dextromethorphan ( centurion laboratories pvt . ltd . , vadodara , india ) , msg ( desmo exports limited , mumbai , india ) , gaba ( sigma chemicals , mumbai , india ) , calcium kit ( erba diagnostics mannheim gmbh , solan , hp , india ) and sodium and potassium kit ( crest biosystems , verna , goa , india ) were used in the present study . they were housed in clean polypropylene cages under standard conditions of temperature ( 252 ) and 12 h lightdark cycle and fed with standard diet ( gold mohur , lipton india ltd . ) and water ad libitum . for the preparation of extract , stem bark of pp was collected from the institute 's botanical garden and was identified and authenticated by a botanist . the coarsely powdered pp stem was extracted with ethanol ( 70 - 80 ) in a soxhlet extractor , concentrated in reduced pressure and stored in a desiccator ( percent yield was 6.91 ) . group 1 ( normal ) rats were administered with an aqueous suspension of 1% w / v cmc , ( 5 ml / kg , p.o . ) . dextromethorphan was prepared in 1% cmc for oral administration for the use in groups 2 to 5 . the doses of plant extract , dextromethorphan and msg were selected based on previous literature . group 2 rats were treated with msg ( 2 g / kg , i.p . ) . group 3 rats were administered with an ethanol extract of pp ( 200 mg / kg , p.o . ) after 1 h of treatment with msg ( 2 g / kg , i.p . ) . group 4 rats were administered with an ethanol extract of pp ( 400 mg / kg , p.o . ) after 1 h of treatment with msg ( 2 g / kg , i.p . ) . p.o . ) after 1 h of treatment with msg ( 2 g / kg , i.p . ) . the general behavioural changes , if any , were observed for 30 min daily after the administration of msg . on 8 day , the rats were evaluated for locomotor and muscle relaxant activity . locomotor activity was evaluated by using actophotometer . in this , animals of all the groups were placed in actophotometer for 10 min and the score was recorded . animals of all the groups were placed on the rotating rods and the time of animal fall from the rotating rods was recorded . on 9 day , rats were sacrificed and brain was quickly isolated and stored at 20. blood samples were centrifuged to get clear serum for estimating gaba level . the brain tissue 10% w / v was homogenised with tris  hcl buffer ( ph 7.4 ) using handheld homogeniser ( remi homogeniser , india ) . homogenised samples were centrifuged at 5000 rpm for 10 min ( remi centrifuge , india ) . briefly , 1 ml of homogenate was added to 1 ml of 10% trichloroacetic acid ( tca ) and centrifuged . one millilitre of supernatant was treated with 3 ml of phosphate buffer ( ph 8.0 ) and 0.5 ml of ellmans reagent . the amount of glutathione was calculated using the extension coefficient value of 13,600/m / cm . briefly , 0.1 ml of the tissue homogenate was added with 2.0 ml of the tca  tba hcl reagent ( 15% w / v tca , 0.375% w / v tba and 0.25 n hcl ) . the contents were boiled for 15 min , cooled and centrifuged at 1000 rpm for 10 min . the absorbance of clear supernatant was read at 535 nm and malondialdehyde concentration of the sample was calculated using extinction coefficient of 1.5610/m / cm . the estimation of superoxide dismutase ( sod ) was determined by the method of kakkar et al .. briefly , 0.1 ml of the sample was mixed with 1.2 ml of sodium pyrophosphate buffer ( ph 8.3 , 0.052 m ) , 0.1 ml of 186 m of phenazine methasulphate ( pms ) and 0.3 ml of 300 m nitro blue tetrazolium ( nbt ) . the mixture was incubated at 30 for 90 s. the reaction was stopped by the addition of 0.1 ml glacial acetic acid . the reaction mixture was stirred vigorously and shaken with 4 ml of n - butanol . the mixture was allowed to stand for 10 min , centrifuged to separate the butanol layer . the colour intensity of the chromogen was measured against n - butanol at 560 nm using spectrophotometer . sod activity was defined as its concentration required to decrease the rate of reaction by 50% in 1 min under the assay conditions . the reaction mixture contained 1.0 ml phosphate buffer ( ph 7.0 ) , 0.1 ml of tissue homogenate ( supernatant ) and 0.4 ml of 0.2 m h2o2 . the reaction was stopped by the addition of 2.0 ml of dichromate - acetic acid reagent . colour intensity was measured colorimetrically at 620 nm and cat activity expressed as micromoles of h2o2 consumed / min / mg protein . the concentration of electrolytes like calcium , sodium and potassium in the brain was measured by using commercial kits by erba diagnostics ( for calcium ) and crest biosystems ( for sodium and potassium ) . the gaba level in serum was assayed by paper chromatography as mentioned by mishra et al . with modifications . the serum sample ( 100 l ) was added to 1.5 ml of absolute alcohol and centrifuged at 3000 g for 15 min . fifty microlitre of the sample was used for paper chromatography using the mobile phase containing n - butanol , glacial acetic acid and water . optical density of eluted sample was