw current traces in Fig. 2. The peak component had a half-maximal activation of
16 ± 3 mV (n = 3), and the sustained component had a half-maximal activation of
14 ± 3 mV (n = 4) in barium saline. The two components were also similarly affected by holding potential, as seen in the inactivation curves and left inset of raw current traces in Fig. 2. The peak component had a half-maximal voltage of inactivation of
30 ± 5 mV (n = 4) and the sustained component of
31 ± 5 mV (n = 4) in barium saline. The similar voltage dependence of both portions of the total calcium current suggests that the calcium current is generated by one type of calcium channel. It is also consistent with two or more types of calcium channel with similar voltage dependence of activation and inactivation.

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| FIG. 4.
Comparison of the affinity of 3 blockers for calcium current. Each curve is taken from a different cultured neuron. Curves are fit to data with the equation y = 1/{1 + exp[(logdose IC50)/c]}.
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| FIG. 5.
Dihydropyridines selectively block calcium-activated outward current. A: the time course and voltage dependence of outward current blocked by nicardipine is the same as that blocked by manganese or BAPTA injection. Ai: normalized difference current traces represent subtraction of treatments that block calcium current from control current. Data are taken from +20 mV and a holding potential of 40 mV. Aii: normalized current-voltage plots measured at the peak current. B: in a single cell, nifedipine blocks outward current identically to a zero-calcium saline. Bi: raw current traces at 5 mV from a holding potential of 30 mV. Bii: a current-voltage plot of the same experiment, measured at the end of the voltage pulse. Numbers refer to the order in which the salines were applied.
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Calcium channel blockers. To define a pharmacological profile for net somatic calcium current and potentially distinguish different components of current, we applied a range of standard calcium channel blockers. Net somatic calcium current was completely blocked by divalent cations such as cadmium (1 mM, n = 12) and manganese (10 mM, n = 6). Calcium current was also blocked by several organic calcium channel blockers. Nifedipine and nicardipine, two dihydropyridines, blocked calcium current completely and reversibly (Fig. 3,A and B).
-Agatoxin IVA, a fraction of funnel web spider venom, blocked calcium current irreversibly (Fig. 3C). Washes were done over 5-25 min.
-Conotoxin GVIA did not affect the calcium current (Fig. 3D). Examples of curve fits for experiments on single neurons are shown in Fig. 4. IC50's fit to all data points for nifedipine, nicardipine and
-Agatoxin IVA, were 48 µM (n = 12), 17 µM (n = 10), and 933 nM (n = 10), respectively (Fig. 4). These values are higher than values reported to block vertebrate calcium currents (Randall and Tsien 1995
).
The peak and sustained portions of calcium current were not pharmacologically distinguishable. The toxins that blocked calcium current blocked both components at the same concentrations (Fig. 3, A-C).
CALCIUM-DEPENDENT OUTWARD CURRENT.
We examined calcium-dependent outward current in cultured cell bodies. Besides being an important regulator of excitability in many cell types (Bielefeldt and Jackson 1993
; el Manira et al. 1994
; Robitaille and Charlton 1992
; Sah and McLachlan 1992
; Viana et al. 1993
), including STG neurons (Golowasch and Marder 1992
; Graubard and Hartline 1991
; Harris-Warrick et al. 1992
; Zhang et al. 1995
), calcium-dependent outward current can be a sensitive assay for calcium current in stomatogastric neurons (Hartline and Graubard 1992
). A calcium-dependent outward current that is largely carried by potassium ions (Hartline and Graubard 1992
) can be voltage clamped from the cell bodies of intact (Graubard and Hartline 1991
) and ligated (Hartline et al. 1993
) stomatogastric neurons. We wished to determine whether a component of the total outward current in cultured crab stomatogastric neurons was similarly calcium dependent, and whether calcium-dependent outward current was sensitive to dihydropyridines.
