Psychopharmacology (2012) 219:213224 DOI 10.

1007/s00213-011-2389-y

ORIGINAL INVESTIGATION

The role of estrogen and testosterone in female rats in behavioral models of relevance to schizophrenia
Andrea Gogos & Perrin Kwek & Maarten van den Buuse

Received: 10 January 2011 / Accepted: 17 June 2011 / Published online: 29 July 2011 # Springer-Verlag 2011

Abstract Rationale The sex steroid hormone, estrogen, may play a protective role in schizophrenia. We previously found that estrogen treatment inhibited serotonin-1A (5-HT1A) and dopamine D2 receptor-mediated disruptions of prepulse inhibition (PPI), a measure of sensorimotor gating which is deficient in schizophrenia. Objectives The present study aimed to further explore the role of sex steroid hormones in schizophrenia. Part 1 of this study examined whether estrogen could inhibit PPI disruption induced by the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801. Part 2 investigated whether the functionally protective effect of estrogen occurs in another animal model of schizophrenia, amphetamineinduced locomotor hyperactivity. Part 3 compared our previous PPI findings in estrogen-treated rats, to treatment with testosterone. Methods Female SpragueDawley rats were ovariectomized (OVX) or sham-operated. Some OVX rats received silastic implants filled with either a low (E20) or high dose (E100) of estradiol, or a low (T5) or high dose (T20) of testosterone, for at least 2 weeks before behavioral testing.
A. Gogos (*) : P. Kwek : M. van den Buuse Behavioural Neuroscience Laboratory, The Mental Health Research Institute of Victoria, 155 Oak Street, Parkville, Victoria 3052, Australia e-mail: agogos@mhri.edu.au A. Gogos Centre for Neuroscience, University of Melbourne, Parkville, Victoria, Australia M. van den Buuse Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia

Results The disruption of PPI caused by MK-801 (0.1 mg/ kg) was significantly reduced by treatment with estradiol (E20 and E100). However, estradiol treatment did not alter amphetamine-induced (0.25 and 0.5 mg/kg) locomotor hyperactivity, in terms of distance traveled, ambulation, or vertical counts. In contrast to estrogen, testosterone treatment did not affect disruption of PPI after administration of 8-OH-DPAT (0.5 mg/kg) or apomorphine (0.3 mg/kg). Testosterone treatment significantly enhanced the MK-801induced (0.1 mg/kg) PPI disruption. Conclusions Estrogen is functionally protective against 5HT1A-, dopamine D2-, and NMDA receptor-induced PPI disruptions, while testosterone treatment enhances NMDA receptor-mediated PPI disruptions. Keywords Estrogen . Estradiol . Testosterone . Prepulse inhibition . Schizophrenia . Locomotor hyperactivity . 8-OH-DPAT . Apomorphine . MK-801 . Female rats

Introduction Men and women with schizophrenia differ in terms of age-at-onset, symptom severity, and functional outcome (Hfner et al. 1993; Szymanski et al. 1995). As women have a later age-at-onset and are generally not as unwell as men, it has been suggested that the female sex steroid hormone, estrogen, plays a protective role in schizophrenia (Hfner et al. 1993; Seeman and Lang 1990). There are a number of epidemiological, clinical, and animal studies that support this hypothesis (for example, Castle et al. (1995) Gogos and Van den Buuse (2004), Kulkarni et al. (2001), and Riecher-Rossler et al. (1994)). However, other studies have suggested that the gender differences may instead be attributed to the male sex steroid hormone, testosterone,

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playing a detrimental role in schizophrenia (Beratis et al. 1994; Salokangas 1995). For example, testosterone levels dramatically increase and peak during adolescence and then progressively decrease with age (Wilson 1996), which mimics the pattern for the onset of schizophrenia. The exact mechanism of action of sex hormones in schizophrenia remains unknown, but we suggest that they modulate neurotransmitter systems implicated in schizophrenia, such as the serotonin and dopamine systems (Gogos et al. 2010b; Sanchez et al. 2010). In order to examine a possible interaction between sex hormones and neurotransmitters, this study will measure prepulse inhibition (PPI) in rats. PPI is an operational measure of sensorimotor gating or information processing. PPI can be reliably measured across species and can be used as an animal model with relevance to schizophrenia as patients with schizophrenia have reduced PPI (Braff et al. 2001; Swerdlow et al. 2008). Several studies have implicated dopamine and serotonin in the regulation of PPI (Geyer et al. 2001). For example, treatment with the dopamine D1/D2 receptor agonist, apomorphine, or the 5-HT1A/5-HT7 receptor agonist, 8-hydroxy-di-propylaminotetralin (8-OHDPAT), caused a disruption of PPI in rats (Gogos et al. 2010b). We recently showed that the 8-OH-DPAT- and apomorphine-induced disruption of PPI could be inhibited by treatment with estrogen in female rats (Gogos et al. 2010b). Similarly, we showed in healthy women that the PPI disruption caused by treatment with the 5-HT1A receptor partial agonist, buspirone, could be inhibited by treatment with estrogen (Gogos et al. 2006). Our results support the notion that estrogen exerts a functional protection against mechanisms that disrupt PPI, in this case 5-HT1A receptor and dopamine D2 receptor activation. In the present study, we first aimed to examine whether this protective effect of estrogen might also occur with another neurotransmitter strongly implicated in schizophrenia, namely glutamate. Evidence suggesting an involvement of the glutamatergic system in schizophrenia includes that drugs acting as antagonists at the Nmethyl-D-aspartate (NMDA) glutamate receptor, such as phencyclidine, induce schizophrenia-like symptoms in healthy individuals and worsen symptoms in schizophrenia patients (Tamminga 1999). It has been proposed that NMDA receptor hypofunction is involved in the symptoms and course of schizophrenia (Olney et al. 1999). In the present study, we used the non-competitive NMDA receptor antagonist, MK-801 (or dizocilpine), which is known to disrupt PPI (Geyer et al. 2001) and assessed whether estrogen can modify its effect. The second aim of this study was to explore whether the protective effect of estrogen is also found for another behavior relevant to schizophrenia. Psychostimulant-induced

locomotor hyperactivity in rats is commonly used as a model for psychosis due to the similarity of the brain mechanisms involved, specifically increased dopamine release in subcortical dopaminergic areas (Van den Buuse 2010). We previously found that castrated male rats showed reduced 8-OH-DPAT-induced PPI disruption compared with sham-operated rats, and testosterone treatment reversed this effect (Gogos and Van den Buuse 2003). This suggests that castration protects against 5-HT1A receptor-mediated disruption of PPI by removing the source of testosterone, supporting the notion of a detrimental role of testosterone in schizophrenia. However, a number of clinical studies have suggested that testosterone treatment in women may improve cognitive function (Aleman et al. 2004; Shah et al. 2006; Wisniewski et al. 2002). For example, in healthy post-menopausal women, testosterone therapy improved visual and verbal memory and simple concentration (Shah et al. 2006). Further, high levels of testosterone were positively correlated with spatial ability in women with schizophrenia (Bergemann et al. 2008). Not all studies however support a therapeutic effect of testosterone; other studies showed no improvements with testosterone treatment in post-menopausal women (Kocoska-Maras et al. 2011; Moller et al. 2010). Nevertheless, given that cognitive deficits in schizophrenia have been consistently reported and are considered the most debilitating and intractable symptoms of schizophrenia (Aleman et al. 1999; Gogos et al. 2010a; Saykin et al. 1994), it is possible that testosterone treatment may improve cognition in women with schizophrenia. In the present study, we therefore aimed to examine the effect of testosterone treatment on drug-induced PPI disruption in female rats (aim 3). In summary, the present study aimed to further explore the role of estrogen and testosterone in female rats in animal models with relevance to schizophrenia, specifically PPI and locomotor hyperactivity. We investigated (1) the effect of estrogen on MK-801-induced PPI disruption, (2) the effect of estrogen on amphetamine-induced locomotor hyperactivity, (3) the effect of testosterone on 8-OH-DPAT-, apomorphine-, and MK-801-induced PPI disruption.

Methods Animals We used 128 female SpragueDawley rats in this study (Monash Animal Services, Monash University, VIC, Australia). The rats were 12 weeks of age at the time of surgery. They were housed in groups of two to three in standard rat cages, with free access to standard pellet food and tap water. The rats were maintained on a 12-h light

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dark cycle (lights on at 0630), at a constant temperature of 22 2C. All surgical techniques, treatments, and experimental protocols were in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1990) set out by the National Health and Medical Research Council of Australia. Surgery Ovariectomy surgery was performed as previously described (Gogos et al. 2010b). Briefly, rats were anesthetized using an isofluorane/oxygen gas mixture and placed upon a heat pad in the prone position. Rats received a subcutaneous (s.c.) injection of 5 mg/kg of the non-steroidal, anti-inflammatory analgesic, carprofen (Zenecarp; Heriot AgVet, VIC, Australia). A 23-cm midline incision was made through the skin above the lower back, followed by an incision through the abdominal wall. The ovaries were bilaterally located and removed and the incisions closed. Intact rats were sham operated. During the ovariectomy procedure, rats were s.c. implanted with silastic implants at the nape of the neck as previously described (Gogos and Van den Buuse 2004). Briefly, these implants (Dow Corning, I.D. 1.98 mm, O.D. 3.18 mm; Futuremedics Australia, VIC, Australia) were either empty or filled with crystalline steroid hormones. Estrogen implants were 5 mm long, and filled with either 100% estradiol (17-estradiol; Sigma Chemical Company, MO, USA) or a 20% estradiolcholesterol mixture (5Cholesten-3-OL; Steraloids Inc., RI, USA). Testosterone implants were either 5 mm or 20 mm in length and filled with 100% testosterone (4-Androsten-17-ol-3-one; Sigma). These implant sizes were based on the literature and our previous findings, and were aimed at producing physiological levels of testosterone of intact female and male rats, respectively (Albert et al. 1989; Dannan et al. 1986; Gogos and Van den Buuse 2003; Milsted et al. 1998). Behavioral experiments commenced 2 weeks after surgery. At least 3 days after completion of experiments, rats were euthanized using a lethal dose of pentobarbitone and then decapitated. The uterus was removed, weighed, and inspected for any abnormalities. Behavioral experiments Prepulse inhibition of the acoustic startle response was measured with eight automated startle chambers (SR-Lab; San Diego Instruments, San Diego, CA, USA) as previously described (Gogos and Van den Buuse 2003). Briefly, rats were placed individually into a transparent Plexiglas cylinder in a sound-attenuating cabinet. The PPI session comprised 80 trials presented with variable intervals (827 s), including 32

pulse-alone trials (four blocks of eight 115 dB trials) and 40 prepulsepulse trials. Prepulsepulse trials consisted of a prepulse (PP) of an intensity of 2, 4, 8, 12, or 16 dB above the 70 dB background (eight of each), followed 100 ms later by the startle pulse. Startle data were measured using all four blocks of pulse-alone trials. The % PPI was calculated as [(pulse-alone trials startle response amplitude prepulse pulse trials startle amplitude)/(pulse-alone trials startle amplitude)]100%. The middle 16 pulse-alone trials were used to calculate % PPI. Locomotor activity was measured using eight automated photocell cages (ENV-520, MED Associates, VT, USA), as previously described (Kusljic et al. 2005). Briefly, the position of the rat at any time was detected with 16 evenly spaced infrared sources and sensors on each of the four sides of the monitor, measuring x, y, and z axes movements. Several types of behavioral responses were recorded, including total distance traveled, ambulatory counts, vertical counts, and stereotypic counts. For the distance traveled measurement, movements within a virtual box of 44 beams around the rat are eliminated, thus providing a filtered total distance moved. Ambulatory counts were defined as at least four consecutive beam breaks within a 500-ms period. Vertical counts/rearing were defined as a z beam break. Stereotypy was counted as small, repetitive movements within a virtual 44 box around the rat. During the experiments, the rats were placed in the locomotor photocell cages for 30 min, to establish baseline locomotor activity and allow habituation to the test environment, after which they were injected and locomotor activity recorded over a further 90 min. Experimental protocol All drugs were dissolved or diluted in saline to doses that were selected on the basis of the literature and preliminary experiments. All drugs were administered in a volume of 1 ml/kg. In a randomized, cross-over protocol, all rats in each experiment received all treatments, with 34 days allowed between each experiment. This allowed for within-animal statistical analysis and also reduced the total number of animals required. The drugs, 8-OH-DPAT (()-8-hydroxy-2-(dipropylamino) tetralin, Tocris, Bristol, UK), apomorphine (R-()-apomorphine hydrochloride hemihydrate, Sigma), and MK-801 (dizocilpine, (5R,10S)-(+)-5-methyl-10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate; Sigma), were administered s.c. in the flank 10 min prior to the rat being placed in the PPI chamber. The dopamine releaser, amphetamine (D-amphetamine hemisulfate salt, Sigma), was administered s.c. in the flank 30 min after the rat was placed in the locomotor monitor.

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Experiment 1 Female rats were randomly chosen to become ovariectomized (OVX) and receiving an empty implant (n = 12), OVX rats implanted with the 20% estradiol mixture (E20, n =12), or OVX rats treated with the 100% estradiol implant (E100, n =8). Rats in this experiment were tested for PPI after administration of saline, 0.02 and 0.1 mg/kg MK-801, and 0.5 mg/kg 8-OHDPAT (as a control). Experiment 2 Female rats were randomly chosen to become sham-operated controls receiving an empty implant, OVX rats receiving an empty implant, E20-treated OVX rats, or E100treated OVX rats (n =8 in each group). Rats in this experiment were tested for locomotor activity after treatment with saline and 0.25 and 0.5 mg/kg amphetamine. Experiment 3 Female rats were randomly chosen to become sham-operated controls receiving an empty implant, OVX rats receiving an empty implant, OVX rats implanted with the 5 mm testosterone implant (T5), or OVX rats receiving the 20 mm testosterone implant (T20); n =8 in each group. One cohort of rats (part A) was tested for PPI after administration of saline, 0.5 mg/kg 8-OHDPAT, and 0.3 mg/kg apomorphine. A second cohort of rats (part B) was tested for PPI after administration of saline and 0.02 and 0.1 mg/kg MK-801.

For experiment 3A, a four testosterone/hormone group (Sham, OVX, T5, T20)3 drug (saline, 8-OH-DPAT, apomorphine)5 prepulse intensity ANOVA was used. For experiment 3B, a four testosterone/hormone group3 MK801/dose (0, 0.02, and 0.1 mg/kg of MK-801)5 prepulse intensity ANOVA was used. For startle amplitude (experiments 3A and 3B), a four testosterone/hormone group3 dose4 block ANOVA was used. For locomotor activity (experiment 2), data were analyzed in 5-min intervals. The 30-min pre-injection data across the three sessions was compared and there were no dose or group differences in distance traveled, ambulatory, vertical, or stereotypic counts; these data were therefore excluded from subsequent analysis. The 5-min interval during which rats were injected was also excluded from data analysis. For each of the locomotor activity measures, a four estradiol/hormone group (Sham, OVX, E20, E100)3 amphetamine/dose (0, 0.25, and 0.5 mg/kg amphetamine)17 time (5-min intervals in the 90 min post-injection) ANOVA was used. Locomotor activity data are graphically presented as the total 90-min post-injection data.

Results Body and uterus weights Estrogen-treated rats

Statistical analysis All data were expressed as meanstandard error of the mean (SEM) and analyzed using the statistical software package SYSTAT 9.0 (SPSS Inc., Chicago, IL, USA). Body weight and uterus weight were analyzed with one-way analysis of variance (ANOVA) for group, with Bonferronicorrected post-hoc analysis. PPI and startle data were analyzed with a two-way ANOVA with repeated measures where appropriate. Main effects of prepulse intensity or startle block were always observed and will not be reported unless there were relevant interactions with other factors. Significant main effects and interactions were further explored with pair-wise ANOVAs. Average % PPI was used for graphical presentation only and reflects the average of the five prepulse intensities. For experiment 1, a three estradiol/hormone group (OVX, E20, E100)3 MK-801/dose (0, 0.02, and 0.1 mg/ kg of MK-801)5 prepulse intensity (PP2PP16) ANOVA was used, followed by a three estradiol/hormone group2 8-OH-DPAT/dose (0 and 0.5 mg/kg of 8-OH-DPAT)5 prepulse intensity ANOVA. For startle amplitude, a three estradiol/hormone groupdose (three doses for MK-801; two doses for 8-OH-DPAT)4 block (four blocks of eight 115 dB pulse-alone trials) ANOVA was used. In both experiments 1 and 2 (Table 1), there were no significant differences in the body weight of groups at the time of surgery within an experiment. There were significant estradiol/hormone group differences in body weight gain by the end of the experiment (experiment 1, F(2,29) =103.5, p < 0.001; experiment 2, F (3,28) = 30.6, p < 0.001). The Bonferroni-corrected post-hoc analysis revealed that compared with untreated OVX rats, weight gain was significantly reduced in E20- and E100-treated OVX rats (experiments 1 and 2; Table 1). Further, compared to sham-operated (intact) rats, weight gain was significantly enhanced in untreated OVX rats (experiment 2). Uterus weight was also significantly different between the groups (uterus weight as a % of body weight: experiment 1, F(2,29) =29.1, p <0.001; experiment 2, F(3,28) =18.1, p <0.001). Compared with untreated OVX rats, E20- and E100-treated OVX rats had significantly larger uteri (experiments 1 and 2; Table 1). Testosterone-treated rats In experiments 3A and 3B (Table 1), there were no significant differences in the body weight of groups at the time of surgery. There were significant testosterone/hormone group differences in body weight gain by the end of the experiment

