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1Department of Biology, Georgia State University, Atlanta, Georgia
Submitted 28 November 2006; accepted in final form 12 January 2007
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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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) and in homeostatic regulation of cellular properties that allow bursting to occur (Marder and Goaillard 2006). The level of intracellular Ca2+ can be affected by the opening of voltage-gated Ca2+ channels and by synaptically driven inputs that lead to increases in intracellular Ca2+. Synaptically driven inputs (including neuromodulatory inputs) can cause a direct influx of Ca2+ through ligand-gated channels (Fucile 2004; Nayak et al. 1999), alter Ca2+ influx through voltage-gated channels (Kloppenburg et al. 2000; Ladewig et al. 2004; Oikawa et al. 2005) or alter the release of Ca2+ from intracellular stores (Berridge 1998). Determining the contributions of these mechanisms to Ca2+ signaling in CPG neurons during bursting is a first step toward understanding the control of Ca2+ dynamics during motor pattern generation.
The CPG underlying the escape swimming behavior of the opisthobranch mollusk Tritonia diomedea provides an opportunity to study Ca2+ signaling in a well-defined neuronal network. The CPG circuit contains three types of interneurons: the serotonergic dorsal swim interneurons (DSIs), cerebral interneuron 2 (C2), and ventral swim interneuron B (VSI) (Fig. 1). The swim is an episodic behavior; the neurons do not produce the rhythmic swim motor pattern (SMP) until it is triggered by sensory input. During the SMP, the CPG neurons fire three to eight rhythmic bursts of action potentials with a period of 
7 s. The Tritonia swim CPG has been described as a network oscillator; none of the neurons can generate rhythmic membrane potential oscillations in isolation, rather rhythmic activity arises through network interactions (Getting 1989).
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,b; Katz et al. 1994; Sakurai and Katz 2003). In fact, serotonin is necessary and sufficient for initiation of the SMP: application of a serotonin receptor antagonist prevents the SMP from occurring, whereas intracellular stimulation of individual DSIs or bath application of serotonin can initiate an SMP (Fickbohm and Katz 2000; McClellan et al. 1994). It is possible that the release of serotonin from the DSIs or the release of other unidentified neuromodulators could allow the SMP to occur by contributing to Ca2+ signaling in VSI and C2. In this study, we first observed the Ca2+ signals in VSI and C2 during the SMP. Then, to determine the proportion of those Ca2+ signals that could be accounted for by membrane potential excursions during the SMP, we voltage-clamped VSI and C2 in turn and measured the Ca2+ signals produced by playing back the voltage recordings of these neurons during the SMP as the voltage command. Thus VSI and C2 reproduced the same membrane potential excursions, but did not receive the same synaptic and neuromodulatory inputs as during the SMP. Our data show that SMP membrane potential excursions can completely account for the SMP Ca2+ signals in VSI but not in C2.
Portions of this work have been published previously in abstract form (Hill and Katz 2005).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Katz and Frost 1995a). The isolated brain, consisting of the fused cerebropleural ganglion and the pedal ganglia, was pinned to a silicone elastomer (Sylgard)-coated 35-mm petri dish, desheathed, and superfused with chilled (10°C) normal saline, containing (in mM) 420 NaCl, 10 KCl, 10 CaCl2, 50 MgCl2, 10 D-glucose, and 10 HEPES, pH 7.4.
Neurons were impaled with glass microelectrodes filled with 3 M KCl (8–12 M
). VSI was identified on the basis of the following characteristics: soma location (1 cell layer below the surface of the ventral side of the pleural ganglion), activity during the SMP, intrinsic properties such as the presence of A-current (Getting 1983), and the presence of its action potential in the pedal-pedal connective (PdN 6). C2 was identified on the basis of its soma location, appearance, and activity during an SMP (Getting 1983; Getting et al. 1980; Taghert and Willows 1978). The SMP was evoked by electrical stimulation of a body wall nerve, pedal nerve 3 (7–10 V, 2-ms pulses, 10 Hz for 1–1.5 s).
After identification, either VSI or C2 was re-impaled with a microelectrode containing 2.5 mM Ca2+ green 1 (CG-1) or 2.5 mM Oregon Green bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) 1 (OGB-1) dissolved in dH2O (both from Molecular Probes, Eugene, OR). The calcium indicator was iontophoresed using –1.0 to –7.0 nA, 500-ms pulses at 1 Hz for 10–40 min. After a successful injection, the soma always appeared pink to magenta in color. To allow for complete diffusion of the dye into distal regions of the neuron, the brain was then left for a few hours to overnight, constantly superfused with normal saline at 8–9°C. To image the neurites in the pedal ganglion, neuronal somata on the ventral surface of the pedal ganglion were removed with fine forceps. This allowed for visualization of the neurites of VSI or C2 in the neuropil and did not impair the SMP activity.
For Ca2+ imaging, the petri dish was transferred to a fixed stage Zeiss Axioskop 2 microscope outfitted with a Zeiss LSM 510 confocal laser scanning system (Zeiss, Jena, Germany). The filled VSI or C2 was impaled again with a fine tipped electrode filled with 3 M KCl. For Ca2+ imaging, an Argon laser (488 nM) was used to excite CG-1 or OGB-1, and a band-pass emission filter (500 to 550 nm) was used. Either a x5 air objective (0.25 N.A.) or a x20 water-immersion objective (0.7 N.A.) was used for imaging. After selecting a frame of interest, a time series of that frame was acquired (2–4 Hz, 2.5 µs pixel dwell time, 512 x 512 pixels/frame). A delay of 50–100 ms between frames was introduced to reduce photo-damage and phototoxicity.
To measure changes in Ca2+, regions of interest (ROIs) were selected, and the raw fluorescence data were saved as a text file. Correction for background fluorescence was made by averaging the values in two or more regions not stained by the Ca2+ indicator and subtracting those values from the ROIs of indicator stained regions. The change in fluorescence (
F/Fo) was calculated with respect to the average baseline fluorescence (Fo; measured for 10 s) in each ROI prior to nerve stimulation. The time constant of decay (
) of the calcium signal was measured from the peak of the final burst in the SMP.
For electrophysiological recordings, we used an AxoClamp 2B amplifier (Axon Instruments, Union City, CA), connected by a 1401micro AD converter [Cambridge Electronic Design (CED), Cambridge, UK] to a PC running Spike2 software (CED) for collection and analysis of data. An output pulse from the LSM was used to trigger a stimulator (A-M Systems, Carlsborg, WA), synchronizing the acquisition of imaging and electrophysiology data. In each preparation several SMPs were elicited by stimulation of pedal nerve 3 (7–10 V, 2-ms pulses, 10 Hz for 1–1.5 s). Each stimulus resulted in a slightly different SMP in terms of spike frequency and number of swim cycles. We usually selected the SMP with the most swim cycles for replaying in voltage clamp. For voltage-clamp recordings of VSI or C2, we used discontinuous single-electrode voltage clamp (dSEVC). The sampling rate was usually set at 0.7 kHz, and the gain was set at 0.7 nA/mV. We continuously monitored the quality of the clamp on an oscilloscope with a fast sweep speed. For playing back the swim voltage waveform, we modified the baseline of the voltage recording to be 0 mV. The membrane potential of the neuron was held at its natural resting potential, and the modified swim episode was used as the voltage command. We usually replayed the SMP two to three times with each resulting in virtually identical Ca2+ signals.
For measurement of the time constant of decay (), curves were fit to single exponentials using SigmaPlot (Jandel Scientific, San Rafael, CA). Statistical analyses were performed using SigmaStat (Jandel Scientific), and a P value of <0.05 was considered a significant difference. Error bars represent SE.
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RESULTS |
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The VSI soma is located in the pleural ganglion, and it has an extended geometry with at least two distinct arborizations (Fig. 1B). A primary neurite extends anteriorly and projects laterally to the ipsilateral pedal ganglion. VSI has secondary neurites proximal to the soma in the pleural ganglion and secondary neurites in the pedal ganglion. It is not known whether VSI receives inputs in both regions and it is not known where VSI synapses with its postsynaptic follower neurons.
During the SMP, VSI and the other swim CPG neurons fire bursts of action potentials riding on underlying membrane potential oscillations (Fig. 2, bottom). Ca2+ signals imaged during the SMP showed rhythmic oscillations that corresponded to the action potential bursts recorded at the soma of VSI (Fig. 2). Rhythmic Ca2+ signals were recorded in both proximal (pleural ganglion; Fig. 2A) and distal (pedal ganglion) regions (Fig. 2B) of VSI.
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To determine the extent to which the Ca2+ signal in VSI was caused by the membrane potential excursions during the SMP, VSI was voltage-clamped (dSEVC) and its own membrane potential recorded during the SMP was played back as the voltage command. In this way, the VSI membrane potential reproduced the same pattern that it exhibited during the SMP without the influence of synaptic and neuromodulatory inputs. This protocol effectively reactivated VSI without activating the rest of the network to produce the SMP (Fig. 3). In this example, two rhythmically active pedal ganglion neurons were recorded during the SMP: a ventral flexion neuron (VFN) and a dorsal flexion neuron A (DFN-A) (Hume et al. 1982). When the VSI swim membrane potential excursions were recreated via voltage-clamp (Fig. 3), the DFN-A no longer displayed rhythmic activity. The VFN was directly driven by VSI but showed greatly reduced activity compared with its activity during the SMP. Extracellularly recorded rhythmic activity was also greatly curtailed, leaving only the VSI action potentials in pedal nerve 6. Similar results were observed in three preparations. These results suggest that replaying the swim motor pattern in VSI did not activate the CPG.
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F/Fo curve was integrated from the time of the stimulus to 60 s after the end of the SMP. There were no significant differences between the Ca2+ signals produced by playback of the membrane potential and those imaged during the SMP [Fig. 4, A and B; P > 0.05 in all cases (paired t-test)].
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To determine the relative contribution to the Ca2+ signal of the action potentials and the underlying membrane potential depolarization during the SMP, we used each separately as the voltage command and measured the respective Ca2+ signals (Fig. 5). In the different regions of VSI, there were differences in the extent to which the spikes alone contributed to the Ca2+ signals. In proximal primary neurites, the action potential waveforms alone reproduced 75.5% of the integrated amplitude of Ca2+ signals produced by the full SMP waveform (n = 2), whereas in the proximal secondary neurites, spikes alone accounted for 60.1% of the signal (n = 2; Fig. 5A, i, green traces, and ii). In proximal regions of VSI, the underlying SMP membrane potential oscillations, produced Ca2+ signals that were 35.0 and 31.7% of the Ca2+ signals produced by the full SMP waveform (primary and secondary neurites, respectively; both n = 3; Fig. 5A, i, magenta traces, and ii). Thus most of the Ca2+ signal during the SMP in the proximal regions of VSI was spike-mediated, but the subthreshold membrane potential oscillations contributed substantially.
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Rhythmic Ca2+ signals showed spatial heterogeneity in C2 during the SMP
Unlike VSI, C2 has its soma in the cerebral ganglion and projects contralaterally to the pedal ganglion. It has almost no arborizations in the cerebral ganglion but has extensive arborizations in the contralateral pedal ganglion. As with VSI, it is unknown where C2 receives synaptic inputs or contacts it postsynaptic followers.
Rhythmic Ca2+ signals were observed in all areas of C2 during the SMP (Fig. 6). In distal (pedal ganglion) regions of C2, the Ca2+ signals decayed more rapidly in the secondary neurites than in the primary neurites [Table 1, P < 0.05 (Kruskal-Wallis 1-way ANOVA on ranks with Dunns multiple pairwise comparisons)]. Although the time constant of decay of the signals in the proximal (cerebral ganglion) primary neurite was about twice that of the distal primary neurite, the difference did not show statistical significance [Table 1, P > 0.05 (Kruskal-Wallis 1-way ANOVA on ranks with Dunns multiple pairwise comparisons)]. Furthermore, although there was a trend for the peak amplitude to be largest in the distal secondary neurite and smallest in the proximal primary neurite, there were no significant differences between the peak Ca2+ signals in any of regions of C2 [Table 1, P = 0.109 (1-way ANOVA)].
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To test whether the Ca2+ signals imaged in C2 during the SMP could be accounted for by membrane potential excursions, we performed the same procedure as previously described for VSI of voltage-clamping (dSEVC) C2 at the soma and replaying the membrane potential of the neuron recorded during the SMP. As with VSI, this did not cause the network to generate the SMP (Fig. 7). A DSI and a pedal ganglion neuron that burst as a DFN-A (Hume et al. 1982) that had been rhythmically active during the SMP were only minimally affected during the replay of the SMP membrane potential excursions in C2 (Fig. 7; similar results were observed in 3 preparations). Also during the SMP, large rhythmic units were observed in pedal nerve 6 that were absent during the SMP replay in C2. However, C2 propagating action potentials generated by the replay were observed in the recording of pedal nerve 6 (Fig. 7).
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To test whether the differences between the SMP Ca2+ signals and the voltage-clamp-induced Ca2+ signals in all regions of C2 could be caused by space-clamp problems, we sought to voltage clamp the distal primary neurite of C2. In numerous attempts, we managed to impale the distal neurite of C2 only three times, and of those, only once could we maintain the recording long enough to voltage clamp the distal neurite. We first recorded the SMP voltage and Ca2+ signals in the distal neurites of C2, and then voltage-clamped the distal primary neurite and recreated the SMP voltage excursions. We found that this completely recreated the SMP Ca2+ signals (Fig. 9), something that we never observed while voltage clamping at the soma of C2. Although we were able to perform this technically difficult experiment only once, this result was clear and suggests that space-clamp issues rather than synaptic inputs could be the cause of the discrepancies between the SMP and voltage-clamp Ca2+ signals observed while clamping C2 at the soma.
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The proportion of the Ca2+ signal that can be accounted for by the action potentials in C2 was less than in VSI. In proximal regions of C2, the spikes alone produced only 12.6 and 17.9% (ipsi- and contralateral, respectively, both n = 5) of the Ca2+ signals produced by the full SMP waveform (Fig. 10A,i green traces, ii). The subthreshold membrane potential oscillations accounted for a greater proportion of the Ca2+ signal: 42.1 and 36.8% (ipsi- and contralateral, respectively, both n = 5) in the proximal regions of C2 (Fig. 10A, i, magenta traces, and ii).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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We recorded rhythmic Ca2+ signals in all regions of two Tritonia CPG neurons (VSI and C2) during the SMP. The Ca2+ signals oscillated in time with the membrane potential signals recorded during the SMP. Similar time-locking of Ca2+ signals to membrane potential signals has been reported in other rhythmically active systems (Backsai et al. 1995; Ladewig and Keller 2000; Ross and Graubard 1989; Viana Di Prisco and Alford 2004). In both VSI and C2, Ca2+ signals during the SMP exhibited more rapid dynamics in secondary neurites compared with primary neurites. These differences may reflect differences in Ca2+ buffering and/or extrusion or could indicate that Ca2+ signals are mainly generated in the secondary neurites.
Membrane potential excursions can fully account for the SMP Ca2+ signal in VSI but not C2
We found that the SMP Ca2+ signals in all regions of VSI could be completely reproduced by recreating the SMP voltage signal via voltage clamp at the soma. In contrast, voltage clamp at the soma of C2 could not completely reproduce the amplitude of the SMP Ca2+ signals in all regions of C2. Space-clamp issues or synaptic inputs to C2 during the SMP could account for the discrepancy between the SMP Ca2+ signals and the Ca2+ signals produced by voltage clamp at the soma of C2. At any rate, in both neurons recreating the SMP voltage signals faithfully reproduced the differences in Ca2+ dynamics (i.e., primary vs. secondary neurite) observed in during the SMP even when the voltage signals were generated in the somata instead of in the neuropil.
The VSI result was somewhat unexpected; we hypothesized that neuromodulatory or synaptically driven Ca2+ signals would contribute to the Ca2+ signal recorded during the SMP. Dynamic biochemical signaling has been speculated to play a potentially integral role in the generation of the Tritonia SMP (Clemens and Katz 2003). Therefore we expected that VSI would receive at least some synaptic inputs during an SMP that would have produced some of the SMP Ca2+ signal. However, our data show that rather than Ca2+ driving the voltage oscillations, the voltage oscillations drive the entire Ca2+ signal recorded in VSI during rhythmic bursting. Thus in VSI, Ca2+ entry via voltage-gated Ca2+ channels appears to be responsible for the entire Ca2+ signal during rhythmic bursting. It remains unknown whether Ca2+ entry via voltage-gated Ca2+ channels leads to Ca2+-induced Ca2+ release from internal stores. Finally, it is possible that synaptic and/or neuromodulatory inputs during the SMP could contribute to Ca2+ signaling in highly restricted microdomains near synaptic terminals.
Synaptic inputs to C2 during the SMP or space-clamp issues may underlie the discrepancy between the SMP Ca2+ signals and those produced by the SMP replay
In one experiment, we were successful at holding the recording long enough to voltage clamp the distal neurite of C2 and replay the SMP voltage signal. This did recreate the SMP Ca2+ signals in both the primary and secondary neurites of C2, something we never observed with voltage clamp at the soma. This result suggests that space-clamp issues could be the cause of the discrepancy between the SMP Ca2+ signals and those produced by replaying the SMP voltage signal at the soma of C2. However, because we were not able to voltage clamp the distal neurite of C2 more than once, these results must be interpreted with caution.
It remains a possibility that synaptic inputs to C2 during the SMP could contribute to Ca2+ signaling. First of all, a neurotransmitter/modulator (such as serotonin) could activate metabotropic receptors, leading to second-messenger cascades that result in localized Ca2+ signals (i.e., release of Ca2+ from internal stores or Ca2+ influx) without causing any voltage signals. In the leech Retzius neuron, activation of metabotropic serotonin receptors induces an influx of Ca2+ through Ca2+ channels located mainly in the dendrites (Beck et al. 2002). Further, activation of metabotropic receptors can lead to an augmentation of Ca2+-induced Ca2+ release from internal stores. In lamprey reticulospinal axons, activation of metabotropic glutamate receptors leads to a rapid release of Ca2+ from intracellular stores in a Ca2+-dependent manner (Cochilla and Alford 1998). These Ca2+ signals would not be reproduced by the voltage signal alone in the absence of the synaptic inputs that triggered the second-messenger cascade that produced or augmented the Ca2+ signals. Second, synaptic inputs during the SMP could enhance Ca2+ entry into C2 through voltage-gated Ca2+ channels. Biogenic amines have been shown to both increase and decrease Ca2+ entry through voltage-gated channels. In mouse hypoglossal neurons, serotonin decreases Ca2+ influx through voltage-activated Ca2+ channels (Ladewig et al. 2004), and in the lobster STG (stomatogastric ganglion), dopamine increases voltage-activated Ca2+ currents in some CPG neurons and decreases it in others (Johnson et al. 2003). Imaging experiments showed that in one lobster STG neuron dopamine decreases voltage-activated Ca2+ entry in presynaptic terminals (Kloppenburg et al. 2000). Nicotine can also increase Ca2+ entry through voltage-gated Ca2+ channels (Oikawa et al. 2005). Thus during an SMP, synaptic inputs to C2 could potentially enhance Ca2+ entry through voltage-activated Ca2+ channels. In fact, recent experiments have shown that stimulation of one DSI (causing synaptic release of serotonin) enhances spike-mediated Ca2+ signaling in distal regions of C2 (Hill and Katz 2006). Finally, receptors, such as nicotinic ACh, NMDA, and 5-HT3 receptors, themselves have been shown to be permeable to Ca2+ (Cochilla and Alford 1999; Fucile 2004; Nayak et al. 1999; Single and Borst 2001). Synaptic inputs to C2 during an SMP could thus directly lead to Ca2+ influx into C2 via Ca2+-permeable receptors.
Spikes versus underlying waveform—contribution to the full Ca2+ signals in VSI and C2
VSI and C2 differed in the contribution of action potentials to the Ca2+ signals. In the distal regions of VSI, the action potentials alone accounted for 75–95% of the Ca2+ signal, whereas the underlying potential had almost no effect. In proximal regions of VSI, the underlying potential contributed more to the Ca2+ signal, but still less than the contribution of the action potentials. These differences could reflect regional differences in the distribution of Ca2+ channels: for example, in distal regions there could be a greater density of high-threshold Ca2+ channels, whereas in proximal regions of VSI, the relative density of low-threshold Ca2+ channels could be higher.
In proximal regions of C2, the situation was reversed—the underlying potential contributed more to the Ca2+ signal than did the action potentials. These data indicate that in proximal regions of C2 there may be a greater density of low-threshold Ca2+ channels than high-threshold Ca2+ channels. Furthermore, the action potentials recorded in the soma of C2 tended to be smaller then those recorded in the soma of VSI, suggesting that the action potentials may be generated farther away from the soma in C2 than in VSI. In the distal regions of C2, the action potentials contributed more to the Ca2+ signal than did the underlying potential, suggesting that in distal regions of C2 there could be a greater relative density of high-threshold Ca2+ channels than low-threshold Ca2+ channels. In all regions of C2, the action potentials or the underlying waveform when used separately as the voltage command resulted in Ca2+ signals that, when added together, were much smaller than the Ca2+ signal that resulted from using the full SMP waveform as the voltage command. Thus in C2, there seems to be a supra-linear summation of Ca2+ signals from these two sources when they are presented together. Possibly, the high-threshold Ca2+ channels in C2 need to be, in essence, "primed" by the sub-threshold potential to permit a large volume of Ca2+ entry.
