Regulation of neurogenesis in the adult mammalian brain [Elektronische Ressource] / vorgelegt von Christiana Marie Cooper-Kuhn
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Regulation of neurogenesis in the adult mammalian brain [Elektronische Ressource] / vorgelegt von Christiana Marie Cooper-Kuhn

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REGULATION OF NEUROGENESISIN THE ADULT MAMMALIAN BRAINDISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DERNATURWISSENSCHAFTEN (DR. RER. NAT.) DERNATURWISSENSCHAFTLICHEN FAKULTÄT III - BIOLOGIE UNDVORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURGvorgelegt vonChristiana Marie Cooper-Kuhn aus Boulder, Colorado USA2003Promotionsgesuch eingereicht am: 18.02.2003Die Arbeit wurde angeleitet von: Prof. rer. nat. Stephan Schneuwly und Prof. Dr. med.Jürgen Winkler in der Klinik und Poliklinik für Neurologie der Universität RegensburgPrüfungsausschuß: Vorsitzender: - Prof. Dr. Karl Kunzelmann1. Prüfer (Erstgutachten) – Prof. Dr. Stephan Schneuwly2. Prüfer (Zweitgutachten) – Prof. Dr. Jürgen Winkler3. Prüfer – Prof. Dr. Inga Neumann2It is only when we forget all our learning that we begin to know.” – Thoreau“If you have built castles in the air, your work need not be lost. That is where theyshould be. Now put the foundation under them.” - Thoreau3INDEXREGULATION OF NEUROGENESIS IN THE ADULT MAMMALIAN BRAIN................................................1LIST OF ABBREVIATIONS .................................................................................................................................8INTRODUCTION.................................................................................................................................................10GENERATION OF NEW NEURONS IN THE ADULT MAMMALIAN BRAIN .....................................................

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Published 01 January 2003
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REGULATION OF NEUROGENESIS
IN THE ADULT MAMMALIAN BRAIN
DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER
NATURWISSENSCHAFTEN (DR. RER. NAT.) DER
NATURWISSENSCHAFTLICHEN FAKULTÄT III - BIOLOGIE UND
VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG
vorgelegt von
Christiana Marie Cooper-Kuhn aus Boulder, Colorado USA
2003Promotionsgesuch eingereicht am: 18.02.2003
Die Arbeit wurde angeleitet von: Prof. rer. nat. Stephan Schneuwly und Prof. Dr. med.
Jürgen Winkler in der Klinik und Poliklinik für Neurologie der Universität Regensburg
Prüfungsausschuß: Vorsitzender: - Prof. Dr. Karl Kunzelmann
1. Prüfer (Erstgutachten) – Prof. Dr. Stephan Schneuwly
2. Prüfer (Zweitgutachten) – Prof. Dr. Jürgen Winkler
3. Prüfer – Prof. Dr. Inga Neumann
2It is only when we forget all our learning that we begin to know.” – Thoreau
“If you have built castles in the air, your work need not be lost. That is where they
should be. Now put the foundation under them.” - Thoreau
3INDEX
REGULATION OF NEUROGENESIS IN THE ADULT MAMMALIAN BRAIN................................................1
LIST OF ABBREVIATIONS .................................................................................................................................8
INTRODUCTION.................................................................................................................................................10
GENERATION OF NEW NEURONS IN THE ADULT MAMMALIAN BRAIN .....................................................................10
Subventricular Zone/Olfactory Bulb Neurogenesis ..................................................................................11
Hippocampal Neurogenesis ......................................................................................................................13
DEVELOPMENTAL ASPECTS OF ADULT NEUROGENESIS.....................................................................................15
PHYLOGENETIC AADULT N........................................................................................17
Invertebrates ..............................................................................................................................................17
Non-mammalian vertebrates .....................................................................................................................17
Mammals....................................................................................................................................................18
FUNDAMENTALS OF ADULT NEUROGENESIS ......................................................................................................19
REGULATING ADULT NEUROGENESIS: WHICH STIMULI MAKE A DIFFERENCE?...................................................23
Systemic Influences:..................................................................................................................................23
Extracellular cues:......................................................................................................................................25
OVERALL GOAL................................................................................................................................................27
SPECIFIC AIMS..................................................................................................................................................27
OVERVIEW .........................................................................................................................................................29
EXPERIMENTS...................................................................................................................................................30
EXPERIMENT I - CRITICAL EVALUATION OF BRDU LABELING...............................................................30
OBJECTIVE .......................................................................................................................................................30
INTRODUCTION TO EXPERIMENT........................................................................................................................30
EXPERIMENTAL DESIGN ....................................................................................................................................32
RESULTS AND DISCUSSION ...............................................................................................................................32
SUMMARY.........................................................................................................................................................41
EXPERIMENT II - IMPAIRED ADULT NEUROGENESIS IN MICE LACKING THE TRANSCRIPTION
FACTOR E2F1....................................................................................................................................................42
OBJECTIVE .......................................................................................................................................................42
INTRODUCTION TO EXPERIMENT........................................................................................................................43
EXPERIMENTAL DESIGN ....................................................................................................................................45
RESULTS........................................................................................................................................................45
Loss of E2F1 affects size of adult mice and brain weight........................................................................45
Neuroanatomy of adult E2F1-deficient animals........................................................................................46
4Loss of E2F1 leads to less proliferation in dentate gyrus and subventricular zone................................48
Loss of E2F1 results in significant decrease of neurons in dentate gyrus and olfactory bulb................50
Loss of E2F1 results in decreased cell death in dentate gyrus and olfactory bulb granule cell layers.
....................................................................................................................................................................51
DISCUSSION..................................................................................................................................................53
Functional significance of E2F1 in other animal models..........................................................................53
Cell death in regions of adult neurogenesis..............................................................................................53
Postnatal phenotype of E2F1-deficiency ..................................................................................................54
EXPERIMENTS III & IV - NEUROTRANSMITTERS AND THEIR POSITION IN THE GAME OF ADULT
NEUROGENESIS ...............................................................................................................................................56
EXPERIMENT III – CHOLINERGIC DENERVATION LESION........................................................................57
OBJECTIVE .......................................................................................................................................................57
INTRODUCTION TO EXPERIMENT........................................................................................................................58
EXPERIMENTAL DESIGN ....................................................................................................................................59
RESULTS........................................................................................................................................................60
ICV injections of 192IgG-saporin result in selective elimination of cholinergic neurons in medial
septum........................................................................................................................................................60
Decrease of Neurogenesis in Dentate Gyrus after Cholinergic Denervation..........................................60
Cholinergic Denervation causes increase in Non-neuronal Cells in the Dentate Gyrus ........................62
Cholinergic Denervation causes an increase in Cell Death in the Dentate Gyrus..................................62
Decrease of Neurogenesis in Olfactory Bulb after Cholinergic Denervation ..........................................63
Cholinergic Denervation causes an increase in Cell Death in the Periglomerular Layer.......................65
DISCUSSION..................................................................................................................................................65
EXPERIMENT IV – NORADRENERGIC DENERVATION LESION................................................................68
OBJECTIVE .......................................................................................................................................................68
INTRODUCTION TO EXPERIMENT........................................................................................................................68
EXPERIMENTAL DESIGN ....................................................................................................................................69
RESULTS........................................................................................................................................................69
Decrease of Neurogenesis in Dentate Gyrus after Noradrenergic Denervation.....................................70
Noradrenergic Denervation causes decrease in Non-neuronal Cells in the Dentate Gyrus..................71
Cell Death in the Dentate Gyrus is not altered by Noradrenergic Denervation ......................................71
Increased Periglomerular Neurogenesis in Olfactory Bulb after Noradrenergic Denervation................72
Noradrenergic Denervation causes a Decrease in Cell Death in the Olfactory Granule Cell Layer......73
DISCUSSION..................................................................................................................................................74
EXPERIMENT V – VASCULAR ENDOTHELIAL GROWTH FACTOR INDUCES NEUROGENESIS IN
THE ADULT BRAIN ...........................................................................................................................................76
5OBJECTIVE .......................................................................................................................................................76
INTRODUCTION TO EXPERIMENT........................................................................................................................76
EXPERIMENTAL DESIGN ....................................................................................................................................78
RESULTS AND DISCUSSION ......................................................................................................................78
VEGF stimulates olfactory neurogenesis..................................................................................................78
VEGF stimulates hippocampal neurogenesis...........................................................................................79
VEGF decreases cell death in SVZ/OB and hippocampus......................................................................80
VEGF does not stimulate proliferation of glial cells..................................................................................81
VEGF does not induce neovascularization of the ventricle wall or the dentate gyrus............................81
EXPERIMENT VI – TRANSIENT EXPRESSION OF DOUBLECORTIN DURING ADULT
NEUROGENESIS ...............................................................................................................................................83
OBJECTIVE .......................................................................................................................................................83
INTRODUCTION TO EXPERIMENT........................................................................................................................83
EXPERIMENTAL DESIGN ....................................................................................................................................84
RESULTS........................................................................................................................................................84
Doublecortin expression during adult hippocampal neurogenesis..........................................................84
Doublecortin expression in the aging dentate gyrus. ...............................................................................88
Doublecortin expression in the SVZ/OB system ......................................................................................89
Doublecortin expression in the aging olfactory bulb.................................................................................92
DISCUSSION..................................................................................................................................................92
DCX expression in adult neural precursor cells? .....................................................................................93
DCX expression in mature neurons?........................................................................................................94
DCX, a transient marker for neuronal committed precursor cells............................................................95
CONCLUDING DISCUSSION............................................................................................................................97
NEUROGENESIS: A BALANCE OF PROLIFERATION AND APOPTOSIS.....................................................................99
COMPARISON OF THE DIFFERENT EXPERIMENTAL STRATEGIES: .......................................................................100
DISEASE-RELATED MODELS AND THE CLINICAL PERSPECTIVE.........................................................................101
Traumatic brain lesion..............................................................................................................................101
Epilepsy/Seizure ......................................................................................................................................102
Hypoxia/Ischemia.....................................................................................................................................102
OUTLOOK .......................................................................................................................................................104
MATERIALS AND METHODS.........................................................................................................................106
ANIMALS, INJECTIONS, AND TISSUE PREPARATION..........................................................................................106
Experiment I - Critical evaluation of BrdU labeling.................................................................................106
Experiment II - Impaired adult neurogenesis in mice lacking the transcription factor E2F1.................106
Experiment III - Cholinergic Denervation Lesion....................................................................................107
6Experiment IV – Noradