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Br. J. Pharmac. (1986), 88, 269-275

Effect oflong term amineptine treatment on pre- and postsynaptic mechanisms in rat brain A. Ceci, S. Garattini, M. Gobbi & T. Mennini' Istituto di Ricerche Farmacologiche 'Mario Negri', Via Eritrea, 62-20157 Milan, Italy

1 The effect of amineptine and its two metabolites on monoamine uptake, release and receptor binding was studied in vitro. 2 Amineptine and its two metabolities did not displace labelled ligands for known neurotransmitters and drug receptor sites. 3 Amineptine and its two metabolities did not influence [3HJ-5-hydroxytryptamine ([3HJ-5-HT) uptake or release by rat brain synaptosomes. Amineptine inhibited [3HJ- and [3H]- noradrenaline ([3HI-NA) accumulation, with IC50 values of 1.4 and 10 pM, respectively. The effect was retained, though with lower efficacy, by the two metabolites. 4 Amineptine released [3H]-dopamine from preloaded synaptosomes. Metabolite 1 had no effect on catecholamine release, and metabolite 2 was about half as active as the parent compound on [3H]- dopamine release. 5 The releasing effect of amineptine on [3HJ-dopamine was potentiated by pretreatment, suggesting that the drug acts on the cytoplasmic neurotransmitter pool. 6 Chronic treatment with amineptine (20 mg kg-', twice daily for 15 days followed by a 3 days drug withdrawal period) resulted in a decrease of [3H]-spiperone binding sites in striatum, and of [3H]- dihyroalprenolol and [3H]-clonidine in cortex. 7 Chronic treatment with amineptine reduced basal [3H]-dopamine accumulation in striatal synaptosomes, without affecting [3IH-NA or [3H]-5-HT accumulation. 8 The adaptive changes in the pre- and postsynaptic dopamine mechanisms observed after long term treatment with amineptine are consistent with the drug acting as an indirect dopamine agonist.

9 The down regulation of 1- and a2-noradrenoceptors observed after long term amineptine treatment may play a role in the activity of the drug.

Introduction Amineptine, an antidepressant drug (Samanin et al., Amineptine differs from other agents in its 1977; Roster, 1979) effective in several screening tests side chain, which is 7-aminoheptanoic acid (Figure 1). for antidepressant activity (Poignant, 1979) and par- Kinetically amineptine is rapidly adsorbed and ticularly on the Porsolt immobility test (Borsini et al., metabolized, to form two majormetabolites (Figure 1) 1981), is characterized by a selective effect on the resulting from P-oxidation of the lateral chain, giving system (Samanin et al., 1977; Dankova rise therefore to a tricyclic agent with 5-aminoeptanoic et al., 1977). Like other agonists, acid (metabolite 1) or 3-aminopentanoic acid amineptine increases locomotor activity and induces (metabolite 2) (Sbarra et al., 1979; 1981). stereotyped movements and hyperthermia in rats In this study an attempt was made to characterize (Samanin et al., 1977), it appears to inhibit dopamine further the mechanism of action by investigating the uptake (Algeri et al., 1978); it raises rat striatal effects of the drug and its two metabolites on uptake, homovanillic acid (HVA) levels (Samanin et al., 1977; release and binding of 3H-monoamines from synap- Dankova et al., 1977) and reduces dopamine turnover tosomal preparations in vitro. Moreover, since the (Algeri et al., 1978), without significantly affecting 3,4- effect of become evident only after dihydroxyphenylacetic acid (DOPAC). repeated drug treatment, we have studied 3H-monoanine uptake, release and binding ex vivo, after chronic aminep- 'Author for correspondence. tine administration. c) The Macmillan Press Ltd 1986 270 A. CECI et al. 3~~ NH NH NH (CH2)6-COOH (CH2)4-COOH (CH2)2-COOH Amineptine Metabolite 1 Metabolite 2

Figure 1 Chemical structure of amineptine and metabolites

Methods final volume 1.0 ml) containing 10mg of tissue (hip- pocampus) and [3H]-5-HT (sp.act. 27 Ci mmol', Ex vivo experiments Radiochemical Centre) (0.10-15 nM) was incubated for 15 min at 37°C. Non-specific binding was deter- Rats (Male Sprague Dawley CD COBS, Charles mined in the presence of 1I1OM unlabelled 5-HT. For River, Italy, average weight 200 g) received i.p. ami- [3H]-dihyroalprenolol (DHA) binding to P-noradren- neptine (20 mg kg- ') or saline twice daily for 15 days. oceptors (Bylund & Snyder, 1976) incubation buffer Three days after the end of treatment animals were (0.05 MTris HCl, pH 7.7, 0.1% ascorbic acid, 1O1gM killed by decapitation, brain regions were quickly pargyline, final volume I ml) containing 10mg of dissected and immediately used for synaptosomal tissue (cortex) and [3HJ-DHA (sp.act. 34 Ci mmol ', experiments or stored at - 80'C until binding assays. NEN) (0.9-7.5 nM) was incubated for 15 min at 25°C. For in vitro experiments either naive or saline-treated Non-specific binding was determined in the presence animals were used. of 1 gM (±)-propranolol. For [3H]-prazosin (a,-NA) (Greengrass & Bremner, 1979) binding incubation Binding assays buffer (0.05 M Tris HCl, pH 7.7, 0.1% ascorbic acid, 1O1M pargyline, final volume 1 ml) containing Crude membrane preparations used for all binding 10mg of tissue (cortex) and [3H]-prazosin (sp.act. assays, were obtained as described by Bennett & 18.8 Ci mmol-', NEN) (0.1-7nM) was incubated for Snyder (1976) and Nelson et al. (1978). The brain 30 min at 25C. Non-specific binding was determined regions were homogenized in 50 vol of cold Tris-HCl in the presence of 3 g1M phentolamine. os2-Noradren- buffer 0.05 M pH 7.4 using an ultra Turrax TP 18-10 oceptors were measured by [3H]-clonidine binding (2 x 20 s) and centrifuged at 50,000g for 10 min. The (Greenberg et al., 1976). Incubation buffer (0.05 M pellets were resuspended in cold Tris buffer, incubated Tris HCl, pH 7.7, 0.1% ascorbic acid, 10 gAM pargyline, at 37°C for 10 min and centrifuged twice more as final volume 1 ml) containing 10 mg of tissue (cortex) above. For [3H]-spiperone binding to dopamine2-re- and [3H]-clonidine (sp.act. 65.0 Ci mmol-', NEN) ceptors (Creese et al., 1978) incubation buffer (0.05 M (1.0-15 nM) was incubated for 20 min at 25°C. Non- Tris HCI, pH 7. 1, 0.1% ascorbic acid, 10 1M pargyline, specific binding was determined in the presence of 120mM NaCl, 5mM KC1, 2mM CaCl2, I mM MgCI2, 1001M noradrenaline (NA). In vitro affinities for final volume 0.5 ml) containing 2.5 mg of tissue dopamine agonist sites (Creese & Snyder, 1978) ben- (striatum) and [3H]-spiperone (sp.act. 23 Ci mmol-', zodiazepine- (Ehlert et al., 1981), GABAA-(Herschel NEN) (0.010-1.4 nM) was incubated for 15 min at & Baldessarini, 1979), histamine1-(Tan Tran et al., 37°C. Non-specific binding was determined in the 1978) and antidepressant-(Raisman et al., 1980; 1982) presence of 1 gM (+ )-. For [3H]- receptors were determined by using [3H]-ADTN, [3H]- binding to 5-hydroxytryptamine2-(5-HT2) receptors flunitrazepam, [3H]-GABA, [3H-], [3H]- (Leysen et al., 1982), incubation buffer (0.05 M Tris and [3H]-desmethylimipramine, respec- HCl, pH 7.7, final volume 1 ml) containing 5mg of tively. All ligands were obtained from NEN. tissue (cortex) and [3H]-ketanserin (sp.act. 77 Ci For all binding assays incubation was stopped by mmol ', NEN (0.3-5 nM) was incubated for 15 min at the addition ofice-cold Tris buffer followed by rapid 37°C. Non-specific binding was determined in the vacuum filtration through Whatman GF/B glass fibre presence of 1 JiM methysergide. 5-HT1 binding sites filters and three additional washes with Tris buffer. were measured by [3H] 5-HT binding (Nelson et al., Filters were counted in 10ml of Filter Count (Pack- 1978). Incubation buffer (0.05 M Tris HCl, pH 7.7, ard) scintillator in a Beckman liquid scintillation 0.1% ascorbic acid, 10I1M pargyline, 4mM CaC12, spectrometer L.S. 7500 with counting efficiency of ADAPTIVE CHANGES AFTER LONG TERM AMINEPTINE 271

about 40%. trol 20 min - c.p.m. drug 20 min)/c.p.m. control Binding parameters were calculated by non linear Omin x 100 fitting of data from saturation experiments with 6-8 For superfusion experiments, 0.8ml portions of different concentrations of3H-ligands, or from inhibi- synaptosomes preloaded with 0.1ItM [3H]-dopamine tion experiments with 5 different inhibitor concentra- were filtered through cellulose nitrate filters. Filters tions; using an HP 85 desk computer (Benfenati & were put at the bottom of superfusion chambers, and Guardabasso, 1984). superfused with Krebs-Henseleit buffer at 0.5 ml min-'. Effluent was collected in counting vials Accumulation andrelease by brain synaptosomes (every 5min). At the end of superfusion (50 min) radioactivity remaining on the filters was also counted Purified synaptosomes were obtained from pooled and used for calculation oftotal radioactivity present. cortex (5-HT and NA) or striata (dopamine) as Data were calculated as % of radioactivity in each described by Gray & Whittaker (1962) and diluted sample over total radioactivity present at the begin- with Krebs-Henseleit buffer containing 0.25 mM par- ning ofsuperfusion. More detailed information about gyline to obtain a final protein concentration of the methods used in studies of monamine uptake and 0.5-1 mg ml l . For accumulation studies, 0.6 ml sam- release is given elsewhere (Mennini et al., 1978; 1981) ples were incubated at 30C (or 4°C to assess passive [3H]-NA was obtained from NEN, [3H]-5-HT and diffusion) with 0.1 iM 3H-monamines for 5min. In [3H]-dopamine from the Radiochemical Centre. vitro amineptine was added during a 5 min pre-incuba- Specific activities were as follows: [3HI-NA 9.4 Ci tion period. The reaction was stopped by cooling the mmol '; [3H]-dopamine 9.0 Ci mmol'; [3H]-5- tubes in ice and adding 0.5 ml of ice-chilled Krebs- HT 28.7 Ci mmol '. Henseleit buffer. Samples were then filtered through cellulose nitrate filters (0.65 JLM pore size; Societa Italiana di Microfiltrazione), washed with Krebs-Hen- Results seleit buffer, dissolved in 5 ml of Atomlight (NEN) and counted for radioactivity in a Beckman LS 7500 In vitro experiments liquid scintillator counter. Percentage accumulation inhibition was calculated on net values (30'C-4°C). Amineptine and its two metabolites, up to a concen- The amineptine concentration giving 50% inhibition tration of 10 ^sM, were unable to displace the following (IC50) was determined by non-linear fitting of dose- ligands from brain membrane preparations of naive response curves, with 5 drug concentrations. rats: [3H]-5-HT, [3H]-spiperone (cortex and striatum), For release experiments, synaptosome suspensions [3HJ-ATDN, [3H1-prazosin, [3HJ-clonidine, [3H]- prelabelled by incubation with 0.1I M 3H-monamines DHA, [3H1-GABA, [31H]-mepyramine, [3H]-flun- for 15 min were centrifuged at 17,000 g for 10 min, and itrazepam, [3H]-imipramine and [3H]-desmeth- the pellets were gently resuspended in fresh Krebs- ylimipramine (data not shown). Henseleit buffer. Portions of0.6 ml were preincubated In saline-treated animals, amineptine significantly 5 min at 37°C to allow equilibration. At the end ofthis inhibited [3HJ-dopamine accumulation (IC501.4I1M) period (O time) drugs were added and the mixtures and at higher concentrations, [3H]-NA accumulation were then incubated for 20 min. Samples were filtered (IC50 lOIM) (Table 1 top half). These effects were and counted as described for accumulation studies. retained, with lower efficacy, by the two metabolites % release was calculated as follows: (c.p.m. con- (data not shown). Amineptine 10I1M also released

Table 1 Effect of chronic amineptine on 3H-monoamine accumulation (basal and amineptine-inhibited)

[3H1-5-HT [3H-NA [ H1-DA Treatment Control Amineptine Control Amineptine Control Amineptine (10 11M) (lo pM) (10 gIM)

Saline 1.77 ± 0.07 1.83 ± 0.07 0.367 ± 0.037 0.175 ± 0.01a 1.97 ± 0.03 0.33 ± 0.09a Chronic 1.74 ± 0.06 1.72 ± 0.1 0.33 ± 0.02 0.153 ± 0.Oa 0.68 ± 0.02b 0.557 ± 0.02k

Data (pmol minm' mg- ' protein) are means ± s.d. of 4 replications. Amineptine was given in vivo 20mg kg- i.p., twice daily for 15 days; in vitro amineptine was tested at IO jM final concentration. aDifferent from respective control: bdifferent from saline-treated; 2 ways ANOVA; P <0.05 Tukey's test. The final concentrations of 3H-monoamines were: 5-HT and NA 0. IjIM; dopamine 0.05 pM. 272 A. CECI et al.

Table 2 Effect of chronic amineptine on 3H-monoamine release (basal and amineptine-stimulated)

[3H1-5-HT [3HI-NA [3H1-DA Control Control Amineptine Control Control Amineptine Control Control Amineptine 0 min 20mi 10 m20min 0min 20 min 10gM20 min 0 min 20 min 10 M 20 min Saline 3.6± 0.5 2.9± 0.1 2.3±0.1 1.41 ±0.06 0.71 ±0.05 0.80± 0.07 10.1±0.3 6.7±0.3 5.2±0.44b Chronic 3.1± 0.7 1.8±0.7 2.0±0.3 1.34±0.09 0.69±0.01 0.76± 0.02 7.4± 0.3a 4.9± 0.3a 3.5+0.2ab Data (pmol mg-' protein) are means ± s.d. of 4 replications. Amineptine was given in vivo 20mg kg' i.p., twice daily for 15 days. In vitro amineptine was tested at 1O gzM, final concentration. 'Different from saline-treated; bdifferent from respective control 20 min; two ways ANOVA, P < 0.01, Tukey's test.

[3H]-dopamine from preloaded synaptosomes but was not affect basal [3H]-5-HT accumulation, and con- inactive on [3H]-NA release (Table 2 top half). At firms that, when added in vitro at 1O tM, it did not equimolar concentrations, metabolite I had no effect inhibit [3H]-5-HT accumulation in rat cortical synap- on 3H-catecholamine release; metabolite 2 slightly tosomes. Chronic amineptine treatment did not alter (6.1 ± 0.2%) enhanced [3H]-NA release and was only basal [3H]-NA accumulation nor the sensitivity of the about half as active as the parent compound on [3H]- noradrenergic carrier to 10 M amineptine added in dopamine release (7.4 ± 0.08%). Neither amineptine vitro (about 50% inhibition), as shown in Table 1. nor its metabolites influenced [3H]-5-HT accumula- Basal [3H]-dopamine accumulation was significantly tion (Table 1 top half) or release (Table 2 top half) by decreased in synaptosomes from rats chronically brain synaptosomes of saline-treated rats. Table 3 treated with amineptine (Table 1), as was the inhibit- shows that the releasing effect of amineptine on [3H]- ing activity of 10 M amineptine added in vitro. While dopamine was potentiated by reserpine pretreatment, the IC50 of amineptine in saline-treated rats was like that of (+ )-, reported for compar- 1.4AM, in drug-treated animals only 30% inhibition of ison. In fact, 1 pM amineptine did not increase [3H]- accumulation could be obtained with 30 M aminep- dopamine release in normal synaptosomes, but it tine (data not shown). stimulated [3H]-dopamine release in synaptosomes Figure 3 shows the kinetic characteristics of [3H]- from reserpine-treated rats. The releasing effect of dopamine accumulation in striatal synaptosomes 10 M amineptine was about half that of I IsM from saline or chronic amineptine-treated rats. The amphetamine in normal and reserpinized synap- apparent affinities of the neuronal carrier for [3HJ- tosomes. dopamine were similar in the two experimental groups Figure 2 shows that the effect of 10 pM amineptine (about 0.4 gM), but the maximum number of uptake on [3H]-dopamine release seen in saline-treated rats is sites was significantly lower in amineptine-treated also evident in superfused synaptosomes, where inter- animals (about 50% of control value). ference due to inhibition is minimized. Chronic amineptine treatment did not affect [3H]-5- HT or [3H]-NA release and did not seem to affect Ex vivo experiments either basal or amineptine-stimulated [3H]-dopamine release in incubated synaptosomes (Table 2). Table 1 shows that chronic amineptine treatment did However, in superfusion experiments, a tendency to

Table 3 Effect of in vitro added amineptine on [3HJ-dopamine ([3HI-DA) release from striatal synaptosomes

In vivo treatment % release induced during 20 min incubation Drug Concentration Normal rats Reserpine-treated rats B:A A B (+ )-Amphetamine 10-6M 31 ± 3** 48 ± 2** 1.55 Amineptine 10-6M NS 13 ± 6** Amineptine lo-0M 15 ± 3** 23 ± 5** 1.53

[3H]-dopamine release was studied in striatal synaptosome incubated in vitro as described in Methods. Reserpine- treated groups were injected with 10 mg kg-' i.p. 18 h before the experiment. [3H]-DA accumulation in synaptosomes from normal and reserpine-treated rats was 4,300 ± 8,500 and 30,000 ± 4,500 d.p.m. per sample, respectively (P<0.01, Student's t test). **P<0.01 significantly different from controls (Duncan's test). ADAPTIVE CHANGES AFTER LONG TERM AMINEPTINE 273

2000 - a) 0._ co c -5 = a 0-0 <: 0 1000 - E

60 9 Time (min) 0.023 0.047 0.09 0.182 Figure 2 Effect of amineptine (1AM) added in vitro or [3HIDA [wUMJ chronic amineptine treatment on [3H]-dopamine release Figure 3 Effect ofchronic amineptine treatment on from superfused synaptosomes. Data were calculated as [3H]- dopamine accumulation. Each point is the mean ± s.d. of the cumulative percentage of total radioactivity (see two replications: (-) = saline-treated rats; % Methods). x = time of superfusion (min); y = of [3H]- (0) = chronic amineptine-treated rats. The kinetic dopamine released. Each point is the mean of three parameters, with the relative standard deviation, = replications, varying less than 10%. (-) saline-treated obtained by non-linear fitting of the data using a HP-85 rats; (-)saline + IO amineptine in vitro; gM computer were: Saline: V,,. = 1 1939 ± 1136 fmol mg-' (A) = chronic amineptine-treated rats. protein min-'; Km = 0.43 ± 0.05 JIM. Chronic aminep- tine: V,,,. = 5447 ±2531 fmol mg' protein min'; higher spontaneous [3H]-dopamine release was found P <0.05 (Student's t test); Km = 0.47 ± 0.28 AM. in synaptosomes from chronically amineptine-treated anjmals (Figure 2). Table 4 shows that chronic amineptine treatment with rat brain synaptosomes have established that significantly reduced the maximum number ofbinding amineptine inhibits the accumulation of dopamine sites for [3H]-spiperone in striatum, as well as those for and, at ten times higher concentrations, that of NA, and a2- and a-NA ligands in rat cortex. No significant without affecting 5-HT accumulation. The inhibition effects were observed at 5-HT1-, 5-HT2- and cx,-NA exerted by amineptine was shared, but to a lesser binding sites. extent by its two metabolites. Release ofmonoamines by amineptine was limited to dopamine, without any effect on NA and 5-HT. Again, the two metabolites Discussion were less effective than the parent drug. These results point to amineptine as an indirect Amineptine and its two metabolites had no effect in dopamine agonist. However, in vitro studies with vitro on a number of receptor sites. Of particular synaptosomal preparations do not clarify whether a interest is the lack ofeffect on [3HJ-imipramine binding drug decreases amine accumulation inside the nerve sites, in spite of the similarity in chemical structure. It terminal by reducing its uptake, increasing release, or is also important to consider that amineptine and its both. Our results with superfused synaptosomes, metabolites have no affinity for dopamine binding where interference due to reuptake inhibition is min- sites which means that direct agonism of the drug at imized (Raiteri et al., 1974), indicate that amineptine is dopamine receptors can be excluded. In vitro studies a true dopamine releaser. Moreover, its effect of

Table 4 Effect of chronic amineptine on neurotransmitter receptor binding

Treatment 5-HT, 5-HT2 DA2 (xi-NA P-NA

Saline KD 4.6 ± 0.9 0.8 ± 0.0 0.4 ± 0.0 0.2 ± 0.1 2.4 ± 0.1 2.0 ± 0.0 Bax 18.4 ± 3.3 15.2 ± 0.7 19.9 ± 0.9 6.7 ± 0.8 6.7 ± 0.1 12.4 ± 0.6 Chronic KD 4.1 ± 0.7 0.8 ± 0.0 0.3 ± 0.1 0.2 ± 0.1 2.6 ± 0.1 2.2 ± 0.1 amineptine Bmax 18.9 ± 2.1 15.0 ± 0.3 15.3 ± 0.6* 6.3 ± 0.4 5.6 ± 0.2* 11.0 ± 0.9t I KD (nM) and B,,,.. (pmol g- tissue) are means ± s.d. of4 animals and were calculated by non-linear fitting of binding data as described in methods. *P < 0.01; t P < 0.05: Student's 1 test. 274 A. CECI et al. releasing dopamine is, like that of ( + )-amphetamine, It might be argued that the enhanced release of[3H]- potentiated in synaptosomes from reserpine dopamine in superfused synaptosomes is a con- pretreated animals, suggesting that it acts on an extra- sequence of the diminished [3H]-dopamine accumula- vescicular pool of dopamine. Whether it also acts by tion after long-term amineptine treatment, which inhibiting the neuronal uptake carrier for dopamine is results in a considerably lower absolute amount of difficult to establish only on the basis of in vitro [3HJ-dopamine released. Further experiments are in experiments; however, in vivo amineptine prevents the progress to check this. It can be excluded that the depletion of brain dopamine following 6-hydroxy- decreased dopamine accumulation is caused by drug dopamine lesions, increases 3-methoxytyramine for- or metabolites present in the brain tissue, since mation and reduces dopamine turnover (Garattini et treatment was suspended three days before the ex- al., 1985), suggesting that inhibition of dopamine periment in order to avoid interference due to residual uptake had probably occurred (Ponzio et al., unpubli- drug concentrations. shed). It seems therefore that the presynaptic carrier for Amineptine concentrations effective in vitro dopamine is modified by long-term amineptine treat- (1-10IM) are in the range of those found in the brain ment, resulting in an apparent reduction of the of rats given pharmacologically active doses of the maximum number of uptake sites. While changes in drug (20mg kg-') (Sbarra et al., 1979). sensitivity of pre- and postsynaptic receptors com- That amineptine acts as an (indirect) dopamine monly occur after long-term drug treatment, (Creese receptor agonist is also shown by the fact that repeated & Sibley, 1981; Raiteri et al., 1982) modifications of treatment induced down-regulation of dopamine re- the neuronal uptake carrier have not been frequently ceptor sites in striatum. The density of P-noradren- reported. Chronic antidepressant treatment results in oceptors was also reduced in the rat cortex after adaptive changes in the presynaptic monoaminergic repeated amineptine, a finding common to the system (Sugrue, 1981), principally in a compensatory majority of antidepressant agents (Charney et al., variation in the turnover rate of these amines. It has 1981), as was the number of x2-noradrenoceptors, a been recently reported that dopamine reuptake is much more selective effect of antidepressants. functionally coupled with hydroxylase It is difficult to explain the effects of chronic activity (Maura & Raiteri, 1982). Therefore the amineptine on noradrenoceptors since, unlike observed reduction in dopamine accumulation might amphetamine, in vitro it inhibits NA accumulation reflect adaptive changes in presynaptic dopamine only at high concentrations and in in vivo it does not function following an amineptine-induced decrease in protect against 6-hydroxydopamine-induced NA de- dopamine synthesis. It is ofinterest to note that in vivo, pletion (Garattini et al., 1985). Moreover, it apparent- amineptine lost its effect on brain dopamine metabol- ly does not influence the noradrenergic at single doses ism after three weeks of daily treatment, as indicated (Ponzio, unpublished results). Whether the effect is by the fact that a challenge with 40 mg kg-' did not related to stimulation of the dopaminergic system elicit the usual rise in striatal homovanillic acid remains to be established. (Garattini et al., 1985). An interesting observation arising from the present In conclusion the results of this study are consistent study is that the basal accumulation of dopamine in with the hypothesis that amineptine increases synaptosomes from rats chronically treated with dopaminergic transmission by presynaptic action, and amineptine was significantly decreased, as was the indicate that long-term amineptine treatment induces ability of in vitro added amineptine to inhibit adaptive modifications in pre- and postsynaptic dopamine accumulation. The reduced basal mechanisms which may be responsible for its phar- accumulation of dopamine could be due to enhanced macological effects. release, as indicated by the fact that spontaneous release was increased in superfused synaptosomes This work was partly supported by the CNR (National from amineptine-treated animals. This result is at Research Council, Rome, Italy), Contract No. 84.01115.95. variance with those obtained by incubating synap- Amineptine and the two metabolites were generously don- tosomes, since spontaneous and amineptine- ated by Servier Laboratories, Neuilly sur Seine, France. The stimulated dopamine release were not modified by authors thank Carlo Taddei for his valuable participation in long-term treatment with the drug. this work.

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