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A Comparison of the Performance and Application Differences Between Manual and Automated Patch-Clamp Techniques
Send Orders of Reprints at [email protected] Current Chemical Genomics, 2012, 6, 87-92 87 Open Access A Comparison of the Performance and Application Differences Between Manual and Automated Patch-Clamp Techniques Xiao Yajuan, Liang Xin and Li Zhiyuan* Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China Abstract: The patch clamp technique is commonly used in electrophysiological experiments and offers direct insight into ion channel properties through the characterization of ion channel activity. This technique can be used to elucidate the in- teraction between a drug and a specific ion channel at different conformational states to understand the ion channel modu- lators’ mechanisms. The patch clamp technique is regarded as a gold standard for ion channel research; however, it suffers from low throughput and high personnel costs. In the last decade, the development of several automated electrophysiology platforms has greatly increased the screen throughput of whole cell electrophysiological recordings. New advancements in the automated patch clamp systems have aimed to provide high data quality, high content, and high throughput. However, due to the limitations noted above, automated patch clamp systems are not capable of replacing manual patch clamp sys- tems in ion channel research. While automated patch clamp systems are useful for screening large amounts of compounds in cell lines that stably express high levels of ion channels, the manual patch clamp technique is still necessary for study- ing ion channel properties in some research areas and for specific cell types, including primary cells that have mixed cell types and differentiated cells that derive from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). -
Patch-Clamp Study of Y-Aminobutyric Acid Receptor C1
Proc. Natl. Acad. Sci. USA Vol. 85, pp. 9336-9340, December 1988 Neurobiology Patch-clamp study of y-aminobutyric acid receptor C1- channels in cultured astrocytes (glia/benzodiazepine/ion channel) JOACHIM BORMANN*t AND HELMUT KETTENMANNt *Max-Planck-Institut fur biophysikalische Chemie, Am Fassberg, D-3400 Gottingen, Federal Republic of Germany; and tInstitut fPir Neurobiologie, Universitat Heidelberg, Im Neuenheimer Feld 504, D-6900 Heidelberg, Federal Republic of Germany Communicated by Gerald D. Fischbach, August 22, 1988 ABSTRACT The membrane channels operated by y- receptors, did not induce any reaction in the astrocytes; aminobutyric acid (GABA) were studied in cultured astrocytes GABAA receptor antagonists, such as bicuculline and picro- from rat cerebral hemispheres by using patch-clamp tech- toxin, blocked the effect of GABA (12). Benzodiazepine niques. The channel properties appeared to be very similar, in receptor agonists and antagonists showed up further similar- many respects, to those present in neuronal cell membranes. ities to neurons, whereas the action of inverse agonists The Cl-selective channels were activated after the sequential revealed differences between neurons and glial cells (14). binding of two GABA molecules to the receptor, as deduced In the present study, we have obtained direct evidence for from the slope of the dose-response curve. Single-channel the existence of GABA-activated channels in glial cells. By currents displayed multiple conductance states of 12 pS, 21 pS, using patch-clamp techniques, we have compared the prop- 29 pS, and 43 pS, with the main-state conductance being 29 pS. erties of GABA receptor channels in cultured astrocytes to The gating properties could be described by a sequential those described for neurons. -
An Improved Voltage Clamp Circuit Suitable for Accurate Measurement of the Conduction Loss of Power Electronic Devices
