Brain Research, 588 (1992) 261-269 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
261
BRES 17995
Changes in brain catecholamines and dopamine uptake sites at different stages of MPTP parkinsonism in monkeys G u i l l e r m o M. Alexander, R o b e r t J. Schwartzman, Laine Brainard, Sharon W. G o r d o n ,
and John R. Grothusen Department of Neurology, Jefferson Medical College, Philadelphia, PA 19107 (USA) (Accepted 24 March 1992)
Key words: Catecholamine; Dopamine uptake site; 1-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine; Parkinsonian primate
1-Methyi-4-,phenyl-l,2,3,6-tetrahydropyridine (MPTP) has been shown to produce parkinsonism in primates. We have studied the changes in brain catecholamines and the distribution of desipramine insensitive mazindol binding sites in MPTP parkinsonian primates at different levels of parkinsonism. Thirty-seven monkeys (Macaca fascicularis) were utilized in this study. Twelve naive animals received no treatment and served as controls. Twenty-five animals were rendered parkinsonian with serial injections of MPTP. All animals were Iliven scored neurologic examinations throughout the study. Their movement was quantitated in an activity box. The animals were sacrificed 30-360 days after their last MPTP injectlon. The clinical exam of the MPTP parkinsonian monkeys demonstrated mildly to severely affected animals. There was an exponential decrease in brain catecholamine levels with increased clinical parkinsonism. The MPTP parkinsonian animals showed the greatest decrease (67-99.8%) in tissue dopamine levels in the caudate nucleus. The putamen followed closely in severity (48-99.8%) and the nucleus accumbens was much less affected (0-40%). The percent reduction of norepinephrine in the anterior pole of the frontal cortex (0-48%) was similar in degree to the decreased dopamine levels in the nucleus accumhens. Mazindol binding was decreased 30-98% in the caudate nucleus, 20-97% in the putamen, 0-26% in the nucleus accumbens, 80-96% in the substantia nigra pars compacta and 49-94% in the ventral tegmental area. In the striatum, the decreased mazindol binding was more pronounced laterally and posteriorly. In each animal, there was good correlation between tissue dopamine levels and the number of mazindol binding sites.
INTRODUCTION
The model of Parkinson's disease produced by the intravenous administration of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) is now well established in man and non-human primates 5-7'~3'~4. The most striking pathological feature of this model is the loss of dopaminergic neurons in the pars compacta of the substantia nigra (SNpc) with a concomitant reduction of dopamine and dopamine uptake sites in the caudate nucleus and putamen 6'9'11'16. MPTP parkinsonism in primates also results in the loss of dopaminergic cells in the ventral tegmcntal area (VTA) and retrorubral field 15'2° as well as noradrenergic neurons in the locus coeruleus 1°'~5. This study examines the loss of dopamin¢ in the caudate nucleus, putamen and nucleus accumbens as
well as the loss of norepinephrine in ~he anterior pole of the frontal cortex in monkeys with different degrees of MPTP parkinsonism. In some of these animals, we have also examined the changes in the pattern of dopamine uptake sites in the caudate nucleus, putamen, nucleus accumbens, SNpc and VTA. MATERIALS AND METHODS Thirty-seven male monkeys (Macaca fascicularis)were utilized in this study. Twelve animals received no MPTP treatment and served as controls. Twenty-five animals were rendered parkinsonian with daily injections (0.25-0.50 mg/kg) of MPTP (Research Biochemicals, Natick, MA). The number of injections and the total amount of MPTP given each animal are tabulated in Table II. These animals have been used in our laboratory for various studies during the past five years l'2'4'8'tT"2t- 24. Some data was not available in some animals; missing data appears as a period in the tables. As part of an ongoing study, two controls and five MPTP monkeys received chronic LDOPA treatment (25/100 mg carbidopa/L-DOPA) twice a day.
Correspondence: G.M. Alexander, Department of Neurology, Jefferson Medical College, 1025 Walnut Street, Philadelphia, PA 19107, USA. Fax: (1)(215) 955-5515.
262 TABLE !
Brain catecholamines in control monkeys C a t e c h o l a m i n e levels are in n a n o m o l e s p e r g r a m o f tissue wet weight. ( + ) Chronic L - D O P A t r e a t m e n t (25/1.00 mg. c a r b i d o p a / L - D O P A ) twice a day.
Animal number
Caudate nucleus dopamine (n -~ 10)
Putamen dopamine (n = I0)
Nucleus accumberts dopamine (n = 6)
Anterior cortex norepinephrine ~n -~ 6)
M76 + M73 M67 M38 M69 M68 M36 BMI2
81.700 80.600 73.400 79.100 71.200 79.800 62.400 62.400
110.000 81.400 86.400 73.400 79.900 61.300 -
37.500 32.100 57.300 43.000 32.600 -
0.819 0.881 0.724 0.956 1.293 -
,~o ~n,~ ~o.Ju u
'"
Clinical score (n-- 12)
.
M72 +
~u . ' -Ar~ ~ ~u
,u ~ -,~r~ J.~u u
M34 BMI0 M37
53.500 -
64.100 50.400 47.000
M e a n + S.E.M.
70.05 + 3.3
71.91 + 5.9
n
0.000 1.000 0.O00 0.000 0.000 0.000 0.000 0.000
0.648
0.000
-
-
0.000
-
-
0.500 0.500
0.89 + 0.09
0.33 + 0.09
38.46 + 4.30
S o m e d a t a from these animals is included in this study. N e u r o l o g i c a l e x a m i n a t i o n s were p e r f o r m e d on all animals prior to M P T P injections and once a w e e k a f t e r M P T P injections.
T h e n e u r o l o g i c e x a m i n a t i o n e v a l u a t e d cranial n e r v e function, muscle t o n e , s e n s o r i m o t o r integration, t r e m o r , fine m o v e m e n t a n d posture. T h e cranial nerve e x a m i n a t i o n e v a l u a t e d eye m o v e m e n t s ,
T A B L E II
Brain catecholamines in MPTP parkinsonian monkeys C a t e c h o l a m i n e levels are in n a n o m o l e s p e r g r a m of tissue wet weight. ( + ) Chronic L - D O P A t r e a t m e n t ( 2 5 / 1 0 0 rag. c a r b i d o p a / L - D O P A ) twice a day. C a u d a t e and p u t a m e n d o p a m i n e levels are significantly lower than controls P < 0.0001 ( • ) . In m o n k e y s with clinical scores g r e a t e r t h a n 4, the nucleus a c c u m b e n s d o p a m i n e levels are significantly lower t h a n controls P < 0.05 ( * ) a n d the a n t e r i o r c o r t e x n o r e p i n e p h r i n e levels a r e significantly lower than controls P < 0.01 ( * * ) ,
Animal number
Caudate nucleus dopamine (n = 22)
Putamen dopamine (n = 22)
Nucleus accumbens dopamine (n = 5)
Anterior cortex norepinephrine (n = 6)
M57 M48 M42 M56 M54 M44 M52 M51 M50 M47 M63 M64 M66 M74 + M70 + M53 M49 M46 M55 M60
21,200 17.300 22,700 ! 3.400 11.000 7.600 3.800 1.300 1.060 1.730 2.790 1.120 1.060 0.890 0.920 0.620 0.550 0.510 0.790
36,900 29.600 . 18.200 16,000 9,800 11.200 3,070 4.970 5.200 3.000 1.930 2.510 1.910 2.020 1.820 1.440 1.190 1.110 0.610
42.900 -
0.925 -
* * * * * • • • * • * • • • • • • • •
* * * * ¢ • • * • * * • • • • • • • •
.
. 26.100 * 30.420 * 23.100 * -
Clinical score (n = 18) 2.000 2.500
. 1.010 0.489 * * 0.546 * * 0.560 * * _ _ _
3.500 5.500 _ 7.500 _ 9.000 5.500 10.500 6.000 8.000 11.000 4.500 14.500 11.000 14.500 12.000 _ 13.500 _ 14.500
M71 + M61 M62
0.170 • 0.260 • 0.170 •
0.760 • 0.280 • 0.140 •
23.900 * -
0.447 * * _ _
M e a n + S.E.M.
