O estudo foi financiado pela Universidade de Linköping e do Conselho Municipal de Östergötland.

1. Ovariectomia pela Rota Dorsal <ol><li> Anestesiar o animal, por exemplo, utilizando 1,5% de isoflurano (4% para a indução) em um óxido de oxigénio / nitroso 30% de mistura / 70%. Colocar o animal na posição de bruços em uma placa de aquecimento ligada a um termómetro rectal. É importante ter uma fina camada de isolamento entre o animal e da placa, e que o sistema de termómetro feed-back mantém a temperatura do corpo dentro de 37 ± 0,5 ° C. Aplicar gel de protecção dos olhos e administrar 5 mg de peso corporal Rimadyl / kg diluído em solução salina por via subcutânea no aspecto lateral do pescoço do animal para o alívio da dor pós-operatória. </li><li> Shave área de 4 * 4 cm (3 * 3 cm para ratos) cephally da crista ilíaca. Lave cuidadosamente a área raspada com por exemplo Jodopax antes de cobrir o animal com uma folha de 4 4 ​​cm * cirurgia de abertura. </li><li> Faça uma incisão de 2-3 cm da linha média e sem rodeios dissecar a pele da fáscia subjacente. Um cm lateral da linha média, fazer uma outra incisão through a fáscia. Bluntly dissecar lateralmente no âmbito do presente fáscia como superficialmente quanto possível, até atingir a cavidade abdominal. </li><li> Com uma pinça, pegue o tecido adiposo que rodeia o ovário na cavidade abdominal e puxe-o para fora. Identificar o ovário e ligadura dos cornos uterinos e vasos 0,5-1 cm proximal desta estrutura. Cortar o ovário e tecido adiposo ligados e voltar a colocar a tecido remanescente para dentro da cavidade abdominal. Fazer outra incisão na fáscia contralateral e repita o procedimento. </li><li> Fechar as feridas, de preferência usando uma sutura de monofilamento, e deixar o animal acordado da anestesia. Colocar o animal numa gaiola aquecida (25-27 ° C) durante pelo menos 2 horas após a cirurgia. Os animais devem ser alojados separadamente os primeiros dias pós-operatório. É fundamental que a gaiola é frequentemente limpas / substituídas durante a fase de recuperação. Vinte e quatro horas após a cirurgia, os animais injectar por via subcutânea com outro 5 mg de peso corporal Rimadyl / kg diluído emsolução salina. </li></ol> 2. A administração subcutânea de 17β-estradiol através Cápsulas Silastic <ol><li> Misturar bem a quantidade desejada de 17β-estradiol em óleo de sésamo. Para concentrações fisiológicas em ratos e ratinhos, recomendamos 180 mcg / mL e 18-36 mcg / mL, respectivamente. As cápsulas de placebo são preenchidos com óleo de sésamo puro. </li><li> Corte 3 cm (2 cm para ratos) comprimentos de tubos de silastic e 5 mm (3 mm para camundongos) comprimentos de varas aplicador de madeira para ligar. Usando uma pinça, espremer uma tomada de madeira no interior da cápsula de silastic antes de injectar a solução de óleo 17β-estradiol/sesame para encher todo o comprimento. Suavemente tampar a cápsula de silastic na extremidade aberta com uma outra tomada de madeira (os tampões devem permanecer permanentemente na cápsula). Incubar as cápsulas na solução 17β-estradiol remanescente durante a noite antes do implante. </li><li> Implante da cápsula pode ser realizada durante a anestesia utilizada em ovariectomia em ratos. Para mice, no entanto, um período de recuperação de 14-dias após a ovariectomia é fortemente recomendada para minimizar o risco da cápsula saliente através da pele frágil. </li><li> Após as preparações cirúrgicas como na 1.1, ou após o fechamento da incisão ovariectomia, fazer a barba e lavar a 2 * A área de 2 cm no dorso do pescoço do animal. Faça uma incisão de 1 cm e sem rodeios dissecar uma bolsa subcutânea caudolaterally, com amplo espaço para a cápsula. Seque a cápsula de silastic (que, até este ponto do tempo foi incubado na solução de 17β-estradiol) e suavemente instalar-lo através da incisão, a qual é subsequentemente fechado por uma sutura. É essencial para se certificar de que as estirpes de cápsula pele a tão pouco quanto possível, particularmente na área próxima da ferida. Desde a produção de cápsulas para a implantação, é importante que a contaminação é evitada. </li></ol> 3. Administração perorai de 17β-estradiol através da Nutella Nut creme- <ol><li> A dissolução completa 17β-estradiol em óleo de gergelim, e em seguida, misture o óleo de gergelim com a Nutella. Sugerimos que cada porção diária contém 28 ug 17β-estradiol, 5 uL de óleo de sésamo e 1 Nutella g por quilograma de peso corporal para os ratos e 1,12 ug 17β-estradiol, 0,312 óleo de sésamo e 60 uL Nutella mg por cada rato 30-g. Placebo porca creme-se preparado de forma idêntica excepto sem a 17β-estradiol. </li><li> Treinar os animais para comer sem hormônios porca creme para cinco dias anteriores ao experimento. Os três primeiros dias de treinamento, os ratos devem ser otimamente treinados em grupos (aproximadamente 0,5 g de Nutella 2-5 animais), em gaiolas de origem. Nos quarto e quinto dias de treinamento, os animais são colocados em gaiolas separadas antes de receber a porção diária para assemelhar-se a situação experimental. </li><li> Durante a experiência, os animais são colocados em gaiolas separadas para cada ocasião de alimentação, após o que a porção Nutella é servido em pequenas azulejos de cerâmica. Se a formação foi realizada como acima, oexperiência ur é que mais de 95% dos ratos e ratinhos aceitar a porca de creme, e uma vez totalmente habituado, a maioria dos animais consomem-lo dentro de segundos. </li></ol> 4. Os resultados representativos Soro fisiológico 17β-estradiol concentrações, que os métodos actuais destinados a reproduzir, são aproximadamente 5-140 pg / mL para ratos e 5-35 pg / mL para ratinhos 4,6. As cápsulas subcutâneas silastic de acordo com as instruções acima, quando administrado a 300 g fêmeas Sprague-Dawley, produzir diminuição lenta 17β-estradiol concentrações de soro de cerca de 40 pg / mL dia 2 ao 10 dia pg / mL 35. No entanto, imediatamente após a administração, um pico de concentração no soro de cerca de 1000 pg / mL pode ser esperado, caindo para a gama fisiológica dentro de 8 horas (Figura 1A) 4,5. As cápsulas de silastic para ratinhos descritos acima, com 36 ug 17β-estradiol/mL, quando administrada a 25 g fêmea C57BL / 6, tornam conc soroentrations que diminuem lentamente a partir de aproximadamente 90 pg / mL dia 2-25 pg / mL dia 35. Isto é provavelmente precedido por um pico semelhante como foi encontrado nos ratos, embora isso não tem sido investigada (Figura 1B) 6. O método Nutella perorai com as dosagens acima descrito, quando administrado a 300 g fêmeas Sprague-Dawley, torna níveis fisiológicos de 17β-estradiol com flutuações diárias entre 10-70 pg / mL (Figura 2A) 4. Para ratinhos usando a dosagem acima, os níveis diários flutuar entre aproximadamente 20 e 120 pg / mL (Figura 2B) 6. <img alt="A Figura 1" src="/files/ftp_upload/4013/4013fig1.jpg" /> <st<html> . Rong&gt; Figura 1 gráficos que apresentam estilizados 17β-estradiol concentrações em ratos (A, n = 10 em vez de pontos até 24 h, n = 15 em posteriores de tempo pontos) 4,5 e ratinhos (B, n = 10) 6, após o implante de cápsula. As cápsulas de silastic produzir um pico inicial curta das concentrações séricas de até 1000 pg / mL, mas diminuem dentro de horas para níveis inferiores a 100 pg / mL. Para as próximas semanas 4-5, as cápsulas rendimento estável, embora constantemente decrescente, as concentrações séricas. As áreas sombreadas correspondem a fisiológicas 17β-estradiol concentrações no soro em ratos fêmeas intactos e ratinhos, respectivamente. Mesmo que não visualmente demonstrado nos gráficos, há variabilidade inter-individual nas concentrações 17β-estradiol, tornando os coeficientes de variação de 27-183%, em ratos e de 28 a 115% em ratos com o método de cápsula de silastic (média muito baixa concentrações muitas vezes geram altos coeficientes de variação). iles/ftp_upload/4013/4013fig2.jpg &quot;/&gt; A Figura 2 gráficos que apresentam estilizados 17β-estradiol concentrações em ratos (A, n = 15). 4 e ratinhos (B, n = 10) 6 como uma consequência da administração hormona diariamente em Nutella. O método processa Nutella perorai picos diários, no entanto, de longe, não tão elevada como produzido por via subcutânea ou métodos intravenosas. Este imita a farmacocinética da terapia de substituição hormonal humano, eo tratamento perorai pode prosseguir durante o tempo que for necessário, mesmo se o exemplo acima apresentar apenas um período inicial de 96 horas. As áreas sombreadas correspondem a fisiológicas 17β-estradiol concentrações no soro em ratos fêmeas intactos e ratinhos, respectivamente. Mesmo embora não visualmente demonstrado nos gráficos, há variabilidade inter-individual nas concentrações 17β-estradiol, tornando os coeficientes de variação de 40-132%, em ratos e de 13 a 219% em ratos com o método Nutella (muito baixas concentrações médias muitas vezes geram altacoeficientes de variação).

<ol>
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Os autores não têm nada a revelar.

