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The stress of intruding: Reduction by chlordiazepoxide
Physiology& Behavior, Vol. 33, pp. 345-347. Copyright©PergamonPress Ltd., 1984. Printedin the U.S.A.
0031-9384/84$3.00 + .00
The Stress of Intruding: Reduction by Chlordiazepoxide S A N D R A E. F I L E
M R C Neuropharmacology Group, Department of Pharmacology, The School o f Pharmacy University o f London, Brunswick Square, London W C I N lAX, England R e c e i v e d 14 D e c e m b e r 1983 FILE, S. E. The stress of intruding: Reduction by chlordiazepoxide. PHYSIOL BEHAV 33(3) 345--347, 1984.--Rats placed as intruders in a long-established colony showed significant elevations of plasma corticosterone after 10-20 minutes of intruding. This rise was prevented by 5 days pretreatment with chlordiazepoxide (5 mg/kg). The rats resident in the colony had significantly higher basal corticosterone concentrations compared with singly housed rats but the same as rats housed in groups of six in a normal cage. The resident rats showed no elevation of plasma corticosterone in response to 20 minutes of an intruder in the colony. Rats
Corticosterone
Aggression
Housing
Benzodiazepines
COLONY housed wild rat strains vigorously attack intruders [2, 3, 7], and, although the attacks may be less vigorous, consistent attacks on intruders can also be observed in colonies of laboratory rats [5, 6, 11], even if the colony consists of all male rats [8]. This test situation provides a clear separation between attack and defensive behaviors [4]. Drug treatment of a rat placed as an intruder into an established colony can modify the ensuing agonistic encounter. Whereas acute administration of low doses of benzodiazepines had little effect on agonistic encounters, after 5 days of benzodiazepine treatment the pattern of interaction was modified [8]. There were fewer fights, less time was spent fighting and the residents directed fewer bites and aggressive acts at the intruder; in turn, the intruder submitted less and initiated more interactions with the residents. The purpose of the present experiment was to study the change in plasma corticosterone in the intruder rat, in response to being placed in an established colony, and to see whether this response was modified by chronic benzodiazepine treatment. The benzodiazepine chosen was chlordiazepoxide in a dose of 5 mg/kg for 5 days since this has been found to modify intruder/resident agonistic encounters [8] and to reduce the plasma corticosterone response to novelty stress [9,10]. For comparison, the plasma corticosterone response of the resident rats to an intruder was also measured. METHOD
Rat Colony A rat colony of 12 male hooded Lister rats (from Olac Ltd., Bicester) was established 9 months prior to this experiment and had received experience of intruders prior to this experiment. The rats had food and water freely available and were housed in a three-story enclosure, with each floor 91x60 cm, the top floor 15 cm high and the others 20.5 cm
345
high. There were tunnels, nest boxes, stair cases and a tree (see [8]). The colony was housed in a room with lights on from 0700 to 1800 hr. The rats weighed 350 g when the colony was started and 450 g at the time of this experiment. Less conflict occurred spontaneously in this colony than had been found in previous colonies, but fighting always occurred over competition for chocolate. A pilot study on 6 intruders showed that most of the aggression occurred in the first 12 min and little occurred after 24 min (see Fig. 1). The durations of the test periods were based on these results.
Drugs Chlordiazepoxide hydrochloride (CDP, Roche Products Ltd) was dissolved in deionised water to a concentration of 2.5 mg/ml. Daily IP injections of CDP (5 mg/kg) or water (as control) were given for 5 days before the intruder test. On the day of test all intruder rats were injected IP with CDP or water, as appropriate, 30 min before test.
Procedure Rats (approximately 250 g) were randomly allocated 24 each to control and CDP treatments. They were singly housed for 5 days before test in a room with lights on 0700 to 1800 hr. Within each group rats were randomly allocated, six to each of the following test conditions: home-cage or 5, I0 or 20 minutes in the colony. On the test day each intruder rat was placed in the middle floor of the colony for 5, 10 or 20 min, then immediately removed and lightly anaesthetised with ether. A blood sample was taken by direct cardiac puncture (this procedure taking 15-30 sec). The blood was collected in heparinised plastic tubes, centrifuged at 3,000 rpm and stored at -20°C for later fluorimetric assay [13]. After each 20 min intruder test a resident rat was also removed, marked for later identification, and a blood sample taken. Three intruder tests
346
I:11 E
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12 13 18 19-2t, 25-30 Minutes
0-6 in
7-12
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~R~
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5~-18 19 26 2 5 - 3 0
colony
FIG. 1. Distribution of number of ftghts (wrestling, kicking, pushing or biting) and number of boxing (both rats on hind-paws and forepaw boxing) episodes during a 30 minutes exposure of an intruder rat to an established colony.
