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Neuropeptide-immunoreactive cells and fibers in the developing primate cerebellum
Developmental Brain Research, 51 (1990) 19-25 Elsevier
19
BRESD 50991
Neuropeptide-immunoreactive cells and fibers in the developing primate cerebellum Akiko Yamashita, Motoharu Hayashi, Keiko Shimizu and Kiyoshi Oshima Department of Physiology, Primate Research Institute, Kyoto University. Aichi (Japan) (Accepted 13 June 1989)
Key words: Somatostatin; Cholecystokinin; Substance P; Primate; Cerebellum; Ontogeny; Immunohistochemistry
Using the avidin-biotin-peroxidase immunohistochemical method, we studied the distributions of somatostatin (SOM)-, cholecystokinin-8 (CCK-8)- and substance P (SP)-like immunoreactivities in the cerebellum of macaque monkeys at embryonic day 120 (El20), El40, newborn, postnatal day 60 (P60) and adults. During the embryonic stages, there were many SOM-, CCK- and SP-immunoreactive structures in the external granular layer, Purkinje cell layer and white matter. SP-immunoreactive mossy fibers and their terminals were distributed in the granular layer and white matter. During these stages, there were SOM-immunoreactive Purkinje cells, Golgi cells and a few cells in the molecular layer, and CCK-immunoreactive Purkinje cells and cells in the molecular layer. At the newborn stage, all of the immunoreactivities in the external granular layer decreased and the number of immunoreactive fibers increased in the white matter. At P60 stage, SOM- and CCK-immunoreactive fibers were observed around Purkinje cells, which seem to be the fiber terminals of basket cells. Many SOM, CCK and SP fibers were distributed in the white matter. In the adult stage, we observed little neuropeptide-immunoreactivity in the cerebellum. The high concentration of the neuropeptide-immunoreactive fibers and cells in the earlier stages suggests that the neuropeptides may be involved in the development of the primate cerebellar cortex. INTRODUCTION In the m a m m a l i a n central nervous system, the cerebellar cortex is a relatively simple system which contains 6 types of n e u r o n a l cells (Purkinje cells, Golgi cells, basket cells, stellate cells, Lugaro cells and granule cells) and neuroglial cells. The cerebellum has two main inputs (mossy and climbing fibers) and one output (axon of Purkinje cells). The physiological (for review see ref. 17) and anatomical (for review see ref. 27) aspects of this organ have been studied in detail. Furthermore, the developmental patterns of cells in the cerebellum have been thoroughly examined in the monkey central nervous system 2°-22'29-32. These reports indicate that the cerebellum is a good system for the study of the development of neurotransmitters and neuromodulators. In the adult m a m m a l i a n cerebellum, it is known that there are only small amounts of neuropeptides such as somatostatin (SOM), cholecystokinin-8 (CCK-8) and substance P (SP) 2-14. Moreover, it has been reported that there are no cells containing messenger R N A s that encode SOM 6, CCK 35 or SP 36 in the adult rat cerebellum. However, in the various central nervous systems, there are some reports indicating that younger animals have more neuropeptides than adult monkeys ln'11 or rats TM 16.25,2~. Recently, Hayashi 7'9 found high concentrations of
SP and SOM in the cerebellum of chick embryos and fetal monkeys by the radioimmunoassay method. To date, there has been no report on the cellular distribution of neuropeptides in developing monkey cerebellum. In the present study, we examined the distribution of SOM, CCK and SP immunoreactivities in the m o n k e y cerebellum at embryonic day 120 ( E l 2 0 ) , E l 4 0 , n e w b o r n (Nb), postnatal day 60 (P60) and adult (Ad) stages using the avidin-biotin complex immunohistochemical method. Preliminary studies have been reported elsewhere 39"4~J. MATERIALS AND METHODS
Antisera The biochemical specificities of rabbit anti-SOM, anti-SP and anti-CCK antisera have been described previouslys'll. In the case of CCK, we used an IgG fraction of the anti-CCK antiserum as the first antibody. The purification of the IgG fraction was performed as follows: solid ammonium sulfate was added to the CCK-antiserum and precipitates between 40 and 70% saturation were collected by centrifugation at 10,000 g for 30 rain. The precipitates were dissolved in 0.015 M Tris-phosphate buffer, pH 8.3, and the solution was dialyzed against the same buffer. The dialyzed solution was applied to a DEAE-cellulose column. The column was eluted with the equilibrating buffer and the peak fraction was used as the IgG fraction. Experimental animals A summary of the macaque monkeys (Macaca fuscata fuscata, M.
Correspondence: M. Hayashi, Department of Physiology, Primate Research Institute, Kyoto University, Inuya.ma, Aichi 484, Japan.
20 mulatta and M. fascicularis) used in this study is shown in Table I. Colchicine (500/~g/kg) was injected into the lateral ventricle of one adult monkey two days before sacrifice. We chose 5 different stages, El20 (120 + 3), El40 (140 + 2), Nb, P60 and Ad. To determine the embryonic days of fetal monkeys, we used the timed mating method and measured the length of the head axis and crown-rump length using an ultrasonic transmission method (Sonolayergraph, Toshiba, Japan). The fetuses were obtained from halothane-anesthetized pregnant monkeys by a Caesarean section. We termed the normally delivered monkeys which were born following 160-170 gestational days as Nb.
lmmunohistochemical procedures
and coverslipped. Some sections were stained by Cresylecht violet (Nissl staining) to define the cellular arrangement. To check the specificity of these immunohistochemical methods, we replaced the first anti-peptide antibodies with normal rabbit serum or with each antiserum pre-incubated with each synthetic peptide (Peptide Institute, Osaka, Japan) at a concentration of 10 nmol/ml. The immunoreactivities disappeared except in the pia mater, blood cells and blood vessel. RESULTS
El20
All the animals were deeply anesthetized with sodium pentobarbital (Nembutal, 35 mg/kg i.p.) and heparin sodium (1000 units/ml) was injected into the left ventricle (0.2 ml for fetus, newborn and P60 and 2 ml for adult). Then they were perfused through the heart with ice-cold 0.15 M NaC1 followed by ice-cold Zamboni's fluid (2% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4). The brains were dissected and cut into blocks. The blocks were immersed in 30% sucrose containing 0.02% sodium azide in phosphate-buffered saline (PBS), pH 7.4, at 4 °C after overnight postfixation in Zamboni's fluid at 4 °C. Forty/~m-thick sections were cut with a frozen sliding microtome (Yamato Koki, Tokyo) or with a Vibratome (Lancer, U.S.A.). The free-floating sections were pre-incubated in methanol containing 0.3% H20 2 for 20 min and in PBS-C (0.2% normal goat serum, 0.1% carrageenan (Sigma), 0.2% Triton X-100 and 0.02% sodium azide in PBS) for 1 h at room temperature. The sections were incubated in antisera at 4 °C. The final dilution of the first antibodies were 1:2000 for anti-SOM antiserum, 1:6400 for anti-SP antiserum and 1:640 for the anti-CCK lgG fraction. The incubation times were 72 h (SOM and CCK) and 24 h (SP). The sections were then incubated in a second antibody (Vectastain ABC kit (Vector Labs., Burlingame, CA)) for 24 h at 4 °C. The immunoreactive sites were visualized by avidin-biotin complex-peroxidase method using the ABC kit. A solution of 0.3% HzO 2, 20/~g/ml 3,3"-diaminobenzidine tetrahydrochloride (Nakarai Chemicals) in 0.05 M Tris-HCl buffer, pH 7.6, was used as the substrate for the peroxidase. The sections were mounted on gelatin-coated slide glass, dehydrated in ethanol, cleared in xylene
TABLE I
Summary of experimental monkeys used in this study M.f., Macaca fascicularis (Crab-eating monkey); M.m., M. mulatta (Rhesus monkey); M.f.f., M. fuscatafuscata (Japanese monkey); F, female; M, male; M.f.*, was a cholchicine-treated animal.
Age
Sex
Species
Head axis (cm)
El20 El20 El40 El40 El40 El40 Nb Nb Nb Nb P60 P60 Ad Ad Ad Ad Ad
M M F F M M F F M F F M F F F M M
M.f. M.f. M.f. M.f. M.f. M.f. M.f. M.m. M.m. M.f.f. M.f. M.m. M.f. M.f, M.f.* M.f.f. M.f.f.
3.9x5.0 3.9x5.1 4.3x6.0 4.6x5,7 4.5x5,7 4.3x5.4 4.6x6.2 5.2x6.4 5.1x6,7 5.2×6.4 5.3x6.6 5.3x6.9
The peptide-immunoreactive structures were distributed in the external granular layer and in the Purkinje
cell layer (Fig. la-c). We found a few SOM- and CCK-immunoreactive cells of about 10-20/tm in diameter in the molecular layer. In the granular layer, there were SP-immunoreactive fibers and their terminals, which seemed to be mossy fibers (Fig. lc). E140
The immunoreactivities of the 3 peptides increased in
the external granular layer and Purkinje cell layer (Fig. ld-f). We also found peptide-immunoreactive fibers in the white matter. We found lateral arrangements of SOM- and CKK-immunoreactive fibers and terminaMike structures in the external granular layer and the molecular layer (Figs. ld,e and 3a). In the molecular layer, there were SOM- and CCK-immunoreactive cells which were the same type as at El20 (Figs. 2a and 3b (arrow)). There were fewer CCK-immunoreactive cells in the molecular layer than SOM-immunoreactive cells. We also observed many SOM- and CCK-immunoreactive Purkinje cells and pericellular plexus surrounding them (Figs. 2b and 3a). There were SOM-immunoreactive cells with a diameter of about 20/~m in the granular layer. From the shape of the cells, they are probably Golgi cells (Fig. 2c). Nb
The immunoreactive structures in the cerebellar cortex decreased in number (Fig. lg,h), There were SOM- and CCK-immunoreactive Purkinje cells and many SOMimmunoreactive Goigi cells (Fig. lg). No SOM- and CCK-immunoreactive cells were observed in the molecular layer. There were also many SP-immunoreactive mossy fibers (Fig. li). P60 The immunoreactivity further decreased in the cerebellar cortex and peptide-immunoreactive fibers were
distributed in the Purkinje cell layer and white matter (Fig. l j-I). In contrast, the SP-immunoreactive fibers increased in the white matter (Fig. 11). In the granular layer and white matter, we observed some SP-immuno-
21 gative Purkinje cells surrounded by SOM- and CCKimmunoreactive fibers, which may be the fibers and their terminals of basket cells (Figs. 2d and 3c (arrows)).
reactive fiber terminals which were probably degenerating mossy fibers. There were no peptide-immunoreactive cells in the cerebellum, although there were immunone-
g
q
Fig. 1. The distribution patterns of peptide immunoreactivities in the cerebellar cortex, a: SOM, El20. b: CCK, El20. c: SP, El20; d: SOM, E140. e: CCK, E140. f: SP, E140. g: SOM, Nb. h: CCK, Nb. i: SP, Nb. j: SOM, P60. k: CCK, P60. l: SP, P60. Bar = 50 pm.
22
h
Fig. 2. The SOM-immunoreactive cells in the molecular layer (a) and Purkinje cells (b) at El40. SOM-immunoreactive Golgi cell in the granule layer at Nb (c). SOM-immunoreactive fiber terminals which surrounded the Purkinje cell (arrow) at P60 (d). Bar = 50 #m.
Ad At adulthood, we observed no peptide-immunoreactive cells and few fibers in the cerebellum. DISCUSSION
General considerations In the adult macaque monkey, the cerebellum had few SOM-, CCK- and SP-immunoreactive fibers and no immunoreactive cells, which is consistent with previous studies TM. In contrast, at early stages, the cerebellum contained many immunoreactive fibers and cells. This is in agreement with the decrease of the concentration of SOM and SP in the monkey cerebellum determined by the radioimmunoassay method 7. Decreases in neuropeptide immunoreactivity have been reported in primate cerebral cortex during development 3"1°']1"38. In contrast, neurotransmitters such as ACh and G A B A seem to
increase in the developing primate central nervous system 3'7't1'13. These findings suggest that neuropeptides not only act as neurotransmitters but also are involved in the development of the primate central nervous system. In the macaque monkey cerebellum, migration of the granule cells is completed and the external granular layer disappears at about P9029, Neuropeptide irnmunoreactivity was detected in the external granular layer and Purkinje cell layer where migrating immature granule cells exist. Moreover, until the newborn stages, there were radial CCK- and SOM-immunoreactive fibers and their terminal-like structures in the molecular layer. These neuropeptides may contribute to the development of the cerebellum, especially in the migration or maturation of granule cells.
Neuropeptides and GABA In the developing rat cerebellum, Inagaki et al. ~6
23
C: m
Fig. 3. The CCK-immunoreactive Purkinje cells and radial arrangement of immunoreactive structures in the external granular layer and molecular layer at El40 (a). The CCK-immunoreactive cell in the molecular layer at El40 (arrow) (b). The CCK-immunoreactive fiber terminals which surrounded the Purkinje cells (arrows) at P60 (c). Bar = 50 ~m.
reported some SOM-immunoreactive cells in the cerebellar medulla and white matter. However, in the developing primate cerebellum, we did not observe SOM-immunoreactive cells in the white matter. However, SOM-immunoreactive Golgi cells, Purkinje cells and some cells in the molecular layer were observed. These discrepancies may reflect species differences or the varying sensitivities of the immunohistochemical methods. We also found CCK-immunoreactive Purkinje cells and cells in the molecular layer. These are the first findings of CCK-immunoreactive cells in the developing cerebellum. At P60, SOM- and CCK-immunoreactive fibers and terminals were distributed around the Purkinje cells. Since basket cells are known to terminate around the Purkinje cells, SOM- and CCK-immunoreactive
structures around the Purkinje cells are probably the fiber terminals of basket cells. SOM- and CCK-immunoreactive cells in the molecular layer at the embryonic stage may be developing basket cells. Basket cells, Golgi cells and Purkinje cells are known to be v-aminobutyric acid (GABA)- or glutamic acid decarboxylase (GAD)positive neurons 26'33. In the cerebral cortex of monkeys 12 and cats 34, almost all the CCK and SOM cells have been reported to be G A B A - o r GAD-immunoreactive. Therefore, during the embryonic stages, CCK and SOM may co-exist with G A B A in a subpopulation of basket cells, Golgi cells and Purkinje cells in the monkey cerebellum. Further immunohistochemical studies are still needed. In the monkey cerebellum 7, the G A D activity increased between El20 and adulthood. These results indicate that the number of GABAergic neurons in-
24 creases during ontogenesis of the primate cerebellum. Caddy et al. 5 have reported that the number of Purkinje cells does not change during development. Furthermore, transmitter plasticity in the nervous system has been described during development and maturity 4. Therefore, the change in the expression of phenotype from peptidergic to G A B A e r g i c may occur in Purkinje cells between the E l 2 0 and adult stage. In the central nervous system, there is some evidence of cell death of the interneurons in the course of development 5'37. Therefore, the presence of SOM- and CCK-immunoreactivities in the Golgi and basket cells during the early stages suggests (1) that the CCK- and SOM-phenotype may disappear by cell death, or (2) that the change in expression from neuropeptides to G A B A may occur in these interneurons. S P in mossy fibers In this study, we observed SP-immunoreactive mossy fibers only in the embryonic and newborn stages in the m o n k e y cerebellum. Inagaki et al. 15 have reported SP-immunoreactive fibers which could be traced from white matter into the granular layer in the developing rat cerebellum, and these may be developing mossy fibers. Moreover, Korte et al. 23 have described SP-immunoreactive mossy fibers in the adult red-eared turtle (Chrys e m y s scripta elegans). They also mentioned the existence
ABBREVIATIONS ACh Ad ChAT CCK E120 E140 eg GABA
acetylcholine adult choline acetyltransferase cholecystokinin embryonic day 120 embryonic day 140 external granular layer y-aminobutylic acid
of SP-immunoreactive mossy fibers in the adult frog. The existence of SP-immunoreactive mossy fibers has not been described in the adult mammalian cerebellum. These results indicate that the loss of SP-immunoreactive mossy fibers occurs during evolution. Neurogenesis does not occur in the central nervous system of adult mammals. However, recent findings show the occurrence of neurogenesis in adult lizards z4 and birds ~. The existence of SP in reptilian and mammalian developing cerebellum suggests that SP may be involved in neurogenesis. The subpopulation of the mossy fibers contains choline acetyltransferase (ChAT)-immunoreactivity in rabbits TM and humans 19. In a previous study 7, C h A T activity in the monkey cerebellum increased and SP immunoreactivity decreased reciprocally between E l 2 0 and adulthood. These findings suggest that the SP-immunoreactive mossy fibers develop before the cholinergic mossy fibers. SP-immunoreactive mossy fibers may disappear as the cholinergic mossy fibers develop. A n o t h e r explanation for this p h e n o m e n o n is the change of expression from SP to acetylcholine in mossy fibers during ontogeny. Acknowledgements. We wish to express our thanks to Dr. K. Kubota, Department of Neurophysiology, Primate Research Institute and Dr. S.C. Fujita, Mitsubishi-Kasei Institute of Life Science for valuable discussion. This work was supported by a grant from the Ministry of Health and Welfare of Japan.
GAD g m Nb P60 PBS pc SOM SP WM
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glutamic acid decarboxylase granular layer molecular layer newborn postnatal day 60 phosphate-buffered saline Purkinje cell layer somatostatin substance P white matter
rat brain with in situ hybridization histochemistry, J. Comp. Neurol., 273 (1988) 558-572. Hayashi, M., Ontogeny of glutamic acid decarboxylase, tyrosine hydroxylase, choline acetyltransferase, somatostatin and substance P in monkey cerebellum, Dev. Brain Res., 32 (1987) 181-186. Hayashi, M., Edgar, D. and Thoenen, H., The development of substance P, somatostatin and vasoactive intestinal potypeptide in sympathetic and spinal sensory ganglia of the chick embryo, Neuroscience, 10 (1983) 31-39. Hayashi, M. and Oshima, K., Ontogeny of substance P, somatostatin and vasoactive intestinal polypeptide in the cerebellum and cerebrum of the chick embryo, Neurosci. Res., 1 (1984) 427-436. Hayashi, M. and Oshima, K., Neuropeptides in cerebral cortex of macaque monkey (Macaca fuscata fuscata): regional distribution and ontogeny, Brain Res., 364 (1986) 360-368. Hayashi, M., Yamashita, A., Shimizu, K. and Oshima, K., Ontogeny of cholecystokinin-8 and glutamic acid decarboxylase in cerebral neocortex of macaque monkey, Exp. Brain Res., 74 (1989) 249-255.
25 12 Hendry, S.H.C., Jones, E.G., DeFelipe, J., Schmechel, D., Brandon, C. and Emson, P.C., Neuropeptide-containing neurons of the cerebral cortex are also GABAergic, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 6526-6530. 13 Huntley, G.W., Hendry, S.H.C., Killackey, H.P., Chalupa, L.M. and Jones, E.G., Temporal sequence of neurotransmitter expression by developing neurons of fetal monkey visual cortex, Dev. Brain Res., 43 (1988) 69-96. 14 H6kfelt, T., Johansson, O., Ljungdahl, ~., Lundberg, J.M. and Schultzberg, M., Peptidergic neurons, Nature (Lond.), 284 (1980) 515-521. 15 Inagaki, S., Sakanaka, M., Shiosaka, S., Senba, E., Takagi, H., Takatsuki, K., Kawai, Y., Matsuzaki, T., Iida, H., Hara, Y. and Tohyama, M., Experimental and immunohistochemical studies on the cerebellar substance P of the rat: localization, postnatal ontogeny and way of entry to the cerebellum, Neuroscience, 7 (1982) 639-645. 16 Inagaki, S., Shiosaka, S., Takatsuki, K., Iida, H., Sakanaka, M., Senba, E., Hara, Y., Matsuzaki, T., Kawai, Y. and Tohyama, M., Ontogeny of somtatostatin-containing neuron system of the rat cerebellum including its fiber connections: an experimental and immunohistochemical analysis, Dev. Brain Res., 3 (1982) 509-527. 17 Ito, M., The Cerebellum and Neural Control, Raven, New York, 1984. 18 Kan, K.-S.K., ChaD, L.-P. and Eng, L.E, Immunohistochemical localization of choline acetyltransferase in rabbit spinal cord and cerebellum, Brain Res., 146 (1978) 221-229. 19 Kan, K.-S.K., ChaD, L.-P. and Forno, L.S., Immunohistochemical localization of choline acetyltransferase in the human cerebellum, Brain Res., 193 (1980) 165-171. 20 Kornguth, S.E., Anderson, J.W. and Scott, G., Observations on the ultrastructure of the developing cerebellum of the Macaca mulatta, J. Cornp. Neurol., 130 (1967) 1-24. 21 Kornguth, S.E., Anderson, J.W. and Scott, G., The development of synaptic contacts in the cerebellum of Macaca mulatta, J. Comp. Neurol., 132 (1968) 531-546. 22 Kornguth, S.E. and Scott, G., The role of climbing fibers in the formation of Purkinje cell dendrites, J. Comp. Neurol., 146 (1972) 61-82. 23 Korte, G.E., Reiner, A. and Karten, H.J., Substance P-like immunoreactivity in cerebellar mossy fibers and terminals in the red-eared turtle Crysemys scripta elegans, Neuroscience, 5 (1980) 903-914. 24 Lopez-Garcia, C., Molowny, A., Garcia-Verdugo, J.M. and Ferrer, I., Delayed postnatal neurogenesis in the cerebral cortex of lizards, Dev. Brain Res., 43 (1988) 167-174. 25 McGregor, G.P., Woodhams, P.L., O'Shaughnessy, D.J., Ghatei, M.A., Polak, J.M. and Bloom, S.R., Developmental changes in bombesin, substance P, somatostatin and vasoactive intestinal polypeptide in the rat brain, Neurosci. Lett., 28 (1982) 21-27.
26 Oertel, W.H., Schmechel, D.E., Mugnaini, E., Tappaz, M.L. and Kopin, I.J., Immunocytochemical localization of glutamate decarboxylase in rat cerebellum with a new antiserum, Neuroscience, 6 (1981) 2715-2735. 27 Palay, S.L. and Chan-Palay, V., Cerebellar Cortex, Springer, Berlin, 1974. 28 Quirion, R. and Dam, T.-V., Ontogeny of substance P receptor binding sites in rat brain, J. Neurosci., 6 (1986) 2187-2199. 29 Rakic, P., Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electronmicroscopic study in Macacus rhesus, J. Comp. Neurol., 141 (1971) 283-312. 30 Rakic, P., Extrinsic cytological determinations of basket and stellate cell dendritic pattern in the cerebellar molecular layer, J. Cornp. Neurol., 146 (1972) 335-354. 31 Rakic, P., Kinetics of proliferation and latency between final cell division and onset of differentiation of cerebellar stellate and basket neurons, J. Cornp. NeuroL, 147 (1973) 523-546. 32 Rakic, P., Genetic and epigenetic determinates of local neuronal circuits in the mammalian central nervous system. In F.O. Schmitt and F.G. Worden (Eds.), The Neurosciences: 4th Study Program, MIT Press, Cambridge, MA, 1979, pp. 109-127. 33 Saito, K., Barber, R., Wu, J.-Y., Matsuda, T., Roberts, E. and Vaughn, J.E., Immunohistochemical localization of glutamic acid decarboxylase in rat cerebellum, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 269-273. 34 Somogyi, P., Hodgson, A.J., Smith, A.D., Nunzi, M.G., Gorio, A. and Wu, J.-Y., Different populations of GABAergic neurons in the visual cortex and hippocampus of cat contain somatostatin- or cholecystokinin-immunoreactive material, J. Neurosci., 4 (1984) 2590-2603. 35 Voigt, M.M. and Uhl, G.R., Preprocholecystokinin mRNA in rat brain: regional expression includes thalamus, Mol. Brain Res., 4 (1988) 247-253. 36 Warden, M.K. and Young III, W.S., Distribution of cells containing mRNAs encoding substance P and neurokinin B in the rat central nervous system, J. Comp. Neurol., 272 (1988) 90-113. 37 Williams, R.W. and Rakic, P., Elimination of neurons from the rhesus monkey's lateral geniculate nucldus during development, J. Comp. Neurol., 272 (1988) 424-436. 38 Yamashita, A., Hayashi, M., Shimizu, K. and Oshima, K., Ontogeny of somatostatin in cerebral cortex of macaque monkey: an immunohistochemical study, Dev. Brain Res., 45 (1989) 103-111. 39 Yamashita, A., Hayashi, M., Shimizu, K. and Oshima, K., Ontogeny of neuropeptides in monkey cerebellar cortex, Dev. Growth Diff., 30 (1988) 421. 40 Yamashita, A., Hayashi, M., Shimizu, K. and Oshima, K., Distribution of neuropeptide immunoreactive cells and fibers in the developing monkey cerebellum, Neurosci. Res., Suppl. in Dress.
19
BRESD 50991
Neuropeptide-immunoreactive cells and fibers in the developing primate cerebellum Akiko Yamashita, Motoharu Hayashi, Keiko Shimizu and Kiyoshi Oshima Department of Physiology, Primate Research Institute, Kyoto University. Aichi (Japan) (Accepted 13 June 1989)
Key words: Somatostatin; Cholecystokinin; Substance P; Primate; Cerebellum; Ontogeny; Immunohistochemistry
Using the avidin-biotin-peroxidase immunohistochemical method, we studied the distributions of somatostatin (SOM)-, cholecystokinin-8 (CCK-8)- and substance P (SP)-like immunoreactivities in the cerebellum of macaque monkeys at embryonic day 120 (El20), El40, newborn, postnatal day 60 (P60) and adults. During the embryonic stages, there were many SOM-, CCK- and SP-immunoreactive structures in the external granular layer, Purkinje cell layer and white matter. SP-immunoreactive mossy fibers and their terminals were distributed in the granular layer and white matter. During these stages, there were SOM-immunoreactive Purkinje cells, Golgi cells and a few cells in the molecular layer, and CCK-immunoreactive Purkinje cells and cells in the molecular layer. At the newborn stage, all of the immunoreactivities in the external granular layer decreased and the number of immunoreactive fibers increased in the white matter. At P60 stage, SOM- and CCK-immunoreactive fibers were observed around Purkinje cells, which seem to be the fiber terminals of basket cells. Many SOM, CCK and SP fibers were distributed in the white matter. In the adult stage, we observed little neuropeptide-immunoreactivity in the cerebellum. The high concentration of the neuropeptide-immunoreactive fibers and cells in the earlier stages suggests that the neuropeptides may be involved in the development of the primate cerebellar cortex. INTRODUCTION In the m a m m a l i a n central nervous system, the cerebellar cortex is a relatively simple system which contains 6 types of n e u r o n a l cells (Purkinje cells, Golgi cells, basket cells, stellate cells, Lugaro cells and granule cells) and neuroglial cells. The cerebellum has two main inputs (mossy and climbing fibers) and one output (axon of Purkinje cells). The physiological (for review see ref. 17) and anatomical (for review see ref. 27) aspects of this organ have been studied in detail. Furthermore, the developmental patterns of cells in the cerebellum have been thoroughly examined in the monkey central nervous system 2°-22'29-32. These reports indicate that the cerebellum is a good system for the study of the development of neurotransmitters and neuromodulators. In the adult m a m m a l i a n cerebellum, it is known that there are only small amounts of neuropeptides such as somatostatin (SOM), cholecystokinin-8 (CCK-8) and substance P (SP) 2-14. Moreover, it has been reported that there are no cells containing messenger R N A s that encode SOM 6, CCK 35 or SP 36 in the adult rat cerebellum. However, in the various central nervous systems, there are some reports indicating that younger animals have more neuropeptides than adult monkeys ln'11 or rats TM 16.25,2~. Recently, Hayashi 7'9 found high concentrations of
SP and SOM in the cerebellum of chick embryos and fetal monkeys by the radioimmunoassay method. To date, there has been no report on the cellular distribution of neuropeptides in developing monkey cerebellum. In the present study, we examined the distribution of SOM, CCK and SP immunoreactivities in the m o n k e y cerebellum at embryonic day 120 ( E l 2 0 ) , E l 4 0 , n e w b o r n (Nb), postnatal day 60 (P60) and adult (Ad) stages using the avidin-biotin complex immunohistochemical method. Preliminary studies have been reported elsewhere 39"4~J. MATERIALS AND METHODS
Antisera The biochemical specificities of rabbit anti-SOM, anti-SP and anti-CCK antisera have been described previouslys'll. In the case of CCK, we used an IgG fraction of the anti-CCK antiserum as the first antibody. The purification of the IgG fraction was performed as follows: solid ammonium sulfate was added to the CCK-antiserum and precipitates between 40 and 70% saturation were collected by centrifugation at 10,000 g for 30 rain. The precipitates were dissolved in 0.015 M Tris-phosphate buffer, pH 8.3, and the solution was dialyzed against the same buffer. The dialyzed solution was applied to a DEAE-cellulose column. The column was eluted with the equilibrating buffer and the peak fraction was used as the IgG fraction. Experimental animals A summary of the macaque monkeys (Macaca fuscata fuscata, M.
Correspondence: M. Hayashi, Department of Physiology, Primate Research Institute, Kyoto University, Inuya.ma, Aichi 484, Japan.
20 mulatta and M. fascicularis) used in this study is shown in Table I. Colchicine (500/~g/kg) was injected into the lateral ventricle of one adult monkey two days before sacrifice. We chose 5 different stages, El20 (120 + 3), El40 (140 + 2), Nb, P60 and Ad. To determine the embryonic days of fetal monkeys, we used the timed mating method and measured the length of the head axis and crown-rump length using an ultrasonic transmission method (Sonolayergraph, Toshiba, Japan). The fetuses were obtained from halothane-anesthetized pregnant monkeys by a Caesarean section. We termed the normally delivered monkeys which were born following 160-170 gestational days as Nb.
lmmunohistochemical procedures
and coverslipped. Some sections were stained by Cresylecht violet (Nissl staining) to define the cellular arrangement. To check the specificity of these immunohistochemical methods, we replaced the first anti-peptide antibodies with normal rabbit serum or with each antiserum pre-incubated with each synthetic peptide (Peptide Institute, Osaka, Japan) at a concentration of 10 nmol/ml. The immunoreactivities disappeared except in the pia mater, blood cells and blood vessel. RESULTS
El20
All the animals were deeply anesthetized with sodium pentobarbital (Nembutal, 35 mg/kg i.p.) and heparin sodium (1000 units/ml) was injected into the left ventricle (0.2 ml for fetus, newborn and P60 and 2 ml for adult). Then they were perfused through the heart with ice-cold 0.15 M NaC1 followed by ice-cold Zamboni's fluid (2% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4). The brains were dissected and cut into blocks. The blocks were immersed in 30% sucrose containing 0.02% sodium azide in phosphate-buffered saline (PBS), pH 7.4, at 4 °C after overnight postfixation in Zamboni's fluid at 4 °C. Forty/~m-thick sections were cut with a frozen sliding microtome (Yamato Koki, Tokyo) or with a Vibratome (Lancer, U.S.A.). The free-floating sections were pre-incubated in methanol containing 0.3% H20 2 for 20 min and in PBS-C (0.2% normal goat serum, 0.1% carrageenan (Sigma), 0.2% Triton X-100 and 0.02% sodium azide in PBS) for 1 h at room temperature. The sections were incubated in antisera at 4 °C. The final dilution of the first antibodies were 1:2000 for anti-SOM antiserum, 1:6400 for anti-SP antiserum and 1:640 for the anti-CCK lgG fraction. The incubation times were 72 h (SOM and CCK) and 24 h (SP). The sections were then incubated in a second antibody (Vectastain ABC kit (Vector Labs., Burlingame, CA)) for 24 h at 4 °C. The immunoreactive sites were visualized by avidin-biotin complex-peroxidase method using the ABC kit. A solution of 0.3% HzO 2, 20/~g/ml 3,3"-diaminobenzidine tetrahydrochloride (Nakarai Chemicals) in 0.05 M Tris-HCl buffer, pH 7.6, was used as the substrate for the peroxidase. The sections were mounted on gelatin-coated slide glass, dehydrated in ethanol, cleared in xylene
TABLE I
Summary of experimental monkeys used in this study M.f., Macaca fascicularis (Crab-eating monkey); M.m., M. mulatta (Rhesus monkey); M.f.f., M. fuscatafuscata (Japanese monkey); F, female; M, male; M.f.*, was a cholchicine-treated animal.
Age
Sex
Species
Head axis (cm)
El20 El20 El40 El40 El40 El40 Nb Nb Nb Nb P60 P60 Ad Ad Ad Ad Ad
M M F F M M F F M F F M F F F M M
M.f. M.f. M.f. M.f. M.f. M.f. M.f. M.m. M.m. M.f.f. M.f. M.m. M.f. M.f, M.f.* M.f.f. M.f.f.
3.9x5.0 3.9x5.1 4.3x6.0 4.6x5,7 4.5x5,7 4.3x5.4 4.6x6.2 5.2x6.4 5.1x6,7 5.2×6.4 5.3x6.6 5.3x6.9
The peptide-immunoreactive structures were distributed in the external granular layer and in the Purkinje
cell layer (Fig. la-c). We found a few SOM- and CCK-immunoreactive cells of about 10-20/tm in diameter in the molecular layer. In the granular layer, there were SP-immunoreactive fibers and their terminals, which seemed to be mossy fibers (Fig. lc). E140
The immunoreactivities of the 3 peptides increased in
the external granular layer and Purkinje cell layer (Fig. ld-f). We also found peptide-immunoreactive fibers in the white matter. We found lateral arrangements of SOM- and CKK-immunoreactive fibers and terminaMike structures in the external granular layer and the molecular layer (Figs. ld,e and 3a). In the molecular layer, there were SOM- and CCK-immunoreactive cells which were the same type as at El20 (Figs. 2a and 3b (arrow)). There were fewer CCK-immunoreactive cells in the molecular layer than SOM-immunoreactive cells. We also observed many SOM- and CCK-immunoreactive Purkinje cells and pericellular plexus surrounding them (Figs. 2b and 3a). There were SOM-immunoreactive cells with a diameter of about 20/~m in the granular layer. From the shape of the cells, they are probably Golgi cells (Fig. 2c). Nb
The immunoreactive structures in the cerebellar cortex decreased in number (Fig. lg,h), There were SOM- and CCK-immunoreactive Purkinje cells and many SOMimmunoreactive Goigi cells (Fig. lg). No SOM- and CCK-immunoreactive cells were observed in the molecular layer. There were also many SP-immunoreactive mossy fibers (Fig. li). P60 The immunoreactivity further decreased in the cerebellar cortex and peptide-immunoreactive fibers were
distributed in the Purkinje cell layer and white matter (Fig. l j-I). In contrast, the SP-immunoreactive fibers increased in the white matter (Fig. 11). In the granular layer and white matter, we observed some SP-immuno-
21 gative Purkinje cells surrounded by SOM- and CCKimmunoreactive fibers, which may be the fibers and their terminals of basket cells (Figs. 2d and 3c (arrows)).
reactive fiber terminals which were probably degenerating mossy fibers. There were no peptide-immunoreactive cells in the cerebellum, although there were immunone-
g
q
Fig. 1. The distribution patterns of peptide immunoreactivities in the cerebellar cortex, a: SOM, El20. b: CCK, El20. c: SP, El20; d: SOM, E140. e: CCK, E140. f: SP, E140. g: SOM, Nb. h: CCK, Nb. i: SP, Nb. j: SOM, P60. k: CCK, P60. l: SP, P60. Bar = 50 pm.
22
h
Fig. 2. The SOM-immunoreactive cells in the molecular layer (a) and Purkinje cells (b) at El40. SOM-immunoreactive Golgi cell in the granule layer at Nb (c). SOM-immunoreactive fiber terminals which surrounded the Purkinje cell (arrow) at P60 (d). Bar = 50 #m.
Ad At adulthood, we observed no peptide-immunoreactive cells and few fibers in the cerebellum. DISCUSSION
General considerations In the adult macaque monkey, the cerebellum had few SOM-, CCK- and SP-immunoreactive fibers and no immunoreactive cells, which is consistent with previous studies TM. In contrast, at early stages, the cerebellum contained many immunoreactive fibers and cells. This is in agreement with the decrease of the concentration of SOM and SP in the monkey cerebellum determined by the radioimmunoassay method 7. Decreases in neuropeptide immunoreactivity have been reported in primate cerebral cortex during development 3"1°']1"38. In contrast, neurotransmitters such as ACh and G A B A seem to
increase in the developing primate central nervous system 3'7't1'13. These findings suggest that neuropeptides not only act as neurotransmitters but also are involved in the development of the primate central nervous system. In the macaque monkey cerebellum, migration of the granule cells is completed and the external granular layer disappears at about P9029, Neuropeptide irnmunoreactivity was detected in the external granular layer and Purkinje cell layer where migrating immature granule cells exist. Moreover, until the newborn stages, there were radial CCK- and SOM-immunoreactive fibers and their terminal-like structures in the molecular layer. These neuropeptides may contribute to the development of the cerebellum, especially in the migration or maturation of granule cells.
Neuropeptides and GABA In the developing rat cerebellum, Inagaki et al. ~6
23
C: m
Fig. 3. The CCK-immunoreactive Purkinje cells and radial arrangement of immunoreactive structures in the external granular layer and molecular layer at El40 (a). The CCK-immunoreactive cell in the molecular layer at El40 (arrow) (b). The CCK-immunoreactive fiber terminals which surrounded the Purkinje cells (arrows) at P60 (c). Bar = 50 ~m.
reported some SOM-immunoreactive cells in the cerebellar medulla and white matter. However, in the developing primate cerebellum, we did not observe SOM-immunoreactive cells in the white matter. However, SOM-immunoreactive Golgi cells, Purkinje cells and some cells in the molecular layer were observed. These discrepancies may reflect species differences or the varying sensitivities of the immunohistochemical methods. We also found CCK-immunoreactive Purkinje cells and cells in the molecular layer. These are the first findings of CCK-immunoreactive cells in the developing cerebellum. At P60, SOM- and CCK-immunoreactive fibers and terminals were distributed around the Purkinje cells. Since basket cells are known to terminate around the Purkinje cells, SOM- and CCK-immunoreactive
structures around the Purkinje cells are probably the fiber terminals of basket cells. SOM- and CCK-immunoreactive cells in the molecular layer at the embryonic stage may be developing basket cells. Basket cells, Golgi cells and Purkinje cells are known to be v-aminobutyric acid (GABA)- or glutamic acid decarboxylase (GAD)positive neurons 26'33. In the cerebral cortex of monkeys 12 and cats 34, almost all the CCK and SOM cells have been reported to be G A B A - o r GAD-immunoreactive. Therefore, during the embryonic stages, CCK and SOM may co-exist with G A B A in a subpopulation of basket cells, Golgi cells and Purkinje cells in the monkey cerebellum. Further immunohistochemical studies are still needed. In the monkey cerebellum 7, the G A D activity increased between El20 and adulthood. These results indicate that the number of GABAergic neurons in-
24 creases during ontogenesis of the primate cerebellum. Caddy et al. 5 have reported that the number of Purkinje cells does not change during development. Furthermore, transmitter plasticity in the nervous system has been described during development and maturity 4. Therefore, the change in the expression of phenotype from peptidergic to G A B A e r g i c may occur in Purkinje cells between the E l 2 0 and adult stage. In the central nervous system, there is some evidence of cell death of the interneurons in the course of development 5'37. Therefore, the presence of SOM- and CCK-immunoreactivities in the Golgi and basket cells during the early stages suggests (1) that the CCK- and SOM-phenotype may disappear by cell death, or (2) that the change in expression from neuropeptides to G A B A may occur in these interneurons. S P in mossy fibers In this study, we observed SP-immunoreactive mossy fibers only in the embryonic and newborn stages in the m o n k e y cerebellum. Inagaki et al. 15 have reported SP-immunoreactive fibers which could be traced from white matter into the granular layer in the developing rat cerebellum, and these may be developing mossy fibers. Moreover, Korte et al. 23 have described SP-immunoreactive mossy fibers in the adult red-eared turtle (Chrys e m y s scripta elegans). They also mentioned the existence
ABBREVIATIONS ACh Ad ChAT CCK E120 E140 eg GABA
acetylcholine adult choline acetyltransferase cholecystokinin embryonic day 120 embryonic day 140 external granular layer y-aminobutylic acid
of SP-immunoreactive mossy fibers in the adult frog. The existence of SP-immunoreactive mossy fibers has not been described in the adult mammalian cerebellum. These results indicate that the loss of SP-immunoreactive mossy fibers occurs during evolution. Neurogenesis does not occur in the central nervous system of adult mammals. However, recent findings show the occurrence of neurogenesis in adult lizards z4 and birds ~. The existence of SP in reptilian and mammalian developing cerebellum suggests that SP may be involved in neurogenesis. The subpopulation of the mossy fibers contains choline acetyltransferase (ChAT)-immunoreactivity in rabbits TM and humans 19. In a previous study 7, C h A T activity in the monkey cerebellum increased and SP immunoreactivity decreased reciprocally between E l 2 0 and adulthood. These findings suggest that the SP-immunoreactive mossy fibers develop before the cholinergic mossy fibers. SP-immunoreactive mossy fibers may disappear as the cholinergic mossy fibers develop. A n o t h e r explanation for this p h e n o m e n o n is the change of expression from SP to acetylcholine in mossy fibers during ontogeny. Acknowledgements. We wish to express our thanks to Dr. K. Kubota, Department of Neurophysiology, Primate Research Institute and Dr. S.C. Fujita, Mitsubishi-Kasei Institute of Life Science for valuable discussion. This work was supported by a grant from the Ministry of Health and Welfare of Japan.
GAD g m Nb P60 PBS pc SOM SP WM
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