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Endocrinology Vol. 144, No. 10 4466-4477
Copyright © 2003 by The Endocrine Society

Dendritic Growth and Spine Formation in Response to Estrogen in the Developing Purkinje Cell

Hirotaka Sakamoto, Yukio Mezaki, Hanako Shikimi, Kazuyoshi Ukena and Kazuyoshi Tsutsui

Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521 Japan; and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo 150-0002, Japan

Address all correspondence and requests for reprints to: Kazuyoshi Tsutsui, Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan. E-mail: tsutsui{at}hiroshima-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neurosteroids are synthesized de novo in the brain, and the cerebellar Purkinje cell is a major site for neurosteroid formation. We have demonstrated that the Purkinje cell possesses intranuclear receptor for progesterone and actively produces progesterone de novo from cholesterol only during rat neonatal life, when cerebellar cortical formation occurs dramatically. We have further demonstrated that progesterone promotes dendritic growth, spinogenesis, and synaptogenesis via its receptor in this neuron in the neonate. On the other hand, estrogen may also play an important role in the process of cerebellar cortical formation, because the neonatal rat Purkinje cell possesses estrogen receptor (ER)ß. However, estrogen formation in the neonatal cerebellum is still unclear. In this study, we therefore analyzed the biosynthesis and action of estrogen in Purkinje cells during neonatal life. RT-PCR-Southern and in situ hybridization analyses showed that Purkinje cells expressed the key enzyme of estrogen formation, cytochrome P450 aromatase, in neonatal rats. A specific enzyme immunoassay for estradiol further indicated that cerebellar estradiol concentrations in the neonate were significantly higher than those in the prepuberty and adult. Both in vitro and in vivo studies with newborn rats showed that estradiol promoted dose-dependent dendritic growth of Purkinje cells. Estradiol also increased the density of Purkinje dendritic spines. These effects were inhibited by the ER antagonist tamoxifen. These results suggest that estradiol in the developing Purkinje cell promotes dendritic growth and spinogenesis via ERß in this neuron. Estradiol as well as progesterone may contribute to the growth of Purkinje cells during the cerebellar cortical formation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROSTEROIDS ARE NOW known to be steroids that are synthesized de novo from cholesterol in the central and peripheral nervous systems of vertebrates through mechanisms at least partly independent of peripheral steroidogenic glands, such as the adrenal and gonads (for reviews, see Refs. 1, 2, 3). To analyze neurosteroid actions in the brain, we need data on the specific synthesis of neurosteroids in particular sites of the brain at particular times. We have demonstrated recently that neuronal neurosteroidogenesis occurs in the brain and indicated that the Purkinje cell, a typical cerebellar neuron, possesses steroidogenic enzymes [i.e. cytochrome P450 side-chain cleavage enzyme (P450scc) and 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4-isomerase (3ß-HSD)] and produces pregnenolone, pregnenolone sulfate, and progesterone in a variety of vertebrates, including mammals (4, 5, 6, 7, 8, 9, 10, 11). Furthermore, in the rat, this neuron actively produces progesterone as a product of an increase of 3ß-HSD activity during neonatal life (7, 8).

It is well known that in the rat cerebellum, marked morphological changes occur during neonatal life (12, 13). Rat Purkinje cells differentiate just after birth, and the formation of the cerebellar cortex becomes complete in the neonate, when cerebellar progesterone is high (8). Therefore, progesterone synthesized de novo in the developing Purkinje cells may be involved in the cerebellar cortical formation during neonatal life. In fact, we have further shown that progesterone promotes dendritic growth, spinogenesis, and synaptogenesis of the Purkinje cell during the rat neonatal period (14, 15). Interestingly, intranuclear receptors for progesterone (PR) were also expressed highly in the Purkinje cell of neonatal rats (14, 16). Thus, it is considered that progesterone acts directly on Purkinje cells through intranuclear receptor-mediated mechanisms to promote Purkinje dendritic growth, spinogenesis, and synaptogenesis (14, 15). Such genomic actions of progesterone may be essential for the formation of the cerebellar neuronal circuit during neonatal development.

In the classical concept, estradiol, as well as progesterone, is known to be a sex steroid and acts on brain tissues through intranuclear receptor-mediated mechanisms to regulate reproductive behavior (17, 18). On the other hand, there is evidence of estrogen actions on nonreproductive functions, such as the response of cerebellar Purkinje cells to the excitatory neurotransmitter, glutamate (19, 20), although previous studies have failed to demonstrate estrogen-concentrating cells (21) or estrogen receptor (ER){alpha} mRNA (22) in the adult rat cerebellum. Recently, a second ER was cloned from the rat prostate and termed ERß (23). Interestingly, the expression of ERß in the Purkinje cell of neonatal and adult rats (24, 25, 26, 27) has suggested that estradiol acts on Purkinje cells via ERß to exert some physiological effects during cerebellar development. In addition, developing Purkinje cells may produce not only progesterone (7, 8) but also estradiol, because active estrogen formation in the gonads does not occur during neonatal life.

With these findings as a background, we first investigated the expression of cytochrome P450 aromatase (P450arom), which is a key enzyme of estrogen formation, in Purkinje cells using neonatal rats. Subsequently, neonatal changes in the estradiol concentration were measured in the cerebellum. Finally, to clarify the action of estradiol in Purkinje cells during neonatal life, we analyzed the effects of estradiol on dendritic growth and spine development of Purkinje cells by both in vitro and in vivo studies using newborn rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male and female rats of the Fisher strain maintained in this laboratory were mated and housed in a temperature-controlled room (25 ± 2 C) under a daily photoperiod of 14-h light/10-h dark cycle (lights on at 0600 h). Newborn and neonatal rats at various ages and sexually mature rats at 60 d of age were used in this study. The experimental protocol was approved in accordance with the Guide for the Care and Use of Laboratory Animals prepared by Hiroshima University (Higashi-Hiroshima, Japan).

RT-PCR analysis of P450arom mRNA
To determine the expression of mRNA encoding for rat P450arom in the cerebellum, RT-PCR analysis was performed using rat tissue collected during neonatal development and from adults according to our previous method (7, 8, 14, 28). In this study, rats at 0, 3, 7, 14, 21, and 60 d of age (n = 4 at each age, of both sexes) were killed between 1000 and 1100 h. Total RNA from the cerebellum of each rat was isolated by the guanidinium thiocyanate-phenol-chloroform extraction method (29). For PCR, an aliquot of the cDNA solution corresponding to 1 µg of the initial total RNA was used as a template in a 25-µl reaction mixture containing recombinant Ex Taq polymerase (Takara Shuzo, Kyoto, Japan). After denaturation at 95 C for 3 min, the mixture was subjected to 35 thermal cycles in a programmed temperature-control system (PC808; ASTEC, Fukuoka, Japan) as follows: denaturation at 94 C for 30 sec, primer annealing at 55 C for 30 sec, and extension at 72 C for 30 sec. After the thermal cycling, the mixture was additionally incubated at 72 C for 10 min. A 5-µl aliquot of each sample was electrophoresed through a 2% agarose gel. To confirm the identity of the amplified fragment, the gels were subjected to Southern analysis with a digoxigenin (DIG)-labeled oligonucleotide probe. Two P450arom mRNA transcripts are present; one of these is a full-length transcript (it contains the entire 5'-coding region) that probably encodes a functional enzyme, and the other transcript is a truncated variant (it seems not to contain portions of the initial 5'-coding exons) (30, 31, 32). Oligonucleotides used as PCR primer and probe for mRNA detection, based on nucleotide sequences of rat P450arom (30, 31, 32) and rat ß-actin (33), were as follows: P450arom (heme) sense primer, 5'-CTGAACATCGGAAGAATGCACAGGC-3'; P450arom (heme) antisense primer, 5'-ATTTCCACAATGGGGCTGTCCTCAT-3'; P450arom (heme) probe, 5'-TTCCATGTGAAGACATTGCA-3'. These primers, which are identical and complementary to a common sequence of P450arom enzyme and its truncated variant (heme-binding region), give a 284-bp amplified fragment of the rat P450arom gene (31): P450arom (full-length) sense primer, 5'-GGAAATGCTGAACCCCATGCA-3'; P450arom (full-length) antisense primer, 5'-CAGACTCTCATGAACTCTCC-3'; P450arom (full-length) probe, 5'-ATCTTCCATACCAGGTC-3'. These primers gave a 255-bp amplified fragment located in exons 2 and 3, which are able to detect a full-length P450arom gene (34, 35): ß-actin sense primer, 5'-GAGACCTTCAACACCCCAGC-3'; and ß-actin antisense primer, 5'-CACAGAGTACTTGCGCTCAG-3'. The ß-actin primers give a 645-bp amplified fragment (33).

Each RT-PCR analysis was repeated four times with independently extracted RNA samples from different animals. We compared sex and developmental differences of the P450arom mRNA expression. After scanning the band corresponding to P450arom mRNA (full-length), we measured the band density using a National Institutes of Health (NIH) Image software package and estimated the standardized value by calculating the ratio to internal control.

In situ hybridization of P450arom mRNA
The site of P450arom expression in the cerebellum was localized by in situ hybridization. Eight male rats at 8 and 60 d of age (n = 4 each group) were deeply anesthetized with chloroform before transcardial perfusion with PBS (10 mM phosphate buffer and 140 mM NaCl, pH 7.3), followed by fixative solution [4% paraformaldehyde (PFA) in PBS]. After dissection from the skull, brains were postfixed overnight (16–18 h) in a similar fixative solution at 4 C, followed by cryoprotection in cooled sucrose solution (30% sucrose in PBS). Ten-micrometer parasagittal sections of the cerebella were cut using a cryostat at -20 C and placed onto 3-aminopropyltriethoxysilane-coated slides. Ovarian sections (10 µm in thickness) from mature rats served as a positive control.

In situ hybridization was carried out according to our previous method (8, 10) with a DIG-labeled antisense RNA probe. The DIG-labeled antisense RNA probe was produced with a RNA labeling kit (Roche Diagnostics, Basal, Switzerland) from a part of ovarian P450arom cDNA (complementary to nucleotides 1261–1544). Control for specificity of the in situ hybridization of P450arom mRNA was performed using the DIG-labeled sense RNA probe, which is complementary to a sequence of antisense probe.

Enzyme immunoassay (EIA) of estradiol-17ß
To measure estradiol-17ß levels in the cerebellum and plasma of neonatal (5 d of age), prepubertal (21 d of age), and adult rats (60 d of age), 16 males of three different ages were killed (n = 8 at 5 d of age, n = 4 at 21 and 60 d of age) between 1000 and 1200 h. Trunk blood was collected into heparinized tubes and centrifuged at 1800 x g for 20 min at 4 C. Plasma was stored at -80 C. To secure a sufficient volume of plasma for assay from the neonatal rats (5 d of age), plasma from two animals was pooled to form a single sample. Immediately after blood collection, cerebella were removed and weighed. Cerebella of neonatal rats (5 d of age) were also pooled from two animals as a single sample. Subsequently, cerebella at each age were frozen in liquid nitrogen. There were four plasma and cerebellar samples for each age group.

Extraction of estradiol-17ß was performed according to a method described previously (7, 8, 14, 27, 36, 37). To measure the estradiol-17ß concentration, aliquots of organic extracts were assayed in an estradiol EIA by using an Estradiol EIA Kit (Cayman Chemical, Ann Arbor, MI) consisting of estradiol-17ß conjugated with acetylcholinesterase and a specific antiserum against estradiol-17ß. The antiserum used in this assay cross-reacted with estrone at 4%, with estriol at 0.57%, with testosterone at 0.1%, with 5{alpha}-dihydrotestosterone at 0.1%, and with estradiol-17{alpha} at less than 0.1%; and therefore, no chromatographic purification of estradiol-17ß was performed. The interassay coefficient of variation was less than 10%, and the least detectable amount was 0.033 pmol/ml.

Slice culture of cerebella
Organotypic slice cultures of cerebella were conducted according to our previous method (14, 15). In brief, cerebella of male pups at 5 d of age were used, and vermal parasagittal slices (400-µm thick) were aseptically cut on a microslicer. Cultures of cerebellar slices were conducted according to a static interface method of organotypic cerebral or hippocampal cultures (38, 39). The culture medium was a modification of medium described previously (39, 40) and composed of a 1:1 mixture of DMEM and Ham’s F-12 (Sigma, St. Louis, MO), supplemented with insulin (5 µg/ml; Sigma), apo-transferrin (100 µg/ml; Sigma), putrescine (100 µM; Sigma), sodium selenite (30 nM), D-glucose (6 mg/ml), penicillin G potassium (100 U/ml), and streptomycin sulfate (100 µg/ml). The culture medium contained 5% fetal bovine serum for the first 2 d of culture [2 d in vitro (2 DIV)]. In this study, steroids were excluded from the medium composition. Cultures were maintained at 37 C in an atmosphere of humidified 95% air-5% CO2.

In vitro steroid treatment
To investigate morphological changes of Purkinje cells induced by estradiol-17ß, estradiol-17ß was applied to organotypic cerebellar slice cultures for 3 d after 2 DIV, and cultures were fixed at 5 DIV. Crystalline estradiol-17ß (Sigma) was dissolved into absolute ethanol and applied to the culture medium at various concentrations (0.1, 1, and 10 nM). The final concentration of ethanol was adjusted to 0.001% (vol/vol) in all estradiol-treated and control (vehicle alone) groups. All cultures were fixed in 2% PFA, 2.5% glutaraldehyde, and 15% saturated picric acid (vol/vol) in 0.1 M phosphate buffer (PB; pH 7.3) overnight at 4 C and subjected to immunocytochemical labeling of Purkinje cells using a calcium-binding protein (calbindin) antibody as described below, followed by the morphological analysis of Purkinje cells.

In vivo steroid treatment
To investigate estrogen effects on morphological changes of Purkinje cells in vivo, we used estradiol benzoate (EB; Sigma), which is more stable than estradiol-17ß. In the present in vivo study, we further compared morphological changes of Purkinje cells induced by EB with those by progesterone, because progesterone promotes the dendritic growth of Purkinje cells (14, 15). EB (5 or 50 µg/25 µl), progesterone (5 or 50 µg/25 µl), or EB plus progesterone (5 µg/25 µl each) dissolved in sesame oil was injected into the cerebrospinal fluid around the posterior vermal lobe (IX) of the cerebellum of pups of both sexes once per day, for 4 d, during 6–9 d of age, when the receptors for both estrogen (ERß) and progesterone (PR) in the Purkinje cell were expressed highly (14, 16, 26). Pups receiving injections of the vehicle alone (sesame oil) served as controls. At 10 d of age, pups were deeply anesthetized with chloroform before transcardial perfusion with PBS, followed by fixative solution [2% PFA, 2.5% glutaraldehyde, and 15% saturated picric acid (vol/vol) in PB]. Vermal cerebella were dissected out and sectioned parasagittally at 50-µm thickness with a microslicer before immunostaining with calbindin antibody.

To confirm the effect of endogenous estrogen on Purkinje dendritic growth, tamoxifen (Sigma), an ER antagonist, was further injected into pups in this study. Tamoxifen dissolved in sesame oil (500 µg/25 µl) or EB (5 µg/25 µl) plus tamoxifen (500 µg/25 µl) dissolved in sesame oil was injected as described above. Pups receiving injections of the vehicle alone (sesame oil) served as controls. Pups at 10 d of age were also used for morphological analyses.

Cellular labeling of Purkinje cells with calbindin or DiI
Purkinje cells were identified by immunostaining with a mouse monoclonal antibody raised against a calcium-binding protein, calbindin-D28k (Sigma), according to our previous method (14, 15). Cerebellar sections and slice cultures were prepared as described above and processed for immunocytochemistry. After elimination of endogenous peroxidase activity with 3% H2O2 and blocking nonspecific binding components with 1% normal horse serum and 1% BSA, the sections and slice cultures were immersed overnight at 4 C with the monoclonal antibody against calbindin at a dilution of 1:50,000. Immunoreactive products were detected with an avidin-biotin kit (Vectastain Elite Kit; Vector Laboratories, Burlingame, CA), followed by diaminobenzidine reaction, as previously described (7, 11, 14, 15, 41).

Purkinje cells were further identified by retrograde labeling with a fluorescent dye, DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) (Molecular Probes, Eugene, OR) according to the method previously reported (42). For retrograde labeling of Purkinje cells, pups receiving injections of the vehicle or tamoxifen were prepared as described above. At 10 d of age, pups were deeply anesthetized with chloroform before transcardial perfusion with PBS, followed by fixative solution (4% PFA in PB). After dissection from the skull, vermal cerebella were postfixed overnight (16–18 h) in a similar fixative solution at 4 C, and a small incision was made in the region of the cerebellar deep nucleus, and DiI was applied using a fine tungsten needle with a small amount of its crystals on the tip. Because Purkinje cells project massively to the cerebellar deep nucleus, most Purkinje cells were labeled randomly in this study. One microliter of Triton X-100 was injected into incisions. Cerebella were then washed in PBS, embedded in 6% agar to prevent diffusion of the dye, and incubated in 4% PFA in PB, in the dark, for 10–15 d at 37 C. After the incubation, the preparations were then washed in PBS and sectioned parasagittally at 50-µm thickness with a microslicer. Sections were then rinsed twice, mounted on 3-aminopropyltriethoxysilane-coated slides, and examined under a fluorescence microscope (Nikon, Melville, NY).

In vitro morphological analysis of Purkinje cells
In vitro morphological analysis was conducted according to our previous method (14). In brief, the whole area, cross-sectional soma area, and perimeter of Purkinje cells were measured in each selected calbindin-immunostained slice using an NIH Image software package. This study measured the whole area, cross-sectional soma area, and perimeter of Purkinje cells by using camera lucida drawings. To measure the dendritic area of Purkinje cells, the cross-sectional soma area was deducted from the whole area of each cell.

To analyze the effects of estradiol-17ß (10-8 M) on Purkinje dendritic morphology, the number of dendritic spines per unit length of dendrite and total dendritic length per cell of Purkinje cells were further measured according to our previous method (14). In brief, the selected segment was traced (magnification, x1200) with a camera lucida drawing tube, all of the dendritic spines visible along that segment were counted, the length of each segment was also measured from its camera lucida drawing with an NIH Image software package, and data were then expressed as the number of spines per 50 µm dendrite. Three to five dendritic segments per cell and at least six Purkinje cells per slice were analyzed.

In vivo morphological analysis of Purkinje cells
Male and female pups injected with EB, progesterone, or EB plus progesterone were used for morphological analyses. The length of molecular layer was evaluated as a parameter of the maximal Purkinje dendritic length (see Fig. 5Go), because Purkinje cell dendrites at 10 d of age were well developed and were not clearly distinguishable from neighboring immunoreactive cells. At least 16 regions from four calbindin-immunostained sections per animal were randomly selected in the vermal lobe IX around the site of in vivo injection. The maximal dendritic length was measured using an ocular micrometer.



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  		FIG. 5. Morphological comparison of Purkinje cell dendrites from vehicle-, EB-, and progesterone-treated groups: in vivo study. Parasagittal sections of neonatal cerebellum at 10 d of age were immunostained for calbindin (lobe IX). Male pups received daily injections of the vehicle (A), EB (B), or progesterone (C) for 4 d during 6–9 d of age. Bars, 20 µm. M, molecular layer; P, Purkinje cell layer.

 
To verify the action of endogenous estrogen on Purkinje dendritic growth, morphological analyses were performed on pups injected with the antiestrogen tamoxifen during 6–9 d of age, when both P450arom and ERß expressions in the Purkinje cell were maximal. The length of molecular layer was also evaluated as a parameter of the maximal Purkinje dendritic length as described above.

Statistical analyses
Data obtained by RT-PCR analysis of P450arom, EIA of estradiol-17ß and Purkinje morphological analyses after in vitro treatment with estradiol-17ß at various doses, and in vivo treatment with EB, progesterone, EB plus progesterone, tamoxifen, or EB plus tamoxifen were analyzed by one-way ANOVA. If significant by these ANOVAs, the analyses were followed by Duncan’s multiple range test (43). Differences in the dendritic spine number and total dendritic length of Purkinje cells after in vitro treatment with estradiol-17ß were analyzed by Student’s t test (43).

All in vitro treatment groups were composed of the cultured slices from multiple animals (at least five animals), and all slices were obtained from different animals and separately cultured in the individual chamber. Statistical comparisons of in vitro studies (see Fig. 4Go and Table 3Go) were based on the individual slice as the unit of analysis, because the physiological environment in tissue culture becomes independent of the animal of origin. Statistical analyses of in vivo studies were based on the individual animal.



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  		FIG. 4. Dose-response of estradiol-17ß: in vitro study. Perimeter (A), soma area (B), and dendrite area (C) of Purkinje cells were measured after immunostaining for calbindin. Estradiol-17ß administration in vitro increased, in a dose-related manner, the perimeter and dendrite area of Purkinje cells, unlike their soma area. Each dot and the vertical line represent the mean ± SEM (n = 5 slices from five individual males in each group). Data were derived from 80 Purkinje cells in each group. **, P < 0.01 vs. vehicle (by one-way ANOVA, followed by Duncan’s multiple range test).

 

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  		TABLE 3. Effects of estradiol-17ß on the number of dendritic spines and total dendritic length of Purkinje cells

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neonatal changes in P450arom mRNA expression in cerebellum
In the present study, the expression of mRNA encoding for P450arom in the rat cerebellum was first examined during neonatal development and in the adult by RT-PCR analysis. The diencephalon from the adult male rat was used as a positive control tissue, and the same amount of cDNA was used in the RT-PCR. The initial RNA amount used in the RT-PCR was adjusted spectrophotometrically. RT-PCR for ß-actin was performed as a control experiment (Fig. 1EGo). We first conducted RT-PCR analysis using primers for the heme-binding region during cerebellar development (Fig. 1Go, A and B). Gel electrophoresis of the RT-PCR product for the heme-binding region of P450arom gene identified a single band of 284 bp corresponding to a common sequence for the full-length and variant transcripts in the cerebellum (Fig. 1AGo). Serial Southern hybridization confirmed that this band was P450arom mRNA (heme-binding region) specific (Fig. 1BGo). Interestingly, cerebellar expression of the message increased between d 0 and d 3 of neonatal life in both sexes (Fig. 1Go, A and B). The expression in both male and female cerebellum tended to decrease thereafter (Fig. 1Go, A and B). We further conducted RT-PCR analysis using primers for the 5'-coding region (specific for the full-length transcript) during cerebellar development (Fig. 1Go, C and D). Gel electrophoresis of the RT-PCR product for the 5'-coding region of P450arom gene identified a single band of 255 bp, which is specific for the full-length transcript, in the cerebellum (Fig. 1CGo). Serial Southern hybridization confirmed that this band was also P450arom mRNA (5'-coding region)-specific (Fig. 1DGo). The expression pattern of the 5'-coding region of P450arom gene (Fig. 1Go, C and D) was very similar to that of the heme-binding region (Fig. 1Go, A and B) during cerebellar development. In this present study, qualitative PCR analysis was further performed. The standardized density of P450arom mRNA was estimated by calculating the ratio to each internal control (ß-actin mRNA) in the same sample (Table 1Go). As shown in Table 1Go, the expression of P450arom mRNA increased during neonatal life, suggesting an age-dependent expression. In contrast, no sex difference in the P450arom mRNA expression was detected in the cerebellum (Fig. 1Go, A–D, and Table 1Go). The expression of P450arom mRNA in the neonatal cerebellum was almost similar to that in the adult diencephalon (Fig. 1Go, A–D).



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  		FIG. 1. RT-PCR analysis of P450arom mRNA in the male and female cerebella at 0, 3, 7, 14, 21, and 60 d of age. A, Result of gel electrophoresis of RT-PCR products for the heme-binding region of rat P450arom (284 bp). B, Identification of the band by Southern hybridization using DIG-labeled oligonucleotide probe for the heme-binding region of rat P450arom. C, Result of gel electrophoresis of RT-PCR products for the 5'-coding region of rat P450arom (255 bp). D, Identification of the band by Southern hybridization using DIG-labeled oligonucleotide probe for 5'-coding region of rat P450arom. cDNA corresponding to 1 µg total RNA extracted from each cerebellar tissue was used for a PCR, and a 5-µl aliquot of each sample was applied on one lane. Diencephalic tissue served as a positive control, and the same amount of cDNA was used in the RT-PCR. The lane labeled No cDNA was performed without template as the negative control. E, Result of the RT-PCR for ß-actin as the internal control, in which PCR cDNA corresponding to 1 µg total RNA was used as a template. RT-PCR experiments were repeated four times using independently extracted RNA samples from different animals. Each experiment produced similar results.

 

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  		TABLE 1. Neonatal changes in P450arom mRNA expressions (full-length) in the cerebellum

 
Cellular localization of P450arom mRNA expression in neonatal cerebellum
In situ hybridization of P450arom mRNA was examined in the cerebellum of male rats using RNA probes for rat P450arom mRNA. The ovary was used as a positive control. Preliminary observations using the ovary of adult rats confirmed the validity of the in situ hybridization technique as follows: DIG-labeled RNA antisense probes for P450arom mRNA gave an intense reaction in the granulosa cell, a typical cell expressing P450arom but not in the thecal/interstitial cells; the control staining using RNA sense probes resulted in a complete absence of the signal.

Subsequently, the site of P450arom mRNA expression in the cerebellum was examined in neonatal rats at 8 d of age (Fig. 2AGo). P450arom mRNA was highly expressed in Purkinje cells (Fig. 2Go, A and D) and external granule cells (Fig. 2AGo) in the cerebellar cortex. The localization of these reactive cells was confirmed by Nissl-staining (Fig. 2Go, C and F). The control staining using RNA sense probes resulted in a complete absence of the signal in Purkinje cells (Fig. 2Go, B and E) and external granule cells (Fig. 2BGo) at 8 d of age. In contrast, the expression of P450arom mRNA was mostly detected in Purkinje cells and a few large cells scattered in the internal granule cell layer at 60 d of age (Fig. 2Go, G and J). This was also abolished by using RNA sense probes (Fig. 2Go, H and K). The location of Purkinje cells in the cerebellum was characterized by Nissl-staining in the adult rat (Fig 2Go, I and L).



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  		FIG. 2. In situ hybridization using the cerebellar cortex of male rats at 8 d (A–F) and 60 d of age (G–L) (adult). DIG-labeled RNA antisense probes for P450arom mRNA (A, D, G, and J) or the sense probes (B, E, H, and K) were used. Histology of the cerebellar cortex was shown by Nissl staining (C, F, I, and L). The blocked areas in A–C and G–I are enlarged in D–F and J–L, respectively. A–C and G–I are of the same low magnification, and D–F and J–L are of the same high magnification. At 8 d of age, P450arom mRNA was expressed in Purkinje cells (arrows, A and D) and external granule cells (small arrowheads, A). In the cerebella of adults, P450arom mRNA was also expressed in Purkinje cells (arrows in J) and a few large cells scattered in the internal granule cell layer (large arrowheads in J). In situ hybridization was repeated independently four times using different animals and produced similar results. EG, External granule cell layer; M, molecular layer; P, Purkinje cell layer; G, internal granule cell layer. Bars, 50 µm.

 
Neonatal change in estradiol-17ß in cerebellum
In this study, estradiol-17ß was measured in the cerebellum of neonatal (5 d of age), prepubertal (21 d of age), and adult (60 d of age) male rats using a specific estradiol EIA. The estradiol-17ß concentration in the cerebellum at 5 d of age was much greater than those at 21 and 60 d of age (P < 0.01; Table 2Go). At 5 d of age, the plasma estradiol-17ß concentration was significantly lower than that in the cerebellum (P < 0.05; Table 2Go). At 21 and 60 d of age, the examined concentrations were not significantly different between the cerebellum and plasma (Table 2Go).


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  		TABLE 2. Neonatal changes in estradiol-17ß concentrations in the cerebellum and plasma

 
Purkinje cell development induced by in vitro administration of estradiol-17ß
Estradiol-17ß is shown in high levels in the cerebellum during neonatal life (see Table 2Go). To investigate whether this steroid is involved in the growth of Purkinje cells, morphological changes of Purkinje cells were measured, after treatment with estradiol-17ß, using cerebellar slice cultures of newborn male rats. Initially, estradiol-17ß (10 nM) was added to cerebellar cultures in serum-free medium for 3 d, after a 2-d incubation period with 5% fetal bovine serum to abolish the excessive cell loss in cultured cerebellar slices. The morphology of the Purkinje cells in the estradiol-treated group (Fig. 3BGo) was compared with that in the vehicle-treated group (Fig. 3AGo). Morphological analysis revealed that estradiol-17ß administration induced the dendritic outgrowth of Purkinje cells (Fig. 3Go, A and B). Estradiol-17ß administration increased, in a dose-related way, the perimeter (Fig. 4AGo) and dendrite area (Fig. 4CGo) of Purkinje cells (n = 5 slices from 5 individual males in each dose) with a threshold concentration ranging between 1 and 10 nM, indicating that estrogen actions were within the physiological range observed during normal neonatal cerebellar development (see Table 2Go). However, estradiol-17ß did not influence the cross-sectional Purkinje cell body area after estradiol-17ß treatment at any dose (Fig. 4BGo).



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  		FIG. 3. Dendritic morphology of Purkinje cells and its modulation by estradiol-17ß treatment: in vitro study. (A–D) Cerebellar cultures from newborn male rats grown for 5 DIV and immunostained for calbindin. (A and B) Purkinje cell dendrites in the estradiol-17ß group (B; 10 nM estradiol-17ß treatment) seemed to be well developed, compared with the vehicle group (A). Higher magnification of the apical dendrites of Purkinje cells shows the greater density of dendritic spines in the estradiol-17ß group (D), compared with the vehicle group (C). Arrowheads indicate presumptive spine-like structures. Bars, 20 µm in A and B and 5 µm in C and D.

 
To analyze the effect of estradiol-17ß (10 nM) on Purkinje dendritic spine morphology, we further measured the dendritic spine number (Fig. 3Go, C and D) of Purkinje cells in cultured slice. As shown in Table 3Go, estradiol-17ß administration resulted in significant increases not only in the total length of dendrites (P < 0.01) but also in the number of dendritic spines per unit length of dendrite (50 µm) (P < 0.01) of Purkinje cells (n = 5 slices from 5 individual males in each treatment).

Purkinje cell development induced by in vivo administration of EB
Although administration of estradiol-17ß promoted the outgrowth of Purkinje cell dendrites in vitro, an in vivo effect of estrogen is still unclear. Therefore, this experiment was designed to verify estrogen action on Purkinje dendritic outgrowth in vivo, and the effect was compared with that of progesterone. EB or progesterone was directly injected at 5 µg/d during 6–9 d of age, when Purkinje cells express the receptors for both estrogen (ERß) and progesterone (PR). It has been previously confirmed that the cerebellar concentration of treated steroids was preferentially increased by local injection, compared with systemic injection (14). The morphology of Purkinje cells was compared among the groups of EB, progesterone, and vehicle (n = 4 in each group) after immunostaining for calbindin (Fig. 5Go). As shown in Fig. 5BGo, in vivo administration of EB to male pups promoted the dendritic outgrowth of Purkinje cells. A similar effect was evident in the progesterone-treated group (Fig. 5CGo). Therefore, we evaluated the length of molecular layer as a parameter of the maximal Purkinje dendritic length (see Fig. 5Go). There was no sex difference in the stimulatory effect of EB and progesterone on the maximal Purkinje dendritic length (Table 4Go). Subsequently, the dose dependency of EB and progesterone was tested at the two different doses, 5 µg/d (low dose) and 50 µg/d (high dose), and the data obtained from male and female pups were analyzed together. Both doses of EB and progesterone caused significant increases in the maximal Purkinje dendritic length, and the stimulatory effect of EB at the low dose (5 µg/d) tended to be greater than that of progesterone (n = 4 in each group; Fig. 6AGo).


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  		TABLE 4. Comparison of the steroid effect on maximal Purkinje dendritic length

 


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  		FIG. 6. Quantitative analysis of the length of molecular layer as a parameter of maximal Purkinje dendritic length: in vivo study. A, Dose-response of EB and progesterone. Each dot and the vertical line represent the mean ± SEM (n = 4 males in each group). **, P < 0.01 vs. vehicle; {dagger}, P < 0.05 vs. high dose (EB treatment) (by one-way ANOVA, followed by Duncan’s multiple range test). B, Effect of EB, progesterone, and EB plus progesterone on the maximal Purkinje dendritic length. Each column and the vertical line represent the mean ± SEM (n = 4 animals in each group). **, P < 0.01 vs. vehicle; {dagger}, P < 0.05 vs. progesterone group (by one-way ANOVA, followed by Duncan’s multiple range test).

 
We further examined the combined effect of EB and progesterone at the low dose (5 µg/d) on the Purkinje dendritic outgrowth. Administration of EB alone (5 µg/d) or progesterone alone (5 µg/d) induced a significant increase in the maximal Purkinje dendritic length (P < 0.01 vs. vehicle group) (Fig. 6BGo), and EB tended to be more effective than progesterone at the same low dose (5 µg/d) (Fig. 6BGo). Combined treatments with EB (5 µg/d) and progesterone (5 µg/d) also induced a significant increase (P < 0.01) in the maximal Purkinje dendritic length, compared with the vehicle-treated group, which was greater (P < 0.05) than that in the progesterone-treated group (Fig. 6BGo). However, this combined effect was not synergistic (Fig. 6BGo).

Inhibition of Purkinje cell development by anti-estrogen
This experiment was designed to verify the action of endogenous estrogen in the cerebellum on Purkinje dendritic growth using the ER antagonist, because cerebellar estrogen levels were high during neonatal life (see Table 2Go). Tamoxifen, an antagonist of ER (both {alpha} and ß subtypes), was directly injected at 500 µg/d during 6–9 d of age, when cerebellar expression of ERß is high (26) and cerebellar cortical formation is most marked (12, 13). The dendritic morphology of Purkinje cells at 10 d of age was compared between vehicle- and tamoxifen-treated groups after immunostaining for calbindin (Fig. 7Go). Immunocytochemistry with anticalbindin antibody revealed well-organized Purkinje dendritic arbors and normally arranged Purkinje somata in both groups at lower magnification (Fig. 7Go, A and B). However, distal Purkinje dendrites of the tamoxifen-treated animals appeared sparse at higher magnification (Fig. 7DGo), and the labeled dendritic spine was lacking (Fig. 7FGo), unlike the dendrites in the vehicle-treated animals (Fig. 7Go, C and E). We confirmed the view of the dendritic spines with another stain, because the treatment may have altered the labeling for calbindin. Therefore, Purkinje cells were identified by retrograde labeling with a fluorescent dye, DiI (Fig. 7Go, G and H). Application of DiI to the cerebellar deep nucleus resulted in the appearance of many retrogradely labeled Purkinje cells in each vermal lobe. The retrogradely labeled Purkinje cells (lobe IX) in tamoxifen-treated animals (Fig. 7HGo) were also devoid of dendritic spine-like structures, unlike the dendrites in the vehicle-treated animals (Fig. 7GGo). In this study, retrograde tracing experiments were repeated independently in four animals (in each group) 10–15 d after application of DiI and provided similar results. No significant difference in DiI labeling was found among different incubation periods (10–15 d) after application of DiI.



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  		FIG. 7. Morphological comparison of Purkinje cell dendrites from vehicle- and tamoxifen-treated groups: in vivo study. Lower magnification of a part of the cerebellar vermal lobe IX in the vehicle-treated (A) and tamoxifen-treated (B) groups after immunostaining for calbindin. Distal Purkinje dendrites of the tamoxifen-treated animals appeared sparse at higher magnification (D), and the labeled dendritic spine-like structure was lacking (F), unlike the dendrites in the vehicle-treated animals (C and E). Purkinje cells were also identified by retrograde labeling with a fluorescent dye, DiI (G and H). The retrogradely labeled Purkinje cells (lobe IX) from tamoxifen-treated animals (H) were also devoid of dendritic spine-like structures, unlike the dendrites in the vehicle-treated animals (G). Bars, 200 µm in A and B, 50 µm in C and D, and 20 µm in E–H. VIII, lobe VIII; IX, lobe IX.

 
Finally, the length of molecular layer was analyzed as a parameter of maximal Purkinje dendritic length at 10 d of age after the treatments with EB, tamoxifen, and EB plus tamoxifen (n = 4 in each group) after immunostaining for calbindin. Treatment with EB alone (5 µg/d) induced a significant increase in the maximal Purkinje dendritic length (P < 0.01 vs. vehicle group) (Fig. 8Go), whereas tamoxifen alone at a concentration of 500 µg/d (100 times greater than the EB concentration) significantly decreased the maximal Purkinje dendritic length, compared with the vehicle-treated group (P < 0.05) (Fig. 8Go). Combined treatments with EB (5 µg/d) and tamoxifen (500 µg/d) revealed that tamoxifen abolished the estrogen-induced dendritic growth of Purkinje cells (P < 0.05 vs. estradiol group) (Fig. 8Go).



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  		FIG. 8. Effect of EB and tamoxifen on the maximal Purkinje dendritic length: in vivo study. Each column and the vertical line represent the mean ± SEM (n = 4 animals in each group). *, P < 0.05; **, P < 0.01 vs. vehicle; {dagger}, P < 0.05 vs. EB group (by one-way ANOVA, followed by Duncan’s multiple range test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this present study, we first demonstrated that Purkinje cells highly expressed the P450arom mRNA in the neonate, suggesting its age-dependent expression. In addition, estradiol levels in the neonate were higher in the cerebellum than in the plasma, but this difference disappeared with aging. Furthermore, treatment of cerebellar cultures with exogenous estradiol promoted the dendritic growth and spine formation of Purkinje cells in a dose-dependent manner, with active doses being in the range of physiological levels of estradiol measured in the cerebellum. Similar morphological effects were also obtained by the in vivo treatment with estradiol. In contrast, the blockage of action of endogenous estrogens by treatment with the ER antagonist tamoxifen had reversed effects on the Purkinje cell morphology. Taken together, these results suggest that estradiol contributes to the growth of Purkinje cells during neonatal life.

The present RT-PCR analyses using primers for the heme-binding region (complementary to a common sequence for the full-length and variant transcripts) and 5'-coding region (specific for the full-length transcript) revealed that the expression patterns of both transcripts were very similar during cerebellar development. Therefore, the full-length mRNA encoding for P450arom, a key enzyme of estrogen formation, in the cerebellum, was expressed with a high level only during the neonatal period. To our knowledge, this is the first observation of an age-dependent expression of P450arom mRNA in the cerebellum. The increased expression of P450arom in the neonatal cerebellum was consistent with the estradiol EIA results, indicating that the cerebellar estradiol concentration in the neonate was maximal, and significantly higher than that in the plasma. Taken together, it is likely that the rat cerebellum expresses P450arom and produces estradiol during neonatal life. Our present data from estradiol EIA suggested that transient estrogen formation in the cerebellum may occur during neonatal life. However, we cannot rule out the possibility that estradiol produced in the peripheral steroidogenic organs is accumulated by means of the binding protein(s) in the cerebellum, because the gonads of neonatal rats may also secrete estradiol (44, 45, 46). Therefore, we need precise biochemical analyses to investigate whether the high concentration of estradiol in the neonatal cerebellum is attributable to an increase of the enzymatic activity of P450arom. In this study, we further characterized the site showing the P450arom expression by in situ hybridization. Preliminary observations using the ovary supported the validity of the in situ hybridization technique, because the staining of P450arom mRNA was localized in the granulosa cell, a typical ovarian cell expressing P450arom, and the control staining using RNA sense probes resulted in a complete absence of the signal. Interestingly, the expression of P450arom mRNA in the cerebellum was restricted to Purkinje cells and external granule cells in neonatal rats. Accordingly, the Purkinje cell may be an active site of estrogen formation in the cerebellum during neonatal life. We have previously demonstrated that the Purkinje cell possesses several kinds of steroidogenic enzymes, such as P450scc, 3ß-HSD, 5{alpha}-reductase, and 3{alpha}-hydroxysteroid oxidoreductase, and produces pregnenolone, pregnenolone sulfate, progesterone, and 3{alpha},5{alpha}-tetrahydroprogesterone, a progesterone metabolite, in the neonatal rat (3, 7, 8, 47, 48). Recently, we have further shown the expression and activity of cytochrome P450 17{alpha}-hydroxylase/c17,20-lyase (P45017{alpha},lyase), which converts pregnenolone to dehydroepiandrosterone or progesterone to androstenedione, an immediate precursor of estrogen formed by P450arom, in the Purkinje cell of avian species (10). The present and previous studies, taken together, support de novo synthesis of estrogen from cholesterol in the Purkinje cell.

We examined the action of estradiol on the growth of Purkinje cells because dramatic morphological changes occur in the cerebellum during neonatal life, concomitant with an increase in the cerebellar estradiol concentration. In vitro treatment with estradiol using cerebellar slice cultures from newborn rats resulted in the promotion of dendritic outgrowth of Purkinje cells. This stimulatory effect occurred in a dose-dependent manner, with a threshold concentration ranging between 1–10 nM, suggesting that the estrogen action was within the physiological range observed in neonatal rats under normal cerebellar development (see Table 2Go). In contrast, there was no evidence for an effect of estradiol on Purkinje somata. These results were consistent with in vivo experiments in which administration of EB, a stable form of estradiol, to newborn rats induced dendritic outgrowth of Purkinje cells. In addition, in vivo administration of the antagonist of ER, tamoxifen, to newborn rats decreased the dendritic outgrowth of Purkinje cells, suggesting a stimulatory action of endogenous estradiol on Purkinje cell dendrites during neonatal life. Furthermore, the action of estradiol on Purkinje cell dendrites was inhibited by tamoxifen in vivo by combined administration of estradiol and tamoxifen. Thus, both in vitro and in vivo studies suggest that a high level of cerebellar estradiol during neonatal life is essential for the promotion of dendritic growth of the Purkinje cell.

In addition to dendritic growth, in vitro treatment with estradiol using cerebellar slice cultures from newborn rats also increased the density of dendritic spines on Purkinje cells. Therefore, it is possible that estradiol promotes not only dendritic growth but also spinogenesis in the developing Purkinje cell. Dendritic spines are thought to represent postsynaptic sites (49). Because changes in the number of dendritic spines during development or in response to experimentally induced injury are positively correlated with changes in the number of synapses (50), we consider that morphological changes in dendritic spines observed in this study may reflect changes in the number of synapses. Indeed, we have reported recently that progesterone produced in neonatal Purkinje cells promotes synaptogenesis as well as dendritic growth and spinogenesis during cerebellar development (14, 15). To investigate whether estradiol induces the promotion of synaptogenesis in Purkinje cells during development, electron microscopic analyses are now in progress. On the other hand, it has been reported that estradiol promotes synaptogenesis in other brain regions, such as hippocampus (51, 52, 53, 54, 55, 56) and hypothalamus (57, 58).

To understand the mode of action of estradiol, the identification of ER in neonatal cerebellum is essential. It has been reported that the neonatal rat Purkinje cell expresses ERß (25). Consequently, estradiol may act directly on the Purkinje cell through ERß-mediated mechanisms, to promote the dendritic growth and spinogenesis in Purkinje cells during cerebellar cortical formation. The hypothesis postulated here is supported by the present finding with the antiestrogen tamoxifen, which inhibited the estrogen action on Purkinje cell dendrites. It is known that this antiestrogen binds to ERs (ER{alpha} and ERß) and activates transcription via activating protein-1 response elements (59) while blocking transcriptional activation through the classical estrogen response element and not producing any agonist effect via this pathway (60, 61). Thus, it is considered that the antiestrogen tamoxifen used in this study blocks transcriptional activation of ERß in the developing Purkinje cell. The absence of almost all the dendritic spine-like structures in Purkinje cells after the treatment with tamoxifen suggests that estradiol acts on Purkinje cells via ERß to induce not only dendritic growth but also dendritic spine formation. However, estradiol might act on Purkinje cells via nonnuclear ERs. According to Smith et al. (19, 20), locally applied estrogens and antiestrogens can alter glutamate-evoked excitation of Purkinje cells. It has also been suggested that the effect of estradiol on hippocampal CA1 pyramidal cell dendrite spine density requires the activation of NMDA receptors in adult female rats (54). Such nongenomic estrogen actions may lead to alterations in gene expression. On the other hand, recent ultrastructural studies have revealed extranuclear ER{alpha} immunoreactivity within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes (55). This study suggests nongenomic action of estrogen via ER{alpha} in this brain region. Future study is needed to demonstrate whether the promotion of dendritic growth and spine formation by estradiol is attributable to a nongenomic action?

Neurotrophins are attractive candidate regulators of Purkinje cell dendrite and spine development. It has been reported that neurotrophic factors, such as brain-derived neurotrophic factor or neurotrophin-3, are highly expressed in the developing cerebellum and are critical for proper development of Purkinje cells and granule cells (62, 63, 64, 65, 66). Accordingly, it can be supposed that estradiol and/or progesterone may induce the expression of some neurotrophic factors that directly promote the dendritic growth and spine development in the Purkinje cell during neonatal life. Future study is required to clarify the neurotrophic factor(s) involving in Purkinje cell development.


   Acknowledgments
 
We thank Dr. George E. Bentley (Department of Biology, University of Washington, Seattle) for his valuable discussion and for reading the manuscript.


   Footnotes
 
This work was supported, in part, by Grants-in-Aid for Scientific Research 12440233, 12894021, 13210101, and 15207007 (to K.T.) from the Ministry of Education, Science, Sports and Culture, Japan. H.S. is supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists.

Present address for H.S.: Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.

Abbreviations: DIG, Digoxigenin; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; DIV, d in vitro; EB, estradiol benzoate; EIA, enzyme immunoassay; ER, estrogen receptor; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4-isomerase; P450arom, cytochrome P450 aromatase; P450scc, cytochrome P450 side-chain cleavage enzyme; PFA, paraformaldehyde; PR, progesterone receptor.

Received March 10, 2003.

Accepted for publication July 8, 2003.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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