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Lu Yin, Ali H. Cetin, Ying Geng, Robin Sharma, Kamran Ahmad, Edward M. Callaway, David R. Williams, William H. Merigan; In Vivo Optical Recording From Mouse Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5597. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We demonstrate in vivo optical recording of retinal ganglion cell (RGC) calcium responses to visual stimulation, imaged at high spatial resolution with a fluorescence adaptive optics scanning laser ophthalmoscope (FAOSLO).
Mouse RGCs were sparsely transduced with the genetically encoded calcium indicator G-CaMP3 by retrograde transport of a glycoprotein-deleted rabies viral vector injected into the superior colliculus. The calcium response of each cell was imaged with a FAOSLO (excitation, 488 nm; emission, 503-538 nm). We activated ganglion cells by stimulating UV-sensitive cones with temporal modulation of UV light (365 nm), or middle-wavelength sensitive cones with temporal modulation of the 488 nm laser. Mean fluorescence intensity (F) was measured in regions of interest manually selected for each ganglion cell soma. The response of ganglion cells was calculated as the percentage of fluorescence change from baseline fluorescence level (ΔF/F0). The same ganglion cells were recorded from over multiple days.
The viral vector produced high levels of expression of G-CaMP3 in mouse RGCs, permitting imaging with light levels that are not damaging to the retina. We recorded the calcium response of many ganglion cells simultaneously at 5 Hz, which is more than adequate to capture the time course of the calcium response. Within the imaged population, ganglion cells show a variety of responses, which differ in their polarities, amplitudes and waveforms. The time course of the G-CaMP3 response was similar to that shown in vitro (Borghuis et al., 2011). Ganglion cell responses were consistent across days. Rabies transduced ganglion cells eventually degenerated and their light responses diminished approximately 8 days after vector injection.
This method has the following advantages over in vitro recording methods: (1) it will be possible to discover the functional role of different retinal ganglion cell classes for central visual processing because the visual system is intact; (2) it allows repeated imaging of single cells over time, so that cell behavior can be more thoroughly characterized or developmental changes assessed; (3) it enables recording from large numbers of RGCs in parallel. This method will be further enhanced by incorporating two-photon imaging, and may eventually be applicable to primates.
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