The Selective Serotonin Reuptake Inhibitor Citalopram Induces the Storage of Serotonin in Catecholaminergic Terminals

Transcription

1 /02/ $7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 302, No. 1 Copyright 2002 by The American Society for Pharmacology and Experimental Therapeutics 34462/ JPET 302: , 2002 Printed in U.S.A. The Selective Serotonin Reuptake Inhibitor Citalopram Induces the Storage of Serotonin in Catecholaminergic Terminals HEBERTO SUAREZ-ROCA and LUIGI X. CUBEDDU Pharmacology Section, Instituto de Investigaciones Clinicas, School of Medicine, University of Zulia, Maracaibo, Venezuela (H.S.-R.); Clinical Pharmacology and Neuropharmacology Units, Department of Pharmacology, School of Pharmacy, Central University of Venezuela, Caracas, Venezuela (L.X.C.); and Nova Southeastern University, Health Professions Division, Fort Lauderdale, Florida (L.X.C.) Received February 13, 2002; accepted March 7, 2002 This article is available online at ABSTRACT We investigated whether selective inhibition of serotonin (5- hydroxytryptamine; 5-HT) transporter with citalopram leads to accumulation of 5-HT in catecholaminergic neurons. In the rabbit olfactory tubercle, citalopram (1 10 M) inhibited [ 3 H]5- HT uptake; however, the maximal degree of inhibition achieved was 70%. Addition of nomifensine (1 10 M) was required for complete inhibition of [ 3 H]5-HT uptake. In slices labeled with 0.1 M [ 3 H]5-HT, cold 5-HT ( M) induced a large increase in the efflux (release) of stored [ 3 H]5-HT, an effect blocked by coperfusion with 1 M citalopram. Similar concentrations ( M) of norepinephrine (NE) or dopamine (DA) failed to release [ 3 H]5-HT. When labeling with 0.1 M [ 3 H]5-HT was carried out in the presence of citalopram, 1) low concentrations of 5-HT failed to release [ 3 H]5-HT; 2) DA and NE were more potent and effective in releasing [ 3 H]5-HT than in control slices; 3) coperfusion of NE, DA, or 5-HT with citalopram enhanced the release of [ 3 H]5-HT induced by the catecholamines but not by 5-HT; and 4) coperfusion of NE or DA with nomifensine antagonized NE- and DA-evoked [ 3 H]5-HT release, with a greater effect on NE than on DA. These results suggest that in the rabbit olfactory tubercle, where there is coexistence of 5-HT, NE, and DA neurons, inhibition of the 5-HT transporter led to accumulation of 5-HT in catecholaminergic terminals. Thus, during treatment with selective serotonin uptake inhibitors (SSRIs), 5-HT may be stored in catecholaminergic neurons acting as a false neurotransmitter and/or affecting the disposition of DA and/or NE. Transmitter relocation may be involved in the antidepressant action of SSRIs. Despite their lower affinity for serotonin (5-hydroxytryptamine; 5-HT), there is experimental evidence to suggest that catecholaminergic neurons may take up and store 5-HT. Indeed, 5-HT-immunoreactive fibers in the pars intermedia of the rat pituitary gland can be eliminated by treatment with 6-hydroxydopamine, a neurotoxin selective for catecholamine neurons (Saland et al., 1986). Interestingly, Thind and coworkers (1987) observed the presence of 5-HT staining in tyrosine hydroxylase-immunoreactive fibers in the suprachiasmatic nuclei and ventromedial hypothalamus 24 h after infundibular stalk section. These authors proposed that catecholaminergic terminals might take up 5-HT coming from cut 5-HT terminals. In a study by Feuerstein and coworkers (1986), prolonged incubations of caudate nucleus slices with [ 3 H]5-HT led to labeling of catecholaminergic terminals, which was prevented by the NE/DA uptake inhibitor nomifensine but not by the selective 5-HT reuptake inhibitor 6-nitroquipazine. The above observations suggest that storage of 5-HT may occur in catecholamine-containing neurons. Based on the above described findings, we proposed that in brain areas where there is a coexistence of catecholaminergic and serotonergic terminals, 5-HT could accumulate in catecholaminergic neurons if the 5-HT neuronal transporter (neuronal uptake pump) is inhibited with a selective 5-HT transporter inhibitor. To test this hypothesis, we employed the olfactory tubercle of the rabbit because of its high density of 5-HT, DA, and NE terminals (Dahlstrom and Fuxe, 1964; Conzolazione and Cuello, 1982; Fallon and Moore, 1987). In addition, the olfactory tubercle is a limbic region of easy access and could be a relevant site of action for antidepressant drugs (Rosin et al., 1995). Citalopram was used as the selective serotonin reuptake inhibitor (SSRI) (Sitte et al., 2000). The experimental rationale was based on the assumption that incubation with low concentrations of [ 3 H]5-HT results in selective labeling of 5-HT terminals, whereas incubation of [ 3 H]5-HT in the presence of citalopram would favor labeling of catecholaminergic neurons. In addition, based on the higher affinity of the 5-HT terminals for 5-HT, we proposed that nonradioactive 5-HT should be more potent than NE and DA in displacing [ 3 H]5-HT, when the [ 3 H]5-HT is stored in serotonergic terminals. Such specificity should be lost if the 5-HT would be stored in catecholaminergic terminals. Our findings suggest that catecholaminergic terminals take up 5-HT when the 5-HT uptake pump is inhibited with the SSRI, citalopram. ABBREVIATIONS:5-HT, serotonin; NE, norepinephrine; DA, dopamine; SSRI, selective serotonin reuptake inhibitor; ANOVA, analysis of variance. 174

2 Citalopram and 5-HT Storage in Catecholaminergic Neurons 175 Materials and Methods Male New Zealand rabbits (2 3.5 kg) were sacrificed by decapitation. The brains were quickly removed, and olfactory tubercles were immediately dissected on ice in a cold room at 4 C, following these landmarks: diagonal band of Broca (caudal-medial border), beginning of the olfactory tract (rostral border), lateral olfactory tract (lateral border), and medial forebrain bundle (dorsal border). Next, the tissues were chopped into 0.4-mm slices by means of a McIlwain tissue chopper (Mickle Laboratory Engineering Co., Ltd., Surrey, UK). Approximately, from 20 to 30 slices were obtained from both olfactory tubercles of one rabbit. Uptake of [ 3 H]5-HT. Olfactory tubercle slices were incubated for 30 min at 37 C with 0.1 M[ 3 H]5-HT in 4 ml of superfusion medium. The medium composition was 118 mm NaCl, 4.8 mm KCl, 1.3 mm CaCl 2, 1.2 mm MgSO 4, 25 mm NaHCO 3, 1.2 mm KH 2 PO 4, 0.03 mm disodium EDTA, 0.57 mm ascorbic acid, and 11 mm glucose. The medium was continuously gassed with 95% O 2 and 5% CO 2, and the ph was set at 7.4. The slices were incubated with [ 3 H]5-HT either in the presence or in the absence of the 5-HT-uptake pump inhibitor citalopram (1 or 10 M) and/or the NE/DA uptake pump inhibitor nomifensine (1 or 10 M). Subsequently, the slices were washed three times with 4 ml of cold (4 C) superfusion medium, and the radioactivity present in the slices was quantified by liquid scintillation spectrometry. Displacement of Stored [ 3 H]5-HT. Immediately after incubation, two olfactory tubercle slices were transferred to a nylon mesh mounted on a polypropylene cylinder. The cylinders were then placed in 1-ml glass superfusion chambers. The slices were superfused with prewarmed oxygenated superfusion medium (37 C) at 1 ml/min. Fraction collection (5-min fractions) started after a 75-min washout period with drug-free medium. After obtaining a basal efflux of radioactivity for 20 min, slices were superfused with stepwise increasing concentrations (from 1 nm to 30 M) of either 5-HT, DA, or NE at sequential intervals of 20 min. In a group of experiments, the effects of 5-HT, NE, or DA were studied in the presence of either 1 M citalopram or 1 M nomifensine. In these experiments, perfusion with citalopram or nomifensine started 30 min before collection of baseline samples (50 min before perfusion with monoamines) and continued until the end of the experiment. Radioactivity in the superfusate-fractions (efflux) and in the slices (at the end of the experiment) was quantified by liquid scintillation spectrometry. Because of differences in the extent of labeling observed between control slices and those labeled in the presence of citalopram, the efflux results were always corrected by the amount of radioactivity present in the slices. The efflux results were expressed as fractional release, i.e., as a percentage of the tissue 3 H content. The radioactivity (total 3 H) collected in the superfusion samples consisted of a mixture of unchanged [ 3 H]5-HT and their respective 3 H-metabolites (Fozard and Berry, 1976). In this study, for convenience, the collected [ 3 H] efflux was simply referred to as displacement of [ 3 H]5-HT. Statistics. Inhibition of [ 3 H]5-HT uptake by citalopram and/or nomifensine was expressed in absolute values (disintegrations per minute per milligram of tissue) and as a percentage of inhibition employing as control values the tissue content obtained in the absence of uptake inhibitors (controls). The efflux of [ 3 H]5-HT was expressed in absolute values (dpm/5 min) and as a fraction of the amount of radioactivity present in the tissue (slices) at the time of sample collection. One-way analysis of variance (ANOVA) was used to establish overall effect of uptake pump inhibitors on uptake of [ 3 H]5-HT, using different treatments (concentrations and combinations) as the levels. Repeated measurement one-way ANOVA was used to determine overall ability of increasing concentrations of nonradioactive monoamines to displace [ 3 H]5-HT, serving monoamine concentrations as a repeated factor. Two-way ANOVA for a repeated measurement design was used to determined differences in [ 3 H]5-HT displacement induced by nonradioactive monoamines in the presence or absence of uptake pump inhibitors, with uptake condition (with or without inhibition) as the cross-factor and monoamine concentrations as the repeated measure factor. In cases where the two-way ANOVA revealed significant interaction between the two factors, data were again analyzed with repeated measurement one-way ANOVA. Statistically significant effects detected by ANOVA were further analyzed using Duncan s multiple range test. Significance was assumed at P Results Effect of Citalopram and Nomifensine on [ 3 H]5-HT Uptake. In slices of the olfactory tubercle, the SSRI citalopram (1 10 M) inhibited the uptake of 0.1 M [ 3 H]5-HT to a maximum of 71 2% (Table 1). Comparable degrees of uptake inhibition were observed with 1 and 10 M citalopram (Table 1). However, nomifensine alone (1 10 M) weakly inhibited [ 3 H]5-HT uptake. Complete inhibition of [ 3 H]5-HT uptake ( %) was achieved with a combination of 1 M citalopram and 10 M nomifensine (Table 1). Release of [ 3 H]5-HT from Superfused Slices Labeled under Control Conditions (Absence of Uptake Inhibitor). In control slices preloaded with 0.1 M [ 3 H]5-HT (with no citalopram in the incubation mixture), superfusion with low concentrations of nonradioactive 5-HT ( M) increased the efflux of 3 H (release) in a concentration-dependent manner (P ) (Fig. 1). Nearly 8% ( %) of tissue radioactivity was released by 0.1 M 5-HT, and radioactivity increased to 34 2% with 1 M 5-HT (Fig. 1A). Neither DA nor NE at low concentrations ( M) evoked the release of [ 3 H]5-HT (Fig. 1A). The increase in 3 H efflux induced by 5-HT was greatly antagonized by coperfusion with 1 M citalopram (P ) (Fig. 2). These results suggest that when the olfactory tubercle slices are incubated with [ 3 H]5-HT in the absence of any drugs (controls), the radioactive amine most likely labels the 5-HT stores. Release of [ 3 H]5-HT from Slices Labeled with [ 3 H]5- HT in the Presence of the SSRI, Citalopram. In this set of experiments, the slices were incubated with 0.1 M [ 3 H]5- HT in the presence of citalopram (1 M). The objective was to determine whether 5-HT could be stored in other monoaminergic terminals when its neuronal uptake transport was blocked with citalopram. After the labeling period, citalopram was removed by extensive washout during the continuous superfusion of the slices. Once the basal efflux had leveled off to a monoexponential decay rate, sample collection TABLE 1 [ 3 H]5-HT uptake in slices of rabbit olfactory tubercle: effects of citalopram and nomifensine Slices were incubated with 1 M[ 3 H]5-HT in the presence or absence of nomifensine, citalopram, or of a combination of both inhibitors. Inhibition of [ 3 H]5-HT uptake was expressed as a percentage of control slices not exposed to the uptake inhibitors. Treatment [ 3 H]5-HT Uptake % Inhibition Control (without drug) 63,000 1,664 1 M nomifensine 56,136 5,294 (N.S.) *,# 10 M nomifensine 52,756 3,274* *,# 1 M citalopram 22, ** ** 10 M citalopram 18, ** ** 1 M citalopram 1 M 5,436 34**,# **,# nomifensine 1 M citalopram 10 M nomifensine 36 1**,# **,# N.S., not significantly different from control values. * Significantly different from control values (P 0.05). ** Significantly different from control values (P 0.001). # Significantly different from 1 M citalopram (P 0.001).

3 176 Cubeddu et al. Fig. 2. Citalopram inhibits 5-HT-induced released of [ 3 H]5-HT. Slices were labeled with 0.1 M [ 3 H]5-HT in the absence of citalopram. Immediately after incubation, the slices were extensively washed with superfusion medium (37 C) and placed on superfusion chambers. Superfusion rate was 1 ml/min. Fraction collection (5-min fractions) started after a 75-min washout period with drug-free medium. After obtaining a basal efflux of radioactivity for 20 min, slices were superfused with stepwise increasing concentrations (20 min each) of 5-HT (controls). In another set of experiments, the releasing effects of 5-HT were evaluated in the presence of 1 M citalopram. In these experiments, citalopram was added to the superfusion medium 30 min prior to initiating the superfusion with 5-HT. Ordinate, increase in [ 3 H]5-HT efflux induced by 5-HT expressed as a percentage of tissue radioactivity. Abscissa, log molar concentration of 5-HT. Release in the presence of citalopram was significantly different from in controls. Significantly different from controls at, P 0.01;, P Fig. 1. Release of [ 3 H]5-HT induced by 5-HT, DA, and NE from slices labeled with [ 3 H]5-HT in the absence and in the presence of citalopram. Slices were incubated with 0.1 M [ 3 H]5-HT in the absence (A) or presence (B) of 1.0 M citalopram. Immediately after incubation, the slices were extensively washed with superfusion medium (37 C) and placed on superfusion chambers. Superfusion rate was 1 ml/min. Fraction collection (5-min fractions) started after a 75-min washout period with drug-free medium. After obtaining a basal efflux of radioactivity for 20 min, slices were superfused with stepwise increasing concentrations (20 min each) of either 5-HT, DA, or NE. The efflux of [ 3 H]5-HT (total [ 3 H]) was quantified and expressed as a percentage of tissue radioactivity (ordinates). The abscissas depict the log molar concentration of nonradioactive 5-HT, DA, and NE. was initiated. Compared with control slices, low concentrations of nonradioactive 5-HT ( M) in slices labeled in the presence of citalopram were no longer able to evoke the release of [ 3 H]5-HT. Higher concentrations of 5-HT ( M) were now required to induce significant displacement of [ 3 H]5-HT (Fig. 3). Indeed, in control slices, 0.1 M 5-HT released % and 1.0 M released 34 2% of tissue radioactivity. In slices labeled in the presence of citalopram, 0.1 M 5-HT failed to evoke release, and 1.0 M released only % of tissue radioactivity (P 0.001) (compare Fig. 1, A and B). In slices labeled in the presence of citalopram, superfusion with NE or DA at concentrations as low as 0.3 and 1 M evoked significant increases in the release of [ 3 H]5-HT (Fig. 1B). However, in control experiments (labeling without citalopram), 1 and 3 M DA and NE failed to evoke the release of [ 3 H]5-HT (Fig. 1A). In addition, in slices labeled in the presence of citalopram, when citalopram was coperfused with DA or NE, it potentiated the releasing effects of both catecholamines (Fig. 3). Both DA and NE (3 10 M) evoked a much greater release of [ 3 H]5-HT when perfused in the presence of citalopram (P ) (Fig. 3). On the other hand, when citalopram was coperfused with 5-HT, it induced a very small parallel shift to the right in the concentration-release curve for 5-HT (P ) (Fig. 3). In slices labeled in the presence of citalopram, nomifensine reduced NE, DA, and 5-HT-evoked release of [ 3 H]5-HT (Fig. 4). Nomifensine antagonized the releasing effect of NE to a greater extent than that of DA (Fig. 4). Although for both catecholamines, nomifensine seemed to induced a parallel shift to the right, the release evoked by 5-HT was decreased, but only at the highest concentrations, 3 to 10 M (P ) (Fig. 4). Discussion Incubation of control rabbit olfactory tubercle slices with concentrations of [ 3 H]5-HT (0.1 M) close to the K m for the neuronal uptake ( nm) (Richelson and Pfenning, 1984; O Reilly and Reith, 1988), led to selective labeling of 5-HT terminals. This statement is based on the following experimental facts (present study): 1) only submicromolar concentrations of 5-HT, but not DA or NE, displaced radioactivity from slices preloaded with [ 3 H]5-HT, 2) 5-HT-evoked release of [ 3 H]5-HT was markedly antagonized by 1 M citalopram, and 3) nomifensine (a preferential NE/DA uptake

4 Citalopram and 5-HT Storage in Catecholaminergic Neurons 177 Fig. 3. Effects of citalopram on the release of [ 3 H]5-HT induced by DA, NE, and 5-HT from slices labeled with [ 3 H]5-HT in the presence of citalopram. In this set of experiments, the slices were loaded with 0.1 M [ 3 H]5-HT in the presence of 1 M citalopram. Immediately after incubation, the slices were extensively washed with superfusion medium (37 C) and placed on superfusion chambers. Superfusion rate was 1 ml/min. Fraction collection (5-min fractions) started after a 75-min washout period with drug-free medium. After obtaining a basal efflux of radioactivity for 20 min, slices were superfused with stepwise increasing concentrations (20 min each) of either DA, NE, or 5-HT. In another set of experiments, the releasing effects of DA, NE, and 5-HT were evaluated in the presence of 1 M citalopram, which was added to the superfusion medium 30 min before initiating the superfusion with DA, NE, or 5-HT. Ordinates, increase in [ 3 H]5-HT efflux induced by DA, NE, or 5-HT expressed as a percentage of tissue radioactivity. Abscissa, log molar concentration of DA, NE, or 5-HT. Significantly different from experiments in the absence of citalopram at, P 0.01;, P Fig. 4. Effects of nomifensine on the release of [ 3 H]5-HT induced by DA, NE, and 5-HT from slices labeled with [ 3 H]5-HT in the presence of citalopram. In this set of experiments, the slices were loaded with 0.1 M [ 3 H]5-HT in the presence of 1 M citalopram. Immediately after incubation, the slices were extensively washed with superfusion medium (37 C) and placed on superfusion chambers. Superfusion rate was 1 ml/min. Fraction collection (5-min fractions) started after a 75-min washout period with drug-free medium. After obtaining a basal efflux of radioactivity for 20 min, slices were superfused with stepwise increasing concentrations (20 min each) of either DA, NE, or 5-HT. In another set of experiments, the releasing effects of DA, NE, and 5-HT were evaluated in the presence of 1 M nomifensine, which was added to the superfusion medium 30 min before initiating the superfusion with DA, NE, or 5-HT. Ordinates, increase in [ 3 H]5-HT efflux induced by DA, NE, or 5-HT expressed as a percentage of tissue radioactivity. Abscissa, log molar concentration of DA, NE, or 5-HT. Significantly different from experiments in the absence of nomifensine at, P 0.01;, P inhibitor) had negligible (10 15%) effects in inhibiting 5-HT uptake. Our conclusion is based on the specificity of the drugs employed and on the premise that 5-HT and catecholamine neuronal pumps do not take up 5-HT metabolites (Caccia et al., 1992; Manfridi et al., 1992; Wong et al., 1995). Thus, the tissue labeling and the displacement of 3 H from the slices most likely derive from unchanged [ 3 H]5-HT. The concentration of 1 M citalopram employed in this study is 1000-fold higher than its K i for 5-HT uptake, and 4 and 28 times lower than its K i for NE and DA uptake, respectively (Richelson and Pfenning, 1984; Sitte et al., 2000), indicating that citalopram is selective for the 5-HT transporter. Consequently, 1 M citalopram should have blocked 5-HT uptake with negligible effects on neuronal catecholamine transporters. Nomifensine, on the other hand, selectively inhibits NE and DA transporters. Its small inhibitory effect on [ 3 H]5-HT accumulation may result from a slight effect on 5-HT transport, since nomifensine K i for 5-HT uptake is about 1.2 M (Richelson and Pfenning, 1984). A striking finding of this study was the inability of high micromolar concentrations of citalopram to produce a complete block of [ 3 H]5-HT uptake. Olfactory tubercle slices incubated in the presence of concentrations of citalopram as high as 10 M could still take up and retain considerable amounts of [ 3 H]5-HT (30 40% of the amount of radioactivity taken up by control slices). The concentration of citalopram used should have been sufficient to block completely the neuronal 5-HT transporter, since it was 5,000- to 10,000-fold higher than the K i for the 5-HT uptake (Richelson and Pfenning, 1984; O Reilly and Reith, 1988). The lack of complete inhibition of [ 3 H]5-HT uptake by citalopram suggests that when the 5-HT uptake is blocked, [ 3 H]5-HT may be labeling non-5-ht terminals. This is supported by the observation that addition of nomifensine (combined treatment with 1 M citalopram and 1 10 M nomifensine) completely blocked the labeling of the slices. Because nomifensine preferentially inhibits NE and DA transporters, our findings suggest that the labeling occurring when [ 3 H]5-HT is incubated in the presence of high concentrations of citalopram may be due to storage of [ 3 H]5-HT in catecholaminergic terminals. We thus

5 178 Cubeddu et al. propose that when the 5-HT neuronal transporter (highest affinity site for 5-HT) is blocked, 5-HT may accumulate in sites of lower affinity, such as the DA and NE terminals. Further support for this view derives from experiments on the displacement of stored [ 3 H]5-HT by nonradioactive monoamines. As previously discussed, when the labeling of the rabbit olfactory tubercle slices with radioactive 5-HT is conducted in the absence of drugs or treatments (controls), tissue [ 3 H]5-HT is selectively displaced by submicromolar concentrations of nonradioactive 5-HT. However, in slices preloaded with [ 3 H]5-HT in the presence of citalopram, the radioactivity retained by the slices (after extensive washout) is released only by higher concentrations of 5-HT. In addition, concentrations of DA and NE that failed to evoke [ 3 H]5- HT efflux from control slices were effective in evoking [ 3 H]5- HT efflux from slices labeled in the presence of citalopram. The marked reduction of the ability of 5-HT to displace [ 3 H]5- HT, associated with a potentiation of the [ 3 H]5-HT releasing action of DA and NE, further suggests that when the access of 5-HT to its nerve terminals is hindered by an SSRI, 5-HT may be stored in catecholaminergic terminals. In slices preloaded in the presence of citalopram, the increase in efflux of [ 3 H]5-HT induced by DA and NE was not inhibited but enhanced by coperfusion with citalopram. This finding suggests that when the slices are labeled in the presence of citalopram, DA and NE release [ 3 H]5-HT from catecholaminergic terminals and not from 5-HT nerve terminals. If DA and NE had released [ 3 H]5-HT from 5-HT terminals, citalopram would have inhibited the increase in 3 H efflux induced by the catecholamines. The fact that coperfusion with citalopram enhanced [ 3 H]5-HT evoked by DA and NE indicates that part of the [ 3 H]5-HT released by DA and NE is taken up by the 5-HT terminals (i.e., [ 3 H]5-HT efflux is less than actual [ 3 H]5-HT release). Therefore, the increase in [ 3 H]5-HT efflux induced by DA and NE in the presence of citalopram best reflects the amount of [ 3 H]5-HT released from catecholaminergic terminals by these monoamines. Under these conditions, NE and DA are 3 to 10 times more potent than 5-HT in evoking [ 3 H]5-HT release, further supporting the view that DA and NE were releasing [ 3 H]5-HT from catecholaminergic terminals. Experiments employing cold 5-HT as a releasing agent indicate that the efflux of [ 3 H]5-HT evoked by micromolar concentrations of cold 5-HT results from two effects: 1) displacement of stored [ 3 H]5-HT and 2) inhibition of [ 3 H]5-HT reuptake into 5-HT nerve terminals by competition with cold 5-HT. Therefore, even if cold 5-HT has a lower affinity than DA and NE to release [ 3 H]5-HT from catecholaminergic terminals, any [ 3 H]5-HT released by cold 5-HT would contribute to the efflux of radioactivity because it would not be taken up by the 5-HT terminals. This dual action of cold 5-HT accounts for the negligible effects of citalopram on [ 3 H]5-HT efflux evoked by cold 5-HT, since both citalopram and cold 5-HT would competitively inhibit the reuptake of [ 3 H]5-HT. The observation that nomifensine, an inhibitor of the catecholamine neuronal transporter, antagonized the increase in efflux of [ 3 H]5-HT evoked by DA, NE, and 5-HT, suggests that the three monoamines were releasing [ 3 H]5-HT from catecholaminergic terminals. All the above-described findings support the view that incubation of olfactory tubercle slices with [ 3 H]5-HT in the presence of citalopram leads to accumulation of [ 3 H]5-HT in catecholaminergic neurons. Information, albeit indirect, about a possible preferential accumulation of [ 3 H]5-HT in NE neurons derives from experiments conducted in the presence of nomifensine. In slices preloaded with [ 3 H]5-HT in the presence of citalopram, nomifensine produced a much greater antagonism of the releasing effect of NE than of DA. These results may in part be explained by the greater affinity of nomifensine for NE than for the DA transporter. In fact, the K i of nomifensine to inhibit NE uptake is 10 times lower than its K i for inhibition of DA uptake (Richelson and Pfenning, 1984). However, it is also possible that differences in density and spatial distribution between dopaminergic and noradrenergic neurons in relationship to the serotonergic neurons may also account for the findings. A closer anatomical association between noradrenergic and serotonergic terminals, than of dopaminergic with serotonergic terminals, may also account for the greater effects of nomifensine on NE than on DA. In summary, we propose that when the 5-HT uptake pump is blocked with citalopram, extracellular [ 3 H]5-HT is shunted into NE and possibly DA terminals, where it could exert multiple actions. 5-HT may by competition inhibit DA and NE uptake and metabolism, and be stored in synaptic vesicles from which it could be coreleased with the catecholamines, acting as a false neurotransmitter. In agreement with our findings, the in vivo microdialysis application of clomipramine, a 5-HT uptake inhibitor, increases DA efflux from the striatum (Santiago et al., 1998). This releasing effect was inhibited by nomifensine, suggesting that when 5-HT uptake is blocked, 5-HT may be shunted to DA neurons inducing DA release (Santiago et al., 1998). 5-HT release from DA neurons has also been demonstrated in the intermediate lobe of the rat pituitary by a classical calcium-dependent exocytotic mechanism (Vanhatalo and Soinila, 1995). Notably, the release of 5-HT as a false neurotransmitter can have functional postsynaptic consequences. For example, canine coronary adrenergic nerves can take up 5-HT secreted from platelets and subsequently release it to cause contraction of the coronary arteries (Palkovits et al., 1986). In the central nervous system, chronic selective blockade of 5-HT uptake increases functional and biochemical DA parameters in rats, such as DA agonist-induced psychomotor response (Maj, 1990) and aggressiveness (Maj et al., 1979), DA receptor number, and DA agonist efficacy in the DA mesolimbic system (Serra et al., 1992). Although these findings may be explained by other mechanisms, the possibility that some of these effects may be related to accumulation of 5-HT in DA neurons and false neurotransmission cannot be ruled out. For example, compensatory changes in catecholaminergic neurons, especially up-regulation of postsynaptic DA and/or NE receptors, may occur if 5-HT instead of DA or NE is released from DA and/or NE neurons. If so, such an effect may account for some of the mood-elevating effects of antidepressants. The mild amphetamine-like effects of some SSRIs may be due to sensitization of the DA system induced by prolonged false neurotransmission and/or by increased extracellular levels of DA and NE due to release and uptake inhibition by shunted 5-HT. In fact, acute inhibition of DA uptake in superfused rat striatal synaptosomes by amineptine increases the overflow of DA, 5-HT, and NE (Garattini and Mennini, 1989), suggesting that selective blockade of catecholamine uptake may cause a similar interaction with other monoamine terminals.

6 Citalopram and 5-HT Storage in Catecholaminergic Neurons 179 References Caccia S, Fracasso C, Gerattini S, Guiso G, and Sarati S (1992) Effects of short-and long-term administration of fluoxetine on the monoamine content of rat brain. Neuropharmacology 31: Conzolazione A and Cuello AC (1982) CNS serotonin pathways, in Biology of Serotoninergic Transmission (Osborne NN ed) pp 29 61, Wiley, New York. Dahlstrom A and Fuxe K (1964) Localization of monoamines in the lower brain stem. Experientia (Basel) 20: Fallon JH and Moore RY (1987) Catecholamine innervation of the basal forebrain. III. Olfactory bulb, anterior olfactory nuclei, olfactory tubercle and piriform cortex. J Comp Neurol 180: Feuerstein TJ, Hertting G, Lupp A, and Neufang B (1986) False labelling of dopaminergic terminals in the rabbit caudate nucleus: uptake and release of [ 3 H]-5- hydroxytryptamine. Br J Pharmacol 88: Fozard JR and Berry JL (1976) Interactions between antimigraine drugs and a high affinity uptake and storage mechanism for 5-hydroxytryptamine. Pharmacology 14: Garattini S and Mennini T (1989) Pharmacology of amineptine: synthesis and updating. Clin Neuropharmacol 12:S13 S18. Maj M (1990) Behavioral effects of antidepressant drugs given repeatedly on the dopaminergic system, in Dopamine and Mental Depression (Gessa GL and Serra G eds) pp , Pergamon Press, Oxford. Maj M, Mogilnicka E, and Klimek V (1979) The effect of repeated administration of antidepressant drugs on the responsiveness of rats to catecholamine agonists. J Neural Transm 44: Manfridi A, Clavenna A, and De Simoni MG (1992) Serotonin uptake inhibition: in vivo effect of sertraline in rats. Neurosci Lett 139: O Reilly CA and Reith ME (1988) Uptake of [ 3 H]-serotonin into plasma membrane vesicles from mouse cerebral cortex. J Biol Chem 263: Palkovits M, Mezey E, Chiueh CG, Krieger DT, Gallatz K, and Brownstein MJ (1986) Serotonin-containing elements of the rat pituitary intermediate lobe. Neuroendocrinology 42: Richelson E and Pfenning M (1984) Blockade by antidepressants and related compounds of biogenic amine uptake into rat brain synaptosomes: most antidepressants selectively block norepinephrine uptake. Eur J Pharmacol 104: Rosin DL, Melia K, Knorr AM, Nestler EJ, Roth RH, and Duman RS (1995) Chronic imipramine administration alters the activity and phosphorylation state of tyrosine hydroxylase in dopaminergic regions of rat brain. Neuropsychopharmacology 12: Saland LC, Wallace JA, and Comunas F (1986) Serotonin-immunoreactive nerve fibers of the rat pituitary: effects of anticatecholamine and antiserotonin drugs on staining patterns. Brain Res 368: Santiago M, Matarredona ER, Machado A, and Cano J (1998) Influence of serotoninergic drugs on in vivo dopamine extracellular output in rat striatum. J Neurosci Res 52: Serra G, Collu M, D Aquila PS, and Gessa GL (1992) Role of the mesolimbic dopamine system in the mechanism of action of antidepressants. Pharmacol Toxicol 71: Sitte HH, Scholze P, Schloss P, Pifl C, and Singer EA (2000) Characterization of carrier-mediated efflux in human embryonic kidney 293 cells stably expressing the rat serotonin transporter: a superfusion study. J Neurochem 74: Thind KK, Boggan JE, Song T, and Goldsmith PC (1987) Immunostaining reveals accumulation of serotonin and coexistence with tyrosine hydroxylase in hypothalamic neurons of acutely stalk-sectioned baboons. Neuroendocrinology 45: Vanhatalo S and Soinila S (1995) Release of false transmitter serotonin from the dopaminergic nerve terminals of the rat pituitary intermediate lobe. Neurosci Res 22: Wong PT, Feng H, and Teo WL (1995) Interaction of the dopaminergic and serotonergic systems in the rat striatum: effects of selective antagonists and uptake inhibitors. Neurosci Res 23: Address correspondence to: Luigi X. Cubeddu, Nova Southeastern University, HPD, 3200 S. University Dr., Ft. Lauderdale, FL lcubeddu@nova.edu