Differences in Ca2+ dynamics in primary versus secondary neurites in both VSI and C2
We observed differences in the temporal dynamics of Ca2+ signals in primary versus secondary neurites in VSI and C2 with the secondary neurites of both neurons exhibiting faster Ca2+ dynamics than the primary neurite. These differences in the decay rate of the Ca2+ signals could be, in part, due to differences in the morphology of the secondary neurites compared with the primary neurites. The secondary neurites have a much larger surface area to volume ratio than do the primary neurites. This could lead to more rapid extrusion of Ca2+ in the secondary neurites than in the primary neurites. Similar differences in temporal dynamics of Ca2+ signaling have been reported in other systems. In crab STG neurons, Ca2+ signals in fine branches exhibit differential temporal dynamics, but no Ca2+ signals were observed in the axon or soma, presumably due to a lack of voltage-gated Ca2+ channels in those regions (Ross and Graubard 1989). Lobster STG neuronal somata show slower Ca2+ dynamics than do fine branches (Levi et al. 2003). Also, mouse hypoglossal neuron dendritic Ca2+ signals have faster recovery kinetics than somatic Ca2+ signals (Ladewig and Keller 2000).
The differences in the temporal nature of the Ca2+ signals that we observed in VSI and C2 primary and secondary neurites could have functional significance. For instance, presumptive synaptic regions of VSI and C2 (secondary neurites) could have more extensive Ca2+ buffering or extrusion than the axon (primary neurite). Because Ca2+ is known to play an intimate role in neurotransmitter release, intracellular Ca2+ homeostasis may be essential in synaptic regions of VSI and C2. Furthermore, intracellular Ca2+ is known to activate Ca2+-activated K+ channels, leading to decreased excitability (El Manira and Wallen 2000; Ladewig and Keller 2000). In synaptic regions, it may be important for such a decrease in excitability to end before the next burst of excitatory synaptic inputs arrives. Thus, in terms of recovery kinetics, the secondary neurites of VSI and C2 may represent separate Ca2+ compartments from the primary neurite.
Area-specific differences in the amplitude of Ca2+ signals in VSI
In VSI, Ca2+ signals were larger in the secondary neurites than in the primary neurites in the proximal regions of the neuron, and Ca2+ signals were also larger in the distal primary neurite than in the proximal primary neurite. Two studies in other systems, using ratiometric calcium indicators found similar amplitude differences. In lamprey spinal cord neurons, Ca2+ signals were found to be larger in distal than proximal dendrites (Viana Di Prisco and Alford 2004), and dendritic Ca2+ signals were larger than somatic Ca2+ signals in mouse hypoglossal neurons (Ladewig and Keller 2000). In VSI, the amplitude differences could reflect differences in the density of voltage-gated Ca2+ channels. Thus, the same voltage signal would elicit a larger Ca2+ signal in the areas containing a higher density of voltage-gated Ca2+ channels than in areas with a low density of such channels. In C2, there were no significant differences in the amplitude of Ca2+ signals in any region of the neuron, possibly indicating a homogeneous distribution of voltage-gated Ca2+ channels.
Ca2+ signaling in VSI and C2 during the SMP
In conclusion, we have shown that Ca2+ signals in two Tritonia CPG neurons during rhythmic bursting are similar in terms of Ca2+ dynamics and but may differ in the source of the Ca2+ signals. In VSI, the SMP Ca2+ signals appear to be purely a result of membrane potential excursions leading to Ca2+ influx via voltage-gated Ca2+ channels. In contrast, synaptic inputs may significantly contribute to Ca2+ signaling in C2 during the SMP.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Katz, Dept. of Biology, Georgia State University, PO Box 4010, Atlanta, GA 30302-4010 (E-mail: pkatz{at}gsu.edu)
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, excitatory synapses; 
, inhibitory synapses. 
, axon of VSI in pedal nerve 6. C: Oregon Green fill of C2 in an unfixed, living preparation. No secondary neurites can be seen in the cerebral ganglia, but secondary neurites are visible in the contralateral pedal ganglion. Unlabeled 
). Differences in temporal dynamics of Ca2+ signals in primary vs. secondary neurites were observed in both proximal (A) and distal (B) regions of VSI. In the primary neurite, the Ca2+ signals summated and stayed above the basal level long after the nerve shock. In the secondary neurites, the Ca2+ signals did not summate and returned back to basal levels faster than in the primary neurite.
