Advisor: Dr. Stephen D. Liberles Erika Kristen Williams

Neural Mechanisms ofMechanosensation Within the Body

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Citation Williams, Erika. 2018. Neural Mechanisms of MechanosensationWithin the Body. Doctoral dissertation, Harvard Medical School.

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iii

Advisor:Dr.StephenD.Liberles ErikaKristenWilliams

Neuralmechanismsofmechanosensationwithinthebody

Abstract

Theabilitytodetectmechanicalforcesplaysacriticalroleinorganismbehaviorand

physiology.Oneofthefundamentalmeansbywhichweinteractwithourenvironmentis

throughtouch,whichincludestheabilitytosensemechanicaleventssuchaspressure,

impact,vibration,andchangesinjointposition.Similarly,oneofthefundamentalcues

usedbyinternalorgansystemstoregulatebehaviorandphysiologicalresponsesis

mechanicalforcewithinthebody.Sensorysystemsintheintestinaltractdetectstretchas

theseorgansfillwithandmovefood,playingapowerfulroleinthemodulationofeating

behavior.Inaddition,sensorysystemsalsomonitortheexpansionandrelaxationofthe

lungsduringbreathingtoregulaterespiration.Similarly,accuratemonitoringofpressure

withinthevascularsystemplaysakeyroleinregulationofcardiovascularfunction.Eating,

breathing,andbloodcirculationconstitutebasicneeds,yetourunderstandingofthe

sensoryneurobiologyincontrolofthesefunctionsislimited.Todate,themolecular

mechanosensorsrequiredremainunknown.However,thediscoveryofthemammalian

mechanosensorPiezo2raisestheinterestingpossibilitythatthismoleculeisnotonly

involvedindetectionofexternalmechanicalcuesinourskin,butmayalsosub-serve

detectionofmechanicalcueswithintheinternalorgansofthebody.

iv

TableofContentsIntroduction...........................................................................................................................................7Mechanosensationininternalorgans.....................................................................................................7ThemammalianmechanosensorPiezo2..............................................................................................25Recordingmechanosensitiveafferentsinthevagus........................................................................30

Experiment1:InVivoimagingofPiezo-2lineageneurons................................................35Introduction...................................................................................................................................................35Methods............................................................................................................................................................35Results..............................................................................................................................................................35Conclusions.....................................................................................................................................................39

Experiment2:InvivoimagingofneuronsthatexpressPiezo2intheadult.................40Introduction...................................................................................................................................................40Methods............................................................................................................................................................40Results..............................................................................................................................................................41Conclusions.....................................................................................................................................................43

Experiment3:DetectionofstretchstimuliinPiezo2knock-outganglia.......................44Introduction...................................................................................................................................................44Methods............................................................................................................................................................44Results..............................................................................................................................................................45Conclusions.....................................................................................................................................................48

Summary..............................................................................................................................................50Bibliography........................................................................................................................................50

v

Glossary

AAV–adeno-associatedvirus

DRG–dorsalrootganglion

GLP1R–GLP1receptor

GPR65–G-proteincoupledreceptor65

IGLE–intraganglioniclaminarending

IMA–intramusculararray

RAR–rapidly-adaptingstretchreceptor

SAR–slowly-adaptingstretchreceptor

siRNA–smallinterferingribonucleicacid

vi

Acknowledgements

Thankyoutomyfamily,theLiberlesLabpastmembersandpresent,theProgramin

Neuroscience,MDPhDProgram,andHSTProgramfortheconstantsupportand

inspiration.Thankyoutosomanyfortechnicalhelpandexpertise,andmostnotablythe

HarvardAnimalFacilities;withoutyounoneofthisworkwouldbepossible.Financial

supportforthisworkwasthroughF30CA177170andNIHMSTP-T32GM007754.

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Introduction

Mechanosensationininternalorgans Internalorgansareinconstantmotion,andsimilarlyconstantlysubjecttoavariety

ofmechanicalforcesofcriticalphysiologicalsignificance.Eatingresultsinaccumulationof

foodinthestomachandmechanicaldistensionofthestomachwall.Neuraldetectionof

stomachstretchplaysacriticalroleinfeedingbehavior.Inaddition,digestioninvolvesthe

mechanicalpropulsionoffoodthroughsphinctersandoverapproximately25feetofadult

humanintestine,aprocesssimilarlymonitoredandregulatedbythenervoussystem.

Breathingischaracterizedbythemechanicalexpansionandrelaxationofthechestcavity

togeneratechangesinpressure,andsubsequentflowofairintoandoutofthelungs.

Sensoryneuronsmonitorrespiratorymovementtoregulatebreathfrequencyanddepth,

importantvariablesforadequateoxygenation,carbondioxideexcretion,andprotectionof

lungtissue.Lastly,thebeatingheartpumpsbloodandgeneratespulsatileflowand

pressurewithinthevasculature.Neuralmonitoringofbloodpressureinitiatesreflex

regulationofheartrateandsubsequentcardiovascularvariablestopreserveadequate

tissueperfusion.

Inthissection,wewillreviewmechanosensationindigestion,respiration,and

circulation,withafocusontransmissionofinformationfromperipheralorganstothe

brainstem.Withineachorgansystem,wewilldiscussphysiologicallyrelevantautonomic

reflexes,neuralresponseproperties,andsensoryneuronanatomy.Oneoftheprimary

neurallinksfrominternalorganstothebrainisthevagusnerve.Therefore,thevagus

nervewillfigureprominentlyinexperimentsanddiscussions,soabriefoverviewofits

generalanatomyandorganizationisprovided.

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TheVagusNerve Thevagusnerveisoneofthemajorbody-to-brainneuralconnections,andmediates

afferentsensoryinformationtransmissionintothebrainstem,aswellasefferentmotor

commandtransmissionouttotargetorgans.Eightypercentofvagalfibersaresensory1,

andeachsensoryneuronextendsoneprojectionintotheperipherytocollectinformation

fromatargetorgan,andasecondprojectionintothebrainstemtotransmittheinformation

tocentralneuralcircuits.Thesensoryneuroncellbodiesresideinganglia,thenodoseand

jugular,locatedadjacenttothebaseoftheskull.Allsensoryneuronsusethe

neurotransmitterglutamatetocommunicatetocentralcircuits.Theremaining20%of

vagalfibersaremotorfibers1.Thecellbodiesofvagalmotorneuronsarelocatedwithin

nucleiinthebrainstem,andsendprojectionsoutfromthebraintoperipheraltarget

organs.Theneurotransmitterusedbythemotorneuronsisacetylcholine.Therefore,the

vagusnervecanbeconsideredananatomicalhighwaysharedbyasensorysystemcarrying

informationcentrally,andamotorsystemcarryingcommandsperipherally.Thesetwo

systemscommunicateclosely;thesensoryterminalsinthebrainstemareimmediately

adjacenttothenucleicontainingthemotorneuroncellbodies,andtheseneuronsare

knowntomakebothdirectandmulti-neuronnetworkconnectionstoeachothertoprovide

reflex-likecontrolofinternalorganstates2-4.Forthepurposesofareviewonperipheral

mechanosensation,wewillfocusontheresponseproperties,anatomy,andphysiological

rolesofvagalsensoryneurons.

Gastrointestinalstretch

Physiology

Mechanicaldistensionofthestomachplaysacriticalroleinregulationoffood

intake.Notonlydoesthisintuitivelycoincidewithnearlyuniversalhumanexperience

followingmeals,butalsocarefulexperimentationover60yearsagohaselaboratedonthe

physiologicalpowerandspecificsensorycuesrelevantforregulationofmealconsumption.

Theearliestexperimentstodemonstratetheimportanceofgastrointestinal

mechanosensationonfeedingbehaviorwereperformedindogswithsurgicallyaltered

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uppergastrointestinaltracts5,6.Gastricfistulasallowedforartificialfillingofthestomach,

whileesophagostomyallowedforartificialdiversionoforallyingestedfoodoutofthe

esophagusandawayfromthestomach,dissociatingoralingestionandchangesinstomach

content.Fillingthestomachviaafistulatoavolumeequivalenttoapproximately40%ofa

normalmealsizejustpriortomealtimedecreasedoralconsumptionbyanequivalent(30-

50%)amount.Thedegreeofintakeinhibitionwasindependentofthecaloricvalueofthe

stomachfiller;intakeinhibitionwasthesameifthestomachwasfilledwithfood,or

caloricallyinertKarayagum,suggestingtherelevantsensorycuewasthemechanical

distensionandnotthechemicalfoodcontent5.Intriguingly,iffoodwasinfusedintothe

stomachatatimeotherthanduringeating,ithadnoimpactonshamfeeding,suggesting

thetemporalrelationshipbetweeningestionandstomachdistensioniscriticalforan

effect6.

Conversely,whenthefoodwasdivertedoutoftheesophagusandpreventedfrom

reachingthestomach,animalsfedforlonger.Furthermore,indiversioncasesinwhichthe

stomachwasdistendedeitherbyreplacementoffoodorbyinflationofarubberlatex

balloon,thiseffectwasreversedandanimalsingestedless5.Fromtheseexperiments,one

canconclude:1)Stomachdistensionisnotnecessarytoterminatefeedingbecauseanimals

willstopfeedingeveniffooddoesnotreachthestomach.2)Stomachdistensiondoes,

however,playanimportantroleinregulatingtheamountoffoodingestedbecausewithout

stomachdistension,animalseatmore,andwithitanimalseatless.3)Themechanical

distensionofthestomach,notthecaloriccontent,istherelevantgastriccueforacute

regulationofmealsize,andthiscuemustoccuratthesametimeasoralingestiontoimpact

acutefeedingbehavior.

Subsequentstudiesinrodentshaveconfirmedthesefindings,andalsodelineated

theeffectsofstomach-derivedversusintestine-derivedpost-ingestioncuesoneating

behavior.Inoneelegantseriesofexperiments7,8,ratsweresurgicallyimplantedwitha

pyloricsphinctercuffandagastriccatheter.Inflationofthepyloriccuffpreventedtransitof

stomachcontentstothesmallintestine,whileinfusionthroughthegastriccatheterallowed

forexperimenter-controlleddeliveryofgastriccontents.Theseexperimentsdemonstrated:

1)asindogs,infusionsinthestomachinhibitedfoodintakeindependentofthecaloric

contentoftheinfusedsolution,and2)incontrast,caloriccontentdidimpactthedegreeof

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feedingsuppressionwhenthepyloriccuffwasleftopenandthecontentscouldpassinto

thesmallintestine7.Boththesefindingsareconsistentwithmechanicallymediated

inhibitionoffoodintakebythestomach,andchemicallymediatedinhibitionoffoodintake

bytheintestine.

Theinhibitionoffoodintakebygastricmechanosensationandintestinal

chemosensationisthoughttobedependentonintactvagalfibers.Animalsconsumelarger

mealsizesaftersurgeriesinwhichvagalinnervationofthestomachisdisrupted,and

infusionofvolumeintothestomachnolongersuppressesfoodintakeinvagotomized

animals9,10.Similarly,thesuppressionofmealsizebychemicalcuesintheintestineis

dependentonanintactvagusnerve11,12,thoughitshouldbenotednotallstudieshave

foundthiseffect13,14.Despitetheshortcomingsofnon-selectivevagotomyexperiments,

theseresultssuggestakeyroleforvagalafferentsinregulationoffeeding,and

demonstrateacleardistinctionbetweenvagalgastricmechanosensorsandintestinal

chemoreceptors.

Theexperimentsinanimalsaredirectlyrelevanttohumans.Surgicalmethodsto

induceweightlossinpeoplebearresemblancetoexperimentalmanipulationsofstomach

distension.Acentralgoalofbariatricsurgeryistoreducestomachcapacitysuchthatthe

sameamountoffoodwillcausegreaterdistension.Reductionofgastriccapacitycanbe

achievedviaseveraldifferentsurgicalalternatives,includingimplantationofavolume-

occupyingballoon(gastricballoon),applicationofaconstrictiveband(banding),stapling

ofthestomachtoreduceitseffectivevolume(verticalbandedgastroplasty),andstapling

andresectionmostofthestomachpouchtoleavearesidualtube-likegastricsleeve(sleeve

gastrectomy)15.Implantationofaballoontypicallyfilledtoavolumeof750-900milliliters

inthestomachofhumanpatientshasbeenshowntoimproveweightlosswhencompared

tonon-operativeinterventions16,17.Similarly,bandingandgastroplastyresultin~15%

weightlossmaintainedover>10years,whereascontrolsubjectsonlylost~2%;these

resultswereaccompaniedbyadecreaseinlong-termmortalitylikelysecondarytothe

reductionincardiovascularriskfactorsanddiabetesassociatedwithobesity18-20.Themost

effective,‘gold-standard’weightlosssurgeryisthegastricbypass,inwhichonlyavery

smallresidualstomachisconnectedtothedistalsmallintestinetobothreducethestomach

volumeandtheabsorptivecapabilitiesoftheintestine20,thoughsleevegastrectomyalone

11

isnearlyaseffectiveinachievingweightloss15.Inshort,surgicalreductionofthestomach

volumeisaneffectiveweightlosstoolinthefaceofthegrowingobesityepidemic,aneffect

likelymediatedinlargepartthroughmanipulationofvagalgastrointestinal

mechanosensation.

Responseproperties

Theimpactofmechanicalstomachdistensiononfeedingpromptedinvestigationof

theoperativesensorymechanisms.Onlyafewyearsfollowingtheinitialcaninephysiology

experimentsdescribedabove,Paintal,Iggo,andcolleaguesdescribedthefirst

electrophysiologicalrecordingsofvagalafferentsinresponsetodistensionofthestomach21,22.Ininitialexperiments,thecervicalvagustrunkwasseriallysub-dividedintofiber

strands,andresponsesineachstrandwererecordedwhilealatexballoonwasinflatedin

thestomach.Usingthismethod,gastricstretch-responsiveunitscouldbeisolatedfrom

unitsresponsivetoheartbeatsortherespiratorycycle,confirmingtheexistenceofa

uniquestomachstretchsensoryneuronclass.Furthermore,theendingsofthesefibers

werethoughttobelikelylocatedinthemuscularstomachwallbecausetheiractivitycould

beevokedbydigitalcompressionofthestomach,butnotbystrokingthemucosalor

peritonealgastricsurfaces21,22.

Gastricmechanoreceptorshaveconsistentresponseproperties.Allfibersresponded

todistensionquickly,exhibitedfiringratesof25-60impulses/secondduringthe

distension,andreturnedtobaselinelevelsofactivityimmediatelyfollowingreliefofthe

distension.Allbutoneofthefiberstestedwereeithernon-adaptingorslowlyadapting,

withlittlechangeinthefiringrateevenduringminuteormulti-minutelongdistensions.

Step-wiseincreasesinthevolumeofdistensiongeneratedalinearincreaseinthefiring

rate,thoughthethresholdofdistensionrequiredforfiberactivationvariedwidely21.

Furthermore,inductionofmuscularcontractionsofportionsofthestomachwallinwhich

thereceptiveendsofthesefiberswerefoundwouldelicitactivityinthegastricreceptors

thatwasevenmorerobustthanthatcausedbypassivedistension22.Therefore,the

relevantcueforfiberactivationisnotintra-gastricpressureperse,butthetensionwithin

themuscularwallofthestomach.Gastricstretchreceptorsarethereforeconsidered‘in-

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series’tensionreceptors,andmonitornotonlypassivetensionofthestomachwallbutalso

activetensiongeneratedbymuscularcontractioninphysiologicallyrelevantranges.A

corollarytotheobservationthatgastricmechanoreceptorsarein-seriesisthattheslow

adaptationobservedintensionreceptorfiringratesmayatleastinpartreflectmuscular

relaxationofthestomachwall,andmaynotbeanintrinsicpropertyofthe

mechanoreceptor.Thesamepropertieswerefoundfordistension-sensitivevagalafferents

thatterminatedintheesophagusorthesmallintestine23.Similarpropertieshavebeen

subsequentlydescribedingastricmechanoreceptorsinmultiplestudies24-27.

Subsequentstudiessoughttoidentifythemolecularmechanismbywhich

mechanicalforcesaretransducedintoaneuralsignalwithinvagalgastrointestinal

mechanoreceptors.However,despitedecadesofrecordings,itisunclearwhethervagal

afferentsthemselvesaredirectlymechanosensitive,orwhethertheyaretransmitting

informationfrommechanosensitiveentericneuronsornon-neuronalgastrointestinalcells.

Threeargumentshavebeencitedtosupporttheideathatvagalafferentsaredirectly

mechanosensitive28.First,thelatencyofresponsefollowingspecificandrapidmechanical

deformationofthemuscularwalloftheesophagusis<6milliseconds,adelayhasbeen

citedastoorapidforamechanismotherthandirectmechanosensation.However,thedelay

betweenfiringofanupstreamneuronanditschemically-coupleddownstreamtargethas

beencalculatedat2millisecondsinthemammalianbrain,suggesting6millisecondsis

morethansufficientforsynaptictransmission,andcastingdoubtonthisclaim29.Second,

extracellularcalciumisconsideredanecessarycomponentforvesiculartransmitter

release,andvagalafferentmechanosensitivityispreservedbothwhenextracellular

calciumisremoved,andinthepresenceofthecalcium-channelblockerCd2+.However,

calcium-dependenttransmissionisnottheonlymeansofcell-cellcommunication;gap

junctionsforexamplewouldallowforrapidandextracellularcalciumindependentsignal

transduction30.Finally,pharmacologicinhibitionofcandidateneurotransmitterreceptors

(e.g.glutamatereceptors,purinergicreceptors)hasnoimpactonvagalmechanosensation,

evidencetakentoexcludearolefortheseputativesecond-messengers,thoughofcourse

thisrepresentsadrasticallyincompletesurveyofpotentialcell-cellsignalingmechanisms.

Insummary,thesedataarenotconclusive,leavingmuchambiguityaboutthetruesiteor

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sitesofmechanosensationinthegastricdistensionsensorypathway.Themolecular

mechanosensor,unsurprisinglyinthiscontext,remainsunknown.

Anatomy

Despiteambiguityaboutthemechanismofmechanotransduction,themorphology

ofdistension-responsivevagalafferentsinthegastrointestinaltractisknown.Isolated

esophagealandstomachpreparationshaveallowedforlocalizationofmechanically

sensitive‘hotspots’,andconcurrentlabelingofvagalfibersinnervatingthosesites31,32.

Hot-spotsintheesophagusandthestomachareenrichedandlocalizedaround

intraganglioniclaminarendings(IGLEs),avagalterminaltypecloselyassociatedwith

myentericneuronganglialocatedbetweenthecircularandlongitudinalmusclelayers.

Consistentwithhot-spotstudies,geneticallylabeleddistension-sensitiveafferents,marked

byexpressionoftheG-protein-coupledreceptorGLP1R,formdenseIGLEterminals

throughoutthegastrointestinaltractandterminatecentrallywithinthemedialnucleusof

thesolitarytract33.

AsmallnumberofstudieshaveexaminedthestructureandlocationsofIGLEsin

hopesofprovidingcluesastothesitesofmechano-transduction.IGLEswereinitially

describedusingsilverstainingmethodsintheesophagus34aslamellatedendings

surroundingandenteringthecapsuleofcollectionsofentericneuroncellbodies.Electron

microscopyofvagalendingsinthesemyentericgangliashowedcloseapproximationof

mitochondria-richIGLEterminalswiththeganglionneuropilandcellbodies,butdidnot

revealaclassicsynapticultrastructureassociatedwiththesecontacts35.Theclose

associationbetweenIGLEsandentericgangliacouldsupportboththeideathatvagal

neuronsreceivesignalsfromprimarilymechanosensitiveentericcells,orthatenteric

gangliacouldprovidecriticalsupporttoprimarilymechanosensitivevagalneurons.

IGLEscanbefoundintheesophagus,stomachandproximalsmallintestine,

consistentwiththevagalresponsepropertiesidentifiedinallthesesites.Thegreatest

densityofIGLEsislocatedinthestomach,withdecreasingdensityalongtheproximal

intestinallength36-39.Someestimatessuggesttherearewellover1000individualIGLE

terminalsinthestomachwall,andthatthoughthedensityofinnervationinthesmall

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intestineislower,thegreatersizeofthatorganwouldmeanalargerabsolutenumberof

intestinalIGLEsthaninthestomach38.ThehighIGLEdensityinthestomachisconsistent

withthephysiologicalimportanceofstomachstretchonfeedingbehavior.Inaddition,the

highnumberofIGLEsinothersitessuggeststhisterminaltypealsotransducesimportant

mechanicalinformationfromgastrointestinalsitesoutsidethestomach.

IGLEs,notably,arenottheonlyvagalterminaltypeinthemuscularwallofthe

gastrointestinaltract.Thesecondterminaltype,namedintra-musculararrays(IMAs),are

notnearlyaswidelydistributedasIGLEsbutratherseemtoterminatearoundthegastro-

esophagealjunction,gastro-duodenalsphincter,andthejunctionbetweentheproximal

andmiddlesegmentsofthecolon36.IMAscoursebetweenthelongitudinalsmoothmuscle

layers,forminglongbranchingparalleltractsthatformsynapse-likecontactswith

intrinsicallyoscillatory,gastriccontractionpace-makingcellstermedinterstitialcellsof

Cajal40.GiventheanatomicalassociationwithICCsandsphincters,IMA-formingneurons

arealsopositedtobemechanosensors,butdefinitiveevidencefortheirresponse

propertiesandphysiologicalroleremainselusive.

Summary

Mechanicaldistensionofthestomachregulatesmealsizeduringfeeding.Anintact

vagusnerveisrequiredforthedetectionofstomachdistensionandreductionofmealsize.

Aselectsubsetofvagalafferentsrespondstostomachstretchwithslowlyadapting

increasesinfiringrateinresponsetobothpassivedistensionandmuscularcontraction.

DistensionsensitivevagalafferentsformIGLE-typeterminalendingsintheesophagus,

stomachandintestinemusclewalls.However,keyquestionsremainabouttheidentityof

therelevantmechanosensorymolecularapparatus,theprimarysiteofmechanosensation,

andthecontributionsofnon-IGLEstomachmuscle-innervatingvagalsensoryafferents.

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Lungstretch

Physiology

Thephysiologicalcontrolofrespirationhasbeenlinkedtothevagusnervesincethe

inceptionoftherespiratoryphysiologyfield.Earlyrespiratoryphysiologistsnotedthat

electricalstimulationofthetransectedvagusnervetrunkcouldinduceeitherexpiratoryor

inspiratoryeffort41.However,resultsweremixedandthephysiologicalrelevanceofthese

observationswasnotclarifieduntiltheworkofHeringandBreuerin1868,whenthe

artificialmanipulationofnerveswasabandonedinfavorofdescriptionofthephysiological

responsestoinflationanddeflationofthelungs42.

Breuernotedthatwhenthelungwasinflated,furtherinspiratorymovementswere

inhibited,andexpirationwaspromoted.Inspiratoryinhibitionoccurreddespite

developmentofprofoundhypoxiaandindependentlyofthechemicalcompositionofthe

gasusedtoinflatethelungs,suggestingtherelevantcuewaslesslikelychemical,andmore

likelymechanical.Thedegreeofinspiratoryinhibitionwasdirectlycorrelatedtothedegree

oflungexpansion.Thereflexalsoseemedtobephysiologicallyrelevantbecauseitcouldbe

observedbothifthelungwereartificiallyinflatedbeyondnormalphysiologicalvolumes,

andalsoifthetracheaweresimplyoccludedatthepeakofnaturalinspiration.Therelevant

stimuluswasthoughttobetheexpansionofthelungitselffortworeasons:1)Expansionof

collapsedlungsalsoblockedinspiratoryeffort,meaningthereflexwasintactevenwhen

intra-thoracicpressureswereabnormallylowandchestwallmovementscouldnot

contribute.2)Introductionofelevatedthoracicpressurethroughatubeinthesideofthe

chestdidnotimpactinspiratoryeffortaslongasthestateofexpansionofthelungswas

heldconstant,meaningthecriticalcuewasthemechanicalmovementofthelungandnot

theexternalpressuretowhichthelungwassubject.Thisreflexisclassicallyreferredtoas

theHering-Breuerinspiratoryreflex,andcanbeabolishedbycuttingbothvagusnerve

trunksintheneck42.

