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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.)

The spinal cord contains an intrinsic locomotor program driven by a central pattern generator that rhythmically activates flexor and extensor limb motor pools. Although long-lasting locomotor activity can be generated pharmacologically, trains of afferent stimuli trigger only few locomotor cycles. The present study investigated whether a new electrical stimulation protocol (termed FListim) could elicit long-lasting fictive locomotion (FL) in the rat spinal cord in vitro. Thus, after first inducing FL by bath application of N-methyl-d-aspartate and serotonin, the recorded waveform obtained from a lumbar ventral root was digitized and then applied to either a lumbar dorsal root or the cauda equina following washout of pharmacological agents. Two FListim cycles were the threshold input to evoke an episode of FL from ventral roots. Longer cycles (up to 1 min) induced sustained FL (up to 1 min) with stereotyped periodicity (2.2 ± 0.5 s), despite changing frequency (2-4 s) or cycle amplitude of FListim. Gradual filtering out of the noise from FListim trace concomitantly decreased the efficiency of FL so that stimulation with equivalent pure sinusoids produced asynchronous, irregular discharges only that could not be converted to FL by adding spontaneous basal activity. This study is the first demonstration that epochs of rhythmic locomotor-like oscillations applied to a dorsal root represent an efficient stimulus to evoke FL as long as they contain the electrophysiological noise produced within FL cycles. These observations suggest novel strategies to improve the efficiency of electrical stimulation delivered by clinical devices for neurorehabilitation after spinal injury.

In the rat spinal cord in vitro, block of synaptic inhibition evokes persistent, regular disinhibited bursting which is a manifestation of the intrinsic network rhythmicity and is readily recorded from ventral roots. This model is advantageous to explore the network mechanisms controlling burst periodicity, and duration. We questioned the relative contribution of K+ conductances to spontaneous rhythmicity by investigating the effects of the broad K+ channel blocker tetraethylammonium (TEA). In TEA (10 mM) solution, bursts occurred at the same rate but became substantially longer, thus showing an unusual dissociation between mechanisms of burst periodicity and duration. In the presence of TEA, electrical stimulation of a single dorsal root or N-methyl-D-aspartate application (5 microM) could, however, fasten bursting associated with immediate decrease in burst length, thus demonstrating maintenance of short-term plasticity. Either riluzole (1 microM) or surgical sectioning that isolated a single spinal segment strongly depressed bursting which could, however, be revived by TEA. In the presence of TEA, the L-type channel blocker nifedipine (20 microM) made bursting faster and shorter. Our data are best explained by assuming that TEA increased network excitability to generate rhythmic bursting, an effect that was counteracted by intrinsic mechanisms, partly dependent on L-type channel activity, to retain standard periodicity. TEA-sensitive mechanisms were, nevertheless, an important process to regulate burst duration. Our results are consistent with the proposal of a hierarchical structural of the central pattern generator in which the circuits responsible for rhythmicity (the clock) drive the discharges of those creating the motor commands (pattern).

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 metabotropic glutamate receptors (mGluRs) to generation of rhythmic discharges. The in vitro spinal cord of the neonatal rat was used as a model to find out if electrical patterns characterized by either alternating or synchronous motor pool discharges (recorded from lumbar ventral roots) required mGluR activation or were modulated by it. Alternating patterns of fictive locomotion (induced by NMDA and 5HT) were slowed down and blocked by the broad spectrum mGluR agonist (+/-)-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (t-ACPD; 5-50 microM) and unaffected by the broad spectrum mGluR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG; 1 mM). The regular, synchronous bursting emerging in the presence of strychnine and bicuculline was accelerated by t-ACPD with a commensurate decrease in single burst length, an effect antagonized by MCPG which per se did not affect bursting. The action of t-ACPD was selectively inhibited by the L-type Ca2+ blocker nifedipine which, however, did not change rhythm acceleration evoked by NMDA. These data suggest that neither alternating nor synchronous oscillatory discharges were apparently dependent on mGluR activation via endogenously released glutamate. However, mGluR activation by the agonist t-ACPD modulated rhythmic patterns, indicating that such receptors are a potential target for pharmacological up- or downregulation of spinal rhythmicity.