research article prenatal exposure to lamotrigine: effects...

Research ArticlePrenatal Exposure to Lamotrigine Effects on PostnatalDevelopment and Behaviour in Rat Offspring

Sekar Sathiya1 Murugan Ganesh2 Periyathambi Kalaivani1 Vijayan Ranju1

Srinivasan Janani3 Bakthavachalam Pramila1 and Chidambaram Saravana Babu1

1 Centre for Toxicology and Developmental Research (CEFT) Sri Ramachandra University Chennai Tamil Nadu 600116 India2Department of Biochemistry Sri Ramachandra University Chennai Tamil Nadu 600116 India3 School of Chemical and Biotechnology Shanmugha Arts Science Technology and Research Academy (SASTRA University)Thanjavur Tamil Nadu 613402 India

Correspondence should be addressed to Chidambaram Saravana Babu ceftpublicationsgmailcom

Received 11 January 2014 Accepted 19 February 2014 Published 14 April 2014

Academic Editors I Berger A M Depino and R Kulesza

Copyright copy 2014 Sekar Sathiya et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Use of antiepileptic drugs (AEDs) in pregnancy warrants various side effects and also deleterious effects on fetal developmentThe present study was carried out to assess the effects of prenatal exposure to lamotrigine (LTG) on postnatal development andbehavioural alterations of offspring Adult male and female Sprague Dawley rats weighing 150ndash180 g b wt were allowed to copulateand pregnancywas confirmed by vaginal cytology Pregnant rats were treatedwith LTG (115 23 and 46mgkg po) fromgestationalday 3 (GND 3) and this treatment continued till postnatal day 11 (PND 11) Offspring were separated from their dam on day 21following parturition LTG at 46mgkg po produced severe clinical signs of toxicity leading to death of dam between GND 15 and17 LTG at 115 and 23mgkg po showed significant alterations in offspringrsquos incisors eruption and vaginal openingwhen comparedto age matched controls LTG (23mgkg po) exposed female offspring expressed hyperactive behaviour and decreased GABA-Areceptor expression when compared to control rats These results reveal that prenatal exposure to LTG may impart differentialpostnatal behavioural alterations between male and female rats which paves way for further investigations

1 Introduction

Use of antiepileptic drugs (AEDs) during pregnancy presentsthe dilemma of minimising the risk of seizure and avoidingadverse effects in the unborn child Various studies revealedthat prenatal exposure of AEDs imparts serious effects oninfants [1ndash3] Further prenatal exposures to AEDs were alsoreported to produce cognitive impairment [4 5] Vigabatrinand valproate were reported to produce neuronal migrationdefect [6] upon prenatal exposure Janz and Fuchs [7]reviewed that exposure to AED during pregnancy increasesthe risk of miscarriage and stillbirth rate However littleattention was paid to the cellular effects of AEDs duringpostnatal development and in adulthood

Lamotrigine (LTG) a phenyltriazine derivative is oneof the most widely used second-generation antiepilepticagents used for both partial and generalised seizures In

pregnant women LTG is reported to produce side effectssuch as rash mania memory and cognitive problems moodchanges runny nose cough nausea indigestion abdominalpain weight loss vaginitis and leukopenia and retardationin development of fetus [8ndash10] Recently exposure to LTGduring pregnancy was shown to impart adverse outcomewithin different developmental domains [11] On the otherhand its use in pregnancy is associated with risk of seizuredeterioration because its clearance is accelerated in preg-nancy [12] This reveals the need of additional informationon the therapeutic window of LTG or its possible toxic effectsin pregnancy

Prenatal exposure to LTG (5ndash20mgkgday) alone andin combination with MX-801 phenobarbital or phenytoinresulted in cell death in the neonatal rat brain [13] ButKatz et al [14] showedno such effects in the same correspond-ing doseThus the effects of LTG during pregnancy offspring

Hindawi Publishing CorporationISRN NeuroscienceVolume 2014 Article ID 163459 8 pageshttpdxdoiorg1011552014163459

2 ISRN Neuroscience

development and behaviour are still needed to be studiedThe present study was undertaken to investigate the effects ofprenatal exposure of LTG on postnatal development and itsimpact on offspring behaviour in rats

2 Materials and Methods

21 Chemicals and Reagents LTG was a kind gift fromMs Sun Pharmaceutical Pvt Ltd Mumbai IndiaCarboxymethyl cellulose (CMC) was procured fromHimedia Mumbai PCR master cycler gradient waspurchased from Genet Bio Korea TRIzol Reagent waspurchased from Sigma Aldrich USA Unless mentioned allother chemicals and reagents were of analytical grade

22 Animal Husbandry and Ethics Approval 20 male and36 female Sprague Dawley rats were used for the studyAnimals were housed in polypropylene cages in a well-ventilated room (air cycles 12ndash15 air changesmin recycleratio 70 30) under an ambient temperature of 23 plusmn 2∘Cand 40ndash65 relative humidity with an artificial photoperiodof 12 h lightdark cycle They were provided with rodentfeed (Provimi Animal Nutrition India Pvt Ltd) and purifiedwater ad libitum Animals were acclimatized for a periodof 7 days to the laboratory conditions prior to initiationof the experiment Guidelines of ldquoGuide for the Care andUse of Laboratory Animalsrdquo (Institute of Laboratory AnimalResources National Academic Press 1996 NIH publicationnumber 85-23 revised 1996) were strictly followed through-out the study Institutional Animal Ethics Committee (IAEC)Sri Ramachandra University Chennai India approved thestudy protocol (IAECXIVSRU992008)

