Changes in GABAA̳ Receptor Expression and Alterations in Ca⁺⁺/Calmodulin-Dependent Protein Kinase II Activity in a Hippocampal Neuronal Culture Model of Epilepsy
Expression levels of GABAA receptor subunits and activity of the Ca++ /calmodulin-dependent protein kinase II (CaM kinase II) enzyme system were evaluated in an in vitro hippocampal neuronal culture model of epilepsy. Treatment of hippocampal neuronal cultures with Mg++-free media for 3 hours result...
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| Format: | Others |
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VCU Scholars Compass
1998
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| Online Access: | http://scholarscompass.vcu.edu/etd/4358 http://scholarscompass.vcu.edu/cgi/viewcontent.cgi?article=5420&context=etd |
| Summary: | Expression levels of GABAA receptor subunits and activity of the Ca++ /calmodulin-dependent protein kinase II (CaM kinase II) enzyme system were evaluated in an in vitro hippocampal neuronal culture model of epilepsy. Treatment of hippocampal neuronal cultures with Mg++-free media for 3 hours results in the induction of an enduring "epileptic" state as evidenced by the expression of spontaneous recurrent seizure (SRS) discharge. The induction of the SRS activity was shown to be a N-methyl-D-aspartate (NMDA) receptor, Ca++-dependent mechanism (Sombati and DeLorenzo, J Neurophys., 73 (4), 1995). Significant and long-lasting decreases in mRNA expression for the GABAA α2 and α5 receptor subunits were observed in association with the induction of SRSs in this model, while levels for α1, β2 and γ2 subunits showed no significant change. Irreversible [3H]-flunitrazepam saturation binding analysis in membrane preparations demonstrated a significant decrease in specific binding in association with the SRS activity observed in this model. No changes in GABAA P subunit immunoreactivity were detected. Selective suppression of the GABAA α2 subunit protein levels in hippocampal neuronal cultures using antisense oligonucleotide technology caused a significant decrease in the amplitude of spontaneous inhibitory postsynaptic currents (sIPSC).
CaM kinase II is highly enriched in the brain and mediates many processes essential to neuronal function and viability. Induction of SRSs in hippocampal cultures were associated with a long-lasting and significant decrease in activity of CaM kinase II. Addition of 2-amino-5-phosphovaleric acid to the low Mg++ treatment solution prevented the decrease in CaM kinase II activity. Suppression of CaM kinase II activity in hippocampal cultures by treatment with either an antisense oligonucleotide specific for a CaM kinase II or KN93 (selective CaM kinase II inhibitor) resulted in significant reductions in IPSC amplitude. This data suggests that CaM kinase II can act to regulate GABAergic inhibitory function in hippocampal cultures. The findings of this study demonstrate long-lasting decreases in GABAA receptor expression and activity of CaM kinase II, which may contribute to the induction of the "epileptic" state of this model. |
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