A role for the cation-chloride cotransporter KCC2 in inhibitory synaptogenesis

Thesis

Reference

A role for the cation-chloride cotransporter KCC2 in inhibitory synaptogenesis

LACOH, Claudia-Marvine

Abstract

Recent data provide a role for KCC2 in dendritic spine formation and, thereby, in excitatory synaptogenesis. Here we investigated whether this cation-chloride cotransporter is involved in inhibitory synapse formation. To visualize inhibitory synapses, we co-electroporated a molecular construct coding for gephyrin, a major component of the postsynaptic protein network in inhibitory synapses, with a plasmid coding for KCC2 into progenitors of layer 2/3 pyramidal neurons by means of in utero electroporation in rats. To reveal detailed neuronal arbor architecture, electroporated neurons were iontophoretically injected using Lucifer Yellow. Confocal microscopy was used to analyze spatial distribution and density of gephyrin clusters along with their relation to dendritic spines. We found out that precocious expression of KCC2 leads to decreased gephyrin cluster densities in pyramidal neurons associated with an increased dendritic spine density. These observations suggest a role for KCC2 in the establishment of excitation/inhibition balance during neural circuitry development.

LACOH, Claudia-Marvine. A role for the cation-chloride cotransporter KCC2 in inhibitory synaptogenesis. Thèse de doctorat : Univ. Genève et Lausanne, 2016, no. Neur. 183

DOI : 10.13097/archive-ouverte/unige:101353 URN : urn:nbn:ch:unige-1013535

Available at:

http://archive-ouverte.unige.ch/unige:101353

DOCTORAT EN NEUROSCIENCES des Universités de Genève

et de Lausanne

UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Directeur de thèse: Professeur Laszlo Vutskits

TITRE DE LA THÈSE

A ROLE FOR THE CATION-CHLORIDE COTRANSPORTER KCC2 IN INHIBITORY SYNAPTOGENESIS

THÈSE Présentée à la Faculté de Médécine de l’Université de Genève

pour obtenir le grade de Docteure en Neurosciences

par

Claudia-Marvine Dede Lacoh du Togo

Thèse N° 183 Genève

Editeur ou imprimeur : Université de Genève Octobre, 2016

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Acknowledgments!

!!

“Do!not!see!what!others!can!bring!you!is!being!narrow!minded!and!ungenerous”!I!do!not!

remember!exactly!where!I!have!heard!this!sentence!but!it!partly!summarizes!these!past!

five!years.!They!were!for!me!a!plethora!of!emotions!of!all!kinds:!joy,!sadness,!serenity,!

deception,! optimism,! disgust,! love,! resignation,! attraction,! outrage,! submission,!

apprehension,! surprise,! anger,! discoveries.! However,! I! am! thankful! for! all! these!

emotions;! they! helped! me! to! be! stronger.! So! I! wanted! to! thank! all! the! people! without!

whom!this!adventure!would!not!have!been!possible.!

!

First,!thanks!to!Professor!Vutskits,!who!gave!me!the!opportunity!to!do!a!thesis!in!his!lab!

and! a! special! thanks! to! Dr.! De! Roo! who! gave! me! considerable! help! in! correcting! my!

thesis.!

!

I!sincerely!want!to!thank!my!lovely!and!dear!Catherine!Fouda.!She!is!the!big!sister!I!have!

never! had,! attentive! to! my! concerns.! Her! thoughtful! advices! were! precious! along! all!

these!years.!I!thank!her!also!for!her!patience!and!help!in!all!westernNblot!experiments.!

These!years!will!not!have!been!the!same!without!her.!

!

Thanks!to!Dr.!JeanNChristophe!Copin!for!sharing!his!knowledge!with!me.!

!

Thanks! to! Dr.! Hubert! Fiumelli! for! teaching! me! the!in#utero! electroporation! procedure!

and!for!his!great!help!in!establishing!the!surgical!platform!in!our!lab.!

!

Thanks!you!to!all!my!colleagues:!

N Adrian!Briner,!for!his!guidance!and!supervision!during!my!master!internship!!

N Teddy!Belem,!for!his!encouragement!and!confidence!in!my!abilities!

N Gréta!Limoni,!for!her!listening!skills!and!her!availability!

N Mari!Virtanen,!from!whom!I!learned!a!lot!

N Michelle!Brunet,!for!her!help!in!building!up!the!surgical!setup!and!during!all!the!

electroporation!procedures!

N Samira!Osterop,!Abdurahman!Mohamad!Mah,!Galil!Mori!for!those!moments!of!fun!

Thanks!to!Aurore!Perrault!and!Natalia!Fernandez!for!the!countless!hours!of!discussions!

and!for!their!emotional!support.!

!

Thanks!to!all!the!members!of!Kiss’!lab,!especially!Beatrice!King,!Cynthia!Saadi,!Elodie!

Husi!for!their!technical!assistance!and!supports.!!

!

Thank!to!all!the!members!of!Dayer’s!and!Jabaudon’s!labs!for!the!discussions!and!

friendships.!

!

Thanks!to!Mme!Christina!Bouldin,!Céline!Brockmann!and!Raquel!Mendez!for!their!help!

in!administrative!procedures.!

!

Thanks!to!all!the!kind!people!I!have!met!in!the!corridors!and!with!whom!I!have!had!the!

opportunity!to!exchange!few!words.!

!

Thanks!to!all!my!family,!my!friends!and!especially!Lilian.!!You!never!stopped!believing!in!

me!and!you!always!managed!to!give!me!a!smile!when!needed!or!not.!A!special!thought!to!

"KNdo"!in!memory!of!our!childhood.!I!am!trying!to!be!as!good!as!your!expectations!by!

giving!the!best!of!myself.!!

!

Finally,! I! would! like! to! dedicate! this! PhD! to! my! parents! Hanou! Yolande! Amevor! and!

Dominique!Amouzou!Lacoh!whose!support!and!love!are!boundless.!They!are!my!armor,!

the!shields!that!allow!me!to!go!forward!without!being!hindered.!I!wanted!to!thank!them!

for!this!unrestricted!love!and!unwavering!strength.!!

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Abstract!

ActivityNdependent! assembly! of! developing! central! nervous! system! circuitry! is! a!

fundamental! requisite! for! proper! neuronal! network! function.! Deciphering! molecular!

candidates! involved! in! circuitry! formation! will! not! only! deepen! our! understanding! on!

brain! function! but,! ultimately,! may! also! lead! to! therapeutic! options.! During! the! past!

decade,! the! cationNchloride! cotransporter! KCC2! emerged! as! an! intriguing! candidate! in!

this! context.! By! regulating! intracellular! chloride! concentration,! KCC2! is! a! major!

determinant! of! the! functional! modalities! of! GABAA! receptorNmediated! neuroN transmission.! Importantly,! recent! observations! suggest! that! KCC2! also! plays! morphoN functional!roles!independently!of!its!ion!transporter!function.!In!this!context,!my!thesis!

work!is!focused!on!the!biology!of!KCC2!during!brain!development!by!asking!two!distinct!

questions:!(i)!Does!general!anaesthesia!affect!the!developmental!expression!pattern!of!

KCC2;!and!(ii)!Does!KCC2!play!a!role!in!the!formation!of!inhibitory!synaptogenesis.!

!

The!first!question,!regarding!the!role!of!general!anaesthesia!in!the!expression!of!KCC2,!

was! motivated! by! seminal! works! suggesting! that! the! developmental! increase! in! the!

expression! pattern! of! KCC2! and! the! concomitant! transition! from! inhibitory! toward!

excitatory! modalities! of! GABAA! receptorNmediated! signalling! is! triggered! by! increased!

GABAergic!activity!during!the!brain!growth!spurt.!Therefore,!we!set!out!the!hypothesis!

that! exposure! of! the! developing! brain! to! general! anaesthetics,! acting! as! positive!

allosteric!modulators!of!GABAA!signalling!may!trigger!the!expression!of!KCC2!and!this,!in!

turn,!could!result!in!a!precocious!functional!transition!of!GABAergic!neurotransmission.!

In!this!part!of!my!thesis!work,!I!have!thus!performed!controlled!anaesthesia!using!either!

propofol,!midazolam!or!ketamine!at!distinct!stages!of!the!brain!growth!spurt!in!Wistar!

rat!pups!and!examined!how!these!anaesthesia!protocols!affect!the!expression!of!KCC2!in!

the!developing!brain.!Results!of!these!experiments!revealed!that!the!applied!anaesthesia!

protocols! did! not! affect! the! physiological! expression! of! KCC2! at! any! of! the!

developmental!stages!investigated.!Importantly,!since!general!anaesthetics!are!powerful!

modulators! of! neuronal! activity,! this! work! also! suggest! that! KCC2! gene! and! protein!

expression!is!not!influenced!in!cortical!neurons!in!an!activityNdependent!manner!during!

normal!development.!

!

The!second!line!of!investigations!of!my!thesis!work!aimed!to!elucidate!whether!and!how!

KCC2!is!implicated!in!inhibitory!synaptogenesis!during!brain!development.!The!rational!

for! this! question! stems! from! previous! series! of! investigations! from! others! as! well! as!

from!our!own!laboratory!demonstrating!a!role!for!KCC2!in!the!formation!of!excitatory!

synaptic! contacts.! However,! before! the! work! conducted! herein,! the! role! of! this! cation!

transporter!in!inhibitory!synaptogenesis!remained!unknown.!To!investigate!this!issue,!

we!have!developed!an!in#vivo!model!allowing!us!to!trace!the!development!of!inhibitory!

synaptic! contacts! onto! pyramidal! cells! in! the! cerebral! cortex.! To! visualize! inhibitory!

synapses,!we!used!a!molecular!construct!coding!for!gephyrin,!a!major!component!of!the!

postsynaptic! protein! network! in! inhibitory! synapses.! This! TomatoNtagged! gephyrin!

construct! was! coNelectroporated! with! a! plasmid! coding! for! KCC2! into! progenitors! of!

layer! II/III! pyramidal! neurons! by! means! of!in!utero! electroporation! at! gestational! day!

17.5! in! Wistar! rats.! In! order! to! reveal! detailed! neuronal! arbor! architecture,!

electroporated!neurons!were!iontophoretically!injected!with!Lucifer!Yellow!fluorescent!

protein.! Confocal! microscopy! was! used! to! analyse! spatial! distribution! and! density! of!

gephyrinNTomato!clusters!along!basal!dendrites!of!layer!II/III!gephyrinNTomato!positive!

pyramidal! cells! with! their! relation! to! dendritic! spines.! We! found! out! that! precocious!

expression!of!KCC2!by!means!of!in#utero!electroporation!led!to!an!overall!decrease!in!the!

number! of! gephyrinNTomato! clusters! on! layer! II/III! pyramidal! neurons! in! the! medial!

prefrontal! cortex.! Spatial! analysis! of! gephyrinNTomato! clusters! distribution! revealed!

that!this!decrease!is!primarily!due!to!the!lower!number!of!gephyrinNTomato!clusters!on!

proximal! dendritic! segments! in! within! a! distance! of! 40! μm! from! the! cell! body.!

Importantly,!an!increased!dendritic!spine!density!accompanied!the!decreased!gephyrinN Tomato! cluster! density! on! these! same! proximal! dendritic! segments! of! layer! II/III!

pyramidal! neurons.! In! conclusion,! precocious! expression! of! KCC2! led! to! decreased!

gephyrinNTomato! clusters! density! in! pyramidal! neurons! along! with! an! increase! in! the!

number!of!dendritic!spines.!Importantly,!precocious!expression!of!a!functional!mutant!of!

KCC2!that!lacks!the!ion!transporter!function!resulted!in!very!similar!results,!suggesting!

thereby! that! the! effect! of! KCC2! on! inhibitory! synaptogenesis! is! independent! of! its! ion!

transporter!function.!These!observations,!along!with!data!demonstrating!an!increase!in!

the! number! of! excitatory! synapses! in! these! same! cells,! suggest! an! ion! transport!

independent!role!for!KCC2!in!the!establishment!of!excitation/inhibition!balance!during!

neural!circuitry!development.!

Résumé!

La!formation!du!circuit!neuronal!basée!sur!l’activité!cérébrale!est!un!élément!inhérent!

au!fonctionnement!du!réseau.!L’analyse!des!molécules!impliquées!dans!la!formation!de!

ce! circuit! va! non! seulement! nous! permettre! d’approfondir! nos! connaissances! sur! le!

fonctionnement! du! cerveau,! mais! pourrait! également! nous! conduire! vers! de! nouvelles!

pistes! thérapeutiques.! Dans! ce! contexte,! le! cotransporteur! cationNchloride! KCC2! s’est!

récemment! révélé! être! une! molécule! prometteuse.! En! régulant! la! concentration!

intracellulaire!en!ions!chlorure,!KCC2!apparaît!comme!l’un!des!acteurs!majeurs!influant!

sur! les! fonctionnalités! de! la! neurotransmission! médiée! par! les! récepteurs! GABAA.! Qui!

plus! est,! de! récentes! données! suggèrent! qu’indépendamment! de! sa! fonction! de!

transporteur!d’ions,!KCC2!jouerait!en!plus!un!rôle!morphoNfonctionnel.!Pour!toutes!ces!

raisons,!mon!projet!de!thèse!se!concentre!sur!l’étude!de!KCC2!durant!le!développement!

du! cerveau.! Ce! projet! est! axé! sur! deux! questions! bien! distinctes! i)! ! EstNce! que!

l’anesthésie! générale! affecte! l’expression! de! KCC2! au! cours! du! développement!?! Et! ii)!

EstNce!que!KCC2!joue!un!rôle!dans!la!formation!des!synapses!inhibitrices!?!!

!

La! première! question! a! été! motivée! par! de! nombreux! travaux! suggérant! que!

l’augmentation! de! l’expression! de! KCC2! au! cours! du! développement! fait! partie! des!

modalités! permettant! la! transition! d’une! transmission! GABAergique! excitatrice! rapide!

vers!une!transmission!GABAergique!inhibitrice!rapide!durant!la!poussée!de!croissance!

du!cerveau.!Par!conséquent,!nous!avons!défini!comme!hypothèse!que!l’exposition!d’un!

cerveau! en! développement! aux! agents! anesthésiants,! qui! agissent! comme! des!

modulateurs! allostériques! positifs! des! récepteurs! GABAA,! pourrait! déclencher!

l’expression!de!KCC2!et!donner!lieu!à!une!transition!précoce!dans!l’action!de!GABA.!Dans!

cette! partie! de! mon! travail,! j’ai! donc! réalisé! des! protocoles! d’anesthésie! avec! soit! le!

propofol,! soit! le! midazolam,! soit! la! kétamine! et! analysé! comment! ces! agents!

anesthésiants! pourraient! impacter! sur! l’expression! de! KCC2! dans! le! cerveau! en!

développement.!Les!résultats!ont!montré!qu’aucun!de!ces!agents!n’affecte!l’expression!

de! KCC2! quelque! soit! l’âge! d’exposition.! Les! anesthésiants! généraux! étant! considérés!

comme!de!puissants!modulateurs!de!l’activité!neuronale,!notre!étude!suggère!ainsi!que!

l’expression!de!KCC2,!que!ce!soit!au!niveau!de!l’expression!du!gène!ou!des!protéines,!ne!

dépend!pas!de!l’activité!neuronale!durant!le!développement.!!

La! seconde! partie! de! mes! expériences! porte! sur! la! question! de! l’implication! de! KCC2!!

dans!la!synaptogenèse!inhibitrice.!Plusieurs!études!réalisées!dans!divers!laboratoires,!y!

compris! le! notre,! ont! démontré! le! rôle! de! KCC2! dans! la! formation! de! synapses!

excitatrices.!Cependant,!la!possibilité!d’un!rôle!de!KCC2!sur!la!formation!des!synapses!

inhibitrices! n’avait! pas! été! étudiée! jusqu’alors.! Pour! répondre! à! cette! question,! nous!

avons! développé! un! modèle!in# vivo! nous! permettant! de! suivre! le! développement! des!

contacts!inhibiteurs!sur!les!cellules!pyramidales!du!cortex!cérébral.!Afin!de!visualiser!les!

synapses! inhibitrices,! nous! avons! utilisé! une! construction! moléculaire! codant! pour!

gephyrin,!un!des!composants!majeurs!du!réseau!protéique!postNsynaptique!des!synapses!

inhibitrices.! Cette! sonde! nommée!Geph/Tom! a! été! coNelectroporée!in# utero! avec! un!

plasmide! codant! pour! KCC2! dans! les! progéniteurs! de! la! couche! II/III! des! neurones!

pyramidaux!au!jour!de!gestation!17,5!chez!le!rat.!Afin!de!révéler!en!détail!l’architecture!

neuronale,!les!neurones!électroporés!ont!été!par!la!suite!injectés!par!iontophorèse!avec!

une!sonde!fluorescente!:!le!Lucifer#Yellow.!Les!images!prises!en!microscopie!confocale!au!

niveau! du! cortex! préfrontal! médian! ont! permis! d’analyser! les! distributions! spatiales!

respectives! des! clusters! de!gephyrin#et! des! épines! dendritiques! le! long! des! dendrites!

basaux! des! cellules! pyramidales.! Nous! avons! découvert! que! l’expression! précoce! de!

KCC2! conduit! à! une! diminution! du! nombre! de!clusters! de!gephyrin! exogène! dans! ces!

neurones.! L’analyse! spatiale! de! la! distribution! de! ces! clusters! a! révélé! que! cette!

réduction! est! principalement! localisée! sur! les! segments! dendritiques! situés! à! une!

distance! inférieure! à! 40! μm! du! corps! cellulaire.! Fait! important,! une! densité! accrue!

d'épines! dendritiques! accompagne! cet! effet! sur! les! mêmes! segments! dendritiques.! En!

conclusion,! l'expression! précoce! de! KCC2! conduit! à! une! diminution! de! la! densité! des!

clusters! de! gephyrin! dans! les! neurones! pyramidaux.! Notablement,! les! mêmes!

expériences! réalisées! avec! un! mutant! de! KCC2! dépourvu! de! sa! fonction! transporteur!

aboutissent!à!des!résultats!similaires.!L’ensemble!de!ces!résultats!suggère!un!rôle!pour!

