Study of mirtazapine antidepressant effects in rats

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1 International Journal of Neuropsychopharmacology (25), 8, Copyright f 25 CINP doi :1.117/S Study of mirtazapine antidepressant effects in rats ARTICLE Riccardo Rauggi, Anna Cassanelli, Anna Raone, Alessandro Tagliamonte and Carla Gambarana Department of Neuroscience, Pharmacology Unit, University of Siena, Siena, Italy Abstract Mirtazapine is a widely used antidepressant and the aim of this study was to further investigate its antidepressant activity in rats. Thus, the efficacy of long-term mirtazapine treatment was assessed in three models of depressive symptoms induced by stress exposure: the acute escape deficit, the chronic escape deficit, and the stress-induced disruption of the acquisition of an appetitive behaviour sustained by a palatable food (vanilla sugar). Administration of mirtazapine for 2 wk prevented the escape deficit development induced by acute exposure to unavoidable stress. This protective effect was antagonized by the administration of a b-adrenergic or a 5-HT1A receptor antagonist just before stress exposure; that is, mirtazapine effect was dependent on functional b-adrenergic and 5-HT1A receptor systems. Repeated stress exposure indefinitely prolongs the condition of escape deficit and a 4-d mirtazapine treatment reversed this model of chronic escape deficit. In a Y-maze satiated rats learn to choose the arm baited with vanilla sugar, and exposure to stress during Y-maze training prevents this learning. Repeated mirtazapine administration completely antagonized the disrupting effect of chronic stress on the acquisition of this instrumental behaviour. We consider these effects to be crucial in the definition of antidepressant activity. Received 14 July 24; Reviewed 31 August 24; Revised 5 November 24; Accepted 1 November 24 Key words: animal models, appetitive behaviour, b-adrenergic receptors, escape deficit, 5-HT1A receptors, stress. Introduction Mirtazapine is a widely used antidepressant drug with a distinctive and rather complex mechanism of action based on its antagonistic activity on pre- and post-synaptic a 2 -adrenergic receptors, with no selectivity between the two, and on 5-HT2A, 5-HT2C and 5-HT3 serotonergic receptors (De Boer, 1996; De Boer et al., 1988; Haddjeri et al., 1996, 1998). Pre-synaptic a 2 -adrenergic receptors are localized in noradrenergic (autoreceptors) and non-noradrenergic (heteroreceptors) terminals where they play an inhibitory role in neurotransmitter release. Thus, their inhibition may result in an increased release of noradrenaline and other neurotransmitters. In particular, the inhibition by mirtazapine of a 2 -autoreceptors in the locus coeruleus enhances the activity of the noradrenergic Address for correspondence : Dr C. Gambarana, Department of Neuroscience, Pharmacology Unit, University of Siena, Via Aldo Moro, 4, 531 Siena, Italy. Tel.: Fax : gambarana@unisi.it neurons that project to the dorsal raphe (De Boer, 1996). This increased adrenergic input is sustained by the stimulation of the a 1 -adrenoceptors located on dendrites of the serotonergic neurons which, in turn, project to the hippocampus (De Boer et al., 1996). The resulting effect is an increased serotonergic transmission in the hippocampus selectively sustained by the stimulation of 5-HT1A receptors (Berendsen and Broekkamp, 1997), as mirtazapine is an antagonist of 5-HT2A, 5-HT2C and 5-HT3 receptors (De Boer et al., 1988). The inhibition of these receptors has been proposed to reduce the frequency of adverse effects such as anxiety, insomnia, sexual dysfunction, and nausea associated with the unselective increase in serotonin neurotransmission produced by SSRIs (Montgomery, 1995). Moreover, mirtazapine shows an inhibitory effect on the hypothalamus pituitary adrenal axis (Laakmann et al., 1999; Schule at al., 22), and it prevents the stress-induced increase in dopamine release in the medial prefrontal cortex (Dazzi et al., 21). Accordingly, in clinical practice mirtazapine appears to be an antidepressant devoid of

2 37 R. Rauggi et al. anxiogenic activity that does not induce sexual dysfunction (Fawcett and Barkin, 1999; Puzantian, 1998; Thompson, 1999). Both these characteristics have been explained in terms of selective 5-HT1A receptor activation (De Boer et al., 1996; Montgomery, 1995). In order to study the efficacy of classical or novel antidepressant drugs, stress-induced behavioural models of depression in rodents are often used (Willner, 1995). We have validated and used three protocols, based on unavoidable stress exposure, that model in rats some of the core symptoms of depression: acute and chronic escape deficit, and the disruption of acquisition of appetitive behaviour sustained by a highly palatable food (Vanilla sugarsustained Appetitive Behaviour, VAB) in animals fed ad libitum (Gambarana et al., 21; Ghiglieri et al., 1997). The three models consistently respond to classical antidepressants (Gambarana et al., 1995a, 21; Ghiglieri et al., 1997). The first model, acute escape deficit (Gambarana et al., 21; Meloni et al., 1993), a modified version of the learned helplessness model (Overmier and Seligman, 1967; Sherman et al., 1982), evaluates the efficacy of a treatment in preventing the development of hyporeactivity to noxious stimuli, evaluated as escape deficit after unavoidable stress exposure. Moreover, in this model the role played by different receptor systems in the protective activity of an antidepressant treatment can be assessed, as it can be inferred by the effects of acute administration of specific receptor agonists or antagonists (Gambarana et al., 21). In fact, we previously demonstrated in this model that the protective effects of a 2-wk treatment with imipramine, fluoxetine or reboxetine are selectively and completely antagonized by the administration of SCH 2339, pindolol, or propranolol respectively (Gambarana et al., 1995a; Grappi et al., 23). The second model is a condition of escape deficit that can be indefinitely sustained by the repeated administration of mild stressors, chronic escape deficit (De Montis et al., 1995; Gambarana et al., 21). This model evaluates the efficacy of a treatment in reverting the escape deficit. Long-term administration of antidepressants, such as imipramine, clomipramine, phenelzine, reboxetine, and fluoxetine, is effective in both the prevention and the reversal of escape deficit (De Montis et al., 1995; Gambarana et al., 21; C. Gambarana, unpublished observations). In rats both the acute escape deficit and the chronic escape deficit model a condition similar to psychomotor retardation that is one of the components of human depression. The third model exploits the fact that exposure to a repeated unavoidable stress prevents the acquisition of an appetitive behaviour aimed at consuming a palatable food in satiated rats (Ghiglieri et al., 1997), and thus, it models a condition of reduced motivation. These three models are useful for studying the processes that underpin these conditions and they could contribute to furthering our understanding of the neurobiological basis of some of the core symptoms of depression. The aim of the present study was to investigate whether repeated treatment with mirtazapine, an antidepressant with an indirect monoaminergic mechanism of action, would show efficacy in these three stress-induced models of core symptoms of depression. This study also examined whether the behavioural effects of repeated mirtazapine treatment in our models were mediated by the same monoaminergic systems that are indirectly activated by the a 2 -adrenergic activity of mirtazapine (De Boer, 1996). Moreover, the possible involvement of the NMDA receptor system in the development of a mirtazapine protective effect was tested by infusing an NMDA receptor antagonist, dizocilpine, during antidepressant treatment. Method Animals Experiments were carried out on male Sprague Dawley rats (Charles River, Calco, Italy) weighing g at their arrival in the vivarium. Rats were housed five per cage (59r38.5r2 cm) for the entire duration of the experiments and they were moved to a different cage or apparatus only for the time required for behavioural manipulation. They were kept in an environment maintained at a constant temperature and humidity, with free access to food and water. A 12 h reverse light/dark cycle (lights off 7: hours, lights on 19: hours) was used. Experiments were carried out from 9: hours to 17: hours under a red light and controlled noise conditions in a testing room separated from and adjacent to the main animal room, under the same conditions of temperature and humidity. Rats were allowed at least 1 wk of habituation to the animal colony and when experimental procedures began they weighed g. The procedures used in this study are in strict accordance with the European legislation on the use and care of laboratory animals (EEC no. 86/69), with the guidelines of the National Institutes of Health on the use and care of laboratory animals, and had the approval of the Italian Minister of Health and of the University of Siena Ethics Committee.

3 Mirtazapine antidepressant effect 371 Antinociceptive tests Pain threshold was assessed by two antinociceptive tests: the tail-flick method according to D Amour and Smith (1941) and the hot-plate test described by Eddy and Leimbach (1953). For the tail-flick test rats were moved to the test room and placed on the tail-flick apparatus (Ugo Basile, Italy). A 1-s cut-off point was employed to prevent tissue damage to the tail. For the hot-plate test rats were moved to the test room, placed on the hot plate apparatus (Ugo Basile) that was maintained at 55.5 xc and response latencies to the licking of the fore or hind paws, shaking or jumping off the surface were measured. Locomotor activity Locomotor activity was measured by an apparatus consisting of eight compartments (4r45r5 cm) with a transparent Perspex cage (23r33r19 cm) in each compartment (Imetronic, Pessac, France). A small lamp present in the compartment lid allowed the experimenter to observe the behaviour of each animal. Motor activity was detected by a system of photocell infrared beams, dividing the floor area into a rear and a front sector. The interruption of two photocell beams belonging to two different sectors was recorded as an ambulatory activity count. Infrared photocell beams placed at a 15 cm height detected rearing activity. For 5 d before the test day rats were placed in the apparatus for 15 min/d. On test day, rats were observed and their locomotor activity was recorded for 3 min, after a 1-min habituation period. Acute escape deficit Apparatus A dark Plexiglas cage (3r6r3 cm) with a floor fitted with stainless-steel rods spaced 1 cm apart was divided into two equal chambers by a dark Plexiglas partition with a 1r1 cm sliding door. One compartment was connected to a S48 Grass stimulator (Grass Instruments, Astro-Med Inc., West Warwick, RI, USA) (electrified chamber), while the other was disconnected from it (neutral chamber). Procedure The experimental procedure consisted of a pre-test session (exposure to an unavoidable stress of the minimum intensity and duration required to induce a reliable behavioural modification) followed 24 h later by an escape test (for the assessment of the stressinduced behavioural modification). Rats spent 3 min a day for 3 d in the experimental cage with the sliding door open to habituate to the test environment, before the first exposure to unavoidable stress. Rats were then exposed to a 5-min pre-test session. Each rat was immobilized with a flexible wire-net; the distal third of the tail was left uncovered, an electrode was applied, and approx. 8 electric shocks (1 mar5 s, 1 every 3 s) were administered. Twenty-four hours later, rats were tested in a shock-escape paradigm in the Plexiglas cage. An electrode connected to the stimulator was applied to the tail and the animal was placed in the electrified chamber. After a 5-min habituation period, the rat received 3 consecutive electric shocks (1 mar5 s), at 3-s intervals. During the delivery of each shock the door connecting the electrified chamber to the neutral one was open. The intensity of the electric shock was graduated in a way that it was almost dispersed through the grid floor and, thus, selectively perceived on the rat s tail. Rats that succeeded in escaping were gently replaced in the electrified chamber, at the end of the 5-s shock period. rats (which had never before received an electric shock) scored between 22 and 3 escapes out of 3 trials. More than 95% of the rats exposed to the pre-test session before the test developed an escape deficit, defined by a score from to 3 escapes out of 3 trials. Chronic escape deficit For the chronic stress procedure, rats were initially exposed to the sequence of pre-test and test trials as described above. Then, each rat: (1) was restrained in a flexible wire net for 1 min starting 48 h after the escape test; (2) received 1 min of restraint plus four unavoidable shocks, 48 h after (1); (3) spent 2 min in the cage where the unavoidable shock had previously been administered, 48 h after (2). By repeating this procedure on alternate days, the escape deficit can be maintained in all rats (De Montis et al., 1995). Induction of VAB Apparatus Two dark Plexiglas boxes (box 1 and 2) were separated by a 1r1 cm sliding door. Box 2 formed the straight arm of a Y-maze (15r4r2 cm for each of the three arms). A vanilla sugar (VS) pellet used as a reinforcer was placed at the end of one of the two divergent arms of the maze. VS pellets were made daily: standard food pellets were crushed by mortar and pestle and the fragments were dampened with water and rolled in VS to obtain regular pellets weighing approximately 15 mg.

