Development of an Optogenetic Method to Stimulate Gamma Motor Neurons In Vitro

Abstract

The muscle spindle is a sensory organ located in the skeletal muscle that is critical for motor control and proprioception. The muscle spindle is innervated by stretch sensitive muscle spindle afferents and gamma motor neurons that control the length of the intrafusal muscle fibers and therefore the sensitivity of the stretch sensitive afferents. However, it has been challenging to study the gamma motor neurons since it is hard to specifically stimulate the gamma but not the alpha motor neurons that control the force generating extrafusal fibers. A previous study showed that alpha motor neurons were recruited from small diameter to large diameter with increasing optical intensities in mice expressing the blue light gated Channelrhodopsin2 (ChR2) in motor neurons (Llewellyn, et al., 2010). This is the reverse recruitment pattern of electrical stimulation. We hypothesized that gamma motor neurons, which are smaller than even the smallest alpha motor neurons, will be recruited first using low optical stimuli. The extensor digitorum longus muscle and sciatic nerve were dissected and placed in a tissue bath with oxygenated synthetic interstitial fluid. The sciatic nerve was attached to an extracellular suction electrode that recorded muscle spindle afferent activity. We used a light guide to deliver blue LED light (470nm; 0.5mW-5 mW) to the end of the nerve. We found that at lower levels of optical stimulation we recruited the gamma motor neurons as evidenced by an increased muscle spindle afferent firing rate and a pause after stimulation, which is typically observed following the release of stretch. Higher optical intensities were required to recruit alpha motor neurons to produce a twitch muscle contraction. Higher frequencies of optical stimulation led to greater increases in muscle spindle afferent firing rates as expected. We are currently testing additional stimulation frequencies and increasing our sample size to understand individual variability. We are also verifying that ChR2 is found in motor neurons only using immunohistochemistry. We will use this technique to study gamma motor neuron function and to produce a more physiological relevant system to study muscle spindle afferents. We can also use this method to screen for intrafusal fiber dysfunction in disease models.