Forced swim test: What about females?

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Neuropharmacology xxx (2015) 1e14

Contents lists available at ScienceDirect

Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm

Q5

Forced swim test: What about females?

Q4

N. Kokras a, b, K. Antoniou c, H.G. Mikail a, V. Kafetzopoulos a, Z. Papadopoulou-Daifoti a, C. Dalla a, * a

Department of Pharmacology, Medical School, University of Athens, Greece First Department of Psychiatry, Eginition Hospital, Medical School, University of Athens, Greece c Department of Pharmacology, Medical School, University of Ioannina, Greece b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 December 2014 Received in revised form 10 March 2015 Accepted 14 March 2015 Available online xxx

In preclinical studies screening for novel antidepressants, male and female animals should be used. However, in a widely used antidepressant test, the forced swim test (FST), sex differences between males and females are not consistent. These discrepancies may discourage the inclusion of females in FST studies. In order to overcome this problem and provide a detailed insight regarding the use of female animals in the FST, we designed the following experiment and we performed a thorough analysis of the relevant literature. Male and female Wistar adult rats were subjected to the FST and sertraline was used as an antidepressant in two doses (10 mg/kg and 40 mg/kg, 3 injections in 24 h). Rodents were subjected in the two FST sessions during all possible combinations of the estrous cycle stages. We found that females exhibited higher levels of immobility than males and this sex difference was alleviated following antidepressant treatment. Sertraline at both doses enhanced swimming in both sexes, but females appeared more responsive to lower sertraline doses regarding immobility levels. Surprisingly, the high sertraline dose enhanced climbing particularly in proestrous and diestrous. Marked sex differences were also observed in the frequency of head swinging, with females exhibiting lower counts than males. Conclusively, when screening for new antidepressants, it is recommended to use standard FST procedures and if possible to include females in all phases of the cycle. Using only one dose of an investigational drug in females in certain phases of the cycle could result to false negative results. © 2015 Published by Elsevier Ltd.

Keywords: Sertraline Depression Estrous cycle Gonadal hormones Rat Sex differences

1. Introduction The U.S. National Institutes of Health recently dictated that experimental studies should be ideally performed on both sexes (Clayton and Collins, 2014) and psychiatric research is in great need of better animal models that are validated in both males and females (Becker et al., 2005; Beery and Zucker, 2011; Dalla et al., 2010; Kokras and Dalla, 2014; Wald and Wu, 2010). The Forced Swim Test (FST) is perhaps the most widely used animal model for investigating the antidepressant potential of current and novel molecules in experimental pharmacological studies (Cryan et al., 2005; Porsolt, 1979). Since depression is twice as common in women as in men and sex differences are present in

* Corresponding author. Department of Pharmacology, Medical School, University of Athens, Mikras Asias 75, Goudi, 11527, Greece. Tel.: þ30 2107462577; fax: þ30 2107462554. E-mail address: [email protected] (C. Dalla).

antidepressant response (Gorman, 2006; Marcus et al., 2005; Sloan and Kornstein, 2003; Thiels et al., 2005), several studies have used both sexes in the FST paradigm (Tables 1e3), although this is not usually the case in research. For this, rats are placed in a cylinder filled with water and they are forced to swim for 15 min. Upon reexposure to this stressful event after 24 h, most rats express enhanced duration of passive behavior (i.e. immobility or floating) and decreased active behaviors (i.e. climbing and swimming). This behavioral response has been simulated with despair or helplessness in depressed humans (Cryan et al., 2002; Kirby and Lucki, 1997). Alternatively, it has been proposed that floating behavior disengages the animal from active forms of stress coping (Slattery and Cryan, 2012). Also, learning and memory properties are probably involved in FST behavioral response (Borsini and Meli, 1988; Thierry et al., 1984). Nevertheless, even though FST has low face validity, it has high predictive validity for antidepressant screening (Slattery and Cryan, 2012). It is well known that antidepressant agents decrease the duration of passive behavior and increase the duration of active

http://dx.doi.org/10.1016/j.neuropharm.2015.03.016 0028-3908/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Kokras, N., et al., Forced swim test: What about females?, Neuropharmacology (2015), http://dx.doi.org/ 10.1016/j.neuropharm.2015.03.016

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N. Kokras et al. / Neuropharmacology xxx (2015) 1e14

Table 1 1A and 1B Sex differences in the Forced Swim Test in published rat studies from 1990 to 2013. Differences between male and female rats are presented in immobility swimming and climbing duration, as well as latency to immobility, when available. The strain, the age of the animals (in weeks) and the phase of the cycle that FST was performed are indicated, when available. Variations in FST methodology, such as tank dimensions (height x diameter in cm), depth (cm) and temperature ( C) of the water, phase of the light (L) or dark (D) cycle that FST was performed, as well as duration (minutes) of one, two or repeated (R) FST sessions are also indicated. Values in parenthesis are not explicitly reported, but assumed from relevant clues or graphs. Strain: NIH ¼ National Institute of Health Rat Strain, WKY ¼ Wistar-Kyoto, F ¼ Fischer 344, SD ¼ SpragueeDawley, H ¼ Holtzman, W ¼ Wistar, LE ¼ Lewis, SHR ¼ Spontaneous Hypertensive, FRL/FSL ¼ Flinders Resistant/Sensitive Line, HAB/LAB ¼ High/Low Anxiety, WAG ¼ Wistar-AlbinoGlaxo, DA ¼ Dark Agouti, SERTKO ¼ Serotonin Transporter Knockout, SS/SR ¼ Swim test Susceptible/Resistant rats, Cycle: P ¼ Proestrous, E ¼ Estrous, D1 ¼ Diestrous I, Q2 D2 ¼ Diestrous II. A Authors

Animals

Females less immobile than Males (Gonzalez et al., 1990) (Alonso et al., 1991) (Walker et al., 1995) (Contreras et al., 1995) (Barros and Ferigolo, 1998) (Brotto et al., 2000) (Consoli et al., 2005) (Brummelte et al., 2006) (Yang et al., 2007) (Jans et al., 2007) (Rubino et al., 2008) (Morrish et al., 2009) (Kokras et al., 2009) (Mourlon et al., 2010) (Verma et al., 2010) (Sterley et al., 2011) (Huynh et al., 2011)

Strain H SD SD W W LE W SD W W SD SD FSL LE SD SHR

Age 12 (8e12) (10e12) 13 13 58 (7e9) 9e10 10e12 18 11 (10e15) 9e12 20 9 5

(Martinez-Mota et al., 2011) (Wegener et al., 2012)

W SD FRL FSL SD LE WMI

13 10e12

(Simpson et al., 2012) (Weathington et al., 2012) (Mehta et al., 2013) Females equally immobile to Males (Orpen and Steiner, 1995)

(Rayen et al., 2011) (Brummelte et al., 2012) (Hong et al., 2012) (Wilkin et al., 2012)

Strain WAG DA F, SHR LE, WKY LE W W W WKY LE SD SS/SR W SD W W W SERTKO SD HAB/LAB SD WKY W LE SHR SD SD SD LE

(Mehta et al., 2013)

WLI

(Fujimoto et al., 2013) (Warner et al., 2013) Females more immobile than Males (Pare and Redei, 1993) (Hill et al., 2003) (Frye and Wawrzycki, 2003)

W SD Strain WKY LE LE

(Armario et al., 1995) (Frye and Walf, 2002) (Papaioannou et al., 2002) (Bellido et al., 2003) (Lee et al., 2003) (Solberg et al., 2003) (Perrot-Sinal et al., 2004) (Poltyrev et al., 2005) (West and Weiss, 2005) (Fujimoto et al., 2006) (Ferguson et al., 2007) (Andrade et al., 2007) (Alves et al., 2008) (Olivier et al., 2008) (Kokras et al., 2009) (Slattery and Neumann 2010) (Verma et al., 2010) (Sterley et al., 2011) (Martinez-Mota et al., 2011) (Izidio et al., 2011)

FST methods

6 9e12 5 13e23 Age 13e23

Cycle P vs. E vs. D1 vs. D2

P vs. E vs. D1 vs. D2 45 28 P vs. E vs. D1 vs. D2

P þ E vs. D1 þ D2

E

Cycle

9 8 13 27 15 12 (8e10) 11e12 13e23 9 12e27 (11e12) 7e8 13e18

Tank 44 16 30 15 50 30 24 50 30

P vs. D1 þ D2

45 40 40 50 45 50 46 43 40 45

28 30 17 20 35 19 20 19.5 19 20

DeptheTemp 19e22 15e23 30e30 18e25 27e25 19e23 15e25 30e25 20e25 30e22 25e25 30e23 40e24 30e24 25/30e25 19e25 30e28

