Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

All animal procedures were conducted in accordance with standard animal ethical guidelines (European Communities Directive 86/609/EU) and approved by the local animal ethics committees. 8 weeks-old Long Evans rats were used for the present study. The animals were obtained from a commercial source (see Table of Materials).

1. Preparation of media and flat-mounting assembly

1. Ames' Medium (1 L)
   1. In a 1 L glass bottle, combine the Ames' Medium powder, 1.9 g/L NaHCO₃, 10 ml of Penicillin/Streptomycin 100x, and 1 L of deionized water (see Table of Materials). Adjust the pH to 7.4 and the osmolarity to 280 mOsm with deionized water or NaHCO₃. Sterilize the solution by filtering it through a 0.2 µm pore size filter under a hood.
      NOTE: Store the sterilized medium at 4 °C. This solution remains stable and can be used for up to 1 month.
2. Mounting membranes
   1. Attach one PTFE porous membrane (see Table of Materials) to a washer using small drops of glue. Allow it to dry for at least 15 min.
   2. To achieve translucency, immerse the membranes in 70% ethanol for 1 min.
   3. Rinse the membranes twice with deionized water to completely remove the ethanol. Store them in deionized water to prevent opacity.

2. GCL labeling and rat retina flat-mounting

NOTE: This labeling method does not differentiate RGCs from displaced amacrine cells. If selective labeling of RGCs is desired, consider using AAVs with RGC-specific promoters¹¹ and/or retrograde labeling through the optic nerve¹². To discriminate between classes of ON- and OFF-center RGCs, classify RGCs based on their light response^13,14, and utilize newer versions of genetically encoded calcium indicators that offer increased sensitivity and the ability to measure single action potentials¹⁵.

