Anti-hypoxic effect of interleukin-10 in hippocampal neurons is mediated by modulation of TASK-1 and TASK-3 channels activity.

It has been shown that anti-inflammatory cytokine interleukin-10 (IL-10) can exert anti-hypoxic effect preventing post-hypoxic neuronal hyperexcitability. Yet, exact mechanisms of IL-10 mediated anti-hypoxic action on neuronal function are not fully understood. We suggested that IL-10 can exert its anti-hypoxic action via modulation of activity of two-pore potassium TASK-1 and TASK-3 channels. To study the involvement of TASK-1 and TASK-3 channels we employed a combination of whole-cell patch clamp and pharmacological inhibitory analysis to assess if IL-10 and brief hypoxic episode can modulate K+ background leak current (Ileak) and membrane input resistance (Rin) in cultured hippocampal neurons. We found that IL-10 in a dose-dependent manner can significantly increase Ileak with concomitant reduction in Rin. Neurons that were exposed to brief hypoxic episode on contrary showed significant decrease in Ileak with concomitant increase in Rin. Pretreatment with IL-10 prior hypoxic episode was able to abolish negative effect of hypoxia on Ileak and Rin. IL-10 potentiating action on Ileak and Rin was occluded by co-addition of selective blockers of TASK-1 and TASK-3 channels - ML365 and PK-THPP. Co-addition of LY294002, an inhibitor of PI3-kinase occluded IL-10 action on Ileak and Rin showing involvement of PI3K-associated pathway in IL-10 mediated regulation of TASK channel function. Our results provide new insights into IL-10 mediated neuroprotective and anti-hypoxic actions showing TASK-1 and TASK-3 channels as downstream targets of this anti-inflammatory cytokine.

Activation of alpha-2 adrenergic receptors stimulates GABA release by astrocytes.

Norepinephrine is one of the key neurotransmitters in the hippocampus, but its role in the functioning of the neuroglial networks remains unclear. Here we show that norepinephrine suppresses NH4 Cl-induced oscillations of the intracellular Ca2+ concentration ([Ca2+ ]i ) in hippocampal neurons. We found that the inhibitory effect of norepinephrine against ammonium-induced [Ca2+ ]i oscillations is mediated by activation of alpha-2 adrenergic receptors. Furthermore, UK 14,304, an agonist of alpha-2 adrenergic receptors, evokes a biphasic [Ca2+ ]i elevation in a minor population of astrocytes. This elevation consists of an initial fast, peak-shaped [Ca2+ ]i rise, mediated by Gißγ subunit and subsequent PLC-induced mobilization of Ca2+ from internal stores, and a plateau phase, mediated by a Ca2+ influx from the extracellular medium through store-operated and TRPC3 channels. We show the correlation between the Ca2+ response in astrocytes and suppression of [Ca2+ ]i oscillations in neurons. The inhibitory effect of UK 14,304 is abolished in the presence of gallein, an inhibitor of Gßγ -signaling. In turn, application of the agonist in the presence of the PLC inhibitor decreases the frequency and amplitude of [Ca2+ ]i oscillations in neurons but does not suppress them. The same effect is observed in the presence of bicuculline, a GABA(A) receptor antagonist. We demonstrate that UK 14,304 application increases the frequency and amplitude of slow outward chloride currents in neurons, indicating the release of GABA by astrocytes. Thus, our findings indicate that the activation of astrocytic alpha-2 adrenergic receptors stimulates GABA release from astrocytes via Gißγ subunit-associated signaling pathway, contributing to the suppression of neuronal activity.

Epileptiform activity promotes decreasing of Ca2+ conductivity of NMDARs, AMPARs, KARs, and voltage-gated calcium channels in Mg2+-free model.

NMDA, AMPA, and kainate receptors are the principal excitatory receptors in the brain. These receptors have been considered as the main targets in the treatment of epilepsy in recent years. This work aimed to determine how the Ca2+ conductivity of ionotropic glutamate receptors and voltage-gated Ca2+ channels changes in an in vitro model of epilepsy. For induction of epileptiform activity, hippocampal neurons were exposed to Mg2+-free medium. It has been shown that removal of Mg2+ from the medium not only removes the block from the NMDA receptors but also stimulates the release of glutamate in a way that is independent of the NMDA receptors. Under these conditions, the structure of the bursts significantly differs from the spontaneous bursts arising in mature hippocampal cultures. We have demonstrated that the frequency and amplitude of Mg2+-free medium-induced Ca2+ oscillations decrease after the 60-min exposure. Besides, the Ca2+ conductivity of ionotropic glutamate receptors and voltage-gated calcium channels significantly reduces. Thus, the decrease of Ca2+ conductivity can be considered as one of the mechanisms of adaptation during epilepsy.

Anti-amyloid activities of three different types of water-soluble fullerene derivatives.

Anti-amyloid activity, aggregation behaviour, cytotoxicity and acute toxicity were investigated for three water-soluble fullerene derivatives with different types of solubilizing addends. All investigated compounds showed a strong anti-amyloid effect in vitrocaused by interaction of the water-soluble fullerene derivatives with the Ab(1-42)-peptide and followed by destruction of the amyloid fibrils. Notably, all of the studied fullerene derivatives showed very low cytotoxicity and low acute toxicity in mice (most promising compound 3 was more than four times less toxic than aspirin). Strong anti-amyloid effect of the fullerene derivatives together with low toxicity reveals high potential of these compounds as drug candidates for treatment of neurodegenerative diseases.

Interleukin-10 Facilitates Glutamatergic Synaptic Transmission and Homeostatic Plasticity in Cultured Hippocampal Neurons.

Anti-inflammatory cytokines are known to exert neuroprotective action ameliorating aberrant neuronal network activity associated with inflammatory responses. Yet, it is still not fully understood if anti-inflammatory cytokines play a significant role in the regulation of synaptic activity under normal conditions. Thus, the aim of our study was to investigate the effect of Interleukin-10 (IL-10) on neuronal synaptic transmission and plasticity. For this we tested the effect of IL-10 on miniature excitatory postsynaptic currents (mEPSC) and intracellular Ca2+ responses using whole-cell patch clamp and fluorescence microscopy in 13-15 DIV primary hippocampal neuroglial culture. We found that IL-10 significantly potentiated basal glutamatergic excitatory synaptic transmission within 15 min after application. Obtained results revealed a presynaptic nature of the effect, as IL-10 in a dose-dependent manner significantly increased the frequency but not the amplitude of mEPSC. Further, we tested the effect of IL-10 on mEPSC in a model of homeostatic synaptic plasticity (HSP) induced by treatment of primary hippocampal culture with 1 µM of tetrodotoxin (TTX) for a 24 h. It was found that 15 min application of IL-10 at established HSP resulted in enhanced mEPSC frequency, thus partially compensating for a decrease in the mEPSC frequency associated with TTX-induced HSP. Next, we studied if IL-10 can influence induction of HSP. We found that co-incubation of IL-10 with 1 µM of TTX for 24 h induced synaptic scaling, significantly increasing the amplitude of mEPSC and Ca2+ responses to application of the AMPA agonist, 5-Fluorowillardiine, thus facilitating a compensatory postsynaptic mechanism at HSP condition. Our results indicate that IL-10 potentiates synaptic activity in a dose- and time-dependent manner exerting both presynaptic (short-term exposure) and postsynaptic (long-term exposure) action. Obtained results demonstrate involvement of IL-10 in the regulation of basal glutamatergic synaptic transmission and plasticity at normal conditions.

Domoic acid suppresses hyperexcitation in the network due to activation of kainate receptors of GABAergic neurons.

Kainate receptors play an important role in the brain. They contribute to postsynaptic depolarization, modulate the release of neurotransmitters such as GABA and glutamate, affect the development of the neuronal network. At the same time, their functions depend not only on the type of neuron expressing them but also on their localization (pre- or postsynaptic). It has been shown in present work that activation of kainate receptors by domoic acid stimulates the secretion of both glutamate and GABA. This effect is observed at a concentration of 100 nM. At higher levels (200-500 nM), domoic acid selectively activates a specific population of GABAergic neurons. The peculiarity of these neurons is increased excitability in the network. This phenomenon can be explained by the weak GABA(A)R-mediated inhibition, as well as by the lower activation threshold of voltage-gated channels. Moreover, activation of these GABAergic neurons by domoic acid leads to the suppression of activity in the network under ammonium-induced hyperexcitation. As shown by inhibitory analysis, this effect is mediated by GABA(A) receptors. The obtained data may be of interest since the suppression of hyperexcitation via the selective activation of GABAergic neurons can be considered as a new potential approach to the treatment of diseases accompanied by increased neuronal activity such as epilepsy, ischemia and hepatic encephalopathy.

Fast changes of NMDA and AMPA receptor activity under acute hyperammonemia in vitro.

It was established in experiments on cell cultures of neurons and astrocytes that ammonium ions at concentrations of 4-8 mM cause hyperexcitation of the neuronal network, as a result of which there is a disturbance of calcium homeostasis, which can lead to the death of neurons. In the present study, we investigated the effect of toxic doses of ammonium (8 mM NH4Cl) on the activity of NMDA and AMPA receptors and the role of these receptors in spontaneous synchronous activity (SSA). In a control experiment in the absence of NH4Cl, SSA is not suppressed by NMDA receptor inhibitors, but is suppressed by AMPA receptor antagonists. In the presence of toxic doses of NH4Cl, SSA is completely inhibited by NMDA receptor inhibitors in 63% of neurons and by AMPA receptor inhibitors in 33% of neurons. After short-term applications of toxic doses of ammonium, the amplitude of the Ca2+ response to 10 µM NMDA increases, and decreases in response to 500 nM FW (agonist of AMPA receptors). NMDA receptor blocker MK-801 (20 µM), competitive antagonist D-AP5 (10 µM) and competitive AMPA receptor antagonist NBQX (2 µM) abolished the activating ammonium mediated effect on the NMDA receptors while only MK-801, but not NBQX, abolished the inhibiting ammonium mediated effect on AMPA receptors. These data indicate that under acute hyperammonemia, the activity of NMDA receptors increases, while the activity of AMPA receptors decreases. This phenomenon could explain such a wide range of toxic effects of ammonium ions mediated by NMDA receptors.

Sarcolemmal α2-adrenoceptors control protective cardiomyocyte-delimited sympathoadrenal response.

Sustained cardiac adrenergic stimulation has been implicated in the development of heart failure and ventricular dysrhythmia. Conventionally, α2 adrenoceptors (α2-AR) have been assigned to a sympathetic short-loop feedback aimed at attenuating catecholamine release. We have recently revealed the expression of α2-AR in the sarcolemma of cardiomyocytes and identified the ability of α2-AR signaling to suppress spontaneous Ca2+ transients through nitric oxide (NO) dependent pathways. Herein, patch-clamp measurements and serine/threonine phosphatase assay revealed that, in isolated rat cardiomyocytes, activation of α2-AR suppressed L-type Ca2+ current (ICaL) via stimulation of NO synthesis and protein kinase G- (PKG) dependent activation of phosphatase reactions, counteracting isoproterenol-induced ß-adrenergic activation. Under stimulation with norepinephrine (NE), an agonist of ß- and α-adrenoceptors, the α2-AR antagonist yohimbine substantially elevated ICaL at NE levels >10nM. Concomitantly, yohimbine potentiated triggered intracellular Ca2+ dynamics and contractility of cardiac papillary muscles. Therefore, in addition to the α2-AR-mediated feedback suppression of sympathetic and adrenal catecholamine release, α2-AR in cardiomyocytes can govern a previously unrecognized local cardiomyocyte-delimited stress-reactive signaling pathway. We suggest that such aberrant α2-AR signaling may contribute to the development of cardiomyopathy under sustained sympathetic drive. Indeed, in cardiomyocytes of spontaneously hypertensive rats (SHR), an established model of cardiac hypertrophy, α2-AR signaling was dramatically reduced despite increased α2-AR mRNA levels compared to normal cardiomyocytes. Thus, targeting α2-AR signaling mechanisms in cardiomyocytes may find implications in medical strategies against maladaptive cardiac remodeling associated with chronic sympathoadrenal stimulation.