The effects of the estrus cycle and citalopram on anxiety-like behaviors and c-fos expression in rats

Pharmacology, Biochemistry and Behavior 124 (2014) 180–187

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The effects of the estrus cycle and citalopram on anxiety-like behaviors and c-fos expression in rats Aslıhan Sayin a,⁎, Okşan Derinöz b, Nevzat Yüksel a, Selda Şahin c, Hayrunnisa Bolay d a

Psychiatry Department of Gazi University Hospital, Ankara, Turkey Pediatric Emergency Department of Gazi University Hospital, Ankara, Turkey Psychiatry Department of Uzunköprü State Hospital, Edirne, Turkey d Neurology Department of Gazi University Hospital, Ankara, Turkey b c

a r t i c l e

i n f o

Article history: Received 17 February 2014 Received in revised form 30 May 2014 Accepted 7 June 2014 Available online 13 June 2014 Keywords: Estrus cycle Ultrasonic vocalization Citalopram Elevated plus maze c-fos expression

a b s t r a c t In rats, hormonal fluctuations during the estrus cycle may have numerous behavioral and neurobiological consequences. The aim of this study was to investigate the effects of estrus cycles and citalopram on behavior, ultrasonic vocalizations, anxiety levels, and c-fos expression in rats. With this aim, the rats were grouped into two: (1) a control group (n = 16) and (2) a citalopram group (n = 16), which received daily intraperitoneal 20 mg/kg citalopram from baseline (D0) to the 10th day (D10). Behavioral analysis and ultrasonic vocalization (USV) recordings were made on D0 and D10. Next, the rats were further subgrouped according to estrus phases identified through a vaginal smear (8 proestrus rats and 8 non-proestrus rats, in each group). The rat's anxiety levels were analyzed with an elevated plus maze (EPM), and their c-fos expression was measured at the cingulate cortex, the amygdala, and the paraventricular thalamic nucleus. Our results showed that the citalopram group showed significantly more grooming behaviors on D10 than the control group (p = 0.002). USVs on D0, D10 and during the EPM did not show any significant differences between the groups. Proestrus rats in the control group showed significantly less anxiety-like behavior during the EPM than the non-proestrus rats in the control group (p = 0.028 for time spent in open arms, and p = 0.011 for entries into open arms). There was no significant difference in anxiety-like behavior between the control and citalopram groups, and between the proestrus and non-estrous rats in the citalopram group. C-fos expression at the amygdala (p = 0.013) and the paraventricular thalamic nucleus (p = 0.014) was significantly inhibited in the citalopram group. We concluded that estrus cycles have a significant effect on anxiety levels in rats, which may be suppressed behaviorally and neurobiologically by citalopram. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Understanding the relationship between the estrus cycle and anxiety is important. Emotional and behavioral symptoms during the premenstrual period can significantly decrease the quality of life of many premenopausal women, as in premenstrual syndrome (PMS) and premenstrual dysphoric disorder (PMDD) (Dennerstein et al., 2012). Biological theories about the etiology of PMS/PMDD include dysregulation of the serotonergic and GABArgic systems, declining levels of ovarian steroid hormones during the late luteal phase of the cycle, endogenous opioid activity in the late luteal phase, and genetic factors (Rapkin and Akopians, 2012). Based on these theories, withdrawal from progesterone and estradiol has been used as an animal model to provoke anxiety and behaviors analogous to PMS and PMDD in rats (Gallo and Smith, 1993; Löfgren et al., 2009). The estrus cycle of rats ⁎ Corresponding author at: Psychiatry Department of Gazi University Hospital Beşevler, Ankara 06500, Turkey. Tel.: +90 532 5840438; fax: +90 312 2025412. E-mail address: [email protected] (A. Sayin).

http://dx.doi.org/10.1016/j.pbb.2014.06.002 0091-3057/© 2014 Elsevier Inc. All rights reserved.

