Formation of the central pattern generator for locomotion in the rat and mouse

Brain Research Bulletin, Vol. 53, No. 5, pp. 661– 669, 2000 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/00/$–see front matter

PII S0361-9230(00)00399-3

Formation of the central pattern generator for locomotion in the rat and mouse Hiroshi Nishimaru and Norio Kudo* Department of Physiology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan [Received 16 May 2000; Revised 4 August 2000; Accepted 11 August 2000] ABSTRACT: It is well known that in the neonatal rat spinal cord preparation, alternating rhythmic bursts in the left and right ventral roots in a given lumbar segment can be induced by bath-application of N-methyl-D-aspartate or 5-hydroxytryptamine. Alternation between L2 and L5 ventral roots on the same side, representing the activity of flexor and extensor muscles, respectively, can be observed as well. In the prenatal period in the rat, alternation between the left and right ventral roots is established between embryonic day (E) 16.5 and E18.5. The alternation between the L2 and L5 ventral roots emerges at E20.5. Recent findings show that locomotor-like rhythmic activity with similar characteristics can be induced in the neonatal mouse preparation. In the lumbar spinal cord in the neonatal mouse, it is likely that the rhythm-generating network is distributed throughout the lumbar region with a rostro-caudal gradient, a situation similar to that in the neonatal and fetal rat spinal cord. With this review we hope to highlight the dramatic changes that neuronal networks generating locomotor-like activity undergo during the prenatal development of the rat. Moreover, the distribution of the neuronal network generating the locomotor rhythm in the neonatal rat and mouse spinal cord is compared. © 2001 Elsevier Science Inc.

after P10, rats are able to walk supporting their body weight with their limbs [68] which is likely to be due to the functional development of hindlimb muscles. However, the CPG for locomotion is functional at earlier stages, because the coordinated movements between left and right hindlimbs during swimming [43] or during L-DOPA-induced air stepping [66] could be observed by P3–5. Moreover, behavioral studies of the rat fetus show that coordinated movement resembling locomotion could be observed before birth as early as E20.5 [1,64]. One promising approach to a better understanding of the CPG is to examine the developmental changes that occur in the spatial and temporal pattern of the locomotor before birth. In an isolated spinal cord preparation with the hindlimb muscles attached taken from neonatal rats, a coordinated pattern of alternating bursts of activity in the antagonistic muscles can be induced by bathapplication of any of the neuroactive substances mentioned above [13,27,37,40]. It is, however, technically difficult to record the activity of a single muscle by extracellular electrodes at the various fetal stages, even in an isolated hindlimb-spinal cord preparation, which is a major obstacle to such studies. Previous studies show that in the neonatal rat spinal cord, locomotor activity in the ventral roots has been shown to correspond to the activity of the antagonistic (flexor and extensor) muscles on the same side, L2/L3 supplying for the activity of flexor muscles and L5 extensor muscles [7,27,34]. We took advantage of this in our studies and recorded from L2/L3 and L5 ventral roots at different stages during fetal development [28,41, 52]. The present review deals with the developmental changes occurring in (1) the spatio-temporal pattern of the locomotor-like activity and (2) the distribution of the rhythm-generating network. Unless otherwise mentioned, we discuss work on the fetal and neonatal rat spinal cord, although where appropriate we make comparisons between the neonatal rat and neonatal mouse.

KEY WORDS: Fetus, Rhythm, Motor activity, Spinal cord, Development.

INTRODUCTION Each of the wide range of rhythmic motor activities in which vertebrates engage, such as swimming or walking, is thought to be generated by the neuronal networks known collectively as the central pattern generator (CPG) located in the spinal cord [21,23]. In the analysis of the neuronal mechanisms that generate and modulate CPG activity, in particular in mammals, the in vitro spinal cord preparation obtained from neonatal rats [aged between postnatal day (P) 0 and P5] has been employed extensively and has yielded much useful information (for recent reviews, see [5,35, 58]). In this preparation, alternating rhythmic bursts in the left and right ventral roots of a given lumbar segment can be induced by bath application of any one of a number of neuro-active substances, including N-methyl-D-aspartate (NMDA), acetylcholine, 5-hydroxytryptamine (5-HT), dopamine and noradrenaline [7,11, 34,36,40,62]. According to behavioral studies using neonatal rats, only at and

PRENATAL DEVELOPMENTAL CHANGES IN THE PHASE RELATIONS OF THE LOCOMOTOR RHYTHM IN THE RAT FETUS Bath application of 5-HT (0.75– 40 ␮M) induces rhythmic bursts in the L2/L3 and L5 ventral roots on both sides in rat fetuses at and after E14.5 [28,45]. Figures 1A–C show the 5-HT-induced rhythm recorded from various ventral roots at E16.5, E18.5 and E20.5. Characteristic features of the spatial pattern of the rhythmic

* Address for correspondence: Norio Kudo, M.D., Ph.D., Department of Physiology, Institute of Basic Medical Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8575, Japan. Fax: ⫹0298-53-3495; E-mail: [email protected]

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NISHIMARU AND KUDO 5-HT induced an alternation of rhythmic bursts between the left and right ventral roots, while the rhythmic bursts in the L2 ventral root were synchronized with those in the L5 ventral root on the same side (Fig. 2B). In E20.5 preparations, 5-HT again induced rhythmic bursts that alternated between the left and right ventral roots (Fig. 2C). However, at this age the rhythmic bursts in the L2/L3 and L5 ventral roots on the same side also alternated. The phase-relations for the rhythmic bursts in various ventral roots were consistent among individual preparations at these ages [28]. Phase-relation data for the activity in the ventral roots on the left and right sides, as well as for different segmental levels, are summarized in Fig. 2D. This graph indicates that while the neuronal mechanisms underlying the alternate left–right rhythm are developed by E18.5, the neuronal circuits inducing alternating activity in L2/L3 and L5 ventral roots develop later and may not be established until E20.5. DEVELOPMENT OF RHYTHM GENERATING CAPABILITY

FIG. 1. 5-Hydroxytryptamine (5-HT)-induced rhythm at embryonic day (E) 16.5, E18.5 and E20.5. Nerve discharges are recorded simultaneously from the right and left L3 ventral roots (R-L3VR, L-L3VR) and the left L5 ventral root (L-L5VR) at E16.5 (A) and E20.5 (C) and from the right and left L2 ventral roots and the left L5 ventral root at E18.5 (B). Concentration of 5-HT used to evoke rhythmic bursts: (A), 10 ␮M; (B), 10 ␮M; (C), 20 ␮M.

bursts in three different ventral roots are illustrated by superimposing integrated records of the activities in these roots in E16.5, E18.5 and E20.5 preparations (Figs. 2A, B and C, respectively). It is clear that the pattern depends on the fetal age. In fact, the rhythmic bursts occurred synchronously in the left and right ventral roots of the same segmental level in E14.5–E16.5 preparations, as shown in Figs. 1A and 2A. Furthermore, at this stage the rhythmic bursts in the L2/L3 and L5 ventral roots also occurred almost synchronously. The phase relation of the rhythmic activity in the left and right ventral roots were variable at E17.5 as shown in Fig. 3A (also see Fig. 2 of [52]). At E18.5, bath application of

As described in the above section, rhythmic bursts occur synchronously in the left and right ventral roots of the same segmental level at the age of E16.5. After splitting the spinal cord down the midline, stable rhythmic activity can be recorded in the ventral roots on each side, although this procedure inevitably disrupts the synchrony of the rhythm between the two sides [52]. This result indicates that the interneuronal circuits that generate the rhythm exist in each half of the spinal cord and that neuronal connections crossing the midline are already in place so as to allow the CPGs on the two sides functioning to interact. Evidence in favour of there being separate CPGs on the two sides at and after E18.5 at which age the neuronal mechanisms underlying the alternate left/ right rhythm have been formed has been obtained by splitting the spinal cord mid-sagitally [52]. In the neonatal rat, the frequency of the rhythm was reduced to 20% of control after the spinal cord had been spilt [40]. Similar results have been obtained from neonatal mouse preparations, in which the frequency of the 5-HT-induced rhythm was reduced, to 60% of the control (Fig. 4B), after splitting the spinal cord. However, in fetal rats, this does not appear to happen: at E16.5– E17.5 the frequency of the rhythm remains relatively unchanged after splitting the spinal cord (Fig. 3A). This is also the case at E18.5, when the rhythm between the two sides is already alternating [52]. Interestingly, in the Xenopus embryo [31] and chick embryo [26], the frequency of the rhythm is unaffected by separation of the spinal cord into the left and right sides, as in the fetal rat, suggesting that autonomy in each half of the spinal cord may be a common feature in immature rhythm-generating networks. DEVELOPMENT OF LEFT–RIGHT ALTERNATION IN THE LOCOMOTOR RHYTHM From studies in the tadpole [63] and the lamprey (for review see [24]) the left/right alternation is thought to be mediated by bilaterally located intersegmental glycinergic commissural interneurons projecting directly to the rhythm-generating interneuronal network on the contralateral side of the cord. Thus, in these species glycinergic commissural interneurons are the major source of the inhibition that is transmitted across the spinal cord. The left/right alternation in the rat spinal cord is also likely to be mediated by glycinergic inhibitory synaptic inputs because the N-methyl-D,Laspartate (NMA)- or 5-HT-induced alternate locomotor-like pattern is changed to a synchrony by concomitant application of strychnine in both the neonatal rat ([12]; for review see [33]) and the rat fetus at and after E18.5 [28,52]. Moreover, intracellular recordings from the neonatal rat spinal cord have shown that

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FIG. 2. Phase-relationship between the rhythmic activity recorded from left/right and L2/L5 ventral roots [a: embryonic day (E) 16.5, b: E18.5, c: E20.5]. The broken line shows the peak of L-L2VR activity at each age. (A)–(C) Representative integrated records of the 5-hydroxytryptamine-induced rhythm in preparations at each embryonic day. (D) Summary diagram showing the phase-lag between rhythmic bursts. The mean phase-lag between the rhythmic bursts in the ipsilateral L2/3 and L5 ventral roots (ordinate) plotted against the mean phase-lag between the rhythmic bursts in the left and right ventral roots of the same segment (abscissa). Each data-point indicates the mean value obtained from individual preparations at E16.5 ({), E18.5 (ⵦ) or E20.5 (E). Error bars represent 1 SD.

motoneurons receive a mainly glycinergic rhythmic synaptic inhibition from the contralateral half of the locomotor network [38]. In that study, using a “longitudinal split-bath” setup, one side of the spinal cord was kept in normal solution, while the contralateral side was exposed to 5-HT and NMDA. This induced rhythmic bursting in the ventral roots on the agonist-exposed side but not on the agonist-free side. L1–L3 motoneurons on the agonist-free side received inhibitory postsynaptic potentials (IPSPs) when rhythmic bursts of activity were occurring on the agonist-exposed side. Strychnine blocked this rhythmic inhibitory input when added to the agonist-free side and intracellular chloride-loading increased the amplitude of the IPSPs, suggesting that they were mediated by glycine receptors and were chloride-dependent. The contribution, if any, made by ␥-aminobutyric acid (GABA), the other major inhibitory transmitter in the spinal cord, to the left/right alternation in the neonatal rat spinal cord has not yet been clarified. Cazalets et al. [5] showed that neither bicuculline (up to 10 ␮M), a GABAA receptor antagonist, nor phaclofen (up to 100 ␮M), a GABAB antagonist, changed the phase relationship in the NMA-induced left/right alternate rhythmic activity. On the other hand, Cowley and Schmidt [12] showed that higher concentrations (10 –25 ␮M) of bicuculline do change the alternate rhythm to a synchronized one. It is possible that both GABA and glycine are involved in the left/right alternation since recent findings show that GABA and glycine are likely to be co-released from individual synaptic vesicles onto inhibitory synaptic sites on spinal cord motoneurons in the neonatal rat [30]. Similar findings have been reported in the fetal rat [19]. Moreover, an immunocytochemical study has shown that while many cells located in the ventral

