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Tryptophan hydroxylase activity and synaptosomal uptake of serotonin in discrete brain regions after midbrain raphe lesions: Correlations with serotonin levels and histochemical fluorescence
165
BRAIN RESEARCH
TRYPTOPHAN HYDROXYLASE ACTIVITY AND SYNAPTOSOMAL U P T A K E OF S E R O T O N I N IN D I S C R E T E BRAIN R E G I O N S A F T E R M I D B R A I N R A P H E LESIONS: C O R R E L A T I O N S W I T H S E R O T O N I N LEVELS A N D H I S T O C H E M I C A L F L U O R E S C E N C E
MICHAEL J. KUHAR, GEORGE K. AGHAJANIAN AND ROBERT H. ROTH Departments of Psychiatry and Pharmacology, Yale University School qf Medicine, and Connecticut Mental Health Center, New Haven, Conn. 06519 (U.S.A.)
(Accepted March 15th, 1972)
INTRODUCTION
The bulk of the serotonin (5-HT) in the rat forebrain appears to be localized to the projections of neurons whose cell bodies are situated in the dorsal and median midbrain raphe nuclei (RN) 6,'. We have recently reported an extensive reduction of forebrain tryptophan hydroxylase, the enzyme thought to be rate-limiting in the biosynthesis of 5-HT from tryptophan 1~, after placement of lesions in the midbrain RN 14, while tyrosine hydroxylase activity in the same animals was unchanged. We have also reported a reduction of uptake of 5-HT into synaptosomes (isolated, intact nerve endings) from whole forebrains of similarly lesioned animals while the uptake of norepinepbrine, dopamine and tryptophan was not reduced 13. These results suggest that the enzyme tryptophan hydroxylase and the uptake mechanism for 5-HT are uniquely, if not predominantly, localized to 5-HT-containing neurons. In this investigation, we have examined regional changes in tryptophan hydroxylase activity and synaptosomal uptake activity before and after placement of midbrain raphe lesions. We have also attempted to correlate these regional changes with 5-HT revels and with the presence of 5-HT-containing axons and nerve terminals as visualized by histochemical fluorescence methods. The utilization of histochemical fluorescence methods has permitted the visualization of various monoamine containing neurons in the central nervous system 6. In contrast to the fluorescence of catecholamine-containing neurons, the fluorescence of serotonin-containing neurons, especially of axons and nerve terminals, has been very weak or even absent in brain regions shown to contain 5-HT by biochemical methods 6,7. It has been reported that pretreatment of animals with L-tryptophan results in selective enhancement of the histochemical fluorescence intensity of raphe neurons in the midbrain 1. We report here a clear visualization of axons and nerve terminals in various regions of the forebrain after a similar pretreatment with a monoamine oxidase inhibitor, pargyline, and L-tryptophan. Braht Research, 44 (1972) 165-176
166
M. 3. KUHAR el al.
M I [H(11)5
Dissection of brain regions [or biochemical studies Charles River male rats, 200-300 g, were decapitated, their brains rapidly removed and placed on a dental wax plate on ice. After removal of the cerebellum, the forebrain was dissected from the brain stem by a section passing between the anterior border of the superior colliculi and the posterior border of the hypothalamus. To obtain cortex, hippocampus and diencephalon (hypothalamus-thalamus), the forebrain was divided by a coronal section at the level of the optic chiasm, and the hippocampus and cortex (predominantly dorsomedial areas) were removed from the larger, posterior portion of the forebrain tbr assay. The remainder of the posterior portion of the forebrain was dissected free of" any remaining striatal and cortical tissue and termed the hypothalamus-thalamus. Both striata (including caudate nucleus, putamen nucleus and most of the globus pallidus nucleus) were removed from whole forebrains after dissecting away the cortex and other forebrain tissue. The weights of the tissues obtained in this way were (mean ~ S.E.M., n): cortex (one side) 290 ~ 16 mg, 9" hippocampus (both sides) 146 j 5 rag, 5; hypothalamus-thalamus 198 12 rag, 5" striatum (both sides) 104 ! 4 mg, 5; and cerebellum, 264 + 8 rag, 5.
Preparation o[ animals with midbrain raphe lesions Animals were lightly anesthetized with chloral hydrate and cathodal lesions were placed in the midbrain RN as previously described 14. The extent of the lesion in each animal was determined by fixing the brain stems of lesioned animals in a 5 ?i~ glutaraldehyde-0.9 ?/,, saline solution, and examining the subsequently obtained serial sections stained with cresyl violet. The extent of these lesions has been previously described 14. Only the data from animals with total midbrain raphe lesions were included in this study. Animals were sacrificed no sooner than 14 days post-lesion, at which time the reduction of serotonin levels, tryptophan hydroxylase activity, and synaptosomal uptake activity in whole forebrain was maximal 13,14.
Serotonin determination Serotonin levels were measured in various brain regions by the utilization of ninhydrin reagent is. Frozen tissue was homogenized in 2.5 ml of 0.1 N HCI, and 0.7 ml of 1 0 ~ ZnSO4 and 0.35 ml of 1 N N a O H were added to the homogenate to precipitate the protein. After centrifugation, the supernatant fluids were brought to pH 10 by the addition of 0.2 ml of a saturated solution of Na2COz and 0.5 ml of 0.5 M borate buffer (pH 10) saturated with NaC1, and shaken thoroughly with 8 ml of n-butanol (Mallinckrodt analytical grade, washed in the following order with equal volumes of 0.1 N NaOH, 0.1 N HC1 and distilled water 3 × ) for 5 min. Seven ml of the butanol phase were again shaken thoroughly with 2.5 ml of 0.1 M borate buffer (pH 10, NaCl-saturated) for 2 rain. Five ml of the butanol phase were then added Brain Research, 44 (1972) 165-176
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to a tube containing 2.5 ml of 0.05 M sodium phosphate buffer (pH 6.5) and 7.5 ml of n-heptane (Mallinckrodt, analytical grade). After shaking for 2 rain, 2 ml of the aqueous phase were added to a tube containing 0.2 ml of 0.1 M ninhydrin, and the mixture was placed in a 75 '~C water bath for 30 rain. The solution, brought to room temperature, was transferred to a quartz cuvette and the fluorescence measured at 490 nm after activation at 385 nm (uncorrected). Blank values were obtained by carrying 0.1 N HCI without tissue through the procedure. The fluorescence intensity was proportional to serotonin concentration over the range 10 ng/ml (twice blank)250 ng/ml. All values were corrected for an extraction efficiency of 80 ~',;].
Tr)y~tol~han hydroxylase activity Tryptophan hydroxylase activity was determined by the CO,, trapping method of Ichyama et al2 with some small modifications as previously described 14.
Synaptosomal uptake activity Synaptosomal uptake activity, a relative measure of the number of intact nerve terminals in a given region, was determined by the method of Coyle and Snyder a as previously described 13. [aH]Serotonin (500mCi/mmole, Amersham-Searle, Des Plaines, Ill., U.S.A.) concentrations in the incubation media were 2.5 ~< 10 s M. Synaptosomal uptake activity in the cerebellum was not measured in nuclei-free homogenates as were the other regions, but rather in whole homogenates. The bulk of the cerebellar uptake activity was found in the nuclear pellet, indicating that 5-HT nerve endings in the cerebellum sediment along with the large mossy fiber endings into the nuclear fractions 10.
Histochemical fluorescence methods These methods utilized modifications3, (~ of the formaldehyde condensation procedure for demonstrating monoamines in freeze-dried tissue. Animals were pretreated with pargyline (100 mg/kg, i.p.) at 4 h and L-tryptophan (100 mg/kg, i.p.) at I h prior to decapitation. The remainder of the procedure has been previously described t. Serotonin fluorescence was distinguishable from catecholamine fluorescence because of its characteristic yellow color and rapid photodecomposition4, 6. RESULTS
Regiona/ re~hwtion o[' 5-HT levels, tryptophan hydroxylase activity and synaptosoma/ uptake activity The cortex, hippocampus, hypothalamus-thalamus, striatum and cerebellum were dissected from brains of control animals and animals with midbrain raphe lesions. Tryptophan hydroxylase activity, 5-HT levels and synaptosomal uptake activ-
Braht Research, 44 (1972) 165-176
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I--'1 CONTROLS ErR RAPHE
0.6
LESIONS
0.4
CORTEX HIPPOCAMPUS HYPOSTRIATUM CEREBELLUM THALAMUS +THALAMUS
Fig. 1. Regional reduction of serotonin levels, tryptophan hydroxylase activity and synaptosomal uptake of serotonin in various brain regions after total, selective destruction of the midbrain RN. Experimental details are supplied in Methods. Results are mean :~:: 1 S.D. Number of animals for both controls and raphe lesions are inside bars. All differences were significant to P --: 0.001 level (one-tailed t-test) except for those of cerebellum which were P < 0.01 and caudate (tryptophan hydroxylase activity) which was P / 0.005.
ity were measured in each of the regions as described in Methods (Fig. 1). The hypothalamus-thalamus, which had the highest control levels of 5-HT and tryptophan hydroxylase activity and a high level of synaptosomal uptake activity, exhibited a 70-90 ~ loss of 5-HT, enzyme activity and synaptosomal uptake activity after placement of lesions. In cortical and hippocampal regions in rats with raphe lesions, there was a 80-90 ~ reduction of 5-HT levels, tryptophan hydroxylase activity and synaptosomal uptake activity. The striatum, a region that exhibited a high level of synaptosomal uptake activity and intermediate levels of 5-HT and enzyme, showed a 4565 ~ reduction of all quantities measured. O f all the regions examined the cerebellum had the lowest control levels of the above measures and exhibited about a 50 ~o reduction of these quantities in lesioned animals. Histochemieal fluorescence studies
After animals were pretreated with pargyline and L-tryptophan and their brain tissue prepared for fluorescence microscopy as described in Methods, it was possible to observe in the midbrain R N intensely fluorescent cells of a characteristic yellow color presumed to contain 5-HT and possibly other indoleaminesl,4,6. Under these Brain Research, 44 (1972) 165-176
7
Fig. 4. Loss of serotonin-containing axons and terminals in the cingulum (C) after midbrain raphe lesions. Lower frame shows tissue from animal with lesion. CC, corpus callosum. Bar, 50/n~.
Fig. 5. Loss of s e t o t o n i n - c o n l a m h l g nc~'xc terminals in tile globus pallidus (GP) after midbrain ruph~." lesions. Lower frame shows tissue from animal with lesions. The d o p a m i n e fluorescence in the ctmdale nucleus {C) is u n c h a n g e d and is hrighi e n o u g h to mask any serolonin fluorescence in H~,a~ region. Bar. 50 ,-m,
MIDBRAIN RAPHE NEURONS
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conditions, it was also possible to observe in certain areas of the brain many fine nerve terminals and axons possessing the same characteristic yellow color. When brain tissue from animals with lesions of the midbrain RN was compared to that of controls, we made several observations regarding these neurons: (1) In the midbrain, the lesions destroyed the fluorescent cells of the midbrain RN. A few fluorescent cells at the periphery of the midbrain RN were spared as was the lateral group of cells near the medial lemniscus (i.e., B9 of Dahlstr6m and Fuxe% (2) The yellow fluorescent fibers from the midbrain RN which enter the medial forebrain bundle in the area of the fasciculus retroflexus were greatly diminished after placement of midbrain RN lesions (Fig. 2), as were the yellow fluorescent terminals in the substantia nigra zona reticulata, pretectal nucleus and superior colliculus. (3) In the hypothalamus, most of the yellow fluorescent nerve terminals in the suprachiasmatic nucleus (Fig. 3) and other areas (e.g. preoptic area and mammillary body) disappear after placement of lesions. Yellow terminals in parts of the thalamus such as habenular, ventral lateral geniculate and reuniens nuclei were greatly diminished. Adjacent to the suprachiasmatic nucleus (Fig. 3), as in other regions, there was no noticeable change in the green fluorescence of nerve terminals and axons presumed to be of the catecholamine type. (4) In the hippocampus and septum, yellow fluorescent fibers and nerve terminals were greatly reduced. In controls, hippocampal fibers are situated in both dorsal and ventral portions of the subiculum and the terminals are mainly in the ventral area. (5) In the cerebral cortex, fluorescent fibers from the anterior portion of the medial forebrain bundle were seen to enter the cingulum via the genu of the corpus callosum. These were greatly reduced after placement of lesions (Fig. 4), as were the yellow fluorescent terminals in the molecular layer. (6) The cortical and basolateral nuclei of the amygdala receive a dense input of yellow fluorescent terminals. These were greatly diminished after placement of lesions. In the caudate nucleus and putamen, it was impossible to distinguish yellow fluorescent axons and terminals because of the intense catecholamine fluorescence. However, yellow fluorescent terminals in the globus pallidus were readily visualized and these disappeared after destruction of the midbrain RN (Fig. 5). (7) The cerebellum had so few fluorescent axons and terminals of this type that it was difficult to judge if there was any change after placement of lesions. DISCUSSION
We have previously found that destruction of the midbrain raphe nuclei results in selective reduction of tryptophan hydroxylase activity, synaptosomal uptake o f 5-HT, and 5-HT levels in whole forebrain~3, la. In this investigation we have examined the regional reduction of tryptophan hydroxylase activity, synaptosomal uptake activity and 5-HT levels after placement of lesions in the midbrain RN. We have also attempted to correlate these reductions with the disappearance of fine, yellow fluorescent axons and nerve terminals in various brain regions after placement of lesions.
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M.J.
l
Fluorescent neurons of this type contain 5-HT and possibly other indoleaminesl,4," The biochemical studies re~.ealed an excellent correlation between 5-HT levels and the levels of enzyme activit? and synaptosomal uptake activity in the various brain regions. After placement of midbrain RN lesions, each of the above levels were reduced similarly in a given region. These results are in agreement with our earlier conclusion that the enzyme tryptophan hydroxylase and the uptake mechanism for 5-HT are predominantly, if not uniquely, associated with 5-HT-containing neuronsla, ~~. The regional levels of 5-HT reported here are roughly similar to those reported by other workers 1~,17,19. A similar correlation between tryptophan hydroxylase activity and 5-HT levels in various regions of the cat brain has been reportedl¢L Tryptophan hydroxylase is believed to be the rate limiting enzyme in the biosynthesis of 5-HT from tryptophan v' and one would therefore expect to find this enzyme localized to the same neuronal compartments that contain 5-HT. The evidence presented here is in accordance with this expectation. The finding that the 5-HT uptake mechanism is localized to the raphe neurons is of interest in terms of the suggestion that the termination of the activity of certain neurotransmitter compounds in the brain may occur by reuptake into presynaptic nerve terminals 11. The uptake of 5-HT observed here in vitro could possibly perform the function of neurotransmitter inactivation in vivo at raphe neuronal terminals. The 5-HT remaining in various regions after electrolytic destruction of the midbrain RN may derive from some few 5-HT neurons situated near the periphery of the RN not destroyed by the lesion, or from other groups of 5-HT-containing cells G. For example, the B9 group of cells, which has a ventrolateral location, was not destroyed by these lesions and appears to project to the forebrain. In general, since the lesions primarily affected the RN, the variations in reduction of 5-HT, enzyme activity and synaptosomal uptake activity in the different regions (e.g. greatest reduction in cortex and hippocampus and least reduction in the cerebellum) probably reflects the degree of innervation of these regions by the cells of the RN as opposed to innervation by other 5-HT-containing cells. Bj6rklund et al. 4 have reported that some neurons in or near the raphe nucleus may contain an indoleamine other than 5-HT. Such neurons would have been included in our lesions, but our biochemical and histochemical data do not bear on the question of the existence of these non-5-HT neurons. By histochemical fluorescence methods, it has been somewhat difficult to see 5-HT-containing axons and terminals in the forebrain without pharmacological or surgical pretreatment 2,<7. It has been reported that pretreatment of animals with L-tryptophan, the amino acid precursor of 5-HT, greatly enhances the fluorescence intensity of the perikarya of 5-HT-containing neurons in the midbrain 1. We report here a clear visualization of projections of these neurons to the hypothalamus, cortex and other areas after pretreatment of animals with L-tryptophan and pargyline. These fluorescent projections are greatly diminished throughout the forebrain after placement of lesions in the midbrain RN, in parallel agreement with the biochemical data. This reduction of fluorescent axons and nerve terminals appeared to be selective in that there was no loss of fluorescence of neurons of the catecholamine type. This is Brain Research, 44 (1972) 165--176
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in agreement with the earlier biochemical findings that there was no reduction of tyrosine hydroxylase activity and no loss of catecholamine uptake into synaptosomes in lesioned animals 13,14. Utilizing animals pretreated with only monoamine oxidase inhibitors, And6n et al. 2 have reported the visualization of 5-HT fibers from the midbrain RN ascending partly through the medial forebrain bundle and passing in a dorsal direction in front of the septal area and into the neocortex. They also observed 5-HT terminals in the globus pallidus, the septal area, the amygdaloid complex, the hypothalamus and the gyrus cinguli. There were marked decreases in the number of 5-HT terminals in these areas after placement of lesions in the medial forebrain bundle. We have not been able to achieve satisfactory results in our laboratory by pretreating animals with only monoamine oxidase inhibitors, but we did find nerve terminals and fibers in those regions as described by And6n et al. '2. We also found, after pretreating animals with c-tryptophan and a monoamine oxidase inhibitor, a clear visualization of 5-HTcontaining nerve terminals in many other regions of the forebrain, including the preoptic areas, habenula and hippocampus. It has been suggested that 5-HT fibers in the neocortex may emanate from interneurons situated above the level of the midbrain s. However, in agreement with Dahlstr6m and Fuxe 6 and And6n et al. 2, no fluorescent interneurons of this type were observed in the forebrain. SUMMARY
We have found an excellent correlation between serotonin (5-HT) levels and the levels of tryptophan hydroxylase activity and synaptosomal uptake of 5-HT in discrete brain regions in the rat. After electrolytic destruction of the midbrain RN, each of the above levels was reduced similarly in a given brain region. These results indicate that the enzyme tryptophan hydroxylase and the uptake mechanism for 5-HT are predominantly, if not uniquely, associated with terminals of the raphe system. We also report a clear visualization of axons and terminals of the raphe neurons by histochemical fluorescence methods in animals pretreated with both L-tryptophan and a monoamine oxidase inhibitor. In agreement with the biochemical data, yellow fluorescent axons and nerve terminals were greatly diminished throughout the forebrain after placement of lesions in the midbrain RN. ACKNOWLEDGEMENTS
This research was supported in part by grants from the National Institutes of Health (MH07114, MH17871, MH14092). G.K.A. is a recipient of a Public Health Service Research Scientist Development Award (MH14459).
REFERENCES I AGHAJANIAN, G. K., AND ASHER, I. M., Histochemical fluorescence of raphe neurons: selective enhancement by tryptophan, Science, 172 (1971) 1159-1162.
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2 ANDI~N, N. E., DAHLSTROM, A., FOXE, K., LARSSON, K., OLSON, L., AND UN~IH~SrLD'r, U. Y.. Ascending monoamine neurons to the telencephalon and dieneephalon, Acta. ph),si,d..wamL, 67 (1966) 313-326. 3 BJORKLUND,A., AND FALCK, B., An improvement of the histochemical fluorescence method for monoamines, J. Histochem. Cytochem., 16 (1968) 717 720. 4 BJORKLUND, A., FALCK, B., AND STENEVI, U., Classification of monoamine neurons in the rat mesencephalon: distribution of a new monoamine neuron system, Brain Research, 32 (1971) 269 286. 5 COVLE, J. T., AND SNVDER, S. H., Catecbolamine uptake by synaptosomes in homogenates of rat brain: stereospecificity in different areas, J. Pharmacol. exp. Ther., 170 (1969) 221-23t. 6 DAHLSTROM, A., AND FUXE, K., Evidence for the existence of monoamine containing neurons in the central nervous system. I. Demonstration of monamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, Suppl. 232 (1965) 1-55. 7 FUXE, K., AND JONSSON, G., A modification of the histochemical fluorescence method for the improved localization of 5-hydroxytryptamine, Histochemie, 11 (1967) 161-166. 8 HEELER, A., AND MOORE, R. Y., Control of brain serotonin and norepinephrine by specific neural systems. In S. GARRATINI AND P. A. SHORE (Eds.), Adv. in Pharmacology, VoL 6A, Academic Press, New York, 1968, pp. 191-206. 9 ICHYAMA, A., NAKAMURA, S., NISHIYUKA, Y., AND HAYASHI, O., Enzymic studies on the biosynthesis of serotonin in mammalian brain, J. biol. Chem., 245 (1970) 1699-1709. 10 ISRAEL,M., AND WHITTAKER~V. P., The isolation of mossy fibre endings from the granular layer of the cerebellar cortex, Experientia (Basel), XXI (1965) 325-326. 11 IVERSEN, L. L., Neuronal uptake processes for amines and amino acids. In E. COSTA AND E. GIACOBINI (Eds.), Biochemistry ~[ Simple Neuronal Models, Raven Press, New York, 1970, pp. 109 132. 12 JEQUIER, E., LOVENBERG,W., AND SJOERDSMA, A., Trytophan hydroxylase inhibition: the mechanism by which p-chlorophenylalanine depletes rat brain serotonin, Molee. Pharmacol., 3 (1967) 274 278. 13 KUHAR, M. J., ROTH, R. H., AND AGHAJANIAN, G. K., Synaptosomes from forebrains of rats with midbrain raphe lesions: selective reduction of serotonin uptake, J. Pharmacol. e.vp. Ther., 181 (1972) 36-45. 14 KUHAR, M. J., ROTH, R. H., AND AGHAJAN~AN,G. K., Selective reduction of tryptophan hydroxylase activity in rat forebrain after midbrain raphe lesions, Brain Research, 35 (1971) 167-176. 15 MAJ, J., GRABOWSKA, M., AND KWIEK, J., The effect of disulfiram, diethyldithiocarbamate and dimethyldithiocarbamate on serotonin and 5-hydroxyindole-3-acetic brain levels in rats, Bioehem. Pharmacol., 19 (1970) 2517-2518. 16 PETERS, D. A. V., MCGEER, P. L., AND MCGEER, E. G., The distribution of tryptophan hydroxylase in cat brain, J. Neurochem., 15 (1968) 1431-1435. 17 SHELLENBERGER,M. K., AND GOROON, J. H., A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine, and 5-hydroxytryptamine from discrete brain areas, Analyt. Biochem., 39 (1971) 356-372. 18 SNYDER, S. H., AXELROD, J., AND ZWEIG, M., A sensitive and specific fluorescence assay for tissue serotonin, Bioehem. Pharmacol., 14 (1965) 831-835. 19 VALZELH, L., AND GARRATINI, S., Biogenic amines in discrete brain areas after treatment with monoamineoxidase inhibitoL J. Neurochem., 15 (1968) 259-261.
Brain Research, 44 (1972) 165-176
BRAIN RESEARCH
TRYPTOPHAN HYDROXYLASE ACTIVITY AND SYNAPTOSOMAL U P T A K E OF S E R O T O N I N IN D I S C R E T E BRAIN R E G I O N S A F T E R M I D B R A I N R A P H E LESIONS: C O R R E L A T I O N S W I T H S E R O T O N I N LEVELS A N D H I S T O C H E M I C A L F L U O R E S C E N C E
MICHAEL J. KUHAR, GEORGE K. AGHAJANIAN AND ROBERT H. ROTH Departments of Psychiatry and Pharmacology, Yale University School qf Medicine, and Connecticut Mental Health Center, New Haven, Conn. 06519 (U.S.A.)
(Accepted March 15th, 1972)
INTRODUCTION
The bulk of the serotonin (5-HT) in the rat forebrain appears to be localized to the projections of neurons whose cell bodies are situated in the dorsal and median midbrain raphe nuclei (RN) 6,'. We have recently reported an extensive reduction of forebrain tryptophan hydroxylase, the enzyme thought to be rate-limiting in the biosynthesis of 5-HT from tryptophan 1~, after placement of lesions in the midbrain RN 14, while tyrosine hydroxylase activity in the same animals was unchanged. We have also reported a reduction of uptake of 5-HT into synaptosomes (isolated, intact nerve endings) from whole forebrains of similarly lesioned animals while the uptake of norepinepbrine, dopamine and tryptophan was not reduced 13. These results suggest that the enzyme tryptophan hydroxylase and the uptake mechanism for 5-HT are uniquely, if not predominantly, localized to 5-HT-containing neurons. In this investigation, we have examined regional changes in tryptophan hydroxylase activity and synaptosomal uptake activity before and after placement of midbrain raphe lesions. We have also attempted to correlate these regional changes with 5-HT revels and with the presence of 5-HT-containing axons and nerve terminals as visualized by histochemical fluorescence methods. The utilization of histochemical fluorescence methods has permitted the visualization of various monoamine containing neurons in the central nervous system 6. In contrast to the fluorescence of catecholamine-containing neurons, the fluorescence of serotonin-containing neurons, especially of axons and nerve terminals, has been very weak or even absent in brain regions shown to contain 5-HT by biochemical methods 6,7. It has been reported that pretreatment of animals with L-tryptophan results in selective enhancement of the histochemical fluorescence intensity of raphe neurons in the midbrain 1. We report here a clear visualization of axons and nerve terminals in various regions of the forebrain after a similar pretreatment with a monoamine oxidase inhibitor, pargyline, and L-tryptophan. Braht Research, 44 (1972) 165-176
166
M. 3. KUHAR el al.
M I [H(11)5
Dissection of brain regions [or biochemical studies Charles River male rats, 200-300 g, were decapitated, their brains rapidly removed and placed on a dental wax plate on ice. After removal of the cerebellum, the forebrain was dissected from the brain stem by a section passing between the anterior border of the superior colliculi and the posterior border of the hypothalamus. To obtain cortex, hippocampus and diencephalon (hypothalamus-thalamus), the forebrain was divided by a coronal section at the level of the optic chiasm, and the hippocampus and cortex (predominantly dorsomedial areas) were removed from the larger, posterior portion of the forebrain tbr assay. The remainder of the posterior portion of the forebrain was dissected free of" any remaining striatal and cortical tissue and termed the hypothalamus-thalamus. Both striata (including caudate nucleus, putamen nucleus and most of the globus pallidus nucleus) were removed from whole forebrains after dissecting away the cortex and other forebrain tissue. The weights of the tissues obtained in this way were (mean ~ S.E.M., n): cortex (one side) 290 ~ 16 mg, 9" hippocampus (both sides) 146 j 5 rag, 5; hypothalamus-thalamus 198 12 rag, 5" striatum (both sides) 104 ! 4 mg, 5; and cerebellum, 264 + 8 rag, 5.
Preparation o[ animals with midbrain raphe lesions Animals were lightly anesthetized with chloral hydrate and cathodal lesions were placed in the midbrain RN as previously described 14. The extent of the lesion in each animal was determined by fixing the brain stems of lesioned animals in a 5 ?i~ glutaraldehyde-0.9 ?/,, saline solution, and examining the subsequently obtained serial sections stained with cresyl violet. The extent of these lesions has been previously described 14. Only the data from animals with total midbrain raphe lesions were included in this study. Animals were sacrificed no sooner than 14 days post-lesion, at which time the reduction of serotonin levels, tryptophan hydroxylase activity, and synaptosomal uptake activity in whole forebrain was maximal 13,14.
Serotonin determination Serotonin levels were measured in various brain regions by the utilization of ninhydrin reagent is. Frozen tissue was homogenized in 2.5 ml of 0.1 N HCI, and 0.7 ml of 1 0 ~ ZnSO4 and 0.35 ml of 1 N N a O H were added to the homogenate to precipitate the protein. After centrifugation, the supernatant fluids were brought to pH 10 by the addition of 0.2 ml of a saturated solution of Na2COz and 0.5 ml of 0.5 M borate buffer (pH 10) saturated with NaC1, and shaken thoroughly with 8 ml of n-butanol (Mallinckrodt analytical grade, washed in the following order with equal volumes of 0.1 N NaOH, 0.1 N HC1 and distilled water 3 × ) for 5 min. Seven ml of the butanol phase were again shaken thoroughly with 2.5 ml of 0.1 M borate buffer (pH 10, NaCl-saturated) for 2 rain. Five ml of the butanol phase were then added Brain Research, 44 (1972) 165-176
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to a tube containing 2.5 ml of 0.05 M sodium phosphate buffer (pH 6.5) and 7.5 ml of n-heptane (Mallinckrodt, analytical grade). After shaking for 2 rain, 2 ml of the aqueous phase were added to a tube containing 0.2 ml of 0.1 M ninhydrin, and the mixture was placed in a 75 '~C water bath for 30 rain. The solution, brought to room temperature, was transferred to a quartz cuvette and the fluorescence measured at 490 nm after activation at 385 nm (uncorrected). Blank values were obtained by carrying 0.1 N HCI without tissue through the procedure. The fluorescence intensity was proportional to serotonin concentration over the range 10 ng/ml (twice blank)250 ng/ml. All values were corrected for an extraction efficiency of 80 ~',;].
Tr)y~tol~han hydroxylase activity Tryptophan hydroxylase activity was determined by the CO,, trapping method of Ichyama et al2 with some small modifications as previously described 14.
Synaptosomal uptake activity Synaptosomal uptake activity, a relative measure of the number of intact nerve terminals in a given region, was determined by the method of Coyle and Snyder a as previously described 13. [aH]Serotonin (500mCi/mmole, Amersham-Searle, Des Plaines, Ill., U.S.A.) concentrations in the incubation media were 2.5 ~< 10 s M. Synaptosomal uptake activity in the cerebellum was not measured in nuclei-free homogenates as were the other regions, but rather in whole homogenates. The bulk of the cerebellar uptake activity was found in the nuclear pellet, indicating that 5-HT nerve endings in the cerebellum sediment along with the large mossy fiber endings into the nuclear fractions 10.
Histochemical fluorescence methods These methods utilized modifications3, (~ of the formaldehyde condensation procedure for demonstrating monoamines in freeze-dried tissue. Animals were pretreated with pargyline (100 mg/kg, i.p.) at 4 h and L-tryptophan (100 mg/kg, i.p.) at I h prior to decapitation. The remainder of the procedure has been previously described t. Serotonin fluorescence was distinguishable from catecholamine fluorescence because of its characteristic yellow color and rapid photodecomposition4, 6. RESULTS
Regiona/ re~hwtion o[' 5-HT levels, tryptophan hydroxylase activity and synaptosoma/ uptake activity The cortex, hippocampus, hypothalamus-thalamus, striatum and cerebellum were dissected from brains of control animals and animals with midbrain raphe lesions. Tryptophan hydroxylase activity, 5-HT levels and synaptosomal uptake activ-
Braht Research, 44 (1972) 165-176
168
xl. i
SEROTONIN LEVELS
~L. I t A R ~ l '~1/
I--'1 CONTROLS ErR RAPHE
0.6
LESIONS
0.4
CORTEX HIPPOCAMPUS HYPOSTRIATUM CEREBELLUM THALAMUS +THALAMUS
Fig. 1. Regional reduction of serotonin levels, tryptophan hydroxylase activity and synaptosomal uptake of serotonin in various brain regions after total, selective destruction of the midbrain RN. Experimental details are supplied in Methods. Results are mean :~:: 1 S.D. Number of animals for both controls and raphe lesions are inside bars. All differences were significant to P --: 0.001 level (one-tailed t-test) except for those of cerebellum which were P < 0.01 and caudate (tryptophan hydroxylase activity) which was P / 0.005.
ity were measured in each of the regions as described in Methods (Fig. 1). The hypothalamus-thalamus, which had the highest control levels of 5-HT and tryptophan hydroxylase activity and a high level of synaptosomal uptake activity, exhibited a 70-90 ~ loss of 5-HT, enzyme activity and synaptosomal uptake activity after placement of lesions. In cortical and hippocampal regions in rats with raphe lesions, there was a 80-90 ~ reduction of 5-HT levels, tryptophan hydroxylase activity and synaptosomal uptake activity. The striatum, a region that exhibited a high level of synaptosomal uptake activity and intermediate levels of 5-HT and enzyme, showed a 4565 ~ reduction of all quantities measured. O f all the regions examined the cerebellum had the lowest control levels of the above measures and exhibited about a 50 ~o reduction of these quantities in lesioned animals. Histochemieal fluorescence studies
After animals were pretreated with pargyline and L-tryptophan and their brain tissue prepared for fluorescence microscopy as described in Methods, it was possible to observe in the midbrain R N intensely fluorescent cells of a characteristic yellow color presumed to contain 5-HT and possibly other indoleaminesl,4,6. Under these Brain Research, 44 (1972) 165-176
7
Fig. 4. Loss of serotonin-containing axons and terminals in the cingulum (C) after midbrain raphe lesions. Lower frame shows tissue from animal with lesion. CC, corpus callosum. Bar, 50/n~.
Fig. 5. Loss of s e t o t o n i n - c o n l a m h l g nc~'xc terminals in tile globus pallidus (GP) after midbrain ruph~." lesions. Lower frame shows tissue from animal with lesions. The d o p a m i n e fluorescence in the ctmdale nucleus {C) is u n c h a n g e d and is hrighi e n o u g h to mask any serolonin fluorescence in H~,a~ region. Bar. 50 ,-m,
MIDBRAIN RAPHE NEURONS
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conditions, it was also possible to observe in certain areas of the brain many fine nerve terminals and axons possessing the same characteristic yellow color. When brain tissue from animals with lesions of the midbrain RN was compared to that of controls, we made several observations regarding these neurons: (1) In the midbrain, the lesions destroyed the fluorescent cells of the midbrain RN. A few fluorescent cells at the periphery of the midbrain RN were spared as was the lateral group of cells near the medial lemniscus (i.e., B9 of Dahlstr6m and Fuxe% (2) The yellow fluorescent fibers from the midbrain RN which enter the medial forebrain bundle in the area of the fasciculus retroflexus were greatly diminished after placement of midbrain RN lesions (Fig. 2), as were the yellow fluorescent terminals in the substantia nigra zona reticulata, pretectal nucleus and superior colliculus. (3) In the hypothalamus, most of the yellow fluorescent nerve terminals in the suprachiasmatic nucleus (Fig. 3) and other areas (e.g. preoptic area and mammillary body) disappear after placement of lesions. Yellow terminals in parts of the thalamus such as habenular, ventral lateral geniculate and reuniens nuclei were greatly diminished. Adjacent to the suprachiasmatic nucleus (Fig. 3), as in other regions, there was no noticeable change in the green fluorescence of nerve terminals and axons presumed to be of the catecholamine type. (4) In the hippocampus and septum, yellow fluorescent fibers and nerve terminals were greatly reduced. In controls, hippocampal fibers are situated in both dorsal and ventral portions of the subiculum and the terminals are mainly in the ventral area. (5) In the cerebral cortex, fluorescent fibers from the anterior portion of the medial forebrain bundle were seen to enter the cingulum via the genu of the corpus callosum. These were greatly reduced after placement of lesions (Fig. 4), as were the yellow fluorescent terminals in the molecular layer. (6) The cortical and basolateral nuclei of the amygdala receive a dense input of yellow fluorescent terminals. These were greatly diminished after placement of lesions. In the caudate nucleus and putamen, it was impossible to distinguish yellow fluorescent axons and terminals because of the intense catecholamine fluorescence. However, yellow fluorescent terminals in the globus pallidus were readily visualized and these disappeared after destruction of the midbrain RN (Fig. 5). (7) The cerebellum had so few fluorescent axons and terminals of this type that it was difficult to judge if there was any change after placement of lesions. DISCUSSION
We have previously found that destruction of the midbrain raphe nuclei results in selective reduction of tryptophan hydroxylase activity, synaptosomal uptake o f 5-HT, and 5-HT levels in whole forebrain~3, la. In this investigation we have examined the regional reduction of tryptophan hydroxylase activity, synaptosomal uptake activity and 5-HT levels after placement of lesions in the midbrain RN. We have also attempted to correlate these reductions with the disappearance of fine, yellow fluorescent axons and nerve terminals in various brain regions after placement of lesions.
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M.J.
l
Fluorescent neurons of this type contain 5-HT and possibly other indoleaminesl,4," The biochemical studies re~.ealed an excellent correlation between 5-HT levels and the levels of enzyme activit? and synaptosomal uptake activity in the various brain regions. After placement of midbrain RN lesions, each of the above levels were reduced similarly in a given region. These results are in agreement with our earlier conclusion that the enzyme tryptophan hydroxylase and the uptake mechanism for 5-HT are predominantly, if not uniquely, associated with 5-HT-containing neuronsla, ~~. The regional levels of 5-HT reported here are roughly similar to those reported by other workers 1~,17,19. A similar correlation between tryptophan hydroxylase activity and 5-HT levels in various regions of the cat brain has been reportedl¢L Tryptophan hydroxylase is believed to be the rate limiting enzyme in the biosynthesis of 5-HT from tryptophan v' and one would therefore expect to find this enzyme localized to the same neuronal compartments that contain 5-HT. The evidence presented here is in accordance with this expectation. The finding that the 5-HT uptake mechanism is localized to the raphe neurons is of interest in terms of the suggestion that the termination of the activity of certain neurotransmitter compounds in the brain may occur by reuptake into presynaptic nerve terminals 11. The uptake of 5-HT observed here in vitro could possibly perform the function of neurotransmitter inactivation in vivo at raphe neuronal terminals. The 5-HT remaining in various regions after electrolytic destruction of the midbrain RN may derive from some few 5-HT neurons situated near the periphery of the RN not destroyed by the lesion, or from other groups of 5-HT-containing cells G. For example, the B9 group of cells, which has a ventrolateral location, was not destroyed by these lesions and appears to project to the forebrain. In general, since the lesions primarily affected the RN, the variations in reduction of 5-HT, enzyme activity and synaptosomal uptake activity in the different regions (e.g. greatest reduction in cortex and hippocampus and least reduction in the cerebellum) probably reflects the degree of innervation of these regions by the cells of the RN as opposed to innervation by other 5-HT-containing cells. Bj6rklund et al. 4 have reported that some neurons in or near the raphe nucleus may contain an indoleamine other than 5-HT. Such neurons would have been included in our lesions, but our biochemical and histochemical data do not bear on the question of the existence of these non-5-HT neurons. By histochemical fluorescence methods, it has been somewhat difficult to see 5-HT-containing axons and terminals in the forebrain without pharmacological or surgical pretreatment 2,<7. It has been reported that pretreatment of animals with L-tryptophan, the amino acid precursor of 5-HT, greatly enhances the fluorescence intensity of the perikarya of 5-HT-containing neurons in the midbrain 1. We report here a clear visualization of projections of these neurons to the hypothalamus, cortex and other areas after pretreatment of animals with L-tryptophan and pargyline. These fluorescent projections are greatly diminished throughout the forebrain after placement of lesions in the midbrain RN, in parallel agreement with the biochemical data. This reduction of fluorescent axons and nerve terminals appeared to be selective in that there was no loss of fluorescence of neurons of the catecholamine type. This is Brain Research, 44 (1972) 165--176
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in agreement with the earlier biochemical findings that there was no reduction of tyrosine hydroxylase activity and no loss of catecholamine uptake into synaptosomes in lesioned animals 13,14. Utilizing animals pretreated with only monoamine oxidase inhibitors, And6n et al. 2 have reported the visualization of 5-HT fibers from the midbrain RN ascending partly through the medial forebrain bundle and passing in a dorsal direction in front of the septal area and into the neocortex. They also observed 5-HT terminals in the globus pallidus, the septal area, the amygdaloid complex, the hypothalamus and the gyrus cinguli. There were marked decreases in the number of 5-HT terminals in these areas after placement of lesions in the medial forebrain bundle. We have not been able to achieve satisfactory results in our laboratory by pretreating animals with only monoamine oxidase inhibitors, but we did find nerve terminals and fibers in those regions as described by And6n et al. '2. We also found, after pretreating animals with c-tryptophan and a monoamine oxidase inhibitor, a clear visualization of 5-HTcontaining nerve terminals in many other regions of the forebrain, including the preoptic areas, habenula and hippocampus. It has been suggested that 5-HT fibers in the neocortex may emanate from interneurons situated above the level of the midbrain s. However, in agreement with Dahlstr6m and Fuxe 6 and And6n et al. 2, no fluorescent interneurons of this type were observed in the forebrain. SUMMARY
We have found an excellent correlation between serotonin (5-HT) levels and the levels of tryptophan hydroxylase activity and synaptosomal uptake of 5-HT in discrete brain regions in the rat. After electrolytic destruction of the midbrain RN, each of the above levels was reduced similarly in a given brain region. These results indicate that the enzyme tryptophan hydroxylase and the uptake mechanism for 5-HT are predominantly, if not uniquely, associated with terminals of the raphe system. We also report a clear visualization of axons and terminals of the raphe neurons by histochemical fluorescence methods in animals pretreated with both L-tryptophan and a monoamine oxidase inhibitor. In agreement with the biochemical data, yellow fluorescent axons and nerve terminals were greatly diminished throughout the forebrain after placement of lesions in the midbrain RN. ACKNOWLEDGEMENTS
This research was supported in part by grants from the National Institutes of Health (MH07114, MH17871, MH14092). G.K.A. is a recipient of a Public Health Service Research Scientist Development Award (MH14459).
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2 ANDI~N, N. E., DAHLSTROM, A., FOXE, K., LARSSON, K., OLSON, L., AND UN~IH~SrLD'r, U. Y.. Ascending monoamine neurons to the telencephalon and dieneephalon, Acta. ph),si,d..wamL, 67 (1966) 313-326. 3 BJORKLUND,A., AND FALCK, B., An improvement of the histochemical fluorescence method for monoamines, J. Histochem. Cytochem., 16 (1968) 717 720. 4 BJORKLUND, A., FALCK, B., AND STENEVI, U., Classification of monoamine neurons in the rat mesencephalon: distribution of a new monoamine neuron system, Brain Research, 32 (1971) 269 286. 5 COVLE, J. T., AND SNVDER, S. H., Catecbolamine uptake by synaptosomes in homogenates of rat brain: stereospecificity in different areas, J. Pharmacol. exp. Ther., 170 (1969) 221-23t. 6 DAHLSTROM, A., AND FUXE, K., Evidence for the existence of monoamine containing neurons in the central nervous system. I. Demonstration of monamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, Suppl. 232 (1965) 1-55. 7 FUXE, K., AND JONSSON, G., A modification of the histochemical fluorescence method for the improved localization of 5-hydroxytryptamine, Histochemie, 11 (1967) 161-166. 8 HEELER, A., AND MOORE, R. Y., Control of brain serotonin and norepinephrine by specific neural systems. In S. GARRATINI AND P. A. SHORE (Eds.), Adv. in Pharmacology, VoL 6A, Academic Press, New York, 1968, pp. 191-206. 9 ICHYAMA, A., NAKAMURA, S., NISHIYUKA, Y., AND HAYASHI, O., Enzymic studies on the biosynthesis of serotonin in mammalian brain, J. biol. Chem., 245 (1970) 1699-1709. 10 ISRAEL,M., AND WHITTAKER~V. P., The isolation of mossy fibre endings from the granular layer of the cerebellar cortex, Experientia (Basel), XXI (1965) 325-326. 11 IVERSEN, L. L., Neuronal uptake processes for amines and amino acids. In E. COSTA AND E. GIACOBINI (Eds.), Biochemistry ~[ Simple Neuronal Models, Raven Press, New York, 1970, pp. 109 132. 12 JEQUIER, E., LOVENBERG,W., AND SJOERDSMA, A., Trytophan hydroxylase inhibition: the mechanism by which p-chlorophenylalanine depletes rat brain serotonin, Molee. Pharmacol., 3 (1967) 274 278. 13 KUHAR, M. J., ROTH, R. H., AND AGHAJANIAN, G. K., Synaptosomes from forebrains of rats with midbrain raphe lesions: selective reduction of serotonin uptake, J. Pharmacol. e.vp. Ther., 181 (1972) 36-45. 14 KUHAR, M. J., ROTH, R. H., AND AGHAJAN~AN,G. K., Selective reduction of tryptophan hydroxylase activity in rat forebrain after midbrain raphe lesions, Brain Research, 35 (1971) 167-176. 15 MAJ, J., GRABOWSKA, M., AND KWIEK, J., The effect of disulfiram, diethyldithiocarbamate and dimethyldithiocarbamate on serotonin and 5-hydroxyindole-3-acetic brain levels in rats, Bioehem. Pharmacol., 19 (1970) 2517-2518. 16 PETERS, D. A. V., MCGEER, P. L., AND MCGEER, E. G., The distribution of tryptophan hydroxylase in cat brain, J. Neurochem., 15 (1968) 1431-1435. 17 SHELLENBERGER,M. K., AND GOROON, J. H., A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine, and 5-hydroxytryptamine from discrete brain areas, Analyt. Biochem., 39 (1971) 356-372. 18 SNYDER, S. H., AXELROD, J., AND ZWEIG, M., A sensitive and specific fluorescence assay for tissue serotonin, Bioehem. Pharmacol., 14 (1965) 831-835. 19 VALZELH, L., AND GARRATINI, S., Biogenic amines in discrete brain areas after treatment with monoamineoxidase inhibitoL J. Neurochem., 15 (1968) 259-261.
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