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The occurrence of cholecystokinin-like immunoreactive neurons in the rat neostriatum: light and electron microscopic analysis
Brain Research, 309 (1984) 346-349 Elsevier
346 BRE20350
The occurrence of cholecystokinin-like immunoreactive neurons in the rat neostriatum: light and electron microscopic analysis HIROSHI TAKAGI 1, HARUO MIZUTA 1, TOSHIJI MATSUDA1, SHINOBU INAGAKI2, KAYOKO TATEISHI3 and TOSHIYUKI HAMAOKA3
12nd Department of Anatomy, Kinki University School of Medicine, Sayama-cho, Minami-kawachi-gun, Osaka, 589, 2Departmentof Neuroanatomy, Institute of Higher Nervous Activity and 3Department of Oncogenesis, Institutefor CancerResearch, Osaka UniversityMedical School, 4-3-57, Nakanoshima, Kitaku, Osaka, 530 (Japan) (Accepted May 1st, 1984)
Key words: basal ganglia - - cholecystokinin - - aspiny neuron
The present study demonstrates the existence of cholecystokinin-like immunoreactive (CCKI) neurons in the rat neostriatum. Light and electron microscopic findings suggest that these CCKI cells correspond to 'medium-size aspiny neurons' classified by Golgi studies. The neostriatum (NST) has been shown to contain a high concentration of cholecystokinin (CCK) by means of radioimmunoassay6, 8. Since immunocytochemical studies have reported that this peptide is present in nerve fibers, but not in cell bodies in the NST 12, CCKI fibers found in the NST are considered to be supplied exclusively from extrastriatal regions such as the piriform cortex 14, amygdala ~4 and mesencephalon 7. In contrast to these views, is the existence of CCK containing neurons as suggested by radioimmunoassay study using the rat 6. The present study will provide morphological evidence for this hypothesis and describe the characteristic features of these cells by light and electron microscopic observations using peroxidase-antiperoxidase method 17. In addition, fine structures of CCK-like immunoreactive boutons in the NST will be described. Twelve Wistar male albino rats weighing 150-300 g were used. Three of them received intraventricular (lateral ventricle) injection of colchicine (70 /~g in 10 ktl sterile saline) 2 days before sacrifice. All the animals were perfused through the heart with saline for 1-2 rain, followed by 200 ml of picric acid (0.2%)paraformaldehyde (4%)-glutaraldehyde (0.05%)
fixative 16 for 30 min at 15 °C. The brain was cut into blocks and placed in the same fresh fixative for 2 - 3 h at 4 °C. Thereafter, small blocks containing the NST were frozen in liquid nitrogen, thawed and cut on a Vibratome (80/~m thick). These sections were subjected to immunocytochemistry and postfixed with OsO 4 as described previously 16. The sections were stained with 1% uranyl acetate at 70% alcohol dehydration state. For correlation between light and electron microscopic studies, the sections were flatembedded on siliconized slides in resin (Epon 812). For light microscopic observation, selected immunoreactive neurons were drawn with a camera lucida and photographed prior to being reembedded for electron microscopy. Ultrathin sections were mounted on formvar-coated single slot (2 × 1 mm) grids and then stained with lead citrate. Electron micrographs were taken at 80 kV on J E O L 100C. The antiserum to CCK-8 was raised in rabbits. The preparation and characteristics of this antiserum have been described elsewhere3, 9. CCK antiserum was used at dilution of 1:2000. The positive structures identified in the present study should be correctly described as showing CCK-like immunoreactivity, but in this paper we will use the simpler term
Correspondence: H. Takagi, 2nd Department of Anatomy, Kinki University School of Medicine, Sayama-cho, Minami-Kawachugun, Osaka, 589, Japan. 0006-8993/84/$03.00© 1984 Elsevier Science Publishers B.V.
347
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.I
Fig. 1. Schematic drawing showing CCK-immunoreactive structures. Large dots indicate the CCKI cells, and small dots, fibers. CAI, capsula interna; CP, nucleus caudatus putamen; GP, globus pallidus. CCK immunoreactive or CCKI. Light microscopic analysis: in addition to the previously reported12 patch distributions of CCKI fibers especially along the medial and ventral border mainly in the caudal NST, CCKI cells were identified in
U
1
d
L, ! _
/
L
Fig. 2. Camera lucida drawing of CCKI cells. All cells were drawn from animals without colchicinepretreatment, d, dorsal; 1, lateral. Scale bar = 20~m.
the caudal NST (about 1-2 cells per section) (Fig. 1). The majority of them were located in the ventral half of the caudal NST. Colchicine treatment resulted in slight increase of detectable CCKI cells. The neurons had round or fusiform perikarya (cross-sectional width of 9/~m and length of 13 ~m) and gave rise to about two dendrite-like processes (Figs. 2, 3A). Spines were only rarely noted on the distal parts of the processes. Electron microscopic analysis: 9 CCKI cells located in the ventral portion of the caudal NST were studied under electron microscopy, including the cells illustrated in Fig. 2. CCKI cells had an oval or elongated nucleus, which was always indented (Fig. 3B). This feature is characteristic of striatal 'aspiny neurons' classified in Golgi studiesl,4,5,18. Furthermore, the paucity of dendritic spines seen under light microscopy was confirmed by serial ultrathin section analysis. Therefore these findings strongly suggest that CCKI cells correspond to 'medium-size aspiny neurons' of Golgi classification. The 'medium-size aspiny neurons' in the NST were further classified into subtypes based upon differences in morphological appearance4An,18. Certain subtypes of this category appear to be identical to each of the chemically characterized cells, such as GABAergicl and peptidergic (substance p2 and somatostatin 19) neurons. Although detailed comparison of immunoreactive neurons with subtypes of the Golgi classified neurons is difficult, at least some of our CCKI cells shared both light and electron microscopic features with spindle shaped medium-size aspiny neurons 18 (cell 4 in Fig. 2) in the same species. This type of Golgi classified neuron gives rise to local axon collaterals that form symmetrical synapses within the NST iS. On the other hand, boutons forming asymmetrical synapses may be of extrinsic origins, since only symmetrical synapses remain intact in the isolated NST 10. Next we attempted to analyze the fine structures of CCKI boutons in the NST in order to determine the type of synapses CCKI fibers form in the NST. Both types of synaptic contacts, asymmetrical (Fig. 4B) and symmetrical (Fig. 4A), were identified between CCKI boutons and the other neuronal elements. The postsynaptic structures of the former type were dendritic shafts and spines, whereas those of the latter were exclusively dendritic shafts, with the former type of contact occurring
348 more frequently. This finding suggests that although C C K I fibers originate partly from striatal C C K I cells, the majority of them are of extrinsic origins. In support of this, r a d i o i m m u n o a s s a y study showed a significant decrease of striatal C C K content after deafferentiation of the NST ~4. In conclusion, the present study indicates that two
striatal C C K neuron systems, extrinsic as well as intrinsic, must be taken into consideration for understanding C C K mechanismS3,15 in the NST. The authors wish to thank Drs. M. T o h y a m a and S. Mori for their interest and critical discussions, and Mrs. M. Nimura for typing the manuscript.
Fig. 3. A: light micrograph of CCKI cell which is labeled cell 4 in Fig. 2. B: low magnification electron micrograph of the CCKI perikaryon shown in A. Note an indented nucleus. Scale bars: A, 10/~m; B, 1.0~m. Fig. 4. Electron micrographs showing (A) symmetrical synaptic contact (arrows) between a CCKI bouton and non-immunoreactive dendritic shaft (d) and (B) asymmetrical contact (open arrow) between CCKI bouton and non-immunoreactive dendritic spine (s). Scale bars = 0.25 am.
349 1 Bolam, J. P., Clarke, D. J., Smith, A. D. and Somogyi, P., A type of aspiny neuron in the rat neostriatum accumulates [3H]y-aminobutyric acid: combination of Golgi-staining, autoradiography, and electron microscopy, J. comp. Neurol., 213 (1983) 121-134. 2 Bolam, J. P., Somogyi, P., Takagi, H., Fodor, I. and Smith, A. D., Localization of substance P-like immunoreactivity in neurons and nerve terminals in the neostriatum of the rat: a correlated light and electron microscopic study, J. Neurocytol., 12 (1983) 325-344. 3 Cho, H. J., Shiotani, Y., Shiosaka, S., Inagaki, S., Kubota, Y., Kiyama, H., Umegaki, K., Tateishi, K., Hashimura, E., Hamaoka, T. and Tohyama, M., Ontogeny of cholecystokinin-8-containingneuron system of the rat: an immunohistochemical analysis. 1. Forebrain and upper brainstem, J. comp. Neurol., 218 (1983) 25-41. 4 DiFiglia, M., Pasik, T. and Pasik, P., Ultrastructure of Golgi-impregnated and gold-toned spiny and aspiny neurons in the monkey neostriatum, J. Neurocytol., 9 (1980) 471-492. 5 Dimova, R., Vuillet, J. and Seite, R., Study of the rat neostriatum using a combined Golgi-electron microscope technique and serial sections, Neuroscience, 5 (1980) 1581-1596. 6 Emson, P. C., Rehfeld, J. F., Langevin, H. and Rossor, M., Reduction in cholecystokinin-like immunoreactivity in the basal ganglia in Huntington's disease, Brain Research, 198 (1980) 497-500. 7 Fallon, J. G., Wang, C., Kim, Y., Canepa, N., Loughlin, S. and Seroogy, K., Dopamine- and cholecystokinin-containing neurons of the crossed mesostriatal projection, Neurosci. Lett., 40 (1983) 233-238. 8 Gilles, C., Lotstra, F. and Vanderhaeghen, J.-J., CCK nerve terminals in the rat striatal and limbic areas originate partly in the brain stem and partly in telencephalic structures, Life Sci., 32 (1983) 1683-1690. 9 Hashimura, E., Shimizu, F., Nishino, T., Imagawa, K., Tateishi, K. and Hamaoka, T., Production of rabbit antibody specific for amino-terminal residues of cholecystokinin oc-
tapeptide (CCK-8) by selective suppression of cross-reactive antibody response, J. immun. Meth., 55 (1982) 375-387. 10 Hassler, R., Chung, J. W., Wagner, A. and Rinne, U., Experimental demonstration of intrinsic synapses in cat's caudate nucleus, Neurosci. Lett., 5 (1977) 117-121. 11 Kemp, J. M. and Powell, T. P. S., The structure of the caudate nucleus of the cat: light and electron microscopy, Phil. Trans. B, 262 (1971) 383-401. 12 Lor6n, I., Alumets, J., Hhkanson, R. and Sundler, F., Distribution of gastrin and CCK-like peptides in rat brain. An immunocytochemical study, Histochemistry, 59 (1979) 249-257. 13 Marshal, R. D., Owen, F., Deakin, J. F. W. and Poulter, M., The effects of cholecystokinin on dopaminergic mechanisms in rat striatum, Brain Research, 277 (1983) 375-376. 14 Meyer, D. K., Beinfeld, M. C., Oertel, W. H. and Brownstein, M. J., Origin of the cholecystokinin-containing fibers in the rat caudate putamen, Science, 215 (1982) 187-188. 15 Meyer, D. K. and Krauss, J., Dopamine modulates cholecystokinin release in neostriatum, Nature (Lond.), 301 (1983) 338-340. 16 Somogyi, P. and Takagi, H., A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry, Neuroscience, 7 (1982) 1779-1783. 17 Sterberger, L. A., Immunocytochemistry, 2nd edn., Wiley, New York, 1979. 18 Takagi, H., Somogyi, P. and Smith, A. D., Aspiny neurons and their local axons in the neostriatum of the rat: a correlated light and electron microscopic study of Golgi-impregnated material, J. Neurocytol., 13 (1984) 239-265. 19 Takagi, H., Somogyi, P., Somogyi, J. and Smith, A. D., Fine structural studies on a type of somatostatin-immunoreactive neuron and its synaptic connections in the rat neostriatum: a correlated light and electron microscopic study, J. comp. Neurol., 214 (1983) 1-16.
346 BRE20350
The occurrence of cholecystokinin-like immunoreactive neurons in the rat neostriatum: light and electron microscopic analysis HIROSHI TAKAGI 1, HARUO MIZUTA 1, TOSHIJI MATSUDA1, SHINOBU INAGAKI2, KAYOKO TATEISHI3 and TOSHIYUKI HAMAOKA3
12nd Department of Anatomy, Kinki University School of Medicine, Sayama-cho, Minami-kawachi-gun, Osaka, 589, 2Departmentof Neuroanatomy, Institute of Higher Nervous Activity and 3Department of Oncogenesis, Institutefor CancerResearch, Osaka UniversityMedical School, 4-3-57, Nakanoshima, Kitaku, Osaka, 530 (Japan) (Accepted May 1st, 1984)
Key words: basal ganglia - - cholecystokinin - - aspiny neuron
The present study demonstrates the existence of cholecystokinin-like immunoreactive (CCKI) neurons in the rat neostriatum. Light and electron microscopic findings suggest that these CCKI cells correspond to 'medium-size aspiny neurons' classified by Golgi studies. The neostriatum (NST) has been shown to contain a high concentration of cholecystokinin (CCK) by means of radioimmunoassay6, 8. Since immunocytochemical studies have reported that this peptide is present in nerve fibers, but not in cell bodies in the NST 12, CCKI fibers found in the NST are considered to be supplied exclusively from extrastriatal regions such as the piriform cortex 14, amygdala ~4 and mesencephalon 7. In contrast to these views, is the existence of CCK containing neurons as suggested by radioimmunoassay study using the rat 6. The present study will provide morphological evidence for this hypothesis and describe the characteristic features of these cells by light and electron microscopic observations using peroxidase-antiperoxidase method 17. In addition, fine structures of CCK-like immunoreactive boutons in the NST will be described. Twelve Wistar male albino rats weighing 150-300 g were used. Three of them received intraventricular (lateral ventricle) injection of colchicine (70 /~g in 10 ktl sterile saline) 2 days before sacrifice. All the animals were perfused through the heart with saline for 1-2 rain, followed by 200 ml of picric acid (0.2%)paraformaldehyde (4%)-glutaraldehyde (0.05%)
fixative 16 for 30 min at 15 °C. The brain was cut into blocks and placed in the same fresh fixative for 2 - 3 h at 4 °C. Thereafter, small blocks containing the NST were frozen in liquid nitrogen, thawed and cut on a Vibratome (80/~m thick). These sections were subjected to immunocytochemistry and postfixed with OsO 4 as described previously 16. The sections were stained with 1% uranyl acetate at 70% alcohol dehydration state. For correlation between light and electron microscopic studies, the sections were flatembedded on siliconized slides in resin (Epon 812). For light microscopic observation, selected immunoreactive neurons were drawn with a camera lucida and photographed prior to being reembedded for electron microscopy. Ultrathin sections were mounted on formvar-coated single slot (2 × 1 mm) grids and then stained with lead citrate. Electron micrographs were taken at 80 kV on J E O L 100C. The antiserum to CCK-8 was raised in rabbits. The preparation and characteristics of this antiserum have been described elsewhere3, 9. CCK antiserum was used at dilution of 1:2000. The positive structures identified in the present study should be correctly described as showing CCK-like immunoreactivity, but in this paper we will use the simpler term
Correspondence: H. Takagi, 2nd Department of Anatomy, Kinki University School of Medicine, Sayama-cho, Minami-Kawachugun, Osaka, 589, Japan. 0006-8993/84/$03.00© 1984 Elsevier Science Publishers B.V.
347
\
.I
Fig. 1. Schematic drawing showing CCK-immunoreactive structures. Large dots indicate the CCKI cells, and small dots, fibers. CAI, capsula interna; CP, nucleus caudatus putamen; GP, globus pallidus. CCK immunoreactive or CCKI. Light microscopic analysis: in addition to the previously reported12 patch distributions of CCKI fibers especially along the medial and ventral border mainly in the caudal NST, CCKI cells were identified in
U
1
d
L, ! _
/
L
Fig. 2. Camera lucida drawing of CCKI cells. All cells were drawn from animals without colchicinepretreatment, d, dorsal; 1, lateral. Scale bar = 20~m.
the caudal NST (about 1-2 cells per section) (Fig. 1). The majority of them were located in the ventral half of the caudal NST. Colchicine treatment resulted in slight increase of detectable CCKI cells. The neurons had round or fusiform perikarya (cross-sectional width of 9/~m and length of 13 ~m) and gave rise to about two dendrite-like processes (Figs. 2, 3A). Spines were only rarely noted on the distal parts of the processes. Electron microscopic analysis: 9 CCKI cells located in the ventral portion of the caudal NST were studied under electron microscopy, including the cells illustrated in Fig. 2. CCKI cells had an oval or elongated nucleus, which was always indented (Fig. 3B). This feature is characteristic of striatal 'aspiny neurons' classified in Golgi studiesl,4,5,18. Furthermore, the paucity of dendritic spines seen under light microscopy was confirmed by serial ultrathin section analysis. Therefore these findings strongly suggest that CCKI cells correspond to 'medium-size aspiny neurons' of Golgi classification. The 'medium-size aspiny neurons' in the NST were further classified into subtypes based upon differences in morphological appearance4An,18. Certain subtypes of this category appear to be identical to each of the chemically characterized cells, such as GABAergicl and peptidergic (substance p2 and somatostatin 19) neurons. Although detailed comparison of immunoreactive neurons with subtypes of the Golgi classified neurons is difficult, at least some of our CCKI cells shared both light and electron microscopic features with spindle shaped medium-size aspiny neurons 18 (cell 4 in Fig. 2) in the same species. This type of Golgi classified neuron gives rise to local axon collaterals that form symmetrical synapses within the NST iS. On the other hand, boutons forming asymmetrical synapses may be of extrinsic origins, since only symmetrical synapses remain intact in the isolated NST 10. Next we attempted to analyze the fine structures of CCKI boutons in the NST in order to determine the type of synapses CCKI fibers form in the NST. Both types of synaptic contacts, asymmetrical (Fig. 4B) and symmetrical (Fig. 4A), were identified between CCKI boutons and the other neuronal elements. The postsynaptic structures of the former type were dendritic shafts and spines, whereas those of the latter were exclusively dendritic shafts, with the former type of contact occurring
348 more frequently. This finding suggests that although C C K I fibers originate partly from striatal C C K I cells, the majority of them are of extrinsic origins. In support of this, r a d i o i m m u n o a s s a y study showed a significant decrease of striatal C C K content after deafferentiation of the NST ~4. In conclusion, the present study indicates that two
striatal C C K neuron systems, extrinsic as well as intrinsic, must be taken into consideration for understanding C C K mechanismS3,15 in the NST. The authors wish to thank Drs. M. T o h y a m a and S. Mori for their interest and critical discussions, and Mrs. M. Nimura for typing the manuscript.
Fig. 3. A: light micrograph of CCKI cell which is labeled cell 4 in Fig. 2. B: low magnification electron micrograph of the CCKI perikaryon shown in A. Note an indented nucleus. Scale bars: A, 10/~m; B, 1.0~m. Fig. 4. Electron micrographs showing (A) symmetrical synaptic contact (arrows) between a CCKI bouton and non-immunoreactive dendritic shaft (d) and (B) asymmetrical contact (open arrow) between CCKI bouton and non-immunoreactive dendritic spine (s). Scale bars = 0.25 am.
349 1 Bolam, J. P., Clarke, D. J., Smith, A. D. and Somogyi, P., A type of aspiny neuron in the rat neostriatum accumulates [3H]y-aminobutyric acid: combination of Golgi-staining, autoradiography, and electron microscopy, J. comp. Neurol., 213 (1983) 121-134. 2 Bolam, J. P., Somogyi, P., Takagi, H., Fodor, I. and Smith, A. D., Localization of substance P-like immunoreactivity in neurons and nerve terminals in the neostriatum of the rat: a correlated light and electron microscopic study, J. Neurocytol., 12 (1983) 325-344. 3 Cho, H. J., Shiotani, Y., Shiosaka, S., Inagaki, S., Kubota, Y., Kiyama, H., Umegaki, K., Tateishi, K., Hashimura, E., Hamaoka, T. and Tohyama, M., Ontogeny of cholecystokinin-8-containingneuron system of the rat: an immunohistochemical analysis. 1. Forebrain and upper brainstem, J. comp. Neurol., 218 (1983) 25-41. 4 DiFiglia, M., Pasik, T. and Pasik, P., Ultrastructure of Golgi-impregnated and gold-toned spiny and aspiny neurons in the monkey neostriatum, J. Neurocytol., 9 (1980) 471-492. 5 Dimova, R., Vuillet, J. and Seite, R., Study of the rat neostriatum using a combined Golgi-electron microscope technique and serial sections, Neuroscience, 5 (1980) 1581-1596. 6 Emson, P. C., Rehfeld, J. F., Langevin, H. and Rossor, M., Reduction in cholecystokinin-like immunoreactivity in the basal ganglia in Huntington's disease, Brain Research, 198 (1980) 497-500. 7 Fallon, J. G., Wang, C., Kim, Y., Canepa, N., Loughlin, S. and Seroogy, K., Dopamine- and cholecystokinin-containing neurons of the crossed mesostriatal projection, Neurosci. Lett., 40 (1983) 233-238. 8 Gilles, C., Lotstra, F. and Vanderhaeghen, J.-J., CCK nerve terminals in the rat striatal and limbic areas originate partly in the brain stem and partly in telencephalic structures, Life Sci., 32 (1983) 1683-1690. 9 Hashimura, E., Shimizu, F., Nishino, T., Imagawa, K., Tateishi, K. and Hamaoka, T., Production of rabbit antibody specific for amino-terminal residues of cholecystokinin oc-
tapeptide (CCK-8) by selective suppression of cross-reactive antibody response, J. immun. Meth., 55 (1982) 375-387. 10 Hassler, R., Chung, J. W., Wagner, A. and Rinne, U., Experimental demonstration of intrinsic synapses in cat's caudate nucleus, Neurosci. Lett., 5 (1977) 117-121. 11 Kemp, J. M. and Powell, T. P. S., The structure of the caudate nucleus of the cat: light and electron microscopy, Phil. Trans. B, 262 (1971) 383-401. 12 Lor6n, I., Alumets, J., Hhkanson, R. and Sundler, F., Distribution of gastrin and CCK-like peptides in rat brain. An immunocytochemical study, Histochemistry, 59 (1979) 249-257. 13 Marshal, R. D., Owen, F., Deakin, J. F. W. and Poulter, M., The effects of cholecystokinin on dopaminergic mechanisms in rat striatum, Brain Research, 277 (1983) 375-376. 14 Meyer, D. K., Beinfeld, M. C., Oertel, W. H. and Brownstein, M. J., Origin of the cholecystokinin-containing fibers in the rat caudate putamen, Science, 215 (1982) 187-188. 15 Meyer, D. K. and Krauss, J., Dopamine modulates cholecystokinin release in neostriatum, Nature (Lond.), 301 (1983) 338-340. 16 Somogyi, P. and Takagi, H., A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry, Neuroscience, 7 (1982) 1779-1783. 17 Sterberger, L. A., Immunocytochemistry, 2nd edn., Wiley, New York, 1979. 18 Takagi, H., Somogyi, P. and Smith, A. D., Aspiny neurons and their local axons in the neostriatum of the rat: a correlated light and electron microscopic study of Golgi-impregnated material, J. Neurocytol., 13 (1984) 239-265. 19 Takagi, H., Somogyi, P., Somogyi, J. and Smith, A. D., Fine structural studies on a type of somatostatin-immunoreactive neuron and its synaptic connections in the rat neostriatum: a correlated light and electron microscopic study, J. comp. Neurol., 214 (1983) 1-16.