Laboratoire de Neurobiologie des Réseaux, Université Bordeaux I and Centre National de la Recherche Scientifique- Unité Mixte de Recherche 5816, 33405 Talence, France
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ABSTRACT |
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Thoby-Brisson, Muriel and John Simmers. Transition to Endogenous Bursting After Long-Term Decentralization Requires De Novo Transcription in a Critical Time Window. J. Neurophysiol. 84: 596-599, 2000. Rhythmic motor pattern generation by the pyloric network in the lobster stomatogastric ganglion (STG) requires neuromodulatory inputs from adjacent ganglia. However, although suppression of these inputs by cutting the stomatogastric nerve (stn) causes the pyloric network to fall silent, network output similar to that expressed when the stn is intact returns after 3-4 days in organ culture. Intracellular recordings from identified pyloric dilator (PD) neurons indicate that the fundamental change underlying rhythm recovery resides with the intrinsic excitability of pyloric neurons themselves, since the prolonged absence of extrinsic modulatory inputs allows the expression of an endogenous oscillatory capability that is maintained in a strictly conditional state when these inputs are present. To examine whether gene transcription was involved in this change in neuronal behavior, we performed in vitro experiments in which the STG was exposed to the RNA-synthesis inhibitor actinomycin D (ACD). ACD (50 µM) incubation at the time of decentralization prevented subsequent reacquisition of PD neuron bursting, but the inhibitor was much less effective when applied at later postdecentralization times, suggesting that the recovery process arises from new protein synthesis triggered when modulatory inputs are first removed. Moreover, in the nondecentralized STG, trans-synaptic modulatory instruction may sustain the conditional pyloric network phenotype by continuously regulating expression of genes responsible for intrinsic neuronal rhythmogenesis.
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INTRODUCTION |
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Motor rhythm-generating networks
depend on neuromodulatory inputs to modify network activity as
behavioral demands change (Marder and Calabrese 1996).
In addition to such short-lasting adaptive actions, modulatory inputs
may also exert long-term trophic influences that continuously regulate
target network operation, in a manner similar to other types of
cell-cell interactions in the peripheral and CNS (Hyatt-Sachs et
al. 1993
; Martinou and Merlie 1991
;
Traynor et al. 1992
; Weiser et al. 1994
).
For example, in the lobster stomatogastric nervous system (STNS), the
pyloric motor network falls silent on elimination of neuromodulatory
inputs from anterior ganglia (Bal et al. 1988
). However,
after several days in organ culture the decentralized network gradually
recovers a pattern-generating capability that no longer depends on
these inputs (Thoby-Brisson and Simmers 1998
). Thus the
prolonged absence of modulatory inputs to the pyloric network allows
the emergence of a modulation-independent rhythmogenic property that is
maintained in a modulation-dependent state when these inputs are present.
To explore the mechanisms that underlie such long-term trans-synaptic
control of pyloric network function, we first compared the endogenous
burst-generating capability of an identified network element, the
pyloric dilator (PD) neuron, before and after short- and long-term
network decentralization. Second, since changes in gene expression and
protein synthesis generally underlie long-lasting plasticity in neural
circuit function (Goelet and Kandel 1986), we assessed
the effect of transcriptional blockade, using the RNA-synthesis
inhibitor actinomycin D (Pedreira et al. 1996
;
Reich and Goldberg 1964
), on the recovery of PD neuron
bursting after decentralization. Moreover, since long-term
modifications in neuronal properties often involve early gene
expression within a narrow time window (Montarolo et al.
1986
; Nguyen et al. 1994
; O'Leary et al.
1995
), we explored the effects of RNA synthesis inhibition on
the reacquisition of PD neuron bursting at discrete intervals during
and after stomatogastric ganglion (STG) decentralization. Our results
suggest that recovery of network rhythmicity arises from a change in
intrinsic excitability of individual pyloric neurons, requiring a
critical period of transcription at the time modulatory inputs are eliminated.
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METHODS |
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Experiments were performed on adult spiny lobsters, Jasus
lalandii. Dissections of the STNS were as described previously
(Thoby-Brisson and Simmers 1998). Briefly, the STNS of
ice-cold anesthetized animals were isolated and pinned out in a Sylgard
(Dow Corning)-lined petri dish under sterile-filtered lobster saline
[composition (in mM): 480 NaCl, 12.75 KCl, 3.9 MgSO4, 13.7 CaCl2-2H2O, and 5 HEPES, pH
7.45]. For long-term organotypic preparations (maintained at 15°C),
glucose (1 g/l), penicillin (35 µg/ml), and streptomycin (50 µg/ml)
were added to the bathing saline, which was renewed daily.
The isolated "combined" STNS consisted of the STG (with motor
nerves; Fig. 1A) attached via
the stomatogastric nerve (stn) to the bilateral commissural ganglia
(CoGs) and single esophageal ganglion (OG). The pyloric network in the
STG was disconnected from descending modulatory inputs by cutting the
stn. Actinomycin D (ACD; Sigma), diluted in lobster saline, was applied
in a Vaseline well constructed around the desheathed STG, for 4 h,
with hourly renewal. Extracellular motor nerve and intrasomatic
recordings were made as previously described (Thoby-Brisson and
Simmers 1998).
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RESULTS |
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The lobster pyloric network is continuously active in the combined
STNS in vitro (Fig. 1A), although when STG inputs from anterior ganglia are eliminated by cutting the stn, the motor network
immediately falls silent (Fig. 1B). However, as previously reported (Golowasch et al. 1999; Thoby-Brisson
and Simmers 1998
), over 3-4 days in organ culture, pyloric
network rhythmicity gradually resumes (Fig. 1C). Thus the
prolonged absence of modulatory input causes a fundamental alteration
in network function, allowing it to generate rhythmicity without the
inputs on which it normally depends.
Changes in intrinsic neuronal excitability underlie this network recovery, as is evident in Fig. 1, D-F, where the ability of PD neurons to express burst-generating properties was tested with intracellular current injection. With intact modulatory inputs (Fig. 1D), the frequency of spontaneous PD oscillations increased with continuous membrane depolarization (Fig. 1D1), and single bursting plateau potentials were triggered with brief current pulses (Fig. 1D2). Soon after modulatory inputs were suppressed, the same neuron ceased to oscillate, even when continuously depolarized (Fig. 1E1), and current pulses failed to elicit active membrane responses (Fig. 1E1). However, four days postdecentralization, an impaled PD neuron (n = 12) expressed strong oscillatory behavior that again displayed voltage-dependent, regenerative responsiveness to tonic (Fig. 1F1) and pulsed (Fig. 1F2) current injection. These changes, which have also been observed in other pyloric cell types following long-term STG decentralization (unpublished data), indicate a crucial alteration in the intrinsic electrical character of these neurons, namely the transition from bursting neurons (Fig. 1, A and D) that are unable to operate without permissive modulatory inputs (Fig. 1, B and E) into chemo-independent bursters that oscillate freely without input (Fig. 1, C and F).
To determine whether this functional plasticity required changes in
gene transcription, the influence of short-lasting ACD exposure on
spontaneous PD neuron bursting in organotypic preparations was tested
initially to establish inhibitor concentrations that did not interfere
with basic neuronal function (Schwartz et al. 1971). As
seen in Fig. 2, 3 µM ACD applied for
4 h on freshly dissected STG had no observable effect at day 5 on
spontaneous PD bursting in either stn intact or stn cut preparations.
At 50 µM, ACD still had no apparent effect on the neuron's capacity to burst at day 5 in all 6 intact STNS tested (Figs. 2 and
3A). By contrast, at this
concentration in 8 of 10 long-term decentralized STG, rhythm recovery
typically seen in untreated (Fig. 3B) or 3 µM ACD-treated
STG failed to occur (Figs. 2 and 3C1). ACD at higher
concentrations (100-350 µM) prevented all rhythmicity by day 5 in
more than 80% of both connected and long-term disconnected STG (Fig.
2). Thus 50 µM ACD blocked RNA synthesis required for reacquisition
of PD bursting following decentralization but not any
"housekeeping" synthesis needed to maintain ongoing activity in the
stn intact preparation.
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Importantly, the suppressive effects of ACD on rhythm recovery were most effective within a specific window around the time of STG decentralization. Whereas ACD applied at the time of stn section completely blocked subsequent PD rhythm recovery (Fig. 3C1), exposures at 4-8 h (Fig. 3C2; n = 8 preparations) and 24-28 h (Fig. 3C3; n = 4) postdecentralization failed to prevent re-expression of at least slow PD neuron bursting by day 5. The mean cycle frequency (±SE) of recovered bursting in all such experiments (0.18 ± 0.04 Hz, e.g., Fig. 3C2; 0.17 ± 0.03 Hz, e.g., Fig. 3C3) was not significantly different (P > 0.05, paired Student's t test) for these two ACD treatment paradigms, although it remained lower than in untreated decentralized controls (0.37 ± 0.19 Hz, n = 5; e.g., Fig. 3B).
Finally, the absence of PD rhythm recovery following RNA synthesis inhibition (as in Fig. 3C1) was not due simply to hyperpolarization of neurons below threshold for voltage-dependent oscillations. On day 5 in transcription blocked preparations (Fig. 3D1; n = 4), experimental depolarization of an otherwise silent PD neuron induced tonic firing only, in contrast to preparations with recovered bursting (e.g., Fig. 3, B, C1, and C2) where injected positive current readily activated PD neuron oscillations (Fig. 3D2).
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DISCUSSION |
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Our results reported here on the PD neuron, together with
observations on other pyloric cell types (unpublished data), indicate that the functional recovery of the lobster pyloric network after long-term suppression of modulatory inputs derives from a fundamental modification in the oscillatory properties of individual neurons whereby they lose their conditional, chemo-dependent character to
become true endogenous oscillators. Although the mechanism(s) by which
the switch from the conditional (stn intact) to nonconditional (long-term stn cut) phenotype is achieved remains unknown, these findings reinforce the previous conclusion (Thoby-Brisson and Simmers 1998) that modulatory inputs normally maintain
expression of the pyloric network in a modulation-dependent state by an
active regulatory process.
Since postdecentralization recovery of pyloric rhythmicity involves de
novo transcription, a plausible interpretation is that the expression
of gene products that would allow endogenous neuronal bursting is
continuously prevented by modulatory input in the intact STNS. We do
not know yet whether the transition from modulation-dependent to
modulation-independent states requires the appearance of entirely new
proteins and/or changes in synthesis of preexisting proteins. However,
the second hypothesis is supported by electrophysiological evidence
that decentralization induces functional alterations in membrane
conductances already present in pyloric neurons (Thoby-Brisson and Simmers 1997; unpublished data). It is also important
to realize that in the present study, RNA-synthesis inhibition was
assessed by applying ACD to the intact pyloric network in which, for
example, the PD neurons remained electrically coupled to the strongly
oscillatory AB interneuron. Therefore, until cell photo-ablation
experiments are performed, conclusions about the specificity of ACD's
action on individual neuron(s) must be drawn with caution.
Finally, the fact that reacquisition of rhythmicity is blocked by an
initial brief exposure to ACD at the time of decentralization suggests
that once triggered, short-lasting, early gene transcription is
followed by a cascade of cellular events (Armstrong and Montminy 1993) that requires several days before the change in neuronal phenotype is finally achieved. This biochemical sequence, indicated here for the first time to underlie persistent, modulatory
input-dependent plasticity in a rhythmic motor system, is reminiscent
of the critical periods necessary for the induction of other long-term
adaptive processes involving de novo macromolecular synthesis both in
the developing (Ribera and Spitzer 1989
) and mature
(Montarolo et al. 1986
; O'Leary et al.
1995
) nervous system. However, the finding that later
applications of ACD continue to influence the reacquisition of pyloric
rhythmicity, as indicated by the fact that in these cases the recovered
rhythm remains slower than that seen under control conditions, suggests
that new gene transcription may continue to contribute to the recovery
process for at least 28 h postdecentralization.
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ACKNOWLEDGMENTS |
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We thank Drs. Denis Combes, Patsy Dickinson, Serge Faumont, and Pierre Meyrand for helpful comments on the manuscript.
This work was supported by the Human Frontier Science Program and a doctoral studentship from the Ministère de l'Enseignement Supérieur et de la Recherche to M. Thoby-Brisson.
Present address of M. Thoby-Brisson: Dept. of Organismal Biology and Anatomy, University of Chicago, 1027 E. 57th St., Chicago, IL 60637.
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FOOTNOTES |
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Address for reprint requests: J. Simmers.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 December 1999; accepted in final form 13 March 2000.
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REFERENCES |
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