Validation of the Zebrafish Pentylenetetrazol …Validation of the Zebrafish Pentylenetetrazol Seizure Model: Behavior Assay for Assessing Anti-Epileptic Drug Efficacy Chongbin Liu

International Journal of Research in Pharmacy and Biosciences

Volume 3 Issue 1 January 2016 PP 24-38

ISSN 2394-5885 (Print) amp ISSN 2394-5893 (Online)

Address for correspondence

liuchongbin1972126com

International Journal of Research in Pharmacy and Biosciences V3 I1January 2016 24

Validation of the Zebrafish Pentylenetetrazol Seizure Model

Behavior Assay for Assessing Anti-Epileptic Drug Efficacy

Chongbin Liuab

Juan Zhoub Yong Zhang

b Taotao Zhu

b Rui Wang

a Chunqi Li

bc

aDepartment of Physiology Huzhou Teachers College Huzhou Zhejiang Province P R China

bZhejiang Provincial Key Lab for Technology and Application of Model Organisms Wenzhou Medical

University Wenzhou Zhejiang Province P R China cHunter Biotechnology Inc Transfarland Hangzhou Zhejiang Province P R China

ABSTRACT

The availability of zebrafish larvae model of epileptic seizure provides opportunities to identify novel

anticonvulsants for treatment of people with epilepsy However the major parameters of zebrafish behavior

assay for assessing anti-epileptic drug efficacy existed disparity which resulted in different results in previous

studies In this study we chose the high medium slow-speed moved distances and the total distances moved for

seizure-like activity quantification in individual wells of a 48-well plate in the dark phase and used this zebrafish

seizure model to assess three commonly prescribed anti-epileptic drugrsquos efficacy and screen bioactive

components from plant extrat Results showed that the high-speed moved distances were given a more

reasonable and sensitive dose-response curve than the total distances in zebrafish larvae model exposed to 10

mM pentylenetetrazole Besides we also optimized the vehicle (dimethyl sulfoxide DMSO) concentration used

in epileptic behavior assay The three anti-epileptic drugs phenytoin valproate sodium and carbamazepine

showed the same efficacy patterns in zebrafish seizure model as in mammalian epileptic models We also found

four positive hits from plant (Vaccinium spp) extract in primary drug screening two hits exhibited

concentration-dependent inhibition of locomotor activity confirming their anticonvulsant characteristics These

results indicated that this zebrafish larvae model could be useful for assessing anti-epileptic drug efficacy

facilitating the primary drug screening and evaluating of effective components in medicinal plants

Key Words Disease Model Epilepsy Methods Pentylenetetrazole Seizure Zebrafish Larvae

INTRODUCTION

Epilepsy is a complex brain disorder marked by recurrent spontaneous epileptic seizures associated

with typical neurobiological and behavioral alterations (Pack et al 2011 Pitkanen and Lukasiuk

2009 Stewart et al 2012) In the past few decades rodent seizure models induced by a broad

spectrum of mechanisms have contributed significantly to our knowledge on epilepsy and

anti-epileptic drugs (AEDs) discovery (Kwan et al 2010 Loumlscher 2011 Loumlscher and Schmidt 2011

Morimoto et al 2004)

Zebrafish larvae have recently emerged as a new species for chemoconvulsant-based models of

epilepsy (Afrikanova et al 2013 Berghmans et al 2007 Ellis and Soanes 2012 Lee et al 2012)

Because of the genetic similarity between zebrafish and humans zebrafish larvae offer significant

advantages for high-throughput drug screening (Eliceiri et al 2011 Howe et al 2013 Jeong et al

2008 Peterson and Macrae 2012) Following on work first described by Baraban et al in 2005

several groups have confirmed the abnormal seizure-like behaviors ictal- and interictal-like electrical

Chongbin Liu et al ldquoValidation of the Zebrafish Pentylenetetrazol Seizure Model Behavior Assay for

Assessing Anti-Epileptic Drug Efficacyrdquo

25 International Journal of Research in Pharmacy and Biosciences V3 I1 January 2016

activity and c-fos expression in brain regions when zebrafish larvae exposed to pentylenetetrazole

(PTZ) (Hortopan et al 2010) With regard to anticonvulsant screens medium- to high-throughput

screening is possible in 96-well format using an automated locomotor tracking system for the

sensitivity to common antiepileptic drugs in this model (Baraban et al 2013) Also several groups

developed a seizure model in zebrafish larvae and made antiepileptic drugs screening from nature

plant (Lee et al 2012 Orellana-Paucar et al 2012) However the major parameters of zebrafish

behavior assay for assessing antiepileptic drug efficacy existed disparity which resulted in different

results in previous studies (Stewart et al 2012) Moreover a question remained as to whether an

AED-induced decrease in locomotion is truly indicative of anticonvulsant activity as some drugs may

impair larval movement through other mechanisms such as general toxicity or sedation So there still

remained some uncertainties to clear before using PTZ-induced zebrafish larvae epileptic seizure

model for anticonvulsant drug screening widely In this study we performed another behavioral

seizure analysis of PTZ-induced seizures refining parameters of the behavioral PTZ assay and

assessing three different antiepileptic drugs efficacy We also used this PTZ-induced seizure model to

discover anticonvulsant compound from plant extract We have demonstrated that this model can be

useful for AEDs primary screening This will also provide an alternative method for test potential

therapeutics of epilepsy

METHODS

Zebrafish Handling

Adult zebrafish (Danio rerio AB strain) were housed in a light- and temperature-controlled

aquaculture facility with a standard 14 10 h lightdark photoperiod Four to five pairs of zebrafish

were set up for nature mating every time On average 200 - 300 embryos were generated Embryos

were collected and staged from multiple AB strain breeding pairs and pooled Following 6 and 24 hpf

(hours post fertilization) in an incubator in E3 media (5 mM NaCl 017 mM KCl 033 mM CaCl2

033 mM MgSO4) unfertilized embryos were removed and the remainder placed in a re-circulating

Tecniplast aquatic system at 28 degC (02 Instant Ocean Salt in deionized water pH 69 - 72

conductivity 480 - 510 μScm and hardness 537 - 716 mgL CaCO3) (Kimmel et al 1995) Also

embryos raised on the system displayed more consistent activity patterns than those reared in a

petri-dish After completing the experiments all larvae were sacrificed through administration of an

overdose of anesthetic (tricaine) The zebrafish facility at Hunter Biotechnology Inc is accredited by

the Association for Assessment and Accreditation of Laboratory Animal Care (AAA LAC)

International

Drug Treatment

Pentylenetetrazole (PTZ Sigma CAS No 54-95-5) was used to induce zebrafish epileptic seizure

Three AEDs carbamazepine (CBZ ACR ORGANICS CAS No 298-46-4) phenytoin (PHT JampK

CAS No 630-93-3) valproate sodium (VPA Sigma CAS No 1069-66-5) and negative control drug

aspirin (Sigma CAS No 50-78-2) were selected for the development and validation of zebrafish

epileptic seizure model Stock solutions were prepared in 100 dimethyl sulfoxide (DMSO) and

stored at -20 degC serial dilutions (1 2 and 3 DMSO) were made before each experiment All

compounds were dissolved in DMSO and diluted in embryo medium to achieve a final DMSO

concentration of 1 wv For zebrafish epilepsy model validation PTZ was added to fish water to

reach the desired concentrations ranging from 125 mM to 30 mM Control embryos were raised in

fish water without PTZ For testing embryo response to anticonvulsants AEDs and aspirin were

added to the fish water to reach the indicated working concentrations 1 hour before the embryos were

exposed to 10 mM PTZ for behavioral analysis and other examinations For AEDs primary screening

the concentrations of plant extract were the maximal tolerable concentration for zebrafish larvae



International Journal of Research in Pharmacy and Biosciences V3 I1 January 2016 26

Behavioral Analysis

5 dpf (days post fertilization) zebrafish larvae were pre-incubated in 100 μl of AED aspirin or vehicle

for 1 h in individual wells of a multi-well plate at 28 degC in the dark In model optimization experiment

a 48-well and a 96-well plate was used respectively For AEDs primary screening from plant extract

we performed in individual wells of a 48-well plate At least eight larvae were used for each group

and every experiment was performed in duplicate The concentrations of AEDs and aspirin used were

described in the results section After the pre-incubation 100 μl of embryo medium or 100 μl of PTZ

solution was added Larvae were allowed to habituate for 10 min in a dark chamber of an automated

tracking device (ZebraBoxTM

apparatus Viewpoint Lyon France) Control groups were embryos of

the same stages without any chemical treatment The locomotor activity was then quantified using

ZebraLab TM

software (Viewpoint Lyon France) (Afrikanova et al 2013) The system consists of an

infrared light source a high-resolution digital video camera to capture larval movements within a

defined time period (60 min in our experimental set-up) and the software to analyze larval locomotor

activity The total and high-speed moved distances were chosen for subsequent efficacy assay

Efficacy of a test drug on epilepsy was calculated based on the formula below

Drug effect on epileptic locomotor activity () = (1-ehicle)Distance(v

rug)Distance(d) times 100

C-Fos Gene Expression Analysis

For RT-PCR 5 dpf larvae were exposed to PTZ PTZ + AEDs or fish water and then the total RNA

was isolated from the whole larvae (n = 50 per group) using single TRI reagent (Sigma catalog

T9424) and cDNA was prepared from 1 μg of the total RNA per group using RT-PCR kits (TOYOBO

catalog FSK-100) in a 20 μl volume Each PCR was carried out in triplicate in a 25 μl volume using

Taq PCR MasterMix kits (Bioteke catalog PR1701 ) on Lifepro Thermal Cycler System (BIOER

China) with standard parameters C-fos cDNA was amplified with forward primer

5rsquo-AACTGTCACGGCGATCTCTT-3rsquo and reverse primer 5rsquo-GCAGGCATGTATGGTTCAGA-3rsquo

(Baraban et al 2005) the predicted size of the amplified cDNA fragment was 1030 bp β-actin used

as internal control were 5-CATCAGCATGGCTTCTGCTCTGTATGG-3 (foward) and

5-GACTTGTCAGTGTACAGAGACACCCT-3 (reverse) All the primers used for RT-PCR analysis

were synthetised by Invitrogen

Statistical Analysis

One-way ANOVA followed by the Dunnettrsquos multiple comparison test was used to compare

differences among groups All statistical analysis were performed using SPSS 160 software (SPSS

USA) and P lt 005 was considered statistically significant For quantitative analysis all data were

presented as mean plusmn SEM and results were statistically compared between drug-treated and

vehicle-treated zebrafish groups

RESULTS

Proconvulsant Treatment

To induce seizures PTZ (125 - 30 mM) was added to fish water PTZ added to fish reliably elicited

distinct seizure-like behaviors in a concentration-dependent manner Zebrafish larvae exhibited signs

of agitation within seconds after exposed to PTZ The larvae swam along the periphery of the

multi-well plate thus displaying stage I seizure-like behavior as described previously (Hortopan et al