23 Groups and Treatment Following acclimatizationfemale rats were introduced with proven breeder duringnight time (1800) and were isolated during day hours (0900)in the ratio of 3 1 respectively for copulation Vaginal smeartest was performed for each female rat every 24 h and thepregnancy were confirmed by the existence of diestrumstage for three consecutive days The day of confirmation ofpregnancy was designated as GND 3 and the pregnant ratwas isolated into a separate cageThe dose of LTGwas arrivedfrom the maximum recommended human dose (500mg) inthe management of epilepsy (httpwwwdrugscom)

Pregnant rats were randomized into 4 groups (6 ineach) based on stratified body weight Group-I receivedvehicle (05 CMC 10mLkg po) Group-II III and IVreceived LTG at 115 23 and 46mgkg po respectively(three-dose regimen was selected to investigate the dosedependent response) Weekly body weight and cumulativefeed and water consumption were measured in dam LTGwas administered from GND 3 and continued till 11 daysfollowing parturition Pregnant rats were allowed to deliverand wean their pups until PND 21 Male and female offspringwere separated from dam on day 22 following delivery

24 Physical Parameters Length of gestation in dam andviability index of the pups were recorded Litter sizes in each

group were restricted to eight pups (4sex) Body weight gainwas measured on postnatal days once in a week till the studytermination The day of occurrence for pinna detachmentincisor eruption eye opening vaginal opening and testesdescent was also recorded

25 Behavioural Function in Male and Female OffspringOffspring were allowed to mature for 90 days with free accessto food and water ad libitum On PND 91 the animals weresubjected to behavioural assessment

251 Anxiety-Elevated Plus Maze Anxiety was assessed inoffspring using elevated plus maze [15] Elevated plus mazewas constructed of black painted wood with two open arms(50 times 10 times 1 cm) and two closed arms (50 times 10 times 40 cm)extending from a common central platform (10 times 10 cm) andelevated to 45 cm above floor level Experiments were carriedout in a sound-attenuated temperature-controlled room andilluminated by two 60w fluorescent light On PND 91 ratswere individually placed in the center of themaze facing openarm Number of entries into open and closed and time spentin open and closed arms were recorded for a period of 5min[16ndash19]

252 Locomotor Function-Open Field Exploratory TestLocomotor behavioural assessment in experimental groupswas performed using open field exploratory test [20] Openfield apparatus consisted of a plywood floor (96 times 96 cm)with highwallsThe entire apparatuswas painted black exceptfor 6mm thick white lines which divided the floor into 16squares On PND 91 each animal was placed at one cornerof the apparatus and observed for next 5min The number ofsquares crossed immobility period (in seconds) and numberof rearing and grooming were recorded

253 Learning- and Memory-Radial Arm Maze Radial armmaze was made of black painted wood finished with apolyurethane coating and consisted of 8 arms with a centerplatform of 60 cm in diameterThe arms were 80 cm long and15 cm wide A disposable plastic cup was placed at each endof the arm to reinforce the sweet smash and all arms werebaited with either a food cup or an empty cup The entireradial arm maze was elevated 50 cm off the floor Visual cueswere located throughout the room to provide spatial cuesThemaze was wiped down with 70 alcohol between each trailTheoffspringwere food-restricted to 85ndash90andmaintainedfor the duration of training and testing period

The maze task was divided into three phases adaptationtraining and retention phases In adaptation phase the ratswere allowed to explore the radial arm maze by 3 times 5mintrials During this period all of the arms of the maze werebaited with the food so that the rats received experiencein gaining a feed reinforcement by completely traversing anarm and reaching into the food cup Time taken by theanimal to move from the starting point (lag period) numberof working memory errors and time taken by the animalto munch the food in all arms were recorded During thetraining period four alternative arms were baited with food

ISRN Neuroscience 3

Table 1 Effect of LTG on dam body weight and feed and water consumption

Treatment Control LTG (mgkg po)115 23 46

Dam body weight (g)GND 3 18200 plusmn 600 16500 plusmn 120 16983 plusmn 356 18369 plusmn 782

GND 7 18700 plusmn 850 17167 plusmn 164 16883 plusmn 412 18050 plusmn 485

GND 14 22750 plusmn 1325 18833 plusmn 560lowast

18553 plusmn 564lowast

17833 plusmn 991lowastlowast


19100 plusmn 916lowastlowast mdash

Cumulative feed consumption (gdam)GND 3ndash6 4075 plusmn 237 3700 plusmn 314 3929 plusmn 137 4137 plusmn 348

GND 7ndash14 8600 plusmn 114 6028 plusmn 426lowastlowast





Cumulative water consumption (mL)GND 3ndash6 4339 plusmn 031 4125 plusmn 232 3977 plusmn 029 4456 plusmn 155

GND 7ndash14 10250 plusmn 059 9654 plusmn 156lowast





119899 = 6group Values are expressed in mean plusmn SEM Significance with Tukeyrsquos test following one way ANOVA is indicated as lowast119875 le 005 and lowastlowast119875 le 001 versuscontrol group GND gestational day LTG lamotrigine

The same arms were remained baited for all training andretention trialsThree 5-minute trials were conducted to eachanimal for ten days The trial was terminated when the rathas entered and eaten from all the four baited arms At theend of the three trials of each day the rat was returned toits home cage In retention period the animal was tested24 h after the final training trials They received three trialsas described under the training procedure Lag time numberof reference memory errors working memory errors correctentries and total time taken by the animal to munch the foodwere recorded [21]

26 GABA-A and GABA-B mRNA Expression by ReverseTranscriptase-Polymerase Chain Reaction (RT-PCR) Afterbehavioural assessment all the experimental animals wereeuthanized using CO

