The authors thank Mrs. Paulette McKenna and Lisa Pezzino in this study for secretarial support, and Mr. Phil Auerbach for technical support. The authors also thank Drs. Michael Ariel and Michael S. Jones (Saint Louis University School of Medicine) for introducing us to the in vitro isolated head preparation. Funding for support of this collaboration was provided by the Department of Biology (Robert S. Chase Fund), the Academic Research Committee, and the Neuroscience Program at Lafayette College. Lastly, this work is dedicated to Mr. Phil Auerbach, who passed away 28 September 2016; he decommissioned a scanning electron microscope and recognized the usefulness of its 5-axis stage for use in this protocol. His friendship and resourcefulness will be missed greatly.

NOTE: Red-eared slider turtles, both male and female, were purchased from a vendor. Turtles were housed in a warm animal suite containing two 60-gallon tubs equipped with brick islands for sunning under 250-W infrared lights. The environment was maintained on a 14/10-h light/dark cycle with the water temperature at 22 &#176;C. Lights were turned on at 6:00 am and turned off at 8:00 pm. The tanks equipped with filtering systems were cleaned weekly, and turtles were fed ad libitum every other day. The care of red-eared slider turtles and all of the following experimental procedures described here32,33 were approved by the Institutional Animal Care and Use Committee (IACUC) at Lafayette College.
1. Equipment Setup
<ol>
	<li>Prepare turtle Ringer&#39;s solution. Add the following to distilled water in this order: sodium chloride 96.5 mM (58.44 g/mol), potassium chloride 2.6 mM (74.56 g/mol), magnesium chloride 2.0 mM (203.31 g/mol), sodium bicarbonate 31.5 mM (84.01 g/mol), dextrose 20.0 mM (180.16 g/mol), concentrated hydrochloric acid to adjust the pH to 7.51, and calcium chloride 4.0 mM (110.98 g/mol) (see Table of Materials). Mix the solution while adding each salt. 
	CAUTION: Concentrated HCl is hazardous (skin, eyes, inhalation, and ingestion hazards).</li>
	<li>Make tips for the suction electrodes from 5-cm-long capillary glass (see Table of Materials), by fire-polishing to different sizes of inner diameters in order to accommodate cranial nerves of varying thickness.
	<ol>
		<li>Use a small file to etch a line across a piece of capillary glass. Place in&#160;tissue paper and break in half.</li>
		<li>Slowly roll one of the ends of the capillary glass into the flame of a Bunsen burner. Periodically examine the tip for size, smoothness, and symmetry using a dissection scope and a fiber optic light source (see Table of Materials). 
		NOTE: For turtles with head widths between 20 and 30 mm, optimal fitting inner diameter sizes typically range from 0.4 to 0.8 mm for the oculomotor nerve (nIII), 0.3 to 0.6 mm for the trochlear nerve (nIV), and 0.2 to 0.4 mm for the abducens nerve (nVI).</li>
	</ol>
	</li>
	<li>Clean and organize Rongeurs, a blunt dissection probe, microscissors, fine forceps, curved forceps, and a scalpel handle with blades (see Table of Materials) for the dissection. 
	NOTE: Sterilization of instruments is optional.</li>
</ol>
2. Anesthesia and Euthanasia
<ol>
	<li>Place the turtle in an ice bucket for 60 min to cryoanesthetize it.</li>
	<li>Euthanize the turtle by decapitation using a small animal guillotine (see Table of Materials).
	<ol>
		<li>Gently pry the jaws of the animal open with a small weighing spatula so that a hook can be inserted and turned to fit under the tip of the upper jaw.</li>
		<li>Pull with steady pressure to extend the animal&#39;s head through the guillotine. Swiftly decapitate the animal.</li>
	</ol>
	</li>
	<li>Place turtle head in a dissection dish. Have enough turtle Ringer&#8217;s solution on hand to irrigate the tissue. Oxygenize the solution with 95/5% O2/CO2 (see Table of Materials).</li>
	<li>Maintain the tissue at 4 &#176;C by placing ice around the outside of the dish.</li>
</ol>
3. Dissection
<ol>
	<li>Use the dissection scope with a fiber optic light source to carry out the dissection.</li>
	<li>Remove the lower jaw. Place a blunt dissection probe through the mouth to provide easier handling of the head. Cut the joint connecting the dentary bone to the cranium with a scalpel. Use rongeurs to pull the lower jaw away from the cranium. Use rongeurs to pull off the skin and muscles from their attachments at the dorsal and lateral regions of the cranium.</li>
	<li>Remove the vertebral column.
	<ol>
		<li>Identify the vertebral column at the caudal end of the cranium. Bend the vertebral column ventrally to expose the spinal cord. Use microscissors to snip the spinal cord. Use rongeurs to remove the vertebral column and other tissue from the cranium by pulling caudally.</li>
	</ol>
	</li>
	<li>Remove the brain from the cranium after cutting the cranial nerves.
	<ol>
		<li>Starting at the foramen magnum, use rongeurs to cut two incisions on the dorsal cranium. Make cuts superficial to avoid damaging the brain beneath.</li>
		<li>Use rongeurs to carefully pull off the dorsal cranium. Use microscissors to remove the meninx to expose the rest of the brain. Remove enough meninx until the olfactory bulbs, in the anterior cranial cavity, can be identified (see Figure 1A). Continue to irrigate the brain with turtle Ringer&#39;s solution, as necessary.</li>
		<li>Use 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.</li>
		<li>Use microscissors to gently push the midbrain toward the midline to expose the cranial nerves; nIII, about 0.6 mm, can be seen in front of nIV, and the diameter of nIV will be slightly less than nIII. Cut nIII and nIV where they attach to the midbrain (see Figure 1B). Repeat this on the other side.</li>
		<li>Cut the left and right optic nerve (nII) with microscissors. Then tilt the brainstem to one side. Observe nVI emerging from the ventral surface near the junction of the pons and the medulla (see Figure 1C); the diameter of nVI is small and approximately 0.3 mm. Cut both the left and right nVI.</li>
		<li>Remove the remaining parts of the brainstem from the turtle with fine forceps and microscissors. Once the cranium is empty, examine the cranial cavity floor. Identify nIII, nIV, and nVI.</li>
	</ol>
	</li>
	<li>Remove the upper and lower eye-lids with fine forceps and microscissors.</li>
</ol>
4. Calibration of Eye Movements
<ol>
	<li>Use a rigid table (see Table of Materials) to support the placement of the gimbal and other instruments. Place the turtle head into the gimbal chuck so that the dorsal surface of the head is parallel to the horizon using a small bubble-level resting across the skull. Roughly position one of the eyes at the center of the gimbal&#39;s horizontal and vertical rotations.</li>
	<li>Place the infrared camera, fitted with an infrared light emitting diode (LED), which is part of the video-based eye tracking system (see Table of Materials), at a viewing distance of approximately 12 cm from the turtle&#39;s eye.
	<ol>
		<li>Angle the camera 45 degrees above the line of sight of the eye. The LED should be at the 11 o&#8217;clock position when looking at the camera lens. Center the LED along the optical axis of the eye. The camera will be slightly off axis (as viewed from above the eye).</li>
		<li>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 eyes (canthi) are at the edges of the horizontal view.</li>
	</ol>
	</li>
	<li>Connect the camera to the video-based eye tracking system to process the data. Split the signal to a DVD-recorder to capture the raw video. Focus the camera to obtain a clear image of the eye. Take care to fine-position the eye at the center of the camera view using the three degrees of linear adjustment (x, y, z) provided with the gimbal.</li>
	<li>Detect the dark pupil by setting the threshold and contrast appropriately using the program provided with the video-based eye tracking system.
	<ol>
		<li>Using the mouse, click on the &#34;Video&#34; menu and under &#34;Mode&#34; select &#34;High Precision&#34; to capture images at a sampling rate of 30 Hz (resolution of 640 pixels x 480 lines). Also under &#34;Video&#34;, select &#34;Dark Pupil&#34; for &#34;Pupil Type&#34; and &#34;Ellipse (rotated ellipse)&#34; for &#34;Pupil Segmentation Method&#34;.</li>
		<li>In the &#34;EyeCamera&#34; window, click on the &#34;Pupil Search Area Adjustment&#34; icon (small vertical rectangle with a dot in the center). Use the mouse to drag out a rectangle that limits an area around the pupil. Avoid dark areas that could be confused with the pupil.</li>
		<li>In the &#34;Controls&#34; window, confirm that boxes for &#34;Auto Image&#34; and &#34;Positive-Lock Threshold-Tracking&#34; are checked. Click on &#34;Auto-Threshold&#34; to optimize the density of scanning, which will show as green dots over the dark pupil.</li>
	</ol>
	</li>
	<li>Calibrate the video-display of the video-based eye tracking program to the rotations of the gimbal 12.5 &#176; (+/-) around its horizontal axis and 10 &#176; (+/-) around its vertical axis.
	<ol>
		<li>In the &#34;Controls&#34; window, click on &#34;Display&#34;. Check the boxes under both &#34;Gaze&#34; and &#34;Stim&#34; for &#34;Gaze Point&#34;, &#34;Calib Region&#34;, and &#34;Geometry Grid&#34;. After checking the boxes under &#34;Geometry Grid&#34;, a window will pop up and say, &#34;The stimulus display geometry must be measured before the geometry grid can be displayed. Do you wish to do this now?&#34; Select &#34;Y&#34; for yes.</li>
		<li>Using the mouse, click on the &#34;Windows&#34; menu and select &#34;Stimulus&#34;. The &#34;Stimulus&#34; window will open showing a vertical and horizontal line crossing at the center of the display. Measure the lengths of the lines to the nearest mm. Press &#34;Esc&#34; on the keyboard to exit the &#34;Stimulus&#34; window.</li>
		<li>Using the mouse, click on the &#34;Stimulus&#34; menu and select &#34;Geometry Setup&#34;. Input the lengths of the lines that were just measured. Adjust the viewing distance so that the degrees/line equal 25 &#176; for the horizontal line and 20 &#176; for the vertical line. Click on the &#34;Store&#34; button and close the window.</li>
		<li>In the &#34;EyeSpace&#34; window, select the number of calibration &#34;Data Point&#34; to be &#34;9&#34;. With the turtle&#39;s eye positioned at the center, click on the center data point and click the &#34;Re-present&#34; button. 
		NOTE: The &#34;Stimulus&#34; window will open, and &#34;Get Ready&#34; will appear at the center of the screen. A box will appear at the center location and then disappear. On its disappearance, the &#34;Stimulus&#34; window will close. The center position should now be calibrated.</li>
		<li>Repeat the procedure by rotating the gimbal right/left, +12.5 &#176;/-12.5 &#176;, and up/down +10 &#176;/-10 &#176; to calibrate the remaining data points.</li>
	</ol>
	</li>
	<li>To calibrate torsional rotation, use the template fitting algorithm provided with the video-based eye tracking program. The algorithm sets a zero position based on markings of the iris and computes an angle of rotation when the markings become offset from the centroid of the pupil.
	<ol>
		<li>Using the mouse pointer, click on the &#34;Windows&#34; menu and select &#34;Torsion&#34;. Click the &#34;START&#34; button in the &#34;Torsion&#34; window. In the &#34;EyeCamera&#34; window, an arc will appear over the image of the eye.</li>
		<li>Adjust the radius, the angle, and the length of the arc using the sliders at a location where irregular markings are present in the iris. Check the boxes for &#34;Real-time graphics&#34; and &#34;Auto-Set after adjust&#34;. If necessary, adjust the brightness and contrast in the Controls window and re-threshold the dark pupil (see step 4). Click the &#34;Set Template&#34; button.</li>
	</ol>
	</li>
	<li>Place a ruler in the same focal plane as the pupil and record the width of the full camera view. The value will be used later to determine the actual width of the pupil.</li>
</ol>
5. Positioning of Suction Electrode on Cranial Nerve to Evoke Eye Movements
<ol>
	<li>Carefully place a pin reference electrode into the connective or muscle tissue remaining on the head.</li>
	<li>Place the suction electrode (see Table of Materials) on the cranial nerve using a micromanipulator and dissection scope mounted on a boom. Use a fiber optic light source to view and guide the placement.
	<ol>
		<li>Match the size of a nerve to a capillary glass tip. Trial and error is necessary to obtain a best fit around the diameter of a nerve (see step 1.2 for size recommendations). Place the glass tip onto the suction electrode. Fill the suction electrode with Ringer&#39;s solution and adjust the volume within the syringe to about half of its capacity.</li>
		<li>Carefully move the glass tip of the electrode using the micromanipulator to a position above the cut-end of the selected nerve. Ensure that Ringer&#39;s solution fills the cranium and the tip is below its surface. If necessary, use modeling clay to dam up locations where the Ringer&#39;s solution is leaking out of the cranium.</li>
		<li>Pull back on the plunger of the syringe. 
		NOTE: The vacuum will draw the nerve into the end of the capillary tip. A good fit is indicated by the nerve remaining within the tip with little or no additional vacuum applied.</li>
	</ol>
	</li>
</ol>
6. Stimulation of Cranial Nerve and Analysis of Eye Movements
<ol>
	<li>Use a general purpose nerve/muscle stimulator with a current isolation device (see Table of Materials) to stimulate the cranial nerve via the suction electrode.
	<ol>
		<li>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.</li>
		<li>Select parameters of the currents using the dials and switches on the stimulator and isolation device. Use a range of currents from 1 to 100 &#181;A, with frequency of 10 to 400 Hz. Use 1- or 2-ms pulses in trains lasting 100, 500, or 1,000 ms.</li>
	</ol>
	</li>
	<li>Record the timing of stimulations. 
	NOTE: Transistor-transistor-logic (TTL) pulses are synchronized with the deliveries of the currents from the stimulator and communicate in real-time via a cable to input channels of the video-based eye tracking system. A software module provided with the video-based eye tracking program controls the communication.
	<ol>
		<li>To visualize the timing of the current applications and their influence on eye movements, click on the &#34;PenPlots&#34; menu. Select &#34;X Gaze Position&#34;, &#34;Y Gaze Position&#34;, &#34;Torsion&#34;, and &#34;Pupil Width&#34; to show real time raw data plots for X and Y eye positions, torsion, and pupil width. Also select the &#34;Seconds &#38; Markers&#34; from the &#34;PenPlots&#34; menu to show a timing plot with tick marks, which appear at 1 s intervals. 
		NOTE: A capital letter &#34;T&#34; will appear marking the onset of the TTL pulse, occurring simultaneously with the current application.</li>
		<li>To store the data of eye movements evoked by currents, click on the &#34;File&#34; menu and select &#34;New Data File&#34; under &#34;Data&#34;. Input a file name and press &#34;Enter&#34;. Saving data can be paused and then restarted using the combination of key commands, &#34;Ctrl&#34; + &#34;p&#34;. When an experimental session is completed, select &#34;Close Data File&#34; off &#34;File&#34; menu under &#34;Data&#34;.</li>
		<li>To help keep track of the type of currents applied, click on the &#34;Windows&#34; menu and select &#34;Data Pad&#34;. The &#34;KeyPad/DataMarker&#34; window will appear. Click on a letter or a number to identify parameters of the current stimulations being delivered to the nerve. 
		NOTE: For example, &#34;X&#34; could stand for 10 &#181;A. Clicking on &#34;X&#34; stores its entry into the data file in real time for post hoc analysis. It also appears on the &#34;PenPlot&#34; for &#34;Seconds &#38; Markers&#34; for ongoing observation.</li>
	</ol>
	</li>
	<li>Analyze data from the eye tracker system.
	<ol>
		<li>Open the saved data file, which is in a text delimited format, into a spread sheet program of choice to organize the data and carry out statistical analysis.
		<ol>
			<li>Convert the pupil width values stored in the file to real sizes in mm.</li>
			<li>Convert the values of X and Y eye positions and torsions to units of degree and use conventions for describing eye movements; therefore, positive directions of rotations: intorsion, elevation, and adduction; and negative directions of rotations: extorsion, depression, and abduction.</li>
		</ol>
		</li>
		<li>Copy the header information onto a new worksheet. This will include values for the screen size (width and height) and the viewing distance. Eight columns of data collected at a rate of 30 Hz will follow below the header information.</li>
		<li>Go back to the worksheet containing the raw data. Do a &#34;Find&#34; for &#34;X&#34; in the last column titled &#34;Marker&#34; to locate where stimulation was applied with 10 &#181;A. Find incidence of &#34;T&#34;, marking the beginning of the current stimulation. Copy 15 rows (0.5 s) of data occurring before &#34;T&#34; and 90 frames after &#34;T&#34; (3.0 s); i.e., 3.5 s total. Paste the data in the new worksheet below the header information.</li>
		<li>Insert a blank column after &#34;PupilWidth&#34;. In the blank column, convert to the calibrated dimension in mm: 
		Horizontal pupil diameter = &#34;PupilWidth&#34; &#215; the dimension of camera view width</li>
		<li>Insert 2 blank columns after both &#34;X_Gaze&#34; and &#34;Y_Gaze&#34;. Normalize positions to the dimensions of the viewing screen: coordinates (0, 0) at the top left of the screen extending to (1, 1) at the bottom right. In the first blank column, translate positions to a coordinate system having (0, 0) at the center of the screen. Include the conversion to the dimensions of the screen in mm: 
		X = (0.5 &#215; width)&#160; &#8211; (&#34;X_Gaze&#34; &#215; width); Y = (0.5 &#215; height) &#8211; (&#34;Y_Gaze&#34; &#215; height) 
		NOTE: The sequence of operation is for the left eye. The sequence will need to be reversed for the right eye in order to follow convention of negativity for abduction and positivity for adduction.</li>
		<li>In the second columns, use trigonometry functions to convert to angles (&#176;) of rotation: 
		horizontal rotation = arctan (X/viewing distance);vertical rotation = arctan (Y/viewing distance)</li>
		<li>Torsion is already shown in units of degrees, but to conform to convention of positivity for intorsion, if measuring is done on the left eye, multiply by -1. For the right eye, no multiplication is necessary. The program codes clockwise rotation as positive.</li>
		<li>Plot the pupil diameter and rotations as a function of time.</li>
	</ol>
	</li>
</ol>

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D., Ariel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+"beat-to-beat"+interval+generator+for+optokinetic+nystagmus.">A "beat-to-beat" interval generator for optokinetic nystagmus.</a> Biol Cybern. 66, 203-216 (1992).</li><li>Keifer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+eye-blink+reflex+model:+Role+of+excitatory+amino+acid+receptors+and+labeling+of+network+activity+with+sulforhodamine.">In vitro eye-blink reflex model: Role of excitatory amino acid receptors and labeling of network activity with sulforhodamine.</a> Exp Brain Res. 97, 239-253 (1993).</li><li>Keifer, J., Armstrong, K. E., Houk, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+classical+conditioning+of+abducens+nerve+discharge+in+turtles.">In vitro classical conditioning of abducens nerve discharge in turtles.</a> J Neurosci. 15, 5036-5048 (1995).</li><li>Rosenberg, A. F., Ariel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+optokinetic+eye+movements+in+turtles+that+incorporates+properties+of+retinal+slip+neurons.">A model for optokinetic eye movements in turtles that incorporates properties of retinal slip neurons.</a> Vis Neurosci. 13, 375-383 (1996).</li><li>Ariel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-loop+optokinetic+responses+of+the+turtle.">Open-loop optokinetic responses of the turtle.</a> Vis Res. 37, 925-933 (1997).</li><li>Anderson, C. W., Keifer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+conditioned+abducens+nerve+responses+in+a+highly+reduced+in+vitro+brainstem+preparation+from+the+turtle.">Properties of conditioned abducens nerve responses in a highly reduced in vitro brainstem preparation from the turtle.</a> J Neurophysiol. 81, 1242-1250 (1999).</li><li>Keifer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+classical+conditioning+of+the+turtle+eyeblink+reflex:+Approaching+cellular+mechanisms+of+acquisition.">In vitro classical conditioning of the turtle eyeblink reflex: Approaching cellular mechanisms of acquisition.</a> Cerebell. 2, 55-61 (2003).</li><li>Zhu, D., Keifer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+controlling+trigeminal+and+auditory+nerve-evoked+abducens+eyeblink+reflexes+in+pond+turtles.">Pathways controlling trigeminal and auditory nerve-evoked abducens eyeblink reflexes in pond turtles.</a> Brain Behav Evol. 64, 207-222 (2004).</li><li>Jones, M. S., Ariel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+unilateral+eighth+nerve+block+on+fictive+VOR+in+the+turtle.">The effects of unilateral eighth nerve block on fictive VOR in the turtle.</a> Br Res. 1094, 149-162 (2006).</li><li>Jones, M. S., Ariel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology,+intrinsic+membrane+properties,+and+rotation-evoked+responses+of+trochlear+motoneurons+in+the+turtle.">Morphology, intrinsic membrane properties, and rotation-evoked responses of trochlear motoneurons in the turtle.</a> J Neurophysiol. 99 (3), 1187-1200 (2008).</li><li>Krenz, J. G., Naylor, G. J. P., Shaffer, H. B., Janzen, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+phylogenetics+and+evolution+of+turtles.">Molecular phylogenetics and evolution of turtles.</a> Mol Phylogenet Evol. 37 (1), 178-191 (2005).</li><li>Dearworth, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+trochlear+nerve+in+eye+abduction+and+frontal+vision+of+the+red-eared+slider+turtle+(Trachemys+scripta+elegans).">Role of the trochlear nerve in eye abduction and frontal vision of the red-eared slider turtle (Trachemys scripta elegans).</a> J Comp Neur. 52, 3464-3477 (2013).</li><li>Dearworth, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pupil+constriction+evoked+in+vitro+by+stimulation+of+the+oculomotor+nerve+in+the+turtle+(Trachemys+scripta+elegans).">Pupil constriction evoked in vitro by stimulation of the oculomotor nerve in the turtle (Trachemys scripta elegans).</a> Vis Neurosci. 26, 309-318 (2009).</li><li>Mead, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFEL+TOUR:+a+description+of+the+introduction+to+FUN+electrophysiology+labs+workshop+at+Bowdoin+College,+July+27-30,+and+the+resultant+faculty+learning+community.">IFEL TOUR: a description of the introduction to FUN electrophysiology labs workshop at Bowdoin College, July 27-30, and the resultant faculty learning community.</a> J Undergrad Neurosci Educ. 5, A42-A48 (2007).</li><li>Jackson, D. C., Ultsch, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+hibernation+under+the+ice+by+turtles+and+frogs.">Physiology of hibernation under the ice by turtles and frogs.</a> J Exp Zool A Ecol Genet Physiol. 313 (6), 311-327 (2010).</li><li>Romano, J. M., Dearworth, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pupil+constriction+evoked+by+stimulation+of+the+ciliary+nerve+in+the+red-eared+slider+turtle+(Trachemys+scripta+elegans).">Pupil constriction evoked by stimulation of the ciliary nerve in the red-eared slider turtle (Trachemys scripta elegans).</a> J Penns Acad Sci. 85, 4-8 (2011).</li><li>Miller, J. M., Robins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraocular-muscle+forces+in+alert+monkey.">Extraocular-muscle forces in alert monkey.</a> Vis Res. 32, 1099-1113 (1992).</li><li>Gamlin, P. D., Miller, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraocular+muscle+motor+units+characterized+by+spike-triggered+averaging+in+alert+monkey.">Extraocular muscle motor units characterized by spike-triggered averaging in alert monkey.</a> J Neurosci Meth. 204, 159-167 (2011).</li><li>Quaia, C., Ying, H. S., Optican, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Viscoelastic+properties+of+passive+eye+muscle+in+primates.+III:+Force+elicited+by+natural+elongations.">The Viscoelastic properties of passive eye muscle in primates. III: Force elicited by natural elongations.</a> PLOS ONE. 5, A236-A254 (2010).</li><li>Anderson, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+primate+oculomotor+plant+revealed+by+effects+of+abducens+microstimulation.">Dynamics of primate oculomotor plant revealed by effects of abducens microstimulation.</a> J Neurophys. 101, 2907-2923 (2009).</li><li>Maxwell, J. H., Harless, M., Morlock, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+surgery.">Anesthesia and surgery.</a> Turtles: Perspective and Research. , 127-152 (1979).</li><li>AVMA Panel on Euthanasia. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Veterinary+Medical+Association.">American Veterinary Medical Association.</a> J Am Vet Med Assoc. 218 (5), 669-696 (2001).</li><li>Clarke, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+the+pupil's+response+to+light+in+the+hooded+rat.">Shaping the pupil's response to light in the hooded rat.</a> Exp Br Res. 176, 641-651 (2007).</li><li>Bennett, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+anesthesia+and+chemical+restraint+in+reptiles.">A review of anesthesia and chemical restraint in reptiles.</a> J Zoo Wild Med. 22 (3), 282-303 (1991).</li><li>Bickler, P. E., Buck, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+Tolerance+in+Reptiles,+Amphibians,+and+Fishes:+Life+with+Variable+Oxygen+Availability.">Hypoxia Tolerance in Reptiles, Amphibians, and Fishes: Life with Variable Oxygen Availability.</a> Ann Rev Physiol. 69, 145-170 (2007).</li></ol>

The authors have nothing to disclose.

Critical Steps:
The critical steps within this protocol are the following: 1) the dissection and the care taken to maintain the viability of the transected nerves; 2) the matching of sizes by the suction electrodes to cranial nerves to provide consistent responses; and 3) the placement of the head in the gimbal to provide adequate calibration of the rotations of the eye.
Troubleshooting:
The dissection can be challenging, but after completing i...

Rationale for Using Red-eared Slider Turtles in Electrophysiological Experiments:
Red-eared slider turtles (Trachemys scripta elegans), are considered one of world&#39;s worst invasive species1 and can indicate that an ecosystem is in trouble. The reason why red-eared slider turtles are so successful is poorly understood but it may in part be due to their tolerant physiology and possession of nervous tissues that can survive under hypoxic conditions2,3,4. Using them for experimentation does not threaten their numbers and with minimal efforts, electrophysiological preparations can remain viable over extended durations, as long as 18 hours5,6. The benefit is similar to the advantage of using invertebrate animals such as crayfish7, which also have the ability to withstand low levels of oxygen8.
Techniques for Measuring Eye Movements:
Approaches to measure eye movements in frontal-eyed animals using non-human primates have been well developed9. The eye rotates in the orbit around three axes: horizontal, vertical, and torsional. The magnetic search coil method is generally considered the most reliable for measuring rotations, but is invasive, requiring small coils to be inserted into the scleras of animals10,11. Video-based systems also can measure rotations and have the advantage of being non-invasive. The development of better cameras along with innovative image processing have enhanced their functionality making video-based systems an attractive alternative to consider12,13,14.
The techniques developed for measuring eye movements in nonmammals have been much less significant. Measures are either low resolution or describe only some of the rotations15,16,17,18. The lack of development can be partially blamed on the difficulty in training nonmammals to follow visual targets. Although eye movements have been well studied in red-eared slider turtles19,20,21,22,23,24,25,26,27,28,29,30, because of the challenge in training animals to track targets, the precise kinematics of their eye movements is poorly understood.
Red-eared slider turtles are generally considered lateral-eyed vertebrates, but because they can fully retract their heads into their shell31, significant occlusion of the lateral visual fields by the carapace occurs32. The result is that their visual line of sight is forced toward the front, making them behave more like frontal-eyed mammals. Therefore, their use as a model for developing approaches for measuring eye movements also offers a unique evolutionary perspective.
The protocol described in this work uses an in vitro isolated head preparation to identify the kinematics of the eye movements in red-eared slider turtles. Brains are dissected from the skulls leaving the cranial nerves intact. Heads are placed into a gimbal to calibrate eye movements and evoke responses by electrical stimulation of the cranial nerves innervating the eye muscles. Measures of rotations by the eyes are done by a video-based system, using software algorithms, which track the dark pupil and the markings of the iris. The preparation provides the opportunity to measure kinematics of both extraocular (i.e., horizontal, vertical, and torsional rotations)32 and intraocular (i.e., pupil changes)33 movements.
Model System for Analysis of Efferent Neural Pathways:
More generally, the approach provides investigators the chance to study how efferent neural signals generate eye movements when muscles start from their relaxed states and in the absence of integrated sensory information processed by the brain32,33. Therefore, the eye kinematics can be examined in a model system in which they are solely processed by the efferent neural pathway leaving the brain and synapsing onto the muscles.

<table><tbody><tr><td>Red-eared slider turtles</td><td>Kons Scientific</td><td>&lt;em&gt;Trachemys scripta elegans&lt;/em&gt;</td><td>Large size (carapace length 15-20 cm)</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich Co. LLC.</td><td>S5886</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich Co. LLC.</td><td>P5405</td><td></td></tr><tr><td>Magnesium choride</td><td>Sigma-Aldrich Co. LLC.</td><td>M7304</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich Co. LLC.</td><td>S5761</td><td></td></tr><tr><td>Dextrose</td><td>Sigma-Aldrich Co. LLC.</td><td>C5767</td><td></td></tr><tr><td>Concentrated hydrochloric acid</td><td>Sigma-Aldrich Co. LLC.</td><td>H7020</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich Co. LLC.</td><td>C7902</td><td></td></tr><tr><td>pH meter</td><td>Oakton</td><td>pH 6+</td><td></td></tr><tr><td>Suction stimulation electrode</td><td>A-M Systems</td><td>573000</td><td>Bipolar suction electrode. Note that 573000 has been replaced with 573050.</td></tr><tr><td>Capillary glass</td><td>A-M systems</td><td>626000</td><td>Single-barrel borosilicate capillary glass without microfilament, length 10 cm, outside diameter 1.0 mm, inner diameter 0.50 mm</td></tr><tr><td>Alternative suction stimulation electrode</td><td>A-M Systems</td><td>573050</td><td>Bipolar suction electrode. Requires larger diameter capillary glass: 627000, outside diameter 1.2 mm, inner diameter 0.68 mm</td></tr><tr><td>Stereoscope</td><td>Lieca</td><td>GZ7</td><td>Magnification range, 10x &amp;ndash; 70x</td></tr><tr><td>Fiber optic light source</td><td>Amscope</td><td>HL250-A</td><td>150W Fiber optical microscope illuminator light box</td></tr><tr><td>Rongeurs</td><td>Carolina Biological Supply Company</td><td>625654</td><td>stainless steel, straight spring, 5.25&quot;</td></tr><tr><td>Blunt dissection probe</td><td>Carolina Biological Supply Company</td><td>627405</td><td>Huber mall probe, double-ended probe and seeker, 6&quot;</td></tr><tr><td>Microscissors</td><td>Carolina Biological Supply Company</td><td>623555</td><td>Iris microdissecting scissors, stainless steel, 0.5&quot; blades, 4.75&quot; long</td></tr><tr><td>Fine forceps</td><td>Sigma-Aldrich Co. LLC.</td><td>F6521</td><td>Jewelers forceps, dumont No. 5, inox alloy, 4.25&quot;</td></tr><tr><td>Curved forceps</td><td>Sigma-Aldrich Co. LLC.</td><td>Z168696</td><td>Medium tip, curved forceps, stainless steel, 4&quot;</td></tr><tr><td>Scalpel handle</td><td>Sigma-Aldrich Co. LLC.</td><td>S2896</td><td>Scalpel handles, No. 3, stainless steel</td></tr><tr><td>Scalpel blade</td><td>Sigma-Aldrich Co. LLC.</td><td>S2771</td><td>Scalpel blades, No. 11, steel</td></tr><tr><td>Guillotine</td><td>Harvard Apparatus</td><td>73-1918</td><td>Kleine guillotine type 7575</td></tr><tr><td>Spatula</td><td>Sigma</td><td>Z648299</td><td>Micro spoon and spatula weighing set. Use small spatula: 5.9&amp;rdquo; long x 0.07&amp;rdquo; diameter handle with square end: 0.17&amp;rdquo; x 1.3&amp;rdquo; long, other end round: 0.17&amp;rdquo; x 1.27&amp;rdquo; long</td></tr><tr><td>Hook</td><td>Autozone</td><td>98069</td><td>SureBilt hook and pick set. Use grinder to dull sharp points of hook to prevent injury to animals mouth.</td></tr><tr><td>95/5% O&lt;sub&gt;2&lt;/sub&gt;/CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas, Inc.</td><td>X02OX95C2003102</td><td>5% Carbon dioxide balance oxygen certified standard gas mixture, size 200 Cylinder, CGA-296</td></tr><tr><td>Regulator</td><td>Airgas, Inc.</td><td>Y11244D296-AG</td><td>Single stage brass 0-100 psi analytical cylinder regulator CGA-296 with needle outlet. Use brass adjustable airline pipe valve to go from 3/8&quot;, inner diameter, vinyl airline tubing connected to regulator to a 3/16&quot;, inner diameter, airline connection going to airstone or glass pasteur pipette.</td></tr><tr><td>Adjustable airline pipe valve</td><td>Doctors Foster and Smith</td><td>CD-12061</td><td>Brass valve</td></tr><tr><td>Rigid table</td><td>Unknown</td><td>Unknown</td><td>Auto-clave door laid on top of a sturdy table. Nine 5&quot; diameter tennis balls isolate vibrations from the top surface of the table.</td></tr><tr><td>5&quot; tennis ball</td><td>Petco Animal Supplies, Inc.</td><td>712868</td><td>Petco Jumbo Pet Tennis Ball: balls are unsliced and held within an integrated frame on the underside part of the autoclave door.</td></tr><tr><td>Alternative vibration isolation table</td><td>Newport Corporation</td><td>INT1-36-6-N</td><td>Rigid vibration control system, integrity 1: Surface dimensions, 3&#039; x 6&#039;</td></tr><tr><td>Gimbal</td><td>ISI, International Scientific Instruments, Inc.</td><td>Stage from SUPER III-A Scanning EM</td><td>5-axis eucentric stage: X, Y, and Z linear movements, &amp;plusmn;20 mm, 0.1 mm precision; Rotations, vertical, &amp;plusmn;10&amp;deg;, and horizontal, &amp;plusmn;12.5&amp;deg;, with 1.25&amp;deg; precision. Note: from decommission instrument.</td></tr><tr><td>Chuck for gimbal</td><td>Unknown</td><td>Unknown</td><td>Chuck from an old microtome of unknown manufacture was machined to fit the shaft of the specimen holder of the Scanning EM stage</td></tr><tr><td>Alternative gimbal</td><td>ThorLabs, Inc.</td><td>GN2/M with MBT602/M</td><td>Dual-axis goniometer (GN2/M) mounted on 3-axis microblock stage with thumbscrew adjusters (MBT602/M): design a chuck to hold turtle head with eye at 12.7 mm above top surface of goniometer (distance to point of rotation)</td></tr><tr><td>Video-based eye tracking system</td><td>Arrington Research, Inc.</td><td>ViewPoint EyeTracker, PC-60</td><td>Tracking method: Infrared video by dark pupil; Black and white camera (Item BC02): 30 Hz, 640 x 480; System requirements: Windows 2000, XP, 7, 8, 8.1, 10; Visual range: Horizontal +/- 44&amp;deg;; vertical +/- 20&amp;deg;; Accuracy ~0.5&amp;deg;; Spatial resolution ~0.15&amp;deg;; Pupil size resolution ~0.03 mm; Eye data: X, Y position of gaze, pupil height and width, torsion, delta time, total time, and regions of interest (ROI); Real-time communication (Item 0022): 4-Channel AnalogOut with eight TTL input channels to mark codes into the data file</td></tr><tr><td>Multi-position magnetic base</td><td>Harbor Freight Tools</td><td>Pittsburg, item #5645</td><td>Magnetic holder reaches up to 12&quot; and produces 45 lbs. of magnetic pull. Use to position camera. Machine thread holes onto the end of the rod to mount cameras.</td></tr><tr><td>Micromanipulator</td><td>Kopf</td><td>900</td><td>5 axis manipulation for mount of suction electrode: X, Y, Z linear travel, 2 axis of rotation</td></tr><tr><td>Dissection scope on boom</td><td>Lieca</td><td>GZ6</td><td>Magnification range, 6.7x &amp;ndash; 40x</td></tr><tr><td>Nerve/muscle stimulator</td><td>Astro-Med Grass Telefactor</td><td>Grass S88</td><td>Dual pulse voltage stimulator: two output channels that can be operated independently or synchronized to generate non-isolated constant voltage pulses (10 mv to 150 V). Pulses can be single (10 &amp;mu;sec to 10 sec), repetitive (0.01 Hz to 1 KHz), and trains (1 ms to 10 s) and synchronized with TTL inputs and output. Send TTL outputs via the output channels of a DB25 connector to the TTL input channels of the ViewPoint EyeTracker. Note: Astro-Med Grass Telefactor is no longer in business.</td></tr><tr><td>Current isolation device</td><td>Astro-Med Grass Telefactor</td><td>PSIU6</td><td>Current stimulus isolation unit: enables safe delivery of constant currents by the S88 to the preparation. The PSIU6 connects by a BNC cable to one of the output channels of the S88. Multiplier switches on the PSIU6 allow the S88 to generate a wide array of current amplitudes ranging from 0.1 &amp;micro;A to 15 mA.</td></tr><tr><td>Alternative nerve/muscle stimulator with isolation</td><td>A-M Systems</td><td>2100</td><td>Isolated Pulse Stimulator: Unit has built-in isolator to produce constant currents.</td></tr></tbody></table>

ocular kinematics measured by in vitro stimulation of the cranial nerves in the turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Figure 1 shows stills of images taken from a video describing the dissection. Images provide typical locations of the nerves prior to cutting from the brain.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56864/56864fig1.jpg" /> 
Figure 1: Stills of images captured from the video of the dissection to show locations of the optic nerve (nII), oculomotor nerve (nIII), trochlear...

Watch this Scientific Journal Video about Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle at JoVE.com

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

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

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

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

ocular-kinematics-measured-vitro-stimulation-cranial-nerves

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Neuroscience

8.4K Views.  Lafayette College. Dit protocol wordt beschreven hoe u een in vitro geïsoleerd schildpad hoofd voorbereiding voor het meten van de kinematica van hun oogbewegingen. Na verwijdering van de hersenen van de schedel, worden craniale zenuwen gestimuleerd met stromingen te kwantificeren rotaties van de ogen en veranderingen in de maten van de leerling.

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

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

Three Dimensional Vestibular Ocular Reflex Testing Using a Six Degrees of Freedom Motion Platform

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the vestibular organ results in mild to severe equilibrium problems, such as vertigo, dizziness, oscillopsia, gait unsteadiness nausea and/or vomiting. A good and frequently used measure to quantify gaze stabilization is the gain, which is defined as the magnitude of compensatory eye movements with respect to imposed head movements. To test vestibular function more fully one has to realize that 3D VOR ideally generates compensatory ocular rotations not only with a magnitude (gain) equal and opposite to the head rotation but also about an axis that is co-linear with the head rotation axis (alignment). Abnormal vestibular function thus results in changes in gain and changes in alignment of the 3D VOR response.
Here we describe a method to measure 3D VOR using whole body rotation on a 6DF motion platform. Although the method also allows testing translation VOR responses 1, we limit ourselves to a discussion of the method to measure 3D angular VOR. In addition, we restrict ourselves here to description of data collected in healthy subjects in response to angular sinusoidal and impulse stimulation.
Subjects are sitting upright and receive whole-body small amplitude sinusoidal and constant acceleration impulses. Sinusoidal stimuli (f = 1 Hz, A = 4&deg;) were delivered about the vertical axis and about axes in the horizontal plane varying between roll and pitch at increments of 22.5&deg; in azimuth. Impulses were delivered in yaw, roll and pitch and in the vertical canal planes. Eye movements were measured using the scleral search coil technique 2. Search coil signals were sampled at a frequency of 1 kHz.
The input-output ratio (gain) and misalignment (co-linearity) of the 3D VOR were calculated from the eye coil signals 3.
Gain and co-linearity of 3D VOR depended on the orientation of the stimulus axis. Systematic deviations were found in particular during horizontal axis stimulation. In the light the eye rotation axis was properly aligned with the stimulus axis at orientations 0&deg; and 90&deg; azimuth, but gradually deviated more and more towards 45&deg; azimuth.
The systematic deviations in misalignment for intermediate axes can be explained by a low gain for torsion (X-axis or roll-axis rotation) and a high gain for vertical eye movements (Y-axis or pitch-axis rotation (see Figure 2). Because intermediate axis stimulation leads a compensatory response based on vector summation of the individual eye rotation components, the net response axis will deviate because the gain for X- and Y-axis are different.
In darkness the gain of all eye rotation components had lower values. The result was that the misalignment in darkness and for impulses had different peaks and troughs than in the light: its minimum value was reached for pitch axis stimulation and its maximum for roll axis stimulation.
Case Presentation
Nine subjects participated in the experiment. All subjects gave their informed consent. The experimental procedure was approved by the Medical Ethics Committee of Erasmus University Medical Center and adhered to the Declaration of Helsinki for research involving human subjects.
Six subjects served as controls. Three subjects had a unilateral vestibular impairment due to a vestibular schwannoma. The age of control subjects (six males and three females) ranged from 22 to 55 years. None of the controls had visual or vestibular complaints due to neurological, cardio vascular and ophthalmic disorders.
The age of the patients with schwannoma varied between 44 and 64 years (two males and one female). All schwannoma subjects were under medical surveillance and/or had received treatment by a multidisciplinary team consisting of an othorhinolaryngologist and a neurosurgeon of the Erasmus University Medical Center. Tested patients all had a right side vestibular schwannoma and underwent a wait and watch policy (Table 1; subjects N1-N3) after being diagnosed with vestibular schwannoma. Their tumors had been stabile for over 8-10 years on magnetic resonance imaging.

A method is described to measure three-dimensional vestibulo ocular reflexes (3D VOR) in humans using a six degrees of freedom (6DF) motion simulator. The gain and misalignment of the 3D angular VOR provide a direct measure of the quality of vestibular function. Representative data on healthy subjects are provided

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the ...

The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish

Zebrafish are a proven model for vision research, however many of the earlier methods generally focused on larval fish or demonstrated a simple response. More recently adult visual behavior in zebrafish has become of interest, but methods to measure specific responses are new coming. To address this gap, we set out to develop a methodology to repeatedly and accurately utilize the optokinetic response (OKR) to measure visual acuity in adult zebrafish. Here we show that the adult zebrafish's visual acuity can be measured, including both binocular and monocular acuities. Because the fish is not harmed during the procedure, the visual acuity can be measured and compared over short or long periods of time. The visual acuity measurements described here can also be done quickly allowing for high throughput and for additional visual procedures if desired. This type of analysis is conducive to drug intervention studies or investigations of disease progression.

Zebrafish have been a valuable tool for many research areas. Here we demonstrate a method to elicit a visual response and calculate a functional visual acuity in the adult zebrafish.

Zebrafish are a proven model for vision research, however many of the  earlier methods generally focused on larval fish or demonstrated a simple  ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury are essential for translating novel therapeutic strategies from rodent models to clinical application due to their closer similarities to humans in size and anatomy. Here we describe some in vivo methods to evaluate the function and structure of the retinal ganglion cells (RGCs) and optic nerve (ON) in large animals, including visual evoked potential (VEP), pattern electroretinogram (PERG) and optical coherence tomography (OCT). Both goat and non-human primate were employed in this study. By presenting these in vivo methods step by step, we hope to increase experimental reproducibility among different labs and facilitate the usage of large animal models of optic neuropathies.

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Here we demostrate several in vivo tests (flash visual evoked potential, pattern electroretinogram and optic coherence tomography) in goat and rhesus macaque to understand the structure and function of the optic nerve and its neurons.

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...

A New Optokinetic Testing Method to Measure Rat Vision

Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement associated with OKN is commonly used as a measure of visual function in rodents. To record OKN responses in normal and experimental rats, a simple and inexpensive apparatus has been developed. This setup uses two tablet screens to display the OKN visual stimulus consisting of high contrast black and white stripes generated using the OKN Stripes Visualization Web Application, a freely available software. The rat is placed inside a clear Plexiglass holder that limits&#160;movement so that the&#160;rat&#39;s head continuously faces the OKN display screen. The position of the rat holder can be changed to adjust the distance between the rat and the display screen. A micro-camera positioned above the rat holder is used to record the rat&#39;s visual activities. These recordings can be used for quantitative assessments. Based on the presence or absence of clear head-tracking, the OKN responses at different spatial frequencies can be determined. The collected data demonstrates a novel technique for reliable measurement of visual acuity in normal and retinal degenerate rats.

The Optokinetic Nystagmus (OKN) behavioral testing method is used for the assessment of visual acuity in rodents. Here a simple method is demonstrated that can be easily set up in research laboratories for a reliable assessment of visual function in both normal and experimental rats.

Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement ...

Evaluating Optokinetic Reflex Responses in Mice: A New High-Precision Protocol

Quantification of Visual Feature Selectivity of the Optokinetic Reflex in Mice

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize retinal images. Due to its importance and robustness, the OKR has been used to study visual-motor learning and to evaluate the visual functions of mice with different genetic backgrounds, ages, and drug treatments. Here, we introduce a procedure for evaluating OKR responses of head-fixed mice with high accuracy. Head fixation can rule out the contribution of vestibular stimulation on eye movements, making it possible to measure eye movements triggered only by visual motion. The OKR is elicited by a virtual drum system, in which a vertical grating presented on three computer monitors drifts horizontally in an oscillatory manner or unidirectionally at a constant velocity. With this virtual reality system, we can systematically change visual parameters like spatial frequency, temporal/oscillation frequency, contrast, luminance, and the direction of gratings, and quantify tuning curves of visual feature selectivity. High-speed infrared video-oculography ensures accurate measurement of the trajectory of eye movements. The eyes of individual mice are calibrated to provide opportunities to compare the OKRs between animals of different ages, genders, and genetic backgrounds. The quantitative power of this technique allows it to detect changes in the OKR when this behavior plastically adapts due to aging, sensory experience, or motor learning; thus, it makes this technique a valuable addition to the repertoire of tools used to investigate the plasticity of ocular behaviors.

Author Spotlight: An Accurate and Quantitative Approach to Study Visual Feature Selectivity of the Optokinetic Reflex in Mice 

Here, we describe a standard protocol for quantifying the optokinetic reflex. It combines virtual drum stimulation and video-oculography, and thus allows precise evaluation of the feature selectivity of the behavior and its adaptive plasticity.

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize ...

Python-Based Tool for Quantitative Optokinetic Reflex Assessment in Animals

PyOKR: A Semi-Automated Method for Quantifying Optokinetic Reflex Tracking Ability

The study of behavioral responses to visual stimuli is a key component of understanding visual system function. One notable response is the optokinetic reflex (OKR), a highly conserved innate behavior necessary for image stabilization on the retina. The OKR provides a robust readout of image tracking ability and has been extensively studied to understand visual system circuitry and function in animals from different genetic backgrounds. The OKR consists of two phases: a slow tracking phase as the eye follows a stimulus to the edge of the visual plane and a compensatory fast phase saccade that resets the position of the eye in the orbit. Previous methods of tracking gain quantification, although reliable, are labor intensive and can be subjective or arbitrarily derived. To obtain more rapid and reproducible quantification of eye tracking ability, we have developed a novel&#160;semi-automated analysis program, PyOKR, that allows for quantification of two-dimensional eye tracking motion in response to any directional stimulus, in addition to being adaptable to any type of video-oculography equipment. This method provides automated filtering, selection of slow tracking phases, modeling of vertical and horizontal eye vectors, quantification of eye movement gains relative to stimulus speed, and organization of resultant data into a usable spreadsheet for statistical and graphical comparisons. This quantitative and streamlined analysis pipeline, readily accessible via PyPI import, provides a fast and direct measurement of OKR responses, thereby facilitating the study of visual behavioral responses.

Author Spotlight: A Streamlined and Accessible Analysis Method to Quantify Optokinetic Reflex Tracking Responses

We describe here PyOKR, a semi-automated quantitative analysis method that directly measures eye movements resulting from visual responses to two-dimensional image motion. A Python-based user interface and analysis algorithm allows for higher throughput and more accurate quantitative measurements of eye-tracking parameters than previous methods.

The study of behavioral responses to visual stimuli is a key component of understanding visual system function. One notable response is the optokinetic ...

Optokinetic Response Assay: A Method to Measure Visual Acuity in Zebrafish

Source: Cameron, D. J., et al. <a href="https://www.jove.com/v/50832/" target="_blank">The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish</a>.&#160;J. Vis. Exp.&#160;(2013)
This video describes the optokinetic response assay in zebrafish. The video measures the reflexive eye movements based on the visual moving stimulus and determines the visual acuity in zebrafish.

Source: Cameron, D. J., et al. The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish.&#160;J. Vis. Exp.&#160;(2013)
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Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

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