FST Sessions L//10 L/15/5 L/15/5 L/15/5 L/15/5 D/15/10 L/15/5 L/15/10 D/15/5 D/5/5 L/15/5 D/15/5 L//5 L/15/5 L/15/5 L/15/5 L/15/5 D/15/5 D/15/5 L/15/5

46 20 60 24

30e24 40e25

50 37

25e24 32e25 23

Tank 47 15

DeptheTemp 25e25

L/15/5 L/5/5R L//6 L/15/5 FST Sessions L/15/5

e 19

18e25

L//15 L//10 L/15/5 L/15/5 L/15/5 L/15/5 L//10 L/15/5 L//15 L/15 L/15/5 L/15/5 L/15/5 L/15/5

27 20 18

40 33 40 e 45 25 60 62 50 60 25 40 50

30 25 60 19 30 30 29 25 19 18

30e30 24 21e37 20e25 25 30e27 30e25 48e26 35e24 50e25 27e25 30e22 30e22

9e12 10 9 5 5 11

50 50 43 40 46 40

19 29 19.5 19 20 18

40e24 30e25 25/30e25 19e25 30e24 20e26

L//5 L//10 D/15/5 L/15/5 D/15/5 L/15/5

4e5 6e7 7 11 13 5 13e23 9 (7) Age 10e12 81 8

50 20 45 28

20e27 30e25 37e25 15/20e25

L//10 L/15/10 L/15/5 L/15/5

35e24 30e24 DeptheTemp 30e25 30e21 30 30

L//6 L/15/5 L//15 L//5 FST Sessions L//15 D/15/10 D//10

P vs. D2

40 18

Cycle P þ E vs. D1 þ D2 D1 þ D2

50 30 46 20 Tank 45 30 45 35 40 27


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Table 1 (continued ) A Authors

Animals

(Solberg et al., 2003) (Drossopoulou et al., 2004) (Zimmerberg et al., 2005) (Wilcoxon et al., 2005) (Wilcoxon and Redei, 2007) (Leussis and Andersen, 2008) (Tonelli et al., 2008) (Dalla et al., 2008) (Pitychoutis et al., 2009) (Andrade et al., 2010) (Negrigo et al., 2011) (Pitychoutis et al., 2011) (Toledo-Rodriguez and Sandi, 2011) (Allen et al., 2012) (Hong et al., 2012) (Kokras et al., 2012) (Wibrand et al., 2013)

F W NIH SD SD SD F W SD W W SD W SD SD W W

FST methods 12 12e13 11 9e10 9e10 5 (11e12) 12e13 12e13 (11e12) 10 13 7 6 10 16 13

45 60 40 45 30 25 60 60 60 22 50 60 46 65

P vs. D2

P þ E vs. D1 þ D2

P vs. D2

P þ E vs. D1 þ D2 P þ E vs. D1 þ D2

25 40e24 30e30 25 45e25 40e25 35e25 40e24 40e24 27e25 25e34 40e24 30e25 48e25 37e25 40e24 30e25

30 38 27 30

22.5 38 38 22 35 25 38 25 25

50 19 50 20

L/15/5 L/15/5 L//10 L/15/5 L/15/5 L/15/5 L/10/5 L/15/5 L/15/5 D/15/5 L/15/5 L/15/5 L/15/5 D/15/5 L/15/5 L/15/5 L/15/5

B Authors

Sex differences

Females less immobile than Males (Gonzalez et al., 1990) (Alonso et al., 1991) (Walker et al., 1995) (Contreras et al., 1995) (Barros and Ferigolo, 1998) (Brotto et al., 2000) (Consoli et al., 2005) (Brummelte et al., 2006) (Yang et al., 2007) (Jans et al., 2007) (Rubino et al., 2008) (Morrish et al., 2009) (Kokras et al., 2009) (Mourlon et al., 2010) (Verma et al., 2010) (Sterley et al., 2011) (Huynh et al., 2011) (Martinez-Mota et al., 2011) (Wegener et al., 2012) (Simpson et al., 2012) (Weathington et al., 2012) (Mehta et al., 2013) Females equally immobile to Males (Orpen and Steiner, 1995) (Armario et al., 1995) (Frye and Walf, 2002) (Papaioannou et al., 2002) (Bellido et al., 2003) (Lee et al., 2003) (Solberg et al., 2003) (Perrot-Sinal et al., 2004) (Poltyrev et al., 2005) (West and Weiss, 2005) (Fujimoto et al., 2006) (Ferguson et al., 2007) (Andrade et al., 2007) (Alves et al., 2008) (Olivier et al., 2008) (Kokras et al., 2009) (Slattery and Neumann 2010) (Verma et al., 2010) (Sterley et al., 2011) (Martinez-Mota et al., 2011) (Izidio et al., 2011) (Rayen et al., 2011) (Brummelte et al., 2012) (Hong et al., 2012) (Wilkin et al., 2012) (Mehta et al., 2013) (Fujimoto et al., 2013)

FM

FM

FM

FM

X

X

X X

X X

X X

X X

X

X

X

X

X X X

X

X X

X FM

FM

FM

FM

X

X X X

X

X X X X

X

X

X

X

X

X X

X X

X

X

X

X X

X X

X X

X

X (continued on next page)


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Table 1 (continued ) B Authors

Sex differences Immobility

(Warner et al., 2013) Females more immobile than Males (Pare and Redei, 1993) (Hill et al., 2003) (Frye and Wawrzycki, 2003) (Solberg et al., 2003) (Drossopoulou et al., 2004) (Zimmerberg et al., 2005) (Wilcoxon et al., 2005) (Wilcoxon and Redei, 2007) (Leussis and Andersen, 2008) (Tonelli et al., 2008) (Dalla et al., 2008) (Pitychoutis et al., 2009) (Andrade et al., 2010) (Negrigo et al., 2011) (Pitychoutis et al., 2011) (Toledo-Rodriguez and Sandi, 2011) (Allen et al., 2012) (Hong et al., 2012) (Kokras et al., 2012) (Wibrand et al., 2013)

FM X X X X X X X X X X X X X X X X X X X X

FM

Climbing X F¼M

FM

FM

were not reducing reliably immobility and were considered false negatives. Even in the modified FST, described by Detke et al., in 1995, SSRIs do not always decrease immobility duration in the FST, hence the need to measure swimming and climbing behaviors and thus increasing the predictive validity of the FST (Detke et al., 1995). From another point of view, several researchers have solely used ovariectomized females with or without estrogen replacement and

Table 2 Effects of the phases of the estrous cycle in the Forced Swim Test in published rat studies between 1990 and 2013. Effects of estrous cycle are presented in immobility swimming and climbing duration, as well as latency to immobility, when available. The strain and the age of the animals (in weeks) are indicated, when available. Variations in FST methodology, such as tank dimensions (cm), depth (cm) and temperature ( C) of the water, phase of the light (L) or dark (D) cycle that FST was performed, as well as duration (minutes) of one, two or repeated (R) FST sessions are also indicated. Values in parenthesis are not explicitly reported, but assumed from relevant clues or graphs. Strain: NIH ¼ National Institute of Health Rat Strain, WKY ¼ Wistar-Kyoto, F ¼ Fischer 344, SD ¼ SpragueeDawley, W ¼ Wistar, LE ¼ Lewis, Cycle: P ¼ Proestrous, E ¼ Estrous, D1 ¼ Diestrous I, D2 ¼ Diestrous II. Authors

Animals

FST methods

Estrous cycle effects on FST

Strain

Age

Tank

Depth-temp

FST sessions

Cycle

Effect

(Alonso et al., 1991) (Pare and Redei, 1993)

SD WKY

(8e12) 10e12

30 15 45 30

15e23 30e25

L/15/5 L//15

P vs. E vs. D1 vs. D2 P þ E vs. D1 þ D2

(Marvan et al., 1996) (Marvan et al., 1997) (Barros and Ferigolo, 1998) (Contreras et al., 1998) (Contreras et al., 2000) (Frye and Walf, 2002) (Zimmerberg et al., 2005)

W W W W W LE NIH

8e10 10e11 13 4 12e13 8 11

61 31 61 31

25 25 27e25 18e23 21e25 30e30 30e30

L/15/5 L/15/5 L/15/5 L//5 L/15/5R L//10 L//10

E E P P P P P

vs. D1 þ D2 vs. D1 þ D2 vs. E vs. D1 vs. D2 þ E vs. D1 þ D2 vs. E vs. D1 vs. D2 vs. D1 þ D2 vs. D2

(Consoli et al., 2005) (Bravo and Maswood, 2006) (Jans et al., 2007) (Andrade et al., 2007) (Schneider and Popik, 2007)

W F W W W

(7e9) 12e18 18 (8e10) (23)

95 25 40 17 25 25

15e25 40e26 30e22 27e25 30e25

L/15/5 D/15/5 D/5/5 L/15/5 D//10

P E P P P

vs. E vs. D1 vs. D2 vs. D1 þ D2 þ E vs. D1 þ D2 vs. D2 vs. D1

(Tonelli et al., 2008) (Andrade et al., 2010) (Craft et al., 2010) (D'Aquila et al., 2010) (Estrada-Camarena et al., 2011)

F W SD SD W

(11) (11e12) 13e27 (13e15) 12e18

60 22 45 40 46

35e25 27e25 30e25 25e30 30e24

L/10/5 D/15/5 L//5R L//10 L/15/5

P P P P P

þ E vs. D1 þ D2 vs. D2 vs. E vs. D1 þ D2 vs. D2 vs. D þ D2

(Allen et al., 2012) (Kokras et al., 2012) (Flores-Serrano et al., 2013)

SD W W

6 16 9e12

65 25 50 19 50 20

48e25 40e24 30e25

D/15/5 L/15/5 L/15/5

P þ E vs. D1 þ D2 P þ E vs. D1 þ D2 P þ E vs. D1 þ D2

No P þ E > D1 þ D2 Immobility D1 þ D2>P þ E Climbing D1 þ D2 > E Immobility D1 þ D2 > E Immobility No PE > D1 þ D2 Latency P þ E > D1 þ D2 Latency D1 þ D2 > P Immobility D2 > P Immobility P > D2 Climbing P þ E > D1 þ D2 Immobility No No No D1 > P Immobility P > D1 Swimming No No No No D1 þ D2 > P Immobility D1 þ D2 > P Swimming No No No

30 30 40 40

50 60 50 60 27 27

22.5 22 35 25 18 20


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Table 3 Sex differences in antidepressant response in the Forced Swim Test in published rat studies between 1990 and 2013. Effects of antidepressant treatment are presented in immobility swimming and climbing duration, as well as latency to immobility, when available. The strain and the age of the animals (in weeks) are indicated, when available. Variations in FST methodology, such as tank dimensions (cm), depth (cm) and temperature ( C) of the water, phase of the light (L) or dark (D) cycle that FST was performed, such as tank dimensions (height diameter in cm), depth (cm) and temperature ( C) of the water, phase of the light (L) or dark (D) cycle that FST was performed, as well as duration (minutes) of one, two or repeated (R) FST sessions are also indicated. Treatment duration (Treat) is indicated in days (d) or injections in 24 h (3inj). Values in parenthesis are not explicitly reported, but assumed from relevant clues or graphs. Strain: SD ¼ SpragueeDawley, W ¼ Wistar, FSL ¼ Flinders Resistant Line, SS/SR ¼ Swim test Susceptible/Resistant rats, Cycle: P ¼ Proestrous, E ¼ Estrous, D1 ¼ Diestrous I, D2 ¼ Diestrous II. Drugs: IMI ¼ imipramine, FLX ¼ fluoxetine, CLM ¼ clomipramine, Q3 DMI ¼ desipramine, AMI ¼ amitriptyline, VEN ¼ venlafaxine, PHE ¼ , BUP ¼ bupropion, SER ¼ sertraline, CIT ¼ citalopram. Authors

Animals

FST methods

Sex differences in antidepressant treatment

Strain

Age

Cycle

Tank

Depth-temp

FST sessions

Drugs

Treat

Male

Female

(Marvan et al., 1997)

W

10e11

E vs. D1 þ D2

61 31

25

L/15/5

CLM

42d

No effect

E: Reduced Immobility

(Contreras et al., 1998) (Consoli et al., 2005) (West and Weiss, 2005)

W

4

P þ E vs. D1 þ D2

30 50 60

18e23

L//5

DMI

21d

No effect

D: No effect Increased Latency

W

(7e9)

P vs. E vs. D1 vs. D2

15e25

L/15/5

CLM

3inj

Reduced Immobility

Reduced Immobility

SS/SR

13e23

62 30

48e26

L//15

14d

(W)

(7e9)

46 20 20

30e25

L/15/5R

FSL SD SD

9e12

50 19

40e24

L//5

13

60 38

40e24

L/15/5

AMI VEN FLX BUP IMI DMI SER 10 SER 25 FLX 5 FLX10 CLM CLM CLM

28d

Reduced Climbing Reduced Climbing No effect Reduced Climbing No effect Increased Climbing Reduced Immobility Reduced Climbing Increased Climbing Reduced Immobility Reduced Immobility No effect Reduced Immobility

No effect No effect No effect No effect No effect Increased Climbing Reduced Immobility Reduced Immobility No effect No effect Increased Latency No effect Reduced Immobility

SD

4e5

50 20

20e27

L//10

FLX

21d

No effect

No effect

SD

6

65 25

48e25

D/15/5

3inj

SD

6

25e24

L/15/5

FLX 5 FLX 10 DMI

3inj

No effect No effect No effect

No effect Increased Swimming No effect

W

13

50 20

30e25

L/15/5

IMI

47d

Reduced Immobility

Reduced Immobility

W

9e12

50 20

30e25

L/15/5

CIT

3inj

No effect

No effect

(Lifschytz et al., 2006) (Kokras et al., 2009) (Pitychoutis et al., 2011) (Rayen et al., 2011) (Allen et al., 2012) (Simpson et al., 2012) (Wibrand et al., 2013) (Flores-Serrano et al., 2013)

P þ E vs. D1 þ D2

P þ E vs. D1 þ D2

in most studies estrogen withdrawal enhances immobility (Estrada-Camarena et al., 2003, 2011; Koss et al., 2012; Stoffel and Craft, 2004). However, ovariectomy aims to model surgical menopause and by extrapolation post-menopausal women with or without hormone replacement therapy, according to whether ovariectomized rodents receive such treatment or not. Less research has been devoted into studying the behavioral response and the effects of antidepressants on intact cycling female rodents, which model women in reproductive age. This is mainly due to problems inherent with monitoring the female estrous cycle. It is often assumed that females have the same behavioral response as males and that fluctuation of gonadal hormones simply obscures behavioral effects (Becker et al., 2005; Wald and Wu, 2010). Importantly, from those studies that dealt with sex differences and estrous cycle effects in the FST, conflicting results emerged, as it is presented in this work (Tables 1e3). Therefore, the unaddressed issue of cycling female's FST performance, at baseline and following treatment, may hold back preclinical research from screening novel molecules in both sexes and from studying the female neurobiology in perhaps the most widely used behavioral test in depression and antidepressant research. Therefore, the present study serves a dual purpose: firstly, to investigate the female FST performance in relation to the estrous cycle and gonadal hormones and secondly, to critically and systematically review current knowledge on male vs. female FST behavior in light of past and present findings. Ultimately, the goal of this work would be to provide guidance on how to use both sexes in

14d 7d 14d

the FST when screening for novel antidepressant molecules and mechanisms. 2. Experimental procedures 2.1. Behavioral testing and treatment Adult male (N ¼ 29) and female (N ¼ 100) Wistar rats, weighing 367 ± 10 and 237 ± 2 g (mean ± s.e.m.) respectively, were group-housed under standard laboratory conditions. The Wistar strain was chosen based on previous studies from our laboratory (Dalla et al., 2005, 2008a; Drossopoulou et al., 2004; Mikail et al., 2012). All experiments were performed in accordance with the EU directive 2010/63. All rats were handled for 4 days for a few minutes before start of the experiments, as proposed before (Slattery and Cryan, 2012). For the modified FST (Cryan et al., 2005; Drossopoulou et al., 2004), individually placed in a cylindrical tank measuring 50 cm height and 20 cm width, which was filled with clean tap water (24 ± 1 C) at a height of 40 cm (Detke and Lucki, 1996). On the first day, rats were forced to swim for a 15min period. Subsequently, rats were carefully dried and before returning to their home cages they were injected i.p. with a lower 10 mg/kg dose, a higher dose of 40 mg/kg of sertraline (Pfizer Hellas, Athens, Greece), or vehicle (10% dimethyl sulfoxide in distilled water). A second and a third i.p. injection was administered 19 and 23 h after the first injection respectively. This widely used subchronic drug administration scheme is repeatedly found effective in rats in the FST (Detke et al., 1995; Sell et al., 2008). Sertraline is a representative molecule from the SSRI class, it has been widely used in the rat FST paradigm and the afore-mentioned doses were based on relevant previous evidence (Detke et al., 1995; Harkin et al., 1999; Mikail et al., 2012). Moreover, we chose to use sertraline as it has been found, in some cases, to have a differential effect on men and women (Baca et al., 2004; Kornstein et al., 2000). One hour after the third injection and 24 h after the first FST session, rats were subjected to a second 5-min FST session, and the total duration of immobility, swimming, climbing and frequency of head swinging were scored as before (Dalla et al., 2008a; Kokras et al., 2012; Pitychoutis et al., 2011), using Kinoscope, an in-house developed software which additionally was capable to re-


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calculate recorded data according to the method of binned scoring as described in Detke et al. (1995). Rats were considered to show immobility when they floated without struggling and making only those movements necessary to keep their heads above the water. Swimming was recorded when they actively swam around in circles (horizontal movement). Climbing was considered when the rats were climbing at the walls of the cylinder (vertical movement). Head swinging (frequency) was recorded when rats exhibited characteristic head-shake-like responses. Furthermore, we calculated the latency to firstly become immobile for 5 s during the FST, as previously described (Carlezon et al., 2002; Kokras et al., 2009). 2.2. Monitoring of the estrous cycle In females, vaginal smears were collected each day to assess the stage of estrus, as previously described (Dalla et al., 2008b; Kokras et al., 2009). Cotton Q-tips were immersed in saline and then guided along the vaginal track to pick up cells. The cells were placed onto slides and stained with 1% Toluidine blue. Stages of the estrous cycle were verified by visualization under a light microscope with 10 magnification. Only adult females that were cycling normally (4e5 day cycles with all stages of estrus) for at least two weeks before the beginning of the experiment were tested in the FST as described above. The vast majority of female Wistar rats, which were screened for the present study, were cycling normally and only 3 rats were excluded. Females in all phases of the estrous cycle were subjected in the first FST session and 24 h later to the second FST session. Thus, all combinations of the estrous cycle phases were represented during the two-day FST protocol (i.e. proestrous in FST pretest e estrous in FST test, similarly estrous-diestrous I, diestrous I-diestrous II and diestrous II-proestrous). Each female was used only once and the number of rats in each phase of the cycle during the second FST session was: estrous (N ¼ 24), diestrous I (N ¼ 23), diestrous II (N ¼ 27) and proestrous (N ¼ 26). 2.3. Gonadal hormones Immediately after the second FST, all rats were sacrificed and the uterus was dissected and weighted for all female rats. Additionally, trunk blood was collected and processed for serum extraction. Estradiol, Progesterone and Testosterone assays were performed using commercially available RIA kits (MP Biomedicals Corticosterone Double Antibody, Costa Mesa, CA, Siemens Coat-A-Count for Total Testosterone and Progesterone and Siemens Double Antibody Estradiol). The detection limits were 0.4 ng/ml, 0.02 ng/ml and 1.4 pg/ml respectively. 2.4. Statistical analysis Results were analyzed with SPSS v.21 (IBM) after controlling that all data met ANOVA requirements. Initially, a two-way ANOVA was performed for all behavioral data with Sex (Male; Female) and Treatment (Vehicle; Low; High) as independent factors, followed by post-hoc pairwise comparisons with Bonferroni type I error correction. In female rats, behavioral data were further analyzed with two-way ANOVA with phases of the estrous cycle (Proestrous; Estrous; Diestrous I; Diestrous II) and Treatment (Vehicle; Low; High) as independent variables, followed by Bonferonni corrected post-hoc comparisons. Associations of behavioral and hormonal data were explored with univariate and multivariate linear regression models. A frequency analysis of all reviewed studies was performed in SPSS in order to produce normal distributions of FST behavioral elements, as depicted on Fig. 3. Distributions were further controlled for normality using the KolmogoroveSmirnov (KeS) non-parametric test. Statistical significance was set at p < 0.05. Results are reported as means ± standard error of mean (SEM).

3. Results 3.1. Behavioral analysis 3.1.1. Comparison of scoring FST methods: total duration versus binned scoring & normal limits Scoring of the original Porsolt FST test consisted in measuring with a stopwatch the total duration of immobility with a theoretical maximum of 300 s for the 5 min test session (Porsolt et al., 1977). When later the modified FST procedure was introduced (Detke et al., 1995), scoring was performed by counting the frequencies of each behavioral element (immobility, swimming, climbing) at the end of each 5-sec period, with a total of 60 counts for all behaviors in 300 s. In the present experiment, we scored behavior using both methods and as seen in Table 4, we found that in females the two scoring methods share an extremely high degree of correlation. In males statistical analysis produced identical results with both methods, with the exception of swimming in vehicle-treated males, which did not correlate. Also, in males, climbing showed a

Fig. 1. Total duration of immobility (a), immobility latency (b), swimming (c), climbing (d), and frequency of head swinging (e) in male and female rats. Female groups are further depicted in the right portion of each graph according to the phase of the estrous cycle (P ¼ Proestrous, E ¼ Estrous, D1 ¼ Diestrous I, D2 ¼ Diestrous II). An asterisk * denotes a significant treatment effect, as evidenced by the ANOVA analysis, between sertraline-treated and the corresponding vehicle-treated group of the same sex or phase of the cycle. An asterisk in parenthesis (*) denotes a statistical significant trend. A cross sign þ indicates a significant sex difference between females and the corresponding male group. N ¼ 7e9 per group, Means ± Standard Error of Mean.

somewhat less robust correlation when comparing the two scoring methods (Table 4). Furthermore, we collected data from all identified studies reporting on control and/or vehicle-treated male and female behavior in the FST and we performed a frequency analysis. As seen in Fig. 3, immobility behavior in male rats follows a normal distribution with most researchers reporting an immobility duration, which falls between 40 and 60% of the 5 min FST session (KeS test p ¼ n.s.). The distribution of studies reporting on female immobility however does not follow a normal distribution (KeS test p ¼ 0.037) with two identified frequency spikes, one between 20 and 40% of time and a second between 60 and 80% of the 5 min FST session.


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effect on immobility levels (Fig. 1a). This was indicated by a twoway ANOVA for total immobility duration with sex and treatment as independent factors, which revealed a significant sex treatment interaction [F(2,121) ¼ 3.592 p ¼ 0.031] and a significant treatment effect [F(2,121) ¼ 23.393 p < 0.001]. Post-hoc pairwise comparisons with Bonferonni correction showed that in males low sertraline dose (10 mg/kg) did not alter immobility duration in the FST, whereas the high sertraline dose (40 mg/kg) marginally reduced immobility duration, in comparison to vehicletreated rats (p ¼ 0.059). On the contrary, in female rats both low and high sertraline doses significantly reduced immobility duration (p < 0.001; p < 0.001, respectively). The same analysis showed that in vehicle-treated rats females displayed more immobility than males (p ¼ 0.028), but no sex differences were detected between male and female rats treated with low and high sertraline dose (Fig. 1a). Moreover, the phase of the estrous cycle affected the response to the low dose of sertraline, whereas the high dose was effective in reducing immobility levels in all phases of the cycle (Fig. 1a). This was indicated by a two-way ANOVA for total immobility with phase of the estrous cycle and treatment as independent factors, which revealed a significant treatment effect [F(2,87) ¼ 54.712 p < 0.001]. Post-hoc testing showed that in females in proestrous and estrous low dose sertraline had no effect in immobility, whereas in diestrous I and II, low sertraline dose significantly reduced immobility duration. The higher sertraline dose significantly reduced immobility in all phases of the estrous cycle (p < 0.001 for all pairwise comparisons) (Fig. 1a). Furthermore, across different phases of the estrous cycle, there were no significant variations in immobility duration, irrespectively of female rats receiving vehicle, low or high sertraline dose.

Fig. 2. Uterus weight (a) and serum levels of estrogens (b), testosterone (c) and progesterone (d). Female groups are further depicted in the right portion of each graph according to the phase of the estrous cycle (P ¼ Proestrous, E ¼ Estrous, D1 ¼ Diestrous I, D2 ¼ Diestrous 2). An asterisk (*) denotes a significant difference, as evidenced by the ANOVA analysis, between sertraline-treated rats and the corresponding vehicletreated group of the same sex or phase of the cycle. A cross sign (þ) indicates a significant sex difference between females and the corresponding male group. N ¼ 7e9 per group, Means ± Standard Error of Mean.

With regards to swimming and climbing, is should be noted that significantly less studies were reporting on those behaviors (35 and 39 studies respectively, in comparison to 62 studies reporting on male and female immobility). With regards to climbing, the distribution of studies was not normal neither for male nor for female rats (KeS p ¼ 0.001 and p ¼ 0.009 respectively) as most researchers of the identified 39 studies agreed that its duration is less than 20% of the FST time, although some researchers are scoring slightly more climbing in females than in males. Differences in distribution are seen also in swimming behavior with reporting on male swimming from 39 studies not being normally distributed (KeS p ¼ 0.012) and reporting on female swimming having a more normal distribution (KeS p ¼ n.s.). In any case, swimming duration is expected to range between 20 and 40% of time for both sexes, with researchers tending to score more swimming in males as seen by the frequency spike between 40 and 60% of time. 3.1.2. Immobility duration Female rats exhibited higher levels of immobility than males and both doses of sertraline reduced immobility in females. However, in males only the high dose produced a marginally significant

3.1.3. Latency to immobility Sertraline treatment had no effect on latency to immobility in males, whereas in females only the high dose was effective in increasing immobility latency in certain phases of the estrous cycle (Fig. 1b). This was indicated by a two-way ANOVA, which revealed a significant treatment main effect [F(2,121) ¼ 12.988 p < 0.001] for the latency to immobility. Follow-up Bonferonni corrected pairwise comparisons showed that in males treatment with both low and high sertraline dose did not alter immobility latency (p > 0.05). In females, treatment with low sertraline dose had no effect, but treatment with high sertraline dose effectively elongated immobility latency (p < 0.001) (Fig. 1b). Moreover, a two-way ANOVA revealed a significant treatment main effect [F(2,87) ¼ 29.908 p < 0.001] and a marginally non-significant interaction [F(6,87) ¼ 1.904 p ¼ 0.089]. Follow-up testing showed that low sertraline dose did not alter immobility latency in any phase of the estrous cycle, but the higher sertraline dose significantly elongated immobility latency in proestrous (p < 0.001), in estrous (p ¼ 0.02), in diestrous II (p < 0.001), but not in diestrous I (Fig. 1b). Furthermore, across different phases of the estrous cycle, there were no significant variations of immobility latency in female rats receiving vehicle or low sertraline dose. Instead, in female rats receiving the high sertraline dose immobility latency was significantly lower in diestrous I, in comparison to diestrous II and proestrous (p ¼ 0.031; p ¼ 0.015) (Fig. 1b). 3.1.4. Swimming duration Sertraline in both doses enhanced swimming duration in both sexes (Fig. 1c). This was indicated by a two-way ANOVA, which a significant treatment main effect for swimming duration [F(2,121) ¼ 21.885 p < 0.001]. Post-hoc testing showed that males treated with both low and high sertraline dose displayed more swimming duration than vehicle-treated rats (p ¼ 0.009; p ¼ 0.018,


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Fig. 3. Number of studies reporting in different percentages of immobility (a, b), swimming (c, d) and climbing (e, f) duration in male and female rats. For each behavioral element, only studies reporting on both male and female behavior and using the standard two-session FST protocol are included (n ¼ 62 studies for immobility, n ¼ 35 for swimming, n ¼ 39 for climbing). Duration of each FST behavioral element is expressed in percentage of the 5 min FST second session, in order to group studies independently of the scoring method.

respectively). Similarly, female rats that received either low or high sertraline dose spent more time swimming than female vehicletreated rats (p < 0.001; p < 0.001, respectively) (Fig. 1c). Regarding sertraline effects during the estrous cycle, the high dose was effective in enhancing swimming duration in all phases of

the cycle, whereas the low dose was effective only in certain phases of the cycle (Fig. 1c). This was indicated by a two-way ANOVA, which revealed significant treatment and estrous cycle main effects [F(2,87) ¼ 41.039 p < 0.001; F(3,87) ¼ 5.154 p ¼ 0.003]. Post-hoc testing showed that rats in diestrous II and proestrous treated


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N. Kokras et al. / Neuropharmacology xxx (2015) 1e14 Table 4 Correlations between total duration scoring and binned scoring for each behavioral element of the Forced Swim Test, as calculated by Pearson's bivariate correlation method. Two or three asterisks denote p < 0.01 and p < 0.001 respectively, ns ¼ not significant. Behavior

Males

Females

Veh

Low

High

Veh

Low

High

Immobility Swimming Climbing

0.987*** 0.612 ns 0.844**

0.978*** 0.996*** 0.995***

0.999*** 0.992*** 0.998***

0.991*** 0.948*** 0.991***

0.993*** 0.971*** 0.996***

0.993*** 0.980*** 0.996***

with low sertraline dose did not differ from vehicle-treated rats, but female rats treated with low sertraline dose during estrous and diestrous I displayed enhanced swimming duration (p ¼ 0.006; p < 0.001 respectively). Instead, the high sertraline dose increased swimming in all phases of the estrous cycle (proestrous: p ¼ 0.003; estrous: p < 0.001; diestrous I: p < 0.001; diestrous II: p ¼ 0.002) (Fig. 1c). Across different phases of the estrous cycle, there were no significant variations of swimming duration in female rats receiving vehicle. In females receiving low sertraline dose, rats in diestrous I had higher swimming duration than rats in diestrous II (p ¼ 0.045). In addition, in females receiving the high sertraline dose, those in diestrous I displayed higher swimming than those in diestrous II and proestrous (p ¼ 0.007; p ¼ 0.045) (Fig. 1c). 3.1.5. Climbing duration The high sertraline dose enhanced climbing duration only in females in certain phases of the cycle, whereas the low dose did not modify climbing duration in both sexes (Fig. 1d). This was indicated by a two-way ANOVA, which revealed a significant treatment main effect [F(2,121) ¼ 7.769 p ¼ 0.001] and post-hoc pairwise comparisons showed that treatment with either low or high sertraline dose did not alter climbing duration in males. In females, low sertraline dose did not alter climbing, but high sertraline dose significantly elongated climbing duration (p < 0.001) (Fig. 1d). Moreover, a twoway ANOVA for climbing duration with phases of the estrous cycle and treatment as independent factors, revealed a significant treatment effect [F(2,87) ¼ 14.182 p < 0.001]. Follow-up Bonferonni tests showed that in all phases of the estrous cycle low sertraline dose did not alter climbing duration. In addition, females treated with high sertraline dose did not display enhanced climbing when in estrous and diestrous I phases of the cycle. Instead, when female rats in diestrous II and proestrous were treated with high sertraline dose, they displayed significantly elongated climbing duration (p ¼ 0.004; p ¼ 0.001, respectively) (Fig. 1d). Across different phases of the estrous cycle, there were no significant variations of climbing, irrespectively of females receiving vehicle, low or high sertraline doses. There was only a nonsignificant trend for females in diestrous I to show a modestly lower climbing duration following high sertraline dose, in comparison to the other phases of the estrous cycle. 3.1.6. Head swinging frequency Males displayed higher head swinging frequency than females and this was decreased by high dose sertraline treatment. On the other hand, low sertraline dose enhanced head swinging only in estrous females (Fig. 1e). This was indicated by a two-way ANOVA, which showed significant sex and treatment main effects [F(1,121) ¼ 16.023 p < 0.001; F(2,121) ¼ 6.238 p ¼ 0.003]. Post-hoc testing showed that only in males, the high sertraline dose reduced head swinging (p ¼ 0.041). In females only the low sertraline dose tended to increase head swings (p ¼ 0.63). As seen in Fig. 1e, in vehicle-treated rats, males displayed more head swings