KCC2! dans! l’établissement! de! la! balance! excitation/inhibition! indépendamment! de! sa!

fonction!transporteur.!!

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List!of!abbreviations!

!

ACC:!! ! ! anterior!cingular!cortex!

AMPA:!! ! acide!alphaNaminoN3NhydroxyN5NméthylN4Nisoxazolepropionique!

ATD:!! ! ! amino!terminal!domain!

BDNF:!! ! brainNderived!neurotrophic!factor!

BMPs:!!! ! bone!morphogenetic!proteins!

Ca²⁺:!! ! ! calcium!

CA1/CA3:!! ! cornu!ammonis!1!and!3!

CAM:!! ! ! cell!adhesion!molecule!

CaMKII:!! ! Ca²⁺/calmodulinNdependent!protein!kinase!II!

CDCN42:!! ! cell!division!cycle!42!

cDNA!:!! ! complementary!deoxyribonucleic!acid!

CNS:!! ! ! central!nervous!system!

CREB:!!! ! CNAMP!response!elementNbinding!protein!

CTD:!! ! ! carboxyl!terminal!domain!

DNA:!! ! ! deoxyribonucleic!acid!

E17.5:!!! ! embryonic!day!17.5!

Eph:!! ! ! ephrin!

EPSP:!!! ! excitatory!postsynaptic!potential!!

GABA:!! ! γNaminobutyric!acid!!

GABAAR:! ! γNaminobutyric!acid!receptor!type!A!

GABABR:!! ! γNaminobutyric!acid!receptor!type!B!

GABACR:!! ! γNaminobutyric!acid!receptor!type!C!

GABARAP:!! ! GABAARNassociated!proteins!!

GD:! ! ! gestational!day!

GDP/GTP:!! ! guanosine!di/triphosphate!!

GDPs:!!! ! giant!depolarizing!potentials!

GephNTom:!! ! gephyrinNTomato!

GFP:!! ! ! green!fluorescent!protein!!

GKAP:!!! ! guanylate!kinaseNassociated!protein!

GlyR:!! ! ! glycine!receptor!

GPI:!! ! ! glycosylphosphatidylinositol!

GSK3β:!! ! glycogen!synthase!kinase!3β!!

Ig:!! ! ! immunoglobulin!

IPSP:!! ! ! inhibitory!postsynaptic!potential!

IUE:!! ! ! in#utero!electroporation!!

KARs:!!! ! kainate!receptors!

KCC2:!!! ! potassium!(K⁺)/chloride!(Cl^N)!cotransporter!!isoform!2!!

KCC2NFL:!! ! KCC2!full!length!

KCC2NΔNTD:!!! KCC2!with!NNterminal!deleted!

Kir2.1:!! ! inwardNrectifier!potassium!ion!channel!

LBD:!! ! ! lingand!binding!domain!

LTD:!! ! ! longNterm!depression!

LTP:!! ! ! longNterm!potentiation!

LY:!! ! ! lucifer!yellow!

MAGUK:!! ! membraneNassociated!guanylate!kinase!

mEPSC:!! ! miniature!excitatory!postsynaptic!currents!

Mg²⁺:!! ! ! magnesium!ion!!

mPFC!:!! ! medial!prefrontal!cortex!

NaCl:!! ! ! sodium!chloride!

NCAM:## # neural!cell!adhesion!molecule#

NKCCs:!! ! sodium!(Na⁺)!potassium!(K⁺)!cotransporters!

NMDA:!! ! NNmethylNDNaspartate!

NSF:!! ! ! NNethlymaleimide!fusion!proteins!

PBS:!! ! ! phosphate!buffer!saline!

PCW:!! ! ! post!conception!weeks!

PH:!! ! ! pleckstrin!homology!

PKA:!! ! ! protein!kinase!A!

PSA:!! ! ! polysialic!acid!

PSD:!! ! ! postsynaptic!density!

PSDN95:!! ! postsynaptic!densityN95!

PKC:!! ! ! protein!kinase!C!

PP1:!! ! ! protein!phosphatase!1!

P20:!! ! ! postnatal!day!20!

RNAi:!!! ! ribonucleic!acid!interference!!

RTNPCR:!! ! reverse!transcription!polymerase!chain!reaction!

Sema:!!! ! semaphorin!

SH3:!! ! ! srcNhomology!3!

sIPSCs:!! ! spontaneous!inhibitory!postsynaptic!currents!

Slit!1:!!! ! slit!homolog!1!!

SNAP:!!! ! soluble!NSF!attachment!proteins!!

SNAREs!:!! ! SNAP!receptors!

SYGN1:!! ! synaptogenesis!1!

SynCAM:!! ! synaptic!adhesion!molecule!

TARPS:!! ! transmembrane!AMPA!receptor!prteins!

TEM:!! ! ! transmission!electron!microscope!

TGFNβ:!! ! transforming!growth!factor!β!

TMD:!! ! ! transmembrane!domain!

Wnt:!! ! ! wingless!int!

WT:!! ! ! wild!type!

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Table!of!contents!

List!of!figures!!

List!of!tables!

Introduction!...!14!

Part!I.!Gatekeepers!for!information!flow!...!16!

1.!Developmental!processes!in!the!central!nervous!system!...!16!

2.!Developmental!timeline!of!postnatal!rodent!synaptogenesis!...!18!

3.!Synaptogenesis!...!20!

3.1.!!Appropriate!target!cell!...!20!

3.2.!!Functional!synapse!...!22!

3.2.1.!PreN!and!postN!sides!assembly!...!22!

3.2.1.1.!CellNcell!adhesion!components!...!22!

3.2.1.2.!Clustering!molecules!...!25!

3.2.2.!Synapse!formation!...!26!

3.2.2.1.!!Presynaptic!specialization!...!26!

3.2.2.2.!!Postsynaptic!specialization!...!26!

Part!II.!Signalling!through!synapses!...!28!

1.!GABAergic!synapses!...!28!

1.1.!GABAA!receptors!...!28!

1.1.!1.!Structure!and!function!...!28!

1.1.2.!Region!dependent!inhibition!(synaptic!versus!extrasynaptic)!...!29!

1.2.!Characteristics!of!GABAergic!transmission!...!30!

1.2.1.!Giant!depolarization!potentials!...!30!

1.2.2.!Developmental!changes!in!GABAergic!transmission!...!30!

2.!Glutamatergic!synapses!...!31!

2.1.!AMPA!and!NMDA!receptors!...!32!

2.1.1.!Structure!and!function!...!32!

2.1.2.!Differences!between!AMPA!and!NMDA!receptors!...!32!

2.2.!Characteristics!of!glutamatergic!transmission!...!33!

2.2.1.!LongNterm!potentiation!and!longNterm!depression!...!33!

2.2.2.!Synergistic!excitatory!actions!of!GABAA!and!NMDA!receptors!...!34!

Part!III.!Detection!of!excitatory!and!inhibitory!postsynaptic!contacts!in!the!CNS!.!35! 1.!Electron!microscopy!...!35!

2.!Immunocyto#(histo)!chemistry!...!36!

2.1.!PSDN95:!a!marker!of!excitatory!postsynaptic!contacts!...!37!

2.1.1.!PSDN95!...!37!

2.1.1.1.!PSDN95!and!NMDA!receptors!...!37!

2.1.1.2.!PSDN95!and!AMPA!receptors!...!38!

2.2.!Gephyrin:!a!marker!of!inhibitory!postsynaptic!contacts!...!38!

2.2.1.!Gephyrin!...!39!

2.2.1.1.!Gephyrin!in!GABAA!receptors!clustering!...!40!

2.2.1.2.!Gephyrin!clustering!...!41!

Part!IV.!KCC2,!a!factor!in!functional!development!of!both!excitatory!and!inhibitory! neurotransmission!...!42!

1.!Structure!...!42!

2.!Expression!...!43!

3.!Function!...!44!

3.1.!KCC2!in!GABA!signalling!...!44!

3.2.!KCC2!at!excitatory!spines!...!44!

4.!Regulation!...!45!

4.1.!Transcription!and!neurotrophic!regulation!...!45!

4.2.!PostNtranslational!regulation!...!46!

5.!Disease!and!treatment!...!47!

Part! V.! General! anaesthesia! and! the! developing! brain:! contextQdependent! modulation!of!neural!plasticity!...!48!

Aims!of!the!thesis!...!50!

Materials!and!methods!...!51!

1.#In#vivo!proteins!overexpression!procedure!...!51!

1.1.!Expression!vectors!for!in#utero!electroporation!...!51!

