Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis
概要
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 interactions1,2,3,4,5,6,7,8,9. Despite their small size (around 10000 neurons/ganglia), there are three main reasons for the development and extensive use of this culture system are i) being the first ganglia in the sympathetic chain, they are larger, and therefore easier to isolate, than the rest of the sympathetic ganglia10; ii) unlike central neurons, the neurons in the SCG are fairly homogeneous with all the neurons being derived from the neural crest, having a similar size, dependence on nerve growth factor and being nor-adrenergic. This makes them a valuable model for morphological and genomic studies10,11 and iii) these neurons can be maintained in a defined serum-free medium containing nerve growth factor for over a month10,12. Perinatal SCG neurons have been extensively used for studying the mechanisms underlying the initiation and maintenance of dendrites2. This is mainly because, although SCG neurons have an extensive dendritic arbor in vivo, they do not extend dendrites in vitro in the absence of serum but can be induced to grow dendrites in the presence of certain growth factors such as bone morphogenetic proteins2,12,13.
This paper describes the protocol for isolating and culturing embryonic rat SCG neurons. Over the past 50 years, primary neuronal cultures from the SCG have been mainly used for morphological studies with a limited number of studies examining the large-scale genomic or proteomic changes. This is mainly due to small tissue size resulting in the isolation of low amounts of DNA or protein, which makes it difficult to perform genomic and proteomic analyses on these neurons. However, in recent years, increased detection sensitivity has enabled development of methods to examine the genome, miRNome and proteome in the SCG neurons during dendritic growth development14,15,16,17. This paper will also describe the method for morphological analysis of these neurons using immunocytochemistry and a protocol to obtain neuronal protein extracts for mass spectrometric analysis.
Protocol
Representative Results
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…
開示
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Faculty Development Fund and Summer Research Program grant at Saint Mary’s College of California. The authors would also like to thank Dr. Pamela Lein at University of California at Davis and Dr. Anthony Iavarone at UC Berkeley Mass spectrometry facility for their advice during the development of these protocols. The authors would also like to thank Haley Nelson in the Office of College Communications at Saint Mary’s College of California for her help with video production and editing.
Materials
| 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 |
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