2010) This was succeeded by rapid lsquowhirlpool-likersquo movement and then followed by a short pause

before swimming in a rapid jerky manner with occasional body-stiffening and loss of posture (larva

turning onto its side or back) These events could be likened to tonic and clonic seizure phases in




mammals (stages I - III) (Berghmans et al 2007) However the results were obviously different

when the locomotor activity of zebrafish seizure model was quantified by different parameters the

high-speed (V﹥20 mms) moved distance was given a more reasonable and sensitive dose-response

curve than the total distance (Figure 1A and B) PTZ at 10 mM was selected for following behavior

test which induced the most significant increase in locomotor activity within 1 h

Figure1A

Figure1B

Figure1 Behavioral profile of zebrafish larvae during a one-hour exposure to vehicle or PTZ

The high-speed moved distance (A) and the total distance moved (B) over the 60 min of video-tracking Values

are given as Means plusmn SEM (n = 8) Asterisks (

) indicate values that are significantly different (P lt 001) using

one-way ANOVA with Dunnettrsquos post-test

In order to avoid the influence on the experimental results of improper use of DMSO serial dilutions

(1 2 and 3 DMSO) were added after PTZ-induced seizure model A 48-well and a 96-well plate

was also used respectively for behavioral analysis Results showed that the high-speed moved

distance decreased significantly in 2 and 3 (P lt 001) DMSO groups when a 48-well plate was

used However the high-speed moved distance decreased significantly (P lt 005 or P lt 001) in all

serial dilutions DMSO when a 96-well plate was used (Figure 2A and B) Therefore our results

demonstrated that if zebrafish larvae were placed in individual well of 96-well microplate there

would be more false positive than in 48-well microplate and even 1 DMSO treated alone the most

used vehicle would give a significant positive effect on zebrafish seizure-like movement




Figure2A

Figure2B

Figure2 Behavioral analysis of adding serial dilutions DMSO after PTZ-induced seizure model

A 48-well plate (A) and a 96-well plate (B) was used for behavioral analysis Values are given as Means plusmn SEM

(n = 8) Asterisks indicate values that are significantly different (P lt 005

P lt 001) using one-way ANOVA

with Dunnettrsquos post-test

Evaluation of the Anticonvulsant Activity of CBZ PHT VPA and Aspirin in the Zebrafish PTZ

Model

The capacity of the investigated drugs to reduce PTZ-induced convulsions in zebrafish larvae was

assessed through quantification of the high-speed moved distance A sample movement plot was given

in Figure 3A-C Individual drug concentrationresponse data were given in Figure 4A-D As

expected PHT and VPA respectively suppressed zebrafish larvae locomotor activity by 74 - 472

and 156 - 166 when comparing with PTZ treated alone group Statistically significant positive

effect on zebrafish larvae seizure-like motility was observed at 300 μM and 1000 μM for PHA (P lt

001) 1000 μM and 3000 μM for VPA (P lt 001) Whereas CBZ promoted zebrafish larvae

movement by 26 at 30 μM and 1167 at 100 μM (P lt 001) and sharply suppressed zebrafish

larvae motility about 948 at 300 μM (P lt 001) when comparing with PTZ treated alone group

Aspirin had no significant effect on zebrafish larvae locomotor activity




Figure3A

Figure3B

Figure3C

Figure3 Sample movement tracking plots for experiments with three AEDs

(A) CBZ treatment of activity changes produced by PTZ (B) PHT treatment of activity changes produced by

PTZ (C) VPA treatment of activity changes produced by PTZ Red indicate speed exceed 20 mms white

indicate speed is less than 4 mms green indicate speed is between 20 mms and 4 mms




Figure4A-1

Figure4A-2

Figure4B-1




Figure4B-2

Figure4C-1

Figure4C-2




Figure4D-1

Figure4D-2

Figure4 Quantitative analysis of behavioral seizures induced by PTZ and of response to AEDs

Values are given as Means plusmn SEM (n = 8) Asterisks (

) indicate values that are significantly different (P lt 001)

using one-way ANOVA with Dunnettrsquos post-test

Assessment of Changes in C-Fos Gene Expression by RT-PCR

Using semi-quantitative approach our experiment showed an obvious increased expression of c-fos

mRNA during PTZ exposure (P lt 001) CBZ at 30 μM and 300 μM could partially rescued

PTZ-evoked abnormal increase of c-fos expression (P lt 005 and P lt 001 respectively) The results

of c-fos gene expression were not well consist with the behavior assay Aspirin had no significant

effect as expected (Figure 5A and B)




Figure5A

Figure5B

Figure5 C-fos expression in zebrafish larvae after exposed to different AEDs

(A) RT-PCR analyses of c-fos expression in zebrafish larvae (B) Semi-quantification of c-fos mRNA by comparing with vehicle treated group Values are given as Means plusmn SEM (n = 50) Asterisks indicate values that are significantly different (

P lt 005

P lt 001) using one-way ANOVA with Dunnettrsquos post-test

Primary Drug Screening from Plant (Vaccinium Spp) Extract in PTZ-Induced Seizure Model

For AEDs primary screening ten different concentrations of plant (Vaccinium spp) extract

compounds were the maximal tolerable concentration for zebrafish larvae 1 DMSO was made

before each experiment In 10 mM PTZ-induced seizure model compound 2 (at 1 M) 5 (at 300 μM)

6 (at 1 M) and 7 (at 1 M) could partially rescued PTZ-evoked abnormal behavior (P lt 005 and P lt

001 respectively) in individual well of 96-well microplate (Figure 6A) The capacity of the four

compounds to reduce PTZ-induced convulsions in zebrafish larvae was also assessed through

quantification of the high-speed moved distance in individual well of 48-well microplate Results

showed that seizure-like swimming pattern was alleviated by the addition of either compound 5

(Cmpd 5) or compound 7 (Cmpd 7) Results also revealed that they exhibited concentration

-dependent inhibition of both locomotor activity (Figure 6B and C)

Figure6A




Figure6B

Figure6C

Figure6 Quantitative analysis of response to VPA and of compounds from plant (Vaccinium spp) extract

(A) A 96-well plate was used for primary durg screening in ten different concentrations compounds (B) The

capacity of the compound 5 to reduce PTZ-induced convulsions in zebrafish larvae in individual well of 48-well

microplate (C) The capacity of the compound 7 to reduce PTZ-induced convulsions in zebrafish larvae in

individual well of 48-well microplateValues are given as Means plusmn SEM (n = 50) Asterisks indicate values that

are significantly different (P lt 005


DISCUSSION

Zebrafish (Danio rerio) has emerged over the last decade as an attractive model for genetic studies

and drug screening (Loumlscher 2011 Mussulini et al 2013) Numerous studies have showed that adult

or zebrafish larvae exposed to PTZ exhibit increased locomotor activity seizure-like behavior and

epileptiform electrographic activity (Afrikanova et al 2013 Hortopan et al 2010 Pineda et al

2011 Wong et al 2010 Vermoesen et al 2011) which have a good correlation between zebrafish

and rodent data (Baxendale et al 2012 Hortopan et al 2010 Mussulini et al 2013

Orellana-Paucar et al 2012) Also many reseachers have used zebrafish seizure model for

anti-epileptic compound discovery and proved that zebrafish can be a very useful model for

anticonvulsant drug screening and early efficacy in vivo assessment (Afrikanova et al 2013

Berghmans et al 2007 Henry et al 2010 Hortopan et al 2010 Johannessen 2008) These models

are currently in existence that subserve particular roles in achieving these aims but all have their

limitations (Henry et al 2010) Moreover the major parameters of zebrafish behavior assay for

assessing anti-convulsant efficacy exist disparity including drug treatment period pre- or




post-treatment light condition microplate format and assessment criteria which result in different

results in different studies So there still remain some uncertainties to clear before using PTZ-induced

zebrafish larvae epileptic seizure model for anti-convulsant drug screening widely

In the present study zebrafish larvae were pre-treated for 1 h before being evoked by PTZ and then

were kept at 28 degC in the dark phase for another 1 h the total and the high-speed moved distances

were chosen for subsequent efficacy assay Also our results showed that if zebrafish larvae were

placed in individual well of 96-well microplate there would be more false positive than in 48-well

microplate and even 1 DMSO treated alone the most used vehicle would give a significant

positive effect on zebrafish seizure-like movement Therefore the 48-well microplate was used in this

research just as Baxendale et al (2012) did Also we used the high-speed moved distances as a

criteria for AEDs efficacy assessment not the total moved distances used by other reseachers

Because we think that the high-speed moved distances were given a more reasonable and sensitive

dose-response curve than the total distances in the proconvulsant treatment experiment Previous

study have proved that when PTZ evoked clonus-like convulsion (stage III) seizure behavior in the

presence of high concentrations zebrafish exhibited stage III and the moved distance reduced when

comparing with stage I and stage II (Baraban et al 2005 2013 Orellana-Paucar et al 2012 Winter

et al 2008) whereas zebrafish kept in high-speed movement in these two earlier stages Therefore

the high-speed moved distance is more typical than the total distance as a index for anticonvulsant

efficacy assessment

Moreover both the behavioral and RT-PCR analysis data showed that drugs were absorbed well by

zebrafish larvae within a defined time period (1 h pretreated and 1 h co-treated with PTZ 2 h totally)

and most false negative results could be avoided if maximum tolerant concentration was used These

results were consistent with Orellana-Paucar et al (2012) and Baxendale et al (2012) data

Therefore for anticonvulsant drug screening it does not need to treat zebrafish larvae more than 2 h

especially in high-throughput screening We kept zebrafish in the dark phase during the whole

experiment as Berghmans et al (2007) did It has been proved that the shift of light and dark could

affect zebrafish locomotor activity and zebrafish being pretreated with PTZ showed a reversal of the

normal response to cycles of light and dark (Ellis and Soanes 2012) So in order to minimize

disruptive factors and the risk of compound photodegradation it is prefered that the experiment

should be performed in the dark phase

The results of the present study were consistent with mammalian experiments and clinical data VPA

acts on a number of targets including GABA glutamine VG sodium and calcium channels (Chang et

al 2012 Johannessen 2008) CBZ primarily acts as an inhibitor of voltage-gated sodium channels

(VGSCs) and likely opposes PTZ-induced activity through a general non-specific decrease in neural

activity as found in a rodent model of mania (Arban et al 2005) Numerous studies have used the

expression of the immediate early genes (IEGs) c-fos and c-Jun as indirect markers of central nervous

system (CNS) neuronal activation particularly in response to acute seizure (Herrera and Robertson

1996 Hidaka et al 2011 So et al 1994) In this present study results showed that CBZ promoted

zebrafish larvae movement at 30 μM and 100 μM and sharply suppressed zebrafish larvae motility at

300 μM Also CBZ at 30 μM and 300 μM could partially rescued PTZ-evoked abnormal increase of

c-fos expression meanwhile CBZ at 100 μM had no significant effect It has been reported that CBZ

intoxication has been associated with aggravation of seizures this may be due to increased levels of

the CBZ-10 11-epoxide metabolite (Chen et al 1999) Our results firstly revealed that CBZ could

promote seizure-like motility in a zebrafish larvae anticonvulsant activity assay Overall PHT and

