Dynamics of changes in neuronal network morphology and development of mitochondria in mechanically damaged primary neuronal culture
Abstract
The aim of the study was (1) to trace morphological changes in a primary neuronal culture during its development and compare these changes with morphological changes in a mechanically damaged culture, and (2) to elucidate the dynamics of mitochondrial formation in normal and damaged cultures. Methods. The development of a primary culture of neurons from the cerebellum of 7-day old rats was recorded at 20-min intervals for 2.5 weeks starting from the cell seeding day with a IncuCyte ZOOM's intravital imaging and analysis system equipped with 20x objective lenses. Images of individual neuronal soma and neurite development were recorded in transmitted light. Mitochondrial formation and generation of electrical transmembrane potential (ΔΨm) were monitored with a potential-sensitive fluorescent probe TMRM (20 nM), which was continuously present in the culture from the moment of seeding. Mechanical brain injury was modeled by applying an approximately one-mm wide scratch to the cell monolayer at 23 hours after plating. Results. Morphological changes in the developing primary neuronal culture (total length of neurites, relative area of soma) were characterized by three phases with different kinetics and duration. TMRM influenced the phase duration and amplitude without changing the number of phases. Mitochondria began developing on the fourth day after plating. Increases in their number and ΔΨm were complete at 10-14 days of culture development. Conclusion. Phases of mitochondrial development were consistent with three phases of morphological changes in the entire culture. During the first 2-3 days following cell plating, the energy supply to the neuronal network was apparently provided by glycolysis since mitochondria did not generate an adequate ΔΨm for ATP synthesis. Axons grow from the intact area into the injured zone mainly in the direction of survived neurons in the scratch zone.
Downloads
References
2. Valtorta F. and Leoni C. Molecular mechanisms of neurite extension. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1999; 354 (1381): 387–94.
3. Mukhin A.G., Ivanova S.A., Knoblach S.M. and Faden A.I. New in vitro model of traumatic neuronal injury: evaluation of secondary injury and glutamate receptor-mediated neurotoxicity. J. Neurotrauma. 1997; 14(9): 651–63.
4. Mukhin A.G., Ivanova S.A., Allen J.W. and Faden A.I. Mechanical injury to neuronal/glial cultures in microplates: Role of NMDA receptors and pH in secondary neuronal cell death. J. Neurosci. Res. 1998; 51(6): 748–58.
5. Cui X., Liu R., Liu Z., Shen X., Wang Q. and Tan X. Cationic Poly-L-Lysine-Fe2O3/SiO2 nanoparticles loaded with small interference RNA: Application to silencing gene expression in primary rat neurons. J. Nanosci. Nanotechnol. 2014; 14(4): 2810–5.
6. Payette D.J., Xie J., Shirwany N. and Guo Q. Exacerbation of Apoptosis of Cortical Neurons Following Traumatic Brain Injury in Par-4 Transgenic Mice. Int. J. Clin. Exp. Pathol. 2008; 1(1): 44–56.
7. Bingham D., John C.M., Panter S.S. and Jarvis G.A. Post-injury treatment with lipopolysaccharide or lipooligosaccharide protects rat neuronal and glial cell cultures. Brain Res. Bull. 2011; 85(6): 403–9.
8. Yu A.C., Lee Y.L. and Eng L.F. Astrogliosis in culture: I. The model and the effect of antisense oligonucleotides on glial fibrillary acidic protein synthesis. J. Neurosci. Res. 1993; 34(3): 295–303.
9. Desclaux M., Teigell M., Amar L., Vogel R., Gimenez y Ribotta M., Privat A. et al. A novel and efficient gene transfer strategy reduces glial reactivity and improves neuronal survival and axonal growth in vitro. PLoS One. 2009; 4(7): e6227.
10. He Y., Li H.L., Xie W.Y., Yang C.Z., Yu A.C.H. and Wang Y. The presence of active Cdk5 associated with p35 in astrocytes and its important role in process elongation of scratched astrocyte. Glia. 2007; 55(6): 573–83.
11. Khodorov B.I., Storozhevykh T.P., Surin A.M., Yuryavichyus A.I., Sorokina E.G., Borodin A.V. et al. The leading role of mitochondrial depolarization in the mechanism of glutamate-induced disruptions in Ca2+ homeostasis. Neurosci. Behav. Physiol. 2002; 32(5): 541–7.
12. Khodorov B.I., Mikhailova M.M., Bolshakov A.P., Surin A.M., Sorokina E.G., Rozhnev S.A. et al. Dramatic effect of glycolysis inhibition on the cerebellar granule cells bioenergetics. Biochem. Suppl. Ser. A Membr. Cell Biol. 2012; 6(2): 186–97.
13. Surin A.M., Khiroug S., Gorbacheva L.R., Khodorov B.I., Pinelis V.G. and Khiroug L. Comparative analysis of cytosolic and mitochondrial ATP synthesis in embryonic and postnatal hippocampal neuronal cultures. Front. Mol. Neurosci. 2013; 5: 1-15.
14. Dumollard R., Carroll J., Duchen M.R., Campbell K. and Swann K. Mitochondrial function and redox state in mammalian embryos. Seminars in Cell and Developmental Biology. 2009; 20(3): 346–53.
15. Nicholls D.G. and Ward M.W. Mitochondrial membrane potential and neuronal glutamate excitotoxicity: Mortality and millivolts. Trends in Neurosciences. 2000; 23(4): 166–74.
16. Duchen M.R., Surin A. and Jacobson J. Imaging mitochondrial function in intact cells. Methods Enzymol. 2003; 361: 353–89.
17. Surin A.M., Sharipov R.R., Krasilnikova I.A., Boyarkin D.P., Lisina O.Yu., Gorbacheva L.R. et al. Disruption of functional activity of mitochondria during MTT assay of viability of cultured neurons. Biokhimiya. 2017; 82(6): 970-984. (in Russian)
18. Gerencser A.A., Chinopoulos C., Birket M.J., Jastroch M., Vitelli C., Nicholls D.G. et al. Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria. J. Physiol. 2012; 590 (12): 2845–71.
19. Trendeleva T.A., Rogov A.G., Cherepanov D.A., Sukhanova E.I., Il’yasova T.M., Severina I.I. et al. Interaction of tetraphenylphosphonium and dodecyltriphenylphosphonium with lipid membranes and mitochondria. Biochem. 2012; 77(9): 1021–8.
20. Dragunow M. High-content analysis in neuroscience. Nature Reviews Neuroscience. 2008; 9(10): 779–88.
21. Mattiazzi Usaj M., Styles E.B., Verster A.J., Friesen H., Boone C. and Andrews B.J. High-Content Screening for Quantitative Cell Biology. Trends in Cell Biology. 2016; 26(8): 598–611.
22. Daub A., Sharma P. and Finkbeiner S. High-content screening of primary neurons: ready for prime time. Current Opinion in Neurobiology. 2009; 19(5): 537–43.
23. Repin V.S., Saburina I.N., Kosheleva N.V., Gorkun A.A., Zurina I.M., Kubatiev A.A. 3D-technology of the formation and maintenance of single dormant microspheres from 2000 human somatic cells and their reactivation in vitro. Kletochnye tekhnologii v biologii i meditsine. 2014; 3: 161-9. (in Russian)
24. Kosheleva N.V., Saburina I.N., Zurina I.M., Gorkun A.A., Borzenok S.A., Nikishin D.A. et al. The technology of obtaining multipotent spheroids from limbal mesenchymal stromal cells for reparation of injured eye tissues. Patologicheskaya fiziologiya i eksperimental'naya terapiya. 2016; 60(4): 160-7. (in Russian)
25. Thangnipon W., Kingsbury A., Webb M. and Balazs R. Observations on rat cerebellar cells in vitro: influence of substratum, potassium concentration and relationship between neurones and astrocytes. Dev. Brain Res. 1983; 11(2): 177–89.
26. Gallo V., Kingsbury A., Balázs R. and Jørgensen O.S. The role of depolarization in the survival and differentiation of cerebellar granule cells in culture. J. Neurosci. 1987; 7(7): 2203–13.
27. Budd S.L. and Nicholls D.G. A reevaluation of the role of mitochondria in neuronal Ca2+ homeostasis. J. Neurochem. 1996; 66(1): 403–11.
28. Sasaki S., Warita H., Abe K. and Iwata M. Impairment of axonal transport in the axon hillock and the initial segment of anterior horn neurons in transgenic mice with a G93A mutant SOD1 gene. Acta Neuropathol. 2005; 110(1): 48–56.
29. Popov V., Medvedev N.I., Davies H.A. and Stewart M.G. Mitochondria form a filamentous reticular network in hippocampal dendrites but are present as discrete bodies in axons: A three-dimensional ultrastructural study. J. Comp. Neurol. 2005; 492(1): 50–65.
30. Knöferle J., Ramljak S., Koch J.C., Tönges L., Asifc A.R., Michel U. et al. TGF-β 1 enhances neurite outgrowth via regulation of proteasome function and EFABP. Neurobiol. Dis. 2010; 38(3): 395–404.
31. Tonges L., Planchamp V., Koch J. C., Herdegen T., Bähr M. and Lingor P. JNK isoforms differentially regulate neurite growth and regeneration in dopaminergic neurons in vitro. J. Mol. Neurosci. 2011; 45(2): 284–93.
