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WINWCP User Guide V3.9 - Spider

1. During initial formation of a glga seal the Pipette option displays pipette resistance computed from V _ pulse O I pulse where Vpuise and Guise are the steady state voltage and current pulse amplitudes The Cell option displays the cell membrane conductance G capacity C and access conductance G computed from G V suis E Else Vaa G 1 G G where l is the initial current at the peak of the capacity transient and T is the exponential time constant of decay of the capacitance current See Gillis 1995 for details Note If G G and C are to be estimated correctly the patch clamp s pipette series resistance compensation and capacity current cancellation features must be turned off A good test to check if WinWCP is set up with the correct input output connections and channel scaling factors is to attach the model cell supplied with most voltage patch clamps and observe the holding potential and current test pulse amplitude and cell parameters correspond with the known values of the model 6 5 Display scaling and sweep triggering The vertical display magnification is automatically adjusted to p Display maintain a visible image of the test pulse within the display area The Auto Scale magnification factor in current use is indicated in the Display box C Manual ax Automatic scaling can be disabled by selecting the Manual option A Free run fixed display magnification can then
2. 8 2 1 Rectangular voltage pulse of fixed size nm This is a simple pulse which does not vary in amplitude and duration between records It has 3 parameters e Initial Delay defines the delay period before the pulse begins e Amplitude defines the pulse amplitude mV e Duration defines the duration of the pulse This element can be used to provide series of stimuli of fixed size or in combination with other elements to provide fixed pre conditioning pulses 8 2 2 Family of rectangular pulses varying in amplitude A This is a rectangular voltage pulse whose amplitude 1s automatically incremented between recording sweeps It has 5 parameters e Initial delay defines the delay period before the pulse begins e Start at Amplitude defines the amplitude of the first pulse in the protocol sequence e Increment by defines the increment to be added to the pulse amplitude between records e Number of increments defines the number of steps in the sequence e Pulse duration determines the duration of the pulse This element is typically used to explore the voltage sensitivity of 1onic conductances by generating records containing the whole cell membrane currents evoked in response to a series of voltage steps to different membrane potentials WinWCP V3 9 User Guide 34 8 2 3 Family of rectangular voltage pulses varying in duration T This is a rectangular voltage pulse whose duration can be automatically incremente
3. WinWCP V3 9 User Guide 16 2 Axon Instruments Digidata 1320 Series Axon Instruments Inc 3280 Whipple Road Union City CA 94587 U S A Tel 510 675 6200 The Digidata 1320 Series 1320A 1322 interfaces consist of self contained mains powered digitiser units with BNC I O sockets attached to the host computer via a SCSI Small Computer Systems Interface interface card and cable A number of versions are available including the 1320A and 1322A The 1322A supports sampling rates up to 500 kHz 16 bit resolution on up to 16 channels It has a fixed input and output voltage range of 10V and supports 4 digital output channels The Digidata 1320 Series is currently supported by WinWCP under Windows 95 98 NT and 2000 2 7 1 Software Installation WinWCP uses Axon s standard software library AxDD132x DLL for the Digidata 1320 Series Details for steps 1 5 can be found in Axon s Digidata 1320 Series Operator s Manual 1 Install the Axon SCSI card in a PCI expansion slot 2 Attach the Digidata 1320 to the SCSI card and switch on the computer and 1320 3 Install the AxoScope software supplied with the Digidata 1320 4 Reboot the computer 5 Run AxoScope to ensure that the software installed OK 6 Run WinWCP and select from its main menu Setup Recording Select Axon Instruments Digidata 1320 from the Laboratory Interface list box WinWCP V3 9 User Guide 17 2 7 2 Signal input output connections Sig
4. 10 2 Making waveform measurements After the records in the data file have been validated select Analysis Measure Waveforms Fasia to open the waveform measurement module on a The module is split five functional sections aa MN ie __ 7 F pages accessed by clicking on the page tab B AA AY Plot Histogram Summary Tables Passes The Analysis page is used to set up the parameters of the waveform analysis and initiate the automatic measurement sequence psu 204 2 9 ace ee a a ad al ae dr FP coe which generates a table of measurements The X Y plot page is used to create X Y graphs of the measurements The Histogram page is used to create frequency histograms of measurement The Summary page presents a summary mean standard deviation etc of the measurements for the series of records analysed The Tables page is used to create tables of results 10 3 Running a waveform analysis sequence The first stage in the measurement process 1s to define and run a waveform analysis sequence on a selected series of records 1 Select the Analysis page by clicking on 1ts page tab 2 Define the range of records to be analysed by selecting All Records to a All records analyse all records or This Record to analyses only the currently amp This record displayed record or Range and enter a range of records C Range 1 10 3 Select the type of records to be measured by selecting an option from th
5. 9 4 Choosing a printer and output format To choose a printer and to select the paper format select File Print Sertap i HE L Pria Print Setup uve come to open the print setup dialog box See eee Wham E phanmig bedded A printer can be selected from the list of currently gt Pao Disisi installed printers The orientation of the plot on the page can be selected as either portrait or landscape iga zji s SP rai La a Paris E lean aed we ee WinWCP V3 9 User Guide 41 9 5 Rejecting flawed records Digitised records cannot all be assumed to be perfect Consequently the visual inspection of records and the elimination of flawed records is an important part of the analysis process If automated waveform measurement procedures are to be applied a mechanism is required for excluding flawed records from the analysis Checking a record s rejected box marks a record as being flawed Rejected records are excluded from automatic waveform curve fitting signal average or leak subtraction calculations Note Pressing the Ctrl R key combination is a quick way of toggling the rejected check box on and off Rejected 9 6 Classifying records Records can also be classified according to the type of signal that they contain by selecting a type from the record s type list box Eight types of records are currently defined TEST LEAK EVOK MINI FAIL TYP1 TYP2 TYP3 Type The EVOK MINI
6. A list of available protocols appears in the Stimulus Program list box allowing quick selection of protocols during a recording session C Stimulus program E ee ee ee ee el WinWCP V3 9 User Guide 32 8 Creating stimulus protocols To create a stimulus protocol file select LT i Setup ES es Stimulus Protocol Editor to open the stimulus editor module A diagram of the z waveform appears in the Waveform display box The 2 0 voltage waveform is shown at the top V om with digital con MN pulse patterns if in use below The duration of the A rele recording sweep 1s shown as a red bar r tava Tee Egr Fieu Lar Seo Le ernie Ba aol M PH mpi haa Click the New button to create a blank protocol ae te 8 1 Building a stimulus protocol The first stage in building a stimulus protocol is to define the number duration and timing of the recording sweeps to be contained in the protocol To set these parameters click on the icon disen gt t _ VV BEE to display the recording sweep parameters table The interval between recording sweeps entry sets the time interval between successive recording sweeps within the protocol Recording sweep 2000 00 Paste Dis Update Interval The duration of recording fame sweep entry sets the duration SmE of the sweep Note Sweep duration must be at least 200 msec shorter than the interval between sweeps to allow time for records to b
7. File Append To display the Append File dialog box Select a file as above for appending Note You can only append files which have compatible record sizes with the same number of channels and samples per channel Append File 2 lx File name C Decay _ Files of type WCP Files WCP x Cancel Open File me Leak qubbraction tach cp Lo fou Teri wis W on tal Contest Tesi wp Look in ZA data y e ex laa EPC Decay wcp la Leak subtraction test wep a NI Test wep fa Quantal Content Test wep fa test wep ia twitch wep I Open as read only 18 3 Importing from foreign data file formats To import records from a non WCP data file select File Import To display the Import File dialog box Select the disk drive and folder from the Look In list Then select the type of data file to be imported from the Files of Type list A list of available files in that type are displayed mort Fie ORT Lookin a data Ej c File name C Decay Files of type Axon Files DAT ABF bd Cancel 7 Open as read only WinWCP V3 9 User Guide 71 Select one of the file names then click the OK button to import the data into a WCP format file Note that the original file is not changed A new WCP format file is created with the same name as the imported file but with the extension WCP 18 3 1 Axon Instruments Data files produced by Axon Instruments pClamp V5 a
8. In order to study a particular current in detail it is usually necessary to eliminate all the other currents from the signal This is often done using pharmacological agents such as TTX to block Na currents TEA to block K currents etc However even when such blocking agents are used there often still remains some residual current in addition to the one under study This current is known as the leak current It usually displays linear time independent properties In some circumstances the leak current is very small and can be ignored However in others it can be as large as the currents under study complicating the analysis of the signal waveforms unless it is removed Although the leak current cannot be removed pharmacological its linear properties permit a digital subtraction approach to be used The current signal can be considered to consist of 3 components LO L 0 L 0 L 12 1 where 1 is the time dependent voltage activated ionic current under study Ij is the leak current and t is transient capacity current due to the charging and discharging of the membrane capacity l t and Jj are always present in the signal and scale linearly with the size of the voltage step However J t only occurs for voltage steps to potentials which activate the voltage sensitive ion channels The Na current for instance is only evoked by depolarising voltage steps to potentials more positive than 60mV It is possible to obtain a record
9. Select the type of records to be measured by selecting an option Ch fenom y from the Type list Select ALL to measure records of any type Type aLL except rejected records Select Manual or Automatic Data Cursors mode In manual mode the region within the signal to which the curve is to be fitted is set manually using a set of 3 cursors on the display In automatic mode the curve fitting region is set automatically Data Cursors te Manual f Automatic If you have selected Automatic Data Cursor mode select On Rise On Decay or Rise Decay to determine whether the cursors are to be placed on the rising phase decaying phase or complete time course of the signal waveform Then enter the levels on the waveform where the cursors are to be placed in the Limits box Default setting is 10 90 placing the cursors at 10 and 90 of peak amplitude on the selected phase If you have selected Manual Data Cursors mode define the curve fitting region by using three vertical cursors two blue marked define the region to which the equation is to be fitted the third green marked t0 defines where the zero time points for the equation is Note that the choice of fitting region depends upon the kind of curve being fitted Sometimes only part of the signal is chosen such as when an exponential curve is to be fitted to the decay phase of the signal In the example shown here the fitting region cursors have been placed on t
10. WinWCP V3 9 User Guide 30 7 1 Trigger modes In general recording sweep s must be synchronised with the start of the signals under study to ensure that the signal is captured within the record and always appears in the same place The trigger mode determines how this synchronisation takes place There are 4 modes Free Run External Trigger Event Detector Stimulus Program You must select a trigger mode appropriate to the type of signal to be recorded and the configuration of your recording system 7 1 1 Free Run The Free Run trigger mode is used for unsynchronised recording Recording sweeps start immediately after the Record button is pressed and continue until the required number of records have been collected Choose the free run mode for simple tests of the laboratory interface and for signals such random ion channel noise where synchronisation is not possible or required 7 1 2 External Trigger Many kinds of electrophysiological signals are evoked by stimulating the cell or tissue using an electrical stimulator In order to record such signals the recording sweep must be synchronised with the stimulator ideally so that the sweeps starts shortly before the cell is stimulated The External Trigger mode links the start of recording sweeps to a trigger pulse applied to the Ext Trigger input of the laboratory interface Choose the External Trigger mode when you are using a stimulator to evoke the signals under study
11. WinWCP V3 9 User Guide 57 11 6 1 Assessing the quality of a curve fit Iterative curve fitting is a numerical approximation technique which is not without its limitations In some circumstances it can fail to converge to a meaningful answer in others the best fit parameters may be poorly defined It is important to make an assessment of how well the function fits the curve before placing too much reliance on the parameters 11 6 2 Does the chosen function provide a good fit to the data One assessment of the goodness of fit is to compare the variance of the residual differences between the best fit function and the data with the background variance of the signal If the function provides a poor fit to the data the residual variance will be significantly greater than the variance of the random background noise on the signal The distribution of the variance as displayed in the residuals plot is also important Deviations should be randomly distributed over the fitted region of the record If the fitted line is consistently higher than the data points in some parts and lower in cthers this indicates that the signal is not well represented by the chosen equation 11 6 3 Are the parameters well defined The aim of most curve fitting exercises is to obtain a well defined set of function parameters e g exponential time constants which characterise the part of the signal being fitted The standard errors of the best fit parameters provide an
12. 4 Connecting WinWCP to your experiment The first step in making a digital recording is to connect the signal outputs from your electrophysiological amplifier to the appropriate analogue inputs of the A D converter in your laboratory interface unit If you plan to use the computer to apply voltage pulse stimuli to the cell you must also connect a D A converter output to the command voltage input of the voltage clamp You may also have to supply a digital trigger pulse to initiate each recording sweep Depending upon the absolute levels produced by the recording device you may need to amplify the signal to make it compatible with the input requirements of your A D converters You may also need to low pass filter the signal and or apply a DC offset to the signal before it can be digitised Two typical recording situations are discussed below 4 1 Example 1 Connecting WinWCP to a patch clamp One of the most common applications for WinWCP is recording from and controlling a whole cell patch clamp experiment Two analogue channels are normally recorded membrane current and voltage and computer generated voltage pulses are applied to the patch clamp command voltage input to stimulate the cell The patch clamp is connected to the computer as follows Ext Trig y Sync Out Patch Clamp V Vcom Out An In Ch 0 An In Ch 1 An In Ch 2 I O Panel Consult the signal connections table for your particular laboratory interface in sections
13. Note the Sync Pulse output of the stimulator must be connected to the external trigger input of the laboratory interface for triggering to occur During a stimulus cycle the sync pulse is produced first triggering the recording sweep and after a delay settable on the stimulator front panel the stimulus itself 7 1 3 Event Detector The event detector mode provides a means of detecting signals as they occur within an incoming analogue signal A threshold based event detection algorithm monitors the incoming signal on one of the input channels An event is detected when the signal deviates by more than a predetermined level from the average baseline level To compensate for slow drifts in the baseline level the threshold level is maintained at a constant distance from the baseline by means of a running average calculation The event detector is configured by setting three parameters If more than one channel is being recorded select the input t Event Detector channel on which events are to be detected from the detection Ch E pea list box eht im Threshold Enter the detection threshold into the Threshold box The L threshold level is expressed as a percentage of the total input range Fretrigger with its polarity determining whether positive or negative going i signals are to be detected The level should be set as small as possible to maximise the likelihood of an event being detected but wit
14. WinWCP V3 9 User Guide 40 Individual channels can also be removed from restored to the display by selecting the menu View Ch lt n gt lt channel name gt 9 3 Printing records To print the records displayed on the screen select File r Calibration Bars Print Typeface arial Size 12 pts To open the print record dialog box Line Width 2 pts Show zero levels M You can set the size of the plotted record on the printed page by adjusting the size of the page margins Use colour Show labels ri The type face used to print text can be selected from Fage Margins the font name list and the type size entered into the Left Right font size box The thickness of the lines used to draw OK Cancel 5 0 cm 60cm Top Bottom 5 0 cm 10 0 cm the signal traces can be set using the line thickness box Vertical and horizontal calibration bars are added to the plot to indicate the units and scaling of the plotted signals You can define the size of the bars by entering values into the calibration bars table The position of the zero level for each plotted trace is indicated by a horizontal dotted line Zero levels can be disabled by un checking show zero levels Plot labelling can be disabled by un checking the show labels check box The use of colours within the plot can be disabled by un checking Use colour When all plot parameters have been set click the OK button to initiate printing
15. WinWCP V3 9 User Guide 42 9 8 Zero levels The signal zero level for a channel can be defined in either of two ways In From record mode it is computed as the average level from a defined portion of each record In Fixed mode it is fixed at a level defined by the user and does not vary from record to record 9 8 1 From record mode x If you want to compute the zero level from a portion of the signal record itself a zero level modes a Move the mouse pointer over the horizontal zero level cursor of the slants channel you want to change The mouse pointer turns into an No averaged up down arrow b Slide the mouse pointer horizontally until it overlies the region of the C Fixed Level record which is to be defined as the zero level E c Click the right hand mouse button to open the zero level dialog box ox cancel d Select the From Record option e The zero level is computed for each record from the average of a series of default 20 samples starting at the sample indicated in the At sample box If you want to change the number of samples averaged to compute the zero level change the value in the No averaged box f Click the OK button to use the new zero level Choose the from record mode when you want to measure transient signals which are superimposed upon a baseline level which may be varying from record to record Synaptic currents such as endplate currents or excitatory postsynaptic currents in n
16. click on the Inactivation properties page To enable the inactivation parameter V halt v slope Tau y halt check the Inactivation in use box Enter the voltage at which the inactivation parameter is at 0 5 in the V half box Enter the inactivation time constant when the membrane potential is at V half in the Tau V half box Enter the voltage sensitivity in the V slope box large values weak voltage sensitivity inactivation IV Inactivation in use h h inf h inf h 0 exp t Tau h 14 Click the Start Simulation button to start the simulation run The Hodgkin Huxley simulation can be used to test the leak subtraction module and the Hodgkin Huxley functions in the curve fitting module WinWCP V3 9 User Guide TI 19 3 Miniature EPSC simulation The miniature EPSC module generates simulated miniature postsynaptic currents exhibiting the stochastic fluctuations associated with the current flow through the population of ion channels opened by a single quantum of transmitter The gating of a single post synaptic ion channel is represented by a simple 4state model Binding of an agonist molecule A with receptor R to form an agonist receptor complex makes 1t possible for the channel to shuttle between a closed state AR an open state AR and a closed desensitised state AR K vind gt k en gt K des gt D A R AR AR AR Tk Tk amp kj unbind close The single channel current time course is gov
17. containing only leak and capacity currents by using a hyperpolarising voltage step or a small depolarising step L L t l 12 2 Scaling this record to account for the differences in the size and or polarity of the voltage step and subtracting it from the test record effectively removes the leak and capacity currents 7 a 12 3 LO 1 0 1 0 V Since the scaling up of small subtraction records also scales up the background noise it is usual to average several subtraction records before scaling and subtracting It is also possible to average the test records WinWCP uses the following general algorithm NV s j l i Vo I t Y L it I t 12 4 Oe L KO Y 1 0 where M is the number of test records averaged and N the number of subtraction records 13 1 Recording protocols for leak subtraction One of the most commonly used leak subtraction protocols is the P N protocol developed by Bezanilla amp Armstrong 1977 For each depolarising test pulse there are N additional subtraction pulses evoked by hyperpolarisng pulses 1 Nth the amplitude of the test pulse WinWCP s stimulus generator can be configured to produce the necessary sequence of test and leak subtraction recording sweeps by selecting the P N Mode leak subtraction option see section 8 This causes the stimulus generator to produce additional scaled down and inverted stimulus pulse waveforms for evoking the linear leak currents without the voltage ac
18. shown on the right shows the distribution of peak amplitudes for a series of 200 simulated endplate currents see section 19 1 It consists of 50 equaksized bins over the range O to 15 nA i e a bin width of 0 3 nA The height of each bin represents the number of records containing a signal with a peak amplitude falling within that bin range 10 7 1 Customising histograms If you want to alter the X or Y axis range scaling or labels click the Set Axes button to open the Set Axes Range Labels dialog box Axis limits and tick spacing are initially set to default values based upon the min max range of the data You can change the axis limits by entering new values for into Min Max and Tick spacing boxes for the X and Y axes An axis can be made Linear or Logarithmic by selecting the option from its Scale list Labels for the X and Y axes and a title for the plot can be entered into the Labels boxes nm Pedi A m Histogram Peaki Channel enom y Wo bins 50 Range g Lower Jo Upper 15 CO Percentage Co Cumulative a ati Bin yin OH anma E mE The style of rectangle used to plot the histogram bins can be changed using the Bin Style WinWCP V3 9 User Guide 50 options Select No Fill to display bins as rectangular outlines Solid Fill to fill the bins in with a solid colour and Hatched Fill for bins filled with a diagonal lines You can define the
19. 23 02 98 log experimental log select 1 09 32 49 WinvvCeP Started File 09 33 26 WinWwCP Started Inspect Log File 09 33 40 Mew file CADELPHMAWINVOPINAS M we po 09 33 44 Hodgkin Huxley Voltage Clamp Simulation 09 33 44 GMax 10 0 n5 4 09 33 44 GLeak 0 5 n8 09 33 44 GSeries 100 0 nS 09 33 44 Cm 10 0 pF 09 33 44 VRev 40 0 rr 09 33 44 mv half 25 0 mv H 09 33 44 mVoslope 10 0 rm 09 33 44 m Tau 1ms 09 33 44 m Power factor 3 0 09 33 44 h Y Half 30 0 mv 09 33 44 h Y Slope 10 0 mv 09 33 44 h Tau 10ms 09 33 48 16 records WinWCP V3 9 User Guide 74 19 Simulations The simulation modules can be used to generate data files containing simulated waveforms with known characteristics which can be used to test the operation of the measurement and analysis modules Three kinds of waveform can be simulated nerve evoked EPSCs voltage activated ionic currents and spontaneous miniature EPSCs 19 1 Nerve evoked EPSCs The nerve evoked EPSC module generates a series of nerve evoked excitatory post synaptic currents EPSCs or potentials EPSPs When the nerve is stimulated a random number of transmitter quanta are released from a pool of size n with each quantum having a probability p of release The number of quanta released per stimulus follows a binomial distribution The EPSC waveform can be made to decay following a single or double exponential function Random background noise with a gaussian distribution can be added to t
20. 4 Measurement variables The sequence number of the record within the data file Group The number of the group to which the record belongs used by leak subtraction module The time relative to the first record in the file that the record was acquired Average The average signal level within the analysis region The integral of the signal level within the analysis region Peak The peak absolute positive or negative depending in peak measurement mode used signal level within the analysis region The variance of the signal within the analysis region Rise Time The time taken for the signal to rise from lo hi 10 90 default of peak time units The maximum rate of rise during the rising phase of the signal Latency The time delay between the zero time cursor and the point at which the signal has risen to 10 of peak T X The time taken for the signal to fall from its peak value to X set by user of peak T 90 The time taken for the signal to fall from its peak value to 10 of peak Baseline Signal level computed from the zero level measurement region but measured relative to true zero levels of input channel WinWCP V3 9 User Guide 46 10 5 Plotting X Y graphs of measurement variables The X Y Plot page can be used to create graphs of the measurements obtained from the analysis run Any measurement variable from any channel can be plotted against any other as a Y vs X graph To plot a graph 1 Sel
21. 5 1 Creating a data file To create a new data file to hold your New Data File El ES recordings select from the menu Sawein E wrwcr Ea FE al el yprot laa Power 1401 Digtest wep WinRT 95 a Power 401 Digtest wep a Zexp1 00ch wep Simulated 2 exp 50 channels wep a Micro 1401 Digtest wep a Simulated single exponential mepsc a Micro1401 Digtest wep Simulated two exponential mepsc w la NS Variance Test wcp test 1 wep File New To get the New Data File dialog box shown here El Select the disk and folder into which the file teme is to be placed using the Save In list box Save asiype wc Files WCF Cancel WinWCP data files have the extension extension wcp 5 2 Setting recording parameters To set the number of channels to be recorded recording Aec a Cierra coh bra lios fable i Reca Duran Ch Ame Wants Wns duration and other parameters select the option Ei CN Setup n Recording Sweep roll sacl to display the Setup dialog box Samping Int Vota gee Bige Trace color MN plien P Tiana O LES 5 2 1 No Channels raid A anal indicar MIA Sets the number of analogue input channels you intend on Sane i to record from WinWCP supports a maximum of 8 channels Channels are always acquired in sequence from Ch 0 upwards 1 e No Channels 1 selects Ch 0 No Channels 2 selects Ch 0 amp Ch 1 etc WinWCP V3 9 User Guide 25 5 2 2
22. 64 15 1 QUANTAL CONTENT DIRECT METHOD ccc ccccssssscceessssccecessssceccessssccesssseeccesseesceessseeeeeesseeeeeeeeas 64 15 2 QUANTAL CONTENT VARIANCE METHOD ad 64 15 3 QUANTAL CONTENT FAILURES METHOD at idad 64 15 4 BINOMIAL ANALY SIS a diia 64 15 5 CORRECTION FOR NON LINEAR SUMMATION OF POTENTIALS cc scccccssssceccesssseccessseeceessseeeeees 65 15 6 QUANTAL CONTENT CALCULATION PROCEDURE cccsssscccessssceceesssseccessseeccessseeeceessseeeeessaeeeeeeeas 65 16 SYNAPTIC CURRENT DRIVING FUNCTION ANALYSIS occcncccnccnoocinocsonsoaccnccorcconoconocens 67 17 EDITING DIGITISED SIGNAL RECORDS coocnccinccnocioocnocoococcnccoccocconcorocoroconoconocon ccoo cconcconcnnss 69 17 1 SHIFTING THE SIGNAL HORIZONTALLY cccccccessssecceesssccecesssececcessseccesssseeccesseeeceessseeeeessaeeeeeeseas 69 17 2 SHIFTING THE SIGNAL VERTICALES o 69 17 3 SCALING TAE SIGNAL ra te api 69 17 4 STIMULUS ARTEFACT REMOVAL 00d AA tt 69 17 5 UNDOING OR ACCEPTING CHANGES cuidas 69 18 DATA PILES iii ia a E a 70 18 1 OPENING A EXISTING WCP DATA FIES 0 A ii 70 18 2 APPENDINGA WCEP DATA REE aida 70 18 3 IMPORTING FROM FOREIGN DATA FILE FORMAT S ccccccconononnnnncnnnnnnnnnnnncnonannnnnnnncononnnnnnonocnnnnnnnnnncnnnnns 70 HOLE AROMAS UIC SA AS 71 18 3 2 Cambra ge Electronice Deoh is 71 TOD BAS CLIT CM INES sna saa O aed hl da salah aa 71 POSSI BUA OL TINS ai cess aed lee lena E acne da de ate a a A 72 18 4 EXPORTING
23. I P See Note 2 Digital sync in PEO A DigitalOu IS EN Digo PAOG l4e l3 PAL S 15 9 13 LES Diga PA2 s l6g 13 LA Dig3 JPA E AAA EN SAA OOOO PAS s 19 9 13 PAG 5 20 0 13 PAT 6218 13 ig 17 0 PA4 s 18 8 13 19 19 9 Note 1 Sync Pulse Out must be connected to Recording Sweep External Trigger I P when recording signals using the Stimulus Program trigger mode and for the Seal Test option For 1200 series boards only Sync Pulse Out must also be connected to Digital Sync In when the digital output pattern options Dig 0 Dig 7 in the pulse protocol are in use Note 2 An active high TTL pulse on this input triggers the start a stimulus program which has been set up with the External Stimulus Trigger Y option 1200 Series boards need the pulse to be 10 ms in duration or longer 2 5 3 Troubleshooting WinWCP V3 9 User Guide 13 National Instruments cards can be used with a number of different types of input output panels BNC 2090 BNC 2110 or CB 68 terminal panel and can also be configured in software to handle the analogue input channels in a number of different ways differential referenced single ended and non referenced single ended Some combinations of setting can lead to signal levels apparently drifting or going off scale In differential mode the boards analogue input channels are paired together and subtracted e g Ch 0 Ch 7 Ch 1 Ch 8 etc In referenced single ended mode
24. Record Duration Sets the default duration of each recording sweep Set it to a value that is approximately 50 longer than the time course of the signals that you intend to record 5 2 3 No Samples Channel Sets the number of samples to be acquired per input channel within the recording sweep The minimum is 256 samples per channel and increments are in units of 256 The maximum is 29952 No Channels Note that the more samples acquired per record the larger the size of the data files produced 5 2 4 Sampling Interval Displays the time between A D samples acquired from each input channel It is determined by the Record Duration and the No Samples Channel Record Duration Sampling Interval _ No Samples C hannel It is important to choose a sampling interval which is small enough to ensure that a sufficient number of samples are acquired during the most rapidly changing phases of the signals being recorded For most types of signal 1024 samples channel and a record duration approximately 50 longer than the signal time course provide satisfactory results However note that some signals such as cardiac ventricular action potentials can combine long time courses 200 300 ms with very rapid rising phases 1 2 ms In such circumstances 8192 or more samples channel might be required to accurately represent the rising phase As discussed in section 4 2 to avoid aliasing artefacts the analogue signals should be
25. The software is supplied as found and the user is advised to verify that the software functions appropriately for the purposes that they choose to use it An acknowledgement of the use of the software in publications to which it has contributed would be gratefully appreciated by the author John Dempster Department of Physiology amp Pharmacology Strathclyde Institute for Biomedical Sciences University of Strathclyde 27 Taylor St GLASGOW G4 ONR Scotland Tel 0 141 548 2320 Fax 0 141 552 2562 E mail j dempsterOstrath ac uk WinWCP V3 9 User Guide 6 2 Introduction amp Software Installation WinWCP is a data acquisition and analysis program for handling signals from whole cell electrophysiological experiments These may include whole cell patch clamp experiments single and two microelectrode voltage clamp studies or simple membrane potential recordings Whole cell signals are produced by the summation of currents through the usually large population of ion channels in the cell membrane and thus consist of relatively smooth current or potential waveforms The amplitude and time course of such signals contain information concerning the kinetic behaviour of the underlying ion channels and other cellular processes which can be extracted by the application of a variety of waveform analysis techniques WinWCP provides in a single program the data acquisition and experimental stimulus generation features necessary to
26. also supported by the pCLAMP electrophysiology software package Inputs to and outputs from the board are via BNC connectors on an I O box connected to the board via a shielded ribbon cable In order to use WinWCP with a Digidata 1200 the following computer system resources must be available for use by the Digidata 1200 e I O port address 320 33F Hex e DMA channels 5 and 6 WinWCP currently supports the Digidata 1200 under Windows 95 and 98 only Windows NT and 2000 support is not available at present 2 6 1 Software Installation 1 Install the Digidata 1200 card into an ISA computer expansion slot and attach it to its BNC I O panel using the shielded ribbon cable supplied with the card 2 Click the Windows Start button and select Install Digidata 1200 driver Win95 98 from within the WinWCP group in the Programs menu Follow the instructions to install the Digidata 1200 Windows device driver WinWCP does not use the standard Axon Instruments Digidata 1200 device driver 3 Reboot the computer 4 Run WinWCP and select from its main menu Setup Recording Select Axon Instruments Digidata 1200 from the Laboratory Interface list box WinWCP V3 9 User Guide 15 2 6 2 Signal input output connections Signal input and output connections are made via the BNC sockets on the front and rear of the Digidata 1200 I O box Analogue inputs as oo Sanog SSS S Analogue oupuis YO Command voltage out AnalogueOw0 Trig
27. and enter the number of that channel in the Telegraph Ch Box 3 Enter the minimum gain setting of the patch clamp in the V Units box for Ch 0 If a CED 1902 computer controllable amplifier is in use this option can also be used to read its gain setting No Telegraph Ch is required in this case WinWCP V3 9 User Guide 27 6 Monitoring input signals amp patch pipette seal test After a data file has been created and input channel parameters defined you can monitor the signals appearing on each channel using the signal monitor pipette seal test module This module provides a reaktime oscilloscope display and digital readout of the signal levels on the cell membrane current and voltage channels Co de ee bed ee miam Se ee A test pulse can also be generated for monitoring pipette resistance in patch clamp papm experiments ja To open the monitor seal test module select EE Lt from the menu E Bee Record r e Monitor Pipette Seal Test ES eee TS An oscilloscope trace showing the current signal on each input channel is displayed ee ee ee eea O RT 6 1 Select current and voltage channels Select the input channels which contain the current and voltage signals by selecting the current channel in the current list Current and the voltage channel in the voltage list Voltage 6 2 Command voltage divide factor Most voltage and patch clamp amplifiers divide down their p com Divide Factor c
28. and FAIL types are used in the quantal analysis of synaptic currents or potentials see section 15 to indicate whether a record is a nerve evoked spontaneous or a nerve evoked transmission failure event Note Types can be selected quickly using the Ctrl T Ctrl L Ctrl E Ctrl M Ctrl F keys TEST and LEAK are used in the digital leak subtraction process see section 13 and used to distinguish respectively normal records containing voltage activated currents and records containing leak currents to be scaled and subtracted from the TEST records TYP1 TYP2 and TYP3 are general purpose user defined record types 9 7 Cursor measurement of signal levels To measure the signal at any point on the displayed record use the mouse to drag the vertical readout cursor to the desired part of the trace Fine positioning of the cursor can be achieved by pressing the lt or gt arrow keys with the mouse pointer over the selected cursor The signal level of the trace s at the cursor position is displayed at the bottom of the window below the cursor Time measurements are made relative to the start of each record and in brackets relative to the location of the t 0 cursor Signal levels are measured relative to each channel s horizontal zero level cursor Cursor measurements can be written to the WinWCP log file by clicking the Save F1 button The Centre Cursor button places the readout cursor to the centre of the displayed region
29. and its correction can be found in McLachlan amp Martin 1981 15 6 Quantal content calculation procedure The following procedure can be used to calculate the quantal content of a series of synaptic currents or potentials which have been recorded and stored in a digitised data file 1 Using the record display module inspect each record in the data file and classify it as being either a nerve evoked signal EVOK a spontaneous miniature event MINI or Gn experiments where the probability of transmitter release 1s low a nerve stimulus which has failed to release any quanta FAIL You should also mark any records containing artefacts as REJECTED 2 Use the waveform measurements module to calculate the waveform parameters for ALL of the records with the intention of measuring the signal peak amplitude Take care to exclude the nerve stimulus artefact 3 Select Mural Ars Analysis Ma Quantal Content q Binaria Record Fangs invoke the quantal analysis modul P Baran to invoke the quantal analysis module sto j 1 Select the type of analysis Choose Poisson eem if there are no MINI records available and Evoked ewani Tea TEST i Guardun avent expected to be low Otherwise choose stent the transmitter release probability is sale re Binomial Uger anami Aum pd A Moda Ps E h f d b d s h AAA cenizas Rasing Fea re El Comardon Enter the range of records to be used in the na PMO Soni pod Fur anal
30. and time dependent open probability of the nicotinic acetylcholine receptor channel Biophys J 57 723 731 Gillis K 1995 Techniques for membrane capacity measurements In Single channel recording 2 ed ed B Sakmann amp E Neher Plenum Press McLachlan E M amp Martin A R 1981 Non linear summation of endplate potentials in the frog and mouse J Physiol 311 307 324 Sigworth F J 1980 The variance of sodium current fluctuations at the node of Ranvier J Physiol 307 97 129 Standen N B Gray P T A amp Whitaker M J eds 1987 The Plymouth Workshop Handbook The Company of Biologists Ltd Cambridge Traynelis S F Silver R A amp Cull Candy S G 1993 Estimated conductance of glutamate receptor channels activated during EPSCs at the cerebellar mossy fibre granule cell synapse Neuron 11 279 289 WinWCP V3 9 User Guide 719 21 Appendix WCP data file structure This appendix provides a detailed specification of the internal structure of the WCP data file The WCP data file is designed to store digitised 16 bit integer binary records of analogue signals the associated scaling information required to reconstitute actual signal levels validation information entered by the user and measurements generated by WCP analysis modules A WCP file can contain up to 2 separate records assuming there is sufficient disk space each record containing up to 8 channels and each channel containing a multipl
31. be entered in the box Amp Gain 0 0070 vipa Note The laboratory interface Sync Pulse output must be connected to the External Trigger Input for test pulses and monitor sweeps to be generated see section 4 1 If you wish to monitor input signals without generating test pulses check the Free run check box allowing sweeps to occur without the Sync Pulse External Trigger connection WinWCP V3 9 User Guide 29 7 Making a recording After a data file has been created and an appropriate set of recording parameters defined select Record Record to disk to enter the recording module The display area of the screen acts like a digital oscilloscope showing traces of the signals as they are recorded Ermy E LITE EA EAT abp LETE Acro ders ee a To collect a series of records 1 Enter a line of text identifying the purpose of the recording in the Ident box optional 2 Enter the number of records to be collected in the Records Required box A Records 500 3 Set the trigger mode see below for details Trigger Mode Free Run t External Trigger C Event Detector Ch cnom Threshold Pretrigger 30 0 Hpi 7 Stimulus program STEPS 4 Make sure that the Save to File box is checked 5 Start recording by clicking the Record button 6 If you want to stop recording before the number of records in the Records Required box have been collected click the Stop button
32. be used to compute the driving function from synaptic current records such as endplate currents To carry out a driving function analysis 1 2 3 4 5 6 1 8 9 Record a series of stimulus evoked synaptic currents Use the signal averaging module to create an average synaptic current from the set of raw records Use the curve fitting module to fit an exponential function to the decay phase of the averaged synaptic current Fit the function from around 95 5 of the decay phase excluding the 5 around the peak where the transmitter is still being release Racor To open the driving function module select Renan cae Analysis Bossi Driving Function Do Teseestorars If there are several channels in the record select the channel containing the signal to be Aicard transformed from the Ch list ae Polntiat Set the range of records to be transformed Select All Records for all the records in the file This Record for the currently displayed record only or select Range and enter a um carios range of records into the box Enter the cell holding potential in mV that the currents were recorded at in the Holding Potential box and the reversal potential of the post synaptic current in the Reversal Potential box The driving function is expressed in units of conductance unit time The holding and reversal potentials are required to convert from current to conductance T
33. colour wed for the solid fill by clicking the Colour box and selecting a colour from the palette The Full Borders check box determines whether the outline is drawn completely around each bar or just where bars do not overlap 10 7 2 Printing the histogram To print the displayed histogram select 9 Le el Fil arial We Print Point Size E pts To open the Print dialog box Click the Print button to L line Trickness 2 pts plot the graph Y Cancel Page Margins Lett Snem Right 50m ston 10 7 3 Copying the histogram data points to the Windows clipboard The numerical values of the X Y data points which generate the histogram can be copied to the clipboard by selecting Edit Copy Data The data is placed on the clipboard as a table of data values in tab text format defining the histogram There are 4 values per row and one row for every bin in the histogram Each row has the format lt Bin Lower Limit gt lt tab gt lt Bin Mid point gt lt Bin Upper Limit gt lt tab gt lt Bin Count gt lt cr gt lt lf gt 10 7 4 Copying an image of the histogram to the Windows clipboard An image of the histogram plot can be copied to the clipboard by selecting Typeface Image Size Ed it arial width es 600 pixels Copy Image Point Size 12 pts i i gt Lines 500 pixel to open the copy image dialog box Uie Thickness 7 pis 500 pixels The dimensions pixels of the bit map which will ho
34. for the active display record can be copied to the clipboard by selecting Edit Copy Data The data is placed on the clipboard as a table containing the scaled values for each sample in the record in the measurement units defined for each channel The table is stored in tab text format allowing the data to be copied into programs such as spreadsheets and graph plotting packages using an Edit Paste command Note that die to limitations in the capacity of the Windows clipboard data points may be skipped to keep the size of the copied record within clipboard storage limits 9 9 2 Copying the displayed image The signal record s on the display can be copied to the clipboard as a bit mapped image by selecting Edit Copy Image Ce arial to open the copy image dialog box Size 12 pixels The dimensions of the bit map which will hold the image Line Width 2 pixels can be set using the width and height image size boxes The Show zero levels Y A 3 r Use colour E more pixels used in the bit map the better the quality of the Show labels Y image Calibration bars zero levels and text font size and ey line thickness can be set in the same way as for a printed With image OK Cua 1000 pixels Height 1000 pixels When the image parameters have been set click the OK button to copy the image to the clipboard 9 10 Smoothing the displayed records A digital low pass filter
35. form of keywords one word per line as follows KEY lt value gt lt cr gt lt lf gt where lt value gt is a number or text depending on the parameter and lt cr gt lt lf gt are the carriage return and line feed characters WinWCP V3 9 User Guide 80 A typical header block from a file with 2 channels contains the following keywords VER 6 4 lt cr gt lt lf gt NE SZ sers lT NR 50 lt ers lt Lt gt NBH 2 lt cr gt lt lf gt NBA 1 lt cr gt lt lf gt NBD 4 lt cr gt lt 1f gt AD 5 0000 lt er gt lt Lf gt ADCMAX 2047 lt cr gt lt lf gt NP 512 lt cr gt lt lf gt DT 1600 lt cr gt lt lf gt NZ 10 lt cr gt lt lLf gt YNO mM lt er lt f gt YUO 0A LOr lt li gt Y SO 146F 02 lt cr gt lt LE gt YGU 167E 04 lt cr gt lt LE gt YA0 1997 lt 6r gt lt L gt YOO 0 lt cr gt lt 1f gt YRO 2 YN L VmM lt cr gt lt LE gt VULSmy Cire lt li gt YSi 244 lt er gt lt li gt YGL 104 0 lt GrE gt lt Lf gt YZI 2046 lt cecrS lt I gt YO1 L lt cr gt lt li gt YR1 0 lt cr gt lt l1f gt TUSmMS SEC bi ID Cell G lt er gt lt LI gt WCEP version number No of channels per record No of records in the file No of 512 byte sectors in file header block No of 512 byte sectors in a record analysis block No of 512 byte sectors in a record data block A D converter input voltage range V Maximum A D sample value No of A D samples per channel A D sampling interval s No o
36. indication of this A large standard error indicates that a parameter is poorly defined by the data and can be varied significantly with little effect on the goodness of fit Such a situation typically arises when there is insufficient information contained in the signal waveform to adequately define the function For instance in the case of exponential functions the waveform data must be of sufficient duration to contain at least one time constant of the exponential function before an accurate estimate can be obtained Similarly it proves difficult to accurately estimate the time constants of multiple exponential functions when they differ by less than a factor of 5 It is worth noting that the parameter standard errors discussed above are computed from the Hessian matrix by the curve fitting program and are not true estimates of experimental standard error since they take no account of inter cell other variability In addition they only provide a lower bound to the estimate of the standard error in parameter value It can be shown by simulation that if the random noise on the experimental signals is correlated then the variability of fitted parameters may be substantially greater than suggested by the computed parameter standard error The error in parameter estimation can be a complex function of the parameter values and the signabnoise ratio of the data It is therefore wise to test the curve fitting procedure using simulated waveforms wit
37. its main menu Setup Recording If you are using Instrutech s old device driver software as supplied with the EPC 9 and downloadable from www instrutech com select Instrutech ITC 16 Old Driver OR Instrutech ITC 18 Old Driver from the Laboratory Interface list box depending upon which interface you have installed Note Instrutech have also introduced a new software driver and library which supports the ITC 16 ITC 18 and ITC 1600 If you are using this library select Instrutech ITC 16 18 New driver from the Laboratory Interface list box 2 5 3 Instrutech ITC 16 18 Troubleshooting WinWCP requires Instrutech s combined device driver library ITCMM DLL released late 2001 It may not work with earlier libraries WinWCP V3 9 User Guide 20 3 Using WCP An Overview Ca dc o di Fa al e Ps ig we Diii Phat Pi fie Ede No Boi a e e Help WinWCP consists of a variety of program modules for recording and analysing wiew ec era a electrophysiological signals These wa 1 An UA 3 i modules are accessed via the main program a mm ae a 2 menu on the program s title bar and appear N as independent sub windows enclosed oe ere AMAT within the main WinWCP window a la E T i PP e R pan I I CN The File menu provides the standard Windows functions for creating opening E and closing data file printing and import ee ee and export to non native data formats A a jii O AS The Edit menu
38. permits data to be copied to the Windows clipboard The View menu provides options for magnifying and selecting the type of record being displayed The Record menu invokes the digital recording module for recording analogue signals to disk and the seal test module for monitoring the sealing of patch pipettes to cells The Setup menu provides options for setting the numbers of analogue channels to be recorded recording sweep duration and other parameters voltage clamp command voltage stimulus patterns and controlling external amplifiers The Analysis menu provides access to a range of analysis modules which can be applied to the digitised signals stored on file These include e A waveform analysis module for the automatic calculation of waveform parameters peak and average amplitude area rise time rate of rise time to 50 and 90 decay and variance Results can be plotted as X Y graphs or as histograms e A curve fitting module for fitting exponential and other curves to waveform transients e A signal averaging module e A leak subtraction module e A non stationary variance analysis module e A quantal content analysis module e A driving function analysis The Simulations menu provides access to modules for creating simulated end plate currents and voltage activated sodium currents The Windows menu selects between active windows The Help menu provides access to the WinWCP Help files WinWCP V3 9 User Guide 21
39. the records inverted or scaled in amplitude Regions of the record containing stimulus or other artefacts can also be blanked out To modify the digitised signal contained in a record select Analysis Signal Editor to open the Edit Record module Records can be displayed using the Record selection slider bar Select the signal channel to be edited from the Channel list Select the This Record option to apply editing operations to the currently displayed record only All Records to change all records in the data file or select Range and enter a specific range of records 17 1 Shifting the signal horizontally To shift the signal leftwards or rightwards enter the distance to Shift shifted in time units in the X Shift box and click the Left arrow or Right arrow button to shift the signal ae 17 2 Shifting the signal vertically Y Shif alpina Enter the distance to shifted in the units of the selected signal channel in the Y Shift box and click the Up arrow or Down arrow button to shift the signal 17 3 Scaling the signal Y Scale Scale by Enter the scaling factor in the Y Scale box and click the Scale By button Note Scaling by 1 inverts the signal 17 4 Stimulus artefact removal Stetact Removal Select the region of the signal record containing the artefact using the 394984 Region region selection cursors The limits of the region to be modified are 0 0977 23 8 ms indicated
40. the box When synaptic currents are being analysed select the Scale to Peak option to scale the average current to the peak amplitude of each current record before the subtraction to produce the residual variance Select No Scaling if the unscaled average is to be subtracted Optional If the start of each signal varies significantly from record to record it can be re aligned with average current before subtraction to produce the residual Set the Alignment mode to On Positive Rise for positive going signal and On Negative Rise for negative signals Select the region of the signal waveform the decay phase in the case of synaptic currents to be used in the o t vs I t plot using the analysis region cursors from the X Axis list and Variance from the Y Axis list then click the New Plot button region of the graph to fitted using the analysis region cursors b select Parabola from the curve fitting Ist c click the Fit Curve button d set the initial parameter guesses optional and click the OK button curve fitting results box along with an estimate of the background variance Vb unrelated to channel activity WinWCP V3 9 User Guide 64 15 Quantal analysis of transmitter release The quantal analysis module can be used to estimate the quantal content of neuromuscular nerve evoked endplate currents or potentials and other forms of synaptic signal using the either the direct method variance method and me
41. the fixed scaling factor entered in the box below Fixed mode is required when the record does not contain a voltage channel If you have selected the From Voltage scaling mode use the Vrest and Vyoia display cursors to define the measurement points on the voltage trace used to compute the voltage scaling Vyoig 18 placed over the holding voltage level and Vres 1s placed over the mid point of the test voltage An average of 20 samples around each measurement point is used to compute the voltage levels Select Do subtraction To initiate the leak subtraction process For each group of records the LEAK and TEST records are averaged scaled and subtracted using equation 12 4 Each group is condensed down to one leak subtracted record that is stored in a SUB file with the same name as the data file These records can then be displayed and analysed using the View Records Waveform analysis and Curve fitting modules by selecting View Leak subtracted WinWCP V3 9 User Guide 62 14 Non stationary noise analysis The non stationary noise module analyses the random fluctuations in the decay of ion channel currents providing an estimate of single channel current and total number of channels in the fluctuating population For a cell containing a population of n ion channels each capable of passing a current i the mean whole cell current t is I t i n p t 13 1 where p t is the probability of a channel being open at time
42. 0 10 7 5 Fitting gaussian curves to the NiStO QAM oi ceeccccescccessecsercccesscesssecseseeseceessseessseseseesssasessnesens 51l 10 8 SUMMARIES OF RESUE TS E di 52 10 9 TABUEATING LISTS OERESUL O Sada 52 11 CURVE FITTEN Gum RAROS 53 WinWCP V3 9 User Guide 4 11 1 INTRODUCTION sli ias 53 11 2 FITTING CURVES TO DIGITISED SIGNAL Sec tonna aa a a 53 11 3 RUNNING A CURVE FITTING SEQUENCE cui A anaes 54 11 4 CURVE FIT RESCUE Sonata dd adas 55 11 5 PLOTTING AND TABULATING RESULT S ita 55 11 6 EOUATIOON SS 00d SO areas 56 IlG ASSeSSING the Guality A nreti a nn te ve a doe nS 57 11 6 2 Does the chosen function provide a good fit to the data oooooococinocinocononnnnonorannnnncnnnnono a7 LOS Arethe parameters Well den dt ea EOT 27 1164 Are all the parameters meaningful ruriini A EAEE ees 57 12 SIGNAL AVERAGING cosita siia a a a i 58 12 1 FRINCIPLES OF SIGNAL AVERAGING daa all 58 122 CREATING SONATA VERAGES oomai A AA TON 58 12 3 VIEWING AVERAGED DATA RECORDS cuidas 59 13 DIGITAL SUBTRACTION OF LEAK CURRENTS onnocincccnoccnoncnocorocionccniccorcconocconccrnocannccnonss 60 13 1 RECORDING PROTOCOLS FOR LEAK SUBTRACTION c ccccccssssssseccceeeesssseceeeeeesssseeeeeeeessssseeeeceeees 60 13 2 SUBTRACTING LEAK CURREN Susa Dada 61 14 NON STATIONARY NOISE ANALYSIS cccccsccsieisscescccscecssecscscssscscusttestessscessenstcascstscecsesusssatscaseess 62 15 QUANTAL ANALYSIS OF TRANSMITTER RELEASE occccinococsonssssssssiss
43. 01 must be installed on the computer The installation procedure is as following but see CED documentation for details 1 Install the CED interface card in a PC expansion slot and attach it to the CED 1401 via the ribbon cable supplied or attach to USB port for USB versions 2 Insert the CED 1401 installation CD and run the program SETUP to install the CED1401 SYS device driver and 1401 commands 3 Ensure that the CED 1401 is switched on and then reboot your computer 4 Test the CED interface by running the program c 1401 utils try1401w exe and clicking the button Run Once If the CED 1401 tests check out OK run WinWCP and select from its main menu Setup Recording Select Cambridge Electronic Design from the Laboratory Interface list box Note The latest versions of the above software can be obtained from CED s Web site www ced co uk See Troubleshooting section if you have a CED 1401 with 10V A D or D A ranges WinWCP V3 9 User Guide 9 2 4 2 Signal input output connections Analogue signal I O connections are made via BNC sockets on the front panel of the CED 1401 units WinWCP channel CED 1401 1401 Micro 1401 Analogue inputs Ch 0 ADC Input 0 ADC Input 0 Ch Ch 2 Ch 3 Ch 4 ADC Input Ch 5 ACh ooo Ch ADC Tmpat E Ch7 ACh Sis SSS Analogue ouipuis __ MicrolPower 401 Command voltage out Triggers Y Sync pulse out DAC Output 0 DAC Output 1 See Note 1 Recording Sweep Event Input 4 Trigge
44. 2 4 2 2 5 2 or 2 6 2 and make the following connections The membrane current J output from the patch clamp is connected to WinWCP input channel Ch 0 The membrane potential Vm is fed into WinWCP input channel Ch 1 Voltage stimulus pulses generated at the WinWCP Command voltage output are passed to the Command voltage input V m of the patch clamp The patch clamp gain telegraph output is connected to WinWCP input channel Ch 2 A synchronisation pulse is generated at the WinWCP Sync pulse out and passed to WinWCP External trigger input so that recording sweeps can be synchronised with voltage pulse generation Note This connection is very important otherwise stimulus pulse generation and the seal test functions will not work WinWCP V3 9 User Guide 22 4 2 Example 2 Recording endplate potentials with WinWCP The recording of endplate potentials EPPs from skeletal muscles or EPSPs from neurons presents an experimental situation where some signal conditioning may be required before digitisation Intracellular EPPs are often no more than 510mV in amplitude and miniature EPPs only 1mV Most recording devices such as the Axoclamp Axon Instruments or WPI 705 World Precision Instruments provide no more than X10 amplification and sometimes only unity gin at their voltage outputs Typically laboratory interface units are not sensitive enough to directly measure such small voltages Most of the laboratory interfaces sup
45. AYED RECORDS a ias 43 10 AUTOMATIC MEASUREMENT OF SIGNAL WAVEFORMS essescessesocscescssossosoossessessesse 44 10 1 PREPARATION FOR WAVEFORM ANALYSIG 0csscccsssessecesscesssescoccccccsssscccceccesssssccesscessssssccesscesseees 44 10 2 MAKING WAVEFORM MEASUREMENT Sonnir a a a a 44 10 3 RUNNING A WAVEFORM ANALYSIS SEQUENCE ccccsssssssccccesssccccccccesssssceeeccceeessseeeceeccesssseeeeeceeees 44 10 4 MEASUREMENT VARIABLES ia siii rada 45 10 5 PLOTTING X Y GRAPHS OF MEASUREMENT VARIABLES ccccccccsssssceceessceccessssecccsssseeccesseeeeees 46 10 5 1 CUSTOM SING TES ED cala 46 10 6 CLASSIFYING RECORDS BY WAVEFORM MEASUREMENT CRITERIA ccsssccccesssseccessseeccessseeeeees 46 ROW BPI TTI CVG oasis esata teas anche eae os ia as T sea ashe aaa i 47 10 6 2 Copying the graph data points to the Windows clipboard ooociccnnnnnnnoninnancnnrnnonnnnnnn noes 47 10 6 3 Copying an image of the graph to the Windows clipboard ooooconnnccnnncccnnoncnncnnnnnnnonnnnos 47 1004 Filttinga Curve 1O1Ne PIDA aos 48 10 7 PLOTTING HISTOGRAMS OF MEASUREMENT VARIABLEG csssscccceesessssecececeesssseeceeceesssseeeeeeeeees 49 10 7 1 CUSLONMU STING T STOL TOM S seer dd 49 LOT es TRINS MERITOS Mi aaa 50 10 7 3 Copying the histogram data points to the Windows clipboard oooconiccnnncinnnnnonannnnnnnos 50 10 7 4 Copying an image of the histogram to the Windows clipboard oooooconccinnoconncnnnnnnnnnnnos 5
46. Conductance properties page and enter the maximum conductance for the voltage activated current being modelled in the Max conductance box 7 Enter the reversal potential for the voltage activated conductance in the Reversal potential box 8 Enter the cell s non voltage dependent leak conductance in the Leak conductance box 9 Enter the access conductance of the patch pipette used to patch clamp the cell in the Pipette conductance box Note that if the pipette access conductance is less than 5X the cell membrane conductance then pipette series resistance artefacts will occur 10 Enter the power to which the activation parameter m is to be raised to in the Activation power factor box Default 3 typical of sodium currents 11 Enter the cell capacity in the Cell capacity box This determines the size of the capacity current artifact at the beginning and end of the voltage step 12 If you want to change the voltage sensitivity of the activation Activation parameter m click on the m m inf m inf m 0 expi tTau m Activation properties page Enter the voltage at which the activation ynat V slope Tau v half parameter is at 0 5 in the V half box Enter the activation time constant when the membrane potential is at V half in the Tau V half box Enter the voltage sensitivity in the V slope box large values weak voltage sensitivity 13 If you want to change the voltage sensitivity of the inactivation parameter h
47. Microelectrode ampilter Vcom Out amplifier Low pass filter An In Ch 0 An In Ch 1 I O Panel A differential amplifier has two inputs and and amplifies the difference between them The voltage output from the microelectrode amplifier is fed into the input while a DC voltage level is fed into the input from a potentiometer The resting membrane potential can thus be subtracted from the signal by adjusting the potentiometer The signal is then amplified low pass filtered and passed to analogue input channel O of the laboratory interface The low pass filter 1s used to prevent the artefact of digital recording known as aliasing from occurring by eliminating signal components at frequencies greater than half the A D converter sampling frequency known as the Nyquist frequency Without such filtering high WinWCP V3 9 User Guide 23 frequency components appear falsely superimposed aliased on true lower frequencies For example if a signal is being sampled at a rate of 20kHz the filter cut off must be set no higher than 10kHz Lower cut off frequencies can be used however and act to smooth the signal The low pass filter should be of a design which does not distort the signal time course an 8 pole Bessel filter being a common choice Further details on filtering and other aspects of signal conditioning can be found in Dempster 1993 The circuit also shows a typical situation where a stimulator such as a Grass
48. OLTAGE CHANNELS stes eii 21 6 2 CONIMAND VOL FACE DIVIDE FACTOR cosita lisa 2 6 3 CELL HOLDING VOLTAGE AND TEST PULSES iridica se n n A e 27 6 4 CURRENT AND VOLTAGE READOUT S arra ds 28 6 5 DISPLAY SCALING AND SWEEP FRIGGERIN Ein aia 28 MAKING A2 REC OR DIN Gusana Selene a dani asirios 29 7 1 TRIGGER MODES dida 30 elas VE AE 1 E A IE E o e 30 ji NA ESTER Troe ioe ia Mica a a tar ee Las hah a ples ase 30 WinWCP V3 9 User Guide 3 Fisa EVENT CIO idas 30 7 1 4 Sumu uS PTOS A Ue ca a ea a hernia aa 31 8 CREATING STIMULUS PROTOCOLS essccisc nosoniocionaonaconiocoanosaconccc nacaacicdana cani cecacicacanasadosconiiianis 32 8 1 BUIEDING A STIMUEUS P ROTO COD siieu ida 32 8 2 CREATING A VOLTAGE ST IMULUS WAVEFORM cccsccccssssscccessssccceesseeeeceesseeeceesseeccesseaeeccesseeecees 33 6 2 1 Rectangular voltage pulse Of fixed S1ZC cccccsccccsssecesccesccesssesssseseeesesseeseesssseecsssesseessseeensnees 33 6 2 2 Family of rectangular pulses varying in amplitude ooooconnncnnncnnonannnnnanononnnnnnn non nnonnnnnnno 33 8 23 Family of rectangular voltage pulses varying in dUratl0N oooococcccncnnnnnnnnonnnnonnnnnnnnnnnnnnnno 34 8 2 4 Seres Of TECLANGUIOF VOLAR PUSE T E E 34 8 2 5 AM ea ne ee er ey ee nan er eee ee 34 8 2 6 Dili Sed GHALO GUC WIVEO Ma Sa A 35 8 3 CREATING A DIGITAL ST IMULUS PATTERN a it A EEE 35 8 3 1 DISTA pulse FINCA CQUTOTION espace a 36 8 3 2 Family of digital pulse varying in duration ccccc
49. S44 is being used to excite the nerve of a nerve muscle preparation Digital recording sweeps are synchronised with the stimulus pulses by connecting the Sync Out output of the stimulator to the external trigger input of the laboratory interface Note External trigger inputs are usually designed to be triggered by 5V digital TTL signals while older Grass stimulators produce 12V signals so some form of signal level conversion may be necessary here also Signal conditioning amplifiers and filters are available from a number of suppliers Some examples include the Frequency Devices 902LPF a self contained 8 pole Bessel low pass filter with a differential amplifier input and gains of X1 X3 16 and X10 and tbe Neurolog range of modular amplifiers and filters produced by Digitimer Ltd Computer controllable signals conditioners are also now available such as the Axon Instruments CyberAmp and the CED 1902 WinWCP V3 9 User Guide 24 5 Configuring WinWCP for a recording session WinWCP digitises analogue signals from your experiments as series of discrete records equivalent to oscilloscope sweeps Up to 8 separate input channels can be acquired per record Each record can hold up to a total of 29952 sample points Before making a digital recording for the first time you must do the following 1 Create a data file to hold your recordings 2 Define the number of analogue channels number of samples per channel etc for the recording
50. Strathclyde Electrophysiology Software Whole Cell Program WCP for Windows V3 9 User Guide c John Dempster 1997 2004 12 2 2004 1 Ze WinWCP V3 9 User Guide 2 Contents CONDITIONS OF USE saracii cies sateatsostebssenstiaesscsantascsexssssesueautelacs 5 INTRODUCTION SOFTWARE INSTALLATION eesessosseccessesccsccscoesoesocsoesocsossocooesessosoosoessessesoeso 6 2l INSTALLATION PROCEDUR Pestini dd a tiiease 7 22 INSTALLING THE W IN W CP SOFTWARE ai sissostcasiecncsancseteccsesduscoassescacsuasasnzetasdsenscdessedeumeasapbaawacabeeedandees 7 23s HARDWARE REQUIREMENTS ii 7 2 4 CAMBRIDGE ELECTRONIC DESIGN INTERFACES ccccssssscccessssccceessececeessaceeceessneeccessaeeccssseeecesees 8 2 4 1 SOJLW ONE TASTE a 8 24 2 SLONGLANPUL OULDUL CONTICCH ONS ssoi de 9 2 4 3 UP OUDTCSIVO OLIN SUL Siocon ease chad da is 10 2 3 NATIONAL INSTRUMENTS INTERFACE CARDG ccccccsssssssecccesssssccecceeeessseeeseeeesssseeeeeceesssseeeeeceeses 11 ZIA SONWATE MAHON Ohno al Beast aa a ote a a a a a a 11 LARS SUQNGL npu OULDUG CONVE ONS ca 12 A se TIO LESA bc 13 2 6 AXON INSTRUMENTS DIGIDATA TU eee ait ees 14 ZO de SSOP WAT OAT SUA ON oi assist A A N 14 2 0 2 SLONGLINPUL OULDUL CONVE CONE 15 LEO PU OUDV CSN OOM O aliada 15 Det AXON INSTRUMENTS DIGIDATA 1320 SERIES ccccccccsssssccessssscceeessccccessseeeceesseeccessseeeccesseseecees 16 Ded SONWare INSTA A E 16 Did ozs SONA IN PUL OULDUL CONNEC CONS diia 17 Dle TO
51. TO FOREIGN DATA FILES ati dio esate 13 18 5 EXPERIMENT LOG TELE e cala 73 19 SIMULA TIONS oniiir oaas A A a aaa 74 19 1 NERVE EVOKED EPSG Suetena iS 74 19 2 VOLTAGE ACTIVATED CURRENTS SIMULATION cccececcccsccccccceeeessssssssseeecesecccesecseeessesesnsseeessesees 75 19 3 MINIATURE EPSC SIMULATION accenno ais TI 20 REFERENCES oea eiia a ia eaaa aSa a Ea aa inci 78 21 APPENDIX WCP DATA FILE STRUCTURE 0 c ccccccccscosssesscessecsssesseossecscecscecssecssonseesseess 79 WinWCP V3 9 User Guide 5 1 Conditions of Use The Strathclyde Electrophysiology Software package is a suite of programs for the acquisition and analysis of electrophysiological signals developed by the author at the department of Physiology amp Pharmacology University of Strathclyde At the discretion of the author the software is supplied free of charge to academic users and others working for non commercial non profit making organisations Commercial organisations may purchase a license to use the software from the University of Strathclyde contact the author for details The author retains copyright and all rights are reserved The user may use the software freely for their own research but should not sell or pass the software on to others without the permission of the author Except where otherwise specified no warranty is implied by either the author or the University of Strathclyde concerning the fitness of the software for any purpose
52. ULL SNOOT sis dics pasa bile A N uta lotic iene sien 17 2 8 INSTRUTECH TLC AS adri alla 18 2e Instrutech ITC 16 18 I O Panel Connections necrosi ciiis iaiia 18 2 8 2 Installing software support for the Instrutech ITC 16 1 8 ccccccscccssccessceeseesseessseesseesseeesees 19 2 8 3 Instr tech ll C 10 18 Troubleshootin oanien te WR 19 USING WCP AN OVERVIEW sccssccscssisccssesteasseasscusactasestitascsciacestuvasasssasscisosuiosdsscasenesosunevesiaaseccteawesieds 20 CONNECTING WINWCP TO YOUR EXPERIMENT essessessescesocsoesocsoesossossocccecoccoscoscescoesoesoesse 21 4 1 EXAMPLE 1 CONNECTING WINWCP TO A PATCH CLAMP ccccssssssecesssceccessssecccsssseeccessneeeeees 21 4 2 EXAMPLE 2 RECORDING ENDPLATE POTENTIALS WITH WINWCP doccccccccccncononccccccncnnancncnincnnn 22 CONFIGURING WINWCP FOR A RECORDING SESSION essessesccsoescesossococescssosoceccssesosssesse 24 5 1 CREATING A DATA Eta 24 5 2 SETTING RECORDING PARAMETERS ia 24 IAS A LR CRONE Saren e o E A vsieaisuscheashavevoteabesideeudssecsbaaeteiaas 24 J22 ROCA o aia 25 JA INO AMPLES CRANE lors aa ii ety 23 D2 IMPI STC VL A eva savin tat exes A a wiaevosseceass 23 D2 PPD CONVCTICT Volare KONTE ia sia scale h en a A US 23 5 2 6 IR AREE 1 INEI E AEAEE N A EAE AE EE A AE ONAT E A EA E TE E 25 5 2 7 CranneliC alba OR TADA St 26 5 2 6 TITS AE A N E S 26 MONITORING INPUT SIGNALS PATCH PIPETTE SEAL TES Fooonncinnccnocsnnocoocconocinoccns 21 6 1 SELECT CURRENT AND V
53. algorithm can be used to smooth the Low pass filter displayed signal To enable to filter select a cut off frequency 2048 0 He from the Low pass filter list The low pass filter set in the display module also acts upon the records in waveform measurement curve fitting and other modules Note that the record on the data file is preserved filtering takes place when the record 1s read from the file WinWCP V3 9 User Guide 44 10 Automatic measurement of signal waveforms The automatic waveform measurement module provides a means of automatically making series of standard measurements on the digitised signals Ten basic amplitude and duration measurements can be made on each channel and stored with each record The results for each record are displayed on screen In addition sets of measurement variables can be plotted against each other or compiled into histograms A summary report showing mean value and standard errors for the measurement sets can also be produced 10 1 Preparation for waveform analysis If the results of an automated measurement procedure are to have meaning it is essential to ensure that the data supplied in the signal records are of good quality 1 e free of artefacts or other imperfections Each record in the data file to be analysed should therefore be visually inspected using the record display module see section 9 and rejected if it contains an artefact This is a time consuming but essential process
54. an 3 Select the variables to be included E tap e En gt Laera irmi LI QMII A a 1n the summary by ticking or un Eien T 4 115 ESA Of ja EJ fl 1 1 1 1 1 E Peak Tah ore 6015 iin OO 14 Aal me ticking the appropriate variable tick E AR TAg BARE 1103 APLI 5 box a A E E A table like that shown here will be displayed 10 9 Tabulating lists of results The Tables page is used to display lists of measurements in tabular form To display lists of data Seer dere ma Berd ma 1 Select the Tables page by clicking on its tab 2 Define one or more data columns by double clicking on the top row of the selected column to get the Set Variable dialog box Set Variable ES Record 0 and select the variable to be listed and the channel from which it 1s obtained To print out a copy of a summary report or table of results on the printer select File Print To copy the report or table to the Windows clipboard select Edit Copy Data WinWCP V3 9 User Guide 53 11 Curve Fitting 11 1 Introduction The curve fitting module allows a number mathematical functions to be fitted to digitised signal waveforms A mathematical model consists of a general equation representing the time course of the signal or part of the signal under study For instance the decay of many signals e g endplate currents can be represented by an exponential function f Aexp where A is the amplitude of the signal and qT is th
55. ate kjes gt O produce mEPSCs with biexponential decays If kaes 0 monoexponential decays result 6 Enter the standard deviation of recording background noise in the Backg Noise box If low pass filtering is to be applied to the mEPSC select the Low p filter On option and enter the cut off frequency in the box Random baseline drift can be added to each record by entering a non zero value in the Drift Max box 7 Click the Start Simulation button to start the simulation run WinWCP V3 9 User Guide 78 20 References Brown K M amp Dennis J E 1972 Derivative free analogs of the Levenberg Marquardt and Gauss algorithms for non linear least squares approximation Numerische Mathematik 18 289 297 Bezanilla F amp Armstrong C M 1977 Inactivation of the sodium channel I Sodium current experiments J Gen Physiol 70 548 566 De Koninck Y amp Mody I 1994 Noise analysis of miniature IPSCs in adult rat brain slices properties of synaptic GABAA receptor channels J Neurophysiol 71 1318 35 Dempster J 1986 The use of the driving function in the analysis of endplate current kinetics J Neurosci Methods 18 277 285 Dempster J 1993 Computer Analysis of Electrophysiogical Signals Academic Press London ISBN 0 12 208940 5 Dempster J 2001 The Laboratory Computer A Guide for neuroscientists and physiologists Academic Press Dilger J P amp Brett R S 1990 Direct measurement of the concentration
56. atistical degrees of freedom 1 in the fit and the number of iterations it j i Facar took to find the best fit m Faska n ll E 10 7 WinWCP V3 9 User Guide 49 Plotting histograms of measurement variables The Histogram page can be used to create frequency histograms of waveform measurements representing the frequency of occurrence of different values within the total set of measurements It is compiled by splitting up the range of possible values into sets of adjacent bins counting the number of individual measurements falling within each bin then plotting the bins as rectangular bars whose height indicates the number of measurements and position on the X axis indicates the range of values in the bin To plot a histogram 1 Select the Histogram page by clicking on its page tab 2 Select the waveform variable from which the histogram is to be generated from the variable and channel list boxes 3 Enter the number of histogram bins in the No of bins box max 1024 4 Enter the range of variable values which are to be included in the histogram from the lower limit m Lower box to the upper limit in the Upper box 5 If you want the histogram bar height expressed as a percentage of the total number of records tick the Percentage option 6 If you want a cumulative histogram tick the Cumulative option 7 Click the New Histogram button to compile and plot the histogram For example the histogram
57. auses problems when running WinWCP If this error occurs disable the DMA channel by clicking on the CED 1401 icon within the Windows Control Panel and un checking the Enable DMA transfers check box WinWCP uses the commands ADCMEMI CMD MEMDACI CMD and DIGTIM CMD with the CED 1401 ADCMEM GXC MEMDAC GXC and DIGTIM GXC with the CED 1401 plus and ADCMEM ARM MEMDAC ARM and DIGTIM ARM with the CED Micro 1401 All three commands must be available within the 1401 directory Modified CED 1401s with 10V A D or D A ranges CED 1401s interfaces are supplied with 5V A D input and D A output voltage ranges as standard They can however be supplied or modified by the user to have 10V ranges either for the D A outputs alone or for both A D inputs and D A outputs WinWCP cannot detect these modifications but you can indicate to the software that 10V ranges are in use by placing an appropriate flag file into the WinWCP program folder 10V D A Outputs If you have a CED 1401 with 10V D A outputs create a file named CEDDACIOV TXT it does not need to contain anything and place it into the folder c Program File Strathclyde University WinWCP 10V A D Inputs If you have a CED 1401 with 10V A D inputs create a file named CEDADCIOV TXT it does not need to contain anything and place it into the folder c Program File Strathclyde University WinWCP WinWCP V3 9 User Guide 11 2 5 National Instruments interface cards National Instrument
58. between two voltage levels It 1s defined by 4 parameters e Initial delay defines the delay period before the series of pulses begin e Start at amplitude defines the voltage level at the start of the ramp e End at amplitude defines the voltage level at the end of the ramp e Ramp duration defines the time taken for the voltage to slew between the start and end amplitudes Voltage ramps provide a means of rapidly generating the steady state current voltage relationship for an ionic conductance Note that the ramp generated by the computer is not truly linear but consists of a staircase of fine steps These steps can be smoothed out by low WinWCP V3 9 User Guide 35 pass filtering the voltage stimulus signal before it 1s fed into the patch clamp 8 2 6 Digitised analogue waveform ime Digitised analogue waveforms which have been previously acquired by WinWCP or synthesised by another program can be used as a waveform element To insert a digitised waveform into the protocol 1 Select the source of the waveform and copy it to the Windows clipboard Waveforms may be copied from a WinWCP signal record using the Edit Copy Data menu option or from a spreadsheet or similar program 2 Drag a digitised analogue waveform icon from the toolbox and drop it into the protocol list 3 Insert the waveform into the protocol by clicking the Paste button The waveform appears in the waveform display and its data points appear in t
