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 elect rical 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 immu nostaining with light and confocal microscopy showed that the somata and de ndrites of oxytocinergic neurons were contacted by numerous synapses, visua lized by their reaction to the synaptic markers, synaptophysin or synapsin, Many were GABAergic, displaying immunoreactivities for glutamic acid decar boxylase or gamma-aminobutyric acid (GABA); others were enriched in glutama te immunoreactivity. Such afferents presumably arose from GABA- or glutamat e-immunoreactive neurons, respectively, with distinct and characteristic mo rphologies and topographies. A few dopaminergic boutons (tyrosine hydroxyla se- or dopamine-immunopositive) impinged on OT neurons; they arose from dop amine-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-relate d 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 inn ervated by hypothalamic dopaminergic neurons. Finally, they confirm the exi stence of homotypic OT synaptic contacts which derive from local OT neurons .