www.elsevier.com/locate/brainres

Brain Research 1000 (2004) 148–155

Research report

Expression analysis of brain-derived neurotrophic factor (BDNF) mRNA

isoforms after chronic and acute antidepressant treatment

Mario Altieria,*, Francesca Marinib, Roberto Arbana, Giovanni Vitullia, Birger O. Janssona,1

aCentre of Excellence for Drug Discovery, Psychiatry, GlaxoSmithKline Research Centre, via Fleming 4, I-37135 Verona, ItalybDepartment of Medicine and Public Health, Section of Pharmacology, School of Medicine, University of Verona,

Policlinico Borgo Roma, I-37134 Verona, Italy

Accepted 18 December 2003

Abstract

The neurotrophin brain-derived neurotrophic factor (BDNF) is considered to be a key factor for neuronal survival, differentiation and

plasticity. According to a proposed hypothetical model BDNF expression might play a central role in the pathogenesis of depression. The

BDNF gene is rather complex in its structure and it can express four different mRNA isoforms by alternative splicing, each producing the

same protein. This might reflect fine tuning of gene regulation by different signalling networks. Since the BDNF gene has been reported to be

upregulated by antidepressants, the expression of the four BDNF mRNA isoforms was measured by real-time quantitative RT-PCR in rat

hippocampi after chronic and acute treatment with the antidepressant drug fluoxetine and GR205171, a selective NK-1 receptor antagonist

with anxiolytic-like properties. The aim of this study was to test the hypothesis of differential regulation of the mRNA isoforms by those

compounds. Our results indicate that the expression of BDNF mRNA isoforms is not affected by chronic or acute treatment with fluoxetine or

GR205171.

D 2004 Elsevier B.V. All rights reserved.

Theme: Development and regeneration

Topic: Neurotrophic factors: expression and regulation

Keywords: Gene expression; Alternative splicing; Hippocampus; Depression; Fluoxetine; Rat

1. Introduction in combination with other genetic and environmental factors

Brain-derived neurotrophic factor (BDNF) belongs to the

family of neurotrophins [2], secreted polypeptides which

regulate neuronal survival [8] differentiation and plasticity

[22]. The regulation of BDNF has been proposed to be

involved in the pathogenesis of depression. According to

this model [6], chronic stress leads to a long-term decreased

expression of BDNF, which makes hippocampal neurons

(CA3 pyramidal cells, CA1 pyramidal cells and dentate

gyrus granule cells) more vulnerable to several types of

neuronal insult with consequent atrophy or damage of these

cellular types. In the long term this pathological mechanism,

0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2003.12.028

* Corresponding author. Tel.: +39-45-921-8548; fax: +39-45-921-

8047.

E-mail address: mario.2.altieri@gsk.com (M. Altieri).1 Present address: Affibody AB, Box 20137, SE-161 02 Bromma,

Sweden.

could result in the development of depressive disorders [6].

Chronic (but not acute) treatment with major classes of

antidepressants has been reported to increase the expression

of the rat BDNF gene in CA3 and dentate gyrus, [14,15] and

to change the BDNF immunoreactivity in rat hippocampus

in a dose dependent manner [24], thus directly linking

BDNF to the mechanism of antidepressant action. An

increased expression of BDNF could reverse the proposed

pathological mechanism by improving the survival of hip-

pocampal neurons [6] and restoring hippocampal neuro-

genesis [11].

The rat BDNF gene comprises five exons: I, II, III, IV

and V (Fig. 1).

Exons I, II, III and IV are non-coding (exon I contains an

extra in-frame initiation codon) and the whole protein open

reading frame (ORF) is encoded within exon V. Four

different mRNA isoforms can be expressed by splicing each

of the non-coding exons to exon V [23].

Fig. 1. The structure of the rat BDNF gene with its mRNA transcripts. The gene structure is shown at the top, with boxes representing the exons and lines

representing the introns. Broken lines represent introns larger than 10 kb. A full box represents the region coding for the mature protein, while a hatched box

represents the region coding for the prepro-BDNF protein. The position of the CRE-like element is indicated. The eight transcripts are shown at the bottom,

with the alternatively spliced gene regions represented as in the gene structure. Adapted from reference [23].

M. Altieri et al. / Brain Research 1000 (2004) 148–155 149

These transcripts are expressed by four independent

promoters each controlled by a different signalling network

[23]. The exon III-specific mRNA isoform is the major

Ca2 + inducible form in cortical neurons and it is regulated

by the transcription factor CREB. (A CRE-like element is

present in the exon III promoter) [21]. Each non-coding

exon has one promoter located in its 5V flanking region [21].In addition, each transcript can either have a longer or a

shorter 3V-UTR (untranslated region), which is also encoded

in exon V [23]. Therefore, each mRNA isoform can either

be long (4.4 kb) or short (1.8 kb), using two alternative

polyadenylation sites located within the untranslated region

[23].

With this peculiar structure the BDNF gene can express

up to eight different types of mRNA molecules, all

producing the same protein. It is likely that this transcrip-

tional complexity allows a very precise regulation poten-

tially responding to signalling cascades in different cell

types.

We hypothesised that antidepressants might differentially

regulate the expression of the four BDNF promoter-specific

mRNA transcripts. Exon I mRNA transcript was indeed

reported to be significantly increased in the rat hippocam-

pus by chronic treatment with the antidepressant drug

tranylcypromine while exon II transcript was not affected

[18]. However, extensive studies on how antidepressants

might specifically regulate the expression of BDNF mRNA

isoforms were not yet available in the literature. Very

recently, during the preparation of this manuscript, a work

on the effect of antidepressants and electroconvulsive

seizure on the rat BDNF mRNA isoforms was published

[5].

With the aim of testing our hypothesis, we therefore

measured the expression of the four BDNF promoter-spe-

cific mRNA transcripts in rat hippocampi after acute and

chronic treatment with fluoxetine, a selective serotonin

reuptake inhibitor (SSRI), and GR205171, a selective NK-

1 receptor antagonist. [7,12] Fluoxetine is a benchmark

SSRI, widely prescribed as antidepressant, reported to

upregulate BDNF mRNA and to increase neurogenesis in

rat hippocampus [11,15]. NK-1 receptor antagonists have

been recently proposed as potential therapeutic agents in the

treatment of anxiety and depression [10] and have been

hypothesised to stimulate hippocampal neurogenesis [17].

In this work, we used real-time quantitative RT-PCR, which

is considered one of the most precise and sensitive methods

for the quantification of gene expression [20].

2. Materials and methods

2.1. Animals and drug treatments:

Male Sprague–Dawley rats (200–250 g, Charles Riv-

er, Italy) were housed on a 12-h light/dark cycle (light at

6:00 am), with free access to food and water. The animals

were divided into six experimental groups (n = 4): three

groups received a chronic drug treatment (21 days) while

the other three received an acute treatment (1 day). Rats were

orally administered fluoxetine (5mg/kg once a day),

GR205171 (5mg/kg twice a day) or vehicle (sterile water).

Plasma samples from parallel groups (n= 3) were taken on

days 1, 9 and 21, respectively, for pharmacokinetic monitor-

ing. Peak levels (Cmax) were measured 2–5 hours after

M. Altieri et al. / Brain Research 1000 (2004) 148–155150

administration. Fluoxetine and GR205171 minimum levels

(Cmin) were measured respectively 24 and 8 h after adminis-

tration. Norfluoxetine (N-demethylated fluoxetine metabo-

lite) levels were measured on day 21 only. Drug levels were

measured using high-performance liquid chromatography

coupled to mass-spectrometry (HPLC-MS). The GR205171

dose was increased to 10mg/kg twice a day on day 15 in order

to keep the plasma level of the drug constant throughout the

treatment, since a tendency to decrease was observed on day

9. The animals were sacrificed by decapitation 24 h after the

last drug administration. Hippocampi were dissected from the

left side of brains immediately after sacrifice, quickly frozen

in dry ice and stored at � 80 jC.This work was performed under a Project License

obtained according to Italian law (art. 7, Legislative Decree

No. 116, 27 January 1992), which acknowledged the

European Directive 86/609/EEC, and was fully compliant

with GlaxoSmithKline policy on the care and use of

laboratory animals and related codes of practice.

