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

Abstract

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."

Nicotine-Like Effects of the Neonicotinoid Insecticides Acetamiprid and Imidacloprid on Cerebellar Neurons from Neonatal Rats

“…We confirmed that the excited small round cells were immunoreactive for L1 (right column of Fig. 3), which is known to be a cerebellar granule cell marker [19].…”

“…Almost all of these small neurons were identified to be cerebellar granule cells based on their morphology and that they were stained by anti-neural cell adhesion molecule L1 (Fig. 1A) [19]. The cerebellar cultures also contained a few large, Tuj1-positive Purkinje neurons (about 1%, arrow in Fig.…”

“…We identified these excited neurons to be granule cells, as they are small in size, round-shaped, and immunoreactive for L1 [19]. During the perinatal stage in rodents and humans, transient expression of nAChRs has been observed in the cerebellum [14].…”

Topography, Cell Response, and Nerve Regeneration

“…It appears that this choice depends both on substrate dimension and cell type, and it may be that cellular processes of a certain type resist conforming to features which are too closely spaced and too deep, and instead cross between adjacent features, resulting in perpendicular contact guidance. This particular phenomenon has also been reported in vivo (69), and in two-dimensional (2D), three-dimensional (3D), microexplant and organotypic cerebellar cultures (70). …”

Roles of Chondroitin Sulfate and Dermatan Sulfate in the Formation of a Lesion Scar and Axonal Regeneration after Traumatic Injury of the Mouse Brain

“…Cerebellar neurons were prepared from neonatal rat cerebella that were dissected and digested with papain. 22 The mixed cultures were cultivated for 2 days, fixed with 4% PFA, and immunostained with each specific antibody under equal conditions. All the co-culture experiments were performed four times, and representative results are shown in the Figures.…”