5.04-t-1.5
6.99+2.1
29.28+3.63
0.66+0.10
8.63:!:1.00
263 vocalization, swallowing, and facial movements. Sensorimotor integration was tested by examining touch-placing, grasp avoidance, and threat response. Tremor was rated on severity and the number of extremities involved. Free activity in these animals was measured with a primate activity box designed in our laboratory. The box utilizes two infrared emitter-sensor pairs. The number of times the infrared light beams are interrupted is recorded oil an IBM-XT computer. Each animal was evaluated for a 24 hour period at least twice prior to MPTP injections. For free movement evaluation each parkinsonian monkey was compared to his own pre-parkinsonian level The free movement score was assigned a value between 0 and 4: a reduction in free movement greater than 90% = 4.0; 80-89% = 3.5; 70-79% = 3.D; 60-69% = 2.5; 50-59% ~ 2.0; 40-49% = 1.5; 30-39% - 1.0; 20-29% --0.5; and 19% or less = 0.0. A composite neurological score was obtained using free movement and six clinical parameters: bradykinesia, face and eye movements, tremor, muscle tone, sensorimotor integration, and posture. Each clinical parameter was assigned a value between 0 and 2: zero was unaffected; 0.5 was minimally affected; 1.0 was mildly affected; 1.5 was moderately affected; and 2.0 was severely affected. The composite score range was 0 to 16, with zero being normal in all calegories and 16 being severely affected in all categories. The animals were sacrificed by an intravenous overdose of sodium pentobarbital. The brain was removed quickly (less than 10 min after sacrifice), frozen in isopentane cooled with dry ice to -35°C, then stored at -70°C. For ligand binding assays the brain was sectioned at 20/zm thickness in a cr,jostat at - 12°C. The sections were picked up on subbed glass slides, dried for 10 min on a warming plate at 40°C, and stored at -20°C. Dopamine uptake sites were labeled with [3H]mazindol in the presence of desipramine n. Mazindol binding assays were performed on 2 0 / z m thick brain sections from 6 naive animals and 7 MPTP parkinsonian monkeys (there were no anterior sections for M65, and no posterior sections for M57). Two of the MPTP monkeys were mildly parkinsonian, one was moderately affected and four were severely affected. These animals were sacrificed 6 months after the last MPTP injection. The brain sections were taken from approximately A20 (20 mm anterior to the internal auditory meatus) and A l l (11 mm anterior to the internal auditory meatus) according to the Szabo and Cowan brain atlas 2s. The 20/zm sections were given two 5-min pre-washes at 4°C in 50 mM Tris buffer pH = 7.9. Total binding was determined using various concentrations (1.0-8.0 n M) of [3H]mazindol (New England Nuclear, Boston, MA) in buffer (50 mM Tris, 300 mM NaCI, 5 mM KCI, pH ~ 7.9) containing 50 nM desipramine (Research Biochemicals, Natick, MA) for 40 min at 40C. Non-specific binding was determined in adjacent sections incubated in buffer containing 10 /zM nomifensine (Research Biochemicals, Natick, MA) t !. After incubation, the sections were given two 1-min post-washes in 40C 50 mM Tris buffer pH--7.9, rinsed in 4°C distilled water (2 dips), dried with cold dry nitrogen and placed on a warming plate for 1 h at 400C. The sections were then placed m light-tight cassettes against Hyperfilm-3H (Amersham, Arlington Heights, IL) along with plastic standards for 17 days. Tissue isotope concentration was determined from the autoradiographs with computer-assisted densitometry 3. Tissue catecholamine levels were determined from punch samples (10-20 mg wet wt.) of the caudate nucleus, putamen, nucleus accumbens and the anterior pole of the frontal cortex. The samples were homogenized in 300/Li of 0.4 M perchloric acid and were then centrifuged. Aliquots of the supernatant were analyzed directly by reverse phase high performance liquid chromatography (HPLC) utilizing electrochemical detection. Chromatography was performed on a Zorbax ODS (4.6 m m x 2 5 cm, DuPont Co., Wilmington, DE) column with a mobile phase consisting of 11% methanol in buffer (50 mM sodium phosphate, 0.2 mM disodium EDTA and 2.5 mM heptanesuifonic acid, pH 3.4). The detector was an ESA model 5100A (ESA, Bedford, MA). Statistical significance between controls and MPTP parkinsonian monkeys was determined by the Student's t-test. Association between variables was evaluated by regression using the method of
least squares. Correlation between variables was estimated by Pearson's product moment (correlation coefficient).
RESULTS
The brain catechoiamine levels and the clinical scores for both naive and MPTP parkinsonian monkeys are tabulated in Tables 1 and II. In the twelve control monkeys the clinical score ranged from 0 to 1. In the two control monkeys (M72 and M76) chronic L-DOPA treatment did not change their clinical score from its value prior to treatment. Nine animals had scores of 0, two had scores of 0.5, and one had a score of 1. The five MPTP parkinsonian monkeys that received chronic L-DOPA treatment demonstrated clinical inprovement (unpublished information). The clinical score used for the five chronic L-DOPA treated MPTP monkeys was their score just prior to L-DOPA treatment. The MPTP monkeys had clinical scores that ranged between 2 and 14.5. These monkeys fell into three broad clinical categories: mildly parkinsonian (clinical score 2-5), moderately parkinsonian (clinical score 6-10), and severely parkinsonian (clinical score greater than 10). Monkeys with clinical scores of 3 or less were almost asymptomatic; a reduction in free movement from pre-MPTP levels and mild bradykinesia distinguished these animals from the controls. The MPTP monkeys showed reduced tissue catecholamine levels in all areas studied. There was a statistically significant reduction in tissue dopamine levels in the caudate nucleus (P < 0.001) and putamen (P < 0.001). The ratio of putamen vs. caudate nucleus tissue dopamine levels (in animals where both were available) was significantly greater (P < 0.05) in the MPTP parkinsonian monkeys as compared to controls (2.16 vs. 1.08). There was an exponential decrease in tissue catecholamine levels with increased parkinsonism. There was good correlation on least square0 exponential regression ( r - 0.82 caudate, r = 0.83 putamen) between clinical score and striatal dopamine levels (Fig. 1). In animals with clinical scores greater than 4, the dopamine levels in the nucleus accumbens and the norepinephrine levels in the frontal cortex were also significantly reduced (P < 0.05 and P < 0.02, respectively). There was also a correlation on least squared exponential regression (r = 0.57 nuc. accumbens, r = 0.67 frontal cortex) between clinical score and the reduction of dopamine levels in the nucleus accumbens and norepinephrine levels in the anterior pole of the frontal cortex (Fig. 2). However, animals with clinical scores greater than 4 demonstrated little further decrease in accumbens dopamine levels of frontal cortex norepinephrine levels.
