| Abstract: | Luminopsins are fusion proteins of luciferase and opsin that allow interrogation of neuronal circuits at different temporal and spatial resolutions by choosing either extrinsic physical or intrinsic biological light for its activation. Building on previous development of fusions of wild-type Gaussia luciferase with channelrhodopsin, here we expanded the utility of luminopsins by fusing bright Gaussia luciferase variants with either channelrhodopsin to excite neurons (luminescent opsin, LMO) or a proton pump to inhibit neurons (inhibitory LMO, iLMO). These improved LMOs could reliably activate or silence neurons in vitro and in vivo. Expression of the improved LMO in hippocampal circuits not only enabled mapping of synaptic activation of CA1 neurons with fine spatiotemporal resolution but also could drive rhythmic circuit excitation over a large spatiotemporal scale. Furthermore, virus-mediated expression of either LMO or iLMO in the substantia nigra in vivo produced not only the expected bidirectional control of single unit activity but also opposing effects on circling behavior in response to systemic injection of a luciferase substrate. Thus, although preserving the ability to be activated by external light sources, LMOs expand the use of optogenetics by making the same opsins accessible to noninvasive, chemogenetic control, thereby allowing the same probe to manipulate neuronal activity over a range of spatial and temporal scales.Optogenetics, which offers precise temporal control of neuronal activity, has been used widely in experimental neuroscience. Although optogenetic probes are indispensable tools, conventionally their application in vivo requires invasive optical fiber implants and thus imposes significant limitations for clinical applications and for applications involving multiple brain regions (1). On the other hand, chemogenetics can modulate neuronal activity throughout the brain using a genetically targeted actuator when combined with a systemically administered small molecule. Although systemic injection of a small molecule is far less invasive than implantation of fiber optics, chemogenetics has its own limitations, such as slow response kinetics and dependence on G protein signaling, which potentially elicits unwanted secondary effects in target neurons (2).Combining the distinct advantages of opto- and chemogenetic approaches would create unprecedented opportunities for interrogation of neural circuits at a wide range of spatial scales. To allow manipulation of activity of dispersed neuronal populations using optogenetic probes without fiber-optic implants, we proposed a different approach where bioluminescence—biological light produced by enzymatic reaction between a protein, luciferase, and its diffusible substrate, luciferin—activates an opsin, which is tethered to the luciferase (3). After injection to the peripheral bloodstream, luciferin reaches a target in the brain because it crosses the blood–brain barrier (4). Light is generated by the luciferase and then activates the opsin, resulting in activation (in case of channelrhodopsins) or inhibition (in case of proton or chloride pumps) of the target neurons. Capitalizing on the major advantage of opsins as powerful generators of electrical current, our approach integrates opto- and chemogenetic methods by preserving conventional photoactivation of opsins where desired, while at the same time providing chemogenetic access to the same molecules, thus allowing manipulation of neuronal activity over a range of spatial and temporal scales in the same experimental animal.Initial proof-of-concept studies showed that Gaussia luciferase (GLuc)-emitted light is able to activate opsins when the two molecules are fused together (luminescent opsin or luminopsin, LMO) (3). Here we report a set of new LMOs, incorporating brighter versions of GLuc, with significantly improved performance. We found that the improved LMOs could modulate neuronal activity via bioluminescence in vitro, ex vivo, and in vivo and could elicit behaviors in freely moving mice. |
