Upregulation of gap junction connexin 32 with epileptiform activity in the isolated mouse hippocampus.
Gap junctions, which serve as intercellular channels providing direct cytoplasmic continuity and ionic current flow between adjacent cells, are constituted by connexin proteins. Using an in vitro model of bicuculline-induced epileptiform activity, we asked whether increased connexin levels occur during epileptiform activity in the intact whole hippocampus, freshly isolated from young (15-day-old) mouse brain. Exposure to bicuculline (10 microM), for 2-10 h, induced persistent changes in electrical activities that included enhanced spontaneous field activity (4 h), an epileptiform response to single electrical stimulation (6 h), and spontaneous epileptiform activity (6 h). These electrophysiological changes were not reversed by up to 60 min perfusion with normal artificial cerebrospinal fluid, but were greatly depressed by the gap junction uncoupler, carbenoxolone (120 microM, 10 min). Data from RNase protection assay and immunoblotting showed that among several detected gap junctions, only connexin 32 was affected. After 2-6 h exposure to bicuculline, the connexin 32 mRNA expression was upregulated to 2-3-fold control (P < 0.01), and its protein level was significantly elevated the following 6 h (P < 0.01), at which time electrophysiologically measured evidence of clearly epileptiform activity was apparent. In addition, the transcription factor, c-fos protein, but not the cAMP response element-binding protein, was also found to be increased at the early stage of bicuculline exposure (2 h) compared to control (P < 0.05).Thus, we have found that exposing the acutely isolated hippocampus to bicuculline, induced increased c-fos protein, followed by increased connexin 32 transcript and protein, and concurrently, persistent epileptiform activity that was depressed by carbenoxolone.
Li
J
J
Bloorview Epilepsy Program, Toronto Western Research Institute, University Health Network, ON, Canada.
Shen
H
H
Naus
C C
CC
Zhang
L
L
Carlen
P L
PL
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D000897:Anti-Ulcer Agents / Q000494:pharmacology; D001640:Bicuculline / Q000494:pharmacology; D002229:Carbenoxolone / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D017630:Connexins / Q000187:drug effects / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000187:drug effects / Q000378:metabolism; D004347:Drug Interactions / Q000502:physiology; D004827:Epilepsy / Q000139:chemically induced / Q000378:metabolism* / Q000503:physiopathology; D018756:GABA Antagonists / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D015151:Immunoblotting; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D023421:Models, Animal; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009924:Organ Culture Techniques; D016760:Proto-Oncogene Proteins c-fos / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology*; D000097002:Gap Junction beta-1 Protein
D000897:Anti-Ulcer Agents; D001640:Bicuculline; D002229:Carbenoxolone; D017630:Connexins; D017362:Cyclic AMP Response Element-Binding Protein; D018756:GABA Antagonists; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D000097002:Gap Junction beta-1 Protein