Background

Optogenetic stimulation or inhibition of peripheral motor nerves has recently emerged as an approach to directly modulate muscle activity in animal models from rodents to non-human primates [1–4]. In this approach, targeted nerves are genetically modified with light-gated ion channels (“opsins”) such as the common channel-rhodopsin2 (ChR2), allowing a nerve to be stimulated or inhibited with specific light wavelengths. Compared to electrical stimulation, this approach has been found to confer benefits, including a more physiological recruitment order of motor units (i.e., small muscle fibers are first recruited with low-intensity optical stimulation, and larger muscle fibers are added at higher intensities) and reduced stimulation-induced muscle fatigue [1,5]. Inhibition of target activity may be achieved using light-sensitive chloride channels or proton pumps (e.g., NpHR3 [6], ArchT [7]), a feat that is more complex with electrical stimulation. Additionally, stimulation of specific downstream nerve targets may be achieved through targeted virus injection, genetic targeting, or opsin selection [8,9]. Taken together, these traits present attractive benefits for the use of this technology in neuroscience or motor rehabilitation applications.

A key step for using this technology is to first biologically label target nerve axons with the desired opsin. Transgenic animals may be used for robust opsin labeling of genetically specified tissues throughout the body, including peripheral nerves for neuromodulation [1,10]. However, transgenic approaches are typically limited to mice, and specificity through these approaches is usually defined by genetic markers, resulting in the labeling of all tissues in the body that meet genetic criteria. Conversely, injection of viral vectors (e.g., adeno-associated virus, AAV) may be used to express optogenetic proteins targeted to tissues based on 1) the relative tropism of the virus (i.e., AAV3 for inner ear cells [11]), 2) genetic specificity tailored by the virus’s expression cassette, and 3) viral biodistribution guided by the route of virus delivery (e.g., broad expression via intravenous injection, focal expression via microinjection in the targeted tissue). These tools provide several knobs through which to tune the specificity or breadth of opsin expression.

In the case of peripheral optogenetic stimulation of motor function, muscle-specific opsin expression and stimulation can be achieved via intramuscular virus injection. For example, intramuscular injection of an AAV6 vector (e.g., AAV6-hSyn-ChR2-eYFP) into the tibialis anterior (TA) muscle of rats can be used to selectively label nerve axons innervating the TA with a light-sensitive opsin (e.g., channel-rhodopsin, ChR2) and a fluorescent tag (e.g., YFP) [2,3]. The human synapsin promoter (hSyn) restricts expression to neural tissue (as opposed to expression in the injected muscle). To label targeted deep peroneal nerve axons innervating just the TA muscle, a primary route would require that virus first crosses from the muscle into the nerve via the neuromuscular junction (NMJ), travel retrogradely up the nerve to motor neurons in the spinal cord, undergo transcription of the viral DNA instructions, and traffic expressed opsin and fluorescent proteins anterogradely back down the nerve axons. Following this injection route, optical stimulation at proximal nerve locations with multiple downstream muscle targets, such as the sciatic nerve, results in stimulation of only the targeted TA muscle [3], a feat of specificity that is challenging with electrical nerve stimulation.

A critical point in this viral transduction pathway is the uptake of the virus from the muscle into the nerve. NMJs are typically arranged in characteristic patterns within a muscle where nerve endings branch out to interface with muscle fibers. Targeting microinjections of virus to follow these NMJ distributions has been shown to increase the uptake of virus and other tracers [12,13]. While injection sites within a muscle may be based on expected NMJ distributions, intersubject variability could potentially limit the success of this approach, especially in larger animals. As an alternative approach, low-level electrical stimulation through intramuscular injection electrodes can be used to localize motor endplates in target muscles. Such an approach has been utilized in human subjects for guiding injections of botulinum toxin, in conditions such as juvenile cerebral palsy, as a fast and minimally invasive way to confirm injection of the targeted muscle and facilitate the paralytic effects of the injection [14,15]. We have previously employed a similar approach for intramuscular AAV injections in non-human primates to achieve optical stimulation of muscle activity [4,16].

Here, our primary purpose is to present a protocol to target AAV injections to motor endplates in hindlimb muscles of rats using electrical stimulation in order to express light-sensitive opsins along their corresponding nerves, thereby allowing light-based stimulation of muscle activity. Following these injections, we describe our common procedures for periodically evaluating a nerve’s optical sensitivity using transdermal laser stimulation to estimate and track the longitudinal time course of functional opsin expression. Finally, we present our standard histology preparations for examining the underlying patterns of opsin expression in nerve and spinal cord samples at the terminal end point of an experiment. These protocols provide a reliable means of achieving robust opsin expression in peripheral motor nerves and optically modulated motor responses. Additionally, these protocols can act as a framework for evaluating the functional opsin expression timelines and histological characteristics of optogenetic vectors with varying serotypes, promoters, and opsins.