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Monoamine fluorescence histochemistry of human post mortem brain
Brain Research, 63 (1973) 231-247
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© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
M O N O A M I N E F L U O R E S C E N C E H I S T O C H E M I S T R Y OF H U M A N POST M O R T E M BRAIN
LARS OLSON, BO NYSTROM AND ,~KE SEIGER
Department of Histology, Karolinska Institutet, Stockholm and Department of Neurosurgery, Akademiska Sjukhuset, Uppsala (Sweden) (Accepted April 5th, 1973)
SUMMARY
The distribution of monoamine-containing neuron systems in adult human brains (including a few cases of mental disease) was studied post mortern by FalckHillarp fluorescence histochemistry. Endogenous intracellular monoamine stores decreased slowly with time after death, but varicose catecholamine-containing nerve terminals could be demonstrated in many regions several hours after death. A large number of catecholamine (CA), and probably 5-hydroxytryptamine (5-HT) nerve terminals retained an active concentration-dependent uptake mechanism over the cell membrane even when the endogenous amines could no longer be visualized. Thus, e.g. the dopamine (DA) nerve terminals of the nucleus caudatus could be clearly visualized by in vitro incubations of caudate slices in Krebs-Ringer buffer containing 10-0 M or 10-5 M a-methyl-noradrenaline up to 7 h after death (longest post mortem time studied). A weak CA fluorescence was observed in some perikarya of the large heavily pigmented neurons of the locus coeruleus and more caudally in the pons, as well as in the substantia nigra. More strongly fluorescent CA nerve cell bodies were found in the hypothalamus close to the third ventricle. CA nerve terminals, probably of the NA type were found in the spinal cord and ilium terminale, throughout the brain stem, and in other subcortical and cortical areas. 5-HT nerve terminals were less frequently seen but had a similar distribution. Diffuse CA fluorescence, probably of the DA type was clearly observed in sections of the nucleus caudatus and putamen, and in the nucleus amygdaloideus centralis, but not in the globus pallidus. The possibility of applying fluorescence histochemistry to post mortem brain material, especially following in vitro amine incubations means access to an inexhaustible source of material permitting a more detailed mapping of the monoamine
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neuron systems in adult man and insight into the involvement of these neuron systems in different human diseases.
I NTRODUCT|ON
In the rat brain, the 3 monoamines dopamine (DA), noradrenaline (NA), and 5-hydroxytryptamine (5-HT) have been shown to be almost exclusively located in specific neuron systems using Falck-Hillarp fluorescence histochemistry14,2°, 21,27-29. In view of the crucial roles suggested to be played by the monoamines in a variety of basic brain functions and the supposed or suggested involvement of a disturbed monoamine neurotransmission in several CNS disorders in man such as Parkinsonism, schizophrenia, affective disorders etc., it is important to be able to study the distribution and concentration of monoamines at the cellular level in man. Falck-Hillarp fluorescence histochemistry can be applied to the human brain using 3 different types of material. Firstly, we have developed a technique which permits easy, rapid and sensitive demonstration of NA nerve terminals in the cerebral cortices using brain smears of tissue obtained at neurosurgery 44. Secondly, using fetal brains, a basic mapping of major monoamine neuron systems has been achieved 46. The third possibility is the use of post mortem brain material. Previous attempts to use post mortem material have had limited success in the sense that positive results have only been obtained in brains processed within 45 rain after death, in this way the presence of catecholamine (CA) fluorescence in the human hypothalamus 17 and diencephalon 22 has been briefly described. As pointed out by De La Torre 22 the outcome of attempts to apply fluorescence histochemistry to post mortem material depends on a number of extraneous factors such as cause of death, drug treatment, and, perhaps most important, the post mortem time. On the other hand, if some of the difficulties can be overcome, post mortem material will no doubt be the most important source of material in order to gain further insight into the cellular distribution of monoamines in the human brain in health and disease. The need for fluorescence histochemical studies of the schizophrenic brain was recently directly expressed by Plum ~ and is further warranted by the hypothesis of Stein and Wise 1,61-64 of a destruction of noradrenergic nerves by endogenous 6-hydroxy-dopamine (6-OH-DA) formation in schizophrenia. The value of fluorescence histochemistry in Parkinsonism with its characteristic neuropathology of neuromelanin pigmentation in the substantia nigra and locus coeruleus and low DA levels of the striatum is self-evident. In the present report we describe the presence of catecholamine and 5-hydroxytryptamine nerve terminals and catecholamine cell bodies in selected regions of the human post mortem brain including preliminary observations on two cases of schizophrenia, one case of Parkinsonism and two cases of mano-depressive illness and describe simple means of detecting the presence of CA nerve terminals even several hours after death, by in vitro incubations of brain slices in amine solutions.
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I--IISTOCHEMISTRY OF HUMAN POST MORTEM BRAIN TABLE I DIAGNOSIS, AGE, SEX AND POST MORTEMTIMESOF ANALYZED MATERIAL Case
Diagnosis
Age
Sex
Post mortem time *
(h) 'Somatic' diseases
F1 F2 F3 F7 F11
Liver cirrhosis, arteriosclerosis Essential hypertension Hypernephroma with CNS metastases Essential hypertension, cerebral hemorrhage Liver cirrhosis
74 84 62 61 70
male male female male male
7.3 5.5 4.5 2.2 3.0
64 58
male male
2.6 1.5
85 63 81 89
female female female female
1.6 1.1 2.5 2.0
71 77 63 62 66
female male female male male
2.0 1.5 3.5 2.5 4.0
'Mental' diseases
F5 F10 F9 F15 F8 F12 F13 F14 F4 F6 TK66
Mano-depressive psychosis + general cardioar teriosclerosis Mano-depressive psychosis, depressive type Schizophrenia with demens, general arteriosclerosis. Duration 70 years. Schizophrenia, duration 38 years, oligophrenia Arteriosclerotic demens Senile demens Arteriosclerotic demens, diabetes mellitus, essential hypertension Psychosis with cerebral arteriosclerosis Presenile demens, cerebral atrophy Presenile psychosis, cerebral atrophy Parkinsonism
* Time lapse between death and beginning of dissection. Total dissection time not longer than 40 min. Thereafter, pieces were kept in ice-cold Ringer solution 1.5-3 h before being frozen and further processed for fluorescence histochemistry.
MATERIAL AND METHODS
Various areas from the brains of 15 patients were obtained between 1.1 and 7.3 h after death through the kind help of Dr. L. Wetterberg, Psychiatric Research Center, Ullerhker Hospital, Uppsala. Sex, age, diagnosis and post mortem times are given in Table I. The procedure followed in the cases with short post mortem times was that of a so-called 'partial autopsy'. Following death, as diagnosed by an independent physician, and after permission of the relatives, dissection was commenced as soon as possible. This procedure has been approved by the staff of the Ullerhker hospital. Small pieces of brain tissue were rapidly prepared, frozen in liquid propane cooled by liquid nitrogen, freeze-dried 51 and further processed for fluorescence histochemical visualization of monoamines according to Falck and Hillarpla,19,27. In several cases (see Tables II and IV) selected brain areas were placed in ice-cold Ringer solution and sent to the laboratory. Slices were then prepared and incubated in vitro for 30 min at 37 °C in a modified Krebs-Ringer solution containing 10 -6 M o r 10 -5 M a-methyl-NA followed by a 10 min rinse in amine-free buffer, all according to
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H a m b e r g e r aa. C o n t r o l s were either taken directly or run for 40 rain in amine-free buffer. In a few cases, slices were p r e i n c u b a t e d in 10 4 M 6 - O H - D A and thereafter i n c u b a t e d for 30 min in 10-SM a - m e t h y l - N A or 5-HT. F o l l o w i n g incubation, smears were p r e p a r e d f r o m p a r t s o f the slices 5~, while the rest was frozen, freeze-dried and further processed as above. The a m o u n t o f lipofuscin is high in neurons a n d glia o f the h u m a n brain a n d its yellow autofluorescence characteristics m a k e it difficult to record specific fluorescence in b l a c k and white m i c r o p h o t o g r a p h s . The typical specific green a n d yellow m o n o a m i n e fluorescence can however be easily recognized in the fluorescence microscope and in c o l o r m i c r o p h o t o g r a p h s in spite o f all autofluorescent granules, especially when using the new T A L 405 n a r r o w b a n d excitation filter a n d a Zeiss 47 b a r r i e r filter. A slight diffusion o f the fluorescent varicosities was often seen a n d the fluorescence faded slowly when illuminated, while that o f the autofluorescent granules r e m a i n e d unchanged. F u r t h e r m o r e , the autofluorescent granules were f o u n d also in smears n o t treated with f o r m a l d e h y d e . RESULTS
E n d o g e n o u s C A fluorescence l o c a t e d in nerve terminals was f o u n d in all b u t one case, the negative case having a p o s t m o r t e m time o f 5.5 h. It is to be n o t e d that only unincubated smears o f the cerebellar a n d cerebral cortex, c a u d a t e nucleus a n d locus coeruleus areas were analyzed in this case. In a n o t h e r case (F1) with a post m o r t e m time o f 7.3 h b o t h e n d o g e n o u s l y fluorescent b r a i n stem C A t e r m i n a l s and an a - m e t h y l - N A - d e p e n d e n t fluorescence o f c a u d a t e nerve terminals were found. In the following, a description o f general findings will be given, disregarding the specific diagnosis. Some c o m m e n t s as to the diseases o f the patients are given in the Discussion.
TABLE II CATECHOLAMINE FLUORESCENCE IN SMEARS OF THE CEREBRAL CORTEX
Slices were prepared and incubated as described in Material and Methods. The amount and fluorescence intensity of the green fluorescent varicosities was estimated using a semi-quantitative scale as described earliers4,36,48. - - , no visible specific fluorescence; +, small; + + , moderate; and + + + , high amount of varicosities. Bracketing the last plus sign indicates that the specimen is in between the lower and the higher value. Number of fluorescent dots and their fluorescence intensity covariates closely in the case of cortex cerebri, and therefore mean estimations of these two parameters are given. Each estimation is based upon 4 different smears. Case no.
F8 F12 TK66 F15
Direct smears
--- - to ( + ) --
Incubations in a-methyl-NA 10 ~M
10-SM
--
(+) +(-÷) +(+) ++
(+)
6-OH-DA 10 4M prior to a-methyl-NA IO-~M
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TABLE III CA
NERVE TERMINAL DENSITY IN SELECTED SUBCORTICAL AREAS AS FOUND BY POST MORTEM FLUORES-
CENCE HISTOCHEMISTRY
Nerve density estimated as in Table II. Each value is based upon the number of cases given. Amount o f nerve terminals
Cases investigated
+ 66-
F12 F5
666-(6-) 6-
F12, F15 F7 F3-F8, F12-F15 F3-F9, F12, F14, F15
6-(6-) 6- 6- 66- 6- 66- 66- 66- 6- 66- (6-) 6- -- 6- * 6- 6- 6-* -6- 66- 6- 6+(6-)
F15 F4-F6, F8, F14 F4, F5, F7, F14 F7-F9, F13 F6 F6 F15 F3-F5, F10, F12-F15 F10, F12, F13 F10, F12, F13 F4, F14 F15 F4-F5, FI4-FI5
Spinal cord
Filum terminale Thoracic gray matter Brain stem
Lower brain stem Bottom of 4th ventricle, caudal part Locus coeruleus area Substantia nigra area Selected hypothalamic areas
Mammillary region Wall of third ventricle Tuber cinereum Infundibulum Nuc. suprachiasmaticus Nuc. supraopticus Stria terminalis Nuc. caudatus Nuc. putamen Globus pallidus Tuberculum olfactorium Nuc, amygdaloideus centralis Bulbus olfactorius
* In these areas a diffuse strong green fluorescence was observed. Individual varicosities are generally not seen in sections nor in smears (cf. Table IV) without pretreatment. Similar observations are well known from animal experiments.
CA nerve terminals
N o , o r very few green fluorescent C A nerve terminals were f o u n d in the cortical areas w i t h o u t p r e t r e a t m e n t with a - m e t h y l - N A . A m o d e r a t e a m o u n t o f fluorescent t e r m i n a l s could be visualized in the cortex cerebri following i n c u b a t i o n in 10-SM a - m e t h y l - N A in all a n a l y z e d cases (Table II). I n sections o f freeze-dried m a t e r i a l they a p p e a r e d in all layers o f the cortex cerebri, b u t were possibly m o s t n u m e r o u s in the m o l e c u l a r layer. T h e t e r m i n a l s were thin with relatively small varicosities. The cerebellar cortex c o n t a i n e d fewer terminals t h a n the cerebral cortex, l o c a t e d in the m o lecular layer as seen in sections o f a - m e t h y l - N A i n c u b a t e d slices. T h e y h a d a similar fluorescence m o r p h o l o g y . N o u p t a k e o f a - m e t h y l - N A c o u l d be detected in the cerebral cortex following p r e i n c u b a t i o n in 6 - O H - D A . In subcortical areas, the green fluorescent nerve t e r m i n a l s were o f v a r y i n g sizes, b u t generally c o n s i d e r a b l y larger a n d m o r e strongly fluorescent t h a n in cortical areas. Such terminals were f o u n d in varying a m o u n t s in a l m o s t all areas investigated ( T a b l e
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i.. ()I.SON ~'l al.
Fig. 1. Varicose green fluorescent CA nerve terminals scattered throughout the lateral wall of the third ventricle. Small dots represent specific formaldehyde-induced fluorescence. Several small cells with heavy accumulations of autofluorescent lipofuscin are also seen (e.g. arrows). Case nr. F5. 10 /,m section of freeze-dried tissue. Fluorescence microphotograph. TAI. 405 filter (see Material and Methods). x 135. Fig. 2. CA cell bodies in the lateral wall of the third ventricle. Five green fluorescent cell bodies, two of which have fluorescent processes, are seen. Lipofuscin accumulation in one negative nerve cell body (arrow). Case nr. F5. 10/~m section of freeze-dried tissue. Fluorescence microphotograph. 340.
I1l). Especially large numbers o f fluorescent terminals were f o u n d in the h y p o t h a l a mus, e.g. close to the third ventricle (Fig. 1) a n d in the t u b e r cinereum (Fig. 3). In these areas long varicose fibers were observed also just beneath the e p e n d y m a l lining a n d a m o n g the e p e n d y m a l cells. A n increased n u m b e r o f fluorescent terminals was f o u n d following a - m e t h y l - N A incubation in the substantia nigra, locus coeruleus area, periventricular areas a n d olfactory bulb. O t h e r subcortical areas s u p p o s e d to c o n t a i n N A fibers have not yet been analyzed after in vitro incubations. The a - m e t h y l N A - i n d u c e d increase in n u m b e r o f visible C A nerve terminals in subcortical areas was less t h a n in the cortical areas. A p a r t f r o m the varicose green fluorescent nerve terminals similar to those found in, e.g. the rat h y p o t h a l a m u s , a n d which have been described above, a second type o f very large, very strongly yellow to green fluorescent r o u n d e d structures was observed t h r o u g h o u t the b r a i n stem. It could not be safely d e t e r m i n e d whether these structures were nerve terminals a l t h o u g h the fact that they were sometimes connected to each o t h e r by thin fluorescent segments and sometimes seemed to be in continuity with m o r e typical C A nerve terminals suggests t h a t this was the case.
HISTOCHEMISTRY OF HUMAN POST MORTEM BRAIN
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iv
Fig. 3. Infundibular recess. A rather dense arrangement of CA nerve terminals is found along the ependymal lining on both sides. The varicosities are of varying sizes. IR = infundibular recess. Case nr. F7. 10 p m section of freeze-dried tissue. Fluorescence microphotograph. × 120. Fig. 4. Nucleus caudatus. A thin slice of tissue was incubated in vitro in 10-~M a-methyl-NA as described in Material and Methods. A strong, diffusely dotted green fluorescence is found throughout the tissue, except for the large non-fluorescent myelinated axon bundles (B) penetrating the nucleus. White dots in these bundles represent autofluorescent glial lipofuscin. Case nr. F15 (schizophrenia). 10 ~m section of freeze-dried slice. Fluorescence microphotograph. × 140.
TABLE IV CATECI-IOLAMINE F L U O R E S C E N C E IN SMEARS OF T H E C A U D A T E N U C L E U S
Procedure as in Table II. All positive fluorescence was in the form of small, uniformly sized, densely packed green fluorescent varicosities similar to DA varicosities as seen in corresponding animal experiments. Case no.
F1 F8 F12 F13 TK66 FI5
Direct smears
Incubations in a-methyl-NA lO-SM
10 5M
---b
++-t-
---
+(+)
+++ +++ + + + + + + + + + +++
6-OH-DA lO-4M prior to a-methyl-NA lO-SM
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l.. OLSON el al.
T I i
J Fig. 5. Slice of caudate nucleus incubated in vitro without and with 10 5M ct-methyl-NA. Following incubation smears were prepared as described in Material and Methods. Without amine in the incubation medium (a) no specific fluorescence was observed. The few white dots seen in (a) are autofluorescent lipofuscin particles derived from cells crushed during smearing. Following ~t-methyI-NA incubation (b) a large number of small strongly green fluorescent varicosities are seen. Case nr. FI 2. Fluorescence microphotograph. ~: 350.
