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Abstract: The potassium-chloride cotransporter 2 (KCC2)-dependent intracellular chloride level determines whether neurons respond to GABA and/or glycine by depolarization or hyperpolarization. However, still unknown is the role of KCC2-dependent chloride homeostasis in regulating the spontaneous activity of neuronal circuits via GABAA receptor (GABAAR) and the glycine receptor (GlyR). In this study, patch-clamp recordings were performed to measure the change of spontaneous neuronal activity in cultured hippocampal neurons. Our results showed that inhibition of KCC2 with furosemide, as well as blockade of GABAAR with bicuculline, significantly enhanced circuit activity. Perfusion with bicuculline further enhanced the effects of furosemide on spontaneous circuit activity, while furosemide did not alter the effects of bicuculline. Surprisingly, blockade of GlyR not only induced obvious tonic currents, but also significantly decreased spontaneous synaptic activity. Moreover, inhibition of KCC2 did not change the depressive effect of strychnine on neuronal circuits. Our findings suggest that KCC2-dependent chloride homeostasis is mainly involved in GABAAR-mediated synaptic inhibition whereas GlyR-mediated tonic action plays a totally different role in regulating hippocampal circuit activity. [Copyright &y& Elsevier]

Abstract: Effects of suppressing the protein expression of Pyk2 on increased tyrosine phosphorylation of N-methyl-d-aspartate (NMDA) receptor subunit 2A (NR2A) after brain ischemia in rat hippocampus were studied with immunoprecipitation and immunoblot. Transient (15min) brain ischemia and reperfusion (I/R) was induced by four-vessel occlusion in Sprague–Dawley (SD) rats. I/R led to increases of tyrosine phosphorylation of NR2A and interaction of Pyk2 and Src kinase with NR2A after 6h of reperfusion. The increases were attenuated by Pyk2 antisense oligonucleotides intracerebroventricularly infused every 24h for 4 days before ischemia, but not missense oligonucleotides or vehicle. The antisense also inhibited the increased auto-phosphorylation of Pyk2 and Src kinase, while the protein expression of NR2A or Src kinase had no obvious change under the above conditions. The data suggested that Pyk2 may be involved in facilitating NR2A tyrosine phosphorylation by Src kinase after I/R. [Copyright &y& Elsevier]