The protective effect of metformin in scopolamine-induced learning and memory impairment in rats

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Original

article

The

protective

effect

of

metformin

in

scopolamine-induced

learning

and

memory

impairment

in

rats

Elif

Aksoz

a,

*

,

Semil

Selcen

Gocmez

b

,

Tugce

Demirtas

Sahin

b

,

Dilek

Aksit

c

,

Hasan

Aksit

d

,

Tijen

Utkan

b

a_Balikesir_University,_Faculty_of_Medicine,_Department_of_Medical_{Pharmacology,}_Cagis_Campus,_Balikesir,_Turkey b

KocaeliUniversity,FacultyofMedicine,DepartmentofMedicalPharmacology,UmuttepeCampus,Kocaeli,Turkey

c

BalikesirUniversity,FacultyofVeterinary,DepartmentofPharmacologyandToxicology,CagisCampus,Balikesir,Turkey

d

BalikesirUniversity,FacultyofVeterinary,DepartmentofBiochemistry,CagisCampus,Balikesir,Turkey

ARTICLE INFO

Articlehistory:

Received14October2018

Receivedinrevisedform17March2019 Accepted16April2019

Availableonline21April2019

Keywords: Metformin Memory Scopolamine Antioxidant pAMPK ABSTRACT

Background:Alzheimer'sdisease(AD)isthemostcommonneurodegenerativediseaseintheworld.One

ofthemostcommonlyprescribedoralantidiabeticdrug,metformin,hasbeenshowntohavebeneﬁcial

effectsonrestoringimpairedcognitivefunction.Inthepresentstudy,weinvestigatedtheeffectsof

metforminon spatial memoryin terms of alleviating scopolamine-induced learningand memory

impairmentsinratsbyusingtheMorriswatermaze(MWM)testandthemodiﬁedelevatedplus-maze

(mEPM) test. Furthermore, we investigated the possible mechanisms of action of metformin in

preventingcognitivedysfunction.

Methods:MaleWistarratsreceivedmetformin(50,100,or200mg/kg/day)viagavagefeedingforthree

weeks.ScopolaminewasadministeredintraperitoneallybeforetheprobestepoftheMWMtestorthe

acquisitionsessionofthemEPMtest.

Results:Thelearningandmemoryimpairmentinducedbyscopolaminewasreversedbymetformin.In

addition,metforminimprovedthelevelofphosphorylatedAMP-activatedprotein kinaseand cAMP

responsiveelementbindingprotein.However,metforminpretreatmenthadnoimpactoninhibitingthe

scopolamine-inducedchangesinacetylcholinelevels.Furthermore,metforminexerteditsantioxidant

effectbysigniﬁcantlyreversingscopolamine-inducedchangesinmalondialdehyde,totalantioxidant

status,andsuperoxidedismutaselevelsinthehippocampus.

Conclusion:Ourresultsindicatethatoneofthemostcommonlyusedantidiabeticdrug,metformin,has

the potential to prevent the development of dementia and be a novel therapeutic drug for the

ameliorationofcognitivedysfunctioninAD.

reserved.

Introduction

Alzheimer's disease (AD) is the most common cause of dementiaintheworld.Dementiadevelopswiththeprogressive lossof memory,language,problem-solving, andothercognitive skillsthataffecttheabilityofapersontoperformdailyactivities [1]. Nearly 50 millionpeople sufferfrom dementia worldwide, withaprojectedincreaseto152millionby2050[2].Themajority of cognitive symptoms in AD are attributed to cholinergic dysfunctioninthebasalforebrain[3].Treatmenttosloworstop thedestructionofneuronsiscurrentlynotavailable.Thecurrent

treatment involves mostly acetylcholinesterase inhibitors to prolongtheavailabilityofacetylcholineatcholinergicsynapses. Unfortunately,drugsareonlysymptomaticallyeffective; further-more,adverseeffectsandlimitedef_ﬁcacyrestricttheirtherapeutic success[4].

Metformin is one of the most commonly prescribed oral antidiabeticmedicationsfortypeIIdiabetes.Metformindecreases insulinresistanceandameliorateshyperglycemiawithoutcausing low blood sugar levels [5]. In addition to improving glucose metabolism, it has antioxidant [6,7] and neuroprotective [8] effects. Furthermore,metforminwas shown topromote neuro-genesisandenhancespatialmemory[9].

Metformin activates AMP-activated protein kinase (AMPK), whichmodulateslongtermpotentiationandmemoryformation [10]. Moreover, thebrain cholinergic system and brain-derived *Correspondingauthor.

E-mailaddress:aksoz@balikesir.edu.tr(E.Aksoz).

http://dx.doi.org/10.1016/j.pharep.2019.04.015

Pharmacological

Reports

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antioxidant status (TAS), superoxide dismutase (SOD), and malondialdehyde(MDA)weremeasuredtoevaluatetheir antioxi-danteffects.Additionally,weassessedtheeffectsofmetforminon thelevelsof moleculessuchaspAMPK,BDNF,and CREBinthe hippocampusofratsinvolvedincognition.

Materialandmethods Subjects

Five-month-oldmaleWistarratswereusedinthisstudy.They were maintained on a standard 12h light/dark cycle at the environmentaltemperature(222C)andwithfreeaccesstotap waterandchowpellets.Behavioralexperimentswereperformed between08:00and13:00 ina semi-soundproof and semi-dark laboratory. The study was approved by the Kocaeli University AnimalResearchEthicsCommittee(30November2014,Number: 11/5-2014).

Drugsandexperimentaldesign

Atotalof80ratswereusedinthestudy.Theyweredividedinto fourmaingroups(n=20pergroup)andeightsubgroups(n=10 pergroup)and weregiven eitherphysiologicalserum(2ml/kg/ day)ormetforminHCl(Metfull,Vitalis,Istanbul,Turkey;50, 100,or 200mg/kg)viagavagefeedingforthreeweeks(Table1). Scopol-amine HCl (Sigma, St Louise, USA; 1mg/kg) was administered intraperitoneally(ip)30minbeforethebehavioralexperimentsto oneofthesubgroupsofeachmaingroup(Fig.1).

Locomotoractivitytest

Locomotoractivitywasevaluatedtoexcludetheadverseeffects of locomotoractivityaltering compounds. A locomotor activity cage(MayCommat,Ankara,Turkey)wasusedforthistest. Morriswatermaze(MWM)test

The circular pool of MWM was divided into four equal quadrants.Aplatform (10cm diameter)was placed 1cm below thesurfaceofthewaterinoneofthequadrantsfortheﬁrstfour days.Thesurfaceofthewaterwascoveredbysmallwhitebeads.At

’s

platformwastakenoutofthepoolduringthistrial. Modiﬁedelevatedplusmaze(mEPM)test

ThetestwasperformedaspreviouslydescribedbyHlinakand Krejci[13].ThemEPMtestwascarriedoutbyusingacross-shaped wooden platformconsisting of two open(5010cm) and two closed(5010cm)armselevated50cmabovetheground.Onthe ﬁrstday,eachratwasplacedatthedistalendoftheopenarm,and thetimeneededtotransfertotheclosedarm(transferlatency,TL) wasrecorded.Themaximumtestdurationwas90s.Afterentering theclosedarm,theratwasreleasedinthemazefor10s.Thetest was repeated on the second dayand the transfer latencywas assessed.

Braintissuesampling

Allratsweresacriﬁcedunderketamine–xylazineanesthesia1h after the MWM test. The wholebrainswere removed and the hippocampiwererapidlyseparated,frozeninliquidnitrogen,and storedat80_C.

Samplepreparation

Weighedtissuesampleswerequicklytreatedwith0.9%NaCl, thenhomogenizedfor1min(2,000rpm/min,1:10w/v)withthe aidofastirrer(StuartSHM1,UK)inPBS(pH7.4)solutioninan ice bath on the day of the experiment. The homogenate and supernatantwereanalyzedforproteinanalysisaccordingtothe Lowrymethod(1951)[14](ShimadzuUV-1800,Japan).

Measurementsoflipidperoxidation

The homogenate MDA levels were detected via the single heatingmethodbasedonthethiobarbituricacid(TBA)reactivityas describedinYoshiokaetal.[15].Brieﬂy,2.5mltrichloroaceticacid solution(TCAA)(20%)and1mlthiobarbituricacid(0.67%)were mixedwith0.5mltissuehomogenateandkeptat95Cinwater bathfor30min.Thereactionmixturewascooledwithtapwater, vortexed,andaddedton-butanol(4ml).Itwasthencentrifugedat 3000rpm for 10min. The absorbance of n-butanol at 535nm was measured after removing the organic layer. The MDA Table1

Themaingroups,subgroupsandthetreatments.

Groups Subgroups n Treatmentforthreeweeks(po) Treatmentbeforethetests(ip) GroupI Controlgroup 10 1ml/kg/daySaline 1ml/kgSaline

SCOgroup 10 1ml/kg/daySaline 1mg/kgScopolamine GroupII Met50group 10 50mg/kg/dayMetforminHCl 1ml/kgSaline

Met50+SCOgroup 10 50mg/kg/dayMetforminHCl 1mg/kgScopolamine GroupIII Met100group 10 100mg/kg/dayMetforminHCl 1ml/kgSaline

Met100+SCOgroup 10 100mg/kg/dayMetforminHCl 1mg/kgScopolamine GroupIV Met200group 10 200mg/kg/dayMetforminHCl 1ml/kgSaline

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concentration was calculated by the MDA-TBA complex absor-bancecoefﬁcient(

e

=1.56105/M/cm)andexpressedas

m

mol/mg tissueproteininthehomogenateor

m

mol/Linserum.Theother half of the homogenate was centrifuged at 5000g for 5min. The supernatant was separated and then other analyses were performed.

Assessmentoftotalantioxidantstatus(TAS)

TAS was detected with a commercially available kit (Total Antioxidant StatusAssay kit, RelAssayDiagnostics, Turkey) by using an automated measurement method. The results are describedasmillimolesofTroloxequivalentpermgtissueprotein inthesupernatant.

Theenzyme-linkedimmunosorbentassay(ELISA)tests

Supernatant and Serum SOD (Rat Superoxide dismutase [Cu-Zn]ELISAkit,FineTest,ER0332,China),Ach(RatAcetylcholine ELISAkit,FineTest,ER1466,China),pAMPK(RatPhosphorylated Adenosine Monophosphate-Activated Protein Kinase ELISA kit, Elabscience,E-EL-R0738,China),BDNF(RatBrainDerived Neuro-trophicFactorELISAkit,FineTest,ER0008,China)andCREB(Rat CyclicAMPResponseElementBindingProteinELISAkit,FineTest, ER0865,China)levelsweremeasuredusingcommerciallyavailable kitsonanELISA-reader(ThermoMultiskanFC,USA).

Statisticalanalysis

Prism 6.0 software(GraphPad Software,Inc., SanDiego, CA, USA)wasusedindataanalysisandresultswereexpressedasthe meanSEM.The acquisitiontest of MWM was evaluatedwith two-way analysis of variance (ANOVA) followed by Bonferroni post-hoc tests. The other results were analyzed with one-way ANOVA followed by Bonferroni post-hoc tests. The results were considered tobe statistically signiﬁcant for p-values less than0.05.

Results

Theeffectofmetforminonthetotallocomotoractivitytest Statistical analysisof thedatademonstrated thatmetformin (50, 100, or 200mg/kg) or scopolamine (1mg/kg), or their combination,didnotsigniﬁcantlymodifythenumberof move-ments[F(7,72)=1.814;p>0.05](Fig.2).

TheeffectofmetforminontheMWMtest

Thelatencytoﬁndthehiddenplatformonthefourtesttrialsof animalsdecreasedfromday1today4(two-wayANOVA,theeffect ofday,F(3,76)=56.87,p<0.05).Asigniﬁcantdecreaseinlatency on each subsequent day of acquisition testing was shown by Bonferroni'spost-hoc comparison test for allgroups (p <0.05) (Fig. 3A). Metformin and control groups were similar in the acquisitiontrial.

DuringtheprobetrialoftheMWMtest,therewasasigniﬁcant differencein latenciesbetweenthescopolaminegroupandthe controlgroupregardingthetimespentinthequadrantwiththe escape platform (one-way ANOVA, F (7,74)=6.341, p < 0.05,

Fig. 3B). Post-hoc comparisons also showed that scopolamine reducedthetime spent intheescape platform'squadrant (p< 0.0001). Metformin (100 and 200mg/kg) + scopolamine co-administration reversed the scopolamine-induced decrease in latencycomparedtoscopolaminealone(p<0.05forMet-100+SCO groupandp<0.0001forMet-200+SCOgroup).

TheeffectofmetformininthemEPMtest

Fig.3showsthefirst-dayandthesecond-daylatencyoftherats. Theretentionlatencywassignificantlydecreasedwithmetformin (50mg/kg)comparedwiththatin thecontrolgroup(p<0.05). Therewasnosignificanteffectofmetforminoritscombination with scopolamine on the first-day latency in the mEPM test [F (7,71)=3.740, p < 0.05; Fig. 4A]. Scopolamine significantly Fig.1.Theexperimentaldesignofthestudy.

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prolongedthesecond-dayretentionlatencycomparedwiththatin the control group (p < 0.05), while metformin pretreatment signiﬁcantlyreversedthiseffectcomparedtoscopolaminealone (p<0.001fortheMet-50+SCOgroup,p<0.05forMet-100+SCO groupandp<0.05forMet-200+SCOgroup).Theretentionlatency was signiﬁcantly decreased with metformin, compared to that withscopolamine alone (p< 0.05) [F (7,72) = 6.171, p <0.05;

Fig.4B].

TheeffectofmetforminonthelevelsofAChinthehippocampus AChlevelsinthehippocampusweresigniﬁcantlydecreasedin thescopolaminegroup(p<0.05),andincombinationgroupsas comparedwiththecontrolgroup[F(7,40)=6,906;p<0.05;Fig.5]. TheeffectofmetforminonthelevelsofpAMPK,CREBandBDNFinthe hippocampus

Levels of pAMPK in the hippocampus were signiﬁcantly decreased in the scopolamine group (p < 0.05). Metformin pretreatmentsigniﬁcantlyincreasedpAMPKlevelscomparedwith scopolaminealone. _Interestingly,metformintreatment alone at 100mg/kgalsomarkedlyincreasedpAMPKlevelscomparedwith thecontrolgroup[F(7,40)=28.43;p<0.05].LevelsofCREBinthe

hippocampus were significantly decreased in the scopolamine group,comparedwiththecontrolgroup.Metforminsignificantly increasedCREBlevelsbothaloneandwithscopolaminecompared withscopolaminealone[F(7,40)=15.50;p<0.05].LevelsofBDNF in the hippocampuswere decreased in the scopolaminegroup compared with the control group [F (7,40)=16.56, p < 0.05]. Metforminpretreatment (100–200mg/kg)significantlyreversed this effect but was still significantly lowerthan in the control (Fig.6).

TheeffectofmetforminonthelevelsofMDA,TAS,andSODinthe hippocampus

MDA levelsweresignificantlyincreasedinthehippocampus of scopolamine group compared with the control group [F(7,40)=133.8,p_<0.05].Metforminalonesigni_ficantlydecreased theMDAlevelsinthehippocampuscomparedwithscopolamine alone. However, pretreatment with metformin significantly attenuated MDAelevation, which didnot returntothe control group values. Scopolamine significantly depleted the levels of antioxidantinthehippocampusincludingTASandSOD,compared with the control group [F (7, 40)=10.62, p < 0.05 and F(7,40)=21.42,p<0.05respectively](Fig.7).Thesechangeswere reversedbymetforminpretreatment.Interestingly,SODlevelsin Fig.3.(A)Effectofmetformin(50,100and200mg/kg)ontheacquisitiontestofMWM.Thereisnoscopolamineadministration.EachvaluerepresentsthemeanSEM (n=10).(B)Effectofmetformin(50,100and200mg/kg),scopolamine(1mg/kg)andthecombinationofdrugsontheprobetestofMWM.Eachvaluerepresentsthe meanSEM(n=10).αp<0.05comparedwiththecontrolgroup;βp<0.05,comparedwiththescopolaminegroup;ep<0.05comparedwiththeMet50+SCOgroup.

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Fig.4.Effectsofmetformin(50,100and200mg/kg),scopolamine(1mg/kg)andthecombinationofdrugson(A)transferlatency1(TL-1)and(B)transferlatency2(TL-2) (n=10)inthemEPMtest.EachvaluerepresentsthemeanSEM(n=10).αp<0.05comparedwiththecontrolgroup;βp<0.05comparedwiththescopolaminegroup.

