Voltage-dependent Ca2+ channels are a major pathway for Ca2+ entry in neurons. We have studied the electrophysiological, pharmacological, and molecular properties of voltage-gated Ca2+ channels in motoneurons of the rat facial nucleus in slices of the brainstem. Most facial motoneurons express both low voltage-activated (LVA) and high voltage-activated (HVA) Ca2+ channel currents. The HVA current is composed of a number of pharmacologically separable components, including 30% of N-type and ∼5% of L-type. Despite the dominating role of P-type Ca2+ channels in transmitter release at facial motoneuron terminals described in previous studies, these channels were not present in the cell body. Remarkably, most of the HVA current was carried through a new type of Ca2+ channel that is resistant to toxin and dihydropyridine block but distinct from the R-type currents described in other neurons.
Using reverse transcription followed by PCR amplification (RT-PCR) with a powerful set of primers designed to amplify all HVA subtypes of the α1-subunit, we identified a highly heterogeneous expression pattern of Ca2+ channel α1-subunit mRNA in individual neurons consistent with the Ca2+ current components found in the cell bodies and axon terminals. We detected mRNA for α1A in 86% of neurons, α1B in 59%, α1C in 18%, α1D in 18%, and α1E in 59%. Either α1A or α1B mRNAs (or both) were present in all neurons, together with various other α1-subunit mRNAs. The most frequently occurring combination was α1Awith α1B and α1E. Taken together, these results demonstrate that the Ca2+ channel pattern found in facial motoneurons is highly distinct from that found in other brainstem motoneurons.
- facial nucleus
- calcium channel
- calcium current
- single-cell RT-PCR
Neuronal Ca2+channels have been subdivided on the basis of their electrophysiological and pharmacological properties into low voltage-activated (LVA) or T-type channels (Huguenard, 1996) and high voltage-activated (HVA) channels, a class that includes L-, N-, P/Q-, and R-types (Hofmann et al., 1994; Dunlap et al., 1995; Wheeler et al., 1995). The HVA channel types are not easily distinguishable from their biophysical properties and have mainly been characterized by their different sensitivities to pharmacological modulators and inhibitory toxins. HVA channels are complexes composed of a pore-forming α1-subunit together with modulatory β-, α2/δ-, and, at least in skeletal muscle, γ-subunits (Hofmann et al., 1994; Catterall, 1995; Dunlap et al., 1995). To date, six HVA α1-subunits have been identified: α1A, α1B, α1C, α1D, α1E, and α1S. Although it is clear that α1B forms the N-type channel, α1C and α1D the L-type channel, and α1S the skeletal muscle channel/voltage sensor, the subunit composition of the P/Q- and R-type channel is less clear. α1A is thought to form the P/Q-type and α1E the R-type. The α1-subunits that compose the T-type channel α1G and α1H have recently been cloned (Perez-Reyes et al., 1998).
In motoneurons, Ca2+ channels are clearly involved in the release of transmitter from the axon at the neuromuscular junction (NMJ). In addition, they are also expressed in the soma and dendrites, where they have been shown to be involved in the control of firing properties, both directly and through the activation of other Ca2+-dependent membrane conductances (Viana et al., 1993). Furthermore, Ca2+ signaling, possibly involving Ca2+ entry through voltage-gated Ca2+ channels, has been implicated as a mechanism involved in the degeneration of motoneurons in amyotrophic lateral sclerosis (ALS) (Uchitel et al., 1988; Llinás et al., 1993;Mosier et al., 1995). Changes in Ca2+ channel types involved in transmitter release at the endplate also accompany axon growth and muscle reinnervation after axotomy of adult motoneurons (Katz et al., 1996).
The Ca2+ channel types present at the nerve terminals of facial motoneurons have been extensively characterized (Uchitel et al., 1992; Protti and Uchitel, 1993; Katz et al., 1996,1997). Although a number of studies describe the Ca2+ channel types present in motoneurons in other regions of the brainstem and in the spinal cord, both in brain slices and in culture (Mynlieff and Beam, 1992; Viana et al., 1993; Umemiya and Berger, 1994, 1995), those in the facial nucleus have not been studied in detail (Umemiya et al., 1993). This precludes the comparison of the Ca2+ channels present in the soma with those at nerve terminals and study of their possible differential distribution. We therefore characterized voltage-gated Ca2+ channels in the facial nucleus of the neonatal rat. The electrophysiological and pharmacological properties were studied using the patch-clamp technique in slices of the brainstem. In addition, we also studied the expression of mRNA for the different α1-subunits of the HVA Ca2+ channel in individual neurons using reverse transcription (RT) followed by PCR amplification of the resulting cDNA.
MATERIALS AND METHODS
Preparation. Neonatal Wistar rats [age: postnatal day 1 (P1)–P7] were decapitated, and the brainstem was removed rapidly and placed in ice-cold saline. Transverse slices (150-250 μm thick) were prepared using a vibrating slicer as described previously (Edwards et al., 1989). After they were cut, the slices were incubated at 37°C for ∼1 hr and thereafter at 25°C until they were transferred to the recording chamber. For some control experiments, rat hippocampal and cerebellar slices were used.
