Development of the septohippocampal projection in vitro

Slices were prepared from septal and hippocampal tissue and co-cultured for periods up to one month. The presence of cholinergic neurons within the septal slices was demonstrated by histochemical staining techniques for acetylcholinesterase or by Golgi-like immunoperoxidase techniques with antibodies raised against the enzyme choline acetyltransferase. Cholinergic fibers originating in the septal explants started to grow radially in all directions. By day 7, the first fibers were seen to reach their target, but maximal hippocampal ingrowth occurred between day 8 and 14 in vitro. Only those fibers reaching the target were maintained, whereas cholinergic fibers growing in other directions degenerated.

Electrophysiological studies showed that cholinergic fibers established functional cholinergic connections with hippocampal pyramidal cells. As a result of septal stimulation, two different potassium currents were inhibited in pyramidal cells: a calcium-independent current, I_M, and a calcium-dependent current, I_AHP, underlying spike afterhyperpolarization.

Application of nerve growth factor (NGF) strongly increased the number of cholinergic fibers which invaded the hippocampal slices and raised the activities of the cholinergic enzymes choline acetyltransferase and acetylcholinesterase, effects which were completely blocked by anti-NGF antibodies. The response of septohippocampal co-cultures to NGF depended on the time of application. During the first two weeks in vitro, NGF elicited sustained increases in enzyme activities, whereas later administration of NGF produced effects which were only maintained for several days.