Estrogen alters c-Fos response to immobilization stress in the brain of ovariectomized rats

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Estrogen alters c-Fos response to immobilization stress in the brain of ovariectomized rats Takashi Ueyama⁎, Tadashi Tanioku, Junya Nuta, Kazuto Kujira, Takao Ito, Saori Nakai, Yoshihiro Tsuruo Department of Anatomy and Cell Biology, Wakayama Medical University, Kimiidera 811-1, Wakayama 641-8509, Japan

A R T I C LE I N FO

AB S T R A C T

Article history:

Estrogen receptors are widely expressed in the brain, where estrogen modulates central

Accepted 7 February 2006

nervous function. In this study, we investigated the effect of estrogen on the emotional

Available online 20 March 2006

stress response in the brain by comparing the CNS patterns of c-Fos expression in response to immobilization stress (IMO) in ovariectomized rats with placebo treatment (OVX + Pla) vs.

Keywords:

ovariectomized rats supplemented with 17β-estradiol (OVX + E2). Increased c-Fos

Emotional stress

immunoreactive neurons in response to IMO were observed in cerebral cortex, septum,

Estrogen

thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with

Heart failure

previous findings. When OVX + E2/Stress were compared with OVX + Pla/Stress, the

c-Fos

numbers of c-Fos immunoreactive cells were significantly lower in the lateral septum,

Takotsubo cardiomyopathy

paraventricular hypothalamic nucleus, dorsomedial hypothalamic nucleus, medial amygdaloid nucleus, lateral periaqueductal gray, laterodorsal tegmental nucleus and locus coeruleus, while they were significantly higher in paraventricular thalamic nucleus and nucleus of the solitary tract. These data suggest that neuronal activities in these areas are influenced bidirectionally by systemic estrogen level. © 2006 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Fax: +81 73 441 0616. E-mail address: [email protected] (T. Ueyama). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.02.008

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Abbreviations: A5, A5 noradrenaline cells A7, A7 noradrenaline cells AcbC, accumbens nucleus, core AcbSH, accumbens nucleus, shell ACTH, adrenocorticotropin AH, anterior hypothalamic area AID + V, agranular insular cortex, dorsal part + ventral part AP, area postrema Arc, arcuate nucleus BST, bed nucleus of the stria terminalis Ce, central amygdaloid nucleus Cg, cingulate cortex CM, central medial thalamic nucleus CRF, corticotropin-releasing factor CVL, caudoventrolateral reticular nucleus DLPAG, dorsolateral periaqueductal gray DMH, dorsomedial hypothalamic nucleus DMPAG, dorsomedial periaqueductal gray DP, dorsal peduncular cortex DRC, dorsal raphe nucleus, caudal part DRD, dorsal raphe nucleus, dorsal part (DRD) E2, 17β-estradiol ER, estrogen receptor GABA, γ-aminobutyric acid IMO, immobilization stress IL, infralimbic cortex LC, locus coeruleus LDTg, laterodorsal tegmental nucleus LH, lateral hypothalamic area LPAG, lateral periaqueductal gray LS, lateral septal nucleus MeAD + PV, medial amygdaloid nucleus, anterodorsal part + posteroventral part MHb, medial habenular nucleus MPA, medial preoptic area NE, norepinephrine NTS, nucleus of the solitary tract OVX, ovariectomy PH, posterior hypothalamic area Pir, piriform cortex Pla, placebo PVA, paraventricular thalamic nucleus, anterior part PVH, paraventricular hypothalamic nucleus PVT, paraventricular thalamic nucleus PVP, paraventricular thalamic nucleus, posterior part

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RPa, raphe pallidus nucleus RVL, rostroventrolateral reticular nucleus VMH, ventromedial hypothalamic nucleus ZI, zona incerta

1.

Introduction

Estrogen receptors (ERα and ERβ) are widely expressed in the central nervous system (Laflamme et al., 1998; Shughrue et al., 1997, 1998; Shughrue and Merchenthaler, 2001). Recent clinical and basic research studies have suggested that estrogen plays a crucial role in sexual behavior, learning and memory processes, protection against ischemic insults and modulation of autonomic nervous function (Li and Shen, 2005; Merchenthaler et al., 2003; Östlund et al., 2003). Therefore, reduction of estrogen levels following menopause may increase the vulnerability of women to stress while estrogen supplementation attenuates the exaggerated response to stress or sympathoadrenal activity (Komesaroff et al., 1999; Vongpatanasin et al., 2001; Weitz et al., 2001). A special type of acute cardiac attack called “Takotsubo” (Japanese for an octopus trapping pot with a round bottom and a narrow neck) cardiomyopathy, “Ampulla” cardiomyopathy or “Transient left ventricular apical ballooning” similar to acute myocardial infarction has been discovered (Akashi et al., 2003; Bybee et al., 2004; Kurisu et al., 2002; Tsuchihashi et al., 2001; Wittstein et al., 2005). This is characterized by: (1) sudden onset of chest symptoms, (2) reversible akinesis around the left ventricle (LV) apex and hyperkinesis around the LV outflow, (3) electrocardiographic (ECG) changes (ST elevation), (4) minimal myocardial enzymatic release, (5) no significant stenosis of coronary arteries. The backgrounds of these patients are very unique and have the following two characteristics: (1) the attack is triggered by emotional or physical stress, and (2) it occurs predominantly in elderly (postmenopausal) females. Although the etiology of this syndrome is yet to be clarified, increase of serum norepinephrine, epinephrine and neuropeptide Y levels in the onset of takotsubo cardiomyopathy compared with acute myocardial infarction suggests that the exaggerated sympathoadrenal activation triggered by stress is the primary cause of this cardiomyopathy (Akashi et al., 2003; Wittstein et al., 2005). Immobilization stress (IMO) in the rat provides a wellknown animal model of emotional stress, which activates the hypothalamic–pituitary–adrenocortical system and the sympathoadrenal system (Kvetnansky et al., 1995). We have succeeded in developing a model of this clinical condition in rats (Ueyama, 2004). The characteristic changes such as elevation of the ST segment in the ECG, the reversible LV apical ballooning in LVG and the induction of immediate early genes in the heart were cancelled by pretreatment with combined blockade of α- and β-adrenoceptors, suggesting that enhanced sympathoadrenal outflow is involved in these cardiac changes (Ueyama et al., 1999, 2000, 2002). We also found that the increase of serum 17β-estradiol (E2) improved the IMO induced left ventricular dysfunction and tachycardia, and downregulated the expression of c-fos mRNA in the left

ventricle (Ueyama et al., 2003; Ueyama, 2004). Effects of E2 on the improvement of the IMO induced-left ventricular dysfunction and downregulation of c-fos mRNA levels in the left ventricle have been reconfirmed by another researcher (F. Ishikura, unpublished results). Our recent data suggest that low levels of estrogen may augment the reactivity to stress via modulation of autonomic nervous function, resulting in the high incidence of takotsubo cardiomyopathy in postmenopausal females. Indeed, peripheral and central injections of E2 attenuated the cardiovascular response by increasing vagal tone and decreasing sympathetic nervous activity (He et al., 1998; Saleh et al., 2000a,b, 2001). Supplemental treatment of E2 to ovariectomized rats also attenuated the stress-induced elevation of plasma ACTH levels, blood pressure and heart rate (Dayas et al., 2000; Morimoto et al., 2004; Serova et al., 2005). It is well known that estrogen directly affects the cardiovascular system by modulation of endothelial nitric oxide synthase (Chambliss and Shaul, 2002). Though direct effects of estrogen on the cardiovascular system can be involved in these cardiac changes, we considered that estrogen might also decrease the sympathoadrenal outflow and thereby improve the emotional stress-induced cardiac dysfunction. To determine the brain regions in which estrogen modifies the stress response in this animal model, we used c-Fos expression as a marker of neuronal activation. Increased numbers of c-Fos immunoreactive neurons in response to IMO stress were observed in cerebral cortex, septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings (Bubser and Deutch, 1999; Ceccatelli et al., 1989; Chen and Herbert, 1995; Chowdhury et al., 2000; Cullinan et al., 1995; Ma and Morilak, 2004; Palmer and Printz, 1999; Senba and Ueyama, 1997). Reduction of c-Fos immunoreactivity in the brain by supplemental estrogen treatment in response to other stress protocols such as swimming stress or restriction stress was demonstrated (Dayas et al., 2000; Rachman et al., 1998). Our present data now demonstrate that neuronal activities in discrete regions where estrogen receptors are also expressed are modified by chronic estrogen treatment. A preliminary report of this study was presented in abstract form (Ueyama et al., 2005). The possible physiological significance of these results is discussed.

2.

Results

Serum E2 levels were significantly increased in OVX + E2 (mean ± SEM; OVX + Pla; 7.8 ± 1.1 pg/ml, n = 6 vs. OVX + E2; 184.4 ± 20.3 pg/ml, n = 6, P < 0.001). In response to stress, heart rate was significantly increased in OVX + Pla (control; 389 ± 15 bpm → stress; 542 ± 7 bpm, n = 6, P < 0.01) and OVX + E2 (control; 355 ± 22 bpm → stress; 496 ± 17 bpm, n = 6, P < 0.01). Heart rate in control was not significantly different

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between OVX + Pla/Control and OVX + E2/Control (P = 0.15) but the heart rate in stress was significantly higher in OVX + Pla/ Stress than in OVX + E2/Stress (P < 0.05). c-Fos immunoreactive neurons were observed in the cerebral cortex, thalamus, hypothalamus, pons and medulla. The mean densities of c-Fos immunoreactive neurons in specific brain regions are provided in Table 1. Increased numbers of c-Fos immunoreactive neurons in response to

IMO stress (OVX + E2/Stress vs. OVX + E2/Control, or OVX + Pla/ Stress vs. OVX + Pla/Control) were observed in the following regions; infralimbic cortex (IL), agranular insular cortex, dorsal part + ventral part (AID + V), piriform cortex (Pir), dorsal peduncular cortex (DP), lateral septal nucleus (LS), anterior hypothalamic area (AH), lateral hypothalamic area (LH), paraventricular hypothalamic nucleus (PVH), paraventricular thalamic nucleus (PVT), dorsomedial hypothalamic nucleus

Table 1 – The mean densities of c-Fos immunoreactive neurons in specific brain regions OVX + E2/Control IL (Bregma 2.20) DP (Bregma 2.20) Pir (Bregma 2.20) AID + V (Bregma 2.20) Cg (Bregma 0.48) LS (Bregma 0.48) AcbSH (Bregma 0.48) AcbC (Bregma 0.48) BST (Bregma − 0.30) MPA (Bregma − 0.30) AH (Bregma − 1.88) LH (Bregma − 1.88) PVA (Bregma − 1.88) PVH (Bregma − 1.88) PVT (Bregma − 2.56) DMH (Bregma − 2.56) MeAD + PV (Bregma − 2.56) Ce (Bregma − 2.56) PVP (Bregma − 3.14) MHb (Bregma − 3.14) CM (Bregma − 3.14) ZI (Bregma − 3.14) VHM (Bregma − 3.14) Arc (Bregma − 3.14) PH (Bregma − 3.80) DLPAG (Bregma − 7.30) DMPAG (Bregma − 7.30) LPAG (Bregma − 7.30) DRD (Bregma − 7.30) LPAG (Bregma − 8.72) DMPAG (Bregma − 8.72) VLPAG (Bregma − 8.72) A7 (Bregma − 8.72) DRC (Bregma − 8.72) A5 (Bregma − 8.72) LDTg (Bregma − 8.72) A5 (Bregma − 10.04) LC (Bregma − 10.04) RVL (Bregma − 12.72) RPa (Bregma − 12.72) AP (Bregma − 14.08) NTS (Bregma − 14.08) CVL (Bregma − 14.60)

1.30 2.24 4.73 2.21 1.88 0.81 1.83 2.10 1.86 6.03 2.93 1.67 6.92 4.12 6.82 3.23 2.00 3.09 7.38 4.60 3.45 1.63 3.83 4.90 2.93 1.47 2.31 1.55 3.39 6.50 4.89 3.21 3.13 4.48 3.65 4.59 2.68 1.12 0.45 6.56 1.44 0.83 0.20

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

a, b

1.158 0.57 a 0.57 a 0.78 a 0.28 0.21 a 0.26 0.26 0.26 1.03 b 0.48 a 0.29 a 0.64 0.51 a 0.45 a 0.32 a 0.20 a 0.27 a 0.96 0.68 a 0.39 b 0.24 0.71 a 0.88 0.35 a 0.20 a 0.50 0.18 0.35 a 0.66 a 0.53 a 0.26 0.44 a 0.28 a 0.69 0.44 a 0.37 0.25 a 0.05 a 0.91 0.31 a 0.09 a, c 0.03 a

OVX + Pla/Control 2.87 2.59 7.60 3.88 2.81 1.49 2.11 1.96 1.84 3.45 2.38 1.67 6.72 6.18 5.68 3.60 2.15 3.21 5.67 4.15 0.90 1.07 3.28 5.33 2.93 1.99 3.03 1.37 3.09 6.30 3.35 3.73 2.75 4.30 3.51 6.30 1.89 1.63 0.47 4.66 1.36 1.28 0.40

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

b

0.67 0.48 c 1.74 c 1.02 c 1.01 0.57 c 0.38 0.38 0.46 0.38 b 0.39 c 0.29 c 0.27 c 0.76 c 0.40 0.57 c 0.30 a, d 0.26 c 0.34 0.97 c 0.30b,c 0.16 c 0.58 c 1.08 0.35 c 0.37 c 0.49 0.19 c 0.57 c 0.70 c 0.59 c 0.35 0.16 c 0.50 c 0.25 0.62 c 0.30 0.29 c 0.13 c 1.01 c 0.32 c 0.17 b, c 0.04 c

OVX + E2/Stress 3.83 5.41 10.80 5.85 3.49 2.40 2.68 2.98 2.58 5.46 4.88 4.47 8.32 12.13 8.72 5.97 3.77 4.37 7.67 8.67 3.75 2.28 6.73 8.40 4.83 3.14 3.59 2.30 10.69 9.16 9.10 4.13 4.90 8.05 4.18 6.60 2.01 4.22 0.99 9.76 6.34 2.46 1.61

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

The results are expressed as cell number per 1 × 104 μm2. Data are shown as the mean ± SEM. a P < 0.05 between OVX + E2/Control and OVX + E2/Stress. b P < 0.05 between OVX + E2/Control and OVX + Pla/Control. c P < 0.05 between OVX + Pla/Control and OVX + Pla/Stress. d P < 0.05 between OVX + E2/Stress and OVX + Pla/Stress. ⁎ P < 0.05, significance of F value. ⁎⁎ P < 0.01, significance of F value. ⁎⁎⁎ P < 0.001, significance of F value. ⁎⁎⁎⁎ P < 0.0001, significance of F value.

a

0.24 0.25 a 0.55 a 0.70 a 0.33 0.22 a, d 0.36 0.78 0.15 0.23 0.44 a 0.24 a 0.69 1.14 a, d 0.37 a, d 0.36 a, d 0.41 a, d 0.46 a 0.67 0.89 a 0.28 0.46 0.92 a 1.83 0.19 a 0.34 a 0.55 0.22 d 1.26 a 0.98 a 1.56 a 0.28 0.51 a 0.71 a 0.70 0.58 a, d 0.25 0.88 a, d 0.09 a 1.64 1.06 a 0.16 a, d 0.20 a

OVX + Pla/Stress 4.04 4.98 13.24 6.49 4.02 4.31 2.38 2.42 2.57 4.57 4.52 3.75 8.50 16.58 6.80 7.82 6.57 5.32 5.78 7.62 4.13 2.223 7.38 7.88 4.03 3.17 3.93 3.26 9.64 9.85 8.91 4.95 4.90 7.64 4.65 8.75 2.42 6.16 1.19 11.91 5.30 1.84 1.53

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.44 0.24 c 1.22 c 0.68 c 0.95 0.44 c, d 0.13 0.39 0.22 0.54 0.56 c 0.40 c 0.68 c 1.84 c, d 0.34 d 0.27 c, d 0.70 c, d 0.35 c 0.74 0.50 c 0.54 c 0.21 c 1.00 c 0.75 0.32 c 0.07 c 0.45 0.34c, d 0.72 c 0.28 c 0.62 c 0.79 0.57 c 0.54 c 0.49 0.80 c, d 0.38 0.80 c, d 0.33 c 2.11 c 0.71 c 0.15 c, d 0.19 c

F values 11.241 ⁎⁎⁎ 17.412 **** 15.15 **** 6.598 ** 1.626 15.241 **** 1.367 0.804 1.684 4.778 * 6.627 ** 21.677 **** 2.423 23.462 **** 10.355 ⁎⁎⁎ 29.503 **** 22.944 **** 9.499 ⁎⁎⁎ 2.176 8.07 ** 14.195 **** 3.897 * 6.267 ** 2.144 5.833 ** 9.345 ⁎⁎⁎ 2.047 12.558 ⁎⁎⁎ 22.025 **** 6.779 ** 9.741 ⁎⁎⁎ 2.445 6.588 ** 14.319 **** 0.785 7.506 ⁎⁎⁎ 1.384 28.103 **** 4.849 * 4.825 * 13.963 **** 25.744 **** 28.613 ****

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(DMH), medial amygdaloid nucleus, anterodorsal part + posteroventral part (MeAD + PV), central amygdaloid nucleus (Ce), medial habenular nucleus (MHb), central medial thalamic nucleus (CM), zona incerta (ZI), ventromedial hypothalamic nucleus (VMH), posterior hypothalamic area (PH), dorsolateral periaqueductal gray (DLPAG), dorsomedial periaqueductal gray (DMPAG), lateral periaqueductal gray (LPAG), dorsal raphe nucleus, dorsal part (DRD), A7 noradrenaline cells (A7), dorsal raphe nucleus, caudal part (DRC), laterodorsal tegmental nucleus (LDTg), locus coeruleus (LC), rostroventrolateral reticular nucleus (RVL), raphe pallidus nucleus (RPa), area postrema (AP), nucleus of the solitary tract (NTS) and caudoventrolateral reticular nucleus (CVL). Effects of stress with estrogen supplementation (OVX + E2/ Stress vs. OVX + Pla/Stress) were observed in LS, PVT, PVH, DMH, MeDA + PV, LPAG, LDTg, LC and NTS. In LS, PVH, DMH, MeDA + PV, LPAG, LTDg and LC, lower numbers of c-Fos immunoreactive neurons were observed in OVX + E2/Stress compared with OVX + Pla/Stress, while higher numbers of cFos immunoreactive neurons were noted in PVT and NTS. Representative photomicrographs are illustrated in Fig. 1 (LS), Fig. 2 (PVH), Fig. 3 (DMH), Fig. 4 (MeDA + PV), Fig. 5

Fig. 2 – Representative photographs showing the effects of IMO stress and estrogen treatment on c-Fos immunoreactive neurons in PVH. Increase of c-Fos immunoreactive neurons was observed in response to IMO stress. When OVX + E2/Stress was compared with OVX + Pla/Stress, c-Fos immunoreactive neurons in parvocellular PVH were small in number. PaMP: paraventricular hypothalamic nucleus, medial parvocellular part. PaLM: paraventricular hypothalamic nucleus, lateral magnocellular. Scale bars, 100 μm.

(LC), Fig. 6 (NTS) and a schematic illustration showing the effects of estrogen and the effect of stress is shown in Fig. 7.

3.

Discussion

3.1. Evaluation of cardiac function and c-Fos expression in response to stress

Fig. 1 – Representative photographs showing the effects of IMO stress and estrogen treatment on c-Fos immunoreactive neurons in LS. Increase of c-Fos immunoreactive neurons was observed in response to IMO stress. When OVX + E2/Stress was compared with OVX + Pla/Stress, c-Fos immunoreactive neurons were small in number. LSI: lateral septal nucleus, intermediate. LSV: lateral septal nucleus, ventral part. Scale bars, 100 μm.

Clinical studies (Komesaroff et al., 1999; Vongpatanasin et al., 2001; Weitz et al., 2001) as well as animal experiments (Dayas et al., 2000; Morimoto et al., 2004; Saleh et al., 2000a,b, 2001; Serova et al., 2005) demonstrated that estrogen modulates cardiovascular reactivity by increasing vagal tone and decreasing sympathetic nervous activity. In fact, heart rate response was attenuated in OVX + E2/Stress compared with OVX + Pla/Stress in accordance with our previous findings (Ueyama et al., 2003), suggesting that treatment with E2 affects the autonomic nervous activity in response to stress.

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3.2.

Lateral septal nucleus

Kubo et al. demonstrated that cholinergic stimulation of M1/ M3 muscarinic receptors in LS caused sympathetic activation and increased blood pressure, whereas stimulation of GABAA receptors inhibited the IMO-induced pressure response (Kubo et al., 2002, 2003). In accordance with these physiological results, viral transneuronal methods revealed that the LS was multisynaptically connected to the sympathoadrenal system (Westerhaus and Loewy, 2001). Neurons expressed ERα and ERβ in LS project to PAG (Tsukahara and Yamanouchi, 2002). From our observations, the lower c-Fos response in the OVX + E2 group suggests that treatment with estrogen attenuates the stress-induced sympathoadrenal outflow from LS.

3.3.

Paraventricular hypothalamic nucleus

The PVH is considered to be a key integrative center for neuroendocrine and autonomic controls. Chronic treatment with estrogen attenuated IMO stress-induced c-Fos expression in parvocellular PVH in accordance with previous observations

Fig. 3 – Representative photographs showing the effects of IMO stress and estrogen treatment on c-Fos immunoreactive neurons in DMH. Increase of c-Fos immunoreactive neurons was observed in response to IMO stress. When OVX + E2/Stress was compared with OVX + Pla/Stress, c-Fos immunoreactive neurons were small in number. 3V: The third ventricle. mt: mammillothalamic tract. f: fornix. Scale bars, 100 μm.

The regions where a significant increase of c-Fos immunoreactive neurons was observed in this study were almost identical with other studies (Bubser and Deutch, 1999; Ceccatelli et al., 1989; Chen and Herbert, 1995; Chowdhury et al., 2000; Cullinan et al., 1995; Ma and Morilak, 2004; Palmer and Printz, 1999; Senba and Ueyama, 1997), affirming the appropriateness of our experimental procedures. In this study, we found that treatment with estrogen modified c-Fos expression levels following IMO stress in discrete regions, such as LS, PVT, PVH, DMH, MeDA + PV, LPAG, LDTg, LC and NTS. These regions are areas of the brain known to contain high or moderate densities of estrogen receptors (ERs) (Laflamme et al., 1998; Shughrue et al., 1997, 1998; Shughrue and Merchenthaler, 2001). These regions are also involved in the regulation of various autonomic functions, including control of cardiovascular reflexes. Accordingly, our findings suggest that estrogen can potentially modulate the autonomic response of these brain regions to stress. The possible physiological and anatomical involvement of these regions in the stress response is discussed below.

Fig. 4 – Representative photographs showing the effects of IMO stress and estrogen treatment on c-Fos immunoreactive neurons in MeDA + PV. Increase of c-Fos immunoreactive neurons was observed in response to IMO stress. When OVX + E2/Stress was compared with OVX + Pla/Stress, c-Fos immunoreactive neurons were small in number. MeDA: medial amygdaloid nucleus, anterodorsal part. MePV: medial amygdaloid nucleus, posteroventral part. opt: optic tract. Scale bars, 100 μm.

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the IMO-induced neuronal activity in the PVH, resulting in an attenuated response of the hypothalamic–pituitary–adrenocortical system and sympathoadrenal system. Taken together, it is evident that estrogen-ER systems in the PVH are deeply involved in the response to stress.

3.4.

Fig. 5 – Representative photographs showing the effects of IMO stress and estrogen treatment on c-Fos immunoreactive neurons in LC. Increase of c-Fos immunoreactive neurons was observed in response to IMO stress. When OVX + E2/Stress was compared with OVX + Pla/Stress, c-Fos immunoreactive neurons were small in number. Me5: mesencephalic nucleus of 5. Scale bars, 100 μm.

(Dayas et al., 2000; Rachman et al., 1998). These results parallel the diminished plasma ACTH response and blood pressure response to emotional stress (Dayas et al., 2000; Morimoto et al., 2004; Serova et al., 2005). ERβ is predominantly expressed in the PVH (Laflamme et al., 1998; Shughrue et al., 1997, 1998; Shughrue and Merchenthaler, 2001). Robust corticotropinreleasing factor (CRF)- and ERβ-double positive neurons were observed in the caudal PVH, and dorsal and ventral zones of the medial parvocellular PVH. These are regions known to be involved in regulation of the autonomic nervous system (Laflamme et al., 1998). PVH CRF neurons and medullary catecholamine neurons in NTS and RVLM were reciprocally activated in response to psychological stress (Dayas et al., 2001a, 2004). Fifty percent of RVLM-projecting PVH neurons especially in the ventromedial parvocellular (PaV) and the posterior parvocelluar (PaPo) subnuclei expressed ERβ immunoreactivity (Stern and Zhang, 2003). Though expression of ERα was low in the PVH, IMO stress or fasting increased ERα immunoreactive neurons in the PVH (Estacio et al., 1996). Catecholaminergic inputs from NTS to PVH mediated the induction of ERα expression in the PVH (Estacio et al., 2004). E2 treatment in the ovariectomized rats resulted in a decrease in the number of ERβ immunoreactive neurons and mRNA (Patisaul et al., 1999; Suzuki and Handa, 2004) and in the number of ERα immunoreactive neurons in the PVH (Gréco et al., 2001). Microinjection of an ER antagonist into the PVH reduced the stress-induced corticosterone response, and ERβ mRNA levels in the PVH were upregulated by corticosterone (Isgor et al., 2003; Suzuki and Handa, 2004). These previous observations indicate that the levels of ERα and ERβ expression are modified by the complex interaction among stress, glucocorticoid and estrogen levels in the PVH. Though it is not clear whether c-Fos positive neurons observed in this study are ER positive neurons, lower c-Fos responses in OVX + E2 group suggest that chronic treatment with estrogen reduces

Medial amygdaloid nucleus

The medial amygdaloid nucleus is involved in the psychological response to stress. For example, restraint stress-induced increase in blood pressure was inhibited by the injection of a GABAA receptor agonist, muscimol into the medial amygdaloid nucleus (Kubo et al., 2004). Dayas reported that the medial amygdaloid nucleus but not the central amygdaloid nucleus is a critical site for activation of medial parvocellular PVH CRF neurons in response to psychological stress (Dayas et al., 1999, 2001b; Dayas and Day, 2002). Both ERα and ERβ are expressed in the medial amygdaloid nucleus (Laflamme et al., 1998; Shughrue et al., 1997, 1998; Shughrue and Merchenthaler, 2001). Expression of ERα and ERβ in the medial amygdaloid nucleus was downregulated by estrogen treatment as also shown in the PVH (Gréco et al., 2001; Osterlund et al., 1998; Suzuki and Handa, 2004). The lower c-Fos response in OVX + E2/Stress group suggests that chronic treatment with estrogen reduces the IMO-induced neuronal activity in the medial amygdaloid nucleus, resulting in the low response in PVH CRF neurons.

3.5. Dorsomedial hypothalamic nucleus and periaqueductal gray The DMH also plays a key role in the cardiovascular changes associated with emotional stress (DiMicco et al., 2002). DMH neurons receive inputs from the hypothalamus, ventral

Fig. 6 – Representative photographs showing the effects of IMO stress and estrogen treatment on c-Fos immunoreactive neurons in NTS. Increase of c-Fos immunoreactive neurons was observed in response to IMO stress. When OVX + E2/Stress was compared with OVX + Pla/Stress, c-Fos immunoreactive neurons were large in number. AP: area postrema. SolC: nucleus of the solitary tract, commissural. SolDM: nucleus of the solitary tract, dorsomedial. sol: solitary tract. CC: central canal. 12: hypoglossal nucleus. Scale bars, 100 μm.

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Fig. 7 – Schematic representation of the brain areas showing the alteration of c-Fos expression by the effects of estrogen supplementation (left halves of sections) and the effects of IMO stress (right halves of sections) in the 13 representative coronal sections arranged anterior–posteriorly. The areas showing statistical significant differences between OVX + E2/Stress and OVX + Pla/Stress (left halves of sections), or between OVX + E2/Stress and OVX + E2/Control (right halves of sections) are colored black, and those showing no statistic significances are colored gray. The schematic figures and stereotactic reference points (distance from the bregma) were modified from the atlas of Paxinos and Watson (1998) with permission.

subiculum, IL, LS, bed nucleus of the stria terminalis, PAG, parabrachial nucleus and ventrolateral medulla (Thompson et al., 1996) and they send a dense projection to PVH (Thompson and Swanson, 1998). Inhibition of neurons in the DMH by the GABAA receptor agonist, muscimol (Stotz-Potter et al., 1996a,b) or the non-specific ionotropic glutamate receptor antagonist, kynurenate (Soltis and DiMicco, 1992), attenuated the emotional stress-induced elevation of heart rate, blood pressure, plasma ACTH and c-Fos expression in the PVH (Morin et al., 2001). On the other hand, chemical stimulation of the DMH by the GABAA receptor antagonist, bicuculline (Soltis and DiMicco, 1991) or excitatory amino acid agonists (Soltis and DiMicco, 1992) produced an elevation of heart rate, blood pressure and plasma ACTH (Bailey and Dimicco, 2001). DMHinduced tachycardia is mediated by projection to neurons in

the RPa (Samuels et al., 2004; Zaretsky et al., 2003). The projection from the DMH to lateral dorsolateral region of the midbrain PAG is also involved in the increase of blood pressure and heart rate (da Silva et al., 2003). The descending pathway from DMH to RVLM is involved in the pressor and sympathoexcitatory responses but not the tachycardiac response (Fontes et al., 2001; Horiuchi et al., 2004). In both the DMH and LPAG, significant increases of c-Fos immunoreactive neurons in response to IMO stress were observed, and estrogen treatment attenuated this stressinduced increase of c-Fos immunoreactivity. In the RPa, a significant increase of c-Fos immunoreactivity in response to IMO stress was observed only between OVX + Pla/Stress and OVX + Pla/Control but not between OVX + E2/Stress and OXV + E2/Control. In the RVLM, the effects of stress were

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observed in both placebo and estrogen groups but the effects of estrogen were not significant. Though expression of ERα and ERβ is low in the DMH and moderate in the PAG (Laflamme et al., 1998; Shughrue et al., 1997, 1998; Shughrue and Merchenthaler, 2001), our present results suggest that estrogen treatment attenuates the IMO stress-induced neuronal activity in the DMH, LPAG and RPa.

3.6.

Locus coeruleus

The LC-noradrenergic system supplies NE throughout the central nervous system and exerts a widespread influence on neuronal circuits that are essential mediators of alert waking and state-dependent cognitive processes (Berridge and Waterhouse, 2003). ERα is expressed in the LC (Laflamme et al., 1998; Shughrue et al., 1997, 1998; Shughrue and Merchenthaler, 2001), and structure and function of the LC are influenced by sex steroids. Normal adult female rats show greater LC volume and the numbers of NE neurons than males (Luque et al., 1992). In this study, we demonstrated that IMO stress-induced c-Fos expression in the LC was clearly inhibited by chronic treatment with estrogen. This reduction of c-Fos expression in LC following estrogen treatment was confirmed by another researcher in a different stress model (K. Morimoto, unpublished results). This implies that chronic treatment with estrogen attenuates the stress-induced noradrenergic output from LC, which may play a role in the well-known effects of estrogen on memory and cognitive function. In support of this result, it was reported that chronic estrogen treatment increased galanin mRNA but not tyrosine hydroxylase mRNA in the LC (Tseng et al., 1997). As galanin functions as an inhibitory neuromodulator of adrenergic transmission (Tsuda et al., 1989), an increase in galanin expression is likely to lead to reduced noradrenergic transmission throughout the LC distribution of the brain.

3.7.

Nucleus of the solitary tract

Between non-stressed control groups, the number of c-Fos immunoreactive neurons in the NTS was lower in OVX + E2 than in OVX + Pla, while between stressed groups it was higher in OVX + E2 than in OVX + Pla. The numbers of c-Fos-expressing catecholaminergic neurons in NTS were increased in rat during the morning of proestrus compared with disestrus (Condé et al., 1995; Jennes et al., 1992). Moderate levels of ERα are expressed in the commissural part of NTS and low levels of ERβ are expressed in the medial part of NTS (Laflamme et al., 1998). IMO stress or fasting increased ERα immunoreactive neurons in the NTS as well as the PVH (Estacio et al., 1996). Expression of catecholamine biosynthetic enzyme genes in NTS was modified by estrogen (Serova et al., 2005). The number of ERα positive A2 noradrenergic neurons in NTS was not influenced by estrogen, while those of progesterone receptor immunoreactive neurons were increased by estrogen (Haywood et al., 1999). Neuronal discharge in the NTS was rapidly inhibited by E2 (Xue and Hay, 2003). In addition, microinjection of E2 into NTS increased vagal activity and decreased sympathetic activity (Saleh et al., 2000a). Though the colocalization of c-Fos immunoreactive and ERα immunoreactive neurons is not clear, these results indicate that expression of ERα and neuronal activity in NTS is modulated by stress or estrogen.

3.8.

75

Paraventricular thalamic nucleus

The PVT functions as a generalized relay from the medulla, pons and midbrain to the limbic forebrain, and all of them are activated by stress (Bubser and Deutch, 1999; Krout et al., 2002; Otake et al., 2002). The number of c-Fos immunoreactive neurons in PVT (central part) was significantly higher in OVX + E2/Stress than in OVX + Pla/Stress. Though expression of ERα and ERβ is limited in PVT, our data suggest that estrogen treatment might also modulate the reactivity of PVT neurons in response to stress.

3.9. Effects of estrogen on stress-induced c-Fos expression in the brain Additional forebrain areas were activated in response to forced swimming in the study of Rachman et al. (1998) compared with areas activated by IMO. Dayas et al. also reported that estrogen treatment suppressed c-Fos expression in response to noise stress in the PVH and A1 and A2 (noradrenergic) and C2 (adrenergic) neurons but not in pontine catecholamine neurons such as A5, A6 and A7 (Dayas et al., 2000). These discrepancies may be attributed to the differences of stress paradigm, estrogen dosage and other experimental conditions. However, the effects of estrogen in DMH, medial amygdaloid nucleus were consistent with Rachman's observation and those in PVH are in accord with the results of Rachman and Dayas. Cardiovascular reactivity observed by Saleh (Saleh et al., 2000a,b, 2001) was modulated by direct application of estrogen into discrete areas. Expression of catecholamine biosynthetic enzyme genes in adrenal medulla, NTS and LC was modified by estrogen (Serova et al., 2005). Taken together, it can be considered that the medial amygdala–PVH–brainstem noradreneregic system and the LC noradrenergic system are the common pathways by which estrogen modulates various kinds of stress responses. In this study, we suggested the involvement of other nuclei such as LS, DMHlateral PAG and DHM-RPa systems, PVT and LTDg in modulation of the stress response by estrogen. However, precise mechanisms by which chronic estrogen treatment modifies neuronal activity in response to stress have yet to be determined. We did not show the expression of ERs and ERmediated signal transduction in this study. Thus, further studies, for example, using an estrogen receptor antagonist in combination with estrogen supplementation will be required to ascertain that the c-Fos expression is modulated by IMO stress in estrogen-specific manner. In summary, estrogen treatment modified neuronal activity in several central regions in response to IMO stress, an animal model of takotsubo cardiomyopathy. This may in part contribute to the high incidence of this heart attack in the elderly postmenopausal female.

4.

Experimental procedures

4.1.

Tissue preparation

Female Wistar rats, 8-week-old, were purchased from Kiwa Animal Lab. (Wakayama, Japan) and housed in a temperature-

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controlled environment. Under anesthesia with sodium pentobarbital (40 mg/kg), a bilateral ovariectomy (OVX) was performed by ligation and dissection of the ovary (n = 24). Twelve rats were subcutaneously implanted with a pellet containing E2 (0.5 mg, release time 21 days) (Innovative Research of America, Sarasota, FL, USA) (OVX + E2), and the other 12 rats were subcutaneously implanted with the corresponding placebo pellet containing the same vehicle without E2 (Innovative Research of America) (OVX + Pla). Experiments were performed after allowing the rats free access to food and water for 2 weeks. Half of the rats in each group were restrained for 60 min by securing them on their back to a board using adhesive tape (immobilization stress: IMO) (OVX + E2/Stress, n = 6; OVX + Pla/ Stress, n = 6). The other half was kept in their home cage until perfusion (OVX + E2/Control, n = 6; OVX + Pla/Control, n = 6). These rats were deeply anesthetized with sodium pentobarbital (80 mg/kg). Blood was sampled from the left ventricle. Finally, the rats were transcardially perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Heart rates were also recorded during IMO (OVX + E2/ Stress, n = 6; OVX + Pla/Stress, n = 6) or following anesthesia (OVX + E2/Control, n = 6; OVX + Pla/Control, n = 6) using electrocardiograph (FX-4100, Fukuda Denshi Co., Tokyo, Japan). The brains were removed, postfixed in the same fixative overnight at 4 °C, and then cryoprotected in 0.1 M phosphatebuffered saline (PBS) containing 30% sucrose for 3 days at 4 °C. Coronal sections of the brain were cut at 50 μm in a cryostat and collected in groups of five in 0.1 M PBS containing 0.1% sodium azide. All animal manipulations were approved by Wakayama Medical University Animal Research Committee.

4.2.

Estimation of serum E2 levels

Serum E2 levels were estimated with estradiol radioimmunoassay kit (Diagnostic Products Corp., Los Angeles, CA) according to the manufacturer's protocol. Cross-reactivities to estrone and estriol were 4.6% and 0.24%, respectively. No cross-reactivities to other steroids were observed. The minimal limit of detection was 1.4 pg of E2 in 1 ml of serum.

4.3.

(rabbit, Oncogene Science, Ab-5), diluted 1:10,000 with 0.1 M PBS containing 5% normal goat serum for 24–48 h at 4 °C. After washing in PBS, they were incubated with the second antibody (biotinylated goat anti-rabbit antiserum; Vector) diluted 1:200 in PBS for 2 h at room temperature. After rinsing twice with PBS, they were reacted with avidin–biotin– HRP complex (Vector) for 2 h. After washing in 0.1 M Tris– HCl-buffered saline (pH 7.5), they were incubated in 0.05% diaminobenzidine solution containing nickel ammonium sulfate (0.2%) for 5 min. The sections were washed in PBS and mounted on slides.

4.4.

Data analysis

All cell counts were performed on coded slides by a single observer who was unaware of the treatments. To adjust the anterior–posterior levels of interest, two-three sections from each animal were examined, and one section was selected for counting the Fos-immunoreactive neurons in each nucleus of interest. The analysis was performed by light microscopy Nikon Y-FL (Nikon Corp., Tokyo, Japan) connected to a digital camera DXM1200 (Nikon). The digitized images were transferred to a Dell personal computer, and c-Fos immunoreactive neurons were counted with the aid of anatomical mapping and neuron tracing software, Neurolucida® with NeuroExplorer™ Ver 5.05.4 (MicroBright Field, Inc., Williston, VT). In short, the border of each nucleus was traced, and c-Fos immunoreactive neurons within the area were marked on the computer. The densities of c-Fos-immunoreactive neurons in the discrete nuclei were calculated, and the results were expressed as cell number per 1 × 104 μm2. Statistical analysis was performed by two-way ANOVA followed by Fisher's protected least significant difference test, or Student's t test using StatView software (Abacus Concepts, Berkeley, CA).

Acknowledgments We thank Dr. Arthur D. Loewy (St. Louis, MO) and Dr. Edith D. Hendley (Burlington, VT) for helpful comments and careful reading of the manuscript. This work was supported in part by Grants-in-Aid from Japan Society for the Promotion of Science (15590603).

Immunohistochemistry

Serial sections collected in groups of five from each of anterior–posterior levels of the brain (bregma levels (in mm): 2.20, 0.48, −0.30, −1.88, −2.56, −3.14, −3.80, −7.30, −8.72, −10.04, −12.72, −14.08, −14.60) (Paxinos and Watson, 1998) were selected and processed for immunohistochemistry. The sections included in this analysis were selected to cover the regions in which induction of c-fos mRNA or c-Fos immunoreactivity in response to emotional stress had been observed in previously published studies (Bubser and Deutch, 1999; Ceccatelli et al., 1989; Chen and Herbert, 1995; Chowdhury et al., 2000; Cullinan et al., 1995; Ma and Morilak, 2004; Palmer and Printz, 1999; Senba and Ueyama, 1997) and in our preliminary study (Ueyama et al., 2005). The sections were incubated with primary anti c-Fos serum

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