Functional activation of punch-cultured magnocellular neuroendocrine cellsby glutamate receptor subtypes

To provide a simple means to isolate and study the cellular functions of sm all groups of neurons, we developed a modified 'punch' culture procedure th at facilitates acute and long-term in vitro physiological studies. Primary 'punch' cultures of magnocellular neuroendocrine cells (MNCs) from the supr aoptic nucleus (SON) were established and the basic physiological effects o f subtype-specific glutamate receptor agonists were characterized. MNCs fro m the punch cultures established a mature morphology in culture with extens ive outgrowth of axons and varicosities. After 8 days, a single cultured SO N punch produced an average of 10.0 +/- 2.1 pg AVP and contained an average of 222 +/- 53 vasopressin-neurophysin immunoreactive cells. Patch clamp re cordings from MNCs demonstrated the presence of N-methyl-D-aspartate (NMDA) -sensitive and DL, alpha-amino-3-hydroxy-5-methylisoxazole propionic acid ( AMPA)-receptors. Stimulation of metabotropic receptors with 1S,3R ACPD indu ced acute or gradual increases in intracellular calcium. NMDA, AMPA and met abotropic receptors all contributed to the secretion of vasopressin from th e punch cultures with an agonist rank order potency of: NMDA (10 mu M) > AM PA (1 mu M) = 1S, 3R ACPD (100 mu M) > kainate (10 mu M). This culture prep aration should be useful for cellular studies of small groups of neuroendoc rine and other cells. (C) 1999 Published by Elsevier Science B.V. All right s reserved.