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InternshipProgramReport

Department„SynapsesandCircuits“

Neuroscience,Ophthalmology,&RareDiseases(NORD)

RochePharmaceuticalResearchandEarlyDevelopment(pRED)

RocheInnovationCenterBasel

MeropiKarakioulakiUndergraduateBiologyStudent,Intern

Supervisor:Dr.FatihaFjeldskaar

6July-6October2015

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ContentsPreface.................................................................................................................3

Acknowledgements..............................................................................................3

Introduction.........................................................................................................3

MethodologyandTechniques..............................................................................8

Results..................................................................................................................9

Conclusions........................................................................................................16

Bibliography.......................................................................................................17

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PrefaceDuring the summer of 2015, I had the opportunity to work for 3 months (6th July- 6thOctober)atHoffmann-LaRoche,at theDepartment “SynapsesandCircuits”, in the labofFatiha Fjeldskaar. My project was to test a number of different anti-GABA-A receptorantibodies inorder to identifywhichantibodyandunderwhichexperimental conditionsprovides the best staining of its antigen target, when used in immunocytochemistry,immunohistochemistryandwesternblottechniques.TheexperimentswereperformedinhumaninducedPluripotentStem(iPS)celllinesaswellasinmicecorticalprimarycultures(E16).Duringmyinternship,Ialsogotacquaintedwithotherresearchtechniques,suchascalciumimaging,primarymousecorticalcellcultureandhumaniPScellculture.

Thisinternshipgavemetheopportunitytoapprehendthestructureandorganizationofaglobalpharmaceuticalleadercompanyandalsotounderstanditsprinciplesandobjectives.Additionally,Ihadthechancetoobtainanumberofdifferentskills,suchusexperimentalplanning, troubleshooting, optimization of experimental conditions and evaluation ofresults.Last,butnot least,duringmythreemonth internship, Ihadthechancetoattendvarious seminars and info-talks and get in touchwithmany scientists from all over theworld,whobecameagreatsourceof inspirationandprovidedmewithhelpfulguidanceformyfuturestepsinscience.

AcknowledgementsIwouldliketothankmysupervisor,Dr.FatihaFjeldskaar,forgivingmetheopportunitytoworkinherlabandgetaquatintedwiththeworldofneuroscience.Moreover,Iwouldliketo expressmy gratitude to Isabell Spindler, Norak Be and especially to Livia Takacs fortheirguidanceandsupportduringmystayatRoche.

IntroductionGABA is themajor inhibitoryneurotransmitter in themammaliannervoussystem[1]. IntheCNSGABAisprimarilyproducedbyinhibitoryneuronsandreleasedduringthefiringofactionpotentials[2].However,GABAmayalsobeproducedbyglialcells(e.g.astrocytes)and is important in the developing of the CNS (cell proliferation, differentiation andmigration)[3-8].

The biological actions of GABA aremediated by ionotropic (GABA-A) andmetabotropic(GABA-B)receptors.TheGABA-Areceptorsare ligand-gatedchloridechannelscomposedofsubunits,fromarepertoireofatleast16distinctsubunits(α1-6,β1-3,γ1-3,δ,ε,π,ρ1-3,θ).Theassemblyoffiveofthesesubunitsformsachloridechannel[9-15].Thecombinationof the above-described subunits generates a large diversity of receptors with differentproperties[16].

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Physiologically,whenGABAbindstotheGABA-Areceptor,itenhancestheconductanceofions,particularlychlorideandbicarbonate.Theincreaseofthesenegativelychargedionsinthecellhyperpolarizesthemembranerestingpotential,leadingtotheinhibitionofcellfiring [16]. Some of the GABA-A receptor subunits have functional roles inneurodevelopment [16]. Each of them has a unique regional and temporal expressionprofile during different developmental stages [17]. The in vivo, ex vivo and in vitrolocalizationofGABA-AandGABA-BreceptorsinglialcellsoftheCentralNervousSystem(CNS)andPeripheralNervousSystem(PNS)isshowninTable1[18].

Table1:InVivoandInVitroLocalizationofGABA-AandGABA-BReceptorsinGlialCellsofCNSandPNS[18].

Alterationsinthesubunitgeneexpressionresultinchangesintheligandbindingaffinities,composition and properties of the GABA-A receptors. Those properties differ fromembryonicorearlypostnatalbraintoadultbrain.IntheratCNSforexample,theα2,α3,α5and β3 subunits aremainly expressed in the embryonic and early postnatal cortex andthalamus, as theα1,α4,β2andδ subunits aremainlyexpressed in theadult cortexandthalamus.Ontheotherhand,theexpressionoftheγ2subunitremainsunchangedduringthedifferentdevelopmentalstages[19,20](Table2).

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Table2: SummaryofprimaryGABA-Areceptor subunitmRNAsexpressed in selectedregionsofperinatalandadultratbrain[19].

Autismisasevereneurodevelopmentaldisordercharacterizedbysocialdeficits,languageabnormalities and repetitive behavior [21]. In the brain of subjects with autism anabnormal pattern of expression of GABA-A receptor genes is frequently being reported,suggesting that there might be a link between GABA system deregulation and thisneurodevelopmental disease [22-25]. Moreover, one of the most commonly reportedchromosomal loci where microdeletion / microduplication Copy Number Variations(CNVs) have been observed in autism, is the chromosome 15q11-q13 region, whichcontainsanumberofgenescodingforparticularsubunitsoftheGABA-Areceptor,liketheβ3,α5andγ3subunits[16].

TheGABA-A receptor is the target of several drugs of clinical significance,most notablyhypnotic,sedative,anesthetic,anti-convulsiveandanxiolyticagents[26].Thebest-knownanxiolytic agent is diazepam and its more modern versions all targeting the allostericbenzodiazepine(BDZ)modulatorysiteofthereceptor[27].

Furthermore, someGABA-A receptor subunits, such as the GABA-A alpha 5 subunit, areverified as targets for a number of drugs against psychiatric diseases. For example,RO4938581andBasmisanil(developmentalcodenames:RG-1662,RO5186582)arebothnegative allosteric modulators of the GABA-A alpha 5 subunit and are currently underdevelopment byRoche for the treatment of cognitive impairment associatedwithDownSyndrome [28-34].Another example isMRK-016, also anegative allostericmodulatoroftheGABA-Aalpha5subunit,whichproducesrapidantidepressanteffectsinratmodelsofdepression[35].Moreover,SKF-97,541,acompoundthatmainlyactsasaselectiveGABA-Breceptoragonisthassedativeeffectsinanimalstudiesandiswidelyusedinresearchasitwouldpotentially treatvarious typesofdrugaddiction [36-40].On theotherhand,CGP-

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7930,BHFFandGS-39783arepositiveallostericmodulatorsoftheGABA-Breceptorthathaveanxiolyticeffectsinanimalstudies[41-50].

Fortheabovereasons,itisveryimportanttohavetheappropriatetoolsforinvestigatingtheexpressionoftheGABAreceptorsubunitsinhumancelllinesandmouseprimarycellcultures.Inordertoattainthosetools,aseriesofantibodytestswasconducted,inordertodetermine themost suitable antibody and to establish the best experimental conditions(type of blocking buffer, antibody dilution etc.) that would allow us to investigate theexpressionofdifferentGABA-Areceptorsubunits,inbothhumaninducedpluripotentstem(iPS) cell lines and mice cortex primary cultures by immunocytochemistry,immunohistochemistryandwesternblot techniques.Theantibodies thatwe testedwereeitherproducedwithinRoche(24differentmonoclonalantibodiesderivedfrommicethatwere immunized with the α5 subunit of the GABA receptor) or were commerciallyavailableforotherGABA-Asubunits(α1,α4,β1,β2,β3,γ2,γ3,δ).

Immunocytochemistry for the localizationofvariousGABA-Areceptorsubunitshasbeenpreviouslyperformedinmiceandratneuronalcells.TherepresentativeresultsfromsuchexperimentsareshowninFigure1.

Figure1:a)Stainingofhippocampusneuronswithanti-GABA-Aalpha5subunitantibody fromSynapticSystems,Cat.No. 224503, dilution 1/500 (red) [51]. b) Staining of SHSY5Y cells with anti-GABA-A apha 5 subunit antibody fromAbcam,Cat.No.10098(green)[52].c)Stainingof298Tcellswithanti-GABA-Aalpha5subunitantibodyfromAbcam,Cat.No. 175195, dilution: 1/50 (red) [53]. d) Staining with anti-GABA-A alpha 5 subunit antibody from Arviva, Cat. No.:ARP30687_P050,dilution:1/100(red)[54].e)f)StainingoftheGABA-Aalpha5subunit(red)andGAD65(green)inthedentategyruswithanti-GAD65antibodyfromDevelopmentalStudiesHybridomaBank,dilution:1/200andanti-GABA-Aalpha5subunitfromAbcam,Cat.No.:ab10098,dilution:1/200[46].g)StainingofGABA-Aalpha5andGAD65proteinsinCA3pyramidalcellswithanti-GABA-Aalpha5subunitandanti-GAD65antibodiesfromfromDevelopmentalStudiesHybridomaBankattheUniversityofIowa,dilution:1/50[54].

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Additionally,westernblotanalysisforthedetectionofvariousGABA-Areceptorsubunitshasbeenpreviouslyperformedinmiceandratneuronalcells.TherepresentativeresultsfromsuchexperimentsasshowninFigure2.

Figure2:a)Westernblotofforebrainlysatesfromwild-typeandalpha1knockoutanimals,usingapproximately5-7μgof tissue per lane. Blots were incubated overnight at 2-8° C with anti-GABA-A receptor alpha 1 subunit, C-Terminusdiluted1/1000.TheantibodyfromRandDsystems,Cat.No:PPS022,labelledthe~51kDaalpha1subunitoftheGABA-Areceptor(Control).Thelabelingwasabsentfromalysatepreparedfromalpha1knockoutanimals(K/O).b)Westernblotofratbrain(hippocampal) lysatesusingapproximately5-7μgof tissue.Blotswere incubatedovernightat2-8°Cwithanti-GABA-Areceptoralpha4subunit,C-Terminusdiluted1/1000.TheantibodyfromRandDSystems,Cat.No.PPS025labelledthe~64kDaalpha4subunitoftheGABA-Areceptor.c)Westernblotof7μgofratcerebellarandhippocampallysates.TheantibodyfromRandDSystems,Cat.No.PPS030labelledthe~55kDabeta1subunitoftheGABA-Areceptor,diluted1/1000.d)WesternblotanalysisofGABA-Agamma3subunitin15μgofratbraincelllysateusingtheGeneTexantibody,Cat.No.:GTX31014,dilution:1/1000.e)Westernblotof5-7μgratbrain (cerebellum) lysates.Theblotwasincubatedovernightat2-8°Cwithanti-GABA-Abeta2subunitantibodyfromRandDSystems,Cat.No.PPS031,diluted1/1000.Theantibodylabeledthe~50-56kDabeta2subunitoftheGABA-Areceptorf)WesternblotofratbrainlysatewithanRandDSystemsantibody,Cat.No.PPS072,showingspecific immunolabelingof theapproximately44-47kDagamma2subunitof theGABA-Areceptor,diluted1/1000.g)Westernblotofmousecerebellar lysates fromwild type(Control) and delta knockout (KO) animals with R and D Systems antibody, Cat. No. PPS090, showing specificimmunolabelingof the~52kDadeltasubunitof theGABA-Areceptor,diluted1/1000.The labelingwasabsent fromalysate prepared fromknockout animalsh)WesternBlotwithGABA-A receptor beta 3 antibody fromNovus, Cat.No.:NB300-199,diluted1/1000at5-7μgofmousecerebellumlysatesfromwildtype(Control)andbeta3knockout(beta3K/O)animalsshowingspecificimmunolabelingofthe~53kbeta3-subunitoftheGABA-Areceptorinthewildtypebutnotinthebeta3K/Oanimals.

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MethodologyandTechniques1) Immunocytochemistry

Thecells, growing in96-wellplates,were initially fixedwith4%paraformaldehyde (PFA)and3%sucroseinphosphatebufferedsaline(PBS).Thentheunspecificsiteswereblockedwithblockingbuffer (10%Donkeyserumorbovineserumalbumin (BSA),0.2%Triton inPBS).Forthestaining,50μloftheprimaryantibodyinblockingbufferwereaddedtoeachwell,andthecellswereincubatedovernightat4oC.Theprimaryantibodywaswashedwith0.05%Triton inPBSand50μlof thesecondaryantibody(1/200) inblockingbufferwereaddedineachwell(Donkeyanti-ChickenIgGAlexa647,Donkeyanti-MouseIgGAlexa594,Donkeyanti-RabbitIgGAlexa488,Donkeyanti-RabbitIgGCy3,Donkeyanti-GoatIgGAlexa488,Donkey anti- Guinea Pig IgG Cy3, Jackson ImmunoResearch). Incubationwas carriedoutfor1.5hoursatroomtemperatureandthen50μlofDAPI(1/10.000)inPBS-Triton0.2%wereaddedtoeachwellandincubatedforfurther10minutes.Thecellswerefinallywashedwith0.05%TritoninPBS.FortheplatescanningweusedthePerkinElmerOperetta®HighContentImagingSystemandtheimageswereobtainedusingtheIntuitiveHarmony®HighContentImagingandAnalysisSoftware.

2) WesternBlotThecorticalembryonic(E16)andpostnatal(P30)tissueswerehomogenizedwith10XRipalysis buffer (Millipore, 24μl per mg of tissue), protease inhibitor (Complete, EDTA-free,Roche) and phosphatase inhibitor (PhosSTOP Phosphatase Inhibitor Cocktail Tablets,Roche)intheRocheMagnaLyserInstrument,at7000rpmfor40sec.Inordertodeterminetheproteinconcentration, the sampleswerecentrifugedat14.000rpm for15min,at4oCand the supernatant was collected and used in the Pierce™ BCA Protein Assay (ThermoScientific).Theconcentrationoftheproteinswasquantifiedatthemicroplatereader,usingtheSoftMax®ProSoftware.Thesampleswerethendilutedto1XPBSand2Xloadingdye,sothattheyreachaconcentrationof2μg/μl.Finally,thesampleswereboiledat95oCfor5-10minutes. For the SDS-PAGE electrophoresiswe used boiled (at 95oC for 10minutes) andnon-boiled samples (at 37oC for 30 minutes), which ran in 1X MES running buffer (LifeTechnologies),at60Vfor30minutesand150Vfor1hour.Theproteinmarkersusedwerethe Chameleon Duo Pre-stained protein ladder (LI-COR) for fluorescentwestern blotting,andamixtureof4:5MagicMark(LifeTechnologies):SeeBluePlus2(LifeTechnologies)forchemiluminescentwesternblotting.Forprotein transferweperformedbothsemi-dryandwettransfer.Forthesemi-drytransferweusednitrocellulosemembranesandtheiBlot®7-MinuteBlottingSystem(LifeTechnologies).ForthewettransferweusedPVDFmembranesin1XNuPAGE-NP006TransferBuffer (LifeTechnologies)with10%Methanolat100Voltsfor 1 hour. For fluorescent western blot the unspecific sites were blocked with Odysseyblocking buffer (LI-COR) for 1 hour. The antibodies were then diluted in 1:1 Odysseyblockingbuffer:0.2%Tween-20inPBS(secondaryantibodies:1:10.000,Donkeyanti-RabbitIgG IRDye700DX,Donkeyanti-Goat IgG IRDye800,Donkeyanti-Mouse IgG IRDye700DX,Rockland)andthemembraneswerewashedwith0.05%Tween20inPBS,beforescanning.For chemiluminescent western blot we blocked the unspecific sites with 0.1% Tween

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20/TBS/5%BSA, inwhichwe alsodiluted the antibodies (secondary antibodies: 1:5000,Peroxidase conjugated AffiniPure Goat anti-Rabbit and anti-Mouse IgG, JacksonImmunoResearch).Themembraneswerewashedwith0.1%Tween-20 inTBSandbeforescanningtheLumi-LightWesternBlottingSubstrate(Roche)wasadded.Forthefluorescentwestern blot scanning we used the Odyssey Infrared Imaging System (LI-COR) and theImageStudioSoftware(LI-COR).ForthechemiluminescentwesternblotscanningweusedtheChemiDocMPSystem(BioRad)andtheImageLabSoftware(BioRad).

3) ImmunohistochemistryThebrainsofmiceembryos(E16)wereincubatedwithPFA,10%sucroseand0.02%azidefor24hoursat4oC.Wethenobtained18μmthickslicesoftissuesfromembryo(E16)brainsusing the cryostatmicrotome (Leica CM 30505). The unspecific sites of the tissues wereblockedwithblockingbuffer(10%Donkeyserum,0.2%TritoninPBS).Forthestaining,weadded200μloftheprimaryantibodyinblockingbuffertoeachslide,andthetissueswereincubatedovernightat4oC.Theprimaryantibodywaswashedwith0.05%TritoninPBSand200μlofthesecondaryantibodyinblockingbufferwereaddedineachwell(Donkeyanti-MouseIgGAlexa594,Donkeyanti-RabbitIgGCy3,Donkeyanti-GoatIgGAlexa488,JacksonImmunoResearch). The secondary antibody (1:500)was incubated for 1.5 hours at roomtemperatureandthen200μlofDAPI(1/5.000)inPBS-Triton0.2%wereaddedtoeachslideandincubatedfor10minutes.Inordertocoverthefluorescentdye-stainedtissuesweusedthe Fluoromount™ Aqueous Mounting Medium (SIGMA-ALDRICH). The cells were finallywashedwith0.05%TritoninPBS.ForthescanningoftheslidesweusedtheLeicaConfocalMicroscope SP5 (Leica Microsystems) and the images were obtained using the LeicaApplicationSuiteSoftware(LeicaMicrosystems).

Results1) Anti-GABA-Aalpha5inhouseproducedantibodies

We initially tested 24 different anti-GABA-A alpha 5 subunit antibodies, which wereproduced in Roche. The antibodies, which gave the best results and the experimentalconditionsinvolvedareshowninTable3.

Antibody Species Company Celltype BlockingBuffer DilutionGABACVV1/9a,5.6mg/mlinPBS

Mouse InHouse

HumaniPScellline BSA 1/100GABACVV1/2a,6.1mg/mlinPBS Mousecorticalculture(E16) DonkeySerum 1/100GABACVV1/2a,6.1mg/mlinPBS HumaniPScellline BSA 1/100GABACQM1/4a,3.5mg/mlinPBS HumaniPScellline DonkeySerum 1/100GABACQM1/4a,3.5mg/mlinPBS Mousecorticalculture(E16) BSA 1/100GABACQM1/4c-1,1.6mg/mlinPBS Mousecorticalculture(E16) BSA 1/500GABACVV1/10b*,1.6mg/mlinPBS Mousecorticalculture(E16) DonkeySerum 1/100GABACVV1/10b*,1.6mg/mlinPBS HumaniPScellline DonkeySerum 1/100GABACVV1/10b*,1.6mg/mlinPBS HumaniPScellline BSA 1/100

Table3:Thedifferentanti-GABA-Aalpha5subunitantibodiesthatprovideduswiththebeststainingimages,whentestedindifferentcelltypes,blockingbuffersanddilutions.

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Thestainingimagesoftheabove-describedantibodiesareshowninFigure3.

Figure3:1a)b)StainingwithGABACVV1/9aantibody,stockconcentration:5.6mg/mlinPBS,dilution:1/100(red),DAPI,dilution:1/10000(darkblue)andanti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000 (light blue) in human iPS cell line, having used BSA blocking buffer.2a) b) Stainingwith 2. GABA CVV 1/2aantibody, stock concentration: 6.1mg/ml in PBS, dilution: 1/100 (red),DAPI, dilution: 1/10000 (dark blue) and anti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000(lightblue)inmousecorticalculture(E16), having used donkey serum blocking buffer. 3a) b) Staining with 15. GABA CQM 1/4a antibody, stockconcentration: 3.5mg/ml in PBS, dilution: 1/100 (red), DAPI, dilution: 1/10000 (dark blue) and anti-MAP2 antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000(lightblue)inHumaniPScellline,havinguseddonkeyserum blocking buffer. 4a) b) Staining with 15. GABA CQM 1/4a antibody, stock concentration: 3.5 mg/ml in PBS,dilution:1/100(red),DAPI,dilution:1/10000(darkblue)andanti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot: 400849, dilution: 1/5000 (light blue) in mouse cortical culture (E16), having used BSA blocking buffer. 5a) b)Staining with 17. GABA CQM 1/4c-1 antibody, stock concentration: 1.6 mg/ml in PBS, dilution: 1/500 (red), DAPI,dilution:1/10000(darkblue)andanti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000(light blue) in mouse cortical culture, having used BSA blocking buffer. 6a) b) Staining with 24. GABA CVV 1/10b*antibody, stock concentration: 1.6mg/ml in PBS, dilution: 1/100 (red),DAPI, dilution: 1/10000 (dark blue) and anti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000(lightblue)inmousecorticalculture(E16), having used donkey serum blocking buffer. 7a) b) Staining with 24. GABA CVV 1/10b* antibody, stockconcentration: 1.6 mg/ml in PBS, dilution: 1/100 (red), DAPI, dilution: 1/10000 and anti-MAP2 antibody fromNeuromics, Cat. No.: CH22103, Lot: 400849, dilution: 1/5000 (light blue) in human iPS cell line, having used donkeyserum blocking buffer.8a) b) Staining with 24. GABA CVV 1/10b* antibody, stock concentration: 1.6mg/ml in PBS,dilution:1/100(red),DAPI,dilution:1/10000(darkblue)andanti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000(lightblue)inhumaniPScellline,havingusedBSAblockingbuffer.

