G-clamp v2 User Manual - University of Pittsburgh
Contents
1. Hi start PA Close Module Al 1 1 0 11 0 12 0 13 0 16341 Time alase Foy OTE moda La The Traces page shows the most recently acquired trace while the experiment is still in progress and all acquired traces of the selected channel after the experiment ends Time Sa rm thresh gsyn eur ry Si an l Plot 1 The History page plots the threshold g y values of consecutive experiments vs real time This chart has a buffer which can hold up to 200 pairs of threshold 2syn and time values after which the oldest pair becomes replaced with the newest pair with each new experiment The buffer can also be emptied with Clear Chart 27 By selecting more than one event file the behavior of a cell to a combination of several virtual synaptic inputs can be tested For example the effect of the after hyperpolarization following one or more action potentials on threshold gsyn can be tested by using one event file and giving these synapses supra threshold strength in combination with another event file which contains the synapse to be tested When using more than one event file the Variable property can only be assigned to one event file The first occurrence of a synaptic event in the event file for which Variable has been enabled sets the start time for the action potential identifying algorithm and allows action potentials generated earlier by synaptic events in other event files to be ignored Exp Set up start G scalin2. function on the column to set all event times to the same number of digits after the decimal point i e sub millisecond resolution Use Save As to save the spreadsheet as a tab delimited text file to the G clamp event file folder G clamp Event Files Open the file with Notepad Scroll down to the end of the file and delete any empty lines after the last event time Save the file Change the file name extension to DAT if you haven t done so in step 3 already rename the file so that it has a meaningful name If you want to combine the events from several DAT files into one EVT file open all DAT files with Microsoft Excel and use copy paste to copy all time values without 0 s into a single column of one spreadsheet Use the Sort Ascending function on the column before proceeding with step 2 above 34 11 Offline Utilities These are LabVIEW VIs written for offline analysis of G clamp data files Files with analysis results created by these VIs are written to the same directory which contained the G clamp data files analyzed 11 1 G clamp FileUtilities Master vi This VI serves as the gateway to the analysis VIs proper It is used to select G clamp data files and to feed these files into the selected file utility G clamp FileUtilities Master vi JJ File Edit View Project Operate Tools Window Help Contents of Count APs in GAI file uf E D Data 2007 012407 cic1 Modified ET gth 1 24 2007 11
3. 37 The output of this VI consists of indicators on the front panel mean Vm Ih mV pA APs in current segment Total of APs and two ASCII text files GAI Vm APs per segment txt and GAI Individual APs txt GAI Individual APs txt contains three columns of data The 1 column contains the locations time relative to start of recording minus Baseline Duration eech of all peaks or valleys detected The 2 column contains the interspike intervals and the 3 column contains the peak amplitudes A new set of three columns is added for each gai file processed GAI Vm APs per segment txt contains one column of data for each gai file processed The 1 value corresponds to mean Vm Ih mV pA the 2 value to Settling Time s the 3 value to segment length s the AN value to of segments and the remaining values to the number of peaks valleys found in each segment 11 3 G clamp Retrieve GTH file Results vi This VI retrieves the threshold 2syn value from a gth file together with some other useful information File Edit View Project Operate Tools Window Help del 100 1 1 1 1 1 1 1 1 1 1 0 000 0 100 0 200 0 300 0 400 0 500 0 600 0 700 0 800 0 900 1 000 Time sec Time sec 16115536 gth HE gi Amplitude File 1 of 12 The VI displays and works only on the voltage traces of a gth file Part of each trace can be defined as baseline with Baseline Start sec and Baseline Duration sec A mean value
4. experiment can cause memory problems on the host computer the waveform graph on the Trace page shows no more than 1 million data points Every trace or zoomed in section of a trace larger than 1 million data points will be decimated until it is less than 1 million Sot p 120 0000 Duration s 41 008 gon delay s 10 000 goff s 3909 Sample Rate liz 9008 DC current pA 0 0 Repeats 73 Parameters Event File Kinetic Erev m omg J 5Hz 4s_repeat prim 210events evt Bullfrog B cell nAchR Ny 0 00 3 20 00 Event File Kinetic Erev mV g ns ES 4s_repeat_new 3sec 560events evt Bullfrog B cell nAchR RS SF 5 40 With gon delay s and goff s an initial and a final part of the recording episode can be excluded from virtual conductance synaptic and or non synaptic activity and or the DC current command see below Exp Set up Traces History start 120 000 1 H 1 1 10 15 20 25 fa ll alx BZ lt 30 Start HET Duration s 4099 gon delay s 1 000 goff s 3099 Sample Rate kHz 10 000 DC current pA 500 0 Repeats 1 Parameters Event File Kinetic Erev mV g ns ES 5 fogB A Event File Kinetic fr el 15 2 Time rr es ee ed Data files generated by the Synaptic Gain module are of type gea 31 9 4 Strip Chart The Strip Chart module is an acquisition only module that records voltage and current traces for as long as the user wishes It w
5. is derived from all data points in the baseline part of the voltage traces and from the mean values of all voltage traces in a gth file a final mean value and standard deviation is calculated The VI also determines a mean action potential peak value by averaging the peak values of all action potentials found AP Search Start can be used to exclude conditioning action potentials prior to the test synapse Action potentials are found using the same algorithm and parameters threshold mV width datapoints as described in 38 section 11 2 for G clamp Count APs in GAI file vi All parameters can be changed until Set is pressed which initiates processing of the current file The output of this VI consists of an ASCII text file GTH Results txt which contains one line of tab delimited data for each gth file processed The columns in this file contain 1 column time of recording hh mm ss this time format is MS Excel compatible allowing plotting of results vs time 2 column mean membrane potential during baseline period mV 3 column SD of membrane potential during baseline period mV 4 column threshold 2syn nS St column mean action potential peak mV 11 4 G clamp Get Membrane Test Results vi This VI is an off line version of the Membrane Test function available in G clamp The latter is more geared towards a speedy analysis by averaging only a limited number of current responses and by using a simple least square fit
6. location in the project name data type delete them and create them new Next re link every instance of a network shared variable in all VIs in the project to the newly created variables Finally create the source distributions Main Target VI and Target Dynamic VIs again and copy the created files again to the embedded controller see above After a LabVIEW G clamp crash on the host computer G clamp is not able any more to connect to the embedded controller o Sometimes crashes result in changes to the file e ni rt ini on the embedded controller e g in deletion of the line server tcp enabled True It is therefore useful to have a working copy of ni rt ini available
