title: Live imaging of microtubule dynamics in organotypic hippocampal slice cultures.
abstract: The microtubule (MT) cytoskeleton plays an active role during different phases of neuronal development and is an essential structure for stable neuronal morphology. MTs determine axon formation, control polarized cargo trafficking, and regulate the dynamics of dendritic spines, the major sites of excitatory synaptic input. Defects in MT function have been linked to various neurological and neurodegenerative diseases and recent studies highlight neuronal MTs as a potential target for therapeutic intervention. Thus, understanding MT dynamics and its regulation is of central importance to study many aspects of neuronal function. The dynamics of MT in neurons can be studied by visualizing fluorescently tagged MT plus-end tracking proteins (+TIPs). Tracking of +TIP trajectories allows analyzing the speeds and directionality of MT growth in axons and dendrites. Numerous labs now use +TIP to track growing MTs in dissociated neuron cultures. This chapter provides detailed methods for live imaging of MT dynamics in organotypic hippocampal slice cultures. We describe protocols for culturing and transducing organotypic slices and imaging MT dynamics by spinning disk confocal microscopy.
authors.affiliation: Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Philipp
authors.initials: P
authors.lastname: Schätzle
authors.affiliation: Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Lukas C
authors.initials: LC
authors.lastname: Kapitein
authors.affiliation: Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Casper C
authors.initials: CC
authors.lastname: Hoogenraad
mesh_terms: D000818:Animals; D001369:Axons / Q000378:metabolism*; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism*; D003712:Dendrites / Q000378:metabolism*; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000254:growth & development; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D009924:Organ Culture Techniques; D051381:Rats; D013194:Staining and Labeling; D014161:Transduction, Genetic
chemical_list: D008869:Microtubule-Associated Proteins
keywords: Dynamic microtubule; Lentivirus; Neuron; Organotypic slice culture; Spinning disk confocal