Entorhinal axons project to dentate gyrus in organotypic slice co-culture.

We have demonstrated the formation of entorhinodentate projections by axons arising from explants of embryonic mouse entorhinal cortex or slices of postnatal rat entorhinal area co-cultured in contact with slices of postnatal rat hippocampus in roller tube and static culture. Species-specific markers (Thy-1 alleles and M6) showed that the most dense part of the projection was to the outer part of the molecular layer of the dentate gyrus (i.e. excluding the commissural-association zone). Retrograde axonal transport of fluorescent tracers placed in the dentate gyrus labelled a densely packed superficial layer of stellate cells in the entorhinal cortex. Anterograde axonal transport of biocytin placed in the entorhinal cortex showed that the entorhinodentate fibres formed typical parallel bundles oriented at right angles to the dentate granule cell dendrites and had short-stalked boutons. The formation of entorhinodentate synapses was confirmed in the electron microscope by electron-dense degeneration after cutting the previously formed connection between the co-cultures. Synaptic transmission was demonstrated by extracellular recording of postsynaptic field potentials after entorhinal stimulation. The entorhinal fibres also projected to the hippocampal stratum lacunosum-moleculare of fields CA1 and CA3, and were present in the outer part of the stratum oriens of the subiculum; in some cases they perforated the pyramidal cell layer of the subiculum. We conclude that the necessary molecular and tissue organizational signals for the formation of an entorhinodentate projection are present in tissues maintained in organotypic slice co-culture, and remain effective in the cross-species mouse-to-rat situation.

Selective growth of hippocampal neurites on cryostat sections of rat brain.

Dissociated rat hippocampal neurons were cultured on horizontal cryostat sections from neonatal and adult rat brain and their growth patterns visualized by brightfield and interference contrast microscopy. Cells adhered to the sections as individuals or in small clusters and grew extensive neurites. Neurites grew over all areas of neonatal sections without apparent selectivity. For adult sections, however, neurites grew almost exclusively on areas of grey matter: there was no neurite growth on areas of white matter, irrespective of the location of that white matter within the brain. The transition from the neonatal to the adult pattern of growth occurred for sections from animals aged between 14 and 21 days.

A silver impregnation technique for normal axons in the human central nervous system for celloidin and epon sections, with substitutes for soft tap water.

An improved silver technique has been developed for human CNS axons in sections from celloidin blocks that resist impregnation because of prolonged storage in alcohol. This method also gives consistently good impregnation of recently fixed material, and thus is suitable for routine use. Slightly modified, the method is also successful with osmicated Epon embedded sections. The quality of silver impregnation in methods using tap water in the reducing solutions varies in different laboratories. Having established that hard water is essential, substitutes for soft water were sought and found.