Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 99, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/52588
Keywords
Neuroscience; Issue 99; Ca2+ biosensor; two-photon Ca2+ imaging; cell death; retinal slice preparation; retinal degeneration
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Funding
- Deutsche Forschungsgemeinschaft (DFG) (Werner Reichardt Centre for Integrative Neuroscience Tubingen) [EXC 307, KFO 134]
- German Federal Ministry of Education and Research (BMBF) (BCCN Tubingen) [FKZ 01GQ1002]
- European Union (DRUGSFORD) [HEALTH-F2-2012-304963]
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Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (slices) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows in-slice measurement of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.
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