Name: dissection and coronal slice preparation of developing mouse pituitary gland
Uploaded: 2017-11-16T13:00:00.000+00:00
Duration: 6 min 53 s
Description: Watch this Scientific Journal Video about Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland at JoVE.com

This work was supported by the grants from National Natural Science Foundation of China (31201086, 31470759 and 31671219) and Shanghai Natural Science Foundation (12ZR1436900).

C57BL/6 mice are bred in specific pathogen-free conditions. All animal experimental methods are in compliance with the guidelines approved by the Animal Care and Ethics Committee at Second Military Medical University.
1. Dissection of Postnatal Developing Pituitary Gland
<ol>
	<li>Perform anesthesia: Place neonatal mice (P0 - P5) on crushed ice to induce hypothermia. For mice older than 5 days of age, inject 5% urethane (30 &#181;L/g of body weight) intraperitoneally to induce anesthesia. Assess responses to tail/toe pinches. Proceed only after the mouse is unresponsive to the noxious stimuli.</li>
	<li>Secure the mouse in the supine position (lying on the back with face upward) by gently taping the forepaws and hindpaws to a work surface that is capable of being pinned (i.e., Styrofoam) inside a chemical fume hood.</li>
	<li>Perform transcardial perfusion.
	<ol>
		<li>Cut the mouse ribcage with surgical scissors to expose the heart (and other thoracic organs).</li>
		<li>Secure the beating heart with blunt forceps and insert a 26G (0.45 mm) needle into the left ventricle. The needle is attached to a 10 mL syringe filled with heparinized phosphate buffered saline (PBS; supplemented with 5 mg/mL sodium nitrite and 10 U/mL heparin, pH 7.4, warm to 37 &#176;C before use). Immediately cut the right atrium with fine scissors and begin to perfuse the body with the warm PBS until the fluid exiting the right atrium is clear. 
		NOTE: Approximate 5 - 10 mL PBS is needed for neonatal mice and 10 - 20 mL PBS for adult mice.</li>
		<li>Keep the needle in place and change to another syringe filled with 4% paraformaldehyde (PFA; pH 7.4). Perfuse 10 mL and 20 mL of fixative for neonatal and P21 mouse, respectively. 
		Caution: PFA is toxic. It may cause skin and respiratory irritations, and eye damage. Wear gloves and work under a chemical hood.</li>
	</ol>
	</li>
	<li>Decapitate the PFA-fixed mouse and cut the skull bone open with scissors.
	<ol>
		<li>Gently lift the hindbrain from the base of the skull with small forceps. At the first sign of the sella turcica, stop lifting but hold the hindbrain, cut the pituitary stalk and nerve fibers connecting to the base of the brain with fine scissors. This step is critical to ensure that the pituitary is not lifted away with the brain.</li>
		<li>Then keep lifting the brain and remove the whole brain to expose the pituitary gland fully. Cut the entire sella region including the pituitary gland, lateral trigeminal nerves, and the beneath sphenoid bone with scissors (approximately 3 x&#160;5 mm2).</li>
	</ol>
	</li>
	<li>Put the dissected tissue into a 35 mm dish or one well of a 6-well plate containing 2 mL cold 4% PFA (pH 7.4). Fix the tissue at 4 &#176;C for 40 min for P0 - P7 pituitaries, 1 h for P14 pituitaries, 1.5 h for P21 - P28 pituitaries, and 3 h for adult pituitaries.</li>
	<li>Further dissociation of pituitary
	<ol>
		<li>Wash the fixed tissue with 10 mL PBS, 5 changes, 15 min each. The neonatal pituitary gland together with its surrounding tissues and the beneath sphenoid bone can be processed directly to dehydration. However, pituitary glands from mice older than 5 days should be dissociated further under a stereo microscope.</li>
		<li>Put the fixed tissue into a 35 mm dish containing 1 mL PBS. For P5 - P14 pituitaries, remove the connective membranes between the nerves and the bone with fine forceps and scissors, and carefully isolate the pituitary gland together with lateral trigeminal nerves as a whole but leaving the sella turcica.The isolated gland and nerves are connected by connective membranes with a &#34;H&#34;-like appearance (Figure 1B).</li>
		<li>For P21 and adult pituitaries, remove the nerves and connective membranes around the pituitary and free the gland from the surrounding tissues. Wrap the pituitary tissue with lens cleaning tissue paper and put it into an embedding cassette. 
		NOTE: Wrapping the tiny pituitary with lens cleaning tissue paper helps to prevent any potential loss of specimens and preserve tissue integrity in the following paraffin-embedding procedure. It may not be necessary to wrap the pituitary if it is processed in a small bottle in the cryo-embedding procedure.</li>
	</ol>
	</li>
</ol>
2. Paraffin Embedding and Sectioning
<ol>
	<li>Dehydrate the specimens in embedding cassettes with 50%, 65%, 75%, and 95% ethanol solutions for 15 min each. Subsequently dehydrate with 3 changes of pure ethanol, 3 min each for P0 - P4 glands, 4 min each for P5 - P14 glands, and 5 min each for P21 and older glands. Clear with xylene, 3 changes, 3 min each for P0 - P4 glands, 4 min each for P5 - P14 glands, and 5 min each for P21 and older glands. Infiltrate with molten paraffin wax, 3 changes, 7 min each for P0 - P4 glands, and 8 min twice followed by 6 min once for P5 and older glands. 
	NOTE: The optimal parameters for tissue processing can be empirically adjusted. To obtain high quality sections, insufficient or over dehydration should be avoided. The same is true for clearing the tissue using xylene.</li>
	<li>Orientation when embedding
	<ol>
		<li>On a tissue embedding console system, remove the specimen from the cassette and place it into a base mold half-filled with molten paraffin wax. Proper orientation of the embedding pituitary gland is essential for satisfying coronal sections.</li>
		<li>For P21 and adult pituitary glands, position the pituitary gland with its short axis perpendicular to the bottom surface of a base mold (the section plane). Use sphenoid bones and trigeminal nerves as anatomic landmarks for P0 - P4 and P5 - P14 pituitaries, respectively. Orient specimens with their trigeminal nerves or transversal lamina of sphenoid bones perpendicular to the bottom surface of a base mold.</li>
		<li>Gently hold the tissue in the desired position with warmed fine forceps until the wax becomes semi-solid on a cooling plate. Top up the mold with molten paraffin wax. Allow the paraffin block to cool until the wax is fully solidified. 
		NOTE: The pituitary at embryonic day 18.5 (E18.5) can be treated in the same way as the neonatal pituitary. But the pituitaries (Rathke&#39;s pouches) younger than E16.5 should be embedded with the entire head and oriented with the sagittal axis (from mouth to occiput) perpendicular to the bottom surface of a base mold.</li>
	</ol>
	</li>
	<li>Chill the paraffin block at -20 &#176;C for 10 - 20 min and then cut it into thin slices (4 &#181;m thickness is preferred) with a microtome. To get satisfactory coronal sections, fine tune the position of paraffin blocks during sectioning. Mount the tissue slices onto polylysine-coated glass slides to prevent detachment of slices in the following procedures. 
	NOTE: Alternatively, specimens can be dehydrated in 15%, 30% (W/V) sucrose solutions (in PBS) at 4 &#176;C until they sink to the bottom, embedded in cryo-embedding compound using the same orientation method, and quickly frozen on dry ice. Then cut the frozen tissue blocks into slices of 8 - 10 &#181;m using a cryostat. The morphology of cryo-sections, however, is often inferior to paraffin sections.</li>
</ol>
3. H&#38;E Staining and Immunofluorescence Labeling
<ol>
	<li>Warm the slides on a heat block at 55 &#176;C and dewax with xylene, 2 changes, 4 min each. Dehydrate the samples with 95% ethanol twice and 75% ethanol twice, 3 min each. Wash the slides with distilled water for 5 min on a shaker.</li>
	<li>For H&#38;E staining, stain the sections in alum hematoxylin solution for 6 min, rinse in running tap water for 2 min, differentiate with 0.2% ammonia water for 45 s, and re-rinse in running water for 2 min. Then dehydrate the sections with 95% ethanol for 1 min and stain with eosin for 2 min. Sequentially dehydrate, clear, and mount.</li>
	<li>Immunofluorescence labeling
	<ol>
		<li>Treat the sections with methanol containing 3% H2O2 and 0.01% NaN3 for 20 min at room temperature to inactivate endogenous peroxidases. Retrieve antigens by boiling sections in the retrieval buffer (1 mM EDTA and 1 mM Tris-HCl, pH 7.4) in a pressure cooker for 2 min. Incubate the sections with blocking buffer (5% goat serum in PBS) for 30 min at 37 &#176;C.</li>
		<li>Incubate sections with rabbit anti-growth hormone (GH) antibody (1:2,000) or rabbit anti-glial fibrillary acidic protein (GFAP) antibody (1:2,000) diluted in blocking buffer overnight at 4 &#176;C.</li>
		<li>Incubate sections with secondary antibody (goat anti-rabbit IgG-HRP, 1:300 in blocking buffer) for 35 min at 37 &#176;C.</li>
		<li>Amplify the signals using Biotinyl Tyramide (1:50 in Tris buffered saline containing 0.3% H2O2) for 15 min at room temperature, and visualize with Streptavidin-Alexa594 or 488 (1:300 in blocking buffer, 30 min, 37 &#176;C).</li>
		<li>Counterstain the nuclei with DAPI (50 mg/L in PBS) for 5 min at room temperature. 
		NOTE: It may not be necessary to amplify the signal using Tyramide Signal Amplification (TSA) system. Alternatively, a fluorescence-conjugated secondary antibody can be used to visualize the signal.</li>
	</ol>
	</li>
	<li>Acquire images using a fluorescence microscope equipped with DAPI, Texas Red, and FITC filter sets, &#215;4/0.13 to &#215;40/0.75 objectives and 5.0 megapixel camera. Use the 360, 488, and 594 nm laser wavelengths for imaging DAPI, Alexa 488-, and Alexa 594-stained sections, respectively. Optimize the laser power (0.8 was used generally) and exposure time to the desired levels for each channel. Capture the image under the control of image acquisition software and overlay the fluorescence images subsequently using image processing software.</li>
</ol>

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	<li>Fauquier, T., Lacampagne, A., Travo, P., Bauer, K., Mollard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hidden+face+of+the+anterior+pituitary.">Hidden face of the anterior pituitary.</a> Trends Endocrinol Metab. 13 (7), 304-309 (2002).</li><li>Haedo, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pituitary+function+by+cytokines.">Regulation of pituitary function by cytokines.</a> Horm Res. 72 (5), 266-274 (2009).</li><li>Zhu, X., Gleiberman, A. S., Rosenfeld, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+pituitary+development:+signaling+and+transcriptional+networks.">Molecular physiology of pituitary development: signaling and transcriptional networks.</a> Physiol Rev. 87 (3), 933-963 (2007).</li><li>Bancalari, R. E., Gregory, L. C., McCabe, M. J., Dattani, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+gland+development:+an+update.">Pituitary gland development: an update.</a> Endocr Dev. 23, 1-15 (2012).</li><li>Kelberman, D., Rizzoti, K., Lovell-Badge, R., Robinson, I. C., Dattani, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+pituitary+gland+development+in+human+and+mouse.">Genetic regulation of pituitary gland development in human and mouse.</a> Endocr Rev. 30 (7), 790-829 (2009).</li><li>Peker, S., Kurtkaya-Yapicier, O., Kilic, T., Pamir, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsurgical+anatomy+of+the+lateral+walls+of+the+pituitary+fossa.">Microsurgical anatomy of the lateral walls of the pituitary fossa.</a> Acta Neurochir (Wien). 147 (6), 641-648 (2005).</li><li>Wolpert, S. M., Molitch, M. E., Goldman, J. A., Wood, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size,+shape,+and+appearance+of+the+normal+female+pituitary+gland.">Size, shape, and appearance of the normal female pituitary gland.</a> AJR Am J Roentgenol. 143 (2), 377-381 (1984).</li><li>Carbajo-Perez, E., Watanabe, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+proliferation+in+the+anterior+pituitary+of+the+rat+during+the+postnatal+period.">Cellular proliferation in the anterior pituitary of the rat during the postnatal period.</a> Cell Tissue Res. 261 (2), 333-338 (1990).</li><li>Nantie, L. B., Himes, A. D., Getz, D. R., Raetzman, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+in+postnatal+pituitary+expansion:+proliferation,+progenitors,+and+cell+specification.">Notch signaling in postnatal pituitary expansion: proliferation, progenitors, and cell specification.</a> Mol Endocrinol. 28 (5), 731-744 (2014).</li><li>Tzou, S. C., Landek-Salgado, M. A., Kimura, H., Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+pituitary+immunogen+for+the+induction+of+experimental+autoimmune+hypophysitis.">Preparation of mouse pituitary immunogen for the induction of experimental autoimmune hypophysitis.</a> J Vis Exp. (46), (2010).</li><li>Budry, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Related+pituitary+cell+lineages+develop+into+interdigitated+3D+cell+networks.">Related pituitary cell lineages develop into interdigitated 3D cell networks.</a> Proc Natl Acad Sci U S A. 108 (30), 12515-12520 (2011).</li><li>Wilson, D. B., Wyatt, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+the+pituitary+gland+in+neonatal+little+(lit)+mutant+mice.">Histopathology of the pituitary gland in neonatal little (lit) mutant mice.</a> Histol Histopathol. 7 (3), 451-455 (1992).</li><li>Jonkers, B. W., Sterk, J. C., Wouterlood, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardial+perfusion+fixation+of+the+CNS+by+means+of+a+compressed-air-driven+device.">Transcardial perfusion fixation of the CNS by means of a compressed-air-driven device.</a> J Neurosci Methods. 12 (2), 141-149 (1984).</li><li>Cao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZBTB20+is+required+for+anterior+pituitary+development+and+lactotrope+specification.">ZBTB20 is required for anterior pituitary development and lactotrope specification.</a> Nat Commun. 7, 11121 (2016).</li></ol>

