Enhanced gap junctional communication (GJC) between neurons is considered a major factor underlying the neuronal synchrony driving seizure activity. In addition, the hippocampal sharp wave ripple complexes, associated with learning and seizures, are diminished by GJC blocking agents. Although gap junctional blocking drugs inhibit experimental seizures, they all have other non-specific actions. Besides interneuronal GJC between dendrites, inter-axonal and inter-glial GJC is also considered important for seizure generation. Interestingly, in most studies of cerebral tissue from animal seizure models and from human patients with epilepsy, there is up-regulation of glial, but not neuronal gap junctional mRNA and protein. Significant changes in the expression and post-translational modification of the astrocytic connexin Cx43, and Panx1 were observed in an in vitro Co ++ seizure model, further supporting a role for glia in seizure-genesis, although the reasons for this remain unclear. Further suggesting an involvement of astrocytic GJC in epilepsy, is the fact that the expression of astrocytic Cx mRNAs (Cxs 30 and 43) is several fold higher than that of neuronal Cx mRNAs (Cxs 36 and 45), and the number of glial cells outnumber neuronal cells in mammalian hippocampal and cortical tissue. Pannexin expression is also increased in both animal and human epileptic tissues. Specific Cx43 mimetic peptides, Gap 27 and SLS, inhibit the docking of astrocytic connexin Cx43 proteins from forming intercellular gap junctions (GJs), diminishing spontaneous seizures. Besides GJs, Cx membrane hemichannels in glia and Panx membrane channels in neurons and glia are also inhibited by traditional gap junctional pharmacological blockers. Although there is no doubt that connexin-based GJs and hemichannels, and pannexin-based membrane channels are related to epilepsy, the specific details of how they are involved and how we can modulate their function for therapeutic purposes remain to be elucidated.

Some forms of seizure activity can be stopped by gap junctional (GJ) blockade. Here, we found that GJ blockers attenuate hippocampal seizure activity induced by a novel seizuregenic protocol using Co 2+ . We hypothesized that this activity may occur because of the altered expression of connexin (Cx) and/or pannexin (Panx) mRNAs and protein.We found a 1.5-, 1.4-, and 2-fold increase in Panx1, Panx2, and Cx43 mRNAs, respectively. Significant post-translational modifications of the proteins Cx43 and Panx1 were also observed after the Co 2+ treatment. No changes were observed in the presence of tetrodotoxin, indicating that seizure activity is required for these alterations in expression, rather than the Co 2+ treatment itself. Further analysis of the QPCR data showed that the Cx and Panx transcriptome becomes remarkably re-organized. Pannexin (Panxs 1 and 2) and glial connexin mRNA became highly correlated to one another; suggesting that these genes formed a transcriptomic network of coordinated gene expression, perhaps facilitating seizure induction. These data show that seizure activity up-regulates the expression of both glial and neuronal GJ mRNAs and protein while inducing a high degree of coordinate expression of the GJ transcriptome. We have shown previously that mechanisms involving activity-dependent facilitation of GJ communication may play a major role in Co 2+ -induced epileptiform discharges (He et al. 2009). To further understand the underlying cellular mechanism of the Co 2+ -induced seizure activity, the expression levels of Cx and Panx mRNA and protein were analyzed using quantitative RT-PCR (QRT-PCR) and western blotting in an in vitro mouse hippocampal model. Materials and methodsAll experimentation conducted in this study has been reviewed and approved by the animal care committee of our institution.Preparation of mouse hippocampal tissues C57BL/6 male mice (postnatal 15 days; Charles River Laboratories, Montreal, QC, Canada) were decapitated under isoflurane anesthesia, and their brains were quickly dissected out and maintained in an ice-cold, oxygenated artificial cerebrospinal fluid (ACSF in mM: 125 NaCl, 3.5 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 2 CaCl 2 , 1.3 MgSO 4 , and 10 glucose) at pH 7.4 when aerated with 95% O 2 -5% CO 2 for a few minutes before further dissection. For preparing whole hippocampal isolates (Wu et al. 2002), we used a fine glass probe and gently separated the dentate gyrus from the CA1 area, and then removed the dentate gyrus area while preserving the CA3-CA1 tissues. The whole hippocampal isolates were maintained in warmed ACSF (31-32°C) for 1 h before further experimental manipulations. Co 2+ treatmentWhole hippocampal isolates (left and right) from C57BL/6 male mice were prepared and pre-incubated separately in warmed ACSF (31-32°C) for 1 h before further experimental manipulations. Then, 100 lM CoCl 2 (Sigma-Aldrich, Oakville, ON, Canada) was added to the ACSF of one of the isolates. Both control (without cobalt) and treated hippocampal isolates were continu...

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