region of the spinal cord express GABA-immunoreactivity as early as E14, this declines towards birth [42]. These GABA-immunoreactive cells might include commissural neurons sending axons to the contralateral side across the ventral commisure, since it has been found that a substantial number of these cells are glutamic acid decarboxylase immunoreactivity during the early fetal period [53]. Thus, it is likely that GABA-mediated synaptic transmission is involved in the interconnections between the CPGs on the left and right sides of the spinal cord, at least during the fetal period. GABAA receptors are certainly involved in mediating motor activities in the spinal cord during the fetal period such as periodic spontaneous bursts that are synchronous between the two sides [48]. The fact that the of NMA- or 5-HT-induced alternate locomotor-like pattern can be changed to a synchronous one by concomitant application of strychnine [12,28,52] suggests that the excitatory connections that serve to synchronize the rhythmic motor activity between the two sides are overridden by the predominant glycinergic inhibitory connections. Bracci et al. [3] showed that simultaneous application of bicuculline and strychnine induces regular rhythmic bursts in the lumbar motoneurons in the isolated rat spinal cord preparation. These bursts, which are synchronized between the left and right ventral roots of the same segment, are blocked by bath-application of 6-cyano-7-nitroquinoxaline-2,3dione, a non-NMDA receptor antagonist, or DL-5-aminophosphonovalerate, an NMDA receptor antagonist. However it is still not clear whether these rhythmic activities induced by bath-application of strychnine and bicuculline are generated by (1) basically the same network involved in locomotor-like rhythmic activity or by

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FIG. 3. Effects of mid-sagittal splitting of the spinal cord (A) and bath-application of strychnine to the unsplit spinal cord (B) on N-methyl-D-aspartate-induced rhythmic motor activity at embryonic day (E) 17.5. Integrated ventral root discharges before (Aa) and after splitting (Ab) in the same preparation. Integrated ventral root discharges in the left (LVR) and right lumbar ventral roots (RVR) are shown. Bath-application of 20 ␮M N-methylD,L-aspartate (NMA) evoked rhythmic motor activity both before (Ba) and after bathapplication of 15 ␮M strychnine (Bb). The graphs show burst number (ordinate) against the elapsed time which is measured from the onset of an arbitrary burst in each episodes. In (Bb), bath-application of strychnine seems to slow-down the frequency of the rhythm. However, this is not always the case (see Fig. 17.2 of [59]).

(2) a different one. The authors [3] discussed that the results showing that 5-HT and NMDA are able to accelerate the strychnine- and bicuculline-induced rhythmic activity in a dose-dependent manner indirectly supports the former possibility. Moreover, the network capable of generating the rhythm is likely to be distributed throughout the lumber spinal cord that is similar to locomotor network. However, the interpretation of these results may not be so simple because strychnine at high levels (⬎1 ␮M) is rather a non-specific drug and blocks a variety of ion channels (for references, see [14]) including K⫹ channels. Also bicuculline (5– 60 ␮M) blocks the small conductance K⫹ channels, which affects the after hyperpolarization of the firing neuron [15,67] which may potentiate the burst firing of spinal neurons as well [17]. Further investigations, particularly at the cellular level are required to solve this question. Nevertheless, this study by Bracci et al. [3] suggests that ionotropic glutamate receptors mediate the rhythmic activity and the excitatory synaptic connections between the left and right sides of the spinal cord. During fetal stages as early as E16.5, the left and

right sides seem to be coupled with excitatory synaptic connections as well [52]. These excitatory influences are likely to be conveyed by the commissural interneurons already present in the spinal cord by this stage [61]. The transition from a synchronous to an alternate left/right pattern of NMA- and 5-HT-induced rhythmic motor activity seems to occur at E17.5. At this age, the left/right pattern of rhythmic burst in the ventral roots is variable even within a single episode [52]. Frequently, the rhythm exhibits different frequencies on the two sides, as shown in Figs. 3Ac and 3Bc. As is the case at earlier and later stages, independent interneuronal circuits generating the rhythm exist in each half of the spinal cord at this age as well (Fig. 3Ab,d). The lack of a phase relationship between the rhythmic activities on the two sides seems to suggest that left and right CPGs have no neuronal interconnections and behave independently at this stage (E17.5). However, the finding that bath-application of strychnine converted the variable pattern into a synchrony in the left and right ventral roots, as shown in Fig. 3B,

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FIG. 4. Locomotor-like activity recorded after splitting mid-sagittal of the spinal cord of the neonatal rat (A) and neonatal mouse (B). (Aa) Electromyographic (EMG) recordings from the left tibialis anterior (TA; upper trace) and left gastrocnemius (G; lower trace). The right side of the spinal cord had been removed as shown in the schematic drawing on the right. N-methyl-D,L-aspartate (NMA) concentration, 20 ␮M. Only the L4 and L5 ventral roots, which contain a mixture of axons innervating flexor and extensor muscles, were kept attached to the muscles. (Ab) EMG recordings from TA and G after the left spinal cord was further transected at the L3 and L6 segments, leaving only the L4 and L5 segments intact (as shown in the schematic drawing on the right). (B) Integrated ventral root discharges in the L2 and L5 lumbar ventral roots of the neonatal mouse before (a) and after (b) splitting the spinal cord are shown. The rhythm was induced by bath-application of 5-HT (20 ␮M).

suggests that there are glycinergic synaptic connections present between the two sides at E17.5. The variable phase-relationship at E17.5 may be due to the presence of immature inhibitory interactions between the neuronal circuits on the two sides. Such immature interconnections might impede, to a variable extent, the phasic rhythm induced in one side by the other. However, it is noteworthy that at exactly this age the excitatory synaptic transmission mediated by glycine and GABA changes into an inhibitory one in the neuronal networks within the spinal cord. At earlier stages (E14.5–E16.5), excitatory drive generating periodic spontaneous bursts in lumbar motoneurons is likely to involve glycine and GABAA receptor-mediated synaptic transmission [48]. The depolarizing response to glycine and GABA is enough to excite the neuron, possibly due to the large positive shift in the Cl⫺ equilibrium potential during this period. It has been shown that the high intracellular Cl⫺ concentration is due to inwardly-directed Na⫹/K⫹/2Cl⫺ co-transport in the rat neocortex [9] and brainstem [32], as well as in the Xenopus larva spinal cord [55]. Strychnine, however, does not block the spontaneous bursts at E17.5 [48]. Interestingly similar developmental changes can be seen in other neuronal networks in the rat lumbar cord during this period. Wu et al. [70] have shown that while strychnine and bicuculline block dorsal-root-evoked responses at E16 –17, the responses at E18 –E19 are enhanced. They also showed that the amplitude of the depolarizations induced by glycine and GABA decreases towards birth. It is probable that this developmental decrease in the magnitude of the GABA- and glycine-induced depolarizations correlates with an increase in the expression of an outwardly directed Cl⫺ pump (Cl⫺-extruding K⫹/Cl⫺ co-transporter: KCC2) as it does in the developing rat hippocampus [56]. In that study, on the pyramidal neurons of the rat hippocampus, the authors showed an ontogenetic change in GABAA receptor-medi-

ated responses from depolarizing to hyperpolarizing, a change that is likely to be related to a developmental induction of the expression of KCC2. The effects produced by glycine- and presumably GABA-mediated synaptic inputs could be either excitatory or inhibitory in a given population of spinal neurons in this transitional period, depending on the maturity of the inputs. This might explain the variable pattern shown by the locomotor rhythm at E17.5. Although these amino acids still depolarize motoneurons at birth, they are likely to exert inhibitory effects by shunting the excitatory currents and reducing the excitability of the motoneuron [18] (also see [38]). The transient excitatory and depolarizing effects of glycine and GABA in the immature neuronal networks including the CPG for locomotion and the reflex pathways could be favorable for elevation of the intracellular Ca2⫹ concentration via voltage gated Ca2⫹ channels [49]. This might play an important role in establishing the future inhibitory synaptic pathway along side with the maturation of connectivity in the lumbar spinal cord. In summary, during the prenatal period in the rat, alternation between the left and right side of the spinal cord during locomotorlike activity is established between E16.5 and E18.5. The developmental changes in glycine- and GABA-mediated synaptic transmission from excitatory to inhibitory are likely to be involved in this process. DEVELOPMENT OF A L2–L5 ALTERNATION IN THE LOCOMOTOR RHYTHM More than a decade ago, we showed that a lumbar spinal half-cord can generate locomotor-like activity in hindlimb muscles on the same side [40]. As illustrated in Fig. 4A, in such preparations taken from neonatal rats, bath-application of NMA induces

666 alternating rhythmic activity in flexor (tibialis anterior) and extensor (gastrocnemius) muscles. In a spinal cord preparation from the neonatal mouse, as well as in one from the neonatal rat, alternating rhythmic activity between the ipsilateral L2 and L5 ventral roots can be induced by bathapplication of 5-HT (Fig. 4B). This alternating pattern persists even after splitting the spinal cord and the pattern is similar to that of the electromyographic recording shown in Fig. 4A. As mentioned above, it is thought that the activity in the L2 and L5 ventral roots represents the flexor and extensor muscle activity, respectively, on the same side [7,27,37]. However, a morphological study has shown that both of these segments contain flexor-muscle motoneurons and extensor-muscle motoneurons in adult rats [47]. In fact, flexor activity, in addition to extensor activity, could be also recorded in the L5 ventral root in a small number of preparations (H. Nishimaru and N. Kudo, unpublished observations). On the other hand, none of the preparations exhibited any detectable extensor activity in L2. The fact that the motoneuronal activity in these two segments seems predominantly to represent either flexor or extensor activity during the locomotor-like rhythm might reflect a large preponderance of one type of motoneuron population in a given segment or different excitabilities within a given segment in the motoneurons innervating individual muscles in these preparations. Our studies disclosed that motor activity alternating between different segments first appeared at E20.5, later than the onset of left/right alternation (Figs. 1 and 2; [28,41]). These results thus strongly suggest that the neuronal circuits regulating coordinated motor activity in the flexors and extensors of a given limb begin to function at around E20.5. Because the 5-HT-induced rhythmic activity is alternate between the left and right sides of both the L2 and L5 segments at E18.5 [28], there might be a difference in maturation between commissural connections and the rostro-caudal linkage. The bursts occurring on the left and right sides are completely synchronous (phase lag: 0.00) as early as E16.5 (Figs. 2A,D) indicating that the connection between the left and right neural circuits seems to be quite tight at this stage. On the other hand, the phase-lag between the bursts is 0.1 in the ipsilateral L2 and L5 ventral roots at the same stage, decreasing to 0.01 two days later at E18.5 (Figs. 2B,D). This indicates that rostro-caudal connections develop later than the commissural connections. In the spinalreflex pathway, weak intersegmental connections between the lumbar segments before E18.5 has been reported. For example, in the dorsal-root-evoked reflex the latency of the evoked response in the L5 ventral root after stimulation of the L3 dorsal root at E17.5 is less than 60% of the latency at E16.5 [57]. These responses are abolished by removing calcium ions from the perfusate, indicating that they are mediated mainly by immature synaptic connections rather than via gap junctions (which are present among the cells in the spinal cord by this age [8]). Similar immaturity of the intersegmental inhibitory synaptic connections could give rise to the delay in the onset of L2–L5 alternation which is established at E20.5. At E18.5, bath-application of 5-HT induces synchronous rhythmic activity between L2 and L5 ventral roots. Because there is already inhibitory coupling between the left and right sides at this stage, L2–L5 alternation is not likely to be initiated by a simple change in the effect of glycine and GABA on motoneurons from excitatory to inhibitory (i.e., decrease of intracellular Cl⫺ concentration in motoneurons). The most likely explanation is that inhibitory connections between L2 and L5 are formed after E18.5 and start to function at E20.5. Coincidentally, in the amplitude of the dorsal-root-evoked reflex including those evoked by stimulation of the dorsal root in different segments is not affected by strychnine before E19.5 [57]. At and after E19.5, however, strych-

