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Keywords:

  • Dopamine receptor;
  • gene expression;
  • neuronal cultures;
  • VNTR

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

The dopamine receptor D4 (DRD4) gene includes several variable number of tandem repeat loci that have been suggested to modulate DRD4 gene expression patterns. Previous studies showed differential basal activity of the two most common variants of a tandem repeat (120 bp per repeat unit) located in the 5′ region adjacent to the DRD4 promoter in human cell lines. In this communication, we further characterized the ability of this polymorphic repeat to elicit tissue-, allele- and stimuli-specific transcriptional activity in vitro. The short and long variants of the DRD4 5′ tandem repeat were cloned into a luciferase reporter gene construct containing the SV40 promoter. The luciferase constructs were cotransfected with expression vectors of two ubiquitously expressed human transcription factors (TFs), CCCTC-binding factor (CTCF) and upstream stimulatory factor 2 (USF2), into human cell lines and primary cultures of neonate rat cortex and luciferase activity measured. Overexpression with these TFs resulted in differential cell- and allele-specific transcriptional activities of the luciferase constructs. The results of our experiments show that variants of this tandem repeat in the 5′ promoter of the DRD4 gene will direct differential reporter gene transcriptional activity in a cell-type-specific manner dependent on the signal pathways activated.

The dopamine D4 receptor gene (DRD4) encodes for a G protein-coupled receptor that regulates dopamine neurotransmission and is expressed in prefrontal cortex, limbic area and retinal tissue (Oak et al. 2000). Changes in D4 receptors and DRD4 mRNA have been observed in frontal cortex of schizophrenic and depressed brains, in addition to lymphocytes of heroin and alcohol abusers. Research has therefore focused on genetic variants affecting DRD4 gene expression as risk-associated parameters for such conditions (Czermak et al. 2004; Li et al. 2004; Matsumoto et al. 1996; Nakajima et al. 2007; Smith 2010; Xiang et al. 2008).

Kamakura et al. (1997) identified the minimal promoter to be located between −591 and −123 upstream of the DRD4 translational start site. The 5′ regulatory region of the gene contains genetic variation implicated in both transcription control and risk factor for specific disorders; these include single nucleotide polymorphisms (SNPs), e.g. at positions −521 and −616, and variable number of tandem repeats (VNTR) of 120-bp repeats units, located 1240 bp upstream of the DRD4 translational start site (Seaman et al. 1999) also termed as ‘tandem duplication’ in the literature. However, alleles with up to four repeat units have been identified (Kereszturi et al. 2007). Therefore, we refer to this VNTR as the ‘5′ region VNTR’. D'Souza et al. (2004) have previously demonstrated that two most common variants [long ‘L’, two repeat units (240 bp) and short ‘S’, one repeat unit (120 bp)] of this VNTR could elicit differential gene expression in human cell lines. Furthermore, Ronai et al. (2004) demonstrated that the ‘L’ allele increases the binding affinity for Sp1 in capillary electrophoretic mobility shift assay (CEMSA), factor previously suggested to regulate DRD4 expression. However, association studies between this VNTR polymorphism and behavioural traits and disorders have revealed both positive and negative results.

To further document the transcriptional profiles of variants of the VNTR at the 5′ region of the DRD4 gene, we tested whether VNTR allelic variation could elicit differential, neuronal-specific gene expression using primary cultures of frontal cortex from rat neonates. In addition, we investigated whether the 5′ VNTR long (L) and short (S) variants cloned into a luciferase reporter gene vector (referred to as D4Lp and D4Sp, respectively) exhibit allele- and stimuli-specific transcriptional activity following cotransfection with transcription factors (TFs), CCCTC-binding factor (CTCF) and upstream stimulatory factor 2 (USF2), in the human neuroblastoma SH-SY5Y, SK-N-MC and the human embryonic kidney HEK-293 cell lines.

Our inspection of the primary sequence identified two E-boxes adjacent to the 3′ end of the repeat element in the DRD4 5′ VNTR (Fig. 1). The CTCF and USF2 proteins were chosen as they represent examples of ubiquitously expressed TFs, previously shown to mediate allele-specific gene transcription via interaction with VNTRs and E-boxes located in VNTRs (e.g. Ali et al. 2010; Haddley et al. 2011; Klenova et al. 2004; Paterson et al. 1995; Roberts et al. 2007). Also, these two proteins have been shown to exert transcriptional regulatory activity of enhancers in the same cell models chosen for our experiments, and have been reported not to interact with the same plasmid backbone (e.g. pGL3p) used for our experiments.

image

Figure 1. Potential TFBS are affected by the presence of the 5′ VNTR. In the 120-bp sequence, nucleotides highlighted in bold and underlined indicate putative binding sites for TFs (shown below the sequence). E-boxes (indicated by rectangles) were identified by eye, and identifications were based on previously reported consensus sequence (e.g. Paterson et al. 1995).

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The human cell lines and primary cultures of neonate rat frontal cortex were chosen as DRD4 gene expression has been confirmed in these cells and cultures previously (D'Souza et al. 2004; Ronai et al. 2004).

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

In silico search of TF-binding sites in the DRD4 5′ region VNTR sequence

To investigate the potential functional implications of the presence of a VNTR to the TF binding in this 5′ region, the 120-bp sequence was subject to in silico analysis, using the publicly available TRANSFAC® public database (http://www.biobase-international.com) via the AliBaba 2.1 program (Grabe 2002). Alibaba 2.1 was set to detect known consensus binding sequences for TF using the following parameters: minimum matrix conservation (similarity between the consensus binding site for a TF and a potential binding site in the query sequence) = 70%, minimum number of homologous sites (the minimum number of sites of which a matrix is build) = 4, factor class level (the classification of TFs in the TRANSFAC database is hierarchical and includes six levels, from family of TFs to splice variants) = 4 and similarity of the sequence to the matrix = 1. The sequence was explored further by visual inspection for identification of E-box consensus sequence. The results of both visual and in silico analysis are shown in Fig. 1.

Generation of the D4Sp and D4Lp reporter gene constructs cloned into pGL3p

The 5′ region VNTR was amplified from two plasmids bearing long ‘L’ and short ‘S’ variants [as described in D'Souza et al. (2004)] using polymerase chain reaction (PCR) with the following primers: D4120-f 5′-ggggtacccct CTG GGA GAG AAG AAA CTT-3′ and D4120-r 5′-ccgctcgagcg ACA GGA CAA GGT CAC-3′. These primers were designed to incorporate flanking restriction sites for Acc65I (forward primer) and XhoI (reverse primer) (highlighted in bold font in the primer sequences) one at either end of the primers, to facilitate restriction digest and cloning into the multiple cloning site, in the reporter gene vector pGL3p (Promega, Southampton, UK) generating the D4Sp (short variant in pGL3p) and D4Lp (Long variant in pGL3p) plasmid constructs. The fragments were cloned in front of the SV40 promoter, which drives firefly transcription of the luciferase gene. Briefly, conditions for PCR comprised: a reaction mix containing 50 ng of DNA template, 1 µm of each primer, 2.5 U Diamond DNA polymerase (Bioline, London, UK), 1× Diamond polymerase buffer, 0.2 mm of each dNTP, 2 mm MgCl2, 1 m betaine (Sigma-Aldrich, Poole, UK) and dH2O to a final volume of 50 µl per reaction. The PCR was performed for 35 cycles consisting of a 95°C denaturing step (1 min), 55°C annealing step (1 min) and a 72°C extension step (1 min) in a Px2 thermal cycler (Thermo Scientific, Hampshire, UK). After ligation, we transformed chemically competent E. coli (DH5-alpha strain, Invitrogen, Paisley, UK). Transformations were spread on ampicillin-selective LB agar plates. A total of 30–50 colonies (per plasmid-construct ligation) were subcultured in LB broth and plasmid DNA was extracted using a DNA miniprep kit (Qiagen, West Sussex, UK). Selection of positive clones was followed by diagnostic restriction digest (using XhoI and Acc65I) to test for the presence of inserts of approximate 324 (S) and 444 bp (L). Exact sequence and direction of cloned fragments into the multiple cloning site of the pGL3p vector was determined by Sanger sequencing, conducted in a 3130 Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA) using commercially available sequencing primers (RVprimer4 and GLP2, Promega) following the manufacturer's instructions (Applied Biosystems).

Cell culture, transfections and luciferase assay

Male Wistar albino rats (2–7 days old) (purchased at the University of Liverpool animal facility) were used to generate primary cell cultures from cortical brain tissue. All animals were culled under local and national schedule one guidelines. All procedures were carried out according to the UK Home Office regulations. Briefly, cortices were collected in dissection solution Hank's Buffered Salt Solution (Invitrogen-Gibco, Paisley, UK). Tissue was dissociated enzymatically with trypsin–ethylenediaminetetraacetic acid (Sigma-Aldrich) and mechanically with fire-polished Pasteur pipettes. Cells were subsequently plated into poly-d-lysine (100 ng/ml)-coated 24-well plates (5 × 105 cells/well) (Sigma-Aldrich) containing medium I [Dulbecco's Modified Eagle's Medium (with 4.5 g/l glucose and l-glutamine), 10% foetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin] for 7 h at 37 C in a humidified atmosphere containing 5% CO2. Medium was changed to Neurobasal-A containing 2% B27 supplement, 2 mm l-glutamine and 1 µg/ml of gentamycin, and cells were incubated overnight prior to transfection. All culture cell media and cell culture reagents were purchased from Invitrogen-Gibco. Reporter constructs (1 µg) or pGL3p (1 µg) and 10 ng of modified pMLuc-2 (renilla luciferase, for use as an internal control, also from Promega) were cotransfected into cultures using Turbofect (for transformed human cell lines) or EXGen 500 transfection reagent (for rat cortical cultures) following the manufacturer's guidelines (both transfection reagents from Fermentas, Hanover, Germany). Cultures of human SH-SY5Y, SK-N-MC and HEK-293 cells were maintained as adherent monolayers and cultured as previously described (D'Souza et al. 2004). Internal negative (pGL3basic plasmid without a promoter or an enhancer) and positive (pGl3control plasmid bearing a SV40 virus enhancer and promoter, both from Promega) controls were included in each transfection experiment in primary cultures and clonal cell lines (luciferase levels generated by these controls are detailed in Table 1).

Table 1. Recorded luciferase values generated by control plasmids pGL3basic and pGL3control (Promega) obtained after transfecting (1 µg) in SH-SY5Y, SK-N-MC and HEK-293 cells
 pGL3_controlpGL3_basic
  1. Values are averages based on three independent transfections (each with three replicates).

SHSY5Y11.13 ± 6.90.05 ± 0.04
SK-N-MC66.91 ± 22.150.03 ± 0.004
HEK-29327.9 ± 9.390.02 ± 0.05

For cotransfection with CTCF or USF2, rat and human cells were transfected with 1 µg of D4Sp or D4Lp reporter gene and 1 µg of expression vector h-CTCF/h-USF2. Cells were harvested and assayed using the Dual Luciferase Reporter Assay System (Promega). In brief, 100 µl of renillin and firefly luciferase were added to 40 µl of cell lysate. Luminescence generated at primary cultures experiment was measured using a Glomax 96 microplate luminometer (Promega) (1 second of integration time). For the human cell line experiments, the luminescence was measured using the luminometer Fluoroskan Ascent FL (Labsystems, Helsinski, Finland) using similar conditions. Mean and SEM (standard error of the mean) were calculated from the results of three independent experiments performed in triplicate or quadruplicate.

Estimation of effect of overexpression on reporter gene constructs activity

There were three steps of normalization for each experiment. From each well, we obtained two sets of values derived from the Firefly and Renilla luciferase assays. In step 1, each Firefly luciferase value was normalized to its corresponding Renilla luciferase values. In step 2, we normalized values from step 1 by the average normalized values from the pGL3p construct. In the next step, in order to compare the fold changes in transcriptional activity between the different cotransfection experiments, the values from step 2 of each cotransfection experiment were normalized by the average value of the corresponding luciferase constructs (e.g. D4Sp + CTCF/D4Sp). The basal activities of D4Sp, D4Lp and pGL3p were expressed as 1.0.

Statistical analysis

Significant differences in luciferase activity registered in cotransfected cells were determined using a two-tailed Student's t-test (*P > 0.05; **P < 0.01; ***P < 0.001).

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

To exemplify the potential effect of the tandem repeat caused by the 5′VNTR in the DRD4 gene, we searched for putative TF-binding sites (TFBS) using AliBaba 2.1 (Fig. 1). This in silico analysis suggested the presence of six binding sites for different types of TFs, including: specificity protein 1 (Sp1), activating protein 2 (AP2), olfactory neuron-specific TF (Olf1), E47 protein (E47), Ying Yang 1 TF (YY1), Neurofibromin 1 (NF1) and CCAAT/enhancer binding protein alpha (C/EBP alpha). In addition, visual inspection of the sequence revealed the presence of E-boxes. The consensus sequence for the E-box element is CANNTG, with a palindromic canonical sequence of CACGTG (indicated by rectangular box in Fig. 1), which are typical sites for binding of the heterodimer complex USF1/USF2.

Transcriptional activities of the 5′ region VNTR of the DRD4 gene are repressed by overexpression of CTCF in primary cultures of neonate rat cortex

We established whether the two DRD4 5′ VNTR constructs cloned into a reporter gene plasmid pGL3p, termed as D4Lp and D4Sp, were capable of supporting reporter gene expression under basal culture conditions in primary cultures of neonate rat cortex (Fig. 2). In these experiments, D4Sp and D4Lp supported no luciferase expression.

image

Figure 2. Transcriptional activities of the DRD4 5′ VNTR variants in cortical cultures. Basal transcriptional levels of the D4Lp and D4Sp constructs were calculated as the transcriptional activities supported by each VNTR construct over pGL3p activity. Regulatory effects of CTCF were calculated as the fold change (expressed as a percentage) relative to reporter activity of each respective D4Sp and D4Lp construct at basal conditions.

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We then tested the effect of overexpression of the human CTCF protein on transcriptional activities of D4Sp and D4Lp constructs. Our cotransfection experiments showed that the basal transcriptional activities of D4Lp and D4Sp constructs were significantly repressed when CTCF was overexpressed compared with basal conditions (86% and 79%, respectively, Student's t-test, control + CTCF vs. D4Sp + CTCF, P < 0.001; control + CTCF vs. D4Lp + CTCF, ***P < 0.001, df = 4, Fig. 2). However, there were no significant differences in the levels of repression induced by CTCF overexpression between the D4Lp and D4Sp constructs in this cell model (Student's t-test, P = 0.06).

Differential activity of VNTR constructs is specific to human neuronal cell types

We tested the effect of overexpression of TFs USF2 and CTCF on the transcriptional activities of the D4Lp and D4Sp in a different cell environment by cotransfecting constructs in cultures of human neuroblastoma cell lines SK-N-MC and SH-SY5Y (Fig. 3a,b) and in the non-neuronal HEK-293 cells (Fig. 3c). In brief, the results of our experiments showed that overexpression of either CTCF or USF2 elicited allele-specific luciferase expression of the D4Lp and D4Sp constructs, in both the SH-SY5Y and SK-N-MC cells but not HEK-293 cells. In SK-N-MC cultures (Fig. 3a), cotransfection with USF2 correlates with a significant upregulation of both D4Sp and D4Lp transcriptional activities compared with constructs transfected alone (upregulation of D4Sp = 4.5-fold and of D4Lp = 13.79-fold, Student's t-test, **P < 0.01, df = 4). This allele-specific activation is not observed for overexpression of CTCF.

image

Figure 3. The DRD4 5′ VNTR variants exhibit stimulus-specific transcriptional activity in human neuronal cell types. Putative regulatory effects of CTCF and USF2 on the activities of the D4Sp and D4Lp constructs on cultures of SK-N-MC cells (a), SH-5YSY cells (b) and HEK-293 cells cultures (c). Effects of CTCF or USF2 were calculated as the fold change (expressed as a percentage) relative to reporter activity of each respective D4Sp and D4Lp construct at basal conditions.

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The experiments in SH-SY5Y cells (Fig. 3b) showed that overexpression of CTCF and USF2 correlated with allele-specific effects on the transcriptional activity of both D4Sp and D4Lp constructs. In brief, cotransfection with USF2 induced similar levels of upregulation of the transcriptional activities of both constructs (2.6- and 1.9-fold increase, respectively, compared with basal activity). Upregulation induced by overexpression of USF2 was only significant for D4Sp (**P < 0.01). Overexpression of CTCF induced upregulation of D4Sp and D4Lp transcriptional activities in 5.5- and 1.6-fold, respectively (compared with basal transcriptional levels). As above, this upregulation was only significant for D4Sp (Student's t-test, ***P < 0.001, df = 4). Furthermore, the difference between fold increase induced by CTCF overexpression on D4Sp and D4Lp activity was statistically significant (Student's t-test, +++P < 0.001, df = 4).

In cultures of the human embryonic kidney cell line HEK-293 (Fig. 3c), cotransfection experiments with CTCF and USF2 induced significantly upregulated transcriptional activities of both D4Sp and D4Lp constructs. CTCF overexpression was accompanied by the greatest enhancement of luciferase expression (compared with basal levels, effect of CTCF on D4Sp = 4.7-fold increase and in D4Lp = 3.8-fold increase, Student's t-test, ***P < 0.001, df = 4), whereas USF2 induced mild upregulation of D4Sp and D4Lp transcriptional activities (1.4-fold increase on both constructs, ***P < 0.001, df = 4). However, there was no allele-specific effect on the upregulation of luciferase activity induced by CTCF or USF2 overexpression in HEK-293 cells.

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

In this study, we characterized the two most common alleles of the DRD4 5′ VNTR (‘L’ and ‘S’) transcriptional response to challenges (i.e. overexpression of TFs) in two different cellular models. Our findings show that each allele responds differently to challenges, and this ability to modify their transcriptional profile was specific to human neuronal cell types tested and not observed in neonate rat primary cultures of cortical neurons.

In the primary neuronal cultures, D4Sp and D4Lp acted as repressor domains of the basal transcriptional activity supported by the SV40 promoter when cotransfected with CTCF. Replication of the same experiment in human clonal cells showed the ability of these D4Sp and D4Lp constructs to act as enhancers of the reporter gene expression. We did not observe differences between basal luciferase levels generated by the D4Sp and D4Lp constructs in any of the three cell lines, nor in the primary cultures. These results contrast with previous studies (D'Souza et al. 2004) and likely reflect the use of a different plasmid construct backbone.

Allele-specific transcriptional activity was observed when the D4Lp and D4Sp constructs were exposed to stimuli in human clonal cells of neuronal origin only. In SK-N-MC cells, USF2 overexpression induced considerable stronger upregulation of D4Lp than that of D4Sp transcriptional activities (luciferase levels increased 13.79- vs. 4.5-fold, respectively). In SH-SY5Y cells, only the transcriptional activities of the D4Sp construct were significantly regulated by overexpression of CTCF and USF2. In HEK-293 cells, the overexpression of CTCF or USF2 resulted in significant increase of luciferase activities with D4Sp and D4Lp constructs. However, in contrast to the observed effect in SK-N-NC and SH-SY5Y cells, in HEK-293 there was no allele-specific effect of the 5′ region VNTR in the DRD4 gene.

The data reported here show that DRD4 5′ VNTR variants act as repressors of luciferase reporter gene activity in primary cultures of rat neonate cortex. This is distinct from their activity in human neuroblastoma cells in which they enhance luciferase reporter gene activity. The findings lend support to the hypothesis that the DRD4 5′ VNTR has a role as a dynamic regulatory element of gene expression. The authors do not imply that the observed effects of overexpression of CTCF or USF2 on the transcriptional activities of the D4Lp and D4Sp constructs indicate a direct interaction between CTCF/USF2 and this VNTR locus. The aim of the cotransfection experiments is simply to exemplify how sequence variation conferred by the presence of this VNTR could potentially generate differences in the transcriptional profile of DRD4 gene expression, when exposed to stimuli in a different cell types. The action of the TFs could be direct or indirect.

The specific binding of CTCF/USF2 to the DRD4 promoter 5′ VNTR is outside the scope of this report, and confirmation of such interaction or interaction with other factors should be conducted by appropriate assays, i.e. electrophoretic mobility shift assay (EMSA) or chromatin immunoprecipitation (ChIP). Indeed, in silico analysis of the 120-bp unit forming the VNTR using the AliBaba 2.0 program revealed the putative presence of several known TFs: Sp1, E47, Olf1, Ap2, YY1, NF1, E-boxes and C/EBPα (Fig. 1). The interaction of most of these TFs with the DRD4 gene and this VNTR has not been shown. However, our preliminary analysis (Fig. 1) suggests that the presence of a VNTR in this locus could potentially direct differential regulation of the DRD4 gene and that there are a number of potential candidate TFs that could mediate that effect in response to challenge as previously suggested (D'Souza et al. 2004; Ronai et al. 2004; Seaman et al. 1999). However, prediction of TFBS is based on sequence similarity, and therefore often inaccurate (Tompa et al. 2005).

Suggestions of CTCF/USF2-DRD4 5′ VNTR interactions have been implicated at this locus as recent data from CTCF ChIP-seq (Rosenbloom et al. 2010) conducted in a variety of cells showed the presence of CTCF binding. Moreover, such wide variability in the magnitude and direction of the response to CTCF and USF2 overexpression exhibited by the D4Lp and D4Sp constructs is indicative of regulation in trans. In other words, the cell-specific composition and concentration of TFs were affected by the regulatory cascade triggered by overexpression of CTCF or USF2, and therefore are likely to be responsible for the diversity of transcriptional response shown by the DRD4 5′ VNTR variants.

In this report, overexpression of USF2 or CTCF did not have a significant impact on basal expression levels supported by pGL3p. This is in agreement with previous reports from our group (e.g. Ali et al. 2010; Roberts et al. 2007). Therefore, we conclude that the variations in luciferase activity exhibited by the plasmids bearing the short (S) and long (L) variants of the VNTR are not caused by interactions with the plasmid pGL3p backbone.

In the light of our previous and present results, we suggest that the duplication of a 120-bp sequence upstream of the DRD4 promoter would affect potential recruitment of TFs, potentially modulating promoter activity and therefore contributing to fine-tuning of DRD4 gene expression in neuronal tissue. It is interesting but nonetheless speculative that the action of CTCF, which is known to modulate epigenetic parameters, could mediate medium- to long-term changes in the expression of the endogenous DRD4 gene and warrants further investigation. Importantly, variation in the transcriptional profile of this VNTR locus could contribute to variation in DRD4 gene expression patterns in human populations, and potentially contribute to variation of dopaminergic-related phenotypes.

Transcriptional regulation directed by DRD4 polymorphisms is far from defined. For example, one study showed the common SNP polymorphism (−521 C/T) to affect promoter activity in Y79 cells (Kereszturi et al. 2006). However, further studies found no functional transcriptional effect of this SNP on DRD4 promoter constructs in several neuronal and non-neuronal human cells (Kereszturi et al. 2006; Okuyama et al. 2000) or in post-mortem brain tissue (Simpson et al. 2010). Furthermore, we have recently reported the presence of a transcriptional regulator in the first intron of the DRD4 gene (Paredes et al. 2011).

Given that the association of the 5′ VNTR polymorphism with neuropsychiatric disorders is conflicting (e.g. Kereszturi et al. 2006; Nakajima et al. 2007; Todd et al. 2001), it is possible that this VNTR could contribute to DRD4 gene regulation, at specific developmental contexts, or under specific stimuli. Therefore, our study highlights the importance of addressing environmental and developmental factors and dynamics of transcriptional regulation when carrying out genetic association studies.

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  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
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Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

J.P.Q. acknowledges the BRC for funding for the studies carried on rat frontal cortex cells. U.M.D. and U.M.P. thank the SGDP Centre funds for research for funding the project on the functional studies in human cell lines. The authors do not declare any conflict of interest.