Pharmacology, Biochemistry and Behavior 166 (2018) 27–34

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Pharmacology, Biochemistry and Behavior

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Early postnatal treatment with clomipramine induces female sexual T behavior and estrous cycle impairment ⁎ Tania Molina-Jiméneza, Ofelia Limón-Moralesb, Herlinda Bonilla-Jaimec, a Posgrado en Biología Experimental, Universidad Autónoma Metropolitana-Iztapalapa, Apartado, Postal 55 535, C.P. 09340 Ciudad de México, Mexico b Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, Av Universidad 3000, Cd. Universitaria, Coyoacán, 04510 Ciudad de México, Mexico c Departamento de Biología de la Reproducción, Universidad Autónoma Metropolitana-Iztapalapa, Apartado Postal 55 535, C.P. 09340 Ciudad de México, Mexico

ARTICLE INFO ABSTRACT

Keywords: Administration of clomipramine (CMI), a tricyclic antidepressant, in early stages of development in rats, is Clomipramine considered an animal model for the study of depression. This pharmacological manipulation has induced be- Depression havioral and physiological alterations, i.e., less pleasure-seeking behaviors, despair, hyperactivity, cognitive Female sexual behavior dysfunction, alterations in neurotransmitter systems and in HPA axis. These abnormalities in adult male rats are Estrous cycle similar to the symptoms observed in major depressive disorders. One of the main pleasure-seeking behaviors affected in male rats treated with CMI is sexual behavior. However, to date, no effects of early postnatal CMI treatment have been reported on female reproductive cyclicity and sexual behavior. Therefore, we explored CMI administration in early life (8–21 PN) on the estrous cycle and sexual behavior of adult female rats. Compared to the rats in the early postnatal saline treatment (CTRL group), the CMI rats had fewer estrous cycles, fewer days in the estrous stage, and longer cycles during a 20-day period of vaginal cytology analysis. On the behavioral test, the CMI rats displayed fewer proceptive behaviors (hopping, darting) and had lower lordosis quotients. Also, they usually failed to display lordosis and only rarely manifested marginal or normal lordosis. In contrast, the CTRL rats tended to display normal lordosis. These results suggest that early postnatal CMI treatment caused long-term disruptions of the estrous cycle and female sexual behavior, perhaps by alteration in the hypotha- lamic-pituitary-gonadal (HPG) axes and in neuronal circuits involved in the regulation of the performance and motivational of sexual behavior as the noradrenergic and serotonergic systems.

1. Introduction increased the syntheses of serotonin and decreased the activity of the monoamine-oxidase, the enzyme that is involved in the catabolism of Depression is the most common psychiatric disorder, as it affects monoamines (Gundlah et al., 2002; Hiroi et al., 2006; Lu et al., 2003). approximately 17% of the population (Duman, 2014). It is character- Moreover, noradrenergic system participates in the ovarian-steroid ized by a profound loss of interest (anhedonia), dysregulation of affect feedback of secretory luteinizing hormone (Miller and Zhu, 1995; and mood, decreased libido, cognitive dysfunction, fatigue, and sleep Szawka et al., 2013, 2007). These data suggest an interaction between and appetite disorders (American Psychiatric Association, 2013). Sig- steroid hormones and neurotransmitter systems, which have a partici- nificantly, women run a lifetime risk of suffering depression twice as pation in the neurobiology of depression. high as that of men (Kessler et al., 2003; Kessler and Bromet, 2013; Animal models are useful tools for studying the neurobiology of Marcus et al., 2005). Their predisposition to depression involves social, depression and screening new molecules with antidepressant activity psychosocial and biological factors like hormonal fluctuations such as (Valvassori et al., 2013; Yan et al., 2010). Most of such models employ estrogen and progesterone that it has been linked to the reproductive different types of stressors to induce depressive-like behaviors, though cycle and premenstrual syndrome, with associated mood disturbances, some involve long-term manipulations that promote a predisposition to regularly recurs during the luteal phase of each menstrual (ovarian) developing depression, such as early postnatal administration of clo- cycle (Noble, 2005; Sassarini, 2016). It is well known that estrogen mipramine (CMI) (Willner and Mitchell, 2002). interacts with neurotransmitter systems. For instance, estrogens Administering CMI, a tricyclic antidepressant, in early life is

⁎ 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, D.F. México, Mexico. E-mail address: [email protected] (H. Bonilla-Jaime). https://doi.org/10.1016/j.pbb.2018.01.004 Received 9 September 2017; Received in revised form 28 January 2018; Accepted 29 January 2018 Available online 31 January 2018 0091-3057/ © 2018 Elsevier Inc. All rights reserved. T. Molina-Jiménez et al. Pharmacology, Biochemistry and Behavior 166 (2018) 27–34 considered a neurodevelopmental model for the study of depression It is important to mention that serotonergic system is critical in that has high face validity (Willner and Mitchell, 2002). Depression is early neurodevelopment due to its involvement in neuronal prolifera- induced by administering CMI, a tricyclic antidepressant that inhibits tion, migration, differentiation and synaptogenesis (Deneris and serotonin and noradrenaline reuptake during the early postnatal stage Gaspar, 2017). Early in life, at the 12-postnatal day (PN), SERT is also (8–21 PN). This pharmacological manipulation induces several beha- expressed in non-serotonergic neurons from thalamus, somatosensory vioral and physiological alterations that are analogous to the sympto- cortex and corticolimbic structures matology observed in major depression disorder (Feng and Ma, 2003; An increased in SERT expression occurs in hippocampus to regulate Justel et al., 2011; Vijayakumar and Meti, 1999; Vogel et al., 1990a; process such as neurogenesis neutrophil formation, axon myelination Yannielli et al., 1998). Male rats treated in early postnatal stage with and synaptogenesis (Glover and Clinton, 2016; Millard et al., 2017). CMI show hyperactivity (Hartley et al., 1990; Hilakivi et al., 1984; Moreover, serotonergic system and adrenergic are involved in sexual Mirmiran et al., 1983; Yannielli et al., 1999), abnormalities in sleep differentiation and in HPG axis development and the hypothalamus is characterized by a shorter latency to REM and increased REM frag- radiated by the axons from the cell bodies of the raphe nuclei (Döhler, mentation, to mention only a few effects (Savelyev et al., 2012; Vogel 1991; Jarzab and Döhler, 1984). In addition, noradrenergic neurons of et al., 1990b). Male rats also exhibit less aggressive behavior (Vogel the brainstem finished the differentiation during embryonic period but et al., 1988), decreased pleasure- and reward-seeking behaviors (e.g. the formation of their neuronal terminals finished until the third week intracranial self-stimulation) (Vogel et al., 1990c), and cognitive al- of postnatal period (Murrin et al., 2007). Then, manipulation of neu- terations that may affect learning and memory (Bhagya et al., 2008). rotransmitter system during early life, especially serotonergic system, On the forced swimming test, CMI rats display greater immobility ac- could lead to several alterations in adulthood. companied by reduced active swimming behavior, and a possible al- Studies with humans, show that the exposition to antidepressants teration in the serotonergic system (Bhagya et al., 2008; Bonilla-Jaime during gestation and or postnatal period increased risk of low birth et al., 1998; Vázquez-Palacios et al., 2005; Velazquez-Moctezuma and weight, neonatal abstinence syndrome, cardiac defects and fine and Diaz Ruiz, 1992). In fact, observations show that CMI rats exhibit al- gross motor alterations (Belik, 2008; Oberlander et al., 2010). Some terations in neurotransmission, especially in the serotonergic, dopami- longitudinal and retrospective studies show motor alterations, presence nergic and noradrenergic systems (Andersen et al., 2002; Bhagya et al., of anxiety, low social and emotional behaviors, higher risk of autism 2011; Hansen and Mikkelsen, 1998; Yavari et al., 1993). spectrum disorder and attention deficit hyperactivity disorder in chil- Early postnatal CMI administration also induces abnormalities in dren (Boukhris et al., 2016; Figueroa, 2010; Hanley et al., 2015); while male sexual behavior. Although CMI rats display more mounts, the in early adolescence increased the risk of autism spectrum disorder and number of intromissions and ejaculations tends to diminish. Additional depression (Harrington et al., 2014; Malm et al., 2016). Nevertheless, findings include increased latencies to mounting, intromitting and clinical data about the effect of early exposition to antidepressant on ejaculating, and a lower post-ejaculation latency, all of which are in- reproductive parameters is unknown. dicators of motivation (Bonilla-Jaime et al., 1998; Feng et al., 2001; Some animal studies reported that perinatal exposure to fluoxetine Neill et al., 1990; Vogel et al., 1996). Moreover, CMI rats have been (SSRI, a selective serotonin reuptake inhibitor) seems to foster pro- seen to spend less time in proximity to a female incentive, a finding that ceptive and receptive behaviors in young females (Rayen et al., 2014), corroborates the lack of sexual motivation (Limón-Morales et al., 2014). although exposure during gestation and lactation seems to produce a These negative effects on male sexual behavior and performance and on delay in reaching puberty (Dos Santos et al., 2016). Hence, it is possible the motivational component are reflected in a reduced libido and the that female rats treated with CMI in the early postnatal stages develop failure to reach orgasm and ejaculation (Hendrick et al., 2000). It is alterations in these physiological and behavioral reproductive para- important to mention that most studies that have used the early post- meters, given that males treated with CMI in early stages show a di- natal CMI administration model have involved only male rats. Female minished capacity for pleasure-seeking behaviors such as sexual activity rats treated with CMI in early life have exhibited anxiety-like behaviors (Bonilla-Jaime et al., 1998; Feng et al., 2001; Neill et al., 1990; Limón- and reduced levels of monoamines in the limbic region (Andersen et al., Morales et al., 2014). Thus, the aim of this study was to evaluate the 2002), but there are no reports on the effects of early postnatal CMI effect of the early postnatal CMI administration on the estrous cycle and administration on pleasure-seeking behaviors in females like sexual female sexual behavior. behavior. Female sexual behavior involves a series of behavioral, interactional 2. Materials and methods and postural adjustments that are classified as proceptive and receptive (Beach, 1976; Erskine, 1989; Sakuma, 1995). Proceptive behaviors are 2.1. Animals and early postnatal clomipramine treatment defined as a complex series of appetitive activities that estrous females display to encourage the male to mate. They include hopping and Six pregnant Wistar rats were obtained from the vivarium at the darting (Beach, 1976; Erskine, 1989), and indicate the intensity of Universidad Autónoma Metropolitana. Three days after delivery, which sexual motivation (Uphouse, 2014). Receptive behaviors, meanwhile, is not a late period of adoption (Barbazanges et al., 1996; Darnaudéry involve the postural changes that are necessary and sufficient to allow et al., 2004), the female pups were separated from their biological copulation with a potent male. This is also known as lordosis behavior mothers and lodged with foster mothers, with the aim the litter size was (Beach, 1976; Fabre-Nys et al., 2003). same (n = 6) (Bonilla-Jaime et al., 1998, 2003; Neill et al., 1990; Sexual behavior and ovulation is regulated by the HPG axis and Vázquez-Palacios et al., 2005; Vogel et al., 1990a, 1990b). Since ma- serotoninergic system. In fact, lordosis reflex depends on estrogens ternal care differs by sex of the pub, male pups were set aside for a priming while progesterone is required for procreativity and to facil- different experiment and cross-fostered with diff erent mothers to those itate the response of lordosis (Blaustein, 2008; Erskine, 1989; Ogawa used for the females (Moore and Chadwick-Dias, 1986; Richmond and et al., 1994), while the serotonergic system exert a dual action on sexual Sachs, 1984). From postnatal (PN) day 8–21, the female pups in the behavior (facilitation or inhibition) through different signaling path- experimental group (CMI) were treated with clomipramine (15 mg/kg; ways (serotonergic receptors family and serotonin transporter (SERT) 0.1 ml, sc, twice per day). This dose was chosen due to its effectiveness (Angoa-Pérez and Kuhn, 2015; Uphouse, 2014, 2000), where some in producing the behavioral and physiological abnormalities required drugs which increased serotonin levels inhibits proceptive and re- for the study protocol (Andersen et al., 2002; Bonilla-Jaime et al., 1998; ceptive responses (Uphouse, 2014, 2000). Additionally, hormonal fa- Hartley et al., 1990; Vázquez-Palacios et al., 2005; Velazquez- cilitation of lordosis behavior involves the expression of adrenergic Moctezuma et al., 1993; Vogel et al., 1990a, 1990b). The female pups in receptors in preoptic area and hypothalamus (Etgen, 2003). the control group received subcutaneous injections of saline in the same

28 T. Molina-Jiménez et al. Pharmacology, Biochemistry and Behavior 166 (2018) 27–34 volume, also twice daily (09:00 and 21:00 h). At PN day 25, the pups Spiteri et al., 2012). Males were alternated in order to avoid ejacula- were separated from their foster mothers and housed in groups (6 rats tion. During testing, the incidence of hopping and darting behaviors per cage from the same treatment group) in Plexiglas boxes was evaluated before the female received 10 mounts from the male. (45 cm × 30 cm × 30 cm) under a reversed 12/12 h light/dark cycle Hopping and darting were taken as indicative parameters of pro- (lights on 20:00–8:00 a.m.) with ad libitum access to food and water. At ceptivity (Madlafousek and Hlinak, 1977), while lordosis behavior was the age of three months, the two groups were subjected to the different taken to indicate receptivity. Lordosis was defined as the act of a female experimental manipulations. All procedures were performed in strict lowering her back while raising her tail to expose her genitals and so accordance with the Official Mexican Standards, NOM-062-ZOO-1999 facilitate insertion of the penis (Felicio et al., 1989; Morali and Beyer, (2001) and the National Institute of Health Guide for the Care and Use 1979). This behavior was assessed by determining lordosis intensity, of Laboratory Animals (NRC, 2011). and lordosis quotient (LQ = number of lordosis/10 mounts). Intensity was scored on the 3-point scale proposed by Hardy and Debold (1971) 2.2. Experimental design where a) 0 = no lordosis; b) 1 = marginal lordosis, characterized by slight flexing of the spine and raising of the head and hips; c) To evaluate the effect of the early postnatal CMI administration on 2 = normal lordosis, which consists in flexion of the spine and head to the reproductive behavior and physiology of these female rats, we an angle of approximately 30° from the floor with the front paws placed analyzed their sexual behavior and estrous cycles. Vaginal smears were slightly forward and hind legs stiffly straightened; and, d) 3 = ex- taken from the rats in both groups (CTRL: n = 10; CMI: n = 10) daily in aggerated lordosis, characterized by pronounced spinal flexion with the the morning (at the same hour) for 27 days to determine the phases of head at an angle ≥ 45 from the floor. All tests were videotaped for later the estrous cycle. The first 7 days served as a habituation period to analysis of the sexual behavior of all subjects. eliminate the influence of handling. We then continued to monitor the estrous cycle daily in the morning (at the same hour) for 20 days in both 2.6. Statistical analysis experimental groups. A week after finalizing the analysis of these smears, we evaluated The estrous cycle was analyzed using a Student's t-test to compare female sexual behavior in a circular arena. Initially, a sexually-experi- the CTRL and CMI groups in terms of the number of days in the estrous enced male was placed in the arena for 5 min for acclimation. After this and diestrus phases and the number of estrous cycles experienced 5-min habituation period, each female rat in the estrous phase was during 20 days. The proportion of rats that presented 1–5 regular or placed in the arena with the male and their proceptive (hopping, extended cycles in 20 days was evaluated with a Chi-square test. darting) and receptive (lordosis) behaviors were scored every 10 Proceptive behaviors (hopping, darting) and the LQ were also mounts by the male. analyzed by a t-test, but lordosis intensity was assessed with a two-way ANOVA. The factors were the early postnatal treatment (CTRL, CMI), 2.3. Drugs and type of lordosis (0: no lordosis; 1, marginal lordosis; 2, normal lordosis; 3, exaggerated lordosis). When p values reached < 0.05, a Clomipramine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) Student-Newman-Keuls post hoc test was used to determine the source was dissolved in a freshly-prepared 0.9% saline solution and injected of significance. Results are presented as mean ± 1 standard error subcutaneously at a volume of 0.1 ml/weight. (SEM). All analyses were performed with the SigmaPlot 10 pack.

2.4. Vaginal cytology 3. Results

To evaluate estrous cyclicity, we obtained daily vaginal smears. 3.1. Estrous cycle Vaginal secretions were collected 2 h after the onset of the dark period, under red light (40 W). Samples were collected with a glass medicine Analysis of the estrous cycles revealed between-group differences in dropper filled with saline solution (NaCl 0.9%) by inserting the tip the mean cycles [t = 2.84, 18 gl, p < 0.01]. The CMI group presented gently –not deeply– into the rat's vaginal orifice. All smears were placed fewer estrous cycles (3.60 ± 0.16) than the CTRL group (4.2 ± 0.13) on microscope slides and observed under light microscopy with 10× during the 20-day period (Fig. 1). Also, early postnatal CMI treatment and 40× objective lenses (Marcondes et al., 2002). Four phases were reduced the number of days of vaginal cell cornification –indicative of determined according to vaginal cytology: proestrus (round, nucleated the stage of estrous– compared to CTRL [t = 2.34, 18 gl, p < 0.03], no cells); estrus (cornified cells); metestrus or diestrus I (mixture of round, nucleated cells, cornified cells and leucocytes); and diestrus or diestrus II (predominance of leucocytes) (Freeman, 1988). The estrous cycles were then classified as follows: a) regular, characterized by 4 or 5 days with either 1–2 days of estrous or 2–3 days of diestrus; b) extended, characterized by the presence of 3–4 days of estrous or 4–5 days of diestrus; and 3) abnormal, characterized by over 4 consecutive days of estrous or 6 days of diestrus (Goldman et al., 2007).

2.5. Behavioral testing

2.5.1. Female sexual behavior Testing of sexual behavior was performed in a circular Plexiglas arena (diameter: 50 cm; height: 50 cm) during the dark phase between 12:00 and 15:00 h. The testing room was dimly lit in red (40 W). To evaluate the female rats' sexual behavior, we used sexually-experienced males that were allowed to explore the arena for 5 min with no female Fig. 1. Effect of neonatal CMI treatment on the number of estrous cycles during a 20-day present. Females in the estrous phase (determined by the vaginal period. A decrease in the average of number of cycles occurred in the 20 days of vaginal smears) were then placed in the arena and the males were allowed to cytology analysis due to neonatal CMI treatment. *p < 0.05 compared to the control perform 10 mounts (Beyer et al., 1995; Sánchez-Montoya et al., 2010; group. Student's t-test. Mean ± SEM, CTRL: control; CMI: clomipramine.

29 T. Molina-Jiménez et al. Pharmacology, Biochemistry and Behavior 166 (2018) 27–34

Fig. 2. Average number of days in the estrous and diestrus stages during a 20-day period in female rats treated in early life with CMI. Evaluation of vaginal cytology shows that neonatal administration of clomipramine decreased the number of days in the estrous phase (A), but did not affect the days in vaginal diestrus (B). Student's t-test. *p < 0.05 compared to the control group. Mean ± SEM, CTRL: con- trol; CMI: clomipramine.

Fig. 3. Effect of neonatal CMI administration on the cycli- city of adult female rats during a 20-day period. A) A higher proportion of rats treated with CMI had two or three reg- ular cycles, while a lower proportion presented four regular cycles. Most of the rats in the CTRL group had four regular cycles. B) The analysis of extended cycles shows that a higher proportion of CMI rats had one extended cycle in the 20-day period than in the CTRL group. Chi-square test. *p < 0.05 compared to the control group. CTRL: control; CMI: clomipramine; 5C, 4C, 3C, 2C, 1C: indicates the number of estrous cycle presented in 20-day period.

between-group modifications were observed in the number of days in groups [t = 4.90, 18 gl, p < 0.001]. Here, the CMI rats showed a lower the diestrus stage [t = −1.41, 18 gl, p < 0.17] (see Fig. 2). LQ than controls, as they achieved a LQ of 100 (see Fig. 5). Fig. 3 shows the proportion of subjects that had regular or extended The analysis of lordosis intensity revealed differences between cycles during the 20-day period of smear analysis. When the estrous treatments (CTRL vs CMI), but these did not reach the level of statistical cycle was classified as regular and extended, we found that 40% of the significance [F(1, 72) = 0.063, p < 0.80, NS]. However, statistically- rats exposed to an early postnatal treatment of CMI had two regular significant differences did exist with respect to the type of lordosis [F(1, cycles, but that none of the female rats in CTRL presented only 2 cycles. 72) = 10.19, p < 0.001]. Moreover, the interaction of early postnatal The other 40% of the CMI rats had 3 regular cycles, compared to only treatment × lordosis was significant [F(1, 72) = 25.17, p < 0.001]. The 10% of the rats in the CTRL group (Xi = 22.42, p < 0.001). Also, only post hoc test indicated that most of the CMI rats failed to display lordosis 20% of the CMI rats had four regular cycles, though most of the control behavior when the male rat mounted; indeed, when those female rats rats (80%) presented four regular cycles (Xi = 69.62, p < 0.001). The showed lordosis, it was generally of the marginal or normal type (1, 2; rest of the CTRL females (10%) had five regular cycles, but no CMI rat p < 0.05). In contrast, the CTRL rats displayed more normal lordosis presented this during the 20-day period of smear analysis. (2) and, though to a lesser extent, also marginal and exaggerated lor- Regarding extended cycles (Fig. 3b), 80% of the rats exposed to an dosis (1, 3; p < 0.05) (see Fig. 6). early postnatal administration of CMI presented one extended cycle, compared to 20% of the CTRL rats (Xi = 69.62, p < 0.001). 4. Discussion

3.2. Behavioral measures Our findings are the first to show that early postnatal administration of CMI produces long-term alterations in the estrous cyclicity and re- 3.2.1. Sexual behavior productive behavior on female rats. The estrous cycle is a repetitive Fig. 4 shows the effect of early postnatal CMI treatment on female process characterized by changes in the levels of estradiol and proges- proceptive behavior. The t-test indicated that the CMI females displayed terone secreted by the ovarian follicles that can be detected by the less darting [t = 3.37, 18 gl, p < 0.003, Panel A] and hopping presence or absence of different cells in vaginal cytology analyses (Cora [t = 2.69, 18 gl, p < 0.01, Panel B] than CTRL. et al., 2015). Normally, it lasts for 4–5 days (Marcondes et al., 2002; The lordosis quotient (LQ) also differed between the experimental Westwood, 2008), but modifications in the reproductive function might

30 T. Molina-Jiménez et al. Pharmacology, Biochemistry and Behavior 166 (2018) 27–34

Fig. 4. Effect of neonatal CMI treatment on proceptive be- haviors. Rats treated with CMI displayed less A) hopping; and, B) darting, behavior during ten mounts performed by the male. Student's t-test. *p < 0.05. Mean ± SEM, CTRL: control; CMI: clomipramine.

controls. No changes were seen in the total number of days of diestrus due to the early postnatal CMI treatment in the 20-day period. Cycles were classified as regular and extended because we did not detect any changes in the total number of days of diestrus caused by the early postnatal CMI treatment during those 20 days. We did find, however, that most CMI rats had two or three regular cycles characterized by the presence of 4–5 days with either 1–2 days of estrous or 2–3 of diestrus (Goldman et al., 2007). Also, a high pro- portion of those rats had at least one extended cycle during the 20-day period of smear analysis. In contrast, most of the CTRL rats had 4 consecutive regular cycles during the period. Previous studies have reported that the offspring of mothers exposed to perinatal fluoxetine administration (from mating through pregnancy and weaning) tend to have longer cycles with a prolonged estrous stage, a greater number of

Fig. 5. Effect of neonatal CMI treatment on the lordosis quotient (LQ) in adult female rats. follicles in the ovary, and an alteration that consists in the presence of The females treated with CMI showed a lower lordosis quotient. Student's t-test. genes that regulate serotonin signaling and action in the ovary (Moore *p < 0.05 compared to the control group. Mean ± SEM, CTRL: control; CMI: clomi- et al., 2015). Finally, no modifications in estrous cyclicity were detected pramine. due to gestational and lactational exposure to fluoxetine (Dos Santos et al., 2016). Thus, our results indicate that CMI treatment in early life disrupts the estrous cycle by increasing the presence of extended cycles, decreasing the total number of vaginal estrous days and shrinking the cycle itself. Since this cycle is regulated by hormones, when it was analyzed provided an index of the functional status of the hypotha- lamic-pituitary-ovarian axis (Goldman et al., 2007). Thus, the extended cycle observed in the CMI rats consisted in the presence of a persistent diestrus stage characterized by low plasma hormone concentrations (Butcher et al., 1974; Feder, 1981; Freeman, 1988). These results sug- gested a possible alteration at the HPG level since serotonergic system is involved in the development of this axis. For instance, serotonergic system has an important participation in developmental due to its in- volvement in cell division, migration, differentiation and synaptogen- esis. It helps to build the proper wiring of circuits in the brain such as thalamocortical, corticolimbic, and somatosensory circuits as well as HPG axis (Deneris and Gaspar, 2017; Millard et al., 2017). Some reports Fig. 6. Effect of neonatal CMI treatment on the intensity of lordosis in adult female rats. indicated that a stimulation of the synthesis of serotonin during post- The rats treated with CMI had more events in which they failed to display lordosis. Also, natal period inhibited the expression of females and male sexual be- the CMI rats had fewer displays of marginal (1) or normal (2) dorsal flection than con- havior in adulthood (Döhler, 1991; Jarzab and Döhler, 1984). In short, trols, which displayed normal lordosis (2) more often and had fewer events of marginal fl (1) or exaggerated (3) lordosis. ANOVA followed by Student-Newman-Keuls post hoc test, animals exposed to uoxetine or citalopram during gestation or post- *p < 0.05 compared to controls; **p < 0.05 compared to 1, 2, 3; &p < 0.05 compared natal period had a reduction in SERT expression in raphe nuclei, hy- to 3; +p < 0.05 compared to 0, 1, 3; °p < 0.05 compared to 0. Mean ± SEM, CTRL: pothalamus hippocampus, amygdala and cortex in adulthood (Bock control; CMI: clomipramine; 0: no lordosis; 1: marginal lordosis; 2: normal lordosis; 3: et al., 2005). Furthermore, a postnatal exposition to reboxetine (selec- exaggerated lordosis. tive norepinephrine reuptake inhibitor) increased the expression of SERT in frontal cortex. Also, perinatal exposition to fluoxetine in- occur due to the administration of drugs or chemicals (Goldman et al., creased the expression of 5-HT1A receptor, tryptophan hydroxylase 2007; Yuan and Foley, 2002). This study found that early postnatal CMI (Tph) and Ets transcription factor of Pet-1 in the ovary, which indicates treatment (8–21 PN days) caused alterations in the estrous cycle; al- and increasing of serotonergic innervation and signaling in the ovaries terations that were manifested as a reduced presence of estrous cycles caused by the fluoxetine exposure. Moreover, an increased in the ex- with a decrease in the number of vaginal estrous days compared to pression of beta estrogen receptors (ERβ) was found as well as changes

31 T. Molina-Jiménez et al. Pharmacology, Biochemistry and Behavior 166 (2018) 27–34 in the mRNA levels of clock gene. These evidences reflected an altera- the hypothalamus and preoptic area is necessary to facilitate the effect tion in the estrous cycle, an increased in follicles, the presence of of estradiol and progesterone on lordosis reflex and luteinizing hor- apoptosis in ovarian cells and a dysregulation in local ovarian circadian mone during preovulatory stage, while a diminution in the nora- regulation (Moore et al., 2015). Then, it seems that serotonergic system drenaline synthesis, turnover or receptors binding inhibit both process, manipulation during early life caused inappropriate environmental lordosis and luteinizing hormone surge (Etgen et al., 1999, 1992; Kalra conditions, which could be reflected in long-term alterations. Hence, it and Kalra, 1984). In addition, female sexual behavior is also influenced is possible that the CMI rats suffered an alteration in the hypothalamic- by the serotonergic system and it appears that the regulatory influence pituitary-ovarian axis that was reflected in a disruption of the estrous depends of the signaling pathway activated. For example, serotonergic cycle. agonist acting in 5-HT1A receptors, exerts an inhibitory effect on pro- We also evaluated female sexual behavior, finding that, even when ceptive and lordosis behavior while activation of 5-HT2A/2C receptors the CMI rats were in the estrous stage, they presented less hopping and facilitate lordosis reflex (Guptarak et al., 2010; Uphouse, 2000, 2014). darting behavior than controls. This means that the CMI rats exhibit This dual effect of serotonin has been explained as a mechanism of fewer proceptive behaviors, which facilitate sexual interaction and coordination of lordosis with other physiological events that allows consist in the expression of patterns of approaches to, and withdrawals sexual receptivity occur in optimal condition in order to reproduction from, a male (Beach, 1976; Erskine, 1989). This suggests a reduction in succeed (Uphouse, 2000). In this aspect, our results suggest that early the motivational component of sexual behavior, since approach pat- postnatal CMI treatment likely triggered alterations not only in the terns occur as a consequence of the intensity level of sexual incentive production and response of the steroid hormones but also in nora- motivation (Uphouse, 2014). It is well known that progesterone and drenaline and serotonin signaling which are involved in regulating the estrogen act sinergically in the regulation of the female sexual behavior reproductive function at the central and periphery levels in female rats, (Rubin and Barfield, 1983; Glaser and Barfield, 1984). But is proges- hypothesis that need to be explore. terone which mediates the precopulatory solicitational behavior and Interestingly, some research has found that rats treated with CMI triggers the lordosis reflex (Erskine, 1989). Moreover, progesterone present hyperactivity in the HPA axis, and that the reactivity of this axis interacts with the serotonin system by reducing extracellular con- is disrupted (Bonilla-Jaime et al., 2003, 2010; Prathiba et al., 1998). In centration of serotonin in mediobasal hypothalamus during the pro- addition, activation of the HPA axis promotes an increase in serum gress towards a sexually receptivity (Uphouse, 2000). Then, it is pos- glucocorticoid concentrations that causes an inhibitory effect on the sible that those circuits involved in the regulation of perceptive HPG axis and reduces reproductive success (Geraghty and Kaufer, behavior were disrupted by the early administration of CMI. Further- 2015). For instance, through activation of the CRH and glucocorticoid more, CMI rats also had a lower LQ than the CTRL rats, and usually receptors expressed in GnRH neurons, glucocorticoids inhibit the failed to display lordosis every time that the male attempted to mount. synthesis and release of GnRH (Bowe et al., 2008; Calogero et al., 1999; Therefore, early postnatal CMI administration not only diminished the Whirledge and Cidlowski, 2010). Also, increased CRH levels lengthened motivational component of female sexual behavior, but also the re- the ovulatory phase and inhibited the LH surge (Breen et al., 2012; ceptive or consummatory behavior, which entails postural changes that Cates et al., 2004), while an increase of corticosterone inhibited the facilitate copulation (Beach, 1976; Fabre-Nys et al., 2003). synthesis of testosterone, estradiol and progesterone (Geraghty and Our results are consistent with those reported for male rats treated Kaufer, 2015; Whirledge and Cidlowski, 2010). Moreover, glucocorti- during early postnatal period with CMI, which show impaired sexual coids are necessary for follicular development, although high con- behavior performance (Bonilla-Jaime et al., 1998, 2003; Feng et al., centrations can lead to ovarian dysfunction (Dorfman et al., 2003; 2001; Neill et al., 1990) accompanied by reduced sexual motivation. In Geraghty and Kaufer, 2015). male rats, administering dihydrotestosterone and estradiol reverted the Together these data support the hypothesis that the disruption of abnormalities observed in their sexual behavior. These findings suggest estrous cyclicity and impairment of sexual behavior may be the con- a possible alteration of the hormonal system (Limón-Morales et al., sequence of an alteration in the HPG axis caused by a prolonged in- 2014). Other studies have reported that developmental exposure to crease of glucocorticoids produced by HPA hyperactivity and also by fluoxetine (1–28 PND) facilitates proceptive and receptive behaviors in possible alteration in serotonergic and noradrenergic systems (neuro- adult female rats (Rayen et al., 2014), but that exposure to this drug transmitter synthesis and signaling pathways), especially in cerebral during gestation and lactation delayed the onset of puberty, though structures involved in sexual behavior and in periphery organs. without disrupting reproductive cyclicity (Dos Santos et al., 2016). Thus, it may be that the differing e ffects observed on reproductive cy- 5. Conclusion clicity and sexual behavior may be due to the period of administration, dosage, and the type of antidepressant. The present findings provide the first evidence of the effects of the The estrous cycle requires the sequential secretion of estradiol and early postnatal administration of CMI on the reproductive function of progesterone (Feder, 1984; Pfaust et al., 2015). During this cycle, these adult female rats. Our data suggest that early postnatal CMI treatment two steroid hormones interact synergically to control the onset, quality caused a disruption of the estrous cycle manifested in fewer estrous and duration of female sexual behavior (Blaustein, 2008; Feder, 1984; cycles, fewer days in the estrous stage, and the presence of extended Mani et al., 1994; Olster and Blaustein, 1988; White and Uphouse, cycles, accompanied by reductions in the proceptive and consummatory 2004). Also, sexual behavior is completely absent in ovariectomized components of female sexual behavior. rats, but can be re-established by priming with physiological doses of estrogens followed by progesterone (Blaustein and Feder, 1979; Acknowledgements McEwen et al., 1979; Pfaust et al., 2015; Whalen, 1974). There is no doubt that sexual behavior is highly regulated by HPG axis, never- During this research, TMJ (CVU: 209784) received a scholarship theless, other factors are involved. For instance, the skin hairy stimu- from the program Estancia Posdoctoral Vinculada al Fortalecimiento de lation of the flanks, rumb, tailbase and perineum by the mounting of the la Calidad del Posgrado Nacional (COVIA-0890-2016) granted by male play a crucial role in triggering the lordosis reflex which is fa- Mexico's Consejo Nacional de Ciencia y Tecnología (CONACyT). cilitated by the presence of estrogen and synergized by progesterone (Kow and Pfaff, 1976; Kow et al., 1979; Pfaff et al., 1977), then it is References possible that CMI rats had a lower response to somatosensorial stimu- lation during the mounting performed by the male, perhaps by lower American Psychiatric Association, 2013. Diagnostic and Statistical Manual of Mental levels of steroid hormones. Moreover, an increase of noradrenaline in Disorders, fifth ed. (Arlington, Virginia).

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