Female Flinders Sensitive Line rats show estrous cycle-independent depression-like behavior and altered tryptophan metabolism

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FEMALE FLINDERS SENSITIVE LINE RATS SHOW ESTROUS CYCLE-INDEPENDENT DEPRESSION-LIKE BEHAVIOR AND ALTERED TRYPTOPHAN METABOLISM

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AMANDA ESKELUND, a,b* DAVID P. BUDAC, b CONNIE SANCHEZ, b BETINA ELFVING a AND GREGERS WEGENER a

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a Translational Neuropsychiatry Unit, Institute for Clinical Medicine, Aarhus University, Skovagervej 4, 8240 Risskov, Denmark

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b Lundbeck Research USA, 215 College Road, Paramus NJ 07652, USA

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Abstract—Clinical studies suggest a link between depression and dysfunctional tryptophan (TRP) metabolism. Even though depression is twice as prevalent in women as men, the impact of the estrous cycle on TRP metabolism is not well-understood. Here we investigated 13 kynurenine and serotonin metabolites in female Flinders Sensitive Line (FSL) rats, a genetic rat model of depression. FSL rats and controls (Flinders Resistant Line rats), 12–20 weeks old, were subject to the forced swim test (FST), a commonly used measure of depression-like behavior. Open field was used to evaluate locomotor ability and agoraphobia. Subsequently, plasma and hemispheres were collected and analyzed for their content of TRP metabolites using liquid chromatography-tandem mass spectrometry. Vaginal saline lavages were obtained daily for P2 cycles. To estimate the effects of sex and FST we included plasma from unhandled, naı¨ ve male FSL and FRL rats. Female FSL rats showed a depression-like phenotype with increased immobility in the FST, not confounded by anxiety. In the brain, 3-hydroxykynurenine was increased whereas anthranilate and 5-hydroxytryptophan were decreased. In plasma, anthranilate and quinolinate levels were lower in FSL rats compared to the control line, independent of sex and FST. The estrous cycle neither impacted behavior nor TRP metabolite levels in the FSL rat. In conclusion, the female

FSL rat is an interesting preclinical model of depression with altered TRP metabolism, independent of the estrous cycle. The status of the pathway in brain was not reflected in the plasma, which may indicate that an inherent local, cerebral regulation of TRP metabolism occurs. Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: tryptophan–kynurenine pathway, serotonin pathway, depression, 3-hydroxykynurenine, flinders line rat, estrous cycle. 13

*Correspondence to: A. Eskelund: Translational Neuropsychiatry Unit, Institute for Clinical Medicine, Aarhus University, Skovagervej 4, 8240 Risskov, Denmark. Tel: +45-6178 0009. E-mail addresses: [email protected] (A. Eskelund), [email protected] (D. P. Budac), [email protected] (C. Sanchez), [email protected] (B. Elfving), [email protected] (G. Wegener). Abbreviations: 3-HAA, 3-hydroxyanthranilate (3-hydroxyanthranilic acid); 3-HK, 3-hydroxykynurenin; 5-HT, serotonin (5hydroxytryptamine); 5-HTP, 5-hydroxytryptophan; ANA, anthranilate (anthranilic acid); ANCOVA, univariate analysis of covariance; DI–DII, diestrus I and II (metestrous + diestrous); E, estrus; EDTA, ethylenediaminetetraacetic acid; FRL, Flinders Resistant Line; FSL, Flinders Sensitive Line; FST, forced swim test; K/T, kynurenine/ tryptophan; KYN, kynurenine; KYNA, kynurenate (kynurenic acid); LC–MS/MS, liquid chromatography coupled to tandem mass spectrometry; MANCOVA, multivariate analysis of covariance; MDD, major depressive disorder; NMDAR, N-methyl-D-aspartate receptor; NTA, nicotinamide; P, proestrus; PA, picolinate (picolinic acid); QUIN, quinolinate (quinolinic acid); TRP, tryptophan. http://dx.doi.org/10.1016/j.neuroscience.2016.05.024 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 1

INTRODUCTION

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Major depressive disorder (MDD) is a serious and debilitating mental disorder, estimated to affect the lives of 350 million people worldwide (WHO, 2012). The warrant for more research into the disease and more efficacious treatments is imperative as evidenced by the low remission rates with currently used antidepressants (Trivedi et al., 2006) and the rising prescription rate of these drugs for treatment of MDD (Pratt et al., 2011; Abbing-Karahagopian et al., 2014). The disease is twice as prevalent in women as in men (Holden, 2005) however, in spite of that preclinical research in neuroscience and pharmacology is mainly conducted on male animals (Beery and Zucker, 2011). The etiology and underlying pathophysiology of depression is largely unknown and the symptomatology varies among patients (Goldberg, 2011). However, the conversion of the amino acid tryptophan (TRP) into kynurenine (KYN) is an important pathway in the context of depression (see Fig. 1), as it is found to be dysregulated in subsets of depressed patients (Myint et al., 2007; Raison et al., 2010; Erhardt et al., 2013; Savitz et al., 2015). Stress hormones and inflammation have been shown to increase the formation of KYN from TRP by the enzymes TRP 2,3-dioxygenase (Gibney et al., 2014) and indoleamine 2,3-dioxygenase (Campbell et al., 2014), respectively. Because 95% of the essential amino acid TRP peripherally metabolizes through this pathway (Wolf, 1974; Ga´l and Sherman, 1980) and several of the metabolites cross the blood–brain barrier (Fukui et al., 1991), increased conversion of TRP into KYN kynurenine/tryptophan (K/T ratio) has been suggested as a mechanism underlying the serotonin (5-HT) depletion (Lapin and Oxenkrug, 1969) observed in depression.

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Fig. 1. Metabolism of tryptophan through kynurenine- and serotonin pathway. Above metabolites were measured in female FSL and FRL rats, bold indicate obtained measure in both plasma and brain, whereas the remaining metabolites were only obtained for either one. Enzymes gating the two pathways are indicated in grey.

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Studies in different populations of depressed patients largely concur on increased K/T ratio or increased production of neurotoxic intermediates over the neuroprotective kynurenate (KYNA) (Myint et al., 2007; Raison et al., 2010; Sublette et al., 2011; Hughes et al., 2012; Savitz et al., 2015) although there are conflicting results (Wood et al., 1978; Orlikov et al., 1994). Increased formation of metabolites through the KYN pathway can lead to neural dysfunction by several mechanisms. 3-hydroxykynurenine (3-HK) can generate free radicals and quinolinic acid (QUIN) can induce excitotoxicity being a glutamate receptor N-methyl-Daspartate (NMDAR) agonist (Chiarugi et al., 2001; Guillemin, 2012). However, KYN additionally gives rise to neuroprotective metabolites such as KYNA, which is an antagonist to the NMDAR (Perkins and Stone, 1982). Thus, TRP metabolites have the potential to modulate both serotonergic and glutamatergic neurotransmission, two neurotransmitter systems that are thought to be intimately involved in development of depression (Mu¨ller and Schwarz, 2007; Miller, 2013). Traditionally, female animals have been excluded from preclinical studies to ensure a homogenous sample devoid of the hormonal fluctuations caused by the estrous cycle. Estrogens have been suggested to alter 5-HT synthesis (Hiroi et al., 2006; Bethea et al., 2009) and decrease the KYN pathway metabolism (Wolf et al., 1980; Bender and Totoe, 1984; Shibata and Toda, 1997) possibly by inhibiting the expression (Bethea et al., 2009) or activity of the enzymes (Rose and Brown, 1969). Additionally, estrogens have been suggested to influence TRP metabolism indirectly by acting on the inflammatory system (Kovats, 2015) or the hypotha lamic–pituitary–adrenal axis (Quinn et al., 2014). In the rodent, the estrous cycle lasts 4–5 days and consists of 4 stages: Proestrus (P), estrus (E), metestrus and diestrus (D), although typically met- and diestrus are combined as DI and DII. There is a characteristic surge of progesterone, prolactin, estradiol, follicle stimulating hormone and leuthenizing hormone in the P phase (Smith et al., 1975) and therefore, behavioral changes induced by variations of estrous cycle hormones would be expected to be most evident at this stage.

Flinders Sensitive Line (FSL) rats have been studied as a genetic model of depression for more than 25 years and there is a good face, construct, and predictive validity for male rats (Overstreet and Wegener, 2013), including alterations in the serotonergic system (Overstreet et al., 2005). However, the female counterpart is less investigated and the parallel metabolism of TRP through both 5-HT and KYN is still uncharacterized. Therefore, the aim of the present study was to investigate the potential of female FSL rats as a model of depression and the impact of the estrous cycle on behavior and TRP metabolism. We used the forced swim test (FST) as a model of depression-like behavior and open field to asses locomotor ability and agoraphobia as an indicator for anxiety-related behavior. 13 TRP metabolites were measured in both plasma and brain tissue. Furthermore, as clinical studies often rely on plasma samples, we investigated the alignment between peripheral and central TRP metabolism.

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EXPERIMENTAL PROCEDURES

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Experiment 1

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Animals. Nulliparous, FSL and Flinders Resistant Line (FRL) rats were obtained from the breeding facility maintained at Translational Neuropsychiatry Unit, Aarhus University (Risskov, Denmark). Animals were housed in pairs at 22 ± 1 °C and on a 12-h light/dark cycle (lights on at 06:00 am) with ad libitum access to a standard diet (Purina) and tap water. Cages were enriched with paper nesting material, a gnaw stick, and a hiding place. Male rats were co-housed in the same room. All animal procedures were approved by the Danish National Committee for Ethics in Animal Experimentation (permission id: 2012-15-2934-00254). Experiments were conducted in three cohorts (June, July and February), only the first two cohorts had metabolites measured, but all three cohorts were included in the behavioral study (Table 1). Based on power calculations from a previous study (Wegener et al., 2012), a minimum of 28 animals per strain were intended to be included (a = 0.05, 1 b = 0.8).

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Table 1. Statistical analyses of tryptophan metabolites measured in brain hemispheres from FSL and FRL rats. Data were analyzed by linear mixed model and interaction term excluded from the final reduced model if non-significant. Age was included as covariate and effect of strain, estrous cycle and brain lateralization are shown below FSL (n* = 31) FRL (n* = 28) median ± iqr

Statistical analyses Strain

Estrous cycle

Hemisphere side (L/R)

Hemisphere weights (R/L) (mg)

895 ± 115 n = 60

870 ± 140 n = 55

z = 0.46 p > 0.05

v2 = 6.75 p > 0.05

z = 0.96 p > 0.05

TRP (mg/g tissue) K/T ratio X 1000 KYN (ng/g tissue) 3-HK (ng/g tissue) ANA (ng/g tissue) QUIN (ng/g tissue) KYNA (ng/g tissue) NTA (mg/g tissue) 5-HTP (ng/g tissue) 5-HT (ng/g tissue) 5-HIAA (ng/g tissue)

7.4 ± 1.1 nh = 60 8.6 ± 4.7 nh = 59 59 ± 34 nh = 59 58 ± 14 nh = 60 0.89 ± 0.22 nh = 60 14 ± 4.0 nh = 60 2.6 ± 3.0 nh = 59 56 ± 1.8 nh = 59 7.6 ± 1.8 nh = 60 634 ± 122 nh = 60 631 ± 84 nh = 60

7.2 ± 0.64 nh = 55 8.9 ± 2.6 nh = 54 65 ± 23 nh = 54 43 ± 11 nh = 55 1.5 ± 0.48 nh = 55 15 ± 4.3 nh = 55 2.8 ± 3.0 nh = 54 56 ± 1.7 nh = 54 8.9 ± 3.4 nh = 55 665 ± 126 nh = 55 631 ± 68 nh = 55

p > 0.05 z = 0.15 p > 0.05 z = 0.92 p > 0.05 z = 1.0 p < 0.001a z = 5.6 p < 0.001a z = 10.3 p < 0.05 z = 2.0 p > 0.05 z = 0.29 p > 0.05 z = 1.01 p < 0.01b z = 3.1 p > 0.05 z = 0.05 p > 0.05 z = 0.65

p > 0.05 v2 = 2.24 p > 0.05 v2 = 0.65 p > 0.05 v2 = 0.38 p > 0.05 v2 = 1.64 p > 0.05 v2 = 0.32 p > 0.05 v2 = 0.21 p > 0.05 v2 = 0.35 p > 0.05 v2 = 4.99 p > 0.05 v2 = 0.61 p < 0.05 v2 = 10.6 p < 0.05 v2 = 2.8

p > 0.05 z = 0.82 p > 0.05 z = 0.04 p > 0.05 z = 0.22 p > 0.05 z = 0.51 p > 0.05 z = 0.97 p > 0.05 z = 0.18 p > 0.05 z = 0.45 p > 0.05 z = 0.63 p > 0.05 z = 0.39 p > 0.05 z = 0.75 p > 0.05 z = 0.12

nh refers to the total number of hemispheres included in the analyses after exclusion of outliers. * n refers the number of rats (unit of analysis) of which 1 or 2 hemispheres were included. a p < 0.001 after correction for multiple comparisons. b p < 0.01 after correction for multiple comparisons. 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156

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Experimental design. To investigate the estrous cycle status, vaginal lavages were obtained daily between 8:00–11:00 am for a period of at least 11 days, to allow for a minimum of two regular cycles. Depression-like behavior was assessed using a modified 2-day FST (Porsolt et al., 1977; Slattery and Cryan, 2012), where rats were exposed to a 15-min pre-swim session 24 h before the experimental test day (swim 6 min). To investigate locomotor ability, rats were assessed in an open field prior to the FST on the test day. The open field was additionally used to assess anxiety-like behavior by investigating agoraphobia. We choose to euthanize animals on the same day as FST to ensure that animals were in the same stage of the estrous cycle for both behavior and metabolite measures. The time point 1.5 h was chosen to allow rats to alleviate from some of the stress associated with FST, while remaining under comparable estrous hormonal influence at both FST and decapitation. The median age of the rats were FSL = 99 ± 15 days and FRL = 101 ± 25 days and their weights 3 days prior to FST were FSL = 195 ± 28 g, FRL = 217 ± 27 g. Right and left hemisphere and plasma was obtained and analyzed for content of TRP metabolites (Fig. 1) by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). All behavioral experiments were carried out between 10:00 and 13:30 am. Estrous state determination. The estrous state was determined by visual inspection of the vaginal lavage

under bright field microscopy as previously described (Marcondes et al., 2002; Goldman et al., 2007) using a phase contrast condenser. 10–20 ll 0.9% sterile saline was used to obtain the lavages. P was characterized by the presence of predominantly round, nucleated cells; E by mostly agranular, cornified cells; DI was the first day with leukocytes being present; and any remaining days with leukocytes being present were classified as DII. The estrous state on the test day was used in the analyses. Animals with an irregular cycle were excluded from the study.

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Forced swim test. FST was conducted in a cylinder (diameter 24 cm, height 60 cm) filled with 40 cm of water at a temperature of 24 ± 1 °C and behavior was recorded from the side of the cylinder using digital video equipment. Primarily immobile behavior (defined as motions necessary to keep the head above water or floating) were evaluated manually in 5 s time bins (Slattery and Cryan, 2012) for a total of 5 min, by a researcher blinded to the study. Animals were scored at least twice, until values were within 10 % or less from each other and the average value was used in the analysis.

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Open field. The total distance moved over 5 min in an open field (1 m  1 m) and the time spent in the 25% inner center were measured automatically using EthoVision Ò

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XT11 (Noldus Information Technology) enabled for 1point detection (center). Tissue preparation. Animals were euthanized by decapitation and brains were quickly removed, hemisectioned and flash frozen in 30 °C isopentane. Trunk blood was collected in K3-EDTA coated tubes (Terumo, Venosafe , VF-053STK) standing on ice and centrifuged at 3400g for 10 min at 4 °C. The resulting plasma fraction and brain tissues were stored at 80 °C until analysis. TM

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Experiment 2

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Additionally, we obtained plasma from naı¨ ve, male FSL and FRL rats (n = 6), 12–13 weeks old that had not been handled. Animals were killed and plasma obtained as described in experiment 1.

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Measurements of TRP metabolites by LC–MS/MS

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Samples were prepared and run on LC–MS/MS as previously described (Mazarei et al., 2013) with minor modifications. Tissues and plasma were diluted at a factor  5 (w/v and v/v, respectively) in 0.2% acetic acid containing stable isotope labeled internal standards and homogenized for 5 min using an Omni-Prep Multi-Sample Homogenizer with disposable blades (Fisher Scientific). Internal standard concentrations were 13 50 ng/ml (13C15 C6-KYN, 13C6-3-HK, 2H511N2-TRP, 13 2 KYNA, 13C15 N-QUIN, C -3-HAA, H4-PA, 2H4-NTA) or 4 6 2 25 ng/ml ( H4-5-HT). Samples were filtered in 3-kDa Amicon Ultra filters for 60 min at 13,500g at 4 °C followed by injection onto the LC–MS/MS. Concentrations were estimated on an average of triplicate measurements and coefficients of variation (%CV) were within ±11%. TRP, KYN, 3-HK, 5-hydroxytryptophan (5-HTP), 5-HT, nicotinamide (NTA), anthranilate (ANA), QUIN and KYNA were measured and analyzed for both brain and plasma. In brain tissue, 3-hydroxyanthranilate (3-HAA) and xanthurenate (XAN) were below or bordering the detection limit and picolinate (PA) had an interfering peak, thus these metabolites were only considered for plasma. Contrary, 5-hydroxyindoleacetate (5-HIAA) was only determined for brain due to high % CV in plasma (>15%). Values for brain analyses were converted into ng/g tissue.

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Statistics

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Data were analyzed using Stata Statistical Software: Release 14 (StataCorp. 2015. College Station, TX: StataCorp LP) and age was included as covariate in all tests on female rats. Behavioral tests were analyzed for effects of strain and estrous cycle by a full factorial 2way analysis of covariance (ANCOVA), where immobility, locomotor activity and time spent in center of open field were log-transformed to fulfill the assumption of homoscedasticity. Log transformed brain metabolite levels and weight of hemispheres were compared by linear mixed model with repeated measures using a topdown approach. Fixed effects included strain, estrous cycle, brain region (R/L hemisphere). Since some

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metabolites were not measurable in both plasma and brain, and data therefore not missing at random, plasma metabolites were log transformed and analyzed separately from the brain by a full factorial 2-way multivariate analysis of variance (MANCOVA), followed by appropriate tests. Pearson correlations were performed on log-transformed brain (average) and plasma metabolite concentrations. Metabolites in plasma from male rats were compared by multiple, unpaired t-test and corrected for multiple comparisons as described below. Observations were excluded as outliers if their standardized residuals >3. For data within each analysis, the p - value was adjusted for multiple comparisons by Bonferroni correction (three comparisons in behavior, 11 comparisons for brain metabolites, 14 comparisons for plasma and 18 comparisons in the correlational analyses). Data are shown as median ± interquartile intervals on raw data and *p < 0.05, **p < 0.01, ***p < 0.001 after adjustment for multiple comparisons.

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RESULTS

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Behavior

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After correcting for multiple testing, the FSL rats showed a higher degree of immobility in the FST than the FRL rats (Fig. 2A, F(1,65) = 11.99, p < 0.01). Furthermore, the FSL rats were significantly more active in the open field compared to the FRL rats (Fig. 2B, F(1,63) = 37.4, p < 0.001), but time spent in the center was similar (Fig. 2C, F(1,65) = 0.47, p > 0.05). There was no interaction or effect of estrous state on any of the behavioral measures (strain  estrous state, immobility, F(3,65) = 0.51, p > 0.05; locomotor activity, F(3,63) = 1.15, p > 0.05; time spent in center, F(3,65) = 0.25, p > 0.05; Estrous state, immobility, F(3,65) = 0.14, p > 0.05; locomotor activity, F(3,63) = 2.4, p > 0.05; time spent in center of open field, F(3,65) = 0.68, p > 0.05).

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Brain levels of TRP metabolites

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Overall, 3-HK levels were 35% higher in FSL rats (p < 0.001 after multiplicity correction), whereas ANA and 5-HTP levels were lower (40%, p < 0.001 and 20%, p < 0.05, respectively). Furthermore, there was no effect of estrous cycle on the metabolite levels and there were no indications of hemispheric differences in metabolite levels (Table 1). Fig. 4 shows the average values of the two hemispheres where the altered levels of 3-HK (Fig. 3C) ANA (Fig. 3D) and 5-HTP (Fig. 3G) are clearly noticeable. Outliers were excluded from 4 subjects as described under the statistics section and consisted of the following measurements: FRL, E, left hemisphereNTA = 68.5 mg/g, FSL, P, left hemisphereNTA = 50.2 mg/g, and measurements from two rats (FRL, E, left hemisphere and FSL, D1, left hemisphere) were excluded for both KYN (522.1 mg/g and 741.4 mg/g, respectively), KYNA (53.5 mg/g and 64.3 mg/g, respectively) and corresponding K/T ratio  1000 (69.4 and 97.3, respectively). Additionally, the weight of

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FRL

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AGORAPHOBIA FRL

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C

FSL

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distance walked (meter/5 min)

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LOCOMOTOR ACTIVITY

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time (sec/5 min)

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Strain x Estrous cycle

time spent in center (% of 5 min)

A

FSL

40 30 20 10 0 -10

D2 P E D1

D2 P E D1

Strain x Estrous cycle

Fig. 2. Female FSL rats display depression-like behavior in the forced swim test (FST) independent of estrous cycle. During the 5 min FST, FSL rats spent more time being immobile than their counterpart control, FRL rats (A). The locomotor activity was tested prior to FST for 5 min in an open field where the FSL rats were more active than the FRL rats (B) and there was no change in the time spent in the center of the open field (C).

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the hemispheres did not vary with strain, estrous cycle or age and there was no difference on the weight of the rightand left side hemisphere (Table 1).

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Plasma levels of TRP metabolites

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A two-way MANCOVA demonstrated a significant effect of strain on metabolite levels in female rats (strain, F(12,38) = 20.7, p < 0.001, K = 0.13) and age (F(12,38) = 2.4, p < 0.05, K = 0.57), but not of estrous cycle (Estrous cycle, F(36,113) = 1.1, p > 0.05, K = 0.40) and there was no interaction (strain  estrous cycle, F(36,113) = 0.89, p > 0.05, K = 0.48). Follow-up, multiple one-way ANCOVA for effect of strain (Table 2), showed that ANA (Fig. 4D, F(1,57) = 228.9, p < 0.001), QUIN (Fig. 4F, F(1,57) = 25.0, p < 0.001) and PA (Fig. 4G, F(1,57) = 19.4, p < 0.001) levels were significantly lower in the plasma of the FSL rats compared to the FRL rats (28%, 28% and 58%, respectively). Outliers were excluded from three subjects (FRL, D2NTA = 1814 ng/ml, FSL, D15-HTP = 4.26 ng/ml and FSL, D1KYN, KYNA and K/T ratio, 1134,7 ng/ml, 68.3 ng/ml and 21.6 ng/ml, respectively) (Fig. 5). We compared plasma from naı¨ ve male FSL and FRL rats and only found significant strain differences in QUIN, ANA and NTA, which remained significant after adjustment for multiple comparisons (Fig. 6) (ANA: FSL = 0.9 ± 0.2 ng/ml, FRL = 1.5 ± 0.3 ng/ml, t(df=10) = 5.4, p < 0.01. QUIN: FSL = 109 ± 36 ng/ml, FRL = 176 ± 90 ng/ml, t(df=10) = 3.9, p < 0.05. PA: FSL = 11.9 ± 1.6 ng/ml, FRL = 14.9 ± 3.2 ng/ml, t(df=10) = 1.3, p > 0.05. NTA: FSL = 334 ± 78 ng/ml, FRL = 511 ± 186 ng/ml, t(df=10) = 4.1, p < 0.05).

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Compartmental correlations in female rats

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To assess the alignment between TRP metabolism in the brain and plasma, Pearson’s correlation coefficients were determined for each metabolite. There were significant correlations between TRP, QUIN, 3-HK and ANA (Fig. 7), for the remaining metabolites a linear relations ship was not present (data not shown).

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DISCUSSION

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The inclusion of female animals in preclinical depression research is important given the higher prevalence of depression in women compared to men. Expanding this

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line of research with the scope of improving the translatability from preclinical animal models to the clinic setting will hopefully lead to development of more efficacious treatment options. The present study shows, for the first time, that female FSL rats display a depression-like behavior in the FST compared to FRL rats, independent of estrous cycle. Furthermore, several TRP metabolite levels are altered in female FSL rats and did not fluctuate with estrous cycle stage.

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Female FSL rats display depression-like behavior

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We show that female FSL rats are more immobile in the FST as compared to FRL rats, when planning group sizes according to a power calculation. Large population samples are warranted due to the high level of variance the female FSL rat display in immobility as seen in Figure 2. Because FSL rats showed increased activity in the open field compared to FRL rats, the increased immobility of FSL rats in the FST could not be explained by a difference in basal locomotor activity. Furthermore, since FSL and FRL rats showed the same level of exploration of the center of the open field, the results in the FST were not confounded by an anxiety-like behavior. This corroborates findings for male FSL rats (Overstreet et al., 2005). The estrous cycle did not influence the behaviors in the FST and open field test of FSL or FRL rats. There are conflicting reports on the impact of estrous cycle on the outcome in the FST [reviewed in 40], however, to our knowledge, no studies have found the estrous cycle to impact the FST results in Sprague–Dawley rats, which is the founding strain for the selectively bred FSL and FRL rats (Overstreet and Wegener, 2013).

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Altered TRP metabolism in FSL rats

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We found significantly altered levels of several metabolites in the TRP metabolism in female FSL rats, which did not vary with estrous cycle stage. In the brain, we found increased 3-HK levels, while ANA and 5-HTP levels were decreased compared to FRL rats. In plasma QUIN, PA and ANA were decreased. The increased brain 3-HK concentration in FSL rats is particularly noteworthy, as it concurs with results from studies of another animal model of depression, the chronic mild stress model (Laugeray et al., 2010;

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TRP BRAIN FRL

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KYN BRAIN FRL

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D2 P E D1

Strain x Estrous cycle D2 P E D1

5-HTP BRAIN FRL

D2 P E D1

5 0

(ng/g hemisphere)

FSL

***

80 60 40 20 0

D2 P E D1

D

ANA BRAIN FRL 2.5

D2 P E D1

1.5 1.0 0.5 0.0

D2 P E D1

D2 P E D1

5-HT BRAIN FRL 800 700 600 500 400

I

5 D2 P E D1

NTA BRAIN FSL

FRL

J

50

KYNA BRAIN FRL

FSL

0.7 0.6 0.5 0.4

K

D2 P E D1

D2 P E D1

Strain x Estrous cycle

D2 P E D1

D2 P E D1

FRL

FSL

15

10 5 0

K/T RATIO BRAIN 20

(X 1000)

(ng/g hemisphere)

(mg/g hemisphere)

65

55

FSL

Strain x Estrous cycle

15

60

D2 P E D1

5-HIAA BRAIN

Strain x Estrous cycle

FRL

D2 P E D1

0.8

10

F

FSL

Strain x Estrous cycle

FSL

15

D2 P E D1

D2 P E D1

900

Strain x Estrous cycle

20

0

H

(mg/g hemisphere)

(ng/g hemisphere)

25

QUIN BRAIN FRL

***

2.0

Strain x Estrous cycle

E

FSL

(ng/g hemisphere)

FRL

D2 P E D1

Strain x Estrous cycle

(ng/g hemisphere)

3-HK BRAIN 100

*

10

Strain x Estrous cycle

C

FSL

15

7

50 0

G

9

(ng/g hemisphere)

B

(mg/g hemisphere)

10

D2 P E D1

D2 P E D1

Strain x Estrous cycle

10 5 0

D2 P E D1

D2 P E D1

Strain x Estrous cycle

Fig. 3. Effect of strain but not estrous cycle on brain tryptophan metabolite levels. 3-HK levels were higher in the hemispheres of FSL rats (C), whereas ANA (D) and 5-HTP (G) were lower compared to FRL rats. For graphical representation, the average of the two hemispheres is shown, but the statistical values are obtained from analyses on the individual hemispheres, corresponding to Table 1.

382 383 384 385 386 387 388 389 390 391 392 393

Agudelo et al., 2014). Furthermore, a role for 3-HK has been indicated in depressed patients (Claes et al., 2011; Savitz et al., 2014). We tried to perform correlational analyses with the immobility from FST, but did not find a monotonic relationship with any of the metabolites in brain and plasma (data not shown). The chronically elevated 3HK in FSL rat brain (FSL = 258 nM, FRL = 191 nM) is not above in vitro neurotoxic levels (1–100 lM (Eastman and Guilarte, 1989; Chiarugi et al., 2001)), but higher than those reported by others measuring 3-HK in whole-cell brain homogenate from Sprague–Dawley rats (Heyes, 1988; Zheng et al., 2012) and regional concentrations

are likely to be higher. 3-HK interferes with energy metabolism (Chiarugi et al., 2001; Schuck et al., 2007) and causes cell death by apoptosis and disruption of the mitochondrial membrane potential (Lee et al., 2004) which is particular interesting given that FSL rats have altered mitochondrial plasticity. This includes reduced numbers of mitochondria (Chen et al., 2013) and proteins involved in oxidative phosphorylation (Piubelli et al., 2011), which could suggest that the FSL rat is particular vulnerable to increases in 3-HK. The possible implications of a reduced ANA in depression are unknown, as few studies have focused

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A

B

KYN PLASMA FRL

FRL (ng/ml plasma)

(ng/ml plasma)

600 400 200 D2 P E D1

40 20 0

D2 P E D1

Strain x Estrous cycle

C

XAN PLASMA FRL

FRL

8 6 4 2 0

FRL

D2 P E D1

100 50 0

D2 P E D1

D2 P E D1

6 4 2 0

D2 P E D1

Strain x Estrous cycle

Strain x Estrous cycle

E

FSL

***

8

150

D2 P E D1

ANA PLASMA

FSL

200 (ng/ml plasma)

(ng/ml plasma)

10

D

3-HK PLASMA

FSL

D2 P E D1

Strain x Estrous cycle

(ng/ml plasma)

I

FSL

60

800

0

KYNA PLASMA

FSL

D2 P E D1

D2 P E D1

Strain x Estrous cycle

3-HAA PLASMA FRL

FSL

(ng/ml plasma)

15 10 5 0

D2 P E D1

D2 P E D1

Strain x Estrous cycle

F

FRL

FSL (ng/ml plasma)

300 200 100 0

50

***

400 (ng/ml plasma)

G

QUIN PLASMA

D2 P E D1

20 10

FRL

FSL 20

D2 P E D1

K/T RATIO PLASMA FRL FSL

15

1000

(X 1000)

(ng/ml plasma)

1500

500 0

D2 P E D1

Strain x Estrous cycle

J

NTA PLASMA

***

30

Strain x Estrous cycle

H

FSL

40

0

D2 P E D1

PA PLASMA FRL

D2 P E D1

D2 P E D1

Strain x Estrous cycle

10 5 0

D2 P E D1

D2 P E D1

Strain x Estrous cycle

Fig. 4. Effect of strain but not estrous cycle on plasma kynurenine metabolite levels. ANA (D), QUIN (F), and PA (G) levels were lower compared to FRL rats. ***p < 0.001 after correction for multiple comparisons.

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on the role of this metabolite. Although it has been suggested not to have direct effects on neuronal activity (Stone, 1993), it may still have beneficial impact on the neural environment as a metal chelator with antioxidant properties (Reyes Ocampo et al., 2014; Chobot et al., 2015).

Our results do not corroborate earlier reports of increased 5-HT levels (Zangen et al., 1997; Kokras et al., 2009) or decreased 5-HT synthesis (Hasegawa et al., 2006) in FSL rats compared to FRL rats. This may be explained by our measures being conducted on whole hemispheres as strain differences are brain

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Table 2. Statistical analyses of tryptophan metabolites in plasma, using 2-way MANCOVA and multiple one-way ANCOVAs for follow-up. Age was included as covariate Multiple ANCOVAs of plasma metabolites n

FRL

n

Strain

751 ± 163

31

736 ± 236

26

PA (ng/ml)

20.1 ± 10.9

31

30.2 ± 16.0

27

QUIN (ng/ml)

117 ± 46

31

179 ± 68.1

27

3-HK (ng/ml)

109 ± 35.1

31

108 ± 68.3

27

5-HT (ng/ml)

662 ± 362

31

470 ± 442

27

5-HTP (ng/ml)

1.99 ± 0.60

30

1.83 ± 0.92

27

KYN (ng/ml)

387 ± 179

31

431 ± 213

27

3-HAA (ng/ml)

4.06 ± 3.0

30

5.12 ± 4.8

27

TRP (mg/ml)

48.5 ± 5.4

31

44.6 ± 8.15

27

XT (ng/ml)

4.83 ± 2.84

31

5.84 ± 2.72

27

KYNA (ng/ml)

18.9 ± 7.3

30

26.0 ± 15.0

27

ANA (ng/ml)

1.92 ± 0.66

31

4.70 ± 1.7

27

K/T ratio (X1000)

7.8 ± 3.6

30

9.90 ± 2.4

27

p > 0.05 F(1,56) = 0.40 p < 0.001a F(1,57) = 19.4 p < 0.001a F(1,57) = 25.0 p > 0.05 F(1,57) = 1.3 p < 0.01 F(1,57) = 7.6 p > 0.05 F(1,55) = 0.00 p < 0.05 F(1,56) = 4.7 p > 0.05 F(1,57) = 3.4 p > 0.05 F(1,57) = 2.9 p > 0.05 F(1,57) = 2.0 p < 0.01 F(1,56) = 8.9 p < 0.001a F(1,57) = 228.9 p < 0.01 F(1,56) = 8.49

p < 0.001 after correction for multiple comparisons.

A

B

TRP PLASMA FRL

FRL

C

60 50 40 D2 P E D1

D2 P E D1

FRL

Strain x Estrous cycle

FSL

1500

3 2 1 0

5-HT PLASMA

FSL

4 (ng/ml plasma)

(mg/ml plasma)

70

30

5-HTP PLASMA

FSL

(ng/ml plasma)

a

FSL NTA (ng/ml)

D2 P E D1

D2 P E D1

Strain x Estrous cycle

1000 500 0

D2 P E D1

D2 P E D1

Strain x Estrous cycle

Fig. 5. There were no significant effects of strain or estrous cycle on TRP metabolites along the serotonergic arm of the pathway in plasma.

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region-dependent in previous studies (Zangen et al., 1997; Kokras et al., 2009). Additionally, our analyses are conducted on brain tissue obtained after FST, which is a limitation of our study. However, our measured median 5-HT levels are within the range of previously reported mean 5-HT concentrations for non-swimming FSL rats, both male and female (Kokras et al., 2009). Since earlier studies have shown conflicting report of either increased (Gibney et al., 2014) or decreased (Ara and Bano, 2012) activity of TDO with stress, we could hypothesize that FST may have affected our results. Therefore, we analyzed the plasma metabolite levels in both the females as well as plasma obtained from unhandled, naı¨ ve male counterparts. In both experiments we found decreased plasma QUIN and ANA in FSL rats,

and conclude that this is indeed a strain effect independent of FST and sex. However, the decreased PA in females and lower NTA levels in male rats makes it difficult to conclude whether these strain differences are sex dependent or vary with the experimental setup. Thus, more experiments are warranted to investigate these strain differences further. Nevertheless, it is interesting that reduced levels of QUIN, PA and ANA occur in female FSL rats as they could be involved in the altered the immune system (Espey et al., 1995; Cai et al., 2006; Darlington et al., 2010; Sasaki et al., 2012) found in this strain (Overstreet et al., 2005; Carboni et al., 2010; Strenn et al., 2015). Overall, the FSL rat does not display an increased K/T ratio or 5-HT depletion in our studies. Altered 5-HT

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MALE PLASMA FRL 2.0

300

FRL

*

1.0 0.5

D

PA PLASMA

FSL

200 ng/ml

ng/ml

FRL

FSL

**

1.5

C

QUIN PLASMA

NTA PLASMA

FSL

FRL

20

1000

18

800

16

100

ng/ml

B

ANA PLASMA

ng/ml

A

14 12

600 400

8

0

Naïve males

Naïve males

*

200

10 0

0.0

FSL

Naïve males

Naïve males *

**

Fig. 6. Significant lower levels of QUIN, ANA and NTA in plasma from non-handled, naı¨ ve male FSL rats. p < 0.05, p < 0.01 after correction for multiple comparisons.

B

TRP

LOG(Plasma)

11.0

FRL

11.0

10.9

10.9

10.8

10.8

10.7

10.7 r = 0.13 p > 0.05

10.6

3-HK

FSL

5.5 LOG(Plasma)

A

r = 0.62 p < 0.05

10.6

10.5 10.5 1.6 1.8 2.0 2.2 2.4 1.6 1.8 2.0 2.2 2.4

FRL

5.5 5.0

5.0 4.5

r = 0.56 p < 0.05

D

1.5

1.5

1.0

FRL

1.0 r = 0.85 p < 0.001

0.5

r = 0.75 p < 0.001

0.0 0.0 -7.5 -7.0 -6.5 -6.0 -5.5 -8.0 -7.5 -7.0 -6.5 -6.0 LOG(Brain)

LOG(Plasma)

LOG(Plasma)

2.0

0.5

QUIN

FSL

2.0

r = 0.53 p < 0.05

LOG(Brain)

ANA FRL

4.5

4.0 4.0 -3.6-3.4-3.2-3.0-2.8-2.6 -3.4-3.2-3.0-2.8-2.6-2.4-2.2

LOG(Brain)

C

FSL

FSL

5.5

5.5

5.0

5.0

4.5 4.0 -5.0

r = 0.60 p < 0.05 -4.5

-4.0

-3.5

4.5 4.0 -5.0

r = 0.82 p < 0.001 -4.5

-4.0

-3.5

LOG(Brain)

Fig. 7. Significant correlations between brain and plasma levels for some tryptophan metabolites in FSL and FRL rats; Spearman’s correlation coefficients were determined for each metabolite, however, only TRP, 3-HK, ANA and QUIN showed linear relationship. Data are log-transformed: LOG (brain) on x-axis and LOG (plasma) on y-axis. p values are shown after correction for multiple comparisons. 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464

metabolism may not be evident when examining the whole FSL rat brain, but may be brain-region dependent. Additionally, 5-HT has been found altered in specific brain regions (Cheetham et al., 1989), cerebrospinal fluid and blood samples from depressed patients, though not consistently (Mann, 1999). The K/T ratio is maintained by TRP 2,3-dioxygenase and indoleamine 2,3-dioxygenase, which are inducible by cortiocosteroids or pro-inflammatory cytokines, respectively. Thus, as the FSL rat is a model with inherent depression-like phenotype, the animals have not been exposed to chronic stress or an inflammatory-related insult, which may be necessary to cause a general induction of the whole pathway downstream KYN. Thus, further studies are needed to elucidate the mechanism of these discrete changes found in TRP metabolites of the FSL rat. As the etiology of depression is complex, more stud-

ies examining the TRP metabolism in animal models of depression are warranted.

465

Correlation between the peripheral and central compartments

467

Our study suggests that the TRP metabolism is differently regulated in the periphery and the brain. Correlational analysis showed a strong association between peripheral and the cerebral ANA levels, which is enabled by passive diffusion (Fukui et al., 1991) and could suggest that the peripheral ANA dominate the cerebral levels. Contrary, 3-HK showed a more moderate correlation, which is a metabolite that uses the large neutral long chain carrier for transport across the blood–brain barrier and therefore is susceptible to competing amino acids. The specific upregulation in the brain could be caused

469

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by inherently increased expression of enzymes regulating the level of this enzyme, although further analyses of enzymes and potential regulators of enzymes (e.g. cytokines) are required to elucidate this further. Interestingly, brain and plasma QUIN levels correlated strongly in both strains, although this metabolite does not cross the blood–brain barrier (Fukui et al., 1991). Brain and plasma levels of TRP correlated only significantly in the FSL rat, although there appears to be a tendency for a linear pattern for FRL rats as well. We did not food deprive rats in order to find natural occurring metabolite levels and the FRL rats have increased appetite compared to FSL rats (Overstreet et al., 2005). Thus, the most likely explanation is that FRL rats may have been eating more recent than FSL rats, which could explain why plasma and brain TRP only correlate in FSL rats.

496

No effect of estrous cycle on TRP metabolites

497

533

Importantly, TRP metabolite levels in intact female FSL rat brain or plasma were not altered by the specific stages of the estrous cycle in our study. Estrogenic hormones have been reported to regulate several enzymes of the KYN pathway in vivo in ovariectomized rats (Bender and Totoe, 1984; Xu et al., 2015) and macaque rhesus monkeys (Bethea et al., 2009). Additionally, there is evidence that estradiol may alter 5-HT levels in some brain regions (Hiroi et al., 2006; Bethea et al., 2009). However, common for these studies is that the levels of estrogens were supplemented for a prolonged duration as compared to the natural cycle (15 days– 12 weeks in the rat, corresponding to 5–20 cycles, and 29 days in the monkey, corresponding to one complete cycle). Thus, as indicated by our data the natural cycle may have little if any effect on TRP metabolism in FSL rats after FST. It is possible that altered expression of enzymes does not mirror altered levels of metabolites if this is a compensatory regulation due to increased flux and production of downstream metabolites, not measured in our study. Thus, further studies including enzyme expression are needed to find the underlying explanation between reports of altered enzyme expressions and the unaltered TRP metabolite levels in our experiment. One major limitation of our study is that we allowed random distribution of phases resulting in only one FRL rats in P. Thus, our results of estrous cycle effect on TRP metabolites are strongest for the FSL rat, where five animals were included. Therefore, we additionally tried one-way ANCOVAs within each strain to pick up effects of estrous cycle, disregarding the factorial design of the two-way ANCOVA. However, even with this method there was no effect of the estrous cycle on plasma or brain-average metabolite levels (data not shown).

534

CONCLUSION

535

We have demonstrated a significantly altered TRP metabolism and an E cycle-independent depression-like phenotype of female FSL rats, not confounded by

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536 537

anxiety in the open field test. We found discrete changes of TRP metabolites in both plasma and brain, rather than a general increase of the whole arm of neurotoxic metabolites. These findings included increased cerebral 3-HK, a neurotoxin that is increased in other animal models of depression and decreased ANA and 5-HTP. Plasma levels of ANA and QUIN were reduced in both female rats exposed to FST and naı¨ ve male FSL rats. Overall, our study suggests that the FSL rat is an interesting preclinical model of depression with altered TRP metabolism and that plasma is insufficient for studying the cerebral TRP metabolism, although several metabolites cross the blood–brain barrier.

538

AUTHOR CONTRIBUTIONS

551

All experiments were conducted by AE. DPB assisted in performing LC–MS/MS experiments. GW, CS, BE and AE designed the experiments. All authors assisted in interpretation of data, editing the manuscript and final approval of the manuscript.

552

FUNDING

557

This work was supported by Health Research Fund of Central Denmark Region, Graduate School of Health (Aarhus University), Christian & Ottilia Brorsons travel scholarship for younger scientists, and AU ideas initiative (eMOOD).

558

CONFLICTS OF INTEREST

563

None to declare.

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553 554 555 556

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564

UNCITED REFERENCE

565

Kokras et al. (2015).

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Acknowledgments—We are deeply grateful to Lundbeck Research USA, for providing instrument and materials to conduct the LC–MS/MS analyses. Big thanks also go to Gudrun Winter and Christina W Fisher for their help in executing the behavioral experiments.

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(Accepted 11 May 2016) (Available online xxxx)

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