title: AMPA receptor-induced intracellular calcium response in the paraventricular nucleus is modulated by nitric oxide: calcium imaging in a hypothalamic organotypic cell culture model.
abstract: An organotypic cell culture (OCC) model of the rat hypothalamic paraventricular nucleus (PVN) was established to monitor intracellular calcium levels ([Ca(2+)](i)) of magnocellular neurons in response to glutamate and nitric oxide (NO). The histoarchitectural organization of these cultures was characterized either by immunohistochemical labeling of vasopressin, neuronal nitric oxide synthase (nNOS) and the neuronal marker NeuN or by the enzyme histochemical NADPH-diaphorase staining. A distinct NeuN positive cell population in 14-days old OCC's was confirmed as being the PVN by its vasopressin- and nNOS-immunostained neurons as well as by its NADPH-diaphorase labeling. Life cell imaging was performed using the [Ca(2+)](i) sensor Fluo-4 to measure [Ca(2+)](i) transients in response to bath applications of glutamate, high potassium (60 mM), and ATP. The glutamate-induced [Ca(2+)](i) response was mimicked by AMPA but not NMDA in the PVN. NMDA, however, elicited a [Ca(2+)](i) transient in a different area of the OCC that corresponds to the suprachiasmatic nucleus indicating the potential effectiveness of the stimulus. The AMPA-receptor blocker NBQX abolished the glutamate-induced response in the PVN. An inhibition of endogenous NO production by the NOS inhibitor L-NAME decreased the amplitude of AMPA- and glutamate-induced [Ca(2+)](i) rises. Taken together, these data suggest that AMPA mediates the glutamate-induced [Ca(2+)](i) rises within the PVN, where endogenous NO is able to modulate such glutamate signaling in OCC.
authors.affiliation: Institute of Medical Neurobiology, Otto von Guericke University, Leipziger Strasse 44, D-39120 Magdeburg, Germany.
authors.forename: Sanjoy
authors.initials: S
authors.lastname: Roychowdhury
authors.forename: Julia
authors.initials: J
authors.lastname: Noack
authors.forename: Mario
authors.initials: M
authors.lastname: Engelmann
authors.forename: Gerald
authors.initials: G
authors.lastname: Wolf
authors.forename: Thomas F W
authors.initials: TF
authors.lastname: Horn
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000814:Aniline Compounds / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D018929:Cell Culture Techniques; D018698:Glutamic Acid / Q000494:pharmacology; D006651:Histocytochemistry; D008954:Models, Biological; D016202:N-Methylaspartate / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D010286:Paraventricular Hypothalamic Nucleus / Q000187:drug effects* / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D018091:Receptors, AMPA / Q000378:metabolism*; D013194:Staining and Labeling; D014966:Xanthenes / Q000737:chemistry
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D018091:Receptors, AMPA; D014966:Xanthenes; D009569:Nitric Oxide; D018698:Glutamic Acid; D016202:N-Methylaspartate; D000255:Adenosine Triphosphate; D019001:Nitric Oxide Synthase; D009252:NADPH Dehydrogenase; D011188:Potassium; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester