Developmental segregation of spinal networks driving axial- and hindlimb-based locomotion in metamorphosing Xenopus laevis

Amphibian metamorphosis includes a complete reorganization of an organism's locomotory system from axial-based swimming in larvae to limbed propulsion in the young adult. At critical stages during this behavioural switch, larval and adult motor systems operate in the same animal, commensurate with a gradual and dynamic reconfiguration of spinal locomotor circuitry. To study this plasticity, we have developed isolated preparations of the spinal cord and brainstem from pre- to post-metamorphic stages of the amphibian Xenopus laevis, in which spinal motor output patterns expressed spontaneously or in the presence of NMDA correlate with locomotor behaviour in the freely swimming animal. Extracellular ventral root recordings along the spinal cord of pre-metamorphic tadpoles revealed motor output corresponding to larval axial swimming, whereas postmetamorphic animals expressed motor patterns appropriate for bilaterally synchronous hindlimb flexion–extension kicks. However, in vitro recordings from metamorphic climax stages, with the tail and the limbs both functional, revealed two distinct motor patterns that could occur either independently or simultaneously, albeit at very different frequencies. Activity at 0.5–1 Hz in lumbar ventral roots corresponded to bipedal extension–flexion cycles, while the second, faster pattern (2–5 Hz) recorded from tail ventral roots corresponded to larval-like swimming. These data indicate that at intermediate stages during metamorphosis separate networks, one responsible for segmentally organized axial locomotion and another for more localized appendicular rhythm generation, coexist in the spinal cord and remain functional after isolation in vitro . These preparations now afford the opportunity to explore the cellular basis of locomotor network plasticity and reconfiguration necessary for behavioural changes during development.     

Development of hindlimb locomotor behavior in the frog

Hindlimb motor behavior of the larval frog (tadpole) begins during midlarval life and occurs with increasing frequency until the tail degenerates during metamorphosis. The threshold for hindlimb withdrawal in response to tactile stimulation is low during premetamorphic stages and rises dramatically during metamorphosis. Testing tadpoles in different environments altered the stage of development at which different hindlimb behaviors were first observed but did not change the ontogenetic sequence of behavioral development. However, even under conditions most favorable to hindlimb locomotion, behavioral expression lagged behind electrophysiological expression. The rates of tail beating, hindlimb stepping, and fog kicks are similar to the rate of bursting of tail and hindlimb motoneurons of the isolated nervous system, but their coordination is variable, whereas that recorded from the isolated CNS is fixed. Because neural mechanisms of hindlim locomotion are functional prior to their behavioral use, the basic hindlimb locomotor circuits must develop without benefit of practice or sensory feedback. However, sensory activity modulates coordination and alters the probability that particular behaviors will be expressed. Implications of these results for studies of early behavior in other species, and the problem of inferring neural maturity from behavioral observations, are discussed.     

Electrically evoked locomotor activity in the turtle spinal cord hemi-enlargement preparation

We assessed the locomotor capacity of the left half of the spinal cord hindlimb enlargement in low-spinal turtles. Forward swimming was evoked in the left hindlimb by electrical stimulation of the right dorsolateral funiculus (DLF) at the anterior end of the third postcervical spinal segment (D3). Animals were held by a band-clamp in a water-filled tank so that hindlimb movements could be recorded from below with a digital video camera. Left hindlimb hip and knee movements were tracked while electromyograms (EMGs) were recorded from left hip and knee muscles. In turtles with intact spinal cords, electrical stimulation of the right D3 DLF evoked robust forward swimming movements of the left hindlimb, characterized by rhythmic alternation between hip flexor (HF) and hip extensor (HE) EMG discharge, with knee extensor (KE) bursts occurring during the latter part of each HE-off phase. After removing the right spinal hemi-enlargement (D8-S2), DLF stimulation still evoked rhythmic locomotor movements and EMG bursts in the left hindlimb that included HF-HE alternation and KE discharge. However, post-surgical movements and EMG bursts had longer cycle periods, and movements showed lower amplitudes compared to controls. These results show that (1) sufficient locomotor CPG circuitry resides within the turtle spinal hemi-enlargement to drive major components of the forward swim motor pattern, (2) contralateral circuitry contributes to the excitation of the locomotor CPG for a given limb, and (3) a sufficient portion of the descending DLF pathway crosses over to the contralateral cord anterior to the hindlimb enlargement to activate swimming.     

