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Distribution of noradrenaline and dopamine (3-hydroxytyramine) in the human brain and their behavior in diseases of the extrapyramidal system1
PRD 115
Parkinsonism & Related Disorders Parkinsonism and Related Disorders 4 (1998) 53–57
Distribution of noradrenaline and dopamine (3-hydroxytyramine) in the human brain and their behavior in diseases of the extrapyramidal system1 H. Ehringer, O. Hornykiewicz* Pharmacological Institute of the University of Vienna, Vienna, Austria
1. Introduction Catecholamines are known to occur in the brain of mammals [1,2]. In a fundamental study on the localisation of sympathin, a mixture of noradrenaline and adrenaline, Vogt [3] was able to show that in the brain of the dog sympathin’s concentration in the various brain regions did not correspond to the degree of their vascular supply, i.e. the density of their sympathetic innervation. Thus, the greatest amounts of sympathin were found in areas of the central representation of the sympathetic nervous system, foremost in the hypothalamus, in the stratum griseum centrale, and in the sympathetic centers of the medulla oblongata. Sympathin’s physiologic role in these brain structures has, however, not been definitely established, especially because the same regions also contain high levels of 5-hydroxytryptamine (5-HT). Recently a third catecholamine has gained special importance, namely 3,4-dihydroxyphenylethylalanine (3hydroxytyramine ¼ dopamine), which for some time now has been accepted as the precursor substance of noradrenaline [4–6]. Recently, dopamine has also been shown to occur in the human brain as well as the brain of various other mammalian species ([7,8]). It appears of the highest interest that the greatest amounts of dopamine have been found by Bertler and Rosengren [9] in the corpus striatum, where there is but little noradrenaline and serotonin present. This striking distribution suggests that dopamine, in addition to its significance as the precursor of noradrenaline, plays a physiological role of its own in the functioning of these brain nuclei. If noradrenaline and dopamine have indeed a specific role in brain functioning, a change in the concentration of these compounds should be followed by functional changes in the corresponding regions. In the human, changes in catecholamine levels in brain diseases, especially those affecting the extrapyramidal * Corresponding author: Dr. O. Hornykiewicz, Institute of Biochemical Pharmacology, Borschkegasse 8a, A-1090 Vienna, Austria. Fax: (+431) 4277-9643. 1 This article was originally published in German in the journal Klinische Wochenschrift, volume 38, issue 24, 15 December 1960, pp. 1236–1239.
1353-8020/98/$19.00 q 1998 Elsevier Science Ltd. All rights reserved PII S 13 53 - 80 2 0( 9 8) 0 00 1 2- 1
system, have, to our knowledge, never been reported. Birkmayer earlier suggested that we measure the postmortem 5-HT content of the hypothalamus of patients with Parkinson’s disease. However, this disease is characterized by functional disturbances in those nuclei of the extrapyramidal system where Bertler and Rosengren [9] had detected especially high amounts of dopamine. As we were not in a position to measure simultaneously, in small pieces of tissue, very low amounts of 5-HT, dopamine and noradrenaline, we chose to exclusively study the behaviour of dopamine and noradrenaline in various brain regions. The aim of the present study was as follows: 1. To determine the levels of dopamine and noradrenaline in various regions of the normal human brain. For this purpose we analyzed postmortem brains of 17 neurologically normal patients. 2. To study the catecholamine content during the ontogenesis of the human brain; for this we analyzed the brains of two fetuses. 3. To examine the brains of patients with extrapyramidal symptomatology. Among these were four cases of postencephalitic Parkinsonism; two cases of idiopathic Parkinson’s disease (paralysis agitans); two cases of Huntington’s chorea, as well as various other conditions accompanied by extrapyramidal symptoms.
2. Methodology and material The study was carried out in human brains obtained from the Institute of Pathological Anatomy of the University of Vienna and the Pathology Department of the City Hospital Wien–Lainz.2 The brains were dissected 3–20 h after death according to 2 We express our thanks to Professor Dr H. Chiari, Head of the Institute of Pathological Anatomy of the University of Vienna, Doz Dr W. Birkinayer, Head of the Neurology Section of the City Hospital Wien–Lainz and Professor Dr L. Haslhofer, Head of the Pathology Department of the City Hospital Wien–Lainz for kindly making the brain material available to us as well as for their advice.
54
H. Ehringer, O. Hornykiewicz/Parkinsonism and Related Disorders 4 (1998) 53–57
the following procedure. Several frontal cuts were made through the brain so as to obtain brain slices that permitted good orientation and easy dissection of the discrete regions and nuclei. Care was taken not to include any white matter with the dissected-out nuclei. The non-fixed material taken out in this manner was frozen without delay in tightly closed vials at ¹ 208C, and was only thawed immediately before being processed. The extraction of noradrenaline and dopamine was carried out according to the method of Bertler et al. [10], using perchloric acid and the ion exchange resin Dowex 50 3 8 (200–400 mesh), with the modifications described by Holzer and Hornykiewicz [11]. Noradrenaline was eluted with 6 ml of 1 N HCl followed by the elution of dopamine with 6 ml 2 N HCl. The quantitative estimation of noradrenaline was performed fluorimetrically according to Euler and Floding [12] as modified by De Schaepdryver [13]. The noradrenaline eluates were adjusted to a pH of 3–4 by means of NaHCO 3 (as substance); the oxidation reaction was carried out in an acetate buffer at pH 6. At this pH both noradrenaline and adrenaline are oxidized. However, since by performing the oxidation reaction at pH 3.5 we never could detect any adrenaline in the human brain, in the following the values measured at pH 6 will be referred to as "noradrenaline". The measurements were carried out in a Beckman photofluorimeter using as
standard a quinine solution (0.25 mg/ml in 0.1 N H 2SO 4) and Schott G12 as primary filter and Schott OG4 as secondary filter. The dopamine fraction (2 N HCl) was evaporated to dryness in a vacuum at 55–608C, the residue dissolved in 1 ml distilled water and quantitatively estimated at 529 mm using the colorimetric assay of Euler and Hamberg [14]. Using the non-oxidized extract as blank, the limit of sensitivity of the method is 2 mg. The relative insensitivity of this assay forced us to pool, from several brains, regions of small size and expected low dopamine level and process them as a single sample. This procedure permitted us to record even relatively low dopamine values per gramme tissue.
3. Results 3.1. Distribution of noradrenaline and dopamine in the brains of adult human subjects The results of the 17 analyzed brains of adult subjects are presented in Table 1. The distribution of noradrenaline in our material corresponds basically to the values reported by Vogt [3] for sympathin in the brain of the dog. Also the results on noradrenaline and dopamine obtained by Sano et al. [15] in three analyzed human brains (HF poisoning,
Table 1 Distribution of noradrenaline and dopamine in the brain of adult human subjects Noradrenaline (mg/g wt.) Region analyzed
Telencephalon Caudate nucleus Putamen Globus pallidus Arnygdala Septurn region White matter Diencephalon Thalamus Thalamus (medial) Hypothalamus Mesencephalon Red nucleus Stratum griseum centrale Pretectal region Substantia nigra Rhombencephalon Formatio reticularis Floor of the 4th ventricle Inferior olive Pons Area postrema Cerebellum Dentate nucleus a
Dopamine (mg/g wt.)
Number of brains examined a
Range
Mean
Number of brains examined
Range
Mean
6 7 7(4) 7(3) 4(1) 1
0.06-0.14 0.08-0.14 0.05-0.30 0.08-0.24 – –
0.09 0.12 0.15 0.21 0.31 0.01
10 12 13(6) 5(2) 4 (1) 1
2.7-5.5 2.1-5.3 0.3–1.8 0.5-1.0 – –
3.5 3.7 0.5 0.6 0.3 0.0
5(3) 3(1) 11(5)
0.09-0.14 – 0.80-1.67
0.13 0.22 1.25
8(4) 3(1) 11(5)
0.2-0.4 – 0.5-1.7
0.3 0.4 0.8
7(2)
0.29-0.40
0.30
9(2)
0.5-1.1
0.7
6(1) 5(1) 8(2)
– – 0.20-0.28
0.46 0.12 0.21
6(1) 5(1) 7(1)
– – –
0.5 0.4 0.9
8(3) 7(2)
0.23-0.34 0.29-0.39
0.28 0.35
5(2) 12(3)
0.3-0.6 0.2-1.0
0.6 0.6
4(1) 2(1) 3(1)
– – –
0.11 0.13 2.06
7(2) 2(1) 3(1)
0.4-0.8 – –
0.6 0.2 1.3
4(1)
–
0.06
4(1)
–
, 0.5
In several instances, regions were pooled from a number of brains and assayed as single samples. In such cases, the numbers given in parentheses denote the number of actual estimations, each performed with pooled material. For some regions only an average value obtained from several brains is given.
55
H. Ehringer, O. Hornykiewicz/Parkinsonism and Related Disorders 4 (1998) 53–57 Table 2 Noradrenaline and dopamine in selected regions of the fetal brain Brain region
Noradrenaline (mg/g wt.) Neonatus 48 cm long
Praematurus 40 cm long
Caudate nucleus Putamen Globus pallidus Hypothalamus
0.14 0.15 0.32 1.16
not studied 0.12 0.35 0.92
Dopamine (mg/g wt.) Neonatus 48 cm long 0.3 1.0
Praematurus 40 cm long not studied 1.1 not studied not studied
Table 3 Human brain dopamine and noradrenaline in postencephalitic Parkinsonism Noradrenaline (mg/g wt.)
Dopamine (mg/g wt.)
Brain region Caudate nucleus Putamen Globus pallidus Hypothalamus
Individual results
Mean
Individual results
Mean
0.00-0.02-0.04-0.00 0.01-0.02-0.05-0.04 0.46-0.13-0.20-0.14 -0.27-1.99-0.68
0.02 0.03 0.23 0.98
0.1-0.2-0.0-0.5 0.3-0.5-0.2-0.1 0.1-0.2-0.0 not studied
0.2 0.3 0.1 –
strangulation, cerebral embolism) are analogous to our findings (see also Bertler and Rosengren [16]). The consistency of our results is striking. We could see little difference in the amine levels regardless whether the brains were dissected and frozen 3 h or 20 h post mortem. This made it possible for us to compare these values with the values obtained in other brains that were handled under the same conditions. 3.2. Distribution of noradrenaline and dopamine in discrete brain regions of human fetuses Of the two brains examined, one was of a 48 cm long fetus and the other of a 40 cm long fetus. Tissue samples were taken only from those regions that could be identified with certainty. The results are summarized in Table 2. It is striking that the noradrenaline content of all analyzed regions, especially the hypothalamus, was already within the normal range, whereas the dopamine content in the corpus striatum was distinctly lower than in normal adults. In contrast, in the brain of a 4 month old infant the values for dopamine and noradrenaline were already comparable to the values given in Table 1. 3.3. Dopamine and noradrenaline in the brain of patients with extrapyramidal disorders
and autonomic signs as well as demelanization of the substantia nigra at autopsy. The noradrenaline and dopamine concentrations in the most relevant brain regions of four such cases are presented in Table 3. The other analyzed regions displayed no notable changes compared with normal values and have not been included in the table.3 An especially striking feature in this disorder was the greatly reduced dopamine level in the caudate nucleus and the putamen. In two cases the noradrenaline content in the hypothalamus was also reduced. In this context it should again be emphasized that these brains were handled under exactly the same conditions as the normal brains in Table 1. 3.3.2. Idiopathic Parkinson’s disease Decisive for the diagnosis was the clinical picture of the case, absence of oculogyric crises, no history of encephalitis, and the late onset of the disease. We were in a position to study two such cases. The results are shown in Table 4. Again, it is remarkable that the dopamine values in the corpus striatum were markedly reduced, although not as much as in the brain of the cases with postencephalitic Parkinsonism. The noradrenaline content in one case analyzed was also reduced.
In order to obtain possible indications as to the physiological role of dopamine and noradrenaline in the central nervous system, we examined post mortem brains of 14 patients presenting with extrapyramidal symptoms. Some of these patients also showed vegetative symptoms.
3.3.3. Other extrapyramidal syndromes We also analyzed the brain catecholamines in two cases of Huntington’s chorea, one case of a child with the so-called cerebral damage syndrome as well as five patients who had presented with extrapyramidal symptoms (tremor, rigidity, hyperkinetic condition) of unknown etiology. In none of these cases could we find abnormal catecholamine values.
3.3.1. Postencephalitic Parkinsonism Decisive for the diagnosis of this condition was a history of encephalitis, presence of oculogyric crises, extrapyramidal
3 The dopamine content in the substantia nigra was too low to be measured by our method in single cases; therefore, we were not in a position to analyze this region.
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H. Ehringer, O. Hornykiewicz/Parkinsonism and Related Disorders 4 (1998) 53–57
Table 4 Human brain dopamine and noradrenaline in idiopathic Parkinson’s disease Brain region
Noradrenaline (mg/g wt.) Individual results
Mean
Dopamine (mg/g wt.) Individual results
Mean
Caudate nuleus Putamen Clobus pallidus Hypothalamus
0.06-0.10 0.08-0.06 0.06 0.53
0.08 0.07 not studied not studied
1.9-0.3 1.2-0.3 0.3 not studied
1.1 0.8 not studied not studied
4. Discussion The results of the above presented study can be divided into three groups: topography of dopamine and noradrenaline in the human brain; behaviour of these catecholamines during brain development; and behaviour of dopamine and noradrenaline in diseases of the extrapyramidal system. With regard to the topography of dopamine in the human brain we would like to make the following remarks in addition to what we already have said in Section 3. The fact that substantial amounts of dopamine are found only in the neostriatum speaks in favour of its special function in these nuclei. The high dopamine content of the caudate nucleus and the putamen is surprising considering the findings of Udenfriend and Creveling [17], indicating that the activity of dopamine-b-hydroxylase in these regions is as high as in the hypothalamus. This enzyme is responsible for the hydroxylation of dopamine to noradrenaline. Nevertheless, because the neostriatum is particularly poor in noradrenaline, it has to be assumed that dopamine and the hydroxylating enzyme are localised in different cellular elements, so that dopamine is protected from the action of the enzyme. Of special interest is the opposite behaviour of dopamine and noradrenaline during the embryogenesis of the brain. Whereas in the two analyzed fetuses the dopamine content of the neostriatum was definitely lower than in the adults, in both these cases the noradrenaline content of the hypothalamus was already quite normal. These findings may perhaps be explained by what is known about the embryogenesis of the brain. According to Spatz [18] the diencephalon attains a considerable degree of maturity already at the end of the third lunar month, whereas the differentiation of the neostriatum takes place at a much later stage. It is, therefore, possible that in the less differentiated cells and tissues also the activity of the enzymes necessary for the synthesis and the catabolism of catecholamines is lower than in maturationally more advanced structures. Shimizu and Morikawa [19] demonstrated analogous differences studying the activity of the monoamine oxidase in the developing rat brain. Similar studies about other enzymes involved in the metabolism of catecholamines are not known to us. The dopamine content of the neostriatum in the cases of idiopathic Parkinson’s disease and postencephalitic Parkinsonism analyzed by us was reduced to about onetenth of normal. All these cases presented with an akinetichypertonic syndrome, that is with akinesia, accompanied by
rigidity and mild resting tremor. In contrast, extrapyramidal syndromes of the hyperkinetic type, such as Huntington’s chorea, had, in our study, normal amounts of dopamine. It appears quite reasonable to relate the lack of dopamine in the neostriatum to the akinetic-hypertonic symptoms. The question, however, whether the latter are directly due to the low dopamine cannot be answered by our study. The difficulty in proving this relationship by means of animal experiments lies in the fact that the experimentally induced lack of brain dopamine is invariably accompanied by reduced levels of noradrenaline and 5-HT. The appearance of Parkinson-like symptoms during prolonged treatment with reserpine offers, in this respect, perhaps a certain analogy to our findings. Reserpine reduces, inter alia, also the dopamine level in the brain [8]. Indeed, according to Carlsson et al. [20], it is possible to abolish certain reserpine effects, especially the sedation, in animals by dihydroxyphenylalanine (Dopa), the precursor of noradrenaline and dopamine. Along similar lines might be interpreted the long-known effects of harmala alkaloids, which also increase brain dopamine levels [11], and which have been used with success in Parkinson’s disease [21]. Regarding dopamine, however, there can be no question that we could not see any qualitative difference between postencephalitic Parkinsonism and idiopathic Parkinson’s disease, though the biochemical changes in postencephalitic Parkinsonism were more marked. This is in good agreement with the view of Klaue [22] regarding the pathogenesis of these two syndromes. Based on our results we would like to briefly discuss the question of brain dopamine’s localisation. Whereas in the periphery dopamine is in part localized to chromatin cells [23,24], nothing is known about its localisation in the cells of the neostriatum.4 In this respect, three cell types may in principle be considered: the most numerous small-sized neurones; the less numerous large multipolar (polygonal) neurones; and finally the glia cells. Concerning this question, we should like to make the following suggestion, based on our study: In Huntington’s chorea there occurs, according to literature [25], up to 90% loss of the smallsized neostriatal neurones and a moderate glia proliferation. In spite of this the dopamine content of these nuclei is, 4 It is also not known to which structural cell constituents brain dopamine is bound: it appears certain, however, that only a small portion of the amine exists in a free state in the cytoplasm [26].
H. Ehringer, O. Hornykiewicz/Parkinsonism and Related Disorders 4 (1998) 53–57
according to our findings, quite normal. The small neurones can thus be excluded as the seat of dopamine. There remain the large neurones and glia cells. Of these two cell types we would like to give preference to the large multipolar neurones. In order to furnish direct proof for this view, however, it would be necessary to study diseases in which this type of cell is preferentially affected. In idiopathic Parkinson’s disease and in postencephalitic Parkinsonism the histological changes in the neostriaturn are so negligible as to preclude any significant conclusions. Thus it might be that these two disorders are possibly related to functional changes in the neostriatal cells in connection with disturbed dopamine metabolism. The high degree of dopamine reduction in the neostriatum in idiopathic Parkinson’s disease and postencephalitic Parkinsonism furnishes, apart from the findings of Bertler and Rosengren [9], new evidence for the physiological significance of dopamine in these nuclei. Unfortunately, we could examine only six such cases. Should this finding be confirmed in further cases, it could be regarded as comparable in significance to the histological changes in the substantia nigra. In this case, a particularly great importance would have to be attributed to dopamine’s role in the pathophysiology and symptomatology of idiopathic Parkinson’s disease and postencephalitic Parkinsonism. In addition, this would represent for the first time direct evidence for the physiological role of dopamine in the brain. The reduction of noradrenaline in the hypothalamus in the three cases analyzed may be due to cellular losses frequently found in this region.
5. Summary The distribution of noradrenaline and dopamine in the brain of adult human subjects (post mortem) and of fetuses was studied. In a total of six cases of idiopathic Parkinson’s disease and postencephalitic Parkinsonism the dopamine
57
content of the neostriatum was found to be severely reduced; also the noradrenaline content of the hypothalamus was lower than in the control cases. Based on these findings, the importance of dopamine for the pathophysiology and symptomatology of the above diseases as well as its physiological role in the neostriatum are discussed. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26]
Euler US. Acta Physiol Scand 1946;12:73. Holtz P. Acta Physiol Scand 1950;20:354. Vogt M. J Physiol (Lond) 1954;123:451. Demis DJ, Blaschko H, Welch AD. J Pharmacol 1955;117:208. Hagen P, Welch A. Recent Progr Hormone Res 1955;12:27. Hagen P. J Pharmacol 1956;116:26. Montagu KA. Nature (Lond) 1957;180:244. Carlsson A, Lindqvist M, Magnusson T, Waldeck B. Science 1958;127:471. Bertler A, Rosengren E. Experientia (Basel) 1959;15:10. Bertler A, Carlsson A, Rosengren E. Fysiogr Sa¨llsk Handl 1958;28:121. Holzer G, Hornykiewicz O. Naunyn-Schmiedeberg’s Arch Exp Path Pharmak 1959;237:27. Euler US., Floding I. Acta Physiol Scand 1955;32(Suppl. 118):57. De Schaepdryver AF. Arch Int Phamacodyn 1958;115:233. Euler US, Hamberg U. Acta Physiol Scand 1949;19:74. Sano I, Gamo T, Kakimoto Y, Taniguchi K, Takesada M, Nishinuma K. Biochim Biophys Acta 1959;32:586. Bertler A, Rosengren E. Experientia 1959;15:382. Udenfriend S, Creveling CR. J Neurochem 1959;4:350. Spatz H. Anat Anz Erg 1925;60:54. Shimizu N, Morikawa N. Nature (Lond) 1959;184:650. Carlsson A, Lindqvist M, Magnusson T. Nature (Lond) 1957; 180:1200. Gunn JA. Arch Int Pharmacodyn 1935;50:379. Klaue R. Arch Psychiat Nervenkr 1940;111:251. Bertler A, Falk B, Hillarp A, Rosengren E, Torp A. Acta Physiol Scand 1959;47:251. Eade NR. J Physiol (Lond) 1958;141:183. Hallervorden J. Handbuch der Speziellen Pathologischen Anatomie und Pathologie, Bd. XIII/I/A, 1957:798. Weil-Malherbe H, Bone AD. Nature 1957;180:1050.
Parkinsonism & Related Disorders Parkinsonism and Related Disorders 4 (1998) 53–57
Distribution of noradrenaline and dopamine (3-hydroxytyramine) in the human brain and their behavior in diseases of the extrapyramidal system1 H. Ehringer, O. Hornykiewicz* Pharmacological Institute of the University of Vienna, Vienna, Austria
1. Introduction Catecholamines are known to occur in the brain of mammals [1,2]. In a fundamental study on the localisation of sympathin, a mixture of noradrenaline and adrenaline, Vogt [3] was able to show that in the brain of the dog sympathin’s concentration in the various brain regions did not correspond to the degree of their vascular supply, i.e. the density of their sympathetic innervation. Thus, the greatest amounts of sympathin were found in areas of the central representation of the sympathetic nervous system, foremost in the hypothalamus, in the stratum griseum centrale, and in the sympathetic centers of the medulla oblongata. Sympathin’s physiologic role in these brain structures has, however, not been definitely established, especially because the same regions also contain high levels of 5-hydroxytryptamine (5-HT). Recently a third catecholamine has gained special importance, namely 3,4-dihydroxyphenylethylalanine (3hydroxytyramine ¼ dopamine), which for some time now has been accepted as the precursor substance of noradrenaline [4–6]. Recently, dopamine has also been shown to occur in the human brain as well as the brain of various other mammalian species ([7,8]). It appears of the highest interest that the greatest amounts of dopamine have been found by Bertler and Rosengren [9] in the corpus striatum, where there is but little noradrenaline and serotonin present. This striking distribution suggests that dopamine, in addition to its significance as the precursor of noradrenaline, plays a physiological role of its own in the functioning of these brain nuclei. If noradrenaline and dopamine have indeed a specific role in brain functioning, a change in the concentration of these compounds should be followed by functional changes in the corresponding regions. In the human, changes in catecholamine levels in brain diseases, especially those affecting the extrapyramidal * Corresponding author: Dr. O. Hornykiewicz, Institute of Biochemical Pharmacology, Borschkegasse 8a, A-1090 Vienna, Austria. Fax: (+431) 4277-9643. 1 This article was originally published in German in the journal Klinische Wochenschrift, volume 38, issue 24, 15 December 1960, pp. 1236–1239.
1353-8020/98/$19.00 q 1998 Elsevier Science Ltd. All rights reserved PII S 13 53 - 80 2 0( 9 8) 0 00 1 2- 1
system, have, to our knowledge, never been reported. Birkmayer earlier suggested that we measure the postmortem 5-HT content of the hypothalamus of patients with Parkinson’s disease. However, this disease is characterized by functional disturbances in those nuclei of the extrapyramidal system where Bertler and Rosengren [9] had detected especially high amounts of dopamine. As we were not in a position to measure simultaneously, in small pieces of tissue, very low amounts of 5-HT, dopamine and noradrenaline, we chose to exclusively study the behaviour of dopamine and noradrenaline in various brain regions. The aim of the present study was as follows: 1. To determine the levels of dopamine and noradrenaline in various regions of the normal human brain. For this purpose we analyzed postmortem brains of 17 neurologically normal patients. 2. To study the catecholamine content during the ontogenesis of the human brain; for this we analyzed the brains of two fetuses. 3. To examine the brains of patients with extrapyramidal symptomatology. Among these were four cases of postencephalitic Parkinsonism; two cases of idiopathic Parkinson’s disease (paralysis agitans); two cases of Huntington’s chorea, as well as various other conditions accompanied by extrapyramidal symptoms.
2. Methodology and material The study was carried out in human brains obtained from the Institute of Pathological Anatomy of the University of Vienna and the Pathology Department of the City Hospital Wien–Lainz.2 The brains were dissected 3–20 h after death according to 2 We express our thanks to Professor Dr H. Chiari, Head of the Institute of Pathological Anatomy of the University of Vienna, Doz Dr W. Birkinayer, Head of the Neurology Section of the City Hospital Wien–Lainz and Professor Dr L. Haslhofer, Head of the Pathology Department of the City Hospital Wien–Lainz for kindly making the brain material available to us as well as for their advice.
54
H. Ehringer, O. Hornykiewicz/Parkinsonism and Related Disorders 4 (1998) 53–57
the following procedure. Several frontal cuts were made through the brain so as to obtain brain slices that permitted good orientation and easy dissection of the discrete regions and nuclei. Care was taken not to include any white matter with the dissected-out nuclei. The non-fixed material taken out in this manner was frozen without delay in tightly closed vials at ¹ 208C, and was only thawed immediately before being processed. The extraction of noradrenaline and dopamine was carried out according to the method of Bertler et al. [10], using perchloric acid and the ion exchange resin Dowex 50 3 8 (200–400 mesh), with the modifications described by Holzer and Hornykiewicz [11]. Noradrenaline was eluted with 6 ml of 1 N HCl followed by the elution of dopamine with 6 ml 2 N HCl. The quantitative estimation of noradrenaline was performed fluorimetrically according to Euler and Floding [12] as modified by De Schaepdryver [13]. The noradrenaline eluates were adjusted to a pH of 3–4 by means of NaHCO 3 (as substance); the oxidation reaction was carried out in an acetate buffer at pH 6. At this pH both noradrenaline and adrenaline are oxidized. However, since by performing the oxidation reaction at pH 3.5 we never could detect any adrenaline in the human brain, in the following the values measured at pH 6 will be referred to as "noradrenaline". The measurements were carried out in a Beckman photofluorimeter using as
standard a quinine solution (0.25 mg/ml in 0.1 N H 2SO 4) and Schott G12 as primary filter and Schott OG4 as secondary filter. The dopamine fraction (2 N HCl) was evaporated to dryness in a vacuum at 55–608C, the residue dissolved in 1 ml distilled water and quantitatively estimated at 529 mm using the colorimetric assay of Euler and Hamberg [14]. Using the non-oxidized extract as blank, the limit of sensitivity of the method is 2 mg. The relative insensitivity of this assay forced us to pool, from several brains, regions of small size and expected low dopamine level and process them as a single sample. This procedure permitted us to record even relatively low dopamine values per gramme tissue.
3. Results 3.1. Distribution of noradrenaline and dopamine in the brains of adult human subjects The results of the 17 analyzed brains of adult subjects are presented in Table 1. The distribution of noradrenaline in our material corresponds basically to the values reported by Vogt [3] for sympathin in the brain of the dog. Also the results on noradrenaline and dopamine obtained by Sano et al. [15] in three analyzed human brains (HF poisoning,
Table 1 Distribution of noradrenaline and dopamine in the brain of adult human subjects Noradrenaline (mg/g wt.) Region analyzed
Telencephalon Caudate nucleus Putamen Globus pallidus Arnygdala Septurn region White matter Diencephalon Thalamus Thalamus (medial) Hypothalamus Mesencephalon Red nucleus Stratum griseum centrale Pretectal region Substantia nigra Rhombencephalon Formatio reticularis Floor of the 4th ventricle Inferior olive Pons Area postrema Cerebellum Dentate nucleus a
Dopamine (mg/g wt.)
Number of brains examined a
Range
Mean
Number of brains examined
Range
Mean
6 7 7(4) 7(3) 4(1) 1
0.06-0.14 0.08-0.14 0.05-0.30 0.08-0.24 – –
0.09 0.12 0.15 0.21 0.31 0.01
10 12 13(6) 5(2) 4 (1) 1
2.7-5.5 2.1-5.3 0.3–1.8 0.5-1.0 – –
3.5 3.7 0.5 0.6 0.3 0.0
5(3) 3(1) 11(5)
0.09-0.14 – 0.80-1.67
0.13 0.22 1.25
8(4) 3(1) 11(5)
0.2-0.4 – 0.5-1.7
0.3 0.4 0.8
7(2)
0.29-0.40
0.30
9(2)
0.5-1.1
0.7
6(1) 5(1) 8(2)
– – 0.20-0.28
0.46 0.12 0.21
6(1) 5(1) 7(1)
– – –
0.5 0.4 0.9
8(3) 7(2)
0.23-0.34 0.29-0.39
0.28 0.35
5(2) 12(3)
0.3-0.6 0.2-1.0
0.6 0.6
4(1) 2(1) 3(1)
– – –
0.11 0.13 2.06
7(2) 2(1) 3(1)
0.4-0.8 – –
0.6 0.2 1.3
4(1)
–
0.06
4(1)
–
, 0.5
In several instances, regions were pooled from a number of brains and assayed as single samples. In such cases, the numbers given in parentheses denote the number of actual estimations, each performed with pooled material. For some regions only an average value obtained from several brains is given.
55
H. Ehringer, O. Hornykiewicz/Parkinsonism and Related Disorders 4 (1998) 53–57 Table 2 Noradrenaline and dopamine in selected regions of the fetal brain Brain region
Noradrenaline (mg/g wt.) Neonatus 48 cm long
Praematurus 40 cm long
Caudate nucleus Putamen Globus pallidus Hypothalamus
0.14 0.15 0.32 1.16
not studied 0.12 0.35 0.92
Dopamine (mg/g wt.) Neonatus 48 cm long 0.3 1.0
Praematurus 40 cm long not studied 1.1 not studied not studied
Table 3 Human brain dopamine and noradrenaline in postencephalitic Parkinsonism Noradrenaline (mg/g wt.)
Dopamine (mg/g wt.)
Brain region Caudate nucleus Putamen Globus pallidus Hypothalamus
Individual results
Mean
Individual results
Mean
0.00-0.02-0.04-0.00 0.01-0.02-0.05-0.04 0.46-0.13-0.20-0.14 -0.27-1.99-0.68
0.02 0.03 0.23 0.98
0.1-0.2-0.0-0.5 0.3-0.5-0.2-0.1 0.1-0.2-0.0 not studied
0.2 0.3 0.1 –
strangulation, cerebral embolism) are analogous to our findings (see also Bertler and Rosengren [16]). The consistency of our results is striking. We could see little difference in the amine levels regardless whether the brains were dissected and frozen 3 h or 20 h post mortem. This made it possible for us to compare these values with the values obtained in other brains that were handled under the same conditions. 3.2. Distribution of noradrenaline and dopamine in discrete brain regions of human fetuses Of the two brains examined, one was of a 48 cm long fetus and the other of a 40 cm long fetus. Tissue samples were taken only from those regions that could be identified with certainty. The results are summarized in Table 2. It is striking that the noradrenaline content of all analyzed regions, especially the hypothalamus, was already within the normal range, whereas the dopamine content in the corpus striatum was distinctly lower than in normal adults. In contrast, in the brain of a 4 month old infant the values for dopamine and noradrenaline were already comparable to the values given in Table 1. 3.3. Dopamine and noradrenaline in the brain of patients with extrapyramidal disorders
and autonomic signs as well as demelanization of the substantia nigra at autopsy. The noradrenaline and dopamine concentrations in the most relevant brain regions of four such cases are presented in Table 3. The other analyzed regions displayed no notable changes compared with normal values and have not been included in the table.3 An especially striking feature in this disorder was the greatly reduced dopamine level in the caudate nucleus and the putamen. In two cases the noradrenaline content in the hypothalamus was also reduced. In this context it should again be emphasized that these brains were handled under exactly the same conditions as the normal brains in Table 1. 3.3.2. Idiopathic Parkinson’s disease Decisive for the diagnosis was the clinical picture of the case, absence of oculogyric crises, no history of encephalitis, and the late onset of the disease. We were in a position to study two such cases. The results are shown in Table 4. Again, it is remarkable that the dopamine values in the corpus striatum were markedly reduced, although not as much as in the brain of the cases with postencephalitic Parkinsonism. The noradrenaline content in one case analyzed was also reduced.
In order to obtain possible indications as to the physiological role of dopamine and noradrenaline in the central nervous system, we examined post mortem brains of 14 patients presenting with extrapyramidal symptoms. Some of these patients also showed vegetative symptoms.
3.3.3. Other extrapyramidal syndromes We also analyzed the brain catecholamines in two cases of Huntington’s chorea, one case of a child with the so-called cerebral damage syndrome as well as five patients who had presented with extrapyramidal symptoms (tremor, rigidity, hyperkinetic condition) of unknown etiology. In none of these cases could we find abnormal catecholamine values.
3.3.1. Postencephalitic Parkinsonism Decisive for the diagnosis of this condition was a history of encephalitis, presence of oculogyric crises, extrapyramidal
3 The dopamine content in the substantia nigra was too low to be measured by our method in single cases; therefore, we were not in a position to analyze this region.
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Table 4 Human brain dopamine and noradrenaline in idiopathic Parkinson’s disease Brain region
Noradrenaline (mg/g wt.) Individual results
Mean
Dopamine (mg/g wt.) Individual results
Mean
Caudate nuleus Putamen Clobus pallidus Hypothalamus
0.06-0.10 0.08-0.06 0.06 0.53
0.08 0.07 not studied not studied
1.9-0.3 1.2-0.3 0.3 not studied
1.1 0.8 not studied not studied
4. Discussion The results of the above presented study can be divided into three groups: topography of dopamine and noradrenaline in the human brain; behaviour of these catecholamines during brain development; and behaviour of dopamine and noradrenaline in diseases of the extrapyramidal system. With regard to the topography of dopamine in the human brain we would like to make the following remarks in addition to what we already have said in Section 3. The fact that substantial amounts of dopamine are found only in the neostriatum speaks in favour of its special function in these nuclei. The high dopamine content of the caudate nucleus and the putamen is surprising considering the findings of Udenfriend and Creveling [17], indicating that the activity of dopamine-b-hydroxylase in these regions is as high as in the hypothalamus. This enzyme is responsible for the hydroxylation of dopamine to noradrenaline. Nevertheless, because the neostriatum is particularly poor in noradrenaline, it has to be assumed that dopamine and the hydroxylating enzyme are localised in different cellular elements, so that dopamine is protected from the action of the enzyme. Of special interest is the opposite behaviour of dopamine and noradrenaline during the embryogenesis of the brain. Whereas in the two analyzed fetuses the dopamine content of the neostriatum was definitely lower than in the adults, in both these cases the noradrenaline content of the hypothalamus was already quite normal. These findings may perhaps be explained by what is known about the embryogenesis of the brain. According to Spatz [18] the diencephalon attains a considerable degree of maturity already at the end of the third lunar month, whereas the differentiation of the neostriatum takes place at a much later stage. It is, therefore, possible that in the less differentiated cells and tissues also the activity of the enzymes necessary for the synthesis and the catabolism of catecholamines is lower than in maturationally more advanced structures. Shimizu and Morikawa [19] demonstrated analogous differences studying the activity of the monoamine oxidase in the developing rat brain. Similar studies about other enzymes involved in the metabolism of catecholamines are not known to us. The dopamine content of the neostriatum in the cases of idiopathic Parkinson’s disease and postencephalitic Parkinsonism analyzed by us was reduced to about onetenth of normal. All these cases presented with an akinetichypertonic syndrome, that is with akinesia, accompanied by
rigidity and mild resting tremor. In contrast, extrapyramidal syndromes of the hyperkinetic type, such as Huntington’s chorea, had, in our study, normal amounts of dopamine. It appears quite reasonable to relate the lack of dopamine in the neostriatum to the akinetic-hypertonic symptoms. The question, however, whether the latter are directly due to the low dopamine cannot be answered by our study. The difficulty in proving this relationship by means of animal experiments lies in the fact that the experimentally induced lack of brain dopamine is invariably accompanied by reduced levels of noradrenaline and 5-HT. The appearance of Parkinson-like symptoms during prolonged treatment with reserpine offers, in this respect, perhaps a certain analogy to our findings. Reserpine reduces, inter alia, also the dopamine level in the brain [8]. Indeed, according to Carlsson et al. [20], it is possible to abolish certain reserpine effects, especially the sedation, in animals by dihydroxyphenylalanine (Dopa), the precursor of noradrenaline and dopamine. Along similar lines might be interpreted the long-known effects of harmala alkaloids, which also increase brain dopamine levels [11], and which have been used with success in Parkinson’s disease [21]. Regarding dopamine, however, there can be no question that we could not see any qualitative difference between postencephalitic Parkinsonism and idiopathic Parkinson’s disease, though the biochemical changes in postencephalitic Parkinsonism were more marked. This is in good agreement with the view of Klaue [22] regarding the pathogenesis of these two syndromes. Based on our results we would like to briefly discuss the question of brain dopamine’s localisation. Whereas in the periphery dopamine is in part localized to chromatin cells [23,24], nothing is known about its localisation in the cells of the neostriatum.4 In this respect, three cell types may in principle be considered: the most numerous small-sized neurones; the less numerous large multipolar (polygonal) neurones; and finally the glia cells. Concerning this question, we should like to make the following suggestion, based on our study: In Huntington’s chorea there occurs, according to literature [25], up to 90% loss of the smallsized neostriatal neurones and a moderate glia proliferation. In spite of this the dopamine content of these nuclei is, 4 It is also not known to which structural cell constituents brain dopamine is bound: it appears certain, however, that only a small portion of the amine exists in a free state in the cytoplasm [26].
H. Ehringer, O. Hornykiewicz/Parkinsonism and Related Disorders 4 (1998) 53–57
according to our findings, quite normal. The small neurones can thus be excluded as the seat of dopamine. There remain the large neurones and glia cells. Of these two cell types we would like to give preference to the large multipolar neurones. In order to furnish direct proof for this view, however, it would be necessary to study diseases in which this type of cell is preferentially affected. In idiopathic Parkinson’s disease and in postencephalitic Parkinsonism the histological changes in the neostriaturn are so negligible as to preclude any significant conclusions. Thus it might be that these two disorders are possibly related to functional changes in the neostriatal cells in connection with disturbed dopamine metabolism. The high degree of dopamine reduction in the neostriatum in idiopathic Parkinson’s disease and postencephalitic Parkinsonism furnishes, apart from the findings of Bertler and Rosengren [9], new evidence for the physiological significance of dopamine in these nuclei. Unfortunately, we could examine only six such cases. Should this finding be confirmed in further cases, it could be regarded as comparable in significance to the histological changes in the substantia nigra. In this case, a particularly great importance would have to be attributed to dopamine’s role in the pathophysiology and symptomatology of idiopathic Parkinson’s disease and postencephalitic Parkinsonism. In addition, this would represent for the first time direct evidence for the physiological role of dopamine in the brain. The reduction of noradrenaline in the hypothalamus in the three cases analyzed may be due to cellular losses frequently found in this region.
5. Summary The distribution of noradrenaline and dopamine in the brain of adult human subjects (post mortem) and of fetuses was studied. In a total of six cases of idiopathic Parkinson’s disease and postencephalitic Parkinsonism the dopamine
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content of the neostriatum was found to be severely reduced; also the noradrenaline content of the hypothalamus was lower than in the control cases. Based on these findings, the importance of dopamine for the pathophysiology and symptomatology of the above diseases as well as its physiological role in the neostriatum are discussed. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26]
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