Digiscan activity: Automated measurement of thigmotactic and stereotypic behavior in rats

PharmacologyBtocheml~trv&Beha~tor Vol 27, pp 56%572 ~ Pergamon Journals Ltd, 1987 Pnnted m the U S A

0091-3057/87 $3 00 + 00

Digiscan Activity: Automated Measurement of Thigmotactic and Stereotypic Behavior in Rats P R L

SANBERG,1 S A ZOLOTY, R WILLIS, C D TICARICH, K RHOADS, R P NAGY, S G MITCHELL, A R LAFOREST, J A JENKS, J HARKABUS, D B GURSON, J A FINNEFROCK AND E J BEDNARIK

Behavtmal Neurosctence Laboratory, Department o f Psychology Ohto Umverslty, Athens, OH 45701 and Laboratory o f Behavioral Neutosctence Departments o f Psychiatry, P~ychology, Neutosutgery, and Phystology Untvet stty o f Cmcmnatt College o f Medtc me, Cmcmnatl, OH 45267-0559

SANBERG, P R , S A ZOLOTY, R WILLIS, C D TICARICH, K RHOADS, R P NAGY, S G MITCHELL, A R LAFOREST, J A JENKS, L J HARKABUS, D B GURSON, J A FINNEFROCK AND E J BEDNARIK Dtgtsean a~tt~tty Automated measmement oJ thtemota~ttt and steteotypt¢ beha~loJ m lats PHARMACOL BIOCHEM BEHAV 27(3) 569-572, 1987 --V,sual measures of stereotypy, margin time (thlgmotaxls or wall-huggmg), and center time were correlated with automated measures using a revised 16 beam version of the Dlglscan Ammal Actlwty Momtor System Rats were injected with d-amphetamine (1 25, 2 5, 5 0 and 10 0 mg/kg), scopolamine (1 25 and 2 5 mg/kg) or sahne and drugs were found to increase center Ume and decrease margin t~me m a dose-dependent manner, wzth the maxtmum effect occurring with 1 25 and 2 5 mg/kg, respectively At higher doses, an oppos,te effect was observed Extremely high correlations between v,sual and automated recordings of both margin t,me and center time were found Since thlgmotaxlc or wall-hugging behavior has been used as an indicator of emot~onahty in rats, the results of the present study suggest that these two locomotor variables may be useful additions to the D~g~scan multlvarmte analysis of locomotor behawor It was also found that a redefinmon of stereotyplc behawor ~mproved Its correlation with wsual measurements compared to earher studies Dlglscan Thlgmotax,s Wall-hugging Locomotor behawor Stereotypy

Emotlonallty

T H E open-field paradigm is a c o m m o n l y - u s e d technique for monitoring b e h a v i o r in laboratory animals [1, 6, 9, 13, 16, 18] This technique has been used for small animals to measure ambulation [1], stereotyplc b e h a v i o r [3], e m o t i o n a h t y [6], thlgmotaxls or wall-hugging [l 1], and rearing [8] The effectiveness o f using c o m p u t e r i z e d a u t o m a t e d systems in recording the m o t o r activity of rodents has been successfully d e m o n s t r a t e d [10, 14, 15], and has gained support as a m e a n s of reducing error due to o b s e r v e r biases and e q u i p m e n t calibration [2,15] Until recently, s o m e open-field behaviors, such as thlgmotaxis, have eluded automation The thlgmotactic tendencies o f the rat in open-field situations h a v e long b e e n of interest [2, 4, 11, 13] Findings by R o y c e [13] suggested that tingmotaxis and penetration to the c e n t e r of an open-field serve as indicators o f e m o t i o n a h t y in rats Hall [6] defined emotlonahty as a group o f organic, experimental, and expressive reactions denoting a general upset o r excited state o f the animal Stimulant drugs are k n o w n to exert specific effects

d-Amphetamine

Scopolamine

upon the amount o f time spent in, or the n u m b e r o f entries into, the center of an o p e n field by a rat [2,4] Previous studies have demonstrated that d-amphetamine-treated animals tended to exhibit more entries into the center o f the field, while scopolamine-treated rats rarely m o v e d a w a y from the periphery of the cage [2,4] It has been suggested that scopolamine interferes with an animal's habituation to a novel e n v i r o n m e n t and thus inhibits exploratory b e h a v i o r [5,7] Tins inhibitory effect may be related to an increase in emotlonality in the rat, and subsequently produce a d e c r e a s e in entries into the center of an open field The importance o f thigmotaxls in psychopharmacologlcal r e s e a r c h suggests the n e e d for its incorporation into a u t o m a t e d systems of animal activity Recently, the Dlglscan-16 Animal Activity Monitoring S y s t e m ( O m m t e c h Electronics, Columbus, OH) has added m e a s u r e s of margin and center times witinn the test cage The present study determined the validity o f these m e a s u r e s A m p h e t a m i n e has also been r e p o r t e d to h a v e m a r k e d ef-

1Requests for repnnts should be addressed to Dr Paul R Sanberg, Laboratory of Behavioral Neurosclence, Department of Psychiatry, Umverslty of Cincinnati, CmclnnaU, OH 45267-0559

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Dose ( m g / k g ) FIG 1 Mean Ume spent m the margin or center of Dlglscan-16 Animal Activity monitors m response to d-amphetamine, scopolamine or sahne (vehicle) Open bars represent v~sual observation measurements and hatched bars represent the automated Dlglscan measures Correlation coefficients compare the respective automated to wsual measures

fects on stereotyplc behawor m rodents [8, 12. 15] Sanberg et al [ 1 4 ] tested the effects of 5 0 and 100 mg/kg d-amphetamme on stereotyplc behawor in male SpragueDawley rats, using the earher eight beam version of the Dlgmcan Ammal Actwlty Momtor System Visual ratings of stereotypy, based on the stereotypy rating scale of Creese and Iversen [3], were slgmficantly correlated with automated measures, and revealed that the drug produced slgmficant Increases m stereotyplc behawor In the present study, the 16 beam version of the Dlglscan system was also used to reinvestigate the behavioral effects of d-amphetamine m order to determine whether thin newer system produced a more accurate measure of stereotypy METHOD

Twenty-four male Sprague-Dawley rats (200 to 300 g) were maintained mdwldually in stamless-steel cages (24 × 11 × 20 cm) and kept on a 12 hr hght/dark cycle with food and water available ad lib Prior to behavioral assessment, the rats were placed singly in one of four Dlgmcan Animal Activity Momtor cages and allowed 30 mln for habituation The ammals were then reJected mtrapentoneally (IP) with either 1 25, 2 5, 5 0, or 10 0 mg/kg of d-amphetamine (Sigma, St Louis, MO), or 1 25 or 2 5 mg/kg of scopolamine (Sigma,

St Lores, MO) or sahne m a volume of 1 mg/kg, and replaced into the acUvtty monitor A counter-balanced design was used in which each rat recewed every dose of d-amphetamme, scopolamine, and saline over the course of the experiment, based upon the drug and dosage administered m the first session A rest period of at least 48 hours was alloted between m lect~ons Four animals were tested m each experimental session, and there were two sessions per day (between 11 00 and 16 00 hours) A double-bhnd procedure was used throughout the study Measurement of motor activity began 15 mm after reJection, and continued for a period of 30 mln, at whmh time the dependent measures were recorded on a silent printer Stereotyp~c behavior, margin time, and center time were recorded m an automated manner by the Dxgmcan-16 system, and also by vmual observation The Dlglscan Ammal Activity Monltonng System (Model RXYZCM, Ommtech Electromcs, I n c , Columbus, OH) consmts of vertical and horizontal infrared sensors, 16 per side, surrounding an acryhc activity momtor cage (40x40× 30 5 cm) A slmdar Dlgmcan-8 system has been described in detail previously [15] In the present system, margin time was operationally defined as the animal's center of gravity being within 6 35 cm or less of the cage wall The remaining time in which the rat did not enter thin area of the cage was considered center t~me For visual

DIGISCAN ACTIVITY

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observation, the center of gravity of each animal was marked with black dye on the center of its back in order to assist the observers in monitoring margin and center time One observer was randomly assigned to each rat, and measured margin time with a stop watch The time an animal spent in the margin was subtracted from the total observation time of 30 min in order to calculate center time Stereotypic behavior was visually rated every 15 mln during a 30 min period using the stereotypy rating scale of Creese and Iversen [3] The ratings were 0=asleep or stationary, 1=active, 2=predominantly active but with bursts of stereotyped sniffing or rearing, 3=stereotyped activity such as sniffing along a fixed path in the cage, 4 = stereotyped sniffing or rearing maintained in one location, 5 = stereotyped behavior in one location with bursts of gnawing or licking The raters were unaware of the drugs used The Dlgiscan measure of stereotypic behavior was defined as the time spent breaking the same beam pattern repeatedly (stereotypy time), and the number of episodes of this stereotypic behavior (number of stereotyples) Each episode was separated by at least one second

