Indian Journal of Experimental Biology Vol. 46, October 2008, pp.

704-714

Dose-dependent response of central dopaminergic systems to buspirone in mice

S A Jadhav, R V Gaikwad, R K Gaonkar, V M Thorat, S C Gursale & J J Balsara*
Department of Pharmacology, Krishna Institute of Medical Sciences, Karad 415110, India Received 5 October 2007; revised 14 July 2008 Buspirone, a partial agonist of 5-hydroxytryptamine1A autoreceptors, preferentially blocks the presynaptic rather than the postsynaptic D2 dopamine (DA) receptors. Behavioural effects of a wide dose range of buspirone were therefore studied in mice. Buspirone at 0.625 to 5 mg/kg ip induced stereotyped cage climbing behaviour which was antagonized by pretreatment with haloperidol, alpha-methyl-p-tyrosine and small doses of apomorphine. Buspirone at 10, 20 and 40 mg/kg ip induced catalepsy and antagonized oral stereotypies induced by high doses of apomorphine and methamphetamine and apomorphine-induced cage climbing behaviour. The findings indicate that buspirone at 0.625 to 5 mg/kg selectively blocks the presynaptic mesolimbic D2 DA autoreceptors and releases DA which stimulates the postsynaptic mesolimbic D2 and D1 DA receptors and induces cage climbing behaviour. Buspirone, at 10, 20 and 40 mg/kg blocks the postsynaptic striatal and mesolimbic D2 and D1 DA receptors. Pretreatment with l-tryptophan, dexfenfluramine and fluoxetine antagonized buspirone induced cage climbing behaviour and potentiated buspirone induced catalepsy. Pretreatment with trazodone, mianserin and p-chlorophenylalanine potentiated buspirone induced cage climbing behaviour and antagonized buspirone induced catalepsy. The results indicate that drugs which influence the activity of central serotonergic systems modulate the intensity of buspirone induced cage climbing behaviour and catalepsy. Keywords: Apomorphine, Buspirone, Cage climbing behaviour, Catalepsy, Haloperidol, Methamphetamine, Mice, Serotonergic system, Stereotypy.

The anxiolytic drug buspirone acts as a partial agonist at 5-hydroxytryptamine (5-HT, serotonin) 5-HT1A autoreceptors and as an antagonist at certain postsynaptic 5-HT1A receptor sites1,2. Buspirone also preferentially blocks the presynaptic rather than the postsynaptic D2 dopamine (DA) receptors3-5. While studying the behavioural effects of buspirone in mice, it was observed that the drug produced dosedependent opposite behavioural effects. At smaller doses (0.625-5 mg/kg, ip) buspirone stimulated locomotor activity and induced stereotyped cage climbing behaviour whereas at higher doses (10, 20, 40 mg/kg,ip) it suppressed locomotor activity and induced catalepsy. Induction of stereotyped cage climbing behaviour by buspirone in mice and rats has not been reported in literature. Further, buspirone does not induce catalepsy3,5. The objective of the present study is to elucidate the mechanisms involved in the induction of stereotyped cage climbing behaviour by small doses of buspirone and of catalepsy by its higher doses.
__________ *Correspondent author Telephone: 91-2164-241555 Fax: 91-2164-242170 E-mail: jehangirbalsara@yahoo.co.in

Stereotyped cage climbing behaviour is induced by apomorphine in mice by directly stimulating the postsynaptic mesolimbic D2 and D1 DA receptors6-9. It was thought worthwhile to investigate whether buspirone (0.625-5 mg/kg, ip) induces the cage climbing behaviour by stimulating the postsynaptic mesolimbic D2 and D1 DA receptors either directly or indirectly by releasing DA from the mesolimbic dopaminergic neurons. The effect of pretreatment with haloperidol, alpha-methyl-p-tyrosine and small doses of apomorphine therefore has been investigated on buspirone (0.625-5 mg/kg, ip) induced cage climbing behaviour. Haloperidol is a postsynaptic mesolimbic and striatal D2 and D1 DA receptor blocker10. Alpha-methyl-p-tyrosine is a tyrosine hydroxylase inhibitor, which by inhibiting synthesis, specifically depletes brain catecholamines11. Small doses of apomorphine selectively stimulate the presynaptic mesolimbic and nigrostriatal D2 DA autoreceptors and induce long lasting inhibition of DA synthesis12,13. They thus decrease the intraneuronal stores of DA available for release12,13. Haloperidol induces catalepsy by blocking the postsynaptic striatal D2 and D1 receptors10. Alphamethyl-p-tyrosine, by inhibiting the synthesis of DA

