THE EFFECT OF PERIPHERAL DEAFFERENTATION ON SPECIFIC AXONAL SYSTEMS AND GLIAL ELEMENTS
IN THE MAIN OLFACTORY BULB OF THE RAT
BY
cSHELLEY R. KING,B.Se.
Athesissubmittedto the Schoolof Graduate Studiesin partialfulfilment of the
requirements for thedegreeof Masterof Science
Faculfy of Medicine MemorialUniversity of Newfoundland
1994
St.John's Newfoundland
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;;ABSTRACT
Heterotypic collatera l sprouting of neurotrans mitter syste ms11.15been observed in manyregionsof th eeNSfollowing injury.Theolfacto rybulb(DB)recelvc sdirect input from theolfacto ry epitheliumand itsneur oanarom v,111<1 ncur otr.msmnter conte nthavebeenwell characte rized. Thus,the DB mayprovide<1lIseful co rtical model of lesion-induc edplasticity.The presentstudy hastestedthehypo thl'sis 111011 heterotypic collateralsprouungofspeci fic axonalsyste ms,especially the sororoucrgtc axonsfrom the raphe,willoccur following peripheraldcauercnuuonof Illl' OB.In addition,wehypothesi zedthatan increasein thenumber of astrccvrcs would rxx'ur followingdeafferen tationof the DB. In this study, seroto nin, dop.umne lJela- hydroxy lase,glialfibrillaryacidic protein (GFAP) and5-100 (Scrolorlp.rgic growth facto r)immunocytochemistry wasperforme dto eluc idate therespons e of diHc rt'tll axonalsystems and glialele ments toperipheral deafferentation of the DB.
Deafferentationof theright DBwas achieved by app lica tion ofZIlSO~10til<!
olfactoryepithelium(DE) ofadult malerats and postnatalday (PND) 10 rillpups.The effectivene ssofthelesionwas determinedbythedegree .,f the loss of tvrc sinc hydroxylase(lH)immunoreactivityinthe periglcmer ularce llswhichrequir e the presence of theDEto expresstheTHphe notype. The deafferen tation res ulted in,I
decrease inthe densityof serotone rglc fibresin the deeper regionsofthelesioll(!(J DBin adult animalswhichisinco ntrastto theincrease in densitycvtdcmintho PND 31animals.The den sityofastrocyles expre ssing 5-100,apropo sedsl~roton<~rl;ic
growth factor,didnol correlate with thedensity of seroro nerg!c innervation.In contrasttothe serct onerg!c system,Ihe densityof noradren erglcfibresin thelestoned 013wasincreasedin theadultanimals, but remainedunchan ged in PND31rat pups.
Thedensities of GfA P positiveastrccytesinthe lesfoned OBofadult andPND 31 animals we resim ilar to those ofcontrolanimals. These results suggestthat deafferentation of the OB of adult animals didnotinducehetero typic sprou ting of intact sero tonerglcfibres in thethreeweektimeperiod.On the contrary,the serotonergic fibreswere se lectively decreased, perhapsdue10 the potential ncuroto xtcnv ofzinc10these fibres.It appear s, however, Ihattheseroto nergicfibres in theyo unger animals are capable ofspro utinginrespo nse to OB deafferentation andthisresponsemayberelated to the indirect neuromodulatorv effectsofzinc.
iii
ACKNOWLEDGEMENTS
Iwouldlike10extendmysincere thank s10Dr.JohnMel.canforprovidilll-:me withthe opportunitytoparticipate intheNeu roscience grad uateprogr-un.wnhour hispatience,guidanceand suppo rtsuccessfulcompletionof thisprogram wouldnut have beenpossib le.Iwouldalsoliketo thanktheothermembersof Illy supcrvtsorv committee, Dr. DetlefBiegerandDr. TomScali,fortheir guidanceduringIlly degree programand Dr.GrahamSkanesfor hisadviceregardingstattsttcal,In.lly;is tow,mls thelatter stagesofmyprogram.Iam alsoindebtedto AndreaDarbv-Kingforher technicalassistance as well as friendship throughout myprogr.un.
Acknowledgementisalsodueto Dr.John Mctoan,thoFacultyof Medkiru' and theSchool ofGraduateStudies,MemorialUniversityofNewfoundland, fo r their financialsupport during my degreeprogram.
TABLE OF CONTENTS ABSTRACT..
ACKNOWLEDGEMEN TS TABLE OF CONTENTS
LIST OF FIGURES viii
INTRODUCTION .•. ... 1
1.1 Plasticityof theCentralNervous System 1
1.2 Plasticity of the Serotonergicsystem 6
1.3 Plasticityof the NoradrenergicSystem 8
1.4 Plasticityof the Choline rgicSystem... .. 9 1.5GlialResponse 10Injury oftheCNS ...•. . .. . . 10 t.6 The Olfactory Bulbas a CorticalStructuretoInvestigateLesion-
InducedPlasticity. . . ... .... .. . .. 12
1.6.1 The OlfactoryEpithelium 13
1.6.2 Neuroanatomy of the Olfactory Bulb 14
1.7 NeurotransrnttterContentof the OlfactoryBulo. . . ,•. 17 1.7.1 Neurotransmiltersand Neurcpeptldes Intrinsic to the
OlfactoryBulb. . . ... .... 1B 1.7.2 Serotone rglc Inne rvation oftheOlfactory Bulb 19 1.7.3 Noradrene rgk Innervation of the Olfactory Bulb 21 1.7.4Cholinergic Innervation ofthe Olfactory Bulb .. . 22 1.8PeripheralDenervatton ofthe Olfactory Bulb 23 1.9 EffectsofPeri pheral Denervationonthe OlfactoryBulb... . 25
1.10Objectivesof theStudy. .. .. 27
MUHODS ... 28
2.1 Animals.. 28
2.2 Anaesthesia. 28
2.3 Surgery.. . .. 29
2,4Perfusion , 31
2.5Immunocytochemi stry 32
2.6 ZincStaining... . . ... 34
2.7 Ana lysis.... 36
2.7.1Quantificatio nof theDeafferen tation of the Olfactory
Axons 36
2.7.2Den sity ofImmunoreactive Fibresand Astrocytes.. .. .. 38 2.7.3Relationship Betwe en the DensityofSerotone rgic Fibres
andAstrocvttc Expressionof 5100. . 43 2.7.4 Re lative Opt ical Density Measureme ntsof ZincStaining. 44
(,2 (,5 70 75 RESULTS.•.• •• . .. . . . .•• •••• . • . •••••.. . ... 45 3.1 TyrosineHydroxylaseImmunocytochemistry . 45
3.2Density ofSerotoninImmunoreactive Fibres 51
3.3Density of5·100Immunoreactive Astrocytcs 57
3.4 Correlation ofthe Densityof SerotonergicFibresand Astrocvt!c Expressionof5100 .
3,5DensityofDBHImmunoreactiveFibres.. 3.6Densityof GFAPImmunoreactiveAstrocvtcs , 3.7 ZincStaining ..
DISCUSSION . , ••• .. •..•. 71\
4.1Tissue ShrinkageFollowingDeafferentation 111
4.2 The Effectiveness ofDeafferentation . 1J2
4.3 Differencein the SerotonergicResponseFollowing Peripheral Deafferentationof the 08 inAdult andPND10Animals... 117 4.3.1 Correlation BetweentheDecreasein Serotonerg!c Input
and loss ofPON Axonal Input:A Punctionn l loss... 1m 4.3.2The lack ofCorrelationof5100Expression andthe
Densityof Serotonerg!c Fibres ~1
4.3.4 The Potential EffectofZincon theSerotouerg!c Innervation of the OB•. .... .. . ... ... 'J4 4.3.4.1 Potential Neurotoxic Effect of Zinc on
Serotonergic Fibresin AdultAnimals 94 4.3.4.2PotentIalNeuromodulatoryEffect of Zincand the
Association with Increased Serctonerg!cFibre
Densityin PND 31 Animals 97
4.3.5 Consolidationof FactorsPotentiall y AffectingSerotonemtc
AxonSprou ting 'J'.l
4.4TheEffect of OBDeafferentation onthe DensityofNoradrenergic
Innervation •• 102
4.5lack of Significan tChangeinthe Density of GFAP'Astrocytes Follow ing Deafferentation or the 06.. .. 10 5 4.5.1Additiona l Responses of AstrocytesFollow ing Inj ury. lOll
CONClUSION 1Of)
LIST OF REFERENCES.• III
LIST OF FIGURES
Figure 1. Diagramof the rat skull indicating site oflesion.
Figure2. Cameralucldadrawing of serotonergic fibers in a PND31animal indicatingareasofthe08selectedfor image analysis.
Figure3. Photomicrograph showing loss ofTHimmunoreactivity in adult animals21daysfoll owi ng peripheraldeafferentationof the08.
Figure4. Graph showing the numberof PG cellsper glomer ular profile expressingTHimm unoreactivityin the Gl of the08incontroland lesto ne d adultanimals21days following peripher al deafferentation.
Figure5. Photomicrograph showing loss ofTHimmunoreactivityinPND31 animals21daysfol low ingperipheraldeafferentatio nofthe OB.
Figure6. Graph showingthenumber ofPGcellsper glomerular profi le expressingTHimmunoreactivity inthe Gl of theOB in controland lestonedPND31animals21daysfollowingperipheraldeafferentation.
Figure 7, Photomicrograph of serotonin immunocytochemi stryinthe08of control(A)andlesloned (B,C)adultanimals21 daysfollowing peripheraldeafferentation.
Figure8. Graphof the densityof serotonergtcinnervation ofthe OBin control and lesioned ad ult animals 21 days following peripheral deafferentation.
Figure9. Photomicrograph ofserotoninimmunocytochemistry inthe08of control (A) andlesioned (B,C)PN D 31animals21days follow ing peripheral deafferentation.
Figure10. Graphofthedensityof serotone rgfcinnervationof the08in control and lesloned PND 31 animals21 days following peripheral deafferentation.
figure11. Photomicrographof5100immuno reactiveastrocvtesin the08of control(A)and lesioned(S,C)adultanimals21days follow ing peripheral deafferentation.
vii
Figure12. Graph of the densityof 5100immunoreactiveastrocvtesin tilt' OB of controland lesloned adult animals21davs follow ingpl'riplll-'r.ll deafferentation.
Figure13. Photomicrographof5100immunore activeastrocvres in the DBof co ntrol(A)andle stoned (B,ClPND 31animals21 d.lySfollo wing peripheraldeafferentation.
Figure14 . Graph ofthedensityof510 0immuno reactiveastrocvtcsinthe OB of controlandlesionedPND 31animals21 daysfollowingperiphc r.ll deafferentation.
Figure15. Graphshowinglack ofcorre lation betweenthedensityofserotnl1f'rgk fibresand densityofastrocytesexpressing5100in the OB of.Illlllt animals21daysfollowingperipheraldeafferentat ion.
Figure16. Graphshowinglackofcorrelation between the density ofscrotoncrgfc fibresanddensityofastrocvtesexpressing5100intheDB ofrNO 31 animals21 days following pe ripheral doafferenrauon . Figure17. Photomicrograph ofDBHimmunoreactivityin theDB ofcontrol(A)
and lestoned (B,C) adultan imals 21days followingpcrtphcr.rl deafferentation.
Figure18. Graphof thedensityof noradrenerg!c innervationorthe Oil incontrol
and lesloned adult animals 21 days following peripheral
deafferentation.
Figure19. Photom icrographof DBHimmunoreactivity inthe OB ofcontrol(f;) andlestoned(B,C)PND 31 animals21daysfollowing pcrfphcrat deafferentation.
Figure 20. Graphof thedensityof noradrenergicinnervationoftho OBincontrol and lesloned PND 31 animals 21days following ponpherat deafferentation,
Figure21. Photo micrograph of GFAPimmunoreactiveasrrocytesinthe013 of contro l (Alandlesioned (B,C) ad ult animals21daysfo llowing periphera ldeafferentation.
Figure 22. Graph ofthedensityof GFAPimmunoreactiveastrocyrcsinthe 013of controland lesto nedadult animals21daysfollowingperipheral deafferentat ion,
viii
Figure23. Photomicrograph ofGFAPimmunoreactiveastrocvtesin the DBof control(A) andlesione d (B,C)PND 31 animals 21days following peripheral deafferentation.
Figure!24. Graphof the density ofCFAP immunoreactive astrocytesinthe DBof control andlestoned PND31animals 21days fo llowing periphe ral deafferentation.
Figure25. Photomi crograph of zinc stainingin the 06 of contro l (A) and tenoned (B/C) of adult animals3 days followingintrodu ctionof lnS04intothe nasal cavity.
Figure 26. Graphor Ihe relativeoptical dens ity measu rementsor Iheintensity of zincsta ininginthe DB of contro landlesioned ad ultanimals3 days following introductionof ZnS04intothenasal cavity.
INTRO DUCTIO N
Thisthe sis examinesthe hypothes is lhalhetero typiccolla tcr.nsproUlillg of intact fibresystems, especially theserotonerglcsyste m,wouldoccurfo!lmv inl-\
removalof the afferentinput to theolfactory bulb(DB).Inaddition,W{'hypn lhesi7C'd that astrocvtesmightincreaseinnumber,thusdisplaying anIncrease indl'llsityill re sponse todeafferent ationof theDB.Abriefhistoryofresearch on pl,IS'icily with in thece ntralnervou s system(CNS) will bepresen tedfirst,followedby" review oftlu- phenom enonofsprouting exhibited byneu ronsand theresponses of Ali'l "flprinjury.
In add ition ,thehypothe sesadd ressedinthis thesisand the rationale hchiud1',lCh hypoth esis are presented inconjunct ionwithsimilarstudies oflesion-imlurcd plasticityinoth er regions ofthe CNS.
1. 1 Plastici tyof theCenlralNervousSystem
The abilityof thenervous syste m toregenerateandformnewsyn,lp5(~Sattor injury or damag eto theeNS hasbeenthe focusof much research since the(,'ldy 1950. Thestudy oflesio n-ind ucedplastic ityisimportantin orde rtodotormtnclhl~
mechanismsunderlyingrecovery offunc tionafte rinjuryand 10compare how differe nt region s of theCNSare reorgan ized . Onemean s by whichthebrainreacts 10inju ry isby growth orspro utingof axons.Historically,collate ralsprouting hilS beendefined asthegrow th ofaxo ncollate rals overso medlstancc beforethey conta ct an areathathasbeen denervated byalesion. Hom otypic collateralsproul i n~occu rs
when undamaged fibres,containingthe same transmitterasthe damagedne uro nal system,Increase theirprojections. In contrast,heterotypiccollateralsprouting occurs whenthe fibre system that responds to the damage isdifferentfromthe system that had originallybeen injured.
The phenomenon ofcollatera lsproutingofintactfibreswas first demonstrated in the peripheral nervoussystemIf dds,19 53), however, muchscepticismexisted as 10the abil ityofthe neuronsinthe central nervo us system 10 sprout in responseto injury or deafferentation.The firstevidenceofcollate ral sproutingofintact axons was observedin the peripheralnervoussystemfollowing pa rtialdenervationofthe sympatheticganglion(MurrayandThompson, 1957),whilethefirstevidenc eof sproutingin the CNS was observedafter partialdenervatlonof thespinal cordof the carIUu and Chambers,1958).Since thelate1950'sthe collateralsprout ingresponse oftheCNS.,fter injuryhasbeen welldocumen tedby sever al investigators, usinga varictvof techniques,in severalareasof theeNSincludin g the septum (Raisman, 196 9; RaismanandField, 1973),dentategyrus(lynchet al.1973 ;Matthewset .11.1976),hippocampus(Azmitiaeta1.1978;ZhouandAzmitia,19 86), striatum (Stachow iaket,11.1984;Synde retaI.1985),superiorcollicu lus(RhoadesetaI.1990), lateral geniculatenucleus(Rhoades et a1.1990)andspinalcord (Polistinaet a1.1990;
W,lIlg eta1.1991 ;Marlieret a1.1992l.
The abilityofvariousfibn~systems10sproutinmanyregions of the eNSh.l!' led to thebelief tha t the sproutingphenomeno n underliesrecovery offu nctio n foll o wingeN Slesio ns,Threedifferent systems, th e seroton--gfc,norad roucrg!c.md cho linergicsystems,each withwidely distributed cortical andsubcorucalpmjcrtious, havebeenespeciallyexaminedin relationto sprou ting,Indeed, colt.nora!sprou ting oftra nsplantedserotonergtcneu ronshasbeenimplicatedin recovery of soxu.rl func tionfollow ingsp inalcordinjury (PrtvatetaI.1989),Stmtlarlv,parli.11recovery of moto rbehav ioris attributed10homotypic collateralsproutingof sllpr.\!'pill.ll.mel/or propriospina lprojectio nsin respo nse10deafferentationofthespinalcord(Gold lll'rgcr andMurray, 1974),However,the exactmech anismsunderlying theappearanceof spro uting and functional recoveryhavenotbeenclearlyelucldatcd.
Although axon alsprouting is a pheno menongene rally recognized.Iftminjury tothe eNS,not allfib ressystemsexhibitsproutingfollowinginjury.Controversyalso exists astothe occurrence ofsproutingin certainareas.Inaddition tothereportsor sprouting in the superiorcclltculus and lateralgeniculate nucleus{Rho,nll'sct aI.19 90),lac k ofsproutinghas alsobeenreported inthese structures (l-lickev,11)7.'i;
Stelz nerand Keating,1977; Chow era1.1981)following unilateralenucle atio n.
Sim ilarly, although sproutinghasbeen extens ively rep ortedinrho spinalcord followi ng injury,absenceofsprouting has alsobee nreported(Rustio ni andMolcnailr, 197 5;Beckerman andKerr,19 76; Goldbe rgerandMurray, 1982;Pubol s andBOWP I1, 198 8). Thediscrepancies in reports of spro uting in the sameregionsof the eNS may bedue to the differen tmethods of produ cingthe injury,For instance,chemica!
deafferentationofthe spinalcordprodu ced by ricin injectiondoesnot induce sprouting of fibresinthe dorsal hornU' ubols andBowen,1988),whiledestruction of the primary effercntsto the same regionusinganotherchemical,capsaicin, ind uces ofsproutingofscrotonergicfibres(Polistina ela1.1990).
In add itionto theconflictingreportsof theoccurrenceof sprouting, the response of differentneuronalsystemstothe sameinjury may notbe identica l.Thus, thnreappearstobesome degree ofselectivityinterms of which fibressprout followingin jury, indicating thatsome neuronalsystemsmayexhibit agreaterdegree ofplasticity in responseto a particular lesion.Forinstance, sercronergfcfibres in the r.ltspin.l lcordhave beenreported to sproutafterunilateraldorsal rhizotomy,while the noradre ne rgic inne rvationappears 10 be unaffectedbythe lesion (Wang et aI.199 1).The developmental stage of theanimal atthetime ofinjury may also be an important factor in determining theresponsetothe lesion. Insomeareasof theeNS, the capacityof fibres to sproutis onlyevidentfollowinglesionsin young orneonatal animals. In the striatum,for example.sproutingof theserotonerg!c systemoccursafter dopamine-depletinglesionsin 3 day old rats,butnot whencomparable lesions are performed inadultanimals (Stachowiaket al.198 4;Synderet aI.1985).
Severalmethodshave been used to detectspoutingof fibres fol low ingeNS lesions. Fibredensitymeasurements,obtained using immunocytochemicalstaining, have been usedby various investigatorstodetect lesion-inducedsprouting(Luthma n et a1.1987;Rhoades et a1.1990;Wangeta1.1991;Zhouetal.1991; Marlieret ,11.1992).Additionalmethodsofdetecting sprouting,whichareoften used aloneor
in combination with im munocytochemical techniques. include ruoasurtng biochemical levels of neurotransmittersand/ortheir metabolites (Slachowi.1kpt a1.1984; luthman et at198 7; Zhouet011.1991;Baker et ill.19931andhorseradish peroxidase retrogradetracingof thefibreprojectionsto the denervatedstructure (Synder eta1.1985l. Inthe presentstudy, trumunocvtochemlcal.\ll.llysis is the method used todetectthe responseof fibresystems 10 deafferentationsine"this techni que allowsus to studychanges in the densityof axons.15well.15 their distributionwithinthe OB.
1.2Plasticityof (heSerct one rgtcSystem
The serotone rgtcsystemexhibitsanexce ptionalabllltvtosprout inresponse to injuryof theCN5.Homotyp ic collateralsproutingofserotonerg!c fibreshasbeen demons trated in thehippocampus followingselective chemical lesion ofthe scrotonergicafferentsin the cingulumbundle (Azmitiaetal.1978;Zhou andAzmitia, 1984;Azmitiaand Zhou,1986).Heterotypiccolla teral sprouti ngofthe serotonergic nxonshasbee n establishedinIhe spinalcord followi ng deafferentation(Polistinaet a1.1990; Wang et a1.1991;MarlieretaI.1992).Inaddition,specific dep letio nof dopamine, usingtheneurotoxin 6-hydroxydopamine,inthe striatum(Stachuwiak et ,
11.1984;Synderetal. 1985)andsubstantianigra(Zho u eta1.19CJ1)results in heterotypiccollateralsproutingof serotonerg!c fibres.
Theplasticity observedinthe serotonergicsystem following varioustypesof injuryto theCN5 may be due torelease of trophicfactors. Ofthe many growth factorsIhought to beinvolvedinregeneratio norsproutingofaxons ingene ral,the 5-100 Bprote inis believed 10be specificforthe scrotonergicsystem(Azmitia et al.1990;Wh itaker-Azmitiaet al.1990).
The 5100Bprote inis amember ofthe5100familyof proteinswhicha-e in turnapari ofthe superfamilyof calcium-modulatedproteins (Van Eldik eta1.1982).
The 51006pro teinisdistributed througho ut thene rvou s systemwith the majo rityof
$-1006 belie ved to reside inglia (Bock, 19 78). In the rat brain 51006 immunoreactivityrevealsthattheprotein is strictly localizedto astrocvtes(Boyes et aI.198 6).5100B isthoughttobeproduced(Bock,1978) andsecreted(Suz ukiet
a1.1987; Van Eldik andZimmer,1987)by astrocytcs in the brain. While thespecific function of 5100has not bee n determined, pote ntialneurotrop hic activity ofthe proteinhas been demonstratedln cellcultureexperiments(Klingm,llland Marshak, 1985 ; Klingmanand Hsieh,1987:VanEldiket ,11.1988).Recent surdfoshave proposed that 5100B is a serotonergic growth (actor (Azmili.let .1IYlIJO;
Wh itaker-Azmitiaetal.1990) that isre leasedby stimulat ionof5-HTl"rece ptors011 astroglialcells (Whilaker-Azmitiaet aI.1990).
Thepresent study investigatedthe hypothesis.tharheterotypic coll.ucral sprou ting of intact serotone rglc fibres would occur following ]JNiphl!r.11 deafferentationof the olfactorybulband that potentialchangesillrho scrotonc rgtr innerv ation may,in part,reflect an accompanyingchangein thonumber ofasuocvres expressing5-100.
1.3Plaslicity of theNoradrener gicSystem
like the serotonergicsystem, noradrenergic fibresalsodisplaythe abilityto sproul in responseto injury.Homotypiccollateralsprouting ofintact ncradrenerglc axons has been repo rted following chemical induced axotomv of central nc radrenergfcneurons (Bendeich era1.1978; Kostrzewa et a1.1978;Bjorklundand Llnuvnll,1979;Nakai eta1.198 7; FritschyandCrazanna, 1992) andpartial dcnorvauon of the hipp ocampu s (Gageet a1.1983;Madisonand Davis,1983).
Heterotypiccollateralgrowth of noradrenergicaxons in thelateralgeniculate body (Stcnevi er <11.1972)and septum(Mooreet a1.1971)has beenreportedfollowing unil.lter.l1ablationofthevisual cortexand hippocampus,respectively.
Thesero tonergic and noradrenergicsystemsexhibitthe ability to sprout after various le sionsof the eNS.The responseof thesesystems, however,has not been compared followingperipheraldeafferentationof the clfactorvbulb.Comparing the responseof the twosyste ms tothesame lesionmay be important indeterminingthe extentof reorganizationfollowinginjury.The presentstudy examined thehypothesis lhatasimilar sproutingresponse ofthe serotonergicandnc radrenergtc svstemswould occurfollowing periphera l deafferentationof the olfactorybulb.
1.4Plasticit y oftheCho linergicSystem
Homotypicsprouting ofthe cholinergic syste m hils been obse rved follo wing partialdeafferentation of the hippoc ampus(lynchet,11.1973;Nadlerel .11.1977;
Nadler etat.1977;Gage et a1.1983)and destruction of cholinurglcncuro~lsfollowillA excttotoxlclesions of the nucleusbasalis (WenkandOltorn,191J4).III cml'r.Jsllolilt' plasticityof cholinergicexons observedinthe cerebralcortexfollowinginjrctionof lbotenic acid, anexcttotoxln, into the nucleusbasalts. no evidenceofSlu outing of cholinergicaxonswasobservedafterinjectionof anothe roxcnotoxtn,qlllSqll.ll.III·, intothesamestructure(Henderson,1991).Despitetheseinlcrestil1j:;.111(1pOlt'llli.llly importan t observationsof choline rgicaxonal sproutingin the eNS,1111'rl'Spoml'of thecho line rgic system to peripheraldeafferentatio nofthe OBcouldno t bl' determined in the presentstudy due tothe lackof a reliable market for cholinergic
10 1.5 Glial ResponsetoInjury oftheeNs
Gliosisis aubiquito us process thatoccursintheeNSfollowing injury.
Re acti ve astrocvtes are oflen appare nt inthelocal enviro nme nt of the injuryfollowing
damage10theeNSandmaybe close lyassociatedwith the regrowthof nervefibres.
Reactiveastrocyteshavebeenre ported followingvarious typesofinjurytothe
eNSincluding cerebralslabwounds(Bignami andDahl,1976;Latovet a1.1979;
Mathewsonand Berry,1985;'lekamlv a elal.1988), cryogenic le sions (Amaducci et
aI.1981),laserinjury(Schifferetat.1986), opticnerve enucleation(Trimmerand Wunderlic h, 1990)andtransection ofthe spinalcord(Barrettet aI.1984). Foll ow i ng injury,reactiveastrccvtesmay increase in numberaroundan inj u redMea,eitherdue 10proliferation IBignamiandDahl,1976;Barrell et a1.1984;Mathewson andBerry, 1985;Tekamtveeta1.1988)ormigrati on (Rose etaI.1976).
Glial fibrillaryacidi c protein (GFAP) is an immunocytoche micalmarker specific for theintermediatefilamentsof fibrousastrocyresin the eNS (Eng et .11.197 1; Bignami etal.1972).Identification of asrrccvtesusing GFAP isa reliable and freq uently used metho dofdetecting astrocvtes intheDB(Bailey and Shipley,1993), inotherregions ofth e eNS (BignamiandDahl, 1973;Schiffer et a1.198 6;Takam iya ct <11.1988) andarter injury 10variou ssitesin theeNS (Bignam iand Dahl,1976;
Am aduccieta1.1981; Takamiva et a1.1988),
II The presentstu dy inve sti gatedthe hypothesisthat an lncroascinthe number of astrocvteswould occurin the 06 following peripheraldeafforom.uton.nwrebv resulti ng in an increase in the density of astrocvros expresstng the immun ocytoch emicalmarkerforGFAP.
12 1.6 TheOlfactoryBulbasa CorticalStructur e toInvestigat e Lesion-Induced Plasticity,
The vertebrate ousocrv epitheli um{OEl containsapopu lation ofimmature
stem cellsinthe basal neuroepitheliu mwhichretain the ability10divide and differentiateinto maturereceptorneuro nsthroughc utthelifeofthe animal(Graziedel
..nd Monli-Graziadei, 1978bl.Thus,theolfactoryreceptorne urons that have degeneratedin response to noxio usenvironmenta lstimu lior experimenta llyinduced lesion s arcable10bereplacedby newreceptorneuronsorigina t ing(ro mthebasal cellsintheneuroepithelium(Gra ztadeiandMonti·Grolz/adel ,197B a;Graziadeiand Monti-G razlad el, 198 0).Inaddition,the ncuroanilt omy andneuro tr ansmittercontent ofthe OBhave been well characterized.Theseprope rties,coupledwith therelative caseof accessibi lityofIhe O Bfor experimentalmanipulation,ma ke s theOBa useful corticalsructureforexamining the plasticity of neurotransm itt ersvstemsafter denervation.
Thefo llowing sections briefly outline the structure ofthe olrac1ory neuro epithel iumandmainolfactory bu lbsince familiarilywiththesestru ctures is necessary10understand the methodofde uervatio n usedand the effec ts of peripheral dcne rv ation reportedInthis stu dv.
13 1.6.1TheOlfac to ry Epithelium
The vertebrateolfactory epitheli um(D E) is composedof three cellular elemen ts:(1) sensory or prima ryolfac toryreceptor cells,(2) sustentacular or supportingcellsand(3) basalcells(Mou ltonandBeid ler,1967; Graziadci .md Monti-G raziadei, 1978 a).Therec eptor cellsarebipolarneurons withprOCCSSl'5of varyinglength.The axonsof theprimaryolfactory neuron s (PON)are unmyelinated but aresurround edby glialcells, called ensheathingcells(Ralsman, 1911 5;Doucenc, 1989;Doucette,1993) untiltheyreachthe olfactory bu lb(OB).Theaxousof the PONleavethe DE indiscretebundles. thefiia o/{actoria,and tra velthro ugh tim famina oibrosaofthe ethmoid bon e\0becomethe olfactorynervelayer(ONl).mil termin ateinthe glomer ularlayer (Gl),the secondlayerof the OB.
Thesupp o rting or sustentacularce llsme columnarepi thelialcellsthai provide mecha n icalsupporttothe olfactoryreceptor cellsby ens heathingthe receptors.In addition 10 their roleinsuppo rt,thesecellsalsoserve a secretory functionandIMVp.
beenimplicated inma in taining propernutritionforthe rec eptor cells.Thefunction of the basal cells hasnotbeendea rlyelu cid ated.It appears,howev e r,ihatthcsocells arere pl acement clementswhichare resp o nsible (or therege neration ofthe olfactory recept o r cells afterinjury «(Moulto nandBeidler,1 967; Mcleanan dShipley,1992).
14 1.6.2Neu roan.lli omyoftheOlfactoryBulb
The neuroa natomy ofthe mainolfadorybulb (M O BIhas been well documented IShe phen:l.1972;Switz e retat.1985;McleanandShiple y,199 2).The MOBis ahighlyorganizedla minar structure consistin gofa totalofsevenlayerswith clearlyidentifiableboundaries.The olfactorynervelayer (O Nl)constitutesthefirs!
layer ofthe MOBand consis ts ofth e axonsofPON and glia cells.The individual axo nsofPO Npenetratethecribriformplateandentertheventromedialaspectofthe 08toterminate inseveralglomeruli(Pinching andPowell,1971)withinthe glomerularlayer(Gll,the second layer oftheDB. In the re t,theGLconsistsof cp p roximatcty3000ovoidglomeruli(I'kisami andSafari,19811andis a cellpoor re gi on wilheachglomerulussurroun dedby a relativel ylhinla ye r of juxtaglomerular orperiglo merularcells and gUa.Thedendritesandaxons ofth e juxtaglomeru larcells mayf,lmifywithintheglomeruli (Ca jal,1911).surroundthe glomeru li with inthe periglomerul arregion(Caja l,1911; MacridesandDavis,198 3;Mcl eanet al.1989) orproject10otherareasofthe ipsilateral bulb<Schoe nfeld et aI.198S).The dendrites of the mitralltuftedcellsalsoprojecttotheGland make syn a p tic contactswiththe axonsofthe PON.Subjacent10theGlis ananotherrelativelycellpoo r region,the externalplexiformla\'er(EPl).The EPlconta insmainlysuperficial,mid dle anddeep tu ftedcellsas wellasmede ndritesofmitra l/tuftedandgranulecells.The mitralcell la y er(Mellis irnmedierelvsubjacenttotheEPland isa th in layer co ntainingthe somataof the mitralcells.Themitralcellsha ve a singleapica ldendrite thatpro jects m.l inlytothe Glwhereitmakes connectionswiththeaxonsofthePON and10
1.1 other centrifugalinputsto the OB(She pherd,1972).The mitralcells, .llang withIhl' middle and deep tufted cells(Schoe nfeld and Macrldes,1984)fromthoEPl, constitutethe output cells of theOB.The internalplexiformI,WN(lPUlies immediatelysubjace nt to the Mel anc'containsthe axons and dendrites of rnitr.ll.uul granulecells,respectively.The nextneuronallayerofthe bulb,the gr.muk-cellI,lyl'r (Gel),lies subjacent tothe IPl.The Gelconsistsmainly of thesom.uaofgr.muk- cells arranged in parallelrows.Thegranulecellsdo not haveaxonshuthave ,lpic,ll andbasal dendrites that ramifyin the EPland Gel.respectively(Mori('t,II.IIJILI;
Oronaeta1.1983;Scoll,1986).The subeoe ndvmelzone isIIl(~docpost1.lyertnuu- MOB. Althougha relativelycellpoor region inthe aduh, the subependvmal ...oue is are gionof cell proliferationduring de ve lopment.
The accessoryolfactorybulb(AOB) is located atthe caudal-dorsal region of the olfacto rybulb.Although theAOS is significantlysmaller thai the MOB,till!
cytoa rchitec tureof the two structuresis similar.The ADais alaminate d structure, composed ofrelatively thesame laye rsas theMOB, withtheexception ofthenerve layer and subependvmalzone.Inthe AOB,it is lhevomeronasalnerveth.urransmlu informationfrom the vomeronasalorganin the nasalseptumto the glollll' ruiiintill' accessoryGl. The accessoryGl isle ssdistinct thantheGl inthe MOB stnco till' glomeruli aresmalle r and fewerinnumbe r.Theperlglomeruler cellsin the ar:cessory GLdo notsurroundthe glomeru li,as inthe MOB,butremain oneithe rstdeofthe glo merulianddonot contain dopa m ine. Subjacent to the acccsscrv Gl,is the accessoryEPl,whichisless prominent than itscounterpartin the MOBsincethe
16 cellsof thenextlayer, the accessoryMeL,aremixed inwiththeAEPl.The accessory IPLliesbetweenthe accessoryMelandlateralolfactorytract.The finallayerofthe ADSisthe accessoryGeLwhich is situateddeep to thelatera l olfacto rytract and contains thesametype of cells as theGel oft heMOB (McleanandShipley,1992).
Althoughboth the MOBandAoaareimportan tsubdivisionsofthe olfactory systeminmacrosmancarnmalssuchasrodents,only theeffectsof peripheral dencrvationof theMOB are discussedinthe presentstudy.
17 1.7 Neurotra nsmitterConlentof the OlfactoryBulb
The olfac torybu lb hasnumer ou s intrins ic neuron s c)(p ressinp,v.uious
neurotransmi tterslpeptidesandreceives centrifu galafferen t input sfromnU,lclnry relatedcorticesandsubcor ticalregions.Theolfactory relatedprojecti ons.lrl'linked 10olfactorysensoryandassociation functio ns.The afferentinputs(0thebull>from
the subco rtical regions often have widespreadprojecti ons10other.U(',lSoftlu-eNS andare bel ieved to servea modulatoryfunctio n .Theolfactory rcl .1[ell<:l'ntrifll p,.11 affere nts arise from the anterior olfactor y nucle us, piriform cortex,pcri,1tllygd,lloid cortex, entorhina l cortex,nucleus of thelater alolfactory trac tend,l111ygd,ll,l(MeLt',1Il and Shipley, 1992), while thedorsal and medianraphenuclei,locus cooruleus,mil nucleu s ofthediagonalbandprovidetheothermajor"non-olfactor y" subcor tical sources of afferentinput intothe DB,
Altho ugh theOB rece ivesmany sourcesof afferent input,onlythe seroto nerglc, noradrenergtc andcholine rgicinnervationwillbe describedin the following sections. Thepresentstudy foc useson the respon se oftheseroronc rgtc;md norad re nergic systems to peripheral deafferentatio nofthe DB,De scriptions ofthe other sources of afferentinnervation have beenrecentlyreviewed(McLcl.1I1and Shipley, ,992).
18 1.7.1Neuro trans mitt ersand NcuropeptidesIntrinsictotheOlfactor y Bulb
Thespecific neurotransmitter(s)ofthePONhas not,asyet,been conclusively
determinedalthoughrece nt findingssuggest thatglutamate oraspartateare involved inncurolransmission(Tremble y andShepherd,1994),Carnosine (Sakaiet a1.1988) and olfactorymarke rprotein(M argo l is,1980 ) are twosolublepro teins thatare fairly specificIDthePO N burproba blyarenot involvedin neurotransmission.Caldtonin
gene relatedpeptide(Rosenfeldelal.1983)and substan ceP(Baker,198 6) have been observed in somefibresin the first layerofthe MOB,the ONL. Thespecific origin ofthe latter two peptlde shas not been clearly definedbutisbelievedto be associat edwiththetrigeminalaxons.In the GL,thePGcells havebeenreportedto containnumerousneurotransmitte rsand neuropeoudesincl ud ing dopamine (Serbv ct .11.1991;DavisandMacrides,198 3), GABA(Ribak etal.1977),substanceP (Kosaka et a1.1988l,met-enkephaltn (DavisetaI.1982 ), vasoact iveintestinal peptide (G allet .11.1986;SanidesKohlrauschand Wahle,1990),somatostatin (Scott etaI.19 87), cholec ysto k inin (Seroogyetal.1985; Matsutan ieta1.1988)and aspartic acid(Hal asz, 1987). Althoug h the PG cells contain a wide variety of transmitters and neuropeptides,the exactrole of these substancesin the processi ngofolfac tory informationhasnot beendearlyelucidated. Thetuftedcells of theEPl have been reported to containGABA(Kosaka etal.19B7),cholecystokinin(Shepherd,1972) and V.1SO<1Ctive intestinalpeptide(Gall et al.1986;SanidesKohlrauschandWahle,19 90).
Themuralcells in thenext layer of the OB,theMel,havebeen reported to contain Necetyl-aspartyl-glutamate (8lakely et a1.1987;ffrench-Mullen eta1.1985) and
19 corticotropinre leasing factor (Imakietal.t989).The middle tuftedcellsoftheMel reportedlycontainvasoactive in testinalpeptide(Gallet <1.1.1(86)while thesuperficial to deep tuftedcells have been reportedto containcholecystokinin(Seroogy et aI.1985).AlthoughthelPlcontains relatively few cells,some ofthe cellshave been reported to containvasoactive intestinalpeptide(San idesKohlrausch,IntiW.lhle, 1990). Finally,the cellsin theGelcontainGABA(Ribaket.11.19 77),V.1SO.1C!iVC!
intestinal peptide (SanidesKohlrauschand Wahle,1990),cholecystokinin(Shcplwrd.
1972) and met-enkephalin(DavisetaI.1982).
'.7.2 Serotoner gicInnervationof theOlfactory Bulb
Serctcnergic fib res are widelydistributed throughout the DB.However,the fibres thatoriginatefromthedorsal(DR) and med ian raphe(MRlnuclei Innervate different layers(Mcl ean andShipley, , 987b).Thickfibres, presumablyfromthe MR (MamounasandMolliver,1988),preferentia llyinne rvate the glomeruli,whilethin fibers,presumably from theDR(MamounasandMolliver,1( 08 ),arefoundprimarily intheinframitral regions ofthebulb (Mcl ean and Shipley, 198 7h).
Inthe DB, the densityof serotoninfibresis greates tinthe Gl.Th edensity of fibres in the Glis estimated 10be2-3times greaterthantheotherbulb lavers (Mclea nandShipley,1987a).Althoug hthe specificconnectionsandthe function of theserotonin fibresinthe glomerulihavenotbeen elucidated,the close proximity of thesefibresandthePO Nter minals suggeststhat serotoninmay haveadin..'Ctor modula toryro lein olfactoryfunctio n.Giventhe constantturnoverof synapsesinrho Gl duetothe continualregenera tionofrece ptorcells andtheiraxons in the
20 periph eryand potenti alrole ofseroton ininthe inhib itio n of synapse formation (Haydo nctill.1984), seroroo!n maypotenti allybe involved inregulationof synapse fcnnauonwith in theGl of theOB.Serotoni n mayalsobeassociated witholfactory informati onprocessing since thefibres arehighlyconce ntrated inthe glomeruli, where theIlrst synaptic relay occ urs (romthe DE. In thisrespec t,wehaverecently shown Ih.l1depictionofseroton in fibresin theneonateratpupresu ltsin theinability ofthepups10acquir e orexpress a preferen ce for apeppe rmi nt odor aftera one trial cond itioned odortrain ing parad igm (Mcleaner al. 1993).
21 1.7.3NoredrenergicInnervationof the OlfacloryBulb
The locu s coeru leus (lCl, located in the rostral ports, provides the noradrenerglcinnervation10 thebulb.Intherat,itisestimat edlhalalmost 40%of theneuro ns origi natingfromthe LC project10 thebulb(Shi pley('IilI.19(5).Thr- noradr energlc fibresinnervate alllayersof the 06with the exception of the ONL.
TheIPL andGelreceivethe strongestnoradronergtcinnerva tionwhile the EPL,Ind Me l have amoderate innervation.However,theGl receivesonly ,I(I'W nc radrcnerglc fibres(Mclean and Shipley,1987a; Mcle an andShlplev,199 1). TIl\' physio logicalrole ofnorad renalinehasbeen extens lvelvstud iedintheOil 11lI!Its actualroleis stilldebated. Noradrenaline hasalsobeenimplkal cdinolfacrorv learning sincenorad rene rgicantagon istshave been shown 10impedethe,lhi lilYof newbornrats10form learnedodorpreferen ces andnoradr enorg!cagonlsts tend10 enhancelearning (Sullivan cr aI.1989).
22 1.7.4Cholin ergicInnervation of theOlfactoryBulb
Thecholine rgicprojection10 the bulbarises fromthe rostromed ialportionof
the horizontallimbof the nucleusofthediagon al band(NDB)(Macrides eta1.1981;
Carson,19 84;Zaborszky er al.1986 l.Thecholinergic neur o ns areintermingled with GABAcrgi\.neuron slocatedmainly inthe lateral-caudalportion of theho rizonral limb
ofthe NOB(Zaborszkyet al,1986).The cholinergicinnerv atio nofthe bulb,as revealedby cholinesterasestaining,is concentratedin the Cl, EPl,lPland GeL.The axonal distributionofthe GABAergicinnervation(romthe NOB isharderto determine sincetheperiglomerutarandgranule cells provideintrinsicGABAergic innervation oftheOB.
23 1.8 PeripheralDenervationof theOlfactoryBulb
A variety of methodshave been used to deprivetheolfactory bulb of sellsory innervation. These methods includeprimaryolfactorynerve axotomv,intr.lllilsal irrigationwithzincsulfate(ZnS04)or tritonX-100and cauterizationof the(~xt('rn.ll naris (Alberts,1974). However,external narisclosure, unlike theothermethods
or
sensory deprivation, results in functionalrather thananatomicaldepnvauon (I3rllnjl's and Frazier,1986).
Denervattonind ucedbyintranasalirrigationwithTritonX-lOa orsurglcal manipulationresults indegenerationofaxons of the PON.However,since thestern cells remain undamaged followingTriton X·I00irrigationor axotomv,new prinwy afferentsto the olfactorybulbareable10 be generated withcompleterc gcnc muon reported to occurwithin30-60 days post-treatment(HardingctaI.1977;Gra/i.lliei andMonti-Graziadei,1976a; GraziadeiandMonti-Craz iadel,1980;Mouti-Graziadci et a1.1960;Baker et aI.1983).
Intran asalirrigation or applicationof ZnS0410the OEresultsin coagulation necrosisofthe OE(Smith,19 38;Schultz,1941;Schultz,196 0;Margolis et.11.19 74;
Matulionis,1975a;Harding ela1.1978),Fo llowing necrosis,theOESl'p<Jriltps(ro m the underlyinglaminapropriaand issloughed off(Smith,1938;Schultz,IlJ4 1J. Slnco the stem cells liein thebasal olfactoryepitheliumthey arebelieved 10 be castoff
24 along withthe OE re$ulling in degenerationofthe axons of iheroN.The degenerativeeffects ofZoSO. havebeenreported 10 persist{rom30 days 10 up 1 year followingthelesion(Sch ultz,1960;Mulvaneyand Heist.1900;MalUlio nis.
1975b).ZnSO.application was the method usedtoachieve deafferentationof the 08 inth is studysinceitresultsin longterm degene rationofthe axonsofthePO N.
25 1.9Effectsof Peripheral Dener vation onthe OlfactoryBulb
Destruction of the primary sensoryefferenrsto theolfactorybulbfeslillingfrom axotomv or intranasalirrigation of ZnS04or Trtton
x ·,
00were the preferred methods of use inbehavioralstud ies ofolfacto ry impairedanimals. Thesemeth odswere thoughtto be spec ific10the peripheral nervou s systemwh ilecausinglittle,if.lIlY, direct damage10theeNS.However, degene rationoftheprtmarvsensory .ufcrenrs inducedbythese methodsproducesprofound morphological changesinthe olfactory bulb.Denervation of thebulbis accompan iedby(1) a 30-75' %,decreaseinolf,lClor y bulb weight(Margoliset a1.1974;Hard ingeta1.19 78;Kawan o andMJrgolis,1982;Baker etat.1984);(2) atrophy orshrinkage of theglome rular layer ,lnd tndividu.i l glome ruli (Margo lis elal.19 74;Hardinget a1.1978;Baker et aI.1984);(J)shnnkagc orcompleteabsence of theolfactory nerve layer(Margo lis, 1972;Baker ctaI.1984);
(4) compactedperiglome ru larcells(Margolisetal.t 974).
Inaddi tion tothemorphol ogicalchanges in theDBduetoremoval ofthe primary afferentinputofthe olfactory bulbseveral changes have been reportedin neurotran smitter content. The periglo merularcellsare by far lhe principal dopaminergic containingelementsofthe olfactorybulb(Ha laszcta1.19 8 1;Hal,lsz andShepherd, 1983).Foll owingafferent dene rvationthe PG cellsexhibit n dmstlc redu ctionin tyrosine hydr oxylase (TH)tmmuno reactlvttv (Bakerct a1.1983; Baker el a1.1984;Kream eta1.1984;McleanandShipley,1988; Bakeret a1.198 8;Biffuct al.1990).Accompanyingthe redu ctio n ofTHimmuno reac tivity isa drasticdec rease in TH enzyme activity(Nadieta1.1981;Kawan o and Margolis,198 2 ; Bilkerct
26 a1.1988)anddopamine COA)contentofthedenervated bulb(Nad iet al.19Sl; Kawano andMargolis,1982;Baker et a1.19831. However,redu ction of TH immuno reactivityanddecreased enzyme activity is not due10degen eration of the PC cells(Pinching and Powell,19 71;Graz iadei and Monli-Graziadei.1980)and the THimmunoreactivityrenrmstocontrol levelsuponregenerationofthe afferent innervationfollowingreversiblelesio ns (Nadiel al.1981;KawanoandfvtargoJis.
1982;Baker el a1.1983;BakeretaI.1984).Thus, theexpressionofthe THphenotype appears10bedependent uponthe presenceoftheafferentinputinto the bu lb(Nad i
ctal. 19Bl;Bakerct al.19B3)andis believed tobemediatedatthe transcriptional levelofTHmRNA (Ehrlichet aI.199 0).Theassociation between THand the olfactory nerveexistsduringdeve lopmen t ofthe OBsince thePG cells do notexpr essTH immu nore act ivilyunlilcontacted by the olfactorynerve (Mclean and Shipley,1988).
Incomparison with the reduct ionin OA conten t, the biochemically determi nedleve lsofnorep inephr ine(NE)appear to remain unchan gedfromcontrol levels withanyslight increasesattributed10thedecr easeinthe size of the bulb after deafferen tation (Nadietat.1981; KawanoandMargolis, 1982;Baker etal.1983).II isnotdear,however,ifanysprouting ofthe NEexonsintothe denervatedareas occurredin these studies.
27 1.10 Object ivesoftheStudy
Themainobjectivesof this smdv were to determine: {1l ifperi!llwr,ll denervationinducedplasticity(o r hetero tvolc colhuera l sprout ing)oftheseroton c rgtc systemin differentlayersof theDB; (2)ifthepotentialincrease inthe density of serotonergicinnervation,as aresultof collateral sprouting,reflected au accomp.mvtng increasein astrocyticexpressionof 5-100, aproposed sercto ne rgtcgrowthfactor; OJ if increasesin the number of GFAP+astrocvtesoccurredin the DB asilconsectucnco
of peripheraldeafferentation; (4)ifperipheral denervalion affectedthe noradronorg!c innervationofthe OBi (5)ifdifferencesexist betweenadultandyoung 'll1im.l ls in their axonaland estrocvuc re sponsetodeaffe re ntation ofthe DB.
28 METHODS
2.1 Animals
Adultmale12SC-275g)and postnatal daylen(PNO10l,bothmale and female, Sprague Dawley rats (Charles River) were used inthis study.Theratswere housed inthe anim al carefaci lityin theHealt h Sciences CentreatMemor ialUniversity.The
animalswe re kepi on a12hourlight-dark schedulewithfoodand wateravailab le ed IibifwlJ.Proceduresperformedontheanimals ....'Pre approvedbythelocalan imal ce re ccronuue einaccordance withtheguide linesof the Canadia nAssociation on AnimalCare .
2.2 Anaesth esia
Adultrats were anaesthetizedwithsodiumpentobarbital(40mglkg.i.p.),The rats weregiven0.5011ofatropine (lm glml. Lp.),5-10 minutes priorto being anaesthetized,inorde rto reduce mucou s secretloosduring surge ry. App roximately equalnumbersof PNO 10 male andfemale rat pups from the same litter were .In.lesl hetizedbyhypothermia.Thepups we replacedinanicewate r bathfor app roximatelythree minutesor untiltheyfailed torespondtoapaw pinch.The pups were kept onicethrougho utthesurgery.
29 2.3 Su rgery
Alongitu di nal incisionwas madealongthemidlinebetween til eC'yC'~of till' anaesthetizedanimal. After retraction ofthedermisi15111.111hole wasdrilh-d immediatelyadjacenttothe midlineon the rightside(Fig.I).The dorsum of theri~hl nasalcavity wasopened10expose the conchae andthe rcsptr.uc rv.urd olf,lrtory epithe lium. Unilateraldenervalion of the DEwasachieved byimerlinp,Ct>lfo.ml (Upjohn), saturatedwith20plof a.34MofZnSO~(SigmaChe lllic.l lCo.)inO.!J%
salineintothenasal cavityof PND 10 animals andSOplofthe same solution in,ulull an imals. The cavity was sealedwith a thinlayer of sterileboneW.1X(EthtronLtd.l before closingthe incisionusingstainless steel wo unddips (ClayAdalns)niltlu- ad ultand 6.0 silksutures(EthiccnLId. )on PND 10anima ls.
Adult rat s were kep tunder aheallamp during and,1ftcrsurgery, inorde rto maint a inbodytempe rature, untiltheycomp lete lyrecoverecl fromrheanaosth oue .The rat pupswere revivedby gentle rubbi ngafterre movalfrom icc andplacedunder.1
heatla mpfor approxi matelyonehalf hourbeforebeing returned10themotlu-r.Till' litters were thenculled toeightpupsto ensurestandar dnutrition.
Figuret:Diagramofrat skullindicating siteoiZnSO~application.
30
Site of olfactorybulb
Openingfor ZnS04
application
J1 2,4 Perfusion
Twent y-onedays aftersurgerythe animalsweredeeply.macsthctlzod with sodium pento barbital(80 mg!kg,i.p.land perfusedtr,lIlsc.lrdi,lllywith in'!enid0.9'X, saline(two minutes)followed by4%pararormaldehvde(FisherScientific) cmd0.1'X, glutaraldehyde(FisherScientific) in 0.1Mphosphate buffer(pH 7.4)for 3Durinuu-s (600w1000rnlj.Thebrainswereremovedfromtheskulls, placedin cold flx.ulvefor 1hour and leftovern ighti~cold 20%sucroseina,lMphosphatebuffer,
In orde r tooptimize theserotonintrnmuuocvrochcmtsrrv,11lL'.mlmalswen- givenSOmglkgof pargyline(Fisher Scientific), a monoamineoxidase-inhibitor,,11111 trypto phan (FisherScientific),a precursorto serotonin.in 0.9%salinei.p.,(,Omimrll''>
and30min u tesprior10 perfusion,respectively.
32 2.5Immunocyt oc hemistry
The dayafterper fusion, frozen 30prnthickcorona lsectionsofthe OBwere cuton a cryostat(-20"0and placed imme diately onto chrome alumsubbedslides.
The sections werethen processed for immunocylochemistry(ICC), directlyonthe slides,withantibodies againsttyrosinehyd roxylase(TH), serotonin{S-HTl,dopamine r5·hydroxylase(DBHI, glialfibrillaryacidic protein(GFAP) andS-100. The sou rceand dilutionsof the primary antibodieswereas follows: (1)TH(EugeneTechnical)1:500,
(2) S-H T(INCSlat) 1:3000,(3) DBH (EugeneTechnical)1:600, (4) GFAP (INC Star) 1:100or1:500, and(5) 5-100(INC Star) 1:10or 1:100.Thesectionswere(1) incubatedin primaryantibody in0.2%TritonX-100(SigmaChemica lCc.),0.02%
sodiumazide (fisherScientific),2%no rmal goat se rurn(NGS)inphosphatebuffered saline(PBS) at4"C for 36 hoursin sealed plasticcontainersandthen,(2) rinsed 3 timesfor 5minu tes inPBS onashaker,(3) incubated in biotinylatedgoalanti-rabbit IgG(vectasratnElileKit, Vectorlabs)in aPBS solutioncontaining 0.2 % TritonX·100 for1houratroomtemperature, (4)rinsed 3 times for 5minutes each in PBS, (5) inc uba ted in vectasta!n avid in-biotin-peroxidasecomplex(Vectorlabs) for1hour at room temperature, (6)rinsed 2 timesfor5minute s eachin PBS.Visualizationof the enzyme-labelledantibodieswasachieved by incubatingthe sectionsin0.05%
diaminobc nzidin edihydrochloride tDAB, FisherScientific)with 0.0 3%HZ01in0.1M phospha tebufferf or 5-10 minutes. The slideswerethenrinsed3 timesfor 5minute s
33 each in phosphate buffer, submergedindeionized H~Ofor 10- 15secon ds, dehydra tedandcoverslippedusingpermount (Fishe r Scientific) orprepared forsilver intensification.
Silver inten sificationwas performed inorderto enhancesectionsprocessed forseroto ninICC whenstaining wasconsideredtobeless thanoptimal.Ill1tlwdi'l tdy after the DAB rea ctionthesectionswere(1)rinsed 3timesfor 5 minuteseach in 0.1Mphosphatebufferand then , (2) rinsed 2limes for 15mhuucse.u'hin2'~, sodiumacetate(FisherScientific),(3)incubat ed overn ightinlO'll"thioglvcolic ,Kid (FisherScientific)at4"C,(4)rinsed3 timesfor15minutesinsodium acor.ueand111{'11 (S)incubatedin physicaldeveloperfor10-15minutes.The physicaldeveloper consistedof200mg ammon iumnitrate(MallinckrodO,200mgsilvernttr.uo (fislwr Scientific),1gmtungstostltc icacid(Polysciences,lnc. ),0.4 ml37'll.,fcnnaktcbvdc (FisherScientific) in100rnldistilledHpadded slowly10allequalvo lumeof5'~, sodiu mcarbo nate(Mallinckrodtlin100011distilledHp .Followingimmersionin the physicaldeveloper the slides were(1) rinsed in1%aceticacid(i=islll' rSciflillifiel (or 5minutes, {21rinsed 3 times for 5minuteseachin 2% sod iumaceta te(3)incubated inO.OS%goldchloride for 8-10 minutes al4"C,(4) rinsedoncefor 5 minutes in2'~"
sod iumacetate,(5)rinsed 2timesfor 10 minuteseachin3%sodiumthiosulphatc (Mallinckrodt), (6)rinsed 2times forSminuteseach in2'%. sodiumacetate,.tnd finally(7)rimed3times forSminuteseachin0.1M phos phate buffer. Theslil]!!!>
were thenrinsedindeioni zedH10 ,dehydrated andcoversllppe dlIsioR permocnt.
34 2.6 ZincStaining
Amodi fiedTimms sulphidesilvermethodfor detection of heavy metals (Haug, 1973; Danscher,1981;Slovitcr,1982) was performedonfiveoperated andthree controladu ltani mals 10 determ ineif increasedamo unts ofzincwere present inthe OBas aresult of introduction of ZnS04inlothenasal cavity.
Thre edaysfollow ing surgery,theanimalswerede ep ly anaesthetizedwith sodi umpen toba rb ital(80mg/kg,i.p.l andperfused transcardiallywith 11.7g Na25 (Fischer Scientific) in1000mlof a.1M phosphatebuffer atroom temperaturefor 30 minutesat a rateof 30ml perminute (900· 1000 milfollowed by neutral buffered form alin (90 ml37% formaldeh ydein 810 mla.1Mphosphatebuffer)for25 minutes illa rate of30ml perminute (70 0·800mil.The brain s were removedfromthe skulls and placedinneutral bufferedformalinforapprox imately90minutes.Thebrain s werecutfrozenat30pmthicknessinthecoronal plane on acryostat(-20"0 andthe sectio nswereplacedim mediatelyontochrom alumsubbe dslides,Representative secrtons were takenfrom the OBand anteri or olfac tory nucleus(AO N).The sectio ns were then ai r dried for15 l"inules,fixedin 96% ethanolandhydratedpriortobeing submergedin devel oper,The developerconsistedofthe follo wi ng solutions: (1) protectingcolloi d -250g ofpowderer! gumarabic (ICNBiochemicals)in 500mt deioniz edHp madeupatleast 5 dayspriortouseand filteredthrough gauze,(2) citratebuffer- 25.5gcitricacid (ICN Biochemical s) and 23. 5g sodiumcitrate(ICN Biochemicals)broughtto afinal volumeof100 011withdeionizedHp ,(3)reducing agent-5.67g hvdroquinone(ICNBiochemical s)in 100011deioni zedHpprepared
as
just before use,(4) silverion supply~8.5gslive rnitrate(FisherScientific)in 50rnl deionized H20which mustbe keptin thedark untilused. The developerW.1S preparedby carefully mixing 60rnlof (1), 10 mlof(2),30 rulof {3land add ing 0.5 ml(4)immediately be fo re use.
The slideswereplaced in glassCoplin jars, covered with developerand placedin a H20 bathat 26"C.Theslides weredevelope d for30~70nunutes with"
completesetofslide s foreach anima lremoved from(he developerevery5-10 minutes. The slideswererinsedthoroughlyin H2010 stopthe~t<linillg,dchydr.llcd , immersed briefly inxylene andcoversupoedusing permounr.
36 2.7Analysis
2.7.1Quanlificat ionoftheDeaff erentat ion oftheOlfacto ry Axo ns Deafferent ationoftheolfactoryaxons wasinitiallyconfirmedby visually examiningtheOBsed ionsforlo ss ofTHimmunoread iv ity inthe PGcell s which
IC<luirethepresenceoftheDEinorde r to expresstheTH phenotype (Nad ier al.19Bl;Baker eraI.198 3).This visualanalysis hasbeenthe standardprotocol by severerlaboratories (Hard ingeta1.1978;Nad ieral.196 1; Bakereta1.1983;Bakeret
<11.1984)andwasIhemethodthatwasinitiallyadopted.
Followin g visualexaminati onof 06sectio ns,quantific ationof thenumberof PGcells exhibitingTH Immunoreactlvttvwas performed in the medialaspectofthe
circumference of the Gl which consistently exhibited diminished TH immunoreactivityindeauereotedanimals.PC ce lls expressing THimmunore activity werecounted125Xobjective)withtheaidofanimage analysissystemIBioquant.R
&M8iometri~).Thenu~ofTHimmunoreactivecells that werepresentinthree glomerularprofiles werecounted{rom atotalofthree sectio nsperbulbper animal toobtainanaveragenu mber ofTH immunoreactivecellsperglome rularprofilefor bulbsipsilateralandco ntralateral10the lesionandin non-lesionedcontrol animals.
Inihc bulbs ipsilateraltothe lesion,the PC cellsexp ressingTHimmunoreactivity were countedin the area of the CLwhe revisualexa minationrevea led themost obviousdoefferen taucu,namelythemedial aspectofthecircumferenceoftheGL regardl essif diminished THimmunoreactivityextended throug houtthe entire circumference oftheGL.Cellscountedinbulbscontralateraltothelesion weretaken
37 from thesame areain the Glas cellco untsfor the ipsilatera lbulb. In control,m inl.lls the cellcounts weretaken fromthemedial aspectofthectrcumrcrcnceof theGt, whichoftencorre sponded10 thelesloned areain dcaifercntcdanimals.
38 2.7.2Density of ImmunoreactlveFibresand Astrccst e s
Following verificationof peripheral deafferentation,adjacentsectionsfrom denervated and cont ro lanimalswereselected10 determine the effectofthe lesion onthedensityof innervation of serotonerg!candnoradrenerg tc immu noreactive axonsinthe DB.The effectofthe lesion ondensityof GFAPandS~,00
immuno react iveustrocvteswas alsoexamined.
Immunoreactive axons from controlbulbsand bulbs contra lateral and lpstlatcrnl10the denervatedOB weredrawn using a cameralucida(40Xobjective) attachment to an Olympus microscop e. Immunoreactiveserotonergfcfibres were drawnfromthe glomerularlay erIGl ),externalplexiformlayer(EPL),andinfra EPL region,whichincludedthemitral celllayer (MCl),internalplexiform layer(lPL)and granu lecelllayer(Cel),wh ilenoradrenergicimmun or eactive axonswere drawn from theEPLandInfraEPlre gio n. Similarly, the densityofastrocvtlc cellsexpressing GFAP and5·100immunoreactivitywas determinedinallOBregionsexamined using camerahrcidadrawings(20X objective)of celldistribut ionand image analysis.
The density of tmmuncreacuve fibres (uml lOOpm2) and astrocytes (cells/100pm2)inthe different areas ofthe DBwas quantitate d using the came ra tuctdadraw ingstransferred 10an image analysis system (Binqua nt, R& M Biometrics).A200pm region of the cameralucida drawing,extend ingfrom the begin ningto endofeach re gion ofthe OBexamine d,was selectedfor ana lysis (Fig.2).The cameralu cida dra w ings werethen traced ona digitizi ng tablet
39 (Sum maS ketch , Summa Graph ics)toobtaintheden sity measu reme nts.A101,11ofth ree came ralucidadrawingsper animalpe r bulbregion were used10obtain an,w~r;lge densit y of imm unoreactive fibres andastrocvtesincontro landdenervated.1I1iIl10115.
Tocompensate for shrinkageoftheolfactorybulbresulting from the
deafferentation, density measurements obtained from bulbs ipsilateral and cont ralateraltothelesion were no rmalized withrespect10contro lbulbs.Th eilVer,lgc areaof eachlayerexaminedof thebulb s ipsilate ralandcon tralateraltotill'lesion, foralloperated animals,W.1Sdiv idedby theaverageareaof comparable laver s of tho cont rol anima lsto obtain a normalizat ion factor(NFl.The average (icilsily measurementsof each leslon ed anima lwasthenmultip liedbythenonn.rlfv .ulon factorto obtainthefina l density measur em ent fortheipsilateraland contrala te ralhulh (Rhoades etal.1990).For insta nce,Hthearea oftheipsilat eral bu lbdecreased10711'Y"
ofthe control. the fibredensity in theipsilateralbulbwouldbemultiplicdby0.710 obtain the fina l densitymeasur e ment.
NF= A/ A
Where: (1)Als th eaverageareaof thelayer oftheco ntrolbulb (2) A, isthe averagearea ofthelayerofthe Ipsil atera lor contralatera lbulb
40 Norma lized De nsity... NFX0
Where: (1)NF isthe normalizationfactor
(2) 0isthe density of immunoreactivefibres orcellsin the ipsilate ralorcontralateralbulb
A mufuva nare stiltisticalanalysis(MANOVA) Is ofte n utilize d 10le st hypothesesin volving morethan threedepe nden tvariables.In lhls studythe depen dentvariableswerethedensityof immun o reactivefibres/c e lls in the control, contralateraland ipsilateralbulbs in eachcfthethreeregions of the OBexamined while theinde pe ndentvariable was theZnSO~-inducedlesionof the afferentinput 10the DB.The MANOVAtes tsifthe means ofthedependent variables are equal endindicatesifthe experimenta ltreatmenthada generalizedeffectwhich has asmall probabililYof happeningbychance(Kirk,19 68;Kirk, 1982). A MANQVAwas performed oneach separateareaofthe OB examined,namelythe Gl,EPLandln rre EPlregions,to determineifsignificant differen ces existedamo ngtheno rmaliz ed fibre/ ceil densitymeasureme ntsof thecontrol, coniralatera land ipsilate ral bulbs.If the F·st.atislicgeneratedbytheMANOVAindicated that overallsignifican t differenceswereapparen tbetweenthe control,contralateralandIpsilater al bulbsin each region of the 08thenfu rther statisticalanalysiswasperformedto determin e whic hcomparisonswere sign ificantlydifferent. A t-testmaybeused tocompare
41 population meansof a spe cificse t of hypotheses{Kirk, 1968;Kirk,198 2},Therefore, inIhepresent study an unpaired ,two-tail edt-testW,lSperformed10determineif significan t differ ences existed betwe en the norm••Hzedfibre!ccll density measurem entsof contro lanimals and bulb slpsllateraltothelesion. A pest-hoc Ttlkt'y testmay be used todetermineifthe exp erimentaltreatment result sillsignlflcant differen ces between populationme ans wh en ihe com pansonswereuuphnncd or1101 original lydefinedbya specific setof hypo thesis(Kirk,1968;Kirk, lIJ1l2).Thus,in thisstudya post-ho cTuke y Multi p leCom pariso nslestwas performedto compare the
normalizedfibre/ celldensitiesofcontroland co n tralate ralbulbs.1011thedensity measure ments ob tained forbulbsipsilater al and ca nt rillateraltothele sion. Thell"I.1 were plottedusingSigma Plot (JandelScie n tiflcl.
42 Figu re 2: Camera lucida drawing (40Xobjective) ofimm u noreactive sercto nergicaxons in a PND 31controlanimalshowing the200jim boxed regionofa draw ingselectedfor image analysis.
4]
2.7.3 RelationshipBetweentileDensnvof serotonerglcFibresand Astrocylic Expres sionof 5100
Inorde rto dete rmineif potentialchangesin(he density of sororonorg!c him'S wererelated to a change in the astrccvnc expression of 5100,,111 .matvsls for linl'.lr correlation was performed between thedensityrneasurcmcnts oht.lim'll for serotonerg!c immunoreactivefibres and 5100immunoreactiveaslra..-ytl'sof Il'sioll{'{1 andno n-leston ed controlanimalsinbot h adultandPND 31anlmals. Till' <lIl<llysis ofline a r corre lationwas performedby combining thedensityrnc.rsuromcnts olJt,lilH'{l for seroto ne rgtcfibres and5100 astrocv tes(rom ellIevers of theonex.uuluedin contro lbulbsand bulbs ipsilatera land contralateral to thelesion withadu!rs and PND 31an imalssubjectedto individua lcorre lationanalysis.
44 2. 7.4RelativeOpticalDensityMeasurements of ZincStaining
Animageanalysissystem(Bioquant,R&M Biometrics) was used toobtain relativeoptica l density (R.O.D.)measuremen ts inordertoquantitate the intensit yof zincstaining incontrolanimals and in operatedanimalsthree days after unilateral applica tion ofZnSO~10 the DE.Relativevaluesofoptical densitywereused to controlfo rvenations instaining intensitiesorsectionthickness.The opticaldensity (0. 0. )measurements weredetermined forthe Gl, EPl and infraEPl region ofthree adiacemsectionsforthe cont rolanimals and thelesioned animals inbulbs ipsilateral andcontralaterairothelesion.Foreacnsection ana lyzedthe0.0 . ofthemiddle of theepend yma lzoneW,15takentorepresentthe intensity of the backgroundstaining.
The R.O.D.measurementsofthe Gt,EPl and infraEPl were obtainedby subtracting the0.0.ofthe background(romthee,D.oflheareaof interestand dividingbythe 0.0. of thebackground .This calculatio nresultedin R.O .D. values between0.'\
{wh ile orunreactive(or zinc) and1.0(blackOfdenselyreactivefor zinc).
45 RESULTS
3.1TyrosineHydroxy laseImmunocytochemistry
Asmentioned previously,redu ction ofTHexpressionintill'PC;(vIIs is indicativeofasuccessfullesion oftheolfactoryepithelium .111<1 suhscquont degen eratio n of the afferent olfactoryneu ro nsfro mthe glomerular1.1Yproftho em (Bake r et 011.1983; Baker eta1.1984;Kream eta1.1984;Mclean,HIdShipley,t9l\U; Baker eta1.1988;BiffoetaI.1990).Red uctio n ofTHjmmunorcocuvttv intIll'.rdult animalsdid not alwaysoccur throughoutthe entirectrcumtcrenccof the e;1.intill' OBsectionsexamined.However,redu ctio nsin TH lmrnun nroactivity consistently occurredinthemedialaspectoftheClinsections fromthe rostral.lnd1l1t'(Ii,11 portio ns oftheipsilateralOB(Fig.3a,bl.Cellcounts inthisiHC,'ofthe Gl indic.\tl'd thatin theipsilate ralbulbthe rewas a significantdecrease inthenumber of PCi(:dls expressingtheTHenzymewhencomparedtonon-lesionedcontrols(p<n.uunI).md whencomparedto bulbscontralateral(p<O.OO1)tothelesion(FigA).Inaddition, a significa ntdecrease (p<O.OS)inthe number of PC cellsexpressingTH immunoreactivityinthemedialaspect oftheCL was observedinbulbscontrol.neral tothelesion incomp arison tocontrolanimals (figA).
The loss ofTHstainingin the youngeranimalsW<lSO(tPllmort! widesp H'olCj than thaiobserved in the adultanimals,with almost completeloss ofTHstilinin~
sometimesoccurringthroughouttheentireclrcu mtereocoof theGl inr)N D :II animals. InPND 31 animals(Fig.S)a significantdecreaseoccurredinthenu mber
46 ofPC cells staining(orTHinthe medial aspect ofthe Glin bulbs fpstleteraltc the les ion compared toncn-lesioned controls(p<O.OOOlland bulbs contralateral {p < O.OO l )to the lesion [Fig.G).It.significantdecrease Ip<O.OS)inthe number of PC cellsexpressingTH was alsoapparentin the bulbs ccntrataterel tothelesionin comparison10controlanimals(Fig.61.
In bot hthe adultandPND31animalsthe loss of THimmunoreactivity consistentlyoccurred insectionstaken from therostral andmiddle portionsof theOB
,1I1Umorespecificallyinthemedial regionof the Glineach individual bulb section.
The refore,inorder tooptimize consistency,thedensityoffibresand astroglialcell bodies obtained fromsectionsadjacent to those exhibitingconsistent loss of TH sta ining we retaken from a standard izedlocation,namely themed ialregion of each areaofthe08examined,inbothadultand PND31 animals.
Figure 3:Darkfieldphotomicrographshow ing TH immunnreactlvityInthe glom erularlayer(GUin the contralate ral (AI and ipsilateral (8 )bulbof lesioned adultanimal.Thedecline in theTH immuocr eactivitvintheGl of theipsilatera lbulb(B)isindicative ofsuccessful lesion of theprimaryolfactory neurons.Note theshrinkage ofIheGlandthe absenceof the olfactorynerve layer(ONl)in(B)which also ind icates deafferentation of theOB.TheTHimmunoreactivitymayappearmoreintensein (B) due10theshrinkage of theGl. Magnificationis thesame for(AIand (8)withthesca le barrepresenting50pm.
figure4:ThemeannumberofPG celt, perglomerularprofile pxpr('ssingTH immunoreactivity (±S.E.M.)inadu ltanimals21davsfollowin g peripheral deafferentat ion of the DB. The asterisks(...,p< O.OOliH,1'<0.01;..
p< O.0 5)representsignificantdifferences in the numbe r of PG cells expressing THstaining inlesionedandnon-leslone dcontrol ani mals.
30
25
20
15
10
. .CONTROL(N=5)
r:··~·~JCONTRALATERAL(N= 5)
I~~'-JIPSILATERAL(N=5)
...
GLO MERUL A R LAYER
50 Figure6:The meannumberofPCcetls per glomerularprofileexpressingTH immunoreactivity(±S.E.M.)inPN D 31animals21days followingperipheral deafferentation ofthe OB.The asterisks (•••• p<O.OOl;••,p<O .OI;", p<O.OS)represent significantdifferencesinthe numberofPG cellsexpressing THstaininginlesioned and non-leslonedcontrolanimals.
_ CONTROL (N="')
f2ili
CONTRALATERAL (N:::4)o
IPSILATERAL(N= 4)GLOMERULARLAYER
51 3.2 Den sityof SerotoninImmunoreactiveFibres
In the adult animal, the re was a significant decrease in the density of serotonerg!c fibres in the Gl(p< O.OO 1), EPl (p<O.OO1) and infra EPl(p <O.05) in the bulbs ipsilateral to thelesioncompared to non-lesloned controls 2111,lYS fo llowingdeaffe rentation (Fig.7,8).In theCl,andEPlregio n, nosi~n irk.lnt differencesin se rotone rgicfibre density wereobserved betweenbulbs umlr,ll,llpr,ll and ip silateral tothe lesionor betweenco ntralateral bulbsandnon-lesloncdcontrols.
However, the densitymeasurements obtainedfor the contralate ralbulbs illIhest' region s exhib iteda tendency(albeitnon-significant)tolie hetweenthose vatues obtainedfor the non-lesioned controlanimalsandbulbsipstletcraltothelesion.In contrast 10 the GL and EPLcomparisons,the infraEPL regio n C!xhihitcd 'lsil,\nific;lnt decrease (p < O.OSI in fibredensitybetween the bulbs contralateraland fpstlatcralro thelesio n (Fig.8l.
In contrastto theadultanimals,nosignificantdiffe rence soccurredin the densityof sero tonerg icfibresin the GLand EPLregionsof leslcnedand non-lcsfoned PND 31 an imals(Fig.9,10). In the infra EPL regio n, however,ther e was asignifica nt increase (p <0.05) inthe densityof serctonerglcfibresin thebulbs ipsil,ller ;111oIhe lesioncompared to non-Iesione dcontrolanimals(Fig.IO).Inthe infra EPl reg ion,the mean serotonergtcfibredensity measurementsappeared10bethesameinbulbs contralateral to thelesionand incontrol bulbs, yet asignifica ntdifferenceW.1S observed betweenthe controland the ipsilatera lbulbshulnot betweentho contralateraland ipsilate ralbulbs (Fig.l0).This difference insignificance arisesfrom
52 thediffering.yetnecessary, statisticalmethodsperformed 10 de termine significance betweenthe controlsand the ipsilateralbulbsandbetweentheipsilateral and contralateralbulb sas explainedinIncmethods section.
Figure 7:Darkfieldphotomicrographof serotonergicimmunoreactive fibres inthe DB ofcontrol(A) adultanimals and in thebulbs contralateral(B) and ipsilateral (C)to thelesion in denervatedanimals.Note thedensity of serotoninimmunoreactivefibresappearsgreater in(C)than in(B)and (A) as consequence of shrinkage ofthe layers oftheDB following deafferentation.Magnification isthesamein(A),(6),and(C)withthe scalebarrepresenting 50pm.Abbreviations:glomerular layer(GL), external plexiformlayer (EPL).mitralcelllayer(MCL),granule celllayer (Gel).
Figure8:Mean de nsityofserotonergic immunoreactive fibres(±S.E.h.l)in,Itlull animals 21daysfollowing peripheraldeafferentationof theDB.Thr,1~I~isl~
(h,p< O.Ql;.,p<O.OSIrepresen t asignificantdecrease inthedl'n~ilyof serotonergicfibres inthe bulbs ipsilateral to the lesioncompared10ccuuol bulbsintheCt,EPlandinfraEPlandbetweenthecontral.ucral,mil ipsilateralbulbsin the infraEPlregion.
~ ••
_ CONTROL(N=')
~ CONTRALATERAL(N= 6 )
o
IPSILATERAL(N=6)•
GLOMERULAR LAYER
EXTERNAL PLEXIF'ORM LAYER
INFRAEPL REGION
Figur e9:Darkfieldphotomicrographofserolonergic immunoreactivefibresinthe DB ofcontrol (A)PND 31 animalsandin thebulbscontralateral(B) and ipsilateral(C)to the lesionin denervated animals.Note the densityof serotoninimmunoreactivefibres appears greaterin (C) thanin(B)and(A) asconsequence of shrinkage of thelayers ofthe DB following deafferentation.Magnificationisthe same in(A),(E),and(C)with the scale barrepresenting SOpm.Abbreviations: glomerular layer (GL), externalplexiformlayer(EPl), mitral celllayer (Me l),granulecelllayer (Gel).