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The effects of amphetamine and scopolamine on margin and center time are illustrated in Fig 1 As shown, high correlations were found between visual automated measures of these two variables In addition, an analysis of variance demonstrated that there were no significant differences between automated and visual recordings over all doses of amphetamine and scopolamine All dosage levels of amphetamine were found to have significant effects on margin time, F(4,140)=10 8, p < 0 001, and center time, F(4,140) = 11 8, p <0 001 The same was true for the influence of scopolamine on margin time, F(2,48)=6 8, p < 0 002, and center time, F(2,84)=6 6, p < 0 002 Figure 2 depicts the effects of amphetamine on stereotypic behavior A significant correlation was found between visual and automated recordings of the number of stereotyples (r= 75, p < 0 05), and between visual and automated recordings of stereotypy time (r= 52, p < 0 05) Amphetamine, over all doses, had a significant effect on stereotypy measured visually, F(2,42)=29 l, p < 0 0 0 1 , number of stereotypies, F(2,42)=11 68, p < 0 0 0 1 , and stereotypy time, F(2,42)=22 74, p < 0 001 DISCUSSION

230

Automated and visual recordings of thIgmotactic behavior were found to be highly correlated across all dosage levels of d-amphetamine and scopolamine These high correlations demonstrated the reliability and vahdlty of the 16 beam Diglscan System in recording thlgmotaxls, and indicated the usefulness of automated monitoring techniques to measure margin and center time The effects of amphetamine and scopolamine upon margin and center time appear to be dose-dependent Both drugs showed similar dose-response curves Intermediate doses of

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FIG 2 Mean stereotypy response to d-amphetamine (5 0 mg/kg, squares, 10 0 mg/kg, triangles), and sahne (circles) as measured by a visual rating scale (visual stereotypy) and Dlglscan-16 Ammal Actwlty Momtors (number ofstereotyples and stereotypy time) Correlation coefficient compares the respectwe automated measure to visual stereotypy

572

SANBERG ET AL

d - a m p h e t a m i n e (2 5 a n d 5 0 mg/kg) p r o d u c e d t h e g r e a t e s t Increase m c e n t e r time, while the l o w e s t a n d highest d o s e s o f d - a m p h e t a m i n e (1 25 a n d 10 0 mg/kg) p r o d u c e d the g r e a t e s t i n c r e a s e m t h l g m o t a x l s 1 25 mg/kg s c o p o l a m i n e r e s u l t e d in i n c r e a s e d c e n t e r time, w h d e 2 5 mg/kg r e s u l t e d m m c r e a s e d m a r g i n time T h e results o f the p r e s e n t i n v e s t i g a t i o n differ w i t h t h o s e o f G e y e r et al [4], w h o r e p o r t e d a d e c r e a s e in cente~ e n t r i e s o v e r i n c r e a s i n g d o s a g e levels in s c o p o l a m i n e t r e a t e d a n i m a l s at d o s e s starting at 0 5 mg/kg A s t u d y by S t e w a r t a n d Blain [17] r e p o r t e d t h a t s c o p o l a m i n e p r o d u c e d a n i n v e r t e d U d o s e - r e s p o n s e effect o n activity, w i t h activity p e a k i n g at 1 0 mg/kg It is t h e r e f o r e , p o s s i b l e t h a t a general i n c r e a s e in activity b r o u g h t a b o u t by s c o p o l a m i n e is r e s p o n sible for the i n c r e a s e d c e n t e r e n t r i e s at this d o s e T h e results of this s t u d y c o n c e r n i n g the effects o f a m p h e t a m i n e o n thigm o t a x l s a n d c e n t e r time are c o n s i s t e n t with p r e v i o u s r e p o r t s I2,4] T h e 16 b e a m Dlglscan a n a l y z e r a p p e a r s to also p r o d u c e a c c u r a t e a n d reliable m e a s u r e m e n t s o f s t e r e o t y p l c b e h a v i o r in rats U s e o f this s y s t e m r e s u l t e d in h i g h e r c o r r e l a t i o n s b e t w e e n visual a n d a u t o m a t e d r e c o r d i n g s o f s t e r e o t y p y for a m p h e t a m i n e - t r e a t e d rats t h a n were f o u n d p r e v i o u s l y with the eight b e a m v e r s i o n [14] T h e p r e s e n t s t u d y did not reveal a significant difference b e t w e e n the 5 0 mg/kg a n d 10 0 mg/kg d o s a g e levels o f d - a m p h e t a m i n e with visual o b s e r v a t i o n . h o w e v e r , the 16 b e a m Dlglscan r e c o r d e d a significant differe n c e b e t w e e n t h e s e d o s e s w h e n m e a s u r i n g s t e r e o t y p y time This finding i n d i c a t e d that the a u t o m a t e d m e a s u r e o f s t e r e o t y p y time m a y b e m o r e s e n s i t i v e t h a n visual o b s e r v a tion to d o s e - r e s p o n s e effects F u r t h e r m o r e , t h e use o f stan-

d a r d l z e d m o n i t o r i n g s y s t e m e l i m i n a t e s e x p e r i m e n t e r bias w h i c h m a y influence t h e m e a s u r e m e n t o f d r u g - i n d u c e d s t e r e o t y p y In the s t u d y b y S a n b e r g et al [14], visual o b s e r v a t i o n o f s t e r e o t y p l c b e h a v i o r r e s u l t e d in a significant differe n c e b e t w e e n the 5 0 a n d 10 0 mg/kg d o s e s o f d - a m p h e t a m i n e a n d It is p o s s i b l e t h a t the d i s c r e p a n c y bet w e e n t h e earlier s t u d y a n d the p r e s e n t f l n d m g s w e r e d u e to differences between observers Overall, the p r e s e n t findings i n d i c a t e d t h a t the 16 b e a m Dlglscan is a n I m p r o v e m e n t o v e r t h e eight b e a m v e r s i o n in p r o d u c i n g reliable, v a h d m e a s u r e s o f s t e r e o t y p y , m a r g i n time a n d c e n t e r time T h e addition o f the D l g l s c a n p r o g r a m to m e a s u r e t h l g m o t a c t l c b e h a v i o r will p r o v e useful in det e r m i n i n g the effects o f v a r i o u s drugs u p o n this b e h a v i o r a n d h e n c e t h e i r role in e m o t l o n a h t y in rats T h e i m p r o v e m e n t o f a u t o m a t e d m u l t l v a r m t e t e c h n i q u e s to m o n i t o r l o c o m o t o r beh a v i o r ms certainly a step f o r w a r d in e x p l o r i n g the b e h a v i o r a l pharmacology of movement

ACKNOWLEDGEMENTS This research was performed as a class project m the Department of Psychology Ohio University by the coauthors It was supported by a Huntington's Disease Grant from the Pratt Famdy and Friends, the Hereditary Disease Foundation the Huntington's Disease Foundahon of America, the Ohio Umverslty Research Committee, the Baker Committee, MH40127, and a gift from Omnltech Electronics (Columbus, OH) to P R S We apprecmte the graphic assistance ot G Cohen, L Chau and L Lutton

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10 Meyers, B and R C Wtlchln Some effects of scopolamine on locomotor activity In rats P~vchonom Sc~ 17 174-175, 1969 11 Nauman. D J Open-field behavior of the mongohan gerbd Ps3chomon Scl 10 163-164, 1968 12 Porrlno, L J , G Luognanl, D Dow-Edwards and L Sokoloff Correlation of dose-dependent effects of acute amphetamine admlmstratlon on behavior and local cerebral metabolism m rats Blare Re~ 307 311-320, 1984 13 Royce, J R On the construct vahdlty of open-field measures Pswhol Bull 84 1098-1106. 1977 14 Sanberg, P R , T H Moran, K L Kubos and J T Coyle Automated measurement of stereotyplc behavior in rats Beha~ Neuro~ct 97 830-832, 1983 15 Sanberg, P R , S H Hagenmeyer and M A Henault Automated measurement of multwarlate locomotor behavior in rodents Neulobeha~ To~ltol Tetatol 7 87-94, 1985 16 Stewart, W J Effects of experience m the test enwronment on scopolamine-reduced changes m actlvxty in an exploration box Psvchol Rep 40 251-254, 1977 17 Stewart, W J and S Blare Dose-response effects of scopolamine on activity in an open field Psvchopharmacologm 44 291-295, 1975 18 Walsh R N and R A Cummins The open-fieldtest a critical review P~vchol Bull 83 482-504, 1976