Irrespective of the many strategies focused on dealing with spinal cord injury (SCI), there is still no way to restore motor function efficiently or an adequate regenerative therapy. One promising method that could potentially prove highly beneficial for rehabilitation in patients is to re-engage specific neuronal populations of the spinal cord following SCI. Targeted activation may maintain and strengthen existing neuronal connections and/or facilitate the reorganization and development of new connections. BioLuminescent-OptoGenetics (BL-OG) presents an avenue to non-invasively and specifically stimulate neurons; genetically targeted neurons express luminopsins (LMOs), light-emitting luciferases tethered to light-sensitive channelrhodopsins that are activated by adding the luciferase substrate coelenterazine (CTZ). This approach employs ion channels for current conduction while activating the channels through treatment with the small molecule CTZ, thus allowing non-invasive stimulation of all targeted neurons. We previously showed the efficacy of this approach for improving locomotor recovery following severe spinal cord contusion injury in rats expressing the excitatory luminopsin 3 (LMO3) under control of a pan-neuronal and motor-neuron-specific promoter with CTZ applied through a lateral ventricle cannula. The goal of the present study was to test a new generation of LMOs based on opsins with higher light sensitivity which will allow for peripheral delivery of the CTZ. In this construct, the slow-burn Gaussia luciferase variant (sbGLuc) is fused to the opsin CheRiff, creating LMO3.2. Taking advantage of the high light sensitivity of this opsin, we stimulated transduced lumbar neurons after thoracic SCI by intraperitoneal application of CTZ, allowing for a less invasive treatment. The efficacy of this non-invasive BioLuminescent-OptoGenetic approach was confirmed by improved locomotor function. This study demonstrates that peripheral delivery of the luciferin CTZ can be used to activate LMOs expressed in spinal cord neurons that employ an opsin with increased light sensitivity.

Manipulation of neuronal activity in behaving experimental animals is crucial for elucidating neuronal networks underlying brain function. Both optogenetic¹ and chemogenetic² approaches continue to be highly valuable for identifying the contributions of genetically defined neuron populations to circuit and behavioral outputs. Both methods offer distinct advantages, with optogenetic control of the activity of subsets of neurons at precise temporal scales and slower chemogenetic control of the activity of entire populations of neurons across the brain. Previous work has developed a toolset that integrates opto- and chemogenetic approaches by fusing a light-emitting luciferase to an optogenetic light-responsive element, resulting in a luminescent opsin, or luminopsin (LMO)^{3 – 5} [Fig. 1(a)]. Bioluminescence produced by oxidation of a diffusible luciferin substrate by the luciferase enzyme activates a tethered, nearby opsin. Depending on the biophysical properties of the opsin, the light generated by the luciferase can either excite or inhibit target neurons expressing the LMO. Such integration of opto- and chemogenetic approaches allows manipulation of neural activity over a range of spatial and temporal scales of the same neurons in the same experimental animal. For example, the contribution to behavioral components of the activation of an entire population of neurons can be compared to those of a subset of the same neurons, activating the opsin chemogenetically through bioluminescence or optogenetically through light fibers.⁶

Understanding how specific cell types, circuits, and brain areas are involved in generating perception, action, and cognition requires approaches to experimentally manipulate spatiotemporal patterns of neural activity in genetically defined populations. At present, optogenetic and chemogenetic techniques constitute the major actuators used for this purpose, each with distinct strengths and limitations. Optogenetics notably offers the advantage of close to millisecond precision in the control of neural output.^1,2 However, this approach requires invasive delivery of light, typically only practical to deliver in one to two locations in the brain or body. In comparison, chemogenetic approaches, including most notably designer receptors exclusively activated by designer drugs, DREADDs, provide sustained and minimally invasive modulation of neural populations distributed across much larger areas of the brain or body.^3,4 To combine the strengths of above techniques and increase user flexibility, we have developed a bimodal strategy in which a luciferase-opsin fusion molecule (a luminopsin, LMO; Refs. 5 and 6) integrates opto- and chemogenetic features in a single actuator.⁷ With bioluminescent optogenetics (BL-OG), chemogenetic modulation of neural activity is achieved via delivery of a substrate, coelenterazine (CTZ), that is oxidized by the luciferase in the LMO, producing photons immediately adjacent to (within the same molecule) the optogenetic actuator. This approach allows the use of the same molecule for chemo- and optogenetic control at distinct times in the same animal or individually.^{8 – 11} As one example, Ref. 12 employed LMO3 to drive large-scale chemogenetic excitation of multiple cell types early in development and then revealed changes in local circuit dynamics after this developmental manipulation using optogenetics in the adult animal in vivo.¹² Further, because actuation of BL-OG produces photons, users can track the time window of engagement by measuring this signal. Given the utility of this integrated single actuator approach, we sought to further improve its activation power.