Identified Serotonergic Neurons in the Tritonia Swim CPG Activate Both Ionotropic and Metabotropic Receptors

Stefan Clemens and Paul S. Katz

Department of Biology, Center for Neural Communication and Computation, Georgia State University, Atlanta, Georgia 30303


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Clemens, Stefan and Paul S. Katz. Identified Serotonergic Neurons in the Tritonia Swim CPG Activate Both Ionotropic and Metabotropic Receptors. J. Neurophysiol. 85: 476-479, 2001. Although G-protein-coupled (metabotropic) receptors are known to modulate the production of motor patterns, evidence from the escape swim central pattern generator (CPG) of the nudibranch mollusk, Tritonia diomedea, suggests that they might also participate in the generation of the motor pattern itself. The dorsal swim interneurons (DSIs), identified serotonergic neurons intrinsic to the Tritonia swim CPG, evoke dual component synaptic potentials onto other CPG neurons and premotor interneurons. Both the fast and slow components were previously shown to be due to serotonin (5-HT) acting at distinct postsynaptic receptors. We find that blocking or facilitating metabotropic receptors in a postsynaptic premotor interneuron differentially affects the fast and slow synaptic responses to DSI stimulation. Blocking G-protein activation by iontophoretically injecting the GDP-analogue guanosine 5'-O-(2-thiodiphosphate) (GDP-beta -S) did not significantly affect the DSI-evoked fast excitatory postsynaptic potential (EPSP) but decreased the amplitude of the slow component more than 50%. Injection of the GTP analogues guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma -S) and 5'-guanylyl-imidodiphosphate, to prolong G-protein activation, had mixed effects on the fast component but increased the amplitude and duration of the slow component of the DSI-evoked response and, with repeated DSI stimulation, led to a persistent depolarization. These results indicate that the fast component of the biphasic synaptic potential evoked by a serotonergic CPG neuron onto premotor interneurons is mediated by ionotropic receptors (5-HT-gated ion channels), whereas the slow component is mediated by G-protein-coupled receptors. A similar synaptic activation of metabotropic receptors might also be found within the CPG itself, where it could exert a direct influence onto motor pattern generation.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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Motor pattern generation is generally considered to occur through an interaction of cellular properties and synaptic connectivity within circuits known as central pattern generators (CPGs) (Kiehn et al. 1997; Marder and Calabrese 1996). Neuromodulatory inputs alter cellular and synaptic properties of CPG neurons and thereby modify motor pattern production (Calabrese 1998; Harris-Warrick and Marder 1991; Kiehn and Katz 1999). Although it is well established that second-messenger pathways play important roles in mediating such neuromodulatory actions (for review, see Jonas and Kaczmarek 1999), it has only recently been speculated that second-messenger pathways may also play a direct role in the actual process of motor pattern generation (Katz and Clemens 2001; Katz and Frost 1996).

The CPG underlying the escape swim of the nudibranch mollusk, Tritonia diomedea, contains a set of identified serotonergic neurons, the dorsal swim interneurons (DSIs) (Getting et al. 1980). The DSIs evoke dual component synaptic potentials, consisting of a fast excitatory postsynaptic potential (EPSP) and a slowly rising, slowly falling depolarization on some postsynaptic followers such as the CPG neuron C2 (Getting 1981) and a class of premotor interneurons, the dorsal flexion neurons (DFN-A) (Hume and Getting 1982). The DSIs also evoke neuromodulatory actions on C2, enhancing synaptic strength and cellular excitability (Katz and Frost 1995b, 1997).

Based on the time course of the synaptic actions and the effects of the serotonin reuptake blocker, imipramine, it was proposed that the fast EPSP was mediated by ionotropic receptors and that the slow component was caused by metabotropic (G-protein-coupled) receptors (Katz and Frost 1995a). Serotonin (5-HT) can act both at ionotropic and at metabotropic receptors (Barnes and Sharp 1999). Furthermore both ionotropic (Green et al. 1996) and metabotropic 5-HT receptors (Angers et al. 1998; Li et al. 1995) have also been described in mollusks. We therefore sought to determine the contributions of these receptor types to the different DSI-evoked responses.

We found that the guanine nucleotide analogs primarily affected the amplitude and duration of the DSI-evoked slow potential in the DFN, indicating that the slow component is mediated by G-protein-coupled receptors. It is likely that both ionotropic and metabotropic 5-HT receptors also play an important role within the CPG itself since the DSI synaptic actions on CPG member C2 strongly resemble the dual component potential in DFN (Fickbohm and Katz 2000; Katz and Frost 1995a).

Some of these results have been previously presented in abstract form (Clemens and Katz 1999, 2000).


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Experiments (n = 18 from 12 animals) were performed on adult Tritonia diomedea. Detailed descriptions of the protocols have been published elsewhere (Fickbohm and Katz 2000; Katz and Frost 1995a,b). The isolated CNS was desheathed and perfused with normal saline [which contained (in mM) 420 NaCl, 10 KCl, 10 CaCl2, 50 MgCl2, 10 D-glucose, and 10 HEPES, pH 7.6]. Neurons were identified using both physical and electrophysiological criteria (Getting 1977; Getting et al. 1980). Signals were digitized with a CED 1401 and analyzed with Spike2 software (Cambridge Electronic Design, England). After successful neuronal identification, normal saline was replaced with high divalent cation saline (2.5 × Ca2+ and Mg2+) to increase firing thresholds, thereby decreasing the contribution of polysynaptic pathways.

A DSI and a contralateral DFN were impaled with microelectrodes. The DSI was stimulated with brief (20 ms) depolarizing current pulses (7 nA) that each elicited a single spike. Control and test stimuli consisted of five pulses at 1 or 2 Hz every 60 s. We measured the height of the first EPSP and of the slow component 2 s after the end of the last DSI-evoked spike before and after application of the respective drug. For the pooled data comparisons, the average percent change was then normalized to the average of the HEPES control injection.

Guanosine 5'-O-(2-thiodiphosphate) (GDP-beta -S), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma -S; Trilithium salts), and 5'-guanylyl-imidodiphosphate (GMP-PNP, Trisodium salt), were dissolved in 20 mM HEPES at 10-4 to 10-3 M (all chemicals: Sigma, MO) and injected iontophoretically into the DFN. Injection pulses (-5 to -7 nA, 500-ms duration) were applied at 1 Hz for 10 and 15 min. Stimulus test protocols were begun after recovery to the preinjection membrane potential.


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The serotonergic DSIs evoke an excitatory response in the DFN-A consisting of a fast EPSP followed by a slow depolarizing component (Fig. 1, A and B). Both components appear to be due to a monosynaptic connection from the DSI to the DFN (Hume et al. 1982; Katz and Frost 1995a). The so-called fast EPSP has a time constant of decay of about 0.3 s and is completely dissipated 2 s after a DSI spike. In contrast, the slow component rises gradually with a short train of DSI spikes and takes nearly 20 s to fully decrement following a five-pulse DSI train at either 1 or 2 Hz.



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Fig. 1. Guanine nucleotides affect the slow component of the dorsal swim interneuron (DSI)-evoked synaptic potential in dorsal flexion neuron (DFN). A: prior to drug injection, DSI stimulation (5 pulses, 1 Hz) evokes fast excitatory postsynaptic potentials (EPSPs) in a DFN and a slow summating potential that recovers after about 20 s (Preinjection trace, average response of 3 consecutive stimuli). After injection of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta -S (average response of 3 consecutive stimuli), the height of the slow component (arrows) is decreased, whereas the fast EPSPs are less affected. B: after injection of a GTP analogue [here: 5'-guanylyl-imidodiphosphate (GMP-PNP)] into the DFN, the amplitude of the slow component is increased (average response of 5 consecutive stimuli). Note that after injection, the slow component does not recover within the time window of 30 s to the prestimulus value. C: summary of the effects of the GDP and GTP analogues on DSI-evoked fast and slow synaptic potentials, normalized to the sham injection of the carrier HEPES (responses to HEPES not shown). The fast EPSP was not significantly affected by injection of GDP-beta -S or GMP-PNP, but guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma -S) caused a small yet significant decrease (12 ± 1%). In contrast, the slow component was significantly reduced by GDP-beta -S injection (50 ± 5.4% reduction from control) and significantly enhanced by GMP-PNP (32 ± 2.5% greater than control). GTP-gamma -S slightly increased the slow component by 10 ± 6.8%.

To determine whether either of these DSI-evoked responses was mediated by metabotropic receptors in the DFN, we first blocked G-protein signaling by injecting GDP-beta -S into a DFN. Injection of the carrier (20 mM HEPES) alone caused a slight decrease in both fast and slow components of the DSI-evoked response (fast EPSPs: 95 ± 3% of preinjection, slow potential: 85 ± 3% of preinjection, means ± SE; n = 3, from 2 animals). In contrast, we found that injection of GDP-beta -S caused a much more substantial decrease in the slow component with only a minor reduction in the fast EPSP (Fig. 1A). The effect of GDP-beta -S on the fast component was not significantly reduced from that of the HEPES control alone (Fig. 1C). However, GDP-beta -S significantly decreased the slow component to 50 ± 5.4% of the HEPES control injection. Thus blocking G-protein activation significantly reduced the slow component of the DSI-evoked EPSPs in the DFN but not the fast.

We next tested whether we could enhance either the fast or slow component by injection of the nonhydrolyzable GTP analogues, GMP-PNP (n = 6, from 4 animals) and GTP-gamma -S (n = 4, from 3 animals). Injection of GMP-PNP injection caused no significant change in the amplitude of the fast EPSP (Fig. 1, B and C), whereas injection of GTP-gamma -S caused a small but significant decrease (12 ± 1%) from HEPES control (Fig. 1C). In contrast, GMP-PNP injection significantly increased the amplitude of the slow component by 32 ± 2.5% over the HEPES control injection (Fig. 1, B and C) and GTP-gamma -S also slightly increased its amplitude (10 ± 6.8% increase over control, Fig. 1C).

Injection of both GTP analogues also prolonged the recovery of the slow component (Fig. 1B) and led to a persistent depolarization in response to DSI stimulation (Fig. 2, B and C). This depolarization was dependent on ongoing DSI stimulation since following the recovery after the injection of GTP-gamma -S or GMP-PNP, the membrane potential remained stable. However, when resuming DSI stimulation in successive spike trains, the membrane potential showed a depolarizing trajectory (Fig. 2C). This effect was only observed after GTP-gamma -S or GMP-PNP injection and not after HEPES (not shown) or GDP-beta -S injection (Fig. 2A). Thus it might be due to a continual activation of G-protein pathways resulting from DSI stimulation.



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Fig. 2. GTP analogues lead to a persistent depolarization in DFN following DSI stimulation. A: under control conditions, repetitive DSI stimulus trains (1-min intervals) have no cumulative effect on the DFN membrane potential. After injection of GDP-beta -S into the DFN, repetitive DSI stimulus trains in 1-min intervals still do not have a long-lasting effect on the DFN membrane potential. B: after injection of a GTP analogue (here: GTP-gamma -S), DSI stimulation leads to a persistent depolarization of the DFN membrane potential. C: this postinjection effect is dependent on DSI stimulation. Before injection (open circle ), the repetitive DSI stimulus trains do not lead to any changes in the DFN membrane potential. After injection () of a GTP analogue (here: GMP-PNP), the repetitive DSI stimulation trains lead to a gradual depolarization of the DFN. Measurements of membrane potential were 1-s averages obtained every 60 s, 2 s before the onset of a DSI stimulus.


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Our results support the previously proposed hypothesis that the fast DSI-evoked EPSP in DFN is mediated by ionotropic receptors and the slow DSI-evoked potential is mediated by metabotropic receptors. Here we found that the time course and amplitude of only the slow potential could be enhanced by injecting the postsynaptic neuron with GMP-PNP or GTP-gamma -S. Furthermore injection of GDP-beta -S reduced the amplitude of the slow component without significantly affecting the fast. Thus these results suggest that ionotropic receptors mediate the fast EPSP, whereas metabotropic (G-protein-coupled) receptors mediate the slow potential.

The continuously rising depolarization caused by GTP analogues might result from a subsequent activation of additional G proteins initiated by DSI stimulation rather than a persistent activation of the DSI-activated receptors themselves. We cannot exclude the possibility that metabolic cross talk between different enzymatic (Diversé-Pierlussi et al. 1997) or other, concentration-dependent (Yamoah and Crow 1996) pathways could be responsible for this effect since injection of the GTP analogues might have also activated other metabotropic receptor pathways.

Our data are consistent with the results of Green et al. (1996) who showed that 5-HT can activate fast ligand-gated ion channels in mollusks. The presence of such synaptically activated ionotropic 5-HT receptors in Tritonia supports the notion that 5-HT can mediate fast as well as slow actions in molluscan systems.

Getting (1989a,b) proposed that different time courses of multicomponent synapses in the CPG may play critical roles in producing the motor pattern. Because the DSI to C2 synapse within the CPG behaves very similarly to the DSI to DFN synapse examined in this study (Fickbohm and Katz 2000; Katz and Frost 1995a), it is likely that it also is mediated by a combination of both ionotropic and metabotropic receptors. This could indicate that metabotropic pathways might be directly involved in the generation of the swim motor pattern rather than only in its modulation and therefore actually contribute in shaping the motor output.

A role for metabotropic regulation of intrinsic neuronal properties has also been reported in several vertebrate systems (e.g., Bianchi and Wong 1995; Bongianni et al. 1999; Delgado-Lezama et al. 1997; Krieger et al. 1998). Thus a continuous regulation of the intrinsic properties of CPG neurons via metabotropic receptors could be a more general feature of neuronal networks and a mechanism to control the activity of ongoing network oscillations (Whittington et al. 1995).


    ACKNOWLEDGMENTS

We thank D. Fickbohm for many helpful discussions throughout this work.

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-35371 to P. S. Katz.


    FOOTNOTES

Address for reprint requests: S. Clemens, Dept. of Biology, Center for Neural Communication and Computation, Georgia State University, 24 Peachtree Center Ave., Atlanta, GA 30303 (E-mail: bioscl{at}panther.gsu.edu).

Received 14 August 2000; accepted in final form 16 October 2000.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society