Effects of sex steroids on the expression of strogen in the principal division of the female bed nucleus of the stria terminalis

2013/2014 Cláudia Alexandra da Rocha Nunes Leite Effects of sex steroids on the expression of estrogen receptors in the principal division of the female bed nucleus of the stria terminalis março, 2014 Doutora Susana Isabel Ferreira da Silva de Sá Trabalho organizado de acordo com as normas da revista Cláudia Alexandra da Rocha Nunes Leite Mestrado Integrado em Medicina Área: Anatomia Trabalho efetuado sob a Orientação de: Doutora Susana Isabel Ferreira da Silva de Sá Trabalho organizado de acordo com as normas da revista : Effects of sex steroids on the expression of estrogen receptors in the principal division of the female bed nucleus of the stria terminalis Brain Research março, 2014 Effects of sex steroids on the expression of estrogen receptors in the principal division of the female bed nucleus of the stria terminalis Cláudia Leite * *Corresponding author: Department of Anatomy, Faculty of Medicine, University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319, Porto, Portugal. Phone: ++ 351 22 5513616 Fax: ++ 351 22 5513617 E-mail address: [email protected]om Abbreviations: ANOVA, analysis of variance; BNST, bed nucleus of the stria terminalis; BNSTpr, principal division of the bed nucleus of the stria terminalis; DAB, diaminobenzidine; DPN, diaryl-propionitrile; EB, estradiol benzoate; ER, estrogen receptor; ERα, estrogen receptor alpha; ERα-ir, estrogen receptor alphaimmunoreactive; ERβ, estrogen receptor beta; OVX, ovariectomized; PB, phosphate buffer; PBS, phosphatebuffered saline; P, progesterone; PPT, propyl-pyrazole triol. 2 ABSTRACT Estrogen actions on neurons of the principal division of the bed nucleus of the stria terminalis (BNSTpr) are essential for the regulation of female sexual behavior. Nevertheless, little is known about the effects of estradiol and progesterone (P) in the expression of estrogen receptor alpha (ERα) in this nucleus and the role played by the activation of each nuclear ER. To study this subject, we used unbiased stereological methods to determine the total number of ERα-immunoreactive (ERα-ir) neurons in the BNSTpr of young female rats across the four stages of the estrus cycle and of young ovariectomized rats after exogenous administration of estradiol benzoate (EB) and/or P. In order to identify the specific role played by the selective activation of each ER, young ovariectomized rats were injected with the ERα agonist, propyl-pyrazole triol (PPT) or the ERβ agonist, diaryl-propionitrile (DPN). The results show that the number of ERα-ir neurons is lowest at proestrus, when hormone levels are higher, and that the value seen at proestrus may be mimicked by the administration of EB but not of P. Administration of PPT induced no changes in the number of ERα-ir neurons. Contrariwise, the administration of DPN induced a decrease in the total number of ERαir neurons to values similar to the ones obtained after EB administration. These results suggest that P has no effect in the modulation of ERα expression and that estradiol regulation of ERα in BNSTpr neurons is mediated by activation of ERβ. Keywords: Estrogen receptors; Estrogen receptor agonists; estrus cycle; Bed nucleus of the stria terminalis; Immunohistochemistry; Stereology 3 1. Introduction The principal nucleus of the bed nucleus of the stria terminalis (BNSTpr), also known as the encapsulated part (Young, 1936) or the posterior medial subnucleus of the BNST (De Olmos et al., 1985; Moga et al., 1989), is a small sized densely packed group of round to oval-shaped neurons located in the medial part of the posterior division of the BNST (Ju and Swanson, 1989; Moga et al., 1989). Rostrally, it is a distinct round cluster of neurons partially encapsulated by a cell-poor area and located above the anterior commissure, whereas caudally it forms a large ribbon of cells oriented ventromedially, lateral to the fornix and stria medularis (Ju and Swanson, 1989; Moga et al., 1989). Its neurons express high levels of receptors for sex steroid hormones, namely the estrogen receptor α (ERα) and the ERβ (Laflamme et al., 1998; Shughrue et al., 1997, 1998), androgen receptors (Simerly et al., 1990) and progesterone receptors (Parsons et al., 1982). The BNSTpr, together with the medial nucleus of the amygdala, with whom it shares strong bidirectional connections, is an important component of the accessory olfactory pathway that conveys pheromonal information from the vomeronasal organ to the hypothalamus (Gu et al., 2003; Segovia and Guillamón, 1993) and, therefore, it plays a significant role in the regulation of neuroendocrine and autonomic responses, and of social behaviors (Dong and Swanson, 2004; Gu et al., 2003). The neuroendocrine control of female social behaviors, like reproduction and aggressiveness, seems to be mediated by ERα-dependent mechanisms (Nelson and Chiavegatto, 2001; Ogawa et al., 1998; Rissman et al., 1997). Sex steroid hormone receptors are ligand-activated nuclear transcription factors that regulate the expression of their own receptors. The observation that estradiol administration reduces 4 [3H]estradiol binding and the levels of ERα mRNA and protein in the preoptic area and hypothalamus (Brown et al., 1996; DonCarlos et al., 1995; Li et al., 1993; Osterlund et al., 1998) led to the general belief that estrogens down regulate ERα (reviewed in Blaustein and Erskine, 2002). However, a careful review of the literature revealed that this effect is not apparent from estimates of ERα-positive neuron numbers (Chakraborty et al., 2003). In particular, in the BNST it was shown that estradiol administration decreases the amount of ERα protein measured by intensity of immunocytochemical staining (DonCarlos et al., 1995; Li et al., 1993), but does not alter the number of neurons immunoreactive for ERα estimated from level-matched sections (Gréco et al., 2001). Also, and in dissonance with the effects of exogenous estradiol, the increase of endogenous estrogen and progesterone (P) plasma levels during the proestrus stage of the estrus cycle has been reported either not to interfere or, alternatively, to increase or decrease ERα mRNA and protein expression in several preoptic and hypothalamic nuclei (Shughrue et al., 1992; Zhou et al., 1995). Despite the recognized role of the BNSTpr in the neural circuit that regulates neuroendocrine responses and sexually dimorphic social behaviors that are mediated by ERα activation, very little is known about the effects of estradiol and P on the expression of ERα by its neurons. In addition, to our knowledge, no studies have so far addressed the role played by each ER subtype in the modulation of ERα expression by BNST neurons. Here, we address these questions by estimating, using stereological techniques, the total number of ERα-immunoreactive (ERα-ir) neurons in intact female rats at each phase of the estrous cycle, and in ovariectomized (OVX) rats injected with estradiol benzoate (EB) and P, alone or in sequence, and with the specific agonists of the ERα, propyl-pyrazole triol (PPT) and ERβ, diaryl-propionitrile (DPN). 5 2. Results 2.1. Uterine weights Uterine weight was used as an index of peripheral response to ovarian hormone levels. We found significant overall effects of stage of the estrous cycle (F(3,16) = 31.15, P < 0.0005) and treatment (F(6,28) = 74.74, P < 0.0005) on uterine weights. Uteri were 80% heavier in proestrus than in any other stage of the estrous cycle (Fig. 1A). The uterine weights of rats injected with EB, EB+P, PPT and PPT+DPN were higher (4-7 times) than in oil-, Pand DPN-injected rats. The uteri weights of rats injected with EB or EB+P were also higher (about 1.5 times) than in rats injected with PPT or PPT+DPN (Fig. 1B). 2.2. Influence of stage of the estrous cycle on the expression of ERα The stage of the estrus cycle influenced the total number of ERα-ir neurons in the BNSTpr (F(3,16) = 12.38, P < 0.0005; Fig. 2). The number was lower in rats at proestrus and estrus (25% and 20%, respectively) than in rats at metestrus or diestrus. In addition, the total number of neurons did not differ between proestrus and estrus rats as well as between metestrus and diestrus rats. 2.3. Effect of EB, P, and ERα and ERβ agonists on the expression of ERα The total number of ERα-ir neurons in the BNSTpr of OVX rats was influenced by the administration of EB, P and ER agonists (F(6,28) = 14.87, P < 0.0005; Fig. 3). The total 12 4.4. Immunocytochemical staining of BNST neurons Blocks of brain tissue containing the preoptic area and hypothalamus were mounted on a Vibratome with the rostral surface up and serially sectioned in the coronal plane at 40 µm. Sections containing the BNST were sampled at regular intervals of 120 µm (one out of 3), collected in phosphate-buffered saline (PBS) and, then, transferred to a cryoprotectant solution where they were maintained until processing for immunocytochemical detection of ERα-positive neurons. Because of the high number of animals and sections, one animal from each group was processed at a time for immunohistochemistry in order to minimize variation across groups. Sections were thoroughly washed in PBS to remove the cryoprotectant, treated with 3% H2O2 for 10 min to inactivate endogenous peroxidase, washed again in PBS and blocked with 10% normal goat serum for 45 min. Sections were then incubated for 74 h, at 4º C, in rabbit polyclonal ERα antibody (MC-20, sc-542, Santa Cruz Biotechnology, Germany), at a 1:1000 dilution, followed by incubation, for 1 h, at room temperature with biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA) at a 1:400 dilution. Sections were then incubated in avidin– biotin peroxidase complex (Vectastain Elite ABC Kit; Vector Laboratories), diluted 1:800, for 1 h, at room temperature. After that, they were incubated for 90 s in 0.05% diaminobenzidine (DAB; Sigma–Aldrich) to which 0.01% H2O2 was added, and rinsed with PBS to wash out all DAB. Stained sections were mounted on gelatin-coated slides. After air-drying overnight at room temperature, sections were counterstained with Giemsa solution for the examination of cytoarchitectural details (Fig. 4), dehydrated in a series of ethanol solutions (50%, 70%, 90% and 100%), cleared in xylol, and coverslipped using Histomount (National Diagnostics, Atlanta, GA, USA). 13 4.5. Estimation of the total number of ERα-immunoreactive neurons The total number of ERα-ir neurons was estimated by applying the optical fractionator method (Madeira et al., 1997; West et al., 1991). For this purpose, sections were analyzed using a modified Olympus BH-2 microscope interfaced with a color video camera and equipped with a Heidenhain ND 281 microcator (Traunreut, Germany), a computerized stage, and an object rotator (Olympus, Albertslund, Denmark). A computer fitted with a framegrabber (Screen Machine II, FAST Multimedia, Germany) was connected to the monitor. The boundaries of the BNSTpr were consistently defined based on cytoarchitectonic criteria (Fig. 4B; Ju and Swanson, 1989). By using the C.A.S.T. – Grid system software (Olympus), the fields of view were sampled in each section at regular intervals of 80 µm along the x and y axes. The disector used had a counting frame area of 991 µm2 at the tissue level and a fixed depth of 10 µm. The estimations were performed using a 100× oil immersion lens with a numerical aperture of 1.40. A cell profile was considered immuno-positive for ERα if dark brown reaction product was present within the cell nucleus (Fig. 4C). The mean coefficient of error (Gundersen et al., 1999) of the estimates was 0.06. All counts were made by an observer who was blind to the group assignment. 4.6. Statistical analyses The effect of the stage of the estrus cycle and of the treatment regimen used was assessed by one-way analysis of variance (ANOVA) followed by post-hoc Tukey’s HSD test. A probability value of 0.05 was used to determine statistical significance. 14 Acknowledgments The authors wish to thank Professor José Paulo Andrade for helping with BNST photomicrographs. This work was supported by National Funds through FCT - Fundação para a Ciência e a Tecnologia within the scope of the Strategic Project UI 121 – 2014 (PEst-OE/SAU/UI0121/2014). 15 REFERENCES Blaustein, J.D., Brown, T.J., 1984. Progesterone decreases the concentration of hypothalamic and anterior pituitary estrogen receptors in ovariectomized rats. Brain Res. 304, 225-236. Blaustein, J.D., Erskine, M.S., 2002. Feminine sexual behavior: cellular integration of hormonal and afferent information in the rodent forebrain. In: Pfaff, D.W., Arnold, A.P., Etgen, A.M., Fahrbach, S.E., Rubin, R.T. (Eds), Hormones, brain and behavior, Academic Press, New York, pp. 139-214. Boling, J.L., Blandau, R.J., 1939. The estrogen-progesterone induction of mating responses in the spayed female rat. Endocrinology 25, 359-364. Brown, T.J., MacLusky, N.J., 1994. Progesterone modulation of estrogen receptors in microdissected regions of the rat hypothalamus. Mol. Cell. Neurosci. 5, 283-290. Brown, T.J., Scherz, B., Hochberg, R.B., MacLusky, N.J., 1996. Regulation of estrogen receptor concentrations in the rat brain: effects of sustained androgen and estrogen exposure. Neuroendocrinology 63, 53-60. Butcher, R.L., Collins, W.E., Fugo, N.W., 1974. Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17beta throughout the 4-day estrous cycle of the rat. Endocrinology 94, 1704-1708. Chakraborty, T.R., Hof, P.R., Ng, L., Gore, A.C., 2003. Stereologic analysis of estrogen receptor alpha (ERα) expression in rat hypothalamus and its regulation by aging and estrogen. J. Comp. Neurol. 466, 409-421. De Olmos, J.S., Alheid, G.F., Beltramino, C.A., 1985. Amygdala. In: Paxinos, G. (Ed), The rat nervous system, Vol 1. Forebrain and Midbrain, Academic Press, Orlando, pp. 223-334. 16 DonCarlos, L.L., Malik, K., Morrell, J.I., 1995. Region-specific effects of ovarian hormones on estrogen receptor immunoreactivity. Neuroreport 6, 2054-2058. Dong, H.W., Swanson, L.W., 2004. Projections from bed nuclei of the stria terminalis, posterior division: implications for cerebral hemisphere regulation of defensive and reproductive behaviors. J. Comp. Neurol. 471, 396-433. Frasor, J., Barnett, D.H., Danes, J.M., Hess, R., Parlow, A.F., Katzenellenbogen, B.S., 2003. Response-specific and ligand dose-dependent modulation of estrogen receptor (ER) α activity by ERβ in the uterus. Endocrinology 144, 3159-3166. Gréco, B., Allegretto, E.A., Tetel, M.J., Blaustein, J.D., 2001. Coexpression of ER β with ER α and progestin receptor proteins in the female rat forebrain: effects of estradiol treatment. Endocrinology 142, 5172-5181. Gu, G., Cornea, A., Simerly, R.B., 2003. Sexual differentiation of projections from the principal nucleus of the bed nuclei of the stria terminalis. J. Comp. Neurol. 460, 542-562. Gundersen, H.J.G., Jensen, E.B.V., Kiêu, K., Nielsen, J., 1999. The efficiency of systematic sampling in stereology – reconsidered. J. Microsc. 193, 199-211. Hall, J.M., McDonnell, D.P., 1999. The estrogen receptor β-isoform (ERβ) of the human estrogen receptor modulates ERα transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140, 5566-5578. Ju, G., Swanson, L.W., 1989. Studies on the cellular architecture of the bed nuclei of the stria terminalis in the rat: I. Cytoarchitecture. J. Comp. Neurol. 280, 587-602. Laflamme, N., Nappi, R.E., Drolet, G., Labrie, C., Rivest, S., 1998. Expression and neuropeptidergic characterization of estrogen receptors (ERα and ERβ) throughout 17 the rat brain: Anatomical evidence of distinct roles of each subtype. J. Neurobiol. 36, 357-378. Li, H.Y., Blaustein, J.D., De Vries, G.J., Wade, G.N., 1993. Estrogen-receptor immunoreactivity in hamster brain: preoptic area, hypothalamus and amygdala. Brain Res. 631, 304-312. Lindberg, M.K., Movérare, S., Skrtic, S., Gao, H., Dahlman-Wright, K., Gustafsson, J.A., Ohlsson, C., 2003. Estrogen receptor (ER)-β reduces ERα-regulated gene transcription, supporting a ‘‘ying yang’’ relationship between ERα and ERβ in mice. Mol. Endocrinol. 17, 203-208. Madeira, M.D., Andrade, J.P., Lieberman, A.R., Sousa, N., Almeida, O.F.X., PaulaBarbosa, M.M., 1997. Chronic alcohol consumption and withdrawal do not induce cell death in the suprachiasmatic nucleus, but lead to irreversible depression of peptide immunoreactivity and mRNA levels. J. Neurosci. 17, 1302-1319. Malikov, V., Madeira, M.D., 2013. Regulation of ERa protein expression by 17bestradiol in cultured neurons of hypothalamic ventromedial nucleus. Neurochem. Res. 38, 82-89. Mazzucco, C.A., Walker, H.A., Pawluski, J.L., Lieblich, S.E., Galea, L.A., 2008. ERα, but not ERβ, mediates the expression of sexual behavior in the female rat. Behav. Brain Res. 191, 111-117. McEwen, B.S., Alves, S.E., 1999. Estrogen actions in the central nervous system. Endocr. Rev. 20, 279-307. Meyers, M.J., Sun, J., Carlson, K.E., Marriner, G.A., Katzenellenbogen, B.S., Katzenellenbogen, J.A., 2001. Estrogen receptor-β potency-selective ligands: structure-activity relationship studies of diarylpropionitriles and their acetylene and polar analogues. J. Med. Chem. 44, 4230-4251. 18 Moga, M.M., Saper, C.B., Gray, T.S., 1989. Bed nucleus of the stria terminalis: cytoarchitecture, immunohistochemistry, and projection to the parabrachial nucleus in the rat. J. Comp. Neurol. 283, 315-332. Nelson, R.J., Chiavegatto, S., 2001. Molecular basis of aggression. Trends Neurosci. 24, 713-719. Ogawa, S., Eng, V., Taylor, J., Lubahn, D.B., Korach, K.S., Pfaff, D.W., 1998. Roles of estrogen receptor-alpha gene expression in reproduction-related behaviors in female mice. Endocrinology 139, 5070-5081 Osterlund, M., Kuiper, G.G., Gustafsson, J.A., Hurd, Y.L., 1998. Differential distribution and regulation of estrogen receptor-α and -β mRNA within the female rat brain. Brain Res. Mol. Brain Res. 54, 175-180. Parsons, B., Rainbow, T.C., MacLusky, N.J., McEwen, B.S., 1982. Progestin receptor levels in rat hypothalamic and limbic nuclei. J. Neurosci. 2, 1446-1452. Pfaff, D.W., Sakuma, Y., 1979. Facilitation of the lordosis reflex of female rats from the ventromedial nucleus of the hypothalamus. J. Physiol. 288, 189-202. Pfaus, J.G., Manitt, C., Coopersmith, C.B., 2006. Effects of pelvic, pudendal, or hypogastric nerve cuts on Fos induction in the rat brain following vaginocervical stimulation. Physiol. Behav. 89, 627-636. Pompili, A., Tomaz, C., Arnone, B., Clotilde Tavares, M., Gasbarri, A., 2010. Working and reference memory across the estrous cycle of rat: A long-term study in gonadally intact females. Behav. Brain Res. 213, 10-18. Rissman, E.F., Wersinger, S.R., Taylor, J.A., Lubahn, D.B., 1997. Estrogen receptor function as revealed by knockout studies: neuroendocrine and behavioral aspects. Horm. Behav. 31, 232-243. 19 Sá, S.I., Lukoyanova, E., Madeira, M.D., 2009. Effects of estrogens and progesterone on the synaptic organization of the hypothalamic ventromedial nucleus. Neuroscience 162, 307-316. Sá, S.I., Pereira, P.A., Malikov, V., Madeira, M.D., 2013. Role of estrogen receptor α and β in the induction of progesterone receptors in hypothalamic ventromedial neurons. Neuroscience 238, 159-167. Segovia, S., Guillamón, A., 1993. Sexual dimorphism in the vomeronasal pathway and sex differences in reproductive behaviors. Brain Res. Rev. 18, 51-74. Shughrue, P.J., Bushnell, C.D., Dorsa, D.M., 1992. Estrogen receptor messenger ribonucleic acid in female rat brain during the estrous cycle: a comparison with ovariectomized females and intact males. Endocrinology 131, 381-388. Shughrue, P.J., Lane, M.V., Merchenthaler, I., 1997. Comparative distribution of estrogen receptor-α and -β mRNA in the rat central nervous system. J. Comp. Neurol. 388, 507-525. Shughrue, P.J., Scrimo, P.J., Merchenthaler, I., 1998. Evidence for the colocalization of estrogen receptor-β mRNA and estrogen receptor-α immunoreactivity in neurons of the rat forebrain. Endocrinology 139, 5267-5270. Simerly, R.B., Chang, C., Muramatsu, M., Swanson, L.W., 1990. Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study. J. Comp. Neurol. 294, 76-95. Smith, M.S., Freeman, M.E., Neill, J.D., 1975. The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy. Endocrinology 96, 219-226. 20 Stauffer, S.R., Coletta, C.J., Tedesco, R., Nishiguchi, G., Carlson, K., Sun, J., Katzenellenbogen, B.S., Katzenellenbogen, J.A., 2000. Pyrazole ligands: structure affinity/ activity relationships and estrogen receptor-α-selective agonists. J. Med. Chem. 43, 4934-4947. West, M.J., Slomianka, L., Gundersen, H.J.G., 1991. Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat. Rec. 231, 482-497. Young, N.W., 1936. The nuclear pattern and fiber connections of the noncortical centers of the telencephalon of the rabbit (Lepus cuniculus). J. Comp. Neurol. 65, 295-401. Zhou, Y., Shughrue, P.J., Dorsa, D.M., 1995. Estrogen receptor protein is differentially regulated in the preoptic area of the brain and in the uterus during the rat estrous cycle. Neuroendocrinology 61, 276-283. 21 Figure Legends Fig. 1 - Graphic representation of uterine weights of normal cycling and ovariectomized rats. (A) Uterine weights of rats over the estrous cycle. (B) Uterine weights of rats injected with oil, EB, EB+P, P, PPT, DPN and a combination of PPT and DPN (PPT+DPN). Columns represent means and vertical bars 1 SD. Tukey’s post hoc tests: *P < 0.0005, compared with diestrus, estrus and metestrus; +P < 0.0005, compared with oil, P and DPN groups; #P < 0.0005, compared with PPT and PPT+DPN groups. Fig. 2 - Graphic representation of the effect of the stage of the estrous cycle on the total number of ERα-ir neurons in the BNSTpr. Columns represent means and vertical bars 1 SD. Tukey’s post hoc tests: *P < 0.05, **P < 0.005, compared with diestrus and metestrus group. Fig. 3 - Graphic representation of the effect of hormones and ERα and ERβ agonists on the total number of ERα-ir neurons in the BNSTpr of rats injected with oil, EB, EB+P, P, PPT, DPN and a combination of PPT and DPN (PPT+DPN). Columns represent means and vertical bars 1 SD. Tukey’s post hoc tests: *P < 0.05, **P < 0.005, compared with oil, P and PPT groups; +P < 0.05, compared with PPT+DPN group. Fig. 4 - Coronal section of the female rat brain immunostained for ERα and counterstained with Giemsa solution. (A) Photomicrograph of a coronal section through the BNSTpr at bregma level approx. -0.80 mm. 3V, third ventricle; LV, lateral ventricle, oc, optic chiasm. Scale bar = 2 mm. (B) Higher magnification of the shaded area represented in A. The dashed line marks the BNSTpr limits. f, fornix; sm, stria Agradecimentos Gostaria de agradecer a todos aqueles que colaboraram na composição da presente tese. À Professora Doutora Maria Dulce Cordeiro Madeira, Directora do Departamento de Anatomia da Faculdade de Medicina da Universidade do Porto, agradeço profundamente por me ter aceitado neste local de aprendizagem e investigação, assim como pelas suas insubstituíveis orientações e laboriosas revisões do meu trabalho. À minha Orientadora de Tese de Mestrado, a Professora Doutora Susana Isabel Sá, agradeço carinhosamente por me ter facultado a excelente oportunidade de aprender consigo, tal como pelas suas contínuas instruções e dinâmicas correcções do meu trabalho. Sem esta formidável Orientadora, a concretização desta tese teria sido impossível. Aos Professores Doutores José Paulo Alves Vieira de Andrade e Armando Cardoso, agradeço a pacientíssima colaboração na estruturação iconográfica do meu trabalho. ANEXO Guia de Publicação de Artigos para a Revista Brain Research ABSTRACTS NEUROSCIENCES 116 117 YES MEETING 2013YES GUIDE BOOK PS104 TOTAL NUMBER OF ERα-IMMUNOREACTIVE NEURONS OF THE PRINCIPAL DIVISION OF THE BED NUCLEUS OF THE STRIA TERMINALIS IN FEMALE RAT BRAIN DURING THE ESTROUS CYCLE C. LEITE (1), M.D. MADEIRA (1) AND S.I. S’Ç (1) (1) DEPARTMENT OF ANATOMY, FACULTY OF MEDICINE, UNIVERSITY OF PORTO, PORTUGAL AIM To evaluate the total number of ERα-ir neurons in the BNSTpr of three-month-old female Wistar rats during the estrous cycle. INTRODUCTION The BNST is a relay nucleus of the rostral forebrain closely related to the amygdala. It is extensively connected to some preoptic and hypothalamic nuclei involved in the neuroendocrine regulation of the reproductive behavior [1]. The effects of estrogens on the BNSTpr are essential for its role in the regulation of sexual and defensive behaviors. The BNST is known to express abundantly both types of nuclear estrogen receptors (ER) and their expression is modulated by hormone levels [2,3]. Estrogen receptor content significantly changes over the estrous cycle, with brain levels being highest during metestrus, attenuated on diestrus, and lowest during proestrus and estrus [4]. METHODS The estrous cycle of 3-mo-old rats was monitored daily by vaginal smear cytology. Prior to perfusion, blood samples were taken directly from the heart and estradiol and progesterone serum levels were assayed. The animals were perfused, the brains were sectioned and the uteri were surgically isolated and weighed. The BNST-containing sections were processed for immunohistochemistry using an anti-rabbit ERÔ± antibody. The estimates of the total number of neurons were obtained by using stereological methods. The results were statistically analyzed using a one-way ANOVA. RESULTS The total number of ERα-ir neurons in the BNSTpr significantly altered over the estrous cycle, being about 35% lower during proestrus and estrus when compared with metestrus and diestrus. CONCLUSION These results suggest that gradual combinations of ovarian hormone levels have the ability to modulate the expression of ERα in the neurons of the BNSTpr. On the one hand, the prominent surge in progesterone levels which is observed in proestrus is correlated with a decrease in ERα expression. On the other hand, slight estradiol levels which are observed in metestrus and diestrus are correlated with an increase in ERα expression. This may be a way to the hormone control of the relay mechanism of the BNSTpr for the olfactory information to the hypothalamic nuclei involved in the control of sexual behavior. This work was supported by National Funds through FCT. References: [1] Krettek, J.E. et al (1978) Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat, J Comp Neurol, 178 (2), 225-254. [2] Shughrue, P.J. et al (1997) Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system, J Comp Neurol, 388 (4), 507-525. [3] Zhou, Y. et al (1995) Estrogen receptor protein is differentially regulated in the preoptic area of the brain and in the uterus during the rat estrous cycle, Neuroendocrinology, 61 (3), 276-283. [4] Shughrue, P.J. et al (1992) Estrogen receptor messenger ribonucleic acid in female rat brain during the estrous cycle: a comparison with ovariectomized females and intact males, Endocrinology, 131 (1), 381-8. Tiago Magalhães President 8th YES Meeting Fábio Carneiro Vice-President 8th YES Meeting The organizing committe of the 8th YES Meeting hereby declares that CLAUDIA ALEXANDRA DA ROCHA NUNES LEITE FACULTY OF MEDICINE, UNIVERSITY OF PORTO has participated in the Eighth YES - Young European Scientist - Meeting, from the 19th to the 22nd September 2013, held at CIM - Centro de Investigação Médica, FMUP - Faculdade de Medicina da Universidade do Porto, Portugal as a presenting student and presented the work: total numBer of erë±-immunoreaCtive neurons of the prinCipal division of the Bed nuCleus of the stria terminalis in female rat Brain during the estrous CyCle AUTHOR INFORMATION PACK 7 Feb 2014 www.elsevier.com/locate/brainres 1 BRAIN RESEARCH AUTHOR INFORMATION PACK TABLE OF CONTENTS . XXX . • Description • Audience • Impact Factor • Abstracting and Indexing • Editorial Board • Guide for Authors p.1 p.2 p.2 p.2 p.3 p.5 ISSN: 0006-8993 DESCRIPTION . Brain Research publishes papers reporting interdisciplinary investigations of nervous system structure and function that are of general interest to the international community of neuroscientists. As is evident from the journals name, its scope is broad, ranging from cellular and molecular studies through systems neuroscience, cognition and disease. Invited reviews are also published; suggestions for and inquiries about potential reviews are welcomed. With the appearance of the final issue of the 2011 subscription, Vol. 67/1-2 (24 June 2011), Brain Research Reviews has ceased publication as a distinct journal separate from Brain Research. Review articles accepted for Brain Research are now published in that journal. N.B. Review articles are by invitation only; inquiries and suggestions for reviews should be directed to the Brain Research Editorial Office ([email protected]). In the journal's Table of Contents, published papers will be shown under one of the Section titles listed below. Authors will be given the opportunity to choose the most appropriate section upon manuscript submission. SECTIONS •Cell Biology, Signaling and Synaptic Transmission Senior Editors: Leonard K. Kaczmarek (New Haven, CT, USA), Diane Lipscombe (Providence, RI, USA) Studies investigating the cellular, molecular and genetic bases of structure, function and signaling (both intracellular and intercellular) in nervous systems. •Development, Degeneration and Regeneration, and Aging Senior Editors: Fen-Biao Gao (Worcester, MA, USA), Michael E. Selzer (Philadelphia, PA, USA), Flora M. Vaccarino (New Haven, CT, USA) Studies concerning neuronal and glial development and the formation of the nervous system, molecular and cellular aspects of degeneration and regeneration, and changes associated with the aging brain. •Systems Neuroscience and Behavior Senior Editors: Gary Aston-Jones (Charleston, SC, USA), Leslie C. Griffith (Waltham, MA, USA), David J. Perkel (Seattle, WA, USA) AUTHOR INFORMATION PACK 7 Feb 2014 www.elsevier.com/locate/brainres 2 Studies concerning structure and organization of neural circuits, sensory and motor systems, internal regulatory systems and the control of behaviors. •Cognition and Computation Senior Editors: Francesco Battaglia (Amsterdam, Netherlands), Erich Schröger (Leipzig, Germany), Christina L. Williams (Durham, NC, USA) Studies of the neural mechanisms of cognition and behavior in humans and animal models including basic behaviors and higher mental functions; as well as studies dealing with realistic simulation, analysis and prediction of the structure and functions of nervous systems and individual neuronal and glial elements within nervous systems. •Neurobiology of Disease Senior Editors: Lorraine Iacovitti (Philadelphia, PA, USA), Jae-Young Koh (Seoul, Korea), Brian A. MacVicar (Vancouver, Canada), Peter H. Reinhart (Boston, MA, USA), J. Paul Taylor (Memphis, TN, USA) Studies whose primary focus is on clinically diseased nervous systems or disease models, including molecular, cellular, systems and behavioral approaches and analysis of therapeutic interventions. •Reviews Senior Editor: Irwin B. Levitan (Philadelphia, PA, USA) Invited reviews on all aspects of nervous system structure and function. The editors welcome suggestions for specific review topics. TYPES OF PAPERS 1.Research Reports reporting results of original fundamental research in any branch of the brain sciences. Papers describing new methods or significant developments of recognised methods which provide significant insight into the structure or function of the nervous system, the pathophysiology of a disease, or its treatment may also be submitted. Articles should be written in sufficient detail to allow others to verify/replicate the described methods. 2. Reviews: Reviews are by invitation only. Inquiries and suggestions for reviews should be directed to the Brain Research Editorial Office ([email protected]). Brain Research will also regularly publish thematic special issues highlighting important new developments in neuroscience research. AUDIENCE . Neuroscientists, neurophysiologists, neuroendocrinologists, neurochemists, neuroanatomists, neuropharmacologists, neurologists. IMPACT FACTOR . 2012: 2.879 © Thomson Reuters Journal Citation Reports 2013 ABSTRACTING AND INDEXING . BIOSIS Chemical Abstracts Current Contents/Life Sciences EMBASE Elsevier BIOBASE MEDLINE® Pascal et Francis (INST-CNRS) PsycINFO Psychological Abstracts RIS (Reference Update) Scopus AUTHOR INFORMATION PACK 7 Feb 2014 www.elsevier.com/locate/brainres 3 EDITORIAL BOARD . Editor-in-Chief Irwin B. Levitan, Philadelphia, PA, USA Deputy Editor-in-Chief Gary Aston-Jones, Charleston, SC, USA J. Paul Taylor, Memphis, TN, USA Senior Editors Gary Aston-Jones, Charleston, SC, USA Francesco Battaglia, Nijmegen, Netherlands Fen-Biao Gao, Worcester, MA, USA Mary Heinricher, Portland, OR, USA Lorraine Iacovitti, Philadelphia, PA, USA Leonard Kaczmarek, New Haven, CT, USA Jae-Young Koh, Seoul, South Korea Brian MacVicar, Vancouver, BC, Canada Peter Reinhart, Cambridge, MA, USA Erich Schröger, Leipzig, Germany Michael Selzer, Philadelphia, PA, USA J. Paul Taylor, Memphis, TN, USA Flora Vaccarino, New Haven, CT, USA Barry Waterhouse, Philadelphia, PA, USA Christina Williams, Durham, NC, USA Editorial Board S. Ackerman, Bar Harbor, ME, USA C. Alain, Toronto, ON, Canada K. Alho, Helsinki, Finland C.J. Aoki, New York, NY, USA L.H.M.C. Arckens, Leuven, Belgium E.C. Azmitia, New York, NY, USA F. Barceló, Palma de Mallorca, Spain R.T. Bartus, San Diego, CA, USA M. Baudry, Los Angeles, CA, USA R.J. Bodnar, Flushing, NY, USA D. Boire, Quebec, QC, Canada C. Borlongan, Tampa, FL, USA P. Chan, Beijing, China J.D. Cohen, Princeton, NJ, USA J.F. Cryan, Cork, Ireland C.G. Cusick, New Orleans, LA, USA J. DeFelipe, Madrid, Spain M. Dragunow, Auckland Mail Centre, Auckland, New Zealand S. Einheber, New York, NY, USA P. Emson, Babraham, Cambridge, UK P. Gabbott, Milton Keynes, Bucks, UK C.M. Gall, Irvine, CA, USA L-M. Garcia-Segura, Madrid, Spain F. Georges, Bordeaux Cedex, France A. Gintzler, Brooklyn, NY, USA U. Heinemann, Berlin, Germany T.L. Hodgson, Exeter, UK T. Hökfelt, Stockholm, Sweden B.L. Jacobs, Princeton, NJ, USA J. Kaiser, Frankfurt, Germany L.A. Krubitzer, Davis, CA, USA J.C. LaManna, Cleveland, OH, USA C. Lance-Jones, Pittsburgh, PA, USA J.D. Levine, San Francisco, CA, USA E.H. Lo, Charlestown, MA, USA G. Lubec, Vienna, Austria C.L. Masters, Melbourne, VIC, Australia J.F. McGinty, Charleston, SC, USA D. Morilak, San Antonio, TX, USA G.D. Mower, Louiseville, KY, USA AUTHOR INFORMATION PACK 7 Feb 2014 www.elsevier.com/locate/brainres 4 E.J. Mufson, Chicago, IL, USA K.A. Nave, Göttingen, Germany E.J. Nestler, MD, PhD, New York, NY, USA R.L. Neve, Belmont, MA, USA K. Noguchi, Hyogo, Japan J. Oberdick, Columbus, OH, USA K.C.H. Pang, East Orange, NJ, USA G.W. Pasternak, New York, NY, USA G. Perry, San Antonio, TX, USA R. Pinaud, Rochester, NY, USA J. Raber, Portland, OR, USA M.N. Rajah, Verdun, QC, Canada M.G.P. Rosa, Clayton, Australia A. Ryabinin, Portland, OR, USA J. Rüsseler, Magdeburg, Germany K. Schilling, Bonn, Germany C. Schmitz, Maastricht, Netherlands R.S. Sloviter, Tucson, AZ, USA D.A. Steindler, Gainsville FL, USA R.L. Stornetta, Charlottesville, VA, USA M. Tohyama, Suita-Shi, Osaka, Japan D. Trotti, Philadelphia, PA, USA L.L. Uphouse, Denton, TX, USA E.J. Van Bockstaele, Philadelphia, PA, USA J.C. Vickers, Hobart, TAS, Australia M.W. Vogel, Baltimore, MD, USA V. Walsh, MD, London, England, UK X. Wang, Charlestown, MA, USA I.S. Zagon, Hershey, PA, USA X. Zhang, Shanghai, China J. Zuo, Memphis, TN, USA AUTHOR INFORMATION PACK 7 Feb 2014 www.elsevier.com/locate/brainres 5 GUIDE FOR AUTHORS . INTRODUCTION Brain Research publishes papers reporting interdisciplinary investigations of nervous system structure and function that are of general interest to the international community of neuroscientists. As is evident from the journals name, its scope is broad, ranging from cellular and molecular studies through systems neuroscience, cognition and disease. Invited reviews are also published; suggestions for and inquiries about potential reviews are welcomed. Note: With the appearance of the final issue of the 2011 subscription, Vol. 67/1-2 (24 June 2011), Brain Research Reviews has ceased publication as a distinct journal separate from Brain Research. Review articles accepted for Brain Research are now published in that journal. In the journals Table of Contents, published papers will be shown under one of the Section titles listed (in bold type) below. Authors will be given the opportunity to choose the most appropriate section upon manuscript submission. SECTIONS Cell Biology, Signaling and Synaptic Transmission Senior Editors: Leonard K. Kaczmarek (New Haven, CT, USA), Diane Lipscombe (Providence, RI, USA) Studies investigating the cellular, molecular and genetic bases of structure, function and signaling (both intracellular and intercellular) in nervous systems. Cognition and Computation Senior Editors: Francesco P. Battaglia (Amsterdam, Netherlands), Erich Schröger (Leipzig, Germany), Christina L. Williams (Durham, NC, USA) Studies of the neural mechanisms of cognition and behavior in humans and animal models including basic behaviors and higher mental functions; as well as studies dealing with realistic simulation, analysis and prediction of the structure and functions of nervous systems and individual neuronal and glial elements within nervous systems. Development, Degeneration and Regeneration, and Aging Senior Editors: Fen-Biao Gao (Worcester, MA, USA), Michael E. Selzer (Philadelphia, PA, USA), Flora M. Vaccarino (New Haven, CT, USA) Studies concerning neuronal and glial development and the formation of the nervous system, molecular and cellular aspects of degeneration and regeneration, and changes associated with the aging brain. Neurobiology of Disease Senior Editors: Lorraine Iacovitti (Philadelphia, PA, USA), Jae-Young Koh (Seoul, Korea), Brian A. MacVicar (Vancouver, Canada), Peter H. Reinhart (Boston, MA, USA), J. Paul Taylor (Memphis, TN, USA) Studies whose primary focus is on clinically diseased nervous systems or disease models, including molecular, cellular, systems and behavioral approaches and analysis of therapeutic interventions. Reviews Senior Editor: Irwin B. Levitan (Philadelphia, PA, USA) Invited reviews on all aspects of nervous system structure and function. The editors welcome suggestions for specific review topics. . Systems Neuroscience and Behavior Senior Editors: Gary Aston-Jones (Charleston, SC, USA), Leslie C. Griffith (Waltham, MA, USA), David J. Perkel (Seattle, WA, USA) Studies concerning structure and organization of neural circuits, sensory and motor systems, internal regulatory systems and the control of behaviors. TYPES OF PAPERS AUTHOR INFORMATION PACK 7 Feb 2014 www.elsevier.com/locate/brainres 6 1.Research Reports reporting results of original fundamental research in any branch of the brain sciences. Papers describing new methods or significant developments of recognised methods which provide significant insight into the structure or function of the nervous system, the pathophysiology of a disease, or its treatment may also be submitted. Articles should be written in sufficient detail to allow others to verify/replicate the described methods. 2. Reviews: Reviews are by invitation only. Inquiries and suggestions for reviews should be directed to the Brain Research Editorial Office ([email protected]). Brain Research will also regularly publish thematic special issues highlighting important new developments in neuroscience research. The Neuroscience Peer Review Consortium Brain Research is a member of the Neuroscience Peer Review Consortium (NPRC). The NPRC has been formed to reduce the time expended and, in particular, the duplication of effort by, and associated burden on reviewers involved in the peer review of original neuroscience research papers. It is an alliance of neuroscience journals that have agreed to accept manuscript reviews from other Consortium journals. By reducing the number of times that a manuscript is reviewed, the Consortium will reduce the load on reviewers and Editors, and speed the publication of research results. If a manuscript has been rejected by another journal in the Consortium, authors can submit the manuscript to Brain Research and indicate that the referees' reports from the first journal be made available to the Editors of Brain Research. It is the authors' decision as to whether or not to indicate that a set of referee's reports should be forwarded from the first journal to Brain Research. If an author does not wish for this to happen, the manuscript can be submitted to Brain Research without reference to the previous submission. No information will be exchanged between journals except at the request of authors. However, if the original referees' reports suggested that the paper is of high quality, but not suitable for the first journal, then it will often be to an author's advantage to indicate that referees' reports should be made available. Authors should revise the original submission in accordance with the first journal's set of referee reports, reformat the paper to Brain Research's specification and submit the paper to Brain Research with a covering letter describing the changes that have been made, and informing the Editors that the authors will ask for the referee's reports to be forwarded from the first Consortium journal. The authors then must contact the first journal, and ask that reviews be forwarded, indicating they have submitted to Brain Research, and providing the new manuscript ID number. The Editors of Brain Research will use forwarded referees' reports at their discretion. The Editors may use the reports directly to make a decision, or they may request further reviews if they feel such are necessary. Visit http://nprc.incf.org for a list of Consortium journals, as well as further information on the scheme. Contact Details for submission Submission of manuscripts to Brain Research is entirely online at http://ees.elsevier.com/bres. Queries about the submission or editorial processes may be directed to the Brain Research Editorial Office, Elsevier, 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA; Fax: (1)-619-699.6850, Email: [email protected] BEFORE YOU BEGIN Ethics in publishing For information on Ethics in publishing and Ethical guidelines for journal publication see http://www.elsevier.com/publishingethics and http://www.elsevier.com/journal-authors/ethics. Policy and ethics The work described in your article must have been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans http://www.wma.net/en/30publications/10policies/b3/index.html; EC Directive 86/609/EEC AUTHOR INFORMATION PACK 7 Feb 2014 www.elsevier.com/locate/brainres 13 Citation in text Please ensure that every reference cited in the text is also present in the reference list (and vice versa). Any references cited in the abstract must be given in full. Unpublished results and personal communications are not recommended in the reference list, but may be mentioned in the text. If these references are included in the reference list they should follow the standard reference style of the journal and should include a substitution of the publication date with either 'Unpublished results' or 'Personal communication'. Citation of a reference as 'in press' implies that the item has been accepted for publication. Reference links Increased discoverability of research and high quality peer review are ensured by online links to the sources cited. In order to allow us to create links to abstracting and indexing services, such as Scopus, CrossRef and PubMed, please ensure that data provided in the references are correct. Please note that incorrect surnames, journal/book titles, publication year and pagination may prevent link creation. When copying references, please be careful as they may already contain errors. Use of the DOI is encouraged. Web references As a minimum, the full URL should be given and the date when the reference was last accessed. Any further information, if known (DOI, author names, dates, reference to a source publication, etc.), should also be given. Web references can be listed separately (e.g., after the reference list) under a different heading if desired, or can be included in the reference list. References in a special issue Please ensure that the words 'this issue' are added to any references in the list (and any citations in the text) to other articles in the same Special Issue. Reference formatting There are no strict requirements on reference formatting at submission. References can be in any style or format as long as the style is consistent. Where applicable, author(s) name(s), journal title/book title, chapter title/article title, year of publication, volume number/book chapter and the pagination must be present. Use of DOI is highly encouraged. The reference style used by the journal will be applied to the accepted article by Elsevier at the proof stage. Note that missing data will be highlighted at proof stage for the author to correct. If you do wish to format the references yourself they should be arranged according to the following examples: Reference style Text: All citations in the text should refer to: 1. Single author: the author's name (without initials, unless there is ambiguity) and the year of publication; 2. Two authors: both authors' names and the year of publication; 3. Three or more authors: first author's name followed by 'et al.' and the year of publication. Citations may be made directly (or parenthetically). Groups of references should be listed first alphabetically, then chronologically. Examples: 'as demonstrated (Allan, 2000a, 2000b, 1999; Allan and Jones, 1999). Kramer et al. (2010) have recently shown ....' List: References should be arranged first alphabetically and then further sorted chronologically if necessary. More than one reference from the same author(s) in the same year must be identified by the letters 'a', 'b', 'c', etc., placed after the year of publication. Examples: Reference to a journal publication: Van der Geer, J., Hanraads, J.A.J., Lupton, R.A., 2010. The art of writing a scientific article. J. Sci. Commun. 163, 51–59. Reference to a book: Strunk Jr., W., White, E.B., 2000. The Elements of Style, fourth ed. Longman, New York. Reference to a chapter in an edited book: Mettam, G.R., Adams, L.B., 2009. How to prepare an electronic version of your article, in: Jones, B.S., Smith , R.Z. (Eds.), Introduction to the Electronic Age. E-Publishing Inc., New York, pp. 281–304. Journal abbreviations source Journal names should be abbreviated according to the List of Title Word Abbreviations: http://www.issn.org/2-22661-LTWA-online.php. AUTHOR INFORMATION PACK 7 Feb 2014 www.elsevier.com/locate/brainres 14 Video data Elsevier accepts video material and animation sequences to support and enhance your scientific research. Authors who have video or animation files that they wish to submit with their article are strongly encouraged to include links to these within the body of the article. This can be done in the same way as a figure or table by referring to the video or animation content and noting in the body text where it should be placed. All submitted files should be properly labeled so that they directly relate to the video file's content. In order to ensure that your video or animation material is directly usable, please provide the files in one of our recommended file formats with a preferred maximum size of 50 MB. Video and animation files supplied will be published online in the electronic version of your article in Elsevier Web products, including ScienceDirect: http://www.sciencedirect.com. Please supply 'stills' with your files: you can choose any frame from the video or animation or make a separate image. These will be used instead of standard icons and will personalize the link to your video data. For more detailed instructions please visit our video instruction pages at http://www.elsevier.com/artworkinstructions. Note: since video and animation cannot be embedded in the print version of the journal, please provide text for both the electronic and the print version for the portions of the article that refer to this content. AudioSlides The journal encourages authors to create an AudioSlides presentation with their published article. AudioSlides are brief, webinar-style presentations that are shown next to the online article on ScienceDirect. This gives authors the opportunity to summarize their research in their own words and to help readers understand what the paper is about. More information and examples are available at http://www.elsevier.com/audioslides. Authors of this journal will automatically receive an invitation e-mail to create an AudioSlides presentation after acceptance of their paper. Supplementary data Elsevier accepts electronic supplementary material to support and enhance your scientific research. Supplementary files offer the author additional possibilities to publish supporting applications, highresolution images, background datasets, sound clips and more. Supplementary files supplied will be published online alongside the electronic version of your article in Elsevier Web products, including ScienceDirect: http://www.sciencedirect.com. In order to ensure that your submitted material is directly usable, please provide the data in one of our recommended file formats. Authors should submit the material in electronic format together with the article and supply a concise and descriptive caption for each file. For more detailed instructions please visit our artwork instruction pages at http://www.elsevier.com/artworkinstructions. 3D neuroimaging You can enrich your online articles by providing 3D neuroimaging data in NIfTI format. This will be visualized for readers using the interactive viewer embedded within your article, and will enable them to: browse through available neuroimaging datasets; zoom, rotate and pan the 3D brain reconstruction; cut through the volume; change opacity and color mapping; switch between 3D and 2D projected views; and download the data. The viewer supports both single (.nii) and dual (.hdr and .img) NIfTI file formats. Recommended size of a single uncompressed dataset is 100 MB or less. Multiple datasets can be submitted. Each dataset will have to be zipped and uploaded to the online submission system via the '3D neuroimaging data' submission category. Please provide a short informative description for each dataset by filling in the 'Description' field when uploading a dataset. Note: all datasets will be available for downloading from the online article on ScienceDirect. If you have concerns about your data being downloadable, please provide a video instead. For more information see: http://www.elsevier.com/3DNeuroimaging. Submission checklist The following list will be useful during the final checking of an article prior to sending it to the journal for review. Please consult this Guide for Authors for further details of any item. Ensure that the following items are present: One author has been designated as the corresponding author with contact details: • E-mail address • Full postal address • Phone numbers All necessary files have been uploaded, and contain: • Keywords • All figure captions • All tables (including title, description, footnotes) AUTHOR INFORMATION PACK 7 Feb 2014 www.elsevier.com/locate/brainres 15 Further considerations • Manuscript has been 'spell-checked' and 'grammar-checked' • References are in the correct format for this journal • All references mentioned in the Reference list are cited in the text, and vice versa • Permission has been obtained for use of copyrighted material from other sources (including the Web) • Color figures are clearly marked as being intended for color reproduction on the Web (free of charge) and in print, or to be reproduced in color on the Web (free of charge) and in black-and-white in print • If only color on the Web is required, black-and-white versions of the figures are also supplied for printing purposes For any further information please visit our customer support site at http://support.elsevier.com. AFTER ACCEPTANCE Use of the Digital Object Identifier The Digital Object Identifier (DOI) may be used to cite and link to electronic documents. The DOI consists of a unique alpha-numeric character string which is assigned to a document by the publisher upon the initial electronic publication. The assigned DOI never changes. Therefore, it is an ideal medium for citing a document, particularly 'Articles in press' because they have not yet received their full bibliographic information. Example of a correctly given DOI (in URL format; here an article in the journal Physics Letters B): http://dx.doi.org/10.1016/j.physletb.2010.09.059 When you use a DOI to create links to documents on the web, the DOIs are guaranteed never to change. Online proof correction Corresponding authors will receive an e-mail with a link to our ProofCentral system, allowing annotation and correction of proofs online. The environment is similar to MS Word: in addition to editing text, you can also comment on figures/tables and answer questions from the Copy Editor. Web-based proofing provides a faster and less error-prone process by allowing you to directly type your corrections, eliminating the potential introduction of errors. If preferred, you can still choose to annotate and upload your edits on the PDF version. All instructions for proofing will be given in the e-mail we send to authors, including alternative methods to the online version and PDF. We will do everything possible to get your article published quickly and accurately - please upload all of your corrections within 48 hours. It is important to ensure that all corrections are sent back to us in one communication. Please check carefully before replying, as inclusion of any subsequent corrections cannot be guaranteed. Proofreading is solely your responsibility. Note that Elsevier may proceed with the publication of your article if no response is received. Offprints The corresponding author, at no cost, will be provided with a PDF file of the article via email (the PDF file is a watermarked version of the published article and includes a cover sheet with the journal cover image and a disclaimer outlining the terms and conditions of use). For an extra charge, paper offprints can be ordered via the offprint order form which is sent once the article is accepted for publication. Both corresponding and co-authors may order offprints at any time via Elsevier's WebShop (http://webshop.elsevier.com/myarticleservices/offprints). Authors requiring printed copies of multiple articles may use Elsevier WebShop's 'Create Your Own Book' service to collate multiple articles within a single cover (http://webshop.elsevier.com/myarticleservices/offprints/myarticlesservices/booklets). AUTHOR INQUIRIES For inquiries relating to the submission of articles (including electronic submission) please visit this journal's homepage. For detailed instructions on the preparation of electronic artwork, please visit http://www.elsevier.com/artworkinstructions. Contact details for questions arising after acceptance of an article, especially those relating to proofs, will be provided by the publisher. You can track accepted articles at http://www.elsevier.com/trackarticle. You can also check our Author FAQs at http://www.elsevier.com/authorFAQ and/or contact Customer Support via http://support.elsevier.com. © Copyright 2012 Elsevier | http://www.elsevier.com