Astrocytic Gap Junctions Contribute to Aberrant Neuronal Synchronization in a Mouse Model of MeCP2 Duplication Syndrome / 神经科学通报·英文版

Abnormal synchronous neuronal activity has been widely detected by brain imaging of autistic patients, but its underlying neural mechanism remains unclear. Compared with wild-type mice, our in vivo two-photon imaging showed that transgenic (Tg1) mice over-expressing human autism risk gene MeCP2 exhibited higher neuronal synchrony in the young but lower synchrony in the adult stage. Whole-cell recording of neuronal pairs in brain slices revealed that higher neuronal synchrony in young postnatal Tg1 mice was attributed mainly to more prevalent giant slow inward currents (SICs). Both in vivo and slice imaging further demonstrated more dynamic activity and higher synchrony in astrocytes from young Tg1 mice. Blocking astrocytic gap junctions markedly decreased the generation of SICs and overall cell synchrony in the Tg1 brain. Furthermore, the expression level of Cx43 protein and the coupling efficiency of astrocyte gap junctions remained unchanged in Tg1 mice. Thus, astrocytic gap junctions facilitate but do not act as a direct trigger for the abnormal neuronal synchrony in young Tg1 mice, revealing the potential role of the astrocyte network in the pathogenesis of MeCP2 duplication syndrome.

Effect of KN93 on delayed afterdepolarization and calcium ion in ventricular myocytes of rabbits with heart failure / 中华急诊医学杂志

Objective To observe the effect of KN93, a CaMK Ⅱ inhibitor, on delayed afterdepolarization (DAD) and calcium ion in ventricular myocytes of rabbits with heart failure, and to investigate the effect of CaMK Ⅱ signaling pathway on trigged arrhythmia after heart failure. Methods Thirty male New Zealand White rabbits were randomized(random number) into the sham operated group (sham group), heart failure group (HF group) and heart failure with KN93 group (HF+KN93 group) (n=10 each group). The rabbit heart failure model was established by abdominal aortic constriction combined with aortic valve regurgitation. The ventricular myocytes were isolated by double enzyme digestion. The action potential and the transient inward current (Iti) were recorded by the whole-cell patch-clamp. The intracellular calcium transient was measured by the ion concentration measurement system. The main calcium transporter protein was detected by Western blotting. Data were analyzed by pCLAMP10.2. Statistical analysis was performed using SPSS 17.0. Comparisons among groups were conducted using ANOVA, and SNK-q multiple comparison procedure was utilized for post-hoc analysis.Results (1) After induction of heart failure, DAD and increment of trigger activity (TA) were observedin rabbit ventricular myocytes. Treatment of KN93 with 1.0 μmol/L reduced the events of DAD and TA.(2) After induction of heart failure, Iti densities were increased from -0.12±0.02 pA/pF to -0.95±0.06pA/pF at the polarization potential of -50 mV (n=10, P<0.01). The current densities were reduced to -0.44±0.04 pA/pF after application of 1.0 μmol/L of KN93 (n=10, P<0.01). (3) KN93 led to decrementof intracellular calcium ion concentration and calcium transient amplitude, and acceleration of the decayprocess of calcium transient. (4) KN93 upregulated the expression of pPLN and SERCA2a, increased the uptake of intracellular calcium ion, downregulated the expression of NCX, decreased the Iti, and reduced the occurrence of DAD and TA. Conclusions KN93 can reduce the intracellular calcium ion concentration of the heart failure animal model, and the occurrence of the DAD and TA. CaMK Ⅱ may be a new therapeutic target for arrhythmias in the heart failure.

Ca2+-activated K+ Current in Freshly Isolated c-Kit Positive Cells in Guinea-pig Stomach

This study was designed to isolate Ca2+-activated K+ current (IKCa) and elucidate its physiological significance in freshly isolated interstitial cells of Cajal (ICCs) of guinea-pig stomach. Single ICC was freshly isolated by enzymatically dissociating from myenteric border of gastric antrum free of circular muscles, and conventional whole-cell voltage clamp technique including immunohistochemical techniques were employed to characterize the cells: In myenteric border of gastric antrum, ICC-MY (ICCs from myenteric border) were detected by immunohistochemical reactivity, and single ICC-MY which has many branches was immunohistochemically c-Kit positive. Under K+-rich and 0.1 mM ethylene glycol-bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid pipette solution, ICC produced spontaneous inward current (-256+/-92.2 pA). When step-depolarizing pulse from -80 to +80 mV was applied at holding potential (Vh) of -80 mV, voltage-dependent outward currents were recorded with superimposed spontaneous transient outward currents (STOCs). Both STOCs and outward currents were reversibly affected by tetraethylammonium chloride (TEA) and iberiotoxin (IbTX); 2 mM TEA and 200 nM IbTX completely abolished STOCs and significantly inhibited outward K+ current over the whole potential range tested for current/voltage (I/V) relationship. In addition, TEA delayed repolarization phase of spontaneous inward current. The present results indicate the presence of IKCa in a single ICC, and it might be involved in regulation of repolarizing phase of spontaneous inward current in guinea-pig stomach.

Characterization of Ionic Currents in Human Neural Stem Cells

The profile of membrane currents was investigated in differentiated neuronal cells derived from human neural stem cells (hNSCs) that were obtained from aborted fetal cortex. Whole-cell voltage clamp recording revealed at least 4 different currents: a tetrodotoxin (TTX)-sensitive Na+ current, a hyperpolarization-activated inward current, and A-type and delayed rectifier-type K+ outward currents. Both types of K+ outward currents were blocked by either 5 mM tetraethylammonium (TEA) or 5 mM 4-aminopyridine (4-AP). The hyperpolarization-activated current resembled the classical K+ inward current in that it exhibited a voltage-dependent block in the presence of external Ba2+ (30micrometer) or Cs+ (3micrometer). However, the reversal potentials did not match well with the predicted K+ equilibrium potentials, suggesting that it was not a classical K+ inward rectifier current. The other Na+ inward current resembled the classical Na+ current observed in pharmacological studies. The expression of these channels may contribute to generation and repolarization of action potential and might be regarded as functional markers for hNSCs-derived neurons.

Blockade of intrinsic oscillatory activity of cerebellar Purkinje cells by apamin and nickel

Intracellular recordings of oscillatory firing (bursting activity) were obtained from Purkinje cells (PCs) in rat cerebellar slices. Apamin inhibited post-burst hyperpolarizations (PBHs) progressively and finally terminated oscillatory firing activity of PCs. Apamin did not affect the amplitude or duration of the after-hyperpolarization (AHP) between spikes within the burst. In the voltage clamp mode, apamin shifted the whole-cell, quasi-steady state I/V relationship in an inward direction and abolished the zero slope resistance (ZSR) region by blocking outward current. Nickel (Ni2+) terminated oscillatory activity and also abolished the ZSR region. However, Ni2+ did not have progressive blocking action on the post-burst hyperpolarization before it blocked oscillatory activity. Ni2+ blocked an inward current at potentials positive to approximately -65 mV, which was responsible for the ZSR region and outward current at more negative potentials. These data indicated that oscillatory activity of PCs is sustained by a balance between a slow Ni2+ -sensitive inward current and an apamin-sensitive outward current in the region of ZSR of the whole-cell I/V curve.

Effects of cyclic-GMP on hyperpolarization-activated inward current (I|f) in sino-atrial node cells of rabbit

The aim of present study is to investigate the effects of cGMP on hyperpolarization activated inward current (If), pacemaker current of the heart, in rabbit sino-atrial node cells using the whole-cell patch clamp technique. When sodium nitroprusside (SNP, 80 muM), which is known to activate guanylyl cyclase, was added, If amplitude was increased and its activation was accelerated. However, when If was prestimulated by isopreterenol (ISO, 1 muM), SNP reversed the effect of ISO. In the absence of ISO, SNP shifted activation curve rightward. On the contrary in the presence of ISO, SNP shifted activation curve in opposite direction. 8Br-cGMP (100 muM), more potent PKG activator and worse PDE activator than cGMP, also increased basal If but did not reverse stimulatory effect of ISO. It was probable that PKG activation seemed to be involved in SNP-induced basal If increase. The fact that SNP inhibited ISO-stimulated If suggested cGMP antagonize cAMP action via the activation of PDE. This possibility was supported by experiment using 3-isobutyl-1-methylxanthine (IBMX), non-specific PDE inhibitor. SNP did not affect If when If was stimulated by 20 muM IBMX. Therefore, cGMP reversed the stimulatory effect of cAMP via cAMP breakdown by activating cGMP-stimulated PDE. These results suggest that PKG and PDE are involved in the modulation of If by cGMP: PKG may facilitate If and cGMP-stimulated PDE can counteract the stimulatory action of cAMP.

Study on the inward currents of cardiomyocyte-like cells derived from rat bone marrow mesenchymal stem cells / 第三军医大学学报

Objective To observe the electrophysiological characteristics of the inward current of cardiomyocyte-like cells derived from rat bone marrow mesenchymal stem cells (MSCs). Methods Bone marrow specimens was extracted from SD rats aged 1 month. MSCs were isolated by gradient centrifugation and obtained by adherence culture. The second-passage MSCs were treated with 10 ?mol/L 5-azacytidine and incubated for 24 h. After 4 weeks, cardiomyocyte-like cells were identified by immunocytochemical staining technique, then used to detect the inward current with the whole-cell patch-clamp technique, which results were compared with the normal cardiomyocytes and the MSCs without induction. Results Some cardiomyocyte-like cells were stained positive for troponin T, and the quickly activated and inactivated inward currents were elicited 4 weeks after 5-aza cytidine treatment. The inward currents of cardiomyocyte-like cells recorded in the same square pulse were weaker than that of the normal cardiomyocytes, and the I-V curve of cardiomyocyte-like cells was right-shifted. Conclusion After treated with 5-azacytidine, MSCs differentiate into cardiomyocyte-like cells which express the excitable inward current.

Effects of lotusine on the action potentials in myocardium and slow inward current in cardiac purkinje fibers / 中国药理学通报

AIM To study the effects of Lotusine(Lot), a pure alkaloid extracted from the green seed embryo of Nelumbo nucifera Gaertn, on the action potentials(APs) in myocardium and slow inward current(Isi) in cardiac Purkinje fibers(PF). METHODS standard microelectrode and two microelectrodes voltage clamp techniques were used. RESULTS Lot 1～100 ?mol?L -1 could concentration dependently prolonged APD 20 and APD 90 of fast AP and increase the contractile force(Fc) in guinea pig papillary muscles. In papillary muscles pretreated with reserpine, similar results were also observed but the effect of Lot was weaker than those pretreated without reserpine. In guinea pig left atria, Lot 30 ?mol?L -1 could partly antigoniste the shortening APD effect of acetylcholine. Lot 3～100 ?mol?L -1 could enhance the action potential amplitude(APA)and maximal velocity of phase 0 depolarization ( V max ) of slow AP of papillary muscles induced by high K + (24 mmol?L -1 )and isolated sinoatral node(SAN) pacemaker cells of rabbits with dose dependent manner,But not obvousely short the sinus cycle length of SAN. Moreover, Lot increased the Isi of in canin cardiac PF with with time dependent and dose dependent manner. CONCLUSION The results suggest that Lot has effect of prolongation of APD and increase of Ca 2+ influx and these are very important in contribution to its positive inotropic effect, which may be related to inhibition of phosphodiesterase Ⅲ.