Abstract
Neuronal activity regulates morphology and connectivity of neurons during development. Many aspects of activity-dependent neuronal development such as dendritic arborization, spinogenesis and synaptogenesis are mediated via N-methyl D-aspartate receptor (NMDAR)-dependent signaling mechanisms. Inasmuch as neuronal activity involves activation of voltage-gated sodium channels (VGSCs) primarily, in this study, we used a sodium channel activator, brevetoxin-2 (PbTx-2) to mimic neuronal activity. Brevetoxins interact with binding site 5 on the α subunit of VGSCs. PbTx-2 was found to increase intracellular sodium concentration ([Na+]i) in immature cerebrocortical neurons above the critical threshold for upregulating NMDAR function. Hence PbTx-2 was used to explore the relationship between [Na+]i and NMDAR-dependent structural plasticity in developing cerebrocortical neurons. Exposure to PbTx-2 was found to sensitize immature neurons to NMDA-induced Ca2+ influx through a Src family kinase (SFK)-dependent pathway. The effects of PbTx-2 on upregulating NMDAR function did not involve depolarization of the neurons as explained by the modest membrane potential change in FMP (FLIPR® membrane potential) blue fluorescence assay and confirmed by the cell attached patch recordings. We found that chronic exposure to low concentration of PbTx-2 (30 nM) accelerated the appearance of spontaneous calcium oscillations in cerebrocortical neurons, which could be indicative of enhanced or accelerated functional network and synapse formation. Acute exposure to PbTx-2 increased cell surface expression of NR2B containing NMDARs and TrkB receptors (high affinity receptor for Brain-derived neurotrophic factor, BDNF) as observed in the surface biotinylation assay, demonstrating direct evidence for the ability of PbTx-2 to sensitize neurons for signaling downstream of NMDAR and BDNF. Further, effects of PbTx-2 on various aspects of neuronal plasticity were investigated. We observed that treatment with PbTx-2 enhanced neurite outgrowth, dendritic arborization, spinogenesis and synaptogenesis. All the aspects of neuronal plasticity exhibited a biphasic concentration-response profile with 30 and 100 nM PbTx-2 having the most robust effect. Biphasic nature of these data can be explained by lower concentrations not sufficient enough to initiate signaling and higher concentrations resulting in internalization of VGSCs as shown in the whole cell binding assay. We also found that exposure to NMDA produced a neurite outgrowth response with similar hormetic profile suggesting engagement of similar signaling mechanisms in PbTx-2 and NMDA-induced neuronal morphogenesis. Pharmacological evaluation of PbTx-2 induced neuronal plasticity showed that it was mediated by signaling mechanisms downstream of NMDAR resulting in the activation of CaMKK (Calcium/calmodulin-dependent protein kinase kinase) and CaMKII (Calcium/calmodulin-dependent protein kinase II) pathway. The signaling mechanisms underlying PbTx-2 stimulated neuronal structural plasticity also involved activation of transcription factor CREB and initiation of further nuclear signaling events that resulted in increased BDNF gene expression. Also, PbTx-2 exposure was able to regulate actin dynamics locally by activation of Rho family GTPases, Rac and Cdc42. These findings suggest that the influence of a sodium channel activator on neuronal development involves NMDAR-mediated Calcium/calmodulin-dependent protein kinase (CaMK) signaling with downstream activation of CREB (Cyclic AMP response element binding protein)-dependent transcription of BDNF.