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Magnocellular neurosecretory cells (MNCs) clustered in the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus constitute a major source of oxytocin (OXT) and arginine vasopressin (AVP) peptides, and are among the best described peptidergic neurons in the brain. OXT and AVP are involved in a range of homeostatic processes, social behaviours, emotional processes, and learning. Notably, their actions can be sex-specific, and several sex differences in the anatomies of the OXT and AVP systems have been reported. Nonetheless, possible sex differences in the detailed distributions of MNCs and in their intrinsic electrical properties ex vivo have not been extensively examined. We addressed these issues utilizing immunostaining and patch-clamp ex vivo recordings. Here, we showed that Sprague-Dawley rat PVN AVP neurons are more numerous than OXT cells and that more neurons of both types are present in males. Furthermore, we identified several previously unreported differences between putative OXT and AVP MNC electrophysiology contributing to their partially unique profiles. Notably, elucidation of the highly specific action potential (AP) shapes, with AVP MNCs having a narrower AP and faster hyperpolarizing after-potential (HAP) kinetics than OXT MNCs, allowed unambiguous discrimination between OXT and AVP MNCs ex vivo for the first time. Moreover, the examined electrophysiological properties of male and female MNCs generally overlapped with the following exceptions: higher membrane resistance in male MNCs and HAP kinetics in putative OXT MNCs, which was slower in males. These reported observations constitute a thorough addition to the knowledge of MNC properties shaping their diverse physiological actions in both sexes.

Graphical abstract Trace (A) shows a typical transient [Ca2+]i response induced by 50 mM K+ observed in an AVP-eGFP neurone (inset), Trace (B) shows the spontaneous [Ca2+]i oscillations observed in an AVP-eGFP neurone (inset). The corresponding changes in the fluorescence are shown in C and D, respectively., Highlights • Supraoptic fluorescent vasopressin (AVP-eGFP) and oxytocin (OT-mRFP1) neurones exhibit distinct spontaneous [Ca2+]i oscillations. • Vasopressin triggers [Ca2+]i oscillations, intensifies existing oscillations, and exceptionally stops oscillations. • Hyper- or hypo-osmotic stimuli have an intensifying or inhibitory effect on oscillations, respectively. • Nearly 90% of neurones from 3 or 5-day-dehydrated rats exhibit oscillations. • More than 80% of OT-mRFP1 neurones from 3 to 6-day-lactating rats are oscillatory vs. about 44% in virgins., The magnocellular vasopressin (AVP) and oxytocin (OT) neurones exhibit specific electrophysiological behaviour, synthesise AVP and OT peptides and secrete them into the neurohypophysial system in response to various physiological stimulations. The activity of these neurones is regulated by the very same peptides released either somato-dendritically or when applied to supraoptic nucleus (SON) preparations in vitro. The AVP and OT, secreted somato-dendritically (i.e. in the SON proper) act through specific autoreceptors, induce distinct Ca2+ signals and regulate cellular events. Here, we demonstrate that about 70% of freshly isolated individual SON neurones from the adult non-transgenic or transgenic rats bearing AVP (AVP-eGFP) or OT (OT-mRFP1) markers, produce distinct spontaneous [Ca2+]i oscillations. In the neurones identified (through specific fluorescence), about 80% of AVP neurones and about 60% of OT neurones exhibited these oscillations. Exposure to AVP triggered [Ca2+]i oscillations in silent AVP neurones, or modified the oscillatory pattern in spontaneously active cells. Hyper- and hypo-osmotic stimuli (325 or 275 mOsmol/l) respectively intensified or inhibited spontaneous [Ca2+]i dynamics. In rats dehydrated for 3 or 5 days almost 90% of neurones displayed spontaneous [Ca2+]i oscillations. More than 80% of OT-mRFP1 neurones from 3 to 6-day-lactating rats were oscillatory vs. about 44% (OT-mRFP1 neurones) in virgins. Together, these results unveil for the first time that both AVP and OT neurones maintain, via Ca2+ signals, their remarkable intrinsic in vivo physiological properties in an isolated condition.

In addition to classical synaptic transmission, information is transmitted between cells via the activation of extrasynaptic receptors that generate persistent tonic current in the brain. While growing evidence supports the presence of tonic NMDA current (INMDA) generated by extrasynaptic NMDA receptors (eNMDARs), the functional significance of tonic INMDA in various brain regions remains poorly understood. Here, we demonstrate that activation of eNMDARs that generate INMDA facilitates the α-amino-3-hydroxy-5-methylisoxazole-4-proprionate receptor (AMPAR)-mediated steady-state current in supraoptic nucleus (SON) magnocellular neurosecretory cells (MNCs). In low-Mg(2+) artificial cerebrospinal fluid (aCSF), glutamate induced an inward shift in Iho lding (IGLU) at a holding potential (Vholding) of -70 mV which was partly blocked by an AMPAR antagonist, NBQX. NBQX-sensitive IGLU was observed even in normal aCSF at Vholding of -40 mV or -20 mV. IGLU was completely abolished by pretreatment with an NMDAR blocker, AP5, under all tested conditions. AMPA induced a reproducible inward shift in Iholding (IAMPA) in SON MNCs. Pretreatment with AP5 attenuated IAMPA amplitudes to ~60% of the control levels in low-Mg(2+) aCSF, but not in normal aCSF at Vholding of -70 mV. IAMPA attenuation by AP5 was also prominent in normal aCSF at depolarized holding potentials. Memantine, an eNMDAR blocker, mimicked the AP5-induced IAMPA attenuation in SON MNCs. Finally, chronic dehydration did not affect IAMPA attenuation by AP5 in the neurons. These results suggest that tonic INMDA, mediated by eNMDAR, facilitates AMPAR function, changing the postsynaptic response to its agonists in normal and osmotically challenged SON MNCs.