Die Autoren danken Frau Paulette McKenna und Lisa Pezzino in dieser Studie für Sekretariat und Herr Phil Auerbach für den technischen Support. Die Autoren danken auch DRS. Michael Ariel und Michael S. Jones (Saint Louis University School of Medicine) für die Einführung von uns, die in-vitro- isoliert Kopf Vorbereitung. Unterstützung dieser Zusammenarbeit wurde von der Fakultät für Biologie (Robert S. Chase Fund), Academic Research Committee und die Neurowissenschaften Programm am Lafayette College finanziert. Schließlich ist diese Arbeit Herr Phil Auerbach, gewidmet der 28. September 2016 verstorben; er ein Rasterelektronenmikroskop stillgelegt und erkannte den Nutzen seiner 5-Achs-Bühne für den Einsatz in diesem Protokoll. Seine Freundschaft und Einfallsreichtum werden sehr vermisst werden.

<ol>
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R., Subramaniam, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balanced+interactions+in+ganglion-cell+receptive+fields.">Balanced interactions in ganglion-cell receptive fields.</a> Vis Neurosci. 16, 319-332 (1999).</li><li>Dearworth, J. R., Granda, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplied+functions+unify+shapes+of+ganglion-cell+receptive+fields+in+retina+of+turtle.">Multiplied functions unify shapes of ganglion-cell receptive fields in retina of turtle.</a> J Vis. 2 (3), 204-217 (2002).</li><li>Nesbit, S. C., Van Hoof, A. G., Le, C. C., Dearworth Jr, J. 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Wichtige Schritte:
Die entscheidenden Schritte innerhalb dieses Protokolls sind die folgenden: 1) die Zerlegung und die Sorgfalt, die zur Aufrechterhaltung der Lebensfähigkeit der durchtrennten Nerven; (2) die passenden Größen von saugelektroden, Hirnnerven, konsistente Antworten bereitzustellen; und 3) die Platzierung des Kopfes in den Gimbal, adäquate Kalibrierung der Umdrehungen des Auges zu bieten.
Fehlerbehebung:
Die Dissektion kann ei...

Begründung für die Verwendung von rot-eared Slider Schildkröten in elektrophysiologischen Experimenten:
Rot-eared Slider Schildkröten (ist Scripta Elegans), gelten als eine der weltweit schlimmsten invasiven Arten1 und können angeben, dass ein Ökosystem in Schwierigkeiten ist. Rot-eared Slider Schildkröten sind deshalb so erfolgreich ist wenig bekannt, aber es kann teilweise durch ihre tolerante Physiologie und des Besitzes von Nervengewebe, die unter hypoxischen Bedingungen2,3,4 überleben können . Verwenden sie für Experimente nicht ihre Zahl gefährdet und mit minimalem Aufwand, elektrophysiologische Vorbereitungen lebensfähig über längere Dauer, so lange wie 18 Stunden5,6 bleiben können. Der Vorteil ist ähnlich wie der Vorteil der Verwendung von wirbellosen Tiere wie Krebse7, die auch geringe Mengen an Sauerstoff8standhalten können.
Techniken zur Messung der Augenbewegungen:
Ansätze zur Messung der Augenbewegungen in Frontal-eyed Tiere mit nicht-menschlichen Primaten sind gut entwickelte9gewesen. Das Auge dreht sich im Orbit um drei Achsen: horizontale, vertikale und Torsionssteifigkeit. Die magnetische Spule Suchmethode gilt allgemein als die zuverlässigste Messung Umdrehungen, aber ist invasiv und erfordert kleine Spulen in den Scleras der Tiere10,11eingefügt werden soll. Video-basierte Systeme können auch Rotationen Messen und haben den Vorteil, nicht-invasiv. Die Entwicklung von besseren Kameras zusammen mit innovativen Bildverarbeitung verbessert ihre Funktionalität, Video-basierte Systeme eine attraktive Alternative zu erwägen,12,13,14.
Techniken entwickelt, die zur Messung der Augenbewegungen in Nonmammals wurden viel weniger bedeutend. Maßnahmen sind entweder niedrige Auflösung oder beschreiben nur einige Umdrehungen15,16,17,18. Der Mangel an Entwicklung kann teilweise auf die Schwierigkeiten bei der Ausbildung Nonmammals, visuellen Ziele Folgen verantwortlich gemacht werden. Obwohl Augenbewegungen auch in rot-eared Slider Schildkröten19,20,21,22,23,24,25 untersucht wurden ,26,27,28,29,30, wegen der Herausforderung in Ausbildung Tiere verfolgen Ziele, die präzise Kinematik des ihre Augenbewegungen ist schlecht verstanden.
Rot-eared Slider Schildkröten gelten in der Regel Lateral-eyed Wirbeltiere, sondern weil sie ihre Köpfe in ihre Schale31vollständig zurückziehen können, erhebliche Okklusion der seitlichen Gesichtsfeld durch die Panzer tritt32. Das Ergebnis ist, dass ihre visuelle Sichtverbindung nach vorne, so dass sie eher wie Frontal-eyed Säugetiere Verhalten gezwungen ist. Ihre Verwendung als Modell für die Entwicklung von Ansätzen zur Messung der Augenbewegungen bietet daher auch eine einzigartige evolutionäre Perspektive.
In dieser Arbeit beschriebene Protokoll verwendet eine in-vitro- isoliert Kopf Vorbereitung um die Kinematik der Augenbewegungen in rot-eared Slider Schildkröten zu identifizieren. Gehirne sind aus den Schädeln der Hirnnerven intakt seziert. Köpfe sind in eine kardanische kalibrieren Augenbewegungen und evozieren Reaktionen durch elektrische Stimulation der Hirnnerven innervieren die Augenmuskeln gelegt. Maßnahmen der Rotationen von den Augen erfolgt durch ein Video-basiertes System mit Software-Algorithmen, die die dunkle Pupille und die Markierungen der Iris zu verfolgen. Die Vorbereitung bietet die Möglichkeit, die Kinematik der beiden extraokulären (d.h., horizontale, vertikale und Torsionssteifigkeit Rotationen) messen32 und intraokularen (d.h. Schüler Änderungen)33 Bewegungen.
Modellsystem zur Analyse der ableitenden Nervenbahnen:
Im Allgemeinen bietet der Ansatz Ermittler die Möglichkeit zu studieren wie ableitenden neuralen Signale generieren Augenbewegungen, wenn Muskeln aus ihren entspannten Zuständen und in Ermangelung von integrierten Sinnesinformationen verarbeitet das Gehirn32beginnen, 33. Daher kann die Auge Kinematik im Modellsystem untersucht werden, in denen sie ausschließlich durch den ableitenden neuronale Weg verlassen das Gehirn und an den muskelsegmenten synapsing verarbeitet werden.

<table><tbody><tr><td>Red-eared slider turtles</td><td>Kons Scientific</td><td>&lt;em&gt;Trachemys scripta elegans&lt;/em&gt;</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 &amp;ndash; 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&quot;</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&quot;</td></tr><tr><td>Microscissors</td><td>Carolina Biological Supply Company</td><td>623555</td><td>Iris microdissecting scissors, stainless steel, 0.5&quot; blades, 4.75&quot; 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&quot;</td></tr><tr><td>Curved forceps</td><td>Sigma-Aldrich Co. LLC.</td><td>Z168696</td><td>Medium tip, curved forceps, stainless steel, 4&quot;</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&amp;rdquo; long x 0.07&amp;rdquo; diameter handle with square end: 0.17&amp;rdquo; x 1.3&amp;rdquo; long, other end round: 0.17&amp;rdquo; x 1.27&amp;rdquo; 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% O&lt;sub&gt;2&lt;/sub&gt;/CO&lt;sub&gt;2&lt;/sub&gt;</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&quot;, inner diameter, vinyl airline tubing connected to regulator to a 3/16&quot;, 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&quot; diameter tennis balls isolate vibrations from the top surface of the table.</td></tr><tr><td>5&quot; 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&#039; x 6&#039;</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, &amp;plusmn;20 mm, 0.1 mm precision; Rotations, vertical, &amp;plusmn;10&amp;deg;, and horizontal, &amp;plusmn;12.5&amp;deg;, with 1.25&amp;deg; 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&amp;deg;; vertical +/- 20&amp;deg;; Accuracy ~0.5&amp;deg;; Spatial resolution ~0.15&amp;deg;; 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&quot; 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 &amp;ndash; 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 &amp;mu;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 &amp;micro;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

