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International Journal of Neuropsychopharmacology International Journal ofThe Neuropsychopharmacology Advance Access published October 31, 2014

Manuscript ID:

Manuscript Type:

Date Submitted by the Author:

IntJNP-14-0237.R1

Regular Research Article

08-Sep-2014

Strube, Wolfgang; University of Munich, Psychiatry Nitsche, Michael; University of Goetttingen, Clinical Neurophysiology Wobrock, Thomas; Kreisklinik Darmstadt -Dieburg, Psychiatry Bunse, Tilmann; University of Munich, Psychiatry Rein, , Bettina; University of Goetttingen, Clinical Neurophysiology Herrmann, Maximiliane; University of Goetttingen, Clinical Neurophysiology Schmitt, Andrea; University of München, Psychiatry Nieratschker, Vanessa; University of Tuebingen, Psychiatry Witt, Stephanie; Central Institute of Mental Health, Medical Faculty Mannheim, Genetic Epidemiology in Psychiatry Rietschel, Marcella; Central Institute of Mental Health, Child and Adolescent Psychiatry and Psychotherapy, Molecular Genetics Laboratory Falkai, Peter; University of Munich, Psychiatry and Psychotherapy Hasan, Alkomiet; University of München, Psychiatry

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Complete List of Authors:

The International Journal of Neuropsychopharmacology

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Journal:

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BDNF-Val66Met-polymorphism impact on cortical plasticity in schizophrenia patients: a proof-of-concept study

Brain stimulation - Other < Treatment

Keywords: Enter up to 5 (minimum 3) keywords that best reflect the research reported in your paper.:

BDNF, tDCS, Schizophrenia, Plasticity

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Research Focus: Choose one or more (maximum 5) that best describe the research focus of your paper:

BDNF-Val66Met-polymorphism impact on cortical plasticity in schizophrenia patients: a proof-of-

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concept study

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Wolfgang Strube1, Michael A. Nitsche2, Thomas Wobrock3,4, Tilmann Bunse1, Bettina Rein4, Maximiliane Herrmann4, Andrea Schmitt1,5, Vanessa Nieratschker6, Stephanie H. Witt7, Marcella Rietschel7, Peter Falkai1, Alkomiet Hasan1 1

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Department of Psychiatry and Psychotherapy, Ludwig Maximilian University, Munich, Germany 2 Department of Clinical Neurophysiology, University of Goettingen, Goettingen, Germany 3 Centre of Mental Health, Darmstadt-Dieburg Clinics, Groß-Umstadt, Germany 4 Department of Psychiatry and Psychotherapy, University of Goettingen, Goettingen, Germany 5 Laboratory of Neuroscience (LIM27), Institute of Psychiatry, University of São Paulo, São Paulo, Brazil 6 Department of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health Mannheim Medical Faculty Mannheim / Heidelberg University, Germany and Department of Psychiatry and Psychotherapy, University of Tuebingen, Tuebingen, Germany 7 Department of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health, Medical Faculty Mannheim / Heidelberg University, Mannheim, Germany.

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Category: Regular Research Article Word count abstract: 249 Word Count main text: 5054 (not including references, tables and figures) References: 65 Tables: 3 Figures: 3 Supplementary Tables: 1

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Corresponding author: Wolfgang Strube, M.D.

Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-University, Munich, Germany Nussbaumstr. 7, D- 80336 Munich, Germany Phone: +49 089 5160 5511; Fax: +49 89 5160 5530 Email: [email protected]

Abstract Background: Brain-derived neurotrophic factor (BDNF) has been shown to be a moderator of

neuroplasticity. A frequent BDNF-polymorphism (Val66Met) is associated with

impairments of cortical plasticity. In patients with schizophrenia, reduced neuroplastic

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responses following non-invasive brain stimulation have been reported consistently. Various

studies have indicated a relationship between the BDNF-Val66Met-polymorphism and motorcortical plasticity in healthy individuals, but schizophrenia patients have yet to be

BDNF-Val66Met-polymorphism

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inhibitory

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schizophrenia patients.

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investigated. The aim of this proof-of-concept study was therefore to test the impact of the

Methods: Cortical plasticity was investigated in 22 schizophrenia patients and 35 healthy controls using anodal and cathodal transcranial direct-current-stimulation (tDCS) applied to the left primary motor cortex. Animal and human research indicates that excitability-shifts

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following anodal and cathodal tDCS are related to molecular long-term-potentiation and longterm-depression. To test motor-cortical excitability before and after tDCS, well-established

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single- and paired-pulse transcranial magnetic stimulation protocols were applied.

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Results: Our analysis revealed increased glutamate-mediated intracortical facilitation in metheterozygotes compared to val-homozygotes at baseline. Following cathodal tDCS, schizophrenia met-heterozygotes had reduced GABA-mediated short-interval intracortical

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inhibition (SICI), whereas healthy met-heterozygotes displayed the opposite effect. The BDNF-Val66Met-polymorphism did not influence single-pulse motor-evoked potential amplitudes after tDCS.

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Conclusions: These preliminary findings support the notion of an association of the BDNFVal66Met-polymorphism with observable alterations in plasticity following cathodal tDCS in schizophrenia patients. This indicates a complex interaction between inhibitory intracortical interneuron-networks, cortical plasticity and the BDNF-Val66Met-polymorphism. Further replication and validation needs to be dedicated to this question to confirm this relationship.

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Key Words: Transcranial direct current stimulation – transcranial magnetic stimulation –

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Brain-derived neurotrophic factor – motor-cortical plasticity – schizophrenia

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1. Introduction Brain-derived neurotrophic factor (BDNF) has been shown to be a moderator of neuroplasticity in the central nervous system through its activity-dependent release, and by balancing the intrasynaptic ratio with its precursor peptide pro-BDNF (Aicardi et al., 2004;

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Woo et al., 2005; Miyamoto, 2006). In healthy subjects, the BDNF G196A polymorphism, which results in a valine to methionine substitution at the amino acid position 66 (Val66Met), has been consistently shown to reduce activity-dependent BDNF-secretion, to be involved in

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the regulation of activity- and stimulus-dependent plasticity and memory processes, and to influence episodic memory and learning processes (Egan et al., 2003; Pezawas et al., 2004;

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Woo et al., 2005; Miyamoto, 2006; Caroni et al., 2012). The impact of this BDNF-Val66Metpolymorphism on global and local (e.g. hippocampus) brain volumes is still subject to discussion (Molendijk et al., 2012). Further studies have addressed the influence of the BDNF-Val66Met-polymorphism on the pathophysiology of

different neuropsychiatric

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disorders including major depressive disorder, bipolar disorder and schizophrenia (Autry and Monteggia, 2012). Post mortem studies in schizophrenia patients have displayed evidence of

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reduced BDNF expression in the prefrontal cortex (Takahashi et al., 2000; Weickert et al.,

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2003; Hashimoto et al., 2005) and the hippocampus (Iritani et al., 2003; Durany and Thome, 2004). However, conflicting evidence has been reported onthe impact of the BDNFVal66Met-polymorphism on the age of onset, symptom severity and MR-based brain

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volumes in schizophrenia (Pezawas et al., 2004; Numata et al., 2006; Naoe et al., 2007; Pillai, 2008; Dutt et al., 2009; Gruber et al., 2012). Given the involvement of BDNF in neurotransmission and memory processes, previous

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studies using non-invasive brain stimulation have examined the relationship between the BDNF-Val66Met-polymorphism and motor-cortical plasticity (Chaieb et al., 2014). Studies using intermittent and continuous theta-burst stimulation showed conflicting findings for either decreased or absent responses in healthy met-allele-carriers (Cheeran et al., 2008; Antal et al., 2010; Jayasekeran et al., 2011) or no differences between val-homozygotes and metallele-carriers (Li Voti et al., 2011; Mastroeni et al., 2013). Related results have been 4

reported for plasticity induction by paired-associative stimulation (PAS), showing either reduced effects of PAS in met-allele-carriers (Cheeran et al., 2008) or no group differences (Witte et al., 2012). With regards to the after-effects of transcranial direct current stimulation (tDCS), two studies using cathodal tDCS showed no differences between val-homozygotes

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and met-carriers (Antal et al., 2010; Di Lazzaro et al., 2012) and a more pronounced increase of cortical excitability in met-carriers following anodal tDCS (Antal et al., 2010). No significant differences were observed following transcranial random noise stimulation (Antal

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et al., 2010). A decreased response of met-carriers was found in a homeostatic plasticity paradigm, pairing cathodal tDCS with 1Hz repetitve transcranial magnetic stimulation

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(repetitive TMS) (Cheeran et al., 2008). The assessment of use-dependent plasticity showed reduced changes in motor cortical excitability in met-carriers (Kleim et al., 2006). In one animal study, Fritsch et al. showed decreased BDNF-secretion and LTP following anodal tDCS in BDNF-mutant mouse M1 slices (Fritsch et al., 2010). Although the origin of these

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conflicting results stands yet to be clarified, these studies suggest an association of the BDNF-Val66Met-polymorphism with the susceptibility to induce plasticity using non-invasive

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brain stimulation.

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In patients with schizophrenia, impairments of neuroplastic responses following different forms of non-invasive brain stimulation have been consistently reported (Oxley et al., 2004; Hasan et al., 2011; Hasan et al., 2013). Despite a likely impact of BDNF on the pathobiology

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of schizophrenia, the effects of the BDNF-Val66Met-polymorphism on cortical plasticity and on cortical excitability following non-invasive brain stimulation in patients with schizophrenia have not yet been investigated. Therefore, the aim of the present proof-of-concept study was

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to address this question for the first time by applying anodal and cathodal tDCS to the primary motor cortex (M1) of schizophrenia patients and healthy controls and by monitoring excitability changes before and after tDCS with single- and paired-pulse TMS paradigms. Paired-pulse TMS paradigms investigate the interaction of a first sub-threshold conditioning stimulus followed by a suprathreshold second test stimulus (Kujirai et al. 1993). Short intervals between both stimuli (2–5ms) result in a subsequent inhibition of the test pulse 5

(short-interval cortical inhibition [SICI]), whereas longer intervals (7–20ms) lead to a facilitation (intracortical facilitation [ICF]) (Kujirai et al. 1993). Pharmacological challenges in healthy subjects strongly indicate that SICI is mediated via GABAA-neurotransmission, whereas ICF is related to glutamatergic neurontransmission (Ziemann et al. 2004). For tDCS,

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animal and human data indicate that the excitability changes following tDCS are long-lasting, N-Methyl-D-Aspartate(NMDA)-dependent and also polarity-dependent, and thus can be

considered to be related to long-term potentiation (LTP, anodal) and long-term depression

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(LTD, cathodal). We first hypothesized that healthy and schizophrenia met-allele-carriers would show reduced cortical inhibition and enhanced cortical facilitation compared to the

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respective val-homozygotes. Second, we hypothesized met-allele-carriers would display less

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plasticity responses following tDCS compared to the val-homozygotes.

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2. Methods 2.1 Subjects In total, the data of 35 healthy controls and 22 schizophrenia patients from two foregoing studies (Hasan et al., 2011; Hasan et al., 2012a) were analysed with regard to BDNF

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genotype. As two different investigators (B.R. or M.H.) performed the experiments for anodal

and cathodal tDCS, each study group was considered to be independent. All schizophrenia patients, but not all healthy controls, received both stimulation polarities (see Table 1). After

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giving written informed consent, genotyping was conducted on 22 participants with schizophrenia (SZ) (8 female, 14 male, mean age = 30.1 years) and 35 healthy control (HC)

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participants (14 female, 21 male, anodal group: mean age = 27.1 years, cathodal group: mean age = 27.8 years). The local ethics committee of the University Medical Centre Goettingen approved the protocol, which was conducted in accordance with the Declaration of Helsinki. Participants with contraindication to TMS or tDCS, such as neurological illness,

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severe brain injury, dermatological disorders, brain tumors, or a history of dementia, and patients with concomitant benzodiazepine or mood stabilizer treatment were excluded. A

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clinical psychiatrist, blinded to the aims of the study, and a member of the study group (T.W.

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or A.H.) made an ICD-10 consensus diagnosis. All participants underwent a standardized test of hand preference (Annett, 1970) and all patients received an assessment of psychopathological symptoms (PANSS;(Kay et al., 1987)), disease severity (CGI;(Guy and

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Bonato, 1976)) and social functioning (GAF;(Endicott et al., 1976)). All schizophrenia patients (apart from three) were treated with antipsychotics (CPZ mean = 356.7 ± 393.5; 12 patients received monotherapy, of these 5 patients were treated with risperidone, 6 with quetiapine

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and 1 with olanzapine). In general, patients did not receive a concomitant medication (e.g. antidepressants, antihypertensive drugs, benzodiazepines).

2.2 TMS procedure The complete protocol has been described previously (Hasan et al., 2011). Briefly summarized, subjects were examined in a comfortable sitting position with arms supported 7

passively. Electromyographic activity (EMG) was recorded by surface electrodes placed over the right first dorsal interosseus muscle (FDI). Raw signals were amplified, bandpass-filtered (2 Hz-10 kHz) and digitized using a standard amplifier (Keypoint portable, Medtronic Co., Denmark). TMS was performed over the left primary motor cortex (M1) with a standard 70

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mmTMS figure-of-eight magnetic coil and a MagPro X 100 magnetic stimulator (Medtronic

Co., Denmark). Throughout all experiments, the coil was held tangentially to the head, with the handle pointing backwards and in a 45° angle lateral to the midline. The stimulation site

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that produced the largest motor evoked potential (MEP) at moderately suprathreshold

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stimulation intensities was defined and marked as the optimal coil position.

2.3 tDCS procedure

Transcranial direct current stimulation (tDCS) was applied after baseline TMS examination. The tonic electrical field with a stimulation intensity of 1mA was induced using a CE-certified

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stimulator (DC-Stimulator-Plus, NeuroConn GmbH, Ilmenau, Germany) through saline soaked rectangular surface sponge-electrodes (7x5 cm) for a duration time of 9 minutes in

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the cathodal and 13 minutes in the anodal condition (Nitsche et al. 2000, Nitsche et al. 2001).

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These standard stimulation durations have been consistently shown to be optimal for the induction of cortical excitability alterations lasting for about 1h after tDCS (Nitsche et al., 2008). The stimulation electrode was placed over the left motor cortex (M1) on the spot of

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the optimal coil position, and the second electrode was placed over the contralateral supraorbital forehead.

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2.4 Cortical excitability

Resting motor threshold (RMT), expressed as a percentage of maximum stimulator output, was defined as the lowest intensity that produced a minimum MEP of 50 µV in the relaxed FDI in at least 5 of 10 trials. Single-pulse MEPs were recorded from the motor cortical representation of the right FDI (MEP-size) to monitor the global effects of tDCS on corticospinal excitability. TMS intensity was adjusted before tDCS to evoke MEPs of 1 mV size on 8

average (S1mV) and was kept unchanged for the after-effect assessment (Hasan et al., 2011). 40 MEPs were recorded before tDCS and 5 min after the stimulation. Follow-up measurements of cortical excitability parameters were started immediately. Cortical silent period (CSP), a measure of cortical inhibition, was obtained by recording from the FDI

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muscle under voluntary contraction while stimulating the contralateral M1 with 120% RMT.

The mean CSP duration was calculated from 10 trials. Short-latency intracortical inhibition, and intracortical facilitation (SICI, ICF) were recorded with a standardized paired-pulse

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protocol (Kujirai et al., 1993) (conditioning stimulus: 80% RMT, test stimulus: intensity that produced resting MEPs averaging 0.7–1.3mV (S1mV), ISIs: 3 and 12). A minimum of 20

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trials with each ISI and 40 trials with the test stimulus alone were performed. RMT and S1mV were adjusted for the paired-pulse protocols and for CSP after tDCS (Chen, 2004).

2.5 BDNF genotyping

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Genomic DNA was extracted from ethylenediaminetetraacetic acid (EDTA) anti-coagulated venous blood samples using the chemagic MSM I system (PerkinElmer Chemagen

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Technologie GmbH; Rodgau; Germany). BDNF Val66Met (rs6265) was genotyped on a

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7900HT Fast Real-Time PCR System (life technologies; Darmstadt; Germany) using TaqMan®SNP Genotyping Assay C-11592758_10 (life technologies; Darmstadt; Germany) and the standard protocol for allelic discrimination. Accuracy was assessed by duplicating

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15% of the original sample, and reproducibility was 100%.

2.6 Statistics

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For statistical analysis, SPSS 22 for Windows was used. Level of significance was set at =0.05. For gender, hand preference and BDNF genotype. Chi2-tests were computed to test

for different distributions between the study groups. One-way analysis of variance (ANOVA) was used to compare mean ages and baseline excitability between the groups. MEP size was calculated as the mean MEP amplitude individually and then interindividually before and after stimulation. As the normality assumption was violated (Kolmogorov–Smirnov-test, p 9

between