Simultaneous Recordings Of Electrical Activity Research Articles

Automated Patch-Clamp and Induced Pluripotent Stem Cell-Derived Cardiomyocytes: A Synergistic Approach in the Study of Brugada Syndrome

The development of high-throughput automated patch-clamp technology is a recent breakthrough in the field of Brugada syndrome research. Brugada syndrome is a heart disorder marked by abnormal electrocardiographic readings and an elevated risk of sudden cardiac death due to arrhythmias. Various experimental models, developed either in animals, cell lines, human tissue or computational simulation, play a crucial role in advancing our understanding of this condition, and developing effective treatments. In the perspective of the pathophysiological role of ion channels and their pharmacology, automated patch-clamp involves a robotic system that enables the simultaneous recording of electrical activity from multiple single cells at once, greatly improving the speed and efficiency of data collection. By combining this approach with the use of patient-derived cardiomyocytes, researchers are gaining a more comprehensive view of the underlying mechanisms of heart disease. This has led to the development of more effective treatments for those affected by cardiovascular conditions.

Glucose stimulates somatostatin secretion in pancreatic δ-cells by cAMP-dependent intracellular Ca2+ release.

Somatostatin secretion from pancreatic islet δ-cells is stimulated by elevated glucose levels, but the underlying mechanisms have only partially been elucidated. Here we show that glucose-induced somatostatin secretion (GISS) involves both membrane potential-dependent and -independent pathways. Although glucose-induced electrical activity triggers somatostatin release, the sugar also stimulates GISS via a cAMP-dependent stimulation of CICR and exocytosis of somatostatin. The latter effect is more quantitatively important and in mouse islets depolarized by 70 mM extracellular K+ , increasing glucose from 1 mM to 20 mM produced an ∼3.5-fold stimulation of somatostatin secretion, an effect that was mimicked by the application of the adenylyl cyclase activator forskolin. Inhibiting cAMP-dependent pathways with PKI or ESI-05, which inhibit PKA and exchange protein directly activated by cAMP 2 (Epac2), respectively, reduced glucose/forskolin-induced somatostatin secretion. Ryanodine produced a similar effect that was not additive to that of the PKA or Epac2 inhibitors. Intracellular application of cAMP produced a concentration-dependent stimulation of somatostatin exocytosis and elevation of cytoplasmic Ca2+ ([Ca2+]i). Both effects were inhibited by ESI-05 and thapsigargin (an inhibitor of SERCA). By contrast, inhibition of PKA suppressed δ-cell exocytosis without affecting [Ca2+]i Simultaneous recordings of electrical activity and [Ca2+]i in δ-cells expressing the genetically encoded Ca2+ indicator GCaMP3 revealed that the majority of glucose-induced [Ca2+]i spikes did not correlate with δ-cell electrical activity but instead reflected Ca2+ release from the ER. These spontaneous [Ca2+]i spikes are resistant to PKI but sensitive to ESI-05 or thapsigargin. We propose that cAMP links an increase in plasma glucose to stimulation of somatostatin secretion by promoting CICR, thus evoking exocytosis of somatostatin-containing secretory vesicles in the δ-cell.

Simultaneous recording of electrical activity and the underlying ionic currents in NG108-15 cells cultured on gold substrate

This paper shows the simultaneous recording of electrical activity and the underlying ionic currents by using a gold substrate to culture NG108-15 cells. Cells grown on two different substrates (plastic Petri dishes and gold substrates) were characterized quantitatively through scanning electron microscopy (SEM) as well as qualitatively by optical and atomic force microscopy (AFM). No significant differences were observed between the surface area of cells cultured on gold substrates and Petri dishes, as indicated by measurements performed on SEM images. We also evaluated the electrophysiological compatibility of the cells through standard patch-clamp experiments by analyzing features such as the resting potential, membrane resistance, ionic currents, etc. Cells grown on both substrates showed no significant differences in their dependency on voltage, as well as in the magnitude of the Na+ and K+ current density; however, cells cultured on the gold substrate showed a lower membrane capacitance when compared to those grown on Petri dishes. By using two separate patch-clamp amplifiers, we were able to record the membrane current with the conventional patch-clamp technique and through the gold substrate simultaneously. Furthermore, the proposed technique allowed us to obtain simultaneous recordings of the electrical activity (such as action potentials firing) and the underlying membrane ionic currents. The excellent conductivity of gold makes it possible to overcome important difficulties found in conventional electrophysiological experiments such as those presented by the resistance of the electrolytic bath solution. We conclude that the technique here presented constitutes a solution to the problem of the simultaneous recording of electrical activity and the underlying ionic currents, which for decades, had been solved only partially.

Rapid Activity Screening of Ion Channels Expressed in Cell-Free Systems Using a Lipid Bilayer Array Pcb-Device

Cell-free expression is a rapidly developing tool for the efficient production of membrane proteins with enormous potential. Here we report on the design of a setup for parallel high-resolution electrophysiological recordings allowing for the screening of functional activity of ion channels synthesized in-vitro in various lipid environments.The developed recording chamber is based on a SU-8 coated printed circuit board containing 4 cavities (50µm) with individual microelectrodes (Micro-Electrode-Cavity-Array (MECA) as well as a common ground electrode. 4 suspended lipid bilayers can be self-assembled on the nonpolar chip surface from phospholipids in organic solvent. The MECA-PCB is connected to a multichannel amplifier capable of simultaneous recording of electrical activity from the ion channels functionally reconstituted into the bilayers with high-bandwidth and low noise (<1 pA rms ≅ 10 kHz).To test the capability of the MECA-PCB-chip, the tetrameric potassium channel KcsA was expressed in-vitro with co-translational insertion into supplied liposomes containing di-myristoyl-glycerophosphocholine or asolectin. The collected proteoliposomes were extruded through 100 nm polycarbonate filter and fused with preformed bilayers. Activity from the single channels inserted in different bilayers was recorded in parallel under identical conditions. We show thatKcsA expressed in-vitro displays the typical single-channel kinetic behavior and sensitivity to blockers (Ba2+) described for the protein isolated from cellular membrane preparations. Channel open times and open probability are shown to be dependent on lipid environment with negatively-charged phospholipids stabilizing the open channel conformation.The combination of the cell-free protein expression system and MECA-PCB-chip electrophysiology allows for functional characterization of the synthesized ion channel within several hours starting from the DNA template. The current PCB-chip design could be easily adapted for higher throughput and automated bilayer formation as recently reported1.1Del Rio Martinez et.al. Small 2014.

Monitoring Population Membrane Potential Signals During Development of the Vertebrate Nervous System.

The functional organization of the vertebrate central nervous system (CNS) during the early phase of development has long been unclear, because conventional electrophysiological means have several technical limitations. First, early embryonic neurons are small and fragile, and the application of microelectrodes is often difficult. Second, the simultaneous recording of electrical activity from multiple sites is limited, and as a consequence, response patterns of neural networks cannot be assessed. Optical recording techniques with voltage-sensitive dyes have overcome these obstacles and provided a new approach to the analysis of the functional development/organization of the CNS. In this review, we provide detailed information concerning the recording of optical signals in the embryonic nervous system. After outlining methodological considerations, we present examples of recent progress in optical studies on the embryonic nervous system with special emphasis on two topics. The first is the study of how synapse networks form in specific neuronal circuits. The second is the study of non-specific correlated wave activity, which is considered to play a fundamental role in neural development. These studies clearly demonstrate the utility of fast voltage-sensitive dye imaging as a powerful tool for elucidating the functional organization of the vertebrate embryonic CNS.

Store-operated Ca2+ entry and depolarization explain the anomalous behaviour of myometrial SR: Effects of SERCA inhibition on electrical activity, Ca2+ and force

In the myometrium SR Ca2+ depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca2+ sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca2+-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca2+ transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20μM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca2+]i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca2+ spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca2+ and depolarization continued a point was reached when electrical and Ca2+ spikes and phasic contractions ceased, and a maintained, tonic force and Ca2+ was produced. Lanthanum, a non-selective blocker of store-operated Ca2+ entry, but not the L-type Ca2+ channel blocker nifedipine (1–10μM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca2+ depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca2+ release is coupled to marked Ca2+ entry, via store operated Ca2+ channels, leading to depolarization and enhanced electrical and mechanical activity.

35. Surface polyelectromyography – Principle of the method and possible use in dysphagia

Surface polyelectromyography (PEMG) represents a specific application of electromyography (EMG) in the functional analysis of movement (”kinesiological EMG”) with the aid of simultaneous recordings of electrical activity from multiple muscles using specialised equipment (typically 16 channels). Swallowing is a very complex function that involves 26 muscles in head and neck. The authors present their experience with PEMG in 12 patients with dysphagia, particularly after a cerebrovascular accident. They used subsurface layer muscle groups (orbicularis oris, masseter muscle, submental and laryngeal muscles) and simple standardised tests (dry swallowing, swallowing 20 ml of water and drinking 100 ml of water without interruption) for a PEMG examination at the beginning and end of an intensive physiotherapy and speech therapy. PEMG findings are presented in practical illustrations of specific cases including software processed EMG signals. PEMG (unlike ”classic EMG”) does not provide information to identify the clinical diagnostic units that cause a disorder. However, it facilitates, in terms of muscle activity, the characterisation of a functional disturbance (acknowledging limitations of the elusive definition for a normal movement pattern) and to monitor the evolution of functional parameters over time including its reaction to various influences (e.g. therapy). Supported by the project Ministry of Health, Czech Republic NT13725–4/2012.

Rapid functional evaluation of beta-cells by extracellular recording of membrane potential oscillations with microelectrode arrays

The membrane potential (V (m)) of beta-cells oscillates at glucose concentrations between ~6 and 25mM, i.e. burst phases with action potentials alternate with silent interburst phases generating so-called slow waves. The slow waves drive oscillations of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) and insulin secretion. The length of the bursts correlates with the amount of insulin release. Thus, the fraction of plateau phase (FOPP), i.e. the percentage of time with burst activity, is an excellent marker for beta-cell function and metabolic integrity. Extracellular voltage changes of mouse islets were measured using a microelectrode array (MEA) allowing the detection of burst and interburst phases. At a non-stimulating glucose concentration (3mM) no electrical activity was detectable while bursting was continuous at 30mM. The glucose concentration-response (determined as FOPP) curve revealed half-maximal stimulation at 12 ± 1mM (Hill equation fit). The signal was sensitive to K(ATP) channel modulators, e.g. tolbutamide or diazoxide. Simultaneous recordings of electrical activity and [Ca(2+)](c) revealed congruent bursts and peaks, respectively. The extracellular recordings are in perfect agreement with more time-consuming intracellular electrical recordings. The results provide a 'proof-of-principle' for detection of beta-cell slow waves and determination of the FOPP using extracellular electrodes in a MEA-based system. The method is facile and provides the capability to study the effects of modulators of beta-cell function including possible anti-diabetic drugs in real time. Moreover, the method may be useful for checking the metabolic integrity of human donor islets prior to transplantation.

Origin and propagation of the slow wave in the canine stomach: the outlines of a gastric conduction system

Slow waves are known to originate orally in the stomach and to propagate toward the antrum, but the exact location of the pacemaker and the precise pattern of propagation have not yet been studied. Using assemblies of 240 extracellular electrodes, simultaneous recordings of electrical activity were made on the fundus, corpus, and antrum in open abdominal anesthetized dogs. The signals were analyzed off-line, pathways of slow wave propagation were reconstructed, and slow wave velocities and amplitudes were measured. The gastric pacemaker is located in the upper part of the fundus, along the greater curvature. Extracellularly recorded slow waves in the pacemaker area exhibited large amplitudes (1.8 +/- 1.0 mV) and rapid velocities (1.5 +/- 0.9 cm/s), whereas propagation in the remainder of the fundus and in the corpus was slow (0.5 +/- 0.2 cm/s) with low-amplitude waveforms (0.8 +/- 0.5 mV). In the antrum, slow wave propagation was fast (1.5 +/- 0.6 cm/s) with large amplitude deflections (2.0 +/- 1.3 mV). Two areas were identified where slow waves did not propagate, the first in the oral medial fundus and the second distal in the antrum. Finally, recordings from the entire ventral surface revealed the presence of three to five simultaneously propagating slow waves. High resolution mapping of the origin and propagation of the slow wave in the canine stomach revealed areas of high amplitude and rapid velocity, areas with fractionated low amplitude and low velocity, and areas with no propagation; all these components together constitute the elements of a gastric conduction system.

Simultaneous recording of electrical activity and contraction from cultured cardiomyocytes

Simultaneous recording of electrical activity and contraction from cultured cardiomyocytes

A hyperpolarizing response induced by glutamate in mouse cerebellar Purkinje cells

In the vertebrate nervous system, glutamate (Glu) receptors are generally known to cause depolarizing responses. We report here a novel type of Glu response in Purkinje neurons of mouse cerebellar slices, namely glutamate-induced hyperpolarization (GH). This response is not due to activation of inhibitory interneurons, because application of tetrodotoxin (TTX), bicuculline, or strychnine did not abolish GH. In addition, GH persisted in a Ca 2+-free or a low-Cl − solution, which rules out the involvement of g K(Ca) or GABA A mechanisms. Quisqualate (Quis) and trans-1-amino-1,3-cyclopentanedicarboxylic acid (tACPD), which are potent and selective agonists, respectively, for the metabotropic Glu receptor (mGluR), failed to induce GH. l-2-Amino-4-phosphonobutyric acid ( l-AP4) was also ineffective. Simultaneous recording of electrical activity and intracellular Ca 2+ concentration ([Ca 2+] i) showed that GH was not accompanied by [Ca 2+] i changes. Voltage clamp experiments showed that GH is due to reduction of a tonically active conductance with a reversal potential around 0 mV. Two possible mechanisms are suggested for GH: (1) changes in the desensitized steady state of ionotropic Glu receptors, or (2) a novel Glu-mediated mechanism.

Optical determination of impulse conduction velocity during development of embryonic chick cervical vagus nerve bundles.

1. Employing an optical method for multiple-site simultaneous recording of electrical activity, we have determined the conduction velocity in cervical vagus nerve bundles isolated from 5- to 21-day-old chick embryos, and investigated its developmental changes. 2. The preparations were stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761), and action potential- (impulse-) related optical signals were elicited by brief stimuli applied to the end of the vagus nerve bundle with a suction electrode. Optical signals were recorded simultaneously from many contiguous regions using a 12 x 12-element photodiode array. 3. The optical signals spread with small delay from the site of stimulation. From the relationship between the delay and distance from the current-applying electrode, conduction velocities were estimated in each tested preparation: the conduction velocity was very small and increased monotonically from about 0.1 m s-1 at 5 days embryonic age to about 0.4 m s-1 by hatching. The increase in the conduction velocity was closely related to a developmental increase in the diameter of the vagus nerve bundle. 4. In addition, we have examined the spread of electrotonic potentials. The space constant was very small (200-450 microns) and increased as development proceeded. 5. Compound optical action signals having two distinct components were also recorded. They often appeared to be concentrated in the preparations from 8- to 12-day-old embryos. The conduction velocity of the second component was slower than that of the first. We suggest that appearance of the second component reflects degeneration of a subset of axons resulting from 'neural cell death' during the development of the vagus nerve.

Role of presynaptic dopamine receptors in regulation of the glutamatergic neurotransmission in rat neostriatum

In experiments with the use of a push-pull cannula and simultaneous recording of electrical activity at the site of perfusion, the release of l-[ 3H]glutamic acid from rat neostriatum induced by K +-depolarization (60mM K + in perfusate) has been shown to be inhibited by replacing Ca 3+ in the perfusion medium by Co 2+. In contrast, release of l-[ 3H]glutamate induced by electrical stimulation of frontal cortex is enhanced by replacement of these cations. Application of dopamine (10 −5–10 −3 M), apomorphine (10 −4 M) or β-phenylethylamine (10 −3 M) as well as stimulation of the substantia nigra enhanced the basal release of l-[ 3H]glutamate. Haloperidol (10 −4 M) completely abolished the effects of apomorphine and β-phenylethylamine and partially abolished the effect of dopamine. The enhancement induced by apomorphine is strongly dependent on the presence of Na + in the perfusion medium. On the other hand, apomorphine (10 −4 M) and β-phenylethylamine (10 −3M) inhibited the release of glutamate induced by electrical stimulation of the frontal cortex and that by K +-depolarization (the latter was shown for apomorphine). This inhibition is also abolished by haloperidol. A possible functional role of endogenous dopamine in the regulation of glutamatergic neurotransmission in rat neostriatum is discussed.

Schistosoma mansoni: Direct method for simultaneous recording of electrical and motor activity

A method utilizing suction electrodes for simultaneous recording of electrical activity and motility of adult male Schistosoma mansoni is described. Spontaneous body contractions, similar to peristaltic waves, occur in an approximately rhythmic manner. Surface electrical potentials are associated with these contractions. Stretch increases the rate of spontaneous contractions. 5-Hydroxytryptamine at a concentration of 10 −6 M increases the contraction rate and also the complexity of the contractions. Carbachol at 10 −5 M decreases the rate at which contractions occur and also decreases the complexity of each contraction burst and at 10 −4 M completely suppresses activity.

Method For Recording Electrical Activity of the Isolated Heart

The simultaneous recording of electrical activity from two sites on an isolated mammalian heart by a simple and effective method is described. The use of agarfilled polyethylene tubing containing cotton wick electrodes offers distinct advantages compared to the usual platinum hook electrodes.

Transmembrane electrical potentials in ventricular tachycardia and fibrillation

Ventricular tachycardia and fibrillation have been induced in the intact ventricle of the guinea pig by the use of aconitine. The micropuncture technique was used to record single fiber electrical activity. Ventricular fibrillation was characterized by irregular action potentials of varying amplitude. Simultaneous recording of electrical activity from two fibers on the ventricular surface revealed partial synchronism of action potentials when the fibers were in close proximity. The synchronism was not evident when the explored fibers were at a distance. The disorganization of guinea pig ventricular fibers during fibrillation was not total. Single fiber electrical alternans was frequently observed during ventricular tachycardia and was associated with mechanical alternans. Mechanical alternans was seen occasionally without demonstrable single fiber electrical alternans. It is suggested that in this circumstance the appropriate fibers were not selected for microelectrode penetration, since it was found that all cells did not participate in electrical alternation in any one preparation. It would appear, therefore, that alternation of the heart, both electrical and mechanical, may be related to alternate variations in behavior of individual fiber membrane and contractile elements.