Abstract
Activity-dependent N-methyl D-aspartate receptor (NMDAR) signaling, gene transcription and protein synthesis play major roles in brain functions that regulate neuronal morphology. Inasmuch as neuronal activity-induced increments in cytoplasmic sodium may augment NMDAR-mediated currents (Rose and Konnerth, 2001; Yu and Salter, 1998; George et al., 2009), we reasoned that intracellular Na+ may function as a signaling molecule and positively regulate neuronal development in immature cerebrocortical neurons. The central hypothesis of this study is that sodium channel activators stimulate neuronal development by elevating [Na+]i , augmenting NMDAR function in presence of activated SFKs, enhancing BDNF release and activating the downstream TrkB signaling. The specific objective of the proposed work is to elucidate the signaling mechanisms by which sodium channel activators influence neuronal morphology in immature cerebrocortical neurons. More specifically, to understand the relationship between increases in [Na+]i and NMDAR, brain-derived neurotrophic factor (BDNF)-TrkB mediated neuronal development.|For these studies, sodium channel activators that increase [Na+]i ,antillatoxin (ATX) and veratridine (VRT) were used as pharmacological tools to determine their potential to mimic neuronal activity. VGSCs activators robustly stimulated neurite outgrowth in a hormetic concentration-response relationship and this enhancement was sensitive to the VGSC antagonist, tetrodotoxin. To unambiguously demonstrate the enhancement of NMDA receptor function by ATX, we recorded single-channel currents from cell-attached patches. ATX was found to increase the open probability of NMDA IV receptors. Na+ dependent upregulation of NMDAR function has been shown to be regulated by Src family kinase (SFK) (Yu and Salter, 1998). The Src kinase inhibitor PP2 abrogated ATX-enhanced neurite outgrowth suggesting a SFK involvement in this response. ATX-enhanced neurite outgrowth was also inhibited by the NMDAR antagonist, MK-801, and the calmodulin dependent kinase kinase (CaMKK) inhibitor, STO-609, demonstrating the requirement for NMDAR activation with subsequent downstream engagement of the Ca2+ dependent CaMKK pathway.|Activity-dependent neuronal development involves N-methyl D-aspartate receptor (NMDAR) mediated calcium influx and brain-derived neurotrophic factor (BDNF)-TrkB signaling. We tested the effect of the VGSC activators on BDNF synthesis and release and TrkB activation in DIV1 cerebrocortical neurons. Inhibition of TrkB receptors and its downstream effector pathways, PI3K, and PLCγ inhibited VRT-enhanced NOG. VRT stimulated phosphorylation of TrkB and its downstream effectors Akt, mTOR, PLCγ1, ERK1/2 and CREB. VRT increased BDNF synthesis and release in a concentration dependent manner; however, VRT stimulation of TrkB phosphorylation displayed a biphasic concentration-response curve. VRT stimulation of BDNF synthesis required VGSCs and NMDARs.|Taken together, these data suggest that VGSC activators seem to be capable of mimicking activity-dependent neuronal development and hence may represent a novel pharmacological strategy to regulate neuronal development through NMDA and neurotrophin receptor-dependent mechanisms.