APPENDIX A - LIMITATIONS IN LTP113E

Note: No measurements were taken on 386 computers because none were available with enough RAM to run LTP113E.

A.1 The LTP program only determines if the computer is fast enough for the data acquisition board during program start-up.

When the LTP program starts up, it automatically determines if the computer is too slow for the chosen data acquisition board, in which case it terminates. However, after this initial ‘speedtest’ the LTP program does not again check if the computer is fast enough for the data acquisition board. (Note that in future LTP programs, this will be tested on each sweep with computers using the Digitata board or having the Pentium (but not the 386 or 486) microprocessor.)

This means that if there was a microprocessor slowdown (which could occur if the microprocessor when into a power saving mode on a laptop computer, or if the ‘Turbo’ speed was inadvertently turned off), a 100 msec sweep would actually take twice as long (200 msec) in real time. This would mean that a 10 msec duration EPSP would actually appear having a 5 msec duration on the ADsweep graph, and the slope would appear twice as large.

If the synaptic potential duration or slope immediately and inexplicably changes by a factor of two, check that the synaptic potential duration is right by independently measuring it with an oscilloscope. Alternatively, if an oscilloscope is not available, restart the LTP program to test the data acquisition board speed again.

We have never seen this happen with our Tandon and Dell computers but it is possible that it could happen on your computer.

A.2 Slight Inaccuracy in Plotting Calculated Amplitudes/Slopes on Waveforms

There may appear to be a slight inaccuracy in plotting the calculated DC Baseline, Amplitude and Slope lines in the ADsweep waveforms because the calculated values and the waveform values were plotted slightly differently.

The ADsweep data points were plotted to obtain maximum speed - all plotting is done in integer values with only one maximum / one minimum point per X pixel plotted. The plotting of calculated DC Baseline, Amplitude and Slope lines is done in floating point on a conventional Cartesian cooardinate system. There may be slight inaccuracies due to floating point roundoff error so that at very low Yaxis sensitivities the DC Baseline, Amplitude and Slope lines drawn on the ADsweep waveform may appear to be one Y pixel off. This is not due to inaccuracy of calculation, but to inaccuracy of plotting.

To convince yourself of this, zoom in on the synaptic waveform of interest by increasing the Yaxis sensitivity as much as possible, and zooming in on the times wanted by setting the AutoReset Timbase capability to Off as suggested in Section 4.7 and Chapter 5.

A.3 When reanalyzing using ADsweep files taken with the Pico board, you should start the program with the Pico board on the command line.

This is just as you ran LTP113E when you were running the experiment in Real Mode DOS. You should do this for running the reanalysis in the both the Real Mode Dos and in the Windows DOS compatibility box. In other words, for doing ranalysis with AD sweeps taken with the Pico board start the LTP program this way:

The reason for this is that 1 bit of accuracy in the AD sweep data will be lost. However, otherwise the results from reanalysis will be exactly the same, and there is no catastrphic problem. This will be fixed in the next LTP version.

A.4 Time to save a 100,000 byte ADsweep file

Because LTP113E saves ADsweep data as ASCII text files, the time to save will be longer than for binary files. Generally this has not been a problem, except for needing to save fairly large files fairly quickly on slow computers. The following values give you a rule of thumb on how fast your computer can save the ASCII ADsweep files. Times are for saving extracellular files of a few mV in each sample (e.g. -x.xxxx mV or 8-9 characters (bytes) per sample when including newline and linefeed line termination characters). Patch clamp files with values in the 1000’s of pA’s per sample would take approximately 33% longer (e.g. -xxxx.xxxx pA or 11-12 bytes per sample when including newline and linefeed line termination characters). Also, the time required to save an ASCII ADsweep file using the 32-bit Windows 98 FAT file system is the same as using the DOS/Win 3.x/Win95/Win98 16-bit FAT file system.

The values for the 400 and 333 MHz Pentium II computers were obtained with LTP113E, all other values were obtained with LTP101M, which has the same code as LTP113E.

A.5 You must have either an S0 or S1 pulse in each ADsweep stimulation

In other words, you cannot set both S0=NONE and S1=NONE (and no Intracellular or Digital graphs) in a sweep in the Stimulation Protocol Dialog Box (Fig. 3.4.1).

A.6 Substantial Input and Output Sample-to-Sample jitter

The Labmaster, Digidata and Pico ADC-42 boards have a substantial amount of sample-to-sample jitter on analog input, and analog-digital output on very slow computers, but somewhat less jitter on faster computers. The sample-to-sample jitter can be approximately measured by measuring the duration of a ‘100 usec’ train pulse (see Section 2.8). Note that an analog input is directly coupled to analog-digital output, so measuring sample-to-sample jitter on output indicates analog input jitter. The measured sample-to-sample output jitter was:

1. Earlier version (LTP092K5), same code as LTP113E
2. The values for the 400 and 333 MHz Pentium II computers were obtained with LTP113E, all other values (unless stated otherwise) were obtained with LTP101M, which has the same code as LTP113E.

For example, on a Dell 486/66MHz with a Digidata board the duration of a ‘100 usec’ pulse can be anywhere from 98.5 usec to 101.5 usec.

For acquiring an analog signal every 100 usec or longer the 2-7 usec of jitter is probably no problem with the waveform analyses performed by LTP113E. The Peak Amplitude, DC Baseline, Cell Resistance and Series Resistance measurements are essentially independent of the 2-7 usec jitter, and the Slope measurement is also probably sufficiently independent of the jitter, because the slope is a linear regression line (least squares fit) calculated using many AD samples. For slope calculations lasting 0.6 to 2.0 msec (the range usually used in our group), 7 to 21 AD samples are used, respectively (sampling every 100 usec). However, each researcher will have to determine whether this level of input sample-to-sample jitter is acceptable in their particular experiment. If it is not acceptable, do not use the LTP113E program.

The same amount of sample-to-sample jitter on analog-digital stimulus pulse output (2-7 usec) is clearly acceptable for the 10’s to 100’s msec long intracellular stimulation pulses. Also, when the Stimulus Isolation Unit (SIU) is merely triggered by the stimulus pulse, the SIU pulse output duration is independently controlled by a knob on the SIU and there is again no problem. However, when the SIU pulse output duration is controlled by the duration of the stimulus pulse (analog controlled SIUs, and gated SIUs), 2-7 usec jitter in a 100 usec long stimulus pulse is probably too much when testing the responses to single pulses, particularly near threshold. However, it is up to each researcher to determine whether this level of output sample-to-sample jitter is acceptable in their particular experiment.

A.7 Fastest LTD Stimulation

The fastest LTP stimulation is dependent on how many samples the ADsweep contains, the type of computer (processor & MHz), whether a disk caching program such as SMARTDRV is installed, and the type of AD board. If a Digidata or Labmaster board is installed, LTD stimulation can be changed in gradations of 0.1 sec, therefore achieving LTD stimulation periods of 0.5 sec (e.g. 2.0 Hz) or faster. However, if a Pico42 board is installed, LTD stimulation can only be changed in gradations of 1.0 sec, therefore achieving LTD stimulation periods no faster than 1.0 sec (e.g. 1.0 Hz).

For computers saving a 50 msec long ADsweep, and with signal averaging on but only saving the averaged (not the raw) sweeps, the fastest LTD sweep frequency for computers having a Labmaster or Digidata board is approximately:

Test this out on your own computer. If you want to increase LTD stimulation frequency try:

1. Reducing the ADsweep length (say to 10-20 msec) so that just the rising phase of the synaptic potential is recorded and saved
2. Save ADsweep data to a RAM drive
3. Stop saving ADsweep data
4. Use a Single Train on a single long duration ADsweep (see Fig. 4.4.7).

A.8 Reanalysis cannot be performed from data written (once) to a read only CD-ROM (CD-R)

Reanalysis cannot be performed from data that, after being written once, can only be read from a CD-ROM (CD-R) or any other disk that cannot also be written to during reanalysis such as a WORM disk.

