Name: ocular kinematics measured by in vitro stimulation of the cranial nerves in the turtle
Uploaded: 2018-06-02T12:30:00
Description: Watch this Scientific Journal Video about Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle at JoVE.com

Los autores agradecen a la Sra. Paulette McKenna y Lisa Pezzino en este estudio para apoyo secretarial y Sr. Phil Auerbach para soporte técnico. Los autores también agradecen los Drs. Michael Ariel y Michael S. Jones (Saint Louis University School of Medicine) para introducirnos a la preparación principal en vitro aislado. Fondos para apoyo de esta colaboración fue proporcionada por el Departamento de Biología (Robert S. Chase fondo), la Comisión de investigación académica y el programa de Neurociencia en la Universidad de Lafayette. Por último, este trabajo está dedicado al Sr. Phil Auerbach, quien falleció el 28 de septiembre de 2016; había desarmado de un microscopio electrónico de barrido y reconoce la utilidad de su etapa de 5 ejes para el uso de este protocolo. Su amistad y su ingenio se echará de menos enormemente.

Nota: Las tortugas resbalador Rojo-espigado, machos y hembras, fueron compradas de un vendedor. Las tortugas fueron alojadas en una suite de animal caliente que contiene dos tinas de 60 galones equipados con islas de ladrillo para asolearse bajo luces infrarrojos 250 W. El ambiente se mantuvo en un ciclo de luz/oscuridad de 14/10-h con la temperatura del agua a 22 ° C. Luces se enciende a las 6:00 y apagar en 20:00. Los tanques equipados con sistemas de filtración se limpiaron semanalmente y tortugas fueron alimentadas...

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Pasos críticos:
Los pasos críticos en este protocolo son los siguientes: 1) la disección y el cuidado para mantener la viabilidad de los nervios seccionados; 2) la correspondencia de los tamaños de los electrodos de succión para los nervios craneales para proporcionar respuestas coherentes; y 3) la colocación de la cabeza en el cardán para proporcionar la calibración adecuada de la rotación del ojo.
Solución de problemas:
La disecció...

Justificación para el uso de las tortugas de orejas rojas Slider en experimentos electrofisiológicos:
Resbalador Rojo-espigado tortugas (Trachemys scripta elegans), son considerados uno de los peores de las especies invasoras del mundo1 y puede indicar que un ecosistema está en problemas. La razón de por qué tienen tanto éxito las tortugas resbalador Rojo-espigado es mal entendida pero en parte puede ser debido a su fisiología tolerante y posesión de los tejidos nerviosos que pueden sobrevivir bajo condiciones hipóxicas2,3,4 . Uso para experimentación no amenaza a sus números y con esfuerzos mínimos, preparaciones electrofisiológicas pueden permanecer viables sobre duraciones extendidas, tanto como 18 horas5,6. El beneficio es similar a la ventaja de usar animales invertebrados como cangrejos7, que también tienen la capacidad de resistir bajos niveles de oxígeno8.
Técnicas para la medición de movimientos de los ojos:
Enfoques para medir los movimientos oculares en animales frontal-eyed con primates no humanos han sido bien desarrolladas9. El ojo gira en la órbita alrededor de tres ejes: horizontal, vertical y torsional. El método de la bobina de búsqueda magnético se considera generalmente el más confiable para medirlas rotaciones, pero es invasivo, que requiere pequeñas bobinas para ser insertado en el scleras de animales10,11. Sistemas de video también pueden medir rotaciones y tienen la ventaja de ser no invasivo. El desarrollo de mejores cámaras con procesamiento de imágenes innovadoras han mejorado su funcionalidad hacer sistemas basados en vídeo una alternativa atractiva al considerar12,13,14.
Las técnicas desarrolladas para la medición de movimientos de los ojos en nonmammals han sido mucho menos importantes. Las medidas son cualquiera baja resolución o describen sólo algunas de las rotaciones15,16,17,18. La falta de desarrollo puede ser parcialmente atribuyó a la dificultad de nonmammals de formación a seguir objetivos visuales. Aunque los movimientos de los ojos han sido bien estudiados en el resbalador Rojo-espigado tortugas19,20,21,22,23,24,25 ,26,27,28,29,30, debido al desafío en animales del entrenamiento para realizar un seguimiento de objetivos, la cinemática exacta de sus movimientos del ojo está mal entendido.
Tortugas resbalador Rojo-espigado generalmente son considerados como vertebrados con ojos laterales, pero porque puede retraer completamente su cabeza en su concha31, obstrucción significativa de los campos de visión lateral por el caparazón produce32. El resultado es que su línea de vista visual es forzado hacia el frente, haciendo que se comporte más como mamíferos frontal-eyed. Por lo tanto, su uso como un modelo para el desarrollo de enfoques para la medición de movimientos de los ojos también ofrece una perspectiva evolutiva única.
El protocolo descrito en este trabajo utiliza una preparación de cabeza en vitro aislado para identificar la cinemática de los movimientos del ojo en las tortugas de orejas rojas slider. Se disecan los cerebros de los cráneos dejando intactos los nervios craneales. Las cabezas se colocan en un cardán para calibrar los movimientos de los ojos y evocar respuestas por estimulación eléctrica de los nervios craneales inervan los músculos del ojo. Medidas de rotaciones por los ojos se realizan mediante un sistema basado en el video, usando algoritmos de software, que la pista la pupila oscura y las manchas del iris. La preparación proporciona la oportunidad de medir cinemática de ambos extraocular (es decir, rotación horizontal, vertical o torsional)32 y intraocular (es decir, cambios de la pupila)33 movimientos.
Sistema modelo para el análisis de vías nerviosas eferentes:
Más generalmente, el enfoque proporciona a los investigadores la oportunidad para estudiar señales neuronales eferentes cómo generar movimientos de los ojos cuando los músculos de sus Estados relajados y en la ausencia de información sensorial integrada, procesada por el cerebro32, 33. Por lo tanto, la cinemática del ojo puede ser examinada en un sistema modelo en el que únicamente son procesados por la vía neural eferente dejando el cerebro y conectado a los músculos.

<table>
	<tbody>
		<tr>
			<td>Red-eared slider turtles</td>
			<td>Kons Scientific</td>
			<td>Trachemys scripta elegans</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 &#8211; 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&#34;</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&#34;</td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Carolina Biological Supply Company</td>
			<td>623555</td>
			<td>Iris microdissecting scissors, stainless steel, 0.5&#34; blades, 4.75&#34; 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&#34;</td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Sigma-Aldrich Co. LLC.</td>
			<td>Z168696</td>
			<td>Medium tip, curved forceps, stainless steel, 4&#34;</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&#8221; long x 0.07&#8221; diameter handle with square end: 0.17&#8221; x 1.3&#8221; long, other end round: 0.17&#8221; x 1.27&#8221; 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% O2/CO2</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&#34;, inner diameter, vinyl airline tubing connected to regulator to a 3/16&#34;, 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&#34; diameter tennis balls isolate vibrations from the top surface of the table.</td>
		</tr>
		<tr>
			<td>5&#34; 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&#39; x 6&#39;</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, &#177;20 mm, 0.1 mm precision; Rotations, vertical, &#177;10&#176;, and horizontal, &#177;12.5&#176;, with 1.25&#176; 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&#176;; vertical +/- 20&#176;; Accuracy ~0.5&#176;; Spatial resolution ~0.15&#176;; 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&#34; 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 &#8211; 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 &#956;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 &#181;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

Después de que los animales son sacrificados, los tejidos comienzan a morir. Las tortugas ofrecen una ventaja debido a un tiempo de supervivencia de sus tejidos, especialmente en comparación con los vertebrados de sangre caliente. Debido a esto, se pueden realizar experimentos en vitro en tortugas para largos períodos de tiempo para investigar las señales neuronales y control de sus acciones de blanco. Con una preparación aislada de cabeza, medimos la cinemática de los movimientos oculares en tortugas, y su modulación por señales eléctricas por los nervios craneales. Después de que el cerebro fue quitado del cráneo, dejando intactos los nervios craneales la cabeza disecada fue colocada en un cardán para calibrar los movimientos de los ojos. Electrodos de vidrio fueron atados a los nervios craneales (oculomotor, trochlear y abducens) y estimulada con corrientes para evocar los movimientos de los ojos. Nos vigila movimientos de los ojos con un video infrarrojo de seguimiento sistema y cuantificada de rotación de los ojos. Corriente de pulsos con una gama de amplitudes, frecuencias, y las duraciones del tren fueron utilizadas para observar efectos sobre las respuestas. Porque la preparación se separa del cerebro, la vía eferente va a objetivos musculares puede examinarse aisladamente para investigar señales neuronales en ausencia de la información sensorial procesada centralmente.

La figura 1 muestra imágenes fijas de imágenes tomadas de un video que describe la disección. Imágenes ofrecen localizaciones típicas de los nervios antes de cortar el cerebro.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56864/56864fig1.jpg"> Figura 1: imágenes de imágenes capturadas del vídeo de la disección para mostrar ubicaciones del nervio óptico (nII), nervi...

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

Este protocolo describe cómo utilizar una en vitro aislado tortuga cabeza preparación para medir la cinemática de los movimientos del ojo. Después del retiro del cerebro del cráneo, los nervios craneales pueden ser estimulados con corrientes cuantificar rotaciones y cambios en el tamaño de la pupila.

Cinemática ocular midiendo In Vitro la estimulación de los nervios craneales en la tortuga

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

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