Cultured crab neurons did express calcium-dependent outward current. Decreasing intracellular calcium by blocking calcium channels with divalent cations such as manganese (n = 10) and by injecting the calcium chelator BAPTA(n = 4) reduced a component of outward current (Fig. 5A). Nicardipine also blocked a component of outward current (Fig. 5A; n = 15). The nicardipine-sensitive component was similar to that blocked by manganese and BAPTA, in both normalized time course (Fig. 5Ai) and in the U-shaped voltage dependence typical of calcium-activated outward current (Hille 1992
) (Fig. 5Aii). This supports the idea that manganese, BAPTA injection, and nicardipine all block the same types of channels in different cells. Even when a divalent cation and a dihydropyridine were applied sequentially in the same cell, the same component of outward current could be blocked, as seen in both raw current traces (Fig. 5Bi) and current-voltage relationships (Fig. 5Bii), further supporting the argument that both treatments affected the same channels.
Dihydropyridines are known to directly affect calcium-dependent potassium channels (Fagni et al. 1994
) as well as other types of potassium channels (Jones and Jacobs 1990
; Lin et al. 1995
; Mlinar and Enyeart 1994
). However, several lines of evidence suggest that dihydropyridines did not directly block potassium currents in cultured stomatogastric cell bodies. First, the relative effectiveness of the two dihydropyridines, nicardipine and nifedipine, was the same for both calcium current and calcium-dependent outward current. For both dihydropyridines, calcium-dependent outward current was more sensitive to block than calcium current, as seen for nicardipine in Fig. 6A. Higher doses of nifedipine than nicardipine were also required to block both currents. These similar relative affinities of the dihydropyridines suggest a similar mechanism of action on calcium current and calcium-dependent outward current. In addition, whether currents were blocked with dihydropyridines or with manganese, the dose-response curves for outward and inward current maintained a constant relationship (Fig. 6B).

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| FIG. 6.
A: dose-response curves for nicardipine block of calcium current ( ) and calcium-dependent outward current ( ). Current measured is total cell current. At the voltage measured (0 mV), calcium-dependent current is the largest outward current; small amounts of other outward current are present. Holding potential was 40 mV. Data were taken from 2 separateexperiments. Lines are fits to the equation y = 1/{1 + exp[(dose IC50)/c]},where c = 0.2. B: comparison of nicardipine block of calcium-activated potassium current ( ) and calcium current ( ). |, the mean IC50 for each set of experiments; for calcium current this is 15.8 ± 1.1 µM, and for calcium-activated potassium current this is 5 ± 1 µM. and , amount of block by MnCl2. The nicardipine data were taken from 3 cells each for calcium current and outward current. The manganese data were taken from 2 cells each for calcium current and outward current. The extracellular calcium concentration was different for calcium current and calcium-activated potassium current.
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Despite these findings, in a number of experiments there was also evidence for a nonselective block of outward current by nifedipine, although not by nicardipine. In several experiments in TEA saline, which blocks calcium-dependent outward current (Hartline and Graubard 1992
), nifedipine irreversibly blocked another component of outward current (Fig. 3B). This irreversible reduction of potassium current was probably not due to rundown over time, because after
10 min in 18 randomly chosen cells, peak current was91 ± 7%, and maintained current was 100 ± 6% of its initial value. We must therefore conclude that nifedipine, although not nicardipine, probably blocks some potassium current directly.
All of these results suggest that one function of dihydropyridine-sensitive current in stomatogastric neuron somata is to activate a large outward current.
Neuromuscular junction
To investigate calcium channels from a different region of stomatogastric neurons, we used two stomatogastric nerve-muscle preparations (Fig. 1B): the cholinergic GM neuron-gm1b muscle and the glutamatergic IC neuron-cv2 muscle. Nerve stimulation of the anterior ventricular nerve (avnfor the IC-cv2) or medial ventricular nerve (mvn for theGM-gm1b) was used to evoke PSPs. PSPs were measured as indirect assays for presynaptic calcium influx. Under control conditions, a single 1-ms stimulus to the nerve containing a stomatogastric motor axon elicited a single excitatory PSP in an innervated muscle fiber (Fig. 1B).
