Autoregulatory effects of oxytocin (OT) and vasopressin (VP) on the activities of identified OT and VP neurons were studied in dissociated cell cultures.
Our aims were to: 1) establish in vitro preparations of dissociated postnatal magnocellular neurons (MCNs) from supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus; 2) identify the OT and VP phenotypes of the electrophysiologically-analyzed MCNs by immunocytochemistry; and 3) study the interactions of OT and VP with amino acid receptors and voltage-gated ion channels. Dissociated SON-MCNs cultured brain astrocytes and maintained for up to five weeks. Patch- clamp methods were used for both whole-cell current clamp and voltage- clamp analyses. Patch-pipettes were filled with various modified intracellular solutions containing 5 mM biocytin. The biocytin was used to identify the recorded cells. Neurotransmitters were applied to the cells through small pipettes by pressure pulses. To identify phenotypes of MCNs, antibodies raised to VP (VA4) and OT (PS36) were used. The best result was obtained using a triple fluorescent dye staining; VP-cells with goat anti-rabbit-FITC, OT-cells with goat anti-mouse Texas red, and Spontaneous activity of intrinsic origin and via synaptic activation were observed in high density, but not in low density MCN cultures. Almost all MCNs were responsive to glutamate and GABA. Glutamate receptor-activated currents were excitatory and GABA receptor-activated currents were inhibitory. Half of the cultured MCNs showed excitatory responses to histamine. Other transmitter candidates (ACh, /-EP, DA, 5HT, CCK-8, enkephalin, GRP, OT, VP, and somatostatin) were examined using the puffing method, but did not induce membrane current flow in MCNs. However, some of these agents (/-EP, CCK-8, OT, VP) did induce small and slow membrane potential changes by bath application. Modulation of the excitatory (KA or NMDA) or inhibitory (GABA) amino acid (10/-4 M) responses by OT and VP was examined by bath-application of 10/-7 M OT or VP. Preliminary results suggested that OT and VP feed back on the MCNs produce activation as well as inhibition.
