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1 -mediated anxiolysis and differing neurobiological responses in 1 2 2 both sexes of a genetic model of depression. 3 4 3 5 6 N. Kokras1,2, I. Sotiropoulos3,4, P.M. Pitychoutis1, O.F.X. Almeida3 and Z. 7 4

8 1 9 5 Papadopoulou-Daifoti 10 11 6 12 13 7 1. Department of Pharmacology, Medical School, University of Athens, Greece 14 15 8 2. First Department of Psychiatry, Eginition Hospital, Medical School, University of 16 17 18 9 Athens, Greece 19 20 10 3. Neuroadaptations Group, Max Planck Institute of Psychiatry, Munich, Germany 21 22 11 4. ICVS Institute, Life and Health Sciences School, University of Minho, Portugal 23 24 12 25 26 27 13 28 29 14 Corresponding Author: 30 31 15 Z. Papadopoulou-Daifoti 32 33 16 Professor and Chair of the Department of Pharmacology 34 35 36 17 Director of Basic Medical Sciences 37 38 18 Medical School, University of Athens 39 40 19 75 Mikras Asias Street, 11527, Goudi, Athens, Greece 41 42 20 Tel: +30-210-7462702; Fax: +30-210-7462554 43 44 21 E-mail: [email protected] 45 46 47 22 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 1 63 64 65

23 1 2 24 Abstract 3 4 25 Disorders such as depression and anxiety exhibit strong sex differences in their 5 6 prevalence and incidence, with women also differing from men in their response to 7 26 8 9 27 . Furthermore, receptors for corticotrophin releasing hormone 10 11 28 (CRHR1) and arginine vasopressin (AVPR1b) are known to contribute to the 12 13 29 regulation of mood and anxiety. In the present study, we compared the anxiety profile 14 15 30 and CRHR1 and AVPR1b expression levels in control Sprague Dawley (SD) rats and 16 17 18 31 rats of the SD-derived Flinders Sensitive Line (FSL), a genetic model of depression. 19 20 32 Additionally, given the apparent sex differences in the therapeutic efficacy of 21 22 33 antidepressants and because antidepressants are commonly used to treat comorbid 23 24 34 anxiety and depressive symptoms, we assessed whether the anxiolytic effects of an 25 26 27 35 occur in a sex-dependent manner. Male and female FSL rats were 28 29 36 treated with citalopram 10mg/kg once daily for 14 days and were then tested in the 30 31 37 open field and the elevated plus maze paradigms. Upon completion of the 32 33 38 behavioural analysis, AVPR1b and CRHR1 expression levels were monitored in the 34 35 36 39 hypothalamus and the prefrontal cortex (PFC) using western blotting. According to 37 38 40 our results, male FSL rats were more anxious than control SD rats, a difference 39 40 41 abolished by citalopram treatment. Baseline anxiety levels were similar in female FSL 41 42 42 and SD rats, and citalopram further reduced anxiety in female FSL rats. Importantly, 43 44 43 whereas citalopram altered AVPR1b expression in the hypothalamus of male FSL 45 46 47 44 rats, its actions on this parameter were restricted to the PFC in female FSL rats. In 48 49 45 both sexes of FSL rats, citalopram did not alter CRHR1 expression in either the 50 51 46 hypothalamus or PFC. Our results demonstrate that antidepressant treatment 52 53 47 reduces anxiety levels in FSL rats of both sexes: the magnitude of treatment effect 54 55 56 48 was related to the starting baseline level of anxiety and the antidepressant elicited 57 58 49 sexually differentiated neurobiological responses in specific brain regions. 59 60 50 61 62 2 63 64 65

51 1 2 52 3 4 53 Keywords 5 6 Antidepressant; Sex differences; Anxiety; Depression; Females; Receptors 7 54 8 9 55 10 11 56 12 13 57 Abbreviations 14 15 58 ANCOVA: Analysis of Co-Variance 16 17 18 59 AVP: Arginine Vasopressin 19 20 60 AVPR1b: Arginine Vasopressin subtype 1b 21 22 61 CRH: Corticotropin Releasing Hormone 23 24 62 CRHR1: Corticotropin Releasing Hormone Receptor subtype 1 25 26 27 63 EPM: Elevated Plus Maze 28 29 64 FSL: Flinders Sensitive Line 30 31 65 HPA: Hypothalamic Pituitary Adrenal 32 33 66 SD: Sprague Dawley 34 35 36 67 SSRI: Selective Serotonin 37 38 68 39 40 69 41 42 70 Article Highlights 43 44 45 71  Male, but not female, Flinders Sensitive Line of rats present anxiety behaviour 46 47 72  Repeated citalopram treatment reduces anxiety levels in both sexes 48 49 73  Citalopram modulates AVPR1b expression in a sex-specific manner 50 51 74  Differing mechanisms converge to produce anxiolysis in FSL rats of both 52 53 54 75 sexes 55 56 76 57 58 59 60 61 62 3 63 64 65

77 1. Introduction 1 2 78 Stress and dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis are 3 4 79 implicated in the pathophysiology of mood and anxiety disorders (Tsigos and 5 6 Chrousos, 2002), which show greater comorbidity and prevalence in women (Kessler 7 80 8 9 81 et al., 1994, Alonso et al., 2004). Two hypothalamic drivers of the HPA axis, 10 11 82 corticotropin releasing hormone (CRH) and arginine vasopressin (AVP), also 12 13 83 influence mood and emotional behaviour through their actions at extra-hypothalamic 14 15 84 sites (Holsboer and Ising, 2008). The actions of CRH are mediated by two receptor 16 17 18 85 subtypes (Lowry and Moore, 2006). The CRH subtype 1 receptor (CRHR1) 19 20 86 modulates context-dependent affective responses and is expressed in the 21 22 87 hypothalamus and extra-hypothalamic areas, including the frontal cortex (Sanchez et 23 24 88 al., 1999). On the other hand, cerebral vasopressin pathways show distinct 25 26 27 89 anatomical and functional patterns (Ermisch et al., 1993); most central actions of this 28 29 90 peptide are mediated by subtype 1b vasopressin receptors (AVPR1b) which are also 30 31 91 expressed in the hypothalamus and frontal cortex (Hernando et al., 2001). 32 33 92 Increasingly, AVPR1b are being implicated in behaviours relevant to mood and 34 35 36 93 anxiety disorders (Surget and Belzung, 2008). Antagonism of either CRHR1 or 37 38 94 AVPR1b results in anxiolytic and antidepressant effects, suggesting these receptors 39 40 95 as alternatives to the current monoaminergic targets (Surget and Belzung, 41 42 96 2008, Binder and Nemeroff, 2010). 43 44 97 Sex differences in the activity of the HPA axis are well known and the sexually 45 46 47 98 differentiated response to stress is increasingly thought to be an important underlying 48 49 99 factor in mood and anxiety disorders (Young, 1998, Goel and Bale, 2009, Young and 50 51 100 Korszun, 2010). Women are nearly twice as likely to suffer from depression (Kessler 52 53 101 et al., 1994, Alonso et al., 2004). Moreover, as compared to men, depressed women 54 55 56 102 make a greater number of suicide attempts, show more somatisation, anger and 57 58 103 hostility, and display increased appetite and weight gain. Importantly, although 59 60 104 melancholic depression occurs equally in both sexes, the anxious and atypical forms 61 62 4 63 64 65

105 of depression are more commonly found in women (Frank et al., 1988, Marcus et al., 1 2 106 2005, Marcus et al., 2008), although the construct of anxious depression has 3 4 107 received some criticism (Nelson, 2008). It is now well-established that 5 6 influence depressive symptoms, including irritability, insomnia, appetite, and general 7 108 8 9 109 physical well-being (Young et al., 2007, Kornstein et al., 2010). Together, these 10 11 110 observations suggest the potential importance of considering the role of sex and the 12 13 111 ovarian steroid milieu in measuring the efficacy of antidepressant therapy. Indeed, 14 15 112 several studies have reported that women respond better to selective serotonin re- 16 17 18 113 uptake inhibitors (SSRI) (Kornstein et al., 2000, Joyce et al., 2003, Baca et al., 2004, 19 20 114 Khan et al., 2005); a similar conclusion was reached in a recent large multicentre trial 21 22 115 of citalopram (Marcus et al., 2008). Nevertheless, some authors have argued that 23 24 116 sex differences in response to antidepressant treatment may be clinically irrelevant 25 26 27 117 (Quitkin et al., 2002, Hildebrandt et al., 2003, Parker et al., 2003, Wohlfarth et al., 28 29 118 2004, Thiels et al., 2005). 30 31 119 Experimental studies from our group and others have consistently demonstrated 32 33 120 increased female vulnerability to the detrimental effects of stress on mood- and 34 35 36 121 anxiety-related behaviours (Patchev and Almeida, 1998, Dalla et al., 2010, 37 38 122 Pitychoutis and Papadopoulou-Daifoti, 2010, Pitychoutis et al., 2010). Furthermore, 39 40 123 our previous studies showed that antidepressants elicit similar, albeit differing in 41 42 124 magnitude, behavioural responses in male and female rats of the Flinders Sensitive 43 44 125 Line (FSL); serotonergic and glutamatergic responses also differ between the two 45 46 47 126 sexes following antidepressant treatment (Kokras et al., 2009a, Kokras et al., 2009b). 48 49 127 FSL rats, derived by selective breeding of Sprague-Dawley (SD) rats, have several 50 51 128 key characteristics which support the face, construct and predictive validity of this 52 53 129 model of depression (Overstreet, 2002). FSL rats exhibit decreased weight and 54 55 56 130 disturbed appetite, have elevated REM sleep, present anhedonia in chronic mild 57 58 131 stress and are less active in novel environments; at the same time they are inherently 59 60 132 more immobile in the forced swim test. Furthermore, in brain limbic areas, FSL rats 61 62 5 63 64 65

133 show significant regional abnormalities in levels of biogenic amines and their 1 2 134 receptors, reduced serotonin synthesis, impaired serotonin-induced dopamine 3 4 135 release, and impairments in their immune system (Overstreet et al., 2005). 5 6 Importantly, chronic and not acute treatment with antidepressants restores those 7 136 8 9 137 observed behavioural and neurobiological abnormalities (Yadid et al., 2000). 10 11 138 Despite the large body of evidence supporting the validity of the FSL model of 12 13 139 depression, it still remains unclear whether FSL rats can model comorbid anxious 14 15 140 depression. Previous research on male FSL showed increased levels of anxiety in 16 17 18 141 the social interaction test (Overstreet, 2002, Janowsky et al., 2004, Lavi-Avnon et al., 19 20 142 2005) but not in the elevated plus maze (EPM) (Schiller et al., 1991, Overstreet et al., 21 22 143 1995). Whereas male FSL rats have been well studied, relatively fewer studies have 23 24 144 focused on female FSL animals. While our previous studies confirmed the validity of 25 26 27 145 female FSL rats as a model of depression (Kokras et al., 2009a, Kokras et al., 28 29 146 2009b), there is no information with respect to their anxiety profile and expression 30 31 147 levels of CRHR1 and AVPR1b. 32 33 148 Based on the aforementioned sex-differences and the documented validity of 34 35 36 149 male and female FSL rats as a model of depression, a first aim of the present study 37 38 150 was to explore the potential suitability of the FSL rat as a model of comorbid anxious 39 40 151 depression. Further, given that male FSL rats were previously reported to be 41 42 152 sensitive to the behavioural actions of CRHR1 and AVPR1b antagonists (Janowsky 43 44 153 et al., 2004, Overstreet et al., 2004, Overstreet et al., 2005) we examined the 45 46 47 154 expression of brain CRHR1 and AVPR1b in male and female FSL rats under 48 49 155 baseline conditions and after repeated citalopram treatment. 50 51 156 52 53 157 2. Experimental Procedures 54 55 56 158 57 58 159 2.1 Animals 59 60 61 62 6 63 64 65

160 Twenty-four adult male and female FSL rats, weighing 275±17 g and 200±15 1 2 161 g, respectively, and aged 10-11 weeks at the beginning of the experiment, were 3 4 162 used. In addition, 24 male and female, similarly aged, Sprague-Dawley rats, weighing 5 6 325±26 g and 245±20 g respectively, were used as controls as previously described 7 163 8 9 164 (Overstreet et al., 2005). Animals were group-housed, according to sex, under 10 11 165 controlled 12:12 light/dark cycles (lights on at 07:00 a.m.) and temperature (222 °C), 12 13 166 with free access to food and tap water. All animal experiments were carried out in 14 15 16 167 accordance with the EEC directive 86/609. 17 18 168 19 20 169 2.2 Oestrous Cycle 21 22 170 In the case of females, a semi-random process controlled for disparities 23 24 25 171 regarding the phases of the oestrous cycle. Specifically, female rats were selected 26 27 172 from a larger pool of experimental animals on the basis of a normal 4-5 day cycle and 28 29 173 assigned to groups on the basis of an equal distribution of oestrous cycle phases; the 30 31 174 latter was monitored by vaginal smears until the day of sacrifice, as described 32 33 175 elsewhere (Becker et al., 2005). Oestrous cycle phases on the day of behavioural 34 35 36 176 testing and sacrifice are reported in Table 1. 37 38 177 39 40 178 2.3 Treatments 41 42 179 FSL and control SD rats were gently handled, daily by the same researcher. 43 44 45 180 Male and female animals were given intra-peritoneal injections of either saline (FSL 46 47 181 n=12; SD n=12) or 10mg/Kg of citalopram (Lundbeck S.A., Denmark; FSL n=12; SD 48 49 182 n=12) during the morning, over 14 days. This dose of citalopram was previously 50 51 183 shown to be effective and has been routinely used in male and female rats 52 53 54 184 (Burghardt et al., 2004, Overstreet et al., 2004, Hasegawa et al., 2005). 55 56 185 57 58 186 2.4 Open Field 59 60 61 62 7 63 64 65

187 Spontaneous activity under novelty stress was measured for 5 min, 1 2 188 approximately 22-26 h after the last injection of saline or citalopram (day 15). As 3 4 189 previously described (Pitychoutis et al., 2009b), all rats were acclimatized to the test 5 6 room for 1 h and thereafter placed in a clear Plexiglas chamber (Med Associates Inc., 7 190 8 9 191 St Albans, VT) measuring 430 x 430 x 300 mm with arrays of 16 x 16 photodetectors, 10 11 192 positioned 2.5 cm and 10 cm above the floor of the chamber. Interruption of adjacent 12 13 193 photobeams provided an index of ambulatory activity (horizontal activity) while 14 15 194 interruption of the upper line of photobeams provided an index of rearing behaviour 16 17 18 195 (vertical activity). The time spent at the centre of the chamber served as an index of 19 20 196 anxiety (Belzung and Griebel, 2001). 21 22 197 23 24 198 2.5 Elevated Plus Maze 25 26 27 199 The Elevated Plus Maze (EPM) test was conducted under dim light, 28 29 200 approximately 1 min after the open field test. The test sequence from the open field 30 31 201 to the elevated plus maze is known to enhance exploratory activity and to improve 32 33 202 the reliability of plus-maze testing (Pellow et al., 1985). Each rat was placed facing 34 35 36 203 an open-arm in the middle of the EPM, which was placed 50 cm above the ground 37 38 204 and consisted of two open (50 x 10 cm) and two identical arms, enclosed within 50 39 40 205 cm high walls with a symmetrical 100 cm2 square area at the centre. The number of 41 42 206 total and open arm entries was recorded for 5 minutes. An index of anxiety was 43 44 207 obtained from the ratio of the time spent in the open arms vs. the total time in the 45 46 47 208 maze (Doremus et al., 2006). Upon completion of the behavioural testing, rats were 48 49 209 returned to their home cages. 50 51 210 52 53 211 2.6 Western blot analysis 54 55 56 212 Twenty minutes after behavioural analysis, all animals were rapidly sacrificed 57 58 213 and the prefrontal cortex and the hypothalamus were dissected out, snap-frozen in 59 60 214 liquid nitrogen and stored at –80° C. Their brains were rapidly removed and for the 61 62 8 63 64 65

215 dissection of the regions of interest a method described in detail by (Heffner et al., 1 2 216 1980) was followed with minor modifications, as described in (Kokras et al., 2009b). 3 4 217 In short, two sagittal sections, rostrally to caudally, separated the prefrontal cortex 5 6 from the frontal lobe and the rest of the brain. Samples were homogenized using a 7 218 8 9 219 Dounce glass homogenizer in lysis buffer containing 100 mM Tris-HCl, 250 mM 10 11 220 NaCl, 1 mM EDTA, 5 mM MgCl2, 1% NP-40, Complete Protease Inhibitor (Roche, 12 13 221 Mannheim, Germany) and Phosphatase Inhibitor Cocktails I and II (Sigma, St. Louis, 14 15 222 MO). Extracts were cleared by centrifugation (14000 g) and protein contents were 16 17 18 223 estimated by Lowry assay. Protein lysates (40ug) were electrophoresed on 10% 19 20 224 acrylamide gels in Laemmli buffer (250 mm Tris-HCl, pH 6.8, containing 4% SDS, 21 22 225 10% glycerol, 2% β-mercaptoethanol, and 0.002% bromophenol blue) and 23 24 226 transferred onto nitrocellulose membranes (Protran BA 85, Schleicher & Schuell, 25 26 27 227 Dassel, Germany). The membranes were blocked in TBS containing 5% non-fat milk 28 29 228 and 0.2% Tween-20 before incubation with anti-AVPR1b (1:1000; AB3510P, 30 31 229 Chemicon, Temecula, CA) and anti-CRHR1 kindly provided by Dr. E.A. Linton 32 33 230 (1:200; Oxford), described in (Castro et al., 1996). For normalization purposes, blots 34 35 36 231 were also probed with anti--actin (1:2000; Chemicon). Following incubation with 37 38 232 horseradish peroxidase–IgG conjugates (Amersham Biosciences, Freiburg, 39 40 233 Germany), antigens were revealed by enhanced chemiluminescence (ECL, 41 42 234 Amersham Biosciences). Band intensities were evaluated by densitometry and all 43 44 45 235 values were normalized and expressed as percentages of control male vehicle – 46 47 236 treated SD rats. 48 49 237 50 51 238 2.7 Corticosterone assay 52 53 54 239 Trunk blood samples were collected at sacrifice and processed to recover 55 56 240 serum (centrifugation at 4000 g, 30 min, 4°C); serum samples were stored at -20° C 57 58 241 before being assayed for corticosterone by a standard radioimmunoassay (MP 59 60 61 62 9 63 64 65

242 Biomedicals, Costa Mesa, CA). The inter- and intra-assay coefficients of variation 1 2 243 were both 8%. 3 4 244 5 6 245 2.8 Statistical Analysis 7 8 9 246 All results presented herein were analyzed with three-way ANCOVA using the 10 11 247 General Linear Model of SPSS version 19 (IBM Corp, Somers, NY, USA) with “strain” 12 13 248 (FSL vs. control SD), “sex” (male vs. female) and “drug” (citalopram vs. vehicle) as 14 15 249 independent variables. Subsequent one-way ANCOVAs were performed to elucidate 16 17 18 250 significant results as indicated by the factorial model. Specifically for corticosterone, 19 20 251 pearson’s correlations with behavioural outcomes were calculated. Oestrous cycle 21 22 252 phase was used as a covariate in all factorial analysis to control variance due to the 23 24 253 female hormonal milieu and to increase the validity of the male/female comparison. 25 26 27 254 In this study, the main effect of oestrous cycle was not significant in any of the 28 29 255 statistical analyses described further on; however this study was not designed to 30 31 256 specifically detect oestrous cycle effects but merely to control for them. A probability 32 33 257 value of p≤0.05 was considered as significant. 34 35 36 258 37 38 259 3. Results 39 40 260 41 42 261 3.1 Behavioural measurements 43 44 262 45 46 47 263 3.1.1 Open Field test 48 49 264 50 51 265 Horizontal activity. Statistical analysis indicated a significant “sex” main 52 53 266 effect, as female FSL and SD rats generally showed higher ambulatory counts than 54 55 56 267 their strain-matched male controls [F(1,39)=27.553 p<0.001]. Furthermore, a significant 57 58 268 “strain” main effect was observed [F(1,39)=9.259 p=0.004], reflecting the lower 59 60 269 ambulatory counts of FSL rats during the 5 min open field test. Male FSL rats 61 62 10 63 64 65

270 displayed considerable lower horizontal activity than their SD counterparts 1 2 271 [F(1,10)=46,507 p<0.001] (Figure 1a). 3 4 272 5 6 273 Vertical Activity. Statistical analysis revealed a significant “sex” main effect 7 8 9 274 [F(1,39)=38.263 p<0.001], as female SD and FSL rats generally had higher vertical 10 11 275 counts than males of the corresponding strains (Figure 1b). 12 13 276 14 15 277 Time in centre. The factorial model revealed significant “strain x sex” and 16 17 18 278 “strain x drug” interactions [F(1,39)=8.344 p=0.006; F(1,39)=10.429 p=0.003]. Further 19 20 279 analysis showed that male, but not female, FSL rats spent less time in the centre of 21

22 280 the arena than their sex-matched SD counterparts [F(1,10)=5.171 p=0.046]. 23 24 281 Citalopram-treated male FSL rats displayed considerably more time in the centre of 25 26 27 282 the arena than vehicle-treated male FSL rats [F(1,10)=27.128 p<0.001]. Interestingly, 28 29 283 citalopram-treated female FSL rats also showed a significant increase in the time 30 31 284 spent in the centre of the arena [F(1,9)=8.749 p=0.016] (Figure 1c). 32 33 285 34 35 36 286 3.1.2 Elevated Plus Maze test 37 38 287 39 40 288 Total arm entries. Statistical analysis revealed a significant “sex” main effect 41 42 289 [F(1,39)=4.288 p=0.045], indicating that female SD and FSL rats made more arm 43 44 290 entries than SD and FSL males, respectively (Figure 2a). 45 46 47 291 48 49 292 Open arm entries. Regarding open arm entries, a significant “sex” main 50

51 293 effect [F(1,39)=6.443 p=0.015] was found, indicating that female SD and FSL rats 52 53 294 make more open arm entries than their male counterparts (Figure 2b). 54 55 56 295 57 58 296 Time in open arms. Statistical analysis uncovered significant “strain x sex” 59 60 297 and “strain x drug” interactions on this parameter [F(1,39)=8.305 p=0.006; 61 62 11 63 64 65

298 F(1,39)=15.002 p<0.001 respectively]. As compared to respective SD controls, male, 1 2 299 but not female, FSL rats spent less time in the open arms of the apparatus 3 4 300 [F(1,10)=126.772 p<0.001]. However, citalopram treatment elongated the time spent in 5 6 the open arms in FSL rats of both sexes [males: F =24.874 p=0.001; females: 7 301 (1,10) 8 9 302 F(1,9)=5.666 p=0.041] (Figure 2c). 10 11 303 12 13 304 3.3 Neurobiological measurements 14 15 305 16 17 18 306 3.3.1 Corticosterone Serum Levels 19 20 307 Statistical analysis revealed that female FSL and SD rats had significantly 21

22 308 higher corticosterone serum levels [F(1,39)=20.314 p<0.001]. Furthermore, male FSL 23 24 309 rats had significantly higher corticosterone levels than control male SD rats 25 26 27 310 [F(1,10)=35,426 p<0.001]. Citalopram treatment mediated a significant decrease in 28 29 311 corticosterone levels in male FSL rats as compared to vehicle treated male FSL rats 30 31 312 [F(1,10)=17.380 p=0.002]. However, citalopram treatment had no effect in female FSL 32 33 313 rats (Figure 3a). Furthermore, Pearson’s correlation analysis showed that 34 35 36 314 corticosterone levels significantly and inversely correlated in males only with the time 37 38 315 spent in the centre of the open field and in the open arms of the EPM [r(24)= -0.590, 39 40 316 p=0.002; r(24)= -656, p<0.001 respectively], as shown in the scatter plot (figure 3b). 41 42 317 These correlations were not significant for female rats. Similarly, a linear regression 43 44 318 model for corticosterone with behavioural and neurobiological measurements as 45 46 47 319 predictors did not produce significant results. 48 49 320 50 51 321 3.3.2. CRHR1 expression levels 52 53 322 Immunoblotting was used to monitor CRHR1 levels in the hypothalamus and 54 55 56 323 PFC. Regarding hypothalamic CRHR1, the analysis revealed a significant “strain” 57 58 324 main effect; male and female FSL rats were found to have significantly lower levels of 59 60 325 CRHR1 expression than SD rats [F(1,39)=11.478 p=0.002] (Figure 4a). Citalopram 61 62 12 63 64 65

326 exhibited no effect on CRHR1 levels. A similar analysis of CRHR1 expression in the 1 2 327 PFC indicated a significant “strain x drug” interaction [F(1,39)=6.617 p=0.014] (Figure 3 4 328 4b). Further analysis showed that, similar to observations in the hypothalamus, the 5 6 prefrontal cortex of male and female FSL rats exhibits lower levels of CRHR1 than 7 329 8 9 330 their SD counterparts [F(1,39)=50.330 p<0.001]. Notably, citalopram treatment resulted 10 11 331 in differential effects in the two rat lines: while the antidepressant failed to produce 12 13 332 any effect in male and female FSL rats, it increased CRHR1 expression in male and 14 15 333 female SD rats [F =7.024 p=0.024; F =5.565 p=0.043] (Figure 4b). 16 (1,10) (1,9) 17 18 334 19 20 335 3.3.3. AVPR1b expression levels 21 22 336 The statistical analysis for AVPR1b hypothalamic expression levels indicated 23 24 337 a significant “strain x sex x drug interaction” [F =9.067 p=0.005] (Figure 4c). 25 (1,39) 26 27 338 Vehicle-treated male, but not female, FSL rats showed higher hypothalamic AVPR1b 28 29 339 expression levels than their SD counterparts [F(1,10)=24.238 p=0.001]. Further 30 31 340 analysis revealed that citalopram lowered hypothalamic AVPR1b expression levels in 32 33 341 male [F(1,10)=8.762 p=0.014], but not female, FSL rats (Figure 4c). Regarding cortical 34 35 36 342 AVPR1b expression levels, the analysis showed a significant “sex x drug” interaction 37 38 343 [F(1,39)=7.163 p=0.011]. Further analysis showed that female FSL rats had lower 39 40 344 cortical AVPR1b than the sex-matched SD rats [F(1,9)=11.440 p=0.008]. Finally, 41 42 345 whereas citalopram did not have a significant effect in males, it robustly increased 43 44 346 cortical AVPR1b in female FSL rats [F =5.674 p=0.041] (figure 4d). 45 (1,9) 46 47 347 48 49 348 4. Discussion 50 51 349 Although depression and anxiety have greater prevalence and different 52 53 350 phenomenology in women, the biological mechanisms underlying such sex 54 55 56 351 differences are not well understood. Likewise, reports on the sex-differentiated 57 58 352 antidepressant response remain controversial. The present study used a well- 59 60 353 validated model of depression and explored its suitability as a model of comorbid 61 62 13 63 64 65

354 anxious depression, taking into consideration sex-differences in anxiety and 1 2 355 depression at baseline and following treatment. In addition, this study explored 3 4 356 possible links between anxiety-like behaviour, corticosterone levels and the 5 6 expression of CRHR1 and AVPR1b in FSL animals under baseline conditions and 7 357 8 9 358 post-antidepressant treatment. 10 11 359 Our results showed that male, but not female, FSL rats are less active and 12 13 360 show greater signs of anxiety than their sex-matched SD controls. These findings are 14 15 361 in agreement with the previously reported anxious profile of male FSL rats in the 16 17 18 362 social interaction test (Overstreet, 2002, Janowsky et al., 2004, Lavi-Avnon et al., 19 20 363 2005). Differences between the present EPM results and those reported previously 21 22 364 (Schiller et al., 1991, Overstreet et al., 1995) may be explained by methodological 23 24 365 differences. In fact, male FSL rats adopt passive coping strategies when challenged 25 26 27 366 in behavioural tests (Overstreet, 2002, Overstreet et al., 2005) and the sequential 28 29 367 behavioural testing along with the repeated injections in this study, possibly provoked 30 31 368 the anxiety-like behavioural response of male FSL rats. 32 33 369 In contrast to males, female rats (of both strains) were generally more active, 34 35 36 370 as reported by others too (Brotto et al., 2000, Romero and Chen, 2004). This is 37 38 371 thought to reflect sex-specific coping strategies and hormonal environment (Palanza, 39 40 372 2001, Dalla et al., 2010); the latter is particularly relevant since gonadal hormones 41 42 373 are known to influence activity in rodents (Nomikos and Spyraki, 1988, Becker et al., 43 44 374 2005) and phenomenology of depression in humans (Kornstein et al., 2010). Rather 45 46 47 375 than focussing on the contribution of individual gonadal hormones using ablation and 48 49 376 hormone replacement strategies (Becker et al., 2005), the present experiments were 50 51 377 designed to average and control for such potentially confounding effects. Thus, while 52 53 378 we previously confirmed a depressive-like phenotype in female FSL rats (Kokras et 54 55 56 379 al., 2009a), it was interesting to find here that FSL females do not express anxiety- 57 58 380 like behaviour. This finding is in contrast to those reported in humans, given that 59 60 61 62 14 63 64 65

381 previous research has established that women present more often anxious 1 2 382 depression than men (Frank et al., 1988, Marcus et al., 2005, Marcus et al., 2008). 3 4 383 Based on the aforementioned observations, we conclude that while the FSL 5 6 rat is a well-validated genetic model for studying depressive-like behaviour, anxiety in 7 384 8 9 385 male FSL rats is dependent on the experimental protocol, hence the previously 10 11 386 conflicting results. In addition, while female rats were not anxious in our study, we 12 13 387 cannot exclude that using other protocols they might also present anxiety. In 14 15 388 summary, our results point towards anxiety being state, rather than trait, dependent 16 17 18 389 in FSL rats; this limits their potential as a model of anxious depression. Interestingly, 19 20 390 should we have used only male FSL rats in our study, the unexpected sex- 21 22 391 differentiated anxious profile of FSL rats, being the polar opposite of the human 23 24 392 condition, would have gone unnoticed. Therefore, in agreement with previous 25 26 27 393 suggestions (Palanza, 2001, Dalla et al., 2010), our findings highlight the importance 28 29 394 of considering both sexes when studying animal models of disease. 30 31 395 With regards to treatment, whereas citalopram did not influence anxiety levels 32 33 396 in control SD rats, the drug robustly reduced anxiety levels in both sexes of the FSL 34 35 36 397 model of depression. The anxiolytic or anxiogenic effect of different SSRI treatments 37 38 398 on male rats has been previously reviewed thoroughly (Borsini et al., 2002) and in 39 40 399 our study, the behaviour of control SD rats was unaffected by this particular dose of 41 42 400 repeated SSRI treatment when rats were tested after a 24 hours wash-out period, in 43 44 401 agreement with similar studies (Wikell et al., 1999). However, behavioural effects 45 46 47 402 have also been reported under different antidepressant doses and experimental 48 49 403 protocols (Kugelberg et al., 2002, Mombereau et al., 2010). It is worth noting that in 50 51 404 FSL rats repeated citalopram treatment did not alter locomotor/exploratory 52 53 405 behavioural indices but selectively affected anxiety-related behavioural responses. 54 55 56 406 Interestingly, the drug-induced responses of male and female FSL rats differed in 57 58 407 magnitude, reflecting their different baseline levels of anxiety. In light of our earlier 59 60 408 studies (Drossopoulou et al., 2004, Dalla et al., 2005, Dalla et al., 2008, Kokras et al., 61 62 15 63 64 65

409 2009a, Kokras et al., 2009b, Pitychoutis et al., 2009a), we conclude that baseline 1 2 410 levels of anxiety in males and females of this model of depression result from sex- 3 4 411 differentiated strategies to cope with identical behavioural challenges. Most 5 6 interestingly, repeated antidepressant treatment masks such differences and 7 412 8 9 413 produces a converging behavioural response. 10 11 414 This conclusion receives further support given that we only found a significant 12 13 415 correlation between corticosterone levels and anxiety in males, although both sexes 14 15 416 displayed reduced anxiety levels after repeated treatment with citalopram. The higher 16 17 18 417 corticosterone levels found in male FSL rats most likely reflect their susceptibility to 19 20 418 distress during handling and subsequent behavioural testing, possibly related to their 21 22 419 impaired serotoninergic transmission (Yadid et al., 2000). Reversal of the latter by 23 24 420 repeated antidepressant treatment attenuates stress-induced drive on the HPA axis, 25 26 27 421 corticosterone and anxiety levels. On the other hand, female rats had higher 28 29 422 corticosterone levels, as consistently reported (Dalla et al., 2010). Curiously, while 30 31 423 female FSL also have an impaired serotoninergic system (Kokras et al., 2009a), and 32 33 424 despite the SSRI-induced behavioural effect, treatment did not attenuate their 34 35 36 425 baseline corticosterone levels. Similar sex differences in the contribution of the HPA 37 38 426 axis to antidepressant actions have been reported (Binder et al., 2009, Horstmann et 39 40 427 al., 2009, McEuen et al., 2009, Goel and Bale, 2010), suggesting that in female FSL 41 42 428 (and perhaps more generally in females), additional mechanisms may influence the 43 44 429 HPA axis, thus obscuring the correlation of corticosterone levels with behavioural 45 46 47 430 outcomes following treatment. 48 49 431 It is known that sex differences in the regulation of corticosteroid secretion, 50 51 432 synthesis and release of hypothalamic CRH and AVP arise during sexual 52 53 433 organization of the brain early in life; on the other hand, the regulation of these 54 55 56 434 hormones remains subject to the influence of fluctuations in gonadal secretory 57 58 435 activity throughout life (Patchev et al., 1999). Importantly, central CRH and AVP 59 60 436 receptors are implicated in mood and anxiety disorders and in the response to 61 62 16 63 64 65

437 antidepressant treatment. Here, we found that, as compared to SD rats, male and 1 2 438 female FSL rats display lower CRHR1 receptors levels at both hypothalamic and 3 4 439 cortical sites. Since CRHR1 has been implicated in depression in humans and 5 6 mouse models (Timpl et al., 1998, Muller et al., 2003), these findings add to the view 7 440 8 9 441 that FSL rats are a good model for understanding the neurobiological basis of 10 11 442 depression. Interestingly, CRHR polymorphisms play an important role in the 12 13 443 therapeutic efficacy of citalopram in anxious depression (Binder et al., 2010) and 14 15 444 other studies have suggested interactions between CRHR1, AVPR1b and serotonin 16 17 18 445 in mediating the actions of established antidepressants (Ishizuka et al., 2010, 19 20 446 Magalhaes et al., 2010). Curiously, the results of the present study show that 21 22 447 citalopram exerts anxiolytic actions in male and female FSL rats without altering 23 24 448 CRHR1 expression levels. Notably, while the results of pharmacological studies in 25 26 27 449 FSL rats implicate CRHR1 in the expression of depressive-like behaviour (Overstreet 28 29 450 et al., 2004, Lavi-Avnon et al., 2005), we found here that citalopram induced changes 30 31 451 in CRHR1 levels in SD animals even though these animals did not show behavioural 32 33 452 responses to the drug. Offering an explanation for these apparent anomalies is 34 35 36 453 difficult but the fact that FSL and SD animals show substantial differences in their 37 38 454 baseline anxiety levels may be an important consideration. Indeed, previous studies 39 40 455 reported that repeated SSRI treatment may affect the temporal co-localization of AVP 41 42 456 and CRH (To et al., 1999, Moncek et al., 2003), initially affecting the CRH 43 44 457 subsystem, and later modifying the AVP subsystem (Keck et al., 2003, Hesketh et 45 46 47 458 al., 2005). 48 49 459 Indeed, our results show that male FSL rats, which display an anxious phenotype 50 51 460 under baseline conditions, express higher levels of hypothalamic AVPR1b as 52 53 461 compared to SD males. Interestingly, we also show that repeated administration of 54 55 56 462 citalopram leads to a downregulation of hypothalamic AVPR1b and, in parallel, 57 58 463 reduced corticosterone and anxiety-like behaviour in male FSL rats. In contrast, 59 60 464 female FSL rats (non-anxious phenotype under basal conditions) exhibit lower 61 62 17 63 64 65

465 cortical AVPR1b mRNA levels than female SD rats. This profile was reversed by 1 2 466 repeated citalopram treatment and the drug led to a further reduction of basal anxiety 3 4 467 levels in female FSL rats without affecting corticosterone levels. Although the 5 6 underlying mechanisms remain elusive at this time, these findings highlight the fact 7 468 8 9 469 that the responses to citalopram are sexually differentiated in FSL animals. Given the 10 11 470 observations that the therapeutic effects of citalopram in males are associated with a 12 13 471 reduction in corticosterone levels, we suggest that the HPA axis plays a critical role in 14 15 472 the regulation of anxiety in males in this sex (see Table 2). Other pathways and 16 17 18 473 mechanisms appear to underlie the actions of citalopram in females; specifically, our 19 20 474 results suggest that modulation of extra-hypothalamic (cortical) AVPR1b may be 21 22 475 important for the anxiolytic actions of citalopram in female FSL rats (see Table 2). 23 24 476 In summary, our results suggest that although male and female FSL rats are a 25 26 27 477 good model for research on depression, they are not suitable for accurately 28 29 478 modelling comorbid anxious depression, as observed in humans. Interestingly 30 31 479 though, SSRI treatment triggers distinct neurobiological mechanisms in male and 32 33 480 female rats of this model of depression. These mechanisms result in similarly 34 35 36 481 converging behavioural response in both sexes, although of different magnitude, 37 38 482 depending on the pre-treatment baseline. It is thus suggested that similar phenotypic 39 40 483 endpoints can arise from divergent neurobiological mechanisms, a concept 41 42 484 previously suggested for central vasopressinergic transmission (De Vries and 43 44 485 Panzica, 2007). It may be further suggested that the neurobiological effects of 45 46 47 486 antidepressants are not strictly predetermined, but rather depend on the substrate 48 49 487 (e.g. male or female brain, depressive and/or anxious status or not). Thus, our 50 51 488 observations raise the question of whether a patient’s clinical response to an SSRI 52 53 489 likewise depends on sexually differentiated neurobiological mechanisms. 54 55 56 490 57 58 491 Acknowledgements 59 60 61 62 18 63 64 65

492 We thank Dieter Fischer for excellent technical assistance, Carola Hetzel for 1 2 493 administrative help, Dr Linton for providing the CRHR1 antibody, and Drs Nuno 3 4 494 Sousa and Alexandre Patchev for their critical comments and discussions. 5 6 Citalopram was kindly gifted by Lundbeck S.A. who nevertheless did not influence 7 495 8 9 496 the study design and interpretation of results from this work. This research was partly 10 11 497 supported through a German-Greek Academic Exchange Programme (DAAD- 12 13 498 IKYDA: D/04/42259), an IBRO studentship to NK, and a Max Planck Society 14 15 499 fellowship to IS; none of these agencies had an influence over the design, outcome 16 17 18 500 or interpretation of the results of this work. 19 20 501 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 19 63 64 65

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759 Tables 1 2 760 3 4 761 Table 1: Oestrous Cycle. Distribution of the phases of the oestrous cycle in female 5 6 FSL and SD rats treated with vehicle and citalopram. 7 762 8 9 763 10 11 764 Table 2: Summary of behavioural and neurobiological changes induced by 12 13 765 citalopram treatment on male and female FSL rats. Repeated citalopram 14 15 766 treatment evoked an anxiolytic behavioural outcome in both male and female FSL 16 17 18 767 rats but this was accompanied by hypothalamic AVPR1b and corticosterone changes 19 20 768 in male FSL rats whereas only cortical AVPR1b changes were identified in female 21 22 769 FSL rats. 23 24 770 25 26 27 771 Figures 28 29 772 30 31 773 Figure 1: Open Field: spontaneous horizontal (a) and vertical activity (b) expressed 32 33 774 in photobeam counts, and time spent in the centre of the arena (c) expressed as 34 35 36 775 percent of total time. Note that male but not female FSL spent less time in centre in 37 38 776 comparison to their SD counterparts, whereas both FSL sexes responded to 39 40 777 citalopram treatment by increasing their time spent in the centre of the arena. All data 41 42 778 shown represent mean ± SEM values; the asterisk (*) represents a significant strain 43 44 779 difference and the hash sign (#) represents a significant drug difference. P<0.05, N=6 45 46 47 780 per group. 48 49 781 50 51 782 Figure 2: Elevated Plus Maze test: number of total (a) and open (b) arm entries, 52 53 783 and time spent in the open arms (c) expressed as percent of total time. Note that 54 55 56 784 male but not female FSL rats spent less time in open arms in comparison to their SD 57 58 785 counterparts but both FSL sexes responded to citalopram treatment by increasing 59 60 786 the percentage of time spent in the open arms. All data shown represent mean ± 61 62 26 63 64 65

787 SEM values; the asterisk (*) represents a significant strain difference and the hash 1 2 788 sign (#) represents a significant drug difference. P<0.05, N=6 per group. 3 4 789 5 6 790 Figure 3: Corticosterone Serum Levels 7 8 9 791 Male but not female FSL rats presented increased corticosterone serum levels and 10 11 792 citalopram treatment successfully abolished this increase in male FSL rats (a). 12 13 793 Increased corticosterone serum levels correlate with lower time in centre and open 14 15 794 arms in males only (b). CORT=Corticosterone, TC=Time in Centre of the Open Field, 16 17 18 795 TOA=Time in Open Arms of the Elevated Plus Mase. All data shown represent mean 19 20 796 ± SEM values .The asterisk (*) represents a significant strain difference and the hash 21 22 797 sign (#) represents a significant drug difference. P<0.05, N=6 per group. 23 24 798 25 26 27 799 Figure 4: CRHR1 and AVPR1b expression levels: Hypothalamic (a) and cortical 28 29 800 (b) CRHR1 and hypothalamic (c) and cortical (d) AVPR1b expression levels of male 30 31 801 and female SD and FSL rats measured by western blot analysis. Representative 32 33 802 blots from each brain region are shown above the respective numeric data in the 34 35 36 803 same order of appearance: M: Males, F: Females, SV: Sprague-Vehicle, SC: 37 38 804 Sprague-Citalopram, FV: Flinder-Vehicle, FC: Flinder-Citalopram. Western blot 39 40 805 numerical data are based on optical density evaluations normalized against actin and 41 42 806 are depicted relative to control values (male vehicle-treated SD rats) as means ± 43 44 807 SEM. An asterisk (*) represents a significant strain difference and a hash sign (#) 45 46 47 808 represents a significant treatment effect. P<0.05, N=6 per group. 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 27 63 64 65 Figure 1 Click here to download Figure: Figure 1 Rev.pdf a Horizontal Activity

1000 Vehicle 800 * Citalopram

600

400 Counts

200

0 SD FSL SD FSL Males Females

b

Vertical Activity 60 Vehicle Citalopram 40

Counts 20

0 SD FSL SD FSL Males Females

c

Time in Center 60 # # Vehicle * Citalopram 40

20 % of total time total % of

0 SD FSL SD FSL Males Females Figure 2 Click here to download Figure: Figure 2 Rev.pdf

a

Total Arm Entries 40 Vehicle Citalopram 30

20 Counts 10

0 SD FSL SD FSL Males Females

b

Open Arm Entries 15 Vehicle Citalopram 10

Counts 5

0 SD FSL SD FSL Males Females

c

Time in Open Arms 60 # # Vehicle * Citalopram 40

20 % of total time total % of

0 SD FSL SD FSL Males Females Click heretodownloadFigure:Figure3Rev.pdf Figure 3

200 a Vehicle

ml) Citalopram 150 * #

rone (ng/ rone 100 ee

50 Corticost 0 SD FSL SD FSL b Males Females Figure 4 Click here to download Figure: Figure 4 Rev.pdf

a HYPOTHALAMUS b PREFRONTAL CORTEX

M: SV SC FV FC F: SV SC FV FC M: SV SC FV FC F: SV SC FV FC CRHR1 CRHR1 actin actin

200 200 Vehicle Vehicle Citalopram Citalopram

its 150 its 150 # s s #

100 100

CRHR1 Level 50 CRHR1 Level 50 Relative O.D. Un Relative O.D. Un

0 0 SD FSL SD FSL SD FSL SD FSL Males Females Males Females

c HYPOTHALAMUS d PREFRONTAL CORTEX M: SV SC FV FC F: SV SC FV FC M: SV SC FV FC F: SV SC FV FC AVPR1b AVPR1b actin actin

200 * # 200 # Vehicle Vehicle Citalopram * Citalopram 150 150

100 100

AVPR1b Levels AVPR1b 50 Levels AVPR1b 50 Relative O.D. Units Relative O.D. Units Relative O.D.

0 0 SD FSL SD FSL SD FSL SD FSL Males Females Males Females Table 1

Vehicle Citalopram FSL SD FSL SD Proestrous 1 2 2 2 Estrous 2 1 1 1 Diestrous I1 2 1 2 Diestrous II 2 1 2 1 Table 2

Males FSL Anxiety Corticosterone HYP AVPR1b PFC AVPR1b Baseline ↑↑ ↑ ↔ Antidepressant ↓↓ ↓ ↔

Females FSL Anxiety Corticosterone HYP AVPR1b PFC AVPR1b Baseline ↔↔ ↔ ↓ Antidepressant ↓↔ ↔ ↑ *Highlights

Article Highlights

 Male, but not female, Flinders Sensitive Line of rats present anxiety behaviour

 Repeated citalopram treatment reduces anxiety levels in both sexes

 Citalopram modulates AVPR1b expression in a sex-specific manner

 Differing mechanisms converge to produce anxiolysis in FSL rats of both

sexes