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Regional changes in brain norepinephrine content in relation to mouse-killing behavior by rats
Brain Research
Bulletin, Vol. 7, PP. 151-155, 1981. Printed in the U.S.A.
Regional Changes in Brain Norepinephrine Content in Relation to Mouse-Killing Behavior by Rats Department
of Pharmacology, Faculty of Pharmaceutical Kyushu University, Fukuoka 812, Japan
Sciences
Received 24 April 1981 YOS~IMURA, H. AND S. UEKI. Regional changes in brain ~ore~inephrine content in relation to mouse-kiiling behavior by ruts. BRAIN RES. BULL 7(2) 151-155, 1981.--Three separate series of experiments were conducted as
follows: isolation housing, bilateral olfactory bufbectomy, and A*-tetrahydr~annabino1 tTHC) admi~stration. All three experimental manipulations produced an increase in the incidence of mouse-killing behavior. In order to elucidate the possible neural mechanisms mediating the killing response, norepinephrine (NE) content was measured in 6 discrete areas of the brain (the cortex, striatum, amygdala, midbrain, hypothalamus, and pons plus medulla oblongata). Following isolation housing, no significant difference in NE levels of any of the brain areas was demonst~ted between the aggregated and isolated rats, nor between the killer and nonkiller rats. The rats with olfactory bulbectomy exhibited high NE content in the hypothalamus as compared with the intact or sham-operated rats, but there was no significant difference between the killer and nonkiller rats. After injection of THC, NE content in both the hypothalamus and pons plus medulla oblongata was decreased independent of the manifestation of killing response. The evidence indicates no regional change in brain NE levels specific to the killing response and suggests that brain NE may not participate in the mediation of mouse-killing behavior. Mouse-killing behavior As-Tetrahydr~~nabinol
Brain norepinephrine
Rats
MOUSE-killing behavior in rats was first reported by Karli in 1956 who found that almost all wild Norway rats kill mice 1121, By contrast, in domesticated rats this behavior is infrequent and various experimenta maniputations are necessary to induce the killing response; for exampIe, prolonged isolation housing [2,25], bilateral olfactory bulbectomy [3,23], or Ag-tetrahydrocannabinol (THC) administration 122,243. As described in previous reports [2,8, 161, however, the behavioral topography of the killing response differs among the experimentally-induced killer rats. The killing response induced by prolonged isolation housing had a similar quality to that observed in natural or spont~eous killer rats: quiet, quick, and eff&ient attack biting to the cervical region with little or no affective display. On the other hand, killer rats with olfactory bulb ablations did not exhibit such a wellorganized killing response. Their killing response had a compulsive, disinhibited quality, and in many cases the killed mice were covered with blood from head to tail. At the introduction of a mouse the THC-treated animal retreated to a corner of the cage and cowered there with startled squeals, and then suddenly attacked the mouse. Moreover, THCinduced killer rats did not release the dead mouse from their mouth, and continued to bite it. Our recent investigation ‘Now at the Department of Pha~acology, the first author.
Copyright
Isolation housing
Olfactory bulbectomy
(unpublished data) indicates that different kinds of manipulations also produce distinctive features in emotionality; isolation-educed killer rats were more reactive in startle response than isolated nonkilIer rats; olfactory bulbectomized rats exhibited high reactivity to external stimuli, and their response to inanimate objects or the experimenter was highly offensive in nature; THC-treated rats vocalized frequently during the measurement of emotion~ity, but both struggle and startle response were not affected by THC. The question which arises is whether or not there exists a common physiological basis mediating the mouse-killing behavior among the expe~ment~ly-induced killer rats. Interestingly, Malick has recentfy reported that the ability of drugs to inhibit killing response differs significantly between the models of killer rats employed [lS]. For the neural mechanisms of mouse-killing behavior, pharmacologic~ studies suggest that brain catechoiamines (CA) may play an inhibitory role in the manifestation of the behavior [ 1, 11, 141: for example, drugs which produce an increase of CA concentration at receptor sites, such as amphetamine and imipramine, can suppress the killing response in the killer rats, while the depletion of CA store with cu-methyl-p-tyrosine or reserpine induces the killing response in the nonkiller rats. This view is further supported by the
School of Medicine, Ehime University, Ehime 791-02, Japan. Reprint requests should be sent to
0 1981 ANKHO
Inte~ational
Inc .-0361-9230/81/0801~1-05$01.~/0
YOSHIMURA finding that lesions of the dorsal noradrenergic bundle facilitate mouse~killing behavior in olfactory bulbe~tomized rats [ 183.In addition, Leaf et nt. [14] has reported that direct application of norepinephrine (NE) into the amygdaloid nuclei suppressed some of the mouse-killing behavior. The present study was conducted to investigate the participation of brain NE in the manifestation of the killing response, using three models of killer rats. Male rats were subjected to one of the following manipulations: prolonged isolation housing, bilateral olfactory bulbectomy, and THC administration. Brain NE content was estimated in 6 discrete regions. METHOD Subjects
