A fully automated open-field apparatus incorporating rearing detection

A fully automated open-field apparatus incorporating rearing detection

Physiology & Behavior, Vol. 26, pp. 741-746, 1981. Pergamon Press and Brain Research Publ. Printed in the U.S.A. A Fully Automated Open-Field Apparat...

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Physiology & Behavior, Vol. 26, pp. 741-746, 1981. Pergamon Press and Brain Research Publ. Printed in the U.S.A.

A Fully Automated Open-Field Apparatus Incorporating Rearing Detection P. T. T O M K I N S 1 A N D D. J. O ' D O N O V A N

Physiology Department, University College, Galway, Ireland Received 28 February 1980 TOMKINS, P. T. AND D. J. O'DONOVAN. A fully automated open-field apparatus incorporating rearing detection. PHYSIOL. BEHAV. 26(4) 741-746, 1981.--An open-field instrument is described utilising a versatile rsistance detection system for measuring ambulation in mice and rats. The ambulation detector may sense horizontal and vertical ambulation or horizontal, vertical and diagonal ambulation in the ground plane, depending on mode selection. In addition, it can respond to an animal's presence on particular plates or areas in a 'touch toggle' fashion. A separate circuit records height selected rears by way of capacitance effects. The rearing detection surface consists of a moveable perforated metal grid above the open-field. The complete open-field system is controlled by a process-timer and facilities for 'real time' recording are incorporated. Details of accuracy, reliability and mode of use are reported.

Open-field

Automated

Rearing

Grid

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THE OPEN-FIELD test, which originated over forty years ago, is now employed frequently in animal psychology studies. In recent years it has been adopted by disciplines as diverse as physiology, pharmacology and genetics. When employed in its simplest form the test can become tiring and tedious for the observer, and a degree of subjectivity and bias may be introduced into the collection of results. Obviously if the grosser forms of behaviour such as walking, running, rearing and inactivity can be recorded automatically, then the observer is free to note the more subtle incidents such as grooming, sniffing, scratching and vocalisation. This will reduce the strain on the observer, increase throughput of subjects and make data collection more exact and consistent. A number of activity monitors and automated open-field designs have been described [1, 2, 3, 5, 6, 10, 13, 14, 15, 16, 17, 18]. These range from very simple mechanical designs using the jiggle cage principle to rather sophisticated mechanical systems employing pressure pads and a variety of resistance and capacitance operated touch and proximity sensors [1, 5, 6, 10, 13]. Light and sound activated systems [14,17] also feature prominently in the literature. Activity measurements and the open-field test have been recently reviewed [19]. Commercial activity monitors are generally not suitable for use in the open-field situation as well as being very expensive. The mechanical designs published while offering the advantage of simplicity over their electronic counterparts suffer from the need for rather precise methods of construction, tedious re-calibration before use and generally exhibit a reduction in sensitivity and rapid physical deterioration with repeated use and washings. Published electronic designs have exhibited a number of

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design flaws concerning sensitivity, effects of moisture, mains and r.f. interference. Furthermore most open-field designs published to date have only measured ambulation and associated locomotory behaviours. DESIGN AND CONSTRUCTION

The ambulation detector may be used in three different modes, four input resistance detection (Mode 1), four input individual touch detection (Mode 2) or two input resistance detection (Mode 3). The complete machine is controlled by a process-timer being switched on and off for the selected time, it may be interfaced with a number of recording devices including a suitable micro-computer. It is possible to keep the overall cost, excluding process timer and recording equipment to under fifty pounds. Twenty five 178 mmx 178 minx 1.6 mm aluminium plates mounted on 178 mmx178 minx6.35 mm explanded polystyrene bases are arranged in a 5 x 5 pattern and separated from each other by 6.35 mmx 11.2 mm perspex spacers in a 0.914 mx0.914 mx0.3 m (ID) blockboard and plywood box. The perspex spacers are covered with white plastic rails 12.7 mmx6.35 mm made from lengths of dual sliding door track. This combination prevents urine bridging plates. The whole assembly is held in place by means of a 'tight fit' and clear epoxy resin (see Fig. 4). The walls of the open-field are covered with a white laminate and all edges and corners are filled and sealed. Each plate contains one or more 6 B A x 12.7 mm countersunk bolts with solder tag. Underboard connections are made via these tags and associated screw terminal blocks. A wire from each plate is fed to a patch board on the side of the open-field. The patch board utilises 50 colour coded 2 mm sockets housed in

1To whom reprint requests should be addressed.

Copyright © 1981 Brain Research Publications Inc.--0031-9384/81/040741-06502.00/0

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a suitable vero box. Various plate electrical arrangements can be realised by interconnecting these sockets in the desired manner. F o r example in Mode 1 all A plates can be wired together at the patch board and similarly for B, C and D, composite ' A ' , ' B ' , ' C ' and ' D ' inputs are then fed to the ambulation detector. The rearing detector consists of a 0.914 mx0.914 m perforated zinc grid (mesh size~10) stretched on a wood and stainless steel frame mounted on top of the open-field and hinged at one side. A conveniently placed handle allows easy manipulation while a short soldered lead runs from the grid to the rearing detector. The whole machine is mounted on plastic castors to allow for ease o f cleaning and removal. Reference to Fig. 1 the block diagram indicates the basis of operation. Ambulation Detector Mode 1. In four-input resistance mode the principle is similar to that used in [5]. A potential divider is balanced via VRI so that respective potentials at point ADO V, B = - 1.5 V, C = - 3 . 0 V, and D = - 4 . 5 V (Fig. 2). The detection point at A runs into "a comparator ICIa based on a 3900 currentdifferencing amplifier, the reference current being set by VR2 and VR3.

When an animal is placed across any combination of two plates A - D (and their multiples) its extra resistance unbalances the divider such that point ' A ' moves from OV to a more positive potential. When this reaches or exceeds the reference current, the comparator output goes high and stays high as long as contact is maintained. This signal is filtered by a low-pass filter ICIb, where: K - C2/C1 = 1 The upper cut off frequency=fch= 1

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display hold functions of the counter are controlled by the process-timer. The trigger output also clocks the input of a 4013B flipflop, configured for T-type toggle action. In our application the bistable operates a reed relay driven by Darlington pair TRI and TR2; an LED may be switched in here to indicate operation of the field when no recording equipment is connected. In its simplest form we have used operation of the flip-flop to switch 9 V into an oscillograph recorder. Mode 2. Single plate touch toggle action may be obtained using the above circuit by careful adjustment of VR2 and VR3 until the comparator is just on edge. Inputs A-D will then be found to have independent, individual touch toggle action. This effect is due to the experimental subject acting as an aerial for mains 50 Hz hum. On touching a plate A-D, 50 Hz noise is coupled to the high impedance input lines, thus switching the 'sensitive' comparator. Its action is obviously reduced if the subject and/or the device is grounded. With this arrangement it is necessary to ensure good layout and adequate screening to prevent interference by or to the circuit. Obvious uses for such single plate activation are in the detection of animals on particular plates, e.g., start/goal plates in a maze or the walls of the open-field for wall/escape rears. Should this particular attribute of the instrument not be required at all, then a capacitor approximately 0.1 p.F inserted between the 'A' detection point and the grounded negative supply will effectively remove most hum coupling. It may be more convenient to arrange for this capacitor to be switched in and out as required (see Fig. 2).

Mode 3. If the plates are wired in an ABABA manner [ 13] and the field A and B outputs fed directly to A' B' then the device will score all horizontal and vertical ambulation. It will not respond to diagonal activity since this is between plates of the same potential. Inputs are fed directly to the filter front end by disconnecting the comparator/divider network via 1C2, a 4016B quad bilateral switch. The combined control points of IC2 are turned on and offby $3, a single-pole changeover switch coupled to the supply rails. When an animal is placed across an AJB junction, the small leakage current that flows through the subject is amplified by ICIb. With gain adjustable by VR4, as previously indicated the operation of the subsequent circuit is as before. Although inputs are by linear IC's and high value resistors the subsequent CMOS circuitry, and p.c.b, layout ensure the device is immune to noise (unless deliberately introduced as in Mode 2). As well as being perfectly adequate for many open-field situations, the two-input resistance mode is also suited to detection in alleyways, some forms of maze, and general direction orientated behaviours. Currents measured at the inputs are all low and most unlikely to effect the animal. Mode 1: A l l A-D combinations<10/zA Mode 3:10/zA-100/zA dependent on sensitivity setting. Rearing Detection While touch switches might respond to body resistance, body capacitance or the body coupled noise of mains 50 Hz hum, true proximity detectors only employ the last two phenomena. Commercial activity monitors frequently use

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the concept of 'animal capacitance' influencing the radiated electromagnetic flux of tuned oscillator transformer coils, often placed under a standard laboratory cage. Previously published designs have often incorporated the animal into the dielectric of a large capacitator using a proximity meter [4, 7, 8]. The rearing monitor described here has its origins in a previous publication [22]. A 318 operational amplifier IC5 operates in a Wein-bridge oscillator arrangement, C7, C10, R22 and 426 constitute the bridge with associated feed-back paths (Fig. 3). Oscillation occurs at the frequency where the impedance of the capacitators equals the resistors in the wein-bridge arms; with the values shown this is around 50 kHz. The 318 has better bandwidth, slew rate and drift characteristics than the ubiquitous 741. It is used here without external compensation. A rat approaching the grid couples-<300 pF to one side of the bridge, thus unbalancing it. As the sine wave collapses or decreases, the rectified 'drop out' voltage is passed to a comparator IC6a, the output of which is squared up by trigger IC6b. The counter input is taken from this point via an optoisolator. The requisite voltage level for the counter being supplied from an independent battery supply. The photo transistor output 'pulse' has its risetime reduced via IC7a, a 4093B CMOS trigger. Before being fed to the counter via a IK pull-up resistor the signal is inverted by IC7b. The trigger also feeds a toggle flip-flop IC6c +d with complementary output states Q, Q, only one is used here to operate a relay and LED, through other logic circuitry could be interfaced at 0 if necessary. Amplitude stabillsation in the oscillator is achieved by the incorporation of TR4 an FET in the negative feedback loop [9,21]. The rectified output from IC5 being used to provide bias for TR4 via the charge held in C6. The gate voltage effectively determines the source-drain resistance. Thus if the amplitude starts to decrease the gate voltage will decrease causing the source-drain resistance to decrease which in turn leads to an increase in amplifier loop gain, re-

establishing the status-quo. This A.G.C. cannot cope with the capacitance loading of the Wein-bridge and therefore does not affect the instruments response, but merely maintains a stable sine-wave between switching. The point at which the feedback loop conducts is obviously dependent upon amplitude and the value of ZD2. Instrument sensitivity being inversely proportional to the ZD2 value; 4.7 V has been found to be quite adequate for our needs. Sine-wave distortion is largely determined by amplifier open-loop gain and by the response of the negative feedback loop-filter R21, C6. It may effectively be trimmed by VR6. The rearing detection point is set by a combination of VR8, VR9 and VR10. The comparator reference current is preset by VR10 to a suitable level and thereafter generally does not need altering again. VR8 in the positive feedback path balances the bridge within a certain range (VR8 is 10 turns with count/dial), while VR9 adjusts the oscillator output level via AC coupling, this is the primary sensitivity control. The monitor has two inputs 'E' and 'F'. These represent two levels of sensitivity [22]. The grid must be attached to 'F' but selected areas of, for example, the open-field wall could be connected to 'E' using metal foil beneath the wall laminate cover. If it is required that only central rears be recorded then the grid periphery may be blocked out with suitable thick insulation. Alternatively a variety of different interchangeable grids may be used. It is necessary for reliable operation that the negative rail of the rearing detector be earthed. The ambulation detector negative rail must however not be earthed, since rearing detection sensitivity will be markedly decreased. Rather than derive power from a transformer with multiple secondaries and suitable regulators it was thought to be cheaper and simpler for the two detectors to have separate power supplies. A stabilised battery supply for the ambulation detector and a simple mains derived stabilised supply for the rearing detector.

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Process Timer F e w details will be given here since commercial examples are common in most laboratories today. The one used here is based upon the ZN1034E precision timing IC [23], a chip using digital and analogue techniques, requiring few external components. Time selection is made by switching in an appropriate resistor and capacitor. All control functions are performed via suitable relays and the high and low Q.Q. outputs of the ZN1034E. Depression of the start button changes open-field illumination from dim red to bright white, turns on a white noise generator (plus associated filter and amplifier), releases hold function on counters and clears displays to allow counting, operates chart recorder motor. At the end of the set time, the sequence is automatically reversed.

Reliability and Accuracy The machine in one form or another has been in frequent use now for almost a year and has not suffered any electronic or mechanical failures, Lost scores due to 'urine bridging' has been found to be negligible, providing plates and spacers are kept very clean and smooth. Scores cannot be lost due to animals walking only along spacers. This would tend to occur in a previous design [5]. Correlation coefficients between observer and machine scores for twenty male inbred W A G rats approximately 150 days of age were: Mode 1:r=0.991 Mode 3 : r = 0 . 9 3 5

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The rearing monitor when correctly set has been found to give a 100% detection rate. To examine the possibility that the rearing grid influenced the incidence of rearing, a further fifteen W A G females (from the same litters as above) were scored for high rears with and without the grid in position. No significant difference could be detected.

The Open-Field in Use In practice the open-field sits inside a three-sided screen made from three 1.2 m x 1.2 m plywood sheets painted white. The grid being hinged rests against one side when not in use. Field illumination is provided by two 200 W bulbs either side of the field; mean illumination at floor level being reasonably uniform. The grid diffuses the light to some degree.

746

TOMKINS AND ()'DONOVAN

Amplified white noise is passed to two speakers at opposite ends of the room, noise levels at the field floor are approximately 50 dB, this is sufficient to mask background noises. In operation the test animal is brought into the room in a small plastic box (with which he has been previously familiarised). The field is illuminated by dim red light, the grid lifted and the subject placed in the bottom right-hand corner facing out. The start button is pressed and the observer places himself behind the screen. During the course of a three minute test, the incidence of grooming, sniffing, scratching, etc., may be noted on the event channel of the chart recorder. A typical trace is shown in Fig. 5. At the end of the test period the animal is retrieved and returned to its quarters. The field is cleaned with a 1% solution of Harris B.A.S. cleaner (Harris Biological Ltd.) and dried before the next subject is brought in. Use of a chart recorder with a time-tracer channel is obviously a simple if rather expensive way of 'real-time' re-

cording. Event encoders-decoders have been described in the literature [12] and data from the open field could be collated by such devices quite easily. The instrument may be interfaced to a whole variety of such recording devices, allowing intra-trial as well as inter-trial analyses for use in multifactorial and/or emotionality studies. Work is currently in progress exploring the use of a high frequertcy low power oscillator for maintaining uniform subjecVplate contact, modification of the open-field using capacitance touch switches and keyboard encoding techniques and additions to the rearing circuitry to register height of rearing. Full construction details for the system are available from the first author. ACKNOWLEDGEMENTS We would like to thank Dolores Tierney very much for typing this paper.

REFERENCES

1. Chambers, P. L. and J. J. Salmons. An electronic animal activity recorder. Experientia 22: 127-128, 1966. 2. Denenberg, V. M., T. Gartner and M. M. Yers. Absolute measurement of open-field activity in mice. Physiol. Behav. 15: 505509, 1975. 3. Dutrieux, A., A. Platel and B. DeWeer. Analyse automatique du comportement exploratoire chez le rat: Actographie par utilisation d'un circuit ferme de television. Physiol. Behav. 21: 721-726, 1978. 4. Garg, M. The effect of nicotine on rearing in two strains of rats. Life Sci. 7: 421-429, 1978. 5. Giulian, D. and G. Silverman. Solid-state animal detection system: Its applications to open-field activity and freezing behaviour. Physiol. Behav. 14: 109-112, 1975. 6. Giulian, D., C. T. Snowdon and L. S. Kran. A completely automated closed field maze series for rats. Physiol. Behav. 13: 181-187, 1974. 7. Gregory, K. and E. Liebeft. An examination of sex and strain difference in the rearing response to a novel environment. Activitas nervosa superior 9: 2-7, 1967. 8. Holland, H. C., B. D. Gupta and E. Weldon. A note on rearing and an environmental constraint. Activitas nervosa superior g: 140-144, 1966. 9. Jung, W. G. I.C. Op-Amp Cook Book. Indianapolis, In.: Howard W. Sams and Co., 1975, pp. 367-370. 10. Lasiter, P. S. An inexpensive contact-sensing device. Behav. Meth. Res. lnstrum. 11: 58, 1979. 11. Lat, J. Learning, conditioning and retention. Proc. 2nd Int. Pharmac. Meeting, Prague, Vol. I. Oxford: Pergamon, 1965, pp. 47-63. 12. Magyar, R. L. and J. R. Fitzsimmons. A multichannel, portable "real time" event encoder-decoder for laboratory and field experiments. Meth. lnstrurn. Behav. Res. 11: 47-50, 1979.

13. Morgret, M. K. and P. R. Albee. An automated open field apparatus utilising an improved resistance detection circuit. Meth. lnstrum. Behav. Res. 6: 327-328, 1974. 14. Moross, G. G. and G. J. Kaufman. Activity monitor for small animals. Physiol. Behav. 16: 493-495, 1976. 15. Pfister, H. P., R. R. Mudge and A. O. Harcombe. A multipurpose activity platform utilised in the open-field setting. Meth. Instrum. Behav. Res. 10: 21-22, 1978. 16. Porter, J. J., J. M. Mudy and A. M. Furber. A pressureactivated open field apparatus for rodents. Meth. lnstrum. Behay. Res. 11: 59-60, 1979. 17. Schenck, P. E., H. Van de Giessen, A. Koos Slob, and J. J. Van der Werff ten Bosch. An automated device for measuring locomotor activity in rats. Meth. Instrum. Behav. Res. 10: 552-556, 1978. 18. Siegel, P. S. A simple electronic device for the measurement of the gross bodily activity of small animals. J. Psychol. 21: 227236, 1946. 19. Silverman, P. Animal Behaviour in the Laboratory. London: Chapman and Hall, 1978, pp. 79-92. 20. Svensson, T. H. and G. Thieme. An investigation of a new instrument to measure motor activity of small animals. Psychopharmacology 14: 157-163, 1969. 21. Linear Applications 1. National Semiconductors (UK) Ltd., Lake Field Industrial Est., Greenock, Scotland, 1973, AN20I-AN20-12. 22. Proximity Switch. Electronics Today Int. (UK) 7: 75-78, 1978. 23. Precision Timer I.C. ZN1034E. Ferranti Ltd., Electronic Components Division, Gem Hill, Oldham, England, Data/Applications, Issue 5, April, 1977. 24. Universal Count/Display Circuit ZN1040E. Ferranti Ltd., Electronics Components Division, Gem Hill, Oldham, England, Data Sheet, Issue 3, June, 1975.