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1Department of Zoology, University of Otago, Dunedin, New Zealand; 2Arizona Research Laboratories Division of Neurobiology, University of Arizona, Tucson, Arizona; and 3Zoologisches Institut II, Lehrstuhl Tierphysiologie, Universität zu Köln, Germany
Submitted 6 October 2004; accepted in final form 9 November 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Oland et al. 1996). Here, we examine intrinsic properties that contribute to the characteristic form of electrical activity apparent in these developing neurons: their ability to generate prolonged membrane depolarizations or plateau potentials.
In holometabolous insects such as moths, the architecture of the ALs changes dramatically during metamorphic development. Manduca larvae hatch from eggs and pass through five larval instars before undergoing metamorphosis from larva to pupa to adult. Pupal development can be divided into 18 stages, each of which lasts 
1 day (Sanes and Hildebrand 1976a, b; Tolbert et al. 1983). At the onset of pupal development (pupal stages 1 and 2; Sanes and Hildebrand 1976a, b), olfactory receptor cells are born in the antennal epithelium. These primary sensory afferent neurons extend axons toward the ALs of the brain, where they trigger the formation, late in pupal stage 3, of subunits of synaptic neuropil called glomeruli (Hildebrand 1985; Tolbert et al. 1983). The glomeruli develop in a lateral-to-medial wave that crosses the AL neuropil (Malun et al. 1994). Each glomerulus is invaded by local interneurons, the processes of which are restricted to the AL neuropil, and by AL projection (output) neurons, which transfer information from the AL to higher-order centers, such as the mushroom bodies of the brain. Centrifugal neurons contribute also to the complex, highly structured glomerular neuropil of the ALs. A large serotonin-immunoreactive neuron enters the developing AL around pupal stage 6 and sends a dense tuft of arbors into each glomerulus (Kent et al. 1987; Oland et al. 1995). Previous reports have shown that serotonin (5HT) affects the growth (Mercer et al. 1996a) as well as the excitability (Kloppenburg and Heinbockel 2000; Kloppenburg and Hildebrand 1995; Kloppenburg et al. 1999; Mercer et al. 1995, 1996b) of Manduca AL neurons.
Rapid changes in AL morphology coincide temporally with changes in the electrophysiological properties and response characteristics of AL neurons (Mercer and Hildebrand 2002a, b). Action potentials in neurons from Manduca ALs early in metamorphosis are generally small in amplitude, long in duration, and Ca2+ dependent, but as development proceeds, they become larger in amplitude, shorter in duration, and increasingly Na+ dependent. Developmental changes in voltage-gated and Ca2+-dependent ionic currents contribute to the emergence of cell type–specific response characteristics in the cells (Mercer and Hildebrand 2002a, b).
During metamorphosis, including critical stages of glomerulus formation, electrical activity can be detected in antennal nerve (sensory afferent) fibers and in AL neurons of the moth (Mercer and Hildebrand 2002a; Oland et al. 1996). In AL neurons, this activity is characterized by prolonged membrane depolarizations that resemble plateau potentials (Mercer and Hildebrand 2002a). Here we confirm the presence of plateau potential–generating mechanisms in Manduca AL neurons and show that the formation and maintenance of plateau potentials in developing AL neurons of the moth is Ca2+ dependent.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Animals were reared on an artificial diet (modified from that of Bell and Joachim 1976) and maintained at 25°C and 50–60% relative humidity under a long-day photoperiod regimen (17-h light/7-h dark). AL neurons were either dispersed and maintained in culture or examined in situ using semi-intact brain preparations. The majority of cells examined in this study were from pupae at developmental stages 6–12 (n = 86), but small numbers of cells were also examined from early (stage 4; n = 5) and late (stages 14–16, n = 4) stages of metamorphosis.
AL neurons in vitro
Cells were maintained in vitro according to methods described previously by Hayashi and Hildebrand (1990). Brains removed from cold-anesthetized pupae were placed into sterile culture saline containing (in mM) 149.9 NaCl, 3 KCl, 3 CaCl2, 0.5 MgCl2, 10 TES [N-tris-(hydroxymethyl)-methyl-2-aminoethane sulfonic acid], and 11 D-glucose and 6.5 g/l lactalbumin hydrolysate (GIBCO), 5 g/l TC Yeastolate (DIFCO), 10% fetal bovine serum (FBS, Hyclone), 100 units/ml penicillin, and 100 µg/ml streptomycin, and adjusted to pH 7 and 360 mOsm. ALs were dissected from moth brains and transferred into Hanks' Ca2+- and Mg2+-free buffered salt solution (GIBCO) containing 0.5 mg/ml collagenase (Worthington) and 2 mg/ml Dispase (Boehringer Mannheim) for 2 min at 37°C to dissociate the tissue, which was dispersed by trituration with a fire-polished Pasteur pipette. Enzyme treatment was terminated by centrifuging cells, first through 6 ml of culture-saline solution and then through the same volume of culture medium (see Culture medium). Dissociated cells were allowed to settle and adhere to the surface of culture dishes coated with Concanavalin A (200 µg/ml, Sigma) and laminin (2 µg/ml, Collaborative Research). The dishes were placed in a humidified incubator at 26°C, and the cells were maintained for a minimum of 5 days and a maximum of 7 days in culture.
Culture medium
The following were added to 500 ml of Leibovitz's L15 medium (GIBCO): 10% FBS, 185 mg 
-ketoglutaric acid, 200 mg fructose, 350 mg glucose, 335 mg malic acid, 30 mg succinic acid, 1.4 g TC Yeastolate, 1.4 g lactalbumin hydrolysate, 0.01 mg niacin, 30 mg imidazole, 100 µg/ml streptomycin, 100 units/ml penicillin, 1 µg/ml 20-hydroxyecdysone (Sigma), and 2.5 ml stable vitamin mix (Mains and Patterson 1973). A 5-ml stock solution of vitamin mix consists of 15 mg aspartic acid, 15 mg cystine, 5 mg 
-alanine, 0.02 mg biotin, 2 mg vitamin B12, 10 mg inositol, 10 mg choline chloride, 0.05 mg lipoic acid, 5 mg p-aminobenzoic acid, 25 mg fumaric acid, 0.4 mg coenzyme A, 15 mg glutamic acid, and 0.5 mg phenol red. The medium was adjusted to pH 7 and 350 mOsm and filter-sterilized prior to use. The majority of recordings reported in this study were from Rick Rack (RR) neurons and Proximal Branching (PB) neurons, two morphologically distinct sets of AL neurons in vitro identified elsewhere as putative projection (output) neurons and putative local interneurons, respectively (Hayashi and Hildebrand 1990; Oland and Hayashi 1993). Because no cell-type specific differences were identified in this study, details of cell morphology are not included in this paper.
AL neurons in situ
Electrical activity was also examined in developing projection (output) neurons in situ using semi-intact brain preparations (Kloppenburg et al. 1999; Mercer and Hildebrand 2002a). The cell bodies of these neurons (n = 5) were located in the medial group of AL neuronal somata. To aid the removal of glial cells that envelop the somata, the brain was placed for 3–5 min in enzyme (0.5 mg/ml collagenase and 2 mg/ml Dispase). The preparation was rinsed thoroughly with insect saline solution (see Electrophysiological recording) and mounted with fine pins in a dish lined with Sylgard (Dow Corning).
Electrophysiological recording
Patch-clamp recordings in whole cell configuration (Hamill et al. 1981) were used to examine the electrical properties of AL neurons. Electrodes with resistances of 1–2 M
were made from borosilicate glass (100-µl micropipettes, 1.71 mm OD, 1.32 mm ID; VWR Scientific, West Chester, CA) and filled with a solution containing (in mM) 150 K-aspartate, 5 NaCl, 2 MgCl2, 1 CaCl2, 11 EGTA, and 10 HEPES (pH7), and adjusted to 330 mOsM with mannitol. Cells were viewed with an IMT-2 inverted microscope (Olympus) equipped with Hoffman modulation contrast optics. To facilitate the formation of high-resistance (gigaohm) seals, culture medium was replaced with insect saline solution containing (in mM) 100 NaCl, 4 KCl, 6 CaCl2, 5 D-glucose, and 10 HEPES (pH 7), adjusted to 360 mOsm with mannitol, prior to recording. Cells were continuously superfused with fresh saline solution throughout the recording period, and junction potentials were nullified prior to seal formation. To obtain whole cell recordings, light suction and brief high-voltage pulses were used to rupture the cell membrane beneath the recording electrode. Recordings were made using an AxoPatch 1B amplifier (Axon Instruments, Union City, CA), and data were acquired and analyzed using pClamp 6 software (version 6.02, Axon Instruments). Membrane responses were sampled at intervals of 100 µs and were filtered at 2 kHz with a low-pass 4-pole Bessel filter. Linear leakage currents were subtracted on-line from all records. Electrical activity and plateau potential properties of the cells were examined under current clamp. Recordings under voltage clamp were used to identify currents underlying the generation of plateau potentials in the cells.
