Progesterone withdrawal effects in the open field test can be predicted by elevated plus maze performance

Hormones and Behavior 50 (2006) 208 – 215 www.elsevier.com/locate/yhbeh

Progesterone withdrawal effects in the open field test can be predicted by elevated plus maze performance Magnus Löfgren a , Inga-Maj Johansson a , Bengt Meyerson b , Per Lundgren a , Torbjörn Bäckström a,⁎ a

Department of Clinical Science, Obstetrics and Gynecology, Umeå Neurosteroid Research Center, Building 5B, 5th floor, Umeå University Hospital, SE-901 85 Umeå, Sweden b Department of Neuroscience, Division of Pharmacology. Box 593, BMC SE-751 24 Uppsala, Sweden Received 21 November 2005; revised 9 March 2006; accepted 9 March 2006 Available online 4 May 2006

Abstract Allopregnanolone (3alpha-hydroxy-5alpha-pregnane-20-one) is a ring-A-reduced metabolite of progesterone, which is naturally produced during the luteal phase of the menstrual cycle, during pregnancy and by stressful events. The steroid hormone inhibits neural functions through increased chloride ion flux through the GABAA receptor. The effects and subsequent withdrawal symptoms are similar to those caused by alcohol, benzodiazepines and barbiturates. This study examined the withdrawal effects of progesterone with regards to the influence of individual baseline exploration and risk taking. Rats were tested on the elevated plus maze (EPM) before hormonal treatment, in order to evaluate differences in risk taking and exploration of open and elevated areas. Treatment consisted of ten consecutive once a day progesterone or vehicle s.c. injections. On the last day of treatment, estradiol was injected in addition to progesterone, followed by a 24-h withdrawal before testing in the open field test (OF). Progesterone-treated rats showed a withdrawal effect of open area avoidance in the OF. The vehicle-treated control rats showed strong correlations between the EPM and OF parameters. This relationship was not found for the progesterone group at withdrawal. Rats with greater numbers of open arm entrance in the EPM pretest showed an increased sensitivity to progesterone withdrawal (PWD) compared to rats with low exploration and risk taking. The results indicate that the effects of PWD relate to individual exploration and risk taking. Furthermore, the possible analogy of PWD and PMS/PMDD in relation to individual traits is discussed. © 2006 Elsevier Inc. All rights reserved. Keywords: Allopregnanolone; Progesterone; Exploration; Risk taking; GABAA receptor; Rat; Withdrawal; Open field; Elevated plus maze

Introduction Withdrawal effects after treatment with GABAA receptor active drugs is a well-known phenomenon occurring after a period of tolerance (Saunders and Ho, 1990; Wafford, 2005). The most common GABAA receptor stimulating drugs are the benzodiazepines, barbiturates and alcohol, all which are potent GABAA receptor modulators with comparable withdrawal effects (Little, 1991; Tanaka et al., 1991; Cagetti et al., 2003). Progesterone effects are similar to that of GABAA receptor acting drugs, and withdrawal has been shown to produce insensitivity to ⁎ Corresponding author. Fax: +46 90 776006. E-mail address: [email protected] (T. Bäckström). 0018-506X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2006.03.002

the potentiating effects of the benzodiazepine lorazepam on GABA-gated chloride ion currents (Costa et al., 1995). The effects of progesterone are most likely caused by allopregnanolone (3alpha-OH-5alpha-pregnan-20-one or 3alpha, 5alpha-THP) (Smith et al., 1998), which is a ringA-reduced metabolite of progesterone with similar effects on the GABAA receptor as alcohol, barbiturates and benzodiazepines (Majewska et al., 1986; Paul, 1992; Grobin et al., 1998). Allopregnanolone strongly enhances GABA-evoked current at the GABAA receptor (Majewska et al., 1986; Harrison et al., 1987; Lambert et al., 1990), which increases chloride ion flux (Canonaco et al., 1993), and consequently raises the inhibitory tone of the CNS (Purdy et al., 1990). Furthermore, allopregnanolone exhibits withdrawal properties

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in a comparable manner to other GABAA modulatory drugs (Smith et al., 1998), and withdrawal has been shown to upregulate the GABAA receptor alpha-4 subunit expression, which occur in conjunction with anxiety characteristics (Moran et al., 1998; Gulinello et al., 2001; Hsu and Smith, 2003; Gulinello et al., 2003). Several animal behavior models have been used to study withdrawal effects from progesterone. Most of them focus on behaviors analogous to anxiety, such as decreased latency and increased duration of defensive burial, along with increased avoidance of the open arms in the EPM (Gallo and Smith, 1993). Repeated progesterone administration has further been shown to produce insensitivity to the sedative effects of lorazepam on locomotor activity, along with an increased expression of the GABAA receptor alpha-4 subunit (Moran et al., 1998). Another prominent effect of progesterone is the increase of seizure susceptibility after discontinuation of chronic progesterone treatment (Moran and Smith, 1998). Combining progesterone and estradiol has also been proven effective, and subsequent withdrawal from chronic treatment produces immobility in the forced swim test (Galea et al., 2001). The behavioral effects of progesterone are likely to be produced, wholly or partially by its metabolite allopregnanolone. The effects of allopregnanolone related to the GABAA receptor do not seem to be tied to sex, since allopregnanolone modulates GABAA receptor subunit expression and behavior in both males and females (Corpechot et al., 1993; Concas et al., 1998; Gomez et al., 1998). Furthermore, withdrawal from diazepam or allopregnanolone (Stock et al., 1999; Gulinello et al., 2002) does not show any sex differences. The withdrawal effects from progesterone and concomitant withdrawal from allopregnanolone have been used as an animal model to mimic the premenstrual syndrome (PMS) and premenstrual dysphoric disorder (PMDD) (Gallo and Smith, 1993; Griffiths and Lovick, 2005). In humans, elevated levels of allopregnanolone are present during the luteal phase of the menstrual cycle, pregnancy and during stress. Both PMS and PMDD have been linked to changes in sensitivity towards pregnanolone, benzodiazepines and alcohol (Sundstrom et al., 1997a,b, 1998; Nyberg et al., 2004). For a greater understanding of substances acting on the GABAA receptor, individual and genetic differences as well as previous experiences ought to be incorporated into the analysis (Parmigiani et al., 1998). Since animals with differences in anxiety characteristics have been shown to react with different sensitivity to anxiolytic drugs (FernandezTeruel et al., 1991; Gendron and Brush, 1996), and there are prominent strain differences with regard to baseline exploration activity and effects of diazepam (Ramos et al., 1997). Furthermore, genetic differences in anxiety-related behavior patterns also show diversity in GABAA receptor sensitivity (Robertson et al., 1978; Chapouthier et al., 1991; Clement et al., 1997). Differences within strains have also been shown, where more active and exploring animals display a heightened sensitivity to diazepam (Crawley and Davis, 1982; Mathis et al., 1994). Allopregnanolone exhibit a similar

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connection to behavioral differences, since less anxious mice strains are more sensitive to the effects (Finn et al., 1997). The effects of allopregnanolone exhibit a similar relationship between phenotype and drug responsiveness as other substances acting on the GABAA receptor. The objective of this study was to evaluate the effects of withdrawal from long-term progesterone treatment with special regard to the effects of progesterone in relation to individual traits.

Materials and method Animals 20 male Wistar rats (Scanbur, BK Sollentuna, Sweden) were used, with a mean weight of 227 ± 2 g at the beginning of the experiment. The rats were housed in pairs in acrylic cages measuring 56 × 34 × 19 cm. Their tails were marked with a permanent marker for identification. The subjects were kept on a reverse light/dark cycle (10/14-h light/dark), with lights on at 9 PM and off at 7 AM. Food and water were supplied ad libitum. Two similar animal facilities were used during the experiment, both were kept at 21°C and 40– 50% humidity. The Animal Experiment Ethical Committee in Umeå, Sweden, approved the protocols.

Treatment The subjects were divided into two groups (n = 10). The control group received vehicle (sesame oil) and a single dose of estradiol (10 μg/kg), and the experimental group received daily progesterone (5 mg/kg) and a single dose of estradiol (10 μg/kg). Progesterone (5 mg/ml) and estradiol (40 μg/ml) were dissolved in sesame oil. The treatments were randomized within each cage pair. Injections were administrated subcutaneously at 0800–0900 AM for 10 consecutive days. On the last day of treatment, in addition to progesterone/ vehicle, all rats were given estradiol (10 μg/kg) intraperitoneally (Gallo and Smith, 1993). Progesterone and estradiol were obtained from Sigma Aldrich, St. Louis, MO, USA.

Elevated plus maze (EPM) The elevated plus maze (Sandown scientific TM, Hampton, UK) was constructed of black polypropylene plastic and elevated 74 cm above the floor. Each maze arm extended 50 cm from the junction area, which measured 10 × 10 cm. The arms were 10 cm wide, and the open maze arms had 1-cmhigh lips extending 26 cm from the junction area. The closed maze arms had walls extending 40 cm above the runway floor. The testing chamber was illuminated by ceiling sited red florescent light located above the EPM intersection. Each runway had 2 IR beams, which were located 4.5 and 6.5 cm from the end of the junction area. The IR beams were connected to an 8channel IR controller via an interface cabinet to a computer. The data were analyzed using the MED-PC IV software (MED Associates Inc., St. Albans, VA, USA). A complete maze arm entrance required a 6.5-cm passage onto an arm, breaking both IR beams. An exploratory behavior was registered if the rat moved outwards from the junction area without passing both beams. The recorded parameters were number of entries onto the open and closed maze arms, percentage time spent on each arm, number of non-complete entrances (open and closed arm exploration) onto each arm, and total activity (added arm entries and arm explorations).

Open field The open field apparatus consisted of a round black circular Fiberglass arena with 60-cm walls and a diameter of 180 cm. A video camera monitored

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Table 1 Mean and ± SEM for the pre-treatment EPM test (% time out of 10 min, frequencies per 10 min) and the post-treatment withdrawal OF test (% time out of 15 min) parameters Control group

PWD group

Mean

±SEM

Mean

±SEM

EPM Open arm entrance % Open arm time Open arm explore Closed arm entrance % Closed arm time Closed arm explore % Junction time Total activity

6.40 6.96 69.70 18.00 54.26 13.30 39.18 107.40

1.12 1.35 9.05 1.31 4.58 1.21 3.74 10.45

9.00 13.56 65.33 16.78 48.98 13.67 37.46 104.78

1.62 2.66 7.02 1.00 4.28 1.39 2.01 8.81

OF % Time in zone A % Time in zone B % Time in zone C % Time in zone A + B % Thigmotaxis time % Thigmotaxis path Path length (m) Speed (cm/s)

0.99 7.54 91.47 8.53 66.43 52.76 117.07 13.10

0.22 1.08 1.25 1.25 2.98 3.17 3.66 0.43

0.82 3.91 ⁎⁎ 95.27 ⁎ 4.73 ⁎ 75.59 ⁎ 62.44 ⁎ 108.17 12.11

0.20 0.39 0.52 0.52 2.01 2.24 7.47 0.86

progesterone-treated group had one outlier which was excluded from the data analysis. For EPM open arm time, the outlier was 2.05 standard deviations above the mean, and for OF percentage of time in zone B 2.70 standard deviations above the mean. Including the outlier did not abolish the significant effects of treatment or the influence of pretreatment risk taking and exploration on the effect of treatment. Data are shown as mean and ± SEM. The statistical analysis was performed using the non-parametric two-tailed Mann-Whitney test and Pearson's correlation. A one-way ANOVA was used to compare the regression lines (Fig. 1), P<0.05 was considered significant.

