Lo studio è stato finanziato da Linköping University e il Consiglio della Contea di Östergötland.

1. Ovariectomia attraverso la via dorsale <ol><li> Anestetizzare la animale, ad esempio utilizzando 1,5% isoflurano (4% per l&#39;induzione) in ossigeno / ossido nitroso 30% / miscela di 70%. Posizionare l&#39;animale in posizione prona su una piastra riscaldante collegata ad un termometro rettale. È importante avere un sottile strato di isolamento tra l&#39;animale e la piastra, e che il feed-back termometro mantiene la temperatura corporea entro 37 ± 0,5 ° C. Applicare il gel per la protezione degli occhi e Rimadyl somministrare 5 mg / kg di peso corporeo, diluito in soluzione salina per via sottocutanea nella regione laterale del collo dell&#39;animale per alleviare il dolore post-operatorio. </li><li> Radere un 4 * 4 cm (3 * 3 cm per topi) area cephally dalla cresta iliaca. Lavarsi accuratamente rasata con per Jodopax esempio, prima di coprire l&#39;animale con un 4 4 * foglio di intervento chirurgico di apertura cm. </li><li> Eseguire un&#39;incisione 2-3 cm linea mediana e senza mezzi termini sezionare la cute dalla fascia sottostante. Uno cm lateralmente alla linea mediana, fare un altro thro incisioneugh la fascia. Schiettamente sezionare lateralmente sotto questa fascia superficialmente quanto possibile fino a raggiungere la cavità addominale. </li><li> Con una pinzetta, afferrare il tessuto adiposo che circonda l&#39;ovaio nella cavità addominale ed estrarla delicatamente. Identificare l&#39;ovaio e legare le corna uterine e dei vasi 0,5-1 cm prossimalmente di questa struttura. Tagliare il ovaie e tessuto adiposo ligato e rimettere il restante tessuto nella cavità addominale. Fai un&#39;altra incisione nella fascia controlaterale e ripetere la procedura. </li><li> Chiudere le ferite, preferibilmente utilizzando una sutura monofilamento, e lasciare che l&#39;animale sveglia dall&#39;anestesia. Posizionare l&#39;animale in una gabbia riscaldata (25-27 ° C) per almeno 2 ore dopo l&#39;intervento. Gli animali devono essere alloggiati separatamente i primi giorni dopo l&#39;intervento. E &#39;fondamentale che la gabbia è frequentemente pulite / sostituite durante la fase di recupero. Ventiquattro ore dopo l&#39;intervento, gli animali iniettare per via sottocutanea con un altro Rimadyl 5 mg / kg di peso corporeo, diluito insalina. </li></ol> 2. La somministrazione sottocutanea di 17β-estradiolo attraverso capsule silastic <ol><li> Mescolare la quantità desiderata di 17β-estradiolo in olio di sesamo. Per le concentrazioni fisiologiche in ratti e topi, si consiglia di 180 mcg / ml e 18-36 mg / mL, rispettivamente. Capsule di placebo sono riempiti con olio di sesamo puro. </li><li> Tagliare 3 cm (2 cm per topi) la lunghezza dei tubi silastic e 5 mm (3 mm per i topi) lunghezze di bastoncini di legno per collegare applicatori. Utilizzando pinze, stringere un tappo di legno nella capsula silastic prima di iniettare la soluzione 17β-estradiol/sesame olio per riempire l&#39;intera lunghezza. Delicatamente tappare la capsula silastic alla fine aperta con un altro tappo di legno (i tappi sono tenuti a restare permanentemente nella capsula). Incubare le capsule restanti 17β-estradiolo soluzione durante la notte prima dell&#39;impianto. </li><li> L&#39;impianto della capsula può essere eseguita durante l&#39;anestesia utilizzato a ovariectomia nei ratti. Per mice, tuttavia, un periodo di 14 giorni di recupero dopo ovariectomia Si raccomanda di minimizzare il rischio della capsula sporgente attraverso la pelle fragile. </li><li> Dopo i preparativi chirurgici come in 1.1, o dopo la chiusura l&#39;incisione ovariectomia, barba e lavare a * 2 2 zona centimetri sulla parte dorsale del collo dell&#39;animale. Eseguire un&#39;incisione 1 cm e senza mezzi termini sezionare una tasca sottocutanea caudolaterally, con ampio spazio per la capsula. Pulire la capsula silastic (che fino a questo punto temporale è stato incubato nella 17β-estradiolo soluzione) e delicatamente installare attraverso l&#39;incisione, che viene successivamente chiusa da una sutura. È fondamentale per assicurarsi che i ceppi capsula la pelle il meno possibile, in particolare nella zona vicino alla ferita. Da produzione di capsule per l&#39;impianto, è importante che sia evitata la contaminazione. </li></ol> 3. Somministrazione orale di 17β-estradiolo tramite il Nut-crema Nutella <ol><li> Accuratamente sciogliere 17β-estradiolo in olio di sesamo, e poi mescolare l&#39;olio di sesamo con la Nutella. Si raccomanda che ogni porzione giornaliera contiene 28 mcg 17β-estradiolo, 5 olio di sesamo e 1 pl Nutella g per chilogrammo di peso corporeo per i ratti e 1,12 mcg 17β-estradiolo, 0,312 olio di sesamo e 60 microlitri Nutella mg per ogni 30 g mouse. Placebo nut-crema è preparata in modo identico, tranne senza il 17β-estradiolo. </li><li> Formare gli animali a mangiare senza ormoni nut-crema per cinque giorni precedenti l&#39;esperimento. I primi tre giorni di formazione, i topi devono essere formati in modo ottimale in gruppi (circa 0,5 g di Nutella 2-5 animali), nelle loro gabbie a casa. Nei giorni in quarta e quinta della formazione, gli animali vengono messi in gabbie separate prima di ricevere la quota giornaliera per assomigliare alla situazione sperimentale. </li><li> Durante l&#39;esperimento, gli animali vengono messi in gabbie separate per ogni occasione alimentazione, dopo che la porzione Nutella viene servita su piccole piastrelle ceramiche. Se la formazione è stata effettuata come sopra, our esperienza è che oltre il 95% dei ratti e topi accettare il dado-crema, e una volta completamente abituato, la maggior parte degli animali che consumano in pochi secondi. </li></ol> 4. Risultati rappresentativi Fisiologici siero concentrazioni di 17β-estradiolo, che i metodi attuali mirano a riprodurre, sono circa 5-140 pg / mL per topi e 5-35 pg / mL per i topi 4,6. Le capsule sottocutanee silastic secondo le istruzioni di cui sopra, se somministrato a 300 g femmina Sprague-Dawley, producono lentamente diminuendo le concentrazioni sieriche 17β-estradiolo di circa 40 pg / mL giorno 2 al 10 pg / mL giorno 35. Tuttavia, subito dopo la somministrazione, un picco di concentrazione sierica circa 1000 pg / mL può prevedere, cadendo per l&#39;intervallo fisiologico entro 8 ore (Figura 1A) 4,5. Le capsule silastic per topi descritti in precedenza con 36 pg 17β-estradiol/mL, quando somministrato a 25 g femmina C57BL / 6 topi, rendono siero concentrations che lentamente diminuire da circa 90 pg / ml 2 giorni a 25 pg / mL 35 giorni. Questo è probabilmente preceduta da un picco simile è stata trovata nei ratti, anche se questo non è stato studiato (figura 1B) 6. Il metodo Nutella via orale con i dosaggi sopra descritto, quando somministrato a 300 g femmina Sprague-Dawley, rende livelli fisiologici di 17β-estradiolo con fluttuazioni giornaliere comprese tra 10-70 pg / ml (Figura 2A) 4. Per topi usando il dosaggio sopra, i livelli giornalieri oscillano tra circa 20 e 120 pg / mL (Figura 2B) 6. <img alt="Figura 1" src="/files/ftp_upload/4013/4013fig1.jpg" /> <st<html> Rong.&gt; Figura 1 grafici stilizzati che presentano concentrazioni di 17β-estradiolo nel ratto (A; n = 10 punti in-time fino a 24 h, n = 15 in un momento successivo i punti) 4,5 e topi (B, n = 10) 6 capsula dopo l&#39;impianto. Le capsule silastic producono un breve picco iniziale di concentrazioni sieriche fino a 1000 pg / mL, ma calo in poche ore a livelli inferiori a 100 pg / mL. Per le prossime settimane 4-5, le capsule resa stabile, anche se in costante diminuzione, le concentrazioni sieriche. Le aree ombreggiate corrispondono a concentrazioni sieriche fisiologiche 17β-estradiolo in intatti femmine di ratto e nel topo, rispettivamente. Anche se non visivamente dimostrato nei grafici, non vi è variabilità inter-individuale nelle concentrazioni di 17β-estradiolo, rendendo coefficienti di variazione 27-183% nei ratti e dal 28 al 115% nei topi con il metodo della capsula silastic (media molto bassa concentrazioni spesso generano elevati coefficienti di variazione). iles/ftp_upload/4013/4013fig2.jpg &quot;/&gt; Figura 2 grafici stilizzati presentano 17β-estradiolo concentrazioni nei ratti (A, n = 15). 4 e topi (B, n = 10) 6 a seguito di somministrazione giornaliera di ormone Nutella. Il metodo Nutella perorale rende picchi giornalieri, ma non di gran lunga più alto prodotto da sottocutanea o endovenosa metodi. Questo imita la farmacocinetica della terapia ormonale sostitutiva umana, e il trattamento per via orale può procedere per il tempo necessario, anche se l&#39;esempio precedente solo presentare un primo 96 ore. Le aree ombreggiate corrispondono a concentrazioni sieriche fisiologiche 17β-estradiolo in intatti femmine di ratto e nel topo, rispettivamente. Anche se non visivamente dimostrato nei grafici, non vi è variabilità inter-individuale nelle concentrazioni di 17β-estradiolo, rendendo coefficienti di variazione 40-132% nei ratti e dal 13 al 219% nei topi con il metodo della Nutella (concentrazioni medie molto basse spesso generano altacoefficienti di variazione).

<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. A., Sokol, K., Smith, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safe+and+effective+method+for+chronic+17beta-estradiol+administration+to+mice.">Safe and effective method for chronic 17beta-estradiol administration to mice.</a> Contemp. Top Lab Anim. Sci. 42, 33-35 (2003).</li></ol>

Gli autori non hanno nulla da rivelare.

Le modalità di somministrazione sopra riportati in studi precedenti hanno dimostrato superiore al commerciali a lento rilascio pellets e le iniezioni in termini di produzione di fisiologiche concentrazioni sieriche di 17β-estradiolo. Mentre il pellets a lento rilascio producono molto elevati e prolungati picchi di concentrazione sierica, le iniezioni causano estreme, fluttuazioni ormonali giornaliere e richiedono ripetute stressante la gestione degli animali 4-6. L&#39;importanza di scegliere le modalità d...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Rimadyl, 50 mg / mL </td><td> Pfizer </td><td> 462986 </td><td> Analgesia postoperatoria dopo ovariectomia. </td></tr><tr><td> Lubrithal eye gel </td><td> Leo Laboratories </td><td></td><td> Protezione degli occhi durante l&#39;intervento chirurgico. </td></tr><tr><td> Jodopax veterinario </td><td> Pharmaxim </td><td></td><td> Chirurgia disinfezione. </td></tr><tr><td> Vicryl filo di sutura 4-0 </td><td> Johnson &amp; Johnson </td><td></td><td> Ferita di sutura e legatura dell&#39;ovaio. </td></tr><tr><td> 17β-estradiolo,&gt; 98% </td><td> Sigma </td><td> E8875-1G </td><td> In olio di sesamo per le capsule silastic e Nutella. </td></tr><tr><td> Olio di sesamo </td><td> Sigma </td><td> S3547-1L </td><td> Dissolvere il 17β-estradiolo. </td></tr><tr><td> Tubazione di silastic, ID / OD: 1.575/3.175 millimetri </td><td> Dow Corning </td><td> 508-008 </td><td> Produzione di capsule silastic. </td></tr><tr><td> Applicatore bastone di legno </td><td> Selefatrade </td><td> 12-0783 </td><td> Riduzione delle capsule silastic. </td></tr><tr><td> Nutella </td><td> Ferrero </td><td></td><td> Media per via orale 17β-estradiolo. </td></tr></tbody></table>

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

Gli estrogeni sono una famiglia di ormoni sessuali femminili, con una gamma eccezionalmente ampia di effetti. Quando i ratti e topi sono utilizzati nella ricerca estrogeni sono comunemente ovariectomizzati al fine di asportare la produzione di ormoni a ciclo rapido, che sostituisce il 17β-estradiolo esogena. Vi è, tuttavia, la mancanza di consenso per quanto riguarda come l&#39;ormone deve essere somministrato per ottenere fisiologiche concentrazioni sieriche. Questo è fondamentale in quanto il livello di 17β-estradiolo / amministrazione metodo influenza profondamente i risultati sperimentali 1-3. Abbiamo in una serie di studi hanno caratterizzato i diversi modi di 17β-estradiolo amministrazione, scoprendo che sottocutanei silastic capsule per via orale e per-nut-crema Nutella sono superiori al disponibili in commercio a lento rilascio pellets (prodotto dalla società di ricerca innovativa d&#39;America) e iniezioni giornaliere in termini di produzione di fisiologiche concentrazioni sieriche di 17β-estradiolo 4-6. Tra le advantages del dado-crema metodo, che precedentemente è stato usato per la somministrazione di buprenorfina 7, che quando è utilizzato per somministrazione di estrogeni assomiglia perorale terapia di sostituzione ormonale e non è invasivo. Le capsule silastic sottocutaneo sono convenienti e producono più stabili le concentrazioni sieriche. In questo articolo video contiene step-by-step dimostrazioni di ovariectomia e 17β-estradiolo sostituzione ormonale da capsule silastic e Nutella per via orale in ratti e topi, seguiti da una riflessione su aspetti importanti delle procedure amministrative.

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

Ovariectomia e la sostituzione successiva di 17β-estradiolo per mezzo di capsule silastic e Nutella per via orale sono dimostrate in ratti e topi.

Sostituzione ovariectomia e 17β-estradiolo in ratti e topi: A dimostrazione visiva

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 Views.  Linköping University. Ovariectomia e la sostituzione successiva di 17β-estradiolo per mezzo di capsule silastic e Nutella per via orale sono dimostrate in ratti e topi.

Video: 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 sistema magnetico Tether per Indagare visivo e olfattivo di controllo mediata Volo 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. ...

Ricerca

Biologia

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.

Dimostrazione di Allodinia cutanea In associazione con dolore pelvico cronico

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.

Unicellulari registrazioni di aspirazione da Fotorecettori Cone mouse

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.

Un approccio pratico alla mappatura genetica Destino inducibile: una guida visiva a Mark e Pista Cellule 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.

Contenzione manuale e comuni Itinerari Compound amministrazione in topi e ratti

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.

Necroscopia diagnostica e selezionati Collezione di tessuti e campioni di ratti e topi

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.

Progettazione e analisi del comportamento delle preferenze di temperatura e del suo ritmo circadiano in Drosophila

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.

Fisiologia Lab Dimostrazione: filtrazione glomerulare in un ratto

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.

La convalida di un modello di topo per Disrupt LINC Complessi in modo specifico-Cell

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 Rilevamento e analisi di Rb sumoilazione di proteine in cellule umane

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