Transition to Endogenous Bursting After Long-Term Decentralization Requires De Novo Transcription in a Critical Time Window

Muriel Thoby-Brisson and John Simmers

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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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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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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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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Fig. 1. Functional recovery of lobster pyloric network rhythmicity and associated changes in pyloric dilator (PD) neuron bursting after long-term stomatogastric ganglion (STG) decentralization in vitro. A: pyloric rhythm recorded extracellularly on pd motor nerve and intrasomatically from PD and lateral pyloric constrictor (LP) neurons in freshly dissected intact stomatogastric nervous system (STNS) (see schematic). B: 1 h after cutting the stomatogastric nerve (stn) the pyloric network is silent. C: same preparation on day 5 in organ culture. The long-term decentralized pyloric network re-expresses coordinated rhythmic activity. D-F: PD neuron activity under same conditions as A-C, respectively. In stn intact control (D), depolarizing current injection (i) increases burst frequency (D1), and single plateau potentials are triggered by brief current pulses (D2). Immediately after stn section (E), injected current elicits only tonic PD firing (E1) and short current pulses fail to trigger single oscillations (E2). F: 5 days postdecentralization, a different PD neuron expresses spontaneous oscillations that are sensitive to both tonic (F1) and pulsed (F2) current injection. pdn, pyloric dilator nerve; CoG, commissural ganglion; OG, esophageal ganglion.


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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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Fig. 2. Effects of actinomycin D (ACD) concentration on the PD neuron's ability to generate rhythmicity in connected (gray bars) and long-term disconnected (black bars) STG in organ culture. Each bar represents the percentage of preparations (number of trials indicated above each bar) expressing continuous or episodic spontaneous PD neuron bursting on day 5 in vitro following a 4 h exposure of the STG to the designated ACD concentration on day 1.



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Fig. 3. ACD treatment in a critical time window prevents recovery of PD neuron bursting. A-C: for each preparation (schematic at left), the pdn was recorded (right) after 5 days in organ culture. A: PD neuron bursting at day 5 in an intact STNS after STG exposure to 50 µM ACD during 4 h on day 1 in vitro (see protocol schematic). B: recovered PD neuron bursting in a control long-term decentralized STG. C: ACD effects at different postdecentralization times. STG incubation with 50 µM ACD for 4 h at time of stn section on day 1 prevented PD bursting on day 5 (C1), whereas the same treatment from 4-8 (C2) and 24-28 h (C3) postdecentralization did not prevent rhythm recovery. In C1, incubation was started 30 min before stn section to maximize the inhibitor's presence at the time of decentralization. Horizontal bars above protocols in A and C indicate ACD application; arrows in B and C indicate stn transection. D: in an ACD recovery-blocked preparation (as in C1), depolarization of a silent PD neuron evokes only tonic firing (D1), whereas in a recovered ACD-treated preparation (as in C2 and C3), depolarizing current drives burst-generating oscillations (D2).

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).


    DISCUSSION
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ABSTRACT
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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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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