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Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins
Published on: August 15, 2017
Hippocampal seizures alter the expression of the pannexin and connexin transcriptome
Shanthini Mylvaganam1, Liang Zhang, Chiping Wu
1Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada.
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.
Insights
Seizure activity increases the expression of gap junction (GJ) proteins and their messenger RNAs (mRNAs) in the brain. This coordinated gene expression may play a role in facilitating seizure induction.
Area of Science:
- Neuroscience
- Molecular Biology
- Epilepsy Research
Background:
- Gap junctional (GJ) blockade can inhibit certain seizure activities.
- The role of connexins (Cx) and pannexins (Panx) in seizure generation is not fully understood.
Purpose of the Study:
- To investigate the effect of a novel seizure-inducing protocol using cobalt (Co2+) on hippocampal GJ mRNA and protein expression.
- To determine if seizure activity itself, rather than the inducer, drives changes in GJ gene expression.
Main Methods:
- Induction of hippocampal seizures using a Co2+ protocol.
- Quantitative PCR (qPCR) to measure Cx and Panx mRNA levels.
- Analysis of protein expression and post-translational modifications.
- Assessment of gene expression changes in the presence of tetrodotoxin.
Main Results:
- Co2+-induced seizures significantly increased Panx1, Panx2, and Cx43 mRNA levels (1.5, 1.4, and 2-fold, respectively).
- Significant post-translational modifications were observed for Cx43 and Panx1 proteins.
- Tetrodotoxin blocked these expression changes, confirming seizure activity as the trigger.
- qPCR data revealed a coordinated re-organization of the Cx and Panx transcriptome, with high correlation between Panx1, Panx2, and glial connexin mRNAs.
Conclusions:
- Seizure activity up-regulates both glial and neuronal GJ mRNAs and proteins.
- A coordinated expression network of the GJ transcriptome is induced during seizures.
- This coordinated gene expression may contribute to seizure induction.
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