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

The overall goal of this procedure is to record light stimulus evoked calcium signals from cone photoreceptors in the mouse retina using a fluorescent biosensor. This is accomplished by isolating the retina from a transgenic mouse line that expresses genetically encoded calcium sensor exclusively in the cone photoreceptors. The second step is to cut vertical slices of the retinal tissue.

Next, the retinal slices are mounted on the cover slips and placed under a two photon microscope. The final step is to record calcium signals from the individual synaptic terminals of cone photoreceptors in response to light stimulation. Ultimately, the recorded calcium signals are analyzed offline.

With this method, we can record the activity of many corn food receptors simultaneously at subcellular resolution. This is the main advantage over existing techniques such as electrical, single cell recordings, which are more difficult and time consuming. So the question that we would like to address is if and how calcium is involved in photoreceptor degeneration.

This method allows us to determine if calcium influx is driving photoreceptor degeneration or if it is a result of the fact that the cell is dying. Generally, individuals new to this method will struggle mainly because retina light sensitive weekly needs to be preserved and therefore all the delicate steps, including retina, dissection and slice ion, have to be carried out under green red light. To begin this procedure, transfer a mouse eyeball to a Petri dish containing freshly carb oxygenated extracellular solution.

Pierce the eyeball at any point along the border between the cornea and the sclera with a sharp injection needle. Next, hold the eye at the sclera with a pair of forceps and gently insert one scissors blade into the hole. Cut along the aura serrata to separate the anterior part of the eye from the eye cup.

Subsequently cut the eye cup radially at the position of the pen mark to indicate the dorsal position on the retina. Then position the eye cup such that the dorsal cut points away. Grab the sclera on the left and the right side of the eye cup by inserting the forceps tips between the retina and the sclera.

Afterward, detach the retina by gently inverting the scleral part of the iica. Finally, cut the optic nerve to free the retina from the sclera. Hold the edge of the retina with a pair of forceps and gently remove debris and vitreous body from the retinal surface using a second pair of forceps.

When the retinal surface is clean, make three additional shorter radial cuts. Now slowly immerse a glass slide into the extracellular solution close to the tissue. Gently pull the retina onto the glass slide with a ganglion cell side up by grabbing the edge to reduce mechanical damage and folding of the tissue.

Next, cut a rectangle of about one by two millimeters out of the selected retinal region using a curved scalpel blade wipe off excess solution around the tissue. Then place the pre-prepared nitrocellulose fiber membrane on top of the retina piece, such that the ganglion cell side adheres to the membrane. Immediately add a drop of extracellular medium onto the membrane to firmly attach the tissue to it.

Transfer the membrane mounted retinal tissue to the slicing chamber containing fresh extracellular solution. Then cut the retina into vertical slices of 200 micrometer thickness. Using a tissue chopper glue, a single membrane mounted slice to a glass cover slip pre-prepared with high vacuum grease.

Keep the glass surface below the retina and free from grease. After that, add a drop of extracellular solution to each cover slip mounted slice in the holding chamber at room temperature. Introduce carbogen to the chamber by bubbling a small water reservoir and to keep the atmosphere in the holding chamber.Humidified.

Allow the slices to rest in the holding chamber for 10 to 15 minutes before transferring them one by one to the recording chamber. Transfer a slice from the holding chamber to the recording chamber and immediately start perfusing it with a carbonated extracellular solution. Maintain a perfusion flow rate of two milliliters per minute and a temperature of 37 degrees Celsius.

In the recording chamber. Use a 20 x 0.95 NA water immersion objective and a CCD camera in combination with an infrared LED below the recording chamber. To locate the retinal slice, start the two photon imaging system as indicated by the manufacturer.

Next, turn on the laser and set it to 860 nanometers. Turn on the two detection channels for fluorescence imaging of ECFP and citrine. Next, using the image acquisition software, control the two photon microscope to scan and select a row of cone terminals for recording set image acquisition to 128 by 16 pixel images or a similar configuration, restrict the scanned area to the cone terminals to avoid bleaching of photo pigments in outer segments.

Turn on the laser. Allow the cones to adapt to the scanning laser and the stimulus background light for 20 to 30 seconds before presenting light stimuli or applying pharmacological agents. Now start the presentation of the arbitrary stimuli.

The stimuli are generated by modulating the intensity of the two LEDs over time, using a microprocessor board controlled by customized software. Then start recording the two fluorescence channels simultaneously using the respective image acquisition software. The recordings were focused on the synaptic terminals of cone photoreceptors in the retinal slices.

This is an example of a recorded region with seven individual terminals. Fluorescence was recorded using two channels, one for the fre scepter citrine, and one for the fret donor.ECFP.Shown. Here are the light evoke changes in citrine and ECFP fluorescence recorded in a single region of interest.

And here are the calcium responses of a cone terminal to a series of one second bright light flashes in five second intervals. This figure shows the exemplary quantitative analysis of light evoked cone calcium responses. And here are the light evoked calcium responses from a single cone terminal.

This figure shows the determination of the mean calcium response. Resting calcium level represents the baseline prior to light stimulation and the response size is ra, which is the area between the resting calcium level and the peak amplitude, and between the kinetics of response onset and offset. This procedure can be further extended.

For example, photoreceptor calcium signals can be major under diverse pharmacological and large stimulation conditions to investigate like adaptation or study different steps of photo transduction in detail. This technique allows to investigate calcium dynamics in cone photoreceptors under normal physiological conditions. And this is particularly relevant for understanding primary and secondary cone photoreceptor degeneration in human blinding diseases such as acro topia or retinitis pigmentosa.