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Neonatal noradrenaline depletion prevents the transition of Bergmann glia in the developing cerebellum
Journal of Chemical Neuroanatomy 14 (1998) 167 – 173
Neonatal noradrenaline depletion prevents the transition of Bergmann glia in the developing cerebellum I. Podkletnova a, H. Alho b,* b
a Laboratory of Neurobiology, Medical School, Uni6ersity of Tampere, P.O. Box 607, 33101 Tampere, Finland Department of Mental Health and Alcohol Research, National Public Health Institute, P.O. Box 719, 00101 Helsinki, Finland
Accepted 12 December 1997
Abstract The influence of neonatal administration of 6-hydroxydopamine (6-OHDA) on the cell proliferation in cerebellum was studied using 10–30 days-old rats. Compared to their littermates, treated rats had poor ability in searching, skills performance and orienting in the new environment. Elimination of noradrenergic terminals by 6-OHDA led to a delay in granular cell migration. The secondary foliation in neo-cerebellum was absent. The Bergmann glial cells were abnormally located, structurally different and did not form the intimate association with Purkinje cells. Our findings indicate that without noradrenergic influence neurones and glial cells do not proliferate normally and noradrenaline may act as an important trophic factor also for Bergmann glial cells. © 1998 Elsevier Science B.V. All rights reserved. Keywords: 6-Hydroxydopamine; Noradrenergic; Proliferation; Rat
1. Introduction It is well recognised that, in addition to their role in synaptic communication, neurotransmitters act as important trophic factors controlling nervous system development (Lauder, 1985; Whitaker-Azmitia, 1991; Kobayashi et al., 1995; Mohanakumar et al., 1995). Noradrenergic system provides one of the best characterised models for evaluating the role of neurotransmitter stimulation in target cell differentiation and in the establishment of receptor-mediated responses (Rowe et al., 1993; Wagner et al., 1995). Cerebellum of rat receives only noradrenergic (not dopaminergic) input, and undergoes its peak of proliferation postnatally (Jacobson, 1978). Because of Abbre6iations: NA, noradrenaline; PD, postnatal day; 5-HT, 5-hydroxytryptamine; 6-OHDA, 6-hydroxydopamine. * Corresponding author. Tel.: +358 9 1333339; fax: + 358 9 1332781. 0891-0618/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0891-0618(98)00006-4
these properties cerebellum provides the unique possibility to study the structural and functional changes after neonatal elimination of noradrenergic terminals. 6-Hydroxydopamine (6-OHDA), a neurotoxin with selective effects on catecholamine neurones, has been widely used to produce noradrenaline (NA) depletion. Similar administration of 5-HT antiserum prevents the development of 5-HT system in the rat brain (Mohanakumar et al., 1995). Repeated injections of 6-OHDA during early postnatal life can be used to produce permanent depletion of NA in the cerebellum. It has been shown that neonatal brain lesion by 6-OHDA produces cerebellar dysfunction and reduction in the amount of granular cells (Lovell, 1982; Podkletnova et al., 1996). Since the Bergmann glial cells are essential for the transition of granular cells, we studied the maturation and localisation of Bergmann glia after neonatal 6-OHDA treatment, employing immunocytochemistry and electron microscopy.
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2. Materials and methods
2.1. Subjects A total of 36 of young Sprague-Dawley rats were used. The earlier studies had indicated that there are no sex differences in the effect of neonatal treatment with 6-OHDA (Podkletnova et al., 1996) Pups from six litters were randomly pooled and divided into two groups on the day of birth, designated as postnatal day 0 (PD0). The first (experimental) group received the 6-OHDA (Sigma) injection (100 mg/kg) subcutaneously in a volume of 0.1 ml in 0.9% saline containing 0.04% L-ascorbic acid (Sigma) at PD1, PD2, and PD3. The second (control) group of rats received an equal volume of the solvent. After injection, the pups were marked and returned to the lactating mothers. Three experimental and three control pups were maintained with every mother in a plastic cage with free access to food and water. The animals were kept in a room with a constant 12:12 h light/dark cycle. The weight of the rats and their behaviour in the home cage were monitored as previously described (Podkletnova et al., 1996) on a daily basis from the day of birth until weaning.
