A brain slice model for in vitro analyses of astrocytic gap junction and connexin43 regulation: actions of ischemia, glutamate and elevated potassium.
Brain slices prepared from adult rats and maintained for up to 3 h in vitro were used to investigate the effects of pharmacological treatments on the phosphorylation state, immunolabelling characteristics and ultrastructural localization of astrocytic gap junctions and connexin43 (Cx43). Slices deprived of glucose/oxygen to mimic ischemia or those exposed to 1 mM glutamate for 1 h exhibited Cx43 dephosphorylation, epitope masking and gap junction internalization as revealed by Western blotting and Cx43 immunolocalization with various antibodies. Treatment with 15 mM K+ caused Cx43 dephosphorylation without junction internalization. The effects of glutamate and K+ were completely blocked by the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist 2-amino-5-phosphonovalerate (APV), which acts largely on neuronal NMDA receptors, suggesting neuronal mediation of glial gap junction responses to these treatments. Astrocytes contained a dephosphorylated form of Cx43 with a typical migration profile at 41 kDa as well as novel, apparently dephosphorylated or partially phosphorylated, forms migrating at 43 kDa. These results indicate that slices prepared from adult brain can serve as a convenient model to investigate the molecular basis and receptor-mediated mechanisms underlying astrocytic Cx43 responses that have been observed in vivo following cerebral ischemia or neural activation. These processes can be related in part to neuronal regulation of astrocytic gap junctional coupling state, which is also amenable to analysis in brain slices.
Nagy
J I
JI
Department of Physiology, Faculty of Medicine, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 3J7. nagyji@ms.umanitoba.ca
Li
W E
WE
D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000502:physiology*; D001921:Brain / Q000187:drug effects / Q000502:physiology* / Q000503:physiopathology; D018031:Connexin 43 / Q000378:metabolism*; D017629:Gap Junctions / Q000187:drug effects / Q000502:physiology*; D018698:Glutamic Acid / Q000494:pharmacology; D020925:Hypoxia-Ischemia, Brain / Q000503:physiopathology*; D066298:In Vitro Techniques; D008959:Models, Neurological; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000502:physiology
D018031:Connexin 43; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D015763:2-Amino-5-phosphonovalerate; D011188:Potassium