Functional significance of motoneuronal persistent inward currents for locomotor activity

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Persistent inward currents (PICs) are excitatory (depolarizing) voltage-sensitive ionic currents in neurons that, once evoked, raise cell excitability to a given stimulus, and continue to flow after cessation of the excitatory stimulus that evoked them. Through PICs, spinal motoneurons exhibit two levels of excitability (termed “bistability”), and this elevated excitability caused by PICs can be turned on through the activation of specific calcium and sodium channels. PICs originate in the dendritic region, primarily through L-type calcium channels, their behaviour is dependent primarily on the monoamines serotonin and norepinephrine, and they seem to be evoked during locomotion and other types of movement, in animals and humans. We do not know how “turning on” of these PICs influences the expression of the forces of the innervated muscle fibers, which are prone to the effects of stimulation frequency-dependent potentiation, depression, and fatigue. In addition, whether motoneurons innervating muscle fibers of different “types” (fast fatiguing, fast fatigue-resistant, and slow) express PICs of differing amplitudes and time is unknown. Finally, we do not know if chronic increase in neuromuscular activity level causes changes in PICs, in the expression of the proteins that cause them, and in the resultant force expression by the innervated muscle fibers which also change with increased activity. The proposed research will determine the functional significance of PICs in rat hindlimb motoneurons, and motor units. In phase 1, I will measure the amplitude and time course of activation/inactivation of PICs in rat motoneurons, and determine their functional impact on the forces evoked in the innervated muscle fibers in response to injection of currents into the innervating motoneuron, by recording motoneuron electrophysiological and muscle force responses simultaneously. This will be done across the spectrum of motoneurons innervating different muscle fiber “types" (fast, slow), to determine of PICs vary systematically, and their impact on muscle force generation. In phase 2, I will determine the role played by specific ion channel and receptor proteins in the PIC phenomenon in motoneurons, by selectively down-regulating individual proteins (L-type calcium channel CaV1.3, serotonin receptor 5HT-2A, -2C, and -1A, alpha-1 adrenoceptor) using short hairpin RNA sequences delivered to motoneurons via lentivirus vectors. This will tell us if different motor unit “types” have different PIC-related protein profiles, and will also allow us to estimate the characteristics of the sodium conductance in PICs when the calcium channel is eliminated. I will also determine using this technology if PICs are important for determining basic spinal locomotion patterns in a fictive locomotion model. In phase 3, I will determine if increased physical activity for 16 weeks, including daily treadmill training and voluntary exercise wheel activity, affects PICs, and changes the expression of proteins underlying PICs. The latter will be performed by sampling motoneurons in frozen spinal cord sections in order to measure gene expression in motoneurons innervating distinct hindlimb muscles that differ in fiber-type populations. The results will improve our understanding of the impact of PICs on the functional response of the target cell (in this case, muscle fibers), and of the significance of neuromuscular heterogeneity in sculpting movements using PICs. Information will help us understand how our neuromuscular system becomes "supercharged" when initiating and performing locomotor movements, what the implications are for optimization of neuromuscular performance, and shed light on what might occur in conditions such as aging and sedentarism.