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Effects of globus pallidus lesions and Parkinson's disease on brain glutamic acid decarboxylase
BRAIN RESEARCH
425
EFFECTS OF GLOBUS PALLIDUS LESIONS AND PARKINSON'S DISEASE ON BRAIN GLUTAMIC ACID DECARBOXYLASE
P. L. McGEER, E. G. McGEER, J. A. WADA ANDE. JUNG Kinsmen Laboratory of Neurological Research, Department of Psychiatry, University of British Columbia, Vancouver 8 (Canada)
(Accepted March 10th, 1971)
INTRODUCTION Although the role of gamma-aminobutyric acid (GABA) in the central nervous system is unknown, information is accumulating suggesting that it may be a neurotransmitter. The unequal distribution in brain of GABA, and of the enzyme responsible for its synthesis, glutamic acid decarboxylase (GAD), is consistent with this hypothesis. High levels of GABA and GAD in structures associated with the extrapyramidal system have been repeatedly noted, with the highest levels reported being in the globus pallidus and substantia nigra in several mammalian speciesl,7,s,zs,~L We report here further evidence associating GAD with extrapyramidal function. Lesions to the region of the globus pallidus in cats caused a decrease in GAD in the substantia nigra. Both the globus pallidus and substantia nigra of individuals who died with Parkinson's disease were found to have lower levels of GAD in relation to the cerebral cortex than the same areas in patients who died with other diseases in hospital or in individuals who were killed suddenly in accidents. MATERIALSAND METHODS Extensive lesions to the region of the globus pallidus were produced in a series of 6 cats. Stereotaxic coordinates were from FI 1 to F15 mm in the anterior-posterior plane. Laterally, they extended from 5 to 9.5 mm and horizontally from --0.5 to --2.5 depending on the frontal level33. The cats were sacrificed between 15 and 41 days following lesion placement. The lesions were all examined grossly at the time of dissection. In one case the brain was fixed for histological study instead of being dissected for biochemical assay. Frozen sections of the fixed brain were cut and alternate sections were stained for cholinesterase20 and for cells with cresyl violet. In every case, the lesion spared the caudate nucleus and the substantia nigra completely. The lesions were nevertheless rather massive and encroachment on the Brain Research, 32 (1971)425--431
426
p.L. MCGEERet al.
medial part of the putamen and the lateral part of the internal capsule was definite in the fixed specimen and probable in the other five. Histochemical staining showed the globus pallidus to be completely destroyed. Adjacent pathways running between the cortex and midbrain, the basal ganglia and thalamus, and the striatum and midbrain obviously could have been damaged in all the cats. Cellular depopulation in the substantia nigra on the side of the lesion was evident in the fixed specimen despite the fact that the structure was grossly spared. Apparently the cellular degeneration was retrograde in nature. Glutamic acid decarboxylase was measured by a microradiometric method involving measurement of a4CO2 produced on incubation of tissue homogenate with L[l-14C]glutamic acid (30-60,000 disint./min, spec. act. 60/~Ci/mmole). Radioactive CO2 was trapped on hyamine hydroxide soaked paper and counted 7. Choline acetyltransferase was measured by a modification of the method of Fonnum m in which 60-182/~g of tissue, activated by treatment with 0.5 ~ Triton X-100, was incubated with radioactive [14C]acetyl--coenzyme A (50,000 disint./min, spec. act. 2.2 #Ci/ #mole). The incubation medium was 50 m M in phosphate, p H 7.4, 1 l m M in choline and in ethylenediaminetetraacetic acid, 290 m M in NaCI, 6 m M in sucrose, 0.1 m M in eserine and contained 0.6 mg/ml albumin (total volume 0.08 ml). As previously reported ~6 the [a4C]acetylcholine was absorbed onto a CG-50 ion exchange column and eluted with 3 ml of 4 N acetic acid. The eluate was counted. Acetylcholinesterase was measured by a colorimetric method which assays the amount of acetylcholine destroyed during an hour long incubation2< Tyrosine hydroxylase was measured by the method previously described eT, with the exception that for the substantia nigra the assay mixture contained no phosphate but instead was 0.2 M in acetate, p H 6.0, 1 0 4 M in DMPH4, 3 • 10 -4 M in FeSO4, and 0.1 M in 2-mercaptoethanol. RESULTS
The biochemical data for globus pallidus lesioned cats are summarized in Table I. G A D was decreased to 39 ~ of the control value in the substantia nigra on the TABLE
I
ENZYME LEVELS IN DISCRETE CAT BRAIN AREAS ON LESIONED SIDE AS ~oo OF CONTROL SIDE FOLLOWING UNILATERAL OLOBUS PALLIDUS LESIONS
Brain area
Enzyme % ± S.D.* Glutamic acid decarboxylase
Substantia nigra 39 4- 4.4 (5) Caudate 95.3 4- 6.0 (4) Cerebellum 110.6 ± 5.9 (5) Hippocampus 95.2 ± 4.8 (5)
Tyrosine hydroxylase
Choline acetyltransferase
Acetylcholinesterase
49.2 ± 17.6 (4) 23.7 ± 7.8 (3) ---
105.8 ± 18.6 (5) 101.7 ± 9.0 (4) -93.5 ± 5.0 (4)
88.8 ± 7.7 (5) 92.3 _q_6.2 (4) 99.3 ± 6.0 (5) 93.0 ± 8.6 (5)
* Number of animals indicated in brackets following values. Brain Research, 32 (1971) 425-431
EXTRAPYRAMIDAL
GAD
427
AND PARKINSON¢S DISEASI~
lesioned side. The absolute values for G A D in the substantia nigra were 22.2 ± 0.5 /zmoles/h/g on the control side v e r s u s 9.76 ~: 1.36 on the lesioned side. The difference was highly significant (P < 0.001). The absolute value for G A D in the caudate was 12.4 + 0.6 #moles/h/g, in the hippocampus 6.9 + 1.2 and in the cerebellum 11.1 ± 0.7. There was no significant change between the control and lesioned sides in these areas. Tyrosine hydroxylase was reduced to 49.29/0 and 23.7 9/0 of the control values in the substantia nigra and caudate respectively, while choline acetyltransferase and cholinesterase were not significantly affected in either structure. Lesions to the substantia nigra4,9,1a, 15 and globus pallidusS,9,17,24 have long been reported in Parkinson's disease. To test whether there might be any abnormality in G A D metabolism in this condition, the G A D content of various brain areas was measured in human postmortem brains. Tissue was obtained from Parkinson's disease patients, patients dying in hospitals with other illnesses, and Coroner's cases after sudden death. The glutamic acid decarboxylase values varied from brain to brain but the frontal cortical areas in any given brain were found to be highly comparable with one another. For each brain, 3 cortical areas in the frontal lobe were averaged. The G A D level in the frontal cortex was 9.42 :k 0.47 (n = 6) pmoles/g/h for Coroner's cases, 5.49 :k 1.44 (n -~ 6) for hospital cases, and 3.24 ± 0.47 (n = 6) for Parkinson's disease cases. The values for accident victims were almost identical with human cortical values obtained at biopsy in patients undergoing surgery for epilepsy. It is doubtful what significance can be attached to these differences. The accident victims tended to be younger (average age 31 as compared to 72 for the other groups) and some of the brains were dissected only 3 h after death. The average time between death and brain dissection was 8 h for the group of accident victims, 11 h for those suffering from such various illnesses as cancer, pneumonia, emphysema and heart failure, and 9 h for the Parkinsonian cases, who, like the other hospital cases, nearly all suffered a terminal pneumonia. Much more needs to be learned regarding the postmortem stability of GAD, the effects of prolonged coma prior to death, and the influence of age before any interpretation could be given of these overall differences. Although there was considerable variation in G A D activity from brain to brain, the relative activities of various areas tended to be more closely comparable. The ratio of the activity of each brain area to the activity in the frontal cortex, was therefore TABLE II GLUTAMIC ACID DECARBOXYLASEIN VARIOUSHUMAN BRAIN AREASAS ~oo OF AVERAGEOF 3 FRONTAL CORTEX AREAS 4- S.D.* Brain area
Parkinson
Other illness
Accident
Substantia nigra Globus pallidus Caudate Putamen Hippocampus
53 ± 8 (6) 83 4- 8 (6) 924-13(6) 107 4- 19 (6) 42 4- 3 (6)
170 4- 22 (6) 151 4- 21 (5) 1044-10(5) 89 4- 8 (5) 46 4- 6 (4)
205 139 91 99 60
-¢- 6 (4) -4- 17 (5) 4- 6(6) 4- 10 (6) 4- 9 (5)
* Number of brains indicated in brackets following value. Brain Research, 32 (1971) 425-431
428
P.L.
MCGEER e l t//~
calculated. On this basis, there was no significant difference between Parkinsonian and non-Parkinsonian patients for most areas, as illustrated in Table II by the data for the caudate, putamen, and hippocampus. The substantia nigra and globus pallidus, however, had reduced GAD in Parkinsonian patients. The substantia nigra had a level of only 53 ~ of the cortex in Parkinsonian patients while it was 205 o/in accident cases /o and 170~ in patients dying in other illnesses. Similarly the globus pallidus had a level of 83~ of the cortex in Parkinsonian patients but was 139~ in accident cases and 151 ~ in other illnesses. Further studies will be required to assess the significance of the findings. DISCUSSION
The etiology and pathogenesis of Parkinson's disease remain a mystery. Attention has focussed in recent years on the upset in dopamine metabolism and a possible involvement of the nigral-striatal dopaminergic tract 3. The finding of a possible alteration in glutamic acid decarboxylase metabolism suggests that the biochemical pathology may be considerably broader. As far as GABA is concerned, further research is necessary to elucidate its role. The iontophoretic effects of the substance when applied to single neurons 21 and the subcellular localization of GAD and GABA-aminotransferase1s,2'~,2~,32 argue in favor of a neurotransmitter function. If GABA is a neurotransmitter, then the GAD data following globus pallidus lesions could be explained on the basis of interruption of a gabaminergic pathway. If, on the other hand, GABA is associated with neuronal or glial function only in a metabolic way, then the drop in GAD might reflect a general cellular depopulation following the lesion. The drop in tyrosine hydroxylase in both the substantia nigra and the caudate in 3 cats with globus pallidus lesions would be consistent with damage to the known dopaminergic pathway between these two structures 2,28,3°. Since GAD was affected only in the substantia nigra and not in the caudate, the drop in value of this enzyme must have been unrelated to the dopaminergic pathway. The fact that neither choline acetyltransferase nor cholinesterase were affected in either the substantia nigra or caudate would imply that there is no major cholinergic pathway between the globus pallidus and either the substantia nigra or the caudate. It would also imply that a direct major cholinergic pathway between the caudate and substantia does not exist, or if it does exist, travels through a route that was spared by the globus pallidus lesions. A minor route could, of course, exist and not be revealed by the techniques employed in this study. Many authors have described pathways between the substantia nigra and the globus pallidus. The presence of rostrally directed fibres from the substantia nigra to the globus pallidus has been repeatedly confirmed6,12,31,aL The presence of caudally directed fibres from the globus pallidus to substantia nigra has been disputed. SomO 4,19 regard such fibres as established although others 30,34 have been unable to confirm their presence. Brain Research, 32 (1971) 425--431
EXTRAPYRAMIDAL
GAD
AND PARKINSON'S DISEASE
429
Following axotomy, enzyme deterioration takes place. Initially enzymes associated with synaptosomes or other distal structures are affected, but eventually enzymes associated with the cells of origin disappear as well. The 15-41 day time lag between lesioning and sacrifice of the cats was such that total disappearance of some tracts took place. This was evidenced by the cellular dropout in the substantia nigra. Thus the drop in substantia nigra GAD could reflect deterioration of tracts coming into or leaving the substantia nigra, or both. Further experiments will be needed to determine whether any of these might depend on GABA as a neurotransmitter. Gabaminergic pathways have already been suggested for other areas of brain. One proposal is that Purkinje cells of the cerebellum elaborate GABA as an inhibitory transmitter at their terminals. This is supported by Fonnum's report 11 that lesions to Purkinje cells in the cerebellum cause a decrease in GAD in Deiters' nucleus in the brain stem. SUMMARY
Cats with unilateral lesions in the region of the globus pallidus showed a highly significant loss (to 39 ~ ) of glutamic acid decarboxylase (GAD) activity in the substantia nigra on the lesioned side. GAD was unaffected in the caudate nucleus or other structures. Tyrosine hydroxylase was reduced to 49 ~ of the control values in the substantia nigra and to 23.7 ~o in the caudate, while choline acetyltransferase and cholinesterase were not significantly affected in either structure. The data suggest that the lesion interrupted the known dopaminergic pathway between caudate and substantia nigra and interfered with another pathway concerned with GABA metabolism involving the substantia nigra but not the caudate. There appeared to be no major cholinergic pathway coursing through the lesioned area. The brains of accident victims and of non-Parkinsonian hospital patients dying with various illnesses showed the expected high GAD activities in the substantia nigra and globus pallidus (1.5 times that of the cortex). The brains of Parkinsonian patients, on the other hand, showed.decreased GAD levels in these structures. The GAD in the substantia nigra was only 53 ~o of that in the cortex, and the globus pallidus was only 83 ~. The caudate nucleus GAD was approximately equal to that of the cortex in all 3 groups. These data suggest the selective deterioration of a GAD containing cellular system in Parkinson disease which involves the substantia nigra and globus pallidus. ACKNOWLEDGEMENTS
This research was supported in part by grants from the Medical Research Council of Canada (MA-4013, MA-3633 and MA-2504) and the National Institutes of Health (NB-02812). We wish to thank Mrs. Virginia Wickson for technical assistance and the staff of the Pearson, Lions Gate, Royal Columbian, Shaughnessy and Vancouver General Hospitals as well as the Coroner's Offices in Vancouver and New Westminster for assistance in obtaining human tissue. Brain Research, 32 (1971) 425-431
430
p.L. McGEER et al.
REFERENCES 1 ALBERS,R. W., AND BRADY, R. O., Distribution of glutamic decarboxylase in the nervous system of the Rhesus monkey, J. biol. Chem., 234 (1959) 926-928. 2 ANDI~N,N. E., DAHLSTROM,A., FUXE, K., AND LARSSON,K., Mapping out of catecholamine and 5-hydroxytryptamine neurones innervating the telencephalon and diencephalon, Life Sci., B4 (1965) 1275-1279. 3 BARBEAU,A., AND McDOWELL, F. H. (Eds.), L-DOPA and Parkinsonism, Davis, Philadelphia, Pa., 1970. 4 BENDA, C. E., AND COBB, S., On the pathogenesis of paralysis agitans (Parkinson's disease), Medicine (Baltimore), 21 (1942) 95-142. 5 BIELSCHOWSKY, M., Einige Bemerkungen zur normalen and pathologischen Histologie des Schweif- und Linsenkerns, J. Psychol. Neurol. (Lpz.), 25 (1919) l - l l . 6 CARPENTER,M. B., AND STROM1NGER,N. L., Efferent fibers of the subthalamic nucleus in the monkey. A comparison of the efferent projections of the subthalamic nucleus, substantia nigra and globus pallidus, Amer. J. Anat., 121 (1967) 41-72. 7 CHALMERS,A., MCGEER, E. G., WICKSON, V., AND MCGEER, P. L., Distribution of glutamic acid decarboxylase in the brain of various mammalian species, Comp. gen. Pharmacol., 1 (1970) 385-390. 8 FAHN, S., AND COTE, L. J., Regional distribution of gamma-aminobutyric acid (GABA) in brain of the Rhesus monkey, J. Neurochem., 15 (1968) 209-213. 9 DENNY-BRowN,D., Diseases of the basal ganglia. Their relation to disorders of movement, The Lancet, (1960) 1099-1105; 1155-1162. 10 FONNUM,F., Radiochemical micro assays for the determination of choline acetyltransferase and AcChE, Biochem. J., 115 (1969) 465-472. 11 FONNUM,F., STORM-MATmSEN,J., ANDWALBER6,F., GABA synthesis in Purkinje axon terminals in cat, Abstracts of the 2nd lnt. Meeting of the Int. Soc. Neurochem., (1969) 170. 12 Fox, C. A., AND SCHMITZ,J. T., The substantia nigra and the entopeduncular nucleus in the cat, J. comp. Neurol., 80 (1944) 323-334. 13 GREENFIELD,J. G., AND BOSANQUET,F. D., Brain stem lesions in Parkinsonism, J. Neurol. Psychiat., 16 (1953) 213-226. 14 GRUNSTEIN,A. M., Zur Frage von den Leitungsbahnen des Corpus striatum, Neurol. Cbl., 30 (1911) 659-665. 15 HASSLER,R., Zur Pathologie der Paralysis agitans und des postenzephalitischen Parkinsonismus, J. Psychol. Neurol. (Leipz.), 48 (1938) 387--483. 16 HASSLER,R., Zur pathologischen Anatomie des senilen und des parkinsonistischen Tremor, J. Psychol. Neurol. (Lpz.), 49 (1939) 193-230. 17 HEATH,J. W., Clinicopathologic aspects of Parkinsonian states, Arch. Neurol. Psychiat. (Chic.), 58 (1947) 484-497. 18 IVERSEN,L. L., AND NEAL, M. J., Subeellular distribution of endogenous and aH-GABA in rat cerebral cortex, Brit. J. Pharmacol., 36 (1969) 206-208. 19 JOHNSON,T. N., AND CLEMENTE, C. O., An experimental study of the fiber connections between the putamen, globus pallidus, ventral thalamus and midbrain tegmentum in cat, J. comp. Neurol., 113 (1959) 83-101. 20 KARNOVSKY,M. J., AND ROOTS, L., A 'direct-coloring' thiocholine method for cholinesterases, J. Histochem. Cytochem., 12 (1964) 219-221. 21 KRNJEVI(~,K., AND SCHWARTZ,S., The action of gamma-aminobutyric acid on cortical neurons, Exp. Brain Res., 3 (1967) 320-336. 22 KURIYAMA,K., ROBERTS,E., AND KAKEFUDA,T., Association of the gamma-aminobutyric acid system with a synaptic vesicle fraction from mouse brain, Brain Research., 8 (1968) 132-152. 23 KURIYAMA,t . , SISKEN,B., ITO, J., SIMONSEN,D. G., HABER,B., AND ROBERTS,E., The ?'-aminobutyric acid system in the developing chick embryo cerebellum, Brain Research, 11 (1968) 412-430. 24 LEWY, F. H., Zur pathologischen Anatomie der Paralysis agitans, Dtsch. Z. Nervenheilk., 50 (1913) 50-55. 25 LOWE, I. P., ROmNS, E., AND EYERMAN,E. S., The fluorometric measurement of glutamic acid decarboxylase and its distribution in brain, J. Neurochem., 3 (1958) 8-18. 26 McGEER, E. G., IKEI)A,H., ASAI~URA,T., ANDWAOA,J. A., Lack of abnormality in brain aromatic amines in rats and mice susceptible to audiogenic seizure, J. Neurochem., 16 (1969) 945-950.
Brain Research, 32 (1971) 425-431
EXTRAPYRAMIDAL G A D AND PARKINSON'S DISEASE
431
27 MCGEER,E. G., GIBSON,S., AND MCGEER,P. L., Some characteristics of brain tyrosine hydroxylase, Canad. J. Biochem., 45 (1967) 1557-1563. 28 McGEER,P. L., WADA,J. A., GIBSON,S., LOESER,C., AND McGEER, E. G., Tyrosine hydroxylase levels in the cat caudate and basal forebrain following selective midbrain lesions. In A. BARBEAU AND A. BRUNETTE(Eds.), Progress in Neurogenetics, Excerpta Medica, Amsterdam, 1969, pp. 294-305. 29 MULLER,P. B., AND LANGEMANN,H., Distribution of glutamic acid decarboxylase activity in human brain, J. Neurochem., 9 (1962) 399-401. 30 POIRtER,L. J., McGEER, E. G., LAROCHELLE,L., McGEER, P. L., BEDARD,P., ANDBOUCHER,R., The effect of brain stem lesions on tyrosine and tryptophan hydroxylases in various structures of the telencephalon of the cat, Brain Research, 14 (1969) 147-155. 31 RANSON,S. W., AND RANSON,S. W., JR., Efferent fibers of the corpus striatum, Res. Publ. Ass. nerv. ment. Dis., 21 (1942) 69-76. 32 SALGANICOFF,L., AND DE ROBERTIS,E., Subcellular distribution of the enzymes of the glutamic acid glutamine and gamma-aminobutyric acid cycles in rat brain, J. Neurochem., 12 (1965) 287-309. 33 SNIDER,R. S., AND NIEMER, W. T., A Stereotaxic Atlas of the Cat Brain, Univ. Chicago Press, Chicago, Ill., 1961. 34 VERHAART,W. J. C., Fiber analysis of the basal ganglia, J. comp. Neurol., 93 (1950) 425--440. 35 WOODnURNE,R. T., CROSBY,E. C., ANDMcCOTrER, R. E., The mammalian midbrain and isthmus regions. Part II. The relations of the tegmentum of the midbrain with the basal ganglia in the Macaca mulatta, J. comp. Neurol., 85 (1946) 67-92.
Brain Research, 32 (1971) 425-431
425
EFFECTS OF GLOBUS PALLIDUS LESIONS AND PARKINSON'S DISEASE ON BRAIN GLUTAMIC ACID DECARBOXYLASE
P. L. McGEER, E. G. McGEER, J. A. WADA ANDE. JUNG Kinsmen Laboratory of Neurological Research, Department of Psychiatry, University of British Columbia, Vancouver 8 (Canada)
(Accepted March 10th, 1971)
INTRODUCTION Although the role of gamma-aminobutyric acid (GABA) in the central nervous system is unknown, information is accumulating suggesting that it may be a neurotransmitter. The unequal distribution in brain of GABA, and of the enzyme responsible for its synthesis, glutamic acid decarboxylase (GAD), is consistent with this hypothesis. High levels of GABA and GAD in structures associated with the extrapyramidal system have been repeatedly noted, with the highest levels reported being in the globus pallidus and substantia nigra in several mammalian speciesl,7,s,zs,~L We report here further evidence associating GAD with extrapyramidal function. Lesions to the region of the globus pallidus in cats caused a decrease in GAD in the substantia nigra. Both the globus pallidus and substantia nigra of individuals who died with Parkinson's disease were found to have lower levels of GAD in relation to the cerebral cortex than the same areas in patients who died with other diseases in hospital or in individuals who were killed suddenly in accidents. MATERIALSAND METHODS Extensive lesions to the region of the globus pallidus were produced in a series of 6 cats. Stereotaxic coordinates were from FI 1 to F15 mm in the anterior-posterior plane. Laterally, they extended from 5 to 9.5 mm and horizontally from --0.5 to --2.5 depending on the frontal level33. The cats were sacrificed between 15 and 41 days following lesion placement. The lesions were all examined grossly at the time of dissection. In one case the brain was fixed for histological study instead of being dissected for biochemical assay. Frozen sections of the fixed brain were cut and alternate sections were stained for cholinesterase20 and for cells with cresyl violet. In every case, the lesion spared the caudate nucleus and the substantia nigra completely. The lesions were nevertheless rather massive and encroachment on the Brain Research, 32 (1971)425--431
426
p.L. MCGEERet al.
medial part of the putamen and the lateral part of the internal capsule was definite in the fixed specimen and probable in the other five. Histochemical staining showed the globus pallidus to be completely destroyed. Adjacent pathways running between the cortex and midbrain, the basal ganglia and thalamus, and the striatum and midbrain obviously could have been damaged in all the cats. Cellular depopulation in the substantia nigra on the side of the lesion was evident in the fixed specimen despite the fact that the structure was grossly spared. Apparently the cellular degeneration was retrograde in nature. Glutamic acid decarboxylase was measured by a microradiometric method involving measurement of a4CO2 produced on incubation of tissue homogenate with L[l-14C]glutamic acid (30-60,000 disint./min, spec. act. 60/~Ci/mmole). Radioactive CO2 was trapped on hyamine hydroxide soaked paper and counted 7. Choline acetyltransferase was measured by a modification of the method of Fonnum m in which 60-182/~g of tissue, activated by treatment with 0.5 ~ Triton X-100, was incubated with radioactive [14C]acetyl--coenzyme A (50,000 disint./min, spec. act. 2.2 #Ci/ #mole). The incubation medium was 50 m M in phosphate, p H 7.4, 1 l m M in choline and in ethylenediaminetetraacetic acid, 290 m M in NaCI, 6 m M in sucrose, 0.1 m M in eserine and contained 0.6 mg/ml albumin (total volume 0.08 ml). As previously reported ~6 the [a4C]acetylcholine was absorbed onto a CG-50 ion exchange column and eluted with 3 ml of 4 N acetic acid. The eluate was counted. Acetylcholinesterase was measured by a colorimetric method which assays the amount of acetylcholine destroyed during an hour long incubation2< Tyrosine hydroxylase was measured by the method previously described eT, with the exception that for the substantia nigra the assay mixture contained no phosphate but instead was 0.2 M in acetate, p H 6.0, 1 0 4 M in DMPH4, 3 • 10 -4 M in FeSO4, and 0.1 M in 2-mercaptoethanol. RESULTS
The biochemical data for globus pallidus lesioned cats are summarized in Table I. G A D was decreased to 39 ~ of the control value in the substantia nigra on the TABLE
I
ENZYME LEVELS IN DISCRETE CAT BRAIN AREAS ON LESIONED SIDE AS ~oo OF CONTROL SIDE FOLLOWING UNILATERAL OLOBUS PALLIDUS LESIONS
Brain area
Enzyme % ± S.D.* Glutamic acid decarboxylase
Substantia nigra 39 4- 4.4 (5) Caudate 95.3 4- 6.0 (4) Cerebellum 110.6 ± 5.9 (5) Hippocampus 95.2 ± 4.8 (5)
Tyrosine hydroxylase
Choline acetyltransferase
Acetylcholinesterase
49.2 ± 17.6 (4) 23.7 ± 7.8 (3) ---
105.8 ± 18.6 (5) 101.7 ± 9.0 (4) -93.5 ± 5.0 (4)
88.8 ± 7.7 (5) 92.3 _q_6.2 (4) 99.3 ± 6.0 (5) 93.0 ± 8.6 (5)
* Number of animals indicated in brackets following values. Brain Research, 32 (1971) 425-431
EXTRAPYRAMIDAL
GAD
427
AND PARKINSON¢S DISEASI~
lesioned side. The absolute values for G A D in the substantia nigra were 22.2 ± 0.5 /zmoles/h/g on the control side v e r s u s 9.76 ~: 1.36 on the lesioned side. The difference was highly significant (P < 0.001). The absolute value for G A D in the caudate was 12.4 + 0.6 #moles/h/g, in the hippocampus 6.9 + 1.2 and in the cerebellum 11.1 ± 0.7. There was no significant change between the control and lesioned sides in these areas. Tyrosine hydroxylase was reduced to 49.29/0 and 23.7 9/0 of the control values in the substantia nigra and caudate respectively, while choline acetyltransferase and cholinesterase were not significantly affected in either structure. Lesions to the substantia nigra4,9,1a, 15 and globus pallidusS,9,17,24 have long been reported in Parkinson's disease. To test whether there might be any abnormality in G A D metabolism in this condition, the G A D content of various brain areas was measured in human postmortem brains. Tissue was obtained from Parkinson's disease patients, patients dying in hospitals with other illnesses, and Coroner's cases after sudden death. The glutamic acid decarboxylase values varied from brain to brain but the frontal cortical areas in any given brain were found to be highly comparable with one another. For each brain, 3 cortical areas in the frontal lobe were averaged. The G A D level in the frontal cortex was 9.42 :k 0.47 (n = 6) pmoles/g/h for Coroner's cases, 5.49 :k 1.44 (n -~ 6) for hospital cases, and 3.24 ± 0.47 (n = 6) for Parkinson's disease cases. The values for accident victims were almost identical with human cortical values obtained at biopsy in patients undergoing surgery for epilepsy. It is doubtful what significance can be attached to these differences. The accident victims tended to be younger (average age 31 as compared to 72 for the other groups) and some of the brains were dissected only 3 h after death. The average time between death and brain dissection was 8 h for the group of accident victims, 11 h for those suffering from such various illnesses as cancer, pneumonia, emphysema and heart failure, and 9 h for the Parkinsonian cases, who, like the other hospital cases, nearly all suffered a terminal pneumonia. Much more needs to be learned regarding the postmortem stability of GAD, the effects of prolonged coma prior to death, and the influence of age before any interpretation could be given of these overall differences. Although there was considerable variation in G A D activity from brain to brain, the relative activities of various areas tended to be more closely comparable. The ratio of the activity of each brain area to the activity in the frontal cortex, was therefore TABLE II GLUTAMIC ACID DECARBOXYLASEIN VARIOUSHUMAN BRAIN AREASAS ~oo OF AVERAGEOF 3 FRONTAL CORTEX AREAS 4- S.D.* Brain area
Parkinson
Other illness
Accident
Substantia nigra Globus pallidus Caudate Putamen Hippocampus
53 ± 8 (6) 83 4- 8 (6) 924-13(6) 107 4- 19 (6) 42 4- 3 (6)
170 4- 22 (6) 151 4- 21 (5) 1044-10(5) 89 4- 8 (5) 46 4- 6 (4)
205 139 91 99 60
-¢- 6 (4) -4- 17 (5) 4- 6(6) 4- 10 (6) 4- 9 (5)
* Number of brains indicated in brackets following value. Brain Research, 32 (1971) 425-431
428
P.L.
MCGEER e l t//~
calculated. On this basis, there was no significant difference between Parkinsonian and non-Parkinsonian patients for most areas, as illustrated in Table II by the data for the caudate, putamen, and hippocampus. The substantia nigra and globus pallidus, however, had reduced GAD in Parkinsonian patients. The substantia nigra had a level of only 53 ~ of the cortex in Parkinsonian patients while it was 205 o/in accident cases /o and 170~ in patients dying in other illnesses. Similarly the globus pallidus had a level of 83~ of the cortex in Parkinsonian patients but was 139~ in accident cases and 151 ~ in other illnesses. Further studies will be required to assess the significance of the findings. DISCUSSION
The etiology and pathogenesis of Parkinson's disease remain a mystery. Attention has focussed in recent years on the upset in dopamine metabolism and a possible involvement of the nigral-striatal dopaminergic tract 3. The finding of a possible alteration in glutamic acid decarboxylase metabolism suggests that the biochemical pathology may be considerably broader. As far as GABA is concerned, further research is necessary to elucidate its role. The iontophoretic effects of the substance when applied to single neurons 21 and the subcellular localization of GAD and GABA-aminotransferase1s,2'~,2~,32 argue in favor of a neurotransmitter function. If GABA is a neurotransmitter, then the GAD data following globus pallidus lesions could be explained on the basis of interruption of a gabaminergic pathway. If, on the other hand, GABA is associated with neuronal or glial function only in a metabolic way, then the drop in GAD might reflect a general cellular depopulation following the lesion. The drop in tyrosine hydroxylase in both the substantia nigra and the caudate in 3 cats with globus pallidus lesions would be consistent with damage to the known dopaminergic pathway between these two structures 2,28,3°. Since GAD was affected only in the substantia nigra and not in the caudate, the drop in value of this enzyme must have been unrelated to the dopaminergic pathway. The fact that neither choline acetyltransferase nor cholinesterase were affected in either the substantia nigra or caudate would imply that there is no major cholinergic pathway between the globus pallidus and either the substantia nigra or the caudate. It would also imply that a direct major cholinergic pathway between the caudate and substantia does not exist, or if it does exist, travels through a route that was spared by the globus pallidus lesions. A minor route could, of course, exist and not be revealed by the techniques employed in this study. Many authors have described pathways between the substantia nigra and the globus pallidus. The presence of rostrally directed fibres from the substantia nigra to the globus pallidus has been repeatedly confirmed6,12,31,aL The presence of caudally directed fibres from the globus pallidus to substantia nigra has been disputed. SomO 4,19 regard such fibres as established although others 30,34 have been unable to confirm their presence. Brain Research, 32 (1971) 425--431
EXTRAPYRAMIDAL
GAD
AND PARKINSON'S DISEASE
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Following axotomy, enzyme deterioration takes place. Initially enzymes associated with synaptosomes or other distal structures are affected, but eventually enzymes associated with the cells of origin disappear as well. The 15-41 day time lag between lesioning and sacrifice of the cats was such that total disappearance of some tracts took place. This was evidenced by the cellular dropout in the substantia nigra. Thus the drop in substantia nigra GAD could reflect deterioration of tracts coming into or leaving the substantia nigra, or both. Further experiments will be needed to determine whether any of these might depend on GABA as a neurotransmitter. Gabaminergic pathways have already been suggested for other areas of brain. One proposal is that Purkinje cells of the cerebellum elaborate GABA as an inhibitory transmitter at their terminals. This is supported by Fonnum's report 11 that lesions to Purkinje cells in the cerebellum cause a decrease in GAD in Deiters' nucleus in the brain stem. SUMMARY
Cats with unilateral lesions in the region of the globus pallidus showed a highly significant loss (to 39 ~ ) of glutamic acid decarboxylase (GAD) activity in the substantia nigra on the lesioned side. GAD was unaffected in the caudate nucleus or other structures. Tyrosine hydroxylase was reduced to 49 ~ of the control values in the substantia nigra and to 23.7 ~o in the caudate, while choline acetyltransferase and cholinesterase were not significantly affected in either structure. The data suggest that the lesion interrupted the known dopaminergic pathway between caudate and substantia nigra and interfered with another pathway concerned with GABA metabolism involving the substantia nigra but not the caudate. There appeared to be no major cholinergic pathway coursing through the lesioned area. The brains of accident victims and of non-Parkinsonian hospital patients dying with various illnesses showed the expected high GAD activities in the substantia nigra and globus pallidus (1.5 times that of the cortex). The brains of Parkinsonian patients, on the other hand, showed.decreased GAD levels in these structures. The GAD in the substantia nigra was only 53 ~o of that in the cortex, and the globus pallidus was only 83 ~. The caudate nucleus GAD was approximately equal to that of the cortex in all 3 groups. These data suggest the selective deterioration of a GAD containing cellular system in Parkinson disease which involves the substantia nigra and globus pallidus. ACKNOWLEDGEMENTS
This research was supported in part by grants from the Medical Research Council of Canada (MA-4013, MA-3633 and MA-2504) and the National Institutes of Health (NB-02812). We wish to thank Mrs. Virginia Wickson for technical assistance and the staff of the Pearson, Lions Gate, Royal Columbian, Shaughnessy and Vancouver General Hospitals as well as the Coroner's Offices in Vancouver and New Westminster for assistance in obtaining human tissue. Brain Research, 32 (1971) 425-431
430
p.L. McGEER et al.
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