264 TABLE Iii
Mazindol binding (anterior) Values are the maximal binding (Bmax) of mazindoi in picomoles per gram of tissue ± S.E.M. Values for the equilibrium binding affinity constant (Kd) are in nanomolar, DL, dorso-lateral; VL, ventro-lateral; DM, dorso-medial; VM, ventro-medial; MVM, most ventro-medial; MM, most medial. The MPTP Parkinsonian group is sub-divided by level of severity into mild, moderate and severe. The two mild parkinsonian monkeys were not combined because the tissue sections from monkey Mild 2 were from a slightly more posterior area (approximately AI8 in the atlas) 25. At this level the nucleus accumbens and the olfactory tubercle are not present.
Structure
Caudate DL Caudate VL Caudate DM Caudate VM Caudate MVM Putamen DL Putamen VL Putamen DM Putamen VM Put•men MM Nuc. accumbens Oil. tubercle Affinity const. (K d)
Controls (n = 6) 587.1 + 24.4 557.6+25.8 640.2 4. 22.2 583.4 + 19.0 434.3 + 29.9 603.0 + 31.9 590.4 + 32.9 551.1 ± 23.1 510.6 + 17.7 406.9 + 17.5 304.8 ± 36.1 298.2 ± 22.2 5.7± 0.4
MPTP Parkinsonian
Mild1
Mild z
Moderate
Severe
(n = 6)
(n = 1)
(n = 1)
(n = 1)
(n = 3)
238.5 279.4 266.6 307.3 242.9 335.9 359.3 374.4 361.9 272.2 232.5 187.9
84.7 138.1 297.9 336.0 176.8 368.1 319.7 423.6 346.3 379.7 -
72.6 90.4 123.7 138.4 212.1 84.6 105.4 114.1 136.7 155.5 149.8 141.1
25.5 + 4.1 26.0+ 5.6 39.8 + 8.8 46.4 + 10.8 168.9 + 24.8 30.7 4. 7.8 61.9 + 24.4 43.5 + 21.3 75.0 ± 25.8 140.7 ± 16.4 172.2 ± 15.9 130.9 4- 23.8
78.7+ 33.8 97.7+41.0 134.6 + 48.9 153.5 + 55.5 218.0 + 30.6 114.9 + 50.1 169.8 + 62.7 157.3 + 61.7 191.2 4. 66.0 204.9 + 41.4 179.8 4.16.4 144.4 ± 17.1 4.6± 0.2
• • • • • • * * • • ** *
• P < 0.001. ** P < 0.01.
The maximum number of mazindol binding sites (Bma x) and its equilibrium binding affinity constant
(K d) were determined by Scatchard analysis (Fig. 3). The results of these studies are tabulated in Tables Ill and IV. In the six control animals, there was no statistical difference ( P > 0 . 1 ) in Bmax between the two chronic ,-DOPA-treated monkeys and the four naive controls, therefore all six animals were used as the control group. There was a statistically significant decrease (P < 0.01) in Bmax in the caudate nucleus, putamen, nucleus accumbens, olfactory tubercle, SNpc and
VTA in the MPTP parkinsonian monkeys as compared tO controls. There was no change in the K d for mazindol between the two groups. In the anterior sections, the decrease in striatal mazindol binding was most pronounced dorsolaterally, with the caudate nucleus more affected than the putamen. The most ventromedial caudate (Cmvm) and the most medial putamen (Prom) were relatively spared even in the severely affected animals, while dorsolateral parts of the nuclei showed significant reductions in mazindol binding, even in the mildly and moderately parkinsonian monkeys
TABLE IV Mazindol binding (posterior) Values are the maximal binding (Bm~) of mazindol in picomoles per gram of tissue 4- S.E.M. The MPTP Parkinsonian group is sub-divided by level of severity into mild, moderate and severe. Values for the equilibrium binding affinity constant (K d) are in nanomolar, DL, dorso-lateral; VL, ventro-lateral; DM, dorso-medial; VM, ventro-medial.
Structure
Caudate DL Caudate VL Caudate DM Caudate VM Caudate tail Putamen DL Putamen VL Putamen DM Putamen VM SNpc lateral SNpc ventral Vent. reg. area Affinity const.(Kd)
P
261
BRES 17995
Changes in brain catecholamines and dopamine uptake sites at different stages of MPTP parkinsonism in monkeys G u i l l e r m o M. Alexander, R o b e r t J. Schwartzman, Laine Brainard, Sharon W. G o r d o n ,
and John R. Grothusen Department of Neurology, Jefferson Medical College, Philadelphia, PA 19107 (USA) (Accepted 24 March 1992)
Key words: Catecholamine; Dopamine uptake site; 1-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine; Parkinsonian primate
1-Methyi-4-,phenyl-l,2,3,6-tetrahydropyridine (MPTP) has been shown to produce parkinsonism in primates. We have studied the changes in brain catecholamines and the distribution of desipramine insensitive mazindol binding sites in MPTP parkinsonian primates at different levels of parkinsonism. Thirty-seven monkeys (Macaca fascicularis) were utilized in this study. Twelve naive animals received no treatment and served as controls. Twenty-five animals were rendered parkinsonian with serial injections of MPTP. All animals were Iliven scored neurologic examinations throughout the study. Their movement was quantitated in an activity box. The animals were sacrificed 30-360 days after their last MPTP injectlon. The clinical exam of the MPTP parkinsonian monkeys demonstrated mildly to severely affected animals. There was an exponential decrease in brain catecholamine levels with increased clinical parkinsonism. The MPTP parkinsonian animals showed the greatest decrease (67-99.8%) in tissue dopamine levels in the caudate nucleus. The putamen followed closely in severity (48-99.8%) and the nucleus accumbens was much less affected (0-40%). The percent reduction of norepinephrine in the anterior pole of the frontal cortex (0-48%) was similar in degree to the decreased dopamine levels in the nucleus accumhens. Mazindol binding was decreased 30-98% in the caudate nucleus, 20-97% in the putamen, 0-26% in the nucleus accumbens, 80-96% in the substantia nigra pars compacta and 49-94% in the ventral tegmental area. In the striatum, the decreased mazindol binding was more pronounced laterally and posteriorly. In each animal, there was good correlation between tissue dopamine levels and the number of mazindol binding sites.
INTRODUCTION
The model of Parkinson's disease produced by the intravenous administration of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) is now well established in man and non-human primates 5-7'~3'~4. The most striking pathological feature of this model is the loss of dopaminergic neurons in the pars compacta of the substantia nigra (SNpc) with a concomitant reduction of dopamine and dopamine uptake sites in the caudate nucleus and putamen 6'9'11'16. MPTP parkinsonism in primates also results in the loss of dopaminergic cells in the ventral tegmcntal area (VTA) and retrorubral field 15'2° as well as noradrenergic neurons in the locus coeruleus 1°'~5. This study examines the loss of dopamin¢ in the caudate nucleus, putamen and nucleus accumbens as
well as the loss of norepinephrine in ~he anterior pole of the frontal cortex in monkeys with different degrees of MPTP parkinsonism. In some of these animals, we have also examined the changes in the pattern of dopamine uptake sites in the caudate nucleus, putamen, nucleus accumbens, SNpc and VTA. MATERIALS AND METHODS Thirty-seven male monkeys (Macaca fascicularis)were utilized in this study. Twelve animals received no MPTP treatment and served as controls. Twenty-five animals were rendered parkinsonian with daily injections (0.25-0.50 mg/kg) of MPTP (Research Biochemicals, Natick, MA). The number of injections and the total amount of MPTP given each animal are tabulated in Table II. These animals have been used in our laboratory for various studies during the past five years l'2'4'8'tT"2t- 24. Some data was not available in some animals; missing data appears as a period in the tables. As part of an ongoing study, two controls and five MPTP monkeys received chronic LDOPA treatment (25/100 mg carbidopa/L-DOPA) twice a day.
Correspondence: G.M. Alexander, Department of Neurology, Jefferson Medical College, 1025 Walnut Street, Philadelphia, PA 19107, USA. Fax: (1)(215) 955-5515.
262 TABLE !
Brain catecholamines in control monkeys C a t e c h o l a m i n e levels are in n a n o m o l e s p e r g r a m o f tissue wet weight. ( + ) Chronic L - D O P A t r e a t m e n t (25/1.00 mg. c a r b i d o p a / L - D O P A ) twice a day.
Animal number
Caudate nucleus dopamine (n -~ 10)
Putamen dopamine (n = I0)
Nucleus accumberts dopamine (n = 6)
Anterior cortex norepinephrine ~n -~ 6)
M76 + M73 M67 M38 M69 M68 M36 BMI2
81.700 80.600 73.400 79.100 71.200 79.800 62.400 62.400
110.000 81.400 86.400 73.400 79.900 61.300 -
37.500 32.100 57.300 43.000 32.600 -
0.819 0.881 0.724 0.956 1.293 -
,~o ~n,~ ~o.Ju u
'"
Clinical score (n-- 12)
.
M72 +
~u . ' -Ar~ ~ ~u
,u ~ -,~r~ J.~u u
M34 BMI0 M37
53.500 -
64.100 50.400 47.000
M e a n + S.E.M.
70.05 + 3.3
71.91 + 5.9
n
0.000 1.000 0.O00 0.000 0.000 0.000 0.000 0.000
0.648
0.000
-
-
0.000
-
-
0.500 0.500
0.89 + 0.09
0.33 + 0.09
38.46 + 4.30
S o m e d a t a from these animals is included in this study. N e u r o l o g i c a l e x a m i n a t i o n s were p e r f o r m e d on all animals prior to M P T P injections and once a w e e k a f t e r M P T P injections.
T h e n e u r o l o g i c e x a m i n a t i o n e v a l u a t e d cranial n e r v e function, muscle t o n e , s e n s o r i m o t o r integration, t r e m o r , fine m o v e m e n t a n d posture. T h e cranial nerve e x a m i n a t i o n e v a l u a t e d eye m o v e m e n t s ,
T A B L E II
Brain catecholamines in MPTP parkinsonian monkeys C a t e c h o l a m i n e levels are in n a n o m o l e s p e r g r a m of tissue wet weight. ( + ) Chronic L - D O P A t r e a t m e n t ( 2 5 / 1 0 0 rag. c a r b i d o p a / L - D O P A ) twice a day. C a u d a t e and p u t a m e n d o p a m i n e levels are significantly lower than controls P < 0.0001 ( • ) . In m o n k e y s with clinical scores g r e a t e r t h a n 4, the nucleus a c c u m b e n s d o p a m i n e levels are significantly lower t h a n controls P < 0.05 ( * ) a n d the a n t e r i o r c o r t e x n o r e p i n e p h r i n e levels a r e significantly lower than controls P < 0.01 ( * * ) ,
Animal number
Caudate nucleus dopamine (n = 22)
Putamen dopamine (n = 22)
Nucleus accumbens dopamine (n = 5)
Anterior cortex norepinephrine (n = 6)
M57 M48 M42 M56 M54 M44 M52 M51 M50 M47 M63 M64 M66 M74 + M70 + M53 M49 M46 M55 M60
21,200 17.300 22,700 ! 3.400 11.000 7.600 3.800 1.300 1.060 1.730 2.790 1.120 1.060 0.890 0.920 0.620 0.550 0.510 0.790
36,900 29.600 . 18.200 16,000 9,800 11.200 3,070 4.970 5.200 3.000 1.930 2.510 1.910 2.020 1.820 1.440 1.190 1.110 0.610
42.900 -
0.925 -
* * * * * • • • * • * • • • • • • • •
* * * * ¢ • • * • * * • • • • • • • •
.
. 26.100 * 30.420 * 23.100 * -
Clinical score (n = 18) 2.000 2.500
. 1.010 0.489 * * 0.546 * * 0.560 * * _ _ _
3.500 5.500 _ 7.500 _ 9.000 5.500 10.500 6.000 8.000 11.000 4.500 14.500 11.000 14.500 12.000 _ 13.500 _ 14.500
M71 + M61 M62
0.170 • 0.260 • 0.170 •
0.760 • 0.280 • 0.140 •
23.900 * -
0.447 * * _ _
M e a n + S.E.M.
5.04-t-1.5
6.99+2.1
29.28+3.63
0.66+0.10
8.63:!:1.00
263 vocalization, swallowing, and facial movements. Sensorimotor integration was tested by examining touch-placing, grasp avoidance, and threat response. Tremor was rated on severity and the number of extremities involved. Free activity in these animals was measured with a primate activity box designed in our laboratory. The box utilizes two infrared emitter-sensor pairs. The number of times the infrared light beams are interrupted is recorded oil an IBM-XT computer. Each animal was evaluated for a 24 hour period at least twice prior to MPTP injections. For free movement evaluation each parkinsonian monkey was compared to his own pre-parkinsonian level The free movement score was assigned a value between 0 and 4: a reduction in free movement greater than 90% = 4.0; 80-89% = 3.5; 70-79% = 3.D; 60-69% = 2.5; 50-59% ~ 2.0; 40-49% = 1.5; 30-39% - 1.0; 20-29% --0.5; and 19% or less = 0.0. A composite neurological score was obtained using free movement and six clinical parameters: bradykinesia, face and eye movements, tremor, muscle tone, sensorimotor integration, and posture. Each clinical parameter was assigned a value between 0 and 2: zero was unaffected; 0.5 was minimally affected; 1.0 was mildly affected; 1.5 was moderately affected; and 2.0 was severely affected. The composite score range was 0 to 16, with zero being normal in all calegories and 16 being severely affected in all categories. The animals were sacrificed by an intravenous overdose of sodium pentobarbital. The brain was removed quickly (less than 10 min after sacrifice), frozen in isopentane cooled with dry ice to -35°C, then stored at -70°C. For ligand binding assays the brain was sectioned at 20/zm thickness in a cr,jostat at - 12°C. The sections were picked up on subbed glass slides, dried for 10 min on a warming plate at 40°C, and stored at -20°C. Dopamine uptake sites were labeled with [3H]mazindol in the presence of desipramine n. Mazindol binding assays were performed on 2 0 / z m thick brain sections from 6 naive animals and 7 MPTP parkinsonian monkeys (there were no anterior sections for M65, and no posterior sections for M57). Two of the MPTP monkeys were mildly parkinsonian, one was moderately affected and four were severely affected. These animals were sacrificed 6 months after the last MPTP injection. The brain sections were taken from approximately A20 (20 mm anterior to the internal auditory meatus) and A l l (11 mm anterior to the internal auditory meatus) according to the Szabo and Cowan brain atlas 2s. The 20/zm sections were given two 5-min pre-washes at 4°C in 50 mM Tris buffer pH = 7.9. Total binding was determined using various concentrations (1.0-8.0 n M) of [3H]mazindol (New England Nuclear, Boston, MA) in buffer (50 mM Tris, 300 mM NaCI, 5 mM KCI, pH ~ 7.9) containing 50 nM desipramine (Research Biochemicals, Natick, MA) for 40 min at 40C. Non-specific binding was determined in adjacent sections incubated in buffer containing 10 /zM nomifensine (Research Biochemicals, Natick, MA) t !. After incubation, the sections were given two 1-min post-washes in 40C 50 mM Tris buffer pH--7.9, rinsed in 4°C distilled water (2 dips), dried with cold dry nitrogen and placed on a warming plate for 1 h at 400C. The sections were then placed m light-tight cassettes against Hyperfilm-3H (Amersham, Arlington Heights, IL) along with plastic standards for 17 days. Tissue isotope concentration was determined from the autoradiographs with computer-assisted densitometry 3. Tissue catecholamine levels were determined from punch samples (10-20 mg wet wt.) of the caudate nucleus, putamen, nucleus accumbens and the anterior pole of the frontal cortex. The samples were homogenized in 300/Li of 0.4 M perchloric acid and were then centrifuged. Aliquots of the supernatant were analyzed directly by reverse phase high performance liquid chromatography (HPLC) utilizing electrochemical detection. Chromatography was performed on a Zorbax ODS (4.6 m m x 2 5 cm, DuPont Co., Wilmington, DE) column with a mobile phase consisting of 11% methanol in buffer (50 mM sodium phosphate, 0.2 mM disodium EDTA and 2.5 mM heptanesuifonic acid, pH 3.4). The detector was an ESA model 5100A (ESA, Bedford, MA). Statistical significance between controls and MPTP parkinsonian monkeys was determined by the Student's t-test. Association between variables was evaluated by regression using the method of
least squares. Correlation between variables was estimated by Pearson's product moment (correlation coefficient).
RESULTS
The brain catechoiamine levels and the clinical scores for both naive and MPTP parkinsonian monkeys are tabulated in Tables 1 and II. In the twelve control monkeys the clinical score ranged from 0 to 1. In the two control monkeys (M72 and M76) chronic L-DOPA treatment did not change their clinical score from its value prior to treatment. Nine animals had scores of 0, two had scores of 0.5, and one had a score of 1. The five MPTP parkinsonian monkeys that received chronic L-DOPA treatment demonstrated clinical inprovement (unpublished information). The clinical score used for the five chronic L-DOPA treated MPTP monkeys was their score just prior to L-DOPA treatment. The MPTP monkeys had clinical scores that ranged between 2 and 14.5. These monkeys fell into three broad clinical categories: mildly parkinsonian (clinical score 2-5), moderately parkinsonian (clinical score 6-10), and severely parkinsonian (clinical score greater than 10). Monkeys with clinical scores of 3 or less were almost asymptomatic; a reduction in free movement from pre-MPTP levels and mild bradykinesia distinguished these animals from the controls. The MPTP monkeys showed reduced tissue catecholamine levels in all areas studied. There was a statistically significant reduction in tissue dopamine levels in the caudate nucleus (P < 0.001) and putamen (P < 0.001). The ratio of putamen vs. caudate nucleus tissue dopamine levels (in animals where both were available) was significantly greater (P < 0.05) in the MPTP parkinsonian monkeys as compared to controls (2.16 vs. 1.08). There was an exponential decrease in tissue catecholamine levels with increased parkinsonism. There was good correlation on least square0 exponential regression ( r - 0.82 caudate, r = 0.83 putamen) between clinical score and striatal dopamine levels (Fig. 1). In animals with clinical scores greater than 4, the dopamine levels in the nucleus accumbens and the norepinephrine levels in the frontal cortex were also significantly reduced (P < 0.05 and P < 0.02, respectively). There was also a correlation on least squared exponential regression (r = 0.57 nuc. accumbens, r = 0.67 frontal cortex) between clinical score and the reduction of dopamine levels in the nucleus accumbens and norepinephrine levels in the anterior pole of the frontal cortex (Fig. 2). However, animals with clinical scores greater than 4 demonstrated little further decrease in accumbens dopamine levels of frontal cortex norepinephrine levels.
264 TABLE Iii
Mazindol binding (anterior) Values are the maximal binding (Bmax) of mazindoi in picomoles per gram of tissue ± S.E.M. Values for the equilibrium binding affinity constant (Kd) are in nanomolar, DL, dorso-lateral; VL, ventro-lateral; DM, dorso-medial; VM, ventro-medial; MVM, most ventro-medial; MM, most medial. The MPTP Parkinsonian group is sub-divided by level of severity into mild, moderate and severe. The two mild parkinsonian monkeys were not combined because the tissue sections from monkey Mild 2 were from a slightly more posterior area (approximately AI8 in the atlas) 25. At this level the nucleus accumbens and the olfactory tubercle are not present.
Structure
Caudate DL Caudate VL Caudate DM Caudate VM Caudate MVM Putamen DL Putamen VL Putamen DM Putamen VM Put•men MM Nuc. accumbens Oil. tubercle Affinity const. (K d)
Controls (n = 6) 587.1 + 24.4 557.6+25.8 640.2 4. 22.2 583.4 + 19.0 434.3 + 29.9 603.0 + 31.9 590.4 + 32.9 551.1 ± 23.1 510.6 + 17.7 406.9 + 17.5 304.8 ± 36.1 298.2 ± 22.2 5.7± 0.4
MPTP Parkinsonian
Mild1
Mild z
Moderate
Severe
(n = 6)
(n = 1)
(n = 1)
(n = 1)
(n = 3)
238.5 279.4 266.6 307.3 242.9 335.9 359.3 374.4 361.9 272.2 232.5 187.9
84.7 138.1 297.9 336.0 176.8 368.1 319.7 423.6 346.3 379.7 -
72.6 90.4 123.7 138.4 212.1 84.6 105.4 114.1 136.7 155.5 149.8 141.1
25.5 + 4.1 26.0+ 5.6 39.8 + 8.8 46.4 + 10.8 168.9 + 24.8 30.7 4. 7.8 61.9 + 24.4 43.5 + 21.3 75.0 ± 25.8 140.7 ± 16.4 172.2 ± 15.9 130.9 4- 23.8
78.7+ 33.8 97.7+41.0 134.6 + 48.9 153.5 + 55.5 218.0 + 30.6 114.9 + 50.1 169.8 + 62.7 157.3 + 61.7 191.2 4. 66.0 204.9 + 41.4 179.8 4.16.4 144.4 ± 17.1 4.6± 0.2
• • • • • • * * • • ** *
• P < 0.001. ** P < 0.01.
The maximum number of mazindol binding sites (Bma x) and its equilibrium binding affinity constant
(K d) were determined by Scatchard analysis (Fig. 3). The results of these studies are tabulated in Tables Ill and IV. In the six control animals, there was no statistical difference ( P > 0 . 1 ) in Bmax between the two chronic ,-DOPA-treated monkeys and the four naive controls, therefore all six animals were used as the control group. There was a statistically significant decrease (P < 0.01) in Bmax in the caudate nucleus, putamen, nucleus accumbens, olfactory tubercle, SNpc and
VTA in the MPTP parkinsonian monkeys as compared tO controls. There was no change in the K d for mazindol between the two groups. In the anterior sections, the decrease in striatal mazindol binding was most pronounced dorsolaterally, with the caudate nucleus more affected than the putamen. The most ventromedial caudate (Cmvm) and the most medial putamen (Prom) were relatively spared even in the severely affected animals, while dorsolateral parts of the nuclei showed significant reductions in mazindol binding, even in the mildly and moderately parkinsonian monkeys
TABLE IV Mazindol binding (posterior) Values are the maximal binding (Bm~) of mazindol in picomoles per gram of tissue 4- S.E.M. The MPTP Parkinsonian group is sub-divided by level of severity into mild, moderate and severe. Values for the equilibrium binding affinity constant (K d) are in nanomolar, DL, dorso-lateral; VL, ventro-lateral; DM, dorso-medial; VM, ventro-medial.
Structure
Caudate DL Caudate VL Caudate DM Caudate VM Caudate tail Putamen DL Putamen VL Putamen DM Putamen VM SNpc lateral SNpc ventral Vent. reg. area Affinity const.(Kd)
P
