Neuroactive Steroids Reverse Tonic Inhibitory Deficits in Fragile X Syndrome Mouse Model

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. A reduction in neuronal inhibition mediated by γ-aminobutyric acid type A receptors (GABAARs) has been implicated in the pathophysiology of FXS. Neuroactive steroids (NASs) are known allosteric modulators of GABAAR channel function, but recent studies from our laboratory have revealed that NASs also exert persistent metabotropic effects on the efficacy of tonic inhibition by increasing the protein kinase C (PKC)-mediated phosphorylation of the α4 and β3 subunits which increase the membrane expression and boosts tonic inhibition. We have assessed the GABAergic signaling in the hippocampus of fragile X mental retardation protein (FMRP) knock-out (Fmr1KO) mouse. The GABAergic tonic current in dentate gyrus granule cells (DGGCs) from 3- to 5-week-old (p21–35) Fmr1KO mice was significantly reduced compared to WT mice. Additionally, spontaneous inhibitory post synaptic inhibitory current (sIPSC) amplitudes were increased in DGGCs from Fmr1 KO mice. While sIPSCs decay in both genotypes was prolonged by the prototypic benzodiazepine diazepam, those in Frm1-KO mice were selectively potentiated by RO15-4513. Consistent with this altered pharmacology, modifications in the expression levels and phosphorylation of receptor GABAAR subtypes that mediate tonic inhibition were seen in Fmr1 KO mice. Significantly, exposure to NASs induced a sustained elevation in tonic current in Fmr1 KO mice which was prevented with PKC inhibition. Likewise, exposure reduced elevated membrane excitability seen in the mutant mice. Collectively, our results suggest that NAS act to reverse the deficits of tonic inhibition seen in FXS, and thereby reduce aberrant neuronal hyperexcitability seen in this disorder

Angiotensin (1-7) Attenuates the Chronotropic Response to Angiotensin II via Stimulation of PTEN in Spontaneously Hypertensive Rat Brain

The pathogenesis of hypertension and its mode of progression are complex, multifactorial and incompletely understood. Several studies have focused on the beneficial effects of peripheral Ang (1-7) in the regulation of cardiovascular functions, showing the counter-regulatory effects of Ang (1-7) against the actions of Ang II in the periphery. However, its actions in the central nervous system are not completely understood. In the present study, our main goal was to determine the central action of Ang (1-7) and its interaction with Ang II in the blood pressure control. Previous studies reported that Ang II produces a greater degree of activation of neuronal cells from brainstem/hypothalamus cultures of SHR versus WKY rats. Our present findings showed that this enhanced action of Ang II was attenuated in co-presence of either Ang (1-7) or PI3-kinase inhibitor. These counter-regulatory effects of Ang (1-7) on Ang II action in SHR neurons were abolished by co-treatment with either A-779, a Mas-R antagonist, or bisperoxovanadium (BPV), a PTEN inhibitor. In addition, incubation of WKY and SHR neurons with Ang (1-7) significantly increased PTEN activity. Chronic treatment with Ang (1-7) or chronic inhibition of PI3K using lentiviral vector significantly abolished the enhanced chronotroic response to Ang II in SHR neurons and significantly enhanced PTEN protein and mRNA expression levels in both WKY and SHR neuronal cultures. To further check the functional implications of our in vitro data, we further studied the interaction between Ang II and Ang (1-7) in the central control of cardiovascular functions. RVLM microinjection of Ang (1-7) or LY-294002 alone did not alter MAP, but reduced the pressor response to Ang II in SHR. Moreover, in compliance with our in vitro data, the inhibitory effect of Ang (1-7) on the pressor response to Ang II in SHR was abolished when co-administered together with A-779 or BPV. The data demonstrated that Ang (1-7) induce PTEN activity and expression via Mas-R, and depresses PI3-kinase-PKB/Akt signal transduction pathway, which lead to the counter-regulatory effect of Ang (1-7) on Ang II induced chronotropic and pressor effect on neuronal activity and cardiovascular functions including MAP and HR in SHR

Analyzing Contract Robustness through a Model of Commitments

Commerce is driven by business contracts. Here, each party to such a contract must be assured that it is robust, fulfilling its goals and avoiding undesirable outcomes. However, real-life business contracts tend to be complex and unamenable both to manual scrutiny and domain- independent scientific methods, making it difficult to provide automated support for determining or improving their robustness. As a result, establishing a contract is nontrivial and adds significantly to the transaction costs of conducting business. If the adoption of multi-agent systems approaches in supporting business interactions is to be viable, we need to develop appropriate techniques to allow such software to reason about contracts in relation to their robustness. To this end, in this paper we propose a powerful approach to assessing the robustness of contracts, and make three main contributions. First, we demonstrate a novel commitment-based formal model for contracts. Second, we define rules to evaluate the robustness of contracts. Third, we offer a methodology for modeling contracts to enable checking them for robustness. We validate these contributions via real-world contracts

The Role of Cell-Penetrating Peptide and Transferrin on Enhanced Delivery of Drug to Brain

The challenge of effectively delivering therapeutic agents to brain has led to an entire field of active research devoted to overcome the blood brain barrier (BBB) and efficiently deliver drugs to brain. This review focusses on exploring the facets of a novel platform designed for the delivery of drugs to brain. The platform was constructed based on the hypothesis that a combination of receptor-targeting agent, like transferrin protein, and a cell-penetrating peptide (CPP) will enhance the delivery of associated therapeutic cargo across the BBB. The combination of these two agents in a delivery vehicle has shown significantly improved (p < 0.05) translocation of small molecules and genes into brain as compared to the vehicle with only receptor-targeting agents. The comprehensive details of the uptake mechanisms and properties of various CPPs are illustrated here. The application of this technology, in conjunction with nanotechnology, can potentially open new horizons for the treatment of central nervous system disorders

Apelin-13 inhibits large-conductance Ca2+-activated K+ channels in cerebral artery smooth muscle cells via a PI3-kinase dependent mechanism.

Apelin-13 causes vasoconstriction by acting directly on APJ receptors in vascular smooth muscle (VSM) cells; however, the ionic mechanisms underlying this action at the cellular level remain unclear. Large-conductance Ca(2+)-activated K(+) (BKCa) channels in VSM cells are critical regulators of membrane potential and vascular tone. In the present study, we examined the effect of apelin-13 on BK(Ca) channel activity in VSM cells, freshly isolated from rat middle cerebral arteries. In whole-cell patch clamp mode, apelin-13 (0.001-1 μM) caused concentration-dependent inhibition of BK(Ca) in VSM cells. Apelin-13 (0.1 µM) significantly decreased BK(Ca) current density from 71.25 ± 8.14 pA/pF to 44.52 ± 7.10 pA/pF (n=14 cells, P<0.05). This inhibitory effect of apelin-13 was confirmed by single channel recording in cell-attached patches, in which extracellular application of apelin-13 (0.1 µM) decreased the open-state probability (NPo) of BK(Ca) channels in freshly isolated VSM cells. However, in inside-out patches, extracellular application of apelin-13 (0.1 µM) did not alter the NPo of BK(Ca) channels, suggesting that the inhibitory effect of apelin-13 on BKCa is not mediated by a direct action on BK(Ca). In whole cell patches, pretreatment of VSM cells with LY-294002, a PI3-kinase inhibitor, markedly attenuated the apelin-13-induced decrease in BK(Ca current density. In addition, treatment of arteries with apelin-13 (0.1 µM) significantly increased the ratio of phosphorylated-Akt/total Akt, indicating that apelin-13 significantly increases PI3-kinase activity. Taken together, the data suggest that apelin-13 inhibits BK(Ca) channel via a PI3-kinase-dependent signaling pathway in cerebral artery VSM cells, which may contribute to its regulatory action in the control of vascular tone

Effect of pertussis toxin and apelin-13 on BK_Ca channels in rat middle cerebral arterial VSM cells.

