Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Striatal GDNF Production Is Independent to Circulating Estradiol Level Despite Pan-Neuronal Activation in the Female Mouse

  • Daniel Enterría-Morales,

    Affiliation Instituto de Biomedicina de Sevilla (IBIS), Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, Sevilla, Spain

    ⨯
  • Ivette López-López,

    Affiliation Instituto de Biomedicina de Sevilla (IBIS), Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, Sevilla, Spain

    ⨯
  • José López-Barneo,

    Affiliations Instituto de Biomedicina de Sevilla (IBIS), Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, Sevilla, Spain, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain

    ⨯
  • Xavier d’Anglemont de Tassigny

    xavier-ibis@us.es

    Affiliations Instituto de Biomedicina de Sevilla (IBIS), Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, Sevilla, Spain, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain

    ⨯

Abstract

Gender difference in Parkinson’s disease (PD) suggests that female sex steroids may promote dopaminergic neuron survival and protect them from degeneration. The glial cell line-derived neurotrophic factor (GDNF) is believed to be dopaminotrophic; thus it is considered as a potential therapeutic target in PD. Additionally, GDNF is endogenously synthetized in the caudate/putamen of humans and striatum in rodents. A neuroprotective role of estrogens on the nigrostriatal pathway via the stimulation of GDNF has been proposed. Since the GDNF-producing parvalbumin (Parv) interneurons express the estrogen receptor alpha in the mouse striatum, we sought to determine whether ectopic estrogenic compound modulates the GDNF synthesis in mice. Using an ovariectomized-estradiol (E2) replacement regimen, which reliably generates a rise of plasma estradiol, we assessed the effects of different levels of E2 on the activation of striatal neuronal populations, and GDNF production. A strong correlation was found between plasma E2 and the expression of the immediate early gene cFos in the striatum, as well as in other cortical regions. However, moderate and high E2 treatments failed to induce any striatal GDNF mRNA and protein synthesis. High E2 only stimulates cFos induction in a low percentage of striatal Parv neurons whereas the majority of cFos-positive cells are medium spiny neurons. Activation of these projecting neurons by E2 suggests a role of circulating sex steroids in the modulation of striatal neural pathways.

Citation: Enterría-Morales D, López-López I, López-Barneo J, d’Anglemont de Tassigny X (2016) Striatal GDNF Production Is Independent to Circulating Estradiol Level Despite Pan-Neuronal Activation in the Female Mouse. PLoS ONE 11(10): e0164391. https://doi.org/10.1371/journal.pone.0164391

Editor: Joohyung Lee, Hudson Institute, AUSTRALIA

Received: May 26, 2016; Accepted: September 23, 2016; Published: October 14, 2016

Copyright: © 2016 Enterría-Morales et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by: Instituto de Salud Carlos iii, Miguel Servet fellowship CP12-03217 (XDADT), and iPFIS studentship (DEM). http://www.eng.isciii.es/ISCIII/es/general/index.shtml. Fundación BBVA (XDADT). http://www.fbbva.es/TLFU/tlfu/ing/home/index.jsp. Fundación Botín (JLB). http://www.fundacionbotin.org/. Centro de Investigación Biomédica en Red, Enfermedades Neurodegenerativas (JLB). http://ciberned.es/en.html. Ministerio de Economía y Competitividad: Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016 (JLB). http://www.idi.mineco.gob.es/portal/site/MICINN/menuitem.7eeac5cd345b4f34f09dfd1001432ea0/?vgnextoid=83b192b9036c2210VgnVCM1000001d04140aRCRD&lang_choosen=en. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Estrogen is an important hormone signal that regulates multiple tissues and functions in the body. Besides modulating the development and regulation of the female reproductive system and secondary sex characteristics, estrogen has a neurotrophic and neuroprotective role in the brain [1,2]. For instance, gender difference exists in the prevalence of patients with Parkinson’s disease (PD), a degenerative disorder mainly characterized by the progressive loss of mesostriatal dopamine (DA) neurons, particularly in the substantia nigra pars compacta (SNpc) [3]. The male population is generally more susceptible to develop PD symptoms such as tremor, dyskinesia and rigidity [4–6]. Likewise, menopause and steroid treatments affect the early symptoms: they usually appear sooner in early-menopausal women while estrogen-based therapies delay PD onset [7].

Multiple studies in rodent and primate PD models showing pro-dopaminergic actions of estrogen reinforced the aforementioned clinical observations [8]. A greater susceptibility of the nigrostriatal dopaminergic pathway to 6-hydroxydopamine (6-OHDA) is observed in male rats [9]. Moreover, estrogenic supplementation usually prevents degeneration of DA neurons after neurotoxin treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mouse and 6-OHDA in rat models [10–12]. The question regarding which estrogen receptor (ER) mediates the estrogen signaling has been well studied. Immunolocalization experiments indicate that nuclear ERβ is hardly expressed in the mouse striatum in comparison to ERα [13]. Moreover, ER knock-out mice, particularly those lacking ERα, are more susceptible to MPTP toxicity [14,15]. Likewise, specific ERα agonists induce protection against MPTP-induced striatal DA depletion, while ERβ agonists have no such efficiency [11,16]. In opposition to the aforementioned findings, absence of neuroprotection by estrogen, or even antidopaminergic effects, have also been described and discussed in PD animal models [12]. For instance, estradiol replacement in ovariectomized (ovx) rats failed to prevent the OHDA-induced DA loss [17].

Since there is no therapy that prevents the loss of DA neurons [18], the neuroprotective action of estrogen is intriguing and understanding its mode of action is important. Indeed, the mechanisms by which estrogen protects the nigrostriatal DA pathway remain relatively unknown. However, it is established that estrogen signaling induces anti-apoptotic genes and growth factors, and inhibits the synthesis of pro-apoptotic genes and pro-inflammatory factors [1]. Among the wealth of potent pro-dopaminergic molecules, glial cell line-derived neurotrophic factor (GDNF) is a potential candidate for mediating the estrogen neurotrophic action. The neurotrophic role of GDNF on catecholaminergic neurons is well recognized [19], however clinical trials assessing exogenous GDNF delivery on PD patients have been unsuccessful thus far. Nonetheless, GDNF is endogenously produced in the brain, particularly in the striatum, and retrogradely transported to the SNpc by the mesencephalic DA neurons of the nigrostriatal pathway [20]. While GDNF deletion effects are controversial [21–23], the results of GDNF elevation are indisputably positive [19]. The most recent and significant data supporting the neuroprotective role of endogenous GDNF on mesencephalic DA neurons were obtained from Gdnf hypermorphic mice (Gdnf hyper) that express twice the normal GDNF levels in the striatum. The subsequent endogenous GDNF elevation increases the number of DA neurons during development, and is retained in adulthood [24]. Importantly, a two-fold elevation in endogenous GDNF levels protects the Gdnf hyper mice from lactacystin toxicity in a PD model, without the side effects associated to ectopic GDNF applications. Thus, recent advances gained from transgenic models suggest that striatal GDNF plays a key role in the maintenance of the nigrostriatal DA pathway and constitutes a potential target to fight against the progressive motor decline associated to PD.

Two recent studies have linked estrogens to GDNF, indicating that the neurotrophic factor synthesis may be modulated by estradiol. Mini-osmotic pumps delivering E2 promoted estrogenic neuroprotection against 6-OHDA-induced nigrostriatal lesion in male rats. This neuroprotection effect of E2 was associated to a fair increase of GDNF protein content in both the substantia nigra and the striatum [25]. Interestingly, in mesencephalic neuron-glia co-cultures, blockade of the GDNF up-regulation by RNA interference or neutralizing antibodies prevents the E2 neuroprotective effect [25]. Additionally, it has been suggested that the G protein-coupled estrogen receptor (GPER) may be involved in the E2-induced GDNF synthesis in primary cultures of embryonic midbrain [26]. These recent advances on the role of estrogen as a neuroprotective factor, and its stimulating action on GDNF, unveil new therapeutic alternatives in PD research. However, in an effort to further understand the interaction of E2 with GDNF, and its putative relevance in PD therapy, it is worth investigating the effect of estrogen in different models. In this context, we chose to work with a model of ovariectomized (ovx) female mouse, whose depletion of endogenous gonadal estrogen was replaced by 17β-estradiol (E2), the most biologically prevalent and active estrogenic compound.

We report here the effect of circulating E2 on the neuronal activation and GDNF levels in the mouse striatum. First, we set out to determine the expression of ERα in striatal interneurons and projection neurons. Secondly, we evaluated the effect of circulating E2 on the striatal GDNF synthesis at both gene expression and protein content. Finally, to further understand the action of high circulating E2 in the striatum, we examined the expression pattern of cFos, an indirect marker of neuronal activity, in the main populations of striatal neurons.

Materials and Methods

Animals

Experiments were undertaken on adult (2 months of age) wild-type C57BL/6J mice supplied by Charles River (France). All mice were housed under a 12:12 h lighting schedule (lights on at 7:00 a.m.) with ad libitum access to food and water. Heterozygous male and female mice (4 months of age) for GDNF (Gdnf+/-) were produced in our animal facility and genotyping was performed as described in Pascual et al. (2008) [21]. All experimental protocols were approved by the Ethical committee of the Virgen Macarena and Virgen del Rocío hospitals (Seville, Spain) under the project licence no. 27-05-15-255 delivered by the Council of Andalusia.

Reagents

We used the following primary antibodies as follows: anti-Akt (9272, Cell Signaling) at 1:1000; anti-phospho(Ser473)-Akt (9271, Cell Signaling) at 1:1000; anti-cFos (PC38, Calbiochem) at 1:10000; anti-Darpp32 (AB10518, Millipore) at 1:2500; anti-ERα (06–935, Millipore) at 1:500; anti-Parvalbumin (PVG214, Swant) at 1:5000. In the immunostaining procedure, we used secondary antibodies anti-rabbit IgG and anti-goat IgG conjugated with Alexa Fluor 488 or Alexa Fluor 568 at 1:1000 (Invitrogen), AffiniPure Fab Fragment Goat Anti-rabbit IgG (H+L) (111-007-003, Jackson Laboratories). In the Western blot protocol, we used HRP-conjugated secondary antibodies anti-rabbit IgG (31460, Thermo Scientific) and anti-mouse IgG (NA931V, GE Healthcare) at 1:10000. The specificity of each antibody used in this study has been extensively tested by other groups and showed specific and expected staining in our hands.

For quantitative PCR, we used the following TaqMan probes (from Thermofisher): Gdnf1-2 (Gdnf exons 1 to 2), Mm00599849_m1; Pvalb (parvalbumin), Mm00443100_m1; Actb (βActin), Mm00443100_m1; Gapdh, Mm99999915_g1; Hmbs, Mm00660262_g1.

17β-estradiol (E8875, Sigma) was used to make up the SILASTIC implants, and β-estradiol 3-benzoate (E-8515, Sigma) for subcutaneous injection.

Ovariectomy / estradiol replacement

To remove the source of circulating gonadal hormones mice were treated as reported previously [27]. In brief, female mice were anesthetized with ketamine / xylasine dosage according to the Cold Spring Harbor Protocols 2006, bilaterally ovariectomized, and given subcutaneous SILASTIC implants containing 17β-estradiol (E2; 1 μg / 20 g of body weight) according to Bronson (1981) [28]. The SILASTIC capsules are made by dissolving crystalline E2 with ethanol, and mixing it with SILASTIC medical adhesive, which is then injected into 1 mm (internal diameter) x 2.125 mm (external diameter) SILASTIC tubing (Dow Corning, USA). Once dried, the tubing is cut to size depending on the weight of the mouse such that a 20 g mouse receives a 1 cm length of tubing (1 μg of E2). Mice were treated with 2.5 mg / kg b.w. Meloxicam i.p. post-surgery, and paracetamol 2 g / l in drinking water during the 10 days following surgery. Three weeks after ovx, mice received an s.c. injection of estradiol benzoate (2 μg / 20 g of b.w.) or vehicle (corn oil) at 12:00 p.m.

Four treatment groups were constituted: i/ group O received SILASTIC implants that contain SILASTIC medical adhesive free of E2 and vehicle 3 weeks later, ii/ group Oi received E2 SILASTIC + vehicle, iii/ group E1 received E2 SILASTIC + one s.c. injection of E2, iiii/ group E5 received E2 SILASTIC + five s.c. injection of E2 during 5 consecutive days.

Mice in groups O, Oi and E1 were sacrificed 24h after s.c. injection of E2 or vehicle. Mice in group E5 were sacrificed 4–5 hours after the last s.c. injection of E2.

Estradiol assay

17-β-estradiol was measured using the Estradiol EIA Kit (582251, Cayman Chemical), following the manufacturer’s recommended protocol, with an intra-assay sensitivity of 10 pg/ml. 50 μL of plasma were assayed in duplicate for each mouse. 17-β-estradiol concentration was calculated using the equation obtained from the standard curve plot, and expressed as pg/ml to perform comparative analysis.

Protein extraction

Protein extracts were prepared in 400μl of lysis buffer / striatum and 600 μl / cortex (pH 7.4, 25 mM Tris, 50mM β-glycerophosphate, 1.5 mM EGTA, 0.5 mM EDTA, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 100 μg/ml PMSF, 1% v/v protease inhibitors cocktail (Sigma, P8350), 1% v/v phosphatase inhibitors cocktail (Sigma, P0044) and 1% Triton X-100) by trituration of the fragments through 22 and 25 gauge needles in succession. Homogenates were incubated on ice for 15 min, followed by centrifugation at 16,000 x g for 15 min. The supernatants were removed and frozen. Protein content was determined using the Bradford method (Bio-Rad, Hercules, CA).

Western blot

15 μg of protein (from striata samples) were run on a 10% SDS-PAGE gel, using Mini-Protean TGX Stain-Free Precast Gels (BioRad), and transferred to PVDF membrane (Immobilon®-P, Millipore). Membrane was blocked in PBS 0.05%, Tween20, 5% BSA for 1 hr at room temperature (RT) and then incubated overnight at 4°C with primary antibody prepared in the same blocking solution. After washing steps, membrane was incubated with horseradish peroxidase-conjugated secondary antibody 1 hr at RT. Blots were revealed with ClarityTM Western ECL substrate (BioRad) and scanned by a luminescent image analyzer (ImageQuant LAS 4000mini, GE Healthcare). Specific bands intensity was quantified as optical density using ImageJ software. The ratio pAkt / Akt was calculated to quantify the Akt phosphorylation level in each group.

Real time quantitative PCR

Total RNAs were isolated with Trizol reagent (Life Technologies) from striatum and cortex, according to the manufacturer’s directions. 500 ng of total RNA was copied to cDNA using QuantiTect Reverse Transcription Kit (Qiagen) in a final volume of 20 μl. Real-time quantitative PCR reactions were performed in a 7500 Fast Real Time PCR System (Life Technologies). PCR reactions were performed in duplicates in a total volume of 20 μl containing 5 μl of cDNA solution and 1 μl of Taqman probe of the specific gene (Life Technologies). Housekeeping genes expression, such as Actb, Gapdh or Hmbs, were also estimated in each sample to normalize the amount of total RNA input in order to perform relative quantifications.

GDNF protein assay

Striatal GDNF protein content was estimated using a commercial enzyme-linked immunosorbant assay (ELISA) kit (GDNF Emax Immunoassay System; Promega). 80 μg of protein were used in the assay. ELISA was performed following the manufacturer’s protocol with minor modifications. Protein extracts of cortex from the same mice were used as negative controls to set background levels since Gdnf is not detected in the mouse cortex (see S1 File). GDNF protein value calculated from cortex samples was subtracted to each individual measurement. The final value expressed as pg/mg total protein was used to perform comparative analysis.

Experiment 1

Six mice per group were sacrificed by overdose injection of thiobarbital 0.6 g / kg b.w. (B. Braun, Jaén, Spain). A 0.5 ml blood sample was taken from the inferior vena cava, mixed with 5 μl EDTA and centrifuged at 10,000 x g for 10 min at 4°C. The plasma supernatant samples were collected and stored at -20°C until assayed. Brain was rapidly removed, washed three times in ice-cold sterile PBS prepared with DEPC H2O, two cortexes and two striata per brain were dissected out on ice-cold sterile plastic plate. Each fragment was placed in a microcentrifuge tube, snap frozen in liquid nitrogen, and stored at -80°C. Cortex tissue dissection mainly includes the somatomotor, somatosensory and auditory areas of the isocortex. Striatum fragment consisted of the whole dorsal striatum.

Experiment 2

3 to 4 female mice per experimental group were used for immunohistochemistry experiments. Mice were deeply anesthetized with thiobarbital as described above and intracardially perfused with phosphate-buffered saline 0.1 M (PBS) followed by 4% paraformaldehyde in PBS pH 7.4. After a 1 hr post fixation step, brains were washed in PBS, cryoprotected in PBS with 30% sucrose, embedded in Tissue-Tek® O.C.T. Compound (Sakura®, Finetek), and frozen on dry ice. Using a cryostat (CM 1950, Leica, Germany), 30 or 50 μm coronal floating sections were obtained and stored in an antifreeze solution (0,9% w/v NaCl, 30% w/v sucrose, 1% w/v Polyvinylpyrrolidone (9003-39-8, Sigma), 30% v/v Ethylene glycol in PBS) at −20°C until use. Primary and secondary antibodies were prepared in PBS with 0.1% Triton X-100, 10% fetal bovine serum and 1 mg/ml bovine serum albumin. Sections were incubated with primary antibodies (Parv, Darpp32, cFos, ERα) overnight at 4°C, and then incubated with secondary anti-IgG antibodies conjugated with Alexa Fluor® 488 or 568 for 2 hrs at room temperature. When a double staining with two rabbit serum antibodies was required, an additional 2 hrs blocking step using unconjugated goat anti-rabbit IgG at 1:1000 was done between the first and second incubations with primary / secondary antibodies. Nuclei were detected by 4’,6’-diamidino-2-phenylindole (Dapi, D9542, Sigma). Fluorescence images were obtained with a BX61 microscope equipped with a DP70 camera (Olympus). We used 5 successive brain sections (150μm between each section) per mouse to take images of 10 regions of interests (ROIs) of the striatum and 5 ROIs of the motor cortex. Photographs of cFos-immunostained slices were analyzed with custom macro automation quantification by ImageJ software to count the number of cFos+ cells. Using the same ROIs, we manually quantified the number of double positive cells in the striatum. Values were reported as cells (PV+ cFos+, Darpp32+ cFos+, PV+ ERα+, Darpp32+ ERα+) per mm3, from the average of the data obtained from all ROIs, the ROI area (0.395 mm2) and the sample thickness (30 or 50 μm).

Experiment 3

Three male and three female mice were used for immunohistochemical mapping of ERα, with the same immunostaining methods as described above.

Statistics

We used Graphpad—Prism 6 software to perform statistical comparisons and to calculate Pearson’s correlation. Before statistical analysis, percentages were subjected to arc-sine transformation to convert them from a binomial to a normal distribution. Comparison between two groups was subjected to an unpaired Student’s t-test. One-way analysis of variance (ANOVA), followed by Tukey post hoc multiple comparisons test, was used to draw comparisons between three or more groups. The level of significance was set at p < 0.05.

Results

Estrogen receptor alpha (ERα) is the ER subtype involved in the E2-mediated neuroprotection [14], and is widely expressed in the mouse brain [13] (Fig A in S1 File). Parvalbumin (Parv) interneurons account for the majority of the GDNF synthesizing cells in the striatum [24,29]. Dual-labeling immunocytochemistry showed the presence of ERα in 94.1 ± 2.5% and 98.4 ± 0.9% of Parv neurons throughout the striatum of female (n = 3) and male (n = 3) respectively (Fig 1A). These data indicate that Parv neurons are equipped with the appropriate estrogen receptor to take part in the neuroprotection mechanism promoted by circulating E2. In addition, we observed the expression of ERα in striatal medium spiny neuron (MSN) by dual staining with dopamine- and cAMP-regulated neuronal phosphoprotein (Darpp32) (Fig 1B), a protein that is specifically expressed in neurons that also express DA receptors [30]. A thorough inspection of male and female striata revealed that nearly every single MSN expresses ERα (94.2 ± 1.9%), as quantified in brains from two female and two male (n = 4) (Fig 1C). Likewise, MSN count for the large majority of the ERα positive cells in the striatum (95.1 ± 0.8%) (Fig 1C), whereas the remaining ERα-expressing cells are presumably interneurons, including Parv neurons (Fig A in S1 File).

thumbnail
Download:
Fig 1. Estrogen receptor alpha (ERα) expression in the mouse striatum.

(A) Photomicrographs show parvalbumin (Parv) (red) and ERα+ (green) staining in male and female coronal sections. Vertical bargraphs on the right indicate the mean percentage ± SEM of the Parv+ cells that are ERα+ (n = 3). (B) microphotograph illustrating ERα (red) expression by Darpp32+ medium spiny neurons (green) in a female brain. Note that ERα is almost exclusively expressed in Darpp32+ cells. (C) Quantification of the Darpp32 and ERα colocalization shows that 94.2 ± 1.9% of the Darpp32+ neurons are immuno-positive for ERα; and 95.1 ± 0.8% of striatal ERα is expressed in Darpp32+ neurons in male and female mice (n = 4, from 2 male and 2 female striata).

https://doi.org/10.1371/journal.pone.0164391.g001

To study the effect of exogenous E2 administration, we divided the animals in four experimental groups labeled O, Oi, E1 and E5 as follows: ovariectomized without any exogenous E2 (O), ovariectomized with basal E2 continuously released from a SILASTIC implant (Oi), ovariectomized with E2 SILASTIC implant + one E2 injection (E1), and ovariectomized with E2 SILASTIC implant + five E2 injections over 5 days (E5) as depicted in Fig 2A. E2 treated mice exhibited an E2-dependant significant increase of blood circulating E2 measured in plasma at sacrifice (Fig 2B). O, 20.8 ± 3.6 pg/ml (n = 5); Oi, 36.0 ± 7.9 pg/ml (n = 6); E1, 138.8 ± 34.4 pg/ml (n = 6); E5, 327.5 ± 80.6 pg/ml (n = 6). Vaginal cytology evaluation at sacrifice indicated that the hormonal replacement was efficient. O mice only presented leucocytes; Oi mice had a mix of leucocytes and large nucleated epithelial cells, whereas mice from E1 and E5 groups presented only cornified cells (Fig B in S1 File), which indicates a high steroidal impregnation as it is normally observed in the estrus stage of the estrous cycle in rodents [31]. Interestingly, Esr1 mRNA expression (gene coding for ERα) did not change upon the increase of blood E2 levels (Fig A in S1 File). Estrogen-mediated neuroprotection in ERα activation induces phosphorylation of the protein kinase Akt [32]. To determine the efficiency of exogenous E2 treatment to promote intracellular activation of ERα signaling in our model, we evaluated the level of Akt phosphorylation (pAkt) in striatal protein samples (Fig 2C). The ratio pAkt / Akt increased with high E2, although this was not significantly different (p = 0.053 between O and E1, n = 6 per group).

thumbnail
Download:
Fig 2. Absence of GDNF modulation by estradiol replacement in ovariectomized female mice.

(A) Mouse experimental model. Ovariectomized (ovx) animals were separated in four groups and received either sham implant (O) or E2 implant (Oi, E1 and E5) at surgery time. Three weeks later, mice received Vehicle (O and Oi), a single s.c. E2 injection (E1), or five E2 injections over 5 days (E5). Animals were sacrificed 24 hours after s.c. injection (O, Oi, E1) or 4–5 hours after the last injection (E5). (B) Vertical bar graph showing the mean ± SEM of plasma E2 in O, Oi, E1 and E5 mice. This shows that the estrogen replacement protocol induced an increase in circulating E2 in ovx mice. (C) Western blot analysis of phosphorylated Akt (pAkt) showing a representative blot (left), and pAkt / Akt ratio is shown with a vertical bar graph (right). The greatest difference is found between O and E1 (p = 0.053). (D) Gdnf mRNA expression, reported as Gdnf1-2 relative to bActin, in the striatum of wild-type (Gdnf+/+) and heterozygous mice (Gdnf+/-). (E) GDNF protein content expressed as pg / mg total protein in Gdnf+/+ and Gdnf+/- striatum. (F) Scatter plot diagram showing the positive correlation (ρ = 0.72, p = 0.002) between Gdnf mRNA (shown in D) and GDNF protein levels (shown in E) in Gdnf+/+ and Gdnf+/- striata. This data shows that GDNF protein concentration measured by ELISA is consistent with Gdnf gene expression level. (G) QPCR analysis of Gdnf expression measured by Gdnf1-2 relative to Parv mRNA (left graph) or Hmbs mRNA (right). (H) ELISA analysis of GDNF protein levels showing no difference between the experimental groups. All data are presented as the mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. N is indicated at the bottom of each bar.

https://doi.org/10.1371/journal.pone.0164391.g002

To examine the relationship between circulating E2 and striatal GDNF, we performed a series of assays to monitor the expression of GDNF at both mRNA and protein levels. First, we tested the specificity of the quantitative RT-PCR using commercial TaqMan probes that amplify a cDNA sequence spanning exons 1 and exons 2. Gdnf+/- mice carrying a single copy of the functional allele are expected to express about half of the normal GDNF levels. Indeed, Gdnf+/- mice (n = 6) showed a significantly reduced striatal Gdnf mRNA content (normalized to bActin gene expression), being 42% of the Gdnf expression measured in Gdnf+/+ striatum (n = 9) (Fig 2D). ELISA was performed to assess the level of GDNF protein in the striatum. Since the adult mouse cortex does not express Gdnf (Fig C in S1 File), cortical proteins were run along with striatal proteins to set the background noise from unspecific binding (Fig C in S1 File). We found that the level of GDNF protein calculated were much lower in Gdnf+/- mice (73 ± 26 pg/mg total protein) than in Gdnf+/+ mice (215 ± 25 pg/mg) (Fig 2E). A significant Pearson’s correlation (ρ = 0.72, p = 0.002) was found between Gdnf mRNA and GDNF protein levels (Fig 2F), which confirms the accuracy of the ELISA method employed in this study to specifically measure GDNF protein. In the ovx mice, no differences in Gdnf mRNA expression were observed among the four experimental groups (n = 5–6 / group) normalized with either Parv or Hmbs gene expression (Fig 2G). Moreover, we observed no variation either in GDNF protein content among the different E2 treatments (n = 6 / group) (Fig 2H). GDNF expression was assessed in two additional separate experiments, and we have never observed any increase of the GDNF levels in high E2 conditions (Fig D in S1 File).

Estrogen activates the proto-oncogene cFos expression in neuronal cell types [33,34]. Immunostaining for cFos followed by unbiased quantification revealed a progressive increase of the number of cFos-positive cells in the striatum and cortex (where ERα is highly present) in parallel with the rising plasma levels of E2 as illustrated in Fig 3A. cFos immunostaining shows relatively low expression in the striatum as compared to neighbor brain regions like the isocortex or the thalamic area (unpublished observations). In mice from group O, the number of cFos cells was 1544 ± 458 per mm3 (n = 3). The E2 SILASTIC implant in Oi mice induced a slight increase of cFos-positive cells (2503 ± 240, n = 4). However, elevated circulating E2 levels significantly stimulate striatal cFos expression (4996 ± 823 and 5591 ± 157 / mm3 in E1 and E5 respectively, n = 4) (Fig 3B). Noticeably, E2 induces cortical cFos expression in a dose-dependent manner (Fig 3B): O, 7646 ± 265 (n = 3); Oi, 9952 ± 1806 (n = 4); E1, 14264 ± 776 (n = 4) and E5, 17368 ± 1273 cFos+ cells / mm3 (n = 4). In addition, there is a significant correlation between striatal and cortical cFos expression (ρ = 0.70, p = 0.004) (Fig 3C). Dual labeling for Parv and cFos in the striatum demonstrated that few Parv neurons express cFos after high E2 stimulation (Fig 3D). O mice had 2.4 ± 1.3%, Oi mice had 2.47 ± 1.22%, E1 had a 5.62 ± 1.72% and E5 had 7.37 ± 3.24% Parv neurons expressing cFos. Surprisingly, we did not see any activation of the ERK1/2 p42/p44 MAPK pathway, a known activator of cFos, in the striatum (Fig E in S1 File). Although there is a trend indicating that circulating E2 induces cFos expression in Parv neurons, in our experiment this did not reach significance (p = 0.33), probably owing to the low number of cFos-positive Parv cells that generate high variability between individuals. The low response of Parv interneurons to high circulating E2, indicated by cFos induction, might help to explain why GDNF level does not vary in our animal model.

thumbnail
Download:
Fig 3. Induction of cFos by E2 in striatum and cortex.

(A) Low magnification photomicrographs of O and E1 female mouse coronal sections illustrate the increase of cFos+ nuclei in the brain under E2 stimulation. Magnified views of striatum and cortex highlight the cFos staining difference between O and E1 mice. (B) Graphs showing the mean ± SEM of cFos+ nuclei in the striatum (left) and cortex (right) from O, Oi, E1 and E5 mice. (C) Correlation chart showing the positive correlation (ρ = 0.70, p = 0.004) between striatal and cortical number of cFos+ nuclei (shown in B). Note the gradual increase of cFos+ cells such as O < Oi < E1 ≤ E5. (D) Dual labeling for cFos (green) and Parv (red) showing the above mentioned increase of cFos+ nuclei in the striatum. Very few Parv+ cells are positive for cFos as indicated by the percentage of Parv+ cFos+ cells in the graph. n.s., not significant. *, p < 0.05; **, p < 0.01. N is indicated at the bottom of each bar.

https://doi.org/10.1371/journal.pone.0164391.g003

Then, we looked for the cell type expressing cFos in response to high E2 and we found that medium spiny neurons (MSN) are the main cells activated by E2 in the striatum (Fig 4A). About 80% of cFos+ cells are Darpp32+ regardless of the E2 treatment (Fig 4B). Along with the general increase of cFos in the striatum, the number of Darpp32+ cells showing cFos induction significantly increases with rising E2: in O mice, 900 ± 193; in Oi mice, 1406 ± 231; in E1 mice, 2752 ± 306; and in E5 mice, 2573 ± 203 Darpp32+ cFos+ cells / mm3 (Fig 4C). Consequently, the percentage of Darpp32+ with cFos+ induction increase with E2: O, 3.3 ± 0.3%; Oi, 9.6 ± 3.6%; E1, 19.2 ± 2.8%; E5, 19.3 ± 1.5% (Fig 4D).

thumbnail
Download:
Fig 4. Induction of cFos by E2 in medium spiny neurons (MSN).

(A) Photomicrographs of the dorsal striatum showing progressive increase of cFos+ (red) expression in Darpp32+ (green) MSN in O, Oi, E1 and E5 groups. (B) Graph showing the percentage of cFos+ nuclei expressed by Darpp32+ MSNs. (C) Graph showing the mean number ± SEM of Darpp32+ cFos+ cells. (D) Graph showing the mean percentage ± SEM of Darpp32+ that are cFos+. All data are presented as the mean ± SEM. *, p < 0.05; **, p < 0.01. N is indicated at the bottom of each bar.

https://doi.org/10.1371/journal.pone.0164391.g004

Discussion

To further study the mechanism involved in the estrogen-mediated neuroprotection of the dopaminergic nigrostriatal pathway, we have evaluated the effect of plasma estrogen on GDNF production in the striatum using a model of ovariectomized (ovx) female mice. We found that virtually every parvalbumin (Parv) interneuron, the cell type producing most of the striatal GDNF [29], expresses the estrogen receptor alpha (ERα), with no sex dimorphism. Surprisingly, high E2 failed to induce GDNF production in ovx mice, and very few Parv interneurons displayed evidence of activation by E2, as evidenced by cFos induction. On the other hand, E2-induced cFos expression was broadly observed in the cortex and the striatum, mainly in medium spiny neurons (MSNs), suggesting a possible role for estrogen in the modulation of basal ganglia circuitry.

We report here the absence of GDNF increase under E2 exposure despite a general activation of ER intracellular pathways in the female adult mouse. These results differ from those obtained in other models, which varied in both experimental design and animal or cell types [25,26]. We used a model of ovx female mouse supplemented with exogenous E2, while others have used 6-OHDA-treated male rats [25] and ventral midbrain primary cultures [26] to show an increase of GDNF protein content by E2 stimulation. The methods to assess GDNF levels also vary between the studies. While other investigators privileged the use of polyclonal antibodies to detect GDNF by western blot [25,26], we favored quantitative RT-PCR to assay Gdnf gene expression, and ELISA to assess GDNF protein levels. In our study, slight variations exist between the different cohorts analyzed, and a decrease of striatal GDNF concentration has been occasionally observed upon high E2. The hormonal regimen used here has proven efficiency based on physiological changes such as vaginal cytology, Akt phosphorylation and cFos induction. Indeed, a 24 hours E2 exposure is enough to induce maximal neuronal activation (cFos) in the striatum. In spite of this, a 15-fold increase of plasma E2 over the baseline repeatedly failed in stimulating GDNF production. A speculative explanation to the lack of action of E2 on GDNF may reside in the absence of intracellular relay to the ERα in the Parv neurons. While ERα is likely to drive the E2 signal leading to neuroprotection by binding to specific response elements [16], a non-genomic action mediated by the G protein-coupled estrogen receptor (GPER) may occur as well. GPER mediates rapid estrogen signaling via neuronal extracellular signal-regulated kinases (ERKs) and phosphatidylinositol-3-kinase (PI3K)-Akt pathways [1,35], which promote neuron survival [36]. Nonetheless, a recent study reported no significant change in striatal GDNF levels in MPTP-treated male mice that were stimulated by E2, ERα, ERβ, and GPER agonists [35]. Also, the absence of ERα signaling lead to lower content in tyrosine hydroxylase and brain-derived neurotrophic factor (BDNF) in the midbrain of ERαKO mice, whereas GDNF remained unaffected [37]. Although obtained from mouse midbrain samples and not striatum, these data suggest that ERα activity may not be linked to GDNF production. With this in mind, the E2–mediated neuroprotection may occur through different pro-survival pathways (see for review [1]), as well as other local trophic factor synthesis, such as BDNF [37,38].

Since Parv interneurons participate to over 90% of the striatal GDNF production [24,29], it may be valuable to study the E2 neuroprotective effect in mice with conditional ERα deletion in the Parv neurons (Parv-Cre / Esr1-flox mouse model). Such model may help to clarify the divergence existing on the interaction between estrogen and GDNF in the neuroprotection of the nigrostriatal DA pathway.

Estrogen receptors (ERs) are expressed throughout the brain and both classical nuclear ERs α and β are found in the striatum [39], although ERβ shows a more restricted topographical expression than ERα [13]. Here we found that ERα is expressed in virtually every single projecting neuron (MSN) in the mouse striatum. High circulating E2 promotes cFos induction in various brain regions, but merely ~ 20% of the MSNs respond to exogenous E2. The sole presence of ERα may not be enough to activate every MSN, thus suggesting heterogeneity in the MSN population whereby some neurons are more sensitive to E2 than others. As yet, the effects of E2 on MSN are not well characterized. Estrogen can rapidly induce cFos expression via activation of both phosphatidylinositol 3-kinase/Akt and ERK1/2 mitogen-activated protein kinase [40,41]. However, we found no activation of the ERK1/2 pathway by ectopic E2 in the striatum. Instead, estrogen may trigger glutamatergic neuron activity in the cortex, since cFos induction is turned on by E2, and therefore indirectly modulate striatal MSN activity. Likewise, E2 is able to influence electrophysiological properties and dendritic plasticity of MSN [42], thus tuning the response of MSN to cortical glutamatergic inputs. Indeed, ERs are paired with metabotropic glutamate receptors (mGluRs) to mediate intracellular signaling such as mitogen-activated protein kinase (MAPK)-dependent CREB phosphorylation leading to transcription regulation in MSN [43]. Also, E2 is required for activity-dependent long-term potentiation in MSN [44], and it may as well affect the basal ganglia indirect pathway by modulating the D2-type DA receptor expression, such as in the rat dorsal striatum [45]. Moreover, E2 activates membrane-localized ERs to modulate DA synaptic neurotransmission [46], and potentiates the amphetamine-induced rotational behavior [47]. It is therefore possible that DA and E2 work together to activate a common cascade of intracellular events such as the cAMP-PKA and ERK pathways that are crucial for LTP. Interestingly, a neuroprotective effect of E2 has been reported in Huntington disease rat model, whereby females have a less severe course of the disease associated to higher resistance of MSNs [48]. In a behavioral perspective, estrogenic modulation of the DA and MSN function may underlie sex differences in motor and sensory functions, and for instance contribute to women’s ability to outperform men at tests of fine motor control and speech articulation [49].

In summary, estradiol treatment to ovariectomized mice was found to activate cFos expression in striatal MSNs, while there was little effect on the GDNF-producing parvalbumin interneurons. As a possible consequence to this, circulating E2 cannot stimulate GDNF production in this animal model. However, our results reinforce a role for estrogen treatment on the activation of a restricted MSN subpopulation that calls for further investigation.

Supporting Information

S1 File.

Fig A. Estrogen receptor alpha (ERα) expression in the female mouse striatum. (A) Fluorescence microphotographs showing the broad ERα immunostaining in the striatum, motor and piriform cortex. (B) Confocal microphotograph of a single 1 micron plane showing ERα staining in parvalbumin (Parv) and medium spiny neurons (Darpp32). Note that ERα is expressed almost exclusively in Darpp32+ cells. (C) ERα expression (Esr1 relative to Hmbs) does not vary between the different E2 treatments. N is indicated at the bottom of each bar. Fig B. Vaginal smears representative cytology from mice in groups O, Oi, E1 and E5. C, cornified cell typical of estrus stage when circulating sex steroids level is high; E, nucleated epithelial cell; L, leucocyte (neutrophil). Fig C. GDNF ELISA specificity. Since adult mouse cortex does not express Gdnf, cortex proteins are used to set the non-specific binding in GDNF ELISA experiment. (A) QPCR analysis of Gdnf1-2 (relative to Actb) in cortex and striatum of the adult female mouse (n = 4). (B) Scattered plot showing raw absorbance (optical density at 450nm) measured in the female mouse cortex (n = 4) and in the striatum of mice from O, Oi, E1, and E5 groups (n = 6). Fig D. Analysis of GDNF levels in the O, Oi, E1 and E5 mice in two separate experiments (A-C and D-H). (A) QPCR analysis of Gdnf1-2 relative to Hmbs in striatum and cortex. (B) QPCR analysis of Gdnf2-3 relative to Hmbs in striatum and cortex. (C) QPCR analysis of Pvalb relative to Hmbs in striatum and cortex. (D, E) QPCR analysis of Gdnf1-2 (D) and Gdnf2-3 (E) relative to Hmbs in the striatum. (F, G) QPCR analysis of Gdnf1-2 (F) and Gdnf2-3 (G) relative to Actb in the striatum. (H) GDNF protein levels in the striatum. n.s., non significant difference. N is indicated at the bottom of each bar. Fig E. Phosphorylation of Erk1/2 (pErk1/2) in the mouse striatum. Western blot analysis of striatal proteins shows no difference between ovx (O), ovx + E2 implant (Oi), ovx + E2 implant + E2 s.c 1 day (E1) and ovx + E2 implant + E2 s.c 5 days (E5). Left, representative blot. Right, bar graph illustrating the pErk1/2 / Erk1/2 ratio. n.s., non significant difference. N is indicated at the bottom of each bar.

https://doi.org/10.1371/journal.pone.0164391.s001

(PDF)

Acknowledgments

The authors would like to thank Dr. Andrew Clarkson (University of Otago, Dunedin, NZ) for providing the SILASTIC implants, Dr. Konstantin Levitsky for helping in confocal microscopy, and members of the IBIS Animal Core Facility for excellent care of the animals.

Author Contributions

  1. Conceptualization: XDADT JLB.
  2. Formal analysis: DEM XDADT.
  3. Funding acquisition: XDADT JLB.
  4. Investigation: DEM ILL XDADT.
  5. Methodology: XDADT DEM.
  6. Project administration: XDADT.
  7. Resources: XDADT ILL DEM.
  8. Supervision: XDADT JLB.
  9. Validation: XDADT.
  10. Visualization: DEM XDADT.
  11. Writing – original draft: XDADT DEM JLB.
  12. Writing – review & editing: XDADT DEM JLB.

References

  1. 1. Arevalo M-A, Azcoitia I, Garcia-Segura LM. The neuroprotective actions of oestradiol and oestrogen receptors. Nat Rev Neurosci. 2015;16(1):17–29. pmid:25423896
  2. 2. Brann DW, Dhandapani K, Wakade C, Mahesh VB, Khan MM. Neurotrophic and neuroprotective actions of estrogen: Steroids. 2007;2:381–405.
  3. 3. Obeso JA, Rodriguez-Oroz MC, Goetz CG, Marin C, Kordower JH, Rodriguez M, et al. Missing pieces in the Parkinson’s disease puzzle. Nat Med. 2010 Jun;16(6):653–61. pmid:20495568
  4. 4. Wooten GF, Currie LJ, Bovbjerg VE, Lee JK, Patrie J. Are men at greater risk for Parkinson’s disease than. J Neurol Neurosurg Psychiatry. 2004;75:637–40. pmid:15026515
  5. 5. Pringsheim T, Jette N, Frolkis A, Steeves TDL. The prevalence of Parkinson’s disease: a systematic review and meta-analysis. Mov Disord. 2014;29(13):1583–90. pmid:24976103
  6. 6. Litim N, Morissette M, Paolo T Di. Neuroactive gonadal drugs for neuroprotection in male and female models of Parkinson’s disease. Neurosci Biobehav Rev. 2015;67:79–88. pmid:26708712
  7. 7. Miller IN, Cronin-Golomb A. Gender differences in Parkinson’s disease: Clinical characteristics and cognition. Mov Disord. 2010;25(16):2695–703. pmid:20925068
  8. 8. Smith KM, Dahodwala N. Sex differences in Parkinson’s disease and other movement disorders. Exp Neurol. 2014;259:44–56. pmid:24681088
  9. 9. Gillies GE, Murray HE, Dexter D, McArthur S. Sex dimorphisms in the neuroprotective effects of estrogen in an animal model of Parkinson’s disease. Pharmacol Biochem Behav. 2004;78(3):513–22. pmid:15251260
  10. 10. Dluzen D. Estrogen decreases corpus striatal neurotoxicity in response to 6-hydroxydopamine. Brain Res. 1997;767(2):340–4. pmid:9367266
  11. 11. D’Astous M, Morissette M, Di Paolo T. Effect of estrogen receptor agonists treatment in MPTP mice: evidence of neuroprotection by an ER α agonist. Neuropharmacology. 2004;47:1180–8. pmid:15567427
  12. 12. Liu B, Dluzen DE. Oestrogen and nigrostriatal dopaminergic neurodegeneration: animal models and clinical reports of Parkinson’s disease. Clin Exp Pharmacol Physiol. 2007;34(7):555–65. pmid:17581209
  13. 13. Mitra SW, Hoskin E, Yudkovitz J, Pear L, Wilkinson HA, Hayashi S, et al. Immunolocalization of estrogen receptor β in the mouse brain: comparison with estrogen receptor α. Endocrinology. 2003;144(5):2055–67. pmid:12697714
  14. 14. Morissette M, Jourdain S, Al Sweidi S, Menniti FS, Ramirez AD, Di Paolo T. Role of estrogen receptors in neuroprotection by estradiol against MPTP toxicity. Neuropharmacology. 2007;52:1509–20. pmid:17420033
  15. 15. Al-Sweidi S, Morissette M, Bourque M, Di Paolo T. Estrogen receptors and gonadal steroids in vulnerability and protection of dopamine neurons in a mouse model of Parkinson’s disease. Neuropharmacology. 2011;61(4):583–91. pmid:21586296
  16. 16. Morissette M, Saux M Le, Astous MD, Jourdain S, Sweidi S Al, Morin N, et al. Contribution of estrogen receptors alpha and beta to the effects of estradiol in the brain. J steroid Biochem Mol Biol. 2008;108:327–38. pmid:17936613
  17. 17. Cordellini M, Piazzetta G, Pinto K, Delattre A, Matheussi F, Carolino R, et al. Effect of different doses of estrogen on the nigrostriatal dopaminergic system in two 6-hydroxydopamine-induced lesion models of Parkinson’s disease. Neurochem Res. 2011;36(6):955–61. pmid:21347839
  18. 18. Kalia L V, Kalia SK, Lang AE. Disease-modifying strategies for Parkinson’s disease. Mov Disord. 2015;30(11):1442–50. pmid:26208210
  19. 19. d’Anglemont de Tassigny X, Pascual A, López-Barneo J. GDNF-based therapies, GDNF-producing interneurons, and trophic support of the dopaminergic nigrostriatal pathway. Implications for Parkinson’s disease. Front Neuroanat. 2015;9:1–15.
  20. 20. Tomac A, Widenfalk J, Lin LF, Kohno T, Ebendal T, Hoffer BJ, et al. Retrograde axonal transport of glial cell line-derived neurotrophic factor in the adult nigrostriatal system suggests a trophic role in the adult. Proc Natl Acad Sci U S A. 1995;92(18):8274–8. pmid:7667281
  21. 21. Pascual A, Hidalgo-Figueroa M, Piruat JI, Pintado CO, Gómez-Díaz R, López-Barneo J. Absolute requirement of GDNF for adult catecholaminergic neuron survival. Nat Neurosci. 2008;11(7):755–61. pmid:18536709
  22. 22. Kopra J, Vilenius C, Grealish S, Härma M, Varendi K, Lindholm J, et al. GDNF is not required for catecholaminergic neuron survival in vivo. Nat Neurosci. 2015;18(3):319–22. pmid:25710828
  23. 23. Pascual A, Lopez-Barneo J. Reply to GDNF is not required for catecholaminergic neuron survival in vivo. Nat Neurosci. 2015;18(3):322–3. pmid:25710829
  24. 24. Kumar A, Kopra J, Varendi K, Porokuokka LL, Panhelainen A, Kuure S, et al. GDNF overexpression from the native locus reveals its role in the nigrostriatal dopaminergic system function. PLOS Genet. 2015;11(12):e1005710. pmid:26681446
  25. 25. Campos FL, Cristovão AC, Rocha SM, Fonseca CP, Baltazar G. GDNF contributes to oestrogen-mediated protection of midbrain dopaminergic neurones. J Neuroendocrinol. 2012;24(11):1386–97. pmid:22672424
  26. 26. Bessa A, Campos FL, Videira RA, Mendes-Oliveira J, Bessa-Neto D, Baltazar G. GPER: A new tool to protect dopaminergic neurons? Biochim Biophys Acta—Mol Basis Dis. 2015;1852(10):2035–41.
  27. 27. Clarkson J, d’Anglemont de Tassigny X, Moreno AS, Colledge WH, Herbison AE. Kisspeptin-GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge. J Neurosci. 2008;28(35):8691–7. pmid:18753370
  28. 28. Bronson FH. The regulation of luteinizing hormone secretion by estrogen: relationships among negative feedback, surge potential, and male stimulation in juvenile, peripubertal, and adult female mice. Endocrinology. 1981;108(2):506–16. pmid:7449740
  29. 29. Hidalgo-Figueroa M, Bonilla S, Gutiérrez F, Pascual A, López-Barneo J. GDNF is predominantly expressed in the PV+ neostriatal interneuronal ensemble in normal mouse and after injury of the nigrostriatal pathway. J Neurosci. 2012;32(3):864–72. pmid:22262884
  30. 30. Hemmings HCJ, Greengard P. DARPP-32, a dopamine- and adenosine 3’:5'-monophosphate-regulated phosphoprotein: regional, tissue, and phylogenetic distribution. J Neurosci. 1986;6(5):1469–81. pmid:3711991
  31. 31. Byers SL, Wiles M V., Dunn SL, Taft RA. Mouse estrous cycle identification tool and images. PLoS One. 2012;7(4):2–6.
  32. 32. D’Astous M, Mendez P, Morissette M, Garcia-Segura LM, Di Paolo T. Implication of the phosphatidylinositol-3 kinase/protein kinase B signaling pathway in the neuroprotective effect of estradiol in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice. Mol Pharmacol. 2006;69(4):1492–8. pmid:16434614
  33. 33. Pfaus JG, Kleopoulos SP, Mobbs C V, Gibbs RB, Pfaff DW. Sexual stimulation activates c-fos within estrogen-concentrating regions of the female rat forebrain. Brain Res. 1993;624:253–67. pmid:8252398
  34. 34. Pleil KE, Glenn MJ, Williams CL. Estradiol alters Fos-immunoreactivity in the hippocampus and dorsal striatum during place and response learning in middle-aged but not young adult female rats. Endocrinology. 2011;152(3):946–56. pmid:21285311
  35. 35. Bourque M, Morissette M, Di Paolo T. Neuroprotection in parkinsonian-treated mice via estrogen receptor alpha activation requires G protein-coupled estrogen receptor 1. Neuropharmacology. 2015;95:343–52. pmid:25892506
  36. 36. Dudek H, Datta S, Franke T, Birnbaum M. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science. 1997;275(5300):661–5. pmid:9005851
  37. 37. Küppers E, Krust A, Chambon P, Beyer C. Functional alterations of the nigrostriatal dopamine system in estrogen receptor-α knockout (ERKO) mice. Psychoneuroendocrinology. 2008;33(6):832–8. pmid:18472350
  38. 38. Al-Sweidi S, Morissette M, Di Paolo T. Estrogen receptors modulate striatal metabotropic receptor type 5 in intact and MPTP male mice model of Parkinson’s disease. J Steroid Biochem Mol Biol. 2016;(2015):1–8.
  39. 39. Küppers E, Beyer C. Expression of estrogen receptor-alpha and beta mRNA in the developing and adult mouse striatum. Neurosci Lett. 1999;276(2):95–8. pmid:10624800
  40. 40. Herdegen T, Leah JD. Inducible and constitutive transcription factors in the mammalian nervous system: control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Brain Res Brain Res Rev. 1998;28:370–490. pmid:9858769
  41. 41. Titolo D, Mayer CM, Dhillon SS, Cai F, Belsham DD. Estrogen facilitates both phosphatidylinositol 3-kinase / Akt and ERK1 / 2 mitogen-activated protein kinase membrane signaling required for long-term neuropeptide Y transcriptional regulation in clonal, immortalized neurons. J Neurosci. 2008;28(25):6473–82. pmid:18562618
  42. 42. Peterson BM, Mermelstein PG, Meisel RL. Estradiol mediates dendritic spine plasticity in the nucleus accumbens core through activation of mGluR5. Brain Struct Funct. 2015;220:2415–22. pmid:24878822
  43. 43. Grove-Strawser D, Boulware MI, Mermelstein PG. Membrane estrogen receptors activate the metabotropic glutamate receptors mGluR5 and mGluR3 to bidirectionally regulate CREB phosphorylation in female rat striatal neurons. Neuroscience. 2010;170(4):1045–55. pmid:20709161
  44. 44. Tozzi A, de Lure A, Tantucci M, Durante V, Giampà C, Di Mauro M, et al. Endogenous 17β -estradiol is required for activity-dependent long-term potentiation in the striatum: interaction with the dopaminergic system. Front Cell Neurosci. 2015;9:1–14.
  45. 45. Lammers CH, D’Souza U, Qin ZH, Lee SH, Yajima S, Mouradian MM. Regulation of striatal dopamine receptors by estrogen. Synapse. 1999;34(3):222–7. pmid:10523759
  46. 46. Becker J. Direct effect of 17 beta-estradiol on striatum: sex differences in dopamine release. Synapse. 1990;5(2):157–64. pmid:2309159
  47. 47. Becker J. Estrogen rapidly potentiates amphetamine-induced striatal dopamine release and rotational behavior during microdialysis. Neurosci Lett. 1990;118(2):169–71. pmid:2125712
  48. 48. Bode FJ, Stephan M, Suhling H, Pabst R, Straub RH, Raber KA, et al. Sex differences in a transgenic rat model of Huntington’s disease: decreased 17b-estradiol levels correlate with reduced numbers of DARPP32+ neurons in males. Hum Mol Genet. 2008;17(17):2595–609. pmid:18502785
  49. 49. Jennings P, Janowsky J, Orwoll E. Estrogen and sequential movement. Behav Neurosci. 1998;112(1):154–9. pmid:9517823