Organotypic Co-Cultures of Rat Locus Coeruleus and Hippocampus

Slices from the brainstem at the level of the locus coeruleus and from the hippocampus of 5 - 7 day old rats were co-cultured using the roller tube technique. After 2 - 6 weeks in vitro the co-cultures were examined with antibodies raised against tyrosine-hydroxylase (TH). The cultures derived from the brainstem consistently contained a bilateral cluster of TH-positive neurons with 3 - 5 long slender dendrites. These neurons typically gave rise to several fine varicose fibres reminiscent of catecholaminergic axons. A morphologically distinct group of TH-positive neurons was detected in the hippocampal slices. The vast majority of them were located in the subicular region and a smaller number in the CA1/CA3 region of the hippocampal explant. TH-positive neurons were also present in mono-cultures of hippocampus or brainstem. In the vast majority of co-cultures, a variable number of TH-immunoreactive fibres of neurons derived from the locus coeruleus grew over considerable distances to terminate finally within the co-cultured hippocampus where they branched to form a diffuse innervation plexus with club-like endings. Even after several weeks in vitro, TH-positive fibres could still be seen exploring sites which were not related to their target, including the cell-free areas surrounding the cultures. Fibres in these outgrowth areas formed whirl-like endings. TH-positive fibres arising from neurons located in the hippocampus, on the other hand, did not branch extensively and never projected over long distances. Nerve growth factor had no apparent trophic effect on TH-positive cells in the hippocampus, the locus coeruleus, or in the co-cultures.