epilepsy is a common and serious chronic neurological disease that affects over 50 million people worldwide .
anti - epileptic drugs ( aeds ) are effective at controlling seizures in many patients , acting through a range of neuronal targets , including ion channels , neurotransmitter release , and receptor mechanisms.1 , 2 however , a third of patients with epilepsy fail to achieve seizure freedom , and current aeds do not show disease - modifying properties .
future treatments for epilepsy need to differentiate from currently marketed drugs , for example , by having a novel mechanism(s ) of action or providing disease - modifying effects.1 , 2 acquired epilepsy is thought to result from disturbances to the expression and function of neurotransmitters and their receptors , ion channels , and signaling components , as well as altered metabolism , neuronal and glial microstructure , neuroinflammation , and other mechanisms.3 , 4 , 5 it is likely that disease - modifying treatments will need to influence multiple genes within a given pathway or in various independent pathways .
potential gene network control points were recently identified and include master regulators of transcription and neuroinflammation , epigenetic factors , and noncoding rnas .
micrornas ( mirnas ) are a conserved family of endogenous small noncoding rnas that regulate protein levels in cells by post - transcriptional control of mrna stability and translation.9 , 10 this is achieved by sequence - specific binding of the mirna to complementary bases in the mrna target .
since this generally requires only a 7- to 8-nucleotide match , individual mirnas can potentially target large numbers of mrnas .
this provides the ability to regulate entire gene networks or multiple biological processes and has been demonstrated in the brain .
microrna-134 ( mir-134 ) was identified as a brain - enriched , neuronally expressed mirna that controls dendrite spine morphogenesis , via repression of lim domain kinase 1 and other targets.14 , 15 this is potentially important for disorders of hyperexcitability , since dendritic spines are contact points for excitatory communication and there is evidence that reorganization of dendrites is a feature of experimental and human epilepsy.16 , 17 we identified mir-134 among upregulated mirnas profiled in areas of hippocampal damage following status epilepticus induced by intra - amygdala kainic acid ( ka ) in mice .
expression of mir-134 is also upregulated after neuronal stimulation and seizures in various other in  vitro and in  vivo models,15 , 19 , 20 , 21 , 22 and mir-134 is overexpressed in the resected human neocortex from patients with intractable temporal lobe epilepsy ( tle ) . silencing mir-134 by intracerebroventricular ( i.c.v . )
injection of locked nucleic acid ( lna ) , cholesterol - tagged antagomirs ( ant-134 ) reduced levels of mir-134 for at least a month and rendered mice refractory to the convulsant effects of both ka and pilocarpine.19 , 22 consistent with known mir-134 targets , lim domain kinase 1 and levels of other mir-134 targets were higher in ant-134-treated mice after status epilepticus and the dendritic spine volume was increased.19 , 22 notably , injection of ant-134 after status epilepticus suppressed the later occurrence of spontaneous recurrent seizures in the intra - amygdala ka model in mice .
there is significant interest in developing mirna - based therapies.23 , 24 there are also barriers to antisense - based approaches and there has been some disengagement of industry from pursuing such efforts .
this is likely to be a problem for any new therapy for epilepsy , particularly one that is not based on traditional small molecule chemistry.1 , 2 pre - clinical development of an mir-134-based treatment for epilepsy might be more attractive if additional important questions are answered .
principally , work to date has only tested ant-134 in mouse models of status epilepticus .
proof of seizure - suppressive effects of ant-134 in a non - status epilepticus model or in another species would bridge the gap in evidence and facilitate pre - clinical development of ant-134-based treatment for epilepsy.26 , 27 here , we evaluated mir-134 expression in the resected hippocampus from patients with pharmacoresistant tle and we tested ant-134 in three different models .
we used the pentylenetetrazol ( ptz ) model in mice , a popular method for screening putative anticonvulsive effects that triggers seizures via -amino butyric acid ( gaba ) blockade.26 , 28 second , the perforant pathway stimulation ( pps ) model , a toxin - free model of acquired epilepsy based on nonconvulsive status epilepticus that avoids the confounding influence of exposing the brain to chemoconvulsants , was used in rats .
finally , we used a high - potassium model of epileptiform activity , using ex  vivo brain slices from rats pre - treated with ant-134 .
our results establish that targeting mir-134 offers broad anticonvulsant and possibly disease - modifying effects in experimental epilepsy , which may encourage pre - clinical development of ant-134 as a treatment for epilepsy .
for this , we analyzed levels of mir-134 in the surgically obtained hippocampus from adults with pharmacoresistant tle and we compared findings to autopsy hippocampal samples from people who died of causes unrelated to neurological disease .
these samples were used previously to analyze expression of other genes , and clinical data for both groups are briefly summarized in tables s1 and s2 .
