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PREMOTONEURONAL ORGANISATION OF SWAlLOWING IN 1HE RAT

By eMirAliHashim.M.Sc.

A thesis submitted10theSchoolofGradua teStudies inpartialfu1fiImeDtof' thefalWremenu for

the degreeof DoctorofPhilosophy

FacultyofMedicine MemorialUnivcnityofNewfoundland

Sept~ber1989

Newfou ndland

To my grandmother(AmmiJoan) and

Myparents(AbbiandBaji)

ABSTRACT

Theoverall aimof this researchwasto examine the role of the neuronal soliterialnetworkinthe neural organization of swallowing.The specific objectives were :1)to map deglutitivcpremolar loci withinthe nucleus tractussolitarii(NTS), 2)to examinetheir pharmacologicalcharacteristicsand 3) trace theirintrabulbar connections. Experimentsweredone on anaesthetised ra ts utilising techniques which includedmicropneumophoresis,neuronaltracingand immunocytochemistry.

Deg.lutitive loci were mappedbylocal applicationsof glutamate;the pharmacology of glutamate receptor subtypes involvedinthe various components of swallowing wereexamined;and both anterogradeandretrograde tracing techniques were employed

The resultsindicate that, within the NlS,pharyngeal lociextend fromthe levelof the obex to 900 um roslrallyoverlappingthe subnuclei ventralis and intermedialis; bothcervical and distal oesophageal lociarecoextensivewiththe subnucleus centraUs of the NTS.

Based on a comparisonof excitatory amino acid agonistpotency andthe effects of N-me1hyl-O-aspartate(NMOA) -selective and non-selectiveantagon istson glutamate -evoked responses, kainate (KA) recep tors were foundlinked to pharyngea lsiteswhereasNMDA receptor swereprefer entiall yassociat edwiththe subnucleuscentralis. Under physiologicalconditions,the responseto NMDA

iii receplor-activatlon in the subnucleuscenlralisappears tobedependenl inpan on anintact cholinergicinput directedatmuscariniccholinoceptors.In contrast,the presumptiveKA receptor-mediatedresponsesat solitarialdeglutitive loci appear independent of an excitatoryserotoninergicinputinsofar as theinvolve menl of excitatory $.HTz and/orS-Hf,creceptorsisconcerned. The $ominput tothe pha1yngealterritoryof thesolitarycomplex originates.atleastinpart,from cells inthe raphe obscures, magnusand pontis nuclei.

Anterogradefibre tracingbythe uscofPlJastolUJlJUIgorisleucoegglutinln revealed dense projectionsfrom the deglutitiveloci tothe pharyngo- and oesophagomolorponon sof the nucleus ambiguus. In addition, solitarial el1erenu.

displayed adistinct pattern ofprojectiontoother motoneuronalpoolscontrolling the musclesof deglutition

va.,

thehypoglossal,the facialanditsaccessory nucleus, the motortrigeminal anditsacussory nucleusand thedorsal vagal motornucleus.

Thus,thephann acological datatogether with theneuroanatomicalevidence of directneuralprojectionsfromdeglutitivclociin thesolital)' complexto themotor nuclei involvedinswallowing lend strong credence 10 thehypothesizedrole of solitarial intemeuroraas generatorsof the deglutitivemotor pattern.

KEY WORDS: SwallowingDeglutition Nucleus tractussolitarii Glutamate NMDA receptors Acetylcholine Serotonin Nucleus ambiguus Premotorneurons

ACKNOWLEDGEMENTS

Iamindebted to mymento r,Dr.Delle!Bicger,forhishelpful scientific discussions and forsuppo rtandencourageme ntin matters bothacademic andnon- academic.

Iamgrateful toDr.RichardS.Neuman forhis amnselandencouragement lhroughOUI my graduateprogramand especially during 1985-1986 asmyacting supervisor.

My heart feltthanksto Dr. Christop herR.Triggle. AssociateDean ofBasic Medical Sciences,forhisrecommendat ion andconsistentsuppo rtin mattersboth financial and moral throughout my graduate program; and for providing transport ation on coldwinter nightsduring1985-1987.

Iamgrateful10Dr.CharlesW.Malsburyfor his counsel as amember of my supervisory committee;and to Dr.D.G.Bryant for statisticaladvice.

Jowemygratitude:

to Dr.Oevhuti Vyas.,Motlur1ndi4,forhergeneroushelpandconstant support throughoutmygraduateprogram and (or herexpert adviceandguidance in immunocytochemical techniques;and

10Mrs.MatteBregerforher kindness.suppon and constantencouragement.

1amvery grateful to Dr.ChesterJ.Michalski, Assistent Dean ofGraduate Studies.FacultyofMedicine. forprovidingbothfinancialand moral suppon

throughout mygraduat e program;and tothepatronsaintof medicalgrad uate studen ts.Mrs.BernadettePower,GraduateStudies, FacultyofMedicine.forher ready help and assistance in all administrative matterspenainingtograduatework and forprovidingfinancialasweDasmoral support.

Iam gratefulto:

Mrs.JanelRobinson for skIllfultech nical assistance;

MB.KimberleyRossfor helpwithwordprocessing and invaluable support insecretarialandadministr ativemailers;

Mrs.Betty GranterandMrs.Therese Thorneforesststaneeinsecret arial andadministrativeworle;

the personnel of Medical Audio-VisualServices, especially Mr. John Crowell andMr.TerryUpshaUfor theirpalience and perseveranceinthedevelop ment and printing ofphotograp hicfilm:Mrs.Sylvia Fickenforherskillful artwork;and Mr.

EugeneRyanforadviceon colourphotogra phy;

the personnel of ComputingServices, Medicine,especiallyMr.S.ScottSmith andMr.RobertG.Ryan.

The Schoolof GraduateStudiesand theFacultyofMedicinefor providing financial support.

Aspecialthanksto all myfriendsand colleaguesin Medicine andespecially to tbe membersofthe Centr e forCataposco logy.

This work woutd not havebeenpossible withoutthelove and support of myuncle.,Mr.SyedAliZaidi(AliMomu)andmysister,Mrs.Abeda Reza(Bibi) to whomIremain foreverindebted

Last.but not least.itiswithinfinilegratitude thatIacknowledge the emotionalsupport providedbyHuma,whichgaveme thestrength and perseverance to realizemygoals.

TABLE OF CONTENTS

ABSTRACf•••• •••.•• •••. .•• • •••• • ••. • .•••• •••• •• .. .• ii

ACKNO\VLEDGEMENTS ••••• •••••••• • • •• ••• ••• ••••••••• tv

TABLE OF CClNTENTS •••• • • •• ••• ••••••••••• •.••..•••••vii

usr

^{OF FIGURES •••.. •••. . .••.• .• • ••••...• • • •••.• •. xil}

UST OFTABLES ••••. •••••. . ..•• ••. •••.•••••• .••.••• xvii

usr

OF ABBREVIATIONS••.••..• ••.••••••••• •. . ..•• • • •xvili

lIm<ODucnoN .

I.Swallowing·an overview •..••, ••• •••.••••• •...•...

II.Neural control ofswallowing•••••..• ••.•.. •• • ••• •...

1.The efferent limb.. . .••• • ••...••••.•.•• •.• • • S 2.Theafferentlimb •• •••••••••• ••• • •••••• ••••• 11 3.The organisational stage ••••• .. •• • •••••. ..•••• 13 lItPbanna cology ofswallowing••••••• • ••••• • • •. ..• •• •• 18 IV.Objectives•. •••••• ••. ..•• • •• •••.• ••• •• • • • • • • • 20

MATERlALS ANDMETHODS •.•...• ..•• .••. . .•••••...•. 23 1. Experimentalprocedures• .••.• ••.• . •. •. .• •••••. • • •• 23 II.Mapping•• ••••••• • • • •••• •••• ••••••• •••..• • • .• 29

viii HI.Phanna cology•• ••...•••••• ••• • •• •• •. •••••• ••.. 30 I. Micropnc umoph oretic applicationof drugs••••••••••• 30 2.Top ical applicationofdrugs•• ••••_ •••.• . •• •• • •• 31 3.Electrical stimulat ion..•• •..••••.•••••.•• •.••• 31 IV.NeuronalTra cing •...•••••••••. ••••••..•• ••• •.. 32 V.Immunocytoche mistry •...• ••. .• • • •.•.••• ••••••••. 36 1.Peroxidase-antiperoxidasemethod(or PHAL .••••• ••• 36 2. Immunon uoresce nce techniquefor5-HT••••••. .•• •• 38 VLChemicalsanddrug solutions•• •• • • ••••••• • • •••• •.•• 40

RESULTS... .• • ••• •• •..••••••• • ••••• •••••••••.••• 42 I.Characterisa tionofthesolitarialdeglutitiveloci••• ••••.• • •• 42 1.Characteristics of evokedrespo nses ••••••• ••••••.• 42 A.Pharyngealrespo nses•• •.• •••••..••••••.• 42 B.Oesophagealresponse s•• .••••• ••. .•.••.•• 45 C.Complet e swallowrespon ses •••••• ....• • . •• 49 2.Resp onsiveloci •••••••.•. .•• ••• • • • •••• • . •• . 49 3.Non-responsiveloci•• • • • • ..••••....• •••. •. •.. 62 II.Pharmacologicobserva tions atsolitarialdeglutitivc loci••••..• 63 1.Effectsofexcita tory amino acidreceptor agents••••... 63 A.Excitato ryamino acidreceptorantagonists •• •••. 63 i)Pharyngeal responses •••• •..••.•.••.. 63 ii)Oesophagea lrespo nses ••• • ••• • • • • • • • 63 iii)Completeswallowrespo nses ••••••••.• 68

a) CSresponses evoked Oyglutam ate microinjeetionsintotheNTS ••.• 68 b) CS respo nsesevoked bysystemic

quipazine •.• ••.••••.••••. 68 c)Pharyngo-oesophageal respo nsesevoked

byelectrical stimulation of the superiorlaryng ealnerve .•••... 72 B.Excitat oryamino acidreceptoragonists .•...•.• 76 i)Pharyngealand CSresponses •• ••••. .•• 76

illOesophagea l respo nses •. ...•. .••...• 80 iii)Glutama tevenusACb (muscarine) •••... 80

2.EffectsofS-}ITreceptoragents •••..•••.. .• • .••• 91 A Ouipazlne-inducedswallowing.• • .•• • •••• ... 91 B.S-HTvenusglutamate ••• ••• ••••••• •••... 96 i)Elicitationof pharyngeal respo nsesbyS-HT. 96 iJ) Effect of 5-HI' on glutamBte-cvokcd

pharyngeal responses • _ • __ • • •••• •• 101 iii) Effectof chemical microstimulationof the

medullary raphe nuclei•••••••• •••• lOS tv)Oesophagealresponses •.• • •• . •• •. .•• lOB v)CSresponses.•. .•• ••••• • • . •.•. . .• 111 til.Neuralconnectionsofsolitarial deglutitivekxi... ... .•._.. 112

I. Retrograde Iracing co mbine d with S·HT immullOC)'tochemislly••.•.. ••.• . •__.•• ••• •• 112 A Injectionsites ••• • ••. •. ••• •... ••••• • • 112 B.Retr ogradely labeledperikarya•.••.. ••••• •••115 C.S-}IT immunoreactivity.•••• • . •••..• • •••.. 115 D.Retrogradely-labeled S·}IT· IRperikarya •••• •••118 2. AnlerogradeTracing. .• •••• •• •••••• • .••••• • •. 121 AInjectionsites ••• •• •••••• •• ••. .•.•.•• •• J21 B.Efferentsfrompharyngealloci ••• • •••• • • ..•• J26 i)The nucleus ambiguus ••• • • • ••••••... J26 ii)The trigeminal motor nucleus •••... J31 iii)Thefacial nuclea rcomplex •• •••. ....• J32 iv)The hypoglossal nucleus ••• ••••. ..•.•132 v)The dorsal vagalmotor nucleus• ••••.••• J31 C. Effercnts fromoesophageal loci• • •••••••. .•• J40 i)The nucleusambiguus.• ••••••••••••• 140 0) The dorsalvagal motornucleus'.•••••••. 143 iii)Other motornuclei ••:... ... . .. .... 143 D.EfferentsfromCSloci•.••••••• ••••• ••..• 146 E. Scluarial projections 10 other pontomeduJlary

regions •• • • • •.••••• •••• •. .• ••• • • • • 146 i)TheNTS• ••••••• •••• •••••••• •••• 153 ii) Nuclei of thespinal trigeminaltract,the

paratrigeminal islands, the principal sensory trigeminal nucleus and the parabrachialcomplex•••••••..•. .• JS3 iii)"Lacrimarneurons •.~•• ••. ..••. .•.• 163 tv)TheinferiorOlivarycomplex •• • •.• .••. J63

F. Unlabeledpontomedullaryregions ••..• • ••~•• 163 G.Controls ••• • ••. . . .••• • ••••••••• •. . . . 166

DISCUSSION•• ••..•• .•• .•...•• .•• . .... .. • •...•.• 169 1. Topography and functional characteristics of solitarial deglutitive

loci •• • •.•• •• ••••••••• ••• •••••...• ••••••••169 1. Responsetypes ••. ..•• •• • ••.•.•.••.. ..•.• •• 169 2.Topography of the solita rial deglutitive loci•• ... ..•.• 171 II.Pharmacologic characterisation of deglutuiveresponse types .•• 178 1. &citatory amino acids•... .... . . .••...•.• .• ••. 178 2.Serotoninergicmechanisms. . .••••• ••..••••••. .. 181 3. Interactio n of GluuunatergicandCholinergiemechanisms. 188 III. Neuralconnectionsof the NTS .•..•...• • • •..•. .••• . 192 1.Methodological considerations.• • ••.• •• •. .... .. •• 192

A Retrograde tracing andS·HTimmunocytochemistry experiments .• ... . . . .•••• ••• ..• •• ••. 192 B. Anterograde tracing experiments •.•• ••. . .•. • 193 2.Serotoninergic afferents to solitarial deglutitive loci... •• 196 3.Efferentsof the NTS •••• •• •• • .. .••••• .. .•••• 201

A Sotitarialdeglutitive efferents to motoneurona l

pools 201

i)The nucleus ambiguus ••• . . .•••... .•• 201 ii)The trigeminalmotor nucleus •.• .•••• • • 204 iii)Thefacialnuclea r complex •• •.•• • • ••. 206 Iv)The hypoglossal nucleus .• • •••••••.. . 201 v) The dorsal motor nucleusofthe vagus .••• 211 B. Solitarial efferents to other pontomedullary

structures ••• •. • • •.•••.•• •.. .•••• •. 213 i) The solitarycomplex ••.• •• •.. ....••. 213 ii) Nuclei of thespinaltrigeminaltract, the

principal trigeminalsensorynucleus, the paratrigeminal islands and the parabrachial

; ;;) "La~~:!le:~u;;';,; ::::::::: :::::::: ~:~

C.Unlabeled pontomedullary regions ;. ... . . .. 218 i)Themedullaryreticular formation 218

il)"Thepontinereticularformation.... . .. 219

D.Proposedcircuitmodel 220

SUMMARYAND CONCLUSIONS.• • •. • ••• ••••.•••••••••• • 224

Future studies 227

LITERATURE CITED •••• • ••• •••• • ••••••• ••. .•• • • • • •• • 229

Figure4 FigureS Figure1 Figure2 FigureJ

xii LIST OF FIGURFS

Pharyngealresponse evokedbjglutamate•••• •• •• ••••• 43 Oesophagealresponsesevokedbyglutamate andACh •• •. 46 Rhythmic oesophageal responses resembling secondary peristalsisevokedbymuscarine•.•••.• ••••• •••••••• 48 Completeswallow clichedbypressureejection DC glutamate . SO Afluorochrome-markedpharyngeal locus inthe rat nucleus tractus solitarii••••• ••••••••.• ..•.•.•. .••.•.•• 52 Figure6 A fluorochrome-markedcervicaloesophageal siteintherat

nucleus tractus solitarii •••••••. ••••• ••••.•• •• •• • 54 Figure7 Auorochrome-markedCSand distal oesophageallociin the

rat nucleus tractussolitarii•••••• • ••. •. • •.•.•.••" 56 Figure8

figure9 Figure 10

Figure11 Figure12 Figure 13

Auorochrom e-mackeddeglutitiveand non-dcglutitivesites •• 58 Map ofdcglut itive loci••••• •••• •• •.••••• ••••• •• 60 Effeeu ofexcitatoryaminoacidreceptor antagonistsat a pharyngealNTS site•• •• • •••••.••• •••• •••• ••• •. 6S Effect ofNMDA receptorblockadeat anoesophagealsite . 66 ECfect ofNMDAreceptorblockade at8CS site 69 Effect of MK-801 on quipazine-induccd automatic swallowing.•• •..•..•.••••••..•.••...••• • • •• 70

xiii Figure 14 Blockadeby OGG of quipazine -inducedswallowing .•• • •• 73 Figure15 Selective blockadebyMK-801of oesophageal componentsof

reflex swallowing••••• ••••••••••.••••••• • • • • •• 75 Figure16 A:.Pote ncy compariso n of excitat oryamino acid agonistsata

pha.r)ngealsite •.• • • •••• • ••••••••••••• • ••••••

n

B:Extentof pha ryngo-oeso phageal progressionof a CS response evoked by pressure ejectionofthethree EAA agonists•••• •.•.. . . .•• •.••••••.• •..• .• ..• • • 77 Figure17 Potencycomparisonof excitat oryaminoacid agonists at an

oesophagealsite .•• • •. • ••• •• • • • •••••• • • •••••• 82 Figure18 Enhancem entof a glutam ate-evo ked oesophagea l responseby

ACh 83

Figure 19 Enhancementofaglutamate-evoked oesophagealresponseby muscarine•• • • • • • ••••.• • • • • ••••••• ••••.• • ••• 84 Figure20 Inhibition of aglueamate-evcked oesophageal responseby

rnethscopolamine•••• •• •••••••••••••••••••• • •• 87 Figure 21 Effect of muscarine receptor blockade on a propulsive

oesophageal response evokedbyglutamate ••••.•••• ••. 89 Figure22 Spontaneousoesophagealcontractions evokedby intravenou s

physostigJTIine ••••• •. ••.•• ••.• ••.•••••• •...•• 92

Figure24 Figure2S Figure 26 Figure27

Figure23 Blockade of quipazine-induced automatic swallowing by ketanserin• • ••• ••••• •••• • • ••••••• •• ••• ••.••• 93 Inhibition of quipazinc:-inducedswallowingby8-OH·DPAT•• 94 Elicitationofpharyngealresponsesby's-HT ••••• • • • ••• 97 Selectivetachyphylaxisof5-HT-evokedresponses ••.•• ••..99 Enhancemenl of5-HT·andglutamate-evokedpharyngeal responsesby syste mic quipazine ..•••• •• •.•••. • ••.• 102 Figure28 Faeilitetion ofaglutamate -evoked pharyngealresponseby

5·HT••••••• • • •• . •••.••.•• • ••. .••. .• ••.• .. 104 Figure29

Figure30 Figure31 Figure32

Figure33

Figure34

Figure35

Selective blockadebyketanseri noffacilitationby5·HT ofa gluta mate-evokedpharyngeal response •• • • •••• •• •• •• • 105 Effect ofS-HTon oesophagealandCS res ponses •••• •• • 109 F1uorogoldinjection site ••• •• • ••••• ••.••. ••••• •• 113 Retrogradely-labeled 5-HT-immunoreadive and immunoreact ive penl:aryaintheraphe obscurusnucleus••• 116 Retrogradely-labeled5-Hr·immunorea etiveperikaryainlhe raphemagnusnucleus.•••.••• • ••.• • •.••• •• •• • •• 119 Gfuremete -responslvepharyngealand oesoph ageal sites of application ofPHAL inthe rat solitary complex •..••.• • 122 A glutam alerespons iveCSsileof applicationof PHAL in the rat SOlitarycomp lex•• •• •..••• ••••. • •. •• • ••• •• • 124

Figure 39 Figure38 Figure")6 Figure37

Photomicrographs showinglectin-filled neurons•••• ••••" 127 Projections10nucleus ambiguus from asolitarial pharyngeal site•.•• ••• •.••.• •••••• ••••. .••• •••••• •••• 129 Projectionsto the facial nucleusfromasoJitaria] pharyngeal site•• • •••• •••••••••• ••••••.• • • • •.••• •. • •• 133 Projections to the hypoglossalnucleus from asclitarial pharyngealsite ••••.••••• •• •••••• •••.. .••.•.• 135 Figure40 Projectio ns to thedorsal motor nucleus ofthe vagusfrom a

solitarial pharyngealsite •••.•• • ••••••••••••••• • • 138 Figure41 Projections to the nucleus ambiguus from a solitarial

oesophageal site •• •••••• •• •••••••••• . ••••• •• • 141 Figure42 Projectionstothe dorsalmalarnucleusof thevagusfrom a

solitarialoesophagealsite •••••••• •••••••••••• ••• 144 Figure43 Projectionstothe nucleus ambiguusfromasolitarialCS

site••••••• •• •••• • •.•••••••••••• •••. •• ••••147 Figure 44 Projectionstothe nucleusambiguusfroma solilarial CS

site••• • •• ••• • •••• ••• •• ••••.•••• •..•••• ••• 149 Figure 45 Projectionsto the accessorylrigeminal motornucleusfrom a

soliterialCS site ••••••••• ••••••• •••••• • •.• •.•151 Figure 46 Projectionsto the subnucleus centralisfrom a solitarial

pharyngealsite •• ••• •• ••• •.• • • ••• • •• . •••••. .• 154

Figure47 ProjectionstotherostrolateralN1Sfromasolitarial pha()'Ilgeal site•• • •••• •••• •••.••••• ••••••••.•.••••••• 156 Figure48 Projectionsto the contralateralNTS ('mirror-image')froma

sclitarialoesophagealsite .•..••. . •• ••.. .•••• •• • •158 Figure49 Projectionsto the parabrachial complex from asolitarial

oesophagealsite •••.•••.••••..••.. ..•••• .•••. 161 Figure 50 Projections to the"lacrimal"neurons from a pharyngeal

solitarialsite••..••..•...•.•••• •••••••••.•••. 164 Figure 51 PHAL Control ... ..•.• .•. ... .. •••...••. 167 Figure52 Summary diagramofsolitarialdeglutitive efferents•• .•• • • 221

xvii LIST OFTABLES

TABLE I Muscles panicipating indeglutition and their sources of innervationintheret,•••• •.• _•.•••••••••• •• . .•• TABLE II VolumeofPluueolu.J vulgarisleucoaggJutinin(pHAL) pressure-

injeczedat glutamate-responsive deglutitivelociin the rat nucleus tractus sclitarii._•... •__•.•• ••• ••••• •• 34 TABLE III Volume ofOuorogokl or latex mlcrcspheres injected at

glutamate-responsiveph81)'l\geal sites intherat nucleus tractus solitarii. ••_••• •• •• •••••••_ • •••••••• •••• ••.• 3S TABLEIV Characteristicsof deglutitiveresponses evokedbyglutamate

micropneumopho resedinto the rat nucleustractus solitarii. • 44 TABLE V Effective dose-range (Picomolcs) of antagonistsat deglutitive

lociintheratnucleus tractus solitarii.••••_• ••••.••.• 64 TABLE VI Equieffective molar doses(EOSO)of EMagonisu· at

dcglutitivelociin the solitariuscomplex of therat,••••• •• 19 TABLEVII Incidence of pharyngo-oesophageaJ progression observed at

deglutitive10ciin rat nucleustractus solitarii..••• • • •• ••• 81

S·IIT 8-OH-DPAT ACh Amb Amb, Amb_K Amb, AP APV AP7 BRL 43694

cc

CE CS CSF

DAB DE

xviii UST OF ABBREVIATIONS

S-hydroxytryptamine (serotonin)

8-hydroxy-2-(di-n-propylamino)tctralin acetylcholine

nucleusambiguus compact formationofAmb semicompactformation ofAmb

looseformation of Amb areapostrema

D,vZ·amino-S-phosphonovalericacid D,L-2-amino-7-phosphonoheptanoic acid

(endc)-N-[9-methyi-9-azabicyclo(3,3,1)non-3-yl}-1-methyl- Indazole-Scarboxamide

central canal cervical oesophagus

completeswallow cerebrospinalfluid dorsal

3,3'·diaminobenzidinetetrachloride distaloesophagus

DGG DMX EAA FTI'C

GF ICS2OS-93O lR IV KA MK-801

MSCP

NMDA aSpv NTS NTS_

NTS,~

PAP PO

Gamma-D-glutamylglycine dorsalmotornucleusof thevagus

excitatoryaminoacid fluoresceinisothiocyanatc gracilefascicle

(3-alpha.troponyl)-lH-indole-).ca rboxylicacidester immunoreactive/-immunoreactivity

(ounbventricle kalnete

(+).S-methyl· lo.l l-dihydro-.sH-dibenzo(a,d]-cyclohepten-S.l~

imine scopolamine mcthylbromide

N-mcthyl·O.aspanatc nucleusofspinaltrigeminaltract nucleustractus solitarii subnucleus centralis ofNTS subnucleusintennedialisofNTS subnucleusventralisof NTS

pharynx

peroxidase-antiperoxidasecomplex parabrachialcomplex

PBS phosphate-bufferedsaline PHAL Phaseolusvulgaris leucoagglutinin

m

parat rigeminalislands

Py Pyramidaltract

QA quisqualate

R(r) respiratoryrate

rostra l SCP

SLN SO SpY

TFMPP Is V.

v:

VII"

vn:

VII"

xn,

scopolamine hydrobromide superior laryngeal nerve superiorolivarycomplex spinaltrigeminaltract t-(3-trifluoromethylphenyl) pipe razine solitary tract

trigeminalmotor nucleus ventromed ial division ofV.

facialnucleus accessoryfacialnucleus facialnerve hypoglossalnucleus

CHAPTER ONE I!ITRODUcnON

LSwallowing-anoveJVicw

According to the 'biogeneticlaw'(Bact,1828; Haeckel1868).ontogeny recapit ulat esphylogeny.Ifthe biogenetic lawwere10holdformotordevelopm ent as wellasformorph ogen esis,itwould predictmotorfunctionsduringontogenyto emergetheearnerthemore ancient the irorigin. The early appeara nceof swallowingduring foetaldevelop ment (Humphr ey,1964;Mistretta and Bradley, 1975)suggeststhe primordialnature of this motor synergy.

Swallowing has been thesubject ofscientific enquiry since antiquity. One ofthefirst authoritativeaccountsislhatofGalen (165-179 A D.) which, although partlybasedupon animalexperimentation,remained entirelyspeculativeand dogmatic, being guidedbydeduclive argumentation.Accuratedescriptionsof the actof swallowingdale backtothe 1600's(Harvey,1628,citedfromDoly, 1968).

Morethan two-and-a-half centurieselapsedbeforeit wasproposedthatthis complexactmustbe underthecontrolof a"centerof deglutition"(Wassilidf,1888 and Marckwald,1889,citedin Dory, 1968;Meltzer,1899). The search forthis elusive swallowingcentreis Sl111continuing.

Comprehensivereviewson deglutitionhavebeen compiledinpreviousyean (Ingelfinger,1958;Doly,1968;Diamantand El-5harkawy, 19TI; Hockmand DL.

1979;Miller,1982, 1986, 1987; Roman,1982, 1986;HiiemaeandCrompton, 1985;

Carpenter,1986;Kennedyand Kent,1988). Therefore, the presentdi:ssenation will notattemptadetailedsurvey of the literaturebut concentrate on themajor currentconceptsof the neuralcontrol of swallowing.

Swallowingmay functionallybe dividedinto threestages, the preparatory (linguopalatal), thepharyngealandthe oesophageal (Magendie,1813, cited from Miller,1982).

In thelingoopalatal stage,foodisrenderedsuilable forswallowingby intraoral manipulation and mastication. Although thisstageof swallowingis predominantlyundervoluntarycontrol, currentevidencesuggests thatparticular patternsoforal stimulifacilitate repetitivejawandtongue movements(e.g.Willigan etal.,l986).

The pharyngealstageismitiated asthebolusmakes contact withspecifJe receptive zonesof the oropharynx(Miller and Shcrrington,1916).Two principal movements arc then performed bythe pharynx.:an elevationof the whole pharyngealtube followedby a descendingperistalticwave (Negus, 1943;1948;

SheltonetDL, 1960). The latterconsists ofsequentialcontractionsofthe pharyngeal constrictors, which propel tbe bolus through the pharynx..

Simulta neously, thelarynx movesup andispulled forwa rd undertherootofthe tongue.

The pharyngealstage ofswallowingischaracterisedbya complex pattern of muscle contractionsthat proceedsinan ell-or-none sequence. Themusclesthat discharge atthe onset ofthepharyngealstage(the "leading complex";Doty and Bosma.19S6)include:superio rconstrictor.palatopharyngeus.palatoglossus, posterior int rinsic tonguemuscles, styloglossus,stylohyoid, geniohyoid andmylohyoid. The activity of thedigastric,genioglossus,geniohyoid and themylohyoidformsatensed floorfortheoralcavityandthetonguebase. Againstthistensed floor,the posterior tonguecontracts and isforcedbackward againstthe food.This posterior movementofthe tongueappeantobecausedinpart bypowerful activity oflhe hyogl05581musculat ure (HiiemaeandCrompton, 1985).

The finalstage of thepharyngealphase isthe propulsionof the bolusfrom thepharynxintotheoesophagus.The pharyngoesophagealjunction,consistingof thecricopha ryngea l muscle oflheinferior pharyngeal constrictor and.themuscles positioningtheaicoid cartilageinto whichtheinferiorpharyngealconstrictor inserts.

norma llyremainsdosedpas.s.ivelybyelasticity ofsurrou ndingtissues,according10 the analysisbyDaty(1968). Whetheror notthecricopharyngealmuscleisalso ton icallyactive remainssomewhatcontroversial. However,it isgenerally agreed tha i thecricopharyngealmusclemustrelaxto allowthe bolus toenter the oesophagus(seeMiller,1982 forreview).

Entry of the:bolus into the oesophagusmarks the initiat ion of the oesophageal stageof swallowing. Meltzer(1899; 1907)divided oesopha geal peristalsisintoprimary and secondary types. The oesophageal componentsof swallowing,consistingor propulsiveoesophagealcontractions,aretermed'primal)"

(or deglutitive) peristalsis.Theoesoph agus alsodisplaysperistalsisindependently of swallowingin respo nse 10a local stimulussuchasballoondiste nsion. Such peristalt ic contractio ns of the oesophagus aretermed 'seco ndary ' peristalsis. Yet another type orperistalsisis seenwhich is termed'tertiary'(Ca nnon, 1907; Jurica, 1926, cited Im mRoman, 1982)or 'auto nomous'(Roman.1982).This istheterm appliedtoperistalt iccontract ionsdisplayedbythesmoot h muscle oesopha gusin the absen ce orextrinsicinnervation.

Whenswallowsare elicitedin quicksuccession,the oesophagealcomponent remainsinhibiteduntil the lastswallowrollOViing whichprimaryperistalsisresumes (Meltzer,1899). Thisphenomenon has been term ed 'deglutitivc inlubition' (HellemansandVantra ppe n,1967;HellemansetaL,1974).

11isgene rallyagreed that,at rest,the loweroesophageal (gastroesophageal) sphincter remainsclosedduetoatoniccontractionof thesphinct eric muscles(see Ingelfinger,1958;HellemansandVantra ppc n, 1974).Relaxationofthissphincteric region occursshon lyafterinitiationor the pharyngealstage (seeDory,1968Ior a reviewor theliterature). The latencyof thisrelaxati onisrelat ively sho rt so thatitinvariably occurswellbeforetheoesopha gea lperistalticwaveapproaches

thearea.Thus, it appearsthat the gastroesophageal junctio nis an entityseparable from the otherstages ofswallowinginsofar as theorganisation of deglutition is con cern ed.This statementisfurther supportedbythe observati on thatdeglutitive inhibition does notextend tothegastroeso pha gealjunction:the tatter remainsopen during repeated swallowinganduntil the oesophagealperistalticwave ofthe last swallowpasses thro ugh the ope ning (Kroneckerand Meltzer,1881,ciled fromDoty, 1968~

n.

Neural controlofswallowing

With therecognnoeof swallowing as a reflexsynergy (Bidder,1865, Blumber g, 1865,Wallerand Prevost, 1870,and Wassilieff,1888,cited from Doty, 1968;Miller andSberri ngto n, 1916),three sepa ratelevelsof the swaltov.ing pathway havetraditionally beendiscerne d :

1.The efferentlimb.

2.The afferent limb.

3.1bc:organisationalstage.

1.The efferentlimb

The efferent limb of the deglutitfve neural pathway represents lhe coord inaled outputof cranialnerves V,YD,IX, X and X1l. Motoneurons

controllingthe oropharyngealandoesophagea l"muscles aredistributed in the brainstem inseveralpoolsbeginningfrom the level of the trigeminalmotornucleus and extending at least asfar caudallyasthefintcenicalsegment of thespinal cord. Withinthis distnbution,twomajorgroupingscanbe distinguished,the dorsally located hypoglossalnucleus and theventrally locatedcolumn of special visceralmotor nucleiinnervating thebranc:hiomeric;andstriated oesophageal musculature. Contrarytothe analysisof Daty(1968),specific grouping of motoneurons active in deglutitionisapparent, at leastv.ithin some

or

the branchiomotornuclei (e.g,the principal columnof thenucleus ambiguus;the accessorynuclei of the facial and trigeminal motornuclei;seeTable I). 1be locations ofmotoneuron s innervating the majormusc:1esparticipating in swallowing in the ratarelistedinTable1.

The source of the extrinsic innervation of the oesophagealsmooth muscle hasbeen asubject of debate.Physiologicalevidence has attested tothepresence ofanextrinsicsourceof nervesupplytothesmoothmuscle oesophagus (JansseN et aL,1916;Janssens,1918;Tieffenbachand Roman, 1972). PhannacologicaJ evidence(Bartlett, 1968; Kamikawa and Shima,1979) indicatestheinnervationto beof thegeneral visceral efferenttypeand,therefore,poinuto thedonal motor nucleus ofthe vagus(DMX) asthesource.Thisissupportedbyhistologicaland tracingstudies(MarinescoandParhen, 1907;Coiland Norgren,1919;Nielet aL, 1980; HudsonandCummings, 1985;Vyasel ez,J987).

TABlEI

Musclespan icipatingin deglutition and theirsources of innervationin the rat.

Muscle LoutloDOf Peripheral

motoneuroDS ntnebranc h

Phary ngeal Semico mpacidivWoD Pharyngul colUtrieton cr eeeteusambiluus branch of

(A mb_K) Xlhnerve

Intrinsic Loose formalion of Recurrent

bryn, e" nuc::leus ambiauus lar yns cal

muscles (Am bl)

Cricothyro id Overlapsrostral Super ior

muscle portionof Amble laryngeal

Stylopharyo · Tipofnucle us IXtb m:fVC acus lnuscle ambiguus

Mylohyoid, Ventromedialdivision POSleriortrunk anterior oftriscminalmotor of mandibular

bcllyo( nuclells(V~ branchof Vlh

dialSuic nerve

Styloh yoid. Accessory facial Slylohyoi dand posterior nucleus (v n :> digutrk rami

bellyof of VlIthnerve.

disu tric

Biel er Ind Hop kins, 1917.

sasamolo, 1979;

Szik elyand Matesz,1982

Su kelya nd Malen, 1982;

Shoh::traand sakai.1983.

TABLEI-coallau"

Musde Locationof Peripheral Reference

motollt aro ns atn_braoth

Styloglossus, Dorsalsubdivision Styloglossaland Krammer tlal..

hyoglossus ofhypoglossal hyoglossal rami 1979.

nucleus (XII,.l ofXllthnerve

Genioglossus ventromedial Genioglossal

subdivisionof XII. ramusof XIIIbnerve

Geniohyoid ventr olateral Geniohyoidal ";K ilamura

subdivision ramusofXllth tlal.,19al

of XII.

Thyrohyoid Spil1lJ port ioo Firslcervica1 Kitamura nol. ,

~~n~~r~::11

^orCI spinai llerves 1913.

Oesophageal compKt division Ce""icalYlluS Bieler

...

striated o( nucleusambil uu.s M~ Hopkins,1917.

muscle (Amb.)

Oesophageal DorsalYlgalmet er XlhDe""e Vyastlol.•19S7.

smooth nucleus(1)

muscle

In.addition to this parasympathetic nerve supply, histonuorescence microscopicdemonstrationofa weDdeveloped adrenergiegroundplexus in the muscularismucosae of thecat orrhesus monkey (Baumgartenand lange,1969) suggests theexistence of an extrinsic sympatheticinnervationof thesmoothmuscle oesophagus. However,the exactsource ofthisinnervationremains obscure.

The originofthecentralnervouscontrolof the loweroesophagealsphincter iseven moreuncertaindue to thepaucityof experimentaldata. Ongoing investigations(VyastlaL,1987;inpreparation)demonstrate that, inthe rat,the spina] ponionof the dorsal vagal motornucleus containsneuronsretrogradely labeledfollowinginjection oftrace r intothe gastroesophagealjunction, thereby indicating this motornucleus10 be thesource of the smooth musclecomponent ofthe ratlower oesophagealsphincter. Itshouldbepointedout that smooth muscleinthelowersphinctericregion oftheoesophagusinterdigitates withstriated musclefibersintherat (Marsh and Bfeger,1987) and the guineapig (Thomasand Trounce, 1960)-

Intrinsic innervationof the oesophagus derives fromtheplexusesof Meissnerand Auerbachwhichare well developed in the smoothmuscle ofthe oesophagus (Kuntz,1947,cited fromRoman,1982). Intramural neuronsofthe myenteric plexus of Auerbach were generally believedtoberelay neurons interposedbetweenthe vagaleffere nt fibers and the smoothmuscle. However, thepresence oftheseIntramuralneuronsalso inthe striated muscle portionofthe

10 oesophagus refutesucha simpleexplanationof theirrole.On the otherhand,the inherent ability of the oesophagea lsmooth muscle todisplayautonomous peristalsis aswen as the resultsfromaplethoraofstudiesonthispre para tion(e.g.Bieger andTriggle,1985;see Roman. 1982forlitera ture)indtealetheprofoundinfluence the inua mural neuronsmust exertonoesophagealmotility.

Several clectroph ysiologicalrecording studieshave beenperformedon the activityof themot oneur on, of thehypoglossal, ambiguusand trigeminalmotor nucleiduring swallowing(Sumi, 1964;1969;Carand Jean,1911;Jean,1972a; 1978;

1984;Amri~l01.,1984; Kesslerand Jean,19858;CarandAmri,1981;Amri and Car,1988:Tomomune andTakat a,1988).Thedata indicat e that the motoneurons displayaburst of spiking activity during swallowingevokedbystimulation of the superior laryngeal nerve (Sl..N)but withsynaptic:delays(S.sms) iliatsuggesttbe pathwaytobepolysynaptic.Inaddition. data suggestthatthesynapticinputs to the moto neurons are not purelyexcitatory; for example. in hypoglossal motoneuroes, a complexcombinationofinhibitionand excitationisoftenobserved duringswallowing(e.g.Tomomu neand Takata,1988). FOllowing a stimulw pattern delivered via the SLN,themotoneurons demonstrate twostagesof depol arisation (Suml,1969):thefirstoftheseisprobab lydue tothepolysynapl ic input whilethe second is accompaniedby greate r dep olerlsauon,displaying a sp iking discharge for201).400 ms.The latterprobablyreprese nts thesynapticdrive ofthe central pattern generato rforswallowing.

11 2.Theafferentlimb

Variousreceptiveregions in the oropharynx can elicit deglutitionupon approp riatestimulation (Miller and Sherrington,1916).Originallydiscoveredby Bidder (1865)andBlumberg (1865).thenerve that elicitsswaUowingmostreadily upon electrical stimulationinmany species inclUdingcat.dog,monkey, sheepand ratis thesuperiorlaryngealnerve branchofthevagus (Daty,1951;Dolyand Bosma.1958; Miller,1972;Jean, 1972a;Biegeret aL,1977;Wecra suriyad aL.

1980). Other nerve routes arealsoeffective ingivingrisetorenexswallowing.

TheIXnerve. when electrica lly stimulated.evokes swallowing (Reid, 1837,cited from MIlle rand Sherrington,1916)thoughlessread ily thanthe SLNinmost species (Doly,1951;Priima,1958.citedfromMiller,1982).Inthe dog.bilateral sectioningof theIXnerveimpaindeglutition (Ogurael aL,1964)butdoes not affect swallowingin human (Ballan tineet aL,1954),indicat ingthe va riat ionbetween species. In the cat.WeerasuriyattaL(1980) showedthatsimultaneo usstimulation oftheIXandthe SLNresultlinspatial summationieoanincreasedresponse (number of swallows) ascompared to stimulat ionof asingle nerve alone.Inthe macaque.theafferentpathwaycarriedbythe Vthnervehas been reportedtobe theprimaryonefortheinitiationof swallowing(Kahn. 1903,citedfromDoty, 1968).

Electrical stimulationoftheIingualis-ehordanerve hasbeenreponedto inhibitswallowing (Millerand Sherrington,1916).

12 Sinceolher~ergies(vomiting.coughing.vocalisation)canalsobe evoked byelect rical stimulationofIhe SLN,itisbelievedthatthesensory code eliciting swallowingcomprises a particular stimulus pattern. Thisissupport edbythe observationthat electricalstimulationof theSLNreleases separate synergies at differentfrequenci eswith a swallowingoptimumof3().SOHzin variousspecies (Doty,1951; Miller. 1972;Weerasuriyaaat,1980)and2040Hzinthe rat (Bieger~IaJ..1911). Exactlywhat comprisesthediverse sensory codes andhow theyarc decipheredbythecentral programs organizingthedifferentsynergies remainsunclear.

Periphera lsensory feedbackisimport ant notonly for triggeringthecentral programfor swallowing, but alsoforcontinually modifyingitdependinguponthe size andconsistencyofthebolus duringitstransitIn the courseofaswallowor aseries ofswallows(e.gHrychshyn and Bumajian,19n). Unlike theoppossum and the rhesus monkey (lanssens~IaL,1976;Janssens, 1978),thedog and baboon (IAnghiandJordan, 1971;Janssenset

at.

^1973;Roman and TIeffenbach,19n;

Diamant andEJ..Sharkav.y,19n forreview)depen d on sensory inputfromthe cervical oesophagusfortheaboral progression of the peristalticwave. Peripheral sensoryfeedbackis especiallyimportant intheelicitationof thepharyngeal stage of neonataldeglutition(Sumi,1967;1975).

Sensory deglutitjveinformation is relayedto thebrainstemmainly viathe IXth and the Xth cranial nervesinmost species. The nucleus tractus solitarii

13 (NTS)isa majorrecipient ofafferent informationfromthe upper alimen tary tract and thoracoabdominal viscera. The affere nttermination fields intheNTSfrom theph8l)'l'LX andthe oesophagus have been very weD characcerized inthe rat (Bieger and Hopkins, 1986;Altschulerel aL,1989).These authors showedthat thepalatopharyngealafferent representation overlaps theinte rmedia te and interstitial subnuclei ofthe NTS whiletheoesophageal afferentaticniscoextensive with the central subnucle usoftheNTS.

In the rat, afferents carriedbythe SUlterminate within theinterstitial dorsalintermedia te,and rostral half of the central subnucleus. In the lamb,SLN afferentationisseenin theinterstitial.medial.vent ralandventrolateralsubnuclei (SweazeyandBradley,1986) andconccMbly includes the intermedialis. Inthe cat,theSL.N was found to project10the dorsolateral, intermediateand interstitial subnuclei(Lucierel aL 1986). Intherabbit.SLN afferentationoverlaps"ce ntral and lateral" portions of the NTS (Hanamon and Smith, 1989), conceivably includingthe subnuclei intermedialis, ventralis,interstitialis and cemraus.

3.The organisational stage

The concept or afunctional swallowing centrewasadvanced a century ago (Wassilieff,1888; Marckwald, 1889; Meltzer, ]899) andhas been describedin neurophysiologicaltermsbyDoty(Doty,1951, 1968;Doryet01.•1967).A centre orpattern generatormaybedefined as"a group of neuronswhose coordinated

14 action produces a stereotypedresponse"(Miller,1982). In otherwords,the inherent organisation of this neuronal circuit producesthepatternedresponseby directingappropriate excitation-inhibitiontothe motoneurcns. The principle of centralpattern generationiseitherwell established or stronglyindicated for a number ofbehaviours throughouttheanimalkingdom (For reviews.see Delcomyn, 1980; Grillner, 1985;Bassler,1986;Harris-Warrick and Flamm,1986). Such behaviours include swimmeret beatingin crayfish (Wiersmaand Ikeda, 1964);

locomotionin lamprey (Orilln cret al,1987;Buchananet aL,1989);locustflight (Wilson,1961);breathing in cat (Wangel aL,1957);and masticatory movemen ts inguineapig(Nozakiet ai, 1986),to namebut afew.

Bydefinition,theoperation ofa pattern generat orshouldnot dependupon peripheral afferent input. During reflex swallowing inadult cats,thebasic sequence of motoneuronalactivation remains essentiallyunchangedfollowing either physical oranaesthetic-inducedsensory deprivation(Doty and Bosma,1956;Miller, 19718). Indeed,the very existenceof a'deglutiloryautomatism' (Bieger ,1981;see below)atteststothe existence of a centralpatte rn generatorforswallowing.

Atthetumofthiscentury,the split-brainstemexperfmentsof Ishihara (1906) gaveriseto some concepts regarding the"swallowing centre". Following midline splitsin themedulla of dogsandrabbits, Ishihara (1906)demonstratedthat swallowingcouldbeelicitedin such preparat ions only ontheside ipsilateraltothe SLNstimulation. From these experimentsemerged the concept thai the

IS swallowingcentreconsists oftwoindependent'half-centres'oneoneachsideof the brelestem,each of whichiscapableof supporting·unilateral swallowing-,a phenomenonthatwas subseque ntlyccnarmedincat andmonkey(Dalyet al., 1967).Thelesion esperiments ofDotynat(1967)suggested. inaddition,that, throughoutthesequence ofswallowing,each'hejf-cenue'excites andinhibits various components ofthe other via cross-cenneceoesthatrun'atleve ls posterior 10the obexandatthe levelofthe trapezoid body'.

Various structureshave beenimputed to subservetheimportant function of coordinatingthefiring ofthedifferentmotone urons so as to generatethe spatiotemporal patternthatcharacterizes the act of IWal1owing.Prominent among suchpostulated-swallowingcentres"arethe medullary ret icularform ation (Daty eloJ.. 1961;Manchanda and Aneja, 1972; Sumi, 1974);the pontine reticular formation (Holsteg ettaL.1983); and thenucleus tractus solitariiand adjacent reticular formation (Jean,1972a.1972b, 1984;Kessler andJea n,1985a).

On the basis oflesion experiments incat,dog and macaque,Dotyttal:

(1967)proposed that the swallowing centreresides within the reticular substance extendingfrom the rostralpoleortheinferior olive10theposter ior pole of the facial motor nucleus.This area corresponds tolhenucleus gigantoc:ellularisof the reticular formatio n, or,accordingtomodern neuroanato mists,spa ns therostr al ventrolateralmedulla, which is believed to containa'tonic vasomotor centre' (Ross tlaL.1984;Rosstlal.,1985).

16 Byemployinganterograde aUloradiographictracinginthecat. Holstc:ge~l gL(1983) found thatin the:caudalpontinetegmentum, anareaislceateddorsal to thesuperiorolivarycomplex that sendsprojections conlralatcrallyto the ventromedial trigeminal,don al group ofambiguus(conceivably,the Amb,, )and ventra lhypoglossal motor Rucki. Based on these findings,itwas propos ed that thelatter pontineareamay correspondto theswallowingcentre.

EJeetcophysiological, neur oanatomical and pharmacological evidence accumulatedthus far hassuppon ed the statusof theNTS as animpona nt neural substrate involvedin the orsanisationofdeglutitionfora numberof reasons. First.

itwas demonstratedinshee pand ratthat,withswallowing elicitedbyelectrical stimulationofthe SLN, some NTS units ('early' neurons)fired during or even priortothe pharyngeal stagewhile ethers('late' and 'very late' neuro ns) dischargedduring the oesophagealstage (Jean, 1972a;Kessler and Jea n,1985a).

The activityof these 'swallowing neurons'remaineduncha nged afterecraraa ucn of the animal. 'Early'neuronswereloca ted eitheraround the solitarytract,Le.

inthernsandthe adjacenlreticularfonnatian(lhe 'dorsalgroup' ) orinthe lat eral reticularfonnationabovethenucleusambiguus (the'ventral group'). The authorssuggestedthatthene urons of the dorsalgroupare involvedininitiating andorganising the sequ ence ofswallowing whilethe ventralgroupof neuro ns are 'switching'neuro nsthat receive their inputfromthedorsal group anddistribute thisinputtothe differentdeglutitivemotoneurons.

17 Second, thereisstrongevidence supportingthevi.._thatthe centralpattern generatorfor swallowing canbebroken downintoatleasttwocomponents governingthepharyngealandoesophageal stages(Jean, 1972b):in sheep,a unilateral lesion placedbetween the solitarytractandtheOMX suppressedthe oesophagealstage or swaUowing elicitedbyelectricalstimulationoftheSLN ipsilateralto thelesion. The integrity of the oesophagealmotorapparatus was ascenainedbythe oesophagealcontractionselicitedupon stimulationof the nucleus ambiguus onthelesioned side. A$well, stimulationof the contralateral SLN producednormal swallowing. Theseresullssuggested thatthe oesophagealpattern generator includes the NTS (Jean,1972b).

Third.the chemicalmicrostimulation and retrograde tracingexperiments of Bieger (1984)provide a precise neuroanatomicellocalisation oftheoesophageal premotarareawithinthe solitarycomplexof the rat.The oesophagea lsubregio n i:L the subnucleuscenrrausoftheNI'S,not only projects directly to the rostral oesophegcmctor portion of thenucleus ambiguus(Bieger, 1984;Rosset aL,1985;

Cunningham and Sawchenko, 1989),but also receives theoesophageal primary afferentinput (Bieger and Hopkins, 1986;AltschuleretoJ.,1989).

Finally, itwasdemonstratedthat'automat ic' swallo'Ningcouldbeelicitedby applicationofS-HT oraS·HT mimetic tothe fou rthventricle(Bieger, 1981);

conceivably, the amine was actingupon underlyingstructures, includingthe solitary complex. Indeed,direct chemicalmicrostimulation,withglutamateand ACh or

18 muscarine,ofstructureswith inthe confinesofthe NTSgaveriseto either complete swallowsor theirconstituent stages. depending upon the sile of stimulation(Bieger, 1984).The latter investigationalso furnishedpharmacological evidenceconfirmingtheklentity oftheoesophagealpattern generator.

Studieson theneuroph arm acology ofdeglutition were instrume ntalingiving

risetothe concept of a 'deglutitoryautomatism' in therat. Inviewofthescant attentionthesedatahave received(e.g.Miller, 1982, 1986,1981;Jean,1984;

Roman,1986), abrierlook at thesepharmacologicfindingsand theirimplications seemsappropriate.

IlLPbannacologyDCswaDowiDg

In 1912,Biegerel QL, descnbed a periodic response consisting of spontaneous twitching of branchiomericmusculaturein the floorof the mouthof theurethane-anaesthetisedra t, Thisphenomenonwastermed oral myoclonic twitchactivity.Central dopaminergicandserotoninergicsystems wereimplicated inthe elicitatio nand maintenance ofthis response.The paradigmwassuccessfully utilisedas amodel system for the evaluation of the centraleffectsofmonoamines (Bieger, 1974;Menonetal., 1976a, 1976b, 1986;Oineschmidtt!l al., 1977;

Oineschmidt and McGuffin, 1978; Tseng, 1978,1979;Tsenge(al., 1978;

Oineschmidl,1979).Thisoralmyoclonic twitchaC1ivitywassubsequently ide ntified

19 as thebuccopbaryngeal component of swallowing (Biegeret aL, 1977). Therefore, theneuropharmacologica ldata fromthestudies listed above apply directlyto swallowing.

Animportantconcept that arises from these investigationsandwhichwas madeexplicitbyBieger (1981)istha tof 'automatic'swallowingor 'deglutilory automatism'in contradistinctiontorc£lc:xswal1owing.Automaticswallowingisa truly centrallygenerated synergy.beingindependent of peripheral sensory influences insofar asits genesisand maintenanceisconcerned (Bieger,1981).

Thelatter investigationdemonstrated thatthe neural generator for serotonincrgically-drivenautomatic:swallO'Yl'ingiscontainedinthe meduUa.possibly withinstructuresintheenvirons ofthe fourth ventricle. However,Kesslerand Jean (198Sb; 1986a; 1986b) have denied any dcglutitiveexcitant action of the monoam ines, including

s·m ,

andascnbedonly inhibitoryeffects of these amines on deglutitionatthe level of theNTS.

Further demonstrationof a deglutitoryautomatismemerged fromthe work of Bieger (1984). Activation ofmuscarinic cholinOteptors within the subnucleus centralis ofthe NTSevokedisolatedoesophagealcontractionswhich resembled secondary peristalsis(Bieger,1984). In addition. oesophageal components of both automatic and ren ex swallowingweresupp ressedfollowingadministrationof antirnuscarinie agents. This finding,together withtheret~detracing of efterentsof the subnucleuscentralis,providestrongevidence to support the

20 contentionthat the latter subnucleus ofthe NTS contains premotorelements forming part of theinternuncialnetwork organisingoesophageal peristalsis.

The study byBieger (1984) directs particular attention to three pharmacologicsubstances: i) glutamate,becauseitiscapableof giving riseto eithera completeswallowing sequenceor isolated parts thereof,dependingupon the siteof applicationwithinthe NTSii)S·HT,because it appears to show selectivityfor eliciting thepharyngealstageandiii)ACh or muscarine,because of theirabilitytoselectively evoke the oesophageal stageof swallowing when applied tothe NTS.

Theforegoing synopsisdraws attention to themany piecesof evidence implicatingthe NTS as animportant substratesupportingdeglutition. However, itisalso evident that analysisof dataso farisnot unequivocal regar ding theexact locationls of thecentralpattern generatorfor swallowing.

IV. Objectives

Thisresearch was aimedat examiningcertainpharmacologicalproperties of. and tracingthe neuralconnections of deglmitivesubstrateswithin thesolitary complexofthe rat inorde r to understandtherole of theNTS intheneural organization of swallowing.Theanaesthetizedratwas chosenas themodel since previous investigations had establishedthevalidity of thisparadigm(Biegeret aL.

21 1972; Bleger, 1974; 1981; 1984; Biegeret aL,1977) and a wealth of neuroanatomical infonnationisavailable on thisspecies(e.g.Kaliaand Sullivan, 1982;Bieger,1984:Shapir o and Miselis.,1985;Bieger and Hopki ns,1986;1981;

Cunningham and Sawche nko, 1989; AltschulereraL. 1989).

The firstimportantste pwas to delineatethe deglutlt iveregionoftheNTS.

The methodofcherokalmicrostimulationbypneumophoresiswasemployedin these investigationsbecauseithadbeen succesfullyused in a previousstudy (Bieger,1984). However,further refinementwasneededto allow microapplicalion of multiplesubstances with a highdegree ofaccuracy andprecision. Two substanceJ. glutamate andacetylcholine(ACh)ormuscarine. were chosen for mapping the dcglutitive silesoftheNTSowing to their reported role asdeglulitive excitants(Bieger,1984).

The secondphaseof the investigations involved identificationof excitatory amino acidreceptor mechanismsmediatingthedeglutitiveactions of glutamate within the solitarialdeglutitiveloci:glutamate had beendemonstratedas a selective deglutitivcexcitant, capable oftriggeringa completeswallow or isolatedparts thereof(Biege r. 1984). Pharmaco logical agentswere usedto charac terize the receptor types mediating thedeglutitiveeffects of glutamate.

Thethirdobjective wasto analysethenon-specificdeglutitive excitantaction of glutamatebycomparisonwithACh (ormusca rine)and 5-HT.BothS-HT end AChwer e known togive riseto 'fictive'or automaticswallowing(Sieger,1991,

22 1984) andcouldbeutilised as pharmacologicaltoots owingto their specificity for eitherthepharyngeal(5-HT) or the oesophageal (muscarine)stageof swallowing (Bieger,1984). In addition,a side issuethatwarrantedclarificationwasthe appa rent disparity between the the excitatory(Bieger, 1981) and inhibitory(Kessler and Jean,1985b) effects of S-Hron deglutition.To locatethe centralsourceof the scrotoninergic inputtothe solitarycomplex. retrograde tracing wasemployed in conjunctionwithimmunocytochemicallocalisationofS-HT.

Thefourth objectivewastotracethe projectionsissuing from the solnarial deglutitivc loci.Previous findings hadsuggested thatthecentralsubnucleus of the NTSisinterposedbetweenthe oesophagealafferentand efferentlimbs(Bieger, 1984;Biegerand Hopkins, 1986; CunninghamandSawchenko,1989; Altschuleret aL,1989) implicating thissubnucleus as theorganiserof oesophagealmotility patterns. Uthe analogyis extendedtotheremainder of the solita rial deglutitive network. theNTS couldbe hypothesisedas thekey substrate involved in the neuralorganization of deglutition. Thus, the tracingof solilarialefferentswas expected not onlyto test thishypothesisbut also to reveal theponlomedullary structuresinvolvedinswallowing. Forthispurpose, the anterogradelytransponed plantlectin,Pnaseotus vulgaris leucoagglutinin (PHAL; Gerfenand Sawchenko, 1984),wasthe tracerof choice.Theapproa ch wasto make discretedeposits of the tracer at identifieddeglutitivelociby the techniqueof micropneumophoresis andtoexamine its presenc einterminals originatingfromthesolitarialdeglut itive sites.

23 CHAYfER 'IWO

MATERIAlS ANDMEff/ODS

LExperimental procedures

Experimentsweredo ne onmale Sprague-Dawleyrats weighing250-450g.

Asurgicalplane of anaesthesiawasinduced eitherby.i)anintraperitoneal(l.p.) injectio nof 20%urethane (1.2 g/kg)orii)aninitialdose ofl.p.sodium pentoba rbital(45 mglkg)followedby intrave nous(loY.) urethane administeredin boluses of15().17Smg/ltg every30 min.

Theanimalwastracheostomi2ed and mountedina stereotaxicframe followingcannulationofthe right external jugularvein for intravenou s infusion of drugs.. The tool hbarwas.5mmbekJwtheintra-aural line. therefore,thehead wasin maximumventroflexionpenniuedbythe apparatus.

Changesinintraluminal pressurewithin thepharynxandtheoesophagus wen: recordedusing miniatureballoon-tipped catheters.P&90 polyethylenetubing (Beclon-Dickenson)wasused to constructthe balloon-tippedcathete rs.Usingthe flameof aspirit lamp,one end ofthetubingwassealedandmelted whileairwas genlly blownthroughtheother end 10produce asmallballoon 5-8mmin diameterand1·1.5cm long. Three such balloon-tipped catheterswere filledwith water andeac h connected 10aStath ampressuretransducer. The balloon

24 catheters were thickly coated with 2%xylocainejelly (AstraPhannaInc.)and insertedintothelalyngopharynr.andthe eeMcal and the supradiaphragmatic oesophagus, respectively. Oesophageal balloonshada diameter ofabout 5 mm while:thephazyngeal balloonwaslarger(diamcter-8mm). Theapp roximate length ofthe ballooncathetersto be insertedwasfirstmarked for each as measured in8number ofanimals. After allowinglheanimal to swallowit,the distaloesophagealballoonwasinserted lr ucthe oesophagus untila slight obstructioncouldbefelt,indicating thepointat which the diaphragm encirclesthe oesophagus. The cervicaloesophageal balloon was securedin placeas soonas the animal swallowedit.ensuringi.positionjustbelowthepharyngo-oesophageal junction.The pharyngealballoon was placed far back intcthe oral cavity,between the pharyngeal palateandthedorsalaspect oftheposteriorlongue.

Acatheter, eonaected externally to apressure transducer,wasinsertedinto the trachealcannula. ColTcsponding intraluminal pressure signals registeredbythe fourStathampressuretransducers were amplifiedand displayedon a pen-recorder (Grass polygraph).Correct placementof (he balloonswasascertainedbyevoldng reOex swallowingbyintra-trachealtactilestimulation.Respiratoryratewasderived fromairway pressure fluctuation and monitoredbymeans ofa tachograph.Rectal temperature wasmaintained between36.5°and37.5⁰C.bymeans of aheatlamp.

Inorder10 exposethe caudal floor of thefourth ventricle and the surroundingstructureson the dorsalmedulla oblongata, asuboccipitalcran iotomy wasperformed.Briefly,a midline incision onthe headwasmade. about2.5em

2S long.running fromthe levelof the animal'sears downtoitsneck. The underlying musculaturewasretracted andthe posterioraspect of theoccipitalbonewas removed. Undervisual control through aWild M6S0stereoeekrcsecpe, apair of fine hooks wereusedforliftingthe duramater and the arachnoidmembranewhile cuttingthemwithapairof mieroscissors. The cerebrospinalOuidwascontinuously drainedusingsmaD wicks made of1Gmw:ipes'tissuepaper.

Forthe microslimulationof deglutiliveneuronsintheNTS,2-,3- or 4·barreledglassmicropipettes (tip0.0.2-5#m) were used. Themicropipettes were pulled fromglasscapillarytubing(0.0.= 1 rom; 1.0.-0.7mm; GlassCompany of America)onan eleclrodepuller (pOL-I,W·PInstruments. Inc.,Conn.)to• shank lengthDC2.5-3.5em. Thelongshanks provided a certaindegree offlexibility thaicould accommodalethemovementsofthe animaldue to breathingand blood pressure pulsations. Drugsolutions. mixedwithdifferen tfluorescentdyesfo r marking ejectionsites (see below). were back·fined into eachbarrelofthe micropipetlcsusing 1mlsyringesfitted with S cmlong, 30 gaugestainless steel needles (Becton-Dickenson).Pressure connections10themicropipettes were made as perthe techniquedescnbcdbyNeuman (1986). BrieDy,stainless steel cannulae made from26 gauge, 318orSI8inchneedles (Becton-Dickenson)were bentinthe middle atanangleof about 4So usinga pair ofnose-pliers. The eennulee were insert edinto eachbarrel of the micropipetteand cemented eitherwith dental acrylic(LD.Caulk Company,Delaware) or with'bazy'glue gel

26 Drug solutions were ejected fromthe micropipeucs

bY

meansofa Picospritzcr II micrcpeeumcpbcresu pump(General Valve Corporation) usinga nitrogenpressuresource.

Pipetteswer e individuallycalibratedbeforeinsertio nintothebrain tissue and after withdrawalaccordingto the techniquedescnbedbyMcCaman,McKenna and Ono (1971). The diameter of the ejecteddropletwasmeasuredwiththeaid of anocular micrometer(Bauschand Lomb. USA). "Thevolume ejectedwith eachpressurepulse (400-500 kPa;50-300 ms)waskeptinthe rangeof 20-100pi (dropletdiameter=34-S7pm).This corresponded toan injection site diameterof 50-100pm(seebelow).

The micropipetteswerefIXedto anelectrode-ho lderassemblytilled at an angleof 28⁰ inthe anteria-post erior plane andcontrolledbya Narishigc micromanipulator. Stereotaxicplacement ofthe micropipetteswithrefer enceto anatomicallandmarks onthe dorsal medullary surfacewas achievedundervisual control throughthesterecmicrcseope, The zeroreferencepointwas ioe ntifiedas asmall ridge markingthe rostralborder of the areapostrema. Thiscorresponded to a pointabout160 ",m rostralto theobex as identifiedhistologically(seebelow).

Swallowing responses were characterisedas:~if a contraction in the pharynx wasnot followedby anoesophagea lperistalticwave;completeswanow

@.if apharyngeal contractio n was folk>wedbyphasic cervical anddistal oesophageal propulsive contra ctions; and cervical oesonhagea lor distaloesoobageaL

27 if • contractile responsewaselicitedprimarilyineither the cervical or distal oesophagus. Pharyngealrespo nseswere considereddcglutitivein natureif.interms of amplilUde. rate of pressureriseand duration,theywere indistinguishablefrom pharyngealcontractions formingpartof a CS.

Attheend ofthe experiment,thebrainwasfixedinsitubyperfusingthe animaleithertranscardiaUy or retrogradely through the abdominal aortawith50 mtof1%magnesiumchloride insaline followedby500m1ofphospha te-b uffered (0.1M)mixtureofparaformaldehyde(4%) and glutaraldeh yde(O.5%).The brain wasremoved andkep t inthefixativeovernight. The brainstemwas blockedfor sectioninginthetransverse planeand the leftsidewasmarkedbyplacingasman knife-cutin the pyramidaltract. 40 pm thick serial sections ofthe medulla oblongata were cut on avib ratomeandcollected on gel-coatedslides. The slides weredriedand ecverslipped wingparaffinoil.Inject ion siteswerevisualized under ultravioletdarldiekt trans-illumination, with the aid of a Zeiss microscopeequipped withexcitation-suppresslonfilters suitable for the dete ctionof the fluorescentdyes.

Photomicrogr3phsof injectionsitC$weretakenon 400ASA colouror black-and·

white filmwith a Winder 35mm cameraattached to thefluorescencemkroscope.

Drawings

o r

thesections were made lJSing a Zeisscameralueida phototebe. The diameterofthe fluorochrome-markedinjection siteswasmeasuredwith theaidofan ocularmicrometer andverifiedbymeasuring thesameincamera lucida drawingsor photomicrographs. With fluorochromeconcentrationsof

28 0.03-0.1% and ejection parametersas outlinedabove.. dye-marked ejection siles werenearly spherical" theirdiametersranging betweenSOand 100p.m. A crude verification ofthesemeasureswasfurtherprovidedbycounting the number of sections containingthefluo rochrome, Nocorrectionwasmadefor shrinkageof thetissue.

The neuroanalomical identificationofstimulationsitesin theflITSwas madewithreference torecentaccountsof the subnuc1ear subdivisions of this region (KaliaandSullivan,1982) and studies of thevtscerotopfcafferent representat ion ofthe upper alimentary tract in the NTS (Biegerand Hopkins, 1986;Altschuleret aL, 1989). Histologically, theobexwasidentified asthepoint where the dorsalwallofthecentral canalmergeswiththeareapostrema. The neuroanatomicalcollection ofDr.D.Bieger wasutilisedfordetermining:i) the rostrocaudal extentof the subnucleus centralis of thetITSfrom aseries of camera lucida drawings of serialtransverse sections through the solitarycomplexinwhich theNTSceoo had been retrograde ly labeledbyan injecdonor a nuoreseemtracer into the oesophagcmo rcrcompact division orthe nucleusambiguus (tromBleger, 1984)ii) thelocationoftheaccessory racial subnucleusinserial sagin al sections of a case inwhich horser adish-peroxKlaschadbeenretrogradelytransported from aninjectionmadeintothe posteriorbellyof thedigastricmuscle.

29

n.

Mapping

For mapping the dcglutilive loci within the NTS, the region systematically exploredwith4-10pmoI of sodiumS-g!utamateand acetyk holine (AQ)and 2·Spmalof D...muscarineejectedfrommultibarreledglassmicro- pipettes in volumesnotexceedingSOpi per pulse.Allobservationswere repeated ina minimumoffM: separa teanimalsand drug-ejecuonswere madeonboth halvesofthemedullain a given prepara tion. Bis·bcnzimide.,nuclearyellow or tluorogold(0.03-0.1%),eitherin aseparate barrelor mixed with oneor more drug solutions conta ined in the micropipette, were usedasmarkersto pennit ultraviolet fluorescencemicroscopicalidentificationof the ejection sites.Mixingthe dyewith the drugs did notappreciably affect the evoked responsesas comparedto those elicitedbythedrugaloneata givenlocus. In addition,ejectionofthedye alone at aconcentra tion ofO.Q3.0.1%didnotaffectsubseq uent responsesevokedbythe drug from thesame sue. Relevantcriteria fortheanalysisof responsiveloci includedlatency ofresponse. pharmacologicalspecificity,anddeglutitivc phase relationship.

30 ID.Pharmacology

1.Micropneumop boreticapp licationofdrup

Pressure pulses of glutamate wereusedfor~tingthe deglutitivelociin theNlS.Glutamatepulses weretypically4-20 pmol,unless staled otherwise, and were appliedinvolumesof 20-100 pI. Glutamatewasemployed as a standardtest substancefor eliciting dcglutitive responsessincei) it canevoke differenttypes of swallowing respon ses, depending uponthesiteofstimulation andii)owingtothe

relatively rapidtermination ofitsaction in braintissue, itisies.sexcitotoxicthan theother excitatoryaminoacids (Olney,1918; Mayer and Westbrook., 1987). A ecmputer-drwen timercircuit was employedinorderto enabletheglulamatc test-pulsestobedelivered at a fixed rate.

rIVe

to eight ecnucl responses spaced at1·2min intervals were obtai ned to ensure n:pr oduClbility before drug interacti ons were exam ined.Controlejections were alsodonewith vehicle (scebelow).Barrin g

aecidentaldisplacementofthe pipette, chemical mierostimulationofOle sites describedin this study yieldedrespo nsesthatremainedstableand reproducible even afterrepetitiveejections (3()'J20 times overaperiodof 30 minutesto 3 hourt)of glulamateor ACh. Intrials wherethe interact ionbetween glutama teand5-KI' was studied,paired drug ejectionswere repeal edup 10J5limes.

Relative potency ofexcitaloryamino acid (EM)agonistsata given silewas evaluatedbydetenniningtheIhresholddoseofeachcompoundrequired 10elicit

31 a responseusing fourdifferentconcentrations,0.1,1,10 and 100mM, keepingthe deliveryvolumeconstant (100pI) andapplyingequimolardosesof the three agonlsts at each site.To ensure reproducibilityata given locusi)responsesto glutama te weretested between the applicationsofdifferentegonlstsandii)each agonistsci wastestedat leasttwice (upto 5 times)and datawererejectedifthe results were inconsistent. Thisprocedu re,therefore,yielded areproducibilityof100% atany given locus.In order10 arriveinan index of relative agonist potency, the effective dose of each agonistcapable of elicilingaresponsein50% of thetotalnumber of testsites wasdeterminedfromaplotoflogmolar doseV5.total percentage of responsive sites(seeTable V).

2.Top ical applicationofdrugs

For theapplicationof drugs10theependymalsurface ofthesolitarycomplex, 5or10 J.ll Hamiltonsyringes wereused.A small cannula tip madefromPE·20 tubingwas attachedtothe tip oftheneedle. Volumesappliedwere typically1 J.ll and did not exceed 5Jll.

3. Electricalstimulation

In a fewexperiments,theeffects of systemicadministrationof drugs on reflexswallowing elicitedbyelectricalstimulationofthesuperior laryngealnerve (SLN) was studied.In these cases,theSLNwas approache d fromtheventralside,