Culturing Rotte sympatiske neuroner fra Embryonic Superior Cervical Ganglia for morfologisk og proteomisk analyse
Summary
Dette papir beskriver isolation og dyrkning af embryonale rotte sympatiske neuroner fra den overlegne livmoderhalskræft ganglier. Det giver også detaljerede protokoller for immunokytokemiske farvning og til forberedelse af neuronale ekstrakter til massespektrometrisk analyse.
Abstract
Sympatiske neuroner fra embryonale rotte overlegen cervikal ganglier (SCG) er blevet brugt som en in vitro model system for perifere neuroner til at studere axonal vækst, axonal handel, synaptogenesis, dendritisk vækst, dendritisk plasticitet og nerve-target interaktioner i co-kultur systemer. Denne protokol beskriver isolering og dissociation af neuroner fra den overlegne cervikale ganglier af E21 rotte embryoner, efterfulgt af forberedelse og vedligeholdelse af ren neuronal kulturer i serum-fri medium. Da neuroner ikke overholder ubestrøget plast, neuroner vil blive dyrket på enten 12 mm glas coverslips eller 6-brønd plader belagt med poly-D-lysin. Efter behandling med et antimitotisk middel (Ara-C, cytosin β-D-arabinofuranoside) genererer denne protokol sunde neuronale kulturer med mindre end 5% ikke-neuronale celler, som kan vedligeholdes i over en måned in vitro. Selv om embryonale rotte SCG neuroner er multipolære med 5-8 dendrites in vivo; under serumfrie forhold strækker disse neuroner kun en enkelt aknoson i kulturen og fortsætter med at være unipolar under hele kulturen. Men, disse neuroner kan induceres til at udvide dendritter i overværelse af kælderen membran ekstrakt, knogle morfoogenetiske proteiner (BMPPs), eller 10% føtal kalv serum. Disse homogene neuronale kulturer kan anvendes til immunokokemisk farvning og til biokemiske undersøgelser. Dette papir beskriver også optimeret protokol for immunokyokemisk farvning for mikrotubule associeret protein-2 (MAP-2) i disse neuroner og til fremstilling af neuronal ekstrakter til massespektrometri.
Introduction
Sympatiske neuroner afledt af embryonale overensne cervikale ganglier (SCG) har været meget udbredt som en primær neuronal kultur system til at studere mange aspekter af neuronal udvikling, herunder vækstfaktor afhængighed, neuron-target interaktioner, neurotransmitter signalering, axonal vækst, dendrite udvikling og plasticitet, synaptogenesis og signalering mekanismer underliggende nerve-target/neuron-glia interaktioner1,,2,,3,4,5,6,7,8,9. På trods af deres lille størrelse (ca. 10000 neuroner/ ganglier), er der tre hovedårsager til udviklingen og omfattende brug af dette kultursystem er i) er den første ganglier i den sympatiske kæde, de er større, og derfor lettere at isolere, end resten af den sympatiske ganglia10; ii) i modsætning til centrale neuroner, neuroner i SCG er temmelig homogen med alle de neuroner, der stammer fra den neurale våbenskjold, der har en lignende størrelse, afhængighed af nerve vækstfaktor og er heller ikke-adrenerge. Dette gør dem til en værdifuld model for morfologiske og genomiskeundersøgelser 10,11 og iii) disse neuroner kan opretholdes i et defineret serumfrit medium, der indeholder nervevækstfaktor i over enmåned 10,12. Perinatal SCG neuroner er blevet flittigt brugt til at studere de mekanismer, der ligger til grund for initiering og vedligeholdelse af dendritter2. Dette skyldes primært, selv om SCG neuroner har en omfattende dendritisk arbor in vivo, de ikke udvide dendritter in vitro i mangel af serum, men kan induceres til at vokse dendritter i nærvær af visse vækstfaktorer såsom knogle morfoogenetiske proteiner2,12,13.
Dette papir beskriver protokollen for isolering og dyrkning embryonale rotte SCG neuroner. I løbet af de sidste 50 år har primære neuronale kulturer fra SCG hovedsagelig været anvendt til morfologiske undersøgelser med et begrænset antal undersøgelser, der undersøger de store genomiske eller proteomiske forandringer. Dette skyldes hovedsagelig lille vævsstørrelse, hvilket resulterer i isolering af små mængder DNA eller protein, hvilket gør det vanskeligt at udføre genomiske og proteomiske analyser af disse neuroner. Men i de seneste år, øget påvisning følsomhed har gjort det muligt at udvikle metoder til at undersøge genom, miRNome og proteome i SCG neuroner under dendritisk vækst udvikling14,15,16,17. Dette papir vil også beskrive metoden til morfologisk analyse af disse neuroner ved hjælp af immunokytokemi og en protokol til at opnå neuronal protein ekstrakter til massespektrometrisk analyse.
Protocol
Representative Results
Discussion
Dette papir beskriver protokollerne for dyrkning sympatiske neuroner fra overlegen livmoderhalskræft ganglier af embryonale rotte hvalpe. Fordelene ved at bruge denne model system er, at det er muligt at opnå en homogen population af neuroner giver en lignende reaktion på vækstfaktorer, og da vækstfaktoren krav til disse neuroner har været godt karakteriseret, er det muligt at dyrke disse neuroner in vitro i definerede medier, under serum-fri betingelser10. Selv om protokollen beskriver proc…
Declarações
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af Fakultetets Udviklingsfond og Summer Research Program tilskud på Saint Mary’s College of California. Forfatterne vil også gerne takke Dr. Pamela Lein ved University of California i Davis og Dr. Anthony Iavarone på UC Berkeley Mass spektrometri facilitet for deres råd under udviklingen af disse protokoller. Forfatterne vil også gerne takke Haley Nelson i Office of College Communications på Saint Mary’s College of California for hendes hjælp med videoproduktion og redigering.
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 |
Referências
- Rees, R. P., Bunge, M. B., Bunge, R. P. Morphological Changes in the neuritic growth cone and target neuron during synaptic junction development in culture. Journal of Cell Biology. 9, (1976).
- Chandrasekaran, V., Lein, P. J. Regulation of Dendritogenesis in Sympathetic Neurons. Autonomic Nervous System. , (2018).
- Higgins, D., Burack, M., Lein, P., Banker, G. Mechanisms of neuronal polarity. Current Opinion in Neurobiology. 7 (5), 599-604 (1997).
- Lein, P., Guo, X., Hedges, A. M., Rueger, D., Johnson, M., Higgins, D. The effects of Extracellular Matrix And Osteogenic Protein-1 on the morphological differentiation of rat sympathetic neurons. International Journal of Developmental Neuroscience. 14 (3), 203-215 (1996).
- Kobayashi, M., Fujii, M., Kurihara, K., Matsuoka, I. Bone morphogenetic protein-2 and retinoic acid induce neurotrophin-3 responsiveness in developing rat sympathetic neurons. Molecular Brain Research. 53 (1-2), 206-217 (1998).
- Burnham, P., Louis, J. C., Magal, E., Varon, S. Effects of ciliary neurotrophic factor on the survival and response to nerve growth factor of cultured rat sympathetic neurons. Biologia do Desenvolvimento. 161 (1), 96-106 (1994).
- Hou, X. E., Li, J. Y., Dahlström, A. Clathrin light chain and synaptotagmin I in rat sympathetic neurons. Journal of the Autonomic Nervous System. 62 (1-2), 13-26 (1997).
- Harris, G. M., et al. Nerve Guidance by a Decellularized Fibroblast Extracellular Matrix. Matrix Biology. , 176-189 (2017).
- Wingerd, K. L., et al. α4 integrins and vascular cell adhesion molecule-1 play a role in sympathetic innervation of the heart. Journal of Neuroscience. 22 (24), 10772-10780 (2002).
- Higgins, D., Lein, P., Osterhout, D. J., Johnson, M., Banker, G., Goslin, K. Tissue culture of autonomic neurons. Culturing Nerve Cells. , 177-205 (1991).
- Lein, P., Johnson, M., Guo, X., Rueger, D., Higgins, D. Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons. Neuron. 15 (3), 597-605 (1995).
- Bruckenstein, D. A., Higgins, D. Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. Biologia do Desenvolvimento. 128 (2), 337-348 (1988).
- Voyvodic, J. T. Development and regulation of dendrites in the rat superior cervical ganglion. The Journal of Neuroscience. 7 (3), 904-912 (1987).
- Garred, M. M., Wang, M. M., Guo, X., Harrington, C. A., Lein, P. J. Transcriptional Responses of Cultured Rat Sympathetic Neurons during BMP-7-Induced Dendritic Growth. PLoS ONE. 6 (7), 21754 (2011).
- Pravoverov, K., et al. MicroRNAs are Necessary for BMP-7-induced Dendritic Growth in Cultured Rat Sympathetic Neurons. Cellular and Molecular Neurobiology. 39 (7), 917-934 (2019).
- Natera-Naranjo, O., Aschrafi, A., Gioio, A. E., Kaplan, B. B. Identification and quantitative analyses of microRNAs located in the distal axons of sympathetic neurons. RNA (New York, N.Y.). 16 (8), 1516-1529 (2010).
- Aschrafi, A., et al. Angiotensin II mediates the axonal trafficking of tyrosine hydroxylase and dopamine β-hydroxylase mRNAs and enhances norepinephrine synthesis in primary sympathetic neurons. Journal of Neurochemistry. 150 (6), 666-677 (2019).
- Ghogha, A., Bruun, D. a., Lein, P. J. Inducing dendritic growth in cultured sympathetic neurons. Journal of Visualized Experiments. (61), 4-8 (2012).
- Caceres, A., Banker, G., Steward, O., Binder, L., Payne, M. MAP2 is localized to the dendrites of hippocampal neurons which develop in culture. Brain Research. 315 (2), 314-318 (1984).
- Guo, X., Rueger, D., Higgins, D. Osteogenic protein-1 and related bone morphogenetic proteins regulate dendritic growth and the expression of microtubule-associated protein-2 in rat sympathetic neurons. Neuroscience Letters. 245 (3), 131-134 (1998).
- Mi, H., et al. PANTHER version 11: Expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements. Nucleic Acids Research. 45, 183-189 (2017).
- Chandrasekaran, V., et al. Retinoic acid regulates the morphological development of sympathetic neurons. Journal of Neurobiology. 42 (4), (2000).
- Courter, L. A., et al. BMP7-induced dendritic growth in sympathetic neurons requires p75 NTR signaling. Developmental Neurobiology. 76 (9), 1003-1013 (2016).
- Lein, P. J., Fryer, A. D., Higgins, D. Cell Culture: Autonomic and Enteric Neurons. Encyclopedia of Neuroscience. , 625-632 (2009).
- Neto, E., et al. Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough of In vitro Tools for Neurobiological Research. Journal of Neuroscience. 36 (46), 11573-11584 (2016).
- Sleigh, J. N., Weir, G. A., Schiavo, G. A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia. BMC Research Notes. 9 (1), 82 (2016).
- Conrad, R., Jablonka, S., Sczepan, T., Sendtner, M., Wiese, S., Klausmeyer, A. Lectin-based isolation and culture of mouse embryonic motoneurons. Journal of Visualized Experiments. (55), e3200 (2011).
- Takeuchi, A., et al. Microfabricated device for co-culture of sympathetic neuron and iPS-derived cardiomyocytes. Proceedings of the Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBS. 2013, 3817-3820 (2013).
- Takeuchi, A., et al. Development of semi-separated co-culture system of sympathetic neuron and cardiomyocyte. Proceedings of the 31st Annual International Conference of the IEEE Engineering in Medicine and Biology Society: Engineering the Future of Biomedicine, EMBC 2009. , 1832-1835 (2009).
- Chandrasekaran, V., Lea, C., Sosa, J. C., Higgins, D., Lein, P. J. Reactive oxygen species are involved in BMP-induced dendritic growth in cultured rat sympathetic neurons. Molecular and Cellular Neuroscience. 67, (2015).
- Kim, W. Y., et al. Statins decrease dendritic arborization in rat sympathetic neurons by blocking RhoA activation. Journal of Neurochemistry. 108 (4), 1057-1071 (2009).
- Dalby, B., et al. Advanced transfection with Lipofectamine 2000 reagent: Primary neurons, siRNA, and high-throughput applications. Methods. 33 (2), 95-103 (2004).
- Szpara, M. L., et al. Analysis of gene expression during neurite outgrowth and regeneration. BMC Neuroscience. 8 (1), 100 (2007).
- Pop, C., Mogosan, C., Loghin, F. Evaluation of rapigest efficacy for the digestion of proteins from cell cultures and heart tissue. Clujul Medical. 87 (4), 5 (2014).
- Vit, O., Petrak, J. Integral membrane proteins in proteomics. How to break open the black box. Journal of Proteomics. 153, 8-20 (2017).