Locomotor and reflex adaptation after partial denervation of ankle extensors in chronic spinal cats

This work investigates the capacity of the spinal cord to generate locomotion after a complete spinal section and its ability to adapt its locomotor pattern after a peripheral nerve lesion. To study this intrinsic adaptive capacity, the left lateral gastrocnemius-soleus (LGS) nerve was sectioned in three cats that expressed a stable locomotion following a complete spinal transection. The electromyograph (EMG) of multiple hindlimb muscles and reflexes, evoked by stimulating the left tibial (Tib) nerve at the ankle, were recorded before and after denervation during treadmill locomotion. Following denervation, the mean amplitude of EMG bursts of multiple hindlimb muscles increased during locomotion, similar to what is found after an identical denervation in otherwise intact cats. Reflex changes were noted in ipsilateral flexors, such as semitendinosus and tibialis anterior, but not in the ipsilateral knee extensor vastus lateralis following denervation. The present results demonstrate that the spinal cord possesses the circuitry necessary to mediate increased EMG activity in multiple hindlimb muscles and also to produce changes in reflex pathways after a muscle denervation. The similarity of changes following LGS denervation in cats with an intact and transected spinal cord suggests that spinal mechanisms play a major role in the locomotor adaptation.     

The development of the dendritic organization of primary and secondary motoneurons in the spinal cord of Xenopus laevis. An HRP study

Summary During embryonic and larval development of the clawed toad, Xenopus laevis, two different populations of motoneurons appear in the spinal cord. In this study the development of primary motoneurons which innervate the axial musculature (used during embryonic locomotion) and of secondary motoneurons which innervate the extremity musculature (used for locomotion during metamorphosis and thereafter) was analyzed with horseradish peroxidase (HRP) as a neuronal marker. After application of HRP to the axial musculature (rostral five postotic myotomes) the first labeled primary motoneurons were found at stage 24/25. During development gradually more labeled neurons were observed. These primary motoneurons send their dendrites into the marginal zone (white matter). At first only dorsal and lateral dendrites develop (stages 25–33), followed by ventral dendrites (stage 37/38). Up till stage 48 the developing dendrites extend throughout the marginal zone. Hereafter the marginal zone increases particularly at the dorsolateral edge, a development which is not followed by the dendrites of the primary motoneurons. The dendrites of mature primary motoneurons (stages 58–62) occupy the ventral and ventrolateral parts of the marginal zone.At stage 48, shortly after the hindlimb bud arises (stage 46, early metamorphosis), the first neurons related to this developing extremity could be labeled in the ventrolateral part of the lumbar spinal cord. At first these secondary motoneurons bear only a few dorsal dendrites of which only the tips reache out in the adjacent white matter. Already at stage 50 these dorsal dendrites have invaded the whole dorsolateral part of the marginal zone. Also the first ventral dendrites were observed at this stage. Later, at stage 53/54 also some ventral dendrites have reached the white matter together with a few lateral dendrites. At these early metamorphic stages already some primary afferent fibers were found making contact with the dorsomedial dendrites. At stage 58 for the first time recurrent axon collaterals were found, which extend into the ventromedial part of the marginal zone. The development of motoneurons in the spinal cord seems to be characterized by two phases: (1) establishment of contacts between motoneurons and target muscles, and (2) subsequent formation of connections of these motoneurons with other nerve cells within the central nervous system. The dendrites of primary motoneurons follow the development of the marginal zone, while dendrites of secondary motoneurons develop into an already well developed marginal zone. Generally, the dendrites of mature motoneurons of the axial musculature were observed in the ventromedial and ventrolateral parts of the marginal zone. The dendrites of themotoneurons which innervate the musculature of the hindlimbs were observed predominantly in the dorsolateral and ventromedial parts of the marginal zone.     

Sequential activation of axial muscles during different forms of rhythmic behavior in man

In humans, studies of back muscle activity have mainly addressed the functioning of lumbar muscles during postural adjustments or rhythmic activity, including locomotor tasks. The present study investigated how back muscles are activated along the spine during rhythmical activities in order to gain insights into spinal neuronal organization. Electromyographic recordings of back muscles were performed at various trunk levels, and changes occurring in burst amplitudes and phase relationships were analyzed. Subjects performed several rhythmic behaviors: forward walking (FW), backward walking (BW), amble walking (where the subjects moved their arms in phase with the ipsilateral leg), walking on hands and knees (HK) and walking on hands with the knees on the edge of a treadmill (Hand). In a final task, the subjects were standing and were asked to swing (Swing) only their arms as if they were walking. It was found that axial trunk muscles are sequentially activated by a motor command running along the spinal cord (which we term “motor waves”) during various types of locomotion or other rhythmic motor tasks. The bursting pattern recorded under these conditions can be classified into three categories: (1) double-burst rhythmic activity in a descending (i.e., with a rostro-caudal propagation) motor wave during FW, BW and HK conditions; (2) double-burst rhythmic activity with a stationary motor wave (i.e., occurring in a single phase along the trunk) during the ‘amble’ walk condition; (3) monophasic rhythmic activity with an ascending (i.e., with a caudo-rostral propagation) motor wave during the Swing and Hands conditions. Our results suggest that the networks responsible for the axial propagation of motor activity during locomotion may correspond to those observed in invertebrates or lower vertebrates, and thus may have been partly phylogenetically conserved. Such an organization could support the dynamic control of posture by ensuring fluent movement during locomotion.     

Physiology and morphology of shared and specialized spinal interneurons for locomotion and scratching

Distinct types of rhythmic movements that use the same muscles are typically generated largely by shared multifunctional neurons in invertebrates, but less is known for vertebrates. Evidence suggests that locomotion and scratching are produced partly by shared spinal cord interneuronal circuity, although direct evidence with intracellular recording has been lacking. Here, spinal interneurons were recorded intracellularly during fictive swimming and fictive scratching in vivo and filled with Neurobiotin. Some interneurons that were rhythmically activated during both swimming and scratching had axon terminal arborizations in the ventral horn of the hindlimb enlargement, indicating their likely contribution to hindlimb motor outputs during both behaviors. We previously described a morphological group of spinal interneurons ("transverse interneurons" or T neurons) that were rhythmically activated during all forms of fictive scratching at higher peak firing rates and with larger membrane potential oscillations than scratch-activated spinal interneurons with different dendritic orientations. The current study demonstrates that T neurons are activated during both swimming and scratching and thus are components of the shared circuitry. Many spinal interneurons activated during fictive scratching are also activated during fictive swimming (scratch/swim neurons), but others are suppressed during swimming (scratch-specialized neurons). The current study demonstrates that some scratch-specialized neurons receive strong and long-lasting hyperpolarizing inhibition during fictive swimming and are also morphologically distinct from T neurons. Thus this study indicates that locomotion and scratching are produced by a combination of shared and dedicated interneurons whose physiological and morphological properties are beginning to be revealed.     

The effect of dorsal root transection on the efferent motor pattern in the cat's hindlimb during locomotion

Mesencephalic cats can walk on a treadmill if the midbrain locomotor region is stimulated. The motor pattern of different hindlimb muscles is similar to that of th intact cat. The present experiments in the mesencephalic preparation test if the complex motor pattern in one hindlimb is causally dependent on the afferent signals arising in the same limb during walking. The electromyographical activity and the movement pattern during locomotion were compared before and after transecting all dorsal root fibres originating from one hindlimb. Flexor and extensor muscles at different joints may retain their general pattern after the dorsal root transection. This applies also to muscles such as the knee flexors, which have a short and early flexor burst and a second burst during the extension phase, and the short toe dorsiflexor , which has an early burst in the transition between flexor and extensor activity. After the dorsal root transection the pattern of activity may become more variable and it can even break down altogether. The present results demonstrate that the central nervous system devoid of phasic afferent inflow from one hindlimb can produce a complex motor output to this limb rather than a motor pattern degraded to a simple alternation between flexors and extensors.     

