Manual itev 90 Intelligent Two-Electrode Voltage Clamp

Transcription

1 Manual 1.04 itev 90 Intelligent Two-Electrode Voltage Clamp

2 HEKA Elektronik Phone +49 (0) 6325 / Dr. Schulze GmbH Fax +49 (0) 6325 / Wiesenstrasse 71 Web Site D Lambrecht/Pfalz sales@heka.com Germany support@heka.com HEKA Electronics Inc. Phone Highway #14 Fax R.R. #2 Web Site Chester, NS B0J 1J0 nasales@heka.com Canada support@heka.com HEKA Instruments Inc. Phone Bellmore Avenue Fax Bellmore, New York Web Site USA ussales@heka.com support@heka.com 2013 HEKA Elektronik Dr. Schulze GmbH COMIMA/2

3 Contents 1 Introduction Support Hotline Description of the Hardware Main Unit Front-panel Connectors Rear-panel Connectors Timing of the AD/DA Channels Current Electrode Potential Electrode Compensation Electrode Installation and Startup Computer Requirements Operating Systems Connecting itev Software Installation Windows Macintosh Calibration The Model Circuit Calibrating itev

4 ii CONTENTS 4 Software Configuration itev Menu Amplifier Window Tabs Gain and Input/Output Controls Protocols Input ADC V Monitor Test Pulse itev Controls Filters Potential Electrode Clamp Controller Compensation Controller Sound Parameters Cell-free itev Test The Model Circuit Tune and Clamp Mode Offset Compensation and Electrode Resistances Impale Cell Current Clamp Voltage Clamp The Demo-1 Protocol

5 CONTENTS iii 5.4 IV Curves with Software and Hardware Leak Correction Cap Sequence for Measuring C m and R m itev and Oocytes Preparation of a TEV Clamp Setup A First Voltage Clamp Experiment New Cell Current Recording Linear Leak Correction Compensation Electrode Inhomogeneous Voltage Clamp Extracellular Current Injection Intracellular Current Injection Application as Stimulation Electrode

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7 Chapter 1 Introduction The itev 90 amplifier is a versatile automated two-electrode voltage and current clamp amplifier providing the additional option of adding an extra current-injecting electrode. Two-electrode voltage clamp (TEV) of Xenopus laevis oocytes is easily applied for the rapid screening of ion channel function, in particular in pharmacological experiments. However, conventional TEV hardware is not straightforwardly operated by technical personnel because adjustment of the electronics requires considerable practical experience. Moreover, the faithful interpretation of experimental data is often compromised by an incomplete control of all parameters determining the voltage-clamp performance. HEKA therefore designed and implemented a hardware/software combination with HEKA s Lih 8+8 AD/DA interface built-in. This arrangement minimizes total recording noise, eliminates compatibility problems and reduces additional equipment expenses and set-up time. The built-in interface utilizes USB 2.0 and high-speed processing technologies without the need for a peripheral PCI card. itev 90 provides complete software control featuring full digital calibration and tuning as well as automatic operation via the electrophysiological data acquisition software Patchmaster. Having digital control of all amplifier functions has distinct advantages over other equipment currently on the market. Patchmaster can store and access variables that describe all

8 2 Introduction of the amplifier settings applicable for an experiment. In addition, computer control lends itself to automation! Such operations with itev 90 include automatic mode switching, automatic measurement of electrode resistances, compensation of offset potentials, and automated transient compensation. In fact, digital control of EVERY adjustable parameter in the amplifier circuitry is implemented, including calibration adjustments. Using itev 90, all operations can be automated in order to speed up the experiment and, more importantly, to guarantee reproducible operation of the amplifier. At the same time, all parameters of the amplifier and data acquisition system are stored with the data for later review. itev 90 furthermore allows monitoring raw signals from various points in the amplifier circuitry. 1.1 Support Hotline If you have any question or suggestions for improvements, please contact HEKA s support team. The best way is to send us an or fax specifying: ˆ Your postal and address (or fax number). ˆ The program name: Patchmaster. ˆ The program version number: v2.63, v2.65. ˆ Your operating system and its version: MacOS 10.8, Windows 7 Prof., etc.. ˆ Your type of computer: Mac G5, Pentium 4, 1.8 GHz, etc.. ˆ Your acquisition hardware, if applicable: itev 90. ˆ Your amplifier, if applicable: itev 90. ˆ The series number and version of your itev 90, version ˆ The questions, problems or suggestions you have. ˆ Under which conditions and how often the problem occurs.

9 1.1 Support Hotline 3 We will address the problem as soon as possible. HEKA Elektronik GmbH Wiesenstrasse 71 D Lambrecht/Pfalz Germany phone: +49 (0) fax: +49 (0) support@heka.com web:

10 4 Introduction

11 Chapter 2 Description of the Hardware The hardware components of the itev 90 series multi-electrode clamp amplifier system consist of: 1. The amplifier main unit with an integrated AD/DA converter board 2. The current electrode headstage 3. The voltage electrode headstage 4. The compensation electrode headstage (optional) 5. A model circuit The amplifier is connected to the computer via a USB 2.0 cable. Specific information about the hardware installation is given below in Installation and Setup, 3 on page Main Unit The main unit of itev 90 contains the power supply, the signal processing electronics, the A/D and D/A converters and the connectors for analog and digital input/output. Essentially all of the calibration adjustments are made by digital switches in the main unit, including those that depend on the properties of components in the probe. itev 90 is factory calibrated. The calibration parameters are stored on the itev 90 main board. Unlike conventional amplifiers, hardware calibration of itev 90 can also be performed by the user as necessary (see Calibration, 3.4 on page 19).

12 6 Description of the Hardware Note: Calibration parameters are unique to each amplifier and headstage combination. Thus, if you exchange the headstage be sure to perform a new hardware calibration Front-panel Connectors Figure 2.1: itev 90 front panel Power Switch: In order to be initialized properly, itev 90 must be switched on before starting the software Patchmaster. Patchmaster, however, allows you to re-initialize the amplifier in case you forgot to turn it on in the first place. Note: Since the calibration settings of the amplifier have been determined for a warmed-up amplifier, switch on the amplifier 30 min before starting an experiment and leave it in tune mode. This will ensure that the amplifier has warmed up to regular working temperature and calibration parameters are most accurate. Chassis Gnd (CHAS): The chassis is connected to the ground line of the power cord, as is typical of most instruments. The Signal Ground (Signal GND) is kept separate from the chassis to avoid ground loops, but is connected to it through a 10 Ω resistor.

13 2.1 Main Unit 7 D/A Outputs: Three D/A channels are provided (0-2) for outputting user defined signals. Note: These are output connectors! Make sure you never feed stimuli into these outputs. A/D Inputs: The built-in interface (Lih 8+8) provides eight A/D input channels. While four of these inputs are internally connected to the itev 90, the remaining A/D inputs are freely available for application programs. For example, the Patchmaster program can use these channels to monitor temperature, pressure or outputs from other sensors. Trigger Input/Output: ˆ In: Input for an external trigger to start data acquisition when the amplifier is waiting for an external trigger. This mode is set in Patchmaster when either Trigger Series or Trigger Sweeps is selected in the Pulse Generator of Patchmaster. ˆ Out 0 through Out 2: Three TTL trigger outputs for synchronization of your TEV clamp experiments with other devices. Current Electrode: This input accepts the multi-pin connector of the headstage for the Current Electrode. Test Stim Input: External stimulus input for Compensation Electrode (alternative to I-cel). Potential Electrode: This input accepts the multi-pin connector of the headstage for the Potential Electrode. VCel: Analog voltage monitor for the potential at the current electrode. Monitor: This output connector is for service operations only. Do not use it! High Voltage LED: Indicates if the compliance voltage of the current electrode exceeds 10 V. Clamp Active LED: Indicates if the clamp control circuitry is active, i.e. the clamp feedback loop is closed.

14 8 Description of the Hardware Clamp Off Switch: Manual switch for turning the clamp circuitry off, i.e. the clamp feedback loop is open. Compensation Electrode: This input accepts the multi-pin connector of the headstage for the Compensation Electrode Rear-panel Connectors Figure 2.2: itev 90 rear panel Power: The internal power supply used in itev 90 is an auto-switching multi-voltage supply that will operate from 90 Volts to 250 Volts. Make sure the itev 90 power cord is plugged into a properly grounded AC receptacle. Digital In / Digital Out: TTL-level, digital input and output lines are available here for the control and monitoring of digital signals. See pin assignments in Appendix I: Technical Data. Digital In/Out: This connector can be used to connect, e.g., a TIB 14 trigger interface box to the itev 90 amplifier. Slave Sync: Connector for accepting a synchronization trigger from another master device. USB: This port is the connection to the USB 2.0 port in the host computer; it allows the computer to communicate with itev 90.

15 2.2 Current Electrode 9 Master Sync: Connector for synchronization of an additional Lih 8+8 interface. Aux DAC: Not supported. Telegraphing Inputs: Not supported. Sound: A 3.5-mm jack is provided for connecting headsets or speakers. Using Patchmaster software, acoustic signals for monitoring electrode potentials or electrode resistances can be output encoded to frequency. Frequency range: 200 Hz to 4 khz Timing of the AD/DA Channels Double-Staggered Acquisition: itev 90 uses a double-staggered acquisition mode. To support cophasic acquisition of the two most relevant signals, e.g. the current and voltage trace of the amplifier, two AD converters are used in parallel. That design results in supporting cophasic acquisition for 2 AD channels. Also, there is no time delay between two DA channels firing and the time of acquisition of two 2 cophasic AD channels. In the design of the Lih 8+8, time delays of 5 µs per pair of AD channels will occur, when acquiring more than 2 channels. 2.2 Current Electrode The Current Electrode headstage of itev 90 is contained in a small enclosure, designed to be mounted on a micromanipulator and directly attached to the recording micropipette. On the probe there are the following connectors: ˆ Output Connector: The standard pipette holder plugs directly into this BNC connector; the center pin is the amplifier output, and the shield is connected to signal ground (GND).

16 10 Description of the Hardware Figure 2.3: Headstage for the Current Electrode Note: Do not touch the probe s output terminal (high voltage up to 120 V!) ˆ Gnd Connector: The black pin jack should be connected to the bath electrode in order to ground the bath solution. 2.3 Potential Electrode Figure 2.4: Headstage for the Potential Electrode The Potential Electrode headstage of itev 90 is contained in a small enclosure, designed to be mounted on a micromanipulator and directly attached to the recording micropipette. On the probe there are the following connectors:

17 2.4 Compensation Electrode 11 ˆ Input Connector: The standard pipette holder plugs directly into this BNC connector; the center pin is the amplifier input, and the shield is driven with the membrane potential V-pel. ˆ Ref Connector: The red pin jack should be connected to the reference electrode. 2.4 Compensation Electrode Figure 2.5: Headstage for the Compensation Electrode The Compensation Electrode headstage of itev 90 is contained in a small enclosure, designed to be mounted on a micromanipulator and directly attached to a recording micropipette or a shield electrode. On the probe there are the following connectors: ˆ Output Connector: The standard pipette holder plugs directly into this BNC connector; the center pin is the amplifier output, and the shield is connected to signal ground (GND). Note: Do not touch the probe s output terminal (high voltage up to 120 V!) ˆ Gnd Connector: The black pin jack should be connected to the bath electrode if used without the current electrode. Otherwise the pin jack must be left unconnected to avoid ground loops.

18 12 Description of the Hardware

19 Chapter 3 Installation and Startup 3.1 Computer Requirements Operating Systems Windows versions itev 90 is supported by Windows 8, Windows 7, Vista, and Windows XP. Throughout this manual we will address all the above Windows versions as Windows. We will explicitly mention the particular Windows versions whenever required Macintosh itev 90 is supported by Macintosh computers running Mac OS X 10.4 or newer. 3.2 Connecting itev itev 90 can be installed into a standard nineteen-inch instrument rack or used as a desktop unit. If installing on a rack, please do not use itev 90 as a shelf to support any other instrument. The itev 90 case was not designed for that and damage to the front panel may result. To minimize noise, it is advisable to mount itev 90 away from devices that emit high-frequency signals (e.g., monitors, power

20 14 Installation and Startup supplies, etc). 2. Connect the headstages for the Current Electrode, Potential Electrode, and Compensation Electrode (optional) to the corresponding connectors at the front panel of itev Connect the power cord to itev 90. The internal power supply used in itev 90 is an auto-switching multi-voltage supply that will operate from 90 Volts to 250 Volts. Make sure that the itev 90 power cord is plugged into a properly grounded AC receptacle. Improper grounding of itev 90 could result in an electrical shock hazard. It is advisable to plug all equipment into a common outlet strip. This will minimize power line induced noise in the system. 4. Connect a headset or active speakers to the Sound socket at the rear panel (optional). 5. Install the USB cable from the USB connector on the rear panel of itev 90, labeled USB, to an available USB 2.0 Hi-Speed port on the computer. This connection should be made directly to the computer s USB 2.0 port and not to a USB HUB. 6. Before powering up, please re-check all connections. With all connections properly established, the power switch will illuminate once itev 90 is powered On. Important note: Please note that the Clamp Active LED will not be illuminated until the acquisition software has initialized the interface. 7. As soon as itev 90 is detected by the host operating system, the appropriate system files will be initialized and itev 90 will be ready for use. Important note: The host operating system treats itev 90 like any flash memory device. Therefore, only standard operating system files are required. This provides ease of installation and flexibility for moving itev 90 from one computer system to another. itev 90 is now connected and ready to go.

21 3.3 Software Installation Software Installation Note: only. itev 90 is supported by the software Patchmaster Windows itev 90 USB Device Driver Upon connecting itev 90 to an USB port of the computer, the required drivers will be installed automatically. Upon successful installation, the amplifier appears as a removable disk named HEKA LIH88 in the Windows Explorer Software Protection Dongle Driver To be able to use Patchmaster, a software protection dongle for the USB port is required. In order for the dongle to work, a driver needs to be installed. The latest version of the dongle driver installer can be found online at: Please navigate to Drivers Dongles. Download the driver, contained in a compressed (*.zip) file and unpack its contents to a folder of your choice. Run HASPUserSetup.EXE inside this folder and follow the installation instructions. You can also find the dongle driver on the HEKA software CD-ROM. After inserting the CD-ROM the HEKA Software installer should start automatically. If not, please run HEKA-CD\Start.exe

22 16 Installation and Startup Figure 3.1: Software protection dongle installation screen Inside the installer window navigate to [Tools & Drivers] then click on the USB dongle icon and follow the installation instructions Patchmaster Application Software To install Patchmaster please make sure the instructions in on the preceding page and on the previous page have completed successfully. Insert the HEKA Software CD-ROM. The installer should start automatically. If not, please run HEKA-CD\Start.exe Inside the installer window navigate to

23 3.3 Software Installation 17 Figure 3.2: Patchmaster installation screen [Installation] then click on the Patchmaster icon and follow the installation instructions Macintosh itev 90 USB Device Driver Upon connecting itev 90 to an USB port of the computer, the required drivers will be installed automatically. Upon successful installation, the amplifier appears as a volume named HEKA LIH88 in the devices section of Finder.

24 18 Installation and Startup Software Protection Dongle Driver To be able to use Patchmaster, a software protection dongle for the USB port is required. In order for the dongle to work, a driver needs to be installed. The latest version of the dongle driver installer can be found online at: Please navigate to Drivers Dongles. Download the driver, contained in a compressed (*.zip) file and unpack its contents to a folder of your choice. Start the installer by double clicking the image file (*.dmg) and then click on Install Sentinel Runtime Environment as shown below. The installation instructions will guide you through the rest of the setup process. You can also find the dongle driver installer on the HEKA Software CD- ROM. Please open Finder and navigate to HEKA-CD Installers Installer for HASP drivers Start the installer by double clicking the image file (*.dmg) as described above Patchmaster Application Software To install Patchmaster please make sure the instructions in on the preceding page and have completed successfully. Please insert the HEKA Software CD-ROM, open Finder and start HEKA-CD Installers Install Patchmaster.app Please choose a location where Patchmaster will be installed on your computer as shown in the dialog below.

25 3.4 Calibration 19 Figure 3.3: Software protection dongle installation screen After having completed the installation process successfully, the following message is shown: Patchmaster software is installed now and ready to run. 3.4 Calibration Calibration of a new amplifier usually is dispensable because the device was calibrated by HEKA before shipment. However, it is advisable to recalibrate itev 90 twice a year or whenever the gain factors of the amplifier are not accurate or offsets become noticeable. Note: The calibration file contains the settings of the digital switches and controls of the amplifier. These are unique to a

26 20 Installation and Startup Figure 3.4: PatchMaster installation screen Figure 3.5: PatchMaster installation screen given combination of amplifier and headstage (probe) and cannot be used for another itev 90. Therefore, you have to recalibrate the amplifier whenever you replace the probe! This is a big advantage of itev 90 since you can use any probe with any amplifier and replacement of a broken probe does not require to return the amplifier to HEKA for re-calibration. Before starting the calibration make sure the amplifier has reached its working temperature. We advice to let itev 90 warm up for 30 minutes after powering the amplifier on, and to leave it in tune mode (see below). For the next steps you will need to connect the model circuit to the headstages The Model Circuit The model circuit connects to the probe inputs of the Potential Electrode and the Current Electrode via a BNC adapter. The red Reference connector of the model cell has to be connected with the red Reference connector of the Potential Electrode. The Chassis connector of the model cell has to be connected with the Chassis ground connector at the front panel of itev 90.

27 3.4 Calibration 21 Figure 3.6: Model circuit with probes The model circuit provides four switches with three positions simulating various conditions typically observed during an electrophysiological experiment on Xenopus laevis oocytes Calibrating itev 90 The calibration procedure can be performed with Patchmaster software. Go to the itev 90 Test and Calibrate menu. This menu contains all the items involved in calibration and generation of scale files. To perform the calibration select Calibrate from the Test and Calibrate menu. Follow the subsequent commands, such as to connect the model circuit and to set it to certain modes. At the end of the calibration, the software will let you know whether the calibration succeeded or failed. On success the program will automatically generate and store a new set of calibration constants. Finally, the software will re-initialize the amplifier. itev 90 is now ready to go.

28 22 Installation and Startup

29 Chapter 4 Software In this section we will describe all control buttons of Patchmaster designated to the operation of itev 90. For all other functions and features of Patchmaster please refer to the respective manual. Upon startup of Patchmaster for the first time, it may request input regarding the connected hardware. Click on itev and Activate AD Board. Patchmaster will then initialize itev 90 and will activate several control windows. The most relevant for the operation of itev 90 is the Amplifier window, typically at the left top corner of the screen. However, there are also some other places in the software where you can find information and commands specific for itev 90 and not available for, e.g., patch-clamp amplifiers. 4.1 Configuration In the hardware section of the Configuration window you can specify if a Compensation Electrode is to be used. Upon activation of the corresponding flag ( Comp. Controller ) the icons necessary to control the Compensation Electrode will appear in the Amplifier window Note: For MacOS: Closing and re-opening of the Amplifier window may be necessary.

30 24 Software 4.2 itev Menu The Patchmaster contains the itev90 menu providing the following functions: Enable Background: Test pulse continues to operate even if Patchmaster is not in the front. Monitor Refresh Interval: A control box will appear to enter the time interval between two measurements of the membrane resistance, as well as I-mon, V-mon, and V-cel. Tracking Interval: Enter interval for tracking pipette resistances. Min. Test Pulse Interval: Enter minimum interval between successive test pulse stimulations. V-cel Re-enable Interval: Whenever the compliance voltage of the Current Electrode exceeds 10 V, electronics used for precisely measuring R-cel is disabled. If the compliance voltage is back to below 10 V, high precision V-cel measurement is enabled automatically after the V-cel Re-enable Interval, to be specified here. R-pip Ranges: Define ranges of electrode resistances, R-pel, R-cel, and optionally R-comp by specifying the upper and lower limit. These limits are used to decide whether the resistances are in an acceptable domain. As long as a resistance is outside the defined range the respective icon field is shown in red. R-cel Parameters: These parameters define the dimensions of squarewave pulses applied in the current clamp mode to measure R-cel (and optionally R-comp). For the recording from Xenopus oocytes these parameters typically do not need to be altered. However, more sensitive preparations or electrodes with much lower or higher resistances than about MΩ may require adjustment. Auto Shutoff Current: Define a maximal current level accepted at the holding potential when turning on the voltage clamp. Upon activating Clamp, the current is measured; if the level is exceeded Patchmaster automatically goes back to Tune mode. This feature may help to prevent electrodes from damage.

31 4.3 Amplifier Window 25 Calibrate itev90: Calibrate the amplifier (see Calibration, 3.4 on page 19. Initialize itev90: Reset all system parameters to default values and initialize hardware. itev info: Display some information on the itev 90 hardware such as the serial number. 4.3 Amplifier Window The Amplifier window contains a virtual front panel of itev 90. functions are grouped in the following subsections. The ˆ Tabs used to select the range of visible icons ˆ Gain and input/output controls ˆ Protocol icons ˆ Input ADC ˆ Test pulse ˆ itev controls ˆ Filter settings ˆ Potential Electrode functions ˆ Clamp Controller functions ˆ Compensation Controller functions (optional) ˆ Sound Tabs

32 26 Software By selecting one of the tab Monitor or Tuning one can restrict the size of the Amplifier window to a subset of functions. Show All will open the full window providing access to all functions Gain and Input/Output Controls Set the amplification factor of itev 90 and, depending on the mode, set the holding voltage (voltage clamp) or the holding current (current clamp). Note, like most settings below, there are always two copies stored, one for voltage clamp and another for current clamp. Gain List of gain settings of the I-mon channel, i.e., it is typically only used in Voltage Clamp mode. Up and down arrow keys can be used to go through the list. V-membrane Holding voltage in Voltage Clamp mode. Left and right arrow keys can be used to change V-membrane by ±10 mv. In voltageclamp mode this field turns into I-membrane. Left and right arrow keys change I-membrane by ±1 µa. I-mon Current passed through the Current Electrode. V-mon Voltage of the Potential Electrode. V-cel Voltage of the Current Electrode. This field can turn into V-comp once Monitor V-comp was selected (see on the next page) Protocols The icons Set-up, New Cell, and Zero Offsets are used to call protocols of equivalent names provided in the Protocol Editor. The user is free to put any content into these protocols. The default functions provided by HEKA are the following:

33 4.3 Amplifier Window 27 Set-up Reset all itev 90 variables to the default values. New Cell Select Tune mode, compensate electrode offset voltages and measure their resistances. Zero Offsets Compensate electrode offset voltages Input ADC Selection of channel to show in the Oscilloscope window. Typically this is I-mon. However, the user also has access to all of the remaining AD channels. Other options are V-mon, V-cel, and I-comp V Monitor When the Compensation Electrode is activated the user can select which voltage is to be displayed in the voltage monitor field above: Monitor V-cel Default setting; monitors the voltage of the Current Electrode. Monitor V-comp Monitors the voltage of the Compensation Electrode Test Pulse The test pulse controls are identical to those used in patch-clamp amplifiers. Test pulses can be turned Off, set to single- or doublepulse mode, or used for noise measurements, i.e., no pulses are output and only current is sampled and r.m.s. noise is calculated and displayed. Amplitude and duration of the test pulses can be set and with a list selection one can specify if current, voltage, or both signals are to be displayed in the Oscilloscope window.

34 28 Software itev Controls These are the most important icons for controlling itev 90. Tune When Tune mode is active the amplifier is in current clamp mode with very small gain - it is effectively inactive. This mode is used to zero potential offsets and to measure electrode resistances. Furthermore, itev 90 settings are selected without actually sending commands to the hardware. For example, the user specifies the Clamp mode to be used upon amplifier activation (Voltage clamp or Current clamp) and sets the holding voltages or holding currents to be applied. Clamp The amplifier is put into an active mode and all settings of this window apply. Make sure both electrodes are properly placed inside a cell before activating Clamp. Typically the user checks if V-pel and V-cel are about the same, i.e., they both are reporting the membrane voltage, before activating the clamp. Off If this icon is hit the amplifier is turned off, i.e., the high-voltage source is inactivated and the feedback loop is opened. Voltage Clamp Select voltage clamp as recording mode. Current Clamp Select current clamp as recording mode. Reset Reset the amplifier setting to default values. (This button appears on the right side of the Amplifier window.) Clear Set all offsets to zero and all ready flags to Off, i.e., all icons for offset potentials and electrode resistances are shown in red to indicate that for this particular experiment that they haven t been compensated or checked, respectively. (This button appears on the right side of the Amplifier window.) Filters

35 4.3 Amplifier Window 29 In this section the filter of the measured current signal (I-mon) is set. In addition, the rise time of the stimulus sent to the controller is limited by specifying a time constant. Current Filter Cutoff frequency of an 4-pole Bessel filter used to process the I-mon signal prior to sampling. Note that this filter is automatically set to appropriate values if the Auto Filter option is turned on in the hardware tab of the Configuration window. Stimulus Filter Before a stimulus is sent to the amplifier it is passed through an RC-element to limit the rise-time and, hence, the size of the capacitive current transients. Depending on the required clamp speed the user can select a time constant ranging from 50 µs to 3 ms. In the Off mode this stimulus filter is bypassed. The rise-time will then be maximal but it may not be well approximated by a single exponential function anymore Potential Electrode The Potential Electrode is used to measure the membrane voltage and to feedback this information to the clamp controller. V-pel A click on the yellow button will perform measurement and compensation of the electrode offset potential. If successfully measured and compensated, the corresponding offset potential value will be displayed in the field on the right. In addition, the field will turn from red to blue to indicate that this electrode is ready for the experiment. The offset voltage can also be set manually. These functions are only accessible in Tune mode in order to avoid accidental changes of the offset during an experiment. R-pel A click on the yellow button will perform a measurement of the electrode resistance. If within an acceptance range (see R-pip Ranges, 4.2 on page 24), R-pel will be displayed in the field on the

36 30 Software right and this field will turn from red to blue to indicate that R-pel was successfully checked before a new experiment. Track Checkbox to activate continuous measurement of R-pel.

37 4.3 Amplifier Window Clamp Controller These icons describe offsets and functions of the Current Electrode and are used to adjust the clamp controller settings. Controller Type Select between P (proportional) or PI (proportional / integral) mode of the clamp controller. Gain Proportional gain of the controller. Tau Time constant (in µs) of the integrator used in the PI-mode. The smaller this value, the faster the clamp controller reaches the desired output value. However, the risk of oscillation increases with decreasing time constant. V-cel A click on the yellow button will perform measurement and compensation of the electrode offset potential. If successfully measured and compensated, the corresponding offset potential value will be displayed in the field on the right. In addition, the field will turn from red to blue to indicate that this electrode is ready for the experiment. The offset voltage can also be set manually. These functions are only accessible in Tune mode in order to avoid accidental changes of the offset during an experiment. E With this button enabling of the Current Electrode can be enforced if it was disabled due to high compliance voltage. R-cel Click on the yellow button will perform a measurement of the electrode resistance. If within an acceptance range (see 4.2 on page 24), R-cel will be displayed in the field on the right and this field will turn from red to blue to indicate that R-cel was successfully checked before a new experiment. Track Checkbox to activate continuous measurement of R-cel.

38 32 Software Compensation Controller These fields are used to configure the so-called Compensation Electrode. This electrode is practically operated like an additional current-injecting electrode in current-clamp mode. Rather than controlling the current according to the stimulation paradigm, it takes I-cel or the test stimulus as an input an tries to clamp that current, weighted with the scale factor. When used as an external shield electrode, this factor could be on the order of When used as additional intracellular electrode, the factor typically is much smaller. A factor of 1.0 means that the Compensation Electrode injects as much current as the Current Electrode. Controller Type Select between Off, P (proportional) or PI (proportional and integrating) mode of the clamp controller. Gain Clamp gain. Note that the compensation electrode runs in currentclamp mode and, hence, a gain of 1-2 should be sufficient. Higher gain may result in oscillation. Tau Time constant (in µs) of the integrator used in the PI-mode. The smaller this value, the faster the compensation clamp controller reaches the desired output value. However, the risk of oscillation increases with decreasing time constant. V-comp A click on the yellow button will perform measurement and compensation of the electrode offset potential. If successfully measured and compensated, the corresponding offset potential value will be displayed in the field on the right. In addition, the field will turn from red to blue to indicate that this electrode is ready for the experiment. The offset voltage can also be set manually. These functions are only accessible in Tune mode in order to avoid accidental changes of the offset during an experiment. E With this button enabling of the Compensation Electrode can be enforced if to was disabled due to high compliance voltage.

39 4.4 Parameters 33 R-comp A click on the yellow button will perform a measurement of the electrode resistance. If within an acceptance range (see 4.2 on page 24), R-comp will be displayed in the field on the right and this field will turn from red to blue to indicate that R-comp was successfully checked before a new experiment. Track Checkbox to activate continuous measurement of R-comp. Scale Set here the fraction of current to be injected via the Compensation Electrode with respect to the selected source (typically I-cel). Scale Offset Field for manual offset correction. Source Select the signal used as a command for the Compensation Electrode. Ctrl. Offset Field for manual offset correction Sound Provided speakers are connected to the computer, Patchmaster will output a sound signal depending on the settings specified here. Encoding Depending on the source (see below) selected, this field is used to specify how the monitored signal is encoded into a sound frequency. For voltages this will be Hz/mV, for resistances Hz/100 kω. Volume Volume of sound output. Source Source of signal do be encoded into sound. The options are: Off (no sound generated), V-mon, V-cel, R-pel, R-cel, R-comp. 4.4 Parameters All variables describing the state of the itev 90 amplifier are going to be stored in the data files, associated to the respective trace. Thus, upon replay all settings can be inspected as shown below.

40 34 Software Figure 4.1: Amplifier tab of Parameter window for a data file acquired with itev 90. The status variables ( ready or - ) specify if the respective control was compensated or checked prior to a new experiment. Important note: When data acquired with itev 90 are to be analyzed with Fitmaster one has to make sure that itev 90 was selected as the amplifier type. Only in this case the Parameter window contains the correct data describing the amplifier state. Thus, it is advised to generate different configuration files ( set files ), when analyzing data from different types of HEKA amplifiers.

41 Chapter 5 Cell-free itev Test The following tutorial will guide you through most of the basic and some of the unique and more sophisticated features of the itev 90 amplifier. At the same time it allows you to check whether the amplifier is functioning properly. You can use the model circuit provided with the amplifier as a substitute for a real TEV clamp recording and explore the virtual front panel of itev 90 supplied in the acquisition software Patchmaster. 5.1 The Model Circuit Figure 5.1: itev 90 model circuit. The switches are used to set the connections of the Potential Electrode (Pel) and the Current Electrode (Cel). Additional switches determine the membrane resistance and capacitance. The model circuit provides the following settings:

42 36 Cell-free itev Test ˆ Electrodes: Disconnect the electrodes ( off ), connect them to ground ( bath ), or insert them into the cell ( on ). Electrode resistances (R pel and R cel ) are 680 kω. ˆ Membrane resistance: R m can be set to infinity ( off ), as well as to 10 or 100 kω. ˆ Membrane capacitance: C m can be set to zero ( off ), as well as to 100 or 220 nf. First, connect the model circuit to the probe input via a BNC adapter and plug the black cable to the black ground connector on the probe. If Patchmaster is not running yet, start the program, which is located in the Patchmaster folder inside the HEKA folder. Windows users might alternatively use the Start button to launch Patchmaster from Programs HEKA. Press the SPACE key to bring up the so-called virtual front panel. It provides a graphical representation of the itev 90 amplifier. The panel lets you control all hardware settings of the amplifier such as gain or filters. Signal display is provided by an oscilloscope-like display.

43 5.2 Tune and Clamp Mode Tune and Clamp Mode itev 90 has a Tune and a Clamp mode. The Tune mode is used to pre-configure the amplifier for current- and voltage-clamp modes without activating them. E.g., you can set the test pulse parameters, filter and stimulus filter settings, gain, holding current or voltage. The intention of the Tune mode is to allow a gentle switch from current to voltage clamp and vice versa without the requirement of manually configuring the amplifier settings Offset Compensation and Electrode Resistances Set both electrodes to bath in the Model Cell. In this configuration, with itev 90 in Tune mode, you will at first compensate the offset potentials. Hit V- pel and V-cel: since you are using the Model Cell, values should be close to zero and the red fields should become blue indicating that the zeroing of the potentials was successful. Then you may want to check the electrode resistances. Hit R-pel and R-cel: the response should be an indication of about 680 kω with both fields indicating ready. The image shows a state in which the Potential Electrode is ready, while the Current Electrode still needs to be adjusted Impale Cell To simulate insertion of the electrode into the cell, set the electrode switches at the Model Cell to on. Now you have to decide if you would like to study the cell in Current Clamp or in Voltage Clamp mode; we start with Current Clamp.

44 38 Cell-free itev Test Current Clamp Select Current clamp and make sure the holding current is set to zero. Set the test pulses to single-pulse mode, an amplitude of 0.5 µa and a duration of 100 ms duration. Make sure the Clamp Controller is set to PI mode and the lowest gain, i.e. 1.1, and the maximal time constant (Tau = 10 ms). Now we simulate a membrane resistance of 100 kω and turn the membrane capacitance off. Make sure the test pulses are running and activate the clamp by selecting Clamp : the controller will now try to inject 0.5 µa for 100 ms intervals. Inspect current and voltage recording and decrease the clamp time constant (e.g. 10 ms and the minimum value of about 40 µs). You should get results as indicated in the figure: I-cel should reach 0.5 µa and V-pel about 50 mv (given R m = 100 kω). Next you may want to leave the clamp in PI mode with a low gain and a short time constant and test the impact of cell capacitance. The figure on the right shows such recordings for C m = off, 100 nf, and 33 nf. You may also test the influence of lowering the membrane resistance to R m = 10 kω: V-pel should now reach about 5 mv. When you set the controller to P mode you will find that a gain of 1.1 is not sufficient to clamp the current to 0.5 µa, and higher gain settings easily cause oscillations. For Current Clamp it is therefore recommended to use PI

45 5.3 The Demo-1 Protocol 39 mode only Voltage Clamp Now try a similar approach for Voltage Clamp mode. Go back to the tab Tuning, set the mode to Voltage Clamp, and set the holding voltage to 0 mv. Make sure the controller is in PI mode with low gain and a long time constant. Set the test pulses to double-pulse mode, the amplitude to 10 mv and the length to 5 ms. The Model Cell should be set to R m = off and C m = 100 nf. Now activate the clamp by selecting Clamp : the controller will now try to hold the voltage at 0 mv and to clamp it to ±10 mv during the test pulses, i.e. it will try to charge the membrane capacitance to such values. The adjustment of the voltage clamp is further described in the subsequent paragraphs using Demo Protocols. 5.3 The Demo-1 Protocol The protocol Demo-1 guides you through the first steps of adjusting the amplifier parameters. After starting the protocol Demo-1, the following message appears asking you to switch the model cell into the starting position, simulating both electrodes being in the bath. After you have configured the model cell appropriately, then you can click Continue. The protocol then initiates the automatic offset compensations on both electrodes. After that it turns on the current-clamp mode.

46 40 Cell-free itev Test While in current clamp you will impale the cell with both electrodes. In this demo the following message asks you to switch the model cell correspondingly. Then the protocol automatically switches to voltage-clamp mode and clamps the cell at -80 mv. Now you can manually tune the clamp parameters Tau and Gain. E.g. increase the Gain of the Clamp Controller until you see the capacitive charging current transients and a more rectangular voltage pulse. In the following example we increased the gain to about 36.

47 5.4 IV Curves with Software and Hardware Leak Correction 41 You may change the test pulse parameters to have a closer look at the rising phase of the voltage. 5.4 IV Curves with Software and Hardware Leak Correction You may execute the pulse generator sequences IV, IV PN, and IV HW to demonstrate different methods of leak compensation. ˆ IV: IV protocol without leak correction ˆ IV PN: IV protocol with p/n software leak correction activated ˆ IV HW: IV protocol with hardware leak subtraction turned on The hardware leak subtraction measures the current transients as done during the p/n method but instead of subtracting it by software it subtracts

48 42 Cell-free itev Test the transient in the hardware before the filter and gain section of the amplifier. This allows leak subtraction at larger gain settings compared with the p/n method.

49 5.5 Cap Sequence for Measuring C m and R m Cap Sequence for Measuring C m and R m By executing the sequence Cap, you can estimate the R m and C m of the cell.

50 44 Cell-free itev Test

51 Chapter 6 itev and Oocytes 6.1 Preparation of a TEV Clamp Setup The following section briefly describes some steps necessary for a TEV clamp experiment on Xenopus laevis oocytes. The cells, which are about 1 mm in diameter, are typically kept in a recording chamber filled with Ringer s solution and can be inspected with a stereo-microscope. As detailed in Figure 6.1, one ground electrode (either Ag/AgCl pellets or thick chlorinated silver wires directly immersed in the bath or connected to the bath via salt bridges) is connected to the reference input of the Potential Electrode headstage and another one to the ground input of the Current Electrode. Potential Electrode and Current Electrode typically are glass electrodes filled with 1-2 M KCl solution, connected to the hardware by means of chlorinated silver wires (e.g., use HEKA s pipette holders: Depending on the application, the tips of the glass electrodes are broken or beveled prior to an experiment to yield a low resistance while keeping the electrodes thin enough in order to minimize damage of the cells upon electrode insertion. Typical electrode resistances are between 0.3 and 0.8 MΩ. If very low-resistance electrodes are required, KCl leaking into the cell can be a serious problem. In such cases electrode tips may be clogged with agar (agarose cushion microelectrodes). Put the cells into the recording chamber. Ideally, the white side is at the top because the large nucleus is in the pigmented hemisphere. However, since glass electrodes give a much better contrast when the pigmented part of the cell is in the background, beginners may put the black side up to have a better visual control of the electrode positioning.

52 46 itev and Oocytes We recommend to position the electrodes such that they impale the cell at ±45 of the normal, both aiming at the center of the cell. While the tip of the Potential Electrode should be positioned very close to the plasma membrane (in order to faithfully report on the membrane potential without much voltage drop in the cytosol), the tip of the current-injecting electrode ideally is at the center of the cell in order to produce a homogeneous voltage drop across the plasma membrane (see Compensation Electrode, 6.3 on page 54). Figure 6.1: Schematic diagram of electrode connection and positioning Make sure itev 90 has warmed up and Patchmaster is turned on. The amplifier should be in Tune mode. In this configuration, position both electrodes such that the tips are immersed in the bath solution. If the electrodes are freshly prepared (i.e. newly chlorinated) one may want to leave them in this position for a couple of minutes to permit offset potentials to stabilize. 6.2 A First Voltage Clamp Experiment Let s suppose you would like to perform a voltage-clamp experiment in order to assay heterologously expressed voltage-gated ion channels. Provided itev 90 was properly calibrated, you can now go ahead and perform the following steps.

53 6.2 A First Voltage Clamp Experiment New Cell The electrodes are in the bath and the next thing to be done is to set relevant program variables to starting values and to compensate the electrode offsets. Typically all these preparing steps can be put into the protocol New Cell. Here we go through the sequence step by step. Depending on what you have done before, you may need to set the holding voltage to a desired value (e.g. to -100 mv in order to avoid channel opening once the voltage-clamp is activated), you may need to reset the clamp gain to a small value, reset the stimulus filter, the current filter, etc. Certainly one should clear all ready flags to indicate that a new experiment is going to start. Then zero the Potential Electrode offset. The offset will be shown and the respective icon will turn from red to ready, i.e. blue, if the auto-zeroing was successful. Do the same with the Current Electrode. Then measure both resistances, i.e. hit R-pel and R-cel. If the resistances are in an acceptable range the respective fields will be indicated as ready. For the first cell of the day you may want to repeat this procedure in order to make sure the electrodes are stable. Still in Tune mode, the electrodes are inserted into the cell. The respective voltages (V-pel und V-cel) are displayed in the Amplifier window. In addition, such voltages can be encoded into a tone using the sound option. When both electrodes are inside the cell (i.e. V-pel and V-cel should be about equal) the voltage clamp loop can be closed by switching from Tune to Clamp mode. Note: Activation of the Clamp mode with at least one electrode being in the bath solution will result in a short-circuit; the full compliance voltage of 120 V will be applied to the Current Electrode and the resulting large current may damage the electrode. Provided there is not much leak current, the next step will be to tune the clamp controller settings. For later documentation it might be good to set the Patchmaster timer to zero at this point; this will ensure that Timer Time always refers to the duration for which the cell was clamped to the respective holding voltage. Experienced users will setup protocols that perform all such initializing tasks with one mouse-click.

54 48 itev and Oocytes Activate the test pulses and make sure both, I-mon and V-mon are displayed in the Oscilloscope. The next aim is to optimize the setting of the clamp controller. This is done in a most straightforward manner if the cell behaves like a linear electronic network. Therefore, the holding voltage should be set to a value at which no non-linear current components are expected. When recording currents from voltage-gated ion channels this typically is between -100 and -80 mv. We assume the controller is in PI mode, i.e. the controller should be able to fully compensate the error signal with the given integration time constant. Starting with a slow integration time constant (9.9 ms), increase the clamp gain to obtain an approximate step response in V-pel. Adjust the speed by decreasing the time constant. Always make sure the clamp does not start to oscillate. An important aspect is the selection of an appropriate Stimulus Filter setting. Depending on the application, a setting between 50 and 3000 µs can be selected. Typically, 200 µs is a good first approach. This implies that a clamp time constant much faster than the Stimulus Filter setting is not meaningful and would just increase noise and would potentially destabilize the clamp. When appropriately adjusted, the step response of V-pel should be characterized by an almost exponential increase with the desired time constant; in addition, the step response should reach a steady-state within the duration of the test depolarization and should not show any overshoot. Note that the adjustment of the clamp time constant in PI mode needs some steady-state current (DC component). For a perfectly tight cell one will only get the capacitive currents for charging the membrane and, hence, no faithful inferences can be made about clamp properties outlasting the current transients of about 1 ms.

55 6.2 A First Voltage Clamp Experiment 49 Figure 6.2: Examples of V pel and I cel recordings for 10 mv test pulse steps for the indicated settings of rise-time (Stimulus Filter), Clamp Gain, and Integration Time Constant. Electrode resistances were about 500 kω. Another criterion to be kept in mind is the current needed to charge the oocyte membrane; the current transients required to clamp the cell membrane voltage should be small enough as to avoid saturation of the amplifier or the AD electronics. Thus, it may be necessary to reduce the current gain accordingly and to further limit the speed of the stimulus by increasing the rise-time in the Stimulus Filter setting. Some examples are shown in Figure Current Recording When the clamp controller is set such that V-pel is following the test pulse stimulus in an acceptable manner, V-membrane can be set to the final value required for the experiment to be performed. Then select a Series from the Pulse Generator pool and start recording. It is advised to always record both, I-mon and V-mon; during subsequent analyses it might be important to know exactly how well/fast the membrane voltage was controlled. Be aware that itev 90 has a built-in 4-pole filter of the I-mon channel. Thus, make sure Auto Filter is activated in the Configuration window and

56 50 itev and Oocytes the stimulation protocol in the Pulse Generator file contains a valid specification of the Filter Factor. In this case, the I-mon filter will be set automatically and no further steps are required. The anti-aliasing filter for the V-mon channel is fixed at about 10 khz and has 4-pole Bessel characteristics. In Figure 6.3 recordings from an oocyte expressing voltage-gated potassium channels are shown. Figure 6.3: Superposition of V pel and I cel recordings from an oocyte expressing voltage-gated potassium channels. Note that no leak correction was applied, and that the transient capacitive currents are off scale. Parameters characterizing itev 90 were: Both electrode resistances = 540 kω, Stimulus Filter = 200 µs, Clamp Mode = PI, Gain = 253, tau = 195 µs Linear Leak Correction For linear correction of leak and capacitive currents the first choice is to use the software p/n correction method provided in the Pulse Generator. Select Leak in the stimulation channel and specify the leak pulse parameters such as Leak Holding, Number of Leak Pulses, Leak Size, and Leak Delay. To minimize the noise generated by subtracting leak currents from the main currents try to optimize the parameters Number of Leaks and Leak Size. Number of Leaks is limited by the time required for leak pulse sampling, while Leak Size has to be small enough as to avoid activation of non-linear current components (i.e., leak pulses must not open ion channels). Figure 6.4 shows an example of software p/n correction.

57 6.2 A First Voltage Clamp Experiment 51 Figure 6.4: Current recording at 40 mv from an oocyte expressing voltagegated potassium channels without (left) and with software p/n leak correction (right). Also shown is the scaled leak current based on 8 leak pulses and a Leak Size of 0.25 (middle). Serious limitations of software p/n correction are: (1) The time required to acquire all leak pulse responses for every trace to be measured. (2) A limitation of the current resolution because of the large current transients that need to be sampled prior to correction. Therefore, a better method may be to eliminate (or at least to reduce) the capacitive currents before filtering and amplification of the current signal and sampling it with the AD board. For this purpose itev 90 features a hardware leak compensation method. The principle of this method is that in parallel to the voltage stimulation paradigm a signal mimicking the expected leak current will be output via a DA channel. This Leak Current is then internally subtracted from the I-mon signal before it is sent to the I-mon filter and amplification stage. The thus hardware-leak corrected current signal is then sampled via the I-mon channel and displayed. For the generation of the Leak Current template there are the following methods: Ad hoc p/n pulses A Leak Current template is sampled using the p/n method in the same way as for software p/n.

58 52 itev and Oocytes Pre-sampled unitary step response A leak template can be constructed from a unitary step response sampled with high resolution before the actual experiment. Such unitary step responses can then be refreshed at intervals selected by the user (e.g. before every Sweep, Series, or larger intervals. Synthetic leak templates Rather than building a leak template based on actually measured current signals, one can try to construct a synthetic leak template assuming (e.g.) a set of weighted exponential processes. This method will not perfectly compensate all current components but it will introduce much less noise than the other methods mentioned above.

59 6.2 A First Voltage Clamp Experiment 53 Figure 6.5: Comparison of software and hardware leak correction. Current recordings for voltage steps from -40 to 20 mv in steps of 10 mv from an oocyte expressing voltage-gated potassium channels. Leak pulse parameters were in all cases: 6 leak pulses and a LeakSize of Top row: I-mon gain was V/µA. left: raw data, no leak correction applied; middle: software p/6 correction applied; right: hardware leak correction applied. Sampling interval was 100 µs for software leak correction, and 5 µs with a compression factor of 20 for hardware leak correction. Bottom row: recordings from the same cell with an increased I-mon gain of V/µA indicating that, under this condition, software leak correction does not work anymore because of saturating transients, while hardware leak correction still provides faithful results. Currently only the method of using Ad hoc p/n pulses is implemented. To activate hardware leak compensation select this option in the Leak section of the Pulse Generator as shown in the figure.

60 54 itev and Oocytes Selection of Hardware Leak will automatically generate the stimulus channel needed to output the leak template. Make sure the Leak Delay is negative because the leak template has to be recorded prior to the main pulse. Be aware that hardware leak compensation will introduce more noise than software leak compensation because there is an additional digitalization and amplification step involved. However, it will allow sampling the step responses with a higher gain than software leak compensation (about a factor of 10), and hence, it will avoid saturation if large current transients are involved (for short rise-time selections). A serious limitation of this method is the time lag between the uncorrected current signal and the leak template signal. Therefore, hardware leak correction only works well if currents are sampled with the maximal speed (sample interval of 5 µs). In this case the time lag of 5 µs will be corrected for internally. As a consequence, the user is advised to use the full sampling speed; to limit the amount of stored data use the option of data compression provided in the Pulse Generator (see dialog above and example in Figure 6.5). 6.3 Compensation Electrode Inhomogeneous Voltage Clamp The current injected via the Current Electrode only clamps the membrane voltage in a homogeneous manner if the electric field lines starting at the Current Electrode tip hit all parts of the plasma membrane with the same density. There are two major obstacles working against that ideal situation. In the first place the oocyte membranes have very many small invaginations (microvilli) such that not all parts of the membrane are readily exposed

61 6.3 Compensation Electrode 55 to the cytosol and the extracellular space. There is not much one can do against these badly accessible membrane fractions. There were some attempts to minimize this problem by swelling the cells, but this certainly cannot be used for routine applications. Another very serious problem arises from the finite conductance of the cytosol. Since the cytosol has a specific resistance, the current injected via the Current Electrode will not only result in a voltage drop across the plasma membrane but also inside the cytosol, i.e. there is a non-negligible series resistance between the Current Electrode and the membrane. As a consequence, the membrane will only experience a homogeneous voltage drop if the point source of current is in the very center of the cell. Deviation from the ideal situation sketched in Figure 6.1 with a superficially injected Potential Electrode and a central Current Electrode results in substantial inhomogeneities of the membrane potential. As outlined in the figures below, in particular under strong current load, e.g. when charging the membrane capacitance, the membrane voltage at the place of the Potential Electrode easily deviates from the potential measured at the opposite side of the cell by 50 mv or more. In Figure 6.6 it is shown how the inhomogeneous voltage clamp is demonstrated: in addition to the Potential Electrode and the Current Electrode, a third voltage-recording electrode is used. The latter electrode is placed at various sites between both standard electrodes, characterized by an angle with respect to the Current Electrode; the potential recorded by that additional electrode is referred to as V m.

62 56 itev and Oocytes Figure 6.6: Schematic diagram of an oocyte and positioning of the Potential Electrode, the Current Electrode, and an additional potential-recording electrode. The sites of secondary potential measurements are indicated (red). Angles are given with respect to the Current Electrode. In Figure 6.7 a typical experiment is shown. Under voltage clamp conditions a voltage step of 100 mv is applied; with the given rise-time and clamp settings this required charging current transients of several 10 µa. When the Potential Electrode as well as the Current Electrode are only inserted into the cell at a depth of about 100 µm there is a substantial inhomogeneity of the membrane potential: V m measured with the third electrode deviates from V pel the more the closer it comes to the Current Electrode (or the farther away from the Potential Electrode V m is measured).

63 6.3 Compensation Electrode 57 Figure 6.7: Recordings of V pel and V m at various sites in the membrane. (A) V pel (black) and V m (colored) at the indicated angles with respect to the Current Electrode for a 100 mv step depolarization. (B) Differences between V m and V pel, measured at the indicated sites of the oocyte membrane. The Potential Electrode and the Current Electrode both penetrated the cell about 100 µm. A solution to this problem is to adopt the ideal positioning of the electrode as sketched in Figure 6.1. This is illustrated in the experiment shown in Figure 6.8. The deeper the Current Electrode is driven into the cell the more homogeneous the membrane potential becomes. The ideal situation is obtained when its tip reaches the cell s center, which is about 600 µm for a cell of about 1.2 mm diameter. A rough estimate for the intracellular series resistance between the difference in distance between the tip of the Current Electrode and the tips of both voltage-recording electrodes is obtained by dividing the maximal voltage difference, V m = V m V pel, by the peak charging current. For V m of 60 mv and a maximal I cel of 30 µa this yields an effective series resistance of 2 kω.

64 58 itev and Oocytes Figure 6.8: Differences between V pel and V m for a voltage step from - 50 to 50 mv. The Potential Electrode was about 100 µm inside the cell. The membrane voltage was additionally measured at 45 with respect to the Current Electrode. (A) V pel (black) and V m (red) for the indicated penetration depths of the Current Electrode. (B) Differences between V m and V pel. Both panels show that V m approximates V pel the best when the Current Electrode is about in the center of the oocyte, i.e., at about 600 µm penetration depth. The current transients required to charge the membrane were about 35 µa in this experiment Extracellular Current Injection To establish an ideal recording configuration as shown above is not always possible because deep insertion of a microelectrode into the oocyte often compromises the cell stability and produces undesired leak currents. According to Baumgartner et al. (Biophysical Journal, 1999, 77: ), under such sub-optimal conditions it should be possible to minimize the voltage errors by a second current-injecting point source placed extracellularly near the Current Electrode. Such a Compensation Electrode is

65 6.3 Compensation Electrode 59 implemented in itev 90. The Compensation Electrode is operated like the Current Electrode, but is restricted to current clamp mode only. Thus, zeroing of the offset potential (V-comp) and determination of the electrode resistance (R-comp), as well as the adjustment of the clamp controller is equivalent to what was described for the Current Electrode. In addition, the user has to specify the source for the controller s input signal (typically this is I-cel) and the scale factor, which determines the output amplitude with respect to the source (see Compensation Controller, on page 32). In the following we briefly describe the use of the Compensation Electrode as an extracellular current source. The aim of an extracellular Compensation Electrode is to limit the voltagedrop close to the Current Electrode by injecting current with the same wave form as I cel into the bath close to the Current Electrode, but magnified by a scale factor. Since the compliance of the Compensation Electrode is the same as that of the Current Electrode, the resistance of the Compensation Electrode has to be considerably lower. In the example shown below we used a blunt glass electrode clogged with agar to yield a resistance of about 50 kω. Figure 6.9: Recording configuration using the itev 90 Compensation Electrode. (A) Schematics of how the electrodes are placed inside or at the oocyte to measure V pel, I cel, V m, and to inject the compensation current, I comp. (B) Image taken through a microscope showing a real situation from the top. The Compensation Electrode was a glass electrode filled with 2 M KCl, and a tip clogged with 0.5 % agar. R comp was 50 kω. Note: The reference of the Compensation Electrode does not need to be connected to the ground pellets in the bath.

66 60 itev and Oocytes In Figure 6.10 the effect of extracellular current injection close to the Current Electrode is illustrated. In this case a scale factor of about 10 was used and the major effects of the compensation current were: ˆ The charging current transient peak (I cel ) was reduced by about a factor of 4.2 ˆ The membrane potential during the charging period was less inhomogeneous as seen by the smaller overshoot in V m. ˆ The rise-time of V pel was reduced, i.e. the membrane potential approached its requested value more quickly. Figure 6.10: Example for the use of the extracellular Compensation Electrode. In voltage-clamp mode the membrane potential of an oocyte was changed from -50 to 50 mv. The superficially impaled Potential Electrode and Current Electrode had resistances of about 500 kω. The black traces show the resulting I cel, V pel and V m, the latter was measured at an angle of 45 with respect to the Current Electrode. The red traces show a similar experiment with an active Compensation Electrode injecting a current 10-times larger than I cel. R comp was 45.6 kω Intracellular Current Injection The Compensation Electrode can also be used as second current-injecting electrode as to (i) increase the overall compliance, i.e. in order to inject

67 6.3 Compensation Electrode 61 more current, and (ii) to improve the homogeneity of current injection. The scale factor for the I comp current injection can be set to values starting from 1; a factor of 2.0 means I comp is about twice the size of I cel. Both applications are illustrated in the examples shown below. Figure 6.11: Recording configuration using the itev 90 Compensation Electrode as additional intracellular current-injecting electrode. (A) Schematics of how the electrodes are placed inside the oocyte to measure V pel, I cel, V m, and to inject current, I comp. (B) Image taken through a microscope showing a real situation from the top.