59. by a horizontal bar along the bottom of the display Enter the Blank signal level to be substituted for the artefact in the Blank Value box a Lona then click the Remove Art button Yale Remove Art 17 5 Undoing or accepting changes To undo editing changes click the Undo button When editing is complete close the Edit Record window to make the changes permanent Note Edit Record acts directly on the digitised signal records and changes made are permanent It is advisable to make a backup copy of the original data file before editing 18 Data files WinWCP V3 9 User Guide 70 WinWCP uses its own custom data file format for storing digitised signal records These files are identified by the file extension WCP Data can also be imported from and exported to files in the Axon Binary Format used by Axon Instruments pClamp program and the Cambridge Electronic Design CFS CED Filing System formats Data can also be imported and exported in the form of ASCII text 18 1 Opening a existing WCP data file To load a previously created WCP data file select File Open to display the Open File dialog box Select the disk drive and folder from the Look In list A list of available WCP files will be displayed Select one of the file names then click the OK button to open the data file for display and analysis 18 2 Appending a WCP data file To append a WCP data on to the end of the currently open file select
60. by waveform measurement criteria The Filter Records option can be used to automatically Result on match et record status ip Accepted Match criteria All Records categorise records as particular types see 8 6 or rejected from analysis based upon waveform measurements To ARR classify the records in a data file Recod E Rejected Set record type l Ch Ch 0lm 7 iver y 1 Click the Filter Records button to open the Filter eee Records dialog box Alo ppb gt Lower limit Cancel CINES WinWCP V3 9 User Guide 47 2 Define the record Match criteria a Click the Variable radio button and select the measurement variable to be used as the record matching criterion from the variable list b Select the input channel for the variable c Define the range of acceptable values by entering appropriate values into the Upper Limit and Lower Limit entry boxes 3 Define the Action to be taken when a record matches the filter criterion To set the record type classification tick the Type box and select TEST LEAK EVOK MINI FAIL TYP1 TYP2 TYP3 from the list To set the record status tick the Record status option and choose Accepted or Rejected 4 When the Apply button is clicked of all records in the data file which match the criterion set by 2 are set to the type and or status defined in 3 10 6 1 Printing the graph To print the displayed graph select z Typeface Fage Margins Fil
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62. ces are currently supported by WinWCP under Windows 95 98 NT and 2000 The ITC 16 and ITC 18 are manufactured by Instrutech Inc 20 Vanderventer Ave Suite 101E Port Washington New York 11050 3752 U S A Telephone 516 883 1300 www instrutech com 2 8 1 Instrutech ITC 16 18 I O Panel Connections Signal input and output connections are made via the BNC sockets on the front of the ITC 16 18 unit Analogue inputs Analogue ouipuis Trigger Sync id Recording Sweep Trig In External Trigger I P Stimulus Program Trig In External Trigger I P See Note 1 Digital Out J Note 1 An active low TTL pulse on this input triggers the start a stimulus program which has been set up with the External Stimulus Trigger Y option WinWCP V3 9 User Guide 19 2 8 2 Installing software support for the Instrutech ITC 16 18 WinWCP uses Instrutech s device interface libraries for the ITC 16 18 family Details for steps 1 3 can be found in the Instrutech Data Acquisition Interface user manual Installation Procedure 1 2 3 4 5 6 Install the Instrutech interface card in an expansion slot Attach the ITC 16 or ITC18 unit to the card Install the Instrutech Device Driver software supplied with the card or downloaded from www instrutech com Reboot the computer Run the Instrutech test program installed with the device driver to test whether the software installed OK Run WinWCP and select from
63. channels are used individually and measured relative to signal ground of the computer In non referenced single ended mode channels are used individually and measured relative to electrical ground of the device being measured The analogue input mode for the card 1s set by right clicking on the card in the Devices amp Interfaces list in the Measure amp Automation Explorer program selecting Properties from the pop up menu then clicking on the AI tab of the Configuring Device dialog box The currently selected input mode is displayed in the Mode list Configuring Device 1 DAOCard 1200 MM 2 eh System Al a0 Acceso OFC Remote Acces Foam Ranga Select he dalani analog npul allro hot Iha ENGE Noreloenced Single Ended T When using the BNC 2090 I O box the mode should be set to referenced single ended with the switches beside each input on the I O panel set to NRSE When using the BNC 2110 T O box the mode should be set to differential since there are only 8 inputs on that box and it is wired for differential input WinWCP V3 9 User Guide 14 2 6 Axon Instruments Digidata 1200 Axon Instruments Inc 3280 Whipple Road Union City CA 94587 U S A Tel 510 675 6200 www axon com The Digidata 1200 1200A and 1200B interface boards fully supports all WinWCP features They have a 330 kHz maximum sampling rates and 4 programmable input voltage ranges 110V 5V 2 5V 1 25V The Digidata 1200 is
64. d between recording sweeps It has 5 parameters e Initial delay defines the delay period before the pulse begins e Amplitude defines the amplitude of the pulse e Pulse duration determines the duration of the pulse e Increment by defines the increment to be added to the pulse duration between records e Number of increments defines the number of steps in the sequence This element is most commonly used as a variable duration preconditioning pulse in 2 or 3 step protocols for investigating inactivation kinetics of Hodgkin Huxley type conductances 8 2 4 Series of rectangular voltage pulses JUL This is a train of rectangular voltage pulses of fixed size It is defined by 5 parameters e Initial delay defines the delay period before the series of pulses begin e Amplitude defines the amplitude of each pulse in the series e Duration defines the duration of each pulse e Pulse interval within train determines the time interval between pulses e Number of pulses defines the number of pulses in the series This element can be used to produce a series of stimuli to observe the effect of repeated application of a stimulus at a high rate It can also be used to produce a train of pe conditioning stimuli if the delay and duration of the recording sweep is adjusted to acquire only the last pulse in the series or a subsequent test pulse defined by another element 8 2 5 Voltage ramp yA This element produces a linear voltage ramp
65. d into the Stimulus Generator for editing by clicking the Open button and selecting a protocol file from the list presented in the Load Stimulus Protocol dialog box 8 9 Stimulus protocol examples A number of example protocols are installed in the vprot folder when WinWCP is installed Steps vpr A family of 12 depolarising 500 msec duration voltage steps ranging from 10 mV to 120 mV TailCur vpr A family of 2step pulse protocol for recording tail currents A 500 msec pre pulse followed by a 60 mV 50 msec duration test pulse The pre pulse steps from 10mV to 120 mV The recording sweep is of 70 msec duration and begins 10 msec before the test pulse A voltage ramp slewing from 100 mV to 100 mV over a period of 1 sec A digitised action potential waveform Digpulse A digital stimulus program controlling digital outputs O and 1 Dig 0 is OFF initially and pulses ON for a period of 50 msec after a delay of 100 msec Dig 0 is ON initially and pulses OFF for 50 msec WinWCP V3 9 User Guide 39 9 Viewing digitised records stored on file To view signal records stored in a data file select from the menu View Raw records to open the record display module Each record in the file can be displayed and measurements made of the signal levels within each channel using a movable cursor The displayed record or superimposed groups of records can be printed out Records can be assessed for the presence Of piss
66. e Type list Select ALL to measure records of any type except rejected records WinWCP V3 9 User Guide 45 4 Set the peak finding mode Select absolute to define the peak value as the largest absolute 1 e positive or negative deviation from the record zero level Select positive to define the peak value as the largest positive deviation Select negative to define the peak value as the largest negative deviation Note Absolute mode should be used for I V curve measurements where both positive and negative going signals may be found in the sequence of records being analysed 5 Set the rate of rise mode to select the differentiation algorithm Forward Diff Quadratic 5 and Quadratic 7 used to calculate the maximum rate of signal rise 6 If want the rise time to be measured over an interval other than the standard 10 90 range enter a new range in the Rise Time box 7 If you want to change the decay for the T x decay time enter a new value into the T x Decay Time box 8 Define the analysis region for each channel by using the pairs of vertical cursors superimposed on the display The analysis region defines the range of samples used to compute the average and integrated signal level and searched to find the peak value 9 To begin the analysis of selected range of records click the Do Analysis button On completion of the analysis the measurements for each Results 2 record appear in the Results table 10
67. e arial Print l l Paint Size 12 pts To open the Print dialog box You can set the size of Lett 5 0 cm Lines Right 5 0 cm Top 50cm the graph on the page adjusting the Left Right Top Line Thickness 3 pis pation adan and Bottom page margin settings Click the OK button to plot the graph Cancel 10 6 2 Copying the graph data points to the Windows clipboard The numerical values of the X Y data points which generate the graph can be copied to the clipboard by selecting Edit Copy Data The data is placed on the clipboard as a table of X Y data pairs in tab text format allowing the data to be copied into programs such as spreadsheets and graph plotting packages using an Edit Paste command 10 6 3 Copying an image of the graph to the Windows clipboard An image of the graph on display can be copied to the clipboard by selecting Typeface Image Size Arial cit Edit Point Size E pts ai Copy Image Lines 500 pixels i i Line Thickness 2 pts to open the Copy Image dialog box The dimensions of the bit map which will hold the image can be set using ok Canen the width and height image size boxes The more pixels used in the bit map the better the quality of the image When the image parameters have been set click the OK button to copy the image to the clipboard WinWCP V3 9 User Guide 48 10 6 4 Fitting a curve to the graph Linear single or double exponential functions can be fitted
68. e decay time constant Expressed as above the equation is quite general applying to any signal depending on the values of the parameters A and T In order to determine whether the equation actually does provide a good model of the signal decay it is necessary to find the parameter values which provides the best match or fit of the theoretical curve to an actual signal This can be done using the process know as iterative curve fitting Starting with initial guesses for the parameters the theoretical curve is compared with the experimental data points the parameters are adjusted to try to improve the fit and the process is repeated until no more improvement can be obtained The quality or goodness of the fit between theoretical curve and the data is determined from the sum of the squared differences S between a set of n data points y i i n and the theoretical curve f i computed at the same sample time points 1 e 2 s Y 00 16 The best fit is found by repeatedly computing S at each iteration adjusting the equation parameters using a strategy to minimise S The best fit parameters are the ones which yield a minimum value of S WinWCP uses a modified Levenberg Marquardt least squares minimisation algorithm the SSQMIN routine developed by Kenneth Brown at the University of Cincimnatti A more detailed discussion of curve fitting algorithms can be found in Chapter 6 of Dempster 1993 11 2 Fitting curves to digitised signa
69. e of 256 samples up to a total of 29952 for the whole record Note All records within a file must be the same size 1 e same number of channels and samples channel For efficiency data is written to the file in units of 512 byte sectors Three kinds of blocks can exist The header block is 1024 bytes in length and contains a list of ASCII format keywords detailing the number of records in the file record size scaling factors etc Signal records are stored in sequence after the header block Each record consists of one 1024 byte analysis block containing validation and analysis results pertaining to the record followed by a data block containing the digitised A D samples The size of the data block is determined by the number of channels and samples channel in the record For instance a data file containing 3 records would have the form Record 1 Record 2 Record 3 La La Lo Lo o The beginning of each record can be determined as a byte offset from the start of the file using the formula Byte Offset 1024 Record Number 1 2 N 1024 Al channels N apen channel where Nchannels 18 the number of channels per record and Nsamples 18 the number of samples per record Header Block The header block contains the information needed to allow a program to determine the size and number of records in the file It is usually the first block to be read when a file is opened File parameters are stored as ASCII text in the
70. e written to the data file The number of repetitions of each waveform entry sets the number of times that a sweep is to be repeated with the same stimulus waveform The delay before start of recording entry allows the start of the recording sweep to be delayed relative to the start of waveform generation The delay incre ment entry increments the above delay between records The holding voltage entry sets the holding voltage to be used between waveform sweeps during the execution of a protocol This value overrides the default holding voltage set in the Seal Test module and Default Settings options The External Stimulus trigger Y N entry allows the stimulus program to be triggered by an external TTL pulse instead of the internal timer When set to Y the Interval between sweeps entry is ignored and the stimulus program begins when a TTL pulse is received on the External Stimulus Trigger Input See interface card connections tables in section 1 WinWCP V3 9 User Guide 33 8 2 Creating a voltage stimulus waveform Waveforms are constructed by dragging waveform elements from the Toolbox and dropping them into the Voltage Command list p Protocol Voltage AAA 5 000001 1120011 002 00021 102000121 02 A single voltage waveform can consist of up 10 separate elements The amplitude and duration for each element is defined in its parameters table which can be made to appear by clicking the element An element can be one of 6 types
71. ect the X Y Plot page by clicking on its page tab 2 Define the variable to be plotted on the X axis by selecting it from the X Axis variable and channel lists 3 Define the variable to be plotted on the Y 3 axis by selecting it from Y Axis variable and channel lists 2 es ma q ida LLG Fan LECI 1 Miaa 2 aaas ke st cee renee ti am 4 Click the New Plot button to plot the graph 10 5 1 Customising the graph If you want to alter the X or Y axis range scaling or labels click the X Axis Y Axis Automatic Automatic Set Axes ha Manual z Manual In fo In fo button to open the Set Axes Range Labels dialog box Mex o Max 00 Tick pa Tick E Scale Linear Scale Linear Axis limits and tick spacing are initially set to default values a S EE xis ecor based upon the range of the data You can change the axis a A Axis Im Peak a nA limits by entering new values for into Min Max and Tick spacing boxes for the X and Y axes ok A An axis can be made Linear or Logarithmic by selecting the a option from its Scale list Labels for the X and Y axes canbe entered into the Labels boxes A type face can be selected for the plot from the Font list and its size defined in the Point Size box The graph can be plotted as a line unconnected markers or both by ticking the Lines and or Markers tick boxes 10 6 Classifying records
72. er Devices amp E 0 Scales Eu Serial Number 0x0 E Software Interfaces Note that the card 1 3 Remote Systems must be installed as Device 1 to work with WinWCP 5 Click on the interface card entry in the Devices amp Interfaces list then click the right hand mouse button and select Test Panel to check if the card is working If the tests check out OK run WinWCP and select from its main menu Setup Recording Select National Instruments interface from the Laboratory Interface list box WinWCP V3 9 User Guide 12 2 5 2 Signal input output connections Signal input and output from National Instruments cards are made via a 50 or 68 way ribbon cable connector on the rear of the card A BNC socketed input output panel BNC 2090 is available from National Instruments for E Series boards Standard screw terminal panels with 50 way ribbon cable sockets can also be obtained from electronic component suppliers The input output connections for 50 pin 1200 and 68 pin E series boards are tabulated below ACH3 s 4 g 9 ACH3 s 30 g 29 ACHT s 8 2 9 ACHT s 57 g 56 Analogue outputs A ee ee Command voltage out DACO OUT s 10 g 11 DACO OUT s 22 g 55 Trigger Sync See Note 1 A AAN Sync pulse out DAC1 OUT s 12 g 11 DACI OUT s 21 g 54 See Note 1 Recording Sweep EXTTRIG s 38 g 50 PFIO TRIG1 s 11 g 44 External Trigger I P See Note 1 Stimulus Program PBO s 22 g 50 PFI1 TRIG2 s 10 g 9 External Trigger
73. erfaces the manufacturer s software support libraries must also be installed and configured before it can be used The full installation procedure consists of the following steps 1 Install the WinWCP software see section 2 2 2 Install the laboratory interface unit and software see section 2 4 2 5 or 2 6 3 Configure WinWCP to work with laboratory interface 4 Attach analogue input output signal cables see section 4 2 2 Installing the WinWCP software To install WinWCP 1 Go to the web page www strath ac uk Departments PhysPharm ses htm and click the WinWCP V3 x x Installation Disk option to download a self extracting archive WinWCP_Vxxx exe containing the WinWCP installation files Store this file in a temporary folder e g c temp on your computer 2 Open the temporary folder and double click the archive to unpack the WinWCP setup program 3 Start the installation program by double clicking the program Setup The setup program creates the directory c Program Files Strathclyde University WinWCP and installs the WinWCP programs files within it You can change the disk drive and directory if you wish 4 To start WinWCP click the Microsoft Windows Start button and select WinWCP V3 1 x from the WinWCP group in the Programs menu 2 3 Hardware requirements To run WinWCP you will require an IBM PC compatible personal computer with at least 16Mbyte of RAM a 66MHz 80486 or better CPU and the Microsoft Window
74. erned by six rate constants rate of binding and unbinding of agonist from receptor Kpina Kunbina rate of channel opening and closure Kopen kaose and the rate of entry and exit from the desensitised state kaes Kuna The mEPSC is generated by summing the individual single channel current time courses for each ion channel opened by the brief pulse of transmitter released from each vesicle time course represented by a decaying exponential function with a time constant of 10 us To create a data file containing simulated mEPSCs 1 Create a new data file to hold the records by selecting File New and entering the name of a new data file 2 Open the simulation module by selecting Simulations a EA Miniature EPSCs 4 T mbr TENN h P I B 3 Enter the number of simulated mEPSC records a J to be created in the No mEPSCs box jeepin m AE pstaa 100 ira 4 Enter the single channel current amplitude for the post synaptic ion channels in the Unitary tig le TE current box Enter the average number of ion ermm fia channels activated when a quantum of toes pon transmitter is released in the No channels box and its standard deviation in the St Dev Box Enter the transmitter release decay time constant into the Transmitter decay box 5 Enter the rates constants which define the ion channel gating properties in the Ion Channel Model boxes Note Models which permit entry into the desensitised st
75. eters dialog box If you want to Fixed Fixed keep a parameter fixed i e not changed by curve fitting id GED z process tick its Fixed box You can also change the me E initial parameter guesses if they appear to be E E unrealistic Click the OK button to fit the curve OK Cancel The best fitting curve is superimposed on the X Y graph in red and the best fit equation parameters are displayed in the Curve Fitting table along with the parameter standard error the residual standard deviation between the fitted and data points statistical degrees of freedom in the fit and the number of iterations it took to find the best fit E B 10 12 14 im Pia fect fue SS DEE is ee e Ppi eis sre Tes DT Hea 11 3 70016477 foc a So 3101 gt OO ea a Empi 137 k ALS 15 uh An Bidai BD Era WinWCP V3 9 User Guide 52 10 8 Summaries of results The Summary page displays a summary report containing the mean values and standard errors for the records which have been analysed To display the summary of results Vo Wee Aare Mitra Arabes ee pit Ho Surrey rato 1 Select the Summary page by og Facundo mn Sd Gee SL Error Min ir clicking on its page tab i a CC ES Tee pu EEN ims 57a acon 7 mT a Time ie lim A TR 400 mu ai 2 Select the channel to be etre cia 1014 03194 001641 4157153 20 summarised from the Channel list E caia tac Veiner cE 517 Dei 05557 Lea TD
76. eurons typically fall into this category 9 8 2 Fixed mode If you want fix the zero level at a constant value to be used for all records in the file a Move the mouse pointer over the horizontal zero level cursor of the channel you want to change The mouse pointer turns into an Zeto level modes up down arrow From Record At sample b Hold down the left mouse button and drag the zero level cursor vertically until it is at the desired level c Click the right mouse button to open the zero level dialog box d Select the Fixed option The vertical position of the fixed baseline is indicated in A D converter units in the Level box You can set the zero level by entering a value ox Cancel e Click the OK button to use the new zero level Choose the fixed mode when you want to make measurements relative to a fixed absolute level Fixed mode is typically used for the membrane potential measurements in voltage patch clamp studies of voltage activated current Note Entering a value of zero into the Level box sets the zero level to the true zero voltage level for the channel WinWCP V3 9 User Guide 43 9 9 Copying records to the Windows clipboard The displayed signal record s can be copied to the Windows clipboard in a variety of formats a data table an image a WinWCP data record 9 9 1 Copying data values Each signal record consists of an array of A D converter sample values A table of data values
77. f samples averaged to calculate a zero level Channel O name Channel 0 units Channel 0 scale factor units bit Channel O gain factor mV units Channel 0 zero level A D bits Channel 0 offset into sample group in data block Channel 1 name Channel 1 units Channel 1 scale factor units bit Channel 1 gain factor mV bit Channel 1 zero level A D bits Channel 1 offset into sample group in data block Time units Experiment identification line Note that 1t should not be assumed that the keywords will follow any particular order Analysis block The first block in each signal record is an analysis block containing a series of internal format variables The first 6 variables provide important classification and scaling information for the record and are detailed as follows Sampling interval 4 byte floating point Max positive limit of A D voltage range 8 x 4 byte floating point 1 per channel WinWCP V3 9 User Guide 81 The remainder of the analysis block contains the results generated for that record by the Waveform analysis and Curve fitting modules Further details of the structure of the analysis block can be obtained by looking at the global pas source code file Data block The data block contains the digitised signals stored in the form of 16 bit binary integers Each A D sample takes up 2 bytes of space The size of the data block is determined by the number of channels and number of samples per channels i
78. ger Sync YO External Trigger I P Stimulus Program Digital Input O See Note 2 extemal Tigger tomo O Dasem PO Sd ooo DisudOouw Sens O oo Deo Demono SSS Degi Dia Or De2 Dado o Des Deos ooo Note 1 Sync Pulse Out must be connected to Recording Sweep External Trigger I P when recording signals using the Stimulus Program trigger mode and for the Seal Test option Note 2 An active high TTL pulse on this input triggers the start a stimulus program which has been set up with the External Stimulus Trigger Y option Note 3 The Digidata 1200 only supports 4 digital output lines 2 6 3 Troubleshooting There are two known problems which will prevent WinWCP from recording from a Digidata 1200 s analogue input channels I O port conflict The Digidata 1200 default I O port addresses span the range 320H 33AH These settings conflict with the default MIDI port setting 330H of Creative Labs Sound Blaster 16 and similar sound cards There are a number of solutions to this problem a Change the Sound Blaster MIDI port setting to a value higher than 33AH b Remove the Sound Blaster card or disable it using the BIOS setup 1f it is built in to the computer motherboard DMA channel conflicts WinWCP requires DMA channels 5 and 6 to support the transfer of data to from PC memory and the Digidata 1200 Many sound cards also make use of DMA 5 and can interfere with the operation of the Digidata 1200
79. h known parameters set spanning the range of values likely to be observed in the experimental data 11 6 4 Are all the parameters meaningful It is also necessary to discriminate between functions which fit the data equally well For instance the question often arises as to whether one two or more exponential functions are needed to fit a signal waveform It is usually obvious from the residual plot when a single exponential does NOT provide a good fit However when a single exponential does fit two or more exponentials will also provide a good fit In such circumstance it is usual to choose the function with the least number of parameters on the principle of parsimony An excess of function parameters also results in the some of the parameters being ill defined with standard errors values often larger than the parameter values themselves A more detailed discussion of the above issues can be found in Dempster 1992 and 2001 WinWCP V3 9 User Guide 58 12 Signal Averaging 12 1 Principles of signal averaging Many electrophysiological signals have poor signaknoise ratios making it difficult to obtain accurate measurements from individual records However if a signal can be made to occur repeatedly digital signal averaging techniques can recover the signal waveform from the background noise The signal average of a series of records is generated by computing the average of each corresponding sample within the records For a se
80. he parameters table The parameters table consists of e Initial delay defines the delay period before the series of pulses begin e A list of data points for the analogue waveform The waveform can be altered by modifying this list There are a number of limitations when using the digitised waveform element a Only one digitised waveform element is permitted per protocol b Digitised waveforms must consist of less than 1000 data points c The sampling interval of the digitised waveform must be greater than 0 1 msec d If digitised waveforms are created with a spreadsheet the data points must be formatted as a pair of columns containing time msecs in the first and amplitude mV in the second E g To Vo La Ya o LO 8 3 Creating a digital stimulus pattern Digital stimulus patterns control up to 8 digital output lines Dig 0 Dig 7 1f these are supported by the laboratory interface Digital stimuli can be controlled smultaneously with the voltage stimulus To create a digital stimulus pattern drag a digital stimulus element from the Toolbox and drop it into the digital pattern list WinWCP V3 9 User Guide 36 TTL Digital Each entry in the list controls one digital output with the left most entry controlling Dig 0 and the right most Dig 7 Each of the 8 outputs can thus have a pattern which is executed simultaneously during the protocol unlike the voltage waveform where elements are executed sequent
81. he decay phase of an endplate current Zero time t0 has been defined at the onset of the signal WinWCP V3 9 User Guide 55 9 If you wish to change the initial values for the equation parameters or fix some parameters so ala they do not change durmg the fit click the Fixed c z Set Parameters z ra E button to open the Set Fitting Parameters dialog ME box If you want to keep a parameter fixed ata set ox value enter the value into the appropriate parameter box and click the Fixed check box 10 When all curve fitting settings have been made click the Do Fit button to initiate the curve fitting sequence The iterative curve fitting process now begins The SSQMIN routine iterates through a variety of trial parameter sets until no more improvement can be obtained The number of iterations are displayed as fitting progresses The best fit 1s usually found within 10 20 iterations Fitting is aborted if the process has not converged to a suitable answer within 100 iterations and can also be aborted by clicking the Abort button 11 4 Curve fit results r Curas ff leer Pa Carr ign pai Hori rara Tarn Pia For each record fitted the best fit curve 1s indicated by a red curve superimposed on the blue signal trace A residuals trace is shown below indicating the difference between the fitted curve and the data The parameters of the best fitting equation are shown in the Results table alo
82. he signal EPSPs can also be simulated including the effects of non linear summation of quantal potentials To create a data file containing simulated EPSCs 1 Create a new data file to hold the records by selecting File New and entering the name of a new data file 2 Open the simulation module by selecting a ESE spain Simulations ee Nerve evoked EPSCs a A E Curae Roti _ TI 3 Enter the number of simulated EPSCs to poes 1 22 01 EIA TE PRE be created in the No records box ranita Retna Misses Release pooier a a i Sas IA 4 Select Currents or Potentials to ges tes 1 itt determine whether simulated currents or MENAMENERNENNI coon rs RET potentials are to be created If you have rima risen nare Bacay pres Paski 10 r dims pee Sms selected potentials enter the resting a amp es z Laere E fs imir potential of the cell in the Resting holding potential box 5 Transmitter release properties Enter the number of quanta available for release in the Release pool n box and the probability of a quantum being release when the nerve is stimulated in the Release prob p box 6 Quantal event properties Set the average peak amplitude of the miniature quantal current in the Peak box and its standard deviation in the St Dev box Enter the standard deviation of the background noise on the signal in the Background noise box 7 Enter the time constant of the EPSC rising phase i
83. he time constant computed by the curve fitting module in step 3 is used to deconvolve the current signal If you wish to use the same time constant for all records rather than using the individual value computed from each record elect the Keep parameters fixed option The basic deconvolution process computes a driving function which represents the rate of change of post synaptic conductance induced by the release of transmitter It 1s also possible to reconvolve this driving function with a different post synaptic current decay function to generate the waveform of the synaptic current that would have existed under these new conditions If you wish to create a simulated current select the Reconvolute waveform option and enter the new time constant in the Reconvolution column 10 Click the Do Transforms button to begin the deconvolution process WinWCP V3 9 User Guide 68 Driving functions are created and stored in a driving function data file DFN extension and can be viewed by selecting Driving Functions from the View menu to display them in the record display module The driving function b computed from a simulated endplate current a is shown below a I 2 0 uS ms 1 0 ms WinWCP V3 9 User Guide 69 17 Editing digitised signal records The record editing module can be used to make modifications to the digitised signal records stored on file The position of signals can be shifted vertically or horizontally within
84. hout producing an excessive number of false WinWCP V3 9 User Guide 31 events due to background noise triggering the detector Values of around 510 are often used but several trials may be necessary before the best level for a particular experiment is found The Pretrigger setting determines the percentage of the record to be collected before the detection point A typical value is 30 7 1 4 Stimulus Program In Stimulus Program mode WinWCP functions as a stimulator as well as a recording device Sequences of recording sweeps are acquired at timed intervals in synchrony with computer generated stimuli applied to the cell The stimuli can be in the form of either command voltage waveforms or digital pulses for controlling valves or other devices Choose Stimulus Program mode when there 1s a need to apply a sequence of voltage pulses in order to stimulate voltage activated ionic currents in cells or apply other complex stimulus patterns Each stimulus pulse is associated with a single recording sweep and the duration or amplitude of any part of a pulse can be incremented between records A complete stimulus protocol thus consists of a series of one or more pulses incremented in amplitude or duration to create a family of pulses Complex stimulus waveforms can be produced including series of rectangular steps ramps and digitised analogue signals Protocols are created using the Stimulus Generator module and stored as protocol files
85. ially There are 3 digital stimulus elements 8 3 1 Digital pulse fixed duration TIL ae This produced a digital pulse on the selected output line of fixed duration It 1s defined by 4 parameters e Initial delay defines the delay before the start of the pulse e Increment delay by defines the amount that the delay is incremented by between recording sweeps e Duration defines the duration of the digital pulse e Invert Signal defines whether the digital pulse is an OFF ON or an ON OFF pulse If set to No the digital line is initially OFF OV and switches to ON 5V during the pulse If set to Yes the digital line is initially ON 5V and switches to OFF OV during the pulse The digital pulse element can be used to switch open or close valves controlling the flow of solutions over a cell Multiple digital outputs can be used to simultaneously open one valve while another is closed 8 3 2 Family of digital pulse varying in duration stn This produced a digital pulse on the selected output line with a duration which is incrementable between records It is defined by 5 parameters e Initial delay defines the delay before the start of the pulse e Starting duration defines the duration of the first pulse in the protocol e Increment by defines the amount that the duration is incremented between records e Number of increments defines the number of increments in the protocol Note that if there are any voltage wavefo
86. l e g Vm Im Units defines the measurement units of the signal e g mV pA etc V Units defines the scaling factors relating the voltage level at the inputs of the A D converter in V to the actual signal levels in each channel in the channel units For instance if the membrane voltage output of your patch clamp supplies a signal which is 10X the measured membrane potential of the cell and the units have been defined as mV then the appropriate V Units setting is 0 01 since the patch clamp voltage output is 0 01 Volts per mV In the case of patch clamp current channels the V Units value is determined by the current gain setting which is usually a switchable value e g if the current output was set at 20 mV pA and the channel units were pA the V Units settings would be 0 02 A typical setup for a patch clamp experiment recording current and voltage channels is shown below Current 1s recorded in channel 0 which is named Im and has units of pA pd Name W Units Units 5 2 8 Amplifiers WinWCP can automatically determine the current Amplifiers gain factor for a number of patch clamp amplifiers a 7 aaa al Ch and use it as the calibration factor for input channel Ch 0 To enable this facility i ahnratnnr Interface fard 1 Select your amplifier from the Amplifiers list Axon Axopatch 1D and 200 are current supported 2 Connect an unused input channel to the Gain Telegraph output of the patch clamp
87. ld seance the image can be set using the Width and Height image size boxes The size and style of the typeface can be set using the Typeface and Size boxes When the image parameters have been set click the OK button to copy the image to the clipboard WinWCP V3 9 User Guide 51 10 7 5 Fitting gaussian curves to the histogram Gaussian probability density functions representing the distribution of discrete populations of events can be fitted to a histogram using non linear least squares curve fitting The number of events expected to be found in each histogram bin for a distribution represented by a mixture of m gaussians is given by y Q where N is the total number of events records w is the histogram bin width and each gaussian i 1s defined by three parameters its mean U standard deviation and the fraction of the total number of events a contained within it To fit a gaussian curve to the displayed histogram 1 Select the number of gaussian functions 1 2 or 3 to be fitted from the equations list NEI SES Fit Curve 2 Define the region within the histogram to which the curve is to be fitted using the pair of vertical analysis region cursors The selected region is indicated by he horizontal bar at the bottom of the O display o ____ 3 Click the Fit Curves button to start the curve fitting process The initial parameter guesses are displayed in the Set Fitting Param
88. lignment is not necessary If alignment is necessary select on positive rise for positive going signals and on negative rise for negative signals 5 Set the alignment search region cursors If records contain stimulus artefacts it may be necessary to restrict the region of the record which is searched for the signal mid point in order to avoid the averages being aligned using the artefacts rather than the true signals The alignment search region is set by moving the two vertical cursors on the display to define the beginning and end of the region containing the signal WinWCP V3 9 User Guide 59 6 To create the averages click Do Averages The averaging process now proceeds An additional digitised data file is created to contain the average record s with the same name as the original data file but with a AVG file extension rather than W CP 12 3 Viewing averaged data records On completion of averaging the record display module is opened to show the average records You can switch the display back and forth between the averages file and the raw data file by selecting View Averaged Records To view the averages and View Raw Records To view the original digitised signal records WinWCP V3 9 User Guide 60 13 Digital subtraction of leak currents Ionic currents recorded using the voltage clamp technique are usually composed of a variety of components mediated by different ionic channels e g Na K Ca Cl etc
89. lling release the quantal content can be calculated from OC log N stimuli 14 3 failures 15 4 Binomial analysis The transmitter release process can often be modelled as a pool of n quanta available for release with each quantum having a probability p of being released when the nerve is stimulated If both evoked and spontaneous signals are available it is possible to calculate estimates for n and p on the assumption that the number of quanta released per stimulus follows a binomial distribution Ave Peak tone 14 4 j AvglPeak VariPeak J n AvgtPeak orea mini WinWCP V3 9 User Guide 65 _ Avg Peak rea n Avg Peak a 14 5 15 5 Correction for non linear summation of potentials Unlike currents recorded under voltage clamp conditions synaptic potentials do not summate linearly Therefore the size of the synaptic potential is not directly proportional to the number of quanta released However given certain assumptions it is possible to correct for the effects of non linear summation using the eqn Peak PAK evoked 14 6 I gt f l Peak voked l V y where Peak o okeg 18 the measured peak amplitude of the evoked synaptic potential V is the cell resting potential V is the reversal potential for the post synaptic ion channels and f is a correction factor for the effects of the cell membrane time constant on synaptic potential amplitude A discussion on non linear summation
90. low pass filtered to remove frequency components greater than half of the sampling rate 1 e reciprocal of the sampling interval 5 2 5 A D Converter Voltage Range Defines the measurable voltage range of the A D converter The range of possible options depends upon the laboratory interface in use The CED 1401 for instance only has a single sensitivity 5V other interfaces such as the Axon Instruments Digidata 1200 have 4 programmable input sensitivities E10V 5V 2 5 and 1 25V In order to get an accurate measure of the amplitude of an analogue signal it is important to ensure that it spans a significant proportion 30 50 of the A D converter s input voltage range By changing the voltage range you can adapt the sensitivity of the A D converter to best match the amplitude of the signals from your experiment 5 2 6 Time Units Determines whether time measurements are presented in units of seconds or milliseconds WinWCP V3 9 User Guide 26 5 2 7 Channel Calibration Table WinWCP can display the signals stored in each input CHSA RE MESS PERE hE channel in the units appropriate to each channel In order to do this correctly the names units and scaling f i i f o 0 0476 information for each channel must be entered into the CS Channel Calibration Table There are 3 entries in the table for each analogue channel Ch Mame VWiUnits Units pA Names contains a 1 4 letter name used to identify the source of the channe
91. ls To open the curve fitted module select Analysis Curve Fit The module is split 5 functional sections pages Curve Fitting X Y Plot Histogram Summary Table accessed by clicking on the page tab Mo A per res Peete tee a ae ee The Fit Curve page is used to select the region of the signal and equation to be fitted and initiate the curve fitting process The X Y plot page is used to create X Y graphs of the measurements WinWCP V3 9 User Guide 54 The Histogram page is used to create frequency histograms of measurement The Summary page presents a summary mean standard deviation etc of the measurements for the series of records analysed The Tables page is used to create tables of results 11 3 Running a curve fitting sequence The first stage in the curve fitting process 1s to select an equation and run a curve fitting sequence on selected parts of a series of records 1 2 3 4 5 6 1 8 Select the Curve Fit page by clicking on its page tab Select the equation to be fitted to the record s Analyse gt DoFit Abort Select the channel containing the signal trace to which the curve is lecavino Exp to be fitted from the Ch list All records Define the range of records to be analysed by selecting All Records This Record to analyse all records or This Record to analyses only the currently C Range displayed record or Range and enter a range of records 1 200
92. make a digital recording of the electrophysiological signals and a range of waveform analysis procedures commonly applied to such signals WinWCP acts like a multi channel digital oscilloscope collecting series of signal and storing them in a data file on magnetic disk Its major features are Recording 8 analogue input channels 29952 samples per recording sweep 2 billion records per data file Stimulus voltage waveform generator 8 TTL digital output lines for operating solenoid controlled valves or other experimental devices TTL External trigger input to synchronise recording sweeps with external events e Digital valve control pattern generator e Spontaneous event detector Analysis Signal averaging Leak current subtraction Automatic waveform amplitude time course measurement Mathematical curve fitting to waveforms Non stationary noise analysis Quantal analysis of synaptic currents Synaptic driving function analysis Synaptic current and Hodgkin Huxley current simulations WinWCP V3 9 User Guide 7 2 1 Installation procedure If you wish to use WinWCP to digitise analogue signals rather than just analyse existing or simulated data files you must have one of the laboratory interface cards supported by WinWCP installed in your computer You must also ensure that the interface card is appropriately configured to work with WinWCP This may involve setting switches or jumpers on the card itself With some int
93. n the Tau rise box and the variability of the time between stimulation and the event in the Latency variability box 8 Enter the time constant of the decay of the EPSC in the Time constant 1 box If a double exponential decay is required tick the Double exponential decay option enter a second time constant in the Time constant 2 box and enter the ratio between the amplitudes of the two decaying exponential components in the Amp 1 Amp 2 box 9 Click the Start Simulation button to start the simulation run WinWCP V3 9 User Guide 75 19 2 Voltage activated currents simulation The voltage activated currents module simulates the currents evoked in response to a series of rectangular voltage steps using the Hodgkin Huxley equations Both activation and inactivation kinetics are modelled Two channels are generated membrane potential and membrane current The model also simulates the effects of patch clamp pipette access conductance on measured currents The currents are modelled by the equation P ps Rotate 1 V V G rev Mm m Mo Jexp max Tau Tau n m where V is the voltage level to which the cell potential is stepped V e 1s the reversal potential for the conductance Gmax 18 the maximal conductance Tau the activation m time constant Tau the inactivation A time constant and p the power to which the activation parameter is raised mp and moo and ho and Ao are the initial and final steady
94. n the record Npp EZEN A 2 bytes channels la If there 1s more than one A D input channel samples are interleaved within the data block For example for 2 channels YO YI YO YI YO nsamples YI nsamples A 3 Different laboratory interfaces supported by WinWCP return multi channel A D samples in different orders The channel interleaving order for a data file is specified by the YOn channel keyword in the file header block The calibrated signal level in the appropriate channel units can be reconstructed using information stored in the header and analysis blocks using V Ya ADC A max where Vmax the maximum positive limit of the A D converter voltage range from analysis block ADCwnax 18 maximum A D sample value at Vmax header block and YG is the calibration factor Volts channel units for channel n header block
95. n version of the command voltage waveform A digital average is obtained from these records and stored as a LEAK record along with the basic TEST record WinWCP V3 9 User Guide 38 You can change the number of pulses used to compute the yesin LEAK record and division factor by altering the values in PM mode div by the boxes shown The default values are 4 leak records with Mone voltage waveform divided by 4 Note that subtraction of the LEAK from TEST records is done using the leak subtraction module See section 13 for details 8 7 Protocol linking Recording normally stops when the requested sequence of records within a protocol is completed Protocols can however be linked Link to next protocol together by selecting a protocol from the Link to next protocol list so that on completion of the first protocol control is transferred to the linked protocol 8 8 Saving and loading stimulus protocols When you have created a stimulus protocol you gt E EEI can save it to a protocol file by clicking the Save wen fae a AS button to get the Save Stimulus Protocol Peon ippo Depicted dialog box e a er rai n am Apoyar pr mm highs iil eur pr Ma CAL bla pr a big Te hi40 p Aj Prema be pr ai i OA ia Cag Tastis vor han enor Stimulus protocols are stored as files with VPR file extensions in the directory C WinWCP vprot Protocol files can be re loade
96. nal input and output connections are made via the BNC sockets on the front of the Digidata 1320 Series digitiser unit Analogue inputs Analogue ouppuis Trigger Sync Recording Sweep Trigger In Start extemal Tigger Stimulus Program Trigger In Start External Trigger I P See Note 1 Digital Out See Note 1 Note 1 The Digidata 1320 Series only supports 4 digital output lines Note 2 An active high TTL pulse on this input triggers the start a stimulus program which has been set up with the External Stimulus Trigger Y option 1200 Series boards need the pulse to be 10 ms in duration or longer 2 7 3 Troubleshooting When multiple analogue input channels are being sampled and the sampling interval is greater than 10 ms samples get mixed up between channels This problem can be seen to occur also with AxoScope suggesting a bug in the Digidata 1320 firmware or AXDD132X DDL library The only limited solution at present is to increase the number of samples per record to ensure that the sampling interval is less than 10 ms WinWCP V3 9 User Guide 18 2 8 Instrutech ITC 16 18 The Instrutech ITC 16 and ITC 18 interfaces consist of self contained 19 rack mountable mains powered digitiser unit with BNC I O sockets attached to the host computer via a digital interface card and cable Both the ITC 16 and ITC 18 support 8 analogue input channels 4 analogue outputs 2 used by WinWCP and 8 digital outputs Both devi
97. nd V6 programs The files should contain episodic data records such as created by the CLAMPEX program Axon data files have a DAT file extension 18 3 2 Cambridge Electronic Design Data files produced by CED s Voltage amp Patch Clamp program in the CFS CED Filing System format CFS files have a DAT file extension 18 3 3 ASCII text files Files with the sample values stored as ASCII text in tab text format The Data File Import dialog box allows you to view the format of the ASCII data to be imported and to specify how it should be imported The data must consist of rows of samples each row terminated by a carriage return line feed pair of ass characters lt cr gt lt lf gt Channel sample values within No of initial rows to ignore each row are separated by lt tab gt characters No ofsignalchannels 1 Mo of samples channel The number of signal channels in the record to be created Time data column is determined from the number of columns in the table Sampling interval with one of the columns usually the first assumed to Time units contain the time 7 Cancel The first data row s in ASCII table often contain labels or AAA identification information which should not be treated as samples To skip o one or more or these lines enter the number to skipped in the No of initial rows to ignore By default column 1 is assumed to contam the time that each block of samples was acquired If
98. ng with the parameter standard error the residual standard deviation between the fitted and data points statistical degrees of freedom in the fit and the number of iterations 1t took to find the best fit 11 5 Plotting and tabulating results X Y graphs hstograms summaries and tables of the best fit equation parameters can be produced using the X Y Plot Histogram Summary and Table pages of the curve fitting module These options are identical in function to the corresponding pages in the waveform measurement module and are therefore not repeated here See sections 9 5 10 7 for details of their use WinWCP V3 9 User Guide 56 11 6 Equations Straight line y x M x C Exponential general Decaying Exp 2 Exponential general 2 Decaying Exps 3 Exponential 3 Decaying Exps Ay Taul Endplate current x x0 x x0 x 0 5 A l e exp de TauR TauD The endplate current rising phase is modelled by error function and the decay with an exponential function x P SAs a y oo TauM X x Al ex A2 ex y x A Olas The time course of activation of a voltage activated current with Hodgkin Huxley kinetics y x A Eo k Pa y Hi a eso a The time course of a current with voltage dependent activation and inactivation following Hodgkin Huxley kinetics e g sodium current Note It is assumed that initially the activation parameter m 0 and inactivation parameter h 1
99. ommand voltage input signals by some factor in the range 10 50 Enter the scaling factor into the Vcom divide factor box WinWCP uses this factor to scale the stimulus voltage output to the D A converter to obtain the correct voltage at the cell Note Axon Instruments amplifiers require a divide factor of 50 while the Heka EPC 7 patch clamp requires a divide factor 10 6 3 Cell holding voltage and test pulses You can control the holding voltage applied to the cell and the amplitude and duration of a test voltage pulse by selecting one of two available test pulse types Pulse 1 Pulse 2 or a holding voltage level without a pulse Pulse pci a F3 3 ce sae The size of each pulse type is set by entering an appropriate value for Amplitude 20 0 mv mi holding voltage and pulse amplitude into the Holding voltage or Amplitude box for each pulse The width of both pulses is defined by the pulse width box art You can switch between pulses by pressing the function key associated with each pulse Pulse 1 F3 Pulse 1 F4 Pulse 1 F5 WinWCP V3 9 User Guide 28 6 4 Current and voltage readouts A readout of the cell membrane holding current and voltage and test pulse amplitude appears at the bottom of the monitor window Tvoltage _ p Curent ee Holding Holding Pipette Cal 0 2 mv Poona Pulse Pulse faioome PAT 01na Resistance 94 8 MOh
100. ormation on the size of each of the file and record header blocks and the number of channels and samples in each data record the import module can extract the signals from the file These details of the data file structure can often be obtained from the user manuals associated with the software which created the data files Note that the sampling interval and other scaling information is discarded by the binary import module The Import settings must be carefully set up to match a ae A A the characteristics of the file being imported ASCII Enter the size of the file header and record header blocks Import settings in the appropriate boxes If no file or record header File header size bytes exists set these values to zero Record header size bytes o Enter the number of input channel in the No of signal 3i8nsienannels channels box and the number of A D samples per Ye Samples channel channel in the No of samples channel box Palla Offset by The WinWCP data file format stores sample values as sanina ena 12 bit binary integer numbers in the range 0 4095 If the import file uses a different number format it may be necessary to scale or add an offset to each sample to transform it to be compatible with the WinWCP format E To scale the signal enter the scaling factor into the Scale ecient by box To add an offset to the signal enter it into the Offset by box Time units Enter the time in
101. ported by WinWCP are fitted with a 12 bit resolution A D converter designed to digitise voltages within the range 5V to 5V This means that during digitisation the A D converter generates a 12 bit binary number proportional to the amplitude of the analogue voltage at its input Twelve bits are sufficient to describe only 4096 discrete level Thus given a 5V input range the smallest voltage difference that can be directly measured is 10000mV 4096bits 2 44mV bit Clearly a 10mV amplitude EPP would be barely observable Such small signals must be amplified to make them span a significant fraction of the input voltage of the A D converter e g 12V Amplification factors in the order of 500 1000 are therefore required Such high amplification factors require that the resting membrane potential is subtracted from the signal before amplification to avoid overloading the A D converter inputs Although the EPP may only be 10mV in amplitude it is superimposed on a 90mV resting potential Amplifying this signal by X500 would theoretically result in a signal with DC level of 45V Most instrumentation amplifiers cannot sustain such a level being limited to 10V output levels In any case signal levels exceeding 20V may damage the input stages of many A D converter The above difficulties can be overcome by using a differential amplifier as shown in the circuit below Stimulator Stim Out Ext Trig Differential Sync Out
102. r In External Trigger I P See Note 1 Stimulus Program Event Input 3 Pin 4 sig 9 gnd External Trigger I P on Events 15 pin female D See Note 3 ee connector Digital sync Input Event Input 2 Pin 3 sig 9 gnd See Note 2 on Events 15 pin female D connector Digital Out 25 pin female Digital Outputs POON Dy Opus socken 2s pin female socke Note 1 Syne Pulse Out must be connected to External Trigger In when recording signals using WinWCP s Stimulus Program trigger mode and for the Seal Test option Sync Pulse Out goes low OV for the duration of the stimulus program Note 2 Syne Pulse Out must also be connected to Digital Sync In when the digital output pattern options Dig 0 Dig 7 in a stimulus program are in use Note 3 An active high TTL pulse on this input triggers the start a stimulus program which has been set up with the External Stimulus Trigger Y option WinWCP V3 9 User Guide 10 2 4 3 Troubleshooting tips Verify that the CED 1401 is working correctly before investigating problems using WinWCP Use the TRY1401W program to test the CED 1401 The CED 1401 ISA card default I O port addresses are at 300H Check that these do not conflict with other cards within the computer The CED 1401 also makes use of DMA channel and an IRQ channel IRQ2 These may also conflict with other cards Some standard 1401 appear to fail the DMA direct memory access test in TRY1401W and this also c
103. re scans through the E xn data file calculates the mean and 3s e P Do Analysis B variance of the peak amplitude of the Close eee aa signal records uses these to obtain Poisson Standard deviation 1 44 nA estimates for the quantal content and Seana spontaneous miniature currents n 10 displays the results in the report window IO Ranga Mean 0 966 na All records Standard deviation 0 0543 nA A copy of the quantal analysis report is Range Quantal content 3 32 direct method also written to the log file ails id Ch cnom gt Im Binomial Analysis Note that you can test the operation of Release Probability 0 339 th dal ent e dul Evoked event Pool size 9 8 e quantal content analysis module aon using simulated endplate currents or 42 an potentials generated by the synaptic Eventsiniile signal simulation module See section Te User entered 19 1 Amplitude Mode amp Currents C Potentials Resting Reversal Correction Standard Dev potential potential Factor Urry WinWCP V3 9 User Guide 67 16 Synaptic current driving function analysis The synaptic current driving function is a measure of the rate of evoked release of transmitter at a synapse If the time course of decay the post synaptic current 1s known the driving function can be computed using a deconvolution process More details of the method can be found in Dempster 1984 WinWCP s driving function module can
104. rm elements in use within the protocol the number of increments defined here must be the same e Invert Signal defines whether the digital pulse is an OFF ON or an ON OFF pulse If set to No the digital line is initially OFF OV and switches to ON 5V during the pulse If set to Yes the digital line is initially ON SV and switches to OFF OV during the pulse WinWCP V3 9 User Guide 37 8 3 3 Train of digital pulses m TTL LLL_ This produces a series of digital pulses of fixed intervals and of fixed duration It is defined by 5 parameters e Initial delay defines the delay before the start of the first pulse in the series e Pulse duration defines the duration of the each pulse in the series e Inter pulse interval defines the time interval between pulses in the series e Number of pulses defines the number of pulses in the series e Invert Signal defines whether the digital pulse is an OFF ON or an ON OFF pulse If set to No the digital line is initially OFF OV and switches to ON 5V during the pulse If set to Yes the digital line is initially ON 5V and switches to OFF OV during the pulse This element can be used to apply a rapid train of stimuli to a cell 8 4 Command voltage divide factor Most voltage and patch clamp amplifiers divide down their command SEAAAAIEADEAEEASEA voltage input signals by some factor Enter the scaling factor into the PEA TEE command voltage divide factor box WinWCP uses
105. s 95 98 NT V4 or 2000 operating system A laboratory interface unit is also required to perform the analogue digital A D and digitabanalogue D A conversion of the signals and stimulus waveforms The following families of laboratory interfaces are supported e Cambridge Electronic Design 1401 1401 plus Micro 1401 Power 1401 e National Instruments Lab PC Lab PC Lab PC 1200 DAQ Card 1200 AT MIO 16E10 and other interfaces supported by the NI DAQ library e Axon Instruments Digidata 1200 or 1320 Series e Instrutech ITC 16 or ITC 18 WinWCP V3 9 User Guide 8 2 4 Cambridge Electronic Design interfaces Cambridge Electronic Design Ltd Science Park Milton Rd Cambridge CB4 4FE Tel 01223 420186 Fax 01223 420488 www ced co uk The CED 1401 series consists of an external microprocessor controlled programmable laboratory interface units attached to the PC via a digital interface card There are 4 main types of CED 1401 in common use CED 1401 CED 1401 plus CED Micro 1401 and CED Power 1401 They all fully support WinWCP s features with the exception that only 4 analogue input channels are available cn the Micro1401 and that the maximum sampling rate for the older CED 1401 is substantially less than the others 2 4 1 Software installation Before WinWCP can use these interface units the CED 1401 device driver CED1401 SYS support library USE1432 DLL and a number of 1401 command files stored in the directory 14
106. s UK 21 Kingfisher Court Hambridge Rd Newbury RG14 5SJ Tel 0635 523545 Fax 0635 523154 OR National Instruments 6504 Bridge Point Parkway Austin Texas 78730 5039 Tel 512 794 0100 Fax 512 794 8411 www ni com WinWCP is compatible with many of the 1200 Series Lab PC and E Series cards supplied by National Instruments It has been tested with the Lab PC Lab PC Lab PC 1200 DAQ Card 1200 PCI MIO 16E 1 PCI MIO 16E 4 and PCI 6024 WinWCP controls the National Instruments interface cards via the company s NIDAQ interface library NIDAQ must therefore be installed before WinWCP can use the interface card WinWCP is compatible with NIDAQ versions 4 9 6 8 running under Windows 95 98 NT V4 2000 or XP 2 5 1 Software installation 1 Install the NIDAQ library from the disks supplied with interface card following the instructions supplied by National Instruments 2 Install the interface card in an expansion slot OYUN CLUDE LUIS LT ULE TIES Las Oe Les Ie WL Loe Led i E DAQCard 1200 Device 1 Measurement amp Automation Explorer 3 Reboot the computer File Edit view Tools Help Properties PalDelete HA Test Panel 9 ae O a E 4 Run National Instruments Configuration 1 F DAQCard 1200 Device Measurement amp E My System Automation Explorer EE Data Neighborhood program You should find the Fo e PW socket i DUN ELA a g i a k levice 1 F card listed und
107. state values of the activation m and inactivation parameter respectively mo and mo and hp and ho are Boltzmann functions of membrane potential Tau Tau are bell shaped functions To create a data file containing simulated voltage activated currents 1 Create a new data file to hold the records by selecting File New and entering the name of a new Hodgkin Huxley Voltage Clamp Simulation _ Start Simulation cose aot EEE ees Voltage protocol Holding voltage data file 2 Open the simulation module by selecting Simulations Hodgkin Huxley 3 Enter the voltage clamp holding voltage into the Holding voltage box the number of simulated voltage steps to be created in the No of steps box and the Voltage step No of steps 4 e Leak subtraction None Divide by N M Noise Background 001 nA o 001 ra Conductance an Y rta gt g G max mp h Max conductance G max Reversal potential V rev Leak conductance G Leak Activation power factor p Pipette conductance G sern 100 nS Cell capacity Cm 10 pF increment in voltage between steps in the Voltage step box 4 If leak subtraction records are to be created select Divide by N and enter the P N divide factor WinWCP V3 9 User Guide 76 5 Enter the standard deviation of the gaussian background noise to be added to the signals in the Background noise box 6 Select the
108. sue as joie aye gee le lyre ems J on interference other artefacts and marked as rejected or assigned with particular record types 9 1 Selecting and displaying records Each record in the data file is numbered in the sequence that it Record was recorded Records can be selected for display using the selection bar to move back or forward through the file You can jump directly to a record by entering its number into the record KI El number box above the selection bar You can also use the Ctrl Plus and Ctrl Minus keys to step forward or backwards through the file To superimpose up to 200 records on the display select View Auto Erase to disable automatic display erasure when records are Selecting the option again re enables auto erase 9 2 Magnifying the display To adjust the display magnification select the channel to be changed by double clicking on the signal trace The selected channel is expanded to fill the display window and the message Zoom In Out appears at the top left indicating that the display is in zoom mode A rectangular box defining the currently selected upper and lower display limits is superimposed on the signal Drag the edges of this box to indicate the new region of the record to be displayed Then double click again to return to normal display mode at the new magnification setting You can set all channels back to mintmum magnification by selecting View Zoom Out All
109. t The variance Ot of the current fluctuations at time t about this mean 1s O 1 in plt 1 plt 132 These two equations can be combined to provide a relationship between O t and I t erra 133 The single channel current i and number of channels n can thus be calculated by fitting the above parabolic function to a plot of o t vs J t during a current transient where p t is changing t can be computed as the average current of a series of transient current records repeated M times all evoked by the same stimulus yy I t 13 4 a The variance 0 1 at each sample point t can similarly be computed from 7 2 o 1 E 13 5 M 1 The method was developed by Sigworth 1981 for voltage activated Na currents It has also been used to study the fluctuations during the rapidly desensitising currents induced by high concentrations of acetylcholine Dilger amp Brett 1990 With modification it can also been applied to synaptic currents The basic non stationary variance approach assumes that the only source of variance arises from the fluctuations of the ion channels that carry the current However synaptic current amplitude can fluctuate due to both ion channels and quantal size content variation Traynelis et al 1993 found a way round this problem by scaling the amplitude of the average current to the peak amplitude of each signal before the subtraction in Eqn 13 5 thus compensating for the quantal
110. t of N records consisting of samples y i 1 n the average record consists of n samples and is given by Avg why 12 1 The location of signals within the record sometimes varies from record to record due to imperfections in the detection of spontaneous signals or fluctuations in stimulus latency In such circumstances averaging corresponding sample points within the record would result in a distorted signal average This problem can be avoided by aligning the signals by the mid points of their rising phases before averaging 12 2 Creating signal averages To compute signal averages from records stored in a SE Po ee digitised data file select a tal Analysis w i Signal Averager Mica drid to open the signal averager module va Femge i 210 in bikia of 101 THe pii Aigi mei mi dosiormend To create the averages 1 Specify the range of records to be averaged by entering the first and last records separated by a In the range box 2 Enter the number of records to be included in each average in the in blocks of box Note The default settings produce a single average record from all the records in the data file 3 You can restrict averaging to a specific type of record by selecting a type from the type list Select ALL to use records of any type except rejected records 4 Set the alignment mode Select No alignment if the position of the signals do not vary within the records and a
111. terval between adjacent samples within each channel in the Sampling interval box Select the units of the time interval from the Time units list Click the OK button to import the data when the import settings are complete WinWCP V3 9 User Guide 73 18 4 Exporting to foreign data files WCP data files can also be exported a number of data file formats To export the currently San e Save in data el ex open data file select File Export aa MinisOl abf To open the Export file dialog box Select the disk drive and folder where the fiename exported file is to be stored from the Look In list The export file name is initially set to the same name as the WCP file but with a DAT file extension Save as type Axon Files DAT 4BF y Cancel Files can currently be exported in Axon Instruments data file formats 18 5 Experiment Log file WinWCP maintains a log file of the operations initiated by the user during the course of recording or analysing an experiment The names of data files created or loaded comments entered stimulus programs used and other events are stored along with the time that the event occurred The log file can be used like an experimenter s notebook to keep a written record of the experiment A new log file is opened on a daily basis with a name in the form dd mm yy log and stored in the WinWCP program directory 1 LiLo A P cat CL d disp lay the Log File C DELPHI WINWCP
112. the time data is contained in a different column enter the correct column number in the Time data column box If there is no time data at all set Time data column 0 The sampling interval is derived from the times of adjacent rows If there is no time data or if the displayed interval is wrong enter the correct value in the Sampling interval box Select the units that the time data is expressed in from the Time units list Click the OK button to import the data when the import settings are complete WinWCP V3 9 User Guide 72 18 3 4 Binary data files If the type of data file being imported does not match any of the known formats the import module reverts to its general purpose Binary import mode The import module assumes that the data has the general format File Record Data Record Data Header Header block Header block At the beginning of the file there is a block of file header data which contains the information on the number of records in the file size of record number and scaling of channels This is followed by one or more records containing the digitised signal series of recording sweeps Each record consists of a record header block containing information associated with the record followed by a data block containing the A D converter samples stored as 16 bit integers 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 Given inf
113. this factor to scale Factor mA the stimulus voltage output to the D A converter to obtain the correct voltage at the cell Axon Instruments amplifiers require a divide factor of 50 while the Heka EPC 7 patch clamp requires a divide factor of 10 8 5 Recording Sweep Trigger Pulse Trigger Pulse Sets the polarity of the trigger pulse used to synchronise the start of a recording sweep with a stimulus protocol and seal test pulses This trigger pulse is produced on D A output channel 1 DAC1 When Trigger Pulse is set to Normal a 1 msec active low pulse TTL is produced 1 e the DAC1 output is at 5V and drops to OV for 1 msec to trigger the recording sweep When Trigger Pulse is set to Inverted a 1 msec active high pulse is produced NOTE The Inverted Trigger Pulse option is provided to allow the DAC1 output to be used to synchronise external devices e g stimulators which require an active high TTL trigger pulse with the start of the recording sweep Otherwise the setting should not be changed from Normal It should be noted that a stimulus protocol with TTL digital pattern outputs will only work with the Normal trigger pulse setting when using CED 1401 series interfaces 8 6 Leak subtraction A protocol can be programmed to add digital leak subtraction records by selecting the P N mode option When this option is set a series of additional sweeps are generated for each record defined in the protocol using an inverted and scaled dow
114. thod of failures In circumstances where both evoked and spontaneous miniature events are available transmitter release parameters n number of available quanta and p probability of release is calculated using binomial analysis 15 1 Quantal content direct method If the data file contains both evoked and miniature signals the direct method of calculating quantal content can be used Avg Peak e OC direct Avg Peak ee 14 1 This is the most accurate method for calculating quantal content 15 2 Quantal content variance method It is not always possible to record the miniature synaptic signals which represent single quanta In such circumstances it may still be possible to calculate quantal content from the variability of the evoked signal Avel Peak y 14 2 Varl Peak Varl Background OC evoked This method is dependent upon the assumption that the number of quanta released follows a Poisson distribution This will only be the case when the probability of release is very low i e p lt 0 1 Since large errors can result if this condition is not satisfied results using the variance method should be treated with caution 15 3 Quantal Content failures method If the quantal release probability is very low a nerve stimulus may occasionally release no quanta at all resulting in intermittent failures to evoked post synaptic signals Again using the assumption of a Poisson distribution contro
115. tivated currents The leak current recordings are averaged and stored in a record marked as a LEAK type The test record is marked as TEST type record The TEST record with its WinWCP V3 9 User Guide 61 associated LEAK record are collected together in a group i e they have the same group number 13 2 Subtracting leak currents To subtract the leak currents from a data file 1 2 3 4 5 6 1 Open the leak current subtraction module by selecting Analysis rr Es A Leak Current Subtraction ss H Saam T Rejectad Select the channel containing the cell membrane Siaa potential from the Voltage channel list ET walega chanel Select the channel containing the cell membrane pon E A ono current from the Current channel list nom Ae E eure ha ft From group From C Froeetole fle FP Freed oo r J T 1 Select the source of the Leak records If records are grouped into LEAK TEST record pairs select From Group This option should be used for P N protocols If the LEAK records are not grouped with the TEST records select From whole file Whole file mode is used when one or more records at the beginning or end of a data file are to be used as LEAK records Note that these records will have to be manually classified as LEAK Set the current Scaling mode Select From Voltage to use the ratio between the TEST and LEAK voltage pulses as the current scaling factor default Select Fixed if you wish to use
116. to an X Y graph using non linear least squares curve fitting To fit a curve to the displayed X Y graph 1 Select the type of curve to be fitted from the fitting equations list Fit Curve Exponential Linear y x M x C exponential y x exp x Tau Ss and two exponential y x Al exp x Taul A2 exp x Tau2 Ss functions can be fitted 2 Define the region within the graph to which the curve is to be fitted using the 4 pair of vertical analysis region cursors The 3 o selected region is indicated by the z horizontal bar at the bottom of the display 7 Oo oO a 3 Define the initial starting point of the fitted y a curve using the x0 cursor 4 Click the Fit Curves button to start the curve fitting process The initial parameter guesses are displayed in Bsramotor the Set Fitting Parameters dialog box If you want to Mie Fisad f A feina AL l o B keep a parameter fixed i e not changed by curve pp p fitting process tick its Fixed box You can also change le pima rT PE the initial parameter guesses if they appear to be E unrealistic Click the OK button to fit the curve The best fitting curve is superimposed on the X Y graph in red and the best fit equation parameters are displayed in the Curve Fitting table along with the 3 parameter standard error the residual z ki standard deviation between the fitted and data points st
117. variation This approach does have limitations and it is worth reading De Koninck amp Mody 1994 if considering using the scaling approach To compute the single channel current and number of channels from a series of ionic current transient 1 Collect a series of 100 200 records containing the transient signal under study 2 3 4 5 6 1 8 9 10 Click the X Y Plot tab to switch to the X Y Plot page poor 11 To plot a o t vs J t curve select Mean 12 To fit Eqn 13 3 to the curve a select the The estimated single channel current Iu and number of channels Nc are displayed in the WinWCP V3 9 User Guide 63 i a mi Select M r an Analysis Non stationary noise analysis to open the non stationary variance analysis module If there is more than one input channel select the channel containing the current signal to be analysed from the Channel list Current signals are displayed blue superimposed upon the average current red for the selected range of records in the upper panel of the display The residual difference between the each record and the average is displayed in the lower panel Optional To use a specific record type only change the Type list from ALL to the selected type TEST LEAK EVOK MINI FAIL TYP1 TYP2 TYP3 Select the All Records option to use all records of the selected type To use a sub range only Select Range and enter the range of record in
118. ysis Select All records if you want to in io use all records in the file or select Range and enter a range of records 3 If there are several channels in the signal record select the channel which contains the signals to be analysed from the Ch list 4 Evoked events Select the EVOK record classification type used to indicate stimulus WinWCP V3 9 User Guide 66 evoked signals from the Type list 5 Quantum events f the file contains miniature events select the Events in file option and select MINI from the Type list If there are no miniature events in the file but you know what the quantal signal amplitude is select User entered and enter the average peak amplitude of the spontaneous miniature signal into the Amplitude box and the standard deviation of peak amplitude in the Standard Dev box 6 Analysis mode If the signals being analysed are currents recorded under voltage clamp conditions select the Currents option If the signals are potentials select the Potentials option and enter the cell resting potential and the reversal potential of he synaptic conductance into the Resting potential and Reversal potential boxes This data is used to apply a correction for the non linear summation effect The Correction factor should be left at the default value 1 unless the appropriate factor for the synapse under study 1s known 7 Click the Do Analysis button to begin the quantal analysis sequence The analysis procedu

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