Neonatal treatment with clomipramine modifies the expression of estrogen receptors in brain areas of male adult rats

Brain Research 1724 (2019) 146443

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Neonatal treatment with clomipramine modifies the expression of estrogen receptors in brain areas of male adult rats

T

Ofelia Limón-Moralesa,f, Marcela Arteaga-Silvab, Julio Cesar Rojas-Castañedac, Tania Molina-Jiménezd, Gabriela Veronica Guadarrama-Cruze, Marco Cerbónf, ⁎ Herlinda Bonilla-Jaimeb, a

Posgrado en Biología Experimental, Universidad Autónoma Metropolitana-iztapalapa, C.P 09340 CDMX, Mexico Departamento de Biología de la Reproducción, Universidad Autónoma Metropolitana-iztapalapa, C.P 09340 CDMX, Mexico c Laboratorio de Biología de la Reproducción, Instituto Nacional de Pediatría, CDMX, Mexico d Facultad de Química Farmacéutica Biológica, Universidad Veracruzana, Circuito Gonzalo Aguirre Beltrán s/n, Zona Universitaria Xalapa, Veracruz, Mexico e Hospital General de México, CDMX, Mexico f Unidad de Investigación en Reproducción Humana Instituto Nacional de Perinatología-facultad de Química, Universidad Nacional Autónoma de México, CDMX, Mexico b

H I GH L IG H T S

CMI treatment modified the ER expression in brain in adulthood. • Neonatal CMI treatment induced a decrease in the number of cells in CA1. • Neonatal • ER expression changes occur in regions related to depression behaviors.

A R T I C LE I N FO

A B S T R A C T

Keywords: Clomipramine Estrogen receptors Central nervous system Depression Male sexual behavior Forced swimming test

The participation of estrogens in depression has been well recognized. To exert its effects, estradiol binds mainly to estrogen receptors ESR1 and ESR2 (α and β, respectively), expressed in brain regions including the hippocampus, limbic regions and hypothalamic nuclei. In rodents, modified estrogen receptors expression in brain areas have been implicated in different signs similar to those observed in depressive patients. Neonatal clomipramine (CMI) treatment is a pharmacological manipulation that generates behavioral and neurochemical changes that persist throughout adulthood and resemble human depression. The aim of this study was to analyze whether CMI neonatal treatment modifies the expression of nuclear ESR1 and ESR2 in the hippocampus, amygdala basolateral (BLA), amygdala medial (MeA), hypothalamic medial preoptic area (mPOA) and raphe nucleus in male rats. Our results indicate that CMI treatment significantly induced an mRNA increase of ESR1 in the hypothalamus, additionally produce a reduction in the mRNA ESR2 expression in raphe accompanied of an increase in hypothalamus and amygdala. CMI treated rats show more immunorreactive cells to ESR1 (ESR1-ir) in mPOA, BLA, MeA, together with a reduction of these cells in the hippocampal CA1 region. Moreover, an increase in the number of immunorreactive cells to ESR2 (ESR2-ir), in BLA and MeA, was observed in CMI treated rats. Additionally, the hippocampal CA2 region and raphe nucleus showed a decrease in these cells. Also, neonatal CMI treatment induced a decrease in the number of cells of the pyramidal layer in CA1. Overall, the results suggest that neonatal CMI treatment in rats (during brain development) induces changes in estrogen receptors in different brain areas involved with the regulation of depressive-like behaviors.

1. Introduction Depressive illness is a heterogeneous syndrome characterized by

sadness, anhedonia (understood as a “loss of pleasure”), decreased libido, cognitive impairment, among others (American Psychiatric Association, 2013). It is a result of disrupted reciprocal interactions

⁎ Corresponding author at: Departamento de Biología de la Reproducción, Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco No. 186, Col. Vicentina, C.P. 09340, Iztapalapa, CDMX, Mexico. E-mail addresses: [email protected] (O. Limón-Morales), [email protected] (H. Bonilla-Jaime).

https://doi.org/10.1016/j.brainres.2019.146443 Received 9 April 2019; Received in revised form 4 September 2019; Accepted 8 September 2019 Available online 09 September 2019 0006-8993/ © 2019 Published by Elsevier B.V.

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serotonergic system (Yavari et al., 1993; Andersen et al., 2002) and some behavioral alterations include despair, reported as an increase in the immobility behavior of the forced swimming test (VelazquezMoctezuma and Diaz-Ruiz, 1992), decreased pleasure-seeking behaviors (less intracraneal self-stimulation and low consume sucrose solution) (Vogel et al., 1990, Bhagya et al., 2008), learning impairment (deteriorate reference memory component of spatial learning in the radial arm maze) (Bhagya et al., 2008), anxiety related behaviors in elevated plus-maze (Yannielli et al., 1999) and a reduction in male sexual behavior (Bonilla-Jaime et al., 1998; Limón-Morales et al., 2014). Sexual impairment was normalized after administration of E2 and dihydrotestosterone (Limón-Morales et al., 2014). The aforementioned evidence could indicate that CMI produces a possible modification in the expression of nuclear ESR1 and ESR2 in CNS. Also, exposing to 5HT reuptake inhibitors during development induced a decrease in neuronal number in frontal lobe (Swerts et al., 2010) and raphe (Silva et al., 2010). So the CMI neonatal treatment could decrease the number of cells in the regions evaluated. The aim of the present study was analyze the effect of neonatal CMI treatment in ESR1 and ESR2 expression mRNA and protein in the hypothalamus (mPOA), hippocampus, amygdala and raphe nuclei, since the information of both techniques is complementary (see experimental procedure). Additionally cell number in these regions was evaluated.

between multiple neural circuits, neurotransmitters and hormones (aan het Rot et al., 2009) and is commonly associated with symptoms of anxiety (Tiller, 2013). Particularly, dysfunction of serotonin (5HT) has been linked to the genesis of this disease (Fakhoury et al., 2016). Interaction between estrogens and 5HT has been widely reported. 5HT levels vary during the menstrual cycle and are decreased with lower levels of 17β-estradiol (E2) (Joffe et al., 1998). In rats, ovariectomy decreases 5HT levels in the amygdala (Izumo et al., 2012) and orchidectomy lowers 5HT concentration in the hypothalamus and hippocampus (Bitar et al., 1991). Additionally, estrogens increase 5HT synthesis by enhancing tryptophan hydroxylase-2 (TPH2) expression and decrease the activity of the monoamine-oxidase (Bethea et al., 2000; Hiroi et al., 2006). In this sense, affective disorders, including premenstrual syndrome, postnatal depression and postmenopausal depression (Albert, 2015) have been associated with low E2 serum levels in women. Interestingly, the incidence of depression in men is greater with age and is associated to a decline in plasma testosterone (Khera, 2013), but the influence of estrogens in depression in men has been poorly studied. Studies show that rodents with low levels of E2 exhibit signs of depression (Dalla et al., 2004; Ordian et al., 2013), anxiety (Morgan and Pfaff, 2002; Filova et al., 2015), deterioration of male sexual behavior (Morali et al., 1977) and cognitive dysfunction (Zameer and Vohora, D, 2017), and exogenous E2 therapy improves some of the symptoms (Morgan and Pfaff, 2002; Ordian et al., 2013; Filova et al., 2015). Steroid hormones, particularly E2, regulate multiple physiological functions in the body, especially in the central nervous system (CNS) (McEwen and Alves, 1999; Cersosimo and Benarroch, 2015; Barth et al., 2015). E2 is synthesized from testosterone by aromatase and mediates its organizational functions in the male CNS such as sexual differentiation of the brain (Sharpe, 1998). They have an activating effect, playing a vital role in modulating the expression of copulatory behavior in rodents as well as in mood, motivation, learning and memory processes (Sharpe, 1998; McEwen and Alves, 1999; Abizaid et al., 2005; Carrier et al., 2015). To exert its actions, E2 binds mainly to two types of estrogen receptors: ESR1 and ESR2 (α and β, respectively) (Shugrue et al., 1997; Kuiper et al., 1997). Recently has been reported a non-nuclear estrogen receptor, known as GPR30 and it has been attributed the rapid actions of estrogen. However, its role in depression has not yet been elucidated (Lu and Herndon, 2017). Both ESR1 and ESR2 are expressed in several brain regions like the hippocampus, amygdala and hypothalamic nuclei (Shugrue et al., 1997; Mitra et al., 2003). In this sense, the expression of estrogen receptors mRNAs and proteins in the hippocampus, amygdala, hypothalamus, and raphe are essential to regulate various functions linked to these areas. Basolateral amygdala (BLA) are implicated in emotional processing of memory by facilitating information storage in the hippocampus (Mercerón-Martínez et al., 2016). Medial amygdala (MeA) and medial preoptic area (mPOA) are involved in the regulation of male sexual behavior (Hull and Dominguez, 2007; Sano et al., 2013). Raphe nuclei have also been implicated in 5HT synthesis and the regulation of depressive symptoms (Li et al., 2018). Some studies have reported that ESR1 null mutant mice (male and female) show abnormalities in sexual behavior (Wersinger et al., 1997; Ogawa et al., 1998). However, ESR2 knockout mice display nearly normal reproductive behaviors (Temple et al., 2003; Ogawa et al., 1999) yet a disruption in mood regulation (Rissman, 2008) and a delay in learning acquisition was also observed (Rissman et al., 2002). Furthermore, some animal depression models show changes in estrogen receptors in the prefrontal cortex, hippocampus and amygdala (Osterlund et al., 1999; Sharma and Thakur, 2015). Neonatal treatment in adult rats with clomipramine (CMI), a tricyclic antidepressant that inhibits 5HT reuptake, leads to behavioral abnormalities resembling human depression (Vogel et al., 1990) and has been used mainly as a depression model with high face (Willner and Mitchell, 2002). Neonatal CMI treatment induces alterations in the

2. Results 2.1. Effect of neonatal CMI treatment on FST and male sexual behavior The immobility, swimming and climbing behaviors displayed in FST by CMI treated rats are shown in Fig. 1. Concerning to immobility behavior at adulthood, CMI treated rats exhibited significantly more immobility behavior [U = 11, 10 df, p < 0.01] accompanied by a significant decrease in swimming behavior [U = 14, 10 df, p < 0.01], without modified the climbing behavior [U = 47, 10 df, p = 0.1542]. With respect to the copulatory parameters (Table 1), the CMI rats show a deterioration of sexual behavior. Only three individuals treated with CMI presented ejaculations in the test of 30 min, while all the control rats reached to ejaculate and CMI rats that ejaculated showed a decrease in the ejaculation frequency [U = 2, 10 df, p < 0.05]. CMI rats, showed an increase in mount latency [U = 6, 10 df, p < 0.01]

Fig. 1. Effect of neonatal administration of CMI on behaviors displayed in forced swimming test (FST). In the group neonatally treated with CMI, findings showed an increase in immobility time and a decrease in swimming behaviors, compared to the group treated with the control group. Mean ± E.E.M, Mann Whitney test; **p < 0.01 vs. control. 2

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Table 1 Male sexual behavior parameters in neonatally CMI treated males. Neonatal CMI treatment caused a decrease of ejaculation frequency but increases in mount, intromission and ejaculation latencies. Mean ± E.E.M, Mann Whitney test; *p < 0.05 vs. control; **p < 0.01 vs. control. Abbreviations: EF = Ejaculation frequency; ML = Mount latency; IL = Intromission latency; EL = ejaculation latency; NM = Number of mounts; NI = Number of intromissions; HR = Hit rate; PR = Refractory period. Mean ± E.E.M, Mann Whitney test; *p < 0.05 vs. control.

