ANTIDEPRESSANT AUGMENTATION WITH BUSPIRONE: EFFECTS ON SEIZURE THRESHOLD AND BRAIN SEROTONIN LEVELS IN MICE

Indian Journal of Pharmacology 2001; 33: 198-202 RESEARCH PAPER ANTIDEPRESSANT AUGMENTATION WITH BUSPIRONE: EFFECTS ON SEIZURE THRESHOLD AND BRAIN SEROTONIN LEVELS IN MICE KHANAM RAZIA, M.A.A. SIDDIQUI, K.K. PILLAI, S.N. PAL Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard, New Delhi - 110062. Manuscript Received: 7.9.2000 Revised: 25.10.2000 Accepted:7.11.2000 SUMMARY Objective: To evaluate the antidepressant augmentation with buspirone (BUS) and to study the effects of antidepressants and their combinations with buspirone on seizure threshold and brain 5-HT levels in mice. Methods: Imipramine (IMI, 16, 8, 4 and 2 mg/kg) and fluoxetine (FLU, 10, 5 and 1 mg/kg) were administered alone and in combination (IMI 2 mg/kg; FLU 1 mg/kg) with buspirone (BUS, 2.5 mg/kg) to evaluate the effect on immobility time in the forced swimming test in mice. The effect of the above s and BUS (20 mg/kg) on the seizure threshold was studied in the Increasing Current Electroshock Seizure (ICES) test in mice. 5-HT levels in the different brain regions (cerebrum and cerebellum) were also determined spectrofluorimetrically, with these s, post ICES. Results: The combinations of IMI (2 mg/kg) and FLU (1 mg/kg) with BUS (2.5 kg/kg) produced significant reduction in the swimming induced immobility in mice. Such combinations did not adversely affect the seizure threshold in the ICES. IMI (16 mg/kg) and BUS (20 mg/kg) significantly decreased the seizure threshold whereas FLU (10 mg/kg) increased the same. ICES and the drugs studied, at their higher doses, moderately enhanced 5-HT levels in the brain regions, but FLU effected a significant increase. Conclusion: Results show that BUS can effectively augment the antidepressant effect of IMI and FLU with no adverse effect on seizure threshold. The ICES and 5-HT data suggest that anticonvulsive effect of FLU may be attributed to the 5-HT reuptake inhibition while the proconvulsant effect of BUS and IMI may be due to an additional influence of these drugs on nor-adrenergic transmission. KEY WORDS Depression buspirone imipramine fluoxetine 5-HT ICES INTRODUCTION Several depressive patients undergoing antidepressant (AD) drug therapy fail to respond to an adequate trial of a particular AD 1. Various strategies have been used for the augmentation of the AD effect in such patients. One such strategy is the addition of Buspirone (BUS) to an AD drug regimen 1,2. Several of the ADs are known to lower the seizure threshold in susceptible patients 3,4. Recently, BUS was also reported to be proconvulsive in mice 5. Hence, a co-administration of BUS with ADs could precipitate convulsions in the susceptible individuals. Therefore, the decision to include BUS as an add-on therapy in AD require a good deal of caution and reasoning. The present study was undertaken to see if BUS, when administered with antidepressants imipramine (IMI) a norepinephrine and serotonin reuptake inhibitor and fluoxetine (FLU) a selective serotonin reuptake inhibitor), would augment their AD effect and/or precipitate convulsions in mice. Various doses of IMI and FLU were first evaluated in Porsolt s Forced swimming test, a model of behavioral depression in rodents 6. The dose that was ineffective in this test was coupled with BUS for further evaluation in the FST for AD augmentation effects and in the Increasing Current Electroshock Seizure (ICES) Test, a model of epilepsy in mice 7, for proconvulsive effects. It has been suggested that the epileptogenic effects of tricyclic ADs are a consequence of enhanced monoaminergic transmission 4,8. Therefore, the effect of the BUS-AD combinations on 5- HT levels in the cerebrum and cerebellum regions was also studied to establish a possible correlation between AD induced seizures and brain 5-HT mechanisms. Correspondence: Shanthi N. Pal e-mail: shanthi_pal@lycos.com

