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Tianeptine treatment induces regionally specific changes in monoamines
BRAIN RESEARCH ELSEVIER
Brain Research 696 (1995) 1-6
Research report
Tianeptine treatment induces regionally specific changes in monoamines Maya Frankfurt a, *, Christina R. McKittrick a, Bruce S. M c E w e n a, Victoria N. Luine b a
Laboratory of Neuroendocrinology, The Rockefeller University, 1230 YorkAve., Box 165, New York, NY 10021, USA b Department of Psychology, Hunter College, 695 Park Ace., New York, NY 10021, USA Accepted 9 May 1995
Abstract
Tianeptine is an atypical tricyclic antidepressant that facilitates serotonin (5-HT) reuptake. Tianeptine (10 mg/kg) or saline was administered intraperitoneally to male rats daily for 4 days. Monoamine levels were measured in micropunches of discrete brain nuclei that are implicated in mood and cognition. In addition, the rates of 5-HT and norepinephrine (NE) accumulation were determined by the pargyline method. Few changes were noted in the 5-HT system. 5-HT levels were increased by short-term tianeptine in the CA3 region of hippocampus, and 5-hydroxyindoleacetic acid (5-HIAA) was increased in the ventromedial nucleus of hypothalamus, while 5-HT turnover was decreased in preoptic area (POA). In addition, short-term tianeptine treatment increased NE levels in POA, parietal sensory cortex (SCTX) and dorsal raphe (DR), and decreased NE in dentate gyrus. NE turnover was also decreased in DR, SCTX and parietal motor cortex. These data suggest that the short-term neural and behavioral actions of tianeptine may be attributable, in part, to alterations of the norepinephrine system. Keywords: Serotonin; 5-Hydroxytryptamine; Norepinephrine; Tianeptine; Antidepressant
I. Introduction
Tianeptine, an atypical tricyclic compound with a novel mechanism of action, is a clinically active antidepressant [8,21]. Whereas classic tricyclic antidepressants and serotonin-selective uptake inhibitors block serotonin (5-hydroxytryptamine, 5-HT) reuptake, tianeptine has been shown to enhance brain 5-HT uptake [14,35]. Decreases in 5-HT-dependent behaviors following tianeptine administration suggest that 5-HT uptake is enhanced in vivo as well [10,28,49] Tianeptine also facilitates 5-HT uptake in rat and human platelets [8,36,37]. With some exceptions [4,23], tianeptine's effects have been reported to be limited to the 5-HT system [35]. Various brain regions appear to be differentially affected by tianeptine. In homogenates of large brain regions, acute tianeptine treatment was shown to increase levels of 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of 5-HT, in brain stem, striatum and cerebral cortex, but not in hippocampus [14]. In contrast, 5-HIAA levels measured by in vivo voltammetry were increased by acute tianeptine in hippocampus and hypothalamus, but not
* Corresponding author. Fax: (1) (212) 327-8634 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 6 6 3 - X
nucleus accumbens [11]. Finally, increased 5-HT uptake was observed in synaptosomes from cortex and hippocampus, but not from brainstem, of rats receiving both acute and repeated administration of tianeptine [35]. The pharmacological profile of tianeptine has called into question traditional theories concerning the mechanism of action of antidepressant drugs, which suggest that the therapeutic actions of antidepressants result from their ability to increase 5-HT (and/or NE) availability in the brain. The present study was designed to examine possible mechanisms by which tianeptine exerts its initial therapeutic effects, by identifying precise targets of tianeptine action. Monoamine content and turnover was measured in micropunches of discrete brain areas of rats receiving 4-day tianeptine treatment. Because, in addition to its antidepressant effects, tianeptine has been shown to enhance attention and learning in animals [12,25], we examined monoamine metabolism in brain regions known to be involved in learning and memory as well as affective state. In a recent study performed in our laboratory, the same parameters were measured following repeated administration of fluoxetine, a 5-HT uptake inhibitor [15]. We hypothesized that fluoxetine and tianeptine might induce some similar biochemical changes, thus providing a mechanistic basis for their common antidepressant effects.
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M. Frankfurt et al./Brain Research 696 (1995) 1-6
2. Materials and methods Male rats ( 2 2 5 - 2 5 0 g) were housed under controlled light (14 h l i g h t / 1 0 h dark) conditions with food and water available ad libitum. All animal procedures were approved by the Rockefeller University A n i m a l Care Committee and were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Animals received intraperitoneal (i.p.) injections of 10 m g / k g tianeptine (in saline, Servier, Paris) or saline for 4 consecutive days. 1 h after the last injection, one half of each treatment group received 75 m g / k g pargyline (Sigma) and the other half received saline 15 min prior to sacrifice by decapitation. Brains were quickly removed and frozen on dry ice. Brains were sectioned ( 3 0 0 / . t m ) on a cryostat ( - 10°C) and thaw-mounted onto glass slides. Brain nuclei were dissected using a 500 ~ m diameter needle, according to method of Pallkovits and Brownstein [38]. Monoamine levels were measured by high performance liquid chromatography (HPLC) as described below. Areas examined were preoptic area (POA), ventromedial hypothalamic nucleus (VMN), CA1 and CA3 of hippocampus, dentate gyrus (DG), parietal motor cortex (MCTX), parietal sensory cortex (SCTX), frontal cortex (FCTX), and dorsal raphe (DR). 2.1. M o n o a m i n e determination
Monoamine levels were measured by HPLC with electrochemical detection as previously described [43] using an E S A m o d e l 5011 detector. The screening electrode was set ai: + 0 . 0 5 V and the detecting electrode at + 0 . 3 5 V. Briefly, punched samples were expelled into 60 /zl of 0.2
M sodium acetate buffer (pH 5) containing 10 -7 M a methyl dopamine as an internal standard and freeze-thawed to release monoamines and metabolites. Sodium acetate buffer and freeze-thawing have been shown to effectively rupture cell membranes and release monoamines without causing the degradation of 5-HT and 5 - H I A A observed with perchloric acid extraction [43]. Samples were centrifuged, and 2 /zl of 1 m g / m l ascorbate oxidase was added to 40 /xl of the supernatant to reduce the solvent front. 40 /xl of sample was injected into a Waters Associates (Milford, M A ) chromatographic system consisting of a W I S P automated injector, a 590 pump and a C18 reverse-phase microBondapak column. Concentrations of compounds were calculated by reference to standards using peak integration with the computer-assisted Waters M a x i m a 820 system. The sample pellet was dissolved in 100 /xl of 0.2 N NaOH for protein measurement [5]. Pargyline irreversibly inhibits monoamine oxidase (MAO), preventing the degradation of both 5-HT and NE; the accumulation of 5-HT [27] has been shown to be linear from 15 to 30 min after pargyline administration in microdissected tissue samples. Application of this method for analysis of NE turnover is subject to limitations in interpretation since NE is also catabolized by catechol-O-methyl transferase (COMT) and exerts negative feedback inhibition on its own synthesis; however, values obtained for NE turnover 15 min after pargyline administration are within the standard error of turnover rates reported for the a methyl-paratyrosine method of NE turnover determination [15,31,43]. 2.2. D a t a analysis
S t e a d y . s t a t e levels of 5-HT, NE and 5 - H I A A were expressed as p g / / ~ g protein and statistical differences
Table 1 Tianeptine effects on serotonin levels and turnover Area i
Treatment
5-HIAA Level (pg//zg protein)
5-HT Level (pg//xg protein)
5-HT Accumulation (pg//xg protein/h)
POA
Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine
0.65 + 0.20 0.76 + 0.17 2.13 +_0.35 7.01 + 1.50 * 0.12 + 0.01 0.11 + 0.01 0.17 + 0.03 0.15 + 0.02 0.22 + 0.03 0.22 + 0.02 0.75 + 0.05 0.63 + 0.04 0.19 + 0.02 0.19 + 0.02 3.25 + 1.60 1.60 + 0.50 2.29 + 0.07 3.92 + 1.00
3.04 + 0.31 3.90 + 0.38 2.84 + 0.30 2.59 + 0.23 1.32 + 0.08 1.30 + 0.08 1.73 + 0.11 2.34 + 0.18 * 0.91 + 0.17 0.72 + 0.16 5.04 + 0.70 3.74 + 0.40 2.29 -t- 0.20 1.90 + 0.15 1.43 + 0.22 1.08 + 0.20 10.95 -1-0.60 11.10 + 0.60
10.2 5:1.9 4.6 _+ 1.9 * 12.6 + 2.1 12.9 + 2.7 6.1 + 0.6 7.0 + 0.7 7.9 + 1.0 7.8 + 1.2 4.5 + 0.8 6.2 + 1.1 99.0 + 9.5 123.5 + 12.6 11.5 + 1.3 12.0 + 2.1 35.0 5= 5.0 47.4 _ 8.9 242.0 5:27.2 192.0 + 29.5
VMN CA1 CA3 DG MCTX SCTX FCTX DR
* Significant (Student's t-test).
M. Frankfurt et al. /Brain Research 696 (1995) 1-6 Table 2 Tianeptine effects on norepinephrine levels and turnover Area
Treatment
NE Level ( p g / / ~ g protein)
NE Accumulation ( p g / / ~ g protein/h)
POA
Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine
10.50+0.60 16.60 + 1.60 14.18 + 2.40 15.78 + 1.80 2.09 + 0.20 2.19+0.30 3.77 -/-0.30 5.05 + 0.70 3.74 + 0.50 2.42 + 0.30 8.28 + 1.50 9.43 + 1.00 1.38+0.10 2.05+0.20 1.23+0.23 1.70+0.20 12.21 + 0.70 16.45 + 1.60
13.6+ 4.7 N.A. 16.0 + 13.0 28.7 + 13.0 0.8 _+ 1.3 1.2+ 1.5 1.8 + 1.6 N.A. N.A. 8.0___ 2.4 39.0 _+ 4.5 21.0_+ 5.8 7.5+ 1.0 4.3_+ 1.2 12.1+ 2.5 14.4+ 3.1 48.6 + 10.0 22.8 + 8.8
VMN CA1 CA3 DG MCTX SCTX FCTX DR
*
*
*
*
* Significant (Student's t-test). N.A., no accumulation following pargyline.
determined with Student's t-test. 5-HT and NE accumulation rates (expressed as pg//xg protein/h) were calculated using least squares analysis to determine the slope of the line generated by amine concentrations at 0 and 15 min after pargyline. Statistical differences in accumulation rates were determined by a t-test modified for the comparison of slopes [53].
3. Results 3.1. 5 - H T levels and turnover
Short-term tianeptine (TIA) treatment had very few effects on 5-HT content and turnover in the regions examined (Table 1). Compared to saline (SAL), 5-HT levels were significantly altered only in CA3 of hippocampus where they were increased by 35%. 5-HIAA, the major metabolite of 5-HT, was increased by 229% in the VMN. As determined by the pargyline method, 5-HT turnover was decreased by 55% in the POA. 3.2. N E levels and turnover
NE metabolism was altered by the tianeptine treatment in several brain regions (Table 2). Levels of NE in DG were decreased by 35%. In addition, NE levels were significantly increased by 58% in POA, by 49% in SCTX and by 35% in DR. Concurrent with the increase in levels, NE accumulation was decreased by 43% in SCTX and by 53% in DR; NE accumulation was decreased by 46% in MCTX as well. The turnover rate in the POA and CA3 of the tianeptine-treated animals could not be calculated,
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since there was no NE accumulation following pargyline administration. NE did accumulate in SAL-treated animals, however, suggesting that tianeptine decreased NE turnover in the POA and CA3.
4. Discussion
Abnormalities in serotonergic and noradrenergic transmitter a n d / o r receptor systems have been the focus of theories on the etiology of depression for almost 30 years [7,9,45]. The therapeutic actions of the tricyclic antidepressants are believed to result from the inhibition of both 5-HT and NE reuptake, while the clinical efficacy of 5-HT selective reuptake inhibitors (SSRIs) such as fluoxetine, have strengthened the case for serotonergic hypotheses of depression [34]. The atypical tricyclic compound, tianeptine, provides a pharmacological paradox because it is a clinically active antidepressant even though it facilitates 5-HT reuptake. The present study, which examined the initial effects of tianeptine treatment on monoamine levels and turnover in specific nuclei of limbic regions of the brain, is part of an ongoing investigation into the neurochemical effects of both short- and long-term antidepressant treatment in order to gain insight into the mechanism of action of these drugs [15,32]. Limbic areas, such as the hippocampus and hypothalamus, are implicated in emotional states and other processes altered in affective disorders, such as sleep, ingestive behaviors and sexual behavior; they also receive heavy 5-HT innervation from the median and dorsal raphe nuclei [24], and are thus likely targets for tianeptine action. Tianeptine. treatment for 4 days had relatively few effects on steady-state levels of 5-HT and its principal metabolite, 5-HIAA. The only change in 5-HT itself was in CA3 of hippocampus, where tianeptine treatment increased 5-HT levels. This relative lack of effects on 5-HT is not surprising, as basal 5-HT levels have been reported to be unchanged by tianeptine in vivo [10,14,36,49] Tianeptine has been shown, however, to attenuate the increase in 5-HT release in frontal cortex following administration of the 5-HT precursor, 5-hydroxytryptophan [10] and in hippocampus following stimulation with potassium ion [48,49]. In this study, 5-HT turnover was also assessed "by comparing the rate of 5-HT accumulation in the presence and absence of the MAO inhibitor, pargyline. Tianeptine significantly decreased 5-HT turnover only in the POA. The increased 5-HIAA levels observed in the VMN after tianeptine treatment may contribute to the increase in extracellular 5-HIAA observed in hypothalamus by De Simoni et al. [11]. Other reports of increases in 5-HIAA in large brain regions are inconsistent, as De Simoni et al. [11] reported increases in hippocampus using microdialysis, while Fattacini et al. [14] measured tissue content of 5-HIAA, and found no changes in hippocampus but did
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M. Frankfurt et al./Brain Research 696 (1995) 1-6
find increases in whole brain stem, striatum and cerebral cortex. It is difficult to directly compare these results with those reported herein, however, due to differences in the size of regions examined and the experimental techniques used. Short-term tianeptine induced several changes in the NE system. Levels of NE were significantly increased in POA, DR and in SCTX. NE accumulation following MAO inhibition was also decreased in DR and SCTX, as well as in MCTX, whereas there was no measurable accumulation of NE in POA and CA3 following tianeptine treatment. While estimation of NE turnover using pargyline is subject to some limitations in interpretation (see Section 2), application of this method has yielded values similar to those reported for these areas using other methods of analysis [31,44]. As the micropunch technique measures monoamines in both the extracellular space and in the nerve terminal, the decreased NE accumulation, combined with increased levels, suggests that NE is not being released into the synapse, where it would be subject to degradation by COMT and MAO. Rather, it appears that NE may be stored in the terminals and not released. This reduction in noradrenergic neuronal activity is supported by the observation that systemic tianeptine decreases the firing rate of locus coeruleus neurons [13]. In contrast, tianeptine reduced NE levels in dentate gyrus. There was detectable accumulation in the tianeptine group but not in the saline-treated group, suggesting that, in the dentate, there is an enhancement of NE release (with subsequent degradation) and synthesis. We recently completed a similar study of the effects of short-term fluoxetine treatment on monoamines, which has revealed several interesting similarities and differences between it and tianeptine [15]. In the VMN, fluoxetine decreased 5-HIAA levels whereas tianeptine increased them, a result that is consistent with their opposing actions on 5-HT reuptake. There were no other regions where both fluoxetine and tianeptine altered the 5-HT system. Both drugs had similar effects on NE in several areas, however. NE accumulation was decreased in MCTX and SCTX by both tianeptine and fluoxetine. Although the decrease in NE turnover in the DR was only significant following tianeptine treatment, both drugs increased NE levels in this region. The NE changes in the DR are particularly important because the majority of 5-HT cell bodies lie in the raphe nuclei; the DR nucleus specifically sends ascending projections to basal ganglia-motor systems, amygdala, hypothalamus and cerebral cortex [24]. NE-mediated influences on 5-HT neuronal activity in DR could therefore result in altered 5-HT transmission throughout the brain and lead to the similar therapeutic effects seen with administration of these drugs. The 5-HT and NE systems are closely linked anatomically and biochemically. Light and electron microscopic studies have shown heavy innervation of the raphe nuclei by noradrenergic afferents [3,30]. Pharmacological studies
have also demonstrated a-adrenoceptor mediated modulation of 5-HT neuronal activity and metabolism: NE has been shown to increase firing activity in dorsal raphe neurons via oq-adrenoceptor activation [1,2,47,51], while a2-adrenoceptor activation inhibits 5-HT synthesis in DR and hippocampus [52] and 5-HT release in DR [16], hippocampus [17,18,46] and cortex [22,40-42]. Therefore, the increased 5-HT levels observed in CA3 of hippocampus following tianeptine treatment may result from disinhibition of 5-HT neuronal activity by NE. Similarly, 5-HT afferents have been shown to project to NE cell bodies in the locus coeruleus [29,39], where they inhibit NE synthesis and release [6,33]. Both NE and 5-HT can also alter adrenoceptor mediated responses in terminal fields [20,26]. Therefore, regionally selective alterations in 5-HT metabolism induced by fluoxetine and tianeptine could, via different anatomical pathways, lead to similar changes in NE activity. In conclusion, the results of this study show that shortterm treatment with the atypical antidepressant, tianeptine, leads to several anatomically specific changes in steadystate levels and turnover rates of both 5-HT and NE. The changes in NE levels and accumulation are similar in direction and anatomical localization to those induced by the 5-HT uptake inhibitor, fluoxetine. This is particularly interesting given that tianeptine and fluoxetine have been shown to be selective for 5-HT reuptake, with no direct effect on NE reuptake [19,37,50]. Thus, it can be speculated that, following the initial (opposite) actions on 5-HT uptake, subsequent neurochemical events may occur that lead to similar changes in NE neurotransmission, providing a point of convergence for tianeptine and fluoxetine that may play an important role in mediating the common therapeutic effects of the two drugs. Preliminary results from our long-term study comparing fluoxetine and tianeptine demonstrate alterations in NE metabolism in several brain regions and therefore support our present findings [32]. Whether the alterations in NE metabolism contribute to beneficial changes in behavior and affect after long-term treatment with these drugs remains to be determined.
Acknowledgements The authors would like to thank Caroline Logan for excellent technical assistance, and Servier, France, for generously providing the tianeptine. This work was supported by GM07524-18 (C.R.M.), HD12011 (V.N.L.) and Servier.
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[47] Trulson, M.E. and Crisp, T., Role of norepinephrine in regulating the activity of serotonin-containing dorsal raphe neurons, Life Sci., 35 (1984) 551-515. [48] Whitton, P.S., Sarna, G.S., Datla, K.P. and Curzon, G., Effects of tianeptine on stress-induced behavioural deficits and 5-HT dependent behaviour, Psychopharmacology, 104 (1991) 81-85. [49] Whitton, P.S., Sarna, G.S., O'Connell, M.T. and Curzon, G., The effect of the novel antidepressant tianeptine on the concentration of 5-hydroxytryptamine in rat hippocampal dialysates in vivo, Neuropharmacology, 30 (1991) 1-4. [50] Wong, D.T., Bymaster, F.P., Horng, J.S. and Molloy, B.B., A new selective inhibitor for uptake of serotonin into synaptosomes of rat brain: 3-(p-trifluoromethylphenoxy)-N-methyl-3-phenylpropylamine (Lilly 110140), J. Pharmacol. Exp. Ther., 193 (1975) 804-811. [51] Yoshimura, M., Higashi, H. and Nishi, S., Noradrenaline mediates slow excitatory synaptic potentials in rat dorsal raphe neurons in vitro, Neurosci. Lett., 61 (1985) 305-310. [52] Yoshioka, M., Matsumoto, M., Togashi, H. Smith, C.B. and Saito, H., a2-adrenoceptor modulation of 5-HT biosynthesis in the rat brain, Neurosci. Lett., 139 (1992) 53-56. [53] Zar, J.H., Biostatistical Analysis, Prentice Hall, Englewood Cliffs, NJ, 1990.
Brain Research 696 (1995) 1-6
Research report
Tianeptine treatment induces regionally specific changes in monoamines Maya Frankfurt a, *, Christina R. McKittrick a, Bruce S. M c E w e n a, Victoria N. Luine b a
Laboratory of Neuroendocrinology, The Rockefeller University, 1230 YorkAve., Box 165, New York, NY 10021, USA b Department of Psychology, Hunter College, 695 Park Ace., New York, NY 10021, USA Accepted 9 May 1995
Abstract
Tianeptine is an atypical tricyclic antidepressant that facilitates serotonin (5-HT) reuptake. Tianeptine (10 mg/kg) or saline was administered intraperitoneally to male rats daily for 4 days. Monoamine levels were measured in micropunches of discrete brain nuclei that are implicated in mood and cognition. In addition, the rates of 5-HT and norepinephrine (NE) accumulation were determined by the pargyline method. Few changes were noted in the 5-HT system. 5-HT levels were increased by short-term tianeptine in the CA3 region of hippocampus, and 5-hydroxyindoleacetic acid (5-HIAA) was increased in the ventromedial nucleus of hypothalamus, while 5-HT turnover was decreased in preoptic area (POA). In addition, short-term tianeptine treatment increased NE levels in POA, parietal sensory cortex (SCTX) and dorsal raphe (DR), and decreased NE in dentate gyrus. NE turnover was also decreased in DR, SCTX and parietal motor cortex. These data suggest that the short-term neural and behavioral actions of tianeptine may be attributable, in part, to alterations of the norepinephrine system. Keywords: Serotonin; 5-Hydroxytryptamine; Norepinephrine; Tianeptine; Antidepressant
I. Introduction
Tianeptine, an atypical tricyclic compound with a novel mechanism of action, is a clinically active antidepressant [8,21]. Whereas classic tricyclic antidepressants and serotonin-selective uptake inhibitors block serotonin (5-hydroxytryptamine, 5-HT) reuptake, tianeptine has been shown to enhance brain 5-HT uptake [14,35]. Decreases in 5-HT-dependent behaviors following tianeptine administration suggest that 5-HT uptake is enhanced in vivo as well [10,28,49] Tianeptine also facilitates 5-HT uptake in rat and human platelets [8,36,37]. With some exceptions [4,23], tianeptine's effects have been reported to be limited to the 5-HT system [35]. Various brain regions appear to be differentially affected by tianeptine. In homogenates of large brain regions, acute tianeptine treatment was shown to increase levels of 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of 5-HT, in brain stem, striatum and cerebral cortex, but not in hippocampus [14]. In contrast, 5-HIAA levels measured by in vivo voltammetry were increased by acute tianeptine in hippocampus and hypothalamus, but not
* Corresponding author. Fax: (1) (212) 327-8634 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 6 6 3 - X
nucleus accumbens [11]. Finally, increased 5-HT uptake was observed in synaptosomes from cortex and hippocampus, but not from brainstem, of rats receiving both acute and repeated administration of tianeptine [35]. The pharmacological profile of tianeptine has called into question traditional theories concerning the mechanism of action of antidepressant drugs, which suggest that the therapeutic actions of antidepressants result from their ability to increase 5-HT (and/or NE) availability in the brain. The present study was designed to examine possible mechanisms by which tianeptine exerts its initial therapeutic effects, by identifying precise targets of tianeptine action. Monoamine content and turnover was measured in micropunches of discrete brain areas of rats receiving 4-day tianeptine treatment. Because, in addition to its antidepressant effects, tianeptine has been shown to enhance attention and learning in animals [12,25], we examined monoamine metabolism in brain regions known to be involved in learning and memory as well as affective state. In a recent study performed in our laboratory, the same parameters were measured following repeated administration of fluoxetine, a 5-HT uptake inhibitor [15]. We hypothesized that fluoxetine and tianeptine might induce some similar biochemical changes, thus providing a mechanistic basis for their common antidepressant effects.
2
M. Frankfurt et al./Brain Research 696 (1995) 1-6
2. Materials and methods Male rats ( 2 2 5 - 2 5 0 g) were housed under controlled light (14 h l i g h t / 1 0 h dark) conditions with food and water available ad libitum. All animal procedures were approved by the Rockefeller University A n i m a l Care Committee and were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Animals received intraperitoneal (i.p.) injections of 10 m g / k g tianeptine (in saline, Servier, Paris) or saline for 4 consecutive days. 1 h after the last injection, one half of each treatment group received 75 m g / k g pargyline (Sigma) and the other half received saline 15 min prior to sacrifice by decapitation. Brains were quickly removed and frozen on dry ice. Brains were sectioned ( 3 0 0 / . t m ) on a cryostat ( - 10°C) and thaw-mounted onto glass slides. Brain nuclei were dissected using a 500 ~ m diameter needle, according to method of Pallkovits and Brownstein [38]. Monoamine levels were measured by high performance liquid chromatography (HPLC) as described below. Areas examined were preoptic area (POA), ventromedial hypothalamic nucleus (VMN), CA1 and CA3 of hippocampus, dentate gyrus (DG), parietal motor cortex (MCTX), parietal sensory cortex (SCTX), frontal cortex (FCTX), and dorsal raphe (DR). 2.1. M o n o a m i n e determination
Monoamine levels were measured by HPLC with electrochemical detection as previously described [43] using an E S A m o d e l 5011 detector. The screening electrode was set ai: + 0 . 0 5 V and the detecting electrode at + 0 . 3 5 V. Briefly, punched samples were expelled into 60 /zl of 0.2
M sodium acetate buffer (pH 5) containing 10 -7 M a methyl dopamine as an internal standard and freeze-thawed to release monoamines and metabolites. Sodium acetate buffer and freeze-thawing have been shown to effectively rupture cell membranes and release monoamines without causing the degradation of 5-HT and 5 - H I A A observed with perchloric acid extraction [43]. Samples were centrifuged, and 2 /zl of 1 m g / m l ascorbate oxidase was added to 40 /xl of the supernatant to reduce the solvent front. 40 /xl of sample was injected into a Waters Associates (Milford, M A ) chromatographic system consisting of a W I S P automated injector, a 590 pump and a C18 reverse-phase microBondapak column. Concentrations of compounds were calculated by reference to standards using peak integration with the computer-assisted Waters M a x i m a 820 system. The sample pellet was dissolved in 100 /xl of 0.2 N NaOH for protein measurement [5]. Pargyline irreversibly inhibits monoamine oxidase (MAO), preventing the degradation of both 5-HT and NE; the accumulation of 5-HT [27] has been shown to be linear from 15 to 30 min after pargyline administration in microdissected tissue samples. Application of this method for analysis of NE turnover is subject to limitations in interpretation since NE is also catabolized by catechol-O-methyl transferase (COMT) and exerts negative feedback inhibition on its own synthesis; however, values obtained for NE turnover 15 min after pargyline administration are within the standard error of turnover rates reported for the a methyl-paratyrosine method of NE turnover determination [15,31,43]. 2.2. D a t a analysis
S t e a d y . s t a t e levels of 5-HT, NE and 5 - H I A A were expressed as p g / / ~ g protein and statistical differences
Table 1 Tianeptine effects on serotonin levels and turnover Area i
Treatment
5-HIAA Level (pg//zg protein)
5-HT Level (pg//xg protein)
5-HT Accumulation (pg//xg protein/h)
POA
Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine
0.65 + 0.20 0.76 + 0.17 2.13 +_0.35 7.01 + 1.50 * 0.12 + 0.01 0.11 + 0.01 0.17 + 0.03 0.15 + 0.02 0.22 + 0.03 0.22 + 0.02 0.75 + 0.05 0.63 + 0.04 0.19 + 0.02 0.19 + 0.02 3.25 + 1.60 1.60 + 0.50 2.29 + 0.07 3.92 + 1.00
3.04 + 0.31 3.90 + 0.38 2.84 + 0.30 2.59 + 0.23 1.32 + 0.08 1.30 + 0.08 1.73 + 0.11 2.34 + 0.18 * 0.91 + 0.17 0.72 + 0.16 5.04 + 0.70 3.74 + 0.40 2.29 -t- 0.20 1.90 + 0.15 1.43 + 0.22 1.08 + 0.20 10.95 -1-0.60 11.10 + 0.60
10.2 5:1.9 4.6 _+ 1.9 * 12.6 + 2.1 12.9 + 2.7 6.1 + 0.6 7.0 + 0.7 7.9 + 1.0 7.8 + 1.2 4.5 + 0.8 6.2 + 1.1 99.0 + 9.5 123.5 + 12.6 11.5 + 1.3 12.0 + 2.1 35.0 5= 5.0 47.4 _ 8.9 242.0 5:27.2 192.0 + 29.5
VMN CA1 CA3 DG MCTX SCTX FCTX DR
* Significant (Student's t-test).
M. Frankfurt et al. /Brain Research 696 (1995) 1-6 Table 2 Tianeptine effects on norepinephrine levels and turnover Area
Treatment
NE Level ( p g / / ~ g protein)
NE Accumulation ( p g / / ~ g protein/h)
POA
Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine Saline Tianeptine
10.50+0.60 16.60 + 1.60 14.18 + 2.40 15.78 + 1.80 2.09 + 0.20 2.19+0.30 3.77 -/-0.30 5.05 + 0.70 3.74 + 0.50 2.42 + 0.30 8.28 + 1.50 9.43 + 1.00 1.38+0.10 2.05+0.20 1.23+0.23 1.70+0.20 12.21 + 0.70 16.45 + 1.60
13.6+ 4.7 N.A. 16.0 + 13.0 28.7 + 13.0 0.8 _+ 1.3 1.2+ 1.5 1.8 + 1.6 N.A. N.A. 8.0___ 2.4 39.0 _+ 4.5 21.0_+ 5.8 7.5+ 1.0 4.3_+ 1.2 12.1+ 2.5 14.4+ 3.1 48.6 + 10.0 22.8 + 8.8
VMN CA1 CA3 DG MCTX SCTX FCTX DR
*
*
*
*
* Significant (Student's t-test). N.A., no accumulation following pargyline.
determined with Student's t-test. 5-HT and NE accumulation rates (expressed as pg//xg protein/h) were calculated using least squares analysis to determine the slope of the line generated by amine concentrations at 0 and 15 min after pargyline. Statistical differences in accumulation rates were determined by a t-test modified for the comparison of slopes [53].
3. Results 3.1. 5 - H T levels and turnover
Short-term tianeptine (TIA) treatment had very few effects on 5-HT content and turnover in the regions examined (Table 1). Compared to saline (SAL), 5-HT levels were significantly altered only in CA3 of hippocampus where they were increased by 35%. 5-HIAA, the major metabolite of 5-HT, was increased by 229% in the VMN. As determined by the pargyline method, 5-HT turnover was decreased by 55% in the POA. 3.2. N E levels and turnover
NE metabolism was altered by the tianeptine treatment in several brain regions (Table 2). Levels of NE in DG were decreased by 35%. In addition, NE levels were significantly increased by 58% in POA, by 49% in SCTX and by 35% in DR. Concurrent with the increase in levels, NE accumulation was decreased by 43% in SCTX and by 53% in DR; NE accumulation was decreased by 46% in MCTX as well. The turnover rate in the POA and CA3 of the tianeptine-treated animals could not be calculated,
3
since there was no NE accumulation following pargyline administration. NE did accumulate in SAL-treated animals, however, suggesting that tianeptine decreased NE turnover in the POA and CA3.
4. Discussion
Abnormalities in serotonergic and noradrenergic transmitter a n d / o r receptor systems have been the focus of theories on the etiology of depression for almost 30 years [7,9,45]. The therapeutic actions of the tricyclic antidepressants are believed to result from the inhibition of both 5-HT and NE reuptake, while the clinical efficacy of 5-HT selective reuptake inhibitors (SSRIs) such as fluoxetine, have strengthened the case for serotonergic hypotheses of depression [34]. The atypical tricyclic compound, tianeptine, provides a pharmacological paradox because it is a clinically active antidepressant even though it facilitates 5-HT reuptake. The present study, which examined the initial effects of tianeptine treatment on monoamine levels and turnover in specific nuclei of limbic regions of the brain, is part of an ongoing investigation into the neurochemical effects of both short- and long-term antidepressant treatment in order to gain insight into the mechanism of action of these drugs [15,32]. Limbic areas, such as the hippocampus and hypothalamus, are implicated in emotional states and other processes altered in affective disorders, such as sleep, ingestive behaviors and sexual behavior; they also receive heavy 5-HT innervation from the median and dorsal raphe nuclei [24], and are thus likely targets for tianeptine action. Tianeptine. treatment for 4 days had relatively few effects on steady-state levels of 5-HT and its principal metabolite, 5-HIAA. The only change in 5-HT itself was in CA3 of hippocampus, where tianeptine treatment increased 5-HT levels. This relative lack of effects on 5-HT is not surprising, as basal 5-HT levels have been reported to be unchanged by tianeptine in vivo [10,14,36,49] Tianeptine has been shown, however, to attenuate the increase in 5-HT release in frontal cortex following administration of the 5-HT precursor, 5-hydroxytryptophan [10] and in hippocampus following stimulation with potassium ion [48,49]. In this study, 5-HT turnover was also assessed "by comparing the rate of 5-HT accumulation in the presence and absence of the MAO inhibitor, pargyline. Tianeptine significantly decreased 5-HT turnover only in the POA. The increased 5-HIAA levels observed in the VMN after tianeptine treatment may contribute to the increase in extracellular 5-HIAA observed in hypothalamus by De Simoni et al. [11]. Other reports of increases in 5-HIAA in large brain regions are inconsistent, as De Simoni et al. [11] reported increases in hippocampus using microdialysis, while Fattacini et al. [14] measured tissue content of 5-HIAA, and found no changes in hippocampus but did
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M. Frankfurt et al./Brain Research 696 (1995) 1-6
find increases in whole brain stem, striatum and cerebral cortex. It is difficult to directly compare these results with those reported herein, however, due to differences in the size of regions examined and the experimental techniques used. Short-term tianeptine induced several changes in the NE system. Levels of NE were significantly increased in POA, DR and in SCTX. NE accumulation following MAO inhibition was also decreased in DR and SCTX, as well as in MCTX, whereas there was no measurable accumulation of NE in POA and CA3 following tianeptine treatment. While estimation of NE turnover using pargyline is subject to some limitations in interpretation (see Section 2), application of this method has yielded values similar to those reported for these areas using other methods of analysis [31,44]. As the micropunch technique measures monoamines in both the extracellular space and in the nerve terminal, the decreased NE accumulation, combined with increased levels, suggests that NE is not being released into the synapse, where it would be subject to degradation by COMT and MAO. Rather, it appears that NE may be stored in the terminals and not released. This reduction in noradrenergic neuronal activity is supported by the observation that systemic tianeptine decreases the firing rate of locus coeruleus neurons [13]. In contrast, tianeptine reduced NE levels in dentate gyrus. There was detectable accumulation in the tianeptine group but not in the saline-treated group, suggesting that, in the dentate, there is an enhancement of NE release (with subsequent degradation) and synthesis. We recently completed a similar study of the effects of short-term fluoxetine treatment on monoamines, which has revealed several interesting similarities and differences between it and tianeptine [15]. In the VMN, fluoxetine decreased 5-HIAA levels whereas tianeptine increased them, a result that is consistent with their opposing actions on 5-HT reuptake. There were no other regions where both fluoxetine and tianeptine altered the 5-HT system. Both drugs had similar effects on NE in several areas, however. NE accumulation was decreased in MCTX and SCTX by both tianeptine and fluoxetine. Although the decrease in NE turnover in the DR was only significant following tianeptine treatment, both drugs increased NE levels in this region. The NE changes in the DR are particularly important because the majority of 5-HT cell bodies lie in the raphe nuclei; the DR nucleus specifically sends ascending projections to basal ganglia-motor systems, amygdala, hypothalamus and cerebral cortex [24]. NE-mediated influences on 5-HT neuronal activity in DR could therefore result in altered 5-HT transmission throughout the brain and lead to the similar therapeutic effects seen with administration of these drugs. The 5-HT and NE systems are closely linked anatomically and biochemically. Light and electron microscopic studies have shown heavy innervation of the raphe nuclei by noradrenergic afferents [3,30]. Pharmacological studies
have also demonstrated a-adrenoceptor mediated modulation of 5-HT neuronal activity and metabolism: NE has been shown to increase firing activity in dorsal raphe neurons via oq-adrenoceptor activation [1,2,47,51], while a2-adrenoceptor activation inhibits 5-HT synthesis in DR and hippocampus [52] and 5-HT release in DR [16], hippocampus [17,18,46] and cortex [22,40-42]. Therefore, the increased 5-HT levels observed in CA3 of hippocampus following tianeptine treatment may result from disinhibition of 5-HT neuronal activity by NE. Similarly, 5-HT afferents have been shown to project to NE cell bodies in the locus coeruleus [29,39], where they inhibit NE synthesis and release [6,33]. Both NE and 5-HT can also alter adrenoceptor mediated responses in terminal fields [20,26]. Therefore, regionally selective alterations in 5-HT metabolism induced by fluoxetine and tianeptine could, via different anatomical pathways, lead to similar changes in NE activity. In conclusion, the results of this study show that shortterm treatment with the atypical antidepressant, tianeptine, leads to several anatomically specific changes in steadystate levels and turnover rates of both 5-HT and NE. The changes in NE levels and accumulation are similar in direction and anatomical localization to those induced by the 5-HT uptake inhibitor, fluoxetine. This is particularly interesting given that tianeptine and fluoxetine have been shown to be selective for 5-HT reuptake, with no direct effect on NE reuptake [19,37,50]. Thus, it can be speculated that, following the initial (opposite) actions on 5-HT uptake, subsequent neurochemical events may occur that lead to similar changes in NE neurotransmission, providing a point of convergence for tianeptine and fluoxetine that may play an important role in mediating the common therapeutic effects of the two drugs. Preliminary results from our long-term study comparing fluoxetine and tianeptine demonstrate alterations in NE metabolism in several brain regions and therefore support our present findings [32]. Whether the alterations in NE metabolism contribute to beneficial changes in behavior and affect after long-term treatment with these drugs remains to be determined.
Acknowledgements The authors would like to thank Caroline Logan for excellent technical assistance, and Servier, France, for generously providing the tianeptine. This work was supported by GM07524-18 (C.R.M.), HD12011 (V.N.L.) and Servier.
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