Name: immunostaining to visualize murine enteric nervous system development
Uploaded: 2015-04-29T13:00:00
Description: Watch this Scientific Journal Video about Immunostaining to Visualize Murine Enteric Nervous System Development at JoVE.com

This work was supported bythe American Pediatric Surgical Association Foundation Award (AG) and the National Institutes of Health K08DK098271 (AG).

The University of Wisconsin Animal Care and Use Committee approved all procedures.
1. Preparation of Solutions
<ol>
	<li>Use 1x phosphate buffered saline (PBS) as dissection buffer and rinsing solution.</li>
	<li>Prepare 30% sucrose by weighing 30 g of sucrose and place into a bottle. Add 99 ml of 1x PBS and add 1 ml 10% sodium azide. Mix thoroughly until all of the sucrose is dissolved. Store at 4 &#176;C until required.</li>
	<li>Prepare Blocking solution by mixing 1x PBS, 3% bovin...

<ol>
	<li>Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+determinants+of+the+independence+and+complexity+of+the+enteric+nervous+system.">Developmental determinants of the independence and complexity of the enteric nervous system.</a> Trends Neurosci. 33 (10), 446-456 (2010).</li><li>Amiel, J., Sproat-Emison, E., 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=Hirschsprung+disease,+associated+syndromes+and+genetics:+a+review.">Hirschsprung disease, associated syndromes and genetics: a review.</a> J Med Genet. 45 (1), 1-14 (2008).</li><li>Erickson, C. S., Barlow, A. 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S., 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=Altered+neuronal+density+and+neurotransmitter+expression+in+the+ganglionated+region+of+Ednrb+null+mice:+implications+for+Hirschsprung's+disease.">Altered neuronal density and neurotransmitter expression in the ganglionated region of Ednrb null mice: implications for Hirschsprung's disease.</a> Neurogastroenterol Motil. , (2013).</li><li>Margolis, K. G., Stevanovic, K., 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=Enteric+neuronal+density+contributes+to+the+severity+of+intestinal+inflammation.">Enteric neuronal density contributes to the severity of intestinal inflammation.</a> Gastroenterology. 141 (2), 588-598 (2011).</li><li>Qu, Z. -. D., Thacker, M., Castelucci, P., Bagy&#225;nszki, M., Epstein, M. L., Furness, 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=Immunohistochemical+analysis+of+neuron+types+in+the+mouse+small+intestine.">Immunohistochemical analysis of neuron types in the mouse small intestine.</a> Cell Tissue Res. 334 (2), 147-161 (2008).</li><li>Bian, X. -. C., Bornstein, J. C., Bertrand, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+transmission+at+functionally+distinct+synapses+in+descending+reflex+pathways+of+the+rat+colon.">Nicotinic transmission at functionally distinct synapses in descending reflex pathways of the rat colon.</a> Neurogastroenterol Motil. 15 (2), 161-171 (2003).</li><li>Johnson, C. D., Epstein, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+and+polyvalent+antiserum+to+chicken+choline+acetyltransferase.">Monoclonal antibodies and polyvalent antiserum to chicken choline acetyltransferase.</a> J Neurochem. 46 (3), 968-976 (1986).</li><li>Tooyama, I., Kimura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+encoded+by+an+alternative+splice+variant+of+choline+acetyltransferase+mRNA+is+localized+preferentially+in+peripheral+nerve+cells+and+fibers.">A protein encoded by an alternative splice variant of choline acetyltransferase mRNA is localized preferentially in peripheral nerve cells and fibers.</a> J Chem Neuroanat. 17 (4), 217-226 (2000).</li><li>Koga, T., Bellier, J. -. P., Kimura, H., Tooyama, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoreactivity+for+Choline+Acetyltransferase+of+Peripheral-Type+(pChAT)+in+the+Trigeminal+Ganglion+Neurons+of+the+Non-Human+Primate+Macaca+fascicularis.">Immunoreactivity for Choline Acetyltransferase of Peripheral-Type (pChAT) in the Trigeminal Ganglion Neurons of the Non-Human Primate Macaca fascicularis.</a> Acta histochemica et cytochemica. 46 (2), 59-64 (2013).</li><li>Sang, Q., Young, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+and+chemical+coding+of+cholinergic+neurons+in+the+small+and+large+intestine+of+the+mouse.">The identification and chemical coding of cholinergic neurons in the small and large intestine of the mouse.</a> Anat Rec. 251 (2), 185-199 (1998).</li><li>Lei, J., Howard, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+deletion+of+Hand2+in+enteric+neural+precursor+cells+affects+its+functions+in+neurogenesis,+neurotransmitter+specification+and+gangliogenesis,+causing+functional+aganglionosis.">Targeted deletion of Hand2 in enteric neural precursor cells affects its functions in neurogenesis, neurotransmitter specification and gangliogenesis, causing functional aganglionosis.</a> Development (Cambridge, England). 138 (21), 4789-4800 (2011).</li><li>Erickson, C. S., Lee, S. J., Barlow-Anacker, A. J., Druckenbrod, N. R., Epstein, M. L., Gosain, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+cholinergic+myenteric+neurons+during+enteric+nervous+system+development:+comparison+of+different+ChAT+fluorescent+mouse+reporter+lines.">Appearance of cholinergic myenteric neurons during enteric nervous system development: comparison of different ChAT fluorescent mouse reporter lines.</a> Neurogastroenterol Motil. 26 (6), 874-884 (2014).</li><li>Teitelbaum, D. H., Caniano, D. A., Qualman, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+of+Hirschsprung's-associated+enterocolitis:+importance+of+histologic+correlates.">The pathophysiology of Hirschsprung's-associated enterocolitis: importance of histologic correlates.</a> J Pediatr Surg. 24 (12), 1271-1277 (1989).</li><li>Aslam, A., Spicer, R. D., Corfield, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Children+with+Hirschsprung's+disease+have+an+abnormal+colonic+mucus+defensive+barrier+independent+of+the+bowel+innervation+status.">Children with Hirschsprung's disease have an abnormal colonic mucus defensive barrier independent of the bowel innervation status.</a> J Pediatr Surg. 32 (8), 1206-1210 (1997).</li><li>Puig, I., Champeval, D., De Santa Barbara, P., Jaubert, F., Lyonnet, S., Larue, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Pten+in+the+mouse+enteric+nervous+system+induces+ganglioneuromatosis+and+mimics+intestinal+pseudoobstruction.">Deletion of Pten in the mouse enteric nervous system induces ganglioneuromatosis and mimics intestinal pseudoobstruction.</a> J Clin Invest. 119 (12), 3586-3596 (2009).</li></ol>

Our laboratory and others have shown that intestinal defects in HSCR are not restricted to the aganglionic colon but extended proximally, even into the ganglionated small intestine5,15,16. These alterations include changes in ENS neuronal density and neurotransmitter phenotype and may account for dysmotility that has been observed in patients with HSCR. We have utilized the above techniques in our efforts to understand the determinants of ENS formation. Specifically, these techniques have been employed to visu...

A functioning Enteric Nervous System (ENS), which controls motility, nutrient absorption, and local blood flow, is essential to life1. The ENS is formed by neural crest cells (NCC) that proliferate, migrate and colonize the gut, where they differentiate into ganglia containing neurons and glial cells. Hirschsprung&#8217;s Disease (HSCR, Online Mendelian Inheritance in Man), a multigeneic congenital disorder with an incidence of 1 in 4,000 live births, can be considered the prototypic disease for studying disrupted ENS formation. In HSCR, NCC fail to migrate to and colonize variable lengths of the distal hindgut2. Additionally, other common gastrointestinal (GI) developmental defects in the pediatric population, such as anorectal malformations, intestinal atresias, and motility disorders are associated with disturbances in basic ENS functions, and are likely associated with subtle, underappreciated, anatomic changes and functional changes in the ENS3-6. Therefore, techniques that allow us to understand the developmental determinants of ENS formation may shed light on the pathogenesis and potential treatment of pediatric GI tract disorders.
Following migration and colonization, NCC differentiates into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons comprise approximately 60% of enteric neurons7, and can be detected by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for the excitatory neurotransmitter acetylcholine. Historically, attempts to visualize cholinergic neurons were confounded by differing antigen specificity of antibodies directed against central nervous system (CNS) ChAT versus peripheral nervous system (PNS) ChAT8-10. However, antibodies directed against placental ChAT recognize both central and peripheral ChAT11-13, and we have recently described techniques that allow for visualization of ENS cholinergic neurons with high sensitivity earlier in development than has been achieved with ChAT reporter lines14.
Here, we present a technique for dissecting, fixing and immunostaining of the murine embryonic GI tract to visualize ENS neurotransmitter expression in neurons. For these studies, we have utilized ChAT-Cre mice mated with R26R:floxSTOP:tdTomato animals to produce ChAT-Cre;R26R:floxSTOP:tdTomato mice (defined as ChAT-Cre tdTomato throughout the manuscript). These animals were then mated with homozygous ChAT-GFP reporter mice, to obtain mice expressing both fluorescent reporters that detect ChAT expression14. These two reporter animals are on a C57BL/6J background and are commercially available (Jackson Laboratories, Bar Harbor, ME).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Oxoid</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Fisher</td>
			<td>BP9221</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>BP1605</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dishes</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence scope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine science tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech, Birmingham, AL</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>16004-330</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>Nikon A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Elements</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

immunostaining to visualize murine enteric nervous system development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

We have previously described the generation of mice expressing both GFP and tdTomato fluorescent reporters that detect ChAT expression14. Briefly, ChAT-Cre mice were mated with R26R:floxSTOP:tdTomato animals to produce ChAT-Cre;R26R:floxSTOP:tdTomato mice (called ChAT-Cre tdTomato). These animals were then mated with homozygous ChAT-GFP reporter mice. Embryos were isolated and tissues were dissected prior to being fixed and immunostained as above, with the antibodie...

Watch this Scientific Journal Video about Immunostaining to Visualize Murine Enteric Nervous System Development at JoVE.com

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

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