Psychopharmacology (2001) 155:434439 DOI 10.

1007/s002130100746

O R I G I N A L I N V E S T I G AT I O N

Abraham Zangen Rachel Nakash David H. Overstreet Gal Yadid

Association between depressive behavior and absence of serotonindopamine interaction in the nucleus accumbens
Received: 8 December 2000 / Accepted: 13 February 2001 / Published online: 21 April 2001 Springer-Verlag 2001

Abstract Rationale: Current hypotheses on the etiology of depression attribute the disorder to alterations in serotonin and norepinephrine neurotransmission. However, the relationship between these alterations and depressive behavior is poorly understood. Conversely, an interaction between the serotonergic and dopaminergic systems in the nucleus accumbens has been established. Since motivation and hedonia have been associated with dopamine release in the nucleus accumbens, we decided to test its modulation by serotonin in relation to depressive-like behavior. Objectives and methods: The extracellular dopamine levels in the nucleus accumbens were studied in vivo in Flinders Sensitive Line (FSL, a rat model of depressive behavior) and control rats, before and after antidepressant treatment. Rats were chronically treated with the antidepressants desipramine (5 mg/kg/day) and paroxetine (7.5 mg/kg/day) for 18 consecutive days. As a measure of depressive behavior we used a modified swim test. The release of dopamine in response to local serotonin application was monitored using the microdialysis technique. Results: Serotonin (0.5 M) facilitated dopamine release in the nucleus accumbens of control rats. In FSL rats, basal extracellular dopamine levels in the nucleus accumbens were 40% lower than in control rats and did not increase in response to serotonin stimulation. However, chronic antidepressant treatment of the FSL rats normalized the serotonindopamine interaction as well as their behavioral deficiencies. Conclusions: The inability of serotonin to stimulate dopamine release in the nucleus accumbens, thereby leading to anhedonia and lack of motivation, may therefore be an essential factor in the onset of depression and a target for modulation by antidepressant drugs.
A. Zangen R. Nakash G. Yadid () Faculty of Life Science, Bar Ilan University, Ramat-Gan, Israel e-mail: yadidg@mail.biu.ac.il Tel.: +972-3-5318123, Fax: +972-3-5351824 D.H. Overstreet Department of Psychiatry, University of North Carolina, Chapel Hill, North Carolina, USA

Keywords Animal model Antidepressant Dopamine Serotonin Microdialysis

Introduction
Alterations in monoamine neurotransmission are associated with depressive disorders and with antidepressant modes of action (Jefferson and Greist 1994; Feldman et al. 1997), however without clarifying the relationship between these alterations and the etiology of depression. Release of dopamine (DA) in the nucleus accumbens has been shown to be associated with motivation and hedonia (Koob and Bloom 1988; Self and Nestler 1995; Wise 1996; Schultz et al. 1997). Therefore, we studied monoamine levels and release in the nucleus accumbens in a rat model of depressive behavior, before and after chronic antidepressant therapy. The genetically selected Flinders Sensitive Line (FSL) of rats was used. These rats exhibit behavioral features characteristic of depression, such as reduced locomotion, reduced activity in the swim test, increased anhedonia in response to chronic mild stress, increased amount and reduced onset of REM (rapid eye movement) sleep, cognitive difficulties, and reduced body weight (for review see Overstreet 1993). Chronic, but not acute, treatment of the FSL rats with antidepressants, such as desipramine, imipramine, sertraline, and fluoxetine, almost abrogates their behavioral manifestations of depression (for review see Yadid et al. 2000b, 2000c). We have previously demonstrated increased tissue content of serotonin (5-HT) and DA in the limbic brain regions of FSL rats (with a six- to sevenfold increase in the nucleus accumbens), which was normalized following chronic antidepressant treatment (Zangen et al. 1997, 1999; Yadid et al. 2000b). It was not surprising, therefore, that mRNA levels for tyrosine hydroxylase (the rate-limiting enzyme for catecholamine biosynthesis) were significantly higher in the ventral tegmental area (the main location of dopaminergic cells that project into the nucleus accumbens) of FSL rats compared with control Sprague Dawley rats (Serova et al. 1998).

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Local application of 5-HT into the nucleus accumbens of Sprague-Dawley rats has previously been shown to stimulate local DA release (Parsons and Justice 1993; De Deurwaerdere et al. 1998). In the present study, basal monoamine release and the interaction between 5-HT and DA in the nucleus accumbens was studied in FSL and control rats before and after chronic antidepressant treatment using the in vivo microdialysis technique in freely moving rats. The behavioral performance of each rat group was measured by means of the swim test. The present study emphasizes the communication between the serotonergic and dopaminergic systems in the nucleus accumbens and has important implications regarding the abnormalities that may mediate depressive behavior.

pumped continuously (1 l/min) through the dialysis probe using a microinjection pump (CMA 100, Carnegie Medicine). The experiments were initiated 2224 h after probe implantation in awake, freely moving rats. The dialysates were collected at 30-min intervals into polyethylene tubes containing 15 l ethylene diamine tetraacetic acid (EDTA)/ethanol (0.02/1%) and stored at 70C until subjected to high-performance liquid chromatography (HPLC) monoamine analysis. After five baseline collections, the aCSF was switched to aCSF containing 0.5 M 5-HT for one fraction (30 min) and then switched back to normal aCSF for five more fractions. At the conclusion of each microdialysis experiment, the rat was euthanased with chloral hydrate and its brain was removed into a 4% paraformaldehyde solution. The position of the probe in the brain was verified by pumping crystal violet via the probe and examining brain sections by light microscopy (Yadid et al. 2000a). Analysis of the monoamines and their metabolites Quantification of DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-HT, and 5-hydroxyindoleacetic acid (5-HIAA) levels in the microdialysates was performed as described previously (Zangen et al. 1999; Yadid et al. 2000a). Briefly, the dialysates were injected directly via a HPLC pump (Model 510, Waters; Milford, MA) onto a column (Merck C-18, 5 m; 4.6 mm ID 250 mm, at 30C) coupled to an electrochemical detector (Model 460 Waters; Milford, Mass.) with an EiCOM CB100 analytical cell (EiCOM; Kyoto, Japan) and the oxidation potential set to 0.77 V. The mobile phase (2 l water containing 0.55 g heptane sulfonic acid, 0.2 g EDTA, 16 ml triethylamine, 12 ml 85% phosphoric acid, and 80 ml acetonitrile; pH 2.6) was pumped at 0.8 ml/min. Statistical analysis Data are expressed as the meanSEM of values obtained from the indicated number of rats. Significance was determined using the independent two-tailed t-test or analysis of variance (ANOVA) with application of the student Newman-Keuls post-hoc test as indicated. A probability value of <0.05 was considered significant.

Materials and methods

Animals and drugs Male FSL rats, selectively bred from Sprague-Dawley rats (Overstreet 1993), and control (Sprague-Dawley) rats (230260 g) were housed two per cage under conditions of constant temperature (22C) and humidity (50%), with a 12-h/12-h light/dark cycle. Food and water were provided ad libitum. All animal procedures were approved by the Bar-Ilan University Animal Care Committee and were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Some rats were injected intraperitoneally with desipramine (5 mg/kg; Sigma), paroxetine (7.5 mg/kg; SmithKline Beecham Pharmaceuticals), or vehicle (0.5 ml saline) once a day for 18 days. The behavioral or neurochemical measurements in antidepressant-treated rats were preformed 2024 h after the last antidepressant injection. Different groups of rats were used for the behavioral, or microdialysis experiments. Swim test A modified forced swim test protocol was conducted in a cylindrical tank (40 cm high and 18 cm in diameter; constructed at BarIlan University), which contained enough water at 25C so that the rat could not touch the bottom with its hind paws. The rat was considered to have stopped swimming when both front paws were immobile. The animals underwent a single 5-min exposure to the swim tank 24 h after the last treatment with an antidepressant drug or saline, since previous studies (Pucilowski and Overstreet 1993; Overstreet et al. 1995) have demonstrated that this protocol is effective in detecting the anti-immobility effects of classical antidepressants and in rejecting psychomotor stimulants such as amphetamine, which typically gives false positive results in the standard forced swim test (Borsini and Meli 1988). The swim tests and the biochemical measurements were performed on different groups of animals in order to avoid the effects of stress on the biochemical measurements. Microdialysis Rats (230250 g) were anesthetized with chloral hydrate (400 mg/kg, i.p.). A microdialysis probe (2 mm length, 20 kDa cutoff value, CMA/10, Carnegie Medicine) was surgically implanted into the nucleus accumbens (1.3 mm anterior and 1.0 mm lateral to the bregma and 7.8 mm ventral to the dura) of each rat using a stereotactic device (David-Kopf Instruments) and cemented to the skull. Artificial cerebrospinal fluid (aCSF; 145 mM NaCl, 1.2 mM CaCl2, 2.7 mM KCl, 1.0 mM MgCl2, pH 7.4) was

