This thesis addresses two major challenges in neuromuscular physiology. The first challenge is to understand how hundreds individually contracting muscle fibers act in concert to produce macroscopic forces and movements. Previous analyses of this issue have been limited by our inability to directly visualize the basic contractile units of striated muscle, called sarcomeres. Our knowledge about sarcomere dynamics has primarily come from in vitro studies of muscle fibers and analysis of optical diffraction patterns obtained from living muscles. Both approaches involve highly invasive procedures and neither allows examination of individual sarcomeres in live subjects. The second challenge was to develop an alternative to electrical stimulation, which is commonly used to excite muscle but reverses the order of motor unit recruitment. Under physiologic conditions small motor units with fatigue resistant muscle fibers are recruited before large motor units with more fatigable muscle fibers. This control scheme allows fine motor control at low levels of muscle activity, and high force production for short bursts. Electrical stimulation of muscle inverts this mechanism by recruiting large, fatigable muscle fibers first, severely limiting therapeutic applications of electrical stimulation.

In this thesis, we report direct visualization of individual sarcomeres and their dynamical length variations using minimally invasive optical microendoscopy to observe second harmonic frequencies of light generated in the muscle fibers of live mice and humans. Using microendoscopes as small as 350 urn in diameter, we imaged individual sarcomeres in both passive and activated muscle. Our measurements permit in vivo characterization of sarcomere length changes that occur with alterations in body posture and visualization of local variations in sarcomere length not apparent in aggregate length determinations. High-speed data acquisition enabled observation of sarcomere contractile dynamics with millisecond-scale resolution. These experiments point the way to in vivo imaging studies demonstrating how sarcomere performance varies with physical conditioning and physiological state, as well as imaging diagnostics revealing how neuromuscular diseases affect contractile dynamics.

This thesis also reports methods to control muscle activity with optical stimulation by way of channelrhodopsin-2 channels genetically inserted into motor axons. We show that optical stimulation of motor units proceeds in the normal physiologic order by comparing several measures of motor unit recruitment, such as conduction latency, contraction and relaxation time, and stimulation threshold, using both an electrical and a LED-based optical cuff for stimulation in transgenic mice. We also show the evidence that these observations are not the result of differences in channel expression among different sizes of motor axons, suggesting that optical stimulation may excite individual neurons based on their size. When this knowledge is combined with genetic targeting strategies it will allow the further flexibility to target specific neuromuscular structures or biochemical pathways for optically-based prosthetics and therapies for neuromuscular diseases.

Together, these new tools will expand the bounds of neuromuscular knowledge by overcoming barriers to current research. The first project resulted in the first direct visualization of individual sarcomeres in humans, making it possible for future clinical studies to elucidate the impact of genetic, pharmacologic or therapeutic effects on sarcomere structure and function in diseases such as cerebral palsy and stoke. The second project produced a new method of stimulating neuromuscular tissue that is amenable to the knowledge of genetic engineering, paving the way for new optogenetic therapies and prosthetics in a wide range of applications for the peripheral nervous system, including upper and lower motor neuron diseases. These novel tools will spawn new branches of research to address previously impenetrable questions and will eventually lead to therapies for neuromuscular diseases.