Dihydropyridine- and Neurotoxin-Sensitive and -Insensitive Calcium Currents in Acutely Dissociated Neurons of the Rat Central Amygdala

Baojian Yu and Patricia Shinnick-Gallagher

Department of Pharmacology and Toxicology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1031

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Yu, Baojian and Patricia Shinnick-Gallagher. Dihydropyridine- and neurotoxin-sensitive and -insensitive calcium currents in acutely dissociated neurons of the rat central amygdala. J. Neurophysiol. 77: 690-701, 1997. The central amygdala (CeA) is an area involved in emotional learning and stress, and identification of Ca2+ currents is essential to understanding interneuronal communication through this nucleus. The purpose of this study was to separate and characterize dihydropyridine (DHP)- and neurotoxin-sensitive and -resistant components of the whole cell Ca2+ current (ICa) in acutely dissociated rat CeA neurons with the use of whole cell patch-clamp recording. Saturating concentrations of nimodipine (NIM, 5 µM), a DHP antagonist, blocked 22% of ICa; this NIM-sensitive (L-type) current was recorded in 68% of CeA neurons. The DHP agonist Bay K 8644 (5 µM) produced a 36% increase in ICa in a similar proportion of CeA neurons (70%). omega -Conotoxin GVIA (CgTx GVIA, 1 µM) in saturating concentrations inhibited 30% of ICa, whereas omega -agatoxin IVA (Aga IVA, 100 nM), in concentrations known to block P-type currents, did not affect ICa. Higher concentrations of Aga IVA (1 µM) alone reduced ICa by 34%, but in the presence of NIM (5 µM) and CgTx GVIA (1 µM) blocked only 18% of ICa. omega -Conotoxin MVIIC (CgTx MVIIC, 250 nM) reduced ICa by 13% in the presence of CgTx GVIA (1 µM). Application of NIM (5 mM), CgTx GVIA (1 µM), and Aga IVA (1 µM) blocked ~67% of ICa. A similar portion (63%) of Ca2+ current was blocked with CgTx MVIIC (250 nM) in the presence of NIM (5 µM) and CgTx GVIA (1 µM). The current resistant to NIM and the neurotoxins represented 37% of ICa, whereas in neurons not having L-type currents the resistant current made up ~53% of ICa (49 ± 2%, mean ± SE). The resistant current activated at around -40 mV and peaked at ~0 mV with half-activation and -inactivation potentials of -17 and -58 mV and slopes for activation and inactivation of -5 and 13 mV, respectively. The resistant current was sensitive to Cd2+ (IC50 = 2.5 µM) and Ni2+ (IC50 = 86 µM), was larger in Ca2+ than in Ba2+ (ratio = 1.31:1), and showed a moderate rate of decay. In summary, our results show that the high-voltage-activated calcium current in rat CeA neurons is composed of at least four pharmacologically distinct components: L-type current (NIM sensitive, 22%), N-type current (CgTx GVIA sensitive, 30%), Q-type current [Aga IVA (1 µM) and CgTx MVIIC sensitive, ~13-18%], and a resistant current (Non-L, -N, and -Q current, 33 ~ 37%), amounting to 37-53% of the total current. The resistant current has some electrophysiological and pharmacological characteristics in common with doe-1, alpha 1E, and R-type calcium currents, but remains unclassified.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The central amygdala (CeA) is a prominent nucleus within the amygdaloid complex (De Olmos et al. 1985). As a major intra-amygdalar target and output structure of the amygdaloid complex (Krettek and Price 1978; McDonald 1991, 1992b; Stefanacci et al. 1992), this nucleus has rich reciprocal connections that allow the CeA to integrate the behavioral, neuroendocrine, and autonomic responses associated with fear or stress-related behaviors. Stimulation of the CeA causes increases in heart rate, blood pressure, respiration, and gastric acid secretion, and elicits flight and fight behavior (Applegate et al. 1983; Bonvalet and Gary BoBo 1972; Galeno and Brody 1983; Gloor et al. 1981; Henke 1985), whereas lesions of the CeA disrupt learned heart rate, respiration, blood pressure, stress-induced gastric ulcer, and startle responses to aversive stimuli (Henke 1980a,b; Johansson et al. 1981; Kapp et al. 1979; Markgraf and Kapp 1991; VandeKar et al. 1991; Zhang et al. 1986). The CeA is also the center of output pathways for the development and expression of aversive emotional learning (Davis 1992; LeDoux 1993, 1994; LeDoux et al. 1990).

Brain slice studies of CeA cells (Nose et al. 1991; Rainnie et al. 1992; Schiess et al. 1993) have shown that low-Ca2+ medium or Ca2+ channel blockers alter the evoked repetitive firing frequency, suggesting that Ca2+-dependent mechanisms influence neuronal firing behavior in the CeA (Schiess et al. 1993). Furthermore, low-voltage-activated (LVA, T-type) Ca2+ currents have been shown to be present in unidentified amygdala neurons (Kaneda and Akaike 1989), whereas pyramidal cells dissociated from the amygdaloid complex possess multiple types of high-voltage-activated (HVA) currents including L-, N-, and P-type currents (Foehring and Scroggs 1994). However, the types of whole cell calcium currents present in CeA neurons have not been described thus far.

On the basis of electrophysiological and pharmacological properties, at least six types of Ca2+ currents have been characterized in neurons. These consist of an LVA (or T-type) current Ca2+ channel and HVA Ca2+ channels that can be classified into at least five native neuronal types on the basis of their pharmacology and five cloned groups according to molecular studies of alpha 1 subunits. The native types include L-, N-, P-, Q-, and R-type Ca2+ channels (Zhang et al. 1993), and the cloned types include alpha 1A, alpha 1B, alpha 1C, alpha 1D, and alpha 1E (see Dunlap et al. 1995 for review; Zhang et al. 1993). HVA Ca2+ currents are best separated with selective pharmacological agents, specifically by dihydropyridine (DHP) agonists and antagonists for L-type Ca2+ channels (Bean 1989; Fox et al. 1987a; Hess 1990; Tsien et al. 1988), by omega -conotoxin GVIA (CgTx GVIA) (Hess 1990; Sher and Clementi 1991; Tsien et al. 1991) for N-type Ca2+ channels, and by a low concentration of omega -agatoxin IVA (Aga IVA) (Brown et al. 1994; Mintz et al. 1992a,b) for P-type channels. Q-type currents, the largest component of HVA currents found in cerebellar granule cells, resemble alpha 1A calcium currents expressed in Xenopus oocytes and are sensitive to higher concentrations of Aga IVA (1 µM) and omega -conotoxin MVIIC (CgTx MVIIC) (Randall and Tsien 1995; Randall et al. 1993; Sather et al. 1993; Zhang et al. 1993). R-type channels are rapidly inactivating and are insensitive to neurotoxins and DHPs (Randall et al. 1993; Zhang et al. 1993). The R-type current has many properties in common with the rat alpha 1E and marine ray doe-1 currents (Soong et al. 1993; Williams et al. 1994; Zhang et al. 1993).

In this study we analyze the components of the Ca2+ current with the use of specific pharmacological agents in neurons of the CeA, an area involved in fear conditioning and in integrating autonomic, behavioral, and neuroendocrine responses to stress.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Cell dissociation

Pregnant female Sprague-Dawley rats at 16-18 days of gestation were purchased from Harlan (Houston, TX). Pups 8-18 days old were used in these studies. The techniques used to prepare 300- to 330-µm brain slices and cell dissociation were similar to that described previously (Yu and Shinnick-Gallagher 1994b). The rat was decapitated with the use of a decapitator (Harvard Bioscience), and the brain was removed and cooled rapidly in dissecting solution (0 ~ 5°C) continuously bubbled with 100% O2. The dissecting solution was composed of (in mM) 120 NaCl, 10 KCl, 2 KH2PO4, 1 CaCl2 × 2 H2O, 6 MgSO4 × 7 H2O, 10 d-glucose, and 10 piperazine-N,N'-bis-[2-ethanesulfonic acid], pH adjusted to 7.3 ~ 7.4 with 10 N NaOH. The osmolarity of the dissecting solution was measured with the use of a 5100 Vapor Pressure Osmometer (Wescor) and adjusted to 300 ~ 310 mosM with sucrose in all experiments. In cold dissecting solution, the brain was cut transversely, posterior to the first branch and anterior to the last branch of the superior cerebral vein; the resulting block of brain tissue was hemisected. Three to four serial coronal slices per hemisphere were sectioned with the use of a Vibroslice (Campden) and incubated in a beaker containing oxygenated dissecting solution at room temperature (22 ~ 25°C) for 20-30 min. The slices were then transferred to an enzyme solution preheated to 34.5°C and incubated for 12-15 min with oxygenation. The enzyme solution consisted of pronase E (20-30 mg, Type XXV, Sigma) and trypsin (18-20 mg, Sigma) in dissecting solution (15 ml). After enzyme treatment, the slices were washed twice and placed in oxygenated dissecting solution at room temperature for another 10 min. Subsequently, the CeA area in each brain slice was carefully removed with a scalpel under a stereo microscope under 60 × 120 magnification. The CeA in serial sections was identified and dissected from surrounding structures; only tissue removed from within its defined borders was used (Fig. 1A). CeA tissue was triturated gently with a series of flame-polished Pasteur pipettes to mechanically dissociate the individual neurons. The dissociated cells were pipetted into the holding section of the recording chamber that was mounted on the stage of a Nikon inverted microscope (Nikon Diaphot).


View larger version (42K):
[in this window]
[in a new window]
 
FIG. 1. Schematic representation of the central amygdala (CeA) dissected from the slice (A) and acutely isolated cells (B). A: schematic view of brain section of 10-day-old rat containing CeA (black-square) was redrawn from Sherwood and Timiras (1970). B: photographs of typical acutely isolated CeA neurons having morphology similar to those used to record Ca2+ currents. The diameters of these 2 cells are 13 × 14 and 15 × 16 µm, respectively. Generally, isolated cells have round somata and few, if any, dendrites. Ba: ×20 magnification. Bb: ×40 magnification. LaA, lateral amygdala; MeA, medial amygdala.

Electrophysiological recordings

The whole cell patch-clamp methodology (Hamill et al. 1981) was employed for recording Ca2+ currents. Patch electrodes were made from Corning 7052 glass (1.5 mm OD, Garner Glass), pulled with the use of a Flaming-Brown micropipette puller (Model P-97, Sutter) or a laser puller (Model P-2000, Sutter), and polished with the use of a Narishige microforge (MF-9, Narishige). The patch electrodes were coated with 20% Sylgard (Dow Corning) before polishing and had resistances of 3-6 MOmega when filled with an internal solution composed of (in mM) 90 cesium acetate, 18 tetraethylammonium chloride, 18 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid], 9 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, d-glucose, 5 Mg-ATP, 0.2 sodium guanosine 5'-triphosphate, and 0.1 leupeptin, pH adjusted to 7.1-7.2 with 1 N CsOH at room temperature, final osmolarity 270-280 mosM. The external solution consisted of 120 mM tetraethylammonium chloride, 3 mM CaCl2, 10 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid], 10 mM CsCl, 5 mM 4-aminopyridine, 10 mM d-glucose, and 2 µM tetrodotoxin. The pH of the external solution was titrated to ~7.4 with 1 N HCl or 1 N CsOH, and the osmolarity adjusted to 320 ± 5 mosM with sucrose. Both the internal and the external solutions were designed to suppress the interfering sodium and potassium currents pharmacologically. The Ca2+ currents recorded in this study are thought to be in the relative physiological range, because the extracellular Ca2+ concentration used (3 mM) is close to "normal" recording conditions (2.5 mM) (Allen et al. 1993). The reference electrode was filled with the same solution as the internal recording solution. Junction potentials of the patch electrodes were checked according to the method described by Neher (1992); the values ranged from 2 to 4 mV with tip diameters of 1.0-1.5 mm.

After a gigaohm seal was established, the membrane under the electrode was ruptured by application of gentle suction to the pipette. The recording was considered acceptable if neurons displayed robust inward sodium currents in the dissecting solution. The cells were voltage clamped with the use of the continuous single-electrode voltage-clamp mode of a Axoclamp-2A amplifier; the capacitance neutralization, gain, and phase controls were adjusted to produce optimal clamp efficiency. Under these conditions, a clamp gain of 8-10 nA/mV could be attained. Unless otherwise noted, all cells were clamped at -70 or -40 mV. After formation of whole cell recording configuration, the cell was lifted off the bottom of the chamber and inserted into an acetate tube (approximate volume of 80 µl) through an access hole in which the pipette coated with Sylgard fit tightly. One end of the tube was connected to a small chamber into which test solutions were introduced. The other end of the tube was connected via a piece of tubing to a solenoid valve. When the valve was opened, solution was pulled through the tube by gravity and, because of the small volume changed, relatively fast bath applications of drugs were obtained. This superfusion system is similar to that used by Akaike et al. (1986).

