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Differential Involvementof Ca

2

Channels inSurvival andNeurite

Outgrowth ofCultured EmbryonicCockroach BrainNeurons PASCAL BENQUET,JANINE LEGUEN, YVESPICHON, ANDFRANC ¸OIS TIAHO

Equipe Canauxet Re´ cepteursMembranairesUnite´Mixte deRecherche 6026-CentreNationaldela Recherche

Scientifique, Universite´deRennes 1,35042 RennesCedex, France Received 5September 2001;accepted innal form15 May2002 Benquet, Pascal,Janine LeGuen, YvesPichon, andFranc ¸ois

Tiaho.Differential involvementof Ca

2 channels insurvival and neurite outgrowthof culturedembryonic cockroachbrain neurons.J Neurophysiol88: 1475-1490,2002; 10.1152/jn.00749.2001.The con- tribution ofvoltage-gated calciumchannels (VGCC)to thedevelop- ment ofcultured embryoniccockroach brainneurons wasassessed using pharmacologicalagents. VGCCcurrents wererecorded using the patch-clamptechnique andwere foundto beblocked dose-depen- dently bymicromolar concentrationsof mibefradil.The activationand inactivation propertiesof thecalcium channelsenable asizeable calcium currentto owat rest(about 30 and20 mVin high- potassium culturemedia). Asexpected, thecytoplasmic-free calcium concentration wasfound torise whenthe extracellularpotassium concentration wasraised from3 to15 and30 mM.The effectsof VGCC blockersand calciumchelators weredifferent infresh andin mature culturesin whichthe neuronswere connectedto eachother to form adened network.In freshcultures, thetwo non-selective VGCC blockers(verapamil andmibefradil) induceda dose-dependent cell deaththat wasproportional totheir blockingeffect onI Ba . This effect couldnot beprevented byaddition offetal calfserum tothe culture medium.A similareffect wasobtained usingintra- orextra- cellular calciumchelating agents(10

M BAPTA-AMor 10mM

EGTA). Quiteunexpectedly, blockadeof theP/Q-like ( -Aga WA- sensitive) componentof thecalcium currentby 500nM of -AgaTx IVA hadno lethaleffect, suggestingthat thecorresponding channels are notinvolved inthe survivalmechanism. Asexpected fromtheir lack ofeffect onI Ba , isradipine,nifedipine, and-CgTx GVIAdid not induce celldeath. Whenthe neuronsstarted growingneurites, their sensitivity tocalcium channelblockade bymibefradil decreased, indicating acorrelation betweenneurite outgrowthand resistanceto calcium depletion.In maturecultures, theneurons becameresistant to mibefradil, verapamil,and BAPTA-AM.However, theseagents, as well as -AgaTx IVA,had asignicant inhibitoryeffect onthe increase indiameter ofthe connectivesthat linkedadjacent clustersof neurons. Thiseffect hasbeen shownto result,in thecase ofmibe- fradil, froman inhibitionof neuriteoutgrowth characterizedby a signicant reductionof thenumber ofprimary neuritesand secondary branchings butnot toa signicantmodication ofthe diameterof individual neurites.These resultssupport theview that,as inverte- brates, calciuminux throughVGCC playsan importantrole in survival andneurite outgrowthof culturedembryonic insectneurons. The differentialcontribution ofthe P/Q-likeand R-like( -Aga WA- sensitive) calciumchannels inthese processesis discussed.

INTRODUCTION

In theCNS ofvertebrates, voltage-gatedcalcium channels (VGCC) areknown tobe implicatedin synapticplasticity and gene transcriptionin additionto their"classical" rolein neu- rotransmission andregulation ofcell excitability(Berridge

1998; Bitoet al.1997; Finkbeinerand Greenberg1998). In

vitro, inthe absenceof neurotrophicfactors, neuronsurvival is rescued byactivation ofcalcium inuxthrough L-typeVGCC (Collins andLile 1989;Gallo etal. 1987;Koike etal. 1989; Murrell andTolkovsky 1993;Yano etal. 1998).It isonly recently thata tyrosinekinase (Trk)-likereceptor hasbeen identied inthe CNSof invertebrates(Lucini etal. 1999;van Kesteren etal. 1998).The difcultiesin identifyingneurotro- phins hasimpeded studiesabout themechanisms ofneuron survival andgrowth ininvertebrates. Despitethe observation that survivaland growthof insectcultured neuronsneeded a culture mediumwith highK (Beadle andHicks 1985), suggesting thatdepolarization ofcell membranemight bea prerequisite, littleis knownabout therole ofVGCC, and therefore calciuminux, onneuronal survivaland neurite outgrowth ininsects.

So far,most VGCCfound oninsect neuronsomata were

found tobe insensitiveto dihydropyridine(DHP) andsensitive to both -CgTx GVIAor -AgaTx IVAtoxin fractions(Ben- quet etal. 1999;Bickmeyer etal. 1994;Wicher andPenzlin

1997). Theyhave alsobeen shownto befunctional earlyin

neuronal development(Baines andBate 1998;Goodman and Spitzer 1979),a ndingthat isconsistent withthe hypothesis that theymight, asin vertebrates,play akey rolein different aspects ofneuronal differentiationsuch assurvival orneurite outgrowth. Thishypothesis isalso consistentwith theobser- vations that,in Drosophila,nullmutation ofthe genescoding for VGCC(Smith etal. 1996)or mutationsreducing the activity ofthe calcium/calmodulin-dependentprotein kinase-II (Grifth 1997)are lethalduring embryogenesis.

We havepreviously shownthat (Benquetet al.1999)

1 ) embryoniccockroach brainneurons inprimary cultureex- press atleast twotype ofhigh voltageactivated (HVA)calcium channel currentsnamed P/Q-like(sensitive to -AgaTx IVA) and R-like(insensitive toDHP andthe 3toxins -CgTx GVIA, -CmTx MVIIC,and -AgaTx IVA)and 2) thephenylalkyl- amine (PAA)verapamil isa non-selectiveblocker sinceit Address forreprint requests:F. Tiaho,Equipe Canauxet Re´ cepteursMem- branaires, UMR-CNRS6026, Universite´ deRennes1,Ba ˆt. 13Campus de Beaulieu, 35042Rennes Cedex,France (E-mail:francois.tiaho@univ- rennes1.fr). The costsof publicationof thisarticle weredefrayed inpart bythe payment of pagecharges. Thearticle musttherefore behereby marked' ' advertisement in accordancewith 18U.S.C. Section1734 solelyto indicatethis fact.

J Neurophysiol

88: 1475-1490,2002; 10.1152/jn.00749.2001.

14750022-3077/02 $5.00Copyright ©2002 TheAmerican PhysiologicalSociety www.jn.org

by 10.220.33.4 on December 31, 2016http://jn.physiology.org/Downloaded from brought to you by COREView metadata, citation and similar papers at core.ac.ukprovided by HAL-Rennes 1

suppressed efciently bothcomponents ofthe calciumchannel current. Todirectly assessthe physiologicalrole ofVGCC in neuronal developmentin insects,we haveanalyzed theeffects of selectiveand non-selectiveblockers usingthe wholecell con guration ofthe patch-clamptechnique andcompared these effects withthose ofthese sameblockers onsurvival and neurite outgrowth. Our experimentsindicate that,as invertebrates, VGCCare involved inneuronal survivaland neuriteoutgrowth. Further- more, theresults suggestthat theactivation ofthe DHP-, -CgTx GVIA-,-CmTx MVIIC-,and -AgaTx IVA-resis- tant currentcomponents (R-like)is importantfor neuronal survival, whereasthe -AgaTx IVA-sensitivecurrent compo- nent (P/Q-like)is selectivelyinvolved inneurite outgrowth.

METHODS

Cell culture

The culturetechnique wasderived fromthat ofChen andLevi- Montalcini (1970),as describedby Beadleand Hicks(1985) and recently modi ed (Angevinet al.2000). Brie y, newlylaid eggswere stored for21

23 daysin anincubator (28-29°C). Theheads ofthe

embryos wereremoved fromthe eggcases, andthe brainsdissected out byremoving thehead capsule.The cellswere dissociatedme- chanically bygentle mechanicaltrituration witha Pasteurpipette ina de ned volumeof culturemedium. Thecultures wereinitiated ina medium (54) containingve partsof Schneider's revisedDro- sophilamedium andfour partsof Eagle's basalmedium containing

100 i.u./mlpenicillin and100

g/ml streptomycincomplemented with

6 mg/ml

L-glutamine and2.5 g/ml fungizone.After 5days, therst (54) culturemedium wasreplaced bya second(L G) medium made ofequal partsof Leibovitz s L-15medium andYunker s mod- i ed Gracemedium containingpenicillin, streptomycin,glutamine, and fungizone,supplemented with10% fetalcalf serum.It shouldbe noted thatboth culturemedia containedhigh K concentrations (16 mM inthe former,30 mMin thelatter), whichfavored thedevelop- ment ofthe cultures.The Ca 2 concentrations were4 mMin therst medium and3 mMin thesecond medium.Low intra-or extracellular calcium concentrationswere obtainedby adding10

M BAPTA-AM

(30-60 min)or 10mM EGTAto theculture media.The resultingfree calcium concentrationwas estimatedto belower than25 and75 nM, respectively, insideand outsidethe cells,assuming K d values of, respectively, 0.2and 0.07

M forthe bindingof calciumto thetwo

chelators. Theculture mediawere obtainedfrom GIBCO-BRL(Cergy

Pontoise, France).

Electrophysiology

Currentsowing throughthe calciumchannels werestudied using the wholecell con guration ofthe patch-clamptechnique (Benquetet al. 1999;Christensen etal. 1988;Hamill etal. 1981).Whenever necessary, theculture mediumwas replacedwith arecording solution containing (inmM)100 tetraethylammonium(TEA)Cl, 70Tris-HCl, 10

4-AP, 10BaCl

2 , 4MgCl 2 , and10 HEPESbuffered atpH 7.2using TEA-OH. Unlessnoted otherwise,the patchelectrodes werelled witha solution containing(in mM)120 CsF,25 CsOH,2 MgCl 2 , 10EGTA, 3

ATP-Mg

2 , 0.5GTP-Tris, and10 HEPESbuffered atpH 7.3using CsOH. Theresistance ofthese electrodesranged from2 to5 M. Voltage-clamp experimentswere performedwith thepatch-clamp am- pli er RK300(Biologic ScienceInstrument, Claix,France). Unlessstated otherwise, theholding potential(HP) was70 mV.All experiments were performedat roomtemperature (20-27°C). ThepClamp (Axon Instruments) 5.5software wasused forstimulation, dataacquisition, and

analysis. Whenevernecessary, thedata wereanalyzed off-lineusing different softwarepackages: Excel(Microsoft), andSigmaplot (JandelScientic). TheStudent 'st-test wasused forstatistical analysis.

The peakcurrent-voltage relationships( I-Vcurve) weretted withquotesdbs_dbs4.pdfusesText_8