Functional activation of punch-cultured magnocellular neuroendocrine cells by glutamate receptor subtypes.
To provide a simple means to isolate and study the cellular functions of small groups of neurons, we developed a modified 'punch' culture procedure that facilitates acute and long-term in vitro physiological studies. Primary 'punch' cultures of magnocellular neuroendocrine cells (MNCs) from the supraoptic nucleus (SON) were established and the basic physiological effects of subtype-specific glutamate receptor agonists were characterized. MNCs from the punch cultures established a mature morphology in culture with extensive outgrowth of axons and varicosities. After 8 days, a single cultured SON punch produced an average of 10.0 +/- 2.1 pg AVP and contained an average of 222 +/- 53 vasopressin-neurophysin immunoreactive cells. Patch clamp recordings 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 induced acute or gradual increases in intracellular calcium. NMDA, AMPA and metabotropic receptors all contributed to the secretion of vasopressin from the punch cultures with an agonist rank order potency of: NMDA (10 microM) > AMPA (1 microM) = 1S,3R ACPD (100 microM) > kainate (10 microM). This culture preparation should be useful for cellular studies of small groups of neuroendocrine and other cells.
Meeker
R B
RB
Department of Neurology and Neurobiology Curriculum, University of North Carolina, Chapel Hill 27599, USA. rbmeeker@med.unc.edu
Curras
M C
MC
Stewart
J
J
Serje
A
A
Al-Ghoul
W
W
D000818:Animals; D001127:Arginine Vasopressin / Q000032:analysis; D002118:Calcium / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D003515:Cycloleucine / Q000031:analogs & derivatives / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D005333:Fetus; D007608:Kainic Acid / Q000494:pharmacology; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009481:Neurophysins / Q000032:analysis; D009490:Neurosecretory Systems / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D051381:Rats; D020318:Rats, Long-Evans; D018091:Receptors, AMPA / Q000502:physiology; D018094:Receptors, Metabotropic Glutamate / Q000819:agonists / Q000502:physiology; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology; D013495:Supraoptic Nucleus / Q000166:cytology / Q000502:physiology*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
D018690:Excitatory Amino Acid Agonists; D009481:Neurophysins; D018091:Receptors, AMPA; D018094:Receptors, Metabotropic Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; D003515:Cycloleucine; C036361:1-amino-1,3-dicarboxycyclopentane; D001127:Arginine Vasopressin; D016202:N-Methylaspartate; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D007608:Kainic Acid; D002118:Calcium