Biophysical and Pharmacological Characterization of Voltage-Dependent Ca2+ Channels in Neurons Isolated From Rat Nucleus Accumbens

Dennis Churchill and Brian A. Macvicar

Neuroscience Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Alberta T2N 4N1, Canada

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Churchill, Dennis and Brian A. MacVicar. Biophysical and pharmacological characterization of voltage-dependent Ca2+ channels in neurons isolated from rat nucleus accumbens. J. Neurophysiol. 79: 635-647, 1998. The nucleus accumbens (NA) has an integrative role in behavior and may mediate addictive and psychotherapeutic drug action. Whole cell recording techniques were used to characterize electrophysiologically and pharmacologically high- and low-threshold voltage-dependent Ca2+ currents in isolated NA neurons. High-threshold Ca2+ currents, which were found in all neurons studied and include both sustained and inactivating components, activated at potentials greater than -50 mV and reached maximal activation at ~0 mV. In contrast, low-threshold Ca2+ currents activated at voltages greater than -64 mV with maximal activation occurring at -30 mV. These were observed in 42% of acutely isolated neurons. Further pharmacological characterization of high-threshold Ca2+ currents was attempted using nimodipine (Nim), omega -conotoxin-GVIA (omega -CgTx) and omega -agatoxin-IVA (omega Aga), which are thought to identify the L, N, and P/Q subtypes of Ca2+ currents, respectively. Nim (5-10 µM) blocked 18%, omega CgTx (1-2 µM) blocked 25%, and omega Aga (200 nM) blocked 17% of total Ca2+ current. Nim primarily blocked a sustained high-threshold Ca2+ current in a partially reversible manner. In contrast, omega CgTx irreversibly blocked both sustained and inactivating components. omega Aga irreversibly blocked only a sustained component. In all three of these Ca2+ channel blockers, plus 5 µM omega -conotoxin-MVIIC to eliminate a small unblocked Q-type Ca2+ current (7%), a toxin-resistant high-threshold Ca2+ current remained that was 32% of total Ca2+ current. This current inactivated much more rapidly than the other high-threshold Ca2+ currents, was depressed in 50 µM Ni2+ and reached maximal activation 5-10 mV negative to the toxin-sensitive high-threshold Ca2+ currents. Thus NA neurons have multiple types of high-threshold Ca2+ currents with a large component being the toxin-resistant "R" component.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The entry of Ca2+ through voltage-dependent Ca2+ channels performs many neuronal functions including the regulation of synaptic transmission, membrane excitability, cell growth/apoptosis, enzyme activity, and gene expression (Augustine et al. 1987; Choi 1995; Ghosh et al. 1994; McCleskey 1994). Neurons express a variety of Ca2+ currents each having distinct physiological properties (Eliot and Johnston 1994; Foehring and Scroggs 1994; Forti et al. 1994; Fox et al. 1987; Miller 1987; Nowycky et al. 1985; Tsien et al. 1988), distribution among and within neuronal types and display a wide range of responses to neuromodulators (Berridge and Dupont 1994; Snutch and Reiner 1992; Tsien et al. 1988, 1991).

Ca2+ currents have been classified into low-threshold (Akaike 1991; Huguenard 1996), and high-threshold sustained and inactivating currents (Bean 1989; Carbone and Swandulla 1989; Kostyuk 1989; Tsien et al. 1988). More recently, high-threshold Ca2+ currents have been grouped into L, N, and P/Q types using biophysical and pharmacological tools; the latter includes peptide toxins found in invertebrate venom in addition to dihydropyridines (DHPs) that have been well-characterized (Tsien et al. 1991). It has been demonstrated in many neurons that DHPs primarily block L-type, omega -conotoxin-GVIA blocks N-type, omega -agatoxin-IVA blocks P/Q-type, and omega -conotoxin-MVIIC blocks N/P/Q-type Ca2+ currents (Hillyard et al. 1992; Mintz et al. 1992b; Sher and Clementi 1991; Tsien et al. 1991). An additional class of neuronal high-threshold Ca2+ current has been named the R-type current by some investigators due to its resistance to block by the available toxins (Brown et al. 1994; Eliot and Johnston 1994; Magee and Johnston 1995; Randall and Tsien 1995).

The nucleus accumbens (NA), as an interface between the limbic and extrapyramidal system, has an integrative role in behavior. It functions in selective attention, secondary reinforcement, and reward (Carlsson and Carlsson 1990a; Pennartz et al. 1994). Interestingly, it may mediate the reinforcing effects of addictive drugs (Koob 1992) and possibly is involved in the pathogenesis and pharmacotherapy of schizophrenia (Andreasen 1994; Carlsson and Carlsson 1990b; Meltzer 1991). Both the NA and the related striatum (involved in motor integration) are composed of GABAergic medium-spiny neurons and receive cortical excitatory input and modulatory dopaminergic input from the ventral midbrain (Gerfen 1988) as well as input of a wide variety of endogenous neuromodulators (Angulo and McEwen 1994).

Neurotransmitter modulation of Ca2+ channel subtypes has been observed in many CNS neurons including the striatum (Surmeier et al. 1995; Tsien et al. 1991; Yan and Surmeier 1996). Therefore, it is possible that addictive drugs and neuroleptics that act on several neurotransmitter systems in the NA may at least partially influence the properties of NA neurons by affecting Ca2+ currents. We used whole cell voltage-clamp techniques to characterize some basic biophysical and pharmacological properties of the Ca2+ currents in cultured and acutely dissociated rat NA neurons. Similar experimental approaches to those used here have been applied on Ca2+ currents in the striatum and several other neuronal types (Bargas et al. 1994; Hoehn et al. 1993; Randall and Tsien 1995; Tsien et al. 1991). We found that NA neurons express T-, L-, N-, P/Q-type Ca2+ currents as well as a rapidly inactivating toxin-resistant high-threshold Ca2+ current. The present study is a necessary step for further investigations into the roles of Ca2+ currents in these neurons.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Cell isolation and culture

NA neurons used in this study were isolated using a technique modified from Kay and Wong (1986) and similar to that used on rat striatal neurons (Hoehn et al. 1993). Five- to 11- or 24- to 32-day-old ether-anesthetized Charles River rat pups (P5-11 or P24-32) of either sex were decapitated, and their brains removed and immersed in ice-cold artificial cerebral spinal fluid (ACSF) that contained (in mM) 124 NaCl, 5 KCl, 1.3 MgCl2, 26.2 NaHCO3, 10 glucose, and 2 CaCl2 [pH 7.35-7.40 and equilibrated with carbogen (95% O2-5% CO2)]. The cerebrum was bisected in the coronal plane with a razor blade, and 400-µm coronal vibratome slices were made. Three to five slices per brain were kept based on identification of the landmarks shown in Fig. 1A. During slicing, the brain and slices were kept in 4°C ACSF. The NA was punched out (see Fig. 1A) with a 16-gauge needle on a 1-ml syringe and transferred to a spinner flask containing 30 ml of ACSF, 1 mM kynurenic acid, 26-28 U/ml papain, and 0.27-0.28 mg/ml L-cysteine (at 32-33°C). Plugs of tissue were stirred gently to prevent settling and bubbled with carbogen for 30-75 min. The tissue was removed to 5 ml of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered Eagle's minimal essential medium (MEM) (cat No. 11090-040; GIBCO BRL, Burlington, ON) and triturated using a fire-polished Pasteur pipette (1-mm tip diam). The cells were centrifuged at 800 rpm for 5 min, and the supernatant replaced with modified SF1C medium (McCarthy and De Vellis 1980), which contained (components from GIBCO BRL and Sigma, St. Louis, MO) 47% MEM, 40% Dulbecco's modified Eagle's medium (DMEM), 10% Hams F-12 nutrient medium (cat No. 11550-027; GIBCO BRL), 0.25% albumin, 100 µg/ml transferrin, 30 nM Na2SeO3, 30 nM triiodothyronine, 25 µg/ml insulin, 200 nM progesterone, 125 nM hydrocortisone, 5 µg/ml superoxide dismutase, 10 µg/ml catalase, 10 ml penicillin/streptomycin, and 1% fetal calf serum (FCS) (pH 7.3). The cells then were plated on Cell Tak-coated (cat No. 40240; Collaborative Biomedical Products, Chicago, IL) glass coverslips (cat No. 12 CIR No. 1 D; VWR, Edmonton, AB) in a multiwell plate (cat No. 3534; Costar, Toronto, ON) and allowed to settle and stick for 30-60 min before using them for <= 8 h.


