Epileptiform activity in hippocampal slice cultures exposed chronically to bicuculline: increased gap junctional function and expression

Abstract

Chronic (18 h) exposure of cultured hippocampal slices to the type-A GABA receptor blocker, bicuculline methiodide (BMI) 10 lM 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 lM). Extracellular recordings in CA1 stratum pyramidale, performed in BMI-free solution, demonstrated that BMIexposed 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 lM), but not its inactive analog, oleanolic acid (100 lM), 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. Keywords: bicuculline, carbenoxolone, connexins, epilepsy, gap junction. Abbreviations used: ACSF, artificial cerebrospinal fluid; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionate; APV, 2-amino-5-phosphonovaleric acid; BMI, bicuculline methiodide; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CREB, cAMP response element binding protein; Cx, connexin; FRAP, fluorescence recovery after photobleaching; IPSC, inhibitory post-synaptic current; MPP, myelin proteolipid protein; PAD, primary afterdischarge; PPD, pair-pulse depression; Syn, synapsin.

Connexin expression by radial glia-like cells is required for neurogenesis in the adult dentate gyrus

“…However, previous work yielded indirect evidence for such a link, by demonstrating that epileptiform activity induces proliferation of RG-like cells in the DG (3) as well as enhanced expression of Cx43 and coupling (30). In addition, several in vitro studies suggest a role of coupling in the control of precursor cell proliferation and migration (10,11,31).…”

Astrocyte dysfunction in temporal lobe epilepsy: K+ channels and gap junction coupling

“…Opposite results have been reported by Takahashi et al (2010) who used the same method in a post-SE rat model of epilepsy (systemic kainate injection) and observed increased coupling of hippocampal astrocytes during the latent period (7-16 days post-SE). Increased coupling has also been found with fluorescence recovery after photobleaching (FRAP) in hippocampal slice cultures chronically exposed to bicuculline (Samoilova et al, 2003). So far, there is only one culture study investigating coupling in astrocytes from human epilepsy.…”

Role of Gap Junction Channel in the Development of Beat-to-Beat Action Potential Repolarization Variability and Arrhythmias

“…Gap junction channels are involved in physiological and pathological processes such as embryonic development, cell differentiation, growth [24][25][26], pathogenesis of neuropathies [27], epilepsy [28], cardiovascular diseases [29], and arrhythmias [30,31]. Numerous densely packed gap junction channels form clusters that directly connect the cytoplasmic compartment of cells [32].…”

“…The antiarrhythmic peptide rotigaptide (molecular formula C 28 protect against enzymatic degradation. Rotigaptide plasma half-life was more than 10 days compared with less than 15 min for AAP10 [106].…”