Modulation of the activity of the locomotor central pattern generator in the rat spinal cord in vitro

The present study has investigated the rhythmic properties of spinal networks in the neonatal rat spinal cord in vitro, by means of intracellular recordings from single motoneurons (MNs) and extracellular recordings from ventral and dorsal roots (VRs;DRs). Distinct subclasses of metabotropic glutamate receptors (mGluRs) on rat spinal neurons mediated complex facilitatory and inhibitory effects. The class I agonist DHPG evoked MN depolarization (via the mGluR1 subtype) mostly at network level and generated sustained, network-dependent oscillations (via the mGluR5 subtype). DHPG also decreased the amplitude of reflex responses induced by DR stimuli, an effect unrelated to depolarization but dependent on glycinergic transmission. Single reflex responses were insensitive to group I mGluRs antagonists, suggesting no phasic activation of group I receptors during this process. Finally, DHPG depressed the glycinergic recurrent IPSP, perhaps by impairing the cholinergic input to Renshaw cells. Thus, the cellular distribution of those mGluRs at strategic circuit connections may determine the functional outcome of the network in terms of excitation or inhibition. Activation of class II or III mGluRs had no direct action on MNs although it strongly blocked evoked synaptic transmission, presumably acting at presynaptic level. To extend our understanding of the network-based properties, which enable a neuronal circuit to produce sustained electrical oscillations, we explored the potential contribution of mGluRs to generate rhythmic discharges. During cumulative depolarization or fictive locomotion, spinal mGluRs were minimally activated by endogenous glutamate, although they could potently modulate these responses once activated by exogenously applied mGluR agonists. Disinhibited bursting was associated with the activation of mGluR1 receptors (facilitating network excitability) and of group II mGluRs (depressing it). 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. 4-AP 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. 4-AP slightly increased input resistance of lumbar MNs, without affecting their action or resting potentials. DR evoked synaptic responses were enhanced by 4-AP without changes in axon conduction. 4-AP accelerated chemically or electrically induced fictive locomotion and facilitated the onset of fictive locomotion in the presence of subthreshold stimuli, that were previously insufficient to activate locomotor patterns. 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. On DRs, 4-AP induced sustained synchronous oscillations 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 network mediated via glutamatergic, glycinergic and GABAergic transmission. Isolated ventral horn areas could not generate 4-AP oscillations, although their intrinsic, disinhibited bursting was accelerated by the substance. Activation of fictive locomotion by either application of neurochmicals or stimulus trains to a single DR reversibly suppressed DR oscillations induced by 4-AP. The present electrophysiological investigation also examined whether the broad spectrum potassium channel blocker tetraethylammonium (TEA) could generate locomotor-like patterns. Low concentrations of TEA induced irregular, synchronous discharges incompatible with locomotion. Higher concentrations evoked alternating discharges between flexor and extensor motor pools, plus a large depolarization of MNs with spike broadening. The alternating discharges were superimposed on slow, shallow waves of synchronous depolarization. Rhythmic alternating patterns were suppressed by blockers of glutamate, GABAA and glycine receptors, disclosing a background of depolarizing bursts inhibited by antagonism of group I mGluRs. Furthermore, TEA also evoked irregular discharges on DRs. The rhythmic alternating patterns elicited by TEA on VRs were relatively stereotypic, had limited synergy with the fictive locomotion induced by DR stimuli, and were not accelerated by 4-AP. Horizontal section of the spinal cord preserved irregular VR discharges and DR discharges, demonstrating that the action of TEA on spinal networks was fundamentally different from that of 4-AP

Afferent Input Induced by Rhythmic Limb Movement Modulates Spinal Neuronal Circuits in an Innovative Robotic In Vitro Preparation

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

Multilevel Analysis of Locomotion in Immature Preparations Suggests Innovative Strategies to Reactivate Stepping after Spinal Cord Injury

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

GABAergic Mechanisms Can Redress the Tilted Balance between Excitation and Inhibition in Damaged Spinal Networks