7. This creates a source distribution in C ni rt startup Dynamic VIs on the host computer of all files required to run the experiment modules from within the master program on the embedded computer Copy the folder C ni rt with all its sub directories and files from the host computer into the root directory C of the embedded controller In the project under My Computer Host G clamp v2 Host lvlib open the file G clamp v2 Host cfg o In the section LastUsed replace the Target entry with the name of your embedded controller o Inthe section Available replace the existing Targets entries with the name of your embedded controller o Edit one of the existing target sections G clamp1 or G clamp2 delete the other one so that the section has the name of your embedded controller and the IP entry has its IP address 15 Tt is not necessary to add the host computer to the VI Server machine access list By default machine access should be unrestricted with the wildcard symbol 46 Trouble Shooting This section gives hints on how to deal with problems we encountered Disclaimer Because we do not completely understand the reason for these problems we do not claim that our solutions will always work or that they are the only possible and or best solutions The embedded controller is seen by MAX but LabVIEW or Internet Explorer MS Explorer FTP clients are unable to connect to the embedded controller o Make sure that embed
8. between the wait 4000 specifying the ramp duration and the wait 800 specifying the post ramp time before the recording episode ends If the midpoint of a ramp differs from 0 mV pA the value for the following command then should be relative to the ramp midpoint Leak Subtraction This script demonstrates the zap command It generates a sine wave The frequency of the sine wave increases at a constant rate from a start value to an end value In this example zap 15 fstart 0 fstop 5 the sine wave starts at 0 Hz and ends at 5 Hz The speed of the modulation is determined by the following wait command therefore in this case 5 Hz 0 Hz 10000 ms 0 5 Hz s Note that after the zap command the value of the next const command is absolute and not relative to the level achieved at the end of the sine wave Data files generated by the Vclamp module are of type ivb Simulate allows observation of the behavior of an implemented non synaptic conductance under voltage clamp conditions The recording amplifier is operated in voltage clamp mode and a script is used to run a voltage clamp protocol e g the protocol to measure I steady state inactivation described above The script together with the holding potential dialed in at the amplifier determine the Vm reading Based on the measured Vm G clamp calculates the current that would flow through the conductance at any time Instead
9. in the list of existing synaptic conductances brings up the parameter field for the conductance For details on the different kinetic models see the preceding section Creating a new synaptic conductance Synaptic Conductance Editor Synaptic Conductance Name NMDAR fall e rise 1 eta exp 1 Um Select action i i tau fall Duration Display Edit existing 7 Jo an ms duu ms OC Bi Ma Vm k gamma Afis mM SR mv Zon mV 1 05002 Existing Synaptic Conductances Bullfrog B cell nAchR A Bullfrog C cell nAchR NMDAR AMPAR Jampiitude eil 5 3 Deleting an existing synaptic conductance Select the conductance to be deleted in the list of existing synaptic conductances Synaptic Conductance Editor Synaptic Conductance Name Select action Delete existing T Existing Synaptic Conductances Bullfrog B cell nAchR A Bullfrog C cell nAchR NMDA R AMPA R Hie JAmpiitude eil 12 6 Seal Test This function which is similar to the seal test function in PClamp serves to monitor seal formation while patching a cell in voltage clamp A unipolar periodic voltage step is given and the current response is monitored As the patch pipette approaches the cell membrane and forms a seal the initially large current response becomes smaller and smaller The trace shown reflects the average of the last 10 current responses and the values shown for seal resistance Rseal and holding c
10. indicating that it is not an active script It is activated by starting to edit it To make any changes to the scripts permanent you have to exit the module by using the Close Module button Any changes made to scripts are lost if you exit G clamp v2 with the Vclamp module still open Setting fstart and fstop to the same value xy yields an unmodulated sine wave of frequency xy 21 Display Channel Vm Leak Subtraction GD im 0 06 amp papi mV 1 6 Dir This script generates a square pulse of 10 mV pA with a delay of 50 ms by first giving the amplifier command const 10 This command level is maintained for 50 ms before another amplifier command const 10 returns the command level to zero The recording episode ends after another 50 ms This pattern is repeated 50 times for 50 with 150 ms breaks in between episodes pause 150 If the last amplifier command does not return the command level back to zero that level will be maintained during the pause until the start of the next episode samplerate 10000 pause 3000 Display Channel Vm Leak Subtraction GD Im This script gives a series of negative square pulses of increasing amplitude The first pulse is 10 mV pA const 10 With increment 10 this value becomes 20 mV pA for the second episode 10 10 20 30 mV pA for the third epsisode 20 10 30 and so on With the last seventh episode
11. of using this value as a command to the amplifier the value is only displayed to the user as the Im trace and optionally saved to file vy Simulate 10 Tt seems like only sine waves symmetrical around 0 mV pA work Using a preceding const xx command to let the sine wave oscillate around xx mV pA does not work 24 Leak Subtraction is only for voltage clamp recordings to correct for a cell s passive membrane current i e leak current G clamp uses P N subtraction in which a series of scaled down versions of the command waveform are generated and the responses to the scaled down versions are accumulated and subtracted from the command response Scaled down versions are used to prevent active currents from being activated The number of these scaled down commands is the N referred to in P N The command waveform pulse P is scaled down by a factor of 1 N and this applied N times to the cell Since leak current has a linear response the Leak accumulated responses of the subsweeps approximate the leak Subtraction current component of the response to the actual command waveform For added flexibility in preventing the occurrence of active currents the polarity of the P N waveform can be reversed by making N in Protocol P N a negative value and by eliciting the scaled down versions from a different holding potential as dialed in at the recording amplifier by shifting it with Vhold mV To speed up the process of ru
12. proceeding to the next file 40 11 5 G clamp Convert stp to gai vi Converts stp files created by the Strip Chart module into gai files The original stp files remain in the folder 41 12 Data files 12 1 Names Data file names have the general form ddhhmmss in which dd is the day of month hh is the hour 24 hour clock mm is the minute ss 1s the second when the experiment finished and indicates the experiment type Vclamp IVB Gsyn Threshold GTH Synaptic Gain GAI All numerical values in the name have a leading zero if in the single digit range 12 2 Content Each G clamp experiment module has its own data file format 12 2 1 Vclamp IVB a SGL giving the number of iterations done 4 SGLs without meaning a SGL giving the episode duration in ms a SGL giving the sample rate in Hz a SGL giving the pause time in ms between epsiodes as often as episodes were performed a l D array of SGLs giving the voltage trace al D array of SGLs giving the current trace 42 12 2 2 Gsyn Threshold GTH aSGL giving the length of the following string astring giving the name of the event file used for the experiment a SGL giving the number of episodes 2SGLs giving the initial upper limit in nS for the search algorithm a SGL giving the pause time in ms from end of one episode to start of next a SGL giving the reversal potential Esey of the virtual synapse a SGL giving the sam
13. was started use the Abort Exp button in the lower right section of the G clamp v2 Host vi front panel This works only for experiments with the modules Vclamp and Threshold gsyn Synaptic Gain experiments can not be aborted and Strip Chart experiments are terminated with the Enough data button in this module s front panel Check the RT Loop Info indicator After each data transmission it indicates performance of the dynamic clamp feedback loop by showing mean cycle length and its standard deviation as well as maximum jitter A mean value larger than the set value indicates consistent failure to perform at the requested rate while a correct mean value but maximum jitter larger than the Y mean indicates an occasional failure which did not affect overall performance The Strip Chart module uses a data acquisition mode unrelated to the dynamic clamp feedback loop and therefore does not evaluate loop performance 2 1 Experiment Logging G clamp v2 keeps a running record of most experimental settings together with time stamps when an experiment is started User comments e g about drug applications can be added at any time This record is maintained in computer memory until one of two conditions is met achange in the data directory or exiting G clamp These two conditions trigger writing the experiment record to a text file in the data directory used last and the start of a new record if a change in data directory occurred Part o
14. 45 AM 24114558 gth 1 24 2007 11 46 AM 24114616 gth 1 24 2007 11 46 AM 24114639 gth 1 24 2007 11 46 AM 24114650 gai 1 24 2007 11 47 AM 24114750 gth 1 24 2007 11 47 AM 24114807 gth 1 24 2007 11 48 AM 24114823 gth 1 24 2007 11 48 AM 24114841 gth 1 24 2007 11 48 AM 24114857 gth 1 24 2007 11 48 AM 24114913 gth 1 24 2007 11 49 AM 24114929 gth 1 24 2007 11 49 AM 24114945 gth 1 24 2007 11 49 AM 24115001 gth 1 24 2007 11 50 AM 24115021 gth 1 24 2007 11 50 AM 24115036 gth 1 24 2007 11 50 AM 24115055 gth 1 24 2007 11 50 AM 24115121 gth 1 24 2007 11 51 AM 24115139 0th 1 24 2007 11 51 AM 24115155 gth 1 24 2007 11 51 AM 24115211 gth 1 24 2007 11 52 AM 241157996 ath 1 74 07 11 57 AM A file dialog opens at VI start or with Change Directory to navigate to a directory containing G clamp data files Press Current Folder to exit the file dialog window All files and subfolders are listed in alphanumerical order File types can be used to limit the file list to display only files of the selected type File Utilities is populated at VI start with the analysis VIs available and used to select an analysis VI Analysis VIs typically are specific for one of the G clamp data file types and the data files that can be processed by the selected analysis VI are shown in the file list on a gray background G clamp FileUtilities Master vi File Edit View Project Operate Tools Window Help Sie E File Utilities Retrieve GTH file Results zi Y D Data 200
15. 7 012407 cici mA Name Modified 24110719 ivb 1 24 2007 11 07 AM 24110823 ivb Geer 11 o AM 7 3 2 0 AM S AM 11 AM 11 AM 11 AM 12 AM 12 AM 13 AM 14 AM 78 07 11 14AM 24111924 vb 1 24 2007 11 19 AM 24112138 ivb PESE 11 21 a My Computer All files selected in the file list will be processed successively by the analysis VI selected when Do it is pressed Once the analysis VI finishes a new data directory file analysis VI selection can be made to process another set of data files Done stops G clamp FileUtilities Master vi 11 2 G clamp Count APs in GAI file vi This VI uses a peak detection function to find action potentials in files created by the Synaptic Gain module or by the Strip Chart module after the stp files have been converted to gai files 36 E G clamp Count APs in GAI file vi File Edit View Project Operate Tools Window Help y el 1 83 o Time s Current segment APs in current segment Total of APs vm mV weh Ps 25 My Computer Analyze toggles the VI from working either on the voltage trace current clamp recording or the current trace e g loose patch voltage clamp recording Accordingly peaks valleys toggles the function from detecting AP peaks or inward current peaks Peaks have to be above threshold mV while valleys have to be below Peak detection is based on an algorithm that fits a quadratic polynomial to sequential groups of data po
16. BRIDGE current clamp and cont SEVC voltage clamp are available y Q Set the MODE control to the same mode as selected on the Y AxoClamp 28 amplifier for correct selection of the Ext Command Only BRIDGE and SEVC Mode are supported by G damp Discont SEVC i seve Wiring Instructions A eS y 10 Vm amplifier output to analog in channel 0 TEVC Im amolifier output to analoa in channel 1 lt 4 2 Model 2400 The Model 2400 amplifier telegraphs several of its settings to the computer Actively used by the G clamp program are the MODE and GAIN settings while the FILTER and Cmembrane settings are only of informative nature to the user The remaining two controls PROBE GAIN and EXTERNAL CLAMP SIGNAL are NOT telegraphed and have to be set by the user for correct scaling of the amplifier output and the amplifier input command The status of the telegraphed amplifier settings are updated automatically each time before an experiment is started and an update can also be initiated with the button Amplifier Settings Update to the right 5 Synaptic Conductance Editor The Synaptic Conductance Editor allows the user to define the kinetics of a synaptic conductance gsyn for use with the experiment modules Threshold gsyn and Synaptic Gain Once a synaptic conductance has been defined its kinetic parameters are preserved in a configuration file and therefore defined synaptic conductances are available from the begin
17. G clamp version 2 0 User Manual Paul H M Kullmann and John P Horn Department of Neurobiology and Center for the Neural Basis of Cognition University of Pittsburgh School of Medicine E 1440 Biomedical Science Tower Pittsburgh PA 15261 http www neurobio pitt edu To download this document and the G clamp program go to http hornlab neurobio pitt edu Contact Information Dr Paul H M Kullmann Phone 412 648 9291 pkullman pitt edu Dr John P Horn Voice 412 648 9429 jph pitt edu Grant Support This document and the G clamp software that it describes were both developed at the University of Pittsburgh with support from National Institutes of Health grant RO1 NS21065 Copyright Information These materials are freely available for non commercial research and educational purposes The authors reserve the copyright and request that others using this material acknowledge its origin Those wishing to publish any of this material or to develop commercial applications must first contact the authors G clamp v1 2003 v1 2 October 1 2004 G clamp v2 February 1 2007 Copyright 2007 Table of Contents LG PROGRAM START epsssscctctcahcctde sessstsicute cd dadecsctiacussasdocccscausautssededdsssoebealdaveesasesbehavbedidasesestevtulectsasesssteleede 4 2 RUNNING AN EXPERIMENT wisissoccocsssescsescssscoosvecscsesnccossceasedesetncssesccassveee coesscnsevssseveddseteaesvevsesesussacseve 5 2 1 EXPERIMENT LOGGING cand
18. I FILE VI ccccccccccccsssscececececsesssuececececsessaaececccscsessaeceseeeesensaaeeeeeeeesenes 35 11 3 G CLAMP RETRIEVE GTH FILE RrsutLrgwvt NEE TE E E 37 11 4 G CLAMP GET MEMBRANE TEST Route 38 11 5 G CLAMP CONVERT STP TOFGAR RE 40 12 IA NN 41 12 1 NAMES a a ta 41 12 2 L GR RE NEE 41 12 2 1 Velamp IVB As A idol 4 12 2 2 Gsyrt Thr shold GIH E 42 12 2 3 Synaptic Gam GAL mio ae a de E E aa Ea E E EN 42 12 2 4 ET E let A ca E E Eu 42 APPENDIX A G CLAMP INSTALLATION csssccssssscccsssccsssscccccssssccscssccccessscccesssacceessscecscsssccessnee 44 FART WAR DAE ITE EEE EEE ETE EIEEE AEE ETE E E ES E A EE 44 Connecting embedded controller and host computer 44 Connecting recording amplifier and break out box BN 20001 44 AxoClamp 2B amplifier UI IA DORT WARE rara ee TROUBLE SHOOTING aiii dia daa 1 Program Start Start the embedded controller Start LabVIEW 8 on the host computer and open the project G clamp lvproj Open G clamp v2 Host vi under My Computer Host G clamp vi Host vi Front Panel EI jo m By ext C Pata Recent recordings 011707 c2c1 j G damp2 Transient Potassium DA Neuron Transient Potassium Bullfrog g E 0 00 Once start up of the embedded controller is complete press the Run button to start G clamp v2 Host vi The VI will go through an initialization process that includes starting G clamp components on the embedded controller and estab
19. as originally developed to record long uninterrupted sequences of spontaneous pacemaking activity of midbrain DA neurons Because no computations are performed and no commands are given to the amplifier recorded data can be sent every second from the embedded controller to the host computer and displayed to the user without interference with the ongoing data acquisition The only parameter that can be set by the user is sample rate Hz After initiating a data acquisition with the Start button the Start and Close Module buttons become disabled and grayed out Data are acquired transferred to and displayed on the host computer in 1 second packets This module always saves the acquired data into a file with the file name extension STP irrespective of the status of the Save Data button in the lower right section of the G clamp user interface The file is created when the first 1 second packet arrives and its name is built from the recording time of this first packet New data are appended to this file as they arrive on the host computer STP files can be converted to a GAI file with the offline utility Convert stp to gai vi Close Module Sample Rate Hz 0 0000 Seconds recorded Display Channel 68 1 H 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 H 1 1 y 0 2 0 4 0 6 0 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48 5 Time Time Talaj The Freeze button stops updating of the waveform graph thus allowing closer inspe
20. ction of the trace currently on display This has no effect on the on going transfer and saving of data 32 10 Event files Event files are new in G clamp v2 and replace the template files used in G clamp v1 Event files are text files with the file extension EVT containing a single column of ascending numbers standing for the timing in ms of virtual synaptic events The type of virtual synapse i e its kinetic is determined by the user with the Kinetic control of the experiment modules Threshold gsyn and Synaptic Gain Thus a single event file can be used for different types of virtual synapses Note that the length of a recording episode is user specified in both experiment modules Threshold gsyn and Synaptic Gain Therefore virtual synaptic events listed in an event file to occur at times after the end of the recording episode are ignored Note also that if an event file contains two identical time values two synaptic events occurring at the same time only one event will start at the specified time and the second one will start in the next iteration of the dynamic clamp loop The delay between the two events therefore will depend on the user specified sampling rate Consider the relation of user specified sample rate and the temporal precision used when creating the event file For example in an event file with a precision of 50 us two events listed to occur at 100 95 and 101 00 ms will start at these times if sampling rate is se
21. ded controller and host computer are on the same subnet If the problem persists try different subnet masks E g on one of our set ups subnet mask 255 255 255 0 for embedded controller and host worked fine while on another we had to change the mask to 255 255 0 0 to make it work G clamp starts fine updating RT Target System Info or amplifier settings if the Model 2400 amplifier module is in use works but when an experiment is started no commands are given to the amplifier and no data are recorded o This seems to be a problem with the deployment of the network shared variables from the host computer to the embedded controller when the host computer is running under an IP address different from the one that was used when the shared variables were created during G clamp development Possible work arounds In the project right click the embedded controller and select Connect This apparently deploys the missing stuff to the embedded controller After the deployment you can safely disconnect With host computer and embedded controller connected directly via a cross over cable change the IP address of the host computer to 136 142 127 88 If you disconnect from the embedded controller and connect to your LAN you have to change back to your network administrator assigned IP address Replace all network shared variables with newly created ones huge PITA Carefully write down for each network shared variable under My Computer Host its properties
22. ent Set up your experiment with the corresponding experiment module for details see the specific experiment module section under Experiment Modules Make sure that any settings in the amplifier module section that have to be set by the user see section Amplifier Modules for details correspond to the real settings at your amplifier Check the control Virtual Conductances on the General page for the status of the conductances green enabled red disabled and the strength of the conductance in nSy These conductances can be used with all experiment modules except the Strip Chart module Check the status of the control Save Data green enabled red disabled The Strip Chart module ignores this control and always saves data to file Closing a module sometimes results in a LabVIEW error message This error is non fatal and can be ignored Select Continue in the error dialog to proceed with the G clamp session The kinetics of these conductances are hard wired into the software and can not be changed while running G clamp v2 Instructions for modifying or adding a conductance are not yet available for G clamp v2 However consulting the Programmer s Guide for G clamp v1 2 http hornlab neurobio pitt edu downloads htm should give you an idea about how to Start the experiment with the Start button on the front panel of the corresponding experiment module If you want to abort the experiment after it
23. for 7 the command level reaches 70 mV pA Note increment 10 is equivalent to decrement 10 Display Channel Vm Leak Subtraction aD Im 8 81 finv This script demonstrates the combination of a pre pulse and a variable test pulse With const 50 the pre pulse is initiated and maintained for 400 ms wait 400 By being relative to the preceding command level const 20 next changes the command level to 70 mV pA 50 20 70 in the first episode With increment 20 the change relative to the pre pulse becomes 40 mV pA in the second episode 60 mV pA in the third episode and so on M type K Current Ya depending on Vh set 1st const command to reach 30 mV Y samplerate 5000 Display Channel Kol Leak Subtraction ab Im EHS This script demonstrates the use of the ramp command The numerical value of the ramp 80 command sets its amplitude 80 mV pA with the start value defined by the preceding amplifier command level In this case set to 40 mV pA with the preceding const 40 The speed of the ramp is 20 s 80 4000 ms as set by the following wait 4000 command At the end of the ramp the amplifier command level returns automatically to a value corresponding to the midpoint of the ramp Note that although in this example the ramp 23 returns to 0 mV pA as it starts at 40 mV pA and ends at 40 mV pA a const 0 command is required in the script
24. for a current clamp command and text in square brackets gives additional information o const xy sets the amplifier command to a new value which remains in effect for the duration of the following wait command o ramp xy initiates a linear change in the amplifier command starting at the previous amplifier command level the speed of the change is determined by the following wait command o zap xy fstart xy fstop xy initiates a frequency modulated sine wave of amplitude xy command to the amplifier the start frequency is set with the numerical value after fstart inHz and the end frequency with the numerical value after fstop in Hz the speed of the frequency modulation is determined by the following wait command All of these commands operate relative to the holding potential voltage clamp or DC current command current clamp dialed in on the recording amplifier The const and ramp commands can be followed by either decrement xy or increment xy with xy denoting the change in the amplifier command which successively changes the effective value of the preceding const or ramp command from episode to episode The change becomes effective with the second episode i e in the first episode the effective amplifier command corresponds to the value of const or ramp To add a new script scroll to the end of the protocol array The last element is grayed out
25. forever for a wrong amount of data from the controller and therefore to become unresponsive Even if the reason for this bug could be figured out we recommend the direct connection Because there is no other network traffic the direct connection is so fast that even tens of Megabytes of data several minutes of experiment with the Synaptic Gain Module can now be transferred to the host computer in almost no time for display and saving Connecting recording amplifier and break out box BNC 2090 AxoClamp 2B amplifier OUTPUT 10 Vm gt analog in 0 OUTPUT Im gt analog in 1 INPUT EXT MEI COMMAND gt analog out 0 INPUT EXT VC COMMAND gt analog out 1 Model 2400 amplifier variable Output gt analog in 8 Fixed Output x 10 Vm gt analog in 9 Fixed Output Im gt analog in 10 Telegraph Output FREQ gt analog in 12 Telegraph Output MODE gt analog in 13 Telegraph Output GAIN gt analog in 14 Telegraph Output Cmembrane gt analog in 15 EXTERNAL command input gt analog out 0 Alternatively according to NI you can also use normal network cables with a hub between embedded controller and host computer We did not test this alternative and can not exclude the possibility that this solution might have the same problems as experienced when going through a LAN 45 Software Extract the content of the file G clamp zip on your drive C This will result in a folder C G clamp with
26. g Close Module Duration s 209 Pause s 1 00 Sample Rate kHz 10 000 Iterations 710 Repeats 71 4 Parameters Event File d Erev mV Variable g ns ae for 1s evt Bullfrog B cell nAchR uf J 0 00 9 SEX j Erev mV Variable g nS Bullfrog B cell nAchR Si S e 9 Haag Erev mV Variable o nS start ATT WER D 1 0 6 0 3 1 Time PEIA alaj l an lt Data files generated by the Threshold gsyn module are of type gth 28 9 3 Synaptic Gain This module uses event files to stimulate a neuron with patterns of virtual synaptic activity and it determines the number of action potentials generated by these synaptic events Traces History start Sof ps 240 0006 Duration s 41 000 gon delay s 0 000 goff s 41 000 Sample Rate kHz 10 000 DC current pA Zog Repeats J1 Parameters Event File 5Hz 4s_repeat prim 210events evt S amp S Event File J 5Hz 4s_repeat_new 3sec 560events evt Event File gt Event File e The controls in the Parameters section have identical function as the corresponding controls in the Threshold gsyn module In this module synaptic strength s is fixed therefore g nS specifies Gan Duration s determines the length of the recording episode Exp Set up Traces History start sot Ua 240 000c Because displaying the potentially huge amount of data acquired with a Synaptic Gain
27. iles 6 3 TEXTEING G CEAMPP V2 sicsucs satislissccesosssecits senntecess ceshes dented sve codnes vaeesuuncecsdeeb cacede nes cs dacessdetesosbessees iaeoa saia 6 4 AMPELTRIE RiEMODU GES aee a aE rE OOo L Erte e Eege deeg 7 4 1 AXO CLAMP 2 EE 7 4 2 MODEL TEE 8 5 SYNAPTIC CONDUCTANCE EDITOR cssssscscsssssssessscccsssscssecccessssssssececescessscssenecesessssssceneceseees 9 dels CREATING A NEW SYNAPTIC CONDUCTANCE ssssssesessessereeetsesseseretesssessereesessereesesesserereessssesrreesess 9 DDD ALPHA TURCO A ei Saved ae Res A ee ee Een 10 5 1 2 double exponential 2 i 2 thi setae Ee ebe e a 10 5 1 3 voliage dependent s s se hice Bites a ada 10 5 2 EDITING AN EXISTING SYNAPTIC CONDUCTIANCE 11 5 3 DELETING AN EXISTING SYNAPTIC CONDUCTANCR 11 6 SEAT TEST RE 12 Tes MEMBRANE TEST iecdedeccesicsbecevecsccstshinss deer deet 14 Talc CALCULA E Le 15 8 BRIDGE TES WEEN 17 9 EXPERIMENT MODULES isssssisccssssssssciss Eddie ENEE EENS edd Reeder dees Zeie 19 9 1 VA WA Sine BEEE OERE Sa coca 19 9 2 THRESHOLD G SYN E AEE EE EEEE iaa 25 9 3 SDAN AREKE TAN CERN 28 9 4 STRIP CHARM LAEE INN 31 10 INCA O ONO 32 10 1 CREATING EVENT FILES ii aiii 32 EE 32 11 OFFLINE DN RTE RR UR piss csccscccdisscscssceccescsdedscocssascasecsacesoossedsescede cons sssescescssoctecscodsecsese cacvessessescobeceat 34 11 1 G CELAMP FILEUTILITIES MASTER VI oi d DEES ENEE Cole Cewek oe Sees eaves 34 11 2 G CLAMP COUNT APS IN GA
28. ing seemingly implying that it is intended only for voltage clamp recordings In fact the purpose of this module is to generate several types of command waveforms for the amplifier irrespective of whether the amplifier is in voltage clamp or dynamic current clamp mode Currently three types of command waveform are available square pulse ramp zap a frequency modulated sine wave These commands can be combined sequentially and command amplitudes can be made to increment or decrement from one iteration to the next Almost all experiment parameters are defined in a script like protocol Protocols are stored in an array of which only one element protocol is visible at any time and this visible protocol is the one that will be executed upon pressing Start To scroll through the array of protocols use the horizontal slider at the bottom of the Scripts window When scrolling through the array of protocols the waveform display shows exactly what command s will be given to the amplifier Similarly editing a protocol in the Scripts window triggers continuous updates of the waveform display If your waveform does not look like what you expect you probably have a syntax error in your protocol Note that no error checking is implemented and therefore execution of a script containing syntax errors could crash G clamp Before explaining some example protocols the general rules of the protocol syntax shall be described All expressions ha
29. ints The number of data points used in the fit is specified by width datapoints Each peak valley found is marked in the waveform graph by a red square An initial part of the trace can be excluded from the peak search with Settling time s If Settling time s gt 0 then this initial part of the trace is used to calculate mean Vm Ih mV pA otherwise mean Vm Ih mV pA yields NaN Not a Number Analysis of the whole trace occurs in consecutive segments The length of these segments can be adjusted with segment length s while of segments informs the user about the total number of segments for a specific segment length Next proceeds to the next segment while Previous goes back to the segment before the current one Any change in Analyze peaks valleys threshold mV width datapoints or Settling time s triggers re analysis of the current trace A change in segment length s triggers re analysis of the trace from its beginning 15 width datapoints is coerced to a value greater than or equal to 3 The value should be no more than about 1 2 of the half width of the peaks valleys and can be much smaller but gt 2 for noise free data Large widths can reduce the apparent amplitude of peaks and shift the apparent location For noisy data this modification is unimportant since the noise obscures the actual peak Ideally width should be as small as possible but must be balanced against the possibility of false peak detection due to noise
30. lding current Ih is determined from the last 25 of the pre voltage step period Steady state current Iss is determined from the last 25 of the voltage step period The transient portion of the current response is fit with a single exponential which gives the time constant tau The area charge Q under the current transient can be used to calculate access resistance Ra and membrane resistance Rm Q1 CV Tau Q3 Iss la AV Ra Rm Ihold at Vhold The total charge Qt under the transient is the sum of the charge Q1 in the transient above the steady state response plus the correction factor Q2 Qt Q1 Q2 QI is found by integrating the charge above Iss Q2 is calculated as AI x tau Iss Ih x tau The small error charge Q3 introduced with the calculation of Q2 is ignored After Qt has been derived Cm can be calculated Cm Qt AVm 1 where AVm is the amplitude of the voltage step In the steady state the relation between AVm and AV is AVm AV x Rm Rt AV x Rm Ra Rm 2 16 where Rt Rm and Ra are the total membrane and access resistances respectively Substituting for AVm from 2 into 1 derives Cm Qt x Rt AV x Rm 3 Substituting 3 for Cm in definition of the time constant Ra x Cm tau Qt x Ra AV yields Ra tau x AV Qt 4 which provides access resistance directly as a function of measured variables The total resistance is calculated from the steady s
31. lishing a communication link between G clamp software running on the host computer and on the embedded controller The final initialization step is the loading of an amplifier module see section Amplifier Modules DO NOT interact with the front panel of G clamp v2 Host vi until the amplifier module has been loaded Transient Potassium Bullfrog P e RT Loop Set the MODE control to the same mode as selected on the mean uS AxoClamp 28 amplifier for correct selection of the Ext Command Only BRIDGE and SEVC Mode are supported by G damp Save Data SD Wiring Instructions 5 ep 15 10 Vm amplifier output to analog in channel 0 Im amolifier output to analoa in channel 1 Mi Abort Exp max jitter Set your data directory either by typing a path into the DataDirectory control on the General page or by using the folder button to the right of the control to open a file dialog box Navigate to the designated data directory and finalize your selection by pressing Current Folder in the dialog box Open up to three experiment modules by selecting them from the list under the menu item ExpModules Each experiment module will be loaded into a sub panel starting on the page Exp Module 1 and continuing with pages Exp Module 2 and Exp Module 3 If you try to load more than three modules a dialog will appear asking you to cancel loading the fourth module or to close one of the already open modules 2 Running an experim
32. n potential the current setting becomes the new lower limit Typically within 10 iterations the procedure zeroes in on threshold gsyn The peak of an action potential has to exceed 0 mV in order to be identified as an action potential The type of synapse is determined by the selection made from the Kinetic control and by the setting for its reversal potential Erev mV The timing of the synaptic event is specified with the control Event File To select an event file click on the folder symbol to the right of the control and select an event file from the file dialog The Variable control has to be enabled green to allow for the iterative adjustment of synaptic strength Disabling Variable red keeps the strength of a synapse constant and is an easy way to test the behavior of a cell repeatedly to the same synaptic input start G scaling Close Module Pause s 11 00 Sample Rate kHz 10 000 Iterations 10 Erev mV J 100 evt Bullfrog B cell nAchR zi Zoo Event File Kinetic Erev mv la Event File Kinetic Erev mV Variable amp 0 00 Event File Kinetic Erev mV Variable Event File Kinetic Erev mV Variable Event File Kinetic Erev mV Variable Event File Kine Erev mV Variable Duration s determines the length of one recording episode Pause s the length of the break from then end of one episode to the start of the next and Iterations determines the number of episodes 26 Exp Set up Traces
33. ning whenever G clamp v2 is started The Synaptic Conductance Editor also allows modification editing of an existing synaptic conductance and its removal deleting from the configuration file The Editor is started from the G clamp v2 Host vi menu Edit gt Synaptic Conductances At start it reads the configuration file and populates the list of existing synaptic conductances with their names The Cancel button stops the Synaptic Conductance Editor and exits it without any changes to the configuration file A new synaptic conductance or modifications to an existing synaptic conductance become available for use immediately after exiting the Synaptic Conductance Editor i e no G clamp restart is required E Synaptic Conductance Editor Synaptic Conductance Name Select action ES Time alaj Amplitude alfy Next the user has to select an action Create new Edit existing Delete existing 5 1 Creating a new synaptic conductance First the user has to select one of three possible kinetic models for the new synaptic conductance alpha function double exponential voltage dependent 10 E Synaptic Conductance Editor Synaptic Conductance Name non NMDA Select action Create new a Select kinetic model alpha function vf HE Amplitude alu Once a kinetic model has been selected a model specific field for setting the model specific parameters becomes visible Any change of a pa
34. nning the leak subtraction the scaled down versions of the command waveform are executed without the delay between episodes that is defined with the pause command in the script Instead brief breaks before Pre Pulse and after Post Pulse each subsweep can be added with the Settling Times ms controls With Leak Subtraction and Save Data enabled G clamp generates two data files One file contains the raw data and the other one contains the leak subtracted data Both files are of type ivb and have a name consisting of the usual recording time stamp ddhhmmss plus leak and test pulses for file with the raw data and leak subtracted for the file with the corrected data 11 S d day h hour m minute s second 25 9 2 Threshold gsyn This module determines the strength of a virtual synaptic conductance required to bring the neuron under observation to action potential threshold It does this in an iterative process in which the strength of the virtual synapse is adjusted depending on whether the previous trial elicited an action potential or not An initial starting value g nS reflecting an upper limit has to be set by the user The procedure operates by setting the strength of the virtual synapse to the arithmetic mean of the upper and a lower limit which is initially zero If this setting elicits an action potential the current setting becomes the new upper limit if it fails to elicit an actio
35. ple rate Hz as often as episodes were performed a SGL giving the strength of the virtual synapse in nS al D array of SGLs giving the voltage trace al D array of SGLs giving the current trace 12 2 3 Synaptic Gain GAI aSGL giving the length of the following string asString listing the template file s used for the experiment aSGL giving 1 sample rate sample rate is in Hz a SGL without any meaning aSGL giving the number of action potentials elicited aSGL giving the length of the traces as of data points anumber of bytes 0s to extend header length to a total of 512 bytes voltage trace SGLs current trace SGLs 12 2 4 Strip Chart STP aDBL of value 1 a DBL giving the number of data channels 2 as many DBLs as indicated by the previous DBL each giving the sample rate 43 as often as 1 sec episodes were recorded for each data channel a 1 D array of SGLs comprising a 1 sec episode Use Convert stp to gai vi in G clamp lvproj My Computer OfflineUtilities to convert stp files to gai files 44 Appendix A G clamp Installation Hardware Connecting embedded controller and host computer Connect both machines directly using a cross over cable DO NOT go through a local area network LAN We found that when going through a LAN transfer of larger amounts of data gt 100 000 bits is not reliable causing the host computer part of the G clamp software to wait
36. r all of the current record can also be marked and with copy paste transferred to other applications 3 Exiting G clamp v2 The Exit button on the front panel of G clamp v2 Host vi stops G clamp v2 Host vi and the G clamp software running on the embedded controller It also saves the current experiment log as well as information about the amplifier module and the data directory to the configuration file for use as start up parameters the next time G clamp v2 is started 4 Amplifier Modules Amplifier modules contain amplifier specific controls required to communicate the recording mode current clamp voltage clamp to the G clamp software The last module used is loaded at program start To change the amplifier module select another amplifier on the General page of the experiment section Currently modules for two amplifiers are available AxoClamp 2B from Molecular Devices formerly Axon Instruments with the x 0 1 LU gain headstage Model 2400 from A M Systems Inc with a headstage that has the 100 MQ 10 GQ feedback resistor combination If you have a different amplifier or one of these amplifiers with a different headstage consult the manual with the set up instructions 4 1 AxoClamp 2B The AxoClamp 2B amplifier has two separate inputs for external current and voltage commands For the G clamp program to use the right command input the user has to set the recording mode in the amplifier module section Note that only
37. rameter triggers an update of the conductance waveform graph which shows the template for the synaptic event that G clamp will use whenever the Threshold gsyn or Synaptic Gain modules execute This template has a finite length which the user has to specify with the Duration parameter 5 1 1 alpha function Zsyn t k x t x exp t tau with k e tau and e the base of the natural logarithm 5 1 2 double exponential Zsyn t k x exp t taurise exp t taufan with k a dimensionless factor that scales the peak to unitary 1 nS conductance 5 1 3 voltage dependent This kinetic model mimics a NMDA receptor conductance and its voltage dependent block by extracellular Mg Zsyn Vm t k x exp t taurise exp t taugn 1 eta x Mg x exp 1 x gamma x Vm with k a dimensionless factor that scales the peak to unitary 1 nS conductance This model as well as the default values of its parameters taUrise 0 67 ms taufa 80 ms eta 0 33 mM gamma 0 06 mV are from Zador Koch amp Brown The voltage dependence of this conductance can be studied by changing the parameter Vm and by toggling the waveform display between conductance g and current I I is calculated assuming a reversal potential of 0 mV Zador A Koch C amp Brown TH 1990 Biophysical model of a Hebbian synapse PNAS 87 6718 6722 11 5 2 Editing an existing synaptic conductance Selecting the conductance to be edited
38. several subfolders Start Measurement amp Automation Explorer MAX and start the Configuration Import Wizard under File gt Import to import the file G clamp v2 nce from C G clamp Main VIs Target to your embedded controller This file contains all the NI DAQmx Scales Global Virtual Channels and Tasks defined for the AxoClamp 2B and Model 2400 recording amplifiers Under Devices and Interfaces complete the sections NI DAQmx Devices and PXI Systems according to your hardware Start LabVIEW 8 and open the LabVIEW project G clamp lvproj from C G clampl In the project right click My Computer and select Properties In the upcoming Options window go to VI Server Machine Access and add the IP address of your embedded controller to allow access In the project right click Project G clamp Ivproj and select New gt Targets and Devices Search for and add your embedded controller to the project Right click your embedded controller and select Properties Under VI Server Configuration check the box TCP IP to enable this protocol Test whether this is really necessary Under My Computer gt Build Specifications select Main Target VI right click and select Build This creates a source distribution in C ni rt startup on the host computer of all files required to run the master program on the embedded computer G clamp v2 Target vi Under My Computer gt Build Specifications select Target Dynamic VIs right click and select Build