includes four phases: estrus, metestrus, diestrus, and proestrus. The highest levels of progesterone and estradiol occur during the proestrus (Marcondes et al., 2002). Natural fluctuations of progesterone and estradiol that occur during the estrus cycle in rats can be used to study recurrent changes in mood, anxiety levels, motivation and motor behaviors that are closely related to premenstrual symptoms in humans (Löfgren et al., 2009). Estradiol and progesterone peak during the proestrus and cause many physiological changes such as increased physiological activity (Kawakami et al., 1970), enhanced plasticity (Teresawa and Timiras, 1968), facilitated long term potentiation (Korol et al., 1994; Warren et al., 1995), and greater synaptic density in the hippocampus (Gould et al., 1990; Woolley and McEwan, 1992). This peak also causes some significant behavioral changes including cognitive enhancement (Frye et al., 2007; Paris and Frye, 2008; Shors et al., 1998; Walf et al., 2006), anti-anxiety-like behaviors (Diaz-Veliz et al., 1997; Frye et al., 2000; Frye and Walf, 2002; Marcondes et al., 2001; Mora et al., 1996), and anti-depression-like behaviors (Pare and Redei, 1993; Marvan et al., 1996). The estrus cycle in rats has also been used to study recurrent

A. Sayin et al. / Pharmacology, Biochemistry and Behavior 124 (2014) 180–187

changes in motor behaviors (Chen et al., 2009; Nin et al., 2012), and substance consumption motivation for alcohol (Roberts et al., 1998; Ford et al., 2002) and for cocaine (Lynch et al., 2000; Feltenstein and See, 2007; Kerstetter et al., 2011). Behaviors induced by these drugs and closely related to cyclic hormonal changes (Quinones-Jonab et al., 1999; Sell et al., 2000) have also been examined. Selective serotonin reuptake inhibitors (SSRIs) are the best established treatment for both PMS and PMDD, whether taken in the luteal phase only or continuously. There is no clear evidence of a difference in effectiveness related to the mode of administration of this treatment (Marjoribanks et al., 2013). SSRIs have been tested for their antidepressant-like and anxiolytic-like effects in animal models, with conflicting results for anxiety (Borsini et al., 2002) but with possible antidepressant-like effects (Pollak et al., 2010). In addition, the anxiolytic-like effects of psychotropic medications may show significant differences at different stages of the estrous cycle (Fernandez-Guasti et al., 1999; Molina-Hernandez et al., 2013). In particular, an increased sensitivity to anxiolytic drugs has been observed during the proestrus stage (Bitran and Dowd, 1996; Molina et al., 2002). Understanding the relationship between the estrus cycle and anxiety, and the effects of SSRIs on this relationship could help us to understand the etiology and treatment of PMS and PMDD, which cause significant disability for many women. Animal studies in this area have repeatedly examined the relationship between the estrus cycle and anxiety-like behavior. But to our knowledge, there have been no studies on the effects of the estrus cycle on ultrasonic vocalizations and c-fos expression. In this study, we aimed to investigate the possible effects of the estrus cycle on rat behavior, anxiety levels, ultrasonic vocalizations and c-fos expression at the amygdale, the cingulated cortex and the paraventricular thalamic nucleus (the primary brain regions related to anxiety), and the effect of citalopram, a well-known SSRI, on these parameters. We had two hypotheses: (1) rats in the proestrus phase of the estrus cycle would show less anxiety-like behavior than rats in other phases of the cycle, probably due to high estradiol and progesterone levels; (2) citalopram would decrease anxiety-like behaviors related to estrus phases. 2. Materials and methods 2.1. Subjects This study was performed in the Neuroscience Laboratory of Gazi University. Ethical approval was obtained from Gazi University Animal Studies Ethical Committee. Thirty-two adult Wistar female rats weighting 200–350 g were used. All the rats were obtained from the Laboratory Animals Breeding and Research Center of Gazi University, and brought to the Neuroscience Animal Research Laboratory of Gazi University. They were handled for weighing and housed, 2–3 animals per cage, as soon as they were brought to the Neuroscience Laboratory, and then left untouched for 3 days of habituation. During the entire experiment, the animals were kept in a closet with a controlled dark–light cycle so that the rats received daylight between 07:00 a.m. and 07:00 p.m. and were kept in darkness between 07:00 p.m. and 07:00 a.m. They received food (Korkuteli Food Industry, Turkey) and water ad libitum. The room temperature was held at 22 ± 2 °C and humidity at 55 ± 5%. The animals were grouped into two: (1) a control group (n = 16), that did not receive any kind of special treatment (such as saline injection) between the first day (D0) and the 10th day (D10); (2) a citalopram group (n = 16), that received daily intraperitoneal 20 mg/kg citalopram at 9:00–9:30 a.m. every morning between D0 and D10. Injections were consistently administered by the same researcher. The mean weight of the rats was 238.25 ± 2.99 (mean ± standard error). The mean weights of the controls (234.00 ± 3.80) and citalopram group (242.50 ± 4.49) showed no statistically significant differences (p = 0.184).

