- Email: [email protected]
Differential effects of 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) on BDNF mRNA expression in rat frontal cortex and hippocampus
Neuroscience Letters 402 (2006) 126–130
Differential effects of 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) on BDNF mRNA expression in rat frontal cortex and hippocampus Rebeca Mart´ınez-Turrillas, Sonia Moyano, Joaqu´ın Del R´ıo, Diana Frechilla ∗ Department of Pharmacology, School of Medicine, University of Navarra, Aptdo. 177, 31080-Pamplona, Spain Received 8 February 2006; received in revised form 20 March 2006; accepted 24 March 2006
Abstract The serotonergic neurotoxin 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) produces rapid serotonin (5-HT) depletion in different areas of the forebrain after acute administration to rats and other animal species. We previously found that 5-HT depletion induced by acute MDMA treatment was transient in the frontal cortex, but not in the hippocampus, and recovery of cortical 5-HT levels correlated with an induction of CRE-binding activity and increased expression of tryptophan-hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis. As the brainderived neurotrophic factor (BDNF) stimulates the growth and sprouting of serotonergic neurons, we sought the possible involvement of this neurotrophin in the region-specific increase in TPH mRNA expression induced by MDMA. We here report that, 24–48 h after acute MDMA treatment, the expression of BDNF in the frontal cortex is increased by ∼33–70%, and the levels of the transcription factor phospho-CREB are also increased. In the hippocampus, however, a time-dependent decrease in BDNF mRNA expression (maximal decrease of ∼73%) is found in all subfields examined 2–7 days after treatment in spite of increased phospho-CREB levels, perhaps as a consequence of corticosterone release by the serotonergic neurotoxin. The differential regulation of BDNF mRNA expression in the two brain regions examined appears to account for the enhanced TPH expression and the recovery of 5-HT levels in the frontal cortex, but not in the hippocampus, after neurotoxin treatment. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: BDNF; MDMA; Serotonin; CREB
The amphetamine derivative, 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”), is a widely used recreational drug that, in rats and other animal species, induces a rapid reduction in the content of brain serotonin (5-HT) [2,22], in the activity of tryptophan-hydroxylase (TPH), the rate-limiting enzyme in 5-HT synthesis [23], and in 5-HT transporter density [2]. On chronic treatment, it has been repeatedly shown that MDMA produces a degeneration of serotonergic nerve terminals [2,19,22]. The cellular and molecular mechanisms involved in MDMA actions have been extensively studied although many aspects remain to be elucidated (see [7] for review). In a previous study [6], we found that acute MDMA treatment produced a rapid reduction of 5-HT content in rat frontal cortex and hippocampus. In the cortex, but not in the hippocampus, 5-HT levels
∗
Corresponding author. Tel.: +34 948 194700; fax: +34 948 194715. E-mail address: [email protected] (D. Frechilla).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.03.055
returned to normal values 48 h after injection, and this recovery was correlated with an increase in binding to the CRE consensus sequence and in mRNA levels of TPH. It is known that the neurotrophin brain-derived neurotrophic factor (BDNF), which is upregulated in response to various types of neuronal injury as a compensatory effect after brain damage [10], augments 5-HT synthesis by enhancing TPH mRNA levels [24]. Many other studies point to the mutual interactions between brain 5HT and BDNF (reviewed in [15]). This neurotrophin influences the survival and function of 5-HT neurons in the rat brain [5,13] whereas blockade of the 5-HT transporter and activation of 5HT receptor subtypes induces transcription of the BDNF gene [15]. Serotonergic neurotoxins such as p-chloroamphetamine (PCA) and 2 -NH2 -MPTP may alter the expression of BDNF mRNA and protein in the rat hippocampus and cortical areas [12,32]. A single study showed that neonatal administration of MDMA moderately increased BDNF protein on postnatal day 21 [11]. On the basis of the previously observed differential changes in 5-HT and TPH mRNA levels [6], the current study
R. Mart´ınez-Turrillas et al. / Neuroscience Letters 402 (2006) 126–130
sought to determine the possible effects of acute MDMA administration on BDNF mRNA expression in the rat frontal cortex and hippocampus. Given that MDMA also increased the activity of protein binding to CRE [6] and the known regulation by cAMP response element-binding protein (CREB) of the BDNFinduced gene expression in the cortex [28], we also looked for eventual changes in the levels of phospho-CREB in response to the neuronal injury caused by MDMA. Male Wistar rats (Harlan, Barcelona, Spain) weighing 200–220 g were used. Animals were housed three to a cage in a temperature-controlled room (22–23 ◦ C) with a 12 h light/dark cycle and had free access to food and water. All procedures were in accordance with the guidelines established by the normative of the European Community of November 24, 1986 (86/609/EEC). Rats were injected with saline (5 ml/kg, i.p.) or with a single dose of MDMA.HCl (10 mg/kg, i.p.) and killed by decapitation at different times. The brains were removed and placed on ice. Western-blot analyses were carried out with fresh tissue from the frontal cortex and hippocampus. In rats assigned to in situ hybridization studies, the brains were immediately frozen in isopentane on dry ice. All tissues were stored at −80 ◦ C until use. The levels of 5-HT were measured in rat brain regions by high-performance liquid chromatography with electrochemical detection, as described [20]. For in situ hybridization, 14 m coronal sections were cut serially with a cryostat at the level of frontal cortex (3.7 mm relative to bregma) and dorsal hippocampus (−3.3 mm relative to bregma), according to the atlas of Paxinos and Watson. The sections were mounted onto Superfrost/Plus slides and then processed for the in situ hybridization of BDNF mRNA as previously described [14]. For detection of BDNF mRNA, an oligonucleotide complementary to bases of the coding region of rat BDNF exon V (Invitrogen Custom primers) was used: 5 -AGT TCC AGT GCC TTT TGT CTA TGC CCC TGC AGC CTT TGG TGT AAC-3 . The probe was 3 -tail labeled with ␣S[35 S]dATP (specific activity >1000 Ci/mmol, Amersham Pharmacia) using terminal deoxynucleotide transferase (Roche). A negative control included hybridization of sections using an oligonucleotide in the sense orientation, which showed minimal background signals. Hybridization densities were measured from films autoradiograms, with labeling densities calibrated relative to film images of 14 C-labeled standards (Amersham Pharmacia), using the Microcomputer Imaging Device (Imaging Research, St Catherines, Ontario, Canada). Densitometric values measured from four sections of each animal were averaged and expressed as nCi/g tissue, and mRNA abundance in drug treated groups was expressed as a percentage of their respective controls. Nuclear protein extracts from rat frontal cortex and hippocampus were prepared as previously described [6,26]. For Western blotting, 25 g of nuclear protein extract were separated onto SDS-polyacrilamide gel (12%). Proteins were transferred to a nitrocellulose membrane using a Trans-Blot® SD semidry (BioRad) system for 30 min at 16 V. The membranes were blocked and incubated at 4 ◦ C overnight with the primary antibody, diluted in 5% non-fat dried milk in TBST:
127
Table 1 Effect of MDMA on 5-HT levels in rat frontal cortex and hippocampus Time after treatment
Frontal cortex
Control 24 h 48 h 7 days
527.0 326.8 502.2 510.3
± ± ± ±
36.2 29.5* 40.5 29.2
Hippocampus 470.2 135.8 142.5 237.0
± ± ± ±
21.5 23.6** 30.1** 20.3**
Animals received saline (controls) or a single injection of MDMA (10 mg/kg). Data, expressed as pg/mg wet tissue, are mean ± S.E.M. (n = 6–8). * P < 0.05 vs. controls (one-way ANOVA followed by post hoc Scheff´ e test). ** P < 0.01 vs. controls (one-way ANOVA followed by post hoc Scheff´ e test).
mouse anti-phospho-CREB (Ser133) (1B6) monoclonal (Cell Signaling Technology® ). After washing, an HRP-conjugated anti-mouse antibody (Dako; dilution 1:1000) was added and incubated for 60 min at room temperature. Immunolabeled protein bands were detected using an enhanced chemilumiscence system (ECL Amersham Pharmacia). The quantification of signals was determined by densitometry. Twenty-four hours later, acute MDMA treatment reduced 5HT levels by ∼38% in the frontal cortex and by ∼72% in the hippocampus. Two days after treatment, cortical levels did not significantly differ from controls, but hippocampal 5-HT levels were reduced to a similar extent (∼70%) and were still significantly reduced by ∼50% one week after MDMA treatment (Table 1). In the frontal cortex, one-way ANOVA revealed a significant effect of MDMA treatment on BDNF mRNA expression [F3,160 = 19.76; P < 0.001]. The levels of BDNF mRNA were significantly increased (∼33%) 24 h after injection and were further increased (∼70%) at the 48 h time point. Seven days after MDMA administration, BDNF mRNA expression returned to control levels (Fig. 1). In the hippocampus, ANOVA also revealed a significant treatment effect on BDNF mRNA expression in CA1 [F3,53 = 218.40; P < 0.001], CA3 [F3,52 = 82.21; P < 0.001], and dentate gyrus [F3,52 = 51.73; P < 0.001]. However, in sharp contrast with the effects observed in the frontal cortex, the levels of BDNF mRNA in the hippocampus were moderately decreased 48 h after MDMA treatment, and were further decreased (∼73%) 7 days after injection (Fig. 2). In the frontal cortex, acute MDMA treatment significantly increased (∼35%) pCREB levels 24 and 48 h later. In the hippocampus, the effect was more pronounced and significantly increased pCREB levels were also detected 24 and 48 h after MDMA injection (Table 2). Cortical and hippocampal cells respond to MDMA by modulating transcription of several genes that may lead to long-term changes in the brain [3,6,29]. The present results show that BDNF gene transcription is increased or decreased by MDMA treatment in the frontal cortex and hippocampus respectively. A modest increase in BDNF protein levels in different forebrain structures (∼19–21%) after chronic treatment of neonatal rats with high MDMA doses (20 mg/kg bid for 10 consecutive days) had been reported [11]. We here show that a single administration of a lower MDMA dose (10 mg/kg) produced much more marked changes in BDNF mRNA expression in adult animals.
128
R. Mart´ınez-Turrillas et al. / Neuroscience Letters 402 (2006) 126–130 Table 2 Effect of MDMA on phospho-CREB levels in nuclear extracts from rat frontal cortex and hippocampus Time after treatment
Frontal cortex
Hippocampus
Control 24 h 48 h
100 ± 8.3 133 ± 7.7* 138 ± 5.6*
100 ± 4.6 143 ± 9.5* 158 ± 12.4**
Animals received saline (controls) or a single injection of MDMA (10 mg/kg). Data are mean ± S.E.M. (n = 5–7) expressed as percentage of optical density of controls. * P < 0.05 vs. controls (one-way ANOVA followed by post hoc Scheff´ e test). ** P < 0.01 vs. controls (one-way ANOVA followed by post hoc Scheff´ e test).
Fig. 1. Effect of a single administration of MDMA (10 mg/kg) on BDNF mRNA expression in the frontal cortex of rats killed at different times after drug administration. Data are mean ± S.E.M. (n = 5–7) expressed as percentage of the hybridization signal of control rats. * P < 0.05, ** P < 0.01 vs. vehicle administered controls (one-way ANOVA followed by post hoc Scheff´e test). Representative autoradiograms are shown at the upper part of the figure.
The changes found with MDMA on BDNF mRNA expression, i.e. increase in the frontal cortex and reduction in the dentate gyrus, are quite similar to those reported after acute administration of another serotonergic neurotoxin, p-
chloroamphetamine (PCA, [32]) with a mechanism of action similar, but not identical, to that of MDMA [27]. In the study with PCA [32], it was hypothesized, in line with a previous study [30], that serotonin release caused by p-chloroamphetamine could enhance neuronal activity through stimulation of 5-HT2 receptors, located on excitatory glutamatergic neurons in the frontal cortex and on inhibitory GABAergic interneurons in the dentate gyrus. However, in the study of Vaidya et al. [30] decreased BDNF mRNA expression was found in the dentate gyrus, but not in CA subfields. Since MDMA-induced decrease in BDNF was similar in all hippocampal subfields examined (Fig. 2), the involvement of 5-HT2 receptors in this effect is unclear. Stimulation of 5-HT receptor subtypes may increase the intracellular concentration of kinases that phosphorylate CREB [15]. Once phosphorylated, CREB interacts with CREB-binding protein and this complex binds to the CRE sequence located in the promoter region of specific target genes, such as the BDNF gene [28]. The activation of this signalling cascade could explain the upregulated levels of phospho-CREB and the subsequent increase in BDNF mRNA expression observed in the rat
Fig. 2. Effect of a single administration of MDMA (10 mg/kg) on BDNF mRNA expression in CA1, CA3 and dentate gyrus of hippocampus of rats killed at different times after drug administration. Data are mean ± S.E.M. (n = 5–7) expressed as percentage of the hybridization signal of control rats. * P < 0.05, ** P < 0.01 vs. vehicle administered controls (one-way ANOVA followed by post hoc Scheff´e test). Representative autoradiograms are shown at the upper part of the figure.
R. Mart´ınez-Turrillas et al. / Neuroscience Letters 402 (2006) 126–130
frontal cortex, which could be a compensatory response to minimize MDMA effects. In this regard, BDNF effectively blocks methamphetamine-induced neuronal death in cortical neurons, through the phosphatidylinositol-3-kinase/Akt pathway [16] and exerts a neuroprotective effect in methamphetamine-induced neurotoxicity on the nigrostriatal dopaminergic system of mice [4]. At variance with the results obtained in the rat frontal cortex, BDNF mRNA expression was significantly downregulated in different hippocampal subfields (CA1, CA3 and dentate gyrus) 48 h after a single dose of MDMA, and was further decreased 7 days after injection. Since BDNF-induced changes are thought to underlie consolidation of synaptic efficacy and hippocampal memory storage [21], it is tempting to speculate on the link between the reduced BDNF mRNA expression in the hippocampus and the amnesic effect of acute MDMA treatment (e.g. [18]). Consonant with this observation, significantly reduced TPH mRNA expression and no recovery of 5-HT levels 48 h after acute MDMA treatment, were previously found in the rat hippocampus [6]. It is of note that BDNF mRNA expression was significantly reduced in the hippocampus 48 h after MDMA in spite of the marked increase at this time point in the levels of the transcription factor pCREB, which regulates BDNF expression [28]. Even though overactivation of CREB may lead to increased repressor activity [31], the increase of pCREB in the hippocampus was not much higher than in the cortex. We speculate that a more likely interpretation for this differential finding is the high vulnerability of the hippocampus to the neurotoxic effects of MDMA. Corticosterone is released by MDMA [1] and it is known that stress or high levels of glucocorticoids may produce hippocampal cell loss (reviewed in [17]). One mechanism probably involved in the neurotoxic effect of stress/glucocorticoids in the hippocampus is the decreased expression of BDNF mRNA [25]. On the contrary, BDNF infusion protects against the stressinduced deficits in memory processes [21]. BDNF expression is strongly regulated by adrenocorticosteroids via activated glucocorticoid (GR) and mineralocorticoid receptors (MR) which can act directly as transcription factors by binding to their common glucocorticoid response element (GRE). Both types of receptors are highly prevalent in the hippocampus. To date, no canonical GRE sequence has been found in the promoter regions of the BDNF gene. However, a putative GRE element has been localised in the BDNF promoter region IV, and GR/MR binding to this region appears to be involved in the changes on BDNF mRNA expression observed after adrenalectomy (ADX) or glucocorticoid treatment [9]. Interestingly, ADX significantly increases BDNF mRNA levels in all subfields of the hippocampus but does not produce any effect in the neocortex. Glucocorticoid treatments significantly reverse the ADX effect in hippocampus without producing any effect in the neocortex, where BDNF expression seems to be largely resistant to the effects of glucocorticoids [8]. In summary, acute MDMA treatment induced time and region-dependent modifications in BDNF mRNA expression in the rat frontal cortex and hippocampus at very early stages after exposure to the drug, suggesting a novel mechanism for the dif-
129
ferential effects of this drug on 5-HT synthesis in these two brain regions. Acknowledgements Supported by grants from EC (QLG3-CT-2002-00809) and MCyT, Spain (BFI-2001-1602). References [1] N. Aguirre, D. Frechilla, A. Garcia-Osta, B. Lasheras, J. Del R´ıo, Differential regulation by methylenedioxyamphetamine of 5-hydroxytryptamine1A receptor density and mRNA expression in rat hippocampus, frontal cortex and brainstem: the role of corticosteroids, J. Neurochem. 68 (1997) 1099–1105. [2] G. Battaglia, S.Y. Yeh, E. O’Hearn, M.E. Molliver, M.J. Kuhar, E.B. De Souza, 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyamphetamine destroy serotonin terminals in rat brain: quantification of neurodegeneration by measurement of [3H] paroxetine-labeled serotonin uptake sites, J. Pharmacol. Exp. Ther. 242 (1987) 911–916. [3] T.J. Beveridge, A.O. Mechan, M. Sprakes, Q. Pei, T.S. Zetterstr¨om, A.R. Green, Effect of 5-HT depletion by MDMA on hyperthermia and Arc mRNA induction in rat brain, Psychopharmacology 173 (2004) 346–352. [4] D.E. Dluzen, The effect of gender and the neurotrophin, BDNF, upon methamphetamine-induced neurotoxicity of the nigrostriatal dopaminergic system in mice, Neurosci. Lett. 359 (2004) 135–138. [5] D. Frechilla, R. Insausti, P. Ruiz-Golvano, A. Garcia-Osta, M.P. Rubio, J.M. Almendral, J. Del Rio, Implanted BDNF-producing fibroblasts prevent neurotoxin-induced serotonergic denervation in the rat striatum, Mol. Brain Res. 76 (2000) 306–314. [6] A. Garcia-Osta, J. Del Rio, D. Frechilla, Increased CRE-binding activity and tryptophan hydroxylase mRNA expression induced by 3,4methylenedioxymethamphetamine (MDMA, “ecstasy”) in the rat frontal cortex but not in the hippocampus, Mol. Brain Res. 126 (2004) 181–187. [7] A.R. Green, A.O. Mechan, J.M. Elliott, E. O’Shea, M.I. Colado, The pharmacology and clinical pharmacology of 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”), Pharmacol. Rev. 55 (2003) 463–508. [8] A.C. Hansson, A. Cintra, N. Belluardo, W. Sommer, M. Bhatnagar, M. Bade, D. Ganten, K. Fuxe, Gluco- and mineralocorticoid receptormediated regulation of neurotrophic factor gene expression in the dorsal hippocampus and the neocortex of the rat, Eur. J. Neurosci. 12 (2000) 2918–2934. [9] A.C. Hansson, W.H. Sommer, M. Metsis, I. Stromberg, L.F. Agnati, K. Fuxe, Corticosterone actions on the hippocampal brain-derived neurotrophic factor expression are mediated by exon IV promoter, J. Neuroendocrinol. 18 (2006) 104–114. [10] R.R. Hicks, V.B. Martin, L. Zhang, K.B. Seroogy, Mild experimental brain injury differentially alters the expression of neurotrophin and neurotrophin receptor mRNAs in the hippocampus, Exp. Neurol. 160 (1999) 469–478. [11] J.B. Koprich, N.G. Campbell, J.W. Lipton, Neonatal 3,4-methylenedioxymethamphetamine (ecstasy) alters dopamine and serotonin neuroquemistry and increases brain-derived neurotrophic factor in the forebrain and brainstem of the rat, Dev. Brain. Res. 147 (2003) 177–182. [12] B.A. Luellen, M.E. Szapacs, C.K. Materese, A.M. Andrews, The neurotoxin 2 -NH(2)-MPTP degenerates serotonin axons and evokes increases in hippocampal BDNF, Neuropharmacology 50 (2006) 297–308. [13] L.A. Mamounas, M.E. Blue, J.A. Siuciak, C.A. Altar, Brain-derived neurotrophic factor promotes the survival and sprouting of serotonergic axons in rat brain, J. Neurosci. 12 (1995) 7929–7939. [14] R. Mart´ınez-Turrillas, J. Del R´ıo, D. Frechilla, Sequential changes in BDNF mRNA expression and synaptic levels of AMPA receptor subunits in rat hippocampus after chronic antidepressant treatment, Neuropharmacology 49 (2005) 1178–1188.
130
R. Mart´ınez-Turrillas et al. / Neuroscience Letters 402 (2006) 126–130
[15] M.P. Mattson, S. Maudsley, B. Martin, BDNF and 5-HT: a dynamic duo in age-related neuronal plasticity and disorders, Trends Neurosci. 27 (2004) 589–594. [16] H. Matsuzaki, K. Namikawa, H. Kiyama, N. Mori, K. Sato, Brainderived neurotrophic factor rescues neuronal death induced by methamphetamine, Biol. Psychiatry 55 (2004) 52–60. [17] B.S. McEwen, Stress and hippocampal plasticity, Annu. Rev. Neurosci. 22 (1999) 105–122. [18] S. Moyano, D. Frechilla, J. Del R´ıo, NMDA receptor subunit and CaMKII changes in rat hippocampus induced by acute MDMA treatment: a mechanism for learning impairment, Psychopharmacology 173 (2004) 337–345. [19] E. O’Shea, R. Granados, B. Esteban, M.I. Colado, A.R. Green, The relationship between the degree of neurodegeneration of rat brain 5-HT nerve terminals and the dose and frequency of administration of MDMA (“ecstasy”), Neuropharmacology 37 (1998) 919– 926. [20] I. Perez-Ota˜no, M.T. Herrero, C. Oset, M.L. De Ceballos, M.R. Luquin, J.A. Obeso, J. Del R´ıo, Extensive loss of brain dopamine and serotonin induced by chronic administration of MPTP in the marmoset, Brain Res. 567 (1991) 127–132. [21] D.T. Radecki, L.M. Brown, J. Mart´ınez, T.J. Teyler, BDNF protects against stress-induced impairments in spatial learning and memory and LTP, Hippocampus 15 (2005) 246–253. [22] G. Ricaurte, G. Bryan, L. Strauss, L. Seiden, C. Schuster, Hallucinogenic amphetamine selectively destroys brain serotonin teminals, Science 229 (1985) 986–988. [23] C.J. Schmidt, V.L. Taylor, Depression of rat brain tryptophan hydroxylase following the acute administration of methylenedioxymetamphetamine, Biochem. Pharmacol. 36 (1987) 4095–4102.
[24] J.A. Siuciak, M.S. Clark, H.B. Rind, S.R. Whittemore, A.F. Russo, BDNF induction of tryptophan hydroxylase mRNA levels in the rat brain, J. Neurosci. Res. 52 (1998) 149–158. [25] M.A. Smith, S. Makino, R. Kvetnansky, R.M. Post, Stress and glucocorticoids affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the hippocampus, J. Neurosci. 15 (1995) 1768–1777. [26] J.L. Sonnenberg, P.F. Macgregor-Leon, T. Curran, J.I. Morgan, Dynamic alterations occur in the levels and composition of transcription factor AP-1 complexes after seizure, Neuron 3 (1989) 359–365. [27] J.E. Sprague, M.P. Johnson, C.J. Schmidt, D.E. Nichols, Studies on the mechanism of p-chloroamphetamine neurotoxicity, Biochem. Pharmacol. 52 (1996) 1271–1277. [28] X. Tao, S. Finkbeiner, D.B. Arnold, A.J. Shaywitz, M.E. Greenberg, Ca2+ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism, Neuron 20 (1998) 709–726. [29] N. Thiriet, B. Ladenheim, M.T. McCoy, J.L. Cadet, Analysis of ecstasy (MDMA)-induced transcriptional responses in the rat cortex, FASEB J. 16 (2002) 1884–1887. [30] V.A. Vaidya, G.J. Marek, G.K. Aghajanian, R.S. Duman, 5-HT2A receptor-mediated regulation of brain-derived neurotrophic factor mRNA in the hippocampus and the neocortex, J. Neurosci. 17 (1997) 2785–2795. [31] Q. Yuan, C.W. Harley, A. Darby-King, R.L. Neve, J.H. McLean, Early odor preference learning in the rat: bidirectional effects of cAMP response element-binding protein (CREB) and mutant CREB support a causal role for phosphorylated CREB, J. Neurosci. 23 (2003) 4760–4765. [32] T.S. Zetterstr¨om, Q. Pei, T.R. Madhav, A.L. Coppell, L. Lewis, D.G. Grahame-Smith, Manipulations of brain 5-HT levels affect gene expression for BDNF in rat brain, Neuropharmacology 38 (1999) 1063–1073.
Differential effects of 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) on BDNF mRNA expression in rat frontal cortex and hippocampus Rebeca Mart´ınez-Turrillas, Sonia Moyano, Joaqu´ın Del R´ıo, Diana Frechilla ∗ Department of Pharmacology, School of Medicine, University of Navarra, Aptdo. 177, 31080-Pamplona, Spain Received 8 February 2006; received in revised form 20 March 2006; accepted 24 March 2006
Abstract The serotonergic neurotoxin 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) produces rapid serotonin (5-HT) depletion in different areas of the forebrain after acute administration to rats and other animal species. We previously found that 5-HT depletion induced by acute MDMA treatment was transient in the frontal cortex, but not in the hippocampus, and recovery of cortical 5-HT levels correlated with an induction of CRE-binding activity and increased expression of tryptophan-hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis. As the brainderived neurotrophic factor (BDNF) stimulates the growth and sprouting of serotonergic neurons, we sought the possible involvement of this neurotrophin in the region-specific increase in TPH mRNA expression induced by MDMA. We here report that, 24–48 h after acute MDMA treatment, the expression of BDNF in the frontal cortex is increased by ∼33–70%, and the levels of the transcription factor phospho-CREB are also increased. In the hippocampus, however, a time-dependent decrease in BDNF mRNA expression (maximal decrease of ∼73%) is found in all subfields examined 2–7 days after treatment in spite of increased phospho-CREB levels, perhaps as a consequence of corticosterone release by the serotonergic neurotoxin. The differential regulation of BDNF mRNA expression in the two brain regions examined appears to account for the enhanced TPH expression and the recovery of 5-HT levels in the frontal cortex, but not in the hippocampus, after neurotoxin treatment. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: BDNF; MDMA; Serotonin; CREB
The amphetamine derivative, 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”), is a widely used recreational drug that, in rats and other animal species, induces a rapid reduction in the content of brain serotonin (5-HT) [2,22], in the activity of tryptophan-hydroxylase (TPH), the rate-limiting enzyme in 5-HT synthesis [23], and in 5-HT transporter density [2]. On chronic treatment, it has been repeatedly shown that MDMA produces a degeneration of serotonergic nerve terminals [2,19,22]. The cellular and molecular mechanisms involved in MDMA actions have been extensively studied although many aspects remain to be elucidated (see [7] for review). In a previous study [6], we found that acute MDMA treatment produced a rapid reduction of 5-HT content in rat frontal cortex and hippocampus. In the cortex, but not in the hippocampus, 5-HT levels
∗
Corresponding author. Tel.: +34 948 194700; fax: +34 948 194715. E-mail address: [email protected] (D. Frechilla).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.03.055
returned to normal values 48 h after injection, and this recovery was correlated with an increase in binding to the CRE consensus sequence and in mRNA levels of TPH. It is known that the neurotrophin brain-derived neurotrophic factor (BDNF), which is upregulated in response to various types of neuronal injury as a compensatory effect after brain damage [10], augments 5-HT synthesis by enhancing TPH mRNA levels [24]. Many other studies point to the mutual interactions between brain 5HT and BDNF (reviewed in [15]). This neurotrophin influences the survival and function of 5-HT neurons in the rat brain [5,13] whereas blockade of the 5-HT transporter and activation of 5HT receptor subtypes induces transcription of the BDNF gene [15]. Serotonergic neurotoxins such as p-chloroamphetamine (PCA) and 2 -NH2 -MPTP may alter the expression of BDNF mRNA and protein in the rat hippocampus and cortical areas [12,32]. A single study showed that neonatal administration of MDMA moderately increased BDNF protein on postnatal day 21 [11]. On the basis of the previously observed differential changes in 5-HT and TPH mRNA levels [6], the current study
R. Mart´ınez-Turrillas et al. / Neuroscience Letters 402 (2006) 126–130
sought to determine the possible effects of acute MDMA administration on BDNF mRNA expression in the rat frontal cortex and hippocampus. Given that MDMA also increased the activity of protein binding to CRE [6] and the known regulation by cAMP response element-binding protein (CREB) of the BDNFinduced gene expression in the cortex [28], we also looked for eventual changes in the levels of phospho-CREB in response to the neuronal injury caused by MDMA. Male Wistar rats (Harlan, Barcelona, Spain) weighing 200–220 g were used. Animals were housed three to a cage in a temperature-controlled room (22–23 ◦ C) with a 12 h light/dark cycle and had free access to food and water. All procedures were in accordance with the guidelines established by the normative of the European Community of November 24, 1986 (86/609/EEC). Rats were injected with saline (5 ml/kg, i.p.) or with a single dose of MDMA.HCl (10 mg/kg, i.p.) and killed by decapitation at different times. The brains were removed and placed on ice. Western-blot analyses were carried out with fresh tissue from the frontal cortex and hippocampus. In rats assigned to in situ hybridization studies, the brains were immediately frozen in isopentane on dry ice. All tissues were stored at −80 ◦ C until use. The levels of 5-HT were measured in rat brain regions by high-performance liquid chromatography with electrochemical detection, as described [20]. For in situ hybridization, 14 m coronal sections were cut serially with a cryostat at the level of frontal cortex (3.7 mm relative to bregma) and dorsal hippocampus (−3.3 mm relative to bregma), according to the atlas of Paxinos and Watson. The sections were mounted onto Superfrost/Plus slides and then processed for the in situ hybridization of BDNF mRNA as previously described [14]. For detection of BDNF mRNA, an oligonucleotide complementary to bases of the coding region of rat BDNF exon V (Invitrogen Custom primers) was used: 5 -AGT TCC AGT GCC TTT TGT CTA TGC CCC TGC AGC CTT TGG TGT AAC-3 . The probe was 3 -tail labeled with ␣S[35 S]dATP (specific activity >1000 Ci/mmol, Amersham Pharmacia) using terminal deoxynucleotide transferase (Roche). A negative control included hybridization of sections using an oligonucleotide in the sense orientation, which showed minimal background signals. Hybridization densities were measured from films autoradiograms, with labeling densities calibrated relative to film images of 14 C-labeled standards (Amersham Pharmacia), using the Microcomputer Imaging Device (Imaging Research, St Catherines, Ontario, Canada). Densitometric values measured from four sections of each animal were averaged and expressed as nCi/g tissue, and mRNA abundance in drug treated groups was expressed as a percentage of their respective controls. Nuclear protein extracts from rat frontal cortex and hippocampus were prepared as previously described [6,26]. For Western blotting, 25 g of nuclear protein extract were separated onto SDS-polyacrilamide gel (12%). Proteins were transferred to a nitrocellulose membrane using a Trans-Blot® SD semidry (BioRad) system for 30 min at 16 V. The membranes were blocked and incubated at 4 ◦ C overnight with the primary antibody, diluted in 5% non-fat dried milk in TBST:
127
Table 1 Effect of MDMA on 5-HT levels in rat frontal cortex and hippocampus Time after treatment
Frontal cortex
Control 24 h 48 h 7 days
527.0 326.8 502.2 510.3
± ± ± ±
36.2 29.5* 40.5 29.2
Hippocampus 470.2 135.8 142.5 237.0
± ± ± ±
21.5 23.6** 30.1** 20.3**
Animals received saline (controls) or a single injection of MDMA (10 mg/kg). Data, expressed as pg/mg wet tissue, are mean ± S.E.M. (n = 6–8). * P < 0.05 vs. controls (one-way ANOVA followed by post hoc Scheff´ e test). ** P < 0.01 vs. controls (one-way ANOVA followed by post hoc Scheff´ e test).
