Target- and Maturation-Specific Membrane-Associated Molecules Determine the Ingrowth of Entorhinal Fibers into the Hippocampus

Abstract

AbstractIn this study the role of membrane-associated molecules involved in entorhinohippocampal pathfinding was examined. First outgrowth preferences of entorhinal neurites were analyzed on membrane carpets obtained from their proper target area, the hippocampus, and compared to preferences on control membranes from brain regions which do not receive afferent connections from the entorhinal cortex. On a substrate consisting of alternating lanes of hippocampal and control membranes, entorhinal neurites exhibited a strong tendency to grow on lanes of hippocampal membrane. These tissue-specific outgrowth preferences were maintained even on membrane preparations from adult brain tissue devoid of myelin. To determine the possible maturation dependence of these membranes, we examined guidance preferences of entorhinal neurites on hippocampal membranes of different developmental stages ranging from embryonic to postnatal and adult. Given a choice between alternating lanes of embryonic (E15–E16) and neonatal (P0–P1) hippocampal membranes, entorhinal neurites preferred to extend on neonatal membranes. No outgrowth preferences were observed on membranes obtained between E19 and P10. From P10 onward there was a reoccurrence of a preference for postnatal membrane lanes when neurites were presented with a choice between P15, P30, and adult membranes (>P60). This choice behavior of entorhinal neurites temporally correlates with the ingrowth of the perforant path into the hippocampus and with the stabilization of this brain area in vivo. Experiments in which postnatal and adult hippocampal membranes were heat inactivated or treated to remove molecules sensitive to phosphatidylinositol-specific phospholipase C demonstrated that entorhinal fiber preferences were controlled in this assay by attractive guidance cues and were independent of phosphatidylinositol-sensitive linked molecules. Moreover, entorhinal neurites displayed a positive discrimination for membrane-associated guidance cues of their target field, thus preferring to grow on membranes from the molecular layer of the dentate gyrus compared with CA3 or hilus membranes. Heat-inactivation experiments indicated that preferential growth of entorhinal axons is due to a specific attractivity of the molecular layer substrate. The data presented demonstrate that outgrowth of entorhinal fibers on hippocampal membranes is target and maturation dependent