TRACING OF THE ENTORHINAL-HIPPOCAMPAL PATHWAY IN-VITRO

In vitro tract tracing allowing for continuous observation of the perf orant path is a crucial prerequisite for experimental studies on the e ntorhinal-hippocampal interaction in an organotypic slice culture cont aining the entorhinal cortex, the perforant path, and the dentate gyru s (OEHSC). We prepared horizontal slices of the temporal entorhinal-hi ppocampal region of the rat on a vibratome, and the perforant path axo ns were traced by application of the fluorescent tracer Mini Ruby on t he entorhinal cortex. After 2 days in vitro (div), the perforant path became visible in most cultures. Entorhinal neurons and single perfora nt fibers could be followed to the outer molecular layers of the denta te gyrus by in vitro fluorescence microscopy and it was possible to mo nitor the perforant path directly over a period of 25 div. Moreover, u ltrastructural analysis proved the existence of traced perforant path boutons forming synapses with spines and dendritic shafts in the outer molecular layers of the dentate gyrus. Transsection of the prelabelle d perforant path in vitro resulted in anterograde degeneration and sub sequent phagocytosis of axonal material by activated microglial cells in the zone of denervation. In conclusion, in vitro tracing demonstrat es the maintenance of the entorhinal-hippocampal pathway in OEHSCs and permits monitoring of dynamic changes in the prelabeled perforant pat h after various lesion paradigms, e.g., transsection or neurotoxin tre atment. This approach permits further studies on the efficacy of neuro protectants, cytokines, and growth factors in the treatment of lesion- induced neuronal degeneration. (C) 1998 Wiley-Liss, Inc.