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.