Development of the raphe-hippocampal projection in vitro

In this study we examined whether the serotonergic raphe-hippocampal projection preserves its characteristic target selectivity for GABAergic interneurons when developing in vitro, in organotypic cultures. Hippocampal slices from one- to three-day-old rats were co-cultured with slices derived from the raphe nuclei of the same animals. After several weeks of in vitro incubation, a large number of raphe fibres--visualized by immunostaining for serotonin--were found to innervate the hippocampal tissue. In our random sample of over 250 serially sectioned boutons--52 of which were completely reconstructed from serial sections--only two were found to form conventional synapses in the electron microscope, and contacted dendritic spines. These results demonstrate that raphe-hippocampal serotonergic afferent are unable to form synaptic contacts with their normal targets in vitro, if explanted one to three days postnatally. Neurons in the afferent and/or target area may have passed a critical age when selective synaptic contacts can be formed, or unknown chemical or electrical signals may be missing under these conditions, which should serve to guide subcortical afferents to their synaptic target elements.