parkinson s disease ( pd ) is one of the most common neurodegenerative disorders and is 
 characterized by progressive loss of nigrostriatal dopaminergic neurons leading to dopamine 
 depletion and motor dysfunction deficits .
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) is known to cause pd - like symptoms by 
 damaging dopaminergic neurons in the substantia nigra ( sn ) , and has been used widely to 
 establish rodent models of pd2 .
the neurotransmitter dopamine plays a critical role in the cellular signaling processes 
 that underlie information transfer in neurons functioning in the nervous system .
dopamine 
 receptors ( dr ) belong to the g - protein - coupled receptor superfamily , and have five 
 isoforms2 .
dopamine receptor subtype 2 
 ( d2dr ) has affinity for dopamine and the dopamine receptor agonists used to treat pd3 .
it plays a major role in long - term 
 depression ( ltd ) , a form of synaptic plasticity that involves integration of glutamatergic 
 and dopaminergic neurotransmission and leads to motor function encoding in the dorsolateral 
 striatum4 .
marked inhibition of the d2dr 
 pathway in the basal ganglia - thalamus - cortical loop leads to parkinsonian features , loss of 
 motor skills , and decreased cell proliferation in the hippocampus5 , 6 .
adult hippocampal neurogenesis is positively affected by neurotrophic factors , such as 
 brain - derived neurotrophic factor ( bdnf ) , nerve growth factor ( ngf ) , and neurotrophin-3 
 ( nt-3 ) . among them , bdnf is involved in learning ability , memory function , and synaptic 
 plasticity7 , 8 .
bdnf is a neurotrophin implicated in neuronal survival and 
 plasticity and functions by binding to the high - affinity receptor , tyrosine kinase b ( trk 
 b)9 .
bdnf - trk b interaction promotes the 
 survival and differentiation of neurons , increases synaptic plasticity , inhibits apoptosis , 
 and improves cognition deficits .
inhibition of bdnf - trk b signaling suppresses neurogenesis 
 in the hippocampal dentate gyrus ( dg)10 .
physical exercise is currently advocated as a behavioral intervention to ameliorate 
 neurological impairment by impeding the neuronal loss that is caused by several 
 neurodegenerative diseases , including pd , and exercise is currently being proposed to have 
 many possible therapeutic benefits11 , 12 .
in addition , several studies have 
 demonstrated the benecial effect of exercise on functional outcomes in patients with 
 pd13 , 14 . in this study
, we investigated the effects of treadmill running on 
 long - term memory and hippocampal neurogenesis in an mptp - induced pd mouse model .
male icr mice weighing 45  3  g ( 10-week - old ) were used in this experiment .
all animal 
 experimental procedures conformed to national institutes of health ( nih ) and korean academy 
 of medical science guidelines .
animals were housed at a controlled room temperature ( 20  
 2  c ) and under a 12-hour light/12-hour dark cycle .
animals were randomly divided into four groups ( n = 10 in 
 each group ) : the probenecid group , probenecid - exercise group , mptp / probenecid ( mptp / p ) 
 group , and mptp / p- exercise group ( mptp / p - ex ) . to generate a chronic pd mouse model ,
the 
 mice were injected with a total of 10 doses of mptp hydrochloride ( 20  mg / kg in saline , s.c . ) 
 in combination with an adjuvant , probenecid ( 250  mg / kg in saline with sodium hydroxide 
 ( naoh ) , i.p . ) , over 5 weeks at 3.5 day intervals .
control mice were treated with the vehicle 
 ( probenecid ) in the same volume and on the same schedule .
mice in the exercise groups ran on 
 a treadmill after the last drug injection for 30  min / day , five times / week for 4 weeks at 
 speeds up to 10 m / min .
non - exercise group was placed on the treadmill ( exer-6 m , columbus , 
 oh , usa ) lane for the same duration as the exercised groups to expose them to similar 
 conditions , but they did not run on the treadmill .
the passive avoidance test was used to evaluate long - term memory after the last exercise 
 session .
the floor of the dark compartment consisted of 
 a stainless steel grid . during the training trial ,
after 30  s of habituation , the small door was opened . immediately after the 
 mouse entered the dark compartment , the small door was closed and
an electric foot shock was 
 delivered to the floor grids for 3 s. after 72 h , the test trial was performed .
the latency 
 was defined as the time taken to enter the dark compartment and place all four paws on the 
 grid .
any latency > 300  s was counted as 300 s. to measure protein expression , the striatum was dissected from deeply anesthetized mice 
 after the passive avoidance test .
sample 
 tissue was lysed in ice - cold lysate buffer and then the mixture was incubated for 30  min at 
 4  c .
protein aliquots ( 40 g ) were separated on sds - polyacrylamide gels and then 
 transferred onto a nitrocellulose membrane ( schleicher & schuell gmbh , dassel , germany ) 
 that was subsequently probed with the following primary antibodies : anti - tyrosine 
 hydroxylase ( th ) , anti - d2dr , anti - bdnf , anti - trk b ( 1:1000 ; santa cruz biotech , ca , usa ) . 

horseradish peroxidase - conjugated anti - rabbit antibody ( 1:3000 ; santa cruz biotech ) for th , 
 d2dr , bdnf , and trk b were used as secondary antibodies .
bands were detected using the 
 enhanced chemiluminescence ( ecl ) detection system ( santa cruz biotech ) . to detect the expression of th in the substantia nigra pars compacta ( snpc ) and striatum , 
 floating tissue sections were incubated in 3% h2o2 for 30  min and 
 pre - treated with a double detection solution ( brdu / neun and brdu / dcx ) .
after pre - treatment , 
 sections were blocked in solution include 1% bsa and 10% horse serum in 0.05  m pbs .
the 
 sections were then incubated with rat anti - mouse th antibody ( 1:1000 ; bd biosciences , san 
 jose , ca , usa ) and brdu antibody ( 1:300 , abcam , biomeda , ca , usa ) .
the sections were 
 incubated with biotinylated anti - mouse igg ( 1:200 ; vector laboratories , burlingame , ca , 
 usa ) .
after brdu staining , counter - staining was performed on the same sections using neun 
 antibody ( 1:300 ; chemicon international , temecula , ca , usa ) and dcx ( 1:200 ; santa cruz 
 biotech ) , respectively .
sections were incubated with a biotinylated anti - mouse igg ( 1:200 ; 
 vector laboratories ) , and processed with the vector elite abc kit ( vector 
 laboratories ) . for visualization ,
sections were reacted with 0.05% 3,3-diaminobenzidine 
 ( dab ) and 0.01% h2o2 in 0.05  m tris buffer ( ph 7.6 ) . between each 
 step ,
lastly , sections were 
 mounted on gelatin - coated slides and cover slipped with mounting medium .
all data were analyzed using an image - pro plus computer - assisted image analysis 
 system ( media cyberbetics inc . , silver spring , md , usa ) .
statistical analysis was performed 
 using one - way anova followed by duncan s post - hoc test , and the results are expressed as the 
 mean  standard error of the mean ( sem ) . the threshold for statistical significance was set 
 at p < 0.05 .
the latency in the passive avoidance task was 282.6  11.68  s , 276.16  16.06  s , 183.0  
 35.44  s , and 269.0  9.86  s in the probenecid , probenecid exercise , mptp / p , and 
 mptp / p - exercise groups , respectively ( p < 0.05 ) .
these results demonstrate that mptp / p 
 treatment decreases memory function and that treadmill exercise protects against this effect 
 ( table 1table 1.effects of treadmill exercise on long - term memory in the passive avoidance 
 testgroupprobenecidprobenecid - exmptp / pmptp / p - exlatency ( s)282.6  11.68276.16  16.06183.0  35.44269.0  9.86ex : exercise ; mptp / p : 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid ; s : 
 secondsp<0.05 compared to the probenecid group , p<0.05 
 compared to the mptp / p group ) .
ex : exercise ; mptp / p : 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid ; s : 
 seconds p<0.05 compared to the probenecid group , p<0.05 
 compared to the mptp / p group in the present study , th and d2dr expression in the ventral midbrain in the probenecid 
 group was set at 1.00 .
the level of th protein was 1.05  0.01 , 0.79  0.01 , and 1.18  0.01 
 in the probenecid , mptp / p , and mptp / p - exercise groups , respectively .
the level of d2dr 
 protein was 1.08  0.03 , 0.61  0.02 , and 0.77  0.02 in the probenecid exercise , mptp / p , 
 and mptp / p - exercise groups , respectively ( p < 0.05 ) ( table 2table 2.effects of treadmill exercise on protein expression ( unit : o.d.)groupprobenecidprobenecid - exmptp / pmptp / p - exth1.000.001.050.010.790.011.180.01d2dr1.000.001.080.030.610.020.770.02bdnf1.000.000.970.020.740.010.870.01trk b1.000.001.100.030.520.070.710.02bdnf : brain - derived neurotrophic factor ; d2dr : dopamine receptors subtype 2 ; ex : 
 exercise ; mptp / p : 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid ; o.d .
: 
 optical density ; th : tyrosine hydroxylase ; trk b : tyrosine kinase bp<0.05 compared to the probenecid group , p<0.05 
 compared to the mptp / p group ) .
bdnf : brain - derived neurotrophic factor ; d2dr : dopamine receptors subtype 2 ; ex : 
 exercise ; mptp / p : 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid ; o.d . : 
 optical density ; th : tyrosine hydroxylase ; trk b : tyrosine kinase b p<0.05 compared to the probenecid group , p<0.05 
 compared to the mptp / p group the number of brdu / dcx - positive cells in the hippocampus was 67.54  9.38/mm , 
 66.60  7.73/mm , 39.30  5.68/mm , and 61.05  7.31/mm in 
 the probenecid , probenecid exercise , mptp / p , and mptp / p - exercise groups , respectively .
the 
 number of brdu / neun - positive cells in the hippocampus was 41.46  5.05/mm , 48.20 
  5.40/mm , 26.63  3.21/mm , and 46.33  5.12/mm in the 
 probenecid , probenecid exercise , mptp / p , and mptp / p - exercise groups , respectively ( p < 
 0.05 ) ( table 3table 3.effects of treadmill exercise on neurogenesis in the hippocampal dentate gyrus 
 ( dg ) ( unit : mm)groupprobenecidprobenecid - exmptp / pmptp / p - exbrdu / dcx67.549.3866.607.7339.305.6861.057.3brdu / neun41.465.0548.205.4026.633.2146.335.1brdu : 5-bromo-2-deoxyuridine ; dcx : doublecortin ; ex : exercise ; mptp / p : 
 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecidp<0.05 compared to the probenecid group , p<0.05 
 compared to the mptp / p group ) .
brdu : 5-bromo-2-deoxyuridine ; dcx : doublecortin ; ex : exercise ; mptp / p : 
 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid p<0.05 compared to the probenecid group , p<0.05 
 compared to the mptp / p group in the present study , bdnf and trk b expression in the ventral midbrain in the probenecid 
 group was set at 1.00 .
the level of bdnf protein was 0.97  0.02 , 0.74  0.01 , and 0.87  
 0.01 in the probenecid exercise , mptp / p , and mptp / p - exercise groups , respectively .
the level 
 of trk b protein was 1.10  0.03 , 0.52  0.07 , and 0.71  0.02 in the probenecid exercise , 
 mptp / p , and mptp / p - exercise groups , respectively ( p < 0.05 ) ( table 2 ) .
previous studies have found that mptp - induced neuronal 
 loss in the sn results in disturbances in memory function and learning impairment16 , 17 . in the present study ,
long - term memory during the passive avoidance 
 test deteriorated in the mptp / p - induced pd mice , and treadmill exercise improved short - term 
 memory .
the beneficial effects of physical exercise on cognitive functions such as spatial 
 learning ability and memory have been reported previously11 .
d2dr is of clinical interest because of its role in movement disorders , such as pd , where 
 d2dr agonists are known to benefit patients18 .
d2dr gene knockout mice demonstrated hypokinesia , bradykinesia , 
 and decreased spontaneous movement19 . 

dopamine receptor antagonists and dopamine depletion have been reported to reduce cell 
 proliferation in the subventricular zone ( svz ) and hippocampal dg of rodents5 . in the present study , expression of d2dr in 
 the ventral midbrain significantly decreased in the mptp - induced pd mice , and treadmill 
 exercise increased d2dr expression in these mice .
hglinger et al.5 reported that an agonist of dopamine d2-like receptors ( d2 , 
 d3 , and d4 receptors ) restored cell proliferation in the svz in a pd rodent model .
fisher et 
 al.13 reported that treadmill exercise 
 reversed the effects of mptp injection on dopamine d2 receptors in the dorsal striatum .
the present study showed that neurogenesis in the hippocampal dg significantly decreased 
 owing to induction of pd , whereas neurogenesis significantly enhanced in response to 
 treadmill exercise .
the observed 
 increase in the expression of brdu - positive and dcx - positive cells in the hippocampal dg 
 indicates that exercise enhanced neuronal plasticity21 .
expression of dcx can be used as a marker for immature neurons ; 
 therefore , the level of dcx expression in the adult brain can be used to measure 
 neurogenesis22 , 23 . in the present study , expression of newborn immature cells in the 
 hippocampal dg
was significantly decreased in the mptp - induced pd mice , while treadmill 
 exercise increased the number of newborn immature cells in these mice .
the increase in the 
 number of dcx - positive cells after treadmill exercise could be owing to the addition of 
 newly generated immature cells22 , 23 .
these results showed that treadmill 
 exercise is not only capable of promoting cellular proliferation but also differentiation of 
 newborn neuroblasts .
physical exercise increases neurogenesis , long - term potentiation , 
 synaptic plasticity , and improves spatial memory24 , 
 25 .
adult hippocampal neurogenesis is affected by neurotrophic factors such as bdnf , ngf , and 
 nt-3 , which are implicated in adult neurogenesis7 .
bdnf has been recognized as a key regulator of synaptic development 
 and plasticity in relation to several neurodegenerative and psychiatric disorders26 . over - expression of bdnf and its receptor 
 trk
b increases neurogenesis , whereas inhibition of bdnf or trk b suppresses 
 neurogenesis27 , 28 .
suppression on bdnf - trk b signaling is associated with the 
 inhibition of neurogenesis in the hippocampal dg20 . in the present study
, we observed that the administration of mptp 
 significantly decreased bdnf and trk b levels in the hippocampus , while treadmill exercise 
 increased bdnf and trk b levels in the hippocampus of mptp - induced pd mice . exercise 
 enhances learning and memory , which is accompanied by increased cell proliferation and 
 survival in the hippocampus via increased bdnf and trk b expression29 , 30 .
it has been 
 reported that bdnf significantly increases neurogenesis in the dg30 . here , we suggest that exercise can overcome pd - induced 
 hippocampal memory impairment by facilitating neurogenesis through enhancement of bdnf 
 expression and prevention of dopaminergic neuronal damage
. therefore , exercise may be a 
 potential therapeutic strategy for the alleviation of memory dysfunction in pd dementia 
 patients .
 [ purpose ] this study aimed to investigate the effect of treadmill exercise on non - motor 
 function , specifically long - term memory , in a 
 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid - induced parkinson s disease mouse 
 model . 
 [ methods ] a mouse model of parkinson s disease was developed by injecting 20  mg / kg 
 of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 250  mg / kg of probenecid ( p ) . 
 we 
 divided in into four groups : probenecid group , probenecid - exercise group , 
 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid group , and 
 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid - exercise group . 
 mice in the 
 exercise groups ran on treadmill for 30  min / day , five times per week for 4 weeks . 
 
 [ results ] latency in the passive avoidance test increased in the 
 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid - exercise group compared with 
 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid group . 
 in addition , the number of 
 5-bromo-2-deoxyuridine / neun - positive cells and 
 5-bromo-2-deoxyuridine / doublecortin - positive cells in the hippocampal dentate gyrus was 
 higher in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid - exercise group than 
 that in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine / probenecid group . 
 these changes 
 were associated with the expression of brain - derived neurotrophic factor in the 
 hippocampus . 
 [ conclusion ] our results suggest that treadmill exercise may improve 
 long - term memory in parkinson s disease mice by facilitating neurogenesis via increased 
 expression of neurotrophic factors . 
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION