The Journal of Neurophysiology Vol. 80 No. 5 November 1998, pp. 2718-2726
Copyright ©1998 by the American Physiological Society

Na⁺-Dependent Neuritic Spikes Initiate Ca²⁺-Dependent Somatic Plateau Action Potentials in Insect Dorsal Paired Median Neurons

Corine Amat¹, Bruno Lapied¹, Andrew S. French², and Bernard Hue¹

¹ Laboratoire de Neurophysiologie Récepteurs et Canaux Ioniques Membranaires, Université d'Angers, F-49045 Angers Cedex, France; and ² Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Amat, Corine, Bruno Lapied, Andrew S. French, and Bernard Hue. Na⁺-dependent neuritic spikes initiate Ca²⁺-dependent somatic plateau action potentials in insect dorsal paired median neurons J. Neurophysiol. 80: 2718-2726, 1998. The origin of plateau action potentials was studied in short-term cultures of dorsal paired median (DPM) neurons dissociated from the terminal abdominal ganglion of the cockroach, Periplaneta americana. Spontaneous plateau action potentials were recorded by intracellular microelectrodes in cell bodies that had neurite stumps. These action potentials featured a fast initial depolarization followed by a plateau. However, only fast spikes of short duration were observed when the cell was hyperpolarized from the resting membrane potential. These two different components of the action potentials could be separated by applying depolarizing current pulses from a hyperpolarized holding potential. Application of 200 nM tetrodotoxin (TTX) abolished both fast and slow phases, but depolarization to the original resting potential by steady current injection triggered slow monophasic action potentials that could be blocked by 3 mM CoCl₂. In contrast, DPM neurons without neurites were not spontaneously active. In these cells, calcium-dependent slow monophasic action potentials were only recorded immediately after impalement or with current pulse stimulation. Immunocytochemical observations showed that dorsal unpaired median (DUM) neuron cell bodies, which are known to exhibit spontaneous sodium-dependent action potentials, reacted with an antibody directed against a synthetic peptide corresponding to the SP19 segment of voltage-activated sodium channels. In contrast, the antibody did not stain DPM neuron cell bodies but gave intense, patchy staining only in the neurite. Whole cell patch-clamp experiments performed on isolated DPM neuron cell bodies without a neurite revealed the presence of an inward current that did not inactivate completly within the duration of the test pulse. This current was insensitive to both 100 nM TTX and sodium-free saline. It was defined as a high-voltage-activated calcium current according to its high threshold of activation (30 mV) and its sensitivity to 1 mM CdCl₂ and 100 nM -conotoxin GVIA. Our findings demonstrate that spontaneous sodium-dependent spikes arising from the neurite are required to initiate slow somatic calcium-dependent action potentials in DPM neurons.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Neurons with different morphological characteristics and physiological roles can be further differentiated on the basis of their firing properties. Among spontaneously active bursting or spiking neurons (Connor 1985), some can be distinguished by their ability to generate complex and long duration action potentials. Intrasomatic recordings have shown that guinea pig inferior olivary neurons (Llinas and Yarom 1981a, 1986), Purkinje cells (Llinas and Sugimori 1980a), and dorsal cochlear nucleus neurons (Manis et al. 1994) are capable of generating spontaneously complex multiphasic or long duration action potentials composed of at least two clearly distinct components, a fast initial depolarization followed by a prolonged slow depolarization. In inferior olivary neurons and Purkinje neurons, the fast initial sodium-dependent depolarizing component is generated in the cell body, whereas the slow calcium-dependent depolarizing component is initiated in dendrites (Llinas and Sugimori 1980b; Llinas and Yarom 1981b). In contrast, rat neocortex pyramidal neurons (Amitai et al. 1993; Kim and Connors 1993; Stuart and Sakmann 1994) and motor cortex neurons (Pockberger 1991) generate both fast and slow components at the dendritic level.

In invertebrate neurons, spontaneous somatic electrical activity, characterized by long duration or plateau action potentials, was described in ganglionic neurons of mollusks Clione limacia (Arshavsky et al. 1986), Helix (Pin et al. 1990), Pleurobranchaea (Gillette et al. 1980), the stick insect Carausius morosus (Orchard and Finlayson 1977), the silkworm Bombyx mori (Miyazaki 1980), the moth Manduca sexta (Carrow et al. 1984), and the larvae lepidopteran Antheraea pernyi (Brookes and Weevers 1988). However, there are no data describing the origin of the inflection on the falling phase of these action potentials.

In the cockroach CNS, four bilaterally paired midline dorsal neurons generating plateau action potentials were recently identified as being part of the dorsal paired median (DPM) neuron group (Amat and Hue 1996). Two of these neurons are located in the central cluster of the terminal abdominal ganglion (TAG) dorsal median cell bodies and two in the anterior cluster. These DPM neurons have been shown to be proctolinergic (Amat et al. 1997). Each DPM neuron cell body, located near the dorsal midline, sends a single axon through the ipsilateral anterior proctodeal nerve to innervate the hindgut. The axon arborizes extensively close to the soma and in the posterior part of the TAG. These four DPM neurons are capable of generating, in situ, spontaneously rhythmic patterns of plateau action potentials at low firing frequency. The plateau action potentials recorded in the soma are composed of a fast initial spike followed by a slow depolarizing phase. However, the exact origin of the biphasic behavior of plateau action potentials (i.e., the localization of ion channels playing a role in the control of the voltage responses that regulate action potential initiation and conduction), the ionic mechanisms underlying each phase, and their respective physiological roles in the generation of spontaneous electrical activity are unknown. Therefore understanding the function of these neurons requires knowledge of the ionic basis of the action potentials and localization of the ion channels to determine where the action potentials are initiated and how they propagate within the neurons. In this study, the complementary approaches of electrophysiology (intracellular microelectrode and patch-clamp techniques) and immunocytochemistry were used to demonstrate that spontaneous neuritic sodium-dependent electrical activity is essential for initiating slow calcium-dependent somatic action potentials.

	METHODS

Abstract Introduction Methods Results Discussion References

Preparation

Adult male cockroaches, Periplaneta americana, were taken from our laboratory colonies, which were maintained under standard conditions (29°C, photoperiod of 12 h light:12 h dark). We performed experiments on neurons isolated from the central (i.e., median) cluster of dorsal cell somata of the TAG of the abdominal nerve cord.

Cell isolation

Separation of adult DPM neuron cell bodies was performed under sterile conditions according to a modified version of the technique developed by Lapied et al. (1989). Briefly, 10 TAGs were excised and dissected to collect only the central parts, which were incubated for 30 or 40 min at 37°C in cockroach saline of the following composition (in mM): 210 NaCl, 3.1 KCl, 5 CaCl₂, 50 sucrose, 10 N-2-hydroxymethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4, containing collagenase (2 mg/ml, type 1 Worthington Biochemical Corporation, Freehold, NJ) and hyaluronidase (2 mg/ml, ICN, Aurora, OH). The median parts of TAG were rinsed in saline without enzymes and mechanically dissociated by repetitive gentle suctions through fire-polished Pasteur pipettes. Dissociated neurons suspended in saline supplemented with fetal calf serum (5% by volume, GIBCO-BRL, Life Technologies, Cergy-Pontoise, France), 50 IU/ml penicillin, and 50 µg/ml streptomycin (GIBCO-BRL, Eragny, France) were allowed to settle onto poly-D-lysine (hydrobromide, MW 70,000-150,000, Sigma Chemicals, L'Isle D'Abeau Chesnes, France) coated tissue culture dishes (Falcon, AES Laboratory, Combourg, France).

Electrophysiology

ACTION POTENTIAL INTRACELLULAR RECORDINGS. All experiments were performed exclusively on DPM neurons, isolated from the central cluster of TAG dorsal neurons, generating plateau action potentials. The electrical activity of DPM neurons was recorded with conventional intracellular microelectrodes (borosilicate capillary tubes, tip resistance 40-60 M, when filled with a solution containing 1/3 of 1 M potassium acetate and 2/3 of 1 M potassium chloride) connected to a VF-180 microelectrode amplifier with current injection capability (Biologic, Claix, France). The electrical activity was displayed on a digital oscilloscope (310, Nicolet Instrument, Madison, WI) and stored on a digital tape recorder (DTR 1202, Biologic) for off-line analysis. Tetrodotoxin (TTX) and cobalt chloride (CoCl₂) were added to the normal saline (described previously) at final concentrations of 200 nM and 3 mM, respectively.

WHOLE CELL CURRENT RECORDINGS. The patch-clamp technique was used in the whole cell configuration (Hamill et al. 1981) to record calcium currents. Patch electrodes were pulled from borosilicate capillary tubes (Clark Electromedical Instruments, Reading, UK) with a Narishige puller and had resistances ranging from 1.8 to 2 M when filled with the pipette solution. Signals were recorded with a RK 300 patch-cell-clamp amplifier (Biologic, Claix, France) and filtered at 3 kHz. The liquid junction potential between the pipette and the superfusing solution was always corrected before formation of a gigaohm seal (5 G). Command potentials were generated by a programmable stimulator (SMP 310, Biologic) or with a PC computer with software control pClamp (version 5.5.1, Axon Instruments, Foster City, CA) connected to a 125-kHz Labmaster DMA data acquisition system (TL-1-125 interface, Axon Instruments). Although most of the capacitance and leak current were electronically compensated at the beginning of each experiment, subtraction of residual capacitance and leak current was performed with an on-line P/4 protocol provided by pClamp. Cells were clamped at a holding potential of 100 mV, and 45-ms test pulses were applied from the holding potential (increments 10 mV) at a frequency of 0.25 Hz. The series resistance value for each experiment (ranging between 3 and 5 M) was obtained from the patch amplifier settings after compensation. Data were displayed on a digital oscilloscope (310, Nicolet Instrument) and stored on the hard disk of the computer (sampling frequency 10 kHz) for off-line analysis.

The solutions used to record whole cell inward currents were designed to eliminate interference from outward potassium currents by the combination of external tetraethylammonium chloride (TEA-Cl) and 4-aminopyridine (4-AP) and by isotonically substituting potassium with cesium in the patch electrode. For these experiments the saline superfusing the cells contained (in mM) 100 NaCl, 3.1 KCl, 4 MgCl₂, 5 CaCl₂, 100 TEA-Cl, 5 4-AP, and 10 HEPES; pH was adjusted to 7.4 with TEA-OH. To demonstrate calcium dependence of the whole cell inward current, all NaCl was replaced with choline chloride, and 100 nM TTX was added to the saline. When necessary, cadmium chloride (CdCl₂) and -conotoxin GVIA were added to the superfusing solution. Patch electrodes were filled with an internal solution containing (in mM) 145 CsCl, 10 CsF, 10 NaCl, 0.5 CaCl₂, 10 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 3 ATP-Mg, 0.2 GTP-Na₂, and 20 HEPES; pH value was adjusted to 7.4 with CsOH. An additional 10 mM phosphocreatine di-Tris was added to this solution immediately before use to minimize rundown of calcium current. All compounds were purchased from Sigma Chemicals except -conotoxin GVIA (Research Biomedical International, Natick, MA).

Experiments were carried out at room temperature (20°C) and results when quantified were expressed as means ± SE.

Immunocytochemistry

The procedures used to obtain the antibodies and test their specificity were described before (French et al. 1993). For light microscope immunocytochemistry, isolated neurons were fixed for 1 h in 4% paraformaldehyde containing 5% (wt/vol) sucrose in phosphate-buffered saline (PBS). To block nonspecific binding of the primary antibody, isolated neurons were treated with 4% bovine serum albumin (BSA) in PBS for 1 h. Primary antiserum, diluted 1:100 in PBS, was applied for 1.5 h. After repeated washing in PBS, the secondary antibody (FITC-labeled swine anti-rabbit IgG, Dakopatts, a/s, Glostrup, Denmark, 1/30 in PBS containing 1% BSA) was applied for 1.5 h in the dark. Isolated neurons were washed in 4% BSA in PBS and mounted on glass slides in glycerol-PBS (70%). Control preparations were treated identically, except that the rabbit antiserum was replaced with preimmune serum from the same rabbit or PBS. Preparations were viewed and photographed through an Olympus compound microscope with an epifluorescence system.

	RESULTS

Abstract Introduction Methods Results Discussion References

Origin of the biphasic spontaneous plateau action potentials in dissociated adult DPM neurons

The first step was to obtain isolated DPM neuron cell bodies and compare their electrophysiological properties with those previously described in situ in the same preparation (Amat and Hue 1996). The diameters of spherical DPM cell bodies ranged from 38 to 45 µm. As illustrated in Fig. 1, A1 and D1, two different morphological forms of DPM neurons could be seen after dissociation, depending on the duration of enzymatic treatment. When the median parts of the TAG were treated for 40 min, the spherical somata of DPM neurons had neurite stumps whose length was approximately twice the soma diameter and that sometimes exhibited short, fine processes (Fig. 1A1). In contrast, incubation for 30 min produced only round somata without neurites or other processes (Fig. 1D1).

View larger version (32K): [in this window] [in a new window]   FIG. 1. Morphological and electrophysiological characteristics of isolated dorsal paired median (DPM) neurons generating plateau action potentials. A1: light photomicrograph of DPM neuron cell body (40-µm diam) isolated from the central part of the terminal abdominal ganglion after enzymatic treatment for 40 min. A thin neurite (arrow) exhibiting fine processes emerged from the soma. A2: spontaneous electrical activity recorded in this DPM soma. B1: spontaneous plateau action potential recorded in DPM soma having a neurite stump (resting membrane potential 45 mV). B2: spontaneous electrical activity recorded when the cell was hyperpolarized (17 mV). The spontaneous electrical activity consisted of a fast and short action potential (~5 ms in duration). B3: superimposed traces of 2 preceding action potentials, revealing a similar time course for both depolarizing phases. C1: spontaneous plateau action potential recorded from a DPM soma with a neurite stump (resting membrane potential 45 mV). C2 and C3: action potentials produced by current stimulation (arrowheads: beginning of stimulations, 0.8 nA, 300 ms in duration) when the membrane is hyperpolarized. C2: hyperpolarization (15 mV) revealed the biphasic properties of the plateau action potentials (i.e., fast initial phase followed by a slow depolarization). C3: 20-mV hyperpolarization induced a complex multiphasic response composed of both fast and slow depolarizing phases. D1: light photomicrograph of DPM neuron soma without neuritic process (42-µm diam) obtained after enzymatic treatment for 30 min. D2: slow monophasic action potential recorded immediately after impalement. D3: superimposed traces of action potentials shown in C1 and D2. Note that the time course of the depolarizing phase was slower than that of the plateau action potential illustrated in C1 (1.6 V.s1 for AP in D2 and 3.9 V.s1 for AP in C1). Scale bar for A1 and D1: 20 µm.

To investigate the origin of the biphasic shape of the action potentials, electrophysiological experiments were performed on isolated DPM neurons from both dissociation methods. Values of the resting membrane potential in DPM neurons possessing a thin neurite were 47.4 ± 2.9 mV (n = 9). As shown in Fig. 1, A2, B1, and C1, these neurons were capable of generating spontaneous plateau action potentials of 77.1 ± 2.4 mV amplitude (n = 9, measured between maximum voltage excursions) with a rapid depolarizing phase (see Table 1). These action potentials corresponding to plateau action potentials were characterized by a duration of 62.4 ± 3.8 ms (n = 9, measured at 50% of the total action potential amplitude). The repetitive firing was always characterized by a regular discharge (2.0 ± 0.2 Hz, n = 7, Fig. 1A2). These electrophysiological properties were similar to those previously recorded from DPM neurons in situ (Amat and Hue 1996).

When the membrane was hyperpolarized (17 mV more negative than the resting membrane potential) by injecting steady current, spontaneous spikes of short duration (<5 ms) devoid of the plateau phase were observed (Fig. 1B2). Spontaneous action potentials occurring at the normal resting potential had a rate of rise similar to that of fast spikes evoked from hyperpolarized membrane potentials (see Table 1 and Fig. 1B3). As shown in Fig. 1C, similar results were obtained by using current stimulation of DPM neurons (0.8 nA, 300 ms in duration). In these experimental conditions, when the membrane was hyperpolarized (15 mV more negative than the resting membrane potential) by applying a steady current a notch appeared on the rising phase, indicating that at least two distinct components were involved, a fast depolarization followed by a slower one (Fig. 1C2). Further hyperpolarization of the membrane (20 mV more negative than the resting membrane potential) allowed better separation of the two components (Fig. 1C3). This complex firing was composed of a fast, short duration initial spike followed by a second action potential that was roughly similar to the plateau action potential illustrated in Fig. 1C2.

DPM neuron cell bodies without neurites (Fig. 1D1) had more depolarized resting membrane potentials than did neurons with attached neurite stumps (31.3 ± 1.6 mV, n = 7) and also exhibited different firing properties. Although it was occasionally possible to record spontaneous slow monophasic action potentials 5 s after the depolarization caused by impalement with the microelectrode (Fig. 1D2), depolarizing current pulses were usually necessary to produce slow action potentials. These slow action potentials 1) had lower amplitudes (62.3 ± 4.5 mV, n = 7), 2) were slightly longer in duration (65.3 ± 6.4 ms, n = 7), and 3) had a slower rising phase than did the plateau action potentials recorded in somata with neurite stumps (Figs. 1, D2 and D3, and Table 1).

View larger version (12K): [in this window] [in a new window]   FIG. 2. Sensitivity of the plateau action potential to tetrodotoxin (TTX) and CoCl2 in a representative DPM neuron exhibiting a neurite stump. A: superimposed action potentials evoked in response to depolarizing current steps (1.5 nA, 220-ms duration) in control solution () and in 200 nM TTX () showing block of plateau action potential. The membrane was hyperpolarized (20 mV). When the membrane potential was returned to its initial resting value, a long duration action potential with a slow rising phase (B) was observed in response to depolarizing current step (1.3 nA, 220-ms duration). This slow action potential was completely blocked when 3 mM CoCl2 was added to the saline containing 200 nM TTX (C).

Ionic species underlying the different components of the plateau action potential

To determine the ionic species underlying the fast and slow components, the effects of the sodium and calcium channel blockers TTX and CoCl₂, respectively, were tested on the electrical activity of isolated DPM neurons (Fig. 2). As illustrated in Fig. 2A, bath application of 200 nM TTX blocked both the fast and slow components of the plateau action potentials evoked by application of a depolarizing current pulse (1.5 nA, 220 ms in duration) when the membrane was hyperpolarized to between 65 and 70 mV. Although spontaneous electrical activity was not observed when the membrane potential was returned to its initial resting value, application of a depolarizing current pulse (1.3 nA, 220 ms in duration) elicited only a slow action potential (Fig. 2B), indicating that TTX suppression of the fast spike caused the disappearance of the slow depolarization. It is interesting to mention that the rate of rise of action potentials recorded under these experimental conditions was very similar to that calculated for action potentials recorded in DPM neurons without neurites (Table 1). These slow action potentials progressively disappeared when the calcium channel blocker CoCl₂ (3 mM) was added to normal saline containing 200 nM TTX. This suggests that calcium ions were involved in the slow depolarizing component (Fig. 2C).

These results demonstrate that the spontaneous plateau action potential is composed of a fast sodium-dependent component that leads to a slow calcium-dependent component. They also suggest a possible different localization of the sodium and calcium channels underlying the biphasic plateau action potential because the slow calcium-dependent depolarizing component was observed in DPM neuron cell bodies either with or without a neurite stump, whereas the sodium-dependent spike was observed only in DPM neurons having a neurite stump. To further substantiate this hypothesis, we used an immunocytochemical method to look for sodium channels in DPM neuron cell bodies and neuritic membranes.

Immunostaining of voltage-dependent sodium channels in cockroach neurons

The antibody was raised against a synthetic peptide corresponding to the highly conserved SP19 segment of the rat brain type I voltage-dependent sodium channel -subunit (Noda et al. 1986). SP19 antibodies have been shown to recognize sodium channel proteins in various vertebrate and invertebrate tissues and organs such as rat brain, heart and skeletal muscle, eel brain and electroplax (Gordon et al. 1988), spider mechanosensory organs (Seyfarth et al. 1995), and insect CNSs from locust (Gordon et al. 1988), cockroach (French et al. 1993; Gordon et al. 1990), grasshopper, fly, and moth (Gordon et al. 1990). In this study, isolated dorsal unpaired median (DUM) neurons were used as a positive control because previous electrophysiological studies reported that isolated DUM neurons preserved their ability to generate spontaneous sodium-dependent action potentials (Lapied et al. 1989,1994).

As illustrated in Fig. 3B, DUM neuron somata devoid of neurites reacted positively with the SP19 antibody (yellow fluorescence). Light microscopy revealed that sodium channels were heterogeneously distributed in DUM neuron cell bodies. SP19 staining with a granular appearance was most intense in the basal region of the soma close to the initial segment. The intensity of staining gradually decreased from this region to the apical pole of the soma, which contained no significant SP19 immunoreactivity. In contrast, spherical DPM neuron somata treated with SP19 antiserum did not exhibit immunoreactivity (Fig. 3C). In this case, SP19 staining was not observed above the green background fluorescence, comparable with control preparations (DUM neurons) in which SP19 antibody was omitted (Fig. 3A). By contrast, the intense staining was only restricted at the neuritic level (Fig. 3D). This intense immunoreactivity was patchy, with some areas showing very little or no staining. These immunocytochemical results supported the electrophysiological data, indicating that voltage-dependent sodium channels were exclusively located on neurites and that DPM neurons without neurites did not express voltage-dependent sodium channels. To reinforce this hypothesis, whole cell patch-clamp experiments were performed, in the voltage-clamp mode, to identify the ionic currents present in isolated DPM neuron cell bodies without neurites.

View larger version (38K): [in this window] [in a new window]   FIG. 3. SP19 immunostaining in short-term cultured cockroach neurons. A: control preparation (isolated DUM neuron) where the antiserum was omitted. Faint green background fluorescence was observed in DUM neuron. B: DUM neuron cell bodies treated with SP19 antibody showed yellow fluorescence. The staining was most intense in the basal region close to the initial segment. C and D: isolated DPM neuron without neurite (C) and exhibiting neuritic process (D) treated with SP 19 antibody. Cell bodies did not exhibit SP19 staining above background fluorescence, whereas intense and patchy immunoreactivity was observed on neurite (D). Scale bars: 20 µm.

Identification of the inward current present in isolated DPM neuron cell bodies without neurites

By using an extracellular saline containing potassium channel blockers (100 mM TEA-Cl and 5 mM 4-AP), an inward current was recorded in response to various depolarizing pulses from a holding potential of 100 mV. Fig. 4A1 illustrates a typical example of this slow inward current evoked by a test pulse of +40 mV. It should be noted that this current did not inactivate completely within the total duration of the test pulse (45 ms).

View larger version (29K): [in this window] [in a new window]   FIG. 4. Whole cell high-voltage-activated (HVA) calcium current in isolated cell bodies of DPM neurons without neurites. A: representative example of inward current elicited by a 45-ms depolarizing voltage step to +40 mV from a holding potential of 100 mV. A1: recordings obtained with a saline containing only potassium channel blockers. Note that the current did not inactivate completely at the end of the pulse. A2: traces recorded in presence of sodium-free saline and 100 nM TTX. B: current-voltage relationships of both peak current (closed circle) and maintained current (closed triangle) amplitudes plotted as a function of test potentials (n = 3). Vertical bars representing means ± SE were shown when larger than symbols. C and D: effects of calcium channel blockers on HVA calcium current. C: blocking action of CdCl2 (1 mM) on calcium current elicited by a 45-ms depolarizing step to +10 mV from a holding potential of 100 mV. D: effect of -conotoxin GVIA (CgTx, 100 nM) on calcium current elicited by a 45-ms depolarizing step to +20 mV from a holding potential of 100 mV. E: histogram representing percentage of remaining peak (open rectangle) and maintained (oblique line rectangle) HVA calcium current amplitudes vs. duration of application of 100 nM -conotoxin GVIA. HVA calcium currents were evoked by a 45-ms test pulses to +20 mV from a holding potential of 100 mV. Scales for A, C, and D are indicated in A.

To determine if the inward current was carried by sodium ions, a sodium-free saline containing 100 nM TTX was used. Superfusion of isolated DPM neuron cell body with this solution did not affect the amplitude of the peak and maintained currents at any potentials tested, indicating that the inward current recorded in Fig. 4A1 was sodium independent and insensitive to TTX (Fig. 4A2). According to our experimental conditions this current was then identified as calcium dependent. Both the peak and maintained current amplitudes of this calcium current were plotted versus membrane potential (Fig. 4B). The calcium current activated at a membrane potential of approximately 30 mV and increased to reach a maximum of 3.11 ± 0.31 nA at +20 mV (n = 3). On the basis of this positive activation threshold, the current was defined as a high-voltage-activated (HVA) calcium current. To provide further evidence for the calcium dependence of this current, specific calcium channel blockers were used. As illustrated in Fig. 4D the inorganic blocker cadmium chloride (CdCl₂, 1 mM) completely abolished the current. Furthermore, the -conotoxin GVIA (100 nM), known to block selectively the N-type calcium channels in vertebrates (De Waard et al. 1996), blocked >90% of the current induced by a test pulse of +20 mV from a holding potential of 100 mV (Fig. 4D). The sensitivities of both peak and maintained HVA calcium currents versus the duration of -conotoxin application are illustrated in Fig. 4E.

As expected from the immunocytochemical experiments, these results confirmed that the somata of isolated DPM cell bodies did not express voltage-dependent sodium current. Furthermore, the positive threshold for activation indicates that this somatic HVA calcium current could be involved in the generation of the slow action potential recorded in DPM cell bodies without neurites.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

We found that isolated DPM neurons are capable of generating spontaneously rhythmic patterns of biphasic plateau action potentials composed of a fast initial phase followed by a slow depolarization. These DPM neurons can be well distinguished electrophysiologically from other insect neurons such as interneurons and motoneurons, which are electrically quiet, or DUM neurons, which generate spontaneous short duration action potentials (Lapied et al. 1994). Furthermore, these electrical patterns do not correspond to the "plateau potentials" previously recorded in some invertebrate and vertebrate neuronal preparations, which were always observed under artificial conditions, (i.e., electrical stimulation) or pretreatment with octopamine (Ramirez and Pearson 1991). Studies on the fast coxal depressor motoneuron (Df) of the cockroach (Hancox and Pitman 1991) and both interneurons and motoneurons of the locust (Ramirez and Pearson 1991) revealed that some insect neurons were able to generate plateau potentials. These plateau potentials were defined by several criteria (Pitman et al. 1993); 1) the duration of the plateau potentials was always longer than the duration of plateau action potentials, 2) they were induced by artificial membrane depolarization (current injection or synaptic stimulation) when the membrane was previously depolarized and were far longer than the triggering stimulus, and 3) these responses could be terminated by a brief hyperpolarizing pulse. Furthermore, plateau potentials in the cockroach motoneuron were calcium dependent and could drive bursts of axonal action potentials, as already observed in both locust and cockroach neurons (Hancox and Pitman 1991; Ramirez and Pearson 1991). From these observations, it is clear that the electrical activity recorded in isolated or in situ DPM neurons (Amat and Hue 1996; Amat et al. 1997) was different from that of Df because each DPM neuron action potential was preceded by a slowly rising phase that led to automatic activity, and this activity was observed under control physiological conditions.

The short-term culture procedure developed here, together with electrophysiology and immunocytochemistry, allowed us to demonstrate for the first time in an insect preparation that calcium-dependent plateau action potentials recorded in the somata are triggered by neuritic sodium-dependent spikes. The somatic calcium-dependent action potentials of DPM neurons also reported in other invertebrate neurons such as Helix U cells (Lux and Hofmeier 1982) or LGC cells in the leech (Johansen et al. 1987) could be separated from biphasic action potentials in DPM neurons having a neurite by treatment with TTX. Under these conditions, a slow calcium-dependent action potential remained, with similar time course to the slow action potential recorded in DPM neurons without neurites. Voltage-clamp experiments allowed us to demonstrate the existence of a HVA calcium current sensitive to cadmium and -conotoxin GVIA. Although different types of calcium currents were already characterized or suspected within other insect neuron cell bodies (Bickmeyer et al. 1994; Byerly and Leung 1988; David and Pitman 1995; Grolleau and Lapied 1996; Mills and Pitman 1997; Pearson et al. 1993), our results seem to indicate that only one type of calcium current is expressed in DPM neuron cell bodies. Indeed there was no evidence of any component of calcium current that could be activated by low voltage (e.g., low voltage-activated calcium current), similar to that known to be involved in the generation of predepolarizing phases in vertebrate as well as invertebrate pacemaker neurons (Bertolino and Llinas 1992; Grolleau and Lapied 1996). From our results it is tempting to suggest that the HVA calcium current was involved in the generation of the slow depolarizing phase of plateau action potential recorded in DPM neuron exhibiting a neurite and/or the slow calcium-dependent phase of action potential recorded in DPM neuron pretreated with TTX.

Neuritic distribution of voltage-dependent sodium channels

Sodium channels in insect nervous system were previously localized by radiolabeled toxins (Gordon et al. 1992; Moskowitz et al. 1991) and immunocytochemistry with antibodies raised against specific peptide corresponding to regions of the sodium channel sequence, which allowed the localization of sodium channels in intact insect neuronal tissue to soma and neurite. These channels were identified in a large pool in the soma, but the density of staining was higher in the neurite and axon than in cell bodies (French et al. 1993; Seyfarth et al. 1995). This study describes the first immunocytochemical distribution of voltage-dependent sodium channels, involved in the generation of neuritic spikes, exclusively on the neurite of an insect neuron. It should be noted that the neurite exhibited punctate regions of immunolabel, reflecting a localized increase in the density of sodium channels. Such patchy distributions were described in other invertebrate (Johnston et al. 1996) as well as vertebrate neurons (Turner et al. 1994). Although the physiological significance of this clustering of sodium channels in the membranes of neurites is unknown, it was suggested that, even in the absence of myelin, clustering may be a way of distributing sodium channels to optimize action potential conduction and that this sodium channel clustering may be a general property of many unmyelinated axons (Johnston et al. 1996; Turner et al. 1994).

Action potential initiating zone

Our results also indicate that the initiation site for spontaneous activity is preferentially situated at the neuritic level for the following reasons; 1) immunocytochemistry failed to stain voltage-dependent sodium channels at the somatic level but did stain them on the neurites, 2) DPM neurons without neurites were never spontaneously active and generated only a slow calcium-dependent action potential after electrical stimulation, and 3) voltage-clamp experiments confirmed the absence of any somatic voltage-dependent sodium current. This indicates that the initiation site for spontaneous activity is preferentially located at the neuritic level.

Previous studies in the X-organ of the crayfish also reported the control of spontaneous or evoked somatic electrical activity by neuritic sodium-dependent spikes. After proximal axotomy (<150 µm from the soma), only a high-threshold somatic slow calcium response could be elicited instead of a sodium/calcium action potential recorded in situ, suggesting that the spike activity was generated at the initial segment of the axon (Onetti et al. 1990). Similarly, intrasomatic recordings of evoked action potentials due to an initiation site remote from the soma were demonstrated in the snail H. aspersa (Zecevic 1996), and the spontaneous electrical response recorded in the lobster mechanoreceptor neuron cell body is due to decremental conduction of depolarization from the dendrites (Combes et al. 1993).

The origin of the spontaneous electrical activity characterized by long-lasting action potentials in invertebrates was not determined, although some studies in mollusk (Arsharsky et al. 1986; Gillette et al. 1980) suggested that cell bodies preserved the capacity to generate similar spontaneous activity when isolated. However, it should be noted that these isolated neurons possessed large parts of their neurites and that application of a constant depolarizing current was often necessary to maintain this spontaneous activity. On the basis of this literature it is difficult to decide the origin of the spontaneous activity and the inflection of the falling phase recorded in these neurons. In mammalian neurons, several studies have shown that the site of action potential initiation is in the axon (for review see Stuart et al. 1997). However, Turner et al. (1991) unexpectedly demonstrated that the site for sodium-dependent spike initiation in rat pyramidal cells is extremely labile and can shift between somatic and dendritic locations. In mammals, calcium channels were rather located on dendrites and sodium channels both on dendrites and soma membranes (Bernardo et al. 1982; Huguenard et al. 1989; Jaffe et al. 1992; Kim and Connors 1993; Magee and Johnston 1995; Regehr et al. 1992; Wong et al. 1979). Although these results generally suggest both somatic and neuritic distributions of sodium channels, our findings clearly demonstrate that DPM neuron cell bodies are devoid of sodium channels and that neuritic sodium channels underlying the spontaneous spikes are essential for triggering spontaneous somatic calcium-dependent action potentials.

Functional role of neuritic sodium spikes and somatic calcium spikes

In contrast with most of insect neurons with nonspiking somata (Burrow 1996), DPM neurons as well as DUM neurons appeared to be rare exceptions as they were capable of generating somatic action potentials. In our case these results raise questions about the possible functional roles of neuritic and somatic action potentials. Neuritic spikes in DPM neurons retrogradely conducted to the soma might act as a booster potential to activate somatic calcium-dependent action potentials. Such retrograde conduction, initiating soma depolarization, was found in pyramidal neurons (Spencer et al. 1961; Turner et al. 1994). The back-propagating spike would provide a retrograde signal to the soma indicating the level of neuronal output. Although the physiological role of the electrical activity in the soma of DPM neurons is unknown, several hypotheses can be suggested. For instance, in insect DUM neurons, Hoyle and Dagan (1978) suggested that action potentials were needed to mobilize transmitter substance in the soma and/or stimulate its manufacture. Furthermore, it was recently reported that stimulation of a quiet insect neuron induced tonic firing composed of plateau action potentials, resulting in the release of its endogenous stored product (Ewer et al. 1997). From other studies (Ewer et al. 1997; Orchard and Finlayson 1977; Pin et al. 1990) it appears that long duration action potentials may cause a prolonged and massive release of neurosecretory material from a single impulse. Because DPM neurons were recently identified as proctolinergic and are thought to modulate proctodeum activity (Amat and Hue 1996; Amat et al. 1997), a similar physiological role could be hypothesized for the plateau action potentials recorded in these neurons.

	ACKNOWLEDGEMENTS

  We thank P. Torkkeli for critical reading of the manuscript and M. Fuentes for typing the manuscript.

  C. Amat was supported by a doctoral fellowship from Region Pays de la Loire. A. S. French was supported by the Medical Research Council of Canada.

	FOOTNOTES

  Address for reprint requests: B. Hue, Laboratoire de Neurophysiologie (RCIM), Université d'Angers, rue haute de reculée, F-49045 Angers, Cedex, France.

  Received 26 January 1998; accepted in final form 27 July 1998.

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