View larger version (106K):
[in this window]
[in a new window]
 
FIG. 1. Example of localization of the nucleus accumbens (NA) for culture (A), phase-contrast micrograph of an acutely isolated NA neuron (B), and epifluorescent micrographs of gamma -aminobutyric acid (GABA)- and beta -tubulin-stained cultured NA neurons (C). A: bright field micrograph taken on a dissection microscope outfitted with a charge-coupled device (CCD) camera of an unstained freshly made 400-µm coronal slice from a 5- to 11-day-old rat brain. Punch of tissue was taken with a 16-gauge blunt hypodermic needle from the region shown by the white dotted line. This region corresponds mostly to the core not the shell region of this nucleus. B: brightfield phase-contrast micrograph taken on an inverted microscope outfitted with a CCD camera of an acutely dissociated NA neuron from a P5- to P11-day-old rat. C: NA neurons were isolated from plugs of tissue as in A and plated on a bed of glia for several days. These were costained with anti-GABA (1) and anti-beta -tubulin antibodies (2) and visualized using rhodamine (GABA) and fluorescein isothiocyanate (FITC; beta -tubulin) secondary antibodies. These neurons are typical of those used for electrophysiological recordings made on cultured cells.

For long-term culture, P5-11 neurons were plated on glass coverslips on a confluent monolayer of glia that were prepared using a method adapted from Rayport et al. (1992) and McCarthy and De Vellis (1980). Glia were isolated from the cerebral cortex of 1-day-old rat pups. The cortex was removed, chopped up, and triturated with a fire-polished Pasteur pipette (1-mm tip diam) and plated onto polyornithine-coated coverslips into modified DMEM (cat No. 310-4080AJ; GIBCO BRL) containing: 0.7 mM NaHCO3, 20 mM HEPES, 10% heat inactivated FCS, and 10 ml of penicillin/streptomycin (cat No. 15070-014; GIBCO BRL) at pH 7.3. Medium was changed twice weekly and then neurons (5-14 days old) were plated onto the confluent glial cultures in modified SF1C medium. The culture medium then was changed only once after 1 day in culture at which time 25 µM 5-fluorodeoxyuridine was added to halt glial growth (Rayport et al. 1992). Cells were maintained under 5% CO2 at 37°C and used within 10 days.

Immunochemistry

Immunohistochemistry was performed on P5-11 cells cultured for 4-7 days with antibodies to beta -tubulin (1:1,000) and gamma -aminobutyric acid (GABA; 1:5,000). Cells were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde for 20 min at 4°C, washed three times in phosphate-buffered saline (PBS) for 10 min each, and then incubated with the primary sera in PBS with 10% normal goat serum (cat No. 16210-015; GIBCO BRL) and 0.3% tritonX with the Ab in PBS for 2 h at 37°C and then washed three times with PBS. The appropriate secondary anti-IgG antibody (anti-rabbit rhodamine-labeled for GABA and anti-mouse fluorescein isothiocyanate (FITC)-labeled for beta -tubulin) was added in PBS at 1:100 and incubated in the dark for 30 min at room temperature. Labeled cells were observed on an upright phase contrast microscope with fluorescence optics (Photomicroscope III; Carl Zeiss, Germany). Images were obtained with a charge-coupled (CCD) camera optically coupled to an image intensifier (KS-1381, Video Scope, Washington, DC) and were digitized using an image processing board (DT3155; Data Translation, Marlboro, MA) in a pentium computer that was controlled using Axon Imaging Workbench software (Axon Instruments, Foster City, CA).

Electrophysiology

For electrophysiology, one coverslip of cells was placed into a 30 mm Petri dish with HEPES-buffered MEM or modified SF1C. The coverslip was broken into pie-shaped pieces by pressing the blunt end of forceps on its center. One piece was removed for electrophysiology, and the rest were replaced in the incubator for later use. The coverslip was placed into a recording chamber composed of a 3-mm-thick sheet of Plexiglas with a 13-mm-diam hole drilled in the middle to form a 400-µl well with a glass coverslip bottom fixed in place with vacuum grease. Extracellular recording solution was gravity-fed into the chamber and siphoned from the bath by suction maintaining a 200-µl volume. The extracellular recording solution for isolating Ca2+ currents contained (in mM) 130 tetraethylammonium (TEA)-Cl, 10 HEPES, 5 4-AP, 5 CaCl2, 10 glucose, 3 KCl, and 300-600 nM tetrodotoxin (pH 7.35 with HCl). This level of external Ca2+ provided good stability of the whole cell patch recordings and increased the size of the measurable currents in these small cells. Standard patch-clamp methods were performed (Hamill et al. 1981) with an Axopatch-1D amplifier (Axon Instruments) and a 486 AT computer. Patch pipettes were pulled from thin-walled 1.5 mm OD capillary tubing (cat No. TW150F-6; WPI, Sarasota, FL) and had ~1-µm tip openings when pulled on a horizontal multistage pipette puller (model P-87; Sutter Instruments, Novato, CA). Some pipettes were coated with a thick coat of beeswax to within 100 µm from the tip and were firepolished (see figure legends for details). These were filled with a solution containing (in mM) 72 Tris-PO4, 25 Tris-base, 11 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 40 TEA-Cl, and 2 Mg-ATP (pH 7.35 with TEA-OH) and yielded pipette resistances of 3-8 MOmega . Gigaseals were formed with suction and fast capacitive transients were subtracted before the whole cell configuration was obtained with more suction. After a stable whole cell recording was obtained, the cell capacitance was recorded digitally and then canceled with the analog circuitry. Series resistance compensation only had a small impact on recordings of peak amplitude and slow inactivation times and was not used. Experiments were conducted at 19-23°C.

Data acquisition and analysis

Data were acquired using an Axon TL-1 DMA interface (Axon Instruments) at a frequency of 83.33 kHz for capacitive transient acquisition and 2.6 kHz for all other data and then filtered with the amplifier's built in three-pole Bessel filter. Ten kilohertz filtering was used to record the capacitance transient example for each cell, and 500 Hz was used for all other data recording. Holding current averaged 20 pA, rarely exceeding 50 pA. The leak current was left unsubtracted or was cadmium subtracted. In all traces shown, the dotted lines on traces mark zero current. Data were saved to disk and analyzed and displayed using Pclamp version 6.0 (Axon Instruments), spreadsheet software (Excel ver5.0; Microsoft), and a graphing program (Prism ver2.0; GraphPad, San Diego, CA). Fits were done using the nonlinear regression routine of Prism. All data are presented as means ± SE, and where appropriate the Student's t-test was used to determine statistical significance (P < 0.05).

