Physiology and Behavior, Vol. 14, pp. 879--881. Brain Research Publications Inc., 1975. Printed in the U.S.A.
BRIEF COMMUNICATION Technique for Producing and Measuring Territorial Aggression using Laboratory M i c e I JOHN B. T H U R M O N D
Department o f Psychology, University o f Louisville, Louisville, K Y 40208 (Received 30 S e p t e m b e r 1974) THURMOND, J. B. Technique for producing and measuring territorial aggression using laboratory mice. PHYSIOL. BEHAV. 14(6) 879-881, 1975. - A method was developed for producing and measuring territorial aggression in male CF-1 laboratory mice using a simple apparatus. The technique is based on data collected from approximately 1000 CF-I mice in order to establish the parameters and optimize the procedures. In this technique the mouse takes up lone residence for 24 hr in a 60 cm square box attached by a tubular runway to a standard mouse (home) cage with food, water, and bedding. After this interval, a naive intruder male CF-1 mouse of the same age is introduced. Under control (no treatment) conditions, 85-90 percent of the resident mice will attack the intruder with a latency of about 5 min, and all residents attacking the intruder are dominant. Dominance or submission is typically decided within the first 20 min of the test. Data on 10 pairs of mice can be collected simultaneously by one observer. Treatments can be assessed in terms of their effects on the production of aggression (percentage of animals attacking) in either the resident or the intruder, and on the level of aggression produced by recording the latency to attack, the frequency of attacks, and the number of animals wounded (showing blood) during the 20 min observation period. The advantages of the technique include the use of a naturally occurring aggressive behavior (as opposed to techniques employing long periods of isolation, shock, or drugs), the highly reliable occurrence of aggression, the ability to study animals exposed to either aggression or defeat, the clear and valid measures of aggression produced, the simple and sturdy apparatus, and the convenience and economy of data collection. Mice
Aggression
Territory
Measurement
Neurochemistry
A n u m b e r of different m e t h o d s have been rather widely used for producing aggression in mice such as electric f o o t shock [ 6 ] , long periods o f isolation [ 7 ] , training fighters [ 3 ] , grouping strange animals t o g e t h e r [4] and pharmacologic agents [ 9 ] . A l t h o u g h territorial f o r m a t i o n has been studied in l a b o r a t o r y mice [ 5 ] , and has been used as a test o f aggression in s o m e studies [ 8 ] , there is n o t at present a c o m m o n l y used standardized test o f aggression based on territoriality. The ideal t e c h n i q u e for assessing aggression should i n c o r p o r a t e the following properties: it should reliably produce clear measures o f aggressive behavior such as threat postures, biting, and fighting, divide the animals unequivocally into categories o f v i c t o r y and defeat, be e c o n o m i c a l and c o n v e n i e n t to use, and it should be based on naturally occurring goal-seeking and c o n s u m m a t o r y acts free of undesirable side effects. The m e t h o d o f p r o d u c i n g and measuring agression described here has yielded results consistent with these characteristics. C o m p a r e d to m e t h o d s e m p l o y i n g prolonged isolation, f o o t shock, and pharma-
cologic agents for inducing aggression, territorial defense is a naturally occurring behavior and should be free o f the changes in biochemistry associated with these m e t h o d s [ 1, 2, 3, 7]. Thus, the t e c h n i q u e r e p o r t e d here for producing and recording aggression in laboratory mice may offer s o m e definite advantages for researchers interested in relating aggressive behavior to changes in brain biochemistry.
Apparatus T w o configurations o f the apparatus used are shown in Fig. 1; t h e y produce essentially identical results but each one has certain advantages. Both consist o f a 60 cm square o p e n field attached via a 4 cm dia. tube to a standard p o l y p r o p y l e n e mouse cage. The tube connecting the o p e n field to the mouse cage was made by forcing a square piece o f 30 ga sheet metal into a cylinder. It can be easily squeezed into a smaller diameter and inserted into the 4 cm dia. holes in the side o f the b o x and in the m o u s e cage, thus
1 Based in part on a paper presented with E. E. Quinton to the Psychonomic Society, November 1972. 879
880
THURMOND
tti I Iti tttlzi/~t.~,ilt,yx t N'F1~,, t.H-IIIIIII
~l1111:llllll~,, '!
