Perfusion by regular Ringer’s solution served as a negative contr

Perfusion by regular Ringer’s solution served as a negative control. Total RNA was extracted after 7 hr and qPCR performed (Figure 1B). We detected significant increases in both KCNQ2 and KCNQ3 mRNA in neurons stimulated by selleck chemical 50 K+ or ACh (Figure 1C). For KCNQ2, the relative

expression levels in neurons treated with high K+ or ACh were 2.05 ± 0.44 (n = 4; p < 0.05), and 1.80 ± 0.19 (n = 4; p < 0.05), respectively. For KCNQ3 transcripts, they were 1.76 ± 0.51 (n = 4; p < 0.05) and 1.56 ± 0.22 (n = 4; p < 0.05), respectively. M-current (IM) amplitudes in SCG neurons were then quantified to assay expression of functional M channels. As in the previous qPCR experiments, neurons were perfused by 50 K+, ACh, or regular Ringer’s solution for 15 min, and after 1, 48, 60, or 72 hr studied under perforated-patch voltage clamp. We did not observe a significant difference of IM amplitudes between neurons treated with regular Ringer’s and 50 K+ solutions 1 hr after stimulation, but we observed significant upregulation

of IM amplitudes in neurons treated with 50 K+ solution after 48, Depsipeptide supplier 60, or 72 hr, indicating that altered expression of M channels is involved. We thus decided to measure IM amplitudes 48–60 hr after stimulation in this paper, and examples of IM traces recorded from such neurons before and after application of the M-channel-specific blocker, XE991 ( Zaczek et al., 1998), are shown in Figure 1D. IM amplitudes were normalized to membrane capacitance and the current density used to indicate expression of functional M channels. In neurons treated with 50 K+ or ACh-containing solutions, IM amplitudes were significantly Florfenicol augmented ( Figure 1E). For neurons treated with regular Ringer’s or 50 K+-containing solutions, the current densities were 0.78 ± 0.10 pA/pF (n = 14) and 1.24 ± 0.12 pA/pF

(n = 14; p < 0.01), respectively. For neurons treated with regular Ringer’s or ACh, the current densities were 0.81 ± 0.06 pA/pF (n = 12) and 1.25 ± 0.15 pA/pF (n = 14; p < 0.01), respectively. NFAT signaling is critical to neural development and axon growth (Graef et al., 2003), as well as transcriptional regulation of several voltage-dependent K+ channels, e.g., upregulation of KV4.2 mRNA in cardiomyocytes (Gong et al., 2006) and downregulation of KV2.1 mRNA in arterial smooth muscle (Amberg et al., 2004). In the rat SCG neurons that we study here, NFATc1–NFATc4 has been shown to be expressed and, when activated, to translocate from cytoplasm to nucleus by electrical stimulation and kinase inhibitors (Hernández-Ochoa et al., 2007). We performed qPCR on SCG neurons and detected transcripts for NFATc1–NFATc4 isoforms (data not shown). We then asked which transcription factors mediate the upregulation of M-channel expression seen here, hypothesizing activity-dependent production of Ca2+/CaN and NFAT activation to be crucial.

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