taken on a spectrophotometer at wavelength of 509 nm and compared with standard gaba solution . the gaba concentration in serum the paraffin sections were prepared and stained with haematoxylin and eosin and examined using light microscopy . resultswere analysed by one - way analysis of variance ( anova ) followed by dunnet 's multiple comparison test using graph pad prism software . group 1 ( normal ) rats were administered with an aqueous suspension of 1% w / v cmc , ( 5 ml / kg , p.o . ) . dextromethorphan was prepared in 1% cmc for oral administration for the use in groups 2 to 5 . the doses of plant extract , dextromethorphan and msg were selected based on previous literature . group 2 rats were treated with msg ( 2 g / kg , i.p . ) . group 3 rats were administered with an ethanol extract of pp ( 200 mg / kg , p.o . ) after 1 h of treatment with msg ( 2 g / kg , i.p . ) . group 4 rats were administered with an ethanol extract of pp ( 400 mg / kg , p.o . ) after 1 h of treatment with msg ( 2 g / kg , i.p . ) . p.o . ) after 1 h of treatment with msg ( 2 g / kg , i.p . ) . the general behavioural changes , if any , were observed for 30 min daily after the administration of msg . on 8 day , the rats were evaluated for locomotor and muscle relaxant activity . locomotor activity was evaluated by using actophotometer . in this , animals of all the groups were placed in actophotometer for 10 min and the score was recorded . animals of all the groups were placed on the rotating rods and the time of animal fall from the rotating rods was recorded . on 9 day , rats were sacrificed and brain was quickly isolated and stored at 20. blood samples were centrifuged to get clear serum for estimating gaba level . the brain tissue 10% w / v was homogenised with tris  hcl buffer ( ph 7.4 ) using handheld homogeniser ( remi homogeniser , india ) . homogenised samples were centrifuged at 5000 rpm for 10 min ( remi centrifuge , india ) . briefly , 1 ml of homogenate was added to 1 ml of 10% trichloroacetic acid ( tca ) and centrifuged . one millilitre of supernatant was treated with 3 ml of phosphate buffer ( ph 8.0 ) and 0.5 ml of ellmans reagent . the amount of glutathione was calculated using the extension coefficient value of 13,600/m / cm . briefly , 0.1 ml of the tissue homogenate was added with 2.0 ml of the tca  tba hcl reagent ( 15% w / v tca , 0.375% w / v tba and 0.25 n hcl ) . the contents were boiled for 15 min , cooled and centrifuged at 1000 rpm for 10 min . the absorbance of clear supernatant was read at 535 nm and malondialdehyde concentration of the sample was calculated using extinction coefficient of 1.5610/m / cm . the estimation of superoxide dismutase ( sod ) was determined by the method of kakkar et al .. briefly , 0.1 ml of the sample was mixed with 1.2 ml of sodium pyrophosphate buffer ( ph 8.3 , 0.052 m ) , 0.1 ml of 186 m of phenazine methasulphate ( pms ) and 0.3 ml of 300 m nitro blue tetrazolium ( nbt ) . the mixture was incubated at 30 for 90 s. the reaction was stopped by the addition of 0.1 ml glacial acetic acid . the reaction mixture was stirred vigorously and shaken with 4 ml of n - butanol . the mixture was allowed to stand for 10 min , centrifuged to separate the butanol layer . the colour intensity of the chromogen was measured against n - butanol at 560 nm using spectrophotometer . sod activity was defined as its concentration required to decrease the rate of reaction by 50% in 1 min under the assay conditions . the reaction mixture contained 1.0 ml phosphate buffer ( ph 7.0 ) , 0.1 ml of tissue homogenate ( supernatant ) and 0.4 ml of 0.2 m h2o2 . the reaction was stopped by the addition of 2.0 ml of dichromate - acetic acid reagent . colour intensity was measured colorimetrically at 620 nm and cat activity expressed as micromoles of h2o2 consumed / min / mg protein . the concentration of electrolytes like calcium , sodium and potassium in the brain was measured by using commercial kits by erba diagnostics ( for calcium ) and crest biosystems ( for sodium and potassium ) . the gaba level in serum the serum sample ( 100 l ) was added to 1.5 ml of absolute alcohol and centrifuged at 3000 g for 15 min . fifty microlitre of the sample was used for paper chromatography using the mobile phase containing n - butanol , glacial acetic acid and water . optical density of eluted sample was taken on a spectrophotometer at wavelength of 509 nm and compared with standard gaba solution . the gaba concentration in serum the paraffin sections were prepared and stained with haematoxylin and eosin and examined using light microscopy . resultswere analysed by one - way analysis of variance ( anova ) followed by dunnet 's multiple comparison test using graph pad prism software . overactivation of nmda receptors by the msg or sodium salt of glutamate exerts excitotoxicity . over activation of nmda along with other glutmate / glycine receptors disturb the calcium homeostasis , which is the key mediator of glutamate - induced excitotoxic neuronal damage . hence in the present study , we have used dextromethorphan against msg - induced neurotoxicity . the accumulation of high intracellular calcium together with increased level of sodium and decreased level of potassium intracellularly triggers a cascade of membrane , cytoplasmic and nuclear events leading to mitochondrial dysfunction and free radical generation resulting in neurotoxicity . in the present study , msg resulted in behavioural and physiological alterations like precipitation of aggressiveness , decreased locomotor activity and loss of muscle strength ( table 1 ) . this observation may be due loss in the hippocampal region of the brain as suggested by histopathological studies . this is known to result in increased levels of glutamate , which leads to aggressive behaviour . treatment with pp as well as standard drug , dextromethorphan did not exhibit any effect on msg - induced aggressiveness and hyperactiveness , which indicates that the msg might be producing behavioural changes through receptors other than nmda receptors . effect of pp on general behaviour , locomotor activity and muscle strength in msg - induced neurotoxicity the msg significantly decreased locomotor activity compared with the normal animals ( table 1 ) . treatment with pp at the dose of 200 and 400 mg / kg as well as standard drug , dextromethorphan significantly reversed the msg - induced decrease in locomotor activity score . the msg is known to impair locomotor activity by causing the damage to the dopaminergic neurons by generating free radicals . dextromethorphan and pp might be overcoming the deleterious effect of msg by protecting the damage by free radicals . treatment with msg significantly decreased the time to fall from the rotating rod compared with normal group ( table 1 ) . this effect might be due to over activation of glutmate pathway leading to neurotoxocity and counter effects on the release of gaba . this is reflected in the decreased level of gaba when treated with msg ( table 2 ) . treatment with pp at the both doses as well as standard drug dextromethorphan significantly increased the time to fall from the rotating rod compared with msg treated group ( table 1 ) . thus , the reversal of msg - induced loss of muscle grip activity with the treatment of pp may be attributed to its antioxidant activity , which provides protection against neurotoxicity as well as its central gaba activity . effect of pp on calcium , sodium , potassium and gaba levels in msg - induced neurotoxicity it is a well - known fact that the reactive oxygen species are involved in the pathogenesis of msg - induced neurotoxicity . in the present study , increase in brain tissue mda level along with decrease in levels of gsh , sod and cat is considered as an indication of the oxidative stress and neuronal damage . significant increase in the level of lipid peroxide ( lpo ) was found in msg treated animals compared with normal animals . the lpo measured by thiobarbituric reactive substances ( tbars ) has been generally accepted as an indicator of oxidative stress . increase in lpo level results in loss of function and integrity of neuronal membranes , which increases nonspecific permeability to ions leading to disruption of membrane structure . the treatment with dextromethorphan and pp dose dependently decreased the lpo levels compared with msg treated group ( table 3 ) , which suggests a protective effect of pp against msg - induced oxidative stress . effect of pp on glutathione , lipid peroxidation catalase and superoxide dismutase level in msg - induced neurotoxicity animals treated with msg significantly decreased the level of gsh compared with normal animals . the gsh is a major scavenger of free radicals in cytoplasm and an important inhibitor of free radical mediated lipid peroxidation . excitotoxicity - induced depletion of glutathione in rat brain tissue is due to the breakdown of the glutamate - cystine antiporter system and thereby reducing the level of gsh as observed in the present study in msg - treated animals . the gsh level was significantly increased in dextromethorphan and pp treated animals at both lower and higher doses compared with msg treated group ( table 3 ) indicating the protection of the neurons against msg - induced neurotoxicity by restoring gsh level . the sod and cat levels were significantly lower in msg treated group compared with normal group . the sod protects the cell against free radical injury by converting o2 radical to h2o2 and prevents the formation of oh radicals through o2 driven fenton reaction . hence , if the activity of cat is not adequate to degrade h2o2 , more h2o2 is converted into toxic hydroxyl radicals . in the present study , significant increase in the sod and cat levels were found in pp treated groups at the dose of 200 and 400 mg / kg and dextromethorphan compared with msg treated group ( table 3 ) . thus , the protective mechanism of pp may be due to its potent antioxidant property . excitotoxicity is a result of over activation of glutamate receptors , that is nmda , ampa and kars leading to increased ca and na levels and decreased k level in the cells resulting to cellular swelling and neuronal death . in msg treated group , the levels of calcium and sodium ions were significantly elevated and that of potassium ions significantly decreased compared with normal group indicating neurotoxicity . the groups treated with pp at the dose of 200 and 400 mg / kg and dextromethorphan significantly decreased the level of ca and na levels and significantly increased the level of k compared with msg treated group and brought the level of these ions near to normal level ( table 2 ) . the gaba level in msg treated group was significantly decreased compared with normal group , which may be due to inhibition of gaba synthesis or destruction of gabaergic neurons . the groups treated with pp at the dose of 400 mg / kg and dextromethorphan significantly increased the level of gaba level compared with msg treated group ( table 2 ) . further , dextromethorphan was used as a standard drug in msg - induced neurotoxicity model . the dextromethorphan is an nmda receptor antagonist and has been reported to possess neuroprotective property . over activation of nmda receptors is responsible for the alteration of these electrolyte levels hence , nmda receptor antagonists may be beneficial in maintaining the levels of these ions . in the present study , both pp and dextromethorphan were found to be effective in maintaining the levels of ca , na and k in the brain indicating that the pp and dextromethorphan act similarly . the results of histopathological study showed that there are no morphological changes of the brain in the normal animals . treated group , marked cerebral oedema , neuronal eosinophilia , nuclear pyknosis and neuronal karyorrhexis were observed . there was no an astrocytic and oligodendroglial change as well as reduced incidence of neuronal eosinophilia , nuclear pyknosis and neuronal karyorrhexis , thereby pp prevented hippocampal damage ( fig . the present study showed the neuroprotective effect of pp , which could be due to the various phytoconstituents such as flavonoids ( furanoflavonoids ) and chalcones as the plants containing these phytoconstituents have been reported to be beneficial in neurodegenerative diseases . it can be concluded that pp exerts neuroprotective property , which may be due to furanoflavonoids or chalcones possessing its potent antioxidant property and either gabaergic or nmda receptor antagonising property . histopathological changes in brain with respect to glial cell proliferation in msg - induced neurotoxicity model exhibited by ( a ) normal , ( b ) msg , ( c ) msg+pp ( 200 mg / kg ) ( d ) msg+pp ( 400 mg / kg ) and e ) msg+dxmp treatment . histopathological changes of hippocampal region of brain in msg - induced neurotoxicity model exhibited by ( a ) normal , ( b ) msg , ( c ) msg+pp ( 200 mg / kg ) ( d ) msg+pp ( 400 mg / kg ) and ( e ) msg+dxmp treatment .
this study was designed to evaluate the neuroprotective activity of ethanol extract of pongamia pinnata stem bark in monosodium glutamate - induced neurotoxicity in rats . 
 neurotoxicity was induced by intraperitoneal injection of monosodium glutamate 2 g per kg body weight daily for 7 days . 
 ethanol extract of pongamia pinnata stem bark ( 200 and 400 mg / kg ) was administered orally after 1 h of monosodium glutamate treatment . 
 dextromethorphan ( 30 mg / kg , p.o . ) was used as standard drug for the comparison . 
 the degree of protection was determined by various behavioural , locomotor , muscle grip activity , lipid peroxidation and measurement of antioxidant status of glutathione , catalase and superoxide dismutase . 
 estimation of calcium , sodium and potassium ions in brain tissue and gamma aminobutyric acid level in serum was carried out . 
 the histopathological study of brain tissue was also carried out . 
 treatment with pongamia pinnata significantly improved monosodium glutamate - induced alteration in behavioural and locomotor activity and muscle strength . 
 significant decrease in lipid peroxidation and increase in glutathione , superoxide dismutase and catalase was observed in pongamia pinnata treated group . 
 further , pongamia pinnata also significantly reduced the monosodium glutamate - induced excitotoxicity by decreasing the level of ca+2 and na+ with concomitant increase in the level of k+ . 
 serum gamma aminobutyric acid level was also increased in pongamia pinnata treated animals . 
 further , the histopathological evidence supports the neuroprotective activity of pongamia pinnata . in conclusion 
 , the present study suggests that the ethanol extract of stem bark of pongamia pinnata possesses significant neuroprotective activity in albino rats .