Identification of plateau potential mechanisms
The following tests described by Russell and Hartline (1982) were used to confirm the presence of plateau-potential mechanisms in developing Manduca AL neurons.
1) Trigger test: brief (20 ms) depolarizing current pulses (0.05–2 nA) were used to identify cells in which it was possible to trigger plateau potentials.
2) Termination test: in cells in which a plateau state could be generated, brief (20 ms) hyperpolarizing current pulses (ca. –0.5 nA) were used to terminate the plateau; to clearly show that termination was induced by injection of hyperpolarizing current, the timing of the hyperpolarizing pulse in successive episodes was shifted progressively closer to the triggering pulse.
3) Threshold test: the pulses used to trigger (0.05–2 nA) or to terminate (–0.05 to –1.0 nA) the plateau state were varied in amplitude systematically to determine whether the triggering and terminating of firing states were threshold phenomena.
4) All-or-none test: stimulus intensity was also varied systematically to determine whether responses were "all-or-none" or graded with stimulus intensity.
5) Symmetrical pulse test: responses to brief (20 ms) symmetrical positive and negative current pulses were compared with examine whether positive (depolarizing) pulses would produce a greater response than negative (hyperpolarizing) pulses in cells exhibiting plateau properties.
Pharmacological analysis of plateau potentials
Routine pharmacological techniques were used to examine the contribution of ionic currents to the generation and maintenance of plateau potentials in the cells. Na+ currents were blocked with TTX (10–7 M) and Ca2+ currents with 5 x 10–4 M CdCl2. In Na+-free solutions, NaCl was replaced with Tris-Cl, and in Ca2+-free solutions, CaCl2 was replaced with BaCl2. K+ currents were blocked by adding 3 x 10–2 M TEA to the solution bathing the cells. Effects on plateau potential formation of bath application of 5HT (50 µM), which reduces K+ current amplitudes in the cells (Kloppenburg et al. 1999; Mercer et al. 1995,1996b), were also examined in this study.
Data analysis
Effects of drug treatment on plateau potential properties (amplitude, duration, and membrane repolarization rate) were examined by comparing measurements taken prior to drug treatment (control) with measurements from the same cells 2–5 min after drug application. For pairwise comparisons, two-tailed Student's t-test were used to assess statistical significance. A significance level of 0.05 was accepted for all tests. Data are presented as means ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Prolonged depolarizations resembling plateau potentials were observed in AL neurons in vitro (Fig. 1A) and in situ, in semi-intact brain preparations (e.g., Fig. 8Bi). The following results confirm the presence of plateau potential–generating mechanisms in these neurons.
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400 ms to several seconds, and was longest in cells at early stages of development. The rate of repolarization of the cell membrane after termination of the plateau was typically slow (<0.03 mV/ms, Table 1A). While similar properties were observed in cells in situ (Table 1B), the plateau potentials in these more mature (stage-10) neurons were generally shorter in duration (however, see Fig. 8). Triggering of the plateau state was a threshold phenomenon, with subthreshold pulses producing no change in state (Fig. 2B). Above threshold, however, the depolarizing level of the plateau was independent of stimulus strength (Fig. 2C).
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NA+ CURRENTS. Bathing cells in Na+-free saline (Fig. 5A), or blocking Na+ channels with TTX (Fig. 5B), hyperpolarized the membrane, reducing the likelihood of triggering a plateau potential. However, if depolarizing current was used to return the membrane potential to the level recorded prior to the application of Na+-free saline or TTX (e.g., Fig. 5B), plateau potentials again could be reliably generated. While neither the formation nor the maintenance of plateau potentials was affected by removal of Na+ ions or blocking Na+ channels with TTX, the amplitude of spikes riding atop the plateau was reduced.
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CA2+ CURRENTS IN ISOLATION. The contribution that Ca2+ currents make to the generation and maintenance of plateau potentials was investigated further by examining Ca2+ currents in isolation (Fig. 7A). Cells in which Na+ and K+ currents had been blocked (see METHODS) continued to exhibit prolonged membrane depolarization in response to brief pulses of depolarizing current (Fig. 7B; Table 3). Prior to termination of the plateau state, the membrane potential sagged toward rest before commencing rapid repolarization around a mean breakpoint voltage of –4.9 ± 1.07 mV. Influxes of Ca2+ were also recorded in these neurons in the absence of experimentally applied depolarizing current pulses (Fig. 7C). These "spontaneous" events were abolished by bath application of 500 µM CdCl2 (data not shown). Blockade of Ca2+ currents with Cd2+ also abolished the characteristic bursts of electrical activity observed in immature Manduca AL neurons (Fig. 1B).
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At late stages of development (stages 12–18), it became increasingly difficult to elicit plateau potentials using brief pulses of depolarizing current alone (e.g., Fig. 8Ai). This change in excitability seemed to be associated with the appearance in the whole cell current profile of prominent, rapidly activating, transient K+ currents (see Mercer and Hildebrand 2002a, b). We have shown elsewhere that these K+ currents can be reduced in amplitude by exposing cells to the neuromodulator 5HT (Kloppenburg et al. 1999; Mercer et al. 1995, 1996b), and we hypothesized that 5HT-induced reduction of outward currents would enhance plateau potential formation in cells at late stages of metamorphosis. To examine this possibility, 5HT (50 µM) was bath-applied for 1–5 min to cells in vitro (stages 14–16; n = 4) or in situ in semi-intact preparations (stage 12; n = 2).
5HT alone did not trigger plateau potentials (data not shown). However, in three of four cells in vitro in which brief (20–200 ms) depolarizing pulses failed to trigger plateau potentials prior to 5HT treatment (Fig. 8Ai), prolonged exposure to 5HT (>3 min) not only increased the number of action potentials elicited by depolarizing current pulses (described in detail elsewhere; Kloppenburg and Heinbockel 2000; Kloppenburg and Hildebrand 1995; Kloppenburg et al. 1999; Mercer et al. 1995, 1996b) but also promoted the cell's entry into a plateau state (Fig. 8Aii). Cells in situ exhibiting bursts of electrical activity also responded to 5HT with increases both in the number of spikes and in the number and duration of plateau potentials recorded in the cells (Fig. 8B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Developing Manduca AL neurons generate prolonged membrane depolarizations that resemble plateau potentials. Here we show that this is an intrinsic property and that it depends on the influx of Ca2+ ions. To confirm the presence of plateau-generating mechanisms, we performed several key tests (Russell and Hartline 1982), including the trigger test, termination test, threshold test, all-or-none test, and symmetrical pulse test. We showed that brief pulses of depolarizing current trigger a plateau state and that plateau potentials can be terminated using brief pulses of hyperpolarizing current. Triggering and terminating of plateau potentials in these cells are threshold phenomena, and both triggering and terminating stimuli result in "all-or-none" responses that show no change in magnitude as stimulus intensity is increased.
Rebound excitation can also trigger entry into a plateau state
In cells in which plateau potentials could be triggered using the rebound excitation from prolonged pulses of hyperpolarizing current, time-dependent rectification produced a depolarizing sag toward the resting membrane potential during the hyperpolarizing pulse. Our results suggest that this rectification is caused by a slowly activating inward current that is activated by hyperpolarization. Slow deactivation of this current on cessation of the hyperpolarizing current pulse could underlie the rebound excitation that triggers the generation of a plateau potential, presumably through the activation of Ca2+ channels in the cells. Full characterization of this hyperpolarization-activated current in Manduca AL neurons awaits further investigation, but its properties, including its sensitivity to blockade by Cs+, resemble those of the hyperpolarization-activated inward current, Ih, described already in many vertebrate and invertebrate neurons (Kiehn and Harris-Warrick 1992b; McCormick and Pape 1990; Pape 1996).
Plateau potentials depend on the influx of Ca2+ ions
Blocking Ca2+ currents in the cells with Cd2+ not only prevented the generation of plateau potentials but also abolished bursts of electrical activity in developing Manduca AL neurons. This indicates that both depend on the activation of Ca2+ channels in the cells. Our results indicate, however, that, in contrast to the formation and maintenance of plateau potentials, termination of the plateau state is not Ca2+ mediated. Replacement of Ca2+ with Ba2+ reduced the duration of plateau potentials, suggesting that their termination is unlikely to involve Ca2+-mediated inactivation of Ca2+ channels (e.g., Gutnick et al. 1989). Our results suggest also that termination of plateau potentials can occur independently of K+ channel activation, because blocking K+ currents with TEA reduced the duration of the plateau state and increased rather than decreased the rate of membrane repolarization. It seems likely that, in the absence (or severe reduction) of K+ channel activation, sustained depolarization inactivates Ca2+ currents that maintain the plateau state.
While activation of K+ channels does not seem to be essential for terminating plateau potentials, we assume that, under normal conditions, K+ channel activation will contribute to membrane repolarization. In the presence of TEA, however, the voltage at which the membrane commences rapid repolarization (the plateau inactivation voltage or "breakpoint") is determined primarily by the voltage dependence of the Ca2+ channels (Reuveni et al. 1993). Ca2+ currents in Manduca AL neurons activate around –40 mV and peak around –10 mV (Mercer and Hildebrand 2002b; Mercer et al. 1995). They are characterized by relatively slow inactivation but undergo steady-state inactivation, being 100% available at –100 mV, 50% available at approximately –40 mV, and fully inactivated at –10 mV and above. The voltage-dependent activation and inactivation properties of these currents are similar to those of Ca2+ currents described in other insect preparations, including fruit fly Drosophila melanogaster (Baines and Bate 1998; Byerly and Leung 1988), cricket Gryllus bimaculatus (Kloppenburg and Hörner 1998), and honey bee Apis mellifera (Grünewald 2003; Kloppenburg et al. 1999; Schäfer et al. 1994). Plateau inactivation voltages recorded in this study under TEA (–7.25 ± 4.6 mV) and for Ca2+ currents in isolation (–5.53 ± 0.4 mV) are consistent also with the voltage-dependent properties reported for Ca2+ currents in developing Manduca leg motoneurons (Grünewald and Levine 1998; Hayashi and Levine 1992), as well as in AL neurons of the moth (Mercer and Hildebrand 2002b; Mercer et al. 1995). Taken together, our results indicate that voltage-activated Ca2+ currents contribute fundamentally to the bistable properties of developing Manduca AL neurons.
Persistent or slowly inactivating Ca2+ currents contribute in a similar way to the maintenance of depolarized plateau potentials in vertebrate neurons (e.g., Carlin et al. 2000; Perrier and Hounsgaard 2000; Seamans et al. 1997; Vergara et al. 2003) and the bistable properties of these neurons allow transient depolarizing inputs, including synaptic inputs, to produce prolonged depolarizations and sustained periods of spiking activity (Kiehn and Eken 1998; Reuveni et al. 1993). Slowly inactivating Na+ currents can play a similar role (Hsiao et al. 1998; Larkum et al. 2001; Schwindt and Crill 1998). However, while there is preliminary evidence to suggest that persistent Na+ currents are expressed in some insect neurons (e.g., Mercer and Hildebrand 2002b; Schäfer et al. 1994), we could find no evidence in this study that Na+ currents play a role in the formation or maintenance of plateau potentials in Manduca AL neurons. Neither the amplitude nor the duration of plateau potentials was altered by removal of Na+ ions from the external medium or by blocking Na+ channels in these cells with TTX.
5HT promotes the formation and maintenance of plateau potentials
A readily identifiable 5HT-immunoreactive neuron invades developing AL glomeruli at pupal stage 6 (Kent et al. 1987; Oland et al. 1995). Ultrastructural studies have shown that, within the glomeruli of adult ALs, most contacts involving this neuron are output synapses (Sun et al. 1993). 5HT applied exogenously to AL neurons in vitro, in situ in semi-intact brain preparations, and in vivo in the brain of the adult moth increases the excitability of AL neurons, exerting its effects through the modulation of K+ currents in the cells (Kloppenburg and Heinbockel 2000; Kloppenburg and Hildebrand 1995; Kloppenburg et al. 1999; Mercer et al. 1995, 1996b). Here we show that, while plateau potentials cannot be triggered by 5HT alone, this neuromodulator increases the likelihood that depolarizing current pulses (and presumably synaptic input) will trigger a cell's entry into a plateau state. This is reminiscent of other cell types, including vertebrate motoneurons that, in the presence of neuromodulators such as 5HT and norepinephrine, can be shifted between two stable modes of firing (Eken and Kiehn 1989; Hounsgaard and Kiehn 1989). This property endows the neurons with a mechanism for translating brief synaptic inputs into long-lasting motor output (reviewed by Kiehn and Eken 1998). 5HT has been shown to increase the excitability of motoneurons in several ways, including through the enhancement of the inward rectifier current, Ih (Kjaerulff and Kiehn 2001). Plateau properties in crustacean motor neurons can also be induced through a dual-conductance mechanism involving 5HT modulation of hyperpolarization-activated inward current, Ih, and Ca2+-dependent outward current (Kiehn and Harris-Warrick 1992a, b). We have shown elsewhere that, in Manduca AL neurons, 5HT modulates a transient A-type current as well as a sustained K+ current that resembles the delayed rectifier, IKV (Kloppenburg et al. 1999; Mercer et al. 1995, 1996b). However, effects of 5HT on Ca2+-activated K+ currents and the hyperpolarization-activated inward current observed in this study have yet to be determined. Whether 5HT modulates Ca2+ currents in developing Manduca AL neurons that show bursts of electrical activity also remains unclear.
Functional significance
There is compelling evidence that early forms of electrical excitability regulate neuronal growth and differentiation (e.g., Baines et al. 2001; Gu and Spitzer 1980; Kater and Mills 1991; Kater et al. 1988; Schilling et al. 1991; Spitzer et al. 1995, 2002) and contribute also to activity-dependent tuning of neuronal connections (e.g., Katz and Shatz 1996; Ruthazer and Stryker 1996; Shatz 1994; Sherrard and Bower 1998). In embryonic Periplaneta neurons, as in vertebrate neurons (e.g., Gallo et al. 1987; Koike et al. 1989; Toescu 1999), Ca2+ influx through voltage-gated Ca2+ channels influences both the survival and differentiation of neurons in culture (Benquet et al. 2001), and in Manduca, elegant studies by Duch and Levine (2000, 2002) suggest that Ca2+ spikes play a key role in postembryonic dendritic remodeling of motor neurons. Here we show that developing AL neurons generate Ca2+-mediated plateau potentials and that influx of Ca2+ ions underlies the characteristic bursts of electrical activity in these cells. The bistable properties of Manduca AL neurons should enable transient depolarizing synaptic inputs to produce prolonged depolarizations and sustained periods of spiking activity, which may also occur spontaneously without exogenous trigger. Changes in intracellular Ca2+ levels resulting from such activity could trigger a diverse array of cellular responses, ranging from modulation of ion channels (Gutnick et al. 1989) to regulation of neuronal gene expression (Berridge 1998; Bito et al. 1997; Brosenitsch and Katz 2001; Finkbeiner and Greenberg 1998). It seems likely that sustained electrical activity in ALs of the moth during critical periods of glomerulus formation and synaptogenesis contribute to the development of this highly structured neuropil. Our results add support also to the growing body of evidence that serotonin contributes to both the development and plasticity of AL neurons in the moth.
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ACKNOWLEDGMENTS |
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Address for reprint requests and other correspondence: A. R. Mercer, Dept. of Zoology, 340 Great King St., Benham Bldg., Rm. 111, Dunedin, New Zealand (E-mail: alison.mercer{at}stonebow.otago.ac.nz)
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REFERENCES |
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