scholarly journals Redox artifacts in electrophysiological recordings

2013 ◽  
Vol 304 (7) ◽  
pp. C604-C613 ◽  
Author(s):  
Jonathan M. Berman ◽  
Mouhamed S. Awayda
Keyword(s):  
Voltage Clamp ◽  
Membrane Conductance ◽  
Chloride Ions ◽  
Experimental Conditions ◽  
Chemical Reagents ◽  
Electrophysiological Recordings ◽  
Electrode Voltage ◽  
Electrophysiological Studies ◽  
Increased Sensitivity ◽  
Chloride Deposition

Electrophysiological techniques make use of Ag/AgCl electrodes that are in direct contact with cells or bath. In the bath, electrodes are exposed to numerous experimental conditions and chemical reagents that can modify electrode voltage. We examined voltage offsets created in Ag/AgCl electrodes by exposure to redox reagents used in electrophysiological studies. Voltage offsets were measured in reference to an electrode separated from the solution by an agar bridge. The reducing reagents Tris-2-carboxyethly-phosphine, dithiothreitol (DTT), and glutathione, as well as the oxidizing agent H2O2used at experimentally relevant concentrations reacted with Ag in the electrodes to produce voltage offsets. Chloride ions and strong acids and bases produced offsets at millimolar concentrations. Electrolytic depletion of the AgCl layer, to replicate voltage clamp and sustained use, resulted in increased sensitivity to flow and DTT. Offsets were sensitive to electrode silver purity and to the amount and method of chloride deposition. For example, exposure to 10 μM DTT produced a voltage offset between 10 and 284 mV depending on the chloride deposition method. Currents generated by these offsets are significant and dependent on membrane conductance and by extension the expression of ion channels and may therefore appear to be biological in origin. These data demonstrate a new source of artifacts in electrophysiological recordings that can affect measurements obtained from a variety of experimental techniques from patch clamp to two-electrode voltage clamp.

Characterization of Transport Activity of SLC11 Transporters in Xenopus laevis Oocytes by Fluorophore Quenching

2021 ◽  
pp. 247255522110041
Author(s):  
Raffaella Cinquetti ◽  
Francesca Guia Imperiali ◽  
Salvatore Bozzaro ◽  
Daniele Zanella ◽  
Francesca Vacca ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Voltage Clamp ◽  
Expression System ◽  
Peptide Transporter ◽  
Xenopus Laevis Oocytes ◽  
Substrate Uptake ◽  
Transport Activity ◽  
Experimental Conditions ◽  
Metal Transporters ◽  
Electrode Voltage

Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. Xenopus laevis oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake. A two-electrode voltage clamp is suitable only for electrogenic proteins, and uptake measurements require the existence of radiolabeled substrates and adequate laboratory facilities. Recently, Dictyostelium discoideum Nramp1 and NrampB were characterized using multidisciplinary approaches. NrampB showed no measurable electrogenic activity, and it was investigated in Xenopus oocytes by acquiring confocal images of the quenching of injected fluorophore calcein. This method is adequate to measure the variation in emitted fluorescence, and thus transporter activity indirectly, but requires long experimental procedures to collect statistically consistent data. Considering that optimal expression of heterologous proteins lasts for 48–72 h, a slow acquiring process requires the use of more than one batch of oocytes to complete the experiments. Here, a novel approach to measure substrate uptake is reported. Upon injection of a fluorophore, oocytes were incubated with the substrate and the transport activity measured, evaluating fluorescence quenching in a microplate reader. The technique permits the testing of tens of oocytes in different experimental conditions simultaneously, and thus the collection of significant statistical data for each batch, saving time and animals. The method was tested with different metal transporters (SLC11), DMT1, DdNramp1, and DdNrampB, and verified with the peptide transporter PepT1 (SLC15). Comparison with traditional methods (uptake, two-electrode voltage clamp) and with quenching images acquired by fluorescence microscopy confirmed its efficacy.

scholarly journals SpikeForest, reproducible web-facing ground-truth validation of automated neural spike sorters

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jeremy Magland ◽  
James J Jun ◽  
Elizabeth Lovero ◽  
Alexander J Morley ◽  
Cole Lincoln Hurwitz ◽  
...  
Keyword(s):  
Ground Truth ◽  
Brain Regions ◽  
Optimal Choice ◽  
Spike Sorting ◽  
Experimental Conditions ◽  
Total Size ◽  
Sorting Algorithms ◽  
Electrophysiological Recordings ◽  
Wide Range ◽  
Electrophysiological Studies

