Imaging Ca²⁺ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Abstract

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 (Ca 2+ ), 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 Ca 2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca 2+ 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 Ca 2+ level. The protocol also allows "in-slice measurement" of absolute Ca 2+ 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 Ca 2+ signaling as well as the potential involvement of Ca 2+ in photoreceptor death and retinal degeneration.

Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration

“…CN02–CN05 were then tested with respect to inhibition of CNGCs and Ca 2+ influx in cone photoreceptors. To this end, we used acute, freshly prepared retinal slices from WT animals expressing the TN-XL Ca 2+ biosensor exclusively in cones ( 27 ), monitored by a two-photon Ca 2+ -imaging approach ( 28 ) ( Fig. 3 A ).…”

Redefining the role of Ca2+-permeable channels in photoreceptor degeneration using diltiazem

“…Light stimulus-evoked Ca 2+ responses were recorded in cone axon terminals using a two-photon (2P) microscope, as previously described [75]. In brief, we used adult (~ P90) transgenic HR2.1:TN-XL mice (for details, see above).…”

Studying a Light Sensor with Light: Multiphoton Imaging in the Retina

“…However, with techniques for targeting and visualising neurons deep in the tissue, whole-mounts are quickly catching up in this respect. In terms of imaging, slices have the additional advantage that layers close to the light-sensitive photoreceptor outer segments, such as the outer plexiform layer (OPL), become more accessible for optical recordings [4,50,51]: with the axis of the excitation laser then running perpendicular to the retinal layering, direct 1P and 2P opsin excitation (see Sections 1.2, 3.4) is substantially reduced. The main downside of the slice is that crucial lateral connections in the retinal network are severed by the sectioning.…”

“…A stepby-step description of mouse retina dissection, including retinal slicing and whole-mount preparation, as well as a list of tools and materials, is available elsewhere (e.g. [50,60]). In the following, we will discuss a few guidelines regarding tissue preparation that are important for successfully imaging light-driven activity in the mouse retina.…”