The polysialylated neural cell adhesion molecule reaches cell surfaces of hypothalamic neurons and astrocytes via the constitutive pathway

Understanding how neurons and glia sort and deliver cell adhesion molecules to their cell surface should provide important clues as to how such molecu les participate in dynamic neuronal functions in the developing and adult b rain. The present study examines translocation of polysialylated neural cel l adhesion molecule (PSA-NCAM), a negative regulator of cell adhesion. in c ells of the rat hypothalamo-neurohypophysial system in which it is expresse d throughout life and which undergo morphological remodelling in response t o stimulation. PSA-NCAM expression in this system does not vary markedly in relation to different conditions of regulated neurosecretion, suggesting t hat the glycoprotein reaches cell surfaces via the constitutive pathway. To study this more directly, we here used immunofluorescence for PSA on NCAM in live, unpermeabilized cells to monitor PSA-NCAM surface expression in or ganotypic slice cultures from postnatal rat hypothalami. Subsequent immunol abelling for oxytocin confirmed that the cultures included magnocellular ox ytocinergic neurons displaying many properties of adult neurosecretory neur ons in situ. In the cultures, immunoreaction for PSA-NCAM was visible on the surface of oxytocinergic and non-oxytocinergic axons. This reaction disappeared after exposure of the cultures to endoneuraminidase, an enzyme which specifically cleaves alpha -2-8-linked PSA from NCAM. PSA-NCAM reappeared on axonal sur faces 4 h after enzyme washout. Such reexpression was visibly not affected by neuronal activity inhibition (blockade of Ca2+ channels with Mn2+, of Na + channels with tetrodotoxin, or of glutamate receptors with 6-cyano-7-nitr oquinoxaline-2.3-dione or D-2 amino-5-phosphonopentanoic acid) or facilitat ion (K+ depolarization or GABA-A receptor blockade with bicuculline). In co ntrast, PSA-NCAM surface translocation was inhibited reversibly by cooling the cultures at 20 degreesC, a procedure which blocks constitutive secretio n and which resulted in accumulation of PSA-NCAM in the cytoplasm of oxytoc inergic and non-oxytocinergic neurons. This treatment also revealed PSA-NCA M in the cytoplasm of underlying astrocytes. Our observations provide direct evidence that PSA-NCAM reaches the cell sur face of hypothalamic neurons and astrocytes via the constitutive pathway, i ndependently of Ca2+ entry and enhanced neuronal activity. Thus, PSA-NCAM i n the hypothalamo-neurohypophysial system would be continuously available t o permit its cells to undergo remodelling whenever the proper stimulus inte rvenes. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserv ed.