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The effect of brain stem lesions on tyrosine and tryptophan hydroxylases in various structures of the telencephalon of the cat
BRAIN RESEARCH
147
T H E E F F E C T OF BRAIN STEM LESIONS ON TYROSINE A N D T R Y P T O P H A N HYDROXYLASES IN VARIOUS S T R U C T U R E S OF T H E T E L E N C E P H A L O N OF T H E CAT
L. J. POIRIER, E. G. M c G E E R , L. LAROCHELLE*, P. L. M c G E E R , P. BEDARD* AND R. B O U C H E R
Laboratoire de Neuropsychiatrie expdrimentale, Ddpartement de Physiologie, Facultd de M~decine, Universitd Laval, Quebec, and Kinsmen Laboratory of Neurological Research, Department of Psychiatry, Faculty of Medicine, University of British Columbia, Vancouver (Canada) (Accepted December 26th, 1968)
INTRODUCTION
Previous studies in the monkey have shown that ventromedial tegmental lesions are associated with a decreased concentration of dopaminO 6, of its metabolite, homovanillic acid 20 and of tyrosine hydroxylasO in the corresponding striatum. Similar lesions in the cat also result in a decreased concentration oftyrosine hydroxylase in the caudate nucleus and septal area s and of dopamine in the striatum 14. Such chemical changes appear to be a consequence of the interruption ofcatecholinergic nerve fibers I which originate in cells of the substantia nigra 13 and the nucleus parabrachialis pigmentosus 14. A decreased concentration of serotonin (5-HT) was also reported in the whole brain of the rat 5 and in various parts of the diencephalon-telencephalon of the cat brain 10 following lesion of the medial forebrain bundle. Ventromedial tegmental lesions also lead to a decreased concentration of serotonin in the corresponding striatum of the monkey 17 and in the striatum 14 and hypothalamus 11 of the cat. The serotonergic fibers appear to originate along the basomed ial part of the tegmentum 3,15. On the other hand, the destruction of the intralaminar nuclei which give rise to an important group of fibers terminating in the striatumg, is does not modify the concentration of dopamine and serotonin in the striatum 2. In the light of these findings, it appeared of particular interest to compare in the same animals the effects of brain stem lesions on the serotonergic and catecholinergic pathways in various parts of the telencephalon. Tryptophan hydro×ylase and tyrosine hydroxylase are the enzymes for the rate-controlling steps in the two biosynthetic routes, and have been shown to be concentrated in those areas of brain 7,12 rich in the respective nerve endings1, a. These enzymes were therefore assayed as * Fellow of the Medical Research Council of Canada.
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indices of the integrity of the neuronal systems, while succinic dehydrogenase activity was determined on the same homogenates as an index of general cellular metabolism. MATERIALS AND METHODS Eleven adult cats of both sexes were used in this study. Unilateral lesions were placed, with the help of a stereotaxic instrument, in the right ventromedial tegmental area of the b r a i n stem in 6 cats ( G r o u p A) a n d in the left i n t r a l a m i n a r nuclei of the t h a l a m u s in 5 cats ( G r o u p B). Lesions were produced under p e n t o b a r b i t a l anesthesia by electrocoagulation using a high-frequency current. Following a postoperative period of 5 weeks, the animals were sacrificed by nitrogen asphyxiation and pieces of tissue of the left a n d right telencephalon were dissected, weighed a n d homogenized in ice-cold 0.25 M sucrose. Tyrosine and trypt o p h a n hydroxylases were determined on aliquots of each h o m o g e n a t e by previously reported methods 6,1~. Succinic dehydrogenase was determined by a modification of the method of Slater and Bonner 22. Duplicate tubes kept in ice were prepared containing homogenate diluted so that there was 10-15 mg of tissue in 0.5 ml of sucrose. To each was added 2.5 ml of a freshly prepared mixture of 1 part 0.2 M sodium succinate, 1 part 0.01 M KaFe(CN)6, 1.5 parts 0.1 M K C N , 1.5 parts water and 7.5 parts 0.2 M potassium phosphate buffer, pH 7.2. One tube was acidified immediately with 0.5 ml of 1 5 ~ trichloroacetic acid a n d both tubes were incubated at 37°C for 20 min, after which the second tube was similarly acidified. The mixtures were centrifuged and the difference in optical densities of the s u p e r n a t a n t s read at 400 m/~. Experiment showed respect to time up to The brain stem used for histological
that this procedure gave conversions which were linear with 30 rain and linear with respect to tissue in the range of 5-20 mg. including the diencephalon was fixed in 10 o / f o r m a l i n and was study based o n serial sections stained with fast blue and basic
fuchsin.
Fig. 1. Transverse section through rostral midbrain of cat 461 showing a right lesion that partially involved the ventromedial tegmental area. Fast blue and basic fuchsin. × 6. Fig. 2. Transverse section through rostral midbrain of cat 462 showing right lesion that invades the lateral part of the ventromedial tegmental area. Same stain, x 6. Fig. 3. Transverse section through caudal midbrain of same animal (462) showing the substantia nigra (Sn) and nucleus parabrachialis pigmentosus (P,pi) on the intact side. Same stain, x 25. Fig. 4. Transverse section through caudal midbrain of same animal (462) showing the area of the substantia nigra (Sn) and nucleus parabrachialis pigmentosus on the side of the lesion. A few cells persist in the caudomedial part of the substantia nigra. Same stain, x 25. Fig. 5. Transverse section through rostral midbrain of cat 481 showing a right lesion that extensively destroyed the ventromedial tegmental area. Same stain. × 6. Figs. 6 and 7. Transverse sections through caudal midbrain of same cat (481) showing the substantia nigra (Sn) caudally to the lesion on the intact (Fig. 6) and lesioned sides (Fig. 7). Same stain. × 25. Fig. 8. Transverse section through caudal midbrain at the level of the large-celled red nucleus of the same cat (481) showing the basomedial part of the tegmentum. Note the marked cell loss on the right side in comparison to the intact (left) side. Ru -- n.ruber; Li = n.linearis. Same stain. ~ 25. Figs. 9 and 10. Transverse sections through diencephalon of cat 890 showing destruction of the left intralaminar nuclei (il) at two different levels. Same stain. × 4 respectively. Brain Research, 14 (1969) 147-155
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150
L.J. POIRIERet al.
RESULTS
Morphological data
In the 6 cats of Group A (Nos. 461,462, 476, 479, 480 and 481) the unilateral right lesion is located in the ventromedial tegmental area of the upper midbrain and caudal hypothalamus. In some animals it invades the subthalamus as well (Figs. 1, 2, 5). In 3 cats (479, 480, 481) the tegmental lesion extended very close to the midline (Fig. 5), while in the other 3 the paramedial area of the ventromedial tegmentum was partially spared. The sparing was slight in cat 476, but more important in cats 461 and 462 (Figs. 1, 2). Correspondingly, most of the nigral neurons located in the right substantia nigra and nucleus parabrachialis pigmentosus were absent as a result of retrograde degeneration in cats 479, 480 and 481 (Figs. 6 and 7). A few nigral cells were present in the caudomedial part of the right substantia nigra in cat 476, and a few clusters of cells were intact in this area in cats 461 and 462 (Figs. 3 and 4). The cell groups, including the nucleus linearis, located along the basomedial part of the tegmentum between the substantia nigra and the red nucleus on the one hand, and the nucleus interpeduncularis on the other hand, showed a severe loss of cells on the right side in cats 479, 480 and 481 (Fig. 8). In cats 461,462 and 476 the same group of neurons showed a diffuse but incomplete cell loss on the right side. In the 5 cats of group B (Nos. 878, 882, 884, 890 and 975) the unilateral lesion extensively damaged the area of the left intralaminar nuclei (Figs. 9 and 10). The latter nuclei include nuclei centrum medianum, parafascicularis, centralis lateralis and paracentralis. In all the animals of group B the substantia nigra, nucleus parabrachialis pigmentosus and the basomedial cell groups of the upper pons and mid brain did not show any cell degeneration. Chemical data
Data are given in Table I on the activities of three enzymes (tyrosine hydroxylase, tryptophan hydroxylase and succinic dehydrogenase) in areas on the side ipsilateral to the lesion as percentages of the activities in the same areas on the side contralateral to the lesion. Measured activities for tyrosine and tryptophan hydroxylase in m#moles per gram of tissue per hour are given in Table II. The areas considered are the caudate and septal area for all cats as well as the anterior perforated substance for the cats of Group A. This third area was not assayed in cats of Group B. DISCUSSION
The contrast in the chemical data between cats of Group A and those of Group B makes it clear that the reductions in tyrosine and tryptophan hydroxylases on the lesioned side in the cats of Group A are not consequences of the operation per se but rather of the position of the lesion. A similar lack of effect on striatal tyrosine hydroxylase has been previously reported following very large lesions in the caudal midbrain s. The lesions made in cats of Group B evidently do not affect the integrity Brain Research, 14 (1969) 147-155
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of the catecholinergic and serotonergic pathways ending in the caudate nucleusz and septal area. In cats of Group A, on the other hand, the activities of both tyrosine and tryptophan hydroxylases are markedly reduced in the caudate nucleus and anterior perforated substance on the side ipsilateral with the ventromedial tegmental lesion. The right septal area also showed a lowered tyrosine hydroxylase activity in 5 out of the 6 cats in Group A and a marked reduction in tryptophan hydroxylase activity in 3 of the 6. These changes are consistent with previous reports on the reduction of striatal dopamine, serotonin and tyrosine hydroxylase in animals with lesions of this kind4,S,14,1~,17,19. It is clear from the data obtained on succinic dehydrogenase in the same tissues that the reductions in hydroxylase activities are not simply reflections of general cellular degradations. There is good correlation between the morphological and chemical data on cats with ventromedial tegmental lesions. The activities of both tyrosine and tryptophan hydroxylases are significantly lower in the right structures of cats 479, 480 and 481 than in the corresponding structures of cats 461,462 and 476. Correspondingly, cats 479, 480 and 481 showed a greater ipsilateral cell loss caudal to the lesion than did cat 476 which, in turn, showed a greater cell loss than cats 461 and 462. A comparison of the concentration oftyrosine hydroxylase in different structures and of the morphological changes in the corresponding side of the brain following unilateral lesions further suggests a somatotopical distribution in the brain stem of the ascending fibers which relate to the synthesis of dopamine. It appears from the present data that the more medially located dopaminergic neurons in the brain stemT M are related to rostral and paramedial structures by fibers which course closer to the midline than those which terminate in more laterally located telencephalic structures. Previous data s on the diverse effects of various brain stem lesions on tyrosine hydroxylase activity in the caudate and septal area are in accord with the distribution of fibers suggested here. The present data confirm previous indications that the ascending serotonergic fibers course more medially and more ventrally in the ventromedial tegmental area than do those related to the synthesis of dopamine 17. The retrograde cellular degeneration at the level of the basomedial tegmentum of the uppzr pons and lower midbrain associated with decreased tryptophan hydroxylase in more rostral structures of the corresponding side is similar to the changes associated with a decreased striatal serotonin following similar lesions14.15,zl. All of the available data indicate that the synthesis of serotonin and dopamine in striatal structures are dependent upon the integrity of separate neuronal systems which originate in cell groups located, respectively, in the basomedial part of the brain stem (for serotonin) and in the substantia nigra and the nucleus parabrachialis pigmentosus (for dopamine). This contrasts with the synthesis of norepinephrine in the hypothalamus which appears to be independent of any intracerebrally coursing pathways terminating in that area 11.
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L.J. POIRIER et al.
SUMMARY
U n i l a t e r a l lesions o f the v e n t r o m e d i a l tegmental a r e a o f the u p p e r brain stem are associated with decreased c o n c e n t r a t i o n s o f tyrosine a n d t r y p t o p h a n hydroxylases w i t h o u t any reduction o f succinic d e h y d r o g e n a s e in different telencephalic structures on the c o r r e s p o n d i n g side o f the b r a i n in the cat. U n i l a t e r a l lesions o f the i n t r a l a m i n a r nuclei o f the t h a l a m u s have no effect on the concentrations o f any o f these enzymes in the same tissues. These results indicate that the syntheses o f d o p a mine and serotonin in the striatal a n d other tissues are d e p e n d e n t on the integrity o f separate neuronal systems originating in specific cell groups o f the brain stem. RESUME
Effet de l~sions du tronc cdrdbral sur l'activit~ des hydroxylases de la tyrosine et du tryptophane au niveau de certaines ~tructures du tdlenc~phale chez le chat A la suite de 16sions unilatdrales int6ressant la r6gion tegmentaire ventrom6diane de la partie sup6rieure d u t r o n c c6r6bral, l ' o n constate une d i m i n u t i o n consid6rable des hydroxylases de la tyrosine et du t r y p t o p h a n e sans modification de la d6hydrog6nase succinique au niveau de diverses structures d u t61enc6phale de m~me c6t6. P a r contre la destruction des n o y a u x i n t r a l a m i n a i r e s du t h a l a m u s ne modifie pas la c o n c e n t r a t i o n de ces enzymes au niveau des m@mes structures. Ces rdsultats sugg~rent que la synth~se de la d o p a m i n e et de la s6rotonine s'61abore aux d6pens de circuits n e u r o n a u × distincts qui o n t leur origine dans des a m a s cellulaires sp6cifiques d u t r o n c c6r6bral. ACKNOWLEDGEMENT
T h i s i n v e s t i g a t i o n w a s s u p p o r t e d by grants ~ o m the M e d i c a l R~search Council of Canada.
REFERENCES 1 ANDEN, N.-E., CARLSSON,A., DAHLSTR6M,A., FUXE, K., HILLARP, N. A., AND LARSSON, K., Demonstration and mapping out of nigro-neostriatal dopamine neurons, Life Sci., 3 (1964) 523-530. 2 BEDARD,P., LAROCHELLE,L., PARENT,A., AND POIRIER,L. J., Effect of lesions of the intralaminar nuclei on the concentration of dopamine and se'rotonin in the striatum of the cat, Arch. Neurol. (Chic.), 20 (1969) 239-242. 3 DAHLSTROM,A., AND FUXE, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Actaphysiol. scand., 62, Suppl. 232(1964) 1-55. 4 GOLDSrEtN,M., ANAGNOSTE,B., OWEN, W. S., AND BATrmTA,A. F., The effects of ventromedial tegmental lesions on the biosynthesis of catecholamines in the striatum, Life Sci., 5 (1966) 21712176. 5 HELLER,A., HARVEY,J., AND MOORE,R., A demonstration of a fall in brain serotonin following central nervous system lesions in the rat, Biochem. Pharmacol., 11 (1962) 859-866.
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6 MCGEER, E. G., GIBSON, S., AND MCGEER, P. L., Some characteristics of brain tyrosine hydrox= ylase, Canad. J. Biochem., 45 (1967) 1557-1563. 7 MCGEER, E. G., GIBSON, G., WADA, J. A., AND MCGEER, P. L., Distribution of tyrosine hydroxylase activity in adult and developing brain, Canad. J. Biochem., 45 (1967) 1943-1959. 8 McGEER, P. L., WADA, J. A., GIBSON, G., LOESER, C., AND MCGEER, E. G., Tyrosine hydroxylase levels in the cat caudate and basal forebrain following selective midbrain lesions, 2nd Int. Congr. Nearogenetics and Neuroophthalmology, Montreal, Sept. 17-22, 1967, in press. 9 MEHLER, W. R., Center median nucleus of Luys. In D. P. PURPURAAND M. D. YAHR (Eds.), The Thalamus, Columbia Univ. Press, New York, 1966, pp. 109-121. 10 MOORE, R. Y., WONG, S.=L. R., AND HEELER, A., Regional effects of hypothalamic lesions on brain serotonin, Arch. NeuroL (Chic.), 13 (1965) 346-354. 11 PARENT, A., SAINT=JACQUES, C., AND POIRIER, L. J., Effect of interrupting the hypothalamic nervous connections on the norepinephrine and serotonin content of the hypothalamus, Exp. Neurol., 23 (1969) 67-75. 12 PETERS,D. A. V., MCGEER, P. L., AND MCGEER, E. G., The distribution of tryptophan hydroxylase in cat brain, J. Neurochem., 15 (1968) 1431-1437. 13 POIRIER, L. J., Experimental and histological study of midbrain dyskinesias, J. Neurophysiol., 23 (1960) 534-551. 14 POIRIER, L. J., SINGH, P., BOUCHER, R., BOUVIER, G., OLIV1ER, A., AND LAROCHELLE,P., Effect of brain lesions on striatal monoamines in the cat, Arch. Neurol. (Chic.), 17 (1967) 601-608. 15 POIRIER, L. J., SINGH, P., SOURKES,T. L., AND BOUCHER,R., Effect of amine precursors on the concentration of striatal dopamine and serotonin in cats with and without unilateral brain stem lesions, Brain Research, 6 (1967) 654-666. 16 POIRIER,L. J., AND SOURKES,T. L., Influence of the substantia nigra on the catecholamine content of the striatum, Brain, 88 (1965) 181-192. 17 POIRIER, L. J., SOURKES, T. L., BOUVIER, G., BOUCHER, R., AND CARABIN, S., Striatal amines, experimental tremor and the effect of harmaline in the monkey, Brain, 89 (1966) 37-52. 18 POWELE, T. P. S., AND COWAN, W. U., A study of the thalamo-striate relations in the monkey, Brain, 79 (1956) 364-390. 19 SEITELBERGER,F., PETSCHE,n., BERNHEIMER,n., UND HORNYKIEWICZ,O., Verhalten des Dopamins (3-Hydroxytyramin) in Nucleus caudatus nach elektrischer Koagulation des Globus pallidus, Naturwissenschaften, 51 (1964) 314-315. 20 SHARMAN, D. F., POIRIER, L. J., MURPHY, G. F., AND SOURKES, T. L., Homovanillic acid and dihydroxyphenylacetic acid in the striatum of monkeys with brain lesions, Canad. J. PhysioL Pharmacol., 45 (1967) 57-62. 21 SING8, P., POIRIER, L. J., AND BOUCHER, R., Effect of monoamine oxidase inhibitors on the concentrations of dopamine and serotonin in the striatum of the cat with and without unilateral brain stem lesions, Canad. J. Physiol. Pharmacol., 45 (1967) 897-904. 22 SEATER, E. C., AND BONNER, W. D., Effect of fluoride on succinic oxidase system, Biochem. J., 52 (1952) 185-196.
Brain Research, 14 (1969) 147-155
147
T H E E F F E C T OF BRAIN STEM LESIONS ON TYROSINE A N D T R Y P T O P H A N HYDROXYLASES IN VARIOUS S T R U C T U R E S OF T H E T E L E N C E P H A L O N OF T H E CAT
L. J. POIRIER, E. G. M c G E E R , L. LAROCHELLE*, P. L. M c G E E R , P. BEDARD* AND R. B O U C H E R
Laboratoire de Neuropsychiatrie expdrimentale, Ddpartement de Physiologie, Facultd de M~decine, Universitd Laval, Quebec, and Kinsmen Laboratory of Neurological Research, Department of Psychiatry, Faculty of Medicine, University of British Columbia, Vancouver (Canada) (Accepted December 26th, 1968)
INTRODUCTION
Previous studies in the monkey have shown that ventromedial tegmental lesions are associated with a decreased concentration of dopaminO 6, of its metabolite, homovanillic acid 20 and of tyrosine hydroxylasO in the corresponding striatum. Similar lesions in the cat also result in a decreased concentration oftyrosine hydroxylase in the caudate nucleus and septal area s and of dopamine in the striatum 14. Such chemical changes appear to be a consequence of the interruption ofcatecholinergic nerve fibers I which originate in cells of the substantia nigra 13 and the nucleus parabrachialis pigmentosus 14. A decreased concentration of serotonin (5-HT) was also reported in the whole brain of the rat 5 and in various parts of the diencephalon-telencephalon of the cat brain 10 following lesion of the medial forebrain bundle. Ventromedial tegmental lesions also lead to a decreased concentration of serotonin in the corresponding striatum of the monkey 17 and in the striatum 14 and hypothalamus 11 of the cat. The serotonergic fibers appear to originate along the basomed ial part of the tegmentum 3,15. On the other hand, the destruction of the intralaminar nuclei which give rise to an important group of fibers terminating in the striatumg, is does not modify the concentration of dopamine and serotonin in the striatum 2. In the light of these findings, it appeared of particular interest to compare in the same animals the effects of brain stem lesions on the serotonergic and catecholinergic pathways in various parts of the telencephalon. Tryptophan hydro×ylase and tyrosine hydroxylase are the enzymes for the rate-controlling steps in the two biosynthetic routes, and have been shown to be concentrated in those areas of brain 7,12 rich in the respective nerve endings1, a. These enzymes were therefore assayed as * Fellow of the Medical Research Council of Canada.
Brain Research, 14 (1969) 147-155
148
L.J. POIRIER dl al.
indices of the integrity of the neuronal systems, while succinic dehydrogenase activity was determined on the same homogenates as an index of general cellular metabolism. MATERIALS AND METHODS Eleven adult cats of both sexes were used in this study. Unilateral lesions were placed, with the help of a stereotaxic instrument, in the right ventromedial tegmental area of the b r a i n stem in 6 cats ( G r o u p A) a n d in the left i n t r a l a m i n a r nuclei of the t h a l a m u s in 5 cats ( G r o u p B). Lesions were produced under p e n t o b a r b i t a l anesthesia by electrocoagulation using a high-frequency current. Following a postoperative period of 5 weeks, the animals were sacrificed by nitrogen asphyxiation and pieces of tissue of the left a n d right telencephalon were dissected, weighed a n d homogenized in ice-cold 0.25 M sucrose. Tyrosine and trypt o p h a n hydroxylases were determined on aliquots of each h o m o g e n a t e by previously reported methods 6,1~. Succinic dehydrogenase was determined by a modification of the method of Slater and Bonner 22. Duplicate tubes kept in ice were prepared containing homogenate diluted so that there was 10-15 mg of tissue in 0.5 ml of sucrose. To each was added 2.5 ml of a freshly prepared mixture of 1 part 0.2 M sodium succinate, 1 part 0.01 M KaFe(CN)6, 1.5 parts 0.1 M K C N , 1.5 parts water and 7.5 parts 0.2 M potassium phosphate buffer, pH 7.2. One tube was acidified immediately with 0.5 ml of 1 5 ~ trichloroacetic acid a n d both tubes were incubated at 37°C for 20 min, after which the second tube was similarly acidified. The mixtures were centrifuged and the difference in optical densities of the s u p e r n a t a n t s read at 400 m/~. Experiment showed respect to time up to The brain stem used for histological
that this procedure gave conversions which were linear with 30 rain and linear with respect to tissue in the range of 5-20 mg. including the diencephalon was fixed in 10 o / f o r m a l i n and was study based o n serial sections stained with fast blue and basic
fuchsin.
Fig. 1. Transverse section through rostral midbrain of cat 461 showing a right lesion that partially involved the ventromedial tegmental area. Fast blue and basic fuchsin. × 6. Fig. 2. Transverse section through rostral midbrain of cat 462 showing right lesion that invades the lateral part of the ventromedial tegmental area. Same stain, x 6. Fig. 3. Transverse section through caudal midbrain of same animal (462) showing the substantia nigra (Sn) and nucleus parabrachialis pigmentosus (P,pi) on the intact side. Same stain, x 25. Fig. 4. Transverse section through caudal midbrain of same animal (462) showing the area of the substantia nigra (Sn) and nucleus parabrachialis pigmentosus on the side of the lesion. A few cells persist in the caudomedial part of the substantia nigra. Same stain, x 25. Fig. 5. Transverse section through rostral midbrain of cat 481 showing a right lesion that extensively destroyed the ventromedial tegmental area. Same stain. × 6. Figs. 6 and 7. Transverse sections through caudal midbrain of same cat (481) showing the substantia nigra (Sn) caudally to the lesion on the intact (Fig. 6) and lesioned sides (Fig. 7). Same stain. × 25. Fig. 8. Transverse section through caudal midbrain at the level of the large-celled red nucleus of the same cat (481) showing the basomedial part of the tegmentum. Note the marked cell loss on the right side in comparison to the intact (left) side. Ru -- n.ruber; Li = n.linearis. Same stain. ~ 25. Figs. 9 and 10. Transverse sections through diencephalon of cat 890 showing destruction of the left intralaminar nuclei (il) at two different levels. Same stain. × 4 respectively. Brain Research, 14 (1969) 147-155
TYROSINE AND TRYPTOPHAN HYDROXYLASES
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150
L.J. POIRIERet al.
RESULTS
Morphological data
In the 6 cats of Group A (Nos. 461,462, 476, 479, 480 and 481) the unilateral right lesion is located in the ventromedial tegmental area of the upper midbrain and caudal hypothalamus. In some animals it invades the subthalamus as well (Figs. 1, 2, 5). In 3 cats (479, 480, 481) the tegmental lesion extended very close to the midline (Fig. 5), while in the other 3 the paramedial area of the ventromedial tegmentum was partially spared. The sparing was slight in cat 476, but more important in cats 461 and 462 (Figs. 1, 2). Correspondingly, most of the nigral neurons located in the right substantia nigra and nucleus parabrachialis pigmentosus were absent as a result of retrograde degeneration in cats 479, 480 and 481 (Figs. 6 and 7). A few nigral cells were present in the caudomedial part of the right substantia nigra in cat 476, and a few clusters of cells were intact in this area in cats 461 and 462 (Figs. 3 and 4). The cell groups, including the nucleus linearis, located along the basomedial part of the tegmentum between the substantia nigra and the red nucleus on the one hand, and the nucleus interpeduncularis on the other hand, showed a severe loss of cells on the right side in cats 479, 480 and 481 (Fig. 8). In cats 461,462 and 476 the same group of neurons showed a diffuse but incomplete cell loss on the right side. In the 5 cats of group B (Nos. 878, 882, 884, 890 and 975) the unilateral lesion extensively damaged the area of the left intralaminar nuclei (Figs. 9 and 10). The latter nuclei include nuclei centrum medianum, parafascicularis, centralis lateralis and paracentralis. In all the animals of group B the substantia nigra, nucleus parabrachialis pigmentosus and the basomedial cell groups of the upper pons and mid brain did not show any cell degeneration. Chemical data
Data are given in Table I on the activities of three enzymes (tyrosine hydroxylase, tryptophan hydroxylase and succinic dehydrogenase) in areas on the side ipsilateral to the lesion as percentages of the activities in the same areas on the side contralateral to the lesion. Measured activities for tyrosine and tryptophan hydroxylase in m#moles per gram of tissue per hour are given in Table II. The areas considered are the caudate and septal area for all cats as well as the anterior perforated substance for the cats of Group A. This third area was not assayed in cats of Group B. DISCUSSION
The contrast in the chemical data between cats of Group A and those of Group B makes it clear that the reductions in tyrosine and tryptophan hydroxylases on the lesioned side in the cats of Group A are not consequences of the operation per se but rather of the position of the lesion. A similar lack of effect on striatal tyrosine hydroxylase has been previously reported following very large lesions in the caudal midbrain s. The lesions made in cats of Group B evidently do not affect the integrity Brain Research, 14 (1969) 147-155
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TYROSINE AND TRYPTOPHAN HYDROXYLASES
153
of the catecholinergic and serotonergic pathways ending in the caudate nucleusz and septal area. In cats of Group A, on the other hand, the activities of both tyrosine and tryptophan hydroxylases are markedly reduced in the caudate nucleus and anterior perforated substance on the side ipsilateral with the ventromedial tegmental lesion. The right septal area also showed a lowered tyrosine hydroxylase activity in 5 out of the 6 cats in Group A and a marked reduction in tryptophan hydroxylase activity in 3 of the 6. These changes are consistent with previous reports on the reduction of striatal dopamine, serotonin and tyrosine hydroxylase in animals with lesions of this kind4,S,14,1~,17,19. It is clear from the data obtained on succinic dehydrogenase in the same tissues that the reductions in hydroxylase activities are not simply reflections of general cellular degradations. There is good correlation between the morphological and chemical data on cats with ventromedial tegmental lesions. The activities of both tyrosine and tryptophan hydroxylases are significantly lower in the right structures of cats 479, 480 and 481 than in the corresponding structures of cats 461,462 and 476. Correspondingly, cats 479, 480 and 481 showed a greater ipsilateral cell loss caudal to the lesion than did cat 476 which, in turn, showed a greater cell loss than cats 461 and 462. A comparison of the concentration oftyrosine hydroxylase in different structures and of the morphological changes in the corresponding side of the brain following unilateral lesions further suggests a somatotopical distribution in the brain stem of the ascending fibers which relate to the synthesis of dopamine. It appears from the present data that the more medially located dopaminergic neurons in the brain stemT M are related to rostral and paramedial structures by fibers which course closer to the midline than those which terminate in more laterally located telencephalic structures. Previous data s on the diverse effects of various brain stem lesions on tyrosine hydroxylase activity in the caudate and septal area are in accord with the distribution of fibers suggested here. The present data confirm previous indications that the ascending serotonergic fibers course more medially and more ventrally in the ventromedial tegmental area than do those related to the synthesis of dopamine 17. The retrograde cellular degeneration at the level of the basomedial tegmentum of the uppzr pons and lower midbrain associated with decreased tryptophan hydroxylase in more rostral structures of the corresponding side is similar to the changes associated with a decreased striatal serotonin following similar lesions14.15,zl. All of the available data indicate that the synthesis of serotonin and dopamine in striatal structures are dependent upon the integrity of separate neuronal systems which originate in cell groups located, respectively, in the basomedial part of the brain stem (for serotonin) and in the substantia nigra and the nucleus parabrachialis pigmentosus (for dopamine). This contrasts with the synthesis of norepinephrine in the hypothalamus which appears to be independent of any intracerebrally coursing pathways terminating in that area 11.
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SUMMARY
U n i l a t e r a l lesions o f the v e n t r o m e d i a l tegmental a r e a o f the u p p e r brain stem are associated with decreased c o n c e n t r a t i o n s o f tyrosine a n d t r y p t o p h a n hydroxylases w i t h o u t any reduction o f succinic d e h y d r o g e n a s e in different telencephalic structures on the c o r r e s p o n d i n g side o f the b r a i n in the cat. U n i l a t e r a l lesions o f the i n t r a l a m i n a r nuclei o f the t h a l a m u s have no effect on the concentrations o f any o f these enzymes in the same tissues. These results indicate that the syntheses o f d o p a mine and serotonin in the striatal a n d other tissues are d e p e n d e n t on the integrity o f separate neuronal systems originating in specific cell groups o f the brain stem. RESUME
Effet de l~sions du tronc cdrdbral sur l'activit~ des hydroxylases de la tyrosine et du tryptophane au niveau de certaines ~tructures du tdlenc~phale chez le chat A la suite de 16sions unilatdrales int6ressant la r6gion tegmentaire ventrom6diane de la partie sup6rieure d u t r o n c c6r6bral, l ' o n constate une d i m i n u t i o n consid6rable des hydroxylases de la tyrosine et du t r y p t o p h a n e sans modification de la d6hydrog6nase succinique au niveau de diverses structures d u t61enc6phale de m~me c6t6. P a r contre la destruction des n o y a u x i n t r a l a m i n a i r e s du t h a l a m u s ne modifie pas la c o n c e n t r a t i o n de ces enzymes au niveau des m@mes structures. Ces rdsultats sugg~rent que la synth~se de la d o p a m i n e et de la s6rotonine s'61abore aux d6pens de circuits n e u r o n a u × distincts qui o n t leur origine dans des a m a s cellulaires sp6cifiques d u t r o n c c6r6bral. ACKNOWLEDGEMENT
T h i s i n v e s t i g a t i o n w a s s u p p o r t e d by grants ~ o m the M e d i c a l R~search Council of Canada.
REFERENCES 1 ANDEN, N.-E., CARLSSON,A., DAHLSTR6M,A., FUXE, K., HILLARP, N. A., AND LARSSON, K., Demonstration and mapping out of nigro-neostriatal dopamine neurons, Life Sci., 3 (1964) 523-530. 2 BEDARD,P., LAROCHELLE,L., PARENT,A., AND POIRIER,L. J., Effect of lesions of the intralaminar nuclei on the concentration of dopamine and se'rotonin in the striatum of the cat, Arch. Neurol. (Chic.), 20 (1969) 239-242. 3 DAHLSTROM,A., AND FUXE, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Actaphysiol. scand., 62, Suppl. 232(1964) 1-55. 4 GOLDSrEtN,M., ANAGNOSTE,B., OWEN, W. S., AND BATrmTA,A. F., The effects of ventromedial tegmental lesions on the biosynthesis of catecholamines in the striatum, Life Sci., 5 (1966) 21712176. 5 HELLER,A., HARVEY,J., AND MOORE,R., A demonstration of a fall in brain serotonin following central nervous system lesions in the rat, Biochem. Pharmacol., 11 (1962) 859-866.
Brain Research, 14 (1969) 147-155
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155
6 MCGEER, E. G., GIBSON, S., AND MCGEER, P. L., Some characteristics of brain tyrosine hydrox= ylase, Canad. J. Biochem., 45 (1967) 1557-1563. 7 MCGEER, E. G., GIBSON, G., WADA, J. A., AND MCGEER, P. L., Distribution of tyrosine hydroxylase activity in adult and developing brain, Canad. J. Biochem., 45 (1967) 1943-1959. 8 McGEER, P. L., WADA, J. A., GIBSON, G., LOESER, C., AND MCGEER, E. G., Tyrosine hydroxylase levels in the cat caudate and basal forebrain following selective midbrain lesions, 2nd Int. Congr. Nearogenetics and Neuroophthalmology, Montreal, Sept. 17-22, 1967, in press. 9 MEHLER, W. R., Center median nucleus of Luys. In D. P. PURPURAAND M. D. YAHR (Eds.), The Thalamus, Columbia Univ. Press, New York, 1966, pp. 109-121. 10 MOORE, R. Y., WONG, S.=L. R., AND HEELER, A., Regional effects of hypothalamic lesions on brain serotonin, Arch. NeuroL (Chic.), 13 (1965) 346-354. 11 PARENT, A., SAINT=JACQUES, C., AND POIRIER, L. J., Effect of interrupting the hypothalamic nervous connections on the norepinephrine and serotonin content of the hypothalamus, Exp. Neurol., 23 (1969) 67-75. 12 PETERS,D. A. V., MCGEER, P. L., AND MCGEER, E. G., The distribution of tryptophan hydroxylase in cat brain, J. Neurochem., 15 (1968) 1431-1437. 13 POIRIER, L. J., Experimental and histological study of midbrain dyskinesias, J. Neurophysiol., 23 (1960) 534-551. 14 POIRIER, L. J., SINGH, P., BOUCHER, R., BOUVIER, G., OLIV1ER, A., AND LAROCHELLE,P., Effect of brain lesions on striatal monoamines in the cat, Arch. Neurol. (Chic.), 17 (1967) 601-608. 15 POIRIER, L. J., SINGH, P., SOURKES,T. L., AND BOUCHER,R., Effect of amine precursors on the concentration of striatal dopamine and serotonin in cats with and without unilateral brain stem lesions, Brain Research, 6 (1967) 654-666. 16 POIRIER,L. J., AND SOURKES,T. L., Influence of the substantia nigra on the catecholamine content of the striatum, Brain, 88 (1965) 181-192. 17 POIRIER, L. J., SOURKES, T. L., BOUVIER, G., BOUCHER, R., AND CARABIN, S., Striatal amines, experimental tremor and the effect of harmaline in the monkey, Brain, 89 (1966) 37-52. 18 POWELE, T. P. S., AND COWAN, W. U., A study of the thalamo-striate relations in the monkey, Brain, 79 (1956) 364-390. 19 SEITELBERGER,F., PETSCHE,n., BERNHEIMER,n., UND HORNYKIEWICZ,O., Verhalten des Dopamins (3-Hydroxytyramin) in Nucleus caudatus nach elektrischer Koagulation des Globus pallidus, Naturwissenschaften, 51 (1964) 314-315. 20 SHARMAN, D. F., POIRIER, L. J., MURPHY, G. F., AND SOURKES, T. L., Homovanillic acid and dihydroxyphenylacetic acid in the striatum of monkeys with brain lesions, Canad. J. PhysioL Pharmacol., 45 (1967) 57-62. 21 SING8, P., POIRIER, L. J., AND BOUCHER, R., Effect of monoamine oxidase inhibitors on the concentrations of dopamine and serotonin in the striatum of the cat with and without unilateral brain stem lesions, Canad. J. Physiol. Pharmacol., 45 (1967) 897-904. 22 SEATER, E. C., AND BONNER, W. D., Effect of fluoride on succinic oxidase system, Biochem. J., 52 (1952) 185-196.
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