<p>Whole-cell BK_Ca currents were recorded in rat middle cerebral arterial VSM cells as described in the Methods. <b>A</b>-<b>D</b>: representative tracings depicting the currents recorded from a single VSM cell under the following sequential treatment conditions: Control (A), followed by superfusion with apelin-13 (0.1 µM, 5 min) (B), washout of apelin-13, superfusion with Pertussis toxin (PT, 100 nM, 5 min) (C), and superfusion with apelin-13 plus Pertussis toxin (D). <b>E</b>: Average whole-cell current-voltage plots of BK_Ca current. <b>F</b>: Bar graphs summarizing the average current density (pA/pF) at +50 mV obtained at each treatment condition described above. Values are mean±SEM (n=6). *<i>P</i><0.05 indicates a significant difference from the corresponding control value.</p

Effects of apelin-13 on the activity of BK_Ca channels recorded from cell-attached patches of rat middle cerebral arterial VSM cells.

<p>Currents were recorded at room temperature with a pipette potential of -40 mV. <b>A</b>: Representative tracings showing the large-conductance K⁺ channel currents recorded from cell-attached patches of VSM cells under the following sequential treatment conditions: Control; apelin-13 (0.1 µM, 5 min); washout. <b>B</b>: Bar graph summarizing the open state probability (NP_o) of BK_Ca channels during each treatment condition described above. *<i>P</i><0.05 indicates a significant difference from the corresponding control value. Values presented are mean±SEM recorded from 9 cells. </p

Effect of PI3-kinase inhibition on the inhibitory action of apelin-13 on BK_Ca channel currents in rat middle cerebral arterial VSM cells.

<p>Whole-cell BK_Ca currents were recorded as described in the Methods. <b>A</b>-<b>D</b>: Representative tracings showing the currents recorded from a single VSM cell under the following sequential treatment conditions: control (A); followed by superfusion with 0.1 µM apelin-13 (B) for 5 min; washout of apelin-13; superfusion with LY-294002 (10 mM, 5 min) (C); and superfusion with apelin-13 plus LY-294002 (D). <b>E</b>: Average whole cell current density-voltage plots of BK_ca current. <b>F</b>: Bar graph summarizing the average current density (pA/pF) at 50 mV obtained at each treatment condition described above. Values are mean±SEM (n=6 cells). *<i>P</i><0.05 indicates a significant difference from the corresponding control value.</p

Effect of apelin-13 on activity of large conductance Ca²⁺-activated K⁺ (BK_Ca ) channels of rat middle cerebral arterial VSM cells.

<p>Whole-cell K⁺ currents were recorded at room temperature in response to successive voltage pulses of 800 ms duration, increasing in 10-mV increments from -70 mV to +50 mV before and after the treatment of apelin-13 (0.001-1 µM). <b>A</b>-<b>D</b>: representative tracings depicting the currents recorded from a single VSM cell before and after treatment with apelin-13 (0.1 µM, 5 min) or iberiotoxin (IBT) (100 nM, 5 min). <b>E</b>: I-V curve plots of BK_Ca currents at baseline and after application of apelin-13 (0.1 µM, 5 min). <b>F</b>: Bar graph summarizing the concentration-dependent effect of apelin-13 (0.001-1 µM) on average current density (pA/pF) at +50 mV. Values are mean±SEM (n=6 to 14 cells). *<i>P</i><0.05 indicates a significant difference from the corresponding control value. <b>G</b>: A representative VSM cell isolated from rat middle cerebral arteries under optical phase and fluorescence imaging. Fluorescence micrographs demonstrate the APJ receptor expression in VSM cells immunostained with anti-alpha smooth muscle actin antibodies (red) and anti-APJ receptor antibodies (green). The overlap of these two images shows that green fluorescence is VSM cell-located. </p

Effect of apelin-13 on BK_Ca channel activity recorded from inside-out patches of VSM cells freshly isolated from rat middle cerebral arteries.

<p>Currents were recorded at room temperature before and after administration of apelin-13 through the recording pipettes. <b>A</b>: Representative tracings depicting the effects of apelin-13 (0.1 µM, 5 min) on the large-conductance K⁺ channel currents recorded from inside-out patches of VSM cells isolated from rat middle cerebral arteries. <b>B</b>: Bar graph summarizing the open state probability (NP_o) of BK_Ca channels before and after administration of apelin-13 (0.1 µM). Values presented are mean±SEM recorded from 7 cells.</p