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 reset the locomotor rhythm consistent with these neurons having access to the central pattern generator. We found that the locomotor rhythm persisted and its frequency was unaltered when the light-gated hyperpolarizing proton pump Archaerhodopsin-3 was used to silence motoneurons during on-going locomotor-like activity. We did find however, that when the light used to activate Archaerhodopsin-3 was turned off, the locomotor rhythm increased in intensity and frequency for about 20 seconds before returning to normal levels. The locomotor rhythm was suppressed when V1 interneurons expressing the excitatory channelrhodopsin-2 were illuminated. Conversely when V1 interneurons were silenced the locomotor rhythm slowed and became more precise. Collectively, these finding suggests that motoneuron discharge does not affect either the presynaptic locomotor drive or its frequency during drug-induced locomotor like activity. However, the results also implicate the V1 interneuronal population a subset of which is activated by motoneurons in the maintenance and organization of the locomotor rhythm. 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 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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