,,
.........
i-m-II III ~tr
ILdl
;llllll
. . . . . I, , ~ I I Illlll
II II
I",,
Ilillllll
11
I1 III11
II,, II 11
III',[[~' |1 I1|1
FIG. 1. Two configurations of the apparatus used for developing the technique.
connecting the two, or it can be easily removed in this manner for cleaning. The floor of the open field boxes is made of hardware cloth with small (approximately 0.6 cm square) holes, which are small enough to prevent the mouse from sticking its head through and getting stuck. The box shown in the top of Fig. 1 was made by nailing together four 2.5 x 10 cm boards at the corners, and attaching the hardware cloth to the bottom. A 60 cm square piece of transparent Plexiglass was placed (not attached) over the top of the open field to keep the mice in the box, and paper ordinarily used to cover rat pans (approximately 60 cm wide) was placed under the open field box for cleaning purposes. The box shown in the bottom of Fig. 1 was made from 30 ga sheet metal; the hardward cloth attached to the bottom was cut slightly smaller than 60 cm square, stretched across the inside bottom of the box, and anchored at 12 equally spaced points 2 cm from the b o t t o m edge. A 62 cm square pan for the b o t t o m was constructed from 30 ga sheet metal with sides 2.5 cm high, covered first with paper and then layered 1 cm deep with bedding. The sheet metal box can easily be placed in the pan and removed for cleaning purposes. The sides of the box are 60 cm high and no top is required to keep the mice from escaping. A 30 cm high tower made of small-holed hardware cloth was anchored to the hardware cloth floor at the center of the box, and provides an 8 cm square platform at the top for introducing the intruder. The tower
also offers the submissive mouse of the pair partial protection from the attacks of the dominant animal. The 4 cm dia. tube connecting the box to the mouse cage is 12 cm long and provides a convenient method for catching the mice at the end of the test: the mouse almost invariably runs into the tube and stops, with its tail sticking out of the end. Procedure The CF-1 mouse (Carworth, New City, New York) was chosen for development of the technique because it provides the homogeniety characteristic of inbred strains, it is very aggressive, and it is inexpensive. Ninety day-old CF-1 male mice were received from Carworth, housed 5 per cage at a room temperature of approximately 21°C, and acclimated to the laboratory on a 12-hr on, 12-hr off, light cycle. They were maintained ad lib on Rat/Mouse Purina Chow and water, and bedded in Litter Green small animal litter which was changed once a week. At the time of the test for territorial aggression, the mice were completely naive, they were 104 days old, and the average mouse weighed 35 g. The mice were not designated as residents or intruders until the time of testing; thus, all mice were housed and treated exactly the same. For aggression testing, 4 cages of mice were randomly selected, 2 cages were randomly designated as residents (I 0 mice), the remaining 2 cages as intruders (10 mice), and each resident was placed in the middle of the open field (on the tower in the boxes containing one) of his respective test box. If the resident mice are placed initially in the home cage, occasionally one of them will fail to enter the open field during the next 24 hr; however, if placed in the open field, they always explore it and end up in the home cage. All 4 cages of mice were transferred to the testing room containing ten identical test boxes, and the residents were placed in their respective test box exactly 2 hr following the onset of the dim phase of the light cycle. A spot blue liquid food dye was rubbed on the top of the resident's head before placing him in the test box for identification during the aggression test. The light cycle in the testing room was identical to that where the mice were housed, and was governed by a 40 W bulb reflected off the ceiling superimposed for 12 hr with the brighter fluorescent lights of the room. On the next day, exactly 24 hr following placement of the resident mice in the test boxes, the 10 intruder mice were removed from their two cages, one each was placed in the center of the open field of the test boxes, and the ensuing aggression was recorded during a 30 rain observation period. The l 0 pairs of mice were observed simultaneously (by standing on a chair in the case of the boxes with high sides), and the following measures were recorded: (a) the latency of attack; (b) the number of attacks during the observation period; (c) the number of animals wounded (showing blood) at the end of the observation period; and (d) the number of residents attacking the intruder. An attack is defined here as a bout of activity lasting up to several seconds during which one animal bites the other one at least once; thus, an attack may consist of a number of bites one animal inflicts on the other during the course of a chase, a fight, or a stationary one-sided attack. Since a tube has been used in other research for testing dominance behavior in mice, it should be pointed out that the intruder placed in the open field is always naive. Thus,
T E R R I T O R I A L AGGRESSION IN MICE
881
exploration on the part of the intruder is limited, and displays o f dominance occur in the open field before the intruder investigates the tube.
Parameters It is advantageous to have approximately 20 resident mice in each experimental condition to show reasonable differences in treatment effects. Mice that are tested at 90 and 104 days of age show comparable levels o f aggression. At both ages, approximately 90 percent of the residents attack intruders with a latency of 5 - 7 min, the resident attacks the intruder about 24 times during the first 15 rain of the test (i.e. 15 min from the m o m e n t the intruder is placed in the test box) and about 16 times during the
second 15 min of the test. Except for the latency to attack which remains constant for those animals who attack, these values are half as great when the residents and intruders are tested at 70 days of age. An IP saline injection administered 60 min before testing reduces "the number of 104 day-old residents attacking to 70 percent; latency and number of attacks are unaffected. Thus, an IP injection of 1.25 mg/kg of Chlorpromazine HC1 administered to 20 resident mice 60 min before testing produces results that are no different from an IP injection of saline. An IP injection of 2.5 mg/kg reduces the number of residents attacking to 35 percent, increases the latency, and decreases the number of attacks thus producing results that are significantly different from the saline injected controls.
REFERENCES 1. Butcher, L. L. and J. Engel. Peripheral factors in the mediation of the effects of L-DOPA on locomotor activity. Z Pharm. Pharmac. 21: 614-616, 1969. 2. Eichelman, Jr., B. S. and N. B. Thoa. The aggressive monoamines. Biol. Psychiat. 6: 143-164, 1973. 3. Eleftheriou, B. E. and R. L. Church. Brain levels of serotonin and norepinephrine in mice after exposure to aggression and defeat. Physiol. Behav. 3: 977-980, 1968. 4. Lycke, E., K. Modigh and B. E. Roos. Aggression in mice associated with changes in the monoamine-metabolism of the brain. Experientia 25: 951-953, 1969. 5. Mackintosh, J. H. Territory formation by laboratory mice. Anim. Behav. 18: 177-183, 1970.
6. Tedeschi, R. E., D. H. Tedeschi, A. Mucha, L. Cook, P. A. Mattis and E. J. Fellows. Effects of various centrally acting drugs on fighting behavior of mice. Z Pharmac. exp. Ther. 125: 28-34, 1959. 7. Valzelli, L. The "isolation syndrome" in mice. Psychopharmacologia 31: 305-320, 1973. 8. Van Oortmerssen, G. A. Biological significance, genetics and evolutionary origin of variability in behaviour within and between inbred strains of mice. (Mus musculusJ. Behaviour 38: 1-92, 1970. 9. Yen, H. C. Y., M. H. Katz and S. Krop. Effects of various drugs on 3, 4-dihydroxyphenylalanine (DL-DOPA) - induced excitation (aggressive behavior) in mice. Toxic. appL Pharmac. 17: 597-604, 1970.
BRIEF COMMUNICATION Technique for Producing and Measuring Territorial Aggression using Laboratory M i c e I JOHN B. T H U R M O N D
Department o f Psychology, University o f Louisville, Louisville, K Y 40208 (Received 30 S e p t e m b e r 1974) THURMOND, J. B. Technique for producing and measuring territorial aggression using laboratory mice. PHYSIOL. BEHAV. 14(6) 879-881, 1975. - A method was developed for producing and measuring territorial aggression in male CF-1 laboratory mice using a simple apparatus. The technique is based on data collected from approximately 1000 CF-I mice in order to establish the parameters and optimize the procedures. In this technique the mouse takes up lone residence for 24 hr in a 60 cm square box attached by a tubular runway to a standard mouse (home) cage with food, water, and bedding. After this interval, a naive intruder male CF-1 mouse of the same age is introduced. Under control (no treatment) conditions, 85-90 percent of the resident mice will attack the intruder with a latency of about 5 min, and all residents attacking the intruder are dominant. Dominance or submission is typically decided within the first 20 min of the test. Data on 10 pairs of mice can be collected simultaneously by one observer. Treatments can be assessed in terms of their effects on the production of aggression (percentage of animals attacking) in either the resident or the intruder, and on the level of aggression produced by recording the latency to attack, the frequency of attacks, and the number of animals wounded (showing blood) during the 20 min observation period. The advantages of the technique include the use of a naturally occurring aggressive behavior (as opposed to techniques employing long periods of isolation, shock, or drugs), the highly reliable occurrence of aggression, the ability to study animals exposed to either aggression or defeat, the clear and valid measures of aggression produced, the simple and sturdy apparatus, and the convenience and economy of data collection. Mice
Aggression
Territory
Measurement
Neurochemistry
A n u m b e r of different m e t h o d s have been rather widely used for producing aggression in mice such as electric f o o t shock [ 6 ] , long periods o f isolation [ 7 ] , training fighters [ 3 ] , grouping strange animals t o g e t h e r [4] and pharmacologic agents [ 9 ] . A l t h o u g h territorial f o r m a t i o n has been studied in l a b o r a t o r y mice [ 5 ] , and has been used as a test o f aggression in s o m e studies [ 8 ] , there is n o t at present a c o m m o n l y used standardized test o f aggression based on territoriality. The ideal t e c h n i q u e for assessing aggression should i n c o r p o r a t e the following properties: it should reliably produce clear measures o f aggressive behavior such as threat postures, biting, and fighting, divide the animals unequivocally into categories o f v i c t o r y and defeat, be e c o n o m i c a l and c o n v e n i e n t to use, and it should be based on naturally occurring goal-seeking and c o n s u m m a t o r y acts free of undesirable side effects. The m e t h o d o f p r o d u c i n g and measuring agression described here has yielded results consistent with these characteristics. C o m p a r e d to m e t h o d s e m p l o y i n g prolonged isolation, f o o t shock, and pharma-
cologic agents for inducing aggression, territorial defense is a naturally occurring behavior and should be free o f the changes in biochemistry associated with these m e t h o d s [ 1, 2, 3, 7]. Thus, the t e c h n i q u e r e p o r t e d here for producing and recording aggression in laboratory mice may offer s o m e definite advantages for researchers interested in relating aggressive behavior to changes in brain biochemistry.
Apparatus T w o configurations o f the apparatus used are shown in Fig. 1; t h e y produce essentially identical results but each one has certain advantages. Both consist o f a 60 cm square o p e n field attached via a 4 cm dia. tube to a standard p o l y p r o p y l e n e mouse cage. The tube connecting the o p e n field to the mouse cage was made by forcing a square piece o f 30 ga sheet metal into a cylinder. It can be easily squeezed into a smaller diameter and inserted into the 4 cm dia. holes in the side o f the b o x and in the m o u s e cage, thus
1 Based in part on a paper presented with E. E. Quinton to the Psychonomic Society, November 1972. 879
880
THURMOND
tti I Iti tttlzi/~t.~,ilt,yx t N'F1~,, t.H-IIIIIII
~l1111:llllll~,, '!
,,
.........
i-m-II III ~tr
ILdl
;llllll
. . . . . I, , ~ I I Illlll
II II
I",,
Ilillllll
11
I1 III11
II,, II 11
III',[[~' |1 I1|1
FIG. 1. Two configurations of the apparatus used for developing the technique.
connecting the two, or it can be easily removed in this manner for cleaning. The floor of the open field boxes is made of hardware cloth with small (approximately 0.6 cm square) holes, which are small enough to prevent the mouse from sticking its head through and getting stuck. The box shown in the top of Fig. 1 was made by nailing together four 2.5 x 10 cm boards at the corners, and attaching the hardware cloth to the bottom. A 60 cm square piece of transparent Plexiglass was placed (not attached) over the top of the open field to keep the mice in the box, and paper ordinarily used to cover rat pans (approximately 60 cm wide) was placed under the open field box for cleaning purposes. The box shown in the bottom of Fig. 1 was made from 30 ga sheet metal; the hardward cloth attached to the bottom was cut slightly smaller than 60 cm square, stretched across the inside bottom of the box, and anchored at 12 equally spaced points 2 cm from the b o t t o m edge. A 62 cm square pan for the b o t t o m was constructed from 30 ga sheet metal with sides 2.5 cm high, covered first with paper and then layered 1 cm deep with bedding. The sheet metal box can easily be placed in the pan and removed for cleaning purposes. The sides of the box are 60 cm high and no top is required to keep the mice from escaping. A 30 cm high tower made of small-holed hardware cloth was anchored to the hardware cloth floor at the center of the box, and provides an 8 cm square platform at the top for introducing the intruder. The tower
also offers the submissive mouse of the pair partial protection from the attacks of the dominant animal. The 4 cm dia. tube connecting the box to the mouse cage is 12 cm long and provides a convenient method for catching the mice at the end of the test: the mouse almost invariably runs into the tube and stops, with its tail sticking out of the end. Procedure The CF-1 mouse (Carworth, New City, New York) was chosen for development of the technique because it provides the homogeniety characteristic of inbred strains, it is very aggressive, and it is inexpensive. Ninety day-old CF-1 male mice were received from Carworth, housed 5 per cage at a room temperature of approximately 21°C, and acclimated to the laboratory on a 12-hr on, 12-hr off, light cycle. They were maintained ad lib on Rat/Mouse Purina Chow and water, and bedded in Litter Green small animal litter which was changed once a week. At the time of the test for territorial aggression, the mice were completely naive, they were 104 days old, and the average mouse weighed 35 g. The mice were not designated as residents or intruders until the time of testing; thus, all mice were housed and treated exactly the same. For aggression testing, 4 cages of mice were randomly selected, 2 cages were randomly designated as residents (I 0 mice), the remaining 2 cages as intruders (10 mice), and each resident was placed in the middle of the open field (on the tower in the boxes containing one) of his respective test box. If the resident mice are placed initially in the home cage, occasionally one of them will fail to enter the open field during the next 24 hr; however, if placed in the open field, they always explore it and end up in the home cage. All 4 cages of mice were transferred to the testing room containing ten identical test boxes, and the residents were placed in their respective test box exactly 2 hr following the onset of the dim phase of the light cycle. A spot blue liquid food dye was rubbed on the top of the resident's head before placing him in the test box for identification during the aggression test. The light cycle in the testing room was identical to that where the mice were housed, and was governed by a 40 W bulb reflected off the ceiling superimposed for 12 hr with the brighter fluorescent lights of the room. On the next day, exactly 24 hr following placement of the resident mice in the test boxes, the 10 intruder mice were removed from their two cages, one each was placed in the center of the open field of the test boxes, and the ensuing aggression was recorded during a 30 rain observation period. The l 0 pairs of mice were observed simultaneously (by standing on a chair in the case of the boxes with high sides), and the following measures were recorded: (a) the latency of attack; (b) the number of attacks during the observation period; (c) the number of animals wounded (showing blood) at the end of the observation period; and (d) the number of residents attacking the intruder. An attack is defined here as a bout of activity lasting up to several seconds during which one animal bites the other one at least once; thus, an attack may consist of a number of bites one animal inflicts on the other during the course of a chase, a fight, or a stationary one-sided attack. Since a tube has been used in other research for testing dominance behavior in mice, it should be pointed out that the intruder placed in the open field is always naive. Thus,
T E R R I T O R I A L AGGRESSION IN MICE
881
exploration on the part of the intruder is limited, and displays o f dominance occur in the open field before the intruder investigates the tube.
Parameters It is advantageous to have approximately 20 resident mice in each experimental condition to show reasonable differences in treatment effects. Mice that are tested at 90 and 104 days of age show comparable levels o f aggression. At both ages, approximately 90 percent of the residents attack intruders with a latency of 5 - 7 min, the resident attacks the intruder about 24 times during the first 15 rain of the test (i.e. 15 min from the m o m e n t the intruder is placed in the test box) and about 16 times during the
second 15 min of the test. Except for the latency to attack which remains constant for those animals who attack, these values are half as great when the residents and intruders are tested at 70 days of age. An IP saline injection administered 60 min before testing reduces "the number of 104 day-old residents attacking to 70 percent; latency and number of attacks are unaffected. Thus, an IP injection of 1.25 mg/kg of Chlorpromazine HC1 administered to 20 resident mice 60 min before testing produces results that are no different from an IP injection of saline. An IP injection of 2.5 mg/kg reduces the number of residents attacking to 35 percent, increases the latency, and decreases the number of attacks thus producing results that are significantly different from the saline injected controls.
REFERENCES 1. Butcher, L. L. and J. Engel. Peripheral factors in the mediation of the effects of L-DOPA on locomotor activity. Z Pharm. Pharmac. 21: 614-616, 1969. 2. Eichelman, Jr., B. S. and N. B. Thoa. The aggressive monoamines. Biol. Psychiat. 6: 143-164, 1973. 3. Eleftheriou, B. E. and R. L. Church. Brain levels of serotonin and norepinephrine in mice after exposure to aggression and defeat. Physiol. Behav. 3: 977-980, 1968. 4. Lycke, E., K. Modigh and B. E. Roos. Aggression in mice associated with changes in the monoamine-metabolism of the brain. Experientia 25: 951-953, 1969. 5. Mackintosh, J. H. Territory formation by laboratory mice. Anim. Behav. 18: 177-183, 1970.
6. Tedeschi, R. E., D. H. Tedeschi, A. Mucha, L. Cook, P. A. Mattis and E. J. Fellows. Effects of various centrally acting drugs on fighting behavior of mice. Z Pharmac. exp. Ther. 125: 28-34, 1959. 7. Valzelli, L. The "isolation syndrome" in mice. Psychopharmacologia 31: 305-320, 1973. 8. Van Oortmerssen, G. A. Biological significance, genetics and evolutionary origin of variability in behaviour within and between inbred strains of mice. (Mus musculusJ. Behaviour 38: 1-92, 1970. 9. Yen, H. C. Y., M. H. Katz and S. Krop. Effects of various drugs on 3, 4-dihydroxyphenylalanine (DL-DOPA) - induced excitation (aggressive behavior) in mice. Toxic. appL Pharmac. 17: 597-604, 1970.