39. t to 20 kHz or a multiple of 20 kHz However if the sampling rate is only 10 kHz temporal precision of 100 us 100 95 will be rounded up to 101 0 The first of these two events will therefore start at 101 0 ms and the second event as described in the preceding paragraph will start at 101 1 ms 10 1 Creating event files Any text editor can be used to create an event file File requirements are The file has to be saved as an ASCII text file The file name extension has to be EVT The file contains only numbers event times arranged in a single column in ascending order Time unit is milliseconds ms No empty lines or formatting codes after the last number with Neurosim To create an event file with Neurosim follow the instructions in the Neurosim manual for creating a template file of virtual synaptic plasticity For each synapse e g primary synapse n secondary synapses Neurosim creates a text file of type DAT which contains the event times for that synapse To use these files as event files in G clamp v2 the DAT files have to be re formatted and re named 12 If real simultaneous start of the two events is required place the time values in separate event files and execute both event files together 4 5 33 Convert the line arrangement of event times in the DAT file to a column arrangement by opening the DAT file with Microsoft Excel Use the Increase Decimal and or Decrease Decimal
40. tate response Rt AV AI Ra Rm 5 from which one obtains Rm Rt Ra 6 17 8 Bridge Test This function serves to monitor and adjust the settings of the bridge circuitry while recording in current clamp from a cell A unipolar periodic current step is given and the voltage response is monitored Because the much smaller capacitance of the pipette charges much faster than the whole cell capacitance the voltage response has two phases The initial almost instantaneous change reflects charging of the pipette capacitance and its amplitude reflects the size of the access resistance By setting the bridge so that the slower response of the whole cell capacitance seems to rise exactly from the pre current pulse level the voltage drop across the access resistance is eliminated The trace shown reflects the average of the last 10 voltage responses EF Bridge Test Liradarataral vunn Time xs 0 08 19 Jamplitude mv Gill If the recording amplifier and the corresponding amplifier module of the G clamp software is in voltage clamp mode a voltage command will be given 7 Detection of this change is facilitated by increasing the cut off frequency of the low pass filter operating on the amplifier s voltage output and by turning up the capacitance neutralization of the amplifier to speed up the charging process Bridge Test 19 9 Experiment Modules 9 1 Vclamp The name of this module is somewhat mislead
41. to determine the time constant of the averaged current response G clamp Get Membrane Test Results vi on the other hand averages as many current responses as supplied with i e as many as are contained in an ivb data file and it uses the Levenberg Marquardt algorithm to determine the least squares set of coefficients in an iterative process For details on the calculations see section 7 Membrane Test and 7 1 Calculations 39 G clamp Get Membrane Test Results vi SEE File Edit View Project Operate Tools Window Help KU To perform the exponential fit to the decay phase of the current transient the function has to be provided with a information about the voltage pulse used and b initial guess coefficients for the fit The voltage pulse has to be defined with Baseline Duration Pulse Duration and Pulse Amplitude Initial Guess Coefficients Fit contains three numerical values The first value corresponds to the initial amplitude of the signal the second value corresponds to the inverse of the time constant of decay and the third value corresponds to the end value ultimately approached by the exponential All user settable parameters can be changed any time Each change triggers a re calculation of the outputs With Done the VI proceeds to the next data file or stops after processing the last data file This VI does not save processing results to file therefore its output Ra Rm Cm tm and Th has to be written down by the user before
42. urrent Ih are based on this average Th is the mean holding current during the last 25 of the pre voltage step period and Rseal is then calculated according to Ohm s Law from the difference between Ih and the last 25 of the current response during the voltage step gt Seal Test 1 1 I Ke 0 00 2 50 5 00 7 50 10 00 12 50 15 00 17 50 20 00 nol Time xs E nee xs BIEL ampitude pA Gg If the recording amplifier and the corresponding amplifier module of the G clamp software is in current clamp mode a current command will be given Seal Test 13 14 7 Membrane Test This function which is similar to the membrane test function in PClamp uses the current response to a unipolar voltage step to calculate several parameters after whole cell configuration has been achieved Membrane Test The fit limits are relative to the difference between peak response and holding current for the upper limit and relative to the difference between peak response and steady state current for the lower limit The trace displayed and all calculations are based on the average of the last 10 responses The button Add to Log creates an entry with the current values in the experiment protocol If the recording amplifier and the corresponding amplifier module of the G clamp software is in current clamp mode a current command will be given 13 7 1 Calculations 4 peak AV pulse Vhold amplitude Ho
43. ve to be typed in lower case In most commands consisting of an expression and a numerical value expression and numerical value have to be separated by exactly one space Each command consists of a separate line Comments can be added to a script by placing the symbol at the beginning of each comment line Required commands for a script are in the following xy denotes a numerical value and text in square brackets gives additional information o samplerate xy sample rate in Hz o pause xy time in ms from end of one episode to start of next o Tor zw indicates start of a recording episode with the duration of the recording episode determined by the Scrolling through the array of protocols while grabbing the slider with the cursor does not always trigger a waveform update In this case move the cursor into the script field and click the left mouse button once 20 sum of all wait commands from here to the end command xy has to be at least 1 o end indicates end of the recording episode o wait xy time in ms before a change in the amplifier command is given at least one wait command has to be within the for xy end sequence to set the duration of the recording episode A wait command can not be followed by another wait command Commands that change the amplifier command are in the following xy denotes the amplitude of the command in mV for a voltage clamp command and in pA
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