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Effects of repeated tianeptine treatment on CRF mRNA expression in non-stressed and chronic mild stress-exposed rats
Neuropharmacology 50 (2006) 824e833 www.elsevier.com/locate/neuropharm
Effects of repeated tianeptine treatment on CRF mRNA expression in non-stressed and chronic mild stress-exposed rats Sung-Jin Kim a, Sang-Ha Park a, Song-hyen Choi a, Bo-Hyun Moon a, Kuem-Ju Lee a, Seung Woo Kang a, Min-Soo Lee b, Sang-Hyun Choi a, Boe-Gwun Chun a, Kyung-Ho Shin a,* a
Department of Pharmacology, College of Medicine, Korea University, Seoul, South Korea b Department of Psychiatry, College of Medicine, Korea University, Seoul, South Korea
Received 22 June 2005; received in revised form 8 November 2005; accepted 6 December 2005
Abstract Accumulating evidence suggests that dysregulation of corticotropin-releasing factor (CRF) may play a role in depression and that this dysregulation may be corrected by antidepressant drug treatment. Here, we examined whether chronic mild stress (CMS) alters CRF mRNA levels in stress-related brain areas including the bed nucleus of the stria terminalis (BNST) and the central nucleus of amygdala (CeA), and whether repeated tianeptine treatment can attenuate CMS-induced changes in CRF mRNA levels. Male rats were exposed to CMS for 19 days, and control animals were subjected to brief handling. Both groups were injected daily with tianeptine or saline. CMS significantly increased CRF mRNA levels in the dorsal BNST (dBNST), but not in other areas. Repeated tianeptine treatment prevented the CMS-induced increase in CRF mRNA levels in the dBNST, and reduced CRF mRNA levels in dBNST in non-stressed controls. Moreover, repeated tianeptine treatment significantly decreased CRF mRNA levels in the ventral BNST and CeA of non-stressed controls as well as CMS-exposed rats. These results show that CMS induces a rather selective increase of CRF mRNA in the dBNST. In addition, these results suggest that repeated tianeptine treatment diminishes the basal activity of CRF neurons and reduces their sensitivity to stress. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Tianeptine; Stress; Corticotropin-releasing factor; Bed nucleus of the stria terminalis; Amygdala; Depression; Anxiety
1. Introduction Corticotropin-releasing factor (CRF) was first described as a hypothalamic neuropeptide that stimulates the secretion of adrenocorticotropin (ACTH) (Vale et al., 1981). In addition to the paraventricular nucleus of hypothalamus (PVN), CRF is found in stress-related brain areas such as the bed nucleus of the stria terminalis (BNST), central nucleus of the amygdala (CeA), and Barrington’s nucleus (Imaki et al., 1991; Morin et al., 1999). CRF functions as a neurotransmitter in the integration of behavioral and autonomic responses to stress (Koob, * Corresponding author. Korea University, College of Medicine, Department of Pharmacology, Sungbuk-gu Anam-dong 5-ka 126-1, Seoul, 136-705 Republic of Korea. Tel.: þ82 2 920 6195; fax: þ82 2 927 0824. E-mail address: [email protected] (K.-H. Shin). 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.12.003
1999). As stress is known to be a predisposing factor for major depression, it is possible that dysregulation of the CRF system may contribute to the pathology of depression (Nemeroff et al., 1984). For example, increased concentrations of CRF in cerebrospinal fluid (CSF) have been reported in depressed patients (Banki et al., 1987). Moreover, the normalization of elevated CRF concentrations in CSF has been reported after successful treatment of depression by fluoxetine (De Bellis et al., 1993) or electroconvulsive shock (Nemeroff et al., 1991). Tianeptine is a serotonin reuptake enhancer (Mennini et al., 1987), and has an antidepressant efficacy similar to that of selective serotonin reuptake inhibitors (SSRIs) (Wilde and Benfield, 1995). Interestingly, chronic administration of tianeptine has been shown to attenuate lipopolysaccharide- or stress-induced increases in plasma ACTH and corticosterone
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levels (Castanon et al., 2003; Delbende et al., 1994). These findings suggest that chronic tianeptine treatment may suppress pituitaryeadrenal axis activation in response to stress. In addition, tianeptine attenuated stress-induced reduction of hypothalamic CRF concentration (Delbende et al., 1994), suggesting that tianeptine exerts antidepressant effects by regulating the pituitaryeadrenal axis and/or CRF neurotransmission in the hypothalamus. A caveat of antidepressant research is the use of normal laboratory rats (Nestler et al., 2002). This concern emerges from the observation that antidepressant treatments in humans without depression cause typical side effects rather than antidepressant effects (Nestler et al., 2002). Thus, the elucidation of mechanisms of antidepressants using normal rats may not reflect the mechanism of antidepressant effects in the depressed brain. To take this possibility into account, we injected tianeptine under a chronic stress condition to imitate the clinical feature of depression. Anhedonia, the loss of interest in or ability to derive pleasure from all or most activities, is one of the two core symptoms of depression according to the Diagnostic and Statistical Manual of Mental Disorders, fourth revision (1994). Since a similar state of anhedonia can be induced in rats by chronic mild stress (CMS) procedures (Moreau et al., 1992; Stout et al., 2000), a CMS model has been used as an animal model of depression. Moreover, it has been shown that increased CRF concentration in the BNST was observed in CMS anhedonic rats (Stout et al., 2000). In the present study, we tested whether CMS increases CRF mRNA expression in stress-related brain areas, and whether repeated tianeptine treatment during CMS procedures may inhibit CMS-induced changes in CRF mRNA. 2. Materials and methods 2.1. Animals Adult male SpragueeDawley rats (Orient, Seoul, South Korea) weighing 190e210 g at the beginning of the CMS exposure were used. Rats were handled daily for 7 days to habituate them to the laboratory environment. Rats were housed three per cage in a humidified room with a 12-h light/dark cycle (lights on at 6:00 AM), and food and water were available ad libitum except during food or water deprivation periods during CMS procedures. All procedures used in this study were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).
2.2. Stress paradigm For CMS, rats were exposed to a variable stress regimen for 19 days as previously described (Moreau et al., 1992; Stout et al., 2000). In brief, the stressor included repeated 1 h periods of confinement in a small cage (24 10 9 cm), one period of continuous overnight illumination, one overnight period of food and water deprivation followed by 2 h access to restricted food (3 g per cage), one overnight period of water deprivation followed by 1 h exposure to an empty bottle, and one overnight period of group housing (6 rats per cage) in a soiled cage (200 ml water in sawdust bedding). Animals were also maintained on a reversed light/dark cycle from Friday evening to Monday morning. The last procedure of CMS consisted of reversed light/dark cycle Friday evening for 12 h. Lights in the animal room were not turned off on Saturday morning after the final CMS procedure was completed. All perfusions were conducted between 11:00 and 14:00 h in order to reduce possible
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circadian changes in CRF peptide levels. Non-stressed control rats were housed in a separate room and were briefly handled each day without any deliberately stressing procedures.
2.3. Antidepressant administration The animals were divided into four groups: saline-treated non-stressed controls (CON þ SAL, n ¼ 9), saline-treated CMS-exposed group (CMS þ SAL, n ¼ 9), tianeptine-treated non-stressed controls (CON þ TIA, n ¼ 9) and tianeptine-treated CMS-exposed group (CMS þ TIA, n ¼ 9). Tianeptine was dissolved in 0.9% saline, which was used as the vehicle. Tianeptine (10 mg/kg) was injected intraperitoneally (i.p.) 30 min before restraint stress each morning (08:30 h) during the 19-day CMS period.
2.4. Body weight, food and water intake measurements For measurement of food and water intake, a different batch of animals was exposed to CMS. Rats were housed individually to determine daily food and water intake. The food hopper and water bottle were removed and weighed at 08:30 h. At this time, rats were removed from their cages, weighed, and then injected with saline or tianeptine. A fresh supply of preweighed food and water was returned 1 h later. Daily food intake (g/day) was calculated by subtracting the weight of the hopper from its initial weight. Daily water intake (ml/day) was determined by subtracting the volume of the bottle from its initial volume. Cages were carefully monitored for evidence of food spillage or grinding. Cumulative food and water intake were calculated from daily food and water intake, which included food and water intake during food or water deprivation periods in CMS-exposed rats. However, mean daily food and water intake were averaged from daily food and water intake except during food and water deprivation periods in CMS-exposed rats as well as in non-stressed rats.
2.5. Histological procedures Rats were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.) and were perfused intracardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PPB, pH 7.2). The brains were fixed in situ for 1 h, removed, post-fixed in PPB for 2 h, and finally placed in 20% sucrose/PPB overnight at 4 C. Serial coronal sections (30 mm) of whole brain were obtained using a freezing microtome and were stored in cryoprotectant solution [30% RNase free sucrose, 30% ethylene glycol, and 1% polyvinyl pyrrolidone (PVP-40) in 100 mM sodium phosphate buffer, pH 7.4] at 20 C.
2.6. Probe labeling The CRF riboprobe was constructed from a 1.2-kb EcoRI fragment of fulllength rat CRF cDNA (a gift from K. Mayo, Northwestern University, Evanston, IL, USA) subcloned into a pBluescript II-SKþ plasmid. Transcription reactions were performed using an Ambion MAXIscript kit (Austin, TX, USA) with SP6 RNA polymerases according to the manufacturer’s instructions. After transcription and removal of the cDNA template with 2 U of DNase, the cRNA probes were recovered using NucTrap minicolumns (Stratagene, La Jolla, CA, USA).
2.7. In situ hybridization Brain sections were permeabilized with proteinase K (1 mg/ml, 37 C, 30 min), treated with acetic anhydride in 0.1 M triethanolamine (pH 8.0), washed in 2 SSC (pH 7.0), and were then transferred into 500 ml of hybridization solution in 24-well culture plates. The hybridization solution was comprised of 50% formamide, 0.01% polyvinyl pyrrolidone, 0.01% Ficoll, 0.01% bovine serum albumin, 50 mg/ml denatured salmon sperm DNA, 250 mg/ml yeast tRNA, 40 mM dithiothreitol (DTT), 10% dextran sulfate, and 35S-labeled CRF cRNA probes at 1 107 cpm/ml. Sections were hybridized with the CRF riboprobe for 18 h at 55 C in the hybridization solution. After overnight
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incubation in a humidified chamber, sections were washed twice for 30 min at 55 C in 4 SSC with 5 mM DTT, treated with RNase A (20 mg/ml, 30 min at 45 C), and washed four times (15 min/wash) at room temperature in 2 SSC containing 5 mM DTT. The sections were then washed twice (30 min/wash) in 0.5 SSC containing 5 mM DTT, once for 30 min in 0.1 SSC containing 5 mM DTT at 50e55 C, and once in 0.1 SSC with 5 mM DTT at room temperature. Sections were then mounted on gelatin-coated microscope slides and air-dried overnight. Tissue sections were exposed to Hyperfilm b-max (Amersham, Arlington Heights, IL, USA) for 3 days. To correct for nonlinearity, 14Clabeled radioactive standards (Amersham, Arlington Heights, IL, USA) were used for calibration.