2.2. RNA isolation

Frozen brain tissues were homogenised in tubes contain-

ing beads (Lysing Matrix D, Q-Biogene USA) and RLT

buffer (Qiagen) supplemented with 1% (v/v) h-mercaptoe-

thanol, using a Ribolyser (Hybaid) apparatus (20 s at speed

6). After the homogenisation was complete, the tubes were

centrifuged in a microfuge (14,000� g, 3 min) at room

temperature and the clear tissue lysate was collected. Total

RNAwas then isolated using the RNeasy Midi Kit (Qiagen)

by following the manufacturer’s protocol. Potential trace

amounts of residual genomic DNA were removed by diges-

tion with RNase-free DNase (Qiagen) included in the

purification method according to the Rneasy Midi/Maxi

Handbook (Qiagen). Purified total RNA was eluted in

RNase-free water and stored at � 80 jC. RNA concentra-

tion was measured in a GeneQuant II spectrophotometer

(Pharmacia) at 260 nm, and the quality of samples was

determined by analysis on 1.2% agarose electrophoresis gels

Table. 1

List of primers

Rat target gene GenBank Accession No. Primers Sequence

h-actin V01217 Forward TGAACC

Reverse CTCATA

GAPDH AF106860 Forward CAAGGT

Reverse GGGCCA

BDNF exon I X67106 Forward GCTGGT

Reverse CCAGGT

BDNF exon II X67106 Forward GGCTGG

Reverse CCGGTG

BDNF exon III X67107 Forward CACTGA

Reverse TGTACT

BDNF exon IV X67107 Forward GGCGCA

Reverse TCAGGG

(containing 0.22 M formaldehyde), after staining with

SYBR Gold (Molecular Probes).

2.3. DNA oligonucleotide primers

Primers specific to rat BDNF isoforms were designed

from public sequences using Primer Express v.1.00 software

(Applied Biosystems) (Table 1), and information on the

gene expression was from reference [23].

The sequence of primers for rat h-actin were obtained

from Sala et al. [19] while the primer sequences for the rat

GAPDH gene were from P. Murdock (Quantitative Expres-

sion Dept. GlaxoSmithKline, Stevenage, UK). All DNA

oligonucleotide primers were custom synthesised by Proligo

Europe.

2.4. cDNA synthesis and RT-PCR

Reverse transcription (RT) reactions were performed in

duplicate as follows: 1 Ag oligo(dT)12–18 primers (Invitro-

gen) and 250 ng random hexamers (Amersham Biosciences )

were hybridised to 300 ng total RNA in 10 Al volume by

heating up to 95 jC for 30 s and quick chilling on ice. First

strand cDNA was then synthesised by incubating the hybri-

dised RNA at 37 jC for 45 min with 20 units RNAguard

(Amersham Biosciences), dGTP, dTTP, dCTP, dATP (1 mM

each), and Superscript II reverse transcriptase (200 units,

Invitrogen) in 20 Al 1�1st strand buffer (Invitrogen) con-

taining 10 mM 1,4-dithio-DL-threitol (DTT). cDNA reactions

were then diluted fivefold in nuclease-free water. The quality

of cDNAwas checked by RT-PCR using rat h-actin specific

primers, which were designed in order to distinguish be-

tween an amplified product from cDNA (393 bp) and a

product from genomic DNA (858 bp). Using a Robocycler

(Stratagene), the RT-PCR reactions were performed in 30

Al 1�Gold buffer (Applied Biosystems), 2 Al cDNA, for-ward and reverse primers (500 nM each), dATP, dCTP,

dGTP, dTTP (250 AM each), 1.5 mM MgCl2, 0.75 units

AmpliTaq Gold DNA polymerase (Applied Biosystems), for

(5V! 3V) Tm (jC) Amplicon size (bp)

CTAAGGCCAACCGTG 63 393 (cDNA) 858

(genomic DNA)

GCTCTTCTCCAGGG 60

CATCCATGACAACTTTG 61 90

TCCACAGTCTTCTG 63

GCAGGAAAGCAAC 60 76

AAGAAAAGCTTCGCC 61

AATAGACTCTTGGCA 61 90

GCTAGATCCTGGA 62

AGGCGTGCGAGTATT 61 83

CCTGTTCTTCAGCAAAGAA 63

GGGACCAGG 57 73

TCCACACAAAGCTC 60

Table 2

Drug levels in rat plasma

Day 1 Day 9 Day 21 Ki

Fluoxetine 2.00

Cmax 97.48 224.64 297.57

Cmin 27.85 20.52 3.66

Norfluoxetine 1.9

Cmax 637.4

Cmin 228.7

GR205171 0.32

Cmax 95.12 70.20 107.07

Cmin 83.08 35.86 31.06

Values (nM) are mean from three animals. Ki values (nM) for fluoxetine, its

N-demethylated metabolite norfluoxetine and GR205171 were derived from

the literature [7,16].