ANTIDEPRESSANT AUGMENTATION AND BRAIN SEROTONERGIC EFFECTS WITH BUSPIRONE 199 MATERIALS AND METHODS Animals: Swiss strain albino mice (25 + 5 gm body weight) of either sex, raised at the Hamdard University Animal House, were used. They were housed in polypropylene cages and maintained on a natural light / dark cycle with free access to food and water. The animals were assigned to various groups with 6 and 8 animals per group for acute and chronic studies, respectively. Drugs: Hydrochloride salts of IMI (Merind Ltd, INDIA), FLU (Torrent Pharmaceuticals, INDIA) and BUS (Cadila Pharmaceuticals INDIA) were used. All drugs were dissolved in normal saline and administered intraperitoneally (i.p), in a volume of 10 ml /kg body weight. Control groups received an equal volume of normal saline. Antidepressant activity: The AD effects of various drugs were evaluated by a modified version of Porsolt s FST 6,9. Briefly, animals were forced to swim individually, for 15 min, in a glass container (11 cm diameter, 15 cm height) containing fresh water up to a height of 6 cm, at a temperature of 22 + 1 o C. This constituted the pre-test session. Twenty-four hours later, in the acute studies, the animals were administered a single dose of either IMI (16,8,4 or 2 mg/kg), FLU (10,5 or 1 mg/kg), BUS (2.5 mg/kg) or a combination of IMI (2 mg/kg) and BUS (2.5 mg/kg) or FLU (1 mg/kg) and BUS (2.5 mg/kg). 30 min after the drug the animals were forced to swim in a similar environment as before, for a period of 5 min in a test - session. The animals attempts to get out of the beaker were interspersed with periods of immobility signifying behavioral despair. The total duration of immobility during the last 4 min of the 5 min test was recorded. In the chronic studies, all drugs were administered for 14 days. On the 13 th day, the animals were subjected to the pre-test of FST. 24 hours later, the last (14 th day) dose was given and immobility in FST was recorded after 30 min. Drug doses (IMI 16 mg/kg, FLU 10 mg/kg) were based on pilot studies. These initial effective antidepressant doses for IMI and FLU were systematically decreased (8,4 and 2 mg/kg IMI and 5 and 1 mg/kg FLU) to arrive at a dose that was ineffective in the FST (2 mg/kg of IMI and 1 mg/kg of FLU). Each drug at its ineffective FST (AD) dose was then administered with 2.5 mg/kg of BUS and evaluated in the FST for AD augmentation. BUS was neither proconvulsive (in ICES) nor an effective AD (in FST) at 2.5 mg/kg and hence this dose was used in all the augmentation studies. Proconvulsive effect: The proconvulsive effects of BUS and that of IMI and FLU, alone and in combination with BUS, was determined in a modification of the ICES test of Kitano et al 7. Two doses each of IMI (16 & 2 mg/kg) and FLU (10 & 1 mg/kg) were used, the higher dose being the effective and the lower dose being the ineffective AD dose as determined earlier. Thus, mice were treated with either IMI (16 or 2 mg/ kg), FLU (10 or 1 mg/kg), BUS (20 or 2.5 mg/kg) or a combination of IMI (2 mg/kg) and BUS (2.5 mg/kg) or FLU (1 mg/kg) and BUS (2.5 mg/kg) in a single dose, i.p, in the acute studies or for 14 days in the chronic studies. 30 min later, on the same day (acute studies) or on the 14 th day (chronic studies), electroshock was delivered via ear electrodes as a single train of pulses for 0.2 sec with a linearly increasing current intensity (initial current 2 ma, increment of 2 ma/2 sec). The current at which tonic hind limb extension occurred was recorded as the seizure threshold current (STC). 5-HT estimation: The 5-HT levels were measured in the cerebrum and cerebellum at the proconvulsive doses of IMI (16 mg/kg) and BUS (20 mg/kg) and anticonvulsive dose of FLU (10 mg/kg) and also at the non- convulsive dose BUS (2.5 mg/kg) with IMI (2 mg/kg) or FLU (1 mg/ kg). 5-HT was estimated at these doses for a comparative evaluation of serotonergic influences at pro-, antiand non-convulsant doses of the different s. All s were given i.p, for 14 days. 30 min after the last dose each animal was challenged with ICES and decapitated. Its brain was removed immediately. The cerebrum and cerebellum were separated, weighed and processed for 5-HT estimations as per the method of Snyder et al 10. 5-HT was extracted into 1-butanol from a salt saturated solution (ph 10), returned to an aqueous solution (ph 7) by the addition of heptane and reacted with ninhydrin to yield a fluorescent product. Fluorescence was measured using a Hitachi spectrofluorometer at 490 mm after activation at 385 mµ (uncorrected). Statistics: Results are expressed as mean + SEM. Statistical analysis of the data was performed using a one-way analysis of variance (ANOVA) and Tukey s test. The P values equal to or less than 0.05 were adjudged statistically significant.

