Hippocampal seizures alter the expression of the pannexin and connexin transcriptome

Abstract

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...

Role of gap junctions in epilepsy

“…In Co 2+ -induced epileptiform discharges, Cx43 mRNA and protein are up-regulated, while the mRNA levels of Cx26, Cx30, Cx32, Cx36, Cx40, Cx45, and Cx47 do not change in the hippocampus [79] . In mouse hippocampal slices, 2-to 10-hour exposure to bicuculline induces marked increases in Cx32 mRNA and protein, with no changes in the protein and mRNA levels of Cx26 and Cx43 [80] .…”

“…-induced epileptiform C57BL/6 male mice In the presence of octanol, no ictal or interictal discharges recorded but discharges Whole hippocampus basal rhythmic activity is evident [79] .…”

“…Schaffer collaterals parahippocampal slices after-discharges; level of dephosphorylated Cx43↓ [78] Co 2+ -induced epileptiform Whole hippocampus C57BL/6 male mice Cx43 mRNA and protein↑; mRNAs of Cx26, Cx30, Cx32, Cx36, Cx40, discharges Cx45 and Cx47 unchanged [79] Bicuculline-induced Whole hippocampus Male C57BL/6 mice After 2-10-h exposure to bicuculline, Cx32 mRNA and protein↑;…”

The cellular life of pannexins

“…Upon induction of hippocampal seizures, there was a coordinated upregulation of Cx43, Panx1, and Panx2 both at the mRNA and protein level, with changes also reported in the post-translational modifications of Panx1 (glycosylation) and Cx43 (phosphorylation) induced upon seizure induction. 30 Furthermore, ectopic expression of Panx3 in differentiated C2C12 cells has also been recently reported to trigger the expression of Cx43, whereas Panx3 knockdown seemed to downregulate Cx43 expression. 5 In the same study, the increase in gap junction function as measured by intercellular calcium wave propagation was attributed to Panx3 gap junction channels because of channel blockage by carbenoxolone treatment.…”

“…9 Interestingly, the level of Panx1 glycosylation was reported to change in the male reproductive tract of rats in response to orchidectomy, 22 as well as after induction of hippocampal seizures by Co 2+ treatment in mouse brains. 30 All these phenomena suggests a role for the glycosylation state regulating pannexin function. One additional post-translational modification has been reported for Panx2 in a study where an ∼80 kDa species expressed in neural progenitor cells was found to be sensitive to hydroxylamine suggesting that Panx2 could be palmitoylated.…”