On- and off-line data acquisition and analysis were performed with the use of a DigiData 1200 interface (Axon Instruments) between a Axoclamp-2A preamplifier and a Gateway 2000 486/33C computer utilizing Pclamp 6.02 software programs (Axon Instruments). Analog signals were also stored as hard copy on a Gould (Model 3400) chart recorder for further analysis. Signals were filtered at 1 or 3 kHz before being digitized with the use of a built-in filter on the recording amplifier. The series resistance (Rs) was calculated as Rs = V/It and determined by fitting the decay of the whole cell capacitative transient in response to a voltage step (V) with the use of the P clamp 6.0 fitting function to obtain the value of It when t (time) = 0; the value of Rs was 5 ~ 10 MOmega (typically 7 MOmega ). Series resistance was not compensated because the currents measured were usually <1 nA, suggesting that the series resistance error was not significant in the present study. In addition, only cells showing rapidly deactivating tail currents were used. Capacitance and leak currents were estimated from the current evoked with depolarizing step commands in the presence of Cd2+ (200 µM) and were digitally subtracted for data analysis. All the experiments were performed at room temperature (22-25°C). Throughout the text, the reduction of the whole cell Ca2+ current (ICa) by Ca2+ channel blockers is expressed as percent of the control ICa obtained in normal recording solution. Traces (not including those used for current-voltage curve) displayed in figures are the averages of two consecutive voltage steps that were generated at 7-s intervals. Student's t-test was used to evaluate the significance of the data and the statistical significance was determined at the level of P <=  0.05. All data are expressed as means ± SE.

Whole cell calcium currents generally run down slowly with time although ATP and guanosine 5'-triphosphate are present in the pipette (Eliot and Johnston 1994; Mintz 1994; Mintz et al. 1992a; Mynlieff and Beam 1992). In the majority of CeA neurons, ICa showed a slow, progressive rundown with a decay rate of ~8% for the first 2-3 min, after which the current stabilized with a ~3-5% per minute rate of decay for baseline control recordings (time 0). ICa was not corrected for rundown in the data analysis.

Drug application

All drugs were applied with the use of a microsuperfusion bath application technique similar to the "concentration-clamp" technique described by Akaike et al. (1986). Nimodipine (NIM) (RBI, Natick, MA) and (±) Bay K 8644 (RBI, Natick, MA) were prepared as concentrated stock solutions in 90% dimethylsulfoxide and protected from light. Final concentration of dimethylsulfoxide was <0.05%, which had no effect when applied alone. The toxins CgTx GVIA (RBI, Natick, MA), CgTx MVIIC (Bachem, Torrance, CA), and Aga IVA (Alomone Labs, Jerusalem, Israel and a gift from Dr. Nicholas Saccomano, Pfizer) were made up as concentrated stocks in distilled water and stored as aliquots at -20°C. The stock solutions of drugs were diluted in the external solution before each experiment; cytochrome C (0.1%) was also added to the solution during testing of Aga IVA (Mintz et al. 1992a).


View larger version (21K):
[in this window]
[in a new window]
 
FIG. 2. Whole cell calcium current (ICa) and its dependence on calcium concentration. A, left: traces of ICa elicited by 100-ms voltage step commands from -70 to 0 mV. A, right: current-voltage relationship for ICa in the same cell. Peak current was recorded at 0 mV in this cell. B: ICa recorded in different extracellular calcium concentrations ([Ca2+]o). B, top left: control traces of ICa recorded in 3 mM extracellular Ca2+ concentration. B, middle left: ICa in 10 mM extracellular Ca2+ concentration. B, right: plot of current-voltage relationship for the peak ICa in 3 mM (bullet ) and 10 mM () calcium in same cell. A 3.3-fold (10 mM) rise in extracellular Ca2+ concentration increased the peak ICa by 20% in this cell. Note that the peak shifted with a change in extracellular Ca2+ concentration. Bottom left traces in A and B: voltage monitor of currents above. Leak currents were subtracted in A but not in B. Holding potential in B: -80 mV.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

ICa in acutely dissociated CeA neurons

These data were obtained from 129 neurons acutely dissociated from the CeAs (Fig. 1A) of 40 rats. Acutely dissociated CeA neurons (Fig. 1, Ba and Bb) in this study ranged in size from 10 to 20 µm with an average diameter of 13 ± 2 µm × 15 ± 2 µm (n = 115). This is in agreement with the previous morphological studies of CeA neurons in the rat: the principal neurons of the CeA are medium sized with a cell body diameter of 15-20 µm long and 11-13 µm wide (Cassell and Gray 1989; McDonald 1982). In the majority of isolated neurons only the soma remained after enzymatic treatment, but in some cells one or two dendrites remained (Fig. 1, Ba and Bb). To ensure better voltage-clamp control, cells with only short processes were selected for study.

Figure 2A illustrates a typical ICa in a dissociated CeA neuron. ICa normally activated at around -40 mV, peaked between 0 and 10 mV, and reversed between +50 and +60 mV when elicited by 100-ms voltage step commands from a holding potential of -70 mV. The current inactivated especially during strong depolarizing steps. With 3 mM external Ca2+ as the charge carrier, the peak ICa in CeA neurons ranged from 129 to 1,275 pA (mean: 477 ± 22 pA; n = 129) when measured from a holding potential of -70 mV with 100-ms voltage step commands to 0 or 10 mV. Raising the external Ca2+ concentration 3.3-fold to 10 mM increased the peak amplitude of ICa by 59 ± 13% (n = 3) and produced a 10-mV positive shift of the peak ICa (Fig. 2B).

This voltage shift was most likely due to a change in the surface negative charge around the Ca2+ channels (Ohmori and Yoshii 1977). ICa was completely blocked by 100 or 200 µM Cd2+ (Figs. 5 and 6) and 5 mM Co2+ (n = 5, not shown). Ni2+ at 1 mM blocked 95 ± 1% of the total Ca2+ current (n = 5, not shown).


View larger version (29K):
[in this window]
[in a new window]
 
FIG. 5. Effect of Aga IVA and omega -conotoxin MVIIC (CgTx MVIIC). A, left: current-time plot of Aga IVA (1 µM). Aga IVA was applied after inhibition of L- and N-type currents with 5 µM NIM and 1 µM CgTx GVIA; NIM and CgTx GVIA reduced ICa by 28%; addition of Aga IVA (1 µM) to NIM (5 µM) plus CgTx GVIA (1 µM) further reduced ICa by 17%, producing 45% inhibition of total ICa. Note that in this cell the resistant current accounted for ~54% of the total ICa (left). A, right: representative traces obtained from the same neuron shown at left. Neuron was held at -70 mV and stepped to 0 mV. B: current-time plot for CgTx MVIIC (250 nM). In this particular cell, CgTx GVIA (1 µM) alone reduced ICa by 42%; addition of CgTx MVIIC (250 nM) to the CgTx GVIA solution further reduced the current by 5%, yielding 47% inhibition of the total ICa. A mixture of CgTx GVIA (1 µM), CgTx MVIIC (250 nM), and NIM (5 µM) produced a 68% reduction in ICa. Addition of 100 nM Aga IVA in the above solution caused no further inhibition. In the presence of CgTx GVIA (1 µM), CgTx MVIIC (250 nM) reduced only 5% of ICa in this cell. The effect of CgTx MVIIC reached the plateau quickly. During the 9-min CgTx MVIIC toxin exposure, a slowly developing block was not apparent. Cd2+ (50 µM) completely blocked the resistant component of ICa. Note the resistant current in this cell accounted for 32% of the total ICa. B, inset: representative traces obtained from the same neuron shown in current-time plot. C: effect of CgTx MVIIC on non-N-type ICa in another cell. CgTx GVIA (1 µM) initially blocked 20% of the total ICa; addition of CgTx MVIIC (250 nM) further reduced ICa by 16%. Leak currents were subtracted; holding potentials were as indicated.


View larger version (20K):
[in this window]
[in a new window]
 
FIG. 6. Inhibition of the DHP- and neurotoxin-resistant calcium current by Cd2+ (A) and Ni2+ (B). Currents were evoked with the use of 100-ms step commands from -70 to 0 mV. 1: CgTx GVIA (1 µM) plus CgTx MVIIC (250 nM) plus NIM (5 µM). A: concentration-response effect of Cd2+ and representative traces recorded from a typical neuron treated sequentially with each antagonist (inset). The estimated half-inactivating concentration (IC50) was 2.5 µM and the Hill coefficient was 1. B: concentration-response curve for Ni2+ showed an estimated IC50 of 86 µM and a Hill coefficient of 0.8; representative traces (inset) recorded from a typical neuron treated with the antagonists. Curve fitting equation: (Imax - I)/Imax = 1/[1 + (IC50/C)n], where C is the concentration of either Cd2+ or Ni2+.

Both LVA and HVA Ca2+ currents could be recorded in CeA neurons depending on the holding potential. We found that in 53% of CeA neurons held at -100 mV (extracellular Ca2+ concentration = 3 mM), voltage step commands to -60 mV evoked a small LVA current (32 ± 7 pA; n = 10; not shown), whereas commands to -50 mV produced a LVA current 1.6 times larger (52 ± 7 pA; n = 10; not shown). These findings are similar to the results of Kaneda and Akaike (1989) in neurons dissociated from the whole amygdaloid complex. Similar step commands (to both -60 and -50 mV) from a holding potential of -70 mV did not elicit a recordable LVA current in any CeA neurons tested (Figs. 2 and 3B). In addition, CeA neurons held at -70 mV were more stable than those held at -80 mV. For this reason, we performed our experiments with -70 mV as the holding potential, a potential close to the resting membrane potential of CeA neurons recorded in slice preparations (-67 mV) (Rainnie et al. 1992; Schiess et al. 1993), suggesting that the recorded ICa would be within the physiological range for activation and inactivation.


View larger version (20K):
[in this window]
[in a new window]
 
FIG. 3. Effects of the dihydropyridines (DHPs). A: effect of nimodipine (NIM). A, left: NIM (5 µM) reduced the peak ICa by 31%; doubling the concentration to 10 µM reduced the peak ICa by 32%. A, right: NIM (5 µM) inhibited the current by 40% in a different neuron held at -40 mV. B: effects of Bay K 8644. B, left: representative traces of ICa recorded in the presence and absence of Bay K 8644 (5 µM). Bottom traces: voltage monitor. B, right: current-voltage relationship of the peak ICa in the presence and absence of Bay K 8644 (5 µM) for the same cell as at left. Bay K 8644 shifted the maximum of the current-voltage relationship to the left by ~10 mV. Note Bay K 8644 began to enhance the current at -50 mV, with the maximum current occurring at approximately -20 mV. Leak currents were subtracted. Holding potential (Vh) = -70 mV.

Effects of DHP antagonist and agonist on ICa

We analyzed the effects of the DHP antagonist NIM, and agonist (±) Bay K 8644, on ICa. DHPs have been reported to affect selectively the L-type Ca2+ current without altering T-, N-, Q-, or P-type Ca2+ currents and have been used as specific tools for testing the presence of the L-type Ca2+ channel (Eliot and Johnston 1994; Fox et al. 1987a,b; Tsien et al. 1988, 1991).

Figure 3 illustrates the effects of a DHP antagonist and agonist on ICa evoked by voltage step commands to 0 mV from a holding potential of -70 mV. NIM (5 µM) reduced ICa in 68% of CeA neurons tested (n = 17 of 25 cells; Fig. 3A, left); in these cells, NIM (5 µM) inhibited 22 ± 2% of the peak ICa. However, in 32% (n = 8 of 25 neurons) of CeA cells, similar concentrations of NIM (5 µM) did not cause a significant inhibition (4 ± 0.6%; P >=  0.05). The block induced by NIM occurred rapidly after application and was partially reversible by wash with control solution.

In a paired group of five neurons, doubling the concentration of NIM from 5 to 10 µM did not cause further inhibition of ICa (27 ± 3% and 29 ± 2%, respectively; P >=  0.05), suggesting that 5 µM NIM is a saturating concentration.

NIM (5 µM) blocked a slightly greater proportion of the peak ICa evoked by voltage step commands to 0 or 10 mV from a more depolarized holding potential (-40 mV; 33 ± 9%; n = 4; Fig. 3A, right) compared with that obtained from a more negative holding potential (-70 mV; 22 ± 2%; n = 17). The NIM-sensitive current at a holding potential of -70 mV was 106 ± 13 pA, whereas that at -40 mV was 78 ± 24 pA. These data suggest that L-type Ca2+ currents compose a larger proportion of the whole cell current elicited from holding potentials of -40 mV and are not substantially inactivated to a large extent at -40 mV, because 74% (78 pA/106 pA) of the current is still recorded.

The DHP agonist Bay K 8644 (5 µM) increased the peak ICa by 33 ± 8% in 70% (n = 7 of 10) neurons tested (Fig. 3B). The remaining 30% of the cells (n = 3 of 10) did not respond to Bay K 8644 (5 ± 2% decrease; n = 3). The increase in the amplitude of ICa with Bay K 8644 was greater at potentials between -50 and 0 mV and less pronounced at potentials more positive than 0 mV (Fig. 3B); the amplitude of the peak ICa in the current-voltage relationship for the Bay K 8644 current peaked at a more negative potential, a 10-mV shift (Fig. 3B, right). The inactivation rate was slightly accelerated in 86% of the sensitive neurons tested (Fig. 3B, left) and ICa deactivation (tail current) was prolonged in two of seven cells. These findings are in agreement with previous reports on cardiac cells, rat sensory neurons, and trigeminal neurons (Carbone et al. 1989, 1990; Huang 1989; Sanguinetti et al. 1986).

Effects of neurotoxins on ICa

We next used neurotoxins to test for the presence of N-, P-, and Q-type Ca2+ currents in CeA neurons. CgTx GVIA, a 27-polypeptide toxin from the venom of the snail Conus geographus (Gray and Olivera 1988; Olivera et al. 1984), is thought to block N-type Ca2+ channels selectively (Dunlap et al. 1995; Tsien et al. 1988, 1991). A different toxin, a 48-amino-acid peptide from funnel web spider Agelenopsis aperta (Mintz et al. 1992b), named Aga IVA, is reported to be a specific antagonist of the P-type Ca2+ channels at very low concentrations (Mintz et al. 1992a,b), but also affects Q-type channels in cerebellar granule cells at higher concentrations (Randall and Tsien 1995). Another omega -conotoxin, CgTx MVIIC, a 26-polypeptide toxin from the marine snail Conus magus (Hillyard et al. 1992), is believed to block Q-type Ca2+ channels (Randall et al. 1993, 1994) as well as N- and P-type channels (Olivera et al. 1994; Randall and Tsien 1995). We tested the effects of these toxins alone and in combination on ICa in CeA neurons.

Application of CgTx GVIA (1 µM) reduced ICa evoked by voltage step commands from -70 mV to 0 or 10 mV by 30 ± 2% (n = 18; Figs. 4, left, and 5C). Doubling the concentration to 2 µM CgTx GVIA produced a similar degree of inhibition (31 ± 5%; n = 8; Fig. 4A, left), suggesting that the CgTx GVIA effect was maximal at the 1 µM concentration in CeA neurons. The effect occurred immediately on application of the toxin and was not reversible after wash with control solution. We found that CgTx GVIA (1 µM) blocked a comparable proportion (33 ± 3%; n = 11) of ICa in neurons held at -40 mV (Fig. 4A, right). However, the CgTx-GVIA-sensitive current obtained at a holding potential of -40 mV (86 ± 15 pA; n = 11) was significantly less than the current obtained at -70 mV (139 ± 18 pA; n = 18), suggesting that an inactivation of N-type channels may occur at a more depolarized holding potential (-40 mV).


View larger version (21K):
[in this window]
[in a new window]
 
FIG. 4. Effects of omega -conotoxin GVIA (CgTx GVIA) and omega -agatoxin IVA (Aga IVA) on ICa. Currents in A and B were recorded from different cells and elicited by 100-ms depolarizing steps to 0 mV from a holding potential of -70 mV. A: effect of CgTx GVIA. A, left: CgTx GVIA (1 µM) irreversibly inhibited ICa by 39%. Increasing the concentration of CgTx GVIA (2 µM) produced only an additional 5% inhibition of ICa in this cell. A, right: CgTx GVIA (1 µM) inhibited the current by 30% in neurons held at -40 mV. B: effect of Aga IVA. B, left: Aga IVA (100 nM) did not significantly affect ICa. Increasing concentration of Aga IVA to 200 nM reduced ICa by 7% in this cell. B, right: Aga IVA (100 nM) had no effect on ICa. Leak currents were subtracted and the holding potentials were as indicated.

At low concentration, Aga IVA did not significantly affect ICa in CeA neurons, as shown in Fig. 4B. Application of Aga IVA (100 nM) for 1 min reduced the peak current by only 3 ± 2% (n = 4; Fig. 4B, left); this percent reduction is equal to that resulting from the rundown of ICa. An Aga IVA effect of similar magnitude (1%) was recorded after 2 min of the toxin application when the neuron was held at -40 mV (n = 2; Fig. 4B, right). Doubling the concentration (200 nM) caused a 6 ± 2% (n = 4) reduction in ICa elicited by step commands to 0 mV from holding potentials of -70 mV (Fig. 4B, left); this effect may be due to an action of the toxin on a Q-type (Randall and Tsien 1995), N-type, or L-type (Pearson et al. 1995) Ca2+ current, because the P-type channel is highly sensitive to low concentrations [half-inhibitory concentration (IC50) = 1 ~ 2 nM] of Aga IVA (Mintz et al. 1992a,b). The lack of a significant effect of a low concentration of Aga IVA on ICa suggested that CeA neurons do not possess P-type channels. For that reason, Aga IVA was not tested further at low concentrations.

Randall and Tsien (1995) recently reported that a Q-type current could be rapidly blocked by high concentrations (1 µM) of Aga IVA. To test the presence of Q-type current in CeA neurons, 1 µM Aga IVA was applied in the presence of CgTx GVIA (1 µM) and NIM (5 µM). In 10 neurons tested, 1 µM Aga IVA (at plateau) further reduced ICa by 18 ± 3% (Fig. 5A). However, Aga IVA (1 µM) alone reduced ICa by 34 ± 7% (n = 4; not shown), significantly larger than the reduction observed in the presence of NIM and CgTx GVIA (18 ± 3%; n = 10; P <=  0.05). The data suggest that Aga IVA may affect other currents as reported in neuronal cells (Pearson et al. 1995).

We also tested the effect of CgTx MVIIC (250nM) on ICa in the presence of 1 µM CgTx GVIA (Fig. 5B). We found the toxin rapidly reduced ICa by 13 ± 2% (n = 9; Fig. 5C) at -70 mV and by 11 ± 2% (n = 8) when the neurons were held at -40 mV. The CgTx-MVIIC-sensitive component measured162 ± 24 pA and 101 ± 30 pA holding at -70 and -40 mV, respectively, suggesting that the CgTx-MVIIC-sensitive component may inactivate at more depolarized potentials. The percent block recorded with CgTx MVIIC was not significantly different from that produced by 1 µM Aga IVA (18 ± 3%; P > 0.05) in the presence of CgTx GVIA and NIM. These results suggest that Q-type currents in CeA neurons may contribute a relatively small portion (13-18%) of the total ICa and that CgTx MVIIC at a concentration of 250 nM may adequately block Q-type currents in CeA neurons.

NIM and neurotoxins blocked distinct components of ICa in CeA neurons

To analyze the selectivity of calcium channel blockers on ICa in CeA neurons, we applied these drugs sequentially in combination. First, in the presence of CgTx GVIA (1 µM) and CgTx MVIIC (250 nM), we found that NIM (5 µM) still reduced ICa by 18 ± 2% (n = 11; Fig. 5B); the magnitude of this response was not significantly different from that produced with NIM alone (22 ± 2%; n = 13; P <=  0.05). These data suggest that these concentrations of the neurotoxins did not occlude the NIM block, and are in agreement with a report showing that CgTx MVIIC did not antagonize the L-type Ca2+ channels in CA1 hippocampal neurons (Hillyard et al. 1992). Conversely, when the effect of CgTx GVIA was tested in the presence of NIM, we found that the toxin (1 µM) still blocked ICa by 31 ± 3% (n = 4). The magnitude of the block produced by the toxin was not significantly different in the presence or absence of NIM (P >= 0.05), suggesting that NIM (5 µM) did not occlude the effect of CgTx GVIA. These data suggest that NIM and CgTx GVIA blocked distinct components of ICa in CeA neurons.

We further tested the interaction between the neurotoxins. After treatment with 1 µM CgTx GVIA, 250 nM CgTx MVIIC only blocked 13 ± 2% (n = 9) of ICa (Fig. 5C). This inhibition was significantly less than that achieved by CgTx MVIIC applied alone (38 ± 6%; n = 7; P <=  0.05; not shown), suggesting that CgTx MVIIC may affect the N-type Ca2+ current. Conversely, we found that 1 µM CgTx GVIA reduced ICa by 11 ± 4% (n = 4) in the presence of CgTx MVIIC (Fig. 6A, inset). Recently it has been shown that CgTx MVIIC produced a nonspecific block of N-, P-, and Q-type Ca2+ currents (Hillyard et al. 1992; Olivera et al. 1994; Sather et al. 1993) and the N-type channel was sensitive to CgTx MVIIC at nanomolar concentrations (>100 nM) (Birnbaumer et al. 1994). The present data further support the notion that CgTx MVIIC may affect N-type Ca2+ currents. Although CgTx MVIIC is less specific, it still blocked 13% of the total Ca2+ current in the presence of a maximally effective concentration of CgTx GVIA (1 µM), suggesting that both neurotoxins blocked distinct Ca2+ currents.

Properties of the DHP- and neurotoxin-resistant current

In the presence of CgTx GVIA (1 µM), NIM (5 µM), and CgTx MVIIC (250 nM), 48.9 ± 2.4% of the total ICa (control: 480 ± 43.4 pA; resistant: 244 ± 25 pA; n = 28) was not blocked. In 18 of these neurons, NIM (5 µM), CgTx GVIA (1 µM), and CgTx MVIIC (250 nM) applied sequentially blocked 63 ± 4% (n = 9 of 18) of ICa recorded in these cells; 37 ± 6% (n = 9) of ICa was resistant to the three blockers (Figs. 6 and 7). When the concentrations of NIM (10 µM), CgTx GVIA (2 µM), and CgTx MVIIC (500 nM) were increased, a similar percentage (36 ± 2%; n = 4 of 18) of resistant current was recorded (not shown). A similar portion of the resistant current was recorded with a high concentration of Aga IVA (1 µM) in the presence of 1 µM CgTx GVIA and 5 µM NIM (Fig. 5A). In 10 cells tested, application of 1 µM CgTx GVIA and 5 µM NIM in combination reduced 49 ± 6% of ICa; addition of 1 µM Aga IVA to the solution blocked 67 ± 5% of ICa, leaving 33 ± 6% unblocked, a percentage similar to that produced by the combination of saturating concentrations of CgTx GVIA, NIM, and CgTx MVIIC (37 ± 6%; n = 9). Furthermore, the resistant component made up 53 ± 6% (n = 5 of 18) of the total ICa in neurons that did not possess L-type Ca2+ currents. When the neurons were held at -40 mV (Fig. 5B), a similar contribution of resistant current (35 ± 4%; n = 8) was recorded in the presence of CgTx GVIA (1 µM), CgTx MVIIC (250 nM), and NIM (5 µM) in CeA neurons having L-type currents. Thus the relative contribution of the resistant current to the total ICa was not dependent on holding potential but was related to the presence of an L-type current.


View larger version (21K):
[in this window]
[in a new window]
 
FIG. 7. Comparison of the resistant currents obtained under similar conditions with equal extracellular concentration of Ca2+ and Ba2+ in 1 CeA neuron. Currents were evoked from a holding potential of -70 mV to a test potential of 0 mV. Treating the neuron with NIM (5 µM), CgTx GVIA (1 µM), and CgTx MVIIC (250 nM) reduced ICa by 56% and revealed a resistant component amounting to 44% of the total current. A: resistant currents recorded from the neuron with 3 mM external Ca2+ or 3 mM external Ba2+ (as indicated). The resistant channels generated a larger current with Ca2+ than with an equivalent molar concentration of Ba2+ as the charge carrier; the Ca:Ba current ratio is 1.29:1 in this cell. B: current-voltage relationship of the resistant current obtained with 3 mM external Ca2+ or 3 mM external Ba2+. The data were obtained from the same neuron as in A. Currents were leak subtracted.

Zhang et al. (1993) described, in cerebellar granule cells, a novel type Ca2+ current, the R-type Ca2+ current, which is resistant to NIM (10 µM), CgTx GVIA (1 µM), CgTx MVIIC (500 nM), and Aga IVA (100 nM) and shows considerable sensitivity to Ni2+ (IC50 = 66 µM) and Cd2+(IC50 = 1.2 µM). The R-type Ca2+ current has pharmacological and electrophysiological characteristics similar to the marine ray doe-1 and the alpha 1E calcium current (Ellinor et al. 1994; Soong et al. 1993; Williams et al. 1994; Zhang et al. 1993). Both alpha 1E and doe-1 show homology in their primary sequences.

We tested the sensitivity of the resistant current to both Cd2+ and Ni2+ in neurons held at -70 mV and stepped to 0 mV. Increasing the concentration of Cd2+ produced a potent block of the resistant current with an estimated IC50 of 2.5 µM (n = 4; Fig. 6A), a value slightly higher than that for the R-type Ca2+ current (IC50 = 1.2 µM) in cerebellar granule cells (Zhang et al. 1993). The resistant current was also sensitive to Ni2+ (Fig. 6B). Analysis of the concentration-response relationship showed that the estimated IC50 for Ni2+ was 86 µM (n = 4); this estimated value is greater than that reported for R-type Ca2+ current (IC50 = 66 µM) (Zhang et al. 1993) in cerebellar granule cells. Furthermore, the resistant current elicited from a holding potential of -40 mV was more sensitive to Ni2+, with an estimated IC50 of 73 µM (n = 4, not shown).


View larger version (13K):
[in this window]
[in a new window]
 
FIG. 8. Voltage dependence of activation and inactivation of the resistant current. For the activation curve, currents were measured at a variety of test potentials (Istep) and were normalized to the maximum current (Imax) from holding potentials of -70 mV. Curve fitting with a Boltzmann equation yielded a V1/2 of -17.1 mV and a k of -5.1 mV (see text). For the inactivation curve, the resistant current was obtained by measuring the current activated at 0 mV from various holding potentials (held for 20 s). The curve was fitted with a Boltzmann equation and described by V1/2 = -58 mV and k = 13.8 mV. Voltage step commands were 100 ms.

We further analyzed characteristics of the resistant current. We recorded the resistant currents with Ca2+ or Ba2+ and found that the resistant current was larger with Ca2+ as the charge carrier than with Ba2. The ratio of Ca2+ current to Ba2+ current was 1.31 ± 0.04:1 (n = 8; Fig. 7) for the resistant channel, which is similar to that observed for alpha 1E channels expressed in Xenopus oocytes (Bourinet et al. 1996). The voltage dependence of activation and inactivation was determined for the resistant current. The resistant channels began to activate at -40 mV and the amplitude of the resistant current increased steeply with depolarization, reaching a peak at between 0 and 10 mV. The activation curve could be fitted with the following Boltzmann equation
<IT>I</IT><SUB>step</SUB>/<IT>I</IT><SUB>max=</SUB>1/{1 + exp[(<IT>V − V</IT><SUB>1/2</SUB>)/<IT>k</IT>]}
where V1/2 is the potential at which Istep/Imax = 0.5 and k is the slope factor of the curve. The value for V1/2 (Istep/Imax) was -17.1 mV and the k value was -5.1 mV (n = 7; Fig. 8). Inactivation of the resistant current was analyzed by measuring the current at 0 mV and varying holding potential between -120 and 0 mV. The current was normalized with respect to the peak resistant current evoked from a holding potential of -120 mV, and plotted as a function of the holding potential. The resistant current began to inactivate at a holding potential of -100 mV and was completely inactivated when the cell was held beyond -10 mV (n = 3; Fig. 8). The inactivation curve could be best fitted with a Boltzmann equation (see above) with V1/2 = -58 mV and k = 13.8 mV. These activation and inactivation properties of the resistant current were similar to those of the R-type Ca2+ current (Zhang et al. 1993) and the alpha 1E (Williams et al. 1994) and rbE-II currents (Soong et al. 1993) recorded in expression systems.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

CeA neurons possess at least five distinct components of ICa

This study shows that ICa of CeA neurons is composed of multiple types of HVA Ca2+ channels, namely, L-, N-, and Q-type but not P-type Ca2+ channels. Additionally, neurons in the CeA possess a major component (37-53%; mean:49 ± 2%) of the total calcium current that is resistant to DHPs and neurotoxins and that has some characteristics similar to those of alpha 1E currents.

L-type Ca2+ current

About 70% of CeA neurons possess a DHP-sensitive Ca2+ current. The DHP antagonist NIM blocked nearly 22% of ICa in those neurons. Approximately 30% of the cells were not sensitive to NIM. NIM blocked a distinct component of ICa, an L-type Ca2+ current, because its effect was not occluded by saturating concentrations of the neurotoxins. The effects of NIM on ICa were rapid, partially reversible, and saturable at 5 µM. A fast onset and partially reversible action of NIM has been demonstrated in other preparations (Eliot and Johnston 1994; Huang 1989; Moyer et al. 1994).

The DHP agonist Bay K 8644 enhanced ICa in 70% of the neurons tested. The remaining 30% of the cells did not respond to Bay K 8644. The proportion of CeA neurons sensitive to DHPs was similar whether an antagonist or agonist was used, suggesting that a certain population (30%) of CeA neurons does not possess L-type Ca2+ channels.

N-type Ca2+ current

In CeA neurons, the CgTx-GVIA-sensitive Ca2+ currents compose ~30% of ICa. A saturating concentration of CgTx GVIA (1 µM) blocked ~30% of ICa whether elicited from a holding potential of -70 or -40 mV. N-type Ca2+ currents account for the second largest component of ICa in CeA neurons; this agrees with the studies showing a high density of [125I]b-CgTx GVIA binding sites in the amygdaloid complex (Takemura et al. 1988). CgTx GVIA did not occlude the effect of NIM on ICa, suggesting that the peptide was not acting on L-type Ca2+ channels. Furthermore, other studies report that CgTx GVIA binds only N-type Ca2+ channels (Olivera et al. 1994) and does not block Q-, P-, and L-type channels (Brown et al. 1994; Eliot and Johnston 1994; Sather et al. 1993; Tsien et al. 1991), suggesting that the effect of CgTx GVIA in CeA neurons is selective for the N-type Ca2+ channels.

P-type Ca+ current

Aga IVA is highly selective for native P-type Ca2+ channels, with an IC50 of 1-2 nM (Mintz et al. 1992a,b). Aga IVA at a concentration of 100 nM did not significantly affect ICa in CeA neurons, whether neurons were held at -70 or -40 mV. Increasing the concentration of the toxin to 200 nM still did not produce a significantly greater inhibition of ICa (6%). Thus the results of the present study suggest that CeA neurons probably do not possess Aga-IVA-sensitive or P-type Ca2+ current sensitive to lower concentrations of Aga IVA (100 nM).

Q-type Ca2+ current

We found that Aga IVA (1 µM) in the presence of saturating concentrations of CgTx GVIA and NIM blocked 18% of the total ICa, suggesting a moderate portion of Q-type currents in CeA neurons (Randall and Tsien 1995). CgTx MVIIC blocked a similar portion (~13%) of ICa at a concentration of 250 nM under similar conditions. These data suggest that 1) the contribution of Q-type current to ICa in CeA neurons is moderate (~13-18%) and 2) CgTx MVIIC may adequately block Q-type currents at a concentration of 250 nM in CeA neurons. The present finding that Q-type Ca2+ currents constitute a rather small portion of the total ICa is consistent with the finding that the number of binding sites for an analogue, CgTx MVIICnle, is relatively low in rat amygdaloid complex (Filloux et al. 1994). These data are further supported by in situ hybridization studies that show little expression of alpha 1A Ca2+ channels in the CeA (Stea et al. 1994). Furthermore, Stea et al. (1994) suggested that both P- and Q-type currents contain the alpha 1A subunit.

We found that CgTx MVIIC had a fast onset of inhibition. Although this rapid onset of action is similar to that of CgTx MVIIC on a non-L-type Ca2+ current in native retinal ganglion cells (Liu and Lasater 1994), the data contrast with those recorded from alpha 1A currents in expression systems (Sather et al. 1993) and from slice preparations (Wheeler et al. 1994). The reasons for these discrepancies are unclear, but there are several possible explanations. First, it is possible that Q-type channels in CeA neurons are not identical to those recorded in oocyte expression systems. The composition of subunits is tissue specific and some subunits are able to facilitate the onset of CgTx-MVIIC-induced block (De Waard and Campbell 1995). Second, the concentration-clamp system used in this study results in an instantaneous exposure of every Ca2+ channel to the toxin under static conditions. Addition of toxin to neurons in a slice or attached to the bottom of a chamber or a slow perfusion system could be factors resulting in differing onsets of action.

R-type Ca2+ current

In CeA neurons, 33-37% of ICa at -70 mV was resistant to NIM, CgTx GVIA, and Aga IVA or CgTx MVIIC. The portion of the resistant current was larger (53%) in neurons that did not posses L-type Ca2+ currents, yielding an overall resistant current of 49% of the total ICa in CeA neurons.

The resistant current is clearly not mediated by LVA channels, because 1) the resistant current could be recorded at holding potentials of -40 mV where LVA currents are inactivated (Fox et al. 1987b; Nowycky et al. 1985), 2) LVA currents were not able to be recorded in CeA neurons held at -70 mV, and 3) the resistant current was Cd2+ sensitive (IC50 = 2.5 µM) whereas LVA channels are less Cd2+ sensitive [IC50 = 650 µM in amygdaloid neurons (Kaneda and Akaike 1989)].

