Search

Locomotor patterns are mainly modulated by afferent feedback, but its actual contribution to spinal network activity during continuous passive limb training is still unexplored. To unveil this issue, we devised a robotic in vitro setup (Bipedal Induced Kinetic Exercise, BIKE) to induce passive pedaling, while simultaneously recording low-noise ventral and dorsal root (VR and DR) potentials in isolated neonatal rat spinal cords with hindlimbs attached. As a result, BIKE evoked rhythmic afferent volleys from DRs, reminiscent of pedaling speed. During BIKE, spontaneous VR activity remained unchanged, while a DR rhythmic component paired the pedaling pace. Moreover, BIKE onset rarely elicited brief episodes of fictive locomotion (FL) and, when trains of electrical pulses were simultaneously applied to a DR, it increased the amplitude, but not the number, of FL cycles. When BIKE was switched off after a 30-min training, the number of electrically induced FL oscillations was transitorily facilitated, without affecting VR reflexes or DR potentials. However, 90 min of BIKE no longer facilitated FL, but strongly depressed area of VR reflexes and stably increased antidromic DR discharges. Patch clamp recordings from single motoneurons after 90-min sessions indicated an increased frequency of both fast- and slow-decaying synaptic input to motoneurons. In conclusion, hindlimb rhythmic and alternated pedaling for different durations affects distinct dorsal and ventral spinal networks by modulating excitatory and inhibitory input to motoneurons. These results suggest defining new parameters for effective neurorehabilitation that better exploits spinal circuit activity., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Locomotion is one of the most complex motor behaviors. Locomotor patterns change during early life, reflecting development of numerous peripheral and hierarchically organized central structures. Among them, the spinal cord is of particular interest since it houses the central pattern generator (CPG) for locomotion. This main command center is capable of eliciting and coordinating complex series of rhythmic neural signals sent to motoneurons and to corresponding target-muscles for basic locomotor activity. For a long-time, the CPG has been considered a black box. In recent years, complementary insights from in vitro and in vivo animal models have contributed significantly to a better understanding of its constituents, properties and ways to recover locomotion after a spinal cord injury (SCI). This review discusses key findings made by comparing the results of in vitro isolated spinal cord preparations and spinal-transected in vivo models from neonatal animals. Pharmacological, electrical, and sensory stimulation approaches largely used to further understand CPG function may also soon become therapeutic tools for potent CPG reactivation and locomotor movement induction in persons with SCI or developmental neuromuscular disorder., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Objectives: Explore the primary characteristics of afferent noisy stimuli, which optimally activate locomotor patterns at low intensity., Materials and Methods: Intracellular and extracellular electrophysiological traces were derived from single motoneurons and from ventral roots, respectively. From these recordings, we obtained noisy stimulating protocols, delivered to a dorsal root (DR) of an isolated neonatal rat spinal cord, while recording fictive locomotion (FL) from ventral roots., Results: We decreased complexity of efficient noisy stimulating protocols down to single cell spikes. Then, we identified four main components within the power spectrum of these signals and used them to construct a basic multifrequency protocol of rectangular impulses, able to induce FL. Further disassembling generated the minimum stimulation paradigm that activated FL, which consisted of a pair of 35 and 172 Hz frequency pulse trains, strongly effective at low intensity when delivered either jointly to one lumbosacral DR or as single simultaneous trains to two distinct DRs. This simplified pulse schedule always activated a locomotor rhythm, even when delivered for a very short time (500 ms). One prerequisite for the two-frequency protocol to activate FL at low intensity when applied to sacrocaudal afferents was the ability to induce ascending volleys of greater amplitude., Conclusion: Multifrequency protocols can support future studies in defining the most effective characteristics for electrical stimulation to reactivate stepping following motor injury., (© 2016 International Neuromodulation Society.)

Study Design: Experimental animal study., Objectives: Epidural stimulation has been used to activate locomotor patterns after spinal injury and typically employs synchronous trains of high-frequency stimuli delivered directly to the dorsal cord, thereby recruiting multiple afferent nerve roots. Here we investigate how spinal locomotor networks integrate multi-site afferent input and address whether frequency coding is more important than amplitude to activate locomotor patterns., Setting: Italy and Belgium., Methods: To investigate the importance of input intensity and frequency in eliciting locomotor activity, we used isolated neonatal rat spinal cords to record episodes of fictive locomotion (FL) induced by electrical stimulation of single and multiple dorsal roots (DRs), employing different stimulating protocols., Results: FL was efficiently induced through staggered delivery (delays 0.5 to 2 s) of low-frequency pulse trains (0.33 and 0.67 Hz) to three DRs at intensities sufficient to activate ventral root reflexes. Delivery of the same trains to a single DR or synchronously to multiple DRs remained ineffective. Multi-site staggered trains were more efficient than randomized pulse delivery. Weak trains simultaneously delivered to DRs failed to elicit FL. Locomotor rhythm resetting occurred with single pulses applied to various distant DRs., Conclusion: Electrical stimulation recruited spinal networks that generate locomotor programs when pulses were delivered to multiple sites at low frequency. This finding might help devising new protocols to optimize the increasingly more common use of epidural implantable arrays to treat spinal dysfunctions.

A new stimulating protocol [fictive locomotion-induced stimulation (FListim)], consisting of intrinsically variable weak waveforms applied to a single dorsal root is very effective (though not optimal as it eventually wanes away) in activating the locomotor program of the isolated rat spinal cord. The present study explored whether combination of FListim with low doses of pharmacological agents that raise network excitability might further improve the functional outcome, using this in vitro model. FListim was applied together with N-methyl-d-aspartate (NMDA) + serotonin, while fictive locomotion (FL) was electrophysiologically recorded from lumbar ventral roots. Superimposing FListim on FL evoked by these neurochemicals persistently accelerated locomotor-like cycles to a set periodicity and modulated cycle amplitude depending on FListim rate. Trains of stereotyped rectangular pulses failed to replicate this phenomenon. The GABA(B) agonist baclofen dose dependently inhibited, in a reversible fashion, FL evoked by either FListim or square pulses. Sustained episodes of FL emerged when FListim was delivered, at an intensity subthreshold for FL, in conjunction with subthreshold pharmacological stimulation. Such an effect was, however, not found when high potassium solution instead of NMDA + serotonin was used. These results suggest that the combined action of subthreshold FListim (e.g., via epidural stimulation) and neurochemicals should be tested in vivo to improve locomotor rehabilitation after injury. In fact, reactivation of spinal locomotor circuits by conventional electrical stimulation of afferent fibers is difficult, while pharmacological activation of spinal networks is clinically impracticable due to concurrent unwanted effects. We speculate that associating subthreshold chemical and electrical inputs might decrease side effects when attempting to evoke human locomotor patterns.

It is not well-studied how the ubiquitous neuromodulator adenosine (ADO) affects mammalian locomotor network activities. We analyzed this here with focus on roles of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)-sensitive A(1)-type ADO receptors. For this, we recorded field potentials from ventral lumbar nerve roots and electrically stimulated dorsal roots in isolated newborn rat spinal cords. At ≥ 25μM, bath-applied ADO slowed synchronous bursting upon blockade of anion-channel-mediated synaptic inhibition by bicuculline (20 μM) plus strychnine (1 μM) and this depression was countered by DPCPX (1 μM) as tested at 100 μM ADO. ADO abolished this disinhibited rhythm at ≥ 500 μM. Contrary, the single electrical pulse-evoked dorsal root reflex, which was enhanced in bicuculline/strychnine-containing solution, persisted at all ADO doses (5 μM-2 mM). In control solution, ≥ 500 μM ADO depressed this reflex and pulse train-evoked bouts of alternating fictive locomotion; this inhibition was reversed by 1 μM DPCPX. ADO (5 μM-2 mM) did not depress, but stabilize alternating fictive locomotion evoked by serotonin (10 μM) plus N-methyl-d-aspartate (4-5 μM). Addition of DPCPX (1μM) to control solution did not change either the dorsal root reflex or rhythmic activities indicating lack of endogenous A(1) receptor activity. Our findings show A(1) receptor involvement in ADO depression of the dorsal root reflex, electrically evoked fictive locomotion and spontaneous disinhibited lumbar motor bursting. Contrary, chemically evoked fictive locomotion and the enhanced dorsal root reflex in disinhibited lumbar locomotor networks are resistant to ADO. Because ADO effects in standard solution occurred at doses that are notably higher than those occurring in vivo, we hypothesize that newborn rat locomotor networks are rather insensitive to this neuromodulator., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Although spinal injury is a major cause of chronic disability, the mechanisms responsible for the lesion pathophysiology and their dynamic evolution remain poorly understood. Hence, current treatments aimed at blocking damage extension are unsatisfactory. To unravel the acute spinal injury processes, we have developed a model of the neonatal rat spinal cord in vitro subjected to kainate-evoked excitotoxicity or metabolic perturbation (hypoxia, aglycemia, and free oxygen radicals) or their combination. The study outcome is fictive locomotion one day after the lesion and its relation to histological damage. Excitotoxicity always suppresses locomotor network activity and produces large gray matter damage, while network bursting persists supported by average survival of nearly half premotoneurons and motoneurons. Conversely, metabolic perturbation simply depresses locomotor network activity as damage mainly concerns white rather than gray matter. Coapplication of kainate and metabolic perturbation completely eliminates locomotor network activity. These results indicate distinct cellular targets for excitotoxic versus dysmetabolic damage with differential consequences on locomotor pattern formation. Furthermore, these data enable to estimate the minimal network membership compatible with expression of locomotor activity.

Acute spinal cord injury evolves rapidly to produce secondary damage even to initially spared areas. The result is loss of locomotion, rarely reversible in man. It is, therefore, important to understand the early pathophysiological processes which affect spinal locomotor networks. Regardless of their etiology, spinal lesions are believed to include combinatorial effects of excitotoxicity and severe stroke-like metabolic perturbations. To clarify the relative contribution by excitotoxicity and toxic metabolites to dysfunction of locomotor networks, spinal reflexes and intrinsic network rhythmicity, we used, as a model, the in vitro thoraco-lumbar spinal cord of the neonatal rat treated (1 h) with either kainate or a pathological medium (containing free radicals and hypoxic/aglycemic conditions), or their combination. After washout, electrophysiological responses were monitored for 24 h and cell damage analyzed histologically. Kainate suppressed fictive locomotion irreversibly, while it reversibly blocked neuronal excitability and intrinsic bursting induced by synaptic inhibition block. This result was associated with significant neuronal loss around the central canal. Combining kainate with the pathological medium evoked extensive, irreversible damage to the spinal cord. The pathological medium alone slowed down fictive locomotion and intrinsic bursting: these oscillatory patterns remained throughout without regaining their control properties. This phenomenon was associated with polysynaptic reflex depression and preferential damage to glial cells, while neurons were comparatively spared. Our model suggests distinct roles of excitotoxicity and metabolic dysfunction in the acute damage of locomotor networks, indicating that different strategies might be necessary to treat the various early components of acute spinal cord lesion.

Intrinsic spinal networks generate a locomotor rhythm characterized by alternating electrical discharges from flexor and extensor motor pools. Because this process is preserved in the rat isolated spinal cord, this preparation in vitro may be a useful model to explore methods to reactivate locomotor networks damaged by spinal injury. The present electrophysiological investigation examined whether the broad spectrum potassium channel blocker tetraethylammonium could generate locomotor-like patterns. Low (50-500 microM) concentrations of tetraethylammonium induced irregular, synchronous discharges incompatible with locomotion. Higher concentrations (1-10 mM) evoked alternating discharges between flexor and extensor motor pools, plus large depolarization of motoneurons with spike broadening. The alternating discharges were superimposed on slow, shallow waves of synchronous depolarization. Rhythmic alternating patterns were suppressed by blockers of glutamate, GABA(A) and glycine receptors, disclosing a background of depolarizing bursts inhibited by antagonism of group I metabotropic glutamate receptors. Furthermore, tetraethylammonium also evoked irregular discharges on dorsal roots. Rhythmic alternating patterns elicited by tetraethylammonium on ventral roots were relatively stereotypic, had limited synergy with fictive locomotion induced by dorsal root stimuli, and were not accelerated by 4-aminopyridine. Horizontal section of the spinal cord preserved irregular ventral root discharges and dorsal root discharges, demonstrating that the action of tetraethylammonium on spinal networks was fundamentally different from that of 4-aminopyridine. These results show that a potassium channel blocker such as tetraethylammonium could activate fictive locomotion in the rat isolated spinal cord, although the pattern quality lacked certain features like frequency modulation and strong synergy with other inputs to locomotor networks.

The mammalian thoracolumbar spinal cord contains all the necessary elements to generate a rhythmic oscillatory activity that is transformed into locomotor commands to agonist and antagonist limb muscles to produce gait at various speed. This motor program is produced by interneurons in the ventral horn and can be readily recorded even with in vitro spinal cord preparations isolated from rats or mice (once dorsal afferents are stimulated or excitatory neuronchemicals applied). The locomotor program is continuously modulated and refined by afferent sensory inputs and by signals descending from brain centers. Nevertheless, this program is not the only type of rhythmic discharge produced by spinal networks. In fact, activation of metabotropic group I glutamate receptors or block of certain K+ currents by 4-aminopyridine generates non-locomotor discharges, and, at the same time, facilitates evoked locomotor activity, which then suppresses any other interfering rhythmicity. These findings suggest that accessory networks, activated by suitable stimuli, might be exploited to restore locomotor activity damaged by a lesion, an obvious goal for neuro-rehabilitation purposes. The structure of the locomotor networks appears to include a rhythm-generating circuit that drives a pattern formation circuit, commanding motoneurons to discharge appropriate signals to skeletal muscles. Studies with the K+-channel blocker tetraethylammonium have indicated that this hierarchical arrangement is preserved in vitro. Hence, isolated spinal cord preparations represent an interesting experimental tool to investigate new mechanisms to upregulate various components of locomotor networks, especially after the induction of experimental lesions.

Recently the K+ channel blocker 4-aminopyridine (4-AP) has been suggested to be useful to improve motor deficits due to spinal cord lesions. There is, however, little basic research support for this action of 4-AP. In this study we have used as a model the neonatal mammalian spinal cord in vitro that generates a rhythmic activity termed fictive locomotion (induced by bath-application of NMDA + 5-HT) with phasic electrical discharges alternating between flexor and extensor motor pools and between left and right motoneurons within the same segment. When 4-AP was added in the presence of sub-threshold concentrations of NMDA + 5-HT, there was facilitation of fictive locomotion which appeared with alternating patterns on all recorded ventral roots (VR). Furthermore, in the presence of 4-AP, weak dorsal root (DR) stimuli, previously insufficient to activate locomotor patterns, generated alternating discharges from various VRs. The present data show that 4-AP could strongly facilitate the locomotor program initiated by neurochemicals or electrical stimuli, indicating that the spinal locomotor network is a very sensitive target for the action of 4-AP.

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., (Copyright 2004 IBRO)