A.9 Some Sweep-to-Sweep Jitter During Rapid LTD Stimulation

There is also some jitter between the start of AD sweeps. For slow speed ADsweep stimulations (e.g. sweeps once every 15 seconds) this is clearly no problem. Even for higher speed LTD ADsweep stimulations (e.g. sweeps of 1-2 Hz or higher) this probably will not be a problem (although measure with an oscilloscope and look at the Amplitude/Calculation *.AM? file to make sure). The LTD sweep-to-sweep jitter presented below was measured as the jitter of the first pulse in a 50 msec long sweep. The signal averaging was On, but only the averaged (not the raw) sweeps were saved to disk), and values to the Amplitude/Calculation file were also saved to disk.

Measure this sweep-to-sweep jitter on your computer. If the jitter is too large:

1. Do a Single Train stimulation on a single long ADsweep (Fig. 4.4.7)
2. Alternatively, you may still be able to stimulate with ‘signal averaging on’ but just set the number of fast LTD sweeps to average (NumSweeps to Avg) to be greater than the number of fast LTD sweeps taken (Total Num Pulses). This will ensure there is no time consuming re-zeroing of arrays that occurs at the end of the sweep averaging.
3. Do your LTD stimulation with an auxiliary stimulator,

A.10 If Digital Output stimulation is ON, then all stimulation graphs (S0, S1, IC as well as digital output graphs) are plotted

A.11 Boundary Problems in Digital Filtering

Digital filtering is not accurate at beginning and ends of an AD sweep trace (where it filters to 0 mV or 0 pA). Because of this, Filtered traces are not presently saved, only Raw or Averaged traces can be saved. However, on-line analysis and off-line reanalysis can be done on Filtered traces provided care is taken not to use data points near either the beginning or end of the AD waveform.

A.12 Labmaster Time-Of-Day Clock

When using the Labmaster board, the Time-Of-Day timer will not work correctly for more than 24 hrs after staring the LTP program.

A.13 Computer / AD board combinations that are too slow

Some combinations of computers and AD boards may not be fast enough to sample at 100 usec ADsample interval. LTP113E works with a Labmaster board on all computers tested. LTP113E also works with a Digidata board on all computers tested, although it was not tested on 386 computers. LTP113E also works with a Pico board on all computers tested, with the one glaring exception of an Acer Pentium notebook computer! - although again it was not tested on 386 computers. This is a good example of how a 486 can be actually faster than a Pentium II with the Pico board!!!

To test the speed of your AD board enter the following at the DOS command line if you are using the Labmaster or Digidata board:

C:\YOURDATA\980704\ ltp113e speed

or this, if you are using the Labmaster or Pico board:

These are some of the results I found:

A.14 Errors occurring when the LTP113E program is started from the C:\ root directory

Even though I say start the program from a data directory you made (see Section 2.3), e.g.:

C:\LTP113E\950407>ltp113e

one researcher started the program in the C: root directory:

C:\>ltp113e

That person found that the LTP program stopped saving data after around 500 ADsweep files (meaning DATA LOSS!!!). Furthermore, when they tried to restart the program they got this INTERNAL ERROR message, and the program stopped:

INTERNAL ERROR: current dirname could not be obtained, exit program.

The error was actually was only due to the fact that the C:\ root directory can only store about 500 files. (In fact, if you save as many files as you can into the C:\ root directory with LTP113E, and then try to save 1 more with an ASCII text editor such as NOTEPAD.EXE, it will say: "Not enough disk space on drive C:" which will be patently false because megabytes of disk space can still be available.) So no more ADsweeps could be saved because the LTP113E program thought the disk was full!

The ‘workaround’ is don’t start the program from the root directory!

APPENDIX B KNOWN BUGS IN LTP113E

B.1 LTP113E cannot run on Tandon 25 or 33MHz 486 computers using the Pico board.