M. Altieri et al. / Brain Research 1000 (2004) 148–155 151

40 cycles (94 jC 1 min, 55 jC 1 min,72 jC 1 min) after an

initial step at 94 jC, 12 min to activate the DNA polymerase.

Only the former small amplicon was detected in all samples

on 4% Nu-Sieve (FMC) agarose gel electrophoresis, but not

the latter larger amplicon, demonstrating no contamination

of genomic DNA (data not shown).

2.5. Real-time quantitative RT-PCR

The amplification reactions were performed in triplicate in

30 Al 1� SYBR Green PCR master mix (Applied Biosys-

tems), 5 Al cDNA, forward and reverse primers (300 nM

each) using an ABI PRISM 7700 Sequence Detector (Ap-

plied Biosystems). The cycling parameters were: 95 jC 15 s,

60 jC 1min, 40 cycles, after one initial step at 95 jC, 10 min,

which was set to activate the AmpliTaq Gold polymerase.

Fig. 2. The expression of BDNF mRNA isoforms reported for each animal. Bars re

hippocampal total RNA sample, each measured in triplicate). Error bars represent t

acute fluoxetine. FlxCh: chronic fluoxetine. GR205171Ac: acute GR205171. GR

Ct (cycle threshold) values were calculated by the SDS

software v1.9 (Applied Biosystems) from fluorescence read-

ings, and these values were converted into copy number per

ng total RNA input, by using a standard curve of serially

diluted amounts of rat genomic DNA (Clontech). Primer

specificity was checked by analysing PCR products from rat

genomic DNA using 4% Nu-Sieve agarose gel electropho-

resis, and the dissociation curve method according to the

Applied Biosystems protocol.

2.6. Data analysis

The average copy number per ng total RNA input was

calculated for each sample (2 RTs per sample, 3 real-time

quantitative RT-PCR reactions per RT). An analysis of

covariance (ANCOVA) was performed on these data. The

expression data for the housekeeping gene GAPDH (refer-

ence gene) were used as covariate under the hypothesis that

it was not affected by drug treatments, in order to remove

the effects due to RNA sample quality from the analysis.

This method has been described in Ref. [1].

3. Results

3.1. Drug plasma levels in animals

Fluoxetine, its N-demethylated metabolite norfluoxetine

and GR205171 resulted to keep their plasma concentration

in treated animals above their Ki throughout the treatment,

as shown in Table 2.

present an average value from 6 datapoints (2 reverse transcriptions of each

he standard deviation. CtrlAc: acute control. CtrlCh: chronic control. FlxAc:

205171Ch: chronic GR205171.

Table 3

Results from ANCOVA analysis

BDNF mRNA isoform SS DF MS F p

Exon I GAPDH 0.029 1 0.029 5.048 0.0382

Treatment 0.103 5 0.021 3.541 0.0224

Error 0.099 17 0.006

Exon II GAPDH 0.000 1 0.000 0.027 0.8715

Treatment 0.055 5 0.011 2.937 0.0433

Error 0.063 17 0.004

Exon III GAPDH 0.083 1 0.083 18.707 0.0005

Treatment 0.018 5 0.004 0.809 0.5589

Error 0.075 17 0.004

Exon IV GAPDH 0.044 1 0.044 9.527 0.0067

Treatment 0.045 5 0.009 1.947 0.1391

Error 0.079 17 0.005

M. Altieri et al. / Brain Research 1000 (2004) 148–155152

3.2. Expression of BDNF mRNA isoforms in rat hippocampi

The expression of BDNF mRNA isoforms is shown in

Fig. 2. We found that exon III mRNAwas the most abundant

form in the rat hippocampus, followed by exon II, exon IV

and exon I (25.2%, 13.7% and 3.7% of exon III, respective-

ly). This expression profile is in good agreement with

Fig. 3. Fold changes from controls (bar centres) and their respective 95% confid

GAPDH), following acute (a) and chronic treatment (b). Control baseline value =

previously published data [23]. The expression of the rat

gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

was measured in the same samples as an internal reference in

order to normalise the data for RNA quantity and quality.

3.3. Statistical analysis of data

The gene expression data were statistically evaluated by

analysis of covariance (ANCOVA), considering the expres-

sion of GAPDH as a covariate, under the hypothesis that it

was not affected by the treatments. The covariance efficien-

cy factor (CEF) for groups was determined to be 0.992, thus

proving independence from treatments. The results from

ANCOVA are shown on Table 3.

