Stimulation-Guided AAV Delivery and Longitudinal Assessment of Optogenetic Expression in Rat Motor Nerves

Optogenetic stimulation of peripheral motor nerves is a promising technique for modulating neural activity via illumination of light-sensitive ion channels known as opsins. Stimulating muscle activity through this method offers many advantages, such as a physiological recruitment order of motor units, reduced fatigue, and target-specific stimulation, which make it a favorable option for use in many neuroscience and motor rehabilitation applications. To enable such optical stimulation, opsin expression in peripheral nerves can be achieved either with transgenic animal models or through injection of viral vectors. In this protocol, we describe a method for driving peripheral nerve opsin expression via intramuscular adeno-associated virus (AAV) injection with the goal of enhancing virus uptake by targeting injections to neuromuscular junctions with electrical stimulation. We also describe procedures for non-invasively assessing functional opsin expression over time with transdermal optical stimulation of opsin-labeled nerves and electromyography (EMG) recordings. The presence of time-locked EMG spikes 4-8 ms after each stimulation pulse demonstrates that functional opsin expression is present at a given assessment time point. Onset of functional optical sensitivity generally occurs 2-4 weeks following virus injection, and sensitivity generally peaks or plateaus between 6-10 weeks. Stimulation sequences such as light intensity, stimulation pulse width, and frequency sweeps provide further information on functional opsin expression at the testing timepoint. The methods presented here can be used for driving functional opsin expression with a standard AAV6 vector commonly used in similar experiments or as a protocol for assessing peripheral nerve opsin expression with novel viral vectors. Key features • Uses electrical stimulation to guide needle placement during intramuscular viral injection. • Drives robust and muscle-specific opsin expression in peripheral motor neurons. • Describes transdermal optical stimulation sequences with varying stimulation light intensity, pulse width, and frequency for longitudinal assessment of opsin expression. • Adaptable for use with multiple viral vectors and target muscles.