Yen F Tai

Rosa Hoshi

Catherine M Brignell

Lisa Cohen

David James Brooks

Helen Valerie Curran

Professor Paola Piccini

Persistent nigrostriatal dopaminergic abnormalities in ex-users of MDMA ('Ecstasy'): an 18F-dopa PET study

Keywords: MDMA, ecstasy, addiction, dopamine, F-dopa, PET

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INTRODUCTION

±3,4-methylenedioxymethamphetamine (MDMA) is an amphetamine derivative which is the main psychoactive compound in 'ecstasy' tablets. Ecstasy is a popular recreational drug and is frequently taken in multiple doses in nightclubs and parties. In the United Kingdom, the prevalence of ecstasy use in the 15-34 age group is 13.6% ((EMCDDA), 2005). Neurotoxicity of MDMA on the serotonergic system, as defined by a reduction in the neuronal or neurochemical markers, has been demonstrated in both animals and human [START_REF] Green | Dopaminergic abnormalities in ex-MDMA users Hayman MAX. TWO MINUTE CLINICAL TEST FOR MEASUREMENT OF INTELLECTUAL IMPAIRMENT IN PSYCHIATRIC DISORDERS[END_REF], though this may be partly reversible after a prolonged abstinence [for review, see [START_REF] Cowan | Neuroimaging research in human MDMA users: a review[END_REF][START_REF] Reneman | Neuroimaging findings with MDMA/ecstasy: technical aspects, conceptual issues and future prospects[END_REF]]. The effects of MDMA on dopaminergic neurons are less certain. Acutely, MDMA releases dopamine from the dopaminergic terminals [START_REF] Colado | Acute and long-term effects of MDMA on cerebral dopamine biochemistry and function[END_REF]. In animals, with the exception of mice and, under certain conditions, rats [START_REF] Commins | Biochemical and histological evidence that methylenedioxymethylamphetamine Dopaminergic abnormalities in ex-MDMA users (MDMA) is toxic to neurons in the rat brain[END_REF]Yuan et al, 2002), MDMA has not been found to cause dopamine neurotoxicity [START_REF] Colado | Acute and long-term effects of MDMA on cerebral dopamine biochemistry and function[END_REF][START_REF] Logan | Differences between rats and mice in MDMA (methylenedioxymethylamphetamine) neurotoxicity[END_REF])unlike amphetamine [START_REF] Melega | Ethological and 6-[18F]fluoro-L-DOPA-PET profiles of long-term vulnerability to chronic amphetamine[END_REF] or methamphetamine [START_REF] Wilson | Striatal dopamine nerve terminal markers in human, chronic methamphetamine users[END_REF]. In humans, some studies have suggested chronic effects of MDMA usage on dopaminergic neurons. One study showed that three weeks after discontinuation of MDMA, the growth hormone response to dopamine agonist bromocriptine was significantly attenuated in ecstasy users compared to controls, possibly reflecting reduced dopamine D2 receptor sensitivity in the hypothalamus [START_REF] Gerra | Effects of (+/-) 3,4-methylene-dioxymethamphetamine (ecstasy) on dopamine system function in humans[END_REF]. There have also been several reports suggesting an association between MDMA usage and subsequent development of parkinsonism [START_REF] Kuniyoshi | MDMA and Parkinsonism[END_REF][START_REF] Mintzer | Dopaminergic abnormalities in ex-MDMA users[END_REF], but no definite causal link has been established.

Dopaminergic abnormalities in ex-MDMA users

The interpretation of many studies involving ecstasy users can be confounded by concomitant polydrug use given that most ecstasy users have also taken other recreational drugs [START_REF] Gouzoulis-Mayfrank | The confounding problem of polydrug use in recreational ecstasy/MDMA users: a brief overview[END_REF]. Previous neuroimaging studies investigating the effects of ecstasy on dopaminergic system have produced heterogeneous results. The relatively short period of abstinence from ecstasy in participants included in these studies also cannot address long-term toxicity. Using 123 I-beta-CIT single-photon emission computed tomography, one study reported no difference in striatal dopamine transporter (DAT) uptake between a group of current MDMA users and polydrug-using controls [START_REF] Semple | Reduced in vivo binding to the serotonin transporter in the cerebral cortex of MDMA ('ecstasy') users[END_REF]. Another study showed an increase in striatal DAT binding in ecstasy users who had been abstinent for at least three weeks compared with controls and ecstasy users who also used amphetamine [START_REF] Reneman | Use of amphetamine by recreational users of ecstasy (MDMA) is associated with reduced striatal dopamine transporter densities: a [123I]beta-CIT SPECT study-preliminary report[END_REF]. Finally, one group used 11 C-WIN35428 positron emission tomography (PET) and did not find any difference in striatal DAT binding between MDMA users who had been abstinent for a mean of 2•75 months and a group of unmatched polydrug users [START_REF] Mccann | Positron emission tomographic studies of brain dopamine and serotonin transporters in abstinent (+/-)3,4-methylenedioxymethamphetamine ("ecstasy") users: relationship to cognitive performance[END_REF].

18 F-dopa PET provides an in vivo marker of presynaptic dopaminergic terminal function [START_REF] Brooks | Imaging end points for monitoring neuroprotection in Parkinson's disease[END_REF]. Using 18 F-dopa PET, we investigated the long-term effects of ecstasy exposure on nigrostriatal dopaminergic function in ex-ecstasy users who have abstained from this drug for at least a year. Aspects of mood and cognition associated with dopaminergic function or with impairment following ecstasy use were also assessed. We included a group of polydrug-using controls to minimise the possible confounding effects of exposure to other recreational drugs.

MATERIALS AND METHODS

Design and participants

We recruited three groups of healthy participants: (1) ex-ecstasy users (EE) who had taken ecstasy on at least 25 occasions but had not taken any for the past 12 months, though they could continue to use other recreational drugs; (2) polydrug-using controls (PC) who were matched to the EE in their recreational drug use but had never taken ecstasy; and (3) drug-naïve controls (DC) who had never taken any recreational drugs except alcohol. The 25 occasions of use of ecstasy was an arbitrary cut-off to exclude very light ex-users from this study. Participants were recruited through magazine advertisements and word of mouth. Participants were not informed of inclusion criteria beforehand, and they were reimbursed travel expenses and £50 for their time. All three groups were matched for age, pre-morbid IQ, level of education and, for the drug-using groups, history of drug use apart from ecstasy.

Inclusion criteria for all groups were that the participants were male, aged 25-50, not taking prescribed psychotropic medication or receiving psychological treatment, no current or history of drug addiction, not being depressed as determined by the Structured Clinical Interview for DSM-IV (SCID) [START_REF] First | Structured Clinical Interview for DSM-IV Axis I Disorders[END_REF], have a score of <18 on the Beck Depression Inventory (BDI), have a score of <55 on the Spielberger Trait Anxiety Scale (STAI), no significant medical illness or serious head injuries in the past, and no history of schizophrenia or parkinsonism among first degree relatives. Participants were Dopaminergic abnormalities in ex-MDMA users also required not to drink more than three units of alcohol in the 24 hours prior to testing and not to use any recreational drugs for at least three days prior to testing (for the drug using groups). Only male volunteers above the age of 25 were included because the local ethics committee has imposed restrictions on exposing young adults and healthy women of childbearing age to ionising radiation. All participants had a neurological examination to detect any signs of parkinsonism. The participants in this study were part of a larger cohort of volunteers who took part in a related neuropsychological study [START_REF] Hoshi | Neurocognitive function in current and ex-users of ecstasy in comparison to both matched polydrug-using controls and drug-naive controls[END_REF], where a separate subgroup also had 11 C-DASB PET to assess serotonin transporter status [START_REF] Selvaraj | Brain serotonin transporter binding in former users of MDMA ('ecstasy')[END_REF].

PET scanning and neuropsychological testing were performed on the same day. Each participant also provided urine and hair samples on the day which were analyzed to corroborate their account of recent and longer term history of drug use respectively. The hair and urine samples were tested using enzyme-multiplied immunoassay to detect the presence of amphetamines, cocaine and methamphetamines groups of drugs, while the urine samples were tested additionally for opiates, benzodiazepines, barbiturates and cannabis. If the initial screen was positive for amphetamines, cocaine, methamphetamines or opiates, the sample was analyzed further by gas/liquid chromatography-mass spectrometry to determine the actual substance present. Most recreational drugs persist in the urine for 48 to 72 hours after ingestion apart from cannabis, which could be detected for two to three weeks after use. Every 1cm of hair length approximates to a one-month window of detection. The duration tested is limited by the length of hair available for analysis [START_REF] Pragst | State of the art in hair analysis for detection of drug and alcohol abuse[END_REF]. Hair analysis was performed by TrichoTech Ltd, Dopaminergic abnormalities in ex-MDMA users Cardiff, UK. If the urine or hair analysis showed that the participants no longer fulfilled the inclusion criteria, then they would be excluded from further analysis.

The study was approved by the local research ethics committee and permission to administer 18 F-dopa was granted by the Administration of Radioactive Substances Advisory Committee of the UK. All participants gave written informed consent to take part in this study in accordance with the Declaration of Helsinki.

PET scanning

All participants had one 18 F-dopa PET. PET was performed in the morning and participants were asked to have a light breakfast at least four hours before imaging and to avoid caffeine for at least 12 hours. Participants arrived at our PET unit at least two hours before the scan, so that they could settle down in a controlled environment. They were given orally 150mg of carbidopa and 400mg of entacapone one hour before the injection of 18 F-dopa to block the activity of peripheral aromatic amino acid decarboxylase and catechol-O-methyl transferase respectively. PET was performed using an ECAT EXACT HR++ (Knoxville, TN) camera. The camera has a transaxial spatial resolution of 4•8±0•2mm and axial resolution of 5•6±0•5mm after image reconstruction. A 5-minute transmission scan was performed prior to injection of 18 F-dopa to correct for tissue attenuation of 511keV γ-radiation. 114•5±4•7 (mean±SD) MBq of 18 F-dopa was injected intravenously over 30 seconds, and the dynamic emission data was acquired as 26 timeframes over 95 minutes.

Dopaminergic abnormalities in ex-MDMA users

Image analysis

The investigator (Y.F.T.) analysing the scans was blinded to the drug use history of the participants.

The 18 F-dopa dynamic images were corrected for between-frame head movements during PET scans by applying a frame-by-frame realignment paradigm [START_REF] Lang | Randomized controlled trial of intraputamenal glial cell line-derived neurotrophic factor infusion in Parkinson disease[END_REF][START_REF] Montgomery | Correction of head movement on PET studies: comparison of methods[END_REF]. Briefly, the non-attenuation corrected dynamic images were first denoised using a Battle-Lemarie wavelet [START_REF] Turkheimer | Multiresolution analysis of emission tomography images in the wavelet domain[END_REF]. Frames 4 to 26 of dynamic scans were co-registered to frame 3 (the first frame with high signal-to-noise ratio) and the transformation matrices were applied to the corresponding frames of the attenuation corrected dynamic images.

18 F-dopa images were analyzed as previously described [START_REF] Whone | A technique for standardized central analysis of 6-(18)F-fluoro-L-DOPA PET data from a multicenter study[END_REF]. Individual parametric images of 18 F-dopa influx rate constant (Ki maps) were created using a standard multiple-time graphical approach [START_REF] Patlak | Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data. Generalizations[END_REF] with an occipital reference input function. An "ADD" image of integrated 18 F-dopa signal from 30 to 90min was also created for each participant. The ADD images were then spatially normalised to an in-house 18 F-dopa template in standard stereotactic (MNI) space using statistical parametric mapping (SPM) software (Wellcome Functional Imaging Laboratory, London). The transformation matrices were then applied to the corresponding Ki maps. A standard region-of-interest object map that outlined putamen, heads of caudate nucleus and ventral striatum was defined on the 18 F-dopa template with magnetic resonance imaging guidance [START_REF] Whone | A technique for standardized central analysis of 6-(18)F-fluoro-L-DOPA PET data from a multicenter study[END_REF]. This object map was then Dopaminergic abnormalities in ex-MDMA users applied to each normalised parametric Ki map to measure individual Ki values. For each subject, the left-and right-sided Ki values were averaged.

Cognitive and mood assessments

The participants in this study form part of a larger cognitive study, and the cognitive and mood assessments used have been described elsewhere [START_REF] Hoshi | Neurocognitive function in current and ex-users of ecstasy in comparison to both matched polydrug-using controls and drug-naive controls[END_REF]. They included, amongst others, tests which are known to tap dopaminergic and serotonergic functions: immediate and delayed prose recall (Rivermead Behavioural Memory Tests; [START_REF] Wilson | The Rivermead Behavioural Memory Test[END_REF]; the Buschke Selective Reminding Task (BSRT; [START_REF] Buschke | Evaluating storage, retention, and retrieval in disordered memory and learning[END_REF]; Go/No-go task [START_REF] Mesulam | Principles of behavioral neurology[END_REF]; Rapid Visual Information Processing [START_REF] Wesnes | Effects of smoking on rapid information processing performance[END_REF]; the Serial Sevens task (Hayman, 1942); semantic and phonemic verbal fluency; the Trail Making Test [START_REF] Reitan | A manual for the administration and scoring of the Trail Making Test[END_REF]; CANTAB spatial working memory [START_REF] Owen | Planning and spatial working memory following frontal lobe lesions in man[END_REF]; CANTAB Stockings of Cambridge [START_REF] Owen | Visuo-spatial short-term recognition memory and learning after temporal lobe excisions, frontal lobe excisions or amygdalo-hippocampectomy in man[END_REF]; and Gibson's spiral maze [START_REF] Gibson | Manual of the Gibson Spiral Maze[END_REF]. Impulsivity was assessed using the Barratt Impulsiveness Scale (BIS; [START_REF] Barratt | Impulsivity: cognitive, behavioural and psychophysiological correlates[END_REF] and aggression was measured using the Aggression Questionnaire (AQ; [START_REF] Buss | The aggression questionnaire[END_REF].

Drug use history

A semi-structured interview was carried out on all participants to ascertain their levels of drug usage. For each drug, a lifetime estimate of amount used was obtained using the formula: (number of days used per month) x 12 x (number of years of regular use) x (dosage per session).

Statistical analysis

Statistical analysis was performed using SPSS v16 (SPSS Software Inc, Chicago, IL).

The Kolmogorov-Smirnov test confirmed the normal distribution of drug-use data. The majority of group differences were assessed using one-way ANOVA. The prose recall task was analyzed using repeated measure ANOVA. Post-hoc comparisons were performed with a Bonferroni correction and the significance level was set at p=0•05. Chisquared tests were used to compare non-parametric data. Pearson's correlations were used to explore the relationship between PET data, drug use and cognitive performance.

Due to the number of correlations performed, α=0•01 was adopted to minimise the probability of type I errors.

RESULTS

Demographic details and drug use history

A total of 46 participants were tested: 17 EE, 16 PC and 13 DC. Several participants were eventually excluded from analysis as they were found not to fulfil the inclusion criteria: three EE (two tested positive for cocaine in urine analysis indicating recent cocaine use, one reported the use of ecstasy four months prior to testing), two PC (one due to scan failure and one tested positive for MDMA in hair analysis) and one DC (tested positive for MDMA in hair analysis). Thus, the final analysis included 14 EE, 14 PC and 12 DC.

One PC was diagnosed with 'depressed mood' by his family doctor more than a year prior to his PET study and had a trial of a selective serotonin reuptake inhibitor for two weeks. He decided to discontinue the medication after that as he felt he was much better.

One-way ANOVA detected a significant group difference in the putamen 18 F-dopa uptake [(F 2, 39 =4.108, p=0•024); table 4 & figure 1]. Post-hoc comparison revealed the EE had 9% higher putamen 18 F-dopa uptake than the DC (p=0•021, corrected). The putamen 18 Fdopa uptake of PC fell between that of EE and DC but there were no significant group differences. There were no significant group differences in 18 F-dopa uptake in the ventral striatum and caudate nucleus, though these were 6% and 5% higher in the EE than the DC. Exclusion of the five participants who tested positive for cocaine and cannabis on hair and urine analysis had minimal impact on the results [putamen 18 F-dopa uptake (F 2, 34 =3.969, p=0•029); EE > DC, p = 0.025, corrected].

To further ensure that the differences seen in the putamen 18 F-dopa uptake were not due to the influences of other recreational drugs which could potentially affect dopaminergic activity, we performed an ANCOVA with putamen 18 F-dopa uptake as the dependent variable, participants' drug-use group as fixed factor and lifetime estimated amount of amphetamine, cocaine and cannabis as covariates. We also included amount of alcohol consumed as a covariate since there were group differences in its consumption.

ANCOVA confirmed a significant effect of group on the putamen 18 F-dopa uptake (F 2,33 =4.116, p=0•025), with the EE having higher values than the DC (p=0.024, corrected). The effects of other recreational drugs on putamen 18 F-dopa uptake were not significant.

Neuropsychological data

Data for two participants (one EE and one DC) on the RVIP and Go/No-Go were lost due to computer failure. A significant group difference was observed on the first trial of the BSRT (F 2,39 =5•85, p=0•006). Post-hoc tests revealed that both the EE (p=0•04) and PC (p=0•01) recalled fewer words than DC. Analysis of the Go/No-Go task (table 5) revealed a significant group difference on the number of hits (χ 2 =7•57, df = 2, p=0•023), with the PC making significantly fewer hits than the EE (p=0•008). A group difference was also found in number of false alarms (F 2,37 =6•16, p=0•005): PC made more false alarms than both EE (p=0•01) and DC (p=0•02). A group difference was observed on reaction times to hits (F 2,37 =0•395, p=0•028). PC reacted faster to hits than EE (p=0•03). There were no significant group differences in other assessments.

Correlations

Ex-ecstasy users group

18 F-dopa uptake in the putamen correlated negatively with the total number of false alarms on the Go/No-Go task (r=-0•815, p=0•001) and correlated positively with reaction time to hits (r=0•761, p=0•003). There was no correlation between striatal 18 F-dopa uptake and the following: lifetime amount of ecstasy or other recreational drugs, time since last use of ecstasy and O-LIFE scores (see below).

Assessment of schizotypy

also suggests that polydrug use could be an important factor. We have not demonstrated any evidence of ecstasy-induced nigrostriatal dopaminergic neurotoxicity.

As mentioned in the introduction, previous neuroimaging studies investigating the effects of ecstasy on nigrostriatal dopaminergic functions produced rather mixed results. There were significant methodological differences, including periods of abstinence and control groups chosen, with our study which made direct comparisons difficult. None of the studies mentioned used hair analyses to corroborate the drug use history of their participants. Semple and colleagues studied current ecstasy users, not ex-users, and they did not include any drug-naïve controls [START_REF] Semple | Reduced in vivo binding to the serotonin transporter in the cerebral cortex of MDMA ('ecstasy') users[END_REF]. McCann and colleagues did not detect any difference in DAT binding in ecstasy users who had been abstinent for 2•75 months (mean) and a group of controls with low-level polydrug use [START_REF] Mccann | Positron emission tomographic studies of brain dopamine and serotonin transporters in abstinent (+/-)3,4-methylenedioxymethamphetamine ("ecstasy") users: relationship to cognitive performance[END_REF]; but they did not include a drug-naïve control group which may partially explain the lack of significant difference in DAT binding. Reneman et al reported significantly higher striatal DAT binding in a group of ecstasy users who had never used amphetamine and who had been abstinent from ecstasy for a mean 3•4 months, compared to a group of ecstasy users who had also used amphetamine and a group of controls who had never used these two drugs [START_REF] Reneman | Use of amphetamine by recreational users of ecstasy (MDMA) is associated with reduced striatal dopamine transporter densities: a [123I]beta-CIT SPECT study-preliminary report[END_REF]. The conclusion of the study focused on the 'lower' DAT binding with amphetamine use, which in fact was not different from the controls, rather than the increase in DAT binding with ecstasy use. The authors proposed that amphetamine, a probable dopamine neurotoxin, in conjunction with ecstasy might be responsible for the lower DAT binding compared to ecstasy users naïve to amphetamine.

Of the three studies quoted above, the latter study had a more similar set-up with our Dopaminergic abnormalities in ex-MDMA users study, with a relatively drug-free control groups. Taken together with their findings, our results are compatible with the presence of a striatal hyperdopaminergic state in execstasy users and its persistence after an abstinence from ecstasy for over 3 years mean suggests that the effects maybe long-lasting.

A post-mortem study found increased homovanillic acid, a dopamine metabolite, in the putamen of the single MDMA chronic user studied compared to drug-naïve controls [START_REF] Kish | Striatal serotonin is depleted in brain of a human MDMA (Ecstasy) user[END_REF], indicating possible increased dopamine turnover. Other studies have also shown increased cerebrospinal fluid and plasma homovanillic acid in patients with psychosis [START_REF] Maas | Schizophrenia, Psychosis, and Cerebral Spinal Fluid Homovanillic Acid Concentrations[END_REF][START_REF] Pickar | Longitudinal measurement of plasma homovanillic acid levels in schizophrenic patients. Correlation with psychosis and response to neuroleptic treatment[END_REF][START_REF] Ramirez-Bermudez | Cerebrospinal fluid homovanillic acid is correlated to psychotic features in neurological patients with delirium[END_REF], a condition linked to hyperdopamnergic state. An increase in striatal 18 F-dopa uptake has been reported in neuropsychiatric conditions particularly schizophrenia and psychosisincluding those with prodromal psychotic symptoms [START_REF] Howes | Molecular imaging studies of the striatal dopaminergic system in psychosis and predictions for the prodromal phase of psychosis[END_REF][START_REF] Howes | Elevated striatal dopamine function linked to prodromal signs of schizophrenia[END_REF]; whilst less consistent findings have been seen in Tourette syndrome and attentional deficit hyperactivity disorder [START_REF] Nikolaus | In vivo imaging of synaptic function in the central nervous system: II. Mental and affective disorders[END_REF], which are mainly disorders of childhood onset. The magnitude of increase in putamen 18 F-dopa uptake in the EE compared to DC in our study is similar to that seen in the at-risk mental state subjects who have prodromal symptoms of schizophrenia [START_REF] Howes | Elevated striatal dopamine function linked to prodromal signs of schizophrenia[END_REF]. The O-LIFE questionnaire in our study did not reveal any significant differences in schizotypy amongst our participants but the results are limited by the variable response rate, and the retrospective and cross-sectional nature of our survey. Chronic ecstasy use may lead to multiple long-term neuropsychiatric consequences including psychosis [START_REF] Mcguire | Diversity of psychopathology associated with use of 3,4-methylenedioxymethamphetamine ('Ecstasy')[END_REF][START_REF] Montoya | Long-term neuropsychiatric consequences of "ecstasy" (MDMA): a review[END_REF][START_REF] Winstock | 103Dopaminergic abnormalities in ex-MDMA users Table 4: Mean (and standard deviations) striatal 18 F-dopa influx rate constant (Ki; min -1 ) in the three groups of participants. Significant comparison: *EE > DC (p=0•021, corrected). Ex-ecstasy users (EE) Polydrug controls (PC) Drug-naïve controls (DC) Caudate[END_REF]. One study looked at substance abuse in Dopaminergic abnormalities in ex-MDMA users patients presenting with first-episode psychosis and found that age at first use of cannabis, cocaine, ecstasy and amphetamine was independently associated with age at first psychotic symptom [START_REF] Barnett | Substance use in a population-based clinic sample of people with first-episode psychosis[END_REF].

The underlying molecular mechanisms for ecstasy-related hyperdopaminergic state are still unclear but our findings suggest they may be related to synergistic or interactive effects with other recreational drugs. The increased striatal 18 F-dopa uptake and DAT binding are consistent with increased dopamine turnover. This may be a reaction to chronic exposure to a dopamine depleting agent or may reflect long-term sensitization of the nigrostriatal dopaminergic system to MDMA, where repeated exposure to a stimulant drug resulted in heightened dopamine release after re-exposure, as demonstrated in vivo with amphetamine one year following limited exposure to the substance [START_REF] Boileau | Modeling sensitization to stimulants in humans: an [11C]raclopride/positron emission tomography study in healthy men[END_REF]. Acutely, MDMA increases DOPA accumulation in the striatum which is blocked by 5-HT2 antagonist ketanserin, suggesting that MDMA activates nigrostriatal dopaminergic pathways via 5-HT2 receptors [START_REF] Nash | Effect of 3,4-methylenedioxymethamphetamine on 3,4-dihydroxyphenylalanine accumulation in the striatum and nucleus accumbens[END_REF]. It is uncertain if this effect persists after repeated dosing or prolonged abstinence but this mechanism could offer a potential explanation for our findings.

An alternative, albeit speculative, explanation for our findings is that there are preexisting differences in the nigrostriatal dopaminergic system between ecstasy and nonecstasy users, i.e. the hyperdopaminergic state leads to a desire to use stimulants such as ecstasy. We do not have baseline or pre-drug use data on these participants. However, a longitudinal multi-modality neuroimaging study did not reveal any difference in the baseline neuroimaging data between subjects who remained ecstasy-naïve versus those who started using ecstasy at follow-up, though dopaminergic function was not measured [START_REF] De Win | Sustained effects of ecstasy on the human brain: a prospective neuroimaging study in novel users[END_REF].

Cognitive assessments revealed minor differences across groups. Both drug-using groups were impaired on the BSRT, indicating they had a shorter immediate memory span. The lack of major cognitive impairment in the EE is in line with other published studies [START_REF] Hoshi | Neurocognitive function in current and ex-users of ecstasy in comparison to both matched polydrug-using controls and drug-naive controls[END_REF][START_REF] Mccann | Positron emission tomographic studies of brain dopamine and serotonin transporters in abstinent (+/-)3,4-methylenedioxymethamphetamine ("ecstasy") users: relationship to cognitive performance[END_REF]. These cognitive tests probably have lower sensitivity than PET in detecting neurochemical abnormalities in the drug users as previous studies have demonstrated reduced serotonin transporters in ecstasy users but without concomitant changes in their cognitive performances [START_REF] Mccann | Positron emission tomographic studies of brain dopamine and serotonin transporters in abstinent (+/-)3,4-methylenedioxymethamphetamine ("ecstasy") users: relationship to cognitive performance[END_REF].

Higher putamen 18 F-dopa uptake in EE correlated with fewer false alarms but a slower reaction time to hits on a choice reaction time task. The significance of these correlations is unclear and it needs to be interpreted with caution since there were no differences between EE and DC in these parameters. We excluded participants with significant anxiety or depression, which can often be seen in chronic drug users. This may also explain the lack of group differences in the personality traits and psychological data in table 1.

There are several potential limitations of our study. As with all studies involving recreational drug use, we have to rely on the participants' subjective recall of their drug use history. We attempted to corroborate that as far as possible with urine and hair analysis but inevitably recall biases or errors could occur. The small number of participants tested positive for cannabis on urine analysis and cocaine on hair analysis indicated that the confounding effects from recent use of other drugs were likely to be small. Excluding these participants from analysis or incorporating other drug use as covariates in the ANCOVA analysis did not alter our study results. The average duration of three months being free from amphetamines, cocaine and methamphetamines groups of drugs as shown on hair analysis also implied that the impact from recent ingestion of these drugs on the dopaminergic system was likely to be small. Nevertheless, we had not been able to test for the longer history of drug use especially with regards to ecstasy to verify that the EE had indeed been abstain from the drug for more than a year. Whilst we tried to minimize the confounding effects of polydrug use by matching EE and PC groups, in reality it was difficult to do so exactly especially when we also had to rely on subjective recalls of the participants. One further limitation is that the purity of recreational drugs taken by the participants in our study cannot be ascertained. Our study included male participants only, and hence the results may not be generalised to female EE. One study has reported that women might be more susceptible than men to MDMAinduced alterations of the serotonergic system (Reneman et al, 2001). The EE showed a modest 9% increase in the putamen 18 F-dopa uptake compared to DC. Whilst this is significantly greater than the striatal 18 F-dopa uptake test-retest variability of about 5% [START_REF] Egerton | The test-retest reliability of 18F-DOPA PET in assessing striatal and extrastriatal presynaptic dopaminergic function[END_REF] and of a similar magnitude seen in at-risk mental state subjects with prodromal symptoms of schizophrenia [START_REF] Howes | Elevated striatal dopamine function linked to prodromal signs of schizophrenia[END_REF], it would be prudent to repeat this in a larger scale study. Finally, we studied participants with a history of recreational drug use only. Therefore, our results may not be applicable to those with drug addiction and heavy use.

Figure 1 :

 1 Figure legends

Table 1 :

 1 Group means (standard deviations) for age, pre-morbid IQ (Spot The Word), depression (BDI), anxiety (STAI), aggression (AQ) and impulsivity (BIS). The range of possible scores for each of the rating scale is listed in the first column. There was no significant difference between groups for any of the items

Ex-ecstasy users Polydrug controls Drug-naïve controls

	Age (yrs)	31•07 (5•62)	30•50 (7•30)	30•58 (8•15)
	Spot The Word (0-60) 51•79 (3•09)	49•79 (4•49)	50•67 (3•98)
	BDI (0-63)	8•07 (6•08)	7•71 (4•29)	5•42 (6•36)
	STAI (20-80)	38•93 (10•64)	37•50 (6•73)	36•25 (8•47)
	AQ (29-145)	72•79 (21•24)	74•36 (13•80)	72•25 (17•75)
	BIS (30-120)	53•50 (13•27)	55•29 (17•53)	52•25 (16•13)

Table 3 :

 3 Group means (standard deviations) for alcohol, cannabis, amphetamine, and cocaine use; and length of hair tested for amphetamines, cocaine and methamphetamines use ‡ (excludes 1 EE and 2 PC who tested positive for cocaine). Significant comparison: *PC > DC (p=0•02, corrected).

Dopaminergic abnormalities in ex-MDMA users

Acknowledgments

We would like to thank Oliver Howes for his helpful comments on the manuscript.

Funding

The work was supported by the Parkinson's Disease Society (UK). RH was supported by a Medical Research Council studentship, YFT was supported by a Wellcome Trust fellowship.

Dopaminergic abnormalities in ex-MDMA users

There was no other history of psychiatry disorder or history of use of psychotropic drugs amongst the participants.

The demographic details of the participants are listed in table 1. There were no group differences in age, pre-morbid IQ (Spot the Word), depression, anxiety, aggression and impulsivity (Table 1), or groups in level of education attained and current employment status (not listed). All participants had a normal neurological examination. The EE have on average been using ecstasy for 4•38 years but have abstained from it for 3•22 years (table 2). There was no significant difference in the drug use pattern of EE and PC (table 3). A group difference was found in units of alcohol consumed per week (F 2,39 =4•479, p=0•018), with the PC reported drinking more than DC (p=0•02, corrected). Three participants tested positive for cocaine on hair analysis: one EE (within last three months based on hair analysis, self-reported last use 5 days ago) and two PC (one PC within last three months, self reported last use 30 days ago; the second PC within last 3.5 months, self reported last use 5 days ago). The remaining participants tested negative on hair analysis. Excluding these three participants, the average length of hair tested was approximately 3cm in all three groups, equivalent to 3 months being free from amphetamines, cocaine and methamphetamines groups of drugs (table 3). Three participants also tested positive for cannabis in urine: one EE (the same participant who also tested positive for cocaine on hair analysis) and two PC.

Striatal 18 F-dopa uptake

Dopaminergic abnormalities in ex-MDMA users

The striatal hyperdopaminergic state found in EE was not predicted a priori. The main disorder in the literature that demonstrates a consistent association with a hyperdopaminergic state is psychosis, usually in the context of schizophrenia [START_REF] Hietala | Presynaptic dopamine function in striatum of neuroleptic-naive schizophrenic patients[END_REF][START_REF] Howes | Molecular imaging studies of the striatal dopaminergic system in psychosis and predictions for the prodromal phase of psychosis[END_REF]. We therefore performed a post-hoc assessment using the Oxford-Liverpool Inventory of Feelings and Experiences (O-LIFE) short scale [START_REF] Mason | Short scales for measuring schizotypy[END_REF] to measure the participants' schizotypy or psychosis-proneness as none of our participants has frank psychotic symptoms. O-LIFE consists of four subscales, Unusual Experiences, Cognitive Disorganisation, Introvertive Anhedonia and Impulsive Nonconformity (table 6). It was administered within three months of completion of their PET scans. The rate of response and the O-LIFE scores are listed in table 6. There were no group differences in the response rates or O-LIFE scores. The relatively poor response rate was probably due to the post-hoc nature of the questionnaire with associated dropouts.

DISCUSSION

This is the first study looking into the long-term effects of ecstasy exposure, in the setting of polydrug use, on nigrostriatal dopaminergic system after a prolonged abstinence. We have detected an increase in the putamen 18 F-dopa uptake in a group of male EE, who had abstained for over three years (mean), compared to DC. The putamen 18 F-dopa uptake of PC lay between that of EE and DC, suggesting that the difference seen between the latter two groups may be due to the combined effects of ecstasy and polydrug use rather than ecstasy alone. The lack of correlation between striatal 18 F-dopa uptake and ecstasy use Dopaminergic abnormalities in ex-MDMA users

The long-term sequelae of our findings are as yet uncertain, and follow-up of the execstasy users would be important to evaluate any development of psychopathology or further changes in dopaminergic function. Nevertheless, our study highlights potential long-term adverse effects from recreational use of a very popular illicit substance, in the setting of polydrug use, with consequent public health implications. Larger scale prospective studies [START_REF] De Win | Sustained effects of ecstasy on the human brain: a prospective neuroimaging study in novel users[END_REF]) would be able to confirm and to further delineate the effects of ecstasy on human nigrostriatal dopaminergic system, whilst avoiding difficulties and limitations frequently encountered with cross-sectional drug studies as outlined above.

Conflicts of interest

DJB serves or has served on scientific advisory boards for CeNes, Synosia, Genentech,


MBBS Nivethida Thirugnanasambandam

MD Jessica Grundey

Kim Adam

Anne Drees

Angela C Skwirba

MD Nicolas Lang

MD Walter Paulus

MD Michael A Nitsche
email: mnitsch1@gwdg.de

M Tech

Nitsche Ma

Nicotinergic impact on focal and non-focal neuroplasticity induced by non-invasive brain stimulation in non-smoking humans

Keywords: neuroplasticity, nicotine, paired associative stimulation, transcranial direct current stimulation, transcranial magnetic stimulation

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Smoking tobacco is the single largest preventable cause of mortality and morbidity [START_REF] Peto | Mortality from tobacco in developed countries: indirect estimation from national vital statistics[END_REF]. Nicotine is the primary constituent of tobacco that is responsible for its addictive properties. Nicotine is the classical agonist at nicotinic acetylcholine receptors (nAchRs) which are ligand-gated cation channels. Studies in animals and humans have shown that nicotine improves attention as well as working and long-term memory [START_REF] Lazzaro | Effects of nicotine on novelty detection and memory recognition performance: double-blind, placebo-controlled studies of smokers and nonsmokers[END_REF][START_REF] Hahn | Nicotine-induced attentional enhancement in rats: effects of chronic exposure to nicotine[END_REF][START_REF] Kumari | Cognitive effects of nicotine in humans: an fMRI study[END_REF]. While many studies focused on the behavioral effects of nicotine in healthy humans and patients [START_REF] Hasselmo | Dynamics of learning and recall at excitatory recurrent synapses and cholinergic modulation in rat hippocampal region CA3[END_REF][START_REF] Sacco | Effects of cigarette smoking on spatial working memory and attentional deficits in schizophrenia: involvement of nicotinic receptor mechanisms[END_REF], very few have investigated the nicotinergic impact on cortical excitability and plasticity, which are the likely neurophysiological basis for the cognitive effects of the substance. For global cholinergic enhancement, it was shown that cholinesterase-inhibitors reduce intracortical inhibition, increase facilitation, and enhance focal, but diminish non-focal facilitatory plasticity in healthy humans [START_REF] Korchounov | Modification of motor cortical excitability by an acetylcholinesterase inhibitor[END_REF][START_REF] Kuo | Focusing effect of acetylcholine on neuroplasticity in the human motor cortex[END_REF]. A study on tobacco smokers, who are under chronic nicotine exposure, revealed enhanced motor cortex inhibition and reduced facilitation [START_REF] Lang | Cortical hypoexcitability in chronic smokers? A transcranial magnetic stimulation study[END_REF]. Nicotine also enhances and prolongs the facilitatory after-effects of intermittent theta burst stimulation in human motor cortex [START_REF] Swayne | The facilitatory effects of intermittent theta burst stimulation on corticospinal excitability are enhanced by nicotine[END_REF]). Thus, global cholinergic and nicotinergic activation might have at least partially dissimilar effects on cortical excitability.

Transcranial direct current stimulation (tDCS) and paired associative stimulation (PAS) are noninvasive brain stimulation techniques that induce neuroplastic cortical excitability alterations (Nitsche andPaulus 2000, 2001;Nitsche et al. 2003a;[START_REF] Stefan | Mechanisms of enhancement of human motor cortex excitability induced by interventional paired associative stimulation[END_REF][START_REF] Wolters | A temporally asymmetric Hebbian rule governing plasticity in the human motor cortex[END_REF].

Both techniques induce NMDA-and calcium-dependent changes of cortical excitability (Nitsche et al. 2003b;[START_REF] Stefan | Mechanisms of enhancement of human motor cortex excitability induced by interventional paired associative stimulation[END_REF][START_REF] Wolters | A temporally asymmetric Hebbian rule governing plasticity in the human motor cortex[END_REF]. tDCS modulates spontaneous neuronal Nicotinergic impact on neuroplasticity Nitsche MA -4 -activity and excitability by either depolarizing or hyperpolarizing neurons. Anodal tDCS induces depolarization that enhances neuronal excitability whereas cathodal tDCS hyperpolarizes neurons, decreasing their excitability levels (Nitsche andPaulus 2000, 2001;Nitsche et al. 2003a). Since tDCS affects all neurons beneath the electrodes, it is thought to induce relatively non-focal plasticity. PAS, on the other hand, induces focal, synapse-specific, timing-dependent, associative neuroplasticity in the targeted neurons. Here an electrical pulse to a mixed peripheral nerve at an intensity which activated somatosensory fibres is followed by a suprathreshold magnetic pulse applied to the corresponding area of the primary motor cortex. Depending on the interstimulus interval, there occurs synchronous or asynchronous activation of somatosensorymotor cortical connections that enhance or reduce excitability respectively [START_REF] Stefan | Mechanisms of enhancement of human motor cortex excitability induced by interventional paired associative stimulation[END_REF].

In the current study we aimed to identify the specific contribution of nicotinic receptors to the cholinergic effect on focal and non-focal neuroplasticity by exploring the effects of nicotine on tDCS-and PAS-generated plasticity in healthy non-smoking humans to improve our comprehension of the cognition-enhancing and addictive properties of this substance. As in the foregoing studies, the motor cortex was taken as a model system in this single blinded, placebocontrolled, partial crossover study because it allows a convenient monitoring of excitability alterations by measuring motor evoked potential (MEP) amplitudes via transcranial magnetic stimulation (TMS).

Materials and methods

Subjects

Forty eight healthy human volunteers participated in the study. All of them were complete nonsmokers, that is, none of them had smoked tobacco for at least 3 years prior to the study. They Nicotinergic impact on neuroplasticity Nitsche MA -5 -did not suffer from any chronic or acute medical illness or any history of neurological/psychiatric diseases, and did not take any chronic or acute medication. This information was obtained by a detailed free personal interview with the subjects. Pregnancy, family history of epilepsy, presence of any metallic implant or cardiac pacemaker was ruled out. All of them were righthanded according to the Edinburgh handedness inventory (Oldfield, 1971). The selection of subjects was not based on their results from previous plasticity experiments in our laboratory; most of them were naïve to the experimental procedure. Table 1 shows the demographic characteristics of the different groups. All subjects gave written informed consent before participating in the study. The experiments were approved by the local Ethics Committee and conformed to the Declaration of Helsinki. Allocation of the subjects to the respective experimental conditions as well as order of sessions was randomized.

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Insert Table 1 here

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Assessing motor cortex excitability

Single transcranial magnetic (TMS) pulses were delivered from a Magstim 200 stimulator (Magstim Company, Whitland, Dyfed, UK) at a frequency of 0.25 Hz with a figure-of-eight shaped coil (diameter of one winding, 70mm; peak magnetic field, 2.2 T). The coil was held tangentially to the scalp at an angle of 45º to the sagittal plane with the coil handle pointing laterally and posterior. This induced a postero-anterior current flow in the brain at an angle that optimally activates the corticospinal system monosynaptically (Di [START_REF] Lazzaro | Effects of nicotine on novelty detection and memory recognition performance: double-blind, placebo-controlled studies of smokers and nonsmokers[END_REF]. Motor evoked potentials (MEPs) were recorded using a surface electromyogram (EMG) set-up. Surface Nicotinergic impact on neuroplasticity Nitsche MA -6 -EMG electrodes (Ag-AgCl) were placed over the abductor digiti minimi muscle (ADM) in a belly-tendon montage. Signals were amplified, band-pass filtered (2Hz -2KHz), digitized (5KHz) and stored in a laboratory computer for offline analysis using Signal software and CED 1401 (Cambridge Electronic Design, Cambridge, UK). MEPs were elicited using single pulse TMS over the motor cortex representation of the ADM. The position of the coil on the scalp, where the stimulus elicited consistently the largest MEP amplitudes for slightly suprathreshold intensity was marked as the motor 'hotspot'. Stimulus intensity was then adjusted in order to obtain peak to peak MEP amplitudes of approximately 1mV (SI1mV). This TMS intensity was kept constant throughout the experiment. The mean MEP amplitude was calculated from at least 20 pulses for baseline, and post-intervention excitability monitoring. The change of the mean MEP amplitude over time reflects alterations of motor cortex excitability.

Transcranial direct current stimulation

Twenty four subjects participated in the tDCS experiments. tDCS was administered by a batterydriven constant current stimulator (Schneider Electronic, Gleichen, Germany) through rubber electrodes covered by saline soaked sponges (35 square cm). One electrode was placed over the motor cortex representation of the right ADM as determined by single pulse TMS and the other electrode over the contralateral supra-orbital region. All subjects received 1mA of either anodal (for 13 min) or cathodal stimulation (for 9 min), combined with nicotine or placebo medication in different experimental sessions. Therefore, twelve subjects received anodal tDCS with nicotine or placebo patches and the remaining twelve received cathodal tDCS with nicotine or placebo patches. This stimulation intensity and duration (13 min anodal tDCS and 9 min cathodal tDCS) generates after-effects on cortical excitability lasting for approximately 60 min Nicotinergic impact on neuroplasticity Nitsche MA -7 -after stimulation [START_REF] Nitsche | Sustained excitability elevations induced by transcranial DC motor cortex stimulation in humans[END_REF]Nitsche et al. 2003a). The two consecutive experimental sessions per subject were separated by at least one week interval.

Paired associative stimulation

Twenty four subjects participated in the PAS experiment. Here a peripheral electrical pulse over the right ulnar nerve at wrist level was followed by a TMS pulse over the motor cortex representation of the ADM at inter-stimulus intervals (ISI) of either 10 (PAS 10) or 25 milliseconds (PAS 25). The peripheral pulse was delivered from a Digitimer D185 multipulse stimulator (Digitimer, Welwyn Garden City, UK) at an intensity of 300% of the sensory perceptual threshold. The suprathreshold magnetic pulse was delivered from a Magstim 200 stimulator with an intensity which elicited MEP amplitudes of approximately SI1mV. The paired pulses were repeated 90 times at a frequency of 0.05 Hz. This protocol induces long-lasting excitability changes in the motor cortex depending on the ISI duration. An ISI of 10 ms induces excitability diminution whereas an ISI of 25 ms induces facilitation [START_REF] Stefan | Mechanisms of enhancement of human motor cortex excitability induced by interventional paired associative stimulation[END_REF][START_REF] Wolters | A temporally asymmetric Hebbian rule governing plasticity in the human motor cortex[END_REF]. The subjects were instructed to count the number of pulses they received at their wrist throughout the whole stimulation duration in order to guarantee sufficient attention to the procedure, which has been shown to be crucial to obtain the intended effects (Stefan et al., 2004).

Pharmacological intervention

Each subject participated in two sessions in randomized order. 30cm 2 nicotine transdermal patches, each containing nicotine 0.83mg/cm 2 releasing 15mg over 16 hours or placebo patches were administered to all subjects in combination with one of the stimulation protocols -anodal tDCS, cathodal tDCS, PAS-10 or PAS-25. By this dosage of nicotine, physiologically and Nicotinergic impact on neuroplasticity Nitsche MA -8 -behaviorally relevant plasma levels are accomplished [START_REF] Tønnesen | A double-blind trial of a 16-hour transdermal nicotine patch in smoking cessation[END_REF]. Subjects received the patch 6 hours before the start of the stimulation. This was the approximate time for the plasma level of nicotine to reach its maximum following application of the patch [START_REF] Nørregaard | Long-term nicotine substitution after application of a 16-hour nicotine patch in smoking cessation[END_REF]). The patch was retained until the end of the last after-measurement of the experiment on the evening of the second day. In order to counteract possible systemic side effects of nicotine, the subjects were instructed to take 20mg domperidone, a peripheral acting dopamine D2-receptor antagonist with antiemetic effects, in case of need.

Course of the experiment

The subjects received either a placebo or nicotine patch, which was adhered to the left upper arm and remained there until the end of the last after-measurement on the following evening. They were given 20mg domperidone and asked to take it orally in case of any side effects.

Unpublished results from our group show that domperidone alone does not have any significant effect on motor cortical excitability. Six hours later, subjects were seated comfortably in a reclined position on a dentist´s chair with proper arm and head rests and asked to relax completely. The EMG electrodes were placed at the right ADM as described above. The motor 'hotspot' was determined over the left motor cortex and marked with a water-proof skin marker, and the TMS intensity needed to induce MEP amplitudes of 1mV (SI1mV) size was determined.

Twenty MEPs were recorded at this stimulus intensity and the mean MEP amplitude was calculated as the baseline. One of the stimulation protocols, either tDCS or PAS, was administered. At least 20 MEPs were recorded immediately following the stimulation (0min) and at time points of 5,10,15,20,25,30,60,90, and 120 min. For the nicotine patch sessions, the after-measurements were also conducted in the evening of the stimulation day and in the Nicotinergic impact on neuroplasticity Nitsche MA -9 -morning and evening of the day following the plasticity induction procedure. We used a waterproof pen to mark the ADM electrodes and TMS coil positions and ensured that these were positioned over the same spot during the whole course of the experiment.

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Insert Figure 1 here

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Data analysis and statistics

The individual means of the 20 MEP amplitudes recorded at each time point were calculated for all subjects. The post-intervention mean MEP amplitudes from each subject were then normalized to the respective individual mean baseline MEP amplitude. The normalized mean MEP amplitudes from all subjects were pooled together and the grand average across subjects for each time bin was calculated.

A repeated measures ANOVA was performed on the normalized data using MEP amplitude as the dependent factor including all time points up to 120 min after stimulation. Drug (Nicotine versus Placebo) and time points were included as within-subjects factors. Stimulation (anodal tDCS/ cathodal tDCS/ PAS-10/ PAS-25) served as between-subjects factors. The Mauchly test was performed to test for sphericity, and the Greenhouse-Geisser correction applied when necessary. Conditional on significant results of the ANOVA, we performed post-hoc comparisons using Student's t-tests (paired, two-tailed, p<0.05, not adjusted for multiple comparisons) where we compared (i) the mean MEP amplitudes at the time points after plasticity induction versus baseline and (ii) the mean MEP amplitudes following nicotine versus placebo at one time point within a stimulation condition. Moreover, we compared absolute baseline MEP Nicotinergic impact on neuroplasticity Nitsche MA -10 -values between the stimulation and drug conditions via Student´s t-tests to exclude a priori differences. Chi square test was performed to look for significant differences in gender distribution between the groups. For comparison of the age of subjects between the groups, Student's t-tests (two -tailed, unpaired, p<0.05) were performed.

Results

All subjects tolerated the experimental procedure well. None of them complained of any side effects of either nicotine or the stimulation. Especially the participants did not complain of any sedative effects of the patch. During the experiment, they were completely alert and relaxed.

Since no systemic side effects of nicotine were perceived and none of the participants needed to take domperidone, the subjects were blinded effectively. Gender distribution did not differ significantly between the various groups tested by chi square test (p = 0.083). There were significant differences in the mean age between some of the groups as tested by Student's unpaired t-tests. However, the maximum difference of mean age between groups was 2.65 years.

Absolute baseline MEP amplitudes did not differ significantly between groups (Student's t-test, two-tailed, paired, p>0.05 for all cases) or medication conditions (Student's t-test, unpaired, twotailed, p = 0.66).

The ANOVA revealed a significant main effect of the between-subjects factor stimulation (F(3,44) = 18.137; p < 0.001), in accordance with different effects of inhibitory and facilitatory tDCS and PAS on MEP amplitudes. The main effects of either nicotine (F(1,44) = 0.093; p = 0.762) or time (F(10,440) = 1.654; p = 0.089) were not significant. However, the interactions between nicotine X stimulation (F(3,44) = 5.498; p = 0.003); time X stimulation (F(30,440) = Nicotinergic impact on neuroplasticity Nitsche MA -11 -3.070; p < 0.001) were significant, showing that nicotine had different effects on the abovementioned stimulation protocols, and that the time course of the effects of these stimulation protocols was not identical. The three-way interaction nicotine X time X stimulation (F(30,440) = 1.848; p = 0.005) was also significant. Thus, application of nicotine patch significantly influenced the after-effects of the different stimulation protocols differently over time.

Nicotinergic impact on tDCS-induced plasticity

As shown by the post hoc t-tests, in the control condition without nicotine, MEPs were significantly enhanced following anodal tDCS for up to 90 minutes. Cathodal tDCS diminished excitability levels significantly also for up to 90 minutes after stimulation. Under nicotine, both anodal and cathodal tDCS-induced after-effects were abolished. The post-hoc test revealed that for anodal tDCS, post-tDCS MEP amplitudes under nicotine were not different from baseline values, but differed significantly from those under placebo medication (table S1). A trendwise reversal of the effects of anodal tDCS from facilitation to inhibition under nicotine did not reach statistical significance (p > 0.073). The excitability diminution induced by cathodal tDCS, as compared to baseline excitability, was also abolished under nicotine; however relative to the placebo medication condition nicotine induced only a trendwise change (Figure 2 A, B, table S1).

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Effect of nicotine on PAS-induced plasticity

As shown by the post hoc tests, PAS induced a significant facilitation of MEP amplitudes following PAS-25 and excitability diminution following PAS-10, lasting for up to 90 or 120 minutes after stimulation (table S1) under placebo medication. Under nicotine, the facilitatory effects of PAS-25 remained significant as compared to the respective baseline MEP for up to 90 min after PAS. MEP amplitudes under nicotine did not differ versus the respective placebo medication condition for up to 90 min after PAS-25, but were significantly larger as compared to placebo 120 min after PAS-25, being in favor for a prolonged excitability enhancement accomplished by PAS-25 under nicotine. The missing difference of PAS-25 under nicotine relative to baseline excitability 120 min after placticity induction might be caused by the relatively large variability of the MEPs at this time point (see Table S1). The inhibitory effect of PAS-10 was abolished under nicotine. Consequently, the post hoc tests revealed no significant differences of the respective MEP amplitudes relative to baseline, but significant differences of the respective MEP amplitudes relative to those under placebo medication (figure 3A,B).

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Insert Figure 3A and 3B here

______________________________________________________________________________ Discussion

The present study shows that in healthy non-smoking individuals nicotine prominently affects neuroplasticity. Our data illustrate that (i) nicotine exposure slightly prolongs or at least preserves the synapse-specific cortical excitability enhancement induced by PAS-25, but Nicotinergic impact on neuroplasticity Nitsche MA -13 -abolishes the PAS-10 induced depression of cortical excitability (ii) the non-focal excitability enhancing after-effect of anodal tDCS and the excitability diminution caused by cathodal tDCS are both abolished (Figure 4). There is also a trendwise reversal of anodal tDCS -induced facilitation to inhibition under nicotine. Hence, we observe a focusing effect of nicotine on facilitatory neuroplasticity and an abolishment of inhibitory plasticity, the latter irrespective of the focality of stimulation.

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Insert Figure 4 here ______________________________________________________________________________ Some of our observations match with those of previous studies. The enhancement or preservation of facilitatory plasticity induced by PAS-25 by nicotine is similar to the effect it had on intermittent theta burst stimulation [START_REF] Swayne | The facilitatory effects of intermittent theta burst stimulation on corticospinal excitability are enhanced by nicotine[END_REF]. The effect of nicotine on facilitatory plasticity is also comparable to the effect of the cholinesterase inhibitor rivastigmine on identical plasticity induction protocols, however its effects on inhibitory plasticity are at variance to those under global cholinergic enhancement under rivastigmine [START_REF] Kuo | Focusing effect of acetylcholine on neuroplasticity in the human motor cortex[END_REF].

Proposed mechanism of action

The focusing effect of nicotine on facilitatory plasticity, i.e. consolidating/preserving focal, PASinduced, but diminishing non-focal tDCS-generated plasticity, is quite similar to that of global cholinergic enhancement via rivastigmine described in a previous study [START_REF] Kuo | Focusing effect of acetylcholine on neuroplasticity in the human motor cortex[END_REF]. A likely explanation for this effect is the different impact of cholinergic activation on recurrent activation of afferent input to cortical neurons. It has been shown that excitatory glutamatergic Nicotinergic impact on neuroplasticity Nitsche MA -14 -synaptic transmission is suppressed by presynaptic inhibition at intrinsic, recurrent synapses, but not at afferent fibre synapses via cholinergic activation [START_REF] Hasselmo | Cholinergic suppression specific to intrinsic not afferent fiber synapses in rat piriform (olfactory) cortex[END_REF]Hasselmo et al., 1995;[START_REF] Vogt | Cholinergic modulation of excitatory synaptic transmission in the CA3 area of the hippocampus[END_REF]. Since PAS induces plasticity by a combination of afferent somatosensory input with a motor cortical stimulus, while tDCS is thought to affect primarily excitability of cortical interneurons [START_REF] Nitsche | Modulating parameters of excitability during Nicotinergic impact on neuroplasticity Nitsche MA -24 -and after transcranial direct current stimulation of the human motor cortex[END_REF], it might be speculated that these specific aspects of the stimulation techniques caused the differences of the effects also in the present experiments. In accordance, in animal experiments an inhibitory effect of the activation of nicotinergic subreceptors on feedforward interneurons has has been shown to prevent LTP induction by inhibiting pyramidal neurons, whereas spike-timing dependent LTP was enhanced (Rosza et al., 2008;[START_REF] Yamazaki | Nicotine-induced switch in the nicotinic cholinergic mechanisms of facilitation of long-term potentiation induction[END_REF], although the latter effect was not shown in all studies [START_REF] Couey | Distributed network actions by nicotine increase the threshold for spike-timingdependent plasticity in prefrontal cortex[END_REF]. Due to the results of the present experiment, this effect of cholinergic activation on facilitatory plasticity in humans can likely be attributed to an impact of nicotinic receptors. Another possible explanation of the results is based on the enhancement of intracellular calcium concentration by nicotinic receptors, e.g. the alpha-7 subreceptor.

Intracellular calcium is a key determinant of plasticity induction, and the after-effects of tDCS and PAS are calcium-dependent (Nitsche et al., 2003b;[START_REF] Stefan | Mechanisms of enhancement of human motor cortex excitability induced by interventional paired associative stimulation[END_REF]. The amount of intracellular calcium determines if inhibitory, facilitatory, or no plasticity is induced. A slight enhancement of intracellular calcium induces LTD, a large enhancement LTP [START_REF] Lisman | Three Ca2+ levels affect plasticity differently: the LTP zone, the LTD zone and no man's land[END_REF].

Medium and very large concentrations of intracellular calcium induce no or convert plasticity, the latter possibly due to an activation of hyperpolarizing calcium channels [START_REF] Misonou | Regulation of ion channel localization and phosphorylation by neuronal activity[END_REF]. Since tDCS induces facilitatory plasticity by tonic depolarization of neurons for some minutes, which might activate voltage-dependent calcium channels, whereas PAS is a phasic stimulation technique, which induces only short lasting depolarization, the amount of Nicotinergic impact on neuroplasticity Nitsche MA -15 -intracellular calcium increase caused by tDCS might be larger than that caused by PAS.

Therefore, a further calcium enhancement via nicotinic receptor activation might enhance the calcium level above the concentration inducing LTP-like plasticity, and therefore result in an abolishment of plasticity in case of tDCS, but not PAS. This effect on different kinds of facilitatory plasticity of nicotine might enhance the signal-to-noise ratio (Hasselmo and Barkai, 1995), which would facilitate the representation of meaningful, synchronous inputs and suppress non-meaningful inputs. Indeed, it has been recently suggested that nicotine improves memory performance via calcium-dependent mechanisms in animals (Biala & Kruk, 2009).

In contrast, nicotine abolished all kinds of inhibitory plasticity irrespective of the specific stimulation protocol. One possible explanation might be that the calcium-enhancing properties of nicotinic receptor activation here resulted in an intracellular calcium level too large to induce LTD-like plasticity. This mechanism of action would also explain the different effect of global cholinergic activation by rivastigmine on inhibitory plasticity, as described in a previous study, where this substance prolonged tDCS-and PAS-generated inhibitory plasticity [START_REF] Kuo | Focusing effect of acetylcholine on neuroplasticity in the human motor cortex[END_REF], because muscarinic receptors inhibit voltage-gated calcium channels [START_REF] Brown | Muscarinic acetylcholine receptors (mAChRs) in the nervous system: some functions and mechanisms[END_REF], and thus might counteract the effects of nicotinic receptors in this case. The effects of nicotine on inhibitory plasticity obtained in the present study are not in accordance with some animal experiments, where nicotinic activation has been shown to be important for LTD induction [START_REF] Partridge | Nicotinic acetylcholine receptors interact with dopamine in induction of striatal long-term depression[END_REF][START_REF] Fujii | Nicotine accelerates reversal of long-term potentiation and enhances long-term depression in the rat hippocampal CA1 region[END_REF], however, the effect of nicotine on LTD seems to depend on the general proneness of the system to inhibitory plasticity [START_REF] Alzoubi | Adult-onset hypothyroidism facilitates and enhances LTD: reversal by chronic nicotine treatment[END_REF][START_REF] Alzoubi | Effect of chronic stress or nicotine on hypothyroidism-induced enhancement of LTD: electrophysiological and molecular studies[END_REF], which likely differs between animal preparations and in vivo studies in humans.

It should be mentioned that these mechanistic explanations of the results are hypothetical presently. Alternative explanations, such as the modification of NMDA receptor-dependent Nicotinergic impact on neuroplasticity Nitsche MA -16 -plasticity by nicotine-dependent alteration of GABAergic activity [START_REF] Couey | Distributed network actions by nicotine increase the threshold for spike-timingdependent plasticity in prefrontal cortex[END_REF], cannot be ruled out. These hypotheses should be tested more directly in future studies, e.g. by altering the activity of nicotinic subreceptors, or calcium channels, in combination with nicotine exposure.

General remarks

The results of the present study demonstrate that nicotine clearly influences neuroplasticity in non-smoking individuals. Nicotine focuses facilitatory plasticity whereas it abolishes inhibitory plasticity. The effects differ from those of non-specific cholinergic activation. The focusing effect of nicotine on facilitatory plasticity might help to explain how this drug improves attention, working memory and long-term memory in animals and humans via enhancing the signal to noise ratio of plasticity. Also the abolition of inhibitory plasticity by nicotine might affect cognitive processes. First, it might shift the net balance of plasticity more into the direction of facilitatory plasticity, and therefore indirectly enhance cognitive performance further. Second, inhibitory plasticity, especially long-term depression (LTD) has been shown to be directly involved in certain forms of learning and memory formation. Collingridge and colleagues (2010) describe a role of LTD in hippocampal-based learning and memory formation, and recognition memory in perirhinal cortex. Since our findings show that nicotine abolishes LTD-like plasticity it could be speculated that nicotine might worsen LTD-dependent forms of learning and memory.

However, an impairment of cognitive functions by nicotine has been rarely described [START_REF] Toledano | Diversity and variability of the effects of nicotine on different cortical regions of the brain -therapeutic and toxicological implications[END_REF], thus further behavioral studies are needed to explore the cognitive effects of nicotine more systematically. Some limiting aspects of this study should be mentioned. Blinding could have been somewhat compromised considering the fact that the experimenters were not blinded to the intervention.

Nicotinergic impact on neuroplasticity

Nitsche MA -17 -However, the data were collected by more than one investigator without notable difference in the results, which probably indicates low experimenter bias, and experimenters were not informed about hypotheses about expected outcomes of the experiments, which should have limited expectancy effects. Although the subjects did not complain of any sedation due to nicotine, the degree of alertness was not explicitly assessed and hence its effect on the measurements cannot be ruled out completely. We did not measure plasma concentrations of nicotine, thus it could be argued that inter-individual differences of the bioavailability of the substance had an impact on the results. However, since we studied a fairly homogenous group of participants, and we induced plasticity during steady state drug concentration, we do not think that variability of plasma concentration of nicotine can explain the results. We studied the effect of only a single dosage of nicotine in the present experiments. Thus it cannot be ruled out that the effect of nicotine on plasticity differs dose-dependently, as shown for other neuromodulators, like dopamine [START_REF] Kuo | Dose-dependent inverted U-shaped effect of dopamine (D2-like) receptor activation on focal and nonfocal plasticity in humans[END_REF][START_REF] Brown | Muscarinic acetylcholine receptors (mAChRs) in the nervous system: some functions and mechanisms[END_REF]. Moreover, since nicotine receptors are rapidly modified by chronic exposure, this study cannot discern between primary effects of nicotine on nicotinic receptors and secondary effects caused by receptor desensitization or upregulation. Moreover, it should not be taken for granted that the effects obtained on motor cortex plasticity, as in the current study, translate exactly to other cortices, where nicotinic receptor density, and subreceptor composition might differ [START_REF] Mcgehee | Regulation of synaptic transmission and plasticity by neuronal nicotinic acetylcholine receptors[END_REF]Gotti et el., 2009) Furthermore the results of a single dosage nicotine application in non-smokers, as performed here, might differ from the effects of nicotine in smokers who are chronically exposed to it. Future studies should address these aspects in larger detail.

Nicotinergic impact on neuroplasticity

Nitsche MA -18 -

Conclusion

The results of this study deliver clear evidence for an important role of nicotine in the formation of neuroplasticity, the likely basis of learning and memory formation, in humans. Via its focusing effect on facilitatory plasticity, nicotine might be an attractive candidate to enhance these processes in neuropsychiatric diseases accompanied by cognitive decline. The abolition of inhibitory plasticity by nicotine also could have a significant impact on some forms of learning and memory, and also affect addictive behavior to some extent. Moreover, its effect on plasticity might be an important mechanism for starting nicotine consumption, addiction and the high probability of relapse in smokers. Interestingly the effects of nicotine on plasticity share some of those of dopamine [START_REF] Kuo | Boosting focally-induced brain plasticity by dopamine[END_REF], which might be an explanation for the frequent nicotine consumption in schizophrenia, in which dopaminergic malfunctioning is an important pathologic mechanism. Clearly, more studies are needed to explore the exact role of nicotine in healthy humans and in those suffering from neuropsychiatric diseases to a larger degree. Moreover the results of this study are important in another aspect. Non-invasive brain stimulation techniques are increasingly used as scientific and therapeutic tools. The results of the present study show that the activity of the nicotinergic system might critically affect the effects of brain stimulation.

This potentially important confounding factor should thus be taken into account in future studies using brain stimulation.

Disclosure/Conflict of interest

The authors declare that, except for income received from my primary employer, no financial support or compensation has been received from any individual or corporate entity over the past

three years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.

Funding:

The study was supported by the Deutsche Forschungsgemeinschaft (DFG grant NI683/4-1 'Towards risk prediction of nicotine dependency by exploring individual limits of cortical neuroplasticity in humans'; NI 683/4-2 "Impact of the nicotinergic alpha7 receptor on cortical plasticity in smokers and nonsmokers") within the DFG priority program 'Nicotine: Molecular and Physiological Effects in Central Nervous System'.

Supplementary information is available at the Neuropsychopharmacology website.

Nicotinergic impact on neuroplasticity

Nitsche MA -26 -

Titles and legends to figures:

Figure 1 Illustration of the experimental design. Each subject underwent two experimental sessions. At the beginning of the session, the subjects first received either a placebo or a nicotine patch. After 6 hours break, baseline (BL) motor evoked potentials (MEPs) of approximately 1 mV amplitude were recorded by single pulse transcranial magnetic stimulation (TMS) over the motor cortical representational area of the abductor digiti minimi muscle (ADM). One of the four stimulation protocols (anodal tDCS/cathodal tDCS/PAS-25/PAS-10) was then applied. After-measurements were made immediately following the stimulation and once every 5 min for the first 30 min, then once every 30 min for 120 min for both sessions. In addition, MEPs were also recorded the same evening, next morning and evening for the nicotine patch sessions.