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

Forfatterne takker fru Paulette McKenna og Lisa Pezzino i denne studien for Administrativ støtte, og Mr. Phil Auerbach kundestøtte. Forfatterne også takke Dr. Michael Ariel og Michael S. Jones (Saint Louis University School of Medicine) for å introdusere oss til i vitro isolert hodet utarbeidelse. Finansiering for støtte av dette samarbeidet ble levert av Institutt for biologi (Robert S. Chase Fund), akademiske forskning komiteen og nevrovitenskap Program på Lafayette College. Til slutt, dette arbeidet er dedikert til Mr. Phil Auerbach, som gikk bort 28 September 2016; Han utrangerte scanning elektron mikroskop anerkjent nytten av 5-aksen stadiet for bruk i denne protokollen. Hans vennskap og oppfinnsomhet vil bli savnet mye.

Merk: Red-eared glidebryteren skilpadder, både mannlige og kvinnelige, ble kjøpt fra en leverandør. Skilpadder ble plassert i en varm dyr pakke inneholder to 60-galle stamper utstyrt med murstein øyene solte under 250 W infrarødt lys. Miljøet var vedlikeholdes på en 14/10-h lys/mørke syklus med temperaturen på 22 ° C. Lysene ble slått på på 6:00 og deaktivert på 8:00 pm. Tankene utstyrt med filtrering systemer ble renset ukentlig, og skilpadder ble matet annonse libitum annenhver dag. Av rød-eared...

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Avgjørende skritt:
De avgjørende skritt i denne protokollen er følgende: 1) dissection og omsorg å beholde levedyktigheten til transected nerver; 2) den tilsvarende størrelser av inntaks elektrodene til Hjernenerve å gi konsistent svar; og 3) plassering av hodet i gimbal å gi tilstrekkelig kalibrering av rotasjoner av øyet.
Feilsøking:
Dissection kan være utfordrende, men etter at det noen ganger, trinnene bør bli relativt rett frem....

Begrunnelsen for å bruke røde-eared glidebryteren skilpadder elektrofysiologiske eksperimenter:
Red-eared glidebryteren skilpadder (Trachemys scripta elegans), regnes som en av verdens verste invasiv Art1 og kan indikere at et økosystem er i trøbbel. Grunnen hvorfor rød-eared glidebryteren skilpadder er så vellykket er dårlig forstått, men det kan delvis skyldes deres tolerant fysiologi og besittelse av nervøs vev som kan overleve under hypoxic forhold2,3,4 . Bruke dem for eksperimentering ikke True deres antall og med minimal innsats, elektrofysiologiske forberedelser kan være levedyktig over lengre varighet, så lenge som 18 timer5,6. Fordelen er fordelen med å bruke virvelløse dyr som kreps7, som også har evne til å tåle lave nivåer av oksygen8.
Teknikker for måling av øyebevegelser:
Tilnærminger til å måle øyebevegelser i frontal-eyed dyr med ikke-menneskelige primater har vært godt utviklet9. Øyet roterer i bane rundt tre akser: vannrett, loddrett og vridningsstivhet. Den magnetiske coil metoden er generelt betraktet som den mest pålitelige for måling rotasjoner, men er invasiv, som krever liten spoler settes inn i scleras av dyr10,11. Video-baserte systemer kan også måle rotasjoner og har fordelen av å ikke-invasiv. Utvikling av bedre kamera med nyskapende bildebehandling har forbedret funksjonaliteten gjør video-basert systemer et attraktivt alternativ å vurdere12,13,14.
Teknikker utviklet for å måle øyebevegelser i nonmammals har vært mye mindre betydning. Tiltak er enten lav oppløsning eller beskrive noen av rotasjoner15,16,17,18. Mangel på utvikling kan delvis skylden på problemer i trening nonmammals å følge visuelle mål. Selv om øyebevegelser har blitt godt studert i rød-eared glidebryteren skilpadder19,20,21,22,23,24,25 ,26,27,28,29,30, på grunn av utfordringen i opplæring dyr å spore mål, presis kinematikken av deres øyebevegelser er dårlig forstått.
Red-eared glidebryteren skilpadder er generelt ansett lateral-eyed virveldyr, men fordi de fullt trekke hodet i deres skall31, betydelig okklusjon av de laterale visuelle feltene av ryggskjoldet oppstår32. Resultatet er at deres visuelle siktelinjen blir tvunget mot fronten, noe som gjør dem oppfører seg mer som frontal-eyed pattedyr. Derfor tilbyr benyttet som en modell for å utvikle metoder for å måle øyebevegelser også et unikt evolusjonært perspektiv.
Protokollen beskrevet i dette arbeidet bruker en i vitro isolert leder forberedelse for å identifisere kinematikken øye bevegelser i rød-eared glidebryteren skilpadder. Hjernen er dissekert fra hodeskaller røres Hjernenerve. Hoder er plassert i gimbal å kalibrere øyebevegelser og fremkalle svar av elektrisk stimulering av Hjernenerve innervating øyemusklene. Mål rotasjoner av øynene gjøres av en video-basert system, bruker programvare algoritmer, som sporer den mørke eleven og merkingen av iris. Forberedelse gir muligheten til å måle kinematikk både extraocular (dvs., vannrett, loddrett og vridningsstivhet rotasjoner)32 og intraokulært (dvs., elev endringer)33 bevegelser.
Modellsystem for analyse av Efferent nervebaner:
Mer generelt gir tilnærmingen etterforskerne sjansen til å studere hvordan efferent nevrale signaler genererer øyebevegelser når musklene begynner fra avslappet stater og i fravær av integrert sensoriske informasjonen behandles av hjernen32, 33. Derfor kan øye kinematikken undersøkes i et modellsystem der de behandles utelukkende av efferent nevrale veien forlater hjernen og synapsing på musklene.

<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

Etter dyr euthanized, begynner deres vev å dø. Skilpadder tilbyr en fordel på grunn av lengre overlevelse tid av deres vev, spesielt sammenlignet med hjertelig-blod virveldyr. Derfor kan i vitro eksperimenter i skilpadder utføres for lengre tid å undersøke thе nevrale signaler og kontroll av sine mål handlinger. Bruker en isolert leder forberedelse, vi målt kinematikken av øyebevegelser i skilpadder og deres modulering av elektriske signaler båret av kraniale nerver. Etter hjernen ble fjernet fra skallen, ble røres Hjernenerve, dissekert hodet plassert i gimbal kalibrere øyebevegelser. Glass elektroder ble knyttet til Hjernenerve (oculomotor, trochlear, og abducens) og stimulert med strøm til å fremkalle øyebevegelser. Vi overvåket øyebevegelser med en infrarød video sporing system og kvantifisert rotasjoner i øynene. Nåværende pulser med en rekke amplituder, frekvenser, og tog varigheter ble brukt til å observere effekter på svar. Fordi preparatet er separert fra hjernen, kan efferent veien skal muskel mål undersøkes isolert undersøke nevrale signalering i fravær av sentralt behandlet sensoriske informasjonen.

Figur 1 viser stillbilder av bilder tatt fra en video som beskriver dissection. Bilder gi vanlige plasseringer for nerver før klippe fra hjernen.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56864/56864fig1.jpg"> Figur 1: stillbilder av bilder tatt fra video av dissection å vise steder av synsnerven (nII), nervus nerve (nIII), trochlear nerve (Salmene) og abducens nerve (V...

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

Denne protokollen beskriver hvordan bruke en i vitro isolert skilpadde leder forberedelse til å måle kinematikken av deres øyebevegelser. Etter fjerning av hjernen fra kraniet stimuleret Hjernenerve med strøm å kvantifisere rotasjoner av øyet og endringer i elev størrelser.

Okulære kinematikk målt ved In Vitro stimulering av Hjernenerve i 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 ...

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

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