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Expression of proenkephalin mRNA in developing cerebellar cortex of the rat: expression levels coincide with maturational gradients in Purkinje cells
Developmental Brain Research, 63 (1991) 63-69 © Elsevier Science Publishers B.V. All rights reserved. 0165-3806/91/$03.50 ADONIS 0165380691513403
63
BRESD 51340
Expression of proenkephalin mRNA in developing cerebellar cortex of the rat: expression levels coincide with maturational gradients in Purkinje cells John G. Osborne 1, Mark S. Kindy 2 and Kurt F. Hauser 1 Departments of Anatomy and Neurobiology 1, and Biochemistry 2, University of Kentucky Medical Center, Lexington, KY 40536-0084 (U.S.A.) (Accepted 9 July 1991) Key words: Purkinje cell; Proenkephalin; Opioid gene expression; Cerebellar development; Endogenous opioid system
The cellular localization of proenkephalin (PE) mRNA expression was systematically examined in midsagittal (vermal) sections of the developing rat cerebellar cortex by in situ hybridization. PE mRNA was initially detected in Golgi cells of postnatal day 7 (PND 7) rats and in each group thereafter. Moreover, PND 7 rats also displayed an intense layer of PE mRNA hybridization signal over the Purkinje cell layer. By PND 14, distinct cellular labeling was observed in a subpopulation of Purkinje cells in all Iobules of the vermis except lobule III. At PND 7 and 14, the area and level of intensity of Purkinje cell associated PE mRNA hybridization signal followed a gradient that was most intense caudally but then decreased rostrally. At PND 21, the proportion of labeled Purkinje cells and the intensity of PE hybridization signal was evenly dispersed between the anterior and posterior lobules of the cerebellar vermis. PE hybridization signal was not detected in the developing neural cells of the external granular layer or the interneurons of the molecular layer in the vermis. These results indicate that the ontogeny of PE mRNA expression in Purkinje cells is developmentally regulated since levels of expression closely follow the chronological order of settling and maturation of these neurons. Based on prior evidence that endogenous opioids inhibit the growth of Purkinje cell dendrites and dendritic spines, PE expression is likely to be important for Purkinje cell maturation. INTRODUCTION Experimental manipulations of endogenous opioid systems (i.e., endogenous opioids and opioid receptors) indicate that opioid neuropeptides modulate neuronal maturation by exerting a negative trophic influence during nervous system development 1°'11'27. In the cerebellum, manipulation of endogenous opioid systems during the first 3 weeks of postnatal life alters the rate and pattern of dendritic growth and spine formation in Purkinje cells 1°'11 as well as the area and volume of the molecular and internal granular layers (IGL) 27. Changes in neuron and glial cell numbers, soma size and cell packing density have also been reported 25'27. Moreover, [3H]thymidine incorporation by neuroblasts of the external granular layer ( E G L ) in the cerebellum of 6-day-old rats is inhibited by systemic administration of [MetS] enkephalin, whereas chronic blockade of opioid receptors with the opioid antagonist naltrexone, is reported to increase [3H]thymidine incorporation compared to untreated controls 28. These experiments suggest that endogenous opioid peptides are normally present and avail-
able to opioid-receptor containing cells of the developing cerebellum in sufficient quantities to tonically inhibit growth. This hypothesis is supported by reports of increased levels of opioid peptides 21"22, proenkephalin (PE) m R N A 16 and opioid binding 21'22 during rat brain development. In the developing cerebellum, enkephalin immunoreactivity is reported to be transiently present within the germinative cells of the E G L in vivo 26 and in vitro 15. Furthermore, ultrastructural analysis of cerebella of preweaning rats has detected enkephalin immunoreactivity in the dendrites, dendritic spines and somata of Purkinje cells 29. Reaction product was also detected in glia as well as in basket and stellate neurons 29. Proenkephalin, the unprocessed peptide precursor, and PE m R N A have been localized in a subpopulation of Purkinje cells using monoclonal antibodies against the intact PE precursor and by in situ hybridization histochemistry, respectively, in 25 day-old rats 18. Recently, astrocyte-enriched cultures from neonatal rat brain 13'17"23, and the cerebellum in particular w'23 have been shown to express PE m R N A by Northern analysis and the cellular location of PE
Correspondence: K.E Hauser, Department of Anatomy and Neurobiology, University of Kentucky Medical Center, 800 Rose Street, Lexington, Kentucky 40536-0084, U.S.A. Fax: (1) (606) 258-5946.
64 m R N A in cerebellar astrocytes has b e e n identified by in situ hybridization 12. The present study examines the temporal expression of PE m R N A in Purkinje cells of preweaning rats. O u r studies indicate that detectable levels of PE m R N A are present in a subpopulation of Purkinje cells at day 14 with an increase in signal intensity and n u m b e r of cells displaying hybridization signal at PND 21. This pattern of m R N A expression appears to follow a caudal to rostral gradient during m a t u r a t i o n with the posterior lobules of the cerebellum displaying the strongest signals initially. MATERIALS AND METHODS Animal and tissue preparation
A total of 23 male Sprague-Dawley rat pups (ages 1, 7, 14, and 21 days) were anesthetized with ether and perfused intracardially with 4% paraformaldehyde (PFA) in phosphate buffer pH 7.25. Cerebella were removed and immersed in fixative overnight at 4 °C then washed 3 times (10 min each) in 0.01 M phosphate buffered saline (PBS) pH 7.25 before cryoprotecting overnight in 20% sucrose in PBS. Frozen sections (10/am thick) were cut in the midsagittal plane in the region of the vermis and thaw mounted onto poly-L-lysine subbed Probe-On slides (Fisher Diagnostics). In situ hybridization
A [35S]-labeledcRNA probe specific for rat PE mRNA was transcribed from a 1070 bp eDNA probe (in pSP64 plasmid, Promega, Madison WI, gift from Dr. M.H. Melner), linearized with BamH1 restriction enzyme and transcribed with SP6 RNA polymerase using [35S]UTP plus unlabeled nucleotides. The probe was subjected to mild alkaline hydrolysis to fragment the RNA (0.1 M Na2CO3/ NaHCO3, pH 10 for 40 min at 60 °C) which produced a range of fragments from 50-200 bp in length. Hybridization procedures were performed using previously described methods sas. Briefly, air-dried sections were treated with Proteinase K (1.0/ag/ml 20 mM Tris pH 8.0) for 10 min then rinsed 5 times (30 min total time) in water treated with 0.1% diethylpyrocarbonate (DPC). Sections were then prehybridized for 2 h at room temperature in buffer containing 50% deionized formamide, 4x SSC, 20 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 10x Denhardt's solution and 0.1 mg/ml salmon sperm DNA. Tissue sections were hybridized for 16 h at 37 °C using a probe concentration of 6 x 105 cpm./section in hybridization buffer (prehybridization buffer plus 100/am dithiothreitol (DTT) and 10% dextran sulfate). After hybridization, the sections were washed 10x (5 min each) at room temperature in 2x SSC containing l0 mM DTI" and treated with RNase A (20 gg/ml 2x SSC) for l0 min. Sections were washed in 2× SSC plus 0.1% SDS for 10 min, 4 x 10 rain in 0.2x SSC plus 0.1% SDS and again in 2x SSC plus 0.1% SDS for 10 min, all at room temperature. After dehydration in graded alcohols and air drying, sections were coated with NTB2 emulsion and exposed for 4 weeks at 4 °C. Finally, the sections were developed at 15 °C in D-19 developer (Kodak) and stained with Ehrlich's hematoxylin. Hybridization was evaluated by visually identifying clusters of silver grains over cell bodies. Controls included sections treated with a 25-fold excess concentration of cold probe, pretreatment with RNase A, and comparison of labeling patterns of in situ hybridization with patterns visualized by immunocytochemical staining for PE peptide products on adjacent sections. To further insure probe specificity, combined techniques of immunocytochemistry, using antisera against [MetS]enkephalin (gift from Dr. B.E. Maley), and in situ hybridization were performed on the same tissue sections taken from 14-day-old rats. Combined immunocytochemistry and in situ hybridization
All buffers and reagents used for immunostaining were prepared
with water that had been treated with 0.1% I)PC. Sections ~cl~ reacted with primary rabbit antisera (I:351XJdilution t against Melenkephalin, previously characterized to have limited cross reacti~ ity with Leu-enkephalin 24, following the avidin-biotin pcroxidasc method (Vectastain; Vector Labs., Burlingamc, CA) with 0.05~,,~ 3-3" diaminobenzidine (DAB) used to visualize the reaction product. Immunoreactivity was not observed in tissue sections trcalcd with antisera preabsorbed with excess [Met 5} and [Leu~]enkephalin (24/~g/ml of antiscra) ensuring immunocytochemical specificity ~t the [MetS]enkephalin antisera. After treatment with DAB the sections were washed ~' ~ i0 rain in DPC water, treated with Proteinase K for I0 rain. thcn washed again in DPC water for 3 × 10 min. To eliminate excessive background, the sections were immersed in 0.1 M triethanolamine pH 8.0 followed by addition of acetic anhydride (while stirring) to a final v/v concentration of 0.5% acetic anhydride. After incubating for 10 min in the acetic anhydridc-triethanolaminc solution, the sections were washed 2 × 5 min in 2× SSC then 3 :,: lt) rain in DPC water followed by the prehybridization stcp as outlined above+
RESULTS Proenkephalin m R N A was not detected in neural cells of the cerebeUar cortex at postnatal day (PND) 1, although labeled n o n - n e u r a l cells were observed in the pial layer as well as in the choroid plexus of the fourth ventricle (not shown). A t PND 7, low intensity hybridization signal was observed over the I G L with a somewhat stronger signal over the Purkinje cell layer whose origin could not be positively identified, although its location suggests that it probably emanates from Purkinje cells and/or immediately adjacent cells such as B e r g m a n n glial cells. This diffuse hybridization signal was identified in all of lobule X and in portions of lobules I through IX (Figs. IB and 2A,B) and appears to represent the onset of PE m R N A expression by these cells. Overall, the PE hybridization signal appeared to be more intense caudally with a gradual caudal to rostral decrease in both area and intensity. A t PND 7, the intensity of PE hybridization signal over the molecular layer and E G L was not above background. A t PND 14, distinct cellular labeling for PE m R N A was observed in a subpopulation of Purkinje cells in all lobules of the vermis with lobules V. VI. VIII and IX appearing to have the greatest proportion of labeled cells (Figs. 1C and 2D). As was observed in the 7 day-old animals, there appeared to be a caudal to rostral gradient with respect to the proportion of Purkinje cells displaying positive hybridization signal for PE m R N A as well as to the intensity of signal observed (Figs. 1C and 2C,D). In addition, diffuse hybridization signal, similar to that observed on PND 7. was also identified over the Purkinje cell layer in regions where no distinct cellular labeling was observed. Also, as was the case with P N D 7 animals, PE hybridization signal over the molecular layer and E G L was not above background. By PND 21, hybridization signal for PE m R N A ex-
65
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Fig. 1. Camera lucida drawings of midsagittal sections of cerebella from postnatal day (PND) 1 (A), PND 7 (B), PND 14 (C), and PND 21 (D) rats illustrate patterns of proenkephalin (PE) hybridization signal over the respective Purkinje cell layers. Large dots represent areas of distinct cellular labeling of Purkinje cells. Small dots represent layers of diffuse PE hybridization signal associated with the Purkinje cell layer. The intensity of PE mRNA expression by Purkinje cells displays a caudal to rostral gradient during maturation. Bar = 1 mm.
pression was detected in a subpopulation of Purkinje cells that appeared to be dispersed evenly between the anterior and posterior lobules of the midsagittally sectioned cerebellar vermis without the caudal to rostral pattern observed in younger animals (Figs. 1D and 2E, F). Although this study focused on the cerebellar cortex with particular emphasis on Purkinje cells, PE mRNA
hybridization signal was also observed over Golgi cells of the IGL (Figs. 2 B - F and 3C) and the deep cerebellar nuclei as early as PND 7 (Fig. 2A). PE hybridization signal was also detected over the nucleus of the solitary tract in PND 14 (not shown) and 21 rats (Fig. 2E) as previously described by Morita et al. 14. There was not, however, any evidence of specific cellular labeling within
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Fig. 3. A,B: brightfield photomicrographs displaying co-localization of proenkephalin (PE) hybridization signal and enkephalin immunoreactivity in Purkinje cells (P) of postnatal day (PND) 14 rats (arrows). Note the greater intensity of labeling over Purkinje cells in (A) as compared to the cells in (B), this demonstrates that even within a given lobule, intensity of hybridization signal for PE mRNA may vary among the Purkinje cells. MOL, molecular layer; IGL, internal granular layer. Bar for A,B = 25 #m. C,D: lobule X from PND 14 rats displaying PE hybridization signal over Golgi cells of the IGL in (C) and adjacent section after treatment with a 25-fold excess of cold probe (D). Bar for C,D = 100 #m.
Fig. 2. Darkfield photomicrographs of proenkephalin (PE) hybridization signal using [3~S]-labeled cRNA probe in midsagittal sections of rat cerebella from postnatal day (PND) 7 (A,B), PND 14 (C,D), and PND 21 (E,F). A: lobule I. Distinct cellular labeling was evident over the deep cerebellar nuclei (DCN), otherwise only a layer of low intensity hybridization signal over Purkinje cell layer (P). B: portions of lobules IX & X. There was an apparent increase in the area and intensity of PE hybridization signal over the Purkinje cell layer in lobules IX and X as compared to (A) lobule I of same section. During development, PE hybridization signal was first observed in cells of the internal granular layer (IGL) which resemble Golgi cells (arrows). C: lobule II. PE hybridization signal was associated with Golgi cells of the IGL but not expressed by Purkinje cells. D: lobule IX of the same section as (C). First evidence of distinct cellular labeling of Purkinje ceils with PE hybridization signal (arrowheads). E,F: lobules II and I respectively (E) and Lobule X (F). At PND 21, PE hybridization signal is more uniformly distributed among Purkinje cells in different lobules, and PE hybridization signal intensity is more similar over Golgi and Purkinje cells. Intense labeling can be seen over cells in the nucleus of the solitary tract (NTS). M, molecular layer; BN, brainstem nuclei. Bars = 100/~m.
68 the molecular layer or E G L of any animal used in this study; a pattern which is consistent with previous studies that examined PE mRNA expression by in situ hybridization in older and adult rats 9'19. Loss of PE mRNA hybridization signal in sections treated with an excess concentration of cold probe (Fig. 3D) or pretreated with RNase A assured specificity of the probe (not shown). Moreover, co-localization of PE hybridization signal and enkephalin immunoreactivity in the same tissue section served as positive controls (Fig. 3A,B). DISCUSSION The aim of this study was to characterize the expression of PE mRNA in neural cells of the developing rat cerebellar cortex and to identify possible relationships between the cellular location of PE gene expression and the cellular targets of opioid action during cerebellar maturation. Animals were chosen whose ages ranged from the day of birth (PND 1) through PND 21 because key stages of the cerebellar cortical maturation occur at these times 1-3. Moreover, by PND 21, Purkinje cell dendrites have begun to elaborate and form synapses with granule cell axons (parallel fibers) similar to those found in the adult cerebellum 3. The results indicate that by PND 7, patches of diffuse hybridization signal (above background) can be identified in the IGL of lobules I through IX and over most of the IGL of lobule X (Figs. 1B and 2A,B). Moreover, in these same areas, a more intense band of silver grains was observed over the Purkinje cell layer (Fig. 2A,B). Although present hybridization conditions in our lab do not permit distinct cellular localization of this discrete layer of silver grains at PND 7, its location indicates probable association with Purkinje cells, glial cells or both. This observation is consistent with previous studies by Spruce et a1.19 who assessed PE mRNA and peptide expression in 25 day-old rats. The relative size and signal intensity of these areas yield a definite caudal to rostral pattern with the larger, more intense regions lying in the posterior lobules of the vermis (Figs. 1B and 2B). This gradient of signal distribution suggests a correlation between PE mRNA expression and the chronological order of Purkinje cell settling and maturation in the rat as has been previously reported by Altman 2 and Altman and Bayer 6 and in mice by Andreoli et al. 7. These studies reported that the Purkinje cells of the cerebellar vermis are produced in a caudal to rostral order with the Purkinje cells of lobule X through VI of the posterior vermis being produced earlier than those in the anterior vermis with the exception of a longitudinal band of early forming Purkinje cells termed the 'vermal wedge'
by Altman 3, Altman and Bayer" and Andreoli et al. This hypothesis is supported by the distribution of hybridization signal observed on sections of PND 14 animals (Figs. 1C and 2C,D). Examination of sections from this age group indicate distinct cellular labeling over a subpopulation of Purkinje cells with a greater proportion of labeled cells and increased intensity of signal observed in the posterior lobules (Figs. IC and 2C,D). The expression of opioids has been associated with a maturational process in the cerebellum of another species. Walker and King 24 have demonstrated medial to lateral gradients of enkephalin immunoreactivity ill afferents in the North American Opossum. By PND 21, there appeared to be a fairly uniform distribution of hybridization signal for PE mRNA in the Purkinje cells of the anterior and posterior regions of the cerebellar vermis (Figs. 1D and 2E,F), There are, however, some variations among the Purkinje cells in the individual lobules within each region. For example, a subpopulation of Purkinje cells in lobules IXb and IXc express PE mRNA at PND 14 but do not at PND 21. This suggests a possible transient expression of PE mRNA by a subpopulation of Purkinje cells which could alter the intensity of signal as well as the number of cells expressing the PE gene at any time during development. The notion that there is transient expression of PE mRNA by Purkinje cells is even more interesting considering the studies which describe transient enkephalin (a posttranslational peptide product of PE) immunoreactivity in E G L cells of the developing cerebellum 26. The spatiotemporal pattern of E G L cell migration and settling closely follows that of Purkinje cells 6. Equally as interesting is the fact that expression of PE mRNA has not been detected in the developing neural progenitor cells of the vermal EGL or their mature counterparts, i.e., the granule cells of the IGL (present study and Spruce et al.l'). This absence of detectable signal suggests that other opioid genes (prodynorphin or proopiomelanocortin) may be involved in the production of transient enkephalin-like immunoreactivit~ in EGL cells26 or that sources of enkephalins originating from outside the EGL cells may be internalized (e.g., receptor-mediated endocytosis) and therefore m~ly bc responsible for the transient appearance of enkephalin immunoreactivity by EGL cells as previously reported by Zagon et al. 2~'. In summary, present data indicate that Purkinje cells, in particular, express PE mRNA early during development and undergo complex patterns of expression during ontogeny. This supports the hypothesis that expression of PE mRNA in Purkinje cells of the developing cerebellar vermis closely follows the chronological order of settling and maturation of these neurons. The evi-
69 dence also suggests that, in light of recent studies link-
global (caudal to rostral) maturational gradients that
ing [MetS]enkephalin (a product of the p r o e n k e p h a l i n gene) to n e u r o n a l growth and development ~°'11"25"27"28
govern PE expression by Purkinje cells. A more detailed
and the fact that endogenous opioids dramatically influ-
expression by Purkinje cells in the developing cerebel-
ence the elaboration of Purkinje cell dendrites and dendritic spines 1°'11, the expression of PE m R N A may be
lum may further clarify the role of PE gene expression by Purkinje cells during development.
spatiotemporal assessment of the patterns of PE m R N A
important for Purkinje cell development. Yet, differences in levels of PE m R N A expression between individual Purkinje cells suggest that subtle differences in m i c r o e n v i r o n m e n t or intrinsic timing impose local constraints on PE expression that may override the more
REFERENCES 1 Altman, J., Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer, J. Comp. Neurol., 145 (1972) 353-398. 2 Altman, J., Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer, J. Cornp. NeuroL, 145 (1972) 399-464. 3 Altman, J., Postnatal development of the cerebellar cortex in the rat. III. Maturation of the components of the granular layer, J. Comp. Neurol., 145 (1972) 465-514. 4 Altman, J. and Bayer, S.A., Embryonic development of the rat cerebellum. I. Delineation of the cerebellar primordium and early cell movements, J. Comp. Neurol., 231 (1985) 1-26. 5 Altman, J. and Bayer, S.A., Embryonic development of the rat cerebellum: II. Transiocation and regional distribution of the deep neurons, J. Comp, Neurol., 231 (1985) 27-41. 6 Altman, J. and Bayer, S.A., Embryonic development of the rat cerebellum. III. Regional differences in the time of origin, migration and settling of Purkinje cells, J. Comp. Neurol., 231 (1985) 42-65. 7 Andreoli, J., Rodier, P. and Langerman, J., The influence of a prenatal trauma on formation of Purkinje cells, Am. J. Anat., 137 (1973) 87-102. 8 Angerer, L.M., Stoler, M.H. and Angerer, R.C., In situ hybridization with RNA probes: an annotated recipe. In K.L. Valentino, J.H. Eberwine and J.D. Barchas (Eds.), In Situ Hybridization. Applications to Neurobiology, Oxford University Press, New York, 1987, pp. 42-70. 9 Harlan, R.E., Shivers, B.D., Romano, G.J., Howells, R.D. and Pfaff, D.W., Localization of preproenkephalin mRNA in the rat brain and spinal cord by in situ hybridization, J. Comp. Neurol., 258 (1987) 159-184. 10 Hauser, K.F., McLaughlin, P.J. and Zagon, I.S., Endogenous opioids regulate dendritic growth and spine formation in the developing rat brain, Brain Research, 416 (1987) 157-171. 11 Hauser, K.E, McLaughlin, P.J. and Zagon, I.S., Endogenous opioid systems and the regulation of dendritic growth and spine formation, J. Comp. Neurol., 281 (1989) 13-22. 12 Hauser, K.E, Osborne, J.G., Stiene-Martin, A. and Melner, M.H., Cellular localization of proenkephalin mRNA and enkephalin peptide products in cultured astrocytes, Brain Research, 522 (1990) 347-353. 13 Melner, M.H., Low, K.G., Allen, R.G., Nielson, C.P., Young, S.L. and Saneto, R.P., The regulation of proenkephalin expression in a distinct population of glial cells, EMBO J., 9 (1990) 791-796. 14 Morita, Y., Zhang, J., Hironaka, T., Tateno, E., Noguchi, K., Sato, M., Kiyama, H. and Tohyama, M., Postnatal development of preproenkephalin mRNA containing neurons in the rat lower brainstem, J. Comp. Neurol., 292 (1990) 193-213.
Acknowledgements. The authors wish to thank Mrs. Aruna Bhat, Drs. Anne Stiene-Martin, S.L. Sanders, K.B. Seroogy and B.E Sisken for assistance and/or helpful comments; Dr. M.H. Melner for providing the cDNA probe and Dr. B.E. Maley for providing enkephalin antisera. Supported by NIDA Grant DA 06204.
15 Osborne, J.G. and Hauser, K.E, Enkephalin immunoreactivity in organotypic cultures of the developing rat cerebellum: evidence for transient opioid expression in vitro, Soc. Neurosci. Abstr., 15 (1989) 278. 16 Rosen, H. and Polakiewicz, R., Postnatal expression of opioid genes in rat brain, Dev. Brain Res., 46 (1989) 123-129. 17 Shinoda, H., Marini, A.M., Cosi, C. and Schwartz, J.P., Brain region and gene specificity of neuropeptide gene expression in cultured astrocytes, Science, 245 (1989) 415-417. 18 Shivers, B.D., Harlen, R.E., Romano, G.J., Howells, R.D. and Pfaff, D.W., Cellular localization of proenkephalin mRNA in rat brain: gene expression in the caudate-putamen and cerebellar cortex, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 62216225. 19 Spruce, B.A., Curtis, R., Wilkin, G.P. and Glover, D.M., A neuropeptide precursor in the cerebellum: proenkephalin exists in subpopulations of both neurons and astrocytes, EMBO J., 9 (1990) 1787-1795. 20 Stiene-Martin, A., Osborne, J.G. and Hauser, K.E, Co-localization of proenkephalin mRNA using cRNA probes and a celltype-specific immunocytochemical marker for intact astrocytes in vitro, J. Neurosci. Methods, 36 (1991) 119-126. 21 Tsang, D., Ng, S.C. and Ho, K.P., Development of methionine-enkephalin and naloxone binding sites in regions of the rat brain, Dev. Brain Res., 3 (1982) 637-644. 22 Tsang, D., Ng, S.C., Ho, K,P. and Ho, W.K.K., Ontogenesis of opiate binding sites and radioimmunoassayable beta-endorphin and enkephalin in regions of the rat brain, Dev. Brain Res., 5 (1982) 257-261. 23 Vilijn, M.H., Vayasse, P.J.J., Zukin, R.S. and Kessler, J.A., Expression of preproenkephalin messenger RNA by cultured astrocytes and neurons, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 6551-6555. 24 Walker, J.J. and King, J.S., Ontogenesis of enkephalinergic afferent systems in the opossum cerebellum, Dev. Brain Res., 48 (1989) 35-58. 25 Zagon, I.S. and McLaughlin, P.J., Increased brain size and cellular content in infant rats treated with an opioid antagonist, Science, 221 (1983) 1179-1180. 26 Zagon, I.S., Rhodes, R.E. and McLaughlin, P.J., Distribution of enkephalin immunoreactivity in germinative cells of developing rat cerebellum, Science, 227 (1985) 1049-1051. 27 Zagon, I.S. and McLaughlin, P.J., Opioid antagonist (naltrexone) modulation of cerebellar development: histological and morphometric studies, J. Neurosci., 6 (1986) 1424-1432. 28 Zagon, I.S. and McLaughlin, P.J., Endogenous opioid systems regulate cell proliferation in the developing rat brain, Brain Res., 412 (1987) 68-72. 29 Zagon, I.S. and McLaughlin, P.J., Ultrastructural localization of enkephalin-like immunoreactivity in developing rat cerebellum, Neuroscience, 34 (1990) 479-489.
63
BRESD 51340
Expression of proenkephalin mRNA in developing cerebellar cortex of the rat: expression levels coincide with maturational gradients in Purkinje cells John G. Osborne 1, Mark S. Kindy 2 and Kurt F. Hauser 1 Departments of Anatomy and Neurobiology 1, and Biochemistry 2, University of Kentucky Medical Center, Lexington, KY 40536-0084 (U.S.A.) (Accepted 9 July 1991) Key words: Purkinje cell; Proenkephalin; Opioid gene expression; Cerebellar development; Endogenous opioid system
The cellular localization of proenkephalin (PE) mRNA expression was systematically examined in midsagittal (vermal) sections of the developing rat cerebellar cortex by in situ hybridization. PE mRNA was initially detected in Golgi cells of postnatal day 7 (PND 7) rats and in each group thereafter. Moreover, PND 7 rats also displayed an intense layer of PE mRNA hybridization signal over the Purkinje cell layer. By PND 14, distinct cellular labeling was observed in a subpopulation of Purkinje cells in all Iobules of the vermis except lobule III. At PND 7 and 14, the area and level of intensity of Purkinje cell associated PE mRNA hybridization signal followed a gradient that was most intense caudally but then decreased rostrally. At PND 21, the proportion of labeled Purkinje cells and the intensity of PE hybridization signal was evenly dispersed between the anterior and posterior lobules of the cerebellar vermis. PE hybridization signal was not detected in the developing neural cells of the external granular layer or the interneurons of the molecular layer in the vermis. These results indicate that the ontogeny of PE mRNA expression in Purkinje cells is developmentally regulated since levels of expression closely follow the chronological order of settling and maturation of these neurons. Based on prior evidence that endogenous opioids inhibit the growth of Purkinje cell dendrites and dendritic spines, PE expression is likely to be important for Purkinje cell maturation. INTRODUCTION Experimental manipulations of endogenous opioid systems (i.e., endogenous opioids and opioid receptors) indicate that opioid neuropeptides modulate neuronal maturation by exerting a negative trophic influence during nervous system development 1°'11'27. In the cerebellum, manipulation of endogenous opioid systems during the first 3 weeks of postnatal life alters the rate and pattern of dendritic growth and spine formation in Purkinje cells 1°'11 as well as the area and volume of the molecular and internal granular layers (IGL) 27. Changes in neuron and glial cell numbers, soma size and cell packing density have also been reported 25'27. Moreover, [3H]thymidine incorporation by neuroblasts of the external granular layer ( E G L ) in the cerebellum of 6-day-old rats is inhibited by systemic administration of [MetS] enkephalin, whereas chronic blockade of opioid receptors with the opioid antagonist naltrexone, is reported to increase [3H]thymidine incorporation compared to untreated controls 28. These experiments suggest that endogenous opioid peptides are normally present and avail-
able to opioid-receptor containing cells of the developing cerebellum in sufficient quantities to tonically inhibit growth. This hypothesis is supported by reports of increased levels of opioid peptides 21"22, proenkephalin (PE) m R N A 16 and opioid binding 21'22 during rat brain development. In the developing cerebellum, enkephalin immunoreactivity is reported to be transiently present within the germinative cells of the E G L in vivo 26 and in vitro 15. Furthermore, ultrastructural analysis of cerebella of preweaning rats has detected enkephalin immunoreactivity in the dendrites, dendritic spines and somata of Purkinje cells 29. Reaction product was also detected in glia as well as in basket and stellate neurons 29. Proenkephalin, the unprocessed peptide precursor, and PE m R N A have been localized in a subpopulation of Purkinje cells using monoclonal antibodies against the intact PE precursor and by in situ hybridization histochemistry, respectively, in 25 day-old rats 18. Recently, astrocyte-enriched cultures from neonatal rat brain 13'17"23, and the cerebellum in particular w'23 have been shown to express PE m R N A by Northern analysis and the cellular location of PE
Correspondence: K.E Hauser, Department of Anatomy and Neurobiology, University of Kentucky Medical Center, 800 Rose Street, Lexington, Kentucky 40536-0084, U.S.A. Fax: (1) (606) 258-5946.
64 m R N A in cerebellar astrocytes has b e e n identified by in situ hybridization 12. The present study examines the temporal expression of PE m R N A in Purkinje cells of preweaning rats. O u r studies indicate that detectable levels of PE m R N A are present in a subpopulation of Purkinje cells at day 14 with an increase in signal intensity and n u m b e r of cells displaying hybridization signal at PND 21. This pattern of m R N A expression appears to follow a caudal to rostral gradient during m a t u r a t i o n with the posterior lobules of the cerebellum displaying the strongest signals initially. MATERIALS AND METHODS Animal and tissue preparation
A total of 23 male Sprague-Dawley rat pups (ages 1, 7, 14, and 21 days) were anesthetized with ether and perfused intracardially with 4% paraformaldehyde (PFA) in phosphate buffer pH 7.25. Cerebella were removed and immersed in fixative overnight at 4 °C then washed 3 times (10 min each) in 0.01 M phosphate buffered saline (PBS) pH 7.25 before cryoprotecting overnight in 20% sucrose in PBS. Frozen sections (10/am thick) were cut in the midsagittal plane in the region of the vermis and thaw mounted onto poly-L-lysine subbed Probe-On slides (Fisher Diagnostics). In situ hybridization
A [35S]-labeledcRNA probe specific for rat PE mRNA was transcribed from a 1070 bp eDNA probe (in pSP64 plasmid, Promega, Madison WI, gift from Dr. M.H. Melner), linearized with BamH1 restriction enzyme and transcribed with SP6 RNA polymerase using [35S]UTP plus unlabeled nucleotides. The probe was subjected to mild alkaline hydrolysis to fragment the RNA (0.1 M Na2CO3/ NaHCO3, pH 10 for 40 min at 60 °C) which produced a range of fragments from 50-200 bp in length. Hybridization procedures were performed using previously described methods sas. Briefly, air-dried sections were treated with Proteinase K (1.0/ag/ml 20 mM Tris pH 8.0) for 10 min then rinsed 5 times (30 min total time) in water treated with 0.1% diethylpyrocarbonate (DPC). Sections were then prehybridized for 2 h at room temperature in buffer containing 50% deionized formamide, 4x SSC, 20 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 10x Denhardt's solution and 0.1 mg/ml salmon sperm DNA. Tissue sections were hybridized for 16 h at 37 °C using a probe concentration of 6 x 105 cpm./section in hybridization buffer (prehybridization buffer plus 100/am dithiothreitol (DTT) and 10% dextran sulfate). After hybridization, the sections were washed 10x (5 min each) at room temperature in 2x SSC containing l0 mM DTI" and treated with RNase A (20 gg/ml 2x SSC) for l0 min. Sections were washed in 2× SSC plus 0.1% SDS for 10 min, 4 x 10 rain in 0.2x SSC plus 0.1% SDS and again in 2x SSC plus 0.1% SDS for 10 min, all at room temperature. After dehydration in graded alcohols and air drying, sections were coated with NTB2 emulsion and exposed for 4 weeks at 4 °C. Finally, the sections were developed at 15 °C in D-19 developer (Kodak) and stained with Ehrlich's hematoxylin. Hybridization was evaluated by visually identifying clusters of silver grains over cell bodies. Controls included sections treated with a 25-fold excess concentration of cold probe, pretreatment with RNase A, and comparison of labeling patterns of in situ hybridization with patterns visualized by immunocytochemical staining for PE peptide products on adjacent sections. To further insure probe specificity, combined techniques of immunocytochemistry, using antisera against [MetS]enkephalin (gift from Dr. B.E. Maley), and in situ hybridization were performed on the same tissue sections taken from 14-day-old rats. Combined immunocytochemistry and in situ hybridization
All buffers and reagents used for immunostaining were prepared
with water that had been treated with 0.1% I)PC. Sections ~cl~ reacted with primary rabbit antisera (I:351XJdilution t against Melenkephalin, previously characterized to have limited cross reacti~ ity with Leu-enkephalin 24, following the avidin-biotin pcroxidasc method (Vectastain; Vector Labs., Burlingamc, CA) with 0.05~,,~ 3-3" diaminobenzidine (DAB) used to visualize the reaction product. Immunoreactivity was not observed in tissue sections trcalcd with antisera preabsorbed with excess [Met 5} and [Leu~]enkephalin (24/~g/ml of antiscra) ensuring immunocytochemical specificity ~t the [MetS]enkephalin antisera. After treatment with DAB the sections were washed ~' ~ i0 rain in DPC water, treated with Proteinase K for I0 rain. thcn washed again in DPC water for 3 × 10 min. To eliminate excessive background, the sections were immersed in 0.1 M triethanolamine pH 8.0 followed by addition of acetic anhydride (while stirring) to a final v/v concentration of 0.5% acetic anhydride. After incubating for 10 min in the acetic anhydridc-triethanolaminc solution, the sections were washed 2 × 5 min in 2× SSC then 3 :,: lt) rain in DPC water followed by the prehybridization stcp as outlined above+
RESULTS Proenkephalin m R N A was not detected in neural cells of the cerebeUar cortex at postnatal day (PND) 1, although labeled n o n - n e u r a l cells were observed in the pial layer as well as in the choroid plexus of the fourth ventricle (not shown). A t PND 7, low intensity hybridization signal was observed over the I G L with a somewhat stronger signal over the Purkinje cell layer whose origin could not be positively identified, although its location suggests that it probably emanates from Purkinje cells and/or immediately adjacent cells such as B e r g m a n n glial cells. This diffuse hybridization signal was identified in all of lobule X and in portions of lobules I through IX (Figs. IB and 2A,B) and appears to represent the onset of PE m R N A expression by these cells. Overall, the PE hybridization signal appeared to be more intense caudally with a gradual caudal to rostral decrease in both area and intensity. A t PND 7, the intensity of PE hybridization signal over the molecular layer and E G L was not above background. A t PND 14, distinct cellular labeling for PE m R N A was observed in a subpopulation of Purkinje cells in all lobules of the vermis with lobules V. VI. VIII and IX appearing to have the greatest proportion of labeled cells (Figs. 1C and 2D). As was observed in the 7 day-old animals, there appeared to be a caudal to rostral gradient with respect to the proportion of Purkinje cells displaying positive hybridization signal for PE m R N A as well as to the intensity of signal observed (Figs. 1C and 2C,D). In addition, diffuse hybridization signal, similar to that observed on PND 7. was also identified over the Purkinje cell layer in regions where no distinct cellular labeling was observed. Also, as was the case with P N D 7 animals, PE hybridization signal over the molecular layer and E G L was not above background. By PND 21, hybridization signal for PE m R N A ex-
65
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Fig. 1. Camera lucida drawings of midsagittal sections of cerebella from postnatal day (PND) 1 (A), PND 7 (B), PND 14 (C), and PND 21 (D) rats illustrate patterns of proenkephalin (PE) hybridization signal over the respective Purkinje cell layers. Large dots represent areas of distinct cellular labeling of Purkinje cells. Small dots represent layers of diffuse PE hybridization signal associated with the Purkinje cell layer. The intensity of PE mRNA expression by Purkinje cells displays a caudal to rostral gradient during maturation. Bar = 1 mm.
pression was detected in a subpopulation of Purkinje cells that appeared to be dispersed evenly between the anterior and posterior lobules of the midsagittally sectioned cerebellar vermis without the caudal to rostral pattern observed in younger animals (Figs. 1D and 2E, F). Although this study focused on the cerebellar cortex with particular emphasis on Purkinje cells, PE mRNA
hybridization signal was also observed over Golgi cells of the IGL (Figs. 2 B - F and 3C) and the deep cerebellar nuclei as early as PND 7 (Fig. 2A). PE hybridization signal was also detected over the nucleus of the solitary tract in PND 14 (not shown) and 21 rats (Fig. 2E) as previously described by Morita et al. 14. There was not, however, any evidence of specific cellular labeling within
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Fig. 3. A,B: brightfield photomicrographs displaying co-localization of proenkephalin (PE) hybridization signal and enkephalin immunoreactivity in Purkinje cells (P) of postnatal day (PND) 14 rats (arrows). Note the greater intensity of labeling over Purkinje cells in (A) as compared to the cells in (B), this demonstrates that even within a given lobule, intensity of hybridization signal for PE mRNA may vary among the Purkinje cells. MOL, molecular layer; IGL, internal granular layer. Bar for A,B = 25 #m. C,D: lobule X from PND 14 rats displaying PE hybridization signal over Golgi cells of the IGL in (C) and adjacent section after treatment with a 25-fold excess of cold probe (D). Bar for C,D = 100 #m.
Fig. 2. Darkfield photomicrographs of proenkephalin (PE) hybridization signal using [3~S]-labeled cRNA probe in midsagittal sections of rat cerebella from postnatal day (PND) 7 (A,B), PND 14 (C,D), and PND 21 (E,F). A: lobule I. Distinct cellular labeling was evident over the deep cerebellar nuclei (DCN), otherwise only a layer of low intensity hybridization signal over Purkinje cell layer (P). B: portions of lobules IX & X. There was an apparent increase in the area and intensity of PE hybridization signal over the Purkinje cell layer in lobules IX and X as compared to (A) lobule I of same section. During development, PE hybridization signal was first observed in cells of the internal granular layer (IGL) which resemble Golgi cells (arrows). C: lobule II. PE hybridization signal was associated with Golgi cells of the IGL but not expressed by Purkinje cells. D: lobule IX of the same section as (C). First evidence of distinct cellular labeling of Purkinje ceils with PE hybridization signal (arrowheads). E,F: lobules II and I respectively (E) and Lobule X (F). At PND 21, PE hybridization signal is more uniformly distributed among Purkinje cells in different lobules, and PE hybridization signal intensity is more similar over Golgi and Purkinje cells. Intense labeling can be seen over cells in the nucleus of the solitary tract (NTS). M, molecular layer; BN, brainstem nuclei. Bars = 100/~m.
68 the molecular layer or E G L of any animal used in this study; a pattern which is consistent with previous studies that examined PE mRNA expression by in situ hybridization in older and adult rats 9'19. Loss of PE mRNA hybridization signal in sections treated with an excess concentration of cold probe (Fig. 3D) or pretreated with RNase A assured specificity of the probe (not shown). Moreover, co-localization of PE hybridization signal and enkephalin immunoreactivity in the same tissue section served as positive controls (Fig. 3A,B). DISCUSSION The aim of this study was to characterize the expression of PE mRNA in neural cells of the developing rat cerebellar cortex and to identify possible relationships between the cellular location of PE gene expression and the cellular targets of opioid action during cerebellar maturation. Animals were chosen whose ages ranged from the day of birth (PND 1) through PND 21 because key stages of the cerebellar cortical maturation occur at these times 1-3. Moreover, by PND 21, Purkinje cell dendrites have begun to elaborate and form synapses with granule cell axons (parallel fibers) similar to those found in the adult cerebellum 3. The results indicate that by PND 7, patches of diffuse hybridization signal (above background) can be identified in the IGL of lobules I through IX and over most of the IGL of lobule X (Figs. 1B and 2A,B). Moreover, in these same areas, a more intense band of silver grains was observed over the Purkinje cell layer (Fig. 2A,B). Although present hybridization conditions in our lab do not permit distinct cellular localization of this discrete layer of silver grains at PND 7, its location indicates probable association with Purkinje cells, glial cells or both. This observation is consistent with previous studies by Spruce et a1.19 who assessed PE mRNA and peptide expression in 25 day-old rats. The relative size and signal intensity of these areas yield a definite caudal to rostral pattern with the larger, more intense regions lying in the posterior lobules of the vermis (Figs. 1B and 2B). This gradient of signal distribution suggests a correlation between PE mRNA expression and the chronological order of Purkinje cell settling and maturation in the rat as has been previously reported by Altman 2 and Altman and Bayer 6 and in mice by Andreoli et al. 7. These studies reported that the Purkinje cells of the cerebellar vermis are produced in a caudal to rostral order with the Purkinje cells of lobule X through VI of the posterior vermis being produced earlier than those in the anterior vermis with the exception of a longitudinal band of early forming Purkinje cells termed the 'vermal wedge'
by Altman 3, Altman and Bayer" and Andreoli et al. This hypothesis is supported by the distribution of hybridization signal observed on sections of PND 14 animals (Figs. 1C and 2C,D). Examination of sections from this age group indicate distinct cellular labeling over a subpopulation of Purkinje cells with a greater proportion of labeled cells and increased intensity of signal observed in the posterior lobules (Figs. IC and 2C,D). The expression of opioids has been associated with a maturational process in the cerebellum of another species. Walker and King 24 have demonstrated medial to lateral gradients of enkephalin immunoreactivity ill afferents in the North American Opossum. By PND 21, there appeared to be a fairly uniform distribution of hybridization signal for PE mRNA in the Purkinje cells of the anterior and posterior regions of the cerebellar vermis (Figs. 1D and 2E,F), There are, however, some variations among the Purkinje cells in the individual lobules within each region. For example, a subpopulation of Purkinje cells in lobules IXb and IXc express PE mRNA at PND 14 but do not at PND 21. This suggests a possible transient expression of PE mRNA by a subpopulation of Purkinje cells which could alter the intensity of signal as well as the number of cells expressing the PE gene at any time during development. The notion that there is transient expression of PE mRNA by Purkinje cells is even more interesting considering the studies which describe transient enkephalin (a posttranslational peptide product of PE) immunoreactivity in E G L cells of the developing cerebellum 26. The spatiotemporal pattern of E G L cell migration and settling closely follows that of Purkinje cells 6. Equally as interesting is the fact that expression of PE mRNA has not been detected in the developing neural progenitor cells of the vermal EGL or their mature counterparts, i.e., the granule cells of the IGL (present study and Spruce et al.l'). This absence of detectable signal suggests that other opioid genes (prodynorphin or proopiomelanocortin) may be involved in the production of transient enkephalin-like immunoreactivit~ in EGL cells26 or that sources of enkephalins originating from outside the EGL cells may be internalized (e.g., receptor-mediated endocytosis) and therefore m~ly bc responsible for the transient appearance of enkephalin immunoreactivity by EGL cells as previously reported by Zagon et al. 2~'. In summary, present data indicate that Purkinje cells, in particular, express PE mRNA early during development and undergo complex patterns of expression during ontogeny. This supports the hypothesis that expression of PE mRNA in Purkinje cells of the developing cerebellar vermis closely follows the chronological order of settling and maturation of these neurons. The evi-
69 dence also suggests that, in light of recent studies link-
global (caudal to rostral) maturational gradients that
ing [MetS]enkephalin (a product of the p r o e n k e p h a l i n gene) to n e u r o n a l growth and development ~°'11"25"27"28
govern PE expression by Purkinje cells. A more detailed
and the fact that endogenous opioids dramatically influ-
expression by Purkinje cells in the developing cerebel-
ence the elaboration of Purkinje cell dendrites and dendritic spines 1°'11, the expression of PE m R N A may be
lum may further clarify the role of PE gene expression by Purkinje cells during development.
spatiotemporal assessment of the patterns of PE m R N A
important for Purkinje cell development. Yet, differences in levels of PE m R N A expression between individual Purkinje cells suggest that subtle differences in m i c r o e n v i r o n m e n t or intrinsic timing impose local constraints on PE expression that may override the more
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Acknowledgements. The authors wish to thank Mrs. Aruna Bhat, Drs. Anne Stiene-Martin, S.L. Sanders, K.B. Seroogy and B.E Sisken for assistance and/or helpful comments; Dr. M.H. Melner for providing the cDNA probe and Dr. B.E. Maley for providing enkephalin antisera. Supported by NIDA Grant DA 06204.
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