Effects of the 5-HT6 receptor agonist ST 1936 on depression- and anhedonia-like experimental models

Behavioural Brain Research 224 (2011) 35–43

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Effects of the 5-HT6 receptor agonist ST 1936 on depression- and anhedonia-like experimental models Simona Scheggi a , Giovanna Marchese a , Franco Borsini b , Fabio Bordi b , Maria Graziella De Montis a,∗ a b

Department of Neuroscience, University of Siena, Via Moro 4, 53100 Siena, Italy Sigma-Tau Industrie Farmaceutiche Riunite SpA, Pomezia, Italy

a r t i c l e

i n f o

Article history: Received 17 January 2011 Received in revised form 20 May 2011 Accepted 22 May 2011 Keywords: Antidepressants 5-HT6 receptor Anhedonia Unavoidable stress Appetitive behavior

a b s t r a c t Serotonin 5-HT6 receptor agonists and antagonists have been proposed as possible useful compounds in the treatment of psychiatric disorders such as depression. This study was aimed at characterizing ST 1936, a new 5-HT6 receptor agonist, as a possible antidepressant/anti-anhedonic drug by studying its effects on three experimental models of depression. These models are based on the behavioral sequelae induced in rats by unavoidable stressors that result in decreased reactivity to avoidable stressors (escape deficit, ED) and an anhedonia-like condition based on the disruptive effect of stress on the competence to acquire an instrumental vanilla sugar-sustained appetitive behavior (VAB). The repeated administration of ST 1936 prevented the development of ED, but did not revert a condition of chronic ED. The protective effect of ST 1936 was antagonized by co-administration of SB 271046, a 5-HT6 receptor antagonist, indicating that the 5-HT6 receptor stimulation is crucial for triggering a plasticity process that resulted in the prevention of ED development. ST 1936 administration in rats undergoing VAB training did not interfere with its acquisition, whereas SB 271046 administered in similar conditions prevented VAB acquisition. Moreover, ST 1936 administration in rats trained in the Y-maze while exposed to a chronic stress protocol consistently antagonized the stress-disrupting effect, and also this effect was antagonized by SB 271046 coadministration. It was concluded that a tonic 5-HT6 receptor activity was crucial for VAB acquisition, and that pharmacological stimulation of 5-HT6 receptors reinstated a stress-reduced hedonic competence with an efficacy similar to that of classical antidepressant drugs. © 2011 Elsevier B.V. All rights reserved.

1. Introduction A large amount of information in recent years has been gathered on serotonin 5-HT6 receptors and their possible physiological role within the central nervous system. A role for this receptor has been established in mechanisms of learning and memory [1–4]. In fact, 5-HT6 receptor antagonists have been proposed for treatment of Alzheimer’s disease and cognitive impairment [5–9] and the effects on cognition could be related to the suggested role played by 5-HT6 transmission in controlling cholinergic function [10,11]. However, also 5-HT6 receptor agonists have been shown to be effective in experiments on memory improvement [1,3,12,13]. Thus experimental findings are not always consistent [14]. The 5-HT6 receptor mRNA expression and the receptor protein are largely confined to the central nervous system, particularly in the striatum, nucleus accumbens, olfactory tubercle, cortex and hippocampus [15–18], and this neuroanatomical distribution has suggested that ligands of this receptor may play a role in mood

∗ Corresponding author. Tel.: +39 0577234069; fax: +39 0577234208. E-mail address: [email protected] (M.G. De Montis). 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.05.019

disorders [19–21]. Accumulated evidence in recent years suggests that serotonin 5-HT6 receptors may be a target for the pharmacological treatment of depression [21]. Different therapeutically effective antidepressant drugs show high affinity for 5-HT6 receptor [19], and a relationship has been proposed to exist between their efficacy and a polymorphism in the 5-HT6 gene [22,23]. 5-HT6 receptor expression appears to be regulated by glucocorticoids in the hippocampus [24], an effect that might further imply a role of these receptors in the pathogenesis of mood disorders. SB 271046, SB 399885, and SB 258585, three rather selective 5-HT6 receptor antagonists have been used in preclinical studies in order to assess the role played by these receptors in mood control [25–27]. The experimental models used were the forced swim test in mice and rats and the tail suspension test in mice, and the results obtained after acute administration of an antagonist alone or associated with an antidepressant compound show that 5-HT6 receptor antagonists seem to exert antidepressant-like effects [28–30]. Conversely, studies performed with compounds, as WAY-181187, WAY-208466 or EMDT that have been described as 5-HT6 receptor agonists indicate antidepressant and anxiolytic effects for the two WAY compounds [31,32] and an antidepressant-like effect of EMDT, with the latter effect antagonized by the administration of a 5-HT6

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antagonist [33]. Moreover, similar to antidepressant drugs administration, 5-HT6 receptor stimulation may result in increased BDNF mRNA expression [34]. To reconcile these apparent incongruities it has been proposed that acute antagonism of 5-HT6 receptors may elicit antidepressant-like effects through an enhancement of brain noradrenergic and/or dopaminergic neurotransmission [35]; an effect that could be exerted mainly through inhibition of 5-HT6 receptors in the hippocampus [36]. In contrast, 5-HT6 receptor agonists may also produce antidepressant-like effects by enhancing extracellular GABA levels and decreasing stimulated glutamatergic neurotransmission in cortical areas [32]. All the antidepressant-like effects elicited by 5-HT6 receptor antagonists and agonists were observed after a short-term or even a single acute administration of each compound tested. The hypotheses on their mechanisms of action were proposed based on these results. Thus, interpretation of this body of data raises two issues. First, while it is well established that the acute administration of antidepressants reduces immobility in behavioral despair tests, this does not necessarily imply that every compound that shows this effect is endowed with antidepressant activity [37]. Claims of antidepressant-like activity are usually supported by evidence of effects after longterm or repeated treatment on behavioral models of depression. Second, the question of whether an organism develops adaptive responses upon repeated administrations of 5-HT6 receptor antagonists or agonists, regardless of the observed initial effects, has not been addressed. This is a relevant issue as antidepressant activity is likely related to the development of adaptive responses to the repeated drug administration. Acute imipramine administration has no effect on the behavioral consequences of an acute unavoidable stress, while it induces intense stress resilience upon long-term administration [38]. Indeed, the stress resilience induced in rats by a 3-week imipramine administration is prevented to occur by the concomitant administration of dizocilpine, which inhibits the development of an NMDA receptor-dependent neuronal plasticity process [39]. The main aim of this study was to further clarify the proposed antidepressant effects of 5-HT6 receptor agonists by studying the effect of acute and repeated administration of ST 1936, a new full agonist that binds to human and rat recombinant 5-HT6 receptors with nanomolar affinity [40–42]. In the present study we investigated the effect of ST 1936 in three experimental models of depression induced by exposure to unavoidable stress that consistently respond to classical antidepressants [43–45]. The first model is an acute escape deficit (ED) condition induced by a single exposure to unavoidable stress and is a modified version of the learned helplessness procedure. Acute ED is also an appropriate tool for assessing the role played by different receptor systems in the protective activity of an antidepressant treatment, inferred by the effects of acute administration of agonists or antagonists, specific for different receptor types [45]. The second model is a condition of chronic ED, which can be indefinitely sustained by the repeated administration of mild stressors. This model allows us to evaluate the capacity of a treatment to revert the ED condition. The third model is based on the disruptive effect of chronic stress exposure on the spontaneous competence to perceive palatable food as an incentive stimulus. Consumption of a palatable food is a widely accepted index of hedonic responsiveness and we use an instrumental behavior based on the reinforcing properties of vanilla sugar (VS) pellets, the earning of which is made contingent on the choice of one of two diverging arms of a Y-maze (VS sustained appetitive behavior, VAB) [42,46]. Experiments are carried out in non food-deprived rats, since the emotional value of food has a prevalent hedonic component in these animals [47]. Exposure to chronic stress during Y-maze training disrupts the animal competence to acquire VAB [44], thus configuring an anhedonia-like condition.

2. Materials and methods 2.1. Animals Experiments were performed on male Sprague-Dawley rats (Charles River, Italy) that were allowed at least 1 week habituation at the animal colony and weighed 200–225 g when the experimental procedures began. Animals were housed five per cage, in an environment maintained at a constant temperature and humidity with free access to food and water. A reverse 12-h light/dark cycle (7:00 a.m. lights off, 7:00 p.m. lights on) was used. Experiments were performed from 9:00 a.m. to 5:00 p.m. 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. The procedures used were in accordance with the European legislation on the use and care of laboratory animals (EEC Council Directive 86/609) and were approved by the University of Siena Ethics Committee. All efforts were made to minimize the number of animals used and their suffering. 2.2. Behavioral tests 2.2.1. Induction of acute escape deficit Escape deficit was induced by the exposure of rats to an unavoidable shock session (pre-test). During the pre-test each rat, immobilized by a flexible wire net, received 80 electric shocks (1 mA × 5 s, one every 30 s) in about 50 min through an electrode connected to an S48 Grass stimulator (Grass Instrument, Astro-Med Inc., West Warwick, RI) and applied to the distal third of the tail. Twenty-four hours later, rats were tested in a shock-escape paradigm in a Plexiglas cage (30 cm × 60 cm × 30 cm; DF Srl, Farmaceutica Engineering, Siena, Italy.) with dark walls and a floor fitted with stainless steel rods. An electrode connected to the stimulator was applied to the tail and the animal was placed in the Plexiglas cage which is divided into two equal chambers (by a dark Plexiglas partition with a 10 cm × 10 cm sliding door), one disconnected from the tail electrode (neutral chamber) and the other connected with it (electrified chamber). After a 5 min habituation period, the animal in the electrified chamber received 30 consecutive electric shocks (1 mA × 5 s), at 30 s intervals. Shocks were delivered, coinciding with a 5 s opening of the door connecting the electrified to the neutral chamber. Rats that succeeded in escaping were gently replaced in the electrified chamber, at the end of the 5-s shock period. More than 95% of the rats exposed to the pre-test session before the test developed an escape deficit, defined by a score of 0–5 escapes out of 30 trials. Naive rats (which had never before received an electric shock) scored between 22 and 30 escapes out of 30 trials [43]. During the week preceding the pre-test animals spend 30 min a day for at least 3 days in the experimental cage with the sliding door open to become familiar with the test environment. 2.2.2. Chronic stress exposure In order to maintain the condition of escape deficit each rat, after pre-test and test, was exposed for 3 weeks to a sequence of mild stressors, each administered on alternate days at 24 h intervals, as follows: (1) restraining in a flexible wire-net for 20 min starting 48 h after the escape test; (2) 10 min of restraint plus 10 min of unavoidable shocks 24 h after (1). By repeating this alternate-day stress procedure, ED condition can be maintained in all rats [48]. 2.2.3. VS pellet preparation VS pellets were prepared daily: standard food pellets (Harlan Italy, Italy) were crushed by mortar and pestle and the fragments were dampened with water and rolled in powdered vanilla sugar (Zucchero Vanigliato, Italy) to obtain pellets weighing approximately 150 mg. 2.2.4. Vanilla sugar-sustained appetitive behavior (VAB) This model is based on the rat’s competence to acquire an instrumental appetitive behavior aimed at earning a palatable food. Animals were non food-deprived, so that palatability was the main food reinforcer. Apparatus: Two dark Plexiglas boxes (boxes 1 and 2) separated by a 10 cm × 10 cm sliding door formed the straight arm of a Y-maze (15 cm × 40 cm × 20 cm for each of the three arms) (Ars Neon, Siena, Italy). For each trial, a vanilla sugar (VS) pellet was placed in a small tray at the end of one of the two diverging arms. Training procedure: Sessions of 10 trials with 15-min intervals between them were administered daily for a total of 10 sessions [44,45]. In the training sessions 1–3 the rat was placed in box 1 and a cue-light signaled the opening of the sliding door. If the rat did not enter box 2 within 60 s, it was returned to the home cage for 15 min. If it entered box 2, it was allowed 60 s to reach the end of one of the two diverging arms. Either the right or the left arm was designated correct, balanced among animals. If the empty arm was chosen (incorrect trial), the rat was returned to its cage for 15 min before the next trial. When the baited arm was chosen (correct trial) the rat was allowed to consume the VS pellet and then returned to its cage for 15 min before the next trial. A trial was defined as incomplete when the rat did not reach the end of one of the two diverging arms.

S. Scheggi et al. / Behavioural Brain Research 224 (2011) 35–43 In each session, the variables were manually recorded and were: number of correct, incorrect, incomplete trials, and the number of VS pellets consumed. 2.2.5. Chronic stress-induced anhedonia Repeated unavoidable stress has a disruptive effect on the acquisition of the VAB in satiated rats [42]. When VAB was studied in repeatedly stressed rats, the training sessions and the chronic stress protocol were administered on alternate days. 2.2.6. Antinociceptive test Pain threshold was assessed by the radiant heat tail-flick method according to D’Amour and Smith [49]. The animals were taken into the test room, placed on the tail flick apparatus (Ugo Basile, Italy), and the radiant heat intensity was adjusted to obtain average latency values of 5 s in control animals. A 10 s cut-off point was employed to prevent tissue damage to the tail. 2.2.7. Locomotor activity Locomotor activity was measured by an apparatus consisting of a mobile rack (height 180 cm, width 100 cm and depth 60 cm) with 8 compartments (h 40 cm, w 45 cm and d 50 cm), into which a transparent Perspex cage (height 19 cm, floor area 23 cm × 33 cm) was placed (Imetronic, Pessac, France). Motor activity was detected by a system of photocell infrared beams, dividing the cage area into two sectors, rear and front sector. A “barrier” of infrared photocell beams, placed at the height of 15 cm, detected vertical activity. The apparatus was connected to a personal computer by an electronic interface, the interruption of two photocell beams was recorded as an ambulatory activity count. Rats were individually placed into the perspex cages and motor activity was detected by a system of infrared photocell beams. Rats were observed for 30 min after a 5-min habituation period [50]. 2.2.8. Drugs ST 1936 2-(5-chloro-2-methyl-1H-indol-3-yl)-N,N-dimethylethanamine-HCl and SB 271046 [5-chloro-N-[4-methoxy-3-(piperazin-1-yl)phenyl]-3-methyl1.benzothiophene-2-sulfonamid-HCl] were synthesized in Sigma-Tau’s Chemistry Department, Sigma-Tau, Pomezia, Italy. The doses of ST 1936 (10–30 mg/kg) are in the range of those utilized to show an increase of brain catecholamines [42]. The dose of SB 271046 (20 mg/kg) was selected based on Valentini et al. [42]. Fluoxetine, SCH 23390, scopolamine were purchased from commercial sources (Sigma–Aldrich, St. Louis), WAY 100635 was a generous gift of Wyeth Pharmaceuticals (Pennsylvania, USA). The doses of SCH 23390, WAY 100635 and scopolamine were selected based on Grappi et al. [51] and Rauggi et al. [52]. All the compounds and vehicle were injected intra peritoneum (i.p.) in a volume of 1 ml/kg rat body weight, with the exception of scopolamine that was administered subcutaneously (s.c.). 2.3. Experimental protocols 2.3.1. Experiment 1: effects of ST 1936 treatment on ED development To study whether ST 1936 treatment was able to prevent stress-induced escape deficit, its effect was examined after acute or repeated administration before unavoidable stress exposure. The acute treatment was performed in order to evaluate similarities or differences with the results present in the literature after acute administration of other 5-HT6 agonists in forced swimming or tail suspension tests. In the ED model classical antidepressants imipramine or fluoxetine are ineffective on ED development after acute administration [38]. Thus, rats were divided into different groups and treated according to the following protocol: Group

Acute treatment

Pre-test

Escape test

Naïve (n = 5)

Vehicle 1 ml/kg 24 h before escape test Vehicle 1 ml/kg 30 min before pre-test ST 1936, 10 mg/kg 30 min before pre-test ST 1936, 30 mg/kg 30 min before pre-test Repeated treatment days 1–14

No

Yes

Stress (n = 12) ST 10 + Stress (n = 8) ST 30 + Stress (n = 8)

Naïve (n = 5) Stress (n = 13) ST 10 + Stress (n = 8) ST 30 + Stress (n = 8) ST 10 (n = 5) ST 30 (n = 5)

Vehicle 1 ml/kg for 2 weeks Vehicle 1 ml/kg for 2 weeks before pre-test ST 1936, 10 mg/kg for 2 weeks before pre-test ST 1936, 30 mg/kg for 2 weeks before pre-test ST 1936, 10 mg/kg for 2 weeks ST 1936, 30 mg/kg for 2 weeks

Yes Yes

Yes Yes

Yes

Yes

Pre-test day 15

Escape test day 16

No Yes

Yes Yes

Yes

Yes

Yes

Yes

No No

Yes Yes

In order to exclude a non-specific analgesic effect or a stimulant activity of repeated ST 1936 treatment, which would constitute a bias in evaluating a reduc-

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tion of behavioral sequelae to a nociceptive stimulus (36), rats treated with ST 1936 or vehicle were tested with the radiant heat tail-flick method few days before stress exposure. Thus, after 12 days of treatment rats repeatedly administered 10 or 30 mg/kg ST 1936 (n = 8 for each dose) and 8 rats from one of the two groups repeatedly treated with vehicle that were used as Control group, were tested for the evaluation of nociceptive threshold. Two days later the same rats were tested for spontaneous locomotor activity. Both locomotor activity and nociceptive threshold were recorded 30 min after the treatment.

2.3.2. Experiment 2A: pharmacological characterization of ST 1936 protective effect on ED development In order to verify whether the observed protective effect was related to a prominent function of a receptor system, rats were divided into different groups and treated according to the following protocol. Since the repeated administration of ST 1936 demonstrated a similar preventive activity on ED development at both 10 and 30 mg/kg, we used for this experiment the lowest effective dose of 10 mg/kg. The 60-min delay for SB 271046 was chosen based on Da Silva Costa et al. [5]; the 30-min delay for the other antagonists was chosen based on Rauggi et al. [52] and Grappi et al. [51]. Group

Repeated treatment days 1–14

Receptor antagonists day 15

Pre-test day 15

Escape test day 16

Naive (n = 6)

Vehicle

No

Yes

Vehicle

Yes

Yes

Vehicle

Yes

Yes

ST 10 + SB + Stress (n = 8)

Vehicle 1 ml/kg Vehicle 1 ml/kg ST 1936, 10 mg/kg ST 1936, 10 mg/kg

Yes

Yes

ST 10 + Way+ Stress (n = 8)

ST 1936, 10 mg/kg

Yes

Yes

ST 10 + SCH + Stress (n = 7)

ST 1936, 10 mg/kg

Yes

Yes

ST 10 + Scop + Stress (n = 8)

ST 1936, 10 mg/kg

SB 271046 20 mg/kg 60 min before pre-test WAY 100635 0.2 mg/kg 30 min before pre-test SCH 23390 0.03 mg/kg 30 min before pre-test Scopolamine 0.1 mg/kg 30 min before pre-test

Yes

Yes

Stress (n = 8) ST 10 + Stress (n = 8)

2.3.3. Experiment 2B: effect of the repeated administration of SB 271046 on ST 1936 protective effect on ED development In order to further clarify the role of 5-HT6 transmission in ST 1936 protective effect the 5-HT6 receptor antagonist SB 271046 was administered concomitant with ST 1936. As for the previous experiment we used the lowest effective dose of 10 mg/kg. Rats were divided into different groups and treated according to the following protocol: Group

Treatment days 1–14

Pre-test day 15

Escape test day 16

Naïve (n = 8) Stress (n = 8) ST 10 + Stress (n = 8) SB + ST + Stress (n = 8) SB + Stress (n = 7)

Vehicle 1 ml/kg Vehicle 1 ml/kg ST 1936, 10 mg/kg

No Yes Yes

Yes Yes Yes

SB 271046, 20 mg/kg 60 min before ST 1936 10 mg/kg SB 271046, 20 mg/kg

Yes

Yes

Yes

Yes

2.3.4. Experiment 3: effect of repeated ST 1936 administration on ED reversal Since a long-term administration of antidepressants such as imipramine, clomipramine, phenelzine, reboxetine and fluoxetine is effective in both the prevention and the reversal of an already established escape deficit [51,38,45], the possible effect of ST 1936 on the reversal of ED was studied. Rats were divided into different groups and treated according to the following protocol. A group of rats treated with fluoxetine (FLX) was included as positive control of antidepressant activity.

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S. Scheggi et al. / Behavioural Brain Research 224 (2011) 35–43

Group

Pretest day 1

Escape test day 2

Treatment days 3–24

Escape test day 25

Naïve (n = 8)

No

No

Yes

Stress (n = 8)

Yes

Yes

Stress + ST 10 (n = 8)

Yes

Yes

Stress + ST 30 (n = 7)

Yes

Yes

Stress + FLX (n = 8)

Yes

Yes

Vehicle 1 ml/kg for 3 weeks Vehicle 1 ml/kg for 3 weeks ST 1936, 10 mg/kg for 3 weeks ST 1936, 30 mg/kg for 3 weeks FLX, 5 mg/kg for 3 weeks

Yes Yes Yes Yes

2.3.5. Experiment 4: effect of repeated ST 1936 treatment on VAB acquisition in control and chronically stressed rats Before studying the possible antianhedonic effect of ST 1936 a preliminary experiment was performed to test the response in the VAB apparatus of rats treated with ST 1936 at 10 and 30 mg/kg according to the following protocol: Group

VAB training days 1–10

Control (n = 8) ST 10 (n = 8) ST 30 (n = 8)

Vehicle 1 ml/kg ST 1936, 10 mg/kg ST 1936, 30 mg/kg

The compound or vehicle were administered daily after the session of VAB training. Since the treatment with ST 1936 did not modify VAB acquisition compared to control rats, in the following experiment the effect of ST 1936 on stress-induced anhedonic effect was studied. Rats were divided into different groups and treated according to the following protocol: Group

Pretest day 1

Escape test day 2

VAB training + stress exposure and treatment days 3–24

Control (n = 8)

No

No

Stress (n = 8) Stress + ST 10 (n = 8) Stress + ST 30 (n = 8)

Yes Yes Yes

Yes Yes Yes

Rats in this group were only exposed to VAB training + Vehicle 1 ml/kg Vehicle 1 ml/kg ST 1936, 10 mg/kg ST 1936, 30 mg/kg

In the “stress” groups treatments began after the initial unavoidable stress exposure and escape test and these rats received the stress procedure or Y-maze training on alternate days for a total of 3 weeks. 2.3.6. Experiment 5: effect of the 5-HT6 receptor antagonist SB 271046 on the antianhedonic activity of ST 1936 Since ST 1936 consistently antagonized the stress-induced anhedonic effect at the highest dose, the following experiment with the 5-HT6 antagonist was carried out. Thirty-six rats were divided into different groups that were then exposed to: Group

Pretest day 1

Escape test day 2

VAB training + stress exposure and treatment days 3–24

Control (n = 8)

No

No

Stress (n = 6) Stress + ST (n = 8) Stress + SB + ST (n = 8)

Yes Yes Yes

Yes Yes Yes

SB (n = 6)

No

No

Rats in this group were only exposed to VAB training + Vehicle, 1 ml/kg Vehicle 1 ml/kg ST 1936, 30 mg/kg ST 1936, 30 mg/kg + SB 271046 20 mg/kg Rats in this group were only exposed to VAB training + SB 271046, 20 mg/kg

Treatments were performed as described for the experiment 4. 2.4. Statistical analysis Statistical analyses were performed on commercially available software (Prism 4.0a, GraphPad Software Inc., USA). Data from the locomotor activity, tail flick test and ED experiments were analyzed using one-way ANOVA followed by Dunnett’s or Bonferroni’s tests when P < 0.05. Data from the VAB experiments were analyzed using one-way or two-way repeated-measures ANOVA (rm-ANOVA) with treatment as the between-subject variable and time or session as the within-subject variable. Post hoc analysis was performed by Dunnett’s or Bonferroni’s tests, when P < 0.05.

Fig. 1. Effects of acute and repeated ST 1936 administration on ED development. For the acute treatment (a) rats received vehicle or ST 1936 (10 or30 mg/kg, i.p.) 30 min before unavoidable stress exposure. Twenty-four hours later they were tested for escape. A group of control rats (Naïve) was tested for escape without unavoidable stress exposure. Scores are expressed as the mean number of escapes ± S.E.M. in 30 consecutive trials. **P < 0.01 Naive group vs. Stress groups; *P < 0.05 Naïve group vs. ST 10 + Stress or ST 30 + Stress groups (Dunnett’s test). For the repeated treatment (b) rats received vehicle or ST 1936 (10 or30 mg/kg, i.p.) once a day for 14 days, then were exposed to the unavoidable stress and the following day they were tested for escape. A group of control rats (Naïve) and two groups of rats treated for 14 days with ST 1936 (10 or 30 mg/kg, i.p.) were tested for escape without unavoidable stress exposure. Scores are expressed as the mean number of escapes ± S.E.M. in 30 consecutive trials. **P < 0.01 Stress group vs. all other groups (Dunnett’s test).

3. Results 3.1. Experiment 1A: effects of ST 1936 acute and repeated treatment on ED development ANOVA analysis of the escape response indicated a difference between the groups both after acute (F3,29 = 7.35; P < 0.001) and repeated treatments (F5,36 = 9.78; P < 0.001). In the “acute treatment” protocol, post hoc analysis (Dunnett’s test) revealed that escape responses were significantly higher only in naïve compared to stressed rats, while the treatment with both doses of ST 1936 induced a non-significant increase in the number of escapes (Fig. 1a). In the “repeated treatment” protocol, escape responses were significantly higher in naïve, and all treated groups compared to the Stress group (Fig. 1b). These results indicate that the repeated administration of ST 1936, exerted a protective effect on the development of escape deficit. 3.2. Experiment 1B: effect of ST 1936 repeated treatment on pain threshold and locomotor activity As described in the experimental protocols rats repeatedly treated with ST 1936 10 or 30 mg/kg, or saline, before being exposed to the stress procedures, after 12 days of treatment were tested to assess their pain threshold. ANOVA analysis indicated that in the tail-flick test, rats treated with ST 1936 at both doses, did

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not show any significant modification in pain threshold compared with the saline treated rats (mean latency value ± S.E.M. Vehicle group: 3.6 ± 0.4 s; ST 1936 10 mg/kg group: 4.1 ± 0.2 s; ST 1936 30 mg/kg group: 3.1 ± 0.4 s). To verify whether repeated ST 1936 treatment had a stimulant activity on locomotion, the same rats were also tested for spontaneous locomotor activity 2 days later. ANOVA analysis indicated non-significant differences in spontaneous locomotor activity between rats in the groups treated with the 2 doses of ST 1936 and rats in the Vehicle group (mean motility counts in 30 min ± S.E.M. for the Vehicle group: 686 ± 42, for the ST 1936 10 mg/kg group: 628 ± 55 and for the ST 1936 30 mg/kg group: 710 ± 95). Thus, the protective effect of ST 1936 cannot be ascribed to an altered perception of nociceptive stimuli or to a generic state of increased motility. 3.3. Experiment 2A: pharmacological characterization of ST 1936 protective effect on ED development In order to verify whether the protective effect of ST 1936 treatment was related to a prominent function of a receptor system during unavoidable stress exposure, 30–60 min before exposure to unavoidable stress, rats treated for 2 weeks with ST 1936 received an acute injection of saline or a selective receptor antagonist. Twenty-four hours later rats were tested for escape. ANOVA analysis of the number of escapes showed a difference between the groups (F6,49 = 10.12; P < 0.001). Post hoc test indicated that only pretreatment with the muscarinic antagonist scopolamine, that per sé did not modify the stress-induced ED (Fig. S1 supplementary material) significantly inhibited the protective effect of ST 1936 (Fig. 2a). 3.4. Experiment 2B: effect of the repeated administration of SB 271046 on ST 1936 protective effect on ED development Since the acute blockade of 5-HT6 receptors during unavoidable stress exposure does not seem to play a crucial role in ST 1936 protective activity, the effect of repeated administration of the 5-HT6 receptor antagonist concomitant with ST 1936 administration was examined. ANOVA analysis of the escape response indicated a difference between the groups (F4,32 = 18.40; P < 0.001). The 2-week treatment with ST 1936 confirmed its protective effect against ED development, protection that was completely abolished by the concomitant treatment with the 5-HT6 receptor antagonist SB 271046 (Fig. 2b).

Fig. 2. Effects of acute and repeated receptor antagonists administration on ST 1936 protective effect on ED development. For the acute treatment (a) rats received vehicle or ST 1936 (10 mg/kg, i.p.) once a day for 14 days. On the 15th day animals repeatedly treated with vehicle or ST 1936 received an acute injection of vehicle (1 ml/kg, i.p.), or SB 271046 (20 mg/kg, i.p.), or WAY 100635 (0.2 mg/kg, i.p.), or SCH 23390 (0.03 mg/kg, i.p.), or scopolamine (0.1 mg/kg s.c.) 30–60 min before unavoidable stress exposure as described in the experimental protocols. The following day these rats and a group of control rats (Naïve) never exposed to unavoidable stress were tested for escape. Scores are expressed as the mean number of escapes ± S.E.M. in 30 consecutive trials. ***P < 0.001 Naïve and ST 10 + Stress groups vs. Stress group; **P < 0.01 ST 10 + Scopol. + Stress group vs. ST 10 + Stress group (Bonferroni’s test). For the repeated treatment (b) rats received vehicle or ST 1936 (10 mg/kg, i.p.) or SB 271046 (20 mg/kg, i.p.) or SB 271046 + ST once a day for 14 days. On the 15th day they were exposed to the unavoidable stress and the following day they were tested for escape. A group of control rats (Naïve) was tested for escape without unavoidable stress exposure. Scores are expressed as the mean number of escapes ± S.E.M. in 30 consecutive trials. **P < 0.01 Stress group vs. Naive and ST + stress groups (Dunnett’s test).

3.5. Experiment 3: effect of repeated ST 1936 administration on ED reversal The reversal of ED has been used to evaluate the actual antidepressant efficacy of ST 1936, since the reversal of an already established behavioral deficit better mimics the clinical condition where antidepressant drugs are used to cure an established depression. In the reversal protocol, a difference among the groups was revealed by ANOVA (F6,48 = 28.44; P < 0.001). Escape responses in the Stress group were significantly lower compared to the Naïve and Stress + FLX groups, and similar to the responses of the Stress + ST 1936 10 and Stress + ST 1936 30 groups (Fig. 3). The results suggest that the efficacy of ST 1936 in this test is not comparable to that of classical antidepressants. In fact, long-term administration of clinically effective antidepressants is successful in both the prevention and the reversal of ED [38]. 3.6. Experiment 4: effect of repeated ST 1936 treatment on VAB acquisition in control and chronically stressed rats The preliminary experiment indicated that treatment with ST 1936 at the doses of 10 or 30 mg/kg did not alter rat’s com-

Fig. 3. Effects of long-term ST 1936 administration on ED reversal. Rats were exposed to the unavoidable stress and the following day they were tested for escape. Twenty-four hours later they began the treatment with vehicle (1 ml/kg, i.p.; Stress) or ST 1936 (10 or 30 mg/kg, i.p.) or fluoxetine (FLX 5 mg/kg, i.p.) once a day for 3 weeks, concomitantly with exposure to chronic stress. A control group of rats received vehicle for 3 weeks, without stress exposure (Naive). At the end of 3 weeks rats were tested for escape. Scores are expressed as mean number of escapes ± S.E.M. in 30 consecutive trials. **P < 0.01 Stress group vs. Naive and Stress + FLX groups (Dunnett’s test).

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Fig. 4. Effects of repeated ST 1936 treatment on VAB acquisition in control and chronically stressed rats. Rats were exposed to the unavoidable stress and the following day they were tested for escape. Twenty-four hours later they began the treatment with vehicle (1 ml/kg, i.p.; Stress) or ST 1936 (10 or 30 mg/kg, i.p. Stress + ST) once a day; the treatment continued for 3 weeks concomitant with chronic stress protocol and VAB training. A control group of rats not exposed to the stress procedure received saline for 3 weeks concomitant with VAB training (CTR). Data are presented as mean ± S.E.M. of the number of correct (a), incorrect (c) and incomplete trials (d), and number of pellets consumed (b).*§ P < 0.05 Stress group vs. CTR and Stress + ST 30 groups; ***P < 0.001 Stress group vs. CTR and §§ P < 0.01 Stress group vs. Stress + ST 30 group (Bonferroni’s test).

petence to acquire VAB (Fig. S1 in Supplementary Material). In the anhedonia experiment analysis by two-way repeated measure ANOVA indicated that the number of correct trials performed and VS pellets consumed were affected by treatment (correct trials: F3,252 = 3.30, P < 0.001; VS pellets consumed; F3,252 = 5.91, P < 0.001), session (correct trials: F9,252 = 6.49, P < 0.001; VS pellets consumed F9,252 = 22.83, P < 0.01); and their interaction (correct trials: F27,252 = 2.42, P < 0.05; VS pellets consumed: F27,252 = 3.00). Analysis of incorrect and incomplete trials indicated no treatment effect, an effect of session (F9,252 = 7.18; P < 0.001 incorrect; F9,252 = 11.57; P < 0.001 incomplete) and interaction (F27,252 = 1.71; P < 0.05 incorrect; F27,252 = 1.73; P < 0.05 incomplete) (Fig. 4c). ST 1936 treatment at the dose of 30 mg/kg restored the response of stressed rats to values similar to those of control animals in the number of correct trials (Fig. 4a) and in the number of VS pellets consumed (Fig. 4b). These results indicate that 5-HT6 receptor agonist ST 1936 possess antianhedonic activity. 3.7. Experiment 5: effect of the 5-HT6 receptor antagonist SB 271046 on the antianhedonic activity of ST 1936 Analysis by two-way repeated measure ANOVA indicated that the number of correct trials performed and VS pellets consumed were affected by treatment (correct trials: F4,279 = 18.43, P < 0.001; VS pellets consumed; F4,279 = 14.70; P < 0.001), session (correct trials: F9,279 = 9.33, P < 0.001; VS pellets consumed F9,279 = 28.95, P < 0.001); and their interaction (correct trials: F36,279 = 1.90, P < 0.05; VS pellets consumed: F36,279 = 3.83; P < 0.001). Moreover, SB 271046 treatment per sé induced a deficit of VAB acquisition (Fig. 5a and b). Analysis of incorrect trials number indicated a

treatment effect, (F4,279 = 9.34; P < 0.001) (Fig. 5c). Analysis of the number of incomplete trials indicated a treatment (F4,279 = 16.67, P < 0.001), session (F9,279 = 6.91; P < 0.001) and interaction effect (F36,279 = 1.49, P < 0.05) (Fig. 5d). In particular, rats in the SB and Stress + ST + SB groups showed a higher number of incomplete trials compared to rats in the ST + Stress and Control groups. These results indicate that the antianhedonic effect of the 5-HT6 receptor agonist ST 1936 is completely prevented by the concomitant treatment with the 5-HT6 receptor antagonist SB 271046. 4. Discussion The results of this study indicate that ST 1936, a rather selective 5-HT6 receptor agonist had no effect on stress-induced hyporeactivity after acute administration; elicited a protective effect towards the ED induced by a single unavoidable stress exposure after repeated administration, but it failed to affect chronic stresssustained ED. Moreover, long-term ST 1936 treatment protected rats from the disrupting effects of chronic stress on VAB learning and this protective activity was prevented by the concomitant administration of the 5-HT6 receptor antagonist SB 271046. The protective effect exerted on acute ED development seems to be mediated by muscarinic receptor activation, since it was selectively antagonized by scopolamine administration soon before unavoidable stress exposure, and it cannot be ascribed to a central stimulant or analgesic activity, since repeated ST 1936 treatment did not modify spontaneous motility or pain threshold. The fact that acute SB 271046 administration before unavoidable stress exposure did not antagonize the ST 1936-induced anti-stress activity, while the daily co-administration of the two compounds did, indicates that 5-HT6 receptor activation is crucial for triggering

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Fig. 5. Effect of the 5-HT6 receptor antagonist SB 271046 on the antianhedonic activity of ST 1936. Rats were exposed to the unavoidable stress and the following day they were tested for escape. Twenty-four hours later they began the treatment once a day with saline (1 ml/kg, i.p.; Stress) or ST 1936 30 mg/kg, i.p. (Stress + ST), or ST 1936, 30 mg/kg + SB 271046 20 mg/kg, i.p. (Stress + ST + SB), or SB 271046 20 mg/kg, i.p. (SB). The treatment continued for 3 weeks concomitant with chronic stress protocol and VAB training. A control group of rats not exposed to the stress procedure received saline for 3 weeks concomitant with VAB training (CTR). Data are presented as mean ± S.E.M. of the number of correct (a), incorrect (c) and incomplete trials (d), and number of pellets consumed (b). ***P < 0.001 CTR and Stress + ST groups vs. Stress, Stress + ST + SB and SB groups (a and b); **P < 0.01 SB group vs. CTR and Stress + ST groups; ***P < 0.001; §§§ P < 0.001 Stress + ST + SB group vs. CTR and Stress + ST groups, respectively (d) (Bonferroni’s test).

a neuronal plasticity process that resulted in the prevention of ED development. At the end of this process the protective effect resulted to be dependent on muscarinic receptor activation and independent of 5-HT6 receptor availability. The acute or short-term administration of selective antagonists of 5-HT6 receptors induces acetylcholine release in the frontal cortex [10,11], a stretching and yawning syndrome mediated by muscarinic receptor stimulation [53], and, inhibition of the rotational behavior in 6-OHDA lesioned rats induced by treatment with the muscarinic antagonists scopolamine and atropine [54]. Thus, the involvement of muscarinic receptors in the protective effect of a long-term treatment with ST 1936 confirms the existence of a 5-HT6 –muscarinic transmission interplay, an aspect that certainly needs further investigations. An analogous phenomenon has been formerly observed in rats repeatedly treated with imipramine – an inhibitor of norepinephrine and 5-HT neuronal membrane transporters with no direct effects on dopamine transmission – that show a protective effect on acute ED development selectively mediated by dopamine D1 receptor activation [43]. The daily ST 1936 administration for 3 weeks did not revert ED in rats exposed to the chronic stress procedure. Thus, this compound has a clear anti-stress activity, but this activity is not comparable to that of classical antidepressants such as imipramine, fluoxetine, reboxetine, etc., that after an average 3-week treatment consistently revert chronic ED [38,44,45]. That is, ST 1936 seems to be endowed with a major antianhedonic and a weak antistress activity when compared to classical antidepressants. Daily ST 1936 administration in rats undergoing the Y-maze training did not interfere with the acquisition of VAB, whereas SB 271046 administered in similar conditions prevented VAB acquisition. Exposure to palatable food is widely used in experimental protocols that evaluate animal’s hedonic competence since it elicits consistent behavioral and neurochemical responses, it is a validated index of hedonic responsiveness and the two bottle choice test is a common test that assesses hedonic reactions to sucrose [55]. We