NISHIMARU AND KUDO nine enhanced the reflex discharges especially those induced by stimulation of different segments. It is likely that connections between the CPGs distributed in the lumbar segment undergoes a similar change. In preparations before E20.5, if the activity of these ventral roots does reflect the activity of flexor and extensor muscles like in the neonates, a simple explanation is that these synchronized rhythmic bursts reflect a co-activation of flexors and extensors, previously shown to be evoked by some putative locomotorinducing substances in the neonatal rat [11], though this might not be a typical locomotor activity. An alternative possibility is that the rhythmic motor activity seen in the different segments may represent flexor motor activity almost exclusively, due to the immaturity of the extensor system at early stages described in the neonatal rats before P15 [69]. However, this is unlikely to be the main factor because behavioral study reveals that co-ordinated locomotor-like activity i.e. repeated movements of alternating flexion and extension, could be observed in the hindlimb at the late fetal period [64]. It is also noteworthy that the motoneuron population may not remain the same in each segment during this period because it has been reported that in the rat lumbar spinal cord, the number of motoneurons shows a dramatic decrease (by approximately 41%) between E15 to P1 due to naturally occurring cell death [51]. Although it is not clear whether these motoneurons are innervating extensor or flexor muscles, such decrease could lead to clearer differentiation between the flexor and extensor motor output in each segment. ROSTRO-CAUDAL GRADIENT IN THE CPG Because the rhythm between L2 and L5 undergoes such dramatic changes in pattern, it seemed interesting to focus on the rostro-caudal organisation of the CPG during the fetal period. The rhythm-generating networks are reported to be distributed throughout the spinal cord in undulatory swimming species such as dogfish [20] and lamprey [10]. This is also the case in species with the different nature of locomotion, such as chick embryo [26] and cats [16]. Similar findings have been made in the neonatal rat as well [4,13,37,39]. Moreover, it should be noted that even a neonatal rat preparation that contains only the L4 and L5 segments of one side with hindlimb muscles attached will display stable rhythmic activity (following bath-application of NMA) that alternates between the tibialis anterior and gastrocnemius muscles (Fig. 4Ab; [40]). In essence, the pattern of the rhythmic activity is similar to that induced in a split preparations containing an entire lumbar halfcord (Fig. 4Aa), indicating that interneuronal circuits producing such rhythmic motor outputs are located within the spinal segments corresponding to the motoneurons (also see [65]). Similar results have been reported for adult cats [25] in which alternating activity in the ankle persists when only the L6 –S1 segments remain intact. In a spinal cord preparation including the lower thoracic (Th 7/8) to the sacral region taken from a fetal rat at E16.5, the correlation between the 5-HT induced rhythmic activity in the L2/L3 and L5 ventral roots can be disrupted by transecting the spinal cord at the mid-lumbar level (L3/L4) [45]. However, rhythmic bursts could still be observed in both upper and lower lumbar ventral roots, showing that each region is capable of generating the rhythm independently. However, after the transection the frequency of the rhythm was greatly reduced (41% of control) in the lower lumbar ventral root, while the rhythm in the upper region was relatively unaffected (95% of control). This indicates that the rostral region is better able to generate the rhythm than the caudal region. Moreover, the activity in the L2 ventral root always pre-

