Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina - PubMed (original) (raw)
Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina
Wei Wei et al. Nat Protoc. 2010 Jul.
Abstract
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.
Conflict of interest statement
COMPETING INTEREST STATEMENT: The authors declare that they have no competing financial interests.
Figures
Figure 1. Setup for two-photon targeted recording of light response from GFP+ retinal ganglion cells
a. (Left) Schematic of the two-photon microscope in the configuration for targeted recording. A mode-locked Ti-sapphire is used to excite the fluorophores at 920 nm (106 mW power into the back of the microscope and 11 mW at the sample). Once a GFP-labeled cell body is identified, transmitted infrared light detected with a CCD camera is used to obtain electrophysiological recordings. INSET: A glass microelectrodes containing Alexa 594 (20 μM in Ames’ medium) was visualized together with the GFP signal from the recorded cell to confirm correct targeting and the integrity of the plasma membrane. GFP+ membrane is visible within the recording electrode during cell-attached recording. Scale bar: 25 μm (Right) Schematic of the two-photon microscope in the light stimulation configuration. Once the desired recording configuration has been obtained, visual stimuli from an OLED are presented to the retina through a plano-convex lens, a 50/50 beam splitter, and the objective. D1 = dichroic 1 (695 nm split, Chroma part # 695dcxxr); D2 = dichroic 2 (585 nm split, Chroma part # 585dcxr); BP1 = band pass 1 (500–550 nm, Chroma part # ET525/50m-2p); BP2 = band pass 2 (600–660 nm, Chroma part # HQ630/60m-2p). Modified from . b. An example photo of the components and layout of the Olympus Fluoview 300 system. c. The layout of the visual stimulation arm. The OLED, a lever-activated threaded iris (optional), and the plano-convex lens are mounted in a 30 mm cage system via a side epifluorescence port of the microscope.
Figure 2
The landmarks in the choroid for marking the orientation of the retina. Under a dissection microscope, an eye cup with the retinal pigment epithelium attached is positioned with the ganglion cell layer facing up and dorsal side facing upward (Top and middle). The nasal/temporal axis is aligned with a horizontal stripe running beneath the optic nerve, with a dark-appearing patch in the ventral side (Lower image). The nasal/temporal side is opposite for left and right eyes. Scale bars: 250 μm
Figure 3. Examples of successful and unsuccessful experiments of two-photon targeted recording from GFP-expressing cells
a–c An example experiment using a Drd4-GFP transgenic mouse, which expressed GFP in nasal-preferring On-OFF direction selective cells. Light-evoked spike responses were obtained from the GFP-positive cell using cell-attached recordings. Alexa 594 was added to the internal solution for labeling the dendritic morphology by two-photon imaging after the recording. a. Light responses to stationary spots. Top trace, cell-attached recordings showing the On and Off spike responses to a 100 μm white spot centered on the soma. Lower graph, spike density histograms of corresponding on & off spike responses (10 repetitions, 50 ms bins). Grey bar represents the time of the spot stimuli. b. Light responses to drifting gratings. Top trace, cell-attached recordings showing the On and Off spike responses to drifting gratings in the preferred direction. Lower graph, direction tuning curves (spike counts during 3s of gratings, 12 directions, 4 repetitions) show nasal preference to drifting gratings. The black arrow indicates the vector sum of spike responses. c. Maximal intensity projections of Z-stack images of the same cell filled with Alexa 594 through a whole cell electrode showing the dendritic arbors in the On and Off sublaminae. Scale bar: 50 μm. d. A cell-attached recording from a GFP-labeled cell from a Drd4-GFP mouse that was exposed to bright light from the halogen bulb through the condenser. The cell exhibited tonic firing, with no light-evoked responses to a 100 μm white spot centered on the soma, presumably due to photobleaching.
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