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Striatal dopamine metabolism in living monkeys examined by positron emission tomography
Brain Research, 280 (1983) 169-171
169
Elsevier
Striatal dopamine metabolism in living monkeys examined by positron emission tomography STEPHEN GARNETS', GUNTER FIRNAU, CLAUDE NAHMIAS and RAMAN CHIRAKAL Department of Nuclear Medicine, McMaster University Medical Centre, Hamilton, Ont. L8N 3Z5 (Canada)
(Accepted August 2nd, 1983) Key words: dopamine - - striatum - - monkey - - [~SF]6-fluoro-l,-dopa- - positron emission tomography
Positron emission tomography, using the dopa analogue [ISF]6-fluoro-c-dopa,has been used to depict the neostriatum in living monkeys. The amount of lSF that accumulated preferentially in the striatum could be augmented by a peripheral decarboxylase inhibitor. Striatal ~8Fcould also be discharged with reserpine. This is the first time that the regional distribution of a neurotransmitter has been demonstrated in monkeys. Positron emission tomography is a relatively new technology that allows the regional distribution of a positron-labeled molecule to be examined in a living animal. The positron labeled molecule is injected i.v. or inhaled. The animal or patient is positioned in the tomograph and several planes are examined concurrently or sequentially so that the 3 dimensional distribution of the positron-emitting molecule within the animal can be determined. Positron emission tomography has been used to measure regional cerebral blood flowlZ, regional cerebral oxygen consumption 7, and local cerebral glucose consumption HI. In this work positron emission tomography has been used to make direct studies of the distribution and metabolism of dopamine in living brain. Cynomolgus monkeys were studied. Their weight ranged from 3.0 to 5.5 kg. Each monkey was anesthetized with a halothane-oxygen mixture. Four monkeys were pretreated with an i.v. injection of the aromatic acid decarboxylase inhibitor Ro4-4046 [25 mg/kg], the other two acted as controls. The anesthetized animals were examined in the McMaster positron emission tomograph 5. The tomographic slice selected for study was parallel to and 1 cm above the orbito-meatal plane. This slice was chosen because it contained the largest proportion of striaturn of any horizontal section of cynomolgus brain (Fig. 2); it was 1 cm thick. Once the monkey was positioned, 11 MBq-[~SF]-6-fluoro-L-dopa (spec. act., 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
3.0 GBq/mmol)3 was injected i.v. and sequential measurements of the distribution of JSF in the brain were made for up to 180 rain. In two monkeys reserpine (4 mg/kg), a drug known to discharge dopamine from its presynaptic storage vesicles, was given i.v. 30 min after [tSF]6-fluoro-L-dopa. In the two monkeys that were not given reserpine an additional experiment with [JSF]2-fluoro-2-deoxy-glucose was done on the day after the [tSF]fluoro-dopa study. After [lSF]6-fluoro-L-dopa was injected the amount of lSF in the striatum increased for 30 rain at which time its preferential concentration was easily seen (Fig. 1). The region of the striatum was determined by comparison with a horizontal slice cut from a cynomolgus head that had been deep frozen (Fig. 2). It was also the region that tallied with the area of greatest central [tSF]fluoro-deoxy-glucose consumption in a comparable slice. By 45 rain 0.1% of the injected lSF was contained in the striatum of the control animals. This was increased to 0.25% in the animals given Ro4-4046. When reserpine was given the striatal accumulation of 18F was reduced by half over the succeeding 120 rain. This is the first time that the localization of an intracerebral neurotransmitter has been demonstrated in a living primate. Previous studies have been confined to measurements of the blood-brain barrier transport of dopa and its global utilization 4. Although the fraction of the injected dose of
170 to influence intracerebral aromatic acid decarboxvlase activity the accumulation of tSF in the striatum was increased 2 to 3-fold. This c o m p a r e s favourably with the increases r e p o r t e d by Bartholini and
Fig. l A positron emission tomograph section of the brain of a cynomolgus monkey obtained 30 min after the i.v, injection ol [18F16-fluoro-L-dopa.The centrally situated striatum is clearly visible. Radioactivity is also seen in muscles that originatc from the skull. [lSF]6-fluoro-L-dopa that accumulated in the caudate and p u t a m e n was only 0.1% of the injected dose. it was sufficient to m a k e clear pictures of the striatal accumulation of dopa. It was also similar to that reported for whole mouse brain following an i.p. injection of [lnC]DL-dopa 11. When the p e r i p h e r a l decarboxylase inhibitor Ro4-4046 was given in a dose too small
Fig. 2. Horizontal section of the head of a cynomolgus monkey taken at the same level as Fig. 1. The anterior margins of striaturn are located 2 mm behind the posterior walls of the orbits.
Pletscher et al. t. who used R04-4046 to augment thc accumulation of [~4C]L-dopa in the brains of rats. The difference between the present work and that m rats may be due to m i n o r functional modifications produced by introducing 18F into the benzene rmg. We ourselves have shown that such changes are small: It is more likely that the divergence reflects species differences in the decarboxylating capacity of the endothelium of the cerebral capillaries(' The fact that reserpine discharged a p p r o x i m a t e l y half of the lSF that had accumulated in the striatum at 30 min is strong evidence to support the notion that [18F]6-fluoro-l,-dopa was stored, a~ its amine, in presynaptic vesicles. More tSF would p r o b a b l y have been discharged had we waited longer, but the physical half-life of ISF [110 mini precludes protracted studies. Positron emission t o m o g r a p h y has previously been used in an attempt to define the intracerebrat dislribution of the r e c e p t o r sites for benzodiazepine ~. These studies were confounded by the large fraction of the dose of p o s i t r o n - l a b e l e d drug that was bound. non-specifically, by reason of its lipophilicity to the brain and other tissues. Thus. when large doses of untabeled drug were given to b a b o o n s p r e t r e a t e d with [tlC]flunitrazepam the concentration of the labeled drug in the blood and the amount of label in the brain rose concomitantly, D o p a itself tins been labeled with c a r b o n - t l in the carboxyl groupv. W h e n the brains of rats into which this agent had been rejected were dissected the striatum contained less c a r b o n - l t than the surrounding brain. This might have been predicted from the known striatal localization of aromatic acid decarboxylase. The investigation has not been followed up. The present study has p r o v e d that a positron-labeled n e u r o t r a n s m i t t e r molecule will localize and can be visualized in a specific brain region. F u r t h e r , the concentration of transmitter in that region can be m a n i p ulated pharmacologically. Similar studies will be possible in man as soon as sufficient quantities of ~SF-labeled d o p a can be synthesized. It s h o u l d t h e n be possible to p r o b e directly the changes in d o p a m m e metabolism that are believed to be associated with disor-
171 ders of l o c o m o t i o n , such as P a r k i n s o n ' s d i s e a s e , a n d
da a n d t h e O n t a r i o M e n t a l H e a l t h F o u n d a t i o n for fi-
d i s o r d e r s of m o o d such as t h e s c h i z o p h r e n i c syn-
n a n c i a l s u p p o r t . W e a r e e s p e c i a l l y g r a t e f u l to Dr. J.
drome.
K u e h n e r a n d M r . J o h n M c K a y w h o p r o v i d e d acceler a t o r facilities for m a n u f a c t u r i n g lSF.
W c t h a n k t h e M e d i c a l R e s e a r c h C o u n c i l of C a n a -
1 Bartholini, G. and Plctscher, A., Cerebral accumulation and metabolism of HC-DOPA after selective inhibition of peripheral decarboxvlasc, J. Pharmacol. exp. Ther., i61 (19{18) 14-20. 2 Firnau, G., Oarnctt, E. S,, Sourkes, T. L. and Missala, K., ]l~Flfluorodopa: a unktuc gamma emitting substrate for dopa dccarboxvlasc, Experientia, 31 ( 1975 ) 1254-1255. 3 Firnau, G.. Shirakal, R., Sood, S. and Garnett. E. S., Aronmtic fluorination with Xcnon difluoride:L-3,4-dihydroxy6-fluor(~phenylalanine, ('anad. J. Chem., 58 (1980) 1449-14511. 4 Garnett, E. S., Firnau, G.. Nahmias, C.. Sood, S. and Belbeck, L., Blood brain barrier transport and cerebral utilization of dopa in monkeys, Amer. J. Physiol., 238 (1980) R3IS-R327. 5 (;arneu, E. S., Nahmias, ('., Kenyon, D. B. and Firnau, (i., A positron emission tomograph to study brain metabolism in man, J. ('anad. As',soc. Rad.. 33 (1982) 172-175, 6 Hardcbo, J. E. and Owman. C. N., Barrier mechanisms for ncurotransmittcr monoamines and their precursors in the blood brain interface, Ann. ,~'eurol., 8 (1980) 1-11. 7 ,hines, T., Cheslcr, D. A. and Ter Pogossian. M. M., 3"he continuous inhalation of oxygen-15 for assessing regional oxygen extraction in the brains of man, Brit. J. Radiol.. 49 (19761 339 343.
8 Maziere, M., Godot, J. M,, Berger, G., Baron, J. C., Comar, D., Cepeda, C., Benini, Ch. and Naquet, R., Positron tomography. A new method for in vivo brain studies of benzodiazepine in animal and man, Advanc. Biochem. Psychopharmacol.. 26 ( 1981 ) 273-286. 9 Reiffers, S., Beerling-van der Molen, H. D.. Vaalberg, W., Tenhoeve. W., Paans, A. M. J., Korf, .I., Woldring, M. G. and Wynberg, H., Rapid synthesis and purification of carbon-I 1 labelled DOPA: a potential agent for brain studies, Int. J. appl. Radial. lsol., 28 (1977) 955-t)58. i0 Reivich, M., Kuhl, D., Wolf, A., Greenberg..1., Phelps, M., Ido. T., Casella, V., Fowler, B., Gallagher, B., Hoffman, E., Alvari, A. and Sokoloff, L., Measurement of local cerebral glucose metabolism, Acta neurol..~cand.. 56 (Suppl) (1977) 19(>191. 11 Wurtman, R. J., Chou, C. and Rose, C., The fate of C 14Dihydroxyphenylalanine (CI4-DOPA) in the whole mousc. J. Pharm. exp. Ther.. 174 {197(})351-356. 12 Yamamoto, Y. L., Thompson, E., Meyer, E,, Nukul, H.. Matsunaga, M. and Feindel, W.. Three dimensional tomographical regional cerebral blood flow in man measured with high efficiency mini-BGO two ring positron device using Krypton 77, Acta neurol, stand.. 6(I (Suppl) (1979) 186-187.
169
Elsevier
Striatal dopamine metabolism in living monkeys examined by positron emission tomography STEPHEN GARNETS', GUNTER FIRNAU, CLAUDE NAHMIAS and RAMAN CHIRAKAL Department of Nuclear Medicine, McMaster University Medical Centre, Hamilton, Ont. L8N 3Z5 (Canada)
(Accepted August 2nd, 1983) Key words: dopamine - - striatum - - monkey - - [~SF]6-fluoro-l,-dopa- - positron emission tomography
Positron emission tomography, using the dopa analogue [ISF]6-fluoro-c-dopa,has been used to depict the neostriatum in living monkeys. The amount of lSF that accumulated preferentially in the striatum could be augmented by a peripheral decarboxylase inhibitor. Striatal ~8Fcould also be discharged with reserpine. This is the first time that the regional distribution of a neurotransmitter has been demonstrated in monkeys. Positron emission tomography is a relatively new technology that allows the regional distribution of a positron-labeled molecule to be examined in a living animal. The positron labeled molecule is injected i.v. or inhaled. The animal or patient is positioned in the tomograph and several planes are examined concurrently or sequentially so that the 3 dimensional distribution of the positron-emitting molecule within the animal can be determined. Positron emission tomography has been used to measure regional cerebral blood flowlZ, regional cerebral oxygen consumption 7, and local cerebral glucose consumption HI. In this work positron emission tomography has been used to make direct studies of the distribution and metabolism of dopamine in living brain. Cynomolgus monkeys were studied. Their weight ranged from 3.0 to 5.5 kg. Each monkey was anesthetized with a halothane-oxygen mixture. Four monkeys were pretreated with an i.v. injection of the aromatic acid decarboxylase inhibitor Ro4-4046 [25 mg/kg], the other two acted as controls. The anesthetized animals were examined in the McMaster positron emission tomograph 5. The tomographic slice selected for study was parallel to and 1 cm above the orbito-meatal plane. This slice was chosen because it contained the largest proportion of striaturn of any horizontal section of cynomolgus brain (Fig. 2); it was 1 cm thick. Once the monkey was positioned, 11 MBq-[~SF]-6-fluoro-L-dopa (spec. act., 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
3.0 GBq/mmol)3 was injected i.v. and sequential measurements of the distribution of JSF in the brain were made for up to 180 rain. In two monkeys reserpine (4 mg/kg), a drug known to discharge dopamine from its presynaptic storage vesicles, was given i.v. 30 min after [tSF]6-fluoro-L-dopa. In the two monkeys that were not given reserpine an additional experiment with [JSF]2-fluoro-2-deoxy-glucose was done on the day after the [tSF]fluoro-dopa study. After [lSF]6-fluoro-L-dopa was injected the amount of lSF in the striatum increased for 30 rain at which time its preferential concentration was easily seen (Fig. 1). The region of the striatum was determined by comparison with a horizontal slice cut from a cynomolgus head that had been deep frozen (Fig. 2). It was also the region that tallied with the area of greatest central [tSF]fluoro-deoxy-glucose consumption in a comparable slice. By 45 rain 0.1% of the injected lSF was contained in the striatum of the control animals. This was increased to 0.25% in the animals given Ro4-4046. When reserpine was given the striatal accumulation of 18F was reduced by half over the succeeding 120 rain. This is the first time that the localization of an intracerebral neurotransmitter has been demonstrated in a living primate. Previous studies have been confined to measurements of the blood-brain barrier transport of dopa and its global utilization 4. Although the fraction of the injected dose of
170 to influence intracerebral aromatic acid decarboxvlase activity the accumulation of tSF in the striatum was increased 2 to 3-fold. This c o m p a r e s favourably with the increases r e p o r t e d by Bartholini and
Fig. l A positron emission tomograph section of the brain of a cynomolgus monkey obtained 30 min after the i.v, injection ol [18F16-fluoro-L-dopa.The centrally situated striatum is clearly visible. Radioactivity is also seen in muscles that originatc from the skull. [lSF]6-fluoro-L-dopa that accumulated in the caudate and p u t a m e n was only 0.1% of the injected dose. it was sufficient to m a k e clear pictures of the striatal accumulation of dopa. It was also similar to that reported for whole mouse brain following an i.p. injection of [lnC]DL-dopa 11. When the p e r i p h e r a l decarboxylase inhibitor Ro4-4046 was given in a dose too small
Fig. 2. Horizontal section of the head of a cynomolgus monkey taken at the same level as Fig. 1. The anterior margins of striaturn are located 2 mm behind the posterior walls of the orbits.
Pletscher et al. t. who used R04-4046 to augment thc accumulation of [~4C]L-dopa in the brains of rats. The difference between the present work and that m rats may be due to m i n o r functional modifications produced by introducing 18F into the benzene rmg. We ourselves have shown that such changes are small: It is more likely that the divergence reflects species differences in the decarboxylating capacity of the endothelium of the cerebral capillaries(' The fact that reserpine discharged a p p r o x i m a t e l y half of the lSF that had accumulated in the striatum at 30 min is strong evidence to support the notion that [18F]6-fluoro-l,-dopa was stored, a~ its amine, in presynaptic vesicles. More tSF would p r o b a b l y have been discharged had we waited longer, but the physical half-life of ISF [110 mini precludes protracted studies. Positron emission t o m o g r a p h y has previously been used in an attempt to define the intracerebrat dislribution of the r e c e p t o r sites for benzodiazepine ~. These studies were confounded by the large fraction of the dose of p o s i t r o n - l a b e l e d drug that was bound. non-specifically, by reason of its lipophilicity to the brain and other tissues. Thus. when large doses of untabeled drug were given to b a b o o n s p r e t r e a t e d with [tlC]flunitrazepam the concentration of the labeled drug in the blood and the amount of label in the brain rose concomitantly, D o p a itself tins been labeled with c a r b o n - t l in the carboxyl groupv. W h e n the brains of rats into which this agent had been rejected were dissected the striatum contained less c a r b o n - l t than the surrounding brain. This might have been predicted from the known striatal localization of aromatic acid decarboxylase. The investigation has not been followed up. The present study has p r o v e d that a positron-labeled n e u r o t r a n s m i t t e r molecule will localize and can be visualized in a specific brain region. F u r t h e r , the concentration of transmitter in that region can be m a n i p ulated pharmacologically. Similar studies will be possible in man as soon as sufficient quantities of ~SF-labeled d o p a can be synthesized. It s h o u l d t h e n be possible to p r o b e directly the changes in d o p a m m e metabolism that are believed to be associated with disor-
171 ders of l o c o m o t i o n , such as P a r k i n s o n ' s d i s e a s e , a n d
da a n d t h e O n t a r i o M e n t a l H e a l t h F o u n d a t i o n for fi-
d i s o r d e r s of m o o d such as t h e s c h i z o p h r e n i c syn-
n a n c i a l s u p p o r t . W e a r e e s p e c i a l l y g r a t e f u l to Dr. J.
drome.
K u e h n e r a n d M r . J o h n M c K a y w h o p r o v i d e d acceler a t o r facilities for m a n u f a c t u r i n g lSF.
W c t h a n k t h e M e d i c a l R e s e a r c h C o u n c i l of C a n a -
1 Bartholini, G. and Plctscher, A., Cerebral accumulation and metabolism of HC-DOPA after selective inhibition of peripheral decarboxvlasc, J. Pharmacol. exp. Ther., i61 (19{18) 14-20. 2 Firnau, G., Oarnctt, E. S,, Sourkes, T. L. and Missala, K., ]l~Flfluorodopa: a unktuc gamma emitting substrate for dopa dccarboxvlasc, Experientia, 31 ( 1975 ) 1254-1255. 3 Firnau, G.. Shirakal, R., Sood, S. and Garnett. E. S., Aronmtic fluorination with Xcnon difluoride:L-3,4-dihydroxy6-fluor(~phenylalanine, ('anad. J. Chem., 58 (1980) 1449-14511. 4 Garnett, E. S., Firnau, G.. Nahmias, C.. Sood, S. and Belbeck, L., Blood brain barrier transport and cerebral utilization of dopa in monkeys, Amer. J. Physiol., 238 (1980) R3IS-R327. 5 (;arneu, E. S., Nahmias, ('., Kenyon, D. B. and Firnau, (i., A positron emission tomograph to study brain metabolism in man, J. ('anad. As',soc. Rad.. 33 (1982) 172-175, 6 Hardcbo, J. E. and Owman. C. N., Barrier mechanisms for ncurotransmittcr monoamines and their precursors in the blood brain interface, Ann. ,~'eurol., 8 (1980) 1-11. 7 ,hines, T., Cheslcr, D. A. and Ter Pogossian. M. M., 3"he continuous inhalation of oxygen-15 for assessing regional oxygen extraction in the brains of man, Brit. J. Radiol.. 49 (19761 339 343.
8 Maziere, M., Godot, J. M,, Berger, G., Baron, J. C., Comar, D., Cepeda, C., Benini, Ch. and Naquet, R., Positron tomography. A new method for in vivo brain studies of benzodiazepine in animal and man, Advanc. Biochem. Psychopharmacol.. 26 ( 1981 ) 273-286. 9 Reiffers, S., Beerling-van der Molen, H. D.. Vaalberg, W., Tenhoeve. W., Paans, A. M. J., Korf, .I., Woldring, M. G. and Wynberg, H., Rapid synthesis and purification of carbon-I 1 labelled DOPA: a potential agent for brain studies, Int. J. appl. Radial. lsol., 28 (1977) 955-t)58. i0 Reivich, M., Kuhl, D., Wolf, A., Greenberg..1., Phelps, M., Ido. T., Casella, V., Fowler, B., Gallagher, B., Hoffman, E., Alvari, A. and Sokoloff, L., Measurement of local cerebral glucose metabolism, Acta neurol..~cand.. 56 (Suppl) (1977) 19(>191. 11 Wurtman, R. J., Chou, C. and Rose, C., The fate of C 14Dihydroxyphenylalanine (CI4-DOPA) in the whole mousc. J. Pharm. exp. Ther.. 174 {197(})351-356. 12 Yamamoto, Y. L., Thompson, E., Meyer, E,, Nukul, H.. Matsunaga, M. and Feindel, W.. Three dimensional tomographical regional cerebral blood flow in man measured with high efficiency mini-BGO two ring positron device using Krypton 77, Acta neurol, stand.. 6(I (Suppl) (1979) 186-187.