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Differential involvement of cholinergic mechanisms in activity and sociability in rats
Neuroscience Letters, 2 (1976) 39--44
39
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
D I F F E R E N T I A L INVOLVEMENT OF CHOLINERGIC MECHANISMS IN ACTIVITY AND SOCIABILITY IN RATS
WARREN STEWART*, DIANA EVANS and DEIRDRE MAYNARD
Department of Psychology, University of Tasmania, Hobart, Tasmania 7001 (Australia) (Received January 20th, 1976) (Accepted January 21st, 1976)
SUMMARY
Four groups of six rats, injected with saline, 0.1, 0.25 or 1.0 mg/kg scopolamine, were tested in a 36 × 36 in. open field for 10 min. Scopolamine differentially affected ambulation, rearing, social contacts and the social and perimeter distances between rats; and increased corner preference. This experiment shows that several c o m p o n e n t responses of activity and sociability need to be recorded if a better understanding of the involvement of cholinergic mechanisms in behaviour is to be obtained.
Muscarinic cholinergic mechanisms are involved in a number of behaviours, for example, in operant responding [9] and activity [2,5--8]. This has been shown from studies using anticholinergic drugs, in particular scopolamine, and solitary tested animals. There has been little investigation, however, of the involvement of cholinergic mechanisms in social behaviour, in particular in sociability: the affiliative tendency between animals in a group. Most studies have traditionally measured one behavioural response and/or used one dosage level. Experimenters have concluded, for example, that scopolamine increases activity, where activity has been measured b y ambulation or b y an automatic device which summates the effects of a drug on many c o m p o n e n t responses of activity, such as ambulation, rearing and grooming. Open fields were generally used as the test environment. Recently, however, it has been shown that scopolamine may decrease various c o m p o n e n t activity responses in certain testing situations [2,6,7]. Also the effects of scopolamine may be described as differential, that is, that the drug affects various component responses of activity differently. Hughes et al. [2], for example, found that 1.0 mg/kg scopolamine increased ambulation and decreased rearing and preference for a novel compartment. Conclusions a b o u t brain-behaviour * N o w at Department of Psychology, La Trobe University, Bundoora, Victoria, 3083, Australia.
40 relationships, in particular the hippocampus, a major site of action of scopolamine [12], may be quite erroneous then, if one dose is used and, more importantly, if one behavioural measure is taken in one test environment [7 ]. A multi-index m e t h o d of recording scopolamine-induced changes in various c o m p o n e n t activity responses, ambulation and rearing, was made possible in the present study by using a video recording system. Replaying the tape of each group's behaviour was necessary. Sociability has been traditionally measured by contacts of one rat to a not he r {e.g., refs. 1 and 3), yet anot her measure may be the inter-animal distance. Both contacts and inter-animal distances can be measured simultaneously from a series of photographs taken of groups of rats [11,12]. F o u r groups of six, 120-day-old, female albino rats were kept in group cages for several weeks prior to the experiment. Ad lib. access to food and water was provided. The open field that was used was 36 X 36 in. with 12 in. high walls and painted black inside with white lines making 6 X 6 in. squares. A video recording system and a 35 m m camera equipped with an electronic flash were used. All of the six rats in each group were injected (1 ml/kg i.p.) with either saline or 0.1, 0.25 or 1.0 mg/kg scopolamine h y d r o b r o m i d e . After the injection, each group was put back in its hom e cage for 10 min. Each group was then placed, one at a time, in the open field. One minute was allowed to elapse before the video recording system was switched on. Photographs were also taken, one every 30 sec for 10 min. The phot ographer stood on a chair with the camera 66 in. above the field. The flash did n o t seem to disturb the rats. The resulting photographs were printed to a size 10 X 8 in. Rats were identified by vivid marker markings on their backs. The videotapes were later analyzed for each group. The n u m b e r of squares entered and rearing responses [8] were recorded for each 2.5 min interval of the 10 min session. Sociability was assessed f r om the photos, as shown in Fig. 1. Social contacts was the co mb in e d total num be r of contacts for each rat per photograph. When rats were clumped together a rat in the clump was said to be in c o n t a c t with all rats in the clump, since it is impossible for any one rat to be in c o n t a c t with all other rats in the clump; for example, rat 2 is t aken as being in c o n t a c t with rats 1, 3 and 4 in Fig. 1. The social distance was measured as the average shortest distance between each rat and its partners, so t hat in Fig. 1 the social distance for rat A is the average of the distances AB, AC and AD. T he perimeter distance measure was the average distance between each rat and every o th er rat around the perimeter of the field. This was calculated since rats are seen to spend the majority of their time around the perimeter. For t he few rats that did n ot o c c u p y a perimeter square the perimeter point at which t h e y were closest was taken for the analysis. Two way analyses of variance with repeated measures over time were performed on the results for each response, the resulting F values are shown in
41
SOCIABILITY ANALYSIS
SOCIAL CONTACTS
SOCIAL DISTANCE
\\ \ \ \
\
PERIMETER
DISTANCE
Fig. 1. T h e t h r e e s o c i a b i l i t y m e a s u r e s a s s e s s e d f r o m p h o t o g r a p h s : s o c i a l a n d p e r i m e t e r d i s t a n c e s b e t w e e n rats.
social contacts and
Table I. Further significant differences (P < 0.05) were found using t-tests [4]. Scopolamine significantly reduced the number of squares entered, in the 1.0 mg/kg treated group, and the frequency of rearing, in the 0.1 and 1.0 mg/kg treated groups. Both these responses decreased over time, as shown in Fig. 2. For social contacts there was a significant drug effect. Both 0.1 and 0.25 mg/kg scopolamine decreased contacts while 1.0 mg/kg increased contacts, as shown in Fig. 3. A significant drug × time interaction appears attributable to a decrease in contacts over time in the 1.0 mg/kg and saline groups yet not in the other groups. The social distance between rats was increased by the 0.25 and 1.0 mg/kg doses. Significant time and drug X time interaction effects were found. Fig. 3 shows that saline treated rats increase the distance between TABLE I F VALUES FOR THE ANALYSES OF VARIANCE FOR THE EFFECT OF SCOPOLAMINE V A R I O U S R E S P O N S E S O F R A T S T E S T E D IN G R O U P S
A (drug) B (time) A ×B
ON
DF
Ambulation
Rearing
Social contacts
Social distance
Perimeter distance
3/16 3/48 9/48
5.30 b 34.20 b 1.13
13.70 b 15-00b 1.67
30.59 b 2.63 c 2.07 a
6.29 b 3.15 a 2.31 a
7.33 b 2.05 1.57
ap < 0.05, b p < 0.01, F (0.05): c = 2.76.
42
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TIME (min)
Fig. 2. Mean squares entered and rearing responses for saline (SAL) and 0.1, 0.25 and 1.0 mg/kg scopolamine treated groups. Fig. 3. Mean social contacts and social and perimeter distances between rats for saline (SAL) and 0.1, 0.25 and 1.0 mg/kg scopolamine treated groups.
each other, while drug groups generally show a decrease in the second 2.5 min interval, followed by an increase. The perimeter distance was decreased by scopolamine. From each of the 20 photos, for a group, an analysis was also performed on the spatial distribution of the rats in the field. It was found that the saline treated rats spent more time in the centre of the field than drugged rats, i.e. not in the outside row of squares around the perimeter (X2 -- 19.1, P < 0.001). Further, all drugged rats showed a higher corner preference than saline treated rats (X 2 = 9.57, P < 0.05), i.e. in the four comer squares. Unlike the normally observed finding that scopolamine increases ambulation and tends to increase rearing [ 5,8] in solitary tested rats, scopolamine decreased these responses in this group testing situation. However, scopolamine
43
has been found to decrease these responses in a complex environment [6]. In the latter case the drug-induced decrease was due to a rate effect, i.e. the level of behaviour in the undrugged rats determines the drug effect, the level being higher in the complex environment than in an open field. The rate effect could also be a causal factor in the group setting, since the level of undrugged activity was considerably higher in the undrugged rats in the present experiment compared to that for solitary tested rats tested in the same apparatus in a previous experiment [5]. This would be expected since rats are normally more active in a social setting compared to a solitary setting [7]. The social test situation as well as the physical test situation [6], therefore, appears to be important in determining drug-induced effects. The three measures of sociability were modified by scopolamine differentially. While sociability was decreased according to the social distance measure, it was increased according to the perimeter distance measure. Furthermore, social contacts were affected differently by different doses. Scopolamine, then, acts differentially on different aspects of sociability, close proximity and gross spatial distribution. The drug-induced increase in corner preference appears related to the increase in social distance and decrease in perimeter distance. The increase in contacts at 1.0 mg/kg also occurs when there is a decrease in ambulation. However, it is not clear which one causes the other, or even if t h e y are necessarily interrelated as some studies maintain [ 1,3]. It may be concluded that cholinergic mechanisms are involved in sociability and t h a t this involvement is of a differential nature like that for activity (ambulation and rearing). The detection of the observed differential drug effects shows the necessity to use a multi-index m e t h o d of recording behaviours [2,5--8]. The concept of differential drug-induced changes will need to be considered more seriously if a correct understanding of the role of cholinergic mechanisms in activity and sociability is to be obtained. REFERENCES 1 Cappell, H., and Latane, B., Effects of alcohol and caffeine on the social and emotional behaviour of the rat, Quart. J. Stud. Alcohol, 30 (1969) 345--356. 2 Hughes, R.N., Blampied, N.M., and Stewart, W.J., Scopolamine-induced changes in activity and reactions to novelty, Pharmacol. Biochem. Behav, (1975) in press. 3 Joy, V., and Latane, B., Autonomic arousal and affiliation in rats, Psychon. Sci. 25 (1971) 299--300. 4 Meyers, L.S., and Grosen, N.E., Behavioural Research: Theory, Procedure and Design, Freeman, San Francisco, Calif., 1974. 5 Stewart, W.J., Size of the environment as a determiner of effects of scopolamine, Psychol. Rep., 37 (1975) 175--178. 6 Stewart, W.J., Environment complexity does affect scopolamine-induced changes in activity, Neuroscience Letters, 1 (1975) 121--125. 7 Stewart, W.J., Activity, Exploration and Sociability in the Rat in Relation to Drugs Altering Cholinergic Mechanisms: Differential Effects of Environment and Testing Procedure, Unpublished Ph.D. Thesis, University of Tasmania, 1975.
44
8 Stewart, W.J., and Blain, S., Dose response effect of scopolamine on activity in an open field, Psychopharmacologia (Berl.), (1975) in press. 9 Stewart, W.J., Blampied, M.M., and Hughes, R.N., The effects of scopolamine on performance on a geometric progressive ratio schedule, Psychopharmacologia (Berl.), 38 (1974) 55--66. 10 Syme, L.A., and Syme, G.J., Effects of chlorpromazine and methamphetamine on sociability in rat~s, Psychopharmacologia (Berl.), 32 (1973) 81--84. l 1 Syme, L.A., and Syme, G.J., Group instability and the social response to methamphet. amine, Pharmacol. Biochem. Behav., 2 (1974) 851--854. 12 Warburton, D.M., and Russell, R.W., Some behavioural effects of cholinergic stimulation in the hippocampus, Life Sci., 8 (1969) 617--628.
39
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
D I F F E R E N T I A L INVOLVEMENT OF CHOLINERGIC MECHANISMS IN ACTIVITY AND SOCIABILITY IN RATS
WARREN STEWART*, DIANA EVANS and DEIRDRE MAYNARD
Department of Psychology, University of Tasmania, Hobart, Tasmania 7001 (Australia) (Received January 20th, 1976) (Accepted January 21st, 1976)
SUMMARY
Four groups of six rats, injected with saline, 0.1, 0.25 or 1.0 mg/kg scopolamine, were tested in a 36 × 36 in. open field for 10 min. Scopolamine differentially affected ambulation, rearing, social contacts and the social and perimeter distances between rats; and increased corner preference. This experiment shows that several c o m p o n e n t responses of activity and sociability need to be recorded if a better understanding of the involvement of cholinergic mechanisms in behaviour is to be obtained.
Muscarinic cholinergic mechanisms are involved in a number of behaviours, for example, in operant responding [9] and activity [2,5--8]. This has been shown from studies using anticholinergic drugs, in particular scopolamine, and solitary tested animals. There has been little investigation, however, of the involvement of cholinergic mechanisms in social behaviour, in particular in sociability: the affiliative tendency between animals in a group. Most studies have traditionally measured one behavioural response and/or used one dosage level. Experimenters have concluded, for example, that scopolamine increases activity, where activity has been measured b y ambulation or b y an automatic device which summates the effects of a drug on many c o m p o n e n t responses of activity, such as ambulation, rearing and grooming. Open fields were generally used as the test environment. Recently, however, it has been shown that scopolamine may decrease various c o m p o n e n t activity responses in certain testing situations [2,6,7]. Also the effects of scopolamine may be described as differential, that is, that the drug affects various component responses of activity differently. Hughes et al. [2], for example, found that 1.0 mg/kg scopolamine increased ambulation and decreased rearing and preference for a novel compartment. Conclusions a b o u t brain-behaviour * N o w at Department of Psychology, La Trobe University, Bundoora, Victoria, 3083, Australia.
40 relationships, in particular the hippocampus, a major site of action of scopolamine [12], may be quite erroneous then, if one dose is used and, more importantly, if one behavioural measure is taken in one test environment [7 ]. A multi-index m e t h o d of recording scopolamine-induced changes in various c o m p o n e n t activity responses, ambulation and rearing, was made possible in the present study by using a video recording system. Replaying the tape of each group's behaviour was necessary. Sociability has been traditionally measured by contacts of one rat to a not he r {e.g., refs. 1 and 3), yet anot her measure may be the inter-animal distance. Both contacts and inter-animal distances can be measured simultaneously from a series of photographs taken of groups of rats [11,12]. F o u r groups of six, 120-day-old, female albino rats were kept in group cages for several weeks prior to the experiment. Ad lib. access to food and water was provided. The open field that was used was 36 X 36 in. with 12 in. high walls and painted black inside with white lines making 6 X 6 in. squares. A video recording system and a 35 m m camera equipped with an electronic flash were used. All of the six rats in each group were injected (1 ml/kg i.p.) with either saline or 0.1, 0.25 or 1.0 mg/kg scopolamine h y d r o b r o m i d e . After the injection, each group was put back in its hom e cage for 10 min. Each group was then placed, one at a time, in the open field. One minute was allowed to elapse before the video recording system was switched on. Photographs were also taken, one every 30 sec for 10 min. The phot ographer stood on a chair with the camera 66 in. above the field. The flash did n o t seem to disturb the rats. The resulting photographs were printed to a size 10 X 8 in. Rats were identified by vivid marker markings on their backs. The videotapes were later analyzed for each group. The n u m b e r of squares entered and rearing responses [8] were recorded for each 2.5 min interval of the 10 min session. Sociability was assessed f r om the photos, as shown in Fig. 1. Social contacts was the co mb in e d total num be r of contacts for each rat per photograph. When rats were clumped together a rat in the clump was said to be in c o n t a c t with all rats in the clump, since it is impossible for any one rat to be in c o n t a c t with all other rats in the clump; for example, rat 2 is t aken as being in c o n t a c t with rats 1, 3 and 4 in Fig. 1. The social distance was measured as the average shortest distance between each rat and its partners, so t hat in Fig. 1 the social distance for rat A is the average of the distances AB, AC and AD. T he perimeter distance measure was the average distance between each rat and every o th er rat around the perimeter of the field. This was calculated since rats are seen to spend the majority of their time around the perimeter. For t he few rats that did n ot o c c u p y a perimeter square the perimeter point at which t h e y were closest was taken for the analysis. Two way analyses of variance with repeated measures over time were performed on the results for each response, the resulting F values are shown in
41
SOCIABILITY ANALYSIS
SOCIAL CONTACTS
SOCIAL DISTANCE
\\ \ \ \
\
PERIMETER
DISTANCE
Fig. 1. T h e t h r e e s o c i a b i l i t y m e a s u r e s a s s e s s e d f r o m p h o t o g r a p h s : s o c i a l a n d p e r i m e t e r d i s t a n c e s b e t w e e n rats.
social contacts and
Table I. Further significant differences (P < 0.05) were found using t-tests [4]. Scopolamine significantly reduced the number of squares entered, in the 1.0 mg/kg treated group, and the frequency of rearing, in the 0.1 and 1.0 mg/kg treated groups. Both these responses decreased over time, as shown in Fig. 2. For social contacts there was a significant drug effect. Both 0.1 and 0.25 mg/kg scopolamine decreased contacts while 1.0 mg/kg increased contacts, as shown in Fig. 3. A significant drug × time interaction appears attributable to a decrease in contacts over time in the 1.0 mg/kg and saline groups yet not in the other groups. The social distance between rats was increased by the 0.25 and 1.0 mg/kg doses. Significant time and drug X time interaction effects were found. Fig. 3 shows that saline treated rats increase the distance between TABLE I F VALUES FOR THE ANALYSES OF VARIANCE FOR THE EFFECT OF SCOPOLAMINE V A R I O U S R E S P O N S E S O F R A T S T E S T E D IN G R O U P S
A (drug) B (time) A ×B
ON
DF
Ambulation
Rearing
Social contacts
Social distance
Perimeter distance
3/16 3/48 9/48
5.30 b 34.20 b 1.13
13.70 b 15-00b 1.67
30.59 b 2.63 c 2.07 a
6.29 b 3.15 a 2.31 a
7.33 b 2.05 1.57
ap < 0.05, b p < 0.01, F (0.05): c = 2.76.
42
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TIME (min)
Fig. 2. Mean squares entered and rearing responses for saline (SAL) and 0.1, 0.25 and 1.0 mg/kg scopolamine treated groups. Fig. 3. Mean social contacts and social and perimeter distances between rats for saline (SAL) and 0.1, 0.25 and 1.0 mg/kg scopolamine treated groups.
each other, while drug groups generally show a decrease in the second 2.5 min interval, followed by an increase. The perimeter distance was decreased by scopolamine. From each of the 20 photos, for a group, an analysis was also performed on the spatial distribution of the rats in the field. It was found that the saline treated rats spent more time in the centre of the field than drugged rats, i.e. not in the outside row of squares around the perimeter (X2 -- 19.1, P < 0.001). Further, all drugged rats showed a higher corner preference than saline treated rats (X 2 = 9.57, P < 0.05), i.e. in the four comer squares. Unlike the normally observed finding that scopolamine increases ambulation and tends to increase rearing [ 5,8] in solitary tested rats, scopolamine decreased these responses in this group testing situation. However, scopolamine
43
has been found to decrease these responses in a complex environment [6]. In the latter case the drug-induced decrease was due to a rate effect, i.e. the level of behaviour in the undrugged rats determines the drug effect, the level being higher in the complex environment than in an open field. The rate effect could also be a causal factor in the group setting, since the level of undrugged activity was considerably higher in the undrugged rats in the present experiment compared to that for solitary tested rats tested in the same apparatus in a previous experiment [5]. This would be expected since rats are normally more active in a social setting compared to a solitary setting [7]. The social test situation as well as the physical test situation [6], therefore, appears to be important in determining drug-induced effects. The three measures of sociability were modified by scopolamine differentially. While sociability was decreased according to the social distance measure, it was increased according to the perimeter distance measure. Furthermore, social contacts were affected differently by different doses. Scopolamine, then, acts differentially on different aspects of sociability, close proximity and gross spatial distribution. The drug-induced increase in corner preference appears related to the increase in social distance and decrease in perimeter distance. The increase in contacts at 1.0 mg/kg also occurs when there is a decrease in ambulation. However, it is not clear which one causes the other, or even if t h e y are necessarily interrelated as some studies maintain [ 1,3]. It may be concluded that cholinergic mechanisms are involved in sociability and t h a t this involvement is of a differential nature like that for activity (ambulation and rearing). The detection of the observed differential drug effects shows the necessity to use a multi-index m e t h o d of recording behaviours [2,5--8]. The concept of differential drug-induced changes will need to be considered more seriously if a correct understanding of the role of cholinergic mechanisms in activity and sociability is to be obtained. REFERENCES 1 Cappell, H., and Latane, B., Effects of alcohol and caffeine on the social and emotional behaviour of the rat, Quart. J. Stud. Alcohol, 30 (1969) 345--356. 2 Hughes, R.N., Blampied, N.M., and Stewart, W.J., Scopolamine-induced changes in activity and reactions to novelty, Pharmacol. Biochem. Behav, (1975) in press. 3 Joy, V., and Latane, B., Autonomic arousal and affiliation in rats, Psychon. Sci. 25 (1971) 299--300. 4 Meyers, L.S., and Grosen, N.E., Behavioural Research: Theory, Procedure and Design, Freeman, San Francisco, Calif., 1974. 5 Stewart, W.J., Size of the environment as a determiner of effects of scopolamine, Psychol. Rep., 37 (1975) 175--178. 6 Stewart, W.J., Environment complexity does affect scopolamine-induced changes in activity, Neuroscience Letters, 1 (1975) 121--125. 7 Stewart, W.J., Activity, Exploration and Sociability in the Rat in Relation to Drugs Altering Cholinergic Mechanisms: Differential Effects of Environment and Testing Procedure, Unpublished Ph.D. Thesis, University of Tasmania, 1975.
44
8 Stewart, W.J., and Blain, S., Dose response effect of scopolamine on activity in an open field, Psychopharmacologia (Berl.), (1975) in press. 9 Stewart, W.J., Blampied, M.M., and Hughes, R.N., The effects of scopolamine on performance on a geometric progressive ratio schedule, Psychopharmacologia (Berl.), 38 (1974) 55--66. 10 Syme, L.A., and Syme, G.J., Effects of chlorpromazine and methamphetamine on sociability in rat~s, Psychopharmacologia (Berl.), 32 (1973) 81--84. l 1 Syme, L.A., and Syme, G.J., Group instability and the social response to methamphet. amine, Pharmacol. Biochem. Behav., 2 (1974) 851--854. 12 Warburton, D.M., and Russell, R.W., Some behavioural effects of cholinergic stimulation in the hippocampus, Life Sci., 8 (1969) 617--628.