Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research

Summary

In this video we perform electroretinogram recording, optic nerve recording, and intraretinal recording with the American horseshoe crab, Limulus Polyphemus. These electrophysiological paradigms can be used for investigating the neural basis of vision in a research or teaching lab.

Abstract

The American horseshoe crab, Limulus Polyphemus is one of the oldest creatures on earth, and the animal continues to play an indispensable role in biomedical research. Not only does their blood contain special cells that scientists use to detect bacteriotoxins in our medicines, but their eyes also contain a neural network that has provided much insight about physiological processes operating in our visual system, such as light adaptation and lateral inhibition. The horseshoe crab remains an attractive model for vision research because the animal is large and hardy for an invertebrate, its retinal neurons are big and easily accessible, its visual system is compact and extensively studied, and its visual behavior is well defined. Moreover, the structure and function of the eyes are modulated on a daily basis by a circadian clock in the animal s brain. In short, the visual system of horseshoe crabs is simple enough to be understood yet complex enough to be interesting.

In this video we present three electrophysiological paradigms for investigating the neural basis of vision that can be performed in vivo with Limulus. They are electroretinogram recording, optic nerve recording, and intraretinal recording. Electroretinogram (ERG) recordings measure with a surface electrode the summed electrical response of all cells in the eye to a flash of light. They can be used to monitor the overall sensitivity of the eye for prolong periods of time. Optic nerve recordings measure the spiking activity of single nerve fibers with an extracellular microsuction electrode. They can be used to study visual messages conveyed from the eye to the brain as well as circadian-clock messages fed back from the brain to the eye. Intraretinal recordings measure with an intracellular microelectrode the voltage fluctuations induced by light in individual cells of the eye. They can be used to elucidate cellular mechanisms of retinal processing.

Protocol

Part 1: Experimental Preparation

Experimental procedures performed on horseshoe crabs have been approved by the Institutional Animal Care and Use Committee at Boston University. Animals are purchased from the Marine Biological Labs (Woods Hole, MA) or other vendor and housed in an aerated saltwater tank in a room exposed to a regulated light-dark cycle. The lighting regimen is important for entraining the crab's circadian clock and cycling the eye daily between its daytime and nighttime states. Immediately before starting any invasive procedures, the animal is chilled in an ice bucket for 10-15 minutes until its motion is slowed and then sec....

Discussion

We have illustrated how to perform ERG recordings, optic nerve recordings, and intraretinal recordings on horseshoe crabs in vivo. The recording techniques each provide different insights into the neural basis of vision, and they can all be used to study retinal function in live animals thanks to the crab's large eyes and hard carapace. Optic nerve activity can even be recorded from freely behaving animals in the ocean with proper construction of electrodes (4). These techniques can also be performed on excised eyes with.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LED	Light source	Newark Inc	33C1292
Suction electrode	Electrode	A-M Systems	573000
XCell 3*4-Channel Extracellular Amplifier	Amplifier	FHC, Inc.	40-40-8B
Intracellular Recording	Amplifier	Cygnus	IR-283A
APM	Neural Spike Discriminator	FHC, Inc.	APM
Bits++	Video Board	Cambridge Research Systems	Bits++
Piezopatch Manipulator	Micropositioner	World Precision Instruments, Inc.	PPM5000
Square Pulse Stimulator	Nerve Stimulator	Grass Technologies	Model S48
P-97	Micropipette Puller	Sutter Instrument Co.	Model P-97
Borosilicate Glass Capillary	Electrode glass	World Precision Instruments, Inc.	1B150-4
Horsesh– crab (Limulus polyphemus)	Animal	Marine Biological Laboratories
Micropipette Puller	Glass Puller	Sutter Instrument Co.	P-97
Zoom Stereoscope	Microscope	Jed Pella Inc.	SMZ-168