Background

Optogenetics has emerged as a potent tool in experimental neuroscience. The expression of light-gated ion channels in neurons can be used to control neuronal activity with great temporal precision, and by targeting of that expression to different neuronal populations the role of that activity can be studied very specifically. Our lab and others have demonstrated that activity-based experimental therapies, such as exercise and electrical stimulation, if applied after peripheral nerve injury will enhance the regeneration of cut axons of motor and sensory neurons (Al-Majed et al., 2000; Brushart et al., 2002; Wood et al., 2012; Thompson et al., 2014). Exercise in the form of treadmill running is a powerful promoter of axon regeneration but it is difficult to separate the effects of exercise-induced activation of injured neurons from other effects, such as alterations in available hormones, changes in muscle properties, modulation of immune cells or activation of uninjured neurons in networks that generate the locomotion. Furthermore, when modeling the effects of exercise on recovery from peripheral nerve injury, it is difficult to determine whether the effect is due to the production of action potentials in the neurons whose axons have been severed or whether exercise may alter the membrane potential of axotomized neurons without directly causing it to fire an action potential. To determine these parameters would be highly challenging in an awake behaving animal running on a treadmill.

Brief electrical stimulation of a cut peripheral nerve also enhances axon regeneration (Elzinga et al., 2015; Gordon and English, 2016). It offers some technical advantages in probing the mechanism of action of exercise on peripheral axon regeneration. A specific peripheral nerve can be unilaterally targeted by applying stimulation to the nerve, and the frequency and duration of stimuli (action potentials) applied to the nerve can be controlled. However, other cell types within a peripheral nerve also respond to electrical stimulation (Schwann cells and probably immune cells), and the conductive nature of tissues can facilitate current spread. The lack of cell-type specificity of electrical stimulation and exercise also have limited some of the mechanistic questions that can be proposed. Here, we describe a way to use optogenetics to apply a cell-type specific activity-based therapy and also to use optogenetics to provide a functional outcome measure (direct muscle response, i.e., the direct M response) following nerve injury.

We used mouse genetics to target the expression of the light-sensitive cation channel, channelrhodopsin 2, selectively in sensory neurons, motoneurons, or both. In the mice we used, only a subset of the neurons with axons in peripheral nerves expressed this opsin, those that also express yellow fluorescent protein (YFP); other neurons acted as internal controls. We then activated the opsin-expressing neurons by application of light to their axons in peripheral nerves. We found that brief optogenetic activation enhanced peripheral axon regeneration regardless of whether motor or sensory neurons were treated individually or synchronously (Figure 1). We also used optogenetics to measure functional recovery using electrophysiology (Ward et al., 2016).

A major advantage of the use of optogenetics in this model system is that activity can be discretely controlled in genetically targeted neuronal populations of interest. Optogenetics can be used to evaluate the mechanism behind the success of activity-based therapies on peripheral axon regeneration. For example, we have also used an inhibitory opsin, Natromonas halorhodopsin, fused to a light-emitting Renilla luciferase and Venus (a protein for bioluminescent resonance energy transfer that amplifies the emitted light) (Tung et al., 2015; Berglund et al., 2016) to inhibit neuronal activity during treadmill exercise. The luciferase oxidizes a small molecule substrate, h-Coelenterazine, to generate yellow photons and activate the halorhodopsin. Thus, after administration of h-Coelenterazine, neurons expressing this iLMO2 fusion protein become hyperpolarized. In motoneurons whose axons were regenerating, we found that inhibiting motoneuron activity during treadmill exercise blocked the enhancing effect of exercise (Jaiswal et al., 2017). Considered together, the results of these optogenetic experiments lead us to conclude that neuronal activity is both necessary and sufficient to promote the regeneration of injured peripheral axons.

Future advances in excitatory and inhibitory opsins, bioluminescence, luminopsins and chemogenetics will continue to facilitate the development of neuromodulation and novel gene therapy approaches. Via chemogenetic manipulation using DREADDs (Cre-dependent excitatory designer receptor exclusively activated by designer drugs), we have shown that subthreshold excitation of motoneurons and sensory neurons is sufficient to enhance their regeneration (Jaiswal et al., 2018). The application of these new optogenetic tools will expand our ability to further define the activity requirements of highly specific neuronal populations.


Figure 1. graphical representation of the mouse model and sciatic nerve. Multiple promoters can be used to selectively express ChR2 (or other opsins) in targeted cell types. In the cartoons of spinal cords and dorsal root ganglia cross-sections, different cell types express ChR2 and are responsive to blue light activation in the periphery. The whole sciatic nerve and its terminal branches are depicted in the cartoon. Using different transgenic mouse models, we have shown that excitatory neuronal stimulation and exercise enhance axon regeneration following sciatic nerve injury, and this effect can be blocked with inhibitory luminopsins.