mouse anti-phospho-CREB (Ser133) (1B6) monoclonal (Cell Signaling Technology® ). After washing, an HRP-conjugated anti-mouse antibody (Dako; dilution 1:1000) was added and incubated for 60 min at room temperature. Immunolabeled protein bands were detected using an enhanced chemilumiscence system (ECL Amersham Pharmacia). The quantification of signals was determined by densitometry. Twenty-four hours later, acute MDMA treatment reduced 5HT levels by ∼38% in the frontal cortex and by ∼72% in the hippocampus. Two days after treatment, cortical levels did not significantly differ from controls, but hippocampal 5-HT levels were reduced to a similar extent (∼70%) and were still significantly reduced by ∼50% one week after MDMA treatment (Table 1). In the frontal cortex, one-way ANOVA revealed a significant effect of MDMA treatment on BDNF mRNA expression [F3,160 = 19.76; P < 0.001]. The levels of BDNF mRNA were significantly increased (∼33%) 24 h after injection and were further increased (∼70%) at the 48 h time point. Seven days after MDMA administration, BDNF mRNA expression returned to control levels (Fig. 1). In the hippocampus, ANOVA also revealed a significant treatment effect on BDNF mRNA expression in CA1 [F3,53 = 218.40; P < 0.001], CA3 [F3,52 = 82.21; P < 0.001], and dentate gyrus [F3,52 = 51.73; P < 0.001]. However, in sharp contrast with the effects observed in the frontal cortex, the levels of BDNF mRNA in the hippocampus were moderately decreased 48 h after MDMA treatment, and were further decreased (∼73%) 7 days after injection (Fig. 2). In the frontal cortex, acute MDMA treatment significantly increased (∼35%) pCREB levels 24 and 48 h later. In the hippocampus, the effect was more pronounced and significantly increased pCREB levels were also detected 24 and 48 h after MDMA injection (Table 2). Cortical and hippocampal cells respond to MDMA by modulating transcription of several genes that may lead to long-term changes in the brain [3,6,29]. The present results show that BDNF gene transcription is increased or decreased by MDMA treatment in the frontal cortex and hippocampus respectively. A modest increase in BDNF protein levels in different forebrain structures (∼19–21%) after chronic treatment of neonatal rats with high MDMA doses (20 mg/kg bid for 10 consecutive days) had been reported [11]. We here show that a single administration of a lower MDMA dose (10 mg/kg) produced much more marked changes in BDNF mRNA expression in adult animals.
128
R. Mart´ınez-Turrillas et al. / Neuroscience Letters 402 (2006) 126–130 Table 2 Effect of MDMA on phospho-CREB levels in nuclear extracts from rat frontal cortex and hippocampus Time after treatment
Frontal cortex
Hippocampus
Control 24 h 48 h
100 ± 8.3 133 ± 7.7* 138 ± 5.6*
100 ± 4.6 143 ± 9.5* 158 ± 12.4**
Animals received saline (controls) or a single injection of MDMA (10 mg/kg). Data are mean ± S.E.M. (n = 5–7) expressed as percentage of optical density of controls. * P < 0.05 vs. controls (one-way ANOVA followed by post hoc Scheff´ e test). ** P < 0.01 vs. controls (one-way ANOVA followed by post hoc Scheff´ e test).
Fig. 1. Effect of a single administration of MDMA (10 mg/kg) on BDNF mRNA expression in the frontal cortex of rats killed at different times after drug administration. Data are mean ± S.E.M. (n = 5–7) expressed as percentage of the hybridization signal of control rats. * P < 0.05, ** P < 0.01 vs. vehicle administered controls (one-way ANOVA followed by post hoc Scheff´e test). Representative autoradiograms are shown at the upper part of the figure.
The changes found with MDMA on BDNF mRNA expression, i.e. increase in the frontal cortex and reduction in the dentate gyrus, are quite similar to those reported after acute administration of another serotonergic neurotoxin, p-
chloroamphetamine (PCA, [32]) with a mechanism of action similar, but not identical, to that of MDMA [27]. In the study with PCA [32], it was hypothesized, in line with a previous study [30], that serotonin release caused by p-chloroamphetamine could enhance neuronal activity through stimulation of 5-HT2 receptors, located on excitatory glutamatergic neurons in the frontal cortex and on inhibitory GABAergic interneurons in the dentate gyrus. However, in the study of Vaidya et al. [30] decreased BDNF mRNA expression was found in the dentate gyrus, but not in CA subfields. Since MDMA-induced decrease in BDNF was similar in all hippocampal subfields examined (Fig. 2), the involvement of 5-HT2 receptors in this effect is unclear. Stimulation of 5-HT receptor subtypes may increase the intracellular concentration of kinases that phosphorylate CREB [15]. Once phosphorylated, CREB interacts with CREB-binding protein and this complex binds to the CRE sequence located in the promoter region of specific target genes, such as the BDNF gene [28]. The activation of this signalling cascade could explain the upregulated levels of phospho-CREB and the subsequent increase in BDNF mRNA expression observed in the rat
Fig. 2. Effect of a single administration of MDMA (10 mg/kg) on BDNF mRNA expression in CA1, CA3 and dentate gyrus of hippocampus of rats killed at different times after drug administration. Data are mean ± S.E.M. (n = 5–7) expressed as percentage of the hybridization signal of control rats. * P < 0.05, ** P < 0.01 vs. vehicle administered controls (one-way ANOVA followed by post hoc Scheff´e test). Representative autoradiograms are shown at the upper part of the figure.
R. Mart´ınez-Turrillas et al. / Neuroscience Letters 402 (2006) 126–130
frontal cortex, which could be a compensatory response to minimize MDMA effects. In this regard, BDNF effectively blocks methamphetamine-induced neuronal death in cortical neurons, through the phosphatidylinositol-3-kinase/Akt pathway [16] and exerts a neuroprotective effect in methamphetamine-induced neurotoxicity on the nigrostriatal dopaminergic system of mice [4]. At variance with the results obtained in the rat frontal cortex, BDNF mRNA expression was significantly downregulated in different hippocampal subfields (CA1, CA3 and dentate gyrus) 48 h after a single dose of MDMA, and was further decreased 7 days after injection. Since BDNF-induced changes are thought to underlie consolidation of synaptic efficacy and hippocampal memory storage [21], it is tempting to speculate on the link between the reduced BDNF mRNA expression in the hippocampus and the amnesic effect of acute MDMA treatment (e.g. [18]). Consonant with this observation, significantly reduced TPH mRNA expression and no recovery of 5-HT levels 48 h after acute MDMA treatment, were previously found in the rat hippocampus [6]. It is of note that BDNF mRNA expression was significantly reduced in the hippocampus 48 h after MDMA in spite of the marked increase at this time point in the levels of the transcription factor pCREB, which regulates BDNF expression [28]. Even though overactivation of CREB may lead to increased repressor activity [31], the increase of pCREB in the hippocampus was not much higher than in the cortex. We speculate that a more likely interpretation for this differential finding is the high vulnerability of the hippocampus to the neurotoxic effects of MDMA. Corticosterone is released by MDMA [1] and it is known that stress or high levels of glucocorticoids may produce hippocampal cell loss (reviewed in [17]). One mechanism probably involved in the neurotoxic effect of stress/glucocorticoids in the hippocampus is the decreased expression of BDNF mRNA [25]. On the contrary, BDNF infusion protects against the stressinduced deficits in memory processes [21]. BDNF expression is strongly regulated by adrenocorticosteroids via activated glucocorticoid (GR) and mineralocorticoid receptors (MR) which can act directly as transcription factors by binding to their common glucocorticoid response element (GRE). Both types of receptors are highly prevalent in the hippocampus. To date, no canonical GRE sequence has been found in the promoter regions of the BDNF gene. However, a putative GRE element has been localised in the BDNF promoter region IV, and GR/MR binding to this region appears to be involved in the changes on BDNF mRNA expression observed after adrenalectomy (ADX) or glucocorticoid treatment [9]. Interestingly, ADX significantly increases BDNF mRNA levels in all subfields of the hippocampus but does not produce any effect in the neocortex. Glucocorticoid treatments significantly reverse the ADX effect in hippocampus without producing any effect in the neocortex, where BDNF expression seems to be largely resistant to the effects of glucocorticoids [8]. In summary, acute MDMA treatment induced time and region-dependent modifications in BDNF mRNA expression in the rat frontal cortex and hippocampus at very early stages after exposure to the drug, suggesting a novel mechanism for the dif-
129
ferential effects of this drug on 5-HT synthesis in these two brain regions. Acknowledgements Supported by grants from EC (QLG3-CT-2002-00809) and MCyT, Spain (BFI-2001-1602). References [1] N. Aguirre, D. Frechilla, A. Garcia-Osta, B. Lasheras, J. Del R´ıo, Differential regulation by methylenedioxyamphetamine of 5-hydroxytryptamine1A receptor density and mRNA expression in rat hippocampus, frontal cortex and brainstem: the role of corticosteroids, J. Neurochem. 68 (1997) 1099–1105. [2] G. Battaglia, S.Y. Yeh, E. O’Hearn, M.E. Molliver, M.J. Kuhar, E.B. De Souza, 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyamphetamine destroy serotonin terminals in rat brain: quantification of neurodegeneration by measurement of [3H] paroxetine-labeled serotonin uptake sites, J. Pharmacol. Exp. Ther. 242 (1987) 911–916. [3] T.J. Beveridge, A.O. Mechan, M. Sprakes, Q. Pei, T.S. Zetterstr¨om, A.R. Green, Effect of 5-HT depletion by MDMA on hyperthermia and Arc mRNA induction in rat brain, Psychopharmacology 173 (2004) 346–352. [4] D.E. Dluzen, The effect of gender and the neurotrophin, BDNF, upon methamphetamine-induced neurotoxicity of the nigrostriatal dopaminergic system in mice, Neurosci. Lett. 359 (2004) 135–138. [5] D. Frechilla, R. Insausti, P. Ruiz-Golvano, A. Garcia-Osta, M.P. Rubio, J.M. Almendral, J. Del Rio, Implanted BDNF-producing fibroblasts prevent neurotoxin-induced serotonergic denervation in the rat striatum, Mol. Brain Res. 76 (2000) 306–314. [6] A. Garcia-Osta, J. Del Rio, D. Frechilla, Increased CRE-binding activity and tryptophan hydroxylase mRNA expression induced by 3,4methylenedioxymethamphetamine (MDMA, “ecstasy”) in the rat frontal cortex but not in the hippocampus, Mol. Brain Res. 126 (2004) 181–187. [7] A.R. Green, A.O. Mechan, J.M. Elliott, E. O’Shea, M.I. Colado, The pharmacology and clinical pharmacology of 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”), Pharmacol. Rev. 55 (2003) 463–508. [8] A.C. Hansson, A. Cintra, N. Belluardo, W. Sommer, M. Bhatnagar, M. Bade, D. Ganten, K. Fuxe, Gluco- and mineralocorticoid receptormediated regulation of neurotrophic factor gene expression in the dorsal hippocampus and the neocortex of the rat, Eur. J. Neurosci. 12 (2000) 2918–2934. [9] A.C. Hansson, W.H. Sommer, M. Metsis, I. Stromberg, L.F. Agnati, K. Fuxe, Corticosterone actions on the hippocampal brain-derived neurotrophic factor expression are mediated by exon IV promoter, J. Neuroendocrinol. 18 (2006) 104–114. [10] R.R. Hicks, V.B. Martin, L. Zhang, K.B. Seroogy, Mild experimental brain injury differentially alters the expression of neurotrophin and neurotrophin receptor mRNAs in the hippocampus, Exp. Neurol. 160 (1999) 469–478. [11] J.B. Koprich, N.G. Campbell, J.W. Lipton, Neonatal 3,4-methylenedioxymethamphetamine (ecstasy) alters dopamine and serotonin neuroquemistry and increases brain-derived neurotrophic factor in the forebrain and brainstem of the rat, Dev. Brain. Res. 147 (2003) 177–182. [12] B.A. Luellen, M.E. Szapacs, C.K. Materese, A.M. Andrews, The neurotoxin 2 -NH(2)-MPTP degenerates serotonin axons and evokes increases in hippocampal BDNF, Neuropharmacology 50 (2006) 297–308. [13] L.A. Mamounas, M.E. Blue, J.A. Siuciak, C.A. Altar, Brain-derived neurotrophic factor promotes the survival and sprouting of serotonergic axons in rat brain, J. Neurosci. 12 (1995) 7929–7939. [14] R. Mart´ınez-Turrillas, J. Del R´ıo, D. Frechilla, Sequential changes in BDNF mRNA expression and synaptic levels of AMPA receptor subunits in rat hippocampus after chronic antidepressant treatment, Neuropharmacology 49 (2005) 1178–1188.
130
R. Mart´ınez-Turrillas et al. / Neuroscience Letters 402 (2006) 126–130
[15] M.P. Mattson, S. Maudsley, B. Martin, BDNF and 5-HT: a dynamic duo in age-related neuronal plasticity and disorders, Trends Neurosci. 27 (2004) 589–594. [16] H. Matsuzaki, K. Namikawa, H. Kiyama, N. Mori, K. Sato, Brainderived neurotrophic factor rescues neuronal death induced by methamphetamine, Biol. Psychiatry 55 (2004) 52–60. [17] B.S. McEwen, Stress and hippocampal plasticity, Annu. Rev. Neurosci. 22 (1999) 105–122. [18] S. Moyano, D. Frechilla, J. Del R´ıo, NMDA receptor subunit and CaMKII changes in rat hippocampus induced by acute MDMA treatment: a mechanism for learning impairment, Psychopharmacology 173 (2004) 337–345. [19] E. O’Shea, R. Granados, B. Esteban, M.I. Colado, A.R. Green, The relationship between the degree of neurodegeneration of rat brain 5-HT nerve terminals and the dose and frequency of administration of MDMA (“ecstasy”), Neuropharmacology 37 (1998) 919– 926. [20] I. Perez-Ota˜no, M.T. Herrero, C. Oset, M.L. De Ceballos, M.R. Luquin, J.A. Obeso, J. Del R´ıo, Extensive loss of brain dopamine and serotonin induced by chronic administration of MPTP in the marmoset, Brain Res. 567 (1991) 127–132. [21] D.T. Radecki, L.M. Brown, J. Mart´ınez, T.J. Teyler, BDNF protects against stress-induced impairments in spatial learning and memory and LTP, Hippocampus 15 (2005) 246–253. [22] G. Ricaurte, G. Bryan, L. Strauss, L. Seiden, C. Schuster, Hallucinogenic amphetamine selectively destroys brain serotonin teminals, Science 229 (1985) 986–988. [23] C.J. Schmidt, V.L. Taylor, Depression of rat brain tryptophan hydroxylase following the acute administration of methylenedioxymetamphetamine, Biochem. Pharmacol. 36 (1987) 4095–4102.
[24] J.A. Siuciak, M.S. Clark, H.B. Rind, S.R. Whittemore, A.F. Russo, BDNF induction of tryptophan hydroxylase mRNA levels in the rat brain, J. Neurosci. Res. 52 (1998) 149–158. [25] M.A. Smith, S. Makino, R. Kvetnansky, R.M. Post, Stress and glucocorticoids affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the hippocampus, J. Neurosci. 15 (1995) 1768–1777. [26] J.L. Sonnenberg, P.F. Macgregor-Leon, T. Curran, J.I. Morgan, Dynamic alterations occur in the levels and composition of transcription factor AP-1 complexes after seizure, Neuron 3 (1989) 359–365. [27] J.E. Sprague, M.P. Johnson, C.J. Schmidt, D.E. Nichols, Studies on the mechanism of p-chloroamphetamine neurotoxicity, Biochem. Pharmacol. 52 (1996) 1271–1277. [28] X. Tao, S. Finkbeiner, D.B. Arnold, A.J. Shaywitz, M.E. Greenberg, Ca2+ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism, Neuron 20 (1998) 709–726. [29] N. Thiriet, B. Ladenheim, M.T. McCoy, J.L. Cadet, Analysis of ecstasy (MDMA)-induced transcriptional responses in the rat cortex, FASEB J. 16 (2002) 1884–1887. [30] V.A. Vaidya, G.J. Marek, G.K. Aghajanian, R.S. Duman, 5-HT2A receptor-mediated regulation of brain-derived neurotrophic factor mRNA in the hippocampus and the neocortex, J. Neurosci. 17 (1997) 2785–2795. [31] Q. Yuan, C.W. Harley, A. Darby-King, R.L. Neve, J.H. McLean, Early odor preference learning in the rat: bidirectional effects of cAMP response element-binding protein (CREB) and mutant CREB support a causal role for phosphorylated CREB, J. Neurosci. 23 (2003) 4760–4765. [32] T.S. Zetterstr¨om, Q. Pei, T.R. Madhav, A.L. Coppell, L. Lewis, D.G. Grahame-Smith, Manipulations of brain 5-HT levels affect gene expression for BDNF in rat brain, Neuropharmacology 38 (1999) 1063–1073.