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2.2. Study design The entire study design is summarized in Table 1. The first day the animals were brought into the lab was considered D0. On D0 and D10 behavioral analyses and ultrasonic vocalization recordings were made, as described below. On D11, a vaginal smear was taken from each animal and all the animals were further sub-grouped in two according to the stage of their estrous cycle: (1) a proestrus group (n = 16): 8 animals from each group showed proestrus according to their vaginal smear; (2) a non-proestrus group (n = 16): 8 animals from each group showed estrus, metestrus, or diestrus according to their vaginal smear. In order to mitigate the possible effects of anxiety caused by the vaginal smear procedure on test performance, all the animals were tested using an elevated plus maze test 2 h after a vaginal smear. Two hours after the EPM test, the animals were decapitated and their brains were removed for immunohistochemistry. 2.3. Citalopram preparation The citalopram was a gift from the Abdi İbrahim Pharmaceutical Company (34467, Sarıyer, Istanbul, Turkey). It was dissolved in 0.9% deionized water, prepared immediately prior to use, and given intraperitoneally (i.p.) at a volume of 0.01 ml/g body weight. The daily dose was 20 mg/kg. This mode of administration and dosage was selected based upon previous dose–response studies (Golembiowska and Dziubina, 2000; Crowley et al., 2006; Wesolowska and Nikiforuk, 2008; Sowa-Kucma et al., 2011). 2.4. Behavioral analysis All behavioral experiments were performed between 07:00 a.m. and 07:00 p.m. On D0 and D10, general behavioral analysis was conducted by an observer who was blind to the treatment group, and also by the Laboratory Animal Behavioral Observation Registration and Analysis System (LABORAS®, Metris, The Netherlands). The animals were not habituated to the test-apparatus before baseline. Direct observation was made for 15 min by one of the authors, who recorded times and durations of behaviors in seconds. Behaviors noted included freezing, bilateral head grooming, right head grooming, left head grooming, body grooming, drinking, eating and sleeping. At the same time, rat behaviors were automatically recorded by a non-invasive behavioral analysis system. This system is recorded by a standard rat cage (polycarnonate/Makrolon type III cage, floor area 840 cm2, height 25 cm/height to food hopper 15 cm), fixed on a platform with several force-displacement transducers, connected to a personal computer (LABORAS®, Metris, The Netherlands). The platform detects and classifies behaviors using vibrations created by captive animals' movements like immobility, locomotor activity and rearing (Quinn et al., 2003, 2006). The rats were free to access food or water located in the standard rat cages, but had to rear to eat or to drink. All experiments were simultaneously recorded by a video-camera system in order to confirm data obtained from the automated analysis system, and to differentiate freezing periods from immobility. 2.5. Ultrasonic vocalization (USV) calls Many vertebrates use species-specific vocalizations to communicate information regarding mother-offspring interactions, mating, mood (fear, pain, distress, aggression, joy etc.), their next likely behavior (approach, avoid, groom) and environmental conditions (presence of predators or the location of food). This information is important to understand animal behaviors under laboratory conditions (Portfors, 2007). Adult rats primarily emit two types of USVs that can be distinguished on the basis of frequency at peak energy. Vocalizations typically referred to as ‘22-kHz vocalizations’ have frequencies between 18 and 32 kHz and a duration of 300–4000 ms and are emitted at a sound pressure level of 65–85 dB (Wohr et al., 2005). Rats emit 22-kHz vocalizations