Total activity represents added closed and open arm explore and entry scores. Significant correlations in bold. ⁎ P < 0.05. ⁎⁎ P < 0.01. the rat's performance and was connected to an image analyzer (HVS Image, Hampton, UK), and data were analyzed using the software HVS Water 2020. The program was used to calculate percentage of the total time spent in each of 3 equally distanced circular zones, A (central zone), B, and C (outermost zone), speed and total distance were also recorded. Percentage of total time spent and percentage of the total distance in the outermost annulus of the arena were designated as thigmotactic movement. The thigmotaxis parameter was defined as movement within 9 cm from the arena wall. The outermost zone (zone C) included the thigmotaxis area. Dim white lights were used in the testing room and the adjacent control room.

Experimental design The animals were habituated for 7 days before the baseline exploration and risk-taking EPM test. Handling was reduced to mere maintenance and cleaning. EPM testing was conducted 24 h prior to commencing drug treatment. The EPM testing schedule was randomized, and the experimenter individually brought animals from the adjoining storage room to the testing chamber. The rats were placed in the junction area facing an open arm. Before each trial, the EPM was thoroughly cleaned with 70% ethanol, which was allowed to fully evaporate before the next subject commenced testing. Each trial extended over 10 min. After EPM testing, the animals were brought to a different storage room, where all injections were carried out. Testing in the OF was performed 24 h after the last injections. Prior to testing, the animals were allowed to habituate to the testing room for 30 min. All animals were brought into the testing room simultaneously, and the subjects remained in their home cages. The experiment lasted from 8 AM until 2 PM, and the testing schedule was randomized across all animals. Before each trial, the open field was cleaned with 70% ethanol, which was allowed to fully evaporate. The rats were carried from the cage to the OF by the experimenter and allowed to explore the open field for 15 min.

Statistical analysis The experimental animals were randomly assigned to 10 days progesterone or vehicle treatment, regardless of the pretreatment EPM scores (Table 1). The

Fig. 1. (A) Regression lines for the control and PWD groups between the number of EPM open arm entries before treatment and percentage time spent in OF zone B 24 h after the last injection (at withdrawal). The regression lines were significantly different between the groups. F(1,18) = 7.35; P = 0.005. For the control group, the frequency of EPM open arm entries prior to treatment correlated with time spent in the OF zone B (r = 0.792. P = 0.006). The progesterone-treated rats with a higher frequency of EPM open arm entries before treatment spent less time in the center of the OF at withdrawal than what was expected from the high EPM-OF correlations in the control rats. The diamond-shaped icon represents an outlier. The vertical line on the abscissa separates the rats into high and low responding subgroups. (B) The histogram illustrates the differences in percentage time spent in OF zone B between PWD and control within the high and low responding subgroups (below and above 6 EPM open arm entries). Within the high responding group, there was a difference between treatment groups (P = 0.006).

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Results Behavior at PWD The two groups performed differently in the open field test at withdrawal from the treatment. The progesterone-treated group had lower mean percentage time spent in OF zone B compared to the control group (P = 0.006), zone A + B (P = 0.016), and higher mean percentage time spent in zone C (P = 0.016). Differences between groups were also found for percentage of time spent in thigmotaxis (P = 0.034) and percentage of path spent in thigmotaxis (P = 0.034). Thus, the rats withdrawn from progesterone spent less time in the inner part of the OF and more time and a larger part of the total distance circling the walls of the open field. Correlations between pretreatment EPM and post-treatment OF parameters The correlations between the EPM and OF parameters for the control group were strong (Table 2). In the progesterone-treated group, the correlation between the pretest EPM open arm entrance and time spent in OF zone B was cancelled at withdrawal. There were positive relationships between EPM open arm entries and percentage of time spent in the OF zone B (P = 0.006, Fig. 1) and zone AB (P = 0.012). As expected, the inverse relationship appeared for EPM open arm entry and percentage time in OF zone C (P = 0.012), the outermost zone in the OF. Correlations were also found between EPM open arm time and percentage time in the OF zone B (P = 0.006), zone A + B (P = 0.012), and a negative correlation with percentage time in the OF zone C (P = 0.012). A negative correlation was also found between the time in the EPM closed arms and percentage time in the OF zone B (P = 0.048). There was further a positive correlation between speed in the OF and open arm

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exploration in the EPM test (P = 0.028). No correlations were found for EPM total activity. For the progesterone-withdrawn group, two negative correlations were found; between OF thigmotaxis path and EPM open arm exploration (P = 0.019, Table 3) and EPM total activity (P = 0.036). Difference in regression between groups The regression lines for the control and progesterone group clearly differed between the number of open arm entries in the pretreatment EPM test (X-axis) and the percentage time spent in OF zone B (Y-axis) (Fig. 1). For the control group, the regression line was determined by the equation: Y = 2.64 + 0.766X, The regression line for the progesterone-withdrawn group was given by Y = 4.02–0.01X, an almost horizontal line. The regression lines were significantly different between the groups, F(1,18) = 7.35; P = 0.005. To clarify the impact of the EPM pretest results, the rats were divided into subgroups of high and low risk taking/exploration, 6 EPM open arm entries was set as the divergence point. The low responding control group showed a mean value of 5.44 ± 0.86 for percentage time spent in OF zone B and 3.97 ± 0.52 for the PWD group. The high responding control group showed a mean value of 9.64 ± 1.52 for percentage time spent in OF zone B, and the PWD group had a mean time of 3.88 ± 0.56. Within the high risktaking/exploring group, there was a difference between treatment groups (P = 0.006, Fig. 1). Discussion The drive to seek information about the immediate environment is common for many species, with various behaviors included in this information search (exploratory activity) (Augustsson and Meyerson, 2004). Such activity is a direct incentive to promote survival and reproductive success

Table 2 Correlations between EPM and OF parameters for the control group

1. % Junction time 2. Closed explore 3. Closed entrance 4. % Closed arm time 5. Open explore 6. Open entrance 7. % Open arm time 8. Total activity 9. % Time in zone A 10. % Time in zone B 11. % Time in zone C 12. % Time zone A + B 13. % Thigmotaxis time 14. % Thigmotaxis path 15. Path length OF 16. Speed OF

1

2

1 0.39 −0.08 −0.97** 0.75* 0.46 0.58 0.74 ⁎ 0.24 0.50 −0.48 0.48 −0.04 −0.00 0.54 0.55

1 0.01 −0.27 0.58 0.03 −0.14 0.62 −0.34 −0.29 0.31 −0.31 −0.15 −0.13 0.27 0.33

Significant correlations in bold. ⁎ P < 0.05. ⁎⁎ P < 0.01. ⁎⁎⁎ P < 0.001.

3

4

5

6

7

8

9

10

11

12

13

14

15 EPM

1 0.00 0.07 0.4 0.19 0.22 −0.32 −0.08 0.16 −0.13 −0.18 −0.21 −0.46 −0.31

1 −0.69 ⁎ 1 −0.63 0.36 1 −0.76 ⁎ 0.25 0.85 ⁎⁎ 1 −0.70 ⁎ 0.98 ⁎⁎⁎ 0.47 0.31 1 −0.32 −0.20 0.37 0.40 −0.21 1 OF −0.64 ⁎ 0.21 0.79 ⁎⁎ 0.79 ⁎⁎ 0.26 0.72 ⁎ 1 0.61 −0.15 −0.75 ⁎ −0.75 ⁎ −0.16 −0.80 ⁎⁎ −0.99 ⁎⁎ 1 −0.61 0.15 0.75 ⁎ 0.75 ⁎ 0.16 0.80 ⁎⁎ 0.93 ⁎⁎ −1 ⁎ 1 −0.80 0.15 0.28 0.28 0.12 0.21 0.32 0.31 0.3 1 −0.10 0.11 0.27 0.27 0.08 3.76 0.32 −0.35 0.35 0.96 ⁎ 1 −0.46 0.56 0.03 0.03 0.47 0.45 0.43 −0.45 0.45 0.35 0.35 1 −0.46 0.69 ⁎ −0.02 −0.09 0.61 0.29 0.36 −0.36 0.36 0.28 0.41 0.97 ⁎⁎

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Table 3 Correlations between EPM and OF parameters for the PWD group

1. % Junction time 2. Closed explore 3. Closed entrance 4. % Closed arm time 5. Open explore 6. Open entrance 7. % Open arm time 8. Total activity 9. % Time in zone A 10. % Time in zone B 11. % Time in zone C 12. % Time zone AB 13. % Thigmotaxis t 14. % Thigmotaxis p 15. Path length OF 16. Speed OF

1

2

1 0.40 0.51 −0.89 ⁎⁎ 0.28 0.58 0.67 0.45 0.43 −0.46 −0.14 0.135 0.62 0.02 −0.66 −0.65

3

4

1 0.35 1 −0.29 −0.45 1 0.09 0.19 −0.41 0.22 0.23 −0.79 ⁎ 0.17 0.34 −0.94 ⁎⁎⁎ 0.31 0.36 −0.57 −0.01 0.23 −0.42 −0.05 0.47 −0.02 0.08 −0.45 0.18 −0.08 0.45 −0.18 0.29 0.48 −0.42 0.08 −0.11 0.31 0.03 0.23 0.66 0.03 0.23 0.67

5

6

7

8

9

10

11

12

13

14

15 EPM

1 0.68 ⁎ 0.44 0.96 ⁎⁎⁎ 0.10 −0.23 0.14 −0.14 −0.34 −0.76 ⁎ −0.01 0.02

1 0.832 ⁎⁎ 0.786 ⁎ 0.53 −0.05 −0.17 0.17 0.12 −0.53 −0.39 −0.39

1 −0.57 −0.42 −0.02 0.18 −0.18 −0.42 0.31 0.66 0.67

1 0.19 1 −0.15 0.42 1 0.04 −0.72 ⁎ −0.93 ⁎⁎⁎ 1 −0.04 0.72 ⁎ 0.93 ⁎⁎⁎ −1⁎ −0.15 0.69 ⁎ 0.44 −0.61 −0.70 ⁎ 0.27 0.15 −0.22 −0.50 −0.30 0.37 −0.16 −0.03 −0.26 0.36 0.17

OF

1 0.61 1 0.22 0.66 1 0.16 −0.34 −0.13 1 0.17 −0.32 −0.11 0.97 ⁎⁎⁎

Significant correlations in bold. t, time; p, path. ⁎ P < 0.05. ⁎⁎ P < 0.01. ⁎⁎⁎ P < 0.01.

and should subsequently be evolutionary conserved over the borders of species. The process of seeking information holds an element of risk assessment that represents an estimate of the potential loss and gain from risk taking (Lima and Dill, 1989). The relationship between risk taking and caution mirrors the emotional reactivity (“anxiety”) of the individual (Roy and Chapillon, 2004). For the laboratory rat, risk seems associated with open and elevated areas, whereas closeness to a wall has qualities of security (Rodgers et al., 1999). This risk/caution relationship is the basis for several behavioral models aimed to measure mental conditions such as anxiety, fear, emotional reactivity, panic reactions etc. (Treit, 1985; Lister, 1990). Two such widely used models are the EPM and OF. In this study, the EPM was used to gain information of individual exploration and risk taking under the pretest conditions. The EPM was chosen as the pretest since it is a well-known method for assessing emotional reactivity in rats (Rodgers et al., 1999). Further, the narrow and elevated arms are likely to produce a more rigid classification of risk taking and exploration than the open area of the OF. The order of the tests was also influenced by the fact that PWD has not previously been studied in the OF, which has otherwise been useful in testing withdrawal effects from other GABAA receptor modulatory drugs (van der Laan et al., 1993). For the control group, the number of EPM open arm entries strongly correlated with the percentage of time spent in the inner parts of the OF. The time interval between the two tests was 10 days; thus, it seems as the willingness for exploration and risk taking in each individual not exposed to progesterone was kept fixed in the adult male rat. The EPM and OF tests have previously been shown to concur in their capability of measuring open area avoidance (Ramos et al., 1997; Aguilar et al., 2002; Carola et al., 2002), and this was further strengthen by the data from the control group in this study. The correlation between the two models still remained

despite repeated vehicle injections, indicating further individual stability even after exposure to mild stressors. Thus, combining these models produces a coherent behavioral profile of exploration and risk taking. The main finding in the present study was the effect of PWD on avoidance of open spaces in the open field and increased time spent close to the wall, i.e., increased thigmotaxis. Thus, withdrawal from long-term progesterone treatment induced a mental condition of increased “anxiety” which reached the same level for all animals. The difference of the regression lines between the number of EPM open arm entries before treatment and percentage time spent in OF zone B 24 h after treatment (i.e., the withdrawal condition) clearly showed that the relationship in the treatment group was eliminated. The effect of the withdrawal was particularly evident in rats initially more exploring and risk taking in response to stimuli of open space and height in the EPM test. The more cautious rats, i.e., those with a lower frequency of EPM open arm entries before progesterone treatment did not show a marked change in avoidance behavior in the OF at withdrawal. Thus, the results suggest that the basal level of exploration and risk taking is of importance for the effects of PWD. The low responders appeared less sensitive to PWD, or they may have been affected in a manner not possible to detect with the OF test. There is a possibility that the low risk experimental animals did not further decrease their risk taking at PWD since the response was already at the lowest measurable level. Another technique might be able to measure PWD in low risk taking and exploring animal with greater sensitivity. The increased “anxiety” behavior after PWD is in line with earlier findings in male rats (Gulinello et al., 2002). However, the influence of baseline exploration and risk taking on the withdrawal effect has to our knowledge not previously been shown. Earlier studies with mice have found greater diazepam

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effects in more active and exploring animals (Crawley and Davis, 1982), suggesting that a withdrawal response in these animals could be stronger compared to less exploring animal. Differences in behavior at baseline can have effects on the experimental outcome. For example, individual differences in reactions to novelty has been found predictive of aggressive behavior (Sgoifo et al., 1996). Furthermore, genetic and individual differences affect aggressive behavior response following administration of GABAA receptor stimulating drugs (Miczek et al., 2003), and more important individual differences exist in estrous-cycle-related aggression in rats. (Melchior et al., 2004). Individual differences do exist, and speculations of the origin can be somewhat overwhelming, along with the interpretations of what a behavior signifies. The EPM is a well-known and validated tool for investing effects of anxiolytic drugs. With this task, increased open arm entries and increased time spent on the open arms together with increased scores of exploration are interpreted as indicative of a less anxious mental state (Montgomery, 1955; Pellow et al., 1985; Reibaud and Böhme, 1993). Differences in spontaneous open arm entrance, open arm time and exploration of the apparatus can be interpreted as differences in the extent to which the EPM elicit fear and exploratory drive. The unprovoked behavior in the EPM can be overtly characterized as differences in risk taking and exploration, which has been referred to as a divergence of coping styles (Koolhaas et al., 1999). Rats with frequent open arm entries do react differently to the general aversive stimuli of the EPM, and the reaction might reflect an agitated mental state promoting an increased avoidance response to escape the apparatus. Risk taking and exploration in the EPM paradigm could be interpreted as an active drive to avoid danger and seek safety, with a probable divergent response to anxiolytic drugs compared to low risk takers. In support of this, high responders to novelty induced locomotor activity in rats have been compared to risk-taking behavior, with higher sensitivity to reinforcing properties of drugs, differences in dopaminergic activity, and prolonged secretion of corticosterone (Piazza et al., 1990; Dellu et al., 1996). There have been several attempts to explain the link between exploration, reaction to novelty and corticosterone, but the subject is still controversial; (Piazza et al., 1990; Piazza et al., 1991; Koolhaas et al., 1999; Rodgers et al., 1999; Kabbaj et al., 2000). The present study was conducted using male rats, previous PWD studies have mainly included females. Therefore, it remains to be shown if the specific exploration and risk-taking trait is predictive of PWD in females as well. Previous studies of diazepam withdrawal has not identified any sex differences (Stock et al., 1999), and more important upregulation of the alpha-4 subunit of the GABAA receptor following PWD has not been shown to be sex-dependent (Gulinello et al., 2002). In humans, withdrawal from progesterone occurs endogenously during premenstrual and postpartum periods. A link between endogenous progesterone decline and response alterations of the GABA system has been shown through increased incidence and severity of seizures during PWD in the luteal phase of the menstrual cycle (Laidlaw, 1956; Backstrom, 1976;

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Herzog et al., 1997). A decrease in the sensitivity of the GABA system has also been observed during the premenstrual period in women with PMDD, as the response to benzodiazepines, pregnanolone and alcohol decreases (Sundstrom et al., 1997a,b; Nyberg et al., 2004). Symptoms from decreasing levels of progesterone are common and bear similarities to withdrawal from GABAA receptor stimulating drugs, and there are clinical examples of individual characteristics influencing the response to GABAA receptor acting drugs (Ben-Porath and Taylor, 2002; Lane et al., 2005). In contrast, normal plasma concentrations of gonadal steroids have been shown to trigger PMS symptoms more readily in susceptible women (Schmidt et al., 1998). Individual differences with regards to risk taking, aggression/ irritability, exploration, novelty seeking, and corticosterone sensitivity could possibly predict effects of PWD during late luteal phase of the menstrual cycle and during the postpartum period, implications of such interactions exist in the clinical literature (Freeman et al., 1995; Berlin et al., 2001). In conclusion, the effects of PWD relate to individual risk taking and exploration were high risk-taking and exploring rats show a greater sensitivity. When assessing the behavioral effects of neurosteroids establishing a behavioral baseline is recommended, since a predisposition of the rat for high risk taking and exploration has valid implications for behavioral reactions to withdrawal from progesterone metabolites. Stable individual differences can be detected through comparing OF and EPM test results, which aid the predictions of withdrawal from GABAA receptor acting progesterone metabolites with regards to open area avoidance. Acknowledgments This work was supported by the EU structural program objective 1, the Swedish Research Council project 11198, and the Umeå University foundations. References Aguilar, R., Gil, L., Flint, J., Gray, J.A., Dawson, G.R., Driscoll, P., GimenezLlort, L., Escorihuela, R.M., Fernandez-Teruel, A., Tobena, A., 2002. Learned fear, emotional reactivity and fear of heights: a factor analytic map from a large F(2) intercross of Roman rat strains. Brain Res. Bull. 57 (1), 17–26. Augustsson, H., Meyerson, B.J., 2004. Exploration and risk assessment: a comparative study of male house mice (Mus musculus musculus) and two laboratory strains. Physiol. Behav. 81 (4), 685–698. Backstrom, T., 1976. Epileptic seizures in women related to plasma estrogen and progesterone during the menstrual cycle. Acta Neurol. Scand. 54 (4), 321–347. Ben-Porath, D.D., Taylor, S.P., 2002. The effects of diazepam (valium) and aggressive disposition on human aggression: an experimental investigation. Addict. Behav. 27 (2), 167–177. Berlin, R.E., Raju, J.D., Schmidt, P.J., Adams, L.F., Rubinow, D.R., 2001. Effects of the menstrual cycle on measures of personality in women with premenstrual syndrome: a preliminary study. J. Clin. Psychiatry 62 (5), 337–342. Cagetti, E., Liang, J., Spigelman, I., Olsen, R.W., 2003. Withdrawal from chronic intermittent ethanol treatment changes subunit composition, reduces synaptic function, and decreases behavioral responses to positive allosteric modulators of GABAA receptors. Mol. Pharmacol. 63 (1), 53–64.

214

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Canonaco, M., Tavolaro, R., Maggi, A., 1993. Steroid hormones and receptors of the GABAA supramolecular complex: II. Progesterone and estrogen inhibitory effects on the chloride ion channel receptor in different forebrain areas of the female rat. Neuroendocrinology 57 (5), 974–984. Carola, V., D'Olimpio, F., Brunamonti, E., Mangia, F., Renzi, P., 2002. Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behaviour in inbred mice. Behav. Brain Res. 134 (1–2), 49–57. Chapouthier, G., Bondoux, D., Martin, B., Desforges, C., Launay, J.M., 1991. Genetic difference in sensitivity to beta-carboline: evidence for the involvement of brain benzodiazepine receptors. Brain Res. 553 (2), 342–346. Clement, Y., Proeschel, M.F., Bondoux, D., Girard, F., Launay, J.M., Chapouthier, G., 1997. Genetic factors regulate processes related to anxiety in mice. Brain Res. 752 (1–2), 127–135. Concas, A., Mostallino, M.C., Porcu, P., Follesa, P., Barbaccia, M.L., Trabucchi, M., Purdy, R.H., Grisenti, P., Biggio, G., 1998. Role of brain allopregnanolone in the plasticity of gamma-aminobutyric acid type A receptor in rat brain during pregnancy and after delivery. Proc. Natl. Acad. Sci. U. S. A. 95 (22), 13284–13289. Corpechot, C., Young, J., Calvel, M., Wehrey, C., Veltz, J.N., Touyer, G., Mouren, M., Prasad, V.V., Banner, C., Sjovall, J., et al., 1993. Neurosteroids: 3 alpha-hydroxy-5 alpha-pregnan-20-one and its precursors in the brain, plasma, and steroidogenic glands of male and female rats. Endocrinology 133 (3), 1003–1009. Costa, A.M., Spence, K.T., Smith, S.S., ffrench-Mullen, J.M., 1995. Withdrawal from the endogenous steroid progesterone results in GABAA currents insensitive to benzodiazepine modulation in rat CA1 hippocampus. J. Neurophysiol. 74 (1), 464–469. Crawley, J.N., Davis, L.G., 1982. Baseline exploratory activity predicts anxiolytic responsiveness to diazepam in five mouse strains. Brain Res. Bull. 8 (6), 609–612. Dellu, F., Piazza, P.V., Mayo, W., Le Moal, M., Simon, H., 1996. Noveltyseeking in rats-biobehavioral characteristics and possible relationship with the sensation-seeking trait in man. Neuropsychobiology 34 (3), 136–145. Fernandez-Teruel, A., Escorihuela, R.M., Tobena, A., Driscoll, P., 1991. Stress and putative endogenous ligands for benzodiazepine receptors: the importance of characteristics of the aversive situation and of differential emotionality in experimental animals. Experientia 47 (10), 1051–1056. Finn, D.A., Roberts, A.J., Lotrich, F., Gallaher, E.J., 1997. Genetic differences in behavioral sensitivity to a neuroactive steroid. J. Pharmacol. Exp. Ther. 280 (2), 820–828. Freeman, E.W., Schweizer, E., Rickels, K., 1995. Personality factors in women with premenstrual syndrome. Psychosom. Med. 57 (5), 453–459. Galea, L.A., Wide, J.K., Barr, A.M., 2001. Estradiol alleviates depressive-like symptoms in a novel animal model of post-partum depression. Behav. Brain Res. 122 (1), 1–9. Gallo, M.A., Smith, S.S., 1993. Progesterone withdrawal decreases latency to and increases duration of electrified prod burial: a possible rat model of PMS anxiety. Pharmacol. Biochem. Behav. 46 (4), 897–904. Gendron, C.M., Brush, F.R., 1996. The effects of extended training and acute administration of an anxiolytic on avoidance learning and intertrial responding in the Syracuse strains of rats. Behav. Genet. 26 (6), 575–580. Gomez, C., Saldivar-Gonzalez, J.A., Rodriguez, R., 1998. Progesterone induces changes in defensive burying in male rats. Proc. West. Pharmacol. Soc. 41, 133–134. Griffiths, J., Lovick, T., 2005. Withdrawal from progesterone increases expression of alpha4, beta1, and delta GABA(A) receptor subunits in neurons in the periaqueductal gray matter in female Wistar rats. J. Comp. Neurol. 486 (1), 89–97. Grobin, A.C., Matthews, D.B., Devaud, L.L., Morrow, A.L., 1998. The role of GABA(A) receptors in the acute and chronic effects of ethanol. Psychopharmacology (Berlin) 139 (1–2), 2–19. Gulinello, M., Gong, Q.H., Li, X., Smith, S.S., 2001. Short-term exposure to a neuroactive steroid increases alpha4 GABA(A) receptor subunit levels in

association with increased anxiety in the female rat. Brain Res. 910 (1–2), 55–66. Gulinello, M., Gong, Q.H., Smith, S.S., 2002. Progesterone withdrawal increases the alpha4 subunit of the GABA(A) receptor in male rats in association with anxiety and altered pharmacology-a comparison with female rats. Neuropharmacology 43 (4), 701–714. Gulinello, M., Orman, R., Smith, S.S., 2003. Sex differences in anxiety, sensorimotor gating and expression of the alpha4 subunit of the GABAA receptor in the amygdala after progesterone withdrawal. Eur. J. Neurosci. 17 (3), 641–648. Harrison, N.L., Majewska, M.D., Harrington, J.W., Barker, J.L., 1987. Structure-activity relationships for steroid interaction with the gammaaminobutyric acidA receptor complex. J. Pharmacol. Exp. Ther. 241 (1), 346–353. Herzog, A.G., Klein, P., Ransil, B.J., 1997. Three patterns of catamenial epilepsy. Epilepsia 38 (10), 1082–1088. Hsu, F.C., Smith, S.S., 2003. Progesterone withdrawal reduces paired-pulse inhibition in rat hippocampus: dependence on GABA(A) receptor alpha4 subunit upregulation. J. Neurophysiol. 89 (1), 186–198. Kabbaj, M., Devine, D.P., Savage, V.R., Akil, H., 2000. Neurobiological correlates of individual differences in novelty-seeking behavior in the rat: differential expression of stress-related molecules. J. Neurosci. 20 (18), 6983–6988. Koolhaas, J.M., Korte, S.M., De Boer, S.F., Van Der Vegt, B.J., Van Reenen, C. G., Hopster, H., De Jong, I.C., Ruis, M.A., Blokhuis, H.J., 1999. Coping styles in animals: current status in behavior and stress-physiology. Neurosci. Biobehav. Rev. 23 (7), 925–935. Laidlaw, J., 1956. Catamenial epilepsy. Lancet 271 (6955), 1235–1237. Lambert, J.J., Peters, J.A., Sturgess, N.C., Hales, T.G., 1990. Steroid modulation of the GABAA receptor complex: electrophysiological studies. Ciba Found. Symp. 153, 56–71 (discussion 71–82). Lane, S.D., Tcheremissine, O.V., Lieving, L.M., Nouvion, S., Cherek, D.R., 2005. Acute effects of alprazolam on risky decision making in humans. Psychopharmacology 181 (2), 364–373 (Berlin). Lima, S.L., Dill, L.M., 1989. Behavioral decisions made under the risk of predation: a review and prospectus. Can. J. Zool. 68, 619–640. Lister, R.G., 1990. Ethologically-based animal models of anxiety disorders. Pharmacol. Ther. 46 (3), 321–340. Little, H.J., 1991. The benzodiazepines: anxiolytic and withdrawal effects. Neuropeptides 19, 11–14. Majewska, M.D., Harrison, N.L., Schwartz, R.D., Barker, J.L., Paul, S.M., 1986. Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science 232 (4753), 1004–1007. Mathis, C., Paul, S.M., Crawley, J.N., 1994. Characterization of benzodiazepine-sensitive behaviors in the A/J and C57BL/6J inbred strains of mice. Behav. Genet. 24 (2), 171–180. Melchior, L.K., Ho, H.P., Olsson, M., Annerbrink, K., Hedner, J., Eriksson, E., 2004. Association between estrus cycle-related aggression and tidal volume variability in female Wistar rats. Psychoneuroendocrinology 29 (8), 1097–1100. Miczek, K.A., Fish, E.W., De Bold, J.F., 2003. Neurosteroids, GABAA receptors, and escalated aggressive behavior. Horm. Behav. 44 (3), 242–257. Montgomery, K.C., 1955. The relation between fear induced by novel stimulation and exploratory behavior. J. Comp. Physiol. Psychol. 48 (4), 254–260. Moran, M.H., Smith, S.S., 1998. Progesterone withdrawal I: pro-convulsant effects. Brain Res. 807 (1–2), 84–90. Moran, M.H., Goldberg, M., Smith, S.S., 1998. Progesterone withdrawal: II. Insensitivity to the sedative effects of a benzodiazepine. Brain Res. 807 (1–2), 91–100. Nyberg, S., Wahlstrom, G., Backstrom, T., Sundstrom Poromaa, I., 2004. Altered sensitivity to alcohol in the late luteal phase among patients with premenstrual dysphoric disorder. Psychoneuroendocrinology 29 (6), 767–777. Parmigiani, S., Ferrari, P.F., Palanza, P., 1998. An evolutionary approach to behavioral pharmacology: using drugs to understand proximate and ultimate mechanisms of different forms of aggression in mice. Neurosci. Biobehav. Rev. 23 (2), 143–153.