Post-hoc analyses (Dunnett’s test, p < 0.05) were per-

formed by comparing acute treatment groups to the acute

control group and chronic treatment groups to the chronic

control group (Fig. 3).

In our experimental conditions chronic treatment with

either fluoxetine or GR205171 did not show any statisti-

cally significant effect on the expression level of the four

BDNF mRNA transcripts (Table 4). Acute treatment of

ence intervals (bar edges), for the BDNF mRNA isoforms (normalized to

1.00.

Table 4

Fold changes with their p-values (Dunnett’s test)

Treatment BDNF mRNA

isoform

Fold change

from control

p-value

Acute fluoxetine Exon I 1.199 0.471

Exon II 1.144 0.542

Exon III 1.035 0.998

Exon IV 0.733a 0.046

Acute GR205171 Exon I 0.764 0.159

Exon II 0.913 0.827

Exon III 0.890 0.730

Exon IV 0.803 0.222

Chronic fluoxetine Exon I 1.068 0.977

Exon II 0.986 1.000

Exon III 1.155 0.564

Exon IV 1.018 1.000

Chronic GR205171 Exon I 0.852 0.582

Exon II 0.953 0.983

Exon III 1.024 1.000

Exon IV 1.048 0.991

a P < 0.05.

M. Altieri et al. / Brain Research 1000 (2004) 148–155 153

the two antidepressants also had no effect, except for a

downregulation of exon IV mRNA by fluoxetine (� 27%,

p = 0.046).

4. Discussion

In this work, the expression of four BDNF mRNA iso-

forms was analysed after chronic and acute treatment with

fluoxetine and GR205171, a specific NK-1 receptor antago-

nist. An expression analysis of the BDNF mRNA isoforms

after treatment with a NK-1 receptor antagonist has never

been reported before. The data indicate that neither chronic

nor acute treatment with these two drugs affected the expres-

sion of BDNF mRNA isoforms in the rat hippocampus. Only

acute fluoxetine had a small but significant negative effect on

the expression of exon IV-specific isoform, but it is not

known if this effect has any biological relevance.

Our investigation did not confirm a previous study,

published by Nibuya et al. [15], who reported an increased

BDNF mRNA expression in the rat hippocampus after

chronic treatment with fluoxetine. We cannot explain the

reason for this different result, but similar discrepancies have

already been observed by the authors of three other pub-

lished studies. Chronic treatment with fluoxetine decreased

BDNF mRNA expression in the rat hippocampus in one case

[13], while it had no significant effect on the expression of

BDNF mRNA in the mouse hippocampus [3]. Finally, in the

very recently published new work, chronic fluoxetine treat-

ment had no significant effect on the expression of the BDNF

mRNA isoforms in the rat hippocampal regions (dentate

gyrus, CA1 and CA3) [5], in full agreement with our work.

Possible methodological or biological factors should be

taken into account in an effort to explain these discordant

results. Technical differences in animal treatment (oral

administration used in this study, minipumps [13], or i.p.

administration [3,5,15]) could result in different drug phar-

macokinetics and therefore alter the exposure to drugs.

Chronic treatment with the antidepressant venlafaxine has

been recently reported to increase BDNF immunostaining at

a lower dose, but to decrease it at a higher dose in rat

hippocampus [24]. Moreover, different animal treatments

could also introduce different level of stress. Chronic

treatment with i.p. injection, for example, could be more

stressful than oral administration. It could be hypothesised

that the association of the treatment factors with different

techniques used to measure gene expression could lead to

very different results. Four different methodologies have

been used to measure gene expression in these studies: real-

time quantitative RT-PCR, used only in our study, Northern

blot [15], RNase protection assay [3] and in situ hybrid-

isation [5,13,15]. Real-time quantitative RT-PCR has the

advantage to be the most sensitive and reliable technique

used in gene quantitation [20] but, as Northern blot and

RNase protection assay, it requires the isolation of RNA

from a whole tissue area, even though it can be very small.

These techniques might fail to detect regional changes in

gene expression if the mRNA under study is diluted in the

RNA from other cellular types. In situ hybridisation, which

works directly on thin tissue slices without the need for

RNA isolation, is more appropriate for the detection of

regional changes in gene expression, even though its many

laborious steps can introduce a high level of variability.

Chronic treatment with fluoxetine increased the expression

of the BDNF gene in all hippocampal subfields (dentate

gyrus, CA3 and CA1) in the study by Nibuya et al. [15], but

only in one subfield (dentate gyrus) in another recently

published work [4], as shown in both studies by in situ

hybridisation. Differences in the treatment protocol have

been taken into consideration by the authors of the latter

study in an effort to explain this discrepancy [4]. Since

Nibuya et al. [15] used both in situ hybridisation and

Northern blot in whole hippocampus, our work should have

necessarily confirmed their results. In addition, since we

used real-time quantitative RT-PCR and we measured the

individual expression of the BDNF mRNA transcripts,

rather than the totality of the mRNA species, our assay

was much more sensitive than Northern blot. However, our

conditions might have induced an upregulation of BDNF

mRNA only in one hippocampal subfield and the real-time

quantitative RT-PCR might have failed to detect this change

in total hippocampal RNA. This possibility might explain

why we could not confirm the observed upregulation of the

BDNF gene after chronic treatment with fluoxetine [15],

and why this finding was not confirmed in the mouse

hippocampus using RNase protection assay [3]. This expla-

nation, however, remains theoretical and highly speculative.

Acute treatment with the antidepressant paroxetine or tra-

nylcypromine was reported to downregulate exon IV BDNF

mRNA specifically in the rat dentate gyrus [9]. In our study

we have also observed a small but statistically significant

downregulation of exon IV BDNF mRNA after acute

M. Altieri et al. / Brain Research 1000 (2004) 148–155154

fluoxetine treatment. Our result was quantitatively similar to

the reported work [9]. Furthermore, in the latest published

study on the effect of antidepressants on the rat BDNF

mRNA isoforms, it was observed, using in situ hybrid-

isation, a small decrease of exon IV BDNF mRNA in the

hippocampal CA1 subfield after acute treatment with fluox-

etine [5]. Although the biological meaning of this effect is

unclear, this would indicate that subtle regional changes in

the BDNF gene expression could have indeed been detected

in our assay. RNase protection assay did not detect any

change in the BDNF mRNA expression in the mouse

hippocampus after chronic treatment with fluoxetine, while

it could measure an upregulation of BDNF mRNA expres-

sion after chronic treatment with desipramine [3]. Therefore

it is unlikely that we could not detect any effect on BDNF

gene expression after chronic treatment with fluoxetine

because of our assay technique.

Finally, a recent work has shown that the time between the

last drug administration and the sacrifice can affect the

expression of the BDNF gene. It was found that chronic

treatment with antidepressants (fluoxetine included) de-

creased the BDNF mRNA expression in the rat hippocampus

at a post drug time of 4 h, while it increased the BDNF

mRNA expression at a post drug time of 24 h [4]. Therefore

this post drug time has become an additional variable to take

into account in order to explain the discrepancies of these

studies. In their very recent work [5], Dias et al. have

considered the post drug time as a possible reason to explain

why they could not confirm the work published by Nibuya et

al. [15]. They did not find any significant effect after chronic

fluoxetine treatment on the expression of the BDNF mRNA

isoforms in the rat hippocampus 2 hours after the last drug

administration [5], while Nibuya et al. [15], measured the

BDNF gene expression in the rat hippocampus 18 h after the

last drug administration. Doses and administration route

(i.p.) were the same in both studies. In our work, however,

we could not detect any significant effect on the expression

of the BDNF mRNA isoforms in the rat hippocampus 24

h after the end of chronic treatment with fluoxetine, given at

the same dose as in the quoted two studies but through an oral

administration route. Therefore, in our case it is unlikely that

the post drug time could account for the different result. In

conclusion, further studies are needed to develop a better

understanding of how BDNF might be involved in the

mechanism of antidepressant action. The effect of different

antidepressant classes on the regulation of BDNF mRNA

isoforms, how dose and post administration time influence

the drug effect on the regulation of the BDNF gene expres-

sion, and how this regulation is related to the antidepressant

mode of action need to be investigated more deeply.

Acknowledgements

The authors wish to thank Matteo Sartori, Lucia Carboni,

(GlaxoSmithKline, Italy) Paolo Repeto and Brian C. Bond

(Biostatistics and Data Sciences, GlaxoSmithKline, UK) for

their expert assistance; Joseph M. Rimland and Enrico

Domenici (GlaxoSmithKline, Italy) for critically reviewing

the manuscript. Enrico Domenici’s support for this work is

also acknowledged.

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