Neuroscience Letters 594 (2015) 127–132
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Effect of sex steroid hormones on the number of serotonergic neurons in rat dorsal raphe nucleus Yuyu Kunimura a,b , Kinuyo Iwata a , Norio Iijima a , Makito Kobayashi b , Hitoshi Ozawa a,∗ a b
Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan Department of Life Science, International Christian University, 3-10-2 Osawa, Mitaka, Tokyo181-8585, Japan
h i g h l i g h t s • • • •
In adult rat brain, sex steroid hormones do not affect serotonin neuron number. These hormones do not affect tryptophan hydroxylase-immunoreactive cell number. Serotonin-immunoreactive signal intensity in dorsal raphe is sexually dimorphic. This subregion-specific sexual dimorphism is independent of sex hormone levels.
a r t i c l e
i n f o
Article history: Received 27 January 2015 Received in revised form 6 March 2015 Accepted 26 March 2015 Available online 28 March 2015 Keywords: 5-Hydroxytryptamine Sex steroid hormone Tryptophan hydroxylase Sex difference Serotonergic system
a b s t r a c t Disorders caused by the malfunction of the serotonergic system in the central nervous system show sexspecific prevalence. Many studies have reported a relationship between sex steroid hormones and the brain serotonergic system; however, the interaction between sex steroid hormones and the number of brain neurons expressing serotonin has not yet been elucidated. In the present study, we determined whether sex steroid hormones altered the number of serotonergic neurons in the dorsal raphe nucleus (DR) of adult rat brains. Animals were divided into five groups: ovariectomized (OVX), OVX + low estradiol (E2), OVX + high E2, castrated males, and intact males. Antibodies against 5-hydroxytryptamine (5-HT, serotonin) and tryptophan hydroxylase (Tph), an enzyme for 5-HT synthesis, were used as markers of 5-HT neurons, and the number of 5-HT-immunoreactive (ir) or Tph-ir cells was counted. We detected no significant differences in the number of 5-HT-ir or Tph-ir cells in the DR among the five groups. By contrast, the intensity of 5-HT-ir showed significant sex differences in specific subregions of the DR independent of sex steroid levels, suggesting that the manipulation of sex steroid hormones after maturation does not affect the number and intensive immunostaining of serotonergic neurons in rat brain. Our results suggest that, the sexual dimorphism observed in the serotonergic system is due to factors such as 5-HT synthesis, transportation, and degradation but not to the number of serotonergic neurons. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Several disorders, such as migraine, depression, and eating disorders, show a sex-specific prevalence. For example, migraines are three times more prevalent in women than in men [1], and women are twice as likely as men to have depression [2]. Ninety percent of eating disorders develop in women [3]. A malfunction of the serotonergic system in the central nervous system has been considered an etiology of these diseases [4–6]. The serotonergic system shows sex differences in mammals. For example, in female rats, serotonin (5-hydroxytryptamine, 5-HT) levels are significantly higher
∗ Corresponding author. Tel.: +81 3 3822 2131x5320; fax: +81 3 5685 6640. E-mail address: [email protected] (H. Ozawa). http://dx.doi.org/10.1016/j.neulet.2015.03.060 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
in the brainstem and limbic forebrain than those in male rats, and 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of 5-HT, also shows significantly higher levels in the several regions of female brain [7]. Conversely, the rate of 5-HT synthesis in women is 52% lower than that in men [8]. In addition, the binding capacity of 5HT2 receptors for 5-HT is higher in men than it is in women [9]. These data suggest a relationship between sex steroid hormones and the serotonergic system. There is considerable evidence for the involvement of sex steroid hormones in the serotonergic system. Estrogen increases the mRNA expression of tryptophan hydroxylase (Tph)-2, which contributes to 5-HT synthesis, in certain subregions of rat median and dorsal raphe nucleus (DR) through the estrogen receptor- (ER) [10,11]. Androgen and estrogen increase 5-HT2A receptor mRNA and the density of ligand-5-HT2A receptor binding sites in both
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female and male rat brains [12]. In addition, DR serotonergic neurons express ER in rats, mice, and monkeys [13–15] while, ER␣ expression in serotonergic neurons has been found in mice [14]. Progesterone receptor (PR) expression in serotonergic neurons has been detected in mice and monkeys [16,17]. Androgen receptors have been observed in the DR of male rats and mice; however, most of these receptors are found in non-5-HT-immunoreactive (ir) cells [14]. Although there are some species differences, this evidence strongly supports the hypothesis that sex steroid hormones modify the brain serotonergic system. Additional evidence that 5-HT neurons are regulated by sex steroid hormones is that, the involvement of 5-HT neurons in the midbrain DR has been reported in the regulation of reproductive activities, such as sexual behavior, ovulation, and pregnancy [18–20]. Thus, numerous studies have examined the relationship between the brain serotonergic system and sex steroid hormones; however, there is no clear evidence for an effect of sex steroid hormones on the number of serotonergic neurons. Because it has been reported that ovariectomy decreased serotonin neuronal number and gene expression in female macaques, it is possible that sex steroid hormones alter the number of serotonergic neurons [21]. Therefore, the aim of this study was to determine whether manipulation of sex steroid hormones alter the number of serotonergic neurons in rats. 2. Materials and methods 2.1. Animals and treatments Adult female and male Wistar rats (200–300 g body weight) were purchased from Kiwa Laboratory Animals Co. Ltd. (Wakayama, Japan) and housed under controlled temperature (24 ± 2 ◦ C) and lighting (lights on from 06:00 to 20:00) conditions with ad libitum access to food and water. The estrous cycle was monitored using daily vaginal smears, and animals showing at least two consecutive 4-day cycles were used. Animals were divided into five groups. [1] OVX group: female rats were bilaterally ovariectomized (OVX) 2 weeks before perfusion. [2] Low estradiol-17 (E2) group: OVX animals immediately received subcutaneously (s.c.) with Silastic tubing (1.57 mm inner diameter; 3.18 mm outer diameter; 37.0 mm in length; Dow Corning, Midland, MI), filled with E2 (Sigma–Aldrich, St. Louis, MO) dissolved in sesame oil at 20 g/ml. In 37.0 mm of overall Silastic tubing length, the length filled with E2 oil was 25.0 mm, and the length of 6.00 mm at both ends were filled with silicone paste (Shin–Etsu Polymer, Tokyo, Japan). This low E2 level produces a negative-feedback level of plasma E2 for 1 week [22]. [3] High E2 group: OVX animals immediately received s.c. with Silastic tubing (1.02 mm inner diameter; 2.16 mm outer diameter; 32.0 mm in length; Dow Corning) filled with crystalline E2 to produce a positive-feedback level of plasma E2 for 3 days [23]. In 32.0 mm of overall Silastic tubing length, the length filled with crystalline E2 was 20.0 mm, and the length of 6.00 mm at both ends were filled with silicone paste. This high E2 level induces daily luteinizing hormone surges in OVX rats. [4] Castration (Cast) group: male rats were bilaterally gonadectomized 2 weeks before perfusion. [5] Intact male group. All surgical procedures were performed under isoflurane anesthesia. All studies were conducted according to the National Institutes of Health’s (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Experimentation Committee, Nippon Medical School. 2.2. Tissue preparation After each treatment was completed, all animals were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.). Animals were perfused transcardially with saline followed by 300 ml of 4%
paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Brains were removed, postfixed in the same fixative solution at 4 ◦ C overnight, and cryoprotected in 30% sucrose in PB at 4 ◦ C for 4 days. Coronal sections (40 m) through the midbrain were cut on a sliding microtome (Leica CM3050, Heidelberg, Germany) and stored in cryoprotectant solution at −25 ◦ C until immunohistochemistry was conducted. 2.3. Immunohistochemistry Sections were washed in 0.1 M PB containing 0.9% NaCl and 0.3% Triton X-100 (PBST) and incubated with PBST containing 10% normal rabbit serum at room temperature (RT) for 1.5 h. Sections were incubated with a goat anti-5-HT antibody (Immunostar, Hudson, WI) diluted 1:5000 in PBST overnight at 4 ◦ C, or with a monoclonal mouse anti-Tph antibody (Sigma–Aldrich) diluted 1:1000 in PBST for 5 days at 4 ◦ C. Sections were incubated with a biotinylated secondary antibody followed by streptavidin conjugated with horseradish peroxidase (HISTOFINE SAB-PO kit, Nichirei Biosciences Inc., Tokyo, Japan) diluted 1:1 in PBST for 2 h at RT each. After each step, sections were washed in PBST. Staining was developed using 3,3’-diaminobenzidine tetrahydrochloride (Sigma–Aldrich) with 0.009% hydrogen peroxide. Sections mounted on glass slides were dehydrated with a graded ethanol series, immersed in xylene, and mounted with Permount (Fisher Scientific, Fair Lawn, NJ). For double fluorescence immunohistochemistry, sections were washed in PBST and incubated with PBST containing 10% normal donkey serum at RT for 1.5 h. Sections were incubated overnight with a monoclonal mouse anti-Tph antibody (Sigma–Aldrich) diluted 1:500 in PBST at 4 ◦ C, followed by the goat anti-serotonin antibody (1:500) for 4 days at 4 ◦ C. Sections were incubated with an Alexa Fluor 488-conjugated donkey anti-mouse IgG antibody (1:500, Invitrogen, Carlsbad, CA) and an Alexa Fluor 568-conjugated donkey anti-goat IgG antibody (1:500, Invitrogen) in PBST for 2 h at RT. After each step, sections were washed in PBST. Thereafter, sections were mounted with VECTASHIELD (Vector Laboratories, Burlingame, CA) on glass slides. 2.4. Image analysis The brain sections containing 5-HT or Tph immunoreactivity were examined under a light microscope (BX-50, Olympus, Tokyo, Japan). The number of DR 5-HT-immunoreactive (ir) cells in 10 sections, between bregma −7.32 and −8.28 mm, and the number of Tph-ir neurons expressed in the section corresponding to bregma −8.40 mm [24], were counted for each rat on a computer display using NIH ImageJ software with the cell counter plug-in (version 10.2). The signal intensity of each DR 5-HT-ir neuron in three sections was verified using photographs. The three sections were collected from the DR, rostral (ROSTRAL), mid (MID), and caudal (CAUDAL), at bregma −7.32, −7.80, and −8.28 mm, respectively [24]. Images were inverted using ImageJ. Since 5-HT immunoreactivity was heterogeneous among subregions in the DR, the DR was divided into three subregions, lateral, dorsal, and ventral regions, and 5-HT immunoreactivity was verified at each subregion of each anatomical level (Fig. 2A–C) [10]. From the ROSTRAL and MID sections, 10 neurons were randomly selected from each subregion. From the CAUDAL sections, 4, 10, and 10 neurons were randomly chosen from the lateral, the dorsal, and the ventral subregions, respectively. The mean gray value of the selected 5-HT-ir neurons was obtained with ImageJ, and was divided by the mean gray value of the background. The number obtained from this calculation was used for statistical analysis.
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Confocal images of 5-HT and Tph immunoreactivity were obtained with an LSM710 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). 2.5. Statistical analysis All data were analyzed using one-way ANOVA followed by Tukey’s test and presented as mean ± standard error of the mean (SEM). All analyses were conducted with SPSS 20. Significance level was set at p < 0.05.
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3. Results The 5-HT-ir cells were observed throughout the DR from bregma −7.32 to −8.28 mm in both sexes (Fig. 1A ). There was no significant difference in the number of 5-HT-ir cells among five groups (Fig. 1C). The signal intensity of 5-HT-ir cells was analyzed at three anatomical levels, ROSTRAL, MID, and CAUDAL, each of which was divided into three subregions, lateral, dorsal, and ventral regions (Figs. 2A–C). At the ROSTRAL level, the 5-HT-ir intensity in
Fig. 1. Effect of sex steroid hormones on the expression of 5-hydroxytryptamine-immunoreactive (5-HT-ir) cells in dorsal raphe (DR) of adult rats. (A) Expression of 5-HT-ir neurons in the DR. Scale bar = 100 m. (B) The representative area shown in Fig. 1A is boxed. (C) Number of 5-HT-ir cells in the DR. Values are means ± SEM. The number in each column indicates the number of animals used. OVX, ovariectomized female; Low E2, ovariectomized female with low dose estradiol; High E2, ovariectomized female with high dose estradiol; Cast, gonadectomized male; Intact, male rats without any treatment; Aq, cerebral aqueduct; CX, cortex.
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Fig. 2. Effect of sex steroid hormones on signal intensity of 5-HT expression in DR. Representative photographs of each section and signal intensity of 5-HT-ir cells in rostral (ROSTRAL) (A), mid (MID) (B), and caudal (CAUDAL) (C) sections. In pictures, lateral, dorsal, and ventral subregions at each level are boxed. Scale bar = 200 m. In bargraphs, values are means ± SEM (n = 4 for each group). Values with identical letters are not significantly different (P > 0.05, one-way ANOVA followed by Tukey’s test). See Fig. 1 for abbreviations.
dorsal region was significantly higher in Cast group than that in OVX and High E2 groups. In ventral region, the 5-HT-ir intensity in Intact group was significantly higher than that in OVX and High E2 groups (Fig. 2A). At the MID level, the 5-HT-ir intensity in all subregions was significantly higher in Intact group than that in OVX and High E2 groups (Fig. 2B). At the CAUDAL level, the 5HT-ir intensity in ventral region was significantly higher in Intact group than that in all female groups (Fig. 2C). The average 5HT-ir intensity in all three subregions was significantly higher in Cast group than that in High E2 group, and significantly higher in Intact group than that in all female groups (OVX, 1.53 ± 0.07; Low E2, 1.60 ± 0.10; High E2, 1.48 ± 0.06; Cast, 1.822 ± 0.05; Intact, 1.95 ± 0.04). Tph has also been used as a marker of serotonergic neurons [10]. Because Tph immunoreactivity was observed in the cell bodies of 5-HT-ir neurons (Fig. 3A), the number of Tph-ir cells was counted. The Tph-ir cells were observed in the DR in both sexes. However, there was no significant difference in the number of Tph-ir cells among all groups (Fig. 3B).
4. Discussion In the present study, we explored the possibility that sex steroid hormones affected the number of 5-HT in neurons because several disorders caused by a malfunction of the brain serotonergic system are more prevalent in women than men. Our results indicated that, the number of 5-HT-ir or Tph-ir cells in the DR was not significantly different among the adult animals exposed to various sex steroid levels. Because serotonin is related to several behavioral and neurophysiological processes [25], it is possible that number of 5-HT neurons hardly varies in the DR. On the other hand, the intensity of the 5-HT immunoreactivity showed a significant difference between males and females in specific regions of the DR, although this sex difference was independent of sex steroid hormone exposure. These results suggest that, protein levels of 5-HT neuron in part of DR may be controlled by other than sex steroid hormones. Sex steroid hormones affect several serotonergic factors that may contribute to sexual dimorphism in the serotonergic system.
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Fig. 3. Effect of sex steroid hormones on the expression of Tph-ir cells in DR. (A) Double fluorescence immunolocalization of 5-HT and tryptophan hydroxylase (Tph). (a) Representative low power view of DR showing 5-HT (red) and Tph (green). Scale bar = 100 m. (b–d) Higher magnification views of the boxed region in (A), showing 5-HT and Tph. Scale bar = 20 m. (B) Representative photographs of DR Tph-ir neurons at bregma −8.40 mm and the number of Tph-ir cells in DR (n = 4). Scale bar = 200 m. Values are means ± SEM. See Fig. 1 for abbreviations.
One of these factors is the 5-HT receptor. Injections of estradiol benzoate into OVX rats induced an acute reduction in brain 5HT1 binding sites [26]. Another serotonergic factor affected by sex steroid hormones is the serotonin transporter (SERT). An E2 injection significantly increased the number of SERT-expressing cells in OVX rats [27]. Therefore, sex steroid hormones may modulate factors such as 5-HT binding sites and transporters rather than the number of neurons. Several plausible mechanisms can explain our present result that no significant difference was observed in the population of serotonergic neurons. First, the length of time after gonadectomy may have been too short to decrease 5-HT neuronal number. One report indicates that long-term OVX decreases 5-HT neuronal number in female macaques [21]. In that study, pubertal macaques were OVX, and the number of 5-HT neurons was counted by identifying Tph2 mRNA, coding rate-limiting enzyme in 5-HT synthesis, 3 years after OVX. We did not examine Tph2 mRNA expression in the present study, but the number of Tph proteinexpressing cells showed no significant differences 2 weeks after OVX. This evidence suggests that the length of time following gonadectomy may be a determinant for the reduction of Tph2 mRNA; however, the results observed in the macaques may also be primate specific. Another reasonable explanation for our result is that the number of 5-HT neurons may be determined before puberty. We examined 5-HT neurons in only the adult rat
because disorders such as depression and migraine manifest after puberty [28,29]. However, prepubertal conditions may be involved in determining the number of 5-HT neurons in the DR, as suggested in several published studies. For example, in monkeys, cortical 5HT levels and receptor numbers peak at 2 months of age [30,31]. In humans, children show higher levels than adults of 5-HIAA in cerebrospinal fluid [32]. Moreover, until the age of five, the capacity of 5-HT synthesis in children is more than double that in adults [33]. Overall, these data suggest that the serotonergic system is more dynamic before than after puberty. Thus, the possibility of prepubertal conditions determining the number of brain 5-HT neurons should be determined in future studies. A final plausible mechanism for the result obtained in the present study is that neuronal modulation of 5-HT may occur through PR. The OVX female animals in this study were not treated with progesterone, and PR has been detected in 5-HT neurons [16,17]. In conclusion, the present study demonstrated that the number of DR 5-HT neurons was not significantly different among animals exposed to different sex steroid levels after maturation. Although, a number of studies have examined the relationship between sex steroid hormones and the serotonergic system, this relationship has not yet been fully elucidated. By investigating the precise relationship between sex steroid hormones and the serotonergic system, adequate treatment, such as hormonal therapy, may be offered for disorders caused by alterations in the serotonergic system. Our
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study provides, fundamental knowledge for better understanding the interactions of sex steroid hormones with 5-HT. Acknowledgements This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan (Nos. S0801035, 22590230 to H.O.), and the MEXT-Supported Program for the Strategic Research Foundation at Private Universities. References [1] B.K. Rasmussen, R. Jensen, M. Schroll, J. Olesen, Epidemiology of headache in a general population – a prevalence study, J. Clin. Epidemiol. 44 (1991) 1147–1157. [2] M.M. Weissman, M. Olfson, Depression in women: implications for health care research, Science 269 (1995) 799–801. [3] N. Piran, Prevention of eating disorders: a review of outcome evaluation research, Isr. J. Psychiatry Relat. Sci. 42 (2005) 172–177. [4] T.D. Brewerton, Toward a unified theory of serotonin dysregulation in eating and related disorders, Psychoneuroendocrinology 20 (1995) 561–590. [5] P. Humphrey, W. Feniuk, M.J. Perren, I.J. Beresford, M. Skingle, E.T. Whalley, Serotonin and migraine, Ann. N. Y. Acad. Sci. 600 (1990) 587–598. [6] M. Naughton, J.B. Mulrooney, B.E. Leonard, A review of the role of serotonin receptors in psychiatric disorders, Hum. Psychopharmacol. 15 (2000) 397–415. [7] M. Carlsson, A. Carlsson, A regional study of sex differences in rat brain serotonin, Prog. Neuropsychopharmacol. Biol. Psychiatry 12 (1988) 53–61. [8] S. Nishizawa, C. Benkelfat, S.N. Young, M. Leyton, S. Mzengeza, C. de Montigny, P. Blier, M. Diksic, Differences between males and females in rates of serotonin synthesis in human brain, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 5308–5313. [9] F. Biver, F. Lotstra, M. Monclus, D. Wikler, P. Damhaut, J. Mendlewicz, S. Goldman, Sex difference in 5HT2 receptor in the living human brain, Neurosci. Lett. 204 (1996) 25–28. [10] N. Donner, R.J. Handa, Estrogen receptor beta regulates the expression of tryptophan–hydroxylase 2 mRNA within serotonergic neurons of the rat dorsal raphe nuclei, Neuroscience 163 (2009) 705–718. [11] R. Hiroi, R.A. McDevitt, J.F. Neumaier, Estrogen selectively increases tryptophan hydroxylase-2 mRNA expression in distinct subregions of rat midbrain raphe nucleus: association between gene expression and anxiety behavior in the open field, Biol. Psychiatry 60 (2006) 288–295. [12] B.E. Sumner, G. Fink, Testosterone as well as estrogen increases serotonin 2A receptor mRNA and binding site densities in the male rat brain, Brain Res. Mol. Brain Res. 59 (1998) 205–214. [13] H. Lu, H. Ozawa, M. Nishi, T. Ito, M. Kawata, Serotonergic neurones in the dorsal raphe nucleus that project into the medial preoptic area contain oestrogen receptor beta, J. Neuroendocrinol. 13 (2001) 839–845. [14] Z. Sheng, J. Kawano, A. Yanai, R. Fujinaga, M. Tanaka, Y. Watanabe, K. Shinoda, Expression of estrogen receptors (alpha, beta) and androgen receptor in serotonin neurons of the rat and mouse dorsal raphe nuclei; sex and species differences, Neurosci. Res. 49 (2004) 185–196.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Effect of sex steroid hormones on the number of serotonergic neurons in rat dorsal raphe nucleus Yuyu Kunimura a,b , Kinuyo Iwata a , Norio Iijima a , Makito Kobayashi b , Hitoshi Ozawa a,∗ a b
Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan Department of Life Science, International Christian University, 3-10-2 Osawa, Mitaka, Tokyo181-8585, Japan
h i g h l i g h t s • • • •
In adult rat brain, sex steroid hormones do not affect serotonin neuron number. These hormones do not affect tryptophan hydroxylase-immunoreactive cell number. Serotonin-immunoreactive signal intensity in dorsal raphe is sexually dimorphic. This subregion-specific sexual dimorphism is independent of sex hormone levels.
a r t i c l e
i n f o
Article history: Received 27 January 2015 Received in revised form 6 March 2015 Accepted 26 March 2015 Available online 28 March 2015 Keywords: 5-Hydroxytryptamine Sex steroid hormone Tryptophan hydroxylase Sex difference Serotonergic system
a b s t r a c t Disorders caused by the malfunction of the serotonergic system in the central nervous system show sexspecific prevalence. Many studies have reported a relationship between sex steroid hormones and the brain serotonergic system; however, the interaction between sex steroid hormones and the number of brain neurons expressing serotonin has not yet been elucidated. In the present study, we determined whether sex steroid hormones altered the number of serotonergic neurons in the dorsal raphe nucleus (DR) of adult rat brains. Animals were divided into five groups: ovariectomized (OVX), OVX + low estradiol (E2), OVX + high E2, castrated males, and intact males. Antibodies against 5-hydroxytryptamine (5-HT, serotonin) and tryptophan hydroxylase (Tph), an enzyme for 5-HT synthesis, were used as markers of 5-HT neurons, and the number of 5-HT-immunoreactive (ir) or Tph-ir cells was counted. We detected no significant differences in the number of 5-HT-ir or Tph-ir cells in the DR among the five groups. By contrast, the intensity of 5-HT-ir showed significant sex differences in specific subregions of the DR independent of sex steroid levels, suggesting that the manipulation of sex steroid hormones after maturation does not affect the number and intensive immunostaining of serotonergic neurons in rat brain. Our results suggest that, the sexual dimorphism observed in the serotonergic system is due to factors such as 5-HT synthesis, transportation, and degradation but not to the number of serotonergic neurons. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Several disorders, such as migraine, depression, and eating disorders, show a sex-specific prevalence. For example, migraines are three times more prevalent in women than in men [1], and women are twice as likely as men to have depression [2]. Ninety percent of eating disorders develop in women [3]. A malfunction of the serotonergic system in the central nervous system has been considered an etiology of these diseases [4–6]. The serotonergic system shows sex differences in mammals. For example, in female rats, serotonin (5-hydroxytryptamine, 5-HT) levels are significantly higher
∗ Corresponding author. Tel.: +81 3 3822 2131x5320; fax: +81 3 5685 6640. E-mail address: [email protected] (H. Ozawa). http://dx.doi.org/10.1016/j.neulet.2015.03.060 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
in the brainstem and limbic forebrain than those in male rats, and 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of 5-HT, also shows significantly higher levels in the several regions of female brain [7]. Conversely, the rate of 5-HT synthesis in women is 52% lower than that in men [8]. In addition, the binding capacity of 5HT2 receptors for 5-HT is higher in men than it is in women [9]. These data suggest a relationship between sex steroid hormones and the serotonergic system. There is considerable evidence for the involvement of sex steroid hormones in the serotonergic system. Estrogen increases the mRNA expression of tryptophan hydroxylase (Tph)-2, which contributes to 5-HT synthesis, in certain subregions of rat median and dorsal raphe nucleus (DR) through the estrogen receptor- (ER) [10,11]. Androgen and estrogen increase 5-HT2A receptor mRNA and the density of ligand-5-HT2A receptor binding sites in both
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female and male rat brains [12]. In addition, DR serotonergic neurons express ER in rats, mice, and monkeys [13–15] while, ER␣ expression in serotonergic neurons has been found in mice [14]. Progesterone receptor (PR) expression in serotonergic neurons has been detected in mice and monkeys [16,17]. Androgen receptors have been observed in the DR of male rats and mice; however, most of these receptors are found in non-5-HT-immunoreactive (ir) cells [14]. Although there are some species differences, this evidence strongly supports the hypothesis that sex steroid hormones modify the brain serotonergic system. Additional evidence that 5-HT neurons are regulated by sex steroid hormones is that, the involvement of 5-HT neurons in the midbrain DR has been reported in the regulation of reproductive activities, such as sexual behavior, ovulation, and pregnancy [18–20]. Thus, numerous studies have examined the relationship between the brain serotonergic system and sex steroid hormones; however, there is no clear evidence for an effect of sex steroid hormones on the number of serotonergic neurons. Because it has been reported that ovariectomy decreased serotonin neuronal number and gene expression in female macaques, it is possible that sex steroid hormones alter the number of serotonergic neurons [21]. Therefore, the aim of this study was to determine whether manipulation of sex steroid hormones alter the number of serotonergic neurons in rats. 2. Materials and methods 2.1. Animals and treatments Adult female and male Wistar rats (200–300 g body weight) were purchased from Kiwa Laboratory Animals Co. Ltd. (Wakayama, Japan) and housed under controlled temperature (24 ± 2 ◦ C) and lighting (lights on from 06:00 to 20:00) conditions with ad libitum access to food and water. The estrous cycle was monitored using daily vaginal smears, and animals showing at least two consecutive 4-day cycles were used. Animals were divided into five groups. [1] OVX group: female rats were bilaterally ovariectomized (OVX) 2 weeks before perfusion. [2] Low estradiol-17 (E2) group: OVX animals immediately received subcutaneously (s.c.) with Silastic tubing (1.57 mm inner diameter; 3.18 mm outer diameter; 37.0 mm in length; Dow Corning, Midland, MI), filled with E2 (Sigma–Aldrich, St. Louis, MO) dissolved in sesame oil at 20 g/ml. In 37.0 mm of overall Silastic tubing length, the length filled with E2 oil was 25.0 mm, and the length of 6.00 mm at both ends were filled with silicone paste (Shin–Etsu Polymer, Tokyo, Japan). This low E2 level produces a negative-feedback level of plasma E2 for 1 week [22]. [3] High E2 group: OVX animals immediately received s.c. with Silastic tubing (1.02 mm inner diameter; 2.16 mm outer diameter; 32.0 mm in length; Dow Corning) filled with crystalline E2 to produce a positive-feedback level of plasma E2 for 3 days [23]. In 32.0 mm of overall Silastic tubing length, the length filled with crystalline E2 was 20.0 mm, and the length of 6.00 mm at both ends were filled with silicone paste. This high E2 level induces daily luteinizing hormone surges in OVX rats. [4] Castration (Cast) group: male rats were bilaterally gonadectomized 2 weeks before perfusion. [5] Intact male group. All surgical procedures were performed under isoflurane anesthesia. All studies were conducted according to the National Institutes of Health’s (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Experimentation Committee, Nippon Medical School. 2.2. Tissue preparation After each treatment was completed, all animals were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.). Animals were perfused transcardially with saline followed by 300 ml of 4%
paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Brains were removed, postfixed in the same fixative solution at 4 ◦ C overnight, and cryoprotected in 30% sucrose in PB at 4 ◦ C for 4 days. Coronal sections (40 m) through the midbrain were cut on a sliding microtome (Leica CM3050, Heidelberg, Germany) and stored in cryoprotectant solution at −25 ◦ C until immunohistochemistry was conducted. 2.3. Immunohistochemistry Sections were washed in 0.1 M PB containing 0.9% NaCl and 0.3% Triton X-100 (PBST) and incubated with PBST containing 10% normal rabbit serum at room temperature (RT) for 1.5 h. Sections were incubated with a goat anti-5-HT antibody (Immunostar, Hudson, WI) diluted 1:5000 in PBST overnight at 4 ◦ C, or with a monoclonal mouse anti-Tph antibody (Sigma–Aldrich) diluted 1:1000 in PBST for 5 days at 4 ◦ C. Sections were incubated with a biotinylated secondary antibody followed by streptavidin conjugated with horseradish peroxidase (HISTOFINE SAB-PO kit, Nichirei Biosciences Inc., Tokyo, Japan) diluted 1:1 in PBST for 2 h at RT each. After each step, sections were washed in PBST. Staining was developed using 3,3’-diaminobenzidine tetrahydrochloride (Sigma–Aldrich) with 0.009% hydrogen peroxide. Sections mounted on glass slides were dehydrated with a graded ethanol series, immersed in xylene, and mounted with Permount (Fisher Scientific, Fair Lawn, NJ). For double fluorescence immunohistochemistry, sections were washed in PBST and incubated with PBST containing 10% normal donkey serum at RT for 1.5 h. Sections were incubated overnight with a monoclonal mouse anti-Tph antibody (Sigma–Aldrich) diluted 1:500 in PBST at 4 ◦ C, followed by the goat anti-serotonin antibody (1:500) for 4 days at 4 ◦ C. Sections were incubated with an Alexa Fluor 488-conjugated donkey anti-mouse IgG antibody (1:500, Invitrogen, Carlsbad, CA) and an Alexa Fluor 568-conjugated donkey anti-goat IgG antibody (1:500, Invitrogen) in PBST for 2 h at RT. After each step, sections were washed in PBST. Thereafter, sections were mounted with VECTASHIELD (Vector Laboratories, Burlingame, CA) on glass slides. 2.4. Image analysis The brain sections containing 5-HT or Tph immunoreactivity were examined under a light microscope (BX-50, Olympus, Tokyo, Japan). The number of DR 5-HT-immunoreactive (ir) cells in 10 sections, between bregma −7.32 and −8.28 mm, and the number of Tph-ir neurons expressed in the section corresponding to bregma −8.40 mm [24], were counted for each rat on a computer display using NIH ImageJ software with the cell counter plug-in (version 10.2). The signal intensity of each DR 5-HT-ir neuron in three sections was verified using photographs. The three sections were collected from the DR, rostral (ROSTRAL), mid (MID), and caudal (CAUDAL), at bregma −7.32, −7.80, and −8.28 mm, respectively [24]. Images were inverted using ImageJ. Since 5-HT immunoreactivity was heterogeneous among subregions in the DR, the DR was divided into three subregions, lateral, dorsal, and ventral regions, and 5-HT immunoreactivity was verified at each subregion of each anatomical level (Fig. 2A–C) [10]. From the ROSTRAL and MID sections, 10 neurons were randomly selected from each subregion. From the CAUDAL sections, 4, 10, and 10 neurons were randomly chosen from the lateral, the dorsal, and the ventral subregions, respectively. The mean gray value of the selected 5-HT-ir neurons was obtained with ImageJ, and was divided by the mean gray value of the background. The number obtained from this calculation was used for statistical analysis.
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Confocal images of 5-HT and Tph immunoreactivity were obtained with an LSM710 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). 2.5. Statistical analysis All data were analyzed using one-way ANOVA followed by Tukey’s test and presented as mean ± standard error of the mean (SEM). All analyses were conducted with SPSS 20. Significance level was set at p < 0.05.
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3. Results The 5-HT-ir cells were observed throughout the DR from bregma −7.32 to −8.28 mm in both sexes (Fig. 1A ). There was no significant difference in the number of 5-HT-ir cells among five groups (Fig. 1C). The signal intensity of 5-HT-ir cells was analyzed at three anatomical levels, ROSTRAL, MID, and CAUDAL, each of which was divided into three subregions, lateral, dorsal, and ventral regions (Figs. 2A–C). At the ROSTRAL level, the 5-HT-ir intensity in
Fig. 1. Effect of sex steroid hormones on the expression of 5-hydroxytryptamine-immunoreactive (5-HT-ir) cells in dorsal raphe (DR) of adult rats. (A) Expression of 5-HT-ir neurons in the DR. Scale bar = 100 m. (B) The representative area shown in Fig. 1A is boxed. (C) Number of 5-HT-ir cells in the DR. Values are means ± SEM. The number in each column indicates the number of animals used. OVX, ovariectomized female; Low E2, ovariectomized female with low dose estradiol; High E2, ovariectomized female with high dose estradiol; Cast, gonadectomized male; Intact, male rats without any treatment; Aq, cerebral aqueduct; CX, cortex.
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Fig. 2. Effect of sex steroid hormones on signal intensity of 5-HT expression in DR. Representative photographs of each section and signal intensity of 5-HT-ir cells in rostral (ROSTRAL) (A), mid (MID) (B), and caudal (CAUDAL) (C) sections. In pictures, lateral, dorsal, and ventral subregions at each level are boxed. Scale bar = 200 m. In bargraphs, values are means ± SEM (n = 4 for each group). Values with identical letters are not significantly different (P > 0.05, one-way ANOVA followed by Tukey’s test). See Fig. 1 for abbreviations.
dorsal region was significantly higher in Cast group than that in OVX and High E2 groups. In ventral region, the 5-HT-ir intensity in Intact group was significantly higher than that in OVX and High E2 groups (Fig. 2A). At the MID level, the 5-HT-ir intensity in all subregions was significantly higher in Intact group than that in OVX and High E2 groups (Fig. 2B). At the CAUDAL level, the 5HT-ir intensity in ventral region was significantly higher in Intact group than that in all female groups (Fig. 2C). The average 5HT-ir intensity in all three subregions was significantly higher in Cast group than that in High E2 group, and significantly higher in Intact group than that in all female groups (OVX, 1.53 ± 0.07; Low E2, 1.60 ± 0.10; High E2, 1.48 ± 0.06; Cast, 1.822 ± 0.05; Intact, 1.95 ± 0.04). Tph has also been used as a marker of serotonergic neurons [10]. Because Tph immunoreactivity was observed in the cell bodies of 5-HT-ir neurons (Fig. 3A), the number of Tph-ir cells was counted. The Tph-ir cells were observed in the DR in both sexes. However, there was no significant difference in the number of Tph-ir cells among all groups (Fig. 3B).
4. Discussion In the present study, we explored the possibility that sex steroid hormones affected the number of 5-HT in neurons because several disorders caused by a malfunction of the brain serotonergic system are more prevalent in women than men. Our results indicated that, the number of 5-HT-ir or Tph-ir cells in the DR was not significantly different among the adult animals exposed to various sex steroid levels. Because serotonin is related to several behavioral and neurophysiological processes [25], it is possible that number of 5-HT neurons hardly varies in the DR. On the other hand, the intensity of the 5-HT immunoreactivity showed a significant difference between males and females in specific regions of the DR, although this sex difference was independent of sex steroid hormone exposure. These results suggest that, protein levels of 5-HT neuron in part of DR may be controlled by other than sex steroid hormones. Sex steroid hormones affect several serotonergic factors that may contribute to sexual dimorphism in the serotonergic system.
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Fig. 3. Effect of sex steroid hormones on the expression of Tph-ir cells in DR. (A) Double fluorescence immunolocalization of 5-HT and tryptophan hydroxylase (Tph). (a) Representative low power view of DR showing 5-HT (red) and Tph (green). Scale bar = 100 m. (b–d) Higher magnification views of the boxed region in (A), showing 5-HT and Tph. Scale bar = 20 m. (B) Representative photographs of DR Tph-ir neurons at bregma −8.40 mm and the number of Tph-ir cells in DR (n = 4). Scale bar = 200 m. Values are means ± SEM. See Fig. 1 for abbreviations.
One of these factors is the 5-HT receptor. Injections of estradiol benzoate into OVX rats induced an acute reduction in brain 5HT1 binding sites [26]. Another serotonergic factor affected by sex steroid hormones is the serotonin transporter (SERT). An E2 injection significantly increased the number of SERT-expressing cells in OVX rats [27]. Therefore, sex steroid hormones may modulate factors such as 5-HT binding sites and transporters rather than the number of neurons. Several plausible mechanisms can explain our present result that no significant difference was observed in the population of serotonergic neurons. First, the length of time after gonadectomy may have been too short to decrease 5-HT neuronal number. One report indicates that long-term OVX decreases 5-HT neuronal number in female macaques [21]. In that study, pubertal macaques were OVX, and the number of 5-HT neurons was counted by identifying Tph2 mRNA, coding rate-limiting enzyme in 5-HT synthesis, 3 years after OVX. We did not examine Tph2 mRNA expression in the present study, but the number of Tph proteinexpressing cells showed no significant differences 2 weeks after OVX. This evidence suggests that the length of time following gonadectomy may be a determinant for the reduction of Tph2 mRNA; however, the results observed in the macaques may also be primate specific. Another reasonable explanation for our result is that the number of 5-HT neurons may be determined before puberty. We examined 5-HT neurons in only the adult rat
because disorders such as depression and migraine manifest after puberty [28,29]. However, prepubertal conditions may be involved in determining the number of 5-HT neurons in the DR, as suggested in several published studies. For example, in monkeys, cortical 5HT levels and receptor numbers peak at 2 months of age [30,31]. In humans, children show higher levels than adults of 5-HIAA in cerebrospinal fluid [32]. Moreover, until the age of five, the capacity of 5-HT synthesis in children is more than double that in adults [33]. Overall, these data suggest that the serotonergic system is more dynamic before than after puberty. Thus, the possibility of prepubertal conditions determining the number of brain 5-HT neurons should be determined in future studies. A final plausible mechanism for the result obtained in the present study is that neuronal modulation of 5-HT may occur through PR. The OVX female animals in this study were not treated with progesterone, and PR has been detected in 5-HT neurons [16,17]. In conclusion, the present study demonstrated that the number of DR 5-HT neurons was not significantly different among animals exposed to different sex steroid levels after maturation. Although, a number of studies have examined the relationship between sex steroid hormones and the serotonergic system, this relationship has not yet been fully elucidated. By investigating the precise relationship between sex steroid hormones and the serotonergic system, adequate treatment, such as hormonal therapy, may be offered for disorders caused by alterations in the serotonergic system. Our
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