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

Summary

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.

Abstract

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.

Introduction

Rationale for Using Red-eared Slider Turtles in Electrophysiological Experiments:

Red-eared slider turtles (Trachemys scripta elegans), are considered one of world'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 conditions^2,3,4. Using them for experimentation does not threaten their numbers a....

Protocol

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 °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 here^32,33 w....

Results

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.

Figure 1: S.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Red-eared slider turtles	Kons Scientific	Trachemys scripta elegans	Large size (carapace length 15-20 cm)
Sodium chloride	Sigma-Aldrich Co. LLC.	S5886
Potassium chloride	Sigma-Aldrich Co. LLC.	P5405
Magnesium choride	Sigma-Aldrich Co. LLC.	M7304
Sodium bicarbonate	Sigma-Aldrich Co. LLC.	S5761
Dextrose	Sigma-Aldrich Co. LLC.	C5767
Concentrated hydrochloric acid	Sigma-Aldrich Co. LLC.	H7020
Calcium chloride	Sigma-Aldrich Co. LLC.	C7902
pH meter	Oakton	pH 6+
Suction stimulation electrode	A-M Systems	573000	Bipolar suction electrode. Note that 573000 has been replaced with 573050.
Capillary glass	A-M systems	626000	Single-barrel borosilicate capillary glass without microfilament, length 10 cm, outside diameter 1.0 mm, inner diameter 0.50 mm
Alternative suction stimulation electrode	A-M Systems	573050	Bipolar suction electrode. Requires larger diameter capillary glass: 627000, outside diameter 1.2 mm, inner diameter 0.68 mm
Stereoscope	Lieca	GZ7	Magnification range, 10x – 70x
Fiber optic light source	Amscope	HL250-A	150W Fiber optical microscope illuminator light box
Rongeurs	Carolina Biological Supply Company	625654	stainless steel, straight spring, 5.25"
Blunt dissection probe	Carolina Biological Supply Company	627405	Huber mall probe, double-ended probe and seeker, 6"
Microscissors	Carolina Biological Supply Company	623555	Iris microdissecting scissors, stainless steel, 0.5" blades, 4.75" long
Fine forceps	Sigma-Aldrich Co. LLC.	F6521	Jewelers forceps, dumont No. 5, inox alloy, 4.25"
Curved forceps	Sigma-Aldrich Co. LLC.	Z168696	Medium tip, curved forceps, stainless steel, 4"
Scalpel handle	Sigma-Aldrich Co. LLC.	S2896	Scalpel handles, No. 3, stainless steel
Scalpel blade	Sigma-Aldrich Co. LLC.	S2771	Scalpel blades, No. 11, steel
Guillotine	Harvard Apparatus	73-1918	Kleine guillotine type 7575
Spatula	Sigma	Z648299	Micro spoon and spatula weighing set. Use small spatula: 5.9” long x 0.07” diameter handle with square end: 0.17” x 1.3” long, other end round: 0.17” x 1.27” long
Hook	Autozone	98069	SureBilt hook and pick set. Use grinder to dull sharp points of hook to prevent injury to animals mouth.
95/5% O2/CO2	Airgas, Inc.	X02OX95C2003102	5% Carbon dioxide balance oxygen certified standard gas mixture, size 200 Cylinder, CGA-296
Regulator	Airgas, Inc.	Y11244D296-AG	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", inner diameter, vinyl airline tubing connected to regulator to a 3/16", inner diameter, airline connection going to airstone or glass pasteur pipette.
Adjustable airline pipe valve	Doctors Foster and Smith	CD-12061	Brass valve
Rigid table	Unknown	Unknown	Auto-clave door laid on top of a sturdy table. Nine 5" diameter tennis balls isolate vibrations from the top surface of the table.
5" tennis ball	Petco Animal Supplies, Inc.	712868	Petco Jumbo Pet Tennis Ball: balls are unsliced and held within an integrated frame on the underside part of the autoclave door.
Alternative vibration isolation table	Newport Corporation	INT1-36-6-N	Rigid vibration control system, integrity 1: Surface dimensions, 3' x 6'
Gimbal	ISI, International Scientific Instruments, Inc.	Stage from SUPER III-A Scanning EM	5-axis eucentric stage: X, Y, and Z linear movements, ±20 mm, 0.1 mm precision; Rotations, vertical, ±10°, and horizontal, ±12.5°, with 1.25° precision. Note: from decommission instrument.
Chuck for gimbal	Unknown	Unknown	Chuck from an old microtome of unknown manufacture was machined to fit the shaft of the specimen holder of the Scanning EM stage
Alternative gimbal	ThorLabs, Inc.	GN2/M with MBT602/M	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)
Video-based eye tracking system	Arrington Research, Inc.	ViewPoint EyeTracker, PC-60	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°; vertical +/- 20°; Accuracy ~0.5°; Spatial resolution ~0.15°; 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
Multi-position magnetic base	Harbor Freight Tools	Pittsburg, item #5645	Magnetic holder reaches up to 12" and produces 45 lbs. of magnetic pull. Use to position camera. Machine thread holes onto the end of the rod to mount cameras.
Micromanipulator	Kopf	900	5 axis manipulation for mount of suction electrode: X, Y, Z linear travel, 2 axis of rotation
Dissection scope on boom	Lieca	GZ6	Magnification range, 6.7x – 40x
Nerve/muscle stimulator	Astro-Med Grass Telefactor	Grass S88	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 μsec 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.
Current isolation device	Astro-Med Grass Telefactor	PSIU6	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 µA to 15 mA.
Alternative nerve/muscle stimulator with isolation	A-M Systems	2100	Isolated Pulse Stimulator: Unit has built-in isolator to produce constant currents.