[PDF] The effect of age on dendrites in the rat superior cervical ganglion

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J.Anal.(1994)184,pp.111-117,with2figuresPrintedinGreatBritainTheeffectofageondendritesintheratsuperiorcervicalganglionTIMOTHYJ.ANDREWS',DAQINGLI2,JAMESHALLIWELL'ANDTIMOTHYCOWEN'DepartmentsofAnatomyandPhysiology,RoyalFreeHospitalSchoolofMedicine,and2NormanSadieLeeResearchCentre,LaboratoryofNeurobiology,NationalInstituteofMedicalResearch,London,UK.(Accepted5August1993)ABSTRACTIntracellularinjectionofabiotinylatedprobeinfixedsuperiorcervicalgangliafollowedbyconfocalmicroscopywasusedtoinvestigatetheeffectsofageonthedendriticarborisationofsympatheticneuronsinratsaged6wk(youngadult),7months(fullygrownadult)and24months(aged).Inaccordancewithotherstudiesconsiderabledendriticgrowthwasobservedduringpostnataldevelopment.However,inoldagedendriticgrowthdidnotcontinue,andsignificantatrophywasobserved.Quantitationofneuronalmorphologyshowedsignificantreductionsinsomasize,totaldendriticlength,numberofbranchpointsandtotalareaofdendriticarborisationinoldage.Unexpectedly,significahtreductionsinthenumbersofprimarydendriteswereobservedinmaturityandinoldage.Concomitantwiththisatrophytherewasanincreaseinage-relatedmorphologicalabnormalities.Thesimilaritiesbetweentheatrophyanddendriticabnormalitiesshownbyouragedneuronsandthoseseeninotherstudiesofyoungadultsympatheticneuronsfollowingaxotomyortrophicfactordeprivationarediscussed.INTRODUCTIONTheimpairmentofthermoregulatorycontrolinagedhumansisanimportantclinicalproblem.Elderlypeopleshowattenuationofsweatingandperipheralvascularreflexeswithagewhichmayresultfromimpairmentsoftheautonomicnervoussystem(Col-lins,1991;Schmidt,1991;Abdel-Rahmanetal.1992).Inasearchforananatomicaldeterminantofautonomicdysfunction,anumberofstudieshaveinvestigatedtheeffectofageonthemorphologyofautonomicneurons(forreviewseeCowen,1993).Theyshowthatoldageaffectsautonomicneuronsinalocallyandtemporallyspecificmanner:celllossinoldagehasbeenreportedinenteric(Santer&Baker,1988;Gabella,1989)butnotsympatheticganglia(Santer,1991a);atrophyoftheautonomicinner-vationofperipheraltargettissueshasbeendescribedinanumberofareasincludingsomesuppliedbythesuperiorcervicalganglion(SCG)(Cowenetal.1982;Dhalletal.1986;Cowen&Thrasivoulou,1990);abnormaldendriticandaxonalprocesseshavebeenseeninageingautonomicgangliaalthoughtheextentoftheselesionsvariesbetweenandwithindifferentganglia(Schmidt,1991).However,atrophyisnotageneralphenomenon(Santer,1991b);indeedinsomeinstanceshypertrophyofautonomicfibreshasbeenobserved(Mioneetal.1988;Andrews&Cowen,1991).Itiswellestablishedthatsympatheticneuronsdependontheirtargettissuesforsurvivalanddevelopment(Purves,1988).Recentstudiessuggestthatthisrelationshipcontinuesinoldage(Ruitetal.1990;Gavazzietal.1992;Gavazzi&Cowen,1993a).Targettissuesregulatetheirinnervatingsympatheticneuronsbymechanismsincludingthelocalproductionoftheproteinnervegrowthfactor(NGF)(Levi-Montalcini,1987;Hendry&Hill,1980).IncreasedtargetsizeandresultingincreasedNGFsynthesisisbelievedtounderliethedendriticgrowthofsym-patheticneuronsthatoccurspredominantlyinthepostnatalperiodbutcontinueswellintoadulthood(Snider,1986;Voyvodic,1987;Purvesetal.1988;Ruit&Snider,1991).Inthisstudywehaveinvesti-CorrespondencetoDrTimCowen,DepartmentofAnatomyandDevelopmentalBiology,RoyalFreeHospitalSchoolofMedicine,RowlandHillStreet,LondonNW32PF,UK.III

112T.J.Andrews,D.Li,J.HalliwellandT.CowenTable1.DevelopmentandageingofsympatheticneuronsfromtheratSCGNo.ofSomasizeTotaldendriticPrimaryAreaofdendriticAgecells(pm2)length(gm)dendritesBranchpointsarborisation(pm2)6wk47660+34894+497.0+0.3817.1+1.29240+7668months451130+58***1325+69***5.9+0.29*22.5+1.4**18824+1276***24months49876+56*836+50***4.7+0.32*17.5+1.3*13126+854***Valuesrepresentmeans+S.E.M.*P<0.05,**P<0.01,***P<0.001;significantlydifferentfromvalueatprecedingage.gatedhowageingaffectsthedendriticarborisationofsympatheticneurons.Domatureandagedsym-patheticneuronscontinuetogrowastheydoindevelopment?Dotheirdendriticarborisationsstabil-isewhengrowthceases?Dotheyatrophyperhapsasaresultoflosttrophicsupportinoldage?Toanswerthesequestions,wehaveusedintracellularinjectionofabiotinylatedprobe,Neurobiotin(Lietal.1990),intofixedsympatheticneuronsfromtheSCGofyoung,adultandagedratswithconfocalmicroscopytovisualiseandquantifythedendriticarborisationoftheseneurons.MATERIALSANDMETHODSMaleSprague-Dawleyratswerekilledat6wk(youngadult),7months(fullygrownadult)and24months(aged)ofage.Inordertomonitoranimalgrowthwemeasuredthelength(nosetoanus;anustoendoftail)oftheanimalsateachagestage.Webelievethatanimallengthmaygiveamoreaccurateindicationofbodysizecomparedwithbodyweightwhichmaybeundulyinfluencedbyincreasedfatdeposits.Thedendriticmorphologiesofsympatheticneuronsfromthesuperiorcervicalganglion(SCG)werestudiedateachagegroup.Twelveratsofeachagegroupwerekilledwithanoverdoseofsodiumpentobarbitone(500mg/kg)andperfusedthroughtheheartwithTyrodesolutionfollowedby4%paraformaldehydeinPIPESbuffer.Agoodperfusionwasnecessaryforproperfixationofthetissue.SCGweredissectedfree,desheathed,postfixedin4%paraformaldehydefor2handstoredinphosphatebufferedsaline(PBS)containingsodiumazideat4°Cuntilused.Gangliawereimmersedina1:10000solutionofDAPI(MolecularProbes,USA)inPBSfor10mininordertovisualisethenucleioftheneuronspriortobeingpinnedoutonaSylgard-coatedculturedish.Thedishwasplacedonthestageofastandardepifluorescencemicroscope(LeitzOrthoplan,Ger-many)equippedwitha100Wmercurylamp.ThedishwasfilledwithPBSsothatfluidjustcoveredtheganglion.Itisimportantthatthegangliaarenotsubmergedtoodeeplyastherefractiveindexoftheliquidcanimpairtheimpalingprocedureandcon-versely,ifliquiddoesnotcovertheganglionitcandryoutcausingirreversiblestructuraldamage.Sympath-eticganglioncellswerevisualisedusingalongworkingdistancex32objective(Leitz;4.5mm,NA=0.4)andimpaledwithglasselectrodes(ClarkeElectro-medical,UK)containingamixtureof4%Neuro-biotin(Vector,UK)and2.5%LuciferYellow(MolecularProbes);electrodeshadresistancesof30-50MQ.Usingtheultravioletfilterblock(LeitzD2cube)theneurons(DAPI)andtheelectrode(LuciferYellow)couldbeseensimultaneously,thuspermittingaccurateplacementofthemicroelectrodetipwithamotorisedmicromanipulator(M.M.,Germany).AsatisfactoryimpalementwascharacterisedbytherapidfillingofthecellwithLuciferYellowfollowingashortburstofhyperpolarisingcurrent.Ifthecellbodyappearedbrightandtheprimarydendriteswerebeginningtofill,depolarisingcurrent(3nA)waspassedfor30mintofillthecellwithNeurobiotin.Carewastakentoimpalecellsasufficientdistancefromeachotherinordertoavoidcross-overofneighbouringdendriticarborisations.Thepopulationofcellsstudiedincludedsuperficialcellsaswellascellssituateddeepwithintheganglion.Aftercellsonbothsidesoftheganglionhadbeeninjectedinthisway,itwasplacedina1:50solutionofStreptavidin-TexasRed(Amersham,UK)dissolvedinPBScontaining0.3%Tritonovernightat4°C,washedinPBSandmountedinanantifademountant(Citifluor,UK).TexasRedfluorescencewasusedinordertonegatetheeffectoftheage-accumulatedpigment,lipofuscin,intheimagingofneurons.Labelledgangliawerestoredat-20°Cuntilused.Neuronswereobservedeitherwithax40(OlympusDPlanApo4OUVPL)orax16(ZeissNeofluar16/0.5)lensdependingontheextentoftheirar-borisation.Neuronswerefirstlocatedusingcon-ventionalfluorescencemicroscopywithfiltersforTexasRed.ConfocalscanninglasermicroscopywasperformedusingaBio-RadMRC600microscopefittedwithakrypton-argonlaser.Theapertureand

AgeingintheratSCGA.YoungB.Adult4>C.AgedFig.1.Neuronalgeometryofrepresentativesuperiorcervicalganglioncellsatdifferentstagesofdevelopmentandageing:young(6wk);adult(7months)andaged(24months).Cellswerechosentorepresentthefullrangeoftotaldendriticlengthsateachtimepoint(seeTable).Cellsarearrangedfromlefttorightinorderofincreasingtotaldendriticlength.Postganglionicaxonsareindicatedwithanasterisk.Imageswereobtainedbybinarising2Dconfocalprojections.Bar,50gmforallpanels.113.f/,,O.d-t-8ANA184

114T.J.Andrews,D.Li,J.HalliwellandT.Cowenthegainsettingsweresetoptimallyforeachneurontomaximisecontrast.Kalmanfilteringwasusedtoremovenonspecificbackgroundnoise.Successive2gmopticalsectionsoftheneuronsweretakeninbytheconfocalmicroscopeandstoredonopticaldisc,creatingfileswhichwerethenusedtogeneratethrough-focusimagesoftheentireneuron.DendritesweretracedinteractivelyonscreenusingBio-Radconfocalsoftwareinordertoanalysetheneuronalmorphology.Totaldendriticlengthwasmeasuredautomaticallyfromthetraces.Branchpointsweredistinguishedfromcrossingdendritesbyexaminingthez-seriesofimages.Somaareawasmeasuredinteractivelybytracingthecircumferenceofthecellsomawhilstdendriticareawasmeasuredbyjoiningthetipsofthosedendriteswhichextendedfurthestfromthesomabystraightlines.Theenclosedareasweremeasuredusingconfocalsoftware.Primarydendritesweredefinedasdendriticprocessesthatarosefromthecellbodyandextendedradiallyforadistancegreaterthanitsdiameter.One-wayANOVAwasusedtocompareagechangesinthedifferentparameters.RESULTSOurresultsshowthatbodylengthincreasesbyabout50%from6wkto7months(nose-anus,18.6-27.25cm;anus-tailend,13.9-21.58cm)butthatnosignificantincreaseinbodylengthoccursfrom7to24monthsofage(nose-anus,27.25-28.12cm;nose-tailend21.58-23.81cm).Thesedatasupportourde-scriptionofthe7monthratasfullygrown.Measure-mentsofdendritesaresummarisedintheTableandimagesofrepresentativeganglioncellsateachagestageareshowninFigure1.Thedendriticarboris-ationswerefilledtothefullextentoftheirfineprocessesandtheaxon(definedasalongunbranchedneuriteprojectingsomedistancefromthecellsoma)couldoftenbetracedforseveralhundredmicro-metresenroutetowardsthepostganglionictrunks.Postnataldevelopment(6wkto7months)Weshowedsignificantgrowthinthedendriticarbor-isationofsympatheticneuronsfromtheSCGduringthepostnatalperiod.Totaldendriticlengthincreasedbyabout50%(P<0.001),somasizebyabout70%(P<0.001)andthenumberofdendriticbranchesbyabout32%(P<0.01).However,incontrasttothegrowthoftheoverallarborisation,weshowedthatthenumberofprimarydendritesonSCGneuronsdidnotincreaseinnumberbutasignificantdecreaseofabout1wasdetected(P<0.05).Ageing(7monthsto24months)InoldagethedendriticarborisationofsympatheticneuronsintheSCGshowedsignificantatrophy.Thetotaldendriticlengthofneuronsdecreasedbyabout40°%(P<0.001),thenumberofdendriticbranchpointsbyabout20%(P<0.05)andthesomasizeshrankbyabout14%(P<0.05).Abnormaldendriticprofileswereseenatallagestagesbutwerein-creasinglyfrequentinoldneurons.Theseabnor-malitiesappearedin2differentforms:distendeddendriticprocessesorcellbodyswellings(Fig.2).Thenumberofneuronsexhibitingabnormalprofileswas23%at6wk,30%at7monthsand50%at24months.DISCUSSIONUnderstandingthecellularmechanismsunderlyingtheimpairmentofhomeostasisinageinganddiseaseisofmajorclinicalimportance.Longitudinalstudieshaveshownaprogressivedeteriorationintheauto-nomicregulatorycapacityofelderlypeople(Collins,1991).Ithasbeensuggestedthatthismaybeduetoimpairmentsoftheautonomicnervoussystem(Schmidt,1991;Abdel-Rahmanetal.1992).Inthisstudywedescribechangesinthedendriticarbor-isationofsympatheticneuronsoftheratSCGduringdevelopmentandageing.Previousstudieshaveshownthatthedendritesofsympatheticneuronsgrowconsiderablythroughoutpostnataldevelopment(Snider,1986;Voyvodic,1987;Ruit&Snider,1991).Ourresultsconfirmthesefindingsbyshowingin-creasesinsomasize,totaldendriticlength,numberofbranchpointsandtotaldendriticarea.However,althoughqualitativeobservationshavebeenmadeonthedendriticmorphologyofagedsympatheticneurons(Schmidt,1991),therehavetoourknowledgebeennoquantitativestudies.WeshowthatinoldagesignificantdendriticatrophyoccursinthesympatheticneuronsoftheratSCG.Somasize,totaldendriticlength,primarydendrites,numbersofbranchpointsanddendriticareawereallsignificantlyreduced.Inadditionto,orperhapsasaconsequenceof,theiratrophyagedsympatheticneuronsexhibitedabnormaldendriticprofileswithincreasedfrequency.Sympatheticneuronsdependuponthetargettissuestheyinnervatefortheirsurvivalandgrowththroughoutpostnatal

AgeingintheratSCGFig.2.PhotomicrographsofconfocalopticalsectionsthroughsuperiorcervicalganglioncellsthathavebeenintracellularlyinjectedwithNeurobiotin.Neuronsareshownfrom2agegroups;young-adult(6wk)(A,B)andagedanimals(24months)(C,D).Therewasanincreasingincidenceofdendriticabnormalitieswithage.Abnormalitiescouldbedefinedasdistendeddendrites(arrows)orcellbodyswellings(asterisk).Bar,25gm.development(Yawo,1987;Voyvodic,1989a,b).Tar-gettissuesinfluencetheirinnervatingsympatheticneuronsbythelocalproductionandreleaseoftheneurotrophin,NGF,whichistakenupandretro-gradelytransportedbytheaxontothecellsoma(Hendry&Hill,1980;Levi-Montalcini,1987).Theabnormaldendriticstructuresandatrophyweseeinagedsympatheticneuronsshowstrongsimilaritieswiththoseseeninstudiesofyoungadultsympatheticneuronsfollowingaxotomy(Yawo,1987)andtheapplicationofanti-NGFantibodies(Ruitetal.1990).Thisraisesthepossibilitythattheneuronalatrophyandabnormaldendriticprofilesweobservemaybearesultofreducedneurotrophicsupportfromthetargets.Adecreaseintheavailabilityofneurotrophicfactorshasbeenpostulatedasacauseofage-relatedneuronalatrophy(Appel,1981;Perez-Poloetal.1990;Gavazzietal.1992;Gavazzi&Cowen,1993b);however,thelinkhasyettobeproven.Furtherstudiesonthesynthesisandavailabilityofneurotrophicfactorsintheagedperipheraltargetsofsympatheticneuronsmayidentifylossoftrophicsupportasthecauseofage-relatedneuronalatrophy.Dendriticarborisationshaveanimportantroleintheintegrationofpresynapticelectricalpotentials(Miller&Jacobs,1984)andindeterminingthenumberofdifferentinputsthatconvergeonindividualcells(Purves&Lichtman,1985).Invivoimagingofsympatheticneuronshasshownthattheirdendriticarborisationsareinacontinualprocessofgrowthandretractionthroughoutpostnataldevelopmentandintoadulthood(Purvesetal.1986).Ourresultsshowthatthisprocessofgrowthandretractionresultsinnetgrowthofdendritesinpostnataldevelopmentandnet8-2115

116T.J.Andrews,D.Li,J.HalliwellandT.Cowenretractioninoldage.Becausemostofthesynapsesinsympatheticgangliaaremadeontothedendrites(Forehand,1985),thischangeinmorphologymustgohandinhandwithachangeinsynapticconnectivity.Theimpliedlossofsynapticinputintheagedneuronsreceivesexperimentalsupportfromstudiesthatshowtheappearanceofdystrophicpresynapticaxonsinagedratsympatheticganglia(Schmidt,1991).Thenumberofsynapsesthatimpingeonsympatheticneuronsisdramaticallydiminishedfollowingpost-ganglionicaxotomy(Matthews&Nelson,1975)ortheapplicationofantiserumtoNGF(Nja&Purves,1977).Thesestudieslendsupporttothehypothesisthatagechangesinsympatheticneuronsmayresultfromreducedneurotrophicsupport.Thisstudyhasbeencarriedoutusingintracellularinjectionoffixedtissueandconfocalmicroscopy.Althoughthedendriticarborisationsweobtainedappearedtobefilledtothefullextentoftheirfineprocesses,theywereabout40%smallerthanthoseobtainedinacomparablestudy(Voyvodic,1987)usingintracellularinjectionofHRPoflivingneurons.Previousstudiesusinglightfixationofsympatheticneuronspriortointracellularinjectionobtaineddendriticarborisationscomparabletothoseseenusinginvivoinjection(Ruitetal.1990;Ruit&Snider,1991)althoughinthesestudiesthetissuewasstillmaintainedunderphysiologicalconditionsincaseofinsufficientfixation.However,althoughthedendriticlengthswerelargertherelativechangesweobtainedcomparedwellwiththoseofVoyvodic'sstudy.Weshowa50°%increaseindendriticlengthduringthepostnataldevelopmentcomparedwitha55%increaseinhisstudy.Weoffer2possibleexplanationsforthedifferentsizeofdendriticarborisationsobtainedinthepresentstudyandthosefromcomparablestudies:(1)theperfusionfixationwehaveusedmaycausesubstantialshrinkagerelativetotheimmersionfix-ationofotherstudies(Voyvodic,1987;Ruitetal.1990);(2)thefixationwehaveusedmayrestricttheaccessofmoleculestothefineprocessesofthedendritictree.Therefore,althoughtheheavyfixationweusedgavesmallerdendriticarborisationsthanthoseseeninsimilarstudiestherelativechangesduringpostnataldevelopmentusingthistechniquecomparedfavourably.Theadvantagesofthistech-niqueareitsflexibilityandsimplicity.ACKNOWLEDGEMENTSWewouldliketothankProfessorG.Raismanforhishelpinestablishingtheintracellulartechnique,ChrisThrasivoulouforhisexperttechnicalassistanceandIsabellaGavazziforhelpfulcriticismofthemanu-script.ThisworkwassupportedbyagrantfromtheWellcomeTrust.TimAndrewswassupportedbyaJuniorResearchFellowshipfromtheRoyalFreeHospitalSchoolofMedicine.Theconfocalmicro-scopewaspurchasedwithagrantfromtheWellcometrust.REFERENCESABDEL-RAHMANTA,COLLINSKJ,COWENT,RUSTINM(1992)Immunohistochemical,morphologicalandfunctionalchangesintheperipheralsudomotorneuro-effectorsysteminelderlypeople.JournaloftheAutonomicNervousSystem37,187-198.ANDREWST,COWENT(1991)Target-specificchangesinautonomicnervefibresinoldage.EuropeanJournalofNeuroscience,Supplement4,1987.APPELSH(1981)Aunifyinghypothesisforthecauseofamyotrophiclateralsclerosis,parkinsonism,andAlzheimerdisease.AnnalsofNeurology10,499-505.COLLINSKJ(1991)Theautonomicnervoussysteminoldage.ReviewsinClinicalGerontology1,337-345.COWENT(1993)Regulationoftheautonomicinnervationofbloodvesselsduringdevelopmentandageing.InVascularInnervationandReceptorMechanisms:NewPerspectives(ed.L.Edvinnson&R.Uddman),pp.25-40.London:AcademicPress.COWENT,HAVENAJ,WENQINC,GALLENDD,FRANCF,BURNSTOCKG(1982)Developmentandageingofperivascularadrenergicnervesintherabbit.Aquantitativefluorescencehistochemicalstudyusingimageanalysis.JournaloftheAuto-nomicNervousSystem5,317-336.COWENT,THRASIvOULOUC(1990)Cerebrovascularnervesinoldratsshowreducedaccumulationof5-hydroxytryptamineandlossofnervefibres.BrainResearch513,237-243.DHALLU,COWENT,HAVENAJ,BURNSTOCKG(1986)Perivascularnoradrenergicandpeptide-containingnervesshowdifferentpatternsofchangeduringdevelopmentandageingintheguinea-pig.JournaloftheAutonomicNervousSystem16,109-126.FOREHANDCJ(1985)Densityofsomaticinnervationonmammalianautonomicganglioncellsisinverselyrelatedtodendriticcomplexityandpreganglionicconvergence.JournalofNeuro-science5,3403-3408.GABELLAG(1989)Fallinthenumberofmyentericneuronsinagingguineapigs.Gastroenterology96,1487-1493.GAVAZZII,ANDREWSTJ,THRASIVOULOUC,COWENT(1992)Influenceoftargettissuesontheirinnervationinoldage:atransplantationstudy.Neuroreport3,717-720.GAVAZZII,COWENT(1993a)Axonalregenerationfromratsympatheticgangliatransplantedinoculoisnotimpairedbyage.ExperimentalNeurology,inpress.GAVAZZII,COWENT(1993b)NGFcaninducea'young'patternofinnervationintransplantedoldcerebralbloodvessels.JournalofComparativeNeurology,inpress.HENDRYIA,HILLCE(1980)Retrogradeaxonaltransportoftargettissue-derivedmacromolecules.Nature287,647-649.LEVI-MONTALcINIR(1987)Thenervegrowthfactor:35yearslater.Science237,1154-1162.LID,SEELEYPJ,BLISSTVP,RAISMANG(1990)Intracellularinjectionofbiocytinintofixedtissueanditsdetectionwithavidin-HRP.NeuroscienceLettersSupplement38,81.MATTHEWSMR,NELSONVH(1975)Detachmentofstructurallyintactnerveendingsfromchromatolyticneuronesoftheratsuperiorcervicalganglionduringthedepressionofsynaptictransmissionind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