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

Författarna tackar fru Paulette McKenna och Lisa Pezzino i denna studie för sekreterarstöd och Mr Phil Auerbach för teknisk support. Författarna också tacka Drs. Michael Ariel och Michael S. Jones (Saint Louis University School of Medicine) för att införa oss till in vitro- isolerade huvud förberedelsen. Finansiering för att stödja detta samarbete lämnades av Institutionen för biologi (Robert S. Chase fonden), utskottet för akademisk forskning och programmet neurovetenskap vid Lafayette College. Slutligen, detta arbete är tillägnad Mr Phil Auerbach, som avled 28 September 2016. Han avvecklade ett svepelektronmikroskop och erkänd nyttan av sin 5-axlig steg för användning i detta protokoll. Hans vänskap och idérikedom kommer att missa mycket.

Obs: Röd-eared reglaget sköldpaddor, både manliga och kvinnliga, köptes från en leverantör. Sköldpaddorna var inrymt i en varm djur suite som innehåller två 60-gallon badkar utrustat med tegel öar för sunning under 250-W infraröd ljus. Miljön bibehölls på en 14/10-h ljus/mörk cykel med vattentemperaturen på 22 ° C. Lamporna var aktiverad vid 06:00 och avstängd kl 8:00 pm. Stridsvagnar utrustade med filtersystem rengjordes varje vecka, och sköldpaddorna matades ad libitum varannan dag. Vård a...

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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. 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Kritiska steg:
Kritiska steg i detta protokoll är följande: 1) dissektion och den omsorg att upprätthålla lönsamheten för transected nerverna; (2) matchning av storlekar av sug elektroderna till kranialnerv att ge konsekventa svar; och 3) placeringen av huvudet i gimbal att tillhandahålla adekvat kalibrering av rotationer av ögat.
Felsökning:
Dissektion kan vara utmanande, men efter att ha avslutat det några gånger, stegen bör bli r...

Motivet för att använda röd-eared reglaget sköldpaddor i elektrofysiologiska experiment:
Röd-eared reglaget sköldpaddor (Trachemys scripta elegans), anses vara en av världens värsta invasiva arter1 och kan indikera att ett ekosystem är i trubbel. Anledningen till röd-eared reglaget sköldpaddor är så framgångsrika är dåligt känd, men det kan delvis bero på deras toleranta fysiologi och innehav av nervvävnader som kan överleva under hypoxisk villkor2,3,4 . Använda dem för experimenterande hotar inte deras antal och med minimala ansträngningar, elektrofysiologiska preparat kan förbli livskraftig över utökade löptider, så länge som 18 timmar5,6. Fördelen är liknande att fördelen med att använda ryggradslösa djur såsom kräftor7, som också har förmågan att tåla låga nivåer av syre8.
Tekniker för att mäta ögonrörelser:
Metoder att mäta ögonrörelser i frontal-eyed djur med icke-mänskliga primater har varit väl utvecklat9. Ögat roterar i omloppsbana kring tre axlar: horisontell, vertikal och torsional. Magnetisk spole sökmetod anses allmänt vara den mest tillförlitliga för mäta rotationer, men är invasiv, som kräver små spolar ska infogas i scleras djur10,11. Video-baserade system kan också mäta rotationer och har fördelen att vara icke-invasiv. Utvecklingen av bättre kameror tillsammans med innovativa bildbehandling har förbättrat deras funktionalitet att göra video-baserade system ett attraktivt alternativ att överväga12,13,14.
De tekniker som utvecklats för att mäta ögonrörelser i nonmammals har varit mycket mindre betydande. Åtgärder är antingen låg upplösning eller beskriver endast några av rotationer15,16,17,18. Avsaknaden av utveckling kan delvis skyllas på svårigheten att utbildning nonmammals att följa visuella mål. Även om ögonrörelser har studerats väl i rött-eared reglaget sköldpaddorna19,20,21,22,23,24,25 ,26,27,28,29,30, på grund av utmaningen i utbildning djur att spåra mål, deras ögonrörelser exakt kinematik är dåligt förstås.
Röd-eared reglaget sköldpaddorna anses i allmänhet lateral-eyed ryggradsdjur, men eftersom de kan fullt flexa sina huvuden i sina skal31, betydande ocklusion av de laterala visuella fält av ryggskölden sker32. Resultatet är att deras visuella siktlinje tvingas mot fronten, vilket gör dem beter sig mer som frontal-eyed däggdjur. Därför, deras användning som en modell för att utveckla metoder för att mäta ögonrörelser erbjuder också ett unikt evolutionära perspektiv.
Protokollet beskrivs i detta arbete använder en in vitro- isolerade huvud förberedelse för att identifiera ögonrörelser i rött-eared reglaget turtles kinematik. Hjärnor är dissekerade från skallar lämnar kranialnerver intakt. Huvuden placeras en gimbal att kalibrera ögonrörelser och framkalla Svaren genom elektrisk stimulering av de kranialnerver innervating ögonmusklerna. Åtgärder av rotationer av ögonen görs av en video-baserade system, använder programvarualgoritmer, som spårar mörka pupillen och märkningarna av iris. Preparatet ger möjlighet att mäta kinematik av båda extraocular (dvs, horisontella, vertikala och torsional rotationer)32 och intraokulära (dvs, elev ändringar)33 rörelser.
Modellsystem för analys av utåtledande nervbanor:
Mer allmänt ger metoden utredarna möjlighet att studera hur efferenta neurala signaler generera ögonrörelser när musklerna börjar från deras avslappnad stater och i avsaknad av integrerade sensorisk information bearbetas av hjärnan32, 33. Därför kan ögat kinematik undersökas i modellsystem där de behandlas enbart av den efferenta neurala vägen lämnar hjärnan och synapsing på musklerna.

<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

När djur är euthanized, börja sina vävnader dö. Sköldpaddorna erbjuda en fördel på grund av en längre överlevnadstid av deras vävnader, särskilt jämfört med varmblodiga ryggradsdjur. På grund av detta, kan in vitro- experiment i turtles utföras för längre tid att undersöka de neurala signalerna och kontroll av deras målinriktade åtgärder. Använda en isolerad huvud förberedelse, Vi mätte kinematik av ögonrörelser i turtles och sin modulering av elektriska signaler bärs av kranialnerver. Efter hjärnan togs bort från skallen, placerades lämnar kranialnerver intakt, dissekerade huvudet i en gimbal kalibrera ögonrörelser. Glaselektroderna bifogades kranialnerver (ögonmuskelförlamningar, trochlear, och abducens) och stimuleras med strömmar att framkalla ögonrörelser. Vi övervakas ögonrörelser med en infraröd video tracking system och kvantifierade rotationer av ögonen. Ström pulser med en rad amplituder, frekvenser, och tåg varaktigheter användes att iaktta effekterna på svaren. Eftersom preparatet är skild från hjärnan, kan efferent vägen kommer att muskel mål granskas isolerat att undersöka neurala signalering i avsaknad av centralt bearbetade sensorisk information.

Figur 1 visar stillbilder av bilder tagna från en video som beskriver dissektion. Bilder ger typiska platser av nerver innan skära från hjärnan.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56864/56864fig1.jpg"> Figur 1: stillbilder av bilder tagna från video av dissektionen till Visa platser av synnerven (nII), ögonmuskelförlamningar nerv (nIII), trochlear nerv (nIV)...

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

Det här protokollet beskriver hur du använder en in vitro- isolerade sköldpaddan huvud förberedelse för att mäta kinematik av deras ögonrörelser. Efter avlägsnande av hjärnan från kraniet, kan kranialnerver stimuleras med strömmar att kvantifiera rotationer av ögat och förändringar i elev storlekar.

Okulär kinematik mätt In Vitro stimulering av kranialnerver i sköldpaddan

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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