During development, thalamocortical axons form arbors primarily in layer 4 of the neocortex. This lamina-specific branch formation was studied in cultures of rat thalamic explants grown next to chemically fixed cortical slices. After a week in vitro, thalamic axons formed branches specifically in the target layer of fixed cortical slices, regardless of the orientation of the ingrowth. This in vitro system permits a direct assessment of contributions of membrane-associated molecules to thalamic axon branch formation. To this end, the present study uses three enzymatic perturbations: chondroitinase, phosphatidylinositol phospholipase C, or the polysialic acid (PSA)-specific endoneuraminidase (endo N). With endo N pretreatment of cortex, the number of branch points was increased significantly, whereas branch tip length was decreased. In addition, the localization of branch points to the target layer was weakened considerably. These features of branch formation were not altered by the other two enzymatic treatments, except that branch tips were shortened by chondroitinase treatment to the same extent as in endo N treatment. These results suggest that membrane-bound components are involved in lamina-specific branch formation of thalamocortical axons, and in particular that PSA moieties contribute to laminar specificity by inhibiting branch emergence in inappropriate layers.
During development, most thalamocortical axons extend through the deep layers to terminate in layer 4 of neocortex. To elucidate the molecular mechanisms that underlie the formation of layer-specific thalamocortical projections, axon outgrowth from embryonic rat thalamus onto postnatal neocortical slices which had been fixed chemically was used as an experimental model system. When the thalamic explant was juxtaposed to the lateral edge of fixed cortical slice, thalamic axons extended farther in the deep layers than the upper layers. Correspondingly, thalamic axons entering from the ventricular side extended farther than those from the pial side. In contrast, axons from cortical explants cultured next to fixed cortical slices tended to grow nearly as well in the upper as in the deep layers. Biochemical aspects of lamina-specific thalamic axon growth were studied by applying several enzymatic treatments to the cortical slices prior to culturing. Phosphatidylinositol phospholipase C treatment increased elongation of thalamic axons in the upper layers without influencing growth in the deep layers. Neither chondroitinase, heparitinase, nor neuraminidase treatment influenced the overall projection pattern, although neuraminidase slightly decreased axonal elongation in the deep layers. These findings suggest that glycosylphosphatidylinositol-linked molecules in the cortex may contribute to the laminar specificity of thalamocortical projections by suppressing thalamic axon growth in the upper cortical layers.
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