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Cell type-specific green fluorescent protein (GFP) expression in the retina has been achieved in an expanding repertoire of transgenic mouse lines, which are valuable tools for dissecting the retinal circuitry. However, measuring light responses from GFP-labeled cells is challenging because single-photon excitation of GFP easily bleaches photoreceptors. To circumvent this problem, we use two-photon excitation at 920 nm to target GFP-expressing cells, followed by electrophysiological recording of light responses using conventional infrared optics. This protocol offers fast and sensitive detection of GFP while preserving the light sensitivity of the retina, and can be used to obtain light responses and the detailed morphology of a GFP-expressing cell. Targeting of a GFP-expressing neuron takes less than 3 min, and the retina preparation remains light sensitive and suitable for recording for at least 8 h. This protocol can also be applied to study retinal neurons labeled with other two photon-excitable fluorophores. It is assumed that potential users of this protocol will have a basic understanding of retinal physiology and patch-clamp recording, which are not described in detail here.

Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina