Evidence for increased expression of the ... - Wiley Online Library

Journal of Neurochemistry, 2005, 94, 875–883

doi:10.1111/j.1471-4159.2005.03192.x

Evidence for increased expression of the vesicular glutamate transporter, VGLUT1, by a course of antidepressant treatment Rosa M. Tordera, Qi Pei and Trevor Sharp University Department of Pharmacology, Oxford, UK

Abstract The therapeutic effect of a course of antidepressant treatment is believed to involve a cascade of neuroadaptive changes in gene expression leading to increased neural plasticity. Because glutamate is linked to mechanisms of neural plasticity, this transmitter may play a role in these changes. This study investigated the effect of antidepressant treatment on expression of the vesicular glutamate transporters, VGLUT1– 3 in brain regions of the rat. Repeated treatment with fluoxetine, paroxetine or desipramine increased VGLUT1 mRNA abundance in frontal, orbital, cingulate and parietal cortices, and regions of the hippocampus. Immunoautoradiography analysis showed that repeated antidepressant drug treatment increased VGLUT1 protein expression. Repeated electroconvulsive shock (ECS) also increased VGLUT1 mRNA

abundance in regions of the cortex and hippocampus compared to sham controls. The antidepressant drugs and ECS did not alter VGLUT1 mRNA abundance after acute administration, and no change was detected after repeated treatment with the antipsychotic agents, haloperidol and chlorpromazine. In contrast to VGLUT1, the different antidepressant treatments did not commonly increase the expression of VGLUT2 or VGLUT3 mRNA. These data suggest that a course of antidepressant drug or ECS treatment increases expression of VGLUT1, a key gene involved in the regulation of glutamate secretion. Keywords: antidepressant, glutamate, vesicular glutamate transporter. J. Neurochem. (2005) 94, 875–883.

Many current antidepressant treatments have immediate actions on monoamine transport and metabolism, but all need a course of treatment of several weeks to achieve their full therapeutic effect. The molecular mechanism underlying the antidepressant effect is not known, but increasing evidence suggests that the drugs act to modulate gene expression and thereby increase neural plasticity (Duman 2002). For example, different classes of antidepressant drugs, when administered repeatedly but not acutely, increase the expression of brain derived neurotrophic factor (BDNF) and its receptor TrkB (Nibuya et al. 1995; Coppell et al. 2003) in hippocampus. Other work has demonstrated that a course of antidepressant drug treatment increases hippocampal and cortical expression of transcription factor immediate early genes (IEGs) such as c-fos (Morinobu et al. 1997; Pei et al. 2003) as well as effector IEGs such as cAMP response element binding protein (CREB; Thome et al. 2000) and activity-related cytoskeletal protein (Arc; Pei et al. 2003). Antidepressant drugs also promote hippocampal neurogenesis (Santarelli et al. 2003). Many of the effects of antidepressant drugs are common to repeated electroconvulsive

shock (ECS), which is thought to model electroconvulsive therapy (Duman and Vaidya 1998). Several lines of evidence suggest that these effects of antidepressant treatment might be linked with an increase in glutamatergic transmission. First, it is well known that glutamate signalling plays a key role in mechanisms of neural plasticity and enhanced glutamatergic transmission has been shown to mediate increases in trophic factor and IEG expression (Lerea 1997; Xiao et al. 2000; Steward and Worley 2001). Although a rise in glutamate function has been associated with decreases in neurogenesis in some

Received December 12, 2004; revised manuscript received February 17, 2005; accepted February 17, 2005. Address correspondence and reprint requests to Dr T. Sharp, University Department of Pharmacology, Mansfield Road, Oxford OX1 3QT, UK. E-mail: [email protected] Abbreviations used: Arc, activity-related cytoskeletal protein; BDNF, brain-derived neurotrophic factor; CREB, cAMP response element binding protein; ECS, elctroconvulsive shock; 5-HT, 5-hydroxytryptamine; IEG, immediate early gene; VGLUT, vesicular glutamate transporter.

2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 875–883

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876 R. M. Tordera et al.

studies (Fuchs and Gould 2000), other studies are consistent with increased neurogenesis (Arvidsson et al. 2001; Luk et al. 2003). Second, it is established that there are strong interactions between monoamine and glutamate systems, and especially relevant in this context is the excitatory effect of 5-hydroxytryptamine (5-HT) on cortical glutamate (e.g. Aghajanian and Marek 1997). Finally, 5-HT modulates each of trophic factor expression (Zetterstro¨m et al. 1999), IEG expression (Pei et al. 2003) and neurogenesis (Santarelli et al. 2003; Banasr et al. 2004). The loading of glutamate into the synaptic vesicles via the vesicular glutamate transporter (VGLUT) is an essential step in glutamatergic synaptic transmission. Recently three vesicular glutamate transporter subtypes have been identified (VGLUT1, VGLUT2 and VGLUT3). Although the VGLUTs are highly homologous and have similar characteristics when loading vesicles with glutamate (Fremeau et al. 2001; Herzog et al. 2001; Takamori et al. 2001; Gras et al. 2002; Varoqui et al. 2002), they have different CNS distributions. VGLUT1 is especially dense in cortical and hippocampal regions, whereas VGLUT2 is rich in the diencephalon (Hisano et al. 2002; Fremeau et al. 2001; Herzog et al. 2001), and VGLUT3 defines a discrete subpopulation of neurones and is often coexpressed with cholinergic, serotonergic or GABAergic markers (Gras et al. 2002; Schafer et al. 2002; Herzog et al. 2004). In preliminary studies, we unexpectedly found that repeated antidepressant drug treatment increased the abundance of VGLUT1 mRNA in several brain regions of the rat. Here we report a detailed account and investigation of these findings.

Experimental procedures Animals Male Sprague-Dawley rats (250–270 g, Harlan-Olac, Bicester, UK) were housed in groups of six and maintained in a temperature (21 ± 1C) and humidity-controlled room on a 12 h light–dark cycle (lights on 07.00 h) with food and water provided ad libitum. All animal experiments were carried out according to Home Office guidelines and the Animal Scientific Procedures Act of 1986 (UK). Drug treatment and electroconvulsive shock Four sets of experiments each comprising three to four groups of rats (n ¼ 6/group) were carried out. (i) Administration of saline, fluoxetine, paroxetine or desipramine (each at 5 mg/kg, i.p.) twice daily (9.00 h and 17.00 h) for 14 days. The antidepressants were administered at doses that have been shown to effectively block 5-HT and noradrenaline reuptake and to achieve plasma drug levels in the therapeutic range (Ainsworth et al. 1998; Hajo´s-Korcsok et al. 2000). (ii) Administration of a single injection of saline, fluoxetine, paroxetine or desipramine (each at 5 mg/kg, i.p.) at 17.00 h.

(iii) Administration of electroconvulsive shock (ECS) or sham either once or five times every other day for 10 days. ECS was administered under halothane anaesthesia (150 V, 50 Hz, 1 s) via ear-clip electrodes, whereas shams were anaesthetized and had electrodes placed but no current was delivered. (iv) Administration of haloperidol (1 mg/kg i.p.), chlorpromazine (15 mg/kg, i.p.) or saline once daily (approximately 17.00 h) for 14 days. These doses would be expected to give a peak dopamine D2 receptor occupancy at least as great as that attained in patients and have previously been shown to produce effects on gene expression (see Law et al. 2004). All four sets of experiments were analysed for VGLUT1 mRNA, and most experiments were analysed for VGLUT2 and VGLUT3 mRNA. The first set of experiments (i) was repeated to confirm the VGLUT1 mRNA data and for a follow up analysis of VGLUT1 protein. All rats were killed 18 h after the last drug injection or ECS. For in situ hybridization studies, rats were killed by cervical dislocation. For immunoautoradiography studies, rats were over-dosed with pentobarbitone (200 mg/kg, i.p.) and perfused with 200 mL of saline (0.9%). Brains were quickly removed, frozen in isopentane ()30C) and stored at )70C prior to use.

In situ hybridization Coronal brain sections (12 lm) were thaw-mounted onto gelatinesubbed slides and pretreated for in situ hybridization histochemistry using previously published methods (Pei et al. 2003). The sequences for antisense 35SdATP-labelled oligonucleotides for VGLUT1–3 (Herzog et al. 2001; Gras et al. 2002) were as follows: (i) VGLUT1, 5¢-GCACTGGGCACAAGGGAAGACTTGCATCTT-3¢; (ii) VGLUT2, 5¢-ACAGATTGCACTTGATGGGACTCTCACGGT-3¢; (iii) VGLUT3, a mix of 5¢-GTAAGATCCCCAGCGAATCTCCCACGGCAT-3¢, 5¢-CAATAGGAGAGGCACCTCAGAGCCCTTAGC-3¢ and 5¢-GGATTCTCTCTGTTGTCTCCGATCCGTCTT-3¢. Radiolabelled oligonucleotide was added to each section (1 · 106 cpm/section) in hybridization buffer and after incubation overnight in a hybridization oven (31–33C), slides were washed in saline sodium citrate buffer at 55C for 3 · 20 min followed by 2 · 60 min at room temperature. Sections were then air-dried and exposed to Biomax film (Amersham, Buckinghamshire, UK) for 3, 6 or 21 days for the VGLUT1, VGLUT2 and VGLUT3 mRNA hybridized sections, respectively. Controls included hybridization of sections using oligonucleotides in the sense orientation and displacement with unlabelled probes. The relative abundance of VGLUT1–3 mRNA in selected areas was determined by densitometric quantification of autoradiograms using an MCID image analysis system (Image Research, St Catharines, Canada). Optical density values were calibrated to 35S tissue equivalents using 14C microscales (Amersham, UK) and the appropriate conversion factor (Miller et al. 1989). Measurements were made at plates 6–7 (frontal, cingulate and orbital cortex), 11–12 (parietal, piriform cortex and caudate putamen), 30–33 (hippocampus) 30–32 (medial thalamic nuclei) and 52–54 (dorsal and medial raphe) of the rat brain atlas of Paxinos and Watson (1986).


VGLUT expression and antidepressants 877

Immunoautoradiography VGLUT1 protein was measured by immunoautoradiography as described by Herzog et al. (2001). Coronal brain sections (12 lm) were mounted on gelatine-subbed slides and stored at )70C until use. On the day of the experiment, sections were fixed with 4% paraformaldehyde, washed with phosphate-buffered saline (3 · 5 min) and incubated with blocking solution containing donkey serum 1%, bovine serum albumin 3% and NaI 2 mM in buffer phosphate-buffered saline. After 1 h, sections were incubated overnight at 4C in the same buffer containing a rabbit antiVGLUT1 antiserum (1:5000, generous gift of Dr El Mestikawy) that has been extensively validated in previous studies (Herzog et al. 2001). Sections were washed with phosphate-buffered saline and then incubated with a secondary antibody donkey anti-rabbit 125IIgG (0.25 lCi/mL; Amersham Bioscience). Sections were exposed to X-ray films (Biomax MR, Amersham) for 2 days. Optical density values of the immunoautoradiograms (MCID image analysis system) were calibrated to 125I tissue equivalents using 125I microscales prepared ‘in house’. Data and statistical analysis Densitometric values were measured from three sections, averaged and expressed as nCi/g tissue. Data are presented as mean ± SEM expressed as a percentage of control values. The autoradiograms presented were scanned under high resolution into Adobe Photoshop (Version 7.0). Both in situ hybridization and immunoautoradiography data were analysed statistically using one-way ANOVA and compared against vehicle groups using post hoc Dunnett’s t-test. Drugs Paroxetine HCl (GlaxoSmithKline, Harlow, UK), fluoxetine HCl (Eli Lilly & Co. Indianapolis, USA), desipramine HCl, chlorpromazine HCl and haloperidol (each from Sigma-Aldrich, Poole, UK) were

Saline

Fluoxetine

Fig. 1 Representative autoradiograms illustrating the effect of repeated administration of antidepressant drugs on vesicular glutamate transporter 1 (VGLUT1) mRNA expression in sections of the rat forebrain. Top row of images represent sections cut at plates 6–7 of the stereotaxic atlas of Paxinos and Watson (1986), middle row of images represent sections cut at plate 11, and bottom row of images

dissolved in saline (0.9%) and administered i.p. in a volume of 1 mL/kg.

Results

Effect of antidepressant treatment on vesicular glutamate transporter 1 mRNA expression The pattern of VGLUT1 mRNA expression in rat brain as illustrated in Fig. 1 (e.g. high abundance in the cerebral cortices and hippocampus, lower abundance in thalamus) agrees with that previously described (Ni et al. 1995; Hisano et al. 2002; Fremeau et al. 2004a). Compared to vehicle controls, rats treated repeatedly with fluoxetine, paroxetine or desipramine (5 mg/kg, i.p. twice daily for 14 days) showed a significant increase in VGLUT1 mRNA abundance in cortical regions (frontal, cingulate, orbital and parietal cortex) and hippocampus (CA1 and dentate gyrus). These findings were confirmed by the analysis of a second set of brains and the data were pooled (Fig. 2). Figure 2 shows that the most marked change in VGLUT1 mRNAwas in the deep layer of the frontal cortex (+35%, +47% and +47% for fluoxetine, paroxetine and desipramine, respectively). Autoradiograms illustrating the effect of antidepressant drugs on VGLUT1 mRNA are given in Fig. 1. Compared to sham controls, rats treated repeatedly with ECS (five times over 10 days) also showed a significant increase (10–25%) in VGLUT1 mRNA in the frontal and orbital cortex and hippocampus (dentate gyrus) (Fig. 3). In contrast, a single injection of fluoxetine, paroxetine or desipramine (5 mg/kg, i.p.) had no effect on VGLUT1

Paroxetine

Desipramine

represent sections cut at plates 32–33. Fluoxetine, paroxetine and desipramine were injected twice daily for 14 days at 5 mg/kg, i.p. Abbreviations: Fr, frontal cortex; Cg, cingulate cortex; Ob, orbital cortex; Par, parietal cortex; Cpu, caudate putamen; Pir, piriform cortex; DG, dentate gyrus.



Fig. 2 Effect of a course of treatment with fluoxetine, paroxetine and desipramine (5 mg/kg, i.p. twice daily for 14 days) on the abundance of vesicular glutamate transporter 1 (VGLUT1) mRNA in rat forebrain regions. Each column represents a mean ± SEM value (n ¼ 12 rats). **p < 0.01, *p < 0.05 vs. saline controls (ANOVA Dunnett’s t-test). Abbreviations: Fr-in, inner layer frontal cortex; Fr-out, outer layer frontal cortex; Ob, orbital cortex; Cg, cingulate cortex; Par-in, inner layer parietal cortex; Par-out, outer layer parietal cortex; Pir, piriform cortex; DG, dentate gyrus.

Fig. 3 Effect of acute and repeated (five times over 10 days) electroconvulsive shock or sham treatment on abundance of vesicular glutamate transporter 1 (VGLUT1) mRNA in rat forebrain regions. Each column represents a mean ± SEM value (n ¼ 6 rats). **p < 0.01, *p < 0.05 vs. sham controls (Student’s t-test). Abbreviations: Fr-in, inner layer frontal cortex; Fr-out, outer layer frontal cortex; Ob, orbital cortex; Cg, cingulate cortex; Par-in, inner layer parietal cortex; Par-out, outer layer parietal cortex; Pir, piriform cortex; DG, dentate gyrus.

mRNA expression compared to saline controls (Fig. 4). Similarly, a single ECS did not alter VGLUT1 compared to sham controls (Fig. 3). Furthermore, in contrast to the antidepressant drugs and ECS, repeated (14 days) administration of the antipsychotic agents chlorpromazine (15 mg/kg, i.p.) or haloperidol (1 mg/kg, i.p.) had no significant effect on VGLUT1 mRNA abundance in cortical regions or hippocampus (Table 1).

Fig. 4 Effect of a single injection of fluoxetine, paroxetine and desipramine (each at 5 mg/kg, i.p.) on abundance of vesicular glutamate transporter 1 (VGLUT1) mRNA in rat forebrain regions. Each column represents a mean ± SEM value (n ¼ 6 rats). Abbreviations: Fr-in, inner layer frontal cortex; Ob, orbital cortex; Cg, cingulate cortex; Pir, piriform cortex; Par-in, inner layer parietal cortex; Par-out, outer layer parietal cortex; DG, dentate gyrus.

Effect of antidepressant treatment on vesicular glutamate transporter 2 mRNA expression The pattern of expression of VGLUT2 mRNA agreed with previous reports (Herzog et al. 2001; Kaneko et al. 2002; Fremeau et al. 2004b). Specifically, the abundance was high in different nuclei of the thalamus and hypothalamus, but low in regions of the cortex and hippocampus (data not shown). As indicated in Table 2, repeated treatment with desipramine (5 mg/kg, i.p. twice daily for 14 days) produced a statistically significant increase of the VGLUT2 mRNA abundance in thalamus (ventroposteromedial nucleus). However, this effect was not detected in animals treated repeatedly with fluoxetine, paroxetine or ECS (Table 2). None of the antidepressant drugs or ECS altered the abundance of VGLUT2 mRNA when administered acutely (Table 2). Effect of antidepressant treatment on vesicular glutamate transporter 3 mRNA expression The distribution of VGLUT3 mRNA was similar to that described previously (Gras et al. 2002). Specifically, VGLUT3 mRNA expression was high in the midbrain raphe nuclei and in scattered spots across the caudate nucleus (data not shown). Expression of VGLUT3 mRNA was not altered in animals treated repeatedly (5 mg/kg, twice a day, 14 days) with either fluoxetine or paroxetine, but a significant increase was detected in the dorsal raphe nucleus after desipramine (Table 3). Effect of antidepressant treatment on vesicular glutamate transporter 1 protein expression The results of the in situ hybridization experiments were followed up using immunoautoradiography. As previously described (Herzog et al. 2001; Kaneko et al. 2002), high



Table 1 Effect of a course of treatment with chlorpromazine and haloperidol on vesicular glutamate transporter 1 (VGLUT1) mRNA expression in the rat brain regions Brain region

Saline

Fr Cg Ob Par-deep Par-Sup Pir CA1 CA3 DG

100 100 100 100 100 100 100 100 100

± ± ± ± ± ± ± ± ±

2 1 2 6 4 5 6 4 5

Chlorpromazine

Haloperidol

95 94 96 107 102 101 97 97 100

88 88 85 99 101 98 99 97 106

± ± ± ± ± ± ± ± ±

3 3 3 6 8 2 5 3 6

± ± ± ± ± ± ± ± ±

7 7 6 5 5 3 4 2 3

Table 2 Effect of a course of antidepressant drug and ECS treatment on VGLUT2 mRNA expression in the rat brain regions PV

Repeated treatment Saline 100 Fluoxetine 103 Paroxetine 102 Desipramine 124 Sham 100 ECS 98 Acute treatment Saline 100 Fluoxetine 95 Paroxetine 97 Desipramine 94 Sham 100 ECS 98

CM

LDVL

Treatment

Fr

Saline Fluoxetine Paroxetine Desipramine

100 105 120 113

Par ± ± ± ±

9 7 11 5

100 115 114 119

CPu ± ± ± ±

7 8 7 11

100 104 105 107

DRN ± ± ± ±

4 4 4 3

100 94 102 134

± ± ± ±

12 10 10 15*

The drugs were administered twice daily for 14 days at a dose of 5 mg/kg i.p. Each column represents a mean ± SEM value (n ¼ 6 rats). *p < 0.05 vs. saline controls (ANOVA Dunnett’s t-test). Abbreviations: Fr, frontal cortex; Par, parietal cortex; Cpu, caudate putamen; DRN, dorsal raphe nucleus.

Saline, chlorpromazine (15 mg/kg, i.p.) and haloperidol (1 mg/kg, i.p.) were administered once daily for 14 days. Rats were killed approximately 20 h after the last injection. Data are given as mean ± SEM values (n ¼ 6) and expressed as a percentage of saline controls. Abbreviations: Fr, frontal cortex; Cg, cingulate cortex; Ob, orbital cortex; Par-deep, parietal cortex deep layer; Par-sup, parietal cortex superficial layer; DG, dentate gyrus.

Treatment

Table 3 Effect of a course of antidepressant drug treatment on VGLUT3 mRNA expression in the rat brain regions

VPM

± ± ± ± ± ±

10 8 18 10 2 5

100 121 133 123 100 91

± ± ± ± ± ±

7 11 20 12 12 16

100 96 101 129 100 97

± ± ± ± ± ±

5 8 17 9 12 18

100 109 101 133 100 90

± ± ± ± ± ±

7 6 17 7* 8 1

± ± ± ± ± ±

3 4 9 6 2 3

100 92 109 102 100 93

± ± ± ± ± ±

4 4 4 9 4 3

100 92 107 94 100 103

± ± ± ± ± ±

2 6 4 10 8 3

100 90 103 94 100 95

± ± ± ± ± ±

3 9 3 7 6 4

Data are given as mean ± SEM values (n ¼ 6) and expressed as a percentage of the appropriate controls. The drugs were administered either once (acute) or twice daily for 14 days (repeated) at a dose of 5 mg/kg i.p. Abbreviations: PV, paraventricular; CM, central medial; LDVL, laterodorsalventrolateral; VPM, ventroposteromedial thalamic nuclei. *p < 0.05 vs. saline controls.

levels of VGLUT1-like immunoreactivity were detected throughout the forebrain including the cerebral cortex, hippocampus and caudate putamen (Fig. 5). In animals treated repeatedly with fluoxetine, paroxetine or desipramine (5 mg/kg, i.p. twice daily for 14 days), VGLUT1 immunoreactivity was increased in frontal, cingu-

late and parietal cortex compared to vehicle controls (Figs 5 and 6). The greatest effect of antidepressants was in the cingulate cortex (+33%, +20% and +32% for fluoxetine, paroxetine and desipramine, respectively). VGLUT1 immunoreactivity also tended to increase in hippocampus, but this effect only reached statistical significance for fluoxetine (dentate gyrus molecular layer) (Fig. 6). Discussion

Glutamate may play a role in the neuroadaptive changes associated with a course of antidepressant treatment (see Introduction). The main finding of the present study is that treatment with different antidepressant drugs (fluoxetine, paroxetine and desipramine), as well as ECS, increases the expression of the vesicular glutamate transporter, VGLUT1. Our data suggest that this effect is both adaptive, in that none of the antidepressants increased VGLUT1 expression after acute administration, and treatment-specific, in that repeated administration of antipsychotic agents (haloperidol and chlorpromazine) had no effect. In comparison to VGLUT1, the different antidepressants did not commonly increase the expression of the two other vesicular glutamate transporters, VGLUT2 and VGLUT3. When administered over 14 days, each of fluoxetine, paroxetine and desipramine elevated VGLUT1 mRNA abundance in specific regions of the anterior cortex (frontal, orbital, cingulate and parietal cortices) as well as hippocampus (CA1 and dentate gyrus subfields). Interestingly, repeated ECS also increased the abundance of VGLUT1 mRNA expression. Whilst the effect of ECS was smaller than that of the drugs, and detected in fewer regions (frontal and orbital cortices, and dentate gyrus of the hippocampus), it is important to note that the measurements were obtained 18 h after the last ECS. Previous studies show that, whereas the effect of repeated ECS on BDNF mRNA is long-lasting, it markedly diminishes over the 24 h postshock period (Zetterstro¨m et al. 1998). Further analysis of the effect of the antidepressant drugs using immunoautoradiography demonstrated that VGLUT1



Saline

Fluoxetine

Fig. 5 Representative immunoautoradiograms illustrating the effect of repeated administration of antidepressant drugs on vesicular glutamate transporter 1 (VGLUT1) protein expression in sections of the rat forebrain. Top row of images represent sections cut at plates 6–7 of the stereotaxic atlas of Paxinos and Watson (1986), middle row of images represent sections cut at plate 11, and bottom row of images

Fig. 6 Effect of repeated administration of fluoxetine, paroxetine and desipramine (5 mg/kg, i.p. twice daily for 14 days) on abundance of vesicular glutamate transporter 1 (VGLUT1) protein in rat forebrain regions measured by immunoautoradiography. Each column represents a mean ± SEM value (n ¼ 6 rats). **p < 0.01, *p < 0.05 vs. saline controls (ANOVA Dunnett’s t-test). Abbreviations: Fr, frontal cortex; Ob, orbital cortex; Cg, cingulate cortex; Cpu, caudate putamen; Par-in, inner layer parietal cortex; Par-out, outer layer parietal cortex; Or, oriens layer; Mol, molecular layer; DG, dentate gyrus.

Paroxetine

Desipramine

represent sections cut at plates 32–33. Fluoxetine, paroxetine and desipramine were injected twice daily for 14 days at 5 mg/kg, i.p. Abbreviations: Fr, frontal cortex; Cg, cingulate cortex; Ob, orbital cortex; Par, parietal cortex; Cpu, caudate putamen; Pir, piriform cortex; DG, dentate gyrus.

protein increased in many of the regions where enhanced VGLUT1 mRNA was detected. The lack of significant increase in VGLUT1 protein in hippocampus despite a rise in VGLUT1 mRNA might reflect a lower sensitivity of the immunoautoradiography compared to the in situ hybridization technique. More likely, the mismatch might be due to the fact that VGLUT1 mRNA labels cell bodies, whereas VGLUT1 protein labels almost exclusively the terminals. The regional pattern of change in VGLUT1 expression was similar for each of the treatments tested. Given that paroxetine and fluoxetine are selective inhibitors of the 5-HT transporter, and that desipramine acts with selectivity for the noradrenaline transporter, the up-regulation of VGLUT1 expression may well be linked to increase in 5-HT and noradrenaline transmission. This idea is consistent with much evidence of interactions between the monoamines and glutamate, and especially evidence of a facilitatory effect of 5-HT on cortical glutamate (Aghajanian and Marek 1997; Pei et al. 2004). Moreover, there is a large body of data suggesting that, as with the rise in VGLUT1 expression, a course of treatment with these drugs is needed to produce the greatest elevation in monoaminergic neurotransmission (Blier and de Montigny 1994; Hajo´s-Korcsok et al. 2000; Hjorth et al. 2000). Similarly, there is evidence that increases



in monoamine transmission occur during a course of ECS (Chaput et al. 1991). Therefore, not only is VGLUT1 expression enhanced by antidepressant treatment, but monoamines may be involved in this process. This is the first study that we are aware of to demonstrate the regulation of VGLUT1 gene expression. However, there is evidence in the literature for the in vivo regulation of other vesicular transporters (vesicular monoamine transporter 2 and vesicular acetylcholine transporter) by psychotropic drugs (Brown et al. 2001; Zucker et al. 2001) and nerve growth factor (Tian et al. 1996), respectively. It is notable that the expression of the other vesicular glutamate transporters, VGLUT2 and VGLUT3, was weakly modulated under conditions that strongly modulated VGLUT1. Thus, with the exception of a small number of changes in VGLUT2 (ventroposteromedial thalamic nucleus) and VGLUT3 (dorsal raphe nucleus) in rats treated with desipramine, these transporters appeared much less responsive to antidepressant treatment compared to VGLUT1. As the distribution of VGLUT1 is very different from that of VGLUT2 and VGLUT3, it is very probable that the latter transporters are regulated by different influences. In the case of desipramine, the changes in VGLUT2 and VGLUT3 might reflect alterations in noradrenergic transmission, but a more detailed pharmacological investigation is necessary to support this idea. The present study did not address the functional significance of the observed increase in VGLUT1 expression, but there is good evidence that this effect would result in a more efficient accumulation of glutamate in the synaptic vesicles and an increased synaptic availability of glutamate during neurotransmission. Thus, it was recently demonstrated that the quantal size of glutamatergic neurones is enhanced with over-expression of VGLUT1 and reduced in the knock-out of VGLUT1 (Wojcik et al. 2004; see also Daniels et al. 2004). Studies on other vesicular transporters (vesicular acetylcholine transporter and vesicular monoamine transporter 2) also show that elevated expression increases in the vesicular contents and the amount of transmitter released per quanta (Song et al. 1997; Pothos 2002). Although there is no consistent evidence that antidepressant-treated animals demonstrate increased glutamate release in in vitro brain tissue preparations (Bouron and Chatton 1999; Michael-Titus et al. 2000; Wang et al. 2003), there is evidence in the present data that the increase in VGLUT1 expression may be regionspecific and difficult to detect using such neurochemical techniques. Our data do not, however, exclude the possibility that the increase in VGLUT1 expression reflects an increase in the number of glutamatergic synapses (synaptogenesis). Thus, several studies have recently shown that repeated treatment with antidepressant drugs and ECS induces expression of genes associated with synaptogenesis in cortex and hippocampus (Drigues et al. 2003; Altar et al. 2004; Rapp et al.

2004), and in the case of ECS there is evidence that it induces axonal sprouting of hippocampal granule cells (Lamont et al. 2001). Moreover, both 5-HT and noradrenaline have been linked to the formation and the maintenance of central synapses (e.g. Whitaker-Azmitia et al. 1995; Matsukawa et al. 2003; Imai et al. 2004). In addition to VGLUT1, the expression of a number of other genes has been found to be up-regulated in cortex/ hippocampus by a course of antidepressant drug and ECS treatment, and these changes have been linked to increased monoamine transmission. These genes include Arc (Pei et al. 2003), c-fos (Morinobu et al. 1997; Pei et al. 2003), CREB (Thome et al. 2000), BDNF (Nibuya et al. 1995; Coppell et al. 2003) and TrkB (Nibuya et al. 1995). All of these genes may be part of a co-ordinated cascade of altered gene expression that mediates a change in neural plasticity leading to an antidepressant effect (see Introduction). An interesting possibility is that the rise in VGLUT1 expression and subsequent increase in glutamatergic transmission (see above) constitutes a trigger for this cascade. This idea is supported by evidence that increased glutamatergic transmission mediates an increase in trophic factor and IEG expression (Lerea 1997; Xiao et al. 2000; Steward and Worley 2001). In conclusion, the present study provides the first evidence that a course of antidepressant treatment increases expression of the vesicular glutamate transporter, VGLUT1, a key gene involved in the regulation of glutamate secretion. This finding may be relevant to preclinical evidence that increased glutamate underpins neuroadaptive responses induced by repeated antidepressant administration (see Introduction), and recent clinical studies suggesting that severe depression is associated with a glutamate deficit in cortex (Michael et al. 2003; Mirza et al. 2004). Acknowledgements We are grateful to Dr Salah El Mestikawy for the generous gift of VGLUT1 antibody and valuable advice regarding the immunoautoradiography methods, and Dr Philip Burnet for supplying tissue from antipsychotic treated animals. This work was supported by the MRC (G9102310, TS), fellowships from the Spanish Government (RT) and the EU Framework 6 Integrated Project NEWMOOD (LSHM-CT-2004-503474).

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