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 molecules participate in dynamic neuronal functions in the developing and adult brain. The present study examines translocation of polysialylated neural cell adhesion molecule (PSA-NCAM), a negative regulator of cell adhesion, in cells of the rat hypothalamo-neurohypophysial system in which it is expressed throughout life and which undergo morphological remodelling in response to stimulation. PSA-NCAM expression in this system does not vary markedly in relation to different conditions of regulated neurosecretion, suggesting that 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 organotypic slice cultures from postnatal rat hypothalami. Subsequent immunolabelling for oxytocin confirmed that the cultures included magnocellular oxytocinergic neurons displaying many properties of adult neurosecretory neurons 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 surfaces 4h after enzyme washout. Such reexpression was visibly not affected by neuronal activity inhibition (blockade of Ca(2+) channels with Mn(2+), of Na(+) channels with tetrodotoxin, or of glutamate receptors with 6-cyano-7-nitroquinoxaline-2,3-dione or D-2-amino-5-phosphonopentanoic acid) or facilitation (K(+) depolarization or GABA-A receptor blockade with bicuculline). In contrast, PSA-NCAM surface translocation was inhibited reversibly by cooling the cultures at 20 degrees C, a procedure which blocks constitutive secretion and which resulted in accumulation of PSA-NCAM in the cytoplasm of oxytocinergic and non-oxytocinergic neurons. This treatment also revealed PSA-NCAM in the cytoplasm of underlying astrocytes. Our observations provide direct evidence that PSA-NCAM reaches the cell surface of hypothalamic neurons and astrocytes via the constitutive pathway, independently of Ca(2+) entry and enhanced neuronal activity. Thus, PSA-NCAM in the hypothalamo-neurohypophysial system would be continuously available to permit its cells to undergo remodelling whenever the proper stimulus intervenes.

Visualization of local afferent inputs to magnocellular oxytocin neurons in vitro.

We recently showed that oxytocin (OT) neurons in organotypic slice cultures obtained from postnatal rat hypothalamus display complex patterns of electrical activity, similar to those of adult magnocellular OT neurons in vivo. Here we used such cultures to investigate the identity and, in particular, the origin of afferent inputs responsible for this activity. Multiple immunostaining with light and confocal microscopy showed that the somata and dendrites of oxytocinergic neurons were contacted by numerous synapses, visualized by their reaction to the synaptic markers, synaptophysin or synapsin. Many were GABAergic, displaying immunoreactivities for glutamic acid decarboxylase or gamma-aminobutyric acid (GABA); others were enriched in glutamate immunoreactivity. Such afferents presumably arose from GABA- or glutamate-immunoreactive neurons, respectively, with distinct and characteristic morphologies and topographies. A few dopaminergic boutons (tyrosine hydroxylase- or dopamine-immunopositive) impinged on OT neurons; they arose from dopamine-positive neurons located along the third ventricle. No noradrenergic profiles were detected. Despite the presence of choline acetyl-transferase (ChAT)-immunoreactive neurons, there were no cholinergic contacts. Lastly, we found oxytocinergic synapses, identified by immunoreaction for OT-related neurophysin and synapsin, contacting OT somata and dendrites. Our observations thus demonstrate that inhibitory and excitatory inputs to OT neurons derive from local intrahypothalamic GABA and glutamate neurons, in close proximity to the neurons. They also reveal that OT neurons are innervated by hypothalamic dopaminergic neurons. Finally, they confirm the existence of homotypic OT synaptic contacts which derive from local OT neurons.

Regulated expression of the cell adhesion glycoprotein F3 in adult hypothalamic magnocellular neurons.

F3, a glycoprotein of the immunoglobulin superfamily implicated in axonal growth, occurs in oxytocin (OT)-secreting and vasopressin (AVP)-secreting neurons of the adult hypothalamo-neurohypophysial system (HNS) whose axons undergo morphological changes in response to stimulation. Immunocytochemistry and immunoblot analysis showed that during basal conditions of HNS secretion, there are higher levels of this glycosylphosphatidyl inositol-anchored protein in the neurohypophysis, where their axons terminate, than in the hypothalamic nuclei containing their somata. Physiological stimulation (lactation, osmotic challenge) reversed this pattern and resulted in upregulation of F3 expression, paralleling that of OT and AVP under these conditions. In situ hybridization revealed that F3 expression in the hypothalamus is restricted to its magnocellular neurons and demonstrated a more than threefold increase in F3 mRNA levels in response to stimulation. Confocal and electron microscopy localized F3 in secretory granules in all neuronal compartments, a localization confirmed by detection of F3 immunoreactivity in granule-enriched fractions obtained by sucrose density gradient fractionation of rat neurohypophyses. F3 was not visible on any cell surface in the magnocellular nuclei. In contrast, in the neurohypophysis, it was present not only in secretory granules but also on the surface of axon terminals and glia and in extracellular spaces. Taken together, our observations reveal that the cell adhesion glycoprotein F3 is colocalized with neurohypophysial peptides in secretory granules. It follows, therefore, the regulated pathway of secretion in HNS neurons to be released by exocytosis at their axon terminals in the neurohypophysis, where it may intervene in activity-dependent structural axonal plasticity.

Electrophysiological studies of oxytocin neurons in organotypic slice cultures.

We have developed organotypic slice cultures derived from postnatal rat hypothalamus which contain well-differentiated oxytocin neurons. Intracellular recordings of identified neurons show that these cultured oxytocin cells exhibit basal electrical properties closely similar to those of magnocellular cells recorded in vivo and in acute in vitro preparations from adult animals. The cultures also include GABAergic and glutamatergic neurons making connections with the oxytocin cells, which strongly suggests that the rich GABAergic and glutamatergic innervations of adult oxytocin neurons in vivo derive largely from local hypothalamic sources. Pharmacological manipulations indicate that the cultured oxytocin neurons present functional GABAA (but not GABAB) receptors, and ionotropic non-NMDA and NMDA receptors, but no metabotropic receptors for glutamate. These synaptic inputs control to a great extent the electrical activity of oxytocin neurons. Of particular interest is our observation that the cultured oxytocin neurons display a recurrent bursting activity which does not appear to result from an endogenous regenerative activity, but from a patterned glutamatergic input. Our preliminary data show that oxytocin plays a facilitatory role in this bursting activity and suggest that such activity is generated within an hypothalamic circuitry.