The authors have nothing to disclose.

For developing murine pituitaries, it has been technically difficult to obtain proper coronal sections due to their tiny and fragile features and unique anatomical characteristics6,8. Some research groups thus chose mid-sagittal sections to analyze the morphology of embryonic and neonatal pituitary11,12. Though the mid-sagittal section of pituitary is also capable of showing anterior, intermediate, and po...

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis1,2. Anatomically, the pituitary gland is a &#39;&#39;two-in-one&#39;&#39; structure consisting of the neurohypophysis and the adenohypophysis. These parts have different embryonic origins and function very differently. The neurohypophysis is derived from the neural ectoderm and secretes oxytocin and antidiuretic hormone. The adenohypophysis originates from Rathke&#39;s pouch and is responsible for the release of hormones including growth hormone, prolactin, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, and melanocyte-stimulating hormone3,4,5.
The pituitary gland rests on the dorsal surface of the sphenoid bone (sella turcica) of the mouse skull and is attached to the floor of the brain by a fragile stalk. It is surrounded laterally by trigeminal nerves and anteriorly by the optic chiasm6,7. The gland has an elongated oval shape with its long axis perpendicular to that of the head. On its dorsal surface, the neurohypophysis and the adenohypophysis can easily be demarcated, with the former occupying the dorsal medial region and the latter extending laterally and ventrally. During postnatal development, the size of pituitary increases rapidly in the first month after birth8,9. Nevertheless, the mouse pituitary is still very small in size with an average weight of 1.9 mg and a long-axis diameter of ~3 mm in adult10, a long-axis diameter of 2 - 2.5 mm at postnatal day 21 (P21), and only 1 - 1.5 mm at P0.
A coronal section is preferred to display both adenohypophysis and neurohypophysis in a single slice of the mouse pituitary. However, some technical skills are required to obtain satisfying coronal sections of pituitary glands from developing mice due to its exceptionally small size and distinct anatomy. In this video article, we demonstrate how to dissect mouse pituitary glands and prepare pituitary coronal sections at different development stages.

<table><tbody><tr><td>&lt;strong&gt;Tools/Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Surgical scissors-straight</td><td>JinZhong</td><td>J21010</td><td>can be purchased from other vendors</td></tr><tr><td>Fine scissors-strainght</td><td>JinZhong</td><td>WA1010</td><td>can be purchased from other vendors</td></tr><tr><td>Blunt forceps</td><td>JinZhong</td><td>JD1020</td><td>can be purchased from other vendors</td></tr><tr><td>Fine forceps</td><td>Dumont</td><td>RS-5015</td><td>for isolation of the pituitary</td></tr><tr><td>26G (0.45mm) needle</td><td>HongDa</td><td></td><td>for transcardial perfusion</td></tr><tr><td>Syringe (1 mL)</td><td>BD</td><td>300841</td><td>can be purchased from other vendors</td></tr><tr><td>Syringe (10 mL)</td><td>BD</td><td></td><td>can be purchased from other vendors</td></tr><tr><td>35mm dish</td><td>Corning</td><td>430165</td><td>can be purchased from other vendors</td></tr><tr><td>Lens cleaning paper</td><td>ShuangQuan</td><td></td><td>can be purchased from other vendors</td></tr><tr><td>Anatomical microscope</td><td>OLYMPUS</td><td>SZX-ILLB2-200</td><td>can be purchased from other vendors</td></tr><tr><td>Embedding cassette</td><td>Thermo Fisher</td><td>22-272423</td><td>can be purchased from other vendors</td></tr><tr><td>Tissue embedding console system</td><td>KEDEE</td><td>KD-BM11</td><td>can be purchased from other vendors</td></tr><tr><td>Microtome</td><td>Thermo Fisher</td><td>HM315R</td><td>can be purchased from other vendors</td></tr><tr><td>Superfrost-Plus slides</td><td>Thermo Fisher</td><td>22-037-246</td><td>can be purchased from other vendors</td></tr><tr><td>Cover glass</td><td>Thermo Fisher</td><td>12-543</td><td>can be purchased from other vendors</td></tr><tr><td>Fluorescence microscope</td><td>OLYMPUS</td><td>BH2-RFCA</td><td>can be purchased from other vendors</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Urethane</td><td>BBI</td><td>EB0448</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9625</td><td>for PBS</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td>for PBS</td></tr><tr><td>Na2HPO4.12H2O</td><td>Sigma</td><td>71650</td><td>for PBS</td></tr><tr><td>K2HPO4</td><td>Sigma</td><td>P2222</td><td>for PBS</td></tr><tr><td>NaNO2</td><td>Sigma</td><td>237213</td><td></td></tr><tr><td>Heparin Sodium Injection</td><td>SPH</td><td>H31022051</td><td>for perfusion saline</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Ethanol</td><td>SCR</td><td>10009218</td><td></td></tr><tr><td>Xylene</td><td>SCR</td><td>10023418</td><td></td></tr><tr><td>Paraffin</td><td>Thermo Fisher</td><td>8330</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma</td><td>H9627</td><td>for H&amp;amp;E staining</td></tr><tr><td>Eosin Y</td><td>Sigma</td><td>E4009</td><td>for H&amp;amp;E staining</td></tr><tr><td>rabbit anti growth hormone (GH)</td><td>National Hormone</td><td></td><td>for immunostaining</td></tr><tr><td>antibody</td><td>Pituitary Program</td><td></td><td></td></tr><tr><td>Rabbit anti-mouse GFAP antibody</td><td>Sigma</td><td>G9269</td><td>for immunostaining</td></tr><tr><td>Goat anti-rabbit IgG, HRP</td><td>Jackson</td><td>111-035-003</td><td>for immunostaining</td></tr><tr><td>TSA system</td><td>NEN Life Science Products</td><td>NEL700</td><td>for immunostaining</td></tr><tr><td>Streptavidin, Alexa Fluor 594</td><td>Thermo Fisher</td><td>S32356</td><td>for immunostaining</td></tr><tr><td>Anti-FITC Alexa Fluor 488</td><td>Thermo Fisher</td><td>A11090</td><td>for immunostaining</td></tr></tbody></table>

dissection and coronal slice preparation of developing mouse pituitary gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

This protocol presents a method to dissect pituitary glands from developing mice. For the neonatal mouse, the whole sella regions containing the pituitary gland, the trigeminal nerves, and the underneath sphenoid bone were dissected out from the skull base. The tiny and delicate pituitary gland remained intact during the process (Figure 1A). For mice older than 5 days, the pituitary glands attached to the lateral trigeminal nerves were then isolated. The gros...

Watch this Scientific Journal Video about Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland at JoVE.com

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

dissection-coronal-slice-preparation-developing-mouse-pituitary

Research

JoVE Journal

Developmental Biology

17.2K Views.  Second Military Medical University. We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

Video: Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Neuroscience

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Medicine

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.