Intracellular Potential Recordings Research Articles

Sensitive and prolonged intracellular electrophysiological recording by three‐dimensional nanodensity regulation

AbstractWith the advancement of micro/nanotechnologies, multielectrodes arrays (MEAs) with different three‐dimensional (3D) micro/nanostructures have been developed to achieve intracellular action potential recording of cardiomyocytes. However, the effect of the 3D micro/nanostructures density on the intracellular recording has not been fully investigated. In this work, 3D tunable nanodensity electrode arrays (TNDEA) are fabricated by hydrothermal synthesis and standard microfabrication to explore the effect of nanodensity regulation on intracellular biosensing. By low‐voltage electroporation, the signal quality of intracellular potentials recorded by the low‐nanodensity TNDEA showed was significantly improved compared to those recorded by the high‐nanodensity TNDEA. The low‐nanodensity TNDEA improved the amplitude (up to 7.7 mV), signal‐to‐noise ratio (SNR) (up to 69.46 dB), recording duration (up to 83 min), and recording yield (∼100%). The 3D nanodensity regulating strategy has achieved sensitive and prolonged intracellular biosensing of action potentials and is expected to be a powerful electrophysiological research tool in the biomedical field.

A biosensing system employing nanowell microelectrode arrays to record the intracellular potential of a single cardiomyocyte

Electrophysiological recording is a widely used method to investigate cardiovascular pathology, pharmacology and developmental biology. Microelectrode arrays record the electrical potential of cells in a minimally invasive and high-throughput way. However, commonly used microelectrode arrays primarily employ planar microelectrodes and cannot work in applications that require a recording of the intracellular action potential of a single cell. In this study, we proposed a novel measuring method that is able to record the intracellular action potential of a single cardiomyocyte by using a nanowell patterned microelectrode array (NWMEA). The NWMEA consists of five nanoscale wells at the center of each circular planar microelectrode. Biphasic pulse electroporation was applied to the NWMEA to penetrate the cardiomyocyte membrane, and the intracellular action potential was continuously recorded. The intracellular potential recording of cardiomyocytes by the NWMEA measured a potential signal with a higher quality (213.76 ± 25.85%), reduced noise root-mean-square (~33%), and higher signal-to-noise ratio (254.36 ± 12.61%) when compared to those of the extracellular recording. Compared to previously reported nanopillar microelectrodes, the NWMEA could ensure single cell electroporation and acquire high-quality action potential of cardiomyocytes with reduced fabrication processes. This NWMEA-based biosensing system is a promising tool to record the intracellular action potential of a single cell to broaden the usage of microelectrode arrays in electrophysiological investigation.

Considerations and recent advances in nanoscale interfaces with neuronal and cardiac networks.

Nanoscale interfaces with biological tissue, principally made with nanowires (NWs), are envisioned as minimally destructive to the tissue and as scalable tools to directly transduce the electrochemical activity of a neuron at its finest resolution. This review lays the foundations for understanding the material and device considerations required to interrogate neuronal activity at the nanoscale. We first discuss the electrochemical nanoelectrode-neuron interfaces and then present new results concerning the electrochemical impedance and charge injection capacities of millimeter, micrometer, and nanometer scale wires with Pt, PEDOT:PSS, Si, Ti, ITO, IrO x , Ag, and AgCl materials. Using established circuit models for NW-neuron interfaces, we discuss the impact of having multiple NWs interfacing with a single neuron on the amplitude and temporal characteristics of the recorded potentials. We review state of the art advances in nanoelectrode-neuron interfaces, the standard control experiments to investigate their electrophysiological behavior, and present recent high fidelity recordings of intracellular potentials obtained with ultrasharp NWs developed in our laboratory that naturally permeate neuronal cell bodies. Recordings from arrays and individually addressable electrically shorted NWs are presented, and the long-term stability of intracellular recording is discussed and put in the context of established techniques. Finally, a perspective on future research directions and applications is presented.

Ultra-Sharp Nanowire Arrays Natively Permeate, Record, and Stimulate Intracellular Activity in Neuronal and Cardiac Networks.

We report innovative scalable, vertical, ultra-sharp nanowire arrays that are individually addressable to enable long-term, native recordings of intracellular potentials. Stable amplitudes of intracellular potentials from 3D tissue-like networks of neurons and cardiomyocytes are obtained. Individual electrical addressability is necessary for high-fidelity intracellular electrophysiological recordings. This study paves the way toward predictive, high-throughput, and low-cost electrophysiological drug screening platforms.

Intracellular cardiomyocytes potential recording by planar electrode array and fibroblasts co-culturing on multi-modal CMOS chip

Intracellular action potential signals reveal enriched physiological information. Patch clamp techniques have been widely used to measure intracellular potential. Despite their high signal fidelity, they suffer from low throughput. Recently, 3D nanoelectrodes have been developed for intracellular potential recording. However, they are limited by scalability, yield, and cost, directly constraining their use in monitoring large number of cells and high throughput applications. In this paper, we demonstrate intracellular potential monitoring of cardiomyocytes using simple 2D planar electrode array in a standard CMOS process without patch clamps or post fabricated 3D nanoelectrodes. This is enabled by our unique cardiomyocytes/fibroblasts co-culturing technique and electroporation. The co-cultured fibroblasts promote tight sealing of cardiomyocytes on electrodes and enable high-fidelity intracellular potential monitoring based on 2D planar electrode. Compared to existing technologies, our platform has a unique potential to achieve an unprecedented combination of throughput, spatiotemporal resolution, and a tissue-level field-of-view for cellular electrophysiology monitoring.

3D Nanoelectrodes for Electrophysiology: How Size Effects Seal Resistance

Three-dimensional (3D) nanoelectrodes fabricated on standard multielectrode array architecture have proven useful in monitoring intracellular electrophysiology of cardiomyocytes. The electrodes’ mechanism of action involves a voltage pulse at the electrode which exceeds the dielectric breakdown of an engulfing cell's membrane, electroporating the cell and dramatically reducing the impedance to intracellular potential recording. The electroporation mechanism is non-invasive as the pores reseal after such recording events. This gives rise to a method which is useful in its ability to multiplex, its technical ease of use, and non-invasiveness.The driving phenomenon which gives this technological gain over planar electrodes is an enhancement of the seal resistance between the electrogenic center (the cell) and the recording center (the nanoelectrodes). Cells in vitro engulf 3D electrodes such that the membrane-electrode distance is much reduced compared to that of the membrane-planar electrode distance. This membrane-electrode distance is dependent upon the geometry of the electrode, but the electrical ramifications of this have not been characterized to date. Herein we explore this dependence by using electrochemical impedance spectroscopy in correlation with reduced-artifact FIB/SEM and electrophysiological measurement of HL-1 cardiomyocytes over an order of magnitude in 3D electrode diameter.

Inhibitory Regulation of Responses of Glutamate Receptors in Frog Motoneurons

Experiments on isolated frog spinal cords (Rana ridibunda) with intracellular recording of potentials from lumbar motoneurons were performed to study interactions between the effects of exogenous excitatory (glutamate and its agonists NMDA, AMPA, kainate) and inhibitory (GABA, glycine) amino acids. Application of glycine and GABA induced reductions in responses to application of glutamate to a mean of 45.8 ± 2.9% (n = 12) and 67.8 ± 3.9% (n = 16), responses to AMPA to 13.9 ± 4.3% (n = 9) and 32.1 ± 8.3% (n = 12), responses to KA to 36.8 ± 8.2% (n = 7) and 48.0 ± 11.8% (n = 6), responses to NMDA to 7.7 ± 3.5% (n = 9) and 18.1 ± 3.8% (n = 14) of control baseline levels respectively. Similar patterns were seen on sequential application of excitatory amino acids (EAA) after glycine and GABA and on application of a mixture of GABA and glycine with an EAA. The specific glycine receptor antagonist strychnine blocked the effect of suppression of EAA responses on the background of glycine but not GABA administration, while the GABAA receptor blocker gabazine decreased the effect of suppression of EAA responses induced by application of GABA. These results point to a difference in the inhibitory influences on excitatory reactions mediated by different types of glutamate receptors and particularly the predominance of suppression of excitatory reactions mediated by NMDA receptors and the relatively weak suppression of responses mediated by kainate receptors. The inhibitory effect of glycine was about twice that of GABA (at equal concentrations).

Organotypic slice culture from human adult ventricular myocardium

Cardiovascular research requires complex and functionally intact experimental models. Due to major differences in the cellular and subcellular composition of the myocardium between species, the use of human heart tissue is highly desirable. To enhance the experimental use of the human myocardium, we established methods for the preparation of vital tissue slices from the adult ventricular myocardium as well as conditions for their long-term preservation in organotypic culture. Human ventricular heart samples were derived from surgical specimens excised during a therapeutic Morrow myectomy and cut into 300 μm thick slices. Slices were either characterized in acute experiments or cultured at a liquid-air interface. Viability and functionality were proven by viability staining, enzyme activity tests, intracellular potential recordings, and force measurements. Precision-cut slices showed high viability throughout 28 days in culture and displayed typical cardiomyocyte action potential characteristics, which enabled pharmacological safety testing on the rapid component of the delayed rectifier potassium current (I(Kr)) and ATP-dependent potassium channels throughout the whole culture period. Constant expression of major ion channels was confirmed by quantitative PCR. Acute slices developed excitation-dependent contractions with a clear preload dependency and a β-adrenergic response. Contractility and myosin light chain expression decreased during the first days in culture but reached a steady state with reactivity upon β-adrenergic stimulation being preserved. Organotypic heart slices represent a multicellular model of the human myocardium and a novel platform for studies ranging from the investigation of molecular interactions to tissue engineering.

Вплив похідних 1,5-бензодіазепіну на електричну активність нейронів Helix albescens Rossm

Using a standard technique for intracellular recording of potentials, we investigated the influence of two new derivatives of 1,5-benzodiazepine-2-one on the electrical activity of neurons of the Helix albescens Rossm. These substances possess neurotropic properties, the effects were concentration dependent in the concentration range 10(-6) M -10(-2) and did not depend on the type of neurons. We defined the threshold, optimum and toxic concentrations for the compounds investigared. Analysis of the total ion current showed that the compounds reduce the rate of inward Na+- and Ca2+ currents and of outward K+-currents.

Differences in the Activation of Inhibitory Motoneuron Receptors in the Frog Rana Ridibunda by GABA and Glycine and Their Interaction

Intracellular recording of potentials was used in isolated spinal cord segments from the frog Rana ridibunda to compare the inhibitory effects of GABA and glycine on the motoneuron membrane. At equal concentrations, the response (a change in membrane potential) to application of glycine was 1.5-2 times greater than the response to GABA in terms of amplitude, and EC(50) values were 0.75 and 1.57 mM, respectively. The response to simultaneous application of GABA and glycine averaged 79.1 +/- 2.4% (n = 19) of the sum of the individual responses and 130.1 +/- 1.5% (n = 19) of the glycine response (partial occlusion). Preliminary application of glycine decreased the GABA response by 85.3 +/- 0.2% (n = 10), while preapplication of GABA decreased the glycine response by only 52.9 +/- 0.3% (n = 11). The glycine and GABA responses were specifically suppressed by strychnine and bicuculline. These results provide evidence that as in mammals, amphibian motoneurons have both glycine (predominantly) and GABA(A) receptors; they also show that asymmetrical cross inhibition can occur.

Structural remodelling of the sinoatrial node in obese old rats

During ageing, the function of sinoatrial node (SAN), the pacemaker of the heart, declines, and the incidence of sick sinus syndrome increases markedly. The aim of the study was to investigate structural and functional remodelling of the SAN during ageing. Rats, 3 and 24 months old (equivalent to young adult and ∼69-year-old humans), were studied. Extracellular potential recording from right atrial preparations showed that (as expected) the intrinsic heart rate was slower in the old animals. It also showed a shift of the leading pacemaker site towards the inferior vena cava in the old animals. Consistent with this, intracellular potential recording showed that slow pacemaker action potentials were more widespread and extended further towards the inferior vena cava in old animals. Immunohistochemistry demonstrated that SAN tissue expressing HCN4, but lacking the expression of Nav1.5 (lack of Nav1.5 explains why pacemaker action potential is slow), was also more widespread and extended further towards the inferior vena cava in the old animals. Immunolabelling of caveolin3 (expressed in cell membrane of cardiac myocytes) demonstrated that there was a hypertrophy of the SAN cells in the old animals. Histology, quantitative PCR, and immunohistochemistry revealed evidence of a substantial age-dependent remodelling of the extracellular matrix (e.g. ∼79% downregulation of genes responsible for collagens 1 and 3 and ∼52% downregulation of gene responsible for elastin). It is concluded that the age- (and/or obesity-) dependent decline in SAN function is associated with a structural remodelling of the SAN: an enlargement of the SAN, a hypertrophy of the SAN cells, and a remodelling of the extracellular matrix.

Ricardo Miledi and the foundations of synaptic and extra‐synaptic neurotransmitter receptor physiology

Ricardo Miledi and the foundations of synaptic and extra‐synaptic neurotransmitter receptor physiology

Mechanisms of electrical coupling between pyramidal cells.

Direct electrical coupling between neurons can be the result of both electrotonic current transfer through gap junctions and extracellular fields. Intracellular recordings from CA1 pyramidal neurons of rat hippocampal slices showed two different types of small-amplitude coupling potentials: short-duration (5 ms) biphasic spikelets, which resembled differentiated action potentials and long-duration (>20 ms) monophasic potentials. A three-dimensional morphological model of a pyramidal cell was employed to determine the extracellular field produced by a neuron and its effect on a nearby neuron resulting from both gap junctional and electric field coupling. Computations were performed with a novel formulation of the boundary element method that employs triangular elements to discretize the soma and cylindrical elements to discretize the dendrites. An analytic formula was derived to aid in computations involving cylindrical elements. Simulation results were compared with biological recordings of intracellular potentials and spikelets. Field effects produced waveforms resembling spikelets although of smaller magnitude than those recorded in vitro. Gap junctional electrotonic connections produced waveforms resembling small-amplitude excitatory postsynaptic potentials. Intracellular electrode measurements were found inadequate for ascertaining membrane events because of externally applied electric fields. The transmembrane voltage induced by the electric field was highly spatially dependent in polarity and wave shape, as well as being an order of magnitude larger than activity measured at the electrode. Membrane voltages because of electrotonic current injection across gap junctions were essentially constant over the cell and were accurately depicted by the electrode. The effects of several parameters were investigated: 1) decreasing the ratio of intra to extracellular conductivity reduced the field effects; 2) the tree structure had a major impact on the intracellular potential; 3) placing the gap junction in the dendrites introduced a time delay in the gap junctional mediated electrotonic potential, as well as deceasing the potential recorded by the somatic electrode; and 4) field effects decayed to one-half of their maximum strength at a cell separation of approximately 20 micron. Results indicate that the in vitro measured spikelets are unlikely to be mediated by gap junctions and that a spikelet produced by the electric field of a single source cell has the same waveshape as the measured spikelet but with a much smaller amplitude. It is hypothesized that spikelets are a manifestation of the simultaneous electric field effects from several local cells whose action potential firing is synchronized.

Electrophysiological characterization of a motilin agonist, GM611, on rabbit duodenal smooth muscle.

Effects of motilin and a newly synthesized erythromycin derivative, GM611, on membrane potential and currents of rabbit duodenal smooth muscle have been investigated by intracellular potential recording and whole cell patch-clamp technique and compared with results from contractile experiments. Motilin and GM611 (0.01-100 nM) dose dependently produced slowly sustained depolarizations (half-maximal effective dose = 0.15 and 3.9 nM for motilin and GM611, respectively) but exhibited biphasic effects on spike activities superimposed on slow waves. With small depolarizations, the number of spike discharges increased, whereas larger depolarizations markedly reduced spike amplitude. Motilin-induced (or GM611-induced) depolarization appeared to be associated with the activation of monovalent cation-selective channels, and the reduction in the spike amplitude appeared mainly to be associated with inhibition of voltage-dependent Ca2+ channels. Furthermore, data from patch-clamp experiments suggested that Ca2+ release occurred from heparin-sensitive internal stores upon stimulation of motilin receptors by these agonists. Possible implications of these electrophysiological effects in motilin- or GM611-induced tonic and phasic contractions have been discussed.

Amiridin and tacrine modulation of the activity and plasticity of the cholinoreceptors of neurons of the common snail: phenomenology and mechanisms.

The influence of amiridin and tacrine on the membrane potential, activity, and plasticity of cholinoreceptors was investigated using the methods of recording of intracellular potentials and transmembrane currents in identified RPa3 and LPa3 neurons of the common snail. Amiridin and tacrine (1-100 mumole/liter) do not exert appreciable influences on the membrane potential of the cells. Both compounds modulate the activity of cholinoreceptors, judging from their influence on the inward current induced by local application of the acetylcholine: the duration of the ACh current increases and its amplitude varies biphasically (a short-latency increase is succeeded by a decrease). Amiridin and tacrine intensify the extinction of the ACh current induced by repeated applications of ACh to the soma. The acetylcholinesterase inhibitor, physostigmine, exerts a similar modulatory influence on the ACh current and on its extinction, and also prevents the manifestation of the effects of amiridin and tacrine. Amiridin and physostigmine directly influence the cholinoreceptors and the ionic membrane channels controlled by them by similarly altering the voltage-current characteristics (VAC) of the ACh current, and by shifting the reversal potential of the ACh current in the negative direction, bringing it closer to the equilibrium potential for chloride ions; this may be interpreted as a result of the nonidentical action of these compounds on the various ionic currents controlled by cholinoreceptors. The hypothesis is advanced that the modulatory influence of amiridin, tacrine, and physostigmine on the activity and plasticity of the cholinoreceptors is governed by their direct membrane-cytoplasmic action.

Role of cyclic AMP-induced Cl conductance in aqueous humour formation by the dog ciliary epithelium.

1. The effects of isoprenaline, a forskolin derivative NKH-477, and dibutyryl cyclic AMP (db cyclic AMP) on the membrane potential, conductance and cell volume of the dog non-pigmented ciliary epithelium (NPE) were investigated by intracellular potential recording, nystatin-perforated patch clamp technique and videomicroscopic cytometry. 2. The resting membrane potential of NPE was about -70 mV in physiological saline and was depolarized by isoprenaline in a dose-dependent manner with an ED50 of about 3 nM. This depolarization was competitively antagonized by the beta-adrenoceptor antagonist, timolol (pA2 = ca. 9) and almost completely blocked by the Cl transport blocker, DIDS. 3. In single dissociated NPE cells, 10 microM isoprenaline induced an inward current and caused a concomitant decrease in cell volume. The reversal potential measurement indicated that this inward current was carried mainly by Cl ion. DIDS (10 microM) abolished both the current and cell volume decrease. 4. NKH-477 (10 microM) or db cyclic AMP (1 mM) also induced an inward current together with a cell volume decrease, the properties of which were similar to those caused by isoprenaline. 5. These results suggest that beta-adrenoceptor stimulation in NPE leads to an increased rate of aqueous humour production by increasing Cl- efflux via an elevation of cyclic AMP and this effect is efficiently blocked by timolol.

Telescience testbed experiments for biomedical studies: Fertilization potential recording of amphibian eggs using tele-manipulation under stereoscopic vision

The telescience testbed experiments were carried out to test and investigate the tele-manipulation techniques in the intracellular potential recording of amphibian eggs. Implementation of telescience testbed was set up in the two separated laboratories of the Tsukuba Space center of NASDA, which were connected by tele-communication links. Manipulators respective for a microelectrode and a sample stage of microscope were moved by computers, of which command signals were transmitted from a computer in a remote control room. The computer in the control room was operated by an investigator (PI) who controlled the movement of each manipulator remotely. A stereoscopic vision of the microscope image were prepared by using a head mounted display (HMD) and were indispensable to the intracellular single cell recording. The fertilization potential of amphibian eggs was successfully obtained through the remote operating system.

Calcium channels and GABA receptors in the early embryonic chick retina.

The properties of calcium channels were studied at the period of neurogenesis in the early embryonic chick retina. The whole neural retina was isolated from embryonic day 3 (E3) chick and loaded with a Ca(2+)-sensitive fluorescent dye (Fura-2). The retinal cells were depolarized by puff application of high-K+ solutions. Increases in intracellular Ca2+ concentrations were evoked by the depolarization through calcium channels. The type of calcium channel was identified as L-type by the sensitivity to dihydropyridines. The Ca2+ response was completely blocked by 10 microM nifedipine, whereas it was remarkably enhanced by 5 microM Bay K 8644. Then we sought a factor to activate the calcium channel and found that GABA could activate it by membrane depolarization at the E3 chick retina. Puff application of 100 microM GABA raised intracellular Ca2+ concentrations, and this Ca2+ response to GABA was also sensitive to the two dihydropyridines. Intracellular potential recordings verified clear depolarization by bath-applied 100 microM GABA. The Ca2+ response to GABA was mediated by GABAA receptors, since the GABA response was blocked by 10 microM bicuculline or 50 microM picrotoxin, and mimicked by muscimol but not by baclofen. Neither glutamate, kainate, nor glycine evoked any Ca2+ response. We conclude that L-type calcium channels and GABAA receptors are already expressed before differentiation of retinal cells and synapse formation in the chick retina. A possibility is proposed that GABA might act as a trophic factor by activating L-type calcium channels via GABAA receptors during the early period of retinal neurogenesis.

Transcellular ionic currents studied by intracellular potential recordings in Neurospora crassa hyphae Transfer of energy from proximal to apical cells

Membrane potentials, input resistances, and electric coupling in the apical parts of N.crassa growing hyphae were recorded with the aid of intracellular microelectrodes. It was revealed that the apical cells were always depolarized by 10 to 30 mV as compared to the adjacent proximal cells. The septal pore maintained an electrical resistance of 4 to 6 MΩ. The calculated values of the endogenous electrical current passing through the septal pore varied between 0.5 and 1 nA. Electrical isolation of the apical cells resulted in their depolarization from 120–150 mV to 40–60 mV, characteristics of the membrane potential value of N.crassa adult hyphae with completely blocked electrogenic pumps. A simultaneous increase in the input resistance value from 15–20 MΩ to 40–80 MΩ was observed. The above data can be explained assuming that H +-ATPase activity was greatly lowered in the apical cells. Thus in the intact hyphae with electrically coupled cells energy is transferred from the proximal hyphal compartments to the apical ones.

Intracellular potentials of microperfused human sweat duct cells

Intracellular potentials of cells from isolated segments of microperfused human sweat ducts were measured in order to determine the electrical profiles of these cells under resting, transporting, and inhibited conditions. Even though the cells are relatively small (ca. 6-8 microns), continuous recordings of intracellular potentials from the same impalement were stable for up to 2 h. In the resting condition in normal Ringer's solution when the lumen of the duct was collapsed and not perfused, the intracellular potential measured across the basal membrane was 34.6 +/- 1.5 mV (n = 31; mean +/- SE). In the same bathing medium, when the duct lumen was also perfused with normal Ringer's solution, the basolateral membrane potential (Vb), the apical membrane potential (Va) and transepithelial potential (Vt) was -33.8 +/- 0.47 mV, -23.7 +/- 0.48 mV and -9.6 +/- 0.9 mV (n = 73), respectively. The average input impedence (Ri) of these cells was 19.6 +/- 0.4 M omega (n = 36). The frequency distribution of Vb was unimodal suggesting that only one functional cell type exists in this tissue. Amiloride (0.1 mM) in the lumen hyperpolarized both Va and Vb by -40.5 +/- 3.6 mV and -33.2 +/- 3.7 mV (n = 15), respectively, with a slight but significant increase in Ri(15%) while abolishing Vt. Removing luminal Cl- depolarized Va by +37.0 +/- 4.2 mV and hyperpolarized Vb by -19.0 +/- 4.2 mV (n = 11). Removing Cl- from the bath hyperpolarized Va by -3.3 +/- 2.3 mV and depolarized Vb by +24.3 +/- 2.7 mV (n = 15).(ABSTRACT TRUNCATED AT 250 WORDS)