-
u
g
v
20
T
E m
~
10
C
took place each day, in an order randomised for drug treatment and, for the 5 and 10 rain tests, for the duration of test. Because blood samples were taken from the resident rats after 20 minutes of an intruder, this test was always conducted last. Blood samples were taken from the rats left in their single home-cages for 40 minutes after IP injection of CDP or water. All testing took place between 0900 and 1030 hr. In order to provide a measure of the resident rats's corticosterone concentrations when undisturbed by intruders, samples were taken from all the rats 1 week after the last intruder test. For comparison, samples were also taken from rats resident in another colony, that had not been disturbed by intruders for at least 4 weeks. Finally, to compare the colony levels with those from normal group-housing, blood samples were taken from 6 rats that had been housed together in plastic cages (32x50x 19 cm), undisturbed, for the previous 2 months. All these samples were collected between 0900 and 1030 hr. RESULTS The corticosterone results were analysed by a two-way analysis of variance with drug treatment (CDP or control) and time in colony (5, l0 or 20 min) as the two factors. From Fig. 2 it can be seen that control rats showed an elevated corticosterone response after l0 or 20 minutes in the colony, whereas this elevation was prevented by the chronic chlordiazepoxide treatment (drug x time interaction F(3,30)= 22.2, p <0.001). The mean number of agonistic encounters that occurred between the control intruder rats and the residents after 5, l0 and 20 min in the colony were 3.4, 10.7 and 16.0, respectively; for the CDP-treated intruders these were 0, 0.2 and 0.3, respectively. It was therefore only possible to correlate the plasma corticosterone concentrations for the control intruders at the l0 and 20 rain test periods. Combining these data, the Spearman rank-order coefficient of correlation between corticosterone concentrations and number of fights was 0.53. After 20 minutes of intruder presence in the colony the resident rats' corticosterone (RI in Fig. 2) was the same level as that of the intruder rat. However, this did not show an increase over that found in the undisturbed condition (RQ). The cortic0sterone concelatrations in the colony rats were significantly higher, than those of the singly housed control
5"
0
•
~
i 0 home cage
5
t
J
10 m i n u l e s in colony
j
20
FIG. 2. Mean plasma corticosterone concentrations (/xg/100 ml) of singly-housed intruder rats placed in the colony for 5, l0 or 20 minutes after chronic treatment with CDP (5 mg/kg for 5 days and 30 minutes before tests O- - -O) or water A--A. Also shown are the corticosterone concentrations for singly housed rats taken straight from their home-cages 40 minutes after CDP or water injection. The corticosterone concentrations for the resident rats in the colony are also shown after 20 minutes of an intruder (RI) or when undisturbed (RQ). For comparison, the corticosterone concentrations of rats group-housed in a normal cage (GQ) are shown.
rats Lo<0.001, two-tailed t-test). The colony used in this experiment did not have unusually high concentrations since the mean concentration for the other colony sampled was 49.6+2.82 /zg/100 ml. Nor was the corticosterone level higher in the colony than in other group housed rats (see GQ in Fig. 2). DISCUSSION Treatment with a low dose of chlordiazepoxide for 5 days prevented the rise in plasma corticosterone that resulted from intruding into an established rat colony, but was without effect on rats left in their home cages. These results are consistent with those previously reported for the effects of chronic chlordiazepoxide in reducing the corticosterone response to other forms of stressful stimuli [9], and also consistent with the reduction in fighting displayed to drug-treated intruders. In the undrugged intruders the correlation between the incidence of fights and the plasma corticosterone response was not high enough to suggest that the former was a major determinant of the latter. The plasma corticosterone concentrations for the rats that remained in their home-cages were the same as those reported in previous experiments [8,9]. Although caution must always be exercised in comparing across experiments it is interesting that the rise in corticosterone after 20 min in the colony was less than that found in rats from the same supplier following 20 minutes exposure to loud tones (74 /zg/100 ml, [9]) or 20 minutes exposure to a brightly-lit room (102/~g/100 ml, [10]).
CORTICOSTERONE IN RAT INTRUDERS
347
H o w e v e r , the c o r t i c o s t e r o n e r e s p o n s e in the intruders was similar in magnitude to that seen in defeated rats, and this elevation r e m a i n e d for 3 hr after the aggressive e n c o u n t e r [12]. This suggests that the c o r t i c o s t e r o n e response to aggression may be characterised by its long duration rather than by its intensity. The relatively small rise in c o r t i c o s t e r o n e in the colony test may be the result o f an apparent opportunity to escape from an attacker, or to an active m e c h a n i s m suppressing the rise in corticosterone. If this was so, a higher initial rise in c o r t i c o s t e r o n e might be seen before the active suppression occurred. N o e v i d e n c e for this was found in our results and therefore any such m e c h a n i s m was fully operational within 10 minutes of the start of the test. Being housed with o t h e r rats is as effective at raising plasma c o r t i c o s t e r o n e c o n c e n t r a t i o n s as being an intruder in
the colony. This elevation in c o r t i c o s t e r o n e from grouphousing has been reported previously [10] and is consistent with the report that e v e n in a neutral a r e n a the p r e s e n c e of a familiar rat will raise c o r t i c o s t e r o n e m o r e than an unfamiliar rat [1]. H o w e v e r it is interesting that there was no elevation of plasma corticosterone in the residents as a result of an intruder's p r e s e n c e in the colony.
ACKNOWLEDGEMENTS SEF is a Wellcome Trust Senior Lecturer. I am grateful to David Holt and Peter Mabbutt for expert technical assistance. This experiment was supported by a grant from the Medical Research Council.
REFERENCES 1. Armario, A., R. Ortiz and J. Balasch. Corticoadrenal and behavioral response to open field in pairs of male rats either familiar or non-familiar to each other. Experientia 39: 1316-1317, 1983. 2. Barnett, S. A. Physiological effects of "'social stress" in the wild rat. 1. The adrenal cortex. J Psychosom Res 3 : i - 1 l, 1958. 3. Barnett, S. A. Grouping and dispersive behaviour among wild rats. In: Aggressive Behaviour, edited by S. Garattini and E. B. Sigg. Amsterdam: Excerpta Medica Foundation, 1969. 4. Blanchard, R. J. and D. C. Blanchard. The colony model: experience counts. Behav Neural Biol 30:109-112, 1980. 5. Blanchard, R. J., D. C. Blanchard, T. Takahashi and J. R. Kelley. Attack and defensive behavior in the albino rat. Anita Behav 25: 622-634, 1977. 6. Blanchard, R. J., T. Takahashi and D. C. Blanchard. Development of intruder attack in colonies of laboratory rats. Anita Learn Behav 5: 365-369, 1977.
7. Calhoun, J. B. The Ecology and Sociology of the Norway Rat. U.S. Public Health Service Pub. No. 1008. Washington, DC: U.S. Govt. Printing Office, 1962. 8. File, S. E. Colony aggression: Effects of benzodiazepines on intruder behavior. Physiol Psychol 10: 413-416, 1982. 9. File, S. E. The rat corticosterone response: Habituation and modification by chlordiazepoxide. Physiol Behav 29: 91-95, 1982. 10. File, S. E. and L. A. Peet. The sensitivity of the rat corticosterone response to environmental manipulations and to chronic chlordiazepoxide treatment. Physiol Behav 25: 753-758, 1980. I1. Flanelly, K. and R. Lore. The influence of females upon aggression in domesticated male rats (Rattus norvegicus). Anita Behav 25: 654-659, 1977. 12. Schuurman, T. Hormonal correlates of agonistic behavior in adult male rats. Prog Brain Res 53: 415--420, 1981. 13. Zenker, N. and D. E. Bernstein. The estimation of small amounts of corticosterone in rat plasma. J Bh~l Chem 231: 695701, 1958.
