In co-cultures prepared from the septum and the hippocampus, cholinergic fibers originating in the septal slices grew into the neighboring hippocampal tissue and established functional cholinergic connections with pyramidal cells. To get further insight into the mechanisms governing cholinergic fiber growth, we have added TTX to the growth medium (2 x 10(-7) M) to block propagated electrical activity. Under these conditions, considerably fewer cholinergic cells appeared to survive. A few cholinergic fibers still invaded hippocampal target tissue, but their number was markedly reduced compared with control cultures. Simultaneous application of NGF together with TTX, however, not only increased enzyme levels and enhanced survival of cholinergic neurons, but also led to hippocampal ingrowth in virtually all septo-hippocampal co-cultures. These data, therefore, suggest, that in the absence of spiking activity, cholinergic fibers are capable of growing into a co-cultured target tissue. To test the specificity of growth of septal cholinergic fibers, we have co-cultured septal slices with slices of various brain areas which in situ lack a major cholinergic innervation, in particular the cerebellum. In the vast majority of such co-cultures, cholinergic fibers remained restricted within the septal slices, without innervating cerebellar tissue. This failure might in part be related to the lack of trophic factors released by the target tissue. We have, therefore, grown septo-cerebellar cultures in the presence and absence of NGF. Following application of 100 ng/ml NGF during the entire growth of the cultures, numerous AChE-positive fibers originating in the septal slices invaded the co-cultured cerebellar slices.(ABSTRACT TRUNCATED AT 250 WORDS)