Zied Oueghlani

Laurent Juvin

François M Lambert

Laura Cardoit

Gilles Courtand

Frédérique Masmejean

Jean-René Cazalets

Grégory Barrière
email: gregory.barriere@u-bordeaux.fr

Serotonergic modulation of sacral dorsal root stimulation-induced locomotor output in newborn rat

Keywords: locomotion, spinal cord, central pattern generator, serotonin, neonatal rat

Introduction

The control of locomotor behaviors relies on dynamic interactions between the supraspinal locomotor centers (from the cortex to brainstem), the spinal central pattern generators (CPGs) for locomotion and segmental sensory afferent information. In rat, the spinal locomotor network begins to develop during the embryonnic stage, is functional at birth but continues to mature up to 3 weeks of age (Brocard et al., 1999a;1999b;[START_REF] Cazalets | Variability as a characteristic of immature motor systems: an electromyographic study of swimming in the newborn rat[END_REF][START_REF] Clarac | The maturation of locomotor networks[END_REF][START_REF] Vinay | Development of posture and locomotion: an interplay of endogenously activities and neurotrophic actions by descending pathways[END_REF]. During this postnatal period, neuromodulatory pathways originating from the brain progressively descend along the spinal cord and mature to reach their adult pattern of innervation between the second and fourth post-natal weeks [START_REF] Vinay | Perinatal development of lumbar motoneurons and their inputs in the rat[END_REF]. Consequently, the operation of the locomotor network evolves over time, as does the repertoire of behaviors it produces, including the expression of adult locomotion from two post-natal weeks [START_REF] Altman | Postnatal development of locomotion in the laboratory rat[END_REF][START_REF] Clarac | The maturation of locomotor networks[END_REF].

The isolated neonatal rat spinal cord preparation provides the opportunity to study the central genesis of locomotor outputs during the first few post-natal days, since this preparation continues to generate in vitro an array of rhythmical motor outputs, including locomotor-like activities, that the CNS produces in vivo. Activation methods include exogenous application of neurotransmitters and neuromodulators [START_REF] Barrière | Peptidergic neuromodulation of the lumbar locomotor network in the neonatal rat spinal cord[END_REF]2004;[START_REF] Beliez | Multiple monoaminergic modulation of posturo-locomotor network activity in the newborn rat spinal cord[END_REF][START_REF] Cazalets | Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat[END_REF][START_REF] Cowley | A comparison of motor patterns induced by Nmethyl-D-aspartate, acetylcholine and serotonin in the in vitro neonatal rat spinal cord[END_REF][START_REF] Juvin | Propriospinal circuitry underlying interlimb coordination in mammalian quadrupedal locomotion[END_REF][START_REF] Kiehn | Spatiotemporal characteristics of 5-HT and dopamineinduced rhythmic hindlimb activity in the in vitro neonatal rat[END_REF][START_REF] Kiehn | Effects of Noradrenaline on Locomotor Rhythm-Generating Networks in the Isolated Neonatal Rat Spinal Cord[END_REF][START_REF] Smith | In vitro brainstem-spinal cord preparations for study of motor systems for mammalian respiration and locomotion[END_REF][START_REF] Smith | Neural mechanisms generating locomotion studied in mammalian brain stem-spinal cord in vitro[END_REF][START_REF] Sqalli-Houssaini | Noradrenergic control of locomotor networks in the in vitro spinal cord of the neonatal rat[END_REF] and electrical stimulation of specific brainstem areas [START_REF] Atsuta | Characteristics of electrically induced locomotion in rat in vitro brain stem-spinal cord preparation[END_REF][START_REF] Juvin | Locomotor rhythmogenesis in the isolated rat spinal cord: a phase-coupled set of symmetrical flexion extension oscillators[END_REF][START_REF] Oueghlani | Brainstem Steering of Locomotor Activity in the Newborn Rat[END_REF][START_REF] Zaporozhets | A reliable technique for the induction of locomotor-like activity in the in vitro neonatal rat spinal cord using brainstem electrical stimulation[END_REF], descending pathways [START_REF] Iwahara | Locomotion induced by spinal cord stimulation in the neonate rat in vitro[END_REF][START_REF] Magnuson | Long-duration, frequencydependent motor responses evoked by ventrolateral funiculus stimulation in the neonatal rat spinal cord[END_REF][START_REF] Magnuson | Locomotor rhythm evoked by ventrolateral funiculus stimulation in the neonatal rat spinal cord in vitro[END_REF] or segmental sensory afferent pathways [START_REF] Juvin | Cervicolumbar coordination in mammalian quadrupedal locomotion: role of spinal thoracic circuitry and limb sensory inputs[END_REF]Lev-Tov et al., 2000;[START_REF] Marchetti | Alternating rhythmic activity induced by dorsal root stimulation in the neonatal rat spinal cord in vitro[END_REF][START_REF] Smith | In vitro brainstem-spinal cord preparations for study of motor systems for mammalian respiration and locomotion[END_REF]. At the lumbar level, where the hindlimb CPG for locomotion is located [START_REF] Cazalets | Localization and organization of the central pattern generator for hindlimb locomotion in newborn rat[END_REF], rhythmical alternations of left-right segmental motor bursts as well as of ipsilateral flexor-like and extensor-like motor bursts recorded from the second (L2) and fifth (L5) lumbar ventral roots respectively, are a major hallmark of so-called "fictive locomotion" [START_REF] Cazalets | Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat[END_REF][START_REF] Kiehn | Spatiotemporal characteristics of 5-HT and dopamineinduced rhythmic hindlimb activity in the in vitro neonatal rat[END_REF].

Tonic electrical stimulations of one of the sacral/coccygeal dorsal roots that notably convey sensory afferent inputs from the tail in rodent, is one relevant method for activating the lumbar locomotor networks in the isolated neonatal rat spinal cord (Lev-Tov et al., 2000). Activation of these networks is mediated by a sacral relay pathway that projects rostrally through the ventral funiculi [START_REF] Cherniak | Shaping the Output of Lumbar Flexor Motoneurons by Sacral Neuronal Networks[END_REF][START_REF] Etlin | Long and short multifunicular projections of sacral neurons are activated by sensory input to produce locomotor activity in the absence of supraspinal control[END_REF]2014;2013;[START_REF] Strauss | Neural Pathways Between Sacrocaudal Afferents and Lumbar Pattern Generators in Neonatal Rats[END_REF]. This pathway is also probably involved in the pro-locomotor action of perineal stimulations below a spinal cord lesion in cat and rat (Alluin et al., 2015;[START_REF] Barbeau | Recovery of locomotion after chronic spinalization in the adult cat[END_REF][START_REF] Fouad | Treadmill training in incomplete spinal cord injured rats[END_REF].

Until now, however, no developmental investigation of this sacral ascending control of the lumbar network has been carried out. Thus, whether this sensory derived activation modality of the lumbar networks remains capable of triggering episodes of fictive locomotion during the first few days after birth, while the spinal neuromodulatory environment is evolving rapidly, remains to be established.

In the present work, we investigated the temporal organization of the rhythmic lumbar motor outputs triggered by a tonic electrical stimulation of a single sacral dorsal root of isolated spinal cord preparations from neonatal (postnatal day (P) 0 to 5) rats [START_REF] Cazalets | Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat[END_REF][START_REF] Smith | In vitro brainstem-spinal cord preparations for study of motor systems for mammalian respiration and locomotion[END_REF]. Specifically, we focused on the motor outputs recorded simultaneously from the L2 and L5 segments that convey motor commands to mostly flexor and extensor muscles, respectively. We found that the nature of motor output pattern induced by the sacral stimulation varies as a function of the developmental stage. Moreover, our data provide evidence that over time, serotonin (5-HT)-mediated modulatory signaling in the spinal cord becomes necessary to convert non-locomotor output into a fully organized locomotory pattern.

Materials and Methods

Experiments were performed on Sprague Dawley newborn rats (n=101) of either sex aged from 0 to 5 days (P0-P5). The repartition of all animals was as followed: In vitro isolated spinal cord preparation.

Pups were anesthetized (Isoflurane, 4% in air) until no reflexes could be elicited in response to tail and toe pinching. After decapitation and removal of the the skin of the back, animals were positioned dorsal side up in a dissecting chamber under continuous perfusion with oxygenated (95% O 2 /5% CO 2 %) artificial cerebrospinal fluid (aCSF), pH 7.4, and containing (in mM): 130 NaCl, 3 KCl, 2.5 CaCl 2 , 1.3 MgSO 4 , 0.58 NaH 2 PO 4 , 25 NaHCO 3 , and 10 glucose. A laminectomy was performed to expose the brainstem and spinal cord, which together were carefully dissected free using fine forceps and micro-scissors under binocular magnification. The preparation was then placed in a recording chamber and superperfused with aCSF. In some experiments, the spinal cord was artificially partitioned using Vaseline walls [START_REF] Cazalets | Localization and organization of the central pattern generator for hindlimb locomotion in newborn rat[END_REF], to restrict the bath application of pharmacological agents to specific spinal cord regions. The watertightness of the barriers was systematically checked at the end of each experiment by observing the movements of methylene blue added to the bathing medium on one side of the Vaseline barriers.

Electrophysiological recordings and analyses

Motor activities were recorded from lumbar ventral roots using pairs of extracellular Vaseline-insulated stainless-steel pin electrodes (figure 1A-C). Extracellular activities were amplified and digitized using an A/D interface (Digidata 1322A, Axon Instruments, CA, USA) driven by Axograph software. Raw extracellular signals were high-pass ( 50Hz) filtered, rectified, and integrated before analysis and rhythmic activity parameters were computed using custom-made routines written in MATLAB (MathWorks). Wavelet transform analyses [START_REF] Grinsted | Application of the cross wavelet transform and wavelet coherence to geophysical time series[END_REF][START_REF] Mor | Analysis of rhythmic patterns produced by spinal neural networks[END_REF][START_REF] Torrence | A Practical Guide to Wavelet Analysis[END_REF] were performed using the MATLAB wavelet coherence package provided by A. Grinsted (http://grinsted.github.io/wavelet-coherence). Each pretreated signal was decomposed in the frequency domain over time using a continuous wavelet transform (Morlet wavelet, 10 octaves per scale). Cross-wavelet and wavelet coherence algorithms were applied to paired, pretreated signals to highlight the common high power frequencies (color coded) and significant correlations (color coded) between each frequency component of signals over time (figure 1D). Phase relationships between signals were extracted from cross-coherence maps that mix the cross-wavelet and wavelet coherence maps [START_REF] Beliez | Multiple monoaminergic modulation of posturo-locomotor network activity in the newborn rat spinal cord[END_REF]. The phase relationships between ventral root activity pairs were identified as "in phase" for normalized values ranging from 0.875 to 0.125, and as "alternating" for normalized values ranging from 0.375 to 0.625. Otherwise, the phase relationships between signal pairs were classified as "irrelevant phase". For convenience, the range of phase relationship values between the different pairs of ventral root signals that fulfill the criteria of a locomotor-like pattern are indicated by blue-shaded, quarter circle segments on polar plots.

Sacral dorsal root stimulation

Trains of stimulation (pulse duration: 0.5ms; frequency: 2Hz; train duration: 30s) were delivered with a Master-8 pulse generator (A.M.P.I.) connected to an ISO-Flex stimulus isolation unit (A.M.P.I.). Trains were applied unilaterally to one S2 sacral dorsal root using a pair of stainless-steel pin electrodes (bipolar stimulation). The threshold was consistently defined as the minimum current for which a single pulse (0.5ms duration) induced a short-latency motor output response recorded from the homonymous (same segment) ipsilateral (occasionally contralateral) ventral root (figure 1B). Pulse amplitudes in test trains were then adjusted from 2 to 5 times the threshold (T) value.

Pharmacology

Drugs were all obtained from Abcam and were applied in the range of concentrations used in previous studies in neonates [START_REF] Cazalets | Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat[END_REF][START_REF] Dunbar | Endogenous extracellular serotonin modulates the spinal locomotor network of the neonatal mouse[END_REF][START_REF] Gordon | Monoaminergic Control of Cauda-Equina-Evoked Locomotion in the Neonatal Mouse Spinal Cord[END_REF][START_REF] Liu | Stimulation of the parapyramidal region of the neonatal rat brain stem produces locomotor-like activity involving spinal 5-HT7 and 5-HT2A receptors[END_REF][START_REF] Nishimaru | 5-Hydroxytryptamine-induced locomotor rhythm in the neonatal mouse spinal cord in vitro[END_REF]Pearlstein et al., 2005;2011) and adult animals as well [START_REF] Lucas-Osma | 5-HT1D receptors inhibit the monosynaptic stretch reflex by modulating C-fiber activity[END_REF]. This included serotonin (5-HT, 5 and 10µM), 8-OH-DPAT (1, 10 and 50 µM; 5-HT1A receptor agonist) quipazine (1, 2 and 5µM; 5-HT2A receptor agonist), ketanserin (10µM; 5-HT2A receptor antagonist), LP44 (5 and 50 µM; 5-HT7 receptor agonist) and SB269970 (10µM; 5-HT7 receptor antagonist). In some preparations, episodes of fictive locomotion were induced by bath-application of a mixture of NMDA (7.5µM) and 5-HT(15µM) [START_REF] Cazalets | Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat[END_REF]. All drugs were prepared in aCSF at their final concentration and bath-applied using a peristaltic pump (flow rate 5mL/min, recording chamber volume 5 mL). The effects of the drugs were monitored from 10 to 20 minutes after reaching the recording chamber (the time estimated for total replacement of the saline solution and diffusion into the tissue). Each drug bath-application was followed by a prolonged washout with normal saline for at least 30 mins.

Immunochemistry

Rats (P0, n=3; P3, n=3; P5, n=3) were deeply anesthetized with intraperitoneal injection of Ketamine (150mg/kg, Imalgene, CentraVet, France) and Xylazine (15mg/kg, Rompun, CentraVet, France), and intracardially perfused with cold NaCl (0.9%) and then cold paraformaldehyde (PFA, 4% in phosphate buffered saline).

Spinal cords were dissected free, post-fixed in 4% PFA for 2 hr, and cryoprotected overnight in 20% PBS-sucrose (at 4°C). Cervical, thoracic and lumbar spinal cord segments were cut in transverse slices (30 µm thick at -20°C) using a cryostat (Leica CM 3000) and mounted on slides. Slides were incubated for 1h30 in a mixture of (1) PBS, (2) 0.3 % triton, (3) 1% bovine serum albumin (BSA) and (4) Thimerosal (antibacterial and antifungal, T5125, Sigma-Aldrich). Slides were then incubated overnight with a primary rabbit anti-5-HT2A receptor antibody (1:300; AB66049;

Abcam-Biochemicals) in a mixture containing PBS, 0.3% triton and 1% BSA. After washing in PBS, sections were incubated for 1h30 with a secondary antibody (Alexa fluor 488 donkey anti-rabbit, 1:500; A21206, Invitrogen) in 0.3% triton and 1% BSA.

Negative controls for the 5-HT2A receptor immunolabelling were obtained by incubating the slices in absence of the primary antibody during the first stage of the process but in the presence of the secondary antibody during the second phase (see figure 9A). Each slide contained slices from P0, P3 and P5 animals, and all slides were processed simultaneously for immunolabelling to ensure uniformity of the labelling conditions.

Image acquisition and analysis

Images were acquired on an epifluorescent microscope (20x/0.70; Olympus IX81) equipped with a camera (Orca ER, Hamamatsu) controlled by Micro-Manager (MM Studio) software. All images were acquired with fixed parameters including exposure time (200 ms, ChAT, excitation 548 nm; 500 ms, 5-HT2A, 488 nm), gain and light power. Images were analyzed with open-source Fiji software [START_REF] Schindelin | Fiji: an opensource platform for biological-image analysis[END_REF] using a home-made macro. For each image, the spinal cord slice was manually delineated and then automatically segmented in 9 regions of interest (ROIs), redistributing the lateral and middle parts of the spinal cord as ventral, intermediate and dorsal. The level of immunolabelling fluorescence of 5-HT2A receptors was systematically lower in P0 preparations. P0 slices on a given slide therefore served as a reference to automatically determine (using the triangle method) thresholds of detection applied to all other slices on that slide. For all three ROIs covering the ventral cord (see figure 9A), the ratio between the area of each ROI and the corresponding area above the threshold was then extracted and pooled together. This allowed normalizing the value as a function of the spinal cord size.

Statistics

Immunohistochemical data were analyzed using Prism software (GraphPad software).

Descriptive statistics were performed on the ratio between the labelled surface area and the total surface of a given region of interest, and are presented as mean percentage ± standard error from the mean (SEM). Comparison between groups was achieved using a Friedman test followed by a Dunn's multiple comparisons test, with the confidence level set at 95%. Circular statistics were performed on raw data using the circular statistic toolbox [START_REF] Berens | CircStat: A MATLAB Toolbox for Circular Statistics[END_REF] for Matlab. Circular data are represented as polar plots on which the direction of the resultant vector r, which is systematically illustrated as a red arrow, indicates the mean angular value (from 0 and 360°), while the length of the vector is a measure of the concentration of the data around that mean.

The non-uniform distribution of the data was further determined using the Raleigh test with a significance level set at p<0.05. Only data showing a non-uniform distribution were represented as polar plots.

Results

Characterization of sacral dorsal root stimulation-induced rhythmic activity

Unilateral tonic electrical stimulation (2Hz for 30s, 1 to 5x threshold) of a single sacral dorsal root (which subsequently will be referred to as sacral DR stimulation, (Lev-Tov et al., 2000) was very effective in triggering rhythmic motor outputs in ventral roots at the lumbar level. Bilateral alternation of motor bursts recorded from the L2 segment (lL2/rL2) was systematically expressed, although ipsilateral flexor-(L2) /extensor-like (L5) motor burst alternation was mainly obtained in preparations obtained from the youngest neonates (P0-P2). This is illustrated in figure 2A1 In contrast to L2 motor bursts that always alternated bilaterally, the alternation of ipsilateral L2/L5 (flexor/extensor) motor bursts was gradually lost during the first five post-natal days (77.78% at P0, n = 7/9 ; 85.71% at P1, n = 12/14 ; 50.00% at P2, n = 10/20 ; 33.33% at P3, n = 9/27 ; 0% at P4/5, n = 0/14). In parallel, the in-phase coupling of the diagonal L5/L2 motor bursts observed between P0 and P2 was progressively lost thereafter. Thus, the capability to express fictive locomotion resulting from sacral afferent stimulation emerges prior to P2, but at subsequent ages through to P5, the ability to evoke two critical components of a fully organized rhythmic pattern -ipsilateral burst alternation and diagonal burst synchrony -gradually fail to occur.

Locomotor promoting action of 5-HT

In preparations that did not express fictive locomotion in response to sacral DR stimulation alone (from 1 to 5 times the threshold), we applied the same stimulation in the presence of a concentration of serotonin (5-HT, 10 µM) that did not by itself trigger any motor activity per se. At this sub-threshold dose, 5-HT promoted the expression of DR stimulus-induced fictive locomotion. This is illustrated for a typical preparation (P3) in figure 3. In normal aCSF, sacral stimulation failed to elicit any rhythmical activity in the left L5 ventral root and no alternating flexor-and extensor like motor bursts on the right side (rL5/rL2 : Φ = 0.87 ± 0.04; figure 3A1-A2). The same stimulation applied in the presence of 5-HT now switched the motor activity into a complete locomotor-like pattern (rL5/rL2 : Φ = 0.41 ± 0.04; lL5/rL2 : Φ = 0.91 ± 0.05; figure 3B1-B2). After washout of the 5-HT, the latter's permissive action was lost and episodes of fictive locomotion with its characteristic burst timing could no longer be induced (figure 3C1-C2). The ability of 5-HT (10 µM) to reestablish a locomotor pattern of activity was observed in 13 out of 14 preparations tested. The mean phase relationships in these 13 preparations were 0.51 ± 0.01 (lL2/rL2), 0.47 ± 0.02 (rL5/rL2) and 0.96 ± 0.03 (lL5/rL2). Not unexpectedly, lowering the concentration of 5-HT to 5µM was less effective, as it was found to reestablish a locomotor pattern in only 2 out of 8 preparations (data not shown).

Because the lack of locomotor activity described above was primarily due to deficits in the activation or organization of the L5 motor outputs, we next addressed whether the pro-locomotor effect of 5-HT was due to a local action at the L5 segmental level, and/or at the level of the locomotor CPG located in rostral segments (T13-L2, [START_REF] Cazalets | Localization and organization of the central pattern generator for hindlimb locomotion in newborn rat[END_REF]. In a series of experiments, we artificially partitioned the spinal cord by placing a watertight Vaseline barrier at the L3 level. This allowed perfusing 5-HT-containing ACSF selectively on either the rostral (containing the spinal CPG) or caudal (L5 motoneurons) part of the spinal cord. As shown in figure 4 from a preparation that did not express any locomotor activity in response to sacral stimulation alone (figure 4A), selective application of 5-HT (10µM) to the caudal cord compartment encompassing the L5 motoneurons did not promote the generation of fictive locomotion (rL5/rL2 : Φ = 0.79 ± 0.08; lL5/rL2 : Φ = 0.15 ± 0.12; figure 4B). In contrast, following its washout (45 min) from the caudal cord, 5-HT applied to the rostral cord (at the CPG level) of the same preparation and at the same dose promoted the expression of locomotor-like activity (rL5/rL2 : Φ = 0.01 ± 0.03; lL5/rL2: Φ = 0.91 ± 0.03 ; figure 4C). This re-establishment of a locomotor pattern when 5-HT applied selectively to the rostral part of the lumbar cord was observed in 7/9 preparations, and in all cases, the permissive effect was reversed after amine washout (figure 4D). Together, our data thus provide evidence that 5-HT becomes capable of enhancing the activation of the extensor component of the locomotor network, by acting directly at the CPG level in the rostral lumbar cord, in response to sacral dorsal root stimulation.

Identification of underlying serotonergic receptors

We next assessed the contribution of the three main serotonergic receptor types that are known to be involved in the control of spinal locomotor circuitry [START_REF] Hochman | 5-HT Receptors and the Neuromodulatory Control of Spinal Cord Function[END_REF][START_REF] Jordan | Descending command systems for the initiation of locomotion in mammals[END_REF][START_REF] Noga | Locomotor-activated neurons of the cat. I. Serotonergic innervation and co-localization of 5-HT7, 5-HT2A, and 5-HT1A receptors in the thoraco-lumbar spinal cord[END_REF], by applying sacral stimulations in the presence of different 5-HT receptor agonists and antagonists.

5-HT2A receptors

We first tested the involvement of 5-HT2A receptors by bath-applying the agonist quipazine. When applied alone at 1, 2 and 5µM, quipazine did not elicit any motor activity (data not shown). From 2µM, quipazine mimicked the permissive action of 5-HT, promoting the expression of a locomotor pattern in 8 out of 10 preparations in response to sacral stimulation. This is illustrated by a representative example taken from a P3 preparation in figure 5. In normal aCSF, sacral stimulation triggered rhythmic activity that did not fulfill fictive locomotor criteria (Figure 5A1), due to a lack of ipsilateral flexor/extensor-like alternating bursting activity (figure 5A2). However, quipazine (2µM) transformed this disorganized sacral stimulation-induced activity into a locomotor pattern with the proper set of alternating burst structures (figure 5B1-B2).

In contrast to 5-HT, quipazine's effect persisted beyond its washout (figure 5C1-C2), and increasing the agonist to 5µM did not improve its potency (figure 5D-E). For the preparations in which quipazine enabled the expression of fictive locomotion, the mean phase relationships between bilateral L2 were 0.51 ± 0.01 (2µM, n=8) and 0.50 ± 0.01 (5µM, n=6) and were 0.57 ± 0.02 (2 µM) and, 0.51 ± 0.04 (5µM) between ipsilateral rL2/rL5 ventral roots.

In a complementary set of experiments, we bath-applied the 5-HT2A antagonist ketanserin (10µM) in the presence of either 5-HT or quipazine, to preparations that did not express fictive locomotion under normal aCSF in response to sacral DR stimulation. In both conditions, ketanserin blocked the pro-locomotor action of 5-HT in all 6 preparations tested (data not shown) and all 5 preparations tested under quipazine (data not shown). Importantly, when applied onto younger preparations that already expressed fictive locomotion in response to sacral stimulation under normal aCSF, ketanserin (10µM) did not alter the temporal organization this locomotor program (n=5/6 preparation). This is shown in figure 6

5-HT7 receptors

In order to study the contribution of 5-HT7 receptors, we first bath-applied the agonist LP44 at 5µM and 50µM onto preparations that did not express a locomotor pattern with sacral stimulation. At these concentrations, LP44 failed to promote any locomotor-like activity in all preparations tested (n=9, data not shown). In addition, the 5-HT7 receptor antagonist SB269970 (10µM) failed to completely block the locomotor pattern that emerged in the presence of 5-HT (10µM) in 8/10 preparations. This is shown in figure 7 for one representative preparation. Under normal aCSF condition, sacral stimulation failed to initiate a sequence of fictive locomotion (figure 7A1). Adding 5-HT (10µM) to the bath in the presence of SB269970 (10µM) did not prevent the pro-locomotor action of 5-HT (figure 7A2), although it was noticeable that the duration of locomotor-like episode did not last for the whole stimulation period in the presence of SB269970. In only 2/10 preparations did the antagonist completely abolish the locomotor-like pattern observed under 5-HT (data not shown).

In young preparations that already expressed fictive locomotion in response to sacral stimulation, adding SB269970 (10µM) did not alter the expression of the locomotor pattern (n=3/3 preparation) as shown in an example of a P2 preparation in figure 7B1-B2. Altogether, these results indicate that at least for our sacral stimulation paradigm, 5-HT7 receptors do not mediate the principle actions of 5-HT on the pattern generating component of the spinal locomotor network.

5-HT1A receptors

Bath application of 8-OH-DPAT, a 5-HT1A agonist, at 1µM (n=6) and 10µM (n=8) failed to promote the expression of fictive locomotion in response to sacral stimulation (data not shown). At a higher concentration (50µM), the agonist even abolished rhythmic activity expressed specifically from the L5 segment (n=9/10 preparations) as illustrated by recordings from one preparation in figure 8. In this example, sacral stimulation triggered a non-locomotor activity pattern during which synchronous motor bursts occurred on both sides of the cord in ipsilateral L2 and L5 ventral roots (figure 8A).

When the same sacral stimulation was applied during bath-application of 8-OH-DPAT, although rhythmic alternating motor bursts still occurred in the L2 segment, the L5 ventral root on the left side fell silent, while activity recorded from L5 on the opposite side became mostly tonic. In order to ascertain whether this apparent agonist-mediated effect on extensor-like activity was not somehow due to a spontaneous decline in lumbar locomotor network operation, we next bath applied NMDA (7.5µM) and 5-HT (15µM) after washing out the 8-OH-DPAT for 30 minutes. In all cases (n=6/6), NMDA/5-HT triggered sustained episodes of fictive locomotion (figure 8C). Thus it is very likely that the 5-HT1A agonist, at high doses, selectively prevents the activation of the L5 extensor motoneurons, in contrast to L2 flexor motoneurons, which remain unaffected.

5-HT2A receptors are present at birth in the rat spinal cord

Because the 5-HT2A receptor-mediated pro-locomotor action observed in response to sacral stimulation progressively emerges during the very first post-natal days, we next addressed whether this might be due to a corresponding progressive expression of 5-HT2A receptors in the spinal cord between P0 and P5. As shown in the immunodetection data of figure 9, 5-HT2A receptors are already present in the caudal lumbar cord (L5) at birth (figure 9B), and specifically, are mostly expressed in the ventral horn, especially in the vicinity of motoneurons (inset of figure 9B). However, by P5, fluorescence labelling, at the same spinal level, clearly becomes increased in the ventral horn and the lateral edge of the ventral cord (figure 9C-D). Consequently, the lack of effect of the 5-HT2A receptor antagonist early after birth (P0-P2) cannot be due to the actual absence of receptors, but its later prevalence in mediating the action of 5-HT could be at least partly due to a maturational increase and refinement of its pattern of expression.

Discussion

The present work investigated the generation of fictive locomotor episodes elicited by unilateral tonic electrical stimulation of a single sacral (S2) dorsal root. We show that during the first 2 post-natal days (P0-P2), sacral DR stimulation triggers a complete locomotor output in most of the preparations tested. After P2, however, the same stimulation protocol mostly induces partial burst patterns and/or with inappropriate, non-locomotor phase relationships. However, at these later stages, 5-HT, especially through the activation of 5-HT2A receptors, enables the lumbar locomotor network to switch sacral stimulation-induced output from non-locomotor to a complete and functionally appropriate locomotor pattern (figure 10).

Characterization of sacral stimulation-induced lumbar motor outputs.

Tonic electrical stimulation of sacrococcygeal sensory inputs, which mimic the action of tail pinching (Lev-Tov and Delvolve, 2000;[START_REF] Lev-Tov | Sensory-induced activation of pattern generators in the absence of supraspinal control[END_REF], was previously shown to induce rhythmical activity in the lumbar cord, although the precise organization of the flexor and extensor-like motor outputs has never been fully described [START_REF] Blivis | Differential effects of opioids on sacrocaudal afferent pathways and central pattern generators in the neonatal rat spinal cord[END_REF][START_REF] Etlin | Characterization of Sacral Interneurons That Mediate Activation of Locomotor Pattern Generators by Sacrocaudal Afferent Input[END_REF]2010;Lev-Tov et al., 2000;Lev-Tov and Delvolve, 2000;[START_REF] Strauss | Neural Pathways Between Sacrocaudal Afferents and Lumbar Pattern Generators in Neonatal Rats[END_REF]. Fictive locomotion in the isolated spinal cord preparation from newborn rat is characterized by a bilateral alternation of segmental motor bursts, together with an alternation of ipsilateral flexor-like (L2) and extensor-like (L5) motor bursts [START_REF] Cazalets | Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat[END_REF][START_REF] Kiehn | Spatiotemporal characteristics of 5-HT and dopamineinduced rhythmic hindlimb activity in the in vitro neonatal rat[END_REF]. Here, we show that although left/right alternation of flexor-like (L2) motor bursts was always expressed, the expression of proper flexor (L2)/extensor-like (L5) motor organization in response to sacral afferent stimulation decreased over the first five postnatal day period. This occurred either because the stimulus-derived activation of L5 motoneurons was not effective or because their activity was not driven properly by the upstream lumbar CPG (for instance see figure 3). In all cases, however, exogenously applied 5-HT enabled sacral stimulation to induce fully organized fictive locomotion.

Importantly, the pro-locomotor action of 5-HT was mediated only when the amine was acting at the level of the lumbar locomotor network itself, with its selective application to the L5 segment remaining ineffective. This strongly suggests that in response to sacral stimulation, 5-HT becomes increasingly critical over time in recruiting the proper set of interneurons driving the L5 motoneurons during locomotion [START_REF] Cazalets | Localization and organization of the central pattern generator for hindlimb locomotion in newborn rat[END_REF].

Developmental aspect of sacral stimulation-induced locomotor outputs

Ipsilateral flexor/extensor alternation has been shown to be established shortly before birth [START_REF] Iizuka | Development of the spatial pattern of 5-HT-induced locomotor rhythm in the lumbar spinal cord of rat fetuses in vitro[END_REF][START_REF] Ozaki | Development of locomotor activity induced by NMDA receptor activation in the lumbar spinal cord of the rat fetus studied in vitro[END_REF]. The capability of sacral stimulation to produce a flexor/extensor-like alternating pattern persisted in most of our preparations during the first 2 postnatal days, but subsequently this capability was generally lost.

Interestingly, our data are in agreement with the findings of [START_REF] Norreel | Reversible disorganization of the locomotor pattern after neonatal spinal cord transection in the rat[END_REF], who showed in neonatal rat spinalized at birth that pinching the tail of P1-P3 animals triggers air-stepping movements with left-right alternating EMG bursts recorded from extensor muscles. In contrast, at a later stage (P6/7), stimulating the tail failed to induce episodes of air-stepping episodes, and accordingly, EMG signals from the extensors muscles were totally disorganized [START_REF] Norreel | Reversible disorganization of the locomotor pattern after neonatal spinal cord transection in the rat[END_REF].

Thus, our data provides evidence that a developmental shift occurs in the mode of operation of the lumbar locomotor network, involving the spinal serotonergic system.

Although a recent study reported that 5-HT is not required to activate the spinal locomotor program early after birth (P0-P2, [START_REF] Oueghlani | Brainstem Steering of Locomotor Activity in the Newborn Rat[END_REF], this maturational process has never been clearly established for intact animals. Indeed, the lumbar locomotor network in vitro is mostly activated using pharmacological [START_REF] Cazalets | Localization and organization of the central pattern generator for hindlimb locomotion in newborn rat[END_REF][START_REF] Cowley | A comparison of motor patterns induced by Nmethyl-D-aspartate, acetylcholine and serotonin in the in vitro neonatal rat spinal cord[END_REF][START_REF] Kiehn | Spatiotemporal characteristics of 5-HT and dopamineinduced rhythmic hindlimb activity in the in vitro neonatal rat[END_REF] or electrical stimulation approaches [START_REF] Antonino-Green | Neurons labeled from locomotor-related ventrolateral funiculus stimulus sites in the neonatal rat spinal cord[END_REF][START_REF] Cheng | Initiation of segmental locomotor-like activities by stimulation of ventrolateral funiculus in the neonatal rat[END_REF][START_REF] Liu | Stimulation of the parapyramidal region of the neonatal rat brain stem produces locomotor-like activity involving spinal 5-HT7 and 5-HT2A receptors[END_REF][START_REF] Magnuson | Locomotor rhythm evoked by ventrolateral funiculus stimulation in the neonatal rat spinal cord in vitro[END_REF]) that bring into play the serotonergic system. For instance, it was shown that activating the locomotor network with 5-HT between P0 and P4 does not produce locomotor patterns that are significantly different [START_REF] Kiehn | Spatiotemporal characteristics of 5-HT and dopamineinduced rhythmic hindlimb activity in the in vitro neonatal rat[END_REF]. It is thus very likely that these activation methods mask any maturational process taking place at the CPG level. It is worth mentioning in this context that in preparations obtained from rat pups spinalised at birth, the capacity of the glutamatergic agonist NMA to trigger episodes of organized rhythmic activity in absence of 5-HT is lost after P3 [START_REF] Norreel | Reversible disorganization of the locomotor pattern after neonatal spinal cord transection in the rat[END_REF].

The innervation pattern of the spinal cord by the descending monoaminergic systems continues to evolve over the 3 first postnatal weeks [START_REF] Bregman | Development of serotonin immunoreactivity in the rat spinal cord and its plasticity after neonatal spinal cord lesions[END_REF]Commissiong, 1983a;1983b;[START_REF] Rajaofetra | Pre-and postnatal development of noradrenergic projections to the rat spinal cord: an immunocytochemical study[END_REF][START_REF] Rajaofetra | Pre-and post-natal ontogeny of serotonergic projections to the rat spinal cord[END_REF][START_REF] Tanaka | Developmental changes in the serotoninergic innervation of hindlimb extensor motoneurons in neonatal rats[END_REF].

Indeed, serotonergic fibers originating from the raphe nuclei and projecting down to the lumbar cord do not attain their "adult" profile of innervation until P21 [START_REF] Bregman | Development of serotonin immunoreactivity in the rat spinal cord and its plasticity after neonatal spinal cord lesions[END_REF][START_REF] Rajaofetra | Pre-and post-natal ontogeny of serotonergic projections to the rat spinal cord[END_REF]. Although often underestimated, monoaninergic neuromodulation is also regulated by pre-synaptic uptake mechanisms which are likely to evolve over time after birth. For instance, the pattern of expression of the 5-HT transporter in the rodent spinal cord evolve over time from the embryonic stages up to several weeks post-natal [START_REF] Brüning | Prenatal development of the serotonin transporter in mouse brain[END_REF][START_REF] Pranzatelli | Plasticity and ontogeny of the central 5-HT transporter: effect of neonatal 5,7-dihydroxytryptamine lesions in the rat[END_REF].

Consequently, this continual evolution of the cord's neuromodulatory environment may confer profound changes in the operation of the spinal network, including the gradual maturation of the motoneurons (Bellinger and Anderson, 1987a;1987b;[START_REF] Cummings | Prenatal and postnatal development of lamina IX neurons in the rat thoracic spinal cord[END_REF][START_REF] Curfs | Postnatal maturation of the dendritic fields of motoneuron pools supplying flexor and extensor muscles of the distal forelimb in the rat[END_REF][START_REF] Dekkers | Early postnatal changes in the somatodendritic morphology of ankle flexor motoneurons in the rat[END_REF][START_REF] Kerai | Effect of transient neonatal muscle paralysis on the growth of soleus motoneurones in the rat[END_REF][START_REF] Tanaka | Developmental changes in the serotoninergic innervation of hindlimb extensor motoneurons in neonatal rats[END_REF][START_REF] Westerga | Structural changes of the soleus and the tibialis anterior motoneuron pool during development in the rat[END_REF], the acquisition of specific membrane

properties and a refinement of their afferent inputs (Brocard et al., 1999b;[START_REF] Cotel | Identified Ankle Extensor and Flexor Motoneurons Display Different Firing Profiles in the Neonatal Rat[END_REF][START_REF] Vinay | Perinatal development of lumbar motoneurons and their inputs in the rat[END_REF]. During this period, moreover, the intrinsic dynamics of network operation are also substantially altered by a chloride homeostasis change that occurs in rats around P3 [START_REF] Stil | Developmental up-regulation of the potassium-chloride cotransporter type 2 in the rat lumbar spinal cord[END_REF]. This leads to a switch in the action of GABA and glycine from depolarizing to hyperpolarizing, precisely at a stage that coincides with our observed change in the capacity of sacral stimulation to recruit all components of the lumbar locomotor network. Thus, all these functional adaptations that take place along with the maturation of the descending pathways may be critical to the acquisition and maturation of postural and locomotor capability [START_REF] Altman | Postnatal development of locomotion in the laboratory rat[END_REF]Brocard et al., 1999a;[START_REF] Clarac | The maturation of locomotor networks[END_REF][START_REF] Vinay | Development of posture and locomotion: an interplay of endogenously activities and neurotrophic actions by descending pathways[END_REF]2005), and an alteration in spinal monoamines early after birth affects spinal locomotor network operation [START_REF] Bos | Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2[END_REF][START_REF] Gackiere | Contribution of the Potassium-Chloride Cotransporter KCC2 to the Strength of Inhibition in the Neonatal Rodent Spinal Cord In Vitro[END_REF][START_REF] Jean-Xavier | Inhibitory Postsynaptic Potentials in Lumbar Motoneurons Remain Depolarizing After Neonatal Spinal Cord Transection in the Rat[END_REF][START_REF] Myoga | Postnatal development of locomotor movements in normal and para-chlorophenylalanine-treated newborn rats[END_REF][START_REF] Nakajima | Prenatal administration of parachlorophenylalanine results in suppression of serotonergic system and disturbance of swimming movements in newborn rats[END_REF][START_REF] Norreel | Reversible disorganization of the locomotor pattern after neonatal spinal cord transection in the rat[END_REF][START_REF] Pearlstein | Serotonin refines the locomotor-related alternations in the in vitro neonatal rat spinal cord[END_REF][START_REF] Pflieger | Postural Modifications and Neuronal Excitability Changes Induced by a Short-Term Serotonin Depletion during Neonatal Development in the Rat[END_REF].

The progressive loss of fictive locomotion in response to sacral stimulation during the first few day after birth reminds the disappearance of spontaneous stepping seen in newborn vertebrates, including humans [START_REF] Barbu-Roth | Why does infant stepping disappear and can it be stimulated by optic flow?[END_REF], as well as the progressive establishment of descending neuromodulatory systems in governing spinal locomotor network operation. In addition, our stimulation paradigm could reveal a developmental switch between the control of the locomotor and postural functions.

Such a mechanism might be involved in the progressive acquisition of pelvis elevation that precedes the expression of voluntary quadrupedal locomotion [START_REF] Clarac | Role of gravity in the development of posture and locomotion in the neonatal rat[END_REF][START_REF] Vinay | Perinatal development of the motor systems involved in postural control[END_REF]. In this context, further work will be needed to address whether sacral stimulation favors the specific recruitment of motoneurons innervating axial or limb muscles at a given developmental stage, and then whether 5-HT is involved in modulating this recruitment pattern [START_REF] Hinckley | Spinal Locomotor Circuits Develop Using Hierarchical Rules Based on Motorneuron Position and Identity[END_REF]. One possibility is that 5-HT would progressively become a major actor in orchestrating the switch from posture to locomotion.

Receptors involved in 5-HT's pro-locomotor action

The contribution of 5-HT2A receptor activation in preparations (mostly after P2) that expressed fictive locomotion only in the presence of 5-HT or quipazine (5-HT2A receptor agonist) is in agreement with previous data showing the importance of this receptor subtype in mediating 5-HT's pro-locomotor action [START_REF] Cazalets | Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat[END_REF][START_REF] Liu | Stimulation of the parapyramidal region of the neonatal rat brain stem produces locomotor-like activity involving spinal 5-HT7 and 5-HT2A receptors[END_REF][START_REF] Madriaga | Modulation of locomotor activity by multiple 5-HT and dopaminergic receptor subtypes in the neonatal mouse spinal cord[END_REF]. 5-HT2A receptors have been shown to promote locomotor activity both in vitro and in vivo in a variety of species (cat, rat, mouse), from fetal to adult stages, in intact as well as spinal cord injured states [START_REF] Antri | Locomotor recovery in the chronic spinal rat: effects of long-term treatment with a 5-HT2 agonist[END_REF][START_REF] Barbeau | Initiation and modulation of the locomotor pattern in the adult chronic spinal cat by noradrenergic, serotonergic and dopaminergic drugs[END_REF][START_REF] Brumley | The serotonergic agonists quipazine, CGS-12066A, and alpha-methylserotonin alter motor activity and induce hindlimb stepping in the intact and spinal rat fetus[END_REF][START_REF] Cazalets | Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat[END_REF][START_REF] Landry | Differential effects of 5-HT1 and 5-HT2 receptor agonists on hindlimb movements in paraplegic mice[END_REF][START_REF] Liu | Stimulation of the parapyramidal region of the neonatal rat brain stem produces locomotor-like activity involving spinal 5-HT7 and 5-HT2A receptors[END_REF][START_REF] Madriaga | Modulation of locomotor activity by multiple 5-HT and dopaminergic receptor subtypes in the neonatal mouse spinal cord[END_REF][START_REF] Mcewen | L-DOPA and quipazine elicit air-stepping in neonatal rats with spinal cord transections[END_REF][START_REF] Sławińska | 5-HT₂ and 5-HT₇ receptor agonists facilitate plantar stepping in chronic spinal rats through actions on different populations of spinal neurons[END_REF][START_REF] Ung | Role of spinal 5-HT2 receptor subtypes in quipazine-induced hindlimb movements after a low-thoracic spinal cord transection[END_REF]. This amine also reinforces locomotor bursts alternations in our preparation [START_REF] Norreel | Reversible disorganization of the locomotor pattern after neonatal spinal cord transection in the rat[END_REF][START_REF] Pearlstein | Serotonin refines the locomotor-related alternations in the in vitro neonatal rat spinal cord[END_REF]. Here, we found that the recruitment of 5-HT2A receptors is a critical component in mediating the recruitment of the extensor module of the spinal locomotor network only few days after birth. Immunolabelling of 5-HT2A receptors showed a predominant expression in the ventral horn already at birth, notably in the vicinity of motoneuronal pools, which is in agreement with data reported from adult rats [START_REF] Cornea-Hébert | Cellular and subcellular distribution of the serotonin 5-HT2A receptor in the central nervous system of adult rat[END_REF][START_REF] Doly | The 5-HT2A receptor is widely distributed in the rat spinal cord and mainly localized at the plasma membrane of postsynaptic neurons[END_REF][START_REF] Maeshima | The cellular localization of 5-HT2A receptors in the spinal cord and spinal ganglia of the adult rat[END_REF][START_REF] Marlier | Autoradiographic mapping of 5-HT1, 5-HT1A, 5-HT1B and 5-HT2 receptors in the rat spinal cord[END_REF][START_REF] Ridet | Direct immunocytochemical localization of 5hydroxytryptamine receptors in the adult rat spinal cord: a light and electron microscopic study using an antiserum[END_REF][START_REF] Thor | Autoradiographic localization of 5hydroxytryptamine1A, 5-hydroxytryptamine1B and 5-hydroxytryptamine1C/2 binding sites in the rat spinal cord[END_REF]. Under our experimental paradigm, the emergent contribution of these receptors is more likely to be due to an increase of their expression during the first postnatal days rather than a de novo appearance. Although 5-HT2A receptors are already known to contribute directly to regulating the activity of extensor motoneurons [START_REF] Gilmore | The excitability of lumbar motoneurones in the neonatal rat is increased by a hyperpolarization of their voltage threshold for activation by descending serotonergic fibres[END_REF][START_REF] Jackson | Receptor subtypes mediating facilitation by serotonin of excitability of spinal motoneurons[END_REF][START_REF] Liu | Stimulation of the parapyramidal region of the neonatal rat brain stem produces locomotor-like activity involving spinal 5-HT7 and 5-HT2A receptors[END_REF][START_REF] Pflieger | Postural Modifications and Neuronal Excitability Changes Induced by a Short-Term Serotonin Depletion during Neonatal Development in the Rat[END_REF][START_REF] Sławińska | 5-HT₂ and 5-HT₇ receptor agonists facilitate plantar stepping in chronic spinal rats through actions on different populations of spinal neurons[END_REF][START_REF] Takahashi | Direct excitation of rat spinal motoneurones by serotonin[END_REF][START_REF] Wang | 5-Hydroxytryptamine responses in neonate rat motoneurones in vitro[END_REF], one can speculate that 5-HT2A receptors are also expressed by interneurons of the pattern-generating level of the locomotor network or premotor motoneurons interposed between the locomotor network and the extensor motoneurons [START_REF] Noga | Locomotor-activated neurons of the cat. I. Serotonergic innervation and co-localization of 5-HT7, 5-HT2A, and 5-HT1A receptors in the thoraco-lumbar spinal cord[END_REF]. Because 5-HT2A receptor activation regulates the expression of the potassium-chloride co-transporter KCC2 involved in chloridemediated inhibitory synaptic signaling [START_REF] Bos | Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2[END_REF][START_REF] Gackière | Serotonergic modulation of post-synaptic inhibition and locomotor alternating pattern in the spinal cord[END_REF][START_REF] Jean-Xavier | Inhibitory Postsynaptic Potentials in Lumbar Motoneurons Remain Depolarizing After Neonatal Spinal Cord Transection in the Rat[END_REF], some of these neurons may be inhibitory interneurons responsible for regulating reciprocal inhibition in the network. Further work will be needed to decipher the expression patterns of this receptor over time at different levels of the spinal cord.

The 5-HT7 receptor subtype is also an important contributor to the prolocomotor action of 5-HT in a variety of preparations and experimental conditions [START_REF] Cabaj | Serotonin controls initiation of locomotion and afferent modulation of coordination via 5-HT7 receptors in adult rats[END_REF][START_REF] Hochman | 5-HT Receptors and the Neuromodulatory Control of Spinal Cord Function[END_REF][START_REF] Landry | Contribution of spinal 5-HT1A and 5-HT7 receptors to locomotor-like movement induced by 8-OH-DPAT in spinal cord-transected mice[END_REF][START_REF] Liu | Spinal 5-HT7 Receptors Are Critical for Alternating Activity During Locomotion: In Vitro Neonatal and In Vivo Adult Studies Using 5-HT7 Receptor Knockout Mice[END_REF][START_REF] Liu | Stimulation of the parapyramidal region of the neonatal rat brain stem produces locomotor-like activity involving spinal 5-HT7 and 5-HT2A receptors[END_REF][START_REF] Madriaga | Modulation of locomotor activity by multiple 5-HT and dopaminergic receptor subtypes in the neonatal mouse spinal cord[END_REF][START_REF] Noga | Locomotor-activated neurons of the cat. I. Serotonergic innervation and co-localization of 5-HT7, 5-HT2A, and 5-HT1A receptors in the thoraco-lumbar spinal cord[END_REF][START_REF] Pearlstein | Serotonin refines the locomotor-related alternations in the in vitro neonatal rat spinal cord[END_REF][START_REF] Sławińska | 5-HT₂ and 5-HT₇ receptor agonists facilitate plantar stepping in chronic spinal rats through actions on different populations of spinal neurons[END_REF]. In the present study, decreasing the involvement of 5-HT7 receptors did not alter the capacity of 5-HT to promote a locomotor organization of ipsilateral flexor/extensor activities, although the duration of locomotor episodes tended to be reduced. While a contribution of these receptors to the organization of the locomotor pattern cannot be excluded [START_REF] Cabaj | Serotonin controls initiation of locomotion and afferent modulation of coordination via 5-HT7 receptors in adult rats[END_REF][START_REF] Liu | Spinal 5-HT7 Receptors Are Critical for Alternating Activity During Locomotion: In Vitro Neonatal and In Vivo Adult Studies Using 5-HT7 Receptor Knockout Mice[END_REF][START_REF] Pearlstein | Serotonin refines the locomotor-related alternations in the in vitro neonatal rat spinal cord[END_REF], our observations favor, early after birth, a role of these receptors in mediating 5-HT's action on the rhythm-generating component of the locomotor network during sacral stimulation [START_REF] Liu | Stimulation of the parapyramidal region of the neonatal rat brain stem produces locomotor-like activity involving spinal 5-HT7 and 5-HT2A receptors[END_REF][START_REF] Madriaga | Modulation of locomotor activity by multiple 5-HT and dopaminergic receptor subtypes in the neonatal mouse spinal cord[END_REF]. 5-HT7 receptor antagonists reversibly triggers a complete hindlimb paraplegia in adult rat [START_REF] Cabaj | Serotonin controls initiation of locomotion and afferent modulation of coordination via 5-HT7 receptors in adult rats[END_REF] and markedly alter locomotor movements in adult mice [START_REF] Liu | Spinal 5-HT7 Receptors Are Critical for Alternating Activity During Locomotion: In Vitro Neonatal and In Vivo Adult Studies Using 5-HT7 Receptor Knockout Mice[END_REF]. This corresponds to the high expression level of this receptor subtype on locomotor-related interneurons [START_REF] Hochman | 5-HT Receptors and the Neuromodulatory Control of Spinal Cord Function[END_REF][START_REF] Jordan | Propriospinal neurons involved in the control of locomotion: potential targets for repair strategies?[END_REF][START_REF] Noga | Locomotor-activated neurons of the cat. I. Serotonergic innervation and co-localization of 5-HT7, 5-HT2A, and 5-HT1A receptors in the thoraco-lumbar spinal cord[END_REF].

Data from the literature also point to a contribution of 5-HT1A receptor activation to locomotor function, especially after a spinal cord injury [START_REF] Antri | Long-lasting recovery of locomotor function in chronic spinal rat following chronic combined pharmacological stimulation of serotonergic receptors with 8-OHDPAT and quipazine[END_REF]2003;[START_REF] Beato | Serotonin-induced inhibition of locomotor rhythm of the rat isolated spinal cord is mediated by the 5-HT1 receptor class[END_REF][START_REF] Cazalets | Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat[END_REF][START_REF] Landry | Contribution of spinal 5-HT1A and 5-HT7 receptors to locomotor-like movement induced by 8-OH-DPAT in spinal cord-transected mice[END_REF]. Here, 8-OH-DPAT was found to inhibit rhythmical motor outputs from the extensor dominated L5 segment.

This action, which differs from the implication of 5-HT7 receptors, is therefore likely be to be mediated through the activation of 5-HT1A receptors. These latter receptors are expressed presynaptically on serotonergic fiber terminations (autoreceptors) and on motoneurons where they were identified on the axonal initial segments (AIS; [START_REF] Cotel | Serotonin spillover onto the axon initial segment of motoneurons induces central fatigue by inhibiting action potential initiation[END_REF]. It has recently been reported that pharmacological application of 8-OH-DPAT significantly inhibits locomotor-like activity in turtle spinal cord preparations [START_REF] Perrier | Intense Activity of the Raphe Spinal Pathway Depresses Motor Activity via a Serotonin Dependent Mechanism[END_REF], partly due to a 5-HT1A receptor-dependent inhibition of motoneuron activity [START_REF] Perrier | Serotonin differentially modulates the intrinsic properties of spinal motoneurons from the adult turtle[END_REF]. This echoes with the observation that in humans, oral administration of a 5-HT1 agonist is thought to mediate a 5-HTdependent process of central fatigue during physical exercise [START_REF] Marvin | The effects of buspirone on perceived exertion and time to fatigue in man[END_REF].

The observation in the present work that 8-OH-DPAT selectively affects extensor-like activity remains unclear. It may be related to the fact that the distribution of 5-HT1A receptors displays a rostro-caudal gradient, with the highest expression level occurring in the more caudal regions of the rat's spinal cord [START_REF] Marlier | Autoradiographic mapping of 5-HT1, 5-HT1A, 5-HT1B and 5-HT2 receptors in the rat spinal cord[END_REF].

Conclusion

This work paves the way for further studies investigating the developmental aspect of monoaminergic neuromodulation of spinal cord sensorimotor networks. A comprehensive understanding of such processes is also of particular interest in the context of spinal cord injuries in which enhancing the re-expression of motor functions (including locomotion) below a spinal cord injury by promoting the integration of sensory inputs with pharmacological agents is among the therapeutic avenues.

Alluin, O., Delivet-Mongrain, H., Rossignol, S., 2015. Inducing hindlimb locomotor recovery in adult rat after complete thoracic spinal cord section using repeated 6.

  5-HT only experiments (n=14), partitionned spinal cord experiments (n=9), quipazine experiments (n=10), ketanserin experiments (n=18), LP-44 experiments (n=9), including 3 for antibody testing). All procedures were conducted in accordance with the local ethics committee of the University of Bordeaux, and followed the European Committee Council directives (2010/63/EU). All efforts were made to minimize animal suffering and the number of animals used.

  figure2D1) and alternating ipsilateral motor bursts between right L5 and L2 (rL5/rL2:

  (A, B) from a P1 preparation in which the patterning of sacral stimulation-induced fictive locomotion remained unaffected after adding ketanserin (10µM) to the bath (control condition: rL5/rL2: Φ = 0.46 ± 0.05; lL5/rL2: Φ = 0.90 ± 0.02; ketanserin condition: rL5/rL2: Φ = 0.48 ± 0.03; lL5/rL2: Φ = 0.92 ± 0.03; figure6A,B, bottom panels). Altogether, these data indicate that 5-HT2A receptor activation is not critical in young preparations (<P2) to shape locomotor output triggered by sacral stimulation, but at later stages this receptor subtype plays a crucial role in activating the extensor component of the spinal locomotor network.

Figure 1 :

 1 Figure 1: Experimental paradigm. (A) Schematic diagram of the isolated

Figure 2 :

 2 Figure 2: Gradual postnatal maturation of sacral stimulation-induced lumbar motor

Figure 3 :

 3 Figure 3: 5-HT promotes the expression of fictive locomotion induced by sacral

Figure 4 :

 4 Figure 4: 5-HT's pro-locomotor action on extensor activity is mediated at the level of

Figure 5 :Figure 8 :

 58 Figure 5: Agonist-mediated activation of 5-HT2A receptors mimicks 5-HT pro-

Figure 9 :

 9 Figure 9: Immunodetection of 5-HT2A receptors in the spinal cord of postnatal rat. (A)

Figure 10 :

 10 Figure 10: Schematic diagrams summarizing the action of sacral DR stimulation alone

  Figure 1

locomotor-related activity in the presence of quipazine (B2-B2). The pro-locomotor action of quipazine persisted after its washout (C1, C2) D, Histograms of the percentage of preparations expressing tonic activity (gray), "in phase" (light blue), "antiphase" (dark blue) or "irrelevant phase" (medium blue) relationships between pairs of lumbar ventral roots (left: lL2 and rL2; middle: rL5 and rL2; right: lL5 and rL2) at different (A1-A2), Integrated lumbar motor activities recorded during sacral stimulation (top) and associated circular plots of the phase relationships between ventral roots in the same P3 preparation. In the control condition, the preparation did not express fictive locomotion (A1). After exogenous application of 5-HT (10µM) in the presence of the pre-applied 5-HT7 receptor antagonist SB269970 (10µM), the preparation generated fictive locomotion in response to the same sacral stimulation (A2). (B1-B2), Fictive


Alan Cotton
email: alan.cotton@unamur.be

Philippe Orban

Samuel Wildemeersch

Pauline Collon

Vincent Hallet

Serge Brouye Re

Active Dewatering Of Limestone Quarry And Drinking Water Production -Feasibility Assessment Using A Groundwater Model

Keywords: Belgium, Quarry dewatering, Peripheral well, Drinking water supply, Groundwater modeling

Lhoist Industries Ltd. plan to deepen the Boverie Quarry (Rochefort, Belgium), in which two Frasnian limestone reefs are exploited. If this project is allowed, the dewatering will dry up the Tridaine gallery that provides drinking water for the city and Saint-Remy's Abbey of Rochefort. Therefore, the project of deepening has to conciliate both quarry dewatering and drinking water production. The solution found by the quarry owner is to achieve the dewatering using three wells, for technical safety or water quality reasons, located in the periphery of the quarry and to distribute the pumped water to both the city and abbey. Hydrogeological characterization and a model using the SUFT3D research code tend to show that quarry dewatering and drinking water production could be achieved during and after the quarry operation, even by using a single well.

Introduction

In Belgium, as in many other states having a small surface and a high population density, mineral industry and drinking water production are closely tied together. Indeed, as lateral expansion of quarries is therefore extremely limited, most of them have to be deepened. In such a case, the bottom of the extraction pit could reach the water table and a dewatering system has to be established to continue the extraction operations. Most often, quarries dewatering is carried out by means of a sump, which is by far the least expensive solution. Like any other kind of dewatering systems, sumps affect the regional hydrogeology and, occasionally, the productivity of nearby groundwater catchments by, for example, drying up the wells or increasing the pumping costs [START_REF] Younger | Mine Water: Hydrology, Pollution, Remediation[END_REF]. In order to mitigate the impacts of quarry dewatering on groundwater catchments, mine water could be used for drinking water production. However, water that is also collected in the sumps could not be of sufficient quality to be used as drinking water without a heavy and very expensive treatment. In those cases, a viable option to reconcile both dewatering and drinking water supply is by means of drilling one or more peripheral wells.

Lhoist Industries Ltd. have chosen this alternative with regard to their project of deepening the Boverie limestone quarry in Rochefort (Belgium). At the site, the mining company operates two Devonian limestone reefs for high-grade lime production. Actually, groundwater contained in these formations is drained by the Tridaine gallery, an old artificial drainage gallery, which will dry up if the deepening project comes true. This drainage gallery provides drinking water for the majority of the 12,500 inhabitants of the city of Rochefort as well as for Saint-Remy's Abbey of Rochefort, for beer brewing.

Even if the quarry owner has already decided to drill three wells, a groundwater flow model has been developed to assess the feasibility of (1) dewatering the quarry with a single well located in its periphery and (2) providing enough water to fulfil the water demand of both the city and abbey.

Context and Project Description

The Boverie Quarry is located in the southern part of Belgium, in the Famenne-Ardenne UNESCO Global Geopark. The quarry lies on a topographic ridge called "Calestienne", which marks the limit between the Fagne-Famenne depression to the north and the Ardenne plateau to the south (fig. 1). The "Calestienne" is made of Givetian and Frasnian limestone and shale formations. Concerning the Frasnian stage (fig. 2), in which the quarry is located, the sedimentary sequence begins with the shale of the Nismes Formation. It continues with the limestone reef of the Arche Member, which changes laterally into the shaly Ermitage Member. The Arche Member is topped by the clayey limestone of the Bieumont Member, which precedes the second limestone reef of the Lion Member. The upper part of this limestone reef is crossed longitudinally by a major fault zone (fig. 2). The Frasnian sedimentary sequence ends with the shale of the Boussu Member, which overlaps the Lion Member. The Boverie Quarry is located in the Arche and Lion Members (fig. 2). Strike and dip are homogeneous with values of N35°E and 45 °NW, respectively.

The Arche and Lion Members are hydraulically connected together in the quarry zone and represent a single aquifer formation. It contains the water table drained by the Tridaine gallery. Due to their lithologies, the surrounding geological formations are relatively impervious and tend to isolate the aquifer. Consequently, the aquifer may be compared to "a big bathtub" with limited inflow from the surrounding geological formations. The main inputs are the recharge (410 mm/year on average) and the infiltration of the Entre-Deux-Falleux (E2F) stream in the Lion Member to the northeast (fig. 2), and the output being the Tridaine gallery (765,000 m 3 /year on average). As highlighted by several pumping tests, the major fault zone in the upper part of the Lion Member induces a higher hydraulic conductivity of the aquifer in this area. Despite this fault zone, no other preferential flow paths, such as karstic phenomena, have been encountered following geophysical investigations or during the quarry operation.

Currently, the bottom of the extraction pit is limited to the altitude of 220 m.a.s.l., nine meters above the elevation of the Tridaine gallery. This drain has a catchment capacity of 50 m 3 /h, from which 10 m 3 /h are permanently attributed to the abbey for beer brewing while the other 40 m 3 /h are used by the city of Rochefort for public drinking water supply. During high water periods, the gallery discharge can increase up to 300 m 3 /h and the excess water overflows in surface water. However, during low water periods, the drain discharge can decrease to 10 m 3 /h, jeopardizing the water supply of the city, which needs to buy water from the regional water company, which in turn is not always able to provide the required volumes. The project of Lhoist Industries Ltd. consists in the deepening of the Boverie Quarry to the altitude of 160 m.a.s.l., drying up the Tridaine gallery as a consequence. To ensure the continuous supply of drinking water, the quarry owner proposed to achieve the dewatering using three peripheral wells. In this way, the water loss during high water periods can be decreased and, last but not least, the city of Rochefort will not suffer of water scarcity during low water periods. The main pumping well (Lion-1) has been drilled in the high hydraulic conductivity zone located in the upper part of the Lion Member, 100 meters northwest from the nearest mining face (fig. 2). The borehole is 156 meters deep (bottom at the altitude of 91 m.a.s.l.), 440 mm wide and equipped with 305 mm wide stainless steel screened tubing. As expected, fractures have been intersected during the drilling operations as well as important water inflows, showing the potential high productivity of the well. It will be used as a single pumping well in the model. In the event of technical failure or water quality issue of this well, two backup wells were drilled around the quarry pit (fig. 2). In order to estimate the water budget for the Arche and Lion Members separately and to assess the quantity of groundwater flowing from the Arche Member to the Lion Member, four subdomains have been introduced. One subdomain corresponds to each limestone reef, one to the more permeable zone of the Ermitage and Bieumont Members in the middle of the extraction pit and one to the rest of these two members. Groundwater exchanges between the different subdomains are described by Fourier internal boundary conditions.

As explained previously, the only solicitations of the model are the recharge and the infiltration of the E2F stream. The analysis of residuals (fig. 3) shows that the model is well calibrated but tends to overestimate the piezometric head when it is low. This trend may be explained by the better calibration during high water periods, which has been done voluntarily to improve the model accuracy during high recharge events. In this way, the dewatering discharge could be ascertained precisely to avoid flooding of the extraction pit.

Figure 3 Residuals of the model calibration, data from five piezometers

Groundwater modeling: Dewatering simulation According to the blueprint of Lhoist Industries Ltd., the deepening of the Boverie Quarry would take place in four phases (tab. 1), each one corresponding to the creation and operation of a new extraction level. During each of these four phases, the bottom of the extraction pit will be lowered by 15 meters. In order to mitigate any potential groundwater contamination and to avoid any risk of flooding, the water table must be kept always at least ten meters below the bottom of the extraction pit. Giving the project specifications, the dewatering of each level should be achieved in one year and the water table should stabilize during the whole levels operation, i.e. 6.5 years. 160 150 Dewatering simulation has been performed in transient conditions for a period of 30 years, with a monthly time step and monthly solicitations. The mean monthly recharge applied to the model has been calculated from the data collected between 2008 and 2017. The same recharge is applied to the model every year until the end of the simulation. Due to a lack of data, the infiltration of the E2F stream is the same one as for the model calibration. As for the recharge, the same solicitation is also applied every year. To represent the drying up of the Tridaine gallery and to avoid water inflow in the model, the corresponding Fourier boundary condition has been deactivated at the beginning of the simulation. As the downstream part of the E2F stream will not drain the groundwater table anymore during the quarry dewatering, the corresponding Fourier boundary condition has been deactivated for the whole simulation.

Modeling results (fig. 4) show that floor dewatering is achieved in one year with a constant pumping rate of 132 m 3 /h, except for phase 1 where the pumping rate is 145 m 3 /h. This higher value required for the first floor dewatering might be explained by the higher drawdown (23 m) needed for the operation of this level, compared to the drawdown of phases 2 to 4 (15 m) (fig. 4a).

On the other hand, the pumping well discharge required for the water table stabilization is constant from phase to phase, with a value of 113 m 3 /h (fig. 4b). The stability of the pumping rate required during levels operation indicates that groundwater lateral inflows coming from the surrounding geological formations should be limited.

In the light of these results, the use of one single peripheral well should be enough to lower the groundwater table everywhere under the Boverie Quarry pit (fig. 4a). It is quite clear that the possibility to achieve the dewatering in such conditions mainly relies on the high hydraulic conductivity of the aquifer formation as well as on the limited lateral inflows from the surrounding geological formations. The model shows that the dewatering of the Boverie Quarry could be achieved while ensuring the drinking water supply of 50 m 3 /h required for the city and Saint-Remy's Abbey of Rochefort. Finally, a dynamic management of the well pumping rate could be done according to the hydrogeological conditions: when the local water supply rate and minimum piezometric level are respected, the well discharge could be adjusted to minimize groundwater wastage to surface water, avoiding over pumping of the aquifer.

Conclusions

The model developed with the SUFT3D research code seems to prove that the deepening project of the Boverie Quarry using one single peripheral well is feasible, owing to the high hydraulic conductivity of the aquifer as well as to the limited lateral inflows. The model has been calibrated, especially for high water periods. It shows that the discharge of the pumping well should be sufficient to guarantee the drinking water supply of both the city and abbey. However, Lhoist Industries Ltd. have already drilled two more wells, for security or water quality reasons. The modeling results still require to be validated by a long-term pumping test.

Figure 1

 1 Figure 1 Location of the Boverie Quarry (star) within the "Calestienne" (after Poulain 2017)

Figure 2

 2 Figure 2 Frasnian geological map with the location of the Boverie Quarry, Tridaine gallery (triangle), Lion-1 pumping well (diamond), backup wells (stars) and piezometers (circles) (DEM Hillshade from SPW) Groundwater modeling: Conceptual model and calibration A groundwater flow model has been developed with the SUFT3D (Saturated and Unsaturated Flow and Transport in 3D) research code, which has already demonstrated its effectiveness in a number of previous studies (e.g. Brouye re et al. 2009; Orban et al. 2010). This code uses the finite elements method to solve flow problems in variably saturated conditions. The greatest strength of the code is its ability to subdivide the model into sub-domains to perform different water budgets in the model. Those sub-domains communicate with each other by means of internal boundary conditions to simulate groundwater flow between the different sub-domains. The model extends over the Arche and Lion Members as well as the part of the Bieumont Member located in the extraction area. A finite element mesh of 27,000 prismatic elements (mean side length of 20 m) and 19,500 nodes, vertically divided in five layers, characterizes the model. The top of the mesh coincides with the topographic surface retrieved from a digital elevation model and the bottom has been arbitrarily set to the altitude of 50 m.a.s.l. in order to represent the future deepening of the quarry (pit bottom at 160 m.a.s.l.) as well as the whole height of the pumping well. The dip of the geological layers is also taken into account. The conditions prescribed at the boundary of the model domain are Fourier external boundary conditions with the exception of the contact between Nismes Formation and Arche Member, which is a no-flow Neumann external boundary condition. The choice of Fourier boundary conditions has been made to represent the probable groundwater lateral inflows that may enter the model as a consequence of the decrease of the piezometric head during the quarry dewatering. A Fourier external boundary condition has also been prescribed for the Tridaine gallery.

  The recharge is represented by a Neumann external boundary condition prescribed on the top face of each element composing the top layer of the model domain. The recharge calculation takes into account the lack of soil and vegetation in the mined area. The infiltration of the E2F stream in the Lion Member is also represented by a Neumann external boundary condition, prescribed on the top face of each element crossed by the stream. As the downstream part of the E2F stream drains the groundwater table during high water periods, a Fourier external boundary condition has been prescribed in this zone. This boundary condition is deactivated during low water periods to avoid water inflow.The model has been divided in ten zones to describe the spatial heterogeneity. Within each of these zones, hydrogeological parameters such as hydraulic conductivity and effective porosity are constant. Three zones correspond to the Arche Member, three more to the Bieumont Member, one to the Lion Member and the last three zones are located along the fault, in the upper part of the Lion Member.Model calibration has been performed over a whole year (May 2010 to May 2011) based on observed data collected in five piezometers spread out in the Arche and Lion Members (fig.2), and with the discharge of the Tridaine gallery. The simulation has been run in transient conditions, with a time step of one day and weekly solicitations. The choice of such a short time step has been made to reproduce the fast response of the aquifer to the recharge, as good as possible. The calibration has been done by trial and error approach, by changing the hydrogeological parameters of the different zones as well as the exchange coefficients of both internal and external boundary conditions.

Figure 4

 4 Figure 4 (A) Dewatering simulation results for pumping well Lion-1 and piezometers located in the quarry; (B) pumping well Lion-1 discharge during the different operation phases (X axis of both graphs are purely indicative)

Table 1

 1 Operation schedule of the Boverie Quarry deepening

	Phase	Pit bottom (m.a.s.l.)	Water table max. altitude (m.a.s.l.)
	1	205	195
	2	190	180
	3	175	165
	4

Acknowledgements

The authors would like to thank Lhoist Industries Ltd. for letting them access freely the Boverie Quarry and for sharing all the hydrogeological data they have gathered over time. The authors also thank the reviewers and Walter Verhaert who helped to improve the quality of this manuscript. Last but not least, they thank Isabelle Bonniver (UNamur) for the comprehensive hydrogeological synthesis of the site and Gae tan Rochez (UNamur) for his unconditional help during the plentiful fieldworks.