0031-9384/84$3.00 + .00
The Stress of Intruding: Reduction by Chlordiazepoxide S A N D R A E. F I L E
M R C Neuropharmacology Group, Department of Pharmacology, The School o f Pharmacy University o f London, Brunswick Square, London W C I N lAX, England R e c e i v e d 14 D e c e m b e r 1983 FILE, S. E. The stress of intruding: Reduction by chlordiazepoxide. PHYSIOL BEHAV 33(3) 345--347, 1984.--Rats placed as intruders in a long-established colony showed significant elevations of plasma corticosterone after 10-20 minutes of intruding. This rise was prevented by 5 days pretreatment with chlordiazepoxide (5 mg/kg). The rats resident in the colony had significantly higher basal corticosterone concentrations compared with singly housed rats but the same as rats housed in groups of six in a normal cage. The resident rats showed no elevation of plasma corticosterone in response to 20 minutes of an intruder in the colony. Rats
Corticosterone
Aggression
Housing
Benzodiazepines
COLONY housed wild rat strains vigorously attack intruders [2, 3, 7], and, although the attacks may be less vigorous, consistent attacks on intruders can also be observed in colonies of laboratory rats [5, 6, 11], even if the colony consists of all male rats [8]. This test situation provides a clear separation between attack and defensive behaviors [4]. Drug treatment of a rat placed as an intruder into an established colony can modify the ensuing agonistic encounter. Whereas acute administration of low doses of benzodiazepines had little effect on agonistic encounters, after 5 days of benzodiazepine treatment the pattern of interaction was modified [8]. There were fewer fights, less time was spent fighting and the residents directed fewer bites and aggressive acts at the intruder; in turn, the intruder submitted less and initiated more interactions with the residents. The purpose of the present experiment was to study the change in plasma corticosterone in the intruder rat, in response to being placed in an established colony, and to see whether this response was modified by chronic benzodiazepine treatment. The benzodiazepine chosen was chlordiazepoxide in a dose of 5 mg/kg for 5 days since this has been found to modify intruder/resident agonistic encounters [8] and to reduce the plasma corticosterone response to novelty stress [9,10]. For comparison, the plasma corticosterone response of the resident rats to an intruder was also measured. METHOD
Rat Colony A rat colony of 12 male hooded Lister rats (from Olac Ltd., Bicester) was established 9 months prior to this experiment and had received experience of intruders prior to this experiment. The rats had food and water freely available and were housed in a three-story enclosure, with each floor 91x60 cm, the top floor 15 cm high and the others 20.5 cm
345
high. There were tunnels, nest boxes, stair cases and a tree (see [8]). The colony was housed in a room with lights on from 0700 to 1800 hr. The rats weighed 350 g when the colony was started and 450 g at the time of this experiment. Less conflict occurred spontaneously in this colony than had been found in previous colonies, but fighting always occurred over competition for chocolate. A pilot study on 6 intruders showed that most of the aggression occurred in the first 12 min and little occurred after 24 min (see Fig. 1). The durations of the test periods were based on these results.
Drugs Chlordiazepoxide hydrochloride (CDP, Roche Products Ltd) was dissolved in deionised water to a concentration of 2.5 mg/ml. Daily IP injections of CDP (5 mg/kg) or water (as control) were given for 5 days before the intruder test. On the day of test all intruder rats were injected IP with CDP or water, as appropriate, 30 min before test.
Procedure Rats (approximately 250 g) were randomly allocated 24 each to control and CDP treatments. They were singly housed for 5 days before test in a room with lights on 0700 to 1800 hr. Within each group rats were randomly allocated, six to each of the following test conditions: home-cage or 5, I0 or 20 minutes in the colony. On the test day each intruder rat was placed in the middle floor of the colony for 5, 10 or 20 min, then immediately removed and lightly anaesthetised with ether. A blood sample was taken by direct cardiac puncture (this procedure taking 15-30 sec). The blood was collected in heparinised plastic tubes, centrifuged at 3,000 rpm and stored at -20°C for later fluorimetric assay [13]. After each 20 min intruder test a resident rat was also removed, marked for later identification, and a blood sample taken. Three intruder tests
346
I:11 E
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L2
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'~
20
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60
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ii
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rc
:
12 13 18 19-2t, 25-30 Minutes
0-6 in
7-12
30
....
,/
40
~R~
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5~-18 19 26 2 5 - 3 0
colony
FIG. 1. Distribution of number of ftghts (wrestling, kicking, pushing or biting) and number of boxing (both rats on hind-paws and forepaw boxing) episodes during a 30 minutes exposure of an intruder rat to an established colony.
-
u
g
v
20
T
E m
~
10
C
took place each day, in an order randomised for drug treatment and, for the 5 and 10 rain tests, for the duration of test. Because blood samples were taken from the resident rats after 20 minutes of an intruder, this test was always conducted last. Blood samples were taken from the rats left in their single home-cages for 40 minutes after IP injection of CDP or water. All testing took place between 0900 and 1030 hr. In order to provide a measure of the resident rats's corticosterone concentrations when undisturbed by intruders, samples were taken from all the rats 1 week after the last intruder test. For comparison, samples were also taken from rats resident in another colony, that had not been disturbed by intruders for at least 4 weeks. Finally, to compare the colony levels with those from normal group-housing, blood samples were taken from 6 rats that had been housed together in plastic cages (32x50x 19 cm), undisturbed, for the previous 2 months. All these samples were collected between 0900 and 1030 hr. RESULTS The corticosterone results were analysed by a two-way analysis of variance with drug treatment (CDP or control) and time in colony (5, l0 or 20 min) as the two factors. From Fig. 2 it can be seen that control rats showed an elevated corticosterone response after l0 or 20 minutes in the colony, whereas this elevation was prevented by the chronic chlordiazepoxide treatment (drug x time interaction F(3,30)= 22.2, p <0.001). The mean number of agonistic encounters that occurred between the control intruder rats and the residents after 5, l0 and 20 min in the colony were 3.4, 10.7 and 16.0, respectively; for the CDP-treated intruders these were 0, 0.2 and 0.3, respectively. It was therefore only possible to correlate the plasma corticosterone concentrations for the control intruders at the l0 and 20 rain test periods. Combining these data, the Spearman rank-order coefficient of correlation between corticosterone concentrations and number of fights was 0.53. After 20 minutes of intruder presence in the colony the resident rats' corticosterone (RI in Fig. 2) was the same level as that of the intruder rat. However, this did not show an increase over that found in the undisturbed condition (RQ). The cortic0sterone concelatrations in the colony rats were significantly higher, than those of the singly housed control
5"
0
•
~
i 0 home cage
5
t
J
10 m i n u l e s in colony
j
20
FIG. 2. Mean plasma corticosterone concentrations (/xg/100 ml) of singly-housed intruder rats placed in the colony for 5, l0 or 20 minutes after chronic treatment with CDP (5 mg/kg for 5 days and 30 minutes before tests O- - -O) or water A--A. Also shown are the corticosterone concentrations for singly housed rats taken straight from their home-cages 40 minutes after CDP or water injection. The corticosterone concentrations for the resident rats in the colony are also shown after 20 minutes of an intruder (RI) or when undisturbed (RQ). For comparison, the corticosterone concentrations of rats group-housed in a normal cage (GQ) are shown.
rats Lo<0.001, two-tailed t-test). The colony used in this experiment did not have unusually high concentrations since the mean concentration for the other colony sampled was 49.6+2.82 /zg/100 ml. Nor was the corticosterone level higher in the colony than in other group housed rats (see GQ in Fig. 2). DISCUSSION Treatment with a low dose of chlordiazepoxide for 5 days prevented the rise in plasma corticosterone that resulted from intruding into an established rat colony, but was without effect on rats left in their home cages. These results are consistent with those previously reported for the effects of chronic chlordiazepoxide in reducing the corticosterone response to other forms of stressful stimuli [9], and also consistent with the reduction in fighting displayed to drug-treated intruders. In the undrugged intruders the correlation between the incidence of fights and the plasma corticosterone response was not high enough to suggest that the former was a major determinant of the latter. The plasma corticosterone concentrations for the rats that remained in their home-cages were the same as those reported in previous experiments [8,9]. Although caution must always be exercised in comparing across experiments it is interesting that the rise in corticosterone after 20 min in the colony was less than that found in rats from the same supplier following 20 minutes exposure to loud tones (74 /zg/100 ml, [9]) or 20 minutes exposure to a brightly-lit room (102/~g/100 ml, [10]).
