Time-Lapse Video Microscopy and Single Cell Tracking to Study Neural Cell Behavior In Vitro

Paniagua-Herranz, Lucía; Gómez‐Villafuertes, Rosa; Agustín-Durán, David de; Gascón, Sergio; Pérez-Sen, Ráquel; Delicado, Esmerilda G.; Miras-Portugal, Marı́a Teresa; Ortega, Felipe

doi:10.1007/7651_2019_219

Cited by 8 publications

(5 citation statements)

References 20 publications

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“…P0899) coated coverslips at a density of 200–300 cells/mm 2 . Time-lapse video microscopy and single cell tracking 62 , 82 – 84 of adult SEZ cultures was performed with a Nikon TE-2000 microscope at a constant temperature of 37 °C and 8% CO 2 . Phase contrast/Bright field images were acquired every 5 min for 5 days using a 20x phase contrast objective (Nikon), a ZYLA from ANDOR camera and a NIS-element from NIKON software.…”

Section: Methodsmentioning

confidence: 99%

Post-transcriptional control of a stemness signature by RNA-binding protein MEX3A regulates murine adult neurogenesis

et al. 2023

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Neural stem cells (NSCs) in the adult murine subependymal zone balance their self-renewal capacity and glial identity with the potential to generate neurons during the lifetime. Adult NSCs exhibit lineage priming via pro-neurogenic fate determinants. However, the protein levels of the neural fate determinants are not sufficient to drive direct differentiation of adult NSCs, which raises the question of how cells along the neurogenic lineage avoid different conflicting fate choices, such as self-renewal and differentiation. Here, we identify RNA-binding protein MEX3A as a post-transcriptional regulator of a set of stemness associated transcripts at critical transitions in the subependymal neurogenic lineage. MEX3A regulates a quiescence-related RNA signature in activated NSCs that is needed for their return to quiescence, playing a role in the long-term maintenance of the NSC pool. Furthermore, it is required for the repression of the same program at the onset of neuronal differentiation. Our data indicate that MEX3A is a pivotal regulator of adult murine neurogenesis acting as a translational remodeller.

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Section: Methodsmentioning

confidence: 99%

Post-transcriptional control of a stemness signature by RNA-binding protein MEX3A regulates murine adult neurogenesis

et al. 2023

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“…Time‐lapse video microscopy and single‐cell tracking of primary SEZ cultures (Schroeder, 2008; Ortega et al , 2013; Ortega & Costa, 2016; Gómez‐Villafuertes et al , 2017; Paniagua‐Herranz et al , 2020) was performed with a cell observer (Carl Zeiss) at a constant temperature of 37°C and 8% CO 2 . Phase contrast images were acquired every 5 min for 6–10 days using a 20× phase contrast objective (Zeiss), an AxioCamHRm camera and a Zeiss AxioVision 4.7 software.…”

Section: Methodsmentioning

confidence: 99%

Astrogenesis in the murine dentate gyrus is a life‐long and dynamic process

et al. 2022

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Astrocytes are highly abundant in the mammalian brain, and their functions are of vital importance for all aspects of development, adaption, and aging of the central nervous system (CNS). Mounting evidence indicates the important contributions of astrocytes to a wide range of neuropathies. Still, our understanding of astrocyte development significantly lags behind that of other CNS cells. We here combine immunohistochemical approaches with genetic fate‐mapping, behavioural paradigms, single‐cell transcriptomics, and in vivo two‐photon imaging, to comprehensively assess the generation and the proliferation of astrocytes in the dentate gyrus (DG) across the life span of a mouse. Astrogenesis in the DG is initiated by radial glia‐like neural stem cells giving rise to locally dividing astrocytes that enlarge the astrocyte compartment in an outside‐in‐pattern. Also in the adult DG, the vast majority of astrogenesis is mediated through the proliferation of local astrocytes. Interestingly, locally dividing astrocytes were able to adapt their proliferation to environmental and behavioral stimuli revealing an unexpected plasticity. Our study establishes astrocytes as enduring plastic elements in DG circuits, implicating a vital contribution of astrocyte dynamics to hippocampal plasticity.

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“…After treatment, cells were tracked using a NIKON TE-2000 microscope and equipped with a long-distance 20x phase contrast objective (Nikon) and a ZYLA camera from ANDOR [ 33 ]. Cells were kept at a constant temperature of 37 °C and in 5% CO 2 , and images were acquired every 5 min over 3 h using the 4.7/NIS-elements software from NIKON.…”

Section: Methodsmentioning

confidence: 99%

Nucleotide-Induced Nanoscale Changes in the Mechanical Properties of Rat Cerebellar Astrocytes: Selective Stimulation and Blocking of the Purinergic Receptor P2X7

Gil-Redondo

Iturri

Trueba

et al. 2022

IJMS

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As members of the family of nucleotide receptors, P2X7 receptors are of particular interest due to their unique structural and pharmacological characteristics. As ATP-gated ionic channels, P2X7 receptors in their activation elicit membrane depolarization; extracellular calcium influx; and activation of several downstream intracellular signaling pathways, some of them independent of the ionic channel activity. Further interactions of P2X7 receptors and cytoskeleton-related proteins have also been confirmed, and we previously described the effects of P2X7 receptor stimulation on the morphology of rat cerebellar astrocytes. In the present work, we used time-lapse video microscopy and atomic force microscopy (AFM) to elucidate the effects of P2X7 receptor stimulation on the morphology, migratory capabilities, and mechanical properties of rat cerebellar astrocytes in vitro. Stimulation of P2X7 receptors with the selective agonist BzATP specifically caused an increase in cell size, motility, and number of membrane protrusions of the astrocytes in culture. These effects were reverted when cells were previously treated with the competitive antagonist of P2X7R, A 438079. AFM analysis also showed an increase in cell stiffness and viscosity after P2X7 receptor stimulation. Surprisingly, these effects on the mechanical properties of the cell were not blocked by the treatment with the antagonist. Fluorescence microscopy analysis of the actin cytoskeleton showed an increase in actin stress fibers after BzATP treatment, an effect that again was not blocked by previous treatment with the antagonist, further confirming that the effects of P2X7 receptors on the cytoskeleton of astrocytes are, at least in part, independent of the ionic channel activity.

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Time-Lapse Video Microscopy and Single Cell Tracking to Study Neural Cell Behavior In Vitro

Cited by 8 publications

References 20 publications

Post-transcriptional control of a stemness signature by RNA-binding protein MEX3A regulates murine adult neurogenesis

Post-transcriptional control of a stemness signature by RNA-binding protein MEX3A regulates murine adult neurogenesis

Astrogenesis in the murine dentate gyrus is a life‐long and dynamic process

Nucleotide-Induced Nanoscale Changes in the Mechanical Properties of Rat Cerebellar Astrocytes: Selective Stimulation and Blocking of the Purinergic Receptor P2X7

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