winwcp v5.3 - documentation & help · winwcp v5.3.8 whole cell electrophysiology analysis...

StrathclydeElectrophysiologySoftware

WinWCPV5.3.8

WholeCellElectrophysiologyAnalysisProgram

(c)JohnDempster,UniversityofStrathclyde1996-2018

Introduction>MainFeaturesofWinWCPWinWCPisadataacquisitionandanalysisprogramforhandlingsignalsfromwhole-cellelectrophysiologicalexperiments:whole-cellpatchclampexperiments,single-andtwo-microelectrodevoltage-clampstudies,simplemembranepotentialrecordings.Whole-cellsignalsareproducedbythesummationofcurrentsthroughthe(usually)largepopulationofionchannelsinthecellmembrane,andthusconsistofrelativelysmoothcurrentorpotentialwaveforms.Theamplitudeandtimecourseofsuchsignalscontaininformationconcerningthekineticbehaviouroftheunderlyingionchannels,andothercellularprocesses,whichcanbeextractedbytheapplicationofavarietyofwaveformanalysistechniques.

WinWCPprovides, in a single program, the data acquisition and experimentalstimulus generation features necessary to make a digital recording of theelectrophysiological signals, and a range of waveform analysis procedurescommonly applied to such signals.WinWCP acts like a multi-channel digital

oscilloscope, collecting series of signal and storing them in a data file onmagneticdisk.Itsmajorfeaturesare:Recording

4-16*analoginputchannels256-8388608*samplesperrecordingsweep.2billionrecordsperdatafile.2-4*channelstimulusvoltagewaveformgenerator. 4-8*digitaloutput lines,foroperatingsolenoidcontrolledvalvesorotherexperimentaldevices.

Externaltriggerinput,tosynchroniserecordingsweepswithexternalevents.Spontaneouseventdetector.*DependinguponlaboratoryinterfaceAnalysisSignalaveraging.Digitalleakcurrentsubtraction.Automaticwaveformamplitude/timecoursemeasurement.Mathematicalcurvefittingtowaveforms.Non-stationarycurrentfluctuationanalysis.Quantalanalysisofsynapticcurrents.Synapticdrivingfunctionanalysis.SynapticcurrentandHodgkin-Huxleycurrentsimulations.

Next

Introduction>ConditionsofUseTheStrathclydeElectrophysiologySoftwarepackageisasuiteofprogramsfortheacquisitionandanalysisofelectrophysiologicalsignals,developedbytheauthorattheStrathclydeInstituteforPharmacy&BiomedicalSciences,UniversityofStrathclyde.At the discretion of the author, the software is supplied free of charge toacademic users and others working for non-commercial, non-profit making,organisations. Commercial organisations may purchase a license to use thesoftwarefromtheUniversityofStrathclyde(contacttheauthorfordetails).Theauthor retains copyright andall rights are reserved.Theusermayuse thesoftwarefreelyfortheirownresearch,butshouldnotsellorpassthesoftwareontootherswithoutthepermissionoftheauthor.Exceptwhereotherwisespecified,no warranty is implied, by either the author or the University of Strathclyde,concerningthefitnessofthesoftwareforanypurpose.The software is supplied "as found" and the user is advised to verify that thesoftware functionsappropriately for thepurposes that theychoose touse it.Anacknowledgement of the use of the software, in publications to which it hascontributed,wouldbegratefullyappreciatedbytheauthor.JohnDempster

StrathclydeInstituteforPharmacy&BiomedicalSciencesUniversityofStrathclyde161CathedralSt.GLASGOWG40REScotland

Tel(0)1415482320Fax.(0)[email protected]

Next

GettingStarted>HardwareRequirementsTorunWinWCPyouwillrequireanIBMPC-compatiblepersonalcomputerwithatleast16MbyteofRAM,a66MHz80486(orbetter)CPU,andtheMicrosoftWindows95,98,NTV4,2000,XP(32bit),VistaorWindows7(32bit)and(64bit)operatingsystems.A laboratory interface unit is required to perform analog-digital (A/D) anddigital-analog (D/A) conversion of the signals and stimulus waveforms. Thefollowingfamiliesoflaboratoryinterfacesaresupported:

CambridgeElectronicDesign1401,1401-plus,Micro1401,Power1401.

NationalInstrumentslaboratoryinterfacecards.

AxonInstrumentsDigidata1200,1320/22,1440/A,1550or1550A.(Note.DataacquisitionisnotsupportedwiththeDigidata1200and132XinterfacesunderWindows7(64bit).

InstrutechITC-16orITC-18.

HekaEPC-9,EPC-10.

BiologicVP500.

TecellaPico,Triton,TritonPlus.

Next

GettingStarted>InstallingWinWCPToinstalltheWinWCPprogramonyourcomputer:1)Gotothewebpagehttp://spider.science.strath.ac.uk/sipbs/page.php?

page=softwareandclicktheWinWCPVxxxSetupFileoptiontodownloadtheWinWCPinstallationprogram(WinWCP_Vxxx_Setup).Storethisfileinatemporaryfolder(e.g.c:\temp)onyourcomputer.

2)Starttheinstallationprogrambydouble-clickingtheprogramWinWCP_Vx.x.x_Setup.Thesetupprogramcreatesthefolderc:\WinWCPandinstallstheWinWCPprogramsfileswithinit.(YoucanchangethediskdriveandlocationoftheWinWCPfolderifyouwish).

3) To startWinWCP, click theMicrosoftWindowsStart button and selectWinWCPVx.x.xfromtheWinWCPgroupintheProgramsmenu.

4) Install the laboratory interface unit with the appropriate device driversoftware and support library supplied with the device (See LaboratoryInterfaces).

5)ConfigureWinWCPtoworkwithlaboratoryinterface.6) Attachanaloginput/outputsignalcablesbetweenamplifierandlaboratory

interface(seesection4).

Next

GettingStarted>LaboratoryInterfaces>NationalInstrumentsInterfaceCardsNationalInstrumentsInc.(www.ni.com)WinWCP is compatible with most National Instruments multifunction dataacquisition cards or devices, including M, X, and E series cards, the Lab-PC/1200seriesandUSBdevices.ThePCI-6221PCIcard(withBNC-2110I/Oboxand2mSHC68-68-EPMcable)or theUSB-6341(withBNCconnectivity)deviceiscurrentlyrecommended.The National Instruments NIDAQ interface library must be installed beforeWinWCPcanusetheinterfacecard.Mostmoderncards(X,MandEseries)aresupportedviatheNIDAQ-MXlibrary.Oldercards(Lab-PC/1200series)requirethe ‘Traditional’ NIDAQ library (V4.9 or earlier) to be installed. WinWCPsupportsbothtypesoflibrary.

Softwareinstallation

1) Install the NIDAQ library from the disks supplied with interface card,followingtheinstructionssuppliedbyNationalInstruments.

2)Installtheinterfacecardinanexpansionslot(orattachaUSBdevice).

3)Rebootthecomputer.

4) Run National Instruments’ Measurement & AutomationExplorer program. You should find the card listed under Devices &Interfaces.NotetheDevicenumber(Dev1,Dev2etc.)ofthecard.

5) Right-clickoverthedeviceandselectSelfTest tocheckthatthedeviceisfunctioningcorrectly.

6)IfthetestscheckoutOK,runWinWCPandselectfromthemainmenu

Setup

LaboratoryInterface

toopentheLaboratoryInterfaceSetupdialogbox

thenselectNationalInstruments(NIDAQ-MX)fromthelistoflaboratoryinterfaceoptions(exceptforLab-PCor1200seriescardsinwhichcaseuseNationalInstruments(NIDAQTrad)).

7)SelectthedevicenumberofthecardlistedintheNIDevices&InterfaceslistfromtheDevicelist(usuallyDev1ifonlyonecardisinstalled).

8) Set theA/D Input mode. If you are using a BNC-2110 or BNC-2090input/outputbox,selectDifferential.(Note.TheSE/DIswitchesonaBNC2090panelmustbesettoDI)IfyouareusingaLab-PCor1200seriescard,settheA/DInputmodetoSingleEnded(RSE).

Signalinput/outputconnectionsSignal input andoutput fromNational InstrumentsPCI cards aremadevia 68pinsocketson therearof thecardattached toBNCsocket input/outputpanels(BNC-2110 or BNC-2090 ) or screw terminal panels by appropriate shieldedcablesavailablefromNationalInstruments.

NationalInstrumentsX,E&MSeriescards

NationalInstrumentsX,E&MSeriescards

AnalogInput I/OPanel ScrewterminalpanelCh.0 ACH0 68,67+62(signal,ground)Ch.1 ACH1 33,67+62Ch.2 ACH2 65,67+62Ch.3 ACH3 30,67+62Ch.4 ACH4 28,67+62Ch.5 ACH5 60,67+62Ch.6 ACH6 25,67+62Ch.7 ACH7 57,67+62AnalogOutput Ch.0 DACOUT0 22,55Ch.1 DACOUT1 21,55TriggerInputs Ext.SweepTrigger PFI0/TRIG1 11,44Ext.StimulusTrigger PFI0/TRIG1(NIDAQ-MX)

PFI1/TRIG2(NIDAQ)11,44(SeeNote1)10.44

DigitalOutput Ch.0 DIO0 52,53Ch.1 DIO1 17,53Ch.2 DIO2 49,53Ch.3 DIO3 47,53Ch.4 DIO4 19,53Ch.5 DIO5 51,53Ch.6 DIO6 16,53Ch.7 DIO7 48,53

Note1.Anactive-highTTLpulseonthisinputtriggersthestartastimulusprogramwhichhasbeensetupwiththeExternalStimulusTrigger=Yoption.ThetriggersignalisappliedtoPFI0whentheNIDAQ-MXinterface library is in use (Laboratory Interface Card = National Instruments (NIDAQ-MX) andPFI1when theTraditionalNIDAQinterface library(forLab-PC/1200cardsonly) is inuse(LaboratoryInterfaceCard=NationalInstruments(NIDAQ)

USB-6000toUSB-6005DevicesTheinput/outputconnectionsforthelowcostUSB6000-6005devicesare

USB6000-6005AnalogInputs ScrewterminalpanelCh.0 AI+0,-0(signal,ground)Ch.1 AI+1,-1(signal,ground)Ch.2 AI+2,-2(signal,ground)Ch.3 AI+3,-3(signal,ground)AnalogOutputs Ch.0 AO0,Gnd(signal,ground)Ch.1 AO1,Gnd(signal,ground)TriggerInputs Ext.SweepTrigger PFI0,Gnd(signal,ground)Ext.StimulusTrigger PFI0+PFI1,Gnd(signal,ground)

Note. PFI0, PFI1 and P1.0 MUST be connected

together for Stimulus Protocols to beproducedand the testpulse togenerated intheSealTestwindow.

DigitalStimulusprotocolsarenotsupportedbythesedevices.

Lab-PC/1200SeriesCardsThe input/output connections for 50 pin Lab-PC and 1200- series boards aretabulatedbelow.Lab-PC/1200CardsAnalogInputs I/OPanel ScrewterminalpanelCh.0 ACH0 1,9(signal,ground)Ch.1 ACH1 2,9Ch.2 ACH2 3,9Ch.3 ACH3 4,9Ch.4 ACH4 5,9Ch.5 ACH5 6,9Ch.6 ACH6 7,9Ch.7 ACH7 8,9AnalogOutputs Ch.0 DAC0 10,11Ch.1 DAC1 12,11(SeeNote1)TriggerInputs Ext.SweepTrigger EXTTRIG 38,50(SeeNote1)Ext.StimulusTrigger PB7 29,50(SeeNote2)DigitalSynch.Input PC6 36,13(SeeNote1)

DigitalOutputs Ch.0 PA0 14,13Ch.1 PA1 15,13Ch.2 PA2 16,13Ch.3 PA3 17,13Ch.4 PA4 18,13Ch.5 PA5 19,13Ch.6 PA6 20,13Ch.7 PA7 21,13

NOTE2Analogoutputchannel1(DAC1)isusedtosynchronisethestartoftheA/DconversionandD/AwaveformgenerationandmustbeconnectedtoEXTTRIGforWinWCPwaveformgenerationfunctionstooperate.Inaddition,ifstimulusprotocolscontainingdigitalouputsarerequiredDAC1&EXTTRIGmustalsobeconnectedtothedigitalsynchronisationinputPC6.

Note.2.Lab-PC/1200seriesboardsneedthepulsetobe10msindurationorlonger.

Troubleshooting

National Instruments cards can be used with a number of different types ofinput/outputpanels (BNC2090,BNC2110orCB-68 terminalpanel)andcanalsobeconfiguredtohandletheanaloginputchannelsinanumberofdifferentways (differential, referenced single ended and non-referenced single ended).Some combinations of settings can lead to input signals drifting or going offscale.Differential mode (DIFF): Analog input channels are paired together andsubtracted(e.g.Ch.0–Ch.7,Ch.1–Ch.8etc.).Referenced single ended mode (RSE): Analog input channels are usedindividuallyandmeasuredrelativetosignalgroundofthecomputer.Non-referencedsingleendedmode (NRSE):Analog input channels areusedindividuallyandmeasured relative to theelectricalgroundof thedevicebeingmeasured.When using the BNC-2110 I/O box, the USB-6221-BNC or USB-6229-BNCUSBinterfacedevicetheWinWCPA/DInputModemustbesetatDifferential.WhenusingtheBNC-2090I/OboxwithitsSE/DIswitchessettoDI(thedefaultsetting)theWinWCPA/DInputModeshouldbesettoDifferential.

Whenusing theBNC-2090 I/Oboxwith itsSE/DI switches set toSEand theRSE/NRSEswitchsettoNRSEtheWinWCPA/DInputModeshouldbesetatSingleEnded(NRSE)

GettingStarted>LaboratoryInterfaces>AxonInstrumentsDigidata1200AxonInstrumentsInc.(nowownedbyMolecularDevices,www.moleculardevices.com)The Digidata 1200, 1200A and 1200B interface boards fully supports allWinWCP features. They have a 330 kHz maximum sampling rates and 4programmable input voltage ranges (10V, 5V, 2.5V, 1.25V). Inputs to andoutputsfromtheboardareviaBNCconnectorsonanI/Obox,connectedtotheboard via a shielded ribbon cable. In order to useWinWCP with a Digidata1200,thefollowingcomputersystemresourcesmustbeavailableforusebytheDigidata1200.

I/Oportaddress320-33F(Hex)

DMAchannels5and7

SoftwareInstallation1) Install theDigidata 1200 card into an ISA computer expansion slot, and

attachit to itsBNCI/Opanelusingtheshieldedribboncablesuppliedwiththecard.

2)InstalltheWinWCPDigidata1200driversoftwareforyourWindowsoperatingsystem,byrunningtheappropriateinstallationbatchfile.

IfyouarerunningWindows95,98orMe,selectWinWCP\Digidata1200Drivers\InstallDigidata1200driver(Win95/98/Me)fromtheProgramsmenu.

IfyouarerunningtheWindowsNT,2000orXP,selectWinWCP\Digidata1200Drivers\InstallDigidata1200driver(WinNT/2000/XP).(Note.WinWCPdoesnotusethestandardAxonInstrumentsDigidata1200devicedriver).


4)RunWinWCPandselectfromthemainmenu

Setup>LaboratoryInterface


thenselectAxonInstruments(Digidata1200)fromthelistoflaboratoryinterfaceoptions.

Signalinput/outputconnections

SignalinputandoutputconnectionsaremadeviatheBNCsocketsonthefrontandrearoftheDigidata1200I/Obox.

Digidata1200AnalogInput I/OPanel NotesCh.0 AnalogIn0 Ch.1 AnalogIn1 Ch.2 AnalogIn2 Ch.3 AnalogIn3 Ch.4 AnalogIn4 Ch.5 AnalogIn5 Ch.6 AnalogIn6 Ch.7 AnalogIn7 AnalogOutput Ch.0 AnalogOut0 Ch.1 AnalogOut1 TriggerInputs Ext.SweepTrigger Gate3 Ext.StimulusTrigger Gate3 SeeNote1DigitalOutput Ch.0 DigitalOut0 SeeNote2Ch.1 DigitalOut1

Ch.2 DigitalOut2 Ch.3 DigitalOut3

Note1.Anactive-highTTLpulseonthisinputtriggersthestartastimulusprogramwhichhasbeensetupwiththeExternalStimulusTrigger=Yoption.

Note2.WinWCPonlysupportsdigitaloutputlines0-3oftheDigidata1200.

Troubleshooting

There are two known problems which will prevent WinWCP from recordingfromaDigidata1200’sanaloginputchannels.

I/Oportconflict.TheDigidata1200defaultI/Oportaddressesspantherange320H-33AH.Thesesettingsconflictwith thedefaultMIDIportsetting(330H)ofCreativeLabs.Sound-Blaster16andsimilarsoundcards.Thereareanumberofsolutionstothisproblem.

1)ChangetheSound-BlasterMIDIportsettingtoavaluehigherthan33AH.

2) RemovetheSound-Blastercard(ordisableitusingtheBIOSsetupifit isbuiltintothecomputermotherboard).

DMAchannelconflicts.WinWCP requiresDMAchannels5 and7 to supportthe transfer of data to/from PCmemory and the Digidata 1200.Many soundcards also make use of DMA 5 and can interfere with the operation of theDigidata1200.

GettingStarted>LaboratoryInterfaces>CambridgeElectronicDesign1401SeriesCambridgeElectronicDesignLtd.(www.ced.co.uk)The CED 1401 series consists of an external microprocessor-controlledprogrammablelaboratoryinterfaceunitsattachedtothePCviaadigitalinterfacecardorUSB.Thereare4maintypesofCED1401incommonuse-CED1401,CED 1401-plus, CED Micro-1401 and CED Power-1401. They are all fullysupportedWinWCPwith the exception that only 4 analog input channels areavailableontheMicro1401andthatthemaximumsamplingrateandnumberofsamples/sweepforthestandardCED1401issubstantiallylessthantheothers.

SoftwareinstallationBefore WinWCP can use these interface units, the CED 1401 device driver(CED1401.SYS), support library (USE1432.DLL), and a number of 1401commandfilesstoredinthedirectory\1401mustbeinstalledonthecomputer.

The installation procedure is as following, but see CED documentation fordetails.

1)InstalltheCEDinterfacecardinaPCexpansionslotandattachittotheCED1401viatheribboncablesupplied(orattachtoUSBportforUSBversions).

2)DownloadtheCED1401StandardWindowsInstallerprogram(WINSUPP.EXE)fromtheCEDwebsite(http://www.ced.co.uk/upu.shtml)andrunittoinstalltheCED1401.SYSdevicedriverand1401commands.

3)EnsurethattheCED1401isswitchedon,andthenrebootyourcomputer.

4)TesttheCEDinterfacebyrunningtheprogram.c:\1401\utils\try1401w.exeandclickingthebuttonRunOnce

5)IfthetestscheckoutOK,runWinWCPandselectfromthemainmenu

Setup/LaboratoryInterface

toopentheLaboratoryInterfaceSetupdialogbox.

IfyouhaveaCED1401withstandardanalog±5Voutputvoltagerange,selectCED1401(16bit)(5V)fromthelistoflaboratoryinterfaceoptions.IfyouhaveaCED1401witha±10Voutputvoltagerange,selectCED1401(16bit)(10V).


AnalogsignalI/OconnectionsaremadeviaBNCsocketsonthefrontpaneloftheCED1401units.CED1401SeriesAnalogInput I/OPanel NotesCh.0 ADCInput0 Ch.1 ADCInput1 Ch.2 ADCInput2 Ch.3 ADCInput3 Ch.4 ADCInput4 ex.Micro1401Ch.5 ADCInput5 ex.Micro1401Ch.6 ADCInput6 ex.Micro1401Ch.7 ADCInput7 ex.Micro1401AnalogOutput Ch.0 DACOutput0 Ch.1 DACOutput1 Sync.Out DACOutput2 CED1401Only(SeeNote1)TriggerInputs Ext.SweepTrigger EventInput4

TriggerInCED1401,CED1401-plusMicro1401,Power1401

Ext.StimulusTrigger EventInput0 DigitalGateI/P EventInput2 DigitalOutput 25wayDsocketCh.0 DigitalOut8 17,13(signal,ground)Ch.1 DigitalOut9 4,13Ch.2 DigitalOut10 16,13

Ch.3 DigitalOut11 3,13Ch.4 DigitalOut12 15,13Ch.5 DigitalOut13 2,13Ch.6 DigitalOut14 14,13Ch.7 DigitalOut15 1,13

NOTE.1STANDARDCED1401ONLY

EventsInputs2,3,and4mustbeconnectedtogetherandconnectedtoDACOutput2,tosynchroniseA/Dsampling,D/AwaveformgenerationanddigitalpulseoutputforWinWCP’sSealTestoptionandrecordingwithstimuluspulseprotocols.

Note2.ATTLpulseon theExt.SweepTrigger input triggers the startof a recording sweepwhenExtTriggersweeptriggermodehasbeenselected.

Note3.Anactive-highTTLpulseontheExt.StimulusTriggerinputtriggersthestartastimulusprogramwhichhasbeensetupwiththeExternalStimulusTrigger=Yoption.

TroubleshootingtipsVerify that the CED 1401 is working correctly, before investigating problemsusingWinWCP.UsetheTRY1401WprogramtotesttheCED1401.WinWCP uses the commands, ADCMEMI.CMD, MEMDACI.CMD andDIGTIM.CMD with the CED 1401; ADCMEM.GXC, MEMDAC.GXC andDIGTIM.GXC with the CED 1401-plus; and ADCMEM.ARM,MEMDAC.ARM and DIGTIM.ARM with the CED Micro-1401. All threecommandsmustbeavailablewithinthe\1401directory.Power1401Thedigitalpatternoutput commandDIGTIMappears tobehavedifferentlyondifferent versions of the Power 1401 resulting in the the digital outputwaveformsinaWinWCPstimulusprotocolbeingproducedincorrectly(On/Offlevels are inverted and pulse durations are incorrect). This problem can becorrected by setting theCEDPOWER1401DIGTIMCOUNTSHIFT entry in the WinWCPlabinterface.xmlfileinc:\winwcpto0.

Stop the WinWCP program (if it is running) and open the filec:\winwcp\labinterface.xmlwiththeNotepadtexteditorandsearchfortheentry:CEDPOWER1401DIGTIMCOUNTSHIFT>1</CEDPOWER1401DIGTIMCOUNTSHIFTChangeitto:CEDPOWER1401DIGTIMCOUNTSHIFT>0</CEDPOWER1401DIGTIMCOUNTSHIFTandsavethefile.StandardCED1401The performance of the original CED 1401 interface unit is very limitedcomparedtolatermodels.Thenumberofsamples/recordislimitedtoatotalof8192andtheachievablesamplingratewhenstimuluspulsesarebeinggeneratedislimitedtoamaximumofaround20kHzdividedbythenumberofchannels.In some circumstances, the sampling rate set by WinWCP can exceed thecapabilities of the standard 1401, resulting in samples between mixed upbetween channels. This problem can be resolved by reducing the number ofsamplesperrecordorbyincreasingthedurationoftherecordingsweep.TheCED1401ISAcarddefaultI/Oportaddressesareat300H.Checkthatthesedonotconflictwithothercardswithinthecomputer.TheCED1401alsomakesuseofDMAchannel1andanIRQchannel(IRQ2).Thesemayalsoconflictwithothercards.Some standard 1401 appear to fail the DMA (direct memory access) test inTRY1401WandthisalsocausesproblemswhenrunningWinWCP.Ifthiserroroccurs,disabletheDMAchannel,byclickingontheCED1401iconwithintheWindowsControlPanelandun-checkingtheEnableDMAtransferscheckbox.

GettingStarted>LaboratoryInterfaces>AxonInstrumentsDigidata1320AxonInstrumentsInc.(nowownedbyMolecularDevices,www.moleculardevices.com)The Digidata 1320 Series (1320A, 1322) interfaces consist of self-contained,mains-powered digitiser units with BNC I/O sockets attached to the hostcomputer via a SCSI (Small Computer Systems Interface) interface card andcable.Anumberofversionsareavailableincludingthe1320Aand1322A.The1322A supports sampling rates up to 500 kHz (16 bit resolution) on up to 16channels. It has a fixed input andoutputvoltage rangeof10Vand supports 4digitaloutputchannels..

SoftwareInstallationWinWCP uses Axon’s standard software library (AxDD132x.DLL) for theDigidata1320Series.Detailsforsteps(1)-(5)canbefoundinAxon’sDigidata1320SeriesOperator’sManual.

1)InstalltheAxonSCSIcardinaPCIexpansionslot.

2) AttachtheDigidata1320totheSCSIcardandswitchonthecomputerand1320.

3)InstalltheAxoScopesoftwaresuppliedwiththeDigidata1320.


5)RunAxoScopetoensurethatthesoftwareinstalledOK.

6)RunWinWCPandselectfromthemainmenu:



thenselectAxonInstruments(Digidata132X)fromthelaboratoryinterfacelist.

Signalinput/outputconnectionsSignalinputandoutputconnectionsaremadeviatheBNCsocketsonthefrontoftheDigidata1320Seriesdigitiserunit.

Digidata132XSeriesAnalogInput I/OPanel NotesCh.0 AnalogIn0 Ch.1 AnalogIn1 Ch.2 AnalogIn2 Ch.3 AnalogIn3 Ch.4 AnalogIn4 Ch.5 AnalogIn5 Ch.6 AnalogIn6 Ch.7 AnalogIn7 AnalogOutput Ch.0 AnalogOut0 Ch.1 AnalogOut1 TriggerInputs Ext.SweepTrigger TriggerInStart Ext.StimulusTrigger TriggerInStart SeeNote1DigitalOutput Ch.0 DigitalOut0 SeeNote2Ch.1 DigitalOut1 Ch.2 DigitalOut2 Ch.3 DigitalOut3

Note1.Anactive-highTTLpulseonthisinputtriggersthestartastimulusprogramwhichhasbeensetupwiththeExternalStimulusTrigger=Yoption.

Note2.TheDigidata1320Seriesonlysupports4digitaloutputlines.

Troubleshooting

When multiple analog input channels are being sampled and the samplinginterval is greater than 10 ms, samples get mixed up between channels. Thisproblem can be seen to occur also with AxoScope, suggesting a bug in theDigidata1320firmwareorAXDD132X.DDLlibrary.Theonlylimitedsolutionat present is to increase the number of samples per record to ensure that thesamplingintervalislessthan10ms.

GettingStarted>LaboratoryInterfaces>MolecularDevicesDigidata1440AMolecularDevicesCorporation(www.moleculardevices.com)

The Digidata 1440A interface consists of self-contained, mains-powereddigitiserunitwithBNCI/OsocketsattachedtothehostcomputerviaaUSB2.0port.The1440Asupportssamplingratesupto250kHz(16bitresolution)onupto16channels.Ithasafixedinputandoutputvoltagerangeof10Vandsupports4analogoutputchannelsand8digitaloutputchannels.

SoftwareInstallationWinWCP uses Axon’s standard software library (AxDD1400.DLL) for theDigidata1400Series.Detailsforsteps(1)-(5)canbefoundinAxon’sDigidata1440AManual.

1) Install theAxoScope (orPCLAMP ) software suppliedwith theDigidata1440.


3)AttachtheDigidata1440AtoaUSBportandturniton.




thenselectMolecularDevices(Digidata1440)fromthelaboratoryinterfacelist.

AnalogInputOffsetCalibrations

TheoffsetcalibrationvaluesfortheDigidata1440analogueinputchannelscanbe modified by creating a file called "Digidata 1440 adc offsets.txt" in thesettingsfolderU:\users\Public\PublicDocuments\SESLabIOcontainingalistofoffsetsforeachchannel.Thefileshouldcontainalistof16offsetvalues(inmV)tobesubtractedfromeachchannelsignal,e.g.

3.54.01.0-1.0...

AfileDigidata1440calibrationfactors.txtcontaining theanalogue inputandoutputoffset andgaincorrection factors incurrentuse in theDigidata1440 isalsocreatedinthesettingsfolderwhenWinWCPisstarted.


SignalinputandoutputconnectionsaremadeviatheBNCsocketsonthefrontoftheDigidata1440Adigitiserunit.

Digidata1440AAnalogInput I/OPanel NotesCh.0 AnalogInput0 Ch.1 AnalogInput1 Ch.2 AnalogInput2 Ch.3 AnalogInput3 Ch.4 AnalogInput4 Ch.5 AnalogInput5 Ch.6 AnalogInput6 Ch.7 AnalogInput7 AnalogOutput Ch.0 AnalogOut0 Ch.1 AnalogOut1 Ch.2 AnalogOut2 Ch.3 AnalogOut3 TriggerInputs Ext.SweepTrigger Start Ext.StimulusTrigger Start SeeNote1

DigitalOutput Ch.0 DigitalOutput0 Ch.1 DigitalOutput1 Ch.2 DigitalOutput2 Ch.3 DigitalOutput3 Ch.4 DigitalOutput4 Ch.5 DigitalOutput5 Ch.6 DigitalOutput6 Ch.7 DigitalOutput7

Note1.Anactive-highTTLpulseonthisinputtriggersthestartofastimulusprogramwhichhasbeensetupwiththeExternalStimulusTrigger=Yoption.

GettingStarted>LaboratoryInterfaces>MolecularDevicesDigidata1550/1550A/1550BMolecularDevicesCorporation(www.moleculardevices.com)TheMolecularDevicesDigidata1550,1550Aand1550Binterfacesconsistsofself-contained,mains-powereddigitiserunitswithBNCI/Osocketsattachedtothehost computervia aUSB2.0port.They support sampling ratesup to500kHz(16bitresolution)onupto8channels.Theyhaveafixedinputandoutputvoltagerangeof+/-10Vandsupport8analogueand8digitaloutputchannels.

SoftwareInstallationWinWCP uses Molecular Device's software libraries (AxDD1550.DLL,AxDD1550A.DLL or AxDD1550B.DLL) and device drivers for the Digidata1550and1550ASeries.

1) Install theAxoScope (orPCLAMP ) software suppliedwith theDigidata1550X.


3)AttachtheDigidata1550seriesinterfaceunittoaUSBportandturniton.



toopentheLaboratoryInterfaceSetupdialogbox.

ThenselectMolecularDevicesDigidata1550ifyouhaveaDigidata1550,MolecularDevicesDigidata1550AororMolecularDevicesDigidata1550BifyouhaveaDigidata1550Bfromthelaboratoryinterfacelist.

Notes.

a) The HumSilencer feature of the Digidata 1550A and B is notcurrentlysupportedbyWinWCP.

b) When a recording is sweep manually terminated (by the userclicking the Stop button in the Record to Disk window) whileWinWCP iswaiting for an externalSTART trigger pulse in theExtTriggered recordingmode orwhen theExternalStimulusTriggersetting in a stimulus protocol is set toY, an additional trigger pulsemust be applied to the Digidata 1550 START input to terminaterecording.

c)Supportforanaloginputchannelre-mapping(intheAICh.columnof the Input Channels table in the Input Channels & AmplifierSetup dialog box) is limited. Recording channels can only beremappedtoHIGHERanaloginputnumbers.


SignalinputandoutputconnectionsaremadeviatheBNCsocketsonthefrontoftheDigidata1550seriesdigitiserunit.

Digidata1550SeriesAnalogInput I/OPanel NotesCh.0 AnalogInput0 Ch.1 AnalogInput1 Ch.2 AnalogInput2 Ch.3 AnalogInput3 Ch.4 AnalogInput4 Ch.5 AnalogInput5 Ch.6 AnalogInput6

Ch.7 AnalogInput7 AnalogOutput Ch.0 AnalogOut0 Ch.1 AnalogOut1 Ch.2 AnalogOut2 Ch.3 AnalogOut3 TriggerInputs Ext.SweepTrigger Start Ext.StimulusTrigger Start SeeNote1DigitalOutput Ch.0 DigitalOutput0 Ch.1 DigitalOutput1 Ch.2 DigitalOutput2 Ch.3 DigitalOutput3 Ch.4 DigitalOutput4 Ch.5 DigitalOutput5 Ch.6 DigitalOutput6 Ch.7 DigitalOutput7


GettingStarted>LaboratoryInterfaces>InstrutechITC-16/18InstrutechCorpwww.instrutech.com(nowhandledbyHekaElectronikGmbH)The Instrutech ITC-16 and ITC-18 interfaces are self-contained, 19” rack-mountable,mains-powereddigitiserunitswithBNCI/Osocketsattachedtothehostcomputerviaadigitalinterfacecardandcable.BoththeITC-16andITC-18support8analoginputchannels,4analogoutputsand8digitaloutputs.

Note.TheInstrutechITC-1600,LIH8+8andUSB-18arenotcurrentlysupportedbyWinWCP.


WinWCPusestheInstrutechdeviceinterfacelibrariesfortheITC-16/18family.Detailsforsteps(1)-(3)canbefoundintheInstrutechDataAcquisitionInterfaceusermanual.

1)InstalltheInstrutechinterfacecardinanexpansionslot.

2)AttachtheITC-16orITC18unittothecard.

3) Install the Instrutech Device Driver software supplied with the card (ordownloadedfromwww.instrutech.com)


5) Run the Instrutech test program installed with the device driver to testwhetherthesoftwareinstalledOK.


SetupLaboratoryInterface


thenselectInstrutechITC-16/18(Newdriver)fromthelaboratoryinterfaceoptionslist.Note,onsystemswithInstrutech’solderdevicedriversoftwareinstalled,itmaybenecessarytoselecteitherInstrutechITC-16(OldDriver)orInstrutechITC-18(OldDriver)dependinguponwhichinterfaceunitisinstalled.

InstrutechITC-16/18:I/OPanelConnections

SignalinputandoutputconnectionsaremadeviatheBNCsocketsonthefrontoftheITC-16/18unit.

InstrutechITC-18AnalogInput I/OPanel NotesCh.0 ADCInput0 Ch.1 ADCInput1 Ch.2 ADCInput2 Ch.3 ADCInput3 Ch.4 ADCInput4 Ch.5 ADCInput5 Ch.6 ADCInput6 Ch.7 ADCInput7 AnalogOutput Ch.0 DACOutput0 Ch.1 DACOutput1 Ch.2 DACOutput2 Ch.3 DACOutput3 TriggerInputs Ext.SweepTrigger TrigIn Ext.StimulusTrigger TrigIn SeeNote1DigitalOutput Ch.0 TTLOutput0 Ch.1 TTLOutput1 Ch.2 TTLOutput2 Ch.3 TTLOutput3


InstrutechITC-16/18:TroubleshootingWinWCP requires Instrutech's combined device driver library ITCMM.DLL(releasedlate2001).Itmaynotworkwithearlierlibraries.

GettingStarted>LaboratoryInterfaces>BiologicVP500Bio-Logic-ScienceInstrumentsSA(www.bio-logic.fr)

The Biologic VP500 is a computer-controlled patch clamp with a built-inlaboratoryinterfaceunit,attachedtothehostcomputerviaaGPIBinterfacebus.It is supported underWindows 95/98,NT and 2000. TheVP500 patch clampfunctions(gain,filtering,capacitycompensation,etc.)canbecontrolledfromavirtualfrontpanelwithinWinWCP.ThecurrentimplementationoftheWinWCPsoftwaresupports

2analoginputchannels(membranecurrentandvoltage)

Commandvoltageoutput


WinWCP uses Biologic’s BLVP500.DLL library (supplied with WinWCP) tocontrolandacquiredatafromtheVP500.

1) InstalltheNationalInstrumentsNIDAQsoftware,suppliedwiththeGPIB

interfacecardandreboot.2)InstalltheGPIBcardintothehostcomputerandreboot.3)CheckusingtheNationalInstrumentsMeasurement&AutomationExplorer

programthattheGPIBhasbeendetectedandisfunctioningcorrectly.4)RunWinWCPandselectfromthemainmenu


toopentheLaboratoryInterfaceSetupdialogbox,

thenselectBiologicVP500fromthelaboratoryinterfaceoptionslist.

BiologicVP500:I/OPanelConnections

No I/Opanel connections arenecessary.All connectionsbetweenpatch clampandlaboratoryinterfaceareinternaltotheVP500.

GettingStarted>LaboratoryInterfaces>TecellaPico/Triton/Triton+TecellaLLC(www.tecella.com)

TheTecellaPico,TritonandTriton+areUSBbasedcomputercontrolledpatchclamp/digitiserunits.ThePicoisasingle-channelpatchclampandtheTriton(8channels)andTriton+(16channels)aremulti-channeldevices.


1) Download and install the Tecella device driverTecellaDriver from theTecelladownloadswebpage(www.tecella.com/downloads).



toopentheLaboratoryInterfaceSetupdialogbox,

thenselectTecellaTriton/Triton+/Picofromthelaboratoryinterfaceoptionslist.

Tecella:I/OPanelConnections

No I/Opanel connections arenecessary.All connectionsbetweenpatch clampandcomputerareeffectedviatheUSBbus.

GettingStarted>LaboratoryInterfaces>HekaPatchClamps&InterfacesHekaElectronikGmbH(www.heka.com)TheHekaEPC-9andEPC-10are a rangeof computer-controlledpatchclampamplifierswithabuilt-in laboratory interfaceunitattachedto thecomputerviaPCIinterfacecardsorUSB.HekaalsosupplyandsupporttheInstrutechrangeoflaboratoryinterfaceunits:ITC-16,ITC-18,ITC-1600andtheirownunit theLIH-88.


1)InstallthedriversandsoftwaresuppliedwithyourpatchclampordownloadandinstalltheEPC/PGdriversfromhttp://www.heka.com/download/download.html.

2) Attach the Heka patch clamp to the computer or install the Instrutechinterfacecard.

3) Locate the calibration filesSCALE-nnnnn.EPC andCFAST-nnnnn.EPC foryourEPC-9/10patchclamp(wherennnnisyourpatch clamp serial number) and copy them to the WinWCPprogramfolder(c:\programfiles\winwcp).

4)RunWinWCPandselectfromthemainmenuSetupLaboratoryInterface


thenselectyourdevicefromthelist(HekaEPC-10,HekaEPC-10plus,Heka

EPC-10-USB,Heka EPC-9, Heka ITC-16, Heka ITC-18, Heka ITC-1600,HekaLIH-88).

5) EPC-9/10 PanelConnections: If you have an EPC-9 or EPC-10 patchclamp, connect a BNC cable betweenFilter 2 andA/D Input 0 and alsobetweenVoltageMonitorandA/DInput1.

6)InstrutechITC-16/18:I/OPanelConnections

SignalinputandoutputconnectionsaremadeviatheBNCsocketsonthefrontoftheITC-16/18units.

InstrutechITC-18AnalogInput I/OPanel NotesCh.0 ADCInput0 Ch.1 ADCInput1 Ch.2 ADCInput2 Ch.3 ADCInput3 Ch.4 ADCInput4 Ch.5 ADCInput5 Ch.6 ADCInput6 Ch.7 ADCInput7 AnalogOutput Ch.0 DACOutput0 Ch.1 DACOutput1 Ch.2 DACOutput2 Ch.3 DACOutput3 TriggerInputs Ext.SweepTrigger TrigIn Ext.StimulusTrigger TrigIn SeeNote1


GettingStarted>Amplifiers>Patch/Voltage-clampAmplifiersOneofthemostcommonWinWCPapplicationsisrecordingfromandcontrollingawhole-cellpatch-orvoltage-clampexperiment.TwoanalogchannelsarenormallyrecordedbyWinWCP(membranecurrentandvoltage),andcomputer-generatedvoltagepulsesareappliedtothepatchclampcommandvoltageinputtostimulatethecell.

WinWCPsupportsupto4patch/voltage-clampamplifiersand,foreachamplifierinuse,upto5analogsignalconnectionsmustbemadebetweentheamplifierandlaboratoryinterface.

Apairoflaboratoryinterfaceanaloginputchannels(designatedtheprimaryandsecondaryinputchannels)arerequiredtorecordthemembranecurrent(Im)andmembranevoltage(Vm)outputsfromtheamplifier.

An analog outputmust be connected to the amplifier command voltage input(Vcom), toprovidecurrent/voltagestimuluswaveforms.Twoadditionalanaloginputsmayberequiredtoreceivetheamplifiergain(GAIN)andvoltage/currentclamp mode (MODE) telegraph signals (Note. Some patch clamps do notsupport gain and/or mode telegraphs, others communicate gain/modeinformationviaUSBorothercommunicationslines.)

WinWCPsupportsmanyof thecommonlyusedmodelsofpatch-andvoltage-clampamplifiersand is able to readgainandmode telegraphsignalsallowingthecurrentandvoltagesignalchannelsfactorstobescaledcorrectly.

The required primary and secondary input channel, command voltage output,gain and mode telegraph signals connections for the amplifiers currentlysupportedbyWinWCPareshowninthetableonthefollowingpage.

GettingStarted>Amplifiers>SignalConnectionsTableThefollowingtableshowssignalconnectionstherequiredfortheWinWCP-suppportedamplifiers.Amplifieroutputs/inputinpanel(A)areconnectedtothelaboratoryinterfaceinput/outputsinthecorrespondingcolumninpanel(B).A) AmplifierInputs/OutputsTypeofAmplifier

PrimaryChannel

SecondaryChannel

MODE GAIN Vcom

Manualgainentry

ImOut VmOut - - VcommandIn

Axopatch1D

ScaledOutput

10Vm - GainTelegraph

Ext.Command

Axopatch200

ScaledOutput

10Vm(VCmode)Im(CCmode)SeeNote.1

ModeTelegraph

GainTelegraph

Ext.Command(frontswitched)

Multiclamp700A

ScaledOutput

RawOutput

- -SeeNote2 Ext.Command

Multiclamp700B

PrimaryOutput

SecondaryOutput

- -SeeNote2 Ext.Command

RK400 Iout 10xVm - - VoltageCommandInput

VP500 - - - - -HekaEPC-7 Current

MonitorVcommMonitor

- - StimInputX10

HekaEPC-8 CurrentMonitor

VcommMonitor

- SeeNote3. StimInputX10

HekaEPC-800 CurrentMonitor

VcommMonitor

ModeTelegraph

GainTelegraph

StimInputX10

CairnOptopatch

GainOut CommandX10Out

Pin9Pin2Gnd37wayDsocket

GainTelegraphOut

Command/10In

WarnerPC501A

Im Vmx10 - GainTelegraph

CMDIn

WarnerPC505B

Im Vmx10 - GainTelegraph

CommandIn

WarnerOC725

IMonitor Vmx10 - GainTelegraph

CommandIn÷10

NPISEC05LX CurrentOutput

PotentialOutput

- Curr.SensitivityMonitor

VCCommandInput/10

AMSystems2400

Output X10Vm ModeTelegraph

GainTelegraph

External÷50

Tecella - - - - - B) DefaultLaboratoryInterfaceInputs/OutputsAmplifer#1 AICh.0 AICh.1 AICh.6 AICh.7 AOCh.0Amplifer#2 AICh.2 AICh.3 AICh.14 AICh.15 AOCh.1Amplifer#3 AICh.4 AICh.5 AICh.13 AICh.12 AOCh.2

Amplifer#4 AICh.6 AICh.7 AICh.10 AICh.11 AOCh.3

Note 1. When the Axopatch 200 is switched from voltage- to current-clampmode, theScaledOutputsignal to theprimarychannel (AICh.0 forAmplifier#1) changes frommembrane current to voltage.To retain a current signal, thesecondary channel (AI Ch.1 for Amplifier #1) ofWinWCPmust be switchedmanuallyfromtheAxopatch20010VmtotheAxopatch200Imoutput(ontherearpanel).Note2.AxonMulticlampamplifierssupporttwoseparateamplifierchannels(1& 2).When using amplifierCh.1, selectMulticlamp 700A/B asAmplifier #1and connect the Ch.1 Scaled and Raw/Secondary outputs to AI Ch.0 and AICh.1, respectively,andAOCh.0 toCh.1EXTCOMMAND.Whenusingbothchannels,selectMulticlamp700A/BalsoasAmplifier#2andconnecttheCh.2Scaled and Raw/Secondary outputs to AI Ch.2 andAI Ch.3 andAOCh.1 toCh.2EXTCOMMAND. (AxonMulticlampamplifierscanonlybe selectedasWinWCPAmplifiers#1and#2.)

The Axon Multiclamp Commander software must be started up and runningbeforeWinWCPisstarted.

Note 3. The Heka EPC-8 gain and mode telegraphs are TTL digital signalsprovidedviaa50wayIDCribboncableontherearoftheEPC-8.Thesignalsinthe table below must be connected to the digital inputs of the laboratoryinterface.HekaEPC-8Digitalgaintelegraphconnections Gain0 Gain1 Gain2 Range0 Range1 GndEPC-850wayIDCsocket 5 7 9 1 17 19DigitalInputChannel 0 1 2 3 4 Gnd

Next

GettingStarted>Amplifiers>ConfiguringAmplifierSupportinWinWCPToconfigureamplifiersupportselectSetupInputChannels&Amplifiers

toopentheInputChannels&AmplifiersSetupdialogbox.

SelecttheAmplifierstabandchoosetheamplifiertobeconfigured(Amplifier#1 if only one amplifier is in use), then select your type of amplifier in theamplifierlist.

The amplifier configuration table shows the default primary and secondaryinput channels, voltage and current command output channels and gain and

modetelegraphinputchannels(ifthesearerequiredbytheamplifier).

The basic (at minimum gain) scaling factors and units for the current andvoltageoutputsandthecurrentandvoltagecommandinputsarealsodisplayed.

Ifrequired,theGainandVoltage/currentclampmodetelegraphinputscanbechangedtoanotheranaloginputchannel.However,ifthisisdone,thephysicalconnectionsbetweentheamplifierandthelaboratoryinterfacemustbeadjustedtomatch.TelegraphscanalsobedisabledbyselectingOffastheinputchannel.Whentelegraphsaredisabled,gainand/orvoltage/currentclampmodecanbeenteredmanuallybytheuser.

Ifnecessary,primaryandsecondarychannelscalingfactors,units,voltage-andcurrent-clampcommandscalingfactorscanbealteredbytheuser.Thisismostlikely to be case for the command scaling factors where where a range ofdivision factors are available on most common patch clamp amplifiers. Thedefault settingsare set to themostcommonlyused stimulus scalings (0.02or0.1) for the amplifier. In the case of amplifierswhere the primary/secondarychannelsswitchoutputsignalsincurrent-orvoltage-clampmode,twodifferentprimaryandsecondarychannelscalingfactorscanbeentered,byselectingtheICLAMPorVCLAMPoptions.

Notethat,inthecaseofamplifiers

Manualamplifierconfiguration

If the amplifier in use is not specifically supported byWinWCP, current andvoltagescalinginformationcanstillbeenteredmanuallybytheuser.Toenterupamanualamplifierconfiguration,select

ManualGainEntry

astheamplifiertypeandenterthefollowinginformation.

PrimaryChannel(Im)

The amplifier membrane current output should be connected to this inputchannel(AICh.0forAmplifier#1).Enterthemeasurementunitsofthecurrent(pA,nA,uA,mA,A)intotheUnitsboxandtheamplifiercurrentscalingfactorof the amplifier atminimum gain inV/units into theScale factor box. Thescalingfactorcantypicallybeobtainedbyreadingtheminimumgainsettingoftherotatablecurrentgainswitch,foundonmostpatch-clampamplifiers(e.g.a

minimumgainsettingsof1mV/pAis equivalent to a scaling factor of0.001V/pA.)

SecondaryChannel(Vm)

The amplifier membrane potentialoutput shouldbe connected to thisinput channel (AI Ch.1 forAmplifier#1). Enter themeasurement units of the current(mV) in the Units box and theamplifier voltage scaling factor, inV/Units,intotheScalefactorbox.Most amplifiers have a fixedscalingfactorof0.01V/mV.

Voltage-clampcommandchannel

Analog output (AO Ch.0 forAmplifier#1) should be connectedto the amplifier command voltageinput. Enter the voltage-clampcommand potential scaling factor(membrane potential/commandvoltage)intotheScale factorbox.(A label specifying thecommandvoltagescaling factorcanusuallybe foundbesidethecommandvoltageinputsocketontheamplifier.Typicalvaluesare0.1V/Vor0.02V/V)

Current-clampcommandchannel

Enter the current-clamp command potential scaling factor (membranecurrent/command voltage) into the Scale factor box. The current clampcommand scaling factor can usually be found in the amplifier user manual.Typical values are between 10-9 A/V and 10-10 A/V. (Usually the currentcommand signal is applied to the same input on the amplifier as the voltagecommand (AO Ch.0 for Amplifier#1), however on some there may be aseparatecurrentcommandinput.)

Saving/LoadingSettings

Amplifierandinputchannelsettingscanbesavedtoasettingsfilebyclickingthe Save Settings button and saving the settings to an XML settings file.Settings can be reloaded from a settings file by clickingLoad Settings andselectingthefile.ThedefaultscalingandchannelsettingsforanamplifiercanberesetbyclickingtheRestoreDefaultSettingsbutton.Defaultsettingsarealsoloadedwhenevertheamplifiertypeischanged.

Next

GettingStarted>Amplifiers>ConfiguringAxoPatch200PatchClampsTheAxon(nowMolecularDevices)Axopatch200A&Bpatchclampsprovidegainandcurrent-/voltage-clampoperatingmodeinformationviagainandmodetelegraphoutputsontherearpaneloftheamplifier.Upto4Axopatch200patchclampscanbesupported.ForeachAxopatch200inuse,connectionsmustbemadebetweentheScaledOutput,10VmandIOutputsignaloutputchannels,GainandModetelegraphchannels,andtheanalogueinputsofthelaboratoryinterface.AconnectionmustalsobeestablishedbetweenananalogueoutputandtheAxopatch200CommandInput.Involtage-clampmode,theScaledOutputchannelmonitorsthecurrentsignalandthe10Vmprovidesthecellmembranepotential.Incurrent-clampmode,theScaledOutputswitchestomembranepotentialandtheIOutputoutputismonitoredforthecurrent.Thecurrent-voltage-clampmodeswitchingbetweenthe10VmandIOutputoutputscanbeaccomplishedeitherbyphysicallychangingthesignalcableattachmentsorbyreassigninganalogueinputsusingsoftware.PhysicallyswitchedSecondaryInputSwitchingToconfigureWinWCPtouseanAxopatch200asAmplifier#1,selectAxopatch200astheamplifiertypeontheAmplifier#1pageoftheInputChannels&AmplifiersSetupdialogbox,asshownbelow.

Thedefaultconnectionsare: Primary

Ch.SecondaryCh.

Axopatch200 ScaledOutput

10Vm

IOutput

ModeTelegraph

GainTelegraph

CommandInput

LaboratoryInterface

AI0 AI1 AI1 AI6 AI7 AO0

Thesecondaryinputchannel(AI1)mustbephysicallyswitchedbetween10VmandImwhenthepatchclampisswitchedfromvoltage-tocurrent-clampmode.Thiscanbeaccomplishedbyphysicallyswappingthecoaxialsignalcablesor

usingaBNCswitchbox.SoftwareswitchedSecondaryChannelSwitchingVoltage/current-clampmodeswitchingofthe10VmandIOutputchannelscanbeaccomplishedautomaticallybyattachingthe10VmandIOutputoutputstodifferentanalogueinputchannelsandremappingtheanalogueinputtothesecondarychannel(Ch.1)usingsoftware.SoftwarechannelswitchingcanbeconfiguredbyselectingtheIClampoptionontheAmplifier#1pageandselectingafreeanalogueinput(e.g.AI4)fortheCh.1SecondaryChannel,asshownbelow.

Theconnectionsforasoftwareswitchedsecondarychannelareshownbelow. PrimaryCh. SecondaryCh. Axopatch200

ScaledOutput

10Vm

IOutput

ModeTelegraph

GainTelegraph

CommandInput

LaboratoryInterface


MultipleAxopatch200amplifiers.

UptothreeadditionalAxopatch200amplifierscanbedefinedasAmplifiers#1-#3.ThedefaultconnectionsforAmplifier#2inatwoamplifierconfigurationareshownbelow Primary

Ch.SecondaryCh.

Axopatch200

ScaledOutput

10Vm

IOutput

ModeTelegraph

GainTelegraph

CommandInput

LaboratoryInterface


ThisisaphysicallyswitchedsecondarychannelconfigurationandassumesthatAmplifier#1isthesame.Toselectasoftwareswitchedconfiguration,thegainandmodetelegraphchannelsmustbemovedtoinputchannels7-15(ifthelaboratoryinterfacesupportsthem)ordisabledbysettingtheGainorModeTelegraphsettingstoOff,freeinguptheinputchannelsforuseascurrent-clampsecondaryinputs.

GettingStarted>Amplifiers>ConfiguringAMS-2400PatchClampsTheA-MSystemsAMS-2400patchclampsprovidegainandcurrent-/voltage-clampoperatingmodeinformationviagainandmodetelegraphoutputsontherearpaneloftheamplifier.Upto4AMS-2400patchclampscanbesupported.ForeachAMS-2400inuse,connectionsmustbemadebetweentheModeOutput,FixedVmandFixedImsignaloutputchannels,GainandModetelegraphchannels,andtheanalogueinputsofthelaboratoryinterface.AconnectionmustalsobeestablishedbetweenananalogueoutputandtheAMS-2400CommandInput.Involtage-clampmode,theModeOutputchannelmonitorsthecurrentsignalandtheFixedVmprovidesthecellmembranepotential.Incurrent-clampmode,theModeOutputswitchestomembranepotentialandtheFixedImoutputismonitoredforthecurrent.Thecurrent-voltage-clampmodeswitchingbetweentheFixedVmandFixedImoutputscanbeaccomplishedeitherbyphysicallychangingthesignalcableattachmentsorbyreassigninganalogueinputsusingsoftware.PhysicallyswitchedSecondaryInputSwitchingToconfigureWinWCPtouseanAMS-2400asAmplifier#1,selectAMS-2400astheamplifiertypeontheAmplifier#1pageoftheInputChannels&AmplifiersSetupdialogbox,asshownbelow.


Ch.SecondaryCh.

AMS-2400 ModeOutput

FixedVm

FixedIm

ModeTelegraph

GainTelegraph

CommandInput

LaboratoryInterfaceAI0 AI1 AI1 AI6 AI7 AO0Thesecondaryinputchannel(AI1)mustbephysicallyswitchedbetweenFixedVmandFixedImwhenthepatchclampisswitchedfromvoltage-tocurrent-clampmode.ThiscanbeaccomplishedbyphysicallyswappingthecoaxialsignalcablesorusingaBNCswitchbox.

SoftwareswitchedSecondaryChannelSwitchingVoltage/current-clampmodeswitchingoftheFixedVmandFixedImchannelscanbeaccomplishedautomaticallybyattachingtheFixedVmandFixedImoutputstodifferentanalogueinputchannelsandremappingtheanalogueinputtothesecondarychannel(Ch.1)usingsoftware.SoftwarechannelswitchingcanbeconfiguredbyselectingtheIClampoptionontheAmplifier#1pageandselectingafreeanalogueinput(e.g.AI4)fortheCh.1SecondaryChannel,asshownbelow.

Theconnectionsforasoftwareswitchedsecondarychannelareshownbelow. PrimaryCh. SecondaryCh. AMS-2400

ModeOutput

FixedVm

FixedIm

ModeTelegraph

GainTelegraph

CommandInput

LaboratoryInterface


MultipleAMS-2400amplifiers.UptothreeadditionalAMS-2400amplifierscanbedefinedasAmplifiers#1-#3.ThedefaultconnectionsforAmplifier#2inatwoamplifierconfigurationareshownbelow Primary

Ch.SecondaryCh.

AMS-2400 ModeOutput

FixedVm

FixedIm

ModeTelegraph

GainTelegraph

CommandInput

LaboratoryInterface


ThisisaphysicallyswitchedsecondarychannelconfigurationandassumesthatAmplifier#1isthesame.Toselectasoftwareswitchedconfiguration,thegainandmodetelegraphchannelsmustbemovedtoinputchannels7-15(ifthelaboratoryinterfacesupportsthem)ordisabledbysettingtheGainorModeTelegraphsettingstoOff,freeinguptheinputchannelsforuseascurrent-clampsecondaryinputs.

GettingStarted>Amplifiers>ConfiguringtheNPIELC_03XSAmplifierTheNPIELC-03XSAmplifierprovidescurrentandvoltageandgaintelegraphoutputsonthefrontpaneloftheamplifier.Upto4NPIELC-03XSamplifierscanbesupported.ForeachELC-03XSinuse,connectionsmustbemadebetweentheCurrentOutput,PotentialOutputandCommandInputsignalchannels,CurrentandPotentialGaintelegraphchannels,andtheanalogueinputsofthelaboratoryinterface.ConnectionsmustalsobeestablishedbetweentwoanalogueoutputsandtheCommandInputandStimulusInputsTheCurrentOutputchannel,attachedtotheWinWCPprimaryinputmonitorsthecurrentsignalinbothvoltage-andcurrent-clampoperatingmodes.Involtageclampmode,thecellpotential,attachedtotheWinWCPsecondaryinput,isobtainedfromCommandInputand,incurrent-clampmodefromthePotentialOutput.Thecurrent-voltage-clampmodeswitchingbetweenthePotentialOutputandCommandInputcanbeaccomplishedeitherbyphysicallychangingthesignalcableattachmentsorbyreassigninganalogueinputsusingsoftware.PhysicallyswitchedSecondaryInputSwitchingToconfigureWinWCPtouseanNPIELC-03XSasAmplifier#1,selectNPIELC-03XSastheamplifiertypeontheAmplifier#1pageoftheInputChannels&AmplifiersSetupdialogbox,asshownbelow.


Ch.SecondaryCh.

ELC-03XS CurrentOutput

VCmodeCommandInput

CCModePotentialOutput

PotentialTelegraph

GainTelegraph

CommandInput

StimulusInput

LaboratoryInterface

AI0 AI1 AI1 AI6 AI7 AO0 AO1

Thesecondaryinputchannel(AI1)mustbephysicallyswitchedbetween

CommandInputandPotentialOutputwhenthepatchclampisswitchedfromvoltage-tocurrent-clampmode.ThiscanbeaccomplishedbyphysicallyswappingthecoaxialsignalcablesorusingaBNCswitchbox.Notethat,involtage-clampmode,CommandInputhastwoconnectionstoit(AO0andAI1).SoftwareswitchedSecondaryChannelSwitchingVoltage/current-clampmodeswitchingoftheCommandInputandPotentialOutputchannelscanbeaccomplishedautomaticallybyattachingtheCommandInputandPotentialOutputoutputstodifferentanalogueinputchannelsandremappingtheanalogueinputtothesecondarychannel(Ch.1)usingsoftware.SoftwarechannelswitchingcanbeconfiguredbyselectingtheIClampoptionontheAmplifier#1pageandselectingafreeanalogueinput(e.g.AI4)fortheCh.1SecondaryChannel,asshownbelow.

Theconnectionsforasoftwareswitchedsecondarychannelareshownbelow.Thedefaultconnectionsare: Primary

Ch.SecondaryCh.

ELC-03XS CurrentOutput

VCmodeCommandInput

CCModePotentialOutput

PotentialTelegraph

GainTelegraph

CommandInput

StimulusInput

LaboratoryInterface

AI0 AI1 AI4 AI6 AI7 AO0 AO1

NotethatCommandInputhastwoconnectionstoit(AO0andAI1).

GettingStarted>Amplifiers>ChannelCalibrationTableWinWCPdisplaysthesignalsstoredineachsignalchannelintheunitsappropriatetoeachchannel.Thenames,unitsandscalinginformationforeachchannelareenteredintothechannelcalibrationtablewhichcanbedisplayedbyselectingtheInputChannelstabintheInputChannels&Amplifiersdialogbox.

The channel calibration tablesettings for the patch clampcurrent and voltage channels areautomatically configured withappropriate names, units andV/Unitsscalingwhenanamplifieris selected from theAmplifiers list. However, whenno amplifier is selected for use(i.e. Amplifier=None) thecalibration information for achannelmustbeentereddirectlyintothetable.

Thereare5entriesinthetableforeachchannel.

Name:A1-4letternameusedtoidentifythesourceofthechannel(e.g.Vm,Im).

V/Units:ThescalingfactorrelatingthevoltagelevelattheinputsoftheA/Dconverter(inV)totheactualsignallevelsineachchannel(intheunitsdefinedintheUnitscolumn).

Units:Themeasurementunitsofthesignal(e.g.mV,pAetc.).

ADCCh.:Theanalog/digitalconverterinputchannelfromwhichthesignalforthischannelisbeingacquired.(Note.Signalchanneltoanaloginputmapping

iscurrentlyonlyavailablewithNationalInstrumentsinterfacecards.)

Amplifier:Indicateswhetheranamplifierhasbeendefinedforthischannel.

For example, if the membrane voltage output of your amplifier supplies asignalwhichis10Xthemeasuredmembranepotentialofthecell,andtheunitshavebeendefinedasmV, then theappropriateV/Units setting is0.01 (sincetheamplifiervoltageoutputis0.01VoltspermV).

Amplifier

DisplayGridsandTimeUnits

TheTimeUnitsoptionsdeterminetheunits(secsormsecs)usedtodisplaytimeintervalsinsignaldisplaywindows.

Next

GettingStarted>Amplifiers>CED1902Amplifier

CambridgeElectronicDesignLtd(www.ced.co.uk)TheCambridgeElectronicDesign1902isacomputer-controlledamplifierwithbuilt-inisolationcircuitsforrecordingECGandEMGsignalsfromhumans,andabridgecircuitforrecordingfromtensionorpressuretransducers.Itcanalsobeusedtorecordextracellularelectricalactivityfromnerveandmuscle.Ithastwoinputs.TheElectrodesinputisanelectricallyisolateddifferentialamplifierinputusedtorecordECG,EMGandsimilarsignals.Isolationmakesitsafetoattachrecordingleadstohumansubjects.

TheTransducerinputisadifferentialamplifierinputusedtorecordfromtransducerssuchasforceandpressuretransducers.Itisnotisolated.

ConnectionsInordertocontroltheamplifier,theCED1902serialcommunicationscablemustbeconnecttoaserialport(COM1orCOM2).TheanalogoutputoftheCED1902shouldalsobeconnectedtoanaloginputCh.0ofthelaboratoryinterface.TheamplifiergainsettingistakenintoaccountbyWinWCPinscalingthesignallevelonthischannel.ControlPanelSelectSetupCED1902Amplifier

toopenCED1902controlpanel

Thefollowingamplifiersettingscanbeconfiguredusingthepanel:COMPort:Selectstheserialportusedtocommunicatewiththe1902.Input:Selectstheamplifierinput;Groundedtoconnecttheinputtoground(i.e.thereisnosignal.),SingleEndedforthetransducerinputinsingledended(i.e.non-differentialmode),NormalDiff+fortheTransducerinputindifferentialamplifiermode,InvertedDiff-forthetransducerinputindifferentialmodewiththesignalinverted,orIsolatedEEGfortheelectricallyisolateddifferentialinput.Note.NormalDiff+modeisusedwhenrecordingfromaforceorpressuretransducerandIsolatedEEGusedwhenmakingEMG,ECGrecordings.AmplifierGain:SetsthegainoftheCED1902amplifier.Therangeofgainsettingsdependsonwhichinputisinuse.WhenusingtheTransducerinputthereare11gainsettings(X1,X3,X10,X30,X100,X300,X1000,X3000,X10000,X30000,X10000).WhenusingtheElectrodesinput(X100,X300,X1000,X3000,X10000,X30000,X100000,X300000,X1000000,X3000000,X1000000).Note.WhenCED1902isselectedastheAmplifierintheRecordingsetupdialogbox,thegainsettingisautomaticallyincludedinthescalingofinputchannel0LowPassFilter:Setsthecut-offfrequencyoftheCED1902’sbuilt-inlowpassfilter.ThefiltercanbesettoNone(outofuse),1000Hz,500Hz,100Hz.Thelowpassfilterremovessignalfrequenciessignalhigherthanthecut-off,smoothingthesignal.HighPassFilter:Setsthecut-offfrequencyoftheCED1902’sbuilt-inhighpassfilter.ThefiltercanbesettoNone(outofuse),50Hz,100Hz,200Hz.Thehighpassfilterremovessignalfrequenciessignallowerthanthecut-off,

removingsteadyandslowlychangedcomponentsofthesignal.ACCoupled:CheckthisboxtomaketheCED1902inputAC(alternatingcurrent)coupled.Inthismodeonlyvariationsinthesignalareallowedthroughtotheamplifier,constant(DC)levelsareblocked.(ACcouplingshouldbeusedwithEMGandECGrecordingsbutnotwithforceorpressuretransducerrecordings.)50HzFilter:Enable/disablesanotchfilterwhichselectivelyremovesfrequenciesaround50Hz.(Usedtoremove50Hzinterferencefrommainspowerlines).DCOffset:TheDCOffsetfacilityaddsorsubtractsaDCvoltagelevelfromtheinput.Theoffsetrangedependsupontheinputmode(NormalDiff+andInvertedDiff-=+/-0.5mV,SingleEnded=+/-500mV,Electrodes=+/-0.1mV).TheoffsetfacilityisoftenusedtocanceloutthestandingDCvoltagesignalfromtensiontransducers.

Next

GettingStarted>Amplifiers>TecellaPatchClampAmplifierControlPanelTheamplifiergain,compensationandcurrent/voltageclampmodeofTecellapatchclampamplifierscanbesetfromthiscontrolpanel.SelectSetupTecellaPatchClamp

todisplaytheTecellaPatchClampcontrolpanel.

Voltage-ClampModeConfig:SelectoneoftheVCLAMPconfigurationstosettheamplifierintovoltage-clampmode

Input:Selectstheamplifierinput(None=Noinput,Head=headstageinput,VModel=100MOhmmodelcell,IModel=1MOhmmodelcell).Gain:Selectstheheadstagefeedbackresistor.Current-ClampModeConfig:SelectoneoftheICLAMPconfigurationstosettheamplifierintocurrent-clampmode

Input:SelectHead=headstageinputorHead+Model=10MOhmmodelcellinparallelwiththeinput.Gain:Selectsthegainofthevoltagechannelamplifier.SelectEnableStimulustoallowcurrentstimulitobeappliedtothecell.BiasCurrectCorrection:Picoamplifiersproduceasmallbiascurrentwhenisselectedresultinginadeflectionofthecellmembranepotentialunlesscompensatedfor.ThiscanbecorrectedbyobservingtheshiftinmembranepotentialwhichoccurswhenEnableStimulusistoggleonandoffandenteringcurrentvalueintotheZero

Correctionfielduntilnowshiftoccurs.Filters

Lowpassfilter:Sliderselectscut-offfrequencyofBessellowpassfilter.CompensationAutopage:Automaticcapacity/leakcurrentcompensation.

ClickAutoCompensatetoautomaticallycompensateforpipette/cellcapacityandcellleakconduction.ClickJunct.Pot.AutoZerotocompensatefortheelectrodejunctionpotentials,settingtheinputcurrenttozero.ClickClearCompensationtosetallcompensationtozero.Options

TicktheUseanalogleaksubtractionoptiontousetheamplifieranalogleakcurrentsubtractioncircuitsintheautomaticleakcurrentcompensation.TicktheUsedigitalleakcurrentsubtractionoptiontousethedigitalleakcurrentintheautomaticleakcurrentcompensation.TicktheUsedigitalartefactremovaloptiontousethedigitalartefactfunctionintheautomaticleakcurrentcompensation.TickApplytoallchannelstoapplyautomaticcompensationtoallamplifierchannels(onlyappliestomulti-channelamplifiers).Compensationcoefficient:Setsthecompensationcoefficientfactor(0=optimalcompensation,>0=under-compensation,<0=over-compensation).Capacitypage:Capacitycompensationsettingsinuse.CanbeadjustedbytheuserorsetautomaticallyusingAutoCompensate.

Resistancepage:Cellleakandpipetteseriesresistancecompensationsettingsinuse.CanbeadjustedbytheuserorsetautomaticallyusingAutoCompensate.

JunctionPot.page:Electrodejunctionpotentialcompensationsettingsinuse.anbeadjustedbytheuserorsetautomaticallyusingJunct.Pot.AutoZero.

ZapCell:ClickZapCelltoapplyavoltagepulseofamplitudesetbyAmplitudeanddurationsetbyDur.

StimulusStreamingModeSelectStimulusStreamingEnabledtogeneratestimuluspulsesaspoint-by-pointwaveforms(ratherthanassetsofdiscretestep/rampcommands).

GettingStarted>Amplifiers>EPC-9/10PatchClampAmplifierTheamplifiergain,compensationandcurrent/voltageclampmodeofTecellapatchclampamplifierscanbesetfromthiscontrolpanel.SelectSetupEPC-9/10PatchClamp

todisplaytheEPC-9/10PatchClampcontrolpanel.

Amp.No.:Selectstheamplifierchanneldisplayedonthepanelwhenamulti-channelamplifierisinuse.

Gain:Selectstheamplifiercurrentgain.Mode:Selectsvoltage-orcurrent-clampmode.TheCCGainandCCTausettingsdeterminegainandresponsetimeofcurrent-clamp.SelecttheGentleModeChangeoptiontochangemodegently.

Filters:Selectsthefilterresponsetypeandcut-offfrequencyofthethetwolowpassfiltersinthecurrentrecordingpathway.Filter1has4fixedsettings,Bessel100kHz,Bessel,30kHz,Bessel10kHzandHQ30kHz.Filter2canbeselectedtohaveeitheraBesselorButterworthresponseandacut-offfrequencybetween0.1and16kHz.(ABesselresponseminimisessignaldistortion(ringing)afterstepchangesinthefilteredsignalwhereasaButterworthresponseprovidesasharpercut-offofhighfrequencies.)

Cfast:Setstheamplitudeandtimeconstantofthefast(i.e.pipette)capacitycompensation.ClicktheAutobuttontoautomaticallysettheCfastcompensation.ClicktheClearbuttontocancelcompensation.

Cslow:Setstheworkingrange,amplitudeandtimeconstantoftheslow(i.e.cell)capacitycompensation.ClicktheAutobuttontoautomaticallysettheCslowcompensation.ClicktheClearbuttontocancelcompensation.

RSCompensation:Setstheresponsespeedandfraction(%)ofseriesresistancecompensation.ClicktheAutobuttontoautomaticallysettheRScompensation.ClicktheClearbuttontocancelcompensation.

Leak:Setstheamountofleakconductancesubtractedfromcurrents.ClicktheAutobuttontoautomaticallysettheleaksubtraction.ClicktheClearbuttontocancelleaksubtraction.

Vpipette:Setsthepipetteandliquidjunctionpotentialcompensationandholdingvoltage.ClicktheAutobuttontoautomaticallysetthepipettecompensation..ClicktheClearbuttontocancelcompensation.

CommandStimulus:Selectthevoltageclampcommandstimulusinputpathandenable/disablelowpassfilteringofstimuluspulses.

GettingStarted>Amplifiers>MolecularDevicesMulticlamp700A/BMolecularDevicesMulticlamp700Aor700BpatchclampsarecontrolledviatheMulticlampCommandercontrolpanelsoftwaresuppliedwiththeamplifierswhichcanbeusedtosetcurrentandvoltagechannelgainandvoltage/current-clampmode.EachMulticlamp700supportstwoseparateamplifiers(Channel1andChannel2)whichrequirestwoWinWCPamplifierchannelstobedefined.WhenaMulticlamp700isinused,bothAmplifier#1andAmplifier#2shouldbedefinedasAxonMulticlamp700A/B.

(Note.IfasecondMulticlampisinuse,Amplifier#3andAmplifier#4shouldbedefinedasAxonMulticlamp700A/B.)AcommunicationslinkbetweentheMulticlampCommandercontrolpanelandWinWCPisautomaticallysetupafterbothMulticlampCommanderandWinWCPprogramsarestarted,allowingWinWCPto

determinecurrentandvoltagechannelscalingfactorsandvoltage/current-clampmodeforeachamplifierchannel.ResettingtheMulticlamp-WinWCPCommunicationsLinkIfMulticlampCommanderisstoppedandrestartedwhileWinWCPisrunning,communicationsbetweentheprogramscanbelost.ItcanbereestablishedbyselectingSetupResetMulticlamp700A/BLink.

GettingStarted>Amplifiers>DCLAMP-DynamicClampWinWCPsupportstheStrathclydeElectrophysiologySoftwareDCLAMPdynamicclampbasedontheNationalInstrumentscRIO-9076RealTimeController.Thedynamicclamppermitstheadditionorsubtractionofasimulatedvoltage-andtime-dependentionicconductanceto/fromapatch-clampedcellincurrent-clampmode.AHodgkin-Huxleyvoltage-dependentioniccurrentissimulatedwithbi-exponentialdecaykineticsissupported.Note.Contactj.dempster@strath.ac.ukfordetailsofthecRIO-9076hardwareandfirmwarerequiredtoimplementDCLAMP.SelectSetup->DCLAMPDynamicClamptoopenthedynamicclampcontrolpanel.

DCLAMPComPort:SelectstheserialportusedtocommunicatewiththecRIO-9076controller.ReversalPotential:ThereversalpotentialofthesimulatedioniccurrentisdefinedintheReversalPotential(Vrev)field.Conductance(Gmax):Themaximumconductance(nS)ofthesimulatedconductanceisdefinedintheConductancefield.

CurrentCommandScaleFactor:Thecurrentcommandscalingfactor(Amps/Volt)ofthepatchclampisenteredintoCurrentCommandScaleFactorfield.EnableInhibitInput(AI2):Tickthisoptiontoenableinhibitionofthesimulatedcurrentinrealtimebya5VsignalappliedtotheAI2inputofthecRIO-9076controller.Conductance:SelecttheAddoptiontoaddthesimulatedcurrenttothecell,SubtracttosubtractitandOfftodisabletheconductance.ActivationParameter(m):Definesthedependenceofsteady-stateandtimeconstantoftheconductanceactivationparameter(m)onmembranepotential.InactivationParameter(h):Definesthedependenceofsteady-stateandtimeconstantoftheconductanceactivationparameter(h)onmembranepotential.FastandslowkineticsaredefinedandtherationoffasttoslowkineticsbytheFastFractionfield.ParameterIncrementing:ConductancemodelparameterscanbeincrementedattheendofastimulusprotocolbyselectingtheAtEndofProtocoloptionorafteranumberofrecordsbyselectingtheAfterNo.ofRecordsoptionandenteringtherequirednumberofrecordsintheassociatedbox.Increments(bothpositiveandnegative)canbeappliedtothemaximumconductance(Gmax),half-maximalvoltagesoftheactivation(m)andinactivation(h)voltage-sensitivitycurvesandactivationandinactivationtimeconstantsbyenteringnon-zerostepsizesintotheincrementstable.

NOTE.Itcantakeupto45secondstocompletelyupdatethevoltage-sensitivityoftheconductancemodelandduringthatperiodtheconductancemodelwillbeinanintermediatestate.Recordsacquiredwithinthefirst45secondsafteramodelchangeshouldbediscounted.UpdatestoGmaxarefasterandarecompletewithin5seconds.Load/SaveSettings:ClicktheSaveSettingsbuttontosavethedynamicclampconductancesettingstoa.DCSsettingsfile.ClicktheLoadSettingsbuttontoloadsettingsfroma.DCSfile.UpdateDynamicClamp:Updatesdynamicclampwithcurrentsettings.Graphs:TheGraphspageshowsthesteadystateactivation-andinactivation-voltageandtheactivationandinactivationtimeconstant-voltagecurvesforthecurrentsetofmodelparameters.ThedatapointsinthesecurvescanbecopiedtotheWindowsclipboardbyclickingCopytoClipboard.

RecordingExperimentalSignals>MonitoringInputSignals&PatchPipetteSealTestYou can monitor the signals appearing on each channel using the signalmonitor/pipette seal test module which provides a real-time oscilloscopedisplay anddigital readout of the signal levels on the cellmembrane currentandvoltagechannels.Atestpulsecanalsobegeneratedformonitoringpipetteresistanceinpatchclampexperiments.Toopenthemonitor/sealtestmodule,selectfromthemenuRecordPipetteSealTest/SignalMonitor

An oscilloscope trace showing the current signal on each input channel isdisplayed.

DisplayscalingThe vertical display magnification is automatically adjusted to maintain avisibleimageofthetestpulsewithinthedisplayarea.Automaticscalingcanbedisabled by un-checking the Auto scale check box allowing the verticalmagnificationforeachchanneltobeexpandedtoaselectedregionbymovingthe mouse to the upper limit of the region, pressing the left mouse button,drawingarectangletoindicatetheregionandreleasingthemousebutton.The

verticalmagnificationcanalsobeadjustedusingthe+-buttonsattherightedgeofeachplot.AmplifiersTheAmplifierselectionboxindicateswhichamplifieriscurrentlyselectedforsealtestandthecurrentandvoltageinputchannelsbeingmonitored.IftwoormoreamplifiersareinusethesealtestcanbeswitchedbetweenamplifiersbyselectingAmplifier#1,#2etc.(SeeAmplifiers)The amplifier voltage/current- clamp mode is indicated by the ClampModeoptions.(Whenmodetelegraphsareoperationalfortheamplifiertheseindicate the actual state of the amplifier.When telegraph information is notavailable the Vclamp and Iclamp buttons must be set by the user to theamplifierclampmode.)The amplifier gain (current channel gain in voltage-clamp mode, voltagechannelgain incurrent-clampmode) is indicated in theAmplifierGainbox.When gain telegraphs are operational these indicate the actual state of theamplifier.When telegraph information is not available the current gainsettingisenteredherebytheuser.Theanalogoutputchannel(s)towhichthetestpulseisappliedisindicatedintheSendPulseTolistofcheckboxes.SelectinganamplifierintheAmplifierselection box, automatically selects the output channel connected to theamplifier stimulus command input. The test pulse output can be routed to adifferent output channel (or to additional channels) by ticking the requiredchannels.CellholdingvoltageandtestpulsesYoucancontroltheholdingvoltageappliedtothecellandtheamplitudeanddurationofa testvoltagepulsebyselectingoneof threeavailable testpulses(Pulse#1,#2,#3).Thesizeofeachpulsetypeissetbyenteringanappropriatevalueforholdingvoltageandpulse amplitude into theHoldingvoltage orAmplitude box foreachpulse.ThewidthofthepulseisdefinedbythePulsewidthbox.You can switch between pulses by pressing the function key associatedwitheachpulse(Pulse#1=F3,Pulse#1=F4,Pulse#1=F5).

CurrentandvoltagereadoutsA readout of the cell membrane holding current and voltage, and test pulseamplitude,appearsatthebottomofthemonitorwindow.ClickingtheSavetoLogbuttonsaves thecurrentPipetteorCell readings tothelogfile.During initial formation of a giga-seal, the Pipette option displays pipetteresistance,computedfrom

where Vpulse and Ipulse are the steady-state voltage and current pulseamplitudes.TheCell (G)pagedisplays thecellmembraneconductance,Gm,capacity,Cm,andaccessconductance,Ga,andtheCell(R)pagedisplaysthecellmembraneresistance,Rm,(1/Gm)capacity,Cm,andaccessresistance,Ra,(1/Ga).Gm,Cm,andGaarecomputedfrom

whereI0 is the initial currentat the startof thecapacity transientand is theexponentialtimeconstantofdecayofthecapacitancecurrent(SeeGillis,1995,for details). I0 ican be estimated either The Ga estimate from optiondetermineshowI0 isestimated(Peak: I0 estimated from thepeakcurrentofthe capacity transient.Exp.Amp.: I0 estimated from amplitude of the fittedexponentialatthestartofthevoltagestep.Note. If Ga, Gm and Cm are to be estimated correctly, the patch clamp’s

pipette series resistance compensation and capacity current cancellationfeaturesmustbeturnedoff.SweepsAveragedThe Sweeps Averaged setting determines the number of test pulse sweepsaveragedtocalculatethedisplayedpipetteandcellparameters(Rpipette,Rm,Gm,Cm,Ra,Ga).Valuescanrangefrom1(noaveraging)to10(averagingofthe10mostrecenttestpulses).ZapClicking the Zap button to apply a single voltage pulse of amplitude anddurationdefinedintheAmplitudeandPulseWidthboxes.Azappulsecanbeusedtoperforatethecellmembraneinacell-attachedpatchtoformawhole-cellpath.

RecordingExperimentalSignals>RecordingSignalsCreatingadatafile

Tocreateanewdatafiletoholdyourrecordings,selectfromthemenuFileNewDataFiletoopentheNewDataFiledialogbox.

Select thediskand folder intowhich the file is tobeplacedusing theSaveInlistbox.WinWCPdatafileshavetheextensionextension".wcp"Afteradatafilehasbeencreated,selectRecordRecordtodisk

toopentheRecordtodiskwindow.

Thedisplayareaofthescreenactslikeadigitaloscilloscope,showingtracesofthesignalsastheyarerecorded.Theamplifiergain(currentchannelgaininvoltage-clampmode,voltagechannelgainincurrent-clampmode)isindicatedintheAmplifierGain/Modebox.Whengaintelegraphsareoperationaltheseindicatetheactualgainsettingsoftheamplifier.Whentelegraphinformationisnotavailablethecurrentgainsettingisenteredherebytheuser.The amplifier voltage- /current-clamp mode is indicated by the

VClamp/IClamp options. (When mode telegraphs are operational for theamplifier these indicate the actual state of the amplifier. When telegraphinformationisnotavailabletheVclampandIclampbuttonsmustbesetbytheusertotheamplifierclampmode.)

Recordingmodes

Ingeneral,recordingsweep(s)mustbesynchronisedwiththestartofthesignalsunder study, toensure that the signal is capturedwithin the recordandalwaysappears in the same place. The trigger mode determines how thissynchronisationtakesplace.Thereare4recordingmodes:FreeRunExternalTriggerEventDetectorStimulusProtocolYou must select a recording mode appropriate to the type of signal to berecordedandtheconfigurationofyourrecordingsystem.

RecordingExperimentalSignals>RecordingModes>FreeRunTheFree Run trigger mode is used forunsynchronised recording. Recordingsweeps start immediately after theRecord button is pressed and continueuntiltherequirednumberofrecordshavebeencollected.Choosethefreerunmodeforsimpletestsofthelaboratoryinterfaceandforsignalswhere synchronisation is not possible orrequired.

RecordingExperimentalSignals>RecordingModes>ExternalTriggerWhen theExt. Triggeredmode is selected,the recording sweep is synchronised to atrigger pulse applied to the external triggerinput of the laboratory interface. If theActive High option is selected, recordingwillbetriggeredbya0V-to-5Vtransitiononthetriggerinput.IftheActiveLowoptionisselected,recordingwillbetriggeredbya5V-to-0V transition. (Note, some laboratoryinterfaces support only one or other of thetwotriggerpolarities.)

Many kinds of electrophysiological signalsare evokedby stimulating the cell or tissue,using an electrical stimulator. In order to record such signals, the recordingsweepmustbesynchronisedwiththestimulator,ideallysothatthesweepsstartsshortlybeforethecellisstimulated.Duringastimuluscycle,thestimulatorsync.pulse is produced first (triggering the recording sweep) and, after a delay(settableonthestimulatorfrontpanel),thestimulusitself.

RecordingExperimentalSignals>RecordingModes>DetectEventsThe Detect Events event detector modeprovides ameans of detecting signals as theyoccur within an incoming analog signal. Athreshold-based event detection algorithmmonitors the incoming signal on one of theinputchannels.Anevent isdetectedwhenthesignal deviates bymore than a predeterminedlevel from the average baseline level. Tocompensate for slow drifts in the baselinelevel, the threshold level is maintained at aconstant distance from the baseline bymeansof a running average calculation. The eventdetector is configured by setting threeparameters.

If more than one channel is being recorded,selecttheinputchannelonwhicheventsaretobedetectedfromthedetectionChannellist.

Enter the detection threshold into theThreshold box. The threshold level isexpressedasapercentageofthetotalinputrange,withitspolaritydeterminingwhetherpositive-ornegative-goingsignalsaretobedetected.Thelevelshouldbesetassmallaspossibletomaximisethelikelihoodofaneventbeingdetected,butwithoutproducinganexcessivenumberof false eventsdue tobackgroundnoisetriggeringthedetector.Valuesofaround5-10%areoftenused,butseveraltrialsmaybenecessarybeforethebestlevelforaparticularexperimentisfound.

ThePretrigger settingdetermines thepercentageof the record tobecollectedbeforethedetectionpoint.Atypicalvalueis30%.

RecordingExperimentalSignals>RecordingModes>StimulusProtocolInStimulus Protocol mode,WinWCP functions as a stimulator as well as arecordingdevice.Sequencesofrecordingsweepsareacquiredattimedintervals,in synchronywith computer-generated stimuli applied to the cell. The stimulicanbeintheformofeithervoltagewaveformsoron/offTTLdigitalpulsesforcontrollingvalvesorotherdevices.Up to4voltagewaveformoutputchannelsaresupportedand4-8TTLdigitaloutputchannels(dependingonthelaboratoryinterface).

Eachstimuluspulseisassociatedwithasinglerecordingsweepandthedurationor amplitude of any part of a pulse can be incremented between records. Acomplete stimulus protocol thus consists of a series of one or more pulses,incremented in amplitude or duration to create a family of pulses. Complexstimulus waveforms can be produced, including series of rectangular steps,ramps, and digitised analog signals. Protocols are created using theStimulus/RecordingProtocolEditor.

ApplyingSingleProtocols

SelecttheSingleoptiontoapplyasinglestimulusprotocol (or linked series of protocols) selectedfrom the list of available protocols in theProtocollist.

By default, protocol files are stored as files withthe.XMLextensioninthefolderc:\winwcp\vprot.TheprotocolfoldercanbechangedbyclickingSetStimulusProtocolFolderandselectinganotherfolder.

ApplyingSequencesofProtocols

Select the List option to applying a sequence of protocols defined by theprotocolexecutionlist,selectedfromProtocolList.

Tocreateanewprotocolexecutionlist:

1)EnteranameforthenewlistandclickNewList.

2) Add protocols to the list by clicking AddProtocoltoListandselectingaprotocolusingtheprotocolfileselectionbox.

Todeletethecurrentlyselectedprotocolexecutionlist,clickDeleteList.

Protocol execution lists are stored as files with.LSTextensionsintheprotocolsfolder.

RecordingExperimentalSignals>SettingtheRecordingSweepSizeandDurationWhenFreeRun,ExternalTriggerorDetectEventsrecordingmodesareselected,thenumberofanaloginputchannels,numberofsweeps,thenumberofdigitalsamplesperchannelandtherecordingsweepdurationneedtobesetintherecordingwindow.(Note,inStimulusProtocolmode,thesearesetintheprotocol.)No.records: Sets the number of recordingsweepstobecollectedNo. input channels: Sets the number ofanaloginputchannelstobeacquired.Channelsarealwaysacquiredinsequencefrom Ch.0 upwards, i.e. No. input channels=1, selects Ch.0; No. inputchannels=2selectsCh.0&Ch.1etc.Record duration: Sets the required duration of the recording sweep. Set thedurationtoavaluethatisapproximately50%longerthanthetimecourseofthesignalsthatyouintendtorecord.

No.samples:Setsthenumberofsamplestobeacquiredperinputchannel.Theminimum is 256 samples per channel and increments are in units of 256.Themaximumrangesfrom16184/No.Channels-1048576/No.Channels,dependingonthelaboratoryinterfaceinuse.

Sampling Interval: The Sampling interval box displays the time betweendigitisedsamplesacquiredfromeachanaloginputchannel.Itisdeterminedfrom

EnteringavalueintotheSamplingintervalbox,willresultinanadjustmentoftheRecordduration.

Itisimportanttouseasamplingintervalwhichissmallenoughtoensurethatasufficient number of samples are acquired during the most rapidly changingphases of the signals being recorded. For most types of signal, 2048samples/channelandarecorddurationapproximately50%longerthanthesignal

timecourseprovidesatisfactoryresults.(However,notethatsomesignals,suchascardiacventricularactionpotentials,cancombinelongtimecourses(200-300ms)withveryrapidrisingphases(1-2ms).Insuchcircumstances,8192ormoresamples/channelmightberequiredtoaccuratelyrepresenttherisingphase.)

To avoid aliasing artefacts, the analog signals should be low-pass filtered toremove frequency components greater than half of the sampling rate (i.e.reciprocalofthesamplinginterval).

RecordingExperimentalSignals>CreatingStimulusProtocols>OpeningtheStimulus/RecordingProtocolEditorTocreateastimulusprotocol,selectSetupStimulus/RecordingProtocolEditor

toopenthestimuluseditormodule.

Upto4voltagewaveformoutputchannels(AO0-AO3)areavailableand8TTLdigitalpulsechannels(DO0-DO7).AdiagramoftheoutputwaveformsappearsintheWaveformdisplaybox.

Tocreateastimulusprotocol,clicktheNewProtocol

buttontocreateablankprotocol.

Next

RecordingExperimentalSignals>CreatingStimulusProtocols>Analog&DigitalOutputChannels

AnalogOutputs(AO):SelectthenumberofanalogoutputchannelstobeusedintheanalogoutputsNo.Channelslist.For eachAOchanneldefined, select the typeof stimulus (VoltageorCurrent)andthestimulusunitsintheStimulusTypelistandtheholdinglevel(thelevelrelativetowhichvoltage/currentstepsareapplied)intheHoldingLevelbox.

DigitalOutputs(DO):SelectthenumberofdigitaloutputchannelstobeusedinthedigitaloutputsNo.Channelslist.

Settheholdingpattern(thedefaultdigitaloutputstatebetweendigitalstimuluspulses)intheHoldingPatterncheckboxes.

(Note, during execution of a stimulus protocol, the analog and digital holdinglevelsoverridethedefaultholdinglevelssetintheSealTestorDefaultSettingswindows).

Next

RecordingExperimentalSignals>CreatingStimulusProtocols>RecordingSettings

Select theRecording tab page to set the number of recording sweeps to beacquired during the protocol and the number of analog channels,samples/channel,duration,andsweeprepetitioninterval.Recording duration: theduration of the recordingsweep. (Note. When theFixed sampling interval optionisselected,thisentryisdefinedby the Sampling intervalsetting.)

Sampling interval: Timeinterval between samples oneach channel. When theFixed option is selected, thesampling interval entered heredefines the duration of theRecordingduration(SamplingintervalxNo.samplesperchannel).WhentheFixedoptionisnotselected,samplingintervalissetbytheRecordingduration(Recordingduration/No.samplesperchannel).

Stimulus repeat period: the time interval between successive recordingsweepsintheprotocol.

No.records:Thenumberofrecordstobeacquiredduringtheprotocol.

No.repeatsper increment:For incrementedstepwaveforms, thenumberoftimesastimulusistoberepeatedwithoutincrementingthestepsize.

No. input channels: The number of analog input channels to be acquired.Channelsarealwaysacquired insequencefromCh.0upwards, i.e.No. inputchannels=1,selectsCh.0;No.inputchannels=2selectsCh.0&Ch.1etc.No.samples: The number of samples to be acquired per input channel. Theminimumis256samplesperchannelincreasinginstepsof256.Themaximumranges from16184/No.Channels - 1048576/No.Channels, dependingon thelaboratoryinterfaceinuse.

External Stimulus trigger (Y/N): Enables the stimulus protocol to betriggeredbyanexternalTTLpulseinsteadoftheinternaltimer.WhensettoY,thestimulusrepeatperiodisignoredandthestimulusprotocolbeginswhenaTTLpulseisreceivedontheExternalStimulusTriggerInput(Seelaboratoryinterfacecardconnectionstablesinsection1.)

Leaksubtractionsettings

A protocol can be programmed to add digital leak subtraction records usingthe P/N mode option. When this option is selected, a series of additionalrecordingsweepsaregeneratedforeachrecorddefinedintheprotocol,usinganinvertedandscaleddownversionofthecommandvoltagewaveform.Adigitalaverageisobtainedfromtheserecordsandstoredinthedatafileasa“LEAK”record,alongwiththebasic“TEST”record.

Youcanchangethenumberofpulsesusedtocomputethe“LEAK”recordanddivisionfactorbyalteringthevaluesintheboxesshown.Thedefaultvaluesare4leakrecordswithvoltagewaveformdividedby–4.Note that subtraction of the “LEAK” from “TEST” records is done using theleaksubtractionmodule.(Seesection13fordetails.)

Protocollinking

Recording normally stops when the requested sequence of records within aprotocoliscompleted.Protocolscan,however,belinkedtogetherbyselectingaprotocolfromtheLinktonextprotocol list,sothatoncompletionofthefirstprotocol,controlistransferredtothelinkedprotocol.

Next

RecordingExperimentalSignals>CreatingStimulusProtocols>AddingStimulusWaveformstotheProtocol

SelecttheStimulustabpagetoaddstimuluswaveformstotheanalogordigitaloutputchannels.

WaveformsareconstructedbydraggingwaveformstepandrampelementsfromtheToolboxanddroppingthemintotheselectedvoltagechannel(AO0-AO4)ordigital(DO0–DO7)outputlist.Aplotoftheresultingstimulusprotocolforeachoutputchannelisshownintheprotocoldisplaypanel.A stimulus waveform on each output channel can consist of up 10 separateelements. The amplitude and duration for each element is defined in its

parameterstablewhichcanbemadetoappearbyclickingontheelement.

Eight analog and 4 digitalwaveform elements are available in the toolbox, asdetailedbelow.

Rectangularvoltagepulseoffixedsize

Asimplepulse,whichdoesnot vary in amplitude anddurationbetweenrecords.Thiselementcanbeusedtoprovideseriesofstimulioffixedsizeor,incombinationwithotherelements,toprovidefixedpre-conditioningpulses. ParametersInitialDelay Delay(attheholdinglevel)beforethepulsebegins.Amplitude Pulseamplitude.Duration Pulseduration.

Familyofrectangularpulsesvaryinginamplitude

Arectangularvoltagepulsewhoseamplitudeisautomaticallyincrementedbetweenrecordingsweeps.Thiselementistypicallyusedtoexplorethevoltage-sensitivity of ionic conductances, by generating records containing thewhole-cell membrane currents evoked in response to a series of voltage steps todifferentmembranepotentials. ParametersInitialDelay Delay(attheholdinglevel)beforethepulsebegins.Amplitude Amplitudeofthefirstpulseintheseries.Amplitude(increment)

Incrementtobeaddedtoamplitudebetweenrecords.

Duration Pulseduration.

Familyofrectangularvoltagepulsesvaryinginduration

A rectangularvoltagepulsewhoseduration is automatically incrementedbetween recording sweeps.Thiselement ismost commonlyusedasavariableduration preconditioning pulse in 2 or 3 step protocols for investigatinginactivationkineticsofHodgkin-Huxleytypeconductances.

ParametersInitialDelay Delay(attheholdinglevel)beforethepulsebegins.Amplitude Pulseamplitude.Duration Pulseduration.Duration(increment) Incrementtobeaddedtodurationbetweenrecords.

Seriesofrectangularvoltagepulses(incrementableinterval)

Atrainofrectangularvoltagepulsesatfixedtimeintervals(incrementablebetweenrecords)andoffixedduration.Thiselementcanbeusedtoproduceaseriesofstimuli toobservetheeffectofrepeatedapplicationofastimulusatahighrate.Itcanalsobeusedtoproduceatrainofpre-conditioningstimuliforasubsequenttestwaveform. ParametersInitialDelay Delay(attheholdinglevel)beforethepulsebegins.Amplitude Pulseamplitude.Duration Pulseduration.Frequency Intervalbetweenpulsesintrain.Repeatperiod(incr.) Incrementinrepeatperiodbetweenrecords.No.repeats Numberofpulsesintrain.Seriesofrectangularvoltagepulses(incrementablerate)

A trainof rectangularvoltagepulsesata fixed frequency(incrementablebetweenrecords)andoffixedduration.Thiselementcanbeusedtoproduceaseriesofstimuli toobservetheeffectofrepeatedapplicationofastimulusatahighrate.Itcanalsobeusedtoproduceatrainofpre-conditioningstimuliforasubsequenttestwaveform. ParametersInitialDelay Delay(attheholdinglevel)beforethepulsebegins.Amplitude Pulseamplitude.Duration Pulseduration.Frequency Frequency(Hz)ofpulseswithintrain.Frequency(incr.) Incrementinfrequencybetweenrecords.No.repeats Numberofpulsesintrain.

Voltageramp

Alinearvoltagerampbetweentwovoltagelevels.Voltagerampsprovideameansof rapidlygenerating thesteadystatecurrent-voltagerelationshipforanionicconductance. (Note that, the rampgeneratedby thecomputer isnot trulylinear,butconsistsofastaircaseoffinesteps.Thesestepscanbesmoothedout,by low-pass filtering thevoltage stimulus signalbefore it is fed into thepatchclamp.) ParametersInitialDelay Delay(attheholdinglevel)beforethepulsebegins.Amplitude Amplitudeatstartoframp.EndAmplitude Amplitudeatendoframp.Duration Rampduration.

Digitisedanalogwaveform

Adigitisedanalogwaveformloadedfromanexternaldatafile. ParametersInitialDelay Delay (at the holding level) before the pulse

begins.FileName Nameoftextfilecontainingdigitisedwaveform.D/Aupdateinterval Time interval between digitised waveform

points.No.Points No.ofdigitisedwaveformdatapointstobeused

inthestimulus.Startingpoint(increment) Increment to be added between records to the

first data point of the digitisedwaveform to beusedinthestimulus.

Scaleby ScalewaveformbyspecifiedfactorScaleby(increment) IncrementinscalingbetweenrecordsOffsetby AddoffsettowaveformOffsetby(increment) IncrementinoffsetbetweenrecordsDigitised waveforms are loaded into the stimulus protocol from text filescontainingthedigitiseddatapoints.Thewaveformdatacanbeformattedeitherasasinglecolumnofamplitudedataorapairofcolumnsoftime(inseconds)

and amplitude (in the stimulus units of the output channel to contain thewaveform)datapointsseparatedby<tab>characters,i.e.

T0V0T1V1…etcToloadadigitisedwaveformfroma textfile,click the buttonnexttotheFileNametableentryandselectthefilecontainingthedigitisedwaveform.

Afterloadingthedata,theNo.Pointsentryintheparametertablesindicatesthenumberofpointsloadedfromthefile.FortwocolumndatafileswhichcontaintimedatatheD/Aupdateintervalissettothetimedifferencebetweenthefirstandsecondrowsofdata.Forsinglecolumndatafiles,D/Aupdateintervalmustbeenteredbytheuser.

SineWave

A burst of sine waves. Amplitude and frequency can beincrementedbetweenrecords.

ParametersInitialDelay Delay(attheholdinglevel)beforethepulsebegins.Amplitude Sinewaveamplitude.Amplitude(incr.) IncrementinamplitudebetweenrecordsDuration Sineburstduration.Frequency Sinewavefrequency(Hz)Frequency(incr.) Incrementinfrequencybetweenrecords.

Emptyanalogelement

Emptyanalogwaveformelement.Dragging this elementon toananalogoutputlist,erasestheelementitisplacedon.

Digitalpulse(fixedduration)

Afixeddurationdigitalpulse.Thiselementcanbeusedtoswitchopenor

closevalvescontrollingtheflowofsolutionsoveracell.Multipledigitaloutputscanbeusedtosimultaneouslyopenonevalvewhileanotherisclosed. ParametersInitialDelay Delay(attheholdinglevel)beforethepulsebegins.State(0=0V,1=5V) Digitaloutputstateduringpulse:0=0V,1=5V.Duration Pulseduration.Familyofdigitalpulse(varyinginduration)

A digital pulse whose duration is automatically incremented betweenrecordingsweeps.

ParametersInitialDelay Delay(attheholdinglevel)beforethepulsebegins.State(0=0V,1=5V) Digitaloutputstateduringpulse:0=0V,1=5V.Duration Pulseduration.Duration(increment) Incrementindurationbetweenrecords.

Trainofdigitalpulses(incrementableinterval)

Atrainofdigitalpulsesatfixedintervals(incrementablebetweenrecords)andoffixedduration.Thiselementcanbeusedtoapplyarapidtrainofstimulitoacell. ParametersInitialDelay Delay(attheholdinglevel)beforethepulsebegins.State(0=0V,1=5V) Digitaloutputstateduringpulse:0=0V,1=5V.Duration Pulseduration.Repeatperiod Intervalbetweenpulseintrain.Repeatperiod(incr.) Incrementinrepeatperiodbetweenrecords.No.repeats Numberofpulsesintrain.

Trainofdigitalpulses(incrementablerate)

A train of digital pulses at a fixed frequency (incrementable betweenrecords)andoffixedduration.Thiselementcanbeusedtoapplyarapidtrainof

stimulitoacell. ParametersInitialDelay Delay(attheholdinglevel)beforethepulsebegins.State(0=0V,1=5V) Digitaloutputstateduringpulse:0=0V,1=5V.Duration Pulseduration.Frequency Frequency(Hz)ofpulseswithintrain.Repeatperiod(incr.) Incrementinfrequencybetweenrecords.No.repeats Numberofpulsesintrain.

Userdefineddigitalwaveform

AdigitalTTLwaveformloadedfromanexternaldatafile. ParametersInitialDelay Delay (at the holding level) before the pulse

begins.FileName Nameoftextfilecontainingdigitisedwaveform.D/Aupdateinterval Time interval between digitised waveform

points.No.Points No.ofdigitisedwaveformdatapointstobeused

inthestimulus.Startingpoint(increment) Increment to be added between records to the

first data point of the digitisedwaveform to beusedinthestimulus.

Digitised waveforms are loaded into the stimulus protocol from text filescontaining theTTLdata points (0 or 1).Thewaveformdata canbe formattedeitherasasinglecolumnofbinarydataorapairofcolumnsoftime(inseconds)andbinarydatapoints(0/1)separatedby<tab>characters,i.e.

T00T11…etcTo load a waveform from a text file, click the button next to the FileNametableentryandselectthefilecontainingthedigitisedwaveform.

Afterloadingthedata,theNo.Pointsentryintheparametertablesindicatesthenumberofpointsloadedfromthefile.FortwocolumndatafileswhichcontaintimedatatheD/Aupdateintervalissettothetimedifferencebetweenthefirst

andsecondrowsofdata.Forsinglecolumndatafiles,D/Aupdateintervalmustbeenteredbytheuser.

Emptydigitalelement

Empty digital waveform element. Dragging this element on to a digitaloutputlist,erasestheelementitisplacedon.

D/AUpdateIntervalThe D/A Update Interval box displays the D/A converter updateintervaltobeusedtoproducetheanalogstimuluswaveformswithintheprotocol.

The interval is normally set toRecording Duration/No. Samples,(seeRecordingSettings)butmaybegreaterthanthisifthelaboratoryinterfacecannotsupportthisD/Aupdaterate,orlessifshortdurationpulses exist within the protocol. If digitised analog waveformelementsexistwithintheprotocol,theupdateintervalissettototheD/Aupdateintervalforthesewaveforms.

Tokeep theD/Aupdate interval fixedataspecifiedvalue,enter theupdate interval into theD/AUpdate Interval box, and tick theFixIntervaloption.(Note.IfthelaboratoryinterfacecannotsupportthisD/Aupdate rate itwill be adjusted to theminimumpossible updateintervalfortheinterface.)

Next

RecordingExperimentalSignals>CreatingStimulusProtocols>GlobalStimulusVariablesTheamplitudeanddurationofwaveformswithinastimulusprotocolcanbedefinedinthewaveformparametertablesusingtheglobalstimulusvariables(G1..G5)insteadoffixedvalues.Thisallowsmultiplewaveformsettingswithinaprotocolorrelatedsetofprotocolstobechangedbyeditingasinglevalue.Globalvariablesarestoredindependentlyofindividualprotocolsandappliedtoallprotocolsreferencingthemwhentheprotocolisrun.Awaveformparametercanbelinkedtoaglobalvariablebyenteringthenameofoneofthefivevariables(G1,G2,G3,G4,G5)intothefieldintheparametertable.

ThevaluesoftheglobalvariablescanbesetinthetableontheGlobalVariablespage.Changingthevalueofthevariableintheglobalvariablestablechangesthewaveformparameterintheprotocolswhichreferencethatvariable.

Notethattheunitsoftheglobalvariablesappliedtowaveformdurationparametersareseconds(ratherthanmsecsasintheprotocols)andforamplitudeparameterstheunitsofthestimulustype.

RecordingExperimentalSignals>CreatingStimulusProtocols>SavingandLoadingStimulusProtocols

Whenyouhavecreatedastimulusprotocol,youcansaveittoaprotocolfilebyclickingtheSaveProtocolAsbuttontoopentheSaveStimulusProtocoldialogbox.

Stimulusprotocolsarestoredasfileswith.XMLfileextensions,inthedirectory,C:\\WinWCP\vprot\Protocol files can be re-loaded for editing, by clicking the Open Protocolbutton,andselectingaprotocolfilefromthelistpresentedintheLoadStimulusProtocoldialogbox.ThefolderusedtostoreprotocolscanbechangedbyclickingtheSetProtocolFolderbutton toopentheprotocolfolderselectiondialogbox,clickingonaafolder,thenclickingtheOpenbutton.

Next

RecordingExperimentalSignals>CreatingStimulusProtocols>ExampleProtocolsAnumberofexampleprotocolsareinstalledinthevprotfolderwithWinWCP.

VSteps 10-100mV100ms.xml

A family of 10 depolarising, 100msec duration,voltagesteps,rangingfrom10mVto100mV.

ISteps10-100pA100ms.xml

Afamilyof10depolarising,100msecduration,currentsteps,rangingfrom10pAto100pA.

tailcurrent.xml A two step pulse protocol for recording tailcurrents.A500msecpre-pulse,followedbya60mV, 50 msec duration test pulse. The pre-pulsestepsfrom10mVto120mV.

Ramp-100..100mV1s.xml Avoltage ramp,slewingfrom–100mVto+100mVoveraperiodof1sec.

sinewave.xml Adigitised1572samplepointsinewaveform.

Digpulse.xml A digital stimulus program controlling digitaloutputs0and1.Dig.0isOFFinitiallyandpulsesONforaperiodof50msec,afteradelayof100msec.Dig. 0 isON initially and pulsesOFF for50msec.

Next

RecordingExperimentalSignals>CreatingStimulusProtocols>DefaultOutputSettings

ThedefaultoutputsettingspanelsetsthedefaultanaloganddigitaloutputholdinglevelswhenastimulusprotocolisNOTinprogress.SelectSetup>DefaultOutputSettingstoopentheDefaultOutputSettingscontrolpanel

AnalogOutputHoldingLevels:Setsthedefaultholdinglevelfortheanalogoutputs.(Note.Whenanamplifierisdefined,thedefaultholdinglevelforthecommandoutputofthatamplifiercanalsobesetbychangingtheholdinglevelinthePipetteSealTestwindow.)DigitalOutputs:SelectingtheappropriateOn/Offswitch,setsthedefaultvoltagelevels(ON=5V,OFF=0V)tobeoutputonthedigitaloutputlines.DefaultFileName:SelecttheIncludeDateoptiontoincludethecurrentdateinauto-createddefaultfilename.EntertextintothePrefixfieldtoaddprefixtexttothefilename.ClicktheApplybuttontoapplythesettingstotheoutputs.

RecordingExperimentalSignals>AmplifierGainSettingThe amplifier gain (current channel gain involtage-clamp mode, voltage channel gain incurrent-clampmode)isindicatedintheAmplifierGains box.Whengain telegraphsareoperationaltheseindicatetheactualstateoftheamplifier.Whentelegraphinformationisnot available the current gain setting is entered here by the user.(SeeAmplifiers)

RecordingExperimentalSignals>ExperimentIdentification/CommentsAlineofoftextidentifyingthepurposeoftherecordingcanbeenteredintotheIdentboxatthetopoftherecordingwindow.

Thislineisstoredinthedatafile.WhentheReturnkeyispressedthecontentsofthelineiswrittentothelogfile.

Short tags (up to8 characters) canbe associatedwith each recordby enteringtextintotheMarkerboxandclickingtheAddbutton.

LogFileAlogfileoftheoperationsinitiatedbytheuserisupdatedduringthecourseofrecordingoranalysinganexperiment.Thenamesofdatafilescreatedorloaded,comments entered, stimulus programs used, and other events are stored alongwith the time that the event occurred. The log file can be used like anexperimenter'snotebooktokeepawrittenrecordoftheexperiment.Anewlogfileisopenedonadailybasiswithanameintheformdd-mm-yy.logandstoredintheWinWCPprogramdirectory.SelectFileInspectLogFile

todisplaytheexperimentallog:

AdditionalnotescanbeaddedtothelogfilebyenteringtextintotheAddNoteboxandclickingtheAddbutton.

RecordingExperimentalSignals>StartingaRecordingAfterselectingarecordingmodeand(ifnecessary)configuringtherecordingsweepsizeandduration,clicktheRecordbuttontobeginrecording

Note,makesurethattheSavetoFileboxischeckedtoenablewritingtothedatafile.Select the Incl. stim protocol in file name option to append thenameofstimulusprotocolbeingappliedtothefilename.Whenthisoption is selectedanewdata file is createdevery timea seriesofrecording sweeps are initiated by pressing theRecord button, orwhenalinkedstimulusprotocolisrun.Ifyouwanttostoprecordingbeforetheselectednumberofrecordshasbeenacquired,clicktheStopbutton.

RecordingExperimentalSignals>On-lineAnalysisTheon-lineanalysiswindowallowsaseriesofmeasurements(signallevelatcursor,average,peakamplitude,10-90%risetimeandmaximumrateofrise)tobemadeonthewaveformrecordedduringeachsweep.Amaximumof10measurementscanbeplotted.

To display the on-line analysis window, click the On-line Analysis WindowOpenbutton.ItcanbeclosedbyclickingtheClosebutton,orclosingtheOn-lineAnalysiswindow.

CreatingMeasurementPlots

Tocreateawaveformmeasurementplot,select:

1)ThetypemeasurementfromtheMeasurementslist,

2) The recording channel to be measured from theChan.list

3) Forpeak, rise time, rateof riseandslope: the cursorpair(C1-C2orC3-C4)definingtheanalysisregionfromtheCursors list and thepolarityof the signal (positive-going, negative-going or absolute (both positive- andnegative-going))fromthePolarityoptions.

4) ForCursor 1, 2, 3 or 4: enter the number of pointsaveragedaboutthemeasurementpointintoPointsAvgd.

5) Forrateofrise: theseriesof samplespoints (1,5,7)usedtocomputerasmoothedrateofchangeintoSmoothing.

6)Optional.ThestimulusprotocolassociatedwiththemeasurementintheForStimProtocollist.(Whenastimulusprotocolisselected,measurementsareonlyaddedtotheplotwhentheprotocolisrunning.)

ThenclicktheAddtoPlotbutton.

To erase themeasurements list, click theClr.Measurements button.To cleartheplot(butnotthemeasurementlist)clicktheClearPlotsbutton.ThedatainthecurrentlydisplayedplotscanbecopiedtotheWindowsclipboardbyclickingtheCopytoClipboardbutton.Waveformmeasurementsarealsowrittentothelogfileastheyareacquired.

MeasurementcursorsThe two pairs of linked vertical cursorsC1-C2 or C3-C4 define the regionwithin the recording sweep where a waveform measurement is made. Theyshouldbeplacedtoincludethewaveformofinterest,butexcludeanystimulusorotherartefactswithintherecordingsweep.

The C0 cursor defines the baseline level preceding thewaveformof interest.PeakandC1-C4cursormeasurementsare made relative to the signal level at C0 when the at 0cursor zero level option is selected, OR relative to the ‘z’horizontalzerolevelcursorwhentheatZcursoroptionisselected.

MeasurementsTheavailablewaveformmeasurementsarelistedinthetablebelow:

MeasurementsPeak(+), Peakpositiveamplitudewithin the regionof the recording

sweepdefinedbythecursors(C1-C2orC3-C4).

Risetime(+) TimetakenforsignaltorisewithinthepercentagerangeofPeak (+), defined in Rise Time Range box (default 10-90%), (TheC0 cursor isused indicate thebaseline levelthesignalisrisingfromandshouldbeplacedbeforetheriseofthewaveform.)

RateofRise(+) MaximumrateofchangeduringrisefrombaselineleveltoPeak(+).

RisingSlope(+)

SlopeofrisingedgeofsignalwithinthepercentagerangeofPeak(+),definedinRiseTimeRangebox(default10-90%),

Peak(-) Peaknegativeamplitudewithintheregionoftherecordingsweepdefinedbythecursors(C1-C2orC3-C4).

Risetime(-) TimetakenforsignaltorisewithinthepercentagerangeofPeak (-), defined in Rise Time Range box (default 10-90%), (TheC0 cursor isused indicate thebaseline levelthesignalisrisingfromandshouldbeplacedbeforetheriseofthewaveform.)

RateofRise(-) MaximumrateofchangeduringrisefrombaselineleveltotoPeak(-).

RisingSlope(-)

SlopeofrisingedgeofsignalwithinthepercentagerangeofPeak(-),definedinRiseTimeRangebox(default10-90%),

Slope The slope of a straight line fitted to the region of therecordingsweepdefinedbythecursors(C1-C2orC3-C4).

Peak(abs) LargestabsolutevalueofPeak(+)andPeak(-).

Risetime(abs) Timetakenforsignaltorisefrom10%abovepre-waveformbaselinelevelto90%ofPeak(abs).(TheC0cursorisusedindicatebaselinelevelandshouldbeplacedbeforetheriseofthewaveform.)

Rate of Rise(abs)

MaximumrateofchangeduringrisefrombaselineleveltoPeak(abs)

Cursor.1 Signal level atC1 cursor position, averaged over PointsAvgd.pointsaroundcursorposition.

Cursor.2 Average signal level atC2 cursor position, averaged overPointsAvgd.pointsaroundcursorposition.



RecordingExperimentalSignals>ZerolevelsThe signal zero level for a channel can be defined in either of two ways. InFromrecordmode,it iscomputedastheaveragelevelfromadefinedportionofeachrecord.InFixedmode,itisfixedataleveldefinedbytheuseranddoesnotvaryfromrecordtorecord.

FromrecordmodeUsethefromrecordzeromodewhenyouwant tomeasuretransient signals, which are superimposed upon a baselinelevelwhichmaybevaryingfromrecordtorecord.Tosetthezero level fromaportionof the signal record itself in fromrecordmode:1) Move themousepointerover thehorizontalzero level

cursor of the channel you want to change. (The mousepointerturnsintoanup/downarrow.)

2) Slidethemousepointerhorizontallyuntilitoverliestheregion of the record which is to be defined as the zerolevel.

3) Click the right-hand mouse button to open the zeroleveldialogbox.

4)SelecttheFromRecordoption.5)Thezeroleveliscomputed,foreachrecord,fromtheaverageofaseriesof

(default=20)samplesstartingatthesampleindicatedintheAtsamplebox.Ifyou want to change the number of samples averaged to compute the zerolevel,changethevalueintheNo.averagedbox.

6)ClicktheOKbuttontousethenewzerolevel.

FixedLevelmode

Use the fixed level zero mode when you want to makemeasurements relative to a fixed absolute level. To fix thezerolevelataconstantvaluetobeusedforallrecordsinthefile:1) Move themousepointerover thehorizontalzero level


2)Holddowntheleftmousebuttonanddragthezerolevelcursorverticallyuntilitisatthedesiredlevel.

3) Click the right- mouse button to open the zeroleveldialogbox.

4)SelecttheFixedoption.Theverticalpositionofthefixedbaseline is indicated(inA/Dconverterunits) in theLevelbox.Youcan setthezerolevelbyenteringavalue.


Fixed mode is typically used for the membrane potential measurements involtage/patchclampstudiesofvoltage-activatedcurrent.(Note.EnteringavalueofzerointotheLevelboxsetsthezeroleveltothetruezerovoltagelevelforthechannel)

DisplayingRecordsStoredonFile>SelectinganddisplayingrecordsToviewsignalrecordsstoredinadatafile,selectfromthemenu.ViewRawrecords

toopentherecorddisplaymodule.

Each record in the filecanbedisplayedandmeasurementsmadeof thesignallevels within each channel using a movable cursor. The displayed record (orsuperimposedgroupsofrecords)canbeprintedout.Recordscanbeassessedforthepresenceof interferenceotherartefactsandmarkedasrejected,orassignedwithparticularrecordtypes.

Eachrecordinthedatafileisnumberedinthesequencethatit was recorded. Records can be selected for display usingthe selectionbar tomovebackor forward through the file.

Youcanjumpdirectlytoarecordbyenteringitsnumberintotherecordnumberbox above the selection bar. (You can also use the Ctrl+Plus andCtrl+Minuskeystostepforwardorbackwardsthroughthefile.)Tosuperimpose(upto200)recordsonthedisplay,selectViewSuperimposeTraces

todisableautomaticdisplayerasure.(Selectingtheoptionagainre-enablesauto-erase.)

DisplayingRecordsStoredonFile>MagnifyingthedisplayThemagnificationofeachplotcanbeexpandedtodisplayaselectedregionofthesignalsbymovingthemousetotheupperlimitoftheregion,pressingtheleftmousebutton,drawingarectangletoindicatetheregionandreleasingthemousebutton.

The vertical magnification and location of the displayed region within therecordingcanalsobeadjustedusingthe

buttons at the right edge of each plot and the horizontal magnification andlocationbythe

buttonsatthebottomedgeoftheplot..

Individualchannelscanbeadded/removedfromthedisplaybyclickingthe

buttonattheleftedgeofeachchannel.Theverticalareaofthedisplaydevotedtoeachchannelcanbeadjustedbydraggingthetop/leftedgeofeachchannelYaxisupordown.

AllchannelscanbesetbacktominimummagnificationbyselectingView

ZoomOut(All)

DisplayingRecordsStoredonFile>PrintingRecordsToprinttherecordsdisplayedonthescreen,selectFilePrint

Toopentheprintrecorddialogbox.

Youcanset thesizeof theplottedrecordontheprintedpage,byadjustingthesizeofthepagemargins.

Thetypefaceusedtoprinttextcanbeselectedfromthefontnamelistandthetypesizeenteredintothefontsizebox.Thethicknessofthelinesusedtodrawthesignaltracescanbesetusingthelinethicknessbox.

Verticalandhorizontalcalibrationbarsareaddedtotheplottoindicatetheunitsandscalingoftheplottedsignals.Youcandefinethesizeofthebarsbyenteringvaluesintothecalibrationbarstable.

Thepositionofthezerolevelforeachplottedtraceisindicatedbyahorizontaldotted line.Zero levelscanbedisabledbyun-checkingshowzero levels. Plotlabellingcanbedisabledbyun-checkingtheshowlabelscheckbox.Theuseofcolourswithintheplotcanbedisabledbyun-checkingUsecolour.

Whenallplotparametershavebeenset,clicktheOKbuttontoinitiateprinting.

Choosingaprinterandoutputformat.

Tochooseaprinterandtoselectthepaperformat,select

FilePrintSetup

toopentheprintsetupdialogbox.

A printer can be selected from the list of currently installed printers. Theorientation of the plot on the page can be selected as either portrait orlandscape.

DisplayingRecordsStoredonFile>Accepting/Rejecting&ClassifyingRecordsRejectingflawedrecordsDigitisedrecordscannotallbeassumedtobeperfect.Consequently, thevisualinspectionofrecords,andtheeliminationofflawedrecords,isanimportantpartoftheanalysisprocess.Ifautomatedwaveformmeasurementproceduresaretobe applied, a mechanism is required for excluding flawed records from theanalysis.

Checking a record’s rejected box marks a record as being flawed. Rejectedrecordsareexcludedfromautomaticwaveform,curvefitting,signalaverageorleak subtraction calculations. (Note. Pressing theCtrl+R key combination is aquickwayoftogglingtherejectedcheckboxonandoff.)

Classifyingrecords

Recordscanalsobeclassifiedaccordingtothetypeofsignalthattheycontain,byselectingatypefromtherecord’stypelistbox.

Eighttypesofrecordsarecurrentlydefined(TEST,LEAK,EVOK,MINI,FAIL,TYP1,TYP2,TYP3).

TheEVOK,MINIandFAIL typesareused in thequantalanalysisofsynapticcurrents or potentials (see section 15) to indicatewhether a record is a nerve-evoked,spontaneous,oranerve-evoked/transmissionfailureevent.(Note.TypescanbeselectedquicklyusingtheCtrl+T,Ctrl+L,Ctrl+ECtrl+M,Ctrl+Fkeys.)TESTandLEAKareusedinthedigitalleaksubtractionprocess(seesection13),and used to distinguish, respectively, normal records, containing voltage-activated currents, and records containing leak currents to be scaled andsubtractedfromtheTESTrecords.

TYP1,TYP2andTYP3aregeneral-purpose,user-definedrecordtypes.

DisplayingRecordsStoredonFile>MeasuringSignalLevelsTomeasurethesignalatanypointonthedisplayedrecord,usethemousetodragtheverticalreadoutcursortothedesiredpartofthetrace.Finepositioningofthecursorcanbeachievedbypressingtheleftorrightarrowkeyswiththemousepointerovertheselectedcursor.Thesignal levelof the trace(s)at thecursorposition isdisplayedat thebottomofeachchannel.Timemeasurementsaremaderelativetothestartofeachrecordand(inbrackets)relativetothelocationofthet=0cursor.Signallevelsaremeasuredrelativetoeachchannel’shorizontalzerolevelcursor.Cursor measurements can be written to theWinWCP log file by clicking theSave(F1) button.TheCentreCursor button places the readout cursor to thecentreofthedisplayedregion.Zerolevels

The signal zero level for a channel can be defined in either of two ways. InFromrecordmode,it iscomputedastheaveragelevelfromadefinedportionofeachrecord.InFixedmode,itisfixedataleveldefinedbytheuseranddoesnotvaryfromrecordtorecord.

FromrecordmodeUsethefromrecordzeromodewhenyouwant tomeasuretransient signals, which are superimposed upon a baselinelevelwhichmaybevaryingfromrecordtorecord.Tosetthezero level fromaportionof the signal record itself in fromrecordmode:1) Move themousepointerover thehorizontalzero level


2) Slidethemousepointerhorizontallyuntilitoverliestheregion of the record which is to be defined as the zerolevel.

3) Click the right-hand mouse button to open the zero

leveldialogbox.4)SelecttheFromRecordoption.5)Thezeroleveliscomputed,foreachrecord,fromtheaverageofaseriesof

(default=20)samplesstartingatthesampleindicatedintheAtsamplebox.Ifyou want to change the number of samples averaged to compute the zerolevel,changethevalueintheNo.averagedbox.


FixedLevelmodeUse the fixed level zero mode when you want to makemeasurements relative to a fixed absolute level. To fix thezerolevelataconstantvaluetobeusedforallrecordsinthefile:1) Move themousepointerover thehorizontalzero level


2)Holddowntheleftmousebuttonanddragthezerolevelcursorverticallyuntilitisatthedesiredlevel.

3) Click the right- mouse button to open the zeroleveldialogbox.

4)SelecttheFixedoption.Theverticalpositionofthefixedbaseline is indicated(inA/Dconverterunits) in theLevelbox.Youcan setthezerolevelbyenteringavalue.


Fixed mode is typically used for the membrane potential measurements involtage/patchclampstudiesofvoltage-activatedcurrent.(Note.EnteringavalueofzerointotheLevelboxsetsthezeroleveltothetruezerovoltagelevelforthechannel)

DisplayingRecordsStoredonFile>CopyingRecordstotheWindowsClipboard

Thedisplayedsignalrecord(s)canbecopiedtotheWindowsclipboardinavarietyofformats–adatatable,animage,aWinWCPdatarecord.CopyingdatavaluesEachsignalrecordconsistsofanarrayofdigitisedsamplevalues.AtableofdatavaluesfortheactivedisplayrecordcanbecopiedtotheclipboardbyselectingEditCopyData

Thedata isplacedon theclipboardasa table,containing thescaledvaluesforeach sample in the record, in themeasurementunitsdefined for eachchannel.The table is stored in tab text format, allowing the data to be copied intoprogramssuchasspreadsheetsandgraphplottingpackages,usinganEdit/Pastecommand.(NotethatduetolimitationsinthecapacityoftheWindowsclipboarddatapointsmaybeskippedtokeepthesizeofthecopiedrecordwithinclipboardstoragelimits.)

Copyingthedisplayedimage

Thesignalrecord(s)onthedisplaycanbecopied to the clipboard as a bit mappedimagebyselectingEditCopyImage

toopenthecopyimagedialogbox.

Thedimensionsofthebitmap,whichwillholdtheimage,canbesetusingthewidthand height image size boxes. The morepixels used in the bit map the better thequality of the image. Calibration bars,zero levels and text font, size and linethicknesscanbesetinthesamewayasforaprintedimage.

Whentheimageparametershavebeenset,clickthe

OKbuttontocopytheimagetotheclipboard.

DisplayingRecordsStoredonFile>SmoothingtheDisplayedRecordsAdigitallow-passfilteralgorithmcanbeusedtosmooththedisplayedsignal.Toenable the low pass filter, tick the filter Active option and enter a cut-offfrequencyintotheCut-offFrequencybox.

The low pass filter set in the display module also acts upon the records inwaveformmeasurement,curvefittingandothermodules.

(Notethatthedigitisedrecordonthedatafileispreserved,filteringtakesplacewhentherecordisreadfromthefile.)

Automatic Waveform Measurement > Making WaveformMeasurements

The automatic waveform measurement module provides a means ofautomaticallymakingseriesofstandardmeasurementson thedigitisedsignals.Tenbasicamplitudeanddurationmeasurementscanbemadeoneachchannelandstoredwitheachrecord.Theresultsforeachrecordaredisplayedonscreen.Inaddition,setsofmeasurementvariablescanbeplottedagainsteachother,orcompiledintohistograms.Asummaryreportshowingmeanvalueandstandarderrorsforthemeasurementsetscanalsobeproduced.SelectAnalysisMeasureWaveforms

toopentheautomaticwaveformmeasurementwindow

TheAnalysispageisusedtosetuptheparametersofthewaveformanalysisandinitiatetheautomaticmeasurementsequence,whichgeneratesatableofmeasurements.

TheX/YplotpageisusedtocreateX-Ygraphsofthemeasurements.TheHistogrampageisusedtocreatefrequencyhistogramsofmeasurement.TheSummarypagepresentsasummary(mean,standarddeviation,etc.)ofthemeasurementsfortheseriesofrecordsanalysed.

TheTablespageisusedtocreatetablesofresults.

AutomaticWaveformMeasurement>RunningaWaveformAnalysis

The first stage in the measurement process is to define and run a waveform

analysissequenceonaselectedseriesofrecords.1)SelecttheAnalysispagebyclickingonitspagetab.2) Define the rangeof records to be analysed, by selectingAll

Recordstoanalyseallrecords,orThisRecordtoanalysesonlythe currently displayed record, orRange and enter a range ofrecords.

3) Select the typeofrecords tobemeasuredbyselectinganoptionfromthe

Type list. Select ALL to measure records of any type (except rejectedrecords).

4) Define theanalysisregion for each channel byusing the pairs of linkedvertical cursors (t=nns – t-yys) superimposed on the display.The analysisregion defines the region within the record within which waveformmeasurementsare tobemade. (Note.Toallow theanalysis region tobesetforeachchannelseparately,unticktheLockChannelscursoroption.Thet=0cursordefinesthezerotimeforthelatencymeasurement.

5) Set thepeakfindingmode. Selectabsolute todefine thepeak value as the largest absolute (i.e. positive or negative)deviationfromtherecordzerolevel.Selectpositivetodefinethepeakvalueasthelargestpositivedeviation.Selectnegativetodefinethepeakvalueasthelargestnegativedeviation.(Note.Absolutemodeshouldbeused for I/V curve measurements where both positive- and negative-goingsignalsmaybe found in the sequenceof recordsbeinganalysed.)Enter thenumberofdatapointstobeaveragedatthepeakintothePointsAvgd.box.

6) If you want to change the % level of the quantilemeasurement enter the percentage in the Q% box. <n>%

measures the amplitude of the data point that is n%of themaximumdatavalue.100%returns themaximumvalueand0%theminimumvalueof thesignalamplitudepointswithintheanalysisregion,

7) Ifyouwant the rise time tobemeasuredoveran intervalotherthanthestandard10%-90%range,enteranewrangeintheRiseTimebox.(Notethatthissettingalsodeterminesthepart of the waveform rising edge used to calculate the slope rate of riseoption.)

8) Set therateof risemode to select thealgorithmused tocalculatetherateofchangeofthesignal.TheForwardDiff.,Quadratic(5),Quadratic(7)optionscomputethemaximumrateofchangewithintheanalysisregion.TheForwardDiffoptioncomputestherateofchangefromthedifferencebetweensuccessivesamplepoints.TheQuadratic(5)andQuadratic(7)optionscomputethesmoothedrateofchangeestimated of 5 and 7 sample point windows respectively. TheLinearSlopeoptioncomputestherateofchangefromtheslopeofastraightline fitted to a section of the rising edge of the signal, defined by theRiseTimesetting(defaultvalue=10%-90%).

9)Ifyouwanttochangethe%decayfortheT.x%decaytime,enteranewvalueintotheT.x%DecayTimebox.

10)Tobegintheanalysisofselectedrangeofrecords,clicktheDoAnalysisbutton.

Oncompletionoftheanalysis,themeasurementsforeachrecordappearintheResultstable.

Measurementvariables

Record Thesequencenumberoftherecordwithinthedatafile.

Group Thenumberofthegrouptowhichtherecordbelongs(usedbyleaksubtractionmodule).

Time(s) Thetime,relativetothefirstrecordinthefile,thattherecordwasacquired.

Average Theaveragesignallevelwithintheanalysisregion.

Area Theintegralofthesignallevelwithintheanalysisregion.

Peak Thepeak(absolute,positiveornegative,dependinginpeakmeasurementmodeused)signallevelwithintheanalysisregion.

Variance Thevarianceofthesignalwithintheanalysisregion.

RiseTime Thetimetakenforthesignaltorisefromlo%-hi%(10%-90%default)ofpeak.(timeunits)

RateofRise Themaximumrateofchangeduringtherisingphaseofthesignal.

Latency Thetimedelaybetweenthezerotimecursorandthepointatwhichthesignalhasrisento10%ofpeak.

T.X% ThetimetakenforthesignaltofallfromitspeakvaluetoX%(setbyuser)ofpeak.

T.90% Thetimetakenforthesignaltofallfromitspeakvalueto10%ofpeak.

Baseline Signallevel,computedfromthezerolevelmeasurementregion,butmeasuredrelativetotruezerolevelsofinputchannel.

%Quantile Thenthquantileofthesignalamplitudedatapointswithintheanalysisregion.

AutomaticWaveformMeasurement>PlottingGraphsofMeasurementsTheX/YPlotpagecanbeused tocreategraphsof themeasurementsobtainedfrom the analysis run. Any measurement variable from any channel can beplottedagainstanyotherasaYvsXgraph.

Toplotagraph:1)SelecttheX/YPlotpagebyclickingonitspagetab.

2) DefinethevariabletobeplottedontheXaxis,byselectingitfromtheXAxisvariableandchannellists.

3) Define the variable to be plotted on the Y axis, by selecting it fromY

Axisvariableandchannellists.

4)ClicktheNewPlotbuttontoplotthegraph.

Conductanceplots

WhenConductance is selected as theYAxis plot variable, theconductance (G) can be computed from a pair of recordingchannelscontainingcurrentandvoltageusingtheformulaG=Ix(V–Vrev)

where I is the peak or averagemeasurement from theCurrentfrom channel,V is the peak or average measurement from theVoltagefrom channel andVrev is the current reversal potential,entered into the Reversal Pot. box. The units in which theconductanceisplotted(S,mS,uS,nSorpS)canbeselectedfromtheUnitslist.

Customisingthegraph

IfyouwanttoaltertheXorYaxisrange,scalingorlabels,clickthe

SetAxes

buttontoopentheSetAxesRange/Labelsdialogbox.Axis limits and tick spacing are initially set todefault values based upon the range of the data.You can change the axis limits by entering newvalues for into Min, Max. and Tick (spacing)boxesfortheXandYaxes.An axis can be made Linear or Logarithmic by selecting the option from its

Scalelist.LabelsfortheXandYaxescanbeenteredintotheLabelsboxes.AtypefacecanbeselectedfortheplotfromtheFontlistanditssizedefinedinthePoint Size box. The graph can be plotted as a line, unconnectedmarkers, orboth,bytickingtheLines,and/orMarkerstickboxes.

Printingthegraph

Toprintthedisplayedgraph,selectFilePrint

ToopenthePrintdialogbox.YoucansetthesizeofthegraphonthepageadjustingtheLeft,Right,Top andBottom pagemargin settings.Click theOKbuttontoplotthegraph.

CopyingthegraphdatapointstotheWindowsclipboard

Thenumerical values of theX,Ydata pointswhichgenerate the graph canbecopiedtotheclipboardbyselectingEditCopyData

ThedataisplacedontheclipboardasatableofX,Ydatapairsintabtextformat,allowing the data to be copied into programs such as spreadsheets and graphplottingpackages,usinganEdit/Pastecommand.

CopyinganimageofthegraphtotheWindowsclipboard

Animageofthegraphondisplaycanbecopiedtotheclipboardbyselecting

EditCopyImage

to open the Copy Image dialog box. Thedimensions of the bit map, which will hold theimage,canbesetusingthewidthandheightimagesizeboxes.Themorepixelsused in the bit map the better the quality of the image. When the imageparameters have been set, click the OK button to copy the image to theclipboard.

AutomaticWaveformMeasurement>ClassifyingRecordsUsingWaveformMeasurementCriteriaTheFilterRecordsoption(ontheX/YPlotpage)

can be used to automatically categorise records as particular types 470, orrejected from analysis, based upon waveform measurements. To classify therecordsinadatafile:

1)ClicktheFilterRecordsbuttontoopentheFilterRecordsdialogbox.

2) Define therecordMatchcriteria. (a)Click theVariable radiobuttonandselect themeasurementvariable tobeusedas the recordmatchingcriterionfromthevariablelist.(b)Selecttheinputchannelforthevariable.(c)DefinetherangeofacceptablevaluesbyenteringappropriatevaluesintotheUpperLimitandLowerLimitentryboxes.

3)DefinetheActiontobetakenwhenarecordmatchesthefiltercriterion.Tosettherecordtypeclassification,ticktheTypeboxandselect(TEST,LEAK,EVOK,MINI, FAIL, TYP1, TYP2, TYP3) from the list. To set the recordstatus,ticktheRecordstatusoptionandchooseAcceptedorRejected.

WhentheApplybuttonisclicked,ofallrecordsinthedatafilewhichmatchthecriterionsetby(2)aresettothetypeand/orstatusdefinedin(3).

AutomaticWaveformMeasurement>FittingaCurvetoaGraphLinear, single-, double-exponential functions or Boltzmann functions can befittedtoanX/Ygraphusingnon-linearleastsquarescurvefitting.

TofitacurvetothedisplayedX/Ygraph:

1)Selectthetypeofcurve(linear,exponential,twoexponentialorBoltzmann)tobefittedfromthefittingequationslist.

2)Definetheregionwithinthegraphtowhichthecurveistobefittedusingthe

pairof(f-f)verticalanalysisregioncursors.Theselectedregionisindicatedbythehorizontalbaratthebottomofthedisplay.

Note.Forexponentialfunctions,alsodefinetheinitialstartingpointofthefittedcurve,usingthex0cursor.

3) Click theFitCurves button to start the curve fitting process.The initialparameterguessesaredisplayedintheSetFittingParametersdialogbox.

If you want to keep a parameter fixed (i.e. not changed by curve fittingprocess)tickitsFixedbox.Youcanalsochangetheinitialparameterguesses,iftheyappeartobeunrealistic.ClicktheOKbuttontofitthecurve.

ThebestfittingcurveissuperimposedontheX/Ygraph(inred)andthebestfitequation parameters are displayed in the Curve Fitting table, along with theparameterstandarderror,theresidualstandarddeviation(betweenthefittedanddatapoints),statisticaldegreesoffreedominthefit,andthenumberofiterationsittooktofindthebestfit.

AutomaticWaveformMeasurement>PlottingWaveformMeasurementHistogramsTheHistogrampagecanbeused tocreate frequencyhistogramsofwaveformmeasurements, representing the frequency of occurrence of different valueswithinthetotalsetofmeasurements.Itiscompiledbysplittinguptherangeofpossible values into sets of adjacent bins, counting the number of individualmeasurementsfallingwithineachbin,thenplottingthebinsasrectangularbars,whoseheightindicatesthenumberofmeasurements,andpositionontheXaxisindicatestherangeofvaluesinthebin.

Toplotahistogram:

1)SelecttheHistogrampagebyclickingonitspagetab.

2) Select thewaveformvariable fromwhich thehistogramis tobegeneratedfromthevariableandchannellistboxes.

3)EnterthenumberofhistogrambinsintheNo.ofbinsbox(max.1024).

4)Entertherangeofvariablevalueswhicharetobeincludedinthehistogram,fromthelowerlimitinLowerboxtotheupperlimitintheUpperbox.

5)IfyouwantthehistogrambarheightexpressedasapercentageofthetotalnumberofrecordstickthePercentageoption.

6)IfyouwantacumulativehistogramticktheCumulativeoption.

7)ClicktheNewHistogrambuttontocompileandplotthehistogram.

Forexample,thehistogram,below,showsthedistributionofpeakamplitudesfora series of 200 simulated endplate currents It consists of 50 equal-sized binsovertherange0.5to1.5nA(i.e.abinwidthof0.2nA).Theheightofeachbinrepresents the number of records containing a signal with a peak amplitudefallingwithinthatbinrange.

Customisinghistograms

IfyouwanttoaltertheXorYaxisrange,scalingorlabels,clickthe

SetAxes

buttontoopentheSetAxesRange/Labelsdialogbox.

Axis limits and tick spacing are initially set to default values based upon themin.-max. range of the data. You can change the axis limits by entering newvaluesforintoMin,Max.andTick (spacing)boxesfor theXandYaxes.Anaxis can be made Linear or Logarithmic by selecting the option from itsScalelist.LabelsfortheXandYaxesandatitlefortheplotcanbeenteredintotheLabelsboxes.ThestyleofrectangleusedtoplotthehistogrambinscanbechangedusingtheBinStyleoptions.SelectNoFill todisplaybins as rectangularoutlines,SolidFilltofillthebinsinwithasolidcolour,andHatchedFillforbinsfilledwithadiagonallines.Youcandefinethecolourusedforthesolidfill,byclickingtheColour box, and selecting a colour from the palette. TheFullBorders checkboxdetermineswhethertheoutlineisdrawncompletelyaroundeachbar,orjustwherebarsdonotoverlap.

PrintingthehistogramToprintthedisplayedhistogram,selectFilePrint

ToopenthePrintdialogbox.

ClickthePrintbuttontoplotthegraph.

CopyingthehistogramdatapointstotheWindowsclipboard

Thenumericalvaluesof theX,Ydatapointswhichgenerate thehistogramcanbecopiedtotheclipboardbyselectingEditCopyData

Thedataisplacedontheclipboardasatableofdatavalues,intabtextformat,definingthehistogram.Thereare4valuesperrow,andonerowforeverybininthehistogram.Eachrowhastheformat

<BinLowerLimit><tab><BinMid-point><BinUpperLimit><tab><BinCount><cr><lf>

CopyinganimageofthehistogramtotheWindowsclipboardAnimageofthehistogramplotcanbecopiedtotheclipboardbyselectingEditCopyImage


Thedimensions(pixels)ofthebitmap,whichwillholdtheimage,canbesetusingtheWidthandHeightimagesizeboxes.ThesizeandstyleofthetypefacecanbesetusingtheTypefaceandSizeboxes.

Whentheimageparametershavebeenset,clicktheOKbuttontocopytheimagetotheclipboard.

AutomaticWaveformMeasurement>FittingGaussianCurvestoHistogramsGaussianprobabilitydensity functions, representing thedistributionofdiscretepopulationsofevents,canbefittedtoahistogramusingnon-linearleastsquarescurvefitting.Thenumberofeventsexpectedtobefoundineachhistogrambinforadistributionrepresentedbyamixtureofmgaussiansisgivenby

whereNisthetotalnumberofevents(records),wisthehistogrambinwidthandeachgaussian,i,isdefinedbythreeparameters,itsmean,i,standarddeviation,

iandthefractionofthetotalnumberofevents,ai,containedwithinit.

Tofitagaussiancurvetothedisplayedhistogram:

1)Selectthenumberofgaussianfunctions(1,2or3)tobefittedfromtheequationslist.

2)Definetheregionwithinthehistogramtowhichthecurveistobefittedusing

thepairofverticalanalysisregioncursors.Theselectedregionisindicatedbythehorizontalbaratthebottomofthedisplay.

3) Click theFitCurves button to start the curve fitting process. The initialparameterguessesaredisplayedintheSetFittingParametersdialogbox.

If you want to keep a parameter fixed (i.e. not changed by curve fittingprocess)tickitsFixedbox.Youcanalsochangetheinitialparameterguesses,iftheyappeartobeunrealistic.ClicktheOKbuttontofitthecurve.

ThebestfittingcurveissuperimposedontheX/Ygraph(inred)andthebestfitequation parameters are displayed in the Curve Fitting table, along with theparameterstandarderror,theresidualstandarddeviation(betweenthefittedanddatapoints),statisticaldegreesoffreedominthefit,andthenumberofiterationsittooktofindthebestfit.

AutomaticWaveformMeasurement>SummaryStatisticsTheSummarypagedisplaysasummaryreportcontainingthemeanvaluesandstandarderrorsfortherecords,whichhavebeenanalysed.

Todisplaythesummaryofresults:1)SelecttheSummarypagebyclickingonitspagetab.2)SelectthechanneltobesummarisedfromtheChannellist.3) Selectthevariablestobeincludedinthesummarybytickingorun-ticking

theappropriatevariabletickbox.

AutomaticWaveformMeasurement>TablesofMeasurementsTheTablespageisusedtodisplaylistsofmeasurementsintabularform.

Todisplaytablesofmeasurements:1)SelecttheTablespagebyclickingonitstab.2)ClicktheClearTablebuttontoerasetheexistingtable.3) Selectthevariablestobeaddedtothetable,byselectingachannel,ticking

thevariablestobeadded,thenclickingtheAddVariablesbutton.

SavingTablestoFileTo copy the table to a text file (tab separated columns), click the Save toFilebuttontoopentheSaveTabledialogboxandenterafilename.PrintingTablesToprintoutacopyofasummaryreportortableofresultsontheprinter,selectFilePrint

CopyingTablestotheClipboardTocopythereportortabletotheWindowsclipboard,selectEditCopyData

CurveFitting>IntroductionAmathematicalmodelconsistsofageneralequationrepresentingthetimecourseofthesignal(orpartofthesignal)understudy.Forinstance,thedecayofmanysignals(e.g.synapticcurrents)canberepresentedbyanexponentialfunction

,

whereAistheamplitudeofthesignalandisthedecaytimeconstant.Expressedasabove,theequationisquitegeneralapplyingtoanysignal,dependingonthevaluesoftheparametersAand.

Inordertodeterminewhethertheequationactuallydoesprovideagoodmodelof thesignaldecay it isnecessary to find theparametervalueswhichprovidesthebestmatch (or fit)of the theoretical curve toanactual signal.Thiscanbedone using the process know as iterative curve fitting. Starting with initialguesses for the parameters, the theoretical curve is compared with theexperimentaldatapoints, theparameters are adjusted to try to improve the fit,andtheprocessisrepeateduntilnomoreimprovementcanbeobtained.

The quality or goodness of the fit between theoretical curve and the data isdeterminedfromthesumofthesquareddifferences(S)betweenasetofndatapoints,y(i)(i=1..n),andthetheoreticalcurve,f(i),computedatthesamesampletimepoints,i.e.

Thebestfit is foundbyrepeatedlycomputingS,ateachiterationadjustingtheequationparametersusingastrategytominimiseS.Thebestfitparametersarethe ones which yield a minimum value of S. WinWCP uses a modifiedLevenberg-Marquardt least squares minimisation algorithm (the SSQMINroutine,developedbyKennethBrownattheUniversityofCincinnatti).(Amoredetailed discussion of curve fitting algorithms can be found in Chapter 6 ofDempster,1993.)

CurveFitting>FittingCurvesSignalWaveformsSelectAnalysisCurveFitting

toopenthecurvefittingwindow,

The module is split 5 functional sections (pages) – Curve Fitting, X/Y Plot,Histogram,Summary,Table-accessedbyclickingonthepagetab.

TheFitCurvepageisusedtoselecttheregionofthesignalandequationtobefittedandinitiatethecurvefittingprocess.

TheX/Y plot page is used to create graphs of the best fit equation

parametersfortheseriesofrecordsanalysed.

TheHistogrampageisusedtocreatefrequencyhistogramsofofthebestfitequationparametersfortheseriesofrecordsanalysed.

TheSummarypagepresentsasummary(mean,standarddeviation,etc.)oftheofthebestfitequationparametersfortheseriesofrecordsanalysed.

TheTablespageisusedtocreatetablesofresults.

FittingCurvestoWaveforms

Tofitamathematicalfunctiontoaselectedregionofasignalwaveform:1)SelecttheCurveFitpagebyclickingonitspagetab.

2)Selecttheequationtobefittedtotherecord(s).

3)Selectthechannelcontainingthesignalwaveformtowhichthecurveistobefitted,fromtheChannellist.

4) Define the range of records to be analysed, by selectingAllRecords toanalyseallrecords,orThisRecord toanalysesonlythecurrentlydisplayedrecord,orRangeandenterarangeofrecords.

5) Select the typeof records tobemeasuredbyselectinganoption from theType list. Select ALL to measure records of any type (except rejectedrecords).

6) SelectManual orAutomatic Data Cursors mode. In manual mode, theregionwithinthesignaltowhichthecurveistobefittedissetmanuallyusinga set of 3 cursors on the display. In automatic mode, the cursors are setautomaticallautomatically.

7) If you have selected Automatic Data Cursor mode, selectOnRise,OnDecayorRise+Decay, todeterminewhetherthecursorsaretobeplacedonthe rising phase, decaying phase, or complete time course of the signalwaveform.Thenenterthelevelsonthewaveformwherethecursorsaretobeplaced in theLimits box. (Default setting is10-90%,placing thecursors at10%and90%ofpeakamplitudeontheselectedphase)

8) IfyouhaveselectedManualDataCursorsmode, define thecurve fitting

regionbyusingthreeverticalcursors–two(blue,marked|)definetheregionto which the equation is to be fitted, the third (green, marked t0) defineswherethezerotimepointsfortheequationis.

(Notethatthechoiceoffittingregiondependsuponthekindofcurvebeingfitted. Sometimes, only part of the signal is chosen, such as when anexponential curve is to be fitted to the decay phase of the signal. In theexampleshownhere,thefittingregioncursorshavebeenplacedonthedecayphaseofanendplatecurrent.Zerotime(t0)hasbeendefinedattheonsetofthesignal.)

9) Ifyouwish tochange the initialvaluesfor theequationparameters,or fixsome parameters so they do not change during the fit, click theSetParametersbuttontoopentheSetFittingParametersdialogbox.

Ifyouwanttokeepaparameterfixedatasetvalue,enterthevalueintotheappropriateparameterboxandclicktheFixedcheckbox.

10)Whenallcurvefittingsettingshavebeenmade,clicktheDoFitbuttontoinitiatethecurvefittingsequence.

The iterative curve fitting process now begins. The SSQMIN routine iteratesthrough a variety of trial parameter sets, until no more improvement can beobtained.Thenumberofiterationsaredisplayedasfittingprogresses.Thebestfit isusuallyfoundwithin10-20iterations.Fittingisabortediftheprocesshasnotconvergedtoasuitableanswerwithin100iterations,andcanalsobeabortedbyclickingtheAbortbutton.

Foreachrecordfitted,thebestfitcurveisindicatedbyaredcurvesuperimposedon the blue signal trace. A residuals trace is shown below indicating thedifference between the fitted curve and the data. The parameters of the bestfittingequationareshownintheResultstable,alongwiththeparameterstandarderror, the residual standard deviation (between the fitted and data points),statisticaldegreesof freedomin the fit,and thenumberof iterations it took tofindthebestfit.

CurveFitting>CurveFitEquations

Theavailablecurvefittingequationsarelistedinthetablebelow.

Straightline

Exponential(general)

DecayingExp.

2Exponential(general)

2DecayingExps.

3Exponential

3DecayingExps.

EPC

The endplate current rising phase ismodelled by an errorfunctionandthedecaywithanexponentialfunction.

EPC(2exp)

The endplate current rising phase ismodelled by an errorfunction and the decay by the sum of two exponentialfunction.

H-H(K)

Thetimecourseofactivationofavoltage-activatedcurrentwithHodgkin-Huxleykinetics

H-H(Na)

The time course of a current with voltage dependentactivation and inactivation following Hodgkin-Huxleykinetics (e.g. sodium current). (Note. It is assumed that,initially, the activation parameter, m=0 and inactivationparameter,h=1.)

CurveFitting>ClassifyingRecordsUsingFittedParameterCriteriaTheFilterRecordsoption(ontheX/YPlotpage)

canbeusedtoautomaticallycategoriserecordsasparticulartypes(see8.6),orrejectedfromanalysis,baseduponbestfitequationparameters.Toclassifytherecordsinadatafile:

1)ClicktheFilterRecordsbuttontoopentheFilterRecordsdialogbox.

2) Define therecordMatchcriteria. (a)Click theVariable radiobuttonandselect themeasurementvariable tobeusedas the recordmatchingcriterionfromthevariablelist.(b)Selecttheinputchannelforthevariable.(c)DefinetherangeofacceptablevaluesbyenteringappropriatevaluesintotheUpperLimitandLowerLimitentryboxes.

3)DefinetheActiontobetakenwhenarecordmatchesthefiltercriterion.Tosettherecordtypeclassification,ticktheTypeboxandselect(TEST,LEAK,EVOK,MINI, FAIL, TYP1, TYP2, TYP3) from the list. To set the recordstatus,ticktheRecordstatusoptionandchooseAcceptedorRejected.

WhentheApplybuttonisclicked,ofallrecordsinthedatafilewhichmatchthecriterionsetby(2)aresettothetypeand/orstatusdefinedin(3).

CurveFitting>PlottingGraphsofBestFitEquationParametersThe X/Y Plot page can be used to create graphs of the best fit equationsparametersobtained from thecurve fit.Anyvariable fromanychannelcanbeplottedagainstanyotherasaYvsXgraph.

Toplotagraph:1)SelecttheX/YPlotpagebyclickingonitspagetab.

2) DefinethevariabletobeplottedontheXaxis,byselectingitfromtheXAxisvariableandchannellists.

3) Define the variable to be plotted on the Y axis, by selecting it fromYAxisvariableandchannellists.

4)ClicktheNewPlotbuttontoplotthegraph.

Customisingthegraph

IfyouwanttoaltertheXorYaxisrange,scalingorlabels,clicktheSetAxes


Axis limits and tick spacing are initially set to default values based upon therangeofthedata.YoucanchangetheaxislimitsbyenteringnewvaluesforintoMin,Max.andTick(spacing)boxesfortheXandYaxes.An axis can be made Linear or Logarithmic by selecting the option from itsScalelist.LabelsfortheXandYaxescanbeenteredintotheLabelsboxes.AtypefacecanbeselectedfortheplotfromtheFontlistanditssizedefinedinthePoint Size box. The graph can be plotted as a line, unconnectedmarkers, orboth,bytickingtheLines,and/orMarkerstickboxes.

PrintingthegraphToprintthedisplayedgraph,select

FilePrint


YoucansetthesizeofthegraphonthepageadjustingtheLeft,Right,TopandBottompagemarginsettings.ClicktheOKbuttontoplotthegraph.

CopyingthegraphdatapointstotheWindowsclipboard

Thenumerical values of theX,Ydata pointswhichgenerate the graph canbecopiedtotheclipboardbyselectingEditCopyData

ThedataisplacedontheclipboardasatableofX,Ydatapairsintabtextformat,allowing the data to be copied into programs such as spreadsheets and graphplottingpackages,usinganEdit/Pastecommand.

CopyinganimageofthegraphtotheWindowsclipboard

Animageofthegraphondisplaycanbecopiedtotheclipboardbyselecting

EditCopyImage

toopentheCopyImagedialogbox.

Thedimensionsofthebitmap,whichwillholdtheimage,canbesetusingthewidth and height image size boxes. Themore pixels used in the bit map thebetterthequalityoftheimage.Whentheimageparametershavebeenset,clicktheOKbuttontocopytheimagetotheclipboard.

CurveFitting>PlottingHistogramsofBestFitEquationParametersTheHistogrampagecanbeused tocreate frequencyhistogramsofwaveformmeasurements, representing the frequency of occurrence of different valueswithinthetotalsetofmeasurements.Itiscompiledbysplittinguptherangeofpossible values into sets of adjacent bins, counting the number of individualmeasurementsfallingwithineachbin,thenplottingthebinsasrectangularbars,whoseheightindicatesthenumberofmeasurements,andpositionontheXaxisindicatestherangeofvaluesinthebin.

Toplotahistogram:

1)SelecttheHistogrampagebyclickingonitspagetab.

2) Select thewaveformvariable fromwhich thehistogramis tobegeneratedfromthevariableandchannellistboxes.

3)EnterthenumberofhistogrambinsintheNo.ofbinsbox(max.1024).

4)Entertherangeofvariablevalueswhicharetobeincludedinthehistogram,

fromthelowerlimitinLowerboxtotheupperlimitintheUpperbox.5)Ifyouwantthehistogrambarheightexpressedasapercentageofthetotal

numberofrecordstickthePercentageoption.6)IfyouwantacumulativehistogramticktheCumulativeoption.

7)ClicktheNewHistogrambuttontocompileandplotthehistogram.

Forexample, thehistogram,below, shows thedistributionof fitteddecay timeconstantsforaseriesof200simulatedendplatecurrentsItconsistsof50equal-sizedbinsovertherange9to12ms(i.e.abinwidthof0.6ms).Theheightofeach bin represents the number of records containing a signal with a peakamplitudefallingwithinthatbinrange.

CustomisinghistogramsIfyouwanttoaltertheXorYaxisrange,scalingorlabels,clickthe

SetAxes


Axis limits and tick spacing are initially set to default values based upon themin.-max. range of the data. You can change the axis limits by entering newvaluesforintoMin,Max.andTick (spacing)boxesfor theXandYaxes.Anaxis can be made Linear or Logarithmic by selecting the option from itsScalelist.LabelsfortheXandYaxesandatitlefortheplotcanbeenteredintotheLabelsboxes.ThestyleofrectangleusedtoplotthehistogrambinscanbechangedusingtheBinStyleoptions.SelectNoFill todisplaybins as rectangularoutlines,SolidFilltofillthebinsinwithasolidcolour,andHatchedFillforbinsfilledwithadiagonallines.Youcandefinethecolourusedforthesolidfill,byclickingtheColour box, and selecting a colour from the palette. TheFullBorders checkboxdetermineswhethertheoutlineisdrawncompletelyaroundeachbar,orjustwherebarsdonotoverlap.

PrintingthehistogramToprintthedisplayedhistogram,selectFilePrint


ClickthePrintbuttontoplotthegraph.

CopyingthehistogramdatapointstotheWindowsclipboardThenumericalvaluesof theX,Ydatapointswhichgenerate thehistogramcanbecopiedtotheclipboardbyselectingEditCopyData

Thedataisplacedontheclipboardasatableofdatavalues,intabtextformat,definingthehistogram.Thereare4valuesperrow,andonerowforeverybininthehistogram.Eachrowhastheformat

<BinLowerLimit><tab><BinMid-point><BinUpperLimit><tab><BinCount><cr><lf>

CopyinganimageofthehistogramtotheWindowsclipboardAnimageofthehistogramplotcanbecopiedtotheclipboardbyselectingEditCopyImage


Thedimensions(pixels)ofthebitmap,whichwillholdtheimage,canbesetusingtheWidthandHeightimagesizeboxes.ThesizeandstyleofthetypefacecanbesetusingtheTypefaceandSizeboxes.

Whentheimageparametershavebeenset,clicktheOKbuttontocopytheimagetotheclipboard.

CurveFitting>SummaryStatisticsTheSummarypagedisplaysasummaryreportcontainingthemeanvaluesandstandard errors of the fitted parameters for the records, which have beenanalysed.

Todisplaythesummaryofresults:1)SelecttheSummarypagebyclickingonitspagetab.2) Selectthevariablestobeincludedinthesummarybytickingorun-ticking

theappropriatevariabletickbox.

CurveFitting>TablesofFittedParametersTheTablespageisusedtodisplaylistsoffittedparametersintabularform.

Todisplaytablesofmeasurements:1)SelecttheTablespagebyclickingonitstab.2)ClicktheClearTablebuttontoerasetheexistingtable.3) If results for failed curve fits are to be included in the table, tick the

IncludeBadFitsoption.4) Selectthevariablestobeaddedtothetable,byselectingachannel,ticking

thevariablestobeadded,thenclickingtheAddVariablesbutton.

SavingTablestoFileTo copy the table to a text file (tab separated columns), click the Save toFilebuttontoopentheSaveTabledialogboxandenterafilename.PrintingTablesToprintoutacopyofasummaryreportortableofresultsontheprinter,selectFile

Print

CopyingTablestotheClipboardTocopythereportortabletotheWindowsclipboard,selectEditCopyData

CurveFitting>AssessingCurveFitQualityIterative curve fitting is a numerical approximation technique, which is notwithout its limitations. In some circumstances, it can fail to converge to ameaningfulanswer,inothersthebestfitparametersmaybepoorlydefined.Itisimportanttomakeanassessmentofhowwellthefunctionfitsthecurvebeforeplacingtoomuchrelianceontheparameters.

Doesthechosenfunctionprovideagoodfittothedata?Oneassessmentofthegoodnessoffitistocomparethevarianceoftheresidualdifferences between the best fit function and the data with the backgroundvarianceofthesignal.Ifthefunctionprovidesapoorfittothedata,theresidualvariance will be significantly greater than the variance of the randombackgroundnoiseonthesignal.Thedistributionofthevarianceasdisplayedinthe residualsplot is also important.Deviations shouldbe randomlydistributedoverthefittedregionof therecord.If thefittedlineisconsistentlyhigherthanthedatapointsinsomepartsandlowerinothersthisindicatesthatthesignalisnotwellrepresentedbythechosenequation.

Aretheparameterswell-defined?Theaimofmostcurvefittingexercisesistoobtainawell-definedsetoffunctionparameters (e.g. exponential time constants)which characterise the part of thesignal being fitted. The standard errors of the best fit parameters provide anindication of this. A large standard error indicates that a parameter is poorlydefined by the data and can be varied significantly with little effect on thegoodness of fit. Such a situation typically arises when there is insufficientinformationcontainedinthesignalwaveformtoadequatelydefinethefunction.Forinstance,inthecaseofexponentialfunctions,thewaveformdatamustbeofsufficient duration to contain at least one time constant of the exponentialfunction before an accurate estimate can be obtained. Similarly, it provesdifficult to accurately estimate the time constants of multiple exponentialfunctionswhentheydifferbylessthanafactorof5.

It is worth noting that the parameter "standard errors" discussed above arecomputedfromtheHessianmatrixbythecurvefittingprogram,andarenottrueestimatesofexperimentalstandarderrorsincetheytakenoaccountofintercellorothervariability.Inaddition,theyonlyprovidealowerboundtotheestimateofthestandarderrorinparametervalue.Itcanbeshown(bysimulation)that,if

therandomnoiseontheexperimentalsignalsiscorrelated,thenthevariabilityoffittedparametersmaybe substantiallygreater than suggestedby thecomputedparameter standard error. The error in parameter estimation can be a complexfunction of the parameter values and the signal-noise ratio of the data. It istherefore wise to test the curve fitting procedure using simulated waveformswithknownparameterssetspanningtherangeofvalueslikelytobeobservedintheexperimentaldata.

Arealltheparametersmeaningful?

Itisalsonecessarytodiscriminatebetweenfunctions,whichfitthedataequallywell. For instance, the question often arises as towhether one, two, ormore,exponentialfunctionsareneededtofitasignalwaveform.ItisusuallyobviousfromtheresidualplotwhenasingleexponentialdoesNOTprovideagoodfit.However,whenasingleexponentialdoesfit,twoormoreexponentialswillalsoprovideagoodfit.Insuchcircumstance,itisusualtochoosethefunctionwiththe least number of parameters, on the principle of parsimony. An excess offunctionparametersalsoresultsinthesomeoftheparametersbeingill-definedwithstandarderrorsvaluesoftenlargerthantheparametervaluesthemselves.AmoredetaileddiscussionoftheaboveissuescanbefoundinDempster(1992)and(2001).

