Ich möchte Christopher Evans und Darren Burke vom Zentrum danken für die Integrative Study of Animal Behaviour (CISAB).

Jacky Dragons Jacky Eidechsen (A. muricatus) wurden in klimatisierten gefangen Einrichtungen statt. Gehäuse wurden von fabrizierten Glasterrarien 60 cm 3 aufgebaut. Gehäuse wurden ebenfalls mit Sydney Sand Substrat, native Laub und natürlichen Holz-Sitzstangen erfüllt. Gehäuse wurden auf der beweglichen Wagen platziert, dass erlaubte Gegenstände in der Mitte aller Betrachtung Monitore platziert zu sein. 10D Lichtzyklus während der simulierten sommerlichen Bedingungen und waren sowohl mit Wärmelampen (125W) und eine angemessene ultravioletten (UV) Licht (300W) während der ersten halben Stunde Tageslicht versorgt: Echsen wurden unter einem 14L gelegt. Echsen wurden auch mit Wasser ad libitum, gefüttert Grillen (Achetus domesticus) bestäubt mit Vitaminen während der nicht-experimentellen Sitzungen und routinemäßig für Parasiten mit verdünnter Dosen von Ivermectin entwurmt zur Verfügung gestellt. Die Ausrüstung für die Wiedergabe verwendet Drei Video-Wiedergabe-Rigs für Stimulus-Präsentation (siehe Abbildung 1). Die zentrale Rigg besteht aus einem iMac (Apple Computer Inc.) mit dot Reize. Die beiden zusätzlichen Rigs bestehen aus einem iMac mit Final Cut Pro HD für Stimulus-Präsentation, dass die Wiedergabe Reize (dh, Wirbellose) enthalten. Das digitale Signal wird der fortschrittlichen digitalen Video-Konverter (Canopus © gt ADVC110, Melbourne, Victoria Australia) für Digital-Analog-Wandlung für die Videowiedergabe geschickt und auf einem Sony Trintron (Sony Corporation, Shinagawa, Tokyo, Japan) Monitor (Series Nr. PVM-14M2A/PVM-14L2/PVM-14N5A). Ein Canon (Modell Nr. MV650i) Digital-Camcorder mit 22fach optischem Zoom (Cannon Inc., Japan) ist direkt über dem Testgelände und Aufzeichnungen Reaktionen auf Reize direkt an einen Sony (Sony Corporation, Shinagawa, Tokyo, Japan) Videorecorder montiert ( Serial No SLV-EZ717AS). Die aufgenommenen Bilder werden zu einem Farbsucher (NEC, Serial Nr. 1892, Kanagawa, Japan) geschickt. In 7,5 Sekunden von Anfang an die Punkte anzuzeigen, ist eine elektronisch erzeugte Klang Marker auf eine Canopus ® ADVC110 und Aufzeichnungen auf VHS geschickt. Video-Wiedergabe ist für PAL-DV-Standard (; horizontale Auflösung 575 Zeilen, 25 Bilder s-1 5:1-Komprimierung) eingestellt. Random-dot Kinematograms (RDKS) RDKS können mit Hilfe VPixx V1.79 (VPixx V1.79, VPixx Technologies Inc. 2002). Design-Impulse mit einer 30 x 20 Grad Feld mit 200 Punkten zu füllen, wobei jeder Punkt 5 x 5 Pixel. Bauen dot Reize mit acht Variationen in Geschwindigkeit, mit acht Ebenen der Kohärenz und zwei Richtungen kombiniert und erzeugt 128 verschiedene Reize. Speed-Parameter wurden in Grad pro Sekunde (° / s) konzipiert. Speed-Varianten wurden 0,5, 2,5, 5, 10, 20, 40, 80 und 160 ° / s. Coherence Prozentsätze wurden 0, 2,5, 5, 10, 20, 40, 80 und 100% entweder in einer links oder rechts gerichtete Bewegung. Exportieren Sie jede Sequenz als Motion JPEG-B-Datei. Sie können jedoch die Gestaltung RDKS, die eine Reihe von Funktionen. Jede Sequenz ist eine insgesamt 15 s Dauer. Die Sequenz beginnt mit 2,5 s von 0% Übereinstimmung mit der Studie drehzahlvariablen abgestimmt. Dieser Zeitraum startet die aktuelle Studie. Dies wird durch 5 s der Variation in der Geschwindigkeit und Kohärenz Kombination gefolgt. Die Sequenz wurde um 7,5 s von 100% in die entsprechende Richtung abgeschlossen. Importieren Sie alle Motion JPEG-B-Sequenzen und bearbeiten in Apple Final Cut Pro HD (Apple Computer Inc.). Fügen Sie eine Sound-Marker bei jedem Intervall. Diese Markierung ist nur hörbar VHS, und wird als Cue verwendet werden, wenn Scoring-Verhalten, wenn die Änderungen in Stimuluspräsentation auftreten. Exportierte jede der 128 Sequenzen, wie Final Cut Pro-Filme. Primäre Verstärker - Mehlwürmer Legen Sie Themen auf einer randomisierten Stahlliste von fünf Versuchen pro Block-Studie (16 Versuche) während des Experiments. Primäre Verstärker sind Mehlwürmer (Tenebrio molitor). Sekundäre Verstärker - Animated Wirbellose Sekundäre Verstärker sind mit dem primären Verstärker gekoppelt werden. Erstellt biologisch sinnvoll Verstärker, wie drei Arten von Computer-animierte Wirbellose: 1) Cricket, 2) mite, und 3) Spinne. Erstellen von Animationen mit Lightwave v8.3 (NewTek Inc., San Antonio, TX, USA) für 15 s in der Länge, mit der ersten 7,5 s von noch Hintergrund, gefolgt von 7,5 s der wirbellosen auf den entsprechenden Bildschirm. Photograph dem Hintergrund der natürlichen Vegetation als JPEG in eah Clip zu importieren. Export jeder Sequenz in einzelne JPEG-Bild-Sequenzen (375 Bilder), und komprimieren Sie in eine Filmdatei mit Final Cut Pro HD für Video-Wiedergabe. Design 50 einzigartige Clips Gewöhnung an die Konditionierung zu verhindern. Allgemeine Vorgehensweise Legen Themen zwischen den Anzeigen überwacht. Zufällige Anordnung von RDK Reize für jedes Thema. Zeige Reize in Blöcken von 16 Studien über 8 Tage. Jeder Versuch wird 15 s mit intertrial Abständen wechselnden 30 bis 60 s. Segment jedem Versuch Abstand in 2,5 s von 0% Kohärenz widerspiegelt Prüfgeschwindigkeit, 5 s der Prüfgeschwindigkeit und die Kohärenz, gefolgt von 7,5 s von 100% in entsprechende Richtung mit der Geschwindigkeit angepasst. Während dieser 5 s kritisch-Periode sind als Pflichtfächer zu reagieren. Rekord abhängig Maßnahmen als Orientierung hin zu einer nach links oder rechts zu überwachen und Latenz zu reagieren. Verwenden Sie nur fünf zufällige Studien mit einem primären Verstärker und verstärken sich gegenseitig Studie mit einem animierten Wirbellosen. Zum Abschluss eines jeden Blocks Versuch, entfernen Sie unterliegen Gehäusen aus dem Testgelände und ein weiteres Gehäuse.

<ol>
	<li>Burghardt, G. M., Gans, C., Tinkle, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+processes+in+reptiles.">Learning processes in reptiles.</a> Biology of the reptilia. 7, 555-681 (1977).</li><li>VPixx, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Instruction Manual. VPixx Techniologies Inc. , 96 (2002).</li></ol>

Die Finanzierung wurde durch die CISAB und Department of Psychology Postgraduate Award der Macquarie University, und dem Australian Research Council unterstützt. Forschung wurde in den ethischen Richtlinien der Macquarie University Animal Ethics Committee (2003/014) und die New South Wales National Parks and Wildlife Services (S11024) aus Australien durchgeführt.

Prüfung der sensorischen Fähigkeiten in Echsenarten war schwierig, und hat vor allem auch ein langwieriger und mühsamer Prozess. Diese Schwierigkeiten ergeben sich aus der Suche nach den richtigen Verstärker oder über eine funktionale Protokoll. Hier gründete ich eine erfolgreiche Protokoll, bei dem Bewegungswahrnehmung in Eidechsen zu testen. Dieses Verfahren kann auch zur Anwendung in der Untersuchung anderer visueller Merkmale sowie andere sensorische Mechanismen. Darüber hinaus kann dieses Verfahren auch auf andere Arten. Die Nü...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>iMac</td>
<td>Computer</td>
<td>Apple Computers, Inc.</td>
<td>&nbsp;</td>
<td>Three systems</td>
</tr>
<tr>
<td>Canopus&#174;</td>
<td>Digital-to-analog converter</td>
<td>Canopus&#174;</td>
<td>ADVC110</td>
<td>&nbsp;</td>
<tr>
<td>Sony Trintron</td>
<td>Colour viewfinder</td>
<td>Sony Corporation</td>
<td>PVM-14M2A, PVM-14L2, PVM-14N5A</td>
<td>Three monitors</td>
</tr>
<tr>
<td>Canon</td>
<td>Digital camcorder</td>
<td>Cannon Inc.</td>
<td>MV650i</td>
<td>Records behavior</td>
</tr>
<tr>
<td>NEC</td>
<td>Colour viewfinder</td>
<td>NEC</td>
<td>1892</td>
<td>View responses</td>
</tr>
<tr>
<td>Sony VCR</td>
<td>VHS recorder</td>
<td>Sony Corporation</td>
<td>SLV-EZ717AS</td>
<td>Record behavior to VHS</td>
</tr>
<tr>
<td>VHS tape</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Generic VHS</td>
<td>Recording media</td>
</tr>
<tr>
<td>VPixx</td>
<td>Software program</td>
<td>VPixx Technologies, Inc.</td>
<td>v1.79</td>
<td>To create RDKs</td>
</tr>
<tr>
<td>Final Cut Pro</td>
<td>Software program</td>
<td>Apple Computer, Inc.</td>
<td>HD</td>
<td>To create playback sequences</td>
</tr>
<tr>
<td>Lightwave&#174; 3D</td>
<td>Software program</td>
<td>NewTek Inc.</td>
<td>v8.3</td>
<td>To create secondary reinforcers</td>
</tr>
<tr>
<td>Mealworm</td>
<td>Tenebrio molitor</td>
<td>Pisces Enterprises LTD</td>
<td>&nbsp;</td>
<td>Primary reinforcer</td>
</tr>
<tr>
<td>Cricket</td>
<td>Achetus domesticus</td>
<td>Pisces Enterprises LTD</td>
<td>&nbsp;</td>
<td>Food for husbandry</td>
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<td>Terrariums</td>
<td>Glass</td>
<td>Crystal Palace Aquariums</td>
<td>&nbsp;</td>
<td>Enclosure material for lizards (60 x 60 x 60 cm)</td>
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<td>Globes</td>
<td>Spotone</td>
<td>Phillips</td>
<td>&nbsp;</td>
<td>125W, Heat lamps for lizards</td>
</tr>
<tr>
<td>Ultraviolet globes</td>
<td>Ultra-Vitalux</td>
<td>Osram</td>
<td>&nbsp;</td>
<td> 300W, UV lamps for lizards</td>
</tr>
<tr>
<td>Herptivite</td>
<td>&nbsp;</td>
<td>Rep-Cal Research Labs</td>
<td>&nbsp;</td>
<td>Vitamins: beta carotene, calcium, and vitamin D3</td>
</tr>		</tbody>
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testing visual sensitivity to the speed and direction of motion in lizards

Testen visuelle Empfindlichkeit in jeder Art bietet grundlegende Informationen über das Verhalten, Evolution und Ökologie. Allerdings Tests Besonderheiten des visuellen Systems bieten mehr empirische Belege für funktionale Anwendungen. Untersuchung der sensorischen System liefert Informationen über die sensorischen Fähigkeiten, Lernen und Gedächtnis Fähigkeit und stellt bekannte Grundlinie Verhalten in die zu Abweichungen Manometer (Burghardt, 1977). Doch im Gegensatz zu Säugetieren oder Vogelgrippe-Systeme, Prüfung für Lernen und Gedächtnis in einem Reptilienarten ist schwierig. Darüber hinaus mit einem operante Paradigma als eine psychophysische Messung der sensorischen Fähigkeiten ist ebenfalls als schwierig. Historisch gesehen haben Reptilienarten schlecht Konditionierung Studien aufgrund von Problemen im Zusammenhang mit Motivation, Physiologie, Stoffwechsel, und die grundlegenden biologischen Eigenschaften reagiert. Hier habe ich zeigen eine operante Paradigma verwendet ein neuartiges Modell Echsenarten, beschreiben die Jacky Drachen (Amphibolurus muricatus) und wie periphere Sensibilität für herausragende Geschwindigkeit und Bewegung Eigenschaften zu testen. Diese Methode verwendet einen innovativen Ansatz zur Beurteilung der Lern-und sensorischen Fähigkeiten in Eidechsen. Ich beschäftige die Verwendung von random-dot kinematograms (RDKS), um die Empfindlichkeit zu beschleunigen und zu manipulieren das Niveau der Signalstärke, indem der Anteil der Punkte bewegen in eine gemeinsame Richtung zu messen. RDKS repräsentieren nicht eine biologisch sinnvolle Impulse greift das visuelle System und ist ein klassisches psychophysischen Werkzeug benutzt, um die Empfindlichkeit beim Menschen und anderen Tieren zu messen. Hier werden RDKS, Eidechsen mit Hilfe von drei Video-Wiedergabe-Systemen angezeigt. Eidechsen sind, um die Richtung (links oder rechts), in denen sie wahrnehmen Punkte zu bewegen auszuwählen. Die Auswahl der geeigneten Richtung wird durch biologisch wichtige Beute Reize, simuliert durch computer-animierten Wirbellose verstärkt.

Watch this Scientific Journal Video about Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards at JoVE.com

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testen visuelle Empfindlichkeit in Echsen mit einem operante Konditionierung, die Video-Wiedergabe von random-dot kinematograms und computer-generierte Wirbellosen beschäftigt.

Testen visueller Sensibilität für die Geschwindigkeit und Richtung der Bewegung in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

testing-visual-sensitivity-to-speed-direction-motion

Research

JoVE Journal

Biology

11.4K Aufrufe.  Macquarie University. Testen visuelle Empfindlichkeit in Echsen mit einem operante Konditionierung, die Video-Wiedergabe von random-dot kinematograms und computer-generierte Wirbellosen beschäftigt.

Serie: Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

Testen der physiologische Barrieren für die virale Transmission in Blattläuse mit Mikroinjektion

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

Forschung

Biologie

Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

Olfaktorische Behavioral Testing in der adulten Maus

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Einzel-Zell-Saug-Recordings aus Maus Zapfen-Photorezeptoren

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Mit der optokinetischen Reaktion auf visuelle Funktion des Zebrafisch-Studie

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Insulin-Injektion und Hämolymphe Extraction die Insulinsensitivität in Adult Measure Drosophila melanogaster</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Design und Analyse des Temperaturpräferenzverhaltens und seines circadianen Rhythmus in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Validierung eines Maus-Modell zu LINC Komplexe Disrupt in einem zellspezifisch

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Eine grafische Benutzeroberfläche für die softwaregestützte Verfolgung der Proteinkonzentration in dynamischen zellulären Vorsprüngen

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

Anwendung des High-Speed-Höchstauflösung Geschwindigkeit Mikroskopie in Live primäre Zilie

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

Protokolle für die Prüfung der Toxizität von neuartigen Insektizide Chemikalien, Mücken

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...