We have been using optogenetics to understand the contributions of motoneuron activity to fictive locomotion in the neonatal mouse. For this purpose, we have constructed mouse lines in which the excitatory channelrhodopsin or the inhibitory archaerhodopsin is expressed in motoneurons or in the spinal neurons that receive motoneuron inputs. Hyperpolarizing extensor motoneurons by exposure to light results in the neurons firing in the wrong, flexor phase of the cycle, because their membrane potential becomes lower than the chloride equilibrium potential thereby rendering rhythmic inhibitory input depolarizing. Whole cell voltage clamp experiments have revealed that some extensor motoneurons receive exclusively inhibitory rhythmic drive whereas some flexor motoneurons receive purely excitatory rhythmic drive. In motoneurons expressing the excitatory channelrhodopsin-2, we found that blue light could trigger or reset the locomotor rhythm consistent with these neurons having access to the central pattern generator. The locomotor rhythm became unstable and its frequency declined when the light-gated hyperpolarizing proton pump Archaerhodopsin-3 was used to silence motoneurons during on-going locomotor-like activity. The light-induced change in frequency was not abrogated by cholinergic antagonists or gap junction blockers, indicating that neither cholinergic interneurons nor gap junctions mediated the effects. We did find however that the frequency changes were completely blocked by the AMPA-receptor antagonist NBQX. These findings are novel and surprising and reveal a completely new role for motoneurons in the operation of the locomotor central pattern generator and will have major implications not only for basic science but also for spinal cord injuries. Renshaw cells are the only known intraspinal interneuronal targets of motoneurons. They belong to the V1 interneuronal population that expresses the transcription factor engrailed-1. To establish if these interneurons were involved in the regulation of locomotor frequency by motoneuronal activity, we introduced Archaerhodopsin or Channelrhodopsin-2 into the V1 population. We found that hyperpolarizing the V1 population slowed the locomotor rhythm. This result mimics the effect of reduced motoneuron discharge thereby implicating Renshaw cells and their synaptic partners in the regulation of locomotor frequency. When V1 interneurons expressing channelrhodopsin2 were activated the rhythm became disorganized and in some experiments stopped. Collectively these results suggest that motoneuronal input to the Renshaw network can modulate the locomotor rhythm. The next stage is to determine if the effects are mediated by all classes of motoneuron or a motoneuronal sub-type. We have evidence that high-threshold motor axons in the ventral roots have to be activated to manifest the excitatory actions of motoneurons. This raises the possibility that S-type motoneurons may mediate these effects. Traditionally, mammalian motoneurons have been thought to exhibit electrical connections only with members of the same motoneuron pool or close functional synergists. By contrast we have found that motoneurons in the L6 segments of the spinal cord are dye-coupled to non-cholinergic interneurons. Evidence that this motoneuron-interneuron network may be important functionally, and more extensive than the L6 segments alone, comes from the observation that spinal networks can generate synchronized rhythmic drive in the absence of chemical synaptic transmission. Bath-application of ruthenium red (RR) after blocking all chemical neurotransmission produces a slow bursting rhythm in motoneurons and interneurons that is synchronous ipsilaterally and contralaterally throughout the spinal cord.

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2017
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Blivis, Dvir; Haspel, Gal; Mannes, Philip Z et al. (2017) Identification of a novel spinal nociceptive-motor gate control for A? pain stimuli in rats. Elife 6:
Falgairolle, Melanie; Puhl, Joshua G; Pujala, Avinash et al. (2017) Motoneurons regulate the central pattern generator during drug-induced locomotor-like activity in the neonatal mouse. Elife 6:
Pujala, Avinash; Blivis, Dvir; O'Donovan, Michael J (2016) Interactions between Dorsal and Ventral Root Stimulation on the Generation of Locomotor-Like Activity in the Neonatal Mouse Spinal Cord. eNeuro 3:
Falgairolle, Melanie; O'Donovan, Michael J (2015) Pharmacological Investigation of Fluoro-Gold Entry into Spinal Neurons. PLoS One 10:e0131430
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Gonzalez-Islas, Carlos; Chub, Nikolai; Garcia-Bereguiain, Miguel Angel et al. (2010) GABAergic synaptic scaling in embryonic motoneurons is mediated by a shift in the chloride reversal potential. J Neurosci 30:13016-20

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