(+)-S-20499 – a potential antidepressant? A behavioural and neurochemical investigation in the olfactory bulbectomised rat

European Neuropsychopharmacology 9 (1999) 21–27

(1)-S-20499 – a potential antidepressant? A behavioural and neurochemical investigation in the olfactory bulbectomised rat C McGrath a

a ,b ,

*, T.R. Norman a

Department of Psychiatry, Austin and Repatriation Medical Centre, Heidelberg, VIC 3084, Australia b Department of Pharmacology, University College, Galway, Ireland Received 27 August 1997; accepted 28 November 1997

Abstract The study was designed to assess the potential antidepressant properties of the 5-HT 1A receptor agonist, (1)-S-20499 (10 mg kg 21 i.p.) in the olfactory bulbectomised (OB) rat. Following 2 weeks of treatment, the rats were tested in the elevated plus maze and the ‘‘open field’’. A characteristic hyperactive response was evident in the OB animals in the ‘‘open field’’ which was reversed following chronic treatment with (1)-S-20499. In the elevated plus maze an increase in the number of open arm entries and the time spent on the open arms was observed, although this failed to reach significance. A significant decrease in b1 receptor affinity was evident following olfactory bulbectomy which was normalised by (1)-S-20499. (1)-S-20499 also significantly reduced the density of 5-HT 2 receptors in the sham operated (SO) animals. These studies demonstrate the usefulness of the OB rat as a screening test for compounds with novel putative mechanisms of action, and highlights the potential antidepressant properties of (1)-S-20499.  1999 Elsevier Science B.V. / ECNP. All rights reserved. Keywords: (1)-S-20499; Olfactory bulbectomy; b1 receptor; 5-HT 2 receptor binding

1. Introduction Removal of the olfactory bulbs in the rat produces a syndrome characterised by increased activity in novel environments such as the ‘‘open field’’ (Janscar and Leonard, 1983; O’Connor and Leonard, 1988), increased muricidal behaviour (Lloyd et al., 1982) and deficits in passive avoidance acquisition (Van Reizen and Leonard, 1990). These behavioural changes have been shown to be reversed by chronic but not acute antidepressant treatment (Van Reizen and Leonard, 1990). Neurochemical changes have also been reported following bulbectomy with alterations in serotonergic, noradrenergic, GABAergic and glutaminergic neurotransmitter systems similar to those found in the human condition of major depressive disorder. Although the precise mechanisms underlying bulbectomy induced changes are unclear, alterations in receptor *Corresponding author. Tel.: 161 3 94965694; fax: 161 3 94590821; e-mail: [email protected]

populations have been found. Increased 5-HT 2 receptor binding has been reported in the cortex of olfactory bulbectomised (OB) animals, and muscarinic receptor density has been shown to be decreased in the amygdala, hippocampus, hypothalamus and the cortex. These alterations have been shown to be reversed by chronic antidepressant treatment (Earley et al., 1994). GABAA receptor densities have been shown to be increased following olfactory bulbectomy (Dennis et al., 1994) and also reversed by chronic antidepressant treatment (Dennis et al., 1994). One neurotransmitter system that has been reported to be important in depression is the adrenergic system particularly as reflected by b1 receptors (Banerjee et al., 1977). These receptors have been reported to be increased in depressed patients and a decrease in the density of these receptors has been reported following chronic antidepressant treatment (Vetulani et al., 1976; Banerjee et al., 1977; Sulser et al., 1978). Reports of alterations in b-adrenoceptor populations in the OB animals have been inconsistent. Tiong and Richardson (1990) reported an increase in

0924-977X / 99 / $ – see front matter  1999 Elsevier Science B.V. / ECNP. All rights reserved. PII: S0924-977X( 97 )00103-X

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C. McGrath, T.R. Norman / European Neuropsychopharmacology 9 (1999) 21 – 27

the affinity of the b-adrenoceptors in the amygdala and hippocampus of the OB animals while in a later study Dennis et al. (1994) found no bulbectomy induced changes in these receptor populations. However, chronic antidepressant treatment did produce a down regulation of badrenoceptors in the frontal cortex of OB animals (Dennis et al., 1994). This finding suggests that the olfactory bulbectomised rat is a useful model for studying the neurochemical effects of chronic antidepressant treatment. The 5-HT system has long been implicated in the pathophysiology of depression (Glassman, 1969). A class of drugs that have shown antidepressant like activity in animal models of depression including the learned helplessness model (Martin et al., 1990), the restraint test (Kennett et al., 1987), the forced swim test (Cervo and Samanin, 1987) and the olfactory bulbectomised rat model (McNamara et al., 1995; Cryan et al., 1997) are the 5-HT 1A receptor agonists. Although these compounds are marketed for the treatment of anxiety, it has been suggested that their anxiolytic properties are mediated through their effects on presynaptic receptors, while the antidepressant activity is exerted through their actions at postsynaptic receptors (Martin et al., 1990). (1)-S-20499 ((1)-8(4[N-(5-methoxychroman-3yl)-8azaspirol [4,5] decane-7,9-dione) is a selective and full agonist at pre- and post-synaptic 5-HT 1A receptors (Kidd et al., 1993). (1)-S-20499 binds to 5-HT 1A receptors in the hippocampus with a Ki value of 0.19 nM (Curle et al., 1994). 5-HT 1A receptors are located both presynaptically as somatodendritic autoreceptors and postsynaptically in the hippocampus and the amygdala (Verge et al., 1986; Palacious et al., 1987). Acutely (1)-S-20499 been shown to produce hypothermia but unlike 8-OH-DPAT, another 5-HT 1A receptor agonist, it does not induce the serotonin syndrome in the rat (Scott et al., 1994). As mentioned earlier, 5-HT 1A receptor agonists have elicited antidepressant like activity in preclinical studies. Martin (1994) found that (1)-S-20499 reduced the number of escape failures in the learned helplessness model and Porsolt et al. (1992) reported that (1)-S-20499 increased the duration of immobility in the tail suspension test. It has been suggested that based on behavioural, electrophysiological and neuropharmacological data, this compound may be a clinically effective anxiolytic / antidepressant agent (Porsolt et al., 1992; Kidd et al., 1993; Barrett et al., 1994). The effect of antidepressant treatment on the hypothermic response induced by the acute administration of 8-OH-DPAT has often been used as a measure of 5-HT 1A receptor function (Hamon et al., 1987). There is still a great deal of controversy as to whether this is a pre- or post-synaptic effect. This hypothermic response has been shown to be attenuated following chronic antidepressant treatment, although the mechanism responsible for this effect depends on the class of antidepressant studied (Frazer and Hensler, 1990). The aim of the present study was to examine the effect

of olfactory bulbectomy on b-adrenoceptor and 5-HT 2 receptor populations and to assess the effect of chronic treatment with (1)-S-20499 on these receptor populations. The behaviour of the animals following olfactory bulbectomy and chronic (1)-S-20499 treatment on the ‘‘open field’’ and the elevated plus maze was also examined. This work was published in part at the CINP congress in Melbourne.

2. Experimental procedure

2.1. Animals and chemicals Male Sprague–Dawley rats (weight 200–250 g) were housed four per cage. They were allowed free access to food and water throughout the course of the experiment and were maintained on a 12:12 light dark cycle (lights on: 08:00 h; lights off: 20:00). [ 3 H] CGP-12177 (specific activity 42.5 Ci / mmol) and 3 [ H] ketanserin (specific activity 80.0 Ci / mmol) were obtained from Dupont, Australia. 8-OH-DPAT was purchased from Australian Laboratory supply. Oxytetracycline powder, Tris, sodium chloride, potassium chloride, copper sulphate, sodium hydroxide and sodium tartrate were purchased from Sigma Chemical Company. (1)-S-20499 was kindly provided by Servier Laboratories (Melbourne, Australia).

