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REPORT
1Department of Neuroscience, Mt. Sinai School of Medicine; and 2Phase Five Communications, New York, New York
Submitted 20 December 2006; accepted in final form 25 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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). B21 is of interest because afferent transmission is controlled via the regulation of spike propagation. Thus when B21 is peripherally activated at its resting membrane potential, peripherally triggered activity is not transmitted to follower neurons. Spikes fail to propagate to the output process. When B21 is centrally depolarized, the failure is relieved, and afferent transmission occurs (Evans et al. 2003b). Under physiological conditions, B21 is rhythmically depolarized during motor programs (Evans et al. 2003a,b; Rosen et al. 2000a; Shetreat-Klein and Cropper 2004). Spike propagation is therefore dynamically regulated so that afferent transmission occurs in a phase-dependent manner. In this study, we sought to determine why spikes fail to propagate when B21 is peripherally activated at its resting membrane potential. We show that propagation fails as impulses are transmitted from B21's peripheral (input) process to the somatic region of the cell. The failure is in part a consequence of the fact that the soma is relatively inexcitable. At resting membrane potential, the inexcitable soma promotes functional compartmentalization. When the propagation failure in B21 is relieved via central depolarization, conduction occurs. Spikes are actively generated in the peripheral (input) process and although transmission through the somatic region is primarily electrotonic, spikes are reinitiated when impulses reach the output process.
In summary, we characterize a mechanism in which spike propagation is determined by the regulation of membrane potential in a relatively inexcitable part of a cell (the soma). This distinguishes the regulation of spike propagation in B21 from other characterized cells where synaptic input is axonal and can initiate activity (Bucher et al. 2003; Goaillard et al. 2004; Meyrand et al. 1992). Although central depolarizations in B21 effectively alter information transfer from the input to the output process, they generally do not directly trigger spiking during feeding motor programs (Evans et al. 2003b). Consequently, information transmission can be regulated without disrupting peripheral encoding of afferent information.
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METHODS |
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16°C in artificial seawater [ASW, containing (in mM) 460 NaCl, 10 KCl, 11 CaCl2, 55 MgCl2, and 10 HEPES, pH 7.6]. Equipment used in current-clamp experiments included; Getting Model 5A amplifiers modified for 100-nA current injection (Getting Instruments, Iowa City, IA), Tektronix AM 502 amplifiers (Tektronix, Wilsonville, OR), and a Tektronix storage oscilloscope (model 5111). Data were digitized [using a Digidata (Axon Instruments, Union City, CA)] and were acquired and analyzed using pClamp version 9 software (Axon Instruments), and a Sony Vaio PCG-GRT Notebook.
Equipment used in single-electrode voltage-clamp (SEVC) experiments included an NPI SEC-05LX amplifier (NPI Electronics GmbH, Tamm, Germany), a Model 410 filter amplifier (Brownlee Precision, San Jose, CA), and a CED Power 1401 AD/DA converter (Cambridge Electronic Design, Cambridge, UK). Scripts, written in Spike II (version 5.15; Cambridge Electronic Design), were used to control the amplifier, acquire the data, and subtract leak current. Voltage steps had durations of 200 ms and were given every 10 s. Between steps, the membrane potential was held at –70 mV.
To record from the somata of neurons we used single-barrel electrodes filled with 3 M potassium acetate and 30 mM potassium chloride. Electrodes were beveled so that their impedances were generally <10 M
. In voltage-clamp experiments, electrodes were coated with silicone elastomer (Sylgard, Dow Corning, Midland, MI). To record from processes, microelectrodes were generally 50 M, and the tip was filled with 3% 5(6)-carboxyfluorescein dye in 0.1 M potassium citrate. Carboxyfluorescein was injected to verify recording sites. During physiological experiments processes were visualized for impalement by injecting Fast Green dye into the B21 soma (Evans et al. 2003b).
In lesion experiments, processes were mechanically severed using a glass micropipette. In current-clamp experiments, we measured the input resistance of the intact cell, and the input resistance of the cell after the medial lesion. Neurons were only tested for excitability if an increase in input resistance was observed. On average cells originally had an input resistance of 3.2 ± 0.3 M. After lesions the input resistance was 6.2 ± 1.2 M; n = 7). Voltage-clamp experiments were only conducted on neurons with an input resistance above the mean observed in current-clamp experiments.
TTX (Sigma-Aldrich, St. Louis, MO) was focally applied to the medial process at a concentration of 10-4 M. In some experiments (n = 2), the TTX was pressure applied via a micropipette with a large tip diameter. Fast Green was included in the TTX solution to monitor the injection. In other experiments (n = 5), the TTX solution was added to a subchamber that pharmacologically separated the ASW perfusing the medial process from the ASW perfusing the rest of B21. Fast Green was used to test for subchamber leaks. Controls did not show any affect of Fast Green on excitability.