The CA fluorescence found in the caudate nucleus and putamen was probably the most constant and reproducible finding. These areas were always typically diffusely green fluorescent in sections leaving penetrating myelinated axon bundles as nonfluorescent islands. The globus pallidus was essentially non-fluorescent. In smears from the caudate (Table IV), a very dense arrangement of small weakly fluorescent dots was found in one case, while 4 other cases were negative (Fig. 5a). Following incubation of caudate slices in 10-SM a-methyl-NA all slices became filled with densely packed strongly fluorescent small CA varicosities as seen both in sections (Fig. 4) and smears (Fig. 5b). Preincubation in 10-4M 6-OH-DA completely prevented the uptake of a-naethyl-NA and no CA nerve terminals were then found. CA cell bodies
Only the locus coeruleus, substantia nigra and wall of the third ventricle have yet been analyzed in detail. In the locus coeruleus, large, heavily brown pigmented nerve cell bodies typical of the area were easily found in the fluorescence microscope. In the majority of these cells no clear-cut specific cytoplasmic fluorescence was observable. In several cases, however, a few cells on each section were weakly to moderately green fluorescent (Fig. 6a). This was especially easy to observe in tangentially cut pigment-free areas of the cytoplasm. Some of the fluorescent cells had fluorescent processes. In the substantia nigra, the majority of the typically heavily neuromelaninpigmented large elongated nerve cell bodies were non-fluorescent just as in the locus coeruleus. Here too, however, a few cells were often found with a weak to moderate
HISTOCHEMISTRY OF HUMAN POST MORTEM BRAIN
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Fig. 6. CA nerve cell bodies in the lower brain stem of an 89-year-old patient, a: large, weakly green fluorescent neurons of the locus coeruleus. Three cells showing large pigment-free areas of CA fluorescent cytoplasm and smaller neuromelanin- and lipofuscin-containing areas (arrows). b: one large polygonal nerve celt of the substantia nigra. The cytoplasm is weakly green fluorescent. Arrow indicates area containing neuromelanin and fluorescent lipofuscin. Case hr. F12. 10 # m section of freeze-dried tissue. Fluorescence microphotograph. × 350.
green fluorescence, observable in pigment-free areas of the cytoplasm (Fig. 6b). Fluorescent processes were sometimes seen to leave the fluorescent nerve cells of the substantia nigra. In the anterior parts of the substantia nigra, green fluorescent axons of a smooth, non-terminal type were observed among the pigmented nerve cell bodies. In the lateral walls of the third ventricle, scattered, medium-sized nerve cells were often found with a clear-cut, moderate to strong CA fluorescence. The cells had fluorescent processes (Fig. 2).
5-HTfluorescence Fibers exhibiting the yellow to brownish yellow, rapidly fading fluorescence typical of fluorophores formed from indolylethylamines such as 5-HT were much less frequently found in the post mortem brains than the CA fibers, and only in cases with short post mortem times. Long, irregularly beaded as well as typically varicose yellow fluorescent fibers were observed, e.g. close to the ependymal lining above the locus coeruleus, superficially in the olfactory tubercle and in the habenula region in case F14. Small numbers of 5-HT nerve terminals were found scattered superficially throughout the brain stem (Fig. 7a) and in the stria terminalis in cases F14 and F15. In case F15 slices of the cortex cerebri and the caudate nucleus were preincubated in 10-4M 6-OH-DA followed by a 30 min incubation in 10-SM 5-HT. A moderate number of thin, weakly yellow fluorescent fibers were then found in the cortex and probably in the caudate while no green fluorescence at all could be seen. A large number of yellow fluorescent irregularly beaded axons were found in the
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L. OLSON gt ill.
Fig. 7. Yellow fluorescent varicose fibers, in all probability containing 5-HT, as seen in 10 Hm sections of freeze-dried brain stem and spinal cord, respectively. Fluorescence microphotographs, a: one thin, weakly yellow fluorescent varicose fiber near the surface in the pontic brain stem (arrows). V vessel with strongly autofluorescent elastin-containing walls. Autofluorescent lipofuscin pigments are seen scattered over the picture. Case nr. F I 5 . × 415. b: ilium terminale. Many ~eakly yellow fluorescent varicose fibers are seen running in parallel bundles. The fluorescence seen on this microphotograph is almost exclusively of the 5-HT type. Case nr. F12. x 335.
central parts o f the filum terminale (Fig. 7b), where they were mixed with green fluorescent axons and surrounded by non-fluorescent myelinated axons. Large ovoid strongly green or yellow fluorescent structures, probably caused by accumulations o f fluorescent axoplasm in axonal swellings, were sometimes seen. Yellow fluorescent nerve cell bodies have not yet been observed. Most areas o f the h u m a n brain are heavily filled with pigments, mostly lipofuscin, present in perivascular cells, other glial elements and neurons. These pigments exhibit a relatively strong, sharply outlined non-fading (or very slowly fading, hours rather than minutes) yellow to brownish fluorescence. Although this fluorescence is easily distinguished from the specific, paraformaldehyde-induced neuronal fluorescence it may partly mask weakly fluorescent nerve terminals, especially of the yellow type. Blood vessels in the pial tissues, in the plexus choroideus and in many other areas contained green fluorescent sympathetic fibers at the medio-adventitial border. The blood vessel innervation will be subject to a more detailed separate report ( N y s t r 6 m and Olson, to be published).
HISTOCHEMISTRY OF HUMAN POST MORTEM BRAIN
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DISCUSSION
The present results convincingly demonstrate that monoamines of the CA and, to a lesser degree, 5-HT type are retained in many neurons in concentrations high enough to permit fluorescence histochemical demonstration in the human brain several hours after death. Furthermore, the uptake mechanism over the nerve cell membrane is still effective when endogenous stores can no longer be demonstrated. Thus, e.g. CA nerve terminals of the DA type in the caudate nucleus and CA nerve terminals probably of the NA type in the pons could be visualized after in vitro incubation in a-methyl-NA more than 7 h after death. Our results regarding the cellular localization of endogenous monoamine stores post mortem are at variance with those of Constantinidis et al. 17 and De La Torre 2~ claiming that more than 45 min after death 'the endogenous amines are hopelessly diffused and histochemical visualization is no longer possible', but the distribution of green fluorescence in the hypothalamus given by Constantinidis et al. 17 is in agreement with our findings. It must be kept in mind, however, that although some areas were found to contain a relatively large number of nerve terminals post mortem in the present study, it may be assumed that these or other areas contained a larger amount of nerves in the living state. This is most probably the case especially in the cortical areas as indicated by the large increase in number of visible CA nerve terminals in these areas by the in vitro amine incubations. Smears of cortical areas can be obtained at neurosurgery and we know from such studies 44 that the number of endogenously visible varicosities is considerable. However, also in slices obtained at neurosurgery there is an increase in the visible number of NA varicosities following a-methyl-NA incubation in vitro. It is clear that the most important post mortem change of the fluorescence histochemical picture is the gradual disappearance of endogenous intraneuronal amines with time. It was noted that nerve terminals close to the ventral surface of the brain and especially close to ependymal linings seemed to be best preserved. Cortical NA nerve terminals were almost always non-fluorescent, while many terminals were found in subcortical areas. This difference may be due to several factors. Thus, the cortical nerve fibers are thinner and the varicosities smaller than in subcortical areas. Furthermore, they probably belong to a separate coeruleo-cortical NA system just as is the case in rats47, 67 and there may have been an increased impulse activity in this system before death. Increases in NA turnover can be demonstrated histochemically in the coeruleo-cortical NA system in the rat following stresslS, 37 or electrical stimulation of the ascending N A axons 5. The large, very strongly fluorescent ovoid structures found scattered throughout subcortical areas and in the ilium terminale were most probably unusually large parts of CA nerve fibers. This type of fluorescence is not found in the rodent brain and may represent a structure typical of the primate brain since similar structures have been reported for the monkey brain 7. At present, we cannot neglect the possibilities that they represent accumulations of axoplasm in the CA nerve fibers due to degenerative phenomena of old age since they are e.g. not found in the human fetal brain 46, or that they represent post mortem swellings of normal fibers. The last explanation, however,
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L. OLSON ('l tl/.
does not seem to account for the large concentrations of CA present in the swellings. The distribution and transmitter identity of the NA, DA and 5-HT neuron systems in the adult rat brain is relatively well knownZ,e°,el, 28 30,49,67. We have recently described the prenatal ontogeny of the monoamine neuron systems in the rat 5°,58 and in man 46. All the major monoamine neuron systems found in the adult rat can be found in the fetal rat and the basic architecture o f m o n o a m i n e neurons in the human fetus is strikingly similar to fetal rat. It may thus be assumed by analogy with the adult rat that the green fluorescence observed, e.g. in the substantia nigra cell bodies and nerve terminals of the nucleus caudatus and putamen, corresponds to DA, while the fluorescent cell bodies of the locus coeruleus and in other regions of the pons and medulla oblongata and the coarse green fluorescent nerve terminals in subcortical areas and fine green fluorescent terminals of the cortices (mainly as seen after in ritro incubations) are of the NA type. Likewise, yellow fluorescence probably represents 5-HT. These assumptions are further supported by comparison with the regional distribution of monoamines and its associated precursors and enzyme systems in the human brain as determined biochemically9,10,24,26, a8 41,56,68. Monoamine nerve cell bodies were difficult to detect in the post mortem material even in brains containing many strongly fluorescent nerve terminals. Thus, only a smaller proportion of the pigmented cell bodies of the substantia nigra and locus coeruleus were clearly fluorescent although in the human fetal brain, large numbers of these cells show a moderate to strong fluorescence 46. There may have been post mortem changes, heavy pigment accumulations may have obscured specific fluorescence in these 50-90-year-old neurons, or they may simply store CA in concentrations below the level of detectability. The CA nerve cell bodies close to the third ventricle which showed a relatively strong fluorescence in the adult brain have also been observed in the fetal human brain 46. The occurrence of pigments in brain tissue has received much attention because of their widespread distribution in the human brain. It is now generally believed that lipofuscin forms in lysosomes 25 and that neuromelanin may form on a lipofuscin infrastructure 23. Characteristically, both pigment types increase in age and occur in larger amounts in higher primates. Peroxidase may have a common role in CA, lipofuscin and neuromelanin synthesis in the brain 45 and the neuromelanin o f e.g. the substantia nigra is formed by DOPA-melanin and possibly dopamine-melanin 43. The close interrelationship between CA and neuromelanin (see also ref. 6) and the close resemblance between the neuromelanin map of the human brain 8,5a and the CA cell groups of the rat 2° made Bazelon et al. 8 suggest that 'the aging catecholamine neuron may accumulate its own identifying marker - - neuromelanin.' Our present results give direct proof of this hypothesis because we have been able to visualize simultaneously in the same neuronal cytoplasm neuromelanin and catecholamine fluorescence. So far, however, we have performed no detailed correlation of CA fluorescence and neuromelanin content throughout the brain. The large amount of lipofuscin in the human brain is present both in neurons and glial elements (for review of literature on neurolipofuscin and neuromelanin, see Braakll). It presents certain problems in monoamine fluorescence histochemistry due
HISTOCHEMISTRY OF HUMAN POST MORTEM BRAIN
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to its fluorescence characteristics t5 having an autofluorescence 1~,a2,35,55 that is strong, yellow and only very slowly fading. However, although black and white microphotographical documentation of monoamine-induced fluorescence is difficult, the identification of specific fluorescence in the microscope is easy due to the differences in morphology, color, fluorescence intensity, fading characteristics, sharpness of borders, etc. The direct demonstration of CA and neuromelanin in the same nerve cell bodies adds to the wealth of information concerning the relationship between loss of neuromelanin and cell bodies in the substantia nigra in Parkinson's disease and the decreased DA levels of the striatum (see refs. 3, 16, 26). Underpigmentation of the substantia nigra also occurs, e.g. in chronic disease in children 60, and it would be interesting to complete this finding with fluorescence histochemistry. The viability of the amine uptake and Concentration mechanism over the nerve terminal membrane post mortem is most promising, since it allows a more detailed mapping of the distribution of monoamine nerve terminals in various areas by simple in vitro incubations of brain slices as demonstrated in the present study. Using a relatively high concentration of a-methyl-NA, which is resistant to the action of monoamine oxidase, CA nerve terminals were clearly visualized in all parts of the brain examined. 6-OH-DA causes selective destruction of central CA nerve fibers 66 and preincubation with this drug completely prevented the uptake of a-methyl-NA. However, an uptake of 5-HT into varicose nerve fibers could still be achieved in 6-OH-DA preincubated slices of the cortex cerebri, suggesting that this may be a method to reveal the distribution of 5-HT nerve terminals. The clinical implications of the present results are manifold. Principally, two types of pathological disturbances of the monoamine neurons can be detected by fluorescence histochemistry. Firstly, there may be abnormal distribution patterns which would be best revealed by in vitro amine incubations. Degeneration of DA and possibly NA nerve terminals may be expected in Parkinsonism and the hypothesis of Stein and Wise1, 61-64 of a degeneration of NA nerve terminals in cortical areas in schizophrenia can be directly tested. Interestingly, no decrease in the number of small CA nerve terminals of the nucleus caudatus was observed in the case of Parkinsonism studied here*. This observation may suggest that the symptoms of Parkinsonism may have a varying genesis although in the majority of cases a more or less severe degeneration of the nigro-striatal DA system should be expected (see refs. 26 and 33). It could not be excluded that the green fluorescent varicosities found in the present case of Parkinsonism were of the NA type although their small size, high density and typical distribution strongly suggest that they were DA varicosities. One (F15)of the two schizophrenia cases studied had a history of schizophrenia from 25 years of age and with a 38-year duration. Oligophrenia may have been part of the clinical picture in this case from the early stages of the disease. The distribution of CA nerve terminals in the cortical and subcortical areas including the striatum complex was similar to * The diagnosis was somewhat unclear since the patient died before the effect of the initiated LDOPA therapy could be evaluated.
244
L. OLSON cl a/.
that in the rest of the present cases, thus not directly supporting the theory of Stein and Wise1, 61-64, a theory that has been seriously questioned 4. It must be pointed out, however, that no quantitative conclusions can be drawn from the present limited material regarding an altered fluorescence histochemical picture in any diseased state. To our knowledge an increased number of monoamine nerve terminals has not yet been suggested in combination with any pathological states of the human brain. This does not seem to be an unlikely possibility, however, following brain damage that causes chronic deafferentation of certain areas in view of the interesting experimental data on collateral sprouting of CA fibers obtained in animals4'~,G.L Secondly, the amount and concentration of monoamines in the nerve terminals may vary, e.g. as predicted by the catecholamine theory of affective disorders (see refs. 13 and 57). Thus, 5-HT has been reported decreased post mortem in brains of depressive suicides ag. In order to be able to evaluate possible decreases of endogenous stores post mortem and to compare fluorescence intensities between patients the post mortem time has to be carefully considered. The present material is too small to permit any other conclusion than the fact that neuronal monoamine stores can indeed be visualized post mortem in many brain areas of mano-depressive patients. We conclude that endogenous intraneuronal CA and, to a lesser degree, 5-HT stores can be visualized in the human brain up to several hours post mortem and that these nerves likewise retain an active uptake concentration mechanism for amines for several hours. By a continued collection of material for fluorescence histochemical analysis it is hoped that a more precise description of the cytoarchitectonics of the monoamine neuron systems in the adult human brain, as well as the possible involvement of these systems in various diseases, will be obtained. ACKNOWLEDGEMENTS
This study was supported by grants from the Swedish Medical Research Council (04X-3185), 'Magnus Bergvalls Stiftelse' and 'Karolinska Institutets Fonder'. The brains were obtained with the kind help of Dr. L. Wetterberg, Psychiatric Research Center, Ullerhker Hospital, Uppsala. We thank Miss Monica Eliasson, Mrs. Barbro Norstedt and Miss Ingrid Str6mberg for skillful technical assistance.
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55 SAMORAJSKI,T., KEEFE,J. R., AND ORDY, J. M., Intracellular localization of lipofuscin age pigments in the nervous system, J. Gerontol., 19 (1964) 262-276. 56 SANO,I., GAMO,I., KAKIMOTO,Y., TAMIGUCHI,K., TAKESADA,M., ANDNISHIMURA,K., Distribution of catechol compounds in human brain, Biochim. biophys. Acta (Amst.), 32 (1959) 586-587. 57 SCHILDKRAUT,J. J., The catecholamine hypothesis of affective disorders: A review of supporting eivdence, Amer. J. Psychiat., 122 (1965) 509-522. 58 SEIGER,/~., AND OLSON, L., Late prenatal ontogeny of central monoamine neurons in the rat: Fluorescence histochemical observations, Z. Anat. Entwickl.-Gesch., 140 (1973). 59 SItAW, D. M., CAMPS,F. E., AND ECCLESTON,E. G., 5-Hydroxytryptamine in the hind-brain of depressive suicides, Brit. J. Psychiat., 113 (1967) 1407-1411. 60 SPENCE,A. M., ANDGILLES,F. H., Underpigmentationof the substantia nigra in chronic disease in children, Neurology (Minneap.), 21 (1971) 386-390. 61 STEIN, L., Neurochemistry of reward and punishment: some implications for the etiology of schizophrenia, J. psychiat. Res., 8 (1971) 345-361. 62 STEIN,L., AND WISE, C. D., Possible etiology of schizophrenia: Progressive damage of the noradrenergic reward mechanism by 6-hydroxydopamine, Science, 171 (1971) 1032-1036. 63 STEIN, L., AND WISE, C. D., 6-Hydroxydopamine, noradrenergic reward, and schizophrenia, Science, 175 (1972) 922-923. 64 STEIN, L., WISE, C. D., AND BERGER, B. D., Noradrenergic reward mechanisms, recovery of function, and schizophrenia. In J. L. McGAUGH(Ed.), The Chemistry of Mood, Motivation and Memory, Plenum Press, New York, 1972, pp. 81-103. 65 STENEVI, U,, BJ/)RKLUND, A., AND MOORE, R. Y., Growth of intact adrenergic axons in the denervated lateral geniculate body, Exp. Neurol., 35 (1972) 290-299. 66 UNGERSTEDT,U., 6-Hydroxydopamine-induced degeneration of central monoamine neurons, Europ. J. Pharmacol., 5 (1968) 107-110. 67 UNGERSTEDT,U., Stereotaxic mapping of the monoamine pathways in the rat brain, Actaphysiol. scand., Suppl. 367 (1971) 1-48. 68 VOGT,M., The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs, J. Physiol. (Lond.), 123 (1954) 451-481.
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© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
M O N O A M I N E F L U O R E S C E N C E H I S T O C H E M I S T R Y OF H U M A N POST M O R T E M BRAIN
LARS OLSON, BO NYSTROM AND ,~KE SEIGER
Department of Histology, Karolinska Institutet, Stockholm and Department of Neurosurgery, Akademiska Sjukhuset, Uppsala (Sweden) (Accepted April 5th, 1973)
SUMMARY
The distribution of monoamine-containing neuron systems in adult human brains (including a few cases of mental disease) was studied post mortern by FalckHillarp fluorescence histochemistry. Endogenous intracellular monoamine stores decreased slowly with time after death, but varicose catecholamine-containing nerve terminals could be demonstrated in many regions several hours after death. A large number of catecholamine (CA), and probably 5-hydroxytryptamine (5-HT) nerve terminals retained an active concentration-dependent uptake mechanism over the cell membrane even when the endogenous amines could no longer be visualized. Thus, e.g. the dopamine (DA) nerve terminals of the nucleus caudatus could be clearly visualized by in vitro incubations of caudate slices in Krebs-Ringer buffer containing 10-0 M or 10-5 M a-methyl-noradrenaline up to 7 h after death (longest post mortem time studied). A weak CA fluorescence was observed in some perikarya of the large heavily pigmented neurons of the locus coeruleus and more caudally in the pons, as well as in the substantia nigra. More strongly fluorescent CA nerve cell bodies were found in the hypothalamus close to the third ventricle. CA nerve terminals, probably of the NA type were found in the spinal cord and ilium terminale, throughout the brain stem, and in other subcortical and cortical areas. 5-HT nerve terminals were less frequently seen but had a similar distribution. Diffuse CA fluorescence, probably of the DA type was clearly observed in sections of the nucleus caudatus and putamen, and in the nucleus amygdaloideus centralis, but not in the globus pallidus. The possibility of applying fluorescence histochemistry to post mortem brain material, especially following in vitro amine incubations means access to an inexhaustible source of material permitting a more detailed mapping of the monoamine
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neuron systems in adult man and insight into the involvement of these neuron systems in different human diseases.