Fig.5. Theeffectofmetforminonscopolamine-inducedchangesinAchlevelsinthehippocampus.DataareexpressedasthemeanSEM(n=6).αp<0.05comparedwith thecontrolgroup;βp<0.05,comparedwiththeSCOgroup;ep<0.05comparedwiththeMet50group.

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responseto100mg/kgmetformin(p_<0.05)andwithscopolamine (p<0.05)weresigniﬁcantlyenhancedcomparedwiththoseinthe controlgroup.

Discussion

The findings presented heredemonstrate that metformin, a commonly used antidiabetic medication, has the potential to preventthe developmentof dementia associated withAD. Our findingspointtofourimportantconclusions.First,weconfirmed thatmetforminhasa protectiveeffectonscopolamine-induced spatial learning and memory impairment in rats by using behavioraltestssuchasMWMandmEPM.Second,ourbehavioral studiesdemonstratethat theprotectiveeffect ofmetformin on cognitivefunctionisnotduetoitseffectonacetylcholineinthe

hippocampus.Third,wereportforthe_firsttimethatmetformin significantly improved CREB and pAMPK levels reduced by scopolamine.ItiswellknownthatCREBisimportantformemory formation [16]. Fourth, we show metformin exerts antioxidant effectsbysignificantlyreversingtheeffectofscopolamineonMDA, TAS,andSODlevelsinthehippocampus.

Age-relateddementiaandmemorydeﬁcitsobservedinADare correlatedwiththelossofcholinergicneurotransmissioninthe basal forebrain [3]. In addition, pharmacological blockage of cholinergicneuronsbyscopolaminecausesimpairmentoflearning and memory in experimental animals [12]. In our study, as expected,scopolaminesigniﬁcantlyimpairedlearningand mem-ory in MWM and mEPM tests. Although metformin treatment alonedidnotaffectcognitivefunction,metforminpretreatment reversedtheimpairmentofspatiallearningandmemoryinduced Fig.6. TheeffectofmetforminonthelevelsofpAMPK,CREB,andBDNFinthehippocampus.DataareexpressedasthemeanSEM(n=6).αp<0.05comparedwiththe controlgroup;βp<0.05comparedwiththeSCOgroup;ep<0.05comparedwiththeMet50group;fp<0.05comparedwiththeMet100group,gp<0.05comparedwith theMet200groupintheirowngroup.

Fig.7.TheeffectofmetforminonthelevelsofMDA,TAS,andSODinthehippocampus.DataareexpressedasthemeanSEM(n=6).α_p_<_0.05_compared_with_the_control

group;βp<0.05comparedwiththeSCOgroup;ep<0.05comparedwiththeMet50group;fp<0.05comparedwiththeMet100group,gp<0.05comparedwiththe Met200groupintheirowngroup.

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byscopolamine. Ourdataareconsistentwithapreviousreport demonstratingthatmetforminandcontrolgroupsweresimilarin theiracquisitionandprobetestsinMWM[9].

Acetylcholine is the main neurotransmitter of cholinergic neurons,andacetylcholinedeficiencyintheADhasbeenknown foryears.Instudiesofscopolamine-inducedamnesicanimals,an increaseinacetylcholinesterase(AChE) activitywasobservedin thebraintissue after scopolamineadministration[17,18]. Anin vitrostudyfoundthatmetformininhibits50%ofAChEactivityin hemolyzedhumanerythrocytes[19].Wefoundthatscopolamine significantly decreased the level of ACh in the hippocampus. Pretreatment with metformin did not significantly inhibit the scopolamine-inducedchangesinthelevelsofACh.Nevertheless, metforminpretreatmentamelioratedscopolamine-induced learn-ingandmemoryimpairmentintheMWMandmEPMtests.Inline withourdata,Mostafaetal.[20]showedthatalthoughmetformin treatment didnot inhibit the scopolamine-induced increase in AChEactivity,itamelioratedscopolamine-inducedimpairmentin MWMtestinrats.Althoughwehavenotstudied acetylcholines-teraseactivitydirectlyin this study,ourresults haveanumber of similarities withthesefindings. Thus, ourdata indicatethat the protective effect of metformin is not due to its effect on acetylcholinelevelsinthehippocampus.

