Disruption of NMDA receptor-mediated regulation of PPI in the maternal immune activation model of schizophrenia is restored by 17 β -estradiol and raloxifene

Maternal immune activation (MIA) during pregnancy is known to increase the risk of development of schizophrenia in the offspring. Sex steroid hormone analogues have been proposed as potential antipsychotic treatments but the mechanisms of action involved remain unclear. Estrogen has been shown to alter N -methyl- D - aspartate (NMDA) receptor binding in the brain. We therefore studied the effect of chronic treatment with 17 β - estradiol, its isomer, 17 α -estradiol, and the selective estrogen receptor modulator, raloxifene, on MIA-induced psychosis-like behaviour and the effect of the NMDA receptor antagonist, MK-801. Pregnant rats were treated with saline or the viral mimetic, poly(I:C), on gestational day 15. Adult female offspring were tested for changes in baseline prepulse inhibition (PPI) and the effects of acute treatment with MK-801 on PPI and locomotor activity. Poly(I:C) offspring had significantly lower baseline PPI compared to control offspring, and this effect was prevented by 17 β -estradiol and raloxifene, but not 17 α -estradiol. MK-801 reduced PPI in control offspring but had no effect in poly(I:C) offspring treated with vehicle. Chronic treatment with 17 β -estradiol and raloxifene restored the effect of MK-801 on PPI. There were no effects of MIA or estrogenic treatment on MK-801 induced locomotor hyperactivity. These results show that MIA affects baseline PPI as well as NMDA receptor-mediated regulation of PPI in female rats, and strengthen the view that estrogenic treatment may have antipsychotic effects.


Introduction
Several epidemiological studies have suggested that prenatal viral and bacterial infections increase the risk of developing schizophrenia in adulthood (Sørensen et al., 2009;Brown and Derkits, 2010).These infections induce inflammatory processes which indirectly affect critical development of the foetal brain (Shi et al., 2005;Piontkewitz et al., 2011).Maternal immune activation (MIA) animal models have since been developed to study underlying mechanisms of this increase in risk, as well as potential early and late treatments (Meyer et al., 2006).In rats and mice, inflammatory sequalae associated with gestational infection result in schizophrenia-like behavioural changes in the offspring in adulthood.Specifically, polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analogue of double-stranded RNA that mimics acute responses to a viral infection (Kimura et al., 1994), increases levels of proinflammatory cytokines including interleukin-1β, interleukin-6 and tumour necrosis factor-α when administered to the pregnant dam (Cunningham et al., 2007).These mediators then result in altered development of the offspring, for example enlarged ventricles and reduced hippocampal size, similar to schizophrenia patients (Piontkewitz et al., 2009).Other developmental and neurobehavioural changes with relevance to schizophrenia in the offspring include enhanced psychosis-like behaviour, cognitive impairment and specific neurochemical changes (Zuckerman et al., 2003;Wolff and Bilkey, 2008;Wolff and Bilkey, 2010;Piontkewitz et al., 2011;Howland et al., 2012;Meehan et al., 2017;Gray et al., 2019;Gogos et al., 2020).Several of these studies have shown altered dopaminergic regulation in these MIA models (Zuckerman et al., 2003;Vuillermot et al., 2010;Luchicchi et al., 2016;Sbisa et al., 2020) but less is known about changes in other neurotransmitter systems implicated in schizophrenia, for example glutamate.
Hypofunction of N-methyl-D-aspartate (NMDA) glutamate receptors in schizophrenia may lead to excessive glutamate release and contribute to psychotic and cognitive symptoms of the illness (Moghaddam and Javitt, 2012;Hu et al., 2015).Rahman et al. (2017) used autoradiography to measure neurochemical indices of NMDA receptor function in the cortex and striatum of male and female offspring from poly(I:C) treated dams.NMDA receptor NR2A subunit binding was elevated, while NR2B binding was unchanged in all brain regions of poly(I:C) offspring.Of note, the increased NR2A binding in the striatum and cortex was found in male, but not female, poly(I:C) offspring (Rahman et al., 2017).These and other studies (Nakamura et al., 2022) show that long-term neurochemical effects of prenatal poly(I:C) treatment on NMDA receptor function are sex-specific and potentially modulated by sex steroid hormones, in line with sex differences in schizophrenia symptoms (Gogos and van den Buuse, 2023).With respect to animal models, while in vivo NMDA receptor function has been widely assessed by administration of the NMDA receptor antagonist, MK-801 (van den Buuse, 2010), surprisingly little is known on MK-801 effects on psychosis-like behaviour following MIA in female rats.
The study of sex differences in clinical and preclinical schizophrenia is of particular relevance since it has been shown that sex steroid hormones, such as 17β-estradiol, and selective estrogen receptor modulators, such as raloxifene, have antipsychotic effects (Gogos et al., 2015;Kulkarni et al., 2015;Weickert et al., 2015;Sbisa et al., 2017).Treatment with estradiol reduced positive, general and total symptoms in a randomised-controlled trial in premenopausal women with treatmentresistant schizophrenia (Kulkarni et al., 2015).In ovariectomised female rats, treatment with 17β-estradiol and raloxifene attenuated acute dopamine-mediated effects on prepulse inhibition (PPI), a measure of sensorimotor gating (Gogos and van den Buuse, 2015;Sbisa et al., 2018), however the effects of these hormone treatments on NMDA receptor-mediated disruption of PPI in the MIA model have not been studied.
Poly(I:C) treatment during mid-late gestation, i.e. gestation day 15, equivalent to the second trimester in humans, has been shown by several studies to cause disruption of baseline PPI (Wolff and Bilkey, 2008;Wolff and Bilkey, 2010;Yee et al., 2011;Howland et al., 2012;Ballendine et al., 2015;Bikovsky et al., 2016;Luchicchi et al., 2016;Gogos et al., 2020).This reduction of baseline PPI was similar in male and female poly(I:C) offspring (Zuckerman et al., 2003;Howland et al., 2012;Gogos et al., 2020).Treatment with 17β-estradiol in female rat poly(I:C) offspring reversed the reduction of baseline PPI (Sbisa et al., 2020).Moreover, while acute treatment with the dopamine receptor agonist, apomorphine, reduced PPI in control rats, it had no effect in poly(I:C) offspring (Sbisa et al., 2020).This suggested PPI in the MIA model is not only characterised by reduced baseline but also shows disruption of its normal dopaminergic regulation.This deficit in the action of apomorphine was reversed by chronic 17β-estradiol treatment (Sbisa et al., 2020).It is unknown if NMDA receptor-mediated changes in PPI are similarly inhibited in poly(I:C) offspring and restored after chronic treatment with estrogen-like compounds.Similarly, it is unclear if the effect of these hormone treatments extends to other models of psychosis-like behaviour in the MIA model, for example locomotor hyperactivity (van den Buuse, 2010).
The aim of this study was to investigate the modulation by estrogenlike compounds of the effects of MK-801 on PPI and locomotor activity in the rat poly(I:C) MIA model of schizophrenia.We compared the effect of 17β-estradiol with that of 17α-estradiol and raloxifene.While less potent at binding to classical estrogen receptors, both 17α-estradiol and raloxifene have been suggested to have significant modulatory effects particularly in the brain (Toran-Allerand et al., 2005;Arevalo et al., 2011).

Animals
We used outbred Long Evans (LE) rats which were obtained from the Florey Institute of Neuroscience and Mental Health (VIC, Australia) or the Australian Resource Centre (WA, Australia).All studies were conducted at the La Trobe University Animal Research and Teaching Facility (VIC, Australia) and were approved by the La Trobe University Animal Ethics Committee (approval number 15-44), in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes set out by the National Health and Medical Research Council of Australia.The rats were housed in groups of 2-4 in individually-ventilated cages (Tecniplast, Italy) at a room temperature of 20-22 • C and a 12/12 h light cycle, with lights on at 7 am.The rats received standard rat chow and tap water ad libitum.

Maternal immune activation
MIA was carried out as described previously (Gogos et al., 2020).Briefly, females were ~4 months of age at the time of mating, which was verified by the presence of a vaginal plug the next morning (gestation day 1, GD1).Dams were subsequently housed with fellow female breeders (4 per cage) until GD15 after which they were randomly allocated to either the poly(I:C) or control condition.While briefly anaesthetised with an isoflurane/oxygen gas mixture, the rats were injected into the tail vein with 4 mg/kg of poly(I:C) (salt weight; polyinosinic-polycytidylic acid potassium salt, P9582, Lot #034M4086V, Sigma Chemical Company, MO, USA, low molecular weight (Mueller et al., 2019)) with the dose based on previous research (Zuckerman et al., 2003;Wolff and Bilkey, 2008;Howland et al., 2012;Meehan et al., 2017).Poly(I:C) was dissolved in saline and prepared fresh the morning of each injection.Control dams received the injection volume of intravenous 1 ml/kg saline.Following treatment on GD15, dams were individually housed (for further details, see supplementary MIA Model Reporting Guidelines Checklist).
Body weight and rectal temperature were taken prior to injection on GD15 and 24 h later.As described previously (Gogos et al., 2020), dams injected with poly(I:C) had significantly decreased body weight and rectal temperature on GD16.All dams delivered pups normally and litters were weaned at postnatal day 21.We used a total of 37 female poly (I:C) offspring from 12 litters and 34 female control offspring from 16 litters.Up to 6 offspring per dam were used, with ≤2 offspring/litter/ group.
Implant sizes were based on our previous findings (Gogos and Van den Buuse, 2004;Gogos and van den Buuse, 2015;Sbisa et al., 2018).Specifically, using liquid chromatography-tandem mass spectrometry, we have shown that this dose of 17β-estradiol produces an average of 54 ± 4 pg/ml of serum 17β-estradiol over a 6-week period (Gogos et al., 2018), similar to physiological levels observed in intact rats (4-107 pg/ ml) (Sun et al., 2016;Gogos et al., 2018).We have not previously measured circulating 17α-estradiol and raloxifene levels in rats implanted with these steroids.However, given the structural similarity between 17β and 17α-estradiol, it is likely that both steroids will enter the circulation at comparable levels.Therefore the implant size was similar for these hormones.The chemical structure of raloxifene is different from that of 17β-estradiol.In humans, raloxifene is widely distributed throughout the body following oral administration but its bio-availability has been reported to be only about 2 % (Hochner- Celnikier, 1999).Therefore the dose of raloxifene here is higher than that of 17β-estradiol and was based on a previous study where we showed that, in ovariectomised rats, this dose of raloxifene attenuated apomorphineinduced disruption of PPI similar to a 17β-estradiol implant (Gogos and van den Buuse, 2015).

Experimental design
We used a pseudo-randomised, crossover protocol, so that rats received saline or MK-801 with at least a 3-day washout period between each testing session (Supplementary Fig. 1).First, starting ten days after implant surgery, rats were tested for PPI twice, 10 min after s.c.administration of saline or 0.08 mg/kg of the glutamate NMDA receptor antagonist, MK-801 (dizocilpine, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate; Sigma).One week later, the same rats were tested for locomotor hyperactivity twice, following a subcutaneous injection of either saline or MK-801 (0.05 mg/kg, Supplementary Fig. 1).The selected doses of MK-801 were expected to reduce PPI and induce locomotor hyperactivity, respectively, based on previous findings (Gogos et al., 2012;Gogos and van den Buuse, 2023).MK-801 was dissolved in saline and administered in a volume of 1 ml/kg.Around one week after the last behavioural test, the rats were euthanised with CO 2 and decapitated.The brain was removed for later analysis and the uterus was dissected and weighed.

Behavioural tests 2.5.1. Prepulse inhibition
Startle responses and PPI were measured using automated startle chambers (SR-Lab; San Diego Instruments, CA, USA), as previously described (Gogos and van den Buuse, 2015;Sbisa et al., 2018;Gogos et al., 2020).Each rat underwent the same PPI protocol consisting of 80 trials presented with variable intervals (8-27 s), including 32 startle pulse-alone trials of 40 msec duration and 115 dB intensity, and 40 prepulse-pulse trials.Each prepulse-pulse trial consisted of a 20 msec prepulse of an intensity of 2, 4, 8, 12, or 16 dB above the 70 dB background (eight per intensity), followed 100 msec later by the startle pulse.There were also 8 'NOSTIM' trials where no sound stimuli were administered, to confirm that there was no non-specific hyperactivity which could influence the PPI results (data not shown).Percentage PPI was calculated as [(pulse-alone trial startle amplitude minus prepulsepulse trial amplitude) / (pulse-alone trials startle amplitude)] × 100 %.

Locomotor activity
Locomotor activity was measured using eight automated photocell chambers (43 cm L × 43 cm W × 31 cm H; ENV-520, MED Associates, VT, USA), which detected the position of the rat within the chamber in consecutive 5 min time blocks via infrared sources and sensors (Gogos and van den Buuse, 2015;Sbisa et al., 2018;Gogos et al., 2020).Rats were placed in the locomotor chamber for 30 min to allow habituation.They were then injected and locomotor activity was recorded for a further 90 min and expressed as total cumulative distance moved (m).

Data analysis
All data are expressed as mean ± standard error of the mean (SEM) and analysed using the statistical software package SPSS Statistics 28 (IBM, IL, USA).Effect size was estimated using partial eta squared (ηp 2 ), where >0.01 is small, >0.06 is medium and >0.138 is large (Richardson, 2011).Results were considered significant if p < 0.05.Body and uterus weight were assessed with analysis of variance (ANOVA) with condition (control or poly(I:C)) and estrogenic treatment (vehicle, 17βestradiol, 17α-estradiol, raloxifene) as the between-subject factors.For PPI analyses, ANOVA was used to analyse the between-subject factors condition (control or poly(I:C)) and estrogenic treatment (vehicle, 17βestradiol, 17α-estradiol, raloxifene).Repeated-measures analyses were used where appropriate to analyse the within-subject factors drug (saline or MK-801), prepulse intensity (5 levels: PP2-PP16) and startle block (four blocks of eight).Significant condition by estrogenic treatment interactions were interrogated by further ANOVAs separated by estrogenic treatment.PPI increased with prepulse intensity and startle amplitudes habituated over the course of the PPI session.These expected changes did not show relevant statistical interactions with poly(I:C) prenatal treatment, estrogenic treatment, or the effect of MK-801.Therefore, data are presented here as average PPI and average startle.Full details of PPI at each prepulse intensity and of startle amplitude habituation are presented in the Supplementary Data section.Similarly, while MK-801 treatment resulted in the expected time-dependent locomotor hyperactivity, the prenatal or estrogenic treatments did not interact with the time course of locomotor hyperactivity, which is therefore expressed as total distance moved over the 90-minute postinjection period.For clarity, data presentation first focuses on baseline PPI and startle amplitudes, followed by analysis of the effect of MK-801 on these parameters and on locomotor activity.

Offspring body and uterus weight
Analysis of offspring body weight on the day of implant surgery or at the end of the experiment showed a significant main effect of condition (F 1,63 = 7.16, p = 0.009, ηp 2 = 0.10, and F 1,63 = 6.29, p = 0.015, ηp 2 = 0.09) with poly(I:C) offspring weighing slightly less than control offspring (control = 175-212 g, poly(I:C) = 168-202 g).There were no differences between the estrogenic treatment groups (Table 1).Importantly, body weight gain was not different among the groups, suggesting that both control and poly(I:C) offspring gained a similar amount of weight (average of control rats = 37 ± 2 g, poly(I:C) rats = 34 ± 2 g).
There was no significant difference in uterus weight between control and poly(I:C) conditions and no significant condition × estrogenic treatment interaction.However, uterus weight was significantly different between treatment groups (absolute weight: F 3,63 = 32.47,p < 0.001, partial ηp 2 = 0.61; weight as a % of body weight: F 3,63 = 31.38,p < 0.001, partial ηp 2 = 0.60) and post hoc comparison showed that uterus weight in 17β-estradiol-treated offspring was significantly higher than all other groups.Neither 17α-estradiol or raloxifene affected uterus weight (Table 1).

Baseline startle amplitudes
Baseline startle data obtained following saline treatment were analysed using a 2 condition (control, poly(I:C)) × 4 treatment (vehicle, 17β-estradiol, 17α-estradiol, raloxifene) ANOVA.There was a significant main effect of chronic estrogenic treatment (F 3,63 = 3.51, p = 0.020, ηp 2 = 0.14) but not of condition, reflecting a trend for startle amplitudes to be lower in hormone-treated rats independent of prenatal condition (Fig. 1B).Startle amplitudes in the 17α-estradiol group were significantly lower than the vehicle group but there were no other group differences in baseline startle (Fig. 1B).

The effect of MK-801 on startle amplitudes
To analyse the effects of acute MK-801 on startle, a 2 condition (control, poly(I:C)) × 4 treatments (vehicle, 17β-estradiol, 17α-estradiol, raloxifene) × 2 drugs (saline, MK-801) × 4 startle blocks repeatedmeasures ANOVA was used.There was a significant main effect of acute drug treatment (F 1,63 = 24.3,p < 0.001, ηp 2 = 0.29), reflecting an increase of startle amplitudes caused by acute administration of MK-801 (Fig. 2C and D).There was also a main effect of hormone treatment (F 3,63 = 3.88, p = 0.013, ηp 2 = 0.16), but no statistical interactions to suggest that the effect of MK-801 was different depending on hormone or poly(I:C) condition (Fig. 2C and D).Average startle was lower in 17αestradiol and raloxifene-treated rats than in vehicle-treated rats (Fig. 2C  and D).Startle amplitudes significantly habituated over the course of the PPI session similarly in all groups (main effect of Block: F 3,189 = 6.27, p < 0.001, ηp 2 = 0.09; for further details on startle habituation, see Supplementary Table 1).

Locomotor hyperactivity
Spontaneous locomotor hyperactivity, measured during the 30 min pre-injection habituation period, was not different between the estrogenic treatment groups or prenatal condition (data not shown).The s.c.injection of MK-801 induced significant locomotor hyperactivity (F 1,63 = 48.4,p < 0.001, ηp 2 = 0.43).While this effect tended to interact with estrogenic treatment (F 3,63 = 2.66, p = 0.055, ηp 2 = 0.11) there was no difference between controls and poly(I:C) offspring (Fig. 3).Further comparison of locomotor distance moved between vehicle-treated and either of the estrogenic-treated groups showed no difference in the effect of MK-801 on prenatal condition (only main effect of drug, data not shown).

Discussion
The main findings of this study were that (1) female poly(I:C) rat offspring showed baseline deficits in PPI that were successfully prevented by chronic treatment with 17β-estradiol and raloxifene, but not 17α-estradiol; (2) the PPI deficits in poly(I:C) offspring were associated with reduced effects of MK-801 which were prevented by 17βestradiol and raloxifene treatment; (3) the effects of MK-801 on startle amplitude and locomotor activity were independent of poly(I:C) condition or estrogenic treatment.None of the hormone treatments altered baseline PPI or the effect of MK-801 on PPI in control rats (see Fig. 1) which could be consistent with these compounds having no effect on PPI in healthy subjects.
In a previous report in female rats (Sbisa et al., 2020) we showed the effectiveness of 17β-estradiol to prevent the reduction of baseline PPI in the poly(I:C) model and here this finding is extended to raloxifene, but not 17α-estradiol.Reduced baseline PPI in poly(I:C) offspring was furthermore associated with diminished propensity of the dopamine receptor agonist, apomorphine, to reduce PPI, and chronic treatment with 17β-estradiol restored this effect of apomorphine (Sbisa et al., 2020).We interpreted these findings as a disruption of normal dopaminergic regulation of PPI in the MIA model, shown by both reduced baseline PPI and lack of apomorphine effects.The present study shows similar results for NMDA receptor-mediated PPI regulation.Thus, the reduction of PPI by acute administration of MK-801 was diminished in poly(I:C) offspring and the MK-801 effect was restored by both 17βestradiol and raloxifene, but not by 17α-estradiol.Importantly, the differential changes in PPI could not be explained by corresponding changes in startle amplitudes, which were independent of poly(I:C) condition and estrogenic treatment.
It may be argued that the apparent restoration of the effect of MK-801 can simply be explained by the reversal of the reduction in baseline PPI, i.e. in poly(I:C) offspring PPI is low and therefore cannot be further reduced by MK-801.This simple explanation is unlikely for the following reasons.Firstly, several of the individual data show that PPI can be much lower than the average in either control or poly(I:C) offspring and therefore the reduced baseline PPI in poly(I:C) offspring does not represent a 'floor' value.Moreover, in previous studies in female and male rats we observed different doses of MK-801 to reduce average PPI to 10-20 % (Gogos et al., 2012;van den Buuse et al., 2017), lower than baseline PPI in any of the groups in the present study.Secondly, in our previous study (Sbisa et al., 2020), treatment with 17βestradiol restored the effect of apomorphine, but not methamphetamine, on PPI, despite the hormone treatment reversing the effect of prenatal condition on baseline PPI.It is therefore more likely that 17β-estradiol and raloxifene reverse disruption of PPI regulation in poly(I:C) offspring that results in both reduced baseline PPI and diminished MK-801 effects.
This means that the MIA rats with the lowest baseline PPI will also show the least effect of MK-801 and these findings cannot easily be disentangled.Future studies should explore whether other pharmacological challenges are able to reduce PPI in the MIA group, despite the reduced baseline PPI in these animals.For example, in a previous study (Choy and van den Buuse, 2008) in a 'two-hit' developmental model including neonatal maternal deprivation and adolescent corticosterone treatment, we observed in male and female rats that apomorphine-induced disruption of PPI was absent.In contrast, PPI disruption caused by acute treatment with the 5-HT1A receptor agonist, 8-OH-DPAT was not altered (Choy and van den Buuse, 2008).Other future studies could include increasing baseline PPI in the MIA rats.If MK-801-induced PPI disruption would still be absent in such animals, this would argue against the simple 'floor' effect explanation.
The primary deficit leading to altered regulation of PPI in MIA rats may be enhanced dopaminergic activity, as has been suggested by others (Vuillermot et al., 2010;Meehan et al., 2017).Dopamine receptor antagonists were able to reverse the PPI deficit in male and female poly(I: C) offspring (Vuillermot et al., 2010).Our previous study showed a strong trend for a dopamine D2 receptor binding density increase in the nucleus accumbens core in female poly(I:C) offspring, which was reversed by chronic 17β-estradiol (Sbisa et al., 2020).However, other previous studies have suggested that NMDA receptor-mediated control of PPI, and therefore possibly the effects of MIA on NMDA receptor regulation of behaviour, are independent of dopaminergic activity (Keith et al., 1991;Bast et al., 2000).A more direct effect of MIA on NMDA receptor function was suggested by microdialysis studies which showed a blunting of MK-801 induced glutamate release in the frontal cortex of male MIA offspring (Roenker et al., 2011).This functional NMDA receptor hypofunction may be at least partly responsible for the reduced baseline PPI and the diminished effect of MK-801 on PPI in the present study.Further studies are required to characterise the molecular and neurochemical changes in dopaminergic and NMDA receptor activity that occur in the MIA model to better understand the spectrum of behavioural changes that have been observed.
These, and our previous (Sbisa et al., 2020) results suggest that 17βestradiol has antipsychotic potential.In the present study we extend this finding to the selective estrogen receptor modulator, raloxifene, which in several studies has been shown to be clinically beneficial in schizophrenia (Weickert et al., 2015;Kulkarni et al., 2016;de Boer et al., 2018;Brand et al., 2023).A meta-analysis showed that raloxifene improved total symptom severity, as well as positive and negative subscales, but there was no effect on cognitive functioning (de Boer et al., 2018).In contrast, in another study, raloxifene improved processing speed and memory in both men and women, suggesting it might be beneficial as an adjunctive treatment for cognitive deficits in schizophrenia (Weickert et al., 2015).Recently, raloxifene was shown, in women, to improve negative symptoms and working memory (Brand et al., 2023).Differences between the findings of these studies could be caused by symptom severity, study design, and raloxifene dose/duration (Weickert and Weickert, 2017).
Because its receptor binding profile is different to that of 17β-estradiol, raloxifene acts as an estrogen receptor antagonist in mammary and uterine tissues, whereas it functions as an estrogen receptor agonist in brain and bone (Sato et al., 1996).We previously showed that raloxifene treatment had similar effects to 17β-estradiol on apomorphine-induced changes in PPI in ovariectomised rats (Gogos and van den Buuse, 2015;Sbisa et al., 2018), although the present findings are the first to show this in the MIA model.Of relevance to the present study, raloxifene treatment was able to reverse changes in NMDA receptor binding in the hippocampus induced by ovariectomy (Cyr et al., 2001).Given the likely multiple sites of action of MK-801 on PPI (see above) it remains unclear through which mechanism raloxifene reverses both the MIA-induced reduction of baseline PPI and restores the acute effect of MK-801.
The chronic administration of 17α-estradiol had no effect on either baseline PPI or on the reduction of PPI following acute administration of MK-801.Potential benefits of 17α-estradiol over 17β-estradiol are that 17α-estradiol does not have the feminizing and cardiac dysfunction sideeffects associated with 17β-estradiol (Stout et al., 2017).Previous studies suggested that 17α-estradiol has lower potency compared to 17βestradiol to bind to classical estrogen receptors (Toran-Allerand, 2005), however this was disputed by other studies (Toran-Allerand et al., 2005) which showed that 17α-estradiol binds to estrogen receptors ERα and ERβ with affinity similar to that of 17β-estradiol and is able to transactivate an estrogen-responsive reporter gene.However, in contrast to 17β-estradiol, 17α-estradiol does not bind to estrogen-binding plasma proteins and its brain levels are unaffected by gonadectomy (Toran-Allerand et al., 2005).These authors concluded that 17α-estradiol is likely synthesized in the brain where it may act on ER-X receptors rather than the classical estrogen receptors (Toran-Allerand et al., 2005).Effects of 17α-estradiol include it dose-dependently modulating contextual fear conditioning in rats (Barha et al., 2010) and increasing cell proliferation in the hippocampus (Barha et al., 2009).17α-estradiol, at similar doses to 17β-estradiol, enhanced visual and place memory in ovariectomised rats (Luine et al., 2003).However, in the present study 17αestradiol, administered chronically at the same dose as 17β-estradiol, had no effect on PPI either at baseline or following acute administration of MK-801.
None of the hormone treatments altered baseline PPI or the effect of MK-801 on PPI in control rats (see Fig. 1).This could be consistent with these compounds having no effect on PPI in healthy subjects.
Methamphetamine-induced locomotor hyperactivity may be used as a behavioural 'read-out' of dopaminergic activity in some brain regions and hence has been proposed as psychosis-like behaviour in animal models (van den Buuse, 2010).In a previous study in ovariectomised rats we showed that administration of 17β-estradiol, but not raloxifene, reduced methamphetamine-induced locomotor hyperactivity (Sbisa et al., 2018).This could suggest differences in the mechanisms of action between the two estrogen analogues, with 17β-estradiol altering dopamine release but raloxifene more likely to act post-synaptically and, hence, independent of dopamine release.In the present study, neither 17β-estradiol nor raloxifene significantly altered the effect of MK-801 on locomotor activity, showing selectivity of their action for some, but not other models of schizophrenia symptoms.Differences in the effects of these steroids on methamphetamine/apomorphine effects vs. MK-801 effects are consistent with separate involvement of dopaminergic and NMDA receptor-mediated mechanisms in schizophrenia-like behaviour in rats.Future studies could include the effect of 17β-estradiol and raloxifene on dopamine release in the brain, for example by micro-dialysis or voltammetry.

Limitations
This study was done only in female rats and it will be of clinical interest to perform similar studies in male rats.One of the advantages of SERMs, such as raloxifene, is that they induce less feminizing side effects compared to 17β-estradiol which hinder the application of sex steroid hormones in males (de Boer et al., 2018).As discussed above, another limitation of this study is that we were not able to separate the effects of hormone treatments on baseline PPI with those on MK-801-induced PPI reduction.We also require further studies to identify molecular and neurochemical mechanisms in the action of 17β-estradiol and raloxifene, specifically dopaminergic and NMDA receptor markers.Finally, it is recognized that PPI is only one endophenotype of schizophrenia and it remains unclear how our results translate to clinical symptoms of the illness where individuals may already be symptomatic before they receive hormone treatment.Moreover, such hormone treatments will likely be adjunctive to on-going antipsychotic drug treatment; our experiments did not include combination treatments of hormone implants and antipsychotics which is a limitation which will have to be addressed in future studies.

Conclusions
These findings extend our previous observations that an MIAinduced decrease of baseline PPI can be reversed by treatment with 17β-estradiol.We found that the selective estrogen receptor modulator, raloxifene, but not the 17β-estradiol isomer, 17α-estradiol, was similarly able to restore baseline PPI.Extending our previous studies which showed that 17β-estradiol was able to restore the effect of apomorphine on PPI, here we showed that both 17β-estradiol and raloxifene were able to restore the effect of MK-801 in MIA.Collectively, these findings show that MIA affects both dopaminergic and NMDA receptor-mediated regulation of PPI in females and these effects on sensorimotor gating were prevented by estrogenic treatment.

Fig. 1 .
Fig. 1.Average (+ SEM) PPI (A) and startle amplitudes (B) after acute injection of saline in female rats that were offspring of control-treated (white bars) or poly(I:C)-treated (stippled bars) dams.Adult offspring were chronically implanted with either vehicle (Veh), 17β-estradiol (17β), 17α-estradiol (17α) or raloxifene (Ral) (n = 7-10/group).Individual data are denoted by open (control) or closed (poly(I:C)) circles.Avg % PPI denotes the average of the five prepulse intensities.Avg startle amplitude denotes the average of the four blocks of startle pulses.*p < 0.05 comparing control and poly(I:C) offspring within the same estrogenic treatment group; # p < 0.05 compared to vehicletreated poly(I:C) offspring; **p < 0.05 for difference with vehicle-treated rats independent of condition.PPI data from vehicle-and 17β-estradiol treated control and poly(I:C) rats were shown previously in(Sbisa et al., 2020) and are included here for comparison.

Fig. 2 .
Fig. 2. Average (+ SEM) PPI (A) and startle amplitudes (B) of female rats that were control or poly(I:C) offspring.These offspring were chronically implanted with vehicle (Veh), 17β-estradiol (17β), 17α-estradiol (17α) or raloxifene (Ral).All rats received acute injection of saline (white bars, circles) or MK-801 (0.08 mg/kg; grey bars, triangles), with individual data denoted by open (control) or closed (poly(I:C)) shapes.Avg % PPI denotes the average of the five prepulse intensities.Avg startle amplitude denotes the average of the four blocks of startle pulses.*p < 0.05 comparing saline and MK-801 within the same condition.Data are mean + SEM of n = 7-10 per group.

Table 1
Body and uterus weight of control and poly(I:C) offspring.
* p < 0.001 for Bonferroni post-hoc comparison to VEH treated rats.