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

Gli autori ringraziano la Signora Paulette McKenna e Lisa Pezzino in questo studio per supporto segretariale e Mr. Phil Auerbach per il supporto tecnico. Gli autori ringraziano anche i dottori Michael Ariel e Michael S. Jones (Saint Louis University School of Medicine) per ci ha spiegato che la preparazione della testa in vitro isolato. Contributi a sostegno di questa collaborazione è stata fornita dal dipartimento di biologia (Robert S. Chase Fund), il comitato accademico di ricerca e il programma di neuroscienze al Lafayette College. Infine, questo lavoro è dedicato a Mr. Phil Auerbach, scomparso 28 settembre 2016; ha un microscopio elettronico a scansione in disuso e riconosciuto l'utilità del suo stadio di 5 assi per l'uso in questo protocollo. L'amicizia e l'intraprendenza ci mancherà enormemente.

Nota: Le tartarughe cursore rosso-eared sia maschile che femminile, sono state acquistate da un fornitore. Le tartarughe sono state alloggiate in una suite di animale calda contenente due 60-gallone vasche dotate di isole di mattoni per prendere il sole sotto le luci a infrarossi 250-W. L'ambiente è stato effettuato su un ciclo di luce/buio 14/10-h con la temperatura dell'acqua a 22 ° C. Luci erano acceso alle 6:00 e spento alle 20:00. I serbatoi dotati di sistemi di filtraggio sono stati puliti settimanalmente e tarta...

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Fasi critiche:
I passaggi critici all'interno di questo protocollo sono i seguenti: 1) la dissezione e la cura per mantenere la vitalità dei nervi transected; 2) la corrispondenza di dimensioni dagli elettrodi aspirazione ai nervi cranici di fornire risposte coerenti; e 3) il posizionamento della testa nella sospensione cardanica per fornire un'adeguata calibrazione delle rotazioni dell'occhio.
Risoluzione dei problemi:
La dissezione può esse...

Spiegazione razionale per usando il cursore rosso-eared tartarughe in esperimenti elettrofisiologici:
Le tartarughe dalle orecchie rosse (Trachemys scripta elegans), sono considerati uno dei peggiore specie invasive1 del mondo e può indicare che un ecosistema è nei guai. Il motivo per cui sono tanto successo tartarughe cursore rosso-eared è capito male ma può essere in parte dovuto la loro fisiologia tollerante e possesso di tessuti nervosi che può sopravvivere in condizioni di ipossia2,3,4 . Li utilizzano per sperimentazione non minacciano i loro numeri e con sforzi minimi, preparazioni elettrofisiologici possono rimanere vitali sopra le durate estese, fino a 18 ore5,6. Il vantaggio è simile al vantaggio dell'utilizzo di animali invertebrati quali gamberi7, che hanno anche la capacità di sopportare i bassi livelli di ossigeno8.
Tecniche per misurare movimenti oculari:
Approcci per misurare movimenti oculari negli animali dagli occhi frontali, uso di primati non umani sono stati ben sviluppato9. L'occhio ruota in orbita intorno a tre assi: orizzontale, verticale e torsionale. Il metodo di ricerca magnetico della bobina è generalmente considerato il più affidabile per la misura di rotazioni, ma è invasivo, che richiedono piccole bobine da inserire le sclere di animali10,11. Sistemi basati su video possono anche misurare rotazioni e hanno il vantaggio di essere non invasivo. Lo sviluppo delle meglio fotocamere con elaborazione immagine innovativa hanno migliorato la loro funzionalità, rendendo i sistemi basati su video alternativa da considerare12,13,14.
Le tecniche sviluppate per misurare i movimenti di occhio in nonmammals sono state molto meno significative. Misure sono entrambi bassa risoluzione o descrivono solo alcune delle rotazioni15,16,17,18. La mancanza di sviluppo può essere parzialmente imputata sulla difficoltà nel nonmammals di formazione da seguire obiettivi visual. Anche se i movimenti oculari sono state ben studiati in cursore rosso-eared tartarughe19,20,21,22,23,24,25 ,26,27,28,29,30, a causa della sfida in addestramento di animali per tenere traccia degli obiettivi, la cinematica precisa dei loro movimenti oculari è scarsamente capito.
Tartarughe del cursore rosso-eared sono generalmente considerate vertebrati laterale-eyed, ma perché essi possono ritirare completamente le loro teste nel loro guscio31, occlusione significativa dei campi visivi laterali dal carapace si verifica32. Il risultato è che la loro linea di vista visivo è costretto verso la parte anteriore, che li rende si comportano più come mammiferi frontale-eyed. Di conseguenza, loro uso come modello per lo sviluppo di approcci per misurare movimenti oculari offre anche una prospettiva evolutiva unica.
Il protocollo descritto in questo lavoro utilizza una preparazione della testa in vitro isolato per identificare la cinematica dei movimenti dell'occhio in tartarughe cursore rosso-eared. Cervelli vengono sezionati dai teschi intatti i nervi cranici. Teste sono inserite in un giunto cardanico per calibrare i movimenti oculari ed evocano risposte da stimolazione elettrica dei nervi cranici che innervano i muscoli oculari. Misure delle rotazioni degli occhi sono fatte da un sistema basato su video, utilizzando algoritmi software, che traccia la pupilla scura e le marcature dell'iride. La preparazione prevede l'opportunità di misurare cinematica di entrambi extraoculare (cioè, orizzontale, verticale e torsionale rotazioni)32 e intraoculare (cioè, modifiche alla pupilla)33 movimenti.
Sistema modello per l'analisi delle vie neurali efferente:
Più in generale, l'approccio fornisce la possibilità di studiare come efferenti segnali neurali generare movimenti oculari quando muscoli iniziano da loro state rilassate e in assenza di informazioni sensoriali integrate elaborate dal cervello32, gli investigatori 33. Di conseguenza, la cinematica di occhio può essere esaminata in un sistema di modello in cui vengono elaborati esclusivamente dal percorso neurale efferente lasciando il cervello e synapsing sui muscoli.

<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

Dopo che gli animali sono euthanized, loro tessuti cominciano a morire. Le tartarughe offrono un vantaggio a causa di un più lungo periodo di sopravvivenza dei loro tessuti, soprattutto se paragonato ai vertebrati a sangue caldo. Per questo motivo, gli esperimenti in vitro in tartarughe possono essere eseguiti per lunghi periodi di tempo per studiare i segnali neurali e il controllo delle loro azioni mirate. Usando una preparazione testa isolata, abbiamo misurato la cinematica dei movimenti oculari in tartarughe, e loro modulazione di segnali elettrici trasportati dai nervi cranici. Dopo che il cervello è stato rimosso dal cranio, intatti i nervi cranici, la testa dissecata fu posto in un giunto cardanico per calibrare i movimenti oculari. Elettrodi di vetro sono stati fissati ai nervi cranici (oculomotore, trocleare e abducente) e stimolato con correnti di evocare movimenti oculari. Abbiamo monitorato i movimenti di occhio con un video a infrarossi tracking system e quantificati rotazioni degli occhi. Impulsi di corrente con una gamma di ampiezze, frequenze, e treno durate venivano utilizzate per osservare gli effetti sulle risposte. Perché la preparazione è separata dal cervello, la via efferente che va agli obiettivi del muscolo può essere esaminata isolatamente per studiare segnalazione neurale in assenza di informazioni sensoriali centralmente elaborate.

La figura 1 Mostra stills di immagini tratte da un video che descrive la dissezione. Immagini forniscono posizioni tipiche dei nervi prima del taglio dal cervello.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56864/56864fig1.jpg"> Figura 1: Stills di immagini catturate dal video della dissezione per mostrare la località del nervo ottico (nII), nervo oculomotore (nIII), nerv...

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

Questo protocollo viene descritto come utilizzare una preparazione testa in vitro isolato tartaruga per misurare la cinematica dei loro movimenti oculari. Dopo la rimozione del cervello dal cranio, nervi cranici può essere stimolati con correnti a quantificare le rotazioni dell'occhio e cambiamenti nelle dimensioni della pupilla.

Oculare cinematica misurata dalla stimolazione In Vitro dei nervi cranici in tartaruga

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

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