To evoke afferent activity, the subradula tissue (SRT) was peripherally stimulated using a mini-speaker controlled by a stimulator (Grass Instruments) (Cropper et al. 1996).
Statistical tests were performed with Kaleidagraph (Synergy Software, Essex Junction, VT). Unless otherwise noted, two group comparisons utilized a paired t-test and n's provided indicate the number of preparations in which data were obtained. Data are reported as means ± SE.
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RESULTS |
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). The lateral process acts as an output region, being the primary point of contact with motor neurons (the B8 cells) (Borovikov et al. 2000; Gardner 1971). Peripherally triggered spikes are therefore transmitted from the medial process of B21 through the soma to the lateral process (Fig. 1A2). When B21 is peripherally activated at its resting potential, spikes fail to propagate to the lateral process (Fig. 1A1) (Evans et al. 2003b). To determine why the propagation failure occurs, we sought to determine whether there are regional differences in B21 excitability.
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). We found that the same is true for the isolated medial process (Fig. 2A1, right, and A2; n = 3; average spike amplitude 48.7 ± 1.9 mV). The somatic region is, however, different. We mechanically isolated this region of B21 progressively, removing the medial process first and the lateral process second. In most (6/7) cells, medial lesions virtually eliminated spiking. In one preparation, small depolarizations were still apparent (Fig. 2B2) that resembled impulses previously observed when spikes were triggered in the lateral process (Evans et al. 2003b). As expected, these depolarizations were eliminated when the lateral process was severed (Fig. 2B3). These data suggest that the somatic region of B21 (including the proximal medial process) is relatively inexcitable. Consistent with this idea are data we obtained in SEVC experiments in lesioned neurons. Net currents evoked by depolarizing steps in the somatic region were outward in normal saline (Fig. 3A; n = 6).
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,b; Shetreat-Klein and Cropper 2004). We hypothesized that these impulses were triggered in the medial process and electrotonically conducted to the soma (Fig. 2B1). [Previous characterizations of the lateral process length constant preclude the possibility that spikes were laterally initiated (Evans et al. 2003b).] To determine whether impulses were medially initiated, we simultaneously recorded from both the soma and the medial process and triggered spiking by injecting current into the soma. Consistent with the idea that spikes were medially triggered, impulses recorded in the medial process were observed prior to impulses recorded from the soma (Fig. 3B1). (The time difference between peak depolarizations was 1.4 ± 0.2 ms; n = 6.) Impulses in the medial process also had greater peak amplitudes (46.1 ± 2.1 vs. 38.1 ± 3.1 mV; t-test, P < 0.05; t = 2.58; df = 5; n = 6). In a second type of experiment, spikes were triggered via somatic current injection, and TTX was selectively applied to the medial process (Fig. 3B2, top). TTX application blocked spike initiation (Fig. 3B2, bottom; n = 7).
Our results suggest that when B21 is peripherally activated at its resting potential, spikes actively propagate in the medial process but that propagation fails in the relatively inexcitable somatic region (Fig. 1A2). Although the lateral process is capable of active spike generation (Evans et al. 2003b), it does not occur, presumably due to the fact that currents generated by medial spike initiation are insufficient to trigger lateral action potentials. When B21 is centrally depolarized prior to peripheral activation, active spiking again occurs through the branch point in the medial process (the T-junction region) and transmission in the somatic region is electrotonic. Events in the lateral process, however, are changed. Currents generated by medial spike initiation are sufficient to trigger active spike initiation.
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DISCUSSION |
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). In this study, we demonstrate that this propagation failure is in part a result of the somatic region of B21 being inexcitable. Thus the conduction failure is not simply a result of the impedance mismatch between the medial process and soma. Cells with excitable somata have been described in Aplysia, as have neurons with inexcitable somata (e.g., Alving 1968; Hurwitz et al. 1994; Tauc 1962a,b). It is therefore currently not clear whether an inexcitable soma is the rule or exception in this mollusk. In general, it is often difficult to localize sites of spike initiation in Aplysia because length constants in neurons can be surprising long. Consequently, somatic current injection often evokes relatively large-amplitude spike-like impulses, but without further analysis, it cannot be determined whether these impulses are remotely triggered.
Our current data do not implicate a specific mechanism as the underlying cause of the somatic inexcitability in B21. It is likely, however, that the soma is characterized by a low density of voltage-gated sodium channels as has been demonstrated in other sensory neurons (e.g., Safronov et al. 2000). Channels may also have an unfavorable distribution. Thus clustering of voltage-gated sodium channels has been demonstrated in axons of Aplysia and is likely to decrease the number of channels required for spike initiation important for propagation (Johnston et al. 1996). In the B21 soma, channel distribution may be uniform. It is important to point out, however, that our voltage-clamp data indicate that the somatic region of B21 does not simply function as a passive load, i.e., outward currents in the soma are activated within presumably physiological ranges. This phenomenon has been described, and implicated as being an important determinant of neuronal excitability (Graubard and Hartline 1991).
The regional biophysical differences in B21 produce a situation where excitable input and output processes are separated by a relatively inexcitable part of the cell. Although this arrangement is a result of a bipolar morphology in B21, unipolar or pseudo-unipolar neurons can be similar if they are characterized by a relatively short "stem" process exiting from the soma (i.e., if electrical affects of the soma are not masked) (Luscher et al. 1994; Weiss et al. 1986). An interposed inexcitable region may be of functional significance in a sensory neuron like B21 because it permits the regulation of afferent transmission without disrupting peripheral encoding (Fig. 1B, 1 vs. 2). Thus when B21 is peripherally activated at its resting potential, spikes are actively initiated in the input process, but currents generated are insufficient to trigger spiking in the next region capable of spike generation, the output process. In contrast, when B21 is centrally depolarized prior to and during peripheral activation, spikes in the input process do trigger spiking in the output process. Somatic depolarization therefore promotes reinitiation of peripherally triggered spikes. Importantly, however, somatic depolarization does not in itself induce additional spikes. Thus we characterize a mechanism whereby synaptic input can regulate afferent transmission without altering the encoding of peripherally generated signals.
Regulation of spike propagation in B21 is likely to be important for sensori-motor integration during feeding. When Aplysia feed, the food grasping organ (the radula) is protracted and retracted and opened and closed (Cropper et al. 2004). Phase relationships between protraction/retraction and opening/closing determine the nature of the behavior. If the radula closes during protraction, food is pushed out (i.e., behavior is egestive). In contrast, if the radula closes during retraction, food is pulled in (i.e., behavior is ingestive). Studies of spike propagation in B21 have focused on afferent transmission to radula closer motor neurons during ingestive activity. Under these conditions, excitatory input from B21 to closer motor neurons will tend to reinforce ingestive activity if it occurs during radula retraction (i.e., the radula will close more tightly so that food will be pulled into the buccal cavity). In contrast, excitatory input to closer motor neurons during protraction will be counterproductive. As expected, we have found that B21 is continuously depolarized throughout the retraction phase of ingestive programs via input it receives from electrically coupled interneurons (Evans et al. 2003a,b; Rosen et al. 2000a,b; Shetreat-Klein and Cropper 2004). Thus the dynamic regulation of spike propagation to B21's lateral process presumably ensures that ingestive behavior is enhanced rather than disrupted.
In conclusion, we demonstrate that the somatic region of the Aplysia mechanoafferent B21 is relatively inexcitable. A consequence of this arrangement is that active spike propagation fails at resting potential. Physiologically this is likely to be important in that it permits functional compartmentalization of B21. Peripherally triggered activity is not transmitted to all B21 follower neurons. This conduction failure can, however, be relieved if B21 is centrally depolarized prior to peripheral activation. Under physiological conditions, this can occur when the somatic region receives electrical synaptic input during feeding motor programs. Because the soma is inexcitable, central depolarizations do not initiate spiking and therefore do not alter the peripherally determined encoding pattern. Somatic depolarizations do, however, promote conduction in B21. The inexcitable B21 soma is therefore used to control transmission of afferent information without altering it.
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GRANTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. C. Cropper, Dept. Neuroscience, Box 1065, Mt. Sinai Medical School, One Gustave L. Levy Place, New York, NY 10029 (E-mail elizabeth.cropper{at}gmail.com)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Borovikov D, Evans CG, Jing J, Rosen SC, Cropper EC. A proprioceptive role for an exteroceptive mechanoafferent neuron in Aplysia. J Neurosci 20: 1990–2002, 2000.
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Cropper EC, Evans CG, Hurwitz I, Jing J, Proekt A, Romero A, Rosen SC. Feeding neural networks in the mollusc Aplysia. Neurosignals 13: 70–86, 2004.
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Evans CG, Jing J, Rosen SC, Cropper EC. Regulation of spike initiation and propagation in an Aplysia sensory neuron: gating-in via central depolarization. J Neurosci 23: 2920–2931, 2003b.
Gardner D. Bilateral symmetry and interneuronal organization in the buccal ganglia of Aplysia. Science 173: 550–553, 1971.
Goaillard JM, Schulz DJ, Kilman VL, Marder E. Octopamine modulates the axons of modulatory projection neurons. J Neurosci 24: 7063–7073, 2004.
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