Signaling pathways involved in NMDA-induced suppression of M-channels in corticotropin-releasing hormone neurons in central amygdala.

Abstract

Glutamate N-methyl-d-aspartate (NMDA) receptors (NMDARs) and Kv7/M channels are importantly involved in regulating neuronal activity involved in various physiological and pathological functions. Corticotropin-releasing hormone (CRH)-expressing neurons in the central nucleus of the amygdala (CeA) critically mediate autonomic response during stress. However, the interaction between NMDA receptors and Kv7/M channels in the CRHCeA neurons remains unclear. In this study, we identified rat CRHCeA neurons through the expression of an AAV viral vector-mediated enhanced green fluorescent protein (eGFP) driven by the rat CRH promoter. M-currents carried by Kv7/M channels were recorded using the whole-cell patch-clamp approach in eGFP-tagged CRHCeA neurons in brain slices. Acute exposure to NMDA significantly reduced M-currents recorded from the CRHCeA neurons. NMDA-induced suppression of M-currents was eliminated by chelating intracellular Ca2+ , supplying phosphatidylinositol 4,5-bisphosphate (PIP2) intracellularly, or blocking phosphoinositide3-kinase (PI3K). In contrast, inhibiting protein kinase C (PKC) or calmodulin did not alter NMDA-induced suppression of M-currents. Sustained exposure of NMDA decreased Kv7.3 membrane protein levels and suppressed M-currents, while the Kv7.2 expression levels remained unaltered. Pre-treatment of brain slices with PKC inhibitors alleviated the decreases in Kv7.3 and reduction of M-currents in CRHCeA neurons induced by NMDA. PKC inhibitors did not alter Kv7.2 and Kv7.3 membrane protein levels and M-currents in CRHCeA neurons. These data suggest that transient activation of NMDARs suppresses M-currents through the Ca2+ -dependent PI3K-PIP2 signaling pathway. In contrast, sustained activation of NMDARs reduces Kv7.3 protein expression and suppresses M-currents through a PKC-dependent pathway.