Results
Behavioural performance in the swim test The immobility score in the swim test (used as a measure of behavioral deficiency; Overstreet et al. 1995; Detke et al. 1997; Weiss et al. 1998) was 96% higher in the FSL than in normal Sprague-Dawley rats. Chronic treatment

Table 1 Immobility during the swim test of Flinders Sensitive Line (FSL) and control rats untreated or chronically treated with desipramine (5 mg/kg/day), paroxetine (7.5 mg/kg/day), or saline for 18 days. MeanSEM values of total immobility in seconds of 12 rats in each group are presented. The swim test was preformed 2224 h after the last injection. Significance was determined by means of analysis of variance followed by the student Newman-Keuls post-hoc test Untreated Control FSL
a P<0.01 b P<0.01

Saline 11311 19416a

Desipramine 8622 8320b

Paroxetine 10820 13214b

9113 17826a

FSL versus control saline versus antidepressant treatment

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levels were not significantly affected by any of the drugs (Fig. 2C, E). 5-HTDA interaction in the nucleus accumbens Local application (via the microdialysis probe) of 0.5 M 5-HT into the nucleus accumbens of the control rats caused a significant increase in local extracellular levels of DA by 22838% of baseline levels. However, no effect of 5-HT on DA levels was observed in the nucleus accumbens of FSL rats (Fig. 3). Other group of rats received chronic treatment with desipramine, paroxetine, or saline for 18 days. On the last day of injection, rats were implanted with microdialysis probes in the nucleus accumbens, and the interaction between 5-HT and DA was studied 2024 h later. Both antidepressant drugs normalized the 5-HTDA interaction in the nucleus accumbens of FSL rats (Fig. 4A). Saline treatment did not have a significant effect on the 5-HTDA interaction in either strain of rat (Fig. 4A, B), and both antidepressants had no significant effect on the 5-HTDA interaction in the control rats (Fig. 4B).

Fig. 1 Basal monoamine levels in dialysates obtained from the nucleus accumbens of Flinders Sensitive Line (FSL) and control rats. Microdialysis was performed in the nucleus accumbens of FSL and control rats. Basal monoamine levels in dialysates obtained from freely moving rats were measured by means of highperformance liquid chromatography. MeanSEM values of ten rats in each group are presented. Significantly different values were detected using the t-test *P<0.01

Discussion
The main finding of the present study was the correlation between depressive behavior and absence of DA responsiveness to 5-HT stimulation in the nucleus accumbens. Chronic antidepressant treatment normalizes both the 5-HTDA interaction and the depressive behavior. Since DA release in the nucleus accumbens is associated with motivation and hedonia (Koob and Bloom 1988; Self and Nestler 1995; Wise 1996; Schultz et al. 1997), the lower basal extracellular levels of DA observed in the nucleus accumbens of FSL rats (Fig. 1) could explain their impaired motivation and anhedonia. However, chronic treatment with desipramine or paroxetine, which normalize the behavioral deficiency (e.g., the immobility score) of FSL rats, did not normalize the extracellular levels of DA in the nucleus accumbens (Fig. 2A). Therefore, no correlation is found between basal extracellular DA levels in the nucleus accumbens and depressive behavior of FSL rats. Although the tissue content (which represents both extracellular and intracellular levels) of DA and 5-HT in the nucleus accumbens of FSL rats has been shown to be 67 times higher than in control rats (Zangen et al. 1997, 1999; Yadid 2000b), the basal extracellular levels of DA were lower in FSL rats and the basal extracellular levels of 5-HT were not significantly different (Fig. 1). This may indicate a relatively decreased dopaminergic and serotonergic firing or increased uptake of DA and 5-HT in the depressed rats. The effect of paroxetine on the basal extracellular levels of 5-HT (Fig. 2B) was expected since paroxetine inhibits 5-HT re-uptake, thereby increasing extracellular levels of 5-HT. The effect of paroxetine on basal extra-

with the antidepressants desipramine (a norepinephrine re-uptake inhibitor) or paroxetine (a 5-HT re-uptake inhibitor) for 18 days normalized the immobility score of the FSL rats, but did not significantly affect the immobility of the control rats (Table 1). Basal extracellular levels of DA, 5-HT, and metabolites The basal extracellular levels of DA and its main metabolites, DOPAC and HVA in the nucleus accumbens of FSL rats were found to be 4050% lower than those in control rats (Fig. 1). However, the basal extracellular levels of 5-HT and its metabolite 5-HIAA in the nucleus accumbens of FSL rats were not significantly different from the control rats (Fig. 1). Chronic treatment with desipramine did not significantly affect the basal extracellular levels of DA in either rat line, while paroxetine decreased DA levels by 55% and 58% in control and FSL rats, respectively, relative to saline-treated rats (Fig. 2A). The effect of chronic antidepressant treatment on basal extracellular 5-HT levels was found to be opposite when comparing desipramine and paroxetine treatments. Desipramine decreased the 5-HT levels in both rat lines by 71% and 74%, while paroxetine increased 5-HT levels by 172% and 291% in control and FSL rats, respectively, relative to salinetreated rats (Fig. 2B). The 5-HIAA levels were decreased by both drugs in both rat lines by 3050%, relative to saline-treated rats (Fig. 2D), while the DOPAC and HVA

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Fig. 2AE Effect of antidepressant treatment on extracellular levels of monoamines in the nucleus accumbens of Flinders Sensitive Line (FSL) and control rats. FSL or control rats were chronically treated with desipramine (5 mg/kg/day), paroxetine (7.5 mg/kg/day), or saline for 18 days. Microdialysis was performed in the nucleus accumbens of FSL and control rats 2024 h after the last antidepressant injection. Basal monoamine levels in dialysates obtained from freely moving rats were measured by means of high-performance liquid chromatography. MeanSEM values of eight rats in each group are presented. Values statistically different from salinetreated rats were determined by means of analysis of variance followed by the student Newman-Keuls post-hoc test. *P<0.01

cellular levels of DA (Fig. 2A) may be explained by the inhibitory effect of the serotonergic system on dopaminergic tone at the level of the origin of the DA system in the ventral tegmental area and the substantia nigra (Kapur and Remington 1996). The effect of desipramine, which is mainly a noradrenaline re-uptake inhibitor on extracellular levels of 5-HT, may be explained by the inhibitory effect of noradrenaline on the serotonergic tone via 1 and 2 adrenoceptors (Baraban and Aghajanian 1980; Tao and Hjorth 1992).

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Fig. 3 Effect of local application of serotonin (5-HT) on extracellular levels of dopamine in the nucleus accumbens of Flinders Sensitive Line (FSL) and control rats. The microdialysis probe was perfused with artificial cerebrospinal fluid (aCSF) before and after a 30-min perfusion with aCSF containing 0.5 M 5-HT. Dialysates were collected at 30-min intervals. The mean dopamine level measured in the four dialysates prior to the 5-HT perfusion was used as the baseline dopamine level. MeanSEM values of ten rats in each group are presented. Values statistically different from baseline were determined by means of analysis of variance with repeated measures over time followed by the student Newman-Keuls post-hoc test. *P<0.01

