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At least 469 records · Page 26Linked to original sources
PubMed1996

Selective carotid angiography during cross-clamping of the descending thoracic aorta in pigs.

OBJECTIVE: To visualize the intra- and extracranial blood vessels during cross-clamping of the descending thoracic aorta. EXPERIMENTAL DESIGN: Experimental animal study. SETTING: Animal laboratory, University Hospital. MATERIALS: Seven pigs. INTERVENTIONS: Cross-clamping of the descending thoracic aorta for 30 minutes. METHOD: Selective carotid angiography was performed before, during and after cross-clamping. Venous filling time of internal- and external jugular veins, diameter of internal and external jugular veins and internal, external and common carotid arteries and contrast intensity were determined. RESULTS: During cross-clamping the blood flow velocity increased as demonstrated by decreased venous filling time and decreased contrast intensity. In addition, the diameter of the external jugular vein increased and the diameter of the internal carotid artery decreased during cross-clamping. No arteriovenous anastomoses could be detected during the experiment. CONCLUSIONS: Our results indicate increased intra- and extracranial blood flow during cross-clamping of the descending thoracic aorta in pigs.

Animals↗
PubMed2006

A novel method for patch-clamp automation.

An increasing demand of the pharmaceutical industry for automated electrophysiological stations for ion channel drug discovery has recently resulted in the development of several commercial platforms for secondary and safety screening of ion channel modulators. These commercial systems have demonstrated an enhanced throughput, however, often at the expense of some quality-sensitive aspects of traditional patch-clamp recordings. To improve data quality and content, we have developed a patch-clamp robot that fully automates manual patch-clamp recordings, including patch pipette handling, gigaseal formation, obtaining whole-cell or perforated-cell configuration, drug application, and data acquisition. Utilization of glass micropipettes results in high-quality electrophysiological recordings with an overall success rate of about 30% in perforated-cell mode. A fast drug application system with low volume requirements (1-1.5 ml) allows the study of ligand-gated ion channels on a millisecond scale. As proof-of-concept, we present two assays developed for voltage-gated human ether-a-go-go-related and ligand-gated alpha(7) nicotinic receptor ion channels. The system throughput was a single concentration-response curve every 30-40 min or 12-17 6-point concentration-response curves daily, representing a significant improvement of typical manual patch-clamp throughput. This system represents an efficient method for patch-clamp automation without the need for a complex and expensive electrophysiological set-up for cell visualization.

Animals↗
PubMedDate not supplied

A single electrode voltage, current- and patch-clamp amplifier with complete stable series resistance compensation.

An input headstage for single electrode voltage and current clamping is described which permits selecting desired operational modes during an experiment. These include cell attached or whole cell patch clamping, single micro-electrode voltage or current clamping, potential recording, iontophoresis and voltammetry. Input electrode series resistance with the electrode inserted in a cell or during whole cell patch clamping can be measured at any time. The unique circuitry allows complete compensation of the series resistance with high frequency response. The input series electrode resistance can also vary considerably around an initially set 100% compensation with intrinsic stability. An accelerator 'supercharger' or 'booster circuit' is included which can shorten the rise time of membrane potential to the command potential by a hundredfold. This improves the recording of early ion channel currents. Feedback resistors are selected remotely for the specific experimental need. Signal resolution in patch clamping is that expected from the thermal noise of the feedback resistor and electrode to membrane seal resistance. This headstage circuitry design thus allows interchangeable modes of operation and parameters during an experiment to obtain optimal conditions for signal detection and frequency response.

Electrodes↗
PubMed2000

Low frequency voltage clamp: recording of voltage transients at constant average command voltage.

We implemented a simple feedback system that modifies the conventional current clamp mode of a patch clamp amplifier so that transient potentials, such as action potentials and synaptic potentials, can be measured as in the usual current clamp, while the average membrane potential is kept constant at a value chosen by the user. The circuit thus works like the current clamp for high frequency signals and like a voltage clamp for low frequency signals. We delineate its transfer properties and give application examples.

Animals↗
PubMed1997

Synaptic excitation in the dorsal nucleus of the lateral lemniscus: whole-cell patch-clamp recordings from rat brain slice.

The synaptic events underlying the excitation of neurons in the rat's dorsal nucleus of the lateral lemniscus were studied by whole-cell patch-clamp recordings in a brain slice preparation of the auditory midbrain. Both current-clamp and voltage-clamp data were obtained with the brain slice submerged in artificial cerebrospinal fluid. The rats were between 21 and 35 days of age at the time the recordings were made. Synaptic responses were evoked by a bipolar stimulating electrode placed on the lateral lemniscus just ventral to the dorsal nucleus. To eliminate glycinergic inhibitory responses, all physiological data were gathered with 0.5 microM strychnine added to the saline bath. Under current-clamp conditions, excitatory postsynaptic potentials could be subdivided into early and late components. The early component produced a single, highly reliable, short-latency spike and the later component produced a more variable, long-latency spike or train of spikes. The non-N-methyl-D-aspartate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, completely blocked the early excitatory postsynaptic potential and its associated action potential. The N-methyl-D-aspartate antagonist, D,L-2-amino-5-phosphonovaleric acid, blocked the later excitatory postsynaptic potential and its action potentials. Typically, both early and late excitatory postsynaptic potentials could be recorded from the same cell, but the early excitatory postsynaptic potential was evoked at lower stimulus levels and had a larger amplitude than the later excitatory postsynaptic potential. Under voltage-clamp conditions, dorsal nucleus of the lateral lemniscus neurons responded to stimulation of the lateral lemniscus with excitatory postsynaptic currents. Outward excitatory postsynaptic currents were recorded with holding potentials that depolarized the cell membrane and inward currents were seen when the cell was hyperpolarized. The current-voltage (I-V) relation of the early peak portion of the excitatory postsynaptic current was nearly linear, whereas the I-V relation of the later excitatory postsynaptic current (12 ms after the peak) was non-linear over the range between -50 and - 100 mV. The outward excitatory postsynaptic current consisted of an early current that was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione and a later current that was blocked by D,L-2-amino-5-phosphonovaleric acid. In artificial cerebrospinal fluid with normal concentrations of Mg2+, the inward excitatory postsynaptic current was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione, but was not affected by D,L-2-amino-5-phosphonovaleric acid. In Mg2+-free artificial cerebrospinal fluid. however, the early component of the inward excitatory postsynaptic current was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione and a later component was blocked by D,L-2-amino-5-phosphonovaleric acid. The results indicate that both N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor-mediated synaptic responses are present in dorsal nucleus of the lateral lemniscus neurons of rats at 21-35 days of age. The N-methyl-D-aspartate component had a longer time-course and a higher threshold than the non-N-methyl-D-aspartate component, and was subject to a voltage-dependent Mg2+ block when the cell's membrane was hyperpolarized. The long-duration N-methyl-D-aspartate component is probably responsible for the prolonged inhibitory effect of dorsal nucleus of the lateral lemniscus neurons on physiological responses in the rat's inferior colliculus.

Action Potentials↗
PubMed2002

Micromolded PDMS planar electrode allows patch clamp electrical recordings from cells.

The patch clamp method measures membrane currents at very high resolution when a high-resistance 'gigaseal' is established between the glass microelectrode and the cell membrane (Pflugers Arch. 391 (1981) 85; Neuron 8 (1992) 605). Here we describe the first use of the silicone elastomer, poly(dimethylsiloxane) (PDMS), for patch clamp electrodes. PDMS is an attractive material for patch clamp recordings. It has low dielectric loss and can be micromolded (Annu. Rev. Mat. Sci. 28 (1998) 153) into a shape that mimics the tip of the glass micropipette. Also, the surface chemistry of PDMS may be altered to mimic the hydrophilic nature of glass (J. Appl. Polym. Sci. 14 (1970) 2499; Annu. Rev. Mat. Sci. 28 (1998) 153), thereby allowing a high-resistance seal to a cell membrane. We present a planar electrode geometry consisting of a PDMS partition with a small aperture sealed between electrode and bath chambers. We demonstrate that a planar PDMS patch electrode, after oxidation of the elastomeric surface, permits patch clamp recording on Xenopus oocytes. Our results indicate the potential for high-throughput patch clamp recording with a planar array of PDMS electrodes.

Coated Materials, Biocompatible↗
PubMed2006

Surrogate indexes vs. euglycaemic-hyperinsulinemic clamp as an indicator of insulin resistance and cardiovascular risk factors in overweight and obese postmenopausal women.

BACKGROUND: There is considerable interest in validating the most convenient method to estimate insulin sensitivity in clinical research protocols that could best indicate cardiovascular risk factors. To address this issue we examined the interrelationships of several cardiovascular risk factors with surrogate indexes such as fasting insulin, the homeostasis model assessment (HOMA), the quantitative insulin sensitivity check index (QUICKI) and the revised QUICKI vs the euglycaemic-hyperinsulinemic (EH) clamp in a non-diabetic overweight or obese postmenopausal female population. DESIGN: Cross-sectional study involving 88 obese postmenopausal women (age: 57.5+/-5.0 yrs; body mass index: 32.52+/-4.4 kg/m2; percent body fat: 46.35+/-4.9%). METHODS: Insulin sensitivity was determined by the EH clamp technique as well as by surrogate indexes such as fasting insulin, HOMA, log HOMA, QUICKI and revised QUICKI. Body composition and body fat distribution were measured using dual energy x-ray absorptiometry and computed tomography, respectively. RESULTS: Correlations between insulin resistance indexes (fasting insulin, revised QUICKI, QUICKI, log HOMA, HOMA) vs glucose disposal were similar (range of r's=0.40 to 0.49), suggesting that no index was superior to another with respect to its relationship with the EH clamp. Correlations between the insulin resistance indexes with plasma lipids were comparable among all indexes, however, systolic blood pressure, visceral fat and C-reactive protein were moderately superior with index vs the EH clamp. CONCLUSION: Surrogate measures of insulin resistance, in particular fasting insulin, are simple tools appropriate for epidemiological studies that can be used as substitutes for the EH clamp to estimate glucose disposal and cardiovascular risk factors in overweight and obese postmenopausal women.

Adipose Tissue↗
PubMed2006

Open-access microfluidic patch-clamp array with raised lateral cell trapping sites.

A novel open-access microfluidic patch-clamp array chip with lateral cell trapping sites raised above the bottom plane of the chip was developed by combining both a microscale soft-lithography and a macroscale polymer fabrication method. This paper demonstrates the capability of using such an open-access fluidic system for patch-clamp measurements. The surface of the open-access patch-clamp sites prepared by the macroscale hole patterning method of soft-state elastic polydimethylsiloxane (PDMS) is examined; the seal resistances are characterized and correlated with the aperture dimensions. Whole cell patch-clamp measurements are carried out with CHO cells expressing Kv2.1 ion channels. Kv2.1 ion channel blocker (TEA) dosage response is characterized and the binding activity is examined. The results demonstrate that the system is capable of performing whole cell measurements and drug profiling in a more efficient manner than the traditional patch-clamp set-up.

Animals↗
PubMed1990

How reliable is the euglycaemic hyperinsulinemic clamp test for the confirmation of autonomous endogenous hyperinsulinemia?

Inhibition of C-Peptide secretion by exogenous insulin was studied during euglycemic clamp in 13 patients with histologically verified causes of organic hyperinsulinaemia (10 with beta cell adenoma; 2 with beta cell carcinoma and 1 with beta cell hyperplasia) and in 10 healthy controls. Euglycemic clamps were performed using artificial endocrine pancreas (Clamp Mode 9:1) while insulin infusion (Humulin Normal-Lilly) rate was 0.1 U/kg BW/h. Blood samples for serum insulin (RIA INEP) and C-Peptide (RIA-Biodata) were taken at 0; 30; 60; 90 and 120 min. Statistical analysis was done using SPSS on IBM-PC with Wilcoxon sum rank test and one way ANOVA. All the patients were studied before the operation and in four of them clamp studies were repeated after the operation. Statistically significant suppression of C-Peptide values in 120 min was established in the control group (p less than 0.05) while there was no significant suppression in insulinoma group (p greater than 0.05), except in one patient with beta cell hyperplasia. Various types of responses (suppression, no change, paradoxical increase) were observed after the operation in the insulinoma group. Possible mechanisms and the meanings of the absence of insulin induced C-Peptide suppression in insulinoma group are discussed. It is concluded that euglycemic hyperinsulinemic clamp study could be useful and a complementary test to other established tests for the confirmation of the diagnosis of insulinoma. Further work on beta cell response after the operation in patients with insulinoma is necessary.

Adenoma↗
PubMed2000

Assessment of insulin sensitivity by 90 min isoglycaemic hyperinsulinaemic glucose clamp in healthy young men.

We aimed to perform a detailed analysis of the isoglycaemic hyperinsulinaemic glucose clamp in relation to the time spent in performing the procedure, and analysed two series performed by independent investigators on different groups (n = 19 and n = 28) of healthy, young men. We calculated glucose disposal rates (GDR) during 20-min periods at different time points during the clamp. There was no difference in 90- and 120-min GDR when comparing the two series. The differences between 90- and 120-min GDR were (mean +/- SD) 0.48 +/- 1.10 mg/kg/min (p = 0.73) and 0.37 +/- 1.05 mg/kg/min (p = 0.71), respectively. The correlations between 90- and 120-min GDR were 0.94 (p < 0.001) and 0.89 (p < 0.001). Correlations between GDR during the second hour of the clamp and fasting plasma insulin ranged from -0.53 (p = 0.020) to -0.55 (p = 0.016) and from -0.44 (p = 0.020) to -0.54 (p = 0.003), respectively, and did not improve after 60 min of clamping. These data suggest that reliable indices of insulin sensitivity in healthy young men may appear even when the isoglycaemic hyperinsulinaemic clamp procedure is shortened from 120 to 90 min. A shorter procedure is time-effective and less expensive, but may be limited to healthy, young Caucasian men.

Adult↗
PubMed2006

Renal glucose excretion as a function of blood glucose concentration in subjects with type 2 diabetes--results of a hyperglycaemic glucose clamp study.

BACKGROUND: The purpose of this study was to investigate renal glucose excretion as a function of blood glucose concentration and to evaluate the within-subject variability and between-subject variability in subjects with type 2 diabetes. METHODS: Twenty-two subjects with type 2 diabetes [age 58 (12) years, diabetes duration 7 (6) years, endogenous creatinine clearance 117 (38) ml min(-1) 1.73 m(-2); median (inter-quartile range, IQR)] underwent two five-period hyperglycaemic glucose clamp experiments at intervals of 7-21 days. Starting from an initial blood glucose level of 12.2 mmol l(-1), subsequent glucose clamp levels were chosen using an algorithm based on urinary glucose concentrations measured at the end of the preceding glucose clamp period. That is, blood glucose was either stepwise decreased or increased depending on whether urinary glucose concentration was above or below 11.1 mmol l(-1), respectively. RESULTS: As expected, increasing the blood glucose from 7.8 to 13.3 mmol l(-1) during the glucose clamps resulted in a steep increase of urinary glucose excretion from 0.06 to 0.77 mmol min(-1). With decreasing blood glucose, a measurable glucosuria persisted up to a blood glucose level of 7.8 mmol l(-1). When defining the (pseudo)threshold for renal glucose excretion (PRT(G)) as the highest blood glucose level during glucose clamps associated with a concomitant glucose concentration in urine of <2.8 mmol l(-1), median (IQR) PRT(G) was 11.0 (1.1) mmol l(-1). The within-subject variability of PRT(G), i.e. the difference between two assessments, was low, 0.1 (0.0) mmol l(-1) while the between-subject variability of PRT(G) was high, ranging from 7.7 to 12.2 mmol l(-1). CONCLUSION: Renal glucose excretion increases in a proportional manner with increasing blood glucose. When decreasing blood glucose to euglycaemic blood glucose levels, glucosuria persists so that the classical concept of a renal threshold for glucose excretion cannot be upheld in subjects with type 2 diabetes.

Adult↗
PubMed1997

The euglycaemic hyperinsulinaemic clamp: an evaluation of current methodology.

1. The recognition of the role of insulin resistance in disease states and the recent development of new drugs that modify insulin-dependent metabolism has led to increased use of the euglycaemic hyperinsulinaemic clamp to measure in vivo insulin sensitivity, but several key aspects of the technique are poorly documented in the literature. 2. We have evaluated the reproducibility and intersubject variation of measurements of insulin sensitivity in groups of insulin-sensitive and insulin-resistant subjects and assessed the effects of hand warning on haemodynamic and metabolic responses. 3. Subjects participated in one of two protocols: (i) 18 healthy male volunteers and 18 patients with hypertension and glucose intolerance were clamped on two occasions, 1 week apart with measurements of insulin sensitivity (M) derived after 120 and 180 min of hyperinsulinaemia; and (ii) six healthy volunteers were clamped on one occasion with simultaneous sampling of antecubital and 'arterialized' (dorsal hand) venous blood for comparison of plasma glucose concentrations and oxygen saturation and a further six volunteers were clamped on two occasions with and without the use of hand warming. 4. Measurements of M derived after 120 min (M120) and 180 min (M180) of hyperinsulinaemia were reproducible: the coefficients of repeatability (mg/kg per min) of M120 and M180 were 1.0 and 0.9 for volunteers and 1.0 and 1.0 for the patient group, respectively. The intersubject variation in insulin stimulus was high: coefficients of variation for M180 were 22% for volunteers compared with 38% for the patient group. In volunteers compared with the patient group, hand warming significantly increased venous oxygen saturations (95 +/- 2 vs 79 +/- 18%, respectively) and glucose concentrations (5.2 +/- 0.2 vs 4.5 +/- 0.4 mmol/L, respectively) and measurements of M were significantly higher using arterialized compared with antecubital venous blood. However, local hand warming was associated with systemic vasodilatation: blood pressure decreased (e.g. 6 mmHg diastolic; P < 0.05) with a compensatory increase in heart rate (8 b.p.m.). 5. In conclusion, clamps of 120 and 180 min duration yielded measurements of M that were reproducible. The technique is much more robust when used in the context of a crossover design because of the significant (20-40%) intersubject variation in M, even among apparently homogeneous male volunteers. Hand warming effectively arterializes venous blood and gives significantly higher M values, but induces systemic vasodilation, which may confound measurements of M.

Adult↗
PubMedDate not supplied

An unbiased glucose clamp method using a variable insulin infusion: its application in diabetic adolescents.

A simple, unbiased insulin-varying glucose clamp program is described. The aim of the program was to utilize a continuously updated array of data to predict insulin requirements for normoglycaemia. In assessing Type 1 diabetes the quantity of insulin required for maintenance of basal euglycaemia can be more clinically informative than other clamp methods. We present a method which uses an iterative computer program to predict changes in insulin infusion rate required for glucose clamping. After initial parameter estimation, the program uses no fixed algorithm but makes predictions according to previous blood glucose responses to infusion rates. The program has flexible data entry, graphic display, and running statistics including mean infusion data, mean glucose levels, and their respective standard deviations. Data for 26 consecutive overnight clamp studies have been analysed. The median coefficient of variation of glucose values at the end of the clamp was 4.1% (range 1.4-12.0%). The mean bias during the last 2 h was 0.20 +/- 0.24 mmol l-1. Cross-correlation showed that insulin had its maximal effect on the rate of decline of glucose after 15 min, and the nadir of glucose occurred 45 min after a change in insulin infusion rate.

Adolescent↗
PubMed1992

The short insulin tolerance test for determination of insulin sensitivity: a comparison with the euglycaemic clamp.

The glucose clamp technique is currently regarded as the standard test for measuring insulin sensitivity against which other methods are compared but is unsuitable for routine screening of patients outside a hospital base. There is thus a need for a simpler test to measure insulin sensitivity. We have therefore compared the glucose disappearance rate KITT in the first 15 min of the insulin tolerance test (ITT) with the M and M/I values derived from the standard euglycaemic clamp in nine normal subjects and eight subjects with Type 2 (non-insulin dependent) diabetes mellitus and coexisting obesity. All subjects underwent the ITT and euglycaemic clamp in random order. Nine subjects later had a repeat ITT to determine the reproducibility of the test. In the ITT, 0.1 U kg-1 body weight, human Actrapid insulin was given as an IV bolus and simultaneous arterialized and venous blood samples were obtained every minute for 15 min. The first order rate constant for the disappearance of glucose KITT over the period 3-15 min was taken as a measure of insulin sensitivity. The euglycaemic clamp was performed with an insulin infusion of 50 mU kg-1 h-1 for 120 min and a variable rate glucose infusion to maintain blood glucose concentration at 0.5 mmol l-1 below fasting level to minimize the effect of endogenous insulin secretion. The ratio of the mean rate of glucose infused (M, mumol kg-1 min-1) to the plasma insulin over the last 30 min of the clamp was taken as a measure of tissue sensitivity to insulin (M/I) assuming endogenous glucose output was suppressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1999

Glucose production during an IVGTT by deconvolution: validation with the tracer-to-tracee clamp technique.

Recently, a new method, based on a two-compartment minimal model and deconvolution [A. Caumo and C. Cobelli. Am. J. Physiol 264 (Endocrinol. Metab. 37): E829-E841, 1993; P. Vicini, G. Sparacino, A. Caumo, and C. Cobelli. Comput. Meth. Prog. Biomed. 52: 147-156, 1997], has been proposed to estimate endogenous glucose production (EGP) from labeled intravenous glucose tolerance test (IVGTT) data. Our aim here is to compare this EGP profile with that independently obtained with the reference method, based on the tracer-to-tracee ratio (TTR) clamp. An insulin-modified (0.03 U/kg body wt infused over 5 min) [6,6-2H2]glucose-labeled IVGTT (0.33 g/kg of glucose) was performed in 10 normal subjects. A second tracer ([U-13C]glucose) was also infused during the test in a variable fashion to clamp endogenous glucose TTR. The TTR clamp was quite successful. As a result, the EGP profile, reconstructed from [U-13C]glucose data with the models of Steele and Radziuk, were almost superimposable. The deconvolution-obtained EGP profile, calculated from [6,6-2H2]glucose data, showed remarkable agreement with that obtained from the TTR clamp. Some differences between the two profiles were noted in the estimated basal EGP and in the initial modalities of EGP inhibition. A high interindividual variability was also observed with both methods in the resumption of EGP to baseline; variability was high in both the timing and the extent of resumption. In conclusion, the use of the two-compartment minimal model of the IVGTT and deconvolution allows the estimation of a profile of EGP that is in very good agreement with that independently obtained with a TTR clamp.

Adult↗
PubMed2003

Implementation of a fast 16-Bit dynamic clamp using LabVIEW-RT.

The dynamic-clamp method provides a powerful electrophysiological tool for creating virtual ionic conductances in living cells and studying their influence on membrane potential. Here we describe G-clamp, a new way to implement a dynamic clamp using the real-time version of the Lab-VIEW programming environment together with a Windows host, an embedded microprocessor that runs a real-time operating system and a multifunction data-acquisition board. The software includes descriptions of a fast voltage-dependent sodium conductance, delayed rectifier, M-type and A-type potassium conductances, and a leak conductance. The system can also read synaptic conductance waveforms from preassembled data files. These virtual conductances can be reliably implemented at speeds < or =43 kHz while simultaneously saving two channels of data with 16-bit precision. G-clamp also includes utilities for measuring current-voltage relations, synaptic strength, and synaptic gain. Taking an approach built on a commercially available software/hardware platform has resulted in a system that is easy to assemble and upgrade. In addition, the graphical programming structure of LabVIEW should make it relatively easy for others to adapt G-clamp for new experimental applications.

Analog-Digital Conversion↗
PubMed1998

Whole cell patch-clamp recordings of rat midbrain dopaminergic neurons isolate a sulphonylurea- and ATP-sensitive component of potassium currents activated by hypoxia.

The effects of brief (2-4 min) hypoxia on presumed dopaminergic "principal" neurons of the rat ventral mesencephalon were investigated by using either intracellular or whole cell patch-clamp recordings in in vitro conditions. Under single-electrode voltage clamp, with sharp microelectrode (Vh -60 mV), a brief hypoxia caused an outward current (hypoOUT) of 110.2 +/- 15.2 (SE) pA (n = 18), which was followed by a posthypoxic outward current (posthypoOUT) of 149.6 +/- 10.6 pA (n = 18). Although the hypoOUT reversed at -83.7 +/- 3.8 mV (n = 18), the posthypoOUT did not reverse. The K+ATP-blocking sulphonylureas tolbutamide (100 microM) and glibenclamide (30 microM), significantly reduced the peak of the hypoOUT by 47.6 +/- 7.7% (n = 16) and 54.18 +/- 7.5% (n = 3), respectively. In contrast, they did not affect the posthypoOUT. Extracellular barium (300 microM to 1 mM) almost abolished the hypoOUT, leaving the posthypoOUT unchanged. The large K+ channel blocker charybdotoxin (10-50 nM), depressed the hypoOUT after tolbutamide treatment. To investigate whether or not cytosolic factors might control the development of the hypoOUT, we dialyzed the principal neurons by patch-clamp recordings (Vh -60 mV). Under whole cell recordings hypoxia evoked an hypoOUT of 70.2 +/- 14.5 pA that reversed polarity at -87.9 +/- 5.1 mV (n = 8). A small posthypoxic response was detected upon reoxygenation in a few neurons (4 out of 14). Three different sulphonylureas, tolbutamide (100 microM), glibenclamide (10-30 microM), and glipizide (100 nM) completely blocked the hypoOUT in patch-clamped neurons. The hypoOUT was also abolished by extracellular BaCl2 (300 microM). When the content of ATP in the dialyzate was raised from 2 to 10 mM no outward current/hyperpolarization was evoked by hypoxia. These data suggest that the hypoOUT, in principal neurons, is a complex response sustained by at least two barium-sensitive components: 1) an ATP-dependent, sulphonylurea-sensitive K+ conductance which could be isolated by the patch-clamp techniques and 2) a K+ conductance remaining after tolbutamide in intracellularly recorded neurons, which is sensitive to charybdotoxin and dependent on dialyzable cytosolic factors.

Adenosine Triphosphate↗
PubMed2006

Population patch clamp improves data consistency and success rates in the measurement of ionic currents.

Present whole-cell patch-clamp methodology has only moderate consistency and throughput, rendering impractical functional measurements on large numbers of ion channel ligands or on large numbers of unknown or mutant channel genes. In the population patch clamp (PPC) described herein, a single voltage-clamp amplifier sums the whole-cell currents from multiple cells at once, each sealed to a separate aperture in a planar substrate well. The resulting ensemble currents are more consistent from well to well, and the success rate for each recording attempt is >95%. The PPC was implemented by modifying the PatchPlate substrate and amplifiers in the IonWorks patch-clamp instrument. The increased data consistency and likelihood of a successful recording in each well, combined with 384-well measurements in parallel, allow the direct electrophysiological recording of thousands of ensemble ionic currents per day. Therapeutic groups in drug discovery programs require this order of throughput to screen directed compound libraries against ion channel targets. The potential for studying the function of large numbers of ion channel mutants may be realized with the technique. The procedure incorporates subtraction methods that correct for expected distortions and also reliably produces data that agree with previous patch-clamp studies.

4-Aminopyridine↗