2.8. Data analysis and statistics Images were captured digitally and CRF mRNA levels were analyzed by outlining the regions of interest, and then quantifying the regional optical densities of autoradiographic film images using Scion Image beta 4.2 software (Scion Corporation, Frederick, MD, USA). Three or four sections from BNST, PVN, CeA and Barrington’s nucleus containing the highest expression were averaged to obtain a single value for each nucleus. The anterior commissure was considered to be the border between the dorsal and ventral BNST (dBNST and vBNST). To correct for nonlinearity, 14C-labeled radioactive standards (Amersham, Arlington Heights, IL, USA) were exposed with tissue sections and used for calibration. Optical densities were obtained from forebrain to hindbrain based on the stereotaxic atlas of Paxinos and Watson (1998). Accordingly, relative to bregma, regions of interest were located as follows: BNST, 0.26 to 0.40 mm; PVN, 1.80 to 2.12 mm; CeA, 2.56 to 3.14 mm; and Barrington’s nucleus, 9.30 to 10.04 mm. Results are expressed as the mean percentage of the values for the saline-treated non-stressed control, and data are reported herein as means S.E.M. Two-way repeated-measures ANOVAs with Bonferroni’s multiple comparison post hoc tests were used to analyze the effects of CMS and repeated tianeptine treatment on body weight changes as well as on cumulative food and water intake during CMS, with two between-subjects factors [CMS (non-stressed control vs. CMS) and repeated tianeptine treatment (saline vs. tianeptine)] and with time as the only within-subject factor. A one-way repeated-measures ANOVA was used with Bonferroni’s post hoc multiple comparison test to analyze separately whether the groups differed from one another. Mean daily food and water intake as well as CRF mRNA level data were similarly analyzed by a two-way ANOVA to detect overall effects of CMS treatment and repeated tianeptine treatment followed by a one-way ANOVA to determine whether there were differences between the individual animal groups. Fisher’s LSD post hoc tests were used to determine the differences between the groups. Significance was determined at the p < 0.05 level.
3. Results 3.1. Effects of repeated tianeptine treatment on CMS-induced body weight changes, food and water intake Body weights were measured daily during the CMS procedures. A consistent reduction in body weight gain was observed in CMS-exposed rats (Fig. 1). A two-way repeated-measures ANOVA with CMS and repeated tianeptine treatment as between-subjects factors and time as the within-subject factor revealed a significant effect of CMS (F1,32 ¼ 61.8, p < 0.001), a significant effect of time (F19,608 ¼ 2052.3, p < 0.001), and a significant interaction of CMS with time (F19,608 ¼ 55.7, p < 0.001) on body weight gain during the CMS procedures (Fig. 1A). There were no significant main effects of repeated tianeptine treatment and no interaction between repeated tianeptine treatment and time. A one-way repeated-measures ANOVA with Bonferroni’s post hoc test showed that non-stressed and
CMS-exposed groups differed regardless of whether they received saline or tianeptine pretreatment (Fig. 1A, CON þ SAL and CON þ TIA vs. CMS þ SAL; CON þ SAL and CON þ TIA vs. CMS þ TIA; F3,32 ¼ 21.7, p < 0.001). These results showed that while CMS decreased weight gain, repeated tianeptine treatment did not influence the body weight changes in either the non-stressed or CMS-exposed groups. CMS significantly decreased cumulative food intake (Fig. 1B, two-way repeated-measures ANOVA: CMS, F1,32 ¼ 88.2, p < 0.001; CMS time, F19,608 ¼ 54.6, p < 0.001) and cumulative water intake (Fig. 1C, two-way repeated-measures ANOVA: CMS, F1,32 ¼ 57.3, p < 0.001; CMS time, F19,608 ¼ 46.0, p < 0.001) compared with non-stressed groups. There was no significant main effect of repeated tianeptine treatment on cumulative food and water intake, suggesting that the tianeptine treatment did not influence cumulative food and water intake (Fig. 1C). In addition, CMS induced a small but significant decrease in mean daily food intake compared with nonstressed groups (Fig. 1D, one-way ANOVA with Fisher’s post hoc LSD test: F1,32 ¼ 11.2, p < 0.01; CON þ SAL: 25.5 0.8 g/day; CON þ TIA: 25.4 0.5 g/day; CMS þ SAL: 23.6 0.5 g/day; CMS þ TIA: 23.6 0.3 g/day), but this CMS-induced decrease in mean daily food intake was not influenced by repeated tianeptine treatment. The patterns of changes in mean daily water intake observed were similar to those seen with food, but the magnitude of decrease in mean daily water intake by the CMS-treated rats was greater than those in food intake (Fig. 1D, one-way ANOVA with Fisher’s post hoc LSD test: F1,32 ¼ 11.8, p < 0.01 vs. CON þ SAL; CON þ SAL: 42.5 1.6 ml/day, CON þ TIA: 43.2 2.7 ml/day, CMS þ SAL: 35.6 0.8 ml/day; CMS þ TIA: 38.7 0.7 ml/day). 3.2. Effects of repeated tianeptine treatment on CRF mRNA levels in the dorsal bed nucleus of the stria terminalis (dBNST) A two-way ANOVA revealed significant main effects of CMS (F1,32 ¼ 38.2, p < 0.001) and of the repeated tianeptine treatment (F1,32 ¼ 16.5, p < 0.001) on CRF mRNA levels in the dBNST. No significant interaction between CMS and the repeated tianeptine treatment was found. A one-way ANOVA showed that there was a significant difference among these groups (Figs. 2 and 4, F3,32 ¼ 18.2, p < 0.001). Post hoc analysis using Fisher’s LSD test demonstrated that CMS significantly increased CRF mRNA levels in the dBNST relative to saline-treated non-stressed controls ( p < 0.001), whereas repeated tianeptine treatment significantly decreased CRF mRNA levels ( p < 0.01). Moreover, repeated tianeptine treatment prevented the CMS-induced increase in CRF mRNA levels in the dBNST (Figs. 2 and 4, p < 0.01). 3.3. Effects of repeated tianeptine treatment on CRF mRNA levels in the ventral bed nucleus of the stria terminalis (vBNST) A two-way ANOVA revealed significant main effects of CMS (F1,32 ¼ 9.4, p < 0.001) and of the repeated tianeptine
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Fig. 1. Mean body weight changes (A), cumulative food intake (g/rat) (B), and cumulative water intake (ml/rat) (C) during CMS procedures (***p < 0.001, comparison between treatments; one-way repeated-measures ANOVA followed by Bonferroni’s post hoc test). Mean daily food and water intake during CMS procedures (D). CMS significantly decreased mean daily food and water intake (*p < 0.05 and ###p < 0.001 vs. CON þ SAL; comparison between treatment, one-way ANOVA followed by Fisher’s LSD test). Group means S.E.M. are shown; n ¼ 9 per group. Abbreviations used: CON þ SAL, saline-treated non-stressed controls; CON þ TIA, tianeptine-treated non-stressed controls; CMS þ SAL, saline-treated CMS-exposed group; CMS þ TIA, tianeptine-treated CMS-exposed group.
treatment (F1,32 ¼ 15.4, p < 0.001) on CRF mRNA levels in the vBNST. No significant interaction between CMS and the repeated tianeptine treatment was found. A one-way ANOVA showed that there was a significant difference among these groups (Figs. 3 and 4, F3,32 ¼ 8.5, p < 0.001). Post hoc analysis using Fisher’s LSD test demonstrated that CRF mRNA levels in the vBNST following CMS did not differ from those of saline-treated non-stressed controls, but repeated tianeptine treatment significantly reduced CRF mRNA levels in the vBNST relative to saline-treated non-stressed control (Figs. 3 and 4, F3,32 ¼ 8.5, p < 0.01). Repeated tianeptine treatment in CMS-exposed rats significantly reduced CRF mRNA levels in the vBNST relative to saline-treated CMS-exposed rats (Figs. 3 and 4, F3,32 ¼ 8.5, p < 0.05). 3.4. Effects of repeated tianeptine treatment on CRF mRNA levels in the CeA, PVN, and Barrington’s nucleus A two-way ANOVA revealed significant main effects of CMS (F1,32 ¼ 4.5, p < 0.05) and of the repeated tianeptine treatment (F1,32 ¼ 18.5, p < 0.001) on CRF mRNA levels in
the CeA. No significant interaction between CMS and the repeated tianeptine treatment was found. A one-way ANOVA showed that there was a significant difference among these groups (Figs. 5 and 6, F3,32 ¼ 7.8, p ¼ 0.001). Post hoc analysis using Fisher’s LSD test demonstrated that CMS did not induce a detectable change in CRF mRNA levels in the CeA, but repeated tianeptine treatment decreased CRF mRNA levels in the CeA relative to saline-treated non-stressed controls (Figs. 5 and 6, F3,32 ¼ 7.8, p < 0.01). Repeated tianeptine treatment in CMS-exposed rats significantly reduced CRF mRNA levels in the CeA relative to saline-treated CMS-exposed rats (F3,32 ¼ 7.8, p < 0.05). CMS did not affect CRF mRNA levels in either the PVN or Barrington’s nucleus ( p > 0.05 vs. salinetreated non-stressed control). Only a trend toward a tianeptineinduced reduction of CRF mRNA levels in the PVN and Barrington’s nucleus was observed (Table 1). 4. Discussion In the present study, CMS significantly increased CRF mRNA levels in the dBNST, whereas there was a trend toward a CMS-induced increase of CRF mRNA levels in the vBNST
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***
CRF mRNA (% of CON+SAL)
160 140
**
**
120 100 80 60 40 20 0 CON+SAL
CON+TIA
CMS+SAL
CMS+TIA
Fig. 2. The effects of CMS and tianeptine treatment on CRF mRNA levels in the dorsal bed nucleus of the stria terminalis (dBNST). CMS increased CRF mRNA levels in the dBNST [***p < 0.001 vs. saline-treated non-stressed control (CON þ SAL)]. Repeated tianeptine treatment prevented the CMSinduced increase in CRF mRNA levels in the dBNST, and reduced CRF mRNA levels in dBNST in non-stressed controls. There was no significant interaction (CMS tianeptine). The results are expressed as percent of mean value from CON þ SAL group and are the mean S.E.M. of nine separate animals in each group. **p < 0.01 and ***p < 0.001; comparison between treatment (one-way ANOVA followed by Fisher’s LSD test).
and CeA. CMS did not affect CRF mRNA levels in the PVN or Barrington’s nucleus, suggesting that the CMS-induced increase in CRF mRNA levels in the dBNST was selective. Repeated tianeptine treatment prevented the CMS-induced increase in CRF mRNA levels in the dBNST, and reduced CRF mRNA levels in dBNST in non-stressed controls. In addition, repeated tianeptine treatment significantly decreased CRF mRNA levels in the vBNST and CeA in non-stressed control as well as in CMS-exposed rats.
CRF mRNA (% of CON+SAL)
160
*
**
140 120 100 80 60 40 20 0 CON+SAL
CON+TIA
CMS+SAL
CMS+TIA
Fig. 3. The effects of CMS and tianeptine treatment on CRF mRNA levels in the ventral bed nucleus of the stria terminalis (vBNST). Repeated tianeptine treatment significantly reduced CRF mRNA levels of the vBNST in nonstressed and CMS-exposed rats. There was no significant interaction (CMS tianeptine). The results are expressed as percent of mean value of CON þ SAL group and are the mean S.E.M. of nine separate animals in each group. *p < 0.05 and **p < 0.01; comparison between treatment (oneway ANOVA followed by Fisher’s LSD test).
4.1. Changes in body weight and feeding associated with CMS or antidepressant CMS significantly decreased mean daily food and water intake relative to non-stressed control animals, with the decrease in mean water intake being more prominent. These reductions in mean daily food and water intake during CMS procedures are related to the decrease in cumulative food and water intake, which could contribute to the body weight loss exhibited by CMS-exposed rats. However, repeated tianeptine treatment did not influence CMS-induced body weight changes or mean daily food and water intake. At present, it is not clear whether decreased food and water intake could be attributed to anhedonia, because we did not measure sucrose intake or preference as previously reported (Willner et al., 1996). However, considering that anhedonia is characterized by a diminished ability to experience pleasure from natural reinforcers such as food, water, and sex, as well as drugs of abuse, it is possible that decreased food and water intake may reflect some aspect of an anhedonic state. This possibility is supported by the fact that the mild stressors used in the present study were based on the study by Stout et al. (2000), in which a gradual development of an anhedonic state was observed as demonstrated by increased threshold for self-stimulation. Thus, although CMS may increase anhedonia, repeated tianeptine treatment did not affect eating and drinking behavior and therefore appeared not to influence anhedonia. Further experiments will be required to elucidate the relationship between tianeptine treatment and anhedonic behavior. 4.2. CRF in the BNST The BNST is considered to be part of the extended amygdala and shares several neuroanatomical and neurochemical similarities with the CeA (Alheid et al., 1995). The BNST contains numerous CRF-immunopositive neuronal cell bodies (Morin et al., 1999) and receives heavy projections from the CeA, which contains CRF (Sakanaka et al., 1986). The BNST has been implicated in the regulation of psychological responses to stress (Spencer et al., 2005) and withdrawal from drugs of abuse (Aston-Jones et al., 1999; Macey et al., 2003). In particular, evidence suggests that CRF neurotransmission in the BNST is linked to anxiety (Davis et al., 1997; Lee and Davis, 1997) and anhedonia (Stout et al., 2000). In the present study, the increase of CRF mRNA levels in the dBNST was similar to a previous report, in which an increase of CRF peptide was observed in the BNST, but not in the CeA, following CMS (Stout et al., 2000). The present results are consistent with previous work demonstrating that CRF mRNA in dBNST, but not in vBNST, was increased in response to psychosocial stress in rats (Makino et al., 1999). Thus CRF mRNA in the dBNST appears to be more responsive to CMS than that in the vBNST. Moreover, it has been demonstrated that longterm treatment with corticosterone increased CRF mRNA levels in the dBNST and CeA (Makino et al., 1994). Interestingly, the dBNST and vBNST have distinct anatomic connections (Dong et al., 2001) and electrophysiological properties
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Fig. 4. Representative autoradiographs of 35S-labeled CRF mRNA in BNST from each treatment group are shown. In CMS-exposed groups, rats were given i.p. injections of saline (CMS þ SAL) or tianeptine (10 mg/kg, CMS þ TIA) before restraint stress in the morning. Non-stressed control rats were handled each day and given daily injections of saline (CON þ SAL) or tianeptine (10 mg/kg, CON þ TIA) in the morning.
drinking behavior. That is, the CMS-induced increase of CRF mRNA levels in dBNST was attenuated by tianeptine treatment while the CMS-induced decrease in food and water intake was not. Although decreased food and water intake may not reflect the whole spectrum of anhedonia, this result raises the possibility that CRF in dBNST may not be related to 140
CRF mRNA (% of CON+SAL)
(Egli and Winder, 2003). For example, dBNST receives inputs from the CeA, postpiriform transition area, the insular region and the paraventricular nucleus of the thalamus, whereas vBNST receives inputs from medial prefrontal cortex, ventral subicular complex of the hippocampus, nucleus accumbens and subfornical area (Dong et al., 2001). While dBNST sends a projection to CeA and vBNST, both of which have been shown to play an important role in fear response behavior, vBNST projects directly to the ventral tegmental area and sends a projection to the paraventricular and anterior periventricular nuclei of hypothalamus, which modulate ACTH and growth hormone release, respectively (Dong et al., 2001). Considering the neuroanatomical organization and the role of CRF in the dBNST, our results seem to suggest that this increase in CRF mRNA in the dBNST may contribute to an aversive emotional state including CMS-induced anxiety or anhedonia. As of yet, however, it has not been determined whether there is a correlation between CRF levels in the BNST and any related behaviors. Studies examining whether CMS produces measurable changes in indices of anxiety have been conflicting. While D’Aquila et al. (1994) did not find evidence for CMS-induced anxiety as measured in the social interaction test or the elevated plus maze, Duncko et al. (2001) more recently obtained a different result using a different CMS model. In addition, a CMS-induced increase in CRF mRNA levels in the dBNST may contribute to anhedonia as shown by decreased daily food consumption and water intake. However, there was a disassociation between the effects of tianeptine treatment on CRF mRNA levels and eating and
120
**
*
100 80 60 40 20 0 CON+SAL
CON+TIA
CMS+SAL
CMS+TIA
Fig. 5. The effects of CMS and tianeptine treatment on CRF mRNA levels in the central nucleus of amygdala (CeA). Repeated tianeptine treatment significantly decreased CRF mRNA levels in the CeA of non-stressed controls as well as CMS-exposed rats. There was no significant interaction (CMS tianeptine). The results are expressed as percent of mean value from CON þ SAL group and are the mean S.E.M. of nine separate animals in each group. *p < 0.05 and **p < 0.01; comparison between treatment (one-way ANOVA followed by Fisher’s LSD test).
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Fig. 6. Representative autoradiographs for CRF mRNA in the CeA from each treatment group are shown. In CMS-exposed groups, rats were given i.p. injections of saline (CMS þ SAL) or tianeptine (10 mg/kg, CMS þ TIA) before restraint stress in the morning. Non-stressed control rats were handled each day and given daily injections of saline (CON þ SAL) or tianeptine (10 mg/kg, CON þ TIA).
anhedonia. Clearly, further experiments will be required to clarify the role of CRF in the BNST in CMS-induced anxiety and anhedonia.
4.3. Anatomic selectivity of CMS effects on CRF in the BNST In the present study, the BNST, in particular the dBNST, was found to be more involved in the response to CMS than was the CeA. This regional difference between the BNST and CeA could be explained by the persistence of the stress response. It may be that stress exposure during the CMS procedures stimulates the CeA briefly, but produces a more sustained stimulation of the dBNST (Walker et al., 2003). This possibility is supported by the finding that chronic immobilization stress resulted in increased dendritic arborization of neurons in the BNST, but not the CeA (Vyas et al., 2003). Thus, chronic stress appears to stimulate the BNST in a more sustained manner than the CeA and as a result to induce morphological changes of neurons in BNST. Furthermore, it has been suggested that neurons in the BNST are
sensitive to food restriction and are related to modulation of feeding (Ciccocioppo et al., 2003) and the circadian rhythm (Amir et al., 2004). If so, then restricted food and changes in the circadian rhythm during the CMS procedures would be expected to affect the BNST more than the CeA. It is not yet known why CMS increases CRF mRNA in the dBNST more than in the CeA or vBNST. One possibility is that increases in plasma corticosterone levels during CMS procedures may selectively induce increases in dBNST CRF mRNA levels. However, this possibility is unlikely, because Table 1 The effects of CMS and tianeptine treatment on CRF mRNA levels in the paraventricular nucleus of the hypothalamus (PVN) and in Barrington’s nucleus CON þ SAL CON þ TIA CMS þ SAL CMS þ TIA PVN 100.0 2.7 Barrington’s nucleus 100.0 3.6
91.8 4.4 92.1 5.1
97.4 3.1 97.1 4.3
100.7 3.9 99.8 3.2
The results are expressed as percent of the mean value of the CON þ SAL group and are the mean S.E.M. of nine animals per group. One-way ANOVA analysis indicated that there were no significant differences among the groups in CRF mRNA levels in either the PVN or Barrington’s nucleus.
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increased plasma corticosterone levels have been reported to increase CRF mRNA in both the dBNST and the CeA (Makino et al., 1994). Alternatively, it is possible that, apart from acute stress, CRF mRNA in the dBNST, vBNST and CeA may have differential sensitivities to the nature of chronic stress. For example, acute stress caused a parallel increase in CRF mRNA levels in the dBNST and CeA (Makino et al., 1994), whereas chronic stress caused varying degrees of response in CRF mRNA in each area (Albeck et al., 1997; Stout et al., 2000). Finally, it has been suggested that CRF in the BNST may coordinate responding to aversive stimuli that cannot be predicted, whereas the CRF in the CeA may coordinate responding to phasic and/or predictable stressors (Walker et al., 2003). Thus according to this view, CMS, which consists of repeated, varied and unpredictable mild stressors, would be expected to increase CRF mRNA in the BNST more than in the CeA. Further studies will be needed to clarify the regional differences in CRF mRNA levels in response to CMS. 4.4. CMS effects on CRF in the CeA, PVN, and Barrington’s nucleus In the present study, we observed only a trend toward an increase of CRF mRNA expression in the CeA in CMS-exposed rats, although chronic social stress using subordinate rats in the visible burrow model was previously reported to produce increased CRF mRNA expression in the CeA (Albeck et al., 1997). Our results are similar to those observed in previous studies with chronic stress-exposed rats (Chappell et al., 1986). Furthermore, our results are consistent with the observation that the same CMS procedures did not affect amygdalar CRF content (Stout et al., 2000). We did not detect CMS-induced changes in CRF mRNA levels in the PVN or Barrington’s nucleus. It has been shown that stressors such as chronic immobilization stress (Marti et al., 1999) or social stress (Albeck et al., 1997) significantly increase CRF mNRA in the PVN and that even procedures similar to CMS increased CRF mRNA levels in the PVN (Duncko et al., 2001). On the other hand, chronic immobilization stress did not increase CRF mRNA levels in the PVN (Ma and Lightman, 1998). These results suggest a possibility that the responses of CRF mRNA in the PVN to chronic stress are positively related to the frequency and intensity of exposure to the stressor (Armario et al., 2004). Although it is not easy to adequately compare the degree of similarity between CMS procedures, it should be noted that there are a few minor procedural differences between our CMS model and that of Duncko et al. (2001). Because we did not apply stress during the weekend except for that associated with changes of circadian rhythm, the frequency of the stressor in the CMS model used by Duncko et al. (2001) was higher than that in our CMS model. Other procedural variations include differences in body weight at the start of CMS procedures (180e200 g vs. 320e 380 g) and the time of sacrifice after the last CMS procedure (5e8 h vs. ~ 72 h). It may be that any of these procedural differences may have contributed to the changes in CRF mRNA in the PVN.
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The present lack of a detectable effect of CMS upon CRF mRNA levels in the Barrington’s nucleus differed from previous findings (Imaki et al., 1991), in which acute and chronic stress with footshock significantly increased CRF mRNA levels in Barrington’s nucleus. As CRF mRNA levels were significantly increased 24 h, but not 2 h, after a single session of footshock stress, it is possible that the time between stress exposure and sacrifice may determine the extent of changes in CRF mRNA levels in Barrington’s nucleus. 4.5. Tianeptine treatment effects on CRF Tianeptine has an antidepressant efficacy similar to that of SSRIs (Wilde and Benfield, 1995), but unlike SSRIs, tianeptine enhances serotonin reuptake (Mennini et al., 1987). In addition, tianeptine can prevent CA3 pyramidal neuron atrophy (Watanabe et al., 1992) and decreases in dentate gyrus granular precursor cell proliferation (Czeh et al., 2001) induced by 3e4 weeks of repeated stress or glucocorticoid administration. These findings suggest that in addition to an antidepressant effect, tianeptine may also have a functional anti-stress effect. In the present study, repeated tianeptine treatment decreased CRF mRNA levels in the CeA and vBNST in nonstressed controls. In addition, there was a trend toward a tianeptine-induced reduction of CRF mRNA levels in the PVN and Barrington’s nucleus. These results suggest that repeated tianeptine treatment may reduce the basal activity of CRF neurons. Moreover, we found that repeated tianeptine treatment reduced CRF mRNA levels in the dBNST of both CMS-exposed and non-stressed control rats. Given that CRF in the BNST may be involved with anxiety (Davis et al., 1997; Lee and Davis, 1997), it is tempting to speculate that increased CRF mRNA in the dBNST of CMS-exposed rats may contribute to increased aversive behavioral outcomes, including anxiety, and that tianeptine treatment prior to stress may reduce CMS-related anxiety. The finding that tianeptine is effective in reducing chronic stress-induced increase in anxiety-like behavior (Pillai et al., 2004) is consistent with this possibility. Interestingly, there is evidence that tianeptine alleviates anxiety symptoms associated with depression (Wilde and Benfield, 1995). Moreover, repeated tianeptine treatment decreased CRF mRNA in the vBNST and CeA in addition to the dBNST, in both CMS-exposed and non-stressed control rats. As CRF in the amygdala has been implicated in mediating conditioned fear responses, tianeptine treatment might reduce conditioned fear behavior via its actions in the CeA. This expectation is consistent with the finding that tianeptine has anxiolytic properties after repeated administration in a model of conditioned freezing (Burghardt et al., 2004). The mechanism by which repeated tianeptine treatment affects CRF mRNA expression in the BNST and CeA is unknown. However, it has been shown that stress induces increases in norepinephrine (NE) release in the BNST (Pacak et al., 1995) and the CeA (Quirarte et al., 1998) as measured by in vivo microdialysis, and that tianeptine can block the restraint stress-induced increase in NE levels in rat frontal cortex (Sacchetti et al., 1993). In addition, NE in the PVN stimulates
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CRF release (Alonso et al., 1986) and increases CRF mRNA levels in the PVN (Itoi et al., 1994). Considering that NE release in the BNST during stress stimulates CRF release in this region, which in turn leads to increases in CRF gene transcription, it is plausible that a reduction in stress-induced NE release similar to that observed in the prefrontal cortex following tianeptine treatment could contribute to the decrease in CRF mRNA expression in associated brain areas including the BNST. However, even if this possibility is taken into consideration, the fact that repeated tianeptine treatment reduced CRF mRNA levels below those of non-stressed controls in the vBNST and CeA suggests that additional variables are involved in the regulation of CRF mRNA in response to tianeptine treatment. Moreover, although the NE transporter is present in both the dBNST and vBNST (Macey et al., 2003), NE terminals from the A1 and A2 medullary NE cell groups project predominantly to the vBNST (Aston-Jones et al., 1999). Further studies will be required to elucidate how repeated tianeptine treatment produces reductions in CRF mRNA expression in related brain areas such as the BNST and CeA. In conclusion, the present results show that CMS induces increases in CRF mRNA levels in the dBNST. Furthermore, it was demonstrated that this increase is attenuated by repeated tianeptine treatment. Repeated tianeptine treatment significantly decreased CRF mRNA levels in the vBNST and CeA, in both CMS-exposed rats and non-stressed controls. These results suggest that repeated tianeptine treatment diminishes the basal activity of CRF neurons and reduces their sensitivity to stress. Acknowledgements This research was supported by a grant from the Brain Research Center of the 21st Century Frontier Research Program, funded by the Korean Ministry of Science and Technology (M103KV010007-03K2201-00720), and Korea Health 21 R&D Project (KPGRN-R-04-04) by the Ministry of Health and Welfare, Republic of Korea. We are grateful to professor Kelly Mayo for his kind gift of rat CRF cDNA. References Albeck, D.S., McKittrick, C.R., Blanchard, D.C., Blanchard, R.J., Nikulina, J., McEwen, B.S., Sakai, R.R., 1997. Chronic social stress alters levels of corticotropin-releasing factor and arginine vasopressin mRNA in rat brain. J. Neurosci. 17, 4895e4903. Alheid, G.F., de Lomos, J.S., Beltramino, C.A., 1995. Amygdala and extended amygdala. In: Paxinos, G. (Ed.), The Rat Nervous System. Academic Press, San Diego, pp. 495e578. Alonso, G., Szafarczyk, A., Balmefrezol, M., Assenmacher, I., 1986. Immunocytochemical evidence for stimulatory control by the ventral noradrenergic bundle of parvocellular neurons of the paraventricular nucleus secreting corticotropin releasing hormone and vasopressin in rats. Brain Res. 397, 297e307. Amir, S., Lamont, E.W., Robinson, B., Stewart, J., 2004. A circadian rhythm in the expression of PERIOD2 protein reveals a novel SCN-controlled oscillator in the oval nucleus of the bed nucleus of the stria terminalis. J. Neurosci. 24, 781e790.
Armario, A., Valles, A., Dal-Zotto, S., Marquez, C., Belda, X., 2004. A single exposure to severe stressors causes long-term desensitisation of the physiological response to the homotypic stressor. Stress 7, 157e172. Aston-Jones, G., Delfs, J.M., Druhan, J., Zhu, Y., 1999. The bed nucleus of the stria terminalis. A target site for noradrenergic actions in opiate withdrawal. Ann. N.Y. Acad. Sci. 877, 486e498. Banki, C.M., Bissette, G., Arato, M., O’Connor, L., Nemeroff, C.B., 1987. CSF corticotropin-releasing factor-like immunoreactivity in depression and schizophrenia. Am. J. Psychiatry 144, 873e877. Burghardt, N.S., Sullivan, G.M., McEwen, B.S., Gorman, J.M., LeDoux, J.E., 2004. The selective serotonin reuptake inhibitor citalopram increases fear after acute treatment but reduces fear with chronic treatment: a comparison with tianeptine. Biol. Psychiatry 55, 1171e1178. Castanon, N., Konsman, J.P., Medina, C., Chauvet, N., Dantzer, R., 2003. Chronic treatment with the antidepressant tianeptine attenuates lipopolysaccharide-induced Fos expression in the rat paraventricular nucleus and HPA axis activation. Psychoneuroendocrinology 28, 19e34. Chappell, P.B., Smith, M.A., Kilts, C.D., Bissette, G., Ritchie, J., Anderson, C., Nemeroff, C.B., 1986. Alterations in corticotropin-releasing factor-like immunoreactivity in discrete rat brain regions after acute and chronic stress. J. Neurosci. 6, 2908e2914. Ciccocioppo, R., Fedeli, A., Economidou, D., Policani, F., Weiss, F., Massi, M., 2003. The bed nucleus is a neuroanatomical substrate for the anorectic effect of corticotropin-releasing factor and for its reversal by nociceptin/orphanin FQ. J. Neurosci. 23, 9445e9451. 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, 12796e12801. D’Aquila, P.S., Brain, P., Willner, P., 1994. Effects of chronic mild stress on performance in behavioural tests relevant to anxiety and depression. Physiol. Behav. 56, 861e867. Davis, M., Walker, D.L., Lee, Y., 1997. Amygdala and bed nucleus of the stria terminalis: differential roles in fear and anxiety measured with the acoustic startle reflex. Philos. Trans. R. Soc. Lond. B Biol. Sci. 352, 1675e1687. De Bellis, M.D., Gold, P.W., Geracioti Jr., T.D., Listwak, S.J., Kling, M.A., 1993. Association of fluoxetine treatment with reductions in CSF concentrations of corticotropin-releasing hormone and arginine vasopressin in patients with major depression. Am. J. Psychiatry 150, 656e657. Delbende, C., Tranchand Bunel, D., Tarozzo, G., Grino, M., Oliver, C., Mocaer, E., Vaudry, H., 1994. Effect of chronic treatment with the antidepressant tianeptine on the hypothalamo-pituitary-adrenal axis. Eur. J. Pharmacol. 251, 245e251. Dong, H.W., Petrovich, G.D., Watts, A.G., Swanson, L.W., 2001. Basic organization of projections from the oval and fusiform nuclei of the bed nuclei of the stria terminalis in adult rat brain. J. Comp. Neurol. 436, 430e455. Duncko, R., Kiss, A., Skultetyova, I., Rusnak, M., Jezova, D., 2001. Corticotropin-releasing hormone mRNA levels in response to chronic mild stress rise in male but not in female rats while tyrosine hydroxylase mRNA levels decrease in both sexes. Psychoneuroendocrinology 26, 77e89. Egli, R.E., Winder, D.G., 2003. Dorsal and ventral distribution of excitable and synaptic properties of neurons of the bed nucleus of the stria terminalis. J. Neurophysiol. 90, 405e414. Imaki, T., Nahan, J.L., Rivier, C., Sawchenko, P.E., Vale, W., 1991. Differential regulation of corticotropin-releasing factor mRNA in rat brain regions by glucocorticoids and stress. J. Neurosci. 11, 585e599. Itoi, K., Suda, T., Tozawa, F., Dobashi, I., Ohmori, N., Sakai, Y., Abe, K., Demura, H., 1994. Microinjection of norepinephrine into the paraventricular nucleus of the hypothalamus stimulates corticotropin-releasing factor gene expression in conscious rats. Endocrinology 135, 2177e2182. Koob, G.F., 1999. Corticotropin-releasing factor, norepinephrine, and stress. Biol. Psychiatry 46, 1167e1180. Lee, Y., Davis, M., 1997. Role of the hippocampus, the bed nucleus of the stria terminalis, and the amygdala in the excitatory effect of corticotropinreleasing hormone on the acoustic startle reflex. J. Neurosci. 17, 6434e6446.
S.-J. Kim et al. / Neuropharmacology 50 (2006) 824e833 Ma, X.M., Lightman, S.L., 1998. The arginine vasopressin and corticotrophinreleasing hormone gene transcription responses to varied frequencies of repeated stress in rats. J. Physiol. 510 (Pt 2), 605e614. Macey, D.J., Smith, H.R., Nader, M.A., Porrino, L.J., 2003. Chronic cocaine self-administration upregulates the norepinephrine transporter and alters functional activity in the bed nucleus of the stria terminalis of the rhesus monkey. J. Neurosci. 23, 12e16. Makino, S., Gold, P.W., Schulkin, J., 1994. Effects of corticosterone on CRH mRNA and content in the bed nucleus of the stria terminalis; comparison with the effects in the central nucleus of the amygdala and the paraventricular nucleus of the hypothalamus. Brain Res. 657, 141e149. Makino, S., Shibasaki, T., Yamauchi, N., Nishioka, T., Mimoto, T., Wakabayashi, I., Gold, P.W., Hashimoto, K., 1999. Psychological stress increased corticotropin-releasing hormone mRNA and content in the central nucleus of the amygdala but not in the hypothalamic paraventricular nucleus in the rat. Brain Res. 850, 136e143. Marti, O., Harbuz, M.S., Andres, R., Lightman, S.L., Armario, A., 1999. Activation of the hypothalamic-pituitary axis in adrenalectomised rats: potentiation by chronic stress. Brain Res. 821, 1e7. Mennini, T., Mocaer, E., Garattini, S., 1987. Tianeptine, a selective enhancer of serotonin uptake in rat brain. Naunyn Schmiedebergs Arch. Pharmacol. 336, 478e482. Moreau, J.L., Jenck, F., Martin, J.R., Mortas, P., Haefely, W.E., 1992. Antidepressant treatment prevents chronic unpredictable mild stress-induced anhedonia as assessed by ventral tegmentum self-stimulation behavior in rats. Eur. Neuropsychopharmacol. 2, 43e49. Morin, S.M., Ling, N., Liu, X.J., Kahl, S.D., Gehlert, D.R., 1999. Differential distribution of urocortin- and corticotropin-releasing factor-like immunoreactivities in the rat brain. Neuroscience 92, 281e291. Nemeroff, C.B., Bissette, G., Akil, H., Fink, M., 1991. Neuropeptide concentrations in the cerebrospinal fluid of depressed patients treated with electroconvulsive therapy. Corticotrophin-releasing factor, beta-endorphin and somatostatin. Br. J. Psychiatry 158, 59e63. Nemeroff, C.B., Widerlov, E., Bissette, G., Walleus, H., Karlsson, I., Eklund, K., Kilts, C.D., Loosen, P.T., Vale, W., 1984. Elevated concentrations of CSF corticotropin-releasing factor-like immunoreactivity in depressed patients. Science 226, 1342e1344. Nestler, E.J., Barrot, M., DiLeone, R.J., Eisch, A.J., Gold, S.J., Monteggia, L.M., 2002. Neurobiology of depression. Neuron 34, 13e25. Pacak, K., McCarty, R., Palkovits, M., Kopin, I.J., Goldstein, D.S., 1995. Effects of immobilization on in vivo release of norepinephrine in the
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bed nucleus of the stria terminalis in conscious rats. Brain Res. 688, 242e246. Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinates, fourth ed. Academic Press, Orlando. Pillai, A.G., Munoz, C., Chattarji, S., 2004. The antidepressant tianeptine prevents the dendritic hypertrophy in the amygdala and increase in anxiety induced by chronic stress in the rat. Abstract # 762.1, Society for Neuroscience. Quirarte, G.L., Galvez, R., Roozendaal, B., McGaugh, J.L., 1998. Norepinephrine release in the amygdala in response to footshock and opioid peptidergic drugs. Brain Res. 808, 134e140. Sacchetti, G., Bonini, I., Waeterloos, G.C., Samanin, R., 1993. Tianeptine raises dopamine and blocks stress-induced noradrenaline release in the rat frontal cortex. Eur. J. Pharmacol. 236, 171e175. Sakanaka, M., Shibasaki, T., Lederis, K., 1986. Distribution and efferent projections of corticotropin-releasing factor-like immunoreactivity in the rat amygdaloid complex. Brain Res. 382, 213e238. Spencer, S.J., Buller, K.M., Day, T.A., 2005. Medial prefrontal cortex control of the paraventricular hypothalamic nucleus response to psychological stress: possible role of the bed nucleus of the stria terminalis. J. Comp. Neurol. 481, 363e376. Stout, S.C., Mortas, P., Owens, M.J., Nemeroff, C.B., Moreau, J., 2000. Increased corticotropin-releasing factor concentrations in the bed nucleus of the stria terminalis of anhedonic rats. Eur. J. Pharmacol. 401, 39e46. Vale, W., Spiess, J., Rivier, C., Rivier, J., 1981. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 213, 1394e1397. Vyas, A., Bernal, S., Chattarji, S., 2003. Effects of chronic stress on dendritic arborization in the central and extended amygdala. Brain Res. 965, 290e 294. Walker, D.L., Toufexis, D.J., Davis, M., 2003. Role of the bed nucleus of the stria terminalis versus the amygdala in fear, stress, and anxiety. Eur. J. Pharmacol. 463, 199e216. Watanabe, Y., Gould, E., Daniels, D.C., Cameron, H., McEwen, B.S., 1992. Tianeptine attenuates stress-induced morphological changes in the hippocampus. Eur. J. Pharmacol. 222, 157e162. Wilde, M.I., Benfield, P., 1995. Tianeptine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in depression and coexisting anxiety and depression. Drugs 49, 411e439. Willner, P., Moreau, J.L., Nielsen, C.K., Papp, M., Sluzewska, A., 1996. Decreased hedonic responsiveness following chronic mild stress is not secondary to loss of body weight. Physiol. Behav. 60, 129e134.
Effects of repeated tianeptine treatment on CRF mRNA expression in non-stressed and chronic mild stress-exposed rats Sung-Jin Kim a, Sang-Ha Park a, Song-hyen Choi a, Bo-Hyun Moon a, Kuem-Ju Lee a, Seung Woo Kang a, Min-Soo Lee b, Sang-Hyun Choi a, Boe-Gwun Chun a, Kyung-Ho Shin a,* a
Department of Pharmacology, College of Medicine, Korea University, Seoul, South Korea b Department of Psychiatry, College of Medicine, Korea University, Seoul, South Korea
Received 22 June 2005; received in revised form 8 November 2005; accepted 6 December 2005
Abstract Accumulating evidence suggests that dysregulation of corticotropin-releasing factor (CRF) may play a role in depression and that this dysregulation may be corrected by antidepressant drug treatment. Here, we examined whether chronic mild stress (CMS) alters CRF mRNA levels in stress-related brain areas including the bed nucleus of the stria terminalis (BNST) and the central nucleus of amygdala (CeA), and whether repeated tianeptine treatment can attenuate CMS-induced changes in CRF mRNA levels. Male rats were exposed to CMS for 19 days, and control animals were subjected to brief handling. Both groups were injected daily with tianeptine or saline. CMS significantly increased CRF mRNA levels in the dorsal BNST (dBNST), but not in other areas. Repeated tianeptine treatment prevented the CMS-induced increase in CRF mRNA levels in the dBNST, and reduced CRF mRNA levels in dBNST in non-stressed controls. Moreover, repeated tianeptine treatment significantly decreased CRF mRNA levels in the ventral BNST and CeA of non-stressed controls as well as CMS-exposed rats. These results show that CMS induces a rather selective increase of CRF mRNA in the dBNST. In addition, these results suggest that repeated tianeptine treatment diminishes the basal activity of CRF neurons and reduces their sensitivity to stress. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Tianeptine; Stress; Corticotropin-releasing factor; Bed nucleus of the stria terminalis; Amygdala; Depression; Anxiety
1. Introduction Corticotropin-releasing factor (CRF) was first described as a hypothalamic neuropeptide that stimulates the secretion of adrenocorticotropin (ACTH) (Vale et al., 1981). In addition to the paraventricular nucleus of hypothalamus (PVN), CRF is found in stress-related brain areas such as the bed nucleus of the stria terminalis (BNST), central nucleus of the amygdala (CeA), and Barrington’s nucleus (Imaki et al., 1991; Morin et al., 1999). CRF functions as a neurotransmitter in the integration of behavioral and autonomic responses to stress (Koob, * Corresponding author. Korea University, College of Medicine, Department of Pharmacology, Sungbuk-gu Anam-dong 5-ka 126-1, Seoul, 136-705 Republic of Korea. Tel.: þ82 2 920 6195; fax: þ82 2 927 0824. E-mail address: [email protected] (K.-H. Shin). 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.12.003
1999). As stress is known to be a predisposing factor for major depression, it is possible that dysregulation of the CRF system may contribute to the pathology of depression (Nemeroff et al., 1984). For example, increased concentrations of CRF in cerebrospinal fluid (CSF) have been reported in depressed patients (Banki et al., 1987). Moreover, the normalization of elevated CRF concentrations in CSF has been reported after successful treatment of depression by fluoxetine (De Bellis et al., 1993) or electroconvulsive shock (Nemeroff et al., 1991). Tianeptine is a serotonin reuptake enhancer (Mennini et al., 1987), and has an antidepressant efficacy similar to that of selective serotonin reuptake inhibitors (SSRIs) (Wilde and Benfield, 1995). Interestingly, chronic administration of tianeptine has been shown to attenuate lipopolysaccharide- or stress-induced increases in plasma ACTH and corticosterone
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levels (Castanon et al., 2003; Delbende et al., 1994). These findings suggest that chronic tianeptine treatment may suppress pituitaryeadrenal axis activation in response to stress. In addition, tianeptine attenuated stress-induced reduction of hypothalamic CRF concentration (Delbende et al., 1994), suggesting that tianeptine exerts antidepressant effects by regulating the pituitaryeadrenal axis and/or CRF neurotransmission in the hypothalamus. A caveat of antidepressant research is the use of normal laboratory rats (Nestler et al., 2002). This concern emerges from the observation that antidepressant treatments in humans without depression cause typical side effects rather than antidepressant effects (Nestler et al., 2002). Thus, the elucidation of mechanisms of antidepressants using normal rats may not reflect the mechanism of antidepressant effects in the depressed brain. To take this possibility into account, we injected tianeptine under a chronic stress condition to imitate the clinical feature of depression. Anhedonia, the loss of interest in or ability to derive pleasure from all or most activities, is one of the two core symptoms of depression according to the Diagnostic and Statistical Manual of Mental Disorders, fourth revision (1994). Since a similar state of anhedonia can be induced in rats by chronic mild stress (CMS) procedures (Moreau et al., 1992; Stout et al., 2000), a CMS model has been used as an animal model of depression. Moreover, it has been shown that increased CRF concentration in the BNST was observed in CMS anhedonic rats (Stout et al., 2000). In the present study, we tested whether CMS increases CRF mRNA expression in stress-related brain areas, and whether repeated tianeptine treatment during CMS procedures may inhibit CMS-induced changes in CRF mRNA. 2. Materials and methods 2.1. Animals Adult male SpragueeDawley rats (Orient, Seoul, South Korea) weighing 190e210 g at the beginning of the CMS exposure were used. Rats were handled daily for 7 days to habituate them to the laboratory environment. Rats were housed three per cage in a humidified room with a 12-h light/dark cycle (lights on at 6:00 AM), and food and water were available ad libitum except during food or water deprivation periods during CMS procedures. All procedures used in this study were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).
2.2. Stress paradigm For CMS, rats were exposed to a variable stress regimen for 19 days as previously described (Moreau et al., 1992; Stout et al., 2000). In brief, the stressor included repeated 1 h periods of confinement in a small cage (24 10 9 cm), one period of continuous overnight illumination, one overnight period of food and water deprivation followed by 2 h access to restricted food (3 g per cage), one overnight period of water deprivation followed by 1 h exposure to an empty bottle, and one overnight period of group housing (6 rats per cage) in a soiled cage (200 ml water in sawdust bedding). Animals were also maintained on a reversed light/dark cycle from Friday evening to Monday morning. The last procedure of CMS consisted of reversed light/dark cycle Friday evening for 12 h. Lights in the animal room were not turned off on Saturday morning after the final CMS procedure was completed. All perfusions were conducted between 11:00 and 14:00 h in order to reduce possible
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circadian changes in CRF peptide levels. Non-stressed control rats were housed in a separate room and were briefly handled each day without any deliberately stressing procedures.
2.3. Antidepressant administration The animals were divided into four groups: saline-treated non-stressed controls (CON þ SAL, n ¼ 9), saline-treated CMS-exposed group (CMS þ SAL, n ¼ 9), tianeptine-treated non-stressed controls (CON þ TIA, n ¼ 9) and tianeptine-treated CMS-exposed group (CMS þ TIA, n ¼ 9). Tianeptine was dissolved in 0.9% saline, which was used as the vehicle. Tianeptine (10 mg/kg) was injected intraperitoneally (i.p.) 30 min before restraint stress each morning (08:30 h) during the 19-day CMS period.
2.4. Body weight, food and water intake measurements For measurement of food and water intake, a different batch of animals was exposed to CMS. Rats were housed individually to determine daily food and water intake. The food hopper and water bottle were removed and weighed at 08:30 h. At this time, rats were removed from their cages, weighed, and then injected with saline or tianeptine. A fresh supply of preweighed food and water was returned 1 h later. Daily food intake (g/day) was calculated by subtracting the weight of the hopper from its initial weight. Daily water intake (ml/day) was determined by subtracting the volume of the bottle from its initial volume. Cages were carefully monitored for evidence of food spillage or grinding. Cumulative food and water intake were calculated from daily food and water intake, which included food and water intake during food or water deprivation periods in CMS-exposed rats. However, mean daily food and water intake were averaged from daily food and water intake except during food and water deprivation periods in CMS-exposed rats as well as in non-stressed rats.
2.5. Histological procedures Rats were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.) and were perfused intracardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PPB, pH 7.2). The brains were fixed in situ for 1 h, removed, post-fixed in PPB for 2 h, and finally placed in 20% sucrose/PPB overnight at 4 C. Serial coronal sections (30 mm) of whole brain were obtained using a freezing microtome and were stored in cryoprotectant solution [30% RNase free sucrose, 30% ethylene glycol, and 1% polyvinyl pyrrolidone (PVP-40) in 100 mM sodium phosphate buffer, pH 7.4] at 20 C.
2.6. Probe labeling The CRF riboprobe was constructed from a 1.2-kb EcoRI fragment of fulllength rat CRF cDNA (a gift from K. Mayo, Northwestern University, Evanston, IL, USA) subcloned into a pBluescript II-SKþ plasmid. Transcription reactions were performed using an Ambion MAXIscript kit (Austin, TX, USA) with SP6 RNA polymerases according to the manufacturer’s instructions. After transcription and removal of the cDNA template with 2 U of DNase, the cRNA probes were recovered using NucTrap minicolumns (Stratagene, La Jolla, CA, USA).
2.7. In situ hybridization Brain sections were permeabilized with proteinase K (1 mg/ml, 37 C, 30 min), treated with acetic anhydride in 0.1 M triethanolamine (pH 8.0), washed in 2 SSC (pH 7.0), and were then transferred into 500 ml of hybridization solution in 24-well culture plates. The hybridization solution was comprised of 50% formamide, 0.01% polyvinyl pyrrolidone, 0.01% Ficoll, 0.01% bovine serum albumin, 50 mg/ml denatured salmon sperm DNA, 250 mg/ml yeast tRNA, 40 mM dithiothreitol (DTT), 10% dextran sulfate, and 35S-labeled CRF cRNA probes at 1 107 cpm/ml. Sections were hybridized with the CRF riboprobe for 18 h at 55 C in the hybridization solution. After overnight
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incubation in a humidified chamber, sections were washed twice for 30 min at 55 C in 4 SSC with 5 mM DTT, treated with RNase A (20 mg/ml, 30 min at 45 C), and washed four times (15 min/wash) at room temperature in 2 SSC containing 5 mM DTT. The sections were then washed twice (30 min/wash) in 0.5 SSC containing 5 mM DTT, once for 30 min in 0.1 SSC containing 5 mM DTT at 50e55 C, and once in 0.1 SSC with 5 mM DTT at room temperature. Sections were then mounted on gelatin-coated microscope slides and air-dried overnight. Tissue sections were exposed to Hyperfilm b-max (Amersham, Arlington Heights, IL, USA) for 3 days. To correct for nonlinearity, 14Clabeled radioactive standards (Amersham, Arlington Heights, IL, USA) were used for calibration.
2.8. Data analysis and statistics Images were captured digitally and CRF mRNA levels were analyzed by outlining the regions of interest, and then quantifying the regional optical densities of autoradiographic film images using Scion Image beta 4.2 software (Scion Corporation, Frederick, MD, USA). Three or four sections from BNST, PVN, CeA and Barrington’s nucleus containing the highest expression were averaged to obtain a single value for each nucleus. The anterior commissure was considered to be the border between the dorsal and ventral BNST (dBNST and vBNST). To correct for nonlinearity, 14C-labeled radioactive standards (Amersham, Arlington Heights, IL, USA) were exposed with tissue sections and used for calibration. Optical densities were obtained from forebrain to hindbrain based on the stereotaxic atlas of Paxinos and Watson (1998). Accordingly, relative to bregma, regions of interest were located as follows: BNST, 0.26 to 0.40 mm; PVN, 1.80 to 2.12 mm; CeA, 2.56 to 3.14 mm; and Barrington’s nucleus, 9.30 to 10.04 mm. Results are expressed as the mean percentage of the values for the saline-treated non-stressed control, and data are reported herein as means S.E.M. Two-way repeated-measures ANOVAs with Bonferroni’s multiple comparison post hoc tests were used to analyze the effects of CMS and repeated tianeptine treatment on body weight changes as well as on cumulative food and water intake during CMS, with two between-subjects factors [CMS (non-stressed control vs. CMS) and repeated tianeptine treatment (saline vs. tianeptine)] and with time as the only within-subject factor. A one-way repeated-measures ANOVA was used with Bonferroni’s post hoc multiple comparison test to analyze separately whether the groups differed from one another. Mean daily food and water intake as well as CRF mRNA level data were similarly analyzed by a two-way ANOVA to detect overall effects of CMS treatment and repeated tianeptine treatment followed by a one-way ANOVA to determine whether there were differences between the individual animal groups. Fisher’s LSD post hoc tests were used to determine the differences between the groups. Significance was determined at the p < 0.05 level.
3. Results 3.1. Effects of repeated tianeptine treatment on CMS-induced body weight changes, food and water intake Body weights were measured daily during the CMS procedures. A consistent reduction in body weight gain was observed in CMS-exposed rats (Fig. 1). A two-way repeated-measures ANOVA with CMS and repeated tianeptine treatment as between-subjects factors and time as the within-subject factor revealed a significant effect of CMS (F1,32 ¼ 61.8, p < 0.001), a significant effect of time (F19,608 ¼ 2052.3, p < 0.001), and a significant interaction of CMS with time (F19,608 ¼ 55.7, p < 0.001) on body weight gain during the CMS procedures (Fig. 1A). There were no significant main effects of repeated tianeptine treatment and no interaction between repeated tianeptine treatment and time. A one-way repeated-measures ANOVA with Bonferroni’s post hoc test showed that non-stressed and
CMS-exposed groups differed regardless of whether they received saline or tianeptine pretreatment (Fig. 1A, CON þ SAL and CON þ TIA vs. CMS þ SAL; CON þ SAL and CON þ TIA vs. CMS þ TIA; F3,32 ¼ 21.7, p < 0.001). These results showed that while CMS decreased weight gain, repeated tianeptine treatment did not influence the body weight changes in either the non-stressed or CMS-exposed groups. CMS significantly decreased cumulative food intake (Fig. 1B, two-way repeated-measures ANOVA: CMS, F1,32 ¼ 88.2, p < 0.001; CMS time, F19,608 ¼ 54.6, p < 0.001) and cumulative water intake (Fig. 1C, two-way repeated-measures ANOVA: CMS, F1,32 ¼ 57.3, p < 0.001; CMS time, F19,608 ¼ 46.0, p < 0.001) compared with non-stressed groups. There was no significant main effect of repeated tianeptine treatment on cumulative food and water intake, suggesting that the tianeptine treatment did not influence cumulative food and water intake (Fig. 1C). In addition, CMS induced a small but significant decrease in mean daily food intake compared with nonstressed groups (Fig. 1D, one-way ANOVA with Fisher’s post hoc LSD test: F1,32 ¼ 11.2, p < 0.01; CON þ SAL: 25.5 0.8 g/day; CON þ TIA: 25.4 0.5 g/day; CMS þ SAL: 23.6 0.5 g/day; CMS þ TIA: 23.6 0.3 g/day), but this CMS-induced decrease in mean daily food intake was not influenced by repeated tianeptine treatment. The patterns of changes in mean daily water intake observed were similar to those seen with food, but the magnitude of decrease in mean daily water intake by the CMS-treated rats was greater than those in food intake (Fig. 1D, one-way ANOVA with Fisher’s post hoc LSD test: F1,32 ¼ 11.8, p < 0.01 vs. CON þ SAL; CON þ SAL: 42.5 1.6 ml/day, CON þ TIA: 43.2 2.7 ml/day, CMS þ SAL: 35.6 0.8 ml/day; CMS þ TIA: 38.7 0.7 ml/day). 3.2. Effects of repeated tianeptine treatment on CRF mRNA levels in the dorsal bed nucleus of the stria terminalis (dBNST) A two-way ANOVA revealed significant main effects of CMS (F1,32 ¼ 38.2, p < 0.001) and of the repeated tianeptine treatment (F1,32 ¼ 16.5, p < 0.001) on CRF mRNA levels in the dBNST. No significant interaction between CMS and the repeated tianeptine treatment was found. A one-way ANOVA showed that there was a significant difference among these groups (Figs. 2 and 4, F3,32 ¼ 18.2, p < 0.001). Post hoc analysis using Fisher’s LSD test demonstrated that CMS significantly increased CRF mRNA levels in the dBNST relative to saline-treated non-stressed controls ( p < 0.001), whereas repeated tianeptine treatment significantly decreased CRF mRNA levels ( p < 0.01). Moreover, repeated tianeptine treatment prevented the CMS-induced increase in CRF mRNA levels in the dBNST (Figs. 2 and 4, p < 0.01). 3.3. Effects of repeated tianeptine treatment on CRF mRNA levels in the ventral bed nucleus of the stria terminalis (vBNST) A two-way ANOVA revealed significant main effects of CMS (F1,32 ¼ 9.4, p < 0.001) and of the repeated tianeptine
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Fig. 1. Mean body weight changes (A), cumulative food intake (g/rat) (B), and cumulative water intake (ml/rat) (C) during CMS procedures (***p < 0.001, comparison between treatments; one-way repeated-measures ANOVA followed by Bonferroni’s post hoc test). Mean daily food and water intake during CMS procedures (D). CMS significantly decreased mean daily food and water intake (*p < 0.05 and ###p < 0.001 vs. CON þ SAL; comparison between treatment, one-way ANOVA followed by Fisher’s LSD test). Group means S.E.M. are shown; n ¼ 9 per group. Abbreviations used: CON þ SAL, saline-treated non-stressed controls; CON þ TIA, tianeptine-treated non-stressed controls; CMS þ SAL, saline-treated CMS-exposed group; CMS þ TIA, tianeptine-treated CMS-exposed group.
treatment (F1,32 ¼ 15.4, p < 0.001) on CRF mRNA levels in the vBNST. No significant interaction between CMS and the repeated tianeptine treatment was found. A one-way ANOVA showed that there was a significant difference among these groups (Figs. 3 and 4, F3,32 ¼ 8.5, p < 0.001). Post hoc analysis using Fisher’s LSD test demonstrated that CRF mRNA levels in the vBNST following CMS did not differ from those of saline-treated non-stressed controls, but repeated tianeptine treatment significantly reduced CRF mRNA levels in the vBNST relative to saline-treated non-stressed control (Figs. 3 and 4, F3,32 ¼ 8.5, p < 0.01). Repeated tianeptine treatment in CMS-exposed rats significantly reduced CRF mRNA levels in the vBNST relative to saline-treated CMS-exposed rats (Figs. 3 and 4, F3,32 ¼ 8.5, p < 0.05). 3.4. Effects of repeated tianeptine treatment on CRF mRNA levels in the CeA, PVN, and Barrington’s nucleus A two-way ANOVA revealed significant main effects of CMS (F1,32 ¼ 4.5, p < 0.05) and of the repeated tianeptine treatment (F1,32 ¼ 18.5, p < 0.001) on CRF mRNA levels in
the CeA. No significant interaction between CMS and the repeated tianeptine treatment was found. A one-way ANOVA showed that there was a significant difference among these groups (Figs. 5 and 6, F3,32 ¼ 7.8, p ¼ 0.001). Post hoc analysis using Fisher’s LSD test demonstrated that CMS did not induce a detectable change in CRF mRNA levels in the CeA, but repeated tianeptine treatment decreased CRF mRNA levels in the CeA relative to saline-treated non-stressed controls (Figs. 5 and 6, F3,32 ¼ 7.8, p < 0.01). Repeated tianeptine treatment in CMS-exposed rats significantly reduced CRF mRNA levels in the CeA relative to saline-treated CMS-exposed rats (F3,32 ¼ 7.8, p < 0.05). CMS did not affect CRF mRNA levels in either the PVN or Barrington’s nucleus ( p > 0.05 vs. salinetreated non-stressed control). Only a trend toward a tianeptineinduced reduction of CRF mRNA levels in the PVN and Barrington’s nucleus was observed (Table 1). 4. Discussion In the present study, CMS significantly increased CRF mRNA levels in the dBNST, whereas there was a trend toward a CMS-induced increase of CRF mRNA levels in the vBNST
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***
CRF mRNA (% of CON+SAL)
160 140
**
**
120 100 80 60 40 20 0 CON+SAL
CON+TIA
CMS+SAL
CMS+TIA
Fig. 2. The effects of CMS and tianeptine treatment on CRF mRNA levels in the dorsal bed nucleus of the stria terminalis (dBNST). CMS increased CRF mRNA levels in the dBNST [***p < 0.001 vs. saline-treated non-stressed control (CON þ SAL)]. Repeated tianeptine treatment prevented the CMSinduced increase in CRF mRNA levels in the dBNST, and reduced CRF mRNA levels in dBNST in non-stressed controls. There was no significant interaction (CMS tianeptine). The results are expressed as percent of mean value from CON þ SAL group and are the mean S.E.M. of nine separate animals in each group. **p < 0.01 and ***p < 0.001; comparison between treatment (one-way ANOVA followed by Fisher’s LSD test).
and CeA. CMS did not affect CRF mRNA levels in the PVN or Barrington’s nucleus, suggesting that the CMS-induced increase in CRF mRNA levels in the dBNST was selective. Repeated tianeptine treatment prevented the CMS-induced increase in CRF mRNA levels in the dBNST, and reduced CRF mRNA levels in dBNST in non-stressed controls. In addition, repeated tianeptine treatment significantly decreased CRF mRNA levels in the vBNST and CeA in non-stressed control as well as in CMS-exposed rats.
CRF mRNA (% of CON+SAL)
160
*
**
140 120 100 80 60 40 20 0 CON+SAL
CON+TIA
CMS+SAL
CMS+TIA
Fig. 3. The effects of CMS and tianeptine treatment on CRF mRNA levels in the ventral bed nucleus of the stria terminalis (vBNST). Repeated tianeptine treatment significantly reduced CRF mRNA levels of the vBNST in nonstressed and CMS-exposed rats. There was no significant interaction (CMS tianeptine). The results are expressed as percent of mean value of CON þ SAL group and are the mean S.E.M. of nine separate animals in each group. *p < 0.05 and **p < 0.01; comparison between treatment (oneway ANOVA followed by Fisher’s LSD test).
4.1. Changes in body weight and feeding associated with CMS or antidepressant CMS significantly decreased mean daily food and water intake relative to non-stressed control animals, with the decrease in mean water intake being more prominent. These reductions in mean daily food and water intake during CMS procedures are related to the decrease in cumulative food and water intake, which could contribute to the body weight loss exhibited by CMS-exposed rats. However, repeated tianeptine treatment did not influence CMS-induced body weight changes or mean daily food and water intake. At present, it is not clear whether decreased food and water intake could be attributed to anhedonia, because we did not measure sucrose intake or preference as previously reported (Willner et al., 1996). However, considering that anhedonia is characterized by a diminished ability to experience pleasure from natural reinforcers such as food, water, and sex, as well as drugs of abuse, it is possible that decreased food and water intake may reflect some aspect of an anhedonic state. This possibility is supported by the fact that the mild stressors used in the present study were based on the study by Stout et al. (2000), in which a gradual development of an anhedonic state was observed as demonstrated by increased threshold for self-stimulation. Thus, although CMS may increase anhedonia, repeated tianeptine treatment did not affect eating and drinking behavior and therefore appeared not to influence anhedonia. Further experiments will be required to elucidate the relationship between tianeptine treatment and anhedonic behavior. 4.2. CRF in the BNST The BNST is considered to be part of the extended amygdala and shares several neuroanatomical and neurochemical similarities with the CeA (Alheid et al., 1995). The BNST contains numerous CRF-immunopositive neuronal cell bodies (Morin et al., 1999) and receives heavy projections from the CeA, which contains CRF (Sakanaka et al., 1986). The BNST has been implicated in the regulation of psychological responses to stress (Spencer et al., 2005) and withdrawal from drugs of abuse (Aston-Jones et al., 1999; Macey et al., 2003). In particular, evidence suggests that CRF neurotransmission in the BNST is linked to anxiety (Davis et al., 1997; Lee and Davis, 1997) and anhedonia (Stout et al., 2000). In the present study, the increase of CRF mRNA levels in the dBNST was similar to a previous report, in which an increase of CRF peptide was observed in the BNST, but not in the CeA, following CMS (Stout et al., 2000). The present results are consistent with previous work demonstrating that CRF mRNA in dBNST, but not in vBNST, was increased in response to psychosocial stress in rats (Makino et al., 1999). Thus CRF mRNA in the dBNST appears to be more responsive to CMS than that in the vBNST. Moreover, it has been demonstrated that longterm treatment with corticosterone increased CRF mRNA levels in the dBNST and CeA (Makino et al., 1994). Interestingly, the dBNST and vBNST have distinct anatomic connections (Dong et al., 2001) and electrophysiological properties
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Fig. 4. Representative autoradiographs of 35S-labeled CRF mRNA in BNST from each treatment group are shown. In CMS-exposed groups, rats were given i.p. injections of saline (CMS þ SAL) or tianeptine (10 mg/kg, CMS þ TIA) before restraint stress in the morning. Non-stressed control rats were handled each day and given daily injections of saline (CON þ SAL) or tianeptine (10 mg/kg, CON þ TIA) in the morning.
drinking behavior. That is, the CMS-induced increase of CRF mRNA levels in dBNST was attenuated by tianeptine treatment while the CMS-induced decrease in food and water intake was not. Although decreased food and water intake may not reflect the whole spectrum of anhedonia, this result raises the possibility that CRF in dBNST may not be related to 140
CRF mRNA (% of CON+SAL)
(Egli and Winder, 2003). For example, dBNST receives inputs from the CeA, postpiriform transition area, the insular region and the paraventricular nucleus of the thalamus, whereas vBNST receives inputs from medial prefrontal cortex, ventral subicular complex of the hippocampus, nucleus accumbens and subfornical area (Dong et al., 2001). While dBNST sends a projection to CeA and vBNST, both of which have been shown to play an important role in fear response behavior, vBNST projects directly to the ventral tegmental area and sends a projection to the paraventricular and anterior periventricular nuclei of hypothalamus, which modulate ACTH and growth hormone release, respectively (Dong et al., 2001). Considering the neuroanatomical organization and the role of CRF in the dBNST, our results seem to suggest that this increase in CRF mRNA in the dBNST may contribute to an aversive emotional state including CMS-induced anxiety or anhedonia. As of yet, however, it has not been determined whether there is a correlation between CRF levels in the BNST and any related behaviors. Studies examining whether CMS produces measurable changes in indices of anxiety have been conflicting. While D’Aquila et al. (1994) did not find evidence for CMS-induced anxiety as measured in the social interaction test or the elevated plus maze, Duncko et al. (2001) more recently obtained a different result using a different CMS model. In addition, a CMS-induced increase in CRF mRNA levels in the dBNST may contribute to anhedonia as shown by decreased daily food consumption and water intake. However, there was a disassociation between the effects of tianeptine treatment on CRF mRNA levels and eating and
120
**
*
100 80 60 40 20 0 CON+SAL
CON+TIA
CMS+SAL
CMS+TIA
Fig. 5. The effects of CMS and tianeptine treatment on CRF mRNA levels in the central nucleus of amygdala (CeA). Repeated tianeptine treatment significantly decreased CRF mRNA levels in the CeA of non-stressed controls as well as CMS-exposed rats. There was no significant interaction (CMS tianeptine). The results are expressed as percent of mean value from CON þ SAL group and are the mean S.E.M. of nine separate animals in each group. *p < 0.05 and **p < 0.01; comparison between treatment (one-way ANOVA followed by Fisher’s LSD test).
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Fig. 6. Representative autoradiographs for CRF mRNA in the CeA from each treatment group are shown. In CMS-exposed groups, rats were given i.p. injections of saline (CMS þ SAL) or tianeptine (10 mg/kg, CMS þ TIA) before restraint stress in the morning. Non-stressed control rats were handled each day and given daily injections of saline (CON þ SAL) or tianeptine (10 mg/kg, CON þ TIA).
anhedonia. Clearly, further experiments will be required to clarify the role of CRF in the BNST in CMS-induced anxiety and anhedonia.
4.3. Anatomic selectivity of CMS effects on CRF in the BNST In the present study, the BNST, in particular the dBNST, was found to be more involved in the response to CMS than was the CeA. This regional difference between the BNST and CeA could be explained by the persistence of the stress response. It may be that stress exposure during the CMS procedures stimulates the CeA briefly, but produces a more sustained stimulation of the dBNST (Walker et al., 2003). This possibility is supported by the finding that chronic immobilization stress resulted in increased dendritic arborization of neurons in the BNST, but not the CeA (Vyas et al., 2003). Thus, chronic stress appears to stimulate the BNST in a more sustained manner than the CeA and as a result to induce morphological changes of neurons in BNST. Furthermore, it has been suggested that neurons in the BNST are
sensitive to food restriction and are related to modulation of feeding (Ciccocioppo et al., 2003) and the circadian rhythm (Amir et al., 2004). If so, then restricted food and changes in the circadian rhythm during the CMS procedures would be expected to affect the BNST more than the CeA. It is not yet known why CMS increases CRF mRNA in the dBNST more than in the CeA or vBNST. One possibility is that increases in plasma corticosterone levels during CMS procedures may selectively induce increases in dBNST CRF mRNA levels. However, this possibility is unlikely, because Table 1 The effects of CMS and tianeptine treatment on CRF mRNA levels in the paraventricular nucleus of the hypothalamus (PVN) and in Barrington’s nucleus CON þ SAL CON þ TIA CMS þ SAL CMS þ TIA PVN 100.0 2.7 Barrington’s nucleus 100.0 3.6
91.8 4.4 92.1 5.1
97.4 3.1 97.1 4.3
100.7 3.9 99.8 3.2
The results are expressed as percent of the mean value of the CON þ SAL group and are the mean S.E.M. of nine animals per group. One-way ANOVA analysis indicated that there were no significant differences among the groups in CRF mRNA levels in either the PVN or Barrington’s nucleus.
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increased plasma corticosterone levels have been reported to increase CRF mRNA in both the dBNST and the CeA (Makino et al., 1994). Alternatively, it is possible that, apart from acute stress, CRF mRNA in the dBNST, vBNST and CeA may have differential sensitivities to the nature of chronic stress. For example, acute stress caused a parallel increase in CRF mRNA levels in the dBNST and CeA (Makino et al., 1994), whereas chronic stress caused varying degrees of response in CRF mRNA in each area (Albeck et al., 1997; Stout et al., 2000). Finally, it has been suggested that CRF in the BNST may coordinate responding to aversive stimuli that cannot be predicted, whereas the CRF in the CeA may coordinate responding to phasic and/or predictable stressors (Walker et al., 2003). Thus according to this view, CMS, which consists of repeated, varied and unpredictable mild stressors, would be expected to increase CRF mRNA in the BNST more than in the CeA. Further studies will be needed to clarify the regional differences in CRF mRNA levels in response to CMS. 4.4. CMS effects on CRF in the CeA, PVN, and Barrington’s nucleus In the present study, we observed only a trend toward an increase of CRF mRNA expression in the CeA in CMS-exposed rats, although chronic social stress using subordinate rats in the visible burrow model was previously reported to produce increased CRF mRNA expression in the CeA (Albeck et al., 1997). Our results are similar to those observed in previous studies with chronic stress-exposed rats (Chappell et al., 1986). Furthermore, our results are consistent with the observation that the same CMS procedures did not affect amygdalar CRF content (Stout et al., 2000). We did not detect CMS-induced changes in CRF mRNA levels in the PVN or Barrington’s nucleus. It has been shown that stressors such as chronic immobilization stress (Marti et al., 1999) or social stress (Albeck et al., 1997) significantly increase CRF mNRA in the PVN and that even procedures similar to CMS increased CRF mRNA levels in the PVN (Duncko et al., 2001). On the other hand, chronic immobilization stress did not increase CRF mRNA levels in the PVN (Ma and Lightman, 1998). These results suggest a possibility that the responses of CRF mRNA in the PVN to chronic stress are positively related to the frequency and intensity of exposure to the stressor (Armario et al., 2004). Although it is not easy to adequately compare the degree of similarity between CMS procedures, it should be noted that there are a few minor procedural differences between our CMS model and that of Duncko et al. (2001). Because we did not apply stress during the weekend except for that associated with changes of circadian rhythm, the frequency of the stressor in the CMS model used by Duncko et al. (2001) was higher than that in our CMS model. Other procedural variations include differences in body weight at the start of CMS procedures (180e200 g vs. 320e 380 g) and the time of sacrifice after the last CMS procedure (5e8 h vs. ~ 72 h). It may be that any of these procedural differences may have contributed to the changes in CRF mRNA in the PVN.
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The present lack of a detectable effect of CMS upon CRF mRNA levels in the Barrington’s nucleus differed from previous findings (Imaki et al., 1991), in which acute and chronic stress with footshock significantly increased CRF mRNA levels in Barrington’s nucleus. As CRF mRNA levels were significantly increased 24 h, but not 2 h, after a single session of footshock stress, it is possible that the time between stress exposure and sacrifice may determine the extent of changes in CRF mRNA levels in Barrington’s nucleus. 4.5. Tianeptine treatment effects on CRF Tianeptine has an antidepressant efficacy similar to that of SSRIs (Wilde and Benfield, 1995), but unlike SSRIs, tianeptine enhances serotonin reuptake (Mennini et al., 1987). In addition, tianeptine can prevent CA3 pyramidal neuron atrophy (Watanabe et al., 1992) and decreases in dentate gyrus granular precursor cell proliferation (Czeh et al., 2001) induced by 3e4 weeks of repeated stress or glucocorticoid administration. These findings suggest that in addition to an antidepressant effect, tianeptine may also have a functional anti-stress effect. In the present study, repeated tianeptine treatment decreased CRF mRNA levels in the CeA and vBNST in nonstressed controls. In addition, there was a trend toward a tianeptine-induced reduction of CRF mRNA levels in the PVN and Barrington’s nucleus. These results suggest that repeated tianeptine treatment may reduce the basal activity of CRF neurons. Moreover, we found that repeated tianeptine treatment reduced CRF mRNA levels in the dBNST of both CMS-exposed and non-stressed control rats. Given that CRF in the BNST may be involved with anxiety (Davis et al., 1997; Lee and Davis, 1997), it is tempting to speculate that increased CRF mRNA in the dBNST of CMS-exposed rats may contribute to increased aversive behavioral outcomes, including anxiety, and that tianeptine treatment prior to stress may reduce CMS-related anxiety. The finding that tianeptine is effective in reducing chronic stress-induced increase in anxiety-like behavior (Pillai et al., 2004) is consistent with this possibility. Interestingly, there is evidence that tianeptine alleviates anxiety symptoms associated with depression (Wilde and Benfield, 1995). Moreover, repeated tianeptine treatment decreased CRF mRNA in the vBNST and CeA in addition to the dBNST, in both CMS-exposed and non-stressed control rats. As CRF in the amygdala has been implicated in mediating conditioned fear responses, tianeptine treatment might reduce conditioned fear behavior via its actions in the CeA. This expectation is consistent with the finding that tianeptine has anxiolytic properties after repeated administration in a model of conditioned freezing (Burghardt et al., 2004). The mechanism by which repeated tianeptine treatment affects CRF mRNA expression in the BNST and CeA is unknown. However, it has been shown that stress induces increases in norepinephrine (NE) release in the BNST (Pacak et al., 1995) and the CeA (Quirarte et al., 1998) as measured by in vivo microdialysis, and that tianeptine can block the restraint stress-induced increase in NE levels in rat frontal cortex (Sacchetti et al., 1993). In addition, NE in the PVN stimulates
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CRF release (Alonso et al., 1986) and increases CRF mRNA levels in the PVN (Itoi et al., 1994). Considering that NE release in the BNST during stress stimulates CRF release in this region, which in turn leads to increases in CRF gene transcription, it is plausible that a reduction in stress-induced NE release similar to that observed in the prefrontal cortex following tianeptine treatment could contribute to the decrease in CRF mRNA expression in associated brain areas including the BNST. However, even if this possibility is taken into consideration, the fact that repeated tianeptine treatment reduced CRF mRNA levels below those of non-stressed controls in the vBNST and CeA suggests that additional variables are involved in the regulation of CRF mRNA in response to tianeptine treatment. Moreover, although the NE transporter is present in both the dBNST and vBNST (Macey et al., 2003), NE terminals from the A1 and A2 medullary NE cell groups project predominantly to the vBNST (Aston-Jones et al., 1999). Further studies will be required to elucidate how repeated tianeptine treatment produces reductions in CRF mRNA expression in related brain areas such as the BNST and CeA. In conclusion, the present results show that CMS induces increases in CRF mRNA levels in the dBNST. Furthermore, it was demonstrated that this increase is attenuated by repeated tianeptine treatment. Repeated tianeptine treatment significantly decreased CRF mRNA levels in the vBNST and CeA, in both CMS-exposed rats and non-stressed controls. These results suggest that repeated tianeptine treatment diminishes the basal activity of CRF neurons and reduces their sensitivity to stress. Acknowledgements This research was supported by a grant from the Brain Research Center of the 21st Century Frontier Research Program, funded by the Korean Ministry of Science and Technology (M103KV010007-03K2201-00720), and Korea Health 21 R&D Project (KPGRN-R-04-04) by the Ministry of Health and Welfare, Republic of Korea. We are grateful to professor Kelly Mayo for his kind gift of rat CRF cDNA. References Albeck, D.S., McKittrick, C.R., Blanchard, D.C., Blanchard, R.J., Nikulina, J., McEwen, B.S., Sakai, R.R., 1997. Chronic social stress alters levels of corticotropin-releasing factor and arginine vasopressin mRNA in rat brain. J. Neurosci. 17, 4895e4903. Alheid, G.F., de Lomos, J.S., Beltramino, C.A., 1995. Amygdala and extended amygdala. In: Paxinos, G. (Ed.), The Rat Nervous System. Academic Press, San Diego, pp. 495e578. Alonso, G., Szafarczyk, A., Balmefrezol, M., Assenmacher, I., 1986. Immunocytochemical evidence for stimulatory control by the ventral noradrenergic bundle of parvocellular neurons of the paraventricular nucleus secreting corticotropin releasing hormone and vasopressin in rats. Brain Res. 397, 297e307. Amir, S., Lamont, E.W., Robinson, B., Stewart, J., 2004. A circadian rhythm in the expression of PERIOD2 protein reveals a novel SCN-controlled oscillator in the oval nucleus of the bed nucleus of the stria terminalis. J. Neurosci. 24, 781e790.
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