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Tianeptine stimulates uptake of 5-hydroxytryptamine in vivo in the rat brain
Neuropharmacology Vol. 29, No. I, pp. I-8, 1990 Printed in Great Britain. All rights reserved
0028-3908190 $3.00+ 0.00 Copyright 0 1990Pergamon Press plc
TIANEPTINE STIMULATES 5HYDROXYTRYPTAMINE IN VII’0
UPTAKE IN THE
OF RAT
BRAIN
C. M. FATTACCINI, F. BOLAP;TOS-JIMENEZ, H. GOZLAN and M. HAMON* INSERM U288, Neurobiologie Cellulaire et Fonctionnelle, FacultC de Medecine Pitit-Salp&triire, 91, boulevard de l’H_lbpital,75634 Paris Cedex 13, France (Accepted 22 Augur
1989)
Summary-The neurochemical effects of the atypical tricyclic antidepressant, tianeptine, were further assessed on central serotoninergic and dopaminergic systems in the rat. Acute treatment with tianeptine (10 mg/kg i.p.) significantly enhanced the levels of metabolites of 5-HT and DA, 5-hydroxyindole acetic acid and dihydroxyphenylacetic acid respectively, in the brain stem, striatum and cerebral cortex. These effects could be prevented by the administration of drugs acting selectively (or preferentially) on serotoninergic systems such as d,l-fenfluramine and 4-methyl-a-ethyl-metatyramine (H75/12), suggesting that the increased metabolism of DA was secondary to a modification of serotoninergic systems in tianeptine-treated rats. In contrast to that found with inhibitors of the uptake of 5-HT, treatment with tianeptine markedly enhanced depletion of S-HT due to administration of H75/12. However, depletion of DA induced by H75/12, was not altered by tianeptine. In vitro measurement of the uptake of [3H]5-HT also confirmed that tianeptine exerted opposite effects to those of classical tricyclic antidepressants, since the in viuo administration of tianeptine (2 x 10 mg/kg i.p.) induced a significant increase in the uptake of [‘HIS-HT in cortical synaptosomes. The fact that both inhibitors of the uptake of 5-HT and tianeptine which, in contrast, enhanced the in viuo uptake of 5-HT, are potent antidepressants, challenges the current hypothesis on the central mechanisms of action of these drugs. Key words-tianeptine, fenfluramine, dopamine, antidepressants.
4-methyl-cc-ethyl-metatyramine
NP29,lL-A
uptake,
of tianeptine, since evidence for an increased uptake of [3H]5-HT in cortical and hippocampal but not mesencephalic synaptosomes from tianeptine-treated rats has been reported by Mennini et al. (1987). Using in uiuo voltammetry, De Simoni, De Luigi, Manfridi and Sokola (1989) recently observed an increased efflux of the 5-HT metabolite, 5-hydroxyindole acetic acid (5-HIAA), in the hippocampus of freely moving rats, treated with tianeptine. Further neuropharmacological investigations have been made in this laboratory, with particular attention to possible alterations of central serotocinergic and dopaminergic neurotransmission, due to acute treatment with tianeptine in rats. In particular, possible changes in the reuptake of 5-HT in vivo after administration of tianeptine were assessed by examining tianeptine-induced alterations in depletion of 5-HT due to administration of H75/12 (6methyl-clethyl-metatyramine) (Carlsson, Corrodi, Fuxe and HBkfelt, 1969). Previous studies have clearly demonstrated the relevance of this approach for exploring the activity of the uptake of 5-HT in ho, since its inhibition, notably by tricyclic antidepressants, is well known to reduce markedly the depletion of amines induced by releasing agents such as H75/12 (Carlsson et al., 1969; Hamon, Bourgoin, Enjalbert, Bockaert, H&y, Ternaux and Glowinski, 1976) and p-chloroamphetamine (Fuller, Snoddy, Perry, Bymaster and Wong, 1978; i)gren, Ross, Holm and Baumann, 1977). By contrast, the enhancing effect of tianeptine
Although behavioral tests in rats (Labrid, Moleyre, Poignant, Malen, Mocaer and Kamoun, 1988; Mocaer, Rettori and Kamoun, 1988) and clinical trials in humans (Defiance, Marey and Kamoun, 1988; Galinowski, OliC, Guelfi, Malka, Dulcire, Kamoun and L6o, 1988; Olit, Guelfi, Malka, Dulcire, Kamoun and LBo, 1988; LI?IOand Deniker, 1988; Casacchia, Sconci, Vespucci and Brancato, 1989) have clearly shown that tianeptine ([chloro3-methyl-6-dioxo-5,5-dihydro-6,ll-dibenzo (c,f) (tiazepine-1,2)yl- 1 l)amino]-7-heptanoic acid, sodium salt) is a tricyclic antidepressant, this drug differs markedly from other tricyclics, such as imipramine and desipramine, since it inhibits the reuptake of neither serotonin (5_hydroxytryptamine, 5-HT) nor noradrenaline (NA) in the central nervous system (CNS) (Mennini, Mocaer and Garattini, 1987; Kato and Weitsch, 1988; Mocaer et al., 1988). Previous neuropharmacological investigations indicated that tianeptine does not bind to any of the subtypes of 5-HT receptors (El Mestikawy, Gozlan, MCnard, Bourgoin and Hamon, 1988; Hamon, Bourgoin and Gozlan, 1989) and does not affect the flow of nerve impulses within serotoninergic neurones of the dorsal raphe nucleus (Dresse and Scuvee-Moreau, 1988). Nevertheless, serotoninergic neurotransmission is probably involved in the central mechanism of action
*To whom all correspondence
(H75/12), serotonin
should be addressed.
I
C. M. FATTACCNI et rd.
2
on H75,‘12-induced depletion of 5-HT and the other biochemical data reported here further supports the idea that tianeptine stimulates the uptake of 5-HT in r+o in the brain of the rat. METHODS
Chemicals [3H]5-HT ([3H]5-Hydroxytryptamine creatinine sulphate generally labelled, 12 Ci/mmol., Amersham International) was purified by ion-exchange chromatography (Hamon, Bourgoin, Jagger and Glowinski, 1974) just before being used for the uptake experiments (see below). Other drugs were: d,l-fenfluramine and tianeptine (Servier), H75/12 (Astra Labkemi AB), pargyline (Abbott), chlorimipramine (Ciba-Geigy). Animals
and treatments
Adult male SpragueeDawley rats (Charles River strain), weighing 250-300 g, were kept in a controlled environment: 24 + 1°C 60% humidity, 12 hr/l2 hr alternate light/dark cycles, with food and water ad libitum, for at least 7 days before being used for the experiments. All drugs were dissolved in saline and injected intraperitoneally in a volume of 1 ml/rat at IO-1 1 a.m. Control rats received saline by the same route of administration and following the same time conditions as for the drugs. All animals were killed by decapitation and the dissection of the brain proceeded at &4’C according to Glowinski and Iversen (1966). Measurements ?f‘SHT, 5-HIAA, dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) Each dissected structure (brain stem, hippocampus, striatum, cerebral cortex) was homogenized at 04 C with a Branson sonifier in 0.5-l .O ml of 0.1 M HCIO,, supplemented with 0.5% NazS,O, and 0.5% disodium ethylene diamine tetra acetic acid (EDTA). Homogenates were centrifuged at 30,000 g for 15 min at 4°C and the supernatants were neutralized using 2 M KH,PO,/K,HPO,, pH 7.6. After a second centrifugation as before. ascorbate oxidase (Boehringer-Mannheim, 0.1 mg/ml) was added to the final supernatants and aliquots (5-10~1) were injected into the high performance liquid chromatography (HPLC) column (Ultrasphere IP, 25cm, 0.46 cm o.d., 5 pm), protected with a Brownleee Newgard precolumn (3 cm, 5 pm). The mobile phase was as follows: 70 mM K,HPO,, 2 mM triethylamine, 1.5 mM octane sulphonic acid, 0.1 mM disodium EDTA and 11% methanol, adjusted to pH 3.10 with solid citric acid. The elution was performed at a flow rate of I ml/min and the potential for electrochemical detection (ED) was settled at 0.65 V (see Hamon, Fattaccini, Adrien, Gallissot, Martin and Gozlan, 1988 for details). Quantitative determinations were made by comparison with appropriate
external integrator.
standards
Measurement
using
a
CR3A
Shimadzu
qf [-‘H]5-HT uptake
Immediately after dissection, each cerebral cortex was homogenized in 10 vol. (v/w) of ice-cold 0.32 M sucrose using a Potter-Elvejhem apparatus fitted with Teflon pestle. Homogenates were centrifuged at 1000 g for 10 min at 4-C and the sedimented material (cell debris, nuclei, etc. .) was discarded. The IOOOg supernatants were recentrifuged at a higher speed: lO,OOOg, 20 min, 4 C and the crude synaptosomal fraction (PZ), sedimenting under these conditions, was resuspended in 10 vol. of ice-cold KrebsHenseleit medium, supplemented with 10 PM pargyline and 0.57 mM ascorbic acid, final pH 7.4 (Hamon, Nelson, Mallat and Bourgoin, 1981). Aliquots (75 ;tl corresponding to 0.3 mg protein) of each suspension were mixed with 0.415 ml of the same medium and the mixtures were preincubated for 4min at 37 C, under a constant atmosphere of 02:COz (95/5%). Blanks consisted of similar samples supplemented with 10 PM chlorimipramine. After the addition of [‘HIS-HT (10 ~1, final concentration: 12.0 nM), the incubation proceeded for 4 min. Assays were stopped by the addition of 3 ml ice-cold medium to each tube and immediate filtration through GF/B (Whatman) filters under slight vacuum. Each filter was then washed with a further 3 ml of ice-cold medittm, dried, and immersed in 4 ml of Aquasol” (New England Nuclear) for counting of radioactivity at 50% efficiency. Radioactivity in blank samples was regularly lo-12% of that in control samples. Triplicate determinations were made for each assay condition. Proteins were measured using the Folin phenol procedure (Lowry, Rosebrough, Farr and Randall, 1951) with bovine serumalbumin as the standard. Data were analyzed statistically by one-way analysis of variance and, in case of significance (P < 0.05), the F test for significant treatment effects was followed by two-tailed Student’s t-test to compare the experimental groups with their respective controls (Snedecor and Cochran, 1967). When the P value (Student’s t-test) was greater than 0.05, a difference was considered to be nonsignificant. RESULTS
(1) EfSects of acute administratior, qf tianeptine on the levels of S-HT, 5-HIAA, DA and DOPAC in various regions of the brain At least during the first 2 hr after the intraperitoneal administration of tianeptine (10 mg/kg), levels of 5-HT remained unaffected in any of the four regions of the brain examined (Table 1). In contrast, levels of 5-HIAA increased significantly in the brain stem (+ 16%) striatum (t 22%) and cerebral cortex (+ 15%) 1 hr after the injection of the drug (Table I). Some increase in levels of 5-HIAA were also noted in the hippocampus (+ 10%) but the amplitude of this
Tianeptine Table I. Effects of treatment
and
5-HT uptake
3
with tianeptine on levels of 5-HT and S-HIAA in various regions of the brain of the rat Saline
Brain
in viuo
Tianeptine 60 min
Tianeptine 120 min
smn
0.508 * 0.017 0.488 + 0.015
0.510 + 0.012 0.565 i 0.01 I*
0.507 f 0.023 0.490 f 0.01 I
0.298 f 0.008 0.383 k 0.020
0.309 * 0.009 0.469 f 0.015*
0.316+0.015 0.466 + 0.023’
5-HT S-HIAA
0.253 rt 0.015 0.246 & 0.009
0.242 k 0.009 0.271 _+0.008
0.264 f 0.007 0.264 * 0.012
Cerebral corlex 5-HT 5-HIAA
0.229 f 0.010 0.201 f 0.008
0.233 k 0.01 I 0.232 k 0.006*
0.226 f 0.010 0.208 i 0.009
5-HT 5-HIAA Striatum
5.HT 5-HIAA Hippocampus
Tianeptine (IO mg/kg) or saline was administered intraperitoneally 60 or 120 min before death. Each value (in pg/g) is the mea” k SEM of IO-18 independent determinations. Levels of 5-HT and 5.HIAA were not significantly different in rats treated with saline 60 or 120 min before death and corresponding values were pooled in a single group of control rats. *P -C 0.05 when compared to respective control values.
effect was too small to reach statistical significance (Table 1). Two hours after the administration of the drug, levels of 5-HIAA were no longer altered, except in the striatum where a significant increase (+22%) was still observed (Table 1). Measurements of levels of DA and DOPAC also revealed significant alterations, due to treatment with tianeptine. As shown in Table 2, levels of DOPAC were increased in the cerebral cortex (+ 27%) and the striatum (+ 17%), 1 hr after the injection of the drug and this effect even persisted 1 hr later in the second region (f 15%). Some increase in concentrations of DOPAC was also noted in the brain stem (+ 13-19%) but the critical level of significance (P = 0.5) was reached neither at 1 nor at 2 hours after the administration of tianeptine (Table 2). In contrast, levels of DA remained unaffected in all regions examined (Table 2) including the hippocampus (not shown). Levels of DOPAC in the hippocampus were too low (< 4.0 rig/g)) for reliable assessment of possible changes due to treatment with tianeptine.
Table 2. Effects of treatment
If the primary effect of tianeptine is to alter the uptake of 5-HT (see Mennini et al., 1987) and possibly the release of S-HT (De Simoni et al., 1989), it should be possible to interfere with tianeptineinduced changes in the metabolism of monoamines by a drug acting selectively both on the uptake and release of 5-HT, such as d,l-fenfluramine (Garattini, Mennini, Bendotti, Invernizzi and Samanin, 1986; Rowland and Carlton, 1986). On this basis, fenfluramine (5 mg/kg i.p.j was administered 20 min before tianeptine (10 mg/kg i.p.) and the levels of 5-HT, S-HIAA, DA and DOPAC were measured 1 hr later in the striatum and cerebral cortex, where the first series of experiments revealed the most significant changes (Tables 1 and 2). Fenfluramine alone reduced the levels of 5-HT in both regions, but the drug-induced change was much more pronounced in
with tianeptine on levels of DA and DOPAC in various regions of the brain of the rat Saline
Brain
(2) Prevention by d,l-fenjuramine of tianeptineinduced changes in levels of 5-HIAA and DOPAC in the striatum and cerebral cortex
Tianeptine 60 min
Tianeptine 120 min
smn
DA DOPAC
0.113 -i; 0.003 0.031 _+0.002
0.116 f 0.003 0.037 * 0.003
0.113~0.005 0.035 * 0.002
7.58 k 0.18 0.92 k 0.05
7.94f0.19 I .08 k 0.03*
8.64 i 0.49 I .06 f 0.03;
0.106 k 0.010 0.033 f 0.003
0.114 f 0.004 0.042 k 0.003’
0.123 *0.018 0.029 ? 0.002
Striatum
DA DOPAC Cerebral DA DOPAC
correx
Tianeptine (IO mg/kg) or saline was administered intraperitoneally 60 or 120 min before death. Each value (in pg/g) is the mea” k SEM of IO-18 independent determinations. Levels of DA and DOPAC were not significantly different in rats treated with saline 60 or 120min before death and corresponding values were pooled in a single group of control rats. *P -C 0.05 when compared to respective values in saline-treated rats.
C‘. M. FATiACClUI
4 Table 3 Effect\ oftianeptme
and.or c/./-fenfluramme on the levels of.‘-HT. and cerebral cortex of rats 5.HT
Slrururlr Sall”e Tlaneptlne Fenfluramme T>aneptme ( Fenfluramine
L’/ rrl.
5.HIAA
0.295 * 0.009 0311 i_OOO7 0 244 k 0.008*
0 342 t 0.016 0.423 k 0.008t 0 361 * 0.008
0.241 i 0.006’
5.HIAA.
DA and DOPAC I” the atrlatum
DA
DOPAC
7.35 i_ 0.18 7.80 * 0.33 7.09 _t 0.27
0 904 * 0.042 1.094~0.031t(+?l%) (1.9 10 _I 0 030
0.377~0.013(+4”~b)
7.08 * 0.24
l01:~0.050(+4%)
0215io.nII 0.222 * 0.013 0.121 r~ 0.008’
0.197 * 0.008 0.227 it O.OlOt (+ 15%) 0 I99 * 0.00s
0.092 ? 0.006 0.101 k 0.008 0.086 + 0.008
0.029 * 0.003 0.038 f 0.003t (+31%) 0.031 * 0.002
0.124~0.011*
0.208 i 0.006 (+ 5%)
0.106~0.016
0.033 ? 0.004 (+6%)
(+ 24%)
1
C‘er<~hrUILOllPU Sali”e Tianeptmc Fenfluramme Tlaneptinc { Fenfluramme >
Fenfluramine (5mg,kg i.p.) at&or tianrptine (IOmg;kg i.p.) were administered 80 and 60min before death. respecti\ely. Control rats were treated with saline instead of the drugs. Lo& (m pg;g) of 5.HT. 5.HIAA, DA and DOPAC are the mea”\ k EM of at least IO independent determmatmns. The percentage mcreases in levels of S-HIAA and DOPAC due to admimstratmn of tianeptine 20 min after saline or fenfluramine. are indicated tn parentheses *P < 0.05 when compared to tahne- or tianeptlne-treated rats. tP < 0.05 when compared to whne-treated rats.
the cerebral cortex (-44%) than in the striatum (- 17%). Other biochemical parameters were unaltered by this treatment (Table 3). Nevertheless, administration of fenfluramine markedly affected tianeptine-induced changes in the metabolism of the monoamines since the significant increases in levels of 5-HIAA and DOPAC due to the latter drug were no longer observed in fenfluramine-pretreated rats (Table 3). In addition, administration of tianeptine also failed to affect levels of 5-HT and DA in the striatum and cerebral cortex after pretreatment with fenfluramine (Table 3). (3) Eflects qf‘ tiuneptine on H 75/12-induced depletion qf‘ 5-HT and DA in carious regions of the brain Preliminary experiments, aimed at drawing doseresponse curves of the depletion of 5-HT by H75/12 led to the selection of the dose of 35 mg/kg. in order to induce approximately a 50% reduction in levels of 5-HT in the hippcampus and cerebral cortex, 90 min after treatment. Under this condition, depletion of 5-HT was significantly less pronounced in the striatum (-35%) and in the brain-stem (-26%) (Fig. I). Furthermore. at the dose of 35mgikg. H75j12 exerted only discrete effects on levels of DA in all areas examined (a maximum reduction by 15% was noted in the brain stem. see Fig. 1). When tianeptine (10 mg/kg i.p.) was administered twice. 20 min before and 20 min after H75,!12 (35 mg/kg i.p.). the depletion of 5-HT due to the latter drug was significantly increased in the four regions of the brain examined. although tianeptine alone did not affect the levels of amines (as compared to those in saline-treated rats) (Fig. 1). In contrast. the same depletion of DA was noted after administration of H75:‘lZ. whether the rats were treated or not with tianeptine (Fig. I). Further experiments with a larger dose of H75,‘12 (lOOmg/kg Lp.), producing a more pronounced decrease in levels of DA (- 55% in the brain stem, -40% in the striatum, -56% in the cerebral cortex). confirmed that tianeptine
(2 x IO mg/‘kg i.p.) did not significantly alter H75/12induced depletion of DA (not shown). At this large dose (lOOmg/kg i.p.), H75/12 produced a dramatic fall in levels of 5-HT (from -72% in the brain stem to -90% in the cerebral cortex) and possible further enhancement of depletion of 5-HT by combined administration of tianeptine could not be reliably assessed. As shown in Figure 2. H75/12 (35 mg/kg i.p.j did not significantly modify the levels of 5-HIAA in the four areas examined, but decreased those of DOPAC in the striatum and cerebral cortex. Conversely, tianeptine (2 x 10 mgjkg i.p.) increased the levels of both metabohtes in the two latter areas and those of DOPAC in the brain stem (Fig. 2). However, these effects were no longer observed in H75/12-treated rats, since levels of 5-HIAA in any of the four areas examined were not significantly different in rats treated with tianeptine + H75j12 or with the latter drug alone (Fig. 2). Therefore, H75/12, like the other 5-HT releasing drug used in this study, e.g. fenfluramine (see Table 3) prevented the enhancing effect of treatment with tianeptine upon the metabolism of 5-HT and DA. (4) li@cts qf treatment with tianeptine on the uptake of [‘HIS-HT in corticcd s:,nuptosomes For this experiment, as for the previous one (3), tianeptine (10 mg/kg i.p.) was injected twice. with a 40 min interval, and the animals were killed 60 min after the second injection. As shown in Figure 3, [3H]5-HT was taken up at a higher rate (+21%) in synaptosomes from the cerebral cortex of tianeptine-treated animals than in those from control rats. DISCUSSION
Previous neuropharmacological studies on tianeptine have shown that this tricyclic antidepressant is neither a monoamine oxidase inhibitor (Kato and
Tianeptine and 5-HT uptake in IGIXI STRIATUM
CEREBRAL
CORTEX
5
HIPPDCAwUS
I
Fig. 1. Effects of tianeptine and/or H75/12 on levels of 5-HT (a) and DA (b) in various regions of the brain of the rat. Rats received either tianeptine alone (lOmg/kg i.p. twice, IlOmin and 70min before death), H75/12 alone (35 mg/kg i.p., 90 min before death) or both treatments, while control rats received saline (i.p.) under the same time conditions. Levels of 5-HT and DA were measured by HPLC-ED as described in Methods and expressed as a percentage of respective control values. Each bar corresponds to the mean & SEM of 6-10 independent determinations. *P i 0.05when compared to saline-treated rats. tP < 0.05when compared to H75j12alone. Absolute values in saline-treated rats (100%) were: for levels of 5-HT: brain stem: 0.497 2 0.018 pg/g; striatum: 0.321 & 0.006 fig/g; cerebral cortex: 0.225 i: 0.017 pg/g; hippocampus: 0.271 + 0.024 ,ug/g-for levels of DA: Brain stem: 0.104 & 0.005 VP/g; striatum: 7.64 i. 0.23 pg/g; cerebral cortex: 0.127 rt 0.015 pg/g.
Weitsch, 1988) not a monoamine uptake inhibitor (Mennini et al., 1987) and indeed, it was found that treatment with tianeptine enhanced the levels of the metabolites, 5-HIAA and DOPAC in brain, an effect opposite to that induced by these inhibitors (Hamon, 1979). in r&o voltammet~ also led recently to the conclusion of an increased metabolism of both .5-HT and DA, in selected regions of the brain in freelymoving rats, treated acutely with tianeptine. Thus, De Simoni er al. (1989) reported a significant increase in the extracellular levels of S-HIAA in the hippocampus of tianept~n~-treated rats and Mennini, Consolo, De Simoni, Samanin and Garattini (1989) provided evidence for increased release and metabolism of DA in the nucleus accumbens and striatum in these animals. However, the latter authors did not observe any change in levels of 5-HIAA in brain after treatment with tianeptine (Mennini et al., 1987). Differences in the strains of rats, used in the laboratory of Mennini et al. (CD-COB) and in this one (Spra~~Dawiey), might account for this discrepancy, which, in any case, should deserve further investigation. In order to examine whether the changes in metaboIism of S-HT and DA, due to treatment with tianeptine, were dependent on each other, or corre-
sponded to two separate effects of the antidepressant, the possible influence of a selective alteration of serotoninergic neurones, by d,~-fenfluramine, on the effects of tianeptine was examined. Fenfluramine is a potent and selective inhibitor of 5-HT uptake and releaser of 5-HT (Maura, Gemignani, Versace, Martire and Raiteri, 1982; Garattini et al., 1986) and systemic administration of this drug produced a signi~cant depletion of 5-HT, with no change in levels of DA and DOPAC in the striatum and However, pretreatment with cerebral cortex. fenfluramine completely prevented the effects of tianeptine on levels of both .5-HIAA and DOPAC suggesting that the latter changes were not independent but linked to each other. Furthermore, as fenfluramine acts selectively on serotoninergic neurones, these data may indicate that tianeptineinduced increases in levels of DOPAC were secondary to some primary modification of S-HT neurotransmission by tianeptine. Further support for this hypothesis was obtained by the use of H75/12, another drug acting primarily on 5-HT neurotransmission (Carlsson et al., 1969). Thus, H75/12, like fenfluramine, prevented the alterations in both .5HIAA and DOPAC normaily induced by treatment with tianeptine.
C.
BRAIN STEM
M.
FAT ~ACCINI ef al.
STRIATUM
CEREBRAL CORTEX
I
HIPPOCAl4PUS
I
H n/12 I35 Wkd
Fig. 2. Effects of tianeptine and/or H75/12 on levels of 5-HIAA (a) and DOPAC (b) in various regions of the brain of the rat. Rats were treated as described in the legend to Figure 1 and the levels of SHIAA and DOPAC were measured by HPLC-ED (see Methods). Data are expressed as a percentage of respective control values. Each bar corresponds to the mean rf:SEM of 610 independent determinations. *P < 0.05 when compared to saline-treated rats. Absolute values in saline-treated rats (iOO%) were for levels of SHIAA: brain stem: 0.407 F 0.019 pgjg; striatum: 0.377 i 0.022 fig/g; cerebral cortex: 0.180 & 0.004 pg./g; hipp~ampus: 0.272 f 0.012 @g/g-for levels of DOPAC: brain stem: 0.029 f 0.001 pg/g; striatum: 0.983 & 0.061 #g/g: cerebral cortex: 0.035 + 0.003 {{g/g.