1. Intravitreal injection
   1. Anesthetize the 8-week-old Long Evans rat with 2% isoflurane/1% O₂ until there is no pedal reflex, and maintain anesthesia with a rat nasal mask (see Table of Materials).
      NOTE: During anesthesia, position the animal on a heating pad to maintain body temperature.
   2. Administer one drop of commercially available eye drops (see Table of Materials) to dilate the pupil.
   3. Before proceeding with surgery, examine the eye for abnormalities using fundoscopy and Optical Coherence Tomography (OCT) with an in vivo retinal imaging system. Apply one drop of Methocel 2% to facilitate cornea-objective contact (see Table of Materials).
      NOTE: If any abnormalities are detected, do not proceed with the subsequent steps for that eye.
   4. Apply one drop of Prescaine as a local anesthetic. Fix the eyelid and limbal conjunctiva with a commercially available suture filament (see Table of Materials). Create a 1 mm sclerotomy 4 mm from the limbus using a 30 G needle.
      1. Attach a 36 G blunt needle to a precision syringe and inject the AAV particles carrying the genetically encoded calcium indicator into the vitreous for 30 s, at a 45° angle. In this study, we used AAV2-CAG-GCaMP5G (7.5 x 10¹¹ GC/mL in HBSS) (see Table of Materials).
         NOTE: AAV constructs that do not encode potentially tumorigenic gene products or toxin molecules and are produced without a helper virus can be handled in Biosafety Level 1 (BSL-1) facilities. Otherwise, if considered biohazardous material under BSL-2 containment, proper precautions must be taken¹⁶. AAVs encoding for GCaMP are considered BSL-1 and do not require manipulation under biosafety cabinets.
   5. Apply one drop of Tobradex (see Table of Materials) to prevent inflammation and as an antibiotic prophylaxis.
   6. If desired, repeat steps 2, 3, and 4 with the other eye.
      NOTE: Check on the animals 12-24 h after surgery to ensure there are no adverse reactions.
   7. Three days after injection, examine the retinal structure using fundoscopy and OCT with an in vivo retinal imaging system (see Table of Materials).
   8. Two weeks after injection, the GCL should emit fluorescence. Assess retinal structure and AAV expression by fluorescence fundoscopy using an in vivo retinal imaging system.
      NOTE: According to Weitz et al.¹², fluorescence from AAV2-CAG-GCaMP5G becomes noticeable at 1-week post-injection and intensifies by 2 weeks. Starting from the fourth week, overexpression of GCaMP induces cytomorbidity. Dying cells exhibit a high baseline fluorescence signal in the nucleus and cytoplasm that does not fluctuate in response to stimulation. In healthy cells, GCaMP expression is confined to the cytoplasm and excluded from the nucleus^7,8,12,17,18. These features can be observed ex vivo during microscopy imaging. The window of gene expression may vary depending on the viral vector and chosen promoter.
2. Retina excision and flat-mounting
   NOTE: Two to three weeks after injection, intravitreally-injected rats are euthanized immediately before the electrophysiology protocol begins, in accordance with standard ethical guidelines (European Communities Directive 86/609/EU) and approved by local ethical committees. Carbon dioxide (CO₂) inhalation is used as the method of euthanasia in this protocol.
   1. Eye enucleation
      1. Gently press the exterior of the orbit using a pair of curved forceps to slightly protrude the eye from the eye socket.
      2. Use a pair of spring scissors to cut the muscles holding the eye and enucleate it, taking care not to puncture the eyeball.
         NOTE: Starting from this step, dissect the retina under a stereo microscope in oxygenated (95% O₂ / 5% CO₂) Ames' Medium.
   2. Retina excision
      1. Use a pair of small curved forceps and fine spring scissors to remove all surrounding tissue from the eyeball.
      2. Take a piece of approximately 3 cm x 3 cm filter paper and place it on the lid of a 3.5 cm dish. Soak the paper with Ames' Medium.
      3. Place the eyeball on top of the paper, with the anterior segment facing the operator. Use a pair of straight forceps to hold the eyeball, positioning them on top of the ora serrata at approximately a 45° angle from the dish surface. Make a small cut with a blade, using the space between the straight forceps as a reference.
      4. Reimburse the eyeball into Ames' Medium. Use a pair of straight forceps and fine spring scissors to separate the anterior and posterior segments of the eye.
      5. Carefully remove the lens using two pairs of straight forceps. Then, separate the retina from the sclera.
      6. Cut the sclera toward the optic nerve using fine spring scissors until the retina is isolated from the eyecup.
      7. Use a fluorescence stereo microscope to identify the region of the retina with the best calcium indicator expression.
         NOTE: The extent of viral spread depends on the success of the intravitreal injection. Achieving fluorescence across large portions of the retina may require practice. The experience of the investigator plays a crucial role in obtaining optimal results.
      8. Using a cut-tip plastic pipette, transfer the selected piece of the retina onto the mounting membrane (mounting membranes steps). Use a pair of straight forceps to flat-mount the retina with the GCL facing up.
      9. With a plastic pipette attached to a 100 µL pipette tip, remove the media to allow the retina piece to adhere to the porous membrane. Flip the assembly onto the MEA so that the GCL rests on top of the electrodes.
      10. Fill the sample bath with oxygenated Ames' Medium.

3. Ex vivo calcium imaging upon electrical stimulation

NOTE: In this work, a proof-of-concept MEA was used for ex vivo experimentation. The custom MEAs were fabricated with 25 µm diameter porous graphene-based electrodes on 500 µm thick borosilicate glass with Ti/Au traces and later insulated with silicon nitride and SU-8 photoresist¹². However, the calcium imaging methods are valid irrespective of the electrode material used for stimulation.

