it is characterized by a complex of neuropathological , biochemical , and behavioral symptoms , that gradually impair memory and the ability to learn , carry out daily activities and behavior .
in particular , ibotenic acid - induced lesions of the nbm in rats produce impairment in acquisition and retention phase of passive avoidance tasks .
studies have shown that nbm has been widely involved in the pathogenesis of ad and is accompanied with cognitive deficits . a number of studies . suggested that exercise in aged rats improves learning and neurogenesis .
so it may slow the onset and progression of learning and memory deficit in ad .
it is proved that regular exercise attenuates motor deficits , increases formation of new neurons , and ameliorates neurological impairment in several neurodegenerative diseases.[1214 ] some studies on transgenic mouse models of ad have showed beneficial effects of exercise on general activity , spontaneous alternation , object recognition memory and , spatial learning performance .
some animal studies have revealed that treadmill running improves spatial and passive avoidance learning in nbm - lesion rats . in previous studies , transgenic and nbm - lesion models , they investigated the effects of physical activity after onset of ad in these models and , there have been no basic researches performed to estimate the preventive effects of exercise .
the aims of this study were to investigate preventive effects of treadmill running in nbm - lesion rats on learning and memory deficit by passive avoidance task in order to examine whether treadmill running can prevent learning impairment in this model of ad .
male wistar rats ( 250 - 300 g ; n = 54 ) were obtained from jondishapour university , ahwaz , iran ) .
the animals were kept in animal house and provided with food and water ad libitum and experienced a 12:12-h light - dark cycle ( 07:00 - 19:00 ) in a temperature controlled environment ( 22  2c ) .
this study was approved by the ethics committee for animal experiments at isfahan university approved the study , and all experiments were conducted in accordance with the international guiding principles for biomedical research involving animals , which was revised in 1985 .
rats were randomly allocated to the following groups : 
 control group ( co ; n = 11 ) : no injection , no exercise.sham operation ( sh ; n = 10 ) : saline ( drug solvent ) was injected in nbm.alzheimer group ( a ; n = 11 ) : ibotenic acid was injected in nbm.exercise before alzheimer group ( e - a ; n = 12 ) : ibotenic acid was injected bilaterally in nbm , and then rats exercised 21 days.exercise group ( e ; n = 10 ) : rats exercised 21 days . 
 
. sham operation ( sh ; n = 10 ) : saline ( drug solvent ) was injected in nbm .
alzheimer group ( a ; n = 11 ) : ibotenic acid was injected in nbm .
exercise before alzheimer group ( e - a ; n = 12 ) : ibotenic acid was injected bilaterally in nbm , and then rats exercised 21 days . exercise group ( e ; n = 10 ) : rats exercised 21 days .
rats were anesthetized with chloral hydrate ( 450 mg / kg , i.p ) and then placed in a stoelting stereotaxic apparatus ( incisor bar  3.3 mm , ear bars positioned symmetrically ) . the scalp was cleaned and incised on the midline and a burr hole was drilled through the skull and ibotenic acid ( cat number 12765 , sigma ) was injected at coordinates of : ap = 1.2 , ml = 3.2 , dv = 7.5 mm from surface skull .
10 g/l of ibotenic acid was injected ( 5 g/l each sides ) with microinjection pump at the speed of 120  lit / h . instead of ibotenic acid solution ,
post - operatively , the rats were given special care until spontaneous feeding was restored .
behavioral tests were conducted four weeks after the surgery and were evaluated blind to the treatments by the observer .
rats in the exercise group run on a treadmill at a speed of 20 - 21 m / min for 60 min daily ( 6 days a week ) , for 3 weeks at  inclination . to familiarize animals with the experimental set up , the treadmill
was switched on and the speed was increased from 5 to 21 m / min and over the course of 6 days , the duration was increased from 10 to 60 min . when exercising rats moved back on the treadmill , electric shocks were sparingly used to impel the animal to run . from week 2 onwards , speed and duration were kept constant at 20 - 21 m / min , 60 min per run after warm up .
the non - runners groups were not put on the treadmill for the same duration of running as runners did .
after 21 days , rats in all exercise groups , were subjected to passive avoidance learning ( pal ) test .
the training apparatus had two compartments comprising a small chamber ( 25  25  20 cm ) and a large dark compartment ( 50  25  20 cm ) .
electric shocks were delivered to the grid floor by an isolated stimulator . at the beginning of the test
, each rat was placed in the apparatus for 5 min to become habituated . on the second day , an acquisition trial was performed ; rats were placed individually in the illuminated chamber . after a habituation period ( 1 min )
then after the rat entered the dark chamber , the door was lowered and an inescapable scrambled single electric shock ( 75v , 0.2 ma , 50hz ) was delivered for 3 second .
latency to cross the dark compartment ( i.e. , pre - shock latency ) was recorded .
after exposure to the foot shock , the rat was removed from the passive avoidance apparatus to its home cage .
the rat was placed in the lighted ( safe ) compartment again with access to the dark compartment without any shock .
the latency to enter the dark compartment was measured ( i.e. , testing latency ) up to a maximum of 300 seconds . in the passive avoidance test , all results were compared using a kruskal - wallis nonparametric one - way analysis of variance corrected for ties , followed by a two - tailed mann - whitney u test .
the comparisons of retention time 24 h ( within groups ) were analyzed by friedman test , followed by a wilcoxon signed ranks test .
after the completion of behavioral tests , the rats were sacrificed and brains were removed and fixed in formalin , and then were sectioned to verify ibotenic acid injection site .
the lesions were reconstructed on standardized sections of the rat brain [ figure 1 ] .
male wistar rats ( 250 - 300 g ; n = 54 ) were obtained from jondishapour university , ahwaz , iran ) .
the animals were kept in animal house and provided with food and water ad libitum and experienced a 12:12-h light - dark cycle ( 07:00 - 19:00 ) in a temperature controlled environment ( 22  2c ) .
this study was approved by the ethics committee for animal experiments at isfahan university approved the study , and all experiments were conducted in accordance with the international guiding principles for biomedical research involving animals , which was revised in 1985 .
rats were randomly allocated to the following groups : 
 control group ( co ; n = 11 ) : no injection , no exercise.sham operation ( sh ; n = 10 ) : saline ( drug solvent ) was injected in nbm.alzheimer group ( a ; n = 11 ) : ibotenic acid was injected in nbm.exercise before alzheimer group ( e - a ; n = 12 ) : ibotenic acid was injected bilaterally in nbm , and then rats exercised 21 days.exercise group ( e ; n = 10 ) : rats exercised 21 days . 
 
. sham operation ( sh ; n = 10 ) : saline ( drug solvent ) was injected in nbm .
alzheimer group ( a ; n = 11 ) : ibotenic acid was injected in nbm .
exercise before alzheimer group ( e - a ; n = 12 ) : ibotenic acid was injected bilaterally in nbm , and then rats exercised 21 days . exercise group ( e ; n = 10 ) : rats exercised 21 days .
rats were anesthetized with chloral hydrate ( 450 mg / kg , i.p ) and then placed in a stoelting stereotaxic apparatus ( incisor bar  3.3 mm , ear bars positioned symmetrically ) . the scalp was cleaned and incised on the midline and a burr hole was drilled through the skull and ibotenic acid ( cat number 12765 , sigma ) was injected at coordinates of : ap = 1.2 , ml = 3.2 , dv = 7.5 mm from surface skull .
10 g/l of ibotenic acid was injected ( 5 g/l each sides ) with microinjection pump at the speed of 120  lit / h . instead of ibotenic acid solution ,
post - operatively , the rats were given special care until spontaneous feeding was restored .
behavioral tests were conducted four weeks after the surgery and were evaluated blind to the treatments by the observer .
rats in the exercise group run on a treadmill at a speed of 20 - 21 m / min for 60 min daily ( 6 days a week ) , for 3 weeks at  inclination . to familiarize animals with the experimental set up ,
the treadmill was switched on and the speed was increased from 5 to 21 m / min and over the course of 6 days , the duration was increased from 10 to 60 min . when exercising rats moved back on the treadmill , electric shocks were sparingly used to impel the animal to run . from week 2 onwards , speed and duration were kept constant at 20 - 21 m / min , 60 min per run after warm up . the non - runners groups were not put on the treadmill for the same duration of running as runners did .
after 21 days , rats in all exercise groups , were subjected to passive avoidance learning ( pal ) test .
the training apparatus had two compartments comprising a small chamber ( 25  25  20 cm ) and a large dark compartment ( 50  25  20 cm ) .
electric shocks were delivered to the grid floor by an isolated stimulator . at the beginning of the test
, each rat was placed in the apparatus for 5 min to become habituated . on the second day , an acquisition trial was performed ; rats were placed individually in the illuminated chamber . after a habituation period ( 1 min ) , the guillotine door was lifted . then after the rat entered the dark chamber , the door was lowered and an inescapable scrambled single electric shock ( 75v , 0.2 ma , 50hz ) was delivered for 3 second .
latency to cross the dark compartment ( i.e. , pre - shock latency ) was recorded .
after exposure to the foot shock , the rat was removed from the passive avoidance apparatus to its home cage .
the rat was placed in the lighted ( safe ) compartment again with access to the dark compartment without any shock .
the latency to enter the dark compartment was measured ( i.e. , testing latency ) up to a maximum of 300 seconds .
in the passive avoidance test , all results were compared using a kruskal - wallis nonparametric one - way analysis of variance corrected for ties , followed by a two - tailed mann - whitney u test .
the comparisons of retention time 24 h ( within groups ) were analyzed by friedman test , followed by a wilcoxon signed ranks test .
after the completion of behavioral tests , the rats were sacrificed and brains were removed and fixed in formalin , and then were sectioned to verify ibotenic acid injection site .
the lesions were reconstructed on standardized sections of the rat brain [ figure 1 ] .
the latency was measured in pre foot shock ( acquisition time ) and 24 h post foot shocks ( retention time ) .
results indicated that the pre - shock latency was the same among all groups , but compared to group alzheimer ( a ) as well as between group a and group exercise - alzheimer ( e - a ) , acquisition time was longer in control ( co ) group ( p < 0.01 , p < 0.05 respectively ) . there was a significant difference between group exercise ( e ) and a ( p < 0.01 ) , as well as group sham ( sh ) and a [ p < 0.05 , figure 2 ] .
comparison of latency to enter the dark chamber before receiving foot shock ( acquisition time ) .
there were significant differences between control ( n= 11 ) and a ( n= 11 ) group ( p < 0.01 ) , as well as a and e ( n= 12 ) group ( p < 0.01 ) .
there were significant differences between a and others ( sh and e - a groups , p < 0.05 ) .
all results were analyzed by kruskal - wallis test followed by mann - whitney u test however , the retention time during testing ( i.e. , testing latency carried out 24 h after receiving foot shock ) was significantly decreased in group a when compared to other groups ( co , sh , e and groups e - a ; p < 0.001 , p < 0.01 , p < 0.001 , p < 0.001 respectively ) .
the retention time changes were not significant between group e and group sh . also , there were not significant differences in retention time between control group , as well as sh and e - a groups [ figure 3 ] .
comparison of latency to enter the dark chamber 24 h after receiving foot shock ( the retention time ) .
the retention time was significantly decreased in the a group compared to others ( control , sh and e - a group , p < 0.001 , p < 0.01 , p < 0.001 respectively ) .
all results were analyzed by kruskal - wallis test followed by mann - whitney u test the results of pre foot shock and post foot shock latency were analyzed by a paired sample to evaluate changes within groups . in this part
, our data showed that there were significant differences in pre and post foot shock latency among all groups [ p < 0.01 , figure 4 ] .
there were significant differences in pre and post foot shock latency in all groups ( p < 0.01 )
the latency was measured in pre foot shock ( acquisition time ) and 24 h post foot shocks ( retention time ) .
results indicated that the pre - shock latency was the same among all groups , but compared to group alzheimer ( a ) as well as between group a and group exercise - alzheimer ( e - a ) , acquisition time was longer in control ( co ) group ( p < 0.01 , p < 0.05 respectively ) . there was a significant difference between group exercise ( e ) and a ( p < 0.01 ) , as well as group sham ( sh ) and a [ p < 0.05 , figure 2 ] .
comparison of latency to enter the dark chamber before receiving foot shock ( acquisition time ) .
there were significant differences between control ( n= 11 ) and a ( n= 11 ) group ( p < 0.01 ) , as well as a and e ( n= 12 ) group ( p < 0.01 ) .
there were significant differences between a and others ( sh and e - a groups , p < 0.05 ) .
all results were analyzed by kruskal - wallis test followed by mann - whitney u test however , the retention time during testing ( i.e. , testing latency carried out 24 h after receiving foot shock ) was significantly decreased in group a when compared to other groups ( co , sh , e and groups e - a ; p < 0.001 , p < 0.01 , p < 0.001 , p < 0.001 respectively ) .
the retention time changes were not significant between group e and group sh . also , there were not significant differences in retention time between control group , as well as sh and e - a groups [ figure 3 ] .
comparison of latency to enter the dark chamber 24 h after receiving foot shock ( the retention time ) .
the retention time was significantly decreased in the a group compared to others ( control , sh and e - a group , p < 0.001 , p < 0.01 , p < 0.001 respectively ) .
all results were analyzed by kruskal - wallis test followed by mann - whitney u test the results of pre foot shock and post foot shock latency were analyzed by a paired sample to evaluate changes within groups . in this part
, our data showed that there were significant differences in pre and post foot shock latency among all groups [ p < 0.01 , figure 4 ] .
there were significant differences in pre and post foot shock latency in all groups ( p < 0.01 )
in the present study , the passive avoidance retention and retrieval function was impaired by ibotenic acid nbm - lesions .
our results showed that treadmill exercise training significantly improves passive avoidance performance in normal and nbm - lesion rats [ figures 2 and 3 ] .
chopin et al . demonstrated significant performance deficits in both passive avoidance and morris water maze tests in bilateral nbm - lesion rats .
they showed that regular exercise significantly attenuated lesion - associated decrease in brain functions . in these studies , animals exercised after onset ad in this animal model . in this research , relationship between treadmill running and preventive effects on learning and memory deficits observed in nbm - lesion rats were investigated .
our results indicated that in comparison to nbm - lesion rats , learning and memory in groups e - a were improved that is , exercise has preventive effects on acquisition and retention time impairment in passive avoidance test in nbm - lesion [ figures 2 and 3 ] .
furthermore , it will postpone memory impairments that may result from some exercise mechanisms such as increase in dopamine and muscarinic receptor density , acetylcholine level , neurotransmitter release in the hippocampus , brain derived neurotrophic factor , bdnf gene expression , neuron proliferation and survival in the animal 's brain . similarly , van praag et al .
also , other researchers suggested that exercise training ( swimming ) increased memory of rats in passive avoidance test , but such increase was temporary after stopping exercise because swimming requires physical effort and compared to treadmill running in rats , was known as an effective stressor . according to our results , treadmill running had beneficial effects on encountering learning and memory deficits with nbm - lesion as animal model of ad and probably can reduce dementia in patients .
exercise effects might be the result of structural and biological changes in brain , which enhance neuron numbers .
a cell proliferation increase or cell death decrease increases the length and number of dendrites connection between neurons , as well as synaptic plasticity in hippocampus , which is involved in learning and memory.[253234 ] it has been suggested that mechanisms for some mentioned effects of exercise including gene expression , increment of neurotrophic factors such as brain derived neurotrophic factor and insulin - like growth factor i , which are important for neuronal survival and differentiation , as well as synaptic plasticity.[3537 ] hence , all of the aforesaid factors may have a pivotal role in learning and memory enhancement by exercise .
our results at the behavioral level ( passive avoidance test ) emphasize the role of treadmill running in the prevention of learning and memory impairments in nbm - lesion rats .
memory deficit caused by nbm lesion was also reversed by treadmill running , suggesting enhancement of learning and memory functions through physical activity .
 background : alzheimer 's disease was known as a progressive neurodegenerative disorder in the elderly and is characterized by dementia and severe neuronal loss in the some regions of brain such as nucleus basalis magnocellularis . 
 it plays an important role in the brain functions such as learning and memory . 
 loss of cholinergic neurons of nucleus basalis magnocellularis by ibotenic acid can commonly be regarded as a suitable model of alzheimer 's disease . 
 previous studies reported that exercise training may slow down the onset and progression of memory deficit in neurodegenerative disorders . 
 this research investigates the effects of treadmill running on acquisition and retention time of passive avoidance deficits induced by ibotenic acid nucleus basalis magnocellularis lesion.methods:male wistar rats were randomly selected and divided into five groups as follows : control , sham , alzheimer , exercise before alzheimer , and exercise groups . 
 treadmill running had a 21 day period and alzheimer was induced by 5 g/l bilateral injection of ibotenic acid in nucleus basalis magnocellularis.results:our results showed that ibotenic acid lesions significantly impaired passive avoidance acquisition ( p < 0.01 ) and retention ( p < 0.001 ) performance , while treadmill running exercise significantly ( p < 0.001 ) improved passive avoidance learning in nbm - lesion rats.conclusion:treadmill running has a potential role in the prevention of learning and memory impairments in nbm - lesion rats . 
INTRODUCTION
METHODS
Animals
Exercise protocol
Passive avoidance learning test
Statistical analysis
Histology
RESULTS
Passive avoidance test in rats
DISCUSSION
CONCLUSION