SubsequentphysiologicalstudieshaverevealedintriguingfeaturesoftheHering-

Breuerreflexthatcomplicatecurrentunderstandingofitsphysiologicalrole.First,a

similarreflexwasalsoinitiallydescribedinwhichholdingthelungsinexhalationwould

preventfurtherexhalation42;afindingthatsparkedmanydecadesofdebateaboutwhether

16

thesameneuralpathwayscouldunderlieboththeinspiratoryandexpiratoryreflexes43,44.

Second,aseriesofexperimentswereundertakentoinvestigatetherelevanceofthereflex

inman,whichrevealedfourintriguingfindings:1)inspiratoryinhibitiontolunginflation

wasobservedininfantsupuntil3-4daysoflife,duringwhichtimethereflexstrength

decreasedandultimatelywaslost45.2)Thereflexcouldonlybeobservedinadultsunder

anesthesiaor,inonesubject,unconsciousfromaheadinjury46,butnotinsleepingadults47.

3)Surgicalinstillationofanerve-blockingagenttothevagustrunkcouldblocktheHering-

Breuerreflexinanesthetizedpatients46.4)Incontrast,inawakesubjects,nerveblock

injectionsaroundtheninthandtenthcranialnervesatthebaseoftheskullresultedinno

changeinbreathing,thoughtheinjectionsintheseexperimentscausedprofound

hypertension(presumablysecondarytobaroreceptorblockade),andinsensitivityto

hypoxia(presumablysecondarytochemoreceptorblockade)48,49.Thesedatatogether

suggestthatthestrengthoftheHering-Breuerreflexchangeswithdevelopment,andis

influencedbysubjects’stateofconsciousness.Thequestionsraisedabouttheroleofthe

reflexinawake,normalhumansremainunanswered.

LunginflationinducesotherreflexeffectsbeyondtheHering-Breuerinhibitionof

inspiration.Twootherprominentreflexeffectsoccurwithlunginflation.First,

physiologicalinspirationcausesanincreaseinheartrate50.However,carefulexperiments

inwhichlungdistensionwasisolatedfromchangesinbloodflowinthecardiopulmonary

systemandfromchemicalchangesinthebloodsecondarytorespirationsuggestedthe

relevantcueinthisreflexisaugmentedcardiacvenousreturn,tobediscussedin

subsequentsections51.Secondly,inspirationalsotriggersareflexdecreaseofsystemic

vascularresistance52.Intheseexperiments,expansionofthelungsbyinjecting

physiologicallyrelevantvolumesofairresultedinadose-dependentsystemicvasodilation,

whilecollapseofthelungsresultedinsystemicvasoconstriction.LikeintheHering-Breuer

reflex,thereductioninsystemicvascularresistancewasindependentofthecomposition

andtemperatureofthegasusedtoinflatethelungs.Theeffectwasalsomaintainedwhen

thepressuresinthecarotidarteriesandtheaortawereheldconstant.Cuttingpulmonary

nervesandchemicallyinhibitingthesympatheticnervoussystematthestellateganglionin

theneckcouldbothabolishthereflex,suggestingitismediatedbycommunicationbetween

thevagusandsympatheticfibers.However,thesamecareinisolatingpulmonaryversus

17

cardiovascularcontributionstothisreflexwasnottakenasinotherexperiments,so

specificconclusionscannotbedrawn53.

Responseproperties

Asinthegastrointestinalstretchfield,thedescriptionoftheHering-Breuerreflex

drovethesearchfortheoperativeneuronalelements,andtheirdiscoveryonlyawaitedthe

developmentofelectrophysiologyequipmentandmethodssensitiveenoughtorecordfrom

singlenervefibers.Adrianwasthefirsttoidentifyindividualpulmonarystretch-sensitive

vagalafferentsin193354.ThepulmonarymechanoreceptorsAdrianidentifiedgenerate

largecurrentsinfast-conductingA-fibers,explainingtheiridentificationover10years

priortotheirmoreelusiveC-fibermediatedgastricmechanoreceptorcounterparts.Work

followingthisinitialdiscoveryidentifiedtwoclassesofmechanosensitivepulmonary

afferents,distinguishedbytheiradaptationrateandthresholdofactivation55.

Slowly-adaptingstretchreceptors(SARs),definedashavingadeclineoffiringrate

oflessthan55%withinthefirsttwosecondsofastretchstimulus,accountfor

approximately50%ofallpulmonaryinflation-sensitivevagalafferents55.SARsarenot

onlyactivatedbyexperimentalinflation,butalsofirecyclicallywiththerespiratorycycle,

canbeactivatedbyforcedlungdeflation,andarealsoactivatedbycontractionofsmooth

musclesurroundingtheairways56.MeasurementoftrachealpressurethresholdsofSAR

activationsuggeststhatatleasthalfofallSARsareactiveduringtherestingrespiratory

cycle57.Intriguingly,inhaledcarbondioxideinhibitsSARstretchsensitivity58,aneffect

mediatedwithinthelungbecauseCO2inhibitsSARsensitivitywithoutchangesinarterial

carbondioxidelevels59,60.However,otherthantheinhibitoryeffectofCO2,SARsare

notablyinsensitivetootherchemicalstimuli61,andtheoxygencontentoftheinhaledor

infusedgasdoesnotimpacttheresponsepropertiesofSARs54.SARsthereforehavea

mechano-specificreceptivefieldtorivaltheirsimilarlyslowlyadaptinggastricstretch

sensitivecousins.ThesepropertiesmakeSARsidealcandidatestomediatetheHering-

Breuerinspiratoryinhibitionreflex.

Incontrast,rapidly-adaptingstretchreceptors(RARs)arealsoactivatedbylung

18

inflation,butaredefinedashavingfiringratesthatdecrementbyover80%withinthefirst

twosecondsofastretchstimulus55.RARsaccountfor40%ofstretch-sensitivepulmonary

afferents,thoughthisvarieswithanimalmodelinvestigated62.RARsexhibitamarkedly

higherthresholdforactivationcomparedtoSARs;nearlynoRARsareactiveduring

breathingatrest,thoughthemajoritywouldbeactivatedatpressuresexhibitedduring

respiratoryeventssuchascoughing57.Inadditiontothesefeatures,RARsarealsosensitive

toanumberofirritatingchemicalmediatorsincludingpollutantssuchasozoneandwood

smoke,adiversityofchemicals,pulmonarycongestion,andintroductionofhypo-osmotic

solutionswithintheairways,earningthemthename‘irritantreceptors’63-67.Thismixed

high-thresholdmechanicalandchemicalreceptivefieldsuggestsfunctionaloverlapwith

lungafferentsthatarepurelychemo-sensitivewithoutanymechanicallysensitiveresponse

propertycomponent61.Forthesereasons,RARsaresuspectedtounderliereflexresponses

ofhyperpneaandbronchoconstrictiontooffensiveinhaledchemicalagents.Inaddition,

RARscanbesensitized,suggestingaroleinreactiveairwaydisease63.

WhileSARsandRARsareconsideredbroadcategoriesofmechanosensitivelung

afferents,detailedrecordingsrevealmanyfiberswithpropertiesintermediatebetween

SARsandRARsthataccountfortheremaining10%ofstretch-sensitiveafferentsperthe

SARandRARdefinitionsprovidedabove55,57,68.Therefore,itmaybemoreaccurateto

characterizefibersasbelongingtoaspectrumofadaptationandchemicalsensitivityrather

thantotwofullydiscreteneurongroups.However,withoutameanstoselectivelyand

specificallymanipulateRARsvs.SARs,theinnervationpatternsandphysiologicalrolesof

eachstretch-sensitivefibertyperemainsunclear.

Recentexperimentshavesuggestedthemolecularidentityofalung

mechanosensor69,andthesedatawillbepresentedindetailwithinthediscussionofthe

discoveryandelaborationofthemammalianmechanosensorPiezo2.

Anatomy

Vagalafferentstravelalongthemajorairwaysandprovideterminalsatalllevelsof

therespiratorytree,fromthetracheatothealveoli.TheanatomicallocationsofSARand

19

RARterminalshavebeeninferredfromfunctionalstudiesofterminalresponseproperties,

andtheirdetailedterminalanatomyinferredbasedonthestructuresofvagalafferents

foundatthesevariousanatomicalsites.

SARterminalsarethoughttobelocatedinthesmoothmusclesurroundingairways63.Threelinesofevidencesupportthisconclusion.First,SARsrespondtodirectmechanical

probingoftheairwaysmoothmuscle,aswellastoairwaysmoothmusclecontraction56,70.

Second,SARscontinuetofunctionafterthemucosaisstrippedfromisolatedexperimental

preparations,butceasetofunctionafterdisruptionofthesmoothmuscle56.Finally,SARs

arenotinhibitedintra-airwayanesthetics,supportingtheconclusionthattheirterminals

areisolatedfromtheairwaylumen56.Myelinatedvagalafferentterminalsinpulmonary

smoothmuscle,inferredfromthepropertieslistedabovetobeSARs,havebeen

characterizedwithbothlightanelectronmicroscopy.Asinothermusclemechanoreceptor

endings,theseterminalslosetheirmyelinsheathclosetotheirterminalsite,andformclose

contactswiththebasallaminaandconnectivetissuewithinthemuscularlayer71,72.The

distributionofSARsfromthetracheatothealveoliseemstoexhibitsomespecies

specificity,withanywherefrom40-90%beinglocatedintheintra-pulmonaryairways

versusinlargeextra-pulmonarysites73.

RARs,incontrast,arethoughttoterminateinthesubmucosaandairwayepithelium,

aconclusiondrawnfromthreepiecesofevidence.First,lightmucosalstrokinginsidethe

airway,aswellasinhalationofchemicalirritants,isabletoelicitresponsesinRARs,while

applicationofintra-airwaylocalanestheticssuchaslignocainecansuppresstheiractivity57,63,74,75.Second,thegreatestareaofRARresponsivenessisaroundthecarinaandlarge

airwaybranchpoints,whichisalsothesiteofgreatestintra-epithelialendingdensity57.

Finally,anintriguingandindirectobservationisthatanimalsthatlackthecoughreflex(e.g.

mice,ferrets),alsohaveveryfewintra-epithelialvagalterminalsandRARs62.The

sensitivityofRARstointrapulmonarycongestion,however,hasalsoraisedthepossibility

theyarefoundclosetolungvascularaswell76.RARsarelocatedinbothlargeandsmall

airways,andtendtohaveanatomicallyrestrictedreceptivefields.Forexample,inflationof

allairwaysandlungtissuedistaltoanRARreceptivesiteidentifiedbygentleintra-airway

20

mucosalstrokingfailstoelicitactivityoftheRAR57.

Intriguingly,geneticallytargetedlabelingofvagalafferentsthatexpressthe

purinergicreceptorP2RY1selectivelylabelsA-fibersthatinnervateneuroepithelialbodies

intheepithelialliningofthelung,aterminaltypethathadbeendescribedpreviouslybut

whosefunctionhasremainedunknown77,78.ActivationofP2RY1neuronsresultsinapnea

mirroringinhibitionofinflationviatheHering-Breuerreflex,andinsituhybridization

showedpartialoverlapbetweenP2ry1andthemechanosensorPiezo2,raisingthe

possibilitythisneuronpopulationisinvolvedintheHering-BreuerreflexandthatPiezo2

couldbetherelevantmechanosensorymolecule.

Summary

Mechanicaldistensionofthelungregulatesbreathingdepthandpatterns.Anintact

vagusnerveisrequiredforthedetectionoflungdistensionandchangesinrespiratory

physiology.Aselectsubsetofvagalafferentsrespondstolungstretch.Onesubsetisformed

ofslowlyadapting,low-threshold,puremechanosensorsthoughttoterminateinthe

smoothmusclesurroundingairwaysandmediatetheHeringBreuerreflex.Othersare

rapidlyadapting,high-threshold,mixedmechano-andchemo-receptorsthoughtto

terminateclosetotheairwayepitheliumandmediatecoughandbronchoconstrictionin

responsetoirritants.Howevermanyconclusionswithinthefielddependupon

assumptionsaboutstructure-functionrelationshipsthatstillrequirerigorousexamination,

andambiguityaboundsconcerningthemolecularmechanisms,anatomy,andphysiological

relevanceoflungmechanoreceptors.

Cardiovascularsystem

Physiology

Thevagalreflexregulationofthecardiovascularsystemholdsafundamentalplace

inthehistoryofneuroscience.OttoLoewiandHenryDalesharedtheNobelPrizein

21

PhysiologyandMedicinein1936forthediscoveryofthefirstneurotransmitter,

acetylcholine,releasedbythemotorfibersofthevagusnervetocauseadecreaseinheart

rate79.Justtwoyearslater,CorneilleHeymanwasawardedthesameprizeforthediscovery

thatsensorynervesinthelargevesselsleavingtheheartmonitorpressureandblood

chemistrytoregulatethecardiopulmonarysystem,inareflexarcthroughthevagalmotor

fibersinvestigatedinDaleandLoewi’swork.Thesecriticalexperimentsdelineatedthe

neuralpathwayofthebaroreceptorreflex,inwhichincreasesinbloodpressurecausea

reflexdecreaseinheartrateonabeat-to-beatbasis,allowingformaintenanceofaconstant

bloodpressure80,81.Thebaroreceptorreflexisimportantbothforpreventionof

hypertension,andofhypotension.Surgicalremovalofvagalafferentsintheaorticarchand

carotidbulbresultsinchronichypertension82.Inaddition,withoutreflexaccommodation

ofbloodpressure,movingfromalyingtoastandingpositionwouldresultinsucha

dramaticafallincerebralperfusionthatitwouldleadtolossofconsciousness.Vaso-vagal

syncopeandorthostasisare,infact,verycommonclinicalcomplaints.Furthermore,

decrementsinthebaroreceptorreflexwithageandtheconcomitantinabilitytoregulate

bloodpressurewithchangesinpositionisassociatedwithincreasedfalls,fractures,and

mortalityinpeople83.

Thebaroreceptorreflexisnottheonlycardiopulmonaryreflexmediatedbyvagal

sensoryfibers.Twootherprominentreflexesarecontrolledbyvagalafferents.Thefirst,

theBainbridgereflex,servestoregulateintravascularvolumestatus.Increasedatrial

volumeresultsintworeflexeffects:1)anincreaseinheartratetomovetheextravenous

bloodthroughtheheart,and2)anincreaseinurineoutputtoreduceintravascular

volume51,84.Initially,inspirationwasthoughtodrivethereflexincreaseinheartrate,as

notedpreviouslyinthediscussionofpulmonaryreflexphysiology.However,becausethe

heart,lungs,andgreatvesselsareallhousedinthethorax,changesinintrathoracic

pressureimpactsthedistributionofbloodinalltheseorgans.Reductionsinintrathoracic

pressureduringinspirationaugmentsthevenousreturnofthebloodtotheheart,and

carefulisolationofthisvariablefrominspirationitselfrevealedthecardiacchamber

dilationtobetherelevantcue51,84.

Thesecondreflex,termedtheBezold-Jarischreflex,isthoughttobeacardio-

protectivereflexinwhichbothmechanicalstretchandapplicationofirritantchemicalcues

22

tothemuscularheartwallcausesareflexbradycardia,hypotension,coronaryartery

dilatation,andapnea85,86.Reductionofheartratereducesmetabolicdemandofcardiac

tissue,systemichypotensionreducestheresistanceagainstwhichcardiacmyocytesneed

topumpblood,andcoronarydilationaugmentsperfusionofcardiactissue.Therefore,

thoughitisuncleartheextenttowhichmechanicalversuschemicalcuesdrivethisreflex,

theresultisreducedworkandimprovedoxygentationofcardiacmyocytes.Howthe

Bezold-Jarischreflexinteractswiththeothercardiopulmonaryreflexestomaintainnormal

bloodpressureandperfusionremainsanareaofuncertainty87.

Responseproperties

Assearchesfortheoperativeneuronalelementsinlungstretchwereunderway,

Adrian’scontemporariesweresimilarlyidentifyingtheneuronalelementswithchangesin

activitysynchronizedwiththebeatingheart.BronkandStellain1932werethefirstto

isolatesingleunitsinthecarotidsinusnervethatfiredcyclicallywithsystoleduringheart

beats,andchangedtheirfiringrateslinearlywithincreasesinbloodpressurefrom

40mmHgto120mmHg,therangeofnormalphysiologicalbloodpressuresintherabbit.

Theydeterminedthatincreasesinwhole-nervefiringratesinresponsetoincreasingblood

pressurewasbothafunctionofrecruitmentofmorefibers,andincreasesinfiringratein

individualfibers.Furthermore,thearterialbaroreceptorstheyidentified,aswithseveral

othervagalstretch-sensitiveelements,areslowly-adapting88.Subsequentstudieshave

confirmedvagalsensoryfibersareexquisitelytunedtopressurechangeswithinthegreat

vesselsexitingthehearttodeliverbloodtothebodyandbrain89-93.Intriguingly,despite

theirslowadaptationrate,vagalbaroreceptorsdoshifttheirbaselineandthresholds

followingprolongedexposuretohyperorhypo-tension,suggestingwhythissensory

apparatuswouldnotbesufficienttocounteractchronichypertensivedisease93.Thestress

historyofthevesselwallimpactsbaroreceptorresponsiveness92.

Mechanosensitiveafferentsinthecardiovascularsystemarenoonlylocatedinthe

majorvessels,butalsodetectmechanicalforcesintheheartwalls.Cardiacreceptorscanbe

furtherclassifiedbasedontheperiodofthecardiaccycleduringwhichtheiractivitypeaks.

Someafferentsaremaximallyactiveduringatrialcontraction,andexhibitincreasesin

23

firingratewithincreasesinheartrate,suggestingsensitivitytoactivechangesinatrialwall

stretch.Otherreceptorsrespondduringatrialfilling,andexhibitreducedactivitywith

increasesinheartrate.Thesereceptorsadaptslowlytochangesinpressure,suggesting

theyactprimarilyasmeasuresofoverallwallstrain.Bothoftheseatria-sensitivefibersare

exclusivelyfast-conductingA-fibers94,95.

Ventricularfibers,incontrast,aremuchlesscommonthanatrialfibers(ofcardiac

mechanosensors,75%arefoundintheatriaand25%intheventricles)andcanbe

classifiedfurtherintotwofunctionallydistinctgroups:onesetrespondsprimarilyto

ventricularcontraction,andtheothertogentleendocardialstroking96,97.Mostventricular

fibersareC-fibers,andalsorespondtointracardiacadministrationofabroadarrayof

chemicalcompounds.Thefunctionalsignificanceofthischemicalsensitivitymayrelateto

reflexesinvolvedinresponsetocardiacstressorischemia98-102.

However,thespecificmolecularmechanismsusedbycardiacmechanosensorsremain

undefined.Dissociatedaorticbaroreceptorneuronsexhibitmechanicallyinducedcurrents

thatcanbeblockedbygadolinium,suggestingthemolecularmechanosensormaybea

stretch-activatedionchannel103.Inaddition,bothamilorideanditsanalogue,benzamil,

inhibitedpressure-evokednerveactivityinbaroreceptorvagalafferents(Drummond,

2001),suggestingthatthechannelmaybearrelationtoENaCfamilymechanoreceptors.

However,alternativepossibilitiesabound,includingevidenceforexamplethatknockoutof

Trpc5attenuatesthebaroreceptorreflex104.Nocandidatetodatehasyetbeenshownto

fullyabolishthereflex.

Anatomy

Vagalafferentsinnervateallfourchambersoftheheart,theaorticarch,andmajor

brancharteriesintheneck.

Endingswithinthecardiacchambersconsistofthreeprimarytypes,1)complexun-

encapsulatedend-netsfoundmostdenselynearjunctionsofthegreatveinsenteringthe

atria,and2)anastomosingnetsoffineneuritesdistributedthroughoutatrialand

ventricularendocardium,someofwhichcloselycontactcardiacmyocytesand3)

terminalssurrounding“smallintenselyfluorescent”cellsofunknownfunctionin

24

cardiacganglia94,105-108.However,notably,withinthecardiacchambersitseemsthatthe

sameneuroncangiverisetomultipleoftheseendingtypes,complicatinginterpretationsof

structure-functionrelationships107.

Theneuronalendingsinthegreatvesselshavesimilarlybeencharacterizedinto

anatomicalsub-types.Approximately85%ofvessel-terminatingfibersdirectlyinnervate

thevesselwall,forming“flower-spray”and“end-net”typeterminals,whiletheremaining

15%terminateadjacenttoother“smallintensely-fluorescent”cellsproximaland

superficialtothewallitself105.Themostdetailedarchitecturalstudyofvagalafferentshas

beenperformedintheaorticarch,thesiteofmostelectrophysiologicallyactive

baroreceptors108.Aorticnervefibersinthisregionconsistbothofmyelinatedand

unmyelinatedfibersthatpassthroughthevesselwalladventitia,coilandterminatewithin

thevesselsmoothmusclemedialayer.Withinthislayer,myelinatedfiberslosetheirmyelin

sheath,andtheaxolemmaisdirectlyincontactwithextracellularconnectivetissue.The

tightapproximationofneuralelementswithelasticelementsofthewallspeakstoa

structure-functionrelationshipwellsuitedfortransductionofmechanicalforcesand

movementofthevesselwall.

Summary Sensorymonitoringofthemechanicalmovementoftheheartandgreatvessels

playsanimportantroleintheregulationofheartrateandbloodpressure.Aselectsubset

ofvagalafferentsrespondstochangesinpressureandstrainduringthecardiaccycle.Vagal

baroreceptors,arethoughttoinnervatethewallsoflargevessels,anddrivereflex

decreasesinheartrateinthesettingofhypertension,andreflexincreasesinthesettingof

hypotension.However,muchambiguityremainsconcerningtherelativerolesofheart

versusvessel-terminatingfibers,fibersofdifferentconductionvelocities,theinterplay

betweenchemicalandmechanicalstimuli,andofcoursethemolecularmechanisms

underlyingdetectionofpressureandstraininthissystem.

25

Conclusions Thevagusnervecontainsseveralafferentclassesthatmonitormechanicalforces

withinthegastrointestinal,respiratory,andcardiovascularsystems.Careful

electrophysiologicalcharacterizationandanatomicaltracinghasbeguntoelucidatesome

oftheterminaltypesandputativephysiologicalrolesoftheseimportantsensorycircuits.

Geneticidentificationofthespecificneuronsinvolvedhasjustbeguntounfold.Vagal

sensoryneuronsexpressingGLP1Rincludethegastrointestinalmechanosensors33.Vagal

sensoryneuronsexpressingP2RY1areA-fibersandinhibitrespiration,raisingthe

possibilitythesearetheslowly-adaptinglungmechanoreceptorsthatunderlietheHering-

Breuerreflex77.However,todate,theidentityofthereceptorresponsiblefor

mechanotransductionitselfininternalsensorysystemsremainsunknown.Discoveryofthe

molecularmechanismsatplaycouldresolvedebatesaboutthesiteofprimary

mechanosensation,moreclearlydefinetheneuronpopulationsinvolved,andalsoprovide

apharmacologicaltargetforpowerfulmanipulationoffundamentalautonomicreflexes.

ThemammalianmechanosensorPiezo2

Discovery Extensiveinvestigationintothemolecularidentityofmechanosensorshasyielded

discoveryofseveralnon-vertebratemechanosensitivechannelfamilies,includingthe

DEG/ENaC,TRP,TMCsandselectK+channels109.ThediscoveryofthePiezofamilyhas

offeredintriguingmolecularlyspecifiedmechanoreceptorsforqueryinknown

mechanosensitiveneuralsystems.

ThemembersofthePiezofamily,Piezo1andPiezo2,wereidentifiedinalarge-scale

screeningeffort110.Culturesofintrinsicallymechanosensitivemammaliancelllineswere

treatedwithsiRNAdesignedtoknockdownexpressionofproteinsofunknownfunction

predictedtocrossthecellmembraneatleasttwice,aconditionconsideredlikelyinarapid

mechanosensitivechannel.The71stcandidatesiRNAtestedtargetedFam38a,nownamed

Piezo1,andresultedinaseveral-foldreductionoftheintrinsicmechano-sensitivecurrent.

StructuralsimilarityledtoidentificationofthecloselyrelatedPiezo2.Overexpressionof

26

theseproteinsinheterologoussystemsconferredmechanosensitivitytootherwise

mechanicallyinsensitivecells.Furthermore,immunohistochemistryrevealedPiezo1tobe

localizedtothecellmembrane,thephysicalsiteofmechanotransduction.Piezo2wasfound

byinsituhybridizationtobeexpressedinasubsetofdorsalrootganglionneurons,and

knockdownofPiezo2inDRGculturesresultedinareductionofcellswithrapidly

inactivatingmechanosensitivecurrents,providingstrongevidencethatPiezo2playsarole

inmechanotransductioninknownmammalianmechanosensitiveneurons.Notably,slowly

inactivatingmechanosensitiveDRGneuronsremainedunperturbedbyknockdownof

Piezo2,suggestingadditionalmammalianmechanoreceptorsremaintobediscovered.

