AXONS REGENERATE WITH CORRECT SPECIFICITY IN HORIZONTAL SLICE CULTUREOF THE POSTNATAL RAT ENTORHINO-HIPPOCAMPAL SYSTEM

We have used slice culture of the entorhino-hippocampal system to inve stigate (1) whether nerve fibres which are cut postnatally are able to regenerate and (2) whether the regenerating fibres are able to establ ish correct selective target specificity in the formation of their ter minal fields. Slices of tissue were taken in the horizontal plane thro ugh the caudo-ventral pole of the cerebral hemisphere of 9- to 10-day- old rats. Such slices maintain the entorhinal cortex in continuity wit h the hippocampus and intervening retrohippocampal areas. However, bec ause of the dorsal inclination of the entorhino-hippocampal projection fibres in situ, the segments of the entorhinal cortex and hippocampus contained within each individual horizontal slice were disconnected f rom each other. During subsequent culture, the formation of fibre conn ections between the entorhinal area and the hippocampal complex was st udied by the extracellular and intracellular anterograde transport of biocytin or biotin dextran, the retrograde transport of biotin dextran or carbocyanine dyes, and by electrical stimulation and recording. Fo r the first 24 h after taking the slice, there were no entorhinal proj ections beyond the deep white matter, and no fibres reached the hippoc ampus or dentate gyrus. After 3 days in culture a small number of grow ing fibres had perforated the subiculum and entered the target areas. Between 6 and 14 days these projections increased and matured. As in t he normal adult brain, entorhinal layer II stellate cells projected co rrectly to the dentate gyrus and hippocampal field CA3, whereas layer III pyramidal cells projected to hippocampal field CA1 and the subicul um. The new fibres grew along both alvear and perforant pathways. Ante rograde and retrograde labelling showed that the reciprocal projection s from the pyramidal cells of the subiculum and CA1 to the entorhinal area had also been severed at the time of taking the slices, and had s imilarly regenerated. Our results demonstrate that by taking tissue sl ices in appropriate planes it is possible to study the regeneration of axons in the tissue environment through which they normally run. This approach avoids the use of coculture and the concomitant difficulties associated with the need for fibres to cross a coculture interface. I n horizontal slices of postnatal tissue, severed fibre projections bet ween the entorhinal cortex and the hippocampal complex can regenerate in both directions and re-establish their correct laminar, pathway and target specificity.