Department of Biology, Center for Neural Communication and Computation, Georgia State University, Atlanta, Georgia 30303
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ABSTRACT |
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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--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-
-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.
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INTRODUCTION |
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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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METHODS |
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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--S), guanosine
5'-O-(3-thiotriphosphate) (GTP-
-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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RESULTS |
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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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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--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-
-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-
-S on the
fast component was not significantly reduced from that of the HEPES
control alone (Fig. 1C). However, GDP-
-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--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-
-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-
-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--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-
-S or
GMP-PNP injection and not after HEPES (not shown) or GDP-
-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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DISCUSSION |
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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--S. Furthermore injection of GDP-
-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
).
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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