Anotherfactorthatmayberelatedtothemechanismofaction ofmetformininmemoryimprovementisAMPK.Severalstudies haveshownthatAMPKactsasalong-termpotentiationmodulator andisnecessaryformemoryformation[10].Wangetal.suggested that metformin could increase neurogenesis via AMPK [9]. Additionally,ithasbeenshownthatAMPKsignificantlyattenuated thedecreaseintheacetylcholinergicneuronsandimprovedthe scopolamine-induced memory impairment in the MWM test [17]. Consistent with previous reports, our study showed that metformin administration significantly enhanced pAMPK and improved scopolamine-induced behavioral deficits during the MWM and mEPM tests. Thus, these findings suggest that metformincanimprovescopolamine-inducedcognitive dysfunc-tionthroughAMPKenhancement.

AMPKisknowntobeimportantforCREBactivationthrough phosphorylation.Activation of CREBcreates neuronal plasticity andmemoryformationbyregulatinghippocampalneurogenesis [16].PhosphorylationofCREBdecreasesinthebrainofADpatients [21].CREBisadownstreamtargetforAMPKandmediatessomeof theeffectsofAMPKongeneexpression[22].However,Huangetal. showedthatoverexpressionofAMPKreducedthelevelofCREB. Therefore, they suggest a dual role of AMPK in the regulation of CREB in the hippocampus, such as decreasing total CREB expressionandincreasingCREBactivityviaphosphorylation[23]. Here, we report for the ﬁrst time that metformin treatment signi_ﬁcantly improved scopolamine-induced reduction in CREB levels,similartothoseofpAMPKlevels.Oneofthelimitationsof our studyis that wewere not able todeterminethe levelsof phosphorylatedCREB.

BDNF,themostrecognizedneurotrophin ofthebrain,isalso importantforlearningandmemory[24].BDNFlevelsarefoundto decreaseinthebrainofADpatients[25].Consistentwithprevious studies, ourstudy showed a dramatic decrease in BDNF levels in thehippocampusinresponse toscopolamineadministration [11,26]. However, metformin pretreatment did not reverse the effectonBDNFlevels.Allardetal.alsoreportedthatmetformin treatmentdecreasedtranscriptionofBDNFinthebrainofmicefed withthehigh-fatdiet[27].Inthisstudy,wefoundthatmetformin preventedimpairmentofspatialmemoryintheMWMtest,despite thereductioninBDNF.

Sinceoxidativestresscontributestothepathogenesisof neuro-degenerativedisorders,wealsoinvestigatedwhethermetformin hadprotectiveeffectsonoxidativestressinthehippocampus[28].

The increase in oxidative stress levels in the brain can impair hippocampalsynapticplasticity,andcreatememorydeficits[29]. Scopolamine-inducedoxidativestressreduced antioxidants and increasedMDAlevelsinthehippocampus[26].TheMDAlevelisa practicalandreliable biomarkeroflipid peroxidation,reflecting thedamagecausedbyreactiveoxygenspecies[30].Wefoundthat scopolamineincreasedtheMDAlevelsanddecreasedtheTASand SODlevelsinthehippocampus.ThesechangesofMDA,TAS,and SOD were significantly attenuated by metformin pretreatment. Ourexperimentconfirmspreviousdataonthebeneficialeffectsof metforminon oxidative stressparameters in the hippocampus [8,9,20].

Inconclusion,wefoundthatmetformintreatmentsignificantly amelioratedthescopolamine-inducedmemorydeficit.Wereport thatscopolaminetreatment decreasedthelevelsofpAMPKand CREB,and administrationof metformineffectively restored the pAMPKandCREBlevelsinthehippocampus.Metformintreatment significantlyincreasedthelevelsofTAS andSOD andincreased anti-oxidant activity by decreasing MDA levels. In the current study,bothoxidativestressandimpairmentsincognitivefunction inducedbyscopolaminewerepreventedbymetformintreatment. Basedonliteratureandourfindings,weproposethatmetformin canbeusedtopreventthedevelopmentofdementiaandcanbea noveltherapeuticdrugfortheameliorationofcognitive dysfunc-tionrelatedtoAD.

Conﬂictsofinterest

Theauthorsdeclarethattheyhavenoconﬂictofinterest. Acknowledgements

ThisworkwassupportedbyBalikesirUniversity[GrantNumber BAP.2016.38]

WewouldliketothankCuneytOzer,responsiblemanagerof ExperimentalMedical Research and ApplicationUnit at Kocaeli University,forthetechnicalsupport.

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