The BioLuminescent-OptoGenetic in vivo Response to Coelenterazine is Proportional, Sensitive and Specific in Neocortex

Abstract

BioLuminescent (BL) light production can modulate neural activity and behavior through coexpressed OptoGenetic (OG) elements, an approach termed 'BL-OG'. Yet, the relationship between BL-OG effects and bioluminescent photon emission has not been characterized in vivo.Further, the degree to which BL-OG effects strictly depend on optogenetic mechanisms driven by bioluminescent photons is unknown. Crucial to every neuromodulation method is whether the activator shows a dynamic concentration range driving robust, selective, and non-toxic effects.We systematically tested the effects of four key components of the BL-OG mechanism (luciferin, oxidized luciferin, luciferin vehicle, and bioluminescence), and compared these against effects induced by the Luminopsin-3 (LMO3) BL-OG molecule, a fusion of slow burn Gaussia luciferase (sbGLuc) and Volvox ChannelRhodopsin-1 (VChR1). We performed combined bioluminescence imaging and electrophysiological recordings while injecting specific doses of Coelenterazine (substrate for sbGluc), Coelenteramide (CTM, the oxidized product of CTZ), or CTZ vehicle. CTZ robustly drove activity in mice expressing LMO3, with photon production proportional to firing rate. In contrast, low and moderate doses of CTZ, CTM, or vehicle did not modulate activity in mice that did not express LMO3. We also failed to find bioluminescence effects on neural activity in mice expressing an optogenetically non-sensitive LMO3 variant. We observed weak responses to the highest dose of CTZ in control mice, but these effects were significantly smaller than those observed in the LMO3 group. These results show that in neocortex in vivo, there is a large CTZ range wherein BL-OG effects are specific to its active chemogenetic mechanism.

Selective Postnatal Excitation of Neocortical Pyramidal Neurons Results in Distinctive Behavioral and Circuit Deficits in Adulthood

“…To accommodate the need for non-invasive stimulation of genetically targeted neural circuits in a postnatally growing brain and temporally precise interrogation of the same circuits in the same animal as an adult, we employed bioluminescent optogenetics (BL-OG) (Berglund et al, 2013(Berglund et al, , 2016Park et al, 2017;Song et al, 2018;Zenchak et al, 2018;Gomez-Ramirez et al, 2019;Yu et al, 2019). Here an optogenetic element can be activated either by light emitted from a tethered luciferase oxidizing a small molecule substrate, or by light emitted from physical sources, such as LEDs or lasers ( Figure 1A).…”

“…We induced hyperexcitation in developing mouse pups once a day during post-natal days 4-14, capturing the window of significant outgrowth and development of key cortical circuits. BL-OG has been used to activate or silence neurons in adult mice and rats, using various routes of administration of CTZ (Tung, Gutekunst and Gross, 2015;Berglund et al, 2016;Park et al, 2017;Gomez-Ramirez et al, 2019;Yu et al, 2019). To test the efficacy of BL-OG in pups, we tested for bioluminescence emission after intraperitoneal CTZ administration.…”

BL‐OG: BioLuminescent‐OptoGenetics

“…The title “BL‐OG” has been increasingly applied for the combined BioLuminescent‐OptoGenetic approach. This term has been used and “published” in grant announcements and conference abstracts for several years, and is now appearing in published papers in this issue (e.g., Gomez‐Ramirez, More, Friedman, Hochgeschwender, & Moore, ). This acronym not only captures the constituent elements of the strategy, it does so with an isomorphic dash, representing the amino‐acid linker that connects a luciferase and an optogenetic element in an LMO.…”

“…Any of these necessary elements could in concept cause changes in neural activity, clouding the interpretation of the driver of the BL‐OG effect. To test the specificity of the BL‐OG approach, Gomez‐Ramirez et al () tested each component systematically and showed that in reasonable concentrations neither CTZ, coelenteramide, solvent, nor bioluminescence alone changed neural activity in isolation. They further showed that changes in neural activity were directly proportional to the amount of bioluminescence emitted.…”

Miniaturized Devices for Bioluminescence Imaging in Freely Behaving Animals

“…5. For comparison, we overlaid time courses acquired using the BLmini and EMCCD in a different experiment ( [11]; Fig. 5A).…”