hypothalamic neuropeptides in the autonomic …...autonomic innervation of gingiva and lip. j transl...

iMedPub Journalswww.imedpub.com Journal of Translational Neurosciences

2016Vol. 1 No. 1: 7

1

Research Article

© Copyright iMedPub | This article is available in: http://translational-neuroscience.imedpub.com/

Eniko Szabo1, Katalin Koves2, Zsolt Boldogkoi3, Agnes Csaki2, Zsolt Lohinai1, Zsuzsanna Toth1 and Philippe Ciofi4

1 DepartmentofConservativeDentistry,FacultyofDentistry,SemmelweisUniversity,Budapest,1085-Hungary

2 DepartmentofAnatomy,HistologyandEmbryology,FacultyofMedicine,SemmelweisUniversity,Budapest,1094-Hungary

3 DepartmentofMedicalBiology,FacultyofMedicine,UniversityofSzeged,6720-Hungary

4 INSERMU862,NeurocentreMagendie,Bordeaux,France

Corresponding author:KatalinKoves

koves.katalin@med

DepartmentofAnatomy,HistologyandEmbryology,FacultyofMedicine,SemmelweisUniversity,Tuzoltou.58,H-1094Budapest,Hungary.

Tel: 3612156920

Citation: SzaboE,KovesK,BoldogkoiZ,et al. HypothalamicNeuropeptidesintheAutonomicInnervationofGingivaandLip. J TranslNeurosci.2016,1:1.

Hypothalamic Neuropeptides in the Autonomic Innervation of Gingiva and Lip

AbstractBackground: Ampleevidence indicates that thedescendingpathways fromthehypothalamusplayaroleintheautonomicregulationofthelowergingivaandlip.Ourgoalwastoidentifytheneurochemicalnatureofthehypothalamicneuronsgiving origin of these multisynaptic descending pathways and additionally tocharacterizethelowermembersofthesepathways.

Methodology: Retrogradely spreading Green Fluorescence Protein (GFP)labeledviruswasinjectedintothelowergingivaorlipofWistarrats.Intactandsympathectomizedratswereincludedintheexperiment.Viruslabelingwaslookedforinfrozensectionsofthehypothalamus,brainstem,upperthoracicspinalcord,superiorcervical,oticandsubmandibularganglia.

Results:Inintactandsympathectomizedrats,labeledneuronsofthehypothalamicparaventricular nucleus showed oxytocin, vasopressin, but not Cholecystokinin(CCK)andCorticotropichormoneReleasingHormone(CRH)immunoreactivities.Intheperifornicalregiontheviruslabeledneuronsshowedorexinimmunoreactivitiy.In intact and sympathectomized rats themembers of the descending pathwaywere further characterized. In sympathectomized rats the labelingwasmissingfromthe locusceruleus, theventrolateralmedulla, theraphenuclei, thespinalcord,butlabelingfurtherexistedinthegigantocellular,thesalivatorynucleiandthehypothalamussuggestingtheirparasympatheticnature.

Conclusion:Ourpaperdemonstratesforthefirsttimethatoxytocin,vasopressinandorexin, but notCCK andCRH, immunoreactivehypothalamicneuronsmayinfluencebothsympatheticandparasympatheticresponsesofthelowergingivaand lip. These are common command neurons.We also summarized the datastep-by-steponthechemicalcharacteristicsofthelowerpartofthedescendingpathway.

Keywords: Transneuronal virus; Tracing technique; Immunohistochemistry;Neurotransmitters;Neuropeptides;Doublelabeling;Confocalmicroscopy;Rats

Abbreviations: DBH: Dopamine β-Hydroxylase; CGRP: Calcitonin Gene-RelatedPeptide;CCK:Cholecystokinin;CRH:CorticotropicHormoneReleasingHormone;G:Gingiva;Gi:GigantocellularReticularNucleus;GFP:GreenFluorescenceProtein;HRP/WGA:HorseradishPeroxidase/WheatGermAgglutinin;IML:IntermediolateralCellColumn;ISN:InferiorSalivatoryNucleus;KPB:PotassiumPhosphateBuffer;L:Lip;LC:LocusCeruleus;L-ENK:Leu-Enkephalin;NOS:NitricOxideSynthase;NPY:Neuropeptide-Y;OG:OticGanglion;ORX:Orexin;OT:Oxytocin;OT-N:Oxytocin-Neurophysin;Pf:PerifornicalRegion;Rmg:RapheMagnus;Rpa:RaphePallidus;PRV: Pseudorabies Virus; PVN: Paraventricular Nucleus; SCG: Superior CervicalGanglion; SER: Serotonin; SMG: Submandibular Ganglion; SP: Substance P; SS:Somatostatin;SSN:SuperiorsalivatoryNucleus;TriG:TrigeminalGanglion;VAChT:VesicularAcetylcholineTransporter;VIP:VasoactiveIntestinalPolypeptide;VLM:VentrolateralMedulla;rVLM:RostralVentrolateralMedulla;VP:Vasopressin;VP-N:VasopressinNeurophysin

Received: March17,2016; Accepted: April07,2016; Published: April11,2016

mailto:koves.katalin@med

2

ARCHIVOS DE MEDICINAISSN 1698-9465

2016Vol. 1 No. 1: 7

Journal of Translational Neurosciences


IntroductionThe transneuronal virus labeling is a suitablemethod to traceneuralpathwayswhichinnervateperipheraltargetorgans.Itwasfoundtwodecadesagothatthepseudorabiesviruscouldonlybetransportedbyneurons.Afterreplicationinaneuronalcellbody,theviruscanenterthenextmemberoftheneuronalchainandisabletodemonstrateneuronal[1].

It iswell known that thepostganglionicparasympatheticfibersinnervating the gingiva (G) or lip (L) tend to join the sensoryormotor nerves [2]. In the humanG the nerve fibers containcalcitonin Gene-Related Peptide (CGRP), Substance-P (SP),Vasoactive Intestinal Peptide (VIP) and Neuropeptide-Y (NPY).ThepresenceofNPYandVIPfibersoccasionallyobservedaroundbloodvesselsoftheGsuggeststhattheyareautonomicinnature[3].Anotherresearchgroupdemonstratednoradrenergicfibersinthelaminapropriaandinthewallofthevessels[4].HorseradishPeroxidase/Wheat Germ Agglutinin (HRP/WGA) administrationinto the molar gland and the lower lip-gingiva of cat showedretrogradelylabeledneuronsintheOticGanglion(OG),SuperiorCervical Ganglion (SCG) and mandibular subdivision of theTrigeminal Ganglion (TriG) [5]. This observation suggests thatthenervefibers found in thegingivaand lipderive fromthesesources.ItwasalsodemonstratedthatthemucousmembraneofthehumanlipcontainsfibersimmunoreactiveforVIP,SP,NPYandNitricOxideSynthase(NOS)[6].Lohinaietal.[7]describedNOSimmunoreactivefibersincatanddoggingivae.

Numerous data accumulated in the literature on the locationof neurons in the central nervous system which are involvedin the autonomic innervation of many viscera including thesuprarenalglandtheovary,thekidney,thecolon,thepenis,theprostate gland and the perineal muscles, the urinary bladder,the salivary and mammary glands [8-20]. As it was predictedby Strack et al. [21], who injected a retrograde pseudorabiesvirus intovarioussympatheticgangliaandtheadrenalglandofrats, thepremotor sympatheticneuronsaregenerally found inseveralcellgroupsthatregulatetheentiresympatheticoutflow:the hypothalamic Paraventricular Nucleus (PVN), perifornicalregion (Pf), A5 noradrenergic cell group, caudal raphe region,rostral Ventrolateral Medulla (VLM), the Locus Ceruleus (LC)and the Barrington's nucleus. Additionally, local interneuronsin laminaeVIIandXof thespinalcordarealso involved in theneuronal chain innervating the preganglionic neurons in theIntermediolateral cell group (IML). They also showed that theretrograde transneuronal viral labeling method could be usedsimultaneously with either neuropeptide or neurotransmittersyntheticenzymeimmunohistochemistry.

Onlyafewdataareavailableintheliteratureontheneuropeptides,locatedinthehypothalamicPVNandPf,andonthoseinvolvedintheregulationoftheautonomicresponsesofperipheralorgans.We observed that a retrograde spreading trans-synaptic virusinjectedinthemammaryglandappearedinthePVNandthePf.Asubpopulationoftheviruslabeledcells inthePVNcontainedOxytocin(OT)[20].

The major goal of this present study was to examine thechemical nature of the neurons, located in the PVN and Pf

sendingautonomic informationthrough thebrainstemand thespinal cord to the lower G and L. The lower members of thedescending pathway were also characterized. We have usedtransneuronal retrograde tracing technique and neuropeptideimmunohistochemistry. We carried out OT, Vasopressin (VP),Cholecystokinin (CCK) and Corticotropic hormone ReleasingHormone (CRH) immunostaining in the hypothalamic sectionscontainingPVN.ThepresenceofviruslabelinginthePfsuggestedthat the virus labeling might also be present in Orexin (ORX)immunoreactivecellswhichwerediscoveredabouttwodecadesago[22-24].

In a part of the animals sympathectomywas carried out. Thisinterventiongaveusthepossibilitytoinvestigateseparatelytheretrograde projection of the parasympathetic innervation ofthe lowerGandL.The reasonwhyweexamined theseanimalgroups was because studies demonstrated neurons in PVNthat are common command neurons of both sympathetic andparasympatheticregulationofthesubmandibulargland.

Materials and MethodAnimalsAdult Wistar female rats (3-4 month old) were used for theexperiments.Theanimalswerekeptina light/darkcycle(lightson at 5:00 and lights off at 19:00) and temperature controlledvivarium (22 ± 2°C). The treatment of the animals was inaccordancewiththerulesof the“Europeanconventionfortheprotection of vertebrate animals used for experimental andotherscientificpurposes”,Strasbourg,1986andTheHungarianGovernment Directive 243/98. Our protocol was approved bythe Local Animal Care and Use Committee (Permission No:22.1/1158/3/2010).

Preparing and injection of the virusThe same geneticallymodified Pseudorabies Virus (PRV) straintermed memGreen-PRV was used for the experiments as inour previous paper [25]. The construction of memGreen-PRVwas previously described [25-27]. The virus expressed GreenFluorescenceProtein(GFP)andthepresenceofviruswaseasilydiscovered using fluorescence microscope. The concentrationoftheviruswas8×108/mLplaqueformingunits.Thespreadingspeedofthevirusinaretrogradedirectionwas1,5mm/h.Undergeneral anesthesia (chloralhydrate35mg/100grbw),with theuseof10µLHamiltonsyringe,4µLofviruscontainingbufferwasinjectedintothelowerGorLand6µLintothosereceivingvirusbothG+L.ThesmallamountofsolutionremainedinlocointheGortheL[27].Theneedlewasleftinsidethestructuresforoneminutetopreventtheleakageofthesolution.Allnervefibersinthe infiltrated area couldpick the virusup. The appearance of virusintheTriGindicatedthesuccessfulspreadingofthevirus.

SympathectomyPrior to the virus inoculation (2-4 days), SCG was removedfrombothsides.Animalsexhibitingptosisandmyosis (signsofsympathectomy) were used for the inoculation. The successof sympathectomywasalsoconfirmedby theabscenceofGFPlabeling in the IML. In this animal group we could separatelyexaminetheparasympatheticneuronalchain.

3© Under License of Creative Commons Attribution 3.0 License


2016Vol. 1 No. 1: 7


Preparing tissues for investigationSix groupswere included in the experiment.Numbers indicatetheanimalswhichsurvived.

Group1.5intactratsrecievedvirusinthelowerG

Group2.5intactratsrecievedvirusinthelowerL

Group3.4intactratsreceivedvirusintobothG+L

Group4.3sympathectomizedratsreceivedvirusinthelowerG

Group5.3sympathectomizedratsreceivedvirusinthelowerL

Group6.2sympathectomizedratsreceivedvirusinthelowerG+L

The animalswere sacrificed 96 h after inoculation.We had touse this survival period leaving enough time for replication ofthe small amount of virus. The animals were anesthetized bychloralhydrate (35mg/100grbw), theanimalswereperfusedwithpotassiumphosphatebuffer(KPB)(0.1M,pH7.4)containing4%paraformaldehyde(Merck,Darmstadt,Germany)throughtheascendingaorta.ThecomponentsofthebufferwerepurchasedfromSigma-Aldrich(St.Louis,MO).TheSubmandibularGanglion(SMG),SCG,upperthoracicpartofthespinalcordwasremoved.Then the head was placed in the stereotaxic instrument, theskullwasopenedandthebrainwastransected1.6mmbehindthe bregma according to the Stereotaxic Atlas [28]. Then thebrain behind the cut, TriG andOGwere removed. The tissueswere postfixed overnight, then from the forebrain only thehypothalamuswas further processed. All tissueswerewashedin 0,1 M KPB and placed in sucrose solution (15%) (Reanal,Budapest, Hungary), then were embedded in Cryomatrix(ThermoShandon,Pittsburg,PA).20µmthicksectionswerecutoncryostat(Cryotome,ThermoShandon,Pittsburg,PA).Theviruslabeling (presenceofGFP)wasmappedandphotographswererecordedbyamulti-photonconfocalmicroscope(Radiance2100Rainbow Multiphoton Imaging System, Bio-Rad Laboratories,USA,coupledtoanEclipseE800microscope,Nikon,USA)usingaZen2012software.

Double labeling immunohistochemistryOneofthefourseriesofthehypothalamicsectionswasstainedforOT-neurophysin(OT-N),VP-neurophysin(VP-N),CCK,CRHorORX immunoreactivity. After washing, the slides were treatedwith 1% Triton X-100 (Reanal) for better penetration of theantibody. OT-N and VP-N antisera were raised in mouse andcharacterized by Ben-Barak et al. [29] andwere obtained as akindgift fromDr.Gainer (NIH).Bothwereapplied inadilutionof1:200.CCKwasraisedinrabbitandcharacterizedbyCiofiandTramu[30].CRHwaspurchasedfromPenninsula(SanCarlos,CA,USA)andused in1:500dilutions.ORXantiserumwas raised ingoat,andpurchasedfromSantaCruzBiotechnology,Inc.(SantaCruz, CA, USA) and used in 1:500 dilutions. Sections of thebrainstem exhibiting GFP labeling were stained for Dopamineβ-Hydroxylase(DBH)(indicatingthepresenceofnoradrenaline),Vesicular Acethylcholine Transporter (VAChT), Somatostatin(SS), Serotonin (SER), Leu-Enkephalin (L-ENK), OT-N or ORXimmunoreactivity.TheupperthreesegmentsofthespinalcordwerestainedforVAChT,DBH,OT-N,VP-N,L-ENK,CRHandORXimmunoreactivities. Sectionsof theSCGwere stained forDBH,

NPY,VAChTorVIPimmunoreactivity.OGandSMGwerestainedforVAChT,NPY,DBHorVIPimmunoreactivity.VIP,SS,NPY,L-ENKandSERantiserawereraisedinrabbitbyGörcsandcharacterizedbyGulyásetal.[31](VIP,SS),Borostyánkoietal.(NPY)[32],LopezCosta et al. [33] (L-ENK, SER) and used in a dilution of 1:500.VAChTantiserumwas raised inguineapigandpurchased fromEMDMillipore(Temecula,CA).Itwasusedinadilutionof1:500.In every casewe used biotinylated second antibodies. In eachcasethefinalreactionproductofimmunostainingwasvisualizedbystreptavidinCy3conjugate(Sigma-Aldrich,St.Louis,MO).

Evaluation of the number of virus labeled neurons in the hypothalamus Fromthehypothalamus,startingatP1,6mmbehindthebregma,4parallelseriesof20µmthickfrontalsectionswereprepared.Ineveryserieswehad16sections(onefromevery100µmthicktissue).Thesectionsweremountedongelatinizedslides.In4-8sections of the first series containing PVN and 12-16 sectionscontaining Pf the number of virus labeled cells were counted.Thesetensectionswereimmunostained.

Evaluation of the number of virus labeled neurons containing OT-N and VP-N in the PVN and ORX in the PfWhenthedoublelabelingwascompletedthenumberofdoublelabeledcellswasalsocounted.

Specificity testsThe neurotropic spreading and exclusively retrogradetransportation of our virus strain were previously tested [25].In the case of inoculation of the animals through the externaljugularveindidnotresultlabelingintheTriG.Thisindicatestheexclusivelyneurotropicspreading.InoculationofGandLresultedinlabelinginthepseudounipolarneuronsoftheTriGbutnotinthesecondorderneurons.Thisfactclearlyshowsthattheviruscould not spread in an anterograde manner. Two specificitytestswerecarriedoutby:1)omittingtheprimaryorsecondaryantibodies prevented the immunostaining, 2) positive controlstainingwiththeantibodies.OccurrenceofOT-N,VP-NandCCKimmunoreactivities in the hypothalamic PVN iswell known forthirty-fourtyyearsandORXinthePfforseventeenyears[24,30,34-36]. The presence of neuropeptides and neurotransmittersynthetizingenzymesinotherinvestigatedorgansincludingOG,SMG,IML37,20,SCG,LC,rVLM,RPa,Gi,ISN,PVNandPFarealsowellestablished[20,22,37-42].

Results Virus labelingWhentheviruswas injected intotheGorLofboth intactandsympathectomizedratslabelingalwaysappearedintheTriG.Thissignal indicated the successful spreading of the virus as itwaspreviouslydemonstrated[25].

Groups1,2and3.Inintactanimalslabelingwasalwaysobservedintheipsilateralstructuresbelongingtotheperipheralautonomicnervoussystem:intheSCG(Figure 1J, 1M, 1P and 1T),theOG

4


2016Vol. 1 No. 1: 7



(Figure 2A, 2D and 2G)oftheanimalsofGroup1andadditionallyintheSMG(Figure 2M, 2P, 2T and 2Z)oftheanimalsofGroup2and3.LabelingwasalsoseenintheipsilateralIMLofthethreeupper segments of the spinal cord (Figure 1D), the LC (Figure 3A),theventralpartoftheGigantocellularReticularNucleus(Gi)(Figure 3J)whicharchesabovethepyramid,salivatorynuclei,therostralVLM(rVLM)(Figure 3D),thePVN(Figure 4A, 4D and 4G) andthePf(Figure 1J)asitwasdescribedinourpreviouspaper[25]. In thehypothalamus thePf and thePVNwere labeledatbothrightandleftsides.InthePVNthelabeledcellswerepresentmainly in themagnocellular but labeled cells also occurred intheperiventricularparvocellularsubdivision. InthePftheviruslabeledcellsarchedabovethefornixandonthemedialside.

Groups 4, 5 and 6. Only those sympathectomized rats werefurther examined where the labeling was missing in the IML.In the brainstem of these latter groups the labeling was onlyobservedinthesalivatorynucleiandtheGi.Inthehypothalamusitwaspresent in thePVNand thePf, but thenumberof viruslabeled cellswas less than in intact rats. In sympathectomizedanimalsthelabelingfurtherexistedintheOGoftheanimalsandadditionallyintheSMGoftheanimalsofGroups5and6.

Double labelingThe description of the descending autonomic pathways startsfromthepremotorthirdorderhypothalamicneuronsdownwardstotheperiphery.

Third order (premotor) neurons in the hypothalamic nuclei: InthehypothalamicPVNofintactratsabouthalfoftheGFPlabeledcells showed OT-N (Figures 4A-4C) or VP-N immunoreactivity(Figures 4D-4F). We have found only one virus labeled cellwhich also showed CRH immunoreactivity (Figures 4G-4I). Thedouble labeled cell is showed by the insert in Figure 4I. CCK immunoreactivity did not colocalizewith virus labeling (Figure 4M). In the Pf, the majority of virus labeled cells exhibitedORX immunoreactivity (Figures 4J-4L). Figures 4N and 4O schematicallyillustratethelocalizationoftheviruslabeledcellsinthePVNandPf.

Quantitative analysis of the distribution of virus labeled neurons and those containing OT-N and VP-N in the PVN and ORX in the Pf (Group 3): ThenumberoftheviruslabeledcellswasevaluatedinthePVNandPfatipsi-andcontralateralsides(Figures 5A-5C). Theirnumberdidnotdiffersignificantlyfromeachotheratthetwo sides of the PVN and Pf. Counting the cells revealed thatabouthalfoftheviruslabeledcellsfoundinthePVNsynthetizedOTorVP(Figure 5D).NearlyallviruslabeledcellsinthePf(Figure 5D)showedORXimmunoreactivity.

Third order (premotor) neurons in the brainstem (Groups 1-3, intact rats): In the LC and the rVLM the virus labeled neuronsexhibited DBH immunoreactivity (Figures 3A-3F, respectively)andreceivedORXfibers(notshown).Intheraphepallidus(RPa)(Figures 3G- 3I)andintheraphemagnus(RMg)(notshown)theviruslabeledcellswereSERimmunoreactive.OT-NfibersenteredtheVLM,butnottheLC(notshown).

Second order (preganglionic) parasympathetic neurons in the brainstem of sympathectomized rats (Groups 4-6): The virus

labeled Gi neurons arching above the pyramid did not exhibitDBH(Figures 3J-3L),L-ENK(Figures 3M-3O), SER,SSandVAChTimmunoreactivities(notshown).However,intheneighborhoodof virus labeled cells many fibers, immunoreactive for theabove-mentioned transmitters and peptides, were observed(not shown). The Gi neurons are common command neuronsfor both sympathetic and parasympathetic outflows, as wedemonstrated previously [25]. Second order neurons of theparasympathetic pathway located in the salivatory nucleiwerestudied in sympathectomized rats. Theneurons in thesenucleiwere immunopositive for VAChT (Figures 3P-3S). Virus labeledcellsrarelyshowedcolocalizationwithDBHimmunoreactivityaswell(Figures 3T-3V).

Second order (preganglionic) sympathetic neurons (Groups 1-3, intact rats): IntheIMLoftheupperthreesegmentsofthespinal cord the preganglionic virus containing cell bodiesweredesintegratedinmanycases;however,whenthecellswereintactwe found colocalizationwith VAChT immunoreactivity (Figures 1A-1C).TheIML,wheretheviruslabelingwasalwaysintensiveinintactrats(Figure 1D),receivedmanynoradrenergic(Figure 1E),lessoxytocinergic(Figure 1F),afewvasopressinergic(Figure 1G), enkephalinergic(Figure 1H),butnotCRHimmunoreactivefibers;however, the posterior horn showed dense CRH fiber labelingindicatingthattheantibodystainingwassuccessful(notshown).OrexinfiberswereverysparseintheIML(Figure 1I).

First order (postganglionic) sympathetic neurons (Groups 1-3, intact rats): IntheSCGmanyviruslabeledcellswerefound.AllviruslabeledpostganglioniccellscontainedDBH(Figures 1J-1L), andmanyofthem,butnotallcontainedNPYimmunoreactivity(Figures 1M-1O). SomeVAChT immunoreactive cellswere alsoseen in the SCG. We have not found colocalization betweenthevirus labelingandVAChTimmunoreactivity;however,manyVAChT immunoreactive fibers surrounded the virus labeledcells (Figures 1P-1S). Very rarely a few VIP cells were foundand there was colocalization between virus labeling and VIPimmunoreactivityaswell(Figures 1T-1V).

First order (postganglionic) parasympathetic neurons (Groups 1-3 intact and Groups 4-6 sympathectomized rats): In theOG,some virus labeled cells was found. All labeled cells showedVAChTimmunoreactivity(Figures 2A-2C)andabouthalfofthemwere NPY immunoreactive (Figures 2D-2F). Many cells wereDBHimmunoreactiveandnearlyallviruseslabeledcellsshowedcolocalization with this immunoreactivity (Figures 2G-2I). VIPimmunoreactivecellswerealsoobservedintheOGandtheviruslabeled cells partially colocalized with this immunoreactivity(Figures 2J-2L).

In theSMG thevirus labeling alsoappeared. The virus labeledcellsshowedVAChT(Figures 2M-2O),NPY(Figures 2P-2S),DBH(Figures 2T-2V)andVIPimmunoreactivities(Figures 2Y and 2Z).

DiscussionInourpreviouspaperbesidesthedemonstrationofthedescendingautonomicpathwaytothelowerGandLweclearlydemonstratedthatnotonlytheLbuttheGalsoreceivedbothsympatheticandparasympatheticinnervation[25].Theparasympatheticfibersof



2016Vol. 1 No. 1: 7


Figure 1 MicrophotographsdemonstratingcolocalizationbetweentheviruslabelingandneurotransmitterorneuropeptideimmunoreactivitiesintheIMLoftheupperthoracicspinalcord(A-I)andtheSCG(J-V).TheslidesarederivedfromtheanimalsbelongingtoGroup3(intact rats). In the IML thevirus labeledcellsalsoexhibitedVAChT immunoreactivity (A-C).Dshowsvirus labelingwithasmallmagnification,E-IshowDBH,OT-N,VP-N,L-ENKorORXimmunoreactivefiber labeling intheIML. InSCGnearlyallvirus labeledcellsexhibitedDBHimmunoreactivity(J-L).AllNPYimmunoreactivecellscontainedviruslabeling(M-O).AlimitednumberofVAChTimmunoreactivecellsoccurredintheSCG.Thesecellsdidnotshowviruslabeling(P-S).SimilarlyaveryfewVIPcellswereobserved.Andthesecellscontainedvirus(T-V).Arrowsinsamepositionindicateviruslabeledcellswhichalsoshowthegivenimmunoreactivity(A-C, J-O andT-V), arrow inDdemonstrates virus labeled cells andfibers, arrowheads indicate immunoreactivefibers in E-I. InRandS asterisks showVAChT immunoreactive cellswhichdidnot contain virus.Abbreviations:DBH:Dopamineβ-Hydroxylase;Gm:Greymatter; IML: Intermediolateral cell column; L-ENK: Leu-Enkephalin;NPY:Neuropeptide-Y;ORX:Orexin;OT-N:OxytocinNeurophysin;SCG:SuperiorCervicalGanglion;VAChT:VesicularAcetylcholineTransporter;VIP:VasoactiveIntestinalPolypeptide;VP-N:Vasopressin-N;WM:WhiteMatter.Scale:30μminJ-V;50μminA-C;200μminDand250μminE-I.

6


2016Vol. 1 No. 1: 7



Figure 2 MicrophotographsshowcolocalizationbetweenviruslabelingandneurotransmitterorneuropeptideimmunoreactivitiesintheOG(A-L)andtheSMG(M-Y).TheslidesderivefromtheanimalsbelongingtoGroup3(intactratsG+L).InOGviruslabelingcolocalizedwithVAChT (A-C),NPY (D-F),DBH (G-I) andVIP (J-L) immunoreactivities. In the SMGvirus labeling also colocalizedwith all thefour immunoreactivities (M-Y).Arrows in sameposition indicatevirus labeledcellswhichalsoshowthegiven immunoreactivityAbbreviation: DBH: Dopamine β-Hydroxylase; NPY: Neuropeptide-Y; OG: Otic Ganglion; SMG: Submandibular Ganglion; VAChT:VesicularAcetylcholineTransporter;VIP:VasoactiveIntestinalPolypeptide.Scale:75μminA-LandP-Y;60μminM-O.



2016Vol. 1 No. 1: 7


Figure 3 Microphotographsillustrateco-localizationbetweenviruslabelingandneurotransmitterorneuropeptideimmunoreactivityintheLC, rVLM,RPa,Giand ISN.Thephotos inA-Iderive fromtheanimalsbelonging toGroup3 (intact ratsG+L)and thephotos inJ-VderivefromanimalsbelongingtoGroup6(sympathectomizedrats). InLC(A-C)andrVLM(D-F)virus labeledcellsshowDBHimmunoreactivity.IntheRPa(G-I)manyviruslabeledcellsexhibitedSERimmunoreactivity.InGitheviruslabeledcellscontainedneitherDBH (J-L) nor L-ENK (M-O) immunoreactivity. In ISN all virus labeled cells showedVAChT (P-S) and some cells showedDBHimmunoreactivity(T-V).Arrowsinthesamepositionindicateviruslabeledcellswhichalsoshowthegivenimmunoreactivity(A-IandP-V).Arrowheadsshowviruslabeledcellswhichdidnotshowthegivenimmunoreactivity(J-O).AsterisksinG-Iindicatethemidline.Alltheothersectionsderivefromtheinjection(ipsilateral)side.Abbreviations:4V:fourthVentricle;7:facialnerve;DBH:Dopamineβ-hydroxylase;Gi: ventralgigantocellular reticularnucleus; ISN: InferiorSalivatoryNucleus; LC: LocusCeruleus;L-ENK:Leu-Enkephalin;py:pyramid;RPa:RaphePallidus; rVLM: rostralVentrolateralMedulla;SER:Serotonin;VAChT:VesicularAcetylcholineTransporter.Scale:100μm.

8


2016Vol. 1 No. 1: 7



Figure 4 : The diagram demonstrates the mean number of virus labeled cells in the hypothalamic PVN and Pf (A-C). There is no significant difference between the ipsi-(right) and contralateral (left) sides of the virus inoculation. There is no considerable difference between the three animal groups (G, L and G+L). D shows the percent of neuropeptide immunoreactive cells compared to the 100% of virus labeled cells in the given nucleus.

Figure 4 Microphotographsillustratecolocalizationbetweenviruslabeling(GFPfluorescence)andneuropeptideimmunoreactivitiesinthePVNandPf.ThephotosderivefromtheanimalsbelongingtoGroup3(intactG+L).InPVNmanyviruslabeledcellsexhibitedOT-N(A-C)andVP-N(D-F)immunoreactivities(seetext).TheviruslabeledcellsoccasionallyshowedcolocalizationwithCRHimmunoreactivity(G-I).TheviruslabelingdidnotcolocalizewithCCKimmunoreactivity(M).InPfthemajorpartofviruslabeledcellsshowedORXimmunoreactivity(J-L).NandOschematicallyillustratethelocalizationofPVN(1.8mmbehindthebregma)andPF(2.5mmbehindthebregma)inthefrontalsectionsofthehypothalamus.AsterisksindicatethelocalizationoftheGFPexpressingneurons.Arrowsinthesamepositionshowviruslabeledcellswhichalsoshowthegivenimmunoreactivity.Abbreviations:AH:anteriorhypothalamicarea;ARC:ArcuateNucleus;CCK:Cholecystokinin;CRH:CorticotropicHormoneReleasingHormone;DM:DorsomedialNucleus;fx:fornix;mth:mammilothalamictract;ORX:Orexin;OT-N:Oxytocin-Neurophysin;Pf:Perifornicalregion;PVN:ParaventricularNucleus;Rch:Retrochiasmaticarea;SO:Supraopticnucleus;VM:VentromedialNucleus;VP-N:Vasopressin-Neurophysin;ZI:Zona Incerta;Scale:100inA-Land50μminM.



2016Vol. 1 No. 1: 7


thelowerGderivefromtheOG,andtheparasympatheticfibersof the lower L derive from both OG and SMG.We traced themembers of the descending sympathetic and parasympatheticpathwaysstep-by-stepusingtransneuronalretrogradespreadingvirus. Both sympathetic and parasympathetic premotor (thirdorder) neurons were located in the hypothalamus and thebrainstem. The second order neurons of the parasympatheticpathwaymaybepresent inthebrainstemandthesympatheticneuronsinthespinalcord.Itwasalsorevealedthattheneuronsof the Gi located above the pyramid are involved in bothsympatheticandparasympatheticdescendingpathways.

Inthepresentworkwefurther investigatedthesepathways. InspiteofthefactthatusingPhaseolusvulgaristracingtechnique,demonstrated that mainly the parvocellular cells of the PVNproject to the intermediolateral cell columnof the spinal cord,observed virus labeling in the magnocellular subdivisions aswellaftervirusinoculationofthesubmandibulargland[34,43].The PVN is devided into 3 magnocellular and 5 parvocellularsubdivisionsand in thepresentexperimentthemajorityof thevirus labeled cells were located in the lateral magnocellularsubdivision [34, 44]. The number of virus labeled neurons inthePVNandPfofanimalswheretheviruswasinjectedintheG(Group1)orintheL(Group2)wasnearlythesame(Figures 5A and 5B)asinthosereceivedvirusinbothplaces(Group3,Figure 5C).Whereasthenumberofviruslabeledcellswasmuchlowerinsympathectomizedrats(Groups4-6).

In this present work we also showed that the hypothalamicpremotor third order autonomic neurons located in the PVNuseOT-NandVP-N(51%vs.41%respectively),butnotCCKand

CRH.Wehavetoconsiderthatthevirusinjectedanimalsdidnotreceive colchicine, which is usually used to demonstrate CRHorCCKcellbodies inPVN.Because thecolchicineprevents theaxonal transportationwe couldnotuse thispretreatment. Thevirus labeled neurons located in the Pf use ORX (about 80%)for influencing autonomic responses of the lower G and L. Itwasdemonstrated threeyearsago thata retrogradespreadingvirus injectedinthemammaryglandof lactatingratsappearedinthePVNandthesubpopulationofviruslabeledneuronsalsoshowedOT-Nimmunoreactivity[20].Geerlingetal.[9]injectedretrogradely transported virus in the suprarenal gland and thekidney.The labelingappeared inORX immunoreactiveneuronsofthePf.TheysupposedthattheORXneuronsregulategeneralsympatheticfunctions.OurdatasuggestthatotherhypothalamicpeptidessuchasOTandVPhavesimilargeneralfunction.

ThefactthatthemeannumberofGFPlabeledcellsisnearlythesameintheanimalsreceivingviruseitherintheGortheL,asinthosereceivingvirus inbothplacesat thesametimesuggests,that the samePVNneuronsparticipate in the innervationofGandLthroughamultisynapticdescendingpathway.ThepresenceofOT-N,VP-NandORXimmunoreactivefiberswasdemonstratedintheIMLofthespinalcordwherethepreganglionicsympatheticneuronsarelocated[45-48].Oxitocinergic,vasopressinergicandorexinergicfibersarealsofoundinthebrainstem[49,50].PVNneuronsdenselyinnervatetheparasympatheticpreganglionicneuronslocatedintheSSNandlessintheISNandsomefibersdescendtoIMLwhichareinvolvedinthetransynapticinnervationoftheGandL[25,51,52].

The amount of data concerning the chemical coding of thehypothalamic parasympathetic command neurons which is

Figure 5 Thediagramdemonstratesthemeannumberofvirus labeledcells inthehypothalamicPVNandPf(A-C).Thereisnosignificantdifferencebetweentheipsi-(right)andcontralateral(left)sidesofthevirusinoculation.Thereisnoconsiderabledifferencebetweenthethreeanimalgroups(G,LandG+L).Dshowsthepercentofneuropeptideimmunoreactivecellscomparedtothe100%ofviruslabeledcellsinthegivennucleus.

10


2016Vol. 1 No. 1: 7



Figure 6 Schematicdrawingdemonstratestheneuropeptidesandneurotransmittersplayingaroleinthedescendingautonomicinnervation

of the lowerGandL.Abbreviations:7n: facialnerve;9n:glossopharyngealnerve;DBH:dopamineβ-hydroxylase; fx: fornix;Gi:Gigantocellular reticularnucleus; IML: Intermediolateral cell column; ISN: Inferior SalivatoryNucleus; LC: Locus ceruleus; L-ENK:Leu-Enkephalin;NPY:Neuropeptide-Y;OG:OticGanglion;ORX:Orexin;OT-N:Oxytocin;Ot:Optictract;Pf:Perifornicalarea;PVN:ParaventricularNucleus;Py:Pyramid;R:Raphe;Rch:Retrochiasmaticarea;SCG:SuperiorCervicalGanglion;SER:Serotonin;SMG:SubmandibularGanglion;SSN:SuperiorSalivatoryNucleus;VAChT:VesicularAcetylcholineTransporter;VIP:VasoactiveIntestinalPolypeptide;rVLM:rostralVentrolateralMedulla;VP-N:Vasopressin-N;WCR:WhiteCommunicatingRamus.

involved in the descending innervation of lower G and L islimited.ArecentpaperbyHettigodaetal. [43],whodescribedthecentralneuronswhichwereinvolvedinbothsympatheticandparasympatheticregulationofthesubmandibulargland,didnotexaminethechemicalnatureoftheseneurons.Theyhypothesizedthat whilst the peripheral sympathetic and parasympatheticpathwayswereseparate,thedistributionofpremotorneuronsinhigherbrainregionsoftenoverlapped.

Our study on the characterization of the members of thedescendingautonomicpathwaystotheGandLsummarizespartiallythedataavailableintheliteratureandwhatwehavefound.Figure 6 schematicallyillustratesthechemicalcodingoftheentirelengthofthedescendingpathways.FromPVNOTandVPfibersdescendtothebrainstemLC,VLM,ISN,SSNandGI.FromLCandVLMnoradrenergicfibers, and directly from PVN oxytocinergic and vasopressinergicfibers,descendtotheIML.FromtheISN,SSNcholinergicandfromGInon-characterizedfibersreachtheoticganglion,fromtheIMLtotheSCGalsocholinergfibersdescend. InOGVAChT,VIP,NPYandDBH,intheSCGDBH,NPYandVIPimmunoreactiveneuronswerefoundthatmayinnervateGandL.

Itwasdemonstratedthatbesidesthewellknownneurotransmitter

and neuropeptides the parasympathetic OG and SMG cellbodies also contain DBH which is a charateristic sympatheticneurotransmitterconvertingenzyme.Hoardandherco-workers[53]foundthatthehalfofthecholinergicneuronsofhumanandprimateintrinsic parasympathetic cardiac ganglia have a dual cholinergic/noradrenergicphenotype.AnotherresearchgroupfoundDBHintheOG.Theysupposedthatthisenzymeisanembryologicalremnantand it has no functional significance [37]. The question ariseswhetheritistruefortheoralstructuresaswell.

Acknowledgement Weareverygrateful toAnnaTakáts forherexcellent technicalassistance.ThisworkwassupportedbytheDepartmentofHumanMorphologyandDevelopmentalBiology,SemmelweisUniversityandtheDepartmentofConservativeDentistryandEndodontics,FacultyofDentistry,SemmelweisUniversity,Budapest,Hungary,Swiss-Hungarian Cooperation Programme grant (SH/7/2/8) toZsoltBoldogkőiandOTKA-KgrantNo112364toZsoltLohinai.Allco-authorshaveagreedtothepublicationofthepaperandtheyhavereportednopotentialconflicts.



2016Vol. 1 No. 1: 7


References 1 EnquistLW(2002)Exploitingcircuit-specificspreadofpseudorabies

virus in the central nervous system: Insights to pathogenesis andcircuittracers.JInfectDis186Suppl2:S209-214.

2 IzumiH,KaritaK(1991)Vasodilatorresponsesfollowingintracranialstimulationofthetrigeminal,facialandglossopharyngealnervesinthecatgingiva.BrainRes560:71-75.

3 Lundy FT, Linden GJ (2004) Neuropeptides and neurogenicmechanismsinoralandperiodontalinflammation.CritRevOralBiolMed15:82-98.

4 Urbanovich VI, Vylegzhanina TA, Maneeva OA, Kuznetsova TE,Ryzhkovskaya EL (1999) Adrenergic innervation of the gingiva inexperimentalperiodontitis.BullExpBiolMed127:564-568.

5 KuchiiwaS,KuchiiwaT(1996)Autonomicandsensoryinnervationofcatmolarglandandbloodvesselsinthelowerlip,gingivaandcheek.JAutonNervSyst61:227-234.

6 FehérE,ZellesT,NagyG(1999)Immunocytochemicallocalisationofneuropeptide-containingnervefibres inhuman labial glands.ArchOralBiol44Suppl1:S33-37.

7 Lohinai Z, Székely AD, Benedek P, Csillag A (1997) Nitric oxidesynthase containing nerves in the cat and dog dental pulp andgingiva.NeurosciLett227:91-94.

8 StrackAM,SawyerWB,PlattKB,LoewyAD(1989)CNScellgroupsregulatingthesympatheticoutflowtoadrenalglandasrevealedbytransneuronalcellbodylabelingwithpseudorabiesvirus.BrainRes491:274-96.

9 GeerlingJC,MettenleiterTC,LoewyAD(2003)Orexinneuronsprojecttodiversesympatheticoutflowsystems.Neuroscience122:541-550.

10 Tóth IE, Banczerowski P, Boldogkoi Z, Tóth JS, Szabó A, et al. (2008)Cerebralneuronsinvolvedintheinnervationofboththeadrenalglandandtheovary:adoubleviraltracingstudy.BrainResBull77:306-311.

11 LiYW,DingZQ,WesselinghSL,BlessingWW(1992)Renalandadrenalsympatheticpreganglionicneuronsinrabbitspinalcord:tracingwithherpessimplexvirus.BrainRes573:147-152.

12 SchrammLP,StrackAM,PlattKB,LoewyAD(1993)Peripheralandcentralpathwaysregulatingthekidney:astudyusingpseudorabiesvirus.BrainRes616:251-262.

13 ReichartA,BoldogkoiZs,LenkeiZ,MedveczkyI,PalkovitsM(2000)Neurochemical characterization of kidney regulating brainstemneurons identified by pseudorabies transneuronal labeling.Neurobiology8:277-80.

14 VizzardMA,BrissonM,deGroatWC(2000)Transneuronallabelingofneuronsintheadultratcentralnervoussystemfollowinginoculationofpseudorabiesvirusintothecolon.CellTissueRes299:9-26.

15 MarsonL,Carson3rdCC(1999)CentralNervousSystemInnervationofthePenis,Prostate,andPerinealMuscles:ATransneuronalTracingStudy.MolUrol3:43-50.

16 Rouzade-Dominguez ML, Miselis R, Valentino RJ (2003) Centralrepresentationofbladderandcolonrevealedbydualtranssynaptictracing in the rat: substrates forpelvic visceral coordination. Eur JNeurosci18:3311-3324.

17 Jansen ASP, Ter Horst GJ, Mettenleiter TC, Loewy AD (1992) CNScell groups projecting to the submandibular parasympatheticpreganglionicneuronsintherat:aretrogradetransneuronalviralcellbodylabelingstudy.BrainRes572:253-260.

18 RezekO,BoldogkoiZ,TombáczD,KovágóC,GerendaiI,etal.(2008)Locationofparotidpreganglionicneurons inthe inferiorsalivatorynucleusandtheirrelationtothesuperiorsalivatorynucleusofrat.NeurosciLett440:265-269.

19 Gerendai I, Tóth IE,KocsisK,BoldogkoiZ,Medveczky I,etal. (2001)TransneuronallabellingofnervecellsintheCNSoffemaleratfromthemammaryglandbyviraltracingtechnique.Neuroscience108:103-118.

20 KövesK,GyörgyiZ,SzabóFK,BoldogkőZ(2012)Characterizationoftheautonomicinnervationofmammaryglandinlactatingratsstudiedbyretrogradetransynapticviruslabelingandimmunohistochemistry.ActaPhysiolHung99:148-158.

21 Strack AM, SawyerWB, Hughes JH, Platt KB, Loewy AD (1989) Ageneral pattern of CNS innervation of the sympathetic outflowdemonstratedbytransneuronalpseudorabiesviralinfections.BrainRes491:156-162.

22 SakuraiT,AmemiyaA,IshiiM,MatsuzakiI,ChemelliRM,etal.(1998)Orexinsandorexinreceptors:afamilyofhypothalamicneuropeptidesandGprotein-coupledreceptorsthatregulatefeedingbehavior.Cell92:573-85.

23 deLeceaL,KilduffTS,PeyronC,GaoX,FoyePE,etal. (1998)Thehypocretins: hypothalamus-specific peptides with neuroexcitatoryactivity.ProcNatlAcadSciUSA95:322-327.

24 DateY,UetaY,YamashitaH,YamaguchiH,MatsukuraS,etal.(1999)Orexins,orexigenichypothalamicpeptides,interactwithautonomic,neuroendocrineandneuroregulatorysystems.ProcNatlAcadSciUSA96:748-753.

25 SzabóE,BoldogkoiZs,CsákiÁ,TóthZs,KövesK(2015)Identificationofautonomicneuronalchainsinnervatinggingivaandlip.AutonomicNeuroscience:BasicandClinical190:10-19.

26 BoldogköiZ,ErdélyiF,FodorI(2000)Aputativelatencypromoter/enhancer(P(LAT2))regionofpseudorabiesviruscontainsavirulencedeterminant.JGenVirol81:415-420.

27 Boldogkoi Zs, Reichart A, Tóth IE, Sik A, Erdélyi F, et al. (2002)Construction of recombinant pseudorabies viruses optimized forlabelingandneurochemicalcharacterizationofneuralcircuitry.BrainReMolBrainRes109:105-118.

28 FenikVB,KubinL (2009)Differential localizationofcarbachol-andbicuculline-sensitivepontinesitesforelicitingREMsleep-likeeffectsinanesthetizedrats.JSleepRes18:99-112.

29 Ben-Barak Y, Russel JT, Whitnall MH, Ozato K, Gainer H (1985)Neurophysininthehypothalamo-hypophysialsystem,I.Productionandcharacterizationofmonoclonalantibodies.JNeurosci5:81-97.

30 Ciofi P, Tramu G (1990) Distribution of cholecystokinin-like-immunoreactiveneuronsintheguineapigforebrain.JCompNeurol300:82-112.

31 Gulyás AI, Görcs TJ, Freund TF (1990) Innervation of differentpeptide-containingneuronsinthehippocampusbyGABAergicseptalafferents.Neuroscience37:31-44.

32 Borostyánkoi ZA, Görcs TJ, Hámori J (1999) ImmunocytochemicalmappingofNPYandVIPneuronal elements in the cat subcorticalvisualnuclei,withspecialreferencetothepretectumandaccessoryopticsystem.AnatEmbryol(Berl)200:495-508.

33 López Costa JJ, Marter S, Görcs TJ, Saavedra JP, Priestley JV(1991) Enkephalin and serotonin innervation of somatostatin-immunoreactivemedullaryreticularformationneurones.BrainRes548:300-304.

12


2016Vol. 1 No. 1: 7



34 SwansonLW,KuypersHGJM(1980)Theparaventricularnucleusofthehypothalamus:Cytoarchitectonicsubdivisionsandorganizationof projections to the pituitary, dorsal vagal complex, and spinalcord as demonstrated by retrograde fluorescence double-labelingmethods.JCompNeurology194:555-570.

35 Zimmerman EA,RobinsonAG,HusainMK,AcostaM, FrantzAG, et al.(1974) Neurohypophysial peptides in the bovine hypothalamus: therelationshipofneurophysinItooxytocin,andneurophysinIItovasopressininsupraopticandparaventricularregions.Endocrinology95:931-936.

36 KissJZ,WilliamsTH,PalkovitsM(1984)Distributionandprojectionsof cholecystokinin-immunoreactive neurons in the hypothalamicparaventricularnucleusofrat.JCompNeurol227:173-181.

37 Hardebo JE, Suzuki N, Ekblad E, Owman C (1992) VasoactiveintestinalpolypeptideandacetylcholinecoexistwithneuropeptideY,dopamine-beta-hydroxylase,tyrosinehydroxylase,substancePorcalcitoningene-relatedpeptideinneuronalsubpopulationsincranialparasympatheticgangliaofrat.CellTissueRes267:291-300.

38 Wojtkiewicz J, Juranek JK, Kowalski I, Bladowski M, Calka J et al.(2011) Immunohistochemical characterization of superior cervicalganglionneuronssupplyingporcineparotidsalivarygland.NeurosciLett500:57-62.

39 Schwarz LA, Luo L (2015) Organization of the locus coeruleus-norepinephrinesystem.CurrBiol25:R1051-1056.

40 Xu ZQ, Hökfelt T (1997) Expression of galanin and nitric oxidesynthase in subpopulationsof serotoninneuronsof the ratdorsalraphenucleus.JChemNeuroanat13:169-187.

41 HökfeltT,MillhornD,SeroogyK,TsuruoY,CeccatelliS,etal.(1987)Coexistenceofpeptideswithclassicalneurotransmitters.Experientia43:768-780.

42 Choy VJ, Watkins WB (1977) Immunocytochemical study ofthe hypothalamo-neurohypophysial system. II. Distribution ofneurophysin,vasopressinandoxytocininthenormalandosmoticallystimulatedrat.CellTissueRes180:467-490.

43 Hettigoda NS, Fong AY, Badoer E,McKinleyMJ, Oldfield BJ, et al.(2015)IdentificationofCNSneuronswithpolysynapticconnectionsto both the sympathetic and parasympathetic innervation of thesubmandibulargland.BrainStructFunct220:2103-2120.

44 Kiss JZ,Martos J,PalkovitsM (1991)Hypothalamicparaventricularnucleus:aquantitativeanalysisofcytoarchitectonicsubdivisionsintherat.JCompNeurol313:563-573.

45 JójártJ,JójártI,BodaK,GálfiM,MihályA,etal.(2009)Distributionof oxytocin-immunoreactive neuronal elements in the rat spinalcord.ActaBiolHung60:333-346.

46 Buijs RM (1978) Intra- and extrahypothalamic vasopressin andoxytocinpathwaysintherat.Pathwaystothelimbicsystem,medullaoblongataandspinalcord.CellTissueRes192:423-435.

47 NilaverG,ZimmermanEA,Wilkins J,Michaels J,HoffmanD,etal.(1980)Magnocellular hypothalamicprojections to the lowerbrainstemandspinalcordoftherat.Immunocytochemicalevidenceforpredominanceoftheoxytocin-neurophysinsystemcomparedtothevasopressin-neurophysinsystem.Neuroendocrinology30:150-8.

48 Llewellyn-Smith IJ, Martin CL, Marcus JN, Yanagisawa M, MinsonJB, et al. (2003)Orexin-immunoreactive inputs to rat sympatheticpreganglionicneurons.NeurosciLett351:115-119.

49 Lee SK, Ryu PD, Lee SY (2013) Differential distributions ofneuropeptides in hypothalamic paraventricular nucleus neuronsprojecting to the rostral ventrolateralmedulla in the rat.NeurosciLett556:160-165.

50 PuskásN,PappRS,GallatzK,PalkovitsM(2010)Interactionsbetweenorexin-immunoreactive fibers and adrenaline or noradrenaline-expressingneuronsofthelowerbrainsteminratsandmice.Peptides31:1589-1597.

51 GeerlingJC,ShinJW,ChimentiPC,LoewyAD(2010)Paraventricularhypothalamicnucleus:axonalprojectionstothebrainstem.JCompNeurol518:1460-1499.

52 Novak P (2007) Central anatomic network, In: Neurobiology ofdisease (Ed. Gilman S.) Elsevier Academic Press, Burlington, SanDiego,London.

53 Hoard JL,HooverDB,MabeAM,BlakelyRD, FengN, et al. (2008)Cholinergic neurons of mouse intrinsic cardiac ganglia containnoradrenergic enzymes, norepinephrine transporters, and theneurotrophin receptors tropomyosin-related kinase A and p75.Neuroscience156:129-142.

hypothalamic neuropeptides in the autonomic …...autonomic innervation of gingiva and lip. j transl...

Documents