Ca²⁺ and frequency dependence of exocytosis in isolated somata of magnocellular supraoptic neurones of the rat hypothalamus

In addition to action potential-evoked exocytotic release at neurohypophysial nerve terminals, the neurohormones arginine vasopressin (aVP) and oxytocin (OT) undergo Ca²⁺-dependent somatodendritic release within the supraoptic and paraventricular hypothalamic nuclei. However, the cellular and molecular mechanisms that underlie this release have not been elucidated. In the present study, the whole-cell patch-clamp technique was utilized in combination with high-time-resolved measurements of membrane capacitance (C_m) and microfluorometric measurements of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) to examine the Ca ²⁺ and stimulus dependence of exocytosis in the somata of magnocellular neurosecretory cells (MNCs) isolated from rat supraoptic nucleus (SON). Single depolarizing steps (≥20 ms) that evoked high-voltage-activated (HVA) Ca²⁺ currents (I _Ca) and elevations in intracellular Ca²⁺ concentration were accompanied by an increase in C_m in a majority (40/47) of SON neurones. The C_m responses were composed of an initial Ca²⁺ -independent, transient component and a subsequent, sustained phase of increased C_m (termed ΔC_m) mediated by an influx of Ca²⁺, and increased with corresponding prolongation of depolarizing step durations (20-200 ms). From this relationship we estimated the rate of vesicular release to be 1533 vesicles s^-1. Delivery of neurone-derived action potential waveforms (APWs) as stimulus templates elicited I _Ca and also induced a ΔC_m, provided (APWs) were applied in trains of greater than 13 Hz. A train of APWs modelled after the bursting pattern recorded from an OT-containing neurone during the milk ejection reflex was effective in supporting an exocytotic ΔC_m in isolated MNCs, indicating that the somata of SON neurones respond to physiological patterns of neuronal activity with Ca²⁺-dependent exocytotic activity.