Attenuation of Stress-induced Behavioral Deficits by Azadirachta Indica (neem): Role of Serotonin

The present study was designed to investigate the effect of Azadirechta indica (Neem) leaf extract on restraint-induced behavioral deficits and changes in serotonin metabolism in rats. Exposure to a single stress decreased food intake, growth rate and elicited anxiogenic-like behavior on an elevated plus maze. Prior administration of neem leaf extract @ 1 ml/kg for 5 days attenuated stress induced behavioral deficits of food intake, growth rate and anxiogenic behavior but the level of anxiety in unrestrained animals was not altered. Restraint-stress did not alter brain tryptophan and 5-hydroxytryptamine (5-HT) levels. 5-hydroxyindolacetic acid (5-HIAA) concentration increased in saline but not in neem injected rats. Administration of neem leaf extract increased brain tryptophan and decreased brain 5-HT concentration in unrestrained animals. The present study showed that neem extract could attenuate anxiogenic and appetite suppressant effects of stress by decreasing brain 5-HT and 5-HIAA concentartion. Introduction Stress acts as a predisposing and precipitating factor in the onset of affective illness specially depression (Brown et al., 1987). Parallel studies on experimental animals show that an uncontrollable stressor produces neurochemical and behavioral deficits. In similar studies it has been shown that an episode of 2 hours restraint stress decreased food intake, growth rate and locomotor activity in rats (Haleem et al., 1988; Samad et al., 2002). On repeated immobilization these behavioral deficits were no longer observed (Haleem & Parveen, 1994). These studies suggested that behavioral adaptation to a stress schedule develops when the same stress is administered repeatedly. A variety of stress stimuli increase the synthesis and turnover of 5-hydroxytryptamine (5-HT, serotonin) in the whole brain and various brain regions of rats (Haleem et al., 1988). On the other hand, when an episode of 2 hours restraint stress challenged to rats which were adapted repeatedly restrained schedule, 5-HT synthesis did not increase (Haleem & Parveen, 1994). Azadirachta indica (Neem) is an evergreen tree in the Indian subcontinent. Leaves and seeds have been used in Ayuredic medicine. Previous studies showed that neem leaves could reduce anxiety and stress when ingested in small quantities (Banerjee, 1992). The present study was designed to monitor the effect of neem leaves extract on restraint-stress induced behavioral deficits. Associated changes of brain serotonin metabolism were also determined. Address for correspondence: E-mail: [email protected] NOREEN SAMAD ET AL., 132 Materials and Methods Animals and treatment: Locally bred Albino Wistar rats weighing 200 to 220 g were used for the experiment. They were caged individually in plastic cages with free access to cubes of standard rodents diet and tap water for 5 days, before starting the experiment. Preparation and dosage of neem extract: Ten g of fresh neem leaves were grinded in 100 ml distilled water and liquid squeezed was centrifuged. The supernatant was injected intraperitoneally (IP) to rats @ 1 ml/kg body weight. Control animals were injected with saline (0.9% NaCl: 1 ml/kg body weight). Experiment protocol: The animals were divided into saline injected and neem injected groups. These animals were injected with neem extract and saline daily for 6 days. On the 4 day open field activity was monitored, after one hour of injection of neem extract or saline. On the 5 day, animals of the two groups were subdivided into restrained and unrestrained groups. One hour after the injection, a group of neem injected animals and another group of saline injected animals were restrained on wire grids for 2 hours. The other group of neem injected and saline injected animals were left unrestrained in their home cages. Cumulative food intake and body weight changes for 24 hours were monitored on the 6 day. Activity on an elevated plus maze were also monitored on the 6 day after the neem extract or saline injection. The animals were decapitated on the 6 day to collect brain samples 2 hours after the neem extract or saline injection. Brain samples were stored at –70° C for the estimation of tryptophan, 5-HT and 5-HIAA by HPLC-EC (Haleem & Parveen, 1994). Restraint stress: The animals were restrained on wire grids of 10′′x9′′ fitted with a Perspex plate of 9′′x6.5′′. Restraining procedure was same as described earlier (Haleem & Parveen, 1994). The animals were restrained between 11:00 a.m. to 1:00 p.m. After 2 hours of restraining period the animals were released and returned to their home cages. Open field activity: The open field apparatus used in the present investigation consisted of a square area 76x76 cm with walls 42 cm high. The floor was divided by lines into 25 equal squares. To determine the activity, a rat was placed in the centre square of the open field and latency to leave the centre square and number of squares crossed with all four paws were scored for 5 minutes as described earlier (Haleem, 1996). The activity of saline injected rats and neem injected rats were monitored in a balanced design to avoid order effect on the 4 day between 11:00 a.m. to 2 p.m. Plus maze activity: The plus maze apparatus used in the present investigation consisted of four arms in which two were open and two were closed. The arms were of identical length (50 cm) and width (10 cm). Arms were joined by central area of 5 cm. The maze was elevated from the floor at a height of 60 cm. To determine the activity, a rat was placed in the centre of plus maze and time spent in the open and closed arms, number of entries in open and closed arm were determined (Samad et al., 2002). The activities of saline injected restrained and unrestrained, and neem injected restrained and unrestrained rats were monitored for 5 min., in a balanced design on the 6 day between 9:00 a.m. to 12:00 a.m. ATTENUATION OF STRESS-INDUCED BEHAVIORAL DEFICITS 133