Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

The overall goal of this electrophysiological protocol is tho provide the ability to measure the kinematics of eye movements of the red-eared slider turtle using an in vitro isolated head preparation. This method can help answer key questions in the ocular-motor field, such as how vision evolved in turtles. The main advantage of this technique is that it does not require training animals to track targets and uses a gimbal to calibrate their eye movements.

Demonstrating this technique is Steven Nesbit, an undergraduate student in my laboratory. Begin with the turtle head in a dissection dish. Have enough turtle ringer solution on hand to irrigate the tissue.

Maintain the tissue at four degrees Celsius, by placing ice around the outside of the dish. Place a blunt dissection probe through the mouth to provide easier handling of the head. Then, while working under a dissection microscope, cut the joint connecting the dentary bone to the cranium with the scalpel.

Next, use rongeurs to pull the lower jaw away from the cranium. Then, pull off the skin and muscles from their attachments at the dorsal and lateral regions of the cranium. After identifying the vertebral column at the cavum end of the cranium, bend the vertebral column ventrally to expose the spinal cord, and use micro scissors to the snip the spinal cord.

Use rongeurs to remove the vertebral column and other tissue from the cranium by pulling caudally. Then, make two superficial cuts on the dorsal cranium starting at the foramen magnum and carefully pull off the dorsal cranium. Use micro scissors to remove the meninges and expose the rest of the brain.

Remove enough meninges to allow identification of the olfactory bulbs in the anterior cranial cavity. Continue to irrigate the brain with turtle ringer solution as necessary. Next, used curved forceps to gently pull the cerebrum caudally and produce slight tension on the cranial nerves.

Carefully cut away and remove the olfactory bulbs and cerebrum with curved forceps. Then use micro scissors to gentle push the midbrain toward the midline and expose the cranial nerves. Cranial nerve three can be seen in front of cranial nerve four.

The diameter of nerve four is slightly less than nerve three. Then cut the left and right ride of the optic nerve, or cranial nerve two. Cut nerves three and four where they attach to the midbrain.

Repeat this on the other side. Tilt the brainstem to one side and observe cranial nerve six, the abducens nerve, emerging from the ventral surface near the junction of the pons and medulla. Cut cranial nerve six on both the left and right side.

Remove the remaining parts of the brainstem from the head with fine forceps and micro scissors. Once the cranium is empty, examine the cranial cavity floor and identify nerves three, four, and six. Lastly, remove the upper and lower eyelids with fine forceps and micro scissors.

Place the turtle head into the gimbal chuck. Then use a small bubble level to check that the dorsal surface of the head is parallel to the horizon. Position one of the eyes at the center of the gimbal's horizontal and vertical rotations.

Angle an infrared camera 45 degrees above the line of sight of the eye. The infrared LED should be at the 11 o'clock position, when looking at the camera lens, to center the LED along the optical access of the eye. Adjust the distance of the camera from the eye so that the camera view is maximally filled by the eyeball.

Ensure that the corners of the eye are at the edges of the horizontal view. Focus the camera to obtain a clear image of the eye. Then use the three degrees of linear adjustment provided with the gimbal to fine position the eye at the center of the camera view.

Detect the duct pupil by setting the threshold and contrast appropriately using the program provided with the video based eye tracking system. Using the mouse, click on Video menu, and under Mode, select High Precision to capture images at a sampling rate of 30 hertz, giving resolution of 640 pixels by 480 lines. Also, under Video, open Pupil Type, and select Dark Pupil, and Ellipse, rotated ellipse, for pupil segmentation method.

In the EyeCamera window, click on the pupil search area adjustment icon. Use the mouse to drag out a rectangle that limits an area around the pupil, avoiding dark areas that could be confused with the pupil. Then, in the Controls window, confirm that boxes for AutoImage, and Positive Lock Threshold Tracking are checked.

Click on AutoThreshold to optimize the density of scanning, which will show as green dots over the dark pupil. Calibrate the video display of the video based eye tracking program to the rotations of the gimbal, and calibrate torsional rotation as described in the text. Place a ruler in the same focal plane as the pupil and record the width of the full camera view.

To position the electrodes, first insert a pin reference electrode into the connective or muscle tissue remaining on the head. Match the size of the nerve to be stimulated to a fire polished capillary glass tip, and place the glass tip onto the suction electrode. Fill the suction electrode with ringer solution, and adjust the volume within the syringe to about half of its capacity.

Ensure that the cranial nerves are clearly visible. Then, while looking through a boom mounted dissection scope, use the micro-manipulator to carefully move the glass tip of the electrode to a position above the cut end of the selected nerve and below the surface of the ringer solution that fills the cranium. Pull back on the plunger of the syringe.

The vacuum will draw the nerve into the end of the capillary tip. Connect the suction electrode to the current isolation device using a cable. Connect the lead from the pin reference electrode to the ground connection of the isolation device.

Use the dials and switches on the stimulator and isolation device to input the stimulation parameters. Use a range of currents from one to 100 mircoamps, with frequency of 10 to 400 hertz. Use one or two millisecond pulses in trains lasting 100, 500, 1000 milliseconds.

To visualize the timing of the current applications and their influence on eye movements, click on PenPlots menu. Select X Gaze Position, Y Gaze Position, Torsion, and Pupil Width to show real time raw data plots x and y eye positions, torsion, and pupil width. Also, select the Seconds and Markers from the PenPlots menu to show the timing plot with tick marks, which appear at one second intervals.

To help keep track of the type of currents applied, click on the Windows menu and select DataPad. The KeyPad, DataMarker window will appear. Click on the letter or number to identify parameters of the current stimulations being delivered to the nerve, and analyze data as described in the protocol text.

This trace shows the mean pupil diameter from six stimulations in one preparation, the dashed lines show standard deviation. The rectangular waveform on the x axis denotes onset and offset of a 100 hertz train of one millisecond pulses, with an amplitude of 50 micro amps. This sketch shows the orientation of the iris line in the eye prior to stimulation.

This image shows a still frame from a representative trial before stimulation. This image shows the eye during stimulation. Here, the eye is seen after stimulation.

While attempting this procedure it's important to take your time during the dissection to match the size of the suction electrode to each cranial nerve, and to place the head appropriately in the gimbal. Following these procedure, other methods, like testing different temperatures, or applying pharmacological agents, including analgesics, can be performed to answer additional questions, like, how these factors influence eye movements and nervous tissue survivability.