1. Set the perfusion system so the oxygenated Ames' Medium constantly perfuses the sample bath at 33 °C at a constant flow rate of 5 mL/min.
2. Using an inverted fluorescence microscope equipped with a fluorescent lamp, a FITC filter cube, and a CMOS camera, inspect the retina for an area where the stimulating electrodes and the fluorescence from GCaMP-expressing cells are visible. A 20x NA 0.75 air objective was used for this study.
   NOTE: To effectively stimulate (and record) the cells with the electrodes, the retina and the electrode need to be in close contact. Thus, cells are visibly in the same focal plane as the electrodes. If it is not the case, repeat the steps of retina excision from step 8 onwards. When using retinas from healthy animal models (with functioning photoreceptors), note that every time the fluorescent lamp is turned on, there will be some evoked responses generated by the light since the retina is light-sensitive to the wavelength used for exciting the GCaMP sensor. These light-induced calcium changes can be used to assess the health status of the tissue. To avoid mixing light with electrical-evoked responses, turn on the fluorescent lamp at least 1 min before starting the image acquisition.
3. To elicit electrical-evoked responses in the GCL, select an electrode to send current-controlled pulses. Set the electrical stimulation parameters in the software of the pulse generator device, such as: shape, amplitude, duration, phase delay, and frequency of pulses to be applied.
   NOTE: Effective stimulus parameters can vary widely from pulse widths of 50 µs to 100 ms, with amplitudes ranging from 0.1 µA to 10 µA. These parameters, along with stimulus frequency, stimulus polarity, number of pulses, and interphase delays, can influence the spatiotemporal response observed by calcium imaging^19,20,21,22. A train of 40 biphasic pulses delivering 1 ms, 2 µA stimulation often generates a visible response in labeled neurons.
4. To synchronize the image acquisition with the stimulation delivery, use the pulse generator as an external trigger to control the start of image acquisition. Connect the camera (see Table of Materials) with the pulse generator using the output trigger signal and set the "Capture Mode" of the camera software to "External Start Trigger". Press Start in the camera software so that it awaits an external trigger to start. Start the image acquisition with the pulse generator software.
   NOTE: The external trigger control might be set up differently for different cameras. This study typically acquired images (512 x 512 pixels, 16-bit grayscale) at 10 frames per second for 1 min while providing bursts of biphasic pulse trains every 10 s. Pulse delivery starts after 10 s, so the first frames in all experiments correspond to spontaneous activity. Depending on the GCaMP sensor and the analysis one will perform, one might need to adjust the recording rate according to the rise and decay times of your calcium indicator⁸. Consider the sensitivity to detect single action potentials of the calcium indicator¹⁵.
5. Save the images with a file name that includes the electrical stimulation parameters applied, such as [Electrode number]_[Pulse Amplitude]_[Pulse duration]_[Pulse Frequency]_Image001.

4. Data analysis

1. ImageJ/FIJI to extract the fluorescence intensity profile over time and the spatial coordinates from the cell somas
   1. Segment the region of interest (ROI) with the "Area Selection Tools" and add it to the ROI Manager (Analyze > Tools > ROI Manager > Add). From the ROI Manager menu, save it as a .zip folder (More > Save).
      NOTE: Typically, the same ROIs can be applied to all stimulation experiments since they correspond to the same FOV.
   2. Select the "Mean gray value" as the parameter to be extracted (Analyze > Set measurements).
   3. Extract the "Mean gray value" from the cell somas by clicking More > Multi Measure. A dialog box will appear. Enable the Measure all 600 slices and One row per slice options to obtain a single table in which columns correspond to ROIs and rows correspond to time frames. Save the generated table as a .xls spreadsheet.
   4. Select the "Centroid" as the parameter to be extracted (Analyze > Set measurements).
   5. Extract the "Centroid" from the ROIs by clicking Measure. The generated table corresponds to the coordinates (X,Y) of the ROIs. Save it as a .xls spreadsheet.
2. Custom-built script to identify cells that respond to the stimuli
   NOTE: MATLAB (see Table of Materials) was used here, but the steps described can be achieved in any programming language. Users can obtain our custom-built script by requesting the corresponding author.
   1. Photobleaching effect correction: To mitigate the background and photobleaching effect, take 15-20 frames from the non-stimulating periods before each burst and fit them to a linear curve [fit (poly1)].
      NOTE: In this case, for a total 600-frame movie in which periodic bursts of pulse trains were sent every 10 s, frames 1:90, 170:190, 270:290, 370:390, 470:490, 570:590 were considered as non-stimulating periods.
   2. Normalize using the formula: (X-min) / (max-min)
   3. Identification of responding cells
      1. Calculate the root mean square (RMS) of the non-stimulating periods from the normalized data. This will be considered as the baseline signal.
      2. Calculate the maximum of the stimulating periods (frames between the non-stimulating periods). In this case, for a total 600-frame movie in which periodic bursts of pulse trains were sent every 10 s, frames 91:169, 191:269, 291:369, 391:469, 491:569 were considered as the stimulating periods.
      3. If the maximum value surpasses the baseline signal by 2.5 times for a specific ROI, tag the cell as responding to that stimulating period. If the cell responds to three out of the five stimulating periods, classify it as a responding cell.