Nonlocomotor and locomotor hindlimb responses evoked by electrical microstimulation of the lumbar cord in spinalized cats

As a preliminary step to using intraspinal microstimulation (ISMS) for rehabilitation purposes, the distribution of various types of hindlimb responses evoked by ISMS in spinal cats (T(13)) is described. The responses to ISMS applied through a single electrode was assessed, before and after an intravenous injection of clonidine (noradrenergic agonist), using kinematics and electromyographic recordings in subacute (5-7 days, untrained) or chronic (3-5 wk trained on a treadmill) spinal cats. ISMS was applied in the dorsal, intermediate and ventral areas of segments L(3)-L(7), from midline to 3 mm laterally. Uni- and bilateral non-locomotor responses as well as rhythmical locomotor responses were evoked. In the subacute cats, ipsilateral flexion was elicited in the dorsal region of L(3)-L(7), whereas ipsilateral extension was evoked more ventrally and mainly in the caudal segments. Dorsal stimuli could induce ipsilateral flexion followed by ipsilateral extension. Sites inducing bilateral flexion and bilateral extension were similarly distributed to those evoking ipsilateral flexion and extension in the rostrocaudal axis but were evoked from more medial sites. Ipsilateral flexion with crossed extension was evoked from intermediate and ventral zones of all segments and lateralities. Unilateral ipsilateral locomotion was rarely observed. Contralateral locomotion was more frequent and mainly evoked medially, whereas bilateral locomotion was evoked exclusively from dorsal regions. With some exceptions, those distribution gradients were similar in the four conditions (subacute, chronic, pre- and postclonidine), but the proportion of each response could vary. The distribution of ISMS-evoked responses is discussed as a function of known localization of interneurons and motoneurons.     

Mid-lumbar segments are needed for the expression of locomotion in chronic spinal cats

In acute experiments performed in decerebrated and spinalized (T13) cats, an intraspinal injection of clonidine, a noradrenergic agonist, restricted to mid-lumbar segments L3-L4, can induce hindlimb locomotion, whereas yohimbine, a noradrenergic antagonist, can block spinal locomotion, and a second spinal lesion at L4 can abolish all locomotor activity. In the present study, we investigated whether the abolition of locomotion after this second spinal lesion was due to an acute spinal shock or to the functional disconnection of the rostral and caudal lumbar segments. In seven cats, first spinalized at T13 and having recovered treadmill locomotion, a second transection was performed at lower lumbar levels. Video and electromyographic recordings were used to evaluate locomotor performance. Results show that after a second transection at L2 or rostral L3 levels, spinal locomotion was maintained; when the second lesion was performed at caudal L3 or L4, all locomotor activity was abolished even after several weeks of attempted locomotor training; vigorous fast paw shakes (FPS) were observed in all cases; and after an intraperitoneal injection of clonidine in cats with a second transection below L4, perineal stimulation induced hyperextension of the hindlimbs but no locomotion. Considering that the main motoneuron pools of the hindlimbs are caudal to L4 and are still functional after the second spinal transection, as evidenced by the presence of FPS, we conclude that the mid-lumbar spinal segments are essential for the specific expression of spinal locomotion but not necessarily for other rhythmic motor patterns.     

Effects of localized intraspinal injections of a noradrenergic blocker on locomotion of high decerebrate cats

Previous studies demonstrated that neuronal networks located in midlumbar segments (L3-L4) are critical for the expression of locomotion in cats following complete spinalization. In the present study the importance of several thoracolumbar segments (T8-L7) for the generation of spontaneous hindlimb locomotion in decerebrate cats was evaluated. Experiments were performed in high decerebrate cats (n = 18) walking spontaneously. Yohimbine, an alpha2-noradrenergic antagonist, was microinjected intraspinally in various thoracolumbar segments. Locomotor performance was evaluated with kinematics and electromyographic (EMG) recordings before and after each injection. When and if spontaneous locomotion (SL) was abolished, skin or perineal stimuli (exteroceptive stimuli) were used to trigger locomotion (exteroceptive-induced locomotion [EL]). Yohimbine injections at L3 or L4 completely inhibited SL and EL. In contrast, injections at T8 did not interfere with SL or EL. Injections at T10, T11, T12, L5, L6, and L7 inhibited SL but EL could still be evoked. Injections at T13, L1, and L2 had similar effects except that the quality of locomotion evoked by exteroceptive stimulation declined. Combined injections at T13, L1, and L2 abolished SL and EL, in contrast to injections restricted to the same individual segments. Simultaneous injections at L5, L6, and L7 also abolished SL but EL could still be induced. These results suggest that noradrenergic mechanisms in L3-L4 segments are involved in the expression of locomotion in decerebrate cats, whereas antagonizing noradrenergic inputs in individual rostral or caudal segments may alter the expression and overall quality of the locomotor pattern without abolishing locomotion.     

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.     

Localization of spinal neurons activated during locomotion using the c-fos immunohistochemical method

The c-fos immunohistochemical method of activity-dependent labeling was used to localize locomotor-activated neurons in the adult cat spinal cord. In decerebrate cats, treadmill locomotion was evoked by electrical stimulation of the mesencephalic locomotor region (MLR). Spontaneous or MLR-evoked fictive locomotion was produced in decerebrate animals paralyzed with a neuromuscular blocking agent. After bouts of locomotion during a 7- to 9-h time period, the animals were perfused and the L3-S1 spinal cord segments removed for immunohistochemistry. Control animals were subjected to the same surgical procedures but no locomotor task. Labeled cells were concentrated in Rexed's laminae III and IV of the dorsal horn and laminae VII, VIII, and X of the intermediate zone/ventral horn after treadmill locomotion. Cells in laminae VII, VIII, and X were labeled after fictive locomotion, but labeling in the dorsal horn was much reduced. In control animals, c-fos labeling was a small fraction of that observed in the locomotor animals. The results suggest that labeled cells in laminae VII, VIII, and X are premotor interneurons involved in the production of locomotion, whereas the laminae III and IV cells are those activated during locomotion due to afferent feedback from the moving limb. c-fos-labeled cells were most numerous in the L5-L7 segments, consistent with the distribution of locomotor activated neurons detected through the use of MLR-evoked field potentials.     

Locomotor-activated neurons of the cat. II. Noradrenergic innervation and colocalization with NEα 1a or NEα 2b receptors in the thoraco-lumbar spinal cord

Norepinephrine (NE) is a strong modulator and/or activator of spinal locomotor networks. Thus noradrenergic fibers likely contact neurons involved in generating locomotion. The aim of the present study was to investigate the noradrenergic innervation of functionally related, locomotor-activated neurons within the thoraco-lumbar spinal cord. This was accomplished by immunohistochemical colocalization of noradrenergic fibers using dopamine-β-hydroxylase or NEα(1A) and NEα(2B) receptors with cells expressing the c-fos gene activity-dependent marker Fos. Experiments were performed on paralyzed, precollicular-postmamillary decerebrate cats, in which locomotion was induced by electrical stimulation of the mesencephalic locomotor region. The majority of Fos labeled neurons, especially abundant in laminae VII and VIII throughout the thoraco-lumbar (T13-L7) region of locomotor animals, showed close contacts with multiple noradrenergic boutons. A small percentage (10-40%) of Fos neurons in the T7-L7 segments showed colocalization with NEα(1A) receptors. In contrast, NEα(2B) receptor immunoreactivity was observed in 70-90% of Fos cells, with no obvious rostrocaudal gradient. In comparison with results obtained from our previous study on the same animals, a significantly smaller proportion of Fos labeled neurons were innervated by noradrenergic than serotonergic fibers, with significant differences observed for laminae VII and VIII in some segments. In lamina VII of the lumbar segments, the degree of monoaminergic receptor subtype/Fos colocalization examined statistically generally fell into the following order: NEα(2B) = 5-HT(2A) ≥ 5-HT(7) = 5-HT(1A) > NEα(1A). These results suggest that noradrenergic modulation of locomotion involves NEα(1A)/NEα(2B) receptors on noradrenergic-innervated locomotor-activated neurons within laminae VII and VIII of thoraco-lumbar segments. Further study of the functional role of these receptors in locomotion is warranted.     

