INTRODUCTION:

Depression is an incapacitating psychiatric disease ¹ and the core symptoms include depressed mood, anhedonia (reduced ability to experience pleasure from natural rewards), irritability, difficulties in concentrating, and abnormalities in appetite and sleep etc^2. Patients with major depression symptoms that are reflected by changes in brain monoamine neurotransmitters specifically, 5-hydroxytryptamine (serotonin, 5-HT) and noradrenaline (nor epinephrine, NE) ^[1,2].Neurobiological evidences both in animal and in human have also indicate the role of monoaminergic systems (catecholamine and serotonin) in the pathophysiology of mental depression. Consistent with this view, most antidepressants exerted their action by elevating synaptic monoamine concentrations^3. At present, there are several types of classical antidepressants used in clinical practice, including tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), monoamine oxidase inhibitors (MAOIs) and serotonin and noradrenaline reuptake inhibitor^4. The success of the SSRIs has been attributed to their ease of use, safety, and broad spectrum of efficacy over a variety of psychiatric disorders.

Psychotropic actions of gabapentin:

Gabapentin, a novel anticonvulsant, it is currently under investigation for possible efficacy in treating psychiatric disorders. Although developed as a γ-amino-butyric acid (GABA) agonist, Naturaly it does not interact with GABA or benzodiazepine receptors, nor it is metabolized to GABA or involved in GABA uptake^5. At the same time, gabapentin does increases GABA turnover and whole blood serotonin levels and its mechanism of action is unclear^6. Animal and clinical studies suggest that gabapentin may have useful psychotropic actions similar to the other anticonvulsants used in psychiatry today. In addition, its efficacy in treating the unipolar and bipolar depression has been evaluated clinically⁷

Serotonin abnormality in depression:

The monoamine serotonin is well established as a neurotransmitter involved in the pathophysiology of, and therapy for, major depressive disorder. Evidence for serotonergic abnormalities in depression comes from a large number of studies showing recurrence of depression after tryptophan depletion in remitted, medication-free, patients, a decrease in cerebrospinal fluid concentrations of the serotonin metabolite 5-hydroxyindolacetic acid in depressed patients with a history of serious suicidal behavior, a decrease in serotonin uptake and transporter binding sites in brain and platelets from depressed subjects, a blunting of neuroendocrine responses to serotonergic stimuli, and changes in the densities of serotonin receptors in prefrontal cortex in depression and suicide⁸. For instance, the symptoms of depressions are relieved by treatment with medications that potentiate serotonergic activity by preventing the reuptake or metabolism of serotonin^9. Related animal studies revealed that the chronic administration of clinically effective antidepressant treatments results in a net enhancement in serotonin neurotransmission^10. Numerous studies of depression and suicide have used in postmortem and neuro imaging techniques to examine serotonin receptors and the serotonin transporter. The strongest evidence from some but not all postmortem studies of serotonin receptors suggests a significant increase in HT_2Areceptors in dorsolateral prefrontalcortex in suicide. More modern investigations have employed brain-imaging techniques, in particular ligand imaging in conjunction with positron emission tomography (PET) and single photon emission tomography (SPET). This enables more direct investigation of 5-HT receptors in the living human brain. These data have provided consistent and convincing evidence that the binding density of 5-HT_1A receptors shows a widespread decrease in depressed patients^11.

Several studies have shown that the number of 5-HT₂ receptors is increased in the postmortem brain of suicide victims and depressed subjects. 5HT₂ receptors also increased in the platelets of depressed patients^12. In animal studies, the number of 5-HT₂ receptors is decreased in the rat frontal cortex following the chronic administration of antidepressants^13. These findings indicate that the down-regulation of 5HT₂ receptors may be related to the antidepressant activity of antidepressants^17..On the other hand, it has become apparent from a number of studies, that the capacity of antidepressants to down-regulate 5-HT₂ receptors is not a property shared by all antidepressants ^[11’13]. Chronic electroconvulsive seizure (ECS) therapy, one of the most efficacious treatments for depression which have reported to increase 5-HT₂ receptors in the rat frontal cortex an effect opposite to that of antidepressants^14.

MATERIALS AND METHODS:

Animals:

Male albino mice (24–27 g) bred at PSG IMS and R animal house, Coimbatore. They were housed 5 per cage (320×180×160 cm) under normal (25±2 °C, 50 % humidity) laboratory conditions, maintained on a 12 hour day–night cycle, with free access to standard food and water. Animals were adapted to laboratory conditions before the test. All behavioral experiments were carried out between 9.00am to 12.00 am. The experimental protocols were approved by the Institutional Animal Ethical Committee (158/99/CPCSEA) and conducted according to the CPCSEA guidelines on the use and care of experimental animals

Drug Administration:

Mice were treated with gabapentine (1, 3 and 6mg/kg), fluoxetine (30 mg/kg) and saline (vehicle) once daily at 9:00 a.m.–10:00 p.m by intera peritoneal administration. In the following studies, FST, TST or OFT in mice was conducted 1 h after the last drug treatment. Food, but not water was withdrawn from the animals 1 hr prior to drug administration.

Behavioral despair models:

Effect of gabapentin on the duration of immobility in FST:

This test was slightly modified version of Porsolt’s forced swim test ^[15].Animals were divided into different groups (n=6) and subjected to pre test session. Twenty four hours later mice were treated with saline (control), fluoxetine 30mg/kg (test), or gabapentin (1, 3, and 6 mg/kg) through i.p route. Thirty minutes later animals were individually subjected to the FST for six minutes and duration of immobility was measured.

Effect of gabapentin on the duration of immobility in TST:

Tail suspension test was derived from the Porsolt’s forced swim test and the results were based on the observation that mouse suspended by its tail shows the alternate period of agitation immobility similar to that observed in FST^16. Mice were administered with different dose of gabapentin (1, 3 and 6 mg/kg, i.p.). Thirty minutes after the drug treated mice were subjected to tail suspension test and the duration of immobility was measured. Separate groups were served as control and standard.

Effect of gabapentin on number of ambulation and rearing in OFT:

This test was performed to assess whether the changes in immobility time were associated with any effect on motor activity. Various groups of animals were treated either with vehicle or gabapentin (1, 3 and 6mg/kg, i.p) and 30 min later animals were placed individually into the open field apparatus no of rearing and ambulation were noted for 5 minutes.

Neurotransmitter Estimation:

Blood samples collection:

To avoid fluctuations in hormone levels due to circadian rhythms, mice were bled at 09:00 a.m. and 10:00 a.m. on the day of sacrifice blood samples were collected by retro orbital route and immediately transferred to eppendroff tubes contains 10% sodium citrate solution(ml/5µl of sodium citrate). All the blood samples were centrifuged at 2000rpm and supernatant liquid was collected and it was stored at −10 °C for biochemical studies.

Estimation of blood samples by using HPLC Method:

Plasma serotonin level was measured by using 10At HPLC systems (shimadzu LC). The mobile phase used for the study was acetonitrile: methanol: sodium acetate (pH 3.5) in the ratio of (10:10:80 % v/v). The stationary phase used was phenomenex C18 (250*4.6mm i.d, 5µ) at the flow rate of 0.7 ml/min and detected at 275nm. The samples were prepared with phenomenex strata solid phase extraction cartridge was conditioned with methanol and water (1ml) sequentially. To this 0.5ml of plasma was added. The cartridge was washed with 2ml of water. The drug was eluted from the cartridge using 0.5ml of a mobile phase. The standard serotonin was for the preparation of calibration curve

Influence of 5HT2 agonist on the gabapantin induced changes in FST:

This experiment was performed to assess the contribution 5HT₂ receptor on the gabapentine induced effect on immobility in FST. Mice, previously given ‘pretest session’ were treated with vehicle or 5HT₂ agonist, (DOI 1mg/kg, i.p.) 30 min prior to the administration of gabapentine (6mg/kg, i.p.) and after 30 min immobility time was measured for 6 min.

Effect of 5HT2 receptor Antagonist on the action of gabapentin in FST:

To evaluate whether pretreatment of 5HT₂ receptor antagonist, cyproheptadine produces any change on the action of gabapentine in FST, mice, which were subjected to the pretest session on the previous day were treated with saline or cyproheptadine (1mg/kg,i.p) through intraperitoneal route. After thirty minutes, gabapentin (1mg/kg i.p.) was administered. The duration of immobility was measured in the test session at 30 min after the administration of gabapentine.

Statistical analysis:

Data is expressed as mean ± SEM and one way ANOVA followed by post hoc Dunnett’s test was used to analyze the data. In all the experiments, p< 0.05 was considered as the significance level between the groups

RESULTS:

Gabapentin decreases the duration of immobility in FST

The effects of acute intraperitonial administration of different doses of gabapentin on immobility time in the FST were shown in table 1. One-way ANOVA revealed that gabapentin (1, 3 and 6mg/kg) induced marked changes in the duration of immobility [F(4,25)=10.838; p<0.01]. Gabapentin (3, 6 mg/kg), dose dependently reduced the duration of immobility when compared to the control group (p<0.01). Whereas gabapentin 1 mg/kg did not showed any significant changes in immobility time (p>0.05).

Table 1: Antidepressant like effect of gabapentin in FST

S. No	Treatment	Duration of immobility in sec (Mean±SEM)
1	Control	234.66± 9.244
2	Flouxetine 30mg/kg	179.5 ± 6.605
3	Gabapentin 1mg/kg	232.21± 7.25
4	Gabapentin 3mg/kg	189.5±7.835*
5	Gabapentin 6mg/kg	185.3±11.97*

Mice (n=6),Data represents Mean ±SEM immobility time in sec of 6 mice per group. *P<0.01, compared to control group.

Reduction of immobility time by gabapentin in TST:

One way ANOVA showed significant effect of gabapentin in TST [F (4, 25) = 12.829; p<0.01]. Post hoc test revealed that gabapentin (1,3 and 6 mg/kg, i.p.) reduced the immobility time significantly (p<0.01) and the effect was dose dependent. However, gabapentin (1 mg/kg, i.p.) did not affect the immobility time. The results are given in table 2.

Table 2: Antidepressant like effect of gabapentin in TST

S. No	Treatment	Duration of immobility in sec (Mean±SEM)
1	Control	236.16± 12.50
2	Flouxetine 30mg/kg	145.5 ±5.602
3	Gabapentin 1mg/kg	223±4.56
4	Gabapentin 3 mg/kg	176.66± 10.5*
5	Gabapentin 6 mg/kg	150.16 ±7.79*

Mice (n=6). Data represents Mean ±SEM immobility time in sec. *P<0.01, compared to control groups.

Gabapentin did not change the open field behavior of mice:

One way ANOVA showed no significant changes in the number of rearing [F (3, 24) = 0.878; p>0.05] and ambulations [F (3, 24) = 0.977; p>0.05] upon the treatment of gabapentin (1, 6 and 12 mg/kg, i.p.) in open field test shown in table 3.

Table 3: Effect of gabapentine on open field behavior

S. NO	Treatment	No of amulation (Mean±SEM)	Rearing (Mean±SEM)
1	Control	42.16±4.963	1.6±0.33
2	Gabapentin1mg/kg	41.0±4.11	1.66±0.432
3	Gabapentin3mg/kg	40.83±2.120	1.5±0.33
4	Gabapentin6mg/kg	41.66±1.54	6.16±0.94

Mice (n=6). Data represents Mean ±SEM no of ambulation, rearing.

Chronic treatment of gabapentin enhanced the plasma serotonin in mice:

One way ANOVA revealed significant effect due to the treatment of gabapentin on plasma serotonin level [F(3,20) =7.83;p<0.01]. Post hoc test revealed that, gabapentin 3mg/kg and 6 mg/kg showed dose dependent increase (p<0.01) in the plasma serotonin. On the other hand, treatment of gabapentin 1mg/kg did not show any significant change in the level of serotonin in plasma as shown in table 4.

Table 4: Effect of gabapentin on plasma serotonin level

SL. NO	Treatment	Plasma serotonin level in µg/ml Mean±SEM
1	Control	20.2±1.2
2	Gabapentin1mg/kg	19.82±0.7
3	Gabapentin3mg/kg	28.02±1.4*
4	Gabapentin6mg/kg	32.12±0.63*

Mice (n=6) Data represents Mean ±SEM. Dennett’s test.*P<0.01, compared to control group

5HT₂ agonist blocks the antidepressant like effect of gabapentin in FST

Administration of 5HT₂ agonist, DOI modified the reduction of immobility caused by the treatment of gabapentin in FST [F (3, 20) = 10.199; p<0.01]. The decrease in duration of immobility induced by the administration of gabapentin (6 mg/kg, i.p) was reversed completely by the pretreatment of 5HT₂ agonist, DOI (1 mg/kg, i.p). Whereas, administration of DOI (1 mg/kg, i.p) itself did not affect the duration of immobility shown in table 5.

Table 5: Effect of 5HT₂ agonist, DOI antagonized the antidepressant like effect of gabapentin in FST

S. NO	Treatment	Duration of immobility in sec (Mean±SEM)
1	Control	234.66± 9.244
2	Vehicle + Gabapentin 6mg/kg	185.3 ±11.97*
3	DOI 1 mg/kg +Vehicle	236.44± 6.77
4	DOI 1 mg/kg + Gabapentin 6mg/kg	230.11 ±6.272

Mice were injected with vehicle(0.9%NaCl), DOI(1mg/kg) 30 min prior to the administration of gabapentin(6mg/kg) or vehicle and 30 min, thereafter immobility time was measured in test session for 6 min. Data represents Mean ±SEM immobility time in sec of 6 mice per group. Dennett’s test. p>0.05, compared to respective control groups. Groups 2, 3 and 4 Vs1

Potentiation of antidepressant like effect of gabapentin by 5HT2 antagonist:

Administration of 5HT2 antagonist, cyproheptadine (1 mg/kg, i.p), prior to the administration of sub-effective dose of gabapentin (1 mg/kg, i.p) significantly (p<0.01) decreased the duration of immobility as compared to the control [F (3, 20) = 8.03, p<0.01] shown in table (6).

