Membrane Potential Recordings Research Articles

Aging Impairs Electrical Conduction Along Endothelium of Resistance Arteries Through Enhanced Ca2+-Activated K + Channel Activation

Intercellular conduction of electrical signals underlies spreading vasodilation of resistance arteries. Small- and intermediate-conductance Ca(2+)-activated K(+) channels of endothelial cells serve a dual function by initiating hyperpolarization and modulating electrical conduction. We tested the hypothesis that regulation of electrical signaling by small- and intermediate-conductance Ca(2+)-activated K(+) channels is altered with advancing age. Intact endothelial tubes (60 µm wide; 1-3 mm long) were freshly isolated from male C57BL/6 mouse (Young: 4-6 months; Intermediate: 12-14 months; Old: 24-26 months) superior epigastric arteries. Using dual intracellular microelectrodes, current was injected (± 0.1-3 nA) at site 1 while recording membrane potential (Vm) at site 2 (separation distance: 50-2000 µm). Across age groups, greatest differences were observed between Young and Old. Resting Vm in Old (-38 ± 1 mV) was more negative (P<0.05) than Young (-30 ± 1 mV). Maximal hyperpolarization to both direct (NS309) and indirect (acetylcholine) activation of small- and intermediate-conductance Ca(2+)-activated K(+) channels was sustained (ΔVm ≈-40 mV) with age. The length constant (λ) for electrical conduction was reduced (P<0.05) from 1630 ± 80 µm (Young) to 1320 ± 80 µm (Old). Inhibiting small- and intermediate-conductance Ca(2+)-activated K(+) channels with apamin+charybdotoxin or scavenging hydrogen peroxide (H2O2) with catalase improved electrical conduction (P<0.05) in Old. Exogenous H2O2 (200 µmol/L) in Young evoked hyperpolarization and impaired electrical conduction; these effects were blocked by apamin+charybdotoxin. Enhanced current loss through Ca2+-activated K+ channel activation impairs electrical conduction along the endothelium of resistance arteries with aging. Attenuating the spatial domain of electrical signaling will restrict the spread of vasodilation and thereby contribute to blood flow limitations associated with advanced age.

β1‐Adrenoceptor stimulation suppresses endothelial IKCa‐channel hyperpolarization and associated dilatation in resistance arteries

In small arteries, small conductance Ca²⁺-activated K⁺ channels (SK(Ca)) and intermediate conductance Ca²⁺-activated K⁺ channels (IK(Ca)) restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI₂-independent, endothelium-derived hyperpolarizing factor response that is the predominate endothelial mechanism for vasodilatation. As neuronal IK(Ca) channels can be negatively regulated by PKA, we investigated whether β-adrenoceptor stimulation, which signals through cAMP/PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation. Rat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [Ca²⁺]i , mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording. Intraluminal perfusion of β-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM-10 μM) without modifying the associated changes in endothelial cell [Ca²⁺]i . The inhibitory effect of β-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the β-adrenoceptor antagonists propranolol (non-selective), atenolol (β₁) or the PKA inhibitor KT-5720, but remained unaffected by ICI 118 551 (β₂) or glibenclamide (ATP-sensitive K⁺ channels channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by β-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IK(Ca) {with 1 μM 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34)}, but not SK(Ca) (50 nM apamin) channels prevented β-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh. In resistance arteries, endothelial cell β₁-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IK(Ca) channels and may be one consequence of raised circulating catecholamines.

HCN channels contribute to serotonergic modulation of chemoreceptors in the retrotrapezoid nucleus