only use non food-deprived rats since in these animals the emotional value of a palatable food has a prevalent hedonic component [47]. Rats are very fond of vanilla sugar (VS) and non food-deprived rats consistently acquire an instrumental behavior (VAB) based on the reinforcing properties of VS pellets, the earning of which is made contingent on the choice of one of the two divergent arms of a Y-maze [45]. Exposure to repeated mild, unavoidable stressors is known to cause a decreased responsiveness to rewards, which is seen in a variety of different experimental paradigms. The effect most commonly reported is a decrease in the consumption of, and preference for, palatable sweet solutions; other effects include a decrease in place preference conditioning and an increase in brain stimulation reward threshold [56]. All of these deficits are reversed by chronic treatment with clinically effective antidepressant drugs [55,57–62]. The decreases in sensitivity to reward observed in the chronic mild stress (CMS) and in the VAB models are most readily interpreted as reflections of an underlying state of stress-induced anhedonia [57,63]. The results on VAB learning seem to disagree with the fact that 5-HT6 receptor antagonists in other experimental conditions have been reported to play a positive role in cognitive tasks. Acute administration of selective 5-HT6 receptor antagonists or antisense oligonucleotides directed against the initiation codon of the 5HT6 receptor improved retention of spatial learning in the Morris water maze [2,64,65] and recognition memory in the novel object discrimination paradigms in rats [6,7,66]. 5-HT6 receptor antagonists have also been found to improve memory consolidation in an autoshaping Pavlovian/instrumental learning task [67,68]. reverse a scopolamine-induced impairment in step-through passive avoidance [64], and also reverse a scopolamine-induced deficit in a social recognition paradigm in rats [3]. However, not all research groups have been able to replicate the pro-cognitive effects of 5HT6 receptor antagonists in the Morris water maze [69,70] or the reversal of scopolamine-induced deficits in a test of contextual fear

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conditioning or the autoshaping task [69). Finally, E-6801, a potent 5-HT6 receptor agonist, improves recognition memory by combined modulation of cholinergic and glutamatergic neurotransmission [1]. Learning to correctly choose the baited arm of a Y-maze evaluates the rat’s competence to discriminate between two possible choices based on the outcome (VS earning). Thus, after a 10-day Y-maze training control rats show to be competent to acquire an instrumental, appetitive goal directed behavior that would likely evolve into a habit after a more prolonged training [71]. The concomitant daily administration of a 5-HT6 receptor agonist did not interfere with VAB acquisition, while the antagonist administration disrupted the learning competence. Thus suggesting that 5-HT6 receptor functional integrity plays a central role in the acquisition of this goal directed behavior, while an additional pharmacological stimulation of 5-HT6 receptors has no apparent effect. Stress-induced disruption of VAB acquisition has been proposed as a model of anhedonia, and we may conclude that repeated ST 1936 administration has a clear anti-anhedonic activity. On the basis of the results on acute ED, it may be hypothesized that ST 1936 triggered an adaptive neuronal response that not only did not interfere with the acquisition of a goal directed behavior, but it also counteracted the disrupting effect of chronic stress exposure on VAB acquisition. These results are analogous to those observed after repeated l-acetyl-carnitine (ALCAR) administration: rats treated for 10 days with ALCAR do not develop acute ED, and this protective effect is mediated through 5-HT1A receptor stimulation [72]. However, ALCAR treatment, like ST 1936 treatment, does not revert the behavioral deficits induced by chronic ED, whereas it reverts the disrupting effect of chronic stress exposure on VAB acquisition [73]. The failure to revert stresssustained chronic ED associated with the capacity to antagonize the disrupting effect of chronic stress exposure on VAB acquisition may find an explanation in the fact that natural rewards, such as palatable foods, provide in rats a general means of stress reduction, likely via structural and/or functional plasticity in the basolateral amygdala [74]. The behavioral modifications induced by ST 1936 observed in our experimental conditions are based on long-term treatments and are difficult to compare with most of the experimental data reported in the literature that were obtained using models, as behavioral despair tests, that respond to acute treatments. The concept of anhedonia does not merely imply the inability to perceive a pleasurable stimulus, but it also includes a reduced capacity to acquire and/or emit behaviors aimed at approaching or earning a reward; that is, the inability to attribute incentive value to a rewarding stimulus. Chronic stress exposure induces these deficits and, thus, it causes anhedonia. As these effects of stress are reversed by chronic treatment with different classical antidepressants, antidepressants are considered medicaments endowed with anti-anhedonic activity. ST 1936 administration completely prevented the disrupting effect of chronic stress exposure on VAB acquisition and, since SB 271046 co-administration completely antagonized this protective effect, it may be concluded that not only a tonic 5-HT6 receptor activity was crucial for VAB acquisition, but that pharmacological stimulation of 5-HT6 receptors reinstated a stress-reduced hedonic competence. In other words, ST 1936 showed an efficacy to counteract chronic stress-induced anhedonia and it may be proposed as a true anti-anhedonic compound.

Conflicts of interest The authors declare that this work was founded by SigmaTau, Industrie Farmaceutiche Riunite SpA, Pomezia, Italy and that, except for income received by their primary employer, no compensation has been received for this study.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbr.2011.05.019.

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