Table 6: Effect of 5HT₂ antagonist, cyproheptadin potentiated the antidepressant like effect of gabapentin in FST

S. NO	Treatment	Duration of immobility in sec (Mean±SEM)
1	Control	234.66± 9.244
2	Vehicle + Gabapentin 1mg/kg	232.21± 7.25
3	Cyproheptadin 1mg/kg + Vehicle	230.23±5.87
4	Cyproheptadin + Gabapentin 1mg/kg	189.5 ±5.285*

Mice were injected with vehicle(0.9%NaCl), Cyproheptadin (1mg/kg) 30 min prior to the administration of gabapentin (1mg/kg) or vehicle and 30 min, thereafter immobility time was measured in test session for 6 min. Data represents Mean ±SEM immobility time in sec of 6 mice per group. Dunnett’s test.*P<0.01, compared to respective control groups.

DISCUSSION:

In the present study, acute administration of gabapentin showed dose dependent anti-immobility effect in FST and TST. The FST and TST have the ability to induce immobility which represents the state behavioral despair in animals and this has been claimed to reproduce the condition similar to the depression in human beings. Reduction in the duration of immobility by a drug was considered, as it possesses antidespair or antidepressant like effect as gabapentin reduced the duration of immobility in FST and TST may possess anti depressant effect.

The antidepressant like effect of gabapentin showed in the present study is in consistent with the previous clinical and preclinical research findings. Generally, the drugs which enhance the psychostimulant effect show false positive results in behavioral despair models and open field test is used to discriminate the psychostimulant action from anti despair effect. However, gabapentin did not change the ambulatory and rearing behavior of animals at the doses effective on FST and TST and this reveals that anti depressant like effect of gabapentin is more specific to the escape directed behavior.

Hence, our study evaluated the role of serotonergic system in the antidepressant like effect of gabapentin by estimating the changes in level of plasma serotonin due to gabapentin treatment using HPLC method. The reports indicated an increase in the plasma serotonin level after the chronic treatment of gabapentin. This result is in line with the previous report that, treatment of gabapentin increases the whole blood serotonin of normal human volunteers, gabapentin modulates the release of plasma serotonin level and the increase in peripheral serotonin paradigmatically to increase in the bioavailability of serotonin, the similarity of central and peripheral (platelet) serotonin receptors, it has been speculated that gabapentin induces alterations in central serotonin metabolism which may be reflected by changes in peripheral serotonin levels^(17).

Although, less attention was paid in the plasma serotonin level as compare to other biomarkers of depression, considerable reports are found on the efficacy of estimating plasma serotonin in evaluating the responsiveness to the antidepressants treatment. These reports include, the antidepressants, clomipramine has shown an initial increase in plasma serotonin level, plasma serotonin level has been shown to increase one day after the treatment of flouxetine, flouxetine treatment responders showed higher level of serotonin in plasma and lower level in platelet, acute treatment of tianeptine has shown an increase in plasma serotonin and decrease in plasma level after chronic treatment. It is evident from the above reports that the antidepressant activity of a drug is associated with changes in plasma serotonin level.

The serotonin hypothesis of depression is continuously modifying, signifying the subtypes of receptors in this concept. Blockade of 5HT₂ receptors seems to have antidepressant effect in preclinical and clinical studies. In order to study the role of 5HT₂ receptor in the antidepressant like effect of gabapentin, we have conducted interactive study with 5HT₂ analogs in FST. Pretreatment of 5HT₂ antagonist, cyproheptadine, potentiated the antidepressant like effect of gabapentine as evident from the decrease in immobility. Conversely, the anti-immobility exhibited by gabapentin was antagonized by the pre-treatment of 5HT₂ agonist, DOI. This result clearly indicates the modulatory role of 5HT₂ in the antidepressant like effect of gabapentin.

