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Immunohistochemical investigations of the influence of reserpine on the serotonin neuron system in the rat brain
Neuroscience Research, 3 (1985) 145-153
145
Elsevier Scientific Publishers Ireland Ltd. NSR 00099
Immunohistochemical Investigations of the Influence of Reserpine on the Serotonin Neuron System in the Rat Brain Norihiko Ihara, Katsuji Nishida, Shuichi Ueda, Mitsuhiro Kawata and Yutaka Sano Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji,Kamikyo-ku, Kyoto 602 (Japan) (Received June 24th, 1985; Revised version received and accepted August 8th, 1985)
Key words: serotonin - - rat - - reserpine - - immunohistochemistry - - PAP
SUMMARY A peroxidase-antiperoxidase immunohistochemical method with serotonin antiserum was employed to investigate the influence of reserpine on serotonin neurons of rats which were sacrificed at various times after inject!on (10 mg/kg i.p.). The disappearance of serotonin immunoreactivity induced by reserpine was detected only in the perikarya after 15 min, and then rapidly proceeded to the terminals. Between 2 and 4 h, immunoreactivity completely disappeared throughout the brain. The immunoreactivity reappeared in the perikarya after 6 h, and progressed toward the terminals gradually. However, there was an obvious difference in the rate of recovery of immunoreactivity between areas. After 7 days, the immunoreactivity returned to control levels.
INTRODUCTION
One of the most pronounced pharmacological effects of reserpine is depletion of biogenic amines in the brain 1"15.Several histochemical studies have examined the effects of reserpine on monoaminergic neurons using the Falck-Hillarp fluorescence method z' 4,5,8. Although this method has proven to be valuable for visualizing catecholaminecontaining structures, it has several disadvantages, for example, low intensity and rapid fluorophore photodecomposition for detection of serotonin neurons. Thus, until recently, there has been little information on the influences of reserpine on the morphology of serotonin neurons. Correspondence: Y. Sano, Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan. 0168-0102/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
146 We have previously used peroxidase-antiperoxidase (PAP) immunohistochemistry with a highly specific serotonin antiserum to demonstrate the distribution, fiber connections and axonal ramifications of serotonin neurons in the central nervous system of various vertebrates in detail 1°'12'17"18'2°'21. These systematic studies clearly showed that an astonishingly large number of processes of serotonin neurons are widely distributed throughout the central nervous system, from the bulbus olfactorius to the caudal end of the spinal cord, with frequent branching and the formation of dense networks. This report documents changes in the distribution of serotonin immunoreactivity in the rat brain at different times after reserpine administration. MATERIALS AND METHODS
Thirty male Wistar rats, weighing 200-300 g, were divided into ten groups, which were sacrificed at 0.25, 0.5, 2, 4, 6, 8, 24, 48, 72 and 168 h after intraperitoneal administration of reserpine (10 mg/kg Apoplon Inj.-Daiichi, Tokyo, Japan). Under Nembutal anesthesia, the rats were perfused via the left cardiac ventricle with heparinized physiological saline, followed by perfusion with a fixative consisting of 4 ~o paraformaldehyde and 0.2 % picric acid in 0.1 M phosphate buffer (pH 7.4) at 4 ° C. The brains were removed from the skulls and further fixed in the perfusion medium for 48 h at 4 ° C. After immersion in 0.1 M phosphate buffer containing 20 ~o sucrose at 4 ° C, the brain blocks were frozen and cut into 20 #m thick sections in the frontal plane with a cryostat. The sections were stored in 0.1 M phosphate-buffered saline containing 0.3% Triton X-100 at 4 °C. The detailed PAP immunohistochemical procedure and the preparation and specificity of anti-serotonin serum used in this study have been reported elsewhere2°. Briefly, the sections were successively incubated in: (1)rabbit serotonin antiserum (1 : 10,000) for 48 h at 4 °C; (2) goat anti-rabbit IgG (Miles-Yeda Ltd., U.S.A., 1 : 200) for 3 h; (3) peroxidase-antiperoxidase complex (Dako, Denmark, 1 : 200) for 1 h and (4) 3,3'-diaminobenzidine (Sigma, U.S.A.) containing H202 for 10 min. Except for step (1), each step was carried out at room temperature. The sections were mounted on glass slides coated with chrome-alum gelatin, treated with 0.05 % OsO4, dehydrated and coverslipped. RESULTS
Within 15 rain after the intraperitoneal injection of reserpine, the immunoreactivity of the perikarya of serotonin neurons, located mainly in the raphe regions of the brainstem, was markedly reduced. However, serotonin neurons in the dorsal area of the nucleus raphe dorsalis, particularly its median and lateral parts, still displayed relatively strong perikaryal immunoreactivity (Fig. 1). The immunoreactivity of processes of serotonin neurons seemed to be unaffected by the reserpine administration at this stage.
147
Fig. i. Nucleus raphe dorsalis. A: control. B: 15 min after intraperitoneal injection of reserpine (10 mg/kg i.p.). The immunoreactivityof the perikarya in the nucleus raphe dorsalis at 15 rain after i.p. (B) was weaker than the control (A). × 88. After 30 min, immunoreactive perikarya of serotonin neurons disappeared throughout the brain stem and the reactivity of the processes of serotonin neurons was diminished. However, immunoreactive, fine varicose fibers were still observed in the medial part of olfactory tubercle, the ventral part of lateral septal nucleus, medial preoptic area, medial amygdaloid nucleus, lateral hypothalamic area, medial forebrain bundle (MFB) in the diencephalon, ventral tegmental area, spinal trigeminal nucleus,
148
Fig. 2. Subcomrnissural organ (asterisk). A: control. B: 30 rain after i.p. At 30 min after i.p. (B), few immunoreactiveserotonin fibers were found except for the supraependymalregions (arrows) on the third ventricle (V). x 300.
facial nucleus, dorsal nucleus of the vagus nerve, ambiguus nucleus, solitary nucleus and supraependymal plexus in the third ventricle, cerebral aqueduct and fourth ventricle (Fig. 2). They were less densely distributed, though, than in control rats. The subsequent time course was characterized by a complete toss of serotonin immunoreactivity, followed by a recovery which began in the perikarya of serotonin neurons and proceeded distally (Fig. 6). At 2 and 4 h after the injection, immunoreactive perikarya and processes of serotonin neurons were absent throughout the brain (Fig. 3A). The initial phases of recovery were evident at 6 h, when weak immunoreactivity was observed in the perikarya of serotonin neurons in the brainstem. In addition, there was less variation in the intensity of the reaction between cell groups, with the exception of neurons in the dorsal area of the nucleus raphe dorsalis, particularly its median and lateral parts, which showed relatively strong reactivity (Fig. 3B). However, immunoreactive processes were not observed at this time. After 8 h, the immunoreactivity of perikarya in the brainstem was greater than 6 h after the injection, and processes were immunostained proximally. Twenty-four hours after reserpine administration, the immunoreactive perikarya in the brainstem were indistinguishable from untreated rats. At this stage, immunoreactive
149
Fig. 3. Nucleus raphe dorsalis at 4 h (A) and 6 h (B) after i.p. At 4 h, there were no immunoreactive structures at all, and at 6 h, the weak immunoreactivitywas observed only in the perikarya. × 88. tract f i b e r s 17 could be traced rostrally toward the MFB at the diencephalic level and caudally toward the lateral part of the inferior olivary complex (Fig. 4). In addition, a sparse distribution of immunoreactive varicose fibers were restricted to the dorsal nucleus of the vagus nerve and solitary nucleus. By 48 h after reserpine administration, immunoreactive tract fibers in the MFB could be traced to the preoptic area. In addition, characteristic networks of t'me varicose fibers were distributed in the suprachiasmatic nucleus, periventricular thalamic nucleus, spinal trigeminal nucleus, facial nucleus,
150
Fig. 4. The lateral part of the inferior olivary complex at 24 h after i.p. The immunoreactivity of the tract fibers was observed following the recovery of the perikarya, x 175.
dorsal nucleus of the vagus nerve, solitary nucleus, the basal portion of the subcommissural organ and supraependymal plexus in the lateral ventricle, third ventricle, cerebral aqueduct and fourth ventricle (Fig. 5). Few immunoreactive fibers were seen in the substantia nigra, corpora quadrigemina and cerebral cortex.
Fig. 5. Suprachiasmatic nucleus at 48 h after i.p. After 48 h, immunoreactive serotonin fibers were detected in the supraehiasmatic nucleus. OC, optic chiasma; V, third ventricle, × 200.
151
0
~
1~
2-4
6
8
24
48
168 HRS
Fig. 6. Schematicdrawingsshowingthe changesof serotonin-immunoreactivitiesin the neuronalperikarya and fibers at 9different times of intraperitoreal injection of reserpine (10mg/kg). The depletion of serotonin-immunoreactivitiesbeginsfirstin the perikaryaand then in the fibers.The recoveryalsooriginates in the perikarya, and then in the fibers of certain areas. By 72 h after administration, the intensity and distribution of serotonin immunoreactivity was almost identical to the untreated rats. Immunoreactive processes, including tract fibers and t'me varicose fibers, were widely distributed throughout the brain, although their density was still less than the control. Finally, 168 h (7 days) after the reserpine injection, recovery was complete at the light microscopic level. DISCUSSION It is generally accepted that reserpine inhibits the uptake of biogenic amines into storage granules by binding irreversibly to the membranes of intraceilular storage organelles, resulting in depletion of intracellular monoamines 3"11,19. Electron microscopy studies have shown that the administration of reserpine causes a significant reduction in the core materials of dense-cored vesicles, and have identified these vesicles as a monoamine storage site in nerve terminals 9"~6. The results of this immunocytochemical study have revealed several new features of the responses of serotonin neurons to reserpine. Studies employing formaldehyde-induced fluorescence (FIF) histochemistry reported that the total cat~holamine fluorescence of both perikarya and terminals disappears rapidly in rats given the same intrapedtoneal dose of reserpine (10 mg/kg), and that recovery occurred first in the pefikarya4,5. However, different groups of cells showed marked differences in the rate of reappearance of the fluorescence. In particular, they reported that the fluorescence of the perikarya of serotonin neurons was unaffected 30 rain after administration, that the yellowish fluorescence disappeared completely between 1 and 8 h, and that at 24 h the perikarya of serotonin neurons appeared normal 4,5. Furthermore, Fuxe 8 reported that only catecholamine terminals in the
152 median eminence showed a noticeable reduction in fluorescence 30 min after reserpine administration, and that the recovery of fluorescence in the terminals was still far from complete even 10 days later. To summarize these findings, Fuxe 8 stated that the time-course of the depletion and the recovery of the serotonin and catecholamine terminals was essentially the same, and that the serotonin terminals in the suprachiasmatic nucleus recovered very slowly, being barely visible 10 days after the injection of reserpine. By contrast, the present study showed that the serotonin neurons display a characteristic depletion and recovery of serotonin after reserpine administration that was distinct from the FIF results. Serotonin within the perikarya disappeared rapidly within 30 min of administration and immunoreactive fine varicose fibers were only observed in some areas. After a complete depletion 2-4 h after reserpine treatment, recovery proceeded gradually from the somata to more distal processes. However, the immunoreactivity within the processes of serotonin neurons did not recover at equal rates from the perikarya toward the terminals in all brain regions. Rather, the recovery began in areas, such as the suprachiasmatic nucleus, periventricular thalamic nucleus, spinal trigeminal nucleus, facial nucleus, dorsal nucleus of the vagus nerve, solitary nucleus, ambiguus nucleus, the basal portion of the subcommissural organ, and supraependymal plexus in the lateral ventricle, third ventricle, cerebral aqueduct and fourth ventricle, then proceeded in other regions of the central nervous system. This discrepancies between our immunocytochemical results and previous histofluorescence studies 4'5'8 may reflect differences in the sensitivity of the methods for detecting serotonin. Since serotonin histofluorescence is very weak and decomposes rapidly under illumination, the most likely factor is the improvement of detectability with immunochemical methods. This improved resolution, then, permits a more sensitive assay of the status of serotonin after experimental manipulation. These observations raise several questions about the actions of reserpine on different intraneuronal pools of serotonin. Although serotonin has been associated with synaptic vesicles in nerve terminals in the central nervous system6"14, its subcellular distribution in perikarya has not been clarified 7"13.Given this paucity of information on the intrasomatic distribution of serotonin, at least two possible explanations can be suggested for the rapid disappearance of serotonin from neuronal perikarya after reserpine administration. First, perikaryal serotonin may be rapidly transported toward the terminals to compensate for reserpine effects on the transmitter distribution in terminals. A second possibility is that intrasomatic serotonin binding proteins 22 may be directly affected by reserpine, diminishing serotonin content in the somata. However, these are clearly only two of many possible explanations, and resolution of this question requires further investigation of the localization of intraperikaryal serotonin.
ACKNOWLEDGEMENTS This work was supported by Grant-in-Aid for Special Project Research (No. 57214028) from the Japanese Ministry of Education, Science and Culture.
153 REFERENCES 1 Brodie, B.B., Olin, J.S., Kuntzman, R.G. and Shore, P.A., Possible interrelationship between release of brain norepinephrine and serotonin by reserpine, Science, 125 (1957) 1293-1294. 2 Carlsson, A., Falck, B. and Hillarp, N.-/~,., Cellular localization of brain monoamines, Acta Physiol. Scand., 56 Suppl. 196 (1962) 1-28. 3 Carlsson, A., Hillarp, N.-A. and Waldeck, B., Analysis of the Mg2÷-ATP dependent storage mechanism in the amine granules of the adrenal medulla, Acta Physiol. Scand., 59 Suppl. 215 (1963) 1-38 4 Dahlstrrm, A. and Fuxe, K., Evidence for the existence ofmonoamine-containing neurons in the central nervous system. I. Demonstration ofmonoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand., 62 Suppl. 232 (1964) 1-55. 5 Dahlstrrm, A., Fuxe, K. and Hillarp, N.-/~., Site of action of reserpine, Acta Pharmacol. ToxicoL, 22 (1965) 227-292. 6 Descarries, L., Beaudet, A. and Watkins, K.C., Serotonin nerve terminals in adult rat neocortex, Brain Res., 100 (1975) 563-588. 7 Descarries, L., Watkins, K.C., Garcia, S. and Beaudet, A., The serotonin in nucleus raphe dorsalis of adult rat: a light and electron microscope radioautographic study, J. Comp. Neurol., 207 (1982) 239-254. 8 Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system. VI. Distribution ofrnonoamine nerve terminals in the central nervous system,Acta Physiol. Scand., 64 Suppl. 247 (1965) 39-85. 9 Grofova, I. and Rinvik, E., Effect of reserpine on large dense-core vesicles in the boutons in the cat's substantia nigra, Neurobiology, 1 (1971) 5-16. 10 Kawata, M., Takeuchi, Y., Ueda, T., Matsuura, T. and Sano, Y., Immunohistochemical demonstration of serotonin-containing nerve fibers in the hypothalamus of the monkey, Macacafuscata, Cell Tiss. Res., 236 (1984) 495-503. I 1 Kirshner, N., Uptake of catecholamines by a particulate fraction of the adrenal medulla, J. Biol. Chem., 237 (1962) 2311-2317. 12 Kojima, M. and Sano, Y., Sexual differences in the topographical distribution of serotonergic fibers in the anterior column of rat lumbar spinal cord, Anat. Embryol., 170 (1984) i 17-121. 13 Nishida, K., Ueda, S. and Sano, Y., Immunohistological studies of masked indoleamine cells in the area postrema of the rat, Histochemistry, 82 (1985) 101-106. 14 Nojyo, Y. and Sano, Y., Ultrastructure of the serotoninergic nerve terminals in the suprachiasmatic and interpeduncular nuclei of rat brain, Brain Res., 149 (1978) 482-488. 15 Pletscher, A., Shore, P.A. and Brodie, B.B., Serotonin as a mediator of reserpine action in brain, J. Pharmacol. Exp. Ther., 116 (1956) 84-89. 16 Sano, Y., Odake, G. and Taketomo, S., Fluorescence microscopic and electron microscopic observations on the tuberolypoplyseal tract, Neuroendocrinology, 2 (1967) 30-42. 17 Sano, Y., Takeuchi, Y., Kimura, H., Goto, M., Kawata, M., Kojima, M., Matsuura, T., Ueda, S. and Yamada, H., Immunohistochemical studies on the processes of serotonin neurons and their ramification in the central nervous system - - with regard to the possibility of the existence of Golgi's rete nervosa diffusa, Arch. HistoL Jpn., 45 (1982) 305-316. 18 Sano, Y., Morphological aspects of the serotonin neuron system in the central nervous system - - an immunohistochemical approach. In Gunma Symposia on Endocrinology, Institute of Endocrinology, Gunma University, Progress and Prospects in Endocrinology, Vol. 20, Center for Academic Publications Japan, Tokyo, 1983, pp. 137-152. 19 Stj~irne, L., Studies of catecholamines uptake storage and release mechanisms, Acta Physiol. Scand., 62 Suppl. 228 (1964) 1-97. 20 Takeuchi, Y., Kimura, H. and Sano, Y., Immunohistochemical demonstration of the distribution of serotonin neurons in the brainstem of the rat and cat, Cell Tiss. Res., 224 (1982) 247-267. 21 Takeuchi, Y., Kimura, H., Matsuura, T. and Sano, Y., Immunohistochemical demonstration of the organization of serotonin neurons in the brain of the monkey (Macacafuscata), Acta Anat., 114 (1982) 106-124. 22 Tamir, H. and Kuhar, M.J., Association of serotonin-binding protein with projections of the midbrain raphe nuclei, Brain Res., 83 (1975) 169-172.
145
Elsevier Scientific Publishers Ireland Ltd. NSR 00099
Immunohistochemical Investigations of the Influence of Reserpine on the Serotonin Neuron System in the Rat Brain Norihiko Ihara, Katsuji Nishida, Shuichi Ueda, Mitsuhiro Kawata and Yutaka Sano Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji,Kamikyo-ku, Kyoto 602 (Japan) (Received June 24th, 1985; Revised version received and accepted August 8th, 1985)
Key words: serotonin - - rat - - reserpine - - immunohistochemistry - - PAP
SUMMARY A peroxidase-antiperoxidase immunohistochemical method with serotonin antiserum was employed to investigate the influence of reserpine on serotonin neurons of rats which were sacrificed at various times after inject!on (10 mg/kg i.p.). The disappearance of serotonin immunoreactivity induced by reserpine was detected only in the perikarya after 15 min, and then rapidly proceeded to the terminals. Between 2 and 4 h, immunoreactivity completely disappeared throughout the brain. The immunoreactivity reappeared in the perikarya after 6 h, and progressed toward the terminals gradually. However, there was an obvious difference in the rate of recovery of immunoreactivity between areas. After 7 days, the immunoreactivity returned to control levels.
INTRODUCTION
One of the most pronounced pharmacological effects of reserpine is depletion of biogenic amines in the brain 1"15.Several histochemical studies have examined the effects of reserpine on monoaminergic neurons using the Falck-Hillarp fluorescence method z' 4,5,8. Although this method has proven to be valuable for visualizing catecholaminecontaining structures, it has several disadvantages, for example, low intensity and rapid fluorophore photodecomposition for detection of serotonin neurons. Thus, until recently, there has been little information on the influences of reserpine on the morphology of serotonin neurons. Correspondence: Y. Sano, Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan. 0168-0102/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
146 We have previously used peroxidase-antiperoxidase (PAP) immunohistochemistry with a highly specific serotonin antiserum to demonstrate the distribution, fiber connections and axonal ramifications of serotonin neurons in the central nervous system of various vertebrates in detail 1°'12'17"18'2°'21. These systematic studies clearly showed that an astonishingly large number of processes of serotonin neurons are widely distributed throughout the central nervous system, from the bulbus olfactorius to the caudal end of the spinal cord, with frequent branching and the formation of dense networks. This report documents changes in the distribution of serotonin immunoreactivity in the rat brain at different times after reserpine administration. MATERIALS AND METHODS
Thirty male Wistar rats, weighing 200-300 g, were divided into ten groups, which were sacrificed at 0.25, 0.5, 2, 4, 6, 8, 24, 48, 72 and 168 h after intraperitoneal administration of reserpine (10 mg/kg Apoplon Inj.-Daiichi, Tokyo, Japan). Under Nembutal anesthesia, the rats were perfused via the left cardiac ventricle with heparinized physiological saline, followed by perfusion with a fixative consisting of 4 ~o paraformaldehyde and 0.2 % picric acid in 0.1 M phosphate buffer (pH 7.4) at 4 ° C. The brains were removed from the skulls and further fixed in the perfusion medium for 48 h at 4 ° C. After immersion in 0.1 M phosphate buffer containing 20 ~o sucrose at 4 ° C, the brain blocks were frozen and cut into 20 #m thick sections in the frontal plane with a cryostat. The sections were stored in 0.1 M phosphate-buffered saline containing 0.3% Triton X-100 at 4 °C. The detailed PAP immunohistochemical procedure and the preparation and specificity of anti-serotonin serum used in this study have been reported elsewhere2°. Briefly, the sections were successively incubated in: (1)rabbit serotonin antiserum (1 : 10,000) for 48 h at 4 °C; (2) goat anti-rabbit IgG (Miles-Yeda Ltd., U.S.A., 1 : 200) for 3 h; (3) peroxidase-antiperoxidase complex (Dako, Denmark, 1 : 200) for 1 h and (4) 3,3'-diaminobenzidine (Sigma, U.S.A.) containing H202 for 10 min. Except for step (1), each step was carried out at room temperature. The sections were mounted on glass slides coated with chrome-alum gelatin, treated with 0.05 % OsO4, dehydrated and coverslipped. RESULTS
Within 15 rain after the intraperitoneal injection of reserpine, the immunoreactivity of the perikarya of serotonin neurons, located mainly in the raphe regions of the brainstem, was markedly reduced. However, serotonin neurons in the dorsal area of the nucleus raphe dorsalis, particularly its median and lateral parts, still displayed relatively strong perikaryal immunoreactivity (Fig. 1). The immunoreactivity of processes of serotonin neurons seemed to be unaffected by the reserpine administration at this stage.
147
Fig. i. Nucleus raphe dorsalis. A: control. B: 15 min after intraperitoneal injection of reserpine (10 mg/kg i.p.). The immunoreactivityof the perikarya in the nucleus raphe dorsalis at 15 rain after i.p. (B) was weaker than the control (A). × 88. After 30 min, immunoreactive perikarya of serotonin neurons disappeared throughout the brain stem and the reactivity of the processes of serotonin neurons was diminished. However, immunoreactive, fine varicose fibers were still observed in the medial part of olfactory tubercle, the ventral part of lateral septal nucleus, medial preoptic area, medial amygdaloid nucleus, lateral hypothalamic area, medial forebrain bundle (MFB) in the diencephalon, ventral tegmental area, spinal trigeminal nucleus,
148
Fig. 2. Subcomrnissural organ (asterisk). A: control. B: 30 rain after i.p. At 30 min after i.p. (B), few immunoreactiveserotonin fibers were found except for the supraependymalregions (arrows) on the third ventricle (V). x 300.
facial nucleus, dorsal nucleus of the vagus nerve, ambiguus nucleus, solitary nucleus and supraependymal plexus in the third ventricle, cerebral aqueduct and fourth ventricle (Fig. 2). They were less densely distributed, though, than in control rats. The subsequent time course was characterized by a complete toss of serotonin immunoreactivity, followed by a recovery which began in the perikarya of serotonin neurons and proceeded distally (Fig. 6). At 2 and 4 h after the injection, immunoreactive perikarya and processes of serotonin neurons were absent throughout the brain (Fig. 3A). The initial phases of recovery were evident at 6 h, when weak immunoreactivity was observed in the perikarya of serotonin neurons in the brainstem. In addition, there was less variation in the intensity of the reaction between cell groups, with the exception of neurons in the dorsal area of the nucleus raphe dorsalis, particularly its median and lateral parts, which showed relatively strong reactivity (Fig. 3B). However, immunoreactive processes were not observed at this time. After 8 h, the immunoreactivity of perikarya in the brainstem was greater than 6 h after the injection, and processes were immunostained proximally. Twenty-four hours after reserpine administration, the immunoreactive perikarya in the brainstem were indistinguishable from untreated rats. At this stage, immunoreactive
149
Fig. 3. Nucleus raphe dorsalis at 4 h (A) and 6 h (B) after i.p. At 4 h, there were no immunoreactive structures at all, and at 6 h, the weak immunoreactivitywas observed only in the perikarya. × 88. tract f i b e r s 17 could be traced rostrally toward the MFB at the diencephalic level and caudally toward the lateral part of the inferior olivary complex (Fig. 4). In addition, a sparse distribution of immunoreactive varicose fibers were restricted to the dorsal nucleus of the vagus nerve and solitary nucleus. By 48 h after reserpine administration, immunoreactive tract fibers in the MFB could be traced to the preoptic area. In addition, characteristic networks of t'me varicose fibers were distributed in the suprachiasmatic nucleus, periventricular thalamic nucleus, spinal trigeminal nucleus, facial nucleus,
150
Fig. 4. The lateral part of the inferior olivary complex at 24 h after i.p. The immunoreactivity of the tract fibers was observed following the recovery of the perikarya, x 175.
dorsal nucleus of the vagus nerve, solitary nucleus, the basal portion of the subcommissural organ and supraependymal plexus in the lateral ventricle, third ventricle, cerebral aqueduct and fourth ventricle (Fig. 5). Few immunoreactive fibers were seen in the substantia nigra, corpora quadrigemina and cerebral cortex.
Fig. 5. Suprachiasmatic nucleus at 48 h after i.p. After 48 h, immunoreactive serotonin fibers were detected in the supraehiasmatic nucleus. OC, optic chiasma; V, third ventricle, × 200.
151
0
~
1~
2-4
6
8
24
48
168 HRS
Fig. 6. Schematicdrawingsshowingthe changesof serotonin-immunoreactivitiesin the neuronalperikarya and fibers at 9different times of intraperitoreal injection of reserpine (10mg/kg). The depletion of serotonin-immunoreactivitiesbeginsfirstin the perikaryaand then in the fibers.The recoveryalsooriginates in the perikarya, and then in the fibers of certain areas. By 72 h after administration, the intensity and distribution of serotonin immunoreactivity was almost identical to the untreated rats. Immunoreactive processes, including tract fibers and t'me varicose fibers, were widely distributed throughout the brain, although their density was still less than the control. Finally, 168 h (7 days) after the reserpine injection, recovery was complete at the light microscopic level. DISCUSSION It is generally accepted that reserpine inhibits the uptake of biogenic amines into storage granules by binding irreversibly to the membranes of intraceilular storage organelles, resulting in depletion of intracellular monoamines 3"11,19. Electron microscopy studies have shown that the administration of reserpine causes a significant reduction in the core materials of dense-cored vesicles, and have identified these vesicles as a monoamine storage site in nerve terminals 9"~6. The results of this immunocytochemical study have revealed several new features of the responses of serotonin neurons to reserpine. Studies employing formaldehyde-induced fluorescence (FIF) histochemistry reported that the total cat~holamine fluorescence of both perikarya and terminals disappears rapidly in rats given the same intrapedtoneal dose of reserpine (10 mg/kg), and that recovery occurred first in the pefikarya4,5. However, different groups of cells showed marked differences in the rate of reappearance of the fluorescence. In particular, they reported that the fluorescence of the perikarya of serotonin neurons was unaffected 30 rain after administration, that the yellowish fluorescence disappeared completely between 1 and 8 h, and that at 24 h the perikarya of serotonin neurons appeared normal 4,5. Furthermore, Fuxe 8 reported that only catecholamine terminals in the
152 median eminence showed a noticeable reduction in fluorescence 30 min after reserpine administration, and that the recovery of fluorescence in the terminals was still far from complete even 10 days later. To summarize these findings, Fuxe 8 stated that the time-course of the depletion and the recovery of the serotonin and catecholamine terminals was essentially the same, and that the serotonin terminals in the suprachiasmatic nucleus recovered very slowly, being barely visible 10 days after the injection of reserpine. By contrast, the present study showed that the serotonin neurons display a characteristic depletion and recovery of serotonin after reserpine administration that was distinct from the FIF results. Serotonin within the perikarya disappeared rapidly within 30 min of administration and immunoreactive fine varicose fibers were only observed in some areas. After a complete depletion 2-4 h after reserpine treatment, recovery proceeded gradually from the somata to more distal processes. However, the immunoreactivity within the processes of serotonin neurons did not recover at equal rates from the perikarya toward the terminals in all brain regions. Rather, the recovery began in areas, such as the suprachiasmatic nucleus, periventricular thalamic nucleus, spinal trigeminal nucleus, facial nucleus, dorsal nucleus of the vagus nerve, solitary nucleus, ambiguus nucleus, the basal portion of the subcommissural organ, and supraependymal plexus in the lateral ventricle, third ventricle, cerebral aqueduct and fourth ventricle, then proceeded in other regions of the central nervous system. This discrepancies between our immunocytochemical results and previous histofluorescence studies 4'5'8 may reflect differences in the sensitivity of the methods for detecting serotonin. Since serotonin histofluorescence is very weak and decomposes rapidly under illumination, the most likely factor is the improvement of detectability with immunochemical methods. This improved resolution, then, permits a more sensitive assay of the status of serotonin after experimental manipulation. These observations raise several questions about the actions of reserpine on different intraneuronal pools of serotonin. Although serotonin has been associated with synaptic vesicles in nerve terminals in the central nervous system6"14, its subcellular distribution in perikarya has not been clarified 7"13.Given this paucity of information on the intrasomatic distribution of serotonin, at least two possible explanations can be suggested for the rapid disappearance of serotonin from neuronal perikarya after reserpine administration. First, perikaryal serotonin may be rapidly transported toward the terminals to compensate for reserpine effects on the transmitter distribution in terminals. A second possibility is that intrasomatic serotonin binding proteins 22 may be directly affected by reserpine, diminishing serotonin content in the somata. However, these are clearly only two of many possible explanations, and resolution of this question requires further investigation of the localization of intraperikaryal serotonin.
ACKNOWLEDGEMENTS This work was supported by Grant-in-Aid for Special Project Research (No. 57214028) from the Japanese Ministry of Education, Science and Culture.
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