The recording chamber containing the slice was placed on the stage of an upright microscope (Axioskop FS, Zeiss, Jena, Germany) and viewed using infrared differential interference contrast video microscopy (Stuart et al., 1993). In early experiments, the facial nucleus was localized by retrograde labeling with the carbocyanine fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes, Eugene, OR). Briefly, rat pups were anesthetized by hypothermia, and a small incision was made behind one ear. The facial nerve was localized, and a suspension of dye [2.5 mg/ml, 20% ethanol, 80% saline with 0.1% bovine serum albumin (also see Mynlieff and Beam, 1992)] was injected into the nerve using a glass micropipette. The incision was sutured. Rats were killed 1-2 d after injection. Slices containing retrogradely labeled facial motoneurons were clearly visible when viewed using epifluorescence (see Fig. 1). In later experiments, unlabeled slices containing the facial nucleus were identified visually under a dissecting microscope using dark-field illumination and in the experimental set up using infrared differential interference contrast videomicroscopy.
Patch-clamp recording. Whole-cell currents were measured using the patch-clamp technique with an EPC 7 or EPC 9 patch-clamp amplifier and Pulse software (Heka, Lambrecht, Germany). Patch pipettes were made of borosilicate glass (Hilgenberg, Malsfeld, Germany) and coated with a silicone resin (GE-Silicones, Bergen op Zoom, The Netherlands). The electrodes had resistances of 2-3 MΩ when filled with the internal solution that contained (in mm): 130 CsCl, 20 TEACl, 1 EGTA, 4 MgATP, 0.4 GTP, and 10 HEPES (titrated to pH 7.2 with CsOH). Leakage correction was performed using a P/4 protocol at a potential of −100 mV. Series resistance compensation (≥70%) was used in all experiments.
Solutions and chemicals. The standard saline contained (in mm): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 20 glucose (pH 7.3 when gassed with 95% O2 and 5% CO2). To record Ca2+ channel currents, this solution was exchanged for one containing (in mm): 105 NaCl, 20 TEACl, 2.5 KCl, 1 or 0.5 BaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 20 glucose, with 0.5 μm tetrodotoxin (TTX), 1 μm strychnine, and 10 μm bicuculline (pH 7.3 when gassed with 95% O2 and 5% CO2). In experiments in which peptide toxins were applied, the bath solution also contained 0.1 mg/ml cytochrome C. Because the addition of cytochrome C reduced the TTX block of Na+ currents, the TTX concentration was raised to 1 μm. ω-Conotoxin (CTx)-GVIA was obtained from Sigma (Deisenhofen, Germany), ω-Conotoxin-MVIIC was from Alomone Labs (Jerusalem, Israel), different batches of ω-agatoxin (Aga)-IV were obtained from Peptide Institute (Osaka, Japan) or Pfizer Research, and ω-Aga-TK was from Alomone Labs. The toxins were prepared as stocks in water and kept frozen at −20°C. Toxins were applied either locally from a pipette located close to the cell, by bath perfusion, or by addition to the bath while the perfusion was stopped. No difference in blocking efficacy was observed with the different application methods. The experiments were all performed at room temperature (20-25°C).
Cellular RNA harvest and RT. The contents of individual neurons were harvested, and mRNA was transcribed into cDNA as described in detail previously (Lambolez et al., 1992; Plant et al., 1997). Thereafter, the tube was stored at −80°C until it was used for PCR amplification. Positive controls, using total RNA from rat brain, and negative controls, using water instead of RNA or without reverse transcriptase, were performed in parallel to the reactions for material isolated from single cells.
First PCR amplification of the rat brain isoforms of the α1-subunit of the HVA Ca2+channel. PCR amplification was performed using partially degenerate primers (see Fig. 3) that amplify fragments of all of the rat brain isoforms of the HVA calcium channel α1-subunits, α1A–α1S. The primers were selected for regions of homology between segment S6 of repeat III and segment S5 of repeat IV (see Fig. 3). The sense and antisense primers used were: Calpha-up [5′-AT(C/T) (A/G)TC ACC TTC CAG GAG CA-3′] and Calpha-lo [5′-GCG TAG ATG AAG AA(A/G/C) AGC AT-3′]. The positions of the primers on the individual sequences in the GenBank are as follows: the Calpha-up (upstream or sense, 20-mer) primer positions (position 1 is the first base of the initiation codon) were 4384 on α1A (P/Q-type: M64373, rat brain), 4252 on α1B (N-type: M92905, rat brain), 3496/3505 on α1Ca/b (L-type: M67516/M67515, rat brain), 3451 on α1D (L-type: M57682, rat brain), 4123 on α1E (R-type: L15453, rat brain), 1054 on α1S (skeletal muscle-type; L04684, rat brain), and the Calpha-lo (downstream or antisense primer, 20-mer) primer positions were 4954 on α1A, 4822 on α1B, 4105/4114 on α1Ca/b, 4072 on α1D, 4702 on α1E, and 1687 on α1S. The lo primer fully matched with α1B, α1D, α1E, and α1S-subunits, but had one mismatch with α1A and two mismatches with α1C (see Fig. 3). The sizes of the amplified fragments calculated from the published sequences were 590 bp for α1A and α1B, 599 bp for α1E, 629 bp for α1C, 641 bp for α1D, and 653bp for α1S. A mixture containing the fragments α1A, α1B, or α1E should appear as a single DNA band on agarose gel electrophoresis. Mixtures of these subunits with α1C, α1D, or α1S should give two separate bands.
The first PCR amplification was performed as described previously (Lambolez et al., 1992; Plant et al., 1997) except that 40 pmol of each primer was used. Before PCR, samples were heated to 94°C for 3 min. Each PCR cycle consisted of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and elongation at 72°C for 1 min. Forty cycles were performed with a programmable thermocycler (Biometra, Göttingen, Germany). After PCR, samples were heated to 72°C for 10 min. Ten microliters of the amplification reaction were run with a molecular weight marker (φX174, HaeIII-digested) on a 1.5% agarose gel stained with ethidium bromide. The product of the first PCR was cut out of the agarose gel and used for a second amplification step.
Second PCR amplification for restriction analysis. To obtain a sufficient amount of α1-subunit fragments for a restriction analysis, a second round of amplification was performed (Lambolez et al., 1992; Plant et al., 1997) using the up and lo primers described above. After the second PCR amplification in a final volume of 100 μl, under the same conditions as those described above, a chloroform–isoamylalcohol extraction and ethanol precipitation were performed. The DNA was then resuspended in 18 μl H2O, and 3 μl aliquots were digested by one of the restriction enzymes (see Figs. 4, 5). Five enzymes were chosen, DrdI,BpmI, HincII, AflII, andAccI (New England Biolabs, Schwalbach, Germany), which selectively cut the brain α1A, α1B, α1C, α1D, and α1E isoform PCR fragments, respectively. The calculated lengths of the fragments generated by the restriction enzymes are shown in Figure 3. The skeletal muscle isoform α1S contained a restriction site for HincII (at position 1527, yielding fragments of 474 and 179 bp) and three sites for BpmI (at positions 1369, 1467, and 1536, giving fragments of 316, 170, 98, and 69 bp). Because of the large number of fragments with BpmI, α1S is easily distinguishable from α1B. The identity of the α1S-subunit can be confirmed by the presence of a single specific restriction site for ClaI (at position 1466), yielding fragments of 413 and 240 bp. To guarantee complete digestion, five units of each enzyme were used for an incubation time of 3 hr. The restriction reaction was then analyzed by electrophoresis on a 2% agarose gel as described above.
Optimization of PCR sensitivity. Using rat brain RNA (see below), PCR conditions were optimized so that a PCR product could be detected from 10 pg of total RNA (see Fig. 4 A), without contamination caused by unspecific amplification. To test the efficiency of the PCR, cDNA was synthesized from 100 ng total RNA by RT (as described above). The cDNA was diluted to 0.01-10 ng/μl using sterile water and used as a template for the PCR. Total RNA from other tissues and specific brain regions were tested to check that tissue-specific product patterns were observed. Furthermore, single cells, known to express a cell-specific mRNA pattern, were also used to test the PCR amplification.
RNA preparation and amplification. Total RNA was prepared from fresh brain (P12 and adult), pancreas (P14–P17), kidney (adult), heart (adult), skeletal muscle (adult), and adrenal gland (adult) of the rat using the RNeasy Midi-Kit (Qiagen, Hilden, Germany). The total RNA was treated with DNase I to exclude genomic DNA contamination of the RNA preparation and possible amplification of genomic DNA. As additional controls, the RT reaction was performed in the absence of reverse transcriptase or RNA. The RT reactions and the amplification and analysis steps were performed as described above, starting with 10 ng of total RNA.
Genomic DNA preparation and amplification. Genomic DNA was prepared from adult rat brain using the genomic DNA isolation kit (Boehringer Mannheim, Mannheim, Germany). PCR amplification was performed with 200 ng of rat genomic DNA.
Biophysical properties of Ca2+channel currents
Experiments were performed in a total of 156 cells, identified in initial experiments by retrograde labeling (Fig.1) and later by inspection of the slice by dark-field illumination followed by infrared differential interference contrast video microscopy. Large Ca2+channel currents were activated by steps in membrane potential from a holding potential of −80 mV to potentials more positive than −70 mV in Ca2+- and Ba2+-containing solutions. In the majority of experiments, Ba2+ was chosen as charge carrier to decrease the contribution of K+ currents incompletely blocked by internal Cs+ and TEA+ and external TEA+. In addition, to improve the control of the membrane potential and decrease series resistance errors caused by large currents, most experiments were performed using 1 or 0.5 mm Ba2+ as the charge carrier. Under these conditions, potential steps from a holding potential of −80 mV revealed two major components of Ca2+ channel current in facial motoneurons (Fig.2 A). A fast transient component of current was activated during steps positive to −70 mV, and a more sustained, slowly inactivating component was activated at potentials more positive than −50 mV. These resemble the LVA (T) and HVA currents in other neuron types, respectively. In 5% of neurons, only HVA currents were observed. In neurons that expressed both LVA and HVA currents, there was no clear change in their relative proportions with age during the first postnatal week. A marked difference in the relative proportions of current should be reflected in the ratio of the current amplitude at −40 mV, where the current is mainly LVA (see below), to that at −10 mV, where the HVA current dominates. There was no significant difference (t test, 0.05 level) in the values obtained during the first postnatal week; all values were ∼0.3 (0.309 ± 0.018, n = 22, at P1–P2, and 0.252 ± 0.027, n = 5, at P6–P7). A change in the holding potential from −80 to −60 mV completely abolished the LVA current component (Fig. 2 B). The remaining current was exclusively HVA and inactivated only slowly and incompletely during 250 msec potential steps. A difference in the time course of the current at the two holding potentials is most clear near −50 mV (Fig. 2 A,B). At potentials more positive than −10 mV, only the amplitude of current, not the time course, was affected by the change in holding potential. Currents at potentials of more than −10 mV, activated from a holding potential of −60 mV, were smaller, but when scaled-up they had time courses similar to those at the same membrane potential but activated from −80 mV. These results suggest that the fast transient component, most clearly seen at more negative potentials, was completely inactivated by the shift in holding potential, whereas the current activated by larger depolarizations was only partially inactivated. Furthermore, the LVA current contributes only a small fraction of the current at potentials positive to −10 mV.
The current–voltage (I–V) relations for the peak inward current at the two holding potentials are shown in Figure2 C. From the I–V relation it is clear that a component of current that activates above −70 mV is abolished by the change in holding potential, shifting the activation threshold close to −50 mV. The maximum inward current was between −10 and −20 mV at both holding potentials. During steps from a holding potential of −80 mV, the maximum inward current was 1537.2 ± 114.0 pA (n = 45) in 1 mm Ba2+and 995.9 ± 64.4 (n = 31) in 0.5 mmBa2+.
The potential dependence of inactivation of both components of current was studied in more detail. Currents were measured during a test pulse to −10 mV, where the current is mainly HVA, preceded by 15 sec conditioning pulses to potentials between −100 and +10 mV. The resulting inactivation curve is shown in Figure 2 D(•). Even after conditioning pulses of this length, inactivation was incomplete, and a noninactivating component remained. For the current inactivated by the 15 sec pulse, a fit of a Boltzmann distribution to the data gave a potential for half-inactivation of −45.5 ± 1.6 mV (n = 7), and a slope factor k of 13.3 ± 1.1. On average, the current that was not inactivated by a 15 sec pulse was 7.5% of the total current. The inactivation of the LVA component was studied using a test pulse to −40 mV preceded by a 500 msec conditioning pulse to potentials between −100 and −45 mV. Currents were reduced at conditioning potentials more positive than −85 mV, and little further reduction of the current was observed at potentials positive to −55 mV. The inactivation curve for the transient component, after subtraction of the current that was not inactivated by a 500 msec pulse, is shown in Figure2 D. On average, 34 ± 2% (n = 5) of the current at −40 mV was resistant to inactivation and was probably HVA. The transient current was half-inactivated at a conditioning potential of −71.6 ± 2.0 mV; the slope factork was 5.3 ± 0.2. These results suggest that most facial motoneurons express LVA (T) and HVA components, the T-type channel being completely inactivated at more positive holding potentials. Figure 2 D also shows the mean activation curve estimated from tail currents at the end of 250 msec depolarizing impulses, where the current is mainly HVA. The midpoint of the activation curve is at a potential of −26 mV.
PCR analysis of Ca2+ channel α1-subunits
Previous studies on Ca2+ channel α1-subunit mRNA expression in single cells have used antisense RNA amplification (Bargas et al., 1994) or have amplified each subunit in separate reactions using nested PCR with specific outer and inner primers (Yan and Surmeier, 1996). In contrast, we designed partially degenerate primers to amplify all of the HVA α1-subunits in the same reaction and then identified the individual subunits that were present by restriction analysis.
The region of the α1-subunits of the Ca2+ channel that was amplified, the primers used, and the details of the restriction analysis are illustrated in Figure3 and outlined in detail in Materials and Methods. Before using RT-PCR on material isolated from single motoneurons, we tested the ability of the primers to amplify fragments of all forms of the HVA Ca2+ channel α1-subunits. For this, total RNA was isolated from tissues known to express different isoforms of the α1-subunits. Using rat brain RNA (see below), PCR conditions were optimized so that a PCR product could be detected from 10 pg of total RNA. Figure4 A shows the result of an experiment in which different amounts of DNA were used in the PCR and shows that a product was still detected with 10 pg. No product was detected using genomic DNA as template or in reactions without reverse transcriptase (Fig. 4 B).
The Ca2+ channel mRNA detected in adult whole brain after RT-PCR amplification and analysis of the amplified fragments using specific restriction enzymes is shown Figure 4 C. In this RNA, α1A, α1B, and α1E were clearly detected; α1C, α1D, and α1S were not detected. The α1-subunits detected are summarized in Table1. The results are consistent with the very widespread expression of the α1A, α1B, and α1E and the lower, spatially more restricted occurrence of α1C and α1D (Tanaka et al., 1995; Ludwig et al., 1997) being reflected in the concentrations of their mRNAs. As shown in Table 1, the pattern was different in RNA isolated from specific regions of the brain. In the adult neocortex and hippocampus, α1A, α1B, α1C, α1D, and α1E were detected as also shown from in situhybridization (Tanaka et al., 1995; Ludwig et al., 1997). In cerebellar RNA, α1A, α1B, and α1E were detected at both P12 and in the adult; α1D was detected only at P12. In the brainstem, only α1A, α1B, and α1E were detected.
