L&#39;étude a été financée par l&#39;Université de Linköping et le Conseil du comté d&#39;Östergötland.

1. L&#39;ovariectomie par la Route dorsale <ol><li> Anesthésier l&#39;animal, par exemple en utilisant 1,5% d&#39;isoflurane (4% pour l&#39;induction) dans un oxyde d&#39;oxygène / protoxyde d&#39;azote de 30% / 70% mélange. Placer l&#39;animal en position couchée sur une plaque de chauffage relié à un thermomètre rectal. Il est important d&#39;avoir une fine couche d&#39;isolation entre l&#39;animal et la plaque, et que le thermomètre de feed-back du système maintient la température du corps au sein de 37 ± 0,5 ° C. Appliquer le gel de protection des yeux et d&#39;administrer 5 mg Rimadyl / kg de poids corporel dilués dans une solution saline sous-cutanée sur la face latérale du cou de l&#39;animal pour soulager la douleur postopératoire. </li><li> Shave a 4 * 4 cm (3 * 3 cm pour les souris) zone cephally de la crête iliaque. Laver soigneusement la zone rasée avec pour Jodopax par exemple avant de couvrir l&#39;animal avec un 4 * 4 feuille de chirurgie cm d&#39;ouverture. </li><li> Faire une incision de 2-3 cm la ligne médiane et carrément disséquer la peau de l&#39;aponévrose sous-jacente. Un cm latérales de la ligne médiane, faire une autre incision through le fascia. Pour dire les choses disséquer latéralement sous ce fascia superficiel que possible jusqu&#39;à la cavité abdominale. </li><li> L&#39;aide de pincettes, récupérer le tissu adipeux qui entoure l&#39;ovaire dans la cavité abdominale et retirez-le doucement. Identifier l&#39;ovaire et ligaturer les trompes utérines et des vaisseaux 0,5-1 cm proximal de cette structure. Couper l&#39;ovaire et le tissu adipeux et ligaturé remettre le tissu restant dans la cavité abdominale. Faites une autre incision dans le fascia controlatéral et répétez la procédure. </li><li> Fermez les plaies, de préférence en utilisant un fil de suture monofilament, et laisser l&#39;animal se réveiller de l&#39;anesthésie. Placer l&#39;animal dans une cage chauffée (25-27 ° C) pendant au moins 2 heures après la chirurgie. Les animaux doivent être logés séparément les premiers jours après l&#39;opération. Il est impératif que la cage est fréquemment nettoyé / remplacé au cours de la phase de récupération. Vingt-quatre heures après la chirurgie, injection sous-cutanée des animaux avec un autre 5 mg Rimadyl / kg de poids corporel dilués dansune solution saline. </li></ol> 2. Administration sous-cutanée de 17β-estradiol par le biais Capsules Silastic <ol><li> Bien mélanger la quantité désirée de 17β-estradiol dans de l&#39;huile de sésame. Pour des concentrations physiologiques chez les rats et les souris, nous vous recommandons 180 mg / ml et 18-36 pg / mL, respectivement. Capsules placebo sont remplis d&#39;huile de sésame pur. </li><li> Couper 3 cm (2 cm pour les souris) de longueur de tube en silastic et 5 mm (3 mm pour les souris) longueurs de bâtons applicateurs en bois pour brancher. Utilisation pince, presser un bouchon en bois dans la capsule silastique avant l&#39;injection de la solution d&#39;huile 17β-estradiol/sesame à remplir toute la longueur. Doucement plafonner la capsule de silastic dans l&#39;extrémité ouverte avec un autre bouchon en bois (les bouchons sont censés rester en permanence dans la capsule). Incuber les capsules dans le reste de 17β-estradiol solution du jour au lendemain avant l&#39;implantation. </li><li> Implantation de la capsule peut être effectuée pendant l&#39;anesthésie utilisée à l&#39;ovariectomie chez les rats. Pour miCE, cependant, une période de récupération de 14 jours après une ovariectomie est fortement recommandée afin de minimiser le risque de la capsule en saillie à travers la peau fragile. </li><li> Après préparations chirurgicales comme dans 1.1, ou après la fermeture de l&#39;incision ovariectomie, de se raser et laver une superficie de 2 * cm 2 sur la face dorsale du cou de l&#39;animal. Faire une incision de 1 cm et carrément disséquer un poche sous-cutanée caudolaterally, avec amplement d&#39;espace pour la capsule. Essuyer la capsule silastique (que jusqu&#39;à ce point de temps a été incubé dans la solution 17β-estradiol) et lentement l&#39;installer à travers l&#39;incision, qui est ensuite fermée par une suture. Il est essentiel de s&#39;assurer que les souches de la peau capsule aussi peu que possible, en particulier dans la zone proche de la plaie. De la production de capsules à l&#39;implantation, il est important que la contamination est évitée. </li></ol> 3. Administration par voie orale de 17β-estradiol par l&#39;intermédiaire du Nutella noix de crème <ol><li> Bien dissoudre 17β-estradiol dans l&#39;huile de sésame, puis mélanger l&#39;huile de sésame avec le Nutella. Nous recommandons que chaque portion contient 28 mg par jour 17β-estradiol, 5 ul d&#39;huile de sésame et 1 g par Nutella kilogramme de poids corporel pour le rat et de 1,12 mg 17β-estradiol, l&#39;huile de sésame 0,312 pi et 60 mg Nutella pour chaque souris de 30 g. Placebo écrou-crème est préparée de manière identique mais sans le 17β-estradiol. </li><li> Former les animaux de manger sans hormones de noix de crème pendant cinq jours avant l&#39;expérience. Les trois premiers jours de la formation, les rats doivent être formés de manière optimale dans des groupes (environ 0,5 g de Nutella à 2-5 animaux), dans leurs cages à domicile. Sur les quatrième et cinquième jours de formation, les animaux sont mis dans des cages séparées avant de recevoir la portion journalière de ressembler à la situation expérimentale. </li><li> Pendant l&#39;expérience, les animaux sont mis dans des cages séparées pour chaque occasion l&#39;alimentation, après quoi la partie Nutella est servi sur de petits carreaux de céramique. Si la formation a été réalisée comme ci-dessus, our l&#39;expérience, c&#39;est que plus de 95% des rats et des souris d&#39;accepter l&#39;écrou-crème, et une fois totalement habitués, la plupart des animaux la consomment en quelques secondes. </li></ol> 4. Les résultats représentatifs Sérum physiologique 17β-estradiol concentrations, ce qui les méthodes actuelles visent à reproduire, sont d&#39;environ 5 à 140 pg / mL pour les rats et les 5-35 pg / mL pour les souris 4,6. Les capsules sous-cutanées silastic selon les instructions ci-dessus, lorsqu&#39;il est administré à 300 g femelles Sprague-Dawley, de produire diminue lentement 17β-estradiol concentrations sériques de l&#39;ordre de 40 pg / ml le jour 2 à 10 pg / ml de 35 jours. Cependant, immédiatement après l&#39;administration, un pic de concentration sérique d&#39;environ 1000 pg / ml peut être prévu, tombant à la gamme physiologique dans les 8 heures (figure 1A) 4,5. Les capsules en silastic pour les souris décrites ci-dessus avec 36 ug 17β-estradiol/mL, lorsqu&#39;il est administré à 25 g C57BL / 6 femelles souris, de rendre le sérum concentréentrations que lentement diminuer d&#39;environ 90 pg / ml Jour 2 au 25 pg / ml Jour 35. Ceci est probablement précédée par un pic similaire a été trouvé chez les rats, même si cela n&#39;a pas été étudiée (figure 1B) 6. La méthode Nutella par voie orale avec les dosages décrits ci-dessus, lorsqu&#39;il est administré à 300 g femelles Sprague-Dawley, rend les niveaux physiologiques de 17β-estradiol avec des fluctuations quotidiennes entre 10-70 pg / mL (figure 2A) 4. Pour les souris en utilisant le dosage ci-dessus, les niveaux quotidiens varient entre environ 20 et 120 pg / mL (figure 2B) 6. <img alt="Figure 1" src="/files/ftp_upload/4013/4013fig1.jpg" /> <st<html> rong.&gt; Figure 1 graphiques stylisés présentant 17β-estradiol concentrations chez le rat (A, n = 10 dans le temps des points jusqu&#39;à 24 h, n = 15 dans les derniers temps les points) 4,5 et souris (B, n = 10) 6 après l&#39;implantation capsule. Les capsules en silastic produisent un pic initiale de courte durée des concentrations sériques jusqu&#39;à 1000 pg / mL, mais diminution dans les heures à des niveaux inférieurs à 100 pg / mL. Pour les prochaines semaines 4-5, les capsules rendement stable, même si en constante diminution, les concentrations sériques. Les zones ombrées correspondent à physiologiques 17β-estradiol concentrations sériques en intactes rats et des souris femelles, respectivement. Même si pas visuellement démontré dans les graphiques, il ya variabilité inter-individuelle des concentrations 17β-estradiol, ce qui rend les coefficients de variation de 27 à 183% chez le rat et de 28 à 115% chez la souris avec la méthode capsule de silastic (moyenne très faible concentrations génèrent souvent des coefficients de variation élevés). iles/ftp_upload/4013/4013fig2.jpg &quot;/&gt; Figure 2 graphiques stylisées présentant 17β-estradiol concentrations chez le rat (A, n = 15). 4 et souris (B, n = 10) 6 à la suite de l&#39;administration journalière de l&#39;hormone de Nutella. La méthode Nutella par voie orale rend pics journaliers, mais pas de loin plus élevé que produit par voie sous-cutanée ou intraveineuse méthodes. Cela imite la pharmacocinétique de l&#39;hormonothérapie de remplacement de l&#39;homme, et le traitement par voie orale peut procéder aussi longtemps que nécessaire, même si l&#39;exemple ci-dessus ne présentent environ 96 heures initiales. Les zones ombrées correspondent à physiologiques 17β-estradiol concentrations sériques en intactes rats et des souris femelles, respectivement. Même si pas visuellement démontré dans les graphiques, il ya variabilité inter-individuelle des concentrations 17β-estradiol, ce qui rend les coefficients de variation de 40 à 132% chez le rat et de 13 à 219% chez la souris avec la méthode Nutella (très faibles concentrations moyennes génèrent souvent de hautles coefficients de variation).

<ol>
	<li>Strom, J. O., Theodorsson, A., Theodorsson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-related+neuroprotective+versus+neurodamaging+effects+of+estrogens+in+rat+cerebral+ischemia:+a+systematic+analysis.">Dose-related neuroprotective versus neurodamaging effects of estrogens in rat cerebral ischemia: a systematic analysis.</a> J. Cereb. Blood Flow Metab. 29, 1359-1372 (2009).</li><li>Strom, J. 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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+long-term+17beta-estradiol+administration+to+mice.">Methods for long-term 17beta-estradiol administration to mice.</a> General and comparative endocrinology. , (2011).</li><li>Kalliokoski, O., Jacobsen, K. R., Hau, J., Abelson, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+concentrations+of+buprenorphine+after+oral+and+parenteral+administration+in+male+mice.">Serum concentrations of buprenorphine after oral and parenteral administration in male mice.</a> Vet. J. 187, 251-254 (2011).</li><li>Simpkins, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogens+may+reduce+mortality+and+ischemic+damage+caused+by+middle+cerebral+artery+occlusion+in+the+female+rat.">Estrogens may reduce mortality and ischemic damage caused by middle cerebral artery occlusion in the female rat.</a> J. Neurosurg. 87, 724-730 (1997).</li><li>Wise, P. M., Dubal, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estradiol+protects+against+ischemic+brain+injury+in+middle-aged+rats.">Estradiol protects against ischemic brain injury in middle-aged rats.</a> Biol. Reprod. 63, 982-985 (2000).</li><li>Saleh, T. M., Cribb, A. E., Connell, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen-induced+recovery+of+autonomic+function+after+middle+cerebral+artery+occlusion+in+male+rats.">Estrogen-induced recovery of autonomic function after middle cerebral artery occlusion in male rats.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, 1531-1539 (2001).</li><li>Bingham, D., Macrae, I. M., Carswell, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detrimental+effects+of+17beta-oestradiol+after+permanent+middle+cerebral+artery+occlusion.">Detrimental effects of 17beta-oestradiol after permanent middle cerebral artery occlusion.</a> J. Cereb. Blood Flow Metab. 25, 414-420 (2005).</li><li>Carswell, H. 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Blood Flow Metab. 24, 298-304 (2004).</li><li>Yong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=17beta-estradiol+potentiates+ischemia-reperfusion+injury+in+diabetic+ovariectomized+female+rats.">17beta-estradiol potentiates ischemia-reperfusion injury in diabetic ovariectomized female rats.</a> Brain Res. 1054, 192-199 (2005).</li><li>Theodorsson, A., Theodorsson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estradiol+increases+brain+lesions+in+the+cortex+and+lateral+striatum+after+transient+occlusion+of+the+middle+cerebral+artery+in+rats:+no+effect+of+ischemia+on+galanin+in+the+stroke+area+but+decreased+levels+in+the+hippocampus.">Estradiol increases brain lesions in the cortex and lateral striatum after transient occlusion of the middle cerebral artery in rats: no effect of ischemia on galanin in the stroke area but decreased levels in the hippocampus.</a> Peptides. 26, 2257-2264 (2005).</li><li>Levin-Allerhand, J. 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Les auteurs n&#39;ont rien à révéler.

Les modes d&#39;administration présentés ci-dessus dans des études antérieures ont prouvé supérieur à libération lente commerciales boulettes et les injections en termes de production physiologiques des concentrations sériques de 17β-estradiol. Alors que l&#39;granulés à libération lente de très haute et prolongée des pics de concentration sérique, les injections sont extrêmes, les fluctuations hormonales quotidiennes et nécessitent des manipulations répétées de stress des animaux 4-6. L&...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Rimadyl, 50 mg / ml </td><td> Pfizer </td><td> 462986 </td><td> L&#39;analgésie postopératoire après ovariectomie. </td></tr><tr><td> Gel pour les yeux Lubrithal </td><td> Laboratoires Leo </td><td></td><td> Protection des yeux pendant la chirurgie. </td></tr><tr><td> Vétérinaire Jodopax </td><td> Pharmaxim </td><td></td><td> Chirurgie de désinfection. </td></tr><tr><td> Vicryl suture 4-0 </td><td> Johnson &amp; Johnson </td><td></td><td> Enroulé de suture et de ligature des ovaires. </td></tr><tr><td> 17β-estradiol,&gt; 98% </td><td> Sigma </td><td> E8875-1G </td><td> Dans l&#39;huile de sésame pour les capsules en silastic et Nutella. </td></tr><tr><td> Huile de sésame </td><td> Sigma </td><td> S3547-1L </td><td> Dissolution de la 17β-estradiol. </td></tr><tr><td> Tube en Silastic, ID / OD: 1.575/3.175 mm </td><td> Dow Corning </td><td> 508-008 </td><td> La production de capsules en silastic. </td></tr><tr><td> Bâtonnet applicateur en bois </td><td> Selefatrade </td><td> 12-0783 </td><td> Le plafonnement des capsules en silastic. </td></tr><tr><td> Nutella </td><td> Ferrero </td><td></td><td> Moyen pour perorale 17β-estradiol. </td></tr></tbody></table>

ovariectomy and 17&beta;-estradiol replacement in rats and mice: a visual demonstration

Les œstrogènes sont une famille des hormones sexuelles féminines avec un spectre exceptionnellement large d&#39;effets. Lorsque les rats et les souris sont utilisés dans la recherche d&#39;œstrogènes, ils sont souvent une ovariectomie en vue de l&#39;ablation de la production d&#39;hormones cycle rapide, le remplacement de la 17β-estradiol exogène. Il ya, cependant, l&#39;absence de consensus sur la façon dont l&#39;hormone doit être administré pour obtenir des concentrations sériques physiologiques. Ce point est crucial puisque le 17β-estradiol niveau / administration méthode influe profondément sur ​​les résultats expérimentaux 1-3. Nous avons dans une série d&#39;études ont caractérisé les différents modes de 17β-estradiol administration, constatant que sous-cutanés capsules en silastic et par-orale de noix de crème Nutella sont supérieurs aux commercialement disponibles à libération lente pellets (produit par la société Research innovante d&#39;Amérique) et injections quotidiennes en termes de production physiologiques des concentrations sériques de 17β-estradiol 4-6. Parmi les advantages de la méthode de noix de crème, qui a déjà été utilisé pour la buprénorphine administration 7, c&#39;est que lorsqu&#39;il est utilisé pour l&#39;administration d&#39;œstrogènes par voie orale il ressemble à l&#39;hormonothérapie substitutive et est non-invasive. Les capsules sous-cutanées en silastic sont pratiques et de produire des concentrations sériques les plus stables. Cet article contient étape vidéo-par-étape des démonstrations de l&#39;ovariectomie et le remplacement d&#39;hormone 17β-estradiol par des capsules en silastic et Nutella par voie orale chez le rat et la souris, suivi par une discussion sur des aspects importants des procédures d&#39;administration.

Watch this Scientific Journal Video about Ovariectomy and 17Î²-estradiol Replacement in Rats and Mice: A Visual Demonstration at JoVE.com

Ovariectomy and 17&amp;#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomie et le remplacement ultérieur de la 17β-estradiol par voie de capsules en silastic et Nutella par voie orale sont mises en évidence chez des rats et des souris.

Remplacement ovariectomie et 17β-estradiol chez les rats et les souris: une démonstration visuelle

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

ovariectomy-17-estradiol-replacement-rats-mice-visual

Research

JoVE Journal

Biology

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

56.7K vues.  Linköping University. Ovariectomie et le remplacement ultérieur de la 17β-estradiol par voie de capsules en silastic et Nutella par voie orale sont mises en évidence chez des rats et des souris.

Vidéo: Ovariectomy and 17&#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

Un système d&#39;attache magnétique d&#39;enquêter sur les visuels et olfactifs Flight Control Mediated chez la drosophile

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

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Biologie

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2).
The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis.
The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3). 
The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

A demonstration of the bedside test for cutaneous allodynia and its clinical implications.

Démonstration de L&#39;allodynie cutanée En association avec la douleur pelvienne chronique

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis.  In some women the severe episodes of cyclic pain change and the ...

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 ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Une approche pratique de la cartographie inductible destin génétique: un guide visuel à Mark et Track Cellules In Vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Manuel de retenue et communes voies d&#39;administration composés chez la souris et le rat

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

Autopsie de diagnostic et de certains tissus et de collecte des échantillons dans les rats et les souris

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

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 ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

Laboratoire de Physiologie et de démonstration: taux de filtration glomérulaire dans un Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

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 ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

In Vivo Détection et analyse des Rb SUMOylation de protéines dans les cellules humaines

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...