there were no between - group differences in patient age , and each group included males and females .
we detected higher levels ( p  = 0.037 ) of mature mir-134 in the tle specimens compared to autopsy samples ( figure  1a ) .
this difference is unlikely to be an artifact of post mortem delay , since mir-134 levels did not decrease in simulated autopsy experiments lasting up to 12  hr . we began by establishing dosing parameters in the mouse ptz model .
generalized tonic - clonic seizures were first reliably elicited at a dose of 60  mg / kg in c57bl/6j mice ( figure  1b ) .
a dose of 80  mg / kg elicited generalized tonic - clonic seizures in all mice and with a more consistent and shorter latency than with the 60 mg / kg dose ( figure  1b ) .
a dose of 100  mg / kg also produced rapid - onset tonic - clonic seizures in all mice but was associated with high mortality ( figure  1b and data not shown ) .
ordinal logistic regression analysis was used to explore the dose dependence of the ptz - induced convulsions .
ptz dose was significantly associated with racine scores ( figure  1c ) . a dose of 80  mg / kg was selected for further experiments .
to obtain molecular evidence for ptz - induced seizure activity in the model , we measured expression of activity - regulated genes in the hippocampus .
transcript levels of fbj osteosarcoma oncogene ( fos ) , a calcium - dependent marker of neuronal activity and activity regulated cytoskeletal - associated protein ( arc ) , another immediate early gene responsive to intense neuronal activity , were strongly increased in the hippocampus 30  min , but not at 4  hr , after ptz - induced seizures ( figures 1d and 1e ) .
there was no evidence of irreversible neuronal injury in tissue sections from mice after ptz , as assessed by fluoro - jade b ( fjb ) and neun ( rna binding protein , fox-1 homolog [ c.  elegans ] 3 ) staining ( data not shown ) .
expression of mir-134 is known to increase in various in  vitro and in  vivo seizure models.15 , 19 , 20 , 21 , 22 we therefore investigated whether ptz - induced convulsions alter mir-134 levels in mice .
taqman mirna assays showed that levels of mature mir-134 were significantly increased in the hippocampus , but not in the cortex , 30  min after ptz - induced generalized tonic - clonic seizures in mice ( figure  1f and data not shown ) .
injection of antagomirs targeting mir-134 ( ant-134 ) in mice.19 , 22 previous work has established a dose of these antagomirs that reduces hippocampal mir-134 levels by over 90% by 24  hr after injection .
accordingly , we tested ptz responses 24  hr after injection of ant-134 and compared them to responses in mice that received a scrambled antagomir ( scr ) . in scr mice , delay to onset of first tonic - clonic seizure after ptz injection was similar to that observed before in control mice ( figure  2a , and compare to figure  1b ) .
thus , the control antagomir did not alter the normal response to ptz in the model .
the time to first ptz - induced tonic - clonic seizure was significantly longer in mice injected with ant-134 compared to scr - injected animals ( figure  2a ) .
the severity of racine - scored ptz - induced seizures was also significantly lower in ant-134- compared to scr - injected mice ( figures 2b and 2c ) .
analysis of electrographically recorded seizure activity determined that electroencephalography ( eeg ) total power was lower in ant-134 mice compared to the control group after ptz ( figure  2d ) .
we next analyzed brain tissue samples from scr- and ant-134-injected mice for evidence of mirna silencing and differences in expression of activity - regulated genes .
analysis of the hippocampus obtained after ptz - induced seizures confirmed that mir-134 expression was lower in ant-134 pre - treated mice compared to scr - treated animals ( figure  3a ) .
ant-134 had no effect on levels of an unrelated mirna ( figure  3b ) .
next , we assessed expression of arc and c - fos as molecular markers of recent neuronal activity to support the clinically scored behavior and eeg differences in ant-134 mice . quantitative real - time pcr analysis revealed increased c - fos expression in the hippocampus of scr - injected mice after ptz - induced seizures ( figure  3c ) .
in contrast , c - fos levels were lower in ant-134-injected mice , consistent with reduced seizure severity ( figure  3c ) .
this difference was also apparent in the neocortex from the same animals ( figure  3c ) .
expression of arc was increased in the hippocampus of scr - injected mice that received ptz .
arc levels were not significantly different in ant-134 mice compared to scr - injected animals in the hippocampus and neocortex after ptz - induced seizures ( figure  3d ) .
we turned next to the pps model in rats , first investigating effects on mir-134 levels .
taqman mirna assays showed that levels of mature mir-134 were not significantly modified in the hippocampus of rats at two different time points ( figure  4a ) .
next , we assessed the effect of silencing mir-134 before stimulation on seizure severity during status epilepticus .
rats were pre - treated with ant-134 or scr ( i.c.v . ) 24  hr before the 8-hr stimulation protocol .
