Lower Resting Membrane Potential Research Articles

Abstract 16250: Latent His-Purkinje Disease Unmasked by Beta Blocker Overdose

Background Symptomatic bradycardia usually has a single mechanism and treatment modality. Identifying the culprit mechanism can be challenging, especially when faced with concurrent pathologies. We present a case of severe symptomatic bradycardia where appropriate management of one mechanism unmasked a second mechanism, with a need for pacemaker placement. Case Presentation A 56-year-old patient presented with beta blocker overdose and severe bradycardia. The initial ECG showed sinus bradycardia with a sinus cycle length of 2060ms and 2:1 AV conduction, with each conducted P wave preceded by a long-coupled junctional escape beat. Atropine resulted in shortening of the junctional escape interval but had no effect on the sinus rate. There was now 1:1 AV conduction, with each P wave being preceded by a faster junctional escape beat. Glucagon resulted in acceleration of the sinus rate to a cycle length of ~820ms (73 bpm) with return of 1:1 AV conduction, a PR interval of 200ms, and no junctional beats. Conducted beats showed a right bundle branch block and left axis deviation. The dependence of AV conduction on a preceding QRS complex within a certain window of time highly suggested infranodal conduction disease manifesting as phase 4 AV block. While normal sinus rates and 1:1 AV conduction were restored with glucagon, the presence of infranodal disease warranted dual chamber pacemaker implantation. In follow-up, with atrial pacing support, there was < 1% ventricular pacing. Discussion Two causes of bradycardia were simultaneously present - sinus bradycardia due to beta blocker overdose, and 2nd degree AV block due to His-Purkinje disease. Phase 4 AV block is a mechanism of paroxysmal AV block (PAVB) in patients with diseased His-Purkinje system. Spontaneous depolarization occurs in diseased fibers during prolonged periods of ventricular pause, such as with slowing of the sinus rate. The sodium channels are blocked during phase 4 of the action potential and therefore the conduction system is incapable of depolarizing due to low resting membrane potentials. In this case, AV 1:1 conduction can only be restored with a premature ventricular contraction. This is an overall under-recognized entity and should be managed with pacemaker implantation.

PB2195 ASSESSMENT OF PLATELET MEMBRANE ALTERATION IN PATIENTS WITH MYELOID METAPLASIA WITH MYELOFIBROSIS

Background:Patients with Myeloid Metaplasia with Myelofibrosis (MMM) present at the onset or during evolution thrombotic complications. In the assessment of thrombosis risk are important the presence of JAK2 mutation as well as platelet function. Receptors that define the status of activated platelets (CD62P, CD36) are better expressed in chronic myeloproliferative neoplasms (MPNs) patients with thrombotic complications. Numerous studies have shown a high oxidative status in MPNs patients, a situation that correlates with a higher thrombotic risk.Aims:The aim of our study was to determine if resting membrane potential could be correlated with alterations in expression of platelet receptors and reactive species production (ROS)MethodsThis retrospective study included 35 patients with MMM (16 male and 19 female), median age 61.1 (95% CI of median 60,71‐72) as well as 15 healthy volunteers. The diagnosis of MMM was made according to World Health Organization (WHO) classification of MPNs. We evaluated the flow cytometry markers of platelet adhesion (CD42a, CD42b), aggregation (CD41, CD61) and CD36. Production of reactive species (ROS) was examined using fluorescence method with DCFDA and was assessed by aria under curve (AUC) in serial measurements during 900 sec. The determination of platelet membrane potential was done by fluorescence method using 3,3′‐dipropyl‐2,2′‐thiadicarbocyanine iodide (DiSC3‐5)Results:The ROS production is significantly higher in MMM group: median value of AUC patient group 12780882862,5000 compared with controls 11621365017,5000, p= 0.04. Patients in advanced phase (grade 5 Hackett of splenomegaly) has high level of ROS production compared with patients diagnosed in early stage (grade 1 or 2 Hackett of splenomegaly) median value: 13178014970 vs 12303298585, p = 0.001. There are no differences between groups that was splitted by type of treatment Ruxolitinib/Hydroxiureea or type of mutation diagnosed JAK2V617F/CALR/no mutation. Patients with MMM present higher resting membrane potential (RMP) compared with controls (median value ‐ 63 mV vs–57.5 mV, p = 0.05). The expression of CD36 receptor was higher in MMM group vs controls: median value 27.91 vs 20.91, p = 0.003. Number of microparticle (MP) derivated from platelet, erythrocyte and endothelial cells are no different between patient and control group. We obtain only lower receptor expression on MP surface for CD41/CD61, CD42b and GlyA in patient group compared with control group, p < 0.05. There are significant correlation between resting membrane potential and expression of CD42b (r ‐0.45, p = 0.04) and GlyA receptors (r ‐0.45, p = 0.04).Summary/Conclusion:Patients with MMM especially those with have a higher level of ROS production especially in advanced phase. The hyperpolarisation of cell macrophages membrane could be associated with high ROS production, no reports are regarding platelet membrane. In our study we did not obtain any significant correlation between RMP and ROS production. The expression of CD 36 is higher in MMM patients but was not correlated with ROS production. It was proved that generation of intracellular ROS is associated with CD36 signaling and represent one start point for thrombosis. Low expression of CD42b and GlyA of microparticle surface is associated with lower RMP that correspond to activated status of platelet membrane. We have to verify these results in higher number of patients in order to find significant correlations

Constitutive and Synaptic Activation of GIRK Channels Differentiates Mature and Newborn Dentate Granule Cells.

Sparse neural activity in the dentate gyrus is enforced by powerful networks of inhibitory GABAergic interneurons in combination with low intrinsic excitability of the principal neurons, the dentate granule cells (GCs). Although the cellular and circuit properties that dictate synaptic inhibition are well studied, less is known about mechanisms that confer low GC intrinsic excitability. Here we demonstrate that intact G protein-mediated signaling contributes to the characteristic low resting membrane potential that differentiates mature dentate GCs from CA1 pyramidal cells and developing adult-born GCs. In mature GCs from male and female mice, intact G protein signaling robustly reduces intrinsic excitability, whereas deletion of G protein-activated inwardly rectifying potassium channel 2 (GIRK2) increases excitability and blocks the effects of G protein signaling on intrinsic properties. Similarly, pharmacological manipulation of GABAB receptors (GABABRs) or GIRK channels alters intrinsic excitability and GC spiking behavior. However, adult-born new GCs lack functional GIRK activity, with phasic and constitutive GABABR-mediated GIRK signaling appearing after several weeks of maturation. Phasic activation is interneuron specific, arising primarily from nNOS-expressing interneurons rather than parvalbumin- or somatostatin-expressing interneurons. Together, these results demonstrate that G protein signaling contributes to the intrinsic excitability that differentiates mature and developing dentate GCs and further suggest that late maturation of GIRK channel activity is poised to convert early developmental functions of GABAB receptor signaling into GABABR-mediated inhibition.SIGNIFICANCE STATEMENT The dentate gyrus exhibits sparse neural activity that is essential for the computational function of pattern separation. Sparse activity is ascribed to strong local inhibitory circuits in combination with low intrinsic excitability of the principal neurons, the granule cells. Here we show that constitutive activity of G protein-coupled inwardly rectifying potassium channels (GIRKs) underlies to the hallmark low resting membrane potential and input resistance of mature dentate neurons. Adult-born neurons initially lack functional GIRK channels, with constitutive and phasic GABAB receptor-mediated GIRK inhibition developing in tandem after several weeks of maturation. Our results reveal that GABAB/GIRK activity is an important determinant of low excitability of mature dentate granule cells that may contribute to sparse DG activity in vivo.

Low Resting Membrane Potential and Low Inward Rectifier Potassium Currents Are Not Inherent Features of hiPSC-Derived Cardiomyocytes.

SummaryHuman induced pluripotent stem cell (hiPSC) cardiomyocytes (CMs) show less negative resting membrane potential (RMP), which is attributed to small inward rectifier currents (IK1). Here, IK1 was measured in hiPSC-CMs (proprietary and commercial cell line) cultured as monolayer (ML) or 3D engineered heart tissue (EHT) and, for direct comparison, in CMs from human right atrial (RA) and left ventricular (LV) tissue. RMP was measured in isolated cells and intact tissues. IK1 density in ML- and EHT-CMs from the proprietary line was similar to LV and RA, respectively. IK1 density in EHT-CMs from the commercial line was 2-fold smaller than in the proprietary line. RMP in EHT of both lines was similar to RA and LV. Repolarization fraction and IK,ACh response discriminated best between RA and LV and indicated predominantly ventricular phenotype in hiPSC-CMs/EHT. The data indicate that IK1 is not necessarily low in hiPSC-CMs, and technical issues may underlie low RMP in hiPSC-CMs.

Aromatase inhibitors augment nociceptive behaviors in rats and enhance the excitability of sensory neurons

Although aromatase inhibitors (AIs) are commonly used therapies for breast cancer, their use is limited because they produce arthralgia in a large number of patients. To determine whether AIs produce hypersensitivity in animal models of pain, we examined the effects of the AI, letrozole, on mechanical, thermal, and chemical sensitivity in rats. In ovariectomized (OVX) rats, administering a single dose of 1 or 5mg/kg letrozole significantly reduced mechanical paw withdrawal thresholds, without altering thermal sensitivity. Repeated injection of 5mg/kg letrozole in male rats produced mechanical, but not thermal, hypersensitivity that extinguished when drug dosing was stopped. A single dose of 5mg/kg letrozole or daily dosing of letrozole or exemestane in male rats also augmented flinching behavior induced by intraplantar injection of 1000nmol of adenosine 5′-triphosphate (ATP). To determine whether sensitization of sensory neurons contributed to AI-induced hypersensitivity, we evaluated the excitability of neurons isolated from dorsal root ganglia of male rats chronically treated with letrozole. Both small and medium-diameter sensory neurons isolated from letrozole-treated rats were more excitable, as reflected by increased action potential firing in response to a ramp of depolarizing current, a lower resting membrane potential, and a lower rheobase. However, systemic letrozole treatment did not augment the stimulus-evoked release of the neuropeptide calcitonin gene-related peptide (CGRP) from spinal cord slices, suggesting that the enhanced nociceptive responses were not secondary to an increase in peptide release from sensory endings in the spinal cord. These results provide the first evidence that AIs modulate the excitability of sensory neurons, which may be a primary mechanism for the effect of these drugs to augment pain behaviors in rats.

Mechanisms of spikelet generation in cortical pyramidal neurons

Unter Spikelets versteht man kleine Depolarisationen mit einer Spike-ahnlichen Wellenform, die man in intrazellularen Ableitungen von verschiedenen Neuronentypen messen kann. In kortikalen Pyramidenzellen wurde ausgepragte Spikelet-Aktivitat nachgewiesen, die erheblich das Membranpotential beeinflussen kann (Crochet et al., 2004; Epsztein et al., 2010; Chorev and Brecht, 2012). Nichtsdestotrotz bleibt der Ursprung von Spikelets in diesen Neuronen unbekannt. In der vorgelegten Arbeit nutzte ich theoretische Modellierung um die Mechanismen von Spikelet-Erzeugung in Pyramidenzellen zu untersuchen. Zuerst sah ich die verschiedenen Hypothesen uber den Ursprung von Spikelets durch. In der Literatur entdeckte ich zwei verschiedene Typen von Spikelets. Diese Arbeit konzentriert sich auf den haufiger vorkommenden Typ von Spikelets, welcher durch relativ grose Amplituden gekennzeichnet ist. Die Eigenschaften dieser Spikelets passen am besten zu einem axonal Erzeugungsmechanismus. Im zweiten Kapitel widmete ich mich der Hypothese, dass somatische Spikelets axonalen Ursprungs mit somato-dendritischen Inputs hervorgerufen werden konnen. Ich identifizierte Bedingungen, die es erlauben ein Aktionspotential (AP) am Initialsegment vom Axon (AIS) zu initiieren, welches sich entlang des Axons ausbreitet, aber kein AP im Soma auslost. Schlieslich simulierte ich extrazellulare Wellenformen von APs und Spikelets und verglich sie mit experimentellen Daten (Chorev and Brecht, 2012). Dieser Vergleich zeigte auf, dass die extrazellularen Wellenformen von Spikelets, die innerhalb einer Zellen am AIS erzeugt werden, gut zu den Daten passen. Zusammenfassend unterstutzen meine Ergebnisse die Hypothese, dass Spikelets in Pyramidenzellen am AIS entstehen. Dieser Mechanismus konnte ein Mittel zum Energiesparen bei der Erzeugung von Output-APs sein. Auserdem konnte dadurch die dendritische Plastizitat, die auf der Ruckwartspropagierung von APs beruht, reguliert werden.%%%%Spikelets are transient spike-like depolarizations of small amplitudes that can be measured in somatic intracellular recordings of many neuron types. Pronounced spikelet activity has been demonstrated in cortical pyramidal neurons in vivo (Crochet et al., 2004; Epsztein et al., 2010; Chorev and Brecht, 2012), influencing membrane voltage dynamics including action potential initiation. Nevertheless, the origin of spikelets in these neurons remains elusive. In thi thesis, I used computational modeling to examine the mechanisms of spikelet generation in pyramidal neurons. First, I reviewed the hypotheses previously suggested to explain spikelet origin. I discovered two qualitatively different spikelet types described in the experimental literature. This thesis focuses on the more commonly reported spikelet type, characterized by relatively large amplitudes of up to 20 mV. I found that the properties of these spikelets fit best to an axonal generation mechanism. Second, I explored the hypothesis that somatic spikelets of axonal origin can be evoked with somato-dendritic inputs. I…

Diverse Kir expression contributes to distinct bimodal distribution of resting potentials and vasotone responses of arterioles.

The resting membrane potential (RP) of vascular smooth muscle cells (VSMCs) is a major determinant of cytosolic calcium concentration and vascular tone. The heterogeneity of RPs and its underlying mechanism among different vascular beds remain poorly understood. We compared the RPs and vasomotion properties between the guinea pig spiral modiolar artery (SMA), brain arterioles (BA) and mesenteric arteries (MA). We found: 1) RPs showed a robust bimodal distribution peaked at -76 and -40 mV evenly in the SMA, unevenly at -77 and -51 mV in the BA and ~-71 and -52 mV in the MA. Ba2+ 0.1 mM eliminated their high RP peaks ~-75 mV. 2) Cells with low RP (~-45 mV) hyperpolarized in response to 10 mM extracellular K+, while cells with a high RP depolarized, and cells with intermediate RP (~-58 mV) displayed an initial hyperpolarization followed by prolonged depolarization. Moderate high K+ typically induced dilation, constriction and a dilation followed by constriction in the SMA, MA and BA, respectively. 3) Boltzmann-fit analysis of the Ba2+-sensitive inward rectifier K+ (Kir) whole-cell current showed that the maximum Kir conductance density significantly differed among the vessels, and the half-activation voltage was significantly more negative in the MA. 4) Corresponding to the whole-cell data, computational modeling simulated the three RP distribution patterns and the dynamics of RP changes obtained experimentally, including the regenerative swift shifts between the two RP levels after reaching a threshold. 5) Molecular works revealed strong Kir2.1 and Kir2.2 transcripts and Kir2.1 immunolabeling in all 3 vessels, while Kir2.3 and Kir2.4 transcript levels varied. We conclude that a dense expression of functional Kir2.X channels underlies the more negative RPs in endothelial cells and a subset of VSMC in these arterioles, and the heterogeneous Kir function is primarily responsible for the distinct bimodal RPs among these arterioles. The fast Kir-based regenerative shifts between two RP states could form a critical mechanism for conduction/spread of vasomotion along the arteriole axis.

Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro – Correlation between contraction force and electrophysiology

Cardiomyocytes from human pluripotent stem cells (hPSC-CM) have many potential applications in disease modelling and drug target discovery but their phenotypic similarity to early fetal stages of cardiac development limits their applicability. In this study we compared contraction stresses of hPSC-CM to 2nd trimester human fetal derived cardiomyocytes (hFetal-CM) by imaging displacement of fluorescent beads by single contracting hPSC-CM, aligned by microcontact-printing on polyacrylamide gels. hPSC-CM showed distinctly lower contraction stress than cardiomyocytes isolated from hFetal-CM. To improve maturation of hPSC-CM in vitro we made use of commercial media optimized for cardiomyocyte maturation, which promoted significantly higher contraction stress in hPSC-compared with hFetal-CM. Accordingly, other features of cardiomyocyte maturation were observed, most strikingly increased upstroke velocities and action potential amplitudes, lower resting membrane potentials, improved sarcomeric organization and alterations in cardiac-specific gene expression. Performing contraction force and electrophysiology measurements on individual cardiomyocytes revealed strong correlations between an increase in contraction force and a rise of the upstroke velocity and action potential amplitude and with a decrease in the resting membrane potential.We showed that under standard differentiation conditions hPSC-CM display lower contractile force than primary hFetal-CM and identified conditions under which a commercially available culture medium could induce molecular, morphological and functional maturation of hPSC-CM in vitro. These results are an important contribution for full implementation of hPSC-CM in cardiac disease modelling and drug discovery.

A TTX-sensitive resting Na+ permeability contributes to the catecholaminergic automatic activity in rat pulmonary vein.

Ectopic activity arising from pulmonary veins (PV) plays a prominent role in the onset of atrial fibrillation in humans. Rat PV cardiac muscle cells have a lower resting membrane potential (RMP) than the left atria (LA) and presents in the presence of norepinephrine an automatic activity, which occurs in bursts. This study investigated the role of Na channels upon the RMP and the catecholaminergic automatic activity (CAA) in PV cardiac muscle. RMP and CAA experiments were performed in male Wistar rat PV. Whole-cell INa was recorded in isolated PV and LA cardiomyocytes. PV has a higher tetrodotoxin (TTX)-sensitive basal Na(+) permeability than the LA, due to a ∼ 5 mV more negative Na window current in the former tissue. TTX, quinidine, and ranolazine (1 to 10 μM each) decreased CAA incidence and arrhythmias by increasing burst intervals because of a reduction of the slope of slow depolarization between bursts. TTX and ranolazine also reduced burst duration. At 1 Hz, 10 μM quinidine, ranolazine, and TTX inhibited peak INa by 33%, 28%, and 98%, respectively. Each reduced the Na window current. There was no evidence for a TTX- or ranolazine-sensitive late Na current. Na channels confer a TTX-sensitive basal Na(+) permeability to rat PV cardiac muscle cells and contribute to the slope of slow depolarization between bursts of CAA. Na channel blockers act mostly via reduction of the Na window current. Ranolazine also has an anti-α1 adrenergic effect, which contributed to its antiarrhythmic effect.

Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors.

Reprogramming of somatic cells into pluripotency stem cell state has opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human-induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here, we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSCs), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for 6 weeks, lt-NES cell-derived neurons displayed neuronal properties such as tetrodotoxin-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential, and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the lt-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, for example, such as epilepsy.

The microbiome is essential for normal gut intrinsic primary afferent neuron excitability in the mouse

The role of intestinal microbiota in the development and function of host physiology is of high interest, especially with respect to the nervous system. While strong evidence has accrued that intestinal bacteria alter host nervous system function, mechanisms by which this occurs have remained elusive. For this reason, we have carried out experiments examining the electrophysiological properties of neurons in the myenteric plexus of the enteric nervous system (ENS) in germ-free (GF) mice compared with specific pathogen-free (SPF) control mice and adult germ-free mice that have been conventionalized (CONV-GF) with intestinal bacteria. Segments of jejunum from 8 to 12 week old GF, SPF, and CONV-GF mice were dissected to expose the myenteric plexus. Intracellular recordings in current-clamp mode were made by impaling cells with sharp microelectrodes. Action potential (AP) shapes, firing thresholds, the number of APs fired at 2× threshold, and passive membrane characteristics were measured. In GF mice, excitability was decreased in myenteric afterhyperpolarization (AH) neurons as measured by a lower resting membrane potential and by the number of APs generated at 2× threshold. The post AP slow afterhyperpolarization (sAHP) was prolonged for GF compared with SPF and CONV-GF animals. Passive membrane characteristics were also altered in GF mice by a decrease in input resistance. Here, we report the novel finding that commensal intestinal microbiota are necessary for normal excitability of gut sensory neurons and thus provide a potential mechanism for the transfer of information between the microbiota and nervous system.

Electrophysiological and Pharmacological Characteristics of Triggered Activity Elicited in Guinea-Pig Pulmonary Vein Myocardium

The pulmonary vein is known as an important source of ectopic beats, initiating frequent paroxysms of atrial fibrillation. We analyzed electrophysiological and pharmacological characteristics of triggered activity elicited in the isolated pulmonary vein from the guinea pig. Immediately after the termination of train stimulation (pacing cycle length of 100 ms), spontaneous activities accompanied with phase-4 depolarization were detected in 43 out of 45 pulmonary vein preparations. Such triggered activities were not observed in the isolated left atrium. The incidence of triggered activity was higher at a shorter pacing cycle length (100 – 200 ms), and the coupling interval was shorter at a shorter pacing cycle length. Verapamil (1 μM), ryanodine (0.1 μM), and pilsicainide (10 μM) suppressed the occurrence of triggered activities. The resting membrane potential of the pulmonary vein myocardium was more positive than that of the left atrium. Carbachol (0.3 μM) hyperpolarized the resting membrane potential and completely inhibited the occurrence of triggered activities. These results suggest that the pulmonary veins have more arrhythmogenic features than the left atrium, possibly through lower resting membrane potential. The electrophysiological and pharmacological characteristics of triggered activity elicited in the pulmonary vein myocardium were similar to those previously reported using ventricular tissues.

TEA-Sensitive Currents Contribute to Membrane Potential of Organ Surface Primo-node Cells in Rats

The primo-vascular (Bonghan) tissue has been identified in most tissues in the body, but its structure and functions are not yet well understood. We characterized electrophysiological properties of the cells of the primo-nodes (PN) on the surface of abdominal organs using a slice patch clamp technique. The most abundant were small round cells (~10 μm) without processes. These PN cells exhibited low resting membrane potential (-36 mV) and did not fire action potentials. On the basis of the current-voltage (I-V) relationships and kinetics of outward currents, the PN cells can be grouped into four types. Among these, type I cells were the majority (69%); they showed strong outward rectification in I-V relations. The outward current was activated rapidly and sustained without decay. Tetraethylammonium (TEA) dose-dependently blocked both outward and inward current (IC(50), 4.3 mM at ± 60 mV). In current clamp conditions, TEA dose-dependently depolarized the membrane potential (18.5 mV at 30 mM) with increase in input resistance. The tail current following a depolarizing voltage step was reversed at -27 mV, and transient outward current like A-type K(+) current was not expressed at holding potential of -80 mV. Taken together, the results demonstrate for the first time that the small round PN cells are heterogenous, and that, in type I cells, TEA-sensitive current with limited selectivity to K(+) contributed to resting membrane potential of these cells.

A rodent model of schizophrenia derived from postmortem studies

There is compelling postmortem evidence that GABA cell dysfunction plays a role in the pathophysiology of schizophrenia (SZ). Based on a unique distribution of postmortem abnormalities in layer II of the anterior cingulate cortex and sectors CA3/2 of the hippocampus, we postulated that afferent fibers from the basolateral amygdala to these sites may contribute to diminished GABAergic modulation in these disorders. To test this hypothesis, picrotoxin (PICRO), a non-competitive antagonist of the GABA-A receptor, is stereotaxically infused the basolateral complex of the amygdala (BLA) to increase the flow of excitatory activity into stratum oriens (SO) of sectors CA3/2 of the hippocampus. This pharmacological manipulation results in a selective reduction of GABAergic interneurons containing parvalbumin, calbindin and calretinin in CA3/2. Using single cell recordings in a hippocampal slide preparation, these changes in PICRO-treated rats seem to be associated with a reduction in evoked and spontaneous inhibitory post-synaptic potentials (sIPSCs) recorded from pyramidal neurons in sector CA3/2, but not CA1. A lower resting membrane potential and an increased action potential firing rate have been recorded in interneurons in the SO of CA2/3, but not CA1. Additionally, currents associated with hyperpolarization-activated cationic channels (Ih), which help to control neuronal firing rates of GABA cells in the hippocampus, were also increased. Overall, these studies support the view that postmortem studies contribute information for the development of empiric models of SZ, ones that can be used as translational tools for elucidating the functional changes that may be present in GABA cell subtypes their molecular regulatory mechanisms in this disorder.

Amygdala-Dependent Regulation of Electrical Properties of Hippocampal Interneurons in a Model of Schizophrenia

Schizophrenia (SZ) involves dysfunction of gamma-aminobutyric acid (GABA)ergic transmission in the hippocampus (HIPP), particularly in sector CA2/3. Previous work using a rodent model of postmortem abnormalities in SZ demonstrated that activation of the basolateral amygdala (BLA) results in decreases of GABA currents in pyramidal neurons of CA2/3 but not CA1. In addition, a decrease of GABA cells has been reported in postmortem studies of the HIPP in SZ. In the present work we tested the hypothesis that BLA activation in this rodent model of SZ leads to changes in the electrical properties of interneurons located in sector CA2/3. Patch clamp recordings in HIPP slices were performed in rat HIPP slices after 15 days of infusion of picrotoxin into the BLA. The intrinsic and firing properties and hyperpolarization-activated currents (Ih) of interneurons were measured in stratum oriens (SO) of CA2/3 and CA1. The BLA activation was associated with a lower resting membrane potential and an increased action potential firing rate in interneurons of CA2/3 but not CA1. Recordings from interneurons further demonstrated an increase of currents associated with hyperpolarization-activated cationic channels (Ih), which help to control neuronal firing rates and oscillatory rhythms. Taken together, these results suggest that the enhanced BLA activity is capable of increasing the excitability of interneurons in SO of CA2/3 and might contribute to GABAergic dysfunction in SZ.

Progressive increase in conduction velocity across human mesenchymal stem cells is mediated by enhanced electrical coupling

The purpose of the study was to investigate the development of electrical transmission across human adult bone marrow-derived mesenchymal stem cells (hMSCs) during long-term co-incubation with cardiomyocytes (CMCs). Neonatal rat CMCs were cultured in multi-electrode array dishes. A conduction block was induced by creating a central acellular channel, yielding two asynchronously beating CMC fields. Enhanced green fluorescent protein (eGFP)-labeled hMSCs from ischemic heart disease patients (n=8), eGFP-labeled hMSCs having RNA interference-mediated connexin43 (Cx43) knockdown (n=6), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil)-labeled CMCs (n=6), or no cells (n=9) were seeded in the channel. Assessment of conduction velocity (CV), Cx expression and localization, gap junctional coupling, and intracellular electrical recordings were performed for up to 14 days. Resynchronization of the two CMC fields occurred within 24 h after seeding of hMSCs. CV across hMSCs increased from 1.4+/-0.4 cm/s at day 7 to 3.5+/-0.1 cm/s (p<0.05) at day 14. CV across seeded CMCs was 16.8+/-0.2 cm/s throughout this period. No resynchronization occurred in the absence of seeded cells. Knockdown of Cx43 in hMSCs abolished conduction across the channel completely. Time-dependent increase of CV across hMSCs was associated with increased Cx43 mRNA and protein expression resulting in increased gap junctional coupling. Intracellular recordings in coupled hMSCs showed increased conducted action potential (AP) amplitude, lower resting membrane potential, and decreased duration of conducted AP (p<0.05, day 14 versus day 1). CV across hMSCs increases progressively after 7 days of co-incubation with CMCs, most likely via improved electrotonic interaction. This is associated with increased Cx43 expression, increased functional gap junctional coupling, and enhanced intercellular electrical coupling between hMSCs and CMCs.

Regulation of the spontaneous contractile activity of the portal vein by the sarcoplasmic reticulum: evidence from the phospholamban gene-ablated mouse

The rapid contraction/relaxation cycles of phasic smooth muscles necessitates intracellular calcium cycling at a more rapid rate than that of tonic smooth muscles. Recent studies suggest that sarcoplasmic reticulum calcium handling is an important determinant of portal vein phasic contractions. We evaluated the importance and role of phospholamban, a protein which inhibits the sarcoplasmic reticulum (SR) calcium ATPase (SERCA), in regulating the contractility of the phasic mouse portal vein. PLB gene ablation significantly reduced the basal frequency of spontaneous mechanical activity and increased force development of the portal vein. Cyclopiazonic acid (CPA), an inhibitor of SERCA, did not significantly affect the spontaneous activity of the wild-type (WT) portal vein. CPA (1 μM) eliminated the differences in frequency and force between the PLB-KO and WT, localizing the effects to the SR. The PLB-KO portal vein had a lower resting membrane potential than WT controls. There were no significant differences between WT and KO responses to charybdotoxin (250 nM), indicating that calcium-activated potassium channels do not contribute to altered KO portal vein contractility. While contractile sensitivity to acetylcholine was not different between WT and PLB-KO portal veins, force generated in response to a given concentration of acetylcholine was significantly greater in the PLB-KO portal vein, both in the absence and presence of CPA. Our results confirm that SR activity can play a major role in modulating the frequency of the spontaneous mechanical activity of portal veins and removal of PLB inhibition of the SR calcium ATPase has significant effects on the spontaneous activity of the portal vein.

Topographical and electrophysiological characteristics of highly excitable S neurones in the myenteric plexus of the guinea-pig ileum.

1. Most intracellular electrical recordings from myenteric neurones have been made from the centre of large ganglia. In this study, we examined the electrophysiological properties of neurones at the corners of large ganglia close to internodal strands and in microganglia. 2. Of 150 neurones in these locations: 111 were tonic S neurones; 9 were phasic S neurones and 30 were AH neurones. 3. Tonic S neurones were characterized by: (i) low resting membrane potentials (-50 +/- 1 mV, mean +/- s.e.m.); (ii) high input impedance (522 +/- 23 MOmega); (iii) low threshold for action potential (AP) generation (0.012 +/- 0.004 nA); (iv) firing of APs throughout a depolarizing pulse (duration <= 1 s) and one to four APs following a hyperpolarizing pulse and (v) spontaneous fast excitatory postsynaptic potentials (FEPSPs). A substantial proportion of tonic S neurones (43 %) also fired APs spontaneously (7.6 +/- 0.6 Hz; range, 0.3-19 Hz). All APs were blocked by tetrodotoxin (1 microM). 4. Tonic S neurones were subclassified, according to their post-stimulus responses, as SAH or SAD neurones. Following a burst of APs, SAH neurones exhibited a prominent after-hyperpolarization (duration, 711 +/- 10 ms) and SAD neurones an after-depolarization (duration, 170 +/- 10 ms). The after-hyperpolarization was reduced in four of ten neurones by apamin (0.3 microM). 5. FEPSPs were evoked in 20 of 38 S neurones by electrical stimulation applied both oral and anal to the recording site. Repetitive stimuli evoked slow excitatory postsynaptic potentials (SEPSPs) in some tonic S neurones. 6. Three functional classes of S neurones were identified after injection of neurobiotin through the recording microelectrode: (i) longitudinal muscle motor neurones, (ii) short circular muscle motor neurones, and (iii) ascending interneurones. 7. In conclusion, there appears to be topographical organization of highly excitable, tonic S neurones within the myenteric plexus, since, in contrast to other S neurones, they can be readily impaled in myenteric ganglia close to internodal strands and in microganglia.

A Novel Tetrodotoxin-Sensitive Na sup + Current in Cultured Human Coronary Myocytes

Voltage-gated Na+ currents (INaS) are usually not found in arterial smooth muscle. We enzymatically isolated myocytes from the media of left coronary arteries of heart transplant patients with ischemic cardiopathy. Using the whole-cell voltage-clamp technique (20 degrees C to 22 degrees C), we detected no INa in any of the freshly isolated myocytes. In contrast, when the cells were grown in culture, we could record a large INa. This INa was characterized by a biexponential decay comprising a fast inactivating and sustained components that could not be separated by their electrophysiological and pharmacological properties. INa activated at depolarizations positive to -50 mV, was maximal at 0 mV, and was available from relatively low resting membrane potentials (half-inactivation at -46 mV). INa was modulated by several ligands known to bind selectively at different sites of Na+ channels. It was blocked with high affinity by tetrodotoxin (IC50, approximately 10 nmol/L) and local anesthetics (bupivacaine and lidocaine; IC50, approximately 100 nmol/L) and by Cd2+ (IC50, approximately 300 mumol/L). INa was modulated by Na+ channel agonists such as toxin AsV from Anemonia sulcata and veratridine, which slowed current kinetics dramatically. In conclusion, human coronary myocytes in culture can express an atypical tetrodotoxin-sensitive INa with a large sustained component, which is expected to contribute to massive Na+ influx into these cells. Phenotypic modulation of the expression of this INa may be related to cell dedifferentiation and proliferation.

Intraoperative Ventricular Tachycardia Responsive to Adenosine

Intraoperative Ventricular Tachycardia Responsive to Adenosine