Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Tariq Brown - Central Michigan University
Co-Author(s): Ute Hochgeschwender, Central Michigan University, Michigan, Mt. Pleasant
Optogenetics is the use of light to manipulate light sensing molecules, opsins. Activation of these light-gated ion channels and pumps, when expressed by neurons, results in depolarization or hyperpolarization of cell membranes. This allows activation and silencing of neuronal circuits in behaving experimental animals via light fibers implanted into the animal’s brain. We proposed a strategy for non-invasive optogenetics by switching out the light source from an invasive physical to a non-invasive biological one, i.e., a light producing protein, or luciferase. The luciferase emits light, activating the optogenetic actuator upon application of its small-molecule substrate, coelenterazine (CTZ). Thus, using ‘biological’ light allows non-invasive photonic control of neurons. Such capability is highly advantageous in long-term animal experiments, or in experiments where all members of a genetically defined neural circuit need to be activated. We hypothesized that efficient activation of optogenetic elements by a luciferase will depend on the light emission of the luciferase. To test this, we engineered fusion proteins of Gaussia luciferase (GLuc), a luciferase from the copepod Gaussia princeps, with channelrhodopsins from algae and proton pumps from fungus. Specifically, we used the wildtype version and mutated forms of GLuc with increased light emission in combination with channelrhodopsin variants from Chlamydomonas (ChR2) and Volvox (VChR1), and the proton pump from Leptosphaeria maculans (Mac). The cloned fusion proteins were expressed in primary cultured neurons and assessed for their effects on activating and silencing neurons. To this end, electrical activity was recorded from neurons before and after administration of the luciferase substrate versus administration of vehicle (controls). We found significant (two-tailed Students’ T-test) differences between the luciferase variants in their ability to activate opsins, with the mutated GLuc variant sbGLuc resulting in the most efficient coupling to opsins. Identification of this relationship between luciferase light emission and opsin activation confirmed our hypothesis and provided us with a tool for in vivo experiments. Ongoing research involves systematic mutation and screening for luciferases with further increased light emission for highly efficient non-invasive optogenetics.
References: Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8: 1263-1268.
Berglund K, Birkner E, Augustine GJ, Hochgeschwender U (2013) Light-Emitting Channelrhodopsins for Combined Optogenetic and Chemical-Genetic Control of Neurons. PLoS One 8(3):e59759.
Berglund K, Clissold K, Li HE, Wen L, Park SY, Gleixner J, Klein ME, Lu D, Barter JW, Rossi MA, Augustine GJ, Yin HH, Hochgeschwender U. Luminopsins integrate physical and biological light sources for opsin activation in vivo. Proc Natl Acad Sci USA 113: E358-67, 2016.
Funder Acknowledgement(s): Funding was provided by an NSF/BAIN EAGER grant to U. Hochgeschwender.
Faculty Advisor: Ute Hochgeschwender, firstname.lastname@example.org
Role: Created and replicated fluorescent protein-containing plasmids, and infected neuronal cells with the synthesized plasmids.