Psychopharmacology (2012) 219:213224 Table 1 Body weight (BW) and uterus weight (UW) of all female rats Surgery BW Weight gain Uterus weight

217

% UW/BW

Experiment 1Estrogen-treated rats (MK-801 and PPI) OVX 2436 774 E20 2406 52# E100 2466 175# Experiment 2Estrogen-treated rats (amphetamine and locomotor activity) Sham-operated 2564 182 OVX 2617 677* E20 24210 106# E100 2519 24# Experiment 3A.Testosterone-treated rats (8-OH-DPAT, apomorphine, and PPI) Sham-operated 2438 274 OVX 2295 685* T5 2309 442*# T20 2318 645* Experiment 3B.Testosterone-treated rats (MK-801 and PPI) Sham-operated 2419 136 OVX 2295 6210* T5 2266 445* T20 2369 574*

0.140.01 1.070.11# 0.980.19# 0.530.08 0.150.01 1.100.19# 1.390.19*# 0.670.11 0.140.01* 0.180.02* 0.230.02* 0.550.11 0.130.01* 0.160.02* 0.320.03*

0.040.00 0.440.04# 0.380.07# 0.190.03 0.050.00 0.430.08*# 0.550.07*# 0.250.04 0.050.00* 0.070.01* 0.080.01* 0.220.04 0.040.00* 0.060.01* 0.110.02*

Table 1 shows that in experiments 1 and 2, rats were either sham-operated, untreated ovariectomized (OVX) rats, or OVX rats treated with either a 20% (E20) or 100% estradiol-filled implant; n =812 per group. In experiment 3 (parts A and B), rats were either sham-operated, untreated OVX rats, or OVX rats treated with either a 5-mm (T5) or 20-mm testosterone-filled implant (T20); n =8 per group. All weights are in grams and are expressed as meanSEM. Weight gain is the difference between body weight on the day of surgery and final body weight *p <0.05 compared with sham-operated rats using Bonferroni-corrected post-hoc analysis; # p <0.05 compared with untreated OVX rats using Bonferroni-corrected post-hoc analysis

(experiment 3A, F(3,28) =20.6, p <0.001; experiment 3B, F(3,28) =10.8, p <0.001), where compared to sham-operated rats, weight gain was significantly enhanced in untreated OVX rats, T5- and T20-treated OVX rats (experiments 3A and 3B; Table 1). Uterus weight was significantly different between the groups (uterus weight as a % of body weight: experiment 3A, F(3,28) =22.3, p <0.001; experiment 3B, F(3,28) =11.7, p <0.001), when compared with shamoperated rats, the other three groups showed reduced uterus weight (Table 1). Experiment 1: estrogen treatment and MK-801-induced PPI disruption Prepulse inhibition When comparing PPI between untreated OVX, E20- and E100-treated OVX rats which were administered saline, 0.02 or 0.1 mg/kg MK-801 (Fig. 1a), there was a significant main effect of MK-801/dose (F(2,58) =54.5, p <0.001) and a doseestradiol/hormone group interaction (F(4,58) =2.6, p = 0.044). There was no main effect of estradiol/hormone group or any other significant interactions. Compared to

saline treatment, MK-801 significantly disrupted PPI at 0.02 mg/kg (F(1,29) =23.1, p <0.001) and 0.1 mg/kg (F(1,29) =88.7, p <0.001). Further, at 0.1 mg/kg MK-801, there was a MK-801/dose estradiol/hormone group interaction (F(2,29) =4.0, p =0.030), reflecting a difference between untreated OVX rats and E20-treated OVX rats (dose group: F(1,22) =6.6, p =0.017) and untreated OVX rats and E100-treated OVX rats (dosegroup: F(1,18) =4.9, p = 0.041). Specifically, treatment with 0.1 mg/kg of MK-801 induced a 70% reduction of average PPI in untreated OVX rats (F(1,11) =74.1, p <0.001), a 42% reduction in E20treated OVX rats (F(1,11) =17.3, p =0.002), and a 44% reduction in E100-treated OVX rats (F(1,7) =18.4, p = 0.004). Thus, although MK-801 significantly disrupted PPI in all three hormone-treated groups, the disruption was markedly reduced in estradiol-treated rats compared to untreated OVX rats (Fig. 1a). There was no significant difference in the effect of MK-801 between E20- and E100treated OVX rats. When comparing PPI in the three estradiol/hormone groups (Fig. 1b), 8-OH-DPAT caused a significant disruption of PPI (8-OH-DPAT/dose: F(1,29) =33.2, p < 0.001; PP: F(4,116) =178.8, p <0.001; 8-OH-DPAT/dose

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(a)
80

Estrogen and MK-801

Saline 0.02 mg/kg MK-801* 0.1 mg/kg MK-801*

Average % PPI

60

was no MK-801/doseestradiol/hormone group interaction, nor a main effect of estradiol/hormone group. Treatment with 8-OH-DPAT did not significantly affect startle amplitude, nor was there a difference in response to 8-OH-DPAT between the groups (Fig. 2b). Experiment 2: estrogen treatment and amphetamine-induced locomotor hyperactivity Distance traveled

40

20

OVX

E20

E100

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80 60

Estrogen and 8-OH-DPAT

Saline 0.5 mg/kg 8-OH-DPAT

Average % PPI

*
40

20

When comparing distance traveled in the four estradiol/ hormone groups which were administered saline and 0.25 and 0.5 mg/kg amphetamine, there was a main effect of time (F(16,448) = 18.5, p < 0.001) and an amphetamine/ dosetime interaction (F(32,896) =7.1, p <0.001; see Fig. 3a for the total 90-min post-injection data). This reflected changes in locomotor activity over the 90-min postinjection period after saline treatment (F(16,448) =7.4, p < 0.001), 0.25 mg/kg amphetamine (F(16,448) =8.7, p <0.001),

OVX

E20

E100

(a)
500

Estrogen and MK-801

Saline 0.02 mg/kg MK-801 0.1 mg/kg MK-801*

Group

Startle amplitude

Fig. 1 Average % PPI of female rats that were either untreated ovariectomized rats (OVX, n =12) or OVX rats treated with either a 20% (E20, n =12) or 100% estradiol-filled implant (E100, n =8). All groups were administered a saline and 0.02 mg/kg and 0.1 mg/kg MK-801, and b saline and 0.5 mg/kg 8-OH-DPAT. All data are expressed as meanSEM. Average % PPI reflects the average of the five prepulse intensities. *p <0.05 compared to saline treatment

400 300 200 100 0

PP: F(4,116) = 3.6, p =0.009). As expected, the effect of 8OH-DPAT was different between the groups (8-OH-DPAT/ dose estradiol/hormone group interaction: F(2,29) = 3.4, p = 0.046). Pair-wise ANOVA comparing untreated and E100-treated OVX rats showed a significant reduction in the effect of 8-OH-DPAT with E100 treatment (8-OHDPAT/dose estradiol/hormone group interaction: F(1,18) = 7.8, p = 0.012). Specifically, treatment with 0.5 mg/kg of 8-OH-DPAT induced a significant 35% reduction of average PPI in untreated OVX rats (F(1,11) =28.9, p < 0.001), but only a non-significant 11% reduction in E100treated OVX rats (Fig. 1b). There were no differences in the effect of 8-OH-DPAT between untreated OVX rats and E20-treated OVX rats. Startle amplitude When comparing startle amplitude in the three estradiol/ hormone groups administered MK-801 (Fig. 2a), there was a main effect of MK-801/dose (F(2,58) =10.2, p <0.001), reflecting that MK-801 significantly increased startle amplitude at 0.1 mg/kg (F(1,29) =16.8, p <0.001). There

OVX

E20

E100

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500

Estrogen and 8-OH-DPAT

Saline 0.5 mg/kg 8-OH-DPAT

Startle amplitude

400 300 200 100 0

OVX

E20

E100

Group

Fig. 2 Startle amplitude (arbitrary units) of female rats that were either untreated ovariectomized rats (OVX, n =12), or OVX rats treated with either a 20% (E20, n =12) or 100% estradiol-filled implant (E100, n =8). All groups were administered a saline and 0.02 mg/kg and 0.1 mg/kg MK-801, and b saline and 0.5 mg/kg 8OH-DPAT. All data are expressed as meanSEM. Startle amplitude reflects the average of the four blocks of 115 dB pulse-alone trials. *p <0.001 compared to saline treatment

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and 0.5 mg/kg amphetamine (F(16,448) =13.8, p <0.001). There was a significant main effect of amphetamine/dose (F(2,56) =132.3, p <0.001). Further ANOVA revealed that both 0.25 mg/kg (F(1,28) =101.7, p <0.001) and 0.5 mg/kg amphetamine (F(1,28) =195.6, p <0.001) significantly increased total distance moved, compared to saline. There was no amphetamine/doseestradiol/hormone group interaction, reflecting the similar response to amphetamine in all four rat groups (Fig. 3a). There were no other significant main effects or interactions. Ambulatory, vertical, and stereotypic counts Similar to distance traveled, there was a main effect of time and an amphetamine/dosetime interaction, for each of the three measurements (all p <0.001). Also, there was a significant main effect of amphetamine/dose, reflecting an increase in counts after 0.25 and 0.5 mg/kg amphetamine, for each of the three measurements (all p <0.001). In terms of ambulatory (Fig. 3b) and vertical (Fig. 3c) counts, there was no amphetamine/doseestradiol/hormone group interaction, reflecting the similar response to amphetamine in all four rat groups. However, for stereotypic counts (Fig. 3d), there was a significant main effect of estradiol/hormone group (F(3,28) =3.2, p =0.038) reflecting lower stereotypic counts between untreated and E100treated OVX rats (F(1,14) =13.0, p =0.003). There was also an amphetamine/doseestradiol/hormone group interacFig. 3 Measures of locomotor activity of female rats (n =8 per group) that were either sham-operated (Sham), untreated ovariectomized rats (OVX), or OVX rats treated with either a 20% (E20) or 100% estradiolfilled implant (E100). All rat groups were administered saline and 0.25 mg/kg and 0.5 mg/kg amphetamine. All data are expressed as meanSEM of the total distance/counts during the 90-min post-injection period. *p <0.001 compared to saline treatment

tion (F(6,56) =2.8, p =0.019). Further pair-wise ANOVAs revealed a difference between E20-treated OVX rats and both sham-operated rats (F(2,28) =4.8, p =0.016) and untreated OVX rats (F(2,28) =5.0, p =0.014), reflecting a reduced effect of amphetamine on stereotypy in E20treated OVX rats (Fig. 3d). Experiment 3A: testosterone treatment and 8-OH-DPAT- and apomorphine-induced PPI disruption Prepulse inhibition When comparing PPI in the four rat groups which were administered saline, 0.5 mg/kg 8-OH-DPAT (Fig. 4a) and 0.3 mg/kg apomorphine (Fig. 4b), there was a significant main effect of drug (F(2,56) =28.7, p <0.001). Therefore, data were separated according to drug treatment. There was a significant main effect of 8-OH-DPAT (F(1,28) =55.2, p <0.001), reflecting the disruption of PPI caused by 8OH-DPAT treatment. There was no 8-OH-DPATtestosterone/hormone group interaction, nor a main effect of group, reflecting a lack of effect of testosterone treatment on 8OH-DPAT-induced PPI disruption (Fig. 4a). There was also a significant PPtestosterone/hormone group interaction (F(12,112) =3.0, p =0.001) and a 8-OH-DPATPP interaction (F(4,112) =4.2, p =0.004). Apomorphine significantly disrupted PPI (main effect of drug: F(1,28) =39.2, p <0.001). There was no apomorphinetestosterone/hormone group

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Testosterone and 8-OH-DPAT

Saline 0.5 mg/kg 8-OH-DPAT*

Average % PPI

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40

20

were separated accordingly. There were no significant effects of 8-OH-DPAT treatment on startle amplitude, nor was there a 8-OH-DPAT testosterone/hormone group interaction (Fig. 5a). There was a significant effect of apomorphine treatment (F(1,28) =17.6, p <0.001), reflecting the ability of apomorphine to increase startle (Fig. 5b). There was no main effect of testosterone/hormone group or an apomorphinetestosterone/hormone group interaction.

Sham

OVX

T5

T20

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80

Testosterone and Apomorphine

Saline 0.3 mg/kg Apomorphine*

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800 600 400 200 0

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Fig. 4 Average % PPI of female rats (n =8 per group) that were either sham-operated (Sham), untreated ovariectomized rats (OVX), or OVX rats treated with either a 5-mm (T5) or 20-mm testosterone-filled implant (T20). All rat groups were administered a saline and 0.5 mg/ kg 8-OH-DPAT, b saline and 0.3 mg/kg apomorphine, or c saline and 0.02 mg/kg and 0.1 mg/kg MK-801. All data are expressed as mean SEM. Average % PPI reflects the average of the five prepulse intensities. *p <0.01 compared to saline treatment

(c)
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interaction, nor any other significant effects reflecting a lack of effect of testosterone treatment on apomorphineinduced PPI disruption (Fig. 4b). Startle amplitude When comparing startle amplitude in the four groups, there was a main effect of drug (F(2,56) =15.0, p <0.001), thus data

Fig. 5 Startle amplitude (arbitrary units) of female rats (n =8 per group) that were either sham-operated (Sham), untreated ovariectomized rats (OVX), or OVX rats treated with either a 5-mm (T5) or 20mm testosterone-filled implant (T20). All rat groups were administered a saline and 0.5 mg/kg 8-OH-DPAT, b saline and 0.3 mg/kg apomorphine, or c saline and 0.02 mg/kg and 0.1 mg/kg MK-801. All data are expressed as meanSEM. Startle amplitude reflects the average of the four blocks of 115 dB pulse-alone trials. *p <0.05 compared to saline treatment

Psychopharmacology (2012) 219:213224

221

Experiment 3B: testosterone treatment and MK-801-induced PPI disruption Prepulse inhibition When comparing PPI in the four groups that were administered saline, 0.02 or 0.1 mg/kg MK-801 (Fig. 4c), there was a significant main effect of MK-801/dose (F(2,56) = 79.5, p <0.001) and a MK-801/dose PP interaction (F(8,224) =2.7, p =0.007). Further ANOVAs revealed that MK-801 significantly disrupted PPI at 0.02 mg/kg (F(1,28) = 14.2, p =0.001) and 0.1 mg/kg (F(1,28) =120.7, p <0.001), compared to saline. There was also a MK-801/dose testosterone/hormone group interaction (F(6,56) =2.5, p = 0.031). When administered 0.1 mg/kg MK-801, there was a difference between sham-operated and T5-treated OVX rats (MK-801/dosetestosterone/hormone group: F(1,14) = 7.9, p =0.014) and sham-operated and T20-treated OVX rats (F(1,14) =8.4, p =0.012), where testosterone treatment enhanced the MK-801-induced PPI disruption (Fig. 4c). There was no main effect of testosterone/hormone group or any other significant interactions. Startle amplitude MK-801 increased startle amplitude (F(2,54) =14.6, p <0.001) and this was significant after treatment with 0.02 mg/kg (F(1,27) =6.7, p =0.015) and 0.1 mg/kg (F(1,28) =27.9, p < 0.001). There was no MK-801/dosetestosterone/hormone group interaction, nor a main effect of testosterone/hormone group (Fig. 5c).

Discussion This study aimed to explore the role of the sex hormones, estrogen and testosterone, in animal models of relevance to schizophrenia. We investigated the effect of estrogen on MK801-induced PPI disruption and on amphetamine-induced locomotor hyperactivity. We found that estrogen treatment (E20 and E100) reduced the MK-801-induced PPI disruption but had little effect on locomotor hyperactivity. We also investigated the effect of testosterone on 8-OH-DPAT-, apomorphine-, and MK-801-induced PPI disruption. We found that testosterone treatment significantly enhanced the MK-801-induced PPI disruption, but had no effect on PPI disruptions induced by administration of 8-OH-DPAT or apomorphine. The present study used only female rats, some of which were OVX and received an implant filled with estradiol or testosterone. Successful ovariectomy of the rats was ascertained by a significant decrease in uterus weight and an increase in body weight gain (Gogos et al. 2010b; Gogos

and Van den Buuse 2004). Estrogen treatment increased uterus weight, reduced body weight gain, and produced levels of circulating estradiol similar to the mean level of estradiol present throughout the estrous cycle (Albert et al. 1991; Gogos et al. 2010b; Gogos and Van den Buuse 2004). Testosterone treatment did not markedly change uterus or body weight compared to untreated OVX rats. The T5 and T20 implants used in the present study were aimed at producing physiological levels of testosterone of intact female and male rats, respectively (Albert et al. 1989; Dannan et al. 1986; Gogos and Van den Buuse 2003; Milsted et al. 1998). In all three experiments of this study, ovariectomy did not have an effect on baseline PPI or locomotor activity. As discussed previously (Gogos and Van den Buuse 2004), we suggest that the removal of the ovaries, and thus the main source of estrogen and progesterone from the intact female rat, does not play a tonic modulatory role in PPI and locomotor activity. MK-801 (dizocilpine), the non-competitive NMDA receptor antagonist, disrupted PPI in all female rat groups, as previously reported (Geyer et al. 2001). Both doses of estrogen significantly reduced this disruption, although they were not able to completely reverse it. As a control, in these same animals we also confirmed and replicated that estrogen treatment could reverse an 8-OH-DPAT-induced PPI disruption. The effect of estrogen on MK-801-induced PPI disruption was not influenced by the increase in startle amplitude, as this was similar in all groups. Support for our findings comes from studies showing that estrogen affects latent inhibition, a measure of selective attention that is disrupted in schizophrenia (Arad and Weiner 2010a, b). This group found that in female OVX rats, treatment with 17-estradiol reversed amphetamine- and MK-801-induced changes latent inhibition, effects considered predictive for activity against the positive and negative/cognitive symptoms of schizophrenia, respectively (Arad and Weiner 2010a, b). Other studies have shown that estrogen has effects on the glutamatergic system, and in particular, the NMDA receptor (Cyr et al. 2001; Gore 2001). For example, estradiol treatment increases NMDA receptor binding in the hippocampus and decreases it in the frontal cortex and striatum (Cyr et al. 2001). These effects could play a role in the reduced action of MK-801 in PPI in estrogen-treated rats in our study. However, it is not clear why estrogen treatment had only a partial effect. It is unlikely that this was due to not having administered enough estradiol, as this dose completely reversed 8-OH-DPAT- and apomorphine-induced PPI disruptions. Instead, it is possible that the administration of MK-801 had downstream effects on other neurotransmitters, and the site of action of estrogen is on these receptors rather than on NMDA receptors. Administration of MK-801 induces dopamine

222

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release in the prefrontal cortex and nucleus accumbens (Lecourtier et al. 2007). Therefore, in the present study, estrogen may be blocking the downstream dopaminergic effects of MK-801 as we have suggested for the effect of 8OH-DPAT on PPI (Gogos et al. 2010b). Alternatively, 5-HT2 receptor antagonists can reverse the MK-801-induced PPI disruption (Geyer et al. 2001) and estrogen can affect 5HT2 receptor-mediated responses (Rubinow et al. 1998; Uphouse 2000). Overall, further studies are required to delineate the exact interaction between estrogen and NMDA receptor function in PPI. Nevertheless, this finding adds support to the notion that estrogen exerts a functional protection against PPI disruption induced by multiple neurotransmitter systems, specifically 5-HT1A, dopamine D2, and NMDA receptor activation. We found that amphetamine-induced locomotor hyperactivity, in terms of distance traveled, was not affected by estrogen treatment. We also measured ambulatory counts, vertical counts, and stereotypy and found that estrogen treatment had a small effect of reducing stereotypy, in line with previous reports (Forgie and Stewart 1994; Van Hartesveldt and Joyce 1986). For example, intra-striatal injections of amphetamine produced locomotor hyperactivity that was not suppressed by acute estradiol (Joyce et al. 1984). Both amphetamine-induced locomotor activity and apomorphineinduced PPI disruption involve dopaminergic innervation in the nucleus accumbens (Koch 1999; Makanjuola and Ashcroft 1982), thus it is unclear why estrogen only had an effect in PPI. We suggest that differential involvement of the nucleus accumbens subregions may play a role. The nucleus accumbens consists of two main subregions, the core and shell, which have distinct anatomical and neurochemical properties (Zahm 2000). Evidence suggests that these subregions differentially mediate locomotor activity and PPI (Boye et al. 2001; Wan and Swerdlow 1996; Zahm 2000), and estrogen may therefore only affect behaviors mediated by a particular nucleus accumbens subregion. Alternatively, we suggest that these druginduced behaviors may have differing underlying mechanisms. Amphetamine is an indirect dopamine receptor agonist, but also stimulates the release of noradrenaline. A recent study showed that amphetamine-induced locomotor hyperactivity could be blocked by the noradrenergic alpha1 receptor antagonist, prazosin (Alsene et al. 2010), suggesting that noradrenaline is also involved in mediating amphetamine-induced activity. Apomorphine is a dopamine D1/D2 receptor agonist; estrogen appears to specifically target the dopamine D2 receptors (Chavez et al. 2010; Gogos et al. 2010b). Part three of this study examined the effect of testosterone treatment on drug-induced PPI disruptions, in female rats. We found that the 8-OH-DPAT- and

apomorphine-induced PPI disruptions were unaffected by testosterone treatment, indicating that testosterone, unlike estrogen, does not modulate 5-HT1A or dopamine D2 receptor mechanisms in PPI. Except for our previous study (Gogos and Van den Buuse 2003), no other study has examined the effect of testosterone on drug-induced disruptions of PPI. We previously showed that testosterone treatment reinstates 8-OH-DPAT-induced PPI disruption in castrated male rats (Gogos and Van den Buuse 2003); however, testosterone did not affect 5-HT1A receptormediated PPI in female OVX rats. We also found that testosterone treatment significantly increased the MK-801induced PPI disruption, suggesting that testosterone enhances NMDA receptor function. Treatment with MK801 also caused an increase in startle amplitude, but this was similar in all groups and therefore does not explain the group difference observed. Bortolato et al. (2008) examined PPI in intact male rats after treatment with the 5-reductase inhibitor, finasteride, which inhibits the conversion of testosterone to dihydrotestosterone (DHT), among other effects. This study found that finasteride reversed apomorphine-induced PPI disruptions but did not alter the effect of MK-801 on PPI (Bortolato et al. 2008), suggesting differential involvement of neuroactive steroids including testosterone, on dopaminergic and glutamatergic modulation of PPI. Another study showed that in female OVX rats, DHT, but not testosterone, prevented MK-801induced neurodegeneration (de Olmos et al. 2008). Further studies are needed to examine the mechanism whereby testosterone can enhance NMDA receptor function, particularly in female rats. Testosterone has two modes of action: one is by binding to the androgen receptor and the other involves being converted to estrogen (Wilson 1996). This study does not specifically address which mode of action testosterone is using to have its effect. However, because estrogen treatment had the opposite effect to testosterone treatment on MK-801-induced PPI disruption, this suggests that the testosterone effect on PPI is occurring via the androgen receptor. Further studies using DHT, an androgen that cannot be converted to estrogen, may elucidate the role of the androgen receptor in mediating glutamatergic modulation of PPI. In summary, the functionally protective effect of estrogen has now been observed against MK-801-induced PPI disruptions as well as 8-OH-DPAT- and apomorphine-induced disruptions. It is likely that the underlying mechanism for each of these drug-induced PPI disruptions involves estrogen modulating a similar downstream pathway, possibly via dopamine D2 receptors. This protective effect of estrogen appears to be selective for PPI, as it was not observed in amphetamine-induced locomotor hyperactivity. Further, testosterone treatment enhanced NMDA receptor-mediated PPI disruptions, but had no effect on 5-HT1A- and dopamine D2

Psychopharmacology (2012) 219:213224

223 locomotor stimulant effects of nicotine and D-amphetamine in rats. Neuropharmacology 40:792805 Braff DL, Geyer MA, Swerdlow NR (2001) Human studies of prepulse inhibition of startle: normal subjects, patient groups, and pharmacological studies. Psychopharmacology 156:234258 Castle DJ, Abel K, Takei N, Murray RM (1995) Gender differences in schizophrenia: hormonal effect or subtypes? Schizophr Bull 21:112 Chavez C, Hollaus M, Scarr E, Pavey G, Gogos A, van den Buuse M (2010) The effect of estrogen on dopamine and serotonin receptor and transporter levels in the brain: an autoradiography study. Brain Res 1321:5159 Cyr M, Ghribi O, Thibault C, Morissette M, Landry M, Di Paolo T (2001) Ovarian steroids and selective estrogen receptor modulators activity on rat brain NMDA and AMPA receptors. Brain Res Brain Res Rev 37:153161 Dannan GA, Guengerich FP, Waxman DJ (1986) Hormonal regulation of rat liver microsomal enzymes. Role of gonadal steroids in programming, maintenance, and suppression of delta 4-steroid 5 alpha-reductase, flavin-containing monooxygenase, and sexspecific cytochromes P-450. J Biol Chem 261:1072810735 de Olmos S, Bueno A, Bender C, Lorenzo A, de Olmos J (2008) Sex differences and influence of gonadal hormones on MK801induced neuronal degeneration in the granular retrosplenial cortex of the rat. Brain Struct Funct 213:229238 Forgie ML, Stewart J (1994) Effect of prepubertal ovariectomy on amphetamine-induced locomotor activity in adult female rats. Horm Behav 28:241260 Geyer MA, Krebs-Thomson K, Braff DL, Swerdlow NR (2001) Pharmacological studies of prepulse inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review. Psychopharmacology 156:117154 Gogos A, Van den Buuse M (2003) Castration reduces the effect of serotonin-1A receptor stimulation on prepulse inhibition in rats. Behav Neurosci 117:14071415 Gogos A, Van den Buuse M (2004) Estrogen and progesterone prevent disruption of prepulse inhibition by the serotonin-1A receptor agonist 8-hydroxy-2-dipropylaminotetralin. J Pharmacol Exp Ther 309:267274 Gogos A, Nathan PJ, Guille V, Croft RJ, Van den Buuse M (2006) Estrogen prevents 5-HT1A receptor-induced disruptions of prepulse inhibition in healthy women. Neuropsychopharmacology 31:885889 Gogos A, Joshua N, Rossell SL (2010a) Use of the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) to investigate group and gender differences in schizophrenia and bipolar disorder. Aust N Z J Psychiatry 44:220229 Gogos A, Kwek P, Chavez C, van den Buuse M (2010b) Estrogen treatment blocks 8-hydroxy-2-dipropylaminotetralin- and apomorphine-induced disruptions of prepulse inhibition: involvement of dopamine D1 or D2 or serotonin 5-HT1A, 5-HT2A, or 5-HT7 receptors. J Pharmacol Exp Ther 333:218227 Gore AC (2001) Gonadotropin-releasing hormone neurons, NMDA receptors, and their regulation by steroid hormones across the reproductive life cycle. Brain Res Brain Res Rev 37:235248 Hfner H, Riecher-Rossler A, An Der Heiden W, Maurer K, Fatkenheuer B, Loffler W (1993) Generating and testing a causal explanation of the gender difference in age at first onset of schizophrenia. Psychol Med 23:925940 Joyce JN, Montero E, Van Hartesveldt C (1984) Dopamine-mediated behaviors: characteristics of modulation by estrogen. Pharmacol Biochem Behav 21:791800 Koch M (1999) The neurobiology of startle. Prog Neurobiol 59:107 128 Kocoska-Maras L, Zethraeus N, Radestad AF, Ellingsen T, von Schoultz B, Johannesson M, Hirschberg AL (2011) A randomized

receptor-mediated PPI disruptions. In conclusion, these findings add support to the notion that estrogen is protective against sensorimotor gating disruptions induced by multiple neurotransmitter systems, specifically 5-HT1A, dopamine D2, and NMDA receptor activation, while testosterone treatment potentiates selective drug-induced PPI disruptions. The present findings may help to explain the gender differences observed in schizophrenia and the beneficial effects of estrogen treatment on symptoms of the illness.
Acknowledgments Funding for this study was provided by the National Health and Medical Research Council of Australia in the form of a Project Grant (ID 509234), a Peter Doherty Fellowship (ID 435690 to AG), and a senior research fellowship (ID 435500 to MvdB); the J. & P. Clemenger Trust; and the Operational Infrastructure Support (OIS) from the Victorian State Government. All experiments in this study were in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1990) set out by the National Health and Medical Research Council of Australia. There are no conflicts of interest to report.

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