Epileptiform activity in hippocampal slice cultures exposed chronically to bicuculline: increased gap junctional function and expression.
Chronic (18 h) exposure of cultured hippocampal slices to the type-A GABA receptor blocker, bicuculline methiodide (BMI) 10 micro m increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by western blotting were also significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c-fos. The rate of fluorescence recovery after photobleaching, an index of gap junctional coupling, was also significantly increased, whereas it was blocked by the gap junctional blocker, carbenoxolone (100 micro m). Extracellular recordings in CA1 stratum pyramidale, performed in BMI-free solution, demonstrated that BMI-exposed cultures possessed synaptic responses characteristic of epileptiform discharges: (i) significantly greater frequency of spontaneous epileptiform discharges, (ii) post-synaptic potentials with multiple population spikes, and (iii) significantly longer duration of primary afterdischarges. Carbenoxolone (100 micro m), but not its inactive analog, oleanolic acid (100 micro m), reversibly inhibited spontaneous and evoked epileptiform discharges. The findings of BMI-induced parallel increases in levels of gap junction expression and function, and the increase in epileptiform discharges, which were sensitive to gap junctional blockers, are consistent with the hypothesis that increased gap junctional communication plays an intrinsic role in the epileptogenic process.
Samoilova
Marina
M
Bloorview Epilepsy Research Laboratory, Division of Cellular and Molecular Biology, Toronto Western Research Institute, Toronto, Ontario, Canada.
Li
Jianxue
J
Pelletier
Marc R
MR
Wentlandt
Kirsten
K
Adamchik
Yana
Y
Naus
Christian C
CC
Carlen
Peter L
PL
D000818:Animals; D001640:Bicuculline / Q000031:analogs & derivatives*; D002229:Carbenoxolone / Q000494:pharmacology; D000072259:Connexin 26; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D017630:Connexins / Q000235:genetics / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004558:Electric Stimulation; D004827:Epilepsy / Q000139:chemically induced / Q000503:physiopathology*; D036681:Fluorescence Recovery After Photobleaching; D018756:GABA Antagonists; D017629:Gap Junctions / Q000187:drug effects / Q000502:physiology*; D015870:Gene Expression / Q000187:drug effects; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism / Q000503:physiopathology*; D066298:In Vitro Techniques; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism; D000442:Octanols / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D000097002:Gap Junction beta-1 Protein
D001640:Bicuculline; D002229:Carbenoxolone; D000072259:Connexin 26; D018031:Connexin 43; D017630:Connexins; D017362:Cyclic AMP Response Element-Binding Protein; D018756:GABA Antagonists; D009419:Nerve Tissue Proteins; D000442:Octanols; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D000097002:Gap Junction beta-1 Protein; C000607044:Gjb2 protein, rat; C017069:bicuculline methiodide