Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research

Hello, I'm Chris Paglia, director of the Visual Information Processing Lab here in the Department of Biomedical Engineering at Boston University. Hi, I'm Jacque Liu, a PhD student in Dr.Paglia lab. The research efforts of our lab are directed at understanding the neural computation that enable animals to see.

For this research, we use one of the oldest creatures on earth, the American horseshoe crab. Now these animals may be living fossils. They continue to play an indispensable role in biomedical research.

Not only does their blood contain special cells that scientists use to detect bacteria toxins in our medicines, but their eyes also contain a neural network that has provided much insight into how our visual system operates. The cellular basis of processes like light adaptation and lateral inhibition were first revealed in studies of the animal's compound lateral eyes, and for this pioneering work, Dr.Keffer Hartline received a Nobel Prize in 1967. The Horseshoe Creb remains an attractive model for vision research because the animal is large and hearty 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'll illustrate three experimental paradigms for investigating the neuro basis of vision that can be performed in vivo with this classic animal model. For vision research, they are electro retinal gram recording optic nerve recording, and intraretinal recording. The horseshoe crabs Are kept in an aerated salt water tank in a room exposed to a regulated light dark cycle.

Before starting any of these recording techniques, the animals chilled in an ice bucket for about 10 minutes and then secured to a wooden platform by placing two stainless steel screws in the pro soma and two in the EPIs soma. The platform is weighted underneath with granite So that it sinks in water. The electroretinogram Or ERG is used to monitor eye sensitivity over time.

It measures the gross electrical response of all cells in the eye. To have flash of light tools needed for this procedure include a screwdriver, petroleum jelly ringer solution, a pipette an LED, A recording chamber, stainless steel screws, and a cotton swap. We use a self-made recording chamber to measure the ERG.

The body is designed to hold a saline reservoir in contact with the eye. The lid contains a silver chloride wire for coupling the conductive solution to an amplifier and a small hole sized to accommodate an LED before attachment. The underside of the chamber is coated with petroleum jelly.

The chambers then secured over the Horseshoe crab eye with two screws filled with saline and capped. The animal is placed in a light tight cage in a tank filled with seawater over the gills. The LED cable is Inserted into the chamber lid.

The signal lead is clipped to the chamber wire and the reference lead to one of the implanted screws, and both leads are connected to the head stage of a high impedance differential amplifier for signal amplification and noise filtering. The cage is then close to plug room light from reaching the animal. The ad stage is connected to the amplifier and the amplifier filters set to pass frequencies below about 10 hertz.

From there, the signal ascend to oscilloscope For viewing and to a data acquisition board for computer analysis and storage, we use a custom made program written in lab view to deliver light stimuli from the LED and to record the ERG signal. The LED is triggered with a 100 millisecond pulse of five vols every 10 minutes. On the left is displayed the ERG waveform to each light flash, and on the right is plotted the peak to peak amplitude of successive flash waveforms for tracking changes in eye sensitivity over time.

With ERG recordings, one can study the effects of light adaptation and circadian neural modulation. The eye conveys visual messages to the brain and the spike responses of its optic nerve fibers. The encoded messages can be studied by recording extracellularly from single nerve fibers.

Circadian clock messages fed back to the eye can also be studied for this method. Tools needed for this procedure include a screwdriver threat arain ringer solution v jurors van scissors, a recording chamber curved forceps, a fine needle probe, a dull scalpel, surgical scissors, and a sectional electrode. The recording chambers designed with a tongue to accommodate the nerve to stir 20 milliliters of blood may be drained from the horse you crab by inserting a 16 gauge needle between the hinge muscles and to the heart.

Exsanguination is not necessary, but makes optic nerve dissection easier with the animal secured to the platform. The location of the optic nerve is estimated by drawing a slightly curved line on the carpus between the lateral and median eyes. A circular hole is then made in the carpus with a refin.

The hole is the same diameter as the chamber bottom. The center of the hole is located about two centimeters anterior to the lateral eye and slightly dorsal to the line so that the nerve runs along the ventral portion of the chamber overlying connective tissue is cleared until a full length of nerve is visible and the exposed nerve is freed from surrounding and underlying tissue. A strand of threat swooped around the nerve and pulled into the chamber through the semi-circular opening in the bottom.

Through this same opening, the nerve is gently guided into the chamber by pulling on the string. As the nerve enters the chamber bottom is pushed into the hole so as to Minimize stretch on the nerve. The chamber chamber's then affixed with two screws and filled with ringer solution.

After Chamber attachment, the animal is placed in a light tide cage in a tank filled with seawater over the gills. The chamber interior is visualized under a stereoscope and cotton is padded around the opening of the bottom to prevent leakage of blood into and ringers out of the chamber. The chamber is refilled with fresh Ringer solution.

Residual tissue is removed with fine banished scissors and tweezers. A small cut is made in the sheath that encapsulates the nerve and the nerve is separated from the sheath with a fine needle probe. The sheath is gradually unwrapped and removed by cutting along the length of The nerve.

A Tiny Fiber bundle is then separated from the nerve using the probe and cut at the end furthest from the eye For afferent fiber recording and at the end closest to the eye. For eend fiber recording, We Use an AM system section electrode filled with Springer solution for recording, which is connected with tubing to a Gilman syringe for suction. The electrode tip is made by fire, polishing the end of a one millimeter diameter, bur silicate glass capillary a B and C connection provides the signal lead and the reference lead is a Silva chloride wire wrapped around the electrode to Reduce noise.

The electrode tip is Positioned into the recording chamber and the cut end of the nerve bundle is drawn into the glass tip via suction. The signal and reference leads are connected to the head stage of a differential amplifier for signal amplification and noise filtering. The amplifier output is passed onto an oscilloscope for viewing and a data acquisition card for computer analysis and storage.

We use a custom made program written in LabVIEW to control light stimuli and a digital spike discriminator to record spike trains. A fiber optic light pipe is employed for single cell illumination, whereas a computer controlled video display is used for pattern stimulation with optic nerve recordings, one can study signal transmission between the eyes and the brain intraretinal. Recording involves inserting a micro electrode directly into cells in the eye and recording the intracellular voltage fluctuations induced by light.

Tools needed for this procedure include a screwdriver, an L-shaped lucid platform. The threaded screw holes a micro electro to over the glass electrode, stainless steel screws, tweezers, and a fine scalpel. After the harsher crab, a secure tree, a wooden platform, a lucid plate with preset screw holes is attached to the carpus with two screws on the side and one on the top.

The animal is placed in a light tight cage in a tank filled with seawater over the gills. A motorized micro positioner is fast into the blade with screws and the positioner arm is aligned over the eye. A batch of glass micro pipettes are pulled from one millimeter outer Diameter or silicate glass, and the tips backfilled By a capillary action.

By placing the pipettes in a vial of three molar potassium chloride solution for a few minutes, the rest of the pipette is filled manually with the salt solution and inserted into a micro electrode holder prefilled with solution to prevent bubble formation. The electrode holder is then inserted into the head stage of an intracellular amplifier affixed to the micro positioner. A section of the retina is exposed by carefully cutting away a tiny square section of the dorsal cornea.

With the scalpel, A drop of Ringer Solution is placed on the exposed tissue to prevent drying, and the micro pipette is advanced towards the retina. When the pipette Tip touches the solution, the current injection mode of the intracellular amplifier is engaged and the electrode impedance is measured micro pipettes with impedances outside the range of 20 to 70 mega ohms are discarded. Those in this range are advanced in micros size, steps into the retina and impaled into cells by vibrating the tip electronically, light stimuli are delivered to impale cells with a fiber optic pipe or video display.

Three types of cells may be encountered in the horseshoe crab eye, ular cells, eccentric cells, and pigment cells, ular cells through a large depolarizing response to light eccentric cells show a train of action potentials riding on a depolarizing response and pigment cells show no light responses at all. The response is observed on an oscilloscope and recorded to a computer with intraretinal recordings. One can study the cellular Basis of vision.

To conclude, we have shown how to perform electroretinogram recordings, optic nerve recordings, and intraretinal recordings on horseshoe crabs in vivo. These methods are widespread in use and provide unique insights into the neural mechanisms of visual information processing with horseshoe crabs. They're not only straightforward to apply in a research lab but can be easily incorporated into a teaching lab on visual neurophysiology.

Thanks for watching and good luck with your Experiments.