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Dendritic spine formation in response to progesterone synthesized de novo in the developing Purkinje cell in rats
Neuroscience Letters 322 (2002) 111–115 www.elsevier.com/locate/neulet
Dendritic spine formation in response to progesterone synthesized de novo in the developing Purkinje cell in rats Hirotaka Sakamoto a,b, Kazuyoshi Ukena a,b, Kazuyoshi Tsutsui a,b,* b
a Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Tokyo 150-0002, Japan
Received 13 December 2001; received in revised form 7 January 2002; accepted 11 January 2002
Abstract The cerebellar Purkinje cell (PC) is a typical site for neurosteroid formation. We have demonstrated that this neuron possesses intranuclear receptor for progesterone and actively synthesizes progesterone de novo from cholesterol only during rat neonatal life, when the formation of the cerebellar cortex occurs dramatically. In this study, we therefore analyzed the effect of progesterone on dendritic spine formation of the PC. In vitro studies using cerebellar slice cultures from newborn rats showed that progesterone increases the density of PC dendritic spines in a dose-dependent manner. This effect was blocked by the progesterone receptor antagonist, RU486. Furthermore, trilostane, a specific inhibitor of progesterone synthesis, inhibited the increase of spine density. These results suggest that progesterone can promote dendritic spine formation, and endogenous progesterone synthesized de novo in the developing PC may induce such an effect. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neurosteroids; Progesterone; 3a,5a-Tetrahydroprogesterone; Dendritic spine formation; Purkinje cell; Cerebellar development
It is now established that the brain synthesizes steroids de novo from cholesterol (for reviews, see [3,5,17]). Such steroids synthesized de novo in the brain as well as other areas of the nervous system are called neurosteroids [6,10]. We have demonstrated that the Purkinje cell (PC), an important cerebellar neuron, actively synthesizes neurosteroids (for review, see [17]). This neuron possesses several steroidogenic enzymes, i.e. cytochrome P450 side-chain cleavage enzyme (P450scc) and 3b-hydroxysteroid dehydrogenase/ D 5-D 4-isomerase (3bHSD), and produces pregnenolone, pregnenolone sulfate and progesterone in both mammals [18,19] and non-mammals [12,16,20]. Interestingly, in the rat, cytochrome P450scc appears in the PC immediately after differentiation and the expression of this enzyme persists during neonatal development into adulthood, indicating a constant production of pregnenolone [18]. In contrast, both the 3bHSD expression and progesterone formation in the rat PC increase during the neonatal period
* Corresponding author. Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, HigashiHiroshima 739-8521, Japan. Tel.: 181-824-24-6571; fax: 181-82424-0759. E-mail address: [email protected] (K. Tsutsui).
[19]. In addition, we have further identified a metabolite of progesterone, 3a,5a-tetrahydroprogesterone (3a,5a-THP), in the cerebellum of neonatal rats [17]. It is well known that rat PCs differentiate just after birth and the formation of the cerebellar cortex becomes complete in the neonate [1,2]. Progesterone and/or 3a,5a-THP may be involved in the cerebellar cortical formation during neonatal life, when cerebellar progesterone and 3a,5a-THP are both high in rats [17,19]. In fact, we have found recently that progesterone promotes dendritic growth and synaptogenesis in rat PCs during neonatal life [13]. We have further indicated the presence of intranuclear receptor for progesterone (PR) in the PC of neonatal rats [13]. Thus, progesterone synthesized de novo in the developing PC may be an essential factor for the formation of the cerebellar neuronal circuit which occurs during neonatal life. Dendritic spines receive .90% of all central nervous system excitatory synapses [8], marking them excellent models for synaptogenesis and for shortand long-term synaptic modifications [14]. To clarify roles of progesterone and its metabolite, 3a,5a-THP, in the formation of the cerebellar neuronal circuit, we analyzed dendritic spine formation of the PC in response to these neurosteroids. Male and female rats of the Fisher strain maintained in this laboratory were mated and housed in a temperature-
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controlled room under a daily photoperiod of 14 h of light and 10 h of darkness. Newborn male rats of 3 and 5 days of age, when endogenous progesterone and its metabolite, 3a,5a-THP, were very low in the cerebellum [17,19], were used for in vivo and in vitro treatments with these neurosteroids. In previous studies [17,19], the expression of 3bHSD in PCs was negligible at 3 days of age, but increased from 7 days to reach a peak at approximately 10 days, and decreased thereafter. Such changes were consistent with changes in cerebellar progesterone [17,19]. 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). Organotypic slice cultures of cerebella were conducted according to our previous method [13]. In brief, cerebella of male pups at 5 days of age were used and vermal parasagittal slices (400 mm thick) were aseptically cut on a microslicer. Cultures of cerebellar slices were conducted according to a static interface method of hippocampal organotypic cultures [15]. The culture medium was composed of a 1:1 mixture of Dulbecco’s modified essential medium and Ham’s F-12 (Sigma, St. Louis, MO), supplemented with insulin (5 mg/ml; Sigma), apo-transferrin (100 mg/ml; Sigma), putrescine (100 mM; Sigma), sodium selenite (30 nM), d-glucose (6 mg/ml), penicillin G potassium (100 U/ ml), and streptomycin sulfate (100 mg/ml). The culture medium contained 5% fetal bovine serum for the first 2 days of culture (2 days in vitro (2 DIV)). Steroids were excluded from the medium composition. Cultures were maintained at 37 8C in an atmosphere of humidified 95% air and 5% CO2. To investigate spine proliferation of PCs induced by progesterone or 3a,5a-THP, these neurosteroids were applied to granuloprival cerebellar cultures for 3 days after 2 DIV and cultures were fixed at 5 DIV. Crystalline progesterone or 3a,5a-THP (Sigma) was dissolved into absolute ethanol and applied to the culture medium at various concentrations (0.1, 1, 10, 100 and 1000 nM (1 mM)). The final concentration of ethanol was adjusted to 0.001% (v/v) in all neurosteroid-treated and control (vehicle alone) groups. The effect of an anti-progestin, RU486 (Biomol, Plymouth Meeting, PA), was examined at a concentration of 1 mM in a progesterone-treated (10 nM) group. RU486 alone was also tested as a control. To investigate the action of endogenous progesterone, the effect of a specific inhibitor of 3bHSD, trilostane (Mochida, Tokyo, Japan), was examined at a concentration of 2 mM. Cultures were treated with trilostane or vehicle once a day for 3 days from 2 DIV. Combined treatment with trilostane (2 mM) and progesterone (10 nM) was also performed in a similar manner. All cultures were fixed in 2% paraformaldehyde (PFA), 2.5% glutaraldehyde (GA) and 15% saturated picric acid (v/v) in 0.1 M phosphate buffer (PB; pH 7.3) overnight at 4 8C. In vivo steroid treatment was performed according to our previous method [13]. In brief, progesterone dissolved in sesame oil (50 mg/25 ml) was injected into the cerebrospinal
fluid around the posterior vermis of male pups once per day for 4 days from 3 days of age. Pups receiving injections of the vehicle alone (sesame oil) served as controls. At 7 days of age, pups were deeply anesthetized with chloroform before transcardial perfusion with phosphate-buffered saline (10 mM PB and 0.14 M NaCl; pH 7.3), followed by fixative solution (2% PFA, 2.5% GA and 15% saturated picric acid (v/v) in PB). Vermal cerebella were dissected out and sectioned parasagittally at 50 mm thickness with a microslicer. PCs were identified by immunostaining with a mouse monoclonal antibody raised against a calcium-binding protein, calbindin-D28k (Sigma) according to our previous method [13]. After elimination of endogenous peroxidase activity with 3% H2O2 (0% for electron microscopic study) and blocking non-specific binding components with 1% normal horse serum and 1% bovine serum albumin, the sections and slice cultures were immersed overnight at 4 8C 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. After dehydration, stained sections and slice cultures were analyzed using a microscope (Olympus Optical, Tokyo, Japan). To analyze the effects of progesterone or 3a,5a-THP, the number of PC dendritic spines per unit length of dendrite was measured as follows: the selected segment was traced (magnification, 1200£) with a camera lucida drawing tube; all 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 mm dendrite. Three to five dendritic segments per cell and at least six PCs per slice were analyzed. The cross-sectional soma area of PCs was also measured in each selected calbindin-immunostained lobe by using an NIHImage software package. For electron microscopy, calbindin-immunocytochemically stained lobe sections of progesterone- and vehicle-treated male pups at 7 days of age were postfixed in 1% osmium tetroxide in 0.1 M PB, dehydrated in ascending grades of ethanol, and then embedded flat in epoxy resin (Quetol-812; Nisshin EM, Tokyo, Japan) according to our previous method [13]. Ultrathin sections (60 nm in thickness) containing calbindin-immunoreactive PC dendrites in lobe IX were collected in slot grids coated with Formvar film, electron-stained with uranyl acetate and lead citrate, and viewed under a H-600A electron microscope (Hitachi, Tokyo, Japan). At least 24 electron microphotographs of random regions in the molecular layer of lobe IX were generated from six different males per each experimental group. The number of spines, defined as postsynaptic processes smaller than 2 mm in diameter, lacking mitochondria, was counted [4]. Differences in the morphological appearance of PCs following treatment with progesterone or 3a,5a-THP were
H. Sakamoto et al. / Neuroscience Letters 322 (2002) 111–115
Fig. 1. Dendritic morphology of PCs and its modulation by progesterone treatment: in vitro study. (A–D) Cerebellar cultures from newborn male rats grown for 5 DIV and immunostained for calbindin. (A,B) PC dendrites in the progesterone (PROG) group (B; 100 nM progesterone) appeared to be well-developed compared with the vehicle group (A). (C,D) Higher magnification of the blocked areas in (A) and (B) shows the greater density of dendritic spines in the PROG group (D) compared with the vehicle group (C). Arrowheads indicate presumptive spine structures. Scale bars: 20 mm in (A,B); and 5 mm in (C,D).
analyzed by a one-way analysis of variance (ANOVA) among different groups. If significance was reached with the ANOVA test, the analysis was followed by Duncan’s multiple range test. All in vitro treatment groups were composed of the cultured slices from multiple animals (at least four animals), and all slices were separately cultured in the individual chamber. Statistical comparisons of the in vitro studies were based on the individual slice as the unit of analysis, because the physiological environment in tissue culture rapidly becomes independent of the animal of origin. Statistical analysis of the in vivo study was based on the individual animal, and differences in the number of dendritic spines per unit area (100 mm 2) were subjected to a Student’s t-test between two different groups. In vitro study: Morphological analysis revealed that initial administration of 100 nM progesterone in vitro induced an increase in the number of PC spines per unit length of dendrite (spine density) (Figs. 1 and 2A). This stimulatory effect of progesterone occurred in a dose-dependent manner with a threshold concentration ranging between 1 and 10 nM (n ¼ 12 slices from four different
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males in each dose; Fig. 2A), indicating that progesterone actions were within the physiological range observed previously during normal cerebellar development [19]. In contrast to progesterone, 3a,5a-THP failed to significantly alter the density of PC dendritic spines with the same dose treatments (n ¼ 12 slices from four different males in each dose; Fig. 2A). Subsequently, we investigated whether the effect of progesterone is blocked by RU486, an antagonist of PR (n ¼ 18 slices from six different males in each group; Fig. 2B). Treatment with 10 nM progesterone alone induced a significant increase in the density of PC dendritic spines (P , 0:05 vs. vehicle group; Fig. 2B), whereas RU486 alone at a concentration of 1 mM (100 times greater than the progesterone concentration) tended to decrease the PC spine density which was not significantly different from that in the vehicle-treated group (Fig. 2B). In contrast, combined treatments with progesterone (10 nM) and RU486 (1 mM) revealed that RU486 abolished the progesterone-induced dendritic spine formation of PCs (P , 0:01 vs. progesterone group; Fig. 2B). The action of endogenous progesterone on dendritic spine formation of PCs was further assessed by the inhibition of progesterone synthesis using trilostane, a specific inhibitor of 3bHSD. Administration of 2 mM trilostane into cerebellar slice cultures from neonatal rats decreased the density of PC dendritic spines (P , 0:05 vs. vehicle group; Fig. 2B). The inhibitory effect of trilostane was not attributed to its toxicity, because combined treatments with trilostane and progesterone increased the density of PC dendritic spines (P , 0:01 vs. trilostane group; Fig. 2B). In contrast to PC dendritic spine density, the crosssectional cell body area of PCs remained unchanged after progesterone treatment (data not shown). In vivo study: Progesterone was directly injected into the cerebrospinal fluid around the posterior vermal lobe (IX) of the cerebellum at 50 mg/day for 4 days during the early neonatal period (from 3 to 6 days of age). This is a period of time when an active formation of endogenous progesterone does not occur in the PC [19]. The number of PC dendritic spines per unit area (spine density) at 7 days of age was compared between the progesterone (n ¼ 6)- and vehicle (n ¼ 6)-treated groups after immunostaining for calbindin. Progesterone administration increased the density of dendritic spines of PCs (Fig. 3A,B). Quantitative electron microscopic analysis also revealed that the density of dendritic spines on PCs (100 mm 2 field) in the progesterone-treated group (n ¼ 6) was significantly larger (P , 0:01) than that in the vehicle-treated group (n ¼ 6; Fig. 3C). We have demonstrated recently that in rats the PC, a cerebellar neuron, possesses the neurosteroidogenic enzymes cytochrome P450scc and 3bHSD and produces pregnenolone, pregnenolone sulfate and progesterone from cholesterol [18,19]. This is the first observation of neuronal neurosteroidogenesis in the mammalian brain. Interestingly, this neuron produced significant amounts of progesterone, as a result of an increase of 3bHSD activity, only during a limited neonatal period, when cerebellar formation occurs
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Fig. 2. (A) Effect of progesterone (PROG) and its metabolite 3a,5a-THP on the density of dendritic spines of PCs: in vitro study. The number of spines per unit length (50 mm) of dendrite of PCs was measured after immunostaining for calbindin. Each dot and vertical line represent the mean ^ SEM (n ¼ 12 slices from four different males in each group). **P , 0:01; *P , 0:05 vs. vehicle group (by one-way ANOVA, followed by Duncan’s multiple range test). (B) Effect of the anti-progestin RU486 and the specific 3bHSD inhibitor trilostane (TRIL) on the density of PC dendritic spines: in vitro study. Each column and vertical line represent the mean ^ SEM (n ¼ 18 slices from six different males in each group). *P , 0:05 vs. vehicle group; ‡‡P , 0:01 vs. 10 28 M PROG group; §§P , 0:01 vs. TRIL group (by one-way ANOVA, followed by Duncan’s multiple range test).
most markedly [1,2]. We therefore examined the effect of progesterone synthesized de novo in the developing PC on its dendritic spine formation, as an important parameter of dendritic morphological changes, in this study. In vitro treatment with progesterone using cerebellar slice cultures from newborn rats increased the density of dendritic spines on PCs. Progesterone promoted dose-dependent dendritic spine formation of the PC with a threshold concentration within the physiological range, i.e. 1–10 nM. These results were consistent with in vivo experiments in which progesterone administration to newborn rats increased the density of dendritic spines of PCs. According to our previous findings [13,19], in vivo administration of progesterone to newborn rats also induced a significant increase in cerebellar progesterone level to a concentration similar to the maximal level observed in neonatal rats at 10 days of age under normal development. Thus, both in vitro and in vivo studies suggest that progesterone may be involved in the promotion of dendritic spine formation of the developing PC. In contrast, the effect of progesterone on PC somata was negligible. Our previous reverse transcription-polymerase chain
Fig. 3. (A,B) Ultrastructural analysis of dendritic spines of PCs: in vivo study. Calbindin-immunoelectron micrographs of PC dendrites in the molecular layers of vermal cerebella (lobe IX). Spine number was increased in progesterone (PROG) administered pups (B) compared with the vehicle group (A). Arrowheads indicate presumptive spine structures. PD, PC dendrite. Scale bars, 1 mm. (C) Quantitative electron microscopic analysis of dendritic spine density per unit area (100 mm 2 field). Each column and vertical line represent the mean ^ SEM (n ¼ 6 males in each group). Data were derived from randomly selected 24 fields (100 mm 2 in each field) of vermal molecular layers in each group. **P , 0:01 (by Student’s t-test).
reaction/Southern and immunocytochemical analyses showed that the intranuclear PR is expressed in PCs during neonatal life [13]. Accordingly, progesterone may act directly on PCs via intranuclear PR to promote dendritic spine formation of the PC during the neonatal period. This hypothesis is supported by the present finding with the antagonist of PR, RU486. The stimulatory action of progesterone on PC dendritic spines was blocked by RU486 in vitro by combined administration of progesterone and RU486. In addition, trilostane, a specific inhibitor of 3bHSD inhibited the formation of dendritic spines of PCs in vitro, suggesting that endogenous synthesis of progesterone contributes to dendritic spine formation. It is therefore possible that progesterone produced in neonatal PCs may promote dendritic spine formation through intranuclear
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receptor-mediated mechanisms during cerebellar development. Dendritic spines are thought to represent postsynaptic sites [7]. Since changes in the number of dendritic spines during development or in response to experimentally induced injury are correlated positively with changes in the number of synapses [11], it is likely that morphological changes in dendritic spines observed in this study reflect changes in the number of synapses. Indeed, we have reported recently that progesterone promotes synaptogenesis in rat PCs by genomic mechanisms during cerebellar cortical formation [13]. It is well known that dramatic morphological changes occur in the rat cerebellum during neonatal life [1,2]. From these morphological findings [1,2], the formation of the rat cerebellar cortex becomes complete through the processes of migration of external granular cells, neuronal and glial growth, and synaptogenesis, during neonatal life, when cerebellar progesterone is high [19]. In the present study, progesterone administration to newborn rats promoted dendritic spine formation in the PC. Therefore, it is considered that endogenous progesterone synthesized de novo in the developing PC may be able to induce such an effect. To draw a firm conclusion, however, we need more precise experiments using the specific inhibitor of progesterone synthesis, trilostane and/or neurosteroidogenic enzyme knock-out animals. It has also been reported that progesterone promotes myelination in the peripheral nervous system via intranuclear PR [9]. Our results in the central nervous system are in harmony with these findings in the peripheral nervous system. In contrast to progesterone, however, we could not detect any significant effect of 3a,5a-THP, a progesterone metabolite, on PC dendritic spine formation. It is known that 3a,5a-THP does not act on the PR directly, instead by functioning directly by modulation of ion-gated channel receptors, such as g-aminobutyric acid A [3]. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology Japan (12440233, 12894021, and 13210101 to K.T.). H.S. is supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. [1] Altman, J., Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer, J. Comp. Neurol., 145 (1972) 353–398. [2] Altman, J., Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer, J. Comp. Neurol., 145 (1972) 399–464. [3] Baulieu, E.-E., Neurosteroids: of the nervous system, by the nervous system, for the nervous system (Review), Recent Prog. Horm. Res., 52 (1997) 1–32. [4] Bravin, M., Morando, L., Vercelli, A., Rossi, F. and Strata, P.,
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Dendritic spine formation in response to progesterone synthesized de novo in the developing Purkinje cell in rats Hirotaka Sakamoto a,b, Kazuyoshi Ukena a,b, Kazuyoshi Tsutsui a,b,* b
a Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Tokyo 150-0002, Japan
Received 13 December 2001; received in revised form 7 January 2002; accepted 11 January 2002
Abstract The cerebellar Purkinje cell (PC) is a typical site for neurosteroid formation. We have demonstrated that this neuron possesses intranuclear receptor for progesterone and actively synthesizes progesterone de novo from cholesterol only during rat neonatal life, when the formation of the cerebellar cortex occurs dramatically. In this study, we therefore analyzed the effect of progesterone on dendritic spine formation of the PC. In vitro studies using cerebellar slice cultures from newborn rats showed that progesterone increases the density of PC dendritic spines in a dose-dependent manner. This effect was blocked by the progesterone receptor antagonist, RU486. Furthermore, trilostane, a specific inhibitor of progesterone synthesis, inhibited the increase of spine density. These results suggest that progesterone can promote dendritic spine formation, and endogenous progesterone synthesized de novo in the developing PC may induce such an effect. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neurosteroids; Progesterone; 3a,5a-Tetrahydroprogesterone; Dendritic spine formation; Purkinje cell; Cerebellar development
It is now established that the brain synthesizes steroids de novo from cholesterol (for reviews, see [3,5,17]). Such steroids synthesized de novo in the brain as well as other areas of the nervous system are called neurosteroids [6,10]. We have demonstrated that the Purkinje cell (PC), an important cerebellar neuron, actively synthesizes neurosteroids (for review, see [17]). This neuron possesses several steroidogenic enzymes, i.e. cytochrome P450 side-chain cleavage enzyme (P450scc) and 3b-hydroxysteroid dehydrogenase/ D 5-D 4-isomerase (3bHSD), and produces pregnenolone, pregnenolone sulfate and progesterone in both mammals [18,19] and non-mammals [12,16,20]. Interestingly, in the rat, cytochrome P450scc appears in the PC immediately after differentiation and the expression of this enzyme persists during neonatal development into adulthood, indicating a constant production of pregnenolone [18]. In contrast, both the 3bHSD expression and progesterone formation in the rat PC increase during the neonatal period
* Corresponding author. Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, HigashiHiroshima 739-8521, Japan. Tel.: 181-824-24-6571; fax: 181-82424-0759. E-mail address: [email protected] (K. Tsutsui).
[19]. In addition, we have further identified a metabolite of progesterone, 3a,5a-tetrahydroprogesterone (3a,5a-THP), in the cerebellum of neonatal rats [17]. It is well known that rat PCs differentiate just after birth and the formation of the cerebellar cortex becomes complete in the neonate [1,2]. Progesterone and/or 3a,5a-THP may be involved in the cerebellar cortical formation during neonatal life, when cerebellar progesterone and 3a,5a-THP are both high in rats [17,19]. In fact, we have found recently that progesterone promotes dendritic growth and synaptogenesis in rat PCs during neonatal life [13]. We have further indicated the presence of intranuclear receptor for progesterone (PR) in the PC of neonatal rats [13]. Thus, progesterone synthesized de novo in the developing PC may be an essential factor for the formation of the cerebellar neuronal circuit which occurs during neonatal life. Dendritic spines receive .90% of all central nervous system excitatory synapses [8], marking them excellent models for synaptogenesis and for shortand long-term synaptic modifications [14]. To clarify roles of progesterone and its metabolite, 3a,5a-THP, in the formation of the cerebellar neuronal circuit, we analyzed dendritic spine formation of the PC in response to these neurosteroids. Male and female rats of the Fisher strain maintained in this laboratory were mated and housed in a temperature-
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 07 7- 0
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controlled room under a daily photoperiod of 14 h of light and 10 h of darkness. Newborn male rats of 3 and 5 days of age, when endogenous progesterone and its metabolite, 3a,5a-THP, were very low in the cerebellum [17,19], were used for in vivo and in vitro treatments with these neurosteroids. In previous studies [17,19], the expression of 3bHSD in PCs was negligible at 3 days of age, but increased from 7 days to reach a peak at approximately 10 days, and decreased thereafter. Such changes were consistent with changes in cerebellar progesterone [17,19]. 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). Organotypic slice cultures of cerebella were conducted according to our previous method [13]. In brief, cerebella of male pups at 5 days of age were used and vermal parasagittal slices (400 mm thick) were aseptically cut on a microslicer. Cultures of cerebellar slices were conducted according to a static interface method of hippocampal organotypic cultures [15]. The culture medium was composed of a 1:1 mixture of Dulbecco’s modified essential medium and Ham’s F-12 (Sigma, St. Louis, MO), supplemented with insulin (5 mg/ml; Sigma), apo-transferrin (100 mg/ml; Sigma), putrescine (100 mM; Sigma), sodium selenite (30 nM), d-glucose (6 mg/ml), penicillin G potassium (100 U/ ml), and streptomycin sulfate (100 mg/ml). The culture medium contained 5% fetal bovine serum for the first 2 days of culture (2 days in vitro (2 DIV)). Steroids were excluded from the medium composition. Cultures were maintained at 37 8C in an atmosphere of humidified 95% air and 5% CO2. To investigate spine proliferation of PCs induced by progesterone or 3a,5a-THP, these neurosteroids were applied to granuloprival cerebellar cultures for 3 days after 2 DIV and cultures were fixed at 5 DIV. Crystalline progesterone or 3a,5a-THP (Sigma) was dissolved into absolute ethanol and applied to the culture medium at various concentrations (0.1, 1, 10, 100 and 1000 nM (1 mM)). The final concentration of ethanol was adjusted to 0.001% (v/v) in all neurosteroid-treated and control (vehicle alone) groups. The effect of an anti-progestin, RU486 (Biomol, Plymouth Meeting, PA), was examined at a concentration of 1 mM in a progesterone-treated (10 nM) group. RU486 alone was also tested as a control. To investigate the action of endogenous progesterone, the effect of a specific inhibitor of 3bHSD, trilostane (Mochida, Tokyo, Japan), was examined at a concentration of 2 mM. Cultures were treated with trilostane or vehicle once a day for 3 days from 2 DIV. Combined treatment with trilostane (2 mM) and progesterone (10 nM) was also performed in a similar manner. All cultures were fixed in 2% paraformaldehyde (PFA), 2.5% glutaraldehyde (GA) and 15% saturated picric acid (v/v) in 0.1 M phosphate buffer (PB; pH 7.3) overnight at 4 8C. In vivo steroid treatment was performed according to our previous method [13]. In brief, progesterone dissolved in sesame oil (50 mg/25 ml) was injected into the cerebrospinal
fluid around the posterior vermis of male pups once per day for 4 days from 3 days of age. Pups receiving injections of the vehicle alone (sesame oil) served as controls. At 7 days of age, pups were deeply anesthetized with chloroform before transcardial perfusion with phosphate-buffered saline (10 mM PB and 0.14 M NaCl; pH 7.3), followed by fixative solution (2% PFA, 2.5% GA and 15% saturated picric acid (v/v) in PB). Vermal cerebella were dissected out and sectioned parasagittally at 50 mm thickness with a microslicer. PCs were identified by immunostaining with a mouse monoclonal antibody raised against a calcium-binding protein, calbindin-D28k (Sigma) according to our previous method [13]. After elimination of endogenous peroxidase activity with 3% H2O2 (0% for electron microscopic study) and blocking non-specific binding components with 1% normal horse serum and 1% bovine serum albumin, the sections and slice cultures were immersed overnight at 4 8C 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. After dehydration, stained sections and slice cultures were analyzed using a microscope (Olympus Optical, Tokyo, Japan). To analyze the effects of progesterone or 3a,5a-THP, the number of PC dendritic spines per unit length of dendrite was measured as follows: the selected segment was traced (magnification, 1200£) with a camera lucida drawing tube; all 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 mm dendrite. Three to five dendritic segments per cell and at least six PCs per slice were analyzed. The cross-sectional soma area of PCs was also measured in each selected calbindin-immunostained lobe by using an NIHImage software package. For electron microscopy, calbindin-immunocytochemically stained lobe sections of progesterone- and vehicle-treated male pups at 7 days of age were postfixed in 1% osmium tetroxide in 0.1 M PB, dehydrated in ascending grades of ethanol, and then embedded flat in epoxy resin (Quetol-812; Nisshin EM, Tokyo, Japan) according to our previous method [13]. Ultrathin sections (60 nm in thickness) containing calbindin-immunoreactive PC dendrites in lobe IX were collected in slot grids coated with Formvar film, electron-stained with uranyl acetate and lead citrate, and viewed under a H-600A electron microscope (Hitachi, Tokyo, Japan). At least 24 electron microphotographs of random regions in the molecular layer of lobe IX were generated from six different males per each experimental group. The number of spines, defined as postsynaptic processes smaller than 2 mm in diameter, lacking mitochondria, was counted [4]. Differences in the morphological appearance of PCs following treatment with progesterone or 3a,5a-THP were
H. Sakamoto et al. / Neuroscience Letters 322 (2002) 111–115
Fig. 1. Dendritic morphology of PCs and its modulation by progesterone treatment: in vitro study. (A–D) Cerebellar cultures from newborn male rats grown for 5 DIV and immunostained for calbindin. (A,B) PC dendrites in the progesterone (PROG) group (B; 100 nM progesterone) appeared to be well-developed compared with the vehicle group (A). (C,D) Higher magnification of the blocked areas in (A) and (B) shows the greater density of dendritic spines in the PROG group (D) compared with the vehicle group (C). Arrowheads indicate presumptive spine structures. Scale bars: 20 mm in (A,B); and 5 mm in (C,D).
analyzed by a one-way analysis of variance (ANOVA) among different groups. If significance was reached with the ANOVA test, the analysis was followed by Duncan’s multiple range test. All in vitro treatment groups were composed of the cultured slices from multiple animals (at least four animals), and all slices were separately cultured in the individual chamber. Statistical comparisons of the in vitro studies were based on the individual slice as the unit of analysis, because the physiological environment in tissue culture rapidly becomes independent of the animal of origin. Statistical analysis of the in vivo study was based on the individual animal, and differences in the number of dendritic spines per unit area (100 mm 2) were subjected to a Student’s t-test between two different groups. In vitro study: Morphological analysis revealed that initial administration of 100 nM progesterone in vitro induced an increase in the number of PC spines per unit length of dendrite (spine density) (Figs. 1 and 2A). This stimulatory effect of progesterone occurred in a dose-dependent manner with a threshold concentration ranging between 1 and 10 nM (n ¼ 12 slices from four different
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males in each dose; Fig. 2A), indicating that progesterone actions were within the physiological range observed previously during normal cerebellar development [19]. In contrast to progesterone, 3a,5a-THP failed to significantly alter the density of PC dendritic spines with the same dose treatments (n ¼ 12 slices from four different males in each dose; Fig. 2A). Subsequently, we investigated whether the effect of progesterone is blocked by RU486, an antagonist of PR (n ¼ 18 slices from six different males in each group; Fig. 2B). Treatment with 10 nM progesterone alone induced a significant increase in the density of PC dendritic spines (P , 0:05 vs. vehicle group; Fig. 2B), whereas RU486 alone at a concentration of 1 mM (100 times greater than the progesterone concentration) tended to decrease the PC spine density which was not significantly different from that in the vehicle-treated group (Fig. 2B). In contrast, combined treatments with progesterone (10 nM) and RU486 (1 mM) revealed that RU486 abolished the progesterone-induced dendritic spine formation of PCs (P , 0:01 vs. progesterone group; Fig. 2B). The action of endogenous progesterone on dendritic spine formation of PCs was further assessed by the inhibition of progesterone synthesis using trilostane, a specific inhibitor of 3bHSD. Administration of 2 mM trilostane into cerebellar slice cultures from neonatal rats decreased the density of PC dendritic spines (P , 0:05 vs. vehicle group; Fig. 2B). The inhibitory effect of trilostane was not attributed to its toxicity, because combined treatments with trilostane and progesterone increased the density of PC dendritic spines (P , 0:01 vs. trilostane group; Fig. 2B). In contrast to PC dendritic spine density, the crosssectional cell body area of PCs remained unchanged after progesterone treatment (data not shown). In vivo study: Progesterone was directly injected into the cerebrospinal fluid around the posterior vermal lobe (IX) of the cerebellum at 50 mg/day for 4 days during the early neonatal period (from 3 to 6 days of age). This is a period of time when an active formation of endogenous progesterone does not occur in the PC [19]. The number of PC dendritic spines per unit area (spine density) at 7 days of age was compared between the progesterone (n ¼ 6)- and vehicle (n ¼ 6)-treated groups after immunostaining for calbindin. Progesterone administration increased the density of dendritic spines of PCs (Fig. 3A,B). Quantitative electron microscopic analysis also revealed that the density of dendritic spines on PCs (100 mm 2 field) in the progesterone-treated group (n ¼ 6) was significantly larger (P , 0:01) than that in the vehicle-treated group (n ¼ 6; Fig. 3C). We have demonstrated recently that in rats the PC, a cerebellar neuron, possesses the neurosteroidogenic enzymes cytochrome P450scc and 3bHSD and produces pregnenolone, pregnenolone sulfate and progesterone from cholesterol [18,19]. This is the first observation of neuronal neurosteroidogenesis in the mammalian brain. Interestingly, this neuron produced significant amounts of progesterone, as a result of an increase of 3bHSD activity, only during a limited neonatal period, when cerebellar formation occurs
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Fig. 2. (A) Effect of progesterone (PROG) and its metabolite 3a,5a-THP on the density of dendritic spines of PCs: in vitro study. The number of spines per unit length (50 mm) of dendrite of PCs was measured after immunostaining for calbindin. Each dot and vertical line represent the mean ^ SEM (n ¼ 12 slices from four different males in each group). **P , 0:01; *P , 0:05 vs. vehicle group (by one-way ANOVA, followed by Duncan’s multiple range test). (B) Effect of the anti-progestin RU486 and the specific 3bHSD inhibitor trilostane (TRIL) on the density of PC dendritic spines: in vitro study. Each column and vertical line represent the mean ^ SEM (n ¼ 18 slices from six different males in each group). *P , 0:05 vs. vehicle group; ‡‡P , 0:01 vs. 10 28 M PROG group; §§P , 0:01 vs. TRIL group (by one-way ANOVA, followed by Duncan’s multiple range test).
most markedly [1,2]. We therefore examined the effect of progesterone synthesized de novo in the developing PC on its dendritic spine formation, as an important parameter of dendritic morphological changes, in this study. In vitro treatment with progesterone using cerebellar slice cultures from newborn rats increased the density of dendritic spines on PCs. Progesterone promoted dose-dependent dendritic spine formation of the PC with a threshold concentration within the physiological range, i.e. 1–10 nM. These results were consistent with in vivo experiments in which progesterone administration to newborn rats increased the density of dendritic spines of PCs. According to our previous findings [13,19], in vivo administration of progesterone to newborn rats also induced a significant increase in cerebellar progesterone level to a concentration similar to the maximal level observed in neonatal rats at 10 days of age under normal development. Thus, both in vitro and in vivo studies suggest that progesterone may be involved in the promotion of dendritic spine formation of the developing PC. In contrast, the effect of progesterone on PC somata was negligible. Our previous reverse transcription-polymerase chain
Fig. 3. (A,B) Ultrastructural analysis of dendritic spines of PCs: in vivo study. Calbindin-immunoelectron micrographs of PC dendrites in the molecular layers of vermal cerebella (lobe IX). Spine number was increased in progesterone (PROG) administered pups (B) compared with the vehicle group (A). Arrowheads indicate presumptive spine structures. PD, PC dendrite. Scale bars, 1 mm. (C) Quantitative electron microscopic analysis of dendritic spine density per unit area (100 mm 2 field). Each column and vertical line represent the mean ^ SEM (n ¼ 6 males in each group). Data were derived from randomly selected 24 fields (100 mm 2 in each field) of vermal molecular layers in each group. **P , 0:01 (by Student’s t-test).
reaction/Southern and immunocytochemical analyses showed that the intranuclear PR is expressed in PCs during neonatal life [13]. Accordingly, progesterone may act directly on PCs via intranuclear PR to promote dendritic spine formation of the PC during the neonatal period. This hypothesis is supported by the present finding with the antagonist of PR, RU486. The stimulatory action of progesterone on PC dendritic spines was blocked by RU486 in vitro by combined administration of progesterone and RU486. In addition, trilostane, a specific inhibitor of 3bHSD inhibited the formation of dendritic spines of PCs in vitro, suggesting that endogenous synthesis of progesterone contributes to dendritic spine formation. It is therefore possible that progesterone produced in neonatal PCs may promote dendritic spine formation through intranuclear
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receptor-mediated mechanisms during cerebellar development. Dendritic spines are thought to represent postsynaptic sites [7]. Since changes in the number of dendritic spines during development or in response to experimentally induced injury are correlated positively with changes in the number of synapses [11], it is likely that morphological changes in dendritic spines observed in this study reflect changes in the number of synapses. Indeed, we have reported recently that progesterone promotes synaptogenesis in rat PCs by genomic mechanisms during cerebellar cortical formation [13]. It is well known that dramatic morphological changes occur in the rat cerebellum during neonatal life [1,2]. From these morphological findings [1,2], the formation of the rat cerebellar cortex becomes complete through the processes of migration of external granular cells, neuronal and glial growth, and synaptogenesis, during neonatal life, when cerebellar progesterone is high [19]. In the present study, progesterone administration to newborn rats promoted dendritic spine formation in the PC. Therefore, it is considered that endogenous progesterone synthesized de novo in the developing PC may be able to induce such an effect. To draw a firm conclusion, however, we need more precise experiments using the specific inhibitor of progesterone synthesis, trilostane and/or neurosteroidogenic enzyme knock-out animals. It has also been reported that progesterone promotes myelination in the peripheral nervous system via intranuclear PR [9]. Our results in the central nervous system are in harmony with these findings in the peripheral nervous system. In contrast to progesterone, however, we could not detect any significant effect of 3a,5a-THP, a progesterone metabolite, on PC dendritic spine formation. It is known that 3a,5a-THP does not act on the PR directly, instead by functioning directly by modulation of ion-gated channel receptors, such as g-aminobutyric acid A [3]. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology Japan (12440233, 12894021, and 13210101 to K.T.). H.S. is supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. [1] Altman, J., Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer, J. Comp. Neurol., 145 (1972) 353–398. [2] Altman, J., Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer, J. Comp. Neurol., 145 (1972) 399–464. [3] Baulieu, E.-E., Neurosteroids: of the nervous system, by the nervous system, for the nervous system (Review), Recent Prog. Horm. Res., 52 (1997) 1–32. [4] Bravin, M., Morando, L., Vercelli, A., Rossi, F. and Strata, P.,
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