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The neural mechanisms of persistent inhibition in the DNLL of the gerbil (Meriones unguiculatus) [Elektronische Ressource] / vorgelegt von Bernadette T. Saunier Rebori

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THENEURALMECHANISMSOFPERSISTENTINHIBITIONINTHEDNLLOFTHEGERBIL(Meriones unguiculatus)DissertationzurErlangungdesGradeseinesDoktorsderNaturwissenschaftenderFakultätfürBiologiederLudwigMaximiliansUniversitätMünchenvorgelegtvonBernadetteT.SaunierReboriMünchen,February2008 Erstgutachter : Prof. Dr. Benedikt Grothe Zweitgutachter : Prof. Dr. Axel Borst Tag d. mündl. Prüfung : 31. März 2008 ERKLÄRUNGIch versichere,daβ ich meineDissertation selbstständig,ohneunerlaubteHilfeangefertigt, und mich dabei keiner anderen als der von mir ausdrücklichbezeichnetenHilfenundQuellenbedienthabe.DieDissertationwurdeinderjetzigenoderähnlichenFormbeikeineranderenHochschule eingereicht und hat noch keinen sonstigen Prüfungszweckengedient._____________________ _______________________(Ort,Datum) (BernadetteT.SaunierRebori)(…) antes de descubrir, en descubrirnos; antesde modelar la Naturaleza, en modelarnos.Forjarnos un cerebro fuerte, un cerebro original,exclusivamente nuestro: he aquí la laborpreliminarabsolutamenteinexcusable. Reglas y consejos sobre la investigación científica. Los tónicos de la voluntad. Santiago Ramón y Cajal.TABLEOFCONTENTSACKNOWLEDMENTS...............

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THENEURALMECHANISMSOFPERSISTENT
INHIBITIONINTHEDNLLOFTHEGERBIL
(Meriones unguiculatus)


Dissertation
zurErlangungdesGradeseinesDoktors
derNaturwissenschaften

derFakultätfürBiologie
derLudwigMaximiliansUniversitätMünchen





vorgelegtvon
BernadetteT.SaunierRebori
München,February2008

























Erstgutachter : Prof. Dr. Benedikt Grothe

Zweitgutachter : Prof. Dr. Axel Borst

Tag d. mündl. Prüfung : 31. März 2008

ERKLÄRUNG

Ich versichere,daβ ich meineDissertation selbstständig,ohneunerlaubteHilfe
angefertigt, und mich dabei keiner anderen als der von mir ausdrücklich
bezeichnetenHilfenundQuellenbedienthabe.
DieDissertationwurdeinderjetzigenoderähnlichenFormbeikeineranderen
Hochschule eingereicht und hat noch keinen sonstigen Prüfungszwecken
gedient.




_____________________ _______________________
(Ort,Datum) (BernadetteT.SaunierRebori)
















(…) antes de descubrir, en descubrirnos; antes
de modelar la Naturaleza, en modelarnos.
Forjarnos un cerebro fuerte, un cerebro original,
exclusivamente nuestro: he aquí la labor
preliminarabsolutamenteinexcusable.

Reglas y consejos sobre la investigación
científica. Los tónicos de la voluntad.
Santiago Ramón y Cajal.

TABLEOFCONTENTS

ACKNOWLEDMENTS...........................................................................................1

1.ABSTRACT .......................................................................................................3

2.INTRODUCTION................................................................................................5
2.1Soundlocalizationinthemammalianbrain................................................5
2.2BinauralprocessingintheDNLL................................................................7
2.3Theprecedenceeffectandechosuppression .........................................10
2.4Synaptictransmission...............................................................................13
2.5TheMongoliangerbilasananimalmodel ...............................................17
2.6Scopeofthestudy....................................................................................18

3.MATERIALSANDMETHODS........................................................................21
3.1Invivorecordings .....................................................................................21
3.1.1Animalpreparation...........................................................................21
3.1.2Stereotaticprocedure.......................................................................21
3.1.3Recordingprocedure .......................................................................22
3.1.4Acousticstimuli ................................................................................23
3.1.5Dataanalysis....................................................................................24
3.1.6Histology ..........................................................................................25
3.2Invitrorecordings .....................................................................................27
3.2.1Brainslicepreparation .....................................................................27
3.2.2Wholecellrecordings.......................................................................31
3.2.3Stimulationofsynapticinputs ..........................................................32
3.2.4Dataanalysis....................................................................................32
3.2.5Statisticalanalysis............................................................................33
3.3Appendix...................................................................................................34
3.3.1Histology ..........................................................................................34
3.3.1.1.Ringersolutioncontainingheparin(foranimalperfusion) ...........34
3.3.1.2.Nisslstaining................................................................................34
I

3.3.2Dataanalysis....................................................................................35
3.3.2.1.Convert.pl.....................................................................................35
3.3.2.2.Lastpeak......................................................................................36
3.3.2.3.Allpeaks.......................................................................................37
3.3.2.4.bpc_Mini.xop................................................................................38

4.RESULTS ........................................................................................................39
4.1BinauralresponsepropertiesofDNLLneurons.......................................39
4.2Invitrophysiology.....................................................................................42
4.2.1 The contralateral DNLL provides the long lasting GABAergic
inhibition ....................................................................................................42
4.2.2 The decay time constant of GABAergic IPSCs can explain the PI
foundinvivoandinvitro ...........................................................................45
4.2.3 The stimulation strength increase in the IPSC kinetics suggest a
possibleroleofspilloverinDNLLlonglastinginhibition...........................46
4.2.4 Presynaptic mechanisms might be involved in extending synaptic
inhibitioninDNLLneurons........................................................................51
4.2.5TheapplicationofalowaffinityGABA receptorantagonistsuggestA
theinvolvementofextrasynapticreceptorsinprolongingsynapticinhibition
inDNLL .....................................................................................................56
4.2.6BlockingGABAclearanceprolongedtheIPSCkinetics..................63
4.2.7ReducingreleaseprobabilitydecreasedtheIPSCskinetics...........68
4.2.8GlycinergicinputsontoDNLLneurons ............................................74

5.DISCUSSION...................................................................................................83
5.1PersistentinhibitionintheDNLLoftheMongoliangerbil.........................83
5.2DNLLcelltypesandtheirphysiologicalpropertiesinvitro.......................85
5.3ThesourceofPIintheDNLL...................................................................87
5.4SpilloverprolongssynapticinhibitionintheDNLL ...................................89
5.5AsynchronousreleaseinDNLLprincipalneuronscontributestoPIathigh
frequencyactivitylevels..................................................................................97
5.6GlycinergicinputsontoDNLLneurons.....................................................99
II