2exposure and cortical brain structures

were collected for GABA-A and GABA-B mRNA expressionBriefly the total RNA was extracted using TRIzol Reagent(Sigma USA) After homogenization the tubes were incu-bated for 10min and centrifuged at 1000 rpm for 5min200120583L of chloroform was added to the supernatant allowedto incubate for 5min at room temperature and centrifugedat 12000 rcf for 20min Then 500 120583L of isopropyl alcoholwas added to the supernatant to precipitate total RNA andcentrifuged at 12000 rcf for 15min following the incubationperiod of 10min Supernatant was decanted carefully andpellet was washed thrice with 75 alcohol and centrifugedat 12000 rcf for 15min and the pellet was dried The pelletwas resuspended in RNase-free water and stored in minus80∘Cuntil use The isolated RNA was allowed to undergo reversetranscription and polymerization reaction to get cDNA usingRT-PCR master cycler gradient (Genet Bio Korea) The geneexpression was analyzed using the bands formed in agarosegel electrophoresis captured using Gel documentation unit(Vilber Laumar Germany) and quantified by Bio ID software

Primers sequence used were as follows GABA-A sense51015840-AAG GAC CCA TGA CAG TGC TC-31015840 antisense51015840-GGC TCC CTT GTC CAC TCA TA-31015840 GABA-B sense

51015840-GCTGGATGGTTACCACATAG-31015840 antisense 51015840-GGTCAC AGG AGC AGT GAT G-31015840 and 120573-actin sense 51015840-TGCTGT CCC TGT ATG CCT CT-31015840 antisense 51015840-AGG TCTTTA CGG ATG TCA ACG-31015840 [22]

27 Statistical Analysis Data were expressed as mean plusmnstandard error of mean (SEM) Mean differences betweenthe groups were analysed by one way ANOVA followedby Tukeyrsquos multiple comparison as post-hoc test P valuele005 was considered to be significant Statistical analysis wasperformed using GraphPad prism 40 (San Diego USA)

3 Results

31 Effect of LTG on Dam Body Weight and Feed andWater Consumption Administration of LTG at 46mgkgpo produced severe signs of toxicity such as hyperesthesiavocalisation recumbency vaginal bleeding nasal dischargeand finally death of the dam between GND 15 and 17A significant decrease in body weight food and waterconsumption was observed in LTG (115 and 23mgkg po)administered dam on GND 14 (F(320) = 581 119875 lt 001F(320) = 4311 119875 lt 001 and F(320) = 1551 119875 lt 001 resp)and 21 (F(215) = 674 119875 lt 001 F(215) = 5523 119875 lt 001 andF(215) = 1308 119875 lt 001 resp) when compared to control(Table 1)

32 Effect of LTG on Physical Parameters in OffspringLTG (115 and 23mgkg po) significantly increased (F(215)= 2383 119875 lt 001) the gestational period when com-pared to control rats No significant difference in littersize between the groups was observed However a non-significant decrease in pups viability index was observed inLTG administered group when compared to control group(Table 2) LTG (115 and 23mgkg po) produced a significantdelay in incisor eruption in both male (F(215) = 696 119875 lt005) and female (F(29) = 636 119875 lt 005) offspring when

4 ISRN Neuroscience

Table 2 Effect of LTG on length of gestation total number of litters and its viability index

Group Treatment Gestational length (Days) Total number of litters Viability index ()I Control 1933 plusmn 033 1000 plusmn 058 10000 plusmn 000

II LTG (115mgkg po) 2225 plusmn 047lowastlowast

675 plusmn 149 8020 plusmn 1590

III LTG (23mgkg po) 2200 plusmn 000lowastlowast

833 plusmn 067 8148 plusmn 1336

IV LTG (46mgkg po) mdash mdash mdash119899 = 6group Values are expressed in mean plusmn SEM Significance with Tukeyrsquos test following one way ANOVA is indicated as lowast119875 le 005 and lowastlowast119875 le 001 versuscontrol group

Table 3 Effect of LTG on physical growth of male and female offspring

Group Treatment Day of pinna detachment Day of incisor eruption Day of eye opening Day of testesdescent

Day of vaginalopeningMale Female Male Female Male Female

I Control 433 plusmn 033 467 plusmn 033 733 plusmn 033 733 plusmn 033 1667 plusmn 033 1600 plusmn 033 633 plusmn 033 3267 plusmn 033

II LTG(115mgkg po) 533 plusmn 033 533 plusmn 033 933 plusmn 033

lowast

900 plusmn 057 1566 plusmn 066 1500 plusmn 067 623 plusmn 167 4057 plusmn 033lowastlowast

III LTG(23mgkg po) 530 plusmn 029 566 plusmn 033 950 plusmn 064

lowast


1533 plusmn 033 1433 plusmn 033 900 plusmn 000 5400 plusmn 100lowastlowast

119899 = 8group (4sex) Values are expressed in mean plusmn SEM Significance with Tukeyrsquos test following one way ANOVA is indicated as lowast119875 le 005 and lowastlowast119875 le 001versus control group

0 7 14 21 28 35 42 49 56 63 70 77 84 91 980

25

50

75

100

125

150

175

200

225

250

ControlLamotrigine (115 mgkg)

Lamotrigine (23 mgkg)

Days

Body

wei

ght o

f mal

e offs

prin

g (g

m)

Figure 1 Effect of LTG on body weight of male offspring

compared to control offspring A similar observation wasrecorded in vaginal opening in female offspring (F(29) =2865 119875 lt 001) There were no significant differences inthe day of pinna detachment eye opening and testes decentobserved between LTG and control offspring (Table 3) Nosignificant difference in body weight was observed in bothmale and female offspring in comparison to control offspringthroughout the study (Figures 1 and 2)

