Aligned neurite bundles of granule cells regulate orientation of Purkinje cell dendrites by perpendicular contact guidance in two-dimensional and three-dimensional mouse cerebellar cultures.
To identify structures that determine the 90 degree orientation of thin espalier dendritic trees of Purkinje cells with respect to parallel fibers (axonal neurite bundles of granule cells) in the cerebellar cortex, we designed five types of two-dimensional and three-dimensional cell and tissue cultures of cerebella from postnatal mice and analyzed the orientation of Purkinje cell dendrites with respect to neurite bundles and astrocyte fibers by immunofluorescence double or triple staining. We cultured dissociated cerebellar cells on micropatterned substrates and preformed neurite bundles of a microexplant culture two-dimensionally and in matrix gels three-dimensionally. Dendrites, but not axons, of Purkinje cells extended toward the neurites of granule cells and oriented at right angles two-dimensionally to aligned neurite bundles in the three cultures. In a more organized explant proper of the microexplant culture, Purkinje cell dendrites extended toward thin aligned neurite bundles not only consistently at right angles but also two-dimensionally. However, in the "organotypic microexplant culture," in which three-dimensionally aligned thick neurite bundles mimicking parallel fibers were produced, Purkinje cell dendrites often oriented perpendicular to the thick bundles three-dimensionally. Astrocytes were abundant in all cultures, and there was no definite correlation between the presence of and orientation to Purkinje cell dendrites, although their fibers were frequently associated in parallel with dendrites in the organotypic microexplant culture. Therefore, Purkinje cells may grow their dendrites to the newly produced neurite bundles of parallel fibers in the cerebellar cortex and be oriented at right angles three-dimensionally mainly via "perpendicular contact guidance."
Nagata
Isao
I
Department of Brain Structure, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183-8526, Japan. nagata@tmin.ac.jp
Ono
Katsuhiko
K
Kawana
Akio
A
Kimura-Kuroda
Junko
J
D000818:Animals; D000831:Animals, Newborn; D064026:Calbindins; D002450:Cell Communication / Q000502:physiology; D002452:Cell Count / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods*; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D018920:Coculture Techniques / Q000379:methods; D003712:Dendrites / Q000502:physiology* / Q000648:ultrastructure; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D046529:Microscopy, Electron, Transmission / Q000379:methods; D039842:Neural Cell Adhesion Molecule L1 / Q000378:metabolism; D016501:Neurites / Q000502:physiology* / Q000648:ultrastructure; D011689:Purkinje Cells / Q000166:cytology* / Q000502:physiology; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
D064026:Calbindins; D005904:Glial Fibrillary Acidic Protein; D039842:Neural Cell Adhesion Molecule L1; D064030:S100 Calcium Binding Protein G; D014404:Tubulin; C462941:beta3 tubulin, mouse