Sequential effects of thyroxine on the developing cerebellum of rats made hypothyroid by propylthiouracil

Brain Research, 161 (1979)469-479 © Elsevier/North-Holland Biomedical Press

469

S E Q U E N T I A L EFFECTS OF T H Y R O X I N E O N T H E D E V E L O P I N G C E R E B E L L U M OF RATS M A D E H Y P O T H Y R O I D BY P R O P Y L T H I O U R A C I L

A. RABII~*, C. FAVRE, M. C. CLAVEL and J. LEGRAND Laboratoire de Physiologie compar(e, Universitd des Sciences et Techniques du Languedoc, 34060 Montpellier Cedex (France)

(Accepted June 8th, 1978)

SUMMARY Young rats made hypothyroid by propylthiouracil (PTU) received a daily physiological dose of thyroxine (T4) from day 0, 4, 6, 8, 10, 11, 12 or 13 and their cerebella were studied on day 14. With the very low doses of T4 used and when the treatment was started at birth, cerebellar development was nearly normal in terms of the parameters studied (cell formation, migration, maturation and death). The effect of T4 on cell formation appeared after two days. With the same latency, T4 induced migration of the newly-formed granule cells. The effects on those processes requiring cell movements over long distances, e.g. the number of cells in the internal granular layer or the thickness of the molecular layer, were longer to appear. The most rapidly affected parameter was the pyknotic index in the internal granular layer. This index was half reduced by T4 after only one day and was completely corrected within only 4 days. The increased cell death in the cerebellum of hypothyroid rats is probably related to the decreased synaptogenetic competence of Purkinje cells. The rapidity of the effect of T4 on the pyknotic index may be related to an important effect of this hormone on the formation of synapses and, more generally, on the mechanisms of neuronal maturation.

INTRODUCTION It is well documented that thyroid hormones are involved in regulation of cell formation and cell maturation in the mammalian developing CNS (for reviews see refs. 3 and 9). In the cerebellum, thyroid hormone deprivation leads to delayed acquisition of the normal number of cells 5, to abnormal maturation of cerebellar cells, * Attach6 de Recherche au C.N.R.S.

470 especially of the Purkinje cells 11, and to increased cell death within the internal granular layera6,19. On the contrary, administration of relatively large doses of triiodo thyronine (T3) or thyroxine (T4) accelerates cerebellar cell maturation and cell formation x°,z3, but finally leads to a reduced number of cells 4,14. Corrective effects of large or small doses of T4 on cerebellar maturation have been demonstrated in young thyroid-deficient rats6,11,12,19. The dose of T4 required in thyroidectomized animals to restore the plasma concentration to normal within a short time after the administration of the hormone has recently been calculated from the study on the development of rat thyroid function by Vigouroux z~,22. Legrand et al. ~4 have shown that treatment of propylthiouracil (PTU)-treated rats with such doses of T4 corrected the histological maturation as well as cell formation in the developing cerebellum, thus demonstrating the physiological role of T4 in structural maturation and cell formation in the developing cerebellum (for review see ref. 13). In the present work, such corrective doses o f T 4 have been injected to young PTU-treated rats from 0, 4, 6, 8, 10, 11, 12 or 13 days of age. Formation, migration, maturation and death of cerebellar cells were studied in 14-day-old animals in order to determine the latency and the sequence of the appearance of the corrective effects of T4 on the main processes of cerebellar maturation. MATERIALS AND METHODS

Animals Offsprings of Wistar rats born on the same day were pooled and divided at random on the day of birth in nursing families of 8 rat pups each. The normal length of gestation was carefully verified and the day of birth was considered as day 0. Normal, thyroid-deficient and thyroid-deficient supplemented with thyroid hormone young rats were studied. The young rats were made hypothyroid by treating the mother with daily doses of 50 mg propylthiouracil, by gastric intubation from the 18th day of gestation until 14 days post-partum. In some litters of PTU-treated animals, the young rats received in addition a daily subcutaneous injection of e-thyroxine from day 0, 4, 6, 8, 10, 11, 12 or 13. The doses ofT4 employed were progressively increasing with age as shown in Fig. 1. More precisely, the doses of T4 were those calculated by Vigouroux 21,z2 to restore to normal the plasma T4 concentration just after the injection; each day, the dose injected corresponded approximately to the amount of hormone normally secreted by the gland. It was previously shown in our laboratory that such a treatment corrected the disturbances in the histological maturation of the cerebellar cortex and in the postnatal increase of the DNA content of the cerebellum in PTU-treated rats 14. All the animals were killed on day 14.

Histological study At the age of 14 days, in each experimental group, three young rats were killed by exsanguination and their cerebellum was fixed in Carnoy fixative and embedded in paraffin. Midsagittal sections of the vermis (5 #m thick) were stained with cresyl-violet. All histological observations and measurements were made on both sides (declive

471

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and culmen) of fissure E. The total area of the cerebellar cortex as well as the areas of the external granular layer, the molecular layer and the internal granular layer were measured by planimetry after projection of the section with a Gillett and Sibert microscope. All the cells in the external granular layer were counted, while in the molecular layer or the internal granular layer the number of cells with the morphological features of migrating or differentiating and mature granule cells was estimated under the microscope in 6 areas randomly chosen and representative of the entire thickness of the layer studied. The mean diameter of granule cell nuclei was also determined and the total number of granule cells in each layer was calculated and corrected according to Abercrombie z. The number of mitoses in the external granular layer, the number of internal granule cells showing a pyknotic nucleus, the number of Purkinje cells whose section passed through the nucleus and the thickness of the molecular layer (15-20 measurements per animal) were also determined. The correction applied for the calculation of the number of Purkinje cells was the same as for the granule cells. The pyknotic index was the ratio, expressed in per cent, of the number of pyknotic nuclei to the uncorrected number of internal granule cells 15. Since the thyroid state has a marked influence on the development of the area of the cerebellar cortex, but has no effect on the total number of Purkinje cellsT, zT, the numbers of cells and mitoses were expressed per Purkinje cell.

Statistical analysis The results were analysed by a one-way analysis of variance (treatment). All paired comparisons of means were made by using the Duncan's multiple range test s . The mean value for each group of PTU-treated animals supplemented with thyroxine was compared with both the normal and the hypothyroid values. A significance level of P < 0.05 was used in the Duncan's test. RESULTS

Corrective effects of T4 on growth of the cerebellar cortex in PTU-treated rats In the 14-day-old PTU-treated rats, the total area of the cerebellar cortex around fissure E was 31% less than in controls. This decrease could be attributed to a reduction in the area of the internal granular layer (--22 %) and especially of the molecular layer (--57 ~o) (Fig. 2; see also Fig. 4 for the thickness of the molecular

472

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Fig. 2. Effect of PTU-treatment on the total area of the cerebellar cortex and on the area of the different layers of the cerebellar cortex around fissure E in midsagittal sections of the cerebellum of 14-day-old rats. egl, external granular layer; ml, molecular layer; igl, internal granular layer. Treatment significance after analysis of variance :P < 0.01. *, significant difference (P < 0.05) with the normal value after Duncan's multiple range test.

layer). On the contrary, the area of the external granular layer was increased by 71 ~o (Fig. 2). These anomalies were corrected, depending on age, by the administration of T4 (Figs. 3 and 4). Treatment initiated at day 8 or 10 when compared with that at earlier ages led to a transient increase in the estimates on the areas of the molecular and internal granular layers (Fig. 3d and f). This was also reflected in the values of internal granular layer to whole cerebellar cortex ratio (Fig. 3g), and was probably due to the fact that, in contrast with the rapid effects observed in midsagittal sections of the cerebellum of rats receiving T4 from day 8 or 10, growth was manifested in the three dimensions of space when treatment was started at birth. It was of interest that T4 replacement initiated at day 10 or later did not prevent the increase in the area of the external granular layer characteristic of the thyroid-deficient state (Fig. 3b).

Effects on cell formation In the thyroid-deficient rats, the area of the external granular layer was increased (Fig. 3b). Since the packing density of cells here was normal, it follows that the total number of cells in the area of the external granular layer around the primary fissure was also increased. The same trend was evident when the number of external granule cells was expressed in terms of the Purkinje cells, although the results ( 1 3 6 ~ of control) did not reach the level of significance set in this study. When treatment with T4 was begun from day 0, 4 or 6, the number of cells in the external granular layer per Purkinje cell was normal. In contrast, T4 given from day 11 led to a marked increase in the number of external granule cells per Purkinje cell (198 ~ of control) (Fig. 5a). The number of mitoses in the external granular layer (expressed per Purkinje cell) was 136 ~ higher in thyroid-deficient than in the normal rats. When treatment with T4 was started before the age of 10 days, the number of mitotic cells was

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Fig. 3. a, b, d, f: total cerebellar cortex (a), external granular layer (b), molecular layer (d) and internal granular layer (f) areas in midsagittal sections of the cerebellum around fissure E. c, e, g: ratios of the area of the external granular layer (c), the molecular layer (e) and the internal granular layer (g) to the total cerebellar cortex area in midsagittal sections of the cerebellum around fissure E. In abscissa, (1) H: 14-day-old PTU-treated animals; 13, 1 2 . . . 0: 14-day-old PTU-treated animals receiving in addition a daily injection of T4 from day 13, 1 2 . . . 0; N: 14-day-old normal animals. (2): corresponding number of days of treatment with T4 prior to killing at 14 days post-partum. Treatment significance after analysis of variance: P < 0.01 for a, b, c, d, e, fand g. *, significant difference (P < 0.05) with the PTU-treated animals after Duncan's multiple range test.

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Fig. 4. Thickness of the molecular layer in midsagittal sections of the cerebellar cortex around fissure E. In abscissa, (1) H: 14-day-old PTU-treated animals; 13, 1 2 . . . 0; 14-day-old PTU-treated animals receiving in addition a daily injection of T4 from day 13, 1 2 . . . 0; N: 14-day-old normal animals. (2): corresponding number of days of treatment with T4 prior to killing at 14 days post-partum. Treatment significance after analysis of variance: P < 0.01. *, significant difference (P < 0.05) with the PTUtreated animals after Duncan's multiple range test.

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Fig. 5. Number of cells in the external granular layer (a), of migrating granule cells in the molecular layer (b) and of granule cells in the internal granular layer (c) expressed per Purkinje cell in midsagittal sections of the cerebellar cortex around fissure E. In abscissa, (1) H: 14-day-old PTU-treated animals; 13, 1 2 . . . 0: 14-day-old PTU-treated animals receiving in addition a daily injection of T4 from day 13, 1 2 . . . 0 ; N: 14-day-old normal animals. (2): corresponding number of days of treatment with T4 prior to killing at 14 days post-partum. Treatment significance after analysis of variance: P < 0.01 for a and c; P < 0.05 for b. *, significant difference (P < 0.05) with the PTU-treated animals after Duncan's multiple range test.

475

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Fig. 6. Number of mitoses per Purkinje cell in midsagittal sections of the external granular layer around fissure E. In abscissa, (1) H: 14-day-old PTU-treated animals; 13, 1 2 . . . 0: 14-day-old PTU-treated animals receiving in addition a daily injection of T4 from day 13, 1 2 . . . 0; N: 14-day-old normal animals. (2): corresponding number of days of treatment with T4 prior to killing at 14 days post-parturn. Treatment significance after analysis of variance: P < 0.01. *, significant difference (P < 0.05) with the PTU-treated animals after Duncan's multiple range test. corrected. I n contrast, when the treatment was started at day 12, the n u m b e r o f mitoses was increased above the hypothyroid value (Fig. 6).

Effects on cell migration It would appear that thyroid h o r m o n e influences cell migration. I n the thyroiddeficient animals, there was a tendency for a reduction in the n u m b e r of migrating cells (per Purkinje cell) in the molecular layer, whereas their n u m b e r was significantly increased after T4 had been given f r o m day 8, 10 or 11 (Fig. 5b). The n u m b e r of internal granule cells (per Purkinje cell) was 32 ~ less in the h y p o t h y r o i d than in the control rats (see also refs. 14 and 17). This decrease was corrected by T4-treatment, the m a x i m u m effect being observed when T4 was given f r o m day 8 (Fig. 5c). The n u m b e r of migrating granule cells in the molecular layer relative to the n u m b e r o f internal granule cells was n o t significantly affected in the h y p o t h y r o i d animals. However, T4 given to these f r o m day 12 triggered a rapid and marked increase (Fig. 7).

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Fig. 7. Ratio of the number of migrating granule cells in the molecular layer to the number of internal granule cells in midsagittal sections of the cerebellum around fissure E. In abscissa, (1) H: 14-day-old PTU-treated animals; 13, 1 2 . . . 0: 14-day-old PTU-treated animals receiving in addition a daily injection ofT4 from day 13, 1 2 . . . 0; N: 14-day-old normal animals. (2): corresponding number of days of treatment with T4 prior to killing at 14 days post-partum. No significant difference after analysis of variance. *, significant difference (P < 0.05) with the PTU-treated animals after Duncan's multiple range test.

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Fig. 8. Pyknotic index in the internal granular layer in midsagittal sections of the cerebellar cortex araund fissure E. In abscissa, (1) H: 14-day-old PTU-treated animals; 13, 12... 0: 14-day-old PTUtreated animals receiving in addition a daily injection of T4 from day 13, 12... 0; N: 14-day-old normal animals. (2): corresponding number of days of treatment with T4 prior to killing at 14 days post-partum. Treatment significance: P < 0.01. *, significant difference (P < 0.05) with the PTUtreated animals after Duncan's multiple range test.

Effects on cell death within the internal granular layer In accordance with previous observationsl6,19, the pyknotic index increased by a factor of about 100 in the internal granular layer of the cerebellar cortex in the thyroiddeficient rats. A single injection o f T 4 on day 13 was sufficient to reduce the number of pyknotic nuclei by a factor of 2. Treatment with T4 from day 10 restored to normal the number of dying cells in the internal granular layer (Fig. 8). DISCUSSION The present work confirmed that low doses of T4 can correct the anomalies in the development of the cerebellar cortex in PTU-induced hypothyroidism. This observation was an important condition for validating the experimental procedure used to determine the latency of the T4 effect. The results obtained in the animals receiving hormone replacement from the day of birth clearly showed that the treatment was effective: none of the 14 parameters studied differed significantly from normal. This confirmed the physiological quality of the doses used. It has been shown that the rate of cell formation was reduced by hypothyroidism during the first two postnatal weeks. There was a delay in comparison with controls in cell acquisition in the cerebellum of thyroid-deficient animals. However, cell multiplication continued until 35 days in these animals while it stopped at 21 days in normals~,11A s. At the age of 14 days, the number of mitoses in the external granular layer was higher in hypothyroid than in normal animals (Fig. 6). Fourteen days seems to be the age when cell formation begins to be higher in hypothyroid rats than in controls. Moreover, the observations on the animals receiving replacement therapy were consistent with previous results on hyperthyroid rats 1°,14,23 and indicated that thyroid hormone can accelerate cell formation in the cerebellum. T4 treatment induced a transient wave of cell multiplication in the external granular layer. The number of mitoses per Purkinje cell was increased by 45 7ooafter treatment with T4 for two days only. Since the number of mitotic cells was unaffected by a single injection of T4 one day before sacrificing the animals, a latency of about two days is necessary for the

477 action of T4 on cell replication (Fig. 6). It seems that a significant number of cells whose differentiation was delayed by hypothyroidism were triggered to divide by T4. This transient wave of newly-formed cells was reflected in an increase in the number of migrating granule cells in the molecular layer and finally in the number of internal granule cells. The greatest number of external granule cells was observed when T4 was given from day 11, i.e. after a latency of about three days (Fig. 5a). The number of migrating granule cells in the molecular layer was increased maximally when T4-treatment was started at day 8 or 10, i.e. after 4--6 days of latency (Fig. 5b). The maximum number of internal granule cells was obtained after a latent period of 6 days (Fig. 5c). The results, therefore, indicate that (1) T4 administered at physiological doses induces cell multiplication in the external granular layer with a delay of 2 days; (2) the newlyformed granule cells remain about one day in the external granular layer; (3) their migration through the molecular layer lasts for about 3 days and (4) they reach the internal granular layer approximately 4 days after their formation. These results are in good agreement with those of Altman 2 using tritiated thymidine to label the cerebellar granule cells in normal developing rats. The most striking and perhaps surprising result was that the parameter most rapidly affected by T4-treatment was the pyknotic index in the internal granular layer (Fig. 8). Lewis et al. 16 have related the increased number of dying cells to the retardation of the dendritic arborization of Purkinje cells demonstrated by Legrand11. They have proposed that due to the hypoplasia of Purkinje cells, a fraction of the granule cells fail to make synaptic contact and dies. Rabi6 et al. 19 have supported this hypothesis by demonstrating that, in thyroid-deficient rats given various doses of T4, the abnormally high cell loss was related to the thickness of the molecular layer, i.e. to the development of the Purkinje cell dendritic arborizations, rather than to the number of granule cells present in the internal granular layer. The 47 ~ decrease of the pyknotic index after injection of a single physiological dose of thyroxine one day before the sacrifice of the animal, as well as the complete correction after only 4 days of treatment, is consistent with a very rapid effect of thyroid hormone on the establishment of synaptic contacts between Purkinje cells and granule cells, and therefore on maturation of Purkinje cells. This is in agreement with the previous observations that three injections of a small dose of T4 (0.20 #g on the 6th, 7th and 8th days) in young hypothyroid rats increased rapidly (in 2--4 days) the protein content of the synaptosomal fraction isolated from the cerebellumz0. The main objective of this work was to study in thyroid-deficient animals the latency of the action of T4 given in physiological doses on the different processes of cerebellar development. Our results showed that all the different processes of cerebellar cortex development were affected within a few days after the administration of physiological doses ofT4. The processes requiring only cell maturation (e.g. a reduction in the pyknotic index) and the processes immediately related to cell differentiation (e.g. the mitotic activity in the external granular layer or the beginning of migration of granule cells) were very rapidly affected (in one or two days). The effects on cell maturation, however, appeared more rapidly (in one day) than those on cell proliferation (in two days). The processes requiring cell movements over long

478 distances (e.g. the n u m b e r o f cells in the m o l e c u l a r o r the internal g r a n u l a r layers, the increase in the areas o f these two layers or in thickness o f the m o l e c u l a r layer) required o f course a longer time to react significantly. The fact t h a t a m o n g all the processes studied, the n u m b e r o f dying cells in the internal g r a n u l a r layer was the m o s t sensitive to T4 underlines the very i m p o r t a n t a n d specific effect o f t h y r o i d h o r m o n e on n e u r o n a l m a t u r a t i o n , a n d m o r e particularly, on the establishment o f synapses between neurones.

ACKNOWLEDGEMENTS The a u t h o r s t h a n k Dr. R. Bal/tzs for helpful discussions a n d revision o f the English manuscript. T h e y are i n d e b t e d to Mr. F. C a r u s o a n d Mrs. C. A i m a r for their skilled technical assistance. This w o r k was s u p p o r t e d by the D . G . R . S . T . ( G r a n t 77-7-0965) a n d the I . N . S . E . R , M . ( G r a n t 75-1-204-6).

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479 15 Lewis, P. D., Cell death in the germinal layers of the postnatal rat brain, Neuropath. appl. Neurobiol., 1 (1975) 21-29. 16 Lewis, P. D., Patel, A. J., Johnson, A. L and Bal~zs, R., Effect of thyroid deficiency on cell acquisition in the postnatal rat brain: a quantitative histological study, Brain Research, 104 (1976) 49-62. 17 Nicholson, J. L. and Altman, J., The effects of early hypo- and hyperthyroidism on the development of rat cerebellar cortex. I. Cell proliferation and differentiation, Brain Research, 44 (1972) 13-23. 18 Patel, A. J., Rabi6, A., Lewis, P. D. and BalS.zs, R., Effect of thyroid deficiency on postnatal cell formation in the rat brain: a biochemical investigation, Brain Research, 104 (1976) 33--48. 19 Rabi6, A., Favre, C., Clavel, M. C. and Legrand, J., Effects of thyroid dysfunction on the development of the rat cerebellum, with special reference to cell death within the internal granular layer, Brain Research, 120 (1977) 521-531. 20 Rabi6, A and Legrand, J., Effects of thyroid hormone and undernourishment on the amount of synaptosomal fraction in the cerebellum of the young rat, Brain Research, 61 (1973) 267-278. 21 Vigouroux, E., Ddveloppement de la Fonction ThyroMienne chez le jeune Rat, Th6se de Doctorat-esSciences, Universit6 Paris VI, 1974. 22 Vigouroux, E., Dynamic study of postnatal thyroid function in the rat, Acta endocr., 83 (1976) 752-762. 23 Weichsel, M. E., Effect of thyroxine on DNA synthesis and thymidine kinase activity during cerebellar development, Brain Research, 78 (1974) 455--465.