6.CONCLUSIONS.............................................................................................101

7.REFERENCES...............................................................................................105

LISTOFABBREVIATIONS ..............................................................................117

CURRICULUMVITAE……………………………………………………………..121
III

INDEXOFTABLESANDFIGURES

Fig.2.1.TheDNLLcircuitry…………………………………………………………..8
Fig.2.2.Theprecedenceeffect…………………………………………….……….11
Fig. 3.2.1. Schematic illustration of DNLL location in the mammalian brain
………………………………………………………………………………….………27
Table3.2.1.ExtracellularsolutionsforslicingandrecordingDNLL………….…28
Table3.2.2.Internalsolutionsforvoltageandcurrentclamprecordings…….....29
Table3.2.3.Drugsandconcentrationsusedduringtheexperiments…………...30
Fig.4.1.PIevokedinvivobyipsilateralstimulation……………………………….40
Fig.4.2.PIevokedinvitrobystimulationoftheCommissureofProbst………...44
Fig.4.3.DecaytimeofevokedIPSCsinvitromimicsPIinvivo……………….…46
Fig.4.4.GABAergicIPSCskineticsdependencyonstimulationstrength…….…49
Fig.4.5.DepressioncurvesofGABAergicIPSCsafterastimulationtrain……...53
Fig.4.6.FrequencydependencyofGABAergicIPSCsafterastimulationtrain
………………………………………………………………………………………….54
Fig.4.7.Effectof100IMTPMPAontheIPSCskinet ics………………………..57
Fig. 4.8. Effect of 200 IM TPMPA on the kinetics of the last IPSC after
stimulationtrain……………………………………………………………………...59
Fig.4.9.Effectof200IMTPMPAonthekineticsof asingleevokedIPSC..…61
Fig.4.10..EffectofalowconcentrationofahighaffinityGABA antagonistonA
theIPSCskinetics…………………………………………………………………...63
Fig.4.11.EffectofNO711ontheIPSCsdecay………………………………….64
Fig.4.12.LowconcentrationsofNNC711prolongsIPSCskinetics…………..66
Fig.4.13.ReducingtheextracellularcalciumreducestheIPSCskinetics…....69
Fig.4.14.ThereductiononextracelluarcalciumreducesGABAspillover…….71
Fig.4.15.CadmiuminducesareductionofGABAspillover……………………73
Fig.4.16.DNLLneuronsreceivebothGABAandglycinergicinputs……….…76
Fig.4.17.DependencyofglycinergicIPSCskineticsonstimulationstrength...79
Fig.4.18.FrequencydependencyofglycinergicIPSCs…………...……………81
IVAknowledments
_____________________________________________________________________


ACKNOWLEDMENTS
IwouldliketothankmysupervisorProf.Dr.BenediktGrotheforgivingmethe
opportunitytojoinhislabandforhisguidanceduringmyPhD.
IalsowanttothankDr.FelixFelmyforhiscontagiousenthusiasmsforscience
andforhispermanentinterestindiscussingexperimentsandgiveadvice.Many
thankstoDr.AchimKlugfortheirsupportandadvice.
Thanksto allpresent and pastmembersof the Grothe group. Manythanksto
Teresa Kindermann, Ben Haßfurth and Gabi Schebesch for their constant
support,theirfriendshipandbecausemytimeinthelabwouldhavebeenless
enjoyable without them. I would also like to thank Dr. Ursula Koch for fruitful
discussionsandadvicethroughoutalltheseyears.IalsothankClaudiaSchulte
and Olga Alexandrova for their incalculable assistance and to Nick Lesica for
proofreadingthethesis!
Quiero agradecer enormemente a todos mis amigos de acá y allá que me
brindaronsuapoyoysucariñodurantetodosestosaños:NicoPlachtaySteffi
Bissiere,MatiOkawayEvaWertheimer,GabiPirk,NatalyO’Leary,MiraKuisle,
ArielMéndezyCaroHaupt,IannaBechten,LuleKargiemann,BarbaraMulinacci,
StefanoPegoraro,JoséÁlvarezCastroymuchosotrosqueseguramenteolvido
mencionarenestemomento.GRACIASporestarahísiempre!!!
Finalmente, mi mayor gratitud es para toda mi familia (incluidos Luli, tíos y
primos)porcuidarmeyquerermetantoyhacermesentirqueladistancianoera
tangrande.Quieroagradecerespecialmenteamisviejosporquegraciasaellos
hoyterminomidoctorado.Amimamáporcreerenmímásdeloqueyomisma
creo y por siempre darme el coraje para seguir adelante y llevar acabo mis
sueños.GraciasGIGANTESamishermanosporapoyarmeenmidecisiónde
irme tan lejos, por ser mis amigos más queridos y por estar siempre cerca.
Graciasamiscuñadosporsuapoyoincondicionalyamishermosossobrinos,
SofiyTobiporsuenormeamoryporlapacienciainfinitadetenerunatíadel
otroladodelocéano.
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