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1Department of Physics, Santa Clara University, Santa Clara, California; 2Volen Center and Biology Department, Brandeis University, Waltham, Massachusetts; and 3Department of Biology, University of Washington, Seattle, Washington
Submitted 26 August 2005; accepted in final form 2 November 2005
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ABSTRACT |
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INTRODUCTION |
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), and the neuron is said to be "bistable." Plateau production is possible when a persistent inward current (PIC) causes the steady-state current–voltage (I–V) relationship to be "N"-shaped such that there are two quasi-stable membrane potentials corresponding to I = 0: a truly stable state near rest and a long-lived depolarized level above the threshold for spike production (Schwindt and Crill 1977, 1980a). The lifetime of the depolarized level varies from system to system, and the duration of the associated persistent firing can last fractions of a second (Lee and Heckman 1996) or many minutes (Egorov et al. 2002). Bistable neurons have been identified in motor, sensory, and central nervous systems and in both vertebrates (Di Prisco et al. 1997; Egorov et al. 2002; Hounsgaard et al. 1984; Hsiao et al. 1998; Morisset and Nagy 1996; Reuveni et al. 1993; Russo and Hounsgaard 1994; Schwindt and Crill 1980a) and invertebrates (Dembrow et al. 2004; DiCaprio 1997; Dickinson and Nagy 1983; Katz and Harris-Warrick 1989; Lechner et al. 1996; Ramirez and Pearson 1991). Regardless of the system, one feature common to all documented bistable neurons is that the biophysical mechanisms responsible for the bistability are located in the somatodendritic rather than axonal region of the neuron (Bennett et al. 1998; Gutman 1991; Heckman et al. 2003; Hounsgaard and Kiehn 1993; Lee and Heckman 1996; Reuveni et al. 1993; Schiller et al. 2000; Schwindt and Crill 1980b; Svirskis et al. 2001).
Bistable motor neurons in both vertebrates and invertebrates share two additional common traits. First, all previously documented occurrences of bistability in motor neurons have involved the action of a neuromodulator (Kiehn 1991), typically a monoamine, that assists in the production of the plateau potential (Conway et al. 1988; Hounsgaard and Kiehn 1985, 1989; Hounsgaard et al. 1988; Hsiao et al. 1998; Kiehn and Harris-Warrick 1992b; Lee and Heckman 1998; Ramirez and Pearson 1991). Second, although recent studies have identified a role for a persistent Na+ current in some systems (Elson and Selverston 1997; Hsiao et al. 1998; Lee and Heckman 1999; Li and Bennett 2003; Powers and Binder 2003), production of plateau potentials in motor neurons often depends on the activation of an L-type Ca2+ current (Carlin et al. 2000; Hounsgaard and Kiehn 1989; Li and Bennett 2003; Russo and Hounsgaard 1994; Svirskis et al. 2001) and in some cases on a Ca2+-activated nonselective cation current ICAN (Morisset and Nagy 1999; Rekling and Feldman 1997; Zhang et al. 1995).
One motor neuron in which the biophysical mechanisms underlying bistability have been studied in detail is the dorsal gastric (DG) neuron in the stomatogastric ganglion (STG) of the crab Cancer borealis. The stomatogastric nervous system (STNS), of which the STG is part, produces both the gastric mill motor pattern that is responsible for movement of teeth within the stomach and the pyloric motor pattern that controls the muscles of the pylorus, a filtering apparatus (Harris-Warrick et al. 1992). The DG neuron is part of the gastric mill circuit (Weimann et al. 1991) and innervates the gastric mill 4 (gm4) muscle [nomenclature from Maynard and Dando (1974)]. In a series of papers, Harris-Warrick and coworkers showed that serotonin released by a stretch-sensitive neuron (Katz and Harris-Warrick 1989) triggers DG plateau potentials by acting directly and indirectly on at least four currents including a Ca2+ conductance and a Ca2+-activated nonselective cation current (Kiehn and Harris-Warrick 1992a,b; Zhang and Harris-Warrick 1995; Zhang et al. 1995).
Herein we also describe observations of bistability in DG, although our findings are distinct from those presented in the earlier studies. Rather than injecting current into the soma, we stimulated extracellularly the nerve containing the DG axon and observed persistent firing of the DG neuron and contractions of the gm4 muscle with enhanced amplitudes and durations. The magnitude of these effects depended on the amplitude of the stimulus. As in the previous inquiries, we found that calcium and calcium-dependent conductances play an essential role in producing the bistability. However, we conclude that the biophysical mechanisms responsible for bistability are located in the axon. Moreover, we saw no evidence that local neuromodulation plays a role in initiating the bistable behavior in this system. Some of these data previously appeared in abstract form (Verley et al. 2005).
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METHODS |
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Adult male Cancer borealis were obtained from seafood suppliers in the Boston, MA area and from the Marine Biological Laboratory (Woods Hole, MA) and maintained in aerated aquaria at 10–12°C. In physiology experiments, we used saline with the following composition (in mM): 440 NaCl, 11 KCl, 13 CaCl2, 26 MgCl2, 5 maleic acid, and 11 Trizma base, pH 7.4–7.5. Low-calcium saline was identical to that just described except the Ca2+ concentration was reduced to 0.1 mM. The modulators proctolin (American Peptide Company, Sunnyvale, CA), TNRNFLRFamide (American Peptide Company), C. borealis tachykinin-related peptide Ia (CabTRP Ia, a gift from M. P. Nusbaum, Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, PA), serotonin (Sigma, St. Louis, MO), and 
-aminobutyric acid (GABA, Sigma) were dissolved in deionized water at 10–3 or 10–2 M and stored at –20°C before being diluted in saline and then bath-applied to the preparation. The peptide [Asn13]orcokinin (orcokinin, Bachem, San Carlos, CA) was dissolved in DMSO at 10–3 M and stored at –20°C before use. Nicardipine (Sigma) and flufenamic acid (FFA, Sigma) stock solutions were made in DMSO immediately before use and then diluted so that the final concentration of DMSO was 0.1%. We confirmed that DMSO at this concentration had no effect on muscle contraction or spike generation. Tetrodotoxin (TTX, Sigma) was prepared from a stock solution (10–4 M) and dissolved in a 750-mM sucrose solution to reach a final concentration of 10–6 M.
Contraction measurements
The gm4 muscle is innervated solely by the single DG motor neuron whose axon projects from the STG to the muscle by the dorsal gastric nerve (dgn) (Weimann et al. 1991). Neuromuscular preparations were dissected from the stomach and pinned flat in 5-ml Sylgard 182–lined (Dow Corning, Midland, MI) petri dishes. For most physiology experiments, the preparation consisted of the gm4 muscle and the dgn, with the nerve cut just below the STG. During recording sessions the bath volume was approximately 3 ml, and the preparation was continuously superfused (4–5 ml/min) with physiological saline cooled by an ice bath to a temperature regulated between 9 and 11°C. During contraction experiments in particular, care was taken to regulate the temperature to within a few tenths of a degree because we observed the amplitude of muscle contractions to be temperature dependent (Birmingham, unpublished observations).
Contractions of the gm4 muscles were measured using a FT03 force transducer (Astro-Med, West Warwick, RI). One insertion of the gm4 muscle was pinned down to the dish, whereas the other was attached to the transducer with a short (about 3 cm) piece of size 6/0 silk suture thread (Fine Science Tools, Foster City, CA). The transducer was positioned so that the muscle was stretched just past its relaxed length. A stainless steel pin stimulation electrode was inserted into an insulating (petroleum jelly) well around the end of the nerve furthest from the muscle. The electrode was driven by a pulse stimulator (Model 2100, A-M Systems, Carlsborg, WA) to induce muscle shortening, and the force transducer measured the increased tension. The transducer signal was amplified (Brownlee Precision Model 440, San Jose, CA) by a factor of 10,000 and recorded using a Digidata 1322A acquisition system (Axon Instruments, Union City, CA).
Intracellular and extracellular recordings
Measurements of excitatory junctional potentials (EJPs) from the gm4 muscle were made using conventional 2 M KAc-filled microelectrodes with resistances of 7–10 M
. The dgn was stimulated extracellularly as during contraction measurements. EJPs were measured with an Axoclamp 2B intracellular amplifier (Axon Instruments), amplified tenfold using the Brownlee amplifier, and recorded using the Digidata acquisition system. For intracellular recordings from the DG neuron, the complete STNS consisting of the paired commissural ganglia (CoGs), the esophageal ganglion (OG), the STG, and a subset of the motor nerves was dissected out of the animal and pinned out in a Sylgard-coated dish. The STG was then desheathed and petroleum jelly wells were placed on the motor nerves. To prevent activation of the gastric mill rhythm by descending modulatory inputs, we built a petroleum jelly well filled with a sucrose/TTX solution (750 mM sucrose, 10–6 M TTX) on the stomatogastric nerve. Intracellular recordings from the DG soma were made using an Axoclamp 2A and 20- to 40-M glass microelectrodes filled with 0.6 M K2SO4 and 20 mM KCl. DG was identified by correlating intracellular recordings with extracellular recordings of spikes in the dgn.
Extracellular measurements from the dgn were made using either pin or glass suction electrodes amplified by an A-M Systems 1700 differential amplifier and recorded using the Digidata acquisition system. In addition to the axon of DG, axons from the gastric mill (GM) motor neurons and the anterior gastric receptor (AGR) (Simmers and Moulins 1988), a muscle stretch receptor that does not innervate gm4, are known to project down the dgn. If present, electrical coupling between the DG, AGR, and/or GM neurons is too weak for any one of these neurons to activate the others (MP Nusbaum, unpublished observations). In extracellular recordings, the largest amplitude unit corresponded to DG spiking. Spontaneous AGR spiking (Simmers and Moulins 1988) was typically observed in extracellular recordings but was sometimes absent in preparations in which the recording was made very close to the gm4 muscle or from one portion of a branched dgn. The number of distinct spikes observed in a particular experiment varied from one to four.
Whole-mount immunohistochemistry
In anatomical studies one of two preparations was used: the STNS containing the dgn cut just anterior to its insertions onto the gm4 muscle or the dgn with the gm4 muscles attached. In either case, the preparation was dissected free of the foregut in chilled (about 10°C) saline and then pinned in a Sylgard 184–lined (Dow Corning) petri dish. The saline differed slightly from that used in physiology experiments in that it was buffered with HEPES acid (10 mM) instead of maleic acid/Trisma base, and its pH was adjusted to 7.4 with NaOH. Preparations were then fixed at 4°C for 12–24 h in a freshly prepared solution of 4% paraformaldehyde (EM grade, catalog No. 15710; Electron Microscopy Sciences, Hatfield, PA) in 0.1 M sodium phosphate (P) buffer (pH 7.4). After fixation, tissues were rinsed five times over nearly 5 h in a solution of P buffer containing 0.3% Triton X-100 (P-Triton) and then incubated for about 72 h in primary antibody diluted to an appropriate working concentration (see Antibodies) in P-Triton containing 10% normal donkey serum (NDS, catalog No. 017-000-121; Jackson ImmunoResearch Laboratories, West Grove, PA). After incubation in primary antibody, tissues were again rinsed five times over nearly 5 h in P-Triton and then incubated in a 1:300 dilution of secondary antibody (see following text) for 12–24 h. As with the primary, incubation in secondary antibody was done in P-Triton containing 10% NDS. After incubation in secondary antibody, preparations were rinsed five times over about 5 h in P and mounted between a glass microscope slide and coverslip using Vectashield mounting medium (catalog No. H-1000; Vector Laboratories, Burlingame, CA). Incubations in primary and secondary antibody were done at 4°C with gentle agitation. All rinses were done at room temperature (18–24°C) without agitation. Secondary antibody incubation was conducted in the dark, as was all subsequent processing. Likewise, slides were stored in the dark at 4°C until examined.
Antibodies
Antibodies to the vesicle-associated proteins synapsin or synaptotagmin were used as general markers for extraganglionic neuropil. This strategy was previously used to identify areas of synaptic/modulatory neuropil in the STNS of a variety of crustacean species (Christie et al. 2004a; Goaillard et al. 2004; Skiebe and Ganeshina 2000; Skiebe and Wollenschlager 2002). The synapsin antibody used was a mouse monoclonal antibody (SYNORF1; Klagges et al. 1996) generated against a glutathione S–transferase-fusion protein containing a portion of a Drosophila synapsin homologue. This antibody was used at a final concentration of 1:100. The synaptotagmin antibody used was a rabbit polyclonal antibody (DSYT-2; Littleton et al. 1993) generated against amino acid residues 134–474 of Drosophila synaptotagmin. This antibody was used at a final concentration of 1:750.
The secondary antibodies used in our experiments were donkey anti-mouse IgG conjugated to Alexa Fluor 488 (catalog No. A-21202; Molecular Probes, Eugene, OR) or Alexa Fluor 594 (catalog No. A-21203; Molecular Probes) and donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (catalog No. A-21206; Molecular Probes) or Alexa Fluor 594 (catalog No. A-21207; Molecular Probes).
Confocal and epifluorescence microscopy
Fluorescently labeled tissue was viewed and data collected using one of two Bio-Rad MRC 600 laser scanning confocal microscopes (Bio-Rad Microscience, Hemel Hempstead, UK), a Bio-Rad Radiance 2000 laser scanning confocal microscope, or a Nikon Eclipse E600 epifluorescence microscope (Tokyo, Japan). Specifications for each of the microscope systems used in this study were previously published, as were methods for figure production (Fu et al. 2005; Messinger et al. 2005).
Statistics
The SigmaStat program (version 3.1; Systat Software, Point Richmond, CA) was used for data analysis. In physiology experiments where the stimulation voltage was changed from 1 to 7 V or when a neuromodulatory substance or pharmacological agent was applied, a paired t-test was used to test for statistical significance. For experiments in which the effects of a neuroactive substance were tested for both stimulation voltages, a two-way ANOVA was used. Pairwise comparisons were then made using the Holm–Sidak method. Error bars on plots correspond to SEs.
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RESULTS |
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In the crustacean STNS, as well as in many other nervous systems, extracellular stimulation is commonly used to produce muscle contractions in neuromuscular preparations. A pin or suction electrode is used to apply voltage pulses across a nerve containing the motor axon, with each pulse eliciting an action potential when the stimulus voltage is above a threshold value. Beyond this threshold, no dependency on stimulation voltage is expected. Nerve-evoked contractions of the gm4 muscle in the crab C. borealis were previously recorded during investigations of neuromodulation of the stomatogastric musculature (Jorge-Rivera and Marder 1996, 1997; Jorge-Rivera et al. 1998). Using an experimental setup nearly identical to that in the studies mentioned earlier, we found the threshold voltage for gm4 contraction to be about 0.5–0.7 V for the 1-ms unipolar pulses used. When we applied a 12.5-Hz train of 1-V pulses, we observed a contraction (Fig. 1) that was qualitatively very similar to that described previously (Jorge-Rivera and Marder 1996, 1997; Jorge-Rivera et al. 1998). The stimulation frequency was chosen to be comparable to that of the DG neuron during a gastric mill rhythm (Beenhakker et al. 2004). The traces above the contraction show simultaneous measurements of extracellular activity in the dgn (recorded at a spot between the stimulation electrode and the muscle) and EJPs recorded intracellularly from a gm4 fiber. Also seen in the extracellular recording is tonic spiking of AGR.
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), which had not been possible when making intracellular measurements from the muscle fiber. Contraction measurements were made 1 min apart and in order of increasing voltage at different amplitudes from 0.1 to 9 V (Fig. 2B). In this experiment the threshold voltage was found to be between 0.4 and 0.5 V. As expected, the 1- and 3-V contractions were indistinguishable, and in each case the muscle relaxed fully within 1 s after the stimulus ceased. The contraction elicited by the 7-V stimulus, on the other hand, was approximately seven times larger in amplitude, and muscle tension persisted for nearly 40 s after the end of the stimulus (Fig. 2A). A plot of the peak tension for each contraction as a function of stimulus voltage is shown in Fig. 2B. The peak tension remained constant for stimulation voltages between 0.5 and 3.5 V and began to increase with the 4-V stimulus. In this experiment, the peak tension actually decreased for stimulation voltages >7 V. This, however, is not representative of other experiments and most likely reflects the fact that the mechanism underlying the enhanced contractions sometimes decreased with repeated stimulation. In many cases, however, the ability to produce enhanced contractions in response to high-voltage stimulation remained largely undiminished after 2–3 h of intermittent stimulation and outlasted the experiment itself.
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Persistent firing of the DG motor neuron contributes to the production of the enhanced contractions
To investigate whether the behavior shown in Fig. 2 originated in the muscle or the nerve, we made simultaneous measurements of contraction, dgn activity, and EJPs in response to high-voltage stimulation. Figure 3 shows recordings in response to a 1-s duration, 12.5-Hz train of 7-V pulses for the same preparation shown in Fig. 1. The simultaneous extracellular nerve recording and intracellular muscle recording demonstrate that the DG neuron fired action potentials for >20 s after the end of the stimulus, with a corresponding increase in the number of EJPs and the duration of the contraction. In this particular experiment the firing rate of AGR increased appreciably, and the expanded extracellular trace reveals two other spikes (one larger and one smaller than the AGR spike) that we assume to be GM spikes. In other experiments the firing rate of AGR remained unchanged after an extracellular stimulation that caused persistent DG spiking (data not shown).