VPA appeared to provide a stronger opposition to the PTZ-induced changes in activity than CBZ This

would seem to reflect their previously defined therapeutic potential as PHT and VPA are

recommended as first line treatments of bipolar disorder while CBZ is only recommended as a second

line treatment (Nivoli et al 2011)




In the present study we used this PTZ-induced zebrafish seizure model and firstly obtained two

anticonvulsant compounds from plant (Vaccinium spp) extract in AEDs primary screening

Vaccinium spp is a kind of perennial defoliate or evergree shrub It grows in many regions including

the Europe Russia North America the Alps Mountain and parts of China It is used to treat

neurological diseases in China but its mechanism is not clear Many natural plant products

possessing large molecular weight will have biological activity when going through in vivo

metabolismTherefore the traditional method of drug screening in vitro sometimes does not apply to

the activities of natural plant products Zebrafish as an in vivo model possessing high speed screening

short design cycle and small dose drug usage it will provide a new research method for further

research and development of the medicinal plants The popularization and application of the zebrafish

model will greatly facilitate the screening and evaluation of effective components in medicinal plants

ACKNOWLEDGEMENT

The authors are grateful to the Zhejiang Province Natural Sciences Foundation (Y2110388) and

Zhejiang Province Technological Research (Y2011C37091) for financial support

REFERENCES

[1] AFRIKANOVA T SERRUYS AS BUENAFE OE CLINCKERS R SMOLDERS I DE WITTE

PA CRAWFORD AD ESQUERRA CV Validation of the zebrafish pentylenetetrazol seizure

model locomotor versus electrographic responses to antiepileptic drugs PLos one 2013 8 1-9

[2] ARBAN R MARAIA G BRACKENBOROUGH K WINYARD L WILSON A GERRARD P

LARGE C Evaluation of the effects of lamotrigine valproate and carbamazepine in a rodent

model of mania Behav Brain Res 2005 158 123-132

[3] BARABAN SC DINDAY MT HORTOPAN G A Drug screening in Scn1a zebrafish mutant

identifies clemizole as a potential Dravet syndrome treatment Nature Commu 2013 4 2410

[4] BARABAN SC TAYLOR MR CASTRO PA BAIER H Pentylenetetrazole induced changes in

zebrafish behavior neural activity and c-fos expression Neurosci 2005 131 759-768

[5] BAXENDALE S HOLDSWORTH CJ MEZA-SANTOSCOY PL HARRISON MR FOX J

PARKIN CA INGHAM PW CUNLIFFE VT Identification of compounds with anti-convulsant

properties in a zebrafish model of epileptic seizures Dis Models Mech 2012 5 773-784

[6] BERGHMANS S HUNT J ROACH A GOLDSMITH P Zebrafish offer the potential for a

primary screen to identify a wide variety of potential anticonvulsants Epilepsy Res 2007 75

18-28

[7] CHANG P ORABI B DERANIEH RM DHAM M HOELLER O SHIMSHONI JA YAGEN B

BIALER M GREENBERG ML WALKER MC WILLIAMS RS The antiepileptic drug

valproic acid and other medium-chain fatty acids acutely reduce phosphoinositide levels

independently of inositol in Dictyostelium Dis Models Mech 2012 5 115-124

[8] CHEN B WANG JF HILL BC YOUNG LT Lithium and valproate differentially regulate brain

regional expression of phosphorylated CREB and c-fos Mol Brain Res 1999 70 45-53

[9] ELICEIRI BP GONZALEZ AM BAIRD A Zebrafish model of the blood-brain barrier

morphological and permeability studies Methods Mol Biol 2011 686 371-378

[10] ELLIS LD SOANES KH A larval zebrafish model of bipolar disorder as a screening platform

for neuro-therapeutics Behav Brain Res 2012 233 450-457

[11] HENRY BL MINASSIAN A YOUNG JW PAULUS MP GEVER MA PERRY W

Cross-species assessments of motor and exploratory behavior related to bipolar disorder

Neurosci Biobehav Review 2010 34 1296-1306

[12] HERRERA DG ROBERTSON HA Activation of c-fos in the brain Prog Neurobiol 1996 50

83-107




[13] HIDAKA N SUEMARU K TAKECHI K Inhibitory effects of valproate on impairment of

Y-maze alternation behavior induced by repeated electroconvulsive seizures and c-fos protein

levels in rat brains Acta Medica Okayama 2011 65 269-277

[14] HORTOPAN GA DINDAY MT BARABAN SC Spontaneous seizures and altered gene

expression in GABA signaling pathways in a mind bomb mutant zebrafish J Neurosci 2010 30

