epilepsy is a neurological disorder that is characterized by recurrent seizures that result from abnormal and synchronous firing of neurons in the brain .
approximately one - third of the patients with epilepsy do not respond to drugs and are said to have intractable epilepsy .
although the precise mechanism of seizure recurrence remains elusive , elucidation of the mechanisms involved in the transformation of a normal brain into one capable of producing recurrent seizures and of maintaining an epileptic state is essential for understanding epileptogenesis and for developing new treatments for epilepsy .
micrornas , as posttranscriptional regulators for up to 60% of proteins , are a major determinant of protein levels in cells .
microrna-132 ( mir-132 ) is significantly upregulated during active synaptogenesis and plays important roles in spine formation and maturation [ 25 ] .
mir-132 also regulates the inflammatory response and neuronal apoptosis after acute brain injury [ 68 ] .
several studies have shown that mir-132 is persistently upregulated during epileptogenesis after acute brain injury [ 913 ] .
because synaptic dysfunction and reorganization are the most important histopathological changes in epileptic foci , we aimed to investigate whether mir-132 plays a role in epileptogenesis by regulating synaptic reorganization .
p250gap is a target of mir-132 and is enriched in the nmda receptor complex of neuronal synapses .
p250gap is a rho family gtpase - activating protein that can interact with a variety of synaptic proteins by inhibiting the activity of downstream rho family gtpases , including rhoa , rac1 , and cdc42 [ 2 , 15 , 16 ] .
it is an important cytoskeletal regulator that is regulated by neuronal activity - related signaling pathways that result in the depolymerization of the cytoskeleton and a reduction in the density and volume of dendritic spines . in the central nervous system ( cns ) , p250gap has been reported to mainly regulate the activity of rac1 and cdc42 .
this study aimed to explore the possible molecular mechanisms of mir-132 and its target , p250gap , during epileptogenesis .
we also aimed to determine how gtpases are regulated by p250gap in the pathological process of epilepsy .
the mice were kept in an animal room at a constant temperature ( 22  1c ) and a 12-h light / dark cycle with free access to food and water .
all experimental procedures were performed in accordance with the international guidelines for the use of animals and the guidelines of the animal care committee of chongqing medical university , china .
hippocampal neurons from 17- to 19-day - old embryonic mice were cultured ( 5  10 cells per square centimeter ) on plates coated with poly - l - lysine ( catalog number p1399 , sigma , usa ) as described previously .
the neurons were then maintained in neurobasal medium ( catalog number 21103 - 049 , gibco , usa ) supplemented with b27 ( catalog number 17504 - 044 , gibco ) and 0.5  mm l - glutamine ( catalog number g3126 , sigma ) .
approximately 1/3 to 1/2 of the culture medium was changed every 3 - 4 days .
ten micromolar cytosine -d - arabinofuranoside ( catalog number c1768 , sigma ) was added to the culture medium at 3 days in vitro ( div3 ) to inhibit the growth of gliocytes .
the cultured neurons were stained at div7 with a neuron - specific marker , microtubule - associated protein 2 ( map2 ) ( catalog number 11267 , abcam , usa ) , to evaluate the purity of the cultured neurons .
only the cultured cells whose purity was higher than 98% were used for the following experiment . at div10 , sreds were induced in the neuronal cultures by exposing the neurons to magnesium - free ( mgf ) medium ( 145  mm nacl , 10  mm hepes , 2.5  mm kcl , 2  mm cacl2 , 10  mm glucose , and 0.001  mm glycine , with the ph adjusted to 7.3 with naoh and the osmolarity adjusted to 280320 mosm with sucrose ) , for 3  h. the sham controls were treated with nonmagnesium - free medium ( non - mgf ) , which is mgf medium supplemented with 1  mm mgcl2 .
sreds are typically observed within 1224  h using patch clamp recordings and can last for the life of the neurons in culture .
this hippocampal neuronal culture model of status epilepticus ( se ) has been well characterized as a useful in vitro model of refractory se . a mir-132 antagomir ( ant-132 )
a nontargeting scrambled sequence ( scr ) was used as a control ( catalog number mir30000067 - 1 - 10 , ribobio , china ) .
p250gap expression was silenced using a lentivirus ( lv - shp250gap ) , and the same lentivirus vector expressing gfp alone ( lv - gfp ) was chosen as a control ( catalog number lvcon077 , genechem , china ) .
neuronal transfection was conducted according to the manufacturer 's instructions , and the culture medium was completely changed 10  h after transfection .
the membrane potentials of neurons were measured with whole - cell current - clamp recordings using a patch clamp amplifier .
a cell culture dish was mounted on the stage of an inverted microscope ( ix-51 , olympus , japan ) , and patch pipettes were filled with an intracellular solution containing 110  mm kasp , 30  mm kcl , 10  mm egta , 10  mm hepes , 5  mm na - atp , 1  mm cacl2 , 2  mm mgcl2 , and 10  mm teacl , with the ph adjusted to 7.3 with csoh and the osmolarity adjusted to 280300 mosm with sucrose .
the pipette resistance in the intracellular solution was 24  m. the pipette resistance and capacitance were compensated electronically after the establishment of a gigaseal .
after the whole - cell capacitance was compensated , recordings were made only when the series resistance was < 20  m. to optimize the success of recording from pyramidal neurons , phase - bright cells were selected based on both size and pyramidal soma .
cultured neurons with small dendritic arborizations , long axons , and soma diameters of 2026  m were selected for the electrophysiological recordings to avoid space clamp artifacts . routinely , 6080% series resistance compensation was employed , continually monitored , and adjusted as required . whole - cell resistance and resting membrane potential
were also monitored before and during the experiments , and a cell was accepted for study only if these parameters remained stable .
whole - cell recordings were performed using an epc-10 amplifier ( heka , germany ) in the current - clamp mode .
data were collected and analyzed using clamp - fit 10.0 software ( axon , usa ) .
reverse transcription ( rt ) reactions were performed using a primescript rt reagent kit ( catalog number rr047a , takara , china ) .
samples were run at 37c for 15  min and 85c for 5  sec , followed by a hold at 4c .
real - time pcr was carried out on a bio - rad real - time pcr system .
the pcr mixture contained 12.5  l of sybr premix ex taq ii , 1  l of 10  m pcr forward primer , 1  l of 10  m pcr reverse primer , 2  l of the rt reaction solution , and 8.5  l of ddh2o .
real - time pcr was performed under the following conditions : stage 1 , 95c for 30  s ; stage 2 , 40 cycles at 95c for 5  s and 60c for 30  s ; and stage 3 , dissociation .
the primers used were ( mir-132 rt ) 5-gtcgtatccagtgcagggtccgaggtattcgcactggatacgaccgacca-3 , ( mir-132 forward primer ) 5-gcggcggtaacagtctacagcc-3 , and ( mir-132 reverse primer ) 5-atccagtgcagggtccgagg-3. the data were analyzed with bio - rad cfx manager software ; the data are represented as the mean 2  standard deviation ( sd ) .
total proteins were extracted using a whole protein extraction kit ( catalog number p0013 , beyotime , china ) .
the total protein concentrations were determined using an enhanced bicinchoninic acid protein ( bca ) assay kit ( catalog number p0012s , beyotime , china ) , and the samples were stored at 20c until use .
the primary antibodies used were goat anti - p250gap ( 1  :  1,000 , catalog number 138167 , santa cruz , usa ) and rabbit anti - cleaved caspase-3 ( 1  :  150 , catalog number # 9661 , cst , usa ) .
the activation of rac1 and cdc42 was measured using a rac1/cdc42 activation assay kit ( catalog number 17 - 441 , millipore , usa ) according to the manufacturer 's protocol .
this assay uses the downstream effector of rac / cdc42 , p21-activated protein kinase ( pak1 ) , to isolate the active gtp - bound form of rac / cdc42 from the sample . the p21-binding domain ( pbd ) of pak1
after the proteins were precipitated , an immunoblot was performed , and the activated rac1 and cdc42 were detected with specific monoclonal antibodies followed by an hrp - conjugated secondary antibody .
a tunel assay roche kit ( catalog number 11684817910 , roche , switzerland ) was used to detect the apoptosis level of cultured hippocampal neurons according to the manufacturer 's instructions . briefly , after cortical neurons were fixed in freshly prepared 4% formaldehyde solution in phosphate - buffered saline ( pbs ) for 20  min at room temperature and permeabilized with 0.2%
triton x-100 for 5  min , they were incubated with 50  l of a tunel reaction mixture for 60  min at 37c in the dark and then rinsed with pbs ( ph 7.4 ) 3 times for 5  min each . to detect the nuclei , the slides were incubated with dapi for 5  min at room temperature in the dark and observed with a fluorescence microscope .
the apoptotic index was expressed as the ratio of the number of tunel - positive neurons to the total number of neurons .
the pilocarpine model has been widely used to simulate human tle . for se induction ,
all the mice were intraperitoneally ( i.p . ) injected with pilocarpine ( 300  mg / kg , 0.1  ml/10  g , i.p . , sigma ) .
atropine sulfate ( 1  mg / kg , i.p . ) was administered 30  min prior to the first dose of pilocarpine .
the mice that developed a stage 4 or 5 seizure ( racine 's scale )   within 2  h after pilocarpine administration were considered kindled in our study .
diazepam ( 10  mg / kg , i.p . ) was given to terminate the convulsions 1  h after se onset .
all of the mice were allowed to recover for 48  h after se and were then intracerebroventricularly ( i.c.v . )
injected with 2  l of saline ( tle group ) , scr-132 ( tle+scr-132 group ) , or ant-132
the dose of ant-132 was 1  nmol , which was achieved by dilution in double - distilled water .
animals in the chronic period with spontaneous recurrent seizures ( srs ) associated with pilocarpine epilepsy were recorded every day for 24  h using a closed circuit video system to detect the class 4 and class 5 seizures at the 6th week .
clinical events with a score below 2 were excluded . a hito golgi - cox optimstain kit ( catalog number htkns1125 , hitobiotec inc . ,
brain tissues obtained from all the mice in the ant-132-treated and scr-132 control groups in our experiment were sectioned into 100-m coronal sections on a cryostat ( leica , germany ) , and the staining process was conducted following the manufacturer 's instructions . for morphometric measurements ,
at least 15 neurons were analyzed for each data point reported , and 3  15  m of dendrites in each neuron was chosen .
the imaris filamenttracer module ( andor technology ; belfast , northern ireland ) was used to detect , quantify , and characterize spine structures .
thin spines were defined as dendritic protrusions with two times mean neck width < spine length and with mean neck width  maximum head width .
mushroom spines were defined as dendritic protrusions with mean head width > mean neck width . to assess the reliability of the counting of dendritic protrusions , a blind study was initially performed .
statistical analyses were performed with spss statistics for windows , version 20.0 ( armonk , ny , usa ) .
differences between the experimental groups were compared using one - way analysis of variance ( anova ) , and student 's t - test was used for comparisons .
it has been reported that the expression level of mature mir-132 is low during the first week in the neonatal rat hippocampus , with a significant increase in mir-132 levels during weeks 24 , which is also a critical period for the development and maturation of spines in rodents . in our study , the expression levels of mir-132 and p250gap in c57bl/6 mice during postnatal d 1 to d 30 were similar to those of previous studies ( figure 1(a ) ) .
we evaluated the chronological expression level of mir-132 and p250gap during the maturation process of cultured hippocampal neurons .
the level of mir-132 increased from div5 to div13 and was maintained at a relatively high level , whereas the expression of p250gap protein was high at div5 but decreased at div15 ( figure 1(b ) ) .
this result indicated that the expression of mir-132 and p250gap might correlate with the process of physiological spine maturation and synaptogenesis .
the expression levels of mir-132 and p250gap at 6  h , 3  d , 5  d , and 7  d after mgf treatment were evaluated to determine whether the levels of mir-132 and p250gap were influenced by the electrophysiological activity of cultured neurons .
the results showed that mir-132 was upregulated 6  h to 7  d following magnesium - free medium treatment , with statistical significance at 6  h , 3  d , and 7  d , while p250gap protein was downregulated from 6  h to 7  d following magnesium - free medium treatment , with statistical significance at 3  d , 5  d , and 7  d ( figures 1(c ) and 1(d ) ) .
the results suggest that mir-132 expression is increased during the active synaptogenesis periods of the immature brain .
moreover , the upregulation of mir-132 in the sred model of hippocampal neurons suggests that the pathological electrical excitability may also influence mir-132 expression , which may correlate with the pathological synaptogenesis of the cns during epileptogenesis .
we used a specific antagonist of mir-132 and rna interference to knock down the expression of mir-132 and p250gap ( figures 2(a ) and 2(b ) ) .
the transfection efficiency was confirmed by qrt - cpr for mir-132 ( figure 2(c ) ) and by wb for p250gap ( figure 2(d ) ) .
the expression level of p250gap has significantly upregulated after ant-132 treatment which indicated that the expression level of p250gap was negatively regulated by mir1 - 132 in our cultured epileptic hippocampal neurons ( figure 2(e ) ) .
rac1 and cdc42 are considered two important promotors of dendritic branching and synaptic plasticity , while rhoa acts in the opposite manner .
p250gap has been reported to mainly regulate the activation of rac1 and cdc42 in the cns .
the pathological plasticity of neuronal spines and synapses is important in the pathological process of epilepsy ; thus , we studied the activation levels of rac1 and cdc42 to explore whether rac1 and cdc42 are regulated by the mir-132/p250gap pathway in our sred model of cultured hippocampal neurons .
first , we found that the activation levels of both rac1 and cdc42 were significantly elevated in mgf - treated hippocampal neurons compared to control hippocampal neurons ( figures 3(a1 ) and 3(a2 ) ) . to determine whether and how rac1 and cdc42 are regulated by the mir-132/p250gap pathway in our cultured hippocampal neurons , we evaluated the activation levels of rac1 and cdc42 in cultured hippocampal neurons when mir-132 or p250gap expression was inhibited .
the results showed that transfection of ant-132 or lv - shp250gap did not significantly affect the activity of rac1 ( figures 3(b1 ) and 3(b2 ) ) , while the activity of cdc42 was significantly inhibited after ant-132 transfection and elevated after lv - shp250gap transfection ( figures 3(c1 ) and 3(c2 ) ) .
furthermore , we obtained similar results in the mgf - treated sred model of cultured hippocampal neurons , while the expression of mir-132 or p250gap was inhibited ( figures 3(d1 ) , 3(d2 ) , 3(e1 ) , and 3(e2 ) ) .
our data suggest that p250gap may primarily function as a gap for cdc42 in this epileptic model of cultured neurons .
to clarify the effect of mir-132 and p250gap on neuronal excitability , we performed electrophysiological evaluations of the mgf medium - treated cultured hippocampal neurons .
our experiment showed that ant-132 treatment significantly decreased the ap frequency , suggesting that ant-132 can significantly inhibited neuronal electrical excitability in our sred model of cultured hippocampal neurons ( figures 4(a2)4(a4 ) ) . in contrast , ant-132 and lv - shp250gap cotreatment significantly increased the ap frequency ( figures 4(a5 ) and 4(a6 ) ) . silencing the overexpression of mir-132 is known to have a neuroprotective effect after acute brain injury [ 8 , 20 ] ; therefore , we asked whether mir-132 regulates neuronal apoptosis via the p250gap pathway .
we first transected the cultured neurons with ant-132 at div7 and then induced the sred model at div10 .
next , we silenced the expression levels of both mir-132 and p250gap to investigate whether p250gap was involved in the neuronal apoptosis effect of mir-132 .
tunel staining ( figures 5(a ) and 5(b ) ) and wb detection of cleaved caspase-3 levels ( figure 5(c ) ) were performed 24  h later to investigate the level of apoptosis .
our results showed that regardless of whether the expression level of p250gap was suppressed mir-132 silencing decreased neuronal apoptosis .
this finding indicates that mir-132 silencing has a neuroprotective effect but that this effect may occur through pathways other than the p250gap pathway .
we then investigated whether mir-132 inhibition could suppress chronic seizures in vivo using a lithium - pilocarpine model .
continuous video monitoring was performed in the 6th week for 7  d. the frequency of chronic seizure was quantified and statistically analyzed by comparing the scr-132 control group ( figure 6(a ) ) and the ant-132 group ( figure 6(b ) ) .
statistical analysis of our behavioral investigation indicated that the ant-132 treatment can protect experimental mice against developing chronic recurrent seizures and can significantly decrease spontaneous seizure frequency ( figure 6(c ) ) .
in general , mushroom - shaped and stubby spines represent more mature and stable spines , while thin spines and filopodia tend to be more plastic and immature ( figure 7(b ) ) .
disrupted maintenance of the dendritic tree and spinogenesis are two important neurophysiological features of epilepsy .
after the behavioral observations were completed , all the brain tissues of the ant-132-treated ( ep+ant-132 ) , scr-132-treated ( ep+scr-132 ) , and nonintervention control ( ep ) groups were excised and golgi - stained to further investigate the morphological effects of ant-132 treatment on the dendritic spine ( figure 7(a ) ) . the total number and different forms of spines in the hippocampal dentate gyrus ( dg ) and ca1 regions were quantified and statistically analyzed to investigate the differences in spine density and morphology in the hippocampus of the experimental mice .
we found that ant-132 treatment could decrease the spine density and elevate the proportion of stable spines ( figure 7(c ) ) , indicating that the ant-132 treatment might help to suppress spine remodeling under the pathological condition of epilepsy .
first , the altered expression of mir-132 and p250gap in vivo and in vitro indicated that mir-132-mediated p250gap activity might be related to the remodeling process of spines .
second , mir-132/p250gap regulates the activity of cdc42 in a hippocampal neuronal culture model of acquired epilepsy , which is possibly the reason for dendritic spine remodeling during epileptogenesis because cdc42 is an important cytoskeletal regulator that has been reported to trigger the outgrowth of peripheral spike - like protrusions called filopodia .
third , ant-132 can decrease neuronal electrical excitability in vitro through p250gap , and ant-132 can decrease chronic recurrent seizure development in vivo .
finally , the neuroprotective effect of ant-132 was once again verified in our experimental model of cultured neurons , and we revealed that this effect does not occur through the p250gap pathway .
mir-132 is an activity - dependent gene that is implicated in synapse formation through regulating the translation of its target , p250gap .
physiological synapse formation in the immature brain and/or a bicuculline - induced increased in synaptic activity can change the expression levels of mir-132 and p250gap .
conversely , altered expression of mir-132 and p250gap can significantly affect dendritic spine density and head size .
epilepsy is a pathological process of synaptic plasticity and excitatory loop formation that follows many types of acute brain injury and that can change neuronal activity [ 14 , 15 ] .
although the upregulation of mir-132 has been observed in several epileptic models in vivo [ 25 ] , no study has clarified the molecular mechanism of mir-132 in the epileptic process .
we have demonstrated the role of mir-132 and its target , p250gap , in an epileptic model of cultured hippocampal neurons .
the p250gap protein can regulate rho gtpase family members , including rhoa , rac1 , and cdc42 , and mediates actin cytoskeleton organization .
rac1/cdc42 promote dendritic branching and growth ; previous studies performed in vivo had inconsistent results about the ability of p250gap to regulate the gtp - loaded state of rac1 and cdc42 [ 16 , 22 ] . here , we investigated the activation levels of the dendritic growth promoting factors rac1/cdc42 and further demonstrated , that under the condition of aberrant neuronal activity of epileptic discharge , mir-132 and p250gap principally regulated the activation level of cdc42 , which indicated that cdc42 might be a more appropriate target for further study about mir-132 and epilepsy . although silencing mir-132 or p250gap has no significant effect on the activation level of rac1 , the activation level of rac1 has also upregulated in our cultured epileptic hippocampal neurons ; further studies are needed to reveal if rac1 has taken part in the epileptogenesis process .
several studies have shown that mir-132 may regulate a significant increase in the density of spines and length of dendrites under the condition of aberrant neuronal activity in vivo [ 2 , 15 ] . in our study
, we attempted to detect the effect of mir-132 on spinous remodeling during epileptogenesis through morphological analysis of the dendritic spines of ant-132-treated pilocarpine - induced chronic epileptic mice .
treatment with ant-132 significantly increased the proportion of mushroom - shaped dendritic spines in the dg and ca1 regions of the hippocampus , which are two critical areas of pathological synaptic formation that leads to epilepsy . because mushroom - shaped stubby dendritic spines are considered more mature and stable , while dendritic spines with other shapes , such as filopodia and thin spines , are immature and more plastic [ 23 , 24 ] ,
our experimental results indicate that ant-132 could be helpful for stabilize and maintaining the proper functioning of the dendritic spines of the hippocampus in the epileptogenesis process .
our electrophysiological study of cultured neurons also confirmed that ant-132 can inhibit the ap frequency of magnesium - free medium - treated neurons and that this effect can be reversed if p250gap is silenced .
these results suggest that ant-132 may regulate neuronal electrical excitation , which is an important pathological change during epileptic formation , via the p250gap pathway .
several studies have reported that mir-132 overexpression can inhibit cell proliferation and promote cellular apoptosis [ 8 , 21 , 25 ] ; however , the underlying molecular mechanism is still not clear . our study verified that mir-132 upregulation promotes neuronal apoptosis in an epileptic model of cultured hippocampal neurons and that the neuroprotective effect of ant-132 still occurs even if p250gap has been inhibited , thus indicating that other pathways are involved in the neuroprotective effect of ant-132
in summary , we found that the expression of mir-132 and its target , p250gap , are relevant to epileptogenesis . silencing mir-132 can decrease chronic recurrent seizures .
mir-132 likely functions through regulating the expression of p250gap , the activation of downstream cdc42 , and the maintenance of the proper form and function of the dendritic spines , which may affect neural electrical activity of neurons and epileptogenesis .
treatment with ant-132 has a neuroprotective effect ; however , this effect is not mediated through the p250gap pathway .
 increasing evidence suggests that epilepsy is the result of synaptic reorganization and pathological excitatory loop formation in the central nervous system ; however , the mechanisms that regulate this process are not well understood . 
 we proposed that microrna-132 ( mir-132 ) and p250gap might play important roles in this process by activating the downstream rho gtpase family . 
 we tested this hypothesis using a magnesium - free medium - induced epileptic model of cultured hippocampal neurons . 
 we investigated whether mir-132 regulates gtpase activity through p250gap and found that cdc42 was significantly activated in our experimental model . silencing mir-132 inhibited the electrical excitability level of cultured epileptic neurons , whereas silencing p250gap had an opposite effect . 
 in addition , we verified the effect of mir-132 in vivo and found that silencing mir-132 inhibited the aberrant formation of dendritic spines and chronic spontaneous seizure in a lithium - pilocarpine - induced epileptic mouse model . finally , we confirmed that silencing mir-132 has a neuroprotective effect on cultured epileptic neurons ; however , this effect did not occur through the p250gap pathway . 
 generally , silencing mir-132 may suppress spontaneous seizure activity through the mir-132/p250gap / cdc42 pathway by regulating the morphology and electrophysiology of dendritic spines ; therefore , mir-132 may serve as a potential target for the development of antiepileptic drugs . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion