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Endocrinology 148(12):5746 –5751 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2007-0567

Influence of Thyroid Hormone and Thyroid Hormone Receptors in the Generation of Cerebellar ␥-Aminobutyric Acid-Ergic Interneurons from Precursor Cells Jimena Manzano, Maria Cuadrado, Beatriz Morte, and Juan Bernal Instituto de Investigaciones Biome´dicas Alberto Sols, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Autonomous University of Madrid, and Center for Biomedical Research on Rare Diseases (CIBERER), 28029 Madrid, Spain Thyroid hormones have important actions in the developing central nervous system. We describe here a novel action of thyroid hormone and its nuclear receptors on maturation of cerebellar ␥-aminobutyric acid (GABA)-ergic interneurons from their precursor cells. In rats, the density of GABAergic terminals in the cerebellum was decreased by hypothyroidism, as shown by immunohistochemistry for the GABA transporter GAT-1. This was due, at least partially, to a decreased number of GABAergic cells, because the number of Golgi II cells in the internal granular layer was decreased. GABAergic interneurons in the cerebellum differentiate from precursors expressing the Pax-2 transcription factor, generated in the subventricular zone of the embryonic fourth ventricle from where they migrate to the cerebellum. Hypothyroidism

T

HYROID HORMONES ARE important in development and function of the central nervous system. Hypothyroidism during maturation leads to structural abnormalities of the brain by affecting developmental processes such as neural cell migration, differentiation, synaptogenesis, and myelination. In many instances, the action of thyroid hormones during brain development can be correlated with regulation of gene expression (for reviews see Refs. 1–3). In adults, thyroid hormones influence mood and behavior, and thyroid dysfunction is often associated with psychiatric manifestations (4). Neurotransmitter systems are affected by deficiency or excess of thyroid hormones, both in developing and in adult animals (5–7), but in most cases the cellular and molecular basis for the action of thyroid hormones on neurotransmission remain obscure. Among the neurotransmitters, recent interest has been paid to the ␥-aminobutyric acid (GABA)-ergic system (6). GABA is the major inhibitory neurotransmitter, regulating excitatory glutamatergic activity; during development, GABA may act as an excitatory transmitter and also as a trophic factor involved in developmental processes, such as cell proliferation, migration, and synaptogenesis (8, 9). It has been suggested that disorders of First Published Online August 30, 2007 Abbreviations: BrdU, 5-Bromo-2-deoxyuridine; GABA, ␥-aminobutyric acid; GAT-1, GABA transporter-1; P8, postnatal d 8; TR␣1, thyroid hormone receptor ␣1 isoform. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

caused both decreased proliferation and delayed differentiation of precursors, with the net effect being an accumulation of immature cells during the neonatal period. The contribution of thyroid hormone receptors was studied by treating hypothyroid rats with T3 or with the thyroid hormone receptor (TR) ␤-selective agonist GC-1. Whereas treatment with T3 reduced the number of precursors to control levels, GC-1 had only a partial effect, indicating that both TR␣1 and TR␤ mediate the actions of T3. Deletion of TR␣1 in mice decreased cerebellar GAT-1 expression and Pax-2 precursor cell proliferation. It is concluded that thyroid hormone, acting through the nuclear receptors, has a major role in the proliferation and further differentiation of the Pax-2 precursors of cerebellar GABAergic cells. (Endocrinology 148: 5746 –5751, 2007)

GABAergic neurotransmission, such as anxiety, are central in the psychiatric manifestations of thyroid dysfunction (6). In rats, thyroid hormone deficiency during the fetal and postnatal periods alters normal development of GABAergic circuits in the cortex and hippocampus and is functionally correlated with a decrease of GABA-dependent inhibition (10, 11). In adult mice, absence of the thyroid hormone receptor ␣1 isoform (TR␣1) or expression of a dominant-negative mutant version of this receptor reduces the density of GABAergic terminals in the hippocampus and correlates with altered behavior (12, 13). Cerebellar development is a classic target of thyroid hormones. In addition to interfering with the major developmental landmarks of granular cell migration and Purkinje cell differentiation, neonatal hypothyroidism also delays the postnatal increase in GABA receptor density (14), and lowers the final number of cerebellar interneurons such as basket cells (15). The cerebellar cortex contains three types of GABAergic interneurons, the basket and stellate cells, located in the molecular layer, and the Golgi II cells, located in the granular layer. They differentiate from precursors expressing the Pax-2 transcription factor arising in the cerebellar ventricular zone by embryonic d 13 in the mouse (16). Golgi II cells and the deep cerebellar nuclei interneurons originate directly from these cells, whereas basket and stellate cells result from differentiation of a subset of Pax-2⫹ precursors that migrate to the cerebellar white matter where they undergo a final round of cell proliferation. In this article, we describe a novel action of thyroid hormone and thyroid

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Manzano et al. • Thyroid Hormone and Cerebellar Interneurons

hormone receptors in the cerebellum, the generation of GABAergic interneurons from their Pax-2⫹ precursors. Materials and Methods Animals and treatments Rats from the Wistar strain grown in our animal facilities were used. Protocols for animal handling were approved by the local institutional Animal Care Committee and followed the rules of the European Union. Animals were under temperature-controlled (22 ⫾ 2 C) and light-controlled (12-h light, 12-h dark cycle; lights on at 0700 h) conditions and had free access to food and water. To induce fetal hypothyroidism, the dams received 0.02% methylmercaptoimidazole (Sigma Chemical Co., St. Louis, MO) and 1% sodium perchlorate in the drinking water, starting from the ninth day after conception and throughout the experimental period (17). Hormone treatments consisted of daily single ip injections of 15 ng T3 (Sigma) per gram body weight or 10 ng/g of the TR␤-selective compound GC-1, kindly provided by Dr. T. S. Scanlan (University of San Francisco). In each case, treatment was started 5 d before animals were killed as described previously (18). Null mice for TR␣1 (19) were kindly provided by Dr. B. Vennstro¨m (Karolinska Institute, Stockholm, Sweden). As wild-type controls, we used BALB/c mice. At least four animals from different litters and for each condition were used in each experiment.

Tissue processing The animals were anesthetized by ip injection of a mixture of ketamine (4 mg/100 g body weight) and mededomidine (15 mg/100 g body weight) and then perfused transcardially, with 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4). The brains were cryoprotected, and 25-␮m sagittal sections were obtained in a cryostat and processed as described (17). For histological staining, we used the Nissl or Richardson’s blue procedures (17).

Immunohistochemistry Immunohistochemistry was performed on free-floating sections as described (17). Primary antibodies were rabbit anti-GABA (1:2000; Abcam plc, Cambridge, UK), anti-GABA transporter-1 (anti-GAT-1) (1:500; Abcam), and anti Pax-2 (1:650; Zymed Laboratories, San Francisco, CA). As secondary antibodies, we used a biotinylated goat antirabbit antibody at a 1:200 dilution (Vector Laboratories, Burlingame, CA) followed by ABC (Vector). Peroxidase was then visualized with 0.05% diaminobenzidine and H2O2. For microscopy, we used a Nikon Eclipse E400 optical microscope (Nikon Corp., Tokyo, Japan) equipped with a Nikon DN100 digital camera.

Cell proliferation Cell proliferation analyses were performed by confocal microscopy. The percentage of mitotic Pax-2 cells in postnatal d 8 (P8) rat sections was analyzed after immunofluorescence. The slices were incubated with rabbit anti-Pax-2 (1:650; Zymed Laboratories, San Francisco, CA) and a mouse antibody against the Ki67 nuclear antigen (1:200; Novocastra Labs Ltd., Newcastle, UK) (20), followed by incubation with biotinylated horse antimouse (Vector). After that, the slices were incubated with Alexa Fluor 594 goat antirabbit and streptavidin Alexa Fluor 488 (1:2000; Molecular Probes, Invitrogen, Carlsbad, CA). For microscopy, we used a Leica TCS SP2 confocal microscope (Leica Microsystems GmBH, Wetzlar, Germany). Because this antibody gave unsatisfactory results in mouse sections, P6 mice were injected with the thymidine analog 5-bromo-2-deoxyuridine (BrdU; Sigma), 0.1 mg/g body weight, and the mice were killed 2 h later. BrdU in Pax-2⫹ cells was detected by double immunofluorescence with the rabbit anti-Pax-2 (Zymed) and the mouse antibody anti-BrdU (1:200; Dako, Carpinteria, CA).

Western blotting Cerebella from four P16 rats or P11 mice were pooled and homogenized in 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate,

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0.1% sodium dodecyl sulfate, 10% glycerol, 1 mm EDTA, 50 mm Tris-HCl (pH 8.0) containing 2 ␮g/ml leupeptin, 2 ␮g/ml aprotinin, and 100 ␮g/ml phenylmethylsulfonyl fluoride. Aliquots of 50 ␮g of the extracts were electrophoresed in 12% polyacrylamide gels and transferred to nitrocellulose blotting membranes (Schleicher & Schuell, Keene, NH). The membranes were washed in 0.5 m NaCl and 20 mm Tris-HCl (pH 7.5) containing 0.1% Tween 20 and then in 5% skimmed milk. Finally, the membranes were incubated with anti-GAT-1 (1:750; Abcam) and anti-␤-tubulin (1:800; Sigma) antibodies and developed with LUMINOL (Santa Cruz Biotechnology, Santa Cruz, CA). For quantification, the films were scanned, and densitometry was measured using the Scion Image program version 4.02 (http://www.scioncorp.com).

Cell counting and data analysis Cells were counted in lobule VIII of the cerebellum after staining with specific antibodies. We used the AnalySIS program (Soft Imaging Systems GmBH, Mu¨nster, Germany) to count all labeled cells in the lobule from binary images of digital microphotographs. Data were from a minimum of five slices of each individual cerebellum, and the values of three to four animals were recorded for each age and condition. The data were analyzed using the statistic GraphPad Prism software (http:// www.graphpad.com). All data are expressed as mean ⫾ se. Statistical analysis used the Student’s t test for pairs and ANOVA for multigroup comparisons.

Results

Among the GABA transporters, GAT-1 is predominantly expressed in the axons and terminals of GABAergic neurons (21, 22). Using GAT-1 immunohistochemistry, we observed a decreased GAT-1 immunoreactivity in cerebellar slices of developing P16 hypothyroid rats. In agreement with other studies (22, 23), GAT-1 immunoreactivity was observed in normal rats (Fig. 1A) as a punctate pattern throughout all layers of the cerebellar cortex with the most prominent staining in the molecular layer and around the bodies of Purkinje cells. In hypothyroid rats, GAT-1 staining was decreased in all layers, with an almost total loss of staining around the Purkinje cell bodies. Western blotting of whole cerebella also showed a reduction of GAT-1 protein (Fig. 1B) in hypothyroid rats. The decreased GAT-1 immunoreactivity could be due to multiple causes, including lower expression of the GAT-1 gene or decreased cell number and/or density of GABAergic terminals. Thyroid hormone promotes dendritic and axonal growth, and effects on GABAergic synapses (14) and fiber density (11) have been previously demonstrated. Additionally we checked whether an effect on the number of GABAergic cells was also implicated. To this end, we performed immunohistochemistry for GABA (Fig. 1C). The GABA antibody stained multiple cell types, including GABAergic interneurons in the molecular layer, Purkinje cells, and Golgi II cells in the internal granular layer. These cells appeared as large darkly stained cells. The total number of Golgi cells was counted in lobule VIII. The results showed that the number of Golgi II cells was reduced in hypothyroid animals (Fig. 1D). Cerebellar GABAergic cells arise from a common precursor expressing the forkhead transcription factor Pax-2 (16). These precursors are generated in the embryonic neuroepithelium of the fourth ventricle, from where they migrate to the developing cerebellum and differentiate into Golgi II cells and interneurons of the deep cerebellar nuclei. A subset of precursors migrates to the cerebellar white matter where

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Manzano et al. • Thyroid Hormone and Cerebellar Interneurons

FIG. 1. A, Immunohistochemistry for GAT-1 in P16 cerebella from normal and hypothyroid rats and Nissl stain of the same sections. Scale bar, 25 ␮m. B, Western blot for GAT-1 and ␤-tubulin as control. C, Immunohistochemistry for GABA in cerebellum of P16 normal control rats and hypothyroid rats. Scale bar, 100 ␮m. D, Total number of Golgi II cells in the internal granular layer (igl) of lobule VIII. C, Control rats; H, hypothyroid rats; ml, molecular layer; pcl, Purkinje cell layer; egl, external granular layer. ***, P ⬍ 0.001.

they undergo new rounds of proliferation (16, 24, 25). The postmitotic cells generated here then migrate into the molecular layer and differentiate into other classes of mature GABAergic cells, the basket and stellate cells. Figure 2 illustrates the distribution of Pax-2-positive GABAergic precursors after counterstaining with Richardson’s blue. The precursor cells appear as dark dots located in the white matter, and in the internal granular and molecular layers. In the process of differentiation, the precursors lose Pax-2 expression and produce GABA. The effect of thyroid hormone deficiency on the number of GABAergic cells could therefore be due to effects on precursor generation, migration, or terminal differentiation. As a first approximation to this problem, we examined the cerebellar slices for Pax-2 immunoreactivity at different times during postnatal development. Figure 3A shows the pattern of Pax-2 staining in lobule VIII at different times of postnatal development. The Pax-2 antibody clearly and specifically stained the nuclei of cells located in the granular and molecular layer, and in the white matter. On P4 the distribution (not shown) and the number (Fig. 2B) of Pax-2⫹ cells was very similar in euthyroid and

FIG. 2. Double staining with Pax-2 antibody and Richardson’s blue in a P6 mouse cerebellum. The plate shows Pax-2-positive cells as black dots distributed in the white matter (wm) and in the internal granular layer (igl)/molecular layer (ml). Scale bar, 100 ␮m. egl, External granular layer.

hypothyroid rats. The number of Pax-2⫹ cells increased thereafter reaching a maximum around P8. This was followed by a sharp decreased with few cells stained on P10 in the white matter and on P16 in the granular and molecular layers. The number of cells present in the white matter was identical in euthyroid and hypothyroid rats from P4 to P8. Thereafter, the loss of Pax-2 immunoreactivity proceeded at a slower rate in the hypothyroid animals. The temporal pattern of Pax-2 immunoreactivity in the granular and molecular layers was similar to that of the white matter, but the difference between euthyroid and hypothyroid animals was already significant on P6. To check whether hypothyroidism influenced precursor cell proliferation, we used confocal microscopy to examine the Pax-2 cells that were positive for the Ki67 nuclear antigen in the white matter. The result of this experiment showed that on P8, around 30% of Pax-2⫹ cells were also positive for Ki67 in the control animals, and 10% in the hypothyroid animals (Fig. 4). Most actions of thyroid hormones are mediated through the nuclear receptors for T3, TR␣1 and TR␤ (26). To examine the relative contribution of each receptor subtype, we compared the effect of T3 with that of the TR␤-selective agonist GC-1 on the number of precursors. Hypothyroid rats were treated with T3 or GC-1, and the number of Pax-2⫹ cells remaining at P16 in the internal granular and molecular layers were counted (Fig. 5). Hypothyroid rats had a higher number of Pax-2⫹ cells than control rats. The number of cells was completely normalized by T3. GC-1 had a partial effect, indicating that both TR␣1 and TR␤ contributed to the effect of T3. To define in more detail the contribution of TR␣1, we examined the effect of deletion of this receptor in mice (TR␣1⫺/⫺ mice (19). Whole cerebellum GAT-1 content was greatly decreased in TR␣1⫺/⫺ mice by Western blot analysis (Fig. 6A). In the wild-type mice, the number of Pax-2⫹ precursors increased postnatally in the white matter as previ-

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Manzano et al. • Thyroid Hormone and Cerebellar Interneurons

Endocrinology, December 2007, 148(12):5746 –5751

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FIG. 3. A, Pax-2 immunohistochemistry of lobule VIII from normal control (C) and hypothyroid (H) rats at different postnatal days; the discontinuous line delimits the white mater. B, Total number of Pax-2⫹ cells in the white matter (wm) and in the combined internal and molecular layers (igl⫹ml) at different postnatal days. The continuous line represents the data from normal rats. Hypothyroid rats are represented by the discontinuous line. Asterisks, P ⬍ 0.05.

ously shown in normal rats (Fig. 6B), with a doubling in cell density from P4 –P6 followed by a decrease during the next days. The increase in cell number was not observed in the white matter of TR␣1⫺/⫺ mice, which displayed similar cell numbers at P4 –P10. To examine whether precursor proliferation was affected by TR␣1 deletion, we administered the thymidine analog BrdU to P6 mice 2 h before killing and then performed a combined BrdU plus Pax-2 immunofluorescence. The number of Pax-2⫹ cells that were also positive for BrdU was much lower in the TR␣1⫺/⫺ mice, indicating a reduced rate of precursor proliferation (Fig. 6C). TR␣1⫺/⫺ mice also had a lower than normal number of Pax-2 precursors in the white matter already on P4, reflecting a decreased precursor generation or migration from the ventricular layer. In the molecular and granular layers of wild-type mice, precursor cell number increased from P8 to P10, probably reflecting the migration of precursors from the white matter (Fig. 6D). This process was also blunted in the absence of TR␣1.

FIG. 4. Immunoflurescence for Pax-2 (red) and the Ki67 antigen (green); the arrowhead shows examples of cells positive with both antibodies, and the arrow shows Pax-2-positive and Ki67-negative cells. Scale bar, 37 ␮m. The right panel shows the number of Pax-2⫹ cells that were positive for the Ki67 nuclear antigen at P8 in the white matter of control (C) and hypothyroid (H) rats. Asterisks, P ⬍ 0.005.

Discussion

We show here that hypothyroidism during the fetal and neonatal periods in the rat is associated with decreased GAT-1 immunoreactivity in the cerebellum, reduced number of Golgi II cells, and delayed disappearance of the Pax-2⫹ precursors of cerebellar GABAergic interneurons. Other cerebellar interneurons, such as basket cells, are also known to be decreased by hypothyroidism (15). These observations suggest that thyroid hormones have a role in the generation of cerebellar interneurons from their precursor cells. The precursor cells can be identified by immunohistochemistry because they express the Pax-2 transcription factor. Pax-2⫹ neuroepithelial cells are already present in the region of the fourth ventricle around embryonic d 7.5 (27) and in the cerebellum by embryonic d 12. Further migration and differentiation originate in the deep cerebellar nuclei interneurons and Golgi II cells in the granular layer. Other GABAergic interneurons, the basket and stellate cells, located in the molecular layer, are originated from Pax-2⫹ precursors that migrate to the white matter where they proliferate and complete their terminal division (16). Thyroid hormones could be involved in any of the steps leading to final differentiation of the interneurons, i.e. proliferation of precursors, migration to the cerebellar cortex, or terminal differentiation. A major role of thyroid hormones

FIG. 5. Effects of T3 or GC-1 treatment of hypothyroid rats on the number of Pax-2⫹ precursors on P16. Hypothyroid rats were treated with single daily injections of T3 or GC-1 starting on P11. Immunohistochemistry for Pax-2 was performed in rats killed 24 h after the last injection. C, Control; H, hypothyroid. Asterisks, P ⬍ 0.05.

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Manzano et al. • Thyroid Hormone and Cerebellar Interneurons

FIG. 6. A, B, and D, Effect of TR␣1 deletion on GAT-1 expression by Western blotting (A) and number of Pax-2 precursors in the white matter (wm) (B) and the granular and molecular layers (igl⫹ml) (D) of wild-type mice (continuous line) and TR␣1 null mice (discontinuous line). ns, Not significant. C, Percentage of BrdU-positive Pax-2 cells in the white matter of wild-type (Wt) and TR␣1 null mice at P6. Asterisks, P ⬍ 0.05.

appears to be the facilitation of precursor differentiation, because in thyroid hormone deficiency, the rate of precursor cell disappearance was decreased, leading to accumulation of precursor cells. The finding of a similar number of precursors in the cerebellum at P4 in normal and hypothyroid rats argues against a major role of thyroid hormones on embryonic precursor proliferation or migration. However, proliferation of precursors in the white matter of neonatal hypothyroid rats was compromised, as shown by the reduced expression of the Ki67 nuclear protein. This is not in contradiction with the finding of a similar number of precursors in the white matter on P8, because this was the net result of precursor proliferation and differentiation, and both seemed to be affected by thyroid hormone deficiency. To explore what receptor type mediates the action of thyroid hormones, we compared the effects of T3 with the TR␤selective compound GC-1 on the number of Pax-2⫹ precursor cells. Because the effect of this compound was not equivalent to that of T3, it is suggested that both TR␣1 and TR␤ are involved in the effect of thyroid hormone on the number of Pax-2⫹ precursors. Other explanations for the different effects of GC-1 and T3 are difficult to discard. It is unknown whether GC-1 uses the same cellular transport systems as T3, and thyroidal status might alter transport of GC-1 but not of T3. However, we have shown previously that the different effects of GC-1 and T3 in the cerebellum correlate with T3 receptor type distribution (18, 28, 29). We paid particular attention to TR␣1 because, as we have shown previously, the developmental alterations present in the cerebellum of hypothyroid animals are in great part due to the interfering properties of unliganded TR␣1 (28). Landmarks of hypothyroidism such as delayed granular cell migration and Purkinje cell differentiation were not observed in TR␣1-deficient mice, even after induction of hypothyroidism. Therefore, a particular developmental effect of hypothyroidism may not reflect a physiological role of thyroid

hormone, because it may be due to interference of unliganded receptors with the normal developmental process. TR␣1-deficient mice showed decreased GAT-1 expression and impaired accumulation of Pax-2 precursors, both in the white matter and in the granular and molecular layers. The major effect of receptor deletion was to decrease the number of precursors both in the white matter and in the granular and molecular layers at most ages. This was likely a consequence of decreased precursor proliferation, as shown by the lower incorporation of BrdU in Pax-2 cells. The late effect of receptor deletion on the number of cells in the granular and molecular layers is probably the consequence of the lower number of migrating precursors generated in the white matter. In conclusion, we have shown a novel effect of thyroid hormones in the rodent central nervous system, the control of proliferation and differentiation of GABAergic cell precursors. Our observations agree with a major role of thyroid hormone and its nuclear receptors in regulation of the GABAergic system, both during development and in adult animals (6, 10 –15). During development, GABA has effects unrelated to neurotransmission and acts as a trophic factor promoting changes of cell proliferation and migration, axonal growth, synapse formation, etc. (9). Therefore, the regulation of interneuron development may contribute also to other developmental actions of thyroid hormone. In particular, we have shown previously that cerebellar astrocyte maturation is severely impaired in hypothyroid rats (30) and in TR␣1-deficient mice (29). Because GABA is known to influence astrocyte morphology and maturation (31), it is tempting to speculate that both are interrelated. Impaired astrocyte maturation could be secondary to altered interneuron formation. Conversely, proliferation of Pax-2 precursors in the white matter and subsequent migration and differentiation could be influenced by trophic factors from surrounding astrocytes.

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Manzano et al. • Thyroid Hormone and Cerebellar Interneurons

Endocrinology, December 2007, 148(12):5746 –5751

Acknowledgments We thank E. Moreno for technical assistance. Received April 30, 2007. Accepted August 17, 2007. Address all correspondence and requests for reprints to: Dr. Juan Bernal, Instituto de Investigaciones Biome´dicas, Arturo Duperier 4, 28029 Madrid, Spain. E-mail: [email protected]. This work was supported by Grants BFU2005-01740 from the Ministry of Education and Science and the European Union Integrated Project CRESCENDO (LSHM-CT-2005-018652). Disclosure Statement: The authors have nothing to disclose.

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