2.2. Tissue preparation and immunohistochemistry For immunohistochemical staining 10, 15 and 20-day old rats were anaesthetised with chloral hydrate (350 mg/kg, i.p.). The systemic circulation was rinsed by cardiac perfusion of 0.9% saline at room temperature, and then perfused with ice-cold fixative (4% paraformaldehyde in 0.1 M PBS, pH 7.4). Brains were postfixed with the same ice-cold fixative for 2 h, washed and stored in PBS, and cut into 25 – 30-mm thick sections with a vibratome. Processing of sections was carried out as we described earlier (Podkletnova and Alho, 1993) and immuno-histochemical staining was carried out with the ABC mouse IgG Kit (VECTASTAIN, USA). The antisera used were mouse monoclonal antiserum to glial fibrillary acidic protein (GFAP, Boehringer Manneheim) diluted 1:1000 and rabbit polyclonal antiserum to diazepam binding inhibitor (DBI) diluted 1:4000. DBI has been shown to localise in cerebellum mainly in Bergmann glial cells (Alho et al., 1990). Antigen – antibody complexes were visualised by incubation with 3-3%-diaminobenzidine (Sigma, 0.2 mg/ml in 0.1 M Tris, pH 7.4) and hydrogen peroxide (0.001%) for 5 min at room temperature. The sections were mounted in Aquamount and observed under a Nikon microphot-FXA (Japan) photomicroscope in a bright field.
2.3. Electron microscopy. A conventional method was employed to prepare
cerebella for electron microscopy observation. Briefly, brains were fixed in 2.5% glutaraldehyde, declive (lobule VIb) were separated from cerebellum, post-fixed with ice-cold 2% osmium tetroxide in 0.1 M PBS (pH 7.4) for 1 h, dehydrated in ethanol and via propylene oxide, embedded in capsules with Epon 812 (Ladd Research Industries, Burlington, VT), and cut into ultra-thin sections with an Ultratome III (LKB, Rockville, MD). The sections were examined with a JEOL (JEM-1200 EX) electron microscope.
2.4. Image analysis Semiquantitative estimation of staining intensity and the number of labelled cells was performed with an image analyzer. The immunoreactive and counterstained microscopic images were scanned with an MTI 2 CCD-72 camera (Dage, Inc., Michigan City, IN), further processed with a DSP-200 MTI image processor (Dage) and calculated on a Macintosh IIx (Apple Computer Inc., Cupertino, CA) equipped with IMAGE software (courtesy of Dr W. Rusband, NIMH, Bethesda, MD). The mean grey value of sections stained with preimmune serum was imposed as the background value, and on the antiserum-stained sections neurones exceeding this value were considered as immunopositive. The number of immunopositive cells (density) was calculated from six animals of each age group and from four different 1000 mm2 areas in three consecutive sections (25 mm distance), and expressed as the mean of all these cells. The density of astrocytes was calculated in granular cell layer, Bergmann cells— in Purkinje cell layer and Bergmann cell fibres—in the upper part of molecular layer. The total area of granular cell layer was calculated from semithin hematoxylineosin stained sections based on the intense staining (clearly distinguished grey value) of granular cells. All results are expressed as means9 S.E.M. Groups were tested for significance with analysis of variance (ANOVA) with repeated measures and Student’s t-test.
3. Results. Behavioural alterations in treated animals were identical as we had reported earlier (Podkletnova et al., 1996). Neonatal 6-OHDA injections caused behavioural deficit in the ability of young rats (10–20 days from birth) to orientate in new environment. The alteration in morpho-anatomical structure of cerebellum from 6-OHDA treated rats (10, 15 and 20 days old) was observed in semi-thin sections of vermis. Neo-cerebellum (lobuli VI, VII, VIII, IX) was smaller
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169
Fig. 1. Low power photomicrograph demonstrating the morphological alterations in cerebellum after 6-OHDA treatment (hematoxylin-eosin counterstaining, magnification ×10). Saggital sections from 15 days old (top) and 20 days old (bottom) vermis of cerebellum. A, C, control cerebellum; B, D, 6-OHDA-lesioned cerebellum.
than that of the control animals, also the secondary foliation was deficient or absent (Fig. 1). The total area of granular cells was significantly smaller in treated animals. The astrocytes and Bergmann glial cells were visualised by using GFAP and DBI antibodies. The density of GFAP-immunopositive astrocytes inside the granular cell layer of altered lobuli did not differ from control cerebella (Table 1). However the amount of GFAP-immunopositive Bergmann glia fibres in declive (lobulus VI) of 6-OHDA treated cerebella was up to 40% reduced compared to the corresponding region of control ones. When DBI antiserum was applied, there was no difference in the density of immunopositive astrocytes in granular cell layer (data not shown), but the density and location of Bergmann glial cells in 6-OHDA treated cerebella were different (Fig. 2). The density of DBI-LI Bergmann cells in lobuli VI of 6-OHDA treated animals at the age of 15 and 20 days
was significantly less in lesioned cerebella than in control ones (Table 2). In control samples, Bergmann glia were situated around Purkinje cells while after 6OHDA treatment, DBI-LI Bergmann cells were located on the border between granular and molecular layers away from Purkinje cells (Fig. 2 and Fig. 3). Lobuli VIa, VIb and VII from cerebellar vermis of 6-OHDA treated and control rats were taken for EM observation. A 200-mm area around Purkinje cells (Pc) were observed. In control samples, Pc bodies were surrounded by protoplasm of Bergmann glial cells. Cross-sections through the body of Purkinje cells of control brain contained 39 1 Bergmann glial cells (Fig. 3A). In 6-OHDA treated cerebella, Pc bodies were free from Bergmann glia protoplasm. The Bergmann glia cells were located in granular layer away from the Purkinje cells (Fig. 3B). The fine structure of 6-OHDA, treated Bergmann glia differed significantly from control cells. In control
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Fig. 2. DBI-LI in declive (lobulus VI) from 20 days old rats. A, control cerebellum, B, 6-OHDA-lesioned cerebellum. Arrows, Purkinje cells; m, molecular layer; g, granular cell layer. Bar = 50 mm.
Table 1 GFAP-LI in glial cells of cerebellum (20 days old) Control
6-OHDA
Lobulus
III
VI
IX
III
VI
IX
Astrocytes Bergman fibers
414948 76 94
3989 50 809 5
463 949 78 94
404 9 45 809 5
333 940 51 9 6*
439 947 72 94
Density of astrocytes in granular cell layer is expressed as the number of immunopositive cells per 1000 mm2. Density of Bergmann fibers in the upper part of molecular layer is expressed as the number of immunopositive fibers per 1000 mm length. * PB0.05.
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171
Fig. 3. Electron microscopy photographs demonstrating cell morphology in cerebellum. A, C, control cerebellum; B, D, 6-OHDA-lesioned cerebellum. Asterisk, Purkinje cells; arrowheads, granular cells; arrows, Bergmann glial cells. (C, D, Bergmann glial cells, high magnification). Bar =2 mm (A, B) and 1 mm (C, D).
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Table 2 DBI-LI in Bergmann glial cells of cerebellum Control
6-OHDA
Lobulus
III
VI
IX
III
VI
IX
10 days old rat 15 days old rat 20 days old rat
7598 789 7 14498
63 9 7 859 8 769 5
82 9 7 88 9 5 106 9 8
53 96 80 96 120 9 7
52 9 7 52 9 6* 22 9 7**
66 9 6 82 9 7 102 9 8
Density of Bergmann cells in the Purkinje cell layer is expressed as the number of immunopositive cells per 1000 mm length. * PB0.5; ** P B0.05.
cerebella, Bergmann glia had multiple protoplasmic processes extending around neighbouring cells. After neonatal 6-OHDA treatment, the shape of nuclei was not round but irregular and the protoplasm of changed Bergmann glia was not so widely spread and not so closely attached to the neighbouring neurones, as it was in control samples (Fig. 3C, D).
4. Discussion Rat cerebellum undergoes its peak of development postnatally (Jacobson, 1978). Bergmann glia proliferate earlier than neurones in cerebellum. Post-natal proliferation of the Bergmann glial cells occurs mainly between PD6 and PD9. Spatial and temporal pattern in post-natal proliferation of glial cells have been shown by 3 [H]thymidine incorporation. In paleocerebellum (lobules I–VIa, VIII–X) labelling index reached the peak on PD 6–7, and in neocerebellum (lobules VIb – VII) on PD 8 – 9 (Shiga et al., 1983). The rich noradrenergic innervation of cerebellum has been demonstrated by unusual thick highly fluorescent dopamine-beta-hydroxylase-like immunoreactive fibres in the external granular layer during the first 2 weeks after birth (Verney et al., 1982). Those fibres are noradrenergic terminals of locus coeruleus neurones. It has been discovered, that noradrenergic input to beta-receptors is necessary for receptor differentiation (Seidler et al., 1995; Wagner et al., 1995). Lesioning the noradrenergic projections or blocking the neurotransmission with a receptor antagonist prevents astrogliosis and glial cell proliferation (Rowe et al., 1993; Hodges-Savola et al., 1996). However there is no earlier observations of Bergmann glia after 6-OHDA treatment. This may be because of the difficulty in identification of these cells and due to the fact that GFAP antiserum does not stain the Bergmann cell bodies. Bergmann glia of the rodent cerebellum is an example where one glial cell population sequentially performs different functions in early and later phases of tissue maturation. During the first 2 weeks after birth, a major function of the Bergmann glia is to guide the migration of granule cells from the proliferative zone on the surface
of the cerebellar cortex to their ultimate resting place deep to the Purkinje cell layer (Das, 1976; Muller et al., 1996). By the third postnatal week, the Bergmann glia become increasingly intimately associated with the rapidly maturing Purkinje cells, whose somata and dendritic trees they ensheathe (Fisher et al., 1993; Reichenbach et al., 1995). Repeated injections of 6-OHDA during first postnatal week of rats produces depletion of NA in cerebellum, disruption of secondary foliation in neocerebellum and reduction in the amount of granular cells. Our observation that areas of the granular cell layer are smaller in 6-OHDA treated brains are in good agreement with the data published earlier (Lovell, 1982). Reduction in the amount of granular cells can be explained as the result of depletion in trophic noradrenergic influence on the differentiation of granule cells. We present here for the first time the evidence that after 6-OHDA treatment Bergmann glial cells are abnormally located and structurally different. The intimate association between Bergmann glia and Purkinje cell also did not occur after neonatal 6-OHDA treatment. This treatment eliminates noradrenergic terminals from the majority of brain structures among which is the cerebellum (Lauder, 1985). In vitro studies provide the evidence that morphology of astroglial cells is controlled by beta-adrenergic receptors (Shain et al., 1987). Astrocytes in vivo differentiate from the early postnatal epithelial-like form to a stellate cells. In the adult, Bergmann glial cells develop complex membrane structures that are in the intimate contact with synaptic regions in the molecular layer, i.e. the synaptic contacts between Purkinje cells and parallel and climbing fibres (Muller et al., 1996). The lack of Bergmann cell transition after elimination of noradrenergic input in cerebellum indicates that noradrenaline may act as important trophic factor for the development of these cells and their functions. Acknowledgements This study was financially supported by the Medical Research Fund of Tampere University Hospital and International Graduate School of Neuroscience, University of Tampere.
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References Alho, H., Bovolin, P., Slobodyansky, E., 1990. Diazepam binding inhibitor (DBI) processing: Immunohistochemical studies in the rat brain. Neurochem. Res. 15, 209–216. Das, G.D., 1976. Differentiation of Bergmann glia cells in the cerebellum: A Golgi study. Brain Res. 110, 199–213. Fisher, M., Trimmer, P., Ruthel, G., 1993. Bergmann glia require continuous association with Purkinje cells for normal phenotype expression. Glia 8, 172–182. Hodges-Savola, C., Rogers, S.D., Chilardi, J.R., Timm, D.R., Manyth, P.W., 1996. Beta-adrenergic receptors regulate astrogliosis and cell profileration in the central nervous system in vivo. Glia 17, 52 – 62. Jacobson M., 1978. Developmental Neurobiology, Plenum Press, New York. Kobayashi, K., Morita, S., Sawada, H., Mizuguchi, T., Yamada, K., Nagatsu, I., Hata, T., Watanabe, Y., Fujita, K., Nagatsu, T., 1995. Targeted disruption of the tyrosine hydroxylase locus results in severe catecholamine depletion and perinatal lethality in mice. J. Biol. Chem. 270, 27235–27243. Lauder J.M., 1985. Roles for neurotransmitters in development: Possible interaction with drugs during the fetal and neonatal periods. In: Marois, M. (Ed.), Prevention of Physical and Mental Congenital Defect, Alan R. Liss, New York, pp. 375–380. Lovell, K.L., 1982. Effects of 6-hydroxydopamine-induced norepinephrine depletion on cerebellar development. Dev. Neurosci. 5, 359 – 368. Mohanakumar, K.P., Mohanty, S., Ganguly, D.K., 1995. Neonatal treatment with 5-HT antiserum alters 5-HT metabolism and function in adult rats. Dev. Neurosci. 7, 238–240. Muller, T., Mo¨ller, T., Neuhaus, J., Kettenmann, H., 1996. Electrical coupling among Bergmann glial cells and its modulation by glutamate receptor activation. Glia 17, 274–284.
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Podkletnova, I., Alho, H., 1993. Ultrasound-amplified immunohistochemistry. J. Histochem. Cytochem. 41, 51 – 56. Podkletnova, I., Raevsky, V., Alho, H., 1996. Reduction of GABAergic transmission and alterations in behavior after 6-OHDA treatment of rats. Dev. Brain Res. 94, 197 – 204. Reichenbach, A., Siegel, A., Richmann, M., Wolff, J.R., Noone, D., Robinson, S.R., 1995. Distribution of Bergmann glial somata and processes: implications for function. J. Hirnforsch. 36, 509– 517. Rowe, S.J., Messenger, N.J., Warner, A.E., 1993. The role of noradrenaline in the differentiation of amphibian embryonic neurons. Development 119, 1343 – 1357. Seidler, F.J., Temple, S.W., McCook, E.C., Slotkin, T.A., 1995. Cocaine inhibits central noradrenergic and dopaminergic activity during the critical developmental period in which catecholamines influence cell development. Dev. Brain Res. 85, 48 – 53. Shain, W., Forman, D.S., Madelian, V., Tuner, J.N., 1987. Morphology of astroglial cells is controlled by b-adrenergic receptors. J. Cell Biol. 105, 2307 – 2314. Shiga, T., Ichikawa, M., Hirata, Y., 1983. Spatial and temporal pattern of postnatal proliferation of Bergmann glial cells in rat cerebellum: an autoradiographic study. Anat. Embryol. 167, 203– 211. Verney, C., Grzanna, R., Farkas, E., 1982. Distribution of dopaminebeta-hydroxylase-like immunoreactive fibers in the rat cerebellar cortex during ontogeny. Dev. Neurosci. 5, 369 – 374. Wagner, J.P., Seidler, F.J., Lappi, S.E., McCook, E.C., Slotkin, T.A., 1995. Role of presypnaptic input in the ontogeny of adrenergic cell signaling in rat brain: b-receptors, adenylate cyclase and c-fos protooncogene expression. J. Pharmacol. Exp. Ther. 273, 415– 426. Whitaker-Azmitia, P.M., 1991. Role of serotonin and other neurotransmitter receptors in brain development: basis for developmental pharmacology. Pharmacol. Rev. 43, 553 – 561.
Neonatal noradrenaline depletion prevents the transition of Bergmann glia in the developing cerebellum I. Podkletnova a, H. Alho b,* b
a Laboratory of Neurobiology, Medical School, Uni6ersity of Tampere, P.O. Box 607, 33101 Tampere, Finland Department of Mental Health and Alcohol Research, National Public Health Institute, P.O. Box 719, 00101 Helsinki, Finland
Accepted 12 December 1997
Abstract The influence of neonatal administration of 6-hydroxydopamine (6-OHDA) on the cell proliferation in cerebellum was studied using 10–30 days-old rats. Compared to their littermates, treated rats had poor ability in searching, skills performance and orienting in the new environment. Elimination of noradrenergic terminals by 6-OHDA led to a delay in granular cell migration. The secondary foliation in neo-cerebellum was absent. The Bergmann glial cells were abnormally located, structurally different and did not form the intimate association with Purkinje cells. Our findings indicate that without noradrenergic influence neurones and glial cells do not proliferate normally and noradrenaline may act as an important trophic factor also for Bergmann glial cells. © 1998 Elsevier Science B.V. All rights reserved. Keywords: 6-Hydroxydopamine; Noradrenergic; Proliferation; Rat
1. Introduction It is well recognised that, in addition to their role in synaptic communication, neurotransmitters act as important trophic factors controlling nervous system development (Lauder, 1985; Whitaker-Azmitia, 1991; Kobayashi et al., 1995; Mohanakumar et al., 1995). Noradrenergic system provides one of the best characterised models for evaluating the role of neurotransmitter stimulation in target cell differentiation and in the establishment of receptor-mediated responses (Rowe et al., 1993; Wagner et al., 1995). Cerebellum of rat receives only noradrenergic (not dopaminergic) input, and undergoes its peak of proliferation postnatally (Jacobson, 1978). Because of Abbre6iations: NA, noradrenaline; PD, postnatal day; 5-HT, 5-hydroxytryptamine; 6-OHDA, 6-hydroxydopamine. * Corresponding author. Tel.: +358 9 1333339; fax: + 358 9 1332781. 0891-0618/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0891-0618(98)00006-4
these properties cerebellum provides the unique possibility to study the structural and functional changes after neonatal elimination of noradrenergic terminals. 6-Hydroxydopamine (6-OHDA), a neurotoxin with selective effects on catecholamine neurones, has been widely used to produce noradrenaline (NA) depletion. Similar administration of 5-HT antiserum prevents the development of 5-HT system in the rat brain (Mohanakumar et al., 1995). Repeated injections of 6-OHDA during early postnatal life can be used to produce permanent depletion of NA in the cerebellum. It has been shown that neonatal brain lesion by 6-OHDA produces cerebellar dysfunction and reduction in the amount of granular cells (Lovell, 1982; Podkletnova et al., 1996). Since the Bergmann glial cells are essential for the transition of granular cells, we studied the maturation and localisation of Bergmann glia after neonatal 6-OHDA treatment, employing immunocytochemistry and electron microscopy.
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2. Materials and methods
2.1. Subjects A total of 36 of young Sprague-Dawley rats were used. The earlier studies had indicated that there are no sex differences in the effect of neonatal treatment with 6-OHDA (Podkletnova et al., 1996) Pups from six litters were randomly pooled and divided into two groups on the day of birth, designated as postnatal day 0 (PD0). The first (experimental) group received the 6-OHDA (Sigma) injection (100 mg/kg) subcutaneously in a volume of 0.1 ml in 0.9% saline containing 0.04% L-ascorbic acid (Sigma) at PD1, PD2, and PD3. The second (control) group of rats received an equal volume of the solvent. After injection, the pups were marked and returned to the lactating mothers. Three experimental and three control pups were maintained with every mother in a plastic cage with free access to food and water. The animals were kept in a room with a constant 12:12 h light/dark cycle. The weight of the rats and their behaviour in the home cage were monitored as previously described (Podkletnova et al., 1996) on a daily basis from the day of birth until weaning.
2.2. Tissue preparation and immunohistochemistry For immunohistochemical staining 10, 15 and 20-day old rats were anaesthetised with chloral hydrate (350 mg/kg, i.p.). The systemic circulation was rinsed by cardiac perfusion of 0.9% saline at room temperature, and then perfused with ice-cold fixative (4% paraformaldehyde in 0.1 M PBS, pH 7.4). Brains were postfixed with the same ice-cold fixative for 2 h, washed and stored in PBS, and cut into 25 – 30-mm thick sections with a vibratome. Processing of sections was carried out as we described earlier (Podkletnova and Alho, 1993) and immuno-histochemical staining was carried out with the ABC mouse IgG Kit (VECTASTAIN, USA). The antisera used were mouse monoclonal antiserum to glial fibrillary acidic protein (GFAP, Boehringer Manneheim) diluted 1:1000 and rabbit polyclonal antiserum to diazepam binding inhibitor (DBI) diluted 1:4000. DBI has been shown to localise in cerebellum mainly in Bergmann glial cells (Alho et al., 1990). Antigen – antibody complexes were visualised by incubation with 3-3%-diaminobenzidine (Sigma, 0.2 mg/ml in 0.1 M Tris, pH 7.4) and hydrogen peroxide (0.001%) for 5 min at room temperature. The sections were mounted in Aquamount and observed under a Nikon microphot-FXA (Japan) photomicroscope in a bright field.
2.3. Electron microscopy. A conventional method was employed to prepare
cerebella for electron microscopy observation. Briefly, brains were fixed in 2.5% glutaraldehyde, declive (lobule VIb) were separated from cerebellum, post-fixed with ice-cold 2% osmium tetroxide in 0.1 M PBS (pH 7.4) for 1 h, dehydrated in ethanol and via propylene oxide, embedded in capsules with Epon 812 (Ladd Research Industries, Burlington, VT), and cut into ultra-thin sections with an Ultratome III (LKB, Rockville, MD). The sections were examined with a JEOL (JEM-1200 EX) electron microscope.
2.4. Image analysis Semiquantitative estimation of staining intensity and the number of labelled cells was performed with an image analyzer. The immunoreactive and counterstained microscopic images were scanned with an MTI 2 CCD-72 camera (Dage, Inc., Michigan City, IN), further processed with a DSP-200 MTI image processor (Dage) and calculated on a Macintosh IIx (Apple Computer Inc., Cupertino, CA) equipped with IMAGE software (courtesy of Dr W. Rusband, NIMH, Bethesda, MD). The mean grey value of sections stained with preimmune serum was imposed as the background value, and on the antiserum-stained sections neurones exceeding this value were considered as immunopositive. The number of immunopositive cells (density) was calculated from six animals of each age group and from four different 1000 mm2 areas in three consecutive sections (25 mm distance), and expressed as the mean of all these cells. The density of astrocytes was calculated in granular cell layer, Bergmann cells— in Purkinje cell layer and Bergmann cell fibres—in the upper part of molecular layer. The total area of granular cell layer was calculated from semithin hematoxylineosin stained sections based on the intense staining (clearly distinguished grey value) of granular cells. All results are expressed as means9 S.E.M. Groups were tested for significance with analysis of variance (ANOVA) with repeated measures and Student’s t-test.
3. Results. Behavioural alterations in treated animals were identical as we had reported earlier (Podkletnova et al., 1996). Neonatal 6-OHDA injections caused behavioural deficit in the ability of young rats (10–20 days from birth) to orientate in new environment. The alteration in morpho-anatomical structure of cerebellum from 6-OHDA treated rats (10, 15 and 20 days old) was observed in semi-thin sections of vermis. Neo-cerebellum (lobuli VI, VII, VIII, IX) was smaller
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169
Fig. 1. Low power photomicrograph demonstrating the morphological alterations in cerebellum after 6-OHDA treatment (hematoxylin-eosin counterstaining, magnification ×10). Saggital sections from 15 days old (top) and 20 days old (bottom) vermis of cerebellum. A, C, control cerebellum; B, D, 6-OHDA-lesioned cerebellum.
than that of the control animals, also the secondary foliation was deficient or absent (Fig. 1). The total area of granular cells was significantly smaller in treated animals. The astrocytes and Bergmann glial cells were visualised by using GFAP and DBI antibodies. The density of GFAP-immunopositive astrocytes inside the granular cell layer of altered lobuli did not differ from control cerebella (Table 1). However the amount of GFAP-immunopositive Bergmann glia fibres in declive (lobulus VI) of 6-OHDA treated cerebella was up to 40% reduced compared to the corresponding region of control ones. When DBI antiserum was applied, there was no difference in the density of immunopositive astrocytes in granular cell layer (data not shown), but the density and location of Bergmann glial cells in 6-OHDA treated cerebella were different (Fig. 2). The density of DBI-LI Bergmann cells in lobuli VI of 6-OHDA treated animals at the age of 15 and 20 days
was significantly less in lesioned cerebella than in control ones (Table 2). In control samples, Bergmann glia were situated around Purkinje cells while after 6OHDA treatment, DBI-LI Bergmann cells were located on the border between granular and molecular layers away from Purkinje cells (Fig. 2 and Fig. 3). Lobuli VIa, VIb and VII from cerebellar vermis of 6-OHDA treated and control rats were taken for EM observation. A 200-mm area around Purkinje cells (Pc) were observed. In control samples, Pc bodies were surrounded by protoplasm of Bergmann glial cells. Cross-sections through the body of Purkinje cells of control brain contained 39 1 Bergmann glial cells (Fig. 3A). In 6-OHDA treated cerebella, Pc bodies were free from Bergmann glia protoplasm. The Bergmann glia cells were located in granular layer away from the Purkinje cells (Fig. 3B). The fine structure of 6-OHDA, treated Bergmann glia differed significantly from control cells. In control
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170
Fig. 2. DBI-LI in declive (lobulus VI) from 20 days old rats. A, control cerebellum, B, 6-OHDA-lesioned cerebellum. Arrows, Purkinje cells; m, molecular layer; g, granular cell layer. Bar = 50 mm.
Table 1 GFAP-LI in glial cells of cerebellum (20 days old) Control
6-OHDA
Lobulus
III
VI
IX
III
VI
IX
Astrocytes Bergman fibers
414948 76 94
3989 50 809 5
463 949 78 94
404 9 45 809 5
333 940 51 9 6*
439 947 72 94
Density of astrocytes in granular cell layer is expressed as the number of immunopositive cells per 1000 mm2. Density of Bergmann fibers in the upper part of molecular layer is expressed as the number of immunopositive fibers per 1000 mm length. * PB0.05.
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171
Fig. 3. Electron microscopy photographs demonstrating cell morphology in cerebellum. A, C, control cerebellum; B, D, 6-OHDA-lesioned cerebellum. Asterisk, Purkinje cells; arrowheads, granular cells; arrows, Bergmann glial cells. (C, D, Bergmann glial cells, high magnification). Bar =2 mm (A, B) and 1 mm (C, D).
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Table 2 DBI-LI in Bergmann glial cells of cerebellum Control
6-OHDA
Lobulus
III
VI
IX
III
VI
IX
10 days old rat 15 days old rat 20 days old rat
7598 789 7 14498
63 9 7 859 8 769 5
82 9 7 88 9 5 106 9 8
53 96 80 96 120 9 7
52 9 7 52 9 6* 22 9 7**
66 9 6 82 9 7 102 9 8
Density of Bergmann cells in the Purkinje cell layer is expressed as the number of immunopositive cells per 1000 mm length. * PB0.5; ** P B0.05.
cerebella, Bergmann glia had multiple protoplasmic processes extending around neighbouring cells. After neonatal 6-OHDA treatment, the shape of nuclei was not round but irregular and the protoplasm of changed Bergmann glia was not so widely spread and not so closely attached to the neighbouring neurones, as it was in control samples (Fig. 3C, D).
4. Discussion Rat cerebellum undergoes its peak of development postnatally (Jacobson, 1978). Bergmann glia proliferate earlier than neurones in cerebellum. Post-natal proliferation of the Bergmann glial cells occurs mainly between PD6 and PD9. Spatial and temporal pattern in post-natal proliferation of glial cells have been shown by 3 [H]thymidine incorporation. In paleocerebellum (lobules I–VIa, VIII–X) labelling index reached the peak on PD 6–7, and in neocerebellum (lobules VIb – VII) on PD 8 – 9 (Shiga et al., 1983). The rich noradrenergic innervation of cerebellum has been demonstrated by unusual thick highly fluorescent dopamine-beta-hydroxylase-like immunoreactive fibres in the external granular layer during the first 2 weeks after birth (Verney et al., 1982). Those fibres are noradrenergic terminals of locus coeruleus neurones. It has been discovered, that noradrenergic input to beta-receptors is necessary for receptor differentiation (Seidler et al., 1995; Wagner et al., 1995). Lesioning the noradrenergic projections or blocking the neurotransmission with a receptor antagonist prevents astrogliosis and glial cell proliferation (Rowe et al., 1993; Hodges-Savola et al., 1996). However there is no earlier observations of Bergmann glia after 6-OHDA treatment. This may be because of the difficulty in identification of these cells and due to the fact that GFAP antiserum does not stain the Bergmann cell bodies. Bergmann glia of the rodent cerebellum is an example where one glial cell population sequentially performs different functions in early and later phases of tissue maturation. During the first 2 weeks after birth, a major function of the Bergmann glia is to guide the migration of granule cells from the proliferative zone on the surface
of the cerebellar cortex to their ultimate resting place deep to the Purkinje cell layer (Das, 1976; Muller et al., 1996). By the third postnatal week, the Bergmann glia become increasingly intimately associated with the rapidly maturing Purkinje cells, whose somata and dendritic trees they ensheathe (Fisher et al., 1993; Reichenbach et al., 1995). Repeated injections of 6-OHDA during first postnatal week of rats produces depletion of NA in cerebellum, disruption of secondary foliation in neocerebellum and reduction in the amount of granular cells. Our observation that areas of the granular cell layer are smaller in 6-OHDA treated brains are in good agreement with the data published earlier (Lovell, 1982). Reduction in the amount of granular cells can be explained as the result of depletion in trophic noradrenergic influence on the differentiation of granule cells. We present here for the first time the evidence that after 6-OHDA treatment Bergmann glial cells are abnormally located and structurally different. The intimate association between Bergmann glia and Purkinje cell also did not occur after neonatal 6-OHDA treatment. This treatment eliminates noradrenergic terminals from the majority of brain structures among which is the cerebellum (Lauder, 1985). In vitro studies provide the evidence that morphology of astroglial cells is controlled by beta-adrenergic receptors (Shain et al., 1987). Astrocytes in vivo differentiate from the early postnatal epithelial-like form to a stellate cells. In the adult, Bergmann glial cells develop complex membrane structures that are in the intimate contact with synaptic regions in the molecular layer, i.e. the synaptic contacts between Purkinje cells and parallel and climbing fibres (Muller et al., 1996). The lack of Bergmann cell transition after elimination of noradrenergic input in cerebellum indicates that noradrenaline may act as important trophic factor for the development of these cells and their functions. Acknowledgements This study was financially supported by the Medical Research Fund of Tampere University Hospital and International Graduate School of Neuroscience, University of Tampere.
I. Podkletno6a, H. Alho / Journal of Chemical Neuroanatomy 14 (1998) 167–173
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