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Alterations in dopaminergic receptors in Huntington's disease
Life Sciences, Vol . 21, pp . 1123-1128 Printed in the U .S .A .
Pergamon Press
ALTERATIONS IN DOPAMINERGIC RECEPTORS IN HUNTINGTON'S DISEASE
T .D . Reisine, J .Z . Fields, L .Z . Stern* P .C . Johnson**, E .D . Birdl, and H .I . Yamamura Departments of Pharmacology, Neurology* and Pathology** College of Medicine, University of Arizona Health Sciences Center, Tucson, Arizona 85724 and 'Department of Neurological Surgery and Neurology, Addenbrookes Hospital, Cambridge, England (Received in final form September 2,
1977)
SUMMARY To detect variations in dopaminergic receptors and cholinergic activity in regions of postmortem Huntington's diseased brains, 3Hspiroperidol binding assays and choline acetyltransferase (ChAc) activities were carried out . A significant reduction in 3H-spiroperidol binding in the caudate nucleus, putamen and frontal cortex of choreic brains was detected which appeared to be due to a decrease in the total number of binding sites rather than to a decrease in affinity of 3H-spiroperidol for the dopaminergic receptor . In choreic brains, there were also significant reductions in ChAc activity in the caudate nucleus and putamen . The decreases of both 3 H-spiroperidol binding and ChAc activity in the neostriatum suggest that the dopaminergic receptors are localized postsynaptically on cholinergic interneurons . Dopaminergic receptor alterations in the basal ganglia may be one of the causes of the abnormal motor movements found in HD while alterations of these receptors in the frontal cortex may be associated with the neuronal degeneration found in that area of choreic brains . INTRODUCTION Huntington's Disease (HD) is an autosomal dominant neurological disorder that is characterized by extrapyramidal motor abnormalities and dementia (1) . Histological evidence indicates that there are neuronal losses in the basal ganglia particularly in the caudate nucleus and cerebral cortex (2-5) . The neuronal loss and resultant atrophy observed in the basal ganglia may be the cause of the movement disorders typical of the disease . Previous neurochemical studies showed a 50% reduction in choline acetyltransferase (ChAc) activity (6,7) and an 85% decrease in glutamic acid decarboxylase (GAD) activity in the basal ganglia of choreic brains (6,8) . Since the activities of these enzymes serve as markers for cholinergic and GABAergic neurons, respectively, the decline in enzyme activities suggests a selective loss of these neurons . In previous studies, changes in neurotransmitter receptor binding were also examined (7,9,11) in the frontal cortex and basal ganglia of postmortem choreic brains . In the frontal cortex of choreic brains no changes were found in the densities of 9-adrenergic, GABAergic, muscarinic cholinergic or serotonergic receptors (9), while significant reductions in serotonergic and muscarinic cholinergic receptor densities were reported in the basal ganglia 1123
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Alterations in Huntington's Disease
Vol . 21, No . 8, 1977
(7,9,10) . In these latter studies, receptor affinity for the binding agents remained constant (7,9) suggesting that the decrease in ligand binding to serotonergic and muscarinic cholinergic receptors is due to a decrease in receptor density ; this may be associated with the degeneration of striatal neurons .
To examine further receptor alterations that may occur in choreic brains we have studied neurotransmitter receptor binding in several regions of postmortem choreic and control brains using 3 H-spiroperidol which we have shown previously to bind specifically to a dopaminergic receptor (12-15) . METHODS AND MATERIALS Post mortem brain tissue came from three adult patients with a diagnosis of Huntington's Disease (adult-onset type) and from sixteen control brains chosen without regard for cause of death except for elimination of cases with infection or malignant disease of the central nervous system . All HD patients had received either perphenazine or haloperidol up to the time of death . The brains were dissected and the regions were frozen immediately after autopsy, which occurred 6-24 hr after death, and stored at -80°C . On the day of the experiment, the brain regions were thawed and the binding studies were performed . Each brain region was homogenized with a Polytron (Brinkman-setting 5 for 30 sec .) to yield a 5% homogenate in a modified Tris-Ringer medium (16), pH 7 .1 at 37°C . An aliquot (0 .1 ml) of this unwashed homogenate was saved for analysis of ChAc activity . The remainder of the homogenate was washed twice by diluting with 15 ml of buffered medium and centrifuging at 48,000 x g for 15 minutes in a Sorvall RC2-B centrifuge . The supernatants were discarded and the pellets were re suspended in Tris buffer to make a 5% homogenate . Protein determinations were performed by the method of Lowry et al . (17) . To determine specific ligand binding of 3 H-spiroperidol, aliquots of homogenates (20 mg tissue) and 3 H-spiroperidol (custom labeled by NEN, Boston, Mass . ; specific activity = 1 Ci/mmole) were incubated in 10 ml of buffer at 37°C . Incubations were routinely carried out for 30 minutes . Each determination was done in quadruplicate with two tubes containing (+)-butaclamol (O .1 JIM) and two lacking the displacer . Each incubation was terminated by rapid vacuum filtration through a GF/B glass fiber filter (Whatman) . Each filter was immediately rinsed with three consecutive 5 ml aliquots of ice cold buffer . Bound 3 H-spiroperidol retained on the filters was extracted with 9 ml of scintillation cocktail (prepared by mixing 2 L toluene, 1 L Triton X-100 and 16 g Omnifluor) in a scintillation vial . Extraction was continued for at least 12 hrs prior to counting the vials on a Searle Mark II liquid scintillation counter (45% efficiency) . Specific 3 H-spiroperidol binding was defined as the difference in the amount of bound 3 H-spiroperidol in the absence and presence of 0 .1 uM (+)-butaclamol . To determine ChAc activity, an aliquot of unwashed homogenate was diluted to a 3 .3% suspension and assayed as described previously (18) . An aliquot (0 .005 ml) of the homogenate was mixed with 0 .025 ml of ChAc cocktail which had been made by mixing 0 .04 ml Na 2 HP0 4 /NaH2 PO4 buffer, pH 7 .4 (0 .2M) ; 0 .17 ml eserine salicylate (0 .001 M) ; 0 .06 ml MgCl 2 (O .1M) ; 0 .12 ml NaCl (3M) ; 0 .12 ml choline chloride (20 mM) and 0 .1 ml 14 C-acetyl CoA (0 .2 mCi/ml) . Incubations were carried out for 20 minutes at 37°C . The reaction was terminated by adding 0 .1 ml of a solution of tetraphenylboron in 3-heptanone (50 mg/ml) ; the mixture was vortexed, plunged into an ice bath for 5 minutes and then centrifuged for 2 minutes in a Beckman Microfuge B . Aliquots (50 ul) from the top, organic layer were transferred to scintillation vials containing 8 ml triton toluene phosphor scintillation cocktail and counted in the manner described above .
Dol . 21, No . 8,
1977
1125
Alterations in Huntington's Disease
Tissue ChAc activity is expressed as nmoles of acetylcholine synthesized/mg protein-hr . The significance of the difference between groups was analyzed using a twotailed Student's "t" test . RESULTS In the control brains specific 3H-spiroperidol binding was highest in the putamen and caudate nucleus followed by intermediate levels of binding in the frontal cortex, globus pallidus, and occipital cortex (Table I) . Lower levels of binding were found in the substantia nigra, hippocampus, thalamus, artlygdala, anterior cerebellar vermis and dentate nucleus . TABLE I SPECIFIC 3 H-SPIROPERIDOL BINDING IN CONTROL AND CHOREIC BRAIN BRAIN REGION
SPECIFIC 3 H-SPIROPERIDOL BINDING (femtomoles/mg protein) Control
%Control
H .C .
PUTAMEN
(16)
274 .6 t 25 .2
(3)
137 .25 ± 35 .7
50*
CAUDATE NUCLEUS
(16)
234 .6 ± 16 .8
(3)
107 .5
± 20 .7
46***
FRONTAL CORTEX
(15)
72 .1 ± 10 .8
(3)
25 .0
±
35*
GLOBUS PALLIDUS
(16)
79 .2 ± 13 .3
(3)
38 .25 ± 18 .8
48
OCCIPITAL CORTEX
(6)
70 .8 ± 31 .0
(3)
24 .3
34
SUBSTANTIA NIGRA
(4)
19 .3 ±
9 .2
(1)
24 .0
HIPPOCAMPUS
(4)
16 .8 ±
0 .6
(3)
13 .0
±
4 .2
78
THALAMUS
(5)
7 .0 ±
4 .4
(3)
6 .3
±
7 .8
90
AMYGDALA
(3)
41 .7 t
6 .7
(3)
15 .3
±
8 .4
37
ANTERIOR CEREBELLAR VERMIS
(5)
13 .4 ±
6 .0
(3)
18 .7
± 12 .8
140
DENTATE NUCLEUS
(3)
5 .3 ±
1 .7
(3)
1 .7
±
32
±
3 .9
9 .6
124
0 .9
Values are mean t S .E .M . from Huntington's Chorea (adult-onset form) and control brains . Values in parentheses are number of brain samples used in the experiments . *** P< .O1) ~P< .05) *
1126
Alterations in Huntington's Disease
Vol . 21, No . 8, 1977
In choreic brains significant decreases in 3 H-spiroperidol binding were seen in the caudate nucleus, putamen and frontal cortex (Table I) . Binding in the other regions of choreic brain were not significantly different from control tissue . The apparent dissociation constant, Kpapp, for 3 H-spiroperidol binding was determined by Scatchard analysis of saturation isotherms . In both the caudate nucleus and putamen of normal and HD brains, the KDapp value was about 0 .25 (± .05) nM . The inhibitory constant (K i ) for (+)-butaclamol inhibition of 3 H-spiroperidol binding in the caudate nucleus and putamen was about 0 .4 nM in both control and choreic brains . Choline acetyltransferase activity in normal brains was highest in the putamen with lower values in the caudate nucleus and globus pallidus (Table II) . All other brain areas had relatively low arfd uniform activity . TABLE II CHOLINE ACETYLTRANSFERASE ACTIVITY IN CONTROL AND CHOREIC BRAIN BRAIN REGION
ChAc ACTIVITY (nmoles/mg protein-hr) Control
H .C .
%Control
PUTAMEN
(14)
70 .4 ± 12 .2
(3)
3 .2 ± 1 .9
CAUDATE NUCLEUS
(14)
41 .0 ±
3 .6
(3)
16 .8 ± 8 .8
FRONTAL CORTEX
(13)
2 .7 ±
0 .3
(3)
3 .5 ± 1 .1
130
GLOBUS PALLIDUS
(15)
9 .5 ±
1 .6
(3)
7 .1 ± 0 .1
75
HIPPOCAMPUS
(5)
4 .0 ±
1 .1
(3)
1 .6 ± 0 .4
40
AMYGDALA
(4)
7 .3 ±
2 .1
(3)
4 .5 ± 0 .7
62
ANTERIOR CEREBELLAR VERMIS
(6)
4 .7 ±
1 .5
(3)
2 .9 ± 0 .7
62
5* 41**
Values are mean ± S .E .M . for Huntington Chorea (adult-onset form) and control brain . Values in parentheses are number of brain samples . (P< .025) *
(P< .05)
The ChAc activity was reduced significantly in the caudate nucleus and putamen of HD brains (Table II) . In the remaining regions examined, ChAc acttivity was not significantly different . DISCUSSION The specific binding of 3 H-spiroperidol to human brain tissue was used to assay for alterations in dopaminergic receptors . Previous studies (12-15) have indicated that 3 H-spiroperidol binds to dopaminergic receptors in both rat and human brain tissue . The binding was saturable, of high affinity and inhibited
Vol . 21, No . 8, 1977
Alterations in Huntington's Disease
1127
by various dopaminergic agents ; it was not displaced by micromolar concentrations of nondopaminergic agents . The relative regional distribution of 3Hspiroperidol was the same in rat and human brain . Therefore, it appears that specific 3H-spiroperidol binding labels a dopaminergic receptor and can be used as an indicator of the density of dopaminergic receptors in human brain . Neuropathological studies have revealed gross atrophy and neuronal loss in the basal ganglia in HD (2-5) . Considering the modulating role that the basal ganglia performs in motor function, degeneration of the striatum is regarded as the pathologic basis for the clinically observed extrapyramidal motor disturbances of HD . Associated with the neuronal degeneration in the striatum are various alterations in the densities of neurotransmitter receptors (6-8) . Our findings reveal, in addition, a significant 50% decrease in 3H-spiroperidol binding in the putamen and caudate nucleus . Since the KD for 3H-spiroperidol binding and the Ki for (+)-butaclamol displacement were similar in choreic and control brains, the decrease in 3H-spiroperidol binding appears to be due solely to a loss in the number of receptor sites . It is of interest to note that binding to dopaminergic receptors in the putamen and caudate of choreic brain decreased despite the chronic pretreatment of the patients with dopaminergic antagonists, a maneuver which has been shown to produce in rats, an increase in the number of binding sites (19,20) . A significant (P< .05) decrease in ChAc activity was also .found in the striatal areas suggesting that at least some of the dopaminergic receptors may be postsynaptic sites on cholinergic neurons and that the loss of striatal dopamine receptors results from the progressive degeneration of these neurons . While the motor disturbances of HD may most likely be attributed to . pathological changes in the basal ganglia, the mental disorder associated with HD, may be the result of neuronal loss and/or dysfunction in the cerebral cortex . Neuropathological studies have indicated that there is diffuse degeneration of cerebral cortical neurons in some choreic brains, particularly in the frontal lobes (3,7) . Previous reports failed to reveal alterations in a variety of receptors or in enzymes necessary for neurotransmitter synthesis in the cerebral cortex of choreic brains (9,10,21) . In the present study, however, we did find in the frontal cortex a 65% decrease in 3H-spiroperidol binding (P< .05) with no significant alteration in ChAc activity . These data suggest that the decrease in the density of dopaminergic receptors in the frontal cortex is associated with the neuronal depletion found in that area of choreic brains . Lastly, the results of our binding studies on several other areas of choreic brains failed to reveal significant alterations in 3 H-spiroperidol binding (Table I) . These data show that the decrease in 3H-spiroperidol binding in choreic brains is selective and is not the result of nonspecific postmortem changes . ACKNOWLEDGEMENT The authors express their sincere appreciation to Tom McManus and David Chapman for their technical assistance . The work was supported in part by grants from the NIMH MH-24257, MH-29840, and a grant-in-aid from the Committee to Combat Huntington's Disease . H .I . Yamamura is a recipient of a Research Scientist Development Award (RSDA), MH-00095 . J .Z . Fields was supported by Individual Post-doctoral Fellowship from the NIMH, (MH-05248) .
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Vol . 21, No . 8, 1977
REFERENCES 1 . 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 .
I . Shoulson and T .N . Chase : Ann . Rev . Med . 26 : 419-426, 1975 W . Blackwood and J .A .N . Corse lis, Greenf: elds Neuropathology, (pp . 882827) . Edward Arnold Ltd ., London (976 E .D . Bird and L .L . Iversen : in Essays in Neurochemistry, M . Youdin and D . Sharman (eds .), Pergamon Press, Oxford 1977 G .W . Bruyn : in Handbook of Clinical Neurology, P .J . Vinken and G .W . Bruyn (eds .) Vol . I pp . 729-734, Raven Press, New York, (1973) G . Kintworth : in Advances in Neurology, A . Barbeau, T .N . Chase and G .W . Paulson (eds .) Vol I . pp . 353-368, Raven Press, New York, (1973) E .D . Bird and L .L . Iversen, Brain 97 : 457-472, 1974 G .J . Wastek, L .Z . Stern, P .C . Johnson, and H .I . Yamamura, Life Sciences 19 : 1033-1040, 1976 W.L . Stahl and P .D . Swanson, Neurology (Minneap) 24 : 813-819, 1974 S .J . Enna, E .D . Bird, J .P . Bennett, Jr ., D .B . Bylund, H .I . Yamamura, L .L . Iversen, and S .H . Snyder, New England Journal of Med . 294 : 1305-1309, 1976 C .R . Hiley and E .D . Bird : Brain Res . 80 : 355-348, 1974 S .J . Enna, L .Z . Stern, G .J . Wastek, and H .I . Yamamura, Life Sciences 20 : 205-212, (1977) J .Z . Fields, T .D . Reisine and H .I . Yamamura Brain Res . i n press 1977 J .Z . Fields, T .D . Reisine, D . Crawford, S .J . Enna and H .I . Yamamura, Trans Amer . Soc . Neurochem . 8 : 193 (1977) T .D . Reisine, J .Z . Fields, E .D . Bird, P . Schreiner, H .I . Yamamura and S .J . Enna, Trans . Soc . for Neurosciences in press, 1977 T .D . Reisine, J .Z . Fields, H .I . Yamamura, .E .D . Bird, E . Spokes, P .S . Schreiner, S .J . Enna, Life Sciences 21 : 335-344, 1977 D .R . Burt, I . Creese, S .H . Snyder, Mol . Pharmacol . 12 : 800-812, 1976 O .H . Lowry, N .J . Rosebrough, A .L . Fair and R .J . Randall, J . Biol . Chem . _193 : 265-275, 1951 M .J . Kuhar, V .H .Sethy, R .H . Roth and G .K . Aghajanian, J . Neurochem . 20 : 581-593, 1973 R.M . Kobayashi, J .Z . Fields, R .E . Hruska, K . Beaumont and H .I . Yamamura, in Animal Models in Psychiatry and Neurology , I . Hanin and E . Usdin (eds .) Pergamon Press, N .Y . in press, 1978 D .R . Burt, I . Creese and S .H . Snyder, Science , 196 : 326-327, 1977 P .L . McGeer and E .G . McGeer, J . Neurochem . 26 : 65-76, 1976 P .L . McGeer and E .G . McGeer, Arch . Nurol . 2T: 265-268, 1971 G .S . Synch, P .A . Lucas and S .A . Deadwyler, Brain Res . 45 : 617-621, 1972
Pergamon Press
ALTERATIONS IN DOPAMINERGIC RECEPTORS IN HUNTINGTON'S DISEASE
T .D . Reisine, J .Z . Fields, L .Z . Stern* P .C . Johnson**, E .D . Birdl, and H .I . Yamamura Departments of Pharmacology, Neurology* and Pathology** College of Medicine, University of Arizona Health Sciences Center, Tucson, Arizona 85724 and 'Department of Neurological Surgery and Neurology, Addenbrookes Hospital, Cambridge, England (Received in final form September 2,
1977)
SUMMARY To detect variations in dopaminergic receptors and cholinergic activity in regions of postmortem Huntington's diseased brains, 3Hspiroperidol binding assays and choline acetyltransferase (ChAc) activities were carried out . A significant reduction in 3H-spiroperidol binding in the caudate nucleus, putamen and frontal cortex of choreic brains was detected which appeared to be due to a decrease in the total number of binding sites rather than to a decrease in affinity of 3H-spiroperidol for the dopaminergic receptor . In choreic brains, there were also significant reductions in ChAc activity in the caudate nucleus and putamen . The decreases of both 3 H-spiroperidol binding and ChAc activity in the neostriatum suggest that the dopaminergic receptors are localized postsynaptically on cholinergic interneurons . Dopaminergic receptor alterations in the basal ganglia may be one of the causes of the abnormal motor movements found in HD while alterations of these receptors in the frontal cortex may be associated with the neuronal degeneration found in that area of choreic brains . INTRODUCTION Huntington's Disease (HD) is an autosomal dominant neurological disorder that is characterized by extrapyramidal motor abnormalities and dementia (1) . Histological evidence indicates that there are neuronal losses in the basal ganglia particularly in the caudate nucleus and cerebral cortex (2-5) . The neuronal loss and resultant atrophy observed in the basal ganglia may be the cause of the movement disorders typical of the disease . Previous neurochemical studies showed a 50% reduction in choline acetyltransferase (ChAc) activity (6,7) and an 85% decrease in glutamic acid decarboxylase (GAD) activity in the basal ganglia of choreic brains (6,8) . Since the activities of these enzymes serve as markers for cholinergic and GABAergic neurons, respectively, the decline in enzyme activities suggests a selective loss of these neurons . In previous studies, changes in neurotransmitter receptor binding were also examined (7,9,11) in the frontal cortex and basal ganglia of postmortem choreic brains . In the frontal cortex of choreic brains no changes were found in the densities of 9-adrenergic, GABAergic, muscarinic cholinergic or serotonergic receptors (9), while significant reductions in serotonergic and muscarinic cholinergic receptor densities were reported in the basal ganglia 1123
112 4
Alterations in Huntington's Disease
Vol . 21, No . 8, 1977
(7,9,10) . In these latter studies, receptor affinity for the binding agents remained constant (7,9) suggesting that the decrease in ligand binding to serotonergic and muscarinic cholinergic receptors is due to a decrease in receptor density ; this may be associated with the degeneration of striatal neurons .
To examine further receptor alterations that may occur in choreic brains we have studied neurotransmitter receptor binding in several regions of postmortem choreic and control brains using 3 H-spiroperidol which we have shown previously to bind specifically to a dopaminergic receptor (12-15) . METHODS AND MATERIALS Post mortem brain tissue came from three adult patients with a diagnosis of Huntington's Disease (adult-onset type) and from sixteen control brains chosen without regard for cause of death except for elimination of cases with infection or malignant disease of the central nervous system . All HD patients had received either perphenazine or haloperidol up to the time of death . The brains were dissected and the regions were frozen immediately after autopsy, which occurred 6-24 hr after death, and stored at -80°C . On the day of the experiment, the brain regions were thawed and the binding studies were performed . Each brain region was homogenized with a Polytron (Brinkman-setting 5 for 30 sec .) to yield a 5% homogenate in a modified Tris-Ringer medium (16), pH 7 .1 at 37°C . An aliquot (0 .1 ml) of this unwashed homogenate was saved for analysis of ChAc activity . The remainder of the homogenate was washed twice by diluting with 15 ml of buffered medium and centrifuging at 48,000 x g for 15 minutes in a Sorvall RC2-B centrifuge . The supernatants were discarded and the pellets were re suspended in Tris buffer to make a 5% homogenate . Protein determinations were performed by the method of Lowry et al . (17) . To determine specific ligand binding of 3 H-spiroperidol, aliquots of homogenates (20 mg tissue) and 3 H-spiroperidol (custom labeled by NEN, Boston, Mass . ; specific activity = 1 Ci/mmole) were incubated in 10 ml of buffer at 37°C . Incubations were routinely carried out for 30 minutes . Each determination was done in quadruplicate with two tubes containing (+)-butaclamol (O .1 JIM) and two lacking the displacer . Each incubation was terminated by rapid vacuum filtration through a GF/B glass fiber filter (Whatman) . Each filter was immediately rinsed with three consecutive 5 ml aliquots of ice cold buffer . Bound 3 H-spiroperidol retained on the filters was extracted with 9 ml of scintillation cocktail (prepared by mixing 2 L toluene, 1 L Triton X-100 and 16 g Omnifluor) in a scintillation vial . Extraction was continued for at least 12 hrs prior to counting the vials on a Searle Mark II liquid scintillation counter (45% efficiency) . Specific 3 H-spiroperidol binding was defined as the difference in the amount of bound 3 H-spiroperidol in the absence and presence of 0 .1 uM (+)-butaclamol . To determine ChAc activity, an aliquot of unwashed homogenate was diluted to a 3 .3% suspension and assayed as described previously (18) . An aliquot (0 .005 ml) of the homogenate was mixed with 0 .025 ml of ChAc cocktail which had been made by mixing 0 .04 ml Na 2 HP0 4 /NaH2 PO4 buffer, pH 7 .4 (0 .2M) ; 0 .17 ml eserine salicylate (0 .001 M) ; 0 .06 ml MgCl 2 (O .1M) ; 0 .12 ml NaCl (3M) ; 0 .12 ml choline chloride (20 mM) and 0 .1 ml 14 C-acetyl CoA (0 .2 mCi/ml) . Incubations were carried out for 20 minutes at 37°C . The reaction was terminated by adding 0 .1 ml of a solution of tetraphenylboron in 3-heptanone (50 mg/ml) ; the mixture was vortexed, plunged into an ice bath for 5 minutes and then centrifuged for 2 minutes in a Beckman Microfuge B . Aliquots (50 ul) from the top, organic layer were transferred to scintillation vials containing 8 ml triton toluene phosphor scintillation cocktail and counted in the manner described above .
Dol . 21, No . 8,
1977
1125
Alterations in Huntington's Disease
Tissue ChAc activity is expressed as nmoles of acetylcholine synthesized/mg protein-hr . The significance of the difference between groups was analyzed using a twotailed Student's "t" test . RESULTS In the control brains specific 3H-spiroperidol binding was highest in the putamen and caudate nucleus followed by intermediate levels of binding in the frontal cortex, globus pallidus, and occipital cortex (Table I) . Lower levels of binding were found in the substantia nigra, hippocampus, thalamus, artlygdala, anterior cerebellar vermis and dentate nucleus . TABLE I SPECIFIC 3 H-SPIROPERIDOL BINDING IN CONTROL AND CHOREIC BRAIN BRAIN REGION
SPECIFIC 3 H-SPIROPERIDOL BINDING (femtomoles/mg protein) Control
%Control
H .C .
PUTAMEN
(16)
274 .6 t 25 .2
(3)
137 .25 ± 35 .7
50*
CAUDATE NUCLEUS
(16)
234 .6 ± 16 .8
(3)
107 .5
± 20 .7
46***
FRONTAL CORTEX
(15)
72 .1 ± 10 .8
(3)
25 .0
±
35*
GLOBUS PALLIDUS
(16)
79 .2 ± 13 .3
(3)
38 .25 ± 18 .8
48
OCCIPITAL CORTEX
(6)
70 .8 ± 31 .0
(3)
24 .3
34
SUBSTANTIA NIGRA
(4)
19 .3 ±
9 .2
(1)
24 .0
HIPPOCAMPUS
(4)
16 .8 ±
0 .6
(3)
13 .0
±
4 .2
78
THALAMUS
(5)
7 .0 ±
4 .4
(3)
6 .3
±
7 .8
90
AMYGDALA
(3)
41 .7 t
6 .7
(3)
15 .3
±
8 .4
37
ANTERIOR CEREBELLAR VERMIS
(5)
13 .4 ±
6 .0
(3)
18 .7
± 12 .8
140
DENTATE NUCLEUS
(3)
5 .3 ±
1 .7
(3)
1 .7
±
32
±
3 .9
9 .6
124
0 .9
Values are mean t S .E .M . from Huntington's Chorea (adult-onset form) and control brains . Values in parentheses are number of brain samples used in the experiments . *** P< .O1) ~P< .05) *
1126
Alterations in Huntington's Disease
Vol . 21, No . 8, 1977
In choreic brains significant decreases in 3 H-spiroperidol binding were seen in the caudate nucleus, putamen and frontal cortex (Table I) . Binding in the other regions of choreic brain were not significantly different from control tissue . The apparent dissociation constant, Kpapp, for 3 H-spiroperidol binding was determined by Scatchard analysis of saturation isotherms . In both the caudate nucleus and putamen of normal and HD brains, the KDapp value was about 0 .25 (± .05) nM . The inhibitory constant (K i ) for (+)-butaclamol inhibition of 3 H-spiroperidol binding in the caudate nucleus and putamen was about 0 .4 nM in both control and choreic brains . Choline acetyltransferase activity in normal brains was highest in the putamen with lower values in the caudate nucleus and globus pallidus (Table II) . All other brain areas had relatively low arfd uniform activity . TABLE II CHOLINE ACETYLTRANSFERASE ACTIVITY IN CONTROL AND CHOREIC BRAIN BRAIN REGION
ChAc ACTIVITY (nmoles/mg protein-hr) Control
H .C .
%Control
PUTAMEN
(14)
70 .4 ± 12 .2
(3)
3 .2 ± 1 .9
CAUDATE NUCLEUS
(14)
41 .0 ±
3 .6
(3)
16 .8 ± 8 .8
FRONTAL CORTEX
(13)
2 .7 ±
0 .3
(3)
3 .5 ± 1 .1
130
GLOBUS PALLIDUS
(15)
9 .5 ±
1 .6
(3)
7 .1 ± 0 .1
75
HIPPOCAMPUS
(5)
4 .0 ±
1 .1
(3)
1 .6 ± 0 .4
40
AMYGDALA
(4)
7 .3 ±
2 .1
(3)
4 .5 ± 0 .7
62
ANTERIOR CEREBELLAR VERMIS
(6)
4 .7 ±
1 .5
(3)
2 .9 ± 0 .7
62
5* 41**
Values are mean ± S .E .M . for Huntington Chorea (adult-onset form) and control brain . Values in parentheses are number of brain samples . (P< .025) *
(P< .05)
The ChAc activity was reduced significantly in the caudate nucleus and putamen of HD brains (Table II) . In the remaining regions examined, ChAc acttivity was not significantly different . DISCUSSION The specific binding of 3 H-spiroperidol to human brain tissue was used to assay for alterations in dopaminergic receptors . Previous studies (12-15) have indicated that 3 H-spiroperidol binds to dopaminergic receptors in both rat and human brain tissue . The binding was saturable, of high affinity and inhibited
Vol . 21, No . 8, 1977
Alterations in Huntington's Disease
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by various dopaminergic agents ; it was not displaced by micromolar concentrations of nondopaminergic agents . The relative regional distribution of 3Hspiroperidol was the same in rat and human brain . Therefore, it appears that specific 3H-spiroperidol binding labels a dopaminergic receptor and can be used as an indicator of the density of dopaminergic receptors in human brain . Neuropathological studies have revealed gross atrophy and neuronal loss in the basal ganglia in HD (2-5) . Considering the modulating role that the basal ganglia performs in motor function, degeneration of the striatum is regarded as the pathologic basis for the clinically observed extrapyramidal motor disturbances of HD . Associated with the neuronal degeneration in the striatum are various alterations in the densities of neurotransmitter receptors (6-8) . Our findings reveal, in addition, a significant 50% decrease in 3H-spiroperidol binding in the putamen and caudate nucleus . Since the KD for 3H-spiroperidol binding and the Ki for (+)-butaclamol displacement were similar in choreic and control brains, the decrease in 3H-spiroperidol binding appears to be due solely to a loss in the number of receptor sites . It is of interest to note that binding to dopaminergic receptors in the putamen and caudate of choreic brain decreased despite the chronic pretreatment of the patients with dopaminergic antagonists, a maneuver which has been shown to produce in rats, an increase in the number of binding sites (19,20) . A significant (P< .05) decrease in ChAc activity was also .found in the striatal areas suggesting that at least some of the dopaminergic receptors may be postsynaptic sites on cholinergic neurons and that the loss of striatal dopamine receptors results from the progressive degeneration of these neurons . While the motor disturbances of HD may most likely be attributed to . pathological changes in the basal ganglia, the mental disorder associated with HD, may be the result of neuronal loss and/or dysfunction in the cerebral cortex . Neuropathological studies have indicated that there is diffuse degeneration of cerebral cortical neurons in some choreic brains, particularly in the frontal lobes (3,7) . Previous reports failed to reveal alterations in a variety of receptors or in enzymes necessary for neurotransmitter synthesis in the cerebral cortex of choreic brains (9,10,21) . In the present study, however, we did find in the frontal cortex a 65% decrease in 3H-spiroperidol binding (P< .05) with no significant alteration in ChAc activity . These data suggest that the decrease in the density of dopaminergic receptors in the frontal cortex is associated with the neuronal depletion found in that area of choreic brains . Lastly, the results of our binding studies on several other areas of choreic brains failed to reveal significant alterations in 3 H-spiroperidol binding (Table I) . These data show that the decrease in 3H-spiroperidol binding in choreic brains is selective and is not the result of nonspecific postmortem changes . ACKNOWLEDGEMENT The authors express their sincere appreciation to Tom McManus and David Chapman for their technical assistance . The work was supported in part by grants from the NIMH MH-24257, MH-29840, and a grant-in-aid from the Committee to Combat Huntington's Disease . H .I . Yamamura is a recipient of a Research Scientist Development Award (RSDA), MH-00095 . J .Z . Fields was supported by Individual Post-doctoral Fellowship from the NIMH, (MH-05248) .
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