Tianeptine increases brain-derived neurotrophic factor expression in the rat amygdala

European Journal of Pharmacology 565 (2007) 68 – 75 www.elsevier.com/locate/ejphar

Tianeptine increases brain-derived neurotrophic factor expression in the rat amygdala Lawrence P. Reagan a,⁎, Robert M. Hendry a , Leah R. Reznikov a , Gerardo G. Piroli a , Gwendolyn E. Wood b , Bruce S. McEwen b , Claudia A. Grillo a a

b

Department of Pharmacology, Physiology and Neuroscience, University of South Carolina School of Medicine, 6439 Garner's Ferry Road, D40, Columbia, SC 29208, United States Harold and Margaret Milliken Hatch Laboratory of Neuroendocrinology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, United States Received 18 October 2006; received in revised form 5 February 2007; accepted 6 February 2007 Available online 20 February 2007

Abstract Chronic restraint stress affects hippocampal and amygdalar synaptic plasticity as determined by electrophysiological, morphological and behavioral measures, changes that are inhibited by some but not all antidepressants. The efficacy of some classes of antidepressants is proposed to involve increased phosphorylation of cAMP response element binding protein (CREB), leading to increased expression of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF). Conversely, some studies suggest that acute and chronic stress downregulate BDNF expression and activity. Accordingly, the aim of the current study was to examine total and phosphorylated CREB (pCREB), as well as BDNF mRNA and protein levels in the hippocampus and amygdala of rats subjected to chronic restraint stress in the presence and absence of the antidepressant tianeptine. In the hippocampus, chronic restraint stress increased pCREB levels without affecting BDNF mRNA or protein expression. Tianeptine administration had no effect upon these measures in the hippocampus. In the amygdala, BDNF mRNA expression was not modulated in chronic restraint stress rats given saline in spite of increased pCREB levels. Conversely, BDNF mRNA levels were increased in the amygdala of chronic restraint stress/tianeptine rats in the absence of changes in pCREB levels when compared to non-stressed controls. Amygdalar BDNF protein increased while pCREB levels decreased in tianeptine-treated rats irrespective of stress conditions. Collectively, these results demonstrate that tianeptine concomitantly decreases pCREB while increasing BDNF expression in the rat amygdala, increases in neurotrophic factor expression that may participate in the enhancement of amygdalar synaptic plasticity mediated by tianeptine. © 2007 Elsevier B.V. All rights reserved. Keywords: Stress; Glucocorticoid; pCREB; Antidepressant; Hippocampus

1. Introduction Stress and exposure to stress levels of glucocorticoids (GCs) are known to play an important role in the etiology, development and progression of a variety of neurological disorders, including major depressive illness. One of the hallmark features of depressive illness is dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis, and stress has been proposed to precipitate depressive episodes in this patient population. In addition to affective symptoms of depression, ⁎ Corresponding author. E-mail address: [email protected] (L.P. Reagan). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.02.023

imaging studies have revealed structural and functional changes in the hippocampus and amygdala of recurrent depressive illness patients. For example, depressive illness patients exhibit decreases in hippocampal volumes (Bremner et al., 2000; Frodl et al., 2002b; MacQueen et al., 2003; Mervaala et al., 2000; Sheline et al., 1999; Steffens et al., 2000; Vythilingam et al., 2002), whereas amygdala volumes may be increased or decreased in major depression patients (Frodl et al., 2002a; Mervaala et al., 2000; Sheline et al., 1998, 1999; von Gunten et al., 2000). Considering that the hippocampus and amygdala are major sites of glucocorticoid action in the CNS, these results suggest that stress may mediate the neuroanatomical alterations observed in recurrent depressive illness patients.

