Serotonin patterns locomotor network activity in ... - Wiley Online Library

Serotonin Patterns Locomotor Network Activity in the Developing Zebrafish by Modulating Quiescent Periods Edna Brustein,1 Mabel Chong,1 Bo Holmqvist,2 Pierre Drapeau1 1

McGill Center for Research in Neuroscience and Departments of Neurology & Neurosurgery, and Biology, McGill University, Montre´al, Que´bec, Canada H3G 1A4

2

Department of Pathology, University of Lund, So¨lvegatan 25 S-221 85 Lund, Sweden

Received 26 February 2003; accepted 30 June 2003

ABSTRACT:

Developing neural networks follow common trends such as expression of spontaneous, recurring activity patterns, and appearance of neuromodulation. How these processes integrate to yield mature, behaviorally relevant activity patterns is largely unknown. We examined the integration of serotonergic neuromodulation and its role in the functional organization of the accessible locomotor network in developing zebrafish at behavioral and cellular levels. Locally restricted populations of serotonergic neurons and their projections appeared in the hindbrain and spinal cord of larvae after hatching (⬇day 2). However, 5-HT affected the swimming pattern only from day 4 on, when sustained spontaneous swimming appeared. 5-HT and its agonist quipazine increased motor output by reducing intervals of inactivity, observed behaviorally (by highspeed video) and in recordings from spinal neurons during fictive swimming (by whole-cell current clamp).

INTRODUCTION The processes underlying the formation of neural networks during development have gained increasing Correspondence to: P. Drapeau ([email protected].). Contract grant sponsor: CIHR (P.D. and E.B.). Contract grant sponsor: NSERC of Canada (P.D.). Contract grant sponsor: Crafoord Foundation (B.H.). Contract grant sponsor: Swedish Medical Research Council (B.H.). © 2003 Wiley Periodicals, Inc. DOI 10.1002/neu.10292

5-HT and quipazine had little effect on the properties of the activity periods, such as the duration of swim episodes and swim frequency. Further, neuronal input resistance, rheobasic current, and resting potential were not affected significantly. The 5-HT antagonists methysergide and ketanserin decreased motor output by prolonging the periods of inactivity with little effect on the active swim episode or neuronal properties. Our results suggest that 5-HT neuromodulation is integrated early in development of the locomotor network to increase its output by reducing periods of inactivity with little effect on the activity periods, which in contrast are the main targets of 5-HT neuromodulation in neonatal and adult preparations. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 303–322, 2003

Keywords: neuromodulation; locomotion; neural network; development; zebrafish

interest with the prospect of complementing and facilitating the understanding of the functional organization of neural circuits in general (Grillner, 2000). Emerging neural networks share basic patterns of organization because they exhibit similar characteristics during maturation, including expression of spontaneous, recurring activity patterns (O’Donovan, 1999; Ben-Ari, 2001) and the integration of neuromodulatory influences (Pfluger, 1999). The extent to which these processes arise independently or are concerted during development has not been resolved, and determining their roles is essential to understand the emergence of network activity. In the context of the 303

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acquisition of neuromodulation during embryogenesis, the gradual innervation of the well-studied spinal locomotor network by descending brainstem serotonergic axons was correlated with the appearance of more complex firing capacities and the expression of intrinsic oscillatory properties in Xenopus and rat motoneurons (Ziskind-Conhaim et al., 1993; Sillar et al., 1995; Scrymgeour-Wedderburn et al., 1997). However, the acquisition of more complex activity patterns does not necessarily translate to a fully functional locomotor pattern. For example, serotonin (5HT) can induce locomotor rhythm in fetal mice and rats (Branchereau et al., 2000a; Nishimaru and Kudo, 2000), yet these animals do not express mature locomotion at birth (Rajaofetra et al., 1989; Branchereau et al., 2000a; Vinay et al., 2000). In addition, the integration of serotonergic influences during embryonic development cannot easily be separated from and may be linked to other developmental processes, such as the gradual expression of different ionic conductances (Sun and Dale, 1998a; Nakayama et al., 1999; Perrier and Hounsgaard, 2000; Spitzer et al., 2000; Vinay et al., 2000). Furthermore, it is not yet evident whether the cellular effects of 5-HT are essential for locomotor neural network maturation, as interfering with 5-HT levels during embryonic development, for example, whether by increasing its levels transgenically (Cazalets et al., 2000) or by depleting it by neurotoxin injection (Nakajima et al., 1998), caused only a transient delay in postnatal locomotor maturation. To better understand the role of serotonergic neuromodulation in the functional organization of developing neural networks, we have followed the development of the serotonergic system and of the locomotor pattern of the early zebrafish as an accessible network model. We examined embryos and larvae by immunocytochemistry for 5-HT and then activated or blocked 5-HT receptors pharmacologically to test the consequences at the behavioral, network, and cellular levels. Zebrafish swim in a simple pattern consisting of brief (⬇0.2 s) episodes of active swimming, each separated by longer and more variable intervals of rest (Fuiman and Webb, 1988; Buss and Drapeau, 2001). In behavioral experiments, the swimming patterns of agarose-embedded embryos and larvae were recorded by fast video, and the durations of swim episodes and rest intervals, as well as the swimming frequency, were compared before and after acute systemic drug injection. Whole-cell current clamp recordings from spinal neurons of paralyzed larvae in vivo (Drapeau et al., 1999) resolved behaviorally relevant bursts of activity separated by periods of inactivity (Buss and Drapeau, 2001), and the active

and passive properties of individual neurons were compared before and after bath application of serotonergic drugs. Our results suggest that 5-HT modulation was integrated very early during development, even before descending 5-HT axons from hindbrain nuclei that contained 5-HT neurons could be detected. We further found that after the emergence of spontaneous swimming, 5-HT could profoundly change the efficacy of the developing neural network for locomotion by promoting a more constant output. Interestingly, this occurred by reducing periods of inactivity, with little effect on the properties of the active (locomotor) periods that are more commonly the target of 5-HT modulation in neonatal and adult preparations. Preliminary reports of this work have been published in abstract form (Brustein et al., 2000, 2001).

METHODS Experiments were performed on zebrafish (Danio rerio) embryos and larvae, raised at 28.5°C (Westerfield, 1995). All experiments were carried out in compliance with the guidelines stipulated by the Canadian Council for Animal Care, McGill and Lund Universities.

Immunocytochemistry The development of the serotonergic system was examined both in whole-mount and in sectioned preparations. To define the time of first 5-HT immunoreactivity, embryos were examined every second hour from 12–55 h and larvae every 6 –12 h until day 5. The embryos and larvae were fixed in paraformaldehyde (4%) in phosphate buffer (0.1 M, pH 7.2) for 4 –16 h at 4°C. For cryosectioning, the fixed embryos and larvae were first rinsed with 0.1 M phosphate buffer and then immersed for 2– 6 h in a mixture containing 25% sucrose and 25% Tissue-Tek diluted in PBS. Embryos or larvae were then freeze-mounted in groups of six to eight in 100% Tissue-Tek, oriented for transverse, sagittal, and horizontal views, and cut into 10 –20 ␮m sections. The sections were collected on chrome-alum gelatin-coated slides. Whole-mounts were permeabilized at room temperature for a total of 2 h in 1% Triton X-100 in PBS, while sections were rinsed for a total of 20 min. Cryosections and whole-mounts were labeled for 5-HT using a standard protocol for fluorescence immunocytochemistry (adapted from Westerfield, 1995). Briefly, the preparations were incubated at 4°C for 16 –24 h in primary polyclonal antibodies against 5-HT (Incstar) diluted 1:500 in PBS-Triton X-100 solution containing 1% bovine serum albumin. After repeating the rinsing procedures, the preparations were incubated (1 h for cryosections and overnight at 4°C for whole-mounts) in rabbit FITC-conjugated secondary antibodies (Jackson, USA), diluted 1:50 in PBS containing 1% bovine serum

5-HT Modulation and Network Maturation albumin. Excluding the primary antiserum from the protocol abolished the labeling (not shown). Analysis and digital photodocumentation were performed using laser confocal scanning and epi-fluorescence microscopy. Confocal images were reconstructed using COMOS software version 7.1 and converted from black and white PICT files to color TIFF files with Confocal Assistant software version 4.02. Scanning parameters are given in the Figure 1 legend. To colocalize the 5-HT immunoreactive elements and motoneurons, double labeling experiments were performed. In these experiments motoneurons were retrogradely labeled a day before fixation by injecting 15% fluoresceindextran (Molecular Probes) into the muscle overlying one somite near the anus and then processed as whole-mounts for 5-HT immunolabeling using TRITC-conjugated secondary antibodies (diluted 1:400; Sigma). The immunofluorescence in the double-labeled specimens was documented using confocal microscopy, and corresponding images (taken at steps of 0.5 ␮m) were superimposed using Adobe Photoshop software.

Behavioral Experiments Embryos [2.3–2.5-(2)-days-old; dechorionated under anesthesia, 0.2% MS-222; Sigma], 3.3–3.5-(3)- and 4 – 4.5-(4)day-old larvae from 3–10 different batches were video recorded (at 60 or 120 Hz) during spontaneous free swimming (total of n ⫽ 40, not illustrated) or after being embedded in low melting point agarose (1%; Gibco BRL) with the tail free to move (total of n ⫽ 75, see Table 1 for exact details). The video images were taken using CCD cameras (Panasonic wv-bp510 or Pulnix TM-640) mounted on a dissection microscope and were registered on a videotape recorder (Panasonic AG-1960). As the swim patterns were similar under both experimental conditions, the pharmacological experiments were undertaken using the “agarose” paradigm, which permitted us to monitor more easily and quantify the swimming behavior and also to compare the behavior of the same larva before and after drug injection. 5-HT (Sigma), quipazine (RBI), methysergide (MT; RBI), ketanserin (KE; Sigma), or vehicle solution were pressure ejected with a Picospritzer (General Valve Co.) using fine glass needles into the pericardial sac of agaroseembedded embryos and larvae. The drugs were dissolved in extracellular Evans solution (pH 7.8, 290 mOsm, for composition see the Cellular Recordings section) with the vital dye Fast green (1%; Sigma) to monitor the quality of the injections. The drug concentrations for the injections (50 ␮M–1 mM) were chosen initially according to a common concentration range reported in the literature for these drugs. We estimate the effective drug concentration to be ⬇15 times less than that of the injected solution based on the injected volume (⬇0.5 ␮L) and the larva’s volume (⬇3 ␮L). These estimated concentrations (3–50 ␮M) are consistent with the effective concentrations used in the bath during our cellular recordings (see Cellular Recordings section). The swimming behavior was documented before, immediately after the injection, and 30 to 60 min later. A time

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code (SMPTE) was recorded onto each video field using a time code generator (TG-50 Horita) to enable offline reconstruction of the temporal distribution of the swim episodes during equal time recording sessions before and after drug injection in the same preparation and to calculate the swim and rest interval durations. Swimming frequency was estimated from the tail beat frequency and was determined as the number of left to right tail alternations per swim episode.

Cellular Recordings Four-day-old larvae demonstrating spontaneous swimming were minimally dissected under anesthesia (0.2% MS-222; Sigma) to expose a length of spinal cord spanning a few somites, as described previously (Drapeau et al., 1999). Whole-cell current-clamp recordings were obtained in vivo from spinal neurons visualized with Hoffman modulation optics (40X water immersion objective), using patch electrodes filled with (in mM): D-gluconic acid 105; KCl 16; MgCl2 2; HEPES 10; EGTA 10; Na3ATP 4, adjusted to pH 7.2, 290 mOsm. SulforhodamineB (2%) was included in the solution for further neuronal identification under fluorescent illumination at the end of the experiment. Motoneurons had a distinct axonal arborization in the muscle bulk whereas interneuronal processes were restricted to the spinal cord. During the experiments the extracellular solution containing anesthesia was replaced by Evans solution (in mM: 134 NaCl; 2.9 KCl; 2.1 CaCl2; 1.2 MgCl2; 10 HEPES; 10 glucose, pH 7.8, 290 mOsm; Drapeau et al., 1999) containing d-Tubocurarine (15 ␮M; Sigma) to paralyze the preparations. After 5–10 min of control recordings, 5-HT (7– 60 ␮M), quipazine (2–10 ␮M), or MT (2.5–25 ␮M) were bath perfused, followed by a period of washout. MT was preferred over KE for these experiments as its effects on swimming, although similar, were more robust and longer lasting. The recordings, which lasted up to an hour, were made at room temperature using an Axopatch 200B amplifier, and data were digitized online at 40 kHz using Clampex 8.0 software (Axon Instruments). Analysis of the fictive swimming pattern was done offline semiautomatically with Clampex 8.0 software and included spinal neurons with constant input resistance (ranging 150 –350 M⍀) and resting potentials more negative than ⫺60 mV. The fictive swimming frequency was evaluated by counting the number of large, rhythmic EPSPs during the synaptic drive for each swim episode, as established previously (Buss and Drapeau, 2001).

Statistical Analysis Most of the experimental results (unless stated otherwise in the text) were not distributed normally and were therefore compared using nonparametric statistical tests (Sigmastats software) such as the Mann-Whitney rank sum test when comparing two experimental groups or the Kruskal-Wallis analysis of variance on ranks when comparing more than two experimental groups. The effects of the drugs on the different swim pattern parameters, such as number of swim

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Table 1 Summary of Histological Results, Swimming Development, and Effects of the Drugs on Behavioral Swimming Pattern Days Postfertilization Development

2 Hindbrain:

5-HT system Spinal cord:

Behavioral pharmacology agonists

antagonists

3

4

Rostral ir-clusters (superior Raphe nuclei) Caudal and dorsal ir-cells Hindbrain ir-cells do not appear to project to the spinal cord Ventral segmentally distributed ir-cells Ventrolateral processes and varicosities The ir-elements are in close proximity to motoneurons No ir is observed in the dorsal spinal cord.

Spontaneous swimming

Rare

Irregular and variable

Robust and consistent

5-HT, quipazine (100 ␮M)

Ineffective (0/22)

Ineffective (2/8)

Increase in number of swim episodes Decrease of rest interval duration (11/15) Decrease in number of swim episodes

Methysergide (50–100 ␮M) Ketanserin (100 ␮M)

nt nt

Ineffective (0/6) nt

Increase of rest interval duration (Fragmentation) (16/24)

Numbers in parentheses indicate proportion of affected embryos or larvae. ir, immunoreactive; nt, not tested.

episodes, their durations, and swim frequency, were taken from equal time sessions and compared for each embryo or larva pre- and postdrug. The statistical results were considered different at the level of p ⬍ 0.05. This statistical information was used to define the number of affected larvae per experimental group (see Table 1). Data from individual larvae are presented using distribution histograms, while the statistical results of pooled data from affected larvae in each experimental group are presented mostly using medians, data ranges, and box plots. Each box plot contains a central line representing the median, the box itself delineates 25–75% of the data, and “error bars” represent 10 –90% of the data range while open circles are outlying data points. The regression lines illustrated in Figure 9 were tested for statistical difference according to Zar (1996).

RESULTS Serotonergic System of the Developing Zebrafish To understand the role of 5-HT in the functional maturation of the zebrafish locomotor network, it was important first to determine the development of the 5-HT system and to characterize its components. 5-HT expression patterns were determined by immunocytochemistry from prehatching embryonic (before day 2) to larval stages (up to day 5), the period when

spontaneous swimming emerges and matures (Fuiman and Webb, 1988; Buss and Drapeau, 2001). 5-HT immunoreactivity could be detected to a limited extent as early as day 1 and included only a few cell bodies in the rostral hindbrain while 5-HT immunoreactive populations in the hindbrain and spinal cord were detected at day 2 (not illustrated). Because a functional role for 5-HT was not observed until day 4, the following description of the 5-HT system focuses on the later stages of development. By day 4, two bilateral populations of 5-HT immunolabeled cells were located in the hindbrain. A large rostral cell population was located just below the cerebellum [Fig. 1(A) and (B) and their corresponding schematic illustrations], while a smaller population was located dorsolaterally in the caudal hindbrain [Fig. 1(C), see also Fig. 2(E)]. The former may correspond to the superior Raphe nuclei of the adult zebrafish (Wullimann et al., 1995), while the latter may be a precursor of the Raphe nuclei complex. At this stage, we could not detect axons extending from the rostral 5-HT hindbrain cluster [Fig. 1(B)], while the descending axonal processes we could detect from the dorsolateral hindbrain 5-HT clusters tapered off after a short distance in the caudal hindbrain [Fig. 1(D)]. The ventral spinal cord also contained a population of bilateral 5-HT neurons [Fig. 2(A,B)] that were segmentally distributed all the way down the trunk

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(not illustrated). The 5-HT neurons had unipolar descending axons [Fig. 2(B)] and correspond to the VeMe interneurons described previously (Hale et al., 2001). These cells were accompanied by a rich intraspinal ventrolateral plexus of 5-HT processes and varicosities [Fig. 2(C), see also Fig. 2(A)], but we did not detect 5-HT immunoreactivity in the dorsal spinal cord [Fig. 2(A)]. To further localize the 5-HT elements in the spinal cord with respect to motoneurons, the latter were retrogradely labeled a day before fixation by injecting fluorescein-dextran into the muscle overlying one somite and then processed as wholemounts for 5-HT immunolabeling (see Methods). Confocal analysis of such double-labeled specimens revealed that the 5-HT cell bodies, which are retained in the adult (Van Raamsdonk et al., 1996), were located in proximity to motoneuron axons as illustrated in the composite images in Figure 2(D), while the more ventrolaterally located 5-HT plexus was in proximity to the motoneuron cell bodies [slightly out of the plane of the image in Fig. 2(D), indicated by arrowheads; for summary see also the corresponding schematic illustration]. The descending axons from the caudal hindbrain 5-HT cluster did not reach the spinal cord and the spinal 5-HT neurons projected caudally and not rostrally [Fig. 2(E,F)]. These observations of detected 5-HT populations are consistent with studies using retrograde labeling (see Discussion). Taken together, these findings suggest that during the period in which the swimming pattern emerges in the zebrafish larva, 5-HT neural populations and plexi are present both in the hindbrain and in the spinal cord.

Serotonergic Modulation of the Swimming Pattern To study the effects of 5-HT on the emerging behavioral locomotor pattern, we tested the effects of 5-HT, its broad spectrum agonist quipazine, and of the 5-HT antagonists KE and MT. These compounds were injected after control behavioral recordings into the pericardial sac of agarose-embedded embryos and larvae with the tail free to move [inset, Fig. 3(A), see Methods]. The injections were made over a range of concentrations (from 50 ␮M to 1 mM) with a final effective drug concentration estimated to be ⬇15 times lower (3–50 ␮M, see Methods). The predrug swimming patterns of the agarose-embedded embryos and larvae were similar to those of free swimmers (Fuiman and Webb, 1988; Buss and Drapeau, 2001; Budick and O’Malley, 2000) and the injection itself did not interfere with the heart beat, whose vigorous activity was noted during the entire experiment as an

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index of vitality. Injection of 5-HT up to 1 mM failed to elicit swimming in quiescent embryos and larvae. Likewise, the different serotonergic drugs had little effect on the rarely expressed swimming pattern of 2-day-old embryos (n ⫽ 22, not illustrated) or the variable swimming pattern of 3-day-old larvae [see Figs. 4(A), 7(E), Table 1; Buss and Drapeau, 2001]. However, all of the drugs, but not vehicle [n ⫽ 6, see Figs. 3(C,Ci), 4(C,D)], had a dramatic effect on the robust swimming pattern of the 4-day-old larva (Buss and Drapeau, 2001), which alternates between 20 – 40 swim episodes per minute, each lasting ⬇0.2 s [see top graphs in Fig. 3(A–C)] with longer and more variable rest intervals [0.7–2 s, see top right histograms in Fig. 3(Ai–Ci)]. The effects of 5-HT on the 4-day-old larval swimming pattern are described in detail below. Effects of 5-HT and Its Agonist Quipazine. Figure 3 illustrates the results of 5-HT, quipazine (100 ␮M), and vehicle injection in three different 4-day-old larvae [Fig. 3(A,Ai), (B,Bi), (C,Ci), respectively]. The temporal distribution plots of swim episodes showed that 2 min following injection of 5-HT or quipazine [Fig. 3(A,B), respectively] there was a prominent increase in the number of swim episodes (each symbol), from 47 to 83 with 5-HT and from 31 to 57 with quipazine. This effect wore off gradually, returning to control values 30 – 60 min later (not illustrated). The number of swim episodes in the vehicle experiment did not change significantly and was 36 and 33 preand postinjection, respectively [Fig. 3(C)]. A summary of the changes in the number of swim episodes across pooled 5-HT/quipazine experiments (n ⫽ 11/ 15) and those of vehicle (n ⫽ 6) is given in Figure 4(A). On average the number of swim episodes after 5-HT/quipazine injection increased twofold (t test, p ⬍ 0.001), while no change was observed across vehicle experiments (n ⫽ 6). Variable changes were observed in the median of swim episode duration after the injections in different larvae. In the example given in Figure 3(A,Ai) (black bars), the median swim episode duration was 0.17 and 0.21 s (p ⬍ 0.001) in control and after 5-HT, respectively. No change was observed in the quipazine experiment [Fig. 3(B,Bi) black bars, median ⫽ 0.22 s], while in the vehicle experiment [Fig. 3(C,Ci) black bars] the medians were 0.22 and 0.23 s pre- and postinjection, respectively (p ⬍ 0.05). The total changes in the distribution of swim episode durations and their medians are illustrated in Figure 4(C) for pooled 5-HT/quipazine data and for vehicle experiments. A small but statistically significant increase was observed in the total median values after the drug

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Figure 1 The 5-HT hindbrain immunoreactive system of the zebrafish larva. Image orientation is rostral to the left and ventral at the bottom, unless indicated otherwise. (A) Photomicrograph and corresponding schematic illustration, taken from a dorsal view, of large 5-HT cell clusters (arrows) in the rostral hindbrain of a 4.5 day (d) whole-mounted (wm) larva. The (*) indicates the eyes. (B) Confocal image and corresponding schematic illustration (wm, 4 day) of the large rostral 5-HT cell clusters shown in (A) (taken from another specimen) and scanned from a lateral angle at 2.5 ␮m steps for total depth of 75 ␮m. (C) Horizontal cryosection (cs, 4 day) illustrating the small dorsolateral 5-HT cell clusters (large arrow) in the caudal hindbrain and their descending processes (small arrow). Arrowhead indicates the midline. (D) Confocal image (lateral view, wm, 4.5 day) of descending processes originating from dorsolateral 5-HT cells illustrated in (C). The processes (scanned at 0.5 ␮m steps, total depth of 2.5 ␮m) taper off (arrow) in the caudal hindbrain.

injections, including those after vehicle injection (p ⬍ 0.001): from 0.19 predrug to 0.21 s post-5-HT/ quipazine and from 0.22 to 0.23 s pre- and postvehicle injections, respectively. Despite these changes in the median values, 90% of the swim episode durations fell into the same range before and after the drug or vehicle injection across experiments. Furthermore, we

observed no change in the swimming frequency (estimated as the tail beat frequency, see Methods) following 5-HT injection [median of 25 Hz, Fig. 4(B)]. The increase in the number of swim episodes after 5-HT or quipazine injection was due to a pronounced decrease (up to 50%) in the rest interval duration [Fig. 3(Ai,Bi) open bars, data from Fig. 3(A) and (B),

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Figure 2 The 5-HT spinal immunoreactive system of the zebrafish larva. Image orientation is rostral to the left and ventral at the bottom, unless indicated otherwise. (A) Spinal cord cross section (4.5 day) showing ventromedial, bilaterally located serotonergic cells (large arrows) and ventrolaterally located serotonergic processes (small arrows). (B) Lateral view with a slight angle (3.5 day, wm) of spinal 5-HT cells (large arrows) and their descending axons (small arrows). Note the contralateral homologues (out of focus) with ventrally located 5-HT cell bodies in the background. The dashed line delineates the ventral border of the spinal cord. (C) Confocal image (lateral view, wm, 4.5 days) of 5-HT processes and varicosities (scanned at 0.1 ␮m steps, total depth of 0.5 ␮m) in the ventrolateral spinal cord. (D) Composite confocal images and corresponding schematic illustration (lateral view, 4 day, wm, scanned at 0.5 ␮m steps, total depth of 3.5 ␮m) of motoneurons retrogradely labeled with fluorescein-dextran (green cell bodies and axons are also indicated by asterisks and arrows, respectively) to illustrate the proximity of 5-HT immunoreactive cells (blue) to the motoneuron axons. Arrowheads indicate the location (slightly out of image plane) of the ventrolateral 5-HT plexus located in close proximity to motoneuron cell bodies. The dashed line delineates the ventral border of the spinal cord. (E) Sagittal section (4.5 day, cs) of the hindbrainspinal cord border showing the dorsolateral 5-HT cells in the hindbrain and a box localizing the magnified image in (F). (F) Magnified confocal image of the boxed region in (E), showing that at 4.5 days, hindbrain 5-HT processes (arrow) do not cross the hindbrain-spinal cord border (dashed line). The most rostral, descending spinal cord 5-HT processes can also be seen (arrowhead). Scale bars ⫽ 50 ␮m.

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Figure 3

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respectively] across all experiments [Fig. 4(D), medians: 1.4 and 0.85 s before and after drug, respectively; p ⬍ 0.001]. No such changes were observed in the vehicle experiments [Fig. 3(Ci) open bars, data from Fig. 3(C), Fig. 4(D) median ⫽ 1.6 and 1.5 s before and after drug, respectively, across n ⫽ 6 experiments]. We further tested the effects of 5-HT and quipazine on the fictive swimming activity patterns recorded from identified spinal neurons in paralyzed larvae (see Methods), consisting mostly of motoneurons (n ⫽ 18), but with representative sample interneurons (n ⫽ 8). This allowed us to examine cellular as well as network properties because the fictive swimming pattern is synaptically driven, thus reflecting network activity (Buss and Drapeau, 2001). Generally, a lower number of fictive swim episodes were observed under control conditions and they were sometimes clustered into groups (Fig. 5 “control”). However, their properties, such as the duration of fictive swimming events and the swimming frequency (estimated as the frequency of large rhythmic EPSPs during the synaptic drive for each period of fictive swimming), resembled the properties of real swimming, as established previously (Buss and Drapeau, 2001). Neither 5-HT nor quipazine evoked fictive swimming in quiet preparations in the range of doses tested; however, in preparations that produced spontaneously fictive swimming (n ⫽ 26), these substances increased the number of swim episodes [Fig. 5(A,B); see additional examples in Figs. 6(C) and 8(B,D)], much like in the behavioral experiments (Fig. 3). However, it is evident from the expanded traces in Figure 5(A,B) (bottom) that the depolariza-

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tion during fictive swim episodes pre- and postdrug was similar in extent. In addition, the fictive swimming frequency was apparently unaffected by 5-HT or quipazine [Fig. 5(Ai,Bi), median ⫽ 32–34 Hz, data taken from (A) and (B), respectively]. The lack of changes in either fictive swim frequency or duration of the fictive swim episodes was consistent across all experiments [Fig. 6(A)]. Further, no significant change was observed in the range or the median of spiking frequencies during fictive swimming measured in four experiments [Fig. 6(B), medians of 56 and 61 Hz pre- and post-combined 5-HT and quipazine data, respectively]. The clear increase in the number of swim episodes under 5-HT indicates an action on the locomotor network. To test whether the integrative membrane properties of individual neurons were affected by 5-HT, the input resistance and the minimal amount of current (rheobase) necessary to evoke spiking were tested. 5-HT did not have a significant effect on the input resistance [Fig. 6(C,E), 98 ⫾ 7% of control, n ⫽ 9], rheobase [Fig. 6(D,E), 101 ⫾ 12% of control, n ⫽ 5], or the resting potential [Fig. 6(E), 102 ⫾ 3%, n ⫽ 9], and, as described above, did not affect the spiking frequency during fictive swimming. Taken together, these results suggest that 5-HT had no obvious effect on the integrative membrane properties of the individual neurons we recorded from. Thus, in both the behavioral and the fictive experiments, 5-HT appeared to modulate primarily the rest intervals rather than the active swim episode. Effects of 5-HT Antagonists. As described above, injection of 5-HT and quipazine showed that the 4-day-old larval (but not earlier) spontaneous locomo-

Figure 3 Effects of 5-HT, quipazine, and vehicle injection on the spontaneous swim pattern of 4-day-old larvae. (A–Ai) An example of the effects of 5-HT injection. (A) Temporal distribution plots of swim episodes before (control, top) and 2 min after 5-HT injection (bottom), showing an increase in the number of swim episodes after drug injection into the pericardial sac. The duration of individual swim episodes (each symbol) is plotted throughout the recordings. Note the relatively constant duration of the swim episodes (⬇0.2 s). The photomicrographs in (A) show a 4-day-old larva (4 mm long) embedded in agarose in natural position with the tail free to move within the cutout agarose (dashed lines) during a swim episode and a rest interval (left and right images, respectively). (Ai) Distribution histograms of swim episode (black bars) and rest interval (open bars) durations before (control, top) and post-5-HT (bottom), showing that rest interval durations decreased while the range of swim episode duration stayed the same [data taken from the example in (A)]. See Results for statistics. (B–Bi) Same format as (A–Ai). Injection of the 5-HT agonist, quipazine (2 min), had the same effects on the swim pattern as 5-HT. This included an increase in the number of swim episodes [compare graphs (B) top and bottom], a decrease in the rest interval duration [(Bi), open bars], and minimal changes in the range of swim episode duration [(Bi), black bars]. See Results for statistics. (C–Ci) Same format as (A–Ai). Injection of vehicle (5 min) did not affect the swim pattern of a 4-day-old larva [compare graphs (C) top and bottom and related histograms in (Ci)].

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Figure 4 Summary of the effects of 5-HT, quipazine, and vehicle on the swim pattern and its characteristics, across experiments. (A) Bar graphs summarizing the effects of 5-HT and quipazine (pooled data across experiments, n ⫽ 11/15, see Methods) and of vehicle (n ⫽ 6) on the number of swim episodes. A twofold increase of control values was observed after 5-HT/quipazine (5-HT/Q) injection in 4-day-old larvae, while no change was observed after vehicle injection or after 5-HT injection in younger 3.5-day-old larvae (n ⫽ 6/8). (B) Box plots (see Methods) illustrating that swimming frequency (tail beat frequency) was not changed after 5-HT injections in 4-day-old larvae (see Results for statistics). (C) Box plots summarizing the distribution of swim episode durations across all experiments before (C) and after 5-HT/quipazine (5-HT/Q, pooled data, n ⫽ 11/15) and vehicle (V, n ⫽ 6). The data distributions show that both under drug and vehicle injection the swim episode duration fell into the same range. However, the median swim episode duration showed a small but significant increase under both conditions (for statistics see Results). Event numbers are given in brackets. (D) Box plots summarizing the distribution of rest interval duration across all experiments before (C) and after 5-HT/quipazine (5-HT/Q, pooled data, n ⫽ 11/15) and vehicle (V, n ⫽ 6). The data distribution shows that after the drug injection there was a significant decrease in rest interval duration after 5-HT/Quipazine across experiments but not after vehicle injection (for statistics see Results). Event numbers are given in parentheses.

5-HT Modulation and Network Maturation

Figure 5 Effects of 5-HT and its agonist quipazine on the neuronal fictive swim pattern. (A,B) Representative traces of fictive swimming activity recorded in motoneurons pre- (control, top) and post-5-HT [(A), center] or quipazine [(B), center], showing an increase in the number of swim episodes postdrug. The triangles and the boxes indicate the location of the expanded traces at the bottom of each column. The left bottom inset superimposes two swim episodes pre- (black traces) and post-5-HT (grey traces) while the right bottom traces in (B) show a number of consecutive fictive swimming episodes pre- (black trace) and post-quipazine (grey trace), to illustrate the similarity in the depolarization extent during fictive swim episodes pre- and postdrugs. (Ai,Bi) Distribution histograms of fictive swimming frequency [data from (A,B), respectively] for control (top) and for 5-HT [(Ai), bottom] or quipazine [(Bi), bottom], showing similar ranges and medians. The insets in (Ai) “control” show a fictive swim episode and its large rhythmic EPSPs (arrows), according to which fictive swim frequency was estimated (see Methods).

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tor pattern was indeed sensitive to serotonergic modulation both behaviorally and during recordings of fictive swimming from spinal neurons. However, these experiments did not indicate whether endogenous 5-HT was essential for locomotor network function. Therefore, we tested the effect of the 5-HT antagonists MT, which blocks 5-HT1-5-HT2 recep-

tors, and KE, which blocks 5-HT2 receptors, behaviorally and during fictive swimming. Both 5-HT1 and 5-HT2 receptor types have been implicated in locomotion in other preparations (Schmidt and Jordan, 2000). Interestingly, the 5-HT2 receptor of the zebrafish larva was recently cloned, and preliminary results showed that bath application of its agonist

Figure 6

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increased pectoral fin activity (Schneider and Fritzky, 2002), although the molecular nature and physiological roles of specific receptor subtypes have yet to be characterized in zebrafish. MT and KE dramatically reduced the number of swim episodes [10 –30% of control, see Fig. 7(E) for pooled data] and caused the appearance of long rest intervals lasting over 30 s [compare Fig. 7(A,B) top and center graphs, the related open histograms in Fig. 7(Ai,Bi), and across all experiments Fig. 7(C) open boxes, median ⫽ 1 and 1.2 s pre- and postdrug, respectively, p ⬍ 0.001]. These antagonists thus fragmented the robust and consistent swimming pattern into clusters of activity. The effects of KE were observed ⬇5 min post-injection and gradually returned to control levels after about 40 min, while MT effects were observed ⬇15 min after the injection and were more consistent, lasting more than an hour [compare Fig. 7(A,B) center and bottom graphs]. Despite the pronounced effect of MT and KE on the swimming pattern, neither the duration of individual swim episodes [Fig. 7(Ai,Bi) black bars, data from (A) and (B), respectively, and pooled data in Fig. 7(C) black boxes, median ⫽ 0.25 s] nor the swimming frequency [Fig. 7(D), median ⫽ 24 Hz] were affected. MT and KE affected the swimming pattern in 67% (n ⫽ 16/ 24) of the experiments (see Table 1), while no effect was apparent in the others. The effects of MT on the fictive swim pattern recorded in spinal neurons (n ⫽ 21) followed the same trend, with a reduced number of swim episodes and appearance of long rest intervals, when MT was applied on its own [Fig. 8(A)] or after application of 5-HT [Fig. 8(B)]. In these lengthy experiments (up to an hour), we sometimes observed a decrease in spike amplitude. The expanded traces at the bottom of Figure 8(A,B) show that the extent of the depolarization

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during the fictive swim episodes was little affected by MT and the fictive swimming frequency was preserved [Fig. 8(C), medians in the control and postMT: 33 and 36 Hz on the left and right plots, respectively], indicating that the network functioned normally during clusters of activity. Interestingly, Figure 8(D) shows that, much like after 5-HT, the input resistance and resting potential were constant in the presence of MT even during the long rest intervals. To better understand the role of 5-HT in determining the activity state of the locomotor network, we further analyzed the clustered swimming pattern observed after MT (n ⫽ 8) for the correlation between the duration of the activity clusters and the interval between clusters (Fig. 9). This type of statistical correlation has been used previously to describe emerging activity in developing networks, dominated by long quiescent periods (O’Donovan, 1999; see Discussion). It is evident that the preceding cluster interval could to some extent predict the cluster duration [Fig. 9(A), r ⫽ 0.51]. In contrast, there was no correlation (r ⫽ 0.09) between the following cluster interval and the cluster duration [Fig. 9(B)]. The regression lines in Figure 9(A) and (B) are statistically different (p ⬍ 0.05; Zar, 1996). Thus, when 5-HT receptors are blocked the length of the quiescent period between clusters of activity may contribute in part to determining their duration and not vice versa. Taken together, the experiments with 5-HT antagonists supported our initial observations with the agonists, suggesting that endogenous 5-HT at these early stages of development acts primarily to modulate the duration of the rest intervals in order to maintain a constant network output with surprisingly little effect on the characteristics of the active swim episode.

Figure 6 Summary of the effects of 5-HT on fictive swimming parameters and on the input resistance, rheobase, and resting potential across experiments. (A) Box plots, illustrating that fictive swim frequency (gray boxes) and fictive swim episode duration (black boxes) were not affected before (C) or after 5-HT application. (B) Spiking frequency distribution showing similar ranges and medians pre- (control, top) and post-5-HT and quipazine (bottom, pooled data from four experiments). The darker bars mark the range of swim frequencies. (C) Representative traces illustrating the constant hyperpolarization amplitude of responses to 50 ms long pulses of ⫺130 pA at 1 Hz, indicating a constant input resistance in the control (left) and after 5-HT (right) during fictive swimming in the same spinal neuron, in spite of a clear increase in the number of fictive swim episodes after 5-HT. (D) Representative depolarizing responses to current steps (50 pA increments) showing that after 5-HT application there was no change in the minimal (rheobase) current needed to evoke spiking pre- (left) and postdrug (right), in the same spinal neuron. (E) The bar graphs summarize the results across all experiments in which input resistance (left bar), rheobase current (Rheo, central bar), and resting potential (right bar) were compared before and after 5-HT. The bars (data normalized to control values) illustrate that these properties showed no obvious change before and after 5-HT application in the neurons.

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Figure 7

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DISCUSSION Our study proposes that endogenous 5-HT is critical in shaping the developing locomotor swimming pattern in zebrafish. Populations of serotonergic neurons and fiber projections were present in both the hindbrain and spinal cord from day 2. However, not until day 4 did 5-HT modulate the robust and consistent swim pattern. The results from behavioral experiments demonstrated that these effects had clear functional relevance, while the results from recordings in spinal neurons during fictive swimming indicated that 5-HT modulated the neural network underlying locomotion. Together, the behavioral and the fictive swimming results suggest that 5-HT targets primarily the properties of the rest intervals, without major effects on the active (swim) period. The implication of 5-HT in the ontogeny of the locomotor pattern will be discussed before considering its role in maintaining locomotor network efficacy.

Serotonin and the Ontogeny of the Locomotor Pattern Despite the presence of 5-HT at early stages, its administration did not evoke or affect the swim pattern earlier than day 4. Similarly, 5-HT does not evoke swimming in Xenopus embryos before hatching

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(Sillar et al., 1995). However, 5-HT can evoke rhythmic activity that is initially bilaterally synchronized in fetal mice and rats (Nishimaru and Kudo, 2000; Branchereau et al., 2000a). In these mammalian preparations the temporal relationship between the arrival of descending 5-HT pathways and the ability to activate the locomotor network remains to be established, as the responses of motoneurons to 5-HT (ZiskindConhaim et al., 1993) and the expression of rhythmic activity precede the innervation of the spinal cord by 5-HT (Rajaofetra et al., 1989; Vinay et al., 2000). Furthermore, suppression of 5-HT synthesis during gestation affected neither the onset nor the time course of 5-HT-induced potentials (Gao and ZiskindConhaim, 1993). Thus, the contribution of other developmental processes that may promote functional changes in the spinal cord, such as changes in neural conductances, cannot be excluded (Sun and Dale, 1998a; Nakayama et al., 1999; Perrier and Hounsgaard, 2000; Spitzer et al., 2000; Vinay et al., 2000). The lack of effects on the locomotor pattern of the zebrafish before day 4 may also be a result of a developmental gradient of 5-HT receptor expression, as suggested for other preparations (reviewed by Schmidt and Jordan, 2000). However, at this time, not much is known about the types of 5-HT receptors or their development in the zebrafish, except for the presence of the 5-HT2 receptor, which was recently

Figure 7 The effects of 5-HT antagonists on the spontaneous swim pattern. (A) Temporal distribution plots of swim episodes before (control, top) and after ketanserin (KE) injection. KE fragmented the robust and consistent swimming pattern into clusters of activity by decreasing the number of swim episodes and causing the appearance of long rest intervals (cluster intervals) lasting over 30 s (5 min, center plot). The effect of KE wore off ⬇40 min later (bottom graph). (B) Temporal distribution plots of swim episodes before (control, top) and after methysergide (MT) injection (center and bottom plots). Much like KE, MT fragmented the robust and consistent swimming pattern into clusters of swim episodes by decreasing the number of swim episodes and causing the appearance of long rest intervals lasting over 30 s (cluster intervals). In contrast to KE, MT effects lasted over an hour (bottom graph). (Ai,Bi) Distribution histograms [data from (A) and (B), respectively] of swim episodes (black bars) and rest interval (open bars) durations, pre- (control, top) and post-injection (bottom) of KE (Ai) or MT (Bi), showing that the swim episode durations were not affected by KE or MT but very long quiescent periods appeared (*). The inset shows the first bin of briefest rest interval durations on an expanded time scale. (C) Box plots illustrating the distribution of swim period (black boxes) and rest interval (open boxes) durations across all experiments pre- (C) and post-KE/MT (pooled data, n ⫽ 16/24), showing that swim episode duration was not affected, but long rest interval outliers (cluster intervals ranging 40 –180 s) appeared after KE/MT. Event numbers are given in parentheses (for statistics see Results). (D) Box plots, illustrating that the swim frequency (tail beat frequency) was not affected before (C) or after MT injection. (E) Bar graphs summarizing the changes in the number of swim episodes (percentage of control) after MT/KE (pooled data, n ⫽ 16/24) or vehicle injection [n ⫽ 6, same data as in Fig. 3(A)] in 4-day-old larvae, as well as after injection of MT in 3.5-day-old larvae (n ⫽ 6/6). The number of swim episodes decreased up to 10 –30% of control values after MT/KE injection in 4-day-old larvae, while no change was observed after vehicle injection or after MT injection in 3.5-day-old larvae.

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Figure 8 Effects of 5-HT antagonists on the neuronal fictive swim pattern. (A) Traces from a motoneuron recording illustrating fictive swimming pre- (control, top), post-MT (middle), and after washout (bottom), showing that MT decreased the number of fictive swim episodes. (B) MT caused clustering (bottom) of the consistent, fictive swimming pattern induced by 5-HT (middle). The triangles in (A) and boxes in (B) indicate the location of the expanded episodes illustrated at the bottom of each column. The inset in (A) superimposes two expanded swim episodes pre- (black) and post-MT (grey), while the traces in the (B) inset show a number of consecutive fictive swim episodes during 5-HT (black traces) and after MT (blue traces) to illustrate the similarity in the depolarization extent during fictive swim episodes pre- and post-drug. (C) Box plots show that the fictive swim frequency across all experiments was preserved under control (C), MT, 5-HT, and washout (W). Event numbers are given in parentheses (see Results for statistics). (D) Responses to 50 ms long pulses of ⫺130 pA at 1 Hz show constant voltage amplitudes, indicating constant input resistance under control, 5-HT, and MT exposure during fictive swimming in the same neuron even during rest intervals (spikes truncated), in spite of clear effects of the drugs on the number of fictive swim episodes.

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Figure 9 Regression analysis of all the data obtained during swimming after MT treatment. Linear regression analysis showing: (A) positive correlation between the cluster duration and the preceding cluster intervals (r ⫽ 0.51) and (B) weak correlation (r ⫽ 0.09) between the following cluster interval and the cluster duration. The two regression equations are statistically different (p ⬍ 0.05, see Methods).

cloned in the larva (Schneider and Fritzky, 2002). The early presence of 5-HT in the zebrafish embryo may be related to induction of other aspects of early locomotor network establishment and development in general, such as differentiation, growth, and synaptogenesis (Lauder, 1993). For example, as 5-HT is suggested to down-regulate gap junctions in other developing networks (Rorig and Sutor, 1996), in zebrafish it may facilitate the transition from an embryonic gap junction-mediated motor network (Saint-Amant and Drapeau, 2001) to a synaptically driven larval locomotor neural network (Buss and Drapeau, 2001). In addition, we found that using 5-HT antagonists did not cause a developmental regression in the locomotor network pattern, but instead altered its activity state. Thus, 5-HT is critical in shaping the locomotor output after an initial organization of the neural network has occurred, much like in Xenopus (Sillar et al., 1995). The close proximity of spinal 5-HT elements to motoneuron cell bodies and axons makes the spinal elements appropriately localized to account for the rapid effects of 5-HT on the locomotor output, as suggested in the lamprey (Wallen et al., 1989). In adult zebrafish there are descending 5-HT projections from the hindbrain coursing to the dorsal spinal cord (Van Raamsdonk et al., 1996). These were not observed in retrograde labeling experiments (Kimmel, 1982; Lee and Eaton, 1991) in the zebrafish larva. This is in agreement with our findings demonstrating the absence of 5-HT immunoreactive spinal projections from larval hindbrain nuclei. It remains possible, however, that 5-HT has a modulatory action within the hindbrain, for example on descending reticulospinal neurons. In the adult zebrafish, much like in many other adult vertebrate, a finer modulatory organization exists that involves both descending Raphe-spinal axons and intraspinal serotonergic cells, each targeting a different group of motor elements (Van

Raamsdonk et al., 1996), but this may start as a mostly local, spinal influence.

Role of 5-HT Modulation 5-HT promoted locomotor activity in the zebrafish larva without any major effects on the characteristics of the active swimming period. This is in contrast to many neonatal and adult preparations in which 5-HT modulates the activity pattern by enhancing its duration and amplitude (Viala and Buser, 1969; HarrisWarrick and Cohen, 1985; Barbeau and Rossignol, 1990; Woolston et al., 1994; McDearmid et al., 1997; Jovanovic et al., 1996; Branchereau et al., 2000b). These changes in the activity period can be of presynaptic origin, as observed in tadpoles (McDearmid et al., 1997; Sun and Dale, 1998b) and in the lamprey (Buchanan and Grillner, 1991; El Manira et al., 1997; Takahashi et al., 2001). Conversely, the 5-HT effects can be of postsynaptic origin due to modulation of ionic conductances underlying the expression of intrinsic cell properties such as plateau potentials (Hounsgaard et al., 1988; Hounsgaard and Kiehn, 1989), intrinsic oscillations (Wallen et al., 1989; Sillar and Simmers, 1994; Scrymgeour-Wedderburn et al., 1997; MacLean et al., 1998), or those controlling firing frequency (Wallen et al., 1989) and excitability (Kjaerulff and Kiehn, 2001). Furthermore, recent results using fetal mouse organotypic cultures depleted of 5-HT revealed an increase in the duration of the rhythmic bursts in the presence of GABA and glycine blockers, suggesting that 5-HT slows the development of inhibitory systems (Branchereau et al., 2002). The lack of apparent effects of 5-HT on the properties of the active periods in the zebrafish larva is likely due to the early developmental stages we examined. It is possible that at this immature stage, the specific con-

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ductances that are normally modulated by 5-HT in more mature preparations (Sillar et al., 1997) have not yet been expressed (Perrier and Hounsgaard, 2000). For example, motoneurons of the zebrafish larva do not show a pronounced postspike afterhyperpolarization (Buss and Drapeau, 2001), which is a common target for 5-HT modulation in the spinal cord (reviewed by Schmidt and Jordan, 2000), and this could explain the absence of changes in swimming frequency. Furthermore, even if the appropriate channels are present, the intracellular signaling mechanisms that make them responsive to 5-HT may not yet be established (Marcus and Carew, 1998). Our data implicate 5-HT in determining the efficacy of the network activity by modulating the duration of the rest intervals. Interestingly, long quiescent periods, such as those observed after blocking endogenous 5-HT in the zebrafish larva, are common to other developing networks and are suggested to occur due to transient changes in network excitability (O’Donovan, 1999). In developing networks a positive correlation was found between the duration of the preceding quiescent interval and the active period, and was attributed to the presence of activity-dependent depression (O’Donovan, 1999; Tabak et al., 2001). Although the spontaneous activities are not directly comparable to the patterned locomotor activity observed in our study, our findings show such (limited) correlation, suggesting that after blocking 5-HT receptors the duration of the quiescent period in the immature zebrafish larva may be influenced by activity-dependent depression. This depression has been attributed both to pre- and postsynaptic origins (Staley et al., 1998; Fedirchuk et al., 1999; Chub and O’Donovan, 2001; Timofeev et al., 2001). Regardless of the role of network depression, we observed neither a gradual decrease in the extent of the depolarization during fictive swim episodes towards the end of a cluster, nor changes in the frequency of the spiking or of EPSPs during each swim episode in the neurons we recorded from, which may exclude the possibility that 5-HT acted presynaptically. In addition, we did not observe changes in the input resistance, rheobasic current, or resting potential during the rest intervals in the spinal neurons we recorded from in the presence of 5-HT or MT, apparently excluding 5-HT modulation of postsynaptic ionic conductances during the quiescent period. Another postsynaptic change that has been suggested to determine the duration of quiescent periods in the developing spinal cord is the redistribution of intracellular chloride (Chub and O’Donovan, 2001). As intracellular chloride is elevated in embryonic neurons, including those of the zebrafish (Saint-Amant

and Drapeau, 2000; Brustein et al., 2003), and is an important source of early network excitability (BenAri, 2001), 5-HT may be implicated in modulating this type of homeostatic regulation. While more work is needed to define the exact mechanism of 5-HT action during the rest interval in the zebrafish larva, our results nevertheless indicate that integration of 5-HT neuromodulation very early during development plays a critical role in the functional organization of the neuronal network. As shown here, after the emergence of spontaneous swimming, 5-HT could profoundly alter the activation state and efficacy of the developing neural network for locomotion by promoting a more constant locomotor output, with little effect on the properties of the active (locomotor) periods that are normally the target of 5-HT modulation in neonatal and adult preparations. 5-HT decreased the rest intervals in order to increase the locomotor output, and most importantly offset the occurrence of activity clusters interrupted by long periods of inactivity, a commonly observed feature of other developing networks subject to activity- dependent depression. As prolonged inactivity is behaviorally detrimental for the zebrafish larva because it depends on constant swimming for survival, the development of serotonergic neuromodulation plays many essential roles. This work was supported by a Canadian Institute of Health Research (CIHR) Fellowship to E.B. We are indebted to Dr. Serge Rossignol and Ing. Philippe Drapeau for video-analysis software. Berit Ellingsen is acknowledged for excellent technical support and G. Laliberte´ and L. Brent for animal care. Drs C. Bourque and J. McDearmid are appreciated for their helpful comments on the manuscript.

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