High-Frequency Field Stimulation of Primary Neurons Enhances Ryanodine Receptor-Mediated Ca²⁺Release and Generates Hydrogen Peroxide, Which Jointly Stimulate NF-κB Activity

Abstract

Neuronal electrical activity increases intracellular Ca(2+) concentration and generates reactive oxygen species. Here, we show that high frequency field stimulation of primary hippocampal neurons generated Ca(2+) signals with an early and a late component, and promoted hydrogen peroxide generation via a neuronal NADPH oxidase. Hydrogen peroxide generation required both Ca(2+) entry through N-methyl-D-aspartate receptors and Ca(2+) release mediated by ryanodine receptors (RyR). Field stimulation also enhanced nuclear translocation of the NF-κB p65 protein and NF-κB -dependent transcription, and increased c-fos mRNA and type-2 RyR protein content. Preincubation with inhibitory ryanodine or with the antioxidant N-acetyl L-cysteine abolished the increase in hydrogen peroxide generation and the late Ca(2+) signal component induced by electrical stimulation. Primary cortical cells behaved similarly as primary hippocampal cells. Exogenous hydrogen peroxide also activated NF-κB-dependent transcription in hippocampal neurons; inhibitory ryanodine prevented this effect. Selective inhibition of the NADPH oxidase or N-acetyl L-cysteine also prevented the enhanced translocation of p65 in hippocampal cells, while N-acetyl L-cysteine abolished the increase in RyR2 protein content induced by high frequency stimulation. In conclusion, the present results show that electrical stimulation induced reciprocal activation of ryanodine receptor-mediated Ca(2+) signals and hydrogen peroxide generation, which stimulated jointly NF-κB activity.

Involvement of ryanodine receptors in neurotrophin-induced hippocampal synaptic plasticity and spatial memory formation

“…Accordingly, incubation of hippocampal cultures with 1 μM ryanodine stimulates Ca 2+ release (Fig. S3A), whereas prolonged incubation with 50 μM ryanodine suppresses it (18,25). Here, we show that cultures preincubated with inhibitory ryanodine (50 μM, 1 h) did not exhibit the above changes in RyR2, RyR3, and PKMζ mRNA, and protein contents produced by 6 h incubation with BDNF ( Fig.…”

“…Amplification was performed using the primers and conditions detailed in Table S1. Immunocytochemistry assays were carried out as described in detail elsewhere (18,25). Brain tissue fixation and immunohistochemistry was performed as described (44), except that coronal slices were incubated at 4°C overnight with anti-RyR3 antibodies.…”

A Feed-Forward Mechanism Involving the NOX Complex and RyR-Mediated Ca2+Release During Axonal Specification

“…To this aim, 1 DIV neurons were cotransfected with a vector encoding Rac1T17N, the dominant-negative isoform of Rac1 (Ridley et al, 1992), and HyPer to evaluate H 2 O 2 levels after 4-CMC stimulation. Moreover, neurons were also cotransfected with HyPer and DNp22 phox constructs to determine ROS levels.…”

“…Here, we propose that ROS may act as signaling molecules targeting downstream mediators. Previous reports suggest that Ca 2ϩ release from the endoplasmic reticulum (ER), particularly that mediated by the ryanodine receptor (RyR), needs the basal activity of NOX2 in muscle and neuronal cells (Espinosa et al, 2006;Bedard and Krause, 2007;Zhang and Forscher, 2009;Riquelme et al, 2011). Moreover, RyR stimulation activates Rac1 and Cdc-42 in HEK293 cells and cerebellar granule neurons, suggesting a RyRdependent mechanism (Jin et al, 2005).…”

DCC functions as an accelerator of thalamocortical axonal growth downstream of spontaneous thalamic activity

“…This CRE variant binds phosphorylated cJun, which in turn represses tlx3 transcription [37]. Thus, Ca 2+ levels can modulate both NF-jB and c-Jun (AP-1) activity [37,41], and an increase in this activity triggered by the switch in Ca 2+ frequency occurring in late thalamic neurons [4] could have opposing effects on the transcription of target genes, such as the increase in Robo1 and the decrease of Dcc transcription, reducing TCA growth. However, the mechanism by which AP-1 transcriptional activity is regulated in thalamic neurons during early and late phases of TCA growth remains to be elucidated.…”