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than females (p < 0.001). The same sex difference was marginally significant in male and female rats treated with low sertraline dose (p ¼ 0.054), but it was not significant in rats treated with the high sertraline dose. Moreover, a two-way ANOVA revealed a significant estrous cycle treatment interaction [F(6,87) ¼ 2.666 p ¼ 0.020] and significant estrous cycle and treatment main effects [F(3,87) ¼ 3.475 p ¼ 0.019; F(2,87) ¼ 7.344 p ¼ 0.001]. Follow-up Bonferonni testing showed that treatment did not alter head swinging frequency in diestrous I, II and proestrous, irrespectively of high or low sertraline dose. However, females in estrous treated with low sertraline dose showed a significant increase in head swinging frequency (p < 0.001), but this was not observed in estrous females receiving high sertraline dose (Fig. 1e). Head swinging frequency was not affected by estrous cycle in vehicle-treated rats and in female rats treated with high sertraline dose. As seen in Fig. 1e, head swinging frequency was affected by estrous cycle only in rats treated with low sertraline dose. In this case, estrous females had more head swings than females in proestrous and diestrous II (p ¼ 0.013; p ¼ 0.005), whereas diestrous II females displayed less head swings than diestrous I females (p ¼ 0.047). 3.2. Gonadal hormones 3.2.1. Estrogen levels Uterus weight was lower in diestous II females than females in all other phases of the estrous cycle (Fig. 2a). This was indicated by a two-way ANOVA, which revealed a significant estrous cycle main effect for uterus weight [F(3,74) ¼ 12.878 p < 0.001], which is indicative of estrogen levels. Post-hoc testing showed that females in diestrous II had significantly lower uterus weight than females in proestrous, estrous and diestrous I (p < 0.001; p < 0.001; p ¼ 0.003, respectively). As seen in Fig. 2a, sertraline treatment had no effect on uterus weight. With regards to estrogen levels, a proper two-way ANOVA with sex and treatment as factors was not possible, because male's estrogen levels were below the detection limit of the RIA. However, as seen in Fig. 2b, it is obvious that females had higher estrogen levels than males. A one-way ANOVA in female rats showed that treatment with low and high sertraline dose had no effect on estrogens, although there was a tendency for a sertraline dose-dependent decrease in estrogen levels (p ¼ 0.090). 3.2.2. Testosterone levels Males had higher testosterone levels than females and both doses of sertraline decreased testosterone levels only in males (Fig. 2c). This was indicated by a two-way ANOVA, which revealed significant sex treatment interaction and significant sex and treatment main effects [F(2,97) ¼ 28.649, p < 0.001; F(1,97) ¼ 78.129, p < 0.001; F(2,97) ¼ 27.433, p < 0.001, respectively). Post-hoc testing showed that in males, treatment with low and high sertraline dose resulted in a marked decrease of testosterone levels (p < 0.001; p < 0.001 respectively), whereas in females sertraline treatment had no effect (Fig. 2c). Further post-hoc testing showed that females had lower testosterone levels than males irrespectively of receiving vehicle, low or high sertraline dose (p < 0.001; p ¼ 0.047; p ¼ 0.045). With regards to the estrous cycle, a two-way ANOVA did not show significant effects of treatment and estrous cycle on testosterone levels in females. 3.2.3. Progesterone levels Females had higher progesterone levels than males and high sertraline dose enhanced progesterone levels even further in


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females in certain phases of the cycle (Fig. 2d). This was indicated by a two-way ANOVA, which revealed a significant main effect of sex [F(1,96) ¼ 19.849, p < 0.001] and post-hoc pairwise comparisons showed that in males sertraline treatment had no effect on progesterone levels. In females, treatment with low sertraline dose had no effect, but treatment with high sertraline resulted in increased progesterone levels (p ¼ 0.007). Pairwise comparisons also showed that females had higher progesterone levels than males, irrespectively of treatment with vehicle, low or high sertraline dose (p ¼ 0.05, p ¼ 0.04, p ¼ 0.001, respectively) (Fig. 2d). Moreover, a two-way ANOVA revealed significant treatment and estrous cycle main effects [F(2,72) ¼ 5.831, p ¼ 0.004; F(3,72) ¼ 9.649, p < 0.001, respectively). Post-hoc pairwise comparisons showed that females in proestrous and diestrous II treated with high sertraline dose displayed enhanced progesterone levels (p ¼ 0.025, p ¼ 0.05, respectively) (Fig. 2d). 3.2.4. Association of FST behaviors with gonadal hormones None of the behavioral indices (immobility, swimming, climbing) correlated significantly with hormonal serum levels and uterus weight. This was further confirmed by several regression models, which consistently showed that behavior is explained exclusively and significantly by treatment and not by sex, phase of the estrous cycle, uterus weight or gonadal hormones. The only exception was head swinging behavior, in which testosterone explained 13% of the variance, sex explained 10%, and estrous cycle 1%, whereas sertraline treatment explained 5% of the variance. 4. Discussion The present study along with a systematic presentation of the relevant literature aimed to identify current problems and provide guidelines regarding the use of female animals in the forced swim test of antidepressant activity. For this, we examined the female behavioral performance in the FST as a function of the estrous cycle at baseline and following treatment with an SSRI. Female Wistar rats exhibited higher levels of immobility during the second FST session than males and this sex difference was alleviated following sertraline treatment, as before (Kokras et al., 2009). In particular, sertraline at both doses enhanced swimming in both sexes, but females appeared more responsive regarding immobility levels. Furthermore, we examined all combinations of the phases of the estrous cycle during pretest and test, in order to obtain a better understanding of the gonadal hormone influence on FST behavior. As seen by analyzing the behavior of vehicle-treated females, the phase of the estrous cycle did not affect baseline performance in the FST. On the other hand, response to sertraline was significantly affected by the phase of the estrous cycle and sertraline had an effect on gonadal hormone levels. The enhanced immobility of females, in comparison to males at baseline is consistent with previous studies from our laboratory using the modified FST paradigm. This finding is suggestive of enhanced expression of despair in female rats or at least sex differences in coping strategies and/or learning and memory (Dalla et al., 2008a; Dalla and Shors, 2009; Drossopoulou et al., 2004; Kokras et al., 2012; McCarthy and Konkle, 2005; Pitychoutis et al., 2009, 2011). However, as seen in Table 1, results on the female FST performance are highly conflicting. One third of identified studies are in agreement with our findings, one-third show the opposite, with females presenting less immobility than males, and the last third of identified studies failed to show any significant sex differences. These conflicting results cannot be attributed to strain differences (Lopez-Rubalcava and Lucki, 2000; Tejani-Butt et al., 2003), as around 30% of the studies use Wistar rats and 30% use SpragueeDawley rats and the direction of the sex difference is

equally distributed among strains. Another factor that could influence FST behavioral performance in males and females is the differential handling between males and females due to the estrous cycle monitoring in females. Indeed, long- but not short-lasting handling has been found to affect immobility levels in male rats (Cannizzaro et al., 2002). However, rats are expected to habituate to repeated handling from the same person (Dobrakovova et al., 1993). Moreover, sex differences are probably related to different methodological approaches and factors influencing FST behavioral response, as suggested elsewhere (Bogdanova et al., 2013). Although, some of the studies on female rodents are based on FST protocols described in excellent papers regarding male rodents (Slattery and Cryan, 2012), others use heavily modified protocols that are not extensively validated and established. As seen by inspecting Table 1, male and female rats of different strains and ages have been used in water tanks of wildly different dimensions. Furthermore, significant variations are observed in the depth of water and to a lesser degree in the water temperature. In particular, most studies showing more immobility in females than in males use a higher depth of water i.e. between 30 and 50 cm. On the other hand, in most studies showing that males have more immobility than females, the water depth varies between 18 and 30 cm. The water depth could play an important role, because the behavior of male and female rats may be different, when animals are touching the bottom of the tank with their hind paws or tail. Nevertheless, many different factors interact in the appearance of a sex difference or not (Bogdanova et al., 2013; Joel and Yankelevitch-Yahav, 2014). Thus, as seen in Table 1, using rats of many different strains, in many different ages, in the light or dark phase of day, in different water tanks, with different water conditions, and with variable FST sessions, deviating from the standard paradigm (two sessions of 15 and 5 min) only contributes to an unclear and confusing situation regarding sex differences in FST. On top of that, other important factors that are influencing the reported levels of passive versus active behaviors are the scoring methods and the criteria used for the definition of different FST behaviors. In the present study, with the use of standard criteria, we show that manual scoring of the duration of immobility, swimming and climbing does not differ from the binned scoring described in (Detke et al., 1995) (Table 4). Also, as depicted on Fig. 3, based on studies with two FST sessions, we attempted to establish a normal range of immobility, swimming and climbing duration in the second FST session. There is a general agreement between most researchers that immobility should fall between 40 and 60% of the 5 min s FST session and swimming and climbing should fall around 20e40% and less than 20% respectively. However, the observed differences in the distribution of those behavioral elements between males and female raises the concern that researchers are differently understanding immobility and swimming in males and females and consensus is high perhaps only in climbing behavior. This is based on our observation that reporting of immobility among researchers follows a normal distribution for males but not for females and inversely, reporting of swimming follows a normal distribution for females but not for males. Although this is indirect and not strong evidence, this observation of ours warrants further investigation on how the FST behavioral elements are scored on male and female rodents and what degree of consensus exists between researchers. Notably, all but one of the identified studies used manual and not automated movement-tracking scoring methods. The development of a reliable automated scoring method for FST in both male and female rodents is another issue that remains to be elucidated. Regarding the estrous cycle, only 20% of 63 studies report on the estrous cycle when comparing male and female rats in the FST (Table 1). In those studies, phases of the estrous cycle are reported


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because the authors were interested in particular comparisons (e.g. grouping together females in proestrous and estrous) or because the authors chose to use females in a particular phase of the cycle (Table 2). Notably, in the majority of studies on females, there is no evidence regarding the estrous cycle and the reader has to assume that cycling females are hopefully normally distributed across all phases. Of course, as noted above, those studies avoided the differential handling between the two sexes due to vaginal swab. As seen in Table 2, we identified 22 previous studies on normal cycling rats undergoing FST where the authors monitored the effects of the estrous cycle. Of those, 12 studies (55%) showed no effect of the estrous cycle on the FST behavioral performance. On the other hand, 10 previous studies (45%) showed some effects, but the power of those studies is questionable and in any case no absolute consensus can be determined: 8 studies revealed that female rats in diestrous displayed more immobility than females in proestrous or estrous, whereas 2 studies showed the opposite effect. Based on present results and previous literature we conclude that in naturally cycling females the estrous cycle effect on FST behavior is small and perhaps negligible. Surprisingly, FST studies with standard antidepressant treatments comparing males and females are limited, as seen on Table 3. We identified 12 previous studies comparing the antidepressant response of male and female rats in the FST (until 2013). Similarly, to the methodology of studies on sex differences, again studies on the sex-dependent antidepressant response, present significant methodological variations. Only 40% of the identified studies reported on the estrous cycle of the female rats. Only 25% implement the standard subacute protocol of 3 injections between the first and second FST session, and chronic antidepressant treatment varied from 7 to 47 days. Importantly one third of the studies in rats have used a single FST session. Thus, it is a daunting task to identify one study replicating the methods and results of another, although as seen on Table 3, most of them identified several sex differences in the antidepressant response. In the present study, sertraline treatment exerted an antidepressant effect in both sexes. In particular, sertraline at low and high doses exerted its antidepressant effect by elongating swimming duration in both males and females. Interestingly, females appeared more responsive to sertraline, because immobility duration was decreased following both low and high sertraline dose, whereas in males only the high dose was marginally effective in reducing immobility levels. This latter finding in males is not surprisingly, because SSRIs do not always decrease immobility in males, even in the modified FST, hence the need to also score swimming (Detke et al., 1995). The higher responsiveness of female rodents to antidepressant activity in the FST has been demonstrated before for tricyclic antidepressants, paroxetine and reboxetine (Caldarone et al., 2003; David et al., 2001; Gunther et al., 2011). Furthermore, it is known that SSRI's enhance swimming duration and not climbing, given that swimming is indicative of serotonergic activity and climbing of catecholaminergic (Detke et al., 1995). However, in the present study, it is observed for the first time that treatment with a high sertraline dose (40 mg/kg) enhances climbing duration selectively in female rats and not in males. Interestingly, this effect was partially observed with sertraline in the seminal study of Detke et al. (Detke et al., 1995) which was using male rats, but had not reached statistical significance. Thus, it can be assumed that sertraline at higher doses may loose its selectivity for the serotonergic transporter and act in dopaminergic and/or noradrenergic transporters as well, and that this phenomenon may be observed at relatively lower doses in females. Regarding the effect of the estrous cycle on antidepressant activity, based on data from the present study (Fig. 1), it can be said that the female response to the antidepressant is closer to the male

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response, when females are tested in diestrous phases of the cycle. In particular, the combination of estrous (FST session 1) and diestrous I (FST session 2) is closer to the expected FST male response. This could be of importance when female rats are used for the screening of new compounds for antidepressant activity. Specifically, the low sertraline dose was totally ineffective in rats subjected in the first FST session during diestrous II and in the second FST session during proestrous. This is probably related to the fact that during subacute sertraline treatment and exposure to the FST stressful events, females were at the transition from low estrogen levels to high estrogen levels (Becker et al., 2005). It has been repeatedly shown before that female rats, when stressed in diestrous II and tested 24 h later in proestrous on an associative learning task, appear particularly sensitive to stress and as a result learning is impaired (Dalla and Shors, 2009; Shors et al., 1998). Similarly, to our findings it has also been shown that when female rats are treated acutely, but not chronically, with estrogens and progesterone in doses producing proestrous levels, antidepressants do not have an effect in the FST (Benmansour et al., 2012; Shah and Frazer, 2014). This has been attributed to estrogen receptor's (ERa) inhibitory effect on the ability of an SSRI to inhibit the SERT (Benmansour et al., 2012). Thus, during screening for new antidepressants with FST, it is possible to loose significant findings, if a new molecule is tested in a low dose and with the use of females mostly in proestrous. Moreover, in the present FST study, when rats were tested during the diestrous II and proestrous phases of the cycle high sertraline dose enhanced the climbing duration, suggesting that this effect is influenced by estrogen/progesterone levels. Interestingly, at the same phases of the cycle, high sertraline dose also enhanced progesterone levels, which have been associated with enhanced climbing duration in the FST (Molina-Hernandez and TellezAlcantara, 2001). In the past, we have also reported an SSRI enhancing effect on progesterone levels with chronic fluoxetine treatment in intact female rats. In that study, progesterone levels associated with swimming in the FST (Kokras et al., 2014). However, in the present study that included both sexes, statistical models failed to show an association of gonadal hormones and FST behavioral performance. The only exemption was the head swinging behavior that is affected by gonadal hormones and exhibits a marked sex difference (Kokras and Dalla, 2014; Kokras et al., 2014). Head swinging frequency is also markedly increased by low sertraline treatment in females only when they are in the estrous phase of the cycle during to the second FST session. In this case, subacute sertraline treatment took place when estrogen levels were still enhanced (i.e. during proestrous and the transition from proestous to estrous). Sex differences were also present in headswinging behavior with females exhibiting much lower frequency than males, as before (Drossopoulou et al., 2004). Sertraline treatment at a low dose did not affect sex differences in head swinging, as reported before for the tricyclic antidepressant clomipramine (Kokras et al., 2009). However, the high sertraline dose decreased head swinging in males and alleviated the apparent sex difference. Notably, this behavior has been linked with postsynaptic serotonergic 5-HT2A receptor activity (Darmani, 1996; Matuszewich and Yamamoto, 2003), pointing to a sex-dependent activity of these receptors. Therefore, it can be speculated that high estrogen levels are required for an SSRI effect on head swinging behavior in females. However, this effect seems to depend on the dose, because a higher sertraline dose results in no increase in head swings. These findings require further investigation, as head swinging in the FST could represent a useful behavioral index of the interaction of gonadal hormones with the serotonergic system. A final interesting finding of the present study is that the subacute sertraline treatment at both low and high doses markedly


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decreases testosterone levels in male animals. This finding can be explained by a recent study in rat liver showing that sertraline treatment in 24 h induces the P450 iso-enzyme CYP2B that is responsible for the 16-hydroxylation of testosterone (Haduch et al., 2008). Thus, presumably in the present study sertraline enhanced testosterone's metabolism and reduced testosterone levels. However, no correlations were present in the present study between hormone levels and duration of immobility and swimming duration. 5. Conclusions Based on present results and on reviewing the literature, it seems that the effect of the estrous cycle on the FST baseline performance of naturally cycling females is small. According to the power of the study, it can be expected that this effect, if existing at all, may go unnoticed. Nevertheless, it is advisable that female groups contain rats in normal distribution of all phases of the estrous cycle, thus avoiding the chance of over-representation of a particular phase and obtain a skewed result. On the other hand, when investigating the effects of established or candidate antidepressant molecules, several caveats may apply when using and/or comparing the female to the male response. Ideally, female rats in every phase of the estrous cycle should be used, so that a clear understanding can be obtained regarding the effects of the investigational drug across the different phases of the estrous cycle. However, such approach would require enormous numbers of female rats. Thus, when researchers are not interested in studying the female FST response to a drug in such detail, different strategies can be applied: if interested in highlighting sex differences in response to an antidepressant, males can be compared to females in proestrous and estrous phases. However, as mentioned above, depending on the dose, this approach could result to a failure to demonstrate an antidepressant effect in proestrous females. On the other hand, if researchers are interested in similar actions between sexes, then females in diestrous I and II can be used. Alternatively, a strategy that would bridge the aforementioned approaches and avoid the use of too many female rats would be to merge females into two groups (proestrous and estrous vs diestrous I and II). This approach would reduce the amount of rats, but would still preserve information on a potential sex-dependent antidepressant response. Finally, dose-dependent studies should be designed, as males and females might be responsive to different doses of the same drug. Importantly, when screening for new molecules, standard FST procedures and scoring methods should be used on both sexes. Funding sources ZPD, CD and NK are supported by a “Large Scale Cooperative Project” (09SYN-21-1003) and VK was supported by “Education and Lifelong Learning, Supporting Postdoctoral Researchers”. Both projects are co-financed by the European Social Fund (ESF) and the General Secretariat for Research and Technology, Greece. Hudu G. Mikail received a fellowship from the Hellenic State Scholarship Foundation. None of these agencies had any influence over any part of this work. Contributors N. Kokras contributed in the design of the study, all experimental procedures, statistical analysis of the results, literature searches and analyses, and wrote the first draft of the manuscript. K. Antoniou contributed in the design and the analysis of the study and provided significant input. H. G. Mikail and V. Kafetzopoulos

