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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Calcium Dye Loading
- Select calcium dyes based on the preference of the individual researcher. Here we use Fluo-8AM or Fluo-4AM. However, this method can be applied to other dyes as well: dissolve calcium dyes in DMSO to a 1 mM solution and 1% block copolymers (e.g., pluronic acid-127) to a final volume of 50 µL and sonicate for 10 min.
- Add solution to aCSF to a final concentration of 10 µM, 0.01% block copolymers for brain slices.
- For adult animals, (>12 weeks), pipette the dyes (50 µL) directly onto the brain slices, and maintain for 75 min to allow better penetration of the dye into the deep layers.
- To ensure adequate oxygenation of the submerged slice during dye incubation, prepare a glass loading chamber with a closed lid (circular jar of diameter 2.5 cm, 3.5 cm height) with calcium dyes diluted in 2.5 mL of aCSF. Oxygenate continuously with 95% O2/5% CO2. Do not bubble.
- Following dye loading, wash the tissue with aCSF and transfer it to the incubation system, slowly reducing the temperature to 15 - 16 °C until it is ready for experimental use.
2. Electrophysiological Recordings and Imaging
- Place tissue in a submerged recording chamber under a microscope and perfuse with oxygenated aCSF at a flow rate of 4 - 5 mL/min either at RT (~22 °C) or physiological temperature (~35 °C). Hold the tissue in place by using a custom-made ''harp,'' made of nylon or gold threads stretched and glued across a U-shaped piece of gold or platinum wire.
- For electrophysiology:
- Prepare recording pipettes from 1.5 mm (1.19 mm ID) borosilicate glass using a micropipette puller to achieve a final resistance of 5-6 MΩ.
- Fill the pipette with 3 - 4 µL of internal solution and visualize cells under infrared Differential Interference Contrast (IR-DIC) using a CCD camera. The intracellular solution should be carefully tailored to each experiment to achieve experimental outcomes.
- Position the pipette while maintaining positive pressure applied through a suction port on the pipette holder on the membrane of a cell using a micromanipulator. No prior membrane scraping is required since the ILM has been removed from the retina. Once the pipette is on the cell, apply gentle negative pressure to the pipette to achieve a gigaohm seal. Then, rupture the cell membrane with a brief amount of negative pressure.
- Make whole-cell current- or voltage-clamp recordings using standard techniques as appropriate.
- Calcium-imaging:
- For a single excitation wavelength of Fluo-4, filter excitation light through a 460 - 490 nm bandpass filter and emitted light through a 515 - 550 nm bandpass filter. Use a high-speed digital camera for the fastest and most sensitive recording. Acquire images as required.
- Measure alterations in fluorescence as a function of time either at a single wavelength (Fluo-4).