Molecular Psychiatry (2007) 12, 612–619 & 2007 Nature Publishing Group All rights reserved 1359-4184/07 $30.00 www.nature.com/mp
LETTERS TO THE EDITOR
Clozapine upregulates the expression of the vesicular GABA transporter (VGAT) in rat frontal cortex Molecular Psychiatry (2007) 12, 612–613; doi:10.1038/sj.mp.4001987
Clozapine is considered the most efficacious antipsychotic drug for the treatment of schizophrenia, possibly because of its effects on a broad array of molecular targets.1 It has recently been shown that the effects of clozapine on the firing of cortical neurons depend on the neurons’ baseline firing rate, inhibiting those with a high firing rate and exciting those with a low firing rate.2 Such a novel effect may conceivably require a concerted action at glutamatergic and GABAergic synapses. Whereas modulatory actions of clozapine on molecules acting at glutamatergic synapses are well established,3,4 very little is known of its action on molecules acting at GABAergic synapses. Decreased GABA synthesis and release are reportedly the crucial steps in the cascade affecting the cortical GABAergic system in schizophrenia.5 We therefore studied whether clozapine affects the expression of the vesicular GABA transporter, VGAT, which is crucial for GABAergic function and contributes to scaling transmitter storage and release at GABAergic synapses.6,7 Animal care and handling were approved by the local Ethical Committee. Treatment regimen (oral administration), doses (0.5 mg/ml in the drinking water) and length of treatment (9 weeks) were as described;4 estimated clozapine plasma levels were 20–60 ng/ml.4 Control rats received water adjusted with diluted acetic acid to match the pH of the drug solution. For immunoblotting, homogenates from frontal cortical regions rostral to the bregma were obtained from four control and four treated animals and processed as described previously.8 Labeled band visualization and densitometric analysis were performed using BioRad Chemidoc and Quantity One software. For immunocytochemistry, four control and four treated rats were perfused transcardially with a flush of saline followed by 4% paraformaldehyde. All procedures were as described in previous studies;8 anti-VGAT antibodies were kindly provided by RH Edwards (UCSF, San Francisco, CA, USA) and used at 1:1500 for immunoblotting and 1:1000 for immunofluorescence. Sections from frontal cortex were examined using a Bio-Rad confocal laser-scanning microscope. Image processing and analysis was performed as described.8 For both immunoblotting and immunocytochemical studies, significance was evaluated by the Mann–Whitney U test.
Immunoblotting studies showed that anti-VGAT antibodies recognized a single band of B57 kDa (Figure 1a), consistent with its known molecular weight.9 Quantitative analysis of independent samples showed that VGAT was significantly increased in clozapine-treated animals (29.378.7%; P < 0.05; Figure 1a). In both control and treated animals, VGAT immunoreactivity (IR) in frontal cortex was characterized by puncta distributed in all cortical layers;9,10 in treated animals, however, VGAT IR was uneven, displaying areas with increased IR. To verify whether these changes occurred in the same regions where the expression and/or function of molecules acting at glutamatergic synapses is altered by clozapine, we studied the expression of VGAT in the regions of diminished expression of glutamate transporter 1 (GLT-1) described in previous studies.4,8 Thus, we analyzed the size of VGAT þ puncta in cortical fields where GLT-1 IR was reduced (‘area of effect’ in Figure 1b) and in those where it was unchanged (‘area of no effect’ in Figure 1b). In frontal cortical regions exhibiting decreased GLT-1 expression following clozapine administration (60.9713.7% vs controls; P < 0.05), the size of VGAT þ puncta was increased by 48.7713.9% (P < 0.05) (Figure 1c), reflecting an increased amount of protein,8 whereas in areas where GLT-1 was unaffected by clozapine, their size was unchanged (Figure 1c). These studies show for the first time that clozapine upregulates VGAT; the quantitative difference between blotting and immunocytochemical studies in all likelihood to be attributed to a ‘dilution effect’, as homogenates used for western studies inevitably contained regions with increased VGAT and regions where the protein was unchanged. The finding that the protein responsible for transferring GABA from the cytoplasm to synaptic vesicles is increased by clozapine is in line with the results of studies showing that GABA synthesis as well as its reuptake are reduced in certain inhibitory neurons in the prefrontal cortex of schizophrenics.5 In addition, the present data show that clozapine-induced upregulation of VGAT occurs in the very regions where clozapine downregulates the main glutamate transporter, GLT-1, thus indicating a possible physiological substrate for its state-dependent action on cortical neurons. Finally, these results strengthen the hypothesis that clozapine’s efficacy depends on its effects on numerous synaptically active molecules.
Letters to the Editor
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References 1 Roth B, Sheffler DJ, Kroeze WK. Nat Rev Drug Discov 2004; 3: 353–359. 2 Homayoun H, Moghaddam B. Biol Psychiatry 2007; 61: 679–687. 3 Leveque J-C, Macias W, Rajadhyaksha A, Carlson RR, Barczak A, Kang S et al. J Neurosci 2000; 20: 4011–4020. 4 Melone M, Vitellaro-Zuccarello L, Vallejo-Illarramendi A, Perez-Samartin A, Matute C, Cozzi A et al. Mol Psychiatry 2001; 6: 380–386. 5 Lewis DA, Gonzalez-Burgos G. Nature Med 2006; 12: 1016–1022. 6 Wojcik SM, Katsurabayashi S, Guillemin I, Friauf E, Rosenmund C, Brose N et al. Neuron 2006; 50: 575–587. 7 De Gois S, Schafer MK, Defamie N, Chen C, Ricci A, Weihe E et al. J Neurosci 2005; 25: 7121–7133. 8 Bragina L, Melone M, Fattorini G, Torres-Ramos M, VallejoIllarramendi A, Matute C et al. J Neurochem 2006; 99: 131–141. 9 Chaudry FA, Reimer RJ, Bellocchio EE, Danbolt NC, Osen KK, Edwards RH et al. J Neurosci 1998; 18: 9733–9750. 10 Minelli A, Alonso-Nanclares L, Edwards RH, DeFelipe J, Conti F. Neuroscience 2003; 117: 337–346.
c-Fos expression identifies brain areas activated in response to nortriptyline Molecular Psychiatry (2007) 12, 613–615; doi:10.1038/sj.mp.4001989
Figure 1 (a) Immunoblotting of VGAT (15 mg of protein) in the frontal cortex of control and clozapine-treated rats. (b) GLT-1/VGAT double-labeled section showing VGAT IR in areas where GLT-1 is downregulated by clozapine (area of effect on GLT-1) and where it is not downregulated (area of no effect on GLT-1). (c) size of VGAT þ puncta in control and treated rats. In a and c, values are expressed as mean7s.e.m. *P < 0.05. Scale bar: 30 mm.
L Bragina, M Melone, G Fattorini and F Conti Dipartimento di Neuroscienze, Universita` Politecnica delle Marche, Torrette di Ancona, Ancona, Italy. E-mail:
[email protected]
This study shows that the administration of nortriptyline induces a pattern of c-Fos expression in the central nervous system of rats that resembles the typical, inverted U-shaped dose–response curve observed in clinical pharmacokinetic studies in humans. This did not occur following the use of desipramine, an alternative noradrenergic antidepressant drug whose clinical efficacy is not associated with this type of dose–response relationship. Previous studies have suggested that there is an optimum range of nortriptyline blood levels, a therapeutic window above or below which depressed patients respond poorly or do not respond to treatment.1,2 Our group has demonstrated that there is a biphasic curvilinear relationship between plasma levels of nortriptyline and behavior, using the tail suspension test,3 an animal model employed to screen for drugs with antidepressant effects. Although noradrenergic drugs are known to induce c-Fos expression in central neurons, only one study has investigated the effects of nortriptyline on the expression of brain c-Fos.4,5 The magnitude of the therapeutic response observed with various doses of antidepressants may reflect differential activation of distinct brain areas for each dose used; therefore, we hypothesized that different doses of nortriptyline might induce different patterns of brain c-Fos expression in rats. Wistar male rats received intraperitoneal injections of nortriptyline or desipramine (5, 10, 25 and Molecular Psychiatry
