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 the neuronal circuitry of the LMMP preparation 13, which can affect the resulting mix of neurons in the final isolation. Additionally, age should be taken into consideration when using this method, as age-related neuronal losses occur in the myenteric plexus that are specific to cholinergic neurons and their corresponding glia, and can be seen as early as 12 months of age 14. Finally, when adapting this protocol to other species of animals, care should be taken to ensure that the myenteric plexus comes away with the longitudinal muscle rather than remaining attached to the circular muscle.
Tissue Used
Neurons and glia can be cultured from both the ileum and colon using this protocol. Ileal isolations are easier to perform due to a greater amount of available tissue and the ease at which the longitudinal muscle separates from the thick circular muscle. A minimum of two animals are needed to occupy 12 wells of the 24-well plate for applications such as electrophysiology or immunocytochemistry. The isolation of colonic material is more problematic. The thick colonic longitudinal muscle is more difficult to separate from the circular muscle. Furthermore, the underlying circular muscle is thinner in the colon, making it more susceptible to tears. Also, the colon is significantly shorter than the ileum so three animals, at minimum, are required to occupy 12 wells of a 24-well plate. Both the ileum and the colon can be harvested and plated separately from the same animal, but additional animals or the use of less cell plating area will be required for the colon isolation.
Duration of Cell Culture
Electrophysiological recordings are optimally performed in isolated enteric neurons/glia after 2 to 6 days in culture; at this time cells are rounded and pliant, ideal conditions for making a whole-cell seal. After a week in culture cells become flatter and more visually differentiated as the cells adhere to the plate. Immunocytochemical experiments are best done when cells are more firmly attached to the plate, around day 10, to prevent cell loss during numerous wash steps. Mouse enteric cells have been maintained in culture for up to three weeks. Patterns of receptor and neurotransmitter expression over time have yet to be studied in these cultures. However, previous studies on neuronal survival and biochemical/morphological differentiation in traditional guinea pig enteric neuron isolations have found that neurons survived up to 15 days and maintain a high degree of histochemical and morphological properties up to three weeks 8, which suggests that neurons derived from other animals sources may do the same.
Cell Yield
The overall yield of cells from this technique is 12 wells of a 24-well plate from two mice when isolating from the ileum. This is a relatively low number of neurons compared to tissue volume, as the myenteric plexus consists of less than 1% of the total gut wall. One drawback to this method is that to increase the number of cells obtained, one must increase the number of animals used. When animal numbers are increased, it is suggested that several lab members collaborate in the first step of the isolation of the LMMP. This reduces the amount of time the cells are in a disrupted state before final plating and this increases cell survival.
Cell Density
As written, this protocol uses two animals to yield an ileal isolation of low-density cell confluence (~10 - 40% measured after one day in culture). Low-density cell plating is ideal in this methodology to reduce the risk of contamination in the final culture. It is also useful when single cell electrophysiology is performed. Cell number can be increased by using more animals or reducing the final plating area, but risk of contamination increases. To off-set this problem when higher cell densities are required, either increased washing steps or the increased use of antibiotics is suggested.
Cell Heterogeneity
Neurons acquired using these protocols are comprised of at least two functionally distinct populations, as indicated by electrophysiological characterization (Figure 3). However, there are many known types of neurons with specific neurochemical coding patterns found in the guinea pig 15 and the same is probably true of the mouse. This cell heterogeneity is both an advantage and disadvantage to this methodology. Cell heterogeneity is beneficial when performing single-cell functional studies, observing cell-cell interactions or in immunocytochemistry, among others. Cell heterogeneity may be particularly advantageous when studying neuron/glia interactions in culture (Figure 3). However, cell heterogeneity is a hindrance to methodologies such as immunoblotting, where changes in protein expression, etc., cannot be contributed to any one cell type or neuron type. Examination of glia will be less problematic as only two subtypes are shown to exist in the LMMP preparation: intraganglionic and intramuscular enteric glia 16. Other cell types may also be present in this culture, such as interstitial cells of Cajal or fibroblasts.
Plating Cells
In this methodology, cells are plated in twelve wells. Occasionally, one or two of these wells will develop contamination in the first day after isolation. If this occurs, the slides from the contaminated wells are promptly discarded and the well is rinsed with 70% ethanol to kill the remaining contamination. This protocol can be modified to plate all of the isolated cells in fewer wells or in a single dish, and as a result, the risk of contamination will increase. Again, when the risks of contamination increase, extra wash steps or increased antibiotic use is suggested. The antibiotic/antimycotic liquid (Gibco) used in the protocol consists of Amphotericin B, Streptomycin, and Penicillin. Other antibiotic combinations or increased concentrations can be used to optimize the reduction of contamination while preparing the rinse and complete neuron media.
Attaching Cells
Cells are plated on coverslips coated with laminin and poly-D-lysine. Laminin is important in the survival of enteric neurons and glia, and encourages neurite outgrowth and neuronal development 17, and as such, is considered essential to this isolation. However, alternative attachment factors to poly-D-lysine, such as ornithine or Matrigel, may be tried as desired.
In conclusion, this isolation provides a mixed culture of enteric neurons and glia suitable for a wide range of study techniques. This methodology is easy to master, inexpensive, and time efficient. It is our hope that this cell model will contribute to the understanding of various pathologies associated with the ENS.

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。

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			<td style="width:79px;">Reagents</td>
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		<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>
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			<td style="width:79px;"> </td>
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		<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>
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		<tr height="43">
			<td height="43" style="height:43px;width:291px;">24-well cell culture plate</td>
			<td style="width:171px;">CELLTREAT</td>
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			<td style="width:79px;">May use any brand</td>
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			<td height="43" style="height:43px;width:291px;">15 ml Sterile Centrifuge Tube</td>
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			<td style="width:79px;">MW 120.4</td>
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			<td style="width:79px;"> </td>
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			<td style="width:137px;">110-001-101HI</td>
			<td style="width:79px;">May use any brand</td>
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			<td style="width:137px;">25030164</td>
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			<td style="width:137px;">8940</td>
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			<td height="43" style="height:43px;width:291px;">2 ml Microcentrifuge Eppendorf tubes</td>
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			<td style="width:137px;">13-864-252</td>
			<td style="width:79px;">May use any brand</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:291px;">Nitrex Mesh 500 &#181;M</td>
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			<td style="width:137px;">100560</td>
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			<td height="43" style="height:43px;width:291px;">Pipette Set</td>
			<td style="width:171px;">Fisher Scientific</td>
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			<td height="43" style="height:43px;width:291px;">Sharpeining Stone</td>
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		<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>
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		<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>
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		<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>
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	</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 will begin to show neurite outgrowth. Specific identification of neurons may still be indistinct at this time. Provided the cells are adherent enough to transfer to an experimental chamber, they will be ready for electrophysiological study after one day of culture. However, the cells are more adherent after two days in culture and ideal for function studies (electrophysiology, calcium imaging) from approximately days 2 - 5.
Morphological features of neuron become distinct after approximately a week in culture (Figure 1). Ideal immunocyctochemical features can be identified after about ten days, when neurons display long projections and grow in an almost 'ganglionic' like fashion interspersed with glia (Figure 2). This staining suggests it may be possible to study the synaptic interactions of these cells using this methodology.
After one day in culture, neurons are recognized by their sine que non or 'defining feature', the action potential (Figure 3). Electrophysiologically, they can be functionally classified into at least two neuronal types: neurons that contain an after-hyperpolarization and increased current/density relationships of sodium and potassium and neurons that do not have an after-hyperpolarization and significantly less sodium and potassium conductance (Figure 4). AHP positive and negative neurons appear to correlate with AH and S neurons seen in previous guinea work, respectively. AHP positive neurons (AH neurons) have multiple long projections originating from around the cell body, while the AHP negative neurons (S neurons) have one long projection which branches many times. This, in conjunction with the immunocytochemical coding in Figure 1, suggests that neuronal population is very heterogeneous.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50688/50688fig1highres.jpg" src="/files/ftp_upload/50688/50688fig1.jpg" /> 
 Figure 1. Immunohistochemical characterization of enteric neurons and glia isolated from the mouse longitudinal muscle. Confocal microscopy revealed neuronal-specific &beta;-III-tubulin (Abcam, rabbit, 1:1,000) staining in whole mount ileal longitudinal muscle (A) preparation from the mouse. Cells isolated from longitudinal muscle/myenteric plexus (LMMP) preparations contain neurons (B; &beta;-III-tubulin, Abcam, rabbit, 1:1,000) that stain positively for calbindin (C) (Chemicon, rabbit, 1:1,000) and calretinin (D) (Swant, rabbit, 1:2,000). Glia cells (E) were visualized with the glia-specific marker GFAP (Chemicon, mouse, 1:500). Antibodies were visualized via appropriate goat secondary antibody Alexa 488 (green, Molecular Probes, 1:1,000)0. Nuclei were visualized using Hoescht 33342 (blue, C-G, 1 &mu;g/ml). No staining was seen when primary antibody was omitted (F). Modified and reprinted from Smith, T.H., et al. Morphine Decreases Enteric Neuron Excitability via Inhibition of Sodium Channels. PLoS One., doi:10.1371/journal.pone.0045251.g001 (2012). <a href="https://www.jove.com/files/ftp_upload/50688/50688fig1large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50688/50688fig2highres.jpg" src="/files/ftp_upload/50688/50688fig2.jpg" /> 
 Figure 2. Neurons and glia isolated from the mouse longitudinal muscle grow in close proximity to one another. Confocal microscopy images indicate the neurons (green, &beta;-III-tubulin, Abcam, rabbit, 1:1,000) and glia (red, GFAP, Chemicon, mouse, 1:500) readily grow adjacent to one another and appear to interact in vitro. <a href="https://www.jove.com/files/ftp_upload/50688/50688fig2large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50688/50688fig3highres.jpg" src="/files/ftp_upload/50688/50688fig3.jpg" /> 
 Figure 3. Electrophysiology of cultured enteric neurons and glia. In current clamp mode, all neurons (A) displayed action potentials upon current injection. Glia (B) do not have action potentials but do display large electrotonic potentials in response to current injection. Protocols in A &amp; B start with a current injection of -0.01 nA and increase to 0.09 nA in eleven 0.01 nA steps. <a href="https://www.jove.com/files/ftp_upload/50688/50688fig3large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/50688/50688fig4highres.jpg" src="/files/ftp_upload/50688/50688fig4.jpg" /> 
 Figure 4. Neurons cultured from the mouse ileum are an electrophysiologically heterogeneous population. In current clamp mode, a current injection of 0.09 nA into neurons results in action potentials. S neurons (A), immediately return to the resting membrane potential following stimulation. AH-type neurons (B) display an afterhyperpolarization (AHP) following stimulation in which the resting membrane potential falls below baseline before slowly returning to the initial value. Modified and reprinted from Smith, T.H., et al. Morphine Decreases Enteric Neuron Excitability via Inhibition of Sodium Channels. PLoS One., doi:10.1371/journal.pone.0045251.g001 (2012).

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 ...

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Universidad San Sebastian

Research

JoVE Journal

Neuroscience

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

26.7K 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

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.

鼠标感觉神经支配的皮肤和四肢骨骼纤维组织的制备及免疫染色

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.

从人类多能干细胞的规范在前脑谷氨酸能神经元

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.

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 ...

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.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

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.

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 ...

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