sensors Article An Improved Voltage Clamp Circuit Suitable for Accurate Measurement of the Conduction Loss of Power Electronic Devices Qiuping Yu, Zhibin Zhao *, Peng Sun, Bin Zhao and Yumeng Cai State Key Laboratory of Alternate Electrical Power System with Renewable Energy Sources, North China Electric Power University, Beijing 102206, China; [email protected] (Q.Y.); [email protected] (P.S.); [email protected] (B.Z.); [email protected] (Y.C.) * Correspondence: [email protected] Abstract: Power electronic devices are essential components of high-capacity industrial converters. Accurate assessment of their power loss, including switching loss and conduction loss, is essential to improving electrothermal stability. To accurately calculate the conduction loss, a drain–source voltage clamp circuit is required to measure the on-state voltage. In this paper, the conventional drain–source voltage clamp circuit based on a transistor is comprehensively investigated by theoretical analysis, simulations, and experiments. It is demonstrated that the anti-parallel diodes and the gate-shunt capacitance of the conventional drain–source voltage clamp circuit have adverse impacts on the accuracy and security of the conduction loss measurement. Based on the above analysis, an improved drain–source voltage clamp circuit, derived from the conventional drain–source voltage clamp circuit, is proposed to solve the above problems. The operational advantages, physical structure, and design guidelines of the improved circuit are fully presented. In addition, to evaluate the influence of component parameters on circuit performance, this article comprehensively extracts three electrical Citation: Yu, Q.; Zhao, Z.; Sun, P.; quantities as judgment indicators. Based on the working mechanism of the improved circuit and Zhao, B.; Cai, Y. -
Lecture 1: an Introduction to Plasticity and Cellular Electrophysiology
Lecture 1: An Introduction To Plasticity and Cellular Electrophysiology MCP 2003 Charge separation across a ATPase + semipermeable Ca++ 3Na ATPase membrane is the 2K+ (mM) basis of excitability. K+ = 140 + Na = 7 ++ + Ca Cl- = 7 3Na (mM) Ca++ = 1 x 10-4 co-transporter K+ =3 Na+ = 140 membrane pores Cl- = 140 with variable Ca++ = 1.5 permeability - + R - + m Cm gK+ gCl- gNa++ Convention: Current direction is defined by the direction of increasing positive charge. Na++ flux into a cell is an inward current. K+ flux out of a cell is an outward current. Cl- flux into a cell is an outward current. A depolarizing current is a net influx of + ions or a net efflux of negative ions. A hyperpolarizing current is a net efflux of + ions or a net influx of negative ions. I Outward rectification: when a membrane allows outward current (net + charge out) V to flow more easily than and inward current. Outward rectification I Inward rectification: when a membrane allows inward current to flow more easily V than an outward current. Inward rectification Methods of Measuring Function In Excitable Membranes Field potentials: measure current sources _ low pass filter and sinks from populations of neurons + across the electrode resistance. _ 0 Brain mV-V - t (sec) + Microelectrode extracellular recording: measures action potentials from a small _ number of neurons. 0 mV pulse amplitude window - Intracellular recording: can measure voltage, t (msec) band pass filter 1KHz-10KHz +40 0 Voltage clamp recording: resting membrane Can pass current to compensate -60 potential for voltage change. In this way voltage is held ~ constant and current applied to _ compensate is a measure of the current flowing. -
Patch Clamping: an Introductory Guide to Patch Clamp
Patch Clamping An Introductory Guide to Patch Clamp Electrophysiology Areles Molleman University of Hertfordshire, UK Patch Clamping Patch Clamping An Introductory Guide to Patch Clamp Electrophysiology Areles Molleman University of Hertfordshire, UK Copyright© 2003JohnWiley&SonsLtd,TheAtrium,SouthernGate,Chichester,WestSussex PO19 8SQ, England Telephone (+44) 1243 779777 Email (for orders and customer service enquiries): [email protected] Visit our Home Page on www.wileyeurope.com or www.wiley.com All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except under the terms of the Copyright, Designs and Patents Act 1988 or under the terms of a licence issued by the Copyright Licensing Agency Ltd, 90 Tottenham Court Road, London W1T 4LP, UK, without the permission in writing of the Publisher. Requests to the Publisher should be addressed to the Permissions Department, John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England, or emailed or [email protected], or faxed to (+44) 1243 770571. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the Publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. OtherWileyEditorialOffices John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA Jossey-Bass, 989 Market Street, San Francisco, CA 94103-1741, USA Wiley-VCH Verlag GmbH, Boschstr. -
