National Institute of Health Grant DA024009, DK046367 &#38; T32DA007027.

All animal care and experimental procedures were in accordance with and approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University.
1. Preparation of Sterile Poly-D-Lysine- and Laminin-Coated Glass Coverslips in 24-Well Plates
<ol>
	<li>All procedures for step 1 are performed in sterile conditions; under a hood, and with sterile reagents. Glass coverslips and double deionized water (ddH2O) should be sterilized in advance. Preparation of plates can be done up to two weeks before neuron isolation.</li>
	<li>Under the hood, use sterile forceps to place autoclaved glass coverslips in 12 wells of a 24-well plate.</li>
	<li>Prepare poly-D-lysine stock ahead of time. Add 50 ml of sterile ddH2O to 5 mg poly-D-lysine. Vortex and store in 3 ml aliquots at -20 &deg;C. Thaw aliquots before use.</li>
	<li>For a final concentration of 1 ml poly-D-lysine stock per 25 cm2: pipette 80 &mu;l of poly-D-lysine stock on top of each glass coverslip (2 cm2). Let solution settle for 10 min.</li>
	<li>Remove poly-D-lysine using vacuum and rinse coverslips 3x with sterile ddH2O.</li>
	<li>Allow plates to dry for at least 2 hr under the hood.</li>
	<li>Plates may be stored at 4 &deg;C or - 20 &deg;C before proceeding to laminin coating.</li>
	<li>Prepare laminin stock ahead of time. Thaw laminin on ice and keep on ice during use. Dilute to a concentration of 50 &mu;g/ml; pay attention to lot concentration; each lot will be different. Depending on lot concentration, approximately 20 ml ddH2O will be added to each vial of laminin. Aliquot (5 ml) and store at -80 &deg;C. Thaw aliquots on ice before use.</li>
	<li>Coat coverslips with 5 &mu;g/cm2 laminin; pipette 200 &mu;l of laminin stock on each coverslip.</li>
	<li>Incubate laminin solution on coverslips for 1 hr.</li>
	<li>Aspirate remaining laminin solution and rinse coverslips once with ddH2O. Avoid scraping surface of coverslips.</li>
	<li>Store plates at 4 &deg;C for up to two weeks.</li>
</ol>
2. Advance Preparation of Neuron Isolation Solutions
<ol>
	<li>Prepare Krebs solution (in mM: 118 NaCl, 4.6 KCl, 1.3 NaH2PO4, 1.2 MgSO4, 25 NaHCO3, 11 glucose, and 2.5 CaCl2). Place 13.79 g NaCl (FW 58.44), 0.686 g KCl (FW 74.55), 0.312 g NaH2PO4 (FW 120), .289 g MgSO4 (FW 120.4), 4.20 g NaHCO3 (FW 84.01), 3.96 g glucose (FW 180.2), and 0.555 g CaCl2 (FW 111) in 2 L ddH20. Chill to 4 &deg;C.</li>
	<li>Prepare rinse media (F12 media with 10% fetal bovine serum (FBS) and antibiotic/antimycotic): To a 500 ml bottle of F12 media, add 50 ml FBS and 5 ml of Antibiotic/Antimycotic 100x Liquid (Gibco).</li>
	<li>Prepare enteric neuron media (Neurobasal A media with B-27, 2 mM L-glutamine, 1% FBS, 10 ng/ml, Glial Derived Neurotrophic Factor (GDNF), and Antibiotic/Antimycotic 100x Liquid). To make GDNF stock, add 1 ml ddH2O to 10 &mu;g GDNF and freeze back in 50 &mu;l aliquots, store at -80 &deg;C. For a 50 ml vial of complete neuron media, combine 47.5 ml Neurobasal A media, 1 ml B-27 (50x), 500 &mu;l FBS, 500 &mu;l 200 mM L-glutamine (Gibco) 50 &mu;l GDNF stock, and 500 &mu;l Antibiotic/Antimycotic 100x Liquid. Media is made in small 50 ml batches to ensure freshness of GDNF and to avoid contamination of large amounts of this expensive cell media mixture.</li>
</ol>
3. Harvest Longitudinal Muscle/Myenteric Plexus (LMMP) Preparation from Mice
<ol>
	<li>Appropriate national and institutional ethics should be in place before performing animal experiments. Adult Swiss Webster mice weighing over 25 g (typically over 8 weeks of age) are housed in groups of 6 prior to experiments. Tissues from two mice are used for the isolation of ileal enteric neurons and glia and three mice are used for isolation of colonic cells.</li>
	<li>Prepare surgical area. Gather small surgical scissors, angled forceps, cotton swabs, three 200 ml glass beakers, and a glass/plastic rod (paintbrush). Ensure all supplies are thoroughly washed and rinsed before use to minimize contamination. This step can be performed the day before the experiment.</li>
	<li>Place Krebs solution on ice and bubble with carbogen (95% oxygen, 5% CO2) for at least 30 min to stabilize pH.</li>
	<li>Turn on various equipment and warm chemicals to stabilize at desired temperatures; heat water bath(s) to 37 &deg;C, cool centrifuge to 4 &deg;C, place Hank's balanced salt solution (HBSS) and 0.5% trypsin in water bath (37 &deg;C). Complete neuron media and rinse media should remain refrigerated.</li>
	<li>Place 150 ml ice cold bubbled Krebs in three 200 ml glass beakers labeled 'dirty', 'clean', and 'LMMP'. Bubble beaker labeled 'LMMP' with carbogen.</li>
	<li>Following approval from ethics committee, euthanize mouse by cervical dislocation or CO2 asphyxiation.</li>
	<li>Place mouse in dorsal recumbence on surgical surface, clean skin with 70% EtOH, and lift abdominal skin using forceps. Using scissors, open abdominal cavity and reveal internal digestive organs.</li>
	<li>Remove the length of the gastrointestinal tract by lifting a section of ileum and revealing its mesentery. Snip through mesentery with scissors to gently remove and unravel ileum and colon, being careful not to pull mesentery from the ileum/colon.</li>
	<li>After the full length of intestine is unraveled, remove the ileum by cutting through the intestine distal to the stomach and proximal to the cecum. Note: this procedure can also be performed with colonic tissue by removing the colon from distal to the cecum to proximal to the anus.</li>
	<li>Tissues can be further divided between proximal and distal colon, and jejenum, and ileum. The rest of this procedure will refer to isolation of the ileum; the procedure is the same for the isolation of colon LMMP. Place the entire ileum in a glass container with ice cold Krebs marked 'dirty'.</li>
	<li>Create a tool to clean the ileum; blunt a large 20 G needle using a filing stone and attach it to a large syringe (10 ml) containing ice cold Krebs.</li>
	<li>Clean fecal matter from the ileum by sectioning the ileum into three or more large pieces. Remove an ileal section from the beaker and place the blunted needle into the end of an ileal piece.</li>
	<li>Gently run Krebs through the gut section until all fecal matter is removed into a separate waste container. Place the cleaned section into the container of Krebs marked 'clean'. Repeat until entire ileum is cleaned.</li>
	<li>To remove the LMMP, cut the ileum into small segments, approximately 2 - 4 cm. Place a segment of ileum on a plastic or glass rod; ileum should fit snuggly but not be loose or taut (~2 mm). Begin removal of the LMMP by gently removing bits of mesentery still attached to the gastrointestinal (GI) tract using a forceps. Prevent the GI tract from rotating around rod by gently pinning the tube to the rod using the thumb.</li>
	<li>Separate the LMMP from the underlying circular muscle; first gently rub the edge of the forceps along the entire line where the mesentery was attached, from top to bottom of the segment, gently creating a gap in the longitudinal muscle. Then gently tease away the longitudinal muscle using a cotton swab wetted with Krebs.</li>
	<li>Begin at the top of the gap in the longitudinal muscle and tease away using the lightest horizontal stroke while applying very light pressure until the longitudinal muscle just begins to separate from the circular muscle; do this down the entire strip along the mesentery attachment point.</li>
	<li>Then gently begin to work around the GI tube; moving from top to bottom and back as the longitudinal muscle is slowly separated from the circular muscle all the way around the tube. When complete, the LMMP will naturally come off the remainder of the GI tube.</li>
	<li>Place the resulting thin strip of longitudinal muscle in the beaker marked 'LMMP'. Repeat for all the segments.</li>
	<li>After all the strips of LMMP have been gathered from one mouse, repeat procedure for any other mice being used. Thoroughly rinse the 'dirty' and 'clean' beakers before re-use.</li>
	<li>Rinse the LMMP 3x to remove biological contamination. Fill three 2 ml Eppendorf tubes with ice cold Krebs. Place the LMMP strips in the first tube and spin for 30 sec at 356 x g in a centrifuge cooled to 4 &deg;C. Carefully remove the supernatant with a pipette and move the LMMP strips to next clean Krebs filled tube. Repeat this procedure for each remaining tube.</li>
</ol>
4. Digest LMMP
<ol>
	<li>Prepare digestion solution by placing 13 mg collagenase type 2 and 3 mg BSA in 10 ml carbogen-bubbled Krebs solution.</li>
	<li>Place segments of rinsed LMMP into digestion solution and use scissors to snip LMMP into tiny pieces.</li>
	<li>Digest LMMP for 60 min at 37 &deg;C in water bath with shaker while being gently bubbled with carbogen.</li>
	<li>After digestion is complete, gather cells by centrifugation for 8 min at 356 x g in centrifuge cooled to 4 &deg; C.</li>
	<li>From this point forward all procedures should be done under sterile conditions in a cell culture hood! All reagents and containers should be sterilized before use.</li>
	<li>During centrifugation, prepare 0.05% trypsin solution by placing 1 ml warmed 0.25% trypsin and 4 ml warmed HBSS into a sterile 50 ml cell culture tube.</li>
	<li>After centrifugation, remove and discard supernatant. Do not use a vacuum; this will likely suck up the cell pellet due to low centrifugation speeds. Carefully remove the cell pellet and place into clean tube with 5 ml of 0.05% trypsin solution.</li>
	<li>Digest cells in 0.05% trypsin solution in 37 &deg;C water bath with shaking for 7 min. Do not exceed 7 min of total trypsin treatment or neurons will perish.</li>
	<li>Neutralize trypsin with 10 ml of cold rinse media after 7 min of trypsin treatment.</li>
	<li>Centrifuge cells for 8 min at 356 x g. Remove and discard media.</li>
	<li>During centrifugation, balance a section of sterilized Nitex mesh on top of a sterile 15 ml cell culture tube.</li>
	<li>After centrifugation, remove and discard supernatant. Gently resuspend cell mixture by triturating in 3 ml complete neuron media. Avoid generating air bubbles during this step and all future steps in which cell mixture is triturated. All triturations should be done very gently. Filter cell solution through Nitex mesh into clean 15 ml cell culture tube.</li>
	<li>Optional: Cap cell culture tube and place in refrigerated Hula mixer (or any mixer that provides rotation) with gentle rolling and tilting for 30 min. This step is optional but recommended.</li>
	<li>Optional: After Hula mixer, add 2 ml cold rinse media to wash cells.</li>
	<li>Gather cells by centrifugation (8 min, 356 x g). Remove and discard supernatant.</li>
	<li>Resuspend cells in 1,200 &mu;l complete neuron media. Triturate cells gently using 1 ml plastic pipette tip. Do not generate air bubbles. Pipette slowly and gently until most chunks are broken up and cells are suspended into liquid.</li>
	<li>Add 750 &mu;l of complete media to each of the 12 wells containing a pre-coated glass coverslip and add 100 &mu;l of the triturated cell solution.</li>
	<li>Incubate neurons in cell culture incubator at 37 &deg;C with 5% CO2.</li>
	<li>Change half of the cell media every 2 days.</li>
	<li>Neurons are ready for electrophysiological recordings after 1 - 2 days in culture.</li>
</ol>

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The authors declare that no major competing interests exist.

Animals Used
This protocol has been optimized for Swiss Webster mice. However, this method is easily adaptable to other small-sized mammals such as rats and to other strains of mice. We have successfully performed preliminary isolations with C57 mice and &mu;-opioid receptor knock-outs. However, it is also possible that other strains of mice may be problematic due to morphological variations in the GI tract. Furthermore, there are known differences between mouse strains (C57Bl/6 vs. Balb/c) in th...

The enteric nervous system (ENS) is vast network of nerves and glia that runs the entire length of the gastrointestinal (GI) tract. The ENS functionally controls all aspects of digestion, including peristalsis, fluid absorption/secretion, sensation of stimuli, etc (for review see 1). It contains over 500 million neurons, more than found in the spinal cord, and contains every neurotransmitter class found in the brain. Furthermore, the ENS is unique in that it can function reflexively without input from the central nervous system 2. Understanding of the ENS is crucial, not only to understand its normal physiological role, but to understand its involvement in a variety of neuropathies which can be congenital (Hirschsprung's disease), acquired (Chagas), secondary to disease states (diabetic gastroparesis), drug-induced (Opioid bowel syndrome), or due to injury (postoperative ileus) 1. In addition, enteric neurons can be a reservoir for viral infection (varicella zoster)3. Because of its similarities to the brain and the high levels of serotonin in the gut, medications aimed at treating central nervous system defects often have unwanted side effects on the ENS 2. It is also noteworthy that many neuropathies such as Alzheimer's disease and Parkinson's disease show similar cellular changes in the enteric neurons long before their appearance in central neurons, making the ENS an accessible model to study the pathogenesis of these diseases 4. Therefore, a thorough understanding of the ENS is a necessity in understanding disease states and preventing/predicting pharmacological side effects.
The neurons of the ENS have been traditionally studied in the guinea pig using wholemount preparations 5-7 or cultured neurons 8. Despite the ease at which neurons can be studied in this large animal, this model has many limitations including lack of genetically modified strains, lack of reagents specific to this species, and the high cost associated with ordering and housing these subjects. The development of a murine enteric nervous system model has the unique advantage of various knock out systems, a vast array of other established methodologies that can be used in conjunction with the cell culture technique, and the ability to provide a validation for the guinea pig model.
The ENS is comprised of three plexi that run the length of the gastrointestinal tract: the outer myenteric plexus (between the longitudinal and circular muscle) which is mainly responsible for the peristaltic actions of the gut, as well as the submucosal and mucosal plexi, (found under and within the mucosa, respectively) which largely controls fluid absorption/secretion and the detection of stimuli 1. This method begins with the isolation of the longitudinal muscle/ myenteric plexus (LMMP) preparation by peeling off the outer muscle layer of the GI tract. This dramatically cuts down on contamination issues that arise when the mucosal layer is involved in the isolation. As a result, this process is ideal for the study of neuronal control of motility rather than secretory actions of the ENS.
The method presented here results in a mixed culture of enteric neurons and glia. At least two different types of neurons are present based on previous electrophysiological and immunocytochemical observations 9. The presence of glia is highly advantageous, as they are not only an important cell type to study in their own right, but they contribute to the survival of the enteric neurons 10 and maintain native receptor expression on the neuronal cell surface 11. Furthermore, deficiencies of enteric glia may lead to abnormal gastrointestinal motility disease states, coined 'neuro-gliopathies' 12. Therefore, the ENS culture presented here results in several cell types that are ripe for investigation.
The advantages to this methodology are ease of isolation, inexpensive tool requirements, and a short time to master the technique by experienced lab personnel. Limitations of the methodology include low overall cell yield from high tissue volumes and the exclusion of ENS neurons from mucosal and submucosal plexi. This procedure will be highly advantageous to scholars specializing in electrophysiology, immunohistochemistry, single-cell PCR, and other methodologies.

<table border="0" cellpadding="0" cellspacing="0" width="676">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;"></td>
			<td style="width:171px;"></td>
			<td style="width:137px;"></td>
			<td style="width:79px;">Reagents</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Fisherbrand Coverglass for Growth Cover Glasses (12 mm diameter)</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">12-545-82</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Poly-D-lysine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:137px;">P6407- 5 mg</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">24-well cell culture plate</td>
			<td style="width:171px;">CELLTREAT</td>
			<td style="width:137px;">229124</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Laminin</td>
			<td style="width:171px;">BD Biosciences</td>
			<td style="width:137px;">354 232</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:291px;">ddH2O</td>
			<td style="width:171px;"> </td>
			<td style="width:137px;"> </td>
			<td style="width:79px;">Can prepare in lab </td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">15 ml Sterile Centrifuge Tube</td>
			<td style="width:171px;">Greiner Bio-one</td>
			<td style="width:137px;">188261</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">50 ml Sterile Centrifuge Tube</td>
			<td style="width:171px;">Greiner Bio-one</td>
			<td style="width:137px;">227261</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">NaCl</td>
			<td style="width:171px;">Fisher BioReagents</td>
			<td style="width:137px;">BP358-212</td>
			<td style="width:79px;">MW 58.44</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">KCl</td>
			<td style="width:171px;">Fisher BioReagents</td>
			<td style="width:137px;">BP366-500</td>
			<td style="width:79px;">MW 74.55</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:291px;">NaH2PO4 .2H2O</td>
			<td style="width:171px;">Fisher Chemicals</td>
			<td style="width:137px;">S369-3</td>
			<td style="width:79px;">MW137.99</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:291px;">MgSO4</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:137px;">M7506-500G</td>
			<td style="width:79px;">MW 120.4</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:291px;">NaHCO3</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:137px;">S6014-5KG</td>
			<td style="width:79px;">MW 84.01</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">glucose</td>
			<td style="width:171px;">Fisher Chemicals</td>
			<td style="width:137px;">D16-1</td>
			<td style="width:79px;">MW 180.16</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">CaCl22H2O</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:137px;">C5080-500G</td>
			<td style="width:79px;">MW 147.02</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">F12 media</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:137px;">11330</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Fetal Bovine Serum</td>
			<td style="width:171px;">Quality Biological Inc.</td>
			<td style="width:137px;">110-001-101HI</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Antibiotic/antimycotic 100x liquid</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:137px;">15240-062</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Neurobasal A media</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:137px;">10888</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">200 mM L-glutamine</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:137px;">25030164</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Glial Derived Neurotrophic Factor (GDNF)</td>
			<td style="width:171px;">Neuromics</td>
			<td style="width:137px;">PR27022</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Sharp-Pointed Dissecting Scissors</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">8940</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Dissecting Tissue Forceps</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">13-812-41</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Cotton-Tipped Applicators</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">23-400-101</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">250 ml Graduated Glass Beaker</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">FB-100-250</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">2 L Glass Erlenmyer flask</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">FB-500-2000</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Plastic rod (child&#39;s paint brush)</td>
			<td style="width:171px;">Crayola</td>
			<td style="width:137px;">05 3516</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:291px;">Carbogen</td>
			<td style="width:171px;">Airgas</td>
			<td style="width:137px;">UN 3156</td>
			<td style="width:79px;">5% CO2</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">10 ml Leur-lock Syringe</td>
			<td style="width:171px;">Becton Dickinson</td>
			<td style="width:137px;">309604</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">21 G x 1 1/2 in. Hypodermic Needle</td>
			<td style="width:171px;">Becton Dickinson</td>
			<td style="width:137px;">305167</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Collagenase type 2</td>
			<td style="width:171px;">Worthington</td>
			<td style="width:137px;">LS004174</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="63">
			<td height="84" rowspan="2" style="height:84px;width:291px;">Bovine Serum Albumin</td>
			<td rowspan="2" style="width:171px;">American Bioanalytical</td>
			<td rowspan="2" style="width:137px;">AB00440</td>
			<td rowspan="2" style="width:79px;"> </td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">2 ml Microcentrifuge Eppendorf tubes</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">13-864-252</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Nitrex Mesh 500 &#181;M</td>
			<td style="width:171px;">Elko Filtering Co</td>
			<td style="width:137px;">100560</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Pipette Set</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">21-377-328</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Sharpeining Stone</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:137px;">NC9681212</td>
			<td style="width:79px;">May use any brand</td>
		</tr>
	</tbody>
</table>
<table border="0" cellpadding="0" cellspacing="0" width="722">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;"></td>
			<td style="width:108px;"></td>
			<td style="width:137px;"></td>
			<td style="width:195px;">Equipment</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:283px;">LabGard ES 425 Biological Safety Cabinet (cell culture hood)</td>
			<td style="width:108px;">Nuaire</td>
			<td style="width:137px;">NU-425-400</td>
			<td style="width:195px;">May use any brand</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">10 L Shaking Waterbath</td>
			<td style="width:108px;">Edvotek</td>
			<td style="width:137px;">5027</td>
			<td style="width:195px;">May use any brand</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:283px;">Microcentrifuge 5417R</td>
			<td style="width:108px;">Eppendorf</td>
			<td style="width:137px;">5417R</td>
			<td style="width:195px;">May use a single larger centrifuge with size adapters</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:283px;">Allegra 6 Series Centrifuge</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:137px;">366816</td>
			<td style="width:195px;">May use any brand</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">HuluMixer Sample Mixer</td>
			<td style="width:108px;">Invitrogen</td>
			<td style="width:137px;">15920D</td>
			<td style="width:195px;"> </td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">AutoFlow Water Jacket CO2 Incubator</td>
			<td style="width:108px;">Nuiare</td>
			<td style="width:137px;">NU-4750</td>
			<td style="width:195px;">May use any brand</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">Analytical Balance Scale</td>
			<td style="width:108px;">Mettler Toledo</td>
			<td style="width:137px;">XS104</td>
			<td style="width:195px;">May use any brand</td>
		</tr>
	</tbody>
</table>

an in-vitro preparation of isolated enteric neurons and glia from the myenteric plexus of the adult mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

Immediately following isolation of LMMP-derived cells, neurons and other cell types will not be readily evident. Living, round cells of indistinct phenotype can be seen as well as tissue detritus from incompletely digested tissue fragments and connective tissue. This flotsam is of no concern and will be largely removed with the first media change in two days. Do not attempt to clean the slides before this as the healthy, viable cells will be removed as well.
After one day in culture, neurons w...

Watch this Scientific Journal Video about An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse at JoVE.com

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

an-vitro-preparation-isolated-enteric-neurons-glia-from-myenteric

Research

JoVE Journal

Neuroscience

26.7K Views.  Virginia Commonwealth University. We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Video: An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

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Studying the Integration of Adult-born Neurons

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

Cochlear Surface Preparation in the Adult Mouse