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Sulfite is a compound commonly used as preservative in foods and pharmaceuticals. Many studies have examined the neurotoxicity of sulfite, but its effect on neuronal calcium homeostasis has not yet been reported. Here, we observed the effect of sulfite on the cytosolic free calcium concentration ([Ca(2+)]i) in cultured cortical neurons using Fura-2/AM based calcium imaging technique. Sulfite (250-1000μM) caused a sustained increase in [Ca(2+)]i in the neurons via a dose-dependent manner. In Ca(2+)-free solution, sulfite failed to increase [Ca(2+)]i. After the depletion of the intracellular calcium store, the effect of sulfite on the [Ca(2+)]i was largely abolished. Pharmacological inhibition of phospholipase C (PLC)-inositol 1,4,5-triphosphate (IP3) signaling pathway blocked sulfite-induced increase of [Ca(2+)]i. Interestingly, antioxidants such as trolox and dithiothreitol, abolished the increase of [Ca(2+)]i induced by sulfite. Exposure to sulfite triggered generation of sulfur- and oxygen-centered free radicals in neurons and increased oxidative stress both in the cultured cortical neurons and the prefrontal cortex of rats. Furthemore, sulfite decreased cell viability in cultured cortical neurons via a calcium-dependent manner. Thus, our current study suggests that the redox-dependent calcium overload triggered by sulfite in cortical neuronsmay be involved in its neurotoxicity., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

Background: The AMP-activated protein kinase (AMPK) is a sensor of cellular energy and nutrient status, with substantial amount of cross talk with other signaling pathways, including its phosphorylation by Akt, PKA, and GSK3β., Aims: Various signaling pathways and energy-consuming transport of glutamate receptors subunits are required in synaptic plasticity. However, it is unknown which energy sensors integrate the signaling pathways in these processes. In this article, we elucidated the role of AMPK activation and GSK3β phosphorylation after HFS during the inducement of early-phase long-term potentiation (E-LTP)., Methods: Synaptic LTP in vivo was induced by high-frequency stimulation (HFS at 200 Hz at a 5-s interval). In addition, phosphorylation of AMPK and glycogen synthase kinase 3β (GSK3β) were measured using Western blotting. The amount of hippocampal AMP, ADP and ATP was measured by HPLC., Results: We showed that the phosphorylation of AMPK and GSK3β was significantly increased by HFS in vivo. HFS-induced AMPK activation occurred via increased (AMP + ADP)/ATP ratio and activation of Ca(2+) /calmodulin-dependent kinase kinase beta (CaMKKβ). Pharmacological inhibition of AMPK by compound C (CC) prevented HFS-induced inhibitory phosphorylation of GSK3β and the induction of LTP in dentate gyrus (DG) area in vivo., Conclusions: Our findings reveal that HFS-triggered AMPK activation phosphorylates GSK3β and induces E-LTP in vivo., (© 2016 John Wiley & Sons Ltd.)

Dopamine, a key neurotransmitter mediating the rewarding effects, exerts some of its effects by modulating neuronal excitability of striatal medium spiny neurons. A D1-like dopamine receptor-dependent phosphatidylinositol signal pathway exists in the striatum, however little is known about its role in the dopaminergic modulation of striatal neuronal excitability. 3-Methyl-6-chloro-7, 8-hydroxy-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83959) is a selective D1 receptor agonist with high-affinity. Here, we observed its effect on the voltage-gated sodium channels (VGSCs) in primary cultured striatal neurons by whole cell patch-clamp technique. We found that SKF83959 induced an inhibition on VGSCs in a dose-dependent manner in striatal neurons (IC50 value: 3.31 ± 0.39 μM), which could be prevented by antagonist of D1 receptor, but not that of D2, α1 adrenergic, or cholinoceptor. The effect of SKF83959 on VGSCs was also prevented by pretreatment with inhibitors of phospholipase C (PLC) and protein kinases C (PKC), but the inositol-1,4,5-phosphate 3 (IP3) antagonist did not occlude SKF83959 (1μM)-induced reduction of VGSCs. These data indicate that SKF83959 inhibits VGSCs in cultured striatal neurons via D1-like receptor-phosphatidylinositol-PKC pathway, which may underlie the dopaminergic modulation on striatal neuronal excitability., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background and Purpose: Impairment of glucose utilization contributes to neuronal degeneration of Alzheimer's disease patients. Cellular glucose utilization can be regulated by calcium-dependent signaling pathways. Resveratrol (RSV) is a plant-derived polyphenol with multiple beneficial effects, including neuroprotection and metabolic improvement. Here, we investigated the effect of RSV on neuronal calcium signal and glucose utilization., Experimental Methods: Primary culture of cortical neurons, calcium imaging, 2-NBDG assay and western blotting were employed to investigate RSV-mediated effects on neuronal calcium signal and glucose utilization., Results: RSV elevated intracellular calcium in cortical neurons via modulation of secondary messenger system including nitrous oxide, cGMP and cAMP. Secondarily, a calcium-dependent enhancement of neuronal glucose utilization after RSV treatment was observed. The effects on neuronal glucose utilization are largely dependent on RSV-induced calcium-dependent AMP-activated protein kinase activation., Conclusion: Our findings show that activation of calcium-dependent signaling pathways by RSV may convey improvements of neuronal glucose utilization., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aim: To identify the mechanisms underlying the elevation of intracellular Ca(2+) level ([Ca(2+)](i)) induced by lowering extracellular glucose in rat hypothalamic arcuate nucleus NPY neurons., Methods: Primary cultures of hypothalamic arcuate nucleus (ARC) neurons were prepared from Sprague-Dawley rats. NPY neurons were identified with immunocytochemical method. [Ca(2+)](i) was measured using fura-2 AM. Ca(2+) current was recorded using whole-cell patch clamp recording. AMPK and GSK3β levels were measured using Western blot assay., Results: Lowering glucose level in the medium (from 10 to 1 mmol/L) induced a transient elevation of [Ca(2+)](i) in ARC neurons, but not in hippocampal and cortical neurons. The low-glucose induced elevation of [Ca(2+)](i) in ARC neurons depended on extracellular Ca(2+), and was blocked by P/Q-type Ca(2+)channel blocker ω-agatoxin TK (100 nmol/L), but not by L-type Ca(2+) channel blocker nifedipine (10 μmol/L) or N-type Ca(2+)channel blocker ω-conotoxin GVIA (300 nmol/L). Lowering glucose level increased the peak amplitude of high voltage-activated Ca(2+) current in ARC neurons. The low-glucose induced elevation of [Ca(2+)](i) in ARC neurons was blocked by the AMPK inhibitor compound C (20 μmol/L), and enhanced by the GSK3β inhibitor LiCl (10 mmol/L). Moreover, lowering glucose level induced the phosphorylation of AMPK and GSK3β, which was inhibited by compound C (20 μmol/L)., Conclusion: Lowering glucose level enhances the activity of P/Q type Ca(2+)channels and elevates [Ca(2+)](i) level in hypothalamic arcuate nucleus neurons via inhibition of GSK3β.

The adipocyte-derived hormone leptin and the pancreatic beta-cell-derived hormone insulin function as afferent signals to the hypothalamus in an endocrine feedback loop that regulates body adiposity. They act in hypothalamic centers to modulate the function of specific neuronal subtypes, such as neuropeptide Y (NPY) neurons, by modifying neuronal electrical activity. To investigate the intrinsic activity of these neurons and their responses to insulin and leptin, we used a combination of morphological features and immunocytochemical technique to identify the NPY neurons of hypothalamic arcuate nucleus (ARC) and record whole cell large-conductance Ca(2+)-activated potassium (BK) currents on them. We found that both of the hormones increase the peak amplitude of BK currents, shifting the steady-state activation curve to the left. The effect of both insulin and leptin can be prevented by pretreatment with inhibitors of tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) but not MAPK. These data indicate that PI3K-mediated signals are the common regulators of BK channels by insulin and leptin and mediated the two hormones' identical activatory effects on ARC NPY neurons. The effect of insulin and leptin together was similar to that of insulin or leptin alone, and leptin or insulin pretreatment did not lead to insulin- or leptin-sensitizing effects, respectively. These intracellular signaling mechanisms may play key roles in regulating ARC NPY neuron activity and physiological processes such as the control of food intake and body weight, which are under the combined control of insulin and leptin.

Methionine and cysteine residues in proteins are the major targets of reactive oxygen species (ROS). The present work was designed to characterize the impact of methionine and cysteine oxidation upon [Ca(2+)](i) in hippocampal neurons. We investigated the effects of H(2)O(2) and chloramine T(Ch-T) agents known to oxidize both cysteine and methionine residues, and 5, 5'-dithio-bis (2-nitrobenzoic acid) (DTNB)--a cysteine-specific oxidant, on the intracellular calcium in hippocampal neurons. The results showed that these three oxidants, 1 mM H(2)O(2), 1 mM Ch-T, and 500 microM DTNB, induced an sustained elevation of [Ca(2+)](i) by 76.1 +/- 3.9%, 86.5 +/- 5.0%, and 24.4 +/- 3.2% over the basal level, respectively. The elevation induced by H(2)O(2) and Ch-T was significantly higher than DTNB. Pretreatment with reductant DTT at 1 mM for 10 min completely prevented the action of DTNB on [Ca(2+)](i), but only partially reduced the effects of H(2)O(2) and Ch-T on [Ca(2+)](i), the reductions were 44.6 +/- 4.2% and 29.6 +/- 6.1% over baseline, respectively. The elevation of [Ca(2+)](i) induced by H(2)O(2) and Ch-T after pretreatment with DTT were statistically higher than that induced by single administration of DTNB. Further investigation showed that the elevation of [Ca(2+)](i) mainly resulted from internal calcium stores. From our data, we propose that methionine oxidation plays an important role in the regulation of intracellular calcium and this regulation may mainly be due to internal calcium stores.

Aim: To observe redox modulation of ion channel in trigeminal ganglion neurons by oxidants and reducing agents., Methods: The effects of oxidants and reducing agents on maxi-conductance calcium-activated potassium channel in cultured rat trigeminal ganglion neurons by using whole-cell patch-clamp technique., Results: Methionine-specific oxidant chloramine-T (Ch-T) 1 mmol/L slightly increased the current amplitude and this enhancement did not antagonized by DTT. In contrast, cysteine-specific reagent 5, 5'-dithio-bis(2-nitrobenzoic acid) (DTNB) 500 micromol/L significantly decreased current amplitude of BK(Ca) channels. The effect was reversed by the reducing agent 2 mmol/L 1, 4-dithio-DL-threitol (DTT)., Conclusion: Reactive oxygen species were definitely involved in regulation of native neuronal function via redox modulation of BK(Ca) channels, which are suggested to play compensatory roles under oxidative stress-related conditions.