Hindlimb stepping movements in complete spinal rats induced by epidural spinal cord stimulation

The locomotor ability of the spinal cord of adult rats deprived of brain control was tested by epidural spinal cord stimulation. The studies were performed on six rats that had a complete spinal cord transection (T7-T9) and epidural electrode implantations 2-3 weeks before testing was initiated. The stimulating epidural electrodes were implanted at the T12-L6 spinal segments. Epidural electrical stimulation of the dorsal surface of the spinal cord at frequencies between 1 and 50 Hz and intensities between 1 and 10 V without any pharmacological facilitation was used. Stimulation at each of the lumbar spinal cord segments elicited some rhythmic activity in the hindlimbs. However, stimulation at most segmental levels usually evoked activity in only one leg and was maintained for short periods of time (< 10s). Bilateral hindlimb locomotor activity was evoked most often with epidural stimulation at 40-50 Hz applied at the L2 segment. A necessary condition for initiation of locomotor activity was providing a specific amount (at least 5%) of body weight support. Therefore, the rat spinal cord isolated from brain control is capable of producing bilateral stepping patterns induced most readily by epidural stimulation applied at the L2 spinal segment. Furthermore, the induced stepping patterns were dependent on sensory feedback associated with weight bearing.     

Monoaminergic establishment of rostrocaudal gradients of rhythmicity in the neonatal mouse spinal cord

Bath application of monoamines is a potent method for evoking locomotor activity in neonatal rats and mice. Monoamines also promote functional recovery in adult animals with spinal cord injuries by activating spinal cord networks. However, the mechanisms of their actions on spinal networks are largely unknown. In this study, we tested the hypothesis that monoamines establish rostrocaudal gradients of rhythmicity in the thoracolumbar spinal cord. Isolated neonatal mouse spinal cord preparations (P0-P2) were used. To assay excitability of networks by monoamines, we evoked a disinhibited rhythm by bath application of picrotoxin and strychnine and recorded neurograms from several thoracolumbar ventral roots. We first established that rostral and caudal segments of the thoracolumbar spinal cord had equal excitability by completely transecting preparations at the L3 segmental level and recording the frequency of the disinhibited rhythm from both segments. Next we established that a majority of ventral interneurons retrogradely labeled by calcium green dextran were active during network activity. We then bath applied combinations of monoaminergic agonists [5-HT and dopamine (DA)] known to elicit locomotor activity. Our results show that monoamines establish rostrocaudal gradients of rhythmicity in the thoracolumbar spinal cord. This may be one mechanism by which combinations of monoaminergic compounds normally stably activate locomotor networks.     

Intraspinal microstimulation excites multisegmental sensory afferents at lower stimulus levels than local alpha-motoneuron responses

Microstimulation within the motor regions of the spinal cord is often assumed to activate motoneurons and propriospinal neurons close to the electrode tip. However, previous work has shown that intraspinal microstimulation (ISMS) in the gray matter activates sensory afferent axons as well as alpha-motoneurons (MNs). Here we report on the recruitment of sensory afferent axons and MNs as ISMS amplitudes increased. Intraspinal microstimulation was applied through microwires implanted in the dorsal horn, intermediate region and ventral horn of the L(5)-L(7) segments of the spinal cord in four acutely decerebrated cats, two of which had been chronically spinalized. Activation of sensory axons was detected with electroneurographic recordings from dorsal roots. Activation of MNs was detected with electromyographic (EMG) recordings from hindlimb muscles. Sensory axons were nearly always activated at lower stimulus levels than MNs irrespective of the stimulating electrode location. EMG response latencies decreased as ISMS stimulus intensities increased, suggesting that MNs were first activated transsynaptically and then directly as intensity increased. ISMS elicited antidromic activity in dorsal root filaments with entry zones up to 17 mm rostral and caudal to the stimulation sites. We posit that action potentials elicited in localized terminal branches of afferents spread antidromically to all terminal branches of the afferents and transsynaptically excite MNs and interneurons far removed from the stimulation site. This may help explain how focal ISMS can activate many MNs of a muscle even though they are distributed in long thin columns.     