CALCIUM CHANNEL BLOCKERS.
Pharmacological profile. The pharmacological profile of neuromuscular transmission was different from that of the cell body calcium current. Nicardipine at 10-30 µM significantly reduced the amplitude of postsynaptic potentials by 19 ± 6% (n = 5; Fig. 7A). Nifedipine at 50-150 µM caused summating bursts of PSPs in response to each stimulus and increased the latency from stimulus to PSP (Fig. 7B), effects not seen with nicardipine or other toxins. Both of these effects could be related to a block of outward currents by nifedipine, because outward currents can be involved in setting both delay to initial spikes and interspike interval (Disterhoft et al. 1993
; Sah and McLachlan 1992
; Viana et al. 1993
). Nifedipine significantly reduced the amplitude of the first PSP by 38 ± 7% (n = 4).

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| FIG. 7.
Organic calcium channel blockers at the stomatogastric ganglion (STG) neuromuscular junction. PSPs are evoked by stimulation of the presynaptic axon and are recorded in a muscle fiber with a single intracellular microelectrode. Two dihydropyridines, nicardipine (A) and nifedipine (B), partially block synaptic transmission. Nifedipine has the additional effect of inducing a burst of PSPs with each stimulus. C: in contrast, -Agatoxin IVA blocks PSPs entirely. D: -Conotoxin GVIA has no effect. E: a combination of both blockers does not always entirely block the PSP. F: summary of blocker effect on PSP amplitude. All PSPs were measured at a 2-Hz stimulation frequency; stimulus artifacts are seen as vertical lines preceding the PSPs. Both dihydropyridines and -Agatoxin IVA significantly reduced PSP amplitude (P < 0.05), as represented by the stars. In addition, the means for both dihydropyridines and -Conotoxin GVIA were significantly different from that for -Agatoxin IVA. The ranges of toxins used were 30-150 µM for nifedipine, 11-25 µM for nicardipine, 64-1,000 nM for -Agatoxin IVA, and 1-10 µM for -Conotoxin GVIA. nif, nifedipine; nic, nicardipine; Aga, -Agatoxin IVA; Ctx, -Conotoxin GVIA.
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| FIG. 8.
Calcium channel blockers do not affect muscle response to direct application of neurotransmitters. Acetylcholine was pressure ejected directly onto muscle fibers, eliciting depolarizing responses. A-C: none of the 3 organic calcium channel blockers changed the muscle responses; in all cases, responses in toxin almost identically superimpose on control responses. B: PSPs elicited by nerve stimulation ride on top of the depolarizing response caused by direct acetylcholine application. C: the flat line is a response to control pressure ejection of saline.
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-Agatoxin IVA reduced the amplitude of PSPs much more effectively than the dihydropyridines; in some cases PSPs disappeared almost entirely (Fig. 7C). Effective doses (100-140 nM but in one case as low as 64 nM) were about an order of magnitude lower than those required to block somatic calcium current. The latency between the stimulus and PSP was not affected by
-Agatoxin-IVA.
-Conotoxin GVIA at 500 nM had no effect on the amplitude or latency of postsynaptic potentials (Fig. 7D).
A combination of both nicardipine and
-Agatoxin-IVA did not always abolish the PSP entirely, suggesting that calcium channels insensitive to block by either of these toxins are involved in transmitter release (Fig. 7E). The effects of all four calcium channel blockers are compared in Fig. 7F.
Location of effect. We performed two sets of experiments to better define the loci of effect of the blockers. In the first, we pressure-ejected acetylcholine directly onto a gm1b muscle fiber to elicit a depolarizing response. Nicardipine (up to 20 µM; n = 3), nifedipine (up to 200 µM; n = 5), and
-Agatoxin IVA (up to 256 nM; n = 3) did not alter this response, suggesting that their effects on PSPs were not due to blockade of postsynaptic ion channels or input resistance changes (Fig. 8).
In the second set of experiments (Fig. 9), we placed an additional extracellular bipolar pin electrode on the nerve leading to the cv2 muscle to record any changes in the activity of action potentials following toxin addition. Before toxin addition, a stimulus to the axon was correlated with a single PSP. Nifedipine changed the excitability of the preparation. It caused a single stimulus to evoke a burst of action potentials, suggesting that one of its effects was directly on the spike-generating membrane of the axon or terminal. Later action potentials in the burst sometimes appeared to be of opposite polarity from the first action potential, suggesting that they were traveling in a retrograde direction (Fig. 9B).
-Agatoxin IVA did not block the extracellularly recorded action potential even when PSPs were decreased in the same preparation (Fig. 9C). Also in the same preparation, TTX blocked both the axonal spike and the PSP entirely, demonstrating that the lack of
-Agatoxin IVA effect was not due to lack of blocker access to the spike-generating region of membrane (Fig. 9C). Because they were obtained from extracellular recordings along the nerve, these results do not rule out an effect of
-Agatoxin IVA at the neuromuscular junction.

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| FIG. 9.
Effects of calcium channel blockers on action potentials measured extracellularly from motor nerves. A: bipolar extracellular recordings showing that action potentials can be clearly distinguished from a stimulus artifact. Responses to sub- and suprathreshold stimuli are superimposed. B: nifedipine causes a burst of action potentials rather than the single normal action potential in response to a single stimulus pulse. This burst parallels the burstlike appearance of the muscle PSPs. Later action potentials in the train appear to be traveling in a retrograde direction; 2 action potentials of opposite polarity are enlarged. C: in contrast, -Agatoxin IVA does not block the action potential in the motor axon, even as the muscle PSPs decline in amplitude. Tetrodotoxin (TTX) does block the action potential, however.
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The different pharmacological sensitivities of the neuromuscular junction and cultured cell bodies coupled with the presynaptic effects of the blockers at the neuromuscular junction are consistent with the existence of two pharmacologically distinct types of calcium channel in stomatogastric neurons.
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DISCUSSION |
There are pharmacologically distinguishable calcium channels in STG neurons
These data strongly support the hypothesis that there are at least two pharmacologically distinguishable types of calcium channel in stomatogastric neurons. The main finding that supports this hypothesis is the reversed sensitivity to calcium channel blockers of the two isolated preparations of stomatogastric neurons. Concentrations of dihydropyridines that block almost all of the calcium current and a large portion of calcium-activated potassium current in isolated cell bodies only modestly reduce the amplitude of neuromuscular transmission. Conversely, concentrations of
-Agatoxin IVA that almost entirely reduce the amplitude of synaptic transmission block very little calcium current in isolated cell bodies.
There are several alternatives regarding the distribution of the two calcium channel types in the STG. First, because the cell bodies were not identified before culturing and all neuromuscular junctions were either GM neuron to gm1b muscle or IC neuron to cv2 muscle, it is possible that different cell types possess different calcium channel types. The two types of calcium channel we observed could be specific to different neuronal types, with the GM and IC neurons having
-Agatoxin IVA-sensitive current in all regions and the neurons examined in culture having high-threshold dihydropyridine-sensitive current in all regions. This would imply differential survival of particular types of neurons in culture, with the GM and IC dying off more than other neuronal types. Such differential survival has not been reported for experiments in which lobster STG neurons were identified before being cultured, and the IC and GM neurons in particular have been successfully cultured in lobster (Panchin et al. 1993
; Turrigiano and Marder 1993
). We therefore favor a second alternative, that the two calcium channel types are differentially distributed within single stomatogastric neurons.