Patch-Clamp Analysis of Spontaneous Synaptic Currents in Supraoptic Neuroendocrine Cells of the Rat Hypothalamus
The Journal of Neuroscience, June 1993, 13(6): 2323-2331 Patch-Clamp Analysis of Spontaneous Synaptic Currents in Supraoptic Neuroendocrine Cells of the Rat Hypothalamus Jean-Pierre Wuarin and F. Edward Dudek Mental Retardation Research Center and the Laboratory of Neuroendocrinology of the Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90024 Spontaneous synaptic currents were recorded in supraoptic Magnocellular neuroendocrine cells of the supraoptic nucleus magnocellular neurosecretory cells using whole-cell patch- have long been considered as a model system for a wide range clamp techniques in the rat hypothalamic slice preparation. of studies on neurosecretion. These neuroendocrine cells syn- Numerous spontaneous excitatory and inhibitory postsyn- thesize the neuropeptide hormones oxytocin and vasopressin aptic currents (EPSCs and IPSCs) were observed in the 27 and transport them, from their cell bodies in the supraoptic and cells recorded. The rate of occurrence of the spontaneous paraventricular nuclei, along their axons to the neurohypophysis currents and the relative proportion of EPSCs versus IPSCs where they are secreted directly in the general circulation. The varied significantly from cell to cell. Miniature EPSCs and supraoptic nucleus has particular advantages for electrophysi- IPSCs were clearly distinguished from background noise in ological studies on neuroendocrine cells because,unlike other TTX (n = 3 cells at 0.5 pglml). The frequency of EPSCs and hypothalamic nuclei, virtually all of these cells project to the IPSCs decreased by approximately 70% and the largest neurohyphophysis and are therefore neuroendocrine. Numerous events were blocked in TTX, but the peaks of the amplitude substances(e.g., ACh, norepinephrine, opioid peptides, dopa- histograms were shifted by only a few picoamperes. -
Mechanism of Positive Allosteric Modulators Acting on AMPA Receptors
The Journal of Neuroscience, September 28, 2005 • 25(39):9027–9036 • 9027 Cellular/Molecular Mechanism of Positive Allosteric Modulators Acting on AMPA Receptors Rongsheng Jin,1 Suzanne Clark,4 Autumn M. Weeks,4 Joshua T. Dudman,2 Eric Gouaux,1,3 and Kathryn M. Partin4 1Department of Biochemistry and Molecular Biophysics, 2Center for Neurobiology and Behavior, and 3Howard Hughes Medical Institute, Columbia University, New York, New York 10032, and 4Department of Biomedical Sciences, Division of Neuroscience, Colorado State University, Fort Collins, Colorado 80523 Ligand-gated ion channels involved in the modulation of synaptic strength are the AMPA, kainate, and NMDA glutamate receptors. Small molecules that potentiate AMPA receptor currents relieve cognitive deficits caused by neurodegenerative diseases such as Alzheimer’s disease and show promise in the treatment of depression. Previously, there has been limited understanding of the molecular mechanism of action for AMPA receptor potentiators. Here we present cocrystal structures of the glutamate receptor GluR2 S1S2 ligand-binding domain in complex with aniracetam [1-(4-methoxybenzoyl)-2-pyrrolidinone] or CX614 (pyrrolidino-1,3-oxazino benzo-1,4-dioxan-10- one), two AMPA receptor potentiators that preferentially slow AMPA receptor deactivation. Both potentiators bind within the dimer interface of the nondesensitized receptor at a common site located on the twofold axis of molecular symmetry. Importantly, the poten- tiator binding site is adjacent to the “hinge” in the ligand-binding core “clamshell” that undergoes conformational rearrangement after glutamatebinding.Usingrapidsolutionexchange,patch-clampelectrophysiologyexperiments,weshowthatpointmutationsofresidues that interact with potentiators in the cocrystal disrupt potentiator function. We suggest that the potentiators slow deactivation by stabilizing the clamshell in its closed-cleft, glutamate-bound conformation. -
The Current-/Voltage-Clamp
SimNeuron Tutorial 1 SimNeuron Current-and Voltage-Clamp Experiments in Virtual Laboratories Tutorial Contents: 1. Lab Design 2. Basic CURRENT-CLAMP Experiments 2.1 Action Potential, Conductances and Currents. 2.2 Passive Responses and Local Potentials 2.3 The Effects of Stimulus Amplitude and Duration. 2.4 Induction of Action Potential Sequences 2.5 How to get a New Neuron? 3. Basic VOLTAGE-CLAMP Experiments 3.1 Capacitive and Voltage-dependent Currents 3.2 RC-Compensation 3.3 Inward and outward currents. 3.4 Changed command potentials. 3.5 I-V-Curves and “Reversal Potential” 3.6 Reversal Potentials 3.7 Tail Currents 4. The Neuron Editor 5. Pre-Settings 2 SimNeuron Tutorial 1. Lab Design We have kept the screen design as simple as possible for easy accessible voltage- and current-clamp experiments, not so much focusing on technical details of experimentation but on a thorough understanding of neuronal excitability in comparison of current-clamp and voltage-clamp recordings. The Current-Clamp lab and Voltage-Clamp lab are organized in the same way. On the left hand side, there is a selection box of the already available neurons with several control buttons for your recordings, including a schematic drawing the experimental set-up. The main part of the screen (on the right) is reserved for the diagrams where stimuli can be set and recordings will be displayed. Initially, you will be in the Current-Clamp mode where you will only see a current-pulse (in red) in the lower diagram while the upper diagram should be empty. Entering the Voltage-Clamp mode you will find a voltage-pulse in the upper diagram (in blue) with an empty lower diagram. -
Ion Channel Pharmacology Under Flow: Automation Via Well-Plate Microfluidics
ORIGINAL ARTICLE Ion Channel Pharmacology Under Flow: Automation Via Well-Plate Microfluidics C. Ian Spencer,1,* Nianzhen Li,1 Qin Chen,1 Juliette Johnson,1 INTRODUCTION Tanner Nevill,1 Juha Kammonen,2,{ and Cristian Ionescu-Zanetti1 he patch clamp technique is known to provide accurate details on the conduction and gating properties of ion 1Fluxion Biosciences, Inc., South San Francisco, California. channels, yet its usefulness for drug discovery is limited by 2Pfizer Global Research and Development, Sandwich, T a characteristically slow data accumulation rate. Accord- United Kingdom. ingly, several types of automated patch clamp (APC) systems have *Present address: The Gladstone Institute of Cardiovascular been devised to enable pharmaceutical laboratories to pursue ion Disease, San Francisco, California. channel targets at relatively high throughput.1–5 In most of the {Present address: Pfizer Neusentis, Cambridge, United Kingdom. commercial automated electrophysiology platforms, glass patch pi- pettes have been replaced by a planar substrate incorporating nu- merous holes of subcellular dimensions.6–9 Cell capture by suction ABSTRACT allows for many patch-clamping sites on the planar surface and Automated patch clamping addresses the need for high-throughput simple perfusion systems have been replaced by fluid-handling ro- screening of chemical entities that alter ion channel function. As a bots. For higher throughput, whole-cell currents are summated from result, there is considerable utility in the pharmaceutical screening populations -
Noninvasive Measurements of the Membrane Potential and Gabaergic Action in Hippocampal Interneurons
The Journal of Neuroscience, April 1, 1999, 19(7):2546–2555 Noninvasive Measurements of the Membrane Potential and GABAergic Action in Hippocampal Interneurons Jos A. H. Verheugen, Desdemona Fricker, and Richard Miles Laboratoire de Neurobiologie Cellulaire et Moleculaire, Institut National de la Sante´ et de la Recherche Me´ dicale U261, Institut Pasteur, 75724 Paris, France Neurotransmitters affect the membrane potential (Vm ) of target a cell-attached patch. Applying the method to interneurons in cells by modulating the activity of receptor-linked ion channels. hippocampal brain slices yielded more negative values for Vm The direction and amplitude of the resulting transmembrane than subsequent whole-cell current-clamp measurements from current depend on the resting level of Vm and the gradient the same cell, presumably reflecting the development of a across the membrane of permeant ion species. Vm , in addition, Donnan potential between cytoplasm and pipette solution in governs the activation state of voltage-gated channels. Knowl- the whole-cell mode. Cell-attached Vm measurements were edge of the exact level of Vm is therefore crucial to evaluate the used to study GABAergic actions in intact CA1 interneurons. In nature of the neurotransmitter effect. However, the traditional 1- to 3-week-old rats, bath-applied GABA inhibited these cells methods to measure Vm , with microelectrodes or the whole-cell by stabilizing Vm at a level depending on contributions from current-clamp technique, have the drawback that the recording both GABAA and GABAB components. In contrast, in 1- to pipette is in contact with the cytoplasm, and dialysis with the 4-d-old animals, only GABAA receptors were activated resulting pipette solution alters the ionic composition of the interior of the in a depolarizing GABA response. -
Voltage and Current Clamp Transients with Membrane Dielectric Loss
VOLTAGE AND CURRENT CLAMP TRANSIENTS WITH MEMBRANE DIELECTRIC LOSS R. FITZHUGH and K. S. COLE From the Laboratory of Biophysics, National Institute ofNeurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20014 ABsrRAcr Transient responses of a space-clamped squid axon membrane to step changes of voltage or current are often approximated by exponential functions of time, corresponding to a series resistance and a membrane capacity of 1.0 M&F/cm2. Curtis and Cole (1938, J. Gen. Physiol. 21:757) found, however, that the membrane had a constant phase angle impedance z = zi(jwcr), with a mean a = 0.85. (a = 1.0 for an ideal capacitor; a < 1.0 may represent dielectric loss.) This result is supported by more recently published experimental data. For comparison with experiments, we have computed functions expressing voltage and current transients with constant phase angle capacitance, a parallel leakage conductance, and a series resistance, at nine values of a from 0.5 to 1.0. A series in powers of ta provided a good approxima- tion for short times; one in powers of tr, for long times; for intermediate times, a rational approximation matching both series for a finite number of terms was used. These computations may help in determining experimental series resistances and parallel leakage conductances from membrane voltage or current clamp data. INTRODUCTION The sequence of events after the application of a step change of potential across a membrane in a voltage clamp is described as: (a) a capacitive transient of current as the change of potential is being established across the capacity of the membrane, followed by (b) the linear instantaneous leakage and other currents produced by ap- preciable ionic conductances, after which (c) the current changes according to the changes of the ionic conductances. -
Voltage-Clamp Analysis of Sodium Channels in Wild-Type and Mutant Drosophila Neurons
The Journal of Neuroscience, October 1988, &IO): 3633-3643 Voltage-Clamp Analysis of Sodium Channels in Wild-type and Mutant Drosophila Neurons Diane K. O’Dowd and Richard W. Aldrich Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305 In this study we describe a preparation in which we examined of neuronal sodium channels.However, direct physiological ex- directly, using tight-seal whole-cell recording, sodium cur- amination of sodium currents in adult and larval Drosophila rents from embryonic Drosophila neurons maintained in cul- neurons has proven difficult due to the small size and relative ture. Sodium currents were expressed in approximately 65% inaccessibility of the neurons. With the goal of identifying re- of the neurons prepared from wild-type Drosophila embryos gions of the Drosophila genome important in the functional when examined at room temperature, 24 hr after plating. expressionof neuronal sodium channels, including both genes While current density was low, other features of the sodium coding for structural components of the channels and those current in wild-type neurons, including the voltage sensitiv- involved in their regulation, we have utilized a preparation in ity, steady-state inactivation, macroscopic time course, and which we could biophysically examine isolated neuronal sodium TTX sensitivity were similar to those found in other excitable currents (Seecof, 1979; Byerly, 1985). In this article we describe cells. Physiological and biochemical evidence has led to the conditions in which voltage-clamped sodium currents can be suggestion that mutations in the nap, seizure, and tip-E loci studied in cultured Drosophila neurons from both wild-type and of Drosophila may affect voltage-dependent sodium chan- mutant embryos.