After the LTP113E program had been running for 5-60 minutes on Tandon 25 or 33MHz 486 computers using the Pico board, the TimeOfDay clock lost count. This meant that the time the ADsweep was taken was invalid, and the Amplitude/Slope calculations could not be properly plotted. However, Labmaster and Digidata boards have been used for several years on Tandon 25MHz 486s without any problems. If you are planning to use the Pico board, check to see that the TimeOfDay remains accurate for many, many hours (see Section 2.3.3 - note that the Pico board does not have to be installed to see that the TimeOfDay is operating correctly).

There is no workaround for using the Pico board on a Tandon 25 or 33 MHz 486 computer. Use a Digidata or Labmaster board instead.

B.2 Pressing the PgUp, PgDn, LeftArrow or RightArrow on some computers causes the LTP program to freeze

When you run a Repeat Slow Sweep protocol (Ctrl-F6) and you press the PgUp, PgDn, LeftArrow or RightArrow keys, the program "freezes" when it comes to do the next stimulation. Then if you hit any key, the next sweep is taken and the clock restarts at the point that it should be at (i.e. if it's frozen for about 5 s then the clock will read 5s when it restarts). The problem doesn't appear if you stay on the main view page and let it run. Once it's been unfrozen by hitting a key then it's fine again until you press a PgUp/PgDn/LeftArrow/RightArrow key again.

The only workaround I know is to use a different, more IBM compatible, computer.

B.3 Error with the TimeOfDay clock on a 200MHz Pentium using a Labmaster board.

The TimeOfDay clock loses track of time on one 200 MHz Pentium computer. The ‘solution’ was to use a Digidata board instead.

B.4 Error plotting AD sweeps on the screen and the LaserJet printer at very high Y-axis sensitivities

This is a display problem on an AD sweep graph (see Fig. 3.1.3) that occurs when the peak-to-peak Y-axis value (in mV or pA) is very small (e.g. sensitivity is high). Generally this is 100 to 1000 times more sensitive than values normally used and occurs at about the level that individual bits (voltage digitization) is seen. The problem is that lines extending beyond the minimum or maximum Y values of the graph are not clipped correctly. This is an error in the graphics display and is not a corruption of the internal data, nor is the data output to the ASCII ADsweep files corrupted.

B.5 On a slow 25MHz 486 computer, pressing a key just before a P0 or P1 ADsweep is about to go can cause an immediate next ADsweep with no delay

This usually happens when a Carriage Return has been pressed to enter a new field or a PgUp or PgDn key has been pressed to show a new window or page. The workaround is to not press a Carriage Return or PgUp/PgDn key just before a sweep is about to be generated.

This has not been noticed on faster computers.

B.6 Sometimes when changing which Amplitude/Slope calculations are to be graphed in the Calculation Page, the appropriate Calculation Graph is not drawn.

If this occurs try saving, and then reloading the protocol file, or try temporarily putting in another Calculation Graph in the AmpFile -> CalculationsToDo dialog box. This error is rare and pretty benign.

APPENDIX C PICO TECHNOLOGY’S ADC-12 BOARD

The Pico ADC-12 board was actually my first low cost AD board. However, the Pico ADC-12 board (which has an input voltage range of 0 to 5 volts) has therefore been replaced by the Pico ADC-42 board (which has an in input voltage range of -5 to +5 volts).

However, there are several Pico ADC-12 boards in use and they will continue to be supported. To use a Pico ADC-12 board, a +2.5 volt offset must to be added to the analog input signal, and this +2.5 volt offset will be calculated as ‘virtual’ 0 volts, and the ‘virtual’ input voltage range of the Pico ADC-12 will therefore be -2.5 to 2.5 volts. Because this offset can be slightly confusing in situations where DC level is important, we are presently only using the Pico ADC-12 board in extracellular LTP experiments.

Like the Pico ADC-42 board, the Pico ADC-12 cannot be automatically detected by the LTP113E program and an additional word must be added to the command line when LTP113E is started in order to use the Pico ADC-12. If the Pico ADC-12 board is attached to the LPT1 port and digital output is also through LPT1 (or LPT2 or LPT3), use the following command:

If the Pico ADC-12 board is attached to the LPT1 port and digital output is through the LPT2 port, (or LPT2 and LPT3) use the following command:

For more information on using the Pico ADC-12 board, see the discussion of the Pico ADC-42 (Section 2.3.3).