In total RNA from regions other than the brain, we detected tissue-specific expression patterns for the mRNAs (Table 1). Thus, in skeletal muscle a fragment was amplified that was cut byHincII into two fragments and by BpmI into multiple fragments, consistent with the pattern expected for α1S (see Materials and Methods), a result that was confirmed using ClaI. In the kidney, we detected mRNA for the α1A- and α1D-subunits. In the heart, α1A, α1B, α1C, and α1D were detected. In mRNA from the pancreas, the neuroendocrine form α1D was clearly detected, together with α1A, α1B, and α1C. In the adrenal gland, the pattern was α1A, α1B, and α1C.
Thus, from RNA in different brain regions and other tissues, the partially degenerate primers amplify DNA fragments that correspond in length to those predicted from the cDNAs of the HVA Ca2+ channel α1-subunits. Furthermore, digestion with restriction enzymes specific for the individual subunits gave restriction fragments of the predicted sizes. We also detected region-specific patterns of α1-subunit RNA expression similar to those expected from previous studies using RT-PCR orin situ hybridization. No amplification was observed using genomic DNA as the template for the PCR reaction. In none of the tissues or regions did we see a PCR product of unexpected length or restriction fragments of unexpected lengths that would suggest the amplification of an unknown subunit. Similarly, after parallel digestion with all restriction enzymes, no fragments were detected that were resistant to digestion, also ruling out unspecific amplification.
We then tested the ability of the RT-PCR method with the chosen primers to detect Ca2+ channel α1-subunits in single neurons in which the cell’s content was harvested through the patch pipette as described previously (Lambolez et al., 1992; Plant et al., 1997). As controls in single cells, we studied the subunit expression in cells known from in situ hybridization to have a characteristic subunit composition. The results of the first PCR reaction and the restriction analysis are shown in Figure5, and the data from all cells that gave a positive result are summarized in Table2. In four cerebellar Purkinje neurons, we detected only mRNA for α1A (Fig. 5 A, Table2). In contrast, in granule cells of the dentate gyrus, which were chosen because the region expresses all brain HVA subunits at moderate to high levels (Tanaka et al., 1995; Ludwig et al., 1997), we detected a more heterogeneous pattern, with all subunits represented in the three cells but clear differences between individual cells. As expected from both electrophysiological measurements, which demonstrate a large component of L-type current (Eliot and Johnston, 1994), and in situ hybridization studies (Tanaka et al., 1995; Ludwig et al., 1997), α1C or α1D were present in all three cells. Figure 5 B shows data from a cell in which α1C and α1D were detected.
In motoneurons of the facial nucleus, we detected mRNA for α1-subunits after the first round of PCR amplification in 22 of 30 neurons. The agarose gels in Figure 5 C,D show the product of the first PCR reaction and the results of the restriction digest after the second PCR for two cells in which different patterns of α1-subunit expression were detected. For the neuron in Figure 5 C, α1A, α1B, and α1E were detected, whereas for that in Figure 5 D, only α1A and α1C were detected. For all neurons studied, the heterogeneous pattern of α1-subunit mRNA expression shown in Table 2 was obtained. The most frequently occurring messages were those for α1A, α1B, and α1E, with α1C and α1Dalso observed in smaller proportion of cells. However, even those mRNAs that were most frequently observed were not detectable in all cells. Thus, α1A was detected in 86% of neurons, α1B in 59%, α1C in 18%, α1Din 18%, and α1E in 59%. As is clear from Table 2, the pattern detected in individual cells was highly variable. However, mRNAs for either α1A or α1B were present in all neurons (Fig. 5 C,D), with those for the other subunits showing a much higher variability. Of the two most dominant subunits, α1A and α1B, α1A was present alone in 41% of neurons and α1B was present alone in 14%. In the remaining 45% of neurons, both α1A and α1B were detected. Surprisingly, two neurons showed the restriction pattern with BpmI andHincII expected for the skeletal muscle isoform of the α-subunit, α1S, in combination with α1A. The identity of the fragment thought to be from α1S was confirmed by its cleavage into fragments of the expected length by ClaI. This subunit is not usually associated with or widely considered to be expressed in the brain, but a fragment has been cloned from rat brain cDNA (Chin et al., 1992). Similar patterns of α1-subunit expression were detected independently of whether the nucleus was harvested together with the cytoplasm, suggesting that genomic DNA is not being amplified from single cells as is also shown above for total RNA. With the primers used, we have no evidence for a new subunit in single motoneurons. Simultaneous digestion of the PCR product with all of the restriction enzymes resulted in a complete disappearance of the parent band (data not shown).
Pharmacological profile of whole-cell Ca2+channel currents
Effects of Ni2+ and Cd2+
Ni2+ (50 μm) did not clearly differentiate between LVA and HVA currents. The current at −40 mV, after a 1 sec prepulse to −100 mV, was inhibited by 40 ± 1% (n = 6), whereas the current at −10 mV, after a 1 sec prepulse to −60 mV, was inhibited by 50 ± 3% (n= 6). At concentrations of 100 or 200 μm, Cd2+ blocked nearly all of the current at −10 mV. With 100 μm Cd2+, the mean inhibition was 92.8 ± 0.5% (n = 4).
Pharmacological characterization of HVA current components
To characterize the HVA component of the Ca2+current in these neurons with respect to different HVA channel types, which are difficult to differentiate from their voltage dependence and kinetics, we used various antagonists. Currents were measured during short depolarizing (10-50 msec) pulses from −80 to −10 mV, the minimum of the I–V relation, applied at 10 or 15 sec intervals.
Effect of ω-CTx-GVIA
The neurotoxin, ω-CTx-GVIA (Olivera et al., 1984), a specific antagonist of N-type Ca2+ channels (Mogul and Fox, 1991; Regan et al., 1991; Olivera et al., 1994), used at concentrations of 1 μm or more, had a clear effect in most cells, producing, at the holding potential used (Stocker et al., 1997), a rapid irreversible block of the Ca2+ channel current, when applied locally or to the bath solution (Fig.6). The inhibition by ω-CTx-GVIA was 24 ± 3% (n = 5) at a concentration of 1 μm and 32 ± 4% (n = 5) at 10 μm, values that are not significantly different (t test, 0.05 level). Only one cell did not respond to ω-CTx-GVIA. It is notable that the time course of the current was largely unaffected by the toxin treatment (Fig.6 A).
Effect of dihydropyridines
Dihydropyridines (DHPs) are modulators of L-type Ca2+ channel currents in neurons. In facial motoneurons, nitrendipine, at the relatively high concentration of 10 μm, had only a very weak but reversible inhibitory effect on Ca2+ channel currents. The block that was small (Fig. 6 B) and thus difficult to measure accurately, was ∼5% (n = 5). Similar effects were seen in two cells with nimodipine (10 μm). No difference was observed in the size of the effect of dihydropyridines when applied before (in three cells) or, as in Figure 6 B, after (four cells) the other specific toxins, suggesting that it inhibited a component of current insensitive to the other toxins. These results suggest that in this preparation only a very small proportion of the current through Ca2+ channels is contributed by flow through L-type channels.
Effect of ω-Aga-IVA
ω-Aga-IVA inhibits P-type Ca2+ currents with a high affinity and Q-type currents with a lower affinity (Randall and Tsien, 1995). Despite the well documented effects on transmitter release at the axon terminals (Uchitel et al., 1992; Protti and Uchitel, 1993; Katz et al., 1996, 1997; Protti and Uchitel, 1997), in the cell bodies of facial motoneurons, ω-Aga-IVA had only very weak effects, if any. With a concentration of 100 nm ω-Aga-IVA or ω-Aga-TK, no block was observed in five cells (Fig.7 A,B), whereas in two others the current slowly decreased during toxin application to ∼75-80% of the control value. In different cells, concentrations of ω-Aga-IVA up to 1 μm had effects similar to those with 100 nm, showing either a small decrease or no effect. In cells in which a small decrease in current was observed, the block could not be removed by repetitive depolarizations (10 pulses of 60 msec at 1 Hz) to +130 mV after washout of the toxin from the bath solution. This is in contrast to the reported effect on P-type channels for which the inhibition is normally relieved by strong depolarization [Mintz et al. (1992) and see below]. When compared with other brainstem motoneurons at similar postnatal ages that show a considerable block by ω-Aga-IVA (Umemiya and Berger, 1994), these effects of ω-Aga-IVA were surprising. To check that the toxin used was active, we performed control experiments in cerebellar Purkinje neurons, in which most of the current is contributed by P-type channels (Llinás et al., 1992; Mintz et al., 1992), at an age (P4–P8) when the cells have few dendritic processes and can be well voltage-clamped. In three Purkinje neurons, ω-Aga-IVA (100 nm) blocked the Ca2+ current by ∼80% (Fig. 7 C,D). The block was not reversed on washout of the toxin from the bath, but it was completely removed by three trains of depolarizations to +130 mV (Fig. 7 C,D) (also see Mintz et al., 1992).
Effects of ω-CTx-MVIIC
As a further test to substantiate the contribution of N- and P/Q-type channels, we also tested the toxin ω-CTx-MVIIC (Hillyard et al., 1992). This toxin inhibits currents through N- and P/Q-type Ca2+ channels, with more rapid effects on N-type than on P- or Q-type channels (Randall and Tsien, 1995; McDonough et al., 1996). When added alone, ω-CTx-MVIIC clearly had a rapid inhibitory effect that resembled that of ω-CTx-GVIA in time course and extent (Fig. 8 A), with no slow component of inhibition. In four cells, a concentration of 1 μm ω-CTx-MVIIC reduced the current by 22 ± 4%, whereas in one other cell the current was unaffected. In contrast, in three cells pretreated with ω-CTx-GVIA (1 μm) for some minutes, which caused a block like that described above (Fig.6 A), ω-CTx-MVIIC (1 μm) had no additional inhibitory effect on the Ca2+ channel current (Fig. 8 B).
The current that remained at −10 mV after inhibition by ω-CTx-MVIIC (Fig. 8 A) or ω-CTx-GVIA (Fig. 6 A) was similar in time course to that before toxin treatment. In the cells that were treated with ω-CTx-MVIIC, the current had decayed to 88.0 ± 0.6% of the peak value at the end of the 50 msec test pulse before toxin treatment and 84.3 ± 0.7% after toxin treatment. Thus, the toxin-resistant component of current at −10 mV does not resemble T-type current (Huguenard, 1996) or the R-type described in cerebellar granule neurons (Randall and Tsien, 1995), both of which inactivate more rapidly. We therefore designate this currentR slow. Measurement of theI–V relation after the application of ω-CTx-MVIIC or ω-CTx-GVIA indicated that both LVA and HVA current components were present after toxin treatment and that the peak of theI–V relation remained at −10 mV (data not shown).
Comparison of pharmacological and RT-PCR data
None of the cells studied showed a clear, reversible response to ω-Aga-IVA, but the α1A-subunit was detected in 86% of neurons (Table 2). In contrast, for the other α1-subunits that can be pharmacologically differentiated, the proportion of cells that responded to the antagonist was higher than that in which the respective α1-subunit was detected. Thus, 87% of neurons responded to either of the conotoxins, whereas α1B was detected in 59%. Responses to dihydropyridines, albeit very small, were observed in 90% of cells, but α1C or α1D was detected in only 36%.
The main findings of this study on Ca2+channels in facial motoneurons are as follows. (1) A major component of the Ca2+ current is carried by a channelR slow, which is insensitive to inhibitory toxins and DHPs but unlike the R-type channel originally described in cerebellar granule neurons. N-, T-, and L-type components are also present. (2) The partially degenerate primers that were designed amplify all known HVA Ca2+ channel α1-subunits and provide a powerful tool to identify the subunits present in a single cell. (3) With single-cell RT-PCR, highly heterogeneous patterns of Ca2+ channel α1-subunit mRNA expression were detected in individual facial motoneurons. The dominant pattern was α1A with α1B and α1E. However, α1C, α1D, and α1S were detected in combination with the other subunits. In contrast, in cerebellar Purkinje neurons, only α1A was detected. (4) Evidence with the toxins ω-Aga-IVA and ω-CTx-MVIIC indicated that P/Q-type Ca2+ channels are not present in the soma of facial motoneurons, whereas previous studies have shown that P-type channels dominate in the control of transmitter release at the nerve terminal. α1A-subunit mRNA that is thought to code for P-type channels was detected in the majority of motoneurons.
Ca2+ current types identified in facial motoneurons
Nearly all neurons expressed LVA and HVA channels. Of the HVA current at −10 mV, ∼30% was contributed by N-type Ca2+ channels, a proportion similar to that in rat hypoglossal motoneurons in slices (29%) (Umemiya and Berger, 1994), but less than that in a previous report on facial motoneurons (∼50% with 50 μm) (Umemiya et al., 1993) and in reports on cultured embryonic rat and mouse spinal motoneurons (42-54%) (Mynlieff and Beam, 1994; Hivert et al., 1995; Viana et al., 1997). L-type channels contributed only a relatively minor component (∼5%), as in hypoglossal motoneurons (6%) (Umemiya and Berger, 1994). The surprising result of this study was that the major component of the Ca2+ current was insensitive to specific inhibitors, including the inhibitor of P/Q-type channels, ω-Aga-IVA, and the inhibitor of N- and P/Q-type channels, ω-CTx-MVIIC, after inhibition of the N-type current. At the low extracellular Ba2+concentration, all of the known channel types should have been blocked by the concentrations of toxins tested. The results with ω-CTx-MVIIC and ω-Aga-IVA, a toxin that had effects similar to those reported previously in cerebellar Purkinje neurons (Mintz et al., 1992), suggest that P/Q-type channels do not contribute significantly to the somatic/dendritic Ca2+ current in facial motoneurons. This finding contrasts with those on HVA Ca2+ channel currents in most motoneurons, even other brainstem motoneurons, studied to date that are inhibited by ω-Aga-IVA [hypoglossal motoneurons: 50% (Umemiya and Berger, 1994); embryonic rat spinal motoneurons: 10-30% (Hivert et al., 1995; Viana et al., 1997; Magnelli et al., 1998)] at concentrations that suggest that they are carried by P-type channels. However, recent studies on the latter preparation report that some (Viana et al., 1997) or as many as 60% of neurons (Magnelli et al., 1998) do not respond to ω-Aga-IVA.
R slow, the current that was resistant to inhibition in facial motoneurons, displayed only little inactivation and thus resembles neither the antagonist-resistant R-type current in cerebellar granule neurons (Randall and Tsien, 1995) nor a residual LVA current. The latter was suggested for the current remaining after block of L-, N-, and P/Q-type channels in hypoglossal motoneurons (Umemiya and Berger, 1994). In contrast, the current remaining at potentials above −30 mV in facial motoneurons was clearly HVA. Interestingly, a slowly inactivating, antagonist-resistant current similar to that described here has also been observed recently and studied in detail in embryonic rat motoneurons (Magnelli et al., 1998). This current had biophysical properties, a divalent cation permeability, and a sensitivity to Ni2+ and Cd2+ more typical of HVA channels.
PCR analysis of Ca2+ channel α1-subunit expression
In total RNA from rat brain, the detected pattern of HVA Ca2+ channel subunits reflected the region-specific expression of RNA shown previously by in situ hybridization (Tanaka et al., 1995; Ludwig et al., 1997). The pattern in other tissues is mainly that expected from previous studies or predicted from the known pharmacological properties of the Ca2+channel currents. In single neurons we also observed a highly cell-specific pattern. Only α1A was detected in cerebellar Purkinje neurons, known from the toxin sensitivity of their Ca2+ channel currents to mainly express P-type channels (Mintz et al., 1992). In contrast, in granule cells of the dentate gyrus and motoneurons of the facial nucleus, which like the majority of central neurons express multiple channel types, the pattern was more heterogeneous. The most frequently detected subunits in facial motoneurons were α1A, α1B, and α1E, in agreement with data from in situ hybridization for this nucleus (Tanaka et al., 1995).
Ca2+ channel α1-subunit expression and localization in motoneurons
Compared with the pharmacological data, the biggest surprise was the large fraction of facial motoneurons in which mRNA for α1A was detected, despite the lack of clear pharmacological evidence for functional P/Q-type channels. This could be explained by the absence of functional P/Q-type channels in the soma and proximal dendrites, with specific segregation of this channel type to the neuromuscular junction, where it has been shown to be involved in the release of transmitter. This interpretation is supported by a report on spinal cord motoneurons (Westenbroek et al., 1998) showing by immunocytochemistry that the α1A-subunit is localized presynaptically at the neuromuscular junction, but that the staining on the soma and dendrites is more consistent with a presynaptic localization. Of the other subunits, α1C, α1D, and α1E were present in the soma, and α1B was present in the soma and dendrites and occasionally at the NMJ. Other immunocytochemical and pharmacological studies confirm the presence and important role of α1A at the NMJ. Thus, an antibody specific for the Ca2+channel α1A-subunit labels the NMJ in the diaphragm preparation of adult rats (Ousley and Froehner, 1994). Furthermore, in studies at the adult mammalian neuromuscular junction, mostly at endplates that are innervated by the facial nerve, transmitter release and presynaptic currents (both Ca2+ and Ca2+-dependent K+ currents) were strongly inhibited by ω-Aga-IVA but unaffected by ω-CTx-GVIA and nitrendipine, suggesting that Ca2+ entry mainly occurs through P-type Ca2+ channels (Uchitel et al., 1992; Protti and Uchitel, 1993; Katz et al., 1996, 1997; Protti and Uchitel, 1997). In neonatal rats (P0–P9), transmitter release from facial motoneuron nerve terminals is also strongly inhibited by ω-Aga-IVA (M. D. Rosato Siri and O. D. Uchitel, unpublished observations). Therefore, it seems likely that motoneurons express different Ca2+ channel types at the axon terminal than at the soma (Fig. 9).
An alternative explanation, which would also account for the antagonist-insensitive current in the soma, is the expression of different forms of channels containing the α1A-subunit at different subcellular sites: a toxin-resistant form of channel in the soma and a toxin-sensitive form at the axon terminal. The auxiliary subunits of the Ca2+ channel, especially the β-subunit, can strongly influence both the expression and functional properties, including the toxin sensitivity, of the α1-subunits (Moreno et al., 1997; Tareilus et al., 1997). Thus, in the absence of the appropriate β-subunit or expression of a different β-subunit in a subcellular region, α-subunits may not be expressed or may have very different properties, respectively. Likewise, antagonist-insensitive channels could result from the α1E-subunit, thought to compose the toxin resistant channels in cerebellar granule neurons (Randall and Tsien, 1995), with slowed inactivation determined by the β-subunit (Parent et al., 1997). mRNA for α1E was detected in many although not all neurons. From our RT-PCR data, we cannot identify the α1-subunit type that corresponds toR slow. The experiments provided no evidence with the primers used for a novel α1-subunit.
The α1B-subunit was not detected in all neurons, although most responded to ω-CTx-GVIA or ω-CTx-MVIIC. This may just result from a low abundance of the mRNA at the time of harvesting the cytoplasm in some cells and not indicate that functional channels are absent. A similar conclusion was reached in another study using single-cell RT-PCR in neostriatal interneurons (Yan and Surmeier, 1996). There, a highly heterogeneous pattern of mRNA expression was detected in individual cells, but the pharmacological profile reflected the functional expression of all of the channel types in each neuron. The detection in facial motoneurons of α1C and α1D mRNAs in only a small proportion of cells most probably reflects their low abundance and their minor contribution to the whole-cell current.
In conclusion, T-, N-, and L-type Ca2+ channels together with a major toxin-resistant componentR slow, which does not resemble the R-type in other neurons, are present in the soma of neonatal facial motoneurons. Although no P/Q-type current was measured in the soma, mRNA for this subunit was dominant. This result, together with the results of studies of Ca2+ channels involved in transmitter release at the nerve terminal, suggests that there are strong subcellular regional differences in Ca2+channel expression in facial motoneurons. Remarkably, the Ca2+ channel pattern found in motoneurons in the facial nucleus is distinctly different from that found in other brainstem motoneurons.
This study was supported by grants from the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Neurotraumatologie Programm) and the Universität des Saarlandes (ZFK-1997). We thank Dr. Félix Viana for his helpful comments on this manuscript and Erle Eilers, Heide Krempel, Nicole Rothgerber, and Reiko Trautmann for technical assistance.
Correspondence should be addressed to Dr. T. D. Plant at his present address: Institut für Pharmakologie, Freie Universität Berlin, 14195 Berlin, Germany.
Dr. Katz’s present address: Departamento de Biologı́a, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria (1428), Buenos Aires, Argentina.
Dr. Uchitel’s present address: Laboratorio de Fisiologia y Biologı́a Molecular, Departamento de Ciencas Biologicas, Facultad de Ciencias Biologicas, Universidad de Buenos Aires, Ciudad Universitaria (1428), Buenos Aires, Argentina.