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Distribution of [14C]amphetamine in mouse brain: An autoradiographic study
399
BRAIN RESEARCH
D I S T R I B U T I O N OF [14C]AMPHETAMINE IN MOUSE BRAIN: AN A U T O R A D I O G R A P H I C STUDY*
G I A N F R A N C O PLACIDI**, D A V I D T. M A S U O K A AND ROBERT W. EARLE
Neuropharmacology Research Laboratory, V. A. Hospital, Sepulveda, Calif. 91343 and Medical Pharmacology and Therapeutics, University of California, Irvine, Calif. 92664 (U.S.A.) (Accepted September 30th, 1971)
INTRODUCTION
Considerable effort has been expended in elucidating the site and mechanism of the CNS stimulating action of amphetamine 12. For these purposes, a variety of electro- and psychophysiological techniques as well as chemical and radiochemical approaches have been applied. Results of the chemical and radiochemical studies indicate that the drug readily penetrates the blood-brain barrier2, in and that the time course of radioactivity in the brain following injection of [14C]amphetamine roughly correlates with the period of pharmacological activity 16. With autoradiographic techniques it is now feasible to delineate more closely the localization of labeled substances in the fine structures of areas of interest, and this study reports application of these methods to the brain through use of [14C]amphetamine. MATERIALSAND METHODS Autoradiography Nine adult Swiss mice weighing 19-26 g were injected in the tail vein with [14C]amphetamine. Seven of the mice were injected with 1.3 #Ci and the remaining two received 0.72/~Ci. Both doses were adjusted so that the total volume injected did not exceed 0.2 ml. Following injection, at 5-, 20- and 60-min time intervals selected to cover the maximum period of drug activity and to permit determination of the relationship between pharmacological activity and radioactivity distribution patterns, the particular animals to be utilized were lightly anesthetized with ether and killed by immersion in hexane cooled with solid carbon dioxide. Three of the animals re* A p~eliminary report was presented at the Second Meeting of the Italian Neuropharmacology Society, June, 1969. ** Present address: Psychiatry Clinic, Univ. of Pisa, Pisa, Italy. Brain Research, 38 (1972) 399-405
400
G.F. PLACIDI et al.
TABLE I DISTRIBUTION OF [14C]AMPHETAMINE IN MOUSE BRAIN 5, 2 0 AND 6 0 MIN AFTER THE INTRAVENOUS INJECTION
OF 1.3 /~Ci
In parentheses number of mice is indicated. Time after drug (min)
5
20
60
Blood (counts/min/ml)
Tissue
Counts/min/g
14,499 :t_ 1,023"*
Whole brain (5) Cortex (2) Cerebellum (2) Brain stem (2) Whole brain (5) Cortex (2) Cerebellum (2) Brain stem (2) Whole brain (3)
104,020 ± 7,330 117,200 101,800 81,300 110,540 ± 10,960 123,000 94,500 95,700 49,860
12,478 ± 1,422
8,020
% Amphet.*
96.5 87.9 93.7 98.0 77.9 96.7
* ~ unchanged amphetamine in tissue. ** Counts/min ± S.D. ceiving the 1.3/~Ci dose were sacrificed at the appropriate intervals following injection and 20 # m thick sagittal sections were prepared for the whole body autoradiographic technique described by Ullberg 15. Brain sections were prepared from the remaining 4 mice receiving the 1.3/zCi dose as follows: Coronal sections of one mouse at 5 min and of two mice at 20 min and horizontal sections of one mouse at 20 min. Coronal sections were also prepared at 20 min from the two mice which received the 0.72 #Ci dose. All sections were exposed to Structurix X-ray film (Gevaert). Radioactivity counting studies
A number of mice, as indicated in Table I, were injected in the tail vein with 1.3/zCi of [14C]amphetamine and killed by decapitation after 5, 20 and 60 min. Blood allowed to flow from the body was collected in tubes containing heparin. The brain was quickly removed, weighed and homogenized in 5 ml of 0.05 N HCl. After removing an aliquot for determination of total radioactivity, the homogenate was centrifuged at 100,000 × g (average) for 30 min at 0°C. Amphetamine was extracted from the supernatant following the procedure of Axelrod 2, and the final extract was placed on Chromagram sheets for thin-layer chromatography as described in the next section. Radioactive standards were run simultaneously to correct for recovery and quenching. Radioactive material
D-[2-14C]Amphetamine was obtained from Smith, Kline and French Laboratories, Philadelphia, through the courtesy of Dr. Glen E. Ullyot. The specific activity was 3/~Ci/mg. Thin-layer chromatography, using Chromagram sheets, cellulose Brain Research, 38 (1972) 399--405
[14C]AMPHETAMINEDISTRIBUTIONIN MOUSE BRAIN
401
d
C/
| - a~
T
"
Fig. 1. a-f, Distribution of radioactivity (dark areas) in coronal sections of mouse brain 20 min after i.v. injection of [14C]amphetamine. 1, Cerebral cortex; 2, hippocampus; 3, thalamus; 4, colliculus superior; 5, corpus geniculatum mediale; 6, nucleus caudatus/putamen; 7, nucleus lateral is septi; 8, cerebellum; 9, wing-shaped structure (see text). No. 6064, E a s t m a n Organic Chemicals, Rochester, N. Y., and developed in a solvent system consisting o f glacial acetic a c i d - w a t e r - n - b u t a n o l (60:15:60) revealed, after exposure to X-ray film, the presence o f a single radioactive spot having an RF (0.91) corresponding to authentic amphetamine. Brain Research, 38 (1972) 399-405
402
G.F. PLACIDI et al.
Fig. 2. Distribution of radioactivity (dark areas) in a horizontal section of mouse brain 20 min after i.v. injection of [14C]amphetamine. (See legend to Fig. 1.) RESULTS After injection of [14C]amphetamine, radioactivity is seen to penetrate readily into the brain. From the radioactive counting studies (Table I), it may be seen that within 5 min the brain concentration of radioactivity is very high and that it remains high even after 20 min. A comparison of counts in blood and other tissue indicates that amphetamine accumulates in the brain well over the blood level. This accumulation was shown, by thin-layer chromatography of brain extracts, to be chiefly unmetabolized amphetamine. Autoradiograms of sagittal, coronal and horizontal sections of the brains of mice indicate that at the selected times the cerebral cortex, hippocampus, thalamus, septum, basal ganglia, brain stem nuclei and cerebellum, in decreasing order, show the highest levels of radioactivity. In addition, an unusual pattern of distribution was consistently observed in the coronal sections after both doses of [14C]amphetamine. Fig. 1a - f (rostral to caudal) are enlargements of autoradiograms of coronal sections of the brain of a mouse 20 min after injection with 1.3 #Ci of the labeled drug. They are an interrupted sequence of sections proceeding caudally from diencephalon to midbrain. The cerebral cortex and hippocampus are highly labeled in all sections. Brain Research, 38 (1972) 399-405
403
[14C]AMPHETAMINE DISTRIBUTION IN MOUSE BRAIN
It is seen in Fig. If that the midbrain is relatively unlabeled except for the medial geniculate bodies. However, just rostral to this section, a highly labeled wing-shaped structure appears in the midbrain (Fig. le) and continues rostrally to include certain areas within the thalamus. The radioactive area appears to pass through parts of the mesencephalic reticular formation and portions of the posterior, ventral, lateral and anterior thalamus. In the horizontal sections (Fig. 2) the caudate nucleus is also seen to be radioactive. DISCUSSION
Studies by Young and Gordon 16 and Goldstein and Anagnoste1° in rat brain and the present thin-layer chromatography results in mice (Table I) indicate that the majority of radioactivity in the brain during the period of pharmacological effect consists of unchanged amphetamine. Thus, the distribution pattern of radioactivity observed in the brain autoradiograms is probably due to [14C]amphetamine and not to metabolites. From the autoradiograms it is seen that the cerebral cortex and hippocampus have high radioactivity. The high concentration of label in the cerebral cortex is in agreement with the counting data of Young and Gordon 16, but they suggested that this accumulation may be non-specific and unrelated to the pharmacological site of amphetamine action. Admittedly, with this autoradiographic technique, a variety of drugs have been found to accumulate in the cerebral cortexL However, since many drugsTwhich have this property do act on the CNS, the possibility that this high accumulation in the cerebral cortex is relevant to the action of amphetamine cannot be excluded. Additional studies are needed and in progress to distinguish nonspecific from specific binding sites of amphetamine. The pattern of amphetamine distribution probably was not a function of brain vascularity since Nair e t al. 13 have presented evidence that cerebellar cortex, colliculi and cerebral cortex (in decreasing order) were among the most highly vascular areas in rat brain. However, the large dose of amphetamine which was employed in this experiment for technical reasons, may have partially influenced the vascularity and the blood flow of the whole brain. The present autoradiograms (Figs. 1 and 2) show the highest radioactivity in the cerebral cortex but much less in the cerebellar cortex and colliculi. Highest activity in cerebral cortex was also confirmed by scintillation counting (Table I). Since there is now adequate evidence that amphetamine releases NE from brain a,4 and blocks uptake of NE by nerve terminals9, accumulation of amphetamine in brain areas containing catecholamine terminals would not be unexpected. Those areas where radioactivity was localized have been demonstrated by the histochemical studies ot Fuxe7, Fuxe e t al. s and Arbuthnott e t al. 1 to contain NE and dopamine terminals and appear to correspond in large measure i n t e r alia, to structures innervated by the dorsal and ventral NE pathway1,8 and to certain dopaminergic structures 7. The involvement of NE and dopamine containing structures in amphetamine stereotypy, locomotion and rage reaction has been reviewed 14. The peculiar autoradiographic pattern of amphetamine localization in the brain Brain Research,
38 (1972) 399-405
404
G.F. PLAC1DIet al.
stem has not been described previously and, in fact, is different from the diffuse, even distribution pattern of caffeine (to be published). This labeled area appears to correspond to the thalamic portion of the brain stem reticular formation and is of particular interest inasmuch as electrophysiological studies have revealed an action of amphetamine at the midbrain level, possibly on the reticular activating system6,11. Although it cannot be determined at this time whether this localization is the cause of or the consequence of stimulation by amphetamine, an interesting possibility is that a functional system for CNS activation appears to have been labeled and delineated. SUMMARY The localization and distribution of [14C]amphetamine in mouse brain has been studied by an autoradiographic technique and by thin-layer chromatography. Thinlayer chromatography revealed that radioactivity in the brain is chiefly due to unchanged amphetamine. The highest content was found in the cerebral cortex, hippocampus, thalamus, septum and basal ganglia. The possible relationship of this localization to the site of action of ampheta mine and the distribution of catecholamine nerve terminals is discussed. ACKNOWLEDGEMENTS We wish to express our gratitude to Dr. Glen Ullyot of Smith, Kline and French Laboratories for the [14C]amphetamine, to Dr. W. G. Clark for his interest and encouragement, and to Dr. C. D. Clemente for his advice on neuroanatomy. The skillful technical assistance of Mr. A. Alcaraz is gratefully acknowledged.
REFERENCES 1 ARBUTHNOTT,G. W., CROW, T. J., FUXE, K., OLSON,L., AND UNGERSTEDT,U., Depletion of catecholamines in vivo induced by electrical stimulation of central monoamine pathways, Brain Research, 24 (1970) 471-483. 2 AXELROD,J., Studies on sympathomimetic amines. II. The biotransformation and physiological disposition of D-amphetamine, o-p-hydroxyamphetamineand o-methamphetamine, J. Pharmacol. exp. Ther., 110 (1954) 315-326. 3 CARLSSON,A., LINDQVIST, M., DAHLSTR6M, A., FUXE, K., AND MASUOKA, D., Effects of the amphetamine group on intraneuronal brain amines in vivo and in vitro, J. Pharm. Pharmaeol., 17 (1965) 521-524. 4 CARR,L. A., ANDMOORE,K. E., Norepinephrine: Release from brain by D-amphetamine in vivo, Science, 164 (1969) 322-323. 5 CASSANO,G. B., AND HANSSON,E., Autoradiographic distribution studies in mice with [I~C]imipramine, Int. J. Neuropsychiat., 2 (1966) 269-278. 6 FUJIMORI, M., AND HIMWICH, H. E., Electroencephalographic analyses of amphetamine and its methoxy derivatives with reference to their sites of EEG alerting in the rabbit brain, Int. J. Neuropharmacol., 8 (1969) 601-613. 7 FuxE, K., Distribution of monoamine nerve terminals in the central nervous system, Actaphysiol. scand., 64, Suppl. 247 (1965) 39-84. Brain Research, 38 (1972) 399-405
[ 14C]AMPHETAMINE DISTRIBUTIONIN MOUSE BRAIN
405
8 FUXE, K., H~)KFELT,T., AND UNGERSTEDT,U., Morphological and functional aspects of central monoamine neurons, Int. Rev. NeurobioL, 13 (1970) 93-126. 9 GLOWINSKI,J., AXELROD,J., AND IVERSEN,L. L., Regional studies of catecholamines in the rat brain. IV. Effects of drugs on the disposition and metabolism of HD-norepinephrine and H a, dopamine, J. Pharmacol. exp. Ther., 153 (1966)30-41. 10 GOLDSTEIN,i . , ANDANAGNOSTE,B., The conversion in vivo of D-amphetamine to (+)-p-hydroxynorephedrine, Biochim. hiophys. Acta ( Amst.) , 107 (1965) 166-168. l l HIEBEL, G., BONVALLET,i . , HUVE, P., AND DELL, P., Analyse neurophysiologique de l'action centrale de la D-amphetamine (Maxiton), Sere. Hdp. Paris, 30 (1954) 1880-1887. 12 INNES, I. R., AND NICKERSON,i . , Drugs acting on postganglionic adrenergic nerve endings and structures innervated by them (Sympathomimetic Drugs). In L. S. GOODMANAND A. GILMAN (Eds.), The Pharmacological Basis of Therapeutics, 4th ed., MacMillan, New York, 1970, pp. 478-523. 13 NAIR,V., PALM,D., AND ROTH, L. J., Relative vasculaiity of certain anatomical areas of the brain and other organs of the rat, Nature (Lond.), 188 (1960) 497-498. 14 RANDRUP,A., AND MUNKVAD,I., Behavioural stereotypies induced by pharmacological agents, Pharmakopsychiat. Neuro-Psychopharmakol. (Stuttgart), I (1968) 18-26. 15 ULLBERG, S., Studies on the distribution and fate of S35 labelled benzylpenicillin in the body, Acta radiol. (Stockh.), Suppl. 118 (1954) 1-110. 16 YOUNG,R. L., AND GORDON, M. W., The disposition of 14C-amphetamine in rat brain, J. Neurochem., 9 (1962) 161-167.
Brain Research, 38 (1972) 399-405
BRAIN RESEARCH
D I S T R I B U T I O N OF [14C]AMPHETAMINE IN MOUSE BRAIN: AN A U T O R A D I O G R A P H I C STUDY*
G I A N F R A N C O PLACIDI**, D A V I D T. M A S U O K A AND ROBERT W. EARLE
Neuropharmacology Research Laboratory, V. A. Hospital, Sepulveda, Calif. 91343 and Medical Pharmacology and Therapeutics, University of California, Irvine, Calif. 92664 (U.S.A.) (Accepted September 30th, 1971)
INTRODUCTION
Considerable effort has been expended in elucidating the site and mechanism of the CNS stimulating action of amphetamine 12. For these purposes, a variety of electro- and psychophysiological techniques as well as chemical and radiochemical approaches have been applied. Results of the chemical and radiochemical studies indicate that the drug readily penetrates the blood-brain barrier2, in and that the time course of radioactivity in the brain following injection of [14C]amphetamine roughly correlates with the period of pharmacological activity 16. With autoradiographic techniques it is now feasible to delineate more closely the localization of labeled substances in the fine structures of areas of interest, and this study reports application of these methods to the brain through use of [14C]amphetamine. MATERIALSAND METHODS Autoradiography Nine adult Swiss mice weighing 19-26 g were injected in the tail vein with [14C]amphetamine. Seven of the mice were injected with 1.3 #Ci and the remaining two received 0.72/~Ci. Both doses were adjusted so that the total volume injected did not exceed 0.2 ml. Following injection, at 5-, 20- and 60-min time intervals selected to cover the maximum period of drug activity and to permit determination of the relationship between pharmacological activity and radioactivity distribution patterns, the particular animals to be utilized were lightly anesthetized with ether and killed by immersion in hexane cooled with solid carbon dioxide. Three of the animals re* A p~eliminary report was presented at the Second Meeting of the Italian Neuropharmacology Society, June, 1969. ** Present address: Psychiatry Clinic, Univ. of Pisa, Pisa, Italy. Brain Research, 38 (1972) 399-405
400
G.F. PLACIDI et al.
TABLE I DISTRIBUTION OF [14C]AMPHETAMINE IN MOUSE BRAIN 5, 2 0 AND 6 0 MIN AFTER THE INTRAVENOUS INJECTION
OF 1.3 /~Ci
In parentheses number of mice is indicated. Time after drug (min)
5
20
60
Blood (counts/min/ml)
Tissue
Counts/min/g
14,499 :t_ 1,023"*
Whole brain (5) Cortex (2) Cerebellum (2) Brain stem (2) Whole brain (5) Cortex (2) Cerebellum (2) Brain stem (2) Whole brain (3)
104,020 ± 7,330 117,200 101,800 81,300 110,540 ± 10,960 123,000 94,500 95,700 49,860
12,478 ± 1,422
8,020
% Amphet.*
96.5 87.9 93.7 98.0 77.9 96.7
* ~ unchanged amphetamine in tissue. ** Counts/min ± S.D. ceiving the 1.3/~Ci dose were sacrificed at the appropriate intervals following injection and 20 # m thick sagittal sections were prepared for the whole body autoradiographic technique described by Ullberg 15. Brain sections were prepared from the remaining 4 mice receiving the 1.3/zCi dose as follows: Coronal sections of one mouse at 5 min and of two mice at 20 min and horizontal sections of one mouse at 20 min. Coronal sections were also prepared at 20 min from the two mice which received the 0.72 #Ci dose. All sections were exposed to Structurix X-ray film (Gevaert). Radioactivity counting studies
A number of mice, as indicated in Table I, were injected in the tail vein with 1.3/zCi of [14C]amphetamine and killed by decapitation after 5, 20 and 60 min. Blood allowed to flow from the body was collected in tubes containing heparin. The brain was quickly removed, weighed and homogenized in 5 ml of 0.05 N HCl. After removing an aliquot for determination of total radioactivity, the homogenate was centrifuged at 100,000 × g (average) for 30 min at 0°C. Amphetamine was extracted from the supernatant following the procedure of Axelrod 2, and the final extract was placed on Chromagram sheets for thin-layer chromatography as described in the next section. Radioactive standards were run simultaneously to correct for recovery and quenching. Radioactive material
D-[2-14C]Amphetamine was obtained from Smith, Kline and French Laboratories, Philadelphia, through the courtesy of Dr. Glen E. Ullyot. The specific activity was 3/~Ci/mg. Thin-layer chromatography, using Chromagram sheets, cellulose Brain Research, 38 (1972) 399--405
[14C]AMPHETAMINEDISTRIBUTIONIN MOUSE BRAIN
401
d
C/
| - a~
T
"
Fig. 1. a-f, Distribution of radioactivity (dark areas) in coronal sections of mouse brain 20 min after i.v. injection of [14C]amphetamine. 1, Cerebral cortex; 2, hippocampus; 3, thalamus; 4, colliculus superior; 5, corpus geniculatum mediale; 6, nucleus caudatus/putamen; 7, nucleus lateral is septi; 8, cerebellum; 9, wing-shaped structure (see text). No. 6064, E a s t m a n Organic Chemicals, Rochester, N. Y., and developed in a solvent system consisting o f glacial acetic a c i d - w a t e r - n - b u t a n o l (60:15:60) revealed, after exposure to X-ray film, the presence o f a single radioactive spot having an RF (0.91) corresponding to authentic amphetamine. Brain Research, 38 (1972) 399-405
402
G.F. PLACIDI et al.
Fig. 2. Distribution of radioactivity (dark areas) in a horizontal section of mouse brain 20 min after i.v. injection of [14C]amphetamine. (See legend to Fig. 1.) RESULTS After injection of [14C]amphetamine, radioactivity is seen to penetrate readily into the brain. From the radioactive counting studies (Table I), it may be seen that within 5 min the brain concentration of radioactivity is very high and that it remains high even after 20 min. A comparison of counts in blood and other tissue indicates that amphetamine accumulates in the brain well over the blood level. This accumulation was shown, by thin-layer chromatography of brain extracts, to be chiefly unmetabolized amphetamine. Autoradiograms of sagittal, coronal and horizontal sections of the brains of mice indicate that at the selected times the cerebral cortex, hippocampus, thalamus, septum, basal ganglia, brain stem nuclei and cerebellum, in decreasing order, show the highest levels of radioactivity. In addition, an unusual pattern of distribution was consistently observed in the coronal sections after both doses of [14C]amphetamine. Fig. 1a - f (rostral to caudal) are enlargements of autoradiograms of coronal sections of the brain of a mouse 20 min after injection with 1.3 #Ci of the labeled drug. They are an interrupted sequence of sections proceeding caudally from diencephalon to midbrain. The cerebral cortex and hippocampus are highly labeled in all sections. Brain Research, 38 (1972) 399-405
403
[14C]AMPHETAMINE DISTRIBUTION IN MOUSE BRAIN
It is seen in Fig. If that the midbrain is relatively unlabeled except for the medial geniculate bodies. However, just rostral to this section, a highly labeled wing-shaped structure appears in the midbrain (Fig. le) and continues rostrally to include certain areas within the thalamus. The radioactive area appears to pass through parts of the mesencephalic reticular formation and portions of the posterior, ventral, lateral and anterior thalamus. In the horizontal sections (Fig. 2) the caudate nucleus is also seen to be radioactive. DISCUSSION
Studies by Young and Gordon 16 and Goldstein and Anagnoste1° in rat brain and the present thin-layer chromatography results in mice (Table I) indicate that the majority of radioactivity in the brain during the period of pharmacological effect consists of unchanged amphetamine. Thus, the distribution pattern of radioactivity observed in the brain autoradiograms is probably due to [14C]amphetamine and not to metabolites. From the autoradiograms it is seen that the cerebral cortex and hippocampus have high radioactivity. The high concentration of label in the cerebral cortex is in agreement with the counting data of Young and Gordon 16, but they suggested that this accumulation may be non-specific and unrelated to the pharmacological site of amphetamine action. Admittedly, with this autoradiographic technique, a variety of drugs have been found to accumulate in the cerebral cortexL However, since many drugsTwhich have this property do act on the CNS, the possibility that this high accumulation in the cerebral cortex is relevant to the action of amphetamine cannot be excluded. Additional studies are needed and in progress to distinguish nonspecific from specific binding sites of amphetamine. The pattern of amphetamine distribution probably was not a function of brain vascularity since Nair e t al. 13 have presented evidence that cerebellar cortex, colliculi and cerebral cortex (in decreasing order) were among the most highly vascular areas in rat brain. However, the large dose of amphetamine which was employed in this experiment for technical reasons, may have partially influenced the vascularity and the blood flow of the whole brain. The present autoradiograms (Figs. 1 and 2) show the highest radioactivity in the cerebral cortex but much less in the cerebellar cortex and colliculi. Highest activity in cerebral cortex was also confirmed by scintillation counting (Table I). Since there is now adequate evidence that amphetamine releases NE from brain a,4 and blocks uptake of NE by nerve terminals9, accumulation of amphetamine in brain areas containing catecholamine terminals would not be unexpected. Those areas where radioactivity was localized have been demonstrated by the histochemical studies ot Fuxe7, Fuxe e t al. s and Arbuthnott e t al. 1 to contain NE and dopamine terminals and appear to correspond in large measure i n t e r alia, to structures innervated by the dorsal and ventral NE pathway1,8 and to certain dopaminergic structures 7. The involvement of NE and dopamine containing structures in amphetamine stereotypy, locomotion and rage reaction has been reviewed 14. The peculiar autoradiographic pattern of amphetamine localization in the brain Brain Research,
38 (1972) 399-405
404
G.F. PLAC1DIet al.
stem has not been described previously and, in fact, is different from the diffuse, even distribution pattern of caffeine (to be published). This labeled area appears to correspond to the thalamic portion of the brain stem reticular formation and is of particular interest inasmuch as electrophysiological studies have revealed an action of amphetamine at the midbrain level, possibly on the reticular activating system6,11. Although it cannot be determined at this time whether this localization is the cause of or the consequence of stimulation by amphetamine, an interesting possibility is that a functional system for CNS activation appears to have been labeled and delineated. SUMMARY The localization and distribution of [14C]amphetamine in mouse brain has been studied by an autoradiographic technique and by thin-layer chromatography. Thinlayer chromatography revealed that radioactivity in the brain is chiefly due to unchanged amphetamine. The highest content was found in the cerebral cortex, hippocampus, thalamus, septum and basal ganglia. The possible relationship of this localization to the site of action of ampheta mine and the distribution of catecholamine nerve terminals is discussed. ACKNOWLEDGEMENTS We wish to express our gratitude to Dr. Glen Ullyot of Smith, Kline and French Laboratories for the [14C]amphetamine, to Dr. W. G. Clark for his interest and encouragement, and to Dr. C. D. Clemente for his advice on neuroanatomy. The skillful technical assistance of Mr. A. Alcaraz is gratefully acknowledged.
REFERENCES 1 ARBUTHNOTT,G. W., CROW, T. J., FUXE, K., OLSON,L., AND UNGERSTEDT,U., Depletion of catecholamines in vivo induced by electrical stimulation of central monoamine pathways, Brain Research, 24 (1970) 471-483. 2 AXELROD,J., Studies on sympathomimetic amines. II. The biotransformation and physiological disposition of D-amphetamine, o-p-hydroxyamphetamineand o-methamphetamine, J. Pharmacol. exp. Ther., 110 (1954) 315-326. 3 CARLSSON,A., LINDQVIST, M., DAHLSTR6M, A., FUXE, K., AND MASUOKA, D., Effects of the amphetamine group on intraneuronal brain amines in vivo and in vitro, J. Pharm. Pharmaeol., 17 (1965) 521-524. 4 CARR,L. A., ANDMOORE,K. E., Norepinephrine: Release from brain by D-amphetamine in vivo, Science, 164 (1969) 322-323. 5 CASSANO,G. B., AND HANSSON,E., Autoradiographic distribution studies in mice with [I~C]imipramine, Int. J. Neuropsychiat., 2 (1966) 269-278. 6 FUJIMORI, M., AND HIMWICH, H. E., Electroencephalographic analyses of amphetamine and its methoxy derivatives with reference to their sites of EEG alerting in the rabbit brain, Int. J. Neuropharmacol., 8 (1969) 601-613. 7 FuxE, K., Distribution of monoamine nerve terminals in the central nervous system, Actaphysiol. scand., 64, Suppl. 247 (1965) 39-84. Brain Research, 38 (1972) 399-405
[ 14C]AMPHETAMINE DISTRIBUTIONIN MOUSE BRAIN
405
8 FUXE, K., H~)KFELT,T., AND UNGERSTEDT,U., Morphological and functional aspects of central monoamine neurons, Int. Rev. NeurobioL, 13 (1970) 93-126. 9 GLOWINSKI,J., AXELROD,J., AND IVERSEN,L. L., Regional studies of catecholamines in the rat brain. IV. Effects of drugs on the disposition and metabolism of HD-norepinephrine and H a, dopamine, J. Pharmacol. exp. Ther., 153 (1966)30-41. 10 GOLDSTEIN,i . , ANDANAGNOSTE,B., The conversion in vivo of D-amphetamine to (+)-p-hydroxynorephedrine, Biochim. hiophys. Acta ( Amst.) , 107 (1965) 166-168. l l HIEBEL, G., BONVALLET,i . , HUVE, P., AND DELL, P., Analyse neurophysiologique de l'action centrale de la D-amphetamine (Maxiton), Sere. Hdp. Paris, 30 (1954) 1880-1887. 12 INNES, I. R., AND NICKERSON,i . , Drugs acting on postganglionic adrenergic nerve endings and structures innervated by them (Sympathomimetic Drugs). In L. S. GOODMANAND A. GILMAN (Eds.), The Pharmacological Basis of Therapeutics, 4th ed., MacMillan, New York, 1970, pp. 478-523. 13 NAIR,V., PALM,D., AND ROTH, L. J., Relative vasculaiity of certain anatomical areas of the brain and other organs of the rat, Nature (Lond.), 188 (1960) 497-498. 14 RANDRUP,A., AND MUNKVAD,I., Behavioural stereotypies induced by pharmacological agents, Pharmakopsychiat. Neuro-Psychopharmakol. (Stuttgart), I (1968) 18-26. 15 ULLBERG, S., Studies on the distribution and fate of S35 labelled benzylpenicillin in the body, Acta radiol. (Stockh.), Suppl. 118 (1954) 1-110. 16 YOUNG,R. L., AND GORDON, M. W., The disposition of 14C-amphetamine in rat brain, J. Neurochem., 9 (1962) 161-167.
Brain Research, 38 (1972) 399-405