I NTRODUCT|ON
In the rat brain, the 3 monoamines dopamine (DA), noradrenaline (NA), and 5-hydroxytryptamine (5-HT) have been shown to be almost exclusively located in specific neuron systems using Falck-Hillarp fluorescence histochemistry14,2°, 21,27-29. In view of the crucial roles suggested to be played by the monoamines in a variety of basic brain functions and the supposed or suggested involvement of a disturbed monoamine neurotransmission in several CNS disorders in man such as Parkinsonism, schizophrenia, affective disorders etc., it is important to be able to study the distribution and concentration of monoamines at the cellular level in man. Falck-Hillarp fluorescence histochemistry can be applied to the human brain using 3 different types of material. Firstly, we have developed a technique which permits easy, rapid and sensitive demonstration of NA nerve terminals in the cerebral cortices using brain smears of tissue obtained at neurosurgery 44. Secondly, using fetal brains, a basic mapping of major monoamine neuron systems has been achieved 46. The third possibility is the use of post mortem brain material. Previous attempts to use post mortem material have had limited success in the sense that positive results have only been obtained in brains processed within 45 rain after death, in this way the presence of catecholamine (CA) fluorescence in the human hypothalamus 17 and diencephalon 22 has been briefly described. As pointed out by De La Torre 22 the outcome of attempts to apply fluorescence histochemistry to post mortem material depends on a number of extraneous factors such as cause of death, drug treatment, and, perhaps most important, the post mortem time. On the other hand, if some of the difficulties can be overcome, post mortem material will no doubt be the most important source of material in order to gain further insight into the cellular distribution of monoamines in the human brain in health and disease. The need for fluorescence histochemical studies of the schizophrenic brain was recently directly expressed by Plum ~ and is further warranted by the hypothesis of Stein and Wise 1,61-64 of a destruction of noradrenergic nerves by endogenous 6-hydroxy-dopamine (6-OH-DA) formation in schizophrenia. The value of fluorescence histochemistry in Parkinsonism with its characteristic neuropathology of neuromelanin pigmentation in the substantia nigra and locus coeruleus and low DA levels of the striatum is self-evident. In the present report we describe the presence of catecholamine and 5-hydroxytryptamine nerve terminals and catecholamine cell bodies in selected regions of the human post mortem brain including preliminary observations on two cases of schizophrenia, one case of Parkinsonism and two cases of mano-depressive illness and describe simple means of detecting the presence of CA nerve terminals even several hours after death, by in vitro incubations of brain slices in amine solutions.
233
I--IISTOCHEMISTRY OF HUMAN POST MORTEM BRAIN TABLE I DIAGNOSIS, AGE, SEX AND POST MORTEMTIMESOF ANALYZED MATERIAL Case
Diagnosis
Age
Sex
Post mortem time *
(h) 'Somatic' diseases
F1 F2 F3 F7 F11
Liver cirrhosis, arteriosclerosis Essential hypertension Hypernephroma with CNS metastases Essential hypertension, cerebral hemorrhage Liver cirrhosis
74 84 62 61 70
male male female male male
7.3 5.5 4.5 2.2 3.0
64 58
male male
2.6 1.5
85 63 81 89
female female female female
1.6 1.1 2.5 2.0
71 77 63 62 66
female male female male male
2.0 1.5 3.5 2.5 4.0
'Mental' diseases
F5 F10 F9 F15 F8 F12 F13 F14 F4 F6 TK66
Mano-depressive psychosis + general cardioar teriosclerosis Mano-depressive psychosis, depressive type Schizophrenia with demens, general arteriosclerosis. Duration 70 years. Schizophrenia, duration 38 years, oligophrenia Arteriosclerotic demens Senile demens Arteriosclerotic demens, diabetes mellitus, essential hypertension Psychosis with cerebral arteriosclerosis Presenile demens, cerebral atrophy Presenile psychosis, cerebral atrophy Parkinsonism
* Time lapse between death and beginning of dissection. Total dissection time not longer than 40 min. Thereafter, pieces were kept in ice-cold Ringer solution 1.5-3 h before being frozen and further processed for fluorescence histochemistry.
MATERIAL AND METHODS
Various areas from the brains of 15 patients were obtained between 1.1 and 7.3 h after death through the kind help of Dr. L. Wetterberg, Psychiatric Research Center, Ullerhker Hospital, Uppsala. Sex, age, diagnosis and post mortem times are given in Table I. The procedure followed in the cases with short post mortem times was that of a so-called 'partial autopsy'. Following death, as diagnosed by an independent physician, and after permission of the relatives, dissection was commenced as soon as possible. This procedure has been approved by the staff of the Ullerhker hospital. Small pieces of brain tissue were rapidly prepared, frozen in liquid propane cooled by liquid nitrogen, freeze-dried 51 and further processed for fluorescence histochemical visualization of monoamines according to Falck and Hillarpla,19,27. In several cases (see Tables II and IV) selected brain areas were placed in ice-cold Ringer solution and sent to the laboratory. Slices were then prepared and incubated in vitro for 30 min at 37 °C in a modified Krebs-Ringer solution containing 10 -6 M o r 10 -5 M a-methyl-NA followed by a 10 min rinse in amine-free buffer, all according to
234
L. OLSOY e t a / .
H a m b e r g e r aa. C o n t r o l s were either taken directly or run for 40 rain in amine-free buffer. In a few cases, slices were p r e i n c u b a t e d in 10 4 M 6 - O H - D A and thereafter i n c u b a t e d for 30 min in 10-SM a - m e t h y l - N A or 5-HT. F o l l o w i n g incubation, smears were p r e p a r e d f r o m p a r t s o f the slices 5~, while the rest was frozen, freeze-dried and further processed as above. The a m o u n t o f lipofuscin is high in neurons a n d glia o f the h u m a n brain a n d its yellow autofluorescence characteristics m a k e it difficult to record specific fluorescence in b l a c k and white m i c r o p h o t o g r a p h s . The typical specific green a n d yellow m o n o a m i n e fluorescence can however be easily recognized in the fluorescence microscope and in c o l o r m i c r o p h o t o g r a p h s in spite o f all autofluorescent granules, especially when using the new T A L 405 n a r r o w b a n d excitation filter a n d a Zeiss 47 b a r r i e r filter. A slight diffusion o f the fluorescent varicosities was often seen a n d the fluorescence faded slowly when illuminated, while that o f the autofluorescent granules r e m a i n e d unchanged. F u r t h e r m o r e , the autofluorescent granules were f o u n d also in smears n o t treated with f o r m a l d e h y d e . RESULTS
E n d o g e n o u s C A fluorescence l o c a t e d in nerve terminals was f o u n d in all b u t one case, the negative case having a p o s t m o r t e m time o f 5.5 h. It is to be n o t e d that only unincubated smears o f the cerebellar a n d cerebral cortex, c a u d a t e nucleus a n d locus coeruleus areas were analyzed in this case. In a n o t h e r case (F1) with a post m o r t e m time o f 7.3 h b o t h e n d o g e n o u s l y fluorescent b r a i n stem C A t e r m i n a l s and an a - m e t h y l - N A - d e p e n d e n t fluorescence o f c a u d a t e nerve terminals were found. In the following, a description o f general findings will be given, disregarding the specific diagnosis. Some c o m m e n t s as to the diseases o f the patients are given in the Discussion.
TABLE II CATECHOLAMINE FLUORESCENCE IN SMEARS OF THE CEREBRAL CORTEX
Slices were prepared and incubated as described in Material and Methods. The amount and fluorescence intensity of the green fluorescent varicosities was estimated using a semi-quantitative scale as described earliers4,36,48. - - , no visible specific fluorescence; +, small; + + , moderate; and + + + , high amount of varicosities. Bracketing the last plus sign indicates that the specimen is in between the lower and the higher value. Number of fluorescent dots and their fluorescence intensity covariates closely in the case of cortex cerebri, and therefore mean estimations of these two parameters are given. Each estimation is based upon 4 different smears. Case no.
F8 F12 TK66 F15
Direct smears
--- - to ( + ) --
Incubations in a-methyl-NA 10 ~M
10-SM
--
(+) +(-÷) +(+) ++
(+)
6-OH-DA 10 4M prior to a-methyl-NA IO-~M
HISTOCHEMISTRY OF HUMAN POST MORTEM BRAIN
235
TABLE III CA
NERVE TERMINAL DENSITY IN SELECTED SUBCORTICAL AREAS AS FOUND BY POST MORTEM FLUORES-
CENCE HISTOCHEMISTRY
Nerve density estimated as in Table II. Each value is based upon the number of cases given. Amount o f nerve terminals
Cases investigated
+ 66-
F12 F5
666-(6-) 6-
F12, F15 F7 F3-F8, F12-F15 F3-F9, F12, F14, F15
6-(6-) 6- 6- 66- 6- 66- 66- 66- 6- 66- (6-) 6- -- 6- * 6- 6- 6-* -6- 66- 6- 6+(6-)
F15 F4-F6, F8, F14 F4, F5, F7, F14 F7-F9, F13 F6 F6 F15 F3-F5, F10, F12-F15 F10, F12, F13 F10, F12, F13 F4, F14 F15 F4-F5, FI4-FI5
Spinal cord
Filum terminale Thoracic gray matter Brain stem
Lower brain stem Bottom of 4th ventricle, caudal part Locus coeruleus area Substantia nigra area Selected hypothalamic areas
Mammillary region Wall of third ventricle Tuber cinereum Infundibulum Nuc. suprachiasmaticus Nuc. supraopticus Stria terminalis Nuc. caudatus Nuc. putamen Globus pallidus Tuberculum olfactorium Nuc, amygdaloideus centralis Bulbus olfactorius
* In these areas a diffuse strong green fluorescence was observed. Individual varicosities are generally not seen in sections nor in smears (cf. Table IV) without pretreatment. Similar observations are well known from animal experiments.
CA nerve terminals
N o , o r very few green fluorescent C A nerve terminals were f o u n d in the cortical areas w i t h o u t p r e t r e a t m e n t with a - m e t h y l - N A . A m o d e r a t e a m o u n t o f fluorescent t e r m i n a l s could be visualized in the cortex cerebri following i n c u b a t i o n in 10-SM a - m e t h y l - N A in all a n a l y z e d cases (Table II). I n sections o f freeze-dried m a t e r i a l they a p p e a r e d in all layers o f the cortex cerebri, b u t were possibly m o s t n u m e r o u s in the m o l e c u l a r layer. T h e t e r m i n a l s were thin with relatively small varicosities. The cerebellar cortex c o n t a i n e d fewer terminals t h a n the cerebral cortex, l o c a t e d in the m o lecular layer as seen in sections o f a - m e t h y l - N A i n c u b a t e d slices. T h e y h a d a similar fluorescence m o r p h o l o g y . N o u p t a k e o f a - m e t h y l - N A c o u l d be detected in the cerebral cortex following p r e i n c u b a t i o n in 6 - O H - D A . In subcortical areas, the green fluorescent nerve t e r m i n a l s were o f v a r y i n g sizes, b u t generally c o n s i d e r a b l y larger a n d m o r e strongly fluorescent t h a n in cortical areas. Such terminals were f o u n d in varying a m o u n t s in a l m o s t all areas investigated ( T a b l e
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i.. ()I.SON ~'l al.
Fig. 1. Varicose green fluorescent CA nerve terminals scattered throughout the lateral wall of the third ventricle. Small dots represent specific formaldehyde-induced fluorescence. Several small cells with heavy accumulations of autofluorescent lipofuscin are also seen (e.g. arrows). Case nr. F5. 10 /,m section of freeze-dried tissue. Fluorescence microphotograph. TAI. 405 filter (see Material and Methods). x 135. Fig. 2. CA cell bodies in the lateral wall of the third ventricle. Five green fluorescent cell bodies, two of which have fluorescent processes, are seen. Lipofuscin accumulation in one negative nerve cell body (arrow). Case nr. F5. 10/~m section of freeze-dried tissue. Fluorescence microphotograph. 340.
I1l). Especially large numbers o f fluorescent terminals were f o u n d in the h y p o t h a l a mus, e.g. close to the third ventricle (Fig. 1) a n d in the t u b e r cinereum (Fig. 3). In these areas long varicose fibers were observed also just beneath the e p e n d y m a l lining a n d a m o n g the e p e n d y m a l cells. A n increased n u m b e r o f fluorescent terminals was f o u n d following a - m e t h y l - N A incubation in the substantia nigra, locus coeruleus area, periventricular areas a n d olfactory bulb. O t h e r subcortical areas s u p p o s e d to c o n t a i n N A fibers have not yet been analyzed after in vitro incubations. The a - m e t h y l N A - i n d u c e d increase in n u m b e r o f visible C A nerve terminals in subcortical areas was less t h a n in the cortical areas. A p a r t f r o m the varicose green fluorescent nerve terminals similar to those found in, e.g. the rat h y p o t h a l a m u s , a n d which have been described above, a second type o f very large, very strongly yellow to green fluorescent r o u n d e d structures was observed t h r o u g h o u t the b r a i n stem. It could not be safely d e t e r m i n e d whether these structures were nerve terminals a l t h o u g h the fact that they were sometimes connected to each o t h e r by thin fluorescent segments and sometimes seemed to be in continuity with m o r e typical C A nerve terminals suggests t h a t this was the case.
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iv
Fig. 3. Infundibular recess. A rather dense arrangement of CA nerve terminals is found along the ependymal lining on both sides. The varicosities are of varying sizes. IR = infundibular recess. Case nr. F7. 10 p m section of freeze-dried tissue. Fluorescence microphotograph. × 120. Fig. 4. Nucleus caudatus. A thin slice of tissue was incubated in vitro in 10-~M a-methyl-NA as described in Material and Methods. A strong, diffusely dotted green fluorescence is found throughout the tissue, except for the large non-fluorescent myelinated axon bundles (B) penetrating the nucleus. White dots in these bundles represent autofluorescent glial lipofuscin. Case nr. F15 (schizophrenia). 10 ~m section of freeze-dried slice. Fluorescence microphotograph. × 140.
TABLE IV CATECI-IOLAMINE F L U O R E S C E N C E IN SMEARS OF T H E C A U D A T E N U C L E U S
Procedure as in Table II. All positive fluorescence was in the form of small, uniformly sized, densely packed green fluorescent varicosities similar to DA varicosities as seen in corresponding animal experiments. Case no.
F1 F8 F12 F13 TK66 FI5
Direct smears
Incubations in a-methyl-NA lO-SM
10 5M
---b
++-t-
---
+(+)
+++ +++ + + + + + + + + + +++
6-OH-DA lO-4M prior to a-methyl-NA lO-SM
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l.. OLSON el al.
T I i
J Fig. 5. Slice of caudate nucleus incubated in vitro without and with 10 5M ct-methyl-NA. Following incubation smears were prepared as described in Material and Methods. Without amine in the incubation medium (a) no specific fluorescence was observed. The few white dots seen in (a) are autofluorescent lipofuscin particles derived from cells crushed during smearing. Following ~t-methyI-NA incubation (b) a large number of small strongly green fluorescent varicosities are seen. Case nr. FI 2. Fluorescence microphotograph. ~: 350.
The CA fluorescence found in the caudate nucleus and putamen was probably the most constant and reproducible finding. These areas were always typically diffusely green fluorescent in sections leaving penetrating myelinated axon bundles as nonfluorescent islands. The globus pallidus was essentially non-fluorescent. In smears from the caudate (Table IV), a very dense arrangement of small weakly fluorescent dots was found in one case, while 4 other cases were negative (Fig. 5a). Following incubation of caudate slices in 10-SM a-methyl-NA all slices became filled with densely packed strongly fluorescent small CA varicosities as seen both in sections (Fig. 4) and smears (Fig. 5b). Preincubation in 10-4M 6-OH-DA completely prevented the uptake of a-naethyl-NA and no CA nerve terminals were then found. CA cell bodies
Only the locus coeruleus, substantia nigra and wall of the third ventricle have yet been analyzed in detail. In the locus coeruleus, large, heavily brown pigmented nerve cell bodies typical of the area were easily found in the fluorescence microscope. In the majority of these cells no clear-cut specific cytoplasmic fluorescence was observable. In several cases, however, a few cells on each section were weakly to moderately green fluorescent (Fig. 6a). This was especially easy to observe in tangentially cut pigment-free areas of the cytoplasm. Some of the fluorescent cells had fluorescent processes. In the substantia nigra, the majority of the typically heavily neuromelaninpigmented large elongated nerve cell bodies were non-fluorescent just as in the locus coeruleus. Here too, however, a few cells were often found with a weak to moderate
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Fig. 6. CA nerve cell bodies in the lower brain stem of an 89-year-old patient, a: large, weakly green fluorescent neurons of the locus coeruleus. Three cells showing large pigment-free areas of CA fluorescent cytoplasm and smaller neuromelanin- and lipofuscin-containing areas (arrows). b: one large polygonal nerve celt of the substantia nigra. The cytoplasm is weakly green fluorescent. Arrow indicates area containing neuromelanin and fluorescent lipofuscin. Case hr. F12. 10 # m section of freeze-dried tissue. Fluorescence microphotograph. × 350.
green fluorescence, observable in pigment-free areas of the cytoplasm (Fig. 6b). Fluorescent processes were sometimes seen to leave the fluorescent nerve cells of the substantia nigra. In the anterior parts of the substantia nigra, green fluorescent axons of a smooth, non-terminal type were observed among the pigmented nerve cell bodies. In the lateral walls of the third ventricle, scattered, medium-sized nerve cells were often found with a clear-cut, moderate to strong CA fluorescence. The cells had fluorescent processes (Fig. 2).
5-HTfluorescence Fibers exhibiting the yellow to brownish yellow, rapidly fading fluorescence typical of fluorophores formed from indolylethylamines such as 5-HT were much less frequently found in the post mortem brains than the CA fibers, and only in cases with short post mortem times. Long, irregularly beaded as well as typically varicose yellow fluorescent fibers were observed, e.g. close to the ependymal lining above the locus coeruleus, superficially in the olfactory tubercle and in the habenula region in case F14. Small numbers of 5-HT nerve terminals were found scattered superficially throughout the brain stem (Fig. 7a) and in the stria terminalis in cases F14 and F15. In case F15 slices of the cortex cerebri and the caudate nucleus were preincubated in 10-4M 6-OH-DA followed by a 30 min incubation in 10-SM 5-HT. A moderate number of thin, weakly yellow fluorescent fibers were then found in the cortex and probably in the caudate while no green fluorescence at all could be seen. A large number of yellow fluorescent irregularly beaded axons were found in the
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L. OLSON gt ill.
Fig. 7. Yellow fluorescent varicose fibers, in all probability containing 5-HT, as seen in 10 Hm sections of freeze-dried brain stem and spinal cord, respectively. Fluorescence microphotographs, a: one thin, weakly yellow fluorescent varicose fiber near the surface in the pontic brain stem (arrows). V vessel with strongly autofluorescent elastin-containing walls. Autofluorescent lipofuscin pigments are seen scattered over the picture. Case nr. F I 5 . × 415. b: ilium terminale. Many ~eakly yellow fluorescent varicose fibers are seen running in parallel bundles. The fluorescence seen on this microphotograph is almost exclusively of the 5-HT type. Case nr. F12. x 335.
central parts o f the filum terminale (Fig. 7b), where they were mixed with green fluorescent axons and surrounded by non-fluorescent myelinated axons. Large ovoid strongly green or yellow fluorescent structures, probably caused by accumulations o f fluorescent axoplasm in axonal swellings, were sometimes seen. Yellow fluorescent nerve cell bodies have not yet been observed. Most areas o f the h u m a n brain are heavily filled with pigments, mostly lipofuscin, present in perivascular cells, other glial elements and neurons. These pigments exhibit a relatively strong, sharply outlined non-fading (or very slowly fading, hours rather than minutes) yellow to brownish fluorescence. Although this fluorescence is easily distinguished from the specific, paraformaldehyde-induced neuronal fluorescence it may partly mask weakly fluorescent nerve terminals, especially of the yellow type. Blood vessels in the pial tissues, in the plexus choroideus and in many other areas contained green fluorescent sympathetic fibers at the medio-adventitial border. The blood vessel innervation will be subject to a more detailed separate report ( N y s t r 6 m and Olson, to be published).
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DISCUSSION
The present results convincingly demonstrate that monoamines of the CA and, to a lesser degree, 5-HT type are retained in many neurons in concentrations high enough to permit fluorescence histochemical demonstration in the human brain several hours after death. Furthermore, the uptake mechanism over the nerve cell membrane is still effective when endogenous stores can no longer be demonstrated. Thus, e.g. CA nerve terminals of the DA type in the caudate nucleus and CA nerve terminals probably of the NA type in the pons could be visualized after in vitro incubation in a-methyl-NA more than 7 h after death. Our results regarding the cellular localization of endogenous monoamine stores post mortem are at variance with those of Constantinidis et al. 17 and De La Torre 2~ claiming that more than 45 min after death 'the endogenous amines are hopelessly diffused and histochemical visualization is no longer possible', but the distribution of green fluorescence in the hypothalamus given by Constantinidis et al. 17 is in agreement with our findings. It must be kept in mind, however, that although some areas were found to contain a relatively large number of nerve terminals post mortem in the present study, it may be assumed that these or other areas contained a larger amount of nerves in the living state. This is most probably the case especially in the cortical areas as indicated by the large increase in number of visible CA nerve terminals in these areas by the in vitro amine incubations. Smears of cortical areas can be obtained at neurosurgery and we know from such studies 44 that the number of endogenously visible varicosities is considerable. However, also in slices obtained at neurosurgery there is an increase in the visible number of NA varicosities following a-methyl-NA incubation in vitro. It is clear that the most important post mortem change of the fluorescence histochemical picture is the gradual disappearance of endogenous intraneuronal amines with time. It was noted that nerve terminals close to the ventral surface of the brain and especially close to ependymal linings seemed to be best preserved. Cortical NA nerve terminals were almost always non-fluorescent, while many terminals were found in subcortical areas. This difference may be due to several factors. Thus, the cortical nerve fibers are thinner and the varicosities smaller than in subcortical areas. Furthermore, they probably belong to a separate coeruleo-cortical NA system just as is the case in rats47, 67 and there may have been an increased impulse activity in this system before death. Increases in NA turnover can be demonstrated histochemically in the coeruleo-cortical NA system in the rat following stresslS, 37 or electrical stimulation of the ascending N A axons 5. The large, very strongly fluorescent ovoid structures found scattered throughout subcortical areas and in the ilium terminale were most probably unusually large parts of CA nerve fibers. This type of fluorescence is not found in the rodent brain and may represent a structure typical of the primate brain since similar structures have been reported for the monkey brain 7. At present, we cannot neglect the possibilities that they represent accumulations of axoplasm in the CA nerve fibers due to degenerative phenomena of old age since they are e.g. not found in the human fetal brain 46, or that they represent post mortem swellings of normal fibers. The last explanation, however,
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L. OLSON ('l tl/.
does not seem to account for the large concentrations of CA present in the swellings. The distribution and transmitter identity of the NA, DA and 5-HT neuron systems in the adult rat brain is relatively well knownZ,e°,el, 28 30,49,67. We have recently described the prenatal ontogeny of the monoamine neuron systems in the rat 5°,58 and in man 46. All the major monoamine neuron systems found in the adult rat can be found in the fetal rat and the basic architecture o f m o n o a m i n e neurons in the human fetus is strikingly similar to fetal rat. It may thus be assumed by analogy with the adult rat that the green fluorescence observed, e.g. in the substantia nigra cell bodies and nerve terminals of the nucleus caudatus and putamen, corresponds to DA, while the fluorescent cell bodies of the locus coeruleus and in other regions of the pons and medulla oblongata and the coarse green fluorescent nerve terminals in subcortical areas and fine green fluorescent terminals of the cortices (mainly as seen after in ritro incubations) are of the NA type. Likewise, yellow fluorescence probably represents 5-HT. These assumptions are further supported by comparison with the regional distribution of monoamines and its associated precursors and enzyme systems in the human brain as determined biochemically9,10,24,26, a8 41,56,68. Monoamine nerve cell bodies were difficult to detect in the post mortem material even in brains containing many strongly fluorescent nerve terminals. Thus, only a smaller proportion of the pigmented cell bodies of the substantia nigra and locus coeruleus were clearly fluorescent although in the human fetal brain, large numbers of these cells show a moderate to strong fluorescence 46. There may have been post mortem changes, heavy pigment accumulations may have obscured specific fluorescence in these 50-90-year-old neurons, or they may simply store CA in concentrations below the level of detectability. The CA nerve cell bodies close to the third ventricle which showed a relatively strong fluorescence in the adult brain have also been observed in the fetal human brain 46. The occurrence of pigments in brain tissue has received much attention because of their widespread distribution in the human brain. It is now generally believed that lipofuscin forms in lysosomes 25 and that neuromelanin may form on a lipofuscin infrastructure 23. Characteristically, both pigment types increase in age and occur in larger amounts in higher primates. Peroxidase may have a common role in CA, lipofuscin and neuromelanin synthesis in the brain 45 and the neuromelanin o f e.g. the substantia nigra is formed by DOPA-melanin and possibly dopamine-melanin 43. The close interrelationship between CA and neuromelanin (see also ref. 6) and the close resemblance between the neuromelanin map of the human brain 8,5a and the CA cell groups of the rat 2° made Bazelon et al. 8 suggest that 'the aging catecholamine neuron may accumulate its own identifying marker - - neuromelanin.' Our present results give direct proof of this hypothesis because we have been able to visualize simultaneously in the same neuronal cytoplasm neuromelanin and catecholamine fluorescence. So far, however, we have performed no detailed correlation of CA fluorescence and neuromelanin content throughout the brain. The large amount of lipofuscin in the human brain is present both in neurons and glial elements (for review of literature on neurolipofuscin and neuromelanin, see Braakll). It presents certain problems in monoamine fluorescence histochemistry due
HISTOCHEMISTRY OF HUMAN POST MORTEM BRAIN
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to its fluorescence characteristics t5 having an autofluorescence 1~,a2,35,55 that is strong, yellow and only very slowly fading. However, although black and white microphotographical documentation of monoamine-induced fluorescence is difficult, the identification of specific fluorescence in the microscope is easy due to the differences in morphology, color, fluorescence intensity, fading characteristics, sharpness of borders, etc. The direct demonstration of CA and neuromelanin in the same nerve cell bodies adds to the wealth of information concerning the relationship between loss of neuromelanin and cell bodies in the substantia nigra in Parkinson's disease and the decreased DA levels of the striatum (see refs. 3, 16, 26). Underpigmentation of the substantia nigra also occurs, e.g. in chronic disease in children 60, and it would be interesting to complete this finding with fluorescence histochemistry. The viability of the amine uptake and Concentration mechanism over the nerve terminal membrane post mortem is most promising, since it allows a more detailed mapping of the distribution of monoamine nerve terminals in various areas by simple in vitro incubations of brain slices as demonstrated in the present study. Using a relatively high concentration of a-methyl-NA, which is resistant to the action of monoamine oxidase, CA nerve terminals were clearly visualized in all parts of the brain examined. 6-OH-DA causes selective destruction of central CA nerve fibers 66 and preincubation with this drug completely prevented the uptake of a-methyl-NA. However, an uptake of 5-HT into varicose nerve fibers could still be achieved in 6-OH-DA preincubated slices of the cortex cerebri, suggesting that this may be a method to reveal the distribution of 5-HT nerve terminals. The clinical implications of the present results are manifold. Principally, two types of pathological disturbances of the monoamine neurons can be detected by fluorescence histochemistry. Firstly, there may be abnormal distribution patterns which would be best revealed by in vitro amine incubations. Degeneration of DA and possibly NA nerve terminals may be expected in Parkinsonism and the hypothesis of Stein and Wise1, 61-64 of a degeneration of NA nerve terminals in cortical areas in schizophrenia can be directly tested. Interestingly, no decrease in the number of small CA nerve terminals of the nucleus caudatus was observed in the case of Parkinsonism studied here*. This observation may suggest that the symptoms of Parkinsonism may have a varying genesis although in the majority of cases a more or less severe degeneration of the nigro-striatal DA system should be expected (see refs. 26 and 33). It could not be excluded that the green fluorescent varicosities found in the present case of Parkinsonism were of the NA type although their small size, high density and typical distribution strongly suggest that they were DA varicosities. One (F15)of the two schizophrenia cases studied had a history of schizophrenia from 25 years of age and with a 38-year duration. Oligophrenia may have been part of the clinical picture in this case from the early stages of the disease. The distribution of CA nerve terminals in the cortical and subcortical areas including the striatum complex was similar to * The diagnosis was somewhat unclear since the patient died before the effect of the initiated LDOPA therapy could be evaluated.
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L. OLSON cl a/.
that in the rest of the present cases, thus not directly supporting the theory of Stein and Wise1, 61-64, a theory that has been seriously questioned 4. It must be pointed out, however, that no quantitative conclusions can be drawn from the present limited material regarding an altered fluorescence histochemical picture in any diseased state. To our knowledge an increased number of monoamine nerve terminals has not yet been suggested in combination with any pathological states of the human brain. This does not seem to be an unlikely possibility, however, following brain damage that causes chronic deafferentation of certain areas in view of the interesting experimental data on collateral sprouting of CA fibers obtained in animals4'~,G.L Secondly, the amount and concentration of monoamines in the nerve terminals may vary, e.g. as predicted by the catecholamine theory of affective disorders (see refs. 13 and 57). Thus, 5-HT has been reported decreased post mortem in brains of depressive suicides ag. In order to be able to evaluate possible decreases of endogenous stores post mortem and to compare fluorescence intensities between patients the post mortem time has to be carefully considered. The present material is too small to permit any other conclusion than the fact that neuronal monoamine stores can indeed be visualized post mortem in many brain areas of mano-depressive patients. We conclude that endogenous intraneuronal CA and, to a lesser degree, 5-HT stores can be visualized in the human brain up to several hours post mortem and that these nerves likewise retain an active uptake concentration mechanism for amines for several hours. By a continued collection of material for fluorescence histochemical analysis it is hoped that a more precise description of the cytoarchitectonics of the monoamine neuron systems in the adult human brain, as well as the possible involvement of these systems in various diseases, will be obtained. ACKNOWLEDGEMENTS
This study was supported by grants from the Swedish Medical Research Council (04X-3185), 'Magnus Bergvalls Stiftelse' and 'Karolinska Institutets Fonder'. The brains were obtained with the kind help of Dr. L. Wetterberg, Psychiatric Research Center, Ullerhker Hospital, Uppsala. We thank Miss Monica Eliasson, Mrs. Barbro Norstedt and Miss Ingrid Str6mberg for skillful technical assistance.
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cholamines in vivo induced by electrical stimulation of central monoamine pathways, Brain Research, 24 (1970) 471-483. 6 BARDEN,H., The histochemical distribution and localization of copper, iron, neuromelanin and lysosomal enzyme activity in the brain of aging rhesus monkey and the dog, J. Neuropath. exp. Neurol., 30 (1971) 650-667. 7 BATTISTA,A., FUXE, K., GOLDSTEIN, M., AND OGAWA, M., Mapping of central monoamine neurons in the monkey, Experientia (Basel), 28 (1972) 688-690. 8 BAZELON, M., FENICHEL, G. M., AND RANDALL, J., Studies on neuromelanin. I. A melanin system in the human adult brain stem, Neurology (Minneap.), 17 (1967) 512-519. 9 BERTLER,A., Occurrence and localization of catecholamines in the human brain, Acta physiol. scand., 51 (1961) 97-107. 10 BERTLER,A., AND ROSENGREN,E., Occurrence and distribution of catecholamines in brain, Acta physiol, scand., 47 (1959) 350-361. 11 BRAAK,H., lJber das Neurolipofuscin in der unteren Olive und dem Nucleus dentatus cerebelli im Gehirn des Menschen, Z. Zellforsch., 121 (1971) 573-592. 12 BRIZZEE,K. R., ANDJOHNSON, F. A., Depth distribution of lipofuscin pigment in cerebral cortex of albino rat, Acta neuropath. (Berl.), 16 (1970) 205-219. 13 BUNNEY,W. E., BRODIE,K. H., MURPHY,D. L., AND GOODW1N,F. K., Studies of alpha-methylpara-tyrosine, L-dopa, and L-tryptophan in depression and mania, Amer. J. Psychiat., 127 (1971) 872-881. 14 CARLSSON,A., FALCK,B., AND HILLARP,N.-/~., Cellular localization of brain monoamines, Acta physiol, scand., 56, Suppl. 196 (1962) 1-28. 15 CHIO, K. S., REISS,U., FLETCHER,B., AND TAPPEL,A. L., Peroxidation of subcellular organelles: formation of lipofuscinlike fluorescent pigments, Science, 166 (1969) 1535-1536. 16 CHRISTENSEN,E., Pathology of extrapyramidal diseases. In S. REFSUM,H.-M. Losslos AND P. D1ETRICHSON(Eds.), The so-called Extrapyramidal System, Scandinavian University Books, Oslo, 1962, pp. 119-126. 17 CONSTANTINIDIS,J., TISSOT,R., DE LA TORRE,J. C., ET GE1SSBUHLER,F., Essai de localisation des monoamines dans l'hypothalamus humain, Path. et Biol., 17 (1969) 361-363. 18 CORRODI,I"I., FUXE, K., LIDBRINK,P., AND OLSON, L., Minor tranquillizers, stress and central catecholamine neurons, Brain Research, 29 (1971) 1-16. 19 CORRODI, H., AND JONSSON, G., The formaldehyde fluorescence method for the histochemical demonstration of biogenic amines. A review on the methodology, J. Histochem. Cytochem., 15 (1967) 65-78. 20 DAHLSTROM,A., AND FUXE, K., Evidence for the existence of monoamine-containingneurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stern neurons, Acta physiol, scand., 62, Suppl. 232 (1964) 1-55. 21 DAHLSTR~M,m., AND FUXE,K., Evidence for the existence of monoamine-containingneurons in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems, Acta physiol, scand., 64, Suppl. 247 (1965) 1-36. 22 DE LA TORRE,J. C., Catecholamines in the human diencephalon: A histochemical fluorescence study, Acta neuropath. (Berl.), 21 (1972) 165-168. 23 DUFEY,P., AND TENNYSON,V., Phase and electron microscopic observations of Lewy bodies and melanin granules in the substantia nigra and locus coeruleus in Parkinson's disease, J. Neuropath. exp. Neurol., 24 (1965) 398-414. 24 EHRINGER,H., UND HORNYKIEWICZ,O., Verteilung von Noradrenalin und Dopamin (3-Hydroxytyramin) im Gehirn des Menschen und ihr Verhalten bei Erkrankungen des extrapyramidalen Systems, Klin. Wschr., 38 (1960) 1236--1239. 25 ESSNER,E., AND NOVIKOFF,A., Human hepatocellular pigments and lysosomes, J. Ultrastruct. Res., 3 (1960) 374-391. 26 FAHN,S., LIBSCH,L. R., AND CUTLER,R. W., Monoamines in the human neostriatum: Topographic distribution in normals and in Parkinsons's disease and their role in akinesia, rigidity, chorea, and tremor, J. neurol. Sci., 14 (1971) 427-455. 27 FALCK,B., HILLARP,N.-A., THmME,G., ANDTORP, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. Histochem. Cytochem., 10 (1962) 348-354. 28 FuxE, K., Evidence for the existence of monoamine-containingneurons in the central nervous system. III. The monoamine nerve terminal, Z. Zellforsch., 65 (1965) 573-596. 29 FuxE, K., Evidence for the existence of monoamine-containingneurons in the central nervous
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