Mobilization of the cell adhesion glycoprotein F3/contactin to axonal surfaces is activity dependent

F3/contactin is a cell adhesion/recognition molecule of the immunoglobulin superfamily implicated in axonal growth. We examined its subcellular distri bution and mobilization to the cell surface in oxytocin- (OT-) secreting ne urons, which express it throughout life and the axons of which undergo acti vity-dependent remodelling. This was performed in hypothalamic organotypic slice cultures containing OT neurons with properties of adult neurosecretor y cells. Immunocytochemistry and immunoblot analysis confirmed that OT neur ons express high levels of F3/contactin in vitro. Light and confocal micros copy of cultures that underwent double immunofluorescence after fixation sh owed F3/contactin immunoreactivity throughout the cytoplasm of OT somata, d endrites and axons, and also in non-OT axons and in putative synaptic bouto ns which contacted OT neurons. By contrast, after treatment of live culture s with anti-F3/contactin antibodies followed by double immunofluorescence f or the glycoprotein and OT, F3/contactin immunoreactivity was visible only on the surface of axons, whether or not OT-immunoreactivity was present. Be cause of its glycosylphosphatidyl-inositol (GPI) linkage, F3/contactin can occur in a membrane-bound or soluble form. As seen from immunocytochemistry of live cells and immunoblot analysis, treatment of cultures with a GPI-sp ecific phospholipase C (GPI-PLC) resulted in loss of F3/contactin immunorea ctivity from all cell surfaces. F3/contactin immunoreactivity reappeared on axonal surfaces within 5 h after enzyme washout. Such re-expression was ac celerated by neuronal activity facilitation (by K+ depolarization or gamma -aminobutyric acid (GABA)-A receptor blockade with bicuculline) and inhibit ed by neuronal activity repression [by blockade of Ca2+, channels with Mn2, Na+ channels with tetrodotoxin (TTX) or excitatory inputs with glutamate antagonists]. Our observations establish therefore that F3/contactin surfac e expression in hypothalamic neurons is polarized to the axons where it occ urs mainly in a GPI-linked form. We also provide direct evidence that exter nalization of F3/contactin depends on Ca2+ entry and neuronal electrical ac tivity. Taken together with our earlier finding that the glycoprotein is lo calized in neurosecretory granules, we demonstrate that F3/contactin is mob ilized to the axonal surface via the activity-dependent regulated pathway, thus arriving at the correct place and time to intervene in activity-depend ent remodelling of axons.