SignalAveraging>IntroductionMany electrophysiological signals have poor signal-noise ratios, making itdifficulttoobtainaccuratemeasurementsfromindividualrecords.However,ifasignalcanbemadetooccurrepeatedly,digitalsignalaveragingtechniquescanrecoverthesignalwaveformfromthebackgroundnoise.

Thesignalaverageofaseriesofrecordsisgeneratedbycomputingtheaverageof each corresponding sample within the records. For a set of N records,consistingofsamples,yi,(i=1,n)theaveragerecordconsistsofnsamples,andisgivenby.

Thelocationofsignalswithintherecordsometimesvariesfromrecordtorecord,due to imperfections in the detection of spontaneous signals or fluctuations instimuluslatency.Insuchcircumstances,averagingcorrespondingsamplepointswithintherecordwouldresultinadistortedsignalaverage.Thisproblemcanbeavoidedbyaligning the signalsby themid-pointsof their risingphasesbeforeaveraging.

SignalAveraging>CreatingSignalAveragesSelectAnalysisSignalAveraging

toopenthesignalaveragingwindow.

Tocreateaseriesofsignalaverages:

1) Specify the rangeof records to be averagedby entering the first and lastrecords,separatedbya“-“,intheRangebox.

2) Select the Sequential option to average sequential groups of records(i,i+1,i+2...i+n-1) or Interleaved to average sets of records interleavedtogether (i,i+n,i+2n,..). Enter the number of records (n) in a sequential orinterleavinggroupintheingroupsofbox.

3) Youcan restrictaveraging toa specific typeof recordbyselectinga typefrom theType list. SelectALL to use records of any type (except rejectedrecords).

4)Setthealignmentmode.SelectNoalignmentifthepositionofthesignalsdonot varywithin the records and alignment is not necessary. If alignment isnecessary,selectonpositiverise forpositive-goingsignalsandonnegativerisefornegativesignals.

5)Setthealignmentsearchregioncursors.Ifrecordscontainstimulusartefacts,itmaybenecessarytorestricttheregionoftherecordwhichissearchedforthe signalmid-point, inorder toavoid theaveragesbeingalignedusing theartefacts rather than the true signals. The alignment search region is set bymoving the two vertical cursors on the display to define the beginning andendoftheregioncontainingthesignal.

6)Tocreatetheaverages,click

DoAverages

Theaveragingprocessnowproceeds.Anadditionaldigitiseddatafileiscreatedcontaining the average record(s),with the samenameas theoriginal data file,but with a ".AVG" file extension rather than ".WCP". On completion ofaveraging,therecorddisplaymoduleisopenedtoshowtheaveragerecords.

Youcanswitchthedisplaybackandforthbetweentheaveragesfileandtherawdatafilebyselecting

ViewAveragedRecords

Toviewtheaverages,andViewRawRecords

Toviewtheoriginaldigitisedsignalrecords.

DigitalLeakCurrentSubtraction>IntroductionIoniccurrentsrecordedusingthevoltageclamptechniqueareusuallycomposedofavarietyofcomponents,mediatedbydifferentionicchannels(e.g.Na,K,Ca,Cletc.).Inordertostudyaparticularcurrentindetail,itisusuallynecessarytoeliminatealltheothercurrentsfromthesignal.Thisisoftendoneusingpharmacologicalagents,suchasTTXtoblockNacurrents,TEAtoblockKcurrents,etc.However,evenwhensuchblockingagentsareused,thereoftenstillremainssomeresidualcurrentinadditiontotheoneunderstudy.Thiscurrent,isknownastheleakcurrent.Itusuallydisplayslineartime-independentproperties.Insomecircumstances,theleakcurrentisverysmallandcanbeignored.However,inothersitcanbeaslargeasthecurrentsunderstudy,complicatingtheanalysisofthesignalwaveformsunlessitisremoved.

Although the leak current cannot be removed pharmacological, its linearproperties permit a digital subtraction approach to be used.The current signalcanbeconsideredtoconsistof3components

where Ii(t) is the time-dependent, voltage-activated, ionic current under study,Ilkistheleakcurrent,andIc(t)istransientcapacitycurrentduetothecharginganddischargingofthemembranecapacity.Ic(t)andIlkarealwayspresentinthesignal, and scale linearlywith the sizeof thevoltage step.However, Ii(t) onlyoccurs for voltage steps to potentials which activate the voltage sensitive ionchannels.TheNacurrent, for instance, isonlyevokedbydepolarisingvoltagesteps topotentialsmorepositive than -60mV. It is possible toobtain a record,containingonly leak and capacity currents, byusing a hyperpolarisingvoltagestep(orasmalldepolarisingstep)

Scalingthisrecordtoaccountforthedifferencesinthesizeand/orpolarityofthevoltagestep,andsubtractingitfromthetestrecord,effectivelyremovestheleakandcapacitycurrents.

Sincethescalingupofsmallsubtractionrecordsalsoscalesupthebackground

noise, it is usual to average several subtraction records before, scaling andsubtracting. It is also possible to average the test records.WinWCP uses thefollowinggeneralalgorithm

whereMisthenumberoftestrecordsaveragedandNthenumberofsubtractionrecords.

DigitalLeakCurrentSubtraction>StimulusProtocolsforLeakSubtractionOneofthemostcommonlyusedleaksubtractionprotocolsistheP/Nprotocol,developedbyBezanilla&Armstrong (1977).Foreachdepolarising testpulse,there are N additional subtraction pulses, evoked by hyperpolarisng pulses1/Nththeamplitudeofthetestpulse.WinWCP's stimulus generator can be configured to produce the necessarysequence of test and leak subtraction recording sweeps, by selecting theP/NMode leak subtraction option in a stimulus protocol (See RecordingSettings).Thiscausesthestimulusgeneratortoproduceadditionalscaleddownand inverted stimulus pulse waveforms for evoking the linear leak currentswithoutthevoltage-activatedcurrents.Theleakcurrentrecordingsareaveragedand stored in a recordmarked as a LEAK type. The test record ismarked asTEST type record. The TEST record, with its associated LEAK record, arecollectedtogetherinagroup(i.e.theyhavethesamegroupnumber).

DigitalLeakCurrentSubtraction>SubtractingLeakCurrentsTosubtracttheleakcurrentsfromadatafile,select

AnalysisLeakCurrentSubtraction

toopentheleakcurrentsubtractionwindow

Todigitallysubtracttheleakcurrentintherecords{

1) SelectthechannelcontainingthecellmembranepotentialfromtheVoltagechannellist.

2) SelectthechannelcontainingthecellmembranecurrentfromtheCurrentchannellist.

3) Select the source of the Leak records. If records are grouped intoLEAK/TESTrecordpairs,selectFromGroup(ThisoptionshouldbeusedforP/Nprotocols).IftheLEAKrecordsarenotgroupedwiththeTESTrecords,selectFromwholefile.(Wholefilemodeisusedwhenoneormorerecordsat thebeginningorendofadatafileare tobeusedasLEAKrecords.NotethattheserecordswillhavetobemanuallyclassifiedasLEAK).

4)SetthecurrentScalingmode.SelectFromVoltagetousetheratiobetween

the TEST and LEAK voltage pulses as the current scaling factor (default).SelectFixed if youwish to use the fixed scaling factor, entered in the boxbelow. (Fixedmode is requiredwhen the recorddoesnot contain a voltagechannel.)

5) TosubtractbothcapacitycurrentandionicleakcurrentselecttheI(cap)+I(leak)option.Tosubtractionicleakcurrentonly,selectI(leak).(TheI(leak)optionproducessubtractedrecordswithlowerbackgroundnoise,butdoesnotremovecapacitycurrenttransients.)

6) If you have selected the From Voltage scaling mode, use theVTest andVHolddisplaycursorstodefinethemeasurementpointsonthevoltagetraceusedtocomputethevoltagescaling.VHoldisplacedovertheholdingvoltagelevelandVTestisplacedoverthemid-pointofthetestvoltage.(Anaverageof 20 samples around each measurement point isusedtocomputethevoltagelevels.).

7)Select

Dosubtraction

Toinitiatetheleaksubtractionprocess.

Foreachgroupofrecords,theLEAKandTESTrecordsareaveraged,scaledandsubtracted using (See Introduction). Each group is condensed down to oneleak-subtracted record that is stored in a .SUB filewith the samenameas the

data file. These records can then be displayed and analysed using the ViewRecords,Waveformanalysis,andCurvefittingmodules,byselectingViewLeaksubtracted

Non-stationaryNoiseAnalysis>Introduction

Thenon-stationarynoisemoduleanalysestherandomfluctuationsinthedecayofionchannelcurrents,providinganestimateofsingle-channelcurrentandtotalnumberofchannelsinthefluctuatingpopulation.Foracellcontainingapopulationofnionchannels,eachcapableofpassingacurrenti,themeanwholecellcurrent,Im(t)is,

1

wherep(t)istheprobabilityofachannelbeingopenattimet.Thevariance,2(t),ofthecurrentfluctuations,attimet,aboutthismeanis,

2

Thesetwoequationscanbecombinedtoprovidearelationshipbetween2(t)andIm(t),

3Thesingle-channelcurrent,i,andnumberofchannels,n,canthusbecalculatedbyfittingtheaboveparabolicfunctiontoaplotof2(t)vsIm(t)duringacurrenttransientwherep(t)ischanging.Im(t) can be computed as the average current of a series of transient currentrecords,repeatedMtimesallevokedbythesamestimulus,

4

Thevariance,2(t),ateachsamplepoint,t,cansimilarlybecomputedfrom

5The method was developed by Sigworth (1981) for voltage-activated Nacurrents. It has also been used to study the fluctuations during the rapidly

desensitisingcurrentsinducedbyhighconcentrationsofacetylcholine(Dilger&Brett,1990).Withmodificationitcanalsobeenappliedtosynapticcurrents.Thebasicnon-stationaryvarianceapproachassumesthattheonlysourceofvariancearisesfromthefluctuationsoftheionchannelsthatcarrythecurrent.However,synaptic current amplitude can fluctuate due to both ion channels and quantalsize/contentvariation.Traynelisetal(1993)foundawayroundthisproblembyscalingtheamplitudeoftheaveragecurrenttothepeakamplitudeofeachsignalbefore the subtraction in Eqn. 5, thus compensating for the quantal variation.ThisapproachdoeshavelimitationsanditisworthreadingDeKoninck&Mody(1994)ifconsideringusingthescalingapproach.

Non-stationaryNoiseAnalysis>AnalysingSynapticCurrentFluctuations

TheWinWCPnon-stationarynoiseanalysismodulecanbeusedtocomputethetimecourseofresidualcurrentfluctuationsbetweenindividualcurrentrecordsandtheaveragecurrenttimecourse,andplotthevarianceofthesefluctuationsvsmeancurrentateachsampletimepointduringthedecayofthecurrent.Theaveragenumberofionchannelscontributingtotheioniccurrentandthesingle-channelcurrentcanthenbeestimatedbyfittingaparabolicfunctiontothevariancevs.meanplot.SelectAnalysisNon-stationarynoiseanalysistoopenthenon-stationaryvarianceanalysiswindow.

Theuppertraceinthesignaldisplaypanelshowsaselectedindividualsignalrecord(blue)withtheaverageofalltherecordsinthefilesuperimposedinred.Thelowertraceshowstheresidualdifference(Res)betweenthesignalrecordandtheaverage.Togenerateavariancevsmeanplotandestimatesingle-channelcurrentandchannelnumberfromaseriesofsynapticcurrentrecords:1)Ifthereismorethanonesignalchannel,selectthechannelcontainingthe

currentsignaltobeanalysedfromtheChannellist.

2) Optional.To use a specific record type only, change theType list fromALLtotheselectedtype(TEST,LEAK,EVOK,MINI,FAIL,TYP1,TYP2,TYP3).

3)SelecttheAllRecordsoptiontouseallrecordsoftheselectedtype.Touseasub-rangeoftherecordswithinthefile,SelectRangeandentertherangeofrecordsinthebox.

4) Ifsynapticcurrentsarebeinganalysed,selecttheScale toPeakoption toscaletheaveragecurrenttothepeakamplitudeofeachcurrentrecordbeforethe subtraction to produce the residual variance. SelectNo Scaling if theunscaledaverageistobesubtracted..

5) Optional.Ifthestartingtimeofeachsignalvariessignificantlyfromrecordtorecord, itcanbere-alignedwiththeaveragecurrentbeforesubtractiontoproduce the residual. Set the Alignment mode to On Positive Rise forpositive-goingsignalandOnNegativeRisefornegativesignals.

6) Select the region of the signalwaveform (the decay phase in the case ofsynapticcurrents)tobeincludedinthevariancevs.meanplotusingthepairof(a-a)analysisregioncursors.

7)ClicktheX/YPlottabtoswitchtotheX/YPlotpage.

SelectMeanfromtheXAxisvariablelistandVariancefromtheYAxislist,thenclicktheNewPlotbuttontogeneratethevariancevsmeanplot,s

8)Tofitaparabolatothecurve:

a)SelectParabolafromthecurvefittinglisttofittheequationy(x)=Vb+Iu*x+x2/Nc(whereIuisthesingle-channelcurrent,NcisthenumberofionchannelsandVbisthebackgroundvariance).

b)Define the region of the variance vsmeanplot to be fitted to, using theanalysisregioncursors(f-f).

c)ClicktheFitCurvebutton,set(optional)theinitialparameterguessesandclicktheOKbuttontofitthecurve.

Thebestfittingparabolaissuperimposed(inred)onthevariancevs.meanplot.Theestimatedsingle-channelcurrent (Iu)andnumberofchannels(Nc)aredisplayedinthecurvefittingresultsbox,alongwithanestimateofthebackgroundvariance(Vb).

QuantalAnalysisofTransmitterRelease>Introduction

Thequantalanalysismodulecanbeusedtoestimatethequantalcontentofneuromuscularnerve-evokedendplatecurrentsorpotentials,andotherformsofsynapticsignal,usingtheeitherthedirectmethod,variancemethod,andmethodoffailures.Incircumstanceswherebothevokedandspontaneousminiatureeventsareavailable,transmitterreleaseparameters,n,(numberofavailablequanta)andp,probabilityofrelease,iscalculatedusingbinomialanalysis.Quantalcontent(directmethod)

Ifthedatafilecontainsbothevokedandminiaturesignals,thedirectmethodofcalculatingquantalcontentcanbeused.

1Thisisthemostaccuratemethodforcalculatingquantalcontent.

Quantalcontent(variancemethod)

Itisnotalwayspossibletorecordtheminiaturesynapticsignals,whichrepresentsinglequanta.Insuchcircumstances,itmaystillbepossibletocalculatequantalcontentfromthevariabilityoftheevokedsignal

2This method is dependent upon the assumption that the number of quantareleased follows a Poisson distribution. This will only be the case when theprobabilityof release isvery low(i.e.p<0.1).Since largeerrorscan result ifthisconditionisnotsatisfied,resultsusingthevariancemethodshouldbetreatedwithcaution.

QuantalContent(failuresmethod)

Ifthequantalreleaseprobabilityisverylow,anervestimulusmayoccasionallyreleasenoquantaatall,resultinginintermittentfailurestoevokedpost-synapticsignals.AgainusingtheassumptionofaPoissondistributioncontrollingrelease,thequantalcontentcanbecalculatedfrom

3

Binomialanalysis

The transmitter release process can often be modelled as a pool of n quantaavailable for release, with each quantum having a probability, p, of beingreleasedwhen thenerve is stimulated. Ifbothevokedand spontaneous signalsareavailable,itispossibletocalculateestimatesfornandp,ontheassumptionthatthenumberofquantareleasedperstimulusfollowsabinomialdistribution.

4

5

Correctionfornon-linearsummationofpotentials

Unlikecurrentsrecordedundervoltage-clampconditions,synapticpotentialsdonotsummatelinearly.Thereforethesizeofthesynapticpotentialisnotdirectlyproportional to the number of quanta released. However, given certainassumptions, it is possible to correct for the effects of non-linear summationusingtheeqn.

6where Peakevoked is the measured peak amplitude of the evoked synapticpotential,Vmisthecellrestingpotential,Vristhereversalpotentialforthepost-synaptic ion channels, and f is a correction factor for the effects of the cellmembranetimeconstantonsynapticpotentialamplitude.(Adiscussiononnon-linearsummationanditscorrectioncanbefoundinMcLachlan&Martin,1981).

QuantalAnalysisofTransmitterRelease>AnalysingQuantalContentofSynapticCurrentsThefollowingprocedurecanbeusedtocalculatethequantalcontentofaseriesof synaptic currents or potentials, which have been recorded and stored in adigitiseddatafile.

a) Using the record display module, inspect each record in the data file andclassify it as being either a nerve-evoked signal, EVOK, a spontaneousminiatureevent,MINI,or (inexperimentswhere theprobabilityof transmitterrelease is low)anervestimuluswhichhas failed to releaseanyquanta,FAIL.(YoushouldalsomarkanyrecordscontainingartefactsasREJECTED.).

b) Use the waveform measurements module to calculate the waveformparameters forALLof the records,with the intention ofmeasuring the signalpeakamplitude(Takecaretoexcludethenervestimulusartefact).

c)SelectAnalysisQuantalContenttoopenthequantalanalysiswindow.

Tocalculatethequantalcontentofthesynapticcurrents:1) Select the type of quantal analysis.ChoosePoisson if there are noMINI

recordsavailableandthetransmitterreleaseprobabilityisexpectedtobelow.OtherwisechooseBinomial.

2) Enter therangeofrecordstobeusedin theanalysis.SelectAllrecords ifyouwant touseallrecordsinthefile,orselectRangeandentera rangeofrecords.

3) If thereareseveralchannels in thesignal record,select thechannelwhichcontainsthesignalstobeanalysed,fromtheCh.list.

4)Evokedevents.SelecttheEVOKrecordclassificationtype,usedtoindicatestimulus-evokedsignals,fromtheTypelist.

5) Quantumevents.If thefilecontainsminiatureevents,selecttheEvents in

fileoptionandselectMINIfromtheTypelist.

If there are nominiature events in the file, but you knowwhat the quantalsignalamplitudeis,selectUserenteredandentertheaveragepeakamplitudeofthespontaneousminiaturesignalintotheAmplitudebox,andthestandarddeviationofpeakamplitudeintheStandardDev.box

6)Analysismode.

If the signals being analysed are currents, recorded under voltage-clampconditions,selecttheCurrentsoption.

If the signals are potentials, select thePotentials option, and enter the cellresting potential and the reversal potential of the synaptic conductance intotheResting potential andReversal potential boxes. This data is used toapply a correction for the non-linear summation effect. The Correctionfactorshouldbeleftatthedefaultvalue(1)unlesstheappropriatefactorforthesynapseunderstudyisknown.

7)ClicktheDoAnalysisbutton,tobeginthequantalanalysis.

The analysis procedure scans through the data file, calculates the mean andvariance of the peak amplitude of the signal records, uses these to obtainestimatesforthequantalcontent,anddisplaystheresultsinthereportwindow.Acopyofthequantalanalysisreportisalsowrittentothelogfile.(Note that you can test the operation of the quantal content analysis moduleusingsimulatedendplatecurrentsorpotentials,generatedbythesynapticsignalsimulationmodule.)

SynapticCurrentDrivingFunctionAnalysis>ComputingaDrivingFunctionThesynapticcurrentdrivingfunctionisameasureoftherateofevokedreleaseoftransmitteratasynapse.Ifthetimecourseofdecaythepost-synapticcurrentisknown,thedrivingfunctioncanbecomputedusingadeconvolutionprocess.MoredetailsofthemethodcanbefoundinDempster(1984).WinWCP’sdrivingfunctionmodulecanbeusedtocomputethedrivingfunctionfromsynapticcurrentrecords,suchasendplatecurrents.

Tocarryoutadrivingfunctionanalysis:

a)Recordaseriesofstimulus-evokedsynapticcurrents.

b)Usethesignalaveragingmoduletocreateanaveragesynapticcurrentfromthesetofrawrecords.

c)Usethecurvefittingmoduletofitanexponentialfunctiontothedecayphaseoftheaveragedsynapticcurrent.Fitthefunctionfromaround95%-5%ofthedecay phase, excluding the 5% around the peak where the transmitter is stillbeingrelease.

d)Select

AnalysisDrivingFunction

toopenthedrivingfunctionwindow

1) Ifthereareseveralchannelsintherecord,selectthechannelcontainingthesignaltobetransformedfromtheCh.list.

2) Set the rangeof records tobe transformed.SelectAllRecords for all therecords in the file,ThisRecord for the currently displayed record only, orselectRangeandenterarangeofrecordsintothebox.

3)Enterthecellholdingpotential(inmV)thatthecurrentswererecordedat,intheHolding Potential box, and the reversal potential of the post-synapticcurrent,intheReversalPotentialbox.(Thedrivingfunctionisexpressedinunits of conductance/unit time. The holding and reversal potentials arerequiredtoconvertfromcurrenttoconductance).

4)Thetimeconstantcomputedbythecurvefittingmoduleinstep(3)isusedtodeconvolvethecurrentsignal.Ifyouwishtousethesametimeconstantforall records (rather than using the individual value computed from eachrecord),selecttheKeepparametersfixedoption.

5) The basic deconvolution process computes a driving function, which

represents the rate of change of post synaptic conductance induced by therelease of transmitter. It is also possible to reconvolve this driving functionwith a different post-synaptic current decay function to generate thewaveform of the synaptic current thatwould have existed under these newconditions.Ifyouwishtocreateasimulatedcurrent,selecttheReconvolutewaveform option and enter the new time constant in theReconvolutioncolumn.

6)ClicktheDoTransformsbuttontobeginthedeconvolutionprocess.

Driving functions are created and stored in a driving function data file (.DFNextension) and can be viewed by selecting Driving Functions from theViewmenutodisplaythemintherecorddisplaymodule.

The driving function (b) computed from a simulated endplate current (a) isshownbelow.

(a)

(b)

SignalRecordEditor>EditingDigitisedSignalRecordsTherecordeditingwindowscanbeusedtomakemodificationstothedigitisedsignalrecordsstoredonfile.Thepositionofsignalscanbeshiftedverticallyorhorizontallywithintherecords, invertedorscaledinamplitude.Regionsoftherecordcontainingstimulusorotherartefactscanalsobeblankedout.

SelectAnalysisSignalEditor

toopentheEditRecordwindow.

SelectingrecordsforeditingRecordscanbedisplayedusingtheRecordselectionsliderbar.Selectthesignalchannel to be edited from theChannel list. Select theThisRecord option toapplyeditingoperations to thecurrentlydisplayedrecordonly,AllRecords tochangeallrecordsinthedatafile,orselectRangeandenteraspecificrangeof

records.ShiftingthesignalhorizontallyToshiftthesignalleftwardsorrightwards,enterthedistancetoshifted(intimeunits)intheXShiftboxandclicktheLeftarroworRightarrowbuttontoshiftthesignal.ShiftingthesignalverticallyEnterthedistancetoshifted(intheunitsoftheselectedsignalchannel)intheYShiftboxandclicktheUparroworDownarrowbuttontoshiftthesignal.Scalingthesignal.EnterthescalingfactorintheYScaleboxandclicktheScaleBybutton.(Note.Scalingby-1invertsthesignal).StimulusartefactremovalSelect the region of the signal record containing the artefact using the regionselection cursors. The limits of the region to be modified are indicated by ahorizontal bar along the bottom of the display. Enter the signal level to besubstituted for the artefact in the Blank Value box, then click the RemoveArt.button.UndoingoracceptingchangesToundoeditingchanges,clicktheUndoAllEditsbuttontorestorethedatatothebackupcopyof theoriginaldata file. (Abackupcopyof thedata file (filename.wcp.BAK)iscreatedwhenthetheEditRecordwindowisopenedforthefirsttime.)

DataFiles>OpeningaWCPDataFileWinWCP uses its own custom data file format for storing digitised signalrecords.Thesefilesareidentifiedbythefileextension“.WCP”ToloadapreviouslycreatedWCPdatafile,select

FileOpenDataFile

todisplaytheOpenFiledialogbox.

SelectthediskdriveandfolderfromtheLookIn list.AlistofavailableWCPfileswillbedisplayed.Selectoneofthefilenames,thenclicktheOKbuttontoopenthedatafilefordisplayandanalysis.

DataFiles>AddingRecordsfromaDataFileToappendrecordsfromaWCPdatafileontotheendofthecurrentlyopendatafile,selectFileAppendDataFile

TodisplaytheAppendFiledialogbox.

SelectthediskdriveandfolderfromtheLookIn list.AlistofavailableWCPfileswillbedisplayed.Selectoneofthefilenames,thenclicktheOKbuttontoappendtherecordsfromthisfileontotheendofthecurrentlyopenthedatafile.

(Note,youcanonlyappendfileswhichhavecompatiblerecordswiththesamenumberofchannelsandsamplesperchannel.)

DataFiles>InterleavingRecordsfromaDataFileTwoWCPdatafilescanbemergedbyalternatelyinterleavingrecordsfromonefilewith the other. (This feature can be useful for leak subtractionwhen leakrecordshavebeenacquiredinanotherfile.)To interleave records fromaWCPdata filewith recordsof thecurrentlyopendatafile,selectFileInterleaveDataFile

TodisplaytheInterleaveFiledialogbox.

SelectthediskdriveandfolderfromtheLookIn list.AlistofavailableWCPfileswillbedisplayed.Selectoneofthefilenames,thenclicktheOKbuttontointerleavetherecordsfromthisfilewiththoseoftheopenthedatafile.

(Note, you can only interleave files which have compatible records with thesamenumberofchannelsandsamplesperchannel.)

DataFiles>ImportingOtherDataFileFormatsToimportrecordsfromoneormorenon-WCPdatafile,selectFileImport

TodisplaytheImportFiledialogbox.

Select thediskdriveand folder from theLookIn list.Thenselect the typeofdata file tobe imported from theFilesofType list.A listofavailable filesofthattypearedisplayed.Selectoneormoreofthefilenames,thenclicktheOKbuttontoimportthedatafile(s)intoa.WCPformatfile.Anew.WCPformatfileiscreatedwiththesamenameastheimportedfilebutwiththeextension.WCP.Whenmorethanonefileisselectedforimport,a.WCPdatafile iscreatedforeachfile in theselection listwith the last file in the listremainingopenanddisplayedbyWinWCP.

Thecurrentlysupporteddatafileformatsarelistedinthetablebelow.

ImportableDataFileFormats

AxonInstruments(*.abf,*.dat)

AxonInstrumentsABF(AxonBinaryFile)formatfilesproducedbythePCLAMPandAxoScopeprograms.

CambridgeElectronicDesignCFS(*.CFS,*.DAT)

CambridgeElectronicDesignCFS(CEDFilingSystem)formatfiles.

SCANFiles(*.SCA)

DatafilesproducedbytheStrathclydeElectrophysiologySCANprogram(anMS-DOSbasedelectrophysiologypackage.

IgorBinaryFiles(*.IBW)

IBW(IgorBinaryWave)filesproducedbytheIGORProsoftwarepackage.

HEKA(*.ASC)

DatarecordsexportedfromtheHekaPULSEsoftware.

DataFiles>ImportingfromASCIITextFilesTo import records fromoneormore text files containing tablesofnumbers inASCIIformat,select

FileImport


SelectthediskdriveandfolderfromtheLookInlist.ThenselectASCIITextathetypeofdatafiletobeimportedfromtheFilesofTypelist.Selectoneormoreof the list filenames, thenclick theOKbutton toopen theASCIIImportdialogboxwhichallowsyoutoviewtheformatofthedatatobeimportedandtospecifyhowitshouldbeimported.When you have (if necessary) adjusted the import configuration, click theOKbutton,toimportthedatawhentheimportsettingsarecomplete.Anew.WCPformatfileiscreatedwiththesamenameastheimportedfilebut

withtheextension.WCP.Whenmorethanonefileisselectedforimport,a.WCPdatafile iscreatedforeachfile in theselection listwith the last file in the listremainingopenanddisplayedbyWinWCP.ImportConfigurationSettings

ColumnSeparator:Select the character used to separate data columns in thefile(<tab>,commaorsinglespacecharacter).No.oftitlelinestoignore:Thefirstdatarow(s)inthetableoftencontainlabelsor identification information,which should not be treated as samples. To skiponeormoreoftheselines,enterthenumbertoskippedintheNo.oftitlelinestoignorebox.

Sampletimes.Ifthefirstcolumninthetablecontainssamplestimes,selecttheSampletimesinfirstcolumnoption toderive thesampling interval fromthetimesofsuccessiverows.Selecttheunitsthatthetimedataisexpressedinfromthe Time units list. If no sample time data is available, select No sample

timesandenterthesamplingintervalintotheSamplingIntervalbox.

TimeUnits:Select theunitsof thetimedatacolumn(secs,msecs,mins)fromtheTimeUnitslist.

No.of timepointspersweep. If a sample timecolumn ispresent, containingtimevaluesincrementingfromzeroforeachseparaterecordingsweepcontainedthedatatable,thesizeoftheWinWCPdatarecordisdeterminedautomaticallyfromwhentimedataresetstozero.Thenumberofdatapointsineachsweepcanalso be setmanually, by selecting theSet record size option and entering therecordsizeintothebox.Channels:EnterthenamesandunitsforeachchannelintotheChannelstable.

DataFiles>ImportingfromRawBinaryDataFiles

WhenaspecificdatafileformatisnotsupportedbyWinWCPitcanstillbepossibletoimportdatausingtherawbinaryimport.Theimportmoduleassumesthatthedatahasthegeneralformat

Atthebeginningofthefile,thereisablockoffileheaderdatawhichcontainstheinformationonthenumberofrecordsinthefile,sizeofrecord,numberandscalingofchannels.ThisisfollowedbyoneormoredatablockscontainingtheA/D converter samples. If more than one input channel has been digitised,samples are interleaved within the data block (e.g.Ch.0,Ch.1,Ch.2,Ch.0,Ch.1.,Ch.2,…).Thesedetailsofthedatafilestructurecanoften be obtained from the user manuals associated with the software, whichcreated the data files. (Note that the sampling interval and other scalinginformationisdiscardedbythebinaryimportmodule.)Theimportsettingsmustbecarefullysetuptomatchthecharacteristicsofthefilebeingimported.Toimportoneormorerawbinarydatafiles,selectFileImport


SelectthediskdriveandfolderfromtheLookInlist.ThenselectRawBinary(*.dat,*.raw)fromtheFilesofTypelist.Alistofavailablefilesinthattypearedisplayed.Selectoneormoreofthefilenames,thenclicktheOKbuttontoopentheRawBinaryImportdialogbox.

Specifytheformatofthedatainthefiletobeimported:Fileheadersize:Enterthesizeofthefileheader(inbytes).Ifthereisnofileheaderenter0.No.ofInputChannel:Enterthenumberofanaloginputchannelsinthefile.

No.ofSamples/Channel:EnterthenumberofA/Dsamplesperchannelintherecord.

SampleFormat:Select the numerical format of the sample data:Float for 4byte floating point numbers or Integer. If Integer has been selected, enter thesizeoftheintegernumber(bytes)inNo.ofbytes/sampleandtheupperlimitofthenumericaldatainMax.Value.

Sampling interval: Enter the time interval between adjacent samples withineach analog channel. Select the units of the time interval from the Timeunitslist.

Channels: Enter the name and measurement units for each channel and thescalingfactortoconvertfromintegervaluetothemeasurementunits..

ClicktheOKbutton,toimportthedatawhentheimportsettingsarecomplete.Anew.WCPformatfileiscreatedwiththesamenameastheimportedfilebutwiththeextension.WCP.Whenmorethanonefileisselectedforimport,a.WCPdatafile iscreatedforeachfile in theselection listwith the last file in the listremainingopenanddisplayedbyWinWCP.

DataFiles>ExportingtoOtherDataFileFormatsWCPdatafilescanalsobeexportedinanumberofdatafileformats.Toexportdatafiles,selectFileExport

ToopentheExportFiledialogbox.

OutputFormat:Selectthedataformatofthefiletobeexportedtofromthelistofoptions.

Record Range: Select the All Records option to export all records in thecurrentlyopenWCPdatafile,orselectRangeandentertherangeofrecordstobeexported.

Channels:Ticktheanaloguesignalchannelstobeexported.

Selectingfilesforexport:Thecurrentlyopendatafileisselectedforexport.Toselectmore files, clickSelectFiles to open theSelectFiles toExportdialogbox, hold down theCtrlkey, and click on the files to be selected, then click

Openwhencomplete.The list of files selected for export aredisplayed in theFilesbox.Note.Alistoffilestobeexportedcanalsobepastedintothisbox.

Whentheexportsettingsarecomplete,clicktheOKbuttontocreatetheexportfile(s).

Thecurrentlysupportedexportfileformatsarelistedinthetablebelow.

ExportDataFileFormats

AxonInstruments(*.abf)

AxonInstrumentsABF(AxonBinaryFile)V1.8formatfiles.

CambridgeElectronicDesignCFS(*.CFS,*.DAT)

CambridgeElectronicDesignCFS(CEDFilingSystem)formatfiles.

ASCIIText Tab-delimitedcolumnsofASCIItext.

(*.txt)

WCP(*.wcp)

StrathclydeElectrophysiologySoftwareWinWCPdatafileformat.

EDR(*.edr)

StrathclydeElectrophysiologySoftwareWinEDRdatafileformat.

IgorBinaryFiles(*.IBW)

IBW(IgorBinaryWave)filesproducedbytheIGORProsoftwarepackage.

MatlabFiles(*.MAT)

.MATformatarraydatafilesreadablebyMatlab

DataFiles>WCPFileStructure

ThisappendixprovidesadetailedspecificationoftheinternalstructureoftheWinWCPdatafile.TheWCPdatafileisdesignedtostoredigitised16bitintegerbinaryrecordsofanalogsignals,theassociatedscalinginformationrequiredtoreconstituteactualsignallevels,validationinformationenteredbytheuserandmeasurementsgeneratedbyWinWCPanalysismodules.

AWCPfilecancontainupto231separaterecords,eachrecordcontainingupto128channels,eachchannelcontainingamultipleof256samples,uptoatotalof1048576forthewholerecord.Note.Allrecordswithinafilemustbethesamesize,i.e.samenumberofchannelsandsamples/channel.

Threekindsofdatablockscanexistwithinthefile.TheheaderblockcontainsalistofASCII-formatkeywords,detailingthenumberofrecordsinthefile,recordsize, scaling factors etc.Signal records are stored in sequenceafter theheaderblock. Each record consists of an analysis block, containing validation andanalysisresultspertainingtotherecord,followedbyadatablockcontainingthedigitisedA/Dsamples.Adatafilethushastheform:

….

Thebeginningofeachsignalrecordcanbedeterminedasabyteoffsetfromthestartofthefileusingtheformula

Offset=HBsize+(Rec#-1)(RABsize+RDBsize)

whereHBsize,RABsizeandRDBsizearethesizes(bytes)oftheheader,recordanalysisanddatablocksrespectivelyandRec#istherecordnumber.

HeaderBlock

The header block contains the information needed to allow a program todeterminethesizeandnumberofrecordsinthefile.Theheaderblockcanvaryinsizebetween1024and16380bytedependingon thenumberofchannels inthefile,accordingtotheformula:

HBsize=(Int(Nchans-1)/8)+1)x1024

FileparametersarestoredasASCIItextintheformofkeywords,onewordperline,asfollowsKEY=<value><cr><lf>where<value>isanumberortextdependingontheparameterand<cr><lf>arethecarriagereturnandlinefeedcharacters.

A typical header block (from a file with 2 channels) contains the followingkeywords.

VER=9WCPdatafileformatversionnumberCTIME=19-05-201015:15:59.010Creationdate/timeRTIME=19-05-201015:15:60.000Date/timeofstartofrecordingRTIMESECS=2000.00Timeofstartofrecordinginseconds(relativetolastsystemboot)NC=2No.ofchannelsperrecord.NR=50No.ofrecordsinthefile.NBH=2No.of512bytesectorsinfileheaderblockNBA=1No.of512bytesectorsinarecordanalysisblockNBD=4No.of512bytesectorsinarecorddatablockAD=5.0000A/Dconverterinputvoltagerange(V)ADCMAX=2047MaximumA/DsamplevalueNP=512No.ofA/DsamplesperchannelDT=.1600A/Dsamplinginterval(s)NZ=10No.ofsamplesaveragedtocalculateazerolevel.TU=msTimeunitsID=Cell1ExperimentidentificationlineChannelscalingfactorsYN0=ImChannel0nameYU0=nAChannel0unitsYG0=.167E+04Channel0gainfactormV/unitsYZ0=1997Channel0zerolevel(A/Dbits)YO0=0Channel0offsetintosamplegroupindatablockYR0=2

YN1=VmChannel1nameYU1=mVChannel1unitsYG1=10.0Channel1gainfactormV/bitYZ1=2048Channel1zerolevel(A/Dbits)YO1=1Channel1offsetintosamplegroupindatablockYR1=0

WaveformmeasurementsettingsTXPERC=0T.x%decaytimesettingsPKPAVG=1No.ofpointstoaveragebefore/afterforpeakmeasurementNon-stationaryvariancesettingsNSVCHAN=0SignalchanneltobeanalysedNSVALIGN=0AlignmentmodeNSVTYPR=0

NSVS2P=FT=ScaleaveragecurrenttopeakbeforesubtractionNSVCUR0=0FirstanalysiscursorpositionNSVCUR1=0SecondanalysiscursorpositionID=

Notethatitshouldnotbeassumedthatthekeywordswillfollowanyparticularorder.

RecordAnalysisBlock

The record analysis block containing a series of internal format variables. Itvaries in size between 1024 and 16380 byte depending on the number ofchannelsinthefile,accordingtotheformula:

RABsize=(Int(Nchans-1)/8)+1)+1)x1024

whereNchansisthenumberofdatachannelsandInt()meansrounddowntothenearestinteger.Theanalysisblockstructureisdetailedbelow:Variable Type ContentsRecordstatus 8xASCIIbytes ACCEPTED,REJECTEDRecordtype 4xASCIIbytes TEST,LEAK,etc.Groupnumber 4bytefloatingpoint RecordleaksubtractiongroupingnumberTimerecorded 4bytefloatingpoint (s)

Samplinginterval 4bytefloatingpoint (s)MaxpositivelimitofA/Dvoltagerange

4bytefloatingpointxNchans (V)

Marker 16xASCIIbytes UserenteredmarkerValues 4bytefloatingpoint

x(Int(Nchans-1)/8)+1)x28

Waveformmeasurementandcurvefittingdatastoragearray.

Datablock

Thedatablockcontainsthedigitisedsignals,storedintheformof16bitbinaryintegers.EachA/Dsampletakesup2bytesofspace.Itssizedependsuponthenumberofdatachannelsinthefile

RDBsize=2xNchansxNsamplesperchan

whereNchans is the number of data channels and Nsamples per chan is thenumber of A/D samples per channel. If there is more than one A/D inputchannel, samples are interleaved within the data block. For example, for 2channels,

Y01Y11Y02Y12.....Y0nsamplesY1nsamples

DifferentlaboratoryinterfacessupportedbyWinWCPreturnmultichannelA/Dsamplesindifferentorders.ThechannelinterleavingorderforadatafileisspecifiedbytheYOn=channelkeywordinthefileheaderblock.

Thecalibratedsignallevelintheappropriatechannelunitscanbereconstructedusinginformationstoredinthefileheaderandtherecordanalysisblocks,using,

whereVmaxthemaximumpositivelimitoftheA/Dconvertervoltagerange(fromrecordanalysisblock),ADCmaxismaximumA/DsamplevalueatVmax.(headerblock)andYGnisthecalibrationfactor(Volts/channelunits)forchanneln(headerblock).

Simulations>Nerve-evokedEPSCSimulation

Thenerve-evokedEPSCmodulegenerates a seriesof nerve-evoked excitatorypost-synaptic currents (EPSCs) or potentials (EPSPs). When the nerve isstimulated a randomnumberof transmitter quanta are released fromapoolofsize,n,with each quantumhaving a probability,p, of release. The number ofquanta released per stimulus follows a binomial distribution. The EPSCwaveform can be made to decay following a single or double exponentialfunction.Randombackgroundnoisewithagaussiandistributioncanbeaddedtothe signal. EPSPs can also be simulated, including the effects of non-linearsummationofquantalpotentials.TocreateadatafilecontainingsimulatedEPSCs:Createanewdatafiletoholdtherecords,byselectingFileNew

andenteringthenameofanewdatafile.SelectSimulationsNerve-evokedEPSCs

toopenthesimulationwindow.

SetthesimulatedEPSCproperties:

No.Records:EnterthenumberofsimulatedEPSCstobecreated.

Current/Potentials: Select the Currents or Potentials option to determinewhethersimulatedcurrentsorpotentialsare tobecreated. Ifyouhaveselectedpotentials, enter the resting potential of the cell. in the Resting/holdingpotentialbox.

TransmitterRelease: Enter the number of quanta available for release in theReleasepool(n)boxand theprobabilityofaquantumbeingreleasewhen thenerveisstimulatedintheReleaseprob(p)box.

Quantaleventproperties:

Amplitude:SettheaveragepeakamplitudeoftheminiaturequantalcurrentinthePeakboxand its standarddeviation in theStDev box.Enter the standard

deviationofthebackgroundnoiseonthesignalintheBackgroundnoisebox.

Rising Phase: Enter the time constant of the EPSC rising phase in theTau(rise)boxandthevariabilityofthetimebetweenstimulationandtheeventintheLatencyvariabilitybox.

DecayPhase:Enter the time constant of the decay of the EPSC in theTimeconstant(1) box. If a double exponential decay is required, tick theDoubleexponential decay option, enter a second time constant in the Timeconstant(2)box,andentertheratiobetweentheamplitudesofthetwodecayingexponentialcomponentsintheAmp(1)/Amp(2)box.

ClicktheStartSimulationbutton,tocreatethesimulatedEPSCrecords.

Simulations>Voltage-activatedCurrentSimulation

Thevoltage-activatedcurrentsmodulesimulatesthecurrentsevokedinresponsetoaseriesofrectangularvoltagesteps,usingtheHodgkin-Huxleyequations.Bothactivationandinactivationkineticsaremodelled.Twochannelsaregenerated,membranepotentialandmembranecurrent.Themodelalsosimulatestheeffectsofpatchclamppipetteaccessconductanceonmeasuredcurrents.TheHodgkin-HuxleysimulationcanbeusedtotesttheleaksubtractionmoduleandtheHodgkin-Huxleyfunctionsinthecurvefittingmodule.Thecurrents,aremodelledbytheequation

whereV is the voltage level to which the cell potential is stepped,Vh is theinitial holding voltage, Vrev is the reversal potential for the conductance,Gmax is the maximal conductance, Taum the activation (m) time constant,Tauhtheinactivation(h)timeconstant,andpthepowertowhichtheactivationparameterisraised,m,(V)andh(V)arethesteady-statevaluesoftheactivationandinactivationparameters,respectively,definedbyBoltzmannfunctions,m(V)andh(V)arebell-shapedfunctions.Tocreateadatafilecontainingsimulatedvoltage-activatedcurrents:Createanewdatafiletoholdtherecords,byselectingFileNew

andenteringthenameofanewdatafile.SelectSimulationsVoltage-activatedcurrents


SetthesimulatedEPSCproperties:

VoltageProtocol:Enterthevoltage-clampholdingvoltageintotheHoldingvoltagebox,thenumberofsimulatedvoltagestepstobecreatedintheNo.ofstepsbox,andtheincrementinvoltagebetweenstepsintheVoltagestepbox.

LeakSubtraction:Ifleaksubtractionrecordsaretobecreated,selectDividebyNandentertheP/Ndividefactor.

Noise:Enterthestandarddeviationofthegaussianbackgroundnoisetobe

addedtothesignalsintheBackgroundnoisebox.

Conductance:Enterthemaximumconductanceforthevoltage-activatedcurrentbeingmodelledintheMax.conductancebox.EnterthereversalpotentialforthevoltageactivatedconductanceintheReversalpotentialbox.Enterthecell’snon-voltagedependentleakconductanceintheLeakconductancebox.EntertheaccessconductanceofthepatchpipetteusedtopatchclampthecellinthePipetteconductancebox.(Notethatifthepipetteaccessconductanceislessthan5Xthecellmembraneconductance,thenpipetteseriesresistanceartefactswilloccur.).EnterthecellcapacityintheCellcapacitybox.Thisdeterminesthesizeofthecapacitycurrentartifactatthebeginningandendofthevoltagestep.

Activation(m):TheActivation(m)settingsgroupdetermines thevoltage andtime dependence of the Hodgkin-Huxley activation parameter, m.Enter thepowertowhichtheactivationparameter,m,istoberaisedtointheActivationpower factor box. (Default =3, typical of sodium currents). The voltagedependenceofsteady-stateactivationissetbytheBoltzmannfunction,minf(V),defined by its voltage of halfmaximal activationV1/2 and slopeVslope. Thevoltagedependenceoftheactivationtimeconstant,tau(V)issetbyabellshapedfunction, which varies the time constant between the minimum, tmin andmaximum tmax,with tmax occurring atV1/2 and the steepness of the cursordefinedbyVslope.

Inactivation(h):TheInactivation(h)settingsgroupdeterminesthevoltageandtime dependence of the activation parameter, h. To enable the inactivationparameter, check the Inactivation in use box. The voltage dependence ofsteady-stateactivationissetbytheBoltzmannfunction,minf(V),definedbyitsvoltage of half maximal activation V1/2 and slope Vslope. The voltagedependence of the activation time constant, tau(V) is set by a bell shapedfunction, which varies the time constant between the minimum, tmin andmaximum tmax,with tmax occurring atV1/2 and the steepness of the cursordefinedbyVslope.Enterthevoltageatwhichtheinactivationparameterisat0.5in the V.half box. Enter the inactivation time constant when the membranepotentialisatV.halfintheTau(V.half)box.EnterthevoltagesensitivityintheV.slopebox(largevalues=weakvoltagesensitivity).

ClicktheStartSimulationbutton,tocreatethesimulatedvoltage-activated

currentrecords.

Simulations>MiniatureEPSCSimulation

The miniature EPSC module generates simulated miniature postsynapticcurrents, exhibiting the stochastic fluctuationsassociatedwith thecurrent flowthrough the population of ion channels opened by a single quantum oftransmitter.Thegatingofasinglepost-synapticionchannelisrepresentedbyasimple4-statemodel.Bindingofanagonistmolecule (A)with receptor (R) toforman agonist-receptor complex,makes it possible for the channel to shuttlebetweenaclosedstate(AR),anopenstate(AR*)andaclosed/desensitisedstate(ARD).

Thesingle-channelcurrenttimecourseisgovernedbysixrateconstants–rateofbindingandunbindingofagonistfromreceptor,(kbind,kunbind)rateofchannelopening and closure (kopen, kclose) and the rate of entry and exit from thedesensitised state (kdes, kund). The mEPSC is generated by summing theindividualsingle-channelcurrent timecourses, foreach ionchannelopenedbythebriefpulseoftransmitterreleasedfromeachvesicle(timecourserepresentedbyadecayingexponentialfunctionwithatimeconstantof10s.

TocreateadatafilecontainingsimulatedmEPSCs:Createanewdatafiletoholdtherecords,byselectingFileNew

andenteringthenameofanewdatafile.SelectSimulationsMiniatureEPSCs


SetthesimulatedmEPSCproperties:

mEPSC:EnterthenumberofsimulatedmEPSCrecordstobecreatedintheNo.mEPSCsbox.Enterthesingle-channelcurrentamplitudeforthepost-synapticionchannelsintheUnitarycurrentbox.EntertheaveragenumberofionchannelsactivatedwhenaquantumoftransmitterisreleasedintheNo.channelsboxanditsstandarddeviationintheSt.Dev.Box.EnterthetransmitterreleasedecaytimeconstantintotheTransmitterdecaybox.

Ion ChannelModel: Enter the rates constants which define the ion channelgatingproperties. (Note.Modelswhichpermit entry into thedesensitised state(kdes>0) produce mEPSCs with biexponential decays. If kdes =0monoexponentialdecaysresult.)

RecordingConditions:Enter the standard deviation of recording backgroundnoise in the Backg. Noise box. If low-pass filtering is to be applied to themEPSC,selecttheLow-pfilterOnoptionandenterthecut-offfrequencyinthebox.Randombaselinedriftcanbeaddedtoeachrecordbyenteringanon-zerovalueintheDrift(Max)box.

ClicktheStartSimulationbutton,createthesimulatedmEPSCcurrentrecords.

COMAutomationInterface>COMAutomationInterface

WinWCPimplementsaCOMautomationserverwhichallowsitsrecordingandsealtestfunctionstobecontrolledfromVBSCRIPTbatchfilesorfromapplicationssuchasMatlabwhichsupportsCOMautomation.ThenameoftheWinWCPautomationobjectisWinWCP.AUTOandisopenedbytheVBSCRIPTcommand

setWCP=CreateObject(“winwcp.auto”)

Recordingfunctions

Recording can be started/stopped, data files created/opened and the recordingtriggermodeandstimuluspulseprotocolsselected.Therecordingmethodsandpropertiesarelistedbelow.

RecordingMethods&Properties

.NewFile(“filename.wcp”) Method Createsanewdatafilewiththesuppliedname

.OpenFile(“filename.wcp”)Method Opensapre-existingdatafilewiththesuppliedname

.StartRecording Method Startsrecordingtodisk.

.StopRecording Method Stopsrecordingtodisk.

.HoldingVoltage R/WProperty

Reads/setstheholdingvoltage(inVolts)appliedtothecell.E.g.WCP.HoldingVoltage=-0.06

.TriggerMode R/WProperty

Read/setstherecordingsweeptriggermode.“F”=Freerun,“E”=Externaltrigger,“D”=EventDetection,“P”=StimulusPulse.E.g.WCP.TriggerMode=“F”

.StimulusProtocol R/WProperty

Read/setstheselectedstimuluspulseprotocol.e.g.WCP.StimulusProtocol=“prot01”

.Status ReadOnlyProperty

ReadsthecurrentoperationalstatusofWinWCP.(0=idle,1=sealtestrunning,2=recordingtodisk)

Sealtestfunctions

WinWCP’s seal test function can be initiated via the command interface andusedtoapplytestpulsestocellsandcalculatethecellmembraneconductance,capacity, access conductance and pipette seal resistance. These measurementscanbereadviathecommandinterfacewhilethesealtestisrunning.

Thesealtestcommandsarelistedbelow:

SealTestMethodsandProperties

.StartSealTest Method Displays the seal test window andappliesthesealtestpulse.

.SealTestPulseAmplitude R/WProperty

Reads/setstheamplitude(Volts)ofthesealtestpulse(e.g. WCP.SealTestPulseAmplitude=0.01)

.SealTestPulseDuration R/WProperty

Reads/sets the duration amplitude (S)ofthesealtestpulse(e.g. WCP.SealTestPulseDuration=0.01)

.SealTestSmoothingFactor R/WProperty

Set cell parameters smoothing factor(0.1-1.0) '1=nosmoothing, '0.1=maximum smoothing (equivalent toaveragingover10pulses

.Vm ReadOnlyProperty

Reads the most recent cell holdingpotential (V) measurement, computedbythesealtest.

.Im ReadOnlyProperty

Returns the most recent cell holdingcurrent (A) measurement, computedbythesealtest.

.Ga ReadOnlyProperty

Reads the most recent cell accessconductance(S)measurement.

.Gm ReadOnlyProperty

Reads themost recent cellmembraneconductance(S)measurement.

.Cm ReadOnlyProperty

Readsthemostrecentcellcapacity(F)measurement.

.Rseal ReadOnlyProperty

Reads the most recent pipette sealresistance()measurement.

Afile(WinWCPVBSCRIPTExample.vbs)containingVBSCRIPTexamplecodecanbefoundinthec:\programfiles\winwcpfolder.

References>References

BrownK.M.&DennisJ.E.(1972)Derivative-freeanalogsoftheLevenberg-MarquardtandGaussalgorithmsfornon-linearleastsquaresapproximation.NumerischeMathematik,18,289-297.Bezanilla F.&ArmstrongC.M. (1977) Inactivation of the sodium channel I.Sodiumcurrentexperiments.J.GenPhysiol.70,548-566.

DeKoninckY&ModyI.(1994)NoiseanalysisofminiatureIPSCsinadultratbrainslices:propertiesofsynapticGABAAreceptorchannels.J.Neurophysiol.71,1318-35.

Dempster J. (1986)Theuseof thedriving function in theanalysisofendplatecurrentkinetics.J.Neurosci.Methods,18,277-285.

Dempster J. (1993) Computer Analysis of Electrophysiological Signals,AcademicPress,London(ISBN0-12-208940-5)

DempsterJ.(2001)TheLaboratoryComputer:AGuideforneuroscientistsandphysiologists,AcademicPress.

Dilger J.P&Brett R.S. (1990) Directmeasurement of the concentration- andtime-dependentopenprobabilityofthenicotinicacetylcholinereceptorchannel.Biophys.J.57,723-731.

Gillis K. (1995) Techniques formembrane capacitymeasurements. In Single-channelrecording,2nded.(ed.B.Sakmann&E.Neher),PlenumPress.

McLachlan E.M. & Martin A.R. (1981) Non-linear summation of endplatepotentialsinthefrogandmouse.J.Physiol.311,307-324.

SigworthF.J.(1980)ThevarianceofsodiumcurrentfluctuationsatthenodeofRanvier.J.Physiol.307,97-129.

Standen N.B., Gray P.T.A. & Whitaker M.J. (eds) (1987) The PlymouthWorkshopHandbook,TheCompanyofBiologistsLtd.,Cambridge.

TraynelisS.F.,SilverR.A.&Cull-CandyS.G.(1993)Estimatedconductanceofglutamate receptor channels activated during EPSCs at the cerebellar mossyfibre-granulecellsynapse.Neuron11,279-289.