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Formation of excess sublobules in the cerebellum of hypothyroid rats
Annals of Anatomy 194 (2012) 329–333
Contents lists available at SciVerse ScienceDirect
Annals of Anatomy journal homepage: www.elsevier.de/aanat
Research article
Formation of excess sublobules in the cerebellum of hypothyroid rats Yoshinao Z. Hosaka, Yoshihiko Neki, Miki Hasebe, Aya Shinozaki, Masato Uehara ∗ Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan
a r t i c l e
i n f o
Article history: Received 12 August 2011 Received in revised form 15 December 2011 Accepted 15 December 2011
Keywords: Cerebellum Development Foliation Hypothyroidism Internal granular layer Rats Sublobules
a b s t r a c t Cerebellar folia may increase in number in hypothyroid rats (Lauder et al., 1974; Hasebe et al., 2008a). In this study, we aimed to confirm the formation of an excess sublobule and to determine whether excess sublobules are consistently formed in conserved positions in hypothyroid rats. Instead of the foliation pattern partitioned by cerebellar fissures, we employed the bifurcation pattern of the internal granular layer for investigation of complexity of the cerebellar cortex in hypothyroid rats. The basic foliation pattern of the cerebellum was intact in hypothyroid rats, but lobules III to IX frequently showed an increase in the number of sublobules. The excess sublobules were mainly found in the folia and along the shallow region of the fissure. In other words, the excess sublobules were not located in random locations but rather in specific locations. The area in the internal granular layer of lobules V to IX was significantly larger than that in control rats. From the increased area of the internal granular layer it may be inferred that internal granular cells increase in number than those in normal rats. In our study, regions within the cerebellum that show an excess of sublobules correlate with regions that show an intermediate to late-forming internal granular layer (Altman, 1969). Our observations fit with the view that excess sublobules are formed by the external granular layer showing prolonged cell proliferation and hypothyroidism predominantly has an adverse impact on the intermediate to late phases in development of the internal granular layer. © 2012 Elsevier GmbH. All rights reserved.
1. Introduction The importance of thyroid hormones in the development of the central nervous system has been extensively studied. In brain development, thyroid hormones are essential for maturation of the developing brain including dendritic and axonal growth, synaptogenesis, neuronal migration and myelination. Furthermore, thyroid hormones control the differentiation of neurons such as pyramidal cells of the neocortex and the hippocampus and Purkinje cells of the cerebellum (see reviews: Chan and Kilby, 2000; Koibuchi and Chin, 2000; Thompson and Potter, 2000; Anderson, 2001; Mussa et al., 2001; Zoeller et al., 2002; Bernal, 2005; Santisteban and Bernal, 2005). The development of the cerebellum occurs largely postnatally in the rat and mouse (Mares and Lodin, 1970; Altman, 1972; Corrales et al., 2004). In the developing rat cerebellum, perinatal hypothyroidism (HT) results in reduction of synaptogenesis between Purkinje cells and parallel fibers due to a reduction in branching and growth of dendritic arborization of Purkinje cells and due to a reduction in length of parallel fibers and in a prolonged persistence of the external granular layer (EGL) (Nicholson and Altman, 1972; Clos et al., 1974; Lauder et al., 1974; Lauder, 1978; Legrand, 1979; Oppenheimer and Schwartz, 1997; Xiao and Nikodem,
∗ Corresponding author. Tel.: +81 857 31 5419; fax: +81 857 31 5419. E-mail address: [email protected] (M. Uehara). 0940-9602/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.aanat.2011.12.005
1998; Koibuchi and Chin, 2000; Thompson and Potter, 2000; Anderson, 2001, 2008). Thus, previous studies on HT have largely focused on Purkinje cells and granule cells of the developing rat cerebellum. Although the gross anatomy of the cerebellum varies from that of a single leaf or dome-like structure, as in amphibians and reptiles, to more complicated shapes in fish, birds and mammals, the cerebellum shares a common pattern in the structure of its cortex. The cerebellum is principally composed of 10 lobules in mammals and 10 folia in birds (Larsell, 1967; Voogd and Glickstein, 1998). In the full-term rat fetus, the cerebellum shows five or six principal lobules and six fissures including four principal fissures (preculminate fissure, primary fissure, fissure secunda, and posterolateral fissure) and two shallow indentations in the vermis. In the postnatal periods, these five principal lobules are further subdivided into ten lobules (Larsell, 1952). There are several theories on the mechanisms underlying the cerebellar foliation. Cerebellar foliation is divided into two phases: (1) establishment of five lobules in the prenatal rat that arise independently of granule cell production and (2) granule cell-dependent foliation in the postnatal rat (Doughty et al., 1998). The species-specific foliation pattern observed in the mature cerebellum suggests that patterning of cerebellar lobules is genetically regulated. In fact, several genes are candidates for pattern formation in the cerebellar foliation (Millen et al., 1994; Chizhikov and Millen, 2003; Lewis et al., 2004; Corrales et al., 2004, 2006; Cheng et al., 2010).
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Fig. 1. Midsagittal section of the vermis in control (a) and HT rats (b). The HT rat cerebellum shows a normal foliation pattern, but some lobules have excess sublobules (stars) or are elongated (arrows). I–X: lobules I to X. Thionine staining. Scale bar: 1 mm.
Foliation of the rat cerebellum (Larsell, 1952; Lauder et al., 1974; Doughty et al., 1998) and the effect of HT on cerebellar development (Schwartz et al., 1997) occur largely in postnatal life. However, there have been few studies on cerebellar foliation under the condition of HT (Lauder et al., 1974; Hasebe et al., 2008a). Lauder et al. (1974) reported that the cerebellum of the hypothyroid rat has an increased number of abnormally shallow fissures. However, their observations, which have been neglected for a long time, do not show the location of increased fissures. Hasebe et al. (2008a) observed excess sublobules of the cerebellar cortex in hypothyroid rat pups induced by methimazole administration to dams. In this study, we aimed to confirm the formation of excess sublobules and to determine whether excess sublobules are consistently formed in specific positions in the hypothyroid rat. Cerebellar lobules and sublobules, which are separated by fissure, certainly have the internal granular layer (IGL) as a core. However, a small protrusion of the IGL does not appear as a sublobule. In this study, we decided to study the bifurcation pattern of the IGL for assessment of the complexity of the cerebellar cortex instead of fissures. 2. Materials and method 2.1. Animals Mated female rats of Crl (CD) SD strain, 11 weeks old, were delivered from Charles River Inc. (Yokohama, Japan) on the 7th gestational day. All rats were kept in controlled dark-light cycles (light on: 7 a.m. to 7 p.m.) and temperature (22 ◦ C). All animal studies were conducted in accordance with principles and procedures approved by Banyu Institutional Animal Care and Use Committee, Japan. 2.2. Experimental HT Maternal animals were rendered hypothyroid by administration of 20 mg/kg/day methimazole (2-mercapto-1-methylimidazole, Sigma–Aldrich Japan, Tokyo, Japan) in drinking water starting on the 17th gestational day. The maternal animals were allowed to deliver pups naturally. The administration of methimazole was continued even during lactation. The pups remained with their
mothers throughout the treatment period. The fetuses and pups had access to the drug by placental transfer, maternal milk and drinking water. Mothers in the control group drank distilled water. In this study, we did not determine the blood concentrations of thyroid hormones. In our previous study, plasma triiodothyronin (T3) and thyroxin (T4) concentrations in hypothyroid dams and their pups of 15 postnatal days were less than the lower limit of quantization (Hasebe et al., 2008a). 2.3. Three-dimensional (3-D) morphology of cerebella Only males at postnatal day 30, when the EGL virtually disappeared in hypothyroid rats, were used in this study. Ten hypothyroid and two normal pups were sacrificed by CO2 and their brains were removed. Each brain was immersed in 10% neutral formalin. After dehydration in graded ethanol series, the cerebella were embedded in celloidin. Sections were serially cut at 80 m in thickness in sagittal planes and stained with thionine. Images of the IGL were cut out of scanned images of the unilateral cerebellum using Adobe Photoshop Limited 5.0 (Adobe Systems, Tokyo, Japan). The 3-D reconstructions were performed using DeltaViewer 2.1.1 (3-D image reconstruction software), a freely distributed personal computer-based program (http://vivaldi.ics.nara-wu.ac.jp/ wada/DeltaViewer/indexj.html). 2.4. Areas of the IGL of midline sagittal regions of the vermis Areal measurements of the IGL in midsagittal sections of the vermis were made for each rat using Image J, a free, open-source image-processing platform and supported by the National Institutes of Health (Bethesda, MD, USA). Student’s t-test was then performed for test of significance. In all cases, p < 0.05 was taken as statistically significant. 3. Results 3.1. Morphology of thionine-stained sections and 3-D images Lobules III to IX frequently had excess sublobules. Lobule III in 8 of 10 cases (8/10) and lobule V (8/10) in the vermis were
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Fig. 2. Apical parts of simple lobule (SIM), crus 2 of the ansiform lobule (C2) and paramedian lobule (PM) of the control (a) were bifurcated in HT rats (b). Stars show the bifurcated parts. C2: crus 2 of the ansiform lobule, PM: paramedian lobule, SIM: simple lobule, scale bar: 1 mm.
bifurcated in the apical parts (Figs. 1 and 3a and b). In lobule IV (6/10), an excess sublobule appeared in its intermediate portion (Figs. 1 and 3a and b). In lobule VI, a small sublobule was more prominent in hypothyroid rats (9/10; Fig. 1). The simple lobule and the crus 2 of the ansiform lobule (the hemispheral expansions of lobule VI) were bifurcated (7/10; Figs. 2 and 3e and f). An excess sublobule (5/10) was found in the base of crus 2 (Fig. 3e and f). Lobule VII and the paramedian lobe (the hemispheral region lateral to lobule VII) were also bifurcated (7/10; Figs. 1–3e and f). Lobule VIII and the copula pyramidis (the hemispheral region lateral to lobule VIII) were bifurcated (Figs. 1 and 3e and f). In lobule IX, sublobules IXa and b (10/10) and IXc (3/10) were elongated (Fig. 1). Sublobule IXb showed a shallow furrow in the apical surface (5/10). Although lobules in the HT cerebellum showed an irregular contour in sections, relatively smooth ridges or furrows in 3-D images covered the surface of the lobules because an excess bulge of the IGL in section formed a smooth ridge in 3-D configuration. 3.2. Areas of the IGL in mid-sagittal sections of the vermis Areas of the IGL were not significantly different from the controls in lobules I to IV and X but were significantly increased in lobules V to IX compared to those in control animals (Fig. 4). 4. Discussion There are many reports on cerebellar development in HT animals, but little attention has been paid to intra- and interlobular differences. The EGL of normal rats increases in area until about 10 postnatal days and then declines and finally disappears by 21 days (Altman, 1969; Nicholson and Altman, 1972; Lewis et al., 1976). The EGL of HT rats show a prolonged proliferation period and only disappear by 30 postnatal days (Hasebe et al., 2008a). Furthermore, mitotic activity in the EGL reaches a peak during the second week of life and then rapidly decreases in normal rats. Meanwhile, in HT rats, it is depressed below the control level at 12 postnatal days but is about four-times higher than that in the controls at 21 postnatal days (Patel et al., 1976). As a result, the number of internal granule
cells (IGC) in the HT cerebellum increases compared to that in the controls (Nicholson and Altman, 1972). Lauder et al. (1974) reported that HT rats have an increased number of abnormally shallow fissures in the cerebellum. They suggest that HT, which prolongs expansion of the EGL and retards cortical growth, as well as causing a retarded maximization of the ratio of cortical to subcortical area, also prolongs the foliation process, leading ultimately to the formation of a significantly greater number of fissures. However, they did not mention the location of the increased number of fissures. Their observations had been neglected until our previous study (Hasebe et al., 2008a). In this study, the IGL in the HT rat cerebellum increased in dimension in lobules V to IX and showed an intricate bifurcation pattern in lobules III to IX. Each inbred strain of the mouse exhibits a characteristic foliation pattern of the cerebellum, which is slight in individual difference (Inouye and Oda, 1980). This strain-specific variation is under genetic control (Cooper et al., 1991; Tanaka and Marunouchi, 2005). As far as I know, there is no report on the variation in the foliation pattern of each inbred strain in the rat. It is suggested, however, that the cerebellar foliation pattern in the rat derived from each inbred strain is also slight in individual difference. Excess sublobules in the HT rat cerebellum in this study exceed that of normal rats in consistency, number and position. Thus, the present study demonstrated that excess sublobules in the rat cerebellum are formed in specific regions by HT. There are intra- and interlobular differences in formation of the IGL in postnatal rats (Altman, 1969) and mice (Yamasaki et al., 2001). Altman (1969) divided the IGL into three groups, early-, intermediate-and late-forming groups, by the period of main occurrence of IGC. Late-forming regions consist of lobules VIb, VII and VIII, intermediate regions consist of lobules IV, V and IX and part of lobules II, III and VIa, and early-forming regions consist of other regions including lobules I and X and most regions facing the sulci. The lobules with excess sublobules in the HT cerebellum correlate directly with the intermediate-and late-forming regions. This corresponds to the irregular IGL with a complex branching pattern that appears after 15 postnatal days in the HT rat cerebellum (Hasebe et al., 2008a). It has been shown that the fetal brain in the rat is unresponsive to thyroid hormone (Schwartz et al., 1997) and that
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Fig. 3. Reconstructed 3-D images of each lobule of controls (a, c, and e) and HT rats (b, d, and f). 3-D images of lobule III (a and b), showing the bifurcated apical part of lobule III (star) in the HT rat. 3-D images of lobule IV and V (c and d), showing an excess sublobule in lobule IV (black arrow) and bifurcation of lobule V (white arrow). 3-D images of the crus 2 (C2) and the paramedian lobule (PM), showing bifurcated apical parts in both lobules and an excess sublobule in the basal part of C2 (arrow).
Fig. 4. Ratio of the area of IGL in midsagittal plane of the vermis between HT and normal rats. The IGLs of lobules V to IX in HT rats are significantly larger than those of normal rats. The data are expressed as mean ± S.D. Black bars: p < 0.05.
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effect of HT on the developing cerebellum appears in the postnatal period. In postnatal development of the IGL, an adverse effect of HT appears predominantly in intermediate to late phases. In foliation of the rat cerebellum, there are two phases of cerebellar folding: (1) establishment of five lobules that arise independently of granule cell production during prenatal development and (2) granule cell-dependent expansion and partitioning of these five principal lobules during postnatal development (Doughty et al., 1998). In this study, the basic foliation pattern was normal and the only addition of sublobules appeared in the HT rat cerebellum. The IGL area was significantly larger in lobules V–IX compare to controls, suggesting an increase in the number of IGC in the HT rat cerebellum. Nicholson and Altman (1972) also observed that the EGL in HT rats shows prolonged cell proliferation and retarded disappearance and terminal increase in granule cell number. Thus, the present study suggests that an excess sublobule could be formed by granule cell-dependent expansion and partitioning of the principal lobules. Purkinje cell-derived sonic hedgehog (shh) normally promotes the proliferation of external granule cells (Wallace, 1999; Wechsler-Reya and Scott, 1999). Overexpressing shh in the mouse cerebellum results in an increase in the overall size of the cerebellum with a thicker and irregular IGL, but the basic foliation pattern is intact. Depletion of the EGL is delayed by overexpression of shh. Morphological differences induced by overexpressing shh are detected after 8 postnatal days (Corrales et al., 2004). An increase in the level and duration of shh signaling leads in formation of an extra fissure in specific positions (Corrales et al., 2006). The cerebellum produced by increased shh signaling is similar to the HT rat cerebellum in appearance of an excess sublobule in specific positions with conservation of the basic foliation pattern and delayed disappearance of the EGL. Another similarity is that morphological differences appear in the intermediate period of postnatal cerebellar development. However, the HT cerebellum does not show an increase in overall size (Hasebe et al., 2008a). In addition, shh expression in HT rats decreases (Hasebe et al., 2008b). Whatever the cause, the cerebellar cortex may show an increase in complexity or number of sublobules by prolonged continuation of the EGL and relatively excessive formation of IGC to the volume of cerebellar medulla. References Anderson, G.W., 2001. Thyroid hormones and the brain. Front. Neuroendocrinol. 22, 1–17. Anderson, G.W., 2008. Thyroid hormone and cerebellar development. Cerebellum 7, 60–74. Altman, J., 1969. Autoradiographic and histological studies of postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rats. J. Comp. Neurol. 136, 269–294. Altman, J., 1972. Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Comp. Neurol. 145, 353–398. Bernal, J., 2005. Thyroid hormones and brain development. Vitam. Horm. 71, 95–122. Chan, S., Kilby, M., 2000. Thyroid hormone and central nervous system development. J. Endocrinol. 165, 1–8. Cheng, Y., Sudarov, A., Szulc, K.U., Sgaier, S.K., Stephen, D., Turnbull, D.H., Joyner, A.L., 2010. The Engrailed homeobox genes determine the different foliation patterns in the vermis and hemispheres of the mammalian cerebellum. Development 137, 519–529. Chizhikov, V., Millen, K.J., 2003. Development and malformations of the cerebellum in mice. Mol. Genet. Metab. 80, 54–65. Clos, J., Crepel, F., Legrand, J., Rabie, A., Vigouroux, E., 1974. Thyroid physiology during the postnatal period in the rat: a study of the development of thyroid function
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Millen, K.J., Wurst, W., Herrup, K., Joyner, A.K., 1994. Abnormal embryonic cerebellar development and patterning of postnatal foliation in two mouse Engrailed-2 mutants. Development 120, 695–706. Mussa, G.C., Mussa, F., Bretto, R., Zambelli, M.C., Silvestro, L., 2001. Influence of thyroid in nervous system growth. Minerva Pediatr. 53, 325–353. Nicholson, J.L., Altman, J., 1972. The effects of early hypo-and hyperthyroidism on development of rat cerebellar cortex. I. Cell proliferation and differentiation. Brain Res. 44, 12–23. Oppenheimer, J.H., Schwartz, H.L., 1997. Molecular basis of thyroid hormonedependent brain development. Endocr. Rev. 18, 462–475. Patel, A.J., Rabie, A., Lewis, P.D., Balazs, R., 1976. Effect of thyroid deficiency on postnatal cell formation in the rat brain: a biochemical investigation. Brain Res. 104, 33–48. Santisteban, P., Bernal, J., 2005. Thyroid development and effect on the nervous system. Rev. Endocr. Metab. Disord. 6, 217–228. 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Contents lists available at SciVerse ScienceDirect
Annals of Anatomy journal homepage: www.elsevier.de/aanat
Research article
Formation of excess sublobules in the cerebellum of hypothyroid rats Yoshinao Z. Hosaka, Yoshihiko Neki, Miki Hasebe, Aya Shinozaki, Masato Uehara ∗ Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan
a r t i c l e
i n f o
Article history: Received 12 August 2011 Received in revised form 15 December 2011 Accepted 15 December 2011
Keywords: Cerebellum Development Foliation Hypothyroidism Internal granular layer Rats Sublobules
a b s t r a c t Cerebellar folia may increase in number in hypothyroid rats (Lauder et al., 1974; Hasebe et al., 2008a). In this study, we aimed to confirm the formation of an excess sublobule and to determine whether excess sublobules are consistently formed in conserved positions in hypothyroid rats. Instead of the foliation pattern partitioned by cerebellar fissures, we employed the bifurcation pattern of the internal granular layer for investigation of complexity of the cerebellar cortex in hypothyroid rats. The basic foliation pattern of the cerebellum was intact in hypothyroid rats, but lobules III to IX frequently showed an increase in the number of sublobules. The excess sublobules were mainly found in the folia and along the shallow region of the fissure. In other words, the excess sublobules were not located in random locations but rather in specific locations. The area in the internal granular layer of lobules V to IX was significantly larger than that in control rats. From the increased area of the internal granular layer it may be inferred that internal granular cells increase in number than those in normal rats. In our study, regions within the cerebellum that show an excess of sublobules correlate with regions that show an intermediate to late-forming internal granular layer (Altman, 1969). Our observations fit with the view that excess sublobules are formed by the external granular layer showing prolonged cell proliferation and hypothyroidism predominantly has an adverse impact on the intermediate to late phases in development of the internal granular layer. © 2012 Elsevier GmbH. All rights reserved.
1. Introduction The importance of thyroid hormones in the development of the central nervous system has been extensively studied. In brain development, thyroid hormones are essential for maturation of the developing brain including dendritic and axonal growth, synaptogenesis, neuronal migration and myelination. Furthermore, thyroid hormones control the differentiation of neurons such as pyramidal cells of the neocortex and the hippocampus and Purkinje cells of the cerebellum (see reviews: Chan and Kilby, 2000; Koibuchi and Chin, 2000; Thompson and Potter, 2000; Anderson, 2001; Mussa et al., 2001; Zoeller et al., 2002; Bernal, 2005; Santisteban and Bernal, 2005). The development of the cerebellum occurs largely postnatally in the rat and mouse (Mares and Lodin, 1970; Altman, 1972; Corrales et al., 2004). In the developing rat cerebellum, perinatal hypothyroidism (HT) results in reduction of synaptogenesis between Purkinje cells and parallel fibers due to a reduction in branching and growth of dendritic arborization of Purkinje cells and due to a reduction in length of parallel fibers and in a prolonged persistence of the external granular layer (EGL) (Nicholson and Altman, 1972; Clos et al., 1974; Lauder et al., 1974; Lauder, 1978; Legrand, 1979; Oppenheimer and Schwartz, 1997; Xiao and Nikodem,
∗ Corresponding author. Tel.: +81 857 31 5419; fax: +81 857 31 5419. E-mail address: [email protected] (M. Uehara). 0940-9602/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.aanat.2011.12.005
1998; Koibuchi and Chin, 2000; Thompson and Potter, 2000; Anderson, 2001, 2008). Thus, previous studies on HT have largely focused on Purkinje cells and granule cells of the developing rat cerebellum. Although the gross anatomy of the cerebellum varies from that of a single leaf or dome-like structure, as in amphibians and reptiles, to more complicated shapes in fish, birds and mammals, the cerebellum shares a common pattern in the structure of its cortex. The cerebellum is principally composed of 10 lobules in mammals and 10 folia in birds (Larsell, 1967; Voogd and Glickstein, 1998). In the full-term rat fetus, the cerebellum shows five or six principal lobules and six fissures including four principal fissures (preculminate fissure, primary fissure, fissure secunda, and posterolateral fissure) and two shallow indentations in the vermis. In the postnatal periods, these five principal lobules are further subdivided into ten lobules (Larsell, 1952). There are several theories on the mechanisms underlying the cerebellar foliation. Cerebellar foliation is divided into two phases: (1) establishment of five lobules in the prenatal rat that arise independently of granule cell production and (2) granule cell-dependent foliation in the postnatal rat (Doughty et al., 1998). The species-specific foliation pattern observed in the mature cerebellum suggests that patterning of cerebellar lobules is genetically regulated. In fact, several genes are candidates for pattern formation in the cerebellar foliation (Millen et al., 1994; Chizhikov and Millen, 2003; Lewis et al., 2004; Corrales et al., 2004, 2006; Cheng et al., 2010).
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Fig. 1. Midsagittal section of the vermis in control (a) and HT rats (b). The HT rat cerebellum shows a normal foliation pattern, but some lobules have excess sublobules (stars) or are elongated (arrows). I–X: lobules I to X. Thionine staining. Scale bar: 1 mm.
Foliation of the rat cerebellum (Larsell, 1952; Lauder et al., 1974; Doughty et al., 1998) and the effect of HT on cerebellar development (Schwartz et al., 1997) occur largely in postnatal life. However, there have been few studies on cerebellar foliation under the condition of HT (Lauder et al., 1974; Hasebe et al., 2008a). Lauder et al. (1974) reported that the cerebellum of the hypothyroid rat has an increased number of abnormally shallow fissures. However, their observations, which have been neglected for a long time, do not show the location of increased fissures. Hasebe et al. (2008a) observed excess sublobules of the cerebellar cortex in hypothyroid rat pups induced by methimazole administration to dams. In this study, we aimed to confirm the formation of excess sublobules and to determine whether excess sublobules are consistently formed in specific positions in the hypothyroid rat. Cerebellar lobules and sublobules, which are separated by fissure, certainly have the internal granular layer (IGL) as a core. However, a small protrusion of the IGL does not appear as a sublobule. In this study, we decided to study the bifurcation pattern of the IGL for assessment of the complexity of the cerebellar cortex instead of fissures. 2. Materials and method 2.1. Animals Mated female rats of Crl (CD) SD strain, 11 weeks old, were delivered from Charles River Inc. (Yokohama, Japan) on the 7th gestational day. All rats were kept in controlled dark-light cycles (light on: 7 a.m. to 7 p.m.) and temperature (22 ◦ C). All animal studies were conducted in accordance with principles and procedures approved by Banyu Institutional Animal Care and Use Committee, Japan. 2.2. Experimental HT Maternal animals were rendered hypothyroid by administration of 20 mg/kg/day methimazole (2-mercapto-1-methylimidazole, Sigma–Aldrich Japan, Tokyo, Japan) in drinking water starting on the 17th gestational day. The maternal animals were allowed to deliver pups naturally. The administration of methimazole was continued even during lactation. The pups remained with their
mothers throughout the treatment period. The fetuses and pups had access to the drug by placental transfer, maternal milk and drinking water. Mothers in the control group drank distilled water. In this study, we did not determine the blood concentrations of thyroid hormones. In our previous study, plasma triiodothyronin (T3) and thyroxin (T4) concentrations in hypothyroid dams and their pups of 15 postnatal days were less than the lower limit of quantization (Hasebe et al., 2008a). 2.3. Three-dimensional (3-D) morphology of cerebella Only males at postnatal day 30, when the EGL virtually disappeared in hypothyroid rats, were used in this study. Ten hypothyroid and two normal pups were sacrificed by CO2 and their brains were removed. Each brain was immersed in 10% neutral formalin. After dehydration in graded ethanol series, the cerebella were embedded in celloidin. Sections were serially cut at 80 m in thickness in sagittal planes and stained with thionine. Images of the IGL were cut out of scanned images of the unilateral cerebellum using Adobe Photoshop Limited 5.0 (Adobe Systems, Tokyo, Japan). The 3-D reconstructions were performed using DeltaViewer 2.1.1 (3-D image reconstruction software), a freely distributed personal computer-based program (http://vivaldi.ics.nara-wu.ac.jp/ wada/DeltaViewer/indexj.html). 2.4. Areas of the IGL of midline sagittal regions of the vermis Areal measurements of the IGL in midsagittal sections of the vermis were made for each rat using Image J, a free, open-source image-processing platform and supported by the National Institutes of Health (Bethesda, MD, USA). Student’s t-test was then performed for test of significance. In all cases, p < 0.05 was taken as statistically significant. 3. Results 3.1. Morphology of thionine-stained sections and 3-D images Lobules III to IX frequently had excess sublobules. Lobule III in 8 of 10 cases (8/10) and lobule V (8/10) in the vermis were
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Fig. 2. Apical parts of simple lobule (SIM), crus 2 of the ansiform lobule (C2) and paramedian lobule (PM) of the control (a) were bifurcated in HT rats (b). Stars show the bifurcated parts. C2: crus 2 of the ansiform lobule, PM: paramedian lobule, SIM: simple lobule, scale bar: 1 mm.
bifurcated in the apical parts (Figs. 1 and 3a and b). In lobule IV (6/10), an excess sublobule appeared in its intermediate portion (Figs. 1 and 3a and b). In lobule VI, a small sublobule was more prominent in hypothyroid rats (9/10; Fig. 1). The simple lobule and the crus 2 of the ansiform lobule (the hemispheral expansions of lobule VI) were bifurcated (7/10; Figs. 2 and 3e and f). An excess sublobule (5/10) was found in the base of crus 2 (Fig. 3e and f). Lobule VII and the paramedian lobe (the hemispheral region lateral to lobule VII) were also bifurcated (7/10; Figs. 1–3e and f). Lobule VIII and the copula pyramidis (the hemispheral region lateral to lobule VIII) were bifurcated (Figs. 1 and 3e and f). In lobule IX, sublobules IXa and b (10/10) and IXc (3/10) were elongated (Fig. 1). Sublobule IXb showed a shallow furrow in the apical surface (5/10). Although lobules in the HT cerebellum showed an irregular contour in sections, relatively smooth ridges or furrows in 3-D images covered the surface of the lobules because an excess bulge of the IGL in section formed a smooth ridge in 3-D configuration. 3.2. Areas of the IGL in mid-sagittal sections of the vermis Areas of the IGL were not significantly different from the controls in lobules I to IV and X but were significantly increased in lobules V to IX compared to those in control animals (Fig. 4). 4. Discussion There are many reports on cerebellar development in HT animals, but little attention has been paid to intra- and interlobular differences. The EGL of normal rats increases in area until about 10 postnatal days and then declines and finally disappears by 21 days (Altman, 1969; Nicholson and Altman, 1972; Lewis et al., 1976). The EGL of HT rats show a prolonged proliferation period and only disappear by 30 postnatal days (Hasebe et al., 2008a). Furthermore, mitotic activity in the EGL reaches a peak during the second week of life and then rapidly decreases in normal rats. Meanwhile, in HT rats, it is depressed below the control level at 12 postnatal days but is about four-times higher than that in the controls at 21 postnatal days (Patel et al., 1976). As a result, the number of internal granule
cells (IGC) in the HT cerebellum increases compared to that in the controls (Nicholson and Altman, 1972). Lauder et al. (1974) reported that HT rats have an increased number of abnormally shallow fissures in the cerebellum. They suggest that HT, which prolongs expansion of the EGL and retards cortical growth, as well as causing a retarded maximization of the ratio of cortical to subcortical area, also prolongs the foliation process, leading ultimately to the formation of a significantly greater number of fissures. However, they did not mention the location of the increased number of fissures. Their observations had been neglected until our previous study (Hasebe et al., 2008a). In this study, the IGL in the HT rat cerebellum increased in dimension in lobules V to IX and showed an intricate bifurcation pattern in lobules III to IX. Each inbred strain of the mouse exhibits a characteristic foliation pattern of the cerebellum, which is slight in individual difference (Inouye and Oda, 1980). This strain-specific variation is under genetic control (Cooper et al., 1991; Tanaka and Marunouchi, 2005). As far as I know, there is no report on the variation in the foliation pattern of each inbred strain in the rat. It is suggested, however, that the cerebellar foliation pattern in the rat derived from each inbred strain is also slight in individual difference. Excess sublobules in the HT rat cerebellum in this study exceed that of normal rats in consistency, number and position. Thus, the present study demonstrated that excess sublobules in the rat cerebellum are formed in specific regions by HT. There are intra- and interlobular differences in formation of the IGL in postnatal rats (Altman, 1969) and mice (Yamasaki et al., 2001). Altman (1969) divided the IGL into three groups, early-, intermediate-and late-forming groups, by the period of main occurrence of IGC. Late-forming regions consist of lobules VIb, VII and VIII, intermediate regions consist of lobules IV, V and IX and part of lobules II, III and VIa, and early-forming regions consist of other regions including lobules I and X and most regions facing the sulci. The lobules with excess sublobules in the HT cerebellum correlate directly with the intermediate-and late-forming regions. This corresponds to the irregular IGL with a complex branching pattern that appears after 15 postnatal days in the HT rat cerebellum (Hasebe et al., 2008a). It has been shown that the fetal brain in the rat is unresponsive to thyroid hormone (Schwartz et al., 1997) and that
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Fig. 3. Reconstructed 3-D images of each lobule of controls (a, c, and e) and HT rats (b, d, and f). 3-D images of lobule III (a and b), showing the bifurcated apical part of lobule III (star) in the HT rat. 3-D images of lobule IV and V (c and d), showing an excess sublobule in lobule IV (black arrow) and bifurcation of lobule V (white arrow). 3-D images of the crus 2 (C2) and the paramedian lobule (PM), showing bifurcated apical parts in both lobules and an excess sublobule in the basal part of C2 (arrow).
Fig. 4. Ratio of the area of IGL in midsagittal plane of the vermis between HT and normal rats. The IGLs of lobules V to IX in HT rats are significantly larger than those of normal rats. The data are expressed as mean ± S.D. Black bars: p < 0.05.
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effect of HT on the developing cerebellum appears in the postnatal period. In postnatal development of the IGL, an adverse effect of HT appears predominantly in intermediate to late phases. In foliation of the rat cerebellum, there are two phases of cerebellar folding: (1) establishment of five lobules that arise independently of granule cell production during prenatal development and (2) granule cell-dependent expansion and partitioning of these five principal lobules during postnatal development (Doughty et al., 1998). In this study, the basic foliation pattern was normal and the only addition of sublobules appeared in the HT rat cerebellum. The IGL area was significantly larger in lobules V–IX compare to controls, suggesting an increase in the number of IGC in the HT rat cerebellum. Nicholson and Altman (1972) also observed that the EGL in HT rats shows prolonged cell proliferation and retarded disappearance and terminal increase in granule cell number. Thus, the present study suggests that an excess sublobule could be formed by granule cell-dependent expansion and partitioning of the principal lobules. Purkinje cell-derived sonic hedgehog (shh) normally promotes the proliferation of external granule cells (Wallace, 1999; Wechsler-Reya and Scott, 1999). Overexpressing shh in the mouse cerebellum results in an increase in the overall size of the cerebellum with a thicker and irregular IGL, but the basic foliation pattern is intact. Depletion of the EGL is delayed by overexpression of shh. Morphological differences induced by overexpressing shh are detected after 8 postnatal days (Corrales et al., 2004). An increase in the level and duration of shh signaling leads in formation of an extra fissure in specific positions (Corrales et al., 2006). The cerebellum produced by increased shh signaling is similar to the HT rat cerebellum in appearance of an excess sublobule in specific positions with conservation of the basic foliation pattern and delayed disappearance of the EGL. Another similarity is that morphological differences appear in the intermediate period of postnatal cerebellar development. However, the HT cerebellum does not show an increase in overall size (Hasebe et al., 2008a). In addition, shh expression in HT rats decreases (Hasebe et al., 2008b). Whatever the cause, the cerebellar cortex may show an increase in complexity or number of sublobules by prolonged continuation of the EGL and relatively excessive formation of IGC to the volume of cerebellar medulla. References Anderson, G.W., 2001. Thyroid hormones and the brain. Front. Neuroendocrinol. 22, 1–17. Anderson, G.W., 2008. Thyroid hormone and cerebellar development. Cerebellum 7, 60–74. Altman, J., 1969. Autoradiographic and histological studies of postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rats. J. Comp. Neurol. 136, 269–294. Altman, J., 1972. Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Comp. Neurol. 145, 353–398. Bernal, J., 2005. Thyroid hormones and brain development. Vitam. Horm. 71, 95–122. Chan, S., Kilby, M., 2000. Thyroid hormone and central nervous system development. J. Endocrinol. 165, 1–8. Cheng, Y., Sudarov, A., Szulc, K.U., Sgaier, S.K., Stephen, D., Turnbull, D.H., Joyner, A.L., 2010. The Engrailed homeobox genes determine the different foliation patterns in the vermis and hemispheres of the mammalian cerebellum. Development 137, 519–529. Chizhikov, V., Millen, K.J., 2003. Development and malformations of the cerebellum in mice. Mol. Genet. Metab. 80, 54–65. Clos, J., Crepel, F., Legrand, J., Rabie, A., Vigouroux, E., 1974. Thyroid physiology during the postnatal period in the rat: a study of the development of thyroid function
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