Postmortem and neuroimaging techniques studies show an increase in 5HT₂ receptor density in suicidal victims and depressed subjects. 5HT₂ receptors also have shown to be increased in the platelets of patients with depression. In animal studies using rats, the number of 5-HT₂ receptors are found to be decreased in the frontal cortex subsequent to the chronic administration of antidepressants. These findings indicate that the down-regulation of 5HT₂ receptors may be related to the antidepressant activity of antidepressant. On the contrary, a rise in 5HT₂ receptors in the rat frontal cortex after chronic electroconvulsive seizure (ECS) therapy has been reported. While, the reason is not known for this opposite response exhibited by chronic electroconvulsive seizure therapy

The significant roles of 5HT_1A and 5HT₃¹¹ receptor mediating signaling in FST were also demonstrated. However, our study did not evaluate the role of these subtypes of 5HT receptors in the antidepressant effect of gabapentin.

CONCLUSION:

Gabapentin elicits antidepressant like effect in behavioral despair models. The antidepressant effect of gabapentin seems to be mediated by enhancement of serotonergic functions probably through down regulating the 5HT₂ receptor functions

ACKNOWLEDGEMENT:

The authors acknowledge PSGIMSandR for providing research support and facilities for carrying out this research work. The authors thankfully acknowledge Mrs.kalaiyarasi for her guidance.

REFERENCES:

1. Aberg-Wistedt A, The antidepressant effects of 5HT uptake inhibitors. Br. J. Psychiatry, 1989, 8; 32-40.

2. Jouvet M, Biogenic amines and the states of sleep. Science. 1969.163; 32M1

3. 3Castrogiovanni P, Blardi P, De Lalla A, Dell'Erba A, Auteri A, Can serotonin and fluoxetine levels in plasma and platelets predict clinical response in depression?. Psychopharmacol Bull. Spring. 2003. 37(2);pp.102-8.

4. Ortiz J, Mariscot C, Alvarez E, Artigas F, Effects of the antidepressant drug tianeptine on plasma and platelet serotonin of depressive patients and healthy controls. J Affect Disord. 1993,29(4);227-34

5. 5.McLean M J,. Clinical pharmacokinetics of gabapentin. Neurology 44 Suppl. 1994, 5; S17–S22

6. Rao M L, Clarenbach P, Vahlensieck M, and Kratzschmar S, Gabapentin augments whole blood serotonin in healthy young men. J. Neural. Transm. 1988, 73; 129–134.

7. Young LT, Robb JC, Hasey GM, MacQueen GM, PatelisSiotis I, Marriott M, Joffe RT, Gabapentin as an adjunctive treatment in bipolar disorder. J. Affect. Disord. 1999, 55; 73–77.

8. Vinod KY, Subhash M N, Lamotrigine induced selective changes in 5-HT1A receptor mediated response in rat brain. Neurochem Int. 2002, 40^th ed;pp.315-9.

9. Amsterdam JD, Fawcett J, Quitkin FM, Reimherr FW, Rosenbaum JF, Michelson D, Hornig RM, Beasley CM, Fluoxetine and norfluoxetine plasma concentrations in major depression: a multicenter study. Am. J. Psychiatry. 1997, 154;963– 969.

10. Bekris S, Antoniou K, Daskas S, Papadopoulou D Z, Behavioural and neurochemical effects induced by chronic mild stress applied to rat strains. Behav Brain Res. 2005, 161;45–59.

11. Marphy DL,Campbell IC,costa JL,The brain serotonergic system in the effect disorders,prog.neuro psyh. 1978,1;1-3.

12. Pande AC, Crockatt JG, Janney CA, Werth JL, Tsaroucha G, Gabapentin in bipolar disorder. A placebo-controlled trial of adjunctive therapy. Bipolar Disord,. 2000, 2;249–255.

13. Wesolowska A ,.In the search for selective ligands of 5-HT₅, 5-HT₆ and 5-HT₇ serotonin receptors Polish J. Pharmacolog. 2002, 54; 327–41

14. Muscat R, Papp M, Willner P, Reversal of stress-induced anhedonia by the atypical antidepressants, fluoxetine and maprotiline. Psychopharmacology; in press.

15. Porsolt, RD., Anton, G., Blavet, N., Jalfre, M , Behavioural despair in rats: a new model sensitive to antidepressant treatments. Eur J Pharmacol. 1978, 47,379–91

16. Porsolt, RD, Le PM, Jalfre, M., Depression: a new animal model sensitive to antidepressant treatments. Nature, 1977, 266,730–2.

17. Wesolowska A, In the search for selective ligands of 5-HT₅, 5-HT₆ and 5-HT₇ serotonin receptors Polish. J. Pharmacology, 2002, 54, 327–41.

Received on 08.07.2011 Modified on 02.08.2011

Research J. Pharm. and Tech. 4(11): Nov. 2011; Page 1702-1706