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The bistability in DG reported by Kiehn and Harris-Warrick (1992b) was induced by current injection into the neuron's soma in the presence of serotonin. In contrast, the behavior illustrated in Figs. 1–3 was observed in preparations in which the soma was not present. To investigate whether the two mechanisms were related, we did experiments in which the connection between the dgn and the STG was preserved so that we could both record from and inject current into the DG cell body. In these experiments, the activity in the stomatogastric nerve was blocked with sucrose/TTX to prevent descending modulatory inputs from inducing the gastric rhythm. Figure 4, A–C shows simultaneous intracellular DG and extracellular dgn recordings in response to three different stimulus protocols. The response to a 5-s-duration injection of current into the soma is shown in Fig. 4A. Although the DG orthodromic instantaneous firing rate was 
20 Hz during the duration of the stimulus, no persistent firing resulted, consistent with previous results in which the induction of plateau properties from the soma required the presence of a neuromodulator (Kiehn and Harris-Warrick 1992b; Weimann et al. 1993). Figure 4B shows that an extracellular 1-V, 50-Hz stimulus depolarized the DG soma but was unable to produce persistent firing. The inset shows that each pulse produced one antidromic spike. When the same preparation was stimulated at 4.5 V, 12.5 Hz, persistent DG firing was induced (Fig. 4C). The inset shows that extra spikes were also elicited between stimulus pulses such that the firing rate was >12.5 Hz during stimulation. Figure 4D summarizes the dependency on stimulation voltage of the DG spiking in five experiments when a 12.5-Hz extracellular stimulus was used. The minimum stimulus voltage for extra spiking varied from 3.5 to 5.5 V. From the threshold up to this voltage, 13 spikes were generated, one for each pulse. In none of these five preparations did stimulation of the dgn with high-frequency (40 Hz), 1-V pulses or depolarization of DG produce persistent firing. For each of the latter measurements, sufficient current was injected to produce spiking of 20 Hz. In two experiments we hyperpolarized the soma to between –75 and –90 mV while stimulating the dgn extracellularly to see whether persistent firing could be affected. The duration of the persistent firing was shortened by 20–80% but could not be suppressed by hyperpolarization of the cell body (Fig. 4E).
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By varying the location of the extracellular stimulation electrode on the dgn, we determined that persistent firing could be induced from only a portion of the nerve. Figure 5A shows that when the electrode was relatively close (about 6 mm) to the STG, persistent firing was elicited by a 5.2-V, 12.5-Hz, 1-s stimulation. In contrast, persistent spiking could not be produced in the same preparation when the stimulation electrode was 15.5 mm away from the STG, even when the stimulus amplitude was increased to 9 V. We did a total of four experiments, and in each case we found persistent firing was never elicited beyond a distance of 10–15 mm from the STG, even by stimulation voltages more than twice as large as those used to generate the persistent firing by stimulating at a more proximal site.
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Persistent DG firing and enhanced gm4 contractions are eliminated in low-Ca2+ saline
Previous studies referenced in the INTRODUCTION identified Ca2+ as having both direct (ICa) and indirect roles (ICAN, neuromodulatory release) in the induction of bistability in many systems. If any of these mechanisms contributes to the generation of persistent DG axonal firing, we should expect the extra spikes to disappear when Ca2+ is removed from the saline. Persistent firing of DG (Fig. 6A) and enhanced contraction of gm4 (Fig. 6B) were abolished when low-Ca2+ saline was placed in the stimulation well but reappeared when the calcium was returned. Sustained firing after the end of the stimulus was entirely suppressed in each of four experiments when low-Ca2+ saline was placed in the well. However, low-Ca2+ saline did not abolish the DG spikes elicited by each pulse (data not shown).
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To investigate whether L-type Ca2+ channels and ICAN were involved in DG axonal bistability, we tested the effects of the L-type calcium channel blocker nicardipine (a dihydropyridine) (Bean 1989; Hurley and Graubard 1998) and flufenamic acid (FFA), a nonspecific Ca2+-activated current-blocking agent (Del Negro et al. 2005; Di Prisco et al. 2000; Gögelein et al. 1990; Guinamard et al. 2004; Lee et al. 1996; Mironov and Langohr 2005; Morisset and Nagy 1999), on the neuromuscular preparation. In all experiments the dgn was not desheathed and agents were applied specifically to the stimulation well, and not to the whole preparation, to prevent direct effects of the drugs on the musculature. When the stimulation electrode was bathed with nicardipine (100 µM), the enhanced contractions induced at 7 V disappeared, and the contractions induced at 1 and 7 V were identical (Fig. 7A). This effect of nicardipine was reversible. Figure 7B summarizes the results of five experiments and shows that the peak tension in control saline at 7 V was significantly different from both the peak tension in control saline at 1 V and that measured in the drug at 7 V (Holm–Sidak method, *P < 0.05). In each of these experiments, the effect of nicardipine took 20 but <35 min to develop. When FFA (100 µM) was applied to the stimulation well, enhanced gm4 contractions disappeared within 5–10 min and completely recovered on washout. A representative experiment is shown in Fig. 7C and a summary in Fig. 7D. Again, the control 7-V peak tension was significantly different from both the 1-V control value and the peak tension at 7 V in the presence of FFA (Holm–Sidak method, n = 6, **P < 0.01). Application of lower concentrations (10 µM) of either nicardipine or FFA reduced but did not completely block the enhancement of the contractions elicited at high stimulation voltages.
