Journal of the Neurological Sciences, 1979, 41 : 397~,09 © Elsevier/North-Holland Biomedical Press
397
D I S T R I B U T I O N OF A D R E N A L I N E - S Y N T H E S I Z I N G E N Z Y M E A C T I V I T Y I N THE HUMAN BRAIN
N. KOPP i, L. DENOROY ~, B. RENAUD 2, J. F. PUJOL a, A. TABIBi and M. TOMMASIi iLaboratoire de Neuropathologie du CHU de Lyon, Facultd de Mddecine A. Carrel, 8, rue Guillaume Paradin, 69372 Lyon C~dex 2; eLaboratoire de Biochimie, tt6pital Neurologique, BP Lyon Montchat, 69394 Lyon C~dex 3, and aGroupe de Recherche de Neurochimie Fonctionnelle 1NSERM U. 171, 8, avenue Rockefeller, 69373 Lyon C~dex 2(France)
(Received 14 November, 1978) (Accepted 9 January, 1979)
SUMMARY A study of the distribution of phenylethanolamine-N-methyl-transferase (PNMT) activity in normal human brain is presented. After a preliminary dissection to separate brain tissue for formalin fixation and tissue designed for biochemical studies, the hemi-brain stem is cut in slices by hand and a cerebral hemisphere is cut on a cryomicrotome. "Punches" are made with an operating microscope. This dissection method was used to study the distribution of P N M T activity in 117 "punches" made on 21 slices obtained from 5 normal h u m a n brains. The caudo-rostral distribution of P N M T activity in C1 and C2 groups was found to be identical in each brain. The distribution of P N M T activity was found to be similar to that in the rat, but, in addition, important activity was found in the substantia nigra, internal pallidum and nucleus accumbens.
INTRODUCTION The anatomy and biochemistry of central monoamine systems are well documented in laboratory animals, but information on such systems in h u m a n brain is rare and implecise. Recent work (Aquilonius et al. !975; McGeer and McGeer 1976; Riederer and Wuketich 1976; Lew et al. 1977; Nagatsu et al. 1977)has shown that it is possible to measure neurotransmitter synthesizing enzymes in human post-mortem brains but studies on such material require some precautions: (i) the use of enzymatic markers This work was supported by a grant (ATP 657897) from INSERM. Address correspondence to: N. Kopp, M.D., H6pital Neurologique, B. P. Lyon Montchat, 69394 Lyon C6dex 3, France.
398 stable enough after death, (ii) a reproducible protocole for precise anatomical dissectio n, sampling and storage, (iii) a closely matched control group (age, cause of death and post-mortem delay). This paper presents a study of the adrenaline synthesizing enzyme, phenylethanolamine-N-methyl transferase (PNMT) activity in discrete areas of normal human brain chosen for their putative physiological importance and their postulated role in monoamine pathways, on the basis of animal work (Dahlstr6m and Fuxe 1964; H6kfelt et al. 1974; McGeer and McGeer 1976). The activity of the adrenaline forming enzyme was measured in order to determine with anatomical precision the organization of adrenergic systems in human brain and to obtain values of activity of this enzyme. M A T E R I A L A N D METHODS
Five human brains were obtained through the Laboratoire d'Anatomie Patholooptic chiasma
infundibulum
~
mammillary ~
body cerebral peduncle
ii
ba si l a r
sulcus
pyramide olive
Fig. 1. The brain stem and the hypothaiamus were separated from cerebrum and cerebellum (aim u): Then the hypothalamus was separated from the brain stem ( • • • ) . Finally, the left portion of brain stem was separated from the right portion of brain stem (o • • ) .
399 gique, H6pital Cardiologique, Lyon. The mean age of patients was 55 4- 6 years, and the mean post-mortem delay was 11.6 4- 2.6 hours. Except for one case patients had normal blood pressure and received no drugs known to modify neurological status, during the 4 months preceding death. Three patients died of myocardial infarction: one died suddenly and the two others died of cardiogenic shock lasting respectively two and five hours. The other two patients died during surgical operation for mitral stenosis. A. Anatomical Dissection Autopsies and dissections were performed by the same person. Special care was taken to avoid distortion, especially by excessive traction on the brain stem. Preliminary dissection Immediately after autopsy the brain was placed on a cork plate. Arteries and arachnoid were carefully stripped off. The splenium of the corpus callosum was then sectioned in order to allow easy access to the pineal gland and the postero-superior part of the third ventricle. The pineal gland was then removed and the right and left habenular nuclei were dissected by resection of the thalamus adjacent to the habenular commissure. Anterior and lateral limits of the hypothalamus were vertically sectioned as indicated on Fig. 1. The lateral sections were extended across the cerebral peduncles perpendicular to their main axis. The brain stem and hypothalamus were then separated from the cerebral hemispheres. The hypothalamus was isolated by a transverse section along the posterior limit of the mammillary bodies. The brain stem was longitudinally sectioned in a plane parallel to and 2 mm away from the medial sagittal plane on the right side of the brain stem (see Fig. 1). The cerebral hemispheres were then separated after total section of the corpus callosum. The right portion of the brain, designed for histological control, was fixed in 10 ~ formalin and the left portion for chemical assays was frozen and stored at - - 8 0 ° C for less than one month. Fifteen hours before final dissection, frozen tissues were brought to --10 °C for preparation of frontal brain slices and dissection of the brain structures.
@ @
@
Fig. 2. Schematic representation of the 21 slices of human brain. Each dark circle indicates the site of sampling ("punches").
400
@
Fig. 3. Brain stem (left portion) is sectioned in 16 planes. Sections marked by circled numbers are represented on Figs. 4 and 5. The 4 caudal sections of medulla oblongata were approximately 1.5 mm thick, the rostral sections approximately 1 mm.
Preparation of 21 frontal brain slices (numbered (1) to (21)) (see Fig. 2) Brain stem was cut into 16 slices (see Fig. 3) by hand with a Lipshaw tissue cutter. Ten serial sections (numbered (1) to (10)) were cut in the upper medulla oblongata: 8 of them at the level of the olive, one immediately above it, and one immediately below. The 6 other sections were located more rostrally, as follows: (11) at the level of the lower 1/6th of the pons, (12) at the middle of the ports, (13) two mm below the emergence of the IVth cranial nerves, (14) at the junction of upper limit of the pons and posterior limit of the cerebral peduncle, (15) at the level of the inferior colliculus and the decussation of the superior cerebellar peduncles and (16) at the level of the superior colliculus and red nucleus. The hypothalamus was sectioned with a tissue cutter (Lipshaw), perpendicular to the sagittal plane, midway between the origin of the pituitary stalk (infundibutum) and the anterior limit of the mammillary bodies. The cerebralhemisphere was cut according to a slight modification of the method of Aquilonius et al. (1975). We used a microtome (MSE large section microtome, according to Gough, France Omnium Scientific Industry, Paris) operated at - - 1 0 °C. Thick sections embedded in carboxy-methyl-ceUulose were cut from their caudal limit to a predetermined appropriate level. At each level, planes (17), (19), (20) and (2 I) two consecutive sections of 250 # m were made. Dissection of the brain structures Samples were "punched" using a hollow needle (2.0 mm internal diameter)
401 under the control of a portable operating microscope (Optikon). Samples from hemispheric areas were taken from two consecutive 250 #m slices, and pooled. Each dissection site was chosen according to the coordinates of Braak (1970) and Olzweski and Baxter (1954) for the brain stem and of Roberts and Hanaway (1970) for the cerebral hemisphere. A preliminary study of the distribution of PNMT activity was made in planes (4) and (8) of the medulla oblongata. The regions of brain stem which exhibited the highest PNMT activity were referred to as "C 1" and "C2" areas, in comparison with the nomenclature established by H6kfelt et al. (1974) and Lew et al. (1977). Medulla oblongata "punches" (see Figs. 2, 3 and 4) were taken in planes (1) to (10). The "CI" area (C1) was dorsal to the mid portion of the inferior olive. It was composed of parts of the lateral reticular nucleus, the nucleus medullae oblongatae lateralis, the subnucleus ventralis, and the nucleus paragiganto cellularis lateralis. The "C2" lateral group (C21) was situated in the area of the dorsal nucleus of the vagus and of the nucleus tractus solitarius; the "C2" medial group (C2m) was located in the area of the hypoglossal nucleus and dorsal nucleus of the vagus. The nucleus raphes obscurus (OB) was in the upper 3/4 of the midline, ventral to C2m. The nucleus raphes pallidus (PA) was situated on the ventral extremity of midline, immediately dorsal to the pyramids. In the pons (planes (11) to (14)) the "punches" (see Figs. 2, 3, 4 and 5) were locatedasfollows: in plane (11) the nucleus raphes magnus (MA) was on the midline of tegmentum slightly ventral to its center (plane 11). In plane (12) the nucleus raphes pontis, ventral part (POv), was on the more ventral part of the midline of tegmentum and the nucleus raphes pontis, dorsal part (POd), was immediately dorsal to POv. In plane (13) the locus coeruleus, caudal part (LCc) and in plane (14) the locus coeruleus, rostral part (LCr) were easily identified because of the pigmentation of the nucleus. The nucleus raphes dorsalis, caudal part (DOc) (plane 13) and nucleus raphes dorsalis, rostral part (DOr) (plane 14) were located on the midline immediately adjacent to the ventral limit of the aqueduct. The nucleus raphes centralis, caudal part (CEc) (plane 13) and the nucleus raphes centralis, rostral part (CEr) (plane 14) were located on the midline, immediately ventral to DOc and DOr. In the mesencephalon (planes (15) and (16)) the "punches" (see Figs. 2, 3 and5) were located as follows: in the substantia nigra 6 caudal punches (SNlc to SN6c) were made in plane (15) and 6 rostral punches (SNlr to SN6r) were made in plane (16) (see Figs. 3 and 5). In these planes samples I, 2, 3 and 4 corresponded to the pars compacta and samples 5 and 6 to the pars reticulata. The interpeduncular nucleus (IP) was sampled on the midline in its most ventral part, in plane (15), and the substantia perforata posterior (SPP) was sampled in the same way, in plane (16). The periaqueductal gray, caudal part (PGc) (plane 15) and periaqueductal gray, rostral part (PGr) (plane 16) were taken in the lateral and ventral part of the periaqueductal substance. The structures called mesencephalic raphe (MR) were sampled in 4 locations on the midline. In plane (15) the caudal and ventral part (MRcv) was located in the middle of the line joining the superior limit of the decussation of superior cerebellar peduncles; the rostral and dorsal part (MRrv) was
402
.
Figs. 4 and 5. Several planes of section are used for the dissection of the brain stem. The right part of all the sections is a photograph of the dissected tissue. The left part of the section is a schematic representation (see text) of structures that can be identified macroscopically on a fresh section of the human brain stem. Abbreviations in white characters represent the localization of punches as described i n t h e text (see Methods). Abbreviations in dark characters symbolize the structures used for the localization of sampling (according to Olszewski and Baxter 1954).
403
A R C = nucleus arcuatus; CI = nucleus colliculi inferioris ; CS = nucleus colliculi superioris; D P C S -- decussatio p e d u n c u l o r u m cerebellorum s u p e r i o r u m ; D X = nucleus dorsalis motorius nervi vagi; F L M -- fasciculus longitudinalis medialis; G C M = griseum centrale mesencephali; I P M nucleus interpeduncularis subnucleus medialis; LC -- locus coeruleus; LL = lemniscus lateralis; L M = lemniscus medialis; M G B = corpus geniculatum mediale; O I D = nucleus olivaris inferior accessorius dorsalis; O I P nucleus olivaris inferior principalis; O I M = nucleus olivaris inferior accessorius medialis; PCI = pedunculus cerebelli inferior; PCS - pedunculum cerebelli superior; P G L = nucleus paragigantocellularis lateralis; P N = nucleus paranigralis; RP -- nucleus ruber, subnucleus parvocellularis; S N C = nucleus substantia nigra, subnucleus compactus; S N R = nucleus substantia nigra, subnucleus reticulata; SOL -- nucleus tractus solitarii; SPP = substantia perforata posterior; X I I = nucleus nervi hypoglossi.
404 located on the middle of the line joining the center of red nuclei, pars parvocellularis, the rostral and dorsal part (MRrd) was dorsal to MRrv. In the hypothalamus 4 samples corresponding to the arcuate nucleus and to the area immediately adjacent (called here paramedial hypothalamus) were dissected from plane (18) (Fig. 2). In the cerebral hemisphere the frontal cortex at the level of callosomarginal sulcus, four samples were taken: parts 1 and 2 in the first frontal gyrus, part 3 in the second frontal gyrus and part 4 in the third frontal gyrus. Three "punches" (upper, medium and lower) were dissected in the nucleus accumbens immediately rostrally to putamen (Roberts and Hanaway 1970); those corresponding to amygdaloid complex were taken at the level where the anterior commissure crosses the putamen and external pallidum (see Fig. 2, plane (19) and Fig. 6). Hippocampal samples (see Fig. 2, plane (17)) were taken in Sommer's sector and in the area resistant to hypoxia, outside Sommer's sector, at level of maximal development of the lateral geniculate body (Roberts and Hanaway 1970). All samples were kept in Eppendorf 1.5 ml polyethylene tubes in a liquid nitrogen container until they were assayed. All manipulations were performed with surgical mask and gloves. After each manipulation tools were washed with a hypochlorite solution according to Traub et al. (1976). Histological controls (haemalum-phloxine-safranin, Mallory's PTAH) were performed on formalin-fixed tissues from both sides. No gross neuropathological features were observed other than those commonly seen in elderly patients (corporea amylacea, discrete arteriolar thickening).
Fig. 6. The 25 sites of "punch" sampling in the basal ganglia, cingulum and white matter (see:text). Symbols: A M = amygdaloid complex; C I N = cinsulate cortex; C L = claustrum; C N = e a r . a t e nucleus; G P = globus pallidus; P U = putamen; W M = white matter.
405 B. Chemical Methods
Tissue samples were homogenized in 5 mM Tris-HCL buffer pH 7.4 containing 0.2 ~ (v/v) of Triton X100. The homogenisation volumes were 200/,1 for the brain stem samples, 150 #1 for the telencephalic and diencephalic samples, and 500/A for the pineal samples. The homogenates were centrifuged (9000 x g, 15 rain) at -k 4 °C. P N M T activity was measured on 50 #1 of supernatant according to the radiometric technique of Saavedra et al. (1974) with minor modification (Renaud et al. 1978). The concentrations of reagents in the incubation medium (volume 100 #1) were: Tris-HC1 buffer pH 8.6:0.2 M; phenylethanolamine: 400 # M and methyl[aH]S adenosylmethionine: 3 . 5 / , M (1.5 #Ci). The incubation was carried out for 60 min. The N-[aH]methylphenylethanolamine formed was extracted by toluene containing 3 ~ of isoamylalcohol (v/v); the solvent was evaporated overnight in a ventilated oven without significant loss of product, and the radioactivity was counted by liquid scintillation. Blanks were prepared by omitting phenylethanolamine from the incubation mixture. As the blank value differed from one sample to another (700-3000 cpm), blanks were determined for all the samples analyzed. The soluble proteins were determined on 10/~1 of supernatant with the Folin phenol reagent (Lowry et al. 1951) using bovine serum albumin (Miles) as standard. The P N M T activity was expressed as pmoles of N-methyl phenylethanolamine formed per hour of incubation and per mg of protein. The results are given as a mean :k SEM. The limit of detection of the assay was arbitrarily fixed to twice the blank value. The term "traces" was used when the P N M T activity was between 1.5 and twice the blank value. RESULTS As shown in Table 1, the highest P N M T activity in the medulla oblongata was found in the Cl area. This value was therefore chosen as a reference (100 ~ activity). The area containing the hypoglossal nucleus and the dorsal vagal nucleus (C2m) exhibited higher P N M T activity than C21. The rostro-caudal distribution of P N M T activity in the Cl area is presented in Fig. 7. The peak of activity for both regions was located immediately rostral to the middle of the inferior olive. A similar rostro-caudal distribution was found for the C2m area. In the upper portion of the brain, high P N M T activity was found in the arcuate nucleus of hypothalamus (145 ~ of the activity found in the Cl area). P N M T activity was also present in the paramedial hypothalamus (70 ~ ) in the rostral part of the substantia nigra (50 ~), caudal locus coeruleus (40 ~), nucleus accumbens (30.8 ~ :k 14.4 when upper and medial parts are pooled in each subject), internal globus pallidus (28 ~ ) and periaqueductal gray (17 ~). On the other hand very low or no activity was found in various raphe nuclei (except for raphe pallidus which contained 20 ~o of the activity of the C1 area) and in other cortical and subcortical areas. The activities found in brain areas, on the whole, were low when compared with the activity found in the adrenal medulla (132 i 20 mol/hr/mg protein, i.e. 600 ~ of P N M T activity in Cl).
406 TABLE 1 DISTRIBUTION OF S I G N I F I C A N T PNMT ACTIVITY IN THE H U M A N BRAIN No PNMT activity was found in : nucleus raphes magnus, nucleus raphes pontis (ventral part), nucleus raphes centralis (caudal part), prefrontal cortex (part 3), deep white matter, hippocampal formation (resistant), pineal gland. Traces of PNMT activity were found in : nucleus raphes obscurus, nucleus raphes pontis (dorsal part), nucleus raphes dorsalis (caudal and rostral part), nucleus raphes centralis (ro3tral part), interpeduncular nucleus, mesencephalic raphe (dorsal caudal and rostral, and ventral caudal and rostral), habenula, prefrontal cortex (part 1,2 and 4), amygdala (part 1,2, 3 and 4), g~rus cinguli (part 1 and 2), caudate nucleus (part 1, 2 and 3), putamen, hippocampal formation (fragile), claustrum (part 1 and 2). Structures
PNMT activity (pmole/hr/mg prot.) (mean 5: SEM)
% of activity of C1 area
C1 area C2m area C21 area Nucleus raphes pallidus Locus coeruleus (caudal part) Locus coeruleus (rostral part) Periaqueductal grey (caudal) Periaqueductal grey (rostral) Substantia perforata (posterior) Left paramedial hypothalamus Right paramedial hypothalamus Left arcuate nucleus Right arcuate nucleus Nucleus accumbens (upper part) Nucleus accumbens (medium part) Nucleus accumbens (lower part) Globus pallidus (part 1) Globus pallidus (part 2) Globus pallidus (part 3) Globus pallidus (part 4) Substantia nigra (caudal) lc 2c 3c 4c 5c 6c Substantia nigra (rostral) lr 2r 3r 4r 5r 6r
8.28 i 0.74 5.58 ± 1.09 1.72 ± 0.52 1.82 ± 0.69 3.28 5:0.91 2.00 ± 1.01 1.11 £ 0.78 1.24 5:0.82 1.90 ± 0.83 5.36 ± 2.13 6.57 5:2.52 11.47 5: 1.83 11.60 ± 3.49 3.55 5:2.56 1.29 5:0.56 Traces 1.05 ± 0.66 0.98 ± 0.68 2.19 5:0.96 2.26 5:0.77
100 by definition 67.1 ± 15.0 21.1 5:6.8 20.8 i 7.6 39.7 5:10.9 25.6 5:13.4 16.1 ~ t 1.1 18.0 5:11.7 24.4 ± 10.8 63.6 5:20.1 76.7 +- 22.7 145.3 5:29.3 144.0 5:37.6 46.4 5:31.9 16.7 - 7.4 15.4 14.2 27.1 28.7
± 9.6 ± 9.6 5:12.2 -3- 9.6
1.15 ± 0.75 1.48 ± 0.63 0 1.14 5:0.72 0 0
14.1 5:8.7 19.1 i 7.8 0 13.9 5:8.6 0 0
3.32 3.56 3.46 3.97 3.73 4.28
39.4 42.9 45.1 52.2 48.6 52.1
5:1.19 5:0.42 5:0.91 5:1.04 ± 0.94 5:0.52
5:12.6 5:3.5 ± 12.8 ± 14.9 5:13.2 5:5.6
DISCUSSION
The dissection technique described above was used to study the distribution o f P N M T activity in 117 areas of 5 human normal brains. The results were reproducible,
407 P NMT activity
300
200
caudo_ rostral direction
Fig. 7. Caudo-rostral distribution of P N M T activity in the " C I " area of the medulla oblongata. For each section the activity is expressed as percentage (mean ± SEM) of the specific activity (per mg protein) of the total group.
partly because of the anatomical precision of the dissection and the stability of the enzyme in spite of a post-mortem delay ranging from 2.5 to 16 hours. The rostrocaudal distribution of PNMT activity in the C1 and C2 areas (Fig. 7) was found to be identical in each brain: for example the maximum enzymatic activity was always found at the same level (slice (6) of the medulla oblongata). In addition, adjacent structures on the same slice exhibited wide differences in PNMT activity (see structures C1, OB, C2m for example). In the present work we have taken special precautions to avoid assay nonspecificity (Saavedra et al. 1973). In order to minimize the measurement of non-specific N-methyl-transferase activity we performed an appropriate blank on each supematant (see Methods). The biochemical specificity of the PNMT assay is confirmed by the observation of a different pattern of regional distribution when compared to the distribution of non-specific N-methyl-transferase previously described in the human and rat brains (Saavedra et al, 1973). Our results demonstrate the heterogeneity of the distribution of PNMT, since the absolute activity varied over several orders of magnitude in the various brain areas analyzed. A heterogenous distribution of PNMT activity has already been suggested in human brain (Lew et al. 1977; Nagatsu et al. 1977; Vogel et al. 1976). The determination of this enzymatic activity after a precise and reproducible dissecting procedure allows a comparison with previous studies performed in the rat (Saavedra et al. 1974) under
408 similar conditions. In the brain stem the high activity f o u n d in the C I and C2m g r o u p s m a y c o r r e s p o n d , as in the rat, to the presence o f adrenergic cell bodies ( H 6 k f e l t et al. 1974). A c c o r d i n g to the present a n d earlier results o f Mefford et al. (1977), N a g a t s u et al. (1977) and Lew et al. (1977), difference between the distribution o f P N M T and that o f a d r e n a l i n e exists in h u m a n brain, p a r t i c u l a r l y in the CI area. A similar discrepancy has also been f o u n d for the rat b r a i n ( S a a v e d r a et al. 1974; Van der Gugten et al. 1976) especially in the C1 a r e a where a low a d r e n a l i n e c o n c e n t r a t i o n and high P N M T activity has been described. Significant P N M T activity was also f o u n d in other structures (presenting, in the rat, adrenergic innervation) such as h y p o t h a l a m u s , locus coeruleus a n d p e r i a q u e d u c t a l gray. S o m e o t h e r areas m a y be i n n e r v a t e d b y adrenergic n e u r o n s in m a n b u t n o t in rat, such as s u b s t a n t i a nigra, internal p a l l i d u m , nucleus accumbens. This confirms and completes previous d a t a o f Lew et al. (1977), a n d N a g a t s u et al. (1977). It can be h y p o t h e s i z e d t h a t the presence o f an adrenergic innervation in b u l b a r reticular f o r m a t i o n s , h y p o t h a l a m i c areas a n d b a s a l ganglia c o u l d play i m p o r t a n t functional roles, including b l o o d pressure control, e n d o c r i n e regulation a n d c o n t r o l o f motility. ACKNOWLEDGEMENTS The a u t h o r s are grateful to Mrs. N. G a y for her technical assistance, Dr. J. A t k i n s o n for reviewing the m a n u s c r i p t a n d to Miss A. L a b r o s s e for typewriting.
REFERENCES Aquilonius, S. M., S. A. Eckern/is and A. Sundvall (1975) Regional distribution of choline acetyltransferase in the human brain - - Changes in Huntington's chorea, J. Neurol. Neurosurg. Psych., 38 : 669-677. Braak, H. (1970) Ober die Kerngebiete des menschlichen Hirnstammes, Part 2 (Die Raph6kerne), Z. Zellforsch., 107 : 123-141. Dahlstr6m, A. and K. Fuxe (1964) Evidence for the existence of monoamine containing neurons in the central nervous system, Part 1 (Demonstration of monoamines in the cell bodies of brain stem neurons), Acta physiol, scand., 62 (Suppl. 232): 1-55. H6kfelt, T., K. Fuxe, M. Goldstein and O. Johansson (1974) Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Res., 66: 235-251. Lew, J. Y., Y. Matsumoto, J. Pearson, M. Goldstein, T. H6kfelt and K. Fuxe (1977) Localization and characterization of phenylethanolamine-N-methyltransferase in the brain of various mammalian species, Brain Res., 119: 199-210. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall 0951) Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 : 265-275. McGeer, P. L. and E. G. McGeer (1976) Enzymes associated with the metabolism of catecholamines acetylcholine and GABA in human controls and patients with Parkinson's disease and Huntington's chorea, J. Neurochem., 26: 65-76. Mefford, I., A. eke, R. N. Adams and G. Jonsson (1977) Epinephrine localization in human brain stem, Neurosci. Lett., 5: 141-145. Nagatsu, T., T. Kate, Y. Numata Sudo, K. Kuta, M. Sane, I. Nagatsu, Y. Kondo, S. lnagaki, R, Iizuka, A. Hori and H. Narabayashi (1977) Phenylethanolamine N,rnethyltransferase and other enzymes of catecholamine metabolism in human brain, Clin. chim. Acta, 75 : 221-232. Olszewski, J; and D. Baxter (1954) Cytoarchitecture of the HumanBrain Stem, Karger, Basel, New York, Renaud, B., S. Fourni6re, L. Denoroy, M. Vincent, J. F. Pujol and J. Sassard (1978) Early increase in phenylethanolamine-N-mgthyltransferaseactivity in a new strain of spontaneously hyl:ertensive rats, Brain Res., 159: 149-159.
409 Riederer, P. and S. Wuketich (1976) Time course of nigrostriatal degeneration in Parkinson disease, J. Neurol. Transm., 38: 277-301. Roberts, M. and J. Hanaway (1970) Atlas of the Human Brain in Section, Lea and Febiger, Philadelphia. Saavedra, J. M., J. T. Coyle and J. Axelrod (1973) The distribution and properties of the nonspecific N-methyltransferase in brain, J. Neurochem., 20: 743-752. Saavedra, J. M., M. Palkovits, M. J. Brownstein and J. Axelrod (1974) Localization of phenylethanolamine-N-methyltransferase in the rat brain nuclei, Nature (Lond.), 248: 695-696. Traub, R. D., D. C. Gajdusek and C. J. Gibbs (1974) Precautions in conducting biopsies and autopsies on patients with presenile dementia, J. Neurosurg., 41 : 394-395. Van der Gugten, J., M. Palkovits, H. L. J. M. Wijnen and D. H. G. Versteeg (1976) Regional distribution of adrenaline in rat brain, Brain Res., 107: 171-174. Vogel, W. H., L. E. Lewis and D. H. Boheme (1976) Phenylethanolamine-N-methyltransferase activity in various areas of human brain, tissues and fluids, Brain Res., 115 : 357-359.
397
D I S T R I B U T I O N OF A D R E N A L I N E - S Y N T H E S I Z I N G E N Z Y M E A C T I V I T Y I N THE HUMAN BRAIN
N. KOPP i, L. DENOROY ~, B. RENAUD 2, J. F. PUJOL a, A. TABIBi and M. TOMMASIi iLaboratoire de Neuropathologie du CHU de Lyon, Facultd de Mddecine A. Carrel, 8, rue Guillaume Paradin, 69372 Lyon C~dex 2; eLaboratoire de Biochimie, tt6pital Neurologique, BP Lyon Montchat, 69394 Lyon C~dex 3, and aGroupe de Recherche de Neurochimie Fonctionnelle 1NSERM U. 171, 8, avenue Rockefeller, 69373 Lyon C~dex 2(France)
(Received 14 November, 1978) (Accepted 9 January, 1979)
SUMMARY A study of the distribution of phenylethanolamine-N-methyl-transferase (PNMT) activity in normal human brain is presented. After a preliminary dissection to separate brain tissue for formalin fixation and tissue designed for biochemical studies, the hemi-brain stem is cut in slices by hand and a cerebral hemisphere is cut on a cryomicrotome. "Punches" are made with an operating microscope. This dissection method was used to study the distribution of P N M T activity in 117 "punches" made on 21 slices obtained from 5 normal h u m a n brains. The caudo-rostral distribution of P N M T activity in C1 and C2 groups was found to be identical in each brain. The distribution of P N M T activity was found to be similar to that in the rat, but, in addition, important activity was found in the substantia nigra, internal pallidum and nucleus accumbens.
INTRODUCTION The anatomy and biochemistry of central monoamine systems are well documented in laboratory animals, but information on such systems in h u m a n brain is rare and implecise. Recent work (Aquilonius et al. !975; McGeer and McGeer 1976; Riederer and Wuketich 1976; Lew et al. 1977; Nagatsu et al. 1977)has shown that it is possible to measure neurotransmitter synthesizing enzymes in human post-mortem brains but studies on such material require some precautions: (i) the use of enzymatic markers This work was supported by a grant (ATP 657897) from INSERM. Address correspondence to: N. Kopp, M.D., H6pital Neurologique, B. P. Lyon Montchat, 69394 Lyon C6dex 3, France.
398 stable enough after death, (ii) a reproducible protocole for precise anatomical dissectio n, sampling and storage, (iii) a closely matched control group (age, cause of death and post-mortem delay). This paper presents a study of the adrenaline synthesizing enzyme, phenylethanolamine-N-methyl transferase (PNMT) activity in discrete areas of normal human brain chosen for their putative physiological importance and their postulated role in monoamine pathways, on the basis of animal work (Dahlstr6m and Fuxe 1964; H6kfelt et al. 1974; McGeer and McGeer 1976). The activity of the adrenaline forming enzyme was measured in order to determine with anatomical precision the organization of adrenergic systems in human brain and to obtain values of activity of this enzyme. M A T E R I A L A N D METHODS
Five human brains were obtained through the Laboratoire d'Anatomie Patholooptic chiasma
infundibulum
~
mammillary ~
body cerebral peduncle
ii
ba si l a r
sulcus
pyramide olive
Fig. 1. The brain stem and the hypothaiamus were separated from cerebrum and cerebellum (aim u): Then the hypothalamus was separated from the brain stem ( • • • ) . Finally, the left portion of brain stem was separated from the right portion of brain stem (o • • ) .
399 gique, H6pital Cardiologique, Lyon. The mean age of patients was 55 4- 6 years, and the mean post-mortem delay was 11.6 4- 2.6 hours. Except for one case patients had normal blood pressure and received no drugs known to modify neurological status, during the 4 months preceding death. Three patients died of myocardial infarction: one died suddenly and the two others died of cardiogenic shock lasting respectively two and five hours. The other two patients died during surgical operation for mitral stenosis. A. Anatomical Dissection Autopsies and dissections were performed by the same person. Special care was taken to avoid distortion, especially by excessive traction on the brain stem. Preliminary dissection Immediately after autopsy the brain was placed on a cork plate. Arteries and arachnoid were carefully stripped off. The splenium of the corpus callosum was then sectioned in order to allow easy access to the pineal gland and the postero-superior part of the third ventricle. The pineal gland was then removed and the right and left habenular nuclei were dissected by resection of the thalamus adjacent to the habenular commissure. Anterior and lateral limits of the hypothalamus were vertically sectioned as indicated on Fig. 1. The lateral sections were extended across the cerebral peduncles perpendicular to their main axis. The brain stem and hypothalamus were then separated from the cerebral hemispheres. The hypothalamus was isolated by a transverse section along the posterior limit of the mammillary bodies. The brain stem was longitudinally sectioned in a plane parallel to and 2 mm away from the medial sagittal plane on the right side of the brain stem (see Fig. 1). The cerebral hemispheres were then separated after total section of the corpus callosum. The right portion of the brain, designed for histological control, was fixed in 10 ~ formalin and the left portion for chemical assays was frozen and stored at - - 8 0 ° C for less than one month. Fifteen hours before final dissection, frozen tissues were brought to --10 °C for preparation of frontal brain slices and dissection of the brain structures.
@ @
@
Fig. 2. Schematic representation of the 21 slices of human brain. Each dark circle indicates the site of sampling ("punches").
400
@
Fig. 3. Brain stem (left portion) is sectioned in 16 planes. Sections marked by circled numbers are represented on Figs. 4 and 5. The 4 caudal sections of medulla oblongata were approximately 1.5 mm thick, the rostral sections approximately 1 mm.
Preparation of 21 frontal brain slices (numbered (1) to (21)) (see Fig. 2) Brain stem was cut into 16 slices (see Fig. 3) by hand with a Lipshaw tissue cutter. Ten serial sections (numbered (1) to (10)) were cut in the upper medulla oblongata: 8 of them at the level of the olive, one immediately above it, and one immediately below. The 6 other sections were located more rostrally, as follows: (11) at the level of the lower 1/6th of the pons, (12) at the middle of the ports, (13) two mm below the emergence of the IVth cranial nerves, (14) at the junction of upper limit of the pons and posterior limit of the cerebral peduncle, (15) at the level of the inferior colliculus and the decussation of the superior cerebellar peduncles and (16) at the level of the superior colliculus and red nucleus. The hypothalamus was sectioned with a tissue cutter (Lipshaw), perpendicular to the sagittal plane, midway between the origin of the pituitary stalk (infundibutum) and the anterior limit of the mammillary bodies. The cerebralhemisphere was cut according to a slight modification of the method of Aquilonius et al. (1975). We used a microtome (MSE large section microtome, according to Gough, France Omnium Scientific Industry, Paris) operated at - - 1 0 °C. Thick sections embedded in carboxy-methyl-ceUulose were cut from their caudal limit to a predetermined appropriate level. At each level, planes (17), (19), (20) and (2 I) two consecutive sections of 250 # m were made. Dissection of the brain structures Samples were "punched" using a hollow needle (2.0 mm internal diameter)
401 under the control of a portable operating microscope (Optikon). Samples from hemispheric areas were taken from two consecutive 250 #m slices, and pooled. Each dissection site was chosen according to the coordinates of Braak (1970) and Olzweski and Baxter (1954) for the brain stem and of Roberts and Hanaway (1970) for the cerebral hemisphere. A preliminary study of the distribution of PNMT activity was made in planes (4) and (8) of the medulla oblongata. The regions of brain stem which exhibited the highest PNMT activity were referred to as "C 1" and "C2" areas, in comparison with the nomenclature established by H6kfelt et al. (1974) and Lew et al. (1977). Medulla oblongata "punches" (see Figs. 2, 3 and 4) were taken in planes (1) to (10). The "CI" area (C1) was dorsal to the mid portion of the inferior olive. It was composed of parts of the lateral reticular nucleus, the nucleus medullae oblongatae lateralis, the subnucleus ventralis, and the nucleus paragiganto cellularis lateralis. The "C2" lateral group (C21) was situated in the area of the dorsal nucleus of the vagus and of the nucleus tractus solitarius; the "C2" medial group (C2m) was located in the area of the hypoglossal nucleus and dorsal nucleus of the vagus. The nucleus raphes obscurus (OB) was in the upper 3/4 of the midline, ventral to C2m. The nucleus raphes pallidus (PA) was situated on the ventral extremity of midline, immediately dorsal to the pyramids. In the pons (planes (11) to (14)) the "punches" (see Figs. 2, 3, 4 and 5) were locatedasfollows: in plane (11) the nucleus raphes magnus (MA) was on the midline of tegmentum slightly ventral to its center (plane 11). In plane (12) the nucleus raphes pontis, ventral part (POv), was on the more ventral part of the midline of tegmentum and the nucleus raphes pontis, dorsal part (POd), was immediately dorsal to POv. In plane (13) the locus coeruleus, caudal part (LCc) and in plane (14) the locus coeruleus, rostral part (LCr) were easily identified because of the pigmentation of the nucleus. The nucleus raphes dorsalis, caudal part (DOc) (plane 13) and nucleus raphes dorsalis, rostral part (DOr) (plane 14) were located on the midline immediately adjacent to the ventral limit of the aqueduct. The nucleus raphes centralis, caudal part (CEc) (plane 13) and the nucleus raphes centralis, rostral part (CEr) (plane 14) were located on the midline, immediately ventral to DOc and DOr. In the mesencephalon (planes (15) and (16)) the "punches" (see Figs. 2, 3 and5) were located as follows: in the substantia nigra 6 caudal punches (SNlc to SN6c) were made in plane (15) and 6 rostral punches (SNlr to SN6r) were made in plane (16) (see Figs. 3 and 5). In these planes samples I, 2, 3 and 4 corresponded to the pars compacta and samples 5 and 6 to the pars reticulata. The interpeduncular nucleus (IP) was sampled on the midline in its most ventral part, in plane (15), and the substantia perforata posterior (SPP) was sampled in the same way, in plane (16). The periaqueductal gray, caudal part (PGc) (plane 15) and periaqueductal gray, rostral part (PGr) (plane 16) were taken in the lateral and ventral part of the periaqueductal substance. The structures called mesencephalic raphe (MR) were sampled in 4 locations on the midline. In plane (15) the caudal and ventral part (MRcv) was located in the middle of the line joining the superior limit of the decussation of superior cerebellar peduncles; the rostral and dorsal part (MRrv) was
402
.
Figs. 4 and 5. Several planes of section are used for the dissection of the brain stem. The right part of all the sections is a photograph of the dissected tissue. The left part of the section is a schematic representation (see text) of structures that can be identified macroscopically on a fresh section of the human brain stem. Abbreviations in white characters represent the localization of punches as described i n t h e text (see Methods). Abbreviations in dark characters symbolize the structures used for the localization of sampling (according to Olszewski and Baxter 1954).
403
A R C = nucleus arcuatus; CI = nucleus colliculi inferioris ; CS = nucleus colliculi superioris; D P C S -- decussatio p e d u n c u l o r u m cerebellorum s u p e r i o r u m ; D X = nucleus dorsalis motorius nervi vagi; F L M -- fasciculus longitudinalis medialis; G C M = griseum centrale mesencephali; I P M nucleus interpeduncularis subnucleus medialis; LC -- locus coeruleus; LL = lemniscus lateralis; L M = lemniscus medialis; M G B = corpus geniculatum mediale; O I D = nucleus olivaris inferior accessorius dorsalis; O I P nucleus olivaris inferior principalis; O I M = nucleus olivaris inferior accessorius medialis; PCI = pedunculus cerebelli inferior; PCS - pedunculum cerebelli superior; P G L = nucleus paragigantocellularis lateralis; P N = nucleus paranigralis; RP -- nucleus ruber, subnucleus parvocellularis; S N C = nucleus substantia nigra, subnucleus compactus; S N R = nucleus substantia nigra, subnucleus reticulata; SOL -- nucleus tractus solitarii; SPP = substantia perforata posterior; X I I = nucleus nervi hypoglossi.
404 located on the middle of the line joining the center of red nuclei, pars parvocellularis, the rostral and dorsal part (MRrd) was dorsal to MRrv. In the hypothalamus 4 samples corresponding to the arcuate nucleus and to the area immediately adjacent (called here paramedial hypothalamus) were dissected from plane (18) (Fig. 2). In the cerebral hemisphere the frontal cortex at the level of callosomarginal sulcus, four samples were taken: parts 1 and 2 in the first frontal gyrus, part 3 in the second frontal gyrus and part 4 in the third frontal gyrus. Three "punches" (upper, medium and lower) were dissected in the nucleus accumbens immediately rostrally to putamen (Roberts and Hanaway 1970); those corresponding to amygdaloid complex were taken at the level where the anterior commissure crosses the putamen and external pallidum (see Fig. 2, plane (19) and Fig. 6). Hippocampal samples (see Fig. 2, plane (17)) were taken in Sommer's sector and in the area resistant to hypoxia, outside Sommer's sector, at level of maximal development of the lateral geniculate body (Roberts and Hanaway 1970). All samples were kept in Eppendorf 1.5 ml polyethylene tubes in a liquid nitrogen container until they were assayed. All manipulations were performed with surgical mask and gloves. After each manipulation tools were washed with a hypochlorite solution according to Traub et al. (1976). Histological controls (haemalum-phloxine-safranin, Mallory's PTAH) were performed on formalin-fixed tissues from both sides. No gross neuropathological features were observed other than those commonly seen in elderly patients (corporea amylacea, discrete arteriolar thickening).
Fig. 6. The 25 sites of "punch" sampling in the basal ganglia, cingulum and white matter (see:text). Symbols: A M = amygdaloid complex; C I N = cinsulate cortex; C L = claustrum; C N = e a r . a t e nucleus; G P = globus pallidus; P U = putamen; W M = white matter.
405 B. Chemical Methods
Tissue samples were homogenized in 5 mM Tris-HCL buffer pH 7.4 containing 0.2 ~ (v/v) of Triton X100. The homogenisation volumes were 200/,1 for the brain stem samples, 150 #1 for the telencephalic and diencephalic samples, and 500/A for the pineal samples. The homogenates were centrifuged (9000 x g, 15 rain) at -k 4 °C. P N M T activity was measured on 50 #1 of supernatant according to the radiometric technique of Saavedra et al. (1974) with minor modification (Renaud et al. 1978). The concentrations of reagents in the incubation medium (volume 100 #1) were: Tris-HC1 buffer pH 8.6:0.2 M; phenylethanolamine: 400 # M and methyl[aH]S adenosylmethionine: 3 . 5 / , M (1.5 #Ci). The incubation was carried out for 60 min. The N-[aH]methylphenylethanolamine formed was extracted by toluene containing 3 ~ of isoamylalcohol (v/v); the solvent was evaporated overnight in a ventilated oven without significant loss of product, and the radioactivity was counted by liquid scintillation. Blanks were prepared by omitting phenylethanolamine from the incubation mixture. As the blank value differed from one sample to another (700-3000 cpm), blanks were determined for all the samples analyzed. The soluble proteins were determined on 10/~1 of supernatant with the Folin phenol reagent (Lowry et al. 1951) using bovine serum albumin (Miles) as standard. The P N M T activity was expressed as pmoles of N-methyl phenylethanolamine formed per hour of incubation and per mg of protein. The results are given as a mean :k SEM. The limit of detection of the assay was arbitrarily fixed to twice the blank value. The term "traces" was used when the P N M T activity was between 1.5 and twice the blank value. RESULTS As shown in Table 1, the highest P N M T activity in the medulla oblongata was found in the Cl area. This value was therefore chosen as a reference (100 ~ activity). The area containing the hypoglossal nucleus and the dorsal vagal nucleus (C2m) exhibited higher P N M T activity than C21. The rostro-caudal distribution of P N M T activity in the Cl area is presented in Fig. 7. The peak of activity for both regions was located immediately rostral to the middle of the inferior olive. A similar rostro-caudal distribution was found for the C2m area. In the upper portion of the brain, high P N M T activity was found in the arcuate nucleus of hypothalamus (145 ~ of the activity found in the Cl area). P N M T activity was also present in the paramedial hypothalamus (70 ~ ) in the rostral part of the substantia nigra (50 ~), caudal locus coeruleus (40 ~), nucleus accumbens (30.8 ~ :k 14.4 when upper and medial parts are pooled in each subject), internal globus pallidus (28 ~ ) and periaqueductal gray (17 ~). On the other hand very low or no activity was found in various raphe nuclei (except for raphe pallidus which contained 20 ~o of the activity of the C1 area) and in other cortical and subcortical areas. The activities found in brain areas, on the whole, were low when compared with the activity found in the adrenal medulla (132 i 20 mol/hr/mg protein, i.e. 600 ~ of P N M T activity in Cl).
406 TABLE 1 DISTRIBUTION OF S I G N I F I C A N T PNMT ACTIVITY IN THE H U M A N BRAIN No PNMT activity was found in : nucleus raphes magnus, nucleus raphes pontis (ventral part), nucleus raphes centralis (caudal part), prefrontal cortex (part 3), deep white matter, hippocampal formation (resistant), pineal gland. Traces of PNMT activity were found in : nucleus raphes obscurus, nucleus raphes pontis (dorsal part), nucleus raphes dorsalis (caudal and rostral part), nucleus raphes centralis (ro3tral part), interpeduncular nucleus, mesencephalic raphe (dorsal caudal and rostral, and ventral caudal and rostral), habenula, prefrontal cortex (part 1,2 and 4), amygdala (part 1,2, 3 and 4), g~rus cinguli (part 1 and 2), caudate nucleus (part 1, 2 and 3), putamen, hippocampal formation (fragile), claustrum (part 1 and 2). Structures
PNMT activity (pmole/hr/mg prot.) (mean 5: SEM)
% of activity of C1 area
C1 area C2m area C21 area Nucleus raphes pallidus Locus coeruleus (caudal part) Locus coeruleus (rostral part) Periaqueductal grey (caudal) Periaqueductal grey (rostral) Substantia perforata (posterior) Left paramedial hypothalamus Right paramedial hypothalamus Left arcuate nucleus Right arcuate nucleus Nucleus accumbens (upper part) Nucleus accumbens (medium part) Nucleus accumbens (lower part) Globus pallidus (part 1) Globus pallidus (part 2) Globus pallidus (part 3) Globus pallidus (part 4) Substantia nigra (caudal) lc 2c 3c 4c 5c 6c Substantia nigra (rostral) lr 2r 3r 4r 5r 6r
8.28 i 0.74 5.58 ± 1.09 1.72 ± 0.52 1.82 ± 0.69 3.28 5:0.91 2.00 ± 1.01 1.11 £ 0.78 1.24 5:0.82 1.90 ± 0.83 5.36 ± 2.13 6.57 5:2.52 11.47 5: 1.83 11.60 ± 3.49 3.55 5:2.56 1.29 5:0.56 Traces 1.05 ± 0.66 0.98 ± 0.68 2.19 5:0.96 2.26 5:0.77
100 by definition 67.1 ± 15.0 21.1 5:6.8 20.8 i 7.6 39.7 5:10.9 25.6 5:13.4 16.1 ~ t 1.1 18.0 5:11.7 24.4 ± 10.8 63.6 5:20.1 76.7 +- 22.7 145.3 5:29.3 144.0 5:37.6 46.4 5:31.9 16.7 - 7.4 15.4 14.2 27.1 28.7
± 9.6 ± 9.6 5:12.2 -3- 9.6
1.15 ± 0.75 1.48 ± 0.63 0 1.14 5:0.72 0 0
14.1 5:8.7 19.1 i 7.8 0 13.9 5:8.6 0 0
3.32 3.56 3.46 3.97 3.73 4.28
39.4 42.9 45.1 52.2 48.6 52.1
5:1.19 5:0.42 5:0.91 5:1.04 ± 0.94 5:0.52
5:12.6 5:3.5 ± 12.8 ± 14.9 5:13.2 5:5.6
DISCUSSION
The dissection technique described above was used to study the distribution o f P N M T activity in 117 areas of 5 human normal brains. The results were reproducible,
407 P NMT activity
300
200
caudo_ rostral direction
Fig. 7. Caudo-rostral distribution of P N M T activity in the " C I " area of the medulla oblongata. For each section the activity is expressed as percentage (mean ± SEM) of the specific activity (per mg protein) of the total group.
partly because of the anatomical precision of the dissection and the stability of the enzyme in spite of a post-mortem delay ranging from 2.5 to 16 hours. The rostrocaudal distribution of PNMT activity in the C1 and C2 areas (Fig. 7) was found to be identical in each brain: for example the maximum enzymatic activity was always found at the same level (slice (6) of the medulla oblongata). In addition, adjacent structures on the same slice exhibited wide differences in PNMT activity (see structures C1, OB, C2m for example). In the present work we have taken special precautions to avoid assay nonspecificity (Saavedra et al. 1973). In order to minimize the measurement of non-specific N-methyl-transferase activity we performed an appropriate blank on each supematant (see Methods). The biochemical specificity of the PNMT assay is confirmed by the observation of a different pattern of regional distribution when compared to the distribution of non-specific N-methyl-transferase previously described in the human and rat brains (Saavedra et al, 1973). Our results demonstrate the heterogeneity of the distribution of PNMT, since the absolute activity varied over several orders of magnitude in the various brain areas analyzed. A heterogenous distribution of PNMT activity has already been suggested in human brain (Lew et al. 1977; Nagatsu et al. 1977; Vogel et al. 1976). The determination of this enzymatic activity after a precise and reproducible dissecting procedure allows a comparison with previous studies performed in the rat (Saavedra et al. 1974) under
408 similar conditions. In the brain stem the high activity f o u n d in the C I and C2m g r o u p s m a y c o r r e s p o n d , as in the rat, to the presence o f adrenergic cell bodies ( H 6 k f e l t et al. 1974). A c c o r d i n g to the present a n d earlier results o f Mefford et al. (1977), N a g a t s u et al. (1977) and Lew et al. (1977), difference between the distribution o f P N M T and that o f a d r e n a l i n e exists in h u m a n brain, p a r t i c u l a r l y in the CI area. A similar discrepancy has also been f o u n d for the rat b r a i n ( S a a v e d r a et al. 1974; Van der Gugten et al. 1976) especially in the C1 a r e a where a low a d r e n a l i n e c o n c e n t r a t i o n and high P N M T activity has been described. Significant P N M T activity was also f o u n d in other structures (presenting, in the rat, adrenergic innervation) such as h y p o t h a l a m u s , locus coeruleus a n d p e r i a q u e d u c t a l gray. S o m e o t h e r areas m a y be i n n e r v a t e d b y adrenergic n e u r o n s in m a n b u t n o t in rat, such as s u b s t a n t i a nigra, internal p a l l i d u m , nucleus accumbens. This confirms and completes previous d a t a o f Lew et al. (1977), a n d N a g a t s u et al. (1977). It can be h y p o t h e s i z e d t h a t the presence o f an adrenergic innervation in b u l b a r reticular f o r m a t i o n s , h y p o t h a l a m i c areas a n d b a s a l ganglia c o u l d play i m p o r t a n t functional roles, including b l o o d pressure control, e n d o c r i n e regulation a n d c o n t r o l o f motility. ACKNOWLEDGEMENTS The a u t h o r s are grateful to Mrs. N. G a y for her technical assistance, Dr. J. A t k i n s o n for reviewing the m a n u s c r i p t a n d to Miss A. L a b r o s s e for typewriting.
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