Name: Video: Rat Retina Excision and Mounting on Porous Membrane for Calcium Imaging
Uploaded: 2023-08-18T14:25:00
Duration: 2 min 42 s
Description: 345 Views. Rat Retina Excision and Mounting on Porous Membrane for Calcium Imaging

rat retina excision and mounting on porous membrane for calcium imaging

Evaluating Calcium Activity in Retinal Ganglion Cells Layer

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.

Author Spotlight: An Innovative Approach to Neural Electrical Stimulation Using Calcium Imaging 

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Watch this Scientific Journal Video about Rat Retina Excision and Mounting on Porous Membrane for Calcium Imaging at JoVE.com

Rat Retina Excision and Mounting on Porous Membrane for Calcium Imaging  

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Research

JoVE Journal

Neuroscience

345 Views. Rat Retina Excision and Mounting on Porous Membrane for Calcium Imaging

Video: Rat Retina Excision and Mounting on Porous Membrane for Calcium Imaging  

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 ...