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The STNS in C. borealis is modulated by a host of neuroactive compounds that are released locally or circulate as hormones. Jorge-Rivera et al. showed that ten of these substances modulate nerve-evoked gm4 contractions. Two substances (histamine, allatostatin-3) decreased gm4 contraction amplitude, whereas eight [crustacean cardioactive peptide (CCAP), dopamine, octopamine, proctolin, red pigment concentrating hormone (RPCH), SDRNFLRFamide, serotonin, and TNRNFLRFamide] increased it (Jorge-Rivera and Marder 1996, 1997; Jorge-Rivera et al. 1998). Although several of the neuromodulators were shown to have subtle effects on the relaxation rate of the gm4 contraction (Jorge-Rivera et al. 1998), there is no evidence that any of them can produce persistent firing or prolonged gm4 contractions.
The three substances that induced the largest (>200%) increase in contraction amplitude in the previous studies were proctolin, serotonin, and TNRNFLRFamide (Jorge-Rivera et al. 1998). In our own experiments, we confirmed that in no case did application of one of these three modulators (10–7 M proctolin, n = 6; 10–5 M serotonin, n = 4; 10–7 M TNRNFLRFamide, n = 5) to the stimulation well and/or outside the well produce prolonged gm4 contractions in response to low (about 1 V)-voltage, 3-s, 12.5-Hz stimulation, although each modulator did increase the contraction amplitude. In the presence of each modulator, the dependency of the contraction on stimulation voltage was very similar to what had been observed under control conditions: contractions were prolonged and were further increased in amplitude at or above the voltages at which enhanced behavior had been observed under control conditions. We also assayed the effects on contraction of three modulators that had not previously been tested: CabTRP Ia (10–7 M, n = 4), GABA (10–5 M, n = 7), and orcokinin (10–7 M, n = 4). None of these triggered bistable behavior. In all experiments in which a neuromodulator was applied to the dgn, the nerve was not desheathed and was cut below the STG.
In the crustacean STNS, a number of extraganglionic neuropil regions have been identified and modulation at these sites documented. Interestingly, despite the extensive mapping of neuromodulators in the STNS of C. borealis, immunohistochemical investigations of the dgn have produced negative results in every instance. Antibodies tested include those for proctolin (Marder et al. 1986), FLRFamide (Marder et al. 1987), tachykinin-related peptide (Goldberg et al. 1988), serotonin (Katz et al. 1989), pigment-dispersing hormone (Mortin and Marder 1991), buccalin and myomodulin (Christie et al. 1994), allatostatin (Skiebe and Schneider 1994); cholecystokin (Christie et al. 1995), GABA (Swensen et al. 2000), orcokinin (Li et al. 2002), histamine (Christie et al. 2004a), and mandibular organ-inhibiting hormone (MOIH) (Hsu et al. 2004).
Recent mass spectrometric measurements revealed the existence of many previously unknown peptides in neurosecretory structures in C. borealis (Li et al. 2003). Clearly, it is possible that an untested neuromodulator could be present in the dgn and released onto the DG axon in the proximal portion of the nerve by a calcium-dependent process. As a first step toward addressing this possibility, we immunolabeled preparations consisting of either the intact STNS containing the dgn cut just anterior to its insertions onto the gm4 muscle or the dgn with the gm4 muscles attached with antibodies directed against the vesicle-associated proteins synapsin or synaptotagmin. Because these proteins are usually found ubiquitously on clear synaptic or dense-core vesicles, respectively, they have been used extensively in the crustacean STNS as markers for synaptic/neurohemal structures (Christie et al. 2004b; Goaillard et al. 2004; Skiebe and Ganeshina 2000; Skiebe and Wollenschlager 2002).
In all STNS preparations examined (n > 6), extensive synapsin labeling was seen throughout the nervous system, including extraganglionic neuropil patches in the stomatogastric and lateral ventricular nerves; however, no immunoreactivity was noted anywhere in the dgn proper in nerve–muscle preparations (n = 4) (Fig. 8, B–F). As expected, however, DG–gm4 neuromuscular junctions did show intense synapsin immunoreactivity (Fig. 8, G and H). Extensive synaptotagmin labeling was similarly observed in multiple locations throughout the nervous system in every STNS preparation examined (n > 6; data not shown); however, no labeling was noted anywhere in the dgn in nerve–muscle preparations labeled with the synaptotagmin antibody (n = 3; data not shown).
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DISCUSSION |
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Axonal bistability is dependent on familiar channels
One of the more impressive examples of bistability occurs in neurons of the rat entorhinal cortex, in which plateau potentials lasting 13 min (Egorov et al. 2002) can be induced. Plateau production was suppressed by nifedipine (a dihydropyridine) (50 µM) or FFA (10 µM), blockers of L-type Ca2+ currents and ICAN, respectively. The bistability described in our study appears to depend on similar currents.
Calcium channels in the crustacean STNS are not easily placed into vertebrate categories (Bean 1989; Ertel et al. 2000; Triggle 1999). Currents that show largely L-type behavior have been identified in a number of crustacean species (Golowasch and Marder 1992; Hurley and Graubard 1998; Johnson et al. 2003; Turrigiano et al. 1995; Zhang and Harris-Warrick 1995); however, their physiological properties and sensitivities to pharmacological or neuromodulatory agents show differences, even within the same species (Johnson et al. 2003). In the lateral pyloric (LP) neuron of C. borealis, ICa has N- or L-type kinetics but is insensitive to nifedipine (Golowasch and Marder 1992). Sensitivity to the dihydropyridines was not tested in the DG cell in C. borealis, and although the current was blocked by Cd2+, as in LP, activation occurred at somewhat lower voltages (Zhang and Harris-Warrick 1995). Our observation that bistability in the DG axon in C. borealis was completely eliminated in the presence of nicardipine (100 µM) is consistent with the pharmacological profile of calcium channels in unidentified STG neurons in Cancer productus, in which nifedipine and nicardipine were effective blockers (Hurley and Graubard 1998). The IC50 for nicardipine found in that study was 17 µM and a complete block of the Ca2+ current was not obtained at the highest concentration (about 30 µM) applied. Although physiological characterization of Ca2+ currents in the STNS has thus far been limited to regions that are accessible from the somata of motor neurons, it is interesting to note that an antibody to L-type channels has been observed to stain discontinuous patches along peripheral motor axons in the spiny lobster Panulirus interruptus (French et al. 2002). We believe that L-type–like calcium channels are also located in motor axons in C. borealis.
The Ca2+-activated nonselective cation current ICAN has been described in only one stomatogastric neuron, specifically the DG neuron in C. borealis (Zhang et al. 1995). We found that bistability in the DG axon was completely eliminated by FFA (100 µM). Sensitivity to FFA was not measured in the DG cell body, nor, to the best of our knowledge, has it been tested in any invertebrate preparation. Thus although we can only speculate about the similarities between the current in the DG axon and the one recorded in the somatodendritic region, we conclude that the axonal current is sufficiently similar to those in vertebrate preparations to be blocked by the same agent. The high FFA concentration required in our experiments may indicate that FFA is a less-effective blocker of ICAN in the DG axon than in other preparations and/or it could reflect the fact that the dgn was not desheathed during application.
The dependency on stimulation voltage of spiking and contractions in the DG/gm4 system is reminiscent of the graded response to repeated electrical stimulation in rat entorhinal cortical neurons (Egorov et al. 2002) and lamprey reticulospinal neurons (Di Prisco et al. 1997, 2000), two systems in which plateau potential production is dependent on ICAN. In the latter case, the frequency and duration of the persistent firing were correlated with intracellular calcium (Di Prisco et al. 1997, 2000), and we suggest that calcium concentration may play a similar role in our system.
Bistability without action by characterized neuromodulators
The negative results in this and previous investigations do not rule out the possibility that an as yet uncharacterized neuroactive substance mediates bistability in the DG axon. However, if an unknown modulator encapsulated in vesicles were present in the dgn, we would have expected positive immunoreactivity to synapsin and/or synaptotagmin, as has been documented in other regions of extraganglionic neuropil in the STNS (Skiebe and Ganeshina 2000; Skiebe and Wollenschlager 2002). The lack of staining for either protein in the dgn argues against the presence of synaptic contacts or extraganglionic neuropil in the nerve. We conclude that, if local release of a neuromodulator is involved in generating the bistability, it occurs in an unconventional manner.
