Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Corey M. Rountree; John B. Troy; Laxman Saggere

doi:10.3791/56645

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

7.4K views

⸱

DOI:

10.3791/56645

⸱

December 19th, 2017

Corey M. Rountree¹ , John B. Troy² , Laxman Saggere¹

¹Department of Mechanical and Industrial Engineering, University of Illinois at Chicago, ²Department of Biomedical Engineering, Northwestern University

Summary

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

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.

Introduction

Photoreceptor degenerative diseases, such as retinitis pigmentosa and age-related macular degeneration, are leading inheritable causes of vision loss and are currently incurable¹^,². Although 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 loss of photoreceptors triggers widespread remodeling throughout the retina but surviving retinal neurons, including the bipolar cells and RGCs, remain intact and relatively functional even....