4 372 R. Rauggi et al. Training procedure The experimental procedure was previously described in detail (Ghiglieri et al., 1997). The day before the first training session rats were allowed a first run in the Y-maze with one of the two arms closed. Each animal was placed in box 1 and 1 s later a 5-s cue-light signalled the opening of the sliding door. Rats were given 2 min to enter box 2 and to reach the end of the open arm where a VS pellet was earned. Training sessions began 24 h later. $ Training sessions 1 3: the rat was placed in box 1 and the cue-light signalled the opening of the sliding door. If the rat did not enter box 2 within 6 s it was returned to the home cage for 15 min. If it entered box 2 it was allowed 6 s to reach the end of one of the two diverging arms. Either the right or the left arm was designated correct, balanced among the animals. If the empty arm was chosen, the rat was returned to the home cage for 15 min before the next trial. When the baited arm was chosen the rat was allowed to consume the VS pellet and then returned to the home cage for 15 min before the next trial. $ The two time periods (time to leave box 1 and time to reach the end of an arm) were progressively reduced throughout training sessions and at session 1 they were set at 1 and 2 s respectively. One training session was administered every other day. Each rat underwent a total of 1 complete trials for each session, at 15-min intervals. The number of correct runways and number of consumed VS pellets out of 1 trials was recorded for each rat. Experimental protocols Effect of a single administration of mirtazapine on acute escape deficit Rats were injected i.p. with saline (1 ml/kg, n=5, ), or mirtazapine (5 or 1 mg/kg, n=5, Mtz, for each group) 3 min before the pre-test session. Twenty-four hours later, they were tested for escape. Five rats were injected with saline and 24 h later they were tested for escape (). Effect of long-term mirtazapine on motor activity and nociceptive threshold Rats were treated with mirtazapine (1 mg/kg. d i.p., n=12, Mtz) or saline (1 ml/kg i.p., n=11, ) for 12 d. Eighteen hours after the last treatment, spontaneous locomotor activity was assessed in order to evaluate a possible motor stimulant activity of mirtazapine, which would constitute a bias in evaluating an escape deficit behaviour. After assessment of locomotor activity, the nociceptive threshold was determined in the same rats with the tail-flick and the hot-plate test. This experiment was carried out to evaluate a possible analgesic activity of mirtazapine, at the spinal or supraspinal level, which would constitute a major bias in evaluating a reduction in or a lack of behavioural sequelae after exposure to noxious stimuli. After the tests, mirtazapine treatment was resumed until day 14. Effect of a 14-day mirtazapine treatment on acute escape deficit On day 15, the same rats used in the above experiment were divided into four groups. Six saline-treated rats () and seven mirtazapine-treated rats (Mtz+stress) were exposed to the pre-test session and 24 h later they were tested for escape. Five saline-treated rats () and five mirtazapine-treated rats (Mtz) were tested for escape on day 16. Pharmacological characterization of the protective effect of mirtazapine on acute escape deficit: experiment A Ten rats treated with saline (1 ml/kg i.p.) for 14 d were used as control groups for experiments A and B; on day 15, five of them were exposed to the pre-test session () and 24 h later they were tested for escape; five rats were only exposed to the escape test (). To verify whether the protective effect of repeated mirtazapine treatment was related to a prominent function of a monoaminergic receptor system, 4 rats were treated with mirtazapine (1 mg/kg. d i.p.) for 14 d. Then, 3 min before exposure to the pre-test: (a) eight rats received a dopamine D1 receptor antagonist, SCH 2339 (.3 mg/kg i.p.); (b) eight rats received a 5-HT1A receptor antagonist, WAY 1635 (.2 mg/kg s.c.); (c) eight rats received a b-adrenergic receptor antagonist, propranolol (5 mg/kg i.p.); (d) eight rats received a muscarinic receptor antagonist, scopolamine (.1 mg/kg s.c.), and eight rats received saline (1 ml/kg). Twenty-four hours after the pre-test, all rats were tested for escape. All drug doses were chosen on the basis of previously published results (Gambarana et al., 1995a, 1999; Grappi et al., 23). Pharmacological characterization of the protective effect of mirtazapine on acute escape deficit: experiment B The non-competitive NMDA receptor antagonist dizocilpine was used in order to establish whether the effect of repeated mirtazapine treatment on the development of the escape deficit was dependent on the activity of the NMDA receptor system. Twenty-four

5 Mirtazapine antidepressant effect 373 rats were implanted with osmotic minipumps (Alzet 22 1 ; Alzet Osmotic Pumps, Durect Corporation, Cupertino, CA, USA) that delivered dizocilpine (.1 mg/kg. d s.c.) for 14 d. During the infusion period, eight rats were injected with mirtazapine (1 mg/kg i.p.) and, 24 h after the end of infusion and treatment, they were exposed to the sequence of pre-test and escape test (Dizocilpine+Mtz+stress). The other 16 animals received saline i.p. twice a day; 24 h after the end of infusion, eight of them were exposed to the sequence of pre-test and escape test (Dizocilpine+ stress) and eight to the escape test only (Dizocilpine). Effect of long-term mirtazapine treatment on the reversal of chronic escape deficit Rats were injected i.p. with saline for 21 or 4 d (1 ml/kg. d,, n=5 for each treatment period) or with mirtazapine (1 mg/kg. d, Mtz, n=1 for each treatment period), and at the end of treatment they were tested for escape. A second group of rats was exposed to pre-test and escape test, and then to the chronic stress procedure for 21 or 4 d, while injected i.p. with saline (1 ml/kg. d, n=5 for each period of stress exposure, Stress) or mirtazapine (1 mg/kg. d, n=1 for each period of stress exposure, Stress+Mtz). Twenty-four hours after the end of the protocol, rats were tested for escape. Effect of long-term mirtazapine treatment on the disruptive effect of stress on VAB acquisition Thirty-two rats were divided into four groups: $ Eight animals () were injected daily with 1 ml/kg of saline i.p.; after 2 wk they began the training protocol, while continuing to receive saline. $ Eight animals (Stress) were injected daily with 1 ml/kg of saline i.p.; after 2 wk, they were administered the pre-test and tested the following day for escape. On day 16 they began training under repeated stress. was also injected daily. $ Eight rats (Mtz+stress) were administered mirtazapine daily (1 mg/kg i.p.). They received the pre-test on day 14 of treatment and the escape test on the following day. On day 16 they began training under repeated stress, while continuing to receive mirtazapine. $ Eight rats (Mtz), pretreated with mirtazapine for 2 wk, began the training protocol on day 16 while continuing mirtazapine treatment. Drugs Scopolamine, SCH 2339, d,l-propranolol, WAY 1635 were dissolved in deionized/distilled water and injected at a volume of 1 ml/kg rat body weight. Dizocilpine was dissolved in.9% saline and released by s.c. implanted osmotic minipumps (Alzet 22 1 ). Mirtazapine was dissolved in HCl and water, and the ph was adjusted with NaOH to 5.5 6; the volume of injection was 1 ml/kg rat body weight. Mirtazapine was kindly donated by N.V. Organon (Oss, The Netherlands). All other drugs and chemicals were purchased from commercial sources. Statistical analysis Statistical analyses were performed on commercially available software (Prism 4.a, GraphPad Software Inc., San Diego, CA, USA). All data are expressed as mean S.E.M. Comparisons were made by one-way analysis of variance (ANOVA); ANOVA was followed by post-hoc Bonferroni s test, when applicable (p<.5). The data obtained in the Y-maze were analysed using two-way repeated-measures ANOVA with treatment and training session as factors; posthoc analysis was performed by Bonferroni s test, when applicable (p<.5). Statistical comparisons between two experimental groups were made by parametric unpaired t test. Results Effect of a single administration of mirtazapine on acute escape deficit Rats received mirtazapine (5 or 1 mg/kg) or saline (1 ml/kg) i.p. 6 min before the inescapable shock session. Twenty-four hours later these three groups and the group were tested for escape. Mirtazapine treatment at both doses did not modify the development of escape deficit in stress-exposed rats (=24.8.9; =2.2.7; Mtz 5 d=3.4 1.; Mtz 1 d=2.6.7). Analysis of the number of escapes by one-way ANOVA showed a significant difference between the groups (F 3,16 =195.2, p<.1) and Bonferroni s test demonstrated that the number of escapes of the group was higher than that of the, Mtz 5 d and Mtz 1 d groups (p<.1, for all comparisons). Effect of long-term mirtazapine on pain threshold and motor activity The spontaneous locomotor activity of rats treated for 12 d with mirtazapine was not different from that of saline-treated rats, both in terms of total locomotion (p=.182, t=1.38) and in the number of rearings (p=.124, t=1.61; Table 1). Subsequently, the

6 374 R. Rauggi et al. Table 1. Effect of repeated mirtazapine treatment on spontaneous locomotor activity Groups n Locomotor activity Number of rearings 14 d treatment Pre-test + Mirtazapine + 24 h Test Mtz Mtz + stress Mtz Rats were treated i.p. with mirtazapine (1 mg/kg. d, Mtz) or saline (1 ml/kg. d, ) for 12 d. Spontaneous locomotor activity was measured as described in the Methods section. Values are the mean S.E.M. of motility counts and number of rearings. Table 2. Effect of mirtazapine treatment on nociceptive threshold Groups n Tail-flick latency (s) Hot-plate latency (s) Mtz Rats were treated i.p. with mirtazapine (1 mg/kg. d, Mtz) or saline (1 ml/kg. d, ) for 12 d. After the assessment of locomotor activity, rats underwent the tail-flick and hot-plate tests. Values are the mean S.E.M. of response latencies measured in seconds. nociceptive threshold was assessed in the two groups of rats. Repeated mirtazapine treatment did not modify the nociceptive threshold in the tail-flick (p=.445, t=.779) or hot-plate tests (p=.65, t=1.98; Table 2). Effect of a 14-d mirtazapine treatment on acute escape deficit After 14 d of treatment, mirtazapine- and salinetreated rats were exposed to the pre-test and escape test. The development of escape deficit was completely prevented by mirtazapine administration (Figure 1). Analysis of the data by one-way ANOVA indicated a significant difference between the groups (F 3,2 =76.2, p<.1) and the number of escapes in the, Mtz, and Mtz+stress groups was higher than that of the group (Bonferroni s test, p<.1, for all comparisons). Pharmacological characterization of the protective effect of mirtazapine on acute escape deficit: experiment A Rats treated with mirtazapine (1 mg/kg. d i.p.) for 14 d received an acute injection of saline or selective receptor antagonists 3 min before exposure to the Number of escapes *** Mtz Mtz + stress Figure 1. Effect of a 14-d mirtazapine treatment on acute escape deficit. Rats received saline (.1 ml/kg i.p.) or mirtazapine (1 mg/kg i.p.) for 14 d. A group of salinetreated () and a group of mirtazapine-treated rats (Mtz+stress) were exposed to the pre-test session and 24 h later tested for escape. A second group of saline-treated () and a group of mirtazapine-treated rats (Mtz) were only tested for escape. Scores are expressed as mean number of escapes S.E.M. in 3 consecutive trials. *** p<.1 compared to the score of the, Mtz and Mtz+Stress groups (post-hoc Bonferroni s test). pre-test. Twenty-four hours later they were tested for escape. The acute administration of 5-HT1A, b-adrenergic, and dopamine D1 receptor antagonists reduced the protective activity of repeated mirtazapine treatment (Figure 2). Analysis of the data by one-way ANOVA indicated a significant difference between the groups (F 6,43 =19.8, p<.1). The number of escapes in mirtazapine-treated rats was reduced by the acute administration of WAY 1635, propranolol, and SCH 2339 (Bonferroni s test, p<.1, p<.1 and p<.5 respectively). The antagonistic effect of SCH 2339 was less pronounced than that of WAY 1635 or propranolol (Bonferroni s test, p<.5 for both comparisons). Pharmacological characterization of the protective effect of mirtazapine on acute escape deficit: experiment B Dizocilpine infusion completely prevented the development of the protective effect of mirtazapine on escape deficit, while it did not modify the reactivity of

7 Mirtazapine antidepressant effect 375 Antagonist 24 h 14 d treatment Pre-test Test + + Mtz + saline Mirtazapine + + Mtz + antagonist Mtz + saline Mtz + WAY 1635 Mtz + propranolol 3 Mtz + SCH 2339 Mtz + scopolamine Number of escapes 2 1 ** * * ** Figure 2. Effect of selective receptor antagonists on the protective effect of mirtazapine on acute escape deficit. Rats received saline (.1 ml/kg i.p.) or mirtazapine (1 mg/kg i.p.) for 14 d. Mirtazapine-treated rats received an acute injection of saline or of selective receptor antagonists (SCH 2339, WAY 1635, propranolol, scopolamine) 3 min before exposure to the pre-test, as described in the Methods section. Twenty-four hours later they were tested for escape. A group of saline-treated rats was exposed to the pre-test and escape test () and another group was only tested for escape (). Scores are expressed as mean number of escapes S.E.M. in 3 consecutive trials. *** p<.1 and * p<.5 compared to the score of the and Mtz+saline groups; p<.5 compared to the score of the Mtz+WAY 1635 and Mtz+propranol groups (post-hoc Bonferroni s test). saline-treated rats to the escape test, or to the sequence of pre-test and escape test (Figure 3). Analysis of the number of escapes showed a significant difference between groups (ANOVA, F 4,29 =15.4, p<.1) with a reduced number of escapes in the Dizocilpine+ Mtz+stress, Dizocilpine+stress, and groups compared to that of the Dizocilpine and groups (Bonferroni s test, p<.1 for all comparisons). Effect of long-term mirtazapine treatment on the reversal of chronic escape deficit and mirtazapine-treated rats exposed or not exposed to the 21- or 4-d stress protocol were tested for escape. In the control groups, the number of escapes showed no statistical difference between the two groups (t test, p=.22), nor was there a difference between the two Stress groups (t test, p=.18). Thus, the number of escapes of the 21- and * 24 h 14 d treatment Pre-test Test Dizocilpine + Dizocilpine Diz. + stress Dizocilpine + mirtazapine + Diz. + Mtz + stress 3 Dizocilpine Diz. + stress 2 Diz. + Mtz + stress Number of escapes 1 *** *** *** Figure 3. Dizocilpine infusion antagonized the protective effect of mirtazapine on acute escape deficit. Rats were infused with dizocilpine (.1 mg/kg. d s.c.) for 14 d. During the infusion period, a group of rats received mirtazapine (1 mg/kg i.p.) and, 24 h after the end of infusion and treatment, was exposed to the sequence of pre-test and escape test (Dizocilpine+Mtz+stress). The other animals received saline i.p.; 24 h after the end of infusion, they were exposed to the sequence of pre-test and escape test (Dizocilpine+stress) or to the escape test only (Dizocilpine). Data are expressed as mean S.E.M. of the number of escapes out of 3 trials. *** p<.1 compared to the score of the and Dizocilpine groups (post-hoc Bonferroni s test). 4-d control groups were pooled (n=1 for both the and Stress groups). A 21-d mirtazapine treatment did not revert the stress-induced escape deficit, while the 4-d treatment was effective (Figure 4). Analysis of the number of escapes in control and mirtazapine-treated groups indicated a significant difference between the groups (ANOVA: F 3,36 =19.81, p<.1). The number of escapes in the Stress group was lower than that of the and Stress+Mtz 4 d groups (Bonferroni s test, p<.1 and p<.1 respectively), and the number of escapes in the Stress+Mtz 4 d group was higher than that of the Stress+Mtz 21 d group (Bonferroni s test, p<.5, Figure 4). Effect of long-term mirtazapine treatment on the disruptive effect of stress on VAB acquisition - and mirtazapine-treated rats trained in the Y-maze acquired VAB, whereas the Stress group did not. Mirtazapine administration completely protected rats from the disrupting effect of stress (Figure 5). Analysis by two-way repeated-measure ANOVAs indicated a significant effect of training on the number of correct choices (F 9,243 =15.1, p<.1) and on the number of VS pellets consumed (F 9,234 =36.6,

8 376 R. Rauggi et al. Test 24 h Chronic stress (21 or 4 d) Pre-test Test 21 or 4 d treatment Test Stress Mirtazapine Stress + Mtz Stress 3 Stress + Mtz 21 d Stress + Mtz 4 d Number of escapes 2 1 *** Figure 4. Effect of long-term mirtazapine treatment on the reversal of chronic escape deficit. Rats were injected i.p. with saline for 21 or 4 d (1 ml/kg. d, ) or with mirtazapine (1 mg/kg. d, Mtz), and then tested for escape. A second group of rats was exposed to pre-test and escape test, and then to the chronic stress procedure for 21 or 4 d, while injected i.p. with saline (Stress) or mirtazapine (Stress+Mtz). Twenty-four hours after the end of the protocol, rats were tested for escape. Scores are expressed as mean number of escapes S.E.M. in 3 consecutive trials. *** p<.1 and ** p<.1 compared to the score of the group; p<.5 compared to the score of the Stress+Mtz 4 d group (post-hoc Bonferroni s test). p<.1). Treatment also had a significant effect on the number of correct choices (F 3,243 =3.11, p<.5) and on the number of VS pellets consumed (F 3,234 = 9.57, p<.1), with a significant treatmentrtraining session interaction for the number of correct choices (F 27,243 =2.34, p<.1) and VS pellets consumed (F 27,234 =2.92, p<.1). The, Mtz, and Mtz+stress rats reached a consistent increase in correct choice frequency after 1 training sessions compared to their initial performance (Bonferroni s test, p<.1 for all comparisons, Figure 5). The performance in the Y-maze of the Stress group was not significantly different at days 1 and 1 of training. Moreover, in the Stress group the number of correct choices was lower from training sessions 4 1 than that in the, Mtz, and Mtz+stress groups (Bonferroni s test, p<.1 for all comparisons), and the number of VS pellets consumed from training sessions 3 1 was lower than in the, Mtz, and Mtz+stress groups (Bonferroni s test, p<.1 for all comparisons). Moreover, the number of correct choices and the number of VS pellets consumed was not significantly different in the Mtz+stress group compared to those of and Mtz groups. The number of VS pellets ** Correct choices consumed differed between the and the Mtz and Mtz+stress groups only at training sessions 7 and 8 (Figure 5). Discussion VS consumption Training session Training session 14 d pretreatment Mirtazapine Treatment + VAB training Chronic stress Stress Mtz Mtz + stress Stress Mtz Mtz + stress Figure 5. Effect of long-term mirtazapine treatment on the disruptive effect of stress on VAB acquisition. Rats were injected daily with saline and after 2 wk they began the Y-maze training, while they were exposed (Stress) or not exposed () to the chronic stress protocol. Another group of rats was administered mirtazapine daily and after 2 wk they began the Y-maze training, while exposed (Mtz+stress) or not exposed (Mtz) to the chronic stress protocol. Scores are expressed as mean S.E.M. of the number of correct choices or number of VS pellets consumed out of 1 trials. *** p<.1 compared to the score at training session 1 for the, Mtz, and Mtz+stress groups; p<.1 compared to the score of the, Mtz, and Mtz+stress groups at the corresponding training session; # p<.5 compared to the score of the group at the corresponding training session (post-hoc Bonferroni s test). Mirtazapine is a potent, unselective a 2 -adrenoceptor antagonist that acts on both pre- and post-synaptic sites (De Boer et al., 1988, 1996; Haddjeri et al., 1996, 1998). Pre-synaptic a 2 -adrenoceptors can be located in somata, dendrites and terminals of noradrenergic neurons where they negatively control adrenergic transmission, or in non-noradrenergic terminals where they inhibit neurotransmitter release. The acute administration of mirtazapine did not interfere with + +

9 the development of acute escape deficit. However, mirtazapine administered daily for 2 wk completely protected rats from the behavioural sequelae of unavoidable stress. Thus, it may be proposed that the pharmacological effects of mirtazapine probably constituted the perturbation that, in the long-term, caused the neuronal plasticity processes necessary for preventing acute escape deficit development. These processes are dependent on NMDA receptor activity, since dizocilpine infusion during mirtazapine treatment prevented the development of the mirtazapine protective effect. The dose of dizocilpine used (.1 mg/kg. 24 h) did not modify the escape response of the Dizocilpine group. Moreover, we previously demonstrated that this dose of dizocilpine prevents the development of the protective effect of imipramine and fluoxetine on the behavioural and neurochemical sequelae of unavoidable stress, but that alone it does not produce apparent behavioural modifications (C. Gambarana et al., unpublished results; Meloni et al., 1993). These results differ from other findings that suggested a potential antidepressant effect of NMDA receptor antagonists (Paul and Skolnick, 23). However, in our escape deficit model even a 3-wk dizocilpine infusion does not prevent the behavioural consequences of unavoidable stress exposure (Meloni et al., 1993) and similar negative results were reported in several models of depression in mice (Panconi et al., 1993). Rats treated for 2 wk with mirtazapine showed unmodified pain threshold and spontaneous motor activity; thus, the protective effect of mirtazapine cannot be ascribed to an altered perception of nociceptive stimuli or to a generic state of increased motility. The protective effect of repeated mirtazapine administration was completely antagonized by the acute inhibition of either 5-HT1A or b-adrenergic receptors during unavoidable stress exposure. A similar protection was observed in rats repeatedly treated with imipramine, fluoxetine, or reboxetine and the protective effect of each of these antidepressants was antagonized by selectively inhibiting dopamine D1, 5-HT1A or b-adrenergic receptors respectively (Gambarana et al., 1995a; Grappi et al., 23). These data provide strong evidence to support the hypothesis that the preventive effect of a repeated antidepressant treatment on acute escape deficit development is mediated by the activation of a single monoaminergic receptor system specific for each compound or class of compounds. This hypothesis is strengthened by the observation that the acute administration of a dopamine D1 receptor agonist, or a Mirtazapine antidepressant effect HT1A receptor agonist, 15 2 min before unavoidable stress exposure completely protects rats from the behavioural sequelae of the stress (Gambarana et al., 1995b; C. Gambarana et al., unpublished results). However, while the protective effect of fluoxetine is completely inhibited by a 5-HT1A receptor antagonist (pindolol or WAY 1635) and only partially by a dopamine D1 receptor antagonist, long-term clomipramine is antagonized 5% by SCH 2339 administration and 5% by pindolol administration (Gambarana et al., 1995a; C. Gambarana et al., unpublished results). That is, the effects of an antidepressant can be mediated by the activation of more than one monoaminergic receptor system. These data confirm that dopamine, norepinephrine and serotonin are all involved in the complex mechanisms underlying animal reactivity to a stressful event. A repeated treatment with SSRIs induces intense down-regulation of the SERT number that results in a reduction of extraneuronal 5-HT clearance consistently more pronounced than that produced by acute transporter inhibition (Benmansour et al., 22). Moreover, longterm fluoxetine administration induces desensitization of pre-synaptic 5-HT1A autoreceptors and sensitization of post-synaptic 5-HT1A receptors (Castro et al., 23). These adaptive mechanisms support the hypothesis that when a prolonged pharmacological stimulus strengthens the response of one or two of the monoaminergic receptor systems controlling reactivity to stress, this effect can become sufficient to prevent the development of a stress-induced behavioural deficit (Gambarana et al., 1995a). It may be hypothesized that the repeated antagonistic effect exerted by mirtazapine on noradrenergic autoreceptors resulted in an increased noradrenergic transmission that opposed the development of stress-induced behavioural modifications in a manner analogous to that observed after chronic reboxetine treatment (Grappi et al., 23), that is, through increased b-adrenoceptor activation. Moreover, the secondary activation of the dorsal raphe neurons (De Boer, 1996) and the inhibition of a 2 -heteroreceptors located in the serotonergic terminals may explain the protective component mediated by 5-HT1A receptor activation, analogous to the mechanism proposed for long-term fluoxetine administration (Gambarana et al., 1995a). Stimulation of 5-HT1A receptors in the mesencephalon is excitatory on dopaminergic neurons (Arborelius et al., 1993; Doherty and Pickel, 21; Lejeune and Millan, 1998; Prisco et al., 1994), and this mechanism may explain the partial protective component of both long-term fluoxetine (Gambarana et al., 1995a) and mirtazapine on acute escape deficit development.

10 378 R. Rauggi et al. Thus, in rat behaviour the present findings confirm the mechanisms underlying long-term mirtazapine effects previously hypothesized and demonstrated in neurochemical and electrophysiological experiments (De Boer et al., 1988, 1996; Haddjeri et al., 1996, 1998). The stress-sustained escape deficit can be completely reverted within 3 wk by continuous treatment with classical antidepressants (imipramine, fluoxetine, clomipramine, phenelzine, reboxetine) in rats concomitantly exposed to the chronic stress protocol (De Montis et al., 1995; Gambarana et al., 21; Grappi et al., 23). We consider this effect to be crucial to the definition of antidepressant activity (Gambarana et al., 21). Thus, we tested whether a 21- or a 4-d mirtazapine treatment would restore the escape response in rats exposed to chronic stress. Stressed rats recovered their escape response only after 4 d of mirtazapine administration, while rats in the Stress group still presented a marked escape deficit. That is, the preventive effect of mirtazapine on the behavioural sequelae of a single unavoidable stress developed faster than its capacity to revert an already established condition of escape deficit. Mirtazapine administration completely antagonized the disrupting effect of repeated stress exposure on the acquisition of an instrumental behaviour sustained by the high palatability of the reinforcer. Rats trained in the Y-maze to earn VS while undergoing a chronic stress procedure, when tested at the end of the training phase showed no improvement in the number of correct choices compared to their initial performance. Rats treated with mirtazapine had a final score similar to that of trained control animals, whether or not they were exposed to stress. Thus, mirtazapine did not modify the spontaneous ability of rats to acquire VAB and it prevented the disruptive effect of stress on the acquisition of this behaviour. To our knowledge, these are the first experimental results to demonstrate mirtazapine activity on the lack of motivation/anhedonia symptoms of depression. The protective effect of an antidepressant in VAB acquisition during stress exposure is observed only when the drug (imipramine or fluoxetine) is administered for 2 3 wk before and then during the whole training phase (C. Gambarana et al., unpublished results). When antidepressant treatment begins at the first Y-maze trial, i.e. after the initial pre-test and escape test sequence, rats never learn VAB (C. Gambarana et al., unpublished results). Thus, it appears that the anti-stress activity of any antidepressant is more pronounced and seems to develop faster when preventive treatment is started before stress exposure. In conclusion, repeated mirtazapine administration completely protected rats from the hyporeactivity induced by unavoidable stress exposure with a complex mechanism that involves b-adrenergic and 5-HT1A receptor stimulation and requires NMDA receptor activity. Mirtazapine reinstated the escape response of rats exposed to the chronic stress protocol, however, this effect developed after a longer treatment compared to classical antidepressant drugs (De Montis et al., 1995; Gambarana et al., 21; Grappi et al., 23). Moreover, mirtazapine completely counteracted the unavoidable stress-induced lack of motivation in VAB as efficiently as classical antidepressants. Acknowledgements The authors thank Ms. Colleen Pisaneschi for language editing of the manuscript and N.V. Organon for the gift of mirtazapine. This study was supported by a grant from the Ministero dell Università e della Ricerca Scientifica (MIUR) and a research grant from Organon Italia S.P.A., Rome, Italy. Statement of Interest One of the authors (A.T.) is the recipient of a research grant from Organon Italia S.P.A., Rome, Italy. References Arborelius L, Chergui K, Murase S, Nomikos GG, Hook BB, Chouvet G, Hacksell U, Svensson TH (1993). The 5-HT 1A receptor selective ligands, (R)-8-OH-DPAT and (S)-UH- 31, differentially affect the activity of midbrain dopamine neurons. Naunyn Schmiedebergs Archives of Pharmacology 347, Benmansour S, Owens WA, Cecchi M, Morilak DA, Frazer A (22). Serotonin clearance in vivo is altered to a greater extent by antidepressant-induced downregulation of the serotonin transporter than by acute blockade of this transporter. Journal of Neuroscience 22, Berendsen HH, Broekkamp CL (1997). Indirect in vivo 5-HT 1A -agonistic effects of the new antidepressant mirtazapine. Psychopharmacology 133, Castro ME, Diaz A, del Olmo E, Pazos A (23). Chronic fluoxetine induces opposite changes in G protein coupling at pre and postsynaptic 5-HT 1A receptors in rat brain. Neuropharmacology 44, D Amour FE, Smith DL (1941). A method for determining loss of pain sensation. Journal of Pharmacology and Experimental Therapeutics 72, Dazzi L, Spiga F, Pira L, Ladu S, Vacca G, Rivano A, Jentsch JD, Biggio G (21). Inhibition of stress- or anxiogenicdrug-induced increases in dopamine release in the rat