Two distinct sources of bistability in DG
Motor neurons in the STG have a standard monopolar invertebrate structure with a single primary neurite that exits the ganglion as an axon or axons (King 1976a,b). Secondary processes branch off this neurite and into the neuropil region of the ganglion where synaptic connections are made. Spikes are generated in the primary neurite and, unlike in classic bipolar vertebrate neurons, the dendritic and axonal regions are not neatly separated by the cell body. In our experiments, persistent firing induced by extracellular stimulation originated in the axon itself at a spot approximately 0.5 cm away from the spike initiation zone for orthodromic firing.
Our results suggest that generation of the axonal plateau potential underlying the persistent firing involves a current or currents whose activation requires a sustained period of depolarization. This would explain why the bistability could or could not be induced by different stimulation protocols. When the amplitude of extracellular stimuli was large enough, the membrane potential would remain sufficiently depolarized between pulses to allow eventual activation of the current involved in the production of persistent firing. In contrast, orthodromic spiking, at least at the frequencies produced by depolarization of the soma, could never activate the current because the membrane would repolarize between the passings of each spike. The observation that hyperpolarization of the DG soma reduced persistent firing shows that the proximal portion of the axon is not electrotonically distant from the soma. This result is not surprising, given the results of a previous study in C. borealis in which hyperpolarizing current injected into the soma of the lateral gastric (LG) neuron controlled the activity of a peripheral spike initiation zone that was several cm from the STG (Meyrand et al. 1992). The likely explanation for this phenomenon in both neurons is a large axonal length constant, such that the voltage decreases very slowly with distance when propagated along the axon. The length constant (
) of the pyloric dilator (PD) cell in the lobster Homarus americanus has been measured to be approximately 2 mm (D Bucher, unpublished observations). If the length constant of the DG axon were close to this value, negative current injection into the soma would produce hyperpolarization of the axon in the spike initiation region (about 0.5 cm or nearly 2–3 from the soma) that would be 5–15% of the somatic value. For the experiment shown in Fig. 4E, the hyperpolarization at the spike initiation site would be –2 to –3 mV. This could influence the duration of the persistent firing because, at the end of the spike train, the membrane potential in that portion of the axon is probably very close to the threshold for spiking. Our inability to generate bistability at distal sites of the axon points to heterogeneity in the DG axon. It appears that only in the section closer to the STG does the axon have the complement of channels necessary for generating bistability.
Induction of bistability in both the DG soma and axon depends on a Ca2+ current with L-type properties and on ICAN; however, neuromodulatory action appears to be required only in the former case. Without a detailed biophysical model of the neuron and/or direct measurements of the axonal currents, we can only speculate as to why this is true. Clearly, the properties of the channels involved in producing these two currents and/or their densities could vary throughout the neuron. In any case, we would expect both local excitability and cellular morphology to play crucial roles in determining the ease of plateau potential production in each region of the DG neuron.
Possible physiological significance of DG axonal bistability
Crustacean axons can possess active conductances beyond those in the Hodgkin–Huxley model (Connor et al. 1977; French et al. 2002) and some axons, like vertebrate dendrites (Heckman et al. 2003; Major and Tank 2004), appear to have signal-processing capabilities. Several previous studies have demonstrated that, in the presence of a neuromodulator, axons in the STNS can modify the spike trains that are transmitted to synaptic targets (Bucher et al. 2003; Goaillard et al. 2004; Meyrand et al. 1992). The bistable behavior we observed in the DG axon did not involve a neuromodulator, but it did require a very strong extracellular stimulation of the dgn that was 5–10 times greater than that needed to elicit spikes. Given the lack of either neuromodulatory release or electrical connections between DG and other axons within the dgn, how could bistable behavior be elicited in vivo? The simplest scenario involves the assistance of a neuromodulatory substance that circulates as a hormone and facilitates activation of the current(s) underlying the plateau. This might be achieved, for example, if the neurohormone shifted the current's activation voltage downward and/or decreased the leak conductance to increase the membrane time constant. A hormonal action of dopamine has similarly been proposed as being responsible for uncovering a peripheral spike initiation zone in a STG motor axon in H. americanus (Bucher et al. 2003). Under the scenario we put forward, it seems possible that bistable behavior in the DG axon and the resulting enhanced gm4 contraction could be elicited at times by the only physiologically relevant electrical stimulus we can identify: orthodromic DG spiking generated during a gastric mill rhythm.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. T. Birmingham, Department of Physics, Santa Clara University, Santa Clara, CA 95053-0315 (E-mail: jbirmingham{at}scu.edu)
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