Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas Marina Cunquero1, Maria Marsal1, Gustavo Castro-Olvera1, Steven T. Walston2, F. Taygun Duvan2, José Gabriel Macias- Montero3, Carles Puigdengoles3, Mokhtar Chmeissani3, Jose A. Garrido2,4, Pablo Loza-Alvarez1 1 2 Institut de Ciències Fotòniques (ICFO), The Barcelona Institute of Science and Technology Catalan Institute of Nanoscience and Nanotechnology 3 (ICN2), CSIC and BIST, Campus UAB Institut de Física d'Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Campus 4 UAB Institució Catalana de Recerca i Estudis Avançats (ICREA) Corresponding Author Abstract Pablo Loza-Alvarez pablo.loza@icfo.eu Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired Citation light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore Cunquero, M., Marsal, M., Castro- vision by inducing visual percepts. One common avenue of investigation involves Olvera, G., Walston, S.T., Duvan, F.T., Macias-Montero, J.G., Puigdengoles, C., the design and production of implantable devices with a flexible physical structure, Chmeissani, M., Garrido, J.A., Loza- housing a high number of electrodes. This enables the efficient and precise generation Alvarez, P. Calcium Imaging In Electrically Stimulated Flat-Mounted of visual percepts. However, with each technological advancement, there arises a Retinas. J. Vis. Exp. (198), e65705, need for a reliable and manageable ex vivo method to verify the functionality of the doi:10.3791/65705 (2023). device before progressing to in vivo experiments, where factors beyond the device's Date Published performance come into play. This article presents a comprehensive protocol for August 18, 2023 studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the DOI rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence 10.3791/65705 signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from URL individual cells within the GCL. By following this procedure, researchers can efficiently jove.com/video/65705 test new stimulation protocols prior to conducting in vivo experiments. Introduction The retina contains photoreceptors, which are cells Pigmentosa (RP) and Age-related Macular Degeneration responsible for sensing light. They capture photons and (AMD) are degenerative diseases characterized by the convert them into nerve impulses. These impulses are then progressive loss of photoreceptors. These retinopathies processed within the retina and transmitted to the visual are among the leading causes of blindness worldwide1 , cortex, resulting in the formation of a visual image1 . Retinitis impacting millions of individuals and having significant Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 1 of 15 License medical, personal, and socioeconomic consequences for binding to calcium, resulting in increased fluorescence9 . patients, healthcare systems, and society as a whole. Recombinant adeno-associated viral vectors (AAVs) are an Furthermore, with the aging population, it is projected that effective means of transducing retinal cells with GCaMP10 . AMD cases will increase by 15% by 20502 . This protocol presents an efficient method that utilizes Currently, numerous research efforts are underway to calcium imaging for testing stimulation protocols of retinal restore vision in patients affected by these conditions3 . implants. Specifically, we focus on ex vivo rat retinal tissue One promising approach is the use of retinal prostheses, and provide detailed step-by-step instructions, from sample which have demonstrated effectiveness in partially restoring acquisition to data analysis. By offering this comprehensive vision4 , 5 . These devices capture light from the visual scene guide, researchers from various backgrounds can embark on and convert it into electrical pulses. These pulses are electrical stimulation experimentation with confidence. delivered through electrodes within a microelectrode array (MEA) implanted in the eye, stimulating the surviving neurons Protocol and bypassing the function of the lost photoreceptors. The All animal procedures were conducted in accordance with activated retinal ganglion cells (RGCs) transmit the output standard animal ethical guidelines (European Communities to the brain, where it is interpreted as visual perception. Directive 86/609/EU) and approved by the local animal ethics However, the main limitations of current implants lie in committees. 8 weeks-old Long Evans rats were used for the the resolution of the electrode-tissue interface6 and the present study. The animals were obtained from a commercial non-selective stimulation of different cell types. Therefore, source (see Table of Materials). to optimize the design of new implantable devices for more efficient vision restoration, it is crucial to understand 1. Preparation of media and flat-mounting how stimulation paradigms can be developed to selectively assembly activate cells in close proximity to the electrodes. 1. Ames' Medium (1 L) Calcium imaging is a widely employed technique for studying 1. In a 1 L glass bottle, combine the Ames' Medium neural activity, offering several advantages over non-optical powder, 1.9 g/L NaHCO3, 10 ml of Penicillin/ methods7 , 8 . Firstly, it provides cellular and subcellular Streptomycin 100x, and 1 L of deionized water (see resolution. Secondly, calcium markers can target specific Table of Materials). Adjust the pH to 7.4 and the cell types. Thirdly, it allows for long-term tracking, and osmolarity to 280 mOsm with deionized water or fourthly, it enables the observation of entire cell populations NaHCO3. Sterilize the solution by filtering it through while distinguishing between active and inactive cells. This a 0.2 µm pore size filter under a hood. method provides indirect evidence of cellular activity with a NOTE: Store the sterilized medium at 4 °C. This temporal resolution in the range of hundreds of milliseconds. solution remains stable and can be used for up to 1 Genetically-encoded fluorescent calcium indicators, such as month. GCaMP sensors, undergo a conformational change upon 2. Mounting membranes Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 2 of 15 License to facilitate cornea-objective contact (see Table of 1. Attach one PTFE porous membrane (see Table of Materials). Materials) to a washer using small drops of glue. NOTE: If any abnormalities are detected, do not Allow it to dry for at least 15 min. proceed with the subsequent steps for that eye. 2. To achieve translucency, immerse the membranes 4. Apply one drop of Prescaine as a local anesthetic. in 70% ethanol for 1 min. Fix the eyelid and limbal conjunctiva with a 3. Rinse the membranes twice with deionized water commercially available suture filament (see Table of to completely remove the ethanol. Store them in Materials). Create a 1 mm sclerotomy 4 mm from deionized water to prevent opacity. the limbus using a 30 G needle. 2. GCL labeling and rat retina flat-mounting 1. Attach a 36 G blunt needle to a precision NOTE: This labeling method does not differentiate RGCs from syringe and inject the AAV particles carrying the displaced amacrine cells. If selective labeling of RGCs is genetically encoded calcium indicator into the desired, consider using AAVs with RGC-specific promoters11 vitreous for 30 s, at a 45° angle. In this study, we and/or retrograde labeling through the optic nerve12 . To used AAV2-CAG-GCaMP5G (7.5 x 1011 GC/ discriminate between classes of ON- and OFF-center RGCs, mL in HBSS) (see Table of Materials). classify RGCs based on their light response13 , 14 , and utilize NOTE: AAV constructs that do not encode newer versions of genetically encoded calcium indicators that potentially tumorigenic gene products or offer increased sensitivity and the ability to measure single toxin molecules and are produced without action potentials15 . a helper virus can be handled in Biosafety Level 1 (BSL-1) facilities. Otherwise, if 1. Intravitreal injection considered biohazardous material under BSL-2 1. Anesthetize the 8-week-old Long Evans rat with containment, proper precautions must be 2% isoflurane/1% O2 until there is no pedal reflex, taken16 . AAVs encoding for GCaMP are and maintain anesthesia with a rat nasal mask (see considered BSL-1 and do not require Table of Materials). manipulation under biosafety cabinets. NOTE: During anesthesia, position the animal on a 5. Apply one drop of Tobradex (see Table of heating pad to maintain body temperature. Materials) to prevent inflammation and as an 2. Administer one drop of commercially available eye antibiotic prophylaxis. drops (see Table of Materials) to dilate the pupil. 6. If desired, repeat steps 2, 3, and 4 with the other eye. 3. Before proceeding with surgery, examine the eye NOTE: Check on the animals 12-24 h after surgery for abnormalities using fundoscopy and Optical to ensure there are no adverse reactions. Coherence Tomography (OCT) with an in vivo retinal imaging system. Apply one drop of Methocel 2% Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 3 of 15 License 7. Three days after injection, examine the retinal 2. Use a pair of spring scissors to cut the muscles structure using fundoscopy and OCT with an in vivo holding the eye and enucleate it, taking care not retinal imaging system (see Table of Materials). to puncture the eyeball. NOTE: Starting from this step, dissect the retina 8. Two weeks after injection, the GCL should emit under a stereo microscope in oxygenated (95% fluorescence. Assess retinal structure and AAV O2 / 5% CO2) Ames' Medium. expression by fluorescence fundoscopy using an in vivo retinal imaging system. 2. Retina excision NOTE: According to Weitz et al.12 , fluorescence 1. Use a pair of small curved forceps and fine from AAV2-CAG-GCaMP5G becomes noticeable at spring scissors to remove all surrounding tissue 1-week post-injection and intensifies by 2 weeks. from the eyeball. Starting from the fourth week, overexpression of 2. Take a piece of approximately 3 cm x 3 cm filter GCaMP induces cytomorbidity. Dying cells exhibit paper and place it on the lid of a 3.5 cm dish. a high baseline fluorescence signal in the nucleus Soak the paper with Ames' Medium. and cytoplasm that does not fluctuate in response 3. Place the eyeball on top of the paper, with to stimulation. In healthy cells, GCaMP expression the anterior segment facing the operator. Use is confined to the cytoplasm and excluded from a pair of straight forceps to hold the eyeball, the nucleus7 , 8 , 12 , 17 , 18 . These features can be positioning them on top of the ora serrata observed ex vivo during microscopy imaging. The at approximately a 45° angle from the dish window of gene expression may vary depending on surface. Make a small cut with a blade, using the viral vector and chosen promoter. the space between the straight forceps as a 2. Retina excision and flat-mounting reference. NOTE: Two to three weeks after injection, intravitreally- 4. Reimburse the eyeball into Ames' Medium. Use injected rats are euthanized immediately before the a pair of straight forceps and fine spring scissors electrophysiology protocol begins, in accordance with to separate the anterior and posterior segments standard ethical guidelines (European Communities of the eye. Directive 86/609/EU) and approved by local ethical committees. Carbon dioxide (CO2) inhalation is used as 5. Carefully remove the lens using two pairs of straight forceps. Then, separate the retina from the method of euthanasia in this protocol. the sclera. 1. Eye enucleation 6. Cut the sclera toward the optic nerve using fine 1. Gently press the exterior of the orbit using a pair spring scissors until the retina is isolated from of curved forceps to slightly protrude the eye the eyecup. from the eye socket. Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 4 of 15 License 7. Use a fluorescence stereo microscope to 1. Set the perfusion system so the oxygenated Ames' identify the region of the retina with the best Medium constantly perfuses the sample bath at 33 °C at calcium indicator expression. a constant flow rate of 5 mL/min. NOTE: The extent of viral spread depends 2. Using an inverted fluorescence microscope equipped on the success of the intravitreal injection. with a fluorescent lamp, a FITC filter cube, and a Achieving fluorescence across large portions of CMOS camera, inspect the retina for an area where the retina may require practice. The experience the stimulating electrodes and the fluorescence from of the investigator plays a crucial role in GCaMP-expressing cells are visible. A 20x NA 0.75 air obtaining optimal results. objective was used for this study. 8. Using a cut-tip plastic pipette, transfer the NOTE: To effectively stimulate (and record) the cells selected piece of the retina onto the mounting with the electrodes, the retina and the electrode need membrane (mounting membranes steps). Use to be in close contact. Thus, cells are visibly in the a pair of straight forceps to flat-mount the retina same focal plane as the electrodes. If it is not the case, with the GCL facing up. repeat the steps of retina excision from step 8 onwards. When using retinas from healthy animal models (with 9. With a plastic pipette attached to a 100 µL functioning photoreceptors), note that every time the pipette tip, remove the media to allow the retina fluorescent lamp is turned on, there will be some evoked piece to adhere to the porous membrane. Flip responses generated by the light since the retina is light- the assembly onto the MEA so that the GCL sensitive to the wavelength used for exciting the GCaMP rests on top of the electrodes. sensor. These light-induced calcium changes can be 10. Fill the sample bath with oxygenated Ames' used to assess the health status of the tissue. To avoid Medium. mixing light with electrical-evoked responses, turn on the 3. Ex vivo calcium imaging upon electrical fluorescent lamp at least 1 min before starting the image stimulation acquisition. NOTE: In this work, a proof-of-concept MEA was used for 3. To elicit electrical-evoked responses in the GCL, select ex vivo experimentation. The custom MEAs were fabricated an electrode to send current-controlled pulses. Set the with 25 µm diameter porous graphene-based electrodes electrical stimulation parameters in the software of the on 500 µm thick borosilicate glass with Ti/Au traces and pulse generator device, such as: shape, amplitude, later insulated with silicon nitride and SU-8 photoresist12 . duration, phase delay, and frequency of pulses to be However, the calcium imaging methods are valid irrespective applied. of the electrode material used for stimulation. 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 Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 5 of 15 License polarity, number of pulses, and interphase delays, can 4. Data analysis influence the spatiotemporal response observed by calcium imaging19 , 20 , 21 , 22 . A train of 40 biphasic pulses 1. ImageJ/FIJI to extract the fluorescence intensity profile delivering 1 ms, 2 µA stimulation often generates a visible over time and the spatial coordinates from the cell somas response in labeled neurons. 1. Segment the region of interest (ROI) with the "Area 4. To synchronize the image acquisition with the stimulation Selection Tools" and add it to the ROI Manager delivery, use the pulse generator as an external trigger (Analyze > Tools > ROI Manager > Add). From the to control the start of image acquisition. Connect ROI Manager menu, save it as a .zip folder (More the camera (see Table of Materials) with the pulse > Save). generator using the output trigger signal and set the NOTE: Typically, the same ROIs can be applied to "Capture Mode" of the camera software to "External all stimulation experiments since they correspond to Start Trigger". Press Start in the camera software so the same FOV. that it awaits an external trigger to start. Start the image 2. Select the "Mean gray value" as the parameter to be acquisition with the pulse generator software. extracted (Analyze > Set measurements). NOTE: The external trigger control might be set up 3. Extract the "Mean gray value" from the cell somas differently for different cameras. This study typically by clicking More > Multi Measure. A dialog box acquired images (512 x 512 pixels, 16-bit grayscale) will appear. Enable the Measure all 600 slices and at 10 frames per second for 1 min while providing One row per slice options to obtain a single table bursts of biphasic pulse trains every 10 s. Pulse delivery in which columns correspond to ROIs and rows starts after 10 s, so the first frames in all experiments correspond to time frames. Save the generated table correspond to spontaneous activity. Depending on the as a .xls spreadsheet. GCaMP sensor and the analysis one will perform, one might need to adjust the recording rate according to the 4. Select the "Centroid" as the parameter to be extracted (Analyze > Set measurements). rise and decay times of your calcium indicator8 . Consider the sensitivity to detect single action potentials of the 5. Extract the "Centroid" from the ROIs by clicking calcium indicator15 . Measure. The generated table corresponds to the coordinates (X,Y) of the ROIs. Save it as a .xls 5. Save the images with a file name that includes spreadsheet. the electrical stimulation parameters applied, such as [Electrode number]_[Pulse Amplitude]_[Pulse 2. Custom-built script to identify cells that respond to the duration]_[Pulse Frequency]_Image001. 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. Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 6 of 15 License mounting of the retina on the MEA (Figure 1-left), (2) 1. Photobleaching effect correction: To mitigate the visualization of calcium activity in the GCL during electrical background and photobleaching effect, take 15-20 stimulation (Figure 1-middle), and (3) extraction, processing, frames from the non-stimulating periods before each and interpretation of the imaging data (Figure 1-right). burst and fit them to a linear curve [fit (poly1)]. NOTE: In this case, for a total 600-frame movie First, as depicted in Figure 1-left, Long Evans rats are in which periodic bursts of pulse trains were intravitreally injected with AAV2-CAG-GCaMP5G prior to sent every 10 s, frames 1:90, 170:190, 270:290, the imaging session. The optimal viral expression for this 370:390, 470:490, 570:590 were considered as non- vector occurs 2 to 3 weeks after injection12 , 18 . After fully stimulating periods. anesthetizing the animal, a pilot hole is made using a 2. Normalize using the formula: (X-min) / (max-min) 30 G needle, and then 5 µL of AAV2-CAG-GCaMP5G is slowly injected into the vitreous using a 36 G blunt needle 3. Identification of responding cells attached to a precision syringe to prevent reflux. During viral 1. Calculate the root mean square (RMS) of the expression, an in vivo retinal imaging system is used to non-stimulating periods from the normalized assess the condition of the retina post-surgery, with OCT data. This will be considered as the baseline images providing detailed visualization of the retinal layers. signal. Once gene expression is achieved, the retina is carefully 2. Calculate the maximum of the stimulating extracted from the eyecup using a stereo microscope and periods (frames between the non-stimulating high-precision dissection tools. From this point onwards, the periods). In this case, for a total 600-frame tissue is manipulated in oxygenated media to preserve the movie in which periodic bursts of pulse trains sample. The excised retina, with the GCL facing up, is then were sent every 10 s, frames 91:169, 191:269, mounted on a platform designed for flat-mounting to ensure 291:369, 391:469, 491:569 were considered as stability and prevent sample floating. The sample is mounted the stimulating periods. on the MEA surface with the GCL facing the electrodes. 3. If the maximum value surpasses the baseline Next, the MEA is mounted on its interface board on an signal by 2.5 times for a specific ROI, tag the cell inverted fluorescent microscope (Figure 1-middle). The as responding to that stimulating period. If the retinal sample is perfused with oxygenated media at 33 °C cell responds to three out of the five stimulating using a perfusion system. The sample can be maintained in periods, classify it as a responding cell. this configuration for several hours. The desired stimulation Representative Results scheme is programmed, and images are acquired at a rate of 10 frames per second. It is recommended to name The protocol described in this study is based on the the movies according to the applied electrical stimulation fluorescence imaging and electrical stimulation studies parameters. Image acquisition should begin before the conducted by Weitz et al.12 . The protocol consists of three main parts: (1) fluorescent labeling of the GCL and flat- Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 7 of 15 License initiation of stimulation to obtain some baseline frames without responsive cells are identified. Responsive cells are defined stimulation, which will serve as a negative control. as those with fluorescence peaks during stimulation that exceeds their baseline by 2.5 times. If a cell responds to three Finally, as illustrated in Figure 1-right, the data is extracted out of the five bursts of stimulation, it is considered responsive from the time-lapse images by segmenting the cell somas. to that specific train of stimulation. Photobleaching effects are corrected by fitting the data, and Figure 1: Overview of the study. Schematic illustration of the protocol for (left) fluorescently labeling the GCL of the retina and sample mounting, (middle) set up preparation for ex vivo recordings with electrical stimulation provided by a MEA, and (right) analyzing the calcium imaging data to classify responsive cells. Please click here to view a larger version of this figure. Intravitreal-injected retina animals should be followed up. In Figure 2A-D, the retina The incidence of complications associated with intravitreal of a healthy injected animal is shown. After two weeks of injections is very low. However, there are some complications injection, the RGCs begin to express fluorescence, which can that can arise from the surgery itself, regardless of the be visualized using fluorescence fundoscopy (Figure 2B,C). injected component. These complications include cataract OCT images provide detailed visualization of the disposition formation, vitreous hemorrhage, elevation of intraocular and thickness of retinal layers (Figure 2D), offering pressure, and endophthalmitis23 . To determine whether higher resolution compared to funduscopy, particularly when these complications are caused by the surgery, the animal assessing retinal detachment. Once the retina is flat-mounted needs to undergo evaluation before the procedure using and imaged using an inverted fluorescence microscope, it funduscopy and OCT. Three days after the injection, the becomes possible to distinguish the cells and axon bundles. Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 8 of 15 License Unlike other calcium indicators, the GCaMP indicator is restricted to the cytoplasm7 , and fluorescence is excluded from the nucleus (Figure 2E). Figure 2: Representative images of the intravitreal-injected retina. (A) Fundoscopy, (B) fluorescence fundoscopy, (C) zoom-in of the fluorescence fundoscopy, (D) OCT image, and (E) epi-fluorescence image of the excised retina mounted on a custom MEA with graphene-based electrodes on 500 µm thick borosilicate glass. In (E), black lines correspond to Ti/Au traces. Scale bars: 115 µm (D) and 100 µm (E). Please click here to view a larger version of this figure. Electrodes and GCL contact same focal plane (Figure 3A). If the cells are not in the same In order to evoke neural responses effectively, it is crucial to focal plane as the electrodes (Figure 3B), it indicates that ensure that the flat-mounted retina is in close contact with the the contact is suboptimal, which will result in less effective surface of the MEA. A simple way to verify this is by visually stimulation. confirming whether the cells and electrodes are located in the Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 9 of 15 License Figure 3: Electrodes and GCL contact. (A) Cells and the electrode (asterisk) in the same focal plane. (B) Cells and electrodes not in the same focal plane, indicating suboptimal contact for electrical stimulation in that area. Please click here to view a larger version of this figure. Ex vivo calcium imaging upon electrical stimulation will determine the appropriate objective magnification to provided by a MEA choose. Typically, for single-electrode stimulation studies with The resulting data from calcium imaging consist of time- diameters ranging from 5 µm to 100 µm, a 20-25x objective lapse images that monitor the neural activity of hundreds magnification is suitable (Figure 4A), providing a FOV of of cells in response to electrical stimulation. Suprathreshold approximately 600 µm x 600 µm. For experiments involving stimuli cause a calcium influx into the cell somas, resulting stimulation with multiple electrodes, a 4-10x objective in a sudden change in fluorescence intensity (Video 1). This magnification may be necessary to assess a wider area protocol enables determining whether an electrode, MEA, of around 2 mm x 2 mm. Responsive cells can be easily and/or stimulation algorithm elicits the desired response in identified by generating a standard deviation image projection neural tissue. The size and pitch of the electrodes on the of the time-lapse movie (Figure 4B and Video 1). MEA, as well as the proportion of tissue being studied, Figure 4: Calcium imaging of the GCL with electrical stimulation provided by a 25 µm-diameter electrode. (A) Maximum projection of a 60 s time-lapse movie and (B) standard deviation projection clearly depicting cells that respond to electrical stimuli from a 25 µm diameter porous graphene-based electrode. The stimulating electrode is indicated with an asterisk. Scale bar: 50 µm. Please click here to view a larger version of this figure. Analysis of the calcium dynamics over time upon corrected calcium traces from the responsive cells. In this controlled stimulation example, five bursts of biphasic pulse trains (cathodic-first, 40 For each identified cell soma, the mean intensity values were cycles, 1 ms duration, 2 µA amplitude) were delivered every extracted over time. Figure 5A shows the photobleaching- 10 s (indicated by black lines) during a 60 s image acquisition. Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 10 of 15 License Within a given experiment, the same five pulse trains are can examine the relationship between the current required applied to test the consistency of the response. The frames to activate the cells and the distance from the stimulating captured during the non-stimulating periods (highlighted in electrode (Figure 5B). As expected, cells located closer to the red) are used to perform a linear fit, correcting for the stimulating electrode require lower current values to evoke a photobleaching effect. response. Once the responding cells are identified, and their coordinates (x,y) are known relative to the stimulating electrode, one Figure 5: Representation of the electrical-evoked responses. (A) Calcium traces of cell somas upon 5 bursts of pulse trains (biphasic, cathodic-first, 40 cycles, 1 ms duration, 2 µA amplitude) every 10 s (black lines) during a 60 s image acquisition. Non-stimulating (red-highlighted frames) and stimulating periods (yellow-highlighted frames) are shown. Traces surpassing the baseline signal (root mean square of the non-stimulating periods) by 2.5 times are considered evoked responses. Cells responding in three out of the five stimulating periods are classified as responding cells. (B) Calcium activity distribution map showing the stimulating electrode (black outlined circle) and cells (gray outlined circle). The color code represents the minimum pulse amplitude necessary to evoke a cellular response. Please click here to view a larger version of this figure. Video 1: Calcium imaging of the GCL with electrical Discussion stimulation provided by a 25 µm-diameter electrode. The protocol described here serves to study the calcium The video displays differences in fluorescence intensity due dynamics occurring in the rat retinal GCL upon electrical to electrical stimulation from a 25 µm diameter porous stimulation provided with a MEA. It is a reliable and graphene-based electrode. The left side shows the original manageable method but requires some training, particularly movie, and the right side shows the standard deviation to uniformly label the GCL efficiently and to mount the projection where responding cells can be easily identified. retina properly to ensure optimal tissue-electrode contact. Scale bar: 50 µm. Please click here to download this Video. This protocol is specific to rodents and needs to be adapted if applied to a different laboratory species. The critical Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 11 of 15 License points, modifications, and limitations of the methodology are The optimal viral expression for AAV2-CAG-GCaMP5G is presented in detail. 2 to 3 weeks after injection12 , 18 . Beyond that timeframe, the nuclei from transfected cells become fluorescent, cells Intravitreal injections stop responding to stimuli, and ultimately die7 , 28 , 29 . This is Injections are widely used for ocular gene delivery, with due to the overexpression of the GCaMP indicator, which is intravitreal injections being the preferred procedure. They translocated into the nucleus. The time window for optimal have been proven to be safer and less invasive compared gene expression will vary depending on the viral vector to subretinal injections, which introduce the molecules of and the chosen promoter30 and needs to be determined interest directly between the photoreceptors and the retinal experimentally before proceeding with this protocol. pigment epithelium (RPE), risking retinal detachment10 . However, there are limitations, especially when performing Tissue-electrode contact these injections in rodent models. The vitreous humor is For optimal and reproducible results, achieving good tissue- gelatinous, hindering viral diffusion. Moreover, the lens in electrode contact is crucial. Poor contact is typically due to rodent eyes is large, making it non-trivial to insert the the natural curvature of the retina. One approach is to cut the needle without scratching it. The precision syringe needles retina into quarters, mount and image one section at a time. are delicate and need to be replaced often. To prevent Small portions of the retina can be better flattened, resulting obstruction, wash them with deionized water before and in more effective contact with the surface of the MEA. Another after every use and replace them regularly. Additionally, potential reason for poor contact is the presence of vitreous inject the content slowly to prevent solution reflux and humor. When conducting stimulation experiments simulating changes in intraocular pressure. Achieving large and uniform an epi-retinal implant, it is important to carefully remove the fluorescence across the retina may require practice. vitreous humor during retina excision as it can act as an insulator to current. Here, a simple method is described to Retinal cell transduction check if the contact is sufficient by visualizing the electrode Viral vectors are an excellent method for in vivo gene delivery, and cells in the same focal plane. and AAVs have been widely used for transducing retinal cells10 . They have been approved as a treatment for some An alternative to ex vivo retinal measurements is to grow retinopathies causing human blindness24 . However, their neurons directly on the surface of the electrodes. Primary carrier capacity is limited to 5 kb, including the required culture of neurons, such as hippocampal neurons31 , can regulatory elements (e.g., the promoter)10 , 25 . There are be useful for initial tests to evaluate the functionality of the multiple serotypes available, each with different tropism. novel stimulating device. However, this approach still requires Choose the most suitable AAV based on the genes to be the use of laboratory animals and does not represent the delivered and the cells to be transduced26 . For labeling the complexity of the retinal network, which is important for RGCs, it is recommended to use AAV227 . evaluating synaptic responses to stimulation. Gene expression window To visualize the cells underneath the electrode and electrode traces, MEAs fabricated with transparent materials such Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 12 of 15 License as indium tin-oxide (ITO) can be used19 , 20 , 32 . In addition to optical measurements, GCL activity upon electrical References stimulation can be assessed through electrical recordings. 1. Bourne, R. R. A. et al. Prevalence and causes of The MEA can be used to record the local field potential (LFP) vision loss in high-income countries and in Eastern and of the tissue. However, this compromises spatial resolution, Central Europe in 2015: Magnitude, temporal trends and as each electrode captures activity from multiple cells projections. British Journal of Ophthalmology. 102 (5), simultaneously (depending on the electrode dimensions). 575-585 (2018). Optical recording overcomes this limitation and offers higher 2. Li, J. Q. et al. Prevalence and incidence of age-related spatial resolution mapping. Its main advantage is the ability to macular degeneration in Europe: a systematic review distinguish between active and inactive cells while measuring and meta-analysis. British Journal of Ophthalmology. a large FOV with single-cell resolution. Among all cellular 104 (8), 1077-1084 (2020). activity reporters, calcium indicators are well-described and most commonly used33 . 3. Roska, B., Sahel, J. A. Restoring vision. Nature. 557 (7705), 359-367 (2018). Disclosures 4. Hornig, R., Velikay-Parel, M. Retina implants. in The authors have no disclosures to add to the manuscript. Implantable Sensor Systems for Medical Applications. 469-496 Elsevier, (2013). Acknowledgments 5. Lewis, P. M. et al. Advances in implantable bionic devices We are thankful to Merche Rivas, Angel Sandoval, Jesús for blindness: a review. ANZ Journal of Surgery. 86 (9), Planagumà, Jordi Cortés, Sandra Ortonobés Lara and Alina 654-659 (2016). Hirschmann (ICFO-Institut de Ciències Fotòniques) for their 6. Weiland, J. D., Walston, S. T., Humayun, M. S. Electrical technical support, to Anna Duarri (VHIR, Vall d'Hebron stimulation of the retina to produce artificial vision. Institute of Research) from the Ophthalmology Research Annual Review of Vision Science. 2, 273-294 (2016). group for their support with the intravitreal injections and the in vivo retinal imaging. 7. Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nature The funding entities that supported this work are: Methods. 6, 875-881 (2009). Fundació CELLEX; Fundació Mir-Puig; Ministerio de 8. Chen, T.-W. et al. Ultrasensitive fluorescent proteins for Economía y Competitividad - Severo Ochoa program for imaging neuronal activity. Nature. 499, 295-300 (2013). Centres of Excellence in R&D (CEX2019-000910-S, [MCIN/ AEI/10.13039/501100011033]); Generalitat de Catalunya 9. Nakai, J., Ohkura, M., Imoto, K. A high signal-to-noise through CERCA program; Laserlab-Europe (EU-H2020 GA Ca2+ probe composed of a single green fluorescent no. 871124); La Caixa Foundation (LCF/HR19/52160003); protein. Nature Biotechnology. 19, 137-141 (2001). and Fondo Social Europeo (PRE2020-095721, M.C.). 10. Sahu, B., Chug, I., Khanna, H. The ocular gene delivery landscape. Biomolecules. 11 (8), 1135 (2021). Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 13 of 15 License ganglion cell soma and threshold reduction. Journal of 11. Hanlon, K. S. et al. A novel retinal ganglion cell promoter Neural Engineering. . 16 (2), 026017 (2019). for utility in AAV vectors. Frontiers in Neuroscience. 11, (2017). 21. Im, M., Fried, S. I. Temporal properties of network- mediated responses to repetitive stimuli are dependent 12. Weitz, A. C. et al. Imaging the response of the retina upon retinal ganglion cell type. Journal of Neural to electrical stimulation with genetically encoded calcium Engineering. 13 (2), 025002 (2016). indicators. Journal of Neurophysiology. 9 (7), 1979-1988 (2013). 22. Jensen, R. J., Ziv, O. R., Rizzo, J. F. Thresholds for activation of rabbit retinal ganglion cells 13. Briggman, K. L., Euler, T. Bulk electroporation and with relatively large, extracellular microelectrodes. population calcium imaging in the adult mammalian Investigative Ophthalmology & Visual Science. 46 (4), retina. Journal of Neurophysiology. 105 (5), 2601-2609 1486-1496 (2005). (2011). 23. Ramos, M. S. et al. Patient-reported complications 14. Baden, T., Berens, P., Franke, K., Román Rosón, M., after intravitreal injection and their predictive factors. Euler, T. The functional diversity of retinal ganglion cells Ophthalmology Retina. 5 (7), 625-632 (2021). in the mouse. Nature. 529, 345-350(2016). 24. Vandenberghe, L. H., Auricchio, A. Novel adeno- 15. Zhang, Y. et al. Fast and sensitive GCaMP calcium associated viral vectors for retinal gene therapy. Gene indicators for imaging neural populations. Nature. 615, Therapy. 19 (2), 162-168 (2012). 884-891 (2023). 25. Zin, E. A., Ozturk, B. E., Dalkara, D., Byrne, L. 16. Meyer, E. L., Jenkins, C., Rengarajan, K. NIH Guidelines C. Developing new vectors for retinal gene therapy. April 2019. Applied Biosafety. 24, Preprint at https:// Cold Spring Harbor Perspectives in Medicine. a041291 doi.org/10.1177/1535676019871146 (2019). (2023). 17. Akerboom, J. et al. Optimization of a GCaMP 26. Haggerty, D. L., Grecco, G. G., Reeves, K. calcium indicator for neural activity imaging. Journal of C., Atwood, B. Adeno-associated viral vectors in Neuroscience. 32 (40), 13819-13840 (2012). neuroscience research. Molecular Therapy-Methods & 18. Chang, Y.-C., Walston, S. T., Chow, R. H., Weiland, Clinical Development. 17, 69-82 (2020). J. D. GCaMP expression in retinal ganglion cells 27. Hellström, M. et al. Cellular tropism and transduction characterized using a low-cost fundus imaging system. properties of seven adeno-associated viral vector Journal of Neural Engineering. 14 (5), 056018 (2017). serotypes in adult retina after intravitreal injection. Gene 19. Weitz, A. C. et al. Improving the spatial resolution of Therapy. 16 (4), 521-532 (2009). epiretinal implants by increasing stimulus pulse duration. 28. Resendez, S. L. et al. Visualization of cortical, subcortical Science Translational Medicine. 7 (318), 1-12 (2015). and deep brain neural circuit dynamics during naturalistic 20. Chang, Y. C., Ghaffari, D. H., Chow, R. H., Weiland, J. mammalian behavior with head-mounted microscopes D. Stimulation strategies for selective activation of retinal Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 14 of 15 License and chronically implanted lenses. Nature Protocols. 11, 566-597 (2016). 29. Yang, Y. et al. Improved calcium sensor GCaMP-X overcomes the calcium channel perturbations induced by the calmodulin in GCaMP. Nature Communications. 9, 1504 (2018). 30. Nieuwenhuis, B. et al. Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters. Gene Therapy. 30, 503-519 (2023). 31. Cunquero, M., Aguilar, E., Loza-Alvarez, P., Planagumà, J. Hippocampal neuronal cultures to detect and study new pathogenic antibodies involved in autoimmune encephalitis. Journal of Visualized Experiments. 184, e63829 (2022). 32. Behrend, M. R., Ahuja, A. K., Humayun, M. S., Chow, R. H., Weiland, J. D. Resolution of the epiretinal prosthesis is not limited by electrode size. IEEE Transactions on Neural Systems and Rehabilitation Engineering. 19 (4), 436-442 (2011). 33. Grienberger, C., Konnerth, A. Imaging calcium in neurons. Neuron. 73 (5), 862-885 (2012). Copyright © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported jove.com August 2023 • 198 • e65705 • Page 15 of 15 License