Je tiens à remercier Christopher Evans et Darren Burke, du Centre pour l&#39;étude intégrative de l&#39;Animal Behaviour (CISAB).

Jacky Dragons Lézards Jacky (A. muricatus) ont eu lieu dans des installations à température contrôlée en captivité. Boîtiers ont été construits des terrariums de verre fabriquée mesurant 60 cm 3. Boîtiers étaient également remplis de substrat de sable Sydney, feuillage indigènes, et en bois naturel perchoirs. Boîtiers ont été placés sur le dessus des chariots mobiles qui a permis aux sujets d&#39;être placé au milieu de tous les moniteurs de visualisation. Lézards ont été placées sous un 14L: 10D cycle de lumière pendant l&#39;été des conditions simulées, et ont été fournis avec deux lampes de la chaleur (125W) et ultraviolet adéquates (UV) éclairage (300W) pendant la première demi-heure de lumière du jour. Lézards ont également été fournis avec de l&#39;eau ad libitum, les grillons nourris (Achetus domesticus) saupoudré de vitamines pendant les sessions non-expérimentales, et régulièrement vermifugés pour les parasites en utilisant des doses diluées de l&#39;ivermectine. Matériel utilisé pour la lecture Trois plates-formes de lecture vidéo sont utilisés pour la présentation du stimulus (voir Figure 1). La plate-forme centrale se compose d&#39;un iMac (Apple Computer Inc) contenant des stimuli point. Les deux plates-formes supplémentaires se composent d&#39;un iMac avec Final Cut Pro HD pour la présentation du stimulus qui contenait des stimuli de lecture (ie, invertébrés). Le signal numérique est envoyé à un convertisseur de vidéo numérique avancé (Canopus ADVC110 © GT, Melbourne, Victoria, Australie) pour le numérique-analogique de conversion pour la lecture vidéo et affichées sur un Trintron Sony (Sony Corporation, Shinagawa, Tokyo, Japon) moniteur (Série n PVM-14M2A/PVM-14L2/PVM-14N5A). Un Canon (modèle n ° MV650i) caméscope numérique avec zoom optique 22X (Cannon Inc, Japon) est monté directement au-dessus de la zone d&#39;essai et les réponses aux stimuli enregistrements directement à un Sony (Sony Corporation, Shinagawa, Tokyo, Japon) magnétoscope ( Serial No. SLV-EZ717AS). Les images enregistrées sont envoyées à un viseur couleur (NEC, série n ° 1892, Kanagawa, Japon). A 7,5 secondes à partir du début de l&#39;affichage des points, un marqueur sonore généré électroniquement est envoyée à un ADVC110 de Canopus ® et enregistrements sur VHS. Lecture vidéo est ajusté pour le PAL-DV standard (compression 5:1; résolution horizontale 575 lignes, 25 trames S-1). Random-dot Kinematograms (RDK) RDK peuvent être développés en utilisant VPixx V1.79 (VPixx V1.79, Technologies Inc VPixx 2002). Stimuli Design avec un champ de 30 x 20 degrés, remplir avec 200 points, avec chaque point de mesure 5 x 5 pixels. Construire des stimuli points avec huit variations de vitesse, combinée avec huit niveaux de cohérence et de deux directions, générant 128 différents stimuli. Paramètres de vitesse ont été conçus en degrés par seconde (° / s). Les variations de vitesse ont été de 0,5, 2,5, 5, 10, 20, 40, 80 et 160 ° / s. Cohérence pourcentages étaient de 0, 2,5, 5, 10, 20, 40, 80 et 100% en soit un mouvement directionnel gauche ou à droite. Exporter chaque séquence comme une Motion JPEG-B fichier. Cependant, vous pouvez concevoir le RDK dont un éventail de fonctionnalités. Chaque séquence est un total de 15 s dans la durée. La séquence commence avec 2,5 s de 0% correspond cohérence avec la vitesse variable procès. Cette période initie le procès en cours. Il est suivi par 5 s de la variation en combinaison de vitesse et la cohérence. La séquence a été complété par 7,5 s de 100% dans la direction appropriée. Importer tous Motion JPEG B-séquences et d&#39;éditer dans Apple Final Cut Pro HD (Apple Computers Inc.) Ajouter un marqueur sonore à chaque intervalle. Ce marqueur est uniquement audible sur VHS, et est utilisé comme un repère lors de la notation comportement lorsque les changements dans la présentation du stimulus se produire. Exporté chacune des 128 séquences des films de Final Cut Pro. Renforçateurs primaires - Vers de farine Placer des sujets sur un calendrier renfort de cinq essais randomisés par essai de bloc (16 essais) pendant l&#39;expérience. Renforçateurs primaires sont des vers de farine (Tenebrio molitor). Renforçateurs secondaires - Invertébrés animé Renforçateurs secondaires sont d&#39;être jumelé avec le renforçateur primaire. Création renforçateurs biologiquement significatives, telles que trois types d&#39;animation par ordinateur invertébrés: 1) le cricket, 2) d&#39;acariens, et 3) d&#39;araignée. Créer des animations en utilisant Lightwave v8.3 (NewTek Inc, San Antonio, TX, USA) pendant 15 s en longueur, avec l&#39;initiale 7,5 s du reste de fond, et suivie par 7,5 s de l&#39;invertébré sur l&#39;écran approprié. Photographie sur fond de végétation naturelle à l&#39;importation au format JPEG dans le clip EAH. Exporter chaque séquence en individuel séquences d&#39;images JPEG (375 cadres), et de compresser en un fichier vidéo en utilisant Final Cut Pro HD pour la lecture vidéo. Conception unique de 50 clips pour prévenir l&#39;accoutumance au paradigme de conditionnement. Procédure générale Placer des sujets entre les moniteurs de visualisation. Randomize présentation de stimuli RDK pour chaque sujet. Montrer les stimuli dans des blocs de 16 essais sur huit jours. Chaque essai est de 15 s avec des intervalles variant de 30 à 60 intertrial s. Segment chaque intervalle d&#39;essai en 2,5 s de 0% réfléchissant la cohérence de la vitesse d&#39;essai, 5 s de tests de vitesse et de cohérence, et suivie par 7,5 s de 100% dans la direction appropriée correspondant à la vitesse. Durant ces 5 s période critique, les sujets sont tenus de répondre. Enregistrement des mesures dépend de l&#39;orientation vers un écran à gauche ou à droite et de latence pour répondre. Utilisez uniquement des cinq essais aléatoires avec un renforçateur primaire et se renforcent mutuellement essai avec un invertébré animés. À la fin de chaque essai de bloc, retirez enclos personne de la zone d&#39;essai, et d&#39;introduire une autre enceinte.

<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>

Le financement a été soutenue par le Prix d&#39;études supérieures et de CISAB Département de psychologie de l&#39;Université Macquarie, et l&#39;Australian Research Council. Des recherches ont été menées dans les directives éthiques de l&#39;Université Macquarie Comité d&#39;éthique animale (2003/014) et le New South Wales National Parks et Wildlife Services (S11024) de l&#39;Australie.

Test de la capacité sensorielle espèce de lézard a été difficile, et a largement été à la fois un processus fastidieux et laborieux. Ces difficultés viennent de trouver le renforçateur corriger ou l&#39;établissement d&#39;un protocole fonctionnel. Ici, j&#39;ai établi un protocole de succès en permettant de tester la perception du mouvement chez les lézards. Cette procédure peut également être applicables dans les tests d&#39;autres caractéristiques visuelles ainsi que d&#39;autres mécanismes sensoriels. En outre, cette ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<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>
</tr>
<tr>
<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>
</tr>
<tr>
<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>
		</table>
		</div>
			<div>
					</div>

testing visual sensitivity to the speed and direction of motion in lizards

Test de sensibilité visuelle de toute espèce fournit des informations de base concernant le comportement, l&#39;évolution et l&#39;écologie. Cependant, les tests caractéristiques spécifiques du système visuel de fournir plus de preuves empiriques pour des applications fonctionnelles. Enquête sur le système sensoriel fournit des informations sur les capacités, l&#39;apprentissage sensoriel et la capacité de la mémoire, et établit le comportement de base connus dans lesquels de mesurer les écarts (Burghardt, 1977). Cependant, contrairement aux systèmes de mammifères ou oiseaux, des tests d&#39;apprentissage et de mémoire dans une espèce de reptile est difficile. Par ailleurs, en utilisant un paradigme opérant comme une mesure de la capacité psychophysique sensorielle est également aussi difficile. Historiquement, les espèces de reptiles ont mal répondu aux essais de conditionnement en raison de problèmes liés à la motivation, la physiologie, le métabolisme, et de base des caractéristiques biologiques. Ici, je montre un paradigme opérant utilisé une nouvelle espèce de lézard modèle, le dragon Jacky (Amphibolurus muricatus) et décrire la façon de tester la sensibilité périphérique à la vitesse et les caractéristiques saillantes de mouvement. Cette méthode utilise une approche innovante de l&#39;apprentissage et l&#39;évaluation de capacités sensorielles chez les lézards. J&#39;emploie l&#39;utilisation de points aléatoires kinematograms (RDK) pour mesurer la sensibilité à la vitesse, et de manipuler le niveau de puissance du signal en changeant la proportion de points se déplaçant dans une direction cohérente. RDK ne représentent pas un stimulus biologique significatif, engage le système visuel, et est un outil classique de psychophysique utilisée pour mesurer la sensibilité des êtres humains et autres animaux. Ici, RDK sont affichés à l&#39;aide de trois lézards systèmes de lecture vidéo. Les lézards sont pour sélectionner la direction (gauche ou droite) dans lequel ils perçoivent points pour se déplacer. Sélection de la direction appropriée est renforcée par des stimuli biologiquement importantes proies, simulé par ordinateur d&#39;animation invertébrés.

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

Test de sensibilité visuelle chez les lézards en utilisant un paradigme de conditionnement opérant, qui emploie la lecture vidéo de points aléatoires kinematograms et générés par ordinateur invertébrés.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

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 vues.  Macquarie University. Test de sensibilité visuelle chez les lézards en utilisant un paradigme de conditionnement opérant, qui emploie la lecture vidéo de points aléatoires kinematograms et générés par ordinateur invertébrés.

Vidéo: 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.

Test de la barrières physiologiques à la transmission virale chez les pucerons avec microinjection

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

Recherche

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.

Olfactive tests comportementaux chez la souris adulte

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.

Enregistrements aspiration simple cellule à partir de cônes photorécepteurs de la souris

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.

En utilisant la réponse optocinétique pour étudier la fonction visuelle du poisson zèbre

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.

Injection d&#39;insuline et d&#39;extraction hémolymphe pour mesurer la sensibilité d&#39;insuline dans les adultes 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.

Conception et analyse du comportement de préférence de température et de son rythme circadien chez la drosophile

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.

Validation d&#39;un modèle de souris de perturber CLIC complexes de façon spécifique à la cellule

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.

Une interface utilisateur graphique pour le suivi assisté par logiciel de la concentration de protéines dans les protrusions cellulaires dynamiques

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.

Application de la microscopie vitesse haut débit Super-résolution en direct cil primaire

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.

Protocoles pour tester la toxicité des nouvelles chimies insecticides aux moustiques

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