Although the different pharmacological sensitivities of the two isolated preparations support the existence of at least two types of calcium channel in stomatogastric neurons, their precise location remains unclear. The location of both dihydropyridine-sensitive and
-Agatoxin IVA-sensitive channels at the neuromuscular junction was determined to be presynaptic. In addition, the fact that neither nicardipine nor
-Agatoxin IVA changed the latency of the PSP suggests that channels blocked by these toxins do not interfere with spike propagation down the axon. The fact that nifedipine did change the latency of the PSP, along with its irreversible block of some outward current in cultured cell bodies,suggests that it directly blocked potassium channels aswell as calcium channels. However, no evidence that we have presented localizes either dihydropyridine-sensitive or
-Agatoxin IVA-sensitive calcium channels to particular areas of presynaptic terminal, or determines their presynaptic mechanisms of action.
Other calcium channel types may exist
The presence of two pharmacologically distinguishable calcium currents in STG neurons suggests that there may be at least two molecularly distinct calcium channel types. Additional calcium channel types could also be present in stomatogastric neurons. There are several reasons that channel types could have escaped our survey. First, we tested only a limited number of toxins. We selected toxins that block several major classes of vertebrate calcium channels; dihydropyridines such as nicardipine and nifedipine block L-type channels (Fox et al. 1987
),
-Conotoxin GVIA blocks N-type channels (McCleskey et al. 1987
), and
-Agatoxin IVA blocks P- and Q-type channels (Mintz et al. 1992
; Randall and Tsien 1995
). At the neuromuscular junction, the combination of
-Agatoxin IVA and dihydropyridines did not always completely block synaptic transmission, indicating that additional channel types may be involved. In cultured stomatogastric cell bodies, the presence of more rapidly and more slowly inactivating components of calcium current could indicate the presence of two high-threshold channel types. In leech heart interneurons, two similarly kinetically distinct components of calcium current correspond to two components of the graded postsynaptic response (Angstadt and Calabrese 1991
) and are thought to be generated by distinct channel types. In stomatogastric neurons, if there are two kinetically different channel types, they would have similar voltage dependence of activation and inactivation. This is possible, because pharmacologically different current types can have similar voltage dependence and kinetics (Lorenzon and Foehring 1995
). Screening additional toxins, especially those characterized on arthropod preparations (Pocock et al. 1992
), may increase the number of distinguishable calcium channel types in STG neurons.
Second, we examined only two regions of STG neurons, the cell body and the neuromuscular junction. Stomatogastric neurons also branch extensively in the ganglion neuropil, which we did not study. Many events that require calcium influx, such as synaptic transmission between stomatogastric neurons and calcium oscillations, occur in the neuropil (Graubard et al. 1983
; Graubard and Ross 1985
; Ross and Graubard 1986
, 1989
). The calcium channels that underlie these events may differ from those described here.
Third, any transient low-voltage-activated current located in cell bodies could have gone unnoticed. Both T-type calcium current and large A-type outward current are typically available for activation from relatively negative holding potentials. The large A-type current could have obscured aT-type current. Indeed, low-threshold current has been reported in cultured P. interruptus STG neurons (Turrigiano et al. 1995
) .
Fourth, our preparations were isolated. Calcium current can be modulated in STG neurons (Zhang and Harris-Warrick 1995
). If the modulatory environment of the intact ganglion selectively permits the expression of additional types of calcium channel, we would not have encountered them.
Stomatogastric calcium channels may be different from vertebrate calcium channels
We used toxins characterized as selective for vertebrate calcium channel types. Although these toxins have distinguished between calcium channel types in stomatogastric neurons, we cannot conclude that the calcium channels that generate them are molecularly similar to those in vertebrates. In studies of vertebrate-characterized toxins across invertebrate phyla, similarity to vertebrate preparations is variable. In some cases, the toxins appear to work in a similar manner and at a similar concentration to vertebrate preparations. For example, in some molluscan preparations,
-Conotoxin GVIA blocks synaptic transmission and nifedipine is effective in blocking calcium current at 10 µM (Table 1) (Edmonds et al. 1990
; Fossier et al. 1993
; Trudeau et al. 1993
). In cockroach dorsal unpaired median neurons,
-Conotoxin GVIA blocks calcium current at 1 µM (Wicher and Penzlin 1997
). In other cases, toxins may work, but at different concentrations than are effective on vertebrate preparations. In STG neurons, the IC50 of nifedipine was near 48 µM, although that of nicardipine was near 20 µM. In other arthropod neurons such as honeybee Kenyon cells and cockroach motor neurons, nifedipine blocks calcium current only at 100 µM (Table 1) (David and Pitman 1995
; Schafer et al. 1994
), similar to STG neurons. In still other cases, as in leech Retzius neurons, a battery of vertebrate calcium channel blockers is entirely ineffective in blocking calcium current (Table 1) (Hochstrate et al. 1995
). There is also molecular evidence that calcium channels in some arthropods may not fall neatly into existing vertebrate categories; a Drosophila calcium channel
-subunit has 78.3% overall similarity with rat brain type D calcium channel (Zheng et al. 1995
), which codes for L-type channels (Hofmann et al. 1994
), but shows many changes in domains thought to be necessary for dihydropyridine binding (Zheng et al. 1995
). In conjunction with this, the current we measured in the stomatogastric cell body was similar to vertebrate L-type current in that it was blocked by dihydropyridines. However, the dihydropyridine block was at a higher concentration and showed no apparent voltage dependence, and the current was also blocked by high concentrations of
-Agatoxin IVA.
STG calcium currents resemble other crustacean currents
SOMATIC CURRENT.
The high-threshold calcium current in cultured cell bodies resembles calcium current seen in other crustacean STG and non-STG preparations. The current had a similar threshold of activation to calcium current from cultured P. interruptus STG neurons (Turrigiano et al. 1995
), intact Cancer borealis stomatogastric lateral pyloric (LP) neurons (Golowasch and Marder 1992
), ligated cardiac ganglion motor neurons in the lobster Homarus americanus (Tazaki and Cooke 1990
), and X-organ cells from Cardisoma carnifex (Meyers et al. 1992
). The presence of transient and sustained components of calcium current seen in our isolated cell bodies also agrees well with results in intact C. borealis LP neurons (Golowasch and Marder 1992
), cultured P. interruptus neurons (Turrigiano et al. 1995
), H. americanus neurons (Takahashi and Momiyama 1993
), and Cardisoma carnifex neurons (Richmond et al. 1995
). The threshold for calcium spikes in P. interruptus (Graubard and Hartline 1991
) and threshold and voltage of half-maximal conductance in intact DG neurons from C. borealis (Zhang and Harris-Warrick 1995
) are more negative than the threshold for cell body current, however. Again, a possible reason for this discrepancy is that we excluded lower-threshold components of calcium current by culturing only cell bodies.
NEUROMUSCULAR CURRENT.
Presynaptic current sensitive to
-Agatoxin IVA has been reported at crayfish opener neuromuscular junction (Araque et al. 1994
), where
-Agatoxin IVA decreases neuromuscular transmission. The time course and dose of
-Agatoxin IVA in our preparation was comparable with this.
Other interesting differences with previous studies were also found at the STG neuromuscular junction. Although our results pointed to a presynaptic effect of nifedipine, nifedipine blocks voltage-dependent calcium channels of crayfish opener muscle (Araque et al. 1994
). These results can be reconciled with each other, however, because our methods of activating postsynaptic acetylcholine receptors would not necessarily depolarize the muscle membrane enough to activate high-voltage-activated calcium channels. In addition, although we found
-Agatoxin IVA to be an extremely effective blocker of synaptic transmission at concentrations as low as 64 nM, a low- to mid-voltage-activated calcium current in crayfish abdominal motor neuron was only maximally blocked by 600 nM of
-Agatoxin IVA (Hong and Lnenicka 1997
). It is interesting to wonder how much molecular variation underlies this variation in calcium currents.
This work represents the first evidence for pharmacologically distinguishable calcium channel types in the STG, a major model system for neural circuits. It is likely that the calcium channels we have identified make important contributions to motor pattern generation, either directly or via regulation of other types of ion channels. The pharmacological tools we have characterized in this work will allow exploration of these contributions at multiple levels, from intact single cells to neural circuits.