In Vivo Two-Photon Calcium Imaging of Hippocampal Neurons in a Mouse Model of Alzheimer´s ...

103 Pages·2013·26.44 MB·English

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Fakultät für Medizin
Institut für Neurowissenschaften

In Vivo Two-Photon Calcium Imaging of
Hippocampal Neurons in a Mouse Model of
Alzheimer´s Disease

Marc Aurel Busche

Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur
Erlangung des akademischen Grades eines
Doctor of Philosophy (Ph.D.)
genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. Roland M. Schmid
Prüfer der Dissertation:
1. apl. Prof. Dr. Helmuth K. H. Adelsberger
2. Univ.-Prof. Dr. Thomas Misgeld
3. Univ.-Prof. Dr. Martin Kerschensteiner,
Ludwig-Maximilians-Universität München
Betreuer: Univ.-Prof. Dr. Arthur Konnerth

Die Dissertation wurde am 17.04.2013 bei der Fakultät für Medizin der Technischen Universität
München eingereicht und durch die Fakultät für Medizin am 21.08.2013 angenommen.
ThehippocampusisaprimetargetofAlzheimer´sdisease(AD),yetlittleisknownabouthow
the disease affects neuronal activity patterns in the hippocampus in vivo. This information
hasbeendifficulttoobtainbecauseoftheinaccessibilityofthehippocampusforcellular-level
functionalimaging. Here,weadvancedtwo-photonmicroscopytowardscalciumimagingof
neuronsintheCA1regionofthehippocampusinvivo. Byusingthismethod,westudiedthe
functionalalterationsofhippocampalneuronsinADtransgenicmice. Wefoundthatplaque-
laden transgenic mice exhibited greater proportions of silent and hyperactive neurons in the
hippocampusthanwildtypemice,consistentwithpreviousfindingsfromcortex. Surprisingly,
already before the deposition of amyloid-ß (Aß) into plaques, there was a massive increase
of hippocampal neuronal activity with many neurons being abnormally hyperactive. Our
findingthatneuronalhyperactivityoccurredindependentofplaqueformationsuggestedthat
soluble species of Aß, and not plaques themselves, might underlie this dysfunction. To test
this hypothesis, we treated plaque-free transgenic mice with the g-secretase inhibitor LY-
411575andfoundthatasingleoraldosereducedsolubleAßlevelsandrestoredhippocampal
neuronalfunctionwithinfewhours. Furthermore,wedemonstratedthatthedirectapplicationof
solubleAßinducedhippocampalhyperactivity. Takentogether,thisthesisadvancestwo-photon
microscopytoenableopticalstudiesofhippocampalneuronalactivityinvivo. Byusingthis
method,werevealedthatneuronalfunctionwasprofoundlyimpairedinthehippocampusof
AD transgenic mice already before plaque formation. Remarkably, the early hippocampal
dysfunction was fully reversed by a single dose of a g-secretase inhibitor indicating that it
was causally related to the action of soluble Aß. This work has resulted in a publication in
theProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmericain2012.
The title of this publication was “Critical role of soluble amyloid-b for early hippocampal
hyperactivityinamousemodelofAlzheimer’sdisease”.
Contents
1 Introduction 7
1.1 Thehippocampus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.1.1 Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.1.2 Functionalorganization . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.2 Invivotwo-photoncalciumimaging . . . . . . . . . . . . . . . . . . . . . . . 11
1.2.1 Two-photonmicroscopy . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.2.2 Two-photonmicroscopesetup . . . . . . . . . . . . . . . . . . . . . . 13
1.2.3 Fluorescencelabeling. . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3 Alzheimer´sdisease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.3.1 Invivoimaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
1.3.2 Neuronaldysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2 Material&Methods 30
2.1 Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.2 Surgeryandlabelingprocedureforhippocampalimaging . . . . . . . . . . . . 30
2.3 Two-photonimaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.4 Imageanalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.5 Cell-attachedrecordings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.6 Treatmentwithg-secretaseinhibitor . . . . . . . . . . . . . . . . . . . . . . . 33
2.7 Dimerapplication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.8 QuantificationofAß . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2
Contents 3
2.9 DetectionofAßplaques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.10 Statisticalanalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3 Results 36
3.1 Imagingneuronalactivityinthehippocampus . . . . . . . . . . . . . . . . . . 36
3.2 Patternsofneuronalactivityinhippocampusandcortex . . . . . . . . . . . . . 41
3.3 ImagingneuronalactivityinthehippocampusofAlzheimertransgenicmice . . 44
3.3.1 AlteredactivityofCA1neuronsinplaque-bearingmice . . . . . . . . 46
3.3.2 EarlyhyperactivityofCA1neuronsinplaque-freemice . . . . . . . . 46
3.3.3 Rescueofneuronalhyperactivitybyacuteg-secretaseinhibition . . . . 56
3.3.4 SolubleAßinducesneuronalhyperactivity . . . . . . . . . . . . . . . 57
4 Discussion 66
4.1 Invivoimagingofhippocampalneurons . . . . . . . . . . . . . . . . . . . . . 67
4.2 Neuronalhyperactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.3 Regionalfunctionalvulnerability . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.4 Clinicalimplications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
5 PublicationsinPeerReviewedJournals 75
Acronyms
Aß amyloid-ß
AAV adeno-associatedvirus
ACSF artificialcerebrospinalfluid
AD Alzheimer´sdisease
AM acetoxymethyl
AP actionpotential
APD avalanchephotodiode
APO apolipoprotein
APP amyloidprecursorprotein
APV D,L-2-amino-5-phosphonovalericacid
CAA cerebralamyloidangiopathy
CA cornuammonis
CNQX 6-cyano-7-nitroquinoxaline-2,3-dione
CT computedtomography
DAB diaminobenzidine
4
Contents 5
DMSO dimethylsulfoxide
EEG electroencephalogram
FDG fluorodeoxyglucose
fMRI functionalmagneticresonanceimaging
GFP greenfluorescentprotein
LIA largeirregularamplitude
LTP long-termpotentiation
MRI magneticresonanceimaging
NA numericalaperture
NPY neuropeptideY
CA cornuammonis
OGB-1 OregonGreenBAPTA-1
PBS phosphate-bufferedsulfate
PET positronemissiontomography
PFA paraformaldehyde
PiB PittsburghcompoundB
PMT photomultipliertube
PS presenilin
ROI regionofinterest
SDS sodiumdodecylsulfate
Contents 6
SIA smallirregularamplitude
SNR signal-to-noise-ratio
SPECT single-photonemissioncomputedtomography
TBS Tris-bufferedsaline
TGA transientglobalamnesia
Tg transgenic
TTX tetrodotoxin
Wt wildtype
1 Introduction
Astheaimofthisthesiswastoadvancetwo-photonmicroscopytowardshighresolutionimaging
ofneuronalactivitypatternsinthehippocampusinvivo,andinparalleltoapplythismethod
to a transgenic mouse model of Alzheimer´s disease (AD), this chapter is subdivided into
threeintroductorysectionswhichprovidethephysicalandbiologicalfundamentalsrequired
to comprehend the results presented in chapter 3. Section 1.1 introduces the basic anatomy
andfunctionalorganizationofthehippocampus. Section1.2givesashortoverviewofbasic
principles underlying two-photon microscopy, followed by a brief review on state of the art
fluorescence labeling techniques used to visualize biological structure and function in vivo.
Finally, section 1.3 describes imaging techniques used in the study of AD. In particular, it
focusesonownpreviouslypublishedresearchinvestigatingtheeffectsofthediseaseatthelevel
ofneuronsandtheirnetworksinvivo.
1.1 The hippocampus
1.1.1 Anatomy
Thehippocampusproper issubdivided inthreeparts: CA3, CA2andCA1(CA comesfrom
cornuammonis). Theotherregionsofthehippocampalformationincludethedentategyrus,
subiculum,presubiculum,parasubiculum,andenthorinalcortex. Dentategyrus,hippocampus
properandsubiculumhaveasinglecelllayerwithless-cellularoracellularlayersaboveand
belowit,whereastheotherregionshaveseveralcellularlayers[Shepherd2004;Andersen2007].
Basiccircuits. Muchoftheneocorticalinputreachesthehippocampusthroughtheenthorinal
7
Introduction 8
cortex,whichcanbeconsideredasthestartingpointoftheintrinsichippocampalcircuit. The
enthorinal cortex receives most of the sensory information via the adjacent perirhinal and
postrhinalcortices(alsotermedparahippocampalcortex). NeuronsinlayerIIoftheenthorinal
cortexprojecttothedentategyrusandtheCA3regionviatheperforantpath. Thegranulecells
ofthedentategyrusprojecttotheCA3regionviathemossyfiberprojections. Thepyramidal
cellsofCA3arethesourceofmajorinputtotheCA1region(viaaxonsthatformtheSchaffer
collaterals). Inaddition,CA3pyramidalcellsarehighlyinterconnected. CA1pyramidalcells
projecttothesubiculum. BothCA1andthesubiculumprojectbacktothedeeplayersofthe
enthorinalcortex. Thebasichippocampalcircuitryseemstobesimilarinhumans, monkeys
and rodents, however some species differences, such as the commissural connections of the
dentategyrus,areespeciallyrelevantforthisthesis. Inrodents,thereisamassivecommissural
systemthatprovidesnearlyone-sixthoftheexcitatoryinputtothedentategyrus[Gottlieband
Cowan 1973], whereas those connections are absent in humans and monkeys [Amaral et al.
1984]. Furthermore,theCA1pyramidalcelllayerbecomesthickerandmoreheterogeneousin
humansandmonkeysascomparedtorats. Inrats,thepyramidalcelllayerisabout5cellsthick,
whereasitcanbemorethan30cellsthickinhumans.
Cellularelements. Theprincipalcelllayerinthehippocampusisthepyramidalcelllayer,
whichistightlypackedinCA1butlooselypackedinCA2andCA3. Deeptothepyramidalcell
layerliesthestratumoriens,whichmainlycontainsthebasaldendritesofthepyramidalcells
andseveralclassesofinterneurons. WithinstratumorienssomeoftheCA3toCA3associational
connections and the CA3 to CA1 Schaffer collaterals are located. Below the stratum oriens
thefibercontaining alveusislocated. Itcontainsaxonsfrom pyramidalneuronsthatproject
to the fimbria/fornix, one of the major outputs of the hippocampus. In CA3, but not in CA2
and CA1, there is a narrow acellular zone, the stratum lucidum, which is located above the
pyramidalcelllayerandwhichcontainsthemossyfibers. Thestratumradiatumislocatedabove
thestratumluciduminCA3andimmediatelyabovethepyramidalcelllayerinCA2andCA1. It
containsCA3toCA3associationalconnectionsaswellastheCA3toCA1Schaffercollaterals.
Introduction 9
Finally,thestratumlacunosum-moleculareisthemostsuperficiallayercontainingfibersfrom
theenthorinalcortex. Theprincipalneuronsinthedentategyrusarethegranulecells,whereas
inthehippocampustheyarethepyramidalcells. Pyramidalcellsexhibitabasaldendritictree
thatextendsintothestratumoriensandanapicaldendritictreethatextendstothehippocampal
fissure. ThelengthandorganizationofCA3pyramidalcellsarevariable,whereasCA1cells
exhibitaremarkablehomogeneityoftheirdendritictree. Additionally, CA1pyramidalcells
areonaveragesmallerthanCA3cells(CA1: around15mmindiameter,CA3: around20–30
mmindiameter). Thedendritesofpyramidalneuronsarecoveredwithspinesontowhichmost
excitatory synapses terminate. Although pyramidal neurons are the most abundant cell type
inthehippocampus,thereisaheterogeneousgroupofinterneuronsscatteredthroughallcell
layers[FreundandBuzsáki1996]. Mostinterneuronsinthehippocampushavelocallyrestricted
targetregions,lackspines,andareGABAergic. Hippocampalinterneuronsareclassifiedonthe
basisoftheirsynaptictargetsasaxo-axoniccells,basketcells,andbistratifiedcells. Axo-axonic
cellssynapseontotheinitialsegmentsofpyramidalneuronsandregulateactionpotential(AP)
initiation. Basketcellssynapseontothesomaofpyramidalneuronsandbistratifiedcellscontact
theapicalorbasaldendritesofpyramidalneurons.
1.1.2 Functionalorganization
Thehippocampalformationplaysakeyroleinlearningandmemory. Damagetothehippocam-
palformationproducesanenduringimpairmentintheabilitytoencodenewinformationinto
long-term-memory. Thisanterogradeamnesticsyndromewasinitiallyobservedinthepatient
H.M.,whounderwentbilateralhippocampalremovalforthetreatmentofintractableepilepsy
[ScovilleandMilner1957;Corkinetal.1997]. Inrecentyearsanumberofreportsaboutother
patientswith bilateraldamageto thehippocampushave been published[Andersen 2007]. It
is important to note that damage to the hippocampal formation also leads to severe memory
impairmentsinrodents,measuredforexamplebyperformanceintheMorriswatermaze,con-
textualfearconditioning,delaynon-matchtosampletestorspatialalternationT-maze. Studies