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

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

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The authors declare that no major competing interests exist.

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

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

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>Fisherbrand Coverglass for Growth Cover Glasses (12 mm diameter)</td><td>Fisher Scientific</td><td>12-545-82</td><td>&amp;nbsp;</td></tr><tr><td>Poly-D-lysine</td><td>Sigma</td><td>P6407- 5 mg</td><td>&amp;nbsp;</td></tr><tr><td>24-well cell culture plate</td><td>CELLTREAT</td><td>229124</td><td>May use any brand</td></tr><tr><td>Laminin</td><td>BD Biosciences</td><td>354 232</td><td>&amp;nbsp;</td></tr><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>Can prepare in lab&amp;nbsp;</td></tr><tr><td>15 ml Sterile Centrifuge Tube</td><td>Greiner Bio-one</td><td>188261</td><td>May use any brand</td></tr><tr><td>50 ml Sterile Centrifuge Tube</td><td>Greiner Bio-one</td><td>227261</td><td>May use any brand</td></tr><tr><td>NaCl</td><td>Fisher BioReagents</td><td>BP358-212</td><td>MW 58.44</td></tr><tr><td>KCl</td><td>Fisher BioReagents</td><td>BP366-500</td><td>MW 74.55</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Chemicals</td><td>S369-3</td><td>MW137.99</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M7506-500G</td><td>MW 120.4</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>S6014-5KG</td><td>MW 84.01</td></tr><tr><td>glucose</td><td>Fisher Chemicals</td><td>D16-1</td><td>MW 180.16</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>C5080-500G</td><td>MW 147.02</td></tr><tr><td>F12 media</td><td>Gibco</td><td>11330</td><td>&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum</td><td>Quality Biological Inc.</td><td>110-001-101HI</td><td>May use any brand</td></tr><tr><td>Antibiotic/antimycotic 100x liquid</td><td>Gibco</td><td>15240-062</td><td>&amp;nbsp;</td></tr><tr><td>Neurobasal A media</td><td>Gibco</td><td>10888</td><td>&amp;nbsp;</td></tr><tr><td>200 mM L-glutamine</td><td>Gibco</td><td>25030164</td><td>&amp;nbsp;</td></tr><tr><td>Glial Derived Neurotrophic Factor (GDNF)</td><td>Neuromics</td><td>PR27022</td><td>&amp;nbsp;</td></tr><tr><td>Sharp-Pointed Dissecting Scissors</td><td>Fisher Scientific</td><td>8940</td><td>May use any brand</td></tr><tr><td>Dissecting Tissue Forceps</td><td>Fisher Scientific</td><td>13-812-41</td><td>May use any brand</td></tr><tr><td>Cotton-Tipped Applicators</td><td>Fisher Scientific</td><td>23-400-101</td><td>May use any brand</td></tr><tr><td>250 ml Graduated Glass Beaker</td><td>Fisher Scientific</td><td>FB-100-250</td><td>May use any brand</td></tr><tr><td>2 L Glass Erlenmyer flask</td><td>Fisher Scientific</td><td>FB-500-2000</td><td>May use any brand</td></tr><tr><td>Plastic rod (child&#039;s paint brush)</td><td>Crayola</td><td>05 3516</td><td>May use any brand</td></tr><tr><td>Carbogen</td><td>Airgas</td><td>UN 3156</td><td>5% CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>10 ml Leur-lock Syringe</td><td>Becton Dickinson</td><td>309604</td><td>May use any brand</td></tr><tr><td>21 G x 1 1/2 in. Hypodermic Needle</td><td>Becton Dickinson</td><td>305167</td><td>May use any brand</td></tr><tr><td>Collagenase type 2</td><td>Worthington</td><td>LS004174</td><td>&amp;nbsp;</td></tr><tr><td>Bovine Serum Albumin</td><td>American Bioanalytical</td><td>AB00440</td><td>&amp;nbsp;</td></tr><tr><td>2 ml Microcentrifuge Eppendorf tubes</td><td>Fisher Scientific</td><td>13-864-252</td><td>May use any brand</td></tr><tr><td>Nitrex Mesh 500 &amp;micro;M</td><td>Elko Filtering Co</td><td>100560</td><td>May use any brand</td></tr><tr><td>Pipette Set</td><td>Fisher Scientific</td><td>21-377-328</td><td>May use any brand</td></tr><tr><td>Sharpeining Stone</td><td>Fisher Scientific</td><td>NC9681212</td><td>May use any brand</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>LabGard ES 425 Biological Safety Cabinet (cell culture hood)</td><td>Nuaire</td><td>NU-425-400</td><td>May use any brand</td></tr><tr><td>10 L Shaking Waterbath</td><td>Edvotek</td><td>5027</td><td>May use any brand</td></tr><tr><td>Microcentrifuge 5417R</td><td>Eppendorf</td><td>5417R</td><td>May use a single larger centrifuge with size adapters</td></tr><tr><td>Allegra 6 Series Centrifuge</td><td>Beckman Coulter</td><td>366816</td><td>May use any brand</td></tr><tr><td>HuluMixer Sample Mixer</td><td>Invitrogen</td><td>15920D</td><td>&amp;nbsp;</td></tr><tr><td>AutoFlow Water Jacket CO2 Incubator</td><td>Nuiare</td><td>NU-4750</td><td>May use any brand</td></tr><tr><td>Analytical Balance Scale</td><td>Mettler Toledo</td><td>XS104</td><td>May use any brand</td></tr></tbody></table>

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

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

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

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

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

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

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

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

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

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Immunology and Infection

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Embryonic Mouse Brain Mixed Cell Cultures to Study Infection and Innate Immunity

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

Author Spotlight: Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

Efficient Extraction of Astrocytes and Microglia from the Adult Mouse Spinal Cord

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

Author Spotlight: Isolation of Glial Cells from the Adult Mouse Spinal Cord 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

Biology

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Source: Zhou, L., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/60347">Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing.</a> J. Vis. Exp. (2019)
The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord

Source: Malon, J. T., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54801">Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for isolating and culturing primary mixed glial cells from adult mouse spinal cord tissue. The protocol involves enzymatic digestion of the tissue, followed by the removal of broken cell membranes and myelin debris to obtain a single-cell population. The cells are then cultured in media optimized for mixed glial cell growth.

1. Preparation of Solutions in a Culture Hood under Aseptic Conditions

	Prepare culture media: complete Dulbecco&#39;s modification of Eagle&#39;s media ...

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

Summary

Explore More Videos

Chapters in this video

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

In Situ Ca²⁺ Imaging of the Enteric Nervous System

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord