Both Val158Met Polymorphism of Catechol-O-Methyltransferase Gene and Menstrual Cycle Affect Prepulse Inhibition but Not Attentional Modulation of Prepulse Inhibition in Younger-Adult Females

Accepted Manuscript Both Val158Met Polymorphism of Catechol-O-Methyltransferase Gene and Menstrual Cycle Affect Prepulse Inhibition but Not Attentional Modulation of Prepulse Inhibition in Younger-Adult Females

Chao Wu, Yu Ding, Biqing Chen, Yayue Gao, Qian Wang, Zhemeng Wu, Lingxi Lu, Lu Luo, Changxin Zhang, Xiaohan Bao, Pengcheng Yang, Langchen Fan, Ming Lei, Liang Li PII: DOI: Reference:

S0306-4522(19)30094-6 https://doi.org/10.1016/j.neuroscience.2019.02.001 NSC 18893

To appear in:

Neuroscience

Received date: Accepted date:

4 September 2018 1 February 2019

Please cite this article as: C. Wu, Y. Ding, B. Chen, et al., Both Val158Met Polymorphism of Catechol-O-Methyltransferase Gene and Menstrual Cycle Affect Prepulse Inhibition but Not Attentional Modulation of Prepulse Inhibition in Younger-Adult Females, Neuroscience, https://doi.org/10.1016/j.neuroscience.2019.02.001

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Both Val158Met Polymorphism of Catechol-O-Methyltransferase Gene and Menstrual Cycle Affect Prepulse Inhibition but Not Attentional Modulation of Prepulse Inhibition in Younger-Adult Females

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(Running Title: COMT, Menstrual Cycle and Prepulse Inhibition)

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Chao Wua,c#, Yu Dingb#, Biqing Chenc, Yayue Gaob, Qian Wangb, Zhemeng Wub, Lingxi Lub, Lu

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Luob, Changxin Zhangb, Xiaohan Baob, Pengcheng Yangb, Langchen Fanb, Ming Leib, Liang Lib,d,*

School of Nursing, Peking University Health Science Center, Beijing, 100191, China

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School of Psychological and Cognitive Sciences and Beijing Key Laboratory of Behavior and

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Mental Health, Speech and Hearing Research Center, Key Laboratory on Machine Perception

PKU-IDG/McGovern Institute for Brain Research, School of Life Sciences, Peking University,

Beijing 100871, China

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(Ministry of Education), Peking University, Beijing 100080, China

Beijing Institute for Brain Disorders, Beijing, China

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The two authors contributed equally to this work and should be co-first authors

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Correspondence: Liang Li, Ph.D. School of Psychological and Cognitive Sciences, Peking University, Beijing 100080, China E-mail: [email protected]

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ABSTRACT Prepulse inhibition (PPI) can be modulated by both the Val158Met (rs4680) polymorphism of the Catechol-O-Methyltransferase (COMT) gene and the menstrual-cycle-related hormone fluctuations, each of which affects the subcortical/cortical dopamine metabolism. PPI can also be

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modulated by attention. The attentional modulation of PPI (AMPPI) is sensitive to psychoses. Whether the Val158Met polymorphism affects the AMPPI in female adults at different

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menstrual-cycle phases is unknown. This study examined whether AMPPI and/or PPI are affected by

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the Val158Met polymorphism in 177 younger-adult females whose menstrual cycles were mutually

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different across the menstruation, proliferative, or secretory phases. The AMPPI was evaluated by comparing PPI under the condition of the auditory precedence-effect-induced perceptual spatial

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separation between the prepulse stimulus and a masking noise (PPIPSS) against that under the

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condition of the precedence-effect-induced perceptual spatial co-location (PPIPSC). The results

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showed that both the menstrual cycle and the COMT Val158Met polymorphism affected both PPIPSC and PPIPSS, but not the AMPPI (difference between PPIPSS and PPIPSC). Moreover, throughout the

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menstrual cycle, both PPIPSC and PPIPSS decreased monotonously in Val/Val-carrier participants. However, the decreasing pattern was not overserved in either Met/Met-carrier or Met/Val-carrier

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participants. Thus, in healthy younger-adult females, PPIPSC and PPIPSS, but not the AMPPI, is vulnerable to changes of ovarian hormones, and the COMT Val158Met polymorphism also has a modulating effect on this menstrual-cycle-dependent PPI variation. In contrast, the AMPPI seems to be more steadily trait-based, less vulnerable to ovarian hormone fluctuations, and may be useful in assisting the diagnosis of schizophrenia in female adults. Keywords: Attentional modulation; Dopamine; Menstrual cycle; Precedence effect; Prepulse inhibition; Spatial separation; estrogen; Val153Met polymorphism.

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ABBREVIATION AMPPI, attentional modulation of prepulse-inhibition; COMT, Catechol-O-Methyltransferase;

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FFRs, frequency-following responses; PSC, perceived spatial co-location;

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PSS, perceived spatial separation.

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INTRODUCTION Prepulse inhibition (PPI) is the suppression of the startle reflex in response to an intense startling stimulus (pulse) when this startling stimulus is shortly preceded by a weaker, non-startling

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sensory stimulus (prepulse) (Hoffman and Ison, 1980). PPI is recognized as a model of sensorimotor gating (Graham, 1975). The prepulse activates the time-linked inhibitory neural processes that

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weaken the motor responses to the subsequent intense stimulation (Swerdlow, et al., 2007). It has

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been well documented that PPI can be markedly enhanced by selective attention to the prepulse

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signal in both humans (Filion and Poje, 2003; Hawk, et al., 2002; Schell, et al., 2000; Thorne, et al., 2005) and laboratory rats (Du, et al., 2009, 2010; Huang, et al., 2007; Lei, et al., 2014; Li, et al.,

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2008; Wu, et al., 2016, 2018; Zou, et al., 2007), indicating that PPI contains not only lower-level involuntary (automatic) processes but also can be top-down modulated by higher-order

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perceptual/cognitive processes (for a review see Li, et al., 2009). PPI deficits have been observed in multiple psychiatric disorders, many of which exhibit

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dysfunctions in several neurotransmission systems, mainly the dopamine system (For reviews see Garcia-Sanchez, et al., 2011; Kohl, et al., 2013; Swerdlow, et al., 2016). For example, disturbances in

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the dopamine function are considered to be essential to the pathogenesis of schizophrenia (O'Tuathaigh, et al., 2012). Impressively, relative to the PPI when the prepulse is ignored, the attentionally modulated PPI (the PPI that occurs when the prepulse is attended) is more related to the psychosis (Hazlett, et al., 2008; Hazlett, et al., 2007) and the symptom severity in the schizophrenia spectrum (Hazlett, et al., 2007; Kohl, et al., 2013). The catechol-O-methyltransferase (COMT) gene has been proposed to be associated with susceptibility for schizophrenia (For reviews see Farrell, et al., 2015; Zai, et al., 2017). The COMT

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gene codes for the COMT enzyme, a protein that degrades catecholamine neurotransmitters and modulates dopamine metabolisms (Schacht, 2016). The Val158Met (rs4680) polymorphism of the COMT gene, one of the well-studied single-nucleotide polymorphisms (SNP) in schizophrenia, has been reported to modulate PPI in both healthy human males (Quednow, et al., 2017; Roussos, et al.,

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2008) and people with schizophrenia (Quednow, et al., 2010). Also, the Met allele (A) is related to

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lower COMT enzymatic activity, therefore related to higher dopamine levels in some brain regions,

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such as the prefrontal cortex. However, the Val allele (G) is related to higher COMT enzymatic activity, therefore related to lower dopamine levels (Stein, et al., 2006). The Roussos et al. (2008)

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study has shown that male adults with the Val/Val genotype have the lowest level of PPI, male adults

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with the Val/Met genotype have the intermediate level of PPI, and male adults with the Met/Met genotype have the highest level of PPI, implying that a lower dopamine level in the prefrontal cortex

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may account for a lower level of PPI in male adults. The COMT enzymes are also involved in

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estrogen synthesis and metabolism (Bates, et al., 1978). However, to date, whether the Val158Met polymorphism affects PPI during the prepulse is actively attended and/or the AMPPI in female adults

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at different menstrual-cycle phases has not been reported in the literature.

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Using a non-attentional paradigm with discrete 20-ms prepulses and 60-120-ms onset-to-onset intervals, the Swerdlow et al. (2016) study has shown a sex-dependent effect of rs4680 on PPI, with lower PPI in male adults carrying one or more Val alleles (d = 0.47), but with the opposite pattern in female adults. Other previous studies have suggested that the menstrual phase-related variability in PPI is likely to be regulated by the fluctuation of ovarian-hormone levels (Gogos, 2013; Kumari, et al., 2010). More PPI in women occurs during the follicular phase compared to the luteal phase (Jovanovic, et al., 2004; Swerdlow, et al., 1997). Moreover, some evidence has indicated that the

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hormone estradiol regulates dopamine synthesis and release, and modifies basal firing rates of dopamine neurons via membrane estrogen receptors (Jacobs and D'Esposito, 2011; Xiao and Becker, 1994), thereby regulating cortical and sub-cortical neural activities (Peper, et al., 2011) and even higher-order perceptual/cognitive functions (Smith, et al., 2014).

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In summary, PPI in adult females is modulated by not only the menstrual cycle, during which the

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dopamine activity within the mesocorticolimbic pathway is regulated by estrogen (Almey, et al.,

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2015; Sotomayor-Zarate, et al., 2014), but also the COMT Val158Met polymorphism that more specifically regulates the prefrontal dopamine level. To date, in adult females, whether the PPI

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magnitude is modulated by the COMT Val158Met polymorphism when the prepulse is actively

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attended and whether there is an interaction between the menstrual cycle and the Val158Met polymorphism on the PPI and/or AMPPI have not been reported in the literature.

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This study was to explore the effects of the COMT Val158Met polymorphism and menstrual

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cycle on PPI (when the prepulse is actively attended) and AMPPI in healthy younger-adult females. Specifically, we first adopted a paradigm for examining attentional modulation of PPI (Du, et al.,

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2009, 2011; Lei, et al., 2018; Yang et al., 2017) based on the auditory precedence-effect-induced

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perceptual spatial separation (PSS, which enhances selective attention to the prepulse) and the precedence-effect-induced perceptual spatial co-location (PSC, which facilitates masking of the prepulse) between the prepulse stimulus and a masking noise (Li et al., 2004; see the Methods for details). Then we examined the difference between the PPI magnitude under the PSS-listening condition (PPIPSS) and the PPI magnitude under the PSC-listening condition (PPIPSC, which was defined as the baseline PPI). Next, we examined the difference in PPI (PPIPSC and PPIPSS) and AMPPI (PPIPSS minus PPIPSC) across different Val158Met-genotype groups and across different

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menstrual-cycle-phase groups. Finally, we explored the interactions between the listening conditions, the COMT Val158Met polymorphism, and the menstrual cycle on AMPPI, PPIPSS, and PPIPSC.

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EXPERIMENTAL PROCEDURES Participants

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In total 243 younger-adult female college students studying at the Chongqing Medical

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University (mean age = 19.0 ± 0.8 years) participated in this study. None of these participants

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showed pure-tone hearing impairments for each ear at the frequencies of 125, 256, 512, 1024, and 2048 Hz (evaluated by medical tuning forks). None of them had a history of brain injury, psychiatric

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disorder, hearing impairments/diseases, or substance dependency.

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Some participants were excluded from data analyses if over half of the startle-alone trials did

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not provide reliably measurable data, or if these participants had more than half of the trials rejected due to marked excess head movement, frequent spontaneous eye movement, and/or drowsiness

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during testing. Participants who had unusual menstrual cycles [the duration of the whole cycle was longer than 35 (28+7) days or shorter than 21 (28-7) days] or a menstrual period longer than one

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week were also removed from data analyses. Finally, 37 participants were excluded due to excess head movements and/or spontaneous eye movements, and 20 participants were excluded for unusual menstrual cycles. The remaining 189 heathy female participants were remained for further analyses of startle response data. None of the 189 female participants reported the use of exogenous hormones. For each participant, the date of the last menstrual onset, the duration of the menstrual period, and the length of the menstrual cycle were reported on the date of PPI testing. The participants were

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divided into three groups according to their phases in the menstrual cycle on the date of PPI testing: menstruation phase, proliferative phase, and secretory phase (Silverthorn, 2016). Note that the secretory phase is the final phase of the uterine cycle and corresponds to the luteal phase of the ovarian cycle. The duration of the luteal phase is relatively stable and generally has an average of 14

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days. Therefore, the time of 14 days before the menstruation onset can be treated as the luteal

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(secretory) phase. The proliferative phase is the time between the menstruation offset and 14 days

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before the next menstruation onset. The estrogen level usually peaks in the ovulation phase of the ovarian cycle (within the proliferative phase of the uterine cycle), whereas the progesterone level

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increases and peaks in the luteal (secretory) phase (Silverthorn, 2016).

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This study was conducted according to the principles in the Declaration of Helsinki, and the experimental procedures were approved by the Committee for Protecting Human and Animal

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Subjects of the Department of Psychology at Peking University. All participants gave written

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informed consent before their participation in this study.

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Acoustic Stimuli and Equipment

The startling pulse was a 40-ms broadband noise burst (0-10 kHz, 100 dB SPL). The

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(non-startling) prepulse stimuli were 150-ms broadband noise bursts (0-10 kHz, 70 dB SPL). During the testing, a broadband background noise (0-10 kHz, 60 dB SPL) was continuously delivered from the two headphones as the masker. All the acoustic stimuli were presented binaurally through headphones (HD265 linear, SENNHEISER, Germany). An electromyographic startle-recording system (Xeye Human Startle Reflex System; Beijing Macroambition S&T Development Co., Ltd, Beijing, China) was used to measure the eye-blink component of the startle reflex by recording EMG activity of the orbicularis oculi muscle directly

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beneath the right eye (positioning two miniature silver/silver chloride electrodes filled with electrode gel, Compumedics Limited, Victoria, Australia) approximately 10 mm apart (edge to edge) and 8 mm below the lower lid margin. A medial electrode was placed directly under the pupil. A ground electrode was placed behind the right ear on the mastoid (< 10 kΩ).

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EMG activity, which was band-pass filtered between 30-1,000 Hz, was analyzed with the

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sampling rate of 1000 Hz for a duration of 500 ms (starting either from the onset of the prepulse

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stimulus under the prepulse-pulse-pairing conditions or from the onset of the startling pulse under the pulse-only condition). Recorded data were not rectified, smoothed, or integrated.

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The peak of the startling response was identified as the highest point within a window from 20 to

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110 ms following the startling-stimulus onset. The startle-response amplitude was the difference (in μV) between the peak and the baseline (baseline: the mean EMG amplitude over the period of 200

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ms prior to the startling-pulse onset).

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The magnitude of PPI was calculated with the following generally used formula: PPI (%) = (amplitude to startling sound alone - amplitude to startling sound preceded by

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prepulse) / (amplitude to startling sound alone) × 100%.

Design and Procedures of Startle Reflex Testing This study used an auditory precedence-effect-based paradigm to present acoustic stimuli for examining PPIPSC, PPIPSS, and AMPPI (Du, et al., 2009, 2010, 2011; Lei, et al., 2014, 2018; Wu, et al., 2016, 2018). What is the precedence effect? In a reverberant environment, when the time delay between the direct wave from the source and a reflection of the direct wave is sufficiently short, listeners will perceive a single fused sound image as coming from the sound source, this

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phenomenon has been called the precedence effect (Freyman, et al., 1999; Litovsky, et al., 1999; Wallach, et al., 1949). The auditory precedence effect can be used to perceptually separate the target sound from a masker sound (i.e., to induce perceived sound-image laterality at the listener’s left ear and/or right ear) without affecting the signal-to-noise (masker) ratio and sound image compactness

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(Li, et al., 2004; Wu, et al., 2005), thereby without affecting the peripheral processing of acoustic

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signals.

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More in detail for this study, both the prepulse and the noise masker were presented binaurally, and the interaural intervals (i.e., the interval times between the two ears) for the prepulse and that for

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the masking noise were 3 ms (which is sufficiently shorter than the echo threshold for the noise).

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Due to the precedence effect, under the PSS condition, the (perceptually fused) prepulse image was perceived as coming from one ear and the (perceptually fused) masking-noise image was perceived

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as coming from the other ear when the leading ear for the prepulse stimulus was different from that

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for the masking noise. Under the PSC condition, a (perceptually fused) prepulse image and a (perceptually fused) masking-noise image were perceived as coming from the same ear, when the

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leading ear for the prepulse was the same one as for the masking noise.

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Note that when the prepulse image and the masker image are (perceptually) co-located as coming from the same side, the masker image always occupies the listener’s attentional focus to the prepulse, leading to a larger masking effect. However, when the prepulse image and the masker image are (perceptually) separated as coming from different sides, the masker image becomes localized outside the listener’s attentional focus to the prepulse. Thus, compared to the PSC condition, introducing the PSS condition can facilitate both the listener’s selective attention to the prepulse signal and the ignorance to the masker, thereby improving the processing of the prepulse

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signal. In this study, the effect of attentional modulation of PPI (AMPPI) was defined as the difference in PPI magnitude between the PSS listening condition and the PSC listening condition (i.e., PPIPSS minus PPIPSC). It should also be noted that unlike some paradigms used in previous studies for examining the

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effect of attentional modulation of PPI (e.g., Hazlett, et al., 2003, which contain both the condition of

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attending to one type of prepulse (e.g., the one with a higher pitch) and the condition of ignoring

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another type of prepulse (e.g., the one with a lower pitch), in the auditory-precedence-effect based paradigm, the listener always tries to attend to the prepulse signal under either the PSS condition or

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the PSC condition.

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It has been known that reliable PPI can occur when the non-startling prepulse stimulus precedes the startling pulse by an interval from 20 to 500 ms (Fleshler, 1965; Graham, 1975). In this

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study, the duration of the prepulse stimulus was set at 150 ms and the offset-to-onset interval

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between the prepulse and startling pulse was 120-ms. Long onset-to-onset intervals have also been approved to be efficient in eliciting inhibition of startle magnitude in previous studies (e.g., Graham,

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1975; Lei, et al., 2018; Yang, et al., 2017). For example, the 270 ms onset-to-onset prepulse-pulse

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interval has been proved to be efficient in the PPI paradigm when the prepulse is actively attended (Lei, et al., 2018; Yang, et al., 2017). Another consideration is that for future investigations combining scalp recordings of event-related potentials (ERPs) to the prepulse (Lei et al., 2018) with measures of PPI, a sufficient long interval between the prepulse and startling pulse is necessary to prevent the overlap between ERPs to the prepulse and ERPs to the startling pulse. Thus, in this study a 120-ms offset-to-onset interval between the prepulse and startling pulse was used. Also, for future investigation combining

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measures of frequency-following responses (FFRs, Wang et al., 2018) to the prepulse stimulus with measures of PPI magnitude, the duration of a prepulse should be sufficiently long (e.g., 200 ms in Wang et al., 2018). In this study the duration of the prepulse stimulus was set at 150 ms. The recording period for each participant was comprised of two blocks. Each block contained 20

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trials, including 8 startling pulse-alone trials (left-ear background noise-masker leading in 4 trials;

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right-ear noise-masker leading in 4 trials), 6 prepulse-pulse pairing trials under the PSS condition

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(left-ear masker leading and right-ear prepulse leading in 3 trials; right-ear masker leading and left-ear prepulse leading in 3 trials), and 6 prepulse-pulse pairing trials under the PSC condition

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(left-ear masker leading and left-ear prepulse leading in 3 trials; right-ear masker leading and

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right-ear prepulse leading in 3 trials), with a pseudorandom presentation order (Figure 1). There was a brief break between the 2 blocks. The inter-trial interval varied between 10 and 22 s (with the

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average of 15 s). The entire testing session (including two blocks) lasted approximately 12 min for

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each participant.

In a trial with only the startling pulse (without a prepulse), the noise masker was presented first,

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and then the startling pulse was presented about 10.1 s after the onset of the noise masker. In a trial

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with both the prepulse and the startling pulse, the prepulse was started about 8 s after the onset of the noise masker (Figure 1). Due to the long leading time of the noise-masker onset, the influence of the noise-masker onset to PPIPSC, PPIPSS, and/or AMPPI could be minimized.

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Before the formal testing, participants were seated upright in a quiet room and received 3 or 4

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pre-testing trials to get familiar with these listening conditions, including the ones with the startling pulse only, the prepulse only (which did not occur in the formal testing), the prepulse-pulse pairing under the PSS condition, and the prepulse-pulse pairing under the PSC condition. For the baseline startle (pulse-alone) trials, in which startling responses could not be identified

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(due to low amplitudes of EMG waves below the identifying threshold) within the time window of

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20-150 ms following the startling-pulse onset, were excluded from data analyses (i.e., these excluded

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trials had no contributions to the across-trial averaged amplitude for a particular stimulation condition). Moreover, some trials were also excluded from analyses under the following situations:

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(1) the peak of the response amplitude was five times larger than the average response EMG level,

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and (2) excessive head movement, frequent spontaneous eyeblinks, and/or drowsiness occurred. Genotyping

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DNA was extracted from peripheral blood of each participant using the QuickGene whole blood genome DNA extract system (Kurabo Industries Ltd., Japan), and was successfully genotyped on the

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HumanOmniZhongHua-8 Beadchip v1.1 (Illumina, Inc., San Diego, CA, USA). Quality control was conducted using PLINK (v1.07) (Purcell, et al., 2007). Individual participants who did not provide

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blood sample for DNA testing were also excluded from data analyses (n = 12) for this study. Finally, both behavioral data and DNA data from 177 female participants were analyzed: 105 of them were with Val/Val (GG) genotypes, 59 of them were with Met/Val (AG) genotypes, and 13 of them were with Met/Met (AA) genotypes (Table 1).

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Statistical Analyses Statistical analyses were conducted using the SPSS 24.0 software. A paired t-test was first conducted to compare the amplitude difference between PPIPSS and PPIPSC. Then, one-way measures analyses of variance (ANOVA) were performed to test the pulse-only startle (PAS) magnitude, PPI

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(averaged across PPIPSS and PPIPSC), and AMPPI across different Val158Met genotype groups and

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different menstrual-cycle-phase groups. Finally, a mixed model ANOVA on PPI or AMPPI was

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conducted to explore interaction effects among the auditory condition (PSS, PSC), the COMT Val158Met genotype, and the menstrual cycle. The Benjamini-Hochberg standard false discovery

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rate (FDR) method was used for correcting p-values for multiple comparisons.

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RESULTS

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A paired t-test showed that PPIPSS was significantly greater than PPIPSC (t = 2.468, p = 0.015; Cohen’s d = 0.26, 95%CI = [0.05, 0.47]; Figure 2A), confirming the perceptual separation effect on

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PPI.

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Moreover, a one-way ANOVA was conducted to compare the PAS, the PPI (averaged across PPIPSS and PPIPSC), and AMPPI across the 3 COMT Val158Met genotype groups. The results showed that the main effect of genotype group was significant for the PPI (F2,174 = 4.705, p = 0.010; pη2 = 0.051; Figure 2B), but was not significant for either the AMPPI (F2,174 = 0.587, p = 0.557; pη2 = 0.007; Figure 2D) or the PAS (F2,174 = 0.461, p = 0.557; pη2 = 0.005; Figure 2C). Post-hoc analyses revealed more PPI in the Met/Met (AA) homozygote carriers than that in the Met/Val (AG) heterozygote carriers (p = 0.032, FDR corrected p = 0.048; Cohen’s d = 0.35, 95%CI = [0.02, 0.57])

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and that in the Val/Val (GG) homozygote carriers (p = 0.011, FDR corrected p = 0.033; Cohen’s d = 0.57, 95%CI = [0.42, 0.89]). Thus, participants with the Met/Met genotype (the AA group) exhibited the highest group-mean PPI magnitude, and those with the Val/Val genotype (the GG group) exhibited the lowest PPI magnitude. However, either the AMPPI magnitude or the PAS magnitude

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was not affected by the Val158Met polymorphism.

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Figure 3 shows the comparisons in PAS, PPI, and AMPPI across the 3 menstruation-phase groups. An ANOVA showed that the main effect of menstruation-phase group was significant for

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PAS (F2,174 = 4.679, p = 0.010; pη2 = 0.051) and PPI (F2,174 = 5.397, p = 0.005; pη2 = 0.058), but not

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significant for AMPPI (F2,174 = 0.087, p = 0.917; pη2 = 0.001). Post-hoc analyses revealed that the PAS magnitude for the proliferative phase group was significantly larger than those of the

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menstruation phase group (p = 0.017, FDR corrected p = 0.026; Cohen’s d = 0.57, 95%CI = [0.11,

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1.02]) and secretory phase group (p = 0.014, FDR corrected p = 0.026; Cohen’s d = 0.57, 95%CI = [0.11, 1.03]) (Figure 3A). The PPI magnitude of the menstruation group was significantly larger than that of the secretory group (p = 0.002, FDR corrected p = 0.006; Cohen’s d = 0.66, 95%CI = [0.29, 1.03]; Figure 3B). Moreover, both PPIPSS and PPIPSC declined monotonously from the menstruation phase, through the proliferative phase, to the secretory phase (Figure 3C). Thus, participants in the menstruation phase exhibited the highest group-mean PPI magnitude, and those in the secretory phase exhibited the lowest PPI magnitude. However, the AMPPI was not affected by the menstrual

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cycle (Figure 3D)

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A mixed-model ANOVA on PPI was conducted to explore the interaction effects between the

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listening condition (PSS, PSC), Val158Met genotype group (Met/Met + Met/Val, and Val/Val), and

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menstruation phase (menstruation phase, proliferative phase, secretory phase). The listening conditions, Val158Met genotype, and menstrual cycle were treated as fixed-effect factors, and

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individuals were treated as random-effect factors. Here, a dominant model (the major allele

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homozygous was given a score of 1, and the heterozygous grouped with the minor allele homozygous was given a score of −1) was fitted for rs4680 (i.e., GG = 1, AG/AA = −1). The results

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showed that none of the interactions among the three factors were significant. The p value for

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genotype by menstrual-cycle interaction with unrestricted covariance was 0.088 and larger than 0.1 for all other interaction effects. It should be noted that the PPI magnitude in the major allele

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homozygous (Val/Val) group showed a decline along the three phases of menstrual cycle. Whereas

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the PPI magnitude in the heterozygous grouped with the minor allele homozygous (Met/Val + Met/Met) appears to exhibit an inverted U-shape-like change along the menstrual cycle, with an increase trend in females who were in the proliferative phase (Figure 4). However, the result of the Lind and Mehlum (2010) test showed that the inverted U-shape pattern was not statistically significant.

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DISCUSSIONS This study, for the first time, examined both the effects of the COMT Val158Met

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polymorphism and the menstrual cycle on PPI (when the prepulse is actively attended) and the

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effects of attentional modulation of PPI (AMPPI) in healthy younger-adult females. The results

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showed that (1) PPI, but not AMPPI, was affected not only by the menstrual cycle but also by the COMT Val158Met polymorphism. The female participants who carried the COMT Met allele

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(associated with higher frontal dopamine levels) had more PPI than the female participants who

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carried the Val allele (associated with lower frontal dopamine levels). Also, the PPI magnitude fluctuated across the menstrual cycle in female participants, with the lowest levels in the secretory

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phase (usually the estrogen level is elevated and the progesterone level is the highest in the secretory

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phase). (2) The group-mean PPI magnitude across all the participants was significantly higher under the perceptual-separation listening condition than that under the perceptual co-location listening

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condition. (3) Female participants with the Val/Val genotype exhibited a monotonous decline in PPI

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magnitude along the 3 phases of the menstrual cycle, whereas this declining pattern disappeared in participants with the either Met/Met or Met/Val genotypes along the menstrual cycle. It should be noted that the PPI paradigm used in this and our previous studies (Lei et al., 2018; Yang et al., 2017) is different from those used in many previous studies. Specifically, this paradigm applies precedence-effect-induced perceptual separation/co-location between the prepulse and a masker to manipulate the selective attention to the prepulse signal. Thus, the magnitude of PPI and that of the attentional modulation effect obtained with the paradigm used in this study might not

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match those obtained in previous studies using the traditional PPI testing paradigms. The results of this study typically showed that both attention-facilitated PPIPSS and attention-distracted PPIPSC were affected by not only the COMT gene but also the menstrual cycle in healthy female participants. However, the effect of attentional modulation of PPI (AMPPI; i.e., the difference between PPIPSS and

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PPIPSC) was not affected by the two dopamine-related factors. The results suggest that the AMPPI in

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healthy females is steadily trait-based and not affected by fluctuations of dopamine and/or exogenous

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hormones.

This study confirms that the Val158Met polymorphism affects PPI in younger women. Previous

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studies using traditional unintentional PPI paradigm have shown that in healthy male adults, Met/Met

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carriers exhibit the highest PPI, Val/Met carriers the intermediate, and Val/Val carriers the lowest (Roussos, et al., 2008). The results of this study appear to support the view that the high dopamine

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level in the prefrontal cortex enhances PPI (Giakoumaki, et al., 2008; Roussos, et al., 2008). Higher

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dopamine activities in the prefrontal cortex may indicate a better protective function for reducing disruptive influences to the processing of prepulse signals.

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Moreover, the results of this study are also consistent with former studies showing that PPI is

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affected by the menstrual cycle (Jovanovic, et al., 2004; Kumari, et al., 2010). It has been suggested that estrogen may be the leading cause of PPI reduction because males have more PPI than females (Aasen, et al., 2005; Swerdlow, et al., 1993, 1997). However, consistent with a previous study reporting that PPI reduction is the most notable during the period corresponding to mid-luteal elevations of both estrogen and progesterone (Swerdlow, et al., 1997), we found that in the secretory-phase (when the estrogen level rises again and the progesterone level gradually peaks), females exhibited the lowest PPI. Some recent studies have also shown that progesterone in fact

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plays a critical role in reducing PPI compared to estrogen (Kumari, et al., 2010) and lower PPI has been found in healthy pregnant women compared to healthy non-pregnant controls (Comasco, et al., 2015). Thus, the PPI change along the menstrual cycle may be regulated by fluctuations in both estrogen and progesterone. One limitation of this study is that the hormone levels in the uterine cycle

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was not directly measured in each of the participants

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Although we did not find significant interactions of the menstrual cycle and the COMT

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Val158Met polymorphism on either the PPI or the AMPPI, the results of this study showed that the female participants with the Val/Val genotype and the female participants with the other two

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genotypes exhibited different patterns in PPIPSS and PPIPSC fluctuations along the menstrual cycle:

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the former showed a marked decline in PPIPSS and PPIPSC from the menstruation phase, through the proliferative phase, to the secretory phase, but the latter showed a tendency with an inverted

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U-shape-like variation in PPIPSS and PPIPSC along the three phases. The results suggest that higher

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dopamine activity in the prefrontal cortex may antagonize or reverse the PPI reduction caused by the elevated estrogen level in the proliferative phase.

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Previous studies have also shown that dopamine antagonists applied to the orbital prefrontal

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cortex reduce PPI (Zavitsanou, et al., 1999). Administration of dopamine agonists or surgical/chemical lesions of the basal ganglia (e.g., the striatum or the nucleus accumbens) decrease PPI in rats (Swerdlow, et al., 1990; Zavitsanou, et al., 1999). Combined with the results of this study, it can be suggested that the proliferative-phase-related higher estrogen level (therefore higher dopamine level) in certain subcortical regions may cause PPI reduction, but the higher prefrontal dopamine level may protect women with the COMT Val158Met Met allele from the PPI reduction. Schizophrenia has been hypothesized to be related to decreased dopamine transmission in the

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prefrontal cortex and increased dopamine activity in the striatum and nucleus accumbens (Howes and Kapur, 2009). It has been found that people with schizophrenia carrying the Val158Met Met/Met genotype exhibit elevated PPI levels compared to the other two genotype carriers (Quednow, et al., 2010), suggesting that the Val158Met Met allele can also protect schizophrenics from PPI reduction

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compared to the Val158Met Val allele. However, the estrogen interaction with dopamine

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transmission in schizophrenia are more complicated (for reviews see Gogos, et al., 2015; Kulkarni, et

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al., 2012). For example, evidence is accumulating that estrogen exerts a protective effect against schizophrenia (Kulkarni, et al., 2012). Estrogen may exert this effect via an interaction with

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dopamine (Hafner, et al., 1991), brain-derived neurotrophic factor (Pillai, 2008), and other

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neurotransmitters (Gogos, et al., 2015). Females with schizophrenia have significantly less PPI compared to normal females (Braff, et al., 2005). However, severe positive symptoms seem to be

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more correlated to PPI deficits in male patients than in female patients (Matsuo, et al., 2016). Thus,

of the effects of estrogen.

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the PPI deficit may not be a sensitive, specific, or reliable index for schizophrenia in females because

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This study reveals that for healthy younger-adult females, the PPI magnitude was significantly

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higher under the PSS condition than that under the PSC condition. The results are consistent with those of previous human studies and animal studies showing that PSS between a prepulse stimulus and a noise masker enhances PPI by further facilitating selective attention to the prepulse (Du, et al., 2009, 2010, 2011; Lei, et al., 2014, 2018; Wu, et al., 2016, 2018; Yang, et al., 2017). However, this study also reveals that the precedence-effect induced attentional modulation effect of PPI (AMPPI) is not sensitive to either the menstrual cycle or the COMT Val158Met polymorphism in healthy younger-adult females, even though the attentional modulation of PPI based on shifts between the

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PSS condition and the PSC conditions can effectively distinguish schizophrenics from healthy controls (Yang et al., 2017). Thus, in healthy women, the AMPPI may be more steadily trait-based and less susceptible to the changes in hormones, such as estrogen and/or progesterone. Future studies need to investigate whether the AMPPI is affected by the menstrual cycle in adult females with

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schizophrenia.

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Previous studies using traditional PPI paradigms have reported that the baseline startle is greater

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in rs4680 Met/Met carriers than that in Val/Val carries (Giakoumaki et al., 2008; Roussos et al., 2009). In this study, we did not find the significant difference in the baseline startle (pulse-alone

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startle response) between the rs4680 polymorphism groups. However, there was a trend that the PAS

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magnitude seems to be greater in Met/Met carriers than other carriers (see Figure 2A). In addition, we found that participants in the proliferative phase had more PAS magnitude than those in the other

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two menstrual groups, which is consistent with the results of a previous study (n = 111) showing that

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a higher acoustic startle response in mid-menstrual-cycle that is probably due to heightened auditory sensitivity (Armbruster, et al., 2014). Some other studies with a smaller sample size (n < 30) did not

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find difference in startle magnitude across menstrual cycle (Kask et al., 2008; Kumari et al., 2010).

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In the future, more studies are needed to examine the results. In summary, this study reveals that the PPI magnitude (under the condition when the prepulse is actively attended), but not the AMPPI, is susceptible to both the menstrual cycle and the Val158Met polymorphisms in healthy younger-adult females. The PPI magnitude decreases with time in a menstrual cycle in the Val158Met Val/Val carrier participants, but this changing pattern along the three phases of the menstrual cycle does not occur in participants with either the Met/Val or the Met/Met genotype. This study indicates that, in healthy younger-adult females, the PPI is vulnerable

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to changes of ovarian hormones and the COMT Val158Met Polymorphism, but the attentional modulation of PPI appears to be more steadily trait-based. In the future it is necessary and important to investigate whether the attentional modulation of PPI is useful for improving the diagnosis of

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schizophrenia in younger-adult females.

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ACKNOWLEDGMENT This work was supported by the Beijing Municipal Science & Tech Commission (Z161100002616017), the National Natural Science Foundation of China (81601168, 31771252),

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and the Beijing Advanced Innovation Center for Genomics at Peking University.

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476-486. doi: 10.1016/j.neuropharm.2006.08.016

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FIGURE LEGENDS Figure 1. Experimental design for measuring PPI magnitudes. Each block comprised of 20 trails [8 trials for startle-alone (red), 6 trials for PPIPSS (blue), and 6 trials for PPIPSC (green)] which were

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presented in pseudorandom order. PSC, perceived spatial co-location; PSS, perceived spatial

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separation.

Figure 2. (A) The group-mean magnitudes of PPI (across 177 participants) under the PSS condition

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were significantly higher than that under the PSC condition. (B) The group-mean and distribution of (C) The group-mean

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PPI (averaged across PPIPSS and PPIPSC) in the 3 Val158Met genotype groups.

magnitude of pulse-only startle response and (D) AMPPI (PPIPSS minus PPIPSC; i.e., PPI-change) in

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the 3 Val158Met genotype groups. AMPPI, attentional modulation of PPI. Error bars indicate the

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standard errors of the mean.

Figure 3. (A) The magnitude of pulse-only startle response and (B) the group-mean PPI and

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distribution of PPI (averaged across PPIPSS and PPIPSC) in the 3 phases of the menstrual (uterine) cycle. (C) Both PPIPSS and PPIPSC decreased monotonously from the menstruation phase, through the proliferative phase, to the secretory phase. (D) PPI change (AMPPI) in the 3 phases of the menstrual cycle. MEN, menstruation; PRO, proliferative phase; SEC, secretory phase. *p < 0.05; **p < 0.01.

Figure 4. Interaction effects among the auditory perceptual attention conditions (PSS, PSC), the

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Val158Met genotype groups (Met/Met + Met/Val, Val/Val), and the 3 phases of menstrual cycle (menstruation period, proliferative phase, secretory phase) on PPI. Note that the estrogen level peaks at the proliferative phase and maintains high in the secretory (luteal) phase, whereas the progesterone

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level increases and peaks in the luteal (secretory) phase (Silverthorn, 2016). *p < 0.05; **p < 0.01.

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Table 1. Distribution of the Val158Met (rs4680) Genotype Groups and the Menstrual Cycle Groups. N

Age

Startle Magnitude (μV±SD)

PPIPSS

PPIPSC

A

(%

(years ± SD)

Pulse-alone

PSS

PSC

(% ± SD)

(% ± SD)

13 (7.3%)

19.1 ± 1.1

122.3 ± 63.5

38.9 ± 44.7

36.6 ± 30.3

68.9 ± 15.5

68.7 ± 20.0

0.2

(AG)

59 (33.3%)

19.0 ± 0.9

106.4 ± 57.7

37.3 ± 27.7

35.6 ± 25.8

59.8 ± 23.9

58.4 ± 25.2

1.

(GG)

105 (59.3%)

18.9 ± 0.8

105.6 ± 60.1

49.7 ± 39.8

46.0 ± 39.5

52.3 ± 26.8

48.9 ± 25.1

3.

ation

43 (24.3%)

19.1 ± 0.9

99.1 ± 52.7

32.1 ± 23.7

29.7 ± 19.9

65.5 ± 20.0

62.3 ± 23.6

3.

tive phase

36 (20.3%)

19.1 ± 0.8

133.5 ± 69.4

50.4 ± 36.4

48.7 ± 41.7

58.4 ± 28.5

56.4 ± 24.0

2.

y phase

98 (55.4%)

18.9 ± 0.8

100.9 ± 56.0

48.2 ± 40.6

51.0 ± 25.6

48.6 ± 25.7

2.

RI

PT

(AA)

NU

SC

Cycle

44.7 ± 36.6

AC

CE

PT E

D

MA

PPIPSS, prepulse inhibition under the listening condition with perceived spatial separation; PPIPSC, prepulse inhibition under the listening condition with perceived spatial co-location. AMPPI, attentional modulation of PPI (i.e., PPIPSS - PPIPSC).

ACCEPTED MANUSCRIPT 37

(1) In healthy younger-adult females, PPI is vulnerable to ovarian hormones changes. (2) Their PPI is modulated by both Val158Met polymorphism and menstrual cycle. (3) Their attentional modulation of PPI is not affected by menstrual cycle.

PT

(4) Their attentional modulation of PPI is not affected by Val158Met polymorphism.

AC

CE

PT E

D

MA

NU

SC

RI

(5) Attentional modulation of PPI is more steadily trait-based.

Figure 1

Figure 2

Figure 3

Figure 4