Numerous data in the literature support the existence of functional interactions between serotonin-
ergic and dopaminergic systems. Some authors favour the idea of a negative influence of serotoninergic neurones upon dopaminergic systems (Emus, Kemp and Cox, 1981; Nico!aou, Garcia-Munoz, Arbuthnott and Eccleston, 1979; Spampinato, Esposito and Samanin, 1985) but others presented evidence of an opposite control (Giambalvo and Snodgrass, 1978: Liston, Franz and Gibb, 1982) and recently, De Simoni, Dal Toso, Fodritto, Sokola and Algeri (1987) clearly demonstrated that the stimulation of serotoninergic neurones produced a signification acceleration of the turnover of DA in the striatum of the rat. Because of these discrepancies, it would be premature to conclude that the increased levels of DOPAC were due to a lower, rather than a higher, serotoninergic tone in tianeptine-treated rats. Direct measurement of the release of 5-HT in uivo would be the most appropriate approach to reach a firm conclusion. Previous studies with tricyclic antidepressants have demonstrated interactions of these drugs with fenfluramine, since Maura ef al. (1982) noted that chlorimipramine prevented fenfluramine-induced release of 5-HT from synaptosomes from the brain of the rat. However, tianeptine clearly behaved differently from chlor~mipramine, since depletion of W-IT,
due to in niao treatment with fenfluramine, was unaltered by prior administration of tianeptine. A useful approach for exploring the possible effect of a given treatment on the release or reuptake of S-HT in t&o, consists of examining the changes due
to this treatment in H75/12-induced depletion of 5-HT (Carlsson et al., 1969). Indeed numerous data demonstrated that inhibitors of the uptake of S-HT prevent (at least partially) the depletion of 5-HT due to 5-HT-releasing drugs, such as H75/12 (Carisson et al., 1969; Hamon et al., 1976) and p-chloroamphetamine (Fuller ef al., 1978; ogren et al., 1977). However, tianeptine exerted the opposite effect, since it enhanced significantly the depletion of 5-HT due to H75/12. Such an effect is expected for a drug having properties opposite to those of imipramine-related antidepressants, e.g. stimulating instead of inhibiting reuptake of 5-HT in uiuo. Alternatively, the enhanced depletion of 5-HT due to tianeptine + H75/12 might be the consequence of some releasing effect of tianeptine, which would add to that of H75/12. However, the latter hypothesis is very unlikely since tianeptine alone did not decrease the levels of 5-HT in any of the four areas of the brain examined here and previous in vitro studies showed that tianeptine (in a large concentration range >, 10 PM), reduced rather than stimulated the outflow of t3H]5-HT from slices of the brain of the rat (Hamon et al., 1989).
Tianeptine and 5-HT uptake in civo
EI
That a drug, stimulating reuptake of 5-HT, such as tianeptine, has antidepressant properties (see references in the introduction) may seem paradoxical as classical tricyclic antidepressants are, in contrast, potent inhibitors of the reuptake of 5-HT. However, the paradox may be more apparent than real, as Barbaccia, Gandolfi, Chuang and Costa (1983) reported an increased uptake of [3H]5-HT in hippocampal slices from rats treated chronically with imipramine or desmethylimipramine. Further studies on the activity of central serotoninergic synapses, after chronic treatment with tianeptine, should decide whether this drug also induces adaptive regulatory mechanisms, leading to a facilitation of serotoninergic transmission, as antidepressants of various chemical series (monoamine uptake inhibitors, monoamine oxidase inhibitors, some /3-adrenergic agonists etc.) do (Blier, De Montigny and Chaput, 1988; De Montigny, 1989).
Tmneptlne (2xlOmg/kg)
!
1
/ *p-z005
Fig. 3. Effects of treatment with tianeptine on the uptake of [)H]S-HT in cortical synaptosomes. Tianeptine (IO mg/kg i.p.) or saline was administered twice, 100 min and 60 min before death and crude synaptosomal (Pz) fractions were prepared from the cerebral cortex of each rat. Uptake of [rH]S-HT was measured at the end of a 4min incubation at 37°C with 12.0 nM of the labelled amine. Each bar corresponds to the mean (in pmol [3H]5-HT taken up per mg protein and per 4 min) + SEM of 8 independent determinations. *P < 0.05 when compared to saline-treated rats.
Therefore, the enhancing effect of treatment with tianeptine on H75/12-induced depletion of 5-HT resulted more probably from a greater capacity for reuptake of 5-HT, than from a greater release of 5-HT in uiuo. Indeed, direct measurement of the uptake of [3H]5-HT confirmed this interpretation, since a significant increase in accumulation of [3H]5-HT was observed in cortical synaptosomes from tianeptine-treated rats. Similar findings were reported by Mennini et al. (1987) who demonstrated that this effect was associated with an increased V,,, and no change in the K, of the synaptosomal process for the uptake of 5-HT. However, the latter authors did find a stimulatory effect of treatment with tianeptine on the uptake of [‘HIS-HT in the cerebral cortex and the hippocampus but not in the mesencephalon (Mennini et al., 1987), whereas it was observed here that H75/12-induced depletion of 5-HT was enhanced in all areas of the brain by the same treatment. No clear explanation can be given to this discrepancy, unless the strain differences, already noted above, might be associated with some (relatively discrete) differential sensitivity to tianeptine. In conclusion, the present data further support the idea that tianeptine acts by stimulating the reuptake of 5-HT in uiuo in brain, since effects opposite to those of inhibitors of the uptake of 5-HT: increase instead of decrease in levels of 5-HIAA, enhancement instead of prevention of H75/124nduced depletion of 5-HT, were noted in tianeptine-treated rats.
Acknowledgements-This research has been supported by grants from INSERM and Institut de Recherches Internationales SERVIER (I.R.I.S.). F. Bolaiios-Jimenez is in receipt of a fellowship from the Mexican Consejo National de Ciencia y Tecnologia (CONACYT-CEFI program).
REFERENCES
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El Mestikawy S., Gozlan H., Mknard F.. Bourgoin S. and Hamon M. (1988) Interactions of the potential antidepressant tianeptine with central serotonin receptors in the rat brain. 5th Inr. .&fee!. Clin. Pharmac. Psychiat., Tromss, p, 31. Ennis C., Kemp J. D. and Cox B. (1981) Characterisation of inhibitory 5-hydroxytryptamine receptors that modulate dopamine release in the striatum. J. Neurochem. 36: 1515~1520. Fuller R. W., Snoddy H. D.. Perry K. W., Bymaster F. P. and Wong D. T. (1978) Importance of duration of drug action in the antagonism of p-chloroamphetamine depletion of brain serotonin-Comparison of fluoxetine and chlorimipramine. Biochem. Pharmac. 27: 193--198. Galinowski A., Oh& J. P., Guelfi J. D., Malka R., D&ire C., Kamoun A. and L6o H. (1988) Interest of long term antidepressant therapy. Experience with tianeptine: a new antidepressant. Psychopharmacology 96: suppl., 195. Garattini S., Mennini T., Bendotti C., Invernizzi R. and Samanin R. (1986) Neurochemical mechanism of action of drugs which modify feeding via the serotoninergic system. Appetite 7: suppl. 1j-38. Giambalvo C. T. and Snodgrass S. T. (1978) Biochemical and behavioral effects of serotonin neurotoxins on the nigro-striatal dopamine system: comparison of injection sites. Bruin Res. 152: 555-566. Glowinski J. and Iversen L. (1966) Regional studies of catecholamines in the rat brain. I-Disposition of jHnorepinephrine, ‘H-dopamine and ‘H-dopa in various regions of the brain. J. Neurochem. 13: 655469. Hamon M. (1979) Mechanisms of action of antidepressant drugs. In: Phurmucology of the Stale.7 OJ Alertness (Passouant P. and Oswald I., Eds), pp. 117-128, Pergamon Press, Oxford. Hamon M., Bourgoin S., Enjalbert A., Bockaert J., H&y F., Ternaux J. P. and Glowinski J. (1976) The effects of quipazine on 5-HT metabolism in the rat brain. NaunySchmiedebergs Arch. Pharmuc. 294: 99-108. Hamon M., Bourgoin S. et Gozlan H. (1989) Effet de la tianeptine sur la libkration in vitro de [‘HIS-HT et sur les divers types de rCcepteurs SCrotoninergiques dans le systtme nerveux central chez le rat. J. Ps.vchiar. Biol. Ther., Mars, pp. 32-35. Hamon M.. Bourgoin S., Jagger J. and Glowinski J. (1974) Effects of LSD on synthesis and release of 5-HT in rat brain slices. Bruin Res. 69: 265-280. Hamon M., Fattaccini C. M.. Adrien J.. Gallissot M. C., Martin P. and Gozlan H. (1988) Alterations of centrai serotonin and dopamine turnover in rats treated with ipsapirone and other 5-hydroxytryptamine,. agonists with potential anxiolytic properties. J. Pharmnc. exp. Ther. 246: 745-752. Hamon M.. Nelson D. L., Mallat M. and Bourgoin S. (1981) Are 5-HT receptors involved in the sprouting of serotoninergic terminals following neonatal 5,7-dihydroxytryptamine treatment in the rat? Neurochem. Int. 3: 69-79.
Kato G. and Weitsch A. F. (1988) Neurochemical profile of tianeptine, a new antidepressant drug. C/in. h’europharmac. 11: suppl. 2, S43-S50. Labrid C., Moleyre J., Poignant J. C., Malen C.. Mocaer E. and Kamoun A. (1988) Structure-activity relationship of tricyclic antidepressants, with special reference to tianeptine. C/in. Neuropharmac. 11: suppl. 2, S21-S31. Liston D. A., Franz D. N. and Gibb J. W. (1982) Biochemical evidence for alteration of neostriatal dopaminergic function by 5,7-dihydroxytryptamine. J. Neurochem. 38: 1329-1335. Lbo H. and Deniker P. (1988) Position of tianeptine among antidepressive chemotherapies. Clin. Neuropharmac. 11: suppl. 2, S97-S102. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193: 265-275. Maura G., Gemignani A., Versace P., Martire M. and Raiteri M. (1982) Carrier-mediated and carrierindependent release of serotonin from isolated central nerve endings. Neurochem. Int. 4: 219-224. Mennini T., Consolo S., De Simoni M. G., Samanin R. and Garattini S. (1989) Neurochemical effects of tianeptine. Inl. Symp. Serotonin-From Ceil Biology to Pharmacology and Therapeutics. Florence, p. 178. Mennini T., Mocaer E. and Garattini S. (1987) Tianeptine, a selective enhancer of serotonin uptake in rat brain. Naunyn-Schmiedebergs Arch. Pharmac. 336: 478482. Mocaer E., Rettori M. C. and Kamoun A. (1988) Pharmacological antidepressive effects and tianeptine-induced 5-HT uptake increase. C/in. Neuropharmac. 11: suppl. 2. S32XS42. Nicolaou N. M., Garcia-Munoz M., Arbuthnott G. W. and Eccleston D. (1979) Interactions between serotonergic and dopaminergic systems in rat brain demonstrated by small unilateral lesions of the raphe nuclei. Eur. J. Pharmac. 57: 295-305. Cigren S. O., Ross S. B., Holm A. C. and Baumann L. (I 977) 5-Hydroxytryptamine and avoidance performance in the rat. Antagonism of the acute effect of p-chloroamphetamine by zimelidine, an inhibitor of 5-hydroxytryptamine uptake. Neurosci. Lert. 7: 331-336. Oli& J. P., Guelfi J. D., Malka R., D&ire C., Kamoun A. et Leo H. (1988) Traitements de longue durte par les antidtpresseurs. La tianeptine: mtthodologie d une &tude au long tours et r&ultats preliminaires. L’Enckphale 14: 379-384. Rowland N. E. and Carlton J. (1986) Neurobiology of an anorectic drug: fenfluramine. Prog. Neurobiol. 27: 1342. Snedecor G. W. and Cochran W. G. (1967) Staristical Methods-Ames. Iowa State College Press. Spampinato U., Esposito E. and Samanin R. (1985) Serotonin agonists reduce dopamine synthesis in the striatum only when the impulse Row of nigro-striatal neurons is intact. J. Neurochem. 45: 980-982.
0028-3908190 $3.00+ 0.00 Copyright 0 1990Pergamon Press plc
TIANEPTINE STIMULATES 5HYDROXYTRYPTAMINE IN VII’0
UPTAKE IN THE
OF RAT
BRAIN
C. M. FATTACCINI, F. BOLAP;TOS-JIMENEZ, H. GOZLAN and M. HAMON* INSERM U288, Neurobiologie Cellulaire et Fonctionnelle, FacultC de Medecine Pitit-Salp&triire, 91, boulevard de l’H_lbpital,75634 Paris Cedex 13, France (Accepted 22 Augur
1989)
Summary-The neurochemical effects of the atypical tricyclic antidepressant, tianeptine, were further assessed on central serotoninergic and dopaminergic systems in the rat. Acute treatment with tianeptine (10 mg/kg i.p.) significantly enhanced the levels of metabolites of 5-HT and DA, 5-hydroxyindole acetic acid and dihydroxyphenylacetic acid respectively, in the brain stem, striatum and cerebral cortex. These effects could be prevented by the administration of drugs acting selectively (or preferentially) on serotoninergic systems such as d,l-fenfluramine and 4-methyl-a-ethyl-metatyramine (H75/12), suggesting that the increased metabolism of DA was secondary to a modification of serotoninergic systems in tianeptine-treated rats. In contrast to that found with inhibitors of the uptake of 5-HT, treatment with tianeptine markedly enhanced depletion of S-HT due to administration of H75/12. However, depletion of DA induced by H75/12, was not altered by tianeptine. In vitro measurement of the uptake of [3H]5-HT also confirmed that tianeptine exerted opposite effects to those of classical tricyclic antidepressants, since the in viuo administration of tianeptine (2 x 10 mg/kg i.p.) induced a significant increase in the uptake of [‘HIS-HT in cortical synaptosomes. The fact that both inhibitors of the uptake of 5-HT and tianeptine which, in contrast, enhanced the in viuo uptake of 5-HT, are potent antidepressants, challenges the current hypothesis on the central mechanisms of action of these drugs. Key words-tianeptine, fenfluramine, dopamine, antidepressants.
4-methyl-cc-ethyl-metatyramine
NP29,lL-A
uptake,
of tianeptine, since evidence for an increased uptake of [3H]5-HT in cortical and hippocampal but not mesencephalic synaptosomes from tianeptine-treated rats has been reported by Mennini et al. (1987). Using in uiuo voltammetry, De Simoni, De Luigi, Manfridi and Sokola (1989) recently observed an increased efflux of the 5-HT metabolite, 5-hydroxyindole acetic acid (5-HIAA), in the hippocampus of freely moving rats, treated with tianeptine. Further neuropharmacological investigations have been made in this laboratory, with particular attention to possible alterations of central serotocinergic and dopaminergic neurotransmission, due to acute treatment with tianeptine in rats. In particular, possible changes in the reuptake of 5-HT in vivo after administration of tianeptine were assessed by examining tianeptine-induced alterations in depletion of 5-HT due to administration of H75/12 (6methyl-clethyl-metatyramine) (Carlsson, Corrodi, Fuxe and HBkfelt, 1969). Previous studies have clearly demonstrated the relevance of this approach for exploring the activity of the uptake of 5-HT in ho, since its inhibition, notably by tricyclic antidepressants, is well known to reduce markedly the depletion of amines induced by releasing agents such as H75/12 (Carlsson et al., 1969; Hamon, Bourgoin, Enjalbert, Bockaert, H&y, Ternaux and Glowinski, 1976) and p-chloroamphetamine (Fuller, Snoddy, Perry, Bymaster and Wong, 1978; i)gren, Ross, Holm and Baumann, 1977). By contrast, the enhancing effect of tianeptine
Although behavioral tests in rats (Labrid, Moleyre, Poignant, Malen, Mocaer and Kamoun, 1988; Mocaer, Rettori and Kamoun, 1988) and clinical trials in humans (Defiance, Marey and Kamoun, 1988; Galinowski, OliC, Guelfi, Malka, Dulcire, Kamoun and L6o, 1988; Olit, Guelfi, Malka, Dulcire, Kamoun and LBo, 1988; LI?IOand Deniker, 1988; Casacchia, Sconci, Vespucci and Brancato, 1989) have clearly shown that tianeptine ([chloro3-methyl-6-dioxo-5,5-dihydro-6,ll-dibenzo (c,f) (tiazepine-1,2)yl- 1 l)amino]-7-heptanoic acid, sodium salt) is a tricyclic antidepressant, this drug differs markedly from other tricyclics, such as imipramine and desipramine, since it inhibits the reuptake of neither serotonin (5_hydroxytryptamine, 5-HT) nor noradrenaline (NA) in the central nervous system (CNS) (Mennini, Mocaer and Garattini, 1987; Kato and Weitsch, 1988; Mocaer et al., 1988). Previous neuropharmacological investigations indicated that tianeptine does not bind to any of the subtypes of 5-HT receptors (El Mestikawy, Gozlan, MCnard, Bourgoin and Hamon, 1988; Hamon, Bourgoin and Gozlan, 1989) and does not affect the flow of nerve impulses within serotoninergic neurones of the dorsal raphe nucleus (Dresse and Scuvee-Moreau, 1988). Nevertheless, serotoninergic neurotransmission is probably involved in the central mechanism of action
*To whom all correspondence
(H75/12), serotonin
should be addressed.
I
C. M. FATTACCNI et rd.
2
on H75,‘12-induced depletion of 5-HT and the other biochemical data reported here further supports the idea that tianeptine stimulates the uptake of 5-HT in r+o in the brain of the rat. METHODS
Chemicals [3H]5-HT ([3H]5-Hydroxytryptamine creatinine sulphate generally labelled, 12 Ci/mmol., Amersham International) was purified by ion-exchange chromatography (Hamon, Bourgoin, Jagger and Glowinski, 1974) just before being used for the uptake experiments (see below). Other drugs were: d,l-fenfluramine and tianeptine (Servier), H75/12 (Astra Labkemi AB), pargyline (Abbott), chlorimipramine (Ciba-Geigy). Animals
and treatments
Adult male SpragueeDawley rats (Charles River strain), weighing 250-300 g, were kept in a controlled environment: 24 + 1°C 60% humidity, 12 hr/l2 hr alternate light/dark cycles, with food and water ad libitum, for at least 7 days before being used for the experiments. All drugs were dissolved in saline and injected intraperitoneally in a volume of 1 ml/rat at IO-1 1 a.m. Control rats received saline by the same route of administration and following the same time conditions as for the drugs. All animals were killed by decapitation and the dissection of the brain proceeded at &4’C according to Glowinski and Iversen (1966). Measurements ?f‘SHT, 5-HIAA, dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) Each dissected structure (brain stem, hippocampus, striatum, cerebral cortex) was homogenized at 04 C with a Branson sonifier in 0.5-l .O ml of 0.1 M HCIO,, supplemented with 0.5% NazS,O, and 0.5% disodium ethylene diamine tetra acetic acid (EDTA). Homogenates were centrifuged at 30,000 g for 15 min at 4°C and the supernatants were neutralized using 2 M KH,PO,/K,HPO,, pH 7.6. After a second centrifugation as before. ascorbate oxidase (Boehringer-Mannheim, 0.1 mg/ml) was added to the final supernatants and aliquots (5-10~1) were injected into the high performance liquid chromatography (HPLC) column (Ultrasphere IP, 25cm, 0.46 cm o.d., 5 pm), protected with a Brownleee Newgard precolumn (3 cm, 5 pm). The mobile phase was as follows: 70 mM K,HPO,, 2 mM triethylamine, 1.5 mM octane sulphonic acid, 0.1 mM disodium EDTA and 11% methanol, adjusted to pH 3.10 with solid citric acid. The elution was performed at a flow rate of I ml/min and the potential for electrochemical detection (ED) was settled at 0.65 V (see Hamon, Fattaccini, Adrien, Gallissot, Martin and Gozlan, 1988 for details). Quantitative determinations were made by comparison with appropriate
external integrator.
standards
Measurement
using
a
CR3A
Shimadzu
qf [-‘H]5-HT uptake
Immediately after dissection, each cerebral cortex was homogenized in 10 vol. (v/w) of ice-cold 0.32 M sucrose using a Potter-Elvejhem apparatus fitted with Teflon pestle. Homogenates were centrifuged at 1000 g for 10 min at 4-C and the sedimented material (cell debris, nuclei, etc. .) was discarded. The IOOOg supernatants were recentrifuged at a higher speed: lO,OOOg, 20 min, 4 C and the crude synaptosomal fraction (PZ), sedimenting under these conditions, was resuspended in 10 vol. of ice-cold KrebsHenseleit medium, supplemented with 10 PM pargyline and 0.57 mM ascorbic acid, final pH 7.4 (Hamon, Nelson, Mallat and Bourgoin, 1981). Aliquots (75 ;tl corresponding to 0.3 mg protein) of each suspension were mixed with 0.415 ml of the same medium and the mixtures were preincubated for 4min at 37 C, under a constant atmosphere of 02:COz (95/5%). Blanks consisted of similar samples supplemented with 10 PM chlorimipramine. After the addition of [‘HIS-HT (10 ~1, final concentration: 12.0 nM), the incubation proceeded for 4 min. Assays were stopped by the addition of 3 ml ice-cold medium to each tube and immediate filtration through GF/B (Whatman) filters under slight vacuum. Each filter was then washed with a further 3 ml of ice-cold medittm, dried, and immersed in 4 ml of Aquasol” (New England Nuclear) for counting of radioactivity at 50% efficiency. Radioactivity in blank samples was regularly lo-12% of that in control samples. Triplicate determinations were made for each assay condition. Proteins were measured using the Folin phenol procedure (Lowry, Rosebrough, Farr and Randall, 1951) with bovine serumalbumin as the standard. Data were analyzed statistically by one-way analysis of variance and, in case of significance (P < 0.05), the F test for significant treatment effects was followed by two-tailed Student’s t-test to compare the experimental groups with their respective controls (Snedecor and Cochran, 1967). When the P value (Student’s t-test) was greater than 0.05, a difference was considered to be nonsignificant. RESULTS
(1) EfSects of acute administratior, qf tianeptine on the levels of S-HT, 5-HIAA, DA and DOPAC in various regions of the brain At least during the first 2 hr after the intraperitoneal administration of tianeptine (10 mg/kg), levels of 5-HT remained unaffected in any of the four regions of the brain examined (Table 1). In contrast, levels of 5-HIAA increased significantly in the brain stem (+ 16%) striatum (t 22%) and cerebral cortex (+ 15%) 1 hr after the injection of the drug (Table I). Some increase in levels of 5-HIAA were also noted in the hippocampus (+ 10%) but the amplitude of this
Tianeptine Table I. Effects of treatment
and
5-HT uptake
3
with tianeptine on levels of 5-HT and S-HIAA in various regions of the brain of the rat Saline
Brain
in viuo
Tianeptine 60 min
Tianeptine 120 min
smn
0.508 * 0.017 0.488 + 0.015
0.510 + 0.012 0.565 i 0.01 I*
0.507 f 0.023 0.490 f 0.01 I
0.298 f 0.008 0.383 k 0.020
0.309 * 0.009 0.469 f 0.015*
0.316+0.015 0.466 + 0.023’
5-HT S-HIAA
0.253 rt 0.015 0.246 & 0.009
0.242 k 0.009 0.271 _+0.008
0.264 f 0.007 0.264 * 0.012
Cerebral corlex 5-HT 5-HIAA
0.229 f 0.010 0.201 f 0.008
0.233 k 0.01 I 0.232 k 0.006*
0.226 f 0.010 0.208 i 0.009
5-HT 5-HIAA Striatum
5.HT 5-HIAA Hippocampus
Tianeptine (IO mg/kg) or saline was administered intraperitoneally 60 or 120 min before death. Each value (in pg/g) is the mea” k SEM of IO-18 independent determinations. Levels of 5-HT and 5.HIAA were not significantly different in rats treated with saline 60 or 120 min before death and corresponding values were pooled in a single group of control rats. *P -C 0.05 when compared to respective control values.
effect was too small to reach statistical significance (Table 1). Two hours after the administration of the drug, levels of 5-HIAA were no longer altered, except in the striatum where a significant increase (+22%) was still observed (Table 1). Measurements of levels of DA and DOPAC also revealed significant alterations, due to treatment with tianeptine. As shown in Table 2, levels of DOPAC were increased in the cerebral cortex (+ 27%) and the striatum (+ 17%), 1 hr after the injection of the drug and this effect even persisted 1 hr later in the second region (f 15%). Some increase in concentrations of DOPAC was also noted in the brain stem (+ 13-19%) but the critical level of significance (P = 0.5) was reached neither at 1 nor at 2 hours after the administration of tianeptine (Table 2). In contrast, levels of DA remained unaffected in all regions examined (Table 2) including the hippocampus (not shown). Levels of DOPAC in the hippocampus were too low (< 4.0 rig/g)) for reliable assessment of possible changes due to treatment with tianeptine.
Table 2. Effects of treatment
If the primary effect of tianeptine is to alter the uptake of 5-HT (see Mennini et al., 1987) and possibly the release of S-HT (De Simoni et al., 1989), it should be possible to interfere with tianeptineinduced changes in the metabolism of monoamines by a drug acting selectively both on the uptake and release of 5-HT, such as d,l-fenfluramine (Garattini, Mennini, Bendotti, Invernizzi and Samanin, 1986; Rowland and Carlton, 1986). On this basis, fenfluramine (5 mg/kg i.p.j was administered 20 min before tianeptine (10 mg/kg i.p.) and the levels of 5-HT, S-HIAA, DA and DOPAC were measured 1 hr later in the striatum and cerebral cortex, where the first series of experiments revealed the most significant changes (Tables 1 and 2). Fenfluramine alone reduced the levels of 5-HT in both regions, but the drug-induced change was much more pronounced in
with tianeptine on levels of DA and DOPAC in various regions of the brain of the rat Saline
Brain
(2) Prevention by d,l-fenjuramine of tianeptineinduced changes in levels of 5-HIAA and DOPAC in the striatum and cerebral cortex
Tianeptine 60 min
Tianeptine 120 min
smn
DA DOPAC
0.113 -i; 0.003 0.031 _+0.002
0.116 f 0.003 0.037 * 0.003
0.113~0.005 0.035 * 0.002
7.58 k 0.18 0.92 k 0.05
7.94f0.19 I .08 k 0.03*
8.64 i 0.49 I .06 f 0.03;
0.106 k 0.010 0.033 f 0.003
0.114 f 0.004 0.042 k 0.003’
0.123 *0.018 0.029 ? 0.002
Striatum
DA DOPAC Cerebral DA DOPAC
correx
Tianeptine (IO mg/kg) or saline was administered intraperitoneally 60 or 120 min before death. Each value (in pg/g) is the mea” k SEM of IO-18 independent determinations. Levels of DA and DOPAC were not significantly different in rats treated with saline 60 or 120min before death and corresponding values were pooled in a single group of control rats. *P -C 0.05 when compared to respective values in saline-treated rats.
C‘. M. FATiACClUI
4 Table 3 Effect\ oftianeptme
and.or c/./-fenfluramme on the levels of.‘-HT. and cerebral cortex of rats 5.HT
Slrururlr Sall”e Tlaneptlne Fenfluramme T>aneptme ( Fenfluramine
L’/ rrl.
5.HIAA
0.295 * 0.009 0311 i_OOO7 0 244 k 0.008*
0 342 t 0.016 0.423 k 0.008t 0 361 * 0.008
0.241 i 0.006’
5.HIAA.
DA and DOPAC I” the atrlatum
DA
DOPAC
7.35 i_ 0.18 7.80 * 0.33 7.09 _t 0.27
0 904 * 0.042 1.094~0.031t(+?l%) (1.9 10 _I 0 030
0.377~0.013(+4”~b)
7.08 * 0.24
l01:~0.050(+4%)
0215io.nII 0.222 * 0.013 0.121 r~ 0.008’
0.197 * 0.008 0.227 it O.OlOt (+ 15%) 0 I99 * 0.00s
0.092 ? 0.006 0.101 k 0.008 0.086 + 0.008
0.029 * 0.003 0.038 f 0.003t (+31%) 0.031 * 0.002
0.124~0.011*
0.208 i 0.006 (+ 5%)
0.106~0.016
0.033 ? 0.004 (+6%)
(+ 24%)
1
C‘er<~hrUILOllPU Sali”e Tianeptmc Fenfluramme Tlaneptinc { Fenfluramme >
Fenfluramine (5mg,kg i.p.) at&or tianrptine (IOmg;kg i.p.) were administered 80 and 60min before death. respecti\ely. Control rats were treated with saline instead of the drugs. Lo& (m pg;g) of 5.HT. 5.HIAA, DA and DOPAC are the mea”\ k EM of at least IO independent determmatmns. The percentage mcreases in levels of S-HIAA and DOPAC due to admimstratmn of tianeptine 20 min after saline or fenfluramine. are indicated tn parentheses *P < 0.05 when compared to tahne- or tianeptlne-treated rats. tP < 0.05 when compared to whne-treated rats.
the cerebral cortex (-44%) than in the striatum (- 17%). Other biochemical parameters were unaltered by this treatment (Table 3). Nevertheless, administration of fenfluramine markedly affected tianeptine-induced changes in the metabolism of the monoamines since the significant increases in levels of 5-HIAA and DOPAC due to the latter drug were no longer observed in fenfluramine-pretreated rats (Table 3). In addition, administration of tianeptine also failed to affect levels of 5-HT and DA in the striatum and cerebral cortex after pretreatment with fenfluramine (Table 3). (3) Eflects qf‘ tiuneptine on H 75/12-induced depletion qf‘ 5-HT and DA in carious regions of the brain Preliminary experiments, aimed at drawing doseresponse curves of the depletion of 5-HT by H75/12 led to the selection of the dose of 35 mg/kg. in order to induce approximately a 50% reduction in levels of 5-HT in the hippcampus and cerebral cortex, 90 min after treatment. Under this condition, depletion of 5-HT was significantly less pronounced in the striatum (-35%) and in the brain-stem (-26%) (Fig. I). Furthermore. at the dose of 35mgikg. H75j12 exerted only discrete effects on levels of DA in all areas examined (a maximum reduction by 15% was noted in the brain stem. see Fig. 1). When tianeptine (10 mg/kg i.p.) was administered twice. 20 min before and 20 min after H75,!12 (35 mg/kg i.p.). the depletion of 5-HT due to the latter drug was significantly increased in the four regions of the brain examined. although tianeptine alone did not affect the levels of amines (as compared to those in saline-treated rats) (Fig. 1). In contrast. the same depletion of DA was noted after administration of H75:‘lZ. whether the rats were treated or not with tianeptine (Fig. I). Further experiments with a larger dose of H75,‘12 (lOOmg/kg Lp.), producing a more pronounced decrease in levels of DA (- 55% in the brain stem, -40% in the striatum, -56% in the cerebral cortex). confirmed that tianeptine
(2 x IO mg/‘kg i.p.) did not significantly alter H75/12induced depletion of DA (not shown). At this large dose (lOOmg/kg i.p.), H75/12 produced a dramatic fall in levels of 5-HT (from -72% in the brain stem to -90% in the cerebral cortex) and possible further enhancement of depletion of 5-HT by combined administration of tianeptine could not be reliably assessed. As shown in Figure 2. H75/12 (35 mg/kg i.p.j did not significantly modify the levels of 5-HIAA in the four areas examined, but decreased those of DOPAC in the striatum and cerebral cortex. Conversely, tianeptine (2 x 10 mgjkg i.p.) increased the levels of both metabohtes in the two latter areas and those of DOPAC in the brain stem (Fig. 2). However, these effects were no longer observed in H75/12-treated rats, since levels of 5-HIAA in any of the four areas examined were not significantly different in rats treated with tianeptine + H75j12 or with the latter drug alone (Fig. 2). Therefore, H75/12, like the other 5-HT releasing drug used in this study, e.g. fenfluramine (see Table 3) prevented the enhancing effect of treatment with tianeptine upon the metabolism of 5-HT and DA. (4) li@cts qf treatment with tianeptine on the uptake of [‘HIS-HT in corticcd s:,nuptosomes For this experiment, as for the previous one (3), tianeptine (10 mg/kg i.p.) was injected twice. with a 40 min interval, and the animals were killed 60 min after the second injection. As shown in Figure 3, [3H]5-HT was taken up at a higher rate (+21%) in synaptosomes from the cerebral cortex of tianeptine-treated animals than in those from control rats. DISCUSSION
Previous neuropharmacological studies on tianeptine have shown that this tricyclic antidepressant is neither a monoamine oxidase inhibitor (Kato and
Tianeptine and 5-HT uptake in IGIXI STRIATUM
CEREBRAL
CORTEX
5
HIPPDCAwUS
I
Fig. 1. Effects of tianeptine and/or H75/12 on levels of 5-HT (a) and DA (b) in various regions of the brain of the rat. Rats received either tianeptine alone (lOmg/kg i.p. twice, IlOmin and 70min before death), H75/12 alone (35 mg/kg i.p., 90 min before death) or both treatments, while control rats received saline (i.p.) under the same time conditions. Levels of 5-HT and DA were measured by HPLC-ED as described in Methods and expressed as a percentage of respective control values. Each bar corresponds to the mean & SEM of 6-10 independent determinations. *P i 0.05when compared to saline-treated rats. tP < 0.05when compared to H75j12alone. Absolute values in saline-treated rats (100%) were: for levels of 5-HT: brain stem: 0.497 2 0.018 pg/g; striatum: 0.321 & 0.006 fig/g; cerebral cortex: 0.225 i: 0.017 pg/g; hippocampus: 0.271 + 0.024 ,ug/g-for levels of DA: Brain stem: 0.104 & 0.005 VP/g; striatum: 7.64 i. 0.23 pg/g; cerebral cortex: 0.127 rt 0.015 pg/g.
Weitsch, 1988) not a monoamine uptake inhibitor (Mennini et al., 1987) and indeed, it was found that treatment with tianeptine enhanced the levels of the metabolites, 5-HIAA and DOPAC in brain, an effect opposite to that induced by these inhibitors (Hamon, 1979). in r&o voltammet~ also led recently to the conclusion of an increased metabolism of both .5-HT and DA, in selected regions of the brain in freelymoving rats, treated acutely with tianeptine. Thus, De Simoni er al. (1989) reported a significant increase in the extracellular levels of S-HIAA in the hippocampus of tianept~n~-treated rats and Mennini, Consolo, De Simoni, Samanin and Garattini (1989) provided evidence for increased release and metabolism of DA in the nucleus accumbens and striatum in these animals. However, the latter authors did not observe any change in levels of 5-HIAA in brain after treatment with tianeptine (Mennini et al., 1987). Differences in the strains of rats, used in the laboratory of Mennini et al. (CD-COB) and in this one (Spra~~Dawiey), might account for this discrepancy, which, in any case, should deserve further investigation. In order to examine whether the changes in metaboIism of S-HT and DA, due to treatment with tianeptine, were dependent on each other, or corre-
sponded to two separate effects of the antidepressant, the possible influence of a selective alteration of serotoninergic neurones, by d,~-fenfluramine, on the effects of tianeptine was examined. Fenfluramine is a potent and selective inhibitor of 5-HT uptake and releaser of 5-HT (Maura, Gemignani, Versace, Martire and Raiteri, 1982; Garattini et al., 1986) and systemic administration of this drug produced a signi~cant depletion of 5-HT, with no change in levels of DA and DOPAC in the striatum and However, pretreatment with cerebral cortex. fenfluramine completely prevented the effects of tianeptine on levels of both .5-HIAA and DOPAC suggesting that the latter changes were not independent but linked to each other. Furthermore, as fenfluramine acts selectively on serotoninergic neurones, these data may indicate that tianeptineinduced increases in levels of DOPAC were secondary to some primary modification of S-HT neurotransmission by tianeptine. Further support for this hypothesis was obtained by the use of H75/12, another drug acting primarily on 5-HT neurotransmission (Carlsson et al., 1969). Thus, H75/12, like fenfluramine, prevented the alterations in both .5HIAA and DOPAC normaily induced by treatment with tianeptine.
C.
BRAIN STEM
M.
FAT ~ACCINI ef al.
STRIATUM
CEREBRAL CORTEX
I
HIPPOCAl4PUS
I
H n/12 I35 Wkd
Fig. 2. Effects of tianeptine and/or H75/12 on levels of 5-HIAA (a) and DOPAC (b) in various regions of the brain of the rat. Rats were treated as described in the legend to Figure 1 and the levels of SHIAA and DOPAC were measured by HPLC-ED (see Methods). Data are expressed as a percentage of respective control values. Each bar corresponds to the mean rf:SEM of 610 independent determinations. *P < 0.05 when compared to saline-treated rats. Absolute values in saline-treated rats (iOO%) were for levels of SHIAA: brain stem: 0.407 F 0.019 pgjg; striatum: 0.377 i 0.022 fig/g; cerebral cortex: 0.180 & 0.004 pg./g; hipp~ampus: 0.272 f 0.012 @g/g-for levels of DOPAC: brain stem: 0.029 f 0.001 pg/g; striatum: 0.983 & 0.061 #g/g: cerebral cortex: 0.035 + 0.003 {{g/g.
Numerous data in the literature support the existence of functional interactions between serotonin-
ergic and dopaminergic systems. Some authors favour the idea of a negative influence of serotoninergic neurones upon dopaminergic systems (Emus, Kemp and Cox, 1981; Nico!aou, Garcia-Munoz, Arbuthnott and Eccleston, 1979; Spampinato, Esposito and Samanin, 1985) but others presented evidence of an opposite control (Giambalvo and Snodgrass, 1978: Liston, Franz and Gibb, 1982) and recently, De Simoni, Dal Toso, Fodritto, Sokola and Algeri (1987) clearly demonstrated that the stimulation of serotoninergic neurones produced a signification acceleration of the turnover of DA in the striatum of the rat. Because of these discrepancies, it would be premature to conclude that the increased levels of DOPAC were due to a lower, rather than a higher, serotoninergic tone in tianeptine-treated rats. Direct measurement of the release of 5-HT in uivo would be the most appropriate approach to reach a firm conclusion. Previous studies with tricyclic antidepressants have demonstrated interactions of these drugs with fenfluramine, since Maura ef al. (1982) noted that chlorimipramine prevented fenfluramine-induced release of 5-HT from synaptosomes from the brain of the rat. However, tianeptine clearly behaved differently from chlor~mipramine, since depletion of W-IT,
due to in niao treatment with fenfluramine, was unaltered by prior administration of tianeptine. A useful approach for exploring the possible effect of a given treatment on the release or reuptake of S-HT in t&o, consists of examining the changes due
to this treatment in H75/12-induced depletion of 5-HT (Carlsson et al., 1969). Indeed numerous data demonstrated that inhibitors of the uptake of S-HT prevent (at least partially) the depletion of 5-HT due to 5-HT-releasing drugs, such as H75/12 (Carisson et al., 1969; Hamon et al., 1976) and p-chloroamphetamine (Fuller ef al., 1978; ogren et al., 1977). However, tianeptine exerted the opposite effect, since it enhanced significantly the depletion of 5-HT due to H75/12. Such an effect is expected for a drug having properties opposite to those of imipramine-related antidepressants, e.g. stimulating instead of inhibiting reuptake of 5-HT in uiuo. Alternatively, the enhanced depletion of 5-HT due to tianeptine + H75/12 might be the consequence of some releasing effect of tianeptine, which would add to that of H75/12. However, the latter hypothesis is very unlikely since tianeptine alone did not decrease the levels of 5-HT in any of the four areas of the brain examined here and previous in vitro studies showed that tianeptine (in a large concentration range >, 10 PM), reduced rather than stimulated the outflow of t3H]5-HT from slices of the brain of the rat (Hamon et al., 1989).
Tianeptine and 5-HT uptake in civo
EI
That a drug, stimulating reuptake of 5-HT, such as tianeptine, has antidepressant properties (see references in the introduction) may seem paradoxical as classical tricyclic antidepressants are, in contrast, potent inhibitors of the reuptake of 5-HT. However, the paradox may be more apparent than real, as Barbaccia, Gandolfi, Chuang and Costa (1983) reported an increased uptake of [3H]5-HT in hippocampal slices from rats treated chronically with imipramine or desmethylimipramine. Further studies on the activity of central serotoninergic synapses, after chronic treatment with tianeptine, should decide whether this drug also induces adaptive regulatory mechanisms, leading to a facilitation of serotoninergic transmission, as antidepressants of various chemical series (monoamine uptake inhibitors, monoamine oxidase inhibitors, some /3-adrenergic agonists etc.) do (Blier, De Montigny and Chaput, 1988; De Montigny, 1989).
Tmneptlne (2xlOmg/kg)
!
1
/ *p-z005
Fig. 3. Effects of treatment with tianeptine on the uptake of [)H]S-HT in cortical synaptosomes. Tianeptine (IO mg/kg i.p.) or saline was administered twice, 100 min and 60 min before death and crude synaptosomal (Pz) fractions were prepared from the cerebral cortex of each rat. Uptake of [rH]S-HT was measured at the end of a 4min incubation at 37°C with 12.0 nM of the labelled amine. Each bar corresponds to the mean (in pmol [3H]5-HT taken up per mg protein and per 4 min) + SEM of 8 independent determinations. *P < 0.05 when compared to saline-treated rats.
Therefore, the enhancing effect of treatment with tianeptine on H75/12-induced depletion of 5-HT resulted more probably from a greater capacity for reuptake of 5-HT, than from a greater release of 5-HT in uiuo. Indeed, direct measurement of the uptake of [3H]5-HT confirmed this interpretation, since a significant increase in accumulation of [3H]5-HT was observed in cortical synaptosomes from tianeptine-treated rats. Similar findings were reported by Mennini et al. (1987) who demonstrated that this effect was associated with an increased V,,, and no change in the K, of the synaptosomal process for the uptake of 5-HT. However, the latter authors did find a stimulatory effect of treatment with tianeptine on the uptake of [‘HIS-HT in the cerebral cortex and the hippocampus but not in the mesencephalon (Mennini et al., 1987), whereas it was observed here that H75/12-induced depletion of 5-HT was enhanced in all areas of the brain by the same treatment. No clear explanation can be given to this discrepancy, unless the strain differences, already noted above, might be associated with some (relatively discrete) differential sensitivity to tianeptine. In conclusion, the present data further support the idea that tianeptine acts by stimulating the reuptake of 5-HT in uiuo in brain, since effects opposite to those of inhibitors of the uptake of 5-HT: increase instead of decrease in levels of 5-HIAA, enhancement instead of prevention of H75/124nduced depletion of 5-HT, were noted in tianeptine-treated rats.
Acknowledgements-This research has been supported by grants from INSERM and Institut de Recherches Internationales SERVIER (I.R.I.S.). F. Bolaiios-Jimenez is in receipt of a fellowship from the Mexican Consejo National de Ciencia y Tecnologia (CONACYT-CEFI program).
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