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Abstract: Increasing experimental and clinical evidence suggests that abnormal glutamate transmission might play a major role in a vast number of neurological disorders. As a measure of glutamatergic excitation, we have studied the acetylcholine (ACh) release induced by N-methyl-d-aspartate (NMDA) receptor stimulation in primary cultured rat ventral horn spinal neurons and we have evaluated the possibility to limit the consequences of the hyperactivation of glutamatergic receptors, by recruiting the inhibitory transmission mediated by GABA and glycine. For this purpose, we have exposed cell cultures, previously loaded with [3H]choline, to NMDA, which increased the spontaneous tritium efflux in a concentration-dependent manner. Tritium release is dependent upon external Ca2+, tetrodotoxin, Cd2+ ions and ω-conotoxin GVIA, but not on ω-conotoxin MVIIC nor nifedipine, suggesting the involvement of N-type voltage-sensitive calcium channels. NMDA-mediated [3H]ACh release was completely prevented by MK-801, 5,7-diclorokynurenic acid and ifenprodil, while it was strongly inhibited by a lower external pH, suggesting that the involved NMDA receptors contain NR1 and NR2B subunits. Muscimol inhibited NMDA-evoked [3H]ACh release and its effect was antagonized by SR95531 and potentiated by diazepam, indicating the involvement of benzodiazepine-sensitive GABAA receptors. Also glycine, via strychnine-sensitive receptors, inhibited the effect of NMDA. It is concluded that glutamate acts on the NMDA receptors situated on spinal motoneurons to evoke ACh release, which can be inhibited through the activation of GABAA and glycine receptors present on the same neurons. These data suggest that glutamatergic overload of receptors located onto spinal cord motoneurons might be decreased by activating GABAA and glycine receptors. [Copyright &y& Elsevier]

Highly varying patterns of electrostimulation (Dynamic Stimulation, DS) delivered to the dorsal cord through an epidural array with 18 independent electrodes transiently facilitate corticospinal motor responses, even after spinal injury. To partly unravel how corticospinal input are affected by DS, we introduced a corticospinal platform that allows selective cortical stimulation during the multisite acquisition of cord dorsum potentials (CDPs) and the simultaneous supply of DS. Firstly, the epidural interface was validated by the acquisition of the classical multisite distribution of CDPs and their input-output profile elicited by pulses delivered to peripheral nerves. Apart from increased EMGs, DS selectively increased excitability of the spinal interneurons that first process corticospinal input, without changing the magnitude of commands descending from the motor cortex, suggesting a novel correlation between muscle recruitment and components of cortically-evoked CDPs. Finally, DS increases excitability of post-synaptic spinal interneurons at the stimulation site and their responsiveness to any residual supraspinal control, thus supporting the use of electrical neuromodulation whenever the motor output is jeopardized by a weak volitional input, due to a partial disconnection from supraspinal structures and/or neuronal brain dysfunctions., Competing Interests: Declaration of Competing Interest VRE, a researcher on the study team holds shareholder interest in Onward and holds certain inventorship rights on intellectual property licensed by The Regents of the University of California to Onward. VRE, and PG, researchers on the study team hold shareholder interest in SpineX Inc. PG is an employee of SpineX Inc. WL holds shareholder interest in Aneuvo as well as inventorship of IPs licensed by The Regents of the University of California to Aneuvo., (Copyright © 2023. Published by Elsevier Inc.)