13718-13728

[15] HOWE K CLARK MD TORROJA CF TORRANCE J BERTHELOT C MUFFATO M

COLLINS JE HUMPHRAY S MCLAREN K MATTHEWS L MCLAREN S SEALY I

CACCAMO M CHURCHER C SCOTT C BARRETT JC KOCH R RAUCH GJ WHITE S

CHOW W KILIAN B QUINTAIS LT GUERRA-ASSUNCAO JA ZHOU Y GU Y YEN J

VOGEL JH EYRE T REDMOND S BANERJEE R CHI J FU B LANGLEY E MAGUIRE

SF LAIRD GK LLOYD D KENYON E DONALDSON S SEHRA H ALMEIDA-KING J

LOVELAND J TREVANION S JONES M QUAIL M WILLEY D HUNT A BURTON J

SIMS S MCLAY K PLUMB B DAVIS J CLEE C OLIVER K CLARK R RIDDLE C

ELIOTT D THREADGOLD G HARDEN G WARE D MORTIMER B KERRY G HEATH P

PHILLIMORE B TRACEY A CORBY N DUNN M JOHNSON C WOOD J CLARK S

PELAN S GRIFFITHS G SMITH M GLITHERO R HOWDEN P BARKER N STEVENS C

HARLEY J HOLT K PANAGIOTIDIS G LOVELL J BEASLEY H HENNNDERSON C

GORDON D AUGER K WRIGHT D COLLINS J RAISEN C DYER L LEUNG K

ROBERTSON L AMBRIDGE K LEONGAMONMLERT D MCGUIRE S GILDERTHORP R

GRIFFITHS C MANTHRAVADI D NICHOL S BARKER G WHITEHEAD S KAY M

BROWN J MUMANE C GRAY E HUMPHRIES M SYCAMORE N BARKER D

SAUNDERS D WALLIS J BABBAGE A HAMMOND S MASHREGHI-MOHAMMADI M

BARR L MARTIN S WRAY P ELLINGTON A MATTHEWS N ELLWOOD M

WOODMANSEY R CLARK G COOPER J TROMANS A GRAFHAM D SKUCE C

PANDIAN R ANDREWS R HARRISON E KIMBERLEY A GARNETT J FOSKER N

HALL R GAMER P KELLY D BIRD C PALMER S GEHRING I BERGER A DOOLEY

CM ERSAN-UumlRUumlN Z ESER C GEIGEEER H GEISLER M KAROTKI L KIM A

KONANTZ J KONANTZ M OBERLANDER M RUDOLPH-GEIGER S TEUCKE M

OSOEGAWA K ZHU B RAPP A WIDAA S LANGFORD C YANG F CARTER NP

HARROW J NING Z HERRERO J SEARLE SM ENRIGHT A GEISLER R PLASTERK

RH LEE C WESTERFIELD M DE JONG PJ ZON LI POSTLETHWAIT JH

NUSSLEIN-VOLHARD C HUBBARD TJ ROEST CROLLIUS H ROGERS J STEMPLE DL

The zebrafish reference genome sequence and its relationship to the human genome Nature

2013 496 498-503

[16] JEONG JY KWON HB AHN JC KANG D KWON SH PARK JA KIM KW Functional and

developmental analysis of the blood-brain barrier in zebrafish Brain Res Bull 2008 75

619-628

[17] JOHANNESSEN LC Antiepileptic drugs in non-epilepsy disorders relations between

mechanisms of action and clinical efficacy CNS Drugs 2008 22 27 -47

[18] KIMMEL CB BALLARD WW KIMMEL SR Stages of embryonic development of the

zebrafish Develop Dyn 1995 203 253-310

[19] KWAN P ARZIMANOGLOU A BERG AT BRODIE MJ ALLEN HAUSER W MATHERN G

MOSHE SL PERUCCA E FRENCH J Definition of drug resistant epilepsy consensus

proposal by the ad hoc Task Force of the ILAE Commission on Therapeutic Strategies Epilepsia

2010 51 1069-1077

[20] LEE GH SUNG SY CHANG WN KAO TT DU HC HSIAO TH SAFO MK FU TF

Zebrafish larvae exposed to ginkgotoxin exhibit seizure-like behavior that is relieved by

pyridoxal-5rsquo-phosphate GABA and anti-epileptic drugs Dis Models Mech 2012 5 785-795




[21] LoumlSCHER W Critical review of current animal models of seizures and epilepsy used in the

discovery and development of new antiepileptic drugs Seizure 2011 20 359-368

[22] LoumlSCHER W SCHMIDT D Modern antiepileptic drug development has failed to deliver ways

out of the current dilemma Epilepsia 2011 52 657-678

[23] MORIMOTO K FAHNESTOCK M RACINE RJ Kindling and status epilepticus models of

epilepsy rewiring the brain Prog Neurobiol 2004 73 1-60

[24] MUSSULINI BH LEITE CE ZENKI KC MORO L BAGGO S RICO EP ROSEMBERG DB

DIAS RD SOUZA TM CALCAGNOTTO ME CAMPOS MM BATTASTINI AM DE

OLIVEIRA DL Seizures induced by pentylenetetrazole in the adult zebrafish a detailed

behavioral characterization PLos one 2013 8 1-9

[25] NIVOLI AM MURRU A GOIKOLEA JM CRESPO JM MONTES JM GONZALEZ-PINTO

A GARCIA-PORTILLA P BOBES J SAIZ-RUIZ J VIETA E New treatment guidelines for

acute bipolar mania a critical review J Affect Disorders 2011 129 14-26

[26] ORELLANA-PAUCAR AM SERRUYS AS AFRIKANOVA T MAES J DE BORGGRAEVE

W ALEN J LEON-TAMARIZ F WILCHES-ARIZABALA IM CRAWFORD AD DE WITTE

PA ESGUERRA CV Anticonvulsant activity of bisabolene sesquiterpenoids of curcuma longa in

zebrafish and mouse seizure models Epilepsy amp Behav 2012 24 14-22

[27] PACK AM REDDY DS DUNCAN S HERZOG A Neuroendocrinological aspects of epilepsy

important issues and trends in future research Epilepsy amp Behav 2011 22 94-102

[28] PETERSON RT MACRAE CA Systematic approaches to toxicology in the zebrafish Ann

Review Pharma Toxicol 2012 52 433-453

[29] PINEDA R BEATTIE CE HALL CW Recording the adult zebrafish cerebral field potential

during pentylenetetrazole seizures J Neurosci Methods 2011 200 20-28

[30] PITKANEN A LUKASIUK K Molecular and cellular basis of epileptogenesis in symptomatic

epilepsy Epilepsy amp Behav 2009 14 16-25

[31] SO EL RUGGLES KH CASCINO GD AHMANN PA WEATHERFORD KW Seizure

exacerbation and status epilepticus related to carbamazepine-10 11-epoxide Ann Neurol 1994

35 743-746

[32] STEWART AM DESMOND D KYZAR E GAIKWAD S ROTH A RIEHL R COLLINS C

MONNIG L GREE J KALUEFF AV Perspectives of zebrafish models of epilepsy What how

and where next Brain Research Bull 2012 87 135-143

[33] VERMOESEN K SERRUYS AS LOYENS E AFRIKANOVA T MASSIE A SCHALLIER A

MICHOTTE Y CRAWFORD AD ESGUERRA CV DE WITTE PA SMOLDERS I

CLINCKERS R Assessment of the convulsant liability of antidepressants using zebrafish and

mouse seizure models Epilepsy amp Behav 2011 22 450-460

[34] WINTER MJ REDFEM WS HAYFIELD AJ OWEN SF VALENTIN JP HUTCHINSON TH

Validation of a larval zebrafish locomotor assay for assessing the seizure liability of early-stage

development drugs J Pharm Toxicol Methods 2008 57 176-187

[35] WONG K STEWART A GILDER T WU N FRANK K GAIKWAD S SUCIU C DILEO J

UTTERBACK E CHANG K GROSSMAN L CACHAT J KALUEFF AV Modeling

seizure-related behavioral and endocrine phenotypes in adult zebrafish Brain Res 2010 1348

209-215




activity and c-fos expression in brain regions when zebrafish larvae exposed to pentylenetetrazole

(PTZ) (Hortopan et al 2010) With regard to anticonvulsant screens medium- to high-throughput

screening is possible in 96-well format using an automated locomotor tracking system for the

sensitivity to common antiepileptic drugs in this model (Baraban et al 2013) Also several groups

developed a seizure model in zebrafish larvae and made antiepileptic drugs screening from nature

plant (Lee et al 2012 Orellana-Paucar et al 2012) However the major parameters of zebrafish

behavior assay for assessing antiepileptic drug efficacy existed disparity which resulted in different

results in previous studies (Stewart et al 2012) Moreover a question remained as to whether an

AED-induced decrease in locomotion is truly indicative of anticonvulsant activity as some drugs may

impair larval movement through other mechanisms such as general toxicity or sedation So there still

remained some uncertainties to clear before using PTZ-induced zebrafish larvae epileptic seizure

model for anticonvulsant drug screening widely In this study we performed another behavioral

seizure analysis of PTZ-induced seizures refining parameters of the behavioral PTZ assay and

assessing three different antiepileptic drugs efficacy We also used this PTZ-induced seizure model to

discover anticonvulsant compound from plant extract We have demonstrated that this model can be

useful for AEDs primary screening This will also provide an alternative method for test potential

therapeutics of epilepsy

METHODS

Zebrafish Handling

Adult zebrafish (Danio rerio AB strain) were housed in a light- and temperature-controlled

aquaculture facility with a standard 14 10 h lightdark photoperiod Four to five pairs of zebrafish

were set up for nature mating every time On average 200 - 300 embryos were generated Embryos

were collected and staged from multiple AB strain breeding pairs and pooled Following 6 and 24 hpf

(hours post fertilization) in an incubator in E3 media (5 mM NaCl 017 mM KCl 033 mM CaCl2

033 mM MgSO4) unfertilized embryos were removed and the remainder placed in a re-circulating

Tecniplast aquatic system at 28 degC (02 Instant Ocean Salt in deionized water pH 69 - 72

conductivity 480 - 510 μScm and hardness 537 - 716 mgL CaCO3) (Kimmel et al 1995) Also

embryos raised on the system displayed more consistent activity patterns than those reared in a

petri-dish After completing the experiments all larvae were sacrificed through administration of an

overdose of anesthetic (tricaine) The zebrafish facility at Hunter Biotechnology Inc is accredited by

the Association for Assessment and Accreditation of Laboratory Animal Care (AAA LAC)

International

Drug Treatment

Pentylenetetrazole (PTZ Sigma CAS No 54-95-5) was used to induce zebrafish epileptic seizure

Three AEDs carbamazepine (CBZ ACR ORGANICS CAS No 298-46-4) phenytoin (PHT JampK

CAS No 630-93-3) valproate sodium (VPA Sigma CAS No 1069-66-5) and negative control drug

aspirin (Sigma CAS No 50-78-2) were selected for the development and validation of zebrafish

epileptic seizure model Stock solutions were prepared in 100 dimethyl sulfoxide (DMSO) and

stored at -20 degC serial dilutions (1 2 and 3 DMSO) were made before each experiment All

compounds were dissolved in DMSO and diluted in embryo medium to achieve a final DMSO

concentration of 1 wv For zebrafish epilepsy model validation PTZ was added to fish water to

reach the desired concentrations ranging from 125 mM to 30 mM Control embryos were raised in

fish water without PTZ For testing embryo response to anticonvulsants AEDs and aspirin were

added to the fish water to reach the indicated working concentrations 1 hour before the embryos were

exposed to 10 mM PTZ for behavioral analysis and other examinations For AEDs primary screening

the concentrations of plant extract were the maximal tolerable concentration for zebrafish larvae