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in the nigrostriatal dopaminergic neurons, produces a lack of DA at postsynaptic striatal D2 and D1 receptor sites and induces catalepsy14. High doses of apomorphine elicit oral stereotypies, characterized by biting, gnawing or licking behaviour, by directly stimulating the postsynaptic striatal D2 and D1 receptors15,16. High doses of methamphetamine induce oral stereotypies by releasing DA from the nigrostriatal dopaminergic neurons with resultant activation by the released DA of postsynaptic striatal D2 and D1 receptors15-17. Haloperidol, as it blocks the postsynaptic striatal D2 and D1 receptors10, antagonizes the oral stereotypies induced by both apomorphine and methamphetamine18. Alpha-methylp-tyrosine decreases the nigrostriatal neuronal stores of DA available for release by methamphetamine14 and thus antagonizes only methamphetamineinduced oral stereotypy18. High doses of buspirone (10, 20, 40 mg/kg, ip) may be inducing catalepsy either by blocking the postsynaptic striatal D2 and D1 receptors or by depleting the intraneuronal stores of DA. The effect of pretreatment with catalepsy inducing high doses of buspirone (10, 20, 40 mg/kg) was therefore studied on apomorphine and methamphetamine induced oral stereotyped behaviour (SB) and also on apomorphine induced cage climbing behaviour. 5-HT containing neurons originating from midbrain raphe nuclei innervate the substantia nigra, the striatum and the nucleus accumbens19. 5-HT, by stimulating the 5-HT2C receptors, decreases the synthesis and release of DA from the mesolimbic and nigrostriatal dopaminergic neurons20,21. Drugs enhancing or reducing central serotonergic neurotransmission modulate the intensity of behaviours dependent on the functional status of the mesolimbic and nigrostriatal dopaminergic systems viz., apomorphine-induced cage climbing behaviour6, neuroleptic-induced catalepsy and DA agonistinduced SB19. It was therefore thought worthwhile to investigate on buspirone (2.5 mg/kg, ip)induced cage climbing behaviour and on buspirone (40 mg/kg, ip) induced catalepsy the effect of pretreatment with ltryptophan, dexfenfluramine, fluoxetine, trazodone, mianserin and p-chlorophenylalanine. L-tryptophan a precursor of 5-HT, was administered systemically to increase brain 5-HT levels22. It was used instead of 5hydroxytryptophan as it contributes more specifically to 5-HT stores within the serotonergic neurons23. Dexfenfluramine, on acute administration, causes a rapid release of 5-HT from brain serotonergic

neurons24. Fluoxetine selectively blocks the neuronal reuptake of 5-HT25 whereas trazodone and mianserin are 5-HT2A/2C receptor antagonists2,26. p-Chlorophenylalanine, a tryptophan hydroxylase inhibitor, specifically depletes brain 5-HT27. Materials and Methods Animals Male albino mice, weighing 20-30 g, bred in Central Animal House facility of the Institute, were used. The animals were housed under standard conditions, maintained on a 12:12 hr light/dark cycle and had free access to food and water upto the time of experimentation. The animals were brought to the department and kept in a noiseless diffusely illuminated laboratory, at least 1 hr before the experiments for acclimatization to the laboratory environment. Animals were randomly distributed into groups of 10 animals each. Each animal was used only once. During experiments, the animals were placed in individual cages made of wire netting, measuring 25 20 15 cm3, 30 min before drug treatment for adaptation to their new environment. All observations were made between 1000 and 1600 hrs at 27o to 30 oC. Observations were made blind with respect to the treatments used. The experimental protocols were approved by the Institutional Animal Ethics Committee and conducted according to the Indian National Science Academy Guidelines for the use and care of experimental animals. Drugs Buspirone hydrochloride (Merind Ltd, Mumbai, India), apomorphine hydrochloride (Sigma, USA), methamphetamine hydrochloride (Sigma, USA), dl-alpha-methyl-p-tyrosine methyl ester hydrochloride (Sigma, USA), l-tryptophan methyl ester hydrochloride (Sigma, USA), dexfenfluramine hydrochloride (Wockhardt, Ltd, India), fluoxetine hydrochloride (Sun Pharmaceuticals, Mumbai, India), trazodone hydrochloride (Protec, Ltd, Mumbai, India), mianserin hydrochloride (Torrent, Ltd, Ahmedabad, India), dl-p-chlorophenylalanine methyl ester hydrochloride (Sigma, USA) and haloperidol (Senorm injection, Sun Pharmaceuticals, Mumbai, India) were used. Haloperidol injection solution was diluted to required strength with distilled water. Remaining drugs were dissolved in distilled water except apomorphine, which was dissolved in distilled water containing 0.2 mg/ml ascorbic acid. All drug solutions were prepared immediately before use and were injected ip in a volume of 1 ml/100 g body

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weight. Doses refer to the forms mentioned. Drug doses, routes of administration and the testing time intervals were selected based on previous studies conducted in the laboratory and those reported in literature. During preliminary studies, it was observed that 0.3125 mg/kg buspirone neither stimulated locomotor activity nor induced stereotyped cage climbing behaviour in mice. At 0.625, 1.25, 2.5 and 5 mg/kg doses buspirone stimulated locomotor activity and induced stereotyped cage climbing behaviour in 100% of the animals tested (n=10 for each dose). The cage climbing behaviour manifested 30 min after drug injection and lasted for about 3 hr. Maximum intensity of stereotyped cage climbing behaviour was observed during 1 to 2 hr time interval after injection. Animals receiving 10, 20 and 40 mg/kg buspirone did not exhibit increase in locomotor activity or cage climbing behaviour. On the contrary they appeared sedated and when tested for catalepsy by the method of Ahtee and Buncombe28 gave a positive cataleptic response without showing loss of righting reflex or apparent change in muscle tone or motor coordination. Doses beyond 40 mg/kg tended to produce clonic convulsions and hence were not used in the present study. Further, as 0.3125 mg/kg dose of buspirone had not induced cage climbing behaviour in mice, doses below 0.3125 mg/kg were therefore not used in the present study. Buspirone-induced cage climbing behaviour in mice Effect of pretreatment with drugs interacting with the central dopaminergic and serotonergic systems on cage climbing behaviour induced by 0.625 to 5 mg/kg buspirone was studied by the method of Costall et al6. Haloperidol (0.1 and 0.2 mg/kg), apomorphine (0.05 and 0.1 mg/kg) and alpha-methyl-p-tyrosine (100 and 200 mg/kg) were injected 30 min, 1 hr and 4 hr prior to buspirone respectively. In another set of experiments l-tryptophan (100 and 200 mg/kg), dexfenfluramine (5 and 10 mg/kg), fluoxetine (5 and 10 mg/kg), trazodone (5 and 10 mg/kg) and mianserin (1.25 and 2.5 mg/kg) were injected 1 hr before buspirone (2.5 mg/kg). p-Chlorophenylalanine (100 mg/kg/day) was administered daily for 4 days, the last dose was given 18 hr before buspirone (2.5 mg/kg). Control groups received 1ml/100g body weight of distilled water ip at specified time intervals before receiving buspirone. Intensity of cage climbing behaviour induced by buspirone was assessed over a 60 min observation period, 1 hr after injection of

