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The temporal and spatial development of CRF binding sites in the postnatal mouse cerebellum
Neuroscience Research 34 (1999) 45 – 50
Rapid communication
The temporal and spatial development of CRF binding sites in the postnatal mouse cerebellum Paul Madtes Jr. b, James S. King a,* a
Department of Cell Biology, Neurobiology and Anatomy, Di6ision of Neuroscience, The Ohio State Uni6ersity, 333 W. 10th A6enue, Columbus, OH 43210, USA b Biology Department, Mount Vernon Nazarene College, Columbus, OH, USA Received 30 November 1998; accepted 24 February 1999
Abstract This study describes the distribution and relative level of labeling of binding sites for corticotrophin releasing factor (CRF) in the postnatal mouse cerebellum. At birth low levels of labeling are present throughout the cerebellum. However, this labeling is most densely distributed in the caudal and lateral aspects of the cerebellum. By P3 CRF binding sites are present throughout the cerebellum, although the greatest level of labeling is in the posterior lobe of the vermis, especially lobules IX and X; this correlates with the early differential pattern of CRF distribution in cerebellar afferents within these same lobules. At P10, the adult pattern of distribution and level of labeling begins to emerge. The presence of CRF and CRF binding sites at birth, and during postnatal growth, suggests that this peptide could play a role in the regulation of developmental events within the cerebellum. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Purkinje cell; Granule cell; CRF binding; Development
1. Introduction Corticotropin releasing factor (CRF) is present in climbing fibers and mossy fibers in the adult mouse cerebellum (King et al., 1997). It has been shown in other species to modulate the responsiveness of Purkinje cells (Bishop, 1990; Fox and Gruol, 1993). In the mouse, CRF is present at birth in punctate profiles that have a uniform distribution throughout the medial to lateral extent of the cerebellum (Overbeck and King, 1999). The early presence of CRF is prior to the onset of dendritic outgrowth and the major phase of synaptogenesis in the mouse cerebellum (Hendelman and Aggerwal, 1980; Mason et al., 1990). Between P0 and P60, CRF-immunoreactive (IR) afferents go through a series * Corresponding author. Tel.: +1-614-292-5475; fax: + 1-614-2927659.
of developmental stages until the adult phenotype of climbing and mossy fibers, as well as the adult pattern of distribution of the two afferent fibers, is achieved (Yamano and Tohyama, 1994; Overbeck and King, 1999). A recent study has shown that peptides such as CRF may play a role in the regulation of neural development (Keegan et al., 1994). However, the presence of CRF alone is not sufficient to establish a functional role during development. If CRF influences the development of cerebellar circuits, then the appropriate sites to which the peptide can bind also must be present. This paper analyzes the temporal and spatial distribution of CRF binding sites in the postnatal cerebellum, as well as the relative level of labeling at different ages. These data on the developmental expression of CRF binding sites are correlated with the immunohistochemical localization and distribution of CRF previously described
0168-0102/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 9 9 ) 0 0 0 2 8 - 0
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P. Madtes, Jr, J.S. King / Neuroscience Research 34 (1999) 45–50
in the postnatal cerebellum of the same species (Yamano and Tohyama, 1994; Overbeck and King, 1999). Litters from C57BL/6J breeding pairs were collected at postnatal ages 0, 3, 5, 7, 10, and 14 for these experiments. These time points were selected in order to correlate the findings with a previous study on the ontogeny of CRF (Overbeck and King, 1999) and with critical events in cerebellar development (Larramendi, 1969; Hendelman and Aggerwal, 1980; Mason et al., 1990). A minimum of three animals was used at each age. Animals were decapitated and their brains were quickly frozen on dry ice. 12-mm-frozen sections were cut on a cryostat (MICROM) in either the sagittal or transverse planes of section, thaw-mounted on gelatinized slides, and stored at −70°C until processed for ligand binding. Sections were lightly stained for Nissl substance and processed for autoradiography as described previously (DeSouza et al., 1985; DeSouza and Kuhar, 1986; Insel, 1990; King et al., 1997). The time course of the appearance of binding sites and their laminar distribution was analyzed on both X-ray film and emulsion-coated coverslips with a light microscope using dark field optics; however, only the data from X-ray film are illustrated in representative photomicrographs. The tissue sections illustrated were exposed for 9 days. Emulsion-coated coverslips (data not shown) were used to determine the specific laminar distribution of the labeling and showed patterns like those found with X-ray film. Since the entire cerebellum from each animal was assayed using ligand binding, comparison of the variation in the level of labeling from section to section and from animal to animal was possible. To demonstrate that there was little variation in non-specific binding either from section to section or from animal to animal, representative examples are illustrated for selected ages (Fig. 1B and Fig. 2B). The level of non-specific binding is barely visible and is uniformly distributed across the entire cerebellum. Thus, the labeling present in the total binding photomicrographs reflects actual binding and is not attributed to variation due to tissue preparation or assay conditions. At P0, CRF binding sites are not uniformly distributed in the cerebellum. Labeling is most dense throughout the posterior cerebellum (Fig. 1A). Anteriorly, silver grains are most densely distributed in the lateral portion of the cerebellum whereas anteromedially, labeling appears to be just above background (Fig. 1C). At P3, when all cerebellar lobules are well defined (Fig. 1D), binding sites are present throughout the cortex; however, labeling is not evident over the cerebellar nuclei (Fig. 1D). Labeling is most dense over lobules IX and X (Fig. 1D). Lobules I – III also show higher levels of label when compared to lobules IV– VIII. This differential rostral – caudal distribution pattern also is evident in lateral regions of the cerebellum (not illustrated). At P5 (Fig. 2A) and P7 (Fig. 2C and
D), CRF binding sites are localized over all the neuronal layers throughout the medial to lateral extent of the cerebellum (Fig. 2A, C and D). However, as at P3, the labeling is densest over lobules IX and X medially at P7(Fig. 2C). Laterally, the flocculus, as well as the apex of the paramedian lobule and lobus simplex (Fig. 2D) are intensely labeled. In addition, labeling just above background is evident over the cerebellar nuclei, suggesting that CRF binding sites are present, but the labeling in the cerebellar nuclei is not as dense as in the cortex (Fig. 2D, CN). By P10 there is a differential distribution of CRF binding sites over the histological layers of the cerebellar cortex (Fig. 3A). This is most clearly seen in lobules VIII and IX of the vermis where the labeling is densest in the internal granule cell layer when compared to the molecular layer and the external granule cell layer (Fig. 3A). This pattern is still evident at P14 (Fig. 3B). Also, at P10 the labeling generally is densest at the apex of the individual cerebellar lobules (Fig. 3A). However, there are only minimal differences in the intensity of labeling between the anterior and posterior lobules (Fig. 3A). This is no longer true at P14, where lobules VII–X are more densely labeled, compared to more anterior lobules (Fig. 3B). To date, many studies have described the distribution of CRF binding sites in the adult mammalian cerebellum. Radioligand binding studies show a relatively high density of binding sites within the cerebellum of the adult opossum (Madtes and King, 1995), mouse (King et al., 1997), rat (DeSouza and Kuhar, 1986), rabbit (Chai et al., 1990), non-human primate (Aguilera et al., 1990) and man (Rivier et al., 1987). In the adult mouse (King et al., 1997) CRF binding sites are found throughout all lobules of the cerebellar cortex. Although silver grains are present over all three histological layers in the adult, labeling in the internal granule cell layer (IGL) is slightly denser than that seen in the molecular or Purkinje cell layers. Further, the densest labeling is located at the apex of most cerebellar lobules. This latter pattern of distribution does not begin to emerge until P10 in the mouse. Even at this age, however, the pattern is not identical to that seen in the adult. For example, in the posterior lobe vermis, the labeling at the apex of each lobule is equivalent to that at the base of the lobule. During postnatal development there is both temporal and spatial overlap in the distribution of the peptide CRF and CRF binding sites. At birth, CRF-IR afferents are present as punctate profiles that are abundant and present throughout the cerebellum (Overbeck and King, 1999). However, CRF binding sites while present in low levels throughout the cerebellum show a greater density of distribution in caudal and lateral aspects of the cerebellum. This disparity in the distribution of CRF and CRF binding sites is no longer evident at P3. By P3, both CRF and its binding sites have their
P. Madtes, Jr, J.S. King / Neuroscience Research 34 (1999) 45–50
greatest distribution in vermal lobules I, II, IX, and X. Several studies have shown that Purkinje cells in these lobules mature earlier than those in other regions of the vermis or the hemispheres (Altman, 1969). Paradoxically, Inouye and Murakami (1980) have shown that the Purkinje cells in these lobules are some of the last to be born. Taken together, these data suggest that the time of birth alone does not predict the temporal maturation of these neurons. Thus, other factors must influence the timing of Purkinje cell differentiation. CRF is one potential factor based on its presence and the presence of CRF binding sites in lobules where
47
Purkinje cells mature early. CRF-IR puncta are most extensive in the ventral vermal folia, although they are distributed across all lobules, as are the binding sites between P3 and P10. After P10, the distribution of CRF changes, as does the pattern of labeling for binding sites. In contrast to the punctate pattern of CRF immunoreactivity seen before P10 the phenotypic characteristics of CRF-immunoreactive climbing and mossy fibers become apparent at this age. In addition, it is at this age that the adult laminar and lobular pattern of distribution of CRF binding sites begins to emerge (King et al., 1997). Therefore, if CRF plays a role in
Fig. 1. The distribution of binding sites for CRF is illustrated in film autoradiograms in the postnatal (P0, P3) cerebellum. At P0 (A and C) transverse sections show label in the posterior cerebellum and in the lateral regions (C, arrows) of the anterior cerebellum (CB). By P3 (D) label is evident throughout the vermis (lobules I–X) and the hemispheres (not illustrated). The densest labeling is evident over lobules IX and X at P3. (B) shows the low level of labeling present for non-specific binding. This level did not vary from section to section or from animal to animal. Therefore, only one representative autoradiogram is shown. Calibration bar, 1 mm and applies to all figures.
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P. Madtes, Jr, J.S. King / Neuroscience Research 34 (1999) 45–50
Fig. 2. The distribution of binding sites for CRF is illustrated in film autoradiograms in the postnatal (P5, P7) cerebellum. A transverse section of a P5 cerebellum (A) illustrates the same pattern of distribution as at earlier ages. At P7, silver grains above background levels are present throughout the medial (C) to lateral (D) extent of the cerebellar cortex. The densest labeling is present in lobules IX and X. Labeling just slightly above background is evident over the cerebellar nuclei (D, CN). (B) shows the low level of labeling present for non-specific binding. This level did not vary from section to section or from animal to animal.
development, these data suggest it would do so prior to P10. Recent studies have focused on using (a) direct transgenic models in which the level of either CRF (StenzelPoore et al., 1992, 1994; Keegan et al., 1994; Heinrichs et al., 1996) or CRF receptor (CRFR1) (Smith et al., 1998) has been altered or (b) indirect transgenic models in which glucocorticoid receptors, a system that is known to interact with CRF neurons (Dijkstra et al., 1998), has been altered. To date, the results of these studies have focused on the function of CRF in the hypothalamic–pituitary axis, evaluating the degree of anxiety generated in response to stress. In particular, Smith et al. (1998) found that transgenic mice deficient in CRF-R1 receptors showed a markedly decreased anxiety response, a loss of a hormonal response to stress. In addition, homozygous mice died within 48 h due to a deficiency in corticosteroids required for lung maturation. These findings indicate CRF is essential for normal development and normal adult function. How-
ever, the impact on cerebellar development has not been analyzed in these transgenic animal models. In summary, the present data clearly indicate that the necessary binding sites essential for CRF interactions during development are present prior to and during critical developmental events (e.g. cell migration, cell survival, synaptogenesis, dendrogenesis) and lend further support to the concept that CRF may play an important role in regulating cerebellar development. Future studies will more precisely define the specific types of CRF receptors present, along with their cellular distribution during development. These data will facilitate the determination of the proposed role for CRF in cerebellar ontogeny.
Acknowledgements This work was supported by NS-08798. Dr Madtes was supported by a Research Opportunity Award IBN-
P. Madtes, Jr, J.S. King / Neuroscience Research 34 (1999) 45–50
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Fig. 3. The distribution of binding sites for CRF is illustrated in film autoradiograms in the postnatal (P10, P14) cerebellum. CRF binding sites are evident throughout the cerebellar cortex at P10 (A) and P14 (B).
9630867 from NSF. The author wishes to thank G.A. Bishop for reading this manuscript and providing critical discussions of the data. We also wish to thank Barbara Diener-Phelan for technical and photographic assistance.
References Aguilera, G., Flores, M., Carvallo, P., Harwood, J.P., Millan, M., Catt, K.J., 1990. Receptors for corticotropin-releasing factor. In: DeSouza, E.B., Nemeroff, C. (Eds.), Corticotropin Releasing Factor: Basic and Clinical Studies of a Neuropeptide. CRC Press, Boca Raton, FL, pp. 153–174. Altman, J., 1969. Autoradiographic and histological studies of postnatal neurogenesis. 3. Dating the time of production and onset of differentiation of cerebellar microneurons in rats. J. Comp. Neurol. 136, 269–293. Bishop, G.A., 1990. Neuromodulatory effects of corticotropin releasing factor on cerebellar Purkinje cells: an in vivo study in the cat. Neuroscience 39, 251–257. Chai, S.Y., Tarjan, E., McKinley, M.J., Paxinos, G., Mendelsohn, F.A.O., 1990. Corticotropin-releasing factor receptors in the rabbit brain visualized by in vitro autoradiography. Brain Res. 512, 60 – 69. DeSouza, E.B., Kuhar, M.J., 1986. Corticotropin-releasing factor receptors: autoradiographic identification. In: Martin, J.B., Barchas, J. (Eds.), Neuropeptides in Neurologic and Psychiatric Disease. Raven, New York, pp. 179–198. DeSouza, E.B., Insel, T.R., Perrin, M.H., Rivier, J., Vale, W., Kuhar, M.J., 1985. Corticotropin-releasing factor receptors are widely distributed within the rat central nervous system. Brain Res. 5, 3189 – 3203. Dijkstra, I., Tilders, F.J., Aguilera, G., Kiss, A., Rabadan-Diehl, C., Barden, N., Karanth, S., Holsboer, F., Reul, J.M., 1998. Reduced activity of hypothalamic corticotropin-releasing hormone neurons in transgenic mice with impaired glucocorticoid receptor function. J. Neurosci. 18, 3909–3918. Fox, E.A., Gruol, D.L., 1993. Corticotropin-releasing factor suppresses the after hyperpolarization in cerebellar Purkinje neurons. Neurosci. Lett. 149, 103–107.
Heinrichs, S.C., Stenzel-Poore, M.P., Gold, L.H., Battenberg, E., Bloom, F.E., Koob, G.F., Vale, W.W., Pich, E.M., 1996. Learning impairment in transgenic mice with central overexpression of corticotropin-releasing factor. Neuroscience 74, 303–311. Hendelman, W.J., Aggerwal, A.S., 1980. The Purkinje Neuron I. A Golgi Study of its development in mouse and in culture. J. Comp. Neurol. 193, 1063 – 1079. Inouye, M., Murakami, U., 1980. Temporal and spatial patterns of purkinje cell formation in the mouse cerebellum. J. Comp. Neurol. 194, 499 – 503. Insel, T.R., 1990. Brain corticotropin-releasing factor and development. In: DeSouza, E.B., Nemeroff, C. Jr. (Eds.), Corticotropin Releasing Factor: Basic and Clinical Studies of a Neuropeptide. CRC Press, Boca Raton, FL, pp. 91 – 106. Keegan, C., Herman, J., Karolyi, I., O’Shea, K., Camper, S., Seasholtz, A., 1994. Differential expression of corticotropin-releasing hormone in developing mouse embryos and adult brain. Endocrinology 134, 2547 – 2555. King, J.S., Madtes, P.C. Jr., Bishop, G.A., Overbeck, T.L., 1997. The distribution of corticotropin-releasing factor (CRF), CRF binding sites and CRF1 receptor mRNA in the mouse cerebellum. Prog. Brain Res. 114, 55 – 66. Larramendi, L.M.H., 1969. Analysis of synaptogenesis in the cerebellum of the mouse. In: Llinas, R. (Ed.), Neurobiology of Cerebellar Evolution and Development. American Medical Association, Education and Research Foundation, Chicago, IL, pp. 803 – 842. Madtes, P.C., King, J.S., 1995. Distribution of corticotropin-releasing factor (CRF) binding sites in the opossum cerebellum. Neuropeptides 28, 51 – 58. Mason, C.A., Christakos, S., Catalano, S.M., 1990. Early climbing fiber interactions with Purkinje cells in the postnatal mouse cerebellum. J. Comp. Neurol. 297, 77 – 90. Overbeck, T.L., King, J.S., 1999. Developmental expression of corticotropin releasing factor in the postnatal murine cerebellum, Dev. Brain Res. (in press). Rivier, C., Brownstein, M., Spiess, J., Rivier, J., 1987. Corticotropin releasing factor as a transmitter in the human olivocerebellar pathway. Brain Res. 415, 347 – 352. Smith, G.W., Aubry, J.M., Dellu, F., Contarino, A., Bilezikjian, L.M., Gold, L.H., Chen, R., Marchuk, Y., Hauser, C., Bentley, C.A., Sawchenko, P.E., Koob, G.F., Vale, W., Lee, K., 1998.
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Corticotropin releasing factor receptor 1-Deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron 20, 1093–1102. Stenzel-Poore, M.P., Cameron, V.A., Vaughn, J.E., Sawchencko, P.E., Vale, W., 1992. Development of Cushing’s syndrome in corticotropin-releasing factor transgenic mice. Endocrinology 130, 3378 – 3386.
Stenzel-Poore, M., Heinrichs, S., Rivest, S., Koob, G., Vale, W., 1994. Overproduction of Corticotropin-releasing factor in transgenic mice: A Genetic Model of Anxiogenic Behavior. J. Neurosci. 14, 2579 – 2584. Yamano, M., Tohyama, M., 1994. Distribution of corticotropin-releasing factor and calcitonin gene-related peptide in the developing mouse cerebellum. Neurosci. Res. 19, 387 – 396.
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Rapid communication
The temporal and spatial development of CRF binding sites in the postnatal mouse cerebellum Paul Madtes Jr. b, James S. King a,* a
Department of Cell Biology, Neurobiology and Anatomy, Di6ision of Neuroscience, The Ohio State Uni6ersity, 333 W. 10th A6enue, Columbus, OH 43210, USA b Biology Department, Mount Vernon Nazarene College, Columbus, OH, USA Received 30 November 1998; accepted 24 February 1999
Abstract This study describes the distribution and relative level of labeling of binding sites for corticotrophin releasing factor (CRF) in the postnatal mouse cerebellum. At birth low levels of labeling are present throughout the cerebellum. However, this labeling is most densely distributed in the caudal and lateral aspects of the cerebellum. By P3 CRF binding sites are present throughout the cerebellum, although the greatest level of labeling is in the posterior lobe of the vermis, especially lobules IX and X; this correlates with the early differential pattern of CRF distribution in cerebellar afferents within these same lobules. At P10, the adult pattern of distribution and level of labeling begins to emerge. The presence of CRF and CRF binding sites at birth, and during postnatal growth, suggests that this peptide could play a role in the regulation of developmental events within the cerebellum. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Purkinje cell; Granule cell; CRF binding; Development
1. Introduction Corticotropin releasing factor (CRF) is present in climbing fibers and mossy fibers in the adult mouse cerebellum (King et al., 1997). It has been shown in other species to modulate the responsiveness of Purkinje cells (Bishop, 1990; Fox and Gruol, 1993). In the mouse, CRF is present at birth in punctate profiles that have a uniform distribution throughout the medial to lateral extent of the cerebellum (Overbeck and King, 1999). The early presence of CRF is prior to the onset of dendritic outgrowth and the major phase of synaptogenesis in the mouse cerebellum (Hendelman and Aggerwal, 1980; Mason et al., 1990). Between P0 and P60, CRF-immunoreactive (IR) afferents go through a series * Corresponding author. Tel.: +1-614-292-5475; fax: + 1-614-2927659.
of developmental stages until the adult phenotype of climbing and mossy fibers, as well as the adult pattern of distribution of the two afferent fibers, is achieved (Yamano and Tohyama, 1994; Overbeck and King, 1999). A recent study has shown that peptides such as CRF may play a role in the regulation of neural development (Keegan et al., 1994). However, the presence of CRF alone is not sufficient to establish a functional role during development. If CRF influences the development of cerebellar circuits, then the appropriate sites to which the peptide can bind also must be present. This paper analyzes the temporal and spatial distribution of CRF binding sites in the postnatal cerebellum, as well as the relative level of labeling at different ages. These data on the developmental expression of CRF binding sites are correlated with the immunohistochemical localization and distribution of CRF previously described
0168-0102/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 9 9 ) 0 0 0 2 8 - 0
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P. Madtes, Jr, J.S. King / Neuroscience Research 34 (1999) 45–50
in the postnatal cerebellum of the same species (Yamano and Tohyama, 1994; Overbeck and King, 1999). Litters from C57BL/6J breeding pairs were collected at postnatal ages 0, 3, 5, 7, 10, and 14 for these experiments. These time points were selected in order to correlate the findings with a previous study on the ontogeny of CRF (Overbeck and King, 1999) and with critical events in cerebellar development (Larramendi, 1969; Hendelman and Aggerwal, 1980; Mason et al., 1990). A minimum of three animals was used at each age. Animals were decapitated and their brains were quickly frozen on dry ice. 12-mm-frozen sections were cut on a cryostat (MICROM) in either the sagittal or transverse planes of section, thaw-mounted on gelatinized slides, and stored at −70°C until processed for ligand binding. Sections were lightly stained for Nissl substance and processed for autoradiography as described previously (DeSouza et al., 1985; DeSouza and Kuhar, 1986; Insel, 1990; King et al., 1997). The time course of the appearance of binding sites and their laminar distribution was analyzed on both X-ray film and emulsion-coated coverslips with a light microscope using dark field optics; however, only the data from X-ray film are illustrated in representative photomicrographs. The tissue sections illustrated were exposed for 9 days. Emulsion-coated coverslips (data not shown) were used to determine the specific laminar distribution of the labeling and showed patterns like those found with X-ray film. Since the entire cerebellum from each animal was assayed using ligand binding, comparison of the variation in the level of labeling from section to section and from animal to animal was possible. To demonstrate that there was little variation in non-specific binding either from section to section or from animal to animal, representative examples are illustrated for selected ages (Fig. 1B and Fig. 2B). The level of non-specific binding is barely visible and is uniformly distributed across the entire cerebellum. Thus, the labeling present in the total binding photomicrographs reflects actual binding and is not attributed to variation due to tissue preparation or assay conditions. At P0, CRF binding sites are not uniformly distributed in the cerebellum. Labeling is most dense throughout the posterior cerebellum (Fig. 1A). Anteriorly, silver grains are most densely distributed in the lateral portion of the cerebellum whereas anteromedially, labeling appears to be just above background (Fig. 1C). At P3, when all cerebellar lobules are well defined (Fig. 1D), binding sites are present throughout the cortex; however, labeling is not evident over the cerebellar nuclei (Fig. 1D). Labeling is most dense over lobules IX and X (Fig. 1D). Lobules I – III also show higher levels of label when compared to lobules IV– VIII. This differential rostral – caudal distribution pattern also is evident in lateral regions of the cerebellum (not illustrated). At P5 (Fig. 2A) and P7 (Fig. 2C and
D), CRF binding sites are localized over all the neuronal layers throughout the medial to lateral extent of the cerebellum (Fig. 2A, C and D). However, as at P3, the labeling is densest over lobules IX and X medially at P7(Fig. 2C). Laterally, the flocculus, as well as the apex of the paramedian lobule and lobus simplex (Fig. 2D) are intensely labeled. In addition, labeling just above background is evident over the cerebellar nuclei, suggesting that CRF binding sites are present, but the labeling in the cerebellar nuclei is not as dense as in the cortex (Fig. 2D, CN). By P10 there is a differential distribution of CRF binding sites over the histological layers of the cerebellar cortex (Fig. 3A). This is most clearly seen in lobules VIII and IX of the vermis where the labeling is densest in the internal granule cell layer when compared to the molecular layer and the external granule cell layer (Fig. 3A). This pattern is still evident at P14 (Fig. 3B). Also, at P10 the labeling generally is densest at the apex of the individual cerebellar lobules (Fig. 3A). However, there are only minimal differences in the intensity of labeling between the anterior and posterior lobules (Fig. 3A). This is no longer true at P14, where lobules VII–X are more densely labeled, compared to more anterior lobules (Fig. 3B). To date, many studies have described the distribution of CRF binding sites in the adult mammalian cerebellum. Radioligand binding studies show a relatively high density of binding sites within the cerebellum of the adult opossum (Madtes and King, 1995), mouse (King et al., 1997), rat (DeSouza and Kuhar, 1986), rabbit (Chai et al., 1990), non-human primate (Aguilera et al., 1990) and man (Rivier et al., 1987). In the adult mouse (King et al., 1997) CRF binding sites are found throughout all lobules of the cerebellar cortex. Although silver grains are present over all three histological layers in the adult, labeling in the internal granule cell layer (IGL) is slightly denser than that seen in the molecular or Purkinje cell layers. Further, the densest labeling is located at the apex of most cerebellar lobules. This latter pattern of distribution does not begin to emerge until P10 in the mouse. Even at this age, however, the pattern is not identical to that seen in the adult. For example, in the posterior lobe vermis, the labeling at the apex of each lobule is equivalent to that at the base of the lobule. During postnatal development there is both temporal and spatial overlap in the distribution of the peptide CRF and CRF binding sites. At birth, CRF-IR afferents are present as punctate profiles that are abundant and present throughout the cerebellum (Overbeck and King, 1999). However, CRF binding sites while present in low levels throughout the cerebellum show a greater density of distribution in caudal and lateral aspects of the cerebellum. This disparity in the distribution of CRF and CRF binding sites is no longer evident at P3. By P3, both CRF and its binding sites have their
P. Madtes, Jr, J.S. King / Neuroscience Research 34 (1999) 45–50
greatest distribution in vermal lobules I, II, IX, and X. Several studies have shown that Purkinje cells in these lobules mature earlier than those in other regions of the vermis or the hemispheres (Altman, 1969). Paradoxically, Inouye and Murakami (1980) have shown that the Purkinje cells in these lobules are some of the last to be born. Taken together, these data suggest that the time of birth alone does not predict the temporal maturation of these neurons. Thus, other factors must influence the timing of Purkinje cell differentiation. CRF is one potential factor based on its presence and the presence of CRF binding sites in lobules where
47
Purkinje cells mature early. CRF-IR puncta are most extensive in the ventral vermal folia, although they are distributed across all lobules, as are the binding sites between P3 and P10. After P10, the distribution of CRF changes, as does the pattern of labeling for binding sites. In contrast to the punctate pattern of CRF immunoreactivity seen before P10 the phenotypic characteristics of CRF-immunoreactive climbing and mossy fibers become apparent at this age. In addition, it is at this age that the adult laminar and lobular pattern of distribution of CRF binding sites begins to emerge (King et al., 1997). Therefore, if CRF plays a role in
Fig. 1. The distribution of binding sites for CRF is illustrated in film autoradiograms in the postnatal (P0, P3) cerebellum. At P0 (A and C) transverse sections show label in the posterior cerebellum and in the lateral regions (C, arrows) of the anterior cerebellum (CB). By P3 (D) label is evident throughout the vermis (lobules I–X) and the hemispheres (not illustrated). The densest labeling is evident over lobules IX and X at P3. (B) shows the low level of labeling present for non-specific binding. This level did not vary from section to section or from animal to animal. Therefore, only one representative autoradiogram is shown. Calibration bar, 1 mm and applies to all figures.
48
P. Madtes, Jr, J.S. King / Neuroscience Research 34 (1999) 45–50
Fig. 2. The distribution of binding sites for CRF is illustrated in film autoradiograms in the postnatal (P5, P7) cerebellum. A transverse section of a P5 cerebellum (A) illustrates the same pattern of distribution as at earlier ages. At P7, silver grains above background levels are present throughout the medial (C) to lateral (D) extent of the cerebellar cortex. The densest labeling is present in lobules IX and X. Labeling just slightly above background is evident over the cerebellar nuclei (D, CN). (B) shows the low level of labeling present for non-specific binding. This level did not vary from section to section or from animal to animal.
development, these data suggest it would do so prior to P10. Recent studies have focused on using (a) direct transgenic models in which the level of either CRF (StenzelPoore et al., 1992, 1994; Keegan et al., 1994; Heinrichs et al., 1996) or CRF receptor (CRFR1) (Smith et al., 1998) has been altered or (b) indirect transgenic models in which glucocorticoid receptors, a system that is known to interact with CRF neurons (Dijkstra et al., 1998), has been altered. To date, the results of these studies have focused on the function of CRF in the hypothalamic–pituitary axis, evaluating the degree of anxiety generated in response to stress. In particular, Smith et al. (1998) found that transgenic mice deficient in CRF-R1 receptors showed a markedly decreased anxiety response, a loss of a hormonal response to stress. In addition, homozygous mice died within 48 h due to a deficiency in corticosteroids required for lung maturation. These findings indicate CRF is essential for normal development and normal adult function. How-
ever, the impact on cerebellar development has not been analyzed in these transgenic animal models. In summary, the present data clearly indicate that the necessary binding sites essential for CRF interactions during development are present prior to and during critical developmental events (e.g. cell migration, cell survival, synaptogenesis, dendrogenesis) and lend further support to the concept that CRF may play an important role in regulating cerebellar development. Future studies will more precisely define the specific types of CRF receptors present, along with their cellular distribution during development. These data will facilitate the determination of the proposed role for CRF in cerebellar ontogeny.
Acknowledgements This work was supported by NS-08798. Dr Madtes was supported by a Research Opportunity Award IBN-
P. Madtes, Jr, J.S. King / Neuroscience Research 34 (1999) 45–50
49
Fig. 3. The distribution of binding sites for CRF is illustrated in film autoradiograms in the postnatal (P10, P14) cerebellum. CRF binding sites are evident throughout the cerebellar cortex at P10 (A) and P14 (B).
9630867 from NSF. The author wishes to thank G.A. Bishop for reading this manuscript and providing critical discussions of the data. We also wish to thank Barbara Diener-Phelan for technical and photographic assistance.
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