200 RESULTS Behavioral: Table 1 describes the effect of IMI, FLU and BUS at different doses in the FST in mice. An overall one way ANOVA (F (8,45) = 32.7, p< 0.01) with subsequent Tukey s test (p <0.05 Vs simple control) showed significant reduction in the immobility time with the higher doses of the drugs in the acute study. Table 2 details the effect of co-administration of BUS and ADs on swimming induced immobility. An overall one way ANOVA showed a significant reduction in immobility both in the acute (F (4,25) = 13.7, p< 0.01) and chronic (F (4,35) = 12.38, p< 0.01) studies. BUS (2.5 mg/kg) when combined with the lower dose of IMI (2 mg/kg, p<0.05, Tukey s test) or FLU (1 mg/kg, p<0.05, Tukey s test) was highly effective in the FST. Effects of ADs on the seizure threshold current (STC) are given in Table 3. BUS (20 mg/kg) and IMI (16 mg/kg) and FLU (10 mg/kg) affected significantly the seizure threshold in the ICES challenged mice (ANOVA: F (8,63) = 17.3, p <0.01 chronic studies) with IMI and BUS decreasing and FLU increasing the threshold (p <0.05, Tukey s test). However, the combinations i.e., BUS (2.5 mg /kg) and IMI (2 mg/kg) or BUS (2.5 mg /kg) and FLU (1 mg / kg), when evaluated in the ICES, did not significantly alter the STC in either acute or chronic studies. Biochemical: The effect of ICES and ADs on 5-HT in different brain regions are described in Table 4. ICES, BUS (20 mg/kg) and IMI (16 mg/kg) marginally (but not significantly) increased 5-HT levels in the cerebrum and cerebellum. However, significant enhancement was seen with FLU (10 mg/kg) Tukey s test; p< 0.05) The combination of BUS with IMI or FLU did not have any significant effect. DISCUSSION In our present study, BUS produced a potent reduction in the swimming induced immobility when combined with those doses of IMI and FLU that failed to affect immobility in mice in the forced-swimming test. In other words, BUS could augment the AD effect of the drugs studied. It may be noted that BUS, on its own, had no effect in FST at this AD augmenting dose. Since an augmentation was seen in the acute as well as in the chronic studies, it is not likely that the observed effects were just a delayed response to the ADs or a nonspecific placebo response. Table 1. Effect of antidepressants on forced swimming test in mice. Immobility (sec) Drug Dose (mg/kg, i.p) Acute (n=6) Chronic(n=8) Simple Control Saline 218 + 4.3 186 + 10.8 Imipramine 16 153 + 5.3* - Imipramine 8 178.5 + 5.8* - Imipramine 4 199.2 + 8.37 - Imipramine 2 216 + 4.2 192.1 + 4.4 Fluoxetine 10 121 + 9.3 * - Fluoxetine 5 174.3 + 5.8 * - Fluoxetine 1 199 + 8.6 188.6 + 4.8 Buspirone 2.5 227 + 7.9 221 + 10.1 *p< 0.05 Vs simple control. Table 2. Augmentation of antidepressant effect with buspirone on forced swimming test in mice. Drug Dose (mg/kg, i.p) Immobility (sec) Acute (n=6) Chronic (n=8) Simple control Saline 218 + 4.3 186 + 10.8 Imipramine 2 216 + 2.2 192.1 + 4.4 Fluoxetine 1 199 + 8.6 188.6 + 4.8 Buspirone + 2.5, 2 167.8 + 7.9 * 130.8 + 15.3 * imipramine Buspirone + 2.5, 1 164.3 + 12.4 @ 126. 3 + 5.4 @ fluoxetine * p<0.05 Vs imipramine; @ p<0.05 Vs fluoxetine The administration of BUS into the dorsal raphe nucleus, a site containing 5-HT IA autoreceptors, is known to reduce behavioral immobility in the FST. Therefore the AD-augmentation with BUS is probably mediated through 5-HT 1A autoreceptors 11. While an initial administration of BUS could reduce the 5-HT release on account of its action on 5-HT 1A autoreceptors on the serotonergic neurons, continued administration may lead to a desensitization of the inhibitory autoreceptors leading to an increased 5- HT release 12. Thus, in the present study, the BUSaugmented response to FLU in the FST may be due

ANTIDEPRESSANT AUGMENTATION AND BRAIN SEROTONERGIC EFFECTS WITH BUSPIRONE 201 to an enhanced CNS serotonergic function. BUS similar to ADs, can bring about a down-regulation of β- receptors 13. β-downregulation depends on adequate serotonergic function14. It is possible that the BUS induced β-downregulation is accelerated in the presence of a serotonin facilitating drug such as FLU, causing the AD augmentation with Bus 15. An intravenous administration of BUS is known to increase the firing rate of the locus ceruleus 16. Thus, it is likely that BUS can activate nor-adrenergic CNS neurons. These nor-adrenergic effects coupled with the serotonergic effects are probably responsible for the BUS-augmentation of the 5-HT-NE reuptake inhibitor, IMI. In the ICES test, in the chronic studies IMI and BUS were proconvulsive at 16 and 20 mg/kg respectively. These effects were consistent with the results from other studies evaluating the seizurogenic effect of these drugs 4,8. However, at their lower doses (IMI 2 mg/kg and BUS 2.5 mg/kg) neither drug reduced the seizure threshold (ST). A combination of BUS with IMI or FLU, at their respective lower doses, did not adversely affect the ST either. FLU, on the other hand, at 10 mg/kg, significantly raised the ST, thus providing further evidence for its anticonvulsant property 17-19. The neurochemical basis for the proconvulsive effects of ADs is not very clear. However, an enhancement of monoamine transmission that occurs with the administration of ADs may play a certain role 4. Since serotonin has been implicated in the regulation of seizure susceptibility 20,21, it is likely that serotonin reuptake blockade is at least one of the mechanisms by which FLU exerts its anticonvulsant action 17,22. While FLU is a selective 5-HT reuptake inhibitor, IMI inhibits the reuptake of both NE and 5-HT. Taken together, the proconvulsive effect of IMI and the anticonvulsant effect of FLU suggest that noradrenergic mechanisms may be responsible for the proconvulsive action while serotonergic mechanisms may be responsible for the anti-convulsive actions of ADs. That nor-adrenergic mechanisms may be important in the proconvulsive effects of ADs is strengthened by our observations with BUS (20 mg/kg) in the ICES because the major metabolite of BUS is the high affinity α 2 -adrenergic receptor antagonist 1-(2- pyrimidyl)-piperazine 23 and α 2 -adrenoceptor antagonists are known to potentiate convulsions in humans 24. ICES increased the 5-HT levels in the brain stem and Table 3. Effect of antidepressants and buspirone on ICES Test in mice. Drug Dose (mg/kg, i.p) Seizure threshold current (ma) Acute (n=6) Chronic (n=8) Simple control Saline 13.0 + 0.9 15.7 + 0.6 Buspirone 20 10.0 + 0.8 9.3 + 0.9* Buspirone 2.5 11.0 + 0.8 15.25 + 0.9 Imipramine 16 10.4 + 0.2 10.0 + 0.6* Imipramine 2 14.6 + 1.0 15.3 + 0.7 Fluoxetine 10 18.0 + 1.5 18.4 + 0.16* Fluoxetine 1 14.3 + 0.8 14.0 + 0.5 Buspirone + 2.5,2 12.0 + 0.5 14.0 + 0.8 imipramine Buspirone + 2.5,1 15.3 + 1.4 15.0 + 1.0 fluoxetine *p< 0.05 Vs simple control. Table 4. Effect of co-administration of buspirone and ADs on brain 5-HT levels in ICES challenged mice. Drug 5-HT (µg/g of wet tissue ) (mg/kg, i.p) n=5 Cerebrum Cerebellum Simple control 0.0154 + 0.002 0.0534 + 0.009 ICES 0.0222 + 0.002 0.0599 + 0.003 Buspirone (20) 0.0361 + 0.003 0.1306 + 0.023 Imipramine (16) 0.0528 + 0.003 0.111 + 0.015 Fluoxetine (10) 0.308 + 0.105* 0.361 + 0.14* Buspirone (2.5) + 0.0217 + 0.002 0.055 + 0.004 imipramine (2) Busprione (2.5) + 0.0201 + 0.002 0.0576 + 0.005 fluoxetine (1) * p< 0.05 Vs simple control. cerebrum in the mouse brain and this ICES-5-HT phenomenon may reflect corrective changes in the brain following an electroshock. IMI, BUS and FLU in their proconvulsive doses further enhanced the shockinduced 5-HT levels. In addition, all 3-drug s

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