Since 5-HIAA is produced by monoamine oxidase (MAO) acting on free 5-HT in the cytosol (which is obtained mostly due to 5-HT re-uptake; Commissiong 1985), the decreased 5-HIAA levels after paroxetine treatment (Fig. 2D) may be explained by reduction of free 5-HT in the cytosol due to 5-HT re-uptake inhibition in both rat lines. A decrease in free 5-HT in the cytosol may also be obtained by desipramine treatment, since desipramine decreased extracellular levels of 5-HT (probably due to decreased serotonergic firing, as mentioned), therefore the total amount of 5-HT that can undergo a re-uptake process is also reduced. The absence of correlation between depressive behavior of FSL rats and the basal extracellular monoamine levels in the nucleus accumbens led us to seek stimulated rather than basal levels. The basic symptoms characterizing the depressed patient are anhedonia and lack of motivation, both of which are expressed in the absence of an immediate response to environmental stimuli (Jefferson and Greist 1994). In an attempt to correlate psychiatric definitions and neurochemical processes, we assumed that the responses to environmental stimuli, which usually cause release of DA in the nucleus accumbens (leading to hedonia and motivation) and its regulation, should be the focus for elucidating the neurochemical etiology of depressive behavior. It does not seem likely that abnormal basal levels of neurochemical substances can explain the behavioral deficiencies. Previously, it has been demonstrated that local application of 5-HT into the nucleus accumbens of Sprague-

Fig. 4 Effect of antidepressant treatment on the serotonin (5-HT) dopamine interaction in the nucleus accumbens of Flinders Sensitive Line (FSL) and control rats. FSL or control rats were chronically treated with desipramine (5 mg/kg/day), paroxetine (7.5 mg/kg/day), or saline for 18 days. Microdialysis was performed as described in Fig. 3. The effect of artificial cerebrospinal fluid (aCSF) containing 0.5 M

5-HT was tested in all groups. MeanSEM values of eight rats in each group are presented. Values statistically different from baseline (a) or differences between the saline and drug-treated groups (b) were determined by means of analysis of variance followed by the student Newman-Keuls post-hoc test. P<0.01. No significant differences were observed between desipramine- and paroxetine-treated rats

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Dawley rats can stimulate local DA release (Parsons and Justice 1993; De Deurwaerdere et al. 1998). These findings were also observed in our control rats, while no effect on extracellular DA levels was observed in FSL rats (Fig. 3). Chronic treatment with the antidepressants desipramine or paroxetine, which almost abrogates the behavioral deficits of FSL rat, normalized the interaction between 5-HT and DA in the nucleus accumbens (Fig. 4A). The observation that the 5-HTDA interaction in the control rats was not significantly affected by chronic antidepressant treatment (Fig. 4B) is compatible with the observations demonstrating that the behavior of normal rats and the mood of healthy human volunteers are not affected by antidepressant treatment (Table 1; Overstreet 1993; Jefferson and Greist 1994). Since the release of DA in the nucleus accumbens was shown to be essential for reward and motivation, we propose that the inability of 5-HT to induce DA release in FSL rats may account for their behavioral deficiencies. Chronic (but not acute) treatment with antidepressant drugs, which is necessary to affect depressive behavior (American Psychiatry Association 1993; Overstreet 1993; Jefferson and Greist 1994), normalizes the 5-HTDA interaction, probably by affecting neuronal plasticity (Nestler and Duman 1995). We suggest that the impaired 5-HTDA interaction in the nucleus accumbens is an essential factor in the etiology of depression, and its normalization by chronic antidepressant treatment gives a new perspective on the mode of action of these drugs.
Acknowledgements The studies presented herein were supported in part by grants from the National Institute for Psychobiology in Israel (no. 3299) and the Ministry of Health, Israel (no. 4917) to Gal Yadid.

References
American Psychiatry Association (1993) Practice guideline for major depressive disorder in adults. Am J Psychiatry 150[Suppl 4]: 126 Baraban JM, Aghajanian GK (1980) Suppression of firing activity of 5-HT neurons in the dorsal raphe by alpha-adrenoceptor antagonists. Neuropharmacology 19:355363 Borsini F, Meli A (1988) Is the forced swimming test a suitable model for revealing antidepressant activity? Psychopharmacology 94:147160 Commissiong JW (1985) Monoamine metabolites: their relationship and lack of relationship to monoamineergic neuronal activity. Biochem Pharmacol 34:11271131 De Deurwaerdere P, Stinus L, Spampinato U (1998) Opposite change of in vivo dopamine release in the rat nucleus accumbens and striatum that follows electrical stimulation of dorsal raphe nucleus: role of 5-HT3 receptors. J Neurosci 18:6528 6538

Detke MJ, Johnson J, Lucki I (1997) Acute and chronic antidepressant drug treatment in the rat forced swimming test model of depression. Exp Clin Psychopharmacol 5:107112 Feldman RS, Meyer JS, Quenzer LF (1997) Principles of Neuropharmacology. Sinauer, Sunderland, Mass Jefferson JW, Greist JH (1994) Mood disorders. In: Hales RE, Yudofsky SC, Talbott JA (eds) Textbook of psychiatry. American Psychiatry Press, Washington DC, pp 465494 Kapur S, Remington G (1996) Serotonin-dopamine interaction and its relevance to schizophrenia. Am J Psychiatry 153:466476 Koob GF, Bloom FE (1988) Cellular and molecular mechanisms of drug dependence. Science 242:715723 Nestler EJ, Duman RS (1995) Intracellular messenger pathways as mediators of neural plasticity. In: Bloom FE, Kupfer DJ (eds) Psychopharmacology: the fourth generation of progress. Raven Press, New York, pp 695704 Overstreet DH (1993) The Flinders sensitive line rats: a genetic animal model of depression. Neurosci Biobehav Rev 17:5168 Overstreet DH, Pucilowski O, Rezvani AH, Janowsky DS (1995) Administration of antidepressants, diazepam and psychomotor stimulants further confirms the utility of Flinders Sensitive Line rats as an animal model of depression. Psychopharmacology 121:2737 Parsons LH, Justice JB Jr (1993) Perfusate serotonin increases extracellular dopamine in the nucleus accumbens as measured by in vivo microdialysis. Brain Res 606:195199 Pucilowski O, Overstreet DH (1993) Effect of chronic antidepressant treatment on responses to apomorphine in selectively bred rat strains. Behav Brain Res 32:471475 Schultz W, Dayan P, Montague PR (1997) A neural substrate of prediction and reward. Science 275:15931599 Self DW, Nestler EJ (1995) Molecular mechanisms of drug reinforcement and addiction. Annu Rev Neurosci 18:463495 Serova L, Sabban E, Zangen A, Overstreet DH, Yadid G (1998) Altered gene expression for catecholamine biosynthetic enzymes and stress response in rat genetic model of depression. Mol Brain Res 63:133138 Tao R, Hjorth S (1992) Alpha-2 adrenoceptor modulation of rat ventral hippocampal 5-hydroxytryptamine release in-vivo. Naunyn-Schmiedeberg's Arch Pharmacol 345:137143 Weiss JM, Cierpial MA, West CHK (1998) Selective breeding of rats for high and low motor activity in a swim test: toward a new animal model of depression. Pharmacol Biochem Behav 61:4966 Wise RA (1996) Neurobiology of addiction. Curr Opin Neurobiol 6:243251 Yadid G, Harvey-White J, Kopin IJ, Goldstein S (2000a) Estimation of striatal dopamine spillover and metabolism in vivo. Neuroreport 11:33673373 Yadid G. Nakash R, Deri I, Grin T, Kinor N, Gispan I, Zangen A (2000b) Elucidation of the neurobiology of depression: insights from a novel genetic animal model. Prog Neurobiol 62:353378 Yadid G, Zangen A, Dmitrochenko A, Overstreet DH, Zohar J (2000c) Screening for new antidepressants with fast onset and long-lasting action. J Drug Dev 50:392399 Zangen A, Overstreet DH, Yadid G (1997) High serotonin and 5hydroxyindoleacetic acid levels in limbic brain regions in a rat model of depression: normalization by chronic antidepressant treatment. J Neurochem 69:24772483 Zangen A, Overstreet DH, Yadid G (1999) Increased catecholamine levels in specific brain regions of a rat model of depression: normalization by chronic antidepressant treatment. Brain Res 824:243250