We used a combination of immunohistochemistry and carbocyanine dye tracing to study neurons and their processes in the human embryonic forebrain, 4 -7 weeks after conception, before the onset of synaptogenesis. We discovered a widespread network of precocious MAP2 (microtubule-associated protein 2)-immunoreactive cells, with long, nonaxonal processes, before the appearance of the cortical plate and the establishment of thalamocortical connectivity. Dye tracing revealed that the processes of these precocious cells form tangential links between intermediate zones of the thalamus, ganglionic eminence, hypothalamus, and cortical preplate. The spatiotemporal distribution and morphology of the precocious neurons in the cortical preplate suggest that they are generated outside the cerebral wall rather than in the local ventricular zone. The first thalamocortical axons and axons of preplate cells extend across diencephalotelencephalic and striatocortical boundaries before the arrival of the first cortical plate neurons. Precocious cells may provide initial communication between subdivisions of the embryonic brain as well as guidance cues for navigation of growing axons and/or transverse neuronal migration.
The aim of the present work was to develop optimal protocols for immunocytochemical reactions for nuclear protein NeuN for light and laser confocal microscopy which avoid the thermal antigen demasking procedure, which degrades the state of the tissue and requires use of expensive adhesive-coated slices. Maximal antigen retention was obtained after fixation in zinc-formalin and Bouin's fluid (maximum one day). Two protocols are proposed allowing the thermal demasking procedure to be avoided during detection of neuron marker NeuN on paraffin sections examined by light and confocal microscopy.
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