© 2001 by The Society for Integrative and Comparative Biology
Serotonin Directly Increases a Calcium Current in Swim Motoneurons of Aplysia brasiliana1
1 Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555-1031
2 Lucent Technologies, Bell Labs, Biological Computational Research Department, 600 Mountain Avenue, P.O. Box 636, Murray Hill, New York 07974-0636
3 
Department of Radiology, Wake Forest University, School of Medicine, Medical Center Blvd., Winston-Salem, North Carolina 27157-1010
4 Marine Biomedical Institute, University of Texas Medical Branch, 301 University Blvd., Galveston, Texas 77555-1069
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Muscle fibers in the swim appendages of the mollusk Aplysia brasiliana are innervated by cholinergic motoneurons. Serotonin (5-HT) causes an increase in amplitude of junctional potentials and muscle contractions at this neuromuscular synapse. We studied motoneurons with intracellular current-clamp recording and single-electrode voltage-clamp analysis to determine the effects of 5-HT on somatic currents in these presynaptic neurons. Serotonin was found to have no effect on action potential duration in motoneurons bathed in normal seawater, and no effect of 5-HT could be detected on K+ currents, indicating that 5-HT does not indirectly enhance calcium currents by prolonging the action potential. Calcium currents were isolated by replacing extracellular sodium with TEA and adding tetrodotoxin and other K+-channel blockers. Under these conditions motoneuron action potentials were greatly prolonged and could be blocked with Co2+ or Cd2+. Addition of 5-HT increased the duration of these Ca2+ spikes by about 35%. In motoneurons studied with voltage clamp, the amine produced a 58% increase in total inward calcium current. Use of the calcium channel blockers nifedipine, nimodipine,
-conotoxin GVIA, and -agatoxin TK revealed that motoneurons express varying amounts of L-, N- and P-like calcium channels, but only an agatoxin-sensitive, P-type channel is sensitive to 5-HT. It is concluded that 5-HT acts directly to increase a P-type Ca2+ current during a normal spike. The resulting increase in intracellular calcium could contribute to an increase in transmitter release and account for the increase in junctional potentials in swim muscles. |
INTRODUCTION |
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SYNOPSISINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Serotonin and other amines have been shown to act as modulators that enhance neuromuscular transmission in vertebrates (Arreola et al., 1987
; Hirai and Koketsu, 1980; Kuba, 1970) and in a variety of arthropod (Delaney et al., 1991; Kravitz et al., 1985; O'Shea and Evans, 1979; Pasztor and Golas, 1993) and molluscan (Satterlie, 1995; Weiss et al., 1978; Yoshida and Kobayashi, 1995; Zoran et al., 1989) preparations. The mechanisms by which amines act to enhance transmission has not been well defined in most neuromuscular preparations, however. We have begun to address this problem in a favorable molluscan preparation.
Aplysia brasiliana is a marine gastropod that swims by rhythmically flapping its wing-like parapodia. Previous studies of this oscillatory activity have characterized its behavioral components and have localized a swim command system to the cerebral ganglion and a central pattern generator (CPG) to each pedal ganglion (Gamkrelidze et al., 1995; von der Porten et al., 1980, 1982). Parapodial muscle fibers are innervated by cholinergic motor neurons (MNs; McPherson and Blankenship, 1991a, b) and by modulatory serotonergic neurons called parapodial-opener-phase (POP) and closer-phase cells (Parsons and Pinsker, 1988). MNs and POP cells are located in the pedal ganglia, but they have no detectable synaptic influence upon one another; both cell types, however, send axons into the peripheral parapodial muscle bed to influence muscle fibers.
Inputs from command neurons and the CPG (Gamkrelidze et al., 1995) drive POP cells and opener-phase MNs to fire phasically in similar (but not synchronous) bursts to open the parapodia in the down-stroke of swimming (McPherson and Blankenship, 1991c; Parsons and Pinsker, 1988). POP cells and exogenously applied serotonin (5-hydroxytryptamine, 5-HT) cause a significant enhancement of motor neuron effects, producing an approximate doubling of the amplitude of MN-induced excitatory junctional potentials (EJPs) and increasing the amplitude of parapodial muscle contractions by an average of 300%; their activity also causes a 40% increase in the rate of muscle relaxation (McPherson and Blankenship, 1991c). Since neither POP cell activity nor exogenously applied 5-HT has any detectable effect on muscle fiber membrane potential in situ or in vitro when applied alone, we predicted that POP cell axon terminals serve to release 5-HT peripherally as a modulator of the effects of cholinergic motoneurons on muscle fibers. To test this, we first looked for possible effects other than changes in muscle membrane potential that 5-HT could have on postjunctional muscle fibers. We characterized a variety of voltage-gated currents, including an L-type calcium current, in dissociated parapodial muscle fibers (Laurienti and Blankenship, 1996a, b). Motoneuron activity (McPherson and Blankenship, 1991c) or pharmacological activation of excitatory acetylcholine receptors in muscle fibers (Laurienti and Blankenship, 1999) can lead to summed junctional responses of 20–30 mV in amplitude, adequate to activate the L-type calcium channel and contractions in these fibers. 5-HT was shown to enhance this L-type calcium current by an average of 42% (Laurienti and Blankenship, 1997). Thus, one of the roles of POP cell activity is to increase the amount of calcium current that can enter muscle fibers through calcium channels opened by the depolarization caused by MNs. This calcium current probably plays a negligible role in the cationic currents contributing to the EJPs themselves, but plays a major role in the large increase in parapodial opener-muscle contractions caused by POP cell firing (Laurienti and Blankenship, 1999).
