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Evaluation of the effect of p-chloroamphetamine on individual catecholaminergic nuclei in the rat brain
279
European Journal o f Pharmacology, 50 (1978) 279--282 © Elsevier/North-Holland Biomedical Press
Short communication EVALUATION OF THE EFFECT OF p-CHLOROAMPHETAMINE ON INDIVIDUAL CATECHOLAMINERGIC NUCLEI IN THE RAT BRAIN V. JOHN MASSARI *,**, YOUSEF TIZABI * and DAVID JACOBOWlTZ
• Department o f Pharmacology, Howard University College o f Medicine, Washington, D.C. 20059, and Laboratory o f Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20014, U.S.A. Received 15 June 1978, accepted 17 June 1978
V.J. MASSARI, Y. TIZABI and D. JACOBOWITZ, Evaluation of the effect of p-chloroamphetamine on individual catecholaminergic nuclei in the rat brain, European J. Pharmacol. 50 (1978) 279--282. Short and long term effects of p-chloroamphetamine (PCA) on levels of catecholamines (CA) in discrete CA cell body and axon terminal areas were investigated. The levels were unaffected, except for a transient fall in dopamine in the arcuate nucleus. These results do not support the suggestion that PCA is neurotoxic to CA cells. p-Chloroamphetamine
Catecholamines
Neurotoxic drug
1. Introduction A variety of biochemical evidence suggests that p-chloroamphetamine (PCA) may be neurotoxic to serotonergic nerves in the central nervous system (CNS). Thus, after the administration of a single i.p. dose o f PCA, a marked fall in brain serotonin (5HT), 5hydroxyindole acetic acid and tryptophan hydroxylase activity lasting for more than four months is observed. A long term decrease in the high affinity uptake of 5HT has also been reported (Sanders-Bush et al., 1975). Morphological studies also indicate that PCA is a serotonin neurotoxin. Thus, PCA causes the degeneration of a small group of serotonergic (B-9) cells in the ventrolateralmesencephalic tegmentum (Harvey et al., 1975). However, PCA is not toxic to most serotonergic cell bodies and axons in the brain (Massari et al., 1978; Massari et al., 1978). Rather, PCA appears to cause the selective destruction of only serotonergic axon terminals (Massari et al., 1978).
** To whom correspondence should be addressed.
Recently, Hattori et al. (1976) have presented criteria for the ultrastructural identificatipn of 4 different types of nerve terminals in the rat neostriatum. After 10--15 mg/kg of PCA observed the degeneration of 2 of these morphologically distinct types of terminals. One type was identical to that seen after injection of 6-hydroxydopamine, a catecholamine (CA) neurotoxin. This was tentatively identified as a dopaminergic nerve terminal. They suggested that the other type of axon terminal was serotonergic, since it was more frequently attacked by PCA, and was easily labelled by intraventricularly injected 3H-5HT. This suggestion that PCA might be toxic to DA nerve terminals was somewhat surprising, although PCA is known to have a brief amphetamine-like effect on CNS CA. Thus, within the first 24 h after PCA administration, the drug can release CA (Strada and Sulser, 1971), and competitively inhibit their reuptake (Sanders-Bush et al., 1975). However, tyrosine hydroxylase activity is unaffected by doses of PCA which are maximally effective in reducing brain 5HT (5--10 mg/kg, Sulser and Sanders-Bush, 1973; Hat-
280
V.J. M A S S A R I E T AL.
tori et al., 1976), and whole brain levels of NE and DA are also not reduced (Steranka, 1976). This biochemical spectrum of effect of PCA and CA is unlike the effect of CA neurotoxins, such as 6-hydroxydopamine. However, previous investigators have generally examined whole-brain or coarsely dissected regions of the brain, and thus may have overlooked potential t o x i c effects of PCA. In this study the effects of PCA on 7 discrete catecholaminergic nuclei have been examined in order to evaluate the possibility that PCA is also toxic to CA cells.
2. Materials and methods 48 male Sprague-Dawley rats weighing 250-300 g and maintained on standard rat chow and water, ad libitum, were randomly injected with either saline or 10 mg/kg PCA. The rats were sacrificed either 3 or 9 day later by decapitation. Brains were rapidly removed, m o u n t e d on specimen plates, and frozen on dry ice. Subsequently the brains were sectioned into 300 p slices in a cryostat main-
tained at --8°C and individual nuclei were removed by the m e t h o d of Palkovits (1973). CA were assayed by the m e t h o d of Coyle and Henry (1973).
3. Results Table 1 and table 2 show the effect of PCA on CA in 5 CA cell b o d y areas and 2 CA axon terminal regions. PCA did not effect levels of CA in the noradrenergic locus coeruleus and A 5 cell b o d y group (nomenclature of DahlstrSm and Fuxe, 1964), or in the central gray catecholamine area, a noradrenergic nerve terminal region. L o w DA levels measured in these nuclei presumably reflect precursor amounts of DA. PCA significantly lowered levels of DA in arcuate nucleus cells by almost 50% 3 days after injection (table 1). By 9 days, however, arcuate DA levels were equal to control values. Otherwise, PCA did not significantly effect levels of CA at any time on the DA cells of the area tegmenti ventralis (A-10) and substantia nigra compacta (A-9), or in the neostriatum (n. caudatus-putamen), a DA terminal region.
TABLE 1 Levels of catecholamines in discrete brain areas 3 days after injection of p - c h l o r o a m p h e t a m i n e 1 (PCA). Region
A-5 A-6 (locus coerulus) A-9 (substantia nigra c o m p a c t a ) A-10 (area t e g m e n t i ventralis) A-12 (aruate nucleus) C G C A (NE terminals) CP ( D A terminals)
N o r e p i n e p h r i n e (pg/pg protein)
D o p a m i n e (pg/pg p r o t e i n )
Control
PCA
Control
PCA
8.3 70.5 7.6 15.7 41.3 42.6 4.2
8.5 65.3 9.8 15.3 29.9 39.7 3.5
1.9 11.6 21.7 27.0 15.8 4.2 60.2
1.8 12.2 26.0 28.2 8.2 6.0 53.8
-+ 0.84 + 12.50 +- 1.05 -+ 1.03 -+ 5.08 +- 4.26 -+ 0.74
+ 1.06 _+ 6.17 +_ 1.68 -+ 1.17 -+ 4.23 -+ 7.44 -+ 0.77
+ 0.28 + 1.99 -+ 1.85 + 2.56 + 2.75 +- 0.37 +- 3.03
+ 0.43 + 2.99 _+ 3.40 + 2.36 +_ 1.16 2 _+ 1.03 _+ 3.93
1 Rats were injected with 10 m g / k g PCA i.p. and sacrificed by decapitation. Catecholamines were assayed in discrete nuclei as described in the text. Abbreviations: CGCA, central gray c a t e c h o l a m i n e area; CP, nucleus caudatus p u t a m e n ; A-5, A-6, A-9, A-10, A-12, n o m e n c l a t u r e according to Dahlstr~m and F u x e , 1964. n = 9--11 for each point. P ( 0.025 S t u d e n t ' s 2-tail t-test.
281
p-CHLOROAMPHETAMINE AND CATECHOLAMINES TABLE 2 Levels of catecholamines in discrete brain areas 9 days after injection of p-chloroamphetamine 1 (PCA). Region
Norepinephrine (pg//.Lgprotein) Control
PCA
A-5 A-6 (locus coerulus) A-9 (substantia nigra compacta) A-10 (area tegmenti ventralis)
10.8 86.9 13.0 15.1
11.0 80.1 12.9 13.4
A-12 (arcuate nucleus) CGCA (NE terminals) CP (DA terminals)
43.2 _+5.02 58.0 _+4.02 4.5 +_0.45
+_0.49 +_6.61 _+2.09 _+1.16
-+ 1.25 -+ 7.89 + 1.98 _+1.36
36.0 -+ 2.84 50.2 + 6.14 4.17 _+0.45
•Dopamine (pg//~g protein) Control
PCA
2.8 16.1 34.2 23.1
2.7 12.9 31.6 26.8
+ 0.42 _+2.06 _+3.39 +- 4.42
15.0 -+ 1.42 4.7 + 0.38 53.7 -+4.98
+ 0.35 + 1.94 -+4.73 + 3.80
14.7 + 1.55 5.5 + 0.51 63.9 -+4.05
1 Rats were injected with 10 mg/kg PCA i.p. and sacrificed by decapitation. Catecholamines were assayed in discrete nuclei as described in the text. Abbreviations: CGCA, central gray catecholamine area; CP, nucleus caudatus putamen; A-5, A-6, A-9, A-10, A-12, nomenclature according to DahlstrSm and Fuxe, 1964. n = 9--11 for each point.
4. Discussion
Acknowledgements
Multiple d r u g effects u n d e r l y t h e initial a c t i o n s o f P C A o n CNS C A a n d 5 H T neurons. F o r 5 H T n e u r o n s , t h e initial phase o f d r u g effects is f o l l o w e d b y an irreversible n e u r o t o x i c phase a p p r o x i m a t e l y 4 d a y s a f t e r injection ( S a n d e r s - - B u s h et al., 1975). E f f e c t s o n CNS C A have b e e n r e p o r t e d t o be transient. It has b e e n suggested ( H a t t o r i et al., 1976), however, that 10--15 mg/kg PCA may cause t h e d e s t r u c t i o n o f D A as well as 5 H T nerve terminals in the n e o s t r i a t u m as s o o n as t w o d a y s a f t e r injection. Since previous bioc h e m i c a l d a t a did n o t suggest t h a t P C A was n e u r o t o x i c t o C A cells, we have e x a m i n e d t h e s h o r t - t e r m (3 d a y ) a n d l o n g t e r m (9 d a y ) effects o f 10 m g / k g P C A o n discrete C A cell a n d terminal regions. P C A l o w e r e d D A levels in t h e a r c u a t e n u c l e u s t r a n s i e n t l y , b u t did n o t o t h e r w i s e significantly e f f e c t C A in a n y region examined. Thus the data do not support the suggestion t h a t P C A is n e u r o t o x i c t o C A cells o r a x o n terminals in t h e CNS. R a t h e r t h e d a t a give f u r t h e r s u p p o r t t o t h e h y p o t h e s i s t h a t P C A m a y be selectively t o x i c t o 5 H T cells in t h e CNS.
This research was supported in part by Fellowship No. 1"F32N505122-02 from the National Institute for Neurological and Communicative Disorders and Stroke to V. John Masseri. References Coyle, J.T. and D. Henry, 1973, Catecholamines in fetal and newborn rat brain, J. Neurochem. 21, 61. DahlstrSm, A. and K. Fuxe, 1964, Evidence for the existence of monoamine containing neurons in the central nervous system. I. Demonstration of monoaminas in the cell bodies of brain stem neurons, Act. Physiol. Scand., Suppl. 62, 1. Harvey, J., S. McMaster and L. Yunger, 1975, pChloramphetamine: selective neurotoxic action in brain, Science 187,841. Hattori, T., P.L. McGeer and E.G. McGeer, 1976, Synaptic morphology in the neostriatum of the rat: possible serotonergic synapse, Neurochem. Res. 1,451. Massari, V.J., Y. Tizabi, Z. Gottesfeld and D.J. Jacobowitz, 1978, A fluorescence histochemicai and biochemical evaluation of the effect of pchloroamphetamine on individual serotonergic neurons in the rat brain, Neuroscience (in press). Massari, V.J., Y. Tizabi and E. Sanders-Bush, 1978, Evaluation of the neurotoxic effects of p-chloroamphetamine: a histological and biochemical study, Neuropharmacology (in press).
282 Palkovits, M., 1973, Isolated removal of hypothalamic or other brain nuclei o f the rat, Brain Res. 59,449. Sanders-Bush, E., J. Bushing and F. Sulser, 1975, Long-term effects of p-chloroamphetarnine and related drugs on central serotonergic mechanisms, J. Pharmacol. Exptl. Therap. 192, 33. Steranka, L.R., Tolerance to the excitatory effect of p-chloroamphetamine on behavior: role of catecholamines and serotonin, Ph.D. Thesis, Vanderhilt University, Nashville, Tennessee.
V.J. MASSARI ET AL. Strada, S. and F. Sulser, 1971, Comparative effects of p~hloroamphetamine and amphetamine on metabolism and in vivo release of 3H-norepinephfine in the hypothalamus, European J. Pharmacol. 15, 45. Sulser, F. and E. Sanders-Bush, 1973, Halogen substitution of amphetamine: biochemical and pharmacological consequences, in: Frontiers in Catecholamine Research, eds. E. Usdin and S. Snyder (Pergamon Press, New York) p. 995.
European Journal o f Pharmacology, 50 (1978) 279--282 © Elsevier/North-Holland Biomedical Press
Short communication EVALUATION OF THE EFFECT OF p-CHLOROAMPHETAMINE ON INDIVIDUAL CATECHOLAMINERGIC NUCLEI IN THE RAT BRAIN V. JOHN MASSARI *,**, YOUSEF TIZABI * and DAVID JACOBOWlTZ
• Department o f Pharmacology, Howard University College o f Medicine, Washington, D.C. 20059, and Laboratory o f Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20014, U.S.A. Received 15 June 1978, accepted 17 June 1978
V.J. MASSARI, Y. TIZABI and D. JACOBOWITZ, Evaluation of the effect of p-chloroamphetamine on individual catecholaminergic nuclei in the rat brain, European J. Pharmacol. 50 (1978) 279--282. Short and long term effects of p-chloroamphetamine (PCA) on levels of catecholamines (CA) in discrete CA cell body and axon terminal areas were investigated. The levels were unaffected, except for a transient fall in dopamine in the arcuate nucleus. These results do not support the suggestion that PCA is neurotoxic to CA cells. p-Chloroamphetamine
Catecholamines
Neurotoxic drug
1. Introduction A variety of biochemical evidence suggests that p-chloroamphetamine (PCA) may be neurotoxic to serotonergic nerves in the central nervous system (CNS). Thus, after the administration of a single i.p. dose o f PCA, a marked fall in brain serotonin (5HT), 5hydroxyindole acetic acid and tryptophan hydroxylase activity lasting for more than four months is observed. A long term decrease in the high affinity uptake of 5HT has also been reported (Sanders-Bush et al., 1975). Morphological studies also indicate that PCA is a serotonin neurotoxin. Thus, PCA causes the degeneration of a small group of serotonergic (B-9) cells in the ventrolateralmesencephalic tegmentum (Harvey et al., 1975). However, PCA is not toxic to most serotonergic cell bodies and axons in the brain (Massari et al., 1978; Massari et al., 1978). Rather, PCA appears to cause the selective destruction of only serotonergic axon terminals (Massari et al., 1978).
** To whom correspondence should be addressed.
Recently, Hattori et al. (1976) have presented criteria for the ultrastructural identificatipn of 4 different types of nerve terminals in the rat neostriatum. After 10--15 mg/kg of PCA observed the degeneration of 2 of these morphologically distinct types of terminals. One type was identical to that seen after injection of 6-hydroxydopamine, a catecholamine (CA) neurotoxin. This was tentatively identified as a dopaminergic nerve terminal. They suggested that the other type of axon terminal was serotonergic, since it was more frequently attacked by PCA, and was easily labelled by intraventricularly injected 3H-5HT. This suggestion that PCA might be toxic to DA nerve terminals was somewhat surprising, although PCA is known to have a brief amphetamine-like effect on CNS CA. Thus, within the first 24 h after PCA administration, the drug can release CA (Strada and Sulser, 1971), and competitively inhibit their reuptake (Sanders-Bush et al., 1975). However, tyrosine hydroxylase activity is unaffected by doses of PCA which are maximally effective in reducing brain 5HT (5--10 mg/kg, Sulser and Sanders-Bush, 1973; Hat-
280
V.J. M A S S A R I E T AL.
tori et al., 1976), and whole brain levels of NE and DA are also not reduced (Steranka, 1976). This biochemical spectrum of effect of PCA and CA is unlike the effect of CA neurotoxins, such as 6-hydroxydopamine. However, previous investigators have generally examined whole-brain or coarsely dissected regions of the brain, and thus may have overlooked potential t o x i c effects of PCA. In this study the effects of PCA on 7 discrete catecholaminergic nuclei have been examined in order to evaluate the possibility that PCA is also toxic to CA cells.
2. Materials and methods 48 male Sprague-Dawley rats weighing 250-300 g and maintained on standard rat chow and water, ad libitum, were randomly injected with either saline or 10 mg/kg PCA. The rats were sacrificed either 3 or 9 day later by decapitation. Brains were rapidly removed, m o u n t e d on specimen plates, and frozen on dry ice. Subsequently the brains were sectioned into 300 p slices in a cryostat main-
tained at --8°C and individual nuclei were removed by the m e t h o d of Palkovits (1973). CA were assayed by the m e t h o d of Coyle and Henry (1973).
3. Results Table 1 and table 2 show the effect of PCA on CA in 5 CA cell b o d y areas and 2 CA axon terminal regions. PCA did not effect levels of CA in the noradrenergic locus coeruleus and A 5 cell b o d y group (nomenclature of DahlstrSm and Fuxe, 1964), or in the central gray catecholamine area, a noradrenergic nerve terminal region. L o w DA levels measured in these nuclei presumably reflect precursor amounts of DA. PCA significantly lowered levels of DA in arcuate nucleus cells by almost 50% 3 days after injection (table 1). By 9 days, however, arcuate DA levels were equal to control values. Otherwise, PCA did not significantly effect levels of CA at any time on the DA cells of the area tegmenti ventralis (A-10) and substantia nigra compacta (A-9), or in the neostriatum (n. caudatus-putamen), a DA terminal region.
TABLE 1 Levels of catecholamines in discrete brain areas 3 days after injection of p - c h l o r o a m p h e t a m i n e 1 (PCA). Region
A-5 A-6 (locus coerulus) A-9 (substantia nigra c o m p a c t a ) A-10 (area t e g m e n t i ventralis) A-12 (aruate nucleus) C G C A (NE terminals) CP ( D A terminals)
N o r e p i n e p h r i n e (pg/pg protein)
D o p a m i n e (pg/pg p r o t e i n )
Control
PCA
Control
PCA
8.3 70.5 7.6 15.7 41.3 42.6 4.2
8.5 65.3 9.8 15.3 29.9 39.7 3.5
1.9 11.6 21.7 27.0 15.8 4.2 60.2
1.8 12.2 26.0 28.2 8.2 6.0 53.8
-+ 0.84 + 12.50 +- 1.05 -+ 1.03 -+ 5.08 +- 4.26 -+ 0.74
+ 1.06 _+ 6.17 +_ 1.68 -+ 1.17 -+ 4.23 -+ 7.44 -+ 0.77
+ 0.28 + 1.99 -+ 1.85 + 2.56 + 2.75 +- 0.37 +- 3.03
+ 0.43 + 2.99 _+ 3.40 + 2.36 +_ 1.16 2 _+ 1.03 _+ 3.93
1 Rats were injected with 10 m g / k g PCA i.p. and sacrificed by decapitation. Catecholamines were assayed in discrete nuclei as described in the text. Abbreviations: CGCA, central gray c a t e c h o l a m i n e area; CP, nucleus caudatus p u t a m e n ; A-5, A-6, A-9, A-10, A-12, n o m e n c l a t u r e according to Dahlstr~m and F u x e , 1964. n = 9--11 for each point. P ( 0.025 S t u d e n t ' s 2-tail t-test.
281
p-CHLOROAMPHETAMINE AND CATECHOLAMINES TABLE 2 Levels of catecholamines in discrete brain areas 9 days after injection of p-chloroamphetamine 1 (PCA). Region
Norepinephrine (pg//.Lgprotein) Control
PCA
A-5 A-6 (locus coerulus) A-9 (substantia nigra compacta) A-10 (area tegmenti ventralis)
10.8 86.9 13.0 15.1
11.0 80.1 12.9 13.4
A-12 (arcuate nucleus) CGCA (NE terminals) CP (DA terminals)
43.2 _+5.02 58.0 _+4.02 4.5 +_0.45
+_0.49 +_6.61 _+2.09 _+1.16
-+ 1.25 -+ 7.89 + 1.98 _+1.36
36.0 -+ 2.84 50.2 + 6.14 4.17 _+0.45
•Dopamine (pg//~g protein) Control
PCA
2.8 16.1 34.2 23.1
2.7 12.9 31.6 26.8
+ 0.42 _+2.06 _+3.39 +- 4.42
15.0 -+ 1.42 4.7 + 0.38 53.7 -+4.98
+ 0.35 + 1.94 -+4.73 + 3.80
14.7 + 1.55 5.5 + 0.51 63.9 -+4.05
1 Rats were injected with 10 mg/kg PCA i.p. and sacrificed by decapitation. Catecholamines were assayed in discrete nuclei as described in the text. Abbreviations: CGCA, central gray catecholamine area; CP, nucleus caudatus putamen; A-5, A-6, A-9, A-10, A-12, nomenclature according to DahlstrSm and Fuxe, 1964. n = 9--11 for each point.
4. Discussion
Acknowledgements
Multiple d r u g effects u n d e r l y t h e initial a c t i o n s o f P C A o n CNS C A a n d 5 H T neurons. F o r 5 H T n e u r o n s , t h e initial phase o f d r u g effects is f o l l o w e d b y an irreversible n e u r o t o x i c phase a p p r o x i m a t e l y 4 d a y s a f t e r injection ( S a n d e r s - - B u s h et al., 1975). E f f e c t s o n CNS C A have b e e n r e p o r t e d t o be transient. It has b e e n suggested ( H a t t o r i et al., 1976), however, that 10--15 mg/kg PCA may cause t h e d e s t r u c t i o n o f D A as well as 5 H T nerve terminals in the n e o s t r i a t u m as s o o n as t w o d a y s a f t e r injection. Since previous bioc h e m i c a l d a t a did n o t suggest t h a t P C A was n e u r o t o x i c t o C A cells, we have e x a m i n e d t h e s h o r t - t e r m (3 d a y ) a n d l o n g t e r m (9 d a y ) effects o f 10 m g / k g P C A o n discrete C A cell a n d terminal regions. P C A l o w e r e d D A levels in t h e a r c u a t e n u c l e u s t r a n s i e n t l y , b u t did n o t o t h e r w i s e significantly e f f e c t C A in a n y region examined. Thus the data do not support the suggestion t h a t P C A is n e u r o t o x i c t o C A cells o r a x o n terminals in t h e CNS. R a t h e r t h e d a t a give f u r t h e r s u p p o r t t o t h e h y p o t h e s i s t h a t P C A m a y be selectively t o x i c t o 5 H T cells in t h e CNS.
This research was supported in part by Fellowship No. 1"F32N505122-02 from the National Institute for Neurological and Communicative Disorders and Stroke to V. John Masseri. References Coyle, J.T. and D. Henry, 1973, Catecholamines in fetal and newborn rat brain, J. Neurochem. 21, 61. DahlstrSm, A. and K. Fuxe, 1964, Evidence for the existence of monoamine containing neurons in the central nervous system. I. Demonstration of monoaminas in the cell bodies of brain stem neurons, Act. Physiol. Scand., Suppl. 62, 1. Harvey, J., S. McMaster and L. Yunger, 1975, pChloramphetamine: selective neurotoxic action in brain, Science 187,841. Hattori, T., P.L. McGeer and E.G. McGeer, 1976, Synaptic morphology in the neostriatum of the rat: possible serotonergic synapse, Neurochem. Res. 1,451. Massari, V.J., Y. Tizabi, Z. Gottesfeld and D.J. Jacobowitz, 1978, A fluorescence histochemicai and biochemical evaluation of the effect of pchloroamphetamine on individual serotonergic neurons in the rat brain, Neuroscience (in press). Massari, V.J., Y. Tizabi and E. Sanders-Bush, 1978, Evaluation of the neurotoxic effects of p-chloroamphetamine: a histological and biochemical study, Neuropharmacology (in press).
282 Palkovits, M., 1973, Isolated removal of hypothalamic or other brain nuclei o f the rat, Brain Res. 59,449. Sanders-Bush, E., J. Bushing and F. Sulser, 1975, Long-term effects of p-chloroamphetarnine and related drugs on central serotonergic mechanisms, J. Pharmacol. Exptl. Therap. 192, 33. Steranka, L.R., Tolerance to the excitatory effect of p-chloroamphetamine on behavior: role of catecholamines and serotonin, Ph.D. Thesis, Vanderhilt University, Nashville, Tennessee.
V.J. MASSARI ET AL. Strada, S. and F. Sulser, 1971, Comparative effects of p~hloroamphetamine and amphetamine on metabolism and in vivo release of 3H-norepinephfine in the hypothalamus, European J. Pharmacol. 15, 45. Sulser, F. and E. Sanders-Bush, 1973, Halogen substitution of amphetamine: biochemical and pharmacological consequences, in: Frontiers in Catecholamine Research, eds. E. Usdin and S. Snyder (Pergamon Press, New York) p. 995.