oxidative stress refers to the cytotoxic consequences of reactive oxygen species ( ros ) , which are generated as byproducts of normal and aberrant metabolic processes that use molecular oxygen . the tripeptide glutathione ( gsh ; -glutamyl - cysteinyl - glycine ) is one of the most abundant cellular thiols . gsh is a major player in cellular defense against ros , because it nonenzymatically scavenges both singlet oxygen and hydroxyl radicals , and is used by glutathione peroxidases and glutathione transferases to limit the levels of certain reactive aldehydes and peroxides within the cell [ 1 , 2 ] . when ros production exceeds the antioxidant defense capacity of the cell , oxidative stress ensues , leading to the damage of dna , proteins , and membrane lipids . the first and the rate - limiting step in the synthesis of gsh is carried out by glutamate - cysteine ligase ( gcl ; ) . the enzyme consists of two subunits , a larger ( 73  kd ) catalytic subunit ( gclc ) and a smaller ( 31  kd ) modifier , or regulatory , subunit ( gclm ) , which are coded by separate genes . gclc alone provides catalytic activity and is the site of gsh feedback inhibition . by lowering the km of gcl for glutamate and raising the ki for gsh , gclm , although enzymatically inactive , plays an important regulatory function , as the holoenzyme ( gclholo ) has higher catalytic efficiency than gclc [ 3 , 4 ] . while disruption of the gclc gene in mice is embryolethal , no overt phenotype is observed upon disruption of the gclm gene in mice [ 4 , 6 , 7 ] . in the absence of gclm , the ability of gclc to synthesize gsh is drastically reduced . in tissues from gclm mice , gsh levels are only 1030% of those found in gclm animals [ 6 , 7 , 9 ] . in brain tissue and cells , gsh levels in gclm mice are 1735% of those present in wild - type mice [ 6 , 10 , 11 ] . gclm mice are more sensitive to the hepatotoxicity of acetaminophen , and neurons and astrocytes isolated from the brain of gclm mice have been shown to be particularly susceptible to the toxicity of agents that increase oxidative stress , such as domoic acid , certain organophosphorus insecticides , methylmercury and pcbs , and polybrominated diphenyl ethers . a relatively common c588 t polymorphism has been discovered in the 5-flanking region of the human gclm gene . individuals carrying the t allele have a lower promoter activity in a luciferase reporter gene assay in response to oxidants and significantly lower plasma gsh levels . these individuals are also at risk for myocardial infarction and present with impairments in nitric oxide - mediated coronary vasomotor function [ 14 , 15 ] . an association between gclm polymorphisms and schizophrenia has also been suggested   but is still controversial [ 17 , 18 ] . nevertheless , individuals with gclm polymorphisms leading to lower gsh levels would be expected to display an enhanced sensitivity to the adverse effects of oxidative stress . the gclm mouse thus represents a useful model for such gclm polymorphisms , amenable for in vitro , as well as in vivo studies . in order to extend in vitro observations to an in vivo situation , an initial behavioral characterization of gclm mice was carried out , to determine whether the genetically determined diminished gsh level would by itself affect behavioral outcomes . indeed , glutathione dysregulation is associated with the etiology and progression of several diseases , including neurotoxic and neurodegenerative disorders [ 19 , 20 ] . all animal use protocols were approved by the institutional animal care and use committee at the university of washington , and experiments were carried out in accordance with the national research council guide for the care and use of laboratory animals , as adopted by the national institutes of health . wild - type and gclm - null ( gclm ) mice of backcrossed c57bl/6j ( b6.129 ) strain background [ 6 , 7 ] were housed in a centralized , aaalac - accredited , and specific pathogen - free facility at the university of washington . mice were maintained in a 12  h light - dark cycle with ad libitum access to food ( standard mouse chow ) and water . male and female mice hemizygous for the gclm deletion ( gclm - hz ) were intercrossed , generating wild - type , gclm , and gclm - hz mice , in the expected mendelian ratios . to genotype pups , genomic dna was isolated from ear punch tissue using a qiagen dneasy kit , and mice were genotyped by pcr amplification of the wild - type and disrupted gclm alleles ( i.e. , amplification of -geo ) , as previously described [ 6 , 7 ] . as seen previously , all pups developed normally and exhibited no differences in phenotypic landmarks compared to wild - type littermates .   at weaning , mice were transferred to the neurobehavioral testing facility and housed two to four per cage for the duration of testing . one wild - type ( total = 12 ) or gclm ( total = 13 ) male mouse , each taken from a different litter , was used for neurobehavioral testing . tests were chosen to investigate possible differences between the two mouse genotypes in sensory functions , motor activity and coordination , and learning and memory . auditory startle and prepulse inhibition of startle were tested at 12 weeks of age using an automated auditory startle chamber ( san diego instruments ) .   during a 15-minute test session , mice were placed in the startle chamber and presented with 30 stimuli at randomized intervals . the stimuli consisted of a 120  db tone , a 120  db tone preceded by a 70  db prepulse , or a  null  stimulus involving no tone . the order of stimulus presentation was first determined using a random number table , after which each mouse received the stimuli in the same order . the startle chambers used a piezoelectric sensor to measure the maximum amplitude ( vmax ) of the startle response after each stimulus and the latency to the maximum startle response ( tmax ) . prepulse inhibition of startle was calculated as the percent inhibition of the auditory startle response by the 70  db prepulse , after subtracting the startle response to the null stimulus [ 22 , 23 ] . a rotarod ( coulbourn instruments ) was used to test motor coordination and cerebellar learning [ 24 , 25 ] at 13 weeks of age . mice were placed on the rotarod cylinder , which accelerated to 5  rpm / min from a baseline rate of 3  rpm . latency to fall off the cylinder was recorded for each of four successive trials , with a 5  min intertrial interval . open - field behavior and locomotor activity were tested at 20 weeks of age using a tru - scan photo beam tracking system ( coulbourn instruments , whitehall pa ) . mice were placed in an open - field arena that was 25.4  cm wide , 25.4  cm deep , and 40.64  cm high , and movements and behaviors were recorded for 15  min using dual sensor rings to measure infrared beam breaks in the horizontal or vertical plane . beams were spaced 1.52  cm apart , providing 0.76  cm spatial resolution . data were collected in 30  sec bins , and totals over the 15  min testing period were calculated as the sum of the 30 individual values . data were also analyzed individually for the first 5  min period , the second 5-min period , and the third 5  min period . specific measures included total number of movements , total movement time , total rest time , ambulatory move time , latency to first movement , latency to first ambulatory movement , total movement distance , ambulatory distance , mean velocity , ambulatory velocity , distance traveled in arena margin and arena center , time spent in arena margin and arena center , number of entries into arena center , time spent in back half and front half of arena , number of entries into back half and front half of arena , number of entries into vertical plane , time spent in vertical plane , number of jumps from floor plane , number of movements in vertical plane , number of stereotypic movements , number of stereotypic episodes , total time of stereotypic behavior , and number of counterclockwise and clockwise center point rotations . data were collected automatically using tru - scan 2.0 software , and raw data were exported for analysis by microsoft excel . a platform - reversal variant of the morris water maze was used to test learning and memory [ 2628 ] beginning at 14 weeks of age . the maze consisted of a 165-gallon ( 624.6  liter ) , circular , galvanized stock tank , 4  ft ( 1.22  meter ) in diameter and 2  ft ( 0.61  meter ) in height , filled with room temperature water .   a 10  cm square plexiglass stand was placed in the tank just below the water level to serve as the escape platform . a polytrack system ( san diego instruments ) was used to track the location of the mice in the maze . mice were trained for seven days , three trials per day , at 30  min intertrial intervals , to acquire the task . on the first trial , mice were dropped randomly at one of the four drop locations and allowed to explore the tank and become familiar with swimming . mice were then guided to the escape platform and were held on the platform for 30 seconds , and then they were taken out of the tank , dried off , and placed under a heat lamp .   on subsequent trials , mice were dropped into the tank and given 60 seconds to find the platform .   once the mice found and climbed onto the platform , the test was stopped and the latency to find the platform was recorded . after the 21-trial acquisition phase , the platform was moved to the opposite quadrant , and mice were tested for an additional 21 trials , with 3 trials per day separated by a 30  min intertrial interval . latency to find the platform was measured as above .   one month following the last reversal trial , the platform was removed , and mice were placed into the tank at a random drop location and allowed to swim for 2 minutes . dwell time in each quadrant , average distance from target ( previous location of platform ) , and number of target crossings were recorded . differences between genotypes were tested for statistical significance by student 's   t - test , followed in some cases by a bonferroni correction for multiple testing . as previously reported [ 6 , 7 ] , gclm mice and wild - type mice were born in the expected mendelian ratios and exhibited no obvious developmental differences during the postnatal developmental period . adult male and female gclm mice had slightly lower bodyweights than wild - type mice ( table 1 ) . only male mice were used for behavioral testing . at 13 weeks of age , motor coordination and cerebellar learning were tested using a rotarod ( figure 1 ) . wild - type and gclm mice both learned the task , and latency to fall off the rotarod increased with each successive trial ( trial 1 versus trials 2 , 3 , and 4 : wild - type , p < .05 ; gclm , p < .01 ) . there were no significant differences in latency between gclm and wild - type mice on trials one ( p = .27 ) , two ( p = .24 ) , three ( p = .13 ) , or four ( p = .60 ) , nor when averaging latencies across all four trials ( p = .42 ) . auditory startle and prepulse inhibition of startle were tested at 12 weeks of age ( figure 2 ) . there were no differences in auditory startle amplitude ( p = .27 ) between wild - type and gclm mice . when the 120  db auditory stimulus was preceded by a 70  db prepulse , the startle amplitude was significantly reduced ( p < .05 ) in both wild - type and gclm mice ( figure 2 ) . there were no differences between wild - type and gclm mice in the magnitude of this prepulse inhibition of startle ( p = .28 ) . latencies to maximum startle were also similar in wild - type and gclm mice for both auditory startle ( 22.6  1.8  msec and 20.8  1.5  msec , resp . ; p = .46 ) and prepulse inhibition of startle ( 31.9  4.2  msec and 25.9  1.8  msec , resp . ; locomotor activity and behavior in an open field were tested at 20 weeks of age , by placing the mice in a small arena and recording movements and behaviors for 15  min ( figure 3 ) . gclm and wild - type mice were nearly identical in all measures ( p > .18 ) , including locomotor activity , total movement time , ambulatory distance traveled , time spent in different areas of the arena , number of stereotypic movements , and number of entries into the vertical plane ( figure 3 ) . learning and memory were tested using a platform - reversal variant of the morris water maze , beginning at 14 weeks of age . gclm mice acquired the task equally as well as wild - type mice ; latency to find the hidden submerged platform decreased ( p < .001 ) over the seven daily testing sessions for both mouse genotypes ( figure 4 ) . platform reversal began on the eighth day , when the platform was moved to the opposite quadrant . both wild - type and gclm mice had difficulty finding the new platform location on the first day , reflected in their increased latencies to find the platform in session eight but were able to learn the new platform location over subsequent training sessions ( figure 4 ; p < .0001 ) . there were no significant differences between wild - type and gclm mice in either acquisition or reversal of the task ( p > .24 ) . while wild - type mice showed a tendency toward increased platform latency on session ten , when compared to gclm mice , this difference was not statistically significant ( p = .24 ) . one month after the end of the platform - reversal testing , retention was tested once for each mouse , using a probe test . mice were placed in the morris water maze and were allowed to search for two minutes in the absence of a platform . there were no differences between genotypes ( p > .11 ) in percent dwell time in the platform quadrant ( wild - type : 23.9  1.9% ; gclm : 19.7  1.8% ) , percent dwell time in the reversal quadrant ( wild - type : 27.8  1.5% ; gclm : 34.0  3.4% ) , or in the average distance from target ( wild - type : 38.7  1.6  cm ; gclm : 35.7  1.6  cm ) . swim speed was also the same in wild - type ( 19.2  0.4  cm / sec ) and gclm ( 18.5  0.4  cm / sec ) mice . genetic deletion of gclm leads to low gsh levels in all tissues including the brain [ 6 , 911 ] . neurons and astrocytes from gclm mice have been shown to be particularly susceptible to the neurotoxic effects of various chemicals , such as domoic acid , polychlorinated biphenyls , methylmercury , certain organophosphorus insecticides , and polybrominated flame retardants , known to elicit oxidative stress [ 6 , 10 , 12 , 13 ] , and preliminary in vivo experiments with the flame retardant bde-47 ( 2,2,4,4-tetrabromodiphenyl ether ) have supported these findings ( giordano and costa , unpublished ) . in order to carry out in vivo exposure studies with such compounds , to ascertain their potential ability to disrupt normal behavior , a behavioral characterization of gclm mice was first needed . results of the present study indicate that gclm mice did not differ from wild - type mice in a number of behaviors aimed at testing spontaneous motor activity , motor coordination , learning and memory , and sensory functions . these tests are normally used in behavioral toxicology to assess adverse effects of chemicals on the nervous system upon adult or developmental exposure to chemicals . the lack of differences between gclm and wild - type mice in these tests would suggest that gsh deficiency does not lead to alterations in nervous system function significant enough to be detected . however , significant differences would be expected upon challenges with exogenous compounds , particularly those which elicit oxidative stress . as such , gclm mice would represent a good model to assess the importance of such gene - environment interactions leading to neurotoxicity . two additional explanations for the lack of differences observed between gclm mice and wild - type mice in the present study may lie in the types of behavioral tests utilized and in the age of the animals . whilst this work was in progress , these investigators not only confirmed some of our findings , but also evidenced additional subtle alterations . as in our study , no differences were found in spatial working memory , spatial reference and learning memory , and spatial reversal learning . however , in other tests , gclm mice ( 46 month - old ) displayed increased novelty - induced exploration , altered behavior during an object recognition task , reduced behavioral inhibition under stress , and lower response to delayed fear conditioning . these subtle behavioral alterations were attributed to oxidative changes in the ventral hippocampus , with no changes in the dorsal hippocampus , and were suggested to be potentially relevant in schizophrenia . however , while an association between gclm polymorphisms and schizophrenia had been previously suggested , more recent studies have challenged this hypothesis [ 17 , 18 ] . in addition , it is possible that there are subtle differences between these two gclm models , since the strategy for gene deletion was slightly different . in the model ,   the first exon of the gclm gene was replaced with a neomycin phosphotransferase ( neo ) in reverse orientation . in the model we have used here , the first exon was replaced with a beta - galactosidase / neomycin phosphotransferase ( -geo ) fusion gene in the forward orientation . while these two gclm models appear to have essentially identical changes in gsh levels , it is still possible that the two different approaches used to construct these models might have resulted in other unforeseen differences in their biology . mice tested in this study were young adults , between 13 and 20 weeks of age . it has been proposed that oxidative stress may contribute to aging by progressively increasing oxidant damage to cells [ 30 , 31 ] . in addition , oxidative stress has been suggested to be involved in neurodegenerative diseases , such as parkinson 's and alzheimer 's diseases and amyotrophic lateral sclerosis [ 19 , 20 , 32 ] . it has been recently shown that fibroblasts from gclm mice undergo premature senescence , as evidenced by altered cell morphology , diminished growth rate , and increased senescence - associated -galactosidase activity . thus , it is plausible that gclm would display altered behaviors , compared to their wild - type counterparts , as they age and thus become a useful model for studies on the effects of altered antioxidant capacity on the aging of the nervous system .
glutathione ( gsh ) is a major player in cellular defense against oxidative stress . 
 deletion of the modifier subunit of glutamate cysteine ligase ( gclm ) , the first and the rate - limiting enzyme in the synthesis of gsh , leads to significantly lower gsh levels in all tissues including the brain . 
 gclm - knockout ( gclm/ ) mice may thus represent a model for compromised response to oxidative stress amenable to in vitro and in vivo investigations . in order to determine whether the diminished gsh content would by itself cause behavioral alterations , a series of behavioral tests 
 were carried out comparing young adult gclm/ with wild - type mice . 
 tests included the rotarod , acoustic startle reflex and prepulse inhibition of the startle reflex , open field behavior , and the platform reversal variant of the morris water maze . 
 results showed no differences between gclm/ and wild - type mice in any of the neurobehavioral tests . 
 however , more subtle alterations , or changes which may appear as animals age , can not be excluded .