Rhythmic phrenic,intercostal and sympathetic activity in relation to limb and trunk motor activity in spinal cats

During L-DOPA-induced fictive spinal locomotion rhythmic activities in nerves to internal intercostal and external oblique abdominal muscles and in phrenic and sympathetic nerves were observed which were always coordinated with locomotor activity in forelimb and hindlimb muscle nerves. A periodicity with longer lasting tonic phases could be induced by cutaneous nerve stimulation or asphyxia. This activity was observed in limb motor nerves as well as in respiratory motor and sympathetic nerves. A slow independent activity of the phrenic and intercostal nerves or the sympathetic nerves, which could be related to a normal respiratory rhythm or independent sympathetic rhythms was not observed. The findings indicate that during fictive spinal locomotion the activity of spinal rhythm generators for locomotion also projects onto respiratory and sympathetic spinal neurones.     

Crossed commissural pathways in the spinal hindlimb enlargement are not necessary for right left hindlimb alternation during turtle swimming

We examined the coordination between right and left hindlimbs during voluntary forward swimming in adult red-eared turtles, before and after midsagittal section of the spinal cord hindlimb enlargement (segments D8-S2) or the enlargement plus the first preenlargement segment (D7-S2). Our purpose was to assess the role of crossed commissural axons in these segments for right-left hindlimb alternation during voluntary locomotion. Midsagittal splitting severed commissural fibers and separated the right and left halves of the posterior spinal cord. Adult turtles (n = 9) were held by a band clamp around the shell in a water-filled tank while digital video of forward swimming was recorded from below and computer analyzed with motion analysis software. In a subset of these animals (n = 5), we also recorded electromyograms from hip extensor and/or hip flexor muscles on both sides. Surprisingly, splitting spinal segments D8-S2 or D7-S2 did not affect the strength of out-of-phase coordination between right and left hindlimbs, although hindlimb movement amplitudes were reduced compared with presurgical controls. These results show that commissural axons in the hindlimb enlargement and preenlargement cord are not necessary for right-left hindlimb alternation during voluntary swimming. We suggest that alternating propriospinal drive from the right and left sides of the forelimb enlargement maintains the out-of-phase coordination of right and left hindlimbs in the bisected-cord preparation. Our data support the hypothesis that descending propriospinal (forelimb-hindlimb) and crossed commissural (hindlimb-hindlimb) spinal cord pathways function together as redundant mechanisms to sustain right-left hindlimb alternation during turtle locomotion.     

Opposing modulatory effects of D1- and D2-like receptor activation on a spinal central pattern generator

The role of dopamine in regulating spinal cord function is receiving increasing attention, but its actions on spinal motor networks responsible for rhythmic behaviors remain poorly understood. Here, we have explored the modulatory influence of dopamine on locomotory central pattern generator (CPG) circuitry in the spinal cord of premetamorphic Xenopus laevis tadpoles. Bath application of exogenous dopamine to isolated brain stem-spinal cords exerted divergent dose-dependent effects on spontaneous episodic patterns of locomotory-related activity recorded extracellularly from spinal ventral roots. At low concentration (2 μM), dopamine reduced the occurrence of bursts and fictive swim episodes and increased episode cycle periods. In contrast, at high concentration (50 μM) dopamine reversed its actions on fictive swimming, now increasing both burst and swim episode occurrences while reducing episode periods. The low-dopamine effects were mimicked by the D2-like receptor agonists bromocriptine and quinpirole, whereas the D1-like receptor agonist SKF 38393 reproduced the effects of high dopamine. Furthermore, the motor response to the D1-like antagonist SCH 23390 resembled that to the D2 agonists, whereas the D2-like antagonist raclopride mimicked the effects of the D1 agonist. Together, these findings indicate that dopamine plays an important role in modulating spinal locomotor activity. Moreover, the transmitter's opposing influences on the same target CPG are likely to be accomplished by a specific, concentration-dependent recruitment of independent D2- and D1-like receptor signaling pathways that differentially mediate inhibitory and excitatory actions.     

Kinematic and EMG determinants in quadrupedal locomotion of a non-human primate (Rhesus)

We hypothesized that the activation patterns of flexor and extensor muscles and the resulting kinematics of the forelimbs and hindlimbs during locomotion in the Rhesus would have unique characteristics relative to other quadrupedal mammals. Adaptations of limb movements and in motor pool recruitment patterns in accommodating a range of treadmill speeds similar to other terrestrial animals in both the hindlimb and forelimb were observed. Flexor and extensor motor neurons from motor pools in the lumbar segments, however, were more highly coordinated than in the cervical segments. Unlike the lateral sequence characterizing subprimate quadrupedal locomotion, non-human primates use diagonal coordination between the hindlimbs and forelimbs, similar to that observed in humans between the legs and arms. Although there was a high level of coordination between hind- and forelimb locomotion kinematics, limb-specific neural control strategies were evident in the intersegmental coordination patterns and limb endpoint trajectories. Based on limb kinematics and muscle recruitment patterns, it appears that the hindlimbs, and notably the distal extremities, contribute more to body propulsion than the forelimbs. Furthermore, we found adaptive changes in the recruitment patterns of distal muscles in the hind- and forelimb with increased treadmill speed that likely correlate with the anatomical and functional evolution of hand and foot digits in monkeys. Changes in the properties of both the spinal and supraspinal circuitry related to stepping, probably account for the peculiarities in the kinematic and EMG properties during non-human primate locomotion. We suggest that such adaptive changes may have facilitated evolution toward bipedal locomotion.     

Spatio-temporal regulation and cleavage by matrix metalloproteinase stromelysin-3 implicate a role for laminin receptor in intestinal remodeling during Xenopus laevis metamorphosis.

The 37-kd laminin receptor precursor (LR) was first identified as a 67-kd protein that binds laminin with high affinity. We have recently isolated the Xenopus laevis LR as an in vitro substrate of matrix metalloproteinase stromelysin-3 (ST3), which is highly upregulated during intestinal metamorphosis in Xenopus laevis. Here, we show that LR is expressed in the intestinal epithelium of premetamorphic tadpoles. During intestinal metamorphosis, LR is downregulated in the apoptotic epithelium and concurrently upregulated in the connective tissue but with little expression in the developing adult epithelium. Toward the end of metamorphosis, as adult epithelial cells differentiate, they begin to express LR. Furthermore, LR is cleaved during intestinal remodeling when ST3 is highly expressed or in premetamorphic intestine of transgenic tadpoles overexpressing ST3. These results suggest that LR plays a role in cell fate determination and tissue morphogenesis, in part through its cleavage by ST3. Interestingly, high levels of LR are known to be expressed in tumor cells, which are often surrounded by fibroblasts expressing ST3, suggesting that LR cleavage by ST3 plays a role in both physiological and pathological processes.     

Afferent control of central pattern generators: experimental analysis of locomotion in the decerebrate cat

Changes in the motor activity of the spinal locomotor generator evoked by tonic and phasic peripheral afferent signals during fictitious locomotion of both slow and fast rhythms were analysed in the cat. The tonic afferent inflow was conditioned by the position of the hindlimb. The phasic afferent signals were imitated by electrical stimulation of hindlimb nerves. The correlation between the kinematics of hindlimb locomotor movement and sensory inflow was investigated during actual locomotion. Reliable correlations between motor activity parameters during fictitious locomotion were revealed in cases of both slow and fast "locomotor" rhythms. The main difference between these cases was that correlations "duration-intensity" were positive in the first and negative in the second case. The functional role of "locomotor" pattern dependence on tonic sensory inflow consisted of providing stability for planting the hindlimb on the ground. For any investigated afferent input the phase moments in the "locomotor" cycle were found, in which an afferent signal caused no rearrangement in locomotor generator activity. These moments corresponded to the transitions between "flexion" and "extension" phases and to the bursts of integral afferent activity observed during real locomotion. The data obtained are compared with the results previously described for the scratching generator. The character of changes in "locomotor" activity in response to tonic and phasic sensory signals was similar to that of such changes in "scratching" rhythm in the case of fast "locomotion". Intensification of the "flexion" phase caused by phasic high-intensity stimulation of cutaneous afferents during low "locomotor" rhythm was changed to inhibition (such as observed during "scratching") when this rhythm was fast. It is concluded that the main regularities of peripheral afferent control for both the locomotor and scratching generators are the same. Moreover, these central pattern generators are just working regimes of a general spinal motor optimal control system containing the intrinsic model of limb movement dynamics. The consequences of this concept and ways of further research are discussed.     

Ontogeny of human locomotor control I. Infant stepping,supported locomotion and transition to independent locomotion

Summary Locomotor patterns of human infants were studied during stepping in the newborn period (first two months of life), during supported locomotion (6–12 months of age) and during independent locomotion in children who just were able to walk by themselves without external support (10–18 months of age). Leg movements, pattern of muscular activity and reaction forces were studied by a computerized system. The locomotor pattern during the newborn period lacked the specific functions that are unique for human plantigrade locomotion. There was no heel strike in front of the body; the foot was placed instead on its forepart straight under the body. Hip and knee joints were hyperflexed during the whole step cycle and flexed synchronously during swing. The specific knee-ankle coordination of human adults was missing. The ankle extensors were activated prior to touch down together with other extensor muscles. There was no propulsive force. A similar immature non-plantigrade pattern recurred after an inactive period. During the subsequent period of supported locomotion there was a gradual transformation of the infantile pattern towards the plantigrade pattern continuing after establishment of independent locomotion. It is suggested that innate pattern generators in the spinal cord produce the infant stepping and also generate the basic locomotor rhythm in adults, but that neural circuits specific for humans develop late in ontogeny and transform the original, non-plantigrade motor activity to a plantigrade locomotor pattern.     

Reticulospinal neurons controlling forward and backward swimming in the lamprey

Most vertebrates are capable of two forms of locomotion, forward and backward, strongly differing in the patterns of motor coordination. Basic mechanisms generating these patterns are located in the spinal cord; they are activated and regulated by supraspinal commands. In the lamprey, these commands are transmitted by reticulospinal (RS) neurons. The aim of this study was to reveal groups of RS neurons controlling different aspects of forward (FS) and backward (BS) swimming in the lamprey. Activity of individual larger RS neurons in intact lampreys was recorded during FS and BS by chronically implanted electrodes. It was found that among the neurons activated during locomotion, 27% were active only during FS, 3% only during BS, and 70% during both FS and BS. In a portion of RS neurons, their mean firing frequency was correlated with frequency of body undulations during FS (8%), during BS (34%), or during both FS and BS (22%), suggesting their involvement in control of locomotion intensity. RS activity was phasically modulated by the locomotor rhythm during FS (20% of neurons), during BS (29%), or during both FS and BS (16%). The majority of RS neurons responding to vestibular stimulation (and presumably involved in control of body orientation) were active mainly during FS. This explains the absence of stabilization of the body orientation observed during BS. We discuss possible functions of different groups of RS neurons, i.e., activation of the spinal locomotor CPG, inversion of the direction of propagation of locomotor waves, and postural control.     

Defining the excitatory neurons that drive the locomotor rhythm in a simple vertebrate: insights into the origin of reticulospinal control

Important questions remain about the origin of the excitation that drives locomotion in vertebrates and the roles played by reticulospinal neurons. In young Xenopus tadpoles, paired whole-cell recordings reveal reticulospinal neurons that directly excite swimming circuit neurons in the brainstem and spinal cord. They form part of a column of neurons (dINs) with ipsilateral descending projections which fire reliably and rhythmically in time with swimming. We ask if, at this early stage of development, these reticulospinal neurons are themselves the primary source of rhythmic drive to spinal cord neurons on each cycle of swimming. Loose-patch recordings in the hindbrain and spinal cord from neurons active during fictive swimming distinguished dINs from other neurons by spike shape. These recordings showed that reticulospinal dINs in the caudal hindbrain (rhombomeres 7–8) fire significantly earlier on each swimming cycle than other, ipsilateral, swimming circuit neurons. Whole-cell recordings showed that fast EPSCs typically precede, and probably drive, spikes in most swimming circuit neurons. However, the earliest-firing reticulospinal dINs spike too soon to be driven by underlying fast EPSCs. We propose that rebound following reciprocal inhibition can contribute to early reticulospinal dIN firing during swimming and show rebound firing in dINs following evoked, reciprocal inhibitory PSPs. Our results define reticulospinal neurons that are the source of the primary, descending, rhythmic excitation that drives spinal cord neurons to fire during swimming. These neurons are an integral part of the rhythm generating circuitry. We discuss the origin of these reticulospinal neurons as specialised members of a longitudinally distributed population of excitatory interneurons extending from the brainstem into the spinal cord.     