buspirone. Animals were individually tested for climbing behaviour taking the percentage of time spent climbing during the 60 min observation period as a measure of climbing (climbing index). Further, the maximum time (min) spent in a single climb throughout the 60 min observation period was also recorded. Catalepsy testing in mice Animals were tested for catalepsy according to the method of Ahtee and Buncombe28 by placing both front paws of the animal over a 4 cm high wooden block. The time elapsing between paw placement and the first movement of either paw (descent latency) was measured in seconds. Animals were tested and evaluated for catalepsy 0.5, 1, 2, 3, 4 and 5 hr after ip injection of buspirone (10, 20 and 40 mg/kg), haloperidol (0.1, 0.2, 0.5 and 1 mg/kg), small doses of apomorphine (0.05 and 0.1 mg/kg), alphamethyl-p-tyrosine (100 and 200 mg/kg), l-tryptophan (100 and 200 mg/kg), dexfenfluramine (5 and 10 mg/kg), fluoxetine (5 and 10 mg/kg), trazodone (5 and 10 mg/kg), mianserin (1.25 and 2.5 mg/kg) or distilled water (1 ml/100 g body weight, control group). Animals treated with pchlorophenylalanine (100 mg/kg/day for 4 days) were tested for catalepsy 16,17,18,19 and 20 hr after administration of the last dose of p-chlorophenylalanine. Control group for p-chlorophenylalanine received 1 ml/100 g body weight of distilled water daily for 4 days and were tested for catalepsy 16, 17, 18, 19 and 20 hr after administration of the last dose. Catalepsy score (descent latency) in sec of each animal in the group, at the respective testing time interval, was taken to compute the mean value of the group for that particular timing. As animals receiving 0.625 to 5 mg/kg buspirone exhibited an increase in locomotor activity and stereotyped cage climbing behaviour they were not tested for catalepsy. Buspirone on high dose apomorphine and methamphetamine-induced SB of OMV in mice Following administration of apomorphine (3 mg/kg) or methamphetamine (6 mg/kg) the intensity of SB was assessed over a 30 sec observation period at 10 min intervals, using the scoring system of Ozawa and Miyauchi29 where periodic sniffing = score 1, continuous sniffing = 2, periodic biting, gnawing or licking = 3 and continuous biting, gnawing or licking = 4. Interrater reliability was calculated from those simultaneous ratings made by two of the authors using the Pearson product- moment correlation (r).

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Interrater correlation coefficient r was found to be 0.96, indicating a high degree of inter-rater agreement. The cumulative stereotyped rating for each animal was determined as the sum of each 10 min score for 90 min for apomorphineinduced SB or 180 min for methamphetamineinduced SB. The cumulative stereotypy score of each mouse in the group was taken to compute the median value of the group. Buspirone (10, 20 and 40 mg/kg) or haloperidol (0.5 mg/kg) was injected 1 hr before apomorphine or methamphetamine. Control groups received 1 ml/100 g body weight of distilled water ip 1 hr before receiving the DA agonists. Buspirone on apomorphine-induced cage climbing behaviour in mice Effect of pretreatment with 10, 20 and 40 mg/kg doses of buspirone on apomorphine (0.5, 0.75 and 1 mg/kg)-induced cage climbing behaviour was studied by the method of Costall et al.6. Animals received either buspirone, haloperidol (0.2 mg/kg) or distilled water (1 ml/100 g, ip) followed 60 min later by apomorphine. They were individually tested for climbing behaviour taking the percentage of time spent climbing during the 30 min period after the first climb as a measure of climbing (climbing index). Further, the maximum time (min) spent in a single climb throughout the duration of the apomorphine effect was also recorded. Effect of pretreatment with 0.625, 1.25, 2.5 and 5 mg/kg doses of buspirone on high dose apomorphine and methamphetamine induced SB of OMV and on apomorphine induced cage climbing behaviour was not studied as at these doses buspirone had stimulated locomotor activity and induced cage climbing behaviour in mice. Buspirone-induced catalepsy in mice L-tryptophan (100 and 200 mg/kg), dexfenfluramine (5 and 10 mg/kg), fluoxetine (5 and 10 mg/kg), trazodone (5 and 10 mg/kg), mianserin (1.25 and 2.5 mg/kg) were injected 1 hr before buspirone (40 mg/kg). pChlorophenylalanine (100 mg/kg/day) was administered daily for 4 days, the last dose was given 18 hr before buspirone (40 mg/kg). Control groups received 1 ml/100 g body weight of distilled water ip at specified time intervals before receiving buspirone. Animals were tested and evaluated for catalepsy 1 hr after buspirone treatment in the same manner as stated in the section Catalepsy testing in mice. Catalepsy score (descent latency) in sec of each animal in the particular group, at the 1 hr testing time interval

following buspirone (40 mg/kg) administration, was taken to compute the mean value of the group. Statistical analysis Data pertaining to induction of catalepsy was analysed by a one-way ANOVA followed by Tukeys multiple comparison test for assessing significance of differences between individual groups at respective testing time intervals. The data on cage climbing behaviour studies were analysed by a one-way ANOVA followed by Tukeys test for post-hoc comparison with the respective control group. Data pertaining to effects of p-chlorophenylalanine on catalepsy and cage climbing behaviour was analysed by the twotailed Students unpaired t-test. The results of studies pertaining to high dose apomorphine and methamphetamineinduced SB of OMV were evaluated statistically by the two-tailed Mann-Whitney U-test for nonparametric data using the individual cumulative stereotypy score. The level of statistical significance chosen was P < 0.05. Results Effect of haloperidol, alpha-methyl-p-tyrosine and small dose apomorphine pretreatment on buspironeinduced cage climbing behaviour in mice (Table 1) Buspirone at 0.625, 1.25, 2.5 and 5 mg/kg doses induced cage climbing behaviour whose intensity was dose-dependent in the dose range of 0.625 to 2.5 mg/kg, reaching maximum at 2.5 mg/kg dose. However, animals treated with 5 mg/kg dose exhibited a significant decrease (P<0.05) in its intensity as compared to those treated with 2.5 mg/kg dose. Pretreatment with 0.1mg/kg haloperidol, 100 mg/kg alpha-methyl-p-tyrosine and 0.05 mg/kg apomorphine abolished the cage climbing behaviour induced by 0.625 mg/kg buspirone. The intensity of cage climbing behaviour induced by 1.25 mg/kg buspirone was significantly decreased (P<0.01) by pretreatment with 0.1 mg/kg haloperidol, 100 mg/kg alpha-methyl-p-tyrosine and 0.05 mg/kg apomorphine, whereas it was abolished by 0.2mg/kg haloperidol, 200 mg/kg alpha-methyl-p-tyrosine and 0.1 mg/kg apomorphine. Pretreatment with 0.1 mg/kg haloperidol, (100 and 200 mg/kg) alpha-methyl-p-tyrosine and (0.05 and 0.1 mg/kg) apomorphine significantly decreased (P<0.01) cage climbing behaviour induced by 2.5 and 5mg/kg buspirone, whereas pretreatment with 0.2mg/kg

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Table 1 Effect of haloperidol (HAL), alpha-methyl-p-tyrosine (AMT) and small dose apomorphine (APO) pretreatment on buspirone (BUS) induced cage climbing behaviour in mice. [Values are means + SE of 10 animals in each group] Treatment (mg/kg, ip) DW + BUS (0.625) HAL (0.1) + BUS (0.625) AMT (100) + BUS (0.625) APO (0.05) + BUS (0.625) DW + BUS (1.25) HAL (0.1) + BUS (1.25) HAL (0.2) + BUS (1.25) AMT (100) + BUS (1.25) AMT (200) + BUS (1.25) APO (0.05) + BUS (1.25) APO (0.1) + BUS (1.25) DW + BUS (2.5) HAL (0.1) + BUS (2.5) HAL (0.2) + BUS (2.5) AMT (100) + BUS (2.5) AMT (200) + BUS (2.5) APO (0.05) + BUS (2.5) APO (0.1) + BUS (2.5) DW + BUS (5) HAL (0.1) + BUS (5) HAL (0.2) + BUS (5) AMT (100) + BUS (5) AMT (200) + BUS (5) APO (0.05) + BUS (5) APO (0.1) + BUS (5)
a

Climbing index (%) 27.9 + 2.4 0.0 0.0 0.0 47.6 + 2.8 14.4 + 1.2b 0.0 19.3 + 1.7 0.0 16.9 + 1.4 0.0 76.9 + 3.1 40.4 + 2.2 6.7 + 1.4
b b b b

Max. time (min) 2.2 + 0.7 0.0 0.0 0.0 7.4 + 0.9 1.2 + 0.3b 0.0 1.6 + 0.5b 0.0 1.4 + 0.6b 0.0 12.2 + 1.1 6.2 + 0.4b 1.3 + 0.7b 5.7 + 0.5b 2.2 + 0.9b 5.1 + 0.6b 2.1 + 0.8b 6.8 + 0.7c 2.3 + 0.5b 0.0 2.1 + 0.4b 1.1 + 0.3b 2.4 + 0.5b 1.2 + 0.7b

catalepsy score of buspirone (10 mg/kg) at 4 and 5 hr testing time interval was not significant (P>0.05), however difference in catalepsy score between control group and group receiving 10 mg/kg buspirone at 0.5 to 3 hr and groups receiving 20 and 40 mg/kg buspirone at 0.5 to 5 hr testing time interval was significant (P<0.01). There was no significant difference in catalepsy score between control group and group receiving 0.1mg/kg haloperidol at all testing time intervals (P>0.05). However, treatment with 0.2 mg/kg haloperidol showed that the difference in catalepsy score was significant (P<0.05) at 0.5 and 2 hr testing time intervals and was highly significant (P<0.01) at 1 hr testing time interval but was insignificant (P>0.05) from 3 to 5 hr testing time intervals. Further, difference in catalepsy score with 0.5 and 1mg/kg haloperidol was highly significant (P<0.01) at all testing time intervals. The difference in catalepsy score between control group and groups receiving apomorphine (0.05 and 0.1 mg/kg) was highly significant (P<0.01) at all testing time intervals. There was no significant difference in catalepsy score between control group and groups receiving alpha-methyl-p-tyrosine (100 and 200 mg/kg) at 0.5 hr and 100 mg/kg at 1 hr. However, the difference was highly significant (P<0.01) at all the remaining testing time intervals. Effects of buspirone and haloperidol pretreatment on apomorphine and methamphetamine induced SB in mice (Table 3) Intensity of SB induced by 3mg/kg apomorphine was significantly decreased (P<0.001) by pretreatment with buspirone 10 and 20 mg/kg, while it was abolished by buspirone 40mg/kg and haloperidol 0.5 mg/kg. However, the intensity of SB induced by 6 mg/kg methamphetamine was significantly decreased (P<0.001) by pretreatment with buspirone 10 to 40 mg/kg and haloperidol 0.5 mg/kg. Effect of buspirone and haloperidol pretreatment on apomorphine induced cage climbing behaviour in mice. (Table 4) Cage climbing behaviour induced by 0.5 mg/kg apomorphine was abolished by pretreatment with 10 mg/kg buspirone and 0.2 mg/kg haloperidol. Cage climbing behaviour induced by 0.75 mg/kg apomorphine was significantly decreased (P<0.001) by pretreatment with 10 mg/kg buspirone, whereas it was abolished by 20 mg/kg buspirone and 0.2 mg/kg haloperidol. Cage climbing behaviour