The average capacitance (CM) and series resistance (RS) for 228 cells was 6.7 ± 3 pF and 15.7 ± 0.3 MOmega . These were determined by measuring the area and height (IPEAK) of capacitive transients (CM = area/VSTEP; RS = VSTEP/IPEAK) recorded using a hyperpolarizing 25-ms-long, 10-mV voltage step (VSTEP) from a holding potential of -100 mV applied every 500 ms (analogue measurement of these values gave similar results). Of the 67 long-term cultured cells from which recordings were made, 32 were judged to have adequate space clamp based on a smoothly increasing rate of activation with depolarization, no obvious jumps in I-V plots or the current during a voltage pulse that could be attributed to delays in activation of the Ca2+ current in different regions of the cell, and good capacitance transient subtraction using the amplifier circuitry. Data from the other 35 cells were not analyzed.

Ca2+ currents in these cells exhibited a slow, progressive decay with repeated pulses (50% decay in 10 min with 5 s between pulses) that resembled rundown but was reduced only partly by having ATP in the electrode. It was reduced significantly by lengthening the time between voltage pulses (<= 7 s) and by using shorter pulse lengths (<50 ms). This observation is similar to the slow inactivation process as described by Murchison and Griffith (1996) in basal forebrain neurons. In addition to this rundown-like decay, a somewhat faster decay occurred on switching the holding voltage (VH) between -100 and -50 mV, recovering on return to VH = -100, that also resembled that described in other neurons (Kay 1991; Keller and Nussinovitch 1996; Murchison and Griffith 1996). Slow inactivation in the nucleus accumbens stabilized in 40-60 s and was taken into account when doing two I-V protocols first at VH = -100 and then at VH = -50 (as in Figs. 2, 5, and 7D) by leaving time for the current to stabilize between the two protocols.


View larger version (35K):
[in this window]
[in a new window]
 
FIG. 2. Whole cell Ca2+ currents and current-voltage (I-V) relationships from an acutely dissociated NA neuron from a P5-11 rat (A-C) and I-V relationships from a cultured NA neuron from a P5-11 rat (D) and an acutely dissociated neuron from a P24-32 rat (E). A: I-V plots for total peak current (VH = -100 mV; square ), sustained peak current (VH = -50 mV; open circle ), and the inactivating current (difference between the other 2 plots; bullet ) for the same cell as in B and C are shown. These curves have been leak subtracted using an off-line leak subtraction method. B: illustration of a set of nonleak-subtracted Ca2+ currents from the same cell as in A elicited by 150-ms step pulses of -120-50 mV (10-mV increments) applied from VH of -100 (top) and -50 mV (bottom). - - -, zero current. C: inactivating currents for the pulse to 0 mV (top) and the pulse to -30 mV (bottom). Each trace was obtained by subtracting the appropriate trace from the sustained currents (VH = -50 mV) from the appropriate trace from the control currents (total current atVH = -100 mV). This example illustrates the presence of high-threshold sustained (HTS; bottom in B) and inactivating currents (HTI; top in C) and a rapidly inactivating T-type current (LTI; bottom in C and see lower threshold inflection in I-V plot in A, bullet , arrow). D and E: I-V plots as described in A for a cultured NA neuron from a P5-11 rat (D) and an acutely dissociated NA neuron from a P24-32 rat (E). A similar range of high-threshold and LTI Ca2+ currents is seen as in A. Due to the higher density of high-threshold currents in the P24-32 neurons compared with the P5-11 neurons and given the relatively smaller LTI expressed in P24-32 neurons (Table 1), LTI is less obvious than in acute and cultured P5-11 neurons but is still present (E, inset, right-arrow).


View larger version (23K):
[in this window]
[in a new window]
 
FIG. 5. Voltage dependence of the normalized Nim-, omega CgTx-, and omega Aga-sensitive components of Ca2+ current at VH = -50 mV. A: I-V curves were generated as in Fig. 2 by collecting a pair of I-Vs before and after toxin application. In this figure, the I-Vs at VH = -50 mV after toxin application were subtracted from the same before toxin application to give the I-V of the toxin-sensitive component. I-V under Nim is the average of 5 neurons and under the other 2 is the average of 6 neurons. All 3 curves activate at around -50 mV, and maximal current occurs at ~0 mV. B: normalized conductance vs. voltage curves calculated from each result set in A and then fit with a single Boltzman curve. Voltages at half-maximal conductance (V0.5) and slopes for each single Boltzmann fit to each data set were: Nim, -8.9 and 6.7 mV; omega CgTx, -8.7 and 7.4 mV; and omega Aga, -9 and 6.6 mV, respectively.


View larger version (38K):
[in this window]
[in a new window]
 
FIG. 7. Current-voltage characteristics of toxin-resistant whole cell Ca2+ current compared with other high-threshold currents and LTI. A: Cd2+-subtracted traces were generated as for Fig. 2 using VH = -100 mV. - - -, zero current. Toxin-sensitive current (top) were obtained by subtracting the control currents (not shown) from the toxin-resistant current (middle: obtained by combined application of 10 µM Nim, 1 µM omega CgTx, 200 nM omega Aga, and 2 µM omega CmTx). Bottom: toxin-resistant current that was blocked by 50 µM Ni2+. B: Cd2+-subtracted ramp currents for the same cell and treatments as shown in A. Voltage was ramped from -120 to 50 mV at 0.033 mV/s. Vertical bars mark peak current to show that toxin-resistant and Ni2+-sensitive currents peak more negatively than the toxin-sensitive currents. C: this graph illustrates I-V plots of leak-subtracted peak LTI current from a different NA neuron than shown in A and B and the I-V of the toxin-resistant current from the same cell as in A and B. For LTI, the data were acquired as in Fig. 2 with paired I-V protocols at VH = -100 mV and VH = -50 mV that were applied before and after application of 25 µM Cd2+. Cd2+ blocked most if not all of the high-threshold current while leaving an unblocked low-threshold component. D: averaged normalized conductance vs. voltage curves generated for LTI from data as shown in C and for toxin-sensitive and toxin-resistant data from ramps as shown in B. For all curves, conductance was calculated from currents using estimated reversal potentials for each cell. Currents were then normalized to maximal current, averaged, and then fit with a single Boltzmann curve, which yielded values for slope and V0.5 of: LTI: 4.6 mV, -42.8 mV (n = 14); toxin-resistant 6.6mV, -10.3 mV (n = 6); and toxin-sensitive 5.7 mV, -3.1 mV(n = 6).

In pharmacological experiments, the effect of each antagonist was assessed as quickly as possible after obtaining a whole cell recording and getting 1-2 min of control responses. To calculate toxin effects, the average peak current of the two to three traces at the end of the toxin application period were measured and normalized to the average of the last two or three traces just before the antagonist was applied. Peak current was taken as the maximal calcium current during a voltage pulse and end-pulse current as the average current during the last 10 ms of the pulse.

Drugs and their delivery

Cell superfusion was achieved with a multichannel, gravity-based, fast-perfusion system with feed from up to eight different solutions fed through PE160 tubing from 60- or 30-ml syringes into a common stainless steel tube of ~200 µm open diameter and ~0.5 µl. The superfusion tube was placed within 500-1,000 µm of the cell and supplied a flow rate of ~0.5 ml/min that achieved solenoid-controlled solution changes of well less than 1 s. Nimodipine (Nim; cat No. N-149; RBI, Natick, MA) was made up as 50-mM stock solution in ethanol and stored at -20°C. All toxins were made up as stock solutions in double-distilled H2O (milli-Q) and stored in single use containers at -20°C and aliquots were thawed and mixed with solutions just before use. omega -Conotoxin-MVIIC (omega CmTx) was a generous gift from George P. Miljanich, PhD (Neurex, Menlo, Park, CA) and was made up as a 5- or 1-mM stock. omega -Conotoxin-GVIA (omega CgTx; cat No. C9915, Sigma) was made up as a 1-mM stock. omega -Agatoxin-IVA (omega Aga) was a generous gift from Nicholas A. Saccomano, PhD (Pfizer, Groton, CT), came as a 91-µM stock in double-distilled H2O, and was stored as 91 or 10-µM stocks. All solutions including control solutions during toxin experiments contained 0.01-0.2% ethanol and 0.1 mg/ml cytochrome c. All other chemicals used in this study were analytic or research grade from BDH (Toronto, ON) and Sigma.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Cell preparation

Figure 1A shows a brightfield micrograph of a coronal slice from a P5-11 rat showing the landmarks used when making tissue punches. The NA has been divided functionally into the shell and the core regions using a variety of techniques (Jongen-Rêlo et al. 1994; Meredith et al. 1992, 1993; Zahm and Brog 1992). The punches we have taken were composed mostly of core neurons and have little chance of being composed of neurons from the striatum. Dissociation resulted in a fairly uniform distribution of 8- to 12-µm-diam cells of which some had short remaining processes. (Fig. 1B). When kept in HEPES-buffered MEM and plated on Cell Tak-coated glass coverslips, these cells could be used for <= 10 h after a settling time of 30-60 min. When plated and cultured on a layer of glia, neurons grew extensive processes and could be used for recordings for <= 10 days (Fig. 1C). Whole cell recordings were made from 152 acutely dissociated NA neurons from P5-11 rats (1-8 h in culture), 67 cultured NA neurons from P5-11 rats (1-14 days in culture on glia), and 9 acutely dissociated NA neurons from P24-32 rats.

Figure 1C shows epifluorescent micrographs of NA neurons cultured for several days. In Fig. 1C1, these cells were immunocytochemically stained with anti-GABA antibody. Figure 1C2 shows the same preparation of neurons stained with the neuron-specific anti-beta -tubulin to show that these are neurons and not glia. Other investigators have shown that the NA is composed 95% of GABAergic neurons (Gerfen 1988; Smith and Bolam 1990).

Biophysical properties of whole cell Ca2+ current

Figure 2B illustrates typical whole cell currents in an acutely dissociated NA neuron from a P5-11 rat. In other experiments (e.g., Figs. 4 and 6) in which 1 mM cadmium (Cd2+) was used to block Ca2+ current, little outward or inward current was apparent showing that contaminating currents were blocked adequately in our experiments. The rate of activation of Ca2+ current increased with depolarization, and the maximally activated inward current occurred at 0-10 mV. In this and many cells, both low- and high-threshold Ca2+ currents were evident. The threshold of activation for low-threshold current was -64.3 ± 0.9 mV,n = 40. 


View larger version (29K):
[in this window]
[in a new window]
 
FIG. 4. Block of whole cell Ca2+ currents by separate application of each of the Ca2+ channel toxins (A-D) and 50 µM Ni2+ (E). Ca2+ current was monitored with a train of 150-ms-long, 0-mV voltage pulses applied from VH = -100 mV every 7 s. Toxins were applied using rapid perfusion after >= 30 s of stable control responses. Once a stable toxin effect was achieved, the toxin was washed from the recording chamber, and then 0.5-1 mM Cd2+ was applied to block all the current. A measurement of peak (open circle ) and end-pulse (bullet ) current was graphed against time of recording (left). square , periods of control solution application; black-square, periods of toxin or Cd2+ application (as labeled). Illustrated for each treatment (right) are a control trace (a), a trace from a period where a stable toxin effect was achieved (b), and the difference between the 2 (- b) to show the kinetics of the toxin-sensitive current. A: nimodipine (Nim, 10 µM) effect was reversible, and the Nim-sensitive current was largely noninactivating. B: omega -conotoxin-GVIA (omega CgTx, 1 µM) irreversibly blocked a large partially inactivating component of the Ca2+ current. C: omega -agatoxin-IVA (omega Aga; 20 and 200 nM) irreversibly blocked a mostly noninactivating component of Ca2+ current. D: omega -conotoxin-MVIIC (omega CmTx, 5 µM) blocked a partially inactivating component in a somewhat reversible manner. E: Ni2+ (50 µM) rapidly and reversibly blocked a component of Ca2+ current, which, in this cell, almost completely inactivated during the 150-ms voltage pulse.


View larger version (39K):
[in this window]
[in a new window]
 
FIG. 6. Averaged effects of Nim, omega CgTx, omega Aga, omega CmTx, and 50 µM Ni2+ on NA Ca2+ current. Currents were elicited by a train of 120-ms, 0-mV commands at 5-s intervals from VH = -100 mV. A: Cd2+-subtracted whole cell Ca2+ currents from an acutely isolated NA neuron from a P5-11 rat. Lowercase letters on each Ca2+ current trace correspond to the letters on the peak data set in the graph in B. - - -, zero current. B: graph of peak (bullet ), end-pulse (square ), and baseline current (diamond ) against time of recording in seconds for the cell shown in A. Drugs were applied by fast perfusion as shown by the labeled bars. C: graph of average percent block by each toxin applied alone or following application of other toxins. square  for Nim and all black-square but that for Nim are from data from A, B, and D. Thus Nim preceded omega CgTx, Nim and omega CgTx preceded omega Aga and Nim, omega CgTx and omega Aga preceded CmTx, and all the toxins preceded Ni2+ application. Other square  are from single application experiments as in Fig. 4 and the black-square for Nim was obtained after application of omega CgTx. In comparing the difference between the individual and group application data, only the results for omega CgTx and omega CmTx (note * on bars) were statistically significant (P < 0.05). Thus Nim partially blocks omega CgTx-sensitive channels. As expected, omega CmTx blocked much more current when applied alone compared with when it was preceded by Nim, omega CgTx, and omega Aga. Although not a perfectly selective blocker of toxin-resistant currents 50 µM Ni2+ appears to be a potent blocker of this current. D: normalized average toxin-sensitive traces obtained from acutely isolated P5-11 NA neurons from experiments as in A and B. These traces were obtained by subtracting a trace from the steady-state period before each toxin application from a trace from the steady-state period during that toxin's application (i.e., Delta ICa2+). All traces were first Cd2+ subtracted and normalized to the control current (i.e., ICa2+con) so that the relative magnitude of the averaged traces reflects the relative magnitude of the blocked current. The small vertical lines evenly spaced along the traces are standard error bars obtained by averaging those specific points along the curve. Standard errors bars for the other points along the curve have been omitted for clarity but are similar in size to those that have been shown.

In this study, we also have separated high-threshold current into its sustained and inactivating components (HTS and HTI, respectively). This was done so that the Ca2+ current components blocked by the different toxins could be described in more detail within an historical context and so that a detailed comparison to the voltage-dependent activation of the toxin-resistant current could be made. In acutely isolated neurons, HTI and HTS components had very similar thresholds of activation of -50 mV (-51.4 ± 0.7 mV, n = 106 and -53.3 ± 0.8 mV, n = 48, respectively). The voltage-dependent properties were examined by running paired current-voltage (I-V) protocols in which currents were elicited by voltage steps of -120 to 50 mV first from a VH of -100 mV and then from -50 mV (Fig. 2, A and B). The inactivating current was obtained by subtracting currents obtained at VH = -50 mV from those obtained at VH = -100 mV (Fig. 2A, bullet  and traces in Fig. 2C). In the cell in Fig. 2, A-C, both low-threshold inactivating (LTI) and HTI inactivate by VH = -50 mV (Fig. 2A, open circle ). The currents remaining at -50 mV were composed of an HTS component (Fig. 2B, bottom). The average voltage at which each component of Ca2+ current was maximal and the average magnitude of the current respectively were as follows: LTI:-34.7 ± 0.9 mV, -13.6 ± 1.1 pA, n = 45 (peak estimated at -30 or -40 mV); HTS: 3.1 ± 0.6 mV, -94.7 ± 7.5 pA, n = 106 (from I-V plot at VH = -50 mV); and HTI: 3.3 ± 0.6 mV, -93.8 ± 5.8 pA, n = 104 (from I-V plot of inactivating current).

Differences have been reported previously in the expression of the various components of Ca2+ currents among acutely dissociated neurons, cultured neurons, and neurons isolated from different aged rats (Lorenzon and Foehring 1995b; Rossi et al. 1994; Thompson and Wong 1991). This was also true for studies conducted on isolated striatal neurons, a structure related to the NA, in which LTI was expressed in young and cultured neurons but not in striatal neurons isolated from older rats (Bargas et al. 1991, 1994; Hoehn et al. 1993). To see if the expression of LTI followed this same pattern in the NA, we also recorded from 9 acutely isolated neurons from P24-32 rats and 32 cultured neurons from P5-11 rats. Figure 2D shows an I-V plot from a typical cultured NA neuron from a P5-11 rat, and Fig. 2E shows a typical result from acutely dissociated NA neurons from a P24-32 rat. The data were acquired and displayed as for the acutely isolated cell from P5-11 rats (Fig. 2, A-C).

The cultured neurons (Fig. 2D) showed the same range of high-threshold currents and an LTI current that were found with the acutely isolated cells from P5-11 rats except that the LTI was found in a higher percentage of cells (Table 1). The average voltage at which each component of Ca2+ current was maximal and the average size of the current respectively were as follows: LTI: -31.7 ± 4.1 mV, -51.1 ± 7.6 pA, n = 24; HTS: 3.0 ± 0.9 mV, -185.5 ± 24.9 pA, n = 32; and HTI: 0.2 ± 1.6 mV, -93.5 ± 15.3 pA, n = 31. Cultured cells, unlike acutely dissociated cells, could be identified occasionally as spiny (SP) or aspiny (ASP). Of 25 cells where such an identification could be performed, 15 were ASP (10- to 20-µm diam cell bodies) and 10 were SP (8-18 µm diam). No difference was found between the ASP and SP morphological types regarding the expression of HTI and HTS (100% occurrence each) and LTI expression (60 and 70% occurrence, respectively). Acutely isolated cells from P24-32 rats (Fig. 2E) also exhibited a similar range of Ca2+ current components except, in this case, it was more difficult to identify LTI given the relatively larger density of the high-threshold components (Table 1). The average voltage at which each component of Ca2+ current was maximal and the average size of the current respectively were as follows: LTI: -35.0 ± 2.2 mV,-26.3 ± 7.7 pA, n = 5; HTS: 4.4 ± 1.8 mV, -173.9 ± 50.7 pA, n = 9; and HTI: 1.7 ± 3.1 mV, -76.8 ± 27.3 pA,n = 9. 

 
View this table:
[in this window] [in a new window]
 
TABLE 1. Current densities of the various biophysically identified components of Ca2+ current in different neuronal preparations

Table 1 summarizes the average densities of the various biophysically identified currents and the percentages at which the different components were found in the three preparations. The average cell capacitances for P5-11 acutely dissociated, P5-11 cultured, and P24-32 acutely dissociated cells respectively were as follows: 6.0 ± 0.4, 7.0 ± 0.5, and 4.0 ± 0.6 pF. In the rest of the paper, cultured neurons mostly were used to acquire data on LTI (Figs. 3 and 7) because LTI was generally larger and more consistently present in these cells, and acutely isolated NA neurons from P5-11 rats were used for the pharmacological studies because the cells were the most easily acquired and voltage clamped.


View larger version (33K):
[in this window]
[in a new window]
 
FIG. 3. Voltage dependence of inactivation of LTI (A and B) and time dependence of recovery of LTI from voltage-dependent inactivation (C and D). A: family of whole cell LTI currents obtained from a cultured NA neuron by applying conditioning voltages of -110 to -40 in 5-mV increments and then a 200-ms test pulse to -30 mV. Five seconds were left between -30 mV pulses, and the voltage was dropped to the next conditioning voltage immediately after the test pulse. These currents were not leak subtracted. B: average voltage dependence of inactivation curve. This curve is the average from 14 NA neurons (pooled from cultured and acutely dissociated neurons from P5-11 rats). Data were normalized to maximum peak current, and LTI currents were taken as the difference between peak and end-pulse current. V0.5 and slope for a single Boltzmann fit were -80 and 6.7 mV, respectively. C: family of Ca2+ currents obtained using a voltage protocol to determine the time dependence of recovery of LTI from voltage-dependent inactivation. A P5-11 NA neuron was clamped 1st at -50 mV to inactivate LTI and then stepped to -100 mV for durations of 50-1,850 ms in 100-ms increments before a 150-ms test pulse to -30 mV was applied to determine the magnitude of recovered current. Voltage was returned to -50 mV after the test pulse and 6 s were left before the next test duration. These currents were not leak subtracted. - - -, zero current D: graph of recovery of LTI from inactivation for 10 neurons. Fraction of recovered current was obtained by taking the difference between the peak and end-pulse current and then graphing it against the duration of the conditioning -100 mV voltage. These data were fit with a single exponential with tau  = 397 ms.

To describe the LTI current in these neurons in more detail, the voltage dependence of inactivation and the time dependence of recovery from inactivation of LTI was examined in Fig. 3. To determine the voltage dependence of inactivation of LTI, -110- to -40-mV conditioning voltages in 5-mV increments preceded a 200-ms test pulse to -30 mV that was applied every 5 s (Fig. 3A). LTI current was measured as the difference between the peak and end-pulse current for each trace and was normalized to the value at -10 mV (open circle ). This averaged normalized current was graphed against the conditioning voltage and then fit with a Boltzmann curve (Fig. 3B). Complete inactivation occurred at around -50 mV and maximal activation occurred just negative to -100 mV. The voltage at half-maximal conductance (V0.5) and slope of the Boltzmann fit were -80 and 6.7 mV, respectively. These values are typical of central neurons (Akaike 1991; Huguenard 1996) and similar to those found for striatal neurons (Hoehn et al. 1993).

Figure 3, C and D, illustrates the time dependence of recovery of LTI from voltage-dependent inactivation. These data were acquired using a voltage protocol in which voltage was held first at -50 mV to inactivate LTI and then stepped to -100 mV for 50-1,850 ms (in 100-ms increments) to reactivate it before a -30-mV test pulse was applied to determine the magnitude of the recovered current. On graphing the average results from 10 cultured cells and then fitting the data with a single exponential curve, the time constant for recovery was determined to be 397 ms; this is also typical of other central neurons (Akaike 1991; Hoehn et al. 1993; Huguenard 1996).

Pharmacological properties of high-threshold Ca2+ current

In this study, we have applied both the DHP Nim and the toxins omega CgTx, omega Aga, and omega CmTx individually and additively to further identify high-threshold current in acutely isolated NA neurons from P5-11 rats. The individual effects of each toxin were tested using trains of pulses (Fig. 4) and pairs of I-V relationships at VH = -100 mV and VH = -50 mV (Fig. 5). Trains were used to determine the kinetics of block and recovery with washout, the degree of block, and the kinetics of the blocked current. Pairs of I-Vs were used with individually applied toxins to determine the voltage dependence and the proportion of HTS versus HTI comprising the blocked currents. Finally, toxins were applied additively to allow some determination of the selectivity of the toxins and to examine the voltage dependence and kinetics of the toxin-resistant component that was found to be distinct from the toxin-sensitive components.

Individual Nim or toxin application---trains of pulses

Figure 4 illustrates results from the individual application of each of the toxins tested (Fig. 4, A-D) as well as 50 µM Ni2+ (Fig. 4E). The blocking effects were monitored using 0-mV voltage pulses from VH = -100 mV that were applied every 7 s.

The DHP Nim (5-10 µM) blocks L-type Ca2+ channels in a variety of central neurons (Aosaki and Kasai 1989; Bean 1989; Hille 1992; Marchetti et al. 1995; Miller 1987; Nowycky et al. 1985; Tsien et al. 1988) including the striatum (Bargas et al. 1994; Hoehn et al. 1993). In NA neurons, 10 µM Nim caused a partially reversible block of Ca2+ current. Nim reduced the Ca2+ current by 17.8 ± 1.5%(n = 21) of the total in 22.5 ± 5.4 s (Fig. 4A). On removal of Nim from the recording chamber, 88.4 ± 1.4% of the blocked current recovered in 34.6 ± 4.8 s (n = 10). A control trace (Fig. 4Aa) was subtracted from a trace obtained in the presence of Nim (Fig. 4Ab), to show the kinetics of the Nim-sensitive current. For the pulse duration used (150 ms), the kinetics of the Nim-sensitive current was largely noninactivating. This aspect of the toxin-sensitive currents is discussed in more detail in the next section.

N-type Ca2+ channels in a variety of central neurons, including the striatum (Bargas et al. 1994; Hoehn et al. 1993), are blocked by the peptide toxin omega CgTx (Kasai et al. 1987; McCleskey et al. 1987; Olivera et al. 1985; Plummer et al. 1989; Tsien et al. 1991). In NA neurons, 1 µM omega CgTx caused an irreversible block of Ca2+ current (Fig. 4B). omega CgTx reduced the total Ca2+ current by 36.0 ± 2.5%(n = 21) in 41.9 ± 4.5 s; 2 µM omega CgTx only blocked an additional 8 ± 4% of current (n = 6), suggesting that 1 µM omega CgTx was probably at saturating concentration. On removal of omega CgTx from the recording chamber, only an average 2.0 ± 0.7% of the blocked current recovered after several minutes of washout. The kinetics of the omega CgTx-sensitive current was partially inactivating.

omega Aga is a potent blocker (~KD = 20 nM) of P-type Ca2+ channels (Llinás et al. 1992; Mintz et al. 1992a,b) and at higher concentrations is thought to block Q-type channels (Randall and Tsien 1995; Sather et al. 1994). omega Aga has been shown to block current in the striatum (Bargas et al. 1994). Figure 4C illustrates the effect on a single NA cell of 200 nM omega Aga, a concentration that may block P- and Q-type channels (Randall and Tsien 1995), and of 20 nM omega Aga, which should affect mostly P-type channels. Twenty nanomolar omega Aga only blocked 7.0 ± 2.0% (n = 5) of the Ca2+ current in 42.4 ± 12.0 s, whereas further addition of 200 nM omega Aga resulted in a 19.3 ± 2.0% (n = 28) reduction in Ca2+ current in 84.1 ± 9.8 s. With removal of 200 nM omega Aga from the recording chamber, there was no recovery of blocked Ca2+ current (1.8 ± 1.2%) after several minutes of superfusion with control solution. The omega Aga-sensitive current had noninactivating kinetics regardless of the concentration used.

omega CmTx is a broader spectrum toxin that blocks N-, P-, and Q-type channels (Hillyard et al. 1992; Liu et al. 1996; McDonough et al. 1996; Stea et al. 1994; Wu and Saggau 1995). It was used in this study instead of higher concentrations of omega Aga to ensure block of Q-type current in later experiments. As shown in Fig. 4D, 5 µM omega CmTx blocked a large component of Ca2+ current in a partially reversible manner. omega CmTx reduced the current 33.0 ± 6.2% in 15 ± 3 s in four cells examined. Removal of omega CmTx caused80.7 ± 7.9% of the current to recover in 46.5 ± 5.1 s. The omega CmTx current was partially inactivating, which is consistent with block of N-, P-, and Q-type current. In these experiments and those in Fig. 7, we were careful to apply omega CmTx for many minutes because its block of omega Aga-sensitive current has been reported to be slow (McDonough et al. 1996).

Previous studies in several other neuron types (Eliot and Johnston 1994; Forti et al. 1994; Magee and Johnston 1995; Randall and Tsien 1995) have suggested that Ni2+ at low concentrations is a potent blocker of the toxin-resistant current. Ni2+ was applied individually to neurons in these experiments to determine its selectivity. Ni2+ rapidly blocked a mostly inactivating current. This is evident from the graph and traces shown in Fig. 4E, which shows a large reduction in the peak current but only a small reduction in end-pulse current. Ni2+ caused a 25.9 ± 3.0% reduction in peak current within 7 s in the nine cells tested. The effect was mostly reversible with recovery of 95.5 ± 2.3% of the blocked current occurring in 62.1 ± 12.3 s. The percent block data shown in this figure (Fig. 4) have been summarized as part of Fig. 6C.

Individual toxin application---current-voltage relationships

Figure 5 illustrates the voltage-dependence properties of the of the Nim-, omega CgTx- and omega Aga-sensitive currents that remain at VH = -50 mV. All three averaged and normalized I-V curves are very similar in their voltage dependence. To compare the relative voltage differences between the individual currents, the voltage dependence of the normalized estimated conductance for each curve is shown in Fig. 5B. The conductance relationship may be contaminated by a non-Ca2+ channel outward current at the apparent reversal potential (Brown et al. 1993; Lorenzon and Foehring 1995a). Each data set (n = 6 for omega CgTx and omega Aga and n = 5 for Nim) was fit with a Boltzmann curve having V0.5 and slopes for data set of Nim, -8.9 and 6.7 mV; omega CgTx, -8.7 and 7.4 mV; and omega Aga, -9 and 6.6 mV. Nim and omega Aga did not depress HTI, however, omega CgTx blocked both a HTS and a HTI component about equally: 67.3 ± 7.3% and 52.0 ± 8.8%, respectively. The omega CgTx-sensitive HTI component not shown in Fig. 5 was virtually identical to the other toxin-sensitive components (V0.5 = -9.0 mV and slope = 6.7 mV). The average percent decay [(peak current - end-pulse current) radical peak × 100] of the omega CgTx-sensitive current during a 150-ms, 0-mV pulse at VH = -50 was 8.6 ± 3.0% (n = 5) and for the inactivating current (traces at VH = -100-mV minus traces at VH = -50 mV) was 33 ± 11%. omega CgTx had no effect on LTI in five LTI-expressing cells, however, 10 µM Nim reduced LTI by 20 ± 6% in four cells in which LTI was expressed.

Group application of Nim and all the toxins

In the remaining experiments, the drugs were applied sequentially and cumulatively to more accurately determine the ratio of pharmacologically separated high-threshold current components and to examine the toxin-resistant current. Nim (5-10 µM), 1-2 µM omega CgTx, 200 nM omega Aga, 2-5 µM omega CmTx, 50 µM Ni2+, and 1 mM Cd2+ were applied according to the bars in Fig. 6B. Toxin concentrations were selected that generally are considered saturating for their respective currents. [omega CmTx (2-5 µM) was included because it is considered to block N- and P-type channels as well as Q-type channels (Hillyard et al. 1992; Randall and Tsien 1995; Sather et al. 1993; Zhang et al. 1993)]. When omega CgTx and omega Aga are used at saturating concentrations, any further blocked current is considered to be of the Q type.

Figure 6 shows a typical example of one such toxin experiment on an acutely isolated NA neuron from a P5-11 rat. Figure 6A shows whole cell Ca2+ current traces selected from the stabilized period during the application. Currents were elicited using a train of 120-ms step commands to 0 mV from VH = -100 mV applied every 5 s. The peak (bullet ), end-pulse (square ), and baseline (diamond ) currents were graphed against time of recording in seconds in Fig. 6B. There was no change in the baseline current showing that the recording was stable. The current was allowed to reach an apparent steady state before the addition of the next toxin. The percent block was calculated by comparing the steady-state periods before and after each toxin application. In this cell, Nim blocked 31%, omega CgTx 15%, omega Aga 24% and omega CmTx 6% of the total current. This left a resistant component of 24%. Ni2+ reduced the toxin-resistant component by a further 12%. A final component of 12% remained that was resistant to all these treatments.

Figure 6D shows average subtracted current traces from <= 37 cells obtained by subtracting a trace from the steady-state period before each toxin application from a trace from the steady-state period after that toxin's application in experiments as in Fig. 6, A and B. All traces were first Cd2+ subtracted and normalized to the control current so that the relative magnitude of the averaged traces reflects the relative magnitude of the blocked current. The percent decay for each current was as follows: 5-10 µM Nim-sensitive, 11% (n = 21); 1-2 µM omega CgTx-sensitive, 16% (n = 37; 200 nM omega Aga-sensitive, 24% (n = 37); 5 µM CmTx-sensitive, 50% (n = 19); toxin-resistant, 66% (n = 19); toxin-resistant/Ni2+-sensitive, 88%; and toxin and Ni2+-resistant, 56%. These results are consistent with known properties of L-, N-, and P-type currents (Tsien et al. 1991). omega -CmTx blocked a small partially inactivating component that is probably an additional part of a Q-type current that remained unblocked by 200 nM omega Aga. The remaining toxin-resistant current and that portion suppressed by Ni2+ were distinct from the toxin-sensitive currents by inactivating much more rapidly.

The average amount of each toxin-sensitive and resistant current obtained from the data in Fig. 6, A, B, and D, is shown by the solid bars in Fig. 6C, which summarizes all the toxin data. In addition, the open bars are results from single and separate application of each toxin from experiments as in Fig. 4 (except for Nim, which is from Fig. 6). The solid bar for Nim is for the effect of Nim after the application of omega CgTx. These results give some information on the selectivity of the toxins and Ni2+. Nim blocked about the same amount of current whether omega CmTx was applied before it or not (18.8 ± 2.2% vs. 17.6 ± 1.6%, respectively). omega CgTx applied alone blocked statistically (P < 0.05) more current (36.0 ± 2.5%) than when Nim was applied before it (26.7 ± 2.0%). There was not a difference when omega Aga was applied alone (19.3 ± 2.0%) or after Nim and omega CgTx (17.4 ± 1.5%) thus neither toxin blocked omega Aga-sensitive channels nonselectively. Ni2+ (50 µM) was not completely selective for the toxin-resistant current because it blocked more current before toxin addition (25.9 ± 3.0%) than after (15.8 ± 3.5%), however, this difference was not statistically different. As expected, omega CmTx blocked statistically more current before (32.9 ± 6.2%) than after application of all the toxins (6.1 ± 0.7%).

Voltage dependence of the toxin-resistant Ca2+ current

Figure 7 examines the voltage dependence of the toxin-resistant current and compares it with the toxin-sensitive current and LTI. Current traces from an acutely isolated NA neuron in response to 150-ms voltage pulses are shown in Fig. 7A. The top set of traces show the current sensitive to combined application of 10 µM Nim, 1 µM omega CgTx, 200 nM omega Aga, and 2 µM omega CmTx. These were obtained by subtracting the control traces from toxin-resistant traces shown in the middle. The toxin-sensitive current has very distinct kinetics compared with the toxin-resistant current in this cell. In Fig. 7B, voltage ramp currents for the same cell in Fig. 7A are shown (obtained using voltage ramps of -120 to 50 mV at 0.33 mV/s applied from VH = -100 mV). The bars on the traces mark the peak current showing that the toxin-resistant and toxin-resistant/Ni2+-sensitive currents peak at voltages more negative than the toxin-sensitive component. A rather large example of an LTI is shown in Fig. 7C (for a different cell than in the rest of this figure) along with an I-V of the toxin-resistant current (Fox et al. 1987; Hille 1992) for the cell shown in Fig. 7, A and B. The I-V for LTI was obtained by isolating LTI from high-threshold currents using its relative resistance to 25 µM Cd2+. The average estimated normalized conductance curves for LTI (black-square), the toxin-resistant current (open circle ), and the toxin-sensitive current (bullet ) are shown in Fig. 7D. These conductance curves are useful for comparing the voltage dependence of the individual currents. There may be errors in estimating the true conductance relationship because of contamination of non-Ca2+ channel outward currents as described earlier for Fig. 5. The estimated conductance curves were fit with single Boltzmann curves, which yielded slopes and V0.5 of 4.6, -42.8 (n = 14); 6.6, -10.3 (n = 6) and 5.7, -3.1 mV(n = 6), respectively.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

This study demonstrates that NA neurons express multiple Ca2+ current subtypes including Nim-, omega CgTx- and omega Aga-sensitive Ca2+ currents, plus a more rapidly inactivating high-threshold toxin-resistant current. There is also a low-threshold rapidly inactivating T-type current (LTI). Identification of high-threshold current subtypes was largely based on generally accepted pharmacological tools. We confirmed other studies that the biophysical differences between high-threshold subtypes are modest (McCleskey 1994; Olivera et al. 1994; Tsien et al. 1991). However, some biophysical characterization was carried out (Fig. 5) to show that the toxin-resistant current differed from LTI (Fig. 7) and the other high-threshold components not only in its kinetics but also in its voltage dependence.

Most NA neurons (90%) are medium spiny GABAergic neurons, the remainder are larger aspiny neurons (Gerfen 1988; Smith and Bolam 1990). Both types were seen in culture and expressed similar currents. NA neuronal cultures were stained with anti-GABA to show that the majority of neurons we isolated were GABAergic. Acutely isolated cells were mostly spherically shaped without processes, with only short processes, or only short newly sprouted processes and could not be further subclassified. The region of brain tissue removed for cell isolation (Fig. 1A) roughly corresponded to the central core region of the NA. A variety of techniques has been used to separate the NA into the inner core and outer shell regions (Jongen-Rêlo et al. 1994; Meredith et al. 1992, 1993; Zahm and Brog 1992). Because each technique yields different maps of the core versus shell location within the NA, it is difficult to further state the functional identity of the neurons used here.

NA neurons isolated from P5-11 rats were chosen for most of this study because these were easily isolated and voltage clamped. However, because work on the related striatal neurons suggested that LTI was expressed only in neurons isolated from young rats (Hoehn et al. 1993) or in cultured cells (Bargas et al. 1991) but not in neurons from >P28 (Bargas et al. 1994), we also examined cultured P5-11 NA neurons and NA neurons from P24-32 rats. Evidence for loss of LTI during development has been found for some neurons (Thompson and Wong 1991) but not others (Fisher et al. 1990; Takahashi et al. 1989). LTI was found in all three NA preparations. It was seen most easily in neurons from P5-11 rats, but also was identified in neurons from P24-32 rats even though it was relatively smaller. The difference between the NA and striatum is borne out by studies in brain slices from adult rats in which low-threshold Ca2+ spikes, which are generated by low-threshold Ca2+ currents, were found in the NA (O'Donnell and Grace 1993, 1995) but not the striatum (Bargas et al. 1989; Pineda et al. 1992).

Toxin-sensitive currents

Saturating Nim blocked 18% of total current in NA neurons in a partially reversible manner. Most of the blocked current was HTS and decayed 11% during a 150-ms voltage pulse similar to the L-type currents examined in other neurons (Bean 1989, 1991; Carbone and Swandulla 1989; Hille 1992; Kostyuk 1989; McCarthy and TanPiengco 1992; Regan et al. 1991). The concentration of Nim used in this study (5-10 µM) was not completely selective for L-type channels. It was found here that Nim reduced LTI by 20%. Similar Nim effects were reported for other neurons (Akaike et al. 1989; Huguenard 1996; McCleskey et al. 1987). Nim also appeared to partially block omega CgTx-sensitive channels. omega CgTx block was reduced significantly after Nim addition, suggesting a partial overlap similar to that found for other neurons (Akaike et al. 1989; McCleskey et al. 1987).

Saturating omega CgTx irreversibly blocked 25% of total NA Ca2+ current that decayed 16% during a voltage pulse and was composed of both HTS and HTI. These properties are typical of those expected for a N-type Ca2+ current that generally is accepted as omega CgTx-sensitive (Aosaki and Kasai 1989; Hess 1990; Kasai et al. 1987; McCleskey et al. 1987; Mintz et al. 1992a; Olivera et al. 1985; Plummer et al. 1989; Tsien et al. 1991). Reduction of both HTS and HTI by omega CgTx has been reported for other neurons (Aosaki and Kasai 1989; Kasai and Neher 1992; Mynlieff and Beam 1992), and on the basis of single-channel studies, it has been suggested that DHP-resistant but omega CgTx-sensitive channels can have both sustained and inactivating kinetics (Aosaki and Kasai 1989; Plummer and Hess 1991; Plummer et al. 1989).

omega Aga (200 nM) was used to test for P/Q-type channels. It irreversibly blocked 17% of total current (mostly HTS with a small inactivating component). In cerebellar Purkinje neurons, omega Aga irreversibly blocks a noninactivating current with high affinity (Llinás et al. 1992; Mintz et al. 1992a,b). More recently it has been shown that Q-type current is blocked reversibly with lower affinity (Randall and Tsien 1995; Zhang et al. 1993). Both currents are identified with class A alpha  subunits, although alpha 1A-expressing oocytes give a more Q-like current with inactivating kinetics and low-affinity omega Aga block (Mori et al. 1991; Sather et al. 1993). Cerebellar granule cells have been clearly shown to express both a small P- and larger Q-type currents (Randall and Tsien 1995). In our experiments, we applied both 20 and 200 nM omega Aga to see if we could determine whether the current expressed in these cells was a Purkinje cell-like P-type current, a Q-type current, or both. omega Aga at 20 nM only blocked a small component of Ca2+ current, which did not vary in its kinetics from the larger current blocked by 200 nM omega Aga. This, plus the further block of current after 200 nM omega Aga by omega CmTx (Fig. 6), suggests that the majority if not all of the omega Aga-sensitive current is probably Q type. If a P-type current similar to that found in Purkinje cells (i.e., blocked with very high affinity) is present in NA neurons, it is a small component of total current. The rundown/slow inactivation of Ca2+ current in these cells made it impractical to pursue the possibility of such a small P-type current.

Toxin-resistant Ca2+ current

The largest single fraction of total current was resistant to the toxins and Nim (32%). It rapidly inactivated (66% decay), had a high-threshold activation range, and had a voltage of maximal activation 5-10 mV more negative than the toxin-sensitive currents. A rapidly inactivating toxin-resistant and Ni2+-sensitive component, which some investigators have named R type, has been a common finding in a variety of neuronal preparations (Brown et al. 1994; Eliot and Johnston 1994; Lorenzon and Foehring 1995a; Magee and Johnston 1995; Pearson et al. 1995; Randall and Tsien 1995). These unique properties support the possibility that this is a distinct current. Indeed, the resistant current in this and other neurons resembles the toxin-resistant current found in alpha 1E-expressing oocytes and HEK cells (Soong et al. 1993; Williams et al. 1994; Zhang et al. 1993). The message for class E alpha -subunit clones is found throughout the CNS including in the NA (Soong et al. 1993). The resistant current is clearly high threshold and thus cannot be the T current. It is unlikely that a portion of the resistant current was composed of unblocked Q-type current because the toxins were applied for 5-10 min in experiments shown in Fig. 7. Presently, the definition of the toxin-resistant current remains one of exclusion. The toxin-resistant current could have different molecular identities in different neurons and possibly be composed of multiple subtypes in individual neurons.

In summary, NA neurons express a variety of high-threshold Ca2+ currents and a LTI Ca2+ current. The high-threshold currents in NA neurons include four pharmacologically distinct subtypes. It is not well understood why neurons express such a wide variety of Ca2+ channels. Certainly these different types, which are being identified increasingly with distinct molecular subtypes (Snutch and Reiner 1992; Snutch et al. 1991), can have different localization within and among cells and can undergo a wide variety of modulation (Berridge and Dupont 1994; Snutch and Reiner 1992; Tsien et al. 1988, 1991). In light of this, we have conducted this study to form a base for more detailed examination of NA Ca2+ current modulation. The goal is to provide further insight into the role of Ca2+ currents in normal NA function and possibly in NA involvement in drug addiction and the treatment of diseases of the basal ganglia such as schizophrenia.

    ACKNOWLEDGEMENTS

  We gratefully acknowledge the useful and critical comments by Drs. S. Barnes and M. F. Wilkinson; proofreading by M.A.R. Johns; and the skillful cell culture and immunostaining by D. Feighan.

  This work was supported by a grant from the Medical Research Council of Canada. D. Churchill is a recipient of an Alberta Heritage Foundation Medical Research (AHFMR) postdoctoral fellowship and B. A. MacVicar is an AHFMR scientist, a MRC senior scientist, and Ciba Geigy chair for schizophrenia research.

    FOOTNOTES

  Address reprint requests to B. A. MacVicar.

  Received 31 January 1997; accepted in final form 22 September 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society