ex vivo calcium imaging of flat-mounted rat retina upon electrical stimulation

评估视网膜神经节细胞层中的钙活性

The retina contains photoreceptors, which are cells responsible for sensing light. They capture photons and convert them into nerve impulses. These impulses are then processed within the retina and transmitted to the visual cortex, resulting in the formation of a visual image1. Retinitis Pigmentosa (RP) and Age-related Macular Degeneration (AMD) are degenerative diseases characterized by the progressive loss of photoreceptors. These retinopathies are among the leading causes of blindness worldwide1, impacting millions of individuals and having significant medical, personal, and socioeconomic consequences for patients, healthcare systems, and society as a whole. Furthermore, with the aging population, it is projected that AMD cases will increase by 15% by 20502.
Currently, numerous research efforts are underway to restore vision in patients affected by these conditions3. One promising approach is the use of retinal prostheses, which have demonstrated effectiveness in partially restoring vision4,5. These devices capture light from the visual scene and convert it into electrical pulses. These pulses are delivered through electrodes within a microelectrode array (MEA) implanted in the eye, stimulating the surviving neurons and bypassing the function of the lost photoreceptors. The activated retinal ganglion cells (RGCs) transmit the output to the brain, where it is interpreted as visual perception. However, the main limitations of current implants lie in the resolution of the electrode-tissue interface6 and the non-selective stimulation of different cell types. Therefore, to optimize the design of new implantable devices for more efficient vision restoration, it is crucial to understand how stimulation paradigms can be developed to selectively activate cells in close proximity to the electrodes.
Calcium imaging is a widely employed technique for studying neural activity, offering several advantages over non-optical methods7,8. Firstly, it provides cellular and subcellular resolution. Secondly, calcium markers can target specific cell types. Thirdly, it allows for long-term tracking, and fourthly, it enables the observation of entire cell populations while distinguishing between active and inactive cells. This method provides indirect evidence of cellular activity with a temporal resolution in the range of hundreds of milliseconds. Genetically-encoded fluorescent calcium indicators, such as GCaMP sensors, undergo a conformational change upon binding to calcium, resulting in increased fluorescence9. Recombinant adeno-associated viral vectors (AAVs) are an effective means of transducing retinal cells with GCaMP10.
This protocol presents an efficient method that utilizes calcium imaging for testing stimulation protocols of retinal implants. Specifically, we focus on ex vivo rat retinal tissue and provide detailed step-by-step instructions, from sample acquisition to data analysis. By offering this comprehensive guide, researchers from various backgrounds can embark on electrical stimulation experimentation with confidence.

作者聚焦：一种使用钙成像进行神经电刺激的创新方法 

电刺激平装视网膜中的钙成像

Although there have been attempts to test retinal implantable devices using ex vivo methods, a thorough and detailed protocol from sample obtention to data analysis has been lacking. With this work, we aim to fill this gap providing researchers from different backgrounds with the necessary tools to confidently embark on neural retinal stimulation experiments. Calcium imaging is a popular technique for studying neural activity that offers several advantages over non-optical methods.It provides cellular resolution and can target the specific cell types. It is particularly useful for testing new MEAs as it allows discrimination between active and inactive cells upon electrical stimulation. We are introducing a robust methodology for studying the responses of the retinal neurons by the use of calcium imaging.This approach could offer insight into selective excitation of the cells, a critical step in developing better stimulation protocols, improving implant performance, and advancing the state of the art. Our laboratory focus on developing advanced microscopy techniques. Currently, we are working in recording calcium dynamics in 3D to gain a better understanding of the negative interaction in the whole tissue.

Watch this Scientific Journal Video about Ex Vivo Calcium Imaging of Flat-Mounted Rat Retina upon Electrical Stimulation at JoVE.com

Ex Vivo Calcium Imaging of Flat-Mounted Rat Retina upon Electrical Stimulation  

电刺激后平装大鼠视网膜的离体钙成像

将GCaMP 5G表达大鼠视网膜安装在MEA上，GCL朝向显微镜载物台上的电极。连接灌注系统并设置参数，以不断向样品浴灌注含氧 AMS 培养基。接下来，使用装有荧光灯、FITC 滤光片和 CMOS 相机的倒置荧光显微镜检查视网膜。寻找刺激电极和表达GCaMP的细胞发出的荧光可见的区域。在脉冲发生器设备软件中，配置电刺激参数，如脉冲的形状、幅度、持续时间、相位延迟和频率。使用输出触发信号将相机连接到脉冲发生器，并将相机软件的拍摄模式设置为外部启动触发。按下相机软件中的启动按钮，等待外部触发器启动。使用脉冲发生器软件启动图像采集过程并保存捕获的图像，确保文件名包含所有施加的电刺激参数。现在打开图像 J 并使用区域选择工具分割感兴趣的区域，将其添加到 ROI 管理器，并使用 ROI 管理器菜单将其保存为 zip 文件夹。通过导航到“更多”并单击“多度量”，从单元格体质中提取平均灰度值。启用“度量所有 600 个切片和每个切片一行”选项，以获取单个表，其中列对应于 ROI，行对应于时间范围。然后使用 measure 命令从 ROI 中提取质心。为了校正照片漂白效果并最小化背景，请在每次连拍前从非刺激间隔中选择大约 15 到 20 帧，并将这些帧拟合到线性曲线上，以确保最佳数据分析。显示了在 60 秒图像采集期间每 10 秒进行五次脉冲序列爆发时细胞体体的钙痕迹。非刺激期的框架用于校正光漂白效果。激活细胞所需的电流与与刺激电极的距离之间的关系表明，靠近刺激电极的细胞需要较低的电流值才能引起响应。

ex-vivo-calcium-imaging-flat-mounted-rat-retina-upon-electrical

Research

JoVE Journal

Neuroscience

Ex Vivo Calcium Imaging of Flat-Mounted Rat Retina upon Electrical Stimulation

265 次观看.  The Barcelona Institute of Science and Technology. 将GCaMP 5G表达大鼠视网膜安装在MEA上，GCL朝向显微镜载物台上的电极。连接灌注系统并设置参数，以不断向样品浴灌注含氧 AMS 培养基。接下来，使用装有荧光灯、FITC 滤光片和 CMOS 相机的倒置荧光显微镜检查视网膜。寻找刺激电极和表达GCaMP的细胞发出的荧光可见的区域。在脉冲发生器设备软件中，配置电刺激参数，如脉冲的形状、幅度、持续时间、相位延迟和频率。?...

视频: Ex Vivo Calcium Imaging of Flat-Mounted Rat Retina upon Electrical Stimulation  

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...

科研

神经科学

Rat Retina Excision and Mounting on Porous Membrane for Calcium Imaging

Two to three weeks before imaging, the vitreous of an anesthetized rat's eye is injected with the viral particles carrying the genetically encoded calcium indicator. Using fundoscopy and OCT, the retinal structure is examined for adverse reactions from the calcium indicator injection. Two weeks after the injection, the ganglion cells layer or GCL exhibited fluorescence emission.Then the retina excision from the euthanized rat is initiated. Take the eyes expressing AAV2-CAG-GCaMP5G and remove all tissue surrounding the eyeball using small curved forceps and fine spring scissors. Next, take a three by three centimeter piece of filter paper.Position it on the cover of a 3.5 centimeter dish and drench the filter paper with AIMS medium. Position the eyeball on the paper with the anterior segment oriented towards the operator. Use a pair of straight forceps to hold the eyeball at approximately a 45 degree angle from the dish surface.Use the gap between the straight forceps to guide an incision with a blade. Then immerse the eyeball into AIMS medium. Use straight forceps and fine spring scissors to separate the anterior and posterior segments of the eye.Remove the lens carefully using two pairs of straight forceps and detach the retina from the sclera. Using fine spring scissors, make an incision in the sclera towards the optic nerve and cut until the retina has been successfully separated from the eye cup. Transfer the excised piece of the retina onto the mounting membrane using a cut tip plastic pipette.Then use a pair of straight forceps to flat mount the retina, ensuring the ganglion cell layer is oriented upwards. Next, use a plastic pipette fitted with a 100 microliter pipette tip to remove the media, facilitating adherence of the retina piece to the porous membrane. Next, flip the assembly onto the micro electrode array or MEA, positioning the GCL on top of the electrodes.Then fill the sample bath with oxygenated AIMS medium.

大鼠视网膜切除术和安装在多孔膜上用于钙成像

Place the GCaMP 5G expressing rat retina mounted on the MEA with the GCL facing the electrodes on the microscope stage. Connect the perfusion system and set the parameters to constantly perfuse the sample bath with the oxygenated AMS medium. Next, inspect the retina using an inverted fluorescence microscope fitted with a fluorescent lamp, an FITC filter, and a CMOS camera.Look for an area where stimulating electrodes and the fluorescence from GCaMP expressing cells are visible. In the pulse generator devices software, configure the electrical stimulation parameters like the shape, amplitude, duration, phase delay, and frequency of pulses for application. Connect the camera to the pulse generator using the output trigger signal and set the camera software's capture mode to external start trigger.Press the start button in the camera software so that it awaits an external trigger to start. Initiate the image acquisition process using the pulse generator software and save the captured images, ensuring the file name includes all the applied electrical stimulation parameters. Now open image J and segment the region of interest using the area selection tools, add it to the ROI manager, and save it as a zip folder using the ROI manager menu.Extract the mean gray value from the cell somas by navigating to more and clicking multi measure. 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 timeframes. Then extract the centroid from the ROIs using the measure command.To correct the photo bleaching effect and minimize the background, select approximately 15 to 20 frames from the non-stimulating intervals before each burst and fit these frames to a linear curve to ensure optimal data analysis. The calcium traces of cell somas upon five bursts of pulse trains every 10 seconds during a 60 second image acquisition are shown. Frames from the non-stimulating periods are used to correct the photo bleaching effect.The relationship between the current required to activate the cells and the distance from the stimulating electrode showed that cells located closer to the stimulating electrode required lower current values to evoke a response.