The resistant current in the CeA neurons exhibited a current-voltage relationship similar to that of R-type currents recorded in cerebellar granule cells (Ellinor et al. 1993; Zhang et al. 1993) and currents in oocytes expressing rat rbE II alpha  subunits (Soong et al. 1993). The half-activation potential (V1/2) of the resistant current was -17.1 mV, which is close to the value obtained in oocytes expressing human Ca2+ currents composed of alpha 1Ealpha 2bbeta 1-3 subunits (V1/2 = -14 mV) (Williams et al. 1994) or rat rbE II alpha  subunits(V1/2 = -24 mV) (Soong et al. 1993). Furthermore, the half-inactivation potential (V'1/2) for the resistant current was -58 mV, which is similar to that of R-type Ca2+ currents recorded from cerebellar granule cells (V'1/2 = -61 mV; Zhang et al. 1993), rat rbE II currents (V'1/2 = -65 mV;Soong et al. 1993), and human alpha 1E Ca2+ currents (alpha 1Ealpha 2bbeta 1-3,V'1/2 = -71 mV) (Williams et al. 1994), although these comparisons are with Ca2+ or Ba2+ as different charge carriers. The resistant current in the CeA neurons also showed a high sensitivity to both Cd2+ and Ni2+. The estimated IC50s for Cd2+ and Ni2+ were 2.5 and 86 µM (73 mM at holding potential of -40 mV), respectively. These IC50 values are fairly closely to those of Cd2+ (1.2 µM; 0.8 µM) and Ni2+ (66 µM; 27.4 µM) for the Ba2+ currents through the respective R-type (Zhang et al. 1993) or alpha 1E subunits (Williams et al. 1994) that have been detected in high density in the amygdala complex (Soong et al. 1993) and the CeA (Williams et al. 1994; Fig. 5), and suggest that the resistant current in CeA neurons may be mediated through R-type or alpha 1E subunits.

The resistant current is larger with the use of Ca2+ as a carrier than Ba2+, with a current ratio of Ca2+ to Ba2+ in CeA neurons of 1.31:1, but human alpha 1E-3alpha 2bbeta 1-3 channels expressed in HEK293 cells exhibit currents ~80% larger with Ba2+ as the charge carrier than with Ca2+ (Williams et al. 1994). Furthermore, Zhang et al. (1993) reported a ratio of Ca2+ to Ba2+ for R-type Ca2+ channels of 0.82:1, although close examination of results (Zhang et al. 1993) (Fig. 7B) shows granule cell R-type Ca2+ currents as slightly larger in Ca2+ than in Ba2+ at different command potentials. In contrast, the peak currents of Ca2+ or Ba2+ for the resistant channels in CeA neurons (1.31:1) are more similar to those of alpha 1E channels expressed in Xenopus oocytes (Bourinet et al. 1996), which have a ratioof 1.3:1. Thus the ratio for peak current of Ca2+ and Ba2+ differ among cloned subunits and R-type and CeA-type resistant currents.

The R-type Ca2+ currents and all alpha 1E-mediated Ca2+ currents mentioned above are relatively rapidly inactivating. Although we did not analyze the decay kinetics in detail, the resistant current in the CeA neurons inactivates more slowly. This discrepancy in inactivating kinetics could be due to a different composition of subunits in CeA neurons. Several studies in oocytes have shown that beta 2 subunits play a critical role in slowing the inactivation rate. For example, beta 2a or beta 2b subunits coexpressed with alpha 1A subunits produce the slowest inactivation rate (Sather et al. 1993; Stea et al. 1994). Similarly, beta 2 subunits coexpressed with T- and N-type alpha 1 subunits slow the inactivation rate of Ca2+ currents (Lacerda et al. 1994). More importantly, beta 2b is effective in slowing the rate of inactivation of currents mediated by doe-1, the structure of which is similar to that of alpha 1E subunits (Ellinor et al. 1993; Zhang et al. 1993). The high density of alpha 1E transcripts within the amygdala (Soong et al. 1993; Williams et al. 1994) (Fig. 5) suggests that alpha 1E subunits may contribute to the resistant current in CeA neurons. Pharmacologically, the CeA resistant current appears to be similar to R-type current in cerebellar granule cells (Zhang et al. 1993) and the doe-1 calcium channel (Ellinor et al. 1993), which are resistant to DHPs and neurotoxins. It is also possible that one Ca2+ channel antagonist may sterically affect the action of another, causing incomplete blockade. Thus, although the CeA resistant current resembles R-type and doe-1-type currents pharmacologically, this current may be composed of one or more subtypes that remain unclassified.

Functional relevance of different Ca2+ channel types in the CeA neurons

CeA neurons express multiple types of Ca2+ channels. In other brain regions both N- and Q-type Ca2+ channels mediate glutamatergic transmission (Lovinger et al. 1994; Wheeler et al. 1994), whereas both P- and N-type Ca2+ channels have been shown to mediate GABAergic, glycinergic, and glutamatergic transmission (Takahashi and Momiyama 1993). In pituitary peptidergic nerve terminals, N- and L-type Ca2+ channels are involved in vasopressin release (Lemos et al. 1994). Because the CeA region is rich in neurons containing diverse neurotransmitters, especially gamma -aminobutyric acid (GABA) (Nitecka and Ben-Ari 1987; Sun and Cassell 1993; Sun et al. 1994), dopamine (Fallon and Ciofi 1992), and numerous neuropeptides such as corticotropin releasing factor (CRF), neurotensin, and enkephalin (Carlsen 1989; Nose et al. 1991), the multiple types of HVA Ca2+ channels in CeA neurons raise the possibility that they may be implicated in the release process of these neurotransmitters, although in some brain neurons, the somal Ca2+ channels are different from those at their terminals (Ahlijianian et al. 1990).

Perhaps the most interesting question remaining is the functional relevance of the DHP- and neurotoxin-resistant channels that can contribute up to ~49% of ICa in CeA neurons. We have found that CRF increases the resistant component of ICa in CeA neurons (Yu and Shinnick-Gallagher 1994a). Because the CeA is a major output structure of the amygdaloid complex (Krettek and Price 1978; McDonald 1991, 1992a,b; Stefanacci et al. 1992), and the CeA projects to nuclei involved in central control of autonomic function, emotional conditioning, and stress, modulation of the somatic Ca2+ channels, perhaps of the resistant type, would affect neuronal functioning in the CeA and ultimately result in behavioral changes associated with aversive learning or stress.

    ACKNOWLEDGEMENTS

  The authors thank Drs. Joel P. Gallagher, Li-Yen M. Huang, Diana L. Kunze, and Aileen K. Ritchie for help and support in this project and for review of this manuscript.

  This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-29265 to P. Shinnick-Gallagher.

    FOOTNOTES

   Present address of B. Yu: Dept. of Physiology and Biophysics, 835 S. Wolcott Ave., Rm. 202 MSB, University of Illinois at Chicago, Chicago, IL 60302-7342.

  Address reprint requests to P. Shinnick-Gallagher.

  Received 19 January 1996; accepted in final form 19 September 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

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