Having shown that serotonergic POP cells can have an influence on neuromuscular transmission by altering properties of the postjunctional element, we now hypothesize that peripherally released 5-HT from POP cells may also influence presynaptic motoneuron terminals to enhance transmitter release and thus account for a significant proportion of the doubling of EJP amplitude seen during POP cell activity.
Based on experiments on neuro-neuronal synapses, there are two major classes of presynaptic mechanism by which synaptic efficacy can be enhanced by a modulatory substance. First, the presynaptic neuron's spike threshold can be lowered or its excitability enhanced, resulting in an increase in action potential frequency and an overall increase of transmitter output per unit time (temporal summation). Serotonin has been demonstrated to enhance excitability and spike output in vertebrate and invertebrate neurons, and it has been suggested that the underlying mechanism may be a decrease in potassium conductance (Cottrell, 1982, 1988; Van der Maelin and Aghajanian, 1980; Wood and Mayer, 1979). However, in parapodial motoneurons, the action potential is initiated at or near the soma and travels to the axon terminal. Thus, any modulatory influence of serotonin on spike production or excitability would have to occur within the CNS. Since no central synaptic connections have been observed between POP cells and motoneurons (McPherson and Blankenship, 1991c), this mechanism is unlikely. Furthermore, previous studies have demonstrated that POP cell firing increases the amplitude of motoneuron-induced excitatory junctional potentials and parapodial muscle contractions while having no effect on the number of motoneuron spikes elicited by a constant-current pulse (McPherson and Blankenship, 1991c).
The second and better studied mechanism for modulators to influence synaptic efficacy is by altering the amount of transmitter released during a single presynaptic action potential. Synaptic modulation of this sort is often mediated through a mechanism in which a voltage-gated calcium current, normally activated during an action potential, is either directly or indirectly altered. Direct modification of Ca2+ channels by serotonin or other amines has now been demonstrated in a variety of preparations. 5-HT (Dunlap and Fischbach, 1981) or noradrenaline (Bean, 1989b) reduces Ca2+ current in vertebrate dorsal root ganglion (DRG) cells, and serotonin also reduces voltage-gated calcium current in dorsal raphe neurons (Penington and Kelly, 1990; Penington et al., 1991). Similarly, histamine directly inhibits calcium currents in Aplysia buccal neurons (Fossier et al., 1994). On the other hand, serotonin has also been shown to enhance voltage-gated Ca2+ currents in Aplysia (Pellmar, 1984; Pellmar and Carpenter, 1980) and Helix neurons (Gerschenfeld et al., 1986; Hill-Venning and Cottrell, 1992). 5-HT (Penington et al., 1991), noradrenaline (Bean, 1989b) and histamine (Fossier et al., 1994) appear to act on N-type or N-type-like channels, while L-type and T-type channels appear not to be affected. Also, there is no consistent evidence about whether amines influence spike duration in these preparations. Dunlap and Fischbach (1981) reported that 5-HT both reduced a calcium current in DRG cells and shortened the duration of action potentials. Spike duration is also shortened in raphe neurons exposed to 5-HT, but this is due to a combination of a decrease in calcium current and an increase in an inward rectifying K+ current (Penington et al., 1992). Gerschenfeld et al. (1986) reported that serotonin increased the duration of calcium spikes in Helix neurons in which K+ currents were blocked; but whether 5-HT affects action potential duration in neurons under normal physiological conditions or in other cells where Ca2+ current is enhanced has not been reported.
Indirect modulation of Ca2+ currents can be achieved by altering the duration of the action potential, a phenomenon well studied in sensory neurons of A. californica (Baxter and Byrne, 1989, 1990; Klein et al., 1982; Klein and Kandel, 1978; Rosen et al., 1989). The broadening of spikes in sensory neurons has been attributed to the closing of particular K+ currents (IKDV and IKS; Baxter and Byrne, 1990; Seigelbaum et al., 1982; Klein et al., 1982). As the spike is broadened, the longer-lasting depolarization results in an increase in voltage-sensitive calcium current (Klein and Kandel, 1978, 1980). Although 5-HT was shown to enhance selectively a slowly inactivating, dihydropyridine-sensitive (L-type) calcium current, this current was found not to participate in the facilitation of transmitter release, or, indeed to be involved in transmitter release at all during normal action potentials; rather a rapidly inactivating (N-type-like) Ca2+ current, insensitive to 5-HT, is responsible for presynaptic facilitation, and the prolonged activation of this current is caused indirectly by the 5-HT-induced depression of K+ currents that broaden the spike (Edmonds et al., 1990).
In this paper we present evidence that 5-HT does cause an increase in a P-type calcium current in parapodial motoneurons, and that the effect is direct; serotonin does not affect the duration of MN action potentials. We speculate that an increase in such a calcium current could contribute to an increase in EJP amplitude. It is to be noted, however, that these experiments were conducted on motoneuron somata. While we suggest that comparable effects would be seen at the MN terminals, we have no evidence for this; nor do we demonstrate the role of a P-type calcium current in transmitter release. The limitations of our interpretations of the results are discussed later in this report.
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Specimens of A. brasiliana, ranging in size from 50–500 g, were collected from Laguna Madre near Port Isabel, Texas. They were housed in our Institute's aquarium facility in large aquaria with recirculating artificial seawater (ASW) at room temperature and fed dried seaweed (laver) daily.
Three kinds of electrophysiological experiments were performed. In the first set, our goal was to examine, using intrasomatic current-clamp recording from identified parapodial motoneurons in situ, whether serotonin could influence action-potential duration and which membrane currents might be involved with 5-HT's effects. In the second set of studies, voltage-clamp analyses of membrane currents and the effects of 5-HT on them were made using isolated motoneuron somata. Finally, a third series of current-clamp experiments were done on motoneurons in situ where the effects of 5-HT on pharmacologically isolated calcium currents were studied. We describe first the initial, "general" experimental preparation and then describe separately the methods unique to the "current-clamp" and "voltage-clamp" experiments.
General
Animals were first anesthetized by injecting isotonic MgCl2 (33 ml/100 g body wt) into the foot sinus. Next, a dorsal incision was made to allow removal of the viscera and abdominal ganglion, but leaving the head ganglia (cerebral, pleurals and pedals) and their nerves intact. Either one or both parapodia were cut free from the body wall, taking care not to cut the parapodial nerves (PPns) that connect the pedal ganglia to the periphery. The preparation was then transferred to a dish composed of two concentric, circular chambers made from transparent plexiglass and lined with silicone elastomer (Sylgard; Dow). The head ring of ganglia was pinned (dorsal side up) to the floor of the small inner circular chamber that had grooves cut into its wall to allow the PPns to extend into a larger-diameter outer chamber housing the parapodium. The nerve slots in the small chamber were then filled with vacuum grease to isolate the solutions in each chamber. This chamber system allowed the two chambers to be perfused with different bathing solutions.
After the ganglia were pinned to the Sylgard, the pedal ganglion to which the parapodium was attached via the PPns was desheathed by the use of fine scissors. The parapodium was pinned along its medial, cut edge near the small chamber to prevent parapodial movements from stretching the PPns. The lateral edge of the parapodium was left free to contract. The chamber containing the parapodium was bathed in normal ASW. Experiments were conducted at room temperature (20–23°C). Under these conditions, the preparations remained viable for up to 12 hr.
Current clamp
In most experiments, during the initial dissection, just before pinning the head-ring ganglia in the recording chamber, these ganglia were immersed in 0.5% glutaraldehyde (in ASW) for 40 sec to decrease TEA-induced contractions of the connective tissue (Rosen et al., 1989). After the ganglia were pinned to the recording chamber floor, the pleural ganglion ipsilateral to the desheathed pedal ganglion was usually removed to expose pedal-ganglion neurons whose visualization was often obscured by this ganglion. To enhance ease of penetration and facilitate drug perfusion, in many experiments the head-ring ganglia were also bathed for 1–2 hr in a solution of 2% Type I collagenase (in ASW).
Intracellular recordings were amplified using an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA). Signals were recorded on a Gould chart recorder and converted to digital signals on a Digidata 1200 (Axon Instruments) and stored on a pc computer using pClamp software (Axon Instruments). A dual-beam oscilloscope was used for continuous monitoring of neural activity. Intracellular electrodes were pulled from omega-dot capillary glass on a horizontal puller. The electrolyte used in the electrodes was KCl (3 M), and electrodes had resistances of 10–30 M
. The bath was grounded through an AgCl-coated silver wire.
In experiments to examine effects of 5-HT on motoneuron spike width, MN action potentials were elicited by passing a 5-msec current pulse through the recording electrode. AP half-widths were measured as the time taken for the AP to repolarize from peak back to pre-spike baseline (Baxter and Byrne, 1990). Briefly, the pre-spike baseline level of membrane potential was measured just before the application of the spike-producing stimulus for each spike and this baseline value was marked on the falling phase of the AP. The AP half-widths used for each neuron were the mean of at least 10 spikes measured in each experimental condition. The pooled data reported represent averages of these values from all neurons tested in each experiment.
The bath solution used was commercial artificial seawater (ASW; Instant Ocean). The drugs used in these experiments were either made as a stock solution in the appropriate solvent and then diluted with ASW, or were added directly to the ASW to yield concentrations that are stated in the text. The solutions in the two chambers were perfused separately, with all drugs being applied to the chamber containing the head ganglia.
Later current-clamp studies on MNs in situ where 5-HT effects on different calcium currents were measured were performed in a similar manner.
Voltage clamp
The experiments progressed in two stages. First, the reduced preparation was used to functionally identify motoneurons. After the head-ring ganglia and parapodia were pinned in the recording chamber, the pedal ganglia were partially desheathed and all the ganglia in the inner recording chamber were treated for 40–50 min with 0.5% Pronase E followed by a 90–120 min exposure to 2% collagenase Type I. This procedure softens the connective tissue surrounding neurons and permits the removal of individual somata (Arshavsky et al., 1986; Panchin et al., 1993). After the enzymes were washed out, putative motoneurons were identified by their topographic location and their ability to cause parapodial contractions (McPherson and Blankenship, 1991a, b). If depolarization-induced spiking of a cell caused reliable and consistent contractions of a region of a parapodium, the neuron was considered to be a motoneuron. These neurons were isolated from the pedal ganglion by gently pulling the impaled cell away from the ganglion using the penetrating electrode. The stretched axon was then severed near the soma using small glass hooks fashioned from micropipettes. After the axon was cut, the motoneuron soma was transferred with a plastic pipette from the original chamber to a much smaller recording chamber (250–300 µl capacity) consisting of the well of a standard glass depression slide. The well was constantly perfused by gravity from a reservoir at a rate of approximately 1 ml/30–40 sec; solution was withdrawn by vacuum through a small glass suction tube. Stock solutions of drugs were added to the perfusion reservoir. The standard perfusion solution (approximating normal sea water) contained the following (in mM): 427 Na+, 10 K+, 10 Ca2+, 499 Cl–, 48 Mg2+, 26 SO42–, 3 HCO3– (pH, 7.6–7.8). In some experiments, 424 mM Na+ ions were substituted for by equimolar amounts of TEA (NaHCO3 was deleted), and Ca2+ ions were sometimes replaced by equimolar amounts of Co2+ or Ba2+ ions. When Ba2+ was substituted for Ca2+, sulfate ions were replaced by Cl–, and HCO3– was eliminated. In these experiments, the sulfate/HCO3 adjustments were also made to the bathing solution used before and after Ba2+ substitution. In most voltage-clamp experiments, in order to eliminate voltage-gated sodium currents and to suppress K+ currents, all the sodium in the solution (424 mM) was replaced with TEA (NaHCO3 was eliminated), and 1 µM tetrodotoxin (TTX) and 10 mM each of 4-aminopyridine (4-AP), of 3,4 di-aminopyridine (3,4-DAP), and 10 mM Hepes solution (with pH adjusted to 7.6–7.8 with Tris Cl) were added to the solution. All salts, chemicals, drugs and toxins were obtained from Sigma, Fisher or Almone Labs.
Motoneuron somata placed in the well of the depression slide were spherical and had diameters of from 30 to not more than 50 µm. Little or no axon was visible. The motoneurons were repenetrated with 3 M KCl-filled microelectrodes (20–25 M). Motoneurons bathed in artificial seawater containing no Na+, 424 mM TEA, 1 µM TTX, 10 mM each of 4-AP, 3,4-DAP, and Hepes, and normal levels of K+, Cl– and Ca2+ had resting potentials of from –40 to –60 mV (some neurons, almost certainly more damaged by the isolation procedure, had resting potentials less negative than –40 mV but such neurons were not used in experiments) and exhibited no spontaneous firing. The uniform spherical shape, lack of axonal processes, and relatively small size of these identified motoneurons lends some confidence to the adequacy of a space clamp. Motoneurons were voltage clamped with the discontinuous single electrode technique at a sampling rate of 4–5 KHz using an Axoclamp 2A amplifier. Currents were filtered before acquisition, typically at 500–1,000 Hz. During figure preparation, records were often filtered further. The limited gain of the single-electrode voltage clamp, combined with the relatively high resistance (and thus limited current-passing ability) of the electrodes used, sometimes resulted in a disparity between command (nominal) and actual membrane voltage during maintained voltage steps. Correction was made for such disparity by recording the actual membrane voltage in parallel with the membrane current and using these values when constructing I–V plots. Electrical activity was monitored on oscilloscopes and a Gould chart recorder and was digitized and stored on a computer using Axon Instruments Axolab 1100 and pClamp software.
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Effects of 5-HT on motoneuron action potentials
As a first step in determining whether 5-HT has an effect on the presynaptic motoneuron, we examined whether serotonin could alter the duration of the motoneuron action potential and what currents might be involved. In four experiments in which 5 µM 5-HT was applied to the ganglia, no statistically significant change was observed in MN spike half-width. In control ASW, the average half-width of somatic action potentials was 3.5 ± 0.32 msec (SEM; n = 4). In the presence of 5-HT, the average spike half-width for the same four neurons was 3.4 ± 0.31 msec. Since 5-HT alone causes no apparent spike broadening, it is unlikely that this drug affects a calcium current through the indirect route of blocking K+ currents and widening the spike. However, 5-HT could have a direct effect on calcium currents that is not detectable under these conditions. Since large K+ currents that participate in the repolarization of the AP often shunt the Ca2+ current, we repeated the above experiments in the presence of 50 mM TEA (Klein and Kandel, 1978, 1980
; Rosen et al., 1989). Action potential half-widths were recorded under four experimental conditions (Fig. 1A, B): control ASW, 50 mM TEA, 50 mM TEA + 5 µM 5-HT, and washout with ASW. Each experiment was performed by recording 10 APs from each neuron in each experimental condition from which a mean spike half-width was obtained. As expected, the AP widens following the application of TEA. Further broadening is observed when TEA is applied in conjunction with 5-HT. Ten minutes following the application of TEA, control spike half-width increased significantly by 36%. The further addition of 5 µM 5-HT increased the TEA spike half-width significantly by 35%. The effects of TEA and 5-HT were both reversible following washout.
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Although 5-HT was capable of broadening the TEA spike, the role of Ca2+ in this response remained undisclosed. If the spike broadening caused by 5-HT in the presence of TEA is due to a direct effect on calcium channels, the effect of 5-HT should be blocked in the presence of cobalt. In five experiments (Fig. 1C), 15 mM Co2+ blocked the 5-HT-induced increase in AP half-width by an average of 97% ± 12% (SEM). Of the five neurons tested, all responded to TEA and 5-HT with increases comparable to those reported in Figure 1A. However, the addition of Co2+ decreased the average TEA spike half-width from 4.4 msec to 2.6 msec. The further addition of 5-HT had no significant effect on the spike half-width. Each neuron responded to 5-HT in the absence of Co2+ with an average spike half-width of 6.2 msec, which was a 30% increase above the TEA spike. Figure 1D illustrates digitized APs from a representative experiment. The TEA + Co2+ spike and the spike recorded in TEA, 5-HT, and Co2+ both had the same spike half-width, demonstrating a 100% block of the 5-HT effect by Co2+. The same neuron was able to respond to 5-HT in the absence of Co2+, with a spike half-width of 6.5 msec. This figure also clearly illustrates that Co2+ decreases the half-width of the TEA spike, demonstrating that Ca2+ is at least one of the currents flowing during the somatic AP.
Application of 5-HT broadened the TEA spike in a dose-dependent manner (Fig. 2). The average effect of 5-HT at 5 µM was a 1.7 msec increase in spike width. At the highest dose tested (50 µM) an increase in AP width was observed, but the curve exhibited a downward phase. This "bell-shaped" dose response curve is typical of other vertebrate and invertebrate 5-HT responses (Hoyer and Boddeke, 1993; Pliska, 1994; Price and Goldberg, 1993; Simmons and Koester, 1986). All subsequent experiments in this series were performed using 5–10 µM 5-HT, since this dose range produced the greatest effect on AP broadening.
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Effects of 5-HT on isolated membrane currents
To examine in more detail the voltage-gated membrane currents present in motoneuron somata and which of these was altered by 5-HT, we attempted to utilize voltage clamping with isolated MNs. Probably because of the strong enzyme treatment, trauma induced by the isolation procedure, and the prolonged protocols for identifying motoneurons, isolating them and then applying a variety of ion solutions and drugs, these were very difficult and demanding experiments. Confirmation of the presence of calcium currents and their enhancement by 5-HT was accomplished, but other experiments were required for characterizing the specific calcium currents involved.
Isolated MNs were superfused with solutions in which all extracellular Na+ was replaced with TEA (424 mM) and to which was added 1 µM TTX, and 10 mM each of 4-AP, 3,4-DAP and Hepes buffer. This solution assured that any latent Na+ currents and most voltage-gated K+ currents were blocked; however, K+ currents were usually not fully suppressed. Ca2+-activated outward currents likely contributed to a portion of the unblocked current, since when Co2+ or Ba2+ was utilized to replace Ca2+, the outward currents seen with depolarization were smaller, but not always completely absent. Such background currents further complicated the analysis of properties of the calcium currents.
In a total of 21 isolated motoneurons studied under these conditions, all were seen to express inward Ca2+ current upon depolarization (Fig. 3A). The total current varied from cell to cell but was always eliminated when extracellular Ca2+ was replaced with 10 mM Co2+ (Fig. 3C). The total inward current began to activate at between –20 and –10 mV and reached a peak between 0 and +20 mV (Fig. 3D). The apparent reversal potential for this current was variable (from +30 to +70 mV). This variability, which obscured the true calcium reversal potential, was probably due to combinations of movement of varying amounts of K+ ions through different Ca2+ channels, and, particularly, activation of Ca2+-dependent outward (K+) currents. The extent to which the Ca2+ current inactivated from cell to cell was variable when depolarizing steps were held for 80 msec to 2 sec. In some cells, an apparent inactivation of inward calcium current was clearly an artifact due to contamination by outward currents not fully blocked by the potassium current blockers and to Ca2+-dependent outward currents. The latter point was documented by experiments in which Ba2+ was substituted for extracellular Ca2+ as the charge carrier (Fig. 3B, D, E). As expected, barium was found to substitute well for calcium as an inward charge carrier, but typically there was little change in total inward current in the presence of Ba2+ (Fig. 3B, D). However, the use of Ba2+ did demonstrate that normal Ca2+ currents induced Ca2+-dependent outward currents that were prevented in the presence of Ba2+ (Fig. 3D, E). Some of the variability in total calcium current and degree of inactivation was due, however, to the fact that different motoneurons expressed varying amounts of different types of calcium currents as discovered when various Ca2+-channel blockers or ionic substitutions were utilized. We applied two different Ca2+-channel blockers in these experiments; one, the dihydropyridine compound nifedipine, a blocker of L-type Ca2+ channels, and, the other, -conotoxin GVIA, a selective blocker for N-type Ca2+ channels (Bean, 1989a; Fox et al., 1987; McCleskey et al., 1987; Sher and Clementi, 1991; Tsien et al., 1988). In most motoneurons, at least two and sometimes three different Ca2+ channels appear to be present and in different ratios in different cells.
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) and Ba2+ (
) currents from the data represented in A–C. Voltage steps were in 10 mV increments from –50 mV to +70 mV. In Ca2+/ASW, the inward current reverses at more negative potentials than in Ba2+/ASW due to Ca2+ current inactivation as well as to Ca2+-dependent outward current activation. E. Superimposed, unsubstracted current records from the same cell obtained in Ca2+/ASW, Ba2+/ASW and Co2+/ASW. The current was produced by long (2 sec) voltage steps from –50 mV to 0. After the Ca2+ current reaches peak, it rapidly decreases in amplitude, likely due to inactivation and to the activation of a Ca2+-dependent outward current. In Ba2+/ASW, the Ca2+-dependent outward current is blocked; therefore, the gradual decrease of Ba2+ current is apparently caused only by its own inactivation. The inward current is completely blocked in 0 Ca2+, Co2+/ASW (Co), which indicates that the inward current is carried through Ca2+ channels. Voltage calibration applies to A–C and EFigure 4 illustrates representative results from 10 motoneurons studied in this way. The data in Figure 4A and B illustrates motoneuron calcium currents which were little affected by nifedipine but were partially blocked by
-conotoxin. In a total of three cells studied, nifedipine (10 µM) on average was able to block about half of the calcium current; Figure 4C illustrates a clear case of partial nifedipine block of calcium currents from a different cell. In other neurons, it was found that while nifedipine might or might not block some portion of the total Ca2+ current, -conotoxin always blocked part of the current. On average, 8 µM -conotoxin blocked 48% of the total calcium current (n = 7). In most motoneurons, however, there exists a calcium current that is insensitive to both nifedipine and -conotoxin (Fig. 4B, triangles).
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Since 5-HT can produce an apparent Ca2+-dependent increase in action potential duration in K+-channel-blocker-poisoned motoneurons in situ (Fig. 1), we examined the effects of 5 µM 5-HT on isolated Ca2+ currents under voltage clamp. In all isolated motoneurons tested (n = 4), 5-HT was seen to increase the calcium current by an average of 58% (Fig. 5A, B). Although there is little if any indication in these figures that serotonin was having a major effect on K+ currents, since, for example, there was no significant change in apparent inactivation or reversal potential of the inward current in the presence of 5-HT, we did test for the effect of 5-HT on neurons when Ca2+ was replaced by Ba2+ (Fig. 5C). Under these conditions, Ca2+-dependent outward currents are not active, so 5-HT could not be indirectly enhancing the inward current by, for example, blocking the outward K+ currents caused by Ca2+ influx. In three such experiments 5-HT increased the peak inward Ba2+ current by 46%. Figure 5D summarizes data from three other experiments in which we examined the effects of 5-HT on background, unblocked outward current in the presence of K+ current blockers (424 mM TEA and 10 mM each of 4AP and 3,4-DAP) and with Ca2+ ions substituted with Co2+. There was no significant difference in the outward currents, indicating that the serotonin effect on Ca2+ currents in motoneurons is not mediated by blocking such "residual" outward currents. In one experiment we examined the effect of 5-HT on total outward current in a motoneuron bathed in ASW in which all Na2+ was replaced with Tris, Ca2+ was replaced with Co2+, but no K+ blockers were present. There was no significant change in total outward current when 5-HT was added. This experiment also supports the view that 5-HT does not influence K+ currents but acts directly on Ca2+ channels.
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Current-clamp experiments reveal 5-HT affects P-type Ca2+ current
Functionally identified motoneurons that had been isolated for voltage clamp studies could not be used for studies to determine which calcium current might be selectively activated by 5-HT. These already partially damaged and "fragile" cells ran down before the several solution changes necessary could be accomplished. We returned, therefore, to current clamp analyses of functionally identified motoneurons in situ in the pedal ganglion. In normal ASW the functionally identified motoneurons responded to short depolarizing pulses with a train of normal action potentials (Fig. 6A). After several minutes in Na+-free artificial sea with the usual concentrations of TTX, 4-AP and 3,4-DAP, the normal action potentials in these neurons became quite prolonged, stabilizing at durations of several hundred ms. That these spikes were Ca2+-dependent was consistently demonstrated by the addition of either Cd2+ or Co2+, each of which completely blocked the action potentials (Fig. 6B).
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A series of experiments was then done wherein calcium spikes in identified motoneurons were studied on exposure to 5-HT. Addition of 10 µM 5-HT to the bath caused an average increase of 37 ± 6.7% (SEM, n = 16) in Ca2+-spike duration (Fig. 7A). With washing, the spike duration returned to pre-5-HT levels. The cell was then exposed to a solution containing either 20 µM nimodipine (another dihydropyridine similar to nifedipine), 2 µM
-conotoxin GVIA, or to 100 nM agatoxin TK, a blocker of P-type calcium channels (Blundon et al., 1995; Llinas et al., 1989; Wright et al., 1996a). Figure 7B, C illustrates that while either nimodipine or -conotoxin could shorten the calcium spike slightly, suggesting the presence of L- and N-type channels, they did not prevent the ability of 5-HT to cause a significant increase in spike duration. 5-HT produced an average increase of 43 ± 6% in spike width when nimodipine was present (n = 10), and a 36 ± 14% increase in the presence of -conotoxin VIA (n = 3). Agatoxin TK, on the other hand, also caused some shortening of the calcium spike, but also prevented the ability of 5-HT to cause spike broadening. 5-HT produced only a 7 ± 20% (n = 5) increase in Ca2+-spike width in the presence of agatoxin. These results suggest that 5-HT's effects on spike prolongation may be due to its ability to selectively increase an agatoxin-sensitive (P-type) calcium current in swim motoneurons.
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0.05, two-tail unpaired t-test). The differences among the three populations of neurons are also not significant (P 
0.05, two-tailed Mann-Whitney rank-sum test). D. The 5-HT-induced prolongation of Ca2+-spike duration was significantly blocked by |
DISCUSSION |
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SYNOPSISINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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These experiments indicate that serotonergic modulation of the parapodial neuromuscular synapse may, at least in part, be due to presynaptic facilitation. Although the most commonly reported mechanism of presynaptic facilitation in Aplysia is AP widening (Baxter and Byrne, 1990
; Klein and Kandel, 1978; Rosen et al., 1989), we did not observe any enhancement of MN spike width by 5-HT in the absence of TEA. These data indicate that, in parapodial motoneurons, 5-HT does not modulate Ca2+ currents indirectly via prolongation of the AP. However, in the presence of TEA, which blocks some of the currents responsible for repolarization of the AP, 5-HT consistently broadened the MN spike. The increase in AP width in the presence of TEA and 5-HT was blocked with Co2+ or Cd2+, suggesting that 5-HT is able to enhance normal Ca2+ currents and thus potentially increase the amount of transmitter released with each spike.
Previous reports of the role of 5-HT in heterosynaptic facilitation at a different (sensorimotor) synapse in Aplysia have demonstrated that 5-HT increases synaptic transmission with a concomitant widening of the AP caused by 5-HT's ability to decrease a K+ current (Baxter and Byrne, 1989, 1990; Klein et al., 1982; Rosen et al., 1989). AP widening which is caused by the closure of K+ channels results in the recruitment of a larger number of voltage-sensitive Ca2+ channels (Klein and Kandel, 1980; Edmonds et al., 1990). In the presence of TEA, this increase in Ca2+ current further broadens the AP. Although the Ca2+-induced broadening of the AP can be blocked by Co2+ (Klein and Kandel, 1978), modulation of the K+ current is the key factor in synaptic facilitation (Klein and Kandel, 1980). Even though 5-HT has been shown to directly modulate a dihydropyridine-sensitive Ca2+ current in these sensory cells, this current is believed not to be responsible for transmitter release or synaptic facilitation (Braha et al., 1993; Edmonds et al., 1990). Furthermore, Klein (1994) suggests that at Aplysia sensorimotor synapses, synaptic augmentation is independent of action potential broadening and may be due to modulation of the secretory process.
We have demonstrated that 5-HT has no direct effect on parapodial MN spike width (and thus likely little effect on K+ currents), and that only in the presence of TEA is 5-HT able to increase MN spike duration. This enhancement of AP width in parapodial motor neurons is likely the effect of an increased Ca2+ component of the AP. In Aplysia RB, LB and LC cells (Pellmar, 1984; Pellmar and Carpenter, 1980) and in Helix neurons (Gerschenfeld et al., 1986; Hill-Venning and Cottrell, 1992), 5-HT has been shown to enhance directly a voltage-sensitive Ca2+ current. In Helix neurons 5-HT produced an increase in spike duration when K+ currents were blocked (Gerschenfeld et al., 1986); 5-HT caused no apparent change in spike width in Aplysia LB and LC neurons in normal ASW (Pellmar and Carpenter, 1980).
Although the spike broadening in parapodial motoneurons can be accounted for fully by an enhancement of Ca2+ currents, we have not eliminated the possibility that 5-HT modulates other somatic currents. However, any enhancement of transmitter release that is the result of increased Ca2+ currents flowing during the AP appears to be the result of direct enhancement by 5-HT of a P-type Ca2+ current. It is unlikely that 5-HT-induced broadening of the TEA spike is the result of closure of Ca2+-activated K+ channels, since 5-HT also enhances Ba2+ currents, when Ca2+-dependent K+ currents are suppressed. A definitive picture of the effect of 5-HT on calcium influx during a spike and the amine's selectivity for calcium versus potassium channels would require action potential waveform clamping and careful use of selective channel blockers and ion substitutes.
Our voltage-clamp and pharmacologic data indicate that the somata of swim motoneurons express a variety of kinds of calcium current in varying proportions from cell to cell. Most motoneurons express L-type, N-type and P-type channels. Only the agatoxin-sensitive, P-type channel appears to be sensitive to 5-HT, however. While it is clear that 5-HT can enhance the amplitude of EJPs (McPherson and Blankenship, 1991c), we have as yet no data to indicate that it is an agatoxin-sensitive P-type calcium channel that acts to increase transmitter release in the presence of 5-HT. Indeed, we have no direct evidence that the currents we are recording in the motoneuron somata are representative of currents that exist in motor terminals, although other workers have successfully argued that somatic currents in molluscan neurons may be reasonably representative of currents present in axon terminals (Klein and Kandel, 1978, 1980; Meech and Standen, 1975; Stinnarke and Tauc, 1973). There certainly is evidence that P-type channels are involved in transmitter release in molluscan and other invertebrate preparations, including the squid giant synapse (Llinas et al., 1989) and crayfish neuromuscular junction (Wright et al., 1996a, b). On the other hand, it is also clear that a variety of P-like channels with different pharmacologic profiles exists in different preparations, and that transmitter release may be triggered by influx of calcium through N-type calcium channels or by release from intracellular stores (Adams and Olivera, 1994; Bean, 1989a; Katz et al., 1995; Llinas et al., 1989; Maubecin et al., 1995; Mintz et al., 1992). Whether P-type channels are involved in enhancement of neuromuscular transmission in the Aplysia swim system, and the relative importance of 5-HT effects on presynaptic P-type channels (transmitter release) versus postsynaptic (muscle-fiber L-type channels) are the directions for future work. The present results do, however, provide new information on a plausible presynaptic mechanism whereby POP cells can influence the relative amount of power, or gain, that can be selectively induced in swim-muscle groups to augment the strength of the swim stroke and to alter swim direction (Gamkrelidze et al., 1995; McPherson and Blankenship, 1991c).
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ACKNOWLEDGMENTS |
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The symposium was supported by National Science Foundation Grant IBN 9905990.
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FOOTNOTES |
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1 From the Symposium Swimming in Opisthobranch Mollusks: Contributions to Control of Motor Behavior presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2000, at Atlanta, Georgia.
2 E-mail: jeblanke{at}utmb.edu
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