2) Commerciallyavailableanti-GABA-Asubunitsantibodies

Furthermore,weperformed testswithvarious commerciallyavailableantibodies for theGABA-A subunits. For these antibodies we employed 3 different techniques: western

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blotting, immunocytochemistry and immunohistochemistry, in order to identify thetechniquethatprovidesthebestresults(Table4).

Antibody Species Company WesternBlot Immunocytochemistry ImmunohistochemistryantiGABA-Aalpha1

Rabbit

RandDsystems *

antiGABA-Aalpha4 RandDsystems nopositivestaining

antiGABA-Abeta1 RandDsystems *

antiGABA-Abeta2 RandDsystems

*

antiGABA-Abeta3 Novusnopositivestaining *

antiGABA-Agamma2 RandDsystems *

antiGABA-Agamma3 GeneTex * *

antiGABA-Adelta RandDsystems

* *

Table 4: Different anti-GABA alpha subunits antibodies tested with different techniques. The technique thatprovidedthebestresultsisindicatedwithanasterisk.

A. WesternBlotanalyses

For the western blotting technique, we performed both fluorescent andchemiluminescence detection methods, in order to determine which detection methodprovidesthebestsignal(Table5).

Antibody Species Company Fluorescent ChemiluminescentantiGABA-Aalpha1

Rabbit

RandDsystems *

antiGABA-Aalpha4 RandDsystems nopositivestaining

antiGABA-Abeta1 RandDsystems *

antiGABA-Abeta2 RandDsystems *

antiGABA-Abeta3 Novus nopositivestaining

antiGABA-Agamma2 RandDsystems *

antiGABA-Agamma3 GeneTex *

antiGABA-Adelta RandDsystems *

Table5:Differentcommerciallyavailableanti-GABAalphasubunitsantibodiestestedinwesternblot.Thedetectionmethodsthatprovidedthebestsignalisindicatedwithanasterisk.

TherepresentativeresultsobtainedundertheoptimalexperimentalconditionsdescribedinTable5,areshowninFigures4-9.

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Figure4:Westernblottestoftheanti-GABA-Aalpha1antibody,usingchemiluminescentdetectionmethod.

Figure5:Westernblottestoftheanti-GABA-Abeta1antibody,usingfluorescentdetectionmethod.

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Figure6:Westernblottestoftheanti-GABA-Abeta2antibody,usingfluorescentdetectionmethod.

Figure7:Westernblottestoftheanti-GABA-Agamma2antibody,usingfluorescentdetectionmethod.

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Figure8:Westernblottestoftheanti-GABA-Agamma3antibody,usingfluorescentdetectionmethod.

Figure9:Westernblottestoftheanti-GABA-Adeltaantibody,usingfluorescentdetectionmethod.

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B. Immunocytochemistry

Using immunocytochemistry, we observed that different GABA-A subunits aredifferentially expressed inneurons and astrocytes inprimarymouse cortex cell cultures(E16),asshowninTable6.

Antibody Species Company PatternofexpressionatICCantiGABA-Aalpha1

Rabbit

RandDsystems cellbody,dendrites

antiGABA-Aalpha4 RandDsystems nopositivestaining

antiGABA-Abeta1 RandDsystems cellbody,astrocytes

antiGABA-Abeta2 RandDsystems cellbody,dendrites,astrocytes

antiGABA-Abeta3 Novus cellbody

antiGABA-Agamma2 RandDsystems cellbody,astrocytes

antiGABA-Agamma3 GeneTex cellbodyofspecificneuronsthatarealsostainedwithNeuNandParvalbumin,astrocytes

antiGABA-Adelta RandDsystems cellbody,astrocytes

Table6:TheexpressionpatternofdifferentGABA-Asubunitsusingvariouscommerciallyavailableantibodiesbyimmunocytochemistry.

Representative images for the expression of different GABA-A subunits in neurons andastrocytesinprimarymousecortexcellcultures(E16),areshowninFigure10.

Figure 10:1a) b) Stainingwith anti- GABA-A alpha 1 antibody, dilution: 1/100 (red), DAPI, dilution: 1/10000 (darkblue)andanti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000(lightblue) inmousecortical culture (E16), havinguseddonkey serumblockingbuffer.2a)b) Stainingwith anti-GABA-Abeta1 antibody,dilution:1/100(red),DAPI,dilution:1/10000(darkblue)andanti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000(lightblue)inmousecorticalculture(E16),havinguseddonkeyserumblockingbuffer.3a)b)Stainingwithanti-GABA-Abeta2antibody,dilution:1/100(red),DAPI,dilution:1/10000(darkblue)andanti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000(lightblue)inmousecorticalculture(E16),having used donkey serumblocking buffer.4a)b) Stainingwith anti- GABA-A beta 3 antibody, dilution: 1/100 (red),DAPI,dilution:1/10000(darkblue)andanti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000(lightblue)inmousecorticalculture(E16),havinguseddonkeyserumblockingbuffer.5a)b)Stainingwithanti-GABA-A gamma 2 antibody, dilution: 1/100 (red), DAPI, dilution: 1/10000 (dark blue) and anti-MAP2 antibody from

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Neuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000 (lightblue) inmouse cortical culture (E16),havinguseddonkey serum blocking buffer. 6a) b) Staining with anti- GABA-A gamma 3 antibody, dilution: 1/100 (red), DAPI,dilution:1/10000(darkblue)andanti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000(lightblue)inmousecorticalculture(E16),havinguseddonkeyserumblockingbuffer.7a)b)Stainingwithanti-GABA-Adeltaantibody,dilution:1/100(red),DAPI,dilution:1/10000(darkblue),anti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000(lightblue)andanti-SynapsinIantibodyfromSantaCruz,Cat.No.:sc-7379,Lot:K3011 (green) inmouse cortical culture (E16), having used donkey serumblocking buffer.8a)b) Stainingwith anti-GABA-Adeltaantibody,dilution:1/100(red),DAPI,dilution:1/10000(darkblue),anti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000(lightblue)andanti-NeuNantibodyfromMillipore,Cat.No.:MAB377(green) inmousecorticalculture(E16),havinguseddonkeyserumblockingbuffer.9a)b)Stainingwithanti-GABA-Adeltaantibody,dilution:1/100(red),DAPI,dilution:1/10000(darkblue),anti-MAP2antibodyfromNeuromics,Cat.No.:CH22103,Lot:400849,dilution:1/5000(lightblue)andanti-ParvalbuminantibodyfromSwant,Cat.No.:PVG-213(076)(green)inmousecorticalculture(E16),havinguseddonkeyserumblockingbuffer.

C. Immunohistochemistry

Immunohistochemistry, inmousebrain tissue sections,wasalsoperformedusingall theabove-describedcommerciallyavailableantibodies(Table4).However,theonlyantibodythatprovidedpositivestainingwastheanti-GABA-Adeltaantibody(Figure11).

Figure11:Stainingwithanti-GABA-Adeltaantibody,dilution:1/200(red),DAPI,dilution:1/5000(darkblue),anti-NeuN fromMillipore, Cat. No.:MAB377, dilution: 1/250 (light blue) and anti-Parvalbumin antibody from Swant, Cat. No.:PVG-213 (076), dilution: 1/ 2000 (green) of the same tissue section obtained from mouse cerebellum, having useddonkeyserumblockingbuffer.

ConclusionsAfterhavingperformedtheabove-describedexperimentaltests,wearenowconfidentthatwe have optimized the available techniques (western blot, immunocytochemistry andimmunohistochemistry) to obtain the best experimental settings thatwould allow us toaccuratelyinvestigatetheexpressionofdifferentGABA-AreceptorsubunitsinhumaniPScelllinesandmousecorticalcultures.

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This information will allow us to investigate precisely, the expression of those GABA-Asubunits in iPScell linesderived fromautisticpatientsand furthermore, tocomparethisexpression pattern in cells obtained from their healthy relatives. This approach willprovide new insights for the involvement of GABA-A subunits in autism and prove thehypothesis that specific GABA-A receptor subunits may be candidate targets forpharmacologicalinterventionstotreatautism.

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