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Klotho (KL) is a glycosyl hydrolase and aging-suppressor gene. Stress is a risk factor for depression and anxiety that are highly comorbid with each other. The aim of this study was to determine KL is regulated by estrogen and plays an important role in sex differences in stress resilience. Our results showed that KL was regulated by estrogen in rat hippocampal neurons in vivo and in vitro and was essential for estrogen-mediated increase in the number of presynaptic vesicular glutamate transporter 1 (Vglut1) positive clusters on the dendrites of hippocampal neurons. The role of KL in sex differences in stress responses was examined in rats using three-week chronic unpredictable mild stress (CUMS). CUMS produced a deficit in spatial learning and memory, anhedonic-like and anxiety-like behaviors in male but not female rats, which was accompanied by a reduction in KL protein levels in the hippocampus of male, but not female rats. This demonstrated the resilience of female rats to CUMS. Interestingly, knockdown of KL protein levels in the rat hippocampus of both sexes caused a decrease in stress resilience in both sexes, especially in female rats. These results suggest that regulation of KL by estrogen plays an important role in estrogen-mediated synapse formation, and KL plays a critical role in the sex differences in cognitive deficit, anhedonic-like and anxiety-like behaviors induced by chronic stress in rats, highlighting an important role of KL in sex differences in stress resilience.

Regenerative inward currents help produce slow oscillations through a negative-slope conductance region of their current-voltage relationship that is well approximated by a linear negative conductance. We used dynamic-clamp injections of a linear current with such conductance, INL, to explore why some neurons can generate intrinsic slow oscillations whereas others cannot. We addressed this question in synaptically isolated neurons of the crab Cancer borealis after blocking action potentials. The pyloric network consists of a distinct pacemaker and follower neurons, all of which express the same complement of ionic currents. When the pyloric dilator (PD) neuron, a member of the pacemaker group, was injected with INL with dynamic clamp, it consistently produced slow oscillations. In contrast, all follower neurons failed to oscillate with INL To understand these distinct behaviors, we compared outward current levels of PD with those of follower lateral pyloric (LP) and ventral pyloric (VD) neurons. We found that LP and VD neurons had significantly larger high-threshold potassium currents (IHTK) than PD and LP had lower-transient potassium current (IA). Reducing IHTK pharmacologically enabled both LP and VD neurons to produce INL-induced oscillations, whereas modifying IA levels did not affect INL-induced oscillations. Using phase-plane and bifurcation analysis of a simplified model cell, we demonstrate that large levels of IHTK can block INL-induced oscillatory activity whereas generation of oscillations is almost independent of IA levels. These results demonstrate the general importance of a balance between inward pacemaking currents and high-threshold K+ current levels in determining slow oscillatory activity.NEW & NOTEWORTHY Pacemaker neuron-generated rhythmic activity requires the activation of at least one inward and one outward current. We have previously shown that the inward current can be a linear current (with negative conductance). Using this simple mechanism, here we demonstrate that the inward current conductance must be in relative balance with the outward current conductances to generate oscillatory activity. Surprisingly, an excess of outward conductances completely precludes the possibility of achieving such a balance.

Regenerative inward currents help produce slow oscillations through a negative-slope conductance region of their current-voltage relationship that is well approximated by a linear negative conductance. We used dynamic clamp injections of a linear current with this conductance, INL, to explore why some neurons can generate intrinsic slow oscillations whereas others cannot. We addressed this question, in synaptically isolated neurons of the crab Cancer borealis, after blocking action potentials. The pyloric network consists of distinct pacemaker group and follower neurons, all of which express the same complement of ionic currents. When the pyloric dilator (PD) neuron, a member of the pacemaker group, was injected with INL using dynamic clamp, it consistently produced slow oscillations. In contrast, the lateral pyloric (LP) or ventral pyloric (VD) follower neurons, failed to oscillate with INL. To understand these distinct behaviors, we compared outward current levels of PD, LP and VD neurons. We found that LP and VD neurons had significantly larger high-threshold potassium currents (IHTK) than PD, and LP had lower transient potassium current, IA. Reducing IHTK pharmacologically enabled both LP and VD neurons to produce oscillations with INL, whereas modifying IA levels did not affect INL-induced oscillations. Using phase-plane and bifurcation analysis of a simplified model cell, we demonstrate that large levels of IHTK can block INL-induced oscillatory activity, whereas generation of oscillations is almost independent of IA levels. These results demonstrate the importance of a balance between inward pacemaking currents and high-threshold K+current levels in determining slow oscillatory activity.

Inflammation is a significant player in the progression of heart failure and has detrimental effects on cardiac function. Prostaglandin (PG)E2, a major proinflammatory prostanoid in the cardiovascular system, is a potent stimulus in inducing intracellular cAMP but minimally affects cardiac contractile function. Here, we show that the PGE2 stimulation attenuates the adrenergic-induced cardiac contractile response in animal hearts. Stimulation with PGE2 leads to stimulatory G protein (Gs)-dependent production of cAMP. However, the induced cAMP is spatially restricted because of its degradation by phosphodiesterase (PDE)4 and cannot access the intracellular sarcoplasmic reticulum (SR) for increasing calcium signaling and myocyte contraction. Moreover, pretreatment with PGE2 significantly inhibits PKA activities at the SR induced by a β-adrenergic agonist, isoproterenol, and subsequently blocks isoproterenol-induced PKA phosphorylation of phospholamban and contractile responses in myocytes. Further analysis reveals that the PGE2-induced cAMP/PKA is sufficient to phosphorylate and activate PDE4D isoforms, which, in turn, spatially inhibits the diffusion of adrenergic-induced cAMP from the plasma membrane to the SR. Inhibition of PDE4 rescues the adrenergic-induced increase in cAMP/PKA activities at the SR, PKA phosphorylation of phospholamban, and contractile responses in PGE2-pretreated myocytes. Thus, this offers an example that one Gs-coupled receptor is able to inhibit the intracellular signaling transduction initiated by another Gs-coupled receptor via controlling the diffusion of cAMP, presenting a paradigm for G protein-coupled receptor (GPCR) signal transduction. It also provides a mechanism for the integration of signaling initiated by different neurohormonal stimuli, as well as long-term effects of chronically circulating proinflammatory factors in myocardium.

The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases Cdc42 and Rab8a are critical regulators of these processes in mice. Conditional ablation of Cdc42 in the mouse intestinal epithelium resulted in the formation of large intracellular vacuolar structures containing microvilli (microvillus inclusion bodies) in epithelial enterocytes, a phenotype reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells, and increased apoptosis. Cdc42 deficiency impaired Rab8a activation and its association with multiple effectors, and prevented trafficking of Rab8a vesicles to the midbody. This impeded cytokinesis, triggering crypt apoptosis and disrupting epithelial morphogenesis. Rab8a was also required for Cdc42-GTP activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data suggest that defects of the stem cell niche can cause MVID. This hypothesis represents a conceptual departure from the conventional view of this disease, which has focused on the affected enterocytes, and suggests stem cell-based approaches could be beneficial to infants with this often lethal condition.

Cardiac remodeling during heart failure development induces a significant increase in the activity of the L-type Ca2+ channel (Cav1.2). However, the effects of enhanced Cav1.2 activity on myocyte excitation-contraction (E-C) coupling, cardiac contractility, and its regulation by the β-adrenergic system are not clear. To recapitulate the increased Cav1.2 activity, a double transgenic (DTG) mouse model overexpressing the Cavβ2a subunit in a cardiac-specific and inducible manner was established. We studied cardiac (in vivo) and myocyte (in vitro) contractility at baseline and upon β-adrenergic stimulation. E-C coupling efficiency was evaluated in isolated myocytes as well. The following results were found: 1) in DTG myocytes, L-type Ca2+ current ( ICa,L) density, myocyte fractional shortening (FS), peak Ca2+ transients, and sarcoplasmic reticulum (SR) Ca2+ content (caffeine-induced Ca2+ transient peak) were significantly increased (by 100.8%, 48.8%, 49.8%, and 46.8%, respectively); and 2) cardiac contractility evaluated with echocardiography [ejection fraction (EF) and (FS)] and invasive intra-left ventricular pressure (maximum dP/d t and −dP/d t) measurements were significantly greater in DTG mice than in control mice. However, 1) the cardiac contractility (EF, FS, dP/d t, and −dP/d t)-enhancing effect of the β-adrenergic agonist isoproterenol (2 μg/g body wt ip) was significantly reduced in DTG mice, which could be attributed to the loss of β-adrenergic stimulation on contraction, Ca2+ transients, ICa,L, and SR Ca2+ content in DTG myocytes; and 2) E-C couplng efficiency was significantly lower in DTG myocytes. In conclusion, increasing Cav1.2 activity by promoting its high-activity mode enhances cardiac contractility but decreases E-C coupling efficiency and the adrenergic reserve of the heart.

Neuronal oscillatory activity is generated by a combination of ionic currents, including at least one inward regenerative current that brings the cell towards depolarized voltages and one outward current that repolarizes the cell. Such currents have traditionally been assumed to require voltage-dependence. Here we test the hypothesis that the voltage dependence of the regenerative inward current is not necessary for generating oscillations. Instead, a current I NL that is linear in the biological voltage range and has negative conductance is sufficient to produce regenerative activity. The current I NL can be considered a linear approximation to the negative-conductance region of the current---voltage relationship of a regenerative inward current. Using a simple conductance-based model, we show that I NL , in conjunction with a voltage-gated, non-inactivating outward current, can generate oscillatory activity. We use phase-plane and bifurcation analyses to uncover a rich variety of behaviors as the conductance of I NL is varied, and show that oscillations emerge as a result of destabilization of the resting state of the model neuron. The model shows the need for well-defined relationships between the inward and outward current conductances, as well as their reversal potentials, in order to produce stable oscillatory activity. Our analysis predicts that a hyperpolarization-activated inward current can play a role in stabilizing oscillatory activity by preventing swings to very negative voltages, which is consistent with what is recorded in biological neurons in general. We confirm this prediction of the model experimentally in neurons from the crab stomatogastric ganglion.

Slow neural oscillations are known to depend on regenerative inward currents. The voltage range in which these currents dominate the neuron's activity is typically limited to the negative-slope conductance range of their IV curve. At low voltages, these currents are not active and at high voltages, outward currents and spike-activity dominate. We find that the contribution of such currents to neural oscillations can be approximated by a linear current with negative slope conductance (INL). Here we explore the minimal set of requirements to produce neural oscillations in the presence of INL with the goal of elucidating how other current types shape the properties of oscillations.

Introduction: It has been long recognized that rodent myoyctes die during long-term primary culture, which limits the use of genetically altered myocytes for signaling studies. Blebbistatin (BLB), a myosin II ATPase inhibitor, has been used to protect rodent myocytes. However, the mechanisms underlying the protective effects of this drug are not clear and are the topics of this study.Materials & Methods: Adult rat ventricular myocytes (ARVM) were isolated and cultured with or without BLB (10μM) and BDM(10mM) for 72 hours. Myocyte death was evaluated by trypan blue staining. The effects of these two drugs on myocyte contraction, intracellular Ca transient ([Ca]i, Indo-1,410/480), SR Ca content, L-type calcium and Na/Ca exchanger currents were studied acutely.Results: 1, Both BDM (61.5± 6.4%) and BLB (74.0±3.2%) promoted myocyte survival in culture at 72 hours (control: 7.0±1.8%); 2. ARVM fractional shortening was reduced by BLB (1.7±0.4%) and BDM (0.5±0.1%, control: 6.5±0.7%); 3, Acutely, the amplitude of [Ca]i (Δ[Ca]i) was depressed by both BDM (0.038±0.005) and BLB (0.065±0.008) comparing to control (0.130±0.010). 4, Diastolic Ca was significantly increased by BLB (0.90±0.06) but not by BDM (0.73±0.06) comparing to control(0.70±0.05). 5. BLB and BDM significantly reduced the SR Ca content(Δ[Ca]i in BLB vs. BDM vs. control: 0.16±0.016, 0.09±0.01, 0.24±0.03). 5. The mechanisms of the protective effect of BDM and BLB are different in that BDM mainly reduced Ca influx through the L-type Ca channel (85% reduction) and Na/Ca exchanger (60% reduction) while BLB inhibited Na/Ca exchanger (100% inhibition) without altering the LTCC(

T-type Ca2+ channels (TTCCs) are expressed in cardiac pacemaker cells and conduction system of mammals. However, the role of TTCCs in heart rate (HR) generation and regulation is not well understood. In the mouse, the major TTCC expressed in the heart is Cav3.1/α1G, and therefore we used Cav3.1/α1G transgenic (TG) and knockout (KO) mice respectively to define the role of TTCC in the heart rate generation and regulation. Methods: Telemetric (conscious) and surface (anesthetized) ECG were used to determine the effect of isoproterenol (ISO) on the HR in vivo. To reduce the complication of in vivo HR regulation, Langendorff ECG was used to measure the response of the HR to ISO. Whole cell voltage clamp was used to measure the ICa-T before and after ISO application on TG myocytes. Results: At baseline, telemetric ECG recording showed no significant difference in HR between the Cav3.1/α1G TG mice (536.3±24.8bpm vs. FVB control: 550.6±15.3bpm), Cav3.1/α1G KO mice (614.4±39.9bpm vs. c57/bl6 control: 603.1±64.5bpm) and control mice was detected. ISO increased the HR rate in conscious mice to the same extent in both TG (41.2±6.9% vs. FVB control: 34.0±3.6%) and KO (22.6±8.8% vs. c57/bl6 control: 22.8±8.5%) mice. However, when the central regulation is depressed (anesthetized) or removed (ex-vivo Langendorff perfusion), the percentage of HR increase after ISO application were significantly enhanced in the TG mice but reduced in KO mice. Cav3.1/α1G T-type Ca2+ currents (ICa-T) in sinoatrial nodal cells was significantly increased by 43±16 % by ISO. Conclusions: Cav3.1/α1G TTCC might not play a major role in basal HR generation but it may play an important role in sympathetic/adrenergic regulation of HR, in which PKA could be an important mediator.