Coding of locomotor phase in populations of neurons in rostral and caudal segments of the neonatal rat lumbar spinal cord

Several experiments have demonstrated that rostral segments of the vertebrate lumbar spinal cord produce a rhythmic motor output more readily and of better quality than caudal segments. Here we examine how this rostrocaudal gradient of rhythmogenic capability is reflected in the spike activity of neurons in the rostral (L(2)) and caudal (L(5)) lumbar spinal cord of the neonatal rat. The spike activity of interneurons in the ventromedial cord, a region necessary for the production of locomotion, was recorded intracellularly with patch electrodes and extracellularly with tetrodes during pharmacologically induced locomotion. Both L(2) and L(5) neurons tended to be active in phase with their homologous ventral root. L(5) neurons, however, had a wider distribution of their preferred phases of activity throughout the locomotor cycle than L(2) neurons. The strength of modulation of the activity of individual L(2) neurons was also larger than that of L(5) neurons. These differences resulted in a stronger rhythmic signal from the L(2) neuronal population than from the L(5) population. These results demonstrate that the rhythmogenic capability of each spinal segment was reflected in the activity of interneurons located in the same segment. In addition to paralleling the rostrocaudal gradient of rhythmogenic capability, these results further suggest a colocalization of motoneurons and their associated interneurons involved in the production of locomotion.     

Effects of Spinal Cord Electrical Stimulation in Patients with Vertebrospinal Pathology

Until now, no scientific neurophysiologic methods of diagnostics and treatment of vertebrospinal pathologies were developed. Previous study showed that electrical stimulation of lumbar segments of the spinal cord in animals with complete spinal cord transection induced a well-coordinated weight-bearing locomotion. The present comparative study of motor activity triggered by electrical epidural stimulation of one or two segments of the spinal cord in spinal patients showed that stimulation of lumbar (L2-L4) or sacral (S2) segments facilitated generation of motor patterns of muscle activity. Combination of electrical stimulation with locomotor training resulted in the appearance of stepping patterns characteristic of normal walking and tonic activity of the muscles needed for body balance maintenance.     

The role of cutaneous afferents in controlling locomotion evoked by epidural stimulation of the spinal cord in decerebrate cats

The effects of the cutaneous input on the formation of the locomotor pattern in conditions of epidural stimulation of the spinal cord in decerebrate cats were studied. Locomotor activity was induced by rhythmic stimulation of the dorsal surface of spinal cord segments L4-L5 at a frequency of 3-5 Hz. Electromyograms (EMG) recorded from the antagonist muscles quadriceps, semitendinosus, tibialis anterior, and gastrocnemius lateralis were recorded, along with the kinematics of stepping movements during locomotion on a moving treadmill and reflex responses to single stimuli. Changes in the pattern of reactions observed before and after exclusion of cutaneous receptors (infiltration of lidocaine solution at the base of the paw or irrigation of the paw pads with chlorothane solution) were assessed. This treatment led to impairment of the locomotor cycle: the paw was placed with the rear surface downward and was dragged along in the swing phase, and the duration of the stance phase decreased. Exclusion of cutaneous afferents suppressed the polysynaptic activity of the extensor muscles and the distal flexor muscle of the ipsilateral hindlimb during locomotion evoked by epidural stimulation of the spinal cord. The effects of exclusion of cutaneous afferents on the monosynaptic component of the EMG response were insignificant.     