1.2.!In#utero#electroporation!(IUE)!...!51!

2.!Labelling!of!layer!II/III!pyramidal!neurons!...!53!

2.1!Preparation!of!brain!slices!...!53!

2.2.!Iontophoretic!post#hoc!single!cell!injections!...!53!

2.3.!Immunohistochemistry!...!54!

3.!Quantification!of!gephyrinNTomato!clusters!and!dendritic!protrusions!...!55!

3.1.!Image!acquisition!and!selection!of!cells!for!analysis!...!55!

3.2.!Reconstruction,!quantification!and!analysis!...!56!

3.2.1.!Quantification!of!gephyrin!clusters!...!56!

3.2.2.!Quantification!of!dendritic!protrusions!...!56!

3.3.!Sholl!analysis!...!58!

4.!Statistics!...!59!

Results!...!60!

1.!Establishment!of!an!in!vivo!model!allowing!quantification!of!inhibitory!and! excitatory!synapses!in!Layer!II/III!medial!prefrontal!cortex!pyramidal!neurons!...!61!

2.!Precocious!expression!of!KCC2!impacts!on!gephyrinNTomato!cluster!density!at! juvenile!stage!...!63!

3.!Precocious!expression!of!KCC2!impacts!on!protrusion!density!and!diameter!at! juvenile!stage!...!65!

4.!Precocious!expression!of!KCC2!leads!to!a!modification!of!the!ratio!of!excitatory! versus!inhibitory!postNsynaptic!elements!at!juvenile!stage!in!a!spatialNdependent! manner!...!67!

5.!The!effect!of!precocious!expression!of!KCC2!on!gephyrinNtomato!cluster!density!at! juvenile!stage!does!not!depend!on!KCC2!chloride!transport!function!...!69!

Discussion!...!71!

Conclusion!and!future!perspectives.!...!79!

References!...!81!

!

!

!

!

!

!

!

!

!

!

!

List!of!figures!

!

!

Figure 1. Timelines of developmental processes in the central nervous system of rats (Rice

and Barone 2000)!...!17!

Figure 2. Timelines of developmental processes in the central nervous system of humans (Rice and Barone 2000).!...!17!

Figure 3. Densities of synapses in the visual cortex of the macaque monkey!...!18!

Figure 4. Contact-mediated recognition proteins and their binding partners.!...!25!

Figure 5. GABAergic inhibitions (Ge, Pradhan et al. 2007).!...!30!

Figure 6. An electron micrograph (EM) of symmetric and asymmetric synapses in cortex (Kuzirian and Paradis 2011).!...!36!

Figure 7. Molecular organization of the PSD of excitatory synapses (Sheng and Kim 2011).37! Figure 8. Postsynaptic organization of inhibitory GABAergic synapses (Sheng and Kim 2011).!...!39!

Figure 9. Schematic representation of gephyrin with structural homology to Moco biosynthetic proteins (Stallmeyer, Schwarz et al. 1999).!...!39!

Figure 10. Regulatory sites on KCC2 protein.!...!42!

Figure 11. Differential development of NKCC1 (pink) and KCC2 (blue) expression in the brain.!...!43!

Figure 12. In utero electroporation procedure.!...!52!

Figure 13. Iontophoretic post hoc single cell injection of Lucifer Yellow in gephyrin positive layer II/III pyramidal neurons.!...!54!

Figure 14.!Immunohistochemistry against Lucifer Yellow injected cells.!...!55!

Figure 15.!Basal arborisation of Lucifer Yellow-injected neurons expressing gephyrin- Tomato.!...!56!

Figure 16. 3D-Reconstruction of LY- injected neurons expressing gephyrin-Tomato.!...!57!

Figure 17.!Illustration of gephyrin-Tomato cluster and dendritic protrusions quantification process.!...!58!

Figure 18. Sholl analysis on both gephyrin-Tomato clusters and dendritic protrusions trees.!59! Figure 19.!Quantification of inhibitory and excitatory synapses in Layer II/III medial prefrontal cortex pyramidal neurons.!...!62!

Figure 20.!Precocious expression of KCC2 impacts on gephyrin-Tomato cluster density at juvenile stage.!...!64!

Figure 21.!!Precocious expression of KCC2 impacts on protrusion density and diameter at juveline stage.!...!66!

Figure 22.!Precocious expression of KCC2 leads to a modification of the ratio of excitatory versus inhibitory post-synaptic elements at juveline stage in a spatial-dependent manner. !...!68!

Figure 23.!The effect of precocious expression of KCC2 on gephyrin-Tomato cluster density at juveline stage does not depend on KCC2 chloride transport function.!...!70!

! ! List!of!tables! ! Tableau 1. Developmental processes analogous in humans and rodents on post-mortem tissues (Semple, Blomgren et al. 2013).!...!19!

Tableau 2. Molecules involved in axon guidance (Chao, Ma et al. 2009).!...!20!

Tableau 3. Cell-cell adhesion components (Benson, Colman et al. 2001).!...!23!

Introduction!!

!

The! central! nervous! system,! composed! of! the! brain! and! the! spinal! cord,! is! a! complex!

network!of!excitable!nerve!cells!that!receive!and!relay!messages!from!and!to!different!

parts! of! the! body! supported! by! glia! cells.! Independently! of! the! size! of! their! brain,! all!

mammals! share! the! same! organizational! and! evolutionary! conserved! stages! in! the!

development! of! the! central! nervous! (CNS)! that! initiates! with! neuronal! proliferation,!

followed!by!their!migration!and!differentiation!in!a!temporoNspatial!way!to!end!up!with!

synapse! formation! and! circuit! refinements.! Synapses! are! highly! specialized! area!

involving!presynaptic!and!postsynaptic!membranes!sites!that!permit!the!transmission!of!

specific! information! from! one! cell! to! another! either! through! gap! junctions! or! by!

neurotransmitters! (electrical! and! chemical! synapses! respectively).! However,! most! of!

neurons!communicate!through!chemical!synapses!separated!into!two!types!and!relying!

two! opposite! forms! of! signals! that! result! in! an! excitatory! or! an! inhibitory!

neurotransmission.! Within! this! context,! glutamate! is! considered! being! the! main!

excitatory! neurotransmitter! whereas! γNaminobutyric! acid! (GABA)! is! rather! the! main!

inhibitory! neurotransmitter! in! the! adult! brain.! Nevertheless,! GABA! has! not! always!

played! this! role.! Early! during! the! development,! GABA! release! and! binding! to! GABAA! receptors!resulted!to!a!depolarizing!action!due!to!the!high!intracellular!concentration!of!

chloride.! But,! as! the! expression! of! the! chloride! extruder! KCC2! gradually! increased!

during! the! development,! it! appeared! a! transition! in! the! action! of! GABA! from!

depolarizing! (excitatory)! to! hyperpolarizing! (inhibitory).! Unexpectedly,! recent!

observations!demonstrated!the!presence!of!KCC2!in!the!vicinity!of!excitatory!synapses!

and!its!contribution!to!their!formation!in!addition!to!its!primary!role!in!the!functional!

transition! of! GABAA! receptorNmediated! neurotransmission! from! excitatory! towards!

inhibitory! modalities.! Still,! whether! and! how! KCC2! might! be! implicated! in! inhibitory!

synaptogenesis!remains!unknown.!To!that!goal,!one!of!the!main!objectives!of!my!thesis!

was! to! study! the! role! of! the! cationNchloride! cotransporter! KCC2! in! inhibitory!

synaptogenesis!of!layer!II/III!pyramidal!neurons!of!the!rat!medial!prefrontal!cortex.!This!

region,!considered!as!important!in!working!memory!and!higher!order!cognitive!tasks,!

has!been!shown!to!be!considerably!sensitive!to!several!types!of!stress.!!

As! part! of! my! thesis! writing,! I! am! going! to! overview! the! different! steps! of!

synaptogenesis!in!the!first!part!before!focussing!on!the!biology!of!GABA!and!glutamate!

signalling!during!central!nervous!system!development!in!the!second!part.!One!important!

aspect! of! my! work! was! the! analysis! of! gephyrin! clusters! as! markers! of! inhibitory!

synapses.! Potential! changes! in! their! number! or! size! could! impact! on! the! inhibitory!

transmitted! signals! and! might! affect! the! excitatoryNinhibitory! balance.! In! that! line,!

dendritic!protrusions!will!be!analysed!in!parallel!as!they!are!considered!as!the!primary!

site!of!excitatory!inputs!to!neurons.!The!third!part!of!my!introduction!will!be!dedicated!

to! the! detection! of! the! excitatory! and! inhibitory! sites! in! the! central! nervous! system.!

There!will!be!a!particular!focus!on!gephyrin!and!its!opposite!counterpart!PSDN95,!often!

used!as!marker!of!excitatory!synapses,!in!order!to!highlight!the!differences!between!the!

two!types!of!synapses.!Finally,!in!the!last!part!of!my!introduction,!I!will!present!in!more!

detail! KCC2! expression! and! regulation! as! well! as! its! functional! and! structural! role! in!

GABA!signalling!and!excitatory!spines!formation!respectively.!Because!of!its!importance,!

a!disruption!in!KCC2!expression!can!lead!to!serious!disorders.!!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!

!!

Part!I.!Gatekeepers!for!information!flow!

!

In!this!chapter,!I!will!describe!how!synaptogenesis!is!part!of!the!normal!developmental!

processes!of!the!central!nervous!system!and!the!mechanisms!by!which!two!cells!manage!

to!form!a!synapse!between!them.!!

1.!Developmental!processes!in!the!central!nervous!system!!

The! central! nervous! system! (CNS)! composed! of! the! brain! and! spinal! cord! starts! its!

formation! early! during! embryogenesis! through! a! process! named! neurulation.! During!

this!process,!the!primary!axis!of!the!embryo,!the!notochord,!induces!the!formation!of!the!

neural! plate! that! invaginates! later! along! its! central! axis! and! forms! the! neural! groove!

with! neural! folds! on! each! side.! As! the! neural! tube! forms,! in! a! zipper! like! manner,! by!

fusion!of!these!neural!folds!from!the!anterior!end!of!the!notochord!to!the!posterior!side,!

it! creates! a! caudalNtoNrostral! gradient! in! development! of! the! brain.! Neural! tube!

formation!is!complete!at!around!gestation!day!(GD)!10.5N11!in!rats!and!from!GD!26!to!28!

in! humans! with! the! anterior! neuropore! closing! first! (rats! GD! 10.5,! humans! GD! 24N26)!

followed!by!the!posterior!neuropore!closing!(rats!GD!11.3,!humans!GD!25N28)!(reviewed!

by!DeSesso,!1996).!Even!though!the!developmental!time!scale!of!the!CNS!is!significantly!

different!between!rats!and!humans!(timeline!in!days!in!rats!versus!weeks!to!months!in!

humans),! the! sequence! of! events! is! comparable! among! species.! Following! the!

neurulation! and! depending! on! the! area! of! the! CNS,! a! sequence! of! overlapping!

developmental! processes! including! proliferation,! migration,! differentiation,!

synaptogenesis,!apoptosis!and!myelination!take!place!(Figure!1N2).!!

!

!!!!!!! !

Figure 1. Timelines of developmental processes in the central nervous system of rats (Rice and Barone 2000)

!!!!!!!!!!! !

Figure 2. Timelines of developmental processes in the central nervous system of humans (Rice and Barone 2000).

!

Synaptogenesis! is! a! process! that! start! early! in! brain! development,! even! before! birth.!

Little! is! ! known! about! embryonic! synaptogenesis,! however,! it! has! been! identified! five!

important! phases! of! synaptogenesis! (Figure! 3)! in! the! visual! cortex! of! the! macaque!

monkey! (Bourgeois! 1997)! that! can! be! extend! to! humans! (Lagercrantz! and! Ringstedt!

RICE AND BARONE

surface ectoderm at the apex of the neural folds to form the neural crest, which will give rise to the sensory ganglia of spinal and cra- nial nerves, Schwann cells (the cells covering peripheral nerves), the meningeal covering of the brain and spinal cord, and some skeletal and muscle components of the head, among other structures. The neural tube begins

to

close in the area of the hindbrain above the origin of the notochord and proceeds anteri- orly and posteriorly, creating

a

caudal-to- rostral gradient in development of the brain.

Neural tube formation is complete at approx- imately GD 10.5-11 in rats and from GD 26 to 28 in humans; the anterior neuropore closes first (rats GD 10.5, humans GD

24-26) and the posterior neuropore closes later (rats GD 11.3, humans GD 25-28) [reviewed by DeSesso (12)].

Interruption of neural development dur- ing this early period can result in severe abnor- malities of the brain and spinal cord. Spina bifida (divided spine) results from defective induction of mesoderm around the notochord that forms the osseous bone of the spine.

There are several types of spina bifida, which range from anomalies in the vertebrae of no significance to severe defects in the spinal cord or brain. Extreme cases of spina bifida (i.e., anencephaly) lead to failure in the closure of the neural tube and severe defects in the spinal cord or brain. For example, failure of neural

r

Birth_

Embryonic Fetal Postnatal

Ovulation Fertilization

_-Fertilimplann

^Functionalorganization

-Implantation

Histogenesis I Organogenesis

GD5-6 GD 15 Adolescence

GD

8-9 GD111

^{GD21-22 PND15 PND}35-45

Neurulation

Proliferation andmigration Differentiationandsynaptogenesis

Apoptosis

Gliogenesis

Myelination

Figure 1.Timelines ofdevelopmental processes in the ^nervoussystem of ratscompared^totiming ^offertilization, organogenesis,andhistogenesis. ModifiedfromVorhees

( 15)

andreprinted^withpermission^{ofPlenumPress.}

tube closure results

in extroversion

of the neural tissue, which then degenerates

as in

anencephaly, wherein the brain

is

represented by

amass

of degenerated neural tissue exposed

on

the surface of the head.

It

has recently been established that

an

increased intake of folic acid during early gestation

or

prenatally decreases the prevalence of neural tube defects

in

offspring (13). Increased risk for spina bifida may depend

on

the mother and/or fetus being homozygous for specific forms of enzymes involved in folate metabolism (14).

Beginning early in the second week of ges-

tation in

rodents (GD 7

in

mouse, GD 9.5 in rats) and the first month of gestation

in

humans, specific areas of the CNS begin

to

form with the neurogenesis and migration of cells in the forebrain, midbrain, and hindbrain.

There follows a sequence of developmental processes including proliferation, migration, differentiation, synaptogenesis, apoptosis, and myelination [Figures 1 (15) and 2 (16)].

Alterations in these processes can result

in severe

congenital abnormalities of the

nervous

system of humans, with a frequency of 0.74-1.89 cases per 1,000 births according to a recent survey (17). These overt abnormali- ties include conditions that produce extremely severe functional deficits and which may be incompatible with life, including anencephaly, hydrocephaly, and herniation of the spinal cord. Significant risk factors associ- ated with these conditions include parental age, toxemia in the mother, threatened inter- ruption of the pregnancy, and prematurity or intrauterine hypotrophy. A literature survey of > 70 studies from various countries (18) found that the incidence of mental retarda- tion is approximately 4 per 1,000 births, although, as stated by the authors, the true prevalence is difficult to determine.

Regional Development of the Rodent and Primate Brain

In general, regional development of the rodent brain proceeds on a timeline of days versus weeks to months in humans, although gross regional development of the brains of rodents and humans is similar. In the case of specific structures, however, there may be differences

in

the relative mass and/or volume of a specific structure between species. Examples include the relatively larger mass of the neocortex and visual system in humans versus that of rats.

Conversely, the relative mass of the olfactory system is larger in rodents than in humans.

The gradients of maturation of developing regions of the nervous system in rats and humans follow the same general sequence, with more caudal regions like the hindbrain {metencephalon and myelencephalon [Figure 3 (15]) developing earlier than the more ros- tral areas like the forebrain (telencephalon and diencephalon) and with the more medial

Environmental HealthPerspectives ^* Vol 108, Supplement 3 ^* June 2000

Figure2.Comparison of timelines for developmental processesinhumans. The prenatal period isscaledin months andthe postnatal developmentis scaled in years. Adapted fromHerschkowitz et al.(16)and reprinted

with

permis- sionofHippokratesVerlagGmbH.

512

RICE AND BARONE

surface

ectoderm

at

the

apex

of

the neural folds

to

form the neural crest,

which will

give

rise to

the sensory ganglia of spinal and

cra-

nial

nerves,

Schwann cells (the cells covering peripheral nerves), the meningeal covering of the brain and spinal cord, and

some

skeletal and muscle components of the head,

among

other

structures.

The neural tube begins

to

close

in

the

area

of the hindbrain above the origin of the notochord and proceeds

anteri-

orly and posteriorly, creating

a

caudal-to- rostral gradient

in

development of the brain.

Neural tube formation

is

complete

at

approx- imately

GD

10.5-11

in rats

and from

GD 26 to 28 in

humans; the

anterior

neuropore closes first (rats

GD

10.5, humans

GD

24-26)

and the

posterior neuropore closes later (rats GD 11.3, humans GD 25-28) [reviewed by

DeSesso

(12)].

Interruption of neural development dur- ing this early period can result

in

severe abnor- malities of the brain and spinal cord. Spina bifida (divided

spine)

results from

defective induction

of mesoderm around the notochord that forms the

osseous

bone of the spine.

There

are

several

types

of

spina

bifida, which range from anomalies

in

the vertebrae of no significance

to severe

defects

in

the spinal cord

orbrain.

Extreme

cases

of spina bifida (i.e., anencephaly)

lead to

failure

in

the closure

of

the neural tube and

severe

defects

in

the spinal cord or brain. For example, failure of neural

r

Birth_

Embryonic Fetal Postnatal

Ovulation Fertilization

_-Fertilimplann

^Functionalorganization -Implantation

Histogenesis I Organogenesis

GD5-6 GD15 Adolescence

GD

8-9 GD111

^{GD21-22 PND 15 PND}35-45

Neurulation

Proliferation and migration Differentiation andsynaptogenesis

Apoptosis

Gliogenesis

Myelination

Figure 1.Timelines ofdevelopmental processes in thenervous system ofratscompared ^totiming ^offertilization, organogenesis,andhistogenesis. Modified from Vorhees(15)^andreprinted^withpermission^ofPlenum Press.

tube closure results in extroversion

of

the

neural tissue, which then degenerates

as in

anencephaly, wherein the brain

is

represented by

amassof

degenerated neural

tissue

exposed

on

the surface of the head.

It

has recently been established

that an

increased

intake of

folic acid during early gestation

or

prenatally decreases

the

prevalence

of neural tube

defects

in

offspring (13). Increased risk for spina

bifida

may depend

onthe

mother

and/or

fetus being homozygous for specific forms of enzymes

involved in folate

metabolism (14).

Beginning early

in

the second week of ges-

tation in rodents

(GD

7 in

mouse,

GD

9.5

in

rats) and the first month of gestation

in

humans, specific

areas of the CNS

begin

to form with the

neurogenesis and migration

of cells in

the forebrain, midbrain,

and

hindbrain.

There

follows

a

sequence

of

developmental processes including proliferation, migration, differentiation, synaptogenesis, apoptosis,

and

myelination [Figures

1

(15)

and 2

(16)].

Alterations

in

these processes

can

result

in severe

congenital abnormalities

of thenervous

system of humans, with

a

frequency of 0.74-1.89

casesper 1,000

births according

to a recent survey

(17). ^These

overt

abnormali-

ties

include conditions that produce extremely

severe

functional deficits and which may be incompatible with life, including anencephaly, hydrocephaly, and herniation

of the

spinal

cord.

Significant

risk factors associ-

ated with these conditions include parental age,

toxemia in the

mother, threatened

inter-

ruption of the pregnancy, and prematurity

or

intrauterine hypotrophy.

A

literature survey of

>

70 studies from

various countries

(18) found that the incidence of mental retarda-

tion is

approximately

4

per 1,000 births, although,

as

stated by the authors, the true prevalence

is

difficult

to

determine.

Regional Development of the Rodent and Primate Brain

In

general, regional development of the rodent brain proceeds

on a

timeline of days versus weeks

to

months in humans, although gross regional development of the brains of rodents and humans

is

similar.

In

the case of specific structures, however, there may be differences

in therelative mass

and/or volume of a specific

structure

between species. Examples include the relatively larger

massof the neocortex and visual

system

in

humans versus that of rats.

Conversely,

the relative mass of the

olfactory

system is

larger

in rodents

than

in

humans.

The

gradients of maturation of developing regions

of the

nervous system

in

rats and humans follow

the same

general sequence,

with more caudal

regions

like

the hindbrain {metencephalon

and

myelencephalon [Figure

3

(15]) developing earlier

than

the

more

ros- tral areas like the forebrain (telencephalon and diencephalon)

and with the

more medial

Environmental Health Perspectives ^* Vol 108, Supplement3 ^* June2000

Figure^2. Comparisonof timelines for developmental processes in humans. The prenatal period is scaled in months andthe postnatal development is scaledinyears. Adapted from Herschkowitz et al.(16)and reprintedwithpermis- sionofHippokratesVerlagGmbH.

512

2001).!!Nascent!synapses!have!been!observed!in!!mouse!primary!visual!cortex!(Li,!Cui!et!

al.!2010)!and!in!rat!temporal!cortex!from!embryonic!day!15!(Konig,!Roch!et!al.!1975).!!

!

!!!!!!!!!!!!!!!!!!!!!!!!! !

!!!!!!!!!!!!!!!!!!!!!!! !

Figure 3. Densities of synapses in the visual cortex of the macaque monkey (Bourgeois 1997).

!

2.!Developmental!timeline!of!postnatal!rodent!synaptogenesis!

During! the! postnatal! period,! there! is! a! huge! increase! of! synaptic! sites,! preferentially!

located! on! small! protrusions! called! filopodia! (Fiala,! Feinberg! et! al.! 1998),! followed!

thereafter!by!an!elimination!or!pruning!stage!of!refinement.!This!scheme!is!shared!by!all!

mammalian! species! from! rodents! to! humans! (Andersen! 2003).! However,! when! the!

whole!process!seems!to!be!achieved!in!the!rat!brain!by!3N4!weeks,!the!period!of!synapse!

proliferation!and!synapse!elimination!is!much!more!longer!in!humans,!at!least!up!to!20!

years!old!(Huttenlocher!1979,!Uylings!and!van!Eden!1990,!Petanjek,!Judas!et!al.!2011).!

In! addition! to! be! ageNdependent! (Huttenlocher! 1979),! the! period! of! synapse!

proliferation! is! also! regionNdependent! as! the! synapse! density! in! the! primary! visual!

cortex! culminates! before! (between! 8N12! months! in! humans)! the! prefrontal! cortex!

synaptic! density! (2N4! years)! (Huttenlocher,! de! Courten! et! al.! 1982,! Lenroot! and! Giedd!

2006)! with,! nevertheless,! a! general! increase! of! synaptic! density! during! the! two! first!

years! up! to! 50%! higher! than! what! observed! in! humans! adults! (Huttenlocher! 1979,!

Herschkowitz,!Kagan!et!al.!1997).!This!crucial!period!of!synaptogenesis!in!humans!can!

be! translated! into! the! first! three! postnatal! weeks! in! rodents.! The! analogies! of! brain!

development! between! humans! and! rodents! are! summarized! in! Table! 1! (Semple,!

Blomgren!et!al.!2013).!!

!

!!

Tableau 1. Developmental processes analogous in humans and rodents on post-mortem tissues (Semple, Blomgren et al. 2013).

!

The!synaptic!pruning!phase!is!a!fundamental!developmental!stage!in!which!the!cortical!

circuitry! is! refined! for! a! more! efficient! transmission,! thus! a! better! adult! cognition.! A!

decrease!from!55%!to!around!10%!in!layer!III!neuronal!density!in!the!prefrontal!cortex!

has! been! observed! in! humans! between! 2! and! 7! years! of! age! (Huttenlocher! 1979),!

followed!by!a!comparable!decrease!in!the!frontal!cortex!density!between!7!and!15!years!

(Lidow,!GoldmanNRakic!et!al.!1991).!Interestingly,!the!synaptic!pruning!follows!the!same!

caudalNtoNrostral!gradient!previously!observed!in!the!developing!brain.!!

!

!

3.!Synaptogenesis!

The!crucial!function!of!synapses!as!gatekeepers!of!information!flow!makes!their!creation!

process! tightly! regulated.! Synaptogenesis! broadly! implies! two! subsequent! steps:! (i)!

axons!finding!their!suitable!targets!from!a!wide!range!of!choices!and!(ii)!the!formation!of!

functional! synapses! conducting! to! effective! information! transfer! (Juttner! and! Rathjen!

2005,! Salie,! Niederkofler! et! al.! 2005,! Waites,! Craig! et! al.! 2005).! After! finding! the!

appropriate! partner! and! before! establishing! a! connection,! the! differentiation! of! the!

junction!in!preN!and!postNsynaptic!sides!is!a!key!step!that!requires!a!cascade!of!signalling!

proteins,! intercellular! cell! adhesion! molecules! and! intracellular! scaffolding! proteins!

(Siddoway,!Hou!et!al.!2014).!All!these!proteins!act!together!in!order!to!sustain!and/or!

settle! the! right! cellular! mechanisms! needed! for! the! adequate! synapse! function! (Lujan,!

Shigemoto!et!al.!2005).!!

3.1.!!Appropriate!target!cell!!

Neuronal! growth! cones! navigate! over! long! distances! along! specific! pathways! to! find!

their!suitable!targets.!These!dynamics!tips!have!receptors!that!make!them!able!to!sense!

the!environment,!resulting!in!attractive!or!repulsive!behaviours!in!response!to!secreted!

cues!or!other!transNmembranes!receptors.!Proceeding!simultaneously!in!a!coordinated!

manner,!these!attractive!or!repellent!signals!guide!the!growth!cone!navigating!between!

the!guideposts!until!it!reaches!its!specific!target! (TessierNLavigne!and!Goodman!1996,!

Chao,!Ma!et!al.!2009).!The!main!axon!guidance!molecules!are!listed!in!the!Table!2!bellow.!!

!

!

Attraction! Repulsion!

chemoattraction! contact!

attraction! chemorepulsion! contact!

repulsion!

PreNsynaptic! Netrin,!Sema,!

BMP,!Wnt!

Ephrin/Eph,!

Sema!

Netrin,!Sema,!

Slit,!Wnt! Ephrin/Eph!

PostNsynaptic! Wnt,!Sema! Ephrin/Eph,!

Sema! Sema! Ephrin/Eph!

Tableau 2. Molecules involved in axon guidance (Chao, Ma et al. 2009).

!

Interestingly,!almost!all!of!these!molecules!have!a!dual!function!as:!!

N! Netrin:! It! is! a! diffusible! component! that! can! act! either! as! a! chemoattractant! or! as! a!

chemorepellent!depending!on!the!receptor!on!which!it!is!acting.!It!has!been!shown!in!C.!

elegans! that! Netrin! plays! its! chemoattractant! role! through! the! DCC/UNCN40! receptor!

whereas! it! becomes! a! chemorepellent! through! the! UNCN5! receptor! (ColonNRamos,!

Margeta!et!al.!2007,!Poon,!Klassen!et!al.!2008).!

N! Ephrin/Eph:! Ephrins! are! membraneNassociated! ligands! that! bind! to! a! subfamily! of!

tyrosine! kinase! receptors,! Eph! receptors,! mediating! a! shortNrange! contact! interaction!

(Dalva,! Takasu! et! al.! 2000,! Kayser,! McClelland! et! al.! 2006).! They! fall! into! two! classes:!

ephrinNAs,!embedded!in!the!membrane!by!a!glycosylphosphatidylinositol!(GPI)!linkage!

and! specific! for! EphA! receptors;! and! ephrinNBs,! which! have! a! transmembrane! domain!

and!bind!EphB!receptors!(Henkemeyer,!Itkis!et!al.!2003).!

N! Semaphorin! (Sema):! Semaphorins! are! a! large! family! of! secreted! guidance! molecules!

first!identified!as!contactNmediated!repellents!(Yamashita,!Morita!et!al.!2007).!There!are!

six!classes!of!Sema!and!some!of!them!are!thought!to!be!chemoattractant!too!(Polleux,!

Morrow! et! al.! 2000).! It! has! been! shown! that! Sema3A! promotes! both! presynaptic! and!

postsynaptic! differentiations,! especially! in! layer! V! of! cerebral! cortex,! when! applied! on!

cultured!cortical!neurons!(Morita,!Yamashita!et!al.!2006,!Yamashita,!Morita!et!al.!2007).!

By! contrast,! Sema3F! is! required! for! synapse! elimination! in! both! hippocampal! and!

cortical!neurons!(Liu,!Low!et!al.!2005,!Low,!Liu!et!al.!2008).!

N! Wnt:! When! secreted! from! the! postsynaptic! differentiations,! Wnts! function! as!

retrograde! signals! to! induce! the! accumulation! of! presynaptic! components! during!

synapse!formation!(Hall,!Lucas!et!al.!2000,!Krylova,!Herreros!et!al.!2002,!AhmadNAnnuar,!

Ciani!et!al.!2006).!

N! Slit:! According! to! some! experiments,! slits! may! have! a! function! in! synapse! formation!

(Godenschwege,! Simpson! et! al.! 2002).! Nevertheless,! as! it! has! been! demonstrated! on!

zebrafish! retinotectal! system,! slit! signalling! could! also! act! as! inhibitor! during! synapse!

formation!(Campbell,!Stringham!et!al.!2007),!leaving!still!unclear!the!actual!role!of!this!

complex.!

N!BMPs:!Bone!morphogenetics!proteins!(BMPs)!are!members!of!the!TGFNβ!superfamily,!

acting!as!inhibitors!of!synapse!formation!(Korolchuk,!Schutz!et!al.!2007,!Wang,!Shaw!et!

al.! 2007,! O'ConnorNGiles,! Ho! et! al.! 2008)! by! impacting! on! neurotransmitter! release!

(Aberle,!Haghighi!et!al.!2002,!Marques,!Bao!et!al.!2002,!McCabe,!Marques!et!al.!2003).!

Their!action!can!be!effective!(Liu!and!Niswander!2005)!or!not!(Eaton!and!Davis!2005)!

through!the!receptor!Smad!(RNSmad)!signalling.!!

3.2.!!Functional!synapse!

As! soon! as! the! growing! axon! has! attained! its! target! area,! its! end! begins! to! divide! into!

several!small!protrusions!and!the!formation!of!synapses!takes!its!onset.!Most!of!them!

are!formed!at!the!end!of!the!axon!but!there!are!also!some!en#passant!synapses!that!take!

place!along!axon!branches,!all!following!two!consecutives!steps:!the!preN!and!postN!sides!

assembly!and!the!synapse!formation.!!

3.2.1.!PreQ!and!postQ!sides!assembly!

The!synaptic!assembly!phase!includes!the!recruitment!of!all!the!components!necessary!

for!the!synapse!formation!in!addition!to!maintain!the!required!preN!and!postNsynaptic!

sides.! In! order! to! ensure! this,! there! is! a! need! of! cellNcell! adhesion! components! and!

clustering!molecules.!!

3.2.1.1.!CellQcell!adhesion!components!

To!form!the!synapses,!neurons!are!mechanically!coupled!to!each!other!in!a!spatial!and!

organized! architecture! through! cell! adhesion! molecules! (CAMs).! They! often! include!

cadherins! and! protocadherin! molecules! from! the! cadherin! family,! N(neural)CAM,!

Syn(synaptic)CAM,! nectin,! integrins,! sidekicks! and! synaptogenesis! 1N2! (SYG1N2)!

molecules! from! the! immunoglobulin! (Ig)! superfamily.! There! are! also! neuroligin!

molecules! that! act! as! ligands! for! βNneurexin! receptors! as! well! as! Ephrin! ligand! and!

receptors! as! they! are! the! main! actors! in! contact! attraction! or! repulsive! mechanisms.!

Each! of! these! molecules! has! their! own! specificities! as! shown! in! the! Table! 3! and! on!

Figure!4!(Benson,!Colman!et!al.!2001).!!

!

!

!

!

!

!

!

!

!

!

Function! Adhesion!Molecule! Class! Place!of!action! Specific!role!

Target!

recognition!

NNcadherin! Cadherin! ! Synaptic!recognition!

Protocadherin! Cadherin! CNS,!spinal!cord! Determination!of!synaptic!

connectivity!

Ephrin/EphR! Other! ! Synaptic!recognition!

Nectin! Ig!

Hippocampal!

pyramidal!

neurons!

Regulation!of!axonNdendrite!

interactions!

!

Induction!and!

maturation!of!

synapse!

SynCAM! Ig!

Hippocampal!

pyramidal!

neurons!

Induction!of!synaptic!

differentiation!

Neuroligin/neurexin! Other!

Hippocampal!

pyramidal!

neurons!

Induction!of!synaptic!

differentiation!

Synaptic!

plasticity!

! NCAM!

! Ig!

! CNS!

!

Regulation!of!new!synapse!

formation!and!structural!

synaptic!plasticity!

Integrins! Other! ! !

Tableau 3. Cell-cell adhesion components (Benson, Colman et al. 2001).

!

!

!

!

!

!

N! Cadherin! superfamily,! named! for! “calciumNdependent! adhesion”! is! typeN1!

transmembrane! proteins! with! more! than! hundred! members! identified! and! has! been!

shown! to! be! involved! in! several! developmental! processes.! The! bound! between! the!

cytoplasmic!domain!of!cadherin!and!hNcatenin,!that!turn!later!to!αNcatenin,!is!the!main!

characteristic!of!cadherin!family.!Contrary!to!cadherin!members,!protocadherins!do!not!

have!the!main!complex!specific!to!cadherin!family.!However,!they!have!the!potential!to!

induce!downstream!signal!transduction!pathways!through!presynaptic!and!postsynaptic!

specificities!(Kohmura,!Senzaki!et!al.!1998,!Wang,!Su!et!al.!2002,!Phillips,!Tanaka!et!al.!

2003).!!

N!NCAM!was!the!first!Ig!superfamily!member!reported!to!mediate!cell!adhesion!and!to!

induce!synaptic!plasticity!in!neuronal!cells!(Stoenica,!Senkov!et!al.!2006).!A!negatively!

charged! carbohydrate! polysialic! acid! (PSA)! carried! by! NCAM! allows! the! biological!

functions! mentioned! above! (Muller,! Wang! et! al.! 1996,! Eckhardt,! Bukalo! et! al.! 2000,!

Kleene!and!Schachner!2004).!Beside,!the!fixation!of!a!ligand!on!integrin!transmembrane!

receptors!results!in!a!subsequent!activation!of!many!intracellular!signalling!initiated!by!

SrcNfamily,!focal!adhesion!kinase,!and!integrinNlinked!kinases!that!can!lead!to!synaptic!

plasticity.! SynCAM,! another! homophilic! cell! adhesion! molecule,! promotes! synapse!

formation!(Biederer,!Sara!et!al.!2002),!especially!SynCAM!1!and!neuroligins!that!are,!to!

date,! the! only! two! known! CAMs! sufficient! to! drive! the! formation! of! presynaptic!

terminals!(Scheiffele,!Fan!et!al.!2000,!Biederer,!Sara!et!al.!2002,!Fu,!Washbourne!et!al.!

2003).!!

N! Neuroligin,! a! transmembrane! protein,! was! discovered! to! bind! to! βNneurexin! protein!

(Ichtchenko,! Hata! et! al.! 1995)! and! such! interaction! is! able! to! induce! the! formation! of!

presynaptic! terminals! onto! nonNneuronal! cells! (Scheiffele,! Fan! et! al.! 2000,! Fu,!

Washbourne! et! al.! 2003)! and! the! recruitment! of! synaptic! vesicles! (Dean,! Scholl! et! al.!

2003).!!

In!addition!to!the!abovementioned!proteins,!sidekicks!(Yamagata!and!Sanes!2010)!and!

SYG!1N2!(Ozkan,!Chia!et!al.!2014)!are!also!shown!to!be!involved!in!synaptogenesis.!!

!!!!!! ! Figure 4. Contact-mediated recognition proteins and their binding partners.

Molecules are positioned across from one another, as they would be in situ. Common intracellular signalling pathways are indicated. All are present in neurons during development, and many have been localized to synapses, where they might collectively contribute to the proteinaceous network observed in the synaptic cleft by electron microscopy. ABP, AMPA-receptor-binding protein; CASK, calcium/calmodulin-dependent serine protein kinase; CNR1, cadherin-related neuronal receptor 1; GPI, glycosyl phosphatidylinositol; GRIP, glutamate-receptor- interacting protein; ICAM, intercellular adhesion molecule; Ig, immunoglobulin; L1, L1 cell adhesion molecule; LMW-PTP, low molecular weight phosphotyrosine phosphatase; NCAM, neural cell adhesion molecule; PDZ–RGS3, regulator of G-protein signalling 3; PI3-kinase, phosphoinositide-3-kinase;

PICK1, protein that interacts with C kinase 1; PSD95, postsynaptic density protein 95; RasGAP, Ras GTPase- activating protein (Benson, Colman et al. 2001).

3.2.1.2.!Clustering!molecules!

In!addition!to!their!homotypic!and!heterotypic!interactions!properties,!CAMs!have!the!

potential!to!attach!other!clustering!molecules!within!the!cytoplasm!in!both!presynaptic!

and!postsynaptic!sides!(Figure!4).!!

3.2.2.!Synapse!formation!!

The!majority!of!synapses!are!chemical!in!the!central!nervous!system.!Normally,!synaptic!

communication! occurs! in! a! specific! temporal! order! starting! with! the! presynaptic!

membrane! depolarization,! then! the! presynaptic! release! of! neurotransmitters! followed!

by! the! neurotransmitter! diffusion! across! the! synaptic! cleft! and! their! binding! to!

postsynaptic!receptors!localized!on!the!cell!membrane.!

3.2.2.1.!!Presynaptic!specialization!!

The!presynaptic!side!of!the!synapse!is!defined!as!a!specialized!part!of!the!axon,!either!at!

the! end! or! along! the! axon! shaft,! with! vesicles! clustering! at! a! specific! area! of! the!

membrane,!the!active!zone.!This!presynaptic!specialization!is!physiologically!considered!

as! a! platform! for! the! docking,! fusion! and! recycling! of! vesicles! containing!

neurotransmitters! (Couteaux! 1963).! Two! categories! of! cytoplasmic! proteins! are!

involved! in! these! processes,! the! NNethylmaleimide! sensitive! fusion! proteins! (NSF)! and!

the!soluble!NSF!attachment!proteins!(SNAP).!NSF!and!SNAPs!on!the!vesicle!membrane!

along!with!SNAP!receptors!(SNAREs)!on!the!presynaptic!membrane!to!ensure!a!proper!

vesicle!attachment!and!initiate!the!fusion!complex.!Once!the!vesicle!has!fused!with!the!

cell!membrane!and!the!contents!released!into!the!synaptic!cleft,!the!vesicle!membrane!

moves!away!from!the!active!zone!via!clathrinNcoated!mechanisms!and!recycled!to!form!

another!synaptic!vesicle!after!the!clathrin!is!removed!or!degraded.!Interestingly,!it!has!

been! shown! that! developing! axons! can! form! rough! synapses! capable! of! releasing!

neurotransmitters!(Henrikson!and!Vaughn!1974,!Prokop,!Landgraf!et!al.!1996)!without!

any!postsynaptic!contacts!(Hume,!Role!et!al.!1983,!Young!and!Poo!1983)!suggesting!that!

the!presynaptic!site!functioning!is!an!inherent!property!of!axons.!!

3.2.2.2.!!Postsynaptic!specialization!

The!ultimate!function!of!the!postsynaptic!site!is!to!bind!neurotransmitters!released!from!

the! presynaptic! terminal! and! transduce! it! into! electrical! and! biochemical! changes.!

Depending! on! “which”! neurotransmitter! is! released! and! “where”! it! is! fixed,! the!

postsynaptic!transducted!signal!not!only!can!be!excitatory!(EPSP)!or!inhibitory!(IPSP)!

but!also!fast!or!slow!(Benardo!1994).!There!are!several!neurotransmitters!in!the!brain!

including! in! majority! the! amino! acids! such! as! glutamate,! aspartate,! glycine! and! γN aminobutyric! acid! (GABA)! and! the! amines! like! norepinephrine,! dopamine,! serotonin,!

acetylcholine! and! histamine! (Mariussen! and! Fonnum! 2001).! Each! of! these!

abovementioned! neurotransmitters! mediates! its! action! either! through! ionotropic!

receptors! (fast! transmission),! metabotropic! receptors! (not! coupled! directly! to! ions!

channels! for! a! slow! transmission)! or! both! (Siegel! and! Fleisher! 1999).! GABA! and!

glutamate!are!the!most!important!neurotransmitters,!present!in!almost!all!parts!of!the!

central!nervous!system!(Ottersen!and!StormNMathisen!1984).!Glutamate!is!the!dominant!

excitatory! transmitter! while! GABA,! although! having! an! opposite! action! of! that! of!

glutamate! in! mature! neurons! (Lujan,! Shigemoto! et! al.! 2004),! has! not! always! had! this!

specific!function!(Stein!and!Nicoll!2003).!!

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Part!II.!Signalling!through!synapses!

!

The!neurotransmitters!released!by!the!presynaptic!area!bind!their!specific!receptors!at!

the! postsynaptic! site,! leading! to! channels! opening,! activating! a! downstream! signalling!

pathway! and! propagating! the! nervous! impulse! throughout! the! cell.! The! ions! flowing!

through! these! receptors! can! induce! hyperpolarization! (inhibitory! synapses)! or!

depolarization!(excitatory!synapses)!of!the!postsynaptic!neuron.!Chemical!synapses!are!

then!defined!by!the!neurotransmitters!to!which!they!are!responsive.!!

1.!GABAergic!synapses!

The! action! of! GABA! at! the! synapses! is! mediated! by! both! ionotropic! (GABAARs! with!

GABACRs)!and!metabotropic!(GABABRs)!receptors!(Farrant!and!Kaila!2007).!The!ligandN gated! ions! channels! (or! ionotropic! receptors)! are! involved! in! the! fast! response! of!

neurons! to! GABA! and! the! metabotropic! GABAB! receptors! (GABABRs)! mediate! slow!

response! to! GABA! through! activation! of! second! messager! (Go! and! Gi)! proteins.! The!

second! type! of! hyperpolarization! is! distinct! from! the! first! one,! mainly! shown! by!

GABAARs,! as! it! is! slower! and! prolonged! (Dutar! and! Nicoll! 1988)! leading! then! to! a!

number!of!different!downstream!consequences!as!an!activation!of!potassiumNpermeable!

ion! channels! (Gahwiler! and! Brown! 1985),! an! inhibition! of! adenylyl! cyclase! and! a!

decrease!in!protein!kinase!A!(PKA)!activity!(Xu!and!Wojcik!1986,!Chalifoux!and!Carter!

2011).! As! part! of! my! subject,! I! will! focus! on! the! predominant! type! of! receptors,! the!

GABAARs.!!

1.1.!GABAA!receptors!

1.1.!1.!Structure!and!function!

All!the!GABAARs!are!heteroNpentameric!chloride!channels!assembled!from!a!huge!family!

of! homologous! subunits! encoded! by! distinct! genes! (Whiting,! Bonnert! et! al.! 1999).! To!

date,!nineteen!subunits!( 1N6,! 1N3,! 1–3,! ,! ,! ,! ,! 1N3)!have!been!identified!

in! the! mammalian! central! nervous! system! (CNS)! by! mean! of!in#situ! hybridization! and!

immunocytochemical! techniques! (Fritschy,! Benke! et! al.! 1992,! Laurie,! Wisden! et! al.!

1992).!The!molecular!assembly!of!each!GABA!receptor!(GABAR)!complex,!based!on!the!

interN! and! intracellular! distribution! of! these! different! subunits,! determines! different!

functional! properties! and! pharmacological! specificities.! In! addition,! each! of! these!