Nachdem Tiere eingeschläfert werden, beginnen ihre Gewebe zu sterben. Schildkröten bieten einen Vorteil wegen eine längere Überlebenszeit von ihrem Gewebe, vor allem im Vergleich zu warmblütige Wirbeltiere. Aus diesem Grund können in-vitro- Experimente in der Schildkröten für längere Zeit zu untersuchen, die neuronale Signale und Kontrolle ihrer Ziel-Aktionen durchgeführt werden. Mit einem isolierten Leiter Vorbereitung, wir Maßen die Kinematik der Augenbewegungen in Schildkröten und deren Modulation durch elektrische Signale, die von Hirnnerven durchgeführt. Nachdem das Gehirn aus dem Schädel entfernt wurde, war intakt die Hirnnerven, seziert Kopf in eine kardanische Augenbewegungen zu kalibrieren platziert. Glaselektroden wurden angebracht, um Hirnnerven (okulomotorischen, trochlear, und rectus) und stimulierte mit Strömungen, Augenbewegungen zu erwähnen. Wir überwachen Augenbewegungen mit einem Infrarot-Video-tracking-System und quantifizierte Rotationen der Augen. Strom-Impulse mit einer Reihe von Amplituden, Frequenzen, und Zug Dauer wurden verwendet, um die Auswirkungen auf die Reaktionen zu beobachten. Da die Vorbereitung vom Gehirn getrennt ist, kann der ableitenden Weg zu Muskel-Ziele isoliert untersuchen neuronale Signalisierung bei fehlender zentral verarbeitet sensorische Informationen untersucht werden.

Abbildung 1 zeigt Standbilder von Bilder aus einem Video beschreibt die Dissektion. Bilder bieten typische Standorte der Nerven vor dem Schneiden aus dem Gehirn.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56864/56864fig1.jpg"> Abbildung 1: Standbilder von Bildern aus dem Video von der Dissektion anzuzeigenden Standorte der Sehnerv (nII), Oculomotor Nerv (nIII), trochlearis...

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

Dieses Protokoll beschreibt, wie eine in-vitro- isoliert Schildkröte Kopf Vorbereitung zu verwenden, um die Kinematik des ihre Augenbewegungen zu messen. Nach der Entnahme des Gehirns aus dem Schädel können Hirnnerven mit Strömungen, Drehungen des Auges und Veränderungen der Pupille Größen quantifizieren angeregt werden.

Okuläre Kinematik gemessen durch In-vitro- Stimulation der Hirnnerven in die Schildkröte

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

ocular-kinematics-measured-vitro-stimulation-cranial-nerves

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

8.4K Aufrufe.  Lafayette College. Dieses Protokoll beschreibt, wie eine in-vitro- isoliert Schildkröte Kopf Vorbereitung zu verwenden, um die Kinematik des ihre Augenbewegungen zu messen. Nach der Entnahme des Gehirns aus dem Schädel können Hirnnerven mit Strömungen, Drehungen des Auges und Veränderungen der Pupille Größen quantifizieren angeregt werden.

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

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

VisioTracker, eine innovative automatisierte Ansatz zur oculomotorius Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

Forschung

Neurowissenschaften

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

Video-Okulographie in Mäuse

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

Three Dimensional Vestibular Ocular Reflex Testing Using a Six Degrees of Freedom Motion Platform

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the vestibular organ results in mild to severe equilibrium problems, such as vertigo, dizziness, oscillopsia, gait unsteadiness nausea and/or vomiting. A good and frequently used measure to quantify gaze stabilization is the gain, which is defined as the magnitude of compensatory eye movements with respect to imposed head movements. To test vestibular function more fully one has to realize that 3D VOR ideally generates compensatory ocular rotations not only with a magnitude (gain) equal and opposite to the head rotation but also about an axis that is co-linear with the head rotation axis (alignment). Abnormal vestibular function thus results in changes in gain and changes in alignment of the 3D VOR response.
Here we describe a method to measure 3D VOR using whole body rotation on a 6DF motion platform. Although the method also allows testing translation VOR responses 1, we limit ourselves to a discussion of the method to measure 3D angular VOR. In addition, we restrict ourselves here to description of data collected in healthy subjects in response to angular sinusoidal and impulse stimulation.
Subjects are sitting upright and receive whole-body small amplitude sinusoidal and constant acceleration impulses. Sinusoidal stimuli (f = 1 Hz, A = 4&deg;) were delivered about the vertical axis and about axes in the horizontal plane varying between roll and pitch at increments of 22.5&deg; in azimuth. Impulses were delivered in yaw, roll and pitch and in the vertical canal planes. Eye movements were measured using the scleral search coil technique 2. Search coil signals were sampled at a frequency of 1 kHz.
The input-output ratio (gain) and misalignment (co-linearity) of the 3D VOR were calculated from the eye coil signals 3.
Gain and co-linearity of 3D VOR depended on the orientation of the stimulus axis. Systematic deviations were found in particular during horizontal axis stimulation. In the light the eye rotation axis was properly aligned with the stimulus axis at orientations 0&deg; and 90&deg; azimuth, but gradually deviated more and more towards 45&deg; azimuth.
The systematic deviations in misalignment for intermediate axes can be explained by a low gain for torsion (X-axis or roll-axis rotation) and a high gain for vertical eye movements (Y-axis or pitch-axis rotation (see Figure 2). Because intermediate axis stimulation leads a compensatory response based on vector summation of the individual eye rotation components, the net response axis will deviate because the gain for X- and Y-axis are different.
In darkness the gain of all eye rotation components had lower values. The result was that the misalignment in darkness and for impulses had different peaks and troughs than in the light: its minimum value was reached for pitch axis stimulation and its maximum for roll axis stimulation.
Case Presentation
Nine subjects participated in the experiment. All subjects gave their informed consent. The experimental procedure was approved by the Medical Ethics Committee of Erasmus University Medical Center and adhered to the Declaration of Helsinki for research involving human subjects.
Six subjects served as controls. Three subjects had a unilateral vestibular impairment due to a vestibular schwannoma. The age of control subjects (six males and three females) ranged from 22 to 55 years. None of the controls had visual or vestibular complaints due to neurological, cardio vascular and ophthalmic disorders.
The age of the patients with schwannoma varied between 44 and 64 years (two males and one female). All schwannoma subjects were under medical surveillance and/or had received treatment by a multidisciplinary team consisting of an othorhinolaryngologist and a neurosurgeon of the Erasmus University Medical Center. Tested patients all had a right side vestibular schwannoma and underwent a wait and watch policy (Table 1; subjects N1-N3) after being diagnosed with vestibular schwannoma. Their tumors had been stabile for over 8-10 years on magnetic resonance imaging.

A method is described to measure three-dimensional vestibulo ocular reflexes (3D VOR) in humans using a six degrees of freedom (6DF) motion simulator. The gain and misalignment of the 3D angular VOR provide a direct measure of the quality of vestibular function. Representative data on healthy subjects are provided

Dreidimensional Vestibuläre Ocular Reflex-Test mit Six Degrees of Freedom Bewegung Platform

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Eine einfache Verhaltenstest zur Prüfung von Visual Function in<em&gt; Xenopus laevis</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Mit optischen Kohärenztomographie und optokinetischen Antwort als strukturelle und funktionelle visuelles System auslesen bei Mäusen und Ratten

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 IslMN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

Ex Vivo Okulomotor Slice Culture von embryonalen GFP-ausdrückenden Mäusen für Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury are essential for translating novel therapeutic strategies from rodent models to clinical application due to their closer similarities to humans in size and anatomy. Here we describe some in vivo methods to evaluate the function and structure of the retinal ganglion cells (RGCs) and optic nerve (ON) in large animals, including visual evoked potential (VEP), pattern electroretinogram (PERG) and optical coherence tomography (OCT). Both goat and non-human primate were employed in this study. By presenting these in vivo methods step by step, we hope to increase experimental reproducibility among different labs and facilitate the usage of large animal models of optic neuropathies.

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Here we demostrate several in vivo tests (flash visual evoked potential, pattern electroretinogram and optic coherence tomography) in goat and rhesus macaque to understand the structure and function of the optic nerve and its neurons.

Im lebenden Organismus Methoden zur Beurteilung der Funktion und Struktur retinaler Ganglienzellen und Sehnerven bei Großtieren

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...

A New Optokinetic Testing Method to Measure Rat Vision

Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement associated with OKN is commonly used as a measure of visual function in rodents. To record OKN responses in normal and experimental rats, a simple and inexpensive apparatus has been developed. This setup uses two tablet screens to display the OKN visual stimulus consisting of high contrast black and white stripes generated using the OKN Stripes Visualization Web Application, a freely available software. The rat is placed inside a clear Plexiglass holder that limits&#160;movement so that the&#160;rat&#39;s head continuously faces the OKN display screen. The position of the rat holder can be changed to adjust the distance between the rat and the display screen. A micro-camera positioned above the rat holder is used to record the rat&#39;s visual activities. These recordings can be used for quantitative assessments. Based on the presence or absence of clear head-tracking, the OKN responses at different spatial frequencies can be determined. The collected data demonstrates a novel technique for reliable measurement of visual acuity in normal and retinal degenerate rats.

The Optokinetic Nystagmus (OKN) behavioral testing method is used for the assessment of visual acuity in rodents. Here a simple method is demonstrated that can be easily set up in research laboratories for a reliable assessment of visual function in both normal and experimental rats.

Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement ...

Bewertung optokinetischer Reflexantworten bei Mäusen: Ein neues hochpräzises Protokoll

Quantification of Visual Feature Selectivity of the Optokinetic Reflex in Mice

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize retinal images. Due to its importance and robustness, the OKR has been used to study visual-motor learning and to evaluate the visual functions of mice with different genetic backgrounds, ages, and drug treatments. Here, we introduce a procedure for evaluating OKR responses of head-fixed mice with high accuracy. Head fixation can rule out the contribution of vestibular stimulation on eye movements, making it possible to measure eye movements triggered only by visual motion. The OKR is elicited by a virtual drum system, in which a vertical grating presented on three computer monitors drifts horizontally in an oscillatory manner or unidirectionally at a constant velocity. With this virtual reality system, we can systematically change visual parameters like spatial frequency, temporal/oscillation frequency, contrast, luminance, and the direction of gratings, and quantify tuning curves of visual feature selectivity. High-speed infrared video-oculography ensures accurate measurement of the trajectory of eye movements. The eyes of individual mice are calibrated to provide opportunities to compare the OKRs between animals of different ages, genders, and genetic backgrounds. The quantitative power of this technique allows it to detect changes in the OKR when this behavior plastically adapts due to aging, sensory experience, or motor learning; thus, it makes this technique a valuable addition to the repertoire of tools used to investigate the plasticity of ocular behaviors.

Autor im Rampenlicht: Ein genauer und quantitativer Ansatz zur Untersuchung der visuellen Merkmalsselektivität des optokinetischen Reflexes bei Mäusen 

Here, we describe a standard protocol for quantifying the optokinetic reflex. It combines virtual drum stimulation and video-oculography, and thus allows precise evaluation of the feature selectivity of the behavior and its adaptive plasticity.

Quantifizierung der visuellen Merkmalsselektivität des optokinetischen Reflexes in Mäusen

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize ...

Okuläre Kinematik gemessen durch In-vitro- Stimulation der Hirnnerven in die Schildkröte

Zusammenfassung

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