Abstract
Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.
Highlights
Photoreceptor degenerative diseases, such as retinitis pigmentosa and age-related macular degeneration, are leading inheritable causes of vision loss and are currently incurable[1,2]
The retina is carefully placed onto the perforated multielectrode array (pMEA) with the ganglion cell side facing the electrodes, and the pMEA secured inside the multielectrode array (MEA) amplifier (Figure 3A), where it can be continually perfused with fresh, oxygenated Ames medium from both the top (Figure 3B) and bottom (Figure 3C) sides
After ensuring the retina has stabilized from surgical trauma, a glass micropipette or multiport device is fitted into a pipette holder (Figure 7A-E) and interfaced with a patch-clamp headstage whose position is controlled by a 3-axis precision micromanipulator
Summary
Photoreceptor degenerative diseases, such as retinitis pigmentosa and age-related macular degeneration, are leading inheritable causes of vision loss and are currently incurable[1,2]. These diseases arise from a variety of specific genetic mutations, photoreceptor degenerative diseases are characterized as a group by the progressive loss of the photoreceptor cells in the retina, which eventually causes blindness. The early successes of electrical stimulation have demonstrated that artificial neurostimulation can be an effective treatment for photoreceptor degenerative diseases This leads one to hypothesize that an even more effective treatment might be achievable by stimulating the retina with neurotransmitter chemicals, the natural www.jove.com agents of communication at chemical synapses. The purpose of the method presented in this paper is to explore the therapeutic feasibility of chemical stimulation, which seeks to mimic the natural system of synaptic communication between retinal neurons, as a biomimetic alternative to electrical stimulation for a retinal prosthesis
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