CON (n = 14) CMI (n = 14)

EF

ML

IL

EL

NM

NI

HR

PR

n = 12 2.9 ± 0.2 n=3 1.3 ± 0.3*

n = 12 12.3 ± 4.2 n=12 272.9 ± 111.4**

n = 12 13.8 ± 4.8 n=9 406.7 ± 93.9 **

n = 12 396.4 ± 63.1 n=3 1151 ± 296.3*

n = 12 4.5 ± 0.8 n=3 12.3 ± 6.8

n = 12 9.9 ± 1.1

n = 12 0.69 ± 0.04 n=3 0.48 ± 0.11

n = 12 401.1 ± 48.3 n=3 332.7 ± 53.05

8.6 ± 1.9

[U = 5, 5 df, p < 0.05] while the mRNA expression of ESR1 was not modified (Panel D) compared to the control group.

intromission latency [U = 0, 10 df, p < 0.01] and ejaculation latency [U = 2, 10 df, p < 0.05], all the rats treated with CMI present mounts but only 9 presented intromissions. Without changes in the other copulatory parameters.

2.3. Changes in the immunorreactive (ir) cells to ESR1 and ESR2 in brain areas

2.2. Effect of CMI on ESR1 and ESR2 mRNA expression in several brain regions.

Fig. 3 shows the ESR1-ir and ESR2-ir cells in mPOA of adult males treated with CMI during neonatal period. There was an increase in the number of ESR1-ir cells [U = 0, 5 df, p < 0.01] in CMI rats, compared to the control group. However, no change were observed in ESR2. In the hippocampus (Fig. 4), neonatal treatment with CMI did not modified the number cells that express both ESR1 and ESR2 in CA3. Yet, a decrease in the number of ESR1-ir cells was observed in CA1 [U = 2, 5 df, p < 0.05] without modifications in ESR2. By contrast, in CA2 no variations were observed in ESR1 with a significant decrease in the number ESR2-ir cells [U = 4, 5 df, p < 0.05]. In the amygdala, some modification was observed in the immunorreactive cell content of ESR1 and ESR2 in rats neonatally exposed to CMI (Fig. 5). CMI treated rats not only had more ESR1-ir cells [U = 0, 5 df, p < 0.05] and ESR2-ir [U = 3.5, 5 df, p < 0.05] in BLA,

The relative mRNA expression levels of ESR1 and ESR2 in the hypothalamus, amygdala, hippocampus and raphe nuclei of adult rats neonatally treated with CMI are shown in Fig. 2. CMI treatment significantly induced an mRNA increase of ESR1 [U = 2.5, 5 df, p < 0.05] and ESR2 [U = 5, 5 df, p < 0.05] in the hypothalamus (Panel A) compared to the saline control. However, CMI treatment did not significantly modify the mRNA expression of ESR1 or ESR2 in the hippocampus in adulthood (Panel B). On the other hand, in the amygdala (Panel C) no change was observed in the expression of ESR1 mRNA due to the effect of the CMI treatment, yet ESR2 significantly increased [U = 2.5, 5 df, p < 0.05]. Additionally, CMI treatment only caused a reduction in the mRNA expression of ESR2 in raphe nuclei

Fig. 2. Effect of neonatal CMI treatment on ESR1 and ESR2 mRNA expression. CMI treatment increase of ESR1 and ESR2 in the hypothalamus, ESR2 increased in amygdala and ESR2 reduction in raphe nuclei. Mean ± E.E.M, Mann Whitney test; *p < 0.05 vs. control. 3

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Fig. 3. ESR1 and ESR2 immunorreactive (ir) cells in medial preoptic area (mPOA). (A) Representative photomicrographs showing ESR1-ir and ESR2-ir cells. (B) CMI treatment increase in the number of ESR1-ir cells on mPOA. Mean ± E.E.M, Mann Whitney test; **p < 0.01 vs. control. Images were taken at 10X (columns a and c) and 40X (columns b and d). Scale bar = 100 μm.

receptors have been associated to the appearance of signs of depression (Ryan et al., 2012). However, the complete role of estrogens in depression, both in animal models and humans, has not been completely elucidated. In the present study, we demonstrated that CMI treatment during neonatal period in rats induced an increase in ESR1-ir cells protein in mPOA, BLA and MeA, and a reduction of these cells in the hippocampal CA1 region. Moreover, we also found an increase in the number of ESR2-ir cells in BLA and MeA. Additionally, the hippocampal CA2 region and raphe nucleus showed a decrease of these cells in adult rats. These results indicate that CMI treatment during neonatal period modulates estrogen receptors expression in adulthood and suggests that this may be associated to behavioral modifications in these animals. To the best of our knowledge, there are no previous studies reporting changes in estrogen receptors resulting from treatment with CMI (or any other antidepressant) during the neonatal period. In addition, we found that in most of the regions the mRNA and protein results were concordant. RT-qPCR and immunohistochemistry techniques could be complementary, immunohistochemistry indicates the exact location of receptor and RT-qPCR displays a higher sensitivity (Sinn et al., 2017) however, does require the maceration of tissues and could mask results in large regions such as the hippocampus. Interestingly, CMI treatment induced an increase in ESR1-ir in mPOA, the most critical targeting site for steroid hormone actions. This area regulates copulatory behavior in males among various rodent species (Hull and Dominguez, 2007). In this regard, the present results

but also an increased number of ESR1-ir cells [U = 4, 5 df, p < 0.05] and ESR2-ir [U = 3, 5 df, p < 0.05] in MeA was observed. With relation to the dorsal raphe, no changes were detected in the number of ESR1-ir cells in CMI treated rats, however a lower number of ESR2-ir [U = 5.13, 5 df, p < 0.01] was detected in this brain area (Fig. 6) [U = 0, 5 df, p < 0.01]. 2.4. Effect of CMI in cell number of brain areas The analysis of cell density in the mPOA, BLA, MeA, hippocampus (CA2 and CA3) and dorsal raphe between the adult male CMI rats versus control rats did not show significant differences (Fig. 7 and Table 2). Nonetheless, neonatal CMI treatment induced a decrease in the number of cells [U = 1.5, 5 df, p < 0.05] of the pyramidal layer in CA1. 3. Discussion and conclusions It has been widely reported that estrogens play an important role in the origin of depression (Solomon and 2009; Albert, 2015). This hormone exerts its effects via its nuclear receptors: ESR1 and ESR2, which are expressed in several tissues including the brain. Particularly, they have been described in the limbic and hypothalamic regions (Shugrue et al., 1997; Mitra et al., 2003) where they participate in the regulation of memory, sexual behavior and mood (Dalla et al., 2004; Morgan and Pfaff, 2002; Filova et al., 2015). Changes in the expression of these 4

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Fig. 4. Effect of neonatal CMI treatment on ESR1 and ESR2 immunorreactive (ir) cell number in hippocampus. (A) Representative photomicrographs showing ESR1ir and ESR2-ir cells. (B) CMI treatment induce a decrease in the number of ESR1-ir cells in CA1 and a significant decrease in the number ESR2-ir cells in CA2. Mean ± E.E.M, Mann Whitney test; *p < 0.05 vs. control. Images were taken at 10X (columns a and c) and 40X (columns b and d). Scale bar = 100 μm.

behavior (Ogawa et al., 1999). It has also been reported that in noncopulatory rats there is a decrease in the number of ESR1-ir cells, which have been associated to altered sexual behavior in these animals (Portillo et al., 2006). Furthermore, Portillo et al., (2006) reported an increase in ESR1-ir cells in MeA in non-copulatory males, which is in line with our results. It has been suggested that cells expressing steroid receptors in mPOA and MeA are activated during copula, and were associated to an increase in the expression of c-fos (Kippin et al., 2003;

show that CMI early treatment deteriorates the male sexual performance, coinciding with previous reports (Bonilla-Jaime et al., 1998; Limón-Morales et al., 2014). Some reports suggest that ESR1 is related to reproductive behavior, while ESR2 is related to non-reproductive functions (Wersinger et al., 1997; Ogawa et al., 1999; Rissman et al., 2002; Tetel and Pfaff, 2010). In fact, ESR1 knockout (ERαKO) mice show disruption in female sexual receptivity, masculine sexual behavior, male aggression, and maternal behavior, but not depression-like 5

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Fig. 5. Number of ESR1 and ESR2 immunorreactive (ir) cells in basolateral amygdala (BLA) and medial amygdala (MeA). (A) Representative photomicrographs showing ESR1-ir and ISR2-ir cells. (B) Neonatal treatment with CMI produces an increase in the number of ESR1-ir and ESR2-ir cells in BLA and MeA. Mean ± E.E.M, Mann Whitney test; *p < 0.05 vs. control. Images were taken at 10X (columns a and c) and 40X (columns b and d). Scale bar = 100 μm.

(Oyola et al., 2012; Sharma and Thakur, 2015), yet this data is still controversial. For instance, a reduction of ESR1 in the MeA reduced anxiety in animal models (Spiteri et al., 2010) and reduced ESR1 mRNA levels were reported in the basomedial nucleus of the amygdala in women with major depressive and bipolar disorders (Perlman et al., 2004). Also, ESR2 knockout mice showed an increase in anxiety responses (Krezel et al., 2001). Interestingly, our results show that an increase in ESR1-ir cells and ESR2-ir cells in MeA and BLA, after neonatal CMI treatment, although the present work does not evaluate anxiety-like behaviors, previous reports show that CMI neonatal treatment was associated to an increase in anxiety in adulthood (Yannielli et al., 1999). BLA and the hippocampus are related to emotional processing of memory (Mercerón-Martínez et al., 2016). It has been reported that memory correlates positively with hippocampal ESR1 levels and alterations in ESR1 expression have been associated to cognitive dysfunction (Foster, 2012). Differential effects of estradiol in behavior have been observed in estrogen receptors knockout mice. For example, E2 treatment to ERαKO improved memory/cognition, suggesting that

Portillo et al., 2006). Also, male rats that show poor sexual behavior (failed to show any copulatory patterns in two 15-min tests) showed significantly reduced ER expression levels in mPOA (Clark et al., 1985). Nevertheless, ESR1 disruption in the mPOA decreased sexual behavior and but not in MeA (Sano et al., 2013) indicating a differential regulation of these regions. ESR2 has been shown to be involved in defeminization in males while has been hypothesized that ESR1 is responsible for masculinization (Kudwa et al., 2006). Changes in expression at this stage of estrogen receptors may affect performance in sexual behavior in adulthood (Varshney and Nalvarte, 2017), however, the molecular mechanisms of action deserve further study. On other hand, BLA has been involved with memory processing of emotional events as well as to anxiety disorders (Hyde et al., 2011), where both ESR1 and ESR2 are expressed (Perlman et al., 2004). In this sense, estrogens have been reported to have both anxiolytic and anxiogenic activity (Lund et al., 2005) depending on the type of receptor that is activated. ESR1 activation has been involved with anxiogenic activities, whereas ESR2 activation has been related to anxiolytic effects 6

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Fig. 6. Effect of neonatal CMI treatment on ESR1 and ESR2 immunorreactive (ir) cell number in dorsal raphe. (A) Representative photomicrographs showing ESR1-ir and ESR2-ir cells. (B) CMI treatment induce a decrease in the number of ESR2-ir cells. Mean ± E.E.M, Mann Whitney test; **p < 0.01 vs. control. Images were taken at 10X (columns a and c) and 40X (columns b and d). Scale bar = 100 μm.

dorsal raphe nuclei, which may be related to the decline in the level of 5HT previously reported in this model (Yannielli et al., 1999) and the altered behaviors observed in the FST in these animals. The ESR1 and ESR2 expression changes throughout the postnatal development in a specific manner for each brain region, presenting transient increases in regions such as cortex (Wilson et al., 2011), hippocampus (Solum and Handa, 2001; Zuloaga et al., 2014) or some hypothalamic nuclei (Zuloaga et al., 2014) in early postnatal life and declines as the animal approaches to puberty. Which could indicate estrogen receptors activation in different brain regions during perinatal critical sensitive period, in order to establish the way in which the behavior in the adult will be displayed (Zuloaga et al., 2014). And a modification in the expression of both receptors caused by CMI neonatal treatment, during this sensitive period could modify different behavior regulated by E2. The current data also show that neonatal CMI treatment does not change the total number of neurons in the most of the regions evaluated. Besides, other authors show that exposing to 5HT reuptake inhibitors during development induced a decrease in neuronal number in frontal lobe (Swerts et al., 2010) and raphe (Silva et al., 2010). In human patients, several authors have demonstrated an association between depressive disorder and changes in the cell density in different brain areas. Underwood et al. (1999) found that suicide victims display an increase in the dorsal raphe cell number. Whereas Baumann et al. (2002) documented a 31% decrease in dorsal raphe neurons. Also, neonatal CMI treatment induced a decrease in cell number of CA1, some studies show that there is a reduction in hippocampal volume from the onset of the first depressive episode (Sheline et al., 1999; Videbech and Ravnkilde, 2004). Hence, the manipulation of the serotoninergic system during neurodevelopment does not alter the number of neurons in these important regions, although it may modify their function or connections.

ESR2 compensates for ESR1 (Bean et al., 2014). CMI treatment in the neonatal period induces a decrease in ESR1-ir cells in CA1, and in the ESR2-ir cells number in CA2 in the hippocampus. In this regard, has been reported that CMI neonatal treatment produces spatial learning impairment in the eight arms test in male rats (Bhagya et al., 2008), however, more research is required in this regard. In addition, CMI treatment induced a decrease in ESR2-ir cells in the nuclei of the raphe. In this regard, it has been proposed that E2 regulates the serotonergic system via ESR2, because the effects of E2 on the serotoninergic system are absent in ESR2 null mice (ERβKO) (Gundlah et al., 2005; Rocha et al., 2005). However, this occurs without modifications in reproductive behavior (Rissman, 2008) yet mood regulation is altered (Rissman, 2008; Walf et al., 2009) concomitantly with a reduction in 5HT concentrations in several brain regions (Imwalle et al., 2005). The present results show that the treatment with CMI produces an increase in the immobility in the FST accompanied by a decrease in the swimming behavior, which agrees with previous reports (VázquezPalacios et al., 2005), these data suggest a possible alteration in the activity of the 5-HT system in CMI rats (Lucki, 1997). In this regard, it has been proposed E2 improved serotonergic neurotransmission. For instance, chronic treatment with E2 increases the expression of the enzyme TPH2 in dorsal raphe nucleus (Hiroi et al., 2006; Donner and Handa, 2009; Hiroi et al., 2016) and decrease MAO expression in areas such as raphe nucleus (Gundlah et al., 2002) resulting in an increase in the 5-HT. Additionally, TPH2 co-localizes mainly with ESR2, specifically in the dorsal raphe nucleus of both sexes (Sheng et al., 2004). Further, more than 90% of ESR2 immunoreactive cells co-express TPH2 in the dorsal raphe nucleus (Nomura et al., 2005). In fact, the administration of ESR2 agonist, diarylpropionitril (DPN) induces an increase TPH2 expression (Donner and Handa, 2009) favoring 5-HT synthesis. The present study shows there is a decrease in the expression of ESR2 in 7

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Fig. 7. Representative photomicrographs showing effect of neonatal CMI treatment on total cells number in coronal sections stained with Nissl. Images were taken at 10X (columns a and c) and 40X (columns b and d). Scale bar = 100 μm.

In conclusion, neonatal CMI treatment induces changes in both mRNA expression and the number of estrogen receptors immunopositive cells in several brain areas during adulthood, and may be associated to some behavioral changes in these animals. This observation is in line with the effects that estrogens exert on the brain, such as masculinization, sexual behavior, mood, among others. Overall, our data demonstrated that CMI administered in the neonatal period induced depression-like behavior in adulthood and may be related with changes in expression of estrogen receptors.

Table 2 Effect of neonatal CMI treatment on total cells number. CMI induced a decrease in the number of cells of the pyramidal layer in CA1. Mean ± E.E.M, Mann Whitney test; *p < 0.05 vs. control. Cell density

mPOA CA1 CA2 CA3 BLA MeA Raphe

Control

CMI

10.25 ± 0.59 13.63 ± 0.23 9.2 ± 0.51 14.94 ± 0.49 13.89 ± 0.52 23.39 ± 0.51 14.83 ± 0.64

10.31 ± 0.4 11.39 ± 0.48* 9.77 ± 0.3 15.23 ± 0.75 13.72 ± 0.86 26.11 ± 1.22 12.19 ± 0.35

8

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4. Materials and methods

dissecting specific nuclei represented difficulty due to size. Incorrect dissection of an area could mask results, as well as not obtaining enough sample (Cabrera-Reyes et al., 2017). Both techniques could be complementary since the RT-qPCR displays a broader dynamic range and higher sensitivity than immunohistochemistry (Sinn et al., 2017) however, does require the maceration of tissues and not show the exact location of the receptor (Cregger et al., 2006). On the other hand, the immunohistochemistry indicates the exact location. In addition, hormone receptor expression measured quantitatively by RT-qPCR and semiquantitatively by immunohistochemistry has been compared, results showed linear correlation (ÓConnor et al., 2010).

4.1. Animals Four pregnant Wistar rats, from the vivarium of the Universidad Autónoma Metropolitana, provided the litters from which male pups were obtained for this investigation. All experiments were carried out in strict accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals (NIH Publications, 2011), legislation for the protection of animals used for scientific purposes (EC Directive 86/609/EEC) and the Official Mexican Norm (NOM-062ZOO-1999) for the production, care and use of lab animals. On postnatal day (PND) 3, the male pups were separated from the females and randomly cross-fostered. All male pups were placed in small litters (n = 6 male pups/mother). Female pups were discarded to experiments. From PND 8 to 21, pups received two subcutaneous injections per day (at 9 am and 6 pm). Clomipramine (CMI) (15 mg/kg body weight, dissolved in 0.1 ml saline solution) was administered to the experimental group, and the vehicle only (0.1 ml of saline solution) to the control group. This dose and administration scheme was chosen for its reported effectiveness in producing behavioral and physiological abnormalities (Vogel et al., 1990; Bonilla-Jaime et al., 1998; LimónMorales et al., 2014). The male pups were weaned at 23 days of age and for the next three months housed in groups of seven each. They were grouped by treatment and maintained under standard conditions. All pups were kept on a 12–12 h light–dark cycle (lights on at 21:00 h, off at 9:00 h), with ad libitum access to food and water.

4.2.1. Forced swimming test The forced swimming test was performed following the procedure described by Detke et al. (1995). The rats were placed individually placed in a cylindrical tank (46 cm high × 20 cm in diameter) containing water 30 cm deep at a temperature of 25 ± 1 °C. Two swimming sessions were conducted between 12:00 and 14:00. On the first day, each rat was forced to swim for 15 min (pre-test), during which it experienced – for the first time – the novel situation. Twenty-four hours later, all animals were subjected to test with duration of 5-min test session to evaluate the effect of CMI neonatal treatment. After a period of vigorous activity, they eventually adopted an immobile posture marked by less struggling (Porsolt et al., 1977). During testing the following behaviors were scored: immobility; the rat simply floated on the water making only those movements necessary to keep its head above water, -is interpreted as low mood or hopelessness- (Porsolt et al., 1977; Velázquez-Moctezuma et al., 1993). Additionally were evaluated active behaviors: a) swimming; when the animal made active swimming motions beyond those necessary to keep its head above water (e.g., moving around the cylinder); and, b) climbing; when the rat presented active movements with the forepaws in and out of the water, usually against the walls of the cylinder. Swimming and climbing are selectively-associated with serotonergic and noradrenergic activity, respectively (Detke et al., 1995; Lucki, 1997). All test sessions were videotaped for later evaluation and scoring was performed by two expert observers. After test, the rats were carefully dried, placed in a drying chamber and then returned to their home cages.

4.2. Experimental procedure At three months of age, in order to validate the behavioral alterations of CMI treated animals, behavioral tests: forced swimming test (for details see Section 4.2.1) and sexual behavior (for details see Section 4.2.2) described below were carried out in both groups (n = 12 per group). One week after the behavioral tests are finished two independent subgroups were formed for each treatment (CMI and control): A) Experiment 1, to determine the mRNA of the ESR1 and ESR2 through Real-time quantitative polymerase chain reaction (RT-qPCR) in hypothalamus, amygdala, hippocampus and raphe nuclei; B) experiment 2, to examine the expression of ESR1 and ESR2 in hypothalamic mPOA, BLA and MeA, hippocampus (CA1, CA2 and CA3) and dorsal raphe nucleus with immunohistochemistry techniques (see Fig. 8). To perform experiment 1, we opted to dissect complete regions because

4.2.2. Male sexual behavior test During these tests, a male was placed in a clear plexiglass cylinder (45 cm diameter) 5 min before introducing a receptive female as the stimulus. The ovariectomized female rats were made sexually receptive by administering estradiol benzoate (Sigma Chemical Co., St. Louis MO, USA, at 10 µg/0.1 ml oil, SC) 48 h before tests and progesterone (Sigma Chemical Co., St. Louis, MO, USA, at 1 mg/0.1 ml oil, SC) was administered 4 h before the testing. Test duration was 30 min and the sexual behavior tests were performed at 5-day intervals. Behavioral testing was performed under dim red lights 3 h after onset of the dark phase. Tests lasted 30 min after presentation of the female and the following parameters were recorded: latency to first mount; latency to first intromission; latency to first ejaculation; number of mounts (mounts with pelvic thrusting); and intromissions (mounts with pelvic thrusting and penile insertion) during the first copulatory series. In addition, ejaculation frequency (number of ejaculations during 30 min of recording), and post-ejaculatory intervals (time between ejaculation and subsequent intromission) were recorded. The hit rate was calculated as follows: ratio between number of intromissions and mounts plus intromissions. The full description of masculine sexual behavior parameters has been published elsewhere (Larsson, 1956; see the review in Guevara-Perez et al., 2011). Three tests of sexual behavior were conducted in order to avoid the effect of inexperience and only results of the last one are shown.

Fig. 8. Time line illustrating the temporal sequence of events in the experimental manipulations conducted with the animals from the CMI treatment to the processing of the samples for different experiments. The postnatal life days (PND) in which each manipulation was performed are indicated. Abbreviations: FST = forced swimming test, MSB = male sexual behavior, RT-qPCR = realtime quantitative polymerase chain reaction.

4.2.3. Real-time quantitative polymerase chain reaction (RT-qPCR) 4.2.3.1. Tissue dissection. The animals of both treatments (control and 9

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could be influenced by hormones (Bonefeld et al., 2008; Filby and Tyler, 2007; de Kok et al., 2004; Edelmann and Auger, 2011). The reaction mixtures were prepared with the TaqMan® Gene Expression Master Mix (Applied Biosystems) in a final volume of 10 μl. Probes were employed in 250 nM (final concentration) and 250 ng of cDNA was added to each PCR. Thermocycling conditions were the following: one cycle at 95 °C for 20 s, 40 cycles at 95 °C for one second and 62 °C for 20 s. Expression analysis was performed with the 2−ΔΔCT method and data are expressed relative to the control sample.

CMI; n = 6) were euthanized by decapitation under deep anesthesia at the beginning of the dark phase, between 9 and 11 am. The cerebral areas were dissected according to previous reports with some modifications (Heffner et al., 1980; Spijker, 2011; Defazio et al., 2015). Immediately after, an incision was made in the midline of the head skin, then a small incision was made in the upper part of the skull starting from the caudal part at the point of the parietal bone, taking care not to cross the brain. After dorsal cranial bones of the skull were removed and the brain was gently taken out from the cranial cavity, brains are rapidly removed from skull. Subsequently, it was rinsed in Milli Q water treated with ice-cold DEPC to remove blood from the surface. The brain was placed on its dorsal surface in a metal plate placed on ice, leaving visible the hypothalamus, which was carefully removed with a spatula. During use, the cutting metal plate was kept cold on crushed ice. After the cerebellum was removed from the brainstem, the midbrain and pons-medulla were separated by a cut at the level to bregma −7.4 mm corresponding to raphe nucleus (Paxinos and Watson, 2005), the tissue removal leaving a square of tissue (2 mm per side) just below the aqueduct. To dissect the amygdala, frontal lobe was removed (5 mm), the midbrain this was placed rostral side up and dissected sub-cortical nuclei within the temporal ventral lobe from bregma (−2.6 mm) a cut was made along the optic tract followed by another cut at approximately 60 deg to form an approximate triangle (Paxinos and Watson, 2005; Edelmann and Auger, 2011). Brain was placed with the ventral side facing the metal plate and the cortex was carefully removed from the great longitudinal fissure, later the two halves of the hippocampus were separated from the rest of the brain (for detail see Spijker, 2011). All dissections are performed according to the “The rat brain in stereotaxic coordinates atlas” (Paxinos and Watson, 2005) with the help of a stereoscopic microscope, under sterile conditions at 4 °C and performed by an experienced researcher. The tissue was stored at −80 °C until homogenization.

4.2.4. Immunohistochemistry 4.2.4.1. Animal perfusion and tissue processing. At three months of age, male rats (n = 6 per group) were randomly selected and anaesthetized with a combination of ketamine (80 mg/kg) and xylazine (10 mg/kg) injected intraperitoneally. By using a continuous infusion pump (Masterflex, Vernon Hills, USA), animals were perfused intracardially with physiological saline solution through a cannula inserted into the left ventricle followed by 4% cold paraformaldehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.4). After perfusion, brains were dissected carefully from the cranial cavity and post-fixed for 3 h in fresh fixative at 4 °C, and then rinsed in PBS. Blocks of tissue containing complete brains were cryoprotected in solutions of 10%, 20% and 30% sucrose in 0.1 M PBS. These tissue samples were subsequently cut on the coronal plane of the middle regions into 40 µm thick sections with a cryostat (CM1850, Leica Microsystems, Germany). The anteroposterior limits (referred to bregma) of the analyzed brain areas were: mPOA (-0.12 mm), hippocampus (-3.24 mm), BLA and MeA (-2.64 mm) and raphe nucleus (-7.44 mm) (Paxinos and Watson, 2005). Which are indicated in the Fig. 9. For each brain nucleus, three medial histological sections that were anatomically matched between subjects were chosen and evaluated to minimize variability. Alternate sections were placed in separate containers with PBS to obtain three separate sets from sections of each brain. Each set was processed for ESR1 or ESR2 immunostaining (3 sections per antibody). To show cellular bodies and identify the brain areas one set of brain sections from each animal were Nissl-stained (Fig. 9) with cresyl violet acetate for 20 min (Sigma, St. Louis, MO, USA), which were used to count the number of cells in the evaluated region. Free-floating sections from each animal were treated with 0.3% hydrogen peroxide (Merck, Germany) solution for 10 min to inhibit endogenous peroxidase activity. Afterwards, the non-specific binding sites were blocked by incubation in 5% bovine serum albumin (BSA; Amersham Biosciences, UK) and 1% Tween 20 in PBS (BSA-Tween-PBS) at room temperature for 2 h. Tissue sections were then incubated with rabbit-polyclonal antibodies for ESR1 (catalog No. H-104, sc-7207) or ESR2 (catalog No. H-150, sc-8974; Santa Cruz Biotechnology, USA), with a dilution of 1:200 in BSA-Tween-PBS, at 4 °C for 72 h. Subsequently, tissue sections were incubated with biotinylated antirabbit IgG (DAKO, USA) at room temperature for 1 h, followed by incubation with streptavidin–horseradish peroxidase (DAKO) at room temperature for 1 h. The antigen-antibody complex was visualized with the 3,3′-diaminobenzidine reaction (DAKO) and the reaction stopped by rinsing tissues in distilled water. Three ten minutes washes in 0.1 M PBS were performed between steps. Tissue sections were mounted on gelatincoated slides, air-dried and cleared with xylene. The coverslip was applied with Entellan™ (Merck). All tissue sections from control and experimental animals were processed at the same time and in parallel to minimize any potential variance in the staining procedure.

4.2.3.2. RNA extraction and quantification. Brain tissue (hypothalamus, amygdala, hippocampus and raphe nuclei) were homogenized separately in a TRIzol® (Gibco) solution containing guanidine thiocyanate, according to manufacturer's instructions. Subsequently, RNA was washed with ethanol, air-dried and resuspended in RNAsefree water. The concentration and purity of total RNA were determined by optical density (OD) at 260/280 nm. Integrity was corroborated by GelRed® fluorescence from RNA that had been separated electrophoretically on 1% agarose gel. For micro-electrophoresis, RNA integrity number (RIN) ≥ 8.5 was considered adequate for experiments. 4.2.3.3. Synthesis of complementary DNA. Complementary DNA (cDNA) synthesis was performed with the RevertAid First Strand cDNA Synthesis Kit from Thermo Scientific (K1622), according to manufacturer’s protocol. Four µg of total RNA were mixed with 100 ng of oligo (dT) and incubated at 65 °C for five minutes. The mixture was placed on ice, adding 5X reaction buffer, RNase inhibitor Ribo-Lock (20 U/l), 10 mM dNTP and M-MuLV RT RevertAid (200 U/l). The reaction was stirred and briefly centrifuged before incubation at 42 °C for 60 min. This process was terminated by heating at 70 °C for five minutes. Samples were stored at −20 °C until further use. 4.2.3.4. Real-time quantitative polymerase chain reaction. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed by using a 96-well plate (Applied Biosystems, Melbourne, Australia) in a thermocycler (Applied Biosystems, 7500 Real-Time PCR System®). The procedure was customized with TaqMan® probes (Applied Biosystems) for ESR1 (Rn01640372_m1) and ESR2 (Rn00562610_m1). Housekeeping hypoxanthine phosphoribosyl transferase1 (HPRT1Rn01527840_m1) was used as reference gene. Recent publications indicate that HPRT1 might be one of the best broad application RTqPCR housekeeping genes, especially when considering genes that

4.2.4.2. Cell counting and immunoreactive analysis. Slides with tissue sections from both groups were randomized and coded to ensure that future analysis was blinded. Images of tissue sections from the middle of the mPOA, BLA, MeA, hippocampus and dorsal raphe nucleus of each 10

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2011) with some modifications. Regions were identified and delimited at 10X as shown in Fig. 9. Different brain nuclei were determined according to Nissl stained brain sections by correspondence to the reference stereotaxic atlas plate and the Bregma level (Paxinos and Watson, 2005) and cell density in these areas was evaluated in a frame of uniform size (500, 000 µm2) at 40X to minimize errors due to some areas have irregular edges. Cell number was expressed in 10,000 µm2. The immune-stained cells was the analysis was manually counted had a minimum immunoreactivity rate of 3:1 in relation to the background OD. For each brain nucleus, three medial histological sections that were anatomically matched between subjects were chosen and evaluated to minimize variability. For all parameters, individual mean values for each subject were obtained from three tissue sections per animal were averaged to have a mean individual value per 10,000 µm2 area. From these data, mean values were calculated for each experimental group. 4.3. Statistical analysis All data are expressed as the mean ± SEM. A D'Agostino and Pearson omnibus test was performed to verify the normality of data obtained. Data from behavioral test and ESR1 and ESR2 (RT-qPCR and immunohistochemistry) were examined with non-parametric tests Mann Whitney test, considering a statistical significance at p < 0.05. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements PhD. Limon-Morales O. thanks for the support to the National Council of Science and Technology (CONACyT) for the funding provided (CVU: 268133) and profit DGAPA, UNAM Post-doctoral Fellowship grant. We thank Pedro Medina Granados for immunohistochemistry training. References aan het Rot, M., Mathew, S.J., Charney, D.S., 2009. Neurobiological mechanisms in major depressive disorder. CMAJ 180 (3), 305–313. https://doi.org/10.1503/cmaj.080697. Abizaid, A., Mezei, G., Thanarajasingam, G., Horvath, T.L., 2005. Estrogen enhances light-induced activation of dorsal raphe serotonergic neurons. Eur. J. Neurosci. 21 (6), 1536–1546. Albert, P.R., 2015. Why is depression more prevalent in women? J. Psychiatry Neurosci. 40 (4), 219–221. American Psychiatric Association, 2013. Diagnostic and Statistical Manual of Mental Disorders, fifth ed. Arlington, Virginia. Andersen, S.L., Dumont, N.L., Teicher, M.H., 2002. Differences in behavior and monoamine laterality following neonatal clomipramine treatment. Dev. Psychobiol. 41 (1), 50–57. Barth, C., Villringer, A., Sacher, J., 2015. Sex hormones affect neurotransmitters and shape the adult female brain during hormonal transition periods. Front. Neurosci. 9, 37. Baumann, B., Bielau, H., Krell, D., Agelink, M.W., Diekmann, S., Wurthmann, C., Trübner, K., Bernstein, H.G., Danos, P., Bogerts, B., 2002. Circumscribed numerical deficit of dorsal raphe neurons in mood disorders. Psychol. Med. 32 (1), 93–103. Bean, L.A., Ianov, L., Foster, T.C., 2014. Estrogen receptors, the hippocampus, and memory. Neuroscientist 20 (5), 534–545. https://doi.org/10.1177/ 1073858413519865. Bethea, C.L., Gundlah, C., Mirkes, S.J., 2000. Ovarian steroid action in the serotonin neural system of macaques. Novartis Found. Symp. 230, 112–130. Bhagya, V., Srikumar, B.N., Raju, T.R., Shankaranarayana Rao, B.S., 2008. Neonatal clomipramine induced endogenous depression in rats is associated with learning impairment in adulthood. Behav. Brain. Res. 187 (1), 190–194. Bitar, M.S., Ota, M., Linnoila, M., Shapiro, B.H., 1991. Modification of gonadectomyinduced increases in brain monoamine metabolism by steroid hormones in male and female rats. Psychoneuroendocrinology 16 (6), 547–557. Bonefeld, B.E., Elfving, B., Wegener, G., 2008. Reference genes for normalization: a study of rat brain tissue. Synapse 62, 302–309. Bonilla-Jaime, H., Retana-Marquez, S., Velazquez-Moctezuma, J., 1998. Pharmacological

Fig. 9. Identification of study areas in coronal sections stained with Nissl. Medial preoptic area (mPOA), basolateral amygdala (BLA), medial amygdala (MeA), third ventricle (3 V), aqueduct (Aq). The analyzed area is indicated with a dashed line. Images were taken at 10X. Scale bar = 200 μm. Adapted from Paxinos and Watson (2005).

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