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In experimental models, chronic restraint stress affects hippocampal and amygdalar synaptic plasticity. For example, chronic stress produces dendritic atrophy of CA3 pyramidal neurons in the rat hippocampus (Watanabe et al., 1992b), as well as dendritic hypertrophy of neurons in the basolateral nucleus of the amygdala (Vyas et al., 2002). Interestingly, stressinduced morphological changes in the hippocampus are inhibited by some antidepressant treatments, including tianeptine, lithium and clomipramine (Czeh et al., 2001; van der Hart et al., 2002; Watanabe et al., 1992a; Wood et al., 2004), while other antidepressants, such as selective serotonin reuptake inhibitors (SSRIs), do not affect stress-induced morphological changes in the hippocampus (Magariños et al., 1999). The efficacy of some classes of antidepressants is proposed to involve increased phosphorylation of cAMP response element binding protein (CREB), leading to increased expression of neurotrophic factors such as brain-derived neurotrophic factor (BDNF). BDNF plays an important role in amygdalar and hippocampal synaptic plasticity (Duman et al., 2000). For example, hippocampal slices treated with BDNF before tetanic stimulation induces long-term potentiation, a cellular correlate of learning and memory, whereas the absence of BDNF engenders only short-term potentiation (Figurov et al., 1996). In the amygdala, BDNF plays a critical role in the neuroplasticity underlying amygdala-dependent fear conditioning (Rattiner et al., 2004). Interestingly, hippocampal BDNF administration also produces antidepressant-like effects, corroborating data suggesting that the mechanisms of action of some antidepressant drugs involve upregulation of hippocampal BDNF levels (Shirayama et al., 2002). Changes in BDNF levels are proposed to modulate hippocampal synaptic plasticity — in particular, stress-induced morphological changes in the hippocampus. Indeed, heterozygous BDNF knock-out mice exhibit hippocampal dendritic atrophy similar to morphological changes observed following chronic stress (McEwen et al., unpublished observations). Previous studies have examined the stress modulation of BDNF expression in the hippocampus with equivocal results (Chao et al., 1998; Duric and McCarson, 2005; Kuroda and McEwen, 1998; Nibuya et al., 1995; Rosenbrock et al., 2005; Ueyama et al., 1997; Vaidya et al., 1999). In view of these observations, the aim of the current study was to examine the phosphorylation state of CREB and the expression of BDNF under chronic stress conditions that produce morphological changes in the rat hippocampus and amygdala. In addition, since other antidepressant drugs like the SSRIs increase hippocampal phosphorylated CREB (pCREB) and BDNF levels (Duman et al., 2000), we also examined the ability of tianeptine to modulate pCREB and BDNF expression in the rat amygdala and hippocampus. 2. Materials and methods 2.1. Animal protocols Adult male Sprague–Dawley rats (CD strain, Charles River) weighing 200–225 g were housed with ad libitum access to food

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and water in accordance with all guidelines and regulations of the University of South Carolina Animal Care and Use Committee. Animals were maintained in a temperature- and light-controlled environment with a 12/12 h light/dark cycle (lights on at 0700 h). Rats were randomly assigned to experimental groups and handled daily for 2 weeks prior to the commencement of stress and drug treatment. Rats were subjected to 6-hour restraint stress daily for 21 days in wire mesh restrainers secured at the head and tail ends with clips as described in our previous studies (Reagan et al., 2004). Before restraint stress, chronic restraint stress rats were given i.p. injections of sterile saline (SS) or 10 mg/kg tianeptine (ST). Non-stressed control rats were handled each day and given daily injections of sterile saline (CS) or tianeptine (CT). Eighteen hours after the final stress session, rats were weighed and decapitated; the brains were quickly removed, the amygdala and hippocampus were microdissected on ice, frozen immediately on dry ice and stored at −70 °C until use. For the in situ hybridization and radioimmunocytochemical analysis, brains were removed, immediately frozen on dry ice, and stored at −70 °C until use. 2.2. In situ hybridization histochemistry In situ hybridization histochemistry studies were performed as described in our previous studies (Reagan et al., 2004; Wood et al., 2004). Briefly, coronal 16 μm rat brain sections were prepared on a cryostat microtome, collected on gelatin-coated, diethylpyrocarbonate (DEPC) treated slides and stored at − 70 °C until hybridization. Before the hybridization reaction, slides were removed from the freezer and air dried at room temperature. Brain sections were fixed in 4% formaldehyde in PBS, acetylated in 0.25% acetic anhydride in triethanolamine– HCl, rinsed in 2X sodium chloride–sodium citrate (SSC) and dehydrated in increasing ethanol washes. Hybridization mix (including [35S] labeled antisense sequences selective for fulllength BDNF) was added at 120 μl per slide. Slides were coverslipped and incubated overnight at 55 °C in a humidified environment. Following hybridization, coverslips were removed and the slides were washed twice in 2X SSC, treated with 20 μg/ml RNase A and washed again in 2X SSC. Slides were then washed twice in 0.2X SSC at 55 °C for 30 min and dehydrated in increasing ethanol washes. Dried sections were exposed to Kodak BioMax MR film for autoradiography. In the hippocampus, the CA1, CA2, and CA3 areas of Ammon's horn and the superior and inferior blades of the dentate gyrus were analyzed. For amygdalar studies, the basolateral nucleus of the amygdala and medial nucleus of the amygdala were included. 2.3. Enzyme-linked immunosorbent assay Commercial enzyme-linked immunosorbent assay (ELISA) kits (BDNF Emax ImmunoAssay System, Promega Corp., Madison, WI) were used to quantify BDNF protein in selected brain regions. Microdissected hippocampi and amygdalas were homogenized and sonicated in 300 ml lysis buffer (137 mM NaCl, 20 mM Tris–HCl [pH8.0], 1% NP40, 10% glycerol, 1 mM phenylmethanesulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 0.5 mM sodium vanadate). The homogenized

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samples were centrifuged at 14,000 ×g for 15 min at 4 °C. The supernatants were recovered and diluted 1:5 in Dulbecco's phosphate-buffered saline (DPBS). Diluted samples were acidified with 1 N HCl to a pH of approximately 2.6 and allowed to incubate for 15 min. Samples were then neutralized with 1 N NaOH to a pH of approximately 7.6 and stored at − 80 °C until use in ELISA according to the manufacturer's instructions. ELISA plates were analyzed at an absorbance wavelength of 450 nm with a Tecan SPECTRAFluor plate reader (Tecan U.S., Inc., Durham, NC). 2.4. Radioimmunocytochemistry As described in our previous studies (Reagan et al., 1999, 2004), coronal rat brain sections were cut on a cryostat microtome at 16 μm and mounted on gelatin-coated slides and stored at −70 °C. Slides were air-dried at room temperature, fixed with 4% formaldehyde in 0.05 M PBS for 30 min at room temperature. Sections were washed and incubated with 1% bovine serum albumin (BSA) in 0.05 M PBS for 30 min at room temperature to reduce non-specific binding. Sections were incubated with antibodies against CREB (Cell Signaling Technology; 1:250) or pCREB (Upstate Biotechnology; 1:500) for 2 h at room temperature. Sections were washed with 1% BSA in 0.05 M PBS and incubated with [35S] labeled anti-rabbit antisera at a dilution of 1:400 in 1% BSA in 0.05 M PBS for 2 h at room temperature. Sections were rinsed, dehydrated, air dried and exposed to Kodak X-OMAT film.

daily administration of tianeptine (stress + tianeptine: ST). Radioimmunocytochemical analysis revealed that CREB levels were increased in the dentate gyrus of SS rats and ST rats compared to CS rats (Fig. 1, Panel A). In agreement with our previous studies using stereology-based approaches (Wood et al., 2004), pCREB levels were increased in the CA2, CA3 and dentate gyrus of rats subjected to stress and given saline (SS; Fig. 1, Panel B). Rats subjected to chronic stress and given daily administration of tianeptine (ST) also exhibited significant increases in pCREB levels in the dentate gyrus. There was an overall effect of stress to increase hippocampal pCREB levels when compared to non-stressed controls (P ≤ 0.02); tianeptine administration had no overall effect upon pCREB in the hippocampus. 3.2. Hippocampal BDNF expression is not modulated by chronic restraint stress or tianeptine The effects of chronic restraint stress and tianeptine administration upon BDNF mRNA expression were examined

2.5. Autoradiographic film analysis and statistical analysis Computer-assisted microdensitometry of autoradiographic images was determined on MCID image analysis system (Imaging Research, Inc., St. Catherines, Canada). For quantitative purposes, microscale 14C standards (Amersham) were exposed on Kodak X-OMAT film and digitized. Grey level/optical density calibrations were performed using a calibrated film strip ladder (Imaging Research, Inc., St. Catherines, Canada). Optical density was plotted as a function of microscale calibration values. The values obtained represent the means of measurements taken from 2 to 3 sections per slide and 2 slides per animal. Statistical analysis for all studies was performed with a one-way ANOVA, followed by a Student–Newman–Keuls post-hoc test, with P b 0.05 as the cutoff for statistical significance. 3. Results 3.1. Chronic restraint stress increases pCREB levels in the rat hippocampus The effects of chronic restraint stress upon CREB expression, as well as the phosphorylation state of CREB (pCREB), were examined in the hippocampus of non-stressed controls given saline (control + saline: CS), rats subjected to chronic restraint stress and given daily administration of saline (stress + saline: SS), non-stressed rats given daily administration of tianeptine (control + tianeptine: CT), and chronic restraint stress rats given

Fig. 1. Autoradiographic analysis of total and phosphorylated CREB in the hippocampus following chronic stress and tianeptine administration. Panel A. CREB protein expression was increased in the dentate gyrus (DG) of rats subjected to chronic stress and given saline (SS) or daily administration of tianeptine (ST) compared to non-stressed controls administered saline (CS). Total CREB expression was not affected in Ammon's Horn (CA1, CA2 or CA3) of any treatment group. Panel B. CREB phosphorylation (pCREB) was increased in CA2, CA3 and the DG of SS rats compared to non-stressed controls (CS), while pCREB levels were increased in the DG of stress-tianeptine rats (ST). ⁎ = P b 0.05 compared to non-stressed controls given saline (CS).

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Fig. 2. Representative pseudo-color autoradiographic film images of BDNF mRNA expression in rat hippocampus and amygdala. Panel A: Radiolabeled antisense probe selective for BDNF detects BDNF mRNA expression in the pyramidal neurons of CA1, CA2 and CA3 of Ammon's Horn, as well as in granule neurons of the dentate gyrus (DG); BDNF mRNA expression is also observed in the basolateral nucleus of the amygdala (BLA) and the medial nucleus of the amygdala (MEA). Panel B: Adjacent section incubated with [35S] labeled sense riboprobe fails to detect BDNF mRNA in rat brain.

in the hippocampus by in situ hybridization histochemistry. In agreement with previous reports, BDNF expression is observed in pyramidal cells of Ammon's horn and granule neurons of the dentate gyrus (Fig. 2). Subsequent autoradiographic film analysis revealed no significant changes in BDNF mRNA expression in any measured region of Ammon's horn or the dentate gyrus (Fig. 3). Hippocampal BDNF protein was measured by an enzyme-linked immunosorbent assay (ELISA) in CS rats, CT rats, SS rats and ST rats. BDNF protein levels were not modulated in the hippocampus of any of the treatment groups when compared with non-stressed control rats (Fig. 4). These results demonstrate that BDNF mRNA or protein expression in the rat hippocampus is not modulated by

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Fig. 4. Analysis by ELISA of BDNF protein levels in the hippocampus of rats subjected to chronic restraint stress in the presence and absence of tianeptine. Statistical analysis revealed that chronic restraint stress and tianeptine administration had no effect on BDNF protein expression in the hippocampus.

stress or tianeptine administration under conditions in which dendritic remodeling is observed. 3.3. Differential effects of stress and tianeptine upon amygdalar pCREB levels Total CREB expression was not modulated in the basolateral nucleus of the amygdala or the medial nucleus of the amygdala in any of the treatment groups (Fig. 5, Panel A). There was no overall effect of stress or drug treatment upon CREB levels in the amygdala. However, pCREB levels were significantly increased in the basolateral nucleus of the amygdala of rats subjected to stress and given daily saline injections (SS) compared to non-stressed controls (CS and CT) and stressed rats given tianeptine (ST; Fig. 5, Panel B). Stress had an overall effect to increase pCREB levels (P ≤ 0.002), while tianeptine administration produced an overall decrease in pCREB levels in the basolateral nucleus of the amygdala (P ≤ 0.02). In the medial nucleus of the amygdala, there were no significant differences in pCREB levels between the treatment groups (Fig. 5, Panel B). However, similar to observations in the basolateral nucleus of the amygdala, tianeptine administration produced an overall decrease in pCREB levels compared to saline-treated rats (P ≤ 0.03); there was no overall effect of stress in the medial nucleus of the amygdala. 3.4. Tianeptine increases BDNF mRNA and protein levels in the rat amygdala

Fig. 3. Autoradiographic image analysis of hippocampal BDNF mRNA expression in rats subjected to chronic restraint stress and tianeptine treatment. CA1, CA2, and CA3 areas of Ammon's horn and the superior and inferior blades of the dentate gyrus were analyzed. Statistical analysis revealed that chronic restraint stress and tianeptine administration had no effect on expression of BDNF mRNA in any hippocampal region measured.

Unlike the hippocampus, the effects of stress and tianeptine administration on BDNF mRNA expression in the rat amygdala have not been well characterized. Accordingly, mRNA expression of BDNF in the basolateral nucleus of the amygdala and medial nucleus of the amygdala were analyzed by in situ hybridization histochemistry; representative pseudo-color images of BDNF mRNA expression in the basolateral nucleus of the amygdala and medial nucleus of the amygdala are shown in Fig. 2. Autoradiographic image analysis revealed that in

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Fig. 6. Autoradiographic image analysis of BDNF mRNA expression in the basolateral nucleus of the amygdala and medial nucleus of the amygdala of rats subjected to chronic restraint stress and tianeptine treatment. Statistical analysis revealed that tianeptine treatment increased BDNF mRNA expression in rats subjected to chronic restraint stress; there was no effect due to chronic restraint stress alone. ⁎ = P b 0.05 compared to non-stressed controls given saline (CS); # = P b 0.05 compared to rats subjected to chronic stress and given saline (SS).

administrations of saline (SS). As described for the hippocampus, these results would suggest that stress-induced morphological changes in the amygdala may not involve modulation of BDNF protein expression. 4. Discussion Fig. 5. Autoradiographic analysis of total and phosphorylated CREB in the basolateral nucleus of the amygdala and medial nucleus of the amygdala following chronic stress and tianeptine administration. Panel A. CREB protein expression was not modulated in any of the treatment groups compared to nonstressed controls given saline (CS) in the basolateral nucleus of the amygdala or medial nucleus of the amygdala. Panel B. Phosphorylated CREB (pCREB) levels were increased in the BLA of rats subjected to chronic stress and given daily administration of saline (SS) compared to non-stressed controls (CS and CT) and stressed rats given daily tianeptine (ST). No changes in pCREB expression were observed in the medial nucleus of the amygdala in any of the treatment groups. # = P b 0.05 compared to rats subjected to chronic stress and given saline (SS).

animals subjected to chronic restraint stress and given daily administration of tianeptine, BDNF mRNA levels were significantly increased in the basolateral nucleus of the amygdala (Fig. 6). Interestingly, BDNF mRNA expression was not modulated in the basolateral nucleus of SS rats or CT rats when compared to non-stressed controls given saline (CS). Similar results were observed in the medial nucleus of the amygdala. Rats subjected to chronic restraint stress and given daily administration of tianeptine (ST) exhibited significant increases in BDNF mRNA expression, while BDNF mRNA expression was not modulated in SS rats or CT rats when compared with CS rats (Fig. 6). The chronic stress and tianeptine-induced effects upon amygdalar BDNF protein levels were examined by ELISA. Daily tianeptine administration significantly increased BDNF protein expression in the rat amygdala irrespective of stress conditions (Fig. 7; P ≤ 0.0001). However, BDNF protein levels were not modulated in the amygdala of stressed rats given daily

The results of the current study demonstrate that chronic (i.e., 21 day) restraint stress increases the phosphorylation of CREB, but does not modulate BDNF protein or mRNA expression in the rat hippocampus and amygdala. These results suggest that BDNF mRNA or protein expression is not modulated during experimental conditions that elicit morphological changes in these regions. Daily administration of tianeptine decreased pCREB levels while simultaneously increasing BDNF mRNA and protein expression in the rat amygdala, antidepressant

Fig. 7. Analysis by ELISA of BDNF protein levels in the amygdala of rats subjected to chronic restraint stress in the presence and absence of tianeptine administration. Statistical analysis revealed that tianeptine treatment increased BDNF protein levels in rats subjected to chronic restraint stress and in nonstressed control rats. ⁎ = P b 0.05 compared to CS; # = P b 0.05 compared to SS.

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mediated effects that were not observed in the hippocampus. These results demonstrate that tianeptine produces regionspecific increases in BDNF expression that may involve distinct mechanism(s) when compared to other antidepressants. While there are limitations associated with the use of experimental stress paradigms as preclinical models of mood disorders (Nestler et al., 2002), animals subjected to repeated stress exhibit many features similar to those observed in depressive illness patients, including changes in body weight and composition, disrupted sleep cycles, impairments in HPA axis function, as well as morphological changes in the hippocampus and amygdala. For example, chronic restraint stress induces atrophy of the apical dendrites of CA3 pyramidal neurons (Watanabe et al., 1992b), as well as retraction of thorny excrescences in the same neurons (Stewart et al., 2005). This experimentally observed atrophy in the hippocampus may be connected with clinically observed reductions in hippocampal volume in human patients with stress-related disorders (Sheline et al., 1996; Starkman et al., 1992). In animal studies, dendritic remodeling of CA3 pyramidal neurons is observed following 21 days, but not 14 days, of restraint stress (Magariños and McEwen, 1995). In contrast, amygdalar pyramidal and stellate neurons in the basolateral amygdala exhibit enhanced dendritic arborization with 10 days of chronic immobilization stress (Vyas et al., 2002). Stress-induced plasticity in the hippocampus, while later in onset than that observed in the amygdala, is reversible after as little as 7 days of removal from stress conditions (Conrad et al., 1999; Luine et al., 1994). In contrast, stress-induced amygdalar hypertrophy is maintained after 21 days of stress-free recovery (Vyas et al., 2004). These findings suggest that there may be some region-specific mechanisms through which stress elicits morphological changes in the hippocampus and amygdala. Brain-derived neurotrophic factor (BDNF) is an important mediator of synaptic plasticity in the central nervous system and has been proposed to participate in stress-induced morphological changes in the rat hippocampus (Duman et al., 2000). Previous studies have shown that adrenal steroids regulate BDNF mRNA in the rat hippocampus. For example, administration of corticosterone decreases BDNF mRNA in the dentate gyrus (Smith et al., 1995b), while removal of adrenal glands upregulates BDNF expression throughout the hippocampus (Chao et al., 1998). Several experiments have provided evidence that BDNF mRNA is downregulated in response to restraint stress over intermediate time courses of 1 to 10 days (Duric and McCarson, 2005; Smith et al., 1995a; Ueyama et al., 1997; Vaidya et al., 1999). However, other studies have revealed that 3 weeks of restraint stress produces no change in BDNF expression in the hippocampus (Kuroda and McEwen, 1998; Rosenbrock et al., 2005). The current findings support these previous studies that examined the effects of 21 days of restraint stress and suggest that morphological changes that occur in the hippocampus after 3 weeks of stress are modulated not by concurrent activity of BDNF, but rather may be modulated through decreases in hippocampal BDNF expression observed with 7–10 days of stress. As such, BDNF may serve as an early precursor in the synaptic plasticity of hippocampal morphology. In addition to potentially serving as a necessary precursor to

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stress-induced morphological changes, BDNF also exhibits transient decreases in expression following acute stress (Murakami et al., 2005) and may thereby contribute to the maintenance of dendritic atrophy in the hippocampus. Alternatively, stress and BDNF may activate parallel pathways that operate independently to modulate neuronal plasticity (see below). Among the mechanisms of action and therapeutic outcomes of antidepressant drugs, including the SSRIs, is an increase in pCREB levels and BDNF expression in the hippocampus. However, the SSRIs fluoxetine and fluvoxamine do not inhibit stress-induced morphological changes in the hippocampus (Magariños et al., 1999), suggesting that the pathways through which stress and BDNF modulate hippocampal morphology and plasticity may be to some degree independent. Indeed, while tianeptine effectively inhibits stress-induced morphological changes in the hippocampus (Magariños et al., 1999; Watanabe et al., 1992a), the current studies demonstrate that tianeptine does not modulate BDNF mRNA or protein levels in this region. Conversely, a recent study in female mice subjected to chronic stress revealed that tianeptine inhibited stressmediated decreases in BDNF expression in the hippocampus as measured by RT-PCR (Alfonso et al., 2006). There are several potential explanations for the discrepancies in these studies that may be associated with the experimental approaches used, as well as sex, species and even strain-related effects of stress in the hippocampus. Nonetheless, the results from Alfonso and co-workers are interesting in relation to the clinical situation since women are more likely to develop depressive illness when compared with men (Earls, 1987). Accordingly, future studies should examine these plasticity-related changes in neurotrophic factor mRNA and protein expression in the female rat hippocampus following stress and antidepressant treatment. Unlike observations in the hippocampus, chronic tianeptine administration increases BDNF protein expression in the amygdala irrespective of stress conditions. These tianeptine mediated increases in BDNF expression are observed in the presence of decreased pCREB levels in the amygdala, suggesting that tianeptine modulates neurotrophic factor expression via distinct mechanisms. Indeed, a variety of drugs used in the treatment of mood disorders, as well as electroconvulsive shock treatment, concurrently increase pCREB and BDNF levels in the rat hippocampus (Duman, 1998). Accordingly, an important question that remains is the mechanisms through which tianeptine produces these region-specific increases in BDNF expression. Moreover, another interesting question is how tianeptinemediated increases in amygdalar BDNF contribute to the ability of this antidepressant to affect stress-induced morphological changes. For example, tianeptine inhibits stress-induced morphological changes in the hippocampus (Czeh et al., 2001; Magariños et al., 1999; Watanabe et al., 1992a) and the amygdala (McEwen and Chattarji, 2004). It is possible that BDNF is more intimately involved in stress-mediated morphological plasticity in the amygdala when compared with the hippocampus. An alternative explanation is that by increasing BDNF levels in the amygdala, tianeptine inhibits stress-mediated morphological changes in the rat amygdala and also inhibits the subsequent morphological changes elicited by stress in the hippocampus. In

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support of this hypothesis, clinical studies suggest that changes in amygdala volume precede decreases in hippocampal volume observed in major depressive illness patients (McEwen, 2003), similar to observations made in experimental models of chronic stress (McEwen and Chattarji, 2004). In summary, the results of the current study demonstrate that tianeptine increases BDNF mRNA and protein expression in the rat amygdala, increases in neurotrophic factor expression that may be relevant to the clinical efficacy of this antidepressant. Future investigations will be necessary to determine whether tianeptine-mediated increases in amygdalar BDNF expression modulates the activity of the reciprocal connections between the hippocampus and amygdala, thereby inhibiting the electrophysiological, neurochemical and neuroanatomical changes elicited by chronic restraint stress in the rat hippocampus. Acknowledgements The authors would like to thank Dr. Alex McDonald and Dr. Marlene Wilson for helpful comments and suggestions. Supported by MH41256 and MH58911 (BSM) and the University of South Carolina Research Foundation (LPR). References Alfonso, J., Frick, L.R., Silberman, D.M., Palumbo, M.L., Genaro, A.M., Frasch, A.C., 2006. Regulation of hippocampal gene expression is conserved in two species subjected to different stressors and antidepressant treatments. Biol. Psychiatry 59, 244–251. Bremner, J.D., Narayan, M., Anderson, E.R., Staib, L.H., Miller, H.L., Charney, D.S., 2000. Hippocampal volume reduction in major depression. Am. J. Psychiatry 157, 115–118. Chao, H.M., Sakai, R.R., Ma, L.Y., McEwen, B.S., 1998. Adrenal steroid regulation of neurotrophic factor expression in the rat hippocampus. Endocrinology 139, 3112–3118. Conrad, C.D., Magariños, A.M., LeDoux, J.E., McEwen, B.S., 1999. Repeated restraint stress facilitates fear conditioning independently of causing hippocampal CA3 dendritic atrophy. Behav. Neurosci. 113, 902–913. Czeh, B., Michaelis, T., Watanabe, T., Frahm, J., de Biurrun, G., van Kampen, M., Bartolomucci, A., Fuchs, E., 2001. Stress-induced changes in cerebral metabolites, hippocampal volume, and cell proliferation are prevented by antidepressant treatment with tianeptine. Proc. Natl. Acad. Sci. U. S. A. 98, 12796–12801. Duman, R.S., 1998. Novel therapeutic approaches beyond the serotonin receptor. Biol. Psychiatry 44, 324–335. Duman, R.S., Malberg, J., Nakagawa, S., D'Sa, C., 2000. Neuronal plasticity and survival in mood disorders. Biol. Psychiatry 48, 732–739. Duric, V., McCarson, K.E., 2005. Hippocampal neurokinin-1 receptor and brain-derived neurotrophic factor gene expression is decreased in rat models of pain and stress. Neuroscience 133, 999–1006. Earls, F., 1987. Sex differences in psychiatric disorders: origins and developmental influences. Psychiatr. Dev. 5, 1–23. Figurov, A., Pozzo-Miller, L.D., Olafsson, P., Wang, T., Lu, B., 1996. Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus. Nature 381, 706–709. Frodl, T., Meisenzahl, E., Zetzsche, T., Bottlender, R., Born, C., Groll, C., Jager, M., Leinsinger, G., Hahn, K., Moller, H.J., 2002a. Enlargement of the amygdala in patients with a first episode of major depression. Biol. Psychiatry 51, 708–714. Frodl, T., Meisenzahl, E.M., Zetzsche, T., Born, C., Groll, C., Jager, M., Leinsinger, G., Bottlender, R., Hahn, K., Moller, H.J., 2002b. Hippocampal changes in patients with a first episode of major depression. Am. J. Psychiatry 159, 1112–1118.

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