analysis of electrographically recorded seizure activity determined that scr - injected rats presented a mean (  sem ) increase of 1,304.3%   94.9% in eeg total power relative to baseline .
this was similar to animals that were stimulated but not receiving the oligonucleotide ( data not shown ) . in rats
pre - treated with ant-134 , the severity of evoked seizures in the pps model was similar : eeg total power during seizures was a mean (  s.e.m ) of 1,244.6%   82.7% ( percent of baseline ) . despite not seeing an acute anticonvulsant effect of ant-134 in the pps model , we were nevertheless interested in whether silencing mir-134 after status epilepticus in the model would alter the emergence of recurrent spontaneous seizures . a key prior
finding was that silencing mir-134 after status epilepticus induced by intra - amygdala ka reduced the subsequent occurrence of spontaneous recurrent seizures by over 90% .
for these studies , rats underwent 8-hr stimulation of the perforant pathway to induce epilepsy and the i.c.v .
rats were then monitored with video - eeg until a first spontaneous seizure was recorded , typically 1025  days after pps , and they were then followed for a further 8  weeks ( figure  4b ) .
epilepsy developed in all scr - injected rats over the expected course , with the first spontaneous seizures appearing 14  weeks after status epilepticus ( figures 4b and 4c ) .
scr - injected rats averaged 20 spontaneous seizures recorded during the 8-week recording time ( figures 4d and 4e ) .
in contrast , no spontaneous seizures were detected over the 8-week period in six of seven rats injected with ant-134 after status epilepticus ( figures 4d4f ) .
epilepsy emerged in only a single rat treated with ant-134 ( figures 4d and 4f ) .
the fisher exact test determined that there was a significant difference in epilepsy development between the groups ( p  = 0.005 ) . in terms of effect size , ant-134 prevented 86% of the risk of epilepsy , with a 95% confidence interval ( ci ) of 12%98% .
an analysis of the duration of spontaneous seizures ( electrographically defined ) revealed that when spontaneous seizures occurred , they were similar between groups .
that is , the average duration of a spontaneous seizure in scr - treated rats was 53.7   14.4  s ( n  = 20 seizures , from five rats ) , which was similar to the duration in epileptic rats in the pps model that did not receive an i.c.v .
injection ( 49.4   15.9 , n  = 20 seizures ) . in the ant-134 rat that developed epilepsy ,
the average duration of a spontaneous seizure was 52.3   16.2  s ( from n  = 12 seizures ) .
having observed a disparity between the acute anti - seizure effects of ant-134 pre - treatment in the ptz and pps models , we sought to examine the effect of ant-134 on naive , non - epileptic tissue and whether the effects were due to long range networks or if local circuitry was sufficient to mediate any seizure protection .
we injected ant-134 in  vivo in rats and subsequently prepared ex  vivo brain slices ( figure  5a ) .
these slices were then exposed to a 9-mm k seizure challenge , to determine whether ant-134 protects against epilepsy in local circuits of the isolated slice in the absence of epileptogenesis .
onset of activity was delayed in ant-134-treated slices by 176  s relative to the control ( figures 5b and 5c ) .
this effect was statistically significant ( mann - whitney u test , p  = 0.002 ,   = 0.025 , power  = 0.87 ) .
we also compared the power of epileptiform activity and duration of seizure - like events in these data ; these effects were not statistically significant ( power : t test , p  = 0.70 ; duration : mann - whitney u test , p  = 0.067 ) .
this finding suggests that ant-134 can confer seizure resistance in naive tissue and thus mediate its effect even in the absence of epileptogenesis .
in addition , preservation of the seizure resistance in isolated slices indicates that at least some of the effects of ant-134 are due to local changes in activity .
the present study provides important additional evidence of the anticonvulsant and disease - modifying effects of lna - based antagomirs targeting mir-134 in animal models of epilepsy .
our study shows that ant-134 is an anticonvulsant in an acute chemoconvulsant model ( ptz in mice ) and ant-134 had anti - epileptogenic activity in the perforant pathway stimulation model of epilepsy in rats .
ant-134 also confers resistance to epileptic activity in naive tissue , reflected as a delay to activity onset following a seizure challenge in ex  vivo brain slices .
we also report that mir-134 levels are elevated in the hippocampus of patients with pharmacoresistant tle .
the therapeutic potential of targeting mir-134 in established epilepsy will require additional studies , but our findings may encourage further research or pre - clinical development of this mirna - based treatment for epilepsy . despite the recent introduction of two additional aeds ( perampanel and brivaracetam ) , new drug development in epilepsy
faces significant challenges.1 , 2 , 32 new targets are required that can treat pharmacoresistant patients or have disease - modifying effects that are not served by current therapies .