11 Mirtazapine antidepressant effect 379 prefrontal cortex by long-term treatment with antidepressant drugs. Journal of Neurochemistry 76, De Boer T (1996). The pharmacologic profile of mirtazapine. Journal of Clinical Psychiatry 57 (Suppl. 4): De Boer TH, Maura G, Raiteri M, de Vos CJ, Wieringa J, Pinder RM (1988). Neurochemical and autonomic pharmacological prophile of the 6-aza-analogue of mianserine, Org 377 and its enantiomers. Neuropharmacology 27, De Boer TH, Nefkens F, Van Velvoirt A (1996). Differences in modulation of noradrenergic and serotonergic transmission by the alpha-2 adrenoceptor antagonists, mirtazapine, mianserin and idazoxan. Journal of Pharmacology and Experimental Therapeutics 277, De Montis MG, Gambarana C, Ghiglieri O, Tagliamonte A (1995). Reversal of stable behavioural modifications through NMDA receptor inhibition in rats. Behavioural Pharmacology 6, Doherty MD, Pickel VM (21). Targeting of serotonin 1A receptors to dopaminergic neurons within the parabrachial subdivision of the ventral tegmental area in rat brain. Journal of Comparative Neurology 433, Eddy NB, Leimbach D (1953). Synthetic analgesics. II Diethinylbutenyl-and dithienylbutylamines. Journal of Pharmacology and Experimental Therapeutics 17, Fawcett J, Barkin RL (1999). Review of the results from clinical studies on the efficacy, safety and tolerability of mirtazapine for the treatment of patients with major depression. Journal of Affective Disorders 51, Gambarana C, Ghiglieri O, Taddei I, Tagliamonte A, De Montis MG (1995a). Imipramine and fluoxetine prevent the stress-induced escape deficits in rats through a distinct mechanism of action. Behavioural Pharmacology 6, Gambarana C, Ghiglieri O, Tagliamonte A, D Alessandro N, De Montis MG (1995b). Crucial role of D 1 dopamine receptors in mediating the antidepressant effect of imipramine. Pharmacology, Biochemistry, and Behavior 5, Gambarana C, Ghiglieri O, Tolu P, De Montis MG, Giachetti D, Bombardelli E, Tagliamonte A (1999). Efficacy of an Hypericum perforatum (St. John s Wort) extract in preventing and reverting a condition of escape deficit in rats. Neuropsychopharmacology 21, Gambarana C, Scheggi S, Tagliamonte A, Tolu P, De Montis MG (21). Animal models for the study of antidepressant activity. Brain Research Protocols 7, Ghiglieri O, Gambarana C, Scheggi S, Tagliamonte A, Willner P, De Montis MG (1997). Palatable food induces an appetitive behaviour in satiated rats which can be inhibited by chronic stress. Behavioural Pharmacology 8, Grappi S, Nanni G, Leggio B, Rauggi R, Scheggi S, Masi F, Gambarana C (23). The efficacy of reboxetine in preventing and reverting a condition of escape deficit in rats. Biological Psychiatry 53, Haddjeri N, Blier P, De Montigny C (1996). Effect of the a 2 -adrenoceptor antagonist mirtazapine on the 5-hydroxytryptamine system in the rat brain. Journal of Pharmacology and Experimental Therapeutics 277, Haddjeri N, Blier P, De Montigny C (1998). Acute and longterm actions of the antidepressant drug mirtazapine on central 5-HT neurotransmission. Journal of Affective Disorders 51, Laakmann G, Schule C, Baghai T, Waldvogel E (1999). Effects of mirtazapine on growth hormone, prolactin, and cortisol secretion in healthy male subjects. Psychoneuroendocrinology 24, Lejeune F, Millan MJ (1998). Induction of burst firing in ventral tegmental area dopaminergic neurons by activation of serotonin (5-HT)1A receptors: WAY 1,635- reversible actions of the highly selective ligands, flesinoxan and S Synapse 3, Meloni D, Gambarana C, De Montis MG, Dal Prá P, Taddei I, Tagliamonte A (1993). Dizocilpine antagonizes the effect of chronic imipramine on learned helplessness in rats. Pharmacology, Biochemistry, and Behavior 46, Montgomery SA (1995). Safety of mirtazapine : a review. International Clinical Psychopharmacology 1 (Suppl. 4) : Overmier JB, Seligman MEP (1967). Effects of inescapable shock upon subsequent escape and avoidance learning. Journal of Comparative Physiology and Psychology 63, Panconi E, Roux J, Altenbaumer M, Hampe S, Porsolt RD (1993). MK-81 and enantiomers: potential antidepressants or false positives in classical screening models? Pharmacology, Biochemistry, and Behavior 46, Paul IA, Skolnick P (23). Glutamate and depression: clinical and preclinical studies. Annals of the New York Academy of Sciences 13, Prisco S, Pagannone S, Esposito E (1994). Serotonindopamine interaction in the rat ventral tegmental area : an electrophysiological study in vivo. Journal of Pharmacology and Experimental Therapeutics 271, Puzantian T (1998). Mirtazapine, an antidepressant. American Journal of Health-System Pharmacy 55, Schule C, Baghai T, Bidlingmaier M, Strasburger C, Laakman G (22). Endocrinological effects of mirtazapine in healthy volunteers. Progress in Neuropsycopharmacology and Biological Psychiatry 26, Sherman AD, Sacquitne JL, Petty F (1982). Specificity of the learned helplessness model of depression. Pharmacology, Biochemistry, and Behavior 16, Thompson C (1999). Mirtazapine versus selective serotonin reuptake inhibitors. Journal of Clinical Psychiatry 6 (Suppl. 17), Willner P (1995). Animal models of depression: validity and applications. Advances in Biochemistry and Psychopharmacology 49,