SubsequentworkprovidedfurtherevidencethatPiezo1andPiezo2wereinfactthe

molecularmechanosensitiveelement111.ExpressionofPiezoproteinsaloneinlipid

bilayerswassufficienttoconfermechanosensitivity.Furthermore,recordingsfrom

membranescontainingPIEZOproteinsshowsingle-channelcurrentsactivatedby

mechanicalforcethatcouldbeinhibitedwithrutheniumred,anon-selectivecationchannel

blocker.Photo-bleachingexperimentssuggestedthatPIEZOformstetramerswithinthecell

membrane.ThesedatatogethersuggestthatPiezo1andPiezo2assembleintopore-

forming,cationchannelscapableofmechanotransductionindependentofanyother

molecularelements.

Invivoproperties

Invertebratemechanosensation

Followingtheirdiscoveryandmolecularcharacterization,subsequentworksought

toelucidatetheinvivoexpressionpatternsandphysiologicalrelevanceofPiezo1and

Piezo2.ThefirststudytodemonstratetheinvivorelevanceofPiezomechanosensorswas

notinmammals,butratherinthefruitflyDrosophilamelanogaster112.ThePiezofamilyhas

onememberthatisexpressedinDrosophila,Dmpiezo.Expressionofafluorescentreporter

underthecontrolofDmpiezoresultedinfluorescenceinavarietyofsensoryneurons,

suggestingthepossibilityofaroleinmechanosensation.Dmpiezoknockoutflies,however,

didnotexhibitdeficitsincoordination,norinthebristlemechanoreceptorpotential,

27

suggestingDmpiezoisnotakeymechanoreceptorinadultflies.Incontrasthowever,

Dmpiezoknockoutlarvaeexhibitedreducedsensitivitytonoxiousmechanicalstimuli.

Isolationandrecodingofppkneurons,knowntobespecificallyimplicatedinlarvalnoxious

mechanicalsensitivity,demonstratedthatDmpiezoknockoutcausescompletelossof

mechanosensitivecurrentsinthisneuronclass.ThereforeDmpiezoplaysaroleinnoxious

mechanosensationinlarvae,makingitoneofthefirstionchannelstohavedemonstrated

bothinvitromechanosensitivityandaroleinbehaviorinvivo.

Vertebratedorsalrootgangliaandskinsensation

Withthedevelopmentofmousegenetictools,severalstudieshaveshownthat

Piezo2playsakeyroleinmultiplemechanosensorymodalitiesinmammals113,114.The

Piezo2proteincanbefoundonsensoryneuronterminalswithintheskin.Piezo2canalso

befoundwithinGolgitendonorgansandmusclespindlefibers,suggestinganadditional

roleinsensationofjointandlimbposition.Concordantwiththeseanatomicalresults,mice

lackingPiezo2showdeficitsindetectionofvibration,lighttouch,hairdeformation,low-

forcemechanicalstimuli,andproprioception,withoutdeficitsinresponsetopainor

temperature.Inaddition,Piezo2expressionisnotlimitedtoneurons;intriguingly,Piezo2

isalsoexpressedinMerkelcells,aspecializedcelltypeassociatedwithmechanosensitive

neuronterminals.Merkelcell-specificknockoutofPiezo2abolishedthe

mechanosensitivityofMerkelcells,andresultedinareductioninmechanosensitive

currentsandbehavioralresponsestolow-forcemechanicalstimuli115.Therefore,itislikely

thatPiezo2actsbothinneuronsandinMerkelcellstomediatelow-force

mechanosensation.

VertebratelunginflationandtheHeringBreuerreflex

Inadditiontoaroleinexternalsensationoftheskinandextremities,Piezo2has

alsobeenimplicatedinmechanosensationintherespiratorysystem69.Constitutive,global

deletionofPiezo2islethal;knockoutpupsarecyanotic,withlowarterialoxygen

saturation,abnormalrespiratoryrhythms,andsmallairspacesonhistologicalexamination

28

ofthelung,resultingindeathwithin24hoursofbirth.Totrytounderstandtherelevant

siteofPiezo2expressionforthislethalphenotype,differentmousedriverlineswereused

toselectivelyknock-outPiezo2intheendotheliumofbloodvessels,neuralcrestderivative

tissuessuchastheneuroepithelialbodiesinthelungandsensoryneuronswithinjugular

ganglia,thetrigeminalgangliaandofcoursethedorsalrootganglia,andincranialplacode-

derivedtissuessuchasthenodoseganglion.Piezo2knockoutspecificallyinthe

endotheliumorinthenodosegangliondidnotimpactpupsurvival.However,Piezo2

knockoutinthejugularganglion,dorsalrootganglion,andtrigeminalganglion

recapitulatedthephenotypeobservedwithglobalPiezo2deletion.Thesedatatogether

suggestthatPiezo2expressioninneuralcrest-derivedtissuesisnecessaryforsurvival,

andthatPiezo2lossfromtheneuralcrestresultsinabnormalpuprespiratorydynamics.

TheseobservationsbegthequestionoftherolePiezo2playsintherespiratory

systemintheadultanimal.Optogeneticactivationofneuronswithinthenodose-jugular

complexthateithercurrentlyexpressorhaveexpressedPiezo2duringdevelopment

resultsinapneaintheadult,implicatingthisneuronpopulationinadultrespiratory

control.ConditionaldeletionofPiezo2intheadultmousenodose,jugular,anddorsalroot

gangliaresultsinareductionofvagusnerveactivityinresponsetoinflationofthelung,a

findingconsistentwiththeroleofPiezo2inmechanosensationeitherwithintheneural

crestorplacode-derivedtissuesofthisstructure.

WhiledeletionofPiezo2fromtheplacode-derivednodosesensoryneuronsisnot

lethal,theseanimalsdoexhibitabnormalrespiratorycyclesasadults.Furthermore,vagal

responsestolungstretcharecompletelyablatedinPiezo2deletioninthenodoseganglion.

Thisresultraisesinterestingquestionsbecauseitsuggeststhatintheadultmouse,allvagal

lungmechanoreceptorsareinthenodoseganglion,andnoneintheneuralcrest-derived

jugularganglion.However,inpupslossofPiezo2intheneuralcrestbutnotthenodoseis

lethal.DoesthismeanthattheroleofPiezo2intheneuralcrestrequiredforsurvivalisnot

relatedtoadultdetectionoflunginflation?Howdothesensoryrolesofjugularversus

nodoseversusextra-vagalneuronsevolveoverthecourseofdevelopment?Ifdetectionof

lunginflationdependsonnodosePiezo2expression,andlossofdetectionoflunginflation

isnon-lethal,whatisthephysiologicalsignificanceofadultlunginflationmonitoring?

WhereelseisPiezo2deletedinbythedriverusedinnodosegangliondeletionexperiments,

29

anddoesthisresultinabnormalitiesthatcouldexplainthelossoflungstretchsensitivityin

theadult?ThedissociationoftheroleofPiezo2inneuralcrest–derivedversuscranial

placode-derivedtissuessuggestsitsub-servesmorethanonefunctionintherespiratory

system.

RoleofPiezo2inhumanmechanosensation

Piezo2playsanimportantroleinhumanmechanosensationanddevelopment.Gain-

of-functionmutationsinPiezo2havebeenshowntounderliearareautosomaldominant

formofdistalarthrogryposischaracterizedbymultipledistalcontractures,

ophthalmoplegia,ptosis,andrestrictivelungdisease116.Twofamilieswereidentifiedwith

similarfeatures;inoneaPiezo2pointmutationresultedinfasterrecoveryfrom

inactivation,andintheotheraPiezo2missensemutationcausedbothfasterrecoveryfrom

inactivationandslowerinactivationkinetics,bothmutationsthatwouldtranslateto

increasedsensitivitytomechanicalforces.Patientssurvivetilladulthoodandarefertile,

buthavecharacteristicfacieswithdeep-seteyes,anddifficultywithmusclemovement

includingoftheeye,back,neck,andbothlargeandsmalljointsoftheextremities.

Incontrast,twopatientsofdifferentancestryhavebeenidentifiedwithcomplete

Piezo2loss-of-functionmutations117.ThePiezo2variantsfoundinthesepatientseitherhad

Piezo2alleleswithprematurestopcodons,oroneallelewithaprematurestopcodonand

anotherwithamissensemutation.Thesevariantswereunabletogenerate

mechanosensitivecurrentswhenexpressedinheterologouscellsystems.

Thetwopatientswiththesemutationsbothpresentedwithasimilarconstellation

ofcharacteristics.Developmentalfeaturesincludedcongenitalhipdysplasia,finger

contractures,footdeformities,severeprogressivescoliosis,hypotonia,delayedwalkingand

headcontrol,andahistoryofshallowbreathingduringinfancy.Asadults,both

demonstratedaprofoundsensoryataxia,resultingindifficultyperformingreachingtasks

andaninabilitytowalkorstandwitheyesclosed,consistentwithdeficitsin

proprioception.Theywerealsounabletofeelavibratingtuningfork,hadseverelyreduced

lighttouchsensitivity,andexhibitedchanceperformanceoftwo-pointdiscriminationon

30

glabrousskin.Temperatureandpainsensitivitywasnormal.Theyalsohadintactlight

touchsensationonareasofhairyskin,suggestingaPiezo2-independentlighttouchsensory

modalityassociatedwithhair.Insummary,thesepatientsexhibitsimilarsensorydeficits

asmicethatlackPiezo2asadults.However,importantly,humanloss-of-functionofPiezo2

isnotnecessarilylethal.Furthercharacterizationofthemedicalhistoryduringinfancyand

characterizationoftherespiratorysystemofthesepatientswouldbeofgreatinterest.

Conclusions Piezo2isamammalianmechanosensorwithdemonstratedimportanceindetection

ofexternalmechanicalcues.Themolecularidentitiesofthemechanosensorsinvolvedin

detectionofinternalcuesremainunknown,thoughthephenotypesofPiezo2mutantmice

andhumansbothsuggestthatPiezo2couldbeinvolvedincriticalinternalphysiological

reflexes.Therefore,wesoughttodetermineiftheneuronsthathaveexpressedorcurrently

expressPiezo2inthenodoseganglionmediatedetectionofinternalstretch,andwhether

Piezo2knockoutcouldalterneuronresponseproperties.

Recordingmechanosensitiveafferentsinthevagus

Introduction SeveralexperimentalgoalsmustbemettoevaluatetheroleofPiezo2inmammalian

internalmechanosensation.Amethodtorecordtheactivityofvagalafferentsofknown

molecularidentityisrequired.Ideally,suchanexperimentaldesignwouldallowfor

simultaneousrecordingsfrommultipleneuronswithinthesameanimal.Similarly,stimulus

deliveryparadigmsmustenablerobustandconsistentactivationofmechanosensitive

vagalafferents.Thesegoalswereachievedusinginvivocalciumimagingwiththewell-

knowngeneticallyencodedcalciumindicatorGCaMP3inthenodoseganglion.These

methodsandresultsareelaboratedelsewhere33.However,forthepurposesof

understandingtheexperimentsherein,abriefsummaryisprovidedbelow.

31

Methods

Transgenicmiceusedforimagingexperimentsweregeneratedtoprovideganglion-

wideexpressionofthegeneticallyencodedcalciumindicatorGCaMP3,eitherthroughuse

ofaVglut2-ires-CrealleletodriveCre-dependentGcamp3expressioninallvagalsensory

neurons,orusingamouselineinwhichGcamp3isconstitutivelyexpressedinallcellsin

themousefrombirth.Thenodose/jugular/petrosalganglioncomplexwassurgically

exposed.Theconnectionstothebrainstemweretransected,andconnectionstothe

peripherypreserved.ImagingwasperformedwithaLeicaconfocalmicroscope,andthe

confocalpinholewasreducedtoavoidoverlappingcellsinthez-plane.Laserpowerdid

notexceed90uWtopreventbleachingandtissuedamage.Imageanalysiswasperformed

firstusingFiji;videoswerealignedsuchthatthesameneuronswereinthesamelocation

throughoutallimagingexperiments,neuronROIsweremanuallyselected,theaverage

intensitywithineachROIcalculatedforeachframe,andthesevalueswereexported.

MatlabwasusedtoconvertrawintensitiesintoaΔF/F,whereΔF/F=(intensity–average

baselineintensity)/averagebaselineintensity.

Recordingofneuronactivityofknownmolecularidentitywasachievedbycrossing

mouselinesthatexpressCrerecombinaseinasubsetofnodoseganglioncells,aCre-

dependenttdTomatoreporter,andtheconstitutiveGCaMP3allele.Becauseallcellsexpress

GCaMP3,andtargetedneuronswerevisualizedbytdTomatoexpression,thisenablesa

directcomparisonofresponsesinCre-positiveandCre-negativeneurons.Intriplecross

animals,thewavelengthrangecollectedforGCaMP3imagingwasrestrictedtoprevent

bleed-throughfromthetdTomatochannel.ThisresultedinbaselineGCaMP3fluorescence

intensitymeasurementsthatweresimilarbetweentdTomato-positiveandtdTomato-

negativeneurons(seedissertationthesis).InmyPhDdissertationwork,thisallowedfor

evaluationoftheresponsepropertiesofnodoseneuronsubsetsdefinedbyexpressionof

themarkersGpr65,Glp1r,P2ry1,Npy2r,andMc4r.

Gastricdistensionwasachievedbyeitheroftwomethods:1)inflationofasurgically

implantedlatexballoonaffixedtoasmallrodentfeedingneedleandsyringe,or2)inflation

ofthestomachwithnitrogengas(flowrate3-6mL/min,7-15secofinflation).Micewere

fastedovernightforgastrointestinalexperiments.Respiratorystimuliweredeliveredvia

32

trachealcannulaconnectedtogastanksthatwoulddeliverafixedgasflowrate.

Grossbloodpressuremanipulationswereattemptedbyrapidlyinjecting1-3mLof

lactatedringersintothemousevascularsupplyviafemoralveincatheter(4mice).

However,thisstimulusfailedtoelicitanyresponsesinthemicetested,andwas

abandoned.

Neuronswerecodedasresponsivetogastrointestinalmechanicalstimuliifeitherof

twocriteriaweremet:1)maximalGCaMP3fluorescencewas>sevenstandarddeviations

abovethebaselinemeanduringthestimulus,or2)ifmeanGCaMP3fluorescencewas>

threestandarddeviationsabovebaselinemeanduringtheentirestimulus.Neuronswere

categorizedasresponsivetointroductionofgasesinthelungiftheyexhibitedincreasesin

fluorescenceintensityduringthestimulusatleastthreestandarddeviationsorgreater

abovea30-secondbaselinemeanintensity.

Results Thedetailsoftheresultsprovidedarediscussedindetailinthedissertation

defense,andinpublication33.Briefly,however,invivocalciumimagingallowsforthe

identificationandcharacterizationofsensoryneuronpopulationsinthevagusnerveinthe

livingmouse.Stomachstretchcausesavolume-dependentrecruitmentofapproximately

17%ofallelectricallyresponsivevagalsensoryneurons(Figure1A).Lunginflation

similarlyrecruitedinaflow-dependentmannerapproximately4%ofvagalsensory

neurons,manyofwhichalsoshowedincreasesinactivitywiththerestingmouse

respiratorycycle(Figure1B,C).Thesemechanicallyresponsiveneuronsubsetswere

activatedindependentofthechemicalcompositionofthestimulusused(Figure1D).Inthe

caseofstomachstretch,thesameneuronswereactivatedbydistensionwithliquiddietor

nitrogengasinflation.Inthecaseoflungstretch,thesameneuronswereactivatedby

inflationwithnitrogen,oxygen,orroomair.Thesemechanicallyresponsivesubsetswere

uniquefromeachother,andfromtheneuronpopulationresponsivetochemicalstimuli

appliedinthesmallintestine(Figure1E).

33

Figure1.Invivocalciumimagingofvagalsensoryneurons.(A)Increasingvolumesofstomachdistension(300,600and900μL)causevolume-dependentrecruitmentofavagalsensoryneuronsubsetasmeasuredbyinvivoGCaMP3fluorescenceintensity.(B)Increasingflowrateofgasesintothelung(low0.5L/min,high1L/min)causedependentrecruitmentofavagalsensoryneuronsubset,asmeasuredbyinvivoGCaMP3fluorescence.(C)Manyofthelungstretchsensitivevagalafferentsshowcyclicalchangesinfluorescenceintensityentrainedtotherestingrespiratorycycle.(D)Therecruitmentofstomachandlung-stretchsensitiveneuronsisindependentofthechemicalidentityofthestimulususedinorganstretch.(E)RepresentativeimagesofGCaMP3fluorescencesignalinavagalganglionfollowingtandemapplicationofstomachstretch(red),intestinalglucose(blue),andlunginflation(green).Allscalebars,50µm.ModifiedfromWilliams,etal2016.

34

Finally,usingthetripletransgenicstrategytolabelspecificneuronsubsetswiththe

fluorescentproteintdTomatoallowedfortheidentificationoftheresponsepropertiesof

theseneuronpopulations.NeuronsmarkedbyexpressionofCreunderthecontrolofGlp1r,

orGLP1Rneurons,accountedformost(85%)ofthestomachstretchresponsivecells

(Figure2A).Incontrast,neuronsmarkedbyexpressionofCreunderthecontrolofGpr65

accountedformost(66%)oftheintestinallychemosensitiveneuronsubset(Figure2B).

Conclusions Thedevelopmentofinvivocalciumimaginginthenodoseganglionallowsfor

identificationofgastricandlungmechanosensitiveneuronpopulations,andconcurrent

identificationofmolecularlydefinedneuronsubtypes.Therefore,invivoimagingprovides

anexcellentexperimentalplatformtoinvestigatetheresponsepropertiesofPiezo2-

markedvagalsensoryafferents.

Figure2.Invivocalciumimagingofmolecularlydefinedvagalsensoryneuronsubsets.(A)RepresentativeimagesofGLP1RneuronsandincreasesinfluorescenceofthecalciumindicatorGCaMP3inresponsetofoodintheintestine,andtostomachstretch.Thestomachstretch,butnottheintestinalfoodresponsiveneuronsarecompletelycontainedwithintheGLP1Rneuronsubset.(B)Conversely,alloftheintestinefoodresponsiveneuronsarecontainedwithintheGPR65neuronsubset.Scalebar,50µm.ModifiedfromWilliamsetal,2016.

35

Experiment1:InVivoimagingofPiezo2

lineageneurons

Introduction IfPiezo2isthemechanosensorforinternalmechanicalstimuli,mechanosensitive

vagalafferentsshouldexpressPiezo2.Totestthishypothesis,weusedinvivocalcium

imagingtoexaminetheresponsepropertiesofneuronsthatexpressedaCre-dependent

tdTomatoreporterinPiezo2-Cremice.

Furthermore,theobservationthatselectiveactivationofP2RY1neuronscauses

apnearaisesthepossibilitythatP2RY1neuronsmayalsoplayaroleindetectionoflung

stretch.Insituhybridizationhasshownpartialoverlapbetweenneuronsthatexpress

P2ry1andPiezo277,furthersuggestingthatperhapstherelevantlung-stretchsensitive

populationconsistsoftheneuronsthatexpressbothP2ry1andPiezo2.Therefore,wealso

soughttoexaminethemechanicalsensitivityoftheP2RY1neuronsubsets.

Methods Wegeneratedthetripleknock-inmouselinePiezo2-ires-Cre;lox-tdTomato;Rosa26-

GCaMP3(Piezo2-GCaMP3*),andP2ry1-ires-Cre;lox-tdTomato;Rosa26-GCaMP3(P2ry1-

GCaMP3*),whichallowforaninvivonodoseganglionimagingapproachinvolving

constitutiveexpressionofthegeneticallyencodedcalciumindicatorGCaMP3,andthe

Piezo2-Cre-dependentexpressionoftdTomato.Imaging,stimulusadministration,and

analysiswereperformedasdescribedpreviously.

Results Piezo2-GCaMP3*micedemonstratedexpressionoftdTomatobroadlyinsatellite

glialcellssurroundingnodoseganglionneurons,andalsowithinasubsetofsensory

neurons.Of283electricallyresponsiveneurons(n=3mice),98(34.6%)expressed

36

tdTomato.P2ry1-GCaMP3*micealsodemonstratedexpressionoftdTomatobroadlyin

satelliteglialcellssurroundingnodoseganglionneurons,aninasubsetofneuronsaswell.

Of211electricallyresponsiveneurons(n=3mice),31(14.7%)expressedtdTomato.

InPIEZO2experiments,seventeenneuronswereidentifiedasresponsivetolung

stretchstimuli,aproportionofthetotalganglion(7.2%)consistentwithpriorestimatesof

thesizeofthelung-stretchresponsivepopulation.Ofthese17lungstretchresponsive

neurons,16werepositivefortdTomato(94%).Conversely,16of84(19%)tdTomato-

positiveneuronswereresponsivetolungstretch.Fromthesedataweconcludethatnearly

alllung-stretchresponsiveneuronshaveexpressedPiezo2,andthatlung-stretch

responsiveneuronsaccountforapproximatelyone-fifthofneuronsthathaveexpressed

Piezo2(Figure3A).

P2RY1

1

31

52

(211)

P2RY1 Merge Lung stretch

PIEZO2 1

100 98

(283)

PIEZO2 Merge Lung stretch

A B

Figure3.PIEZO2neurons,butnotP2RY1neuronscontainlungmechanoreceptors.(A)Representativeimagesandtime-resolvedresponsesof100sensoryneurons,(ΔF/F,colorscale)inresponsetolungstretch(greenbar,15seconds).Nearlyalllung-stretchresponsiveneuronsarecontainedwiththePIEZO2neuronpopulation.(B)Representativeimagesandtime-resolvedresponsesof52sensoryneurons,(ΔF/F,colorscale)inresponsetolungstretch(greenbar,15seconds).Noneofthelung-stretchresponsiveneuronsarecontainedwiththeP2RY1neuronpopulation.Scalebars,50µm.

37

Incontrast,inP2RY1experiments,twenty-threeneuronswereidentifiedas

responsivetolungstretch(11%),aproportionofthetotalganglionsomewhathigherthan

priorestimatesofthesizeofthelungstretchpopulation.Ofthese23lungstretch

responsiveneurons,2werepositivefortdTomato(8.6%),thoughtheresponseamplitudes

ofthesetwoneuronsweremarkedlyreducedcomparedtotdTomatonegativeresponsive

neurons.Fromthesedata,weconcludethattheP2RY1neuronpopulationlargelydoesnot

includelung-stretchsensitivecells(Figure3B).

InPIEZO2experiments,thirty-oneneuronswereidentifiedasresponsiveto

stomachstretch,aproportionoftheganglion(13.1%)consistentwithpriorestimatesof

thesizeofthestomach-stretchresponsivepopulation.Ofthese31stomachstretch

responsiveneurons,usingstandardcut-offstodefinerespondersversusnon-responders,

18(52.9%)werepositivefortdTomato(Figure4A).Anindependent-samplest-testwas

conductedtocomparethemeanresponseamplitude,expressedaspercentchangefrom

baseline,duringstomachstretchintdTomatopositiveversustdTomatonegativestomach-

stretchresponsiveneurons.Therewasasignificantdifferenceintheresponseamplitudes

fortdTomatopositive(M=66,SEM=16)andtdTomatonegative(M=11,SEM=5)

neurons;t(29)=2.83,p=0.008.Therefore,thoughbystandarddefinitionsforresponsive

versusnon-responsiveneurons,onlyhalfofstomach-stretchresponsivecellsaretdTomato

positive,thetdTomatopositiveneuronsweremuchmorestronglyresponsivethanthe

tdTomatonegativeneurons.Conversely,18of84(21%)tdTomatopositiveneuronswere

stomachstretchresponsive.Fromthesedataweconcludethatmoststronglyresponsive

stomachstretchsensitiveneuronshaveexpressedPiezo2,andthatstomach-stretch

responsiveneuronsaccountforanotherone-fifthofneuronsthathaveexpressedPiezo2.

38

Finally,thoughoneofthestomachstretchpreparationsfailedinP2RY1

experiments,datawasanalyzedfromtwoofthethreeanimalstested,yieldingatotalof

181electricallyresponsiveneurons.Ofthese181neurons,32(17.7%)wereresponsiveto

stomachstretch,aproportionsimilartopriorestimatesofthesizeofthestomachstretch

sensitivepopulation.Ofthe32stomachstretchresponsiveneurons,4(13%)werepositive

fortdTomato(Figure4B).Fromthesedataweconcludethatthemajorityofstomach

stretchsensitiveneuronshaveneverexpressedP2ry1.

B

PIEZO2

236

84

1

A PIEZO2 Merge Stomach stretch P2RY1 Merge Stomach stretch

P2RY1

181

1

31

Figure4.PIEZO2neurons,butnotP2RY1neuronscontainstomachmechanoreceptors.(A)Representativeimagesandtime-resolvedresponsesof236sensoryneurons,(ΔF/F,colorscale)inresponsetostomachstretch.Nearlyalllung-stretchresponsiveneuronsarecontainedwiththePIEZO2neuronpopulation.(B)Representativeimagesandtime-resolvedresponsesof181sensoryneurons,(ΔF/F,colorscale)inresponsetostomachstretch.Noneofthestomach-stretchresponsiveneuronsarecontainedwiththeP2RY1neuronpopulation.Allscalebars,50µm.Greenbarsundertherasterplotsindicatetimeofapplicationofstretchstimuli(Piezo220and30seconds,P2ry130seconds).

39

Conclusions NeuronsthathaveexpressedPiezo2atsomepointintheirlifetimeencompass

stomachandlungmechano-sensitivepopulations.Incontrast,neuronsthathaveexpressed

P2ry1intheirlifetimearenotresponsivetostomachorlungmechanicalstimuli.These

findingshavetwokeyimplications.

First,thesedatasupportthehypothesisthatPiezo2couldbethemolecular

mechanosensor.Asacorollary,suchaconclusionwouldalsosuggestthattheneurons

themselvescouldbetheprimarymechanosensorbecausetheyaremarkedbyexpressionof

aknownmolecularmechanosensor.Furtherexperimentsarerequired,however,to

demonstratetherelevanceofthePiezo2moleculeitself.Whilesomedataalreadysupport

theroleofPiezo2inlungmechanosensation,nothingisknownaboutPiezo2ingastric

mechanosensation.

Second,thesedatasupporttheconclusionthePiezo2-positive,P2ry1-negative

neuronscontaintherelevantlung-stretchresponsivepopulation.Thisobservationis

importantfortworeasons.First,itisacleardemonstrationthatP2RY1neuronsarenot

responsiblefortheHeringBreuerreflex,beggingthequestionwhatA-fibersthat

selectivelyinnervateneuroepithelialbodiesandwhocaninducecompletemouseapneaare

doing.P2ry1isauniquemolecularhandleonanunanticipatedvagalneuronsubset.

Second,becauselung-stretchresponsiveneuronsonlycoverasubsetofPIEZO2neurons,

additionalgeneticmarkersarerequiredtomorepreciselydefinethelungstretchsubset.

P2ry1canthereforeserveinfutureexperiments,suchasinanalysisofsingle-celldata,to

restrictinvestigationintoPiezo2-positive,P2ry1-negativeneurontypes.Suchexperiments

mightrevealcriticalmarkersormanipulablefeaturesoflung-stretchsensitiveneuron

subsetstobeexploitedinthefuture.

40

Experiment2:Invivoimagingofneurons

thatexpressPiezo2intheadult

IntroductionNeuronsthathaveexpressedPiezo2atsomepointintheirdevelopmentcontain

nearlyallthelungandstomachstretchresponders.However,acriticalshortcomingof

lineage-tracingexperimentsisthattheydonotdistinguishbetweenhistoric,

developmentalexpressionandactivefunctionalexpressionintheadult.ifPiezo2werethe

mechanosensoritself,onewouldalsoadditionallypredictthatmechanosensitiveneurons

expressPiezo2notonlyatsometimeduringdevelopment,butalsothattheyexpress

Piezo2inadulthood.Totestthishypothesisrequiresanexperimentalparadigminwhich

neuronsthatexpressPiezo2inadulthoodareselectivelylabeled,andinwhichtheir

responsepropertiescanberecordedtostretchstimuli.

MethodsWegeneratedthedoubleknock-inmouselinePiezo2-ires-Cre;Rosa26-GCaMP3,and

usedadeno-associatedvirusestodeliverflex-tdTomatovectorstoallnodosesensory

neurons.ThisresultedinexpressionoftdTomatoonlyinneuronsexpressingPiezo2-Creat

thetimeofinjectioninadulthood.Responsepropertiesoflabeledneuronscouldthenbe

recordedusinginvivocalciumimagingasinpriorexperiments.

Givenconcernsabouttheimpactofviralinjectionsonneuronhealthand

responsiveness,thesameexperimentswereperformedusingGlp1r-ires-Cre;Rosa26-

GCaMP3animalsaspotentialpositivecontrols.GLP1Rneuronshadpreviouslybeenshown

toincludethestomach-stretchresponsiveneuronpopulation33.

Adeno-associatedvirusinjectionswereperformedasdescribedpreviously33,77.

Briefly,miceareanesthetized,theleftnodoseganglionsurgicallyexposed,andAAVmixed

withFastGreendyeisinjectedintothebodyoftheganglion.Injectionsareconsidered

grosslysuccessfulwhentheganglionbodyfillswithbluedye.Theincisionintheneckis

41

closedandtheanimalsallowedtorecover.Imagingexperimentswereperformed6-7days

followinginjectionsinhopesofpreservingneuronviabilitywhileachievingreasonable

amountsofinfectionandconstructexpression.

Results InPiezo2animals,631electricallyresponsiveneuronswereanalyzed(n=7

animals).Ofthese631neurons,45weretdTomatopositive,suggestingacombined

infectionandexpressionefficiencyof7.1%.Whenthesameanalysiswasperformedbased

onlyuponmorphologicalidentificationofcellbodiesratherthanonresponsivenessto

electricalstimulation,811neuronswereidentifiedofwhich72weretdTomatopositive

(8.8%efficiency).ThesimilarityoftdTomatoexpressionfrequencyinallneuronsversusall

electricallyresponsiveneuronssuggeststhattdTomatoexpressionwasnotimpairinggross

neuronresponsiveness.

InGlp1ranimals,441electricallyresponsiveneuronswereanalyzed(n=4animals).

Ofthese441neurons,40weretdTomatopositive,suggestingacombinedinfectionand

expressionefficiencyof9.0%.Thisefficiencyisconcordantwiththeobservationthatinsitu

hybridizationforPiezo2andGlp1rinadultmousenodosegangliaresultsinlabeling

neuronalsubsetsofsimilarsize.

WefirstexaminedthedegreeofoverlapbetweentdTomato-expressingneurons

andstomachstretchresponsiveness.InGlp1rexperiments,71neuronsintotalresponded

tostomachstretch.Ofthe40tdTomato-positiveneuronsinGlp1ranimals,18(45%)were

responsivetostomachstretch(Figure5A).AChi-squaredtestforindependenceshowed

thattherewasasignificantrelationshipbetweentdTomatoexpressionandstretch

responsiveness,X2(2,N=441)=27.203,p<0.01(Figure5B).Thisresultisinaccordwith

theearlierobservationthatGLP1Rneuronscontainnearlyallthestomachstretch

responsiveneurons,andthatadultlabelingofGLP1RneuronslabelsnearlyallIGLE-type

endingsinthestomach33.Theseresultsserveasanimportantproofofconceptandpositive

control.

InPiezo2animalexperiments,54ofthe631neuronsanalyzedwereresponsiveto

stomachstretch(8.6%).Ofthesestomachstretchresponders,5(9.2%)weretdTomato

42

positive(Figure5A).Thesedatasuggesttwoobservations.First,asubsetofadultPiezo2-

expressingneuronscanrespondtostomachstretch.However,wewouldfurtherpredict

thatifPiezo2istherelevantstomachmechanosensor,thereshouldbegreater-than-chance

co-occurrenceoftdTomatoexpressionandstomachstretchresponsiveness.Weperformed

aChi-squaredanalysis,andfoundthattherewasnorelationshipbetweenstomachstretch

andadultPiezo2expression,X2(2,N=631)=0.40,p=0.53(Figure5B).Thesedatadonot

supportthehypothesisthatPiezo2istherelevantadultgastrointestinalmechanosensor.

WenextexaminedthedegreeofoverlapbetweentdTomato-expressingneurons

andlungstretchresponsiveness.OnlytwoGlp1ranimalshadlung-stretchstimuli

administered,resultinginanalysisof169electricallyresponsiveneurons.Seventeen

neuronswereresponsivetolungstretch(10%),aresponseratesimilartoprior

experimentalparadigms.NoneoftheseresponsiveneuronsweretdTomatopositive,

consistentwithpriorworkshowingnooverlapbetweenGLP1Rneuronsubsetslabeled

withgeneticlineage-tracingmethods,andlung-stretchresponsivesubsets.

InPiezo2experiments,ofthe631electricallyresponsiveneurons,29were

responsivetolungstretch(4.6%),similartopreviouslyreportedratesoflung-stretch

Figure5.ExpressionofPiezo2inadulthooddoesnotpreferentiallylabelstomachstretchsensitiveneurons.(A)Representativeimagesofneuronresponsestostomachstretch(green)inGlp1r-iresCreAAV-lox-tdTomatoandPiezo2-iresCreAAV-lox-tdTomatoneurons(pink).Scalebar,50µm.(B)PlotoftheobservednumberoftdTomatoandstomachstretchdouble-positiveneurons(PIEZO2,black;GLP1R,red)versustheexpectednumberofneuronscalculatedbasedoninfectionandresponserates(gray).**p<0.01forX2testforindependence.

43

responsiveness.Ofthese29lungstretchresponsiveneurons,3(10.3%)weretdTomato

positive.AChi-squaredanalysisshowedtherewasnorelationshipbetweenlungstretch

responsivenessandtdTomatoexpression,X2(2,N=631)=0.47,p=0.49.Thisresultis

surprisinginlightofthefindingsthatknockoutofPiezo2impactstheHering-Breuerreflex

andwhole-nerverecordingsofthevagusinresponsetolungstretch.

Conclusions TherelationshipbetweenGlp1r-drivenAAV-tdTomatoexpressionandstomach

stretchresponsivenessconfirmsthatstomach-stretchresponsiveneuronsexpressGlp1rin

adulthood.Moreimportantly,thisobservationsuggeststhatviralinfectionandinvivo

imagingcanbecompatibletechniquesinthisparadigm.However,thefindingthatthere

wasnorelationshipbetweenadultPiezo2-driventdTomatoexpressionandstomach

stretchresponsivenesscastsdoubtonthehypothesisthatPiezo2istherelevantmolecular

mechanosensorinstomachstretch.Rather,thisresultmightsuggestthatPiezo2playsan

importantdevelopmentalroleintheformationofIGLEs,butthatitsexpressionisnot

preservedintoadulthoodinthisneuronpopulation.

However,theobservationthatadultPeizo2-driventdTomatoexpressionandlung

stretchresponsivenessarenotrelatedissurprising.KnockoutofPiezo2frombirthinthe

neuronsubsetimagedintheseexperimentsabolisheswhole-nerveresponsestolung

stretch.Itispossiblethataninsufficientnumberofneuronswasqueriedinthisparadigm

torevealarelationship;lung-stretchresponsivecellsaremuchmorerarethatstomach-

stretchresponders.Despitemultipleimagingpreparations,thisresultdependsheavilyon

datafromlessthan30neurons.Furthermore,therobust-nessofneuronpopulationsto

viralinfectionmaydifferdependingonneurontype,makingthisexperimentsuitableto

examinestomach–stretch,butnotlung-stretchsensitivesubsets.

Alternatively,iflung-stretchresponsiveneuronstrulydonotexpressPiezo2in

adulthood,itwouldsuggestthatPiezo2intheneuronsthemselvesisnotcriticalforlung

mechanosensation.PerhapstheexpressionofPiezo2innon-neuronalelements,suchits

expressioninthenodosesatellitecellsorintargetorgans,issufficienttoconfer

mechanosensitivity.DriversusedtoknockoutPiezo2inpriorexperimentsarenotfully

44

nodose-neuronspecific.Similarly,anadditionalalternativeexplanationisthatPiezo2plays

animportantdevelopmentalroleinlungstretchsensitiveafferentsand/ortheir

surroundingstructuressuchthatitslosswouldconfermechano-insensitivitywithout

Piezo2beingtheactualmechanoreceptor.

Experiment3:Detectionofstretchstimuliin

Piezo2knock-outganglia

Introduction ThehypothesisthatPiezo2actsasthemechanosensorinvagalsensoryafferents

furthermoresuggeststwoadditionalpredictions.First,lossofexpressionofPiezo2should

resultinthelossofmechanicallyevokedresponses.Second,iftheneuronsarethedirect

mechanosensors,theselectivelossofPiezo2intheneuronsthemselvesbutnotinother

tissuesshouldbesufficienttoabolishresponses.Totestthesepredictionsrequiresan

experimentalparadigminwhichPiezo2expressionisdisruptedselectivelyinthenodose

ganglionneurons,andinwhichresponsepropertiescanstillberecorded.

Methods Usingaseriesofmousecrosses,wegeneratedPiezo2fl/fl-GCaMP3*,Piezo2fl/wt-

GCaMP3*,andPiezo2wt/wt-GCaMP3*animals.Anadeno-associatedviruscontainingaCAG-

Cre-mCherryconstructwaspurchasedfromSignaGenlaboratories(AAV1-CAG-Cre-

mCherry,SL101117).InfectionwiththisvectorisintendedtoinducetheexpressionofaCre

recombinaseandmCherryfusionalleletogeneraterednuclearfluorescenceinevery

neuronalsoexpressingCre.ExpressionofCrerecombinaseincellscontainingafloxed

Piezo2allelewouldresultingeneticknockoutofPiezo2.Imagingexperimentswere

45

performed14-21daysafterAAVinjectionsinhopesofprovidingsufficienttimefor

knockdownofPiezo2expression.SuccessfulexpressionofCre,andexcisionofthePiezo2

allelewereconfirmedusingPCRofgangliacollectedaftercompletionofimaging

experiments.

Results TwotothreeweeksfollowinginjectionofAAV1-CAG-Cre-mCherry,nodoseganglia

demonstratedwidespreadandrobustexpressionoftdTomatointheinfectedganglion,but

notintheuninfectedcontralateralganglion.Theredfluorescencewaslocalizedtothecell

nucleus,concordantwiththefluorescentproteinbeingtaggedtonucleus-localizedCre

recombinase.Inthefiveinjectedanimals,278electricallyresponsiveneuronswere

analyzed,ofwhich186(67%)containedamCherry-positivenucleus,suggestinghigh

infectionefficiencyintheseexperiments(Figure6A).PCRofganglionDNAextractedafter

imagingexperimentsshowedpositivereactionsforCrerecombinasefrominfected,butnot

fromuninfectedganglia.Furthermore,PCRreactionsspecificforthesequenceofthe

knockoutPiezo2allelewerepositiveonlyininfectedgangliafrommicecarryingatleast

onefloxedPiezo2allele(Figure6B).TheseresultssuggestnotonlythatCrewas

successfullyexpressed,butalsothatitwasfunctionalandexcisedthefloxedregionof

Piezo2.

46

Figure6.Validationofneuron-specific,adultdeletionofPiezo2.(A)Fluorescentmicroscopyimageofthenodoseganglion14-21daysfollowinginfectionwithAAV1-CAG-Cre-mCherry.showswidespreadexpressionofnuclearmCherry.Scalebar,200µm.ConfocalimagesofneuronswithintheganglionshownuclearmCherrylocalizationScalebarinsets50µm.(B)PolymerasechainreactiondemonstratestheexpressionofCrerecombinaseininfectedbutnotuninfectedganglia,andexcisionofexons43-45ofPiezo2onlyininjectedgangliaofanimalswithafloxedallele.Scalebar,50µm.

47

Offiveanimalssuccessfullytested,twowerePiezo2fl/wt,meaningthatPiezo2

expressionfromonealleleshouldalwaysbepreserveddespiteexpressionofCre.Inthese

animals,107electricallyresponsiveneuronswereanalyzed.Ofthese107neurons,7

(6.5%)wereresponsivetolungstretch,5ofwhichweremCherrypositiveand2ofwhich

weremCherrynegative.Furthermore,19(17.8%)werestomachstretchresponsive,of

which14weremCherrypositiveand5weremCherrynegative.Therefore,Creexpression

didnotabolishmechanosensitiveresponsesinnodosesensoryneurons(Figure7A).Chi-

squaredanalysisshowedtherewasnorelationshipbetweenlungstretchresponsiveness

andtdTomatoexpression,X2(2,N=107)=0.90,p=0.34,norbetweenstomachstretch

responsivenessandmCherryexpressioninanimals,X2(2,N=107)=1.88,p=0.17.These

datasuggestthatinjectionandconstructexpressionitselfdoesnotimpactmechanical

sensitivityofvagalsensoryneurons.

TheremainingthreeanimalstestedwerePiezo2fl/flanimals,meaningthatneurons

expressingmCherryintheseanimalsalsohaveexcisionofbothcopiesofthefloxedPiezo2

geneticsequence.Intheseanimals,171electricallyresponsiveneuronswereanalyzed.Of

these171neurons,25(14%)werestomach-stretchresponsive,ofwhich14weremCherry

positiveand11weremCherrynegative.Furthermore,4(2.3%)werelungstretch

responsive,ofwhich2weremCherrypositiveand2weremCherrynegative.Therefore,

evenwhenbothPiezo2alleleswerefloxed,neuronsexpressingCrerecombinase

maintainedtheirabilitytorespondtomechanicalstimuli(Figure7A).AChi-squared

analysisshowedtherewasnorelationshipbetweenstomachstretchresponsivenessand

mCherryexpressioninPiezo2-floxedanimals,X2(2,N=171)=0.0002,p=0.98,nor

betweenlungstretchresponsivenessandmCherryexpression,X2(2,N=171)=0.07,p=

0.80.Similarly,therewasnoattenuationinstomachstretchresponseamplitudein

mCherry-positiveneuronsinPiezo2fl/flanimals(Figure7B).Rather,mCherry-positive

neuronshadaslightlygreaterresponseamplitude(M=2.1,SD=1.3)thanmCherry-

negativeneurons(M=1.0,SD=1.02),t(22)=0.481,p=0.03.Toofewlungstretch

responsiveneuronswerepresentinthesepreparationsforrigorousstatisticalanalysisof

responseamplitudes,particularlygiventhesmallnumberofanimalstested.

48

Conclusions ThelackofrelationshipbetweenPiezo2excisionandeitherstomachorlungstretch

castsfurtherdoubtontheroleofPiezo2onlungandstomachmechanosensationinvagal

sensoryafferents.However,onetechnicalpointlimitsenthusiasmforthestrengthof

conclusionstobedrawninthisexperiment.WhilegenomicexcisionofthePiezo2exons

couldbeconfirmedgrosslybywhole-ganglionPCR,thisvalidationprovidesonlyaproxy

indicationthatproteinlevelsofPiezo2arealteredusingthisexperimentalmanipulation.

Forexample,thePiezo2knockout-specificPCRreactioncouldbepositiveincasesof

excisionofthefloxedPiezo2regioninonlyanincompletesubsetofneurons.Alternatively,

Figure7.Neuron-specific,adultdeletionofPiezo2doesnotabolishmechanicalresponses.(A)RepresentativeimagesofpreservedstomachandlungstretchresponsesinmCherry-positiveandnegativeneuronsinPiezo2fl/flandPiezo2fl/wtanimals.Scalebar,50µm.(B)Responseamplitudesforallstomachstretch(left)andlungstretch(right)responsiveneuronsinPiezo2fl/flanimals.TracesfromneuronsthatexpressmCherryareinred,whiletracesfromneuronsthatdonotexpressmCherryareinblack.

49

evenifknockoutwerecompleteatthegenomiclevel,thedurabilityofexpressedPiezo2

proteinisunknown,andmayextendbeyondthe2-3weeksbetweeninfectionandimaging

utilizedinthisexperimentalparadigm.However,intriguingly,theonlyotherexperimentto

knockdownPiezo2inadultanimalsgeneratedreductionsinskinsensitivitywithinone

weekofinductionofgenomicexcisionofthefloxedregionofPiezo269,suggestingthe2-3

weekwindowusedintheseexperimentsshouldbesufficient.Unfortunately,toolsto

evaluateproteinexpression,andspecificallyproteinexpressionatdistalsensoryterminals

withintargetorgans,arelacking.Interpretationofresultsmustthereforenecessarilybe

constrainedwiththisshortcominginmind.Theabsenceofarelationshipisanegative

resultthatcouldreflecttruebiology,oratechnicalhurdle.

WithincompletevalidationofPiezo2proteinexpressionknockdowninmindasa

prominenttechnicalshort-comingfordatainterpretation,thesedataarenotconcordant

withthehypothesisthatvagalsensoryneuronPiezo2isthesoleandnecessary

mechanosensorineitherstomachorlungstretch.Thisconclusionagreeswiththelackof

relationshipbetweentheadultexpressionofPiezo2andstomachstretchinprior

experiments,supportingthenotionthatPiezo2doesnotplayaroleintheadultgastric

mechanosensitiveneuronpopulation.

ThelackofrelationshipbetweenneuronalPiezo2knockoutandlungstretch

responsiveness,however,issurprising.ThesinglepriorexperimenttosuggestPiezo2

knockoutinadultsaltersrespiratorydynamicsandattenuatestheHeringBreuerreflex

usedatamoxifen-dependentAdvillin-CredrivertodeletePiezo2fromvagalanddorsalroot

ganglionneurons69.Theknockoutmicehad30%largertidalvolumes,andreachedpeak

expiratoryflowfasterthanwild-typecontrols.Theseanimalsalsohadlowerwholevagal

nerveactivityduringlunginflation.Onedifferencebetweenthisexperimentandvirally

mediatedknockdownisthatAdvillin-CrealsodrivesPiezo2knockoutoutsidethevagus

(e.g.inthedorsalrootganglia),whichcouldimpactrespiratorydynamics.However,this

wouldnotdirectlyexplainlowervagalwhole-nerverecordinglungstretchresponse

amplitudes.Inadditiontotamoxifen-inducedAdvillin-Cre-drivenPiezo2knockout,the

driverPhox2bwasusedtodeletePiezo2frombirthinnodoseneuronsandglia.Knockout

frombirthresultedinabolitionoftheHeringBreuerreflexandvagusnerveresponsesto

lunginflation.However,experimentsthatknockoutagenefrombirthcannotdistinguish

50

betweendevelopmentalandadultimpactofgeneloss.Therefore,thepossibilityremains

that,asinMerkelcellsintheskin,non-vagalPiezo2expressionplaysanimportantrolein

Piezomechanotransduction,andthatknockoutspecificallyinadultvagalsensoryneurons

isinsufficienttoabolishlungstretchmechanosensitivity.

SummaryDetectionofmechanicalforceswithinthebodyplaysacriticalroleinregulationof

organismphysiology.WeinvestigatedwhetherPiezo2,arecentlyidentifiedmammalian

mechanosensor,couldplayaroleindetectionofinternalmechanosensorycues.Wefound

thatlineagetracingofneuronsthathaveexpressedPiezo2labeledthelargemajorityof

lungandstomachmechanosensitiveneuronpopulationsinthenodoseganglionofthe

vagusnerve.However,neuronsthatexpressedPiezo2inadulthooddidnotpreferentially

labelmechanosensitivepopulations.Inaddition,adultandnodoseneuronspecificPiezo2

knockoutfailedtoabolishmechanosensitiveresponsesinnodosesensoryneurons,though

conclusionsfromthisexperimentarelimitedbytechnicalconcerns.Thesedatain

aggregatesuggestPiezo2maynotbethesolemechanosensorwithininternalsensory

systems,andfurthermoreraisethepossibilitythatextra-neuralPiezo2couldplayan

importantroleinthesesystemsasitdoesintheskin,orthatPiezo2playsanimportant

developmentalrole.Furtherexperimentationsuchas1)usingalternativemethodsto

knockoutPiezo2fromvagalsensoryneuronsandassociatedtissues,2)evaluationofadult

PIEZO2neuronanatomyintheperiphery,and3)investigationintotheroleofPiezo2inthe

cardiovascularsystem,wouldshedintriguinglightontotheroleofPIEZO2neurons,and

theroleofPiezo2expressionininternalmechanosensation.

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Advisor: Dr. Stephen D. Liberles Erika Kristen Williams

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