Soutenu par des subventions des Instituts canadiens de recherche en santé, le Réseau canadien contre les accidents, et l&#39;Alberta Heritage Foundation for Medical Research.

Appareil de marche Rotorod Le rotorod était un cylindre rotatif, 4 cm de diamètre, 35 cm fixée au-dessus du sol et fermé par plexiglas transparent. Le cylindre a été rotorod recouverts d&#39;un revêtement en caoutchouc texturé, ce qui facilite la traction. Le plancher de l&#39;appareil était couvert d&#39;une couche de mousse pour éviter les blessures lorsque les animaux ont diminué. Un petit moteur électrique fourni d&#39;alimentation pour allumer l&#39;rotorod via une courroie en caoutchouc. Le rotorod a été mis à tourner une fois tous les 5 s. Les enregistrements vidéo et de la cinématique Une caméra vidéo haute vitesse numérique a été utilisé pour filmer les animaux, en utilisant une vitesse d&#39;obturation d&#39;un 1000 d&#39;une seconde. La face avant, latérale et arrière, vues ont été filmés alors que l&#39;animal se promenait sur le rotorod. Une à deux bras de Nikon, la source de lumière froide MII et un jeu de lumières à fibres optiques ont été utilisées pour fournir un éclairage suffisant pendant l&#39;enregistrement. Les enregistrements ont été analysés image par image à l&#39;aide d&#39;un Sony DSR 20 DV CAM pont. Les images vidéo ont été capturés en utilisant une carte d&#39;acquisition vidéo et l&#39;ordinateur Macintosh. Procédure d&#39;essai et d&#39;analyse Afin d&#39;évaluer les différences entre les mouvements de marche de la 6-OHDA et les rats témoins, les enregistrements vidéo à partir de post-chirurgicale jours 15 - 30 (après tout la récupération de la lésion pourrait s&#39;attendre à être largement complet) ont été examinés par deux indépendants observateurs aveugles à des conditions expérimentales. Le nigrostrié 6-OHDA lésion a été provoquée dans l&#39;hémisphère droit, résultant des dépréciations durables dans l&#39;avant-gauche et des membres postérieurs. Une échelle de notation a été développé pour la posture du taux de rats et de l&#39;intensification des mouvements des membres antérieurs (Whishaw et al., 2003). Cinq composantes de marcher ont été notés: <ol><li> la posture, la cote de la posture a été faite en examinant la position du rat sur ​​le tambour; </li><li> levage et la libération, la partie du cycle de l&#39;intensification dans lequel la patte a été levé à partir du tambour; </li><li> effectuer, la partie du cycle de l&#39;intensification dans laquelle le membre a eu lieu au-dessus du tambour comme le rat avancé; </li><li> l&#39;avance, la portion de la portée dans laquelle le membre a été étendu à reprendre contact avec le tambour, et </li><li> placement, dans laquelle la patte arrière a été placé sur le tambour. </li></ol> Les critères suivants ont été utilisés pour définir un mouvement normal: <ul><li> La posture: Le corps du rat a été orientée sur le tambour avec la tête haute. </li><li> Ascenseur et la libération: L&#39;épaule, le coude et le poignet sont fléchis élever la patte de la surface du tambour. </li><li> Carry: La patte est maintenue au-dessus de la surface du tambour, les chiffres sont vaguement fléchis, et les conseils chiffres sont centrés sur la ligne médiane du corps. Advance: Le membre est tendue vers l&#39;avant avec les chiffres de passer d&#39;une position de demi-fléchie à une position étendue et ouverte. </li><li> Placement: Le membre est étendu à l&#39;épaule, du coude et du poignet et les doigts sont ouverts à placer la patte avant et sur ​​la surface du tambour, avec 2 chiffres (deuxième chiffre de la ligne médiane), situé à peu près au centre de l&#39;est du rat corps. </li></ul> Chaque mouvement a été évalué sur une échelle de 3 points: 1 = / le mouvement ressemblait à celle d&#39;un rat témoin, 0,5 = / le mouvement semblait légèrement anormale, et 0 = / le mouvement était nettement anormal (Whishaw et al, 2003).. Cette échelle d&#39;évaluation a ensuite été utilisé pour évaluer les cinq mouvements intensification de chaque rat à des jours postopératoires 1, 3, 5, 7, 9 et 15. Les scores des cinq mesures ont été moyennées pour chaque rat à chaque jour, et les résultats soumis à une analyse de variance avec des journées comme une mesure répétée. Vue d&#39;ensemble des rotorod marcher dans la 6-OHDA et les rats de contrôle L&#39;échelle de notation mouvement a été appliquée à des séances de tests post-opératoire pour l&#39;analyse de mouvement qualitatif. Pendant les séances d&#39;essai, le groupe lésion 6-OHDA obtenu des scores significativement plus bas que le groupe contrôle, F (1,8) = 33,2, p &lt;0,001, mais il n&#39;y avait aucun effet significatif de la Journée Test, il n&#39;y avait pas un groupe important par l&#39;interaction Journée Test. Les scores de marcher ont ensuite été analysés séparément avec le membre antérieur comme une variable (pour le rat 6-OHDA la patte avant controlatéral à la lésion par rapport au membre antérieur ipsilatéral à la lésion, car les rats témoins, la patte gauche vs la patte droite). Il y avait un effet significatif de la patte avant, F (1,40) = 15,8, p &lt;0,01, et il y avait une interaction significative du Groupe par des membres antérieurs, F (1,40) = 15,0, p &lt;0,01. Suivi tests-t ont indiqué que le membre antérieur controlatéral à la lésion du groupe 6-OHDA obtenu des scores significativement plus faible que la patte avant ipsilatérale à la lésion ou les membres antérieurs des rats témoins, qui ne diffèrent pas. Afin de déterminer quels composants du mouvement a contribué à la mauvaise scores obtenus par le groupe 6-OHDA, les mesures de la postureet les composantes individuelles du cycle de l&#39;intensification de la forelimbs ipsilatérale et controlatérale du groupe 6-OHDA ont été comparés aux valeurs obtenues à partir des rats témoins. Le groupe 6-OHDA différaient dans la posture et de tous les composants intensification faite par le membre controlatéral, mais aucun des composants du membre ipsilatéral (p &lt;0,05). Les différences qualitatives et descriptives dans la posture de contrôle et de 6-OHDA chez le rat Dans un rat de contrôle, la tête est maintenue dans une position horizontale, et l&#39;avant bras soutenant et membres postérieurs chevaucher le tambour formant un arc de soutien. Les rats 6-OHDA généralement marchaient la tête en bas avec leurs pattes avant avec facultés affaiblies (face au spectateur) positionné rapprocher et plus haut sur la surface du tambour que les produits pour les membres homolatéraux ou les membres de rats témoins (voir la vidéo pour un rat de contrôle avec score &quot;1&quot; pour la posture, et un animal lésion 6-OHDA avec un score de «0»). Généralement, l&#39;anomalie de position peuvent être vus sur un regard de la position relative de la tête d&#39;un rat. Forelimb intensification Dans la séquence de contrôle, l&#39;intensification des membres antérieurs est soulevé par la flexion de l&#39;épaule, du coude et du poignet. Comme le membre est levé, les chiffres sont vaguement fléchi, et le membre est soulevé bien au-dessus de la surface du tambour. Lorsque le membre est avancé, il est prorogé au niveau des épaules, du coude et du poignet et comme il est placé les chiffres sont étendues et ouvertes. En comparaison avec les rats témoins, l&#39;amplitude de toutes les composantes du rat DA appauvri controlatéral à la lésion des membres est atténué. Le membre libère le tambour plus tard, est levé moins, et est avancé une distance plus courte que la patte avant le contrôle. En outre, lorsque le membre est levé, les chiffres sont fléchis plus, et quand le membre est placé, les chiffres sont étendues et ouvertes moins que ceux d&#39;un rat témoin. La patte avant la levée du contrôle une fois qu&#39;il atteint le sommet du tambour, le poignet est fléchi à lever la patte et à transmettre, et le poignet est étendue que les avances des membres. En revanche, le rat 6-OHDA est controlatéral à la lésion est forelimb porté plus loin caudalement par le mouvement du tambour, et libère ainsi plus tard avec le poignet reste tendue. Flexion du poignet semble être passif et se produit comme les chiffres sont effectués en arrière par le mouvement du tambour, et quand la patte avant est mis en avant, la patte et l&#39;avant-bras ne sont pas levées clairement hors de la surface du tambour. Le membre antérieur d&#39;un rat contrôle s&#39;étend bien en avant et les chiffres ouverts que les avances des membres. Comme le membre est placé, il est placé avec un mouvement d&#39;arpèges, avec 5 chiffres contact avec la surface du premier tambour, suivi successivement par les chiffres 4, 3 et 2. En revanche, le membre du rat 6-OHDA est levé et étendu moins, les chiffres sont moins étendus, et ils contact avec la surface du tambour presque en même temps une «gifle». La vidéo montre des mouvements des membres antérieurs intensification dans un animal de contrôle qui ont été marqués comme &quot;1&quot;, et l&#39;intensification des mouvements des membres antérieurs chez un animal lésion 6-OHDA qui ont été essentiellement marqué comme &quot;0&quot;. L&#39;intensification des membres postérieurs Il y avait aussi des déficiences au niveau des membres controlatérale à la lésion postérieure du rat 6-OHDA. Comme le membre le contrôle postérieurs recule, elle semble pousser contre le tambour tournant et comme il le fait, les orteils tournent vers l&#39;extérieur. Comme le membre se déplace vers l&#39;avant, le pied est tourné vers l&#39;extérieur. Le membre postérieur du rat 6-OHDA se déplace directement vers l&#39;arrière avec rotation du tambour et si quelque chose, se tourne légèrement vers l&#39;intérieur. Comme le membre se déplace vers l&#39;avant, le pied est porté directement vers l&#39;avant. Le pied de la balançoire rat témoin extérieur et comme il est avancé, les orteils sont maintenus plus élevés que la cheville, et d&#39;étendre les orteils. Lors du placement du rat contrôle, orteil 5 contacts de la surface du premier tambour et le poids est réparti entre les orteils et les palmiers. En revanche, les orteils du rat 6-OHDA sont fléchis au cours transporter et le placement se fait sans un mouvement d&#39;arpèges. Les orteils du rat de contrôle sont libérés dans un schéma inversé arpège que toe 5 est levé une première et orteils est levé dernier. Ce mouvement était absent chez le rat 6-OHDA lésion de la cheville alors maintenu sa position fléchie pendant le mouvement de transport. Encore une fois les mouvements avant le rat 6-OHDA sont atténuées, avec le membre inférieur exorotated, levé moins, et moins avancé que celui de la lutte contre les rats. Comme les rats témoins placer leurs orteils, elles sont étendues et ouvertes, telles que les orteils 5 points latéralement par rapport au corps du rat. Toe 5 contacts de la batterie en premier, suivi par les orteils restent telles que le rat soutenu son poids sur le bout des orteils. Pour le rat 6-OHDA, les orteils sont fléchis peu que le pied est reporté, les orteils sont inférieurs aux horizontal relatif à la cheville, il ya moins d&#39;ouverture des orteils lorsque le pied est placé sur le tambour. Longueur de la foulée est également un peu plus courte que celle des rats témoins. Afin de quantifier les différences entre control et le 6-OHDA mouvements des membres postérieurs, les composantes de la portance et la libération, le transport, l&#39;avancement, et le placement ont été notés sur une échelle de 3 points, comme l&#39;étaient les membres antérieurs. Les mesures ont été faites à partir des enregistrements vidéo réalisés sur la chirurgie d&#39;un jour après 20 ans, et trois cycles étape ont été notés pour chaque rat. Il y avait des différences significatives du Groupe, F (1,8) = 47,6, p &lt;0,001. Suivi t-tests sur les composants ont indiqué que tous les composants des rats témoins ont reçu des scores significativement plus élevés que ne les composantes de la 6-OHDA chez le rat. La vidéo montre les mouvements des membres postérieurs intensification dans un animal de contrôle qui ont été marqués comme &quot;1&quot;. Des membres postérieurs intensification des mouvements dans le 6-OHDA montré dans la vidéo ont été principalement évalué avec un score de «0».

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Le but de la présente étude était d&#39;examiner si le test d&#39;aptitudes de locomotion rotorod rongeurs peuvent être utilisées pour l&#39;examen qualitatif de la posture, l&#39;intensification des membres antérieurs et des membres postérieurs chez les rats l&#39;intensification du système nerveux blessés. Pour l&#39;analyse, les rats avec des épuisements DA unilatérale et les rats témoins ont été enregistrées à partir vidéos vues frontales, latérales et postérieures. Une échelle de notation de la posture et les mouvem...

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified, and another reference was included.
Additional reference:
15. Whishaw IQ, Li K, Whishaw PA, Gorny B, Metz GA. Distinct forelimb and hind limb stepping impairments in unilateral dopamine-depleted rats: use of the rotorod as a method for the qualitative analysis of skilled walking. J Neurosci Meth 126, 13-23 (2003).
Revised Abstract:
High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.
Additional Discussion Paragraph:
A more detailed presentation of the present study has been made (Whishaw et al, 2003), but the present study presents the video support describing the stepping movement in the good and affected limbs of unilateral dopamine-depleted rats. For the analysis, rats with unilateral DA depletions and control rats were video recorded from front, lateral and posterior views. A rating scale of posture and forelimb movements indicated that stepping movements were chronically impaired following surgery. Examination of limb movements indicated that whereas the DA-depleted rats could use the limbs contralateral to the lesion for support, they received minimal use for shifting weight. The results of this study indicate that the rotorod task, in addition to providing quantitative measures of motor impairments, can also provide insights into the qualitative impairments.
Original Abstract:
The rotorod test, in which animals walk on a rotating drum, is widely used to assess motor status in laboratory rodents. Performance is measured by the duration that an animal stays up on the drum as a function of drum speed. Here we report that the task provides a rich source of information about qualitative aspects of walking movements. Because movements are performed in a fixed location, they can readily be examined using high-speed video recording methods. The present study was undertaken to examine the potential of the rotorod to reveal qualitative changes in the walking movements of hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). Beginning on the day following surgery and then periodically over the next two months, the rats were filmed from frontal, lateral, and posterior views as they walked on the rotorod. Behavior was analyzed by frame-by-frame replay of the video records. Rating scales of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in their posture and in the use of the limbs contralateral to the DA-depletion. The contralateral limbs not only displayed postural and movement abnormalities, they participated less in initiating and sustaining propulsion than did the ipsilateral limbs. These findings not only reveal new deficits secondary to unilateral DA-depletion, but also show that the rotorod can provide a robust tool for the qualitative analysis of movement.

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified and another reference was included.

Erratum: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

use of rotorod as a method for the qualitative analysis of walking in rat

Le test rotorod, dans laquelle les animaux marchent sur un tambour rotatif, est largement utilisé pour évaluer l&#39;état du moteur chez les rongeurs de laboratoire. La performance est mesurée par la durée que l&#39;animal reste en place sur le tambour en fonction de la vitesse du tambour. Nous rapportons ici que la tâche fournit une riche source d&#39;informations sur les aspects qualitatifs des mouvements de marche. Parce que les mouvements sont effectués dans un endroit fixe, elles peuvent facilement être examiné en utilisant des méthodes à haute vitesse d&#39;enregistrement vidéo. La présente étude visait à examiner le potentiel de la rotorod pour révéler des changements qualitatifs dans les mouvements de la marche des rats analogique hémi-Parkinson, produit par l&#39;injection de 6-hydroxydopamine (6-OHDA) dans le faisceau droit nigrostrié à épuiser la dopamine nigro (DA ). À compter du jour suivant la chirurgie, puis périodiquement au cours des deux prochains mois, les rats ont été filmées à partir des vues frontales, latérales et postérieures comme ils marchaient sur la rotorod. Comportement est analysé par image par image rediffusion des enregistrements vidéo. Les échelles d&#39;évaluation du comportement de l&#39;intensification indiqué que les rats hémi-Parkinson étaient chroniquement déficients dans leur posture et de l&#39;utilisation des membres controlatérale à l&#39;AD-épuisement. Les membres controlatéraux non seulement affiché des anomalies de posture et du mouvement, ils ont moins participé au lancement et au maintien de propulsion que ne les membres homolatéraux. Ces résultats ne révèlent pas seulement des nouveaux déficits secondaires à unilatérales DA-épuisement des ressources, mais aussi montrer que la rotorod peut fournir un outil robuste pour l&#39;analyse qualitative du mouvement.

Watch this Scientific Journal Video about Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat at JoVE.com

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Le test rotorod est utilisée pour évaluer l&#39;état du moteur dans le mouvement de la marche des rats analogique hémi-Parkinson.

L&#39;utilisation de Rotorod comme une méthode pour l&#39;analyse qualitative de la marche chez le rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

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12.9K vues.  University of Lethbridge. Le test rotorod est utilisée pour évaluer l'état du moteur dans le mouvement de la marche des rats analogique hémi-Parkinson.

Vidéo: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyroséquençage: une méthode simple pour le génotypage précis

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

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The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

La structure du membre antérieur qualifiés Atteindre chez le rat: Un Rating Scale Mouvement

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Le barreau d&#39;échelle Marche Tâche: Un système de notation et son application pratique.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Analyse du développement d&#39;un phénotype morphologique en fonction de la concentration de protéines dans la levure bourgeonnante

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

Microinjection d&#39;embryons Medaka pour être utilisé comme un organisme modèle génétique

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Une méthode simple pour l&#39;imagerie Arabidopsis Feuilles utilisant perfluorodécaline comme un moyen d&#39;imagerie infiltratives

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

L&#39;utilisation de chémostats dans microbienne biologie des systèmes

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Une méthode hybride d&#39;extraction d&#39;ADN pour l&#39;évaluation qualitative et quantitative des communautés bactériennes de la volaille Échantillons de production

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

L&#39;échantillonnage de sang de la veine caudale latérale de la Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...