FORMATION OF THE CPG FOR LOCOMOTION cedes that in the L5 ventral root before transection. Taken together, this evidence points to the rostral region seeming to play the leading role in rhythm generation, at least during this period. This might be due to a rostro-caudal difference in maturation during the prenatal period, a phenomenon seen both during the formation of cutaneous reflexes [46] and in relation to the periodic spontaneous bursts in motoneurons [44]. Such a phenomenon might also determine the sensitivity of the spinal neurons to 5-HT since when 5-HT projections first appear in the lumbar spinal cord at E16 –E17 they are localized to the lateral and ventral funiculi, only growing into the ventral horn by E18 [54,71]. A similar phenomenon is observed in Xenopus tadpoles, in which the locomotor circuitry displays rostro-caudal gradients both of development and of sensitivity to 5-HT in the early post-embryonic stage, a stage which corresponds to the caudal growth of 5-HT-containing fibers projecting from the raphe nucleus [60]. A rostro-caudal gradient in the distribution of the CPG has been shown in a number of developing vertebrates, such as chick embryo [26] and neonatal rats [6,13,37,39] (for review see [35]). Recently we added the neonatal mouse to this list [50]. It has been reported that in an isolated spinal cord preparation taken from mice aged around P10, locomotor activity similar to that recorded in the spinal cord of the neonatal rat could be induced by bath-application of drugs, although in mice at that age it was necessary to use a combination of 5-HT, dopamine and NMDA [29]. We followed this up by demonstrating that bath-application of 5-HT (15–100 ␮M) alone can induce a stable, alternating rhythm between the left and right ventral roots in either the L2 or L5 segments in a preparation taken from younger neonatal mice (aged between P0 to P3). In that study, a spinal cord preparation containing a length of cord from Th11/12 to the sacral region was further transected at the mid-lumbar level (L3/4). After this transection, 5-HT-induced rhythmic activity could still be recorded from the L2 ventral root, while in the L5 ventral root no clear rhythmic activity could be observed. These results suggest that the upper lumbar region have a greater ability to generate a locomotor rhythm than the lower lumbar spinal cord. However, Bonnot et al. [2] using a similar preparation, found that the caudal region of the neonatal mouse lumbar spinal cord was able to generate periodic bursts in L5 on bath-application of bicuculline, a GABAA receptor antagonist, in a Mg2⫹-free medium. This suggests that although it may be relatively weak, an ability to generate rhythmic activity does reside in the caudal lumbar cord in the neonatal mouse. Moreover, we cannot rule out the possibility that there is a regional difference in the sensitivity to 5-HT of the lumbar spinal cord between the mouse and rat. In the rat spinal cord, lower thoracic region needs to be connected to the lumbar cord for 5-HT alone to induce an alternating activity in the hindlimb [13]. In the equivalent mouse preparation, however, this seems not to be the case: bath-application of 5-HT induces alternate rhythmic activity in the L2 ventral root even after the thoracic cord has been removed (Fig. 5). This demonstrates that in the neonatal mouse, the CPG in the lumbar cord can be activated by 5-HT even in the absence of the thoracic cord. This could reflect a due to the difference in the time course of development between rats and mice. Although it is not clear whether this rostro-caudal gradient exists in the mature spinal cord in these species, it could conceivably be a special feature of the rhythmo-genetic network in the spinal cord of the adult animal as well. Indeed, in the neuronal network generating the scratch reflex in the adult cat, a similar rostro-caudal gradient had been reported [16,22]. In that study, the authors showed that although the rhythmo-genetic network for this movement is distributed throughout the lumbar cord, rhythmic activity usually appears in the more caudal segments (L6 –S1) only if the rostral segments (L3–L5) generate rhythmic oscillations.

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FIG. 5. Effect of removing the thoracic cord on the 5-ydroxytryptamine (5-HT)-induced rhythmic activity in the neonatal mouse. The 5-HT-induced locomotor rhythm in L2 and L5 ventral roots (A) could still be observed after transecting the cord at the T13/L1 and L3/L4 segments (B). Schematic drawings of the lumbar spinal cord preparation are shown on the right of each recordings.

SUMMARY In the neonatal rat spinal cord preparation, alternating rhythmic bursts in the left and right ventral roots in a given lumbar segment can be induced by bath-application of NMDA or 5-HT. Alternation between L2 and L5 ventral roots on the same side can be observed as well. In the prenatal period in the rat, alternation between the left and right ventral roots is established between E16.5 and E18.5. The alternation between the L2 and L5 ventral roots, representing the activity of flexor and extensor muscles, respectively, emerges later. It is suggested that decrease of the intracellular Cl⫺ concentration of the lumbar motoneuron which leads to functional maturation of the inhibitory synaptic transmission mediated by glycine and GABA seems to play an important role in the formation of the left and right alternation of the locomotor-like activity. On the other hand, the reciprocal inhibitory pathway meditating the alternation of flexor and extensor muscles appears to develop at later stages. Moreover, in the lumbar spinal cord in the rat fetus as early as E16.5, the rhythm-generating network is distributed throughout the lumbar region with a rostro-caudal gradient, a situation similar to that in the neonatal rat spinal cord. Finally, locomotor-like rhythmic activity similar to that observed in neonatal rat spinal cord can be induced in the equivalent neonatal mouse preparation.

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