The goal of this dissertation was to develop new methods for optical inhibition of motor neuron and muscle activity to enable studies of neuromuscular control and provide therapeutic insights into disorders of the peripheral nervous system. This work was motivated by the need for new treatment options for spastic hypertonia, characterized by abnormal activity in lower motor neurons and involuntary muscle contractions and occurring frequently with cerebral palsy, stroke, and other cerebrospinal injuries. Optogenetic methods have been used widely in the central nervous system to modulate activity of specified neuronal populations in cellular and behavioral studies of neuronal activity, network connectivity, and disease. For applications of optogenetics in the peripheral nervous system, the work of this dissertation focused on the development of new technologies for light delivery to peripheral nerves, modulation of peripheral nerve temperature, and control of excitation and inhibition of motor neuron and muscle activity.

We developed an optogenetics based method that utilizes the chloride pump halorhodopsin (eNpHR2.0) to achieve precise and rapidly reversible optical inhibition of motor neuron and muscle activity in vivo in transgenic Thy1-eNpHR2.0-EYFP mice by illuminating the nerve with green light. We used this method to demonstrate that eNpHR2.0 is capable of intercepting spikes in the axon, optical inhibition is effective at all amplitudes of electrically evoked twitch force, and light power density modulates the degree of inhibition. This study revealed the potential of eNpHR2.0 as a powerful tool for inhibiting motor neuron and muscle activity in the peripheral nervous system.

We developed a second method that utilizes in a new way the cation channel channelrhodopsin-2 (ChR2) to inhibit motor neuron and muscle activity in vivo in the cooled sciatic nerve of Thy1-ChR2-EYFP mice by illuminating the nerve with high frequency blue light pulses or continuous blue light. We identified light pulse frequency and nerve temperature as important variables for achieving inhibition and quantified their effects. This study presents an all-optical, single opsin and single wavelength method to control neuronal excitation and inhibition without requiring co-expression of excitatory and inhibitory opsins. We hypothesized that ChR2 photocycle kinetics are critical to the mechanism that underlies this bifunctionality and compared it to mechanisms that have been proposed for high frequency electrical inhibition. In contrast to electrical inhibition, our method is free from stimulation induced electrical artifacts and thus provides a new approach to reveal long sought insights into these mechanisms. Such experimental evidence will inform further studies to optimize stimulation parameters and pursue clinical applications of high frequency inhibition.

This dissertation contributes new methods for optical inhibition of motor neuron and muscle activity and facilitates studies of neuromuscular control and treatment strategies for disorders of the peripheral nervous system.