Optogenetic tools have immense value in targeting genetically specified excitable cells with high temporal precision throughout the body, and have been used extensively to investigate circuits of the brain. Optogenetics has great potential to analyze the neural circuitry of the peripheral nervous system (PNS) and spinal cord in freely moving animals, but has been limited by the technical difficulties of delivering opsins and light to these heterogeneous systems. In this dissertation, I present novel optogenetic tools for use throughout the nervous system in freely moving animals. Included are viral strategies to target opsins to cutaneous C-fiber nociceptors and peripheral motor neurons as well as methods to deliver light via optical fiber to peripheral nerves, and wirelessly to cutaneous nerve terminals, spinal cord, and motor cortex of the brain. Using these optogenetic tools, we investigated pain and motor control in awake and freely moving mice and rats and provided proof-of-concept for potential therapies for neuropathic pain and paralysis. This technology opens the door for therapeutic development and more advanced investigations of the nervous system.

We developed a system to optogenetically stimulate and inhibit pain in mice using viral targeting of opsins and transcutaneous light delivery. To target unmyelinated nociceptors of the sciatic nerve, we injected adeno-associated virus serotype 6 (AAV6) containing DNA for a stimulatory opsin, channelrhodopsin-2 (ChR2), or an inhibitory opsin, halorhodopsin (NpHR), into the sciatic nerve. Subsequent illumination of the paw with blue or yellow light served to, respectively, activate or inhibit the unmyelinated nociceptors. This system provides for unprecedented temporally precise control over this group of peripheral neurons and serves as a novel pain model for investigations of peripheral pain pathways. This model may also serve as a therapeutic proof-of-principle, as NpHR-mediated inhibition of unmyelinated nociceptors effectively increased mechanical and thermal thresholds to normal levels in mice that had undergone a chronic constriction injury to model neuropathic pain.

We also developed a method to optogenetically stimulate motor neurons in awake and walking rats. Expressing opsins in motor neurons instead of sensory neurons, requires a different opsin targeting strategy. To transduce the motor neurons of specific muscles in the lower hind limb, without transducing the motor neurons of nearby muscles, we injected the targeted muscles with AAV6 virus containing DNA for ChR2. Motor neurons are too deep to be activated with transcutaneous light delivery as was done for pain neurons, and therefore require an implantable light delivery system. To deliver light to the transduced motor neuron axons of the sciatic nerve, we developed a fiber optic cuff which could be implanted and connected to an external patch cable at a skull-mounted ferrule. ChR2-mediated stimulation of the motor neurons produced muscle activation of the targeted muscles while the rats were awake and walking on a treadmill.

To enable effective light delivery in rodents which is neither tethered nor restricted to transcutaneous applications as discussed in the other sections of this dissertation, we developed a very small wirelessly powered implant. This device is two orders of magnitude smaller than previously reported wireless optogenetic systems which are characterized by large, head-mounted receivers. The small size of the device enables light delivery throughout the nervous system, including the brain, spinal cord, and cutaneous nerve terminals. Importantly, these devices improve on fiber optic-tethers and large, heavy wireless receivers by allowing animals to move completely unencumbered during optogenetic stimulation.

This dissertation introduces novel optogenetic tools and techniques for targeted optogenetic manipulation of the PNS and CNS, which will deepen our knowledge of the important circuits that regulate pain, sensation, motor control, and autonomic control. This body of novel technology may also serve as proof-of-concept for optogenetics-based therapies in humans.