Spike sorting is a crucial step in electrophysiological studies of neuronal activity. While many spike sorting packages are available, there is little consensus about which are most accurate under different experimental conditions. SpikeForest is an open-source and reproducible software suite that benchmarks the performance of automated spike sorting algorithms across an extensive, curated database of ground-truth electrophysiological recordings, displaying results interactively on a continuously-updating website. With contributions from eleven laboratories, our database currently comprises 650 recordings (1.3 TB total size) with around 35,000 ground-truth units. These data include paired intracellular/extracellular recordings and state-of-the-art simulated recordings. Ten of the most popular spike sorting codes are wrapped in a Python package and evaluated on a compute cluster using an automated pipeline. SpikeForest documents community progress in automated spike sorting, and guides neuroscientists to an optimal choice of sorter and parameters for a wide range of probes and brain regions.

Ability of sat-1 to transport sulfate, bicarbonate, or oxalate under physiological conditions

2009 ◽  
Vol 297 (1) ◽  
pp. F145-F154 ◽  
Author(s):  
Wolfgang Krick ◽  
Nina Schnedler ◽  
Gerhard Burckhardt ◽  
Birgitta C. Burckhardt
Keyword(s):  
Basolateral Membrane ◽  
Physiological Role ◽  
Experimental Conditions ◽  
Simultaneous Application ◽  
Proximal Tubule Cells ◽  
Sulfate Anion ◽  
Electrode Voltage ◽  
Electrophysiological Studies ◽  
Sat 1

Tubular reabsorption of sulfate is achieved by the sodium-dependent sulfate transporter, NaSi-1, located at the apical membrane, and the sulfate-anion exchanger, sat-1, located at the basolateral membrane. To delineate the physiological role of rat sat-1, [35S]sulfate and [14C]oxalate uptake into sat-1-expressing oocytes was determined under various experimental conditions. Influx of [35S]sulfate was inhibited by bicarbonate, thiosulfate, sulfite, and oxalate, but not by sulfamate and sulfide, in a competitive manner with Ki values of 2.7 ± 1.3 mM, 101.7 ± 9.7 μM, 53.8 ± 10.9 μM, and 63.5 ± 38.7 μM, respectively. Vice versa, [14C]oxalate uptake was inhibited by sulfate with a Ki of 85.9 ± 9.5 μM. The competitive type of inhibition indicates that these compounds are most likely substrates of sat-1. Physiological plasma bicarbonate concentrations (25 mM) reduced sulfate and oxalate uptake by more than 75%. Simultaneous application of sulfate, bicarbonate, and oxalate abolished sulfate as well as oxalate uptake. These data and electrophysiological studies using a two-electrode voltage-clamp device provide evidence that sat-1 preferentially works as an electroneutral sulfate-bicarbonate or oxalate-bicarbonate exchanger. In kidney proximal tubule cells, sat-1 likely completes sulfate reabsorption from the ultrafiltrate across the basolateral membrane in exchange for bicarbonate. In hepatocytes, oxalate extrusion is most probably mediated either by an exchange for sulfate or bicarbonate.

Effects of vasoactive intestinal contractor on voltage-activated Ca2+ currents in feline parasympathetic neurons

1993 ◽  
Vol 265 (6) ◽  
pp. G1158-G1168 ◽  
Author(s):  
T. Nishimura ◽  
J. Krier ◽  
T. Akasu
Keyword(s):  
Voltage Clamp ◽  
Outward Current ◽  
Membrane Conductance ◽  
Receptor Subtypes ◽  
Current Clamp ◽  
External Application ◽  
Parasympathetic Ganglia ◽  
Electrode Voltage ◽  
Etb Receptor

Intracellular current-clamp and single-electrode voltage-clamp techniques were used to study in vitro action potentials and the action of vasoactive intestinal contractor (VIC; 0.03-1 microM) on the high-voltage-activated Ca2+ currents (ICa) of neurons in feline colonic parasympathetic ganglia. In the current-clamp recording mode, action potential amplitude was depressed by cobalt (1 mM) and omega-conotoxin (300 nM) or in nominally Ca(2+)-free Krebs solutions. In the single-electrode voltage-clamp recording mode, the ICa was isolated by blocking the voltage-gated Na+ current with tetrodotoxin (1-3 microM) and by Krebs solutions containing a low Na+ concentration. The voltage-activated K+ currents were blocked by intracellular injection of cesium through a recording electrode filled with 2 M CsCl and external application of tetraethylammonium (30-50 mM) and barium (2 mM). The Ca(2+)-dependent Cl- current was blocked by replacement of Ca2+ (2 mM) with equimolar barium. Anomalous rectification was blocked by external application of 2 mM cesium. The ICa was evoked by depolarizing step commands more positive than -40 mV from holding potentials ranging between -80 and -60 mV. ICa was depressed by cobalt (1 mM), cadmium (100 microM), and omega-conotoxin (500 nM) but not by nifedipine (10 microM), nicardipine (10 microM), and verapamil (10 microM). BAY K 8644 (3-10 microM) also did not affect the ICa. VIC (0.1-1 microM), one of the endothelin (ET) isopeptides, caused an inward current followed by an outward current. The VIC-induced inward and outward currents were associated with an increase and decrease in membrane conductance, respectively. VIC also caused an initial depression followed by a long-lasting augmentation of the ICa. ET-1, ET-2, and ET-3 equally mimicked the action of VIC on both holding current and ICa. These data suggest that VIC activates a receptor-operated channel and modulates the omega-conotoxin-sensitive voltage-activated Ca2+ channels through ETB receptor subtypes of neurons in feline colonic parasympathetic ganglia.

scholarly journals SpikeForest: reproducible web-facing ground-truth validation of automated neural spike sorters

2020 ◽  
Author(s):  
Jeremy F. Magland ◽  
James J. Jun ◽  
Elizabeth Lovero ◽  
Alexander J. Morley ◽  
Cole L. Hurwitz ◽  
...  
Keyword(s):  
Ground Truth ◽  
Brain Regions ◽  
Optimal Choice ◽  
Spike Sorting ◽  
Experimental Conditions ◽  
Total Size ◽  
Electrophysiological Recordings ◽  
Wide Range ◽  
Electrophysiological Studies ◽  
Synthetic Datasets

AbstractSpike sorting is a crucial but time-intensive step in electrophysiological studies of neuronal activity. While there are many popular software packages for spike sorting, there is little consensus about which are the most accurate under different experimental conditions. SpikeForest is an open-source and reproducible software suite that benchmarks the performance of automated spike sorting algorithms across an extensive, curated database of electrophysiological recordings with ground truth, displaying results interactively on a continuously-updating website. With contributions from over a dozen participating laboratories, our database currently comprises 650 recordings (1.3 TB total size) with around 35,000 ground-truth units. These data include extracellular recordings paired with intracellular voltages, state-of-the-art simulated recordings, and hybrid synthetic datasets. Ten of the most frequently used modern spike sorting codes are wrapped under a common Python framework and evaluated on a compute cluster using an automated pipeline. SpikeForest validates and documents community progress in automated spike sorting, and guides neuroscientists to an optimal choice of sorter and parameters for a wide range of probes and brain regions.

scholarly journals Software-guided microscale flow calorimeter for efficient acquisition of thermokinetic data

2021 ◽  
Author(s):  
Timothy Aljoscha Frede ◽  
Marlene Dietz ◽  
Norbert Kockmann
Keyword(s):  
Design Of Experiments ◽  
Process Development ◽  
Reaction Conditions ◽  
Experimental Conditions ◽  
Flow Calorimeter ◽  
Microscale Flow ◽  
Increased Sensitivity ◽  
Important Time ◽  
Optimal Reaction

AbstractFast chemical process development is inevitably linked to an optimized determination of thermokinetic data of chemical reactions. A miniaturized flow calorimeter enables increased sensitivity when examining small amounts of reactants in a short time compared to traditional batch equipment. Therefore, a methodology to determine optimal reaction conditions for calorimetric measurement experiments was developed and is presented in this contribution. Within the methodology, short-cut calculations are supplemented by computational fluid dynamics (CFD) simulations for a better representation of the hydrodynamics within the microreactor. This approach leads to the effective design of experiments. Unfavourable experimental conditions for kinetics experiments are determined in advance and therefore, need not to be considered during design of experiments. The methodology is tested for an instantaneous acid-base reaction. Good agreement of simulations was obtained with experimental data. Thus, the prediction of the hydrodynamics is enabled and the first steps towards a digital twin of the calorimeter are performed. The flow rates proposed by the methodology are tested for the determination of reaction enthalpy and showed that reasonable experimental settings resulted. Graphical abstract A methodology is suggested to evaluate optimal reaction conditions for efficientacquisition of kinetic data. The experimental design space is limited by thestepwise determination of important time scales based on specified input data.

