FACTORS INFLUENCING MOSSY FIBER COLLATERAL SPROUTING IN ORGANOTYPIC SLICE CULTURES OF NEONATAL MOUSE HIPPOCAMPUS

Collateral sprouting of dentate granule cell axons, the messy fibers, occurs in response to denervation, kindling, or excitotoxic damage to the hippocampus. Organotypic slice culture of rodent hippocampal tissu e is a model system for the controlled study of collateral sprouting i n vitro. Organotypic roller-tube cultures were prepared from hippocamp al slices derived from postnatal day 7 mice. The Timm heavy metal stai n and densitometry were used to assay the degree of messy fiber collat eral sprouting in the molecular layer of the hippocampal dentate gyrus . Factors influencing messy fiber collateral sprouting were time in cu lture, positional origin of the slice culture along the septotemporal axis of the hippocampus, and presence of attached subicular-entorhinal cortical tissues. Collateral sprouting in the molecular layer was fir st detected after 6 days in culture and increased steadily thereafter. By 2 weeks considerable sprouting was apparent, and at 3 weeks intens e sprouting was observed within the molecular layer. An intrinsic sept al-to-temporal gradient of collateral sprouting was apparent at 14 day s in culture. To determine whether differential damage to the messy fi bers was the basis for the differences in collateral sprouting along t he septotemporal axis, we made complete transections of the messy fibe r projection as it exited the dentate hilus at various levels along th e septotemporal axis; no differences were found on subsequent collater al sprouting in the dentate molecular layer. Timm-stained hippocampal cultures with an attached entorhinal cortex, a major source of afferen t innervation to the dentate granule cells, displayed significantly le ss collateral sprouting at 10 days in culture compared to that in cult ures from adjacent sections without attached subicular-entorhinal tiss ues present. Thus, time in culture, position along the septotemporal a xis, and presence of afferent cortical tissues influence aberrant neur ite collateral sprouting in organotypic slice cultures of neonatal mou se hippocampus. (C) 1995 Wiley-Liss, Inc.