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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

1. 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.
2. Under the hood, use sterile forceps to place autoclaved glass coverslips in 12 wells of....

Results

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.......

Discussion

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 μ-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.......

Materials

List of materials used in this article

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