Optogenetic methods to stimulate gamma motor neuron axons ex vivo

It is challenging to stimulate gamma motor neurons, which are important regulators of muscle spindle afferent function, without also recruiting alpha motor neurons. Here, we test the feasibility of stimulating gamma motor neuron axons using optogenetics in two transgenic mouse lines. We used an ex vivo muscle-nerve preparation in adult mice to monitor muscle spindle afferent firing, which should increase in response to gamma motor neuron-induced lengthening of the sensory region of the muscle spindle. A force transducer measured alpha motor neuron-mediated twitch contractions. Blue LED light (470 nm; 1-5 mW) was delivered via a light guide to the sciatic nerve. We confirmed that the more slowly conducting gamma motor neurons were recruited first in mice expressing channelrhodopsin 2 in choline acetyltransferase-positive motor neurons, whereas alpha motor neurons required higher optical intensities, enabling co-activation of alpha and gamma motor neurons depending on light intensity. However, this approach cannot isolate gamma motor neuron activity completely. Cre-dependent channelrhodopsin 2 optoactivation using the putative gamma motor neuron marker neuronal PAS domain protein 1 (Npas1) also increased muscle spindle afferent firing rates and caused only small twitch contractions. This provides functional validation that Npas1 is present primarily in gamma motor neurons and can be used to manipulate gamma motor neurons independently. We propose optogenetic stimulation as a promising tool to manipulate gamma motor neuron activity.