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...

肠神经系统（ENS）是神经和神经胶质细胞，贯穿整个胃肠道（GI）的庞大网络。 ENS消化功能控制的各个方面，包括蠕动，流体吸收/分泌，刺激的感觉， 等 （审查见1）。它包含了超过500万个神经元，发现在脊髓以上，包含了每一个神经递质在大脑中发现的类。此外，ENS是独特的，它的功能会本能地从中枢神经系统2没有输入。 ENS的理解是至关重要的，不仅以了解其正常的生理作用，但了解其参与多种神经病变，它可以是先天性的（先天性巨结肠），收购（美洲锥虫病），继发疾病状态（糖尿病性胃轻瘫），药物诱导（阿片类药物的肠易激综合征），或因损伤（术后肠梗阻）1。此外，肠溶性神经元可以是一个重servoir病毒感染（水痘-带状疱疹）3。由于大脑和高水平的5 -羟色胺在肠道中的相似之处，旨在药物治疗中枢神经系统的缺陷通常有不必要的副作用ENS 2。值得注意的是，许多神经病变，如阿尔茨海默氏症和帕金森氏病，没过多久，他们的出现在中央的神经元在肠道神经元的变化显示出类似的细胞，使ENS访问模型来研究这些疾病的发病机理4。因此，ENS的透彻理解谅解疾病状态和药物的副作用的预防和/或预测的必要性。 ENS的神经元，传统上一直研究在豚鼠使用5-7平片制剂或培养的神经元8。尽管在这个大的动物神经元可以很容易地在研究，这种模式有很大的局限性，包括缺乏转基因的菌株，缺乏这个物种的特定试剂，订购和住房这些学科相关的高昂成本。各种磕出系统，一个庞大的阵列，可用于在细胞培养技术结合其他方法，并且能够提供一个验证豚鼠模型小鼠肠神经系统模型的发展具有得天独厚的优势。 ENS包括三个丛运行的胃肠道的长度：外肠肌丛之间的纵向和环形肌是主要负责对肠道蠕动的行动，以及粘膜下层和粘膜的有机玻璃，（发现下和粘膜内，分别），这在很大程度上控制液体的吸收/分泌和刺激1的检测。这种方法开始与纵肌/肌间神经丛（LMMP）准备的隔离脱皮关闭胃肠道外的肌肉层。这极大地减少了污染问题时，涉及粘膜层中的隔离。其结果是，这个过程是理想的，为研究神经元控制的蠕动而不是分泌行动ENS。 这里介绍的方法在肠道神经元和神经胶质细胞混合培养的结果。至少两种不同的类型的神经元时，根据以往的电生理和免疫细胞化学的观察9。神经胶质细胞的存在是非常有利的，因为它们不仅是一种重要的细胞类型来研究自己的权利，但它们有助于肠道神经元10的生存和保持本地受体表达对神经细胞表面11。此外，肠道的神经胶质细胞上的缺陷可能会导致肠胃蠕动异常的疾病状态，创造“神经gliopathies 12。因此，这里提出ENS文化ŗesults在多种细胞类型，是调查的时机已经成熟。 这种方法的优点是便于隔离，廉价的工具要求，很短的时间掌握技术，由经验丰富的实验室工作人员。该方法的局限性包括低整体细胞产量高组织体积和排除ENS神经粘膜和粘膜下丛。此过程将是非常有利的学者在电生理学，免疫组化，单细胞PCR，和其他方法。

<table border="0" cellpadding="0" cellspacing="0" width="676">
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		<col />
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	</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

肠神经系统是一个庞大的网络运行长度的胃肠道功能控制肠胃蠕动的神经元和神经胶质细胞。混居的肌间神经丛的神经元和神经胶质细胞的分离和培养的过程描述。可维持原代培养7天以上，有连接的发展产生的神经元和神经胶质细胞。从底层的鼠标回肠或结肠环形肌和纵行肌条所附的肌间神经丛剥离酶消化。在无菌条件下，分离的神经元和神经胶质细胞的人口内的颗粒下离心将被保留，并镀上盖玻片。 24-48小时内发生，轴突生长和神经元可以通过泛神经标记。两天后，在文化，孤立的神经元动作电位观察膜片钳研究。此外，肠溶性神经胶质细胞也可以是认同“IED GFAP染色。密切同位语的神经元和神经胶质细胞网络形成于5 - 7天。肠溶性神经元，可单独直接使用的方法，如免疫组织化学，电生理，钙成像，单细胞PCR研究。此外，这种程序，可以在转基因动物中进行。这种方法是执行简单，价格低廉。总体而言，这一协议公开肠神经系统的组件容易被操纵的方式，使我们可以更好地发现ENS在正常和疾病状态的功能。

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.

一个<em在体外</em从成年小鼠的肌间神经丛制备隔离肠道神经元和神经胶质

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

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

26.5K 次观看.  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.

视频: 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.

成人出生的神经元的整合研究

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 ...

科研

神经科学

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

从大鼠脑的解剖脑表面血管及相关脑膜和脉络丛的可视描述，

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

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.

一个孤立的半完整的上下游鼠标前庭上皮细胞电生理和高分辨率的双光子显微镜

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.

一<em&gt;在体外</em&gt;成年小鼠肌肉，神经准备留学肌肉传入放电的放电特性

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.

中脑多巴胺神经元的数目在成年小鼠环境的调变

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.

间接神经元的星形胶质细胞共培养试验：一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

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

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

调整在海马Theta乐队<em&gt;体外</em&gt;：从孤立的啮齿动物皮下注射电路记录的方法

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

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.

从胚胎小鼠小脑分离的神经元的主要培养

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.

成年小鼠中的科利耳表面制备

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