CORTICOSTERONE IN RAT INTRUDERS
347
H o w e v e r , the c o r t i c o s t e r o n e r e s p o n s e in the intruders was similar in magnitude to that seen in defeated rats, and this elevation r e m a i n e d for 3 hr after the aggressive e n c o u n t e r [12]. This suggests that the c o r t i c o s t e r o n e response to aggression may be characterised by its long duration rather than by its intensity. The relatively small rise in c o r t i c o s t e r o n e in the colony test may be the result o f an apparent opportunity to escape from an attacker, or to an active m e c h a n i s m suppressing the rise in corticosterone. If this was so, a higher initial rise in c o r t i c o s t e r o n e might be seen before the active suppression occurred. N o e v i d e n c e for this was found in our results and therefore any such m e c h a n i s m was fully operational within 10 minutes of the start of the test. Being housed with o t h e r rats is as effective at raising plasma c o r t i c o s t e r o n e c o n c e n t r a t i o n s as being an intruder in
the colony. This elevation in c o r t i c o s t e r o n e from grouphousing has been reported previously [10] and is consistent with the report that e v e n in a neutral a r e n a the p r e s e n c e of a familiar rat will raise c o r t i c o s t e r o n e m o r e than an unfamiliar rat [1]. H o w e v e r it is interesting that there was no elevation of plasma corticosterone in the residents as a result of an intruder's p r e s e n c e in the colony.
ACKNOWLEDGEMENTS SEF is a Wellcome Trust Senior Lecturer. I am grateful to David Holt and Peter Mabbutt for expert technical assistance. This experiment was supported by a grant from the Medical Research Council.
REFERENCES 1. Armario, A., R. Ortiz and J. Balasch. Corticoadrenal and behavioral response to open field in pairs of male rats either familiar or non-familiar to each other. Experientia 39: 1316-1317, 1983. 2. Barnett, S. A. Physiological effects of "'social stress" in the wild rat. 1. The adrenal cortex. J Psychosom Res 3 : i - 1 l, 1958. 3. Barnett, S. A. Grouping and dispersive behaviour among wild rats. In: Aggressive Behaviour, edited by S. Garattini and E. B. Sigg. Amsterdam: Excerpta Medica Foundation, 1969. 4. Blanchard, R. J. and D. C. Blanchard. The colony model: experience counts. Behav Neural Biol 30:109-112, 1980. 5. Blanchard, R. J., D. C. Blanchard, T. Takahashi and J. R. Kelley. Attack and defensive behavior in the albino rat. Anita Behav 25: 622-634, 1977. 6. Blanchard, R. J., T. Takahashi and D. C. Blanchard. Development of intruder attack in colonies of laboratory rats. Anita Learn Behav 5: 365-369, 1977.
7. Calhoun, J. B. The Ecology and Sociology of the Norway Rat. U.S. Public Health Service Pub. No. 1008. Washington, DC: U.S. Govt. Printing Office, 1962. 8. File, S. E. Colony aggression: Effects of benzodiazepines on intruder behavior. Physiol Psychol 10: 413-416, 1982. 9. File, S. E. The rat corticosterone response: Habituation and modification by chlordiazepoxide. Physiol Behav 29: 91-95, 1982. 10. File, S. E. and L. A. Peet. The sensitivity of the rat corticosterone response to environmental manipulations and to chronic chlordiazepoxide treatment. Physiol Behav 25: 753-758, 1980. I1. Flanelly, K. and R. Lore. The influence of females upon aggression in domesticated male rats (Rattus norvegicus). Anita Behav 25: 654-659, 1977. 12. Schuurman, T. Hormonal correlates of agonistic behavior in adult male rats. Prog Brain Res 53: 415--420, 1981. 13. Zenker, N. and D. E. Bernstein. The estimation of small amounts of corticosterone in rat plasma. J Bh~l Chem 231: 695701, 1958.