As formas de administração apresentadas acima têm em estudos anteriores mostraram-se superiores a comerciais de libertação lenta e as injecções de peletes em termos de produção de fisiológicas concentrações séricas de 17β-estradiol. Enquanto pelotas liberação lenta produzir muito altas e prolongadas picos de concentração de soro, as injeções causam extremos, as flutuações hormonais diárias e exigem manuseio repetido estressante dos 4-6 animais. A importância da escolha de modos de admi...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Rimadyl, 50 mg / mL </td><td> Pfizer </td><td> 462986 </td><td> Analgesia pós-operatória após a ooforectomia. </td></tr><tr><td> Lubrithal gel para os olhos </td><td> Leo Laboratories </td><td></td><td> Proteção dos olhos durante a cirurgia. </td></tr><tr><td> Vet Jodopax </td><td> Pharmaxim </td><td></td><td> Desinfecção cirurgia. </td></tr><tr><td> Sutura Vicryl 4-0 </td><td> Johnson &amp; Johnson </td><td></td><td> A sutura e ligadura do ovário. </td></tr><tr><td> 17β-estradiol,%&gt; 98 </td><td> Sigma </td><td> E8875-1G </td><td> Em óleo de gergelim para cápsulas de silastic e Nutella. </td></tr><tr><td> Óleo de gergelim </td><td> Sigma </td><td> S3547-1L </td><td> Dissolvendo o 17β-estradiol. </td></tr><tr><td> Tubo de Silastic, ID / OD: 1.575/3.175 milímetros </td><td> A Dow Corning </td><td> 508-008 </td><td> Produção de cápsulas de silastic. </td></tr><tr><td> Vareta aplicadora de madeira </td><td> Selefatrade </td><td> 12-0783 </td><td> Nivelamento das cápsulas de silastic. </td></tr><tr><td> Nutella </td><td> Ferrero </td><td></td><td> Médio para peroral 17β-estradiol. </td></tr></tbody></table>

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

Estrogens são uma família de hormonas sexuais femininos com um espectro excepcionalmente ampla de efeitos. Quando os ratos e ratinhos são utilizados na investigação estrogénio são vulgarmente ovariectomizados, a fim de ablação a produção da hormona rapidamente bicicleta, substituindo o exogenamente 17β-estradiol. Há, no entanto, a falta de consenso em relação a como a hormona deve ser administrada para se obter as concentrações séricas fisiológicas. Isto é crucial uma vez que o 17β-estradiol método nível / administração influencia profundamente os resultados experimentais 1-3. Temos em uma série de estudos caracterizados os diferentes modos de administração 17β-estradiol, encontrando que as cápsulas subcutâneas silastic e por-oral Nutella porca creme-são superiores aos disponíveis comercialmente peletes de libertação lenta (produzido pela empresa Research Innovative da América) e injecções diárias, em termos de produção de fisiológicas concentrações séricas de 17β-estradiol 4-6. Entre os advantages do método porca creme, que anteriormente foi utilizado para a administração da buprenorfina 7, é que, quando utilizada para a administração estrogénio que se assemelha a terapia de substituição hormonal perorai e é não-invasivo. As cápsulas subcutâneas silastic são convenientes e produzir as concentrações mais estáveis ​​no soro. Este artigo contém vídeo passo-a-passo demonstrações de ovariectomia e 17β-estradiol reposição hormonal por cápsulas de silastic e Nutella via oral em ratos e camundongos, seguido por uma discussão de aspectos importantes dos procedimentos administrativos.

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 subsequente substituição do 17β-estradiol, por meio de cápsulas de silastic e Nutella perorai são demonstrados em ratos e ratinhos.

Ovariectomia e 17β-estradiol substituição em ratos e camundongos: uma demonstração visual

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 visualizações.  Linköping University. Ovariectomia e subsequente substituição do 17β-estradiol, por meio de cápsulas de silastic e Nutella perorai são demonstrados em ratos e ratinhos.

Vídeo: 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.

Um sistema de acoplamento magnético para Investigar controle de vôo visual e olfativo Mediated em 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. ...

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.

Demonstração de Allodynia Cutânea Em associação com dor pélvica crônica

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.

Gravações de uma única célula de sucção de cones fotorreceptores rato

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.

Uma Abordagem Prática para mapeamento genético induzível Fate: Um Guia Visual para Mark e Track Células 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.

Restrição manual e rotas de Administração composto comum em camundongos e ratos

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.

Necropsia de diagnóstico e coleta de tecido e amostra selecionada em ratos e camundongos

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.

Projeto e Análise do Comportamento de Preferência de Temperatura e seu Ritmo Circadiano em 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.

Laboratório de Fisiologia Demonstração: Taxa de filtração glomerular em um 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.

A validação de um modelo de camundongo para Disrupt Complexos LINC de um modo específico de célula

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.

Na Vivo Detecção e análise de SUMOylation de proteína Rb em células humanas

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