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Table 1 Study design. Day

D0 D1-D9 D10 D11

Control group

Citalopram group

(n = 16)

(n = 16)

Behavioural analysis and ultrasonic vocalization (USV) call recordings Standard caring Behavioural analysis and ultrasonic vocalization (USV) call recordings Vaginal smear Elevated plus maze Brain removal for c-fos analysis

Behavioural analysis and ultrasonic vocalization (USV) call recordings Intraperitoneal 20 mg/kg/day citalopram injection Behavioural analysis and ultrasonic vocalization (USV) call recordings Vaginal smear Elevated plus maze Brain removal for c-fos analysis

in a number of aversive behavioral situations during distressing events and it is assumed that these sounds reflect a negative affective state (Portfors, 2007). The so-called ‘50-kHz vocalizations’ have a frequency at peak energy of 32–96 kHz, and they have a much shorter duration (30–50 ms). Sometimes 50 k-Hz vocalizations are referred to as ‘chirps’ because of their brief duration (Panksepp and Burgdorf, 2000). Rats emit 50 k-Hz vocalizations under nonaversive conditions, including sexual behaviors, play, and manual tactile stimulation (‘tickling’), and it has been suggested that these vocalizations are associated with positive affect for the animal (Portfors, 2007). These vocalizations are inaudible to humans without the use of specialized equipment. Ultrasonic sounds of laboratory animals within a range of 15–100 kHz can be monitored and analyzed by a USV detector system (Sonotrack®, Metris, The Netherlands). Sonotrack uses a hardware bandbass filter (10th order Butterworth filter) with sharp cutoffs at 15 and 100 kHz. This filter prevents aliasing and it also removes almost all environmental sounds. Data are presented without further filtering and smoothing. In Sonotrack, the dB (decibel) scale is relative to a 1 mV (RMS) signal. In the spectrogram, red indicates the strongest signal value (the 50 mV or 35.3 V RMS or 31 dB) and black indicates background noise (this is approximately 10 mV in Sonotrack or 7 mV RMS or 16 dB). The shifts in frequency at the beginning and the end of a vocalization are characteristic of biological sounds. In this study, we used Sonotrack to detect the USV calls of all the animals during D0 and D10 behavioral analysis, as well as during activity in the elevated plus maze. Detected sounds were then grouped into Band I (vocalizations between 18 and 32 kHz, related to distress) and Band II (vocalizations between 32 and 50 kHz, related to positive affect). 2.6. Vaginal smear Vaginal secretions were collected on D11 between 9 am and 11 am, with a plastic pipette filled with 20-μL normal saline (NaCl 0.9%) by inserting the tip superficially into the rat vagina. A drop of vaginal fluid was smeared on a glass slide, and the unstained material was observed under a light microscope, with a 40× phase-contrast objective. Three types of cells were identified: round and nucleated epithelial cells, irregular cells without nuclei or cornified cells, and small round cells or leukocytes. The proportions of these types of cells in a sample determine the estrous cycle phase: proestrus, estrus, metestrus, or diestrus (Marcondes et al., 2002). 2.7. The elevated plus maze The elevated plus maze (EPM) has been described by Pellow et al. (1985) as a simple method of assessing anxiety responses of rodents. The maze used in this study was made of Plexiglass and consisted of two open arms (50 cm long, 10 cm wide) and two closed arms (50 cm long, 10 cm wide, enclosed by 30 cm walls). Each arm is attached to plastic legs elevating it 50 cm off the floor. The animals were not habituated to the test-apparatus before the test. Animals were placed individually in the center of the maze, facing the same closed arm, and allowed 5 min of free exploration. A video camera was placed above

the system in order to record the behavior of the rats, and simultaneously, an observer recorded the number of entries and time spent in each arm. USV calls during this test were recorded by Sonotrack. The maze was thoroughly cleaned after each test with ethanol (70%) for both control and citalopram groups, and there were 10–15 min intervals between each test. Each rat was tested once. The final results were calculated in percentages (Walf and Frye, 2007):

Percentage of closed ðopenÞ arm entries ¼

Percentage of duration spent in closed ðopenÞ arm ðsecondsÞ

¼

Number of closed ½open arm entries  100 Number of total arm entries

Mean time spent in closed ½open arm  100 Total time spent in all arms

This test was performed on D11, 2 h after the vaginal smear (1 week after chronic stress) (see Table 1 for study design). 2.8. Immunohistochemistry (IHC) The animals were anaesthetized with a lethal dose of thiopental sodium 2 h after the EPM. They were perfused transcardially by heparinated saline, followed by a 4% 0.1 M paraformaldehyde solution. Their brains were prepared for c-fos IHC, and cerebral cortical sections taken of the whole cortex, 50 μm thick coronal sections from every 150 μm, were evaluated for c-fos immunoreactivity. Brain sections were incubated overnight with polyclonal rabbit antic-fos antibody (Santa Cruz Biotechnology, USA, SC-52, diluted 1:1000) as previously described in by Shehab et al. (2006, 2011). After rinsing in phosphate-buffered saline (PBS), sections were incubated for 1 h in biotinylated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories, USA, diluted 1:500), and then for another hour in extravidin–peroxidase conjugate (Sigma-Aldrich, Germany, diluted 1:1000). To make visible any c-fos immunoreactivity, sections were incubated for 8 min in a solution of 25 mg diaminobenzidine (DAB, Sigma) in a 50 ml 0.1 M phosphate buffer (PB, pH 7.4) with 7.5 μl hydrogen peroxide (30%) and 1 ml nickel chloride (3.5%) added to intensify the reaction. Finally, sections were rinsed in PB and mounted on gelatin-coated slides. After air drying, sections were dehydrated in graded alcohol, cleared in xylene and mounted with DPX. All antibodies were diluted in PBS containing 0.3% Triton. 2.9. Statistical analysis Statistical analysis was conducted using the SPSS 15.0 package program. Non-parametric tests were used. There was a normal distribution of the data. For comparison between groups, Mann–Whitney U analyses were made. For comparison within each group, Wilcoxon analysis was used. The Pearson correlation test was used to analyze the correlation between EPM performance and c-fos expression. A p value of less than 0.05 was considered statistically significant.

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3.1.1. Comparison between the groups (control X citalopram) On D0 the control and citalopram groups showed significantly different behaviors with regard to bilateral head grooming (p = 0.001) and drinking behaviors (p = 0.002). The citalopram group showed significantly more bilateral head grooming (85.87 ± 11.49 versus 38.93 ± 5.73), and drinking behaviors (2.37 ± 0.65 versus 0.31 ± 0.25) than the control group. On D10 the two groups showed significantly different behaviors with regard to bilateral head grooming (p = 0.01), right head grooming (p = 0.03), and body grooming (p = 0.002). The citalopram group showed significantly more bilateral head grooming (77.31 ± 12.51 versus 40.62 ± 4.90), right head grooming (17.75 ± 5.34 versus 2.25 ± 0.96), and body grooming (67.18 ± 15.58 versus 16.68 ± 6.51) behaviors than control group. 3.1.2. Comparisons within each group (D0 X D10) The control group showed significantly more body grooming behavior on D0 (mean ± standard error 50.37 ± 14.39) than on D10 (16.68 ± 6.51, p = 0.003). There was no statistically significant difference for the citalopram group. 3.2. Ultrasonic vocalizations

Fig. 1. Comparison of behavioral analysis at baseline (D0) and 10th day (D10) of two groups. *: p b 0.05, **: p b 0.01.

None of the animals in either group made USVs during D0 and the EPM. On D10, 2 subjects in the citalopram group made USVs at Band I (mean duration 0.07 s, Fig. 2), while none of the animals in the control group made USVs (Pearson χ2 = 2.133, Fisher's exact test p = 0.484). 3.3. The elevated plus maze

3. Results

The results of EPM are shown in Fig. 3.

3.1. Behavioral analysis Comparisons of behavioral analyses of the subjects on D0 and D10 were made by direct observation and by the Laboras, as shown in Fig. 1.

3.3.1. Comparison between the groups (control X citalopram) There was no statistically significant difference between the control and the citalopram groups with regard to the percentage of time spent

Fig. 2. Ultrasonic vocalizations.

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Fig. 3. Comparison of elevated plus maze performance between groups. *: p b 0.05.

in open arms (MWU = 127.500, p = 0.984) and the percentage of open arm entries (MWU = 109.500, p = 0.456). 3.3.2. Comparison within the groups (proestrus X other) For the control groups, the animals at proestrus stayed significantly longer in open arms (10.76 ± 3.61 versus 2.58 ± 2.58, MWU = 13.000, p = 0.028), and entered significantly more frequently into the open arms (18.37 ± 4.84 versus 2.50 ± 2.50, MWU = 10.000, p = 0.011) than animals at other stages of menstrual cycle. For the citalopram group, there was no statistically significant difference for either parameter. 3.4. c-fos expression c-fos expression was measured at the cingulated cortex, the paraventricular thalamic nucleus (PVT) and amygdala, and the results are shown in Table 2. 3.4.1. Comparisons between the groups (control X citalopram) When all the animals in each group were analyzed without subdividing them according to their menstrual cycle stage, the control and citalopram groups showed significant differences in two brain regions. c-fos expression at the PVT (MWU = 13,5000, p = 0.014) and the amygdala (MWU = 5000, p = 0.013) was significantly lower in

the citalopram group (9.40 ± 4.54 and 6.13 ± 1.87, respectively) than the control group (23.04 ± 1.95 and 19.79 ± 3.50, respectively). For animals at the proestrus stage, c-fos expression for the citalopram group (4.12 ± 2.12) was significantly lower (MWU = 0.000, p = 0.037) than in the control group at the PVT (20.82 ± 1.23). There was no statistically significant difference for animals at other stages of the menstrual cycle. 3.4.2. Comparisons within the groups (proestrus X other) There was no statistically significant difference with regard to c-fos expression within the groups. 3.4.3. Correlation between c-fos expression and EPM performance The results are shown in Table 3. There was no statistically significant correlation between c-fos expression and EPM performance in any case. 4. Discussion The results of this study confirm both our hypotheses. First, rats in the proestrus phase of the menstrual cycle showed less anxiety-like behavior than rats in other phases of the cycle. We based this hypothesis on previous observations that high estradiol and progesterone levels provide an anti-anxiety effect (Mora et al., 1996; Diaz-Veliz et al., 1997; Frye et al., 2000; Marcondes et al., 2001; Frye and Walf, 2002).

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Table 2 Comparison of c-fos expression between the groups. Control

Cingulate cortex

Paraventricular thalamic nucleus

Amygdala

a b

Proestrus Other Total Proestrus Other Total Proestrus Other Total

Citalopram Mean ± SE

MWUa

p

Mean ± SE

MWUa

p

MWUb

p

20.95 36.57 28.16 20.82 25.58 23.04 18.56 21.02 19.79

15.000

0.391

3.000

0.087

21.500

0.452

1.500

0.240

6.000

0.564

4.75 ± 1.98 11.65 ± 2.36 9.58 ± 1.99 4.12 ± 2.12 12.05 ± 6.68 9.40 ± 4.54 3.16 ± 0.83 7.62 ± 2.54 6.13 ± 1.87

2.000

0.348

4.000 15.500 44.500 0.000 5.500 13.500 0.000 2.000 5.000

0.138 0.431 0.203 0.037 0.107 0.014 0.064 0.076 0.013

± ± ± ± ± ± ± ± ±

9.08 17.13 9.15 1.23 3.88 1.95 5.13 5.47 3.50

Mann–Whitney U analysis for comparison within the group (proestrus × other). Mann–Whitney U analysis for comparison between the groups (control × citalopram).

These ovarian hormones reach their peak during the proestrus phase in rats (Marcondes et al., 2002). Our results confirmed this information, since the proestrus rats in our control group showed less anxiety-like behavior as measured by a longer time spent in the open arms of the EPM and more frequent open arm entries than non-proestrus rats in the control group. It has previously been shown that proestrus rats display significantly less anxiety-like behavior than non-proestrus rats, as measured by open field and the EPM tests (Mora et al., 1996; Diaz-Veliz et al., 1997; Frye et al., 2000; Marcondes et al., 2001; Frye and Walf, 2002). Although a few studies confirm an opposite result (Sadeghipour et al., 2007; Nin et al., 2012), taken together with the fact that estradiol and progesterone have anxiolytic properties, we conclude that anxiety levels may decrease at the proestrus phase. Our second hypothesis was that citalopram would decrease anxietylike behavior related to the estrus phase. This hypothesis was also partly confirmed by our results. The control and citalopram groups did not show significant differences with regard to their behavior in the EPM. However, significant differences that were not present in the citalopram group were demonstrated between the proestrus and non-estrous rats in the control group. In the citalopram group, proestrus and nonproestrus rats behaved in similar ways in the EPM. Since one would expect the non-proestrus rats to show more anxiety-like behavior than those in proestrus (as indicated by previous literature and the results of our control group), this result may show that citalopram decreased the anxiety level of non-proestrus rats. It has previously been shown

that the anxiolytic effects of psychotropic medications may show some significant differences at different stages of estrous (Fernandez-Guasti et al., 1999; Molina-Hernandez et al., 2013). In particular, increased sensitivity to anxiolytic drugs has been shown during the proestrus stage (Bitran and Dowd, 1996; Molina et al., 2002). There are conflicting results about the anxiolytic property of SSRIs, and it remains unclear whether SSRIs in general display an anxiogenic- or an anxiolytic-like effect in animal models. In the EPM, acute and/or chronic administration of SSRIs seems to generate an anxiogenic effect, or no effect at all (for a review see Borsini et al., 2002). For citalopram, acute treatment (1 h before the procedure) seems to cause anxiogenic effects, while chronic treatment (14–22 days, usually 10 mg/kg/day) causes anxiolytic effects as measured by the EPM and other anxiety models in rodents (Griebel et al., 1994; Pollier et al., 2000; Burghardt et al., 2004; Kokras et al., 2011). With regard to general behavior, there were some significant differences between the control and citalopram groups. Grooming (head and body) behavior in the citalopram group was significantly more frequent than in the control group. Grooming behavior has been previously related to negative emotions, such as pain and anxiety (Vos et al., 1994; Kalueff and Tuohimaa, 2005; Xu et al., 2008; Yao and Sessle, 2008). Since the citalopram group received daily intraperitoneal citalopram injections, this procedure may have caused pain (especially in the body), and the grooming behavior may have increased because of this pain. For the control group, body grooming was significantly more frequent on D0 than at D10. This result may reflect anxiety in the animals, since

Table 3 Correlation between EPM performance and c-fos expression. Pearson correlation sig. Cingulate cortex

All animals

Total Proestrus Other

Control

Total Proestrus Other

Citalopram

Total Proestrus Other

Paraventricular thalamic nucleus

Amygdala

Duration

Entry

Duration

Entry

Duration

Entry

.220 .314 .066 .857 .408 .166 .184 .547 −.098 .835 .639 .172 −.178 .622 −.695 .511 −.303 .508

.126 .567 .322 .365 .085 .783 .185 .546 .302 .510 .639 .172 −.113 .756 −.695 .511 −.122 .794

.057 .807 .294 .410 −.021 .950 −.086 .760 −.329 .426 .275 .551 .023 .965 −.823 .162 −.163 .837

−.014 .952 .454 .187 −.236 .484 −.014 .952 −.119 .779 .275 .551 .114 .830 −.302 .510 −.108 .892

.155 .596 .640 .171 −.240 .560 .067 .875 .343 .657 .108 .892 .557 .251 .236 .484 .432 .568

.081 .783 .802 .055 −.543 .165 .166 .694 .612 .388 .432 .568 .370 .471 .543 .165 −.006 .994

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D0 was the first time that they were in a Laboras cage, and a new environment may have increased their anxiety. For the citalopram group, there were no significant behavioral differences between D0 and D10. Nonetheless, one must be careful when interpreting these results because grooming behavior could also be related to stereo-typed activity involved in the dopaminergic system rather than to anxiety (Komorowska and Pellis, 2004; Taylor et al., 2010). The novelty of our study was our investigation of the effect of estrus cycle on USVs and c-fos expression in addition to anxiety-like behavior. The USVs between the groups did not show any significant differences, but this result is preliminary since only two USVs were recorded during the entire experiment. Only two animals from the citalopram group made USVs during D10, and these vocalizations were at Band I indicating distress (Portfors, 2007). As explained above, the citalopram group received daily intraperitoneal injections, which may have caused pain, and that pain may have caused distress-related USVs. Another important result of this study was that the control and citalopram groups showed some significant differences with regard to c-fos expression at the amygdala and the PVT, but not at cingulated cortex. In addition, c-fos expression was not correlated with behaviors in the EPM. This result may show that c-fos expression in brain areas related to anxiety is suppressed by citalopram. c-fos is an early gene used as a marker of neural activation (Kovacs, 1998). Numerous c-fos immunohistochemical studies have identified possible brain circuits associated with anxiety and stress. Previous results in relation to the induction of c-fos immunoreactivity after the EPM test, focus on increased activity in specific brain areas, such as the cingulated cortex, the amygdala, the hippocampus, the prefrontal cortex, the thalamic and hypothalamic nuclei, the raphe nucleus, the locus coeruleus, and the septal nucleus (Silveira et al., 1993; Duncan et al., 1996; Albrechet-Souza et al., 2008; Troakes and Ingram, 2009; Galvis-Alonso et al., 2010). Our reason for choosing three specific areas of the brain (cingulated cortex, amygdala, and paraventricular nucleus of thalamus) was that these areas have repeatedly been shown to be activated by both environmentally evoked anxiety and anxiogenic drugs (Singewald et al., 2003). There are only a few studies about the effects of anti-depressant drugs on c-fos immunoreactivity induced by an acute stress, and the results are contradictory. Acute treatment with fluoxetine after restraint stress showed a decrease, while chronic administration increased c-fos immunoreactivity in various brain areas (Lino-de-Oliveira et al., 2001). Chronic administration of sertraline significantly down-regulated the induction of cfos mRNA in response to restraint stress (Morinobu et al., 1995). Acute administration of citalopram increased the conditioned fear stress model induced c-fos expression in the secondary motor cortex, primary somatosensory cortex, and basolateral nucleus of amygdale (Izumi et al., 2006). In our study, chronic administration of citalopram seems to decrease EPM induced c-fos immunoreactivity at the amygdala and the PVT. The primary limitation of this study is related to sub-grouping the animals in only two phases of the estrus cycle: proestrus and nonestrous. The reason for this sub-grouping was that the proestrus phase shows important differences in hormonal levels (estradiol and progesterone peak) distinct from other phases of the cycle (Marcondes et al., 2002). But by sub-grouping the animals into only two groups, we may have missed important differences between estrous, diestrous, and metestrous rats. The second main limitation is that we used only one dosage level of citalopram (20 mg/kg). Having at least one other group using citalopram (such as 10 mg/kg) could help us to differentiate whether the dosage used in this study has a non-specific sedative or stimulant activity, or interacts with normal animal behavior. The third main limitation is that our study lacks a control-vehicle group. This might confound the effect of behavioral results with regard to body-grooming. Still, we believe that our results are important, because to our knowledge this is the first study to investigate the effect of the estrus cycle on USVs and c-fos expression, in addition to the effect of citalopram on these parameters.

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