mirnas have emerged as potential targets of the future via their influence on neuronal microstructure , gliosis , neuroinflammation , and ion channels.8 , 33 silencing mir-134 has produced the most convincing anti - seizure effects to date , reducing status epilepticus in pre - treatment paradigms in the intra - amygdala ka and pilocarpine models and exerting potent long - term effects on spontaneous seizure development.19 , 22 this mirna is also an attractive target , since studies show increased levels of mir-134 within the temporal lobe of adult and pediatric patients with intractable epilepsy.19 , 20 we report here that mir-134 is also elevated in the hippocampus of adults with pharmacoresistant tle , although there are potential confounders with comparing resected material from patients to autopsy control samples . the present study has answered key questions that could facilitate pre - clinical development by showing that ant-134 has anticonvulsant effects in non - status epilepticus models and is disease modifying in another species .
the first important finding was that pre - treatment with ant-134 strongly suppressed seizures in the mouse ptz model .
effects on both clinical behavior , including delaying the time to first convulsive seizure , and electrographic seizure activity were seen .
thus , lowering brain levels of mir-134 can exert anti - excitability effects , and this implies that mir-134 has a role in setting excitability thresholds in the brain .
this is thought to be due to regulation of proteins that control dendritic morphology,14 , 15 although other targets are known .
ant-134 did not fully prevent seizures in the ptz model ; rather , it delayed and minimized their severity , a finding most closely matching the effect of ant-134 in the pilocarpine model of status epilepticus
. it may not be possible to fully prevent all seizures using ant-134 in chemoconvulsant models , but further adjustments to antagomir dose or delivery may yield optimal seizure - suppressive effects .
ptz triggers seizures via gabaergic blockade ; this differs from the mechanism by which status epilepticus is induced by ka and pilocarpine , which work by promoting glutamatergic and cholinergic transmission , respectively .
this suggests that ant-134 may be effective for epilepsies of various etiologies , which , in patients , can vary considerably between trauma , stroke , infection , genetic , and other causes .
our studies also show that ptz - induced convulsions increase brain levels of mir-134 , transcription of which is probably mediated by myocyte enhancer factor 2c .
we did not observe an increase in mir-134 after perforant pathway stimulation in the rat .
this was surprising , although changes to mir-134 levels are not always detected after chemoconvulsive stimuli , and we can not rule out a technical explanation ( e.g. , the time points selected or a subfield - specific effect that was masked by the use of the whole hippocampus ) .
our study included an analysis of the short - term effects of ant-134 on neuronal activity - regulated genes in the ptz model .
expression of genes such as c - fos and arc is a well - established surrogate marker of recent seizure activity , the protein products of which are involved in coordinating synaptic plasticity and adaptation to excessive neuronal stimulation.38 , 39 unexpectedly , only levels of c - fos , and not arc , showed the expected reductions in ant-134 mice after ptz seizures .
it is known that c - fos induction is calcium dependent , whereas arc expression can be induced via calcium - independent pathways.38 , 39 therefore , ant-134 treatment may have reduced seizure severity sufficient to prevent c - fos induction , such as by minimizing n - methyl - d - aspartate ( nmda ) receptor opening , but enough residual neuronal excitation remained to upregulate arc .
these findings provide additional evidence of the scale and nature of seizure suppression by ant-134 and suggest molecular markers that can distinguish effects of mirna manipulations on different components of seizure responses . in the present study
, we observed that pre - treatment of rats with ant-134 did not protect against perforant pathway stimulation - induced status epilepticus .
while there are examples of anticonvulsants producing effects in some but not all models , these findings suggest that ant-134 may have the most clinical value as an anti - epileptogenic treatment .
previous work showed that injection of ant-134 after status epilepticus in mice suppressed the later occurrence of spontaneous seizures by 90% , evidence of a disease - modifying effect in the intra - amygdala ka model . here
, we found nearly identical anti - epileptogenic effects of ant-134 in a different model in rats .
this is the only demonstration , to our knowledge , of any disease - modifying treatment in the pps model in rats .
the pps technique induces hippocampal sclerosis and recurrent spontaneous seizures,29 , 40 and it provides a means to trigger epilepsy without the bias or other issues associated with chemoconvulsants .
ant-134 was injected shortly after triggering status epilepticus in order to model a reasonably realistic clinical scenario , such as a patient being treated shortly after an initial precipitating injury .
ant-134 had dramatic and potent effects , with the majority of ant-134 rats ( six of seven , 86% ) never developing spontaneous recurrent seizures .
notably , spontaneous seizures in the study with the intra - amygdala ka model did occur in all ant-134 mice , albeit with dramatically lower seizure frequency .
this difference may relate to the extent of hippocampal injury or another aspect of the epileptic phenotype , such as the frequency of spontaneous seizures .
thus , presently we see a possible additional therapeutic action of ant-134 , whereby post - treatment is capable of fully preventing epilepsy at least in some models .
we can not , of course , exclude that epilepsy might have developed in some of the ant-134 rats at some point in the future . however , we recorded for 8  weeks beyond the standard latent period .
we further corroborated the anti - seizure effect of ant-134 by using an acute seizure challenge with high potassium in otherwise healthy ( ex  vivo ) tissue .
inhibitory neurotransmission is retained in this model ( in contrast to ptz ) and there is no hippocampal sclerosis ( unlike pps ) , further suggesting that ant-134 can be therapeutic in multiple epileptic syndromes with varying mechanisms .
we observed a delay in the onset of epileptiform bursts in brain slices that had been pre - treated with ant-134 .
this finding suggests that epileptogenesis is not required for ant-134 to mediate its effects on seizure onset .
we did not see any significant changes in the power or duration of epileptiform activity ; however , these parameters are highly variable in brain slices , which can obscure potential effects . in addition
, we propose that ant-134 can mediate seizure protection in the hippocampus via effects on local circuitry , because long range connections would likely be severed during the slicing process .
the anti - epileptogenic actions of ant-134 may be independent of the etiology of the initial precipitating injury , although this will require further testing ( e.g. , in models of trauma or infection - induced epilepsy ) .
in addition , upregulation of mir-134 is not essential for ant-134 to produce anti - epileptogenic effects , since mir-134 levels were not altered after pps - induced status epilepticus .
thus , epilepsy can be dramatically suppressed in two different in  vivo models , and one in  vitro , in two different species .
this provides strong support for and is an argument for further development of ant-134 for epilepsy treatment or prevention .
antagomirs targeting a liver - expressed mirna have been given to humans and were found to be well tolerated and effective against hepatitis c in clinical trials .
there are concerns , obviously , that because ant-134 can alter dendritic spines , it could have effects on cognition . studies to date have not identified such problems , with natural exploratory behavior and performance in a hippocampus - dependent task found to be normal in mice a few days after i.c.v .
injection of ant-134.19 , 22 however , more demanding tests of cognition or learning will need to be performed .
another factor that argues for general safety is that mice and rats have been recorded for at least 2  months after brain injections of ant-134 .
nevertheless , pre - clinical development will necessitate an extensive battery of toxicity and safety testing .
work to date on ant-134 satisfies certain recommendations for testing and evaluating novel therapies for epilepsy , with seizure - suppressive effects demonstrated in five different models and two different species.1 , 2 , 43 , 44 there are , however , additional seizure models that could be used during any future pre - clinical development .
this includes kindling , a popular model that has been successful in identifying aeds with novel mechanisms of action .
while this is not an essential step before pre - clinical development , sex differences in mirna inhibitor responses may be worth investigating in future studies and sexual dimorphism in mirna expression has been reported , although not for mir-134.46 , 47 it will also be of value to understand how ant-134 produces its potent seizure - suppressive effects .
the most likely mechanism is upregulation of one or more proteins due to de - repression of a mir-134 target .
previous work has shown that mir-134 is uploaded into the rna - induced silencing complex after seizures , which facilitates targeting of mrnas , and in  vitro studies suggest that the neuroprotective effects of ant-134 are due to de - repression of lim domain kinase 1 .
ant-134 treatment also increases dendritic spine volume and changes spine number in the hippocampus,19 , 22 which could influence excitability and would also be consistent with a mechanism involving protection of mir-134 targets such as lim domain kinase 1 or pumilio rna - binding family member 2
. altered spine volume and number is consistent with our observation of a robust local effect of suppressing mir-134 on delaying epileptiform activity , although additional effects on long range connections can not be ruled out .
we must also consider that the acute seizure - suppressive effects and long - term anti - epileptogenesis effects may arise from different mechanisms .
ultimately , in  vivo studies will be necessary to determine the anti - seizure mechanism of ant-134 ( e.g. , by analyzing gene expression networks , quantifying the proteome , and applying techniques to validate antagomir - mirna targeting such as polysome shift assays ) .
what are the next steps for translation or pre - clinical development ? an obvious question is whether and how antagomirs could be given to patients .
their large size precludes their passing an intact blood - brain barrier ( bbb ) .
recent work has explored methods to circumvent the bbb , including conjugating antagomirs with brain - penetrating peptides or administering antagomirs via intranasal injection.19 , 51 the bbb may also be open after certain epilepsy - precipitating injuries or it can be physically breached , such as through use of ultrasound . another question is whether ant-134 injection into already epileptic animals could alter the frequency or duration of spontaneous seizures or attendant hippocampal pathology .
success with that approach could provide novel treatment opportunities for the population of drug - resistant patients for whom surgery or other alternatives to aeds are either not possible or not effective currently . in summary ,
the present study provides important additional validation of the potent seizure - suppressive and possibly disease - modifying effects of ant-134 in two different animal models and one brain slice model .
the effectiveness of ant-134 and the potency of seizure suppression suggest a target for drug development in epilepsy that could provide additional effects beyond those provided by any currently marketed drug for epilepsy .
this study was approved by the ethics ( medical research ) committee of beaumont hospital of dublin ( no . 05/18 ) and written informed consent was obtained from all patients .
the control ( autopsy ) hippocampus ( n  = 12 ) was obtained from 12 individuals .
patients ( n  = 12 ) were referred for surgical resection of the temporal lobe by an epileptologist ( n.d . ) following neurological assessment , video - eeg recording , and mri / neuroimaging . each patient was determined to have medically intractable tle with a history of recurring seizures ( table s2 ) .
the hippocampus was obtained and a portion of each specimen was frozen in liquid nitrogen and stored at 70c until use .
a pathologist ( m.a.f . ) assessed the  hippocampus as part of the pathologic evaluation , addressed the degree of hippocampal sclerosis , and described other pathologic changes .
all animal experiments were performed in accordance with the european communities council directive ( 2010/63/eu ) .
procedures in mice were approved by the research ethics committee of the royal college of surgeons in ireland ( rec-842 ) , under license from the ireland health products regulatory authority ( ae19127/001 ) .
procedures in rats were performed under authorization of the philipps university bioethics committee and were approved by the local regulation authority ( regierungsprsidium gieen ) , or they were performed according to the uk animals ( scientific procedures ) 1986 act ( ppl 70 - 7684 ) and approved by local ethical review ( university college london ) .
male adult c57bl/6j mice ( 2025 g ) and male sprague - dawley rats ( 325350  g or 200300 g ; all from harlan ) were used in all studies .
animals were housed in on - site barrier - controlled facilities having a 12-hr/12-hr light / dark cycle with ad libitum access to food and water .
mice were equipped for eeg recordings under surgical anesthesia ( isoflurane ; 5% induction , 1%2% maintenance ) in a mouse - adapted stereotaxic frame .
body temperature was maintained within the normal physiological range with a feedback - controlled heat pad ( harvard apparatus ) .
after a midline scalp incision , partial craniectomies were performed and animals had skull - mounted recording electrodes placed and fixed with dental cement .
a second cohort of mice had eeg electrodes and a guide cannula implanted to allow i.c.v .
this cannula was placed on the dura mater with the following coordinates from the bregma : anterior - posterior ( ap ) ,  + 0.3  mm ; lateral ( l ) ,  + 0.9  mm ; and ventral ( v ) , 2.0  mm .
after recovery from surgery , mice were connected to the lead socket of a swivel commutator , which was connected to a grass twin digital eeg system .
] ) with different doses of ptz ( 60 , 80 , or 100  mg / kg ) . lorazepam ( 8  mg / kg , i.p . ) was administered after the first tonic - clonic seizure to reduce morbidity and mortality .
mice were followed for 30  min or 4  hr after ptz administration for the appearance of seizures by electrographic and behavioral methods .
after the observation period , mice were deeply anesthetized ( pentobarbital ) and transcardially perfused , and their brains were removed for analysis of gene expression or histological assessment .
injection of 0.12  nmol/2  l 3-cholesterol - tagged locked nucleic acid oligonucleotide targeting mir-134 ( ant-134 ) or its respective control , a non - targeting scrambled version of the antagomir ( scr ; 0.12  nmol/2  l ) ( exiqon a / s ) , as previously described.19 , 22 twenty - four hours later , mice were connected to an eeg system and a baseline recording was obtained .
next , mice were injected with ptz ( 80  mg / kg , i.p . ) .
lorazepam ( 8  mg / kg , i.p . ) was administered after the first tonic - clonic seizure to reduce morbidity and mortality .
these mice were followed up for 30  min after ptz administration for the appearance of seizures .
after this period , mice were deeply anesthetized ( pentobarbital overdose ) and transcardially perfused ( pbs ) for further assessments .
under isoflurane ( 5% induction , 3% maintenance ) anesthesia , bipolar stainless - steel stimulating electrodes ( nex-200 ; rhodes medical instruments ) were positioned in the angular bundles of the perforant pathway and custom unipolar recording electrodes ( crafted from 796000 ; a - m systems ) were lowered into the dorsal dentate gyrus .
electrodes and ground screws were connected to miniature wireless transmitters ( ft20 ; data sciences international ) that were implanted subcutaneously on the animal s flank .
plastic connectors ( gs09plg ; ginder scientific ) joined the electrodes with stimulation / recording equipment .
