187

Journal of Physiology (1992), 455, pp. 187-204 With 9 figures Printed in Great Britain

ACTIVATION OF THE CENTRAL PATTERN GENERATORS FOR LOCOMOTION BY SEROTONIN AND EXCITATORY AMINO ACIDS IN NEONATAL RAT

BY J. R. CAZALETS, Y. SQALLI-HOUSSAINI AND F. CLARAC From the Centre National de la Recherche Scientifique, Laboratoire de Neurosciences Fonctionnelles 2, 31 chemin Joseph Aiguier BP 71, 13402 Marseille, cedex 9, France

(Received 29 July 1991) SUMMARY

1. The role of serotonin (5-HT) and excitatory amino-acids (EAAs) in the activation of the neural networks (i.e. the central pattern generators) that organize locomotion in mammals was investigated in an isolated brainstem-spinal cord preparation from the newborn rat. 2. The neuroactive substances were bath applied and the activity of fictive locomotion was recorded in the ventral roots. 3. Serotonin initiated an alternating pattern of right and left action potential bursts. The period of this rhythm was dose dependent, i.e. it decreased from around 10 s at 2 x 10-5 M to 5 s at 10-4 M. The effects of serotonin were blocked by a 5-HT1 antagonist (propanolol) and by 5-HT2 antagonists (ketanserin, cyproheptadine, mianserin). 5-HT3 antagonists were ineffective. The effects of methoxytryptamine, a non-selective 5-HT agonist, mimicked the 5-HT effects. 4. The endogenous EAAs, glutamate and aspartate, also triggered an alternating rhythmic pattern. Their effects were blocked by 2-amino-5-phosphonovaleric acid (AP-5; a N-methyl-D-aspartate (NMDA) receptor blocker) and 6,7-dinitro-quinoxaline-2,3-dione (a non-NMDA receptor blocker). 5. Several EAA agonists (N-methyl-D,L-aspartate (NMA) and kainate) initiated rhythmic activity. The period of the induced rhythm (from 3 to 1 s) was similar with both of these substances but in a range of concentrations which was ten times lower in the case of kainate (10-6 to 5 x 10-6 M) than in that of NMA (10' to 4 x 10-5 M). a-Amino-3-hydroxy-5-methylisoxazole-4-propionate and quisqualate occasionally triggered some episodes of fictive locomotion with a threshold at 6 x 10-7 and 10-5 M,

respectively. INTRODUCTION

In all animals from invertebrates to vertebrates, it has been shown that rhythmic stereotyped movements, particularly locomotor activity, are generated by neuronal networks that have been called central pattern generators (CPGs; Grillner, 1985; Lydic, 1990). From comparative studies, it has emerged that the same general principles govern CPG functions and that the activity of a CPG is under the control of triggering and/or enabling systems that turn on and modulate the network MS 9598

J. R. CAZALETS AND OTHERS 188 activity (Harris-Warrick, 1988). In addition, the working of these CPGs can be considerably modified by direct sensory controls converging with modulatory inputs to elaborate the optimal motor output. In this framework we have been studying the genesis of locomotion in mammals. In studies on locomotion, the CPGs were first detected in the spinal cord using preparations (mainly from the cat) from which descending inputs and sensory feedback had been removed. Under these restrictive conditions, fictive locomotion could be elicited in various ways (either by means of chemical activation with L3,4-dihydroxyphenylalanine (L-DOPA) or sensory stimulation; Grillner, 1986). More recently, using in vitro preparations of lower vertebrates (Dale & Roberts, 1984; Brodin & Grillner, 1985) it was discovered that not only the amines but also the excitatory amino-acids (EAAs) play a crucial role in the activation of the locomotor CPGs of vertebrates. In this paper we investigated the neurochemical control of the CPGs for locomotion using an in vitro isolated brainstem-spinal cord preparation from the newborn rat. This preparation, initially developed by Otsuka & Konishi (1974), was first used for studying respiration (Suzue, 1984; Smith & Feldman, 1987; Errchidi, Hilaire & Monteau, 1990). Due to its small size and the absence of myelin, the spinal cord of the newborn can survive for several hours, due to the passive diffusion of metabolites and gas. One of the main advantages of this preparation is the fact that there is no blood-brain barrier which makes all drugs accessible to the nervous system. Consequently, the effects of transmitters and those of all their agonists or antagonists can be studied directly without any need to use neurotransmitter precursors that cross the blood-brain barrier such as DOPA, and the extracellular medium can be easily controlled. By recording the activity in the ventral roots one can analyse the global motor output which makes it possible to define the expression of the CPG in terms of the motoneuronal discharge produced in response to an exogenously applied transmitter. Using this preparation, it was previously shown that EAAs play an important role in the initiation of locomotor rhythms in mammals (Kudo & Yamada, 1987; Smith, Feldman & Schmidt, 1988; Cazalets, Grillner, Menard, Creimieux & Clarac, 1990a). In addition, we recently observed that 5-HT can also elicit fictive locomotion in the isolated spinal cord (Cazalets et al. 1990a). However, up to now it was assumed that the action of the EAAs in the rat spinal cord was mediated only via N-methyl-Daspartate (NMDA) receptors (Kudo & Yamada, 1987; Smith et al. 1988). With the discovery of new compounds that specifically antagonize non-NMDA (N-methyl-Daspartate) receptors (i.e. kainate and oc-amino-3-hydroxy-5-methylisoxazole-4propionate (AMPA) receptors (Honore, Davies, Dreger, Fletcher, Jacobson, Lodge & Nielsen, 1988) it has become possible to clearly dissociate the effects of EAAs on the various types of receptors. We therefore reconsidered their action in this study and observed that several types of EAA receptors participate in the genesis of locomotor activity. In parallel, we also carried out a systematic study on the effects of serotonin on the motor pattern, and demonstrated that in addition to its classical action on motoneurones, serotonin acts on the premotoneuronal oscillatory network. We studied the effects of serotonin (5-liT) and EAAs together for two reasons; first, because we have observed that close interactions exist between these two systems (Cazalets, Sqalli-Houssaini, Menard, & Creimieux, 1990b); and secondly, because it

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was not clear whether or not 5-HT could activate the locomotor CPGs (Grillner & Shik, 1975; Barbeau & Rossignol, 1990, 1991). METHODS

I)issection The experiments were performed on a total of 150 animals (newborn Wistar rates aged 0-3 days) raised at our laboratory. Before dissecting out the nervous system, they were anaesthetized with ether, decapitated and rapidly eviscerated, and the skin was then removed. They were pinned back in a Sylgard-lined Petri dish perfused with physiological saline containing (mM): NaCl, 129; KCl, 4; CFaC2, 25; MgCI2, 114; NaH2PO4, 0-58; NaHCO3, 25; glucose, 10) adjusted to pH = 7-4 with HCl. The saline was continuously bubbled with a 95% 02 -5% CO2 mixture. The vertebrae were removed and the spinal cord rapidly exposed. Ventral and dorsal roots were sectioned and the nervous system was transferred to a small Sylgard-lined Petri dish (volume around 12 ml). In some cases the hindlimbs were kept attached in order to perform muscle recordings (Cazalets et al. 1990 a). The ventral roots were pinned to ensure accurate positioning of the electrodes. This whole series of experiments was performed with the spinal cord attached to the brainstem. Throughout the experiments, the temperature was kept constant (25 TC). Recordings The stage and the temperature regulation system have been extensively described elsewhere (Sqalli-Houssaini. Cazalets Fabre & Clarac, 1991). Briefly, motoneuronal activity was recorded in the ventral roots using monopolar pin electrodes that were placed in contact with the nerve and insulated with Vaseline. The signals were amplified (x 10000) with high impedance AC amplifiers and displayed on a Gould ES 1000 electrostatic recorder coupled to a Gould monitor. Recordings were stored on a digital 8-channel tape (Biologic DTR 1800). The traces were occasionally integrated, filtered and rectified. Pharmacology The L-aspartate, cyproheptadine. L-glutamate, 5-hydroxy-DL-tryptophan (5-HTP), kainate, 5rnethoxytryptamine. NMDA. the AN-methyl-D.L-aspartate (NMA), quisqualate and serotonin (5HT) were obtained from Sigma. The AMPA HBr, 1-(3-chlorophenyl)piperazine HC1, (± )-8hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), ketanserin tartrate, p-aminophenylethyl-m-trifluoromethylphenyl piperazine (PAPP), 1-(2-methoxyphenyl)piperazine (2-MPP HCl). metoclopramide HCl, mianserin HCl, 1-phenylbiguanide, (-)-propanolol HCR, quipazine maleate, m-trifluoromethylphenylpiperazine hydrochloride (TFMPP HCR), 3-tropanyl-3,5dichlorobenzoate were obtained from Research Biochemical Incorporated (Natick, USA). The RU2469 was a gift from Roussel-Uclaf (Paris, France). For convenience, the drugs were generally prepared at 10-2 M in distilled water, then frozen and stored at -20 'C. They were diluted in the saline to the appropriate concentration prior to use. Less frequently used substances were generally freshly prepared. The drugs were conveyed by a peristaltic pump (Gilson, Villers-le-Bel, France), and the flow of the saline throughout the preparation was kept constant during the whole experiment. All the substances that were shown to elicit fictive locomotion on the combined brainstem-spinal cord preparation were also shown to have the same effect on the isolated lumbar segments. However, as the activity was generally weaker with this reduced preparation we generally worked with the whole spinal cord and brainstem. To test the effects of antagonists, these were first perfused alone before being applied simultaneously with the transmitter. Period measurements Several methodological problems arose as regards the analysis of the results. The first concerned the period measurements. When a substance was applied to the spinal cord, the period of the induced pattern was initially slow and then shortened progressively until a plateau was reached. This corresponded to the progressive replacement of the normal saline until the actual concentration was reached. It generally occurred within 2 to 3 min. The periods were measured only in the plateau corresponding to the real concentration. In some experiments, however, particularly with EAAS, only a systematic decrease in the period occurred with time, until one tonic discharge was recorded, and no plateau was reached. This made the period difficult to evaluate and these experiments were not taken into account.

190

J. R. CAZALETS AND OTHERS RESULTS

Characterization and induction of the fictire locomotor rhythm The locomotor-like activity generated by the isolated spinal cord consisted of alternating bursts of action potentials that could be recorded either in the hindlimb muscles (when the legs were attached). or the lumbar ventral roots. Sequences of locomotor-like activity were evoked by bath application of various transmitters or their agonists. This motor pattern was similar to that recorded in intact newborn rats (Cazalets, Menard, Cremieux & Clarac, 1990b). In Fig. lB the activity of the flexor muscle of the hip, the gluteus superficialis, (GS, see Fig. IA) was recorded during an episode of locomotion induced by 5 x 106 M-NM1)A. This muscle was active during hip flexion (which was observed visually during the alternating flexion and extension of the right and left hindlimbs), with phase relationships that were identical to those observed in the same muscles in intact neonates (Cazalets et al. 1990b). It was also possible to directly monitor the flexor and extensor phase during one cycle by recording the activity from the ventral roots (Fig. 1 C). due to the intersegmental distribution of flexor and extensor motoneurones (Nicolopoulos-Stournaras & Iles, 1983). In this experiment we simultaneously recorded the lumbar ventral roots L2-5 on one side. In L2 and L3 bursts of spikes were observed that were in phase and were identified as extensor units. These bursts in L2 and L3 were in opposition to the activity exhibited in L5 which mainly contains flexor units (Nicolopoulos-Stournaras & Iles, 1983). On the contrary. in L4 a complex discharge was observed that overlapped with the L2-3 and L5 activity (due to the mixing of the flexor and extensor activity). This intersegmental distribution was consistent with anatomical data by other authors (see Discussion). The serotonin-induced rhythm The addition of 5-HT to the saline-induced bursts of spikes alternating between the left and right sides of each lumbar segment (Fig. 2B-D). The 5-HT rhythm was characterized by a high level of motoneuronal activity and even when the bursts were clearly visible, considerable background activity was generally recorded during the interburst intervals. The average of period of the 5-HT-induced cycling was generally kept constant, i.e. there was no progressive decrease or increase in the period with time. We distinguished three categories of rhythms depending on the regularity of the pattern. The classification was based on twenty-seven experiment in which the 5-HT was applied at 10' M. In 41 % of the experiments the rhythm was stable, regular and could last for as long as the drug was applied (although it could also cease after several minutes). In 37 % of the cases tested, the pattern was irregular and fragile with disruptions. In these cases, the rhythm was evoked transiently (for 4 or 5 min), i.e. during continuous application of the drug, the slow alternating pattern turned into a high-frequency discharge the pattern of which was no longer organized. This was perhaps due to desensitization, since it was however possible to elicit fictive locomotion again, with another bath application, after the washing. In 22 % of the experiments 5-HT did not elicit an alternating pattern but only tonic or high frequency activity (see also Cazalets et al. 1990 a). In some cases, the 5-HT also evoked a non-alternating pattern, instead of the alternating one. We also attempted to

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mimick the effects of the 5-HT by using its precursor 5-hydroxy-DL-tryptophan (5HTP). However, 5-HTP has been found to be inefficient at inducing fictive locomotion, whatever its concentration (up to 5 x 10-3 M) or the duration of the bath; application, although in the same experiment 5-HT alone induced an alternating pattern. A

C T L2

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2s 15 Fig. 1. Fictive locomotion in the isolated preparation is induced by bath application of transmitters. A, drawing of the semi-isolated preparation in which the hindlimbs were attached. Electrodes were inserted into the right and left gluteus superficialis (rGS and IGS) which is a hip flexor. The legs could move freely. B, the bath application of NMDA (5 X 10-6 M) elicited alternating bursts of action potentials in the right and left GS, accompanied by alternate flexion and extension of the legs. C, recordings of ipsilateral lumbar ventral roots 2 to 5 (L2-5. in the isolated spinal cord preparation; iL2-5. integrated trace), during bath application of NMA (2 x 10-' M). The dashed lines indicate the onset of a new cycle of extension/flexion. The burst in L2-3 occurred during the extension phase and was phase opposed to the burst in L5 (which occurred during the flexion phase).

A dose-response relationship was observed in the case of serotonin (Fig. 2), although the range of concentration in which the serotonin active was effective was small. The threshold for eliciting a weak tonic activity was 10-6 M (Fig. 2A). At this concentration the serotonin occasionally elicited a very slow (with a 30 s period) but reciprocally organized pattern. A clearly alternating pattern generally emerged at 5 X 10-5 M (Fig. 2B), after which the period decreased from 5 x 10-6 to 10-4 M (Fig. 2B-D). Upon plotting the period of cycling versus the 5-HT concentration, it emerged that a plateau was reached at 1o-4 1w (Fig. 2E). These systematic changes in the 5-HT concentrations did not affect the phase relationships between the two sides which were maintained at 0 5 (i.e. with a strict phase opposition). In order to identify the receptor affected by the 5-HT, we used both antagonists and agonists. In all cases they were tested in at least three different experiments

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in which the 5-HT was active and at concentrations ranging from 10-6 M to 10-4 M. Among the agonists tested, only 5-methoxytryptamine (a non-selective 5-HT agonist) elicited the alternating right and left pattern; whereas selective 5-HT1 agonists such as RU-24969 (5-HTIB), 8-OH-DPAT (5-HT1A), 1-(3-chloroA 5-HT(10-5M) rL3

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Fig. 2. The period of the fictive locomotion varies with the serotonin concentration. A-D, bath application of serotonin at 10-5 M elicited only a very weak tonic activity. From 5 x 10-5 to 10-4 M, an alternating pattern was induced, and the period gradually shortened, reaching a minimum at 10-4 M. E, the dose-response curve started at 2-5 x 10-5 M, which is the threshold concentration for eliciting an alternating pattern with a very long period; the plateau was reached at 10-4 M with a period of 5-6 s. The number of experiments is indicated in parentheses beside each point.

phenyl)piperazine, PAPP, 1-(2-methoxyphenyl)piperazine (2-MPP HCl) and TFMPP triggered no activity. The 5-HT3 receptor agonists 1-phenylbiguanide and quipazine (a non-selective agonist) were also ineffective. Since the agonists had no effects, we further tested the action of putative antagonists. If 5-HT is assumed to exert a direct action on the locomotor CPGs, it is to be expected that the 5-HT antagonists will decrease the 5-HT-induced rhythmicity. Figures 3 and 4 show that in fact the effects of 5-HT are mediated through both the 5-HT, and 5-HT2 receptor types. Figure 3 shows that (-)propanolol, a putative 5-HT1 antagonist (Middlemiss, 1984), blocked the slow rhythmic activity induced by bath application of 5-HT. Under control conditions, no activity was detected (Fig. 3A) but the bath application of 5-HT (Fig. 3B) induced burst activity at a period of 3-9 + 023 (mean + S.D. n = 30). The integrated trace clearly showed this burst activity (irL3). The subsequent application of propanolol together with serotonin led to a drop in the activity, and the period increased

FICTIVE LOCOMOTION IN RAT SPINAL CORD

193

B 5-HT (10-4 M)

A Control

rL3 irL3

C 5-HT (10-4 M) +propanolol (5 x 10-5 M)

D 5-HT (10-4 M) + propanolol (7T5 x 10-5 M)

5s

Fig. 3. Antagonistic effects exerted by propanolol on the 5-HT-induced activity. In a silent system in which the right lumbar ventral root 3 was recorded (A, rL3; irL3: integrated trace) the bath application of serotonin initiated slow rhythmic activity (B, rL3). After wash-out, serotonin was again applied but in the presence of propanolol which led to a considerable increase in the period of the 5-HT-induced activity. Increasing the propanolol concentration completely suppressed the slow motor pattern (C) and only tonic discharge was then recorded (compare to control assay in A). A Control

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Fig. 4. 5-HT2 receptors are involved in the 5-HT response. In an experiment in which 5HT was inducing activity recorded from the ventral roots (B), the addition of the 5-HT2 antagonist ketanserin (C) increased the period of the alternating motor output. Further increasing the ketanserin concentration (D) completely blocked the rhythmic activity induced by 5-HT and only tonic activity remained (compare with control conditions in A). Recovery occurred if 5-HT was applied after washing with normal saline.

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194

threefold (Fig. 3C). When the propanolol concentration was increased (Fig. 3D) all rhythmic activity was abolished and only a tonic discharge remained, as can be seen in comparison with the control conditions (Fig. 3A). The effects of propanolol were generally difficult or impossible to reverse. It is worth noting that (+ )-propanolol did A Control, Asp (5 x 1 0-4 M)

rL2

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C Wash -out, Asp (5 x 1 0-4 M)

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Fig. 5. The aspartame action is mediated through both NMDA and non-NMDA receptors. In an experiment where the aspartate (Asp) induced a strong alternating pattern (A), the addition to the saline of AP-5 (an NMDA receptor antagonist) completely blocked the activity (B). After 20 min wash-out with normal saline, aspartame bath application restored the activity (C). The subsequent addition of DNQX (a non-NMDA receptor blocker) suppressed the aspartate-induced activity (D).

not antagonize the effects of serotonin when used at the same concentration as (propranolol. Figure 4 demonstrates that the 5-HT response is also partly mediated through 5-HT2 receptors. Under control conditions (Fig. 4A) the system was silent but bath application of 5-HT induced an alternating motor output (Fig. 4B). The addition of ketanserin (10'5 M) induced a twofold increase in the period. With further increase in the concentration of ketanserin (5 x 10'5 M, Fig. 4D) all slow rhythmic activity was blocked and only tonic activity remained as can be seen in comparison with the control. The effects of ketanserin were reversible (Fig. 4E). It should be mentioned that with both propanolol and ketanserin, increasing concentrations finally blocked even the tonic activity. Other 5-HT antagonists were tested, namely mianserin and cyproheptadine which also antagonized the action of serotonin in the same way. These molecules were active in the same concentration range, and progressively blocked the 5-HT-induced activity at increasing concentrations. Recovery occurred more rapidly with cyproheptadine than with ketanserin and mianserin. The 5-HT3 antagonists tested (metoclopramide, 3-tropanyl-3,5-dichloro-

FICTIVE LOCOMOTION IN RAT SPINAL CORD1195

benzoate) failed to block the 5-HT-induced activity (not shown). The slowing down of the period of cycling provoked by 5-HT antagonists, therefore strongly suggests that a blockage occurred at the CPG level itself. This obviously does not exclude the possibility that serotonin may also have acted directly on the motoneurones as observed in others studies (see Discussion). A Control Glu (4 x1 0-4 M)

1L3 B Glu (4 x 10-4 M) + AP-5 (2-5 x 10-5 M)

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(3x10-4 M)+DNQX (25x 10-5 M)

5s Fig. 6. The glutamate action is mediated through NMDA and non-NMDA receptors. An alternate pattern induced by glutamate (Glu) and recorded in the right and left lumbar ventral roots (A) was abolished when the NMDA receptor antagonist AP-5 was added to the medium (B). After 20 min wash-out, the activity partially recovered (C). In a different experiment, the glutamate-induced activity (D) was suppressed by the non-NMDA antagonist DNQX (E).

The excitatory amino acid-induced rhythm Bath application of the endogenous excitatory amino acids aspartate and glutamate elicited fictive locomotor activity. The threshold concentration for this action was between 10'4 and 2 x 10'4 M. We did not establish a dose-response curve for the glutamate and aspartate because their effects appeared to be too variable, in comparison with those of their agonists (see Figs 7 and 8). Some qualitative differences were observed between the activity induced by aspartate and glutamate,

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since the aspartate rhythm was generally easier to induce and more organized, i.e. showed stricter alternation between right and left bursts and was not varying with time. In any case, the activity elicited by these transmitters was often weaker and more fragile than the one elicited by their agonists. A NMA(15x10-5M) rL3

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4 x1 0-5 ~~~~2 N MA concentration (M) x 1 0-5

Fig. 7. Involvement of the NMIDA receptors in the genesis of fictive locomotion. A-C, in the same experiment, an increase in the concentration of the NMA (a NMDA receptor agonist) progressively decreased the period of the alternating right and left pattern. Each bath application of NAIA was followed by wash-out with normal saline. D. dose-response curve of the NAIA-induced rhythm. The period was plotted as a function of the NMA concentration. The threshold was 10- M and a plateau (at 1 s) was rapidly reached at 3 x 10-5 ii. The number of experiments is indicated in parentheses beside each point. The use of several antagonists made it possible to establish that the action of the EAAs was mediated through two different classes of receptors (Figs 5 and 6). The widely used antagonist 2-amino-5-phosphonovaleric acid, which acts at the NMDA receptor site blocked the effects of aspartate (Fig. 5B) and those of glutamate (Fig. 6B) in all the experiments (n = 5). The threshold concentration for this blocking effect of AP-5 to occur was 10' M, and partial recovery occurred (Figs 5C and 6C). Likewise, 6.7-dinitro-quinoxaline-2,3-dione (DNQX) a non-NMDA receptor antagonist (acting on kainate and AMPA receptors) blocked the aspartate (Fig. 5D) and the glutamate (Fig. 6D) induced activity in all the experiments (n = 5). No recovery occurred after the application of DNQX (even after more than 1 h of wash-out). At concentrations lower than 10-5 m, a decrease in the cycling was observed prior to complete suppression of the rhythmic activity (as occurred in the case of the 5-HT antagonist action). Since the use of the antagonists suggested that not only NMDA but also kainate

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and/or AMPA receptors were involved in the EAA response, we subsequently explored the action of various agonists of the three classes of receptors. With each of these we attempted to establish a dose-response curve in order to compare their patterns of action. It turned out that the rhythmic activities elicited by all these substances exhibited similar features and could be distinguished in several ways from the activity of serotonin. The main differences concerned the general 'arousal' of the preparation and the period. The background activity during the interburst intervals was generally weaker and the period shorter than with serotonin. Moreover, the EAA rhythm was generally speeded up, until it became disorganized, ending in a tonic discharge, even if the agonist was still present in the saline (while the 5-HT-induced activity generally stopped without any linear increase in its period). The NMDA-induced activity To activate the NMDA receptor we used NMA (see Methods). This substance elicited alternation of activity between right and left, at the level of the same lumbar segment. The threshold was 10-5 M and the range of concentration narrow (10'5 to 4 x 10-5 M). In the same experiment the period progressively decreased at increasing NMA concentrations (Fig. 7A-C). The dose-response curve (Fig. 7D) indicated that the plateau was reached at 3 x 10-5 M. Above 4 x 10-5 M a pattern of tonic or highfrequency activity was obtained. As with 5-HT, the NMA-induced pattern could be divided into three categories, on the basis of twenty-eight experiments in which NMA 2 x 10-5 M was applied. NMA induced a regular pattern in 53 % of experiments. With time, however, the period of the rhythm often shortened, resulting in highfrequency discharges, and a desynchronization of the contralateral activities. In the other 36 % of the experiments, the pattern was unstable and fragile, exhibiting periods of disruption. Some bursts were missing or were not present in all the segments at the same time. In 11 % of the experiments, the NMA elicited no fictive locomotion whatever its concentration.

The kainate-induced activity The kainate also evoked an alternating burst pattern in the ventral roots but was active at tenfold lower concentrations than NMA, i.e. in a range lo6 to 5 x 10'6 M. Above this concentration it evoked only a transient tonic activity, and at 10'5 M it appeared to be non-reversibly toxic. If one considers only the period, there existed no differences between NMA and kainate within the range of concentration at which these substances were active. From lo-6 M, the period decreased when the concentration of kainate was increased (Fig. SA-C). In addition the dose-response curve showed that a plateau was reached between 3 and 4 x 10-6 M (Fig. 8D). In a total of twenty-seven experiments the kainate bath applied at 2 5 x lo-6 M induced a very regular fictive locomotion in 52% of them. Here again the length of the sequence could be variable and it occasionally lasted as long as the drug was applied but as in the case of NMA it generally turned into tonic discharge after a few minutes. In 18 % of the experiments kainate induced irregular rhythm while in 30 % of cases it was unsuccessful at initiating fictive locomotion. Some qualitative differences were observed, with the kainate generally recruiting more units in the interburst intervals than the NMA.

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The quisqualate/AMPA-induced activity The third type of ionotropic EAA receptor is the AMPA receptor (previously named the quisqualate receptor), towards which both AMPA and quisqualate are potent agonists. Their action was tested in preparations in which the other A Kainate (1 0-6 M) D 4rL3

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Fig. 8. Involvement of the kainate receptors in the genesis of fictive locomotion. A-C, the bath application of kainate induced an alternating pattern at a period of around 3 s at 10-6 M (A) and then progressively decreased when the kainate concentration was increased (B and C). D, dose-response curve of kainate. The period of the fictive locomotion induced by kainate was plotted as a function of its concentration. From the threshold (10-6 M) the period decreased from 3 s reaching a minimum around 1 s at 5 x 10-6 M. The number of experiments is indicated in parentheses beside each point.

excitatory amino acids (i.e. NMA, kainate, glutamate and aspartate) were eliciting fictive locomotion. These two agonists were found to be much less efficient than the NMA and kainate at eliciting fictive locomotion. An example of sequences induced by quisqualate is shown in Fig. 9. The bath application of quisqualate triggered an alternating pattern and a dose-dependent effect could be observed (Fig. 9A-C). The threshold concentration for a response to be evoked was 10-5 M. This rhythmic pattern was never maintained however, and it always ended rapidly in tonic discharge (within 1 or 2 min). No dose-response curve could be established for the quisqualate effects, since although a dose-dependent response was occasionally observed during the same experiment (three out of twelve experiments), this was not generally the case, and from one experiment to another there were no consistently reproducible effects (unlike experiments with NMA or kainate). The same phenomenon was observed with AMPA which appeared to be still less efficient at initiating rhythmic patterns. The threshold concentration for a response to be

FICTIVE LOCOMOTION IN RAT SPINAL CORD

199

elicited with AMPA was between 6 and 7 x 10-7 M. The sequence of fictive locomotion induced by AMPA was transient and it was not generally possible to repeatedly initiate these transient sequences during successive applications in the same experiment. A

Quisqualate (2 x 10-5 M)

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B

Quisqualate (3 x 10 5 M)

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C Quisqualate (4 x 1 0-5 M)

3s Fig. 9. Quisqualate induces fictive locomotion in a dose-dependent manner. During the same experiment, successive bath applications of quisqualate induced sequences of fictive locomotion where the period decreased with changes in the quisqualate concentration. Each application was followed by a wash-out. DISCUSSION

The motor output Our main purpose was first to examine how the various transmitters initiate fictive locomotion. For this purpose, we recorded the overall activity in the ventral roots or used semi-isolated preparations, in which the hindlimbs were attached (Fig. 1). This extracellular analysis of the motor output made it possible to study the all-or-none activity of the CPGs for locomotion. Once we had checked that the activity generated by the bath application of transmitter was capable of making the hindlimbs move in an alternating flexion and extension movement, we preferentially used the completely isolated spinal cord, in which only the ventral roots are recorded, since this preparation is technically more convenient. However, even in these reduced preparations. it is possible to determine the flexion and extension phase

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during one cycle, due to the intersegmental distribution of muscle units, as anatomically demonstrated by Nicolopoulos-Stournaras & Iles (1983). These authors located the motoneurones that innervate twenty different hindlimb muscles in the adult rat, using the retrograde transport of peroxidase. This showed that the lumbar ventral roots L5 contain only flexor motor units, while Li-4 convey axons from both flexor and extensor muscles. Our electrophysiological data are consistent with this anatomical distribution along the lumbar segment. In fact, we observed that the bursts of spikes recorded in L5 were phase opposed with those of the L2-3 ventral roots (Figs 1 C and 2B), which appeared to mainly contain extensor motor units. This explains the strict alternation with L5 bursts (although some double bursts, i.e. flexion/extension occasionally occurred in L3). On the other hand, the recordings in L4 were found to have a mixed flexor/extensor content (to such an extent that it was sometimes difficult or impossible to detect any organized pattern at all). The fictive locomotion recorded in the neonatal isolated preparation certainly differs from stepping in intact adult rats. It has several features in common with the pattern recorded in the intact animal, however, since it has been shown that even at these very early stages, pups can exhibit clearly co-ordinated hindlimb movements in vivo (Cazalets et al. 1990b). In these studies, locomotor movements were observed during swimming activity. The phase relationships between the right and left side were found to be the same in the intact animal as in the isolated preparation, i.e. 0 5 which expresses strict alternation. It was shown (Menard, Cremieux & Cazalets, 1991) that the beating period of the limbs increases non-linearly from day 0 to adulthood, forming a plateau between day 6 and 12. From day 0 to 3, however, the period ranges between 1 and 2 Hz. These values are comparable to those obtained in the isolated spinal cord, although it should be noted that during swimming, due to the high degree of difficulty of the task, the observed frequency probably corresponded to what can be called the 'best performance' of the nervous system, probably resulting from the involvement of parallel pathways activating the CPGs. In addition, we observed that in vitro, the period value was not greatly affected at temperatures ranging between 25 and 35 0C (Sqalli-Houssaini et al. 1991). Similar maximum values have also been reported by Smith et al. (1988), Atsuta, Garcia-Rill & Skinner (1990) and Atsuta, Abraham, Iwahara, Garcia-Rill & Skinner (1991) during fictive locomotion induced by electrical or chemical stimulation of the brain stem. In the latter study, these authors examined the various muscle synergies and antagonisms in the in vitro brainstem-spinal cord preparation, after applying electrical stimulation to mesencephalic locomotor region and concluded that the nature of the motor pattern evoked in the in vitro preparation was similar to that recorded in intact animals. The serotonin-induced pattern In the adult rat and other mammals, the serotonin terminals that invade the spinal cord mainly originate in the raphe nucleus in the brainstem (Steinbusch, 1984), although some intrinsic serotonergic spinal system has been detected (Newton & Hamill, 1988). Ontogenic studies on 5-HT distribution have shown that the first serotonergic terminals develop at the lumbar level on day 14 in utero. At birth, the same distribution as in adults is observed, although the innervation is less dense

FICTIV7E LOCOMOTION IN RAT SPINAL CORD

201 (Bregman, 1987; Rajaofetra, Sandillon, Geffard & Privat, 1989). These results argue in favour of the possibility that 5-HT can operate at an early stage. Although a wealth of physiological data has been collected on the role of 5-HT in the regulation of the motor outflow, its cellular targets have not all been identified yet, and we have to assume that it acts on several spinal compartments. One of the main findings obtained here is that 5-HT induces fictive locomotion. Moreover, the induction of an alternating pattern and the dose-dependent increase in the ventral root bursts suggest that serotonin acts directly on the oscillator component of the CPG itself. In the lamprey, it has been shown that serotonin does not initiate fictive swimming, but that it provokes a dose-dependent decrease in the period of fictive swimming induced by D-glutamate (Harris-Warrick & Cohen, 1985). There has been some controversy, however, as to whether 5-HT actually initiates the activity of the locomotor networks in mammals. Viala & Buser (1969, 1971) reported that 5-HTP (as well as DOPA) elicited fictive locomotion in a curarized and decerebrated rabbit; whereas, in the low spinal decerebrate cat Grillner & Shik (1975) reported that 5HTP only increased the muscle tone, but did not actually induce locomotor activity. Working on the chronic spinal cat, Barbeau & Rossignol (1990, 1991) reached the same conclusions as Grillner & Shik, and reported that the serotonergic agonists only enhanced the EMG burst amplitude but fail to trigger locomotion. It is therefore difficult to draw any definitive conclusion one way or the other, or to tell whether the differences between these are due to an interspecies difference (as was suggested by Barbeau and Rossignol). Our results tend, however, to favour the idea that the serotonergic system may trigger locomotion, and suggest that the above discrepancies may be attributed to methodological differences. In fact, we directly compared the effects of 5-HTP (the precursor) with those of 5-HT (the neurotransmitter) in the isolated spinal cord, and the results confirmed that the precursor was indeed inefficient. As regards the results obtained on the rabbit versus the cat, the difference is that Viala and Buser in this study pretreated the animals with nialamide, an inhibitor of the monoamine oxidase, prior to 5-HTP injections. which was not done in the case of the cat. Thus on the basis of these results it can be concluded that in the cat the lack of 5-HTP effects may be due to insufficient activation of the serotonin targets (due to serotonin inactivation). In addition to acting on the premotoneuronal spinal network that organizes the alternating pattern, serotonin has been found to directly depolarize the motoneurones and increase their excitability (Connell & Wallis, 1988, 1989; Jackson & White, 1990; Wang & Dun, 1990) as well as to induce bi-stable properties in cat motoneurones (Hounsgaard, Hultborn, Jespersen, & Kiehn, 1988; Kiehn, 1991). Our data on the pharmacological properties of serotonin are in agreement with previous studies showing that the motoneurone receptors exhibit some of the 5-HT1 and 5HT2 receptor features but with a somewhat different profile (Connell & Wallis. 1989). In fact, Connell and Wallis antagonized the 5-HT-induced depolarization of the motoneurones in hemisected spinal cord of newborn rats, with non-selective (5HT1/5-HT2) antagonist (cyproheptadine, methysergide) or 5-HT2 antagonist (ketanserin), while the 5-HTlA receptor agonist. 8-Oli-DPAT and the 5-HTlB receptor agonist, RU24969 had no effect. Only the non-selective agonist, 5methoxytryptamine. mimicked the action of serotonin. Xre reached the same

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conclusion as the latter authors, sinee none of the 5-HT1 receptor agonists that we used, (1-(3-ehlorophenyl)piperazine HCl, 8-OH-DPAT, PAPP, 1 -2(methoxyphenyl)piperazine (2-MPP HCl), elicited any response. The non-participation of 5-HT3 receptors suggested by Connell & Wallis (1988, 1989) and Wang & Dun (1990) was confirmed since the 5-HT3 antagonists (metoclopramide, 3-tropanyl-3,5-diehlorobenzoate) did not alter the 5-HT response. The EAA-induced rhythm The present findings show that at least two kinds of excitatory amino acid receptors are mainly involved in the genesis of fictive locomotion: those of the NMDA and kainate types. This was demonstrated by blocking the effects of glutamate and aspartate with AP-5 (an NMDA receptor blocker) and DNQX (a kainate and AMPA receptor blocker). In addition, the selective agonists for these two types of receptors (NMA and kainate respectively) elicited fictive locomotion. Our study confirms that the NMDA receptor participates in the genesis of fictive locomotion, in agreement with previous data by Kudo & Yamada (1987) and Smith et al. (1988). Our results partly diverge, however, from those of these authors as regards the role of the kainate receptors in the genesis of locomotor activity. In fact, strong arguments support the idea that kainate receptors play an important role: (1) the blocking of the action of glutamate and aspartate by the non-NMDA receptors (Figs 5 and 6); and (2) the fact that kainate induced fictive locomotion in a dosedependent manner (Fig. 8), in the same period range as NMDA receptor agonists. The action of EAAs in mammals is comparable in some respects to what occurs in the lamprey, where both NMDA and kainate receptors have been found to participate in the induction of the locomotor pattern. Differences exist, however, between the role of the NMDA and kainate systems since in the lamprey it has been demonstrated that the activities generated by NMDA are significantly slower than the kainateinduced rhythm (Brodin & Grillner, 1985). Smith et al. (1988) using NMDA as an agonist have reported that on average, the minimum period was around 1V1 s, which is the same minimum value as the one we obtained using NMA as agonist. The value is also identical to that published by Atsuta et al. (1991), whereas Kudo & Yamada (1987), also using NMA, reached minimal period values as low as 350 ms. This was perhaps due to the fact that they were only using a lower spinal cord preparation as suggested by Atsuta et al. (1991). In any case, the important point is that under the same experimental condition no differences are to be observed whether fictive locomotion is elicited through the NMDA or kainate receptors. XWhile the participation of NMDA and kainate receptors in the activation of the locomotor networks has been established quite unambiguously, the role of the AMPA/quisqualate receptors is less clear. We have seen that these compounds could occasionally trigger an alternate pattern (Fig. 9), but this was neither frequent nor reproducible in the same experiment and lasted only for some tens of seconds. In the lamprey, quisqualate was initially ruled out as a candidate for initiating fictive swimming (Brodin, Grillner & Rovainen, 1985). In a recent study, however, Alford & Grillner (1990) showed that AMPA induced fictive swimming and suggested that the inefficiency of quisqualate may rather have been due to the re-uptake system. In the newborn rat, however, it looks as if the AMPA receptors were not primarily involved in the genesis of the locomotor activity.

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We wish to thank Dr P. Bertucci for critical comments on the manuscript and Dr Jessica Blanc for correcting the English version of the manuscript. REFERENCES

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