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The relevance of computer techniques in microiontophoretic experiments
THE
RELEVANCE OF COMPUTER TECHNIQUES MICROIONTOPHORETIC EXPERIMENTS
IN
G. CLARKE and R. G. HILL* Department
of Pharmacology,
The School of Pharmacy, University London WCIN IAX
of London.
19139 Brunswick
Square.
Little of the work published to date in the field of microiontophoresis has involved the use of computing techniques. Many people have gone some way towards a computer based approach by using logic timing circuits for drug switching and hard wired signal averagers for data evaluation, but possibly feel that there is little to be gained from having a computer in the laboratory. We would like to put forward the view that computers are useful tools in our type of work, but also stress that these machines are not a panacea. The chief attributes of a computer, from the point of view of datd reduction and data evaluation, are that data can be acquired rapidly; it can also be manipulated rapidly and with great accuracy and these procedures are not subject to bias. A peripheral benefit is that tedium is relieved, especially when the experimental protocol requires repetitive measurements. As a computer does not suffer from loss of concentration, long experiments are more likely to provide meaningful results, as the experimenter is freed from routine monitoring and can take a more objective view of the experimental situation. The chief disadvantage of computers, at the moment, is their high initial cost, although this is falling all the time with advances in integrated circuit technology. Other points worth emphasising relate to the particular type of machine involved. For example, a hard wired device, that is to say one that is pre-programmed with individual programmes selected by switches, is very easy and convenient to use; the main disadvantage is that the wired-in programmes seldom do precisely what is required and the user has no way of altering them. However. a machine that uses software, that is programmes that can be written and altered by the experimenter, is capable of almost any operation up to the limits of the programmer’s imagination. The drawback in this case is that writing programmes takes time, chiefly because, in order to preserve the machine’s operating speed, programming is in machine code rather than a high level language, Overall. we feel the balance swings in favour of a computer that utilizes software since most people do not really know the precise nature of the programme they need until the one they are using falls short of the experimental objective. With a hard wired machine there is no choice but to make JO with an inadequate programme; with software one can alter it at will. The ideal device would fall between the two extremes, and appearing on the market at the moment are machines with a combination of hard-wired and programmable features that will obviously be of great interest to workers in our field. Our idea of the basic configuration for a laboratory computer is summarized in Figure 1. The central processor and memory are common to all machines; suffice it to say that * Present address: Bristol HSX ITD.
Department
of Pharmacology.
University 565
of Bristol, The Medical
School,
University
Walk.
G.
566
CLARKI.and R. G. HIM Central processor ond IllfSlO~~ Moss
Reol time clock
I Working
t
storage Input/output device
register
I
Fig. 1. Basic con~~ur~~tion for a laboratory -~--d@tal
Data from the experiment
ADC-analogoe to dipital converter: DAC converter. voltage to experiment from computer leaves at arrow.
computer: t0 awtlngw
enters at upward arrows, downward
enough memory should be available for both programmes and experimental data (we are working at the moment with 8 K of core memory and consider that to be sufficient for most purposes). An essential* in our view, is a mass storage device. We use a magnetic tape unit but disc or drum devices are equally useful. Plotting or punching out data and results during an experiment is inefficient and time consuming. whereas intermediate storage on a device such as magnetic tape is quick and allows immediate re-inspection of data and results under programme control. An input~output device allows commllnication with the computer during an experiment. there being various ways of achieving this. The usual method is via a Teletype: consolc switches may also be useful. In our type of work, the results arc very often in the form of a histogram or other graphical display. It is therefore very useful to have an oscilloscope display on which to observe the development of the experiment and this is interfaced to the central processor via digital to analogue converters (DAC) which may also be used for applying voltages back into the experiment from the computer. The experiment itself is connected at the points indicated by arrows. If the data is in the form of trains of pulses, as it so often is, then it is usual to take it in via a programmable real-time clock. This device both synchronises the central processor and allows timerelated counts to be fed to the working register. If the data is in analogue form. then input is via analogue to digital converters (ADC) which provide numhcrs for the ccutral proccssor to work on. Most modern laboratory computers are modular in construction and peripherals can be added for special purposes or left out if not needed for a more limited application. The type of machine outlined in the block diagram has proved very flexible and more than adequate for our purposes. We have found a computer to be particularly useful in our investigations of the relationship between the convulsive EEG and the firing patterns of single neurones in the cerebral cortex. A combination of signal averaging and post-stimulus time histograms has enabled us to correlate events easily and with a fair degree of precision. Typical results from experiments of this type are shown in Figure 2. A note of caution should he obscrvcd hcrc. how-
Computers
in microiontophore~is
CELL 4
HISTOGRAM OF NEURONAL ACTIVITY
Fig. 2. The left hand side of the figure shows computer generated averages and histograms from a cat with a focal epileptogenic lesion produced by microinjection of penicillin to the cerebral cortex. In the construction of the post stimulus histograms, actlon potential counts were accumulated in 1000 consecutive bins of computer memory for SO sweeps: duration MOmsec. The sweeps were triggered from the amplified ECoG spikes recorded from the microinjection cnnnula in the focus: I I; sweeps of this signal were also averaged and displayed above the histogram. Histogram calibration: horizontal 100 mscc; \erticul 5 counts per bin. The upper trace shows a neurone (cell 4) that is inhibited by the rpileptiform discharge and the lower shows another neurone (cell 7) that is predominantly excited. Photographic records tori-esponding to these computer plots xe shown to the right of the figure. Each photograph is 10 superilnposed s~vccps. rriggered as before. calibration; horizontal 200 msec. vertical 200 pV. although action potential from more than one neurone are visible in the photographs. only the neurone with the hqest action potentials was used to construct the computer post stimulus histogram\.
ever. as such averaged displays may be from a number of neurones. rather than one. Additionally it would be very easy to treat a variation in spike height as a variation in frequency due to the method of input used for the data (i.e. Schmitt triggers). It is always worth including photographic records (as in Fig. 2) in substantiation of the computer generated plot. Information on the trend of firing pattern during the plotting of histograms such as these may well be lost. In cases where such variation is expected. then the type of raster display shown by KELLY and Renaud (1974) is a preferable method of analysis.
568
G. CLAKM and R. G. HILL
Fig. 3. Flow chart. The computer stays in display mode until the occurrence of a timing pulse. This pulse increments a running total which is used to time each counting epoch. Computer operated ~-clays are used to switch the iontophoresis board for applying drugs; the period of each application being a multiple of the counting epoch.
Such histogramming methods as these are well known and it is more likely that advances in the application of computing techniques will be made in the field of experimental control. An example of this type of approach is the iontophoretic control programme generated in our laboratory. It is often necessary in microiontophoretic experiments to alternately reverse the current flow through two or more barrels of a micropipette whilst maintaining a rigid time cycle. This mode of operation is ideally suited for electronic control, since accurate time cycles can be adhered to for asmany combinations as might be required. Thus, provided such an clectronic system has facilities for human interruption, the burden of potentially confusing and fatiguing manual operations can be alleviated. This allows the operator more time to concentrate on the results being obtained, thus enabling critical decisions to be made more easily. Several forms of electronic and mechanical timing devices have been used, with reasonable success, to control microiontophoretic experiments. However, these have mostly been “hardwired” systems with an associated limited number of combinations available for the timing of drug cycles. Since a digital computer requires a number. rather than a switch
setting, to define each parameter, the number of variations and combinations is greatly increased. We are currently using a digital computer to control microiontophoretic experiments. The neuronal action potentials occurring in short time periods or epochs are counted and the values obtained are plotted as a continuous histogram. the duration of each drug application being a multiple of the number of counting epochs. The control programme (see Fig. 3) uses a timing pulse generated every IOmsec by the computer. the timing pulstx being used to generate counting epochs of any desired length. Different channels of the iontophoresis board can be switched on and off via relays. using the counting epochs to generate the drug application cycle. Alternatively. the operator can terminate a drug application prematurely after which the drug cycle will continue. Such a timing process can be used for several drugs alternately or in sequence. At all times. the current values in each counter and the state of the iontophoresis board are displayed on the computer oscilloscope. Although this control programme greatly reduces the task of the operator the advantagcs do not li111) componsatc for the cost of the computer. not- for the time initiall~~ dcdcated to writing the programme. However. it would be beneficial if the results could be analysed simultaneously. since drug effects could be determined more rapidly. Currently. much experimental time is wasted in having to obtain gross effects in order to be sure of a significant change. An ideal system might apply confidence limits to each response as it was obtained. thus enabling small but significant changes in such a response to be rapidly determined. However, the programme needs to be complex in order to make rapid calculations whilst controlling an assortment of laboratory equipment. A study is currently being made in our laboratory to determine the feasibility of simultaneous computer control and analysis. The next stage of development, if both systems prove viable, is the interactive combination of the two programmes. Thus. using a system of feedback control, the computer could decide how much drug to apply to obtain a significant result. However, the onus on the operator not to lose sight of the original objectives would be even greater. After all. a computer can only make the decisions for which it is programmed. The role of the computer in microiontophoretic experiments must be to assist and not to rule. il~~,lo~~Ic~dqc~,,l~nr.s- The authors are grateful to Professor D. W. STRALGHAN for allowing them the USCof ;I BIOMAC 1001 (purchased bj the MRC) and 3 PDP-I?, 30 (purchLlsed by the Wellcome Trust). R.G.H. i.\ supported by the bequest of W. L. Cust:lnce.
KI-LLY. J. S. ud trucii~tc cork\.
REFERENCE RI \.%L;I). L. P. (1973). Physiological idcntiiication ~~,~fi,~~/‘/i~,i.,r~i~c II/~J,/I’ 13: 467 174
of inhibitory
interneuroncs
in the feline pcric-
RELEVANCE OF COMPUTER TECHNIQUES MICROIONTOPHORETIC EXPERIMENTS
IN
G. CLARKE and R. G. HILL* Department
of Pharmacology,
The School of Pharmacy, University London WCIN IAX
of London.
19139 Brunswick
Square.
Little of the work published to date in the field of microiontophoresis has involved the use of computing techniques. Many people have gone some way towards a computer based approach by using logic timing circuits for drug switching and hard wired signal averagers for data evaluation, but possibly feel that there is little to be gained from having a computer in the laboratory. We would like to put forward the view that computers are useful tools in our type of work, but also stress that these machines are not a panacea. The chief attributes of a computer, from the point of view of datd reduction and data evaluation, are that data can be acquired rapidly; it can also be manipulated rapidly and with great accuracy and these procedures are not subject to bias. A peripheral benefit is that tedium is relieved, especially when the experimental protocol requires repetitive measurements. As a computer does not suffer from loss of concentration, long experiments are more likely to provide meaningful results, as the experimenter is freed from routine monitoring and can take a more objective view of the experimental situation. The chief disadvantage of computers, at the moment, is their high initial cost, although this is falling all the time with advances in integrated circuit technology. Other points worth emphasising relate to the particular type of machine involved. For example, a hard wired device, that is to say one that is pre-programmed with individual programmes selected by switches, is very easy and convenient to use; the main disadvantage is that the wired-in programmes seldom do precisely what is required and the user has no way of altering them. However. a machine that uses software, that is programmes that can be written and altered by the experimenter, is capable of almost any operation up to the limits of the programmer’s imagination. The drawback in this case is that writing programmes takes time, chiefly because, in order to preserve the machine’s operating speed, programming is in machine code rather than a high level language, Overall. we feel the balance swings in favour of a computer that utilizes software since most people do not really know the precise nature of the programme they need until the one they are using falls short of the experimental objective. With a hard wired machine there is no choice but to make JO with an inadequate programme; with software one can alter it at will. The ideal device would fall between the two extremes, and appearing on the market at the moment are machines with a combination of hard-wired and programmable features that will obviously be of great interest to workers in our field. Our idea of the basic configuration for a laboratory computer is summarized in Figure 1. The central processor and memory are common to all machines; suffice it to say that * Present address: Bristol HSX ITD.
Department
of Pharmacology.
University 565
of Bristol, The Medical
School,
University
Walk.
G.
566
CLARKI.and R. G. HIM Central processor ond IllfSlO~~ Moss
Reol time clock
I Working
t
storage Input/output device
register
I
Fig. 1. Basic con~~ur~~tion for a laboratory -~--d@tal
Data from the experiment
ADC-analogoe to dipital converter: DAC converter. voltage to experiment from computer leaves at arrow.
computer: t0 awtlngw
enters at upward arrows, downward
enough memory should be available for both programmes and experimental data (we are working at the moment with 8 K of core memory and consider that to be sufficient for most purposes). An essential* in our view, is a mass storage device. We use a magnetic tape unit but disc or drum devices are equally useful. Plotting or punching out data and results during an experiment is inefficient and time consuming. whereas intermediate storage on a device such as magnetic tape is quick and allows immediate re-inspection of data and results under programme control. An input~output device allows commllnication with the computer during an experiment. there being various ways of achieving this. The usual method is via a Teletype: consolc switches may also be useful. In our type of work, the results arc very often in the form of a histogram or other graphical display. It is therefore very useful to have an oscilloscope display on which to observe the development of the experiment and this is interfaced to the central processor via digital to analogue converters (DAC) which may also be used for applying voltages back into the experiment from the computer. The experiment itself is connected at the points indicated by arrows. If the data is in the form of trains of pulses, as it so often is, then it is usual to take it in via a programmable real-time clock. This device both synchronises the central processor and allows timerelated counts to be fed to the working register. If the data is in analogue form. then input is via analogue to digital converters (ADC) which provide numhcrs for the ccutral proccssor to work on. Most modern laboratory computers are modular in construction and peripherals can be added for special purposes or left out if not needed for a more limited application. The type of machine outlined in the block diagram has proved very flexible and more than adequate for our purposes. We have found a computer to be particularly useful in our investigations of the relationship between the convulsive EEG and the firing patterns of single neurones in the cerebral cortex. A combination of signal averaging and post-stimulus time histograms has enabled us to correlate events easily and with a fair degree of precision. Typical results from experiments of this type are shown in Figure 2. A note of caution should he obscrvcd hcrc. how-
Computers
in microiontophore~is
CELL 4
HISTOGRAM OF NEURONAL ACTIVITY
Fig. 2. The left hand side of the figure shows computer generated averages and histograms from a cat with a focal epileptogenic lesion produced by microinjection of penicillin to the cerebral cortex. In the construction of the post stimulus histograms, actlon potential counts were accumulated in 1000 consecutive bins of computer memory for SO sweeps: duration MOmsec. The sweeps were triggered from the amplified ECoG spikes recorded from the microinjection cnnnula in the focus: I I; sweeps of this signal were also averaged and displayed above the histogram. Histogram calibration: horizontal 100 mscc; \erticul 5 counts per bin. The upper trace shows a neurone (cell 4) that is inhibited by the rpileptiform discharge and the lower shows another neurone (cell 7) that is predominantly excited. Photographic records tori-esponding to these computer plots xe shown to the right of the figure. Each photograph is 10 superilnposed s~vccps. rriggered as before. calibration; horizontal 200 msec. vertical 200 pV. although action potential from more than one neurone are visible in the photographs. only the neurone with the hqest action potentials was used to construct the computer post stimulus histogram\.
ever. as such averaged displays may be from a number of neurones. rather than one. Additionally it would be very easy to treat a variation in spike height as a variation in frequency due to the method of input used for the data (i.e. Schmitt triggers). It is always worth including photographic records (as in Fig. 2) in substantiation of the computer generated plot. Information on the trend of firing pattern during the plotting of histograms such as these may well be lost. In cases where such variation is expected. then the type of raster display shown by KELLY and Renaud (1974) is a preferable method of analysis.
568
G. CLAKM and R. G. HILL
Fig. 3. Flow chart. The computer stays in display mode until the occurrence of a timing pulse. This pulse increments a running total which is used to time each counting epoch. Computer operated ~-clays are used to switch the iontophoresis board for applying drugs; the period of each application being a multiple of the counting epoch.
Such histogramming methods as these are well known and it is more likely that advances in the application of computing techniques will be made in the field of experimental control. An example of this type of approach is the iontophoretic control programme generated in our laboratory. It is often necessary in microiontophoretic experiments to alternately reverse the current flow through two or more barrels of a micropipette whilst maintaining a rigid time cycle. This mode of operation is ideally suited for electronic control, since accurate time cycles can be adhered to for asmany combinations as might be required. Thus, provided such an clectronic system has facilities for human interruption, the burden of potentially confusing and fatiguing manual operations can be alleviated. This allows the operator more time to concentrate on the results being obtained, thus enabling critical decisions to be made more easily. Several forms of electronic and mechanical timing devices have been used, with reasonable success, to control microiontophoretic experiments. However, these have mostly been “hardwired” systems with an associated limited number of combinations available for the timing of drug cycles. Since a digital computer requires a number. rather than a switch
setting, to define each parameter, the number of variations and combinations is greatly increased. We are currently using a digital computer to control microiontophoretic experiments. The neuronal action potentials occurring in short time periods or epochs are counted and the values obtained are plotted as a continuous histogram. the duration of each drug application being a multiple of the number of counting epochs. The control programme (see Fig. 3) uses a timing pulse generated every IOmsec by the computer. the timing pulstx being used to generate counting epochs of any desired length. Different channels of the iontophoresis board can be switched on and off via relays. using the counting epochs to generate the drug application cycle. Alternatively. the operator can terminate a drug application prematurely after which the drug cycle will continue. Such a timing process can be used for several drugs alternately or in sequence. At all times. the current values in each counter and the state of the iontophoresis board are displayed on the computer oscilloscope. Although this control programme greatly reduces the task of the operator the advantagcs do not li111) componsatc for the cost of the computer. not- for the time initiall~~ dcdcated to writing the programme. However. it would be beneficial if the results could be analysed simultaneously. since drug effects could be determined more rapidly. Currently. much experimental time is wasted in having to obtain gross effects in order to be sure of a significant change. An ideal system might apply confidence limits to each response as it was obtained. thus enabling small but significant changes in such a response to be rapidly determined. However, the programme needs to be complex in order to make rapid calculations whilst controlling an assortment of laboratory equipment. A study is currently being made in our laboratory to determine the feasibility of simultaneous computer control and analysis. The next stage of development, if both systems prove viable, is the interactive combination of the two programmes. Thus. using a system of feedback control, the computer could decide how much drug to apply to obtain a significant result. However, the onus on the operator not to lose sight of the original objectives would be even greater. After all. a computer can only make the decisions for which it is programmed. The role of the computer in microiontophoretic experiments must be to assist and not to rule. il~~,lo~~Ic~dqc~,,l~nr.s- The authors are grateful to Professor D. W. STRALGHAN for allowing them the USCof ;I BIOMAC 1001 (purchased bj the MRC) and 3 PDP-I?, 30 (purchLlsed by the Wellcome Trust). R.G.H. i.\ supported by the bequest of W. L. Cust:lnce.
KI-LLY. J. S. ud trucii~tc cork\.
REFERENCE RI \.%L;I). L. P. (1973). Physiological idcntiiication ~~,~fi,~~/‘/i~,i.,r~i~c II/~J,/I’ 13: 467 174
of inhibitory
interneuroncs
in the feline pcric-