contributed in some experimental procedures. Z. PapadopoulouDaifoti contributed in the design of the study and provided additional input. C. Dalla contributed and supervised all phases of the present study. All authors contributed to and have approved the final manuscript. Acknowledgements The authors wish to acknowledge Pfizer Hellas for gifting sertraline for this experiment. The authors would like to thank Mrs Despoina Papassava for excellent technical assistance and Mr D. Baltas and F. Theocharis for assistance in developing Kinoscope, an open source freely-available computer program for scoring behavior. References Allen, P.J., D'Anci, K.E., Kanarek, R.B., Renshaw, P.F., 2012. Sex-specific antidepressant effects of dietary creatine with and without sub-acute fluoxetine in rats. Pharmacol. Biochem. Behav. 101, 588e601. Alonso, S.J., Castellano, M.A., Afonso, D., Rodriguez, M., 1991. Sex differences in behavioral despair: relationships between behavioral despair and open field activity. Physiol. Behav. 49, 69e72. Alves, C.J., Magalhaes, A., Summavielle, T., Melo, P., De Sousa, L., Tavares, M.A., Monteiro, P.R., 2008. Hormonal, neurochemical, and behavioral response to a forced swim test in adolescent rats throughout cocaine withdrawal. Ann. N. Y. Acad. Sci. 1139, 366e373. Andrade, S., Silveira, S.L., Arbo, B.D., Batista, B.A., Gomez, R., Barros, H.M., Ribeiro, M.F., 2010. Sex-dependent antidepressant effects of lower doses of progesterone in rats. Physiol. Behav. 99, 687e690. Andrade, S., Silveira, S.L., Gomez, R., Barros, H.M., Ribeiro, M.F., 2007. Gender differences of acute and chronic administration of dehydroepiandrosterone in rats submitted to the forced swimming test. Prog. Neuropsychopharmacol. Biol. Psychiatry 31, 613e621. Armario, A., Gavalda, A., Marti, J., 1995. Comparison of the behavioural and endocrine response to forced swimming stress in five inbred strains of rats. Psychoneuroendocrinology 20, 879e890. Baca, E., Garcia-Garcia, M., Porras-Chavarino, A., 2004. Gender differences in treatment response to sertraline versus imipramine in patients with nonmelancholic depressive disorders. Prog. Neuropsychopharmacol. Biol. Psychiatry 28, 57e65. Barros, H.M., Ferigolo, M., 1998. Ethopharmacology of imipramine in the forcedswimming test: gender differences. Neurosci. Biobehav Rev. 23, 279e286. Becker, J.B., Arnold, A.P., Berkley, K.J., Blaustein, J.D., Eckel, L.A., Hampson, E., Herman, J.P., Marts, S., Sadee, W., Steiner, M., Taylor, J., Young, E., 2005. Strategies and methods for research on sex differences in brain and behavior. Endocrinology 146, 1650e1673. Beery, A.K., Zucker, I., 2011. Sex bias in neuroscience and biomedical research. Neurosci. Biobehav. Rev. 35, 565e572. Bellido, I., Gomez-Luque, A., Garcia-Carrera, P., Rius, F., de la Cuesta, F.S., 2003. Female rats show an increased sensibility to the forced swim test depressive-like stimulus in the hippocampus and frontal cortex 5-HT1A receptors. Neurosci. Lett. 350, 145e148. Benmansour, S., Weaver, R.S., Barton, A.K., Adeniji, O.S., Frazer, A., 2012. Comparison of the effects of estradiol and progesterone on serotonergic function. Biol. Psychiatry 71, 633e641. Bogdanova, O.V., Kanekar, S., D'Anci, K.E., Renshaw, P.F., 2013. Factors influencing behavior in the forced swim test. Physiol. Behav. 118, 227e239. Borsini, F., Meli, A., 1988. Is the forced swimming test a suitable model for revealing antidepressant activity? Psychopharmacology (Berl) 94, 147e160. Bravo, G., Maswood, S., 2006. Acute treatment with 5-HT3 receptor antagonist, tropisetron, reduces immobility in intact female rats exposed to the forced swim test. Pharmacol. Biochem. Behav. 85, 362e368. Brotto, L.A., Barr, A.M., Gorzalka, B.B., 2000. Sex differences in forced-swim and open-field test behaviours after chronic administration of melatonin. Eur. J. Pharmacol. 402, 87e93. Brummelte, S., Lieblich, S.E., Galea, L.A., 2012. Gestational and postpartum corticosterone exposure to the dam affects behavioral and endocrine outcome of the offspring in a sexually-dimorphic manner. Neuropharmacology 62, 406e418. Brummelte, S., Pawluski, J.L., Galea, L.A., 2006. High post-partum levels of corticosterone given to dams influence postnatal hippocampal cell proliferation and behavior of offspring: a model of post-partum stress and possible depression. Horm. Behav. 50, 370e382. Caldarone, B.J., Karthigeyan, K., Harrist, A., Hunsberger, J.G., Wittmack, E., King, S.L., Jatlow, P., Picciotto, M.R., 2003. Sex differences in response to oral amitriptyline in three animal models of depression in C57BL/6J mice. Psychopharmacology (Berl) 170, 94e101. Cannizzaro, C., Martire, M., Steardo, L., Cannizzaro, E., Gagliano, M., Mineo, A., Provenzano, G., 2002. Prenatal exposure to diazepam and alprazolam, but not to


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Strain differences in the behavioral effects of antidepressant drugs in the rat forced swimming test. Neuropsychopharmacology 22, 191e199. Marcus, S.M., Young, E.A., Kerber, K.B., Kornstein, S., Farabaugh, A.H., Mitchell, J., Wisniewski, S.R., Balasubramani, G.K., Trivedi, M.H., Rush, A.J., 2005. Gender differences in depression: findings from the STAR*D study. J. Affect Disord. 87, 141e150. Martinez-Mota, L., Ulloa, R.E., Herrera-Perez, J., Chavira, R., Fernandez-Guasti, A., 2011. Sex and age differences in the impact of the forced swimming test on the levels of steroid hormones. Physiol. Behav. 104, 900e905. Marvan, M.L., Chavez-Chavez, L., Santana, S., 1996. Clomipramine modifies fluctuations of forced swimming immobility in different phases of the rat estrous cycle. Arch. Med. Res. 27, 83e86.


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Neurobehavioral studies of forced swimming: the role of learning and memory in the forced swim test.

The forced swim test as a model of depressive-like behavior.

Anxiolytic effects of buspirone and MTEP in the Porsolt Forced Swim Test.

Alarm substance emitted by rats in the forced-swim test is a low volatile pheromone.

Physiological effects of alarm chemosignal emitted during the forced swim test.

Effects of corticosterone on response consolidation and retrieval in the forced swim test.

Adrenal activity does not mediate alarm substance reaction in the forced swim test.

Ultrasonic vocalizations during intermittent swim stress forecasts resilience in subsequent forced swim and spatial learning tests.

Chronic forced swim stress produces subsensitivity to nicotine.

Forced vital capacity in Malaysian females.

Sex-specific diurnal immobility induced by forced swim test in wild type and clock gene deficient mice.

Influence of enrichment on behavioral and neurogenic effects of antidepressants in Wistar rats submitted to repeated forced swim test.

The effects of ifenprodil on the activity of antidepressant drugs in the forced swim test in mice.

Acute intracerebroventricular inositol does not reverse the effect of chronic lithium treatment in the forced swim test.

The effect of GABAmimetics on the duration of immobility in the forced swim test in albino mice.

The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test.

Maudsley reactive and nonreactive rats in the forced swim test: comparison in fresh water and soiled water.

Neonatal treatment with clomipramine increased immobility in the forced swim test: an attribute of animal models of depression.

Group II mGlu receptor antagonist LY341495 enhances the antidepressant-like effects of ketamine in the forced swim test in rats.

Immobility behavior during the forced swim test correlates with BNDF levels in the frontal cortex, but not with cognitive impairments.

Forced swim test induces divergent global transcriptomic alterations in the hippocampus of high versus low novelty-seeker rats.

Effect of different restraint schedules on the immobility in the forced swim test: modulation by an opiate mechanism.

The influence of caffeine on the activity of moclobemide, venlafaxine, bupropion and milnacipran in the forced swim test in mice.

Inhibition of the CRF1 receptor influences the activity of antidepressant drugs in the forced swim test in rats.