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1、ProtocolsProtocolsfforNeuralorNeuralCellCultureCellCultureTHIRDEDITIONEditedbySergeyFedoroffandArleenRichardsonHumanaPressInVitroAssaysforAxonalGrowth1ChapterOneInVitroAssaysforAxonalGrowthandTargetingValérieCastellaniandJürgenBolz1.INTRODUCTIONInvitroassa
2、ysareimportanttoolsforinvestigatinghowtheintricatenetworkofneuronalcon-nectionsisestablishedduringbraindevelopment.Wehavebeenusingdifferentinvitrosystemstostudythecellularstrategiesinvolvedintheformationofneuronalprojectionsbetweenthecerebralcortexandsubco
3、rticalareas,andtoexaminemechanismsthatcontroltheassemblyoflocalcorti-calcircuits(forreview,seeBolzetal.,1993;Bolz,1996;BolzandCastellani,1997).Resultsfromthesestudiesindicatedthatdiffusibleandmembrane-associatedmoleculesprovidecrucialinfor-mationfortheesta
4、blishmentoflayer-specificextrinsicandintrinsiccorticalconnections.Untilrecently,verylittlewasknownaboutthemolecularnatureofthesesignals.Duringthepastfewyears,however,severalnewgenefamilies(netrins,semaphorins,andephrins)havebeendis-covered,whichcodeforprot
5、einsthatregulatethegrowth,targeting,andsynapseformationofaxonsindifferentregionsofthebrain(forreview,seeTessier-LavigneandGoodman,1996).Basedonthesefindings,invitroassayshavenowbeenusedtodeterminethepossiblecontributionofdefinedmoleculesonthespecificationo
6、fneuronalconnectionsinthedevelopingcortex.Firstresultsindicatethatthesemoleculesexertcelltype-specificeffects,andhaveawideandcomplexrangeofaction.Somemoleculesselectivelyaffectelongationorbranchingorguidanceofaxons,othersaffectanycombinationthereof.Forexam
7、ple,ephrin-A5,aligandforEphreceptortyrosinekinases,actsasarepulsiveaxonalguidancesignalforneuronsintheuppercorticallayers,andasabranch-promotingsignalforaxonsofneuronsinthedeepcorticallayers(Castellanietal.,1998).Othermoleculeshaveopposingeffectsofdifferen
8、tsetsofcorticalneurons:Theyeitherinduceorinhibitaxonalbranching,ortheyattractorrepelgrowingaxons(CastellaniandBolz,1999).Membersofthesemaphoringenefamilycanfunctionasdiffusibleandassubstrate-boundguid-ancesig