Several spinal motor output and essential rhythmic behaviors are controlled by supraspinal structures, although their contribution to neuronal networks for respiration and locomotion at birth still requires better characterization. As preparations of isolated brainstem and spinal networks only focus on local circuitry, we introduced the in vitro central nervous system (CNS) from neonatal rodents to simultaneously record a stable respiratory rhythm from both cervical and lumbar ventral roots (VRs).Electrical pulses supplied to multiple sites of brainstem evoked distinct VR responses with staggered onset in the rostro-caudal direction. Stimulation of ventrolateral medulla (VLM) resulted in higher events from homolateral VRs. Stimulating a lumbar dorsal root (DR) elicited responses even from cervical VRs, albeit small and delayed, confirming functional ascending pathways. Oximetric assessments detected optimal oxygen levels on brainstem and cortical surfaces, and histological analysis of internal brain structures indicated preserved neuron viability without astrogliosis. Serial ablations showed precollicular decerebration reducing respiratory burst duration and frequency and diminishing the area of lumbar DR and VR potentials elicited by DR stimulation, while pontobulbar transection increased the frequency and duration of respiratory bursts. Keeping legs attached allows for expressing a respiratory rhythm during hindlimb stimulation. Trains of pulses evoked episodes of fictive locomotion (FL) when delivered to VLM or to a DR, the latter with a slightly better FL than in isolated cords.In summary, suprapontine centers regulate spontaneous respiratory rhythms, as well as electrically evoked reflexes and spinal network activity. The current approach contributes to clarifying modulatory brain influences on the brainstem and spinal microcircuits during development. Novel preparation of the entire isolated CNS from newborn rats unveils suprapontine modulation on brainstem and spinal networks. Preparation views (A) with and without legs attached (B). Successful fictive respiration occurs with fast dissection from P0-P2 rats (C). Decerebration speeds up respiratory rhythm (D) and reduces spinal reflexes derived from both ventral and dorsal lumbar roots (E)., (© 2023. The Author(s).)

Exercise modifies respiratory functions mainly through the afferent feedback provided by exercising limbs and the descending input from suprapontine areas, two contributions that are still underestimated in vitro. To better characterize the role of limb afferents in modulating respiration during physical activity, we designed a novel experimental in vitro platform. The whole central nervous system was isolated from neonatal rodents and kept with hindlimbs attached to an ad-hoc robot (Bipedal Induced Kinetic Exercise, BIKE) driving passive pedaling at calibrated speeds. This setting allowed extracellular recordings of a stable spontaneous respiratory rhythm for more than 4 h, from all cervical ventral roots. BIKE reversibly reduced the duration of single respiratory bursts even at lower pedaling speeds (2 Hz), though only an intense exercise (3.5 Hz) modulated the frequency of breathing. Moreover, brief sessions (5 min) of BIKE at 3.5 Hz augmented the respiratory rate of preparations with slow bursting in control (slower breathers) but did not change the speed of faster breathers. When spontaneous breathing was accelerated by high concentrations of potassium, BIKE reduced bursting frequency. Regardless of the baseline respiratory rhythm, BIKE at 3.5 Hz always decreased duration of single bursts. Surgical ablation of suprapontine structures completely prevented modulation of breathing after intense training. Albeit the variability in baseline breathing rates, intense passive cyclic movement tuned fictive respiration toward a common frequency range and shortened all respiratory events through the involvement of suprapontine areas. These observations contribute to better define how the respiratory system integrates sensory input from moving limbs during development, opening new rehabilitation perspectives., (© 2023. The Author(s).)

The present review focuses on the physiological states of spinal networks, which are stochastically modulated by continuously changing ensembles of proprioceptive and supraspinal input resulting in highly redundant neural networks. Spinal epidural interfaces provide a platform for probing spinal network dynamics and connectivity among multiple motor pool-specific spinal networks post-injury under in vivo experimental conditions. Continuous epidural low-frequency pulses at low intensity can evoke motor responses of stochastically changing amplitudes and with an oscillatory pattern of modulation. The physiological significance of this oscillatory pattern, intrinsic to "resting" spinal networks and observed in both uninjured and injured locomotor circuits, is unclear. This neural variability among spinal networks appears to be a fundamental mechanism of the network's design and not a "noise" interfering with movement control. Data to date also suggest that the greater the level of stimulation above motor threshold, the greater the loss of modulation over the motor output that is physiologically provided by interneuronal networks, which integrate naturally occurring proprioceptive and cutaneous input generated during movement. Sub-motor threshold spinal electrical stimulation experiments demonstrate a range of functional improvements of multiple physiological systems when used in concert with sensorimotor training after spinal cord injury. Although our understanding of the systemic, cellular and molecular modulatory mechanisms that trigger these activity-dependent adaptive processes remain incomplete, some basic physiological principles have evolved, at least at the systemic and neural network levels and to some degree at the cellular level., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In intact and spinal-injured anesthetized animals, stimulation levels that did not induce any visible muscle twitches were used to elicit motor evoked potentials (MEPs) of varying amplitude, reflecting the temporal and amplitude dynamics of the background excitability of spinal networks. To characterize the physiological excitability states of neuronal networks driving movement, we designed five experiments in awake rats chronically implanted with an epidural stimulating interface, with and without a spinal cord injury (SCI). First, an uninjured rat at rest underwent a series of single electrical pulses at sub-motor threshold intensity, which generated responses that were continuously recorded from flexor and extensor hindlimb muscles, showing an intrinsic patterned modulation of MEPs. Responses were recruited by increasing strengths of stimulation, and the amplitudes were moderately correlated between flexors and extensors. Next, after SCI, four awake rats at rest showed electrically induced MEPs, varying largely in amplitude, of both flexors and extensors that were mainly synchronously modulated. After full anesthesia, MEP amplitudes were largely reduced, although stimulation still generated random baseline changes, unveiling an intrinsic stochastic modulation. The present five cases demonstrate a methodology that can be feasibly replicated in a broader group of awake and behaving rats to further define experimental treatments involving neuroplasticity. Besides validating a new technology for a neural stimulating interface, the present data support the broader message that there is intrinsic patterned and stochastic modulation of baseline excitability reflecting the dynamics of physiological states of spinal networks. NEW & NOTEWORTHY Chronic implants of a new epidural stimulating interface trace dynamics of spinal excitability in awake rats, before and after injury. Motor evoked potentials induced by trains of pulses at sub-motor threshold intensity were continuously modulated in amplitude. Oscillatory patterns of amplitude modulation reduced with increasing strengths of stimulation and were replaced by an intrinsic stochastic tone under anesthesia. Variability of baseline excitability is a fundamental feature of spinal networks, affecting their responses to external input.

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., (© 2021. The Author(s).)

Novel neural stimulation protocols mimicking biological signals and patterns have demonstrated significant advantages as compared to traditional protocols based on uniform periodic square pulses. At the same time, the treatments for neural disorders which employ such protocols require the stimulator to be integrated into miniaturized wearable devices or implantable neural prostheses. Unfortunately, most miniaturized stimulator designs show none or very limited ability to deliver biomimetic protocols due to the architecture of their control logic, which generates the waveform. Most such designs are integrated into a single System-on-Chip (SoC) for the size reduction and the option to implement them as neural implants. But their on-chip stimulation controllers are fixed and limited in memory and computing power, preventing them from accommodating the amplitude and timing variances, and the waveform data parameters necessary to output biomimetic stimulation. To that end, a new stimulator architecture is proposed, which distributes the control logic over three component tiers - software, microcontroller firmware and digital circuits of the SoC, which is compatible with existing and future biomimetic protocols and with integration into implantable neural prosthetics. A portable prototype with the proposed architecture is designed and demonstrated in a bench-top test with various known biomimetic output waveforms. The prototype is also tested in vivo to deliver a complex, continuous biomimetic stimulation to a rat model of a spinal-cord injury. By delivering this unique biomimetic stimulation, the device is shown to successfully reestablish the connectivity of the spinal cord post-injury and thus restore motor outputs in the rat model., Competing Interests: WL and Y-KL hold shareholder interest in Niche Biomedical Inc. P-MW was employed by the company Niche Biomedical Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Culaclii, Wang, Taccola, Yang, Bailey, Chen, Lo and Liu.)

Motoneuron activity is modulated by histamine receptors. While H 1 and H 2 receptors have been widely explored, H 3 histamine receptors (H 3 Rs) have not been sufficiently characterized. This paper targets the effects of the selective activation of H 3 Rs and their expression on the membranes of large ventral horn cells. The application of selective pharmacological agents to spinal cords isolated from neonatal rats was used to identify the presence of functional H 3 Rs on the membrane of physiologically identified lumbar motoneurons. Intra and extracellular recordings revealed that H 3 R agonist, α-methylhistamine, depolarized both single motoneurons and ventral roots, even in the presence of tetrodotoxin, an effect prevented by H 3 R antagonist, thioperamide. Finally, immunohistochemistry located the expression of H 3 Rs on a subpopulation of large cells in lamina IX. This study identifies H 3 Rs as a new exploitable pharmacological target against motor disturbances.

Study Design: Systematic review., Objectives: Over the past decade, an increasing number of studies have demonstrated that epidural spinal cord stimulation (SCS) can successfully assist with neurorehabilitation following spinal cord injury (SCI). This approach is quickly garnering the attention of clinicians. Therefore, the potential benefits of individuals undergoing epidural SCS therapy to regain sensorimotor and autonomic control, must be considered along with the lessons learned from other studies on the risks associated with implantable systems., Methods: Systematic analysis of literature, as well as preclinical and clinical reports., Results: The use of SCS for neuropathic pain management has revealed that epidural electrodes can lose their therapeutic effects over time and lead to complications, such as electrode migration, infection, foreign body reactions, and even SCI. Several authors have also described the formation of a mass composed of glia, collagen, and fibrosis around epidural electrodes. Clinically, this mass can cause myelopathy and spinal compression, and it is only treatable by surgically removing both the electrode and scar tissue., Conclusions: In order to reduce the risk of encapsulation, many innovative efforts focus on technological improvements of electrode biocompatibility; however, they require time and resources to develop and confirm safety and efficiency. Alternatively, some studies have demonstrated similar outcomes of non-invasive, transcutaneous SCS following SCI to those seen with epidural SCS, without the complications associated with implanted electrodes. Thus, transcutaneous SCS can be proposed as a promising candidate for a safer and more accessible SCS modality for some individuals with SCI.

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 the 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 the 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, the effects of A1 antagonism on motor evoked responses after an acute spinal transection was not detected. These studies support the future investigation of the optimal dosage, methods of delivery, and systemic effects of the synergistic application of A1 antagonists and spinal stimulation in the intact and injured spinal cord., (Copyright © 2020 Taccola, Salazar, Apicella, Hogan, Horner and Sayenko.)

An in vitro system of electrical stimulation was used to explore whether an innovative "noisy" stimulation protocol derived from human electromyographic recordings (EMGstim) could promote muscle regeneration. EMGstim was delivered to cultured mouse myofibers isolated from Flexor Digitorum Brevis, preserving their satellite cells. In response to EMGstim, immunostaining for the myogenic regulatory factor myogenin, revealed an increased percentage of elongated myogenin-positive cells surrounding the myofibers. Conditioned medium collected from EMGstim-treated cell cultures, promoted satellite cells differentiation in unstimulated myofiber cell cultures, suggesting that extracellular soluble factors could mediate the process. Interestingly, the myogenic effect of EMGstim was mimicked by exogenously applied ATP (0.1 μM), reduced by the ATP diphosphohydrolase apyrase and prevented by blocking endogenous ATP release with carbenoxolone. In conclusion, our results show that "noisy" electrical stimulations favor muscle progenitor cell differentiation most likely via the release of endogenous ATP from contracting myofibres. Our data also suggest that "noisy" stimulation protocols could be potentially more efficient than regular stimulations to promote in vivo muscle regeneration after traumatic injury or in neuropathological diseases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.)

Spinal motoneurons and locomotor networks are regulated by monoamines, among which, the contribution of histamine has yet to be fully addressed. The present study investigates histaminergic regulation of spinal activity, combining intra- and extracellular electrophysiological recordings from neonatal rat spinal cord in vitro preparations. Histamine dose-dependently and reversibly generated motoneuron depolarization and action potential firing. Histamine (20 µM) halved the area of dorsal root reflexes and always depolarized motoneurons. The majority of cells showed a transitory repolarization, while 37% showed a sustained depolarization maintained with intense firing. Extracellularly, histamine depolarized ventral roots (VRs), regardless of blockage of ionotropic glutamate receptors. Initial, transient glutamate-mediated bursting was synchronous among VRs, with some bouts of locomotor activity in a subgroup of preparations. After washout, the amplitude of spontaneous tonic discharges increased. No desensitization or tachyphylaxis appeared after long perfusion or serial applications of histamine. On the other hand, histamine induced single motoneuron and VR depolarization, even in the presence of tetrodotoxin (TTX). During chemically induced fictive locomotion (FL), histamine depolarized VRs. Histamine dose-dependently increased rhythm periodicity and reduced cycle amplitude until near suppression. This study demonstrates that histamine induces direct motoneuron membrane depolarization and modulation of locomotor output, indicating new potential targets for locomotor neurorehabilitation., (© 2017 Wiley Periodicals, Inc.)

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.)

Tactile hypersensitivity is one of the most debilitating symptoms of neuropathic pain syndromes. Clinical studies have suggested that its presence at early postoperative stages may predict chronic (neuropathic) pain after surgery. Currently available animal models are typically associated with consistent tactile hypersensitivity and are therefore limited to distinguish between mechanisms that underlie tactile hypersensitivity as opposed to mechanisms that protect against it. In this study we have modified the rat model of spared nerve injury, restricting the surgical lesion to a single peripheral branch of the sciatic nerve. This modification reduced the prevalence of tactile hypersensitivity from nearly 100% to approximately 50%. With this model, we here also demonstrated that the Regulator of G protein Signaling 4 (RGS4) was specifically up-regulated in the lumbar dorsal root ganglia and dorsal horn of rats developing tactile hypersensitivity. Intrathecal delivery of the RGS4 inhibitor CCG63802 was found to reverse tactile hypersensitivity for a 1h period. Moreover, tactile hypersensitivity after modified spared nerve injury was most frequently persistent for at least four weeks and associated with higher reactivity of glial cells in the lumbar dorsal horn. Based on these data we suggest that this new animal model of nerve injury represents an asset in understanding divergent neuropathic pain outcomes, so far unravelling a role of RGS4 in tactile hypersensitivity. Whether this model also holds promise in the study of the transition from acute to chronic pain will have to be seen in future investigations., (Copyright © 2016. Published by Elsevier Inc.)

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., (Copyright © 2016. Published by Elsevier Inc.)

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.)

Objectives: Investigate whether electrical stimulation of the spinal cord adapted to trigger locomotor patterns additionally influences dorsal horn networks., Materials and Methods: An in vitro model of isolated neonatal rat spinal cord was used to repetitively deliver electrical stimuli to lumbar dorsal roots and record from homolateral lumbar dorsal roots and ventral roots., Results: Repetitive electrical lumbar dorsal root stimulation can affect both locomotor rhythms derived from ventral neuronal circuits and activity from dorsal neuronal circuits., Conclusion: These data suggest that neuro-electrostimulation protocols can simultaneously activate functionally distinct spinal neuronal circuits., (© 2015 International Neuromodulation Society.)

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 μM) 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.

Noisy waveforms, sampled from an episode of fictive locomotion (FL) and delivered to a dorsal root (DR), are a novel electrical stimulating protocol demonstrated as the most effective for generating the locomotor rhythm in the rat isolated spinal cord. The present study explored if stimulating protocols constructed by sampling real human locomotion could be equally efficient to activate these locomotor networks in vitro. This approach may extend the range of usable stimulation protocols and provide a wide palette of noisy waveforms for this purpose. To this end, recorded electromyogram (EMG) from leg muscles of walking adult volunteers provided a protocol named ReaListim (Real Locomotion-induced stimulation) that applied to a single DR successfully activated FL. The smoothed kinematic profile of the same gait failed to do so like nonphasic noisy patterns derived from standing and isometric contraction. Power spectrum analysis showed distinctive low-frequency domains in ReaListim, along with the high-frequency background noise. The current study indicates that limb EMG signals (recorded during human locomotion) applied to DR of the rat spinal cord are more effective than EMG traces taken during standing or isometric contraction of the same muscles to activate locomotor networks. Finally, EMGs recorded during various human motor tasks demonstrated that noisy waves of the same periodicity as ReaListim, could efficiently activate the in vitro central pattern generator (CPG), regardless of the motor task from which they had been sampled. These data outline new strategies to optimize functional stimulation of spinal networks after injury.

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.