Behavioral Analysis











ZebraLab TM


























RESULTS

















Figure1A

Figure1B


















Figure2A

Figure2B







Model














Figure3A

Figure3B

Figure3C








Figure4A-1

Figure4A-2

Figure4B-1




Figure4B-2

Figure4C-1

Figure4C-2




Figure4D-1

Figure4D-2














Figure5A

Figure5B



P lt 005













Figure6A




Figure6B

Figure6C








DISCUSSION



































efficacy assessment












































ACKNOWLEDGEMENT



REFERENCES
















18-28















83-107









13718-13728





























2013 496 498-503



619-628








2010 51 1069-1077


































35 743-746














209-215




Behavioral Analysis











ZebraLab TM


























RESULTS

















Figure1A

Figure1B


















Figure2A

Figure2B







Model














Figure3A

Figure3B

Figure3C








Figure4A-1

Figure4A-2

Figure4B-1




Figure4B-2

Figure4C-1

Figure4C-2




Figure4D-1

Figure4D-2














Figure5A

Figure5B



P lt 005













Figure6A




Figure6B

Figure6C








DISCUSSION



































efficacy assessment












































ACKNOWLEDGEMENT



REFERENCES
















18-28















83-107









13718-13728





























2013 496 498-503



619-628








2010 51 1069-1077


































35 743-746














209-215









Figure1A

Figure1B


















Figure2A

Figure2B







Model














Figure3A

Figure3B

Figure3C








Figure4A-1

Figure4A-2

Figure4B-1




Figure4B-2

Figure4C-1

Figure4C-2




Figure4D-1

Figure4D-2














Figure5A

Figure5B



P lt 005













Figure6A




Figure6B

Figure6C








DISCUSSION



































efficacy assessment












































ACKNOWLEDGEMENT



REFERENCES
















18-28















83-107









13718-13728





























2013 496 498-503



619-628








2010 51 1069-1077


































35 743-746














209-215




Figure2A

Figure2B







Model














Figure3A

Figure3B

Figure3C








Figure4A-1

Figure4A-2

Figure4B-1




Figure4B-2

Figure4C-1

Figure4C-2




Figure4D-1

Figure4D-2














Figure5A

Figure5B



P lt 005













Figure6A




Figure6B

Figure6C








DISCUSSION



































efficacy assessment












































ACKNOWLEDGEMENT



REFERENCES
















18-28















83-107









13718-13728





























2013 496 498-503



619-628








2010 51 1069-1077


































35 743-746














209-215




Figure3A

Figure3B

Figure3C








Figure4A-1

Figure4A-2

Figure4B-1




Figure4B-2

Figure4C-1

Figure4C-2




Figure4D-1

Figure4D-2














Figure5A

Figure5B



P lt 005













Figure6A




Figure6B

Figure6C








DISCUSSION



































efficacy assessment












































ACKNOWLEDGEMENT



REFERENCES
















18-28















83-107









13718-13728





























2013 496 498-503



619-628








2010 51 1069-1077


































35 743-746














209-215




Figure4A-1

Figure4A-2

Figure4B-1




Figure4B-2

Figure4C-1

Figure4C-2




Figure4D-1

Figure4D-2














Figure5A

Figure5B



P lt 005













Figure6A




Figure6B

Figure6C








DISCUSSION



































efficacy assessment












































ACKNOWLEDGEMENT



REFERENCES
















18-28















83-107









13718-13728





























2013 496 498-503



619-628








2010 51 1069-1077


































35 743-746














209-215




Figure4B-2

Figure4C-1

Figure4C-2




Figure4D-1

Figure4D-2














Figure5A

Figure5B



P lt 005













Figure6A




Figure6B

Figure6C








DISCUSSION



































efficacy assessment












































ACKNOWLEDGEMENT



REFERENCES
















18-28















83-107









13718-13728





























2013 496 498-503



619-628








2010 51 1069-1077


































35 743-746














209-215




Figure4D-1

Figure4D-2














Figure5A

Figure5B



P lt 005













Figure6A




Figure6B

Figure6C








DISCUSSION



































efficacy assessment












































ACKNOWLEDGEMENT



REFERENCES
















18-28















83-107









13718-13728





























2013 496 498-503



619-628








2010 51 1069-1077


































35 743-746














209-215




Figure5A

Figure5B



P lt 005













Figure6A




Figure6B

Figure6C








DISCUSSION



































efficacy assessment












































ACKNOWLEDGEMENT



REFERENCES
















18-28















83-107









13718-13728





























2013 496 498-503



619-628








2010 51 1069-1077


































35 743-746














209-215




Figure6B

Figure6C








DISCUSSION



































efficacy assessment












































ACKNOWLEDGEMENT



REFERENCES
















18-28















83-107









13718-13728





























2013 496 498-503



619-628








2010 51 1069-1077


































35 743-746














209-215






















efficacy assessment












































ACKNOWLEDGEMENT



REFERENCES
















18-28















83-107









13718-13728





























2013 496 498-503



619-628








2010 51 1069-1077


































35 743-746














209-215















ACKNOWLEDGEMENT



REFERENCES
















18-28















83-107









13718-13728





























2013 496 498-503



619-628








2010 51 1069-1077


































35 743-746














209-215









13718-13728





























2013 496 498-503



619-628








2010 51 1069-1077


































35 743-746














209-215































35 743-746














209-215

Documents

Validation of the Zebrafish Pentylenetetrazol …Validation of the Zebrafish Pentylenetetrazol Seizure Model: Behavior Assay for Assessing Anti-Epileptic Drug Efficacy Chongbin Liu