Correct operation of neuronal networks depends on the interplay between synaptic excitation and inhibition processes leading to a dynamic state termed balanced network. In the spinal cord, balanced network activity is fundamental for the expression of locomotor patterns necessary for rhythmic activation of limb extensor and flexor muscles. After spinal cord lesion, paralysis ensues often followed by spasticity. These conditions imply that, below the damaged site, the state of balanced networks has been disrupted and that restoration might be attempted by modulating the excitability of sublesional spinal neurons. Because of the widespread expression of inhibitory GABAergic neurons in the spinal cord, their role in the early and late phases of spinal cord injury deserves full attention. Thus, an early surge in extracellular GABA might be involved in the onset of spinal shock while a relative deficit of GABAergic mechanisms may be a contributor to spasticity. We discuss the role of GABA A receptors at synaptic and extrasynaptic level to modulate network excitability and to offer a pharmacological target for symptom control. In particular, it is proposed that activation of GABA A receptors with synthetic GABA agonists may downregulate motoneuron hyperexcitability (due to enhanced persistent ionic currents) and, therefore, diminish spasticity. This approach might constitute a complementary strategy to regulate network excitability after injury so that reconstruction of damaged spinal networks with new materials or cell transplants might proceed more successfully

ERG conductance expression modulates the excitability of ventral horn GABAergic interneurons that control rhythmic oscillations in the developing mouse spinal cord

During antenatal development, the operation and maturation of mammalian spinal networks strongly depend on the activity of ventral horn GABAergic interneurons that mediate excitation first and inhibition later. Although the functional consequence of GABA actions may depend on maturational processes in target neurons, it is also likely that evolving changes in GABAergic transmission require fine-tuning in GABA release, probably via certain intrinsic mechanisms regulating GABAergic neuron excitability at different embryonic stages. Nevertheless, it has not been possible, to date, to identify certain ionic conductances upregulated or downregulated before birth in such cells. By using an experimental model with either mouse organotypic spinal cultures or isolated spinal cord preparations, the present study examined the role of the ERG current (IK(ERG) ), a potassium conductance expressed by developing, GABA-immunoreactive spinal neurons. In organotypic cultures, only ventral interneurons with fast adaptation and GABA immunoreactivity, and only after 1 week in culture, were transformed into high-frequency bursters by E4031, a selective inhibitor of IK(ERG) that also prolonged and made more regular spontaneous bursts. In the isolated spinal cord in which GABA immunoreactivity and m-erg mRNA were colocalized in interneurons, ventral root rhythms evoked by NMDA plus 5-hydroxytryptamine were stabilized and synchronized by E4031. All of these effects were lost after 2 weeks in culture or before birth in coincidence with decreased m-erg expression. These data suggest that, during an early stage of spinal cord development, the excitability of GABAergic ventral interneurons important for circuit maturation depended, at least in part, on the function of IK(ERG)

Selective antagonism of A1 adenosinergic receptors strengthens the neuromodulation of sensorimotor network during epidural spinal stimulation

Although epidural spinal stimulation (ESS) results in promising therapeutic effects in individuals with spinal cord injury (SCI), its potential to generate functional motor recovery varies between individuals and remains largely unclear. However, both preclinical and clinical studies indicate the capacity of electrical and pharmacological interventions to synergistically increase engagement of spinal sensorimotor networks and regain motor function after SCI. This study explored whether selective pharmacological antagonism of the adenosine A1 receptor subtype synergizes with ESS, thereby increasing motor response. We hypothesized that selective pharmacological antagonism of A1 receptors during ESS would produce facilitatory effects in spinal sensorimotor networks detected as an increased amplitude of spinally-evoked motor potentials and sustained duration of ESS induced activity. Terminal experiments were performed in adult rats using trains of stereotyped pulses at 40 Hz delivered at L5 with local administration to the cord of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). We demonstrated that ESS combined with the blockage of A1 receptors increased the magnitude of the endogenous modulation and postponed the decay of responses that occur during ESS alone. Although DPCPX significantly increased the yield of repetitive stimulation in intact spinal cords, effects of A1 antagonism on motor evoked responses after an acute spinal transection were not detected. These studies support future investigation of the optimal dosage, methods of delivery, and systemic effects of the synergistic application of A1 antagonists and spinal stimulation in intact and injured spinal cord

Nanomolar oxytocin synergizes with weak electrical afferent stimulation to activate the locomotor CPG of the rat spinal cord in vitro.

Synergizing the effect of afferent fibre stimulation with pharmacological interventions is a desirable goal to trigger spinal locomotor activity, especially after injury. Thus, to better understand the mechanisms to optimize this process, we studied the role of the neuropeptide oxytocin (previously shown to stimulate locomotor networks) on network and motoneuron properties using the isolated neonatal rat spinal cord. On motoneurons oxytocin (1 nM-1 \u3bcM) generated sporadic bursts with superimposed firing and dose-dependent depolarization. No desensitization was observed despite repeated applications. Tetrodotoxin completely blocked the effects of oxytocin, demonstrating the network origin of the responses. Recording motoneuron pool activity from lumbar ventral roots showed oxytocin mediated depolarization with synchronous bursts, and depression of reflex responses in a stimulus and peptide-concentration dependent fashion. Disinhibited bursting caused by strychnine and bicuculline was accelerated by oxytocin whose action was blocked by the oxytocin antagonist atosiban. Fictive locomotion appeared when subthreshold concentrations of NMDA plus 5HT were coapplied with oxytocin, an effect prevented after 24 h incubation with the inhibitor of 5HT synthesis, PCPA. When fictive locomotion was fully manifested, oxytocin did not change periodicity, although cycle amplitude became smaller. A novel protocol of electrical stimulation based on noisy waveforms and applied to one dorsal root evoked stereotypic fictive locomotion. Whenever the stimulus intensity was subthreshold, low doses of oxytocin triggered fictive locomotion although oxytocin per se did not affect primary afferent depolarization evoked by dorsal root pulses. Among the several functional targets for the action of oxytocin at lumbar spinal cord level, the present results highlight how small concentrations of this peptide could bring spinal networks to threshold for fictive locomotion in combination with other protocols, and delineate the use of oxytocin to strengthen the efficiency of electrical stimulation to activate locomotor circuits

Neuromodulation of the neural circuits controlling the lower urinary tract

The inability to control timely bladder emptying is one of the most serious challenges among the many functional deficits that occur after a spinal cord injury. We previously demonstrated that electrodes placed epidurally on the dorsum of the spinal cord can be used in animals and humans to recover postural and locomotor function after complete paralysis and can be used to enable voiding in spinal rats. In the present study, we examined the neuromodulation of lower urinary tract function associated with acute epidural spinal cord stimulation, locomotion, and peripheral nerve stimulation in adult rats. Herein we demonstrate that electrically evoked potentials in the hindlimb muscles and external urethral sphincter are modulated uniquely when the rat is stepping bipedally and not voiding, immediately pre-voiding, or when voiding. We also show that spinal cord stimulation can effectively neuromodulate the lower urinary tract via frequency-dependent stimulation patterns and that neural peripheral nerve stimulation can activate the external urethral sphincter both directly and via relays in the spinal cord. The data demonstrate that the sensorimotor networks controlling bladder and locomotion are highly integrated neurophysiologically and behaviorally and demonstrate how these two functions are modulated by sensory input from the tibial and pudental nerves. A more detailed understanding of the high level of interaction between these networks could lead to the integration of multiple neurophysiological strategies to improve bladder function. These data suggest that the development of strategies to improve bladder function should simultaneously engage these highly integrated networks in an activity-dependent manner

Fictive locomotor patterns generated by tetraethylammonium application to the neonatal rat spinal cord in vitro

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\u2013 500 \ub5M) concentrations of tetraethylammonium induced irregular, synchronous discharges incompatible with locomotion. Higher concentrations (1\u201310 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, GABAA 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