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.
Similar content being viewed by others
References
Yuste R. 2015. From the neuron doctrine to neural networks. Nat. Rev. Neurosci. 16, 487–3
Renshaw S. 2017. Immunohistochemistry and Immunocytochemistry. Essential Methods. New Jersey: Wiley-Blackwell.
Burry R.W. 2011. Controls for immunocytochemistry an update. J. Histochem. Cytochem. 59 (1), 6–12.
Glick B., Pasternak J. 2002. Molekuliarnye biotekhnologii. Printsipy i primenenie rekombinantnykh DNK (Molecular biotechnology: Principles and applications of recombinant DNA). M: Mir.
Watson J.D. 2012. The polymerase chain reaction. New York: Springer Science & Business Media.
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.
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
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.
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.
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.
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.
Braet K., Cabooter L., Paemeleire K., Leybaert L. 2004. Calcium signal communication in the central nervous system. Biol. Cell. 96 (1), 79–91.
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.
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
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.
http://www.merckmillipore.com/RU/ru/search/Smart Flare?search=&TrackingSearchType=SB+-+Search+ Result+Search+Box&SearchContextPageletUUID= &SearchTerm=SmartFlare
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.
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
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.
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
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.
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.
Paredes M., Etzler J.C., Watts L.T., Zheng W., Lechleiter J.D. 2008. Chemical calcium indicators. Methods. 46 (3), 143–151.
Richards D.A. 2010. Regulation of exocytic mode in hippocampal neurons by intra-bouton calcium concentration. J. Physiol. 588 (Pt 24), 4927–4936.
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.
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
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
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.
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
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.
Author information
Authors and Affiliations
Corresponding author
Additional information
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.
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S1990747818020095