Dataset Information

Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina.

ABSTRACT: Targeting the photosensitive ion channel channelrhodopsin-2 (ChR2) to the retinal circuitry downstream of photoreceptors holds promise in treating vision loss caused by retinal degeneration. However, the high intensity of blue light necessary to activate channelrhodopsin-2 exceeds the safety threshold of retinal illumination because of its strong potential to induce photochemical damage. In contrast, the damage potential of red-shifted light is vastly lower than that of blue light. Here, we show that a red-shifted channelrhodopsin (ReaChR), delivered by AAV injections in blind rd1 mice, enables restoration of light responses at the retinal, cortical, and behavioral levels, using orange light at intensities below the safety threshold for the human retina. We further show that postmortem macaque retinae infected with AAV-ReaChR can respond with spike trains to orange light at safe intensities. Finally, to directly address the question of translatability to human subjects, we demonstrate for the first time, AAV- and lentivirus-mediated optogenetic spike responses in ganglion cells of the postmortem human retina.

SUBMITTER: Sengupta A

PROVIDER: S-EPMC5090658 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina.

Sengupta Abhishek A Chaffiol Antoine A Macé Emilie E Caplette Romain R Desrosiers Mélissa M Lampič Maruša M Forster Valérie V Marre Olivier O Lin John Y JY Sahel José-Alain JA Picaud Serge S Dalkara Deniz D Duebel Jens J

EMBO molecular medicine 20161102 11

Targeting the photosensitive ion channel channelrhodopsin-2 (ChR2) to the retinal circuitry downstream of photoreceptors holds promise in treating vision loss caused by retinal degeneration. However, the high intensity of blue light necessary to activate channelrhodopsin-2 exceeds the safety threshold of retinal illumination because of its strong potential to induce photochemical damage. In contrast, the damage potential of red-shifted light is vastly lower than that of blue light. Here, we show ...[more]

PMID: 27679671

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Project description:Channelrhodopsins (ChRs), which form a distinct branch of the microbial rhodopsin family, control phototaxis in green algae. Because ChRs can be expressed and function in neuronal membranes as light-gated cation channels, they have rapidly become an important optogenetic tool in neurobiology. While channelrhodopsin-2 from the unicellular alga Chlamydomonas reinhardtii (CrChR2) is the most commonly used and extensively studied optogenetic ChR, little is known about the properties of the diverse group of other ChRs. In this study, near-infrared confocal resonance Raman spectroscopy along with hydrogen-deuterium exchange and site-directed mutagenesis were used to study the structure of red-shifted ChR1 from Chlamydomonas augustae (CaChR1). These measurements reveal that (i) CaChR1 has an all-trans-retinal structure similar to those of the light-driven proton pump bacteriorhodopsin (BR) and sensory rhodopsin II but different from that of the mixed retinal composition of CrChR2, (ii) lowering the pH from 7 to 2 or substituting neutral residues for Glu169 or Asp299 does not significantly shift the ethylenic stretch frequency more than 1-2 cm(-1) in contrast to BR in which a downshift of 7-9 cm(-1) occurs reflecting neutralization of the Asp85 counterion, and (iii) the CaChR1 protonated Schiff base (SB) has stronger hydrogen bonding than BR. A model is proposed to explain these results whereby at pH 7 the predominant counterion to the SB is Asp299 (the homologue to Asp212 in BR) while Glu169 (the homologue to Asp85 in BR) exists in a neutral state. We observe an unusual constancy of the resonance Raman spectra over the broad range from pH 9 to 2 and discuss its implications. These results are in accord with recent visible absorption and current measurements of CaChR1 [Sineshchekov, O. A., et al. (2013) Intramolecular proton transfer in channelrhodopsins. Biophys. J. 104, 807-817; Li, H., et al. (2014) Role of a helix B lysine residue in the photoactive site in channelrhodopsins. Biophys. J. 106, 1607-1617].

Project description:When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, making it difficult to isolate inputs from individual sources with electrical stimulation. However, by using channelrhodopsin assisted circuit mapping (CRACM), this limitation can now be overcome. Here, we report a method to use CRACM to map ascending inputs from lower auditory brainstem nuclei and commissural inputs to an identified class of neurons in the inferior colliculus (IC), the midbrain nucleus of the auditory system. In the IC, local, commissural, ascending, and descending axons are heavily intertwined and therefore indistinguishable with electrical stimulation. By injecting a viral construct to drive expression of a channelrhodopsin in a presynaptic nucleus, followed by patch clamp recording to characterize the presence and physiology of channelrhodopsin-expressing synaptic inputs, projections from a specific source to a specific population of IC neurons can be mapped with cell type-specific accuracy. We show that this approach works with both Chronos, a blue light-activated channelrhodopsin, and ChrimsonR, a red-shifted channelrhodopsin. In contrast to previous reports from the forebrain, we find that ChrimsonR is robustly trafficked down the axons of dorsal cochlear nucleus principal neurons, indicating that ChrimsonR may be a useful tool for CRACM experiments in the brainstem. The protocol presented here includes detailed descriptions of the intracranial virus injection surgery, including stereotaxic coordinates for targeting injections to the dorsal cochlear nucleus and IC of mice, and how to combine whole cell patch clamp recording with channelrhodopsin activation to investigate long-range projections to IC neurons. Although this protocol is tailored to characterizing auditory inputs to the IC, it can be easily adapted to investigate other long-range projections in the auditory brainstem and beyond.

Dataset Information

Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina.

Publications

Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina.

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