Applicability of Live Cell Imaging of mRNA Expression in Combination with Calcium Imaging for in vitro Studies of Neural Network Activity

Abstract

The effectiveness of live cell mRNA detection for neurobiological studies was evaluated. We modified the commercial protocol for the use of RNA detection probes (SmartFlareTM, Merck) for primary hippocampal cultures. It was shown that RNA probes could be used both as an independent evaluation system and in combination with Ca2+ imaging. The complex of these methods provided the unique possibility of performing simultaneous studies of the functional calcium homeostasis of neuronal-glial networks and differential analysis of the plasticity of cells with different levels of mRNA expression.

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References

  1. 1.

    Yuste R. 2015. From the neuron doctrine to neural networks. Nat. Rev. Neurosci. 16, 487–3

    Article  PubMed  CAS  Google Scholar 

  2. 2.

    Renshaw S. 2017. Immunohistochemistry and Immunocytochemistry. Essential Methods. New Jersey: Wiley-Blackwell.

    Google Scholar 

  3. 3.

    Burry R.W. 2011. Controls for immunocytochemistry an update. J. Histochem. Cytochem. 59 (1), 6–12.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. 4.

    Glick B., Pasternak J. 2002. Molekuliarnye biotekhnologii. Printsipy i primenenie rekombinantnykh DNK (Molecular biotechnology: Principles and applications of recombinant DNA). M: Mir.

    Google Scholar 

  5. 5.

    Watson J.D. 2012. The polymerase chain reaction. New York: Springer Science & Business Media.

    Google Scholar 

  6. 6.

    Seferos D.S., Giljohann D.A., Hill H.D., Prigodich A.E., Mirkin C.A. 2007. Nano-flares: Probes for transfection and mRNA detection in living cells. J. Am. Chem. Soc. 129 (50), 15477–15479.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. 7.

    McClellan S., Slamecka J., Howze P., Thompson L., Finan M., Rocconi R., Owen L. 2015. mRNA detection in living cells: A next generation cancer stem cell identification technique. Methods. 82, 47–3

    Article  PubMed  CAS  Google Scholar 

  8. 8.

    Lahm H., Doppler S., Dreßen M., Werner A., Adamczyk K., Schrambke D., Brade T., Laugwitz K.L., Deutsch M.A., Schiemann M., Lange R., Moretti A., Krane M. 2015. Live fluorescent RNA-based detection of pluripotency gene expression in embryonic and induced pluripotent stem cells of different species. Stem Cells. 33 (2), 392–402.

    Article  PubMed  CAS  Google Scholar 

  9. 9.

    Seftor E.A., Seftor R.E., Weldon D.S., Kirsammer G.T., Margaryan N.V., Gilgur A., Hendrix M.J. 2014. Melanoma tumor cell heterogeneity: a molecular approach to study subpopulations expressing the embryonic morphogen nodal. Seminars in Oncology. 41 (2), 259–266.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. 10.

    Halo T.L., McMahon K.M., Angeloni N.L., Xu Y., Wang W., Chinen A.B., Malin D., Strekalova E., Cryns V.L., Cheng C., Mirkin C.A., Thaxton C.S. 2014. NanoFlares for the detection, isolation, and culture of live tumor cells from human blood. Proc. Natl. Acad. Sci. USA. 111 (48), 17104–17109.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. 11.

    Prigodich A.E., Randeria P.S., Briley W.E., Kim N.J., Daniel W.L., Giljohann D.A., Mirkin C.A. 2012. Multiplexed nanoflares: mRNA detection in live cells. Anal. Chem. 84 (4), 2062–2066.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. 12.

    Braet K., Cabooter L., Paemeleire K., Leybaert L. 2004. Calcium signal communication in the central nervous system. Biol. Cell. 96 (1), 79–91.

    Article  PubMed  CAS  Google Scholar 

  13. 13.

    Jercog P., Rogerson T., Schnitzer M.J. 2016. Largescale fluorescence calcium-imaging methods for studies of long-term memory in behaving mammals. Cold Spring Harbor Perspectives in Biology. 8 (5), 1–27.

    Article  CAS  Google Scholar 

  14. 14.

    Carrillo-Reid L., Yang W., Kang Miller J.E., Peterka D.S. Yuste R. 2017. Imaging and optically manipulating neuronal ensembles. Annu. Rev. Biophys. 46, 271–3

    Article  PubMed  CAS  Google Scholar 

  15. 15.

    Vedunova M., Sakharnova T., Mitroshina E., Perminova M., Pimashkin A., Zakharov Yu., Dityatev A., Mukhina I. 2013. Seizure-like activity in hyaluronidase-treated dissociated hippocampal cultures. Front. Cell. Neurosci. 7, article 149.

  16. 16.

    http://www.merckmillipore.com/RU/ru/search/Smart Flare?search=&TrackingSearchType=SB+-+Search+ Result+Search+Box&SearchContextPageletUUID= &SearchTerm=SmartFlare

  17. 17.

    Portioli C., Pedroni M., Benati D., Donini M., Bonafede R., Mariotti R., Perbellini L., Cerpelloni M., Dusi S., Speghini A., Bentivoglio M. 2016. Citrate-stabilized lanthanide-doped nanoparticles: Brain penetration and interaction with immune cells and neurons. Nanomedicine (Lond). 11 (23), 3039–3051.

    Article  CAS  Google Scholar 

  18. 18.

    Kursungoz C., Taş S.T., Sargon M.F., Sara Y., Ortaç B. 2017. Toxicity of internalized laser generated pure silver nanoparticles to the isolated rat hippocampus cells. Toxicol. Ind. Hlth. 33 (7), 555–563. doi 10.1177/0748233717690992

    Article  CAS  Google Scholar 

  19. 19.

    Rosi N.L., Giljohann D.A., Thaxton C.S., Lytton-Jean A.K., Han M.S., Mirkin C.A. 2006. Oligonucleotide-modified gold nanoparticles for intracellular gene regulation. Science. 12 (5776), 1027–1030.

    Article  CAS  Google Scholar 

  20. 20.

    Budik S., Tschulenk W., Kummer S., Walter I., Aurich C. 2017. Evaluation of SmartFlare probe applicability for verification of RNAs in early equine conceptuses, equine dermal fibroblast cells and trophoblastic vesicles. Reproduction, Fertility Dev. doi.org/10.1071/RD16362

    Google Scholar 

  21. 21.

    Zhao Z., Lu R., Zhang B., Shen J., Yang L., Xiao S., Liu J., Suo W.Z. 2012. Differentiation of HT22 neurons induces expression of NMDA receptor that mediates homocysteine cytotoxicity. Neurol. Res. 34 (1), 38–43.

    Article  PubMed  CAS  Google Scholar 

  22. 22.

    Vanden Berghe P. 2004. Fluorescent molecules as tools to study Ca2+ signaling, mitochondrial dynamics and synaptic function in enteric neurons. Verh. K Acad. Geneeskd. Belg. 66 (5–6), 407–425.

    PubMed  CAS  Google Scholar 

  23. 23.

    Paredes M., Etzler J.C., Watts L.T., Zheng W., Lechleiter J.D. 2008. Chemical calcium indicators. Methods. 46 (3), 143–151.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. 24.

    Richards D.A. 2010. Regulation of exocytic mode in hippocampal neurons by intra-bouton calcium concentration. J. Physiol. 588 (Pt 24), 4927–4936.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. 25.

    Zakharov Yu.N., Korotchenko S.A., Kalintseva Ya.I., Potanina A.V., Mitroshina E.V., Vedunova M.V., Mukhina I.V. 2012. Fluorescence analysis of the metabolic activity patterns of a neuronal-glial network. J. Opt. Technol. 79 (6), 348–351.

    Article  Google Scholar 

  26. 26.

    Ivenshitz M., Segal M.J. 2010. Neuronal density determines network connectivity and spontaneous activity in cultured hippocampus Neurophysiol. 104 (2), 1052–1060. doi 10.1152/jn.00914.2009

    Article  Google Scholar 

  27. 27.

    Korkotian E., Botalova A., Odegova T., Galishevskaya E., Skryabina E., Segal M. 2015. Complex effects of aqueous extract of Melampyrum pratense and of its flavonoids on activity of primary cultured hippocampal neurons. J. Ethnopharmacol. 163, 220–3

    Article  PubMed  CAS  Google Scholar 

  28. 28.

    Amor R., McDonald A., Trägårdh J., Robb G., Wilson L., Abdul Rahman N.Z., Dempster J., Amos W.B., Bushell T.J., McConnell G. 2016. Widefield two-photon excitation without scanning: live cell microscopy with high time resolution and low photo-bleaching. PLoS One. 11 (1), e0147115.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. 29.

    Vedunova M.V., Mishchenko T.A., Mitroshina E.V., Mukhina I.V. 2015. TrkB-mediated neuroprotective and antihypoxic properties of Brain-derived neurotrophic factor. Oxidative Med. Cell. Longevity. 9, article ID 453901). doi.org/10.1155/2015/453901

    Google Scholar 

  30. 30.

    Vedunova M.V., Sakharnova T.A., Mitroshina E.V., Shishkina T.V., Astrakhanova T.A., Mukhina I.V. 2014. Antihypoxic and neuroprotective properties of BDNF and GDNF in vitro and in vivo under hypoxic conditions. Sovremennye tehnologii v medicine (Rus.). 6 (4), 38–47.

    Google Scholar 

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Correspondence to M. V. Vedunova.

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Original Russian Text © T.A. Mishchenko, E.V. Mitroshina, T.V. Shishkina, T.A. Astrakhanova, M.V. Prokhorova, M.V. Vedunova, 2018, published in Biologicheskie Membrany, 2018, Vol. 35, No. 2, pp. 104–114.

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Mishchenko, T.A., Mitroshina, E.V., Shishkina, T.V. et al. Applicability of Live Cell Imaging of mRNA Expression in Combination with Calcium Imaging for in vitro Studies of Neural Network Activity. Biochem. Moscow Suppl. Ser. A 12, 170–179 (2018). https://doi.org/10.1134/S1990747818020095

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Keywords

  • RNA probes
  • Ca2+ imaging
  • fluorescence microscopy
  • primary hippocampal cultures
  • neural networks