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) provide a CO2‐dependent drive to breathe, and function as a key locus of respiratory control by integrating information from several respiratory centers including the medullary raphe. Recent evidence showed that mechanisms underlying serotonergic modulation of RTN neurons involves inhibition of KCNQ channels and activation of an unknown inward current. Evidence also shows that RTN neurons produce a hyperpolarization‐activated inward current (Ih) at negative potentials. HCN channels are the molecular correlate of Ih and have a high propensity for neuromodulation. Therefore, we hypothesize that HCN channels contribute to serotonergic modulation of RTN neurons. We investigated this in vitro and in vivo using selective blockers for KCNQ (XE991) and HCN (ZD7288) channels. Membrane potential recordings in rat brain slices show that serotonin application increased chemoreceptor activity by ~1 Hz. Consistent with our previous evidence, XE991 decreased serotonin‐responsiveness by ~50%. The residual serotonin‐sensitivity in XE991 was blocked by ZD7288. In anesthetized rats RTN injection of XE991 blunted serotonin‐responsiveness and together XE991 and ZD7288 completely eliminated the respiratory response to serotonin. These results indicate that HCN channels contribute to serotonergic modulation of RTN chemoreceptors and respiratory drive. Funding sources: CURE Epilepsy Foundation (AVT, DKM), NIH HL104101 (DKM), NS073981 (AVT), and the Sao Paulo Research Foundation (FAPESP) (ACT, TSM).

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) express SK channels with low Ca2+ affinity

Chemosensitive RTN neurons regulate breathing in response to changes in tissue CO2/H+. despite the importance of these cells, little is known regarding molecular determinants of their intrinsic excitability. We have recently determined that RTN chemoreceptors express Ca2+‐activated small conductance K+ (SK) channels, however, the role of these channels in chemoreceptor function is unknown. Here, we use electrophysiological and computational approaches to explore SK channel function in RTN neurons. In brain slices (300 μm thick) from rat pups (P7–12), cell‐attached recordings of membrane potential show that bath application of apamin (selective SK channel blocker) decreased amplitude of the after hyperpolarization (AHP) evoked by depolarizing current injection but did not affect baseline activity. Based on computer simulations, we predict that under baseline conditions and high Ca2+ pump activity, [Ca2+]i should be sufficient to activate SK channels with a typical Ca2+affinity of &lt; 1μM. Therefore, we predict that RTN neurons express SK channels with low Ca2+ affinity possibly due to high casein kinase‐2 (CK2) activity. Consistent with this possibility, we found that TBB (a selective CK2 inhibitor) eliminated spontaneous activity of RTN neurons as expected for activation of a K+ conductance. These results suggest that the contribution of SK channels to activity of RTN neurons is regulated by CK2 activity.

Modulation of pacemaker potentials by pyungwi-san in interstitial cells of cajal from murine small intestine: pyungwi-san and interstitial cells of cajal.

Objective:Pyungwi-san (PWS) plays a role in a number of physiologic and pharmacologic functions in many organs. Interstitial cells of Cajal (ICCs) are pacemaker cells that generate slow waves in the gastrointestinal (GI) tract. We aimed to investigate the beneficial effects of PWS in mouse small-intestinal ICCs.Methods:Enzymatic digestion was used to dissociate ICCs from the small intestine of a mouse. The wholecell patch-clamp configuration was used to record membrane potentials from the cultured ICCs.Results:ICCs generated pacemaker potentials in the GI tract. PWS produced membrane depolarization in the current clamp mode. Pretreatment with a Ca2+ -free solution and a thapsigargin, a Ca2+ -ATPase, inhibitor in the endoplasmic reticulum, eliminated the generation of pacemaker potentials. However, only when the thapsigargin was applied in a bath solution, the membrane depolarization was not produced by PWS. Furthermore, the membrane depolarizations due to PWS were inhibited not by U-73122, an active phospholipase C inhibitor, but by chelerythrine and calphostin C, protein kinase C inhibitors.Conclusions:These results suggest that PWS might affect GI motility by modulating the pacemaker activity in the ICCs.

A new method to infer higher-order spike correlations from membrane potentials

What is the role of higher-order spike correlations for neuronal information processing? Common data analysis methods to address this question are devised for the application to spike recordings from multiple single neurons. Here, we present a new method which evaluates the subthreshold membrane potential fluctuations of one neuron, and infers higher-order correlations among the neurons that constitute its presynaptic population. This has two important advantages: Very large populations of up to several thousands of neurons can be studied, and the spike sorting is obsolete. Moreover, this new approach truly emphasizes the functional aspects of higher-order statistics, since we infer exactly those correlations which are seen by a neuron. Our approach is to represent the subthreshold membrane potential fluctuations as presynaptic activity filtered with a fixed kernel, as it would be the case for a leaky integrator neuron model. This allows us to adapt the recently proposed method CuBIC (cumulant based inference of higher-order correlations from the population spike count; Staude et al., J Comput Neurosci 29(1–2):327–350, 2010c) with which the maximal order of correlation can be inferred. By numerical simulation we show that our new method is reasonably sensitive to weak higher-order correlations, and that only short stretches of membrane potential are required for their reliable inference. Finally, we demonstrate its remarkable robustness against violations of the simplifying assumptions made for its construction, and discuss how it can be employed to analyze in vivo intracellular recordings of membrane potentials.

Role of impaired endothelial cell Ca2+signaling in uteroplacental vascular dysfunction during diabetic rat pregnancy

Diabetes mellitus in pregnancy is associated with impaired endothelium-mediated dilatation of maternal arteries, although the underlying cellular mechanisms remain unknown. In this study, we hypothesized that diabetes during rat gestation attenuates agonist-induced uterine vasodilation through reduced endothelial cell (EC) Ca(2+) elevations and impaired smooth muscle cell (SMC) hyperpolarization and SMC intracellular Ca(2+) concentration ([Ca(2+)]i) responses. Diabetes was induced by an injection of streptozotocin to second-day pregnant rats and confirmed by the development of maternal hyperglycemia. Control rats were injected with a citrate buffer. Fura-2-based measurements of SMC [Ca(2+)]i or microelectrode recordings of SMC membrane potential were performed concurrently with dilator responses to ACh in uteroplacental arteries from control and diabetic pregnant rats. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations in pressurized vessels and small EC sheets were studied as well. Diabetes reduced ACh-induced vasodilation due to a markedly impaired EDHF-mediated response. Diminished vasodilation to ACh was associated with attenuated SMC hyperpolarization and [Ca(2+)]i responses. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations were significantly reduced by diabetes. In conclusion, these data demonstrate that reduced endothelium-mediated hyperpolarization contributes to attenuated uteroplacental vasodilation and SMC [Ca(2+)]i responses to ACh in diabetic pregnancy. Impaired endothelial Ca(2+) signaling is in part responsible for endothelial dysfunction in the uterine resistance vasculature of diabetic rats. Pharmacological improvement of EC Ca(2+) handling may provide an important strategy for the restoration of endothelial function and enhancement of maternal blood flow in human pregnancies complicated by diabetes.

Development of New Fluorescent Voltage Sensor Proteins

Our goal is to develop genetically encoded fluorescent biosensors (FP voltage sensors) and to use them for optical recording of membrane potential in excitable cells. We created a series of FRET (fluorescence resonance energy transfer) based sensors. These constructs contained the voltage sensing domain of CiVSP (Ciona intestinalis voltage sensitive phosphatase) as voltage sensor fused with two fluorescent proteins, UKG (green-emitting fluorescent protein Umi-Kinoko) and mKOK (orange-emitting fluorescent protein Kusabira orange) as donor and acceptor. We used PCR to generate a library of constructs with insertions of the green and orange fluorescent proteins into different regions in CiVSP. Insertion of FRET donor and acceptor at different locations affects plasma membrane expression, FRET signal intensity, and response time constant. We are screening the these FP voltage sensors by examining the FRET signal in response to changes in membrane potential in HEK293 cells. Several novel probes have been identified; some with relatively large signals (>7% ΔF/F) and fast taus (<3 msec). We hope this effort will lead to voltage sensor proteins useful for in vivo recording of membrane potential in neurons.Supported by US NIH Grants DC005259 and NS054270 and grant WCI 2009-003 from the National Research Foundation of Korea.

Aging Restricts Electrical Signaling along Endothelial Tubes via Enhanced Activation of SKCa/IKCa Channels: Role for Oxidative Stress

Electrical conduction in cellular networks is governed by gap junction patency between cells and the open state of membrane ion channels. In light of endothelial cell (EC) dysfunction with aging, we determined whether aging altered electrical signaling. Intact endothelial tubes (width: ≈60μm; length ≥ 2mm) isolated from superior epigastric arteries of Young (4-6 months) and Old (24-26 months) male C57BL/6 mice were studied using dual intracellular microelectrodes. Separation distance between sites of current injection and membrane potential (Vm) recording was 50-2000μm. A reduction in electrical length constant (Δ) from 1630 (Young) to 1320μm (Old) accompanied an increase in restingVm from Young (-28±1mV) to Old (−36±1mV) (n≥9; P<0.05). With similar pEC50 values and maximum Vm responses to acetylcholine (ΔVm Young: 7.3±0.1, −38±3mV; Old: 7.2±0.1, −39±1mV), the conduction amplitude (CA; ΔVm/nA current injection) for intercellular electrical signaling was less (P<0.05) in Old vs. Young (at 500 μm: 5.9±1 vs. 8.9±1 mV/nA; n=6). Despite similar hyperpolarization (∼-35 mV) to direct activation of calcium-activated K+ channels (SKCa/IKCa; KCa2.3 and KCa3.1) with NS309 (1μM), the reduction in CA was greater (P<0.05) in Young vs. Old (−6±1 vs.-4±1 mV/nA; n≥7). Inhibiting SKCa/IKCa (apamin+charybdotoxin) restored conduction in Old to that of Young (at 500 μm:∼10 mV/nA; n=6). To investigate a role for oxidative stress, H2O2 (200 μM) increased Vm to EK (≈-90mV) and impaired CA by ≥ 90% (n=8) in Young; these effects were blocked by apamin+charybdotoxin (n=6). Catalase (500 U/ml) depolarized resting Vm from −38±1 to −28±1mV and increased CA by ≈30% in Old (n=7; P<0.05). Thus restricted spread of electrical signals along endothelium of Old reflects enhanced current dissipation through open SKCa/IKCa in response to oxidative stress. (Support: NIH R01-HL086483, R37-HL041026, F32-HL110701)

Mollie Elizabeth Holman 1930–2010

Mollie Holman was a biophysicist whose work on the autonomic nervous system and the innervation of smooth muscle was seminal in advancing knowledge of its behaviour at a cellular level. She was particularly known for her technical expertise in microelectrode recording of membrane potential from single smooth muscle cells, and the interpretation of their electrical activity, both spontaneous and in response to transmitters released from their autonomic nerves.

Involvement of MAPKs and PLC Pathways in Modulation of Pacemaking Activity by So-Cheong-Ryong-Tang in Interstitial Cells of Cajal from Murine Small Intestine

Purpose. Interstitial cells of Cajal (ICCs) are the pacemaker cells that generate slow waves in the gastrointestinal (GI) tract. We have aimed to investigate the effects of Socheongryong-Tang (SCRT) in ICCs from mouse's small intestine. Methods. The whole-cell patch-clamp configuration was used to record membrane potentials from cultured ICCs. Intracellular Ca2+ ([Ca2+]i) increase was studied in cultured ICCs using fura-2 AM. Results. ICCs generated pacemaker potentials in mouse's small intestine. SCRT produced membrane depolarization in current clamp mode. Y25130 (5-HT3 receptor antagonist) and RS39604 (5-HT4 receptor antagonist) blocked SCRT-induced membrane depolarizations, whereas SB269970 (5-HT7 receptor antagonist) did not. When GDP-β-S (1 mM) was in the pipette solution, SCRT did not induce the membrane depolarizations. [Ca2+]i analysis showed that SCRT increased [Ca2+]i. In the presence of PD98059 (p42/44 MAPK inhibitor), SCRT did not produce membrane depolarizations. In addition, SB203580 (p38 MAPK inhibitor) and JNK inhibitors blocked the depolarizations by SCRT in pacemaker potentials. Furthermore, the membrane depolarizations by SCRT were not inhibited by U-73122, an active phospholipase C (PLC) inhibitor, but by U-73343, an inactive PLC inhibitor. Conclusion. These results suggest that SCRT might affect GI motility by the modulation of pacemaker activity through MAPKs and PLC pathways in the ICCs.

Inferring trial-to-trial excitatory and inhibitory synaptic inputs from membrane potential using Gaussian mixture Kalman filtering

Time-varying excitatory and inhibitory synaptic inputs govern activity of neurons and process information in the brain. The importance of trial-to-trial fluctuations of synaptic inputs has recently been investigated in neuroscience. Such fluctuations are ignored in the most conventional techniques because they are removed when trials are averaged during linear regression techniques. Here, we propose a novel recursive algorithm based on Gaussian mixture Kalman filtering (GMKF) for estimating time-varying excitatory and inhibitory synaptic inputs from single trials of noisy membrane potential in current clamp recordings. The KF is followed by an expectation maximization (EM) algorithm to infer the statistical parameters (time-varying mean and variance) of the synaptic inputs in a non-parametric manner. As our proposed algorithm is repeated recursively, the inferred parameters of the mixtures are used to initiate the next iteration. Unlike other recent algorithms, our algorithm does not assume an a priori distribution from which the synaptic inputs are generated. Instead, the algorithm recursively estimates such a distribution by fitting a Gaussian mixture model (GMM). The performance of the proposed algorithms is compared to a previously proposed PF-based algorithm (Paninski et al., 2012) with several illustrative examples, assuming that the distribution of synaptic input is unknown. If noise is small, the performance of our algorithms is similar to that of the previous one. However, if noise is large, they can significantly outperform the previous proposal. These promising results suggest that our algorithm is a robust and efficient technique for estimating time varying excitatory and inhibitory synaptic conductances from single trials of membrane potential recordings.

Palette of fluorinated voltage-sensitive hemicyanine dyes

Optical recording of membrane potential permits spatially resolved measurement of electrical activity in subcellular regions of single cells, which would be inaccessible to electrodes, and imaging of spatiotemporal patterns of action potential propagation in excitable tissues, such as the brain or heart. However, the available voltage-sensitive dyes (VSDs) are not always spectrally compatible with newly available optical technologies for sensing or manipulating the physiological state of a system. Here, we describe a series of 19 fluorinated VSDs based on the hemicyanine class of chromophores. Strategic placement of the fluorine atoms on the chromophores can result in either blue or red shifts in the absorbance and emission spectra. The range of one-photon excitation wavelengths afforded by these new VSDs spans 440-670 nm; the two-photon excitation range is 900-1,340 nm. The emission of each VSD is shifted by at least 100 nm to the red of its one-photon excitation spectrum. The set of VSDs, thus, affords an extended toolkit for optical recording to match a broad range of experimental requirements. We show the sensitivity to voltage and the photostability of the new VSDs in a series of experimental preparations ranging in scale from single dendritic spines to whole heart. Among the advances shown in these applications are simultaneous recording of voltage and calcium in single dendritic spines and optical electrophysiology recordings using two-photon excitation above 1,100 nm.

Abstract 526: A New Perspective on HDL: Potential Navigation of Blood Pressure via Adrenoreceptor Regulation

Introduction: HDL is known for its anti-atherogenic qualities and serves as an important protective factor against cardiovascular disease. Recent studies suggested that this vasoprotective effect may not merely be based on reverse cholesterol transport. In fact, HDL has been proven to have vasodilatative properties of its own, a significant mechanism we investigated. Materials and Methods: Flow-dependent isometric tension, intracellularly recorded membrane potential and cAMP-cGMP were measured in segments of 20 coronaries from heart transplantations. Results: Flow-dependent vasodilatation was elevated by HDL administration [50 mg/dL] causing a mean relaxing effect of -0.236 g (Table). Application of phentolamine (10 -7 mol/L) and propranolol (10 -7 mol/L) resulted in a reduction of HDL induced vasorelaxation. This reduction was also flow-dependently progressing as flow increased. Interestingly, simultaneous α- and β-blockade did not follow an additive interaction pattern. The interaction seemed to be flow-dependent as well, with β-receptors contributing more to the combined effect at low flow rates and α-receptors to a greater proportion at high flows. Discussion: For the first time, we describe a potential mechanism of HDL-induced vasodilatation. Blood pressure and lipid profile - two independent risk factors of arteriosclerosis - may be linked by multiple HDL effects. We suggest that this vasorelaxation is in part mediated by activation of β-adrenoreceptors and inactivation of α-adrenoreceptors. This may have crucial implications in the clinical management of both hypertension and hyperlipidemia.

Single electrode dynamic clamp with StdpC

Dynamic clamp is a powerful approach for electrophysiological investigations allowing researchers to introduce artificial electrical components into target neurons to simulate ionic conductances, chemical or electrotonic inputs or connections to other cells. Due to the rapidly changing and potentially large current injections during dynamic clamp, problematic voltage artifacts appear on the electrode used to inject dynamic clamp currents into a target neuron. Dynamic clamp experiments, therefore, typically use two separate electrodes in the same cell, one for recording membrane potential and one for injecting currents. The requirement for two independent electrodes has been a limiting factor for the use of dynamic clamp in applications where dual recordings of this kind are difficult or impossible to achieve. The recent development of an active electrode compensation (AEC) method has overcome some of these prior limitations, permitting artifact-free dynamic clamp experimentation with a single electrode. Here we describe an AEC method for the free dynamic clamp software StdpC. The AEC component of StdpC is the first such system implemented for the use of non-expert users and comes with a set of semi-automated configuration and calibration procedures that facilitate its use. We briefly introduce the AEC method and its implementation in StdpC and then validate it with an electronic model cell and in two different biological preparations.

Abstract 166: Interaction Between LDL and Sympathetic Adrenoreceptors

Introduction: LDL is a significant risk factor for cardiovascular disease and is known to impair flow-dependent vasodilatation. Furthermore, it has been demonstrated that LDL can even directly cause vasoconstricton. Recent studies suggested that lower LDL levels, achieved through administration of statins, are correlated with lower blood pressure, independent from pleiotropic statin effects. Our objective was to investigate whether LDL-induced impairment in flow-dilatation was due to an interaction of LDL with sympathetic adrenoreceptors. Materials and Methods: Flow-dependent isometric tension, intracellularly recorded membrane potential and cAMP-cGMP were measured in segments of 22 coronaries from heart transplantations. Results: As compared to Krebs solution, LDL affected flow-dilatation and caused a relative contraction [Δ(T 3 -T 100 ): Krebs 0.459 g; LDL 0.278 g]. Complete blockade of both α- (phentolamine 10 -7 mol/L) and β-receptors (propranolol 10 -7 mol/L) resulted in a ∼50% reduction of flow-dilatation impairment induced by LDL. Blockage of either α- (1) or β-receptors (2) showed smaller effects which added up to the measured effect under blockade of both receptors (4). Similar effects were apparent in the recorded membrane potential of the vascular smooth muscle cells. Discussion: We conclude that the LDL-induced impairment in flow-dependent dilatation is partially due to the interaction between lipoprotein and both α- and β-adrenoreceptors. Since blockade of each receptor resulted in a relative dilatation, we assume that the LDL effect is caused by a stimulation of α-adrenoreceptors and an inhibition of β-adrenoreceptors.

Analysis of excitatory and inhibitory interactions at high temporal resolution in core circuits of the respiratory CPG

The pre-Botzinger complex (pre-BotC) is the essential core component of the brainstem respiratory central pattern generator (rCPG) in mammals. Phasic excitatory and inhibitory synaptic inputs during the respiratory cycle are thought to dynamically shape membrane potential trajectories and spiking patterns of pre-BotC neurons. These synaptic inputs reflect the functionally interacting neuron populations involved in rhythm and inspiratory-expiratory pattern generation. The dynamic patterns of these synaptic inputs have not been systematically studied and characterized experimentally. Accurate temporal reconstruction of the synaptic input patterns is important for testing current models of the rCPG that incorporate specific excitatory and inhibitory circuit mechanisms for rhythm and pattern generation. A recent computational model of the rCPG generating a normal “three-phase” respiratory pattern [1] specified patterns of synaptic excitation and inhibition in the preBotC during the respiratory cycle required to produce the behaviorally distinct phases of inspiration (I) and two phases of expiration (E1, or post-inspiration, and E2). This model has not been rigorously tested by experimental data on dynamic changes in phasic postsynaptic excitatory and inhibitory conductances in different populations of pre-BotC inspiratory and expiratory neurons. We developed analytical techniques that allow quantitative reconstruction of the patterns of synaptic conductances at high temporal resolution from intracellular recordings of membrane potential trajectories. Our approach extends previously proposed methods for extracting dynamic patterns of synaptic input conductances [2,3] and provides nearly continuous readouts of inhibitory and excitatory synaptic conductances in electrophysiologically different cell types throughout the respiratory cycle. The membrane potential trajectories of pre-BotC respiratory neurons were obtained by sharp microelectrode current-clamp recording within in situ perfused brainstem-spinal cord preparations of mature rats, which generate a three-phase respiratory pattern and provide mechanically stable conditions for intracellular recordings in functionally intact brainstem circuits. We distinguished different types of inspiratory and expiratory pre-BotC neurons and analyzed the dynamical changes of excitatory (Ge )a nd inhibitory (Gi) conductances. Inspiratory neurons exhibited strong excitatory inputs (large dynamic increases in Ge) during their spiking phase followed by a wave(s) of inhibitory inputs during the expiratory period with large increases in Gi and temporal patterns consistent with two populations of inhibitory neurons providing inputs that coordinate inspiratory to expiratory and expiratory to inspiratory phase transitions. Expiratory neurons exhibited strong inhibition during the inspiratory phase, consistent with the rhythmic alternation of inspiration and expiration, and changes in Ge indicating reciprocal interactions between two major populations of inhibitory neurons coordinating the generation of two (E1 and E2) phases of expiration. The reconstructed patterns of Ge and Gi are generally consistent with previously proposed circuit architecture [1] and also suggest its extension. The techniques that we have developed for high temporal resolution of postsynaptic conductance changes are likely to have broad application for analysis of synaptic interactions in circuit components whenever intracellular recordings of membrane potentials can be

Transient receptor potential canonical type 3 channels facilitate endothelium-derived hyperpolarization-mediated resistance artery vasodilator activity

Microdomain signalling mechanisms underlie key aspects of artery function and the modulation of intracellular calcium, with transient receptor potential (TRP) channels playing an integral role. This study determines the distribution and role of TRP canonical type 3 (C3) channels in the control of endothelium-derived hyperpolarization (EDH)-mediated vasodilator tone in rat mesenteric artery. TRPC3 antibody specificity was verified using rat tissue, human embryonic kidney (HEK)-293 cells stably transfected with mouse TRPC3 cDNA, and TRPC3 knock-out (KO) mouse tissue using western blotting and confocal and ultrastructural immunohistochemistry. TRPC3-Pyr3 (ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate) specificity was verified using patch clamp of mouse mesenteric artery endothelial and TRPC3-transfected HEK cells, and TRPC3 KO and wild-type mouse aortic endothelial cell calcium imaging and mesenteric artery pressure myography. TRPC3 distribution, expression, and role in EDH-mediated function were examined in rat mesenteric artery using immunohistochemistry and western blotting, and pressure myography and endothelial cell membrane potential recordings. In rat mesenteric artery, TRPC3 was diffusely distributed in the endothelium, with approximately five-fold higher expression at potential myoendothelial microdomain contact sites, and immunoelectron microscopy confirmed TRPC3 at these sites. Western blotting and endothelial damage confirmed primary endothelial TRPC3 expression. In rat mesenteric artery endothelial cells, Pyr3 inhibited hyperpolarization generation, and with individual SK(Ca) (apamin) or IK(Ca) (TRAM-34) block, Pyr3 abolished the residual respective IK(Ca)- and SK(Ca)-dependent EDH-mediated vasodilation. The spatial localization of TRPC3 and associated channels, receptors, and calcium stores are integral for myoendothelial microdomain function. TRPC3 facilitates endothelial SK(Ca) and IK(Ca) activation, as key components of EDH-mediated vasodilator activity and for regulating mesenteric artery tone.

Minireview: Aldosterone Biosynthesis: Electrically Gated for Our Protection

Aldosterone produced by adrenal zona glomerulosa (ZG) cells plays an important role in maintaining salt/water balance and, hence, blood pressure homeostasis. However, when dysregulated, aldosterone advances renal and cardiovascular disease states. Multiple steps in the steroidogenic pathway require Ca(2+), and the sustained production of aldosterone depends on maintained Ca(2+) entry into the ZG cell. Nevertheless, the recorded membrane potential of isolated ZG cells is extremely hyperpolarized, allowing the opening of only a small fraction of low-voltage-activated Ca(2+) channels of the Ca(v)3.x family, the major Ca(2+) conductance on the ZG cell membrane. As a consequence, to activate sufficient Ca(2+) channels to sustain the production of aldosterone, aldosterone secretagogs would be required to affect large decreases in membrane voltage, a requirement that is inconsistent with the exquisite sensitivity of aldosterone production in vivo to small changes (0.1 mm) in extracellular K(+). In this review, we evaluate the contribution of membrane voltage and voltage-dependent Ca(2+) channels to the control of aldosterone production and consider data highlighting the electrical excitability of the ZG cell. This intrinsic capacity of ZG cells to behave as electrical oscillators provides a platform from which to generate a recurring Ca(2+) signal that is compatible with the lengthy time course of steroidogenesis and provides an alternative model for the physiological regulation of aldosterone production that permits both amplitude and temporal modulation of the Ca(2+) signal.

Abstract 108: LDL-Apolipoprotein E Isoform Codetermines Human Coronary Artery Tone

Background: Apolipoprotein E4 (apoE4) is the most significant risk factor known to cause Alzheimer dementia (AD) [1], but also crucial in atherogenesis as pathological correlative of coronary heart disease (CHD) [2]. Therefore, the present investigation aimed at unveiling the role of LDL-apoE isoforms in vascular reactivity, especially in flow-dilatation of human coronaries. Materials and Methods: Isometric tension was measured in segments of human coronary arteries from heart transplantations changing the superfusate flow rate in the sequence 3, 5, 20, 40 and 100 mL/min. The initial tension was 2 g. Krebs solution without (control) and with addition of 100 mg/dL pooled LDL, LDLapoE4/E4 or LDLapoE0/E0 (from an apoE ‘knockout man’) was used as superfusate. Intracellular recordings of membrane potential and determination of cAMP and cGMP concentrations were carried out. Results: Initial tension at 3 mL/min flow (T 3 ): control 1.649 g, pooled LDL 1.739 g, LDLapoE4/E4 1.773 g, LDLapoE0/E0 1.796 g; flow-dependent Δtension (T 100 - T 3 ): control -0.418 g, pooled LDL -0.263 g, LDLapoE4/E4 -0.197 g, LDLapoE0/E0 +0.313 g. Upon a basal flow rate of 3 mL/min, the initial tension showed a graded course both in normal and in deendothelialized preparations: both pooled LDL (+5.5% vs. +4.7%) and LDLapoE4/E4 (+7.5% vs. +5.6%) as well as LDLapoE0/E0 (+8.9% vs. +10.2%) enhanced mechanical force as compared to the controls. Endothelium-denuded segments had basically a higher tone (+5.2%) on grounds of an impaired basal NO release. Latter preparations demonstrated this graded course especially significant during flow-dependent dilatation, which was reversed to constriction by +109.3% under pooled LDL, by +160.1% under LDLapoE4/E4 and by +332.1% under LDLapoE0/E0 compared with the control. Membrane potentials reflected tension: hyperpolarization of the vascular smooth muscle cells preceded relaxation, depolarization preceded contraction. Relating the membrane potential to the corresponding tone revealed a sigmoidal electromechanical coupling curve. For the first time in literature, we report on a chemomechanical coupling curve by correlating the nucleotide concentration with the corresponding tension. Conclusion: LDLapoE4/E4 reversed flow-dependent vasodilatation to constriction and was only exceeded in its deleterious effect by LDL devoid of any apoE. There is growing evidence that binding of apoE4/E4 and apoE0/E0 to the flow sensor proteoheparan sulfate (endothelial syndecan superfamily) leads to nanoplaque build-up [1,2] at the endothelium-blood interface and thus impairs vascular reactivity.