M. Löfgren et al. / Hormones and Behavior 50 (2006) 208–215 Paul, S.M.P.R.H., 1992. Neuroactive steroids. FASEB J. 6, 2311–2322. Pellow, S., Chopin, P., File, S.E., Briley, M., 1985. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J. Neurosci. Methods 14 (3), 149–167. Piazza, P.V., Deminiere, J.M., Maccari, S., Mormede, P., Le Moal, M., Simon, H., 1990. Individual reactivity to novelty predicts probability of amphetamine self-administration. Behav. Pharmacol. 1 (4), 339–345. Piazza, P.V., Maccari, S., Deminiere, J.M., Le Moal, M., Mormede, P., Simon, H., 1991. Corticosterone levels determine individual vulnerability to amphetamine self-administration. Proc. Natl. Acad. Sci. U. S. A. 88 (6), 2088–2092. Purdy, R.H., Morrow, A.L., Blinn, J.R., Paul, S.M., 1990. Synthesis, metabolism, and pharmacological activity of 3 alpha-hydroxy steroids which potentiate GABA-receptor-mediated chloride ion uptake in rat cerebral cortical synaptoneurosomes. J. Med. Chem. 33 (6), 1572–1581. Ramos, A., Berton, O., Mormede, P., Chaouloff, F., 1997. A multiple-test study of anxiety-related behaviours in six inbred rat strains. Behav. Brain Res. 85 (1), 57–69. Reibaud, M., Böhme, G.A., 1993. Evaluation of putative anxiolytics in the elevated plus-maze test. In: Conn, M.P. (Ed.), Paradigms for the Study of Behavior, Methods in Neuroscience. Academic Press, London, pp. 230–240. Robertson, H.A., Martin, I.L., Candy, J.M., 1978. Differences in benzodiazepine receptor binding in Maudsley reactive and Maudsley non-reactive rats. Eur. J. Pharmacol. 50 (4), 455–457. Rodgers, R.J., Haller, J., Holmes, A., Halasz, J., Walton, T.J., Brain, P.F., 1999. Corticosterone response to the plus-maze: high correlation with risk assessment in rats and mice. Physiol. Behav. 68 (1–2), 47–53. Roy, V., Chapillon, P., 2004. Further evidences that risk assessment and object exploration behaviours are useful to evaluate emotional reactivity in rodents. Behav. Brain Res. 154 (2), 439–448. Saunders, P.A., Ho, I.K., 1990. Barbiturates and the GABAA receptor complex. Prog. Drug Res. 34, 261–286. Schmidt, P.J., Nieman, L.K., Danaceau, M.A., Adams, L.F., Rubinow, D.R., 1998. Differential behavioral effects of gonadal steroids in women with

215

and in those without premenstrual syndrome. N. Engl. J. Med. (338), 209–216. Sgoifo, A., de Boer, S.F., Haller, J., Koolhaas, J.M., 1996. Individual differences in plasma catecholamine and corticosterone stress responses of wild-type rats: relationship with aggression. Physiol. Behav. 60 (6), 1403–1407. Smith, S.S., Gong, Q.H., Hsu, F.C., Markowitz, R.S., ffrench-Mullen, J.M., Li, X., 1998. GABA(A) receptor alpha4 subunit suppression prevents withdrawal properties of an endogenous steroid. Nature 392 (6679), 926–930. Stock, H., Ford, K., Biscardi, R., Wilson, M.A., 1999. Lack of sex differences in anxiety behaviors during precipitated benzodiazepine withdrawal in rats. Physiol. Behav. 66 (1), 125–130. Sundstrom, I., Ashbrook, D., Backstrom, T., 1997a. Reduced benzodiazepine sensitivity in patients with premenstrual syndrome: a pilot study. Psychoneuroendocrinology 22 (1), 25–38. Sundstrom, I., Nyberg, S., Backstrom, T., 1997b. Patients with premenstrual syndrome have reduced sensitivity to midazolam compared to control subjects. Neuropsychopharmacology 17 (6), 370–381. Sundstrom, I., Andersson, A., Nyberg, S., Ashbrook, D., Purdy, R.H., Backstrom, T., 1998. Patients with premenstrual syndrome have a different sensitivity to a neuroactive steroid during the menstrual cycle compared to control subjects. Neuroendocrinology 67 (2), 126–138. Tanaka, S., Aoki, K., Satoh, T., Yoshida, T., Kuroiwa, Y., 1991. Characteristic changes of the GABAA receptor in the brain of phenobarbital-dependent and withdrawn rats. Res. Commun. Chem. Pathol. Pharmacol. 73 (3), 269–279. Treit, D., 1985. Animal models for the study of anti-anxiety agents: a review. Neurosci. Biobehav. Rev. 9 (2), 203–222. van der Laan, J.W., Jansen van't Land, C., de Groot, G., 1993. Tolerance and withdrawal after chronic lorazepam treatment in rats. Eur. Neuropsychopharmacol. 3 (4), 521–531. Wafford, K.A., 2005. GABAA receptor subtypes: any clues to the mechanism of benzodiazepine dependence? Curr. Opin. Pharmacol. 5 (1), 47–52.