Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis

Summary

This paper describes the isolation and culturing of embryonic rat sympathetic neurons from the superior cervical ganglia. It also provides detailed protocols for immunocytochemical staining and for preparing neuronal extracts for mass spectrometric analysis.

Abstract

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study axonal growth, axonal trafficking, synaptogenesis, dendritic growth, dendritic plasticity and nerve-target interactions in co-culture systems. This protocol describes the isolation and dissociation of neurons from the superior cervical ganglia of E21 rat embryos, followed by the preparation and maintenance of pure neuronal cultures in serum-free medium. Since neurons do not adhere to uncoated plastic, neurons will be cultured on either 12 mm glass coverslips or 6-well plates coated with poly-D-lysine. Following treatment with an antimitotic agent (Ara-C, cytosine β-D-arabinofuranoside), this protocol generates healthy neuronal cultures with less than 5% non-neuronal cells, which can be maintained for over a month in vitro. Although embryonic rat SCG neurons are multipolar with 5-8 dendrites in vivo; under serum-free conditions, these neurons extend only a single axon in culture and continue to be unipolar for the duration of the culture. However, these neurons can be induced to extend dendrites in the presence of basement membrane extract, bone morphogenetic proteins (BMPs), or 10% fetal calf serum. These homogenous neuronal cultures can be used for immunocytochemical staining and for biochemical studies. This paper also describes optimized protocol for immunocytochemical staining for microtubule associated protein-2 (MAP-2) in these neurons and for the preparation of neuronal extracts for mass spectrometry.

Introduction

Sympathetic neurons derived from embryonic superior cervical ganglia (SCG) have been widely used as a primary neuronal culture system for studying many aspects of neuronal development including growth factor dependence, neuron-target interactions, neurotransmitter signaling, axonal growth, dendrite development and plasticity, synaptogenesis and signaling mechanisms underlying nerve-target/neuron-glia interactions^{1,2,3,4,5,6,7,}<....

Protocol

All procedures performed in studies involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) at Saint Mary’s College of California. The animal care and use guidelines at Saint Mary’s College were developed based on the guidelines provided by Office of Laboratory Animal Welfare at the National Institute for Health (https://olaw.nih.gov/sites/default/files/PHSPolicyLabAnimals.pdf and https://olaw.nih.gov/sites/default/files/Guide-for-the-Care-and-Use-of-Laboratory-Animals.pdf).

1. Preparation of culture media (also referred to as control medium)

1. Add the following ingredients, in the or....

Results

Isolating and maintaining neuronal cultures of embryonic SCG neurons
Dissociated cells from the rat embryonic SCG were plated in a poly-D-lysine coated plate or coverslip and maintained in serum free culture media containing b-nerve growth factor. The dissociated cells containing a mixture of neurons and glial cells look circular upon plating (Figure 1A). Within 24 hours of plating, the neurons extend small axonal processes with glial cells flattening and appearing pha.......

Discussion

This paper describes the protocols for culturing sympathetic neurons from superior cervical ganglia of embryonic rat pups. The advantages of using this model system are that it is possible to obtain a homogeneous population of neurons providing a similar response to growth factors, and since the growth factor requirements for these neurons has been well -characterized, it is possible to grow these neurons in vitro in defined media, under serum-free conditions¹⁰. Although the protocol describes the.......

Materials

Name	Company	Catalog Number	Comments
2D nanoACQUITY	Waters Corporation
Ammonium bicarbonate	Sigma-Aldrich	9830
BMP-7	R&D Systems	354-BP
Bovine Serum Alumin	Sigma-Aldrich	5470
Cell scraper	Corning	CLS-3010
Collagenase	Worthington Biochemical	4176
Corning Costar or Nunc Flat bottomed Cell culture plates	Fisher Scientific	07-200, 140675, 142475
Cytosine- β- D-arabinofuranoside	Sigma-Aldrich	C1768
D-phosphate buffered saline (Calcium and magnesium free)	ATCC	30-2200
Dispase II	Roche	4942078001
Distilled Water	Thermo Fisher Scientific	15230
Dithiothreitol	Sigma-Aldrich	D0632
DMEM - Low glucose + Glutamine, + sodium pyruvate	Thermo Fisher Scientific	11885
Fatty Acid Free BSA	Calbiochem	126609	20 mg/mL stock in low glucose DMEM
Fine forceps Dumont no.4 and no.5	Ted Pella Inc	5621, 5622
Forceps and Scissors for Dissection	Ted Pella Inc	1328, 1329, 5002
Glass coverlips - 12mm	Neuvitro Corporation	GG-12
Goat-Anti Mouse IgG Alexa 488 conjugated	Thermo Fisher Scientific	A32723
Ham's F-12 Nutrient Mix	Thermo Fisher Scientific	11765
Hank's balanced salt soltion (Calcium and Magnesium free)	Thermo Fisher Scientific	14185
Insulin-Selenium-Transferrin (100X)	Thermo Fisher Scientific	41400-045
Iodoacetamide	Sigma-Aldrich	A3221
L-Glutamine	Thermo Fisher Scientific	25030
Leibovitz L-15 medium	Thermo Fisher Scientific	11415064
Mounting media for glass coverslips	Thermo Fisher Scientific	P36931, P36934
Mouse-anti- MAP2 antibody (SMI-52)	BioLegend	SMI 52
Nerve growth factor	Envigo Bioproducts (formerly Harlan Bioproducts)	BT5017	Stock 125 μg/mL in 0.2% Prionex in DMEM
Paraformaldehye	Spectrum Chemicals	P1010
Penicillin-Streptomycin (100X)	Thermo Fisher Scientific	15140
Poly-D-Lysine	Sigma-Aldrich	P0899
Prionex	Millipore	529600	10% solution, 100 mL
RapiGest SF	Waters Corporation	186001861	5 X 1 mg
Synapt G2 High Definition Mass Spectrometry	Waters Corporation
Trifluoro acetic acid - Sequencing grade	Thermo Fisher Scientific	28904	10 X 1 mL
Triton X-100	Sigma-Aldrich	X100
Trypsin	Promega or NEB	V511A, P8101S	100 μg or 5 X 20 mg
Waters Total recovery vials	Waters Corporation	186000385c