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Temporary increase in forebrain norepinephrine turnover in mouse-killing rats
EUROPEAN JOURNAL OF PHARMACOLOGY 21 ( 1973) 372-374. NORTH-HOLLAND PUBLISHING COMPANY
TEMPORARY
INCREASE IN FOREBRAIN
TURNOVER i
NOREPINEPHRINE
IN MOUSE-KILLING
RATS
A.I. SALAMA and M.E. GOLDBERG Department of Pharmacodynamics, Warner.Lambert R esearch Institute, Morris Plains, New Jersey 07950, U.S.A.
Received 8 September 1972
Accepted 9 October 1972
A.1. SALAMA and M.E. GOLDBERG, Temporary increase in forebrain norepinephrine turnover in mouse-killing rats, European J. Pharmacol. 21 (1973) 372-374. Increased forebrain norepinephrine levels and turnover occur in the aggressive mouse-killing rat 2 hr after a killing episode. These neurochemical effects persist for 24 hr and return to normal by 48 hr. Such changes appear to be related to the killing episode (i.e. stress) and unrelated to aggressiveness. Conceivably, activation or induction of tyrosine hydroxylase might be responsible for these effects. Mouse-killing rats Aggression
Stress Tyrosine hydroxylase
1. I n t r o d u c t i o n
A model of aggressive behavior which can be obtained by genetic selection is the mouse-killing rat. This spontaneous interspecific aggression, which is selectively inhibited by amygdaloid lesions, was first described by Karli (1956, 1964). Later, Horovitz et al. (1966) showed that mouse killing ('muricide') could be selectively blocked by certain classes of drugs such as antidepressants and stimulants. Since then, inhibition of this behavior has become increasingly useful in the evaluation of drugs with the aim of finding selective anti-aggressive agents as well as to gain further knowledge of biochemical changes in the central nervous system of aggressive animals. In earlier studies, Goldberg and Salama (1969) and Salama and Goldberg (1970) have reported that the mouse-killing rat has a higher forebrain level of norepinephrine (NE) than control non-killer rats. Concomitant with these changes, the killer rat elicited a higher rate constant for the decline of forebrain H 3norepinephrine (H3NE) when given intraventricularly. Consequently, much higher turnover rates of NE were obtained in these subjects. No such differences
Norepinephrine
were observed in the hindbrain region. In those studies, the rats had been challenged with a mouse approximately 24 hr before use. It was of interest to study the permanency of these neurochemical changes after a single mousekilling episode. This information was required in order to determine whether these changes were due to the stress of killing or were due to the fact t h a t these rats were killers (i.e. aggressive subjects).
2. Materials and methods Male Long Evans rats (Blue Spruce Farms, Altamont, N.Y.) weighing between 1 6 0 - 1 8 0 g were used throughout these studies. All animals were housed individually for approximately 6 weeks before being tested for their ability to kill mice as described by Horovitz et al. (1965). From the colony, rats which showed a positive mouse killing response within 2 min of challenge for 3 consecutive days were selected and designated as killer rats, while those which had not killed the mouse served as control (nonkillers). For the following ten days, both groups of
373
A.L Salama, M.E. Goldberg, Forebrain norepinephrine turnover in mouse-killing rats
rats were left undisturbed before the next presentation of a mouse. Animals were then challenged once more, and all pre-selected killer rats again exhibited their aggressive behavior, while the non-killers failed to do so. Various times later (2, 24, 48 hr and 1 week) groups o f killer and non-killer rats were used to determine forebrain NE levels and turnover rates. Similar studies were conducted with subjects which were housed in groups o f 5 during the 6-week period; these were never challenged with a mouse and will be considered 'normal' rats. Forebrain NE turnover was estimated from the decline of the specific activity (Brodie et al., 1966) after the administration o f d,1[7-3HNE] hydrochloride (5 #Ci/kg, specific activity 9.2 Ci/mmole, New England Nuclear Corp., Boston, Mass.) in the lateral ventricle o f the brain (Noble et al., 1967). Groups of 5 animals each were sacrificed by decapitation at various time intervals as described previously (Salama and Goldberg, 1970). The whole brain was removed, brainstem including cerebellum (hindbrain) was separated from forebrain by a cut above the corpora quadrigemina. The forebrain was then frozen until ready for analysis. NE and H3NE were extracted and estimated as described by Brodie et al. (1966).
3. Results It is apparent from table 1 that no differences in forebrain NE levels or turnover rates were observed
between normal rats and non-killer rats, thus suggesting an absence of effect by isolation u p o n these parameters. However, a significant elevation in steady levels (20%) was observed in killer rats which had been challenged 2 hr prior to study. Together with this increase, there was a faster disappearance o f HaNE (50% as seen from the rate constant o f the decline), and consequently a calculated 95% increase in the turnover rate of this amine compared with its non-killer control. 24 hr after the killing episode, forebrain levels, rate constants and turnover rates o f NE were still significantly elevated. The magnitude o f these changes was declining and approaching normal values. By 48 hr, both steady state levels and turnover rates had returned to control values; similar values were observed in rats tested 1 week after the killing episode.
4. Discussion It was shown that direct sympathetic nerve stimulation elicited an immediate rise in the synthesis of catecholamines (Alousi and Weiner, 1966; Gordon et al., 1966; Sedvall and Kopin, 1967; Dairman et al., 1968). Some studies have suggested that tyrosine hydroxylase, the rate limiting enzyme for NE biosynthesis (Levitt et al., 1965) is an inducible enzyme (Weiner and Rabadjija, 1968). Such induction occurred in the adrenal medulla and sympathetic ganglia after reserpine treatment (Mueller et al., 1969;
Table 1 Turnover of norepinephrine in the forebrain of killer rats after a single killing episode. Rats were given an intraventdculat injection of HaNE and were sacrificed at various times later. Rate constants (k) were calculated from the method of least squares. Turnover rate is the product of k and the steady state levels. There were 10 rats in each group for steady state level determination. Type of animal
hr after killing epidose
Steady state levels (#g/g ± S.E.M.)
Rate constant HaNE decline [k(hr -1) ± S.E.M.)
Turnover rate (#g/g/hr)
Normal Non-killer Non-killer Killer Killer Killer Killer
-
0.47 0.50 0.52 0.62 0.57 0.50 0.50
0.16 0.15 0.17 0.24 0.22 0.18 0.15
0.075 0.075 0.089 0.145 0.126 0.090 0.075
2 24 2 24 48 1 week
± 0.01 ± 0.02 ± 0.01 ± 0.01" ± 0.01" ± 0.01 ± 0.02
* Significantly different from the respective non-killer group p < 0.01.
± 0.01 ± 0.02 ± 0.02 ± 0.02* -+ 0.02* ± 0.03 ± 0.01
374
A.L Salama, M.E. Goldberg, Forebrain norepinephrine turnover in mouse-killing rats
Mandel and Morgan, 1970) and in brain after chlorpromazine administration (Bartholini and Pletscher, 1969). An increase in brain NE levels and turnover have been reported after several forms of experimental stress, such as electroshock and cold stress (Kety et al., 1967; Thoenen, 1970). Recently, Musacchio et al. (1969) related increased NE turnover to an increase in brain tyrosine hydroxylase activity in rats given repeated electroshock seizures. Similarly, Thoenen (1970) demonstrated a short term adaptation involving induction of tyrosine hydroxylase after one day of cold exposure in rats. In the present studies, increased levels and turnover rates of NE were observed within two hours after the killing episode. Conceivably, these changes are related to the stress induced by the killing episode since they were not permanent and lasted for about 24 hr. Since tyrosine hydroxylase activity can be altered by rapid changes in end-product inhibition or substrate availability, and gradual alterations in the amount of this enzyme occur in response to prolonged neural stimulation (Mueller et al., 1969), it is quite possible that the dynamic changes occurring after the killing episode are related to an activation (or induction) of tyrosine hydroxylase. This would be consonant with results obtained in earlier stress studies. Obviously, measurement of tyrosine hydroxylase activity in brain and periphery is required to substantiate this hypothesis. From these studies, we conclude that alterations in NE metabolism which occur in this model of aggressive behavior are a result of the killing episode, and are not related to the fact that these rats are mouse killers.
References Alousi, A. and N. Weiner, 1966, The regulation of norepinephrine synthesis in sympathetic nerves. Effect of nerve stimulation, cocaine and catecholamine-releasing agents, Proc. U.S. Natl. Acad. Sci. 56, 1491. Bartholini, G. and A. Pletscher, 1969, Enhancement of tyrosine hydroxylation within the brain by chlorpromazine, Experientia 25,920. Brodie, B.B., E. Costa, A. Dlabac, N.H. Neff and H.H. Smookler, 1966, Application of steady state kinetics to the estimation of synthesis rate and turnover time of tissue catecholamines, J. Pharmacol. Exptl. Therap. 154, 493.
Dairman, W., R. Gordon, S. Spector, A. Sjoerdsma and S. Udenfriend, 1968, Increased synthesis of catecholamines in the intact rat following administration of c~-adrenergic blocking agents, Mol. Pharmacol. 4,457. Goldberg, M.E. and A.I. Salama, 1969, Norepinephrine turnover and brain monoamine levels in aggressive mousekilling rats, Biochem. Pharmacol. 18,532. Gordon, R., S. Spector, A. Sjoerdsma and S. Udenfriend, 1966, Increased synthesis of norepinephrine and epinephrine in the intact rat during exercise and exposure to cold, J. Pharmacol. Exptl. Therap. 153,440. Horovitz, Z.P., R.W. Ragozzino and R.C. Leaf, 1965, Selective block of rat mouse-killing by antidepressants, Life Sci. 4, 1909. Horovitz, Z.P., J.J. Piala, J.P. High, J.C. Burke and R.C. Leaf, 1966, Effects of drugs on the mouse killing (muricide) test and its relationship to amygdaloid function, Intern. J. Neuropharmacol. 5,405. Karli, P., 1956, The Norway rat's killing response to the white mouse: an experimental analysis, Behavior (Leiden) 10, 81. Karli, P. and M. Vergnes, 1964, Nouvelles donndes sur les bases neurophysiologiques du comportement d'aggression intersp~cifique rat-souris, J. Physiol. (Paris) 56, 384. Kety, S.S., F. Javoy, A. Thierry, L. Julou and J. Glowinski, 1967, A sustained effect of electroconvulsive shock on the turnover of norepinephrine in the central nervous system of the rat, Proc. U.S. Natl. Acad. Sci. 58, 1249. Levitt, M., S. Spector, A. Sjoerdsma and S. Udenfriend, 1965, Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea-pig heart, J. Pharmacol. Exptl. Therap. 148, 1. Mandel, A.J. and M. Morgan, 1970, Amphetamine-induced increase in tyrosine hydroxylase activity, Nature 227, 75. Mueller, R.A., H. Thoenen and J. Axelrod, 1969, Increase in tyrosine hydroxylase activity after reserpine administration, J. Pharmacol. Exptl. Therap. 169, 74. Musacchio, J.M., L. Julou, S.S. Kety and J. Glowinski, 1969, Increase in rat brain tyrosine hydroxylase activity produced by electroconvulsive shock, Proc. U.S. Nat. Acad. Sci. 63, 1117. Noble, E.P., R.J. Wurtman and J. Axelrod, 1967, A simple and rapid method for injecting H3-norepmephrme • . Into . the lateral ventricle of the rat brain, Life Sci. 6, 281. Salama, A.I. and M.E. Goldberg, 1970, Neurochemical effects of imipramine and amphetamine in aggressive mousekilling (muricidal) rats, Biochem. Pharmacol. 19, 2023. Sedvall, G.C. and I.J. Kopin, 1967, Acceleration of norepinephrine synthesis in the rat submaxillary gland in vivo during sympathetic nerve stimulation, Life Sci. 6, 45. Thoenen, H., 1970, Induction of tyrosine hydroxylase in peripheral and central adrenergic neurones by cold exposure of rats, Nature 228, 861. Weiner, N. and M. Rabadjija, 1968, The regulation of norepinephrine synthesis: Effect of puromycin on the accelerated synthesis of norepinephrine associated with nerve stimulation, J. Pharmacol. Exptl. Therap. 164, 103.
TEMPORARY
INCREASE IN FOREBRAIN
TURNOVER i
NOREPINEPHRINE
IN MOUSE-KILLING
RATS
A.I. SALAMA and M.E. GOLDBERG Department of Pharmacodynamics, Warner.Lambert R esearch Institute, Morris Plains, New Jersey 07950, U.S.A.
Received 8 September 1972
Accepted 9 October 1972
A.1. SALAMA and M.E. GOLDBERG, Temporary increase in forebrain norepinephrine turnover in mouse-killing rats, European J. Pharmacol. 21 (1973) 372-374. Increased forebrain norepinephrine levels and turnover occur in the aggressive mouse-killing rat 2 hr after a killing episode. These neurochemical effects persist for 24 hr and return to normal by 48 hr. Such changes appear to be related to the killing episode (i.e. stress) and unrelated to aggressiveness. Conceivably, activation or induction of tyrosine hydroxylase might be responsible for these effects. Mouse-killing rats Aggression
Stress Tyrosine hydroxylase
1. I n t r o d u c t i o n
A model of aggressive behavior which can be obtained by genetic selection is the mouse-killing rat. This spontaneous interspecific aggression, which is selectively inhibited by amygdaloid lesions, was first described by Karli (1956, 1964). Later, Horovitz et al. (1966) showed that mouse killing ('muricide') could be selectively blocked by certain classes of drugs such as antidepressants and stimulants. Since then, inhibition of this behavior has become increasingly useful in the evaluation of drugs with the aim of finding selective anti-aggressive agents as well as to gain further knowledge of biochemical changes in the central nervous system of aggressive animals. In earlier studies, Goldberg and Salama (1969) and Salama and Goldberg (1970) have reported that the mouse-killing rat has a higher forebrain level of norepinephrine (NE) than control non-killer rats. Concomitant with these changes, the killer rat elicited a higher rate constant for the decline of forebrain H 3norepinephrine (H3NE) when given intraventricularly. Consequently, much higher turnover rates of NE were obtained in these subjects. No such differences
Norepinephrine
were observed in the hindbrain region. In those studies, the rats had been challenged with a mouse approximately 24 hr before use. It was of interest to study the permanency of these neurochemical changes after a single mousekilling episode. This information was required in order to determine whether these changes were due to the stress of killing or were due to the fact t h a t these rats were killers (i.e. aggressive subjects).
2. Materials and methods Male Long Evans rats (Blue Spruce Farms, Altamont, N.Y.) weighing between 1 6 0 - 1 8 0 g were used throughout these studies. All animals were housed individually for approximately 6 weeks before being tested for their ability to kill mice as described by Horovitz et al. (1965). From the colony, rats which showed a positive mouse killing response within 2 min of challenge for 3 consecutive days were selected and designated as killer rats, while those which had not killed the mouse served as control (nonkillers). For the following ten days, both groups of
373
A.L Salama, M.E. Goldberg, Forebrain norepinephrine turnover in mouse-killing rats
rats were left undisturbed before the next presentation of a mouse. Animals were then challenged once more, and all pre-selected killer rats again exhibited their aggressive behavior, while the non-killers failed to do so. Various times later (2, 24, 48 hr and 1 week) groups o f killer and non-killer rats were used to determine forebrain NE levels and turnover rates. Similar studies were conducted with subjects which were housed in groups o f 5 during the 6-week period; these were never challenged with a mouse and will be considered 'normal' rats. Forebrain NE turnover was estimated from the decline of the specific activity (Brodie et al., 1966) after the administration o f d,1[7-3HNE] hydrochloride (5 #Ci/kg, specific activity 9.2 Ci/mmole, New England Nuclear Corp., Boston, Mass.) in the lateral ventricle o f the brain (Noble et al., 1967). Groups of 5 animals each were sacrificed by decapitation at various time intervals as described previously (Salama and Goldberg, 1970). The whole brain was removed, brainstem including cerebellum (hindbrain) was separated from forebrain by a cut above the corpora quadrigemina. The forebrain was then frozen until ready for analysis. NE and H3NE were extracted and estimated as described by Brodie et al. (1966).
3. Results It is apparent from table 1 that no differences in forebrain NE levels or turnover rates were observed
between normal rats and non-killer rats, thus suggesting an absence of effect by isolation u p o n these parameters. However, a significant elevation in steady levels (20%) was observed in killer rats which had been challenged 2 hr prior to study. Together with this increase, there was a faster disappearance o f HaNE (50% as seen from the rate constant o f the decline), and consequently a calculated 95% increase in the turnover rate of this amine compared with its non-killer control. 24 hr after the killing episode, forebrain levels, rate constants and turnover rates o f NE were still significantly elevated. The magnitude o f these changes was declining and approaching normal values. By 48 hr, both steady state levels and turnover rates had returned to control values; similar values were observed in rats tested 1 week after the killing episode.
4. Discussion It was shown that direct sympathetic nerve stimulation elicited an immediate rise in the synthesis of catecholamines (Alousi and Weiner, 1966; Gordon et al., 1966; Sedvall and Kopin, 1967; Dairman et al., 1968). Some studies have suggested that tyrosine hydroxylase, the rate limiting enzyme for NE biosynthesis (Levitt et al., 1965) is an inducible enzyme (Weiner and Rabadjija, 1968). Such induction occurred in the adrenal medulla and sympathetic ganglia after reserpine treatment (Mueller et al., 1969;
Table 1 Turnover of norepinephrine in the forebrain of killer rats after a single killing episode. Rats were given an intraventdculat injection of HaNE and were sacrificed at various times later. Rate constants (k) were calculated from the method of least squares. Turnover rate is the product of k and the steady state levels. There were 10 rats in each group for steady state level determination. Type of animal
hr after killing epidose
Steady state levels (#g/g ± S.E.M.)
Rate constant HaNE decline [k(hr -1) ± S.E.M.)
Turnover rate (#g/g/hr)
Normal Non-killer Non-killer Killer Killer Killer Killer
-
0.47 0.50 0.52 0.62 0.57 0.50 0.50
0.16 0.15 0.17 0.24 0.22 0.18 0.15
0.075 0.075 0.089 0.145 0.126 0.090 0.075
2 24 2 24 48 1 week
± 0.01 ± 0.02 ± 0.01 ± 0.01" ± 0.01" ± 0.01 ± 0.02
* Significantly different from the respective non-killer group p < 0.01.
± 0.01 ± 0.02 ± 0.02 ± 0.02* -+ 0.02* ± 0.03 ± 0.01
374
A.L Salama, M.E. Goldberg, Forebrain norepinephrine turnover in mouse-killing rats
Mandel and Morgan, 1970) and in brain after chlorpromazine administration (Bartholini and Pletscher, 1969). An increase in brain NE levels and turnover have been reported after several forms of experimental stress, such as electroshock and cold stress (Kety et al., 1967; Thoenen, 1970). Recently, Musacchio et al. (1969) related increased NE turnover to an increase in brain tyrosine hydroxylase activity in rats given repeated electroshock seizures. Similarly, Thoenen (1970) demonstrated a short term adaptation involving induction of tyrosine hydroxylase after one day of cold exposure in rats. In the present studies, increased levels and turnover rates of NE were observed within two hours after the killing episode. Conceivably, these changes are related to the stress induced by the killing episode since they were not permanent and lasted for about 24 hr. Since tyrosine hydroxylase activity can be altered by rapid changes in end-product inhibition or substrate availability, and gradual alterations in the amount of this enzyme occur in response to prolonged neural stimulation (Mueller et al., 1969), it is quite possible that the dynamic changes occurring after the killing episode are related to an activation (or induction) of tyrosine hydroxylase. This would be consonant with results obtained in earlier stress studies. Obviously, measurement of tyrosine hydroxylase activity in brain and periphery is required to substantiate this hypothesis. From these studies, we conclude that alterations in NE metabolism which occur in this model of aggressive behavior are a result of the killing episode, and are not related to the fact that these rats are mouse killers.
References Alousi, A. and N. Weiner, 1966, The regulation of norepinephrine synthesis in sympathetic nerves. Effect of nerve stimulation, cocaine and catecholamine-releasing agents, Proc. U.S. Natl. Acad. Sci. 56, 1491. Bartholini, G. and A. Pletscher, 1969, Enhancement of tyrosine hydroxylation within the brain by chlorpromazine, Experientia 25,920. Brodie, B.B., E. Costa, A. Dlabac, N.H. Neff and H.H. Smookler, 1966, Application of steady state kinetics to the estimation of synthesis rate and turnover time of tissue catecholamines, J. Pharmacol. Exptl. Therap. 154, 493.
Dairman, W., R. Gordon, S. Spector, A. Sjoerdsma and S. Udenfriend, 1968, Increased synthesis of catecholamines in the intact rat following administration of c~-adrenergic blocking agents, Mol. Pharmacol. 4,457. Goldberg, M.E. and A.I. Salama, 1969, Norepinephrine turnover and brain monoamine levels in aggressive mousekilling rats, Biochem. Pharmacol. 18,532. Gordon, R., S. Spector, A. Sjoerdsma and S. Udenfriend, 1966, Increased synthesis of norepinephrine and epinephrine in the intact rat during exercise and exposure to cold, J. Pharmacol. Exptl. Therap. 153,440. Horovitz, Z.P., R.W. Ragozzino and R.C. Leaf, 1965, Selective block of rat mouse-killing by antidepressants, Life Sci. 4, 1909. Horovitz, Z.P., J.J. Piala, J.P. High, J.C. Burke and R.C. Leaf, 1966, Effects of drugs on the mouse killing (muricide) test and its relationship to amygdaloid function, Intern. J. Neuropharmacol. 5,405. Karli, P., 1956, The Norway rat's killing response to the white mouse: an experimental analysis, Behavior (Leiden) 10, 81. Karli, P. and M. Vergnes, 1964, Nouvelles donndes sur les bases neurophysiologiques du comportement d'aggression intersp~cifique rat-souris, J. Physiol. (Paris) 56, 384. Kety, S.S., F. Javoy, A. Thierry, L. Julou and J. Glowinski, 1967, A sustained effect of electroconvulsive shock on the turnover of norepinephrine in the central nervous system of the rat, Proc. U.S. Natl. Acad. Sci. 58, 1249. Levitt, M., S. Spector, A. Sjoerdsma and S. Udenfriend, 1965, Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea-pig heart, J. Pharmacol. Exptl. Therap. 148, 1. Mandel, A.J. and M. Morgan, 1970, Amphetamine-induced increase in tyrosine hydroxylase activity, Nature 227, 75. Mueller, R.A., H. Thoenen and J. Axelrod, 1969, Increase in tyrosine hydroxylase activity after reserpine administration, J. Pharmacol. Exptl. Therap. 169, 74. Musacchio, J.M., L. Julou, S.S. Kety and J. Glowinski, 1969, Increase in rat brain tyrosine hydroxylase activity produced by electroconvulsive shock, Proc. U.S. Nat. Acad. Sci. 63, 1117. Noble, E.P., R.J. Wurtman and J. Axelrod, 1967, A simple and rapid method for injecting H3-norepmephrme • . Into . the lateral ventricle of the rat brain, Life Sci. 6, 281. Salama, A.I. and M.E. Goldberg, 1970, Neurochemical effects of imipramine and amphetamine in aggressive mousekilling (muricidal) rats, Biochem. Pharmacol. 19, 2023. Sedvall, G.C. and I.J. Kopin, 1967, Acceleration of norepinephrine synthesis in the rat submaxillary gland in vivo during sympathetic nerve stimulation, Life Sci. 6, 45. Thoenen, H., 1970, Induction of tyrosine hydroxylase in peripheral and central adrenergic neurones by cold exposure of rats, Nature 228, 861. Weiner, N. and M. Rabadjija, 1968, The regulation of norepinephrine synthesis: Effect of puromycin on the accelerated synthesis of norepinephrine associated with nerve stimulation, J. Pharmacol. Exptl. Therap. 164, 103.