Recovery of hindlimb locomotion after incomplete spinal cord injury in the cat involves spontaneous compensatory changes within the spinal locomotor circuitry

After incomplete spinal cord injury (SCI), compensatory changes occur throughout the whole neuraxis, including the spinal cord below the lesion, as suggested by previous experiments using a dual SCI paradigm. Indeed, cats submitted to a lateral spinal hemisection at T10-T11 and trained on a treadmill for 3-14 wk re-expressed bilateral hindlimb locomotion as soon as 24 h after spinalization, a process that normally takes 2-3 wk when a complete spinalization is performed without a prior hemisection. In this study, we wanted to ascertain whether similar effects could occur spontaneously without training between the two SCIs and within a short period of 3 wk in 11 cats. One day after the complete spinalization, 9 of the 11 cats were able to re-express hindlimb locomotion either bilaterally (n = 6) or unilaterally on the side of the previous hemisection (n = 3). In these 9 cats, the hindlimb on the side of the previous hemisection (left hindlimb) performed better than the right side in contrast to that observed during the hemispinal period itself. Cats re-expressing the best bilateral hindlimb locomotion after spinalization had the largest initial hemilesion and the most prominent locomotor deficits after this first SCI. These results provide evidence that 1) marked reorganization of the spinal locomotor circuitry can occur without specific locomotor training and within a short period of 3 wk; 2) the spinal cord can reorganize in a more or less symmetrical way; and 3) the ability to walk after spinalization depends on the degree of deficits and adaptation observed in the hemispinal period.     

Axonal projection of descending pathways responsible for eliciting forelimb stepping into the cat cervical spinal cord

Summary The descending pathways responsible for eliciting forelimb stepping are located in the lateral funiculus (Yamaguchi 1986). In order to determine into which spinal segments the descending pathways project and to know the projections and functions of the other descending system, the ventral funicular pathways, we placed various lesions in the cervical spinal cord of decerebrate cats with the lower thoracic cord transected and studied their effects on forelimb stepping evoked by stimulation of the midbrain locomotor region. (1) The lateral funiculus was transected on one side. The operation removes descending input to all the segments caudal to the lesion. Experiments with serial transections from the caudal to rostral segment revealed that stepping activity of the limb on the lesioned side is reduced when the lesion is placed at the level between the C6 and C7 segment and then between C5 and C6. A slight reduction of activity was also observed after a lesion placed between C7 and C8. (2) Consistently, bilateral transection of the lateral funiculus at the level between C5 and C6 abolished stepping movements of both forelimbs. (3) The cervical cord was split in the parasagittal plane through the dorsal root entry. The operation removes the descending input to the segment in which the lesion is placed. The parasagittal lesions from the C1 to C6 did not abolish stepping activity, although a lesion placed between C5 and C6 could slightly affect stepping. The results, (1)–(3) suggest that the lateral funicular pathways project into the spinal segments mainly at the C6–C7 level with some rostrocaudal extension into C5 and C8. (4) Complete transections of the medial part of the spinal cord cut extensor bursts short and raised stepping frequency. Nevertheless, if the lesion at C1–C5 spared the ventromedial part of the ventral funiculus, it did not result in such high-frequency stepping or in weakened extensor activity. In the case of segments caudal to C6, medial transections which spared the corresponding region could result in such stepping. It is suggested that the pathways descending through the ventromedial part of the ventral funiculus in the rostral segments provide extensor activity during stepping. They may change their course in the more dorsal part of the ventral funiculus below the C6 and presumably project into the grey matter of more caudal segments.     

Effects of electrical stimulation of the thoracic spinal cord on bladder and external urethral sphincter activity in the decerebrate cat

Summary Electrical stimulation of the spinal cord above the sacral segments was used to produce coordinated micturition in the paralysed decerebrate cat. Stimulation of the superficial aspect of the dorsolateral funiculus (DLF) within the lower thoracic (T9-T13) segments produced a bladder contraction coordinated with decreased activity in the external urethral sphincter (EUS) branch of the pudendal nerve during which time fluid was expelled. In addition, a similar response was observed with DLF stimulation at the boundary of the L5/L6 segments. At the second cervical spinal segment, however, stimulation of a more lateral and ventral portion of the superficial spinal white matter was the only effective site for producing micturition. The spinal cord-evoked response was comparable to the micturition evoked by electrical stimulation of the pontine micturition centre (PMC) within the brainstem. A bilateral lesion of the dorsal columns (DC) and the dorsolateral funiculi (DLF) at the lower thoracic levels abolished reflex micturition evoked by bladder distension. However stimulation rostral to the lesion, within the PMC or thoracic DLF, continued to produce coordinated bladder and sphincter response during voiding. Stimulation caudal to the lesion produced a decrease in pudendal nerve activity but did not produce a void or bladder pressure change. This reduction in pudendal nerve activity could be abolished with a second lesion of the superficial DLF caudal to the stimulation site. It was concluded that stimulation of the thoracic dorsolateral funiculus activates both ascending and descending fibres which can influence the bladder and/or sphincter muscles. The spinal cordevoked voiding was hypothesized to be due to activation of some portion of the ascending limb of the spinobulbospinal micturition reflex loop. The decreased activity produced by stimulation of the thoracic DLF caudal to a bilateral DC/DLF subtotal cord lesion may be mediated by fibres descending in the dorsolateral funiculus. The possibility that the spinal cord stimulation antidromically activated axons of neurons having segmental collaterals capable of influencing pudendal neural activity cannot be exclused at this time.     

Low micromolar concentrations of 4-aminopyridine facilitate fictive locomotion expressed by the rat spinal cord in vitro

Upregulating the operation of spinal locomotor networks is one mechanism to restore, at least partially, lesion-impaired locomotion. We investigated if the K+ channel blocker 4-aminopyridine (4-AP) could facilitate spinal locomotor networks in addition to its well-known effect on motor nerve conduction. Fictive locomotor patterns were recorded from ventral roots (VRs) of the isolated spinal cord of the neonatal rat. 4-AP (0.1-50 microM) produced synchronous VR oscillations which did not develop into fictive locomotion. These oscillations had network origin, required intact glutamatergic transmission and were probably amplified via electrotonic coupling because of their depression by the selective gap junction blocker carbenoxolone. 4-AP (5 microM) slightly increased input resistance of lumbar motoneurons without affecting their action or resting potentials. Dorsal root (DR) evoked synaptic responses were enhanced (217 +/- 65%) by 5 microM 4-AP without changes in axon conduction. 4-AP (5 microM) accelerated fictive locomotion induced by N-methyl-d-aspartate (NMDA) and serotonin (5-HT) without altering cycle amplitude and facilitated the onset of fictive locomotion in the presence of sub-threshold concentrations of NMDA and 5-HT. Furthermore, in the presence of 4-AP, weak DR stimuli, previously insufficient to activate locomotor patterns, generated alternating VR discharges. Thus, although 4-AP per se could not directly activate the locomotor network of the spinal cord, it could strongly facilitate the locomotor program initiated by neurochemicals or electrical stimuli. These data suggest that the reported improvement by 4-AP in locomotor activity of spinal-injury patients may include activation of locomotor networks when low concentrations of this drug are administered in coincidence with appropriate stimuli.     

Characteristics of the electrical oscillations evoked by 4-aminopyridine on dorsal root fibers and their relation to fictive locomotor patterns in the rat spinal cord in vitro

4-Aminopyridine (4-AP) is suggested to improve symptomatology of spinal injury patients because it may facilitate neuromuscular transmission, spinal impulse flow and the operation of the locomotor central pattern generator (CPG). Since 4-AP can also induce repetitive discharges from dorsal root afferents, this phenomenon might interfere with sensory signals necessary to modulate CPG activity. Using electrophysiological recording from dorsal and ventral roots of the rat isolated spinal cord, we investigated 4-AP-evoked discharges and their relation with fictive locomotor patterns. On dorsal roots 4-AP (5-10 microM) induced sustained synchronous oscillations (3.3+/-0.8 s period) smaller than electrically evoked synaptic potentials, persistent after sectioning off the ventral region and preserved in an isolated dorsal quadrant, indicating their dorsal horn origin. 4-AP oscillations were blocked by tetrodotoxin, or 6-cyano-7-nitroquinoxaline-2,3-dione and d-amino-phosphonovalerate, or strychnine and bicuculline, suggesting they were network mediated via glutamatergic, glycinergic and GABAergic transmission. Isolated ventral horn areas could not generated 4-AP oscillations, although their intrinsic disinhibited bursting was accelerated by 4-AP. Thus, ventral horn areas contained 4-AP sensitive sites, yet lacked the network for 4-AP induced oscillations. Activation of fictive locomotion by either application of N-methyl-D-aspartate and serotonin or stimulus trains to a single dorsal root reversibly suppressed dorsal root oscillations induced by 4-AP. This suppression was due to depression of dorsal network activity rather than simple block of root discharges. Since dorsal root oscillations evoked by 4-AP were turned off when the fictive locomotor program was initiated, these discharges are unlikely to interfere with proprioceptive signals during locomotor training in spinal patients.     

Origin of signals conveyed by the ventral spino-cerebellar tract and spino-reticulo-cerebellar pathway

The "fictitious" scratch reflex was evoked in decerebrate curarized cats by pinna stimulation. Activity of neurons of the ventral spino-cerebellar tract ( VSCT ) from the L4 and L5 segments of the spinal cord as well as of neurons of the spino-reticulo-cerebellar pathway ( SRCP ) from the lateral reticular nucleus of the medulla oblongata was recorded. Cooling and destruction of different parts of the lumbo-sacral enlargement of the spinal cord were performed. Cooling of the L5 or L6 segment abolished the rhythmic activity in the greater part of the spinal hindlimb centre but did not affect the generation of rhythmic oscillations in the remaining (rostral) segments of the lumbo-sacral enlargement. Under these conditions, neither the rhythmic activity of VSCT neurons located rostral to the thermode nor that of SRCP neurons changed. A normal rhythmic activity of SRCP neurons also persisted after destruction of grey matter in the L3 and L4 segments. It can be concluded that activity of these neurons is independent of whichever part of the enlargement generates rhythmic oscillations. From these observations a hypothesis is advanced that the main content of signals conveyed by the VSCT and SRCP to the cerebellum is the information regarding activity of the generator of rhythmic oscillations that is located in the L3-L5 spinal segments.     

Comparison of the effect of intrathecal administration of clonidine and yohimbine on the locomotion of intact and spinal cats

Several studies have shown that noradrenergic mechanisms are important for locomotion. For instance, L-dihydroxyphenylalanine (L-DOPA) can initiate "fictive" locomotion in immobilized acutely spinalized cats and alpha(2)-noradrenergic agonists, such as 2,6,-dichloro-N-2-imidazolidinylid-enebenzenamine (clonidine), can induce treadmill locomotion soon after spinalization. However, the activation of noradrenergic receptors may be not essential for the basic locomotor rhythmicity because chronic spinal cats can walk with the hindlimbs on a treadmill in the absence of noradrenergic stimulation because the descending pathways are completely severed. This suggests that locomotion, in intact and spinal conditions, is probably expressed and controlled through different neurotransmitter mechanisms. To test this hypothesis, we compared the effect of the alpha(2) agonist, clonidine, and the antagonist (16 alpha, 17 alpha)-17-hydroxy yohimbine-16-carboxylic acid methyl ester hydrochloride (yohimbine), injected intrathecally at L(3)--L(4) before and after spinalization in the same cats chronically implanted with electrodes to record electromyograms (EMGs). In intact cats, clonidine (50-150 microg/100 microl) modulated the locomotor pattern slightly causing a decrease in duration of the step cycle accompanied with some variation of EMG burst amplitude and duration. In the spinal state, clonidine could trigger robust and sustained hind limb locomotion in the first week after the spinalization at a time when the cats were paraplegic. Later, after the spontaneous recovery of a stable locomotor pattern, clonidine prolonged the cycle duration, increased the amplitude and duration of flexor and extensor bursts, and augmented the foot drag at the onset of swing. In intact cats, yohimbine at high doses (800--1600 microg/100 microl) caused major walking difficulties characterized by asymmetric stepping, stumbling with poor lateral stability, and, at smaller doses (400 microg/100 microl), only had slight effects such as abduction of one of the hindlimbs and the turning of the hindquarters to one side. After spinalization, yohimbine had no effect even at the largest doses. These results indicate that, in the intact state, noradrenergic mechanisms probably play an important role in the control of locomotion since blocking the receptors results in a marked disruption of walking. In the spinal state, although the receptors are still present and functional since they can be activated by clonidine, they are seemingly not critical for the spontaneous expression of spinal locomotion since their blockade by yohimbine does not impair spinal locomotion. It is postulated therefore that the expression of spinal locomotion must depend on the activation of other types of receptors, probably related to excitatory amino acids.     

Properties of rhythmic activity generated by the isolated spinal cord of the neonatal mouse

We examined the ability of the isolated lumbosacral spinal cord of the neonatal mouse (P0-7) to generate rhythmic motor activity under several different conditions. In the absence of electrical or pharmacological stimulation, we recorded several patterns of spontaneous ventral root depolarization and discharge. Spontaneous, alternating discharge between contralateral ventral roots could occur two to three times over a 10-min interval. We also observed other patterns, including left-right synchrony and rhythmic activity restricted to one side of the cord. Trains of stimuli delivered to the lumbar/coccygeal dorsal roots or the sural nerve reliably evoked episodes of rhythmic activity. During these evoked episodes, rhythmic ventral root discharges could occur on one side of the cord or could alternate from side to side. Bath application of a combination of N-methyl-D,L-aspartate (NMA), serotonin, and dopamine produced rhythmic activity that could last for several hours. Under these conditions, the discharge recorded from the left and right L(1)-L(3) ventral roots alternated. In the L(4)-L(5) segments, the discharge had two peaks in each cycle, coincident with discharge of the ipsilateral and contralateral L(1)-L(3) roots. The L(6) ventral root discharge alternated with that recorded from the ipsilateral L(1)-L(3) roots. We established that the drug-induced rhythm was locomotor-like by recording an alternating pattern of discharge between ipsilateral flexor and extensor hindlimb muscle nerves. In addition, by recording simultaneously from ventral roots and muscle nerves, we established that ankle flexor discharge was in phase with ipsilateral L(1)/L(2) ventral root discharge, while extensor discharge was in phase with ipsilateral L(6) ventral root discharge. Rhythmic patterns of ventral root discharge were preserved following mid-sagittal section of the spinal cord, demonstrating that reciprocal inhibitory connections between the left and right sides of the cord are not essential for rhythmogenesis in the neonatal mouse cord. Blocking N-methyl-D-aspartate receptors, in both the intact and the hemisected preparation, revealed that these receptors contribute to but are not essential for rhythmogenesis. In contrast, the rhythm was abolished following blockade of kainate/AMPA receptors with 6-cyano-7-nitroquinoxalene-2,3-dione. These findings demonstrate that the isolated mouse spinal cord can produce a variety of coordinated activities, including locomotor-like activity. The ability to study these behaviors under a variety of different conditions offers promise for future studies of rhythmogenic mechanisms in this preparation.