Glass Microelectrodes Research Articles

Co-activation of nAChR and mGluR induces γ oscillation in rat medial septum diagonal band of Broca slices

To examine whether co-activation of nAChR and mGluR1 induced γ oscillation (20-60 Hz) in rat medial septum diagonal band of Broca (MSDB) slices. Rat brain sagittal slices containing the MSDB were prepared. Extracellular field potentials were recorded with glass microelectrodes. The nAChR and mGluR1 agonists were applied to the slices to induce network activity. Data analysis was performed off-line using software Spike 2. Co-application of the nAChR agonist nicotine (1 μmol/L) and the mGluR1 agonist dihydroxyphenylglycine (DHPG, 25 μmol/L) was able to induce γ oscillation in MSDB slices. The intensity of nAChR and mGluR1 activation was critical for induction of network oscillation at a low (θ oscillation) or high frequency (γ oscillation): co-application of low concentrations of the two agonists only increased the power and frequency of oscillation within the range of θ, whereas γ oscillation mostly appeared when high concentrations of the two agonists were applied. Activation of mGluR1 and nAChR is able to program slow or fast network oscillation by altering the intensity of receptor activation, which may provide a mechanism for modulation of learning and memory.

Alternans in Rabbit Heart During Acute Regional Ischemia: Optical Mapping and Microelectrode Recordings

T-wave alternans during acute ischemia have been determined as a precursor of ventricular arrhythmias usually resulting in sudden cardiac death. Optical mapping is often used to investigate the mechanisms of alternans in heart preparations. Several hypothesis exist that may explain the formation of optical alternans. One of them is that optical action potential amplitude (OAPA) alternans may indicate the formation of alternating conduction blocks. In our study by using optical mapping simultaneously with microelectrode recordings we aimed to investigate the occurrence of OAPA alternans during acute regional ischemia and to determine the electrical origin underlying the alternans formation. Experiments were performed in Langendorff-perfused rabbit hearts stained with near-infrared dye di-4-ANBDQBS. Optical signals were registered from left ventricle epicardium. The hearts were stimulated by applying 300 ms, and 200 ms pacing cycle. Results showed that OAP started alternate with high and low amplitudes at 200 ms pacing at early ischemia (4-6 minutes). The alternation zone migrated, shrank and finally disappeared with increasing ischemia time. In order to elucidate the mechanism of this phenomenon two glass microelectrodes were used to examine electrical activity by registering action potentials from subepicardial and deeper (3-5 mm) myocardial layers simultaneously. Microelectrode recordings revealed that OAPA alternans is the result of 2:1 conduction blocks in a certain layer whilst the regular response is held in the other layers. During later ischemia (10-15 minutes) deep myocardial layers lose their excitability remaining optical signals only with low amplitude. It explains the disappearing of OAPA alternans. Thus, our experimental data show that OAPA alternans which arise during the early acute ischemia can be caused by alternation of conduction blocks in a certain myocardial layers.Research is funded by the European Social Fund under the Global Grant measure.

Multi-unit recording with iridium oxide modified stereotrodes in Drosophila melanogaster

BackgroundDrosophila is a very favorable animal model for the studies of neuroscience. However, it remains a great challenge to employ electrophysiological approaches in Drosophila to study the neuronal assembly dynamics in vivo, partially due to the small size of the Drosophila brain. Small and sensitive microelectrodes for multi-unit recordings are greatly desired. New methodWe fabricated micro-scale stereotrodes for electrical recordings in Drosophila melanogaster. The stereotrodes were modified with iridium oxide (IrO2) under a highly controllable deposition procedure to improve their electrochemical properties. Electrical recordings were carried out using the IrO2 stereotrodes to detect spontaneous action potentials and LFPs in vivo. ResultsThe IrO2 electrodes exhibited significantly higher capacitance and lower impedance at 1kHz. Electrical recording with the IrO2 stereotrodes in vivo demonstrated an average signal-to-noise ratio (SNR) of 7.3 and a significantly improved LFP sensitivity. 5 types of different neurons recorded were clearly separated. Electrophysiological responses to visual and odor stimulation were also detected, respectively. Comparison with existing method(s)The most widely used electrodes for electrical recording in Drosophila are glass microelectrode and sharpened tungsten microelectrode, which are typically used for single-unit recordings. Although tetrode technology has been used to record multi-neuronal activities from Drosophila, the fabricated IrO2 stereotrodes possess smaller geometry size but exhibited comparable recording signal-to noise ration and better sorting quality. ConclusionsThe IrO2 stereotrodes are capable to meet the requirements of multi-unit recording and spike sorting, which will be a useful tool for the electrophysiology-based researches especially in Drosophila and other small animals.

Topographical representations of taste response characteristics in the rostral nucleus of the solitary tract in the rat

The rostral nucleus of the solitary tract (rNST) is the first-order taste relay in rats. This study constructed topographical distributions of taste response characteristics (best-stimulus, response magnitude, and taste-tuning) from spike discharges of single neurons in the rNST. The rNST is divided into four subregions along the rostrocaudal (RC) axis, which include r1-r4. We explored single-neuron activity in r1-r3, using multibarreled glass microelectrodes. NaCl (N)-best neurons were localized to the rostral half of r1-r3, while HCl (H)-best and sucrose (S)-best neurons showed a tendency toward more caudal locations. NaCl and HCl (NH)-best neurons were distributed across r2-r3. The mean RC values and Mahalanobis distance indicated a significant difference between the distributions of N-best and NH-best neurons in which N-best neurons were located more rostrally. The region of large responses to NaCl (net response >5 spikes/s) overlapped with the distribution of N-best neurons. The region of large responses to HCl extended widely over r1-r3. The region of large responses to sucrose was in the medial part of r2. The excitatory region (>1 spike/s) for quinine overlapped with that for HCl. Neurons with sharp to moderate tuning were located primarily in r1-r2, while those with broad tuning were located in r2-r3. The robust responses to NaCl in r1-r2 primarily contributed to sharp to moderate taste-tuning. Neurons in r3 tended to have broad tuning, apparently due to small responses to both NaCl and HCl. Therefore, the rNST is spatially organized by neurons with distinct taste response characteristics, suggesting that these neurons serve different functional roles.

Transmembrane Current Imaging in the Heart during Pacing and Fibrillation

Recently, we described a method to quantify the time course of total transmembrane current (Im) and the relative role of its two components, a capacitive current (Ic) and a resistive current (Iion), corresponding to the cardiac action potential during stable propagation. That approach involved recording high-fidelity (200 kHz) transmembrane potential (Vm) signals with glass microelectrodes at one site using a spatiotemporal coordinate transformation via measured conduction velocity. Here we extend our method to compute these transmembrane currents during stable and unstable propagation from fluorescence signals of Vm at thousands of sites (3 kHz), thereby introducing transmembrane current imaging. In contrast to commonly used linear Laplacians of extracellular potential (Ve) to compute Im, we utilized nonlinear image processing to compute the required second spatial derivatives of Vm. We quantified the dynamic spatial patterns of current density of Im and Iion for both depolarization and repolarization during pacing (including nonplanar patterns) by calibrating data with the microelectrode signals. Compared to planar propagation, we found that the magnitude of Iion was significantly reduced at sites of wave collision during depolarization but not repolarization. Finally, we present uncalibrated dynamic patterns of Im during ventricular fibrillation and show that Im at singularity sites was monophasic and positive with a significant nonzero charge (Im integrated over 10 ms) in contrast with nonsingularity sites. Our approach should greatly enhance the understanding of the relative roles of functional (e.g., rate-dependent membrane dynamics and propagation patterns) and static spatial heterogeneities (e.g., spatial differences in tissue resistance) via recordings during normal and compromised propagation, including arrhythmias.

American Clinical Neurophysiology Society's standardized critical care EEG terminology: Interrater reliability and 2012 version

American Clinical Neurophysiology Society's standardized critical care EEG terminology: Interrater reliability and 2012 version

Electrophysiological integration and action potential properties of transplanted cardiomyocytes derived from induced pluripotent stem cells

Induced pluripotent stem cell-derived cardiomyocytes (iPSCM) are regarded as promising cell type for cardiac cell replacement therapy. We investigated long-term electrophysiological integration and maturation of transplanted iPSCM, which are essential for therapeutic benefit. Murine iPSCM expressing enhanced green fluorescent protein and a puromycin resistance under control of the α-myosin heavy chain promoter were purified by antibiotic selection and injected into adult mouse hearts. After 6-12 days, 3-6 weeks, or 6-8 months, viable slices of recipient hearts were prepared. Slices were focally stimulated by a unipolar electrode placed in host tissue, and intracellular action potentials (APs) were recorded with glass microelectrodes in transplanted cells and neighbouring host tissue within the slices. Persistence and electrical integration of transplanted iPSCM into recipient hearts could be demonstrated at all time points. Quality of coupling improved, as indicated by a maximal stimulation frequency without conduction blocks of 5.77 ± 0.54 Hz at 6-12 days, 8.98 ± 0.38 Hz at 3-6 weeks and 10.82 ± 1.07 Hz at 6-8 months after transplantation. AP properties of iPSCM became more mature from 6-12 days to 6-8 months after transplantation, but still differed significantly from those of host APs. Transplanted iPSCM can persist in the long term and integrate electrically into host tissue, supporting their potential for cell replacement therapy. Quality of electrical integration improves between 6-12 days and 6-8 months after transplantation, and there are signs of an electrophysiological maturation. However, even after 6-8 months, AP properties of transplanted iPSCM differ from those of recipient cardiomyocytes.

The Effect of Temperature Changes on in vitro Slow Wave Activity in the Equine Ileum

AimsTo evaluate the effect of temperature changes on in vitro slow wave activity of the equine ileum using intracellular recording techniques.MethodsA section of ileum was collected immediately following euthanasia from 9 normal horses euthanased for clinical reasons unrelated to the gastrointestinal tract. Intestinal tissue samples were cut into 1 mm thick sections, pinned out on a Sylgard plate and superfused with warmed, oxygenated Krebs solution. Intracellular recordings of membrane potential were made from smooth muscle cells using glass microelectrodes. All experiments were performed in the presence of a calcium channel blocker to ensure stable impalements. The temperature of the tissue bath was altered during the course of the experiment at a range of 27–41°C. All data were recorded and stored using a computer interfaced acquisition system. A software package was used to analyse the resting potentials, the amplitude, frequency and duration of slow waves.ResultsIn all 9 horses slow wave frequency appeared to be approximately linearly related to the temperature over the range studied increasing by 0.5 cycles/min for each 1 degree increase in temperature (P<0.01). The initial slow wave frequency resumed when the temperature was returned to 37°C. The recovery time appeared to be directly related to the duration for which the temperature had been changed.ConclusionsSlow wave frequency in the equine ileum is highly temperature sensitive.Practical significanceAs post operative ileus is a major cause of morbidity and mortality in the horse, the negative effect of lower temperatures on slow wave activity should be considered. During colic surgery close attention should be paid to minimising extra‐abdominal gut exposure time and keeping the temperature of the intestinal and abdominal lavage fluids at body temperature.Ethical animal researchPost mortem samples obtained with the consent of the owners. Sources of funding: The Norwegian Agricultural Agreement Research Fund, Norsk Rikstoto and the Research Council of Norway as part of the Norwegian/Swedish research collaboration. Competing interests: None.

Spontaneous Electrical Activity and Spikes in the Tail of Marine Cercariae.

Spontaneous electrical activity is recorded in two species of marine cercariae, Cryptocotyle lingua and Himasthla elongata, with different types of swimming—by glass microelectrode recordings. Slow local field potentials (sLFPs) of low amplitude and fast high amplitude action potentials (APs) are found. The shape of the sLFPs is different in the species and correlates with the type of swimming. Fast high amplitude APs are recorded for the first time in cercariae. The limited number of APs included in the swimming pattern of larva suggests a key role for the spiking neurons in initiating the motility pattern in the cercaria and needs further research.

Elevation and variability of acidic sandy soil pH: Amended with conditioner, activator, organic and inorganic fertilizers

Availability of nutrients for plant uptake is directly related to soil pH and as an indicator of soil fertility status. Determination of effects of soil amendments on soil pH should be a necessary part of fertilizer and fertility research. A pot experiment was carried out to determine and compare the effects of biochar, chicken manure, urea and zeolite on soil pH variability and elevation in a sandy loam acidic soil. A modified method was used to determine the soil pH in the pots. Soil pH in pot was measured by a glass micro-electrode and spatial variability was interpolated and mapped by using geographic information system (GIS) + software. Kriged Maps clearly showed the presence of variability and elevation in pH within each treatment. Furthermore, the position of patches with maxima and minima values for pH changed between all treatments used in the experiment. The highest elevation was found in zeolite treated soil followed by urea and biochar. However, a significant decrease was measured in soil pH in chicken dung treated soil. These findings could be the first step towards temporal stability of the pattern of spatial distribution of soil pH affected by the soil amendments (biochar, urea, chicken manure and zeolite). Key words: Biochar, soil pH, urea, zeolite, spatial variability.

Neurons of the A5 region are required for the tachycardia evoked by electrical stimulation of the hypothalamic defence area in anaesthetized rats

In order to assess the possible interactions between the pontine A5 region and the hypothalamic defence area (HDA), we have examined the pattern of double staining for c-Fos protein immunoreactivity (c-Fos-ir) and tyrosine hydroxylase, throughout the rostrocaudal extent of the A5 region in spontaneously breathing anaesthetized male Sprague-Dawley rats during electrical stimulation of the HDA. Activation of the HDA elicited a selective increase in c-Fos-ir with an ipsilateral predominance in catecholaminergic and non-catecholaminergic A5 somata (P < 0.001 in both cases). A second group of experiments was done to examine the importance of the A5 region in modulating the cardiorespiratory response evoked from the HDA. Cardiorespiratory changes were analysed in response to electrical stimulation of the HDA before and after ipsilateral microinjection of muscimol within the A5 region. Stimulation of the HDA evoked an inspiratory facilitatory response, consisting of an increase in respiratory rate (P < 0.001) due to a decrease in expiratory time (P < 0.01). The respiratory response was accompanied by a pressor response (P < 0.001) and tachycardia (P < 0.001). After muscimol microinjection within the A5 region, pressor and heart rate responses to HDA stimulation were reduced (P < 0.01 and P < 0.001, respectively). The respiratory response persisted unchanged. Finally, to confirm functional interactions between the HDA and the A5 region, extracellular recordings of putative A5 neurones were obtained during HDA stimulation. Seventy-five A5 cells were recorded, 35 of which were affected by the HDA (47%). These results indicate that neurones of the A5 region participate in the cardiovascular response evoked from the HDA. The possible mechanisms involved in these interactions are discussed.

Frequency tuning of individual auditory receptors in female mosquitoes (Diptera, Culicidae)

The acoustic sensory organs in mosquitoes (Johnston organs) have been thoroughly studied; yet, to date, no data are available on the individual tuning properties of the numerous receptors that convert sound-induced vibrations into electrical signals. All previous measurements of frequency tuning in mosquitoes have been based on the acoustically evoked field potentials recorded from the entire Johnston organ. Here, we present evidence that individual receptors have various frequency tunings and that differently tuned receptors are unequally represented within the Johnston organ. We devised a positive feedback stimulation paradigm as a new and effective approach to test individual receptor properties. Alongside the glass microelectrode technique, the positive feedback stimulation paradigm has allowed us to obtain data on receptor tuning in females from three mosquito species: Anopheles messeae, Aedes excrucians and Culex pipiens pipiens. The existence of individually tuned auditory receptors implies that frequency analysis in mosquitoes may be possible.

The auditory system of blood-sucking mosquito females (Diptera, Culicidae): Acoustic perception during flight simulation

Mosquitoes hear with their plumose antennae which respond to the air movement caused by sound propagation and conduct vibrations to the Johnston’s organ located at the base of each antenna. Each of the two Johnston’s organs contains several tens of thousands mechanosensory cells which detect the displacements of the flagellum and transform them into electric potentials. Hearing plays a very important role in the reproductive behavior of the male mosquitoes. At the same time, our knowledge of hearing in female mosquitoes is very limited and its functional significance is obscure. In this study we measured the auditory sensitivity of female mosquitoes and investigated how the flight conditions affect their hearing. We studied mosquitoes of three species: Anopheles messeae, Aedes excrucians, and Culex pipiens pipiens. The neuronal responses were recorded with a glass microelectrode from the antennal nerve and the deutocerebral interneurons. Stimulation was applied in two modes: (1) the main stimulus against the background of flight simulation (strong vibration with the typical wingbeat frequency of a given mosquito species) and (2) only the main stimulus without the background stimulation. During the flight simulation, females demonstrated an increased sensitivity to frequencies below 200 Hz. The mean auditory receptor threshold at 80–120 Hz was 45 dB, which was 8 dB lower than that without flight simulation. An additional zone of increased sensitivity was also found at frequencies higher than the simulated wingbeat frequency (the so-called image channel). Our analysis of frequency tuning curves measured from the receptors and auditory interneurons shows that mosquito auditory neuronal complex consists of several subsystems which have different frequency tuning parameters, and suggests the possibility of spectral analysis of sounds. Three hypotheses could be proposed on the function of hearing in female mosquitoes: (1) predator avoidance, (2) detection of moving prey, and (3) intraspecific communication. Each of the hypotheses involves the ability to analyze the sound frequency spectrum and subsequent signal recognition.

Experimental Diabetes Impairs Endothelial IKCa Channel Function in Uteroplacental Arteries from Pregnant Rats

Diabetic pregnancy is associated with adverse fetal complications in part due to impaired uteroplacental blood flow. In this study, we explored the role of endothelial cell (EC) SKCa and IKCa channels in impaired uteroplacental vasodilation during diabetic pregnancy. Diabetes was induced by i.p. injection of streptozotocin on 2nd day of pregnancy. We tested the effects of NS309 and CyPPA on arterial diameters, SMC [Ca2+]i and membrane potential using fura 2‐based fluorescence and glass microelectrodes. Whole‐cell patch clamp methodology was used to characterize SKCa and IKCa channel function in isolated ECs of control and diabetic rats. NS309‐induced vasodilation, SMC [Ca2+]i reduction and hyperpolarization were significantly impaired by diabetic pregnancy. Maximal responses to CyPPA were 50–60% smaller than those to NS309, and were not affected by diabetes. NS309 markedly potentiated endothelial K+ currents in ECs from control and diabetic rats. K+ currents were inhibited by appr. 80% and 20% with charybdotoxin (CTX) and apamin, respectively. Only CTX‐sensitive currents were reduced in diabetic ECs (6.5 ± 1.6 vs. 11.1 ± 1.1 pA/pF at 0 mV; n = 7).In summary, IKCa channels play a predominant role in the control of uteroplacental dilatation. Impaired function of IKCa channels contributes to diabetes‐induced endothelial dysfunction in the maternal uteroplacental circulation.Supported by NIH HL088245

Increased 20-HETE Synthesis Explains Reduced Cerebral Blood Flow But Not Impaired Neurovascular Coupling after Cortical Spreading Depression in Rat Cerebral Cortex

Cortical spreading depression (CSD) is associated with release of arachidonic acid, impaired neurovascular coupling, and reduced cerebral blood flow (CBF), caused by cortical vasoconstriction. We tested the hypothesis that the released arachidonic acid is metabolized by the cytochrome P450 enzyme to produce the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE), and that this mechanism explains cortical vasoconstriction and vascular dysfunction after CSD. CSD was induced in the frontal cortex of rats and the cortical electrical activity and local field potentials recorded by glass microelectrodes, CBF by laser Doppler flowmetry, and tissue oxygen tension (tpO(2)) using polarographic microelectrodes. 20-HETE synthesis was measured in parallel experiments in cortical brain slices exposed to CSD. We used the specific inhibitor HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) to block 20-HETE synthesis. CSD increased 20-HETE synthesis in brain slices for 120 min, and the time course of the increase in 20-HETE paralleled the reduction in CBF after CSD in vivo. HET0016 blocked the CSD-induced increase in 20-HETE synthesis and ameliorated the persistent reduction in CBF, but not the impaired neurovascular coupling after CSD. These findings suggest that CSD-induced increments in 20-HETE cause the reduction in CBF after CSD and that the attenuation of stimulation-induced CBF responses after CSD has a different mechanism. We suggest that blockade of 20-HETE synthesis may be clinically relevant to ameliorate reduced CBF in patients with migraine and acute brain cortex injuries.

Monitoring Ion Activities In and Around Cells Using Ion-Selective Liquid-Membrane Microelectrodes

Determining the effective concentration (i.e., activity) of ions in and around living cells is important to our understanding of the contribution of those ions to cellular function. Moreover, monitoring changes in ion activities in and around cells is informative about the actions of the transporters and/or channels operating in the cell membrane. The activity of an ion can be measured using a glass microelectrode that includes in its tip a liquid-membrane doped with an ion-selective ionophore. Because these electrodes can be fabricated with tip diameters that are less than 1 μm, they can be used to impale single cells in order to monitor the activities of intracellular ions. This review summarizes the history, theory, and practice of ion-selective microelectrode use and brings together a number of classic and recent examples of their usefulness in the realm of physiological study.

Quantification of Transmembrane Currents during Action Potential Propagation in the Heart

The measurement, quantitative analysis, theory, and mathematical modeling of transmembrane potential and currents have been an integral part of the field of electrophysiology since its inception. Biophysical modeling of action potential propagation begins with detailed ionic current models for a patch of membrane within a distributed cable model. Voltage-clamp techniques have revolutionized clinical electrophysiology via the characterization of the transmembrane current gating variables; however, this kinetic information alone is insufficient to accurately represent propagation. Other factors, including channel density, membrane area, surface/volume ratio, axial conductivities, etc., are also crucial determinants of transmembrane currents in multicellular tissue but are extremely difficult to measure. Here, we provide, to our knowledge, a novel analytical approach to compute transmembrane currents directly from experimental data, which involves high-temporal (200 kHz) recordings of intra- and extracellular potential with glass microelectrodes from the epicardial surface of isolated rabbit hearts during propagation. We show for the first time, to our knowledge, that during stable planar propagation the biphasic total transmembrane current (Im) dipole density during depolarization was ∼0.25 ms in duration and asymmetric in amplitude (peak outward current was ∼95 μA/cm2 and peak inward current was ∼140 μA/cm2), and the peak inward ionic current (Iion) during depolarization was ∼260 μA/cm2 with duration of ∼1.0 ms. Simulations of stable propagation using the ionic current versus transmembrane potential relationship fit from the experimental data reproduced these values better than traditional ionic models. During ventricular fibrillation, peak Im was decreased by 50% and peak Iion was decreased by 70%. Our results provide, to our knowledge, novel quantitative information that complements voltage- and patch-clamp data.

Optimisation of Ionic Models to Fit Tissue Action Potentials: Application to 3D Atrial Modelling

A 3D model of atrial electrical activity has been developed with spatially heterogeneous electrophysiological properties. The atrial geometry, reconstructed from the male Visible Human dataset, included gross anatomical features such as the central and peripheral sinoatrial node (SAN), intra-atrial connections, pulmonary veins, inferior and superior vena cava, and the coronary sinus. Membrane potentials of myocytes from spontaneously active or electrically paced in vitro rabbit cardiac tissue preparations were recorded using intracellular glass microelectrodes. Action potentials of central and peripheral SAN, right and left atrial, and pulmonary vein myocytes were each fitted using a generic ionic model having three phenomenological ionic current components: one time-dependent inward, one time-dependent outward, and one leakage current. To bridge the gap between the single-cell ionic models and the gross electrical behaviour of the 3D whole-atrial model, a simplified 2D tissue disc with heterogeneous regions was optimised to arrive at parameters for each cell type under electrotonic load. Parameters were then incorporated into the 3D atrial model, which as a result exhibited a spontaneously active SAN able to rhythmically excite the atria. The tissue-based optimisation of ionic models and the modelling process outlined are generic and applicable to image-based computer reconstruction and simulation of excitable tissue.

Acetylcholine- and Sodium Hydrosulfide–Induced Endothelium-Dependent Relaxation and Hyperpolarization in Cerebral Vessels of Global Cerebral Ischemia–Reperfusion Rat

We investigated the effects of endothelium-derived hyperpolarizing factor (EDHF) and the role of hydrogen sulphide (H2S) in the cerebral vasorelaxation induced by acetylcholine (ACh) in global cerebral ischemia-reperfusion (CIR) rats. CIR was induced by occlusion of bilateral carotid and vertebral arteries. Isolated arterial segments from the cerebral basilar (CBA) and middle artery (MCA) of CIR rats were studied in a pressurized chamber. Transmembrane potential was recorded using glass microelectrodes to evaluate hyperpolarization. In the CIR CBAs and MCAs preconstricted by 30 mM KCl, ACh induced concentration-dependent vasorelaxation and hyperpolarization that were partially attenuated by NG-nitro-l-arginine methyl ester (l-NAME, 30 μM) and l-NAME plus indomethacin (10 μM). The residual responses were abolished by the H2S inhibitor dl-propargylglycine (PPG, 100 μM). The H2S donor NaHS and l-Cys, the substrate of endogenous H2S synthase, elicited similar responses to ACh and was inhibited by tetraethylamonine (1 mM) or PPG. ACh induces EDHF-mediated vasorelaxation and hyperpolarization in rat cerebral arteries. These responses are up-regulated by ischemia-reperfusion while NO-mediated responses are down-regulated. Further, the ACh-induced, EDHF-mediated relaxation, and hyperpolarization and the inhibition of these responses are similar to the H2S-induced responses, suggesting that H2S is a possible candidate for EDHF in rat cerebral vessels.

Abstract 9917: Electrical Coupling and Action Potential Properties of Transplanted Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Comparison with Fetal Cardiomyocytes

A functional integration and maturation of transplanted cardiomyocytes prevents arrhythmias and enhances synergistic contractions of transplanted and host tissue. We compared electrical coupling and action potential (AP) properties of transplanted induced pluripotent stem cell-derived cardiomyocytes (iPSCM), which are regarded as promising cell type for cell therapy, and fetal cardiomyocytes (FCM). Methods: Murine iPSCM expressing eGFP and a puromycin resistance under control of the α-MHC promoter, were purified by antibiotic selection and dissociated at day 16 of differentiation. FCM from transgenic mice expressing eGFP under control of the α-actin promoter were isolated at day 13.5-15.5 p.c. IPSCM and FCM were injected into adult mouse hearts (2 injections, ∼500,000 cells per site). 6-12 days after transplantation, recipients were sacrificed and viable ventricular tissue slices (thickness: 150 μ m) were prepared. Slices were focally stimulated by a unipolar electrode placed in host tissue. AP recordings were performed by glass microelectrodes in transplanted cells, which could be identified by their green fluorescence, and in neighboring host tissue within the slices. Results: Coupling of iPSCM and FCM to host tissue could be clearly demonstrated, 74% of iPSCM and 85% of FCM were coupled. Some transplanted iPSCM and FCM had no conduction blocks even at high stimulation frequencies up to 8 Hz, while others had blocks at lower frequencies. The delay between stimulus and AP upstroke was significantly longer in iPSCM (35.34±8.79 ms) than in FCM (19.84±3.77 ms). In host tissue, the delay was 6.96±1.0 ms. IPSCM had immature AP properties at all time points, while FCM matured and possessed comparable AP properties as host cardiomyocytes after 12 days. Conclusions: IPSCM and FCM are able to integrate electrically into host tissue, but conduction blocks and decelerations occur. The quality of integration is higher in FCM than in iPSCM. AP properties of transplanted iPSCM differ considerably from those of host cardiomyocytes up to day 12 after transplantation, while FCM mature to an adult electrophysiological phenotype.