High-density Multielectrode Array Recordings of Retinal Waves Using An Electrophysiology Platform

Summary

High-density multielectrode arrays (HD-MEAs) are used to study spontaneous retinal waves, which play a crucial role in neural circuit development. This protocol outlines the steps for preparing mouse retinal tissue and performing electrophysiological recordings using HD-MEAs on an electrophysiology platform.

Abstract

Spontaneous retinal waves are a hallmark of retinal network activity during development, playing a crucial role in the formation of the visual system by influencing the refinement of axons, permeability of vasculature, and overall maturation of neural circuits. These waves are commonly studied in ex vivo retinal preparations using multielectrode arrays (MEAs), which enable electrophysiological recordings of large populations of retinal ganglion cell (RGC) activity. MEA-based electrophysiology has become a powerful tool due to its ease of use to rapidly collect high-throughput data, thus making it ideally suited to study retinal activity in a variety of experimental conditions.

In this protocol, we outline the critical steps for preparing retinal tissue for the acquisition of electrophysiological data using a High-Density MEA (HD-MEA) on an electrophysiology platform. The process begins with the careful isolation of intact retinas from neonatal animals under physiological conditions. Once prepared, the retina is carefully mounted onto an HD-MEA chip, which consists of a grid of 26,400 electrodes capable of performing simultaneous extracellular recordings from at least 1,000 RGCs. Recordings can last up to several hours. Ultimately, this methodological approach offers valuable applications in investigating retinal development, disease, and potentially cross-species comparative studies, contributing to broader advancements in neuroscience and vision research.

Introduction

Spontaneous retinal waves are periodic bursts of correlated activity observed in the developing retina before the onset of vision. In mice, the circuits that initiate and propagate retinal waves change rapidly during development, starting embryonically and ending at eye opening (postnatal day 14)¹. As retinal circuits develop, the spatiotemporal properties of retinal waves change dramatically^2,3. Several studies support that those specific spatiotemporal properties instruct the development of the visual system: asynchronous activity between the eyes instructs eye-specific segregation

Protocol

This protocol outlines the steps for isolating and preparing retinal tissue from neonatal mice for MEA recordings of retinal waves using Maxwell Biosystems HD-MEA platform. The procedure is designed to preserve physiological conditions, ensuring the retinal tissue remains structurally intact, free from damage, and properly prepared for optimal electrode contact. These experiments were approved by the Vanderbilt Animal Care and Use Program, under protocol number M2200056-00. The mice (1-2-week-old, both sexes) were housed in a 12 h day/night cycle vivarium and fed a regular chow diet.

1. Preparation of retinal tissue....

Results

High-throughput recordings and analysis of retinal waves with HD-MEAs
We performed an hour-long HD-MEA recording of spontaneous retinal waves (Figure 2). A raster plot of neuronal activity shows the structured pattern of retinal waves, where each dot represents a detected action potential from an individual electrode (Figure 2A, bottom). Summing activity across electrodes results in a single trace, which makes it easier to v.......

Discussion

The protocol described here provides a reproducible and high-throughput method for preparing retinal tissue and performing HD-MEA recordings, offering a robust method to study retinal network activity. HD-MEA technology offers significant advantages over traditional electrophysiological and imaging techniques, particularly in capturing high-throughput data. HD-MEA delivers real-time, millisecond-scale accuracy recordings of spontaneous retinal wave dynamics, enabling precise wave initiation, propagation, and synchronizat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3 mL disposable transfer pipette (with end cut off)	Fisherbarnd	13-711-9CM	Assists in moving retina between petri dishes.
Artificial cerebrospinal fluid (aCSF)			Maintains physiological conditions for retinal tissue; composed of NaCl, KCl, NaH2PO4, NaHCO3, CaCl2, MgCl2, glucose, and bubbled with carbogen.
CaCl2	Fisher Chemicals	C79-500	To prepare artificial cerebrospinal fluid (aCSF)
Carbogen supply (95% O2, 5% CO2)			Used to oxygenate aCSF and maintain tissue viability.
Curved blade scalpel (#10)	Integra	4-110	Used to cut tissue with precision.
Dissection microscope (stereoscope)	Zeiss	Stemi 508	Essential for visualizing and handling retinal tissue.
glucose	Fisher Chemicals	BP350-1	To prepare artificial cerebrospinal fluid (aCSF)
In-line heater	Multichannel System	TC02	Warms aCSF to 32-34°C for optimal conditions.
Ismatec Perfusion System	Ismatec	ISM4208	Maintains continuous flow of oxygenated aCSF.
KCl	Fisher Chemicals	P271-500	To prepare artificial cerebrospinal fluid (aCSF)
KH2PO4	Sigma Aldrich	P5504-100g	To prepare artificial cerebrospinal fluid (aCSF)
MaxOne Recording Unit	Maxwell Biosystems	MX1-BRD	Interface between MaxOne Chip and System
MaxOne System	Maxwell Biosystems	MX1-SYS	Core system for MEA-based electrophysiological recodrings.
MaxOne Tissue Holder with a 3-axis micromanipulator and replaceable inserts	Maxwell Biosystems	MX1-HLD	Ensures precise placement of the retina on the MEA chip.
MEA chip (MX1-S-CHP, MaxWell Biosystems)	Maxwell Biosystems	MX1-S-CHP	High-density microelectrode array for recording neuronal activity.
MgCl2	Fisher Chemicals	M33-500	To prepare artificial cerebrospinal fluid (aCSF)
NaCl	Fisher Chemicals	S271-1	To prepare artificial cerebrospinal fluid (aCSF)
NaHCO3	Fisher Chemicals	S233-500	To prepare artificial cerebrospinal fluid (aCSF)
Needle (30 G x ½)	BD Biosciences	305106	Helps make incisions in cornea.
Neonatal animals (P1-P14; mouse)			Model organisms such as mice, rats, or others used for retinal studies.
PC with MaxLab Live Scope software	HP	Z4	Used for data acquisition and analysis of recordings.
Petri dish (35 mm or 60 mm)	Pyrex	3483E12	Used as a workspace for dissection.
Roboz micro Adson forceps (RS-5232, 4.75” long, 1 x 2 teeth, 0.5 mm tip)	Roboz	RS-5232	Specialized forceps for fine dissection.
Roboz spring scissors (RS-5671, 10 mm cutting edge, 0.15 mm tip width, 3¾" overall length)	Roboz	RS-5671	Precision scissors for cutting delicate tissue.
Single-hair brush			Small paintbrush modified to a single hair for handling delicate tissue.
Two fine-tipped forceps	Roboz	RS-5060	Used for delicate tissue handling.
Whatman filter papers (#1), cut into small pieces	GE Healthcare	1001-042	Used for handling and drying retinal tissue.