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Brain serotonin depletion by p-chlorophenylalanine or lesions of raphe neurons in rats
Physiology, and Behavior, Vol. 10, pp. 809-811. Brain Research Publications Inc., 1973. Printed in the U.S.A.
BRIEF COMMUNICATION Brain Serotonin Depletion by p-Chlorophenylalanine or Lesions of Raphe Neurons in Rats I MICHAEL H. SHEARD
Department o f Psychiatry, Yale University School o f Medicine, and Connecticut Mental Health Center N e w Haven, Connecticut 06519
(Received 19 September 1972) SHEARD, M. H. Brain serotonin depletion by p-chlorophenylalanine or lesions of raphe neurons in rats. PHYSIOL. BEHAV. 10(4) 809-811, 1973.-Male Sprague-Dawley rats were treated with p-chlorophenylaianine (pCPA) and 15-24 hr later their behavior was rated in an observation chamber. Their behavior was compared with rats which had lesions in the dorsal and median raphe nuclei to deplete brain setotonin. In spite of a comparable lowering of brain serotonin, lesioned rats did not display exaggerated sexual or aggressive behavior. p-chlorophenylalanine
Lesion
Raphe
Sex
Aggression
STRIKING sexual and aggressive behavioral effects following p-chlorophenylalanine (pCPA) have recently been described in cats [2, 3, 13] and in rats [7, 9, 10]. Whether a real increase in complete heterosexual activity occurs in rats has been questioned [11]. In order to provide more evidence on the relationship between serotonin and behavior, depletion of brain serotonin was brought about by lesions of the midbrain raphe neurons in rats. In spite of a comparable depression in brain 5-hydroxytryptamine (SHT) brought about by these lesions the animals did not show the increase in sexual and aggressive behavior seen after p-CPA administration. METHOD Male Sprague-Dawley rats (300-325 g) were used in the experiments. Twenty-four rats were given an intraperitoneal injection of p-CPA in the form of the methyl ester (400 mg/kg dissolved in 1.5 ml water). Twenty-four rats received physiological saline and 24 rats had midbrain lesions m a d e in the dorsal and median raphe nuclei. A constant current stimulator delivered 1.5 ~ A for 15 sec via a stainless steel electrode to each site using coordinates from a rat brain atlas [6]. The method of behavioral observation has been described [7]. In brief, each animal was observed in a test cage of dimensions 38x28x29 in., together with another male and female rat and a mouse. In the cage also was a pile of rat chow and a bottle of water. The time spent in
behavioral items of exploration, grooming, sex, aggression and rest was documented over a 20 min period. The p C P A treated animals were tested between 15 and 24 hr after drug injection and lesioned animals were tested on the fourth day postoperatively. At this time 5 H T and 5hydroxyindoleacetic acid (5HIAA) concentrations most closely approximated those in the p C P A treated animals. Following the 20 rain observation period, 16 rats from the p C P A treated group which had demonstrated increased sexual or aggressive activity and 16 randomly selected lesioned rats were killed by decapitation, the brains dissected out and the fore brains analyzed for 5 H T and 5HIAA. Sixteen randomly selected saline treated rats were also analyzed. The assay technique has been described previously [ 8 ]. The midbrain of all lesioned animals were fixed in 5% glutaraldehyde, then 50 ~ sections cut and stained with cresyl violet for histological verification of lesions. RESULTS The behavior of the 16 selected pCPA treated rats differed significantly from the 16 randomly selected controls with an increase in the time spent in grooming, sexual and aggressive behavior (Fig. l white bars). The aggressive behavior seen in these rats is predominantly muricide, although there is also some increased intraspecific aggression. Values for 5HT and 5HIAA in these rats 15-18 hr
i Supported in part by NIMH Grant MH-17856. 809
810
SHEAR[)
following pCPA was 250 ± 30 and 102 +_ 17 ng/g respectively. Raphe lesioned rats showed significantly more restless exploration and significantly less sexual, less grooming and less aggressive behavior than pCPA treated or saline treated controls (Fig. 1 cross hatched bars). They spent the majority of the time in exploring the cage or other animals. They also showed more resting behavior than pCPA treated but less than controls. Values for 5HT and 5HIAA in 16 randomly selected lesioned animals were 242 ± 32 and 96 ± t8 ng/g respectively. There is no significant difference between these values and the values for the pCPA treated rats. The behavior of the 16 control and 16 lesioned rats randomly selected for biochemical analysis did not differ significantly from the behavior of the total group of 24. The behavior of these lesioned rats is best characterized by j u m p y excitability and restless activity. Histological examination of midbrain sections typically revealed destruction involving the dorsal and median raphe nuclei (Fig. 2). Other lesioned rats tested at different times from 1 day up to 3 weeks following raphe lesions failed to show exaggeration of sexual or aggressive behavior. DISCUSSION The results of this experiment show that animals with depletion of 5HT brought about by brain lesions do not show the exaggeration of sexual and aggressive behavior which is frequently seen after pCPA treatment. These results suggest that an intact though inhibited serotonin
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FIG. 1. Effect of p-chlorophenylalanine treated (white bars) (n=t6) and raphe lesioned rats (cross hatched bats) (n---16) compared with saline treated control rats (black bars) (n=16.) on the time spent (seconds, mean ± gEM) in (from left to right): X -©xploration;G grooming; S - sex; A - aggression; and R - rest. The small inset bat graph repreamnts the corrt~ponding lewis o f forebrain 5-hydroxytryptamine (SHT) and 5-hydroxyindoleacetic acid (5HIAA).
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FIG. 2. Photomicrograph of rat midbrain section stained with cresyl violet to show lesion involving the dorsal and midbrain raphe nuclei.
BEHAVIOR pCPA AND RAPHE LESIONS
8'11
system is necessary for the behavioral effects. It is not possible to make any definite statements about the role of norepinephrine in these behavioral changes since it was not measured in this study. However, it has been postulated that the behavioral changes seen with pCPA could be due to an unbalanced action of norepinephrine [ 10]. One point to be considered is that the lesions involved only serotonin containing neurons in areas 7 and 8, leaving are 9 intact [1]. Lesions in all these areas produce animals which are too incapacitated for this kind of behavioral study. Thus in spite of a similarity between the pCPA treated and lesioned rats in brain concentrations of 5HT and 5 HIAA, the lesioned animals have some intact functioning serotonergic neurons. The pCPA treated rats presumably have all serotonin neurons depressed. Another factor which makes the two groups of animals not strictly comparable is the possible development of postsynaptic hypersensitivity in lesioned animals which may compensate for serotonin depletion. This would be comparable to what is k n o w n to happen with lesion of the dopamine pathways. In this study animals tested soon after lesions also failed to develop
excess sexual or aggressive behavior. However, there is the problem in testing animals too soon after lesions that they may not be fully recovered from the nonspecific effects of the operation itself. The absence of exaggerated sexual or aggressive behavior in animals with brain 5HT depleted by lesions, may, of course, be due to some nonspecific effect of the lesion itself. However, an increase in aggressive behavior is known to occur with lesions of comparable size in other sites, e.g. septal region or vcntromedial nucleus of hypothalamus. Finally, it should be noted that pCPA has a number of other actions besides its action on serotonin synthesis, for example increasing uptake of amino acids through blood brain barrier and on sleep [4, 5, 12] as well as having both peripheral and central actions which may account for its behavioral effects. ACKNOWLEDGEMENTS Thanks are due to Miss S. P. Williamsand D. Perum for technical assistance and to C. Pfizer Co., Groton, Connecticut for supplies of p-chlorophenylalanine.
REFERENCES 1. Dahlstrom, A. and K. Fuxe. Evidence for existence of monoamine containing neurons in the central nervous system: demonstration of monoamines in the cell bodies of brain stem neurons. Acta physiol, scand. 62: Suppl. 2,321,1965. 2. Dement, W. The true function of REM sleep together with some observations clarifying the role of serotonin in brain. Reported at Psychiatric Research Meeting, New Haven, Conn. Dec. 1968. 3. Ferguson, J., S. Henrickson, H. Cohen, G. Mitchell, J. Barchas and W. Dement. Hypersexuality and behavioral changes in cats caused by administration of p-chlorophenylalanine. Science 168: 499-501, 1970. 4. Guroff, G. and S. Udenfriend. Studies on aromatic amino acid uptake by rat brain in vivo. J. biol. chem. 237: 803-806, 1962. 5. Koella, W. P., A. Feldstein and J. S. Czicman. The effect of para-chlorophenylalanine on the sleep of cats. Electroenceph. din. Neurophysiol. 25: 481-490, 1968. 6. Konig, J. F. R. and R. A. Kiippel. The Rat Brain. Baltimore, Maryland: The Williamsand Wilkins Co., 1963. 7. Sheard, M. H. The effect of p-chlorophenylalanine on behavior in rats: relation to brain serotinin and 5-hydroxyindoleacetic acid. Brain Res. 15: 524-528, 1969.
8. Sheard, M. H. and G, K. Aghajanian. Stimulation of the midbrain raphe: Effect on serotonin metabolism. J. Pharmac exp. Ther. 163: 425-430, 1968. 9. Shillito, E. The effect of p-chlorophenylalanine on social interactions of male rats. Br. J. Pharmac. Chemother. 36: 193-194, 1969. 10. Tagliamonte, A., P. Tagiiamonte, G. L. Gessa and B. B. Brodie. Compulsive sexual activity induced by p-chlorophenylalanine in normal and pinealectomized male rats. Science 166: 1433-1435, 1969. 11. Whalen, R. E. and W. Luttge. P-Chlorophenylalanine methyl ester: An aphrodisiac? Science 169: 1000-1001, 1970. 12. Yuwiler, A., E. Geller and G. C. Schuster. On the mechanism of the brain serotonin depletion in experimental phenyl. ketonuria. J. biol. Chem. 240:1170-1174, 1965. 13. Zitrin, A., F. A. Beach, J. D. Barchas and W. Dement. Sexual behavior of male cats after administration of p-chlorophenylalanine. Science 170: 868-870, 1970.
BRIEF COMMUNICATION Brain Serotonin Depletion by p-Chlorophenylalanine or Lesions of Raphe Neurons in Rats I MICHAEL H. SHEARD
Department o f Psychiatry, Yale University School o f Medicine, and Connecticut Mental Health Center N e w Haven, Connecticut 06519
(Received 19 September 1972) SHEARD, M. H. Brain serotonin depletion by p-chlorophenylalanine or lesions of raphe neurons in rats. PHYSIOL. BEHAV. 10(4) 809-811, 1973.-Male Sprague-Dawley rats were treated with p-chlorophenylaianine (pCPA) and 15-24 hr later their behavior was rated in an observation chamber. Their behavior was compared with rats which had lesions in the dorsal and median raphe nuclei to deplete brain setotonin. In spite of a comparable lowering of brain serotonin, lesioned rats did not display exaggerated sexual or aggressive behavior. p-chlorophenylalanine
Lesion
Raphe
Sex
Aggression
STRIKING sexual and aggressive behavioral effects following p-chlorophenylalanine (pCPA) have recently been described in cats [2, 3, 13] and in rats [7, 9, 10]. Whether a real increase in complete heterosexual activity occurs in rats has been questioned [11]. In order to provide more evidence on the relationship between serotonin and behavior, depletion of brain serotonin was brought about by lesions of the midbrain raphe neurons in rats. In spite of a comparable depression in brain 5-hydroxytryptamine (SHT) brought about by these lesions the animals did not show the increase in sexual and aggressive behavior seen after p-CPA administration. METHOD Male Sprague-Dawley rats (300-325 g) were used in the experiments. Twenty-four rats were given an intraperitoneal injection of p-CPA in the form of the methyl ester (400 mg/kg dissolved in 1.5 ml water). Twenty-four rats received physiological saline and 24 rats had midbrain lesions m a d e in the dorsal and median raphe nuclei. A constant current stimulator delivered 1.5 ~ A for 15 sec via a stainless steel electrode to each site using coordinates from a rat brain atlas [6]. The method of behavioral observation has been described [7]. In brief, each animal was observed in a test cage of dimensions 38x28x29 in., together with another male and female rat and a mouse. In the cage also was a pile of rat chow and a bottle of water. The time spent in
behavioral items of exploration, grooming, sex, aggression and rest was documented over a 20 min period. The p C P A treated animals were tested between 15 and 24 hr after drug injection and lesioned animals were tested on the fourth day postoperatively. At this time 5 H T and 5hydroxyindoleacetic acid (5HIAA) concentrations most closely approximated those in the p C P A treated animals. Following the 20 rain observation period, 16 rats from the p C P A treated group which had demonstrated increased sexual or aggressive activity and 16 randomly selected lesioned rats were killed by decapitation, the brains dissected out and the fore brains analyzed for 5 H T and 5HIAA. Sixteen randomly selected saline treated rats were also analyzed. The assay technique has been described previously [ 8 ]. The midbrain of all lesioned animals were fixed in 5% glutaraldehyde, then 50 ~ sections cut and stained with cresyl violet for histological verification of lesions. RESULTS The behavior of the 16 selected pCPA treated rats differed significantly from the 16 randomly selected controls with an increase in the time spent in grooming, sexual and aggressive behavior (Fig. l white bars). The aggressive behavior seen in these rats is predominantly muricide, although there is also some increased intraspecific aggression. Values for 5HT and 5HIAA in these rats 15-18 hr
i Supported in part by NIMH Grant MH-17856. 809
810
SHEAR[)
following pCPA was 250 ± 30 and 102 +_ 17 ng/g respectively. Raphe lesioned rats showed significantly more restless exploration and significantly less sexual, less grooming and less aggressive behavior than pCPA treated or saline treated controls (Fig. 1 cross hatched bars). They spent the majority of the time in exploring the cage or other animals. They also showed more resting behavior than pCPA treated but less than controls. Values for 5HT and 5HIAA in 16 randomly selected lesioned animals were 242 ± 32 and 96 ± t8 ng/g respectively. There is no significant difference between these values and the values for the pCPA treated rats. The behavior of the 16 control and 16 lesioned rats randomly selected for biochemical analysis did not differ significantly from the behavior of the total group of 24. The behavior of these lesioned rats is best characterized by j u m p y excitability and restless activity. Histological examination of midbrain sections typically revealed destruction involving the dorsal and median raphe nuclei (Fig. 2). Other lesioned rats tested at different times from 1 day up to 3 weeks following raphe lesions failed to show exaggeration of sexual or aggressive behavior. DISCUSSION The results of this experiment show that animals with depletion of 5HT brought about by brain lesions do not show the exaggeration of sexual and aggressive behavior which is frequently seen after pCPA treatment. These results suggest that an intact though inhibited serotonin
I i
$-H'r.
G
$°HIA/~
T
T
S
A
R
FIG. 1. Effect of p-chlorophenylalanine treated (white bars) (n=t6) and raphe lesioned rats (cross hatched bats) (n---16) compared with saline treated control rats (black bars) (n=16.) on the time spent (seconds, mean ± gEM) in (from left to right): X -©xploration;G grooming; S - sex; A - aggression; and R - rest. The small inset bat graph repreamnts the corrt~ponding lewis o f forebrain 5-hydroxytryptamine (SHT) and 5-hydroxyindoleacetic acid (5HIAA).
e
.V, .,¢
M
ql
T
T
°
FIG. 2. Photomicrograph of rat midbrain section stained with cresyl violet to show lesion involving the dorsal and midbrain raphe nuclei.
BEHAVIOR pCPA AND RAPHE LESIONS
8'11
system is necessary for the behavioral effects. It is not possible to make any definite statements about the role of norepinephrine in these behavioral changes since it was not measured in this study. However, it has been postulated that the behavioral changes seen with pCPA could be due to an unbalanced action of norepinephrine [ 10]. One point to be considered is that the lesions involved only serotonin containing neurons in areas 7 and 8, leaving are 9 intact [1]. Lesions in all these areas produce animals which are too incapacitated for this kind of behavioral study. Thus in spite of a similarity between the pCPA treated and lesioned rats in brain concentrations of 5HT and 5 HIAA, the lesioned animals have some intact functioning serotonergic neurons. The pCPA treated rats presumably have all serotonin neurons depressed. Another factor which makes the two groups of animals not strictly comparable is the possible development of postsynaptic hypersensitivity in lesioned animals which may compensate for serotonin depletion. This would be comparable to what is k n o w n to happen with lesion of the dopamine pathways. In this study animals tested soon after lesions also failed to develop
excess sexual or aggressive behavior. However, there is the problem in testing animals too soon after lesions that they may not be fully recovered from the nonspecific effects of the operation itself. The absence of exaggerated sexual or aggressive behavior in animals with brain 5HT depleted by lesions, may, of course, be due to some nonspecific effect of the lesion itself. However, an increase in aggressive behavior is known to occur with lesions of comparable size in other sites, e.g. septal region or vcntromedial nucleus of hypothalamus. Finally, it should be noted that pCPA has a number of other actions besides its action on serotonin synthesis, for example increasing uptake of amino acids through blood brain barrier and on sleep [4, 5, 12] as well as having both peripheral and central actions which may account for its behavioral effects. ACKNOWLEDGEMENTS Thanks are due to Miss S. P. Williamsand D. Perum for technical assistance and to C. Pfizer Co., Groton, Connecticut for supplies of p-chlorophenylalanine.
REFERENCES 1. Dahlstrom, A. and K. Fuxe. Evidence for existence of monoamine containing neurons in the central nervous system: demonstration of monoamines in the cell bodies of brain stem neurons. Acta physiol, scand. 62: Suppl. 2,321,1965. 2. Dement, W. The true function of REM sleep together with some observations clarifying the role of serotonin in brain. Reported at Psychiatric Research Meeting, New Haven, Conn. Dec. 1968. 3. Ferguson, J., S. Henrickson, H. Cohen, G. Mitchell, J. Barchas and W. Dement. Hypersexuality and behavioral changes in cats caused by administration of p-chlorophenylalanine. Science 168: 499-501, 1970. 4. Guroff, G. and S. Udenfriend. Studies on aromatic amino acid uptake by rat brain in vivo. J. biol. chem. 237: 803-806, 1962. 5. Koella, W. P., A. Feldstein and J. S. Czicman. The effect of para-chlorophenylalanine on the sleep of cats. Electroenceph. din. Neurophysiol. 25: 481-490, 1968. 6. Konig, J. F. R. and R. A. Kiippel. The Rat Brain. Baltimore, Maryland: The Williamsand Wilkins Co., 1963. 7. Sheard, M. H. The effect of p-chlorophenylalanine on behavior in rats: relation to brain serotinin and 5-hydroxyindoleacetic acid. Brain Res. 15: 524-528, 1969.
8. Sheard, M. H. and G, K. Aghajanian. Stimulation of the midbrain raphe: Effect on serotonin metabolism. J. Pharmac exp. Ther. 163: 425-430, 1968. 9. Shillito, E. The effect of p-chlorophenylalanine on social interactions of male rats. Br. J. Pharmac. Chemother. 36: 193-194, 1969. 10. Tagliamonte, A., P. Tagiiamonte, G. L. Gessa and B. B. Brodie. Compulsive sexual activity induced by p-chlorophenylalanine in normal and pinealectomized male rats. Science 166: 1433-1435, 1969. 11. Whalen, R. E. and W. Luttge. P-Chlorophenylalanine methyl ester: An aphrodisiac? Science 169: 1000-1001, 1970. 12. Yuwiler, A., E. Geller and G. C. Schuster. On the mechanism of the brain serotonin depletion in experimental phenyl. ketonuria. J. biol. Chem. 240:1170-1174, 1965. 13. Zitrin, A., F. A. Beach, J. D. Barchas and W. Dement. Sexual behavior of male cats after administration of p-chlorophenylalanine. Science 170: 868-870, 1970.