45.3 + 2.5b 24.2 + 1.9

b

42.6 + 2.3b 21.7 + 1.6b 54.9 + 2.9c 22.8 + 1.8b 0.0 26.5 + 2.2 9.2 + 1.1
b b

21.1 + 1.6b 7.9 + 1.3b

P < 0.05 and bP < 0.01 as compared to respective distilled water pretreated control buspirone groups by ANOVA. cP < 0.05 as compared to distilled water pretreated control buspirone (2.5 mg/kg) group by Students unpaired t-test. DW = Distilled water (1 ml/100 g, ip).

haloperidol significantly decreased (P<0.01) cage climbing behaviour induced by 2.5 mg/kg and abolished that induced by 5mg/kg buspirone. Induction of catalepsy by buspirone, haloperidol, small doses of apomorphine and alpha-methyl-ptyrosine in mice (Table 2) There was a significant treatment and time interval effect for buspirone (10, 20 and 40 mg/kg) at 0.5 to 5 hr. Difference in

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Table 2 Induction of catalepsy by buspirone (BUS), haloperidol (HAL), small doses of apomorphine (APO) and -methyl-p-tyrosine (AMT) in mice. [Values are mean + SE of 10 animals in each group] Group 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Treatment Distilled water Buspirone Buspirone Buspirone Distilled water Haloperidol Haloperidol Haloperidol Haloperidol Distilled water Apomorphine Apomorphine Distilled water -methyl-p-tyrosine -methyl-p-tyrosine Dose (mg/kg) 10 ml/kg 10 20 40 10 ml/kg 0.1 0.2 0.5 1 10 ml/kg 0.05 0.1 10 ml/kg 100 200 Catalepsy score (immobility time in sec) at 0.5 hr 2.2 + 0.13 8.5 + 0.65c 16.6 + 1.17c 32.8 + 1.45c 2.3 + 0.15 2.4 + 0.16 5.6 + 0.34
a b

1 hr 2.4 + 0.16 17.3 + 1.45c 25.4 + 1.25c 41.6 + 1.64c 2.4 + 0.13 2.5 + 0.17 9.2 + 0.49
a c

2 hr 2.3 + 0.15 14.2 + 1.40c 22.3 + 1.21c 38.5 + 1.53c 2.1 + 0.10 2.4 + 0.16 6.3 + 0.40
a b

3 hr 2.1 + 0.10 9.4 + 0.47c 17.5 + 1.25c 33.7 + 1.50c 2.2 + 0.15 2.3 + 0.15 3.1 + 0.18
a a

4 hr 2.3 + 0.15 4.3 + 0.36a 12.4 + 1.12c 28.6 + 1.32c 2.4 + 0.16 2.2 + 0.13 2.7 + 0.15
a a

5 hr 2.2 + 0.13 2.2 + 0.13a 7.2 + 0.75c 23.4 + 1.24c 2.2 + 0.13 2.1 + 0.10a 2.2 + 0.13a 9.2 + 0.59 c 31.9 + 1.24c 2.3 + 0.15
c c

23.6 + 1.16c 47.0 + 1.29c 2.1 + 0.10 17.1 + 0.73 41.2 + 1.45 2.2 + 0.13 2.2 + 0.13a 2.4 + 0.16a
c c

34.2 + 1.37c 57.6 + 1.56c 2.3 + 0.15 27.7 + 1.15 51.7 + 1.60 2.4 + 0.15 2.5 + 0.17a 4.3 + 0.15c
c c

31.0 + 1.31c 54.4 + 1.48c 2.4 + 0.16 35.3 + 1.28 59.4 + 1.83 2.3 + 0.16 6.3 + 0.30c 9.4 + 0.22c
c c

25.4 + 1.24c 48.8 + 1.45c 2.2 + 0.13 36.0 + 1.39 60.2 + 1.77 2.1 + 0.11 10.2 + 0.34c 15.3 + 0.45c
c c

17.8 + 1.04c 41.2 + 1.35c 2.1 + 0.10 34.4 + 1.31 58.3 + 1.72 2.3 + 0.15 14.4 + 0.45c 20.2 + 0.52c

32.2 + 1.22c 56.2 + 1.66c 2.2 + 0.14 18.7 + 0.73c 25.6 + 0.65c

a P > 0.05 as compared to distilled water treated control group at respective 4 and 5 hr testing time intervals of buspirone 10 mg/kg; at 0.5 to 5 hr testing time intervals of haloperidol 0.1 mg/kg; at 3 to 5 hr testing time intervals of haloperidol 0.2 mg/kg; at 0.5 and 1 hr testing time intervals of -methyl-p-tyrosine 100 mg/kg and at 0.5 hr testing time intervals of -methyl-p-tyrosine 200 mg/kg. b P < 0.05 and cP < 0.01 as compared to distilled water treated control group at respective testing time intervals.

induced by 1 mg/kg apomorphine was significantly decreased (P<0.01) by pretreatment with 10 and 20 mg/kg buspirone and abolished by 40 mg/kg buspirone and 0.2 mg/kg haloperidol. Effect of l-tryptophan, dexfenfluramine, fluoxetine, trazodone, mianserin and p-chlorophenylalanine on buspirone induced cage climbing behaviour in mice (Table 5) Cage climbing behaviour induced by 2.5 mg/kg buspirone was significantly antagonised (P<0.01) by pretreatment with l-tryptophan (100 and 200 mg/kg), dexfenfluramine (5 and 10 mg/kg) and fluoxetine (5 and 10 mg/kg), whereas it was significantly potentiated (P<0.01) by trazodone (5 and 10 mg/kg), mianserin (1.25 and 2.5 mg/kg) and p-chlorophenylalanine 400 mg/kg. Effect of l-tryptophan, dexfenfluramine, fluoxetine, trazodone, mianserin and p-chlorophenylalanine on buspirone induced catalepsy in mice (Table 6) Catalepsy score of 40 mg/kg buspirone was significantly increased (P<0.01) by l-tryptophan (100 and 200 mg/kg), dexfenfluramine (5 and 10 mg/kg) and fluoxetine (5 and 10 mg/kg), whereas it was

Table 3 Effect of buspirone (BUS) and haloperidol (HAL) pretreatment on the intensity of stereotyped behaviour (SB) induced by apomorphine (APO) and methamphetamine (MAM) in mice. [Values are medians (and ranges where indicated) of 10 animals in each group] Treatment (mg/kg, ip) DW + APO (3) BUS (10) + APO (3) BUS (20) + APO (3) BUS (40) + APO (3) HAL (0.5) + APO (3) DW + MAM (6) BUS (10) + MAM (6) BUS (20) + MAM (6) BUS (40) + MAM (6) HAL (0.5) + MAM (6) Median SB score 16.5 (16-19) 10.0 (9-13)a 5.5 (4-7)b 0.0 0.0 27.0 (25-29) 20.5 (18-22)a 16.0 (14-17)b 8.5 (7-11)b 7.0 (6-9)b

P values: a< 0.01; b< 0.001 as compared to the respective distilled water pretreated control apomorphine and methamphetamine group by Mann-Whitneys U test. DW = Distilled water (1 ml/100 g, ip).

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Table 4 Effect of buspirone (BUS) and haloperidol (HAL) pretreatment on apomorphine (APO) induced cage climbing behaviour in mice. [Values are means + SE of 10 animals in each group] Treatment (mg/kg, ip) DW + APO (0.5) BUS (10) + APO (0.5) HAL (0.2) + APO (0.5) DW + APO (0.75) BUS (10) + APO (0.75) BUS (20) + APO (0.75) HAL (0.2) +APO (0.75) DW + APO (1) BUS (10) + APO (1) BUS (20) + APO (1) BUS (40) + APO (1) HAL (0.2) + APO (1)
a b

Climbing index (%) 24.8 + 1.9 0.0 0.0 45.9 + 2.6 18.6 + 1.5 0.0 0.0 0.0 70.4 + 2.6 42.2 + 2.1 21.4 + 1.7 0.0 0.0
c c b

Max. time (min) 2.5 + 0.7 0.0 0.0 7.5 + 0.8 1.4 + 0.4 0.0 11.2 + 0.9 4.7 + 0.4 2.2 + 0.6 0.0 0.0
c c a

Table 5 Effect of L-Tryptophan (LT), Dexfenfluramine (DEX), Fluoxetine (FLU), Trazodone (TRA), Mianserin (MIAN) and p-Chloro-phenylalanine (PCPA) on Buspirone (BUS) induced cage climbing behaviour in mice. [Values are mean + SE of10 animals in each group] Treatment (mg/kg, ip) DW + BUS (2.5) LT (100) + BUS (2.5) LT (200) + BUS (2.5) DEX (5) + BUS (2.5) DEX (10) + BUS (2.5) FLU (5) + BUS (2.5) FLU (10) + BUS (2.5) DW + BUS (2.5) TRA (5) + BUS (2.5) TRA (10) + BUS (2.5) MIAN (1.25) + BUS (2.5) MIAN (2.5) + BUS (2.5) DW +BUS (2.5) PCPA (400) + BUS (2.5) Climbing index (%) 77.2 + 2.8 54.1 + 2.4* 48.4 + 2.3* 52.3 + 2.2* 45.6 + 1.9* 53.7 + 2.2* 47.2 + 2.4* 76.1 + 2.7 90.1 + 0.5* 96.7 + 0.4* 91.4 + 0.6* 97.8 + 0.3* 76.8 + 3.6 89.7 + 0.5
a

Max. time (min) 12.4 + 1.2 6.7 + 0.8* 5.9 + 0.6* 6.4 + 0.7* 5.3 + 0.5* 6.2 + 0.8* 5.5 + 0.6* 11.2 + 0.9 17.5 + 0.7* 20.4 + 0.8* 18.7 + 0.7* 21.7 + 1.1* 11.8 + 1.1 18.1 + 0.8a

P values: < 0.01; < 0.001 as compared to the distilled water pretreated control apomorphine (0.75 mg/kg) group by Students unpaired t-test c P < 0.01 as compared to the distilled water pretreated control apomorphine (1 mg/kg) group by ANOVA. DW = Distilled water (1 ml/100 g, ip).

significantly decreased (P<0.05) by trazodone (5 and 10 mg/kg), mianserin (1.25 and 2.5 mg/kg) and pchlorophenylalanine 400 mg/kg. Discussion Pretreatment with haloperidol, alpha-methyl-ptyrosine and small doses of apomorphine antagonized buspirone (0.625, 1.25, 2.5 and 5 mg/kg)induced cage climbing behaviour. Taken together these results indicate that buspirone induces cage climbing behaviour in mice by releasing DA from the mesolimbic dopaminergic neurons with resultant stimulation of the postsynaptic mesolimbic D2 and D1 DA receptors by the released DA. Neuroleptics, by blocking presynaptic D2 DA autoreceptors increase synthesis and release of DA from dopaminergic neurons13. Buspirone, in addition to interacting with the 5-HT1A receptors1,2, selectively blocks the presynaptic D2 DA autoreceptors at doses which do not exert postsynaptic D2 DA receptor blocking activity3,5. Buspirone antagonises the action of apomorphine in two biochemical presynaptic D2 autoreceptor test systems3. This antagonism was exhibited by buspirone at doses which did not block

*Significant as compared with respective distilled water pretreated control Buspirone group by Tukeys post hoc multiple comparison test at P < 0.01. aP < 0.05 as compared to the distilled water pretreated control buspirone (2.5 mg/kg) group by students unpaired t-test. DW = Distilled water (1 ml/100 g, ip)

the postsynaptic striatal D2 receptor mediated apomorphineinduced turning behaviour in rats with unilateral 6-hydroxydopamine lesions of the substantia nigra3. In the present study buspirone at 10, 20 and 40 mg/kg doses antagonized apomorphineinduced cage climbing behaviour in mice suggesting thereby that at these doses it blocks the postsynaptic mesolimbic D2 DA receptors. It is contended that at the smaller doses (0.625 to 5 mg/kg) buspirone, by selectively blocking the presynaptic mesolimbic D2 DA autoreceptors, increases the synthesis and release of DA from the mesolimbic dopaminergic neurons with resultant stimulation of the postsynaptic mesolimbic D2 and D1 DA receptors by the released DA and occurrence of cage climbing behaviour. Such an explanation was put forth by Bhosale et al30 regarding metoclopramide which at lower doses (1.25 and 2.5 mg/kg ip) acts as a presynaptic D2 DA autoreceptor antagonist, while at higher doses (5 mg/kg ip and more) blocks postsynaptic

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Table 6 Effect of L-Tryptophan (LT), Dexfenfluramine (DEX), Fluoxetine (FLU), Trazodone (TRA), Mianserin (MIAN) and p-Chloro-phenylalanine (PCPA) on Buspirone (BUS) induced catalepsy in mice. [Values are mean + SE of10 animals in each group] Study Group Treatment (mg/kg ip) Catalepsy score (descent latency in seconds) 41.6 + 1.6 47.7 + 1.3a 52.3 + 1.4b 53.7 + 1.6b 58.2 +1.4b 47.6 +1.3a 51.4 + 1.5b 42.5 + 1.5 34.1 + 1.3a 32.7 + 1.4a 35.2 + 1.6a 33.2 + 1.6a 41.9 + 1.4 35.1 + 1.6c

1. 2. 3. 4. 5. 6. 7.

DW + BUS (40) LT (100) + BUS (40) LT (200) + BUS (40) DEX (5) + BUS (40) DEX (10) + BUS (40) FLU (5) + BUS (40) FLU (10) + BUS (40) DW + BUS (40) TRA (5) + BUS (40) TRA (10) + BUS (40) MIAN (1.25) + BUS (40) MIAN (2.5) + BUS (40) DW + BUS (40) PCPA (400) + BUS (40)

II

1. 2. 3. 4. 5.

III
a

1. 2.

P < 0.05 and b P < 0.01 as compared to distilled water pretreated control groups by ANOVA. c P < 0.05 as compared to distilled water pretreated control PCPA group by Students upaired t-test. DW = Distilled water (1 ml/100 gm, ip).

mesolimbic and striatal D2 DA receptors in mice and rats31-33. Further, locomotor stimulation induced by a small dose of haloperidol (0.025 mg/kg ip) in mice was explained by Strombom34 on the basis of selective blockade of presynaptic mesolimbic D2 DA autoreceptors by the small dose of haloperidol. The observation, that animals treated with 5 mg/kg dose of buspirone exhibited a significant decrease in the intensity of cage climbing behaviour as compared to the animals receiving 2.5 mg/kg dose of buspirone is explained on the basis that at 5 mg/kg dose buspirone starts exerting postsynaptic mesolimbic D2 DA receptor blocking activity. Consequently the action of DA released by 5 mg/kg buspirone due to presynaptic mesolimbic D2 DA autoreceptor induced blockade is counteracted to some extent with resultant decrease in the intensity of cage climbing behaviour.

The DA hypothesis of schizophrenia states that the psychotic positive symptoms of schizophrenia are due to hyperfunctioning of the mesolimbic dopaminergic system whereas the negative symptoms and cognitive deficits are associated with low DA activity in the prefrontal cortex35. In the present study the observed stereotyped cage climbing behaviour induced by small doses of buspirone (0.625 to 5 mg/kg) may well be due to augmented synthesis and release of DA resulting in enhanced mesolimbic dopaminergic neurotransmission. This finding suggests that buspirone may induce psychosis. The suggestion is supported by clinical reports stating that buspirone at anxiolytic doses has induced psychotic reactions especially in psychosis prone individuals36. Haloperidol induces catalepsy and antagonizes apomorphine and amphetamine induced SB of OMV by blocking the postsynaptic striatal D2 and D1 DA receptors10,15-17. In the present study treatment with 10, 20 and 40mg/kg buspirone induced catalepsy and pretreatment with these doses antagonized apomorphine and methamphetamine induced SB of OMV. The results suggest that buspirone at these doses exerts postsynaptic striatal D2 and D1 DA receptors blocking activity. The observation that pretreatment with 10 to 40mg/kg buspirone antagonized apomorphine induced SB of OMV concurs with the finding of Skolnick et al37 that pretreatment with 10 and 20 mg/kg buspirone antagonized apomorphine-induced oral stereotypies in rats. Furthermore, the observation that buspirone on weight basis is much less potent than haloperidol in inducing catalepsy and in antagonizing DA agonist induced SB of OMV is in agreement with the finding of Cimino et al38 that buspirone displaced [3H] spiperidol from rat striatal binding sites with an IC50 (1.8 10-7M) considerably lower than that of haloperidol (4.7 10-9 M). Cimino et al38 had also reported that buspirone was only a weak inhibitor of DA stimulated adenyl cyclase. The present results taken alongwith the results of Skolnick et al37 and the radioligand binding and biochemical studies of Cimino et al38 do indicate that buspirone at 10 to 40 mg/kg doses acts as an antagonist at the postsynaptic striatal D2 and D1 DA receptor sites. The neuroleptic haloperidol worsens Parkinsonian condition by blocking the postsynaptic striatal D2 DA receptors39. Further, long-term administration of haloperidol in psychotic patients induces tardive dyskinesia39. Increasing the dose of haloperidol

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suppresses the manifestations of tardive dyskinesia by blocking the supersensitive postsynaptic striatal D2 DA receptors39. The present finding that at high doses (10 to 40 mg/kg) buspirone blocks the postsynaptic striatal D2 DA receptors explains the clinical observations that buspirone, at doses higher than the conventional anxiolytic doses, caused worsening of parkinsonian symptoms40,41 and high doses of buspirone were efficacious in the treatment of tardive dyskinesia41. Further, acute administration of buspirone abolished the expression of behavioural dopaminergic supersensitivity in mice withdrawn from chronic haloperidol treatment42. Pretreatment with drugs which enhance central serotonergic neurotransmission viz l-tryptophan, dexfenfluramine, and fluoxetine antagonized buspirone (2.5 mg/kg, ip) induced cage climbing behaviour and potentiated buspirone (40 mg/kg) induced catalepsy. Further, pretreatment with drugs which decrease central serotonergic neurotransmission viz trazodone, mianserin and p-chlorophenylalanine potentiated buspirone (2.5 mg/kg, ip) induced cage climbing behaviour and antagonized buspirone (40 mg/kg, ip) induced catalepsy. The results of this part of the present study thus indicate that drugs interacting with the central serotonergic systems do modulate the intensity of buspirone (2.5 mg/kg, ip) induced cage climbing behaviour and of buspirone (40 mg/kg, ip) induced catalepsy. These findings concur with the reports cited in literature regarding the effects of drugs influencing the central serotonergic systems on apomorphine induced cage climbing behaviour in mice6, methamphetamineinduced SB43-45 and haloperidolinduced catalepsy 4345 in rats. The results are explained on the basis of the recent in vivo microdialysis and electrophysiological studies which indicate that 5-HT, via activation of 5HT2C receptors, decreases the synthesis and release of DA from the mesolimbic and nigrostriatal dopaminergic neurons20,21. It is postulated that pretreatment with l-tryptophan, dexfenfluramine and fluoxetine, by increasing the concentration of 5-HT in the mesolimbic region, activate the 5-HT2C receptors and thereby decrease the synthesis of DA in the mesolimbic dopaminergic neurons. Consequently, the stores of DA in the mesolimbic dopaminergic neurons are decreased. As less DA is now available for release by 2.5 mg/kg, ip buspirone there is antagonism of buspirone induced cage climbing behaviour. Pretreatment with trazodone and mianserin, by

blocking the 5-HT2C receptors, and pretreatment with p-chlorophenylalanine, by decreasing the 5-HT concentration in the mesolimbic region, release the mesolimbic dopaminergic neurons from the inhibitory control of 5-HT. As a result, there is an increase in the synthesis and hence in the intraneuronal stores of DA. As more intraneuronal stores of DA are available for release by buspirone (2.5 mg/kg, ip), there is a resultant potentiation of buspirone induced cage climbing behaviour. Potentiation of buspirone (40 mg/kg, ip) induced catalepsy by pretreatment with ltryptophan, dexfenfluramine and fluoxetine and its antagonism by pretreatment with trazodone, mianserin and p-chlorophenylalanine are explained as follows. It is hypothesized that, as with haloperidol, following the blockade of the pre-and postsynaptic nigrostriatal D2 DA receptors by buspirone there is a compensatory feedback increase of nigrostriatal dopaminergic neuronal activity, which is associated with an allosteric activation of tyrosine hydroxylase. Consequently, there is an increase in the synthesis and release of DA, which counteracts, to some extent, the buspirone induced blockade of the postsynaptic striatal D2 and D1 DA receptors13. Pretreatment with l-tryptophan, dexfenfluramine and fluoxetine by increasing the concentration of 5HT in the nigrostriatal region, activate the 5-HT2C receptors and thereby decrease the synthesis of DA in the nigrostriatal dopaminergic neurons. Consequently, the stores of DA in the nigrostriatal dopaminergic neurons are decreased. Thus less DA is now available for release during the buspirone-induced compensatory feedback increase of nigrostriatal dopaminergic neuronal activity. As a result the buspironeinduced blockade of the postsynaptic striatal D2 and D1 DA receptors is counteracted to a lesser extent with resultant potentiation of buspirone induced catalepsy. Pretreatment with trazodone and mianserin by blocking the 5-HT2C receptors and pretreatment with p-chlorophenylalanine, by decreasing the 5-HT concentration, in the nigrostriatal region, release the nigrostriatal dopaminergic neurons from the inhibitory control of 5-HT. As a result, there is an increase in the synthesis of DA and hence in the intraneuronal stores of DA. As more DA is available for release during the buspironeinduced compensatory feedback increase of nigrostriatal dopaminergic neuronal activity, the buspirone induced blockade of the postsynptic striatal D2 and D1 DA receptors is counteracted to a greater extent, with the resultant antagonism of buspirone induced catalepsy.

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On the basis of the present behavioural study conducted in mice it is concluded that buspirone in the dose range of 0.625 to 5 mg/kg ip exerts a selective presynaptic D2 DA autoreceptor blocking activity while at 10, 20 and 40 mg/kg ip it exerts postsynaptic mesolimbic and striatal D2 and D1 DA receptor blocking activity. Further, the study also demonstrates that drugs which influence the activity of the central serotonergic systems do modulate the intensity of buspirone (2.5 mg/kg, ip) induced cage climbing behaviour and buspirone (40 mg/kg, ip) induced catalepsy. Acknowledgement Authors are grateful to Merind Ltd for the generous gift of buspirone hydrochloride. Thanks are also due to Dr. S.V. Kakade, Ph.D. for statistical analysis. References
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