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 distribution and mobilization to the cell surface in oxytocin- (OT-) secreting neurons, which express it throughout life and the axons of which undergo activity-dependent remodelling. This was performed in hypothalamic organotypic slice cultures containing OT neurons with properties of adult neurosecretory cells. Immunocytochemistry and immunoblot analysis confirmed that OT neurons express high levels of F3/contactin in vitro. Light and confocal microscopy of cultures that underwent double immunofluorescence after fixation showed F3/contactin immunoreactivity throughout the cytoplasm of OT somata, dendrites and axons, and also in non-OT axons and in putative synaptic boutons which contacted OT neurons. By contrast, after treatment of live cultures with anti-F3/contactin antibodies followed by double immunofluorescence for the glycoprotein and OT, F3/contactin immunoreactivity was visible only on the surface of axons, whether or not OT-immunoreactivity was present. Because 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-specific phospholipase C (GPI-PLC) resulted in loss of F3/contactin immunoreactivity from all cell surfaces. F3/contactin immunoreactivity reappeared on axonal surfaces within 5 h after enzyme washout. Such re-expression was accelerated by neuronal activity facilitation (by K+ depolarization or gamma-aminobutyric acid (GABA)-A receptor blockade with bicuculline) and inhibited 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 surface expression in hypothalamic neurons is polarized to the axons where it occurs mainly in a GPI-linked form. We also provide direct evidence that externalization of F3/contactin depends on Ca2+ entry and neuronal electrical activity. Taken together with our earlier finding that the glycoprotein is localized in neurosecretory granules, we demonstrate that F3/contactin is mobilized to the axonal surface via the activity-dependent regulated pathway, thus arriving at the correct place and time to intervene in activity-dependent remodelling of axons.
Pierre
K
K
INSERM U378 Neurobiologie Morphofonctionnelle, Institut François Magendie, University Victor Segalen Bordeaux II, Camille Saint-Saëns, F-33077 Bordeaux Cedex, France.
Dupouy
B
B
Allard
M
M
Poulain
D A
DA
Theodosis
D T
DT
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002448:Cell Adhesion / Q000502:physiology*; D015816:Cell Adhesion Molecules, Neuronal / Q000378:metabolism*; D002450:Cell Communication / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism*; D058966:Contactins; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005260:Female; D005455:Fluorescent Antibody Technique; D018756:GABA Antagonists / Q000494:pharmacology; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D020439:Growth Cones / Q000378:metabolism* / Q000648:ultrastructure; D007031:Hypothalamus / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism; D008297:Male; D009473:Neuronal Plasticity / Q000502:physiology; D009924:Organ Culture Techniques; D010121:Oxytocin / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D021381:Protein Transport / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D026941:Sodium Channel Blockers; D010738:Type C Phospholipases / Q000494:pharmacology
D002121:Calcium Channel Blockers; D015816:Cell Adhesion Molecules, Neuronal; D058966:Contactins; D018691:Excitatory Amino Acid Antagonists; D018756:GABA Antagonists; D017261:Glycosylphosphatidylinositols; D026941:Sodium Channel Blockers; D010121:Oxytocin; D010738:Type C Phospholipases; D011188:Potassium; D002118:Calcium