All the animals employed were male Wistar King-A rats supplied from Kyushu University Institute of Experimental Animals. Upon arrival, rats were housed communally for at least 10 days before each experimental manipulation, and had free access to food and water throughout the experiments unIess otherwise stated. Ablino mice weighing between 20 and 30 g were used as stimuli for mouse-k~ling behavior. The temperature in the vivarium was maintained at 222 l”C, and a 12 hr light-dark cycle (light on at 7:00 a.m. and off at 7%) p.m.) was controlled automatically. In the present experiments, approximately 6% of the rats were spontaneous killer rats and were eliminated from the subjects. A~purarus The isolation cage was made of wire mesh and was divided into 5 compartments, 18x 17x 17 cm. The compartments were separated by opaque plastic partitions. Under these conditions, rats were exposed to social stimuli such as squeaks and odors, but did not have physical contact or visible stimuli. The aggregated animals were housed in a 40x33~ I7 cm cage with plastic walls. Experimental
Design
Three separate series of experiments were conducted as follows: isolation housing, bilateral olfactory bulbectomy, and THC administ~tion. Experiment I. Twenty-four rats weighing between 180 and 200 g were used. The subjects in the isolated groups were housed individually for 30 days in the isolation cages, while the subjects in the aggregated control group were housed communally, 4 animals per cage, for 30 days. The mouse-killing behavior was tested periodically at intervals of 5 days. On the 30th day of isolation, an equal number of animals (n=6) from each group (aggregated control, isolated killer and nonkiller rats) were randomly selected and sacrificed 15 min after the last presentation of a mouse. Experiment 2. Thirty-six rats weighing between 180 and 200 g at the time of surgery were used. The olfactory bulbectomy was performed under pentobarbital anesthesia (40 mg/kg, IP). The olfactory bulbs were bilaterally removed by suction through two holes made in the skull just above the bulbs. The sham-operated animals underwent the same procedure as the bulbectomized rats, but aspiration of the olfactory bulbs was not made. All the subjects including the intact control rats were housed individually in the isolation cages for I4 days. Mouse-killing behavior was tested 3, 7, and 14 days after surgery. On the 14th day, an equal number of animals (n=5) from each group (intact control, sham-
AND UEKI
operated control, bulbectomized killer and nonkiller rats) were randomly selected and sacrificed 15 min after the last presentation of a mouse. The brains of the bulbectomized rats were examined macroscopically to ensure that the bulbs had been completely ablated. Sham-operated animals were also examined for any possible damage. Experiment 3. Twenty-four rats weighing between 120 and 140 g at the time of drug administration were used. All the animals were housed individually in isolation cages and subjected to food deprivation for 24 hrs prior to THC or vehicle administration. Natural THC, supplied by the Department of Pharmacognosy, Kyushu University, was suspended in a 1% aqueous Tween 80 solution and injected intraperitoneally in a dose of 6 mgikg. Mouse-killing behavior was tested 30 min before and 1 hr after THC or vehicle administration. An equal number of animals (n=6) from each group (vehicle control, THC-treated killer and nonkiller rats) were randomly selected and sacrificed 15 min after the last presentation of a mouse. Behuvioral
Testing
Mouse-killing behavior was determined by introducing an albino mouse into the rat’s home cage. If the rat killed the mouse within 5 min it was judged to be a killer; otherwise it was termed a nonkiller. Norepinephrine
Assay
Animals were sacrificed by the near-freezing method of Takahashi and Aprison [ 2 11,using liquid nitrogen. The brain was quickly removed and 6 regions--cortex, striatum, amygdala, midbrain, hypothalamus, and pons plus medulla oblongata-were separated on an ice-cold glass plate according to Giowinski and Iversen [93. After homogenization in 0.4 N perchloric acid, NE was isolated from the supematant by the alumina absorption method of Crout [S] and was estimated fluorometric~ly by the t~hydroxyindole method of Chang (43. Statistical Anulysis Statistical evaluation of the biochemical data was performed by means of an analysis of variance and the Tukey test. Differences in the prounion of killer rats among the three models were evaluated by means of the Chi-Square test.
RESULTS
Table 1 shows the proportion and percentage of killer rats. Although the behavioral topography of killing response differed among the three models, there was no significant difference in the ability of manipulations to induce kiliing response in nonkiller rats. In both isolation-induced and olfactory bul~~tom~ed killer rats, once killing behavior developed, it was constant and did not disappear under repeated measurements. The killing response in THC-treated rats lasted for at least 3 hrs and disappeared 24 hrs after injection of the drug. Experiment
I
The regional changes in brain NE content after isolation housing are shown in Table 2. There was no significant
BRAIN NOREPINEPH~NE
153
AND MOUSE-KILLING TABLE 1 INCIDENCEOF MOUSE-KILLINGBEHAVIORFGLLOWINGTHREE DIFFERENT~AN~~LATIONS No.
