Retinal supplementation augments optogenetic stimulation efficacy in vivo.

OBJECTIVE: Over the last two decades, optical control of neuronal activity in the central nervous system has seen rapid development, demonstrating the utility of optogenetics as both an experimental and therapeutic tool. Conversely, applications of optogenetics in the peripheral nervous system have been relatively constrained by the challenges of temporally variable opsin expression, light penetration and immune attack of non-native opsins. Whilst opsin expression can be increased significantly through high-concentration viral induction, subsequent attack by the immune system causes temporal decay and high variability in electrophysiological response. APPROACH: In this study, we present a method to circumvent the aforementioned challenges by locally supplementing all-trans-retinal (ATR) (via a slow release pellet) to increase tissue photosensitivity in transgenic mice expressing channelrhodopsin 2 (ChR2) in nerves. MAIN RESULTS: In mice supplemented with ATR, we demonstrate enhanced electrophysiological activation and fatigue tolerance in response to optical stimulation for six weeks. SIGNIFICANCE: Local supplementation of ATR enables improved optogenetic stimulation efficacy in peripheral nerves. This method enables greater exploration of neurophysiology and development of clinically-viable optogenetic treatments in the peripheral nervous system.

Closed-loop functional optogenetic stimulation.

Optogenetics has been used to orchestrate temporal- and tissue-specific control of neural tissues and offers a wealth of unique advantages for neuromuscular control. Here, we establish a closed-loop functional optogenetic stimulation (CL-FOS) system to control ankle joint position in murine models. Using the measurement of either joint angle or fascicle length as a feedback signal, we compare the controllability of CL-FOS to closed-loop functional electrical stimulation (CL-FES) and demonstrate significantly greater accuracy, lower rise times and lower overshoot percentages. We demonstrate orderly recruitment of motor units and reduced fatigue when performing cyclical movements with CL-FOS compared with CL-FES. We develop and investigate a 3-phase, photo-kinetic model to elucidate the underlying mechanisms for temporal variations in optogenetically activated neuromusculature during closed-loop control experiments. Methods and insights from this study lay the groundwork for the development of closed-loop optogenetic neuromuscular stimulation therapies and devices for peripheral limb control.

Optogenetic Peripheral Nerve Immunogenicity.

Optogenetic technologies have been the subject of great excitement within the scientific community for their ability to demystify complex neurophysiological pathways in the central (CNS) and peripheral nervous systems (PNS). The excitement surrounding optogenetics has also extended to the clinic with a trial for ChR2 in the treatment of retinitis pigmentosa currently underway and additional trials anticipated for the near future. In this work, we identify the cause of loss-of-expression in response to transdermal illumination of an optogenetically active peroneal nerve following an anterior compartment (AC) injection of AAV6-hSyn-ChR2(H134R) with and without a fluorescent reporter. Using Sprague Dawley Rag2-/- rats and appropriate controls, we discover optogenetic loss-of-expression is chiefly elicited by ChR2-mediated immunogenicity in the spinal cord, resulting in both CNS motor neuron death and ipsilateral muscle atrophy in both low and high Adeno-Associated Virus (AAV) dosages. We further employ pharmacological immunosuppression using a slow-release tacrolimus pellet to demonstrate sustained transdermal optogenetic expression up to 12 weeks. These results suggest that all dosages of AAV-mediated optogenetic expression within the PNS may be unsafe. Clinical optogenetics for both PNS and CNS applications should take extreme caution when employing opsins to treat disease and may require concurrent immunosuppression. Future work in optogenetics should focus on designing opsins with lesser immunogenicity.

Spectrally distinct channelrhodopsins for two-colour optogenetic peripheral nerve stimulation.

Technologies for peripheral nerve stimulation have conventionally relied on the anatomic placement of electrodes adjacent to subsets of sensory fibres or motor fibres that selectively target an end effector. Here, we demonstrate the use of optogenetics to directly target the innervating fibres of an end effector by relying on retrograde transfection of adeno-associated virus serotype 6 to restrict axonal opsin expression to the desired fibre targets. By using an in vivo screen in rats, we identify the first channelrhodopsins as well as a halorhodopsin that respond to red light in the peripheral nerve. Combining two channelrhodopsins with spectrally distinct activation profiles allowed us to drive opposing muscle activity via two-colour illumination of the same mixed nerve. We also show halorhodopsin-mediated reductions in electrically evoked muscle tremor spectrally optimized for deep peripheral nerves. Our non-invasive peripheral neurostimulator with targeted multi-fascicle resolution enables scientific and clinical exploration, such as motor control in paralysis, biomimetic sensation feedback for amputees and targeted inhibition of muscle tremor.

Transdermal optogenetic peripheral nerve stimulation.

OBJECTIVE: A fundamental limitation in both the scientific utility and clinical translation of peripheral nerve optogenetic technologies is the optical inaccessibility of the target nerve due to the significant scattering and absorption of light in biological tissues. To date, illuminating deep nerve targets has required implantable optical sources, including fiber-optic and LED-based systems, both of which have significant drawbacks. APPROACH: Here we report an alternative approach involving transdermal illumination. Utilizing an intramuscular injection of ultra-high concentration AAV6-hSyn-ChR2-EYFP in rats. MAIN RESULTS: We demonstrate transdermal stimulation of motor nerves at 4.4 mm and 1.9 mm depth with an incident laser power of 160 mW and 10 mW, respectively. Furthermore, we employ this technique to accurately control ankle position by modulating laser power or position on the skin surface. SIGNIFICANCE: These results have the potential to enable future scientific optogenetic studies of pathologies implicated in the peripheral nervous system for awake, freely-moving animals, as well as a basis for future clinical studies.

Prefrontal cortical mechanisms underlying delayed alternation in mice.

The prefrontal cortex (PFC) has been implicated in the maintenance of task-relevant information during goal-directed behavior. Using a combination of lesions, local inactivation, and optogenetics, we investigated the functional role of the medial prefrontal cortex (mPFC) in mice with a novel operant delayed alternation task. Task difficulty was manipulated by changing the duration of the delay between two sequential actions. In experiment 1, we showed that excitotoxic lesions of the mPFC impaired acquisition of delayed alternation with long delays (16 s), whereas lesions of the dorsal hippocampus and ventral striatum, areas connected with the PFC, did not produce any deficits. Lesions of dorsal hippocampus, however, significantly impaired reversal learning when the rule was changed from alternation to repetition. In experiment 2, we showed that local infusions of muscimol (an agonist of the GABA(A) receptor) into mPFC impaired performance even when the animal was well trained, suggesting that the mPFC is critical not only for acquisition but also for successful performance. In experiment 3, to examine the mechanisms underlying the role of GABAergic inhibition, we used Cre-inducible Channelrhodopsin-2 to activate parvalbumin (PV)-expressing GABAergic interneurons in the mPFC of PV-Cre transgenic mice as they performed the task. Using whole cell patch-clamp recording, we demonstrated that activation of PV-expressing interneurons in vitro with blue light in brain slices reliably produced spiking and inhibited nearby pyramidal projection neurons. With similar stimulation parameters, in vivo stimulation significantly impaired delayed alternation performance. Together these results demonstrate a critical role for the mPFC in the acquisition and performance of the delayed alternation task.