Psychiatry Research 216 (2014) 418–423

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Effects of melatonin on prepulse inhibition, habituation and sensitization of the human startle reflex in healthy volunteers Emilia K. Lehtinen a,b,c, Ebru Ucar a,b,c, Birte Y. Glenthøj a,b,d, Bob Oranje a,b,d,e,n a Center for Neuropsychiatric Schizophrenia Research (CNSR), Copenhagen University Hospital, Psychiatric Center Glostrup, Ndr. Ringvej 29-67, DK-2600 Glostrup, Denmark b Center for Clinical Intervention and Neuropsychiatric Schizophrenia Research (CINS), Copenhagen University Hospital, Psychiatric Center Glostrup, Denmark c Faculty of Pharmaceutical Sciences, University of Copenhagen, Denmark d Faculty of Health Sciences, Dept. of Neurology, Psychiatry, and Sensory Sciences, University of Copenhagen, Denmark e NICHE, Department of Psychiatry, Brain Center Rudolf Magnus, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands

art ic l e i nf o

a b s t r a c t

Article history: Received 7 December 2012 Received in revised form 13 February 2014 Accepted 19 February 2014 Available online 28 February 2014

Prepulse inhibition of the startle reflex (PPI) is an operational measure of sensorimotor gating, which is demonstrated to be impaired in patients with schizophrenia. In addition, a disruption of the circadian rhythm together with blunted melatonin secretion is regularly found in patients with schizophrenia and it is theorized that these may contribute to their attentional deficits. The aim of this study was to assess the effects of acute melatonin on healthy human sensorimotor gating. Twenty-one healthy male volunteers were administered melatonin or placebo after which their levels of PPI were assessed. Melatonin significantly reduced startle magnitude and ratings of alertness, but did not influence PPI, nor sensitization and habituation. However, when taking baseline scores in consideration, melatonin significantly increased PPI in low scoring individuals while significantly decreasing it in high scoring individuals in low intensity prepulse trialtypes only. In addition, subjective ratings of alertness correlated with PPI. The results suggest that melatonin has only minor influences on sensorimotor gating, habituation and sensitization of the startle reflex of healthy males. The data do indicate a relationship between alertness and PPI. Further research examining the effects of melatonin on these processes in patients with schizophrenia is warranted. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Sensorimotor gating PPI Schizophrenia Electromyography Orbicularis oculi muscle

1. Introduction Deficits in attention and perceptual awareness have long been observed in schizophrenia and, as postulated by McGhie and Chapman (1961), may be due to a malfunction in the neural mechanism that filters sensory information from the environment. Following extensive clinical observation, they theorized that early filtering, or gating, of external sensory information is critical in safeguarding the brain from irrelevant stimuli, allowing processing capacities to be directed only toward the most salient input. Such inhibitory processes would be particularly important for the ability to draw and maintain attention at will, something which is since long found to be impaired in patients suffering from schizophrenia (Kraepelin, 1913; Bleuler, 1937). Sensory and sensorimotor gating deficits are assumed to lead to an over flooding of the higher brain regions, which in turn, is thought to result in n Corresponding author at: Center for Neuropsychiatric Schizophrenia Research (CNSR), Copenhagen University Hospital, Psychiatric Center Glostrup, Ndr. Ringvej 29-67, DK-2600 Glostrup, Denmark. Tel.: þ 45 386 40828; fax: þ45 432 34653. E-mail address: [email protected] (B. Oranje).

http://dx.doi.org/10.1016/j.psychres.2014.02.030 0165-1781 & 2014 Elsevier Ireland Ltd. All rights reserved.

cognitive disturbances and ultimately to psychosis (e.g. Perry et al., 1999). In support of this hypothesis, reduced sensorimotor gating efficiency can also methodically be demonstrated in schizophrenia. One type of operational measure that has been studied in this respect is the prepulse inhibition (PPI) of the startle reflex, which is consistently found to be impaired in patients with schizophrenia (e.g. Braff et al., 1978, 1992). The startle reflex, which is regulated by a relatively simple neural circuit (Koch, 1999), is a universal involuntary defense mechanism to a sudden intense stimulus (usually auditory) and can be inhibited, if the startling stimulus is closely preceded by a weaker stimulus (prepulse) (Graham, 1975). Inhibition of the startle reflex, which is a form of sensorimotor gating, is thought to exist due to the still on-going processing of the information derived from the prepulse, as the two stimuli are maximum 500 ms apart, which is enough to exceed the capacity of the brain to process, or react to, both stimuli (Graham, 1975). Frequently, and in parallel to sensorimotor gating, an individual's sensitization and habituation processes are assessed. Sensitization represents the exponential increase of a response to the same and initially new stimulus, whereas habituation is the opposite, representing

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attenuation of a response to a repetitive stimulus over time. Sensitization typically takes place over the first 2–3 trials of a startle-eliciting stimulus block, followed by a gradual habituation over the remaining trials. There are inconsistent reports as to whether habituation is impaired in patients with schizophrenia; some studies report a reduced habituation in patients compared to healthy controls (Geyer and Braff, 1982; Braff et al., 1992; Ludewig et al., 2003), while others find no such impairment (Mackeprang et al., 2002; Quednow et al., 2006). The literature regarding sensitization is sparse and conflicting with one publication reporting an increased sensitization in medicated patients with schizophrenia (Meincke et al., 2004), whilst another study found a trend for a reduced sensitization in antipsychotic-naïve first episode patients (Aggernaes et al., 2010). Some atypical antipsychotics, such as quetiapine and clozapine, have been demonstrated to improve PPI together with alleviating psychotic symptoms in some patients with schizophrenia (e.g. Kumari et al., 1999, 2000; Oranje et al., 2002; Wynn et al., 2007; Aggernaes et al., 2010), whereas others have not (e.g. Mackeprang et al., 2002; Duncan et al., 2003a, 2003b). However, the effects of atypical as well as typical antipsychotics on the disabling information-processing disturbances as well as psychopathology are unsatisfactory and all antipsychotics have adverse effects contributing to non-compliance and discontinuation of treatment. Accordingly, there is an urgent need for new medical treatments. Furthermore, following antipsychotic discontinuation, patients who also suffer from sleep disturbances are found to be at a greater risk for worsening of psychotic symptoms (Chemerinski et al., 2002; Poulin et al., 2003; Benson, 2006). Sleep deficits are frequently observed in patients with schizophrenia and are often part of the prodromal phase preceding relapse, even when the patients are on medication (Chemerinski et al., 2002). This observation has drawn growing interest in the role of sleep in the pathophysiology of schizophrenia, as reestablishing a healthy circadian rhythm together with its restorative processes is believed to improve clinical outcome. There is evidence suggesting that the cause of these disturbances in sleep architecture is due to low circulating levels of the endogenous sleep promoter melatonin, which functions as the regulator of the circadian sleep-wake cycle: several investigations have detected blunted nocturnal melatonin levels in drug-free as well as medicated patients (Fanget et al., 1989; Monteleone et al., 1992). Although antipsychotic treatment is able to treat some sleep deficits, it does not restore the disturbed melatonin production (Robinson et al., 1991; Monteleone et al., 1997; Suresh Kumar et al., 2007; Anderson and Maes, 2012). A case study by Afonso et al. (2010) found discrepant nocturnal melatonin profiles in a monozygotic twin pair discordant for schizophrenia, suggesting that the reduced melatonin secretion may be a consequence of the pathological process of the disorder and hence may not solely be genetic. Preclinical studies have shown that melatonin receptor knockout mice exhibit reduced sensorimotor gating (Weil et al., 2006). Although a beneficial effect of melatonin on PPI is likely to be mediated by an improvement of a patient's circadian rhythm or sleep composition, it cannot be ruled out that melatonin may have a direct effect on PPI. We previously reported the effects of melatonin on another widely believed measure of sensory filtering of information, i.e. P50 suppression, in a group of healthy males (Ucar et al., 2012), and now report on its effect on PPI, habituation and sensitization in this same group of subjects. To our knowledge, there are no previous reports on the effects of exogenous (nor endogenous) melatonin on the psychophysiology of attention or PPI in humans. However, gaining an understanding of the role of melatonin and its alterations in the neural processes involving attention may be fruitful in order to identify core biological mechanisms underlying the psychopathology of psychosis. Moreover, this knowledge can form a basis for more efficient medical treatments strategies for schizophrenia and for psychotic disorders

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in general. Hence, the aim of the present study was to investigate the acute effect of sustained-release melatonin on PPI of the startle reflex in a group of healthy male volunteers. Based on the above described literature, we expected melatonin to improve sensorimotor gating. 2. Methods The study was approved by the Ethics Committee of the Capital Region, Copenhagen, in regard to the ethical principles of the Declaration of Helsinki II. 2.1. Subjects Twenty-one healthy male volunteers aged between 18 and 30 years were recruited from the capital region of Copenhagen by web advertisement. All included participants were in good physical and mental health as assessed by anamnesis. Exclusion criteria were current medication, history of neurological illness, psychiatric illness in a first degree relative, alcohol or drug abuse and receiving any experimental medication within 30 days of the study start. All volunteers were informed about the experiment in detail and provided a written informed consent before enrollment to the study. Subsequently, they were interviewed with the Schedules for Clinical Assessments in Neuropsychiatry (SCAN) to ensure absence of psychiatric illness and rendered a urine sample, which was evaluated for the content of opiates, cocaine, amphetamine and cannabis. The volunteers were also screened for hearing deficits (at frequencies of 500, 1000 and 6000 Hz and intensities of 20 and 40 dB(a)) and individuals not able to perceive tones under 20 dB(a) were excluded: none of the volunteers had to be excluded. Furthermore, in accordance to our laboratory standards, we defined nonresponders as those subjects who scored less than 20 mV on average on the pulse alone trials; also on these grounds none of the subjects had to be excluded. The mean age of the included participants was 25 (S.D.: 3.0) years and their mean BMI was 23 (S.D.: 1.90). Of the 21 participants, three were tobacco smokers. 2.2. Experimental design In a double-blind, randomized yet balanced, crossover experiment, participants were administered melatonin (4 mg Circadins controlled-release) or placebo (folic acid also known as vitamin B9) in a white opaque capsule on two occasions separated by a minimum interval of 1 week. None of the volunteers had participated in a psychophysiological assessment before. Volunteers were instructed to fast from 11:00 PM the preceding night and to sleep a minimum of 7 h before arriving at the Center for Neuropsychiatric Schizophrenia Research, Glostrup, at 8:00 AM. To avoid acute or withdrawal effects of caffeine and nicotine, subjects were requested to refrain from smoking 1 h prior and from caffeinated drinks, 2 h prior to test start. They had also been asked not to consume alcohol the preceding day. The capsule containing either melatonin or placebo was administered at 8:30 AM. We choose the morning because we were only interested in the acute, direct effects of melatonin on our dependent variables, with as little influence as possible of endogenous levels of melatonin: the levels of endogenous melatonin are low after waking in the morning (Pacchierotti et al., 2001). At 9:00 AM participants were accompanied to a soundproof, electrically shielded experimental room and signal recording was started 90 min after administration of the capsule in order to assure maximum plasma concentration of melatonin (DeMuro et al., 2000). The subjects were subsequently tested in the Copenhagen Psychophysiological Test Battery (CPTB). Besides a PPI paradigm, the CPTB consists of a P50 suppression, selective attention and mismatch negativity paradigm. The CPTB has recently been validated in, amongst others, a large cohort of antipsychotic-naïve and first-episode patients with schizophrenia (Oranje et al., 2008; Jensen et al., 2008; Aggernaes et al., 2010). For reasons of comprehensiveness, the current manuscript will only report on PPI, sensitization and habituation of the human startle reflex. The results of melatonin on P50 suppression were published elsewhere (Ucar et al., 2012) and so will be the results of the other CPTB-tests. Subjective ratings with a visual analog scale (VAS) of alertness were assessed prior to capsule administration and prior to PPI assessment (Bond and Lader, 1974). 2.3. Assessment of PPI, habituation and sensitization The method has been described previously (Aggernaes et al., 2010; Oranje and Glenthoj, 2013). Briefly, subjects were seated in a comfortable armchair in a room with a sound level o 40 dB and situated adjacent to the control room. They were instructed to sit still, to keep their eyes fixed on a spot on the wall directly in front of them and were asked to stay awake. The assessment of PPI and habituation started with 5 min of acclimation to a background noise (70 dB(a) white noise) after which three experimental blocks of stimuli were superimposed on the background noise. Blocks 1 and 3 were used to assess habituation of the acoustic startle reflex. The two blocks were identical and consisted of eight pulse-alone trials (white noise with an intensity of 115 dB(a), and a duration of 20 ms, instant

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rise and fall) with randomized inter-trial intervals between 10 and 20 s. Block 2 consisted of 50 trials presented in a pseudo-randomized order (no trialtype directly followed the same trialtype) to assess PPI. Since it is known that prepulse intensity and inter-stimulus intervals can affect the levels of PPI (Braff et al., 2001), we chose to use two levels of each, i.e. prepulse intensities of 6 and 15 dB(a) (white noise, 20 ms duration) above background, and stimulus onset asynchronies (SOA) of 60 and 120 ms. The inter-trial intervals were randomized between 10 and 20 s. Randomized across the session, 10 pulse-alone and 10 of each prepulse-pulse combination (60 ms/76 dB(a), 60 ms/85 dB(a), 120 ms/76 dB(a), 120 ms/85 dB(a)) were presented. All auditory stimuli were presented by a computer running Presentations (Neurobehavioral Systems Inc., USA) software (soundcard: Creative Extreme Player, Creative Technology Ltd, Singapore) and presented binaurally through stereo insert earphones (Eartone ABR, 1996–2008 Interacoustics A/S, Denmark, C and H Distributors Inc., USA). The software and hardware settings were calibrated by means of an artificial ear (Brüel and Kjær, type 2133, Odin Metrology Inc., USA). Prepulse inhibition assessment took 25 min. Following offline filtering of the data between 25 and 250 Hz, startle amplitude was scored as the highest absolute amplitude in the time-interval 20–100 ms after the startleeliciting pulse, while PPI was expressed as (1  PP/PA)  100%, where PP is the average startle amplitude to prepulse-pulse trials, and PA is the average startle amplitude to pulse-alone trials. Sensitization appeared maximum from trial 1 to trial 2 in block 1. Habituation was studied starting from trial 3 of block 1 until the end of block 3. Please note: The first two trials of block 1 were not included, since they may have been influenced by the process of sensitization. Furthermore, the startle amplitudes to pulse-alone trials in block 2 were not included in the analysis of habituation, since they might have been influenced by PPI effects. Hence, 14 habituation trials were included (6 of block 1, combined with the 8 of block 3). 2.4. Signal recording The eye-blink component of the acoustic startle response was measured by recording electro-myographic (EMG) activity from the right m. orbicularis oculi. Two electrodes were placed under the right eye. The first of these two electrodes was aligned with the pupil, the other positioned just laterally towards the outer canthus of the eye. The EMG recordings were assessed with BioSemis hardware (Biosemi, Amsterdam, the Netherlands). Sampling started immediately before the presentation of an experimental block and lasted until the end of it. All signals were digitized online by a computer at a rate of 2048 Hz, and a low-pass setting of 1/5 of the AD rate. From the VAS rating only the factor “alertness” was taken into account. 2.5. Statistical analysis All statistical analyses were performed with Statistical Package for the Social Sciences (SPSS; version 11.0, SPSS, Chicago, IL). All data were normally distributed (Kolmogorov–Smirnov test), and were therefore analyzed parametrically. Treatment effects on raw amplitude data were analyzed by repeated measures analysis of variance (ANOVA) with within-factors “treatment” (melatonin or placebo) and “trialtype” (76 dB/60 ms, 76 dB/ 120 ms, 85 dB/60 ms, 85 dB/120 ms and pulse alone trials). Treatment effects on PPI were analyzed by repeated measures ANOVA with within-factors “treatment”, “prepulse intensity” (76 dB or 85 dB) and “SOA” (60 ms or 120 ms). Sensitization was analyzed by repeated measures ANOVA with within factors “treatment” and “trial” (amplitude as a response to trial 1 of block 1, and amplitude as a response to trial 2 of block 1). Habitation was analyzed by repeated measures ANOVA with within factors “treatment” and “time”. To avoid alpha-inflation, Student's t-tests were only used whenever the ANOVAs indicated significant results.

Fig. 1. Mean percent PPI for all four different prepulse-pulse trials (i.e. 85 dB/ 120 ms, 85 dB/60 ms, 76 dB/120 ms and 76 dB/60 ms trial) for both treatment sessions. No effect of treatment was found.

interaction effect [F(1,19) ¼0.03; p ¼0.86]. In addition, no order effect was found in the data: those subjects who were administered placebo first followed by melatonin treatment did not score different percentages PPI in any of the 4 trialtypes compared to those who received the treatment in reversed order (p 40.52), nor did their amplitude to pulse alone or prepulse-pulse trials differ (p 40.32). In order to test the influence of melatonin on baseline (placebo session) percentage PPI scores, we split the subjects into two groups for each of the four trialtypes: those with high baseline scores, and those with low, based on their median scores. These post-hoc tests indicated no significant effect of baseline score in the high intensity prepulse trials (85/120 trials: [F(1,18) ¼2.18; p¼ 0.16], 85/60 trials: [F(1,18) ¼ 4.16; p ¼0.06]), but did indicate significant effects of baseline in the low intensity trialtypes: 76/ 120 trials: [F(1,18) ¼4.47; p ¼0.049], 76/60 trials: [F(1,18) ¼15.90; po 0.001]. Further testing revealed no significant effect of melatonin on either high or low scoring individuals in the 76/120 trialtype (p4 0.07). However, in the 76/60 trialtype melatonin significantly decreased PPI in the high scorers (t¼2.30, d.f. ¼ 9, p¼ 0.047), due to significantly decreasing amplitudes to pulse alone trials (t ¼3.11, d.f. ¼9, p¼ 0.013) while not affecting amplitudes to prepulse-pulse trials (t ¼0.15, d.f. ¼ 9, p¼ 0.89); in the low scorers of this trialtype, melatonin significantly increased PPI (t¼ 3.33, d.f. ¼ 9, p ¼0.009), albeit without significantly affecting amplitude to either pulse alone or prepulse-pulse trials (p 40.07).

3. Results

3.2. Sensitization and habituation

3.1. PPI

Sensitization, defined as the increase in startle amplitude from trial one to two in the first block, tended to show a significant effect [F(1,19) ¼ 4.25; p¼ 0.053] but did not reach statistical significance. The average percentage increase in the placebo session was 145% (S.D.: 120), while in the melatonin session it was 123% (S.D.: 82). Neither an effect of treatment was found [F(1,19) ¼0.68; p¼ 0.42], nor an effect of treatment-order (p 40.43). Fig. 2 presents the startle magnitudes under the two treatment conditions in regard to habituation blocks 1 and 3. The ANOVA showed a main effect of time [F(13,7)¼ 4.42; p ¼0.028], indicating that amplitudes indeed reduced over time (habituation). In addition to this standard result, a significant main effect of treatment [F(1,19) ¼5.64; p ¼0.028] was found, indicating that the subjects displayed reduced startle amplitudes under the melatonin treatment compared to placebo. However, no interaction between

Analyses of variance of raw amplitude data (startle magnitude) revealed a main effect of trials indicating that PPI indeed occurred [F(4,16) ¼ 11.48; p o0.001]. Moreover, a tendency for a main effect of treatment was found [F(1,19) ¼ 3.65; p ¼0.071] that did not reach statistical significance, indicating that melatonin tended to reduce all amplitudes, regardless of trialtype. The ANOVA on percentage PPI revealed only expected results: significant main effects of prepulse intensity [F(1,19) ¼ 40.43; p o0.001] and SOA [F (1,19) ¼ 23.23; p o0.001], as well as a significant first order interaction between intensity and SOA [F(1,19) ¼8.07; p ¼0.01], indicating that higher prepulse intensities and longer SOAs elicit more PPI (Fig. 1). No treatment effects were found in the percentage PPI data, i.e. neither a main effect [F(1,19) ¼1.44; p¼ 0.25], nor an

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Table 1 Correlation alertness with PPI. Trialtype

Average %PPI (S.D.)

Correlation with VAS (r)

Significance (p)

85 dB/120 ms 85 dB/60 ms 76 dB/120 ms 76 dB/60 ms

53.24 38.10 40.48 16.76

0.239 0.518 0.191 0.349

0.297 0.016 0.408 0.121

(30.2) (31.6) (25.2) (32.9)

The table shows the average percentage PPI per trialtype in the placebo session, and its correlation to the baseline score of the VAS alertness scale, averaged over both sessions. Only the percentage PPI in response to the 85 dB/60 ms trialtypes showed a significant correlation.

Fig. 2. Startle amplitudes for the 8 trials of blocks 1 and 3 (block 2 consisted of PPI trials). The gradual decrease in amplitudes (habituation) was apparent for both placebo and melatonin sessions (please note: the first two trials of block one were not included in the habituation analyses, due to a possible influence of sensitization). Melatonin significantly reduced the startle amplitudes compared to placebo (treatment main effect), while no significant treatment  time interaction effect was found. Plac: placebo; Mel: melatonin; B: block

statistical significance (t¼3.38, d.f.¼ 20, p¼ 0.003). Furthermore, a significant correlation was found between the level of gating in the 85dB/60 ms trials and the average level of alertness just after arrival (before intake of the placebo or melatonin containing capsule) (r¼0.52; p¼ 0.02, see Fig. 3 and Table 1); Please note: in order to increase power, the VAS-scores on arrival (before intake of the capsule) of the two sessions were combined for this specific analysis (these scores did not differ significantly from each other (t¼ 0.66, d. f.¼20, p¼ 0.52)).

4. Discussion

Fig. 3. Scatterplot, showing a significant positive correlation between the level of alertness before capsule intake and the percentage prepulse inhibition in the 85 dB/60 ms trialtype.

treatment and time was found [F(13,7)¼ 0.89; p ¼0.60], indicating that melatonin did not influence the level of habituation, nor an effect of treatment-order was found (p4 0.07). 3.3. Subjective ratings The mean composite score for alertness of the VAS in the placebo session was 5.84 (S.D.: 0.55) before capsule intake and 90 min later, just before testing this score was 5.67 (S.D.: 0.80). The mean VAS score of the melatonin session before capsule intake was 5.74 (S.D.: 0.81) and 90 min later, just before testing the score was 5.17 (S.D.: 0.94). ANOVA of these scores revealed a main effect of time [F(1,20)¼ 9.05, p ¼0.007], indicating that the subjects were more alert before the intake of the capsules (8:30 AM) compared to the time just before the assessments took place (10:00 AM), regardless of treatment. In addition, a significant first order interaction between treatment and time was observed [F(1,20)¼ 5.60; p ¼0.028], indicating that the subjects were significantly less alert following treatment with melatonin than following treatment with placebo. In fact, further testing with Student's t-tests revealed that only the reduction of alertness in the melatonin session reached

To our knowledge, this is the first study investigating the effects of exogenous melatonin on healthy human sensorimotor gating. Contrary to what was expected, the main findings of this study suggest that melatonin had only minor influence on PPI. Furthermore, melatonin reduced startle magnitude significantly in the habituation trials. This effect of melatonin was also found in the PPI trials, where melatonin had a non-significant tendency (p ¼0.07) to reduce raw amplitudes in the PPI trials in general, which reached statistical significance in the pulse alone trials only in those individuals with high baseline PPI on the 76/60 trialtype. The fact that melatonin significantly reduced PPI in these high scoring subjects in the 76/60 trials by reducing the amplitude to pulse alone trials is more reflecting a sensory registration issue, then a pure reduction in sensorimotor gating. In addition, melatonin reduced subjective ratings of alertness, while no treatment effects on the subjects' levels of sensitization and habituation were found. It is known that melatonin acts via three melatonin receptors M1, M2 and M3 (Reppert et al., 1994, 1995; Nosjean et al., 2000), but is also found to increase dopaminergic D1/D2 activity (Binfare et al., 2010) as well as decrease noradrenergic activity (Mitchell and Weinshenker, 2010). Although the neurochemical mechanism by which PPI is modulated in humans is still not fully understood, there is substantial evidence for involvement of both dopamine D2 receptors as well as noradrenergic receptors in PPI: antagonists of dopamine D2 receptors result in decreased PPI in healthy volunteers (Abduljawad et al., 1998; Kumari et al., 1998; Oranje et al., 2004; Csomor et al., 2008) while the effects of D2-agonists are less consistent, some studies reporting a decrease in PPI (Abduljawad et al., 1998; Schellekens et al., 2010), sometimes depending on the initial PPI scores (Bitsios et al., 2005), while others reporting no effects on PPI of healthy volunteers (Swerdlow et al., 2002a; Swerdlow et al., 2002b). Studies from our own laboratory showed that a relatively high increase of noradrenergic activity in healthy humans (50 mg desipramine) potently reduces PPI (Oranje et al., 2004), presumably by activating noradrenergic-α1 receptors, while mild doses (25–75 mg) of clonidine were found to normalize

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PPI deficits in chronic patients with schizophrenia, presumably by activating the more sensitive noradrenergic-α2 receptors (Oranje and Glenthoj, 2013). The current finding that melatonin had only limited influence on PPI may therefore indicate that activation of melatonergic receptors has no effect on PPI, at least, not in healthy volunteers. In rats pinealectomy disrupts PPI, while melatonin treatment reverses that disruption; however, similar to our study, melatonin showed no effects on PPI of the control rats (Uzbay et al., 2013). Given the literature from above, if melatonin indeed had increased dopaminergic D1 and D2 activity as suggested by (Binfare et al., 2010), then it would have increased PPI. In contrast, if it would have decreased noradrenergic activity (Mitchell and Weinshenker, 2010), then it would have decreased normal levels of PPI. These opposite effects may have canceled each other out, resulting in no effect on PPI at all. In line with previous research involving both animals and humans demonstrating that melatonin inhibits startle-related motor responses (Datta et al., 1981; Schachinger et al., 2008) and decreases subjective ratings of alertness (Lieberman et al., 1984; Dollins et al., 1993), the current data indicated that melatonin reduced startle magnitude and subjective ratings of alertness in the participants compared to placebo treatment. This result is likely to be attributable to the sedative and anxiolytic actions of the hormone, as the startle response is known to be potentiated by fear and or anxiety (Davis, 1993). Various other sedative compounds, such as benzodiazepines (Abduljawad et al., 1997; van Luijtelaar, 2003) but also the atypical antipsychotics clozapine (Graham et al., 2001) and quetiapine (Graham et al., 2004), have demonstrated to reduce raw startle amplitudes in healthy subjects, whereas opposite effects were observed with the anxiogenics amphetamine (Davis et al., 1975; Swerdlow et al., 1990) and yohimbine (Davis and Astrachan, 1981; Morgan et al., 1993). Alternatively as stated above, melatonin increases D1/D2 activity but decreases noradrenergic activity. It is therefore also possible that the reduction in startle magnitude by melatonin is induced via its actions on (one of) these neurotransmitter systems. The level of alertness at first contact on a test day was found to correlate with the level of gating to the 85 db/60 ms trialtype. However, alertness assessed immediately prior to testing did not correlate with PPI. This may be due to the fact that the participants were affected by the course of the experiment or simply because the analysis of the second time point has less power (based on 21 assessments) than the analysis of the first assessment point (based on 42 assessments).Moreover, shorter SOAs ( o60 ms) are thought to elicit purely automatic and pre-attentive processes, whereas longer SOAs (4120 ms) may allow for attentional and conscious processes to take place, which could account for the difference (Dawson et al., 1993; Filion et al., 1993). In line with Schachinger et al. (2008) melatonin did not affect the participants' habituation processes. Furthermore, we did not observe any effect on sensitization. Habituation and sensitization represent forms of non-associative learning, as the startling stimuli are not regarded to be associated with any particular biological event. Although melatonin was found to affect learning and memory in a preclinical study (Rawashdeh et al., 2007), it does not seem to influence these very basic types of learning processes. There are limitations to the study. As we only assessed healthy male subjects given an acute dose of melatonin, the results may not extrapolate to patients with schizophrenia who, presumably, will be given chronic doses of melatonin. Moreover, we assessed PPI and startle amplitude following melatonin administration in the morning, which physiologically is a rather unnatural time to investigate them, as melatonin secretion is usually confined to the dark hours of the night. Besides the obvious, investigating the effects of melatonin in schizophrenia, assessing different dosages, a wider range of experimental conditions and a larger group of

healthy individuals, (perhaps selected on low levels of gating) may also be warranted. Last, due to the exploratory nature of this study, our results were not corrected for multiple testing. In conclusion, the current results provide the first indication that acute exogenous melatonin has little or no effect on sensorimotor gating of healthy young men. Furthermore, the results demonstrated the sedative and/or anxiolytic effects of melatonin by attenuating startle magnitude and the subjective ratings of alertness. In contrast, melatonin was not found to influence the subjects' sensitization and habituation processes. Finally, initial subjective alertness appeared to correlate with PPI of the 85 dB/ 60 ms trialtype. Future research should focus on the effects of melatonin in patients with schizophrenia.

Conflicts of interest The authors declare no conflict of interest.

Acknowledgments The authors would like to thank Lasse Bak, Ph.D., for his assistance with the manuscript.

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