Group
Ex~~ment 1 2
3
Aggregated Isolated Intact Sham-operated Olfactory b~~ctomi~d Vehicle-treated THC-treated
of
No.
%
of
Tested
Killers
8 16 8 7 13 8 16
0 7 0 I 8 0 9
Killers 0 44 0 14 62* 0 56’F
*Intact group vs olfactory bui~ctomi2ed group, x2=5.56, p
TABLE 2 BRAINNGREPINEPH~N~ LEVELSIN RATSFOLLOWINGPROLONGED ISOLATIONHOUSING Isolated Group Brain Areas Cortex Stiiatum Amygdala Midbrain Hy~~~amus Pons + Medulla Oblongata
A~gated Group (n=6) 026 f 0.22 + 0.37 -t 0.54 + 1.41 * 0.54 2
0.01 0.02 0.03 0.04 0.10 0.03
Killer (n=6) 0.27 -c &Of 0.21 + 0.03 0.40 -+ 0.05 0.52 -e 0.08 1.54 r 0.20 0.55 f 0.05
Non~Ier (n=6) 0.26 + 0.22 t 0.36 f 0.55 + 1.46 r 0.55 t
0.02 0.02 0.05 0.03 0.08 0.03
Norep~n~p~ne contents are expressed as ~8 per g of wet tissue; means r S.D.
difference in all brains areas between the aggregated and isolated groups, or between the killer and nonkiller rats.
Table 3 shows the regional changes in brain NE content after bilateral olfactory bulbectomy. Analysis of variance revealed a s~~~c~t difference in the ~ygd~a, F(3,16) =10.92, p
and midbr~, appears to be caused by olfactory b~~ctomy itself since there was no significant d~erence in the hypothalamic NE content between the intact and sh~~~mted control. However, no significant difference in the hypothalamus was found between the killer and nonkiller rats.
The levels of NE in the 6 brain areas after the administration of THC are shown in Table 4. There were si~i~c~t differences in NE levels of the three brain areas among treatment groups: the amygdala, F(2,15)=8.26, pcO.005, hy~th~amus, F(2,15)=4.43,p
154
YOSHIMURA
AND UEKI
TABLE 3 BRAIN NOREPINEPHRINELEVELSIN
RATS FOLLOWING
BILATERAL
Control Group Brain Areas
(n=S) 0.30 + 0.29 t 0.48 t 0.65 + 1.17 + 0.70 t
Cortex
Striatum Amygdala Midbrain Hypothalamus Pons + Medulla Oblongata
0.04 0.04 0.06 0.03 0.09 0.06
BULBECTOMY
Bulbectomized Group
Sham (n=5)
Intact
OLFACTORY
Killer (n=%
0.34 ?z 0.02 0.30 t 0.03 0.58 + O.OPf 0.79 i o.o5t!: 1.29 -f 0.08 0.70 -c 0.03
Nonkiller (n=5)
0.32 2 0.03 0.34 +- 0.03 0.57 + 0.01*1
0.29 i 0.04 0.30 + 0.03 0.65 t 0.03tltt
0.76+ O.OS*B 1.54 t O.O8t§#
0.77? 0.04ty 1.49 t o.o4tlI**
0.76 ? 0.05
0.76 t 0.04
Norepinephrine contents are expressed as wg per g wet tissue; mean 4 S.D. *p
vs Killer; **=Sham vs Nonkiller:
TABLE 4 BRAIN NOREPINEPHRINE LEVELS IN RATS FOLLOWING ADMINISTRATION OF Aa-TETRAHYDROCANNABINOL
THC Group Brain Areas Cortex Striatum Amygdala Midbrain Hypothalamus Pons + Medulla
Vehicle Group (n=6) 0.26 0.32 0.62 0.56 1.57 0.72
2 + 2 ? t +
0.02 0.03 0.02 0.07 0.18 0.05
Killer (n=6) 0.24 0.31 0.63 0.49 1.35 0.59
+ 0.03 + 0.02 r 0.05 ? 0.06 f 0.1o*i r+_0.03t:
Nonkiller (n=6) 0.23 + 0.30 ? 0.53 2 0.48 t 1.37 t 0.60+
0.02 0.02 o.o5*Ul 0.07 0.07*s o.o4v+
Oblongata Norepinephrine contents are expressed as pg per g of wet tissue; means r
S.D.
*p
‘rpco.01. $=Vehicle vs Killer; $=Vehicle vs Nonkiller; lI=Killer vs Nonkiller.
DISCUSSION
The present results showing that all three experimental manipulations could elicit the killing response in nonkiller rats is in general accordance with previous reports although there are considerable differences in the incidence of mouse-killing behavior between different strains of rats [2, 3, 221. The results of Experiment 1 clearly demonstrate that prolonged isolation housing does not alter brain NE levels in all 6 discrete regions examined, and that brain NE levels of the killer rats do not differe from that of the nonkiller rats, a finding which is not always consistent with the earlier report by Goldberg and Salama [lo] describing an increased level of forebrain NE in the killer rats. In their experiment, however, animals were subjected to a restricted feeding schedule in addition to individual housing, and therefore possible effects of food restriction on both killing response and brain NE content cannot be ruled out. “Hunger” has so far been considered one of the motivational factors leading to the manifestation of killing response [ 191, and consequently a
separate design of experiment may be needed for analysis of effect of food deprivation. Several investigators have reported a decrease in the cortical or telencephalic NE content after olfactory bulbectomy [7,20]. In the present study, however, we did not find any change in the cortical NE content. Recently, Edwards [61 demonstrated that NE content of the telencephalic region was significantly decreased only when olfactory system surgery extensively damaged the olfactory peduncle or tubercle. The ablations of the olfactory bulbs in this study were limited to both the main and accessary olfactory bulbs, and did not extend to more central portions. Our data, therefore, appear to support Edwards’ finding, and the extent of brain damage by surgery may be an essential factor for the change in the cortical NE content. The increase of NE level in the hypothalamus does not apparently correlate with the manifestation of killing response when one takes into consideration that there was no significant difference between the killer and nonkiller rats. This hypothalamic change is likely to correlate with other behavioral characteristics such
BRAIN NOREPINEPHRINE
155
AND MOUSE-KILLING
as hyperirritability
which are common to olfactory bulbectomized rats [3,13]. Contrary to the data on olfactory bulbectomy, acute injection of THC produced a decrease in NE levels of both the hypothalamus and pons plus medulla oblongata both in killer and nonkiller rats. This evidence is interesting in light of the fact that bilateral olfactory bulbectomy increases aggressive behavior [3] while acute injection of THC decreases different aggressive behaviors [17]. It should be noted that in the amygdala only nonkiller rats exhibited lower NE level than vehicle-treated rats or THC-treated killer rats. Since a decrease in NE content can be explained as being due to the increased availability of the transmitter, the decrease in amygdaloid NE level found in the present investigation
might be a reflex of activation of the adrenergic system. This may be supported by Leaf’s finding that microinjection of NE into the amygdaloid nuclei can suppress the killing response in spontaneous killer rats. [14]. Since neither common regional change in brain NE levels among the three models of killer rats, nor specific change to killing response was demonstrated in this study, it appears that brain NE may not participate in the mediation of mouse-killing behavior. ACKNOWLEDGEMENT
We wish to thank Professor Kiyoshi Kataoka, the First Department of Physiology, School of Medicine, Ehime University for his helpful criticism of the manuscript and pertinent advice.
REFERENCES 1. Bermond, B., N. E. Van de Poll and H. Van Dis. Reserpine induction of mouse killing in nonkiller rats. Bull. Psychon. Sot. 8: 605-606, 1976. 2. Bernstein, H. and K. E. Moyer. Aggressive behavior in the rat: Effects of isolation and olfactory bulb lesions. Brain Res. 20: 75-84, 1970. 3. Cain, D. P. Olfactory bulbectomy: Neural structures involved in irritability and aggression in the male rat. J. camp. physiol. Psycho/.
86: 213-220,
1974.
4. Chang, C. C. A sensitive method for spectrophotofluorometric assay of catecholamines. Inr. J. Neuropharmac. 3: 643-649,
5.
6. 7.
8. 9.
1964. Crout, J. R., C. R. Creveling and S. Udenfriend. Norepinephrine metabolism in rat brain and heart. J. Pharmac. exp. Ther. 132: 269-277, l%l. Edwards , D. A., A. J. Schlosberg, S. E. McMaster and J. A. Harvey. Olfactory system damage and brain catecholamines in the rat. Brain Res. 121: 121-130, 1977. Eichelman, B., N. B. Thor, N. M. Bugbee and K. Y. Ng. Brain amine and adrenal enzyme levels in aggressive, bulbectomized rats. Physiol. Behav. 9: 483-485, 1972. Fujiwara, M. and S. Ueki. Muricide induced by single injection of AQetrahydrocannabinol. Physiol. Behav. 21: 581~585~ 1978. Glowinski, J. and L. L. Iversen. Regional studies of catecholamines in the rat brain. I. The disposirion of 3H-norepinephrine, “H-dopamine and 3H-dopa in various regions of the brain. J. Neurochem.
13: 655-669,
1966.
10. Goldberg, M. E. and A. I. Salama. Norepinephrine turnover and brain monoamine levels in aggressive mouse-killing rats. Biochem. Pharmac. 18: 532-534, 1969. 11. Horovitz, Z. P., J. J. Piala, J. P. High, J. C. Burke and R. C. Leaf. Effects of drugs on the mouse-killing test and its relationship to amygdaloid function. Int. J. Neuropharmac. 5: 405-411, 1966. 12. Karli,
P. The Norway rat’s killing response to the white mouse: An experimental analysis. Behavior 10: 81-103, 1956. 13. Kumadaki, N., M. Hitomi and S. Kumada. Effects of psychotherapeutic drugs on hyperemotionality of rats in which the olfactory bulb was removed. Jap. J. Pharmac. 17: 659-667, 1%7.
14. Leaf, R. C., L. Lemer and Z. P. Horovitz. The role of the amygdala in the pharmacological and endocrinological manipulation of aggression. In: Aggressive Behavior, edited by S. Garattini and E. B. Sigg. Amsterdam: Excerpta Medica Foundation, 1969, pp. 120-131. 15. Malick, J. B. Pharmacological antagonism of mouse-killing behavior in the olfactory bulb lesion-induced killer rat. Aggress. Behav. 2: 123-130, 1976. 16. Miczek, K. A. Mouse-killing
chronic A9-tetrahydrocannabinol
and motor actvity: Effects of and pilocarpine. Psychophar-
macology 47: 59-64, 1976. 17. Miczek, K. A. As-tetrahydrocannabinol:
Antiaggressive effects in mice, rats and squirrel monkeys. Science 199: 1459-1461, 1978. 18. Oishi, R. and S. Ueki. Facilitation of muricide by dorsal norepinephrine bundle lesions in olfactory bulbectomized rats. Pharmac.
Biochem.
Behav.
8: 133-136,
1978.
19. Paul, L., W. M. Miley and R. Baenningee. Mouse killing by rats: Roles of hunger and thirst in its initiation and maintenance. J. camp. physiol.-Psychol.
76: 242-249,
1971.
20. Pohorecky, L. A. and J. P. Chalmers. Effects of olfactory bulb lesions on brain monoamines. Life Sci. 10: 985-998, 1971. 21. Takahashi, R. and M. H. Aprison. Acetylcholine content of discrete areas of the brain obtained by near-freezing method. J. Neurochem. 11: 887-898, 1964. 22. Ueki, S., M. Fujiwara and N. Ogawa. Mouse-killing behavior (muricide) induced by A8-tetrahydrocannabinol in the rat. Physiol. Behav
. 9: 585-587, 1972.
23. Yoshimura, H., Y. Gomita and S. Ueki. Changes in acetylcholine content in rat brain after bilateral olfactory bulbectomy in relation to mouse-killing behavior. Pharmac. Biochem. Behav. 2: 703-705,
1974.
24. Yoshimura, H., M. Fujiwara and S. Ueki. Biochemical correlates in mouse-killing behavior of the rat: Brain acetylcholine and acetylcholinesterase after administration of As-tetrahydrocannabinol. Brain Res. 81: 567-570, 1974. 25. Yoshimura, H. and S. Ueki. Biochemical correlates in mousekilling behavior of the rat: Prolonged isolation and brain cholinergic function. Pharmac. Biochem. Behav. 6: 193-196, 1977.
Bulletin, Vol. 7, PP. 151-155, 1981. Printed in the U.S.A.
Regional Changes in Brain Norepinephrine Content in Relation to Mouse-Killing Behavior by Rats Department
of Pharmacology, Faculty of Pharmaceutical Kyushu University, Fukuoka 812, Japan
Sciences
Received 24 April 1981 YOS~IMURA, H. AND S. UEKI. Regional changes in brain ~ore~inephrine content in relation to mouse-kiiling behavior by ruts. BRAIN RES. BULL 7(2) 151-155, 1981.--Three separate series of experiments were conducted as
follows: isolation housing, bilateral olfactory bufbectomy, and A*-tetrahydr~annabino1 tTHC) admi~stration. All three experimental manipulations produced an increase in the incidence of mouse-killing behavior. In order to elucidate the possible neural mechanisms mediating the killing response, norepinephrine (NE) content was measured in 6 discrete areas of the brain (the cortex, striatum, amygdala, midbrain, hypothalamus, and pons plus medulla oblongata). Following isolation housing, no significant difference in NE levels of any of the brain areas was demonst~ted between the aggregated and isolated rats, nor between the killer and nonkiller rats. The rats with olfactory bulbectomy exhibited high NE content in the hypothalamus as compared with the intact or sham-operated rats, but there was no significant difference between the killer and nonkiller rats. After injection of THC, NE content in both the hypothalamus and pons plus medulla oblongata was decreased independent of the manifestation of killing response. The evidence indicates no regional change in brain NE levels specific to the killing response and suggests that brain NE may not participate in the mediation of mouse-killing behavior. Mouse-killing behavior As-Tetrahydr~~nabinol
Brain norepinephrine
Rats
MOUSE-killing behavior in rats was first reported by Karli in 1956 who found that almost all wild Norway rats kill mice 1121, By contrast, in domesticated rats this behavior is infrequent and various experimenta maniputations are necessary to induce the killing response; for exampIe, prolonged isolation housing [2,25], bilateral olfactory bulbectomy [3,23], or Ag-tetrahydrocannabinol (THC) administration 122,243. As described in previous reports [2,8, 161, however, the behavioral topography of the killing response differs among the experimentally-induced killer rats. The killing response induced by prolonged isolation housing had a similar quality to that observed in natural or spont~eous killer rats: quiet, quick, and eff&ient attack biting to the cervical region with little or no affective display. On the other hand, killer rats with olfactory bulb ablations did not exhibit such a wellorganized killing response. Their killing response had a compulsive, disinhibited quality, and in many cases the killed mice were covered with blood from head to tail. At the introduction of a mouse the THC-treated animal retreated to a corner of the cage and cowered there with startled squeals, and then suddenly attacked the mouse. Moreover, THCinduced killer rats did not release the dead mouse from their mouth, and continued to bite it. Our recent investigation ‘Now at the Department of Pha~acology, the first author.
Copyright
Isolation housing
Olfactory bulbectomy
(unpublished data) indicates that different kinds of manipulations also produce distinctive features in emotionality; isolation-educed killer rats were more reactive in startle response than isolated nonkilIer rats; olfactory bulbectomized rats exhibited high reactivity to external stimuli, and their response to inanimate objects or the experimenter was highly offensive in nature; THC-treated rats vocalized frequently during the measurement of emotion~ity, but both struggle and startle response were not affected by THC. The question which arises is whether or not there exists a common physiological basis mediating the mouse-killing behavior among the expe~ment~ly-induced killer rats. Interestingly, Malick has recentfy reported that the ability of drugs to inhibit killing response differs significantly between the models of killer rats employed [lS]. For the neural mechanisms of mouse-killing behavior, pharmacologic~ studies suggest that brain catechoiamines (CA) may play an inhibitory role in the manifestation of the behavior [ 1, 11, 141: for example, drugs which produce an increase of CA concentration at receptor sites, such as amphetamine and imipramine, can suppress the killing response in the killer rats, while the depletion of CA store with cu-methyl-p-tyrosine or reserpine induces the killing response in the nonkiller rats. This view is further supported by the
School of Medicine, Ehime University, Ehime 791-02, Japan. Reprint requests should be sent to
0 1981 ANKHO
Inte~ational
Inc .-0361-9230/81/0801~1-05$01.~/0
YOSHIMURA finding that lesions of the dorsal noradrenergic bundle facilitate mouse~killing behavior in olfactory bulbe~tomized rats [ 183.In addition, Leaf et nt. [14] has reported that direct application of norepinephrine (NE) into the amygdaloid nuclei suppressed some of the mouse-killing behavior. The present study was conducted to investigate the participation of brain NE in the manifestation of the killing response, using three models of killer rats. Male rats were subjected to one of the following manipulations: prolonged isolation housing, bilateral olfactory bulbectomy, and THC administration. Brain NE content was estimated in 6 discrete regions. METHOD Subjects
All the animals employed were male Wistar King-A rats supplied from Kyushu University Institute of Experimental Animals. Upon arrival, rats were housed communally for at least 10 days before each experimental manipulation, and had free access to food and water throughout the experiments unIess otherwise stated. Ablino mice weighing between 20 and 30 g were used as stimuli for mouse-k~ling behavior. The temperature in the vivarium was maintained at 222 l”C, and a 12 hr light-dark cycle (light on at 7:00 a.m. and off at 7%) p.m.) was controlled automatically. In the present experiments, approximately 6% of the rats were spontaneous killer rats and were eliminated from the subjects. A~purarus The isolation cage was made of wire mesh and was divided into 5 compartments, 18x 17x 17 cm. The compartments were separated by opaque plastic partitions. Under these conditions, rats were exposed to social stimuli such as squeaks and odors, but did not have physical contact or visible stimuli. The aggregated animals were housed in a 40x33~ I7 cm cage with plastic walls. Experimental
Design
Three separate series of experiments were conducted as follows: isolation housing, bilateral olfactory bulbectomy, and THC administ~tion. Experiment I. Twenty-four rats weighing between 180 and 200 g were used. The subjects in the isolated groups were housed individually for 30 days in the isolation cages, while the subjects in the aggregated control group were housed communally, 4 animals per cage, for 30 days. The mouse-killing behavior was tested periodically at intervals of 5 days. On the 30th day of isolation, an equal number of animals (n=6) from each group (aggregated control, isolated killer and nonkiller rats) were randomly selected and sacrificed 15 min after the last presentation of a mouse. Experiment 2. Thirty-six rats weighing between 180 and 200 g at the time of surgery were used. The olfactory bulbectomy was performed under pentobarbital anesthesia (40 mg/kg, IP). The olfactory bulbs were bilaterally removed by suction through two holes made in the skull just above the bulbs. The sham-operated animals underwent the same procedure as the bulbectomized rats, but aspiration of the olfactory bulbs was not made. All the subjects including the intact control rats were housed individually in the isolation cages for I4 days. Mouse-killing behavior was tested 3, 7, and 14 days after surgery. On the 14th day, an equal number of animals (n=5) from each group (intact control, sham-
AND UEKI
operated control, bulbectomized killer and nonkiller rats) were randomly selected and sacrificed 15 min after the last presentation of a mouse. The brains of the bulbectomized rats were examined macroscopically to ensure that the bulbs had been completely ablated. Sham-operated animals were also examined for any possible damage. Experiment 3. Twenty-four rats weighing between 120 and 140 g at the time of drug administration were used. All the animals were housed individually in isolation cages and subjected to food deprivation for 24 hrs prior to THC or vehicle administration. Natural THC, supplied by the Department of Pharmacognosy, Kyushu University, was suspended in a 1% aqueous Tween 80 solution and injected intraperitoneally in a dose of 6 mgikg. Mouse-killing behavior was tested 30 min before and 1 hr after THC or vehicle administration. An equal number of animals (n=6) from each group (vehicle control, THC-treated killer and nonkiller rats) were randomly selected and sacrificed 15 min after the last presentation of a mouse. Behuvioral
Testing
Mouse-killing behavior was determined by introducing an albino mouse into the rat’s home cage. If the rat killed the mouse within 5 min it was judged to be a killer; otherwise it was termed a nonkiller. Norepinephrine
Assay
Animals were sacrificed by the near-freezing method of Takahashi and Aprison [ 2 11,using liquid nitrogen. The brain was quickly removed and 6 regions--cortex, striatum, amygdala, midbrain, hypothalamus, and pons plus medulla oblongata-were separated on an ice-cold glass plate according to Giowinski and Iversen [93. After homogenization in 0.4 N perchloric acid, NE was isolated from the supematant by the alumina absorption method of Crout [S] and was estimated fluorometric~ly by the t~hydroxyindole method of Chang (43. Statistical Anulysis Statistical evaluation of the biochemical data was performed by means of an analysis of variance and the Tukey test. Differences in the prounion of killer rats among the three models were evaluated by means of the Chi-Square test.
RESULTS
Table 1 shows the proportion and percentage of killer rats. Although the behavioral topography of killing response differed among the three models, there was no significant difference in the ability of manipulations to induce kiliing response in nonkiller rats. In both isolation-induced and olfactory bul~~tom~ed killer rats, once killing behavior developed, it was constant and did not disappear under repeated measurements. The killing response in THC-treated rats lasted for at least 3 hrs and disappeared 24 hrs after injection of the drug. Experiment
I
The regional changes in brain NE content after isolation housing are shown in Table 2. There was no significant
BRAIN NOREPINEPH~NE
153
AND MOUSE-KILLING TABLE 1 INCIDENCEOF MOUSE-KILLINGBEHAVIORFGLLOWINGTHREE DIFFERENT~AN~~LATIONS No.
Group
Ex~~ment 1 2
3
Aggregated Isolated Intact Sham-operated Olfactory b~~ctomi~d Vehicle-treated THC-treated
of
No.
%
of
Tested
Killers
8 16 8 7 13 8 16
0 7 0 I 8 0 9
Killers 0 44 0 14 62* 0 56’F
*Intact group vs olfactory bui~ctomi2ed group, x2=5.56, p
TABLE 2 BRAINNGREPINEPH~N~ LEVELSIN RATSFOLLOWINGPROLONGED ISOLATIONHOUSING Isolated Group Brain Areas Cortex Stiiatum Amygdala Midbrain Hy~~~amus Pons + Medulla Oblongata
A~gated Group (n=6) 026 f 0.22 + 0.37 -t 0.54 + 1.41 * 0.54 2
0.01 0.02 0.03 0.04 0.10 0.03
Killer (n=6) 0.27 -c &Of 0.21 + 0.03 0.40 -+ 0.05 0.52 -e 0.08 1.54 r 0.20 0.55 f 0.05
Non~Ier (n=6) 0.26 + 0.22 t 0.36 f 0.55 + 1.46 r 0.55 t
0.02 0.02 0.05 0.03 0.08 0.03
Norep~n~p~ne contents are expressed as ~8 per g of wet tissue; means r S.D.
difference in all brains areas between the aggregated and isolated groups, or between the killer and nonkiller rats.
Table 3 shows the regional changes in brain NE content after bilateral olfactory bulbectomy. Analysis of variance revealed a s~~~c~t difference in the ~ygd~a, F(3,16) =10.92, p
and midbr~, appears to be caused by olfactory b~~ctomy itself since there was no significant d~erence in the hypothalamic NE content between the intact and sh~~~mted control. However, no significant difference in the hypothalamus was found between the killer and nonkiller rats.
The levels of NE in the 6 brain areas after the administration of THC are shown in Table 4. There were si~i~c~t differences in NE levels of the three brain areas among treatment groups: the amygdala, F(2,15)=8.26, pcO.005, hy~th~amus, F(2,15)=4.43,p
154
YOSHIMURA
AND UEKI
TABLE 3 BRAIN NOREPINEPHRINELEVELSIN
RATS FOLLOWING
BILATERAL
Control Group Brain Areas
(n=S) 0.30 + 0.29 t 0.48 t 0.65 + 1.17 + 0.70 t
Cortex
Striatum Amygdala Midbrain Hypothalamus Pons + Medulla Oblongata
0.04 0.04 0.06 0.03 0.09 0.06
BULBECTOMY
Bulbectomized Group
Sham (n=5)
Intact
OLFACTORY
Killer (n=%
0.34 ?z 0.02 0.30 t 0.03 0.58 + O.OPf 0.79 i o.o5t!: 1.29 -f 0.08 0.70 -c 0.03
Nonkiller (n=5)
0.32 2 0.03 0.34 +- 0.03 0.57 + 0.01*1
0.29 i 0.04 0.30 + 0.03 0.65 t 0.03tltt
0.76+ O.OS*B 1.54 t O.O8t§#
0.77? 0.04ty 1.49 t o.o4tlI**
0.76 ? 0.05
0.76 t 0.04
Norepinephrine contents are expressed as wg per g wet tissue; mean 4 S.D. *p
vs Killer; **=Sham vs Nonkiller:
TABLE 4 BRAIN NOREPINEPHRINE LEVELS IN RATS FOLLOWING ADMINISTRATION OF Aa-TETRAHYDROCANNABINOL
THC Group Brain Areas Cortex Striatum Amygdala Midbrain Hypothalamus Pons + Medulla
Vehicle Group (n=6) 0.26 0.32 0.62 0.56 1.57 0.72
2 + 2 ? t +
0.02 0.03 0.02 0.07 0.18 0.05
Killer (n=6) 0.24 0.31 0.63 0.49 1.35 0.59
+ 0.03 + 0.02 r 0.05 ? 0.06 f 0.1o*i r+_0.03t:
Nonkiller (n=6) 0.23 + 0.30 ? 0.53 2 0.48 t 1.37 t 0.60+
0.02 0.02 o.o5*Ul 0.07 0.07*s o.o4v+
Oblongata Norepinephrine contents are expressed as pg per g of wet tissue; means r
S.D.
*p
‘rpco.01. $=Vehicle vs Killer; $=Vehicle vs Nonkiller; lI=Killer vs Nonkiller.
DISCUSSION
The present results showing that all three experimental manipulations could elicit the killing response in nonkiller rats is in general accordance with previous reports although there are considerable differences in the incidence of mouse-killing behavior between different strains of rats [2, 3, 221. The results of Experiment 1 clearly demonstrate that prolonged isolation housing does not alter brain NE levels in all 6 discrete regions examined, and that brain NE levels of the killer rats do not differe from that of the nonkiller rats, a finding which is not always consistent with the earlier report by Goldberg and Salama [lo] describing an increased level of forebrain NE in the killer rats. In their experiment, however, animals were subjected to a restricted feeding schedule in addition to individual housing, and therefore possible effects of food restriction on both killing response and brain NE content cannot be ruled out. “Hunger” has so far been considered one of the motivational factors leading to the manifestation of killing response [ 191, and consequently a
separate design of experiment may be needed for analysis of effect of food deprivation. Several investigators have reported a decrease in the cortical or telencephalic NE content after olfactory bulbectomy [7,20]. In the present study, however, we did not find any change in the cortical NE content. Recently, Edwards [61 demonstrated that NE content of the telencephalic region was significantly decreased only when olfactory system surgery extensively damaged the olfactory peduncle or tubercle. The ablations of the olfactory bulbs in this study were limited to both the main and accessary olfactory bulbs, and did not extend to more central portions. Our data, therefore, appear to support Edwards’ finding, and the extent of brain damage by surgery may be an essential factor for the change in the cortical NE content. The increase of NE level in the hypothalamus does not apparently correlate with the manifestation of killing response when one takes into consideration that there was no significant difference between the killer and nonkiller rats. This hypothalamic change is likely to correlate with other behavioral characteristics such
BRAIN NOREPINEPHRINE
155
AND MOUSE-KILLING
as hyperirritability
which are common to olfactory bulbectomized rats [3,13]. Contrary to the data on olfactory bulbectomy, acute injection of THC produced a decrease in NE levels of both the hypothalamus and pons plus medulla oblongata both in killer and nonkiller rats. This evidence is interesting in light of the fact that bilateral olfactory bulbectomy increases aggressive behavior [3] while acute injection of THC decreases different aggressive behaviors [17]. It should be noted that in the amygdala only nonkiller rats exhibited lower NE level than vehicle-treated rats or THC-treated killer rats. Since a decrease in NE content can be explained as being due to the increased availability of the transmitter, the decrease in amygdaloid NE level found in the present investigation
might be a reflex of activation of the adrenergic system. This may be supported by Leaf’s finding that microinjection of NE into the amygdaloid nuclei can suppress the killing response in spontaneous killer rats. [14]. Since neither common regional change in brain NE levels among the three models of killer rats, nor specific change to killing response was demonstrated in this study, it appears that brain NE may not participate in the mediation of mouse-killing behavior. ACKNOWLEDGEMENT
We wish to thank Professor Kiyoshi Kataoka, the First Department of Physiology, School of Medicine, Ehime University for his helpful criticism of the manuscript and pertinent advice.
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