2.2. Surgery and drug treatment After a 1 week acclimatization period, bilateral olfactory bulbectomy was performed in rats anaesthetized with a 2.5% w / v solution of 2–2–2 tribromoethanol in saline administered intraperitoneally (Aldrich, Sydney, Australia) essentially as described by Cairncross et al. (1977). Briefly, the head was shaved and a midline sagittal incision made. Two drill holes of 2 mm diameter were made in the skull and the olfactory bulbs were removed by suction. Bleeding was controlled by plugging the holes with haemostatic sponge. Oxytetracycline powder was sprinkled on the wound prior to closure. Sham operated (SO) animals were treated in the same way but the olfactory bulbs were left in tact. The animals were allowed to recover for 14 days following surgery. They were handled daily throughout the recovery period to eliminate any aggressiveness that would otherwise arise. (1)-S-20499 was dissolved in saline in an injection volume of 1 ml kg 21 . (1)-S-20499 (10 mg kg 21 ) was administered intraperitoneally once daily for a period of two weeks. Control animals received injections of vehicle (saline) alone.

2.3. Binding experiments Twenty four hours after the last dose, all rats were

C. McGrath, T.R. Norman / European Neuropsychopharmacology 9 (1999) 21 – 27

sacrificed by decapitation. The brains were removed, rapidly dissected on ice and the frontal cortex was stored at 2708C for binding studies. One cortical hemisphere was used for b 1 and the other cortical hemisphere for 5-HT 2 receptor binding. The tissue was homogenised for approximately 5 s in 500 ml of ice cold Tris–HCl buffer (50 mM Tris, pH 7.4). The samples were then centrifuged at 30 0003g for 10 min at 48C. The supernatant was discarded and the pellet of particulate membrane resuspended in 500 ml of ice cold Tris–HCl incubation buffer and recentrifuged as before. Following a further resuspension and centrifugation the supernatant was decanted and the resulting pellet was resuspended in ice cold Tris–HCl buffer (50 mM, pH 7.4) to form the final membrane suspension. [ 3 H] CGP 12177 binding was carried out in triplicate by incubation of 200 ml of tissue suspension with [ 3 H] CGP 12177 at six concentrations (0.025–0.8 nM) in incubation buffer with atenolol (1 mM) or isoprenaline (1 mM) as the displacing agents, for 2 h at room temperature. Membrane bound radioactivity was determined by filtration under vacuum through Whatman GF / B glass fibre filters presoaked in Tris buffer (50 mM Tris containing 0.1% BSA, pH 7.7) using a Brandell cell harvester. Filters were washed with 5 ml ice cold Tris buffer. Following three washes with 5 ml of Tris BSA buffer, the filter containing the membrane was transferred into 5 ml of Ready Protein scintillation fluid (Beckman Instruments Inc., Melbourne, Australia) and counted using a Packard Scintillation counter (efficiency 63%). Specific binding was defined as the difference between total binding (defined in the presence of Tris) and the binding observed in the presence of atenolol or isoprenaline. Isoprenaline binding is not specific and reflects both b1 and b2 receptor populations. Atenolol is b1 specific and therefore gives b1 binding. Therefore b2 binding was calculated by subtracting b1 binding from the total b-binding. [ 3 H] Ketanserin binding was carried out in triplicate by the incubation of the membrane suspension with six concentrations of [ 3 H] ketanserin, in concentration range of 0.05–12 nM, in incubation buffer in the presence or absence of methysergide (1 mM) for 1 h at 378C. Membrane bound radioactivity was determined by filtration under vacuum through Whatman GF / B glass fibre filters presoaked in Tris buffer (50 mM Tris containing 0.1% BSA, pH 7.7) using a Brandell cell harvester. Filters were washed with 5 ml ice cold Tris buffer. Following three washes with 5 ml of Tris BSA buffer, the filter containing the membrane was transferred into 5 ml of Ready Protein scintillation fluid and counted using a Packard Scintillation counter.

2.4. Protein determination The methods used for protein determination was based on that of Lowry et al. (1951) using bovine serum albumin

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as a standard. The absorbance at 682 nm of the resulting coloured solution is proportional to the amount of protein in the solution.

3. 8-OH-DPAT hypothermic response 8-OH-DPAT (1.5 mg kg 21 ) was administered s.c. 1 h following the last antidepressant treatment. Rectal temperatures were measured by means of a digital thermometer inserted 2 cm into the rectum of each rat. Temperatures were recorded prior to and at 30, 60, 90 and 120 min following 8-OH-DPAT.

3.1. ‘‘ Open field’’ The open field is essentially similar to that described by Gray and Lalljee (1974). It consists of a white circular base, (90 cm diameter) which is divided into 10 cm squares by faint yellow lines. The wall (75 cm high) surrounding the base is made of aluminium sheeting. Illumination is provided by a 60 W bulb, positioned 90 cm above the floor of the apparatus. All measurements were carried out in a darkened room in the morning.

3.2. Elevated plus maze Pellow et al. (1985) validated the elevated plus maze as a test of anxiety. The test is based on the natural aversion of rodents for open spaces. The maze consists of a 1 shaped structure elevated 50 cm from the floor comprising two opposite enclosed and two open arms. The arms are 45 cm long and 10 cm wide. The enclosed arms have sides and ends of 10 cm height while the open arms have no sides or arms. The central square formed by the arms is open. Illumination was provided by a 60 watt bulb positioned 90 cm above the centre of the maze. Entry into an arm is defined as the animal placing all four paws on the arm. The cumulative time spent in and the number of entries made into the open or closed arms were recorded during a 5 min testing session. The plus maze was carefully cleaned after each animal tested.

3.3. Data analysis The behavioural responses were analysed using a Kruskal Wallis analysis of variance followed by a Mann Whitney U-test. The temperature responses were analyzed using an analysis of variance followed by Student Newman Keuls posthoc test. Binding data were analyzed by Scatchard analysis using the EBDA program (McPherson, 1985) and Bmax and Kd values were calculated. Differences between the groups on these parameters were then examined using an analysis of variance (ANOVA).

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Table 1 Effect of chronic treatment with (1)-S-20499 on the behaviour of the olfactory bulbectomised rat in the ‘open field‘ Group

Ambulation

Grooming

Defaecation

SO1vehicle SO1(1)-S-20499 OB1vehicle OB1(1)-S-20499

48 (34–57) 48 (37–51) 84 (69–85)*1 50 (34–79)

1 2 2 0

1 3 2 1

(1–3) (1–2) (1–3) (0–1)

(1–3) (1–5) (1–3) (0–4)

Results are tabulated as group medians. Numbers in parentheses are interquartile ranges. * P,0.05 versus SO1vehicle; 1 P,0.05 versus OB (1)-S-20499. N58.

Table 2 Effect of chronic treatment with (1)-S-20499 on the behaviour of the olfactory bulbectomised rat in the elevated plus maze Group SO1vehicle SO1S-20499 OB1vehicle OB1S-20499

No. open entries

Time spent on open arms (s)

No. closed entries

Time closed arms (s)

1 2 5 5

12 (0–61) 100 (29–200) 43 (14–157) 38 (14–62)

7 5 8 5

169 (140–261) 133 (45–207) 208 (65–232) 121 (101–192)

(0–4) (1–4) (1–7) (3–9)

(4–9) (3–7) (5–9) (3–7)

Results are expressed as group median. Numbers in parenthesis represent the interquartile ranges. N58

4. Results

4.1. ‘‘ Open field’’ Removal of the olfactory bulbs produced a characteristic hyperactivity in the OB animals when placed in the ‘‘open field’’ and compared to the SO operated animals (P,0.05). This hyperactivity was reversed by chronic treatment with (1)-S-20499 (P,0.05) Table 1.

4.2. Elevated plus maze There was a slight increase in the percentage of time spent on the open arms and an increase in the number of open arm entries made by the OB rat (P,0.01). Chronic treatment with (1)-S-20499 had no statistically significant effect on these parameters Table 2.

Fig. 1. Effect of chronic (1)-S-20499 treatment on b1 receptor density in the olfactory bulbectomised rat model of depression. Data represent group mean6S.E.M. N54.

5. 8-OH-DPAT Prior to 8-OH-DPAT challenge there was no effect of lesion or drug on core body temperature in the animals. Results revealed a significant effect of drug on core body temperature following 8-OH-DPAT (1.5 mg kg 21 s.c.). Chronic treatment with (1)-S-20499 significantly attenuated the hypothermic response in both OB and SO animals Table 3.

5.1. Binding results 5.1.1. b1 receptor binding A two-way analysis of variance found no significant alterations in Bmax values between SO and OB animals as a result of surgery alone 32 days after olfactory bulbectomy although there was a trend towards increased b 1 adrenoceptor density in the OB animals. Student Newman Keuls post hoc analysis revealed that chronic treatment with (1)-S-20499 slightly reduced b 1 -adrenoceptor density in the cortex of the SO animals Fig. 1

Table 3 Effect of chronic treatment with (S)-20499 on the hypothermic response to 8-OH-DPAT in the olfactory bulbectomised rat Treatment

N

T0–T30

T0–T60

T0–T90

T0–T120

Vehicle SO S-20499 SO Vehicle OB S-20499 OB

10 11 6 10

21.4760.15 21.1260.18 21.7160.24 21.0660.14

22.0660.17 20.6460.17* 21.8960.24 20.8260.241

21.4260.15 20.1660.15* 21.4260.19 20.4160.331

20.7760.15 20.1060.13* 21.1360.23 20.1760.281

Results are tabulated as group mean6standard error of the mean (S.E.M.). * P,0.05 versus SO1vehicle; 1 P,0.05 versus OB1vehicle. N58.

C. McGrath, T.R. Norman / European Neuropsychopharmacology 9 (1999) 21 – 27

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of drug on 5-HT 2 receptor density. Chronic treatment with (1)-S-20499 significantly reduced 5-HT 2 receptors in the SO animals (P,0.05, Student Newman Keuls posthoc) (Fig. 3). No effect of drug administration was observed on the affinity of 5-HT 2 receptors.

6. Discussion

Fig. 2. Effect of chronic treatment with (1)-S-20499 on b1 receptor affinity in the OB rat. Data represent group mean6S.E.M. *P,05 versus OB1vehicle. N54.

5.1.2. b1 receptor affinity A two-way ANOVA revealed a significant decrease in b 1 -adrenoceptor affinity in the OB vehicle animals when compared to the SO operated vehicles (P,0.05). This alteration in receptor affinity was found to be reversed following chronic treatment with (1)-S-20499. Fig. 2. 5.1.3. 5 -HT2 receptor binding No significant differences in 5-HT 2 receptor populations were found as a result of bulbectomy alone 32 days after surgery. A two-way ANOVA revealed a significant effect

Fig. 3. Effect of chronic (1)-S-20499 on the density of cortical 5-HT 2 receptors. Results are represented as group mean6S.E.M. * P,0.05 vs. SO1vehicle. N54.

In the ‘‘open field’’ apparatus a significant increase in locomotor activity was observed following olfactory bulbectomy in the rat. Clinically effective antidepressant drugs reversed bulbectomy induced behavioural changes, such as increased activity in novel environments (Janscar and Leonard, 1983; O’Connor and Leonard, 1988), a property shared by both typical and atypical antidepressants. Chronic treatment with the 5-HT 1A receptor agonist, (1)-S-20499 significantly reduced the increased locomotor activity evident in the OB animals (P,0.01) in a fashion similar to that observed with antidepressants. This is in line with previous research which has shown that the 5-HT 1A receptor agonist 8-OH-DPAT reverses the hyperactivity in the ‘‘Open field’’ (McNamara et al., 1995). Other 5-HT 1A receptor agonists have demonstrated ‘‘antidepressant-like’’ behaviour in other animal models including the Learned helplessness model (Martin et al., 1990), the restrained stress model (Kennett et al., 1987) and the forced swim test (Cervo and Samanin, 1987). The present results, together with those from other preclinical testing, are suggestive of an antidepressant effect of (1)-S-20499. This finding is important since other 5-HT 1A partial agonists and full agonists such as flesinoxan have been used as antidepressants (Grof et al., 1993). The elevated plus maze was validated as a test of anxiety by Pellow et al. (1985). In the present study no bulbectomy induced behavioural changes were found on this test. However the data in Table 1 do suggest some differences in hyperactivity between the SO and OB animals in the vehicle treated groups. No significant effects of chronic (1)-S-20499 treatment was observed in these animals. This finding is not in line with previous research in our laboratory which has shown a significant increase in open arm entries and the percentage time on the open arms for the OB animals (Song and Leonard, 1994; McGrath, 1996). These findings suggest that the elevated plus maze may not be a reliable test for examining behavioural deficits following olfactory bulbectomy in the rat. Chronic antidepressant treatment has been reported to attenuate the hypothermic response to 8-OH-DPAT in the rat (Frazer and Hensler, 1990). This is reported to be due to an increase in 5-HT neurotransmission. The mechanism responsible for this effect varies depending on the class of antidepressant used. In general, chronic treatment with SSRI’s has been reported to decrease the sensitivity of the presynaptic 5-HT 1A receptor (Frazer and Hensler, 1990), while TCA’s reportedly increase the sensitivity of the

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C. McGrath, T.R. Norman / European Neuropsychopharmacology 9 (1999) 21 – 27

post-synaptic 5-HT 1A receptors (De Montigny, 1984). In the present study chronic treatment with (1)-S-20499 significantly attenuated this hypothermic response. It has been suggested that chronic treatment with 5-HT 1A receptor agonists attenuate the hypothermic response to 8OH-DPAT by desensitising the presynaptic 5-HT 1A receptor (Blier et al., 1987; Kennett et al., 1987). Since it has been shown that the presynaptic 5-HT 1A receptors do not desensitise following repeated treatment with 5-HT 1A receptor agonists (De Montigny and Aghajanian, 1978) these results suggest that a post-synaptic mechanism may be involved in mediating the antidepressant action of (1)-S-20499. The incorporation of the 8-OH-DPAT challenge and the OB rat model into one study design may prove useful for further exploring the mechanism of action of novel antidepressant compounds. As well as changes in the sensitivity of 5-HT 1A receptor following chronic antidepressant treatment, changes in the function and the density of the 5-HT 2 receptors have been reported (Goodwin et al., 1984). Increased 5-HT 2 receptor density has been reported to occur in the platelets of depressed patients (Biegon et al., 1987; Butler et al., 1988) and an increase in the density of these receptors have been reported in the cortex of depressed suicide victims (Biegon et al., 1987). No significant changes in 5-HT 2 receptor density was found 32 days following olfactory bulbectomy although there was a trend towards an increase in the OB animals. Chronic treatment with (1)-S-20499 significantly reduced the density of these receptors in the SO animals and there was a trend towards a reduction in the OB animals. This is consistent with previous research which has shown a decrease in cortical 5-HT 2 receptor density following chronic treatment with mianserin and desipramine in both SO and OB animals (Earley et al., 1994). In the present study the affinity of the b1-adrenoceptor was found to be decreased in the OB animals when compared to the SO animals which was normalised by chronic (1)-S-20499. The results of the present study are at variance with previous research which found no differences between SO and OB animals in b1-adrenoceptor populations (Tiong and Richardson, 1990; Dennis et al., 1994). However, the use of other radioligands to determine b-receptor binding means that the studies are not directly comparable. No significant alterations in the density of b1-adrenoceptors was evident 32 days following olfactory bulbectomy in the rat. This is in line with work by Dennis et al. (1994) who found no effect of bulbectomy on b1-adrenoceptor density. Chronic treatment with (1)-S-20499 had no significant effect on the density of the b1-adrenoceptors although there was a trend in the SO animals for a reduction in b1 density. It is possible however that the lack of significance seen in the present study could be due to the low sample size (N54) and further analysis using larger groups would therefore be necessary. No significant changes were found in b 2 receptors following drug treat-

ment. It has been suggested that the failure of b 2 receptors to down regulate may be due to the fact that b 1 receptors have 10 times greater affinity for norepinephrine than b 2 receptors (Ordway et al., 1988). The results of the present highlight the potential antidepressant activity of (1)-S-20499 when tested in the olfactory bulbectomised rat model of depression and the usefulness of the olfactory bulbectomised rat for screening drugs with putative activity.

Acknowledgements The authors would like to thank Servier (Australia), for providing the drug to perform this study. Sincere thanks to Professor B.E. Leonard, University College, Galway, for providing C. McGrath with the opportunity to work in Australia.

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