APPENDIX D FUTURE LTP PROGRAM CAPABILITIES

D.1.0 LTP version 2.0 (new ADsweep file format)

1. Simultaneous 2 AD channel (1 Intracellular / 1 Extracellular) acquisition; measuring amplitudes/Slopes on 2 AD channels
2. More accurate on-line Rs measurements, which will require higher sampling (20 us rather than 100 us) and many short, averaged Rs pulses.

D.1.1 LTP version 2.1 (Grease Gap experiments, Ischemia)

1. Continuous DC chart recording

D.2.0 LTP version 3.0 (Epilepsy)

1. Capture Spontaneous bursts
2. Saving long ADsweeps in binary (pClamp compatible) files

D.2.1 LTP version 3.1

1. Cheap multi-channel AD board for the PCI bus (to work with PC99 specification computers that do not contain the ISA bus).

D.2.2 LTP version 3.2

1. Analyze many S0 and/or S1 synaptic potentials during one ADsweep.
2. Off-line signal averaging
3. On-line digital filtering while averaging sweeps
4. Work seamlessly in Windows 95/98
5. Fix digital filter boundary problems
6. Autodetect & set ADgain for Axon Instruments AxoPatch

D.3.0 LTP version 4.0

1. Increment/Decrement Protocols to run Input / Output (Stimulus Amplitude / EPSP amplitude) curves, or synaptic and cell I-V curves

D.4 The ISA bus is becoming obsolete

Microsoft and Intel have announced in their PC98 and PC99 specifications that the ISA bus (the bus you plug your Labmaster and Digidata boards into) is going to be dropped. In the PC98 specification (to take effect in July-Dec, 1998) removing the ISA bus is voluntary. In the PC99 specification, removing the ISA bus is manditory (see PC Magazine UK, p.29, July 1997; PC Magazine US, p93, Feb 1998; and PC Magazine UK, p.148, Aug 1998).

This means that LTP113E will not work with Labmaster or Digidata boards in those advanced PC99 compliant computers because these boards require the ISA bus. The only board I currently support that will work in PC99 computers is the Pico board. The advanced PC99 compliant computers will use the PCI bus, and therefore I will try to put in an inexpensive PCI AD board in these ISA bus-less computers.

Unfortunately, DOS is not the only thing that’s getting obsolete these days.

D.5 Windows

The ‘Real’ MS-DOS that the current LTP program needs will be in Windows 98, and Windows 98 will be in common usage for probably another three years. I do not know whether the MS-DOS Mode will be present in the ‘Windows 2000’ (more like ‘2001’ or ‘2002’!) that will result from the merger of Window 95/98 into Windows NT (it will be more like NT than 95/98). That essentially depends on whether home users will still want to run their old 16 and 32-bit DOS games in the fast ‘Real’ MS-DOS Mode of their computer.

Why haven’t I written LTP as a Windows 95/98 program. Actually, the DOS LTP program has been in use in the Bristol Synaptic Plasticity group for over 3 years now, longer than Windows 95 has been out. I decided to continue with DOS development for five reasons:

1. The current LTP program does run on Windows 95/98 computers.
2. A lot of the background code for data acquisition and user interface had already been developed in DOS (the LTP program actually started out as a combination stimulator and epilepsy program long before that).
3. Only Windows 3.1 was available when the LTP program needed to be ready for our group (three years ago), and as far as I’m concerned Windows 3.1 wasn’t very good for data acquisition programs.
4. I didn’t think that an LTP program would use much of the extra capability of Windows.
5. I knew that with DOS I could make the LTP program work with several inexpensive data acquisition boards (for DOS they can be much simpler than for Windows). This makes the total cost of LTP data acquisition software and hardware much cheaper and affordable to almost all LTP researchers.

I don’t know whether I will develop a ‘Windows 2000’ version of the LTP program. If I do, I’m not sure whether I would develop it at the University of Bristol or on my own time, or I may combine my LTP and stimulator code with another commercial or non-commercial Windows data acquisition program. I admit I would like to have a Windows LTP program. The primary reason to develop a Windows ‘2000’ LTP program is that the MS-DOS Mode may not be in ‘Windows 2000’. It would also be nice to have mouse input, have a modern GUI interface, and do word processing on your data acquisition computer as your experiment was running. However, the need to do multitasking data acquisition and analysis is not valid because LTP113E can do that already (see Section 6.3).

I also do not know whether a ‘Windows 2000’ LTP program would continue to be free to academic users. As far as I’m concerned, the main reason to make a data acquisition program free is if the total cost of doing data acquisition (including computer, program and data acquisition board) is substantially reduced. With DOS I can make an LTP system (program and data acquisition board) for a fraction of the cost of a Windows or Macintosh LTP system. In Windows, with the cost of data acquisition boards being several thousand dollars (most so-called Windows data acquisition boards are not adequate), an inexpensive LTP system is presently not achievable. My primary concern is that the researchers without the big grants can do LTP data acquisition inexpensively. For those researchers with big grants, the only difference of whether a program is free or not is merely the inconvenience of having to buy it.

APPENDIX E UPGRADING FROM LTP101M TO LTP113E

1. If you are upgrading from earlier versions of the LTP program, you will have write new *.pro protocol files. The newer LTP113E program will not load protocol files you made using a previous LTP program for safety reasons.

2. Slightly more RAM memory is needed

LTP101M needed at least 4.2 MB of extended (XMS) memory; LTP113E needs at least 4.4MB of extended memory. Computers now with at least 8 MB seem to be required.

3. Known Bugs in LTP101M (from the LTP101M manual) that are now fixed in LTP113E

B.1 When changing some stimulation protocols with the File->Stimulation Protocol dialog box, the correct new pulse regime or stimulation graph does not appear.

B.3 Sometimes not all values are initialized correctly on start-up with a protocol file.

B.4 Pressing F4 (to Stop) during an ADsweep will cause the Program to hang up when ADsweep duration is longer than Time that hitting keyboard stops ADsweeps (Fig. 3.4.5C).

B.8 If a computer is running with a Digidata board installed and you want to do reanalysis in a Windows DOS compatibility box, LTP113E knows it doesn’t need to acquire data but it proceeds to test the speed of the Digidata board anyway. LTP113E then finds the Digidata board slow (e.g. > 95usec per sample, because it is being tested in the DOS compatibility box) and LTP113E terminates itself.

B.10 Error occurring during the start-up of LTP113E that incorrectly indicates the AD board is too slow

4. The following bugs in LTP101M no longer seem to be a problem in LTP113E

B.6 Sometimes the Peak Amplitude, Slope or other calculation points are not plotted.

B.7 When the Pico board is running, trying to plot an pulse ADsweep file hangs up the computer.

B.11 Incorrect plotting on a LaserJet printer with the AutoReset Off, Minimum Time not zero, and Sweep Duration less than maximum

B.13 Error with one Dell 133MHz Pentium P133s with a Digidata Board

B.15 Changing the Digital Filtering Cuttoff Freq Field sometimes doesn’t cause the filtered line to be replotted or the Amp/Slope values to be recalculated

B.16 Sometimes the AmpSlope Values Y/N Field doesn’t work on N

5. The following LTP101M bugs are now considered limitations

B.2 You must have either an S0 or S1 pulse in each ADsweep stimulation (However, in LTP113E it is now impossible to set both S0=NONE and S1=NONE (and no Intracellular or Digital graphs) in a sweep in the Stimulation Protocol Dialog Box (Fig. 3.4.1) ).

B.9 Errors occurring when the LTP113E program is started from the C:\ root directory

6. The following limitations in LTP101M that have been removed from LTP113E

A.11 Only Default factory settings were used for the Base Address of the data acquisition boards

7. The following additional bugs were found in LP101M and have been fixed in LTP113E

In LTP101M, the analog outputs from AnalogOut0 and AnalogOut1 on the Digidata board were too large by 2.4%. This has been corrected in LTP113E.

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