Non-invasive activation of optogenetic actuators

Abstract

The manipulation of genetically targeted neurons with light (optogenetics) continues to provide unprecedented avenues into studying the function of the mammalian brain. However, potential translation into the clinical arena faces a number of significant hurdles, foremost among them the need for insertion of optical fibers into the brain to deliver light to opsins expressed on neuronal membranes. In order to overcome these hardware-related problems, we have developed an alternative strategy for delivering light to opsins which does not involve fiber implants. Rather, the light is produced by a protein, luciferase, which oxidizes intravenously applied substrate, thereby emitting bioluminescence. In proof-of-principle studies employing a fusion protein of a light-generating luciferase to a light-sensing opsin (luminopsin), we showed that light emitted by Gaussia luciferase is indeed able to activate channelrhodopsin, allowing modulation of neuronal activity when expressed in cultured neurons. Here we assessed applicability of the concept in vivo in mice expressing luminopsins from viral vectors and from genetically engineered transgenes. The experiments demonstrate that intravenously applied substrate reaches neurons in the brain, causing the luciferase to produce bioluminescence which can be imaged in vivo, and that activation of channelrhodopsin by bioluminescence is sufficient to affect behavior. Further developments of such technology based on combining optogenetics with bioluminescence - i.e. combining light-sensing molecules with biologically produced light through luciferases - should bring optogenetics closer to clinical applications.

Keywords: Gaussia luciferase; bioluminescence; channelrhodopsin; coelenterazine; imaging; in vivo; transgenic mice; viral vectors.

Figure 1. Light sources for opsins

Optogenetic approaches to control of neuronal activity utilize light-activated photosensitive proteins (microbial opsins; here: channelrhodopsin, ChR), with a fluorescent protein fused to the C-terminus of ChR to allow visualization by fluorescence microscopy (here: yellow fluorescent protein, YFP). The opsin is activated by applying light via fiber optics from an external light source. Fusion of a luciferase (here: Gaussia luciferase, GLuc) to the N-terminus of ChR creates a luminescent opsin, or luminopsin. Application of the GLuc substrate coelenterazine (CTZ) leads to an enzymatic reaction resulting in light (photon) production and opening of the channel. Utilizing this “biological” light source allows non-invasive activation of the opsin.

Figure 2. In vivo imaging of luminopsins

A. Experimental scheme: newborn pups (postnatal day 1) were injected into the right cortex with a glass micropipette, delivering lentivirus containing LMO1 (GLuc-ChR2-EYFP) under control of a CAG promoter. Mice were returned to the nest. After weaning, mice were subjected to in vivo bioluminescence imaging (postnatal day 30). B. Localized infection of cortical neurons was verified by fluorescence microscopy of brain sections of injected mice at the end of the experiments. As LMOs are fused to EYFP, expression can be readily visualized. C. Bioluminescence images obtained from four different mice after intravenous injection of CTZ (200 μM final concentration). Mice were shaved to avoid scattering of light from hair. D. Quantification of photons emitted from the four mice shown in C. Radiance over the regions of interest was calculated.

Figure 3. In vivo behavioral effects of luminopsins

Neonatal mice were injected into the right motorcortex with lentivirus containing LMO2 (GLuc-VChR1-EYFP) under control of a CAG promoter. Control animals received injections without virus in the solution. Mice were returned to the nest and tested 4–6 weeks later. For behavioral testing, mice were placed in a rectangular bin (14 in × 11 in) and allowed to move freely. After intravenous injection of CTZ, mice were placed back in the bin and allowed to continue moving. Trajectories of their movements were captured over ~ 15 seconds with a video camera, and traced manually. There was no apparent difference in movements of the control mouse (blue traces) before and after CTZ. However, mice which received LMO2-expressing virus in the right motor cortex (red traces) displayed repetitive left (contralateral) turns.

Figure 4. Conditional LMO2 mouse

A. The sequence of LMO2 (GLuc-VChR1-EYFP, colored boxes) was inserted in place of the original reporter in the Ai9 vector (gray shapes), creating the LMO2-ROSA targeting vector. A mouse line carrying this allele in the ROSA locus was generated by ES cell targeting and blastocyst injection. Mice carrying this conditional LMO2 allele were subsequently mated to CaMKII3 Cre-expressing mice. Offspring were genotyped for the presence of both the LMO2-ROSA allele and the CaMKII3 Cre transgene. B. Acute horizontal brain slices from mice both positive for CaMKII3 Cre and LMO2 were placed in ACSF in a well of a 24-well plate. Bioluminescence images (pseudo color) overlayed on bright-field images (gray scale) were taken before (upper panel) and after (lower panel) addition of CTZ (100 μM final concentration). C. Fluorescent image showing CaMKII3-directed expression of LMO2 in dentate gyrus granule cells in the hippocampus. ML: molecular layer; GCL: granule cell layer. The dotted rectangle indicates the region shown in high magnification in D and E. D. Higher magnification image of the same slice. E. Bioluminescent image of the same region shown in D, after addition of CTZ to the chamber. F. Time course of bioluminescence. 50 μM of CTZ was superfused during the time indicated by the horizontal bar.