Intraspinal stimulation caudal to spinal cord transections in rats. Testing the propriospinal hypothesis

Many laboratories have reported the successful regeneration of neurons across damaged portions of the spinal cord. Associated improvements in hindlimb locomotor movements have been attributed to the formation of functional neuronal connections with the locomotor central pattern generator (CPG). However, regenerating axons generally extend no more than 10 mm caudal to the lesion sites, terminating about 20 mm short of the lumbar segments thought to contain the CPG. It has therefore tacitly been assumed that the locomotor improvements arose from activation of propriospinal neurons relaying excitation to the CPG. Here we report a test of this assumption, which we call the propriospinal hypothesis. Intraspinal microstimulation (ISMS) was used to activate the putative propriospinal relay neurons. Approximately 2-3 wk after complete spinal cord transection at T8-T9 in rats, an array of six Pt-Ir microwires was chronically implanted in the intermediate and ventral gray matter of T10-T12 segments. ISMS pulse trains with amplitudes of 0.8-0.9 times threshold for activating axial muscles were delivered during open-field locomotor tests (BBB). ISMS significantly increased BBB scores over control tests, but did not produce limb coordination and weight bearing sufficient for locomotion. These results support the main assumption of the propriospinal hypothesis: that neuronal activity elicited in thoracic spinal segments caudal to a complete spinal cord transection may propagate caudally and activate the locomotor CPG.     

Coordinated rhythmic bursting in respiratory and locomotor muscle nerves in the spinal rabbit.

In unanaesthetized, curarized spinal rabbits (C2 level) treated with Niamide and DOPA, rhythmic activities were recorded from the phrenic nerves; close coordination was observed between the phrenic bursts and the locomotor bursts which developed in hindlimb muscle nerves. The frequency of phrenic bursts was reduced after a second spinal transection at the Th12 level, while rhythms in the hindlimb remained unchanged. It thus appears that in the spinal preparation and under certain pharmacological conditions, phrenic bursts generated by the cervico-thoracic spinal cord can be driven by the lumbar generators of locomotion; spinal links thus exist between these hindlimb locomotion generators and spinal interneuronal networks involved in phrenic motoneuronal activation, may be via hindlimb forelimb driving.     

Metachronal coupling between spinal neuronal networks during locomotor activity in newborn rat

In the present study, we investigate spinal cord neuronal network interactions in the neonatal rat during locomotion. The behavioural and physiological relevance of metachronally propagated locomotor activity were inferred from kinematic, anatomical and in vitro electrophysiological data. Kinematic analysis of freely behaving animals indicated that there is a rhythmic sequential change in trunk curvature during the step cycle. The motoneurons innervating back and tail muscles were identified along the spinal cord using retrograde labelling. Systematic multiple recordings from ventral roots were made to determine the precise intrinsic pattern of coordination in the isolated spinal cord. During locomotor-like activity, rhythmic ventral root motor bursts propagate caudo-rostrally in the sacral and the thoracic spinal cord regions. Plotting the latency as a function of the cycle period revealed that the system adapts the intersegmental latency to the ongoing motor period in order to maintain a constant phase relationship along the spinal axis. The thoracic, lumbar and sacral regions were capable of generating right and left alternating motor bursts when isolated. Longitudinal sections of the spinal cord revealed that both the bilateral antiphase pattern observed for the sacral region with respect to the lumbar segment 2 as well as the intersegmental phase lag were due to cross-cord connections. Together, these results provide physiological evidence that the dynamic changes observed in trunk bending during locomotion are determined by the intrinsic organization of spinal cord networks and their longitudinal and transverse interactions. Similarities between this organization, and that of locomotor pattern generation in more primitive vertebrates, suggest that the circuits responsible for metachronal propagation of motor patterns during locomotion are highly conserved.