33 Effect of LTG on Offspring Behaviour

331 Anxiety-Elevated Plus Maze LTG male showed nosignificant difference in number of entries and time spent



0 7 14 21 28 35 42 49 56 63 70 77 84 91 980

20

40

60

80

100

120

140

160

180

200

220

Days

Body

wei

ght o

f fem

ale o

ffspr

ing

(gm

)

Figure 2 Effect of LTG on body weight of female offspring

between open and closed arms However LTG female(23mgkg po) showed a significant increase in number ofentries (F(29) = 514 119875 lt 005) and time spent (F(29) =5857 119875 lt 001) in open arms and decreased number ofentries (F(29) = 2088 119875 lt 001) and time spent (F(29) =5857 119875 lt 001) in closed arms when compared to controlrats (Table 4)

332 Locomotor Function-Open Field Exploratory TestNo significant difference was observed in number ofsquares crossed immobility period and between the exper-imental groups However a significant (F(29) = 407

ISRN Neuroscience 5

Table 4 Effect of LTG on anxiety behaviour in male and female offspring

Group Treatment Sex Number of entries Time spent (sec)Open Closed Open Closed

I Control Male 250 plusmn 050 1025 plusmn 118 11225 plusmn 1294 18775 plusmn 1294

Female 300 plusmn 134 1100 plusmn 084 12240 plusmn 992 17760 plusmn 992

II LTG (115mgkg po) Male 208 plusmn 094 703 plusmn 125 13372 plusmn 2813 16628 plusmn 2813


III LTG (23mgkg po) Male 283 plusmn 048 800 plusmn 073 14733 plusmn 1097 15267 plusmn 1097

Female 1050 plusmn 250lowast





Table 5 Effect of LTG on locomotor function in male and female offspring

Group Treatment Sex Number of squares crossed Immobility period (sec) Number of rearing Number of grooming






Female 11358 plusmn 464 5000 plusmn 1000 5175 plusmn 100lowast

508 plusmn 063

119899 = 8group (4sex) Values are expressed in mean plusmn SEM Significance with Tukeyrsquos test following one way ANOVA is indicated as lowast119875 le 005 versus controlgroup

119875 lt 005) increase in rearing behaviour was observed in LTG(23mgkg po) female offspring when compared to controlgroup (Table 5)

333 Learning- and Memory-Radial Arm Maze There wasno significant difference in lag period number of referenceand working memory errors number of correct entries andtotal time taken by the animal to munch the food recordedbetween the experimental groups (Table 6)

34 Effect of LTG on GABA-A and GABA-B mRNA Expres-sions mRNA expression of GABA-Awas found to be signifi-cantly (F(23) = 1707119875 lt 005) downregulated in LTG femaleoffspring (23mgkg po) when compared to counterpartswhereas no difference in GABA-B expression was observedbetween the experimental groups (Figure 3)

4 Discussion

Use of antiepileptic drugs such as LTG and levetiracetamduring pregnancy has become challenging these days asthey have toxic effects on the developing embryo Severalinvestigations in rat and rabbit models revealed that LTGcrosses the placenta [23] Administration of LTG in theform of single therapy or polytherapy is at a high risk ofdeveloping signs and symptoms of fetal toxicity [24] Thepresent study demonstrates that prenatal exposure of LTGin rats produced severe signs of toxicity in dam and genderdifferential behavior in female offspring

Various studies demonstrated that prenatal LTG exposureat a dose of 250mgkg (half the human equivalent dose)

increases stillbirth and postnatal death of the offspring[25 26] In the current study gestational LTG exposure at46mgkg produced severe toxic signs such as hyperesthesiavocalisation recumbency vaginal bleeding nasal dischargeand finally death of the dam Sidhu et al [27] showed thatlamotrigine administration increases the follicle stimulatinghormone (FSH) and luteinizing hormone (LH) which in turnstimulated estrogen secretion Increased estrogen secretionresults in maturation of graaffian follicle followed by ovu-lation which leads to embryo detachment This may be thepossible reason for the observed fetal death in the presentstudy

In addition LTG induces the secretion of parathyroidhormone (PTH) thereby modulating calcium homeostasisleading to osteoporosis [28] It is well known that osteoge-nesis of the embryo occurs at GND 15ndash21 in rats Increasedserum calcium levels by PTH reduce the fetal osteogenesisduring embryonic development which could also be one ofthe possible factors for observed fetal death at GND 15ndash17in LTG high dose administered rats Decreased body weightgain and feed and water intake of dam treated with LTGrevealing its possible maternal toxic effects Further delayedvaginal opening and incisors eruption in offspring of LTGlower doses group might also be due to its maternal toxiceffects [13]

On the other hand it is reported that LTG administrationalong with the estrogen-containing hormonal contraceptivesreduces the serum LTG concentration Hence the dose has tobe increased when administered with such contraceptives tomaintain its level of action [29] In addition increased estro-gen level induces FSH and secretes progesterone and testos-terone by feedback control process thereby lengthening the

6 ISRN Neuroscience

Table 6 Effect of LTG on learning and memory function in male and female offspring

Group Treatment Sex Lag period Number of referencememory errors

Number ofworking

memory errors

Number ofcorrectentries

Total time taken tomunch the food

I Control Male 100 plusmn 000 183 plusmn 080 042 plusmn 012 225 plusmn 074 8250 plusmn 1904

Female 033 plusmn 020 073 plusmn 037 020 plusmn 013 253 plusmn 051 8893 plusmn 1581

II LTG (115mgkg po) Male 106 plusmn 041 267 plusmn 044 028 plusmn 008 122 plusmn 043 13711 plusmn 1554


III LTG (23mgkg po) Male 125 plusmn 033 300 plusmn 074 071 plusmn 017 125 plusmn 053 14125 plusmn 1568


119899 = 8group (4sex) Values are expressed in mean plusmn SEM

00

02

04

06

08

10

12

MaleFemale

MaleFemale

Control LTG(115 mgkg)

LTG(23 mgkg)

00

02

04

06

08

10

12

14


LTG(23 mgkg)

GABA-

GAB

Lane 1 2 3 4

GA

BA-A

120573-a

ctin

(rel

ativ

e den

sity)

GA

BA-B

120573-a

ctin

(rel

ativ

e den

sity)

120573-

lowast

Figure 3 Effect of LTG on GABA receptors in male and femaleoffspring Lane 1- and 2-Control (male and female offspring resp)Lane 3- and 4-LTG 115mgkg po (male and female offspringresp) Lane 5- and 6-LTG 23mgkg po (male and female offspringresp)lowast denotes 119875 le 005 versus control group

gestational period Although progesterone and testosteronelevels were not measured in dams the increased gestationallength observed in our study of rats who administered LTG atlower doses may be due to impaired luteolysis and increasedprogesterone levels

Earlier studies have shown that blockade of GABA-A receptor in rat brain induces hyperexcitabilityanxiolyticbehaviour in elevated plus maze [30 31] Progesterone andestrogen induce anxiolytic behaviour inC57BL6 femalemice[32] Further estrogen suppresses GABA-A receptor expres-sion thereby slowing down the GABA mediated inhibition[33] In the present study the observed decrease in GABA-Aexpression and hyperactivation behaviour (as evidenced bythe elevated plus maze and open field test) may be due to theoveractivation of G-protein-coupled receptor 30 (GPR30) byestrogen hormone in the female offspring Put together theobservations showed that the anxiolytic behaviour in femaleoffspring but not the male may be due to the defect in GABA-A expression and also the alterations in GPR30 mediatedestrogen secretion

5 Conclusion

In conclusion the present study demonstrates the potentialuntoward effects of LTG in pregnant rats and also its influenceon the postnatal development and gender based differentialbehavioural effects in offspring Our lab is further investigat-ing the mechanisms and reason for these untoward effects soas to utilise the therapeutic benefits of LTG in a safer way

Abbreviations

AED Antiepileptic drugLTG LamotrigineGND Gestational dayPND Postnatal dayGABA-A 120574-Aminobutyric acid A receptorGABA-B 120574-Aminobutyric acid B receptorCMC Carboxymethyl celluloseIAEC Institutional Animal Ethics CommitteeRT-PCR Reverse transcriptase polymerase chain reactionSEM Standard error of meanPTH Parathyroid hormone

ISRN Neuroscience 7

FSH Follicle stimulating hormoneLH Luteinizing hormoneGPR30 G-protein-coupled receptor 30

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors thank Drugs and Pharmaceutical DivisionDepartment of Science and Technology Govt of India forfunding the project Authors express their sincere thanksto Prof Dr S Thanikachalam Director Centre for Toxicol-ogy and Developmental Research (CEFT) Sri RamachandraUniversity (SRU) for providing the facilities to carry outthe project Authors extend their thanks to all technicaland nontechnical staff of CEFT-SRU Chennai for their helpduring conduct of the experiment

References

[1] L B Holmes ldquoThe teratogenicity of anticonvulsant drugs aprogress reportrdquo Journal of Medical Genetics vol 39 no 4 pp245ndash247 2002

[2] K J Meador G A Baker R H Finnell et al ldquoIn uteroantiepileptic drug exposure fetal death and malformationsrdquoNeurology vol 67 no 3 pp 407ndash412 2006

[3] E Perucca ldquoBirth defects after prenatal exposure to antiepilep-tic drugsrdquoTheLancet Neurology vol 4 no 11 pp 781ndash786 2005

[4] N Adab A Jacoby D Smith and D Chadwick ldquoAdditionaleducational needs in children born to mothers with epilepsyrdquoJournal of Neurology Neurosurgery and Psychiatry vol 70 no 1pp 15ndash21 2001

[5] J Vinten N Adab U Kini J Gorry J Gregg and G A BakerldquoNeuropsychological effects of exposure to anticonvulsantmed-ication in uterordquo Neurology vol 64 no 6 pp 949ndash954 2005

[6] J-B Manent I Jorquera I Mazzucchelli et al ldquoFetal exposureto GABA-acting antiepileptic drugs generates hippocampal andcortical dysplasiasrdquo Epilepsia vol 48 no 4 pp 684ndash693 2007

[7] D Janz and U Fuchs ldquoAre anti epileptic drugs harmful duringpregnancyrdquo Deutsche Medizinische Wochenschrift vol 89 pp241ndash248 1964

[8] B R Danielsson K Lansdell L Patmore and T TomsonldquoEffects of the antiepileptic drugs lamotrigine topiramate andgabapentin on hERG potassium currentsrdquo Epilepsy Researchvol 63 no 1 pp 17ndash25 2005

[9] V Des Portes I Souville F Francis et al ldquoSo-called ldquocrypto-genicrdquo partial seizures resulting from a subtle cortical dysgene-sis due to a doublecortin gene mutationrdquo Seizure vol 11 no 4pp 273ndash277 2002

[10] K J Meador G A Baker N Browning et al ldquoCognitivefunction at 3 years of age after fetal exposure to antiepilepticdrugsrdquo The New England Journal of Medicine vol 360 no 16pp 1597ndash1605 2009

[11] G Veiby A K Daltveit S Schjolberg et al ldquoExposure toantiepileptic drugs in utero and child development a prospec-tive population-based studyrdquo Epilepsia vol 54 no 8 pp 1462ndash1472 2013

[12] C T Clark A M Klein J M Perel et al ldquoLamotrigine dosingfor pregnant patients with bipolar disorderrdquo The AmericanJournal of Psychiatry vol 170 no 11 pp 240ndash247 2013

[13] J-B Manent I Jorquera V Franco Y Ben-Ari E Peruccaand A Represa ldquoAntiepileptic drugs and brainmaturation fetalexposure to lamotrigine generates cortical malformations inratsrdquo Epilepsy Research vol 78 no 2-3 pp 131ndash139 2008

[14] I Katz J Kim K Gale and A Kondratyev ldquoEffects of lamot-rigine alone and in combination with MK-801 phenobarbitalor phenytoin on cell death in the neonatal rat brainrdquo Journalof Pharmacology and Experimental Therapeutics vol 322 no 2pp 494ndash500 2007

[15] S Pellow and S E File ldquoAnxiolytic and anxiogenic drug effectson exploratory activity in an elevated plus-maze a novel testof anxiety in the ratrdquo Pharmacology Biochemistry and Behaviorvol 24 no 3 pp 525ndash529 1986

[16] E Appenrodt R Schnabel and H Schwarzberg ldquoVasopressinadministration modulates anxiety-related behavior in ratsrdquoPhysiology and Behavior vol 64 no 4 pp 543ndash547 1998

[17] S E Bowen J L Wiley and R L Balster ldquoThe effects ofabused inhalants on mouse behavior in an elevated plus-mazerdquoEuropean Journal of Pharmacology vol 312 no 2 pp 131ndash1361996

[18] E F Espejo ldquoEffects of weekly or daily exposure to the elevatedplus-maze inmalemicerdquoBehavioural Brain Research vol 87 no2 pp 233ndash238 1997

[19] T Skutella J C Probst U Renner F Holsboer and C BehlldquoCorticotropin-releasing hormone receptor (type I) antisensetargeting reduces anxietyrdquo Neuroscience vol 85 no 3 pp 795ndash805 1998

[20] R G Lister ldquoEthologically-based animal models of anxietydisordersrdquo Pharmacology and Therapeutics vol 46 no 3 pp321ndash340 1990

[21] D S Olton C Collison and M A Werz ldquoSpatial memory andradial armmaze performance of ratsrdquo Learning andMotivationvol 8 no 3 pp 289ndash314 1977

[22] T Tamayama K Maemura K Kanbara et al ldquoExpression ofGABAA and GABAB receptors in rat growth plate chondro-cytes activation of the GABA receptors promotes proliferationof mouse chondrogenic ATDC5 cellsrdquo Molecular and CellularBiochemistry vol 273 no 1-2 pp 117ndash126 2005

[23] P Prakash L V Prabhu M A Nasar R Rai S Madhyasthaand G Singh ldquoLamotrigine in pregnancy safety profile and therisk of malformationsrdquo Singapore Medical Journal vol 48 no10 pp 880ndash883 2007

[24] P A Forcelli M J Janssen L A Stamps C Sweeney S Viciniand K Gale ldquoTherapeutic strategies to avoid long-term adverseoutcomes of neonatal antiepileptic drug exposurerdquo Epilepsiavol 51 no 3 pp 18ndash23 2010

[25] GenRx The Complete Reference for Generic and Brand DrugsMosby St Louis Mo USA 9th edition 1999

[26] T Tomson E Perucca and D Battino ldquoNavigating towardfetal and maternal health the challenge of treating epilepsy inpregnancyrdquo Epilepsia vol 45 no 10 pp 1171ndash1175 2004

[27] J Sidhu S Job S Singh and R Philipson ldquoThe pharma-cokinetic and pharmacodynamic consequences of the co-administration of lamotrigine and a combined oral contra-ceptive in healthy female subjectsrdquo British Journal of ClinicalPharmacology vol 61 no 2 pp 191ndash199 2006

[28] H A Valsamis S K Arora B Labban and S I McFarlaneldquoAntiepileptic drugs and bone metabolismrdquo Nutrition andMetabolism vol 3 article 36 2006

8 ISRN Neuroscience

[29] A Reimers G Helde and E Brodtkorb ldquoEthinyl estradiolnot progestogens reduces lamotrigine serum concentrationsrdquoEpilepsia vol 46 no 9 pp 1414ndash1417 2005

[30] CH BuenoH Zangrossi Jr andMBViana ldquoThe inactivationof the basolateral nucleus of the rat amygdala has an anxiolyticeffect in the elevated T-maze and lightdark transition testsrdquoBrazilian Journal of Medical and Biological Research vol 38 no11 pp 1697ndash1701 2005

[31] S K Sanders S L Morzorati and A Shekhar ldquoPriming ofexperimental anxiety by repeated subthresholdGABAblockadein the rat amygdalardquoBrain Research vol 699 no 2 pp 250ndash2591995

[32] K M Olesen N Ismail E D Merchasin and J D BlausteinldquoLong-term alteration of anxiolytic effects of ovarian hormonesin female mice by a peripubertal immune challengerdquoHormonesand Behavior vol 60 no 4 pp 318ndash326 2011

[33] M J Morrell ldquoEpilepsy in womenrdquo American Family Physicianvol 66 no 8 pp 1489ndash1494 2002

Submit your manuscripts athttpwwwhindawicom

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BioMed Research International


Research and TreatmentSchizophrenia

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Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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2 ISRN Neuroscience

development and behaviour are still needed to be studiedThe present study was undertaken to investigate the effects ofprenatal exposure of LTG on postnatal development and itsimpact on offspring behaviour in rats

2 Materials and Methods

21 Chemicals and Reagents LTG was a kind gift fromMs Sun Pharmaceutical Pvt Ltd Mumbai IndiaCarboxymethyl cellulose (CMC) was procured fromHimedia Mumbai PCR master cycler gradient waspurchased from Genet Bio Korea TRIzol Reagent waspurchased from Sigma Aldrich USA Unless mentioned allother chemicals and reagents were of analytical grade

22 Animal Husbandry and Ethics Approval 20 male and36 female Sprague Dawley rats were used for the studyAnimals were housed in polypropylene cages in a well-ventilated room (air cycles 12ndash15 air changesmin recycleratio 70 30) under an ambient temperature of 23 plusmn 2∘Cand 40ndash65 relative humidity with an artificial photoperiodof 12 h lightdark cycle They were provided with rodentfeed (Provimi Animal Nutrition India Pvt Ltd) and purifiedwater ad libitum Animals were acclimatized for a periodof 7 days to the laboratory conditions prior to initiationof the experiment Guidelines of ldquoGuide for the Care andUse of Laboratory Animalsrdquo (Institute of Laboratory AnimalResources National Academic Press 1996 NIH publicationnumber 85-23 revised 1996) were strictly followed through-out the study Institutional Animal Ethics Committee (IAEC)Sri Ramachandra University Chennai India approved thestudy protocol (IAECXIVSRU992008)

23 Groups and Treatment Following acclimatizationfemale rats were introduced with proven breeder duringnight time (1800) and were isolated during day hours (0900)in the ratio of 3 1 respectively for copulation Vaginal smeartest was performed for each female rat every 24 h and thepregnancy were confirmed by the existence of diestrumstage for three consecutive days The day of confirmation ofpregnancy was designated as GND 3 and the pregnant ratwas isolated into a separate cageThe dose of LTGwas arrivedfrom the maximum recommended human dose (500mg) inthe management of epilepsy (httpwwwdrugscom)

Pregnant rats were randomized into 4 groups (6 ineach) based on stratified body weight Group-I receivedvehicle (05 CMC 10mLkg po) Group-II III and IVreceived LTG at 115 23 and 46mgkg po respectively(three-dose regimen was selected to investigate the dosedependent response) Weekly body weight and cumulativefeed and water consumption were measured in dam LTGwas administered from GND 3 and continued till 11 daysfollowing parturition Pregnant rats were allowed to deliverand wean their pups until PND 21 Male and female offspringwere separated from dam on day 22 following delivery

24 Physical Parameters Length of gestation in dam andviability index of the pups were recorded Litter sizes in each

group were restricted to eight pups (4sex) Body weight gainwas measured on postnatal days once in a week till the studytermination The day of occurrence for pinna detachmentincisor eruption eye opening vaginal opening and testesdescent was also recorded

25 Behavioural Function in Male and Female OffspringOffspring were allowed to mature for 90 days with free accessto food and water ad libitum On PND 91 the animals weresubjected to behavioural assessment

251 Anxiety-Elevated Plus Maze Anxiety was assessed inoffspring using elevated plus maze [15] Elevated plus mazewas constructed of black painted wood with two open arms(50 times 10 times 1 cm) and two closed arms (50 times 10 times 40 cm)extending from a common central platform (10 times 10 cm) andelevated to 45 cm above floor level Experiments were carriedout in a sound-attenuated temperature-controlled room andilluminated by two 60w fluorescent light On PND 91 ratswere individually placed in the center of themaze facing openarm Number of entries into open and closed and time spentin open and closed arms were recorded for a period of 5min[16ndash19]

252 Locomotor Function-Open Field Exploratory TestLocomotor behavioural assessment in experimental groupswas performed using open field exploratory test [20] Openfield apparatus consisted of a plywood floor (96 times 96 cm)with highwallsThe entire apparatuswas painted black exceptfor 6mm thick white lines which divided the floor into 16squares On PND 91 each animal was placed at one cornerof the apparatus and observed for next 5min The number ofsquares crossed immobility period (in seconds) and numberof rearing and grooming were recorded

253 Learning- and Memory-Radial Arm Maze Radial armmaze was made of black painted wood finished with apolyurethane coating and consisted of 8 arms with a centerplatform of 60 cm in diameterThe arms were 80 cm long and15 cm wide A disposable plastic cup was placed at each endof the arm to reinforce the sweet smash and all arms werebaited with either a food cup or an empty cup The entireradial arm maze was elevated 50 cm off the floor Visual cueswere located throughout the room to provide spatial cuesThemaze was wiped down with 70 alcohol between each trailTheoffspringwere food-restricted to 85ndash90andmaintainedfor the duration of training and testing period

The maze task was divided into three phases adaptationtraining and retention phases In adaptation phase the ratswere allowed to explore the radial arm maze by 3 times 5mintrials During this period all of the arms of the maze werebaited with the food so that the rats received experiencein gaining a feed reinforcement by completely traversing anarm and reaching into the food cup Time taken by theanimal to move from the starting point (lag period) numberof working memory errors and time taken by the animalto munch the food in all arms were recorded During thetraining period four alternative arms were baited with food

ISRN Neuroscience 3






























3 Results



4 ISRN Neuroscience




675 plusmn 149 8020 plusmn 1590


833 plusmn 067 8148 plusmn 1336







lowast



lowast




0 7 14 21 28 35 42 49 56 63 70 77 84 91 980

25

50

75

100

125

150

175

200

225

250



Days

Body

wei

ght o

f mal

e offs

prin

g (g

m)







0 7 14 21 28 35 42 49 56 63 70 77 84 91 980

20

40

60

80

100

120

140

160

180

200

220

Days

Body

wei

ght o

f fem

ale o

ffspr

ing

(gm

)




ISRN Neuroscience 5





















508 plusmn 063





4 Discussion






6 ISRN Neuroscience



Number ofworking

memory errors










00

02

04

06

08

10

12

MaleFemale

MaleFemale


LTG(23 mgkg)

00

02

04

06

08

10

12

14


LTG(23 mgkg)

GABA-

GAB

Lane 1 2 3 4

GA

BA-A

120573-a

ctin

(rel

ativ

e den

sity)

GA

BA-B

120573-a

ctin

(rel

ativ

e den

sity)

120573-

lowast




5 Conclusion


Abbreviations


ISRN Neuroscience 7




Acknowledgments


References





























8 ISRN Neuroscience


















Neural Plasticity










Psychiatry Journal









Journal of


ISRN Neuroscience 3






























3 Results



4 ISRN Neuroscience




675 plusmn 149 8020 plusmn 1590


833 plusmn 067 8148 plusmn 1336







lowast



lowast




0 7 14 21 28 35 42 49 56 63 70 77 84 91 980

25

50

75

100

125

150

175

200

225

250



Days

Body

wei

ght o

f mal

e offs

prin

g (g

m)







0 7 14 21 28 35 42 49 56 63 70 77 84 91 980

20

40

60

80

100

120

140

160

180

200

220

Days

Body

wei

ght o

f fem

ale o

ffspr

ing

(gm

)




ISRN Neuroscience 5





















508 plusmn 063





4 Discussion






6 ISRN Neuroscience



Number ofworking

memory errors










00

02

04

06

08

10

12

MaleFemale

MaleFemale


LTG(23 mgkg)

00

02

04

06

08

10

12

14


LTG(23 mgkg)

GABA-

GAB

Lane 1 2 3 4

GA

BA-A

120573-a

ctin

(rel

ativ

e den

sity)

GA

BA-B

120573-a

ctin

(rel

ativ

e den

sity)

120573-

lowast




5 Conclusion


Abbreviations


ISRN Neuroscience 7




Acknowledgments


References





























8 ISRN Neuroscience


















Neural Plasticity










Psychiatry Journal









Journal of


4 ISRN Neuroscience




675 plusmn 149 8020 plusmn 1590


833 plusmn 067 8148 plusmn 1336







lowast



lowast




0 7 14 21 28 35 42 49 56 63 70 77 84 91 980

25

50

75

100

125

150

175

200

225

250



Days

Body

wei

ght o

f mal

e offs

prin

g (g

m)







0 7 14 21 28 35 42 49 56 63 70 77 84 91 980

20

40

60

80

100

120

140

160

180

200

220

Days

Body

wei

ght o

f fem

ale o

ffspr

ing

(gm

)




ISRN Neuroscience 5





















508 plusmn 063





4 Discussion






6 ISRN Neuroscience



Number ofworking

memory errors










00

02

04

06

08

10

12

MaleFemale

MaleFemale


LTG(23 mgkg)

00

02

04

06

08

10

12

14


LTG(23 mgkg)

GABA-

GAB

Lane 1 2 3 4

GA

BA-A

120573-a

ctin

(rel

ativ

e den

sity)

GA

BA-B

120573-a

ctin

(rel

ativ

e den

sity)

120573-

lowast




5 Conclusion


Abbreviations


ISRN Neuroscience 7




Acknowledgments


References





























8 ISRN Neuroscience


















Neural Plasticity










Psychiatry Journal









Journal of


ISRN Neuroscience 5





















508 plusmn 063





4 Discussion






6 ISRN Neuroscience



Number ofworking

memory errors










00

02

04

06

08

10

12

MaleFemale

MaleFemale


LTG(23 mgkg)

00

02

04

06

08

10

12

14


LTG(23 mgkg)

GABA-

GAB

Lane 1 2 3 4

GA

BA-A

120573-a

ctin

(rel

ativ

e den

sity)

GA

BA-B

120573-a

ctin

(rel

ativ

e den

sity)

120573-

lowast




5 Conclusion


Abbreviations


ISRN Neuroscience 7




Acknowledgments


References





























8 ISRN Neuroscience


















Neural Plasticity










Psychiatry Journal









Journal of


6 ISRN Neuroscience



Number ofworking

memory errors










00

02

04

06

08

10

12

MaleFemale

MaleFemale


LTG(23 mgkg)

00

02

04

06

08

10

12

14


LTG(23 mgkg)

GABA-

GAB

Lane 1 2 3 4

GA

BA-A

120573-a

ctin

(rel

ativ

e den

sity)

GA

BA-B

120573-a

ctin

(rel

ativ

e den

sity)

120573-

lowast




5 Conclusion


Abbreviations


ISRN Neuroscience 7




Acknowledgments


References





























8 ISRN Neuroscience


















Neural Plasticity










Psychiatry Journal









Journal of


ISRN Neuroscience 7




Acknowledgments


References





























8 ISRN Neuroscience


















Neural Plasticity










Psychiatry Journal









Journal of


8 ISRN Neuroscience


















Neural Plasticity










Psychiatry Journal









Journal of














Neural Plasticity










Psychiatry Journal









Journal of


research article prenatal exposure to lamotrigine: effects...

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