scholarly journals Extracellular Hco3− Dependence of Electrogenic Na/Hco3 Cotransporters Cloned from Salamander and Rat Kidney

2000 ◽  
Vol 115 (5) ◽  
pp. 533-546 ◽  
Author(s):  
Irina I. Grichtchenko ◽  
Michael F. Romero ◽  
Walter F. Boron
Keyword(s):  
Membrane Potential ◽  
Voltage Clamp ◽  
Xenopus Oocytes ◽  
Rat Kidney ◽  
Outward Current ◽  
Membrane Vesicles ◽  
Disulfonic Acid ◽  
Ambystoma Tigrinum ◽  
Ph Titration ◽  
Electrode Voltage

We studied the extracellular [HCOabstract 3 −] dependence of two renal clones of the electrogenic Na/HCO3 cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔVm) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract 3 −/CO2 (0.33–99 mM HCOabstract 3−, pHo 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na+-dependent hyperpolarization (or outward current). In ΔVm experiments, the apparent Km for HCOabstract 3− of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent Km for HCOabstract 3− of rkNBC was less (6.5 mM). Because it has been reported that SOabstract 3=/HSO abstract 3− stimulates Na/HCO3 cotransport in renal membrane vesicles (a result that supports the existence of a COabstract 3= binding site with which SOabstract 3= interacts), we examined the effect of SOabstract 3=/HSO abstract 3− on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract 4= nor 33 mM SOabstract 3 =/HSOabstract 3− substantially affects the apparent Km for HCO abstract 3−. We also used microelectrodes to monitor intracellular pH (pHi) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract 3 −/0.5% CO2. We found that SO abstract 3=/HSOabstract 3 − did not significantly affect the DIDS-sensitive component of the pHi recovery from the initial CO2 -induced acidification. We also monitored the rkNBC current while simultaneously varying [CO2]o, pHo, and [COabstract 3=]o at a fixed [HCOabstract 3−]o of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract 3=]o . However, a pH titration curve nicely fitted the data expressed as current versus pHo. Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract 3 =, HSOabstract 3−, or COabstract 3=.

scholarly journals No Relationship Between Perceived Health Anomalies and Perceived Experimental Success in Retired Breeder Male Hartley Albino Guinea Pigs

2020 ◽  
Author(s):  
Chandra B. Bain ◽  
Julie M. Settlage ◽  
Grace A. Blair ◽  
Steven Poelzing
Keyword(s):  
Guinea Pig ◽  
Hair Loss ◽  
Guinea Pigs ◽  
Ex Vivo ◽  
Perceived Health ◽  
Electrophysiological Recordings ◽  
Electrophysiological Studies ◽  
Monthly Pattern ◽  
Time Of Year ◽  
Whole Heart

ABSTRACTGuinea pigs used in our laboratory for cardiac research sometimes exhibit physical abnormalities. These issues may abate or intensify during the time they are housed in our facility. After using a guinea pig for research, experimentalists note the apparent health of an animal based on visible features and/or abnormal electrophysiology of the heart. There was an existing anecdotal observation that the health of the Guinea Pigs, and subsequently the experimental success rate, had a seasonal variation; therefore we sought to determine if there is a time of year in which our guinea pigs are more likely to be perceived as unhealthy, and whether any determined monthly pattern correlates with an experimentalist’s ability to complete an experimental protocol. An electronic log was created to record the perceived health of the animal and the ability to complete the experiment successfully. Irregular symptoms included, but were not limited to, severe weight or hair loss and irregularities with the heart found post thoracotomy or during baseline electrophysiological recordings of whole-heart preparations. Animals that did not exhibit significant weight or hair loss, or other ailments were considered “healthy”. Overall, our results indicate that there are no monthly variations in perceived Hartley Albino guinea pig health or correlations with experimental completion rates, suggesting mild hair or weight loss that is common when shipping animals may not significantly affect the ability to conduct ex vivo whole-heart electrophysiological studies.

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