the pps protocol utilized a paradigm designed to evoke and maintain hippocampal seizure activity throughout the stimulation , but not convulsive status epilepticus , which consisted of continuous , bilateral 2-hz paired - pulse stimuli , with a 40-ms interpulse interval , plus a 10-s train of 20  hz single - pulse stimuli delivered once per minute , generated by an s88 stimulator ( grass instruments ) .
all pulses ( 0.1-ms duration ) were delivered at 2024 v , as this voltage reliably evokes granule cell discharging without tissue - damaging hydrolysis.29 , 40 the current associated with these voltages was typically between 15 and 30  a .
no samples displayed any signs of hydrolysis ( e.g. , holes around the stimulating electrodes ) .
as described previously , stimulation for 30  min ( on 2 consecutive days ) required only isoflurane to terminate seizures .
eight - hour stimulation on the third day did not induce convulsive status epilepticus and , therefore , did not require pharmacological termination .
rats were randomly divided in three cohorts to assess the following : ( 1 ) mir-134 expression after pps ( 24  hr , 4  days , and 14  days ) ; ( 2 ) the effect of ant-134 on pps - induced status epilepticus , in which rats were pre - treated with ant-134 or scr ( 0.36  nmol/6  l , i.c.v . ; exiqon a / s ) 24  hr before the 8-hr stimulation ; and ( 3 ) the effect of ant-134 on epileptogenesis , in which rats were injected with 0.36  nmol/6  l ( i.c.v . ) of ant-134 or scr ( exiqon a / s ) immediately after 8-hr stimulation .
five rats were excluded from this study due to practical issues ( e.g. , blocked cannula ) . for statistical purposes
, we pooled samples from 24  hr and 4  days after pps as an  early  time point , while 14-day samples were considered as the  late  time point .
mouse eeg data were analyzed and quantified using labchart 8 software ( ad instruments ) .
ptz - induced seizures were defined as high - amplitude ( > 2 baseline ) high - frequency ( > 5  hz ) polyspike discharges lasting > 5 s. for statistical purposes , we assigned a cut - off time of 1,800  s for those animals that did not present seizures during the observation period . from the eeg recordings ,
we calculated the delay to first seizure , the total power , and the percentage of total power / spectral bands , as previously described.19 , 54 eeg total power was plotted as the percentage of baseline recording ( each animal s eeg power post - seizure compared to its own baseline eeg ) .
clinical behavior was scored using the following adapted racine scale : score 0 , no seizures observed ; score 1 , rhythmic mouth and facial movement ; score 2 , rhythmic head nodding ( bobbing ) ; score 3 , forelimb clonus ; score 4 , rearing and bilateral forelimb clonus ; and score 5 , rearing and falling ( stay fallen on rear side ; tonic - clonic seizures ) .
the highest score reached during every 5  min during the 30  min after ptz was recorded .
for studies in rats , eeg activity during and after pps was amplified and recorded digitally at 2 khz utilizing a powerlab 16/35 and labchart software versions 7 and 8 ( ad instruments ) .
spontaneous granule cell layer activity was recorded continuously ( 24 hr / day ) and stored digitally and automatically in 3-hr epochs . each day , we analyzed the preceding 24  hr of recordings , as well as all events with amplitudes exceeding 120% of average baseline .
continuous ( 24 hour/7  day ) video monitoring utilized edimax  7010w infrared cameras .
video files were captured at 1215  frames / sec and were time - stamped for integration with the electrophysiological data using securityspy surveillance software ( ben software ) and stored digitally . confirmed seizures were scored according to the racine scale .
rats were anesthetized with isoflurane ( 5% induction,2% maintenance as appropriate ) and mounted in a stereotaxic frame .
we injected ant-134 ( 0.12  nmol ) in 1  l te buffer ( life technologies ) at coordinates relative to the bregma : ap , 0.92  mm ; medial - lateral ( ml ) ,  + 1.3  mm , and dorsal - ventral ( dv ) , 3.3  mm to target the lateral ventricle . to allow the rats to fully recover from the analgesic drugs and to coincide with the maximal anti - epileptic effect seen in  vivo
sodium pentobarbital and transcardially perfused with ice - cold sucrose artificial cerebrospinal fluid ( acsf ; in mm : 205 sucrose , 10 glucose , 2.5 kcl , 0.1 cacl2 , 5 mgcl2 , 26 nahco3 , and 1.2 nah2po4.h2o ; osmolarity , 295 mosm ) .
the brain was quickly dissected into ice - cold sucrose acsf and transferred to a campden 700 smz slicer ( campden instruments ) . slices ( 400  m ) were prepared in the horizontal orientation between the bregma at approximately 7.1 and  5.1  mm . for storage ,
slices were submerged in oxygenated normal acsf ( in mm : 125 nacl , 10 glucose , 3 kcl , 1 mgcl2 , 2 cacl2 , 26 nahco3 , and 1.25 nah2po4.h2o ; osmolarity , 290 mosm ) and allowed to recover at room temperature for at least 60  min prior to recordings .
slice recordings were made using a membrane chamber ( scientific systems design ) , a modified submerged chamber , which improves oxygen supply to the tissue and supports greater epileptiform bursts than conventional submerged recording chambers .
slices were continually perfused with acsf , heated to 32c , at a flow rate of 16  ml / min . for local field potential ( lfp ) recordings ,
borosilicate glass recording micropipettes ( resistance , 34 m ) were placed into hippocampal ca1b stratum pyramidale and epileptiform activity was induced by raising the perfusing kcl concentration to 9  mm .
total rna was isolated from brain samples using trizol ( invitrogen ) , as described previously.19 , 54 for mrna qpcr , 1  g total rna was used to generate cdna by reverse transcription using the superscript iii reverse transcriptase enzyme ( invitrogen ) .
the pcr amplification was performed for 50 cycles on a thermocycler ( applied biosystems ) .
quantitative real - time pcr was performed using a lightcycler 1.5 ( roche diagnostics ) in combination with quantitect sybr green pcr kit ( qiagen ) as per the manufacturer s protocol with 25  m of primer pairs used .
specific primers for each gene assayed were purchased from sigma and sequences used were as follows : actin , beta ( actb ) ( forward [ f ] , ggttggccttagggttcagg ; reverse [ r ] , gggtgtgatggtgggaatgg ) ; c - fos ( f , cattcagaccacctcgacaa ; r , ggaattaacctggtgctgga ) ; and arc ( f , agcagcagacctgacatcct ; r , gtgatgccctttccagacat ) .
data were normalized to actb and relative mrna transcript levels were quantified using the ct method . for mirna ,
250  ng rna was reverse transcribed using stem - loop specific primers for mmu - mir-134 ( applied biosystems ) and real - time quantitative pcr was carried out on a 7900ht fast real - time system ( applied biosystems ) using taqman mirna assays .
expression of rnu19 was used for normalization . a relative fold change in expression of mir-134
was determined using the ct method.19 , 56 specific mirna assays were obtained from thermofisher scientific for mir-134 ( i d 001186 ) , mir-19a ( i d 000395 ) , and rnu19 ( i d 001003 ) .
data were normalized to rnu19 and relative mirna expression levels were quantified using the ct method .
statistical analysis was performed using graphpad prism and stata release 14 software , and a p value  < 0.05 was considered significant .
latencies to tonic - clonic seizures were analyzed by the kruskal - wallis test , followed by the nonparametric dunn multiple comparison test when indicated .
total time spent in seizures , mean amplitude , frequency , total power of eeg recordings , and mrna levels were analyzed by the student t test or one - way anova followed by the bonferroni test , depending on the experimental design .
ex  vivo brain slice data were analyzed using the mann - whitney u test .
three comparisons were made from the same data and  ( significance level ) was consequently reduced to 0.025 for these experiments .
, d.f.o . , and n.d . provided human samples and data ; c.r.r . , s.s .
wrote the paper and all co - authors contributed to data interpretation , review , and editing of the final paper .
the royal college of surgeons in ireland filed for a patent on the inhibition of microrna-134 for the treatment of seizure - related disorders and other neurologic injuries .
 current anti - epileptic drugs ( aeds ) act on a limited set of  neuronal targets , are ineffective in a third of patients with epilepsy , and do not show disease - modifying properties . 
 micrornas are small noncoding rnas that regulate levels of proteins by post - transcriptional control of mrna stability and translation . 
 microrna-134 is involved in controlling neuronal microstructure and brain excitability and previous studies showed that intracerebroventricular injections of locked nucleic acid ( lna ) , cholesterol - tagged antagomirs targeting microrna-134 ( ant-134 ) reduced evoked and spontaneous seizures in mouse models of status epilepticus . 
 translation of these findings would benefit from evidence of efficacy in non - status epilepticus models and validation in another species . here , we report that electrographic seizures and convulsive behavior are strongly reduced in adult mice pre - treated with ant-134 in the pentylenetetrazol model . 
 pre - treatment with ant-134 did not affect the severity of status epilepticus induced by perforant pathway stimulation in adult rats , a toxin - free model of acquired epilepsy . 
 nevertheless , ant-134 post - treatment reduced the number of rats developing spontaneous seizures by 86% in the perforant pathway stimulation model and ant-134 delayed epileptiform activity in a rat ex  vivo hippocampal slice model . 
 the potent anticonvulsant effects of ant-134 in multiple models may encourage pre - clinical development of this approach to epilepsy therapy . 
Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest