The study was funded by Link&ouml;ping University and the County Council of &Ouml;sterg&ouml;tland.

1. Ovariectomy via the Dorsal Route
<ol>
	<li>Anesthetize the animal, for example using 1.5% isoflurane (4% for induction) in an oxygen/nitrous oxide 30%/70% mixture. Place the animal in prone position on a heating plate connected to a rectal thermometer. It is important to have a thin layer of isolation between the animal and the plate, and that the thermometer feed-back system keeps the body temperature within 37 &plusmn; 0.5 &deg;C. Apply gel for eye protection and administer 5 mg Rimadyl/kg bodyweight diluted in saline subcutaneously on the lateral aspect of the animal's neck for postoperative pain relief.</li>
	<li>Shave a 4*4 cm (3*3 cm for mice) area cephally from the iliac crest. Thoroughly wash the shaved area with for example Jodopax before covering the animal with a 4*4 cm aperture surgery sheet.</li>
	<li>Make a 2-3 cm midline incision and bluntly dissect the skin from the underlying fascia. One cm lateral of the midline, make another incision through the fascia. Bluntly dissect laterally under this fascia as superficially as possible until reaching the abdominal cavity.</li>
	<li>Using tweezers, grab the adipose tissue that surrounds the ovary in the abdominal cavity and gently pull it out. Identify the ovary and ligate the uterine horns and vessels 0.5-1 cm proximally of this structure. Cut the ovary and ligated adipose tissue and put the remaining tissue back into the abdominal cavity. Make another incision in the contralateral fascia and repeat the procedure.</li>
	<li>Close the wounds, preferably using a monofilament suture, and let the animal awake from anesthesia. Place the animal in a heated cage (25-27 &deg;C) during at least 2 hours after surgery. The animals should be housed separately the first days postoperatively. It is pivotal that the cage is frequently cleaned/replaced during the recovery phase. Twenty-four hours after surgery, inject the animals subcutaneously with another 5 mg Rimadyl/kg bodyweight diluted in saline.</li>
</ol>
2. Subcutaneous Administration of 17&beta;-estradiol through Silastic Capsules
<ol>
	<li>Thoroughly mix the desired amount of 17&beta;-estradiol in sesame oil. For physiological concentrations in rats and mice, we recommend 180 &mu;g/mL and 18-36 &mu;g/mL respectively. Placebo capsules are filled with pure sesame oil.</li>
	<li>Cut 3 cm (2 cm for mice) lengths of silastic tubing and 5 mm (3 mm for mice) lengths of wooden applicator sticks for plugging. Using forceps, squeeze one wooden plug into the silastic capsule before injecting the 17&beta;-estradiol/sesame oil solution to fill the entire length. Gently cap the silastic capsule in the open end with another wooden plug (the plugs are supposed to remain permanently in the capsule). Incubate the capsules in the remaining 17&beta;-estradiol solution overnight before implantation.</li>
	<li>Implantation of the capsule can be performed during the anesthesia used at ovariectomy in rats. For mice, however, a 14-day recovery period after ovariectomy is strongly recommended to minimize the risk of the capsule protruding through the fragile skin.</li>
	<li>After surgical preparations as in 1.1, or after closing the ovariectomy incision, shave and wash a 2*2 cm area on the dorsal aspect of the animal's neck. Make a 1 cm incision and bluntly dissect a subcutaneous pocket caudolaterally, with ample space for the capsule. Wipe the silastic capsule (that until this time-point has been incubated in the 17&beta;-estradiol solution) and gently install it through the incision, which is subsequently closed by a suture. It is pivotal to make sure that the capsule strains the skin as little as possible, particularly in the area close to the wound. From production of capsules to the implantation, it is important that contamination is avoided.</li>
</ol>
3. Peroral Administration of 17&beta;-estradiol via the Nut-cream Nutella
<ol>
	<li>Thoroughly dissolve 17&beta;-estradiol in sesame oil, and then mix the sesame oil with the Nutella. We recommend that each daily portion contains 28 &mu;g 17&beta;-estradiol, 5 &mu;L sesame oil and 1 g Nutella per kilogram body weight for rats and 1.12 &mu;g 17&beta;-estradiol, 0.312 &mu;L sesame oil and 60 mg Nutella for each 30-g mouse. Placebo nut-cream is prepared identically except without the 17&beta;-estradiol.</li>
	<li>Train the animals to eat hormone-free nut-cream for five days prior to the experiment. The first three days of training, the rats should optimally be trained in groups (approximately 0.5 g Nutella to 2-5 animals), in their home cages. On the fourth and fifth days of training, the animals are put in separate cages before receiving the daily portion to resemble the experimental situation.</li>
	<li>During the experiment, the animals are put in separate cages for every feeding occasion, after which the Nutella portion is served on small ceramic tiles. If training has been performed as above, our experience is that more than 95% of rats and mice accept the nut-cream, and once fully habituated, most of the animals consume it within seconds.</li>
</ol>
4. Representative Results
Physiological serum 17&beta;-estradiol concentrations, which the current methods aim to reproduce, are approximately 5-140 pg/mL for rats and 5-35 pg/mL for mice4,6. The subcutaneous silastic capsules according to the instructions above, when administered to 300 g female Sprague-Dawley rats, produce slowly decreasing 17&beta;-estradiol serum concentrations from around 40 pg/mL day 2 to 10 pg/mL day 35. However, immediately after administration, a serum concentration peak around 1000 pg/mL can be expected, falling to the physiological range within 8 hours (Figure 1A)4,5. The silastic capsules for mice described above with 36 &mu;g 17&beta;-estradiol/mL, when administered to 25 g female C57BL/6 mice, render serum concentrations that slowly decrease from approximately 90 pg/mL day 2 to 25 pg/mL day 35. This is probably preceded by a similar peak as was found in the rats, although this has not been investigated (Figure 1B)6.
The peroral Nutella method with the dosages described above, when administered to 300 g female Sprague-Dawley rats, renders physiological levels of 17&beta;-estradiol with daily fluctuations between 10-70 pg/mL (Figure 2A)4. For mice using the above dosage, the daily levels fluctuate between approximately 20 and 120 pg/mL (Figure 2B)6.
<img alt="Figure 1" src="/files/ftp_upload/4013/4013fig1.jpg" /> 
Figure 1. Stylized graphs presenting 17&beta;-estradiol concentrations in rats (A; n=10 in time-points up to 24 h, n=15 in later time-points)4,5 and mice (B; n=10)6 after capsule implantation. The silastic capsules produce a short initial peak of serum concentrations up to 1000 pg/mL, but decrease within hours to levels below 100 pg/mL. For the coming 4-5 weeks, the capsules yield stable, although steadily decreasing, serum concentrations. The shaded areas correspond to physiological 17&beta;-estradiol serum concentrations in intact female rats and mice respectively. Even though not visually demonstrated in the graphs, there is inter-individual variability in the 17&beta;-estradiol concentrations, rendering coefficients of variation from 27 to 183 % in rats and from 28 to 115 % in mice with the silastic capsule method (very low mean concentrations often generate high coefficients of variation).
<img alt="Figure 2" src="/files/ftp_upload/4013/4013fig2.jpg" /> 
Figure 2. Stylized graphs presenting 17&beta;-estradiol concentrations in rats (A; n=15)4 and mice (B; n=10)6 as a consequence of daily hormone administration in Nutella. The peroral Nutella method renders daily peaks, however not by far as high as produced by subcutaneous or intravenous methods. This mimics the pharmacokinetics of human hormone replacement therapy, and the peroral treatment can proceed for as long as needed, even if the example above only present an initial 96 hours. The shaded areas correspond to physiological 17&beta;-estradiol serum concentrations in intact female rats and mice respectively. Even though not visually demonstrated in the graphs, there is inter-individual variability in the 17&beta;-estradiol concentrations, rendering coefficients of variation from 40 to 132 % in rats and from 13 to 219 % in mice with the Nutella method (very low mean concentrations often generate high coefficients of 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. O., Theodorsson, E., Holm, L., Theodorsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+methods+for+administering+17beta-estradiol+to+ovariectomized+rats+result+in+opposite+effects+on+ischemic+brain+damage.">Different methods for administering 17beta-estradiol to ovariectomized rats result in opposite effects on ischemic brain damage.</a> BMC Neurosci. 11, 1471-2202 (2010).</li><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=Hormesis+and+Female+Sex+Hormones.">Hormesis and Female Sex Hormones.</a> Pharmaceuticals. 4, 15 (2011).</li><li>Isaksson, I. M., Theodorsson, A., Theodorsson, E., 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. 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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. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+17beta-estradiol+upon+stroke+damage+in+stroke+prone+and+normotensive+rats.">Differential effects of 17beta-estradiol upon stroke damage in stroke prone and normotensive rats.</a> J. Cereb. 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>

The authors have nothing to disclose.

The administration modes presented above have in earlier studies proved superior to commercial slow-release pellets and injections in terms of producing physiological serum concentrations of 17&beta;-estradiol. While the slow-release pellets produce very high and prolonged serum concentration peaks, the injections cause extreme, daily hormone fluctuations and require repeated stressful handling of the animals 4-6. The importance of choosing adequate administration modes is highlighted by recent studies indicat...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Rimadyl, 50 mg/mL</td>
 <td>Pfizer</td>
 <td>462986</td>
 <td>Postoperative analgesia after ovariectomy.</td>
 </tr>
 <tr>
 <td>Lubrithal eye gel</td>
 <td>Leo Laboratories </td>
 <td>&nbsp;</td>
 <td>Eye protection during surgery.</td>
 </tr>
 <tr>
 <td>Jodopax vet</td>
 <td>Pharmaxim </td>
 <td>&nbsp;</td>
 <td>Surgery disinfection.</td>
 </tr>
 <tr>
 <td>Vicryl 4-0 suture</td>
 <td>Johnson &amp; Johnson</td>
 <td>&nbsp;</td>
 <td>Wound suture and ovary ligation.</td>
 </tr>
 <tr>
 <td>17&beta;-estradiol, &gt;98%</td>
 <td>Sigma</td>
 <td>E8875-1G</td>
 <td>In sesame oil for silastic capsules and Nutella.</td>
 </tr>
 <tr>
 <td>Sesame oil</td>
 <td>Sigma</td>
 <td>S3547-1L</td>
 <td>Dissolving the 17&beta;-estradiol.</td>
 </tr>
 <tr>
 <td>Silastic tubing, ID/OD: 1.575/3.175 mm</td>
 <td>Dow Corning</td>
 <td>508-008</td>
 <td>Production of silastic capsules.</td>
 </tr>
 <tr>
 <td>Wooden applicator stick</td>
 <td>Selefatrade</td>
 <td>12-0783</td>
 <td>Capping of silastic capsules.</td>
 </tr>
 <tr>
 <td>Nutella</td>
 <td>Ferrero</td>
 <td>&nbsp;</td>
 <td>Medium for peroral 17&beta;-estradiol.</td>
 </tr>
 </tbody>
</table>

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

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 commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

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

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

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

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

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57.0K Aufrufe.  Linköping University. Ovarektomie und anschließender Austausch von 17β-Östradiol durch Silastic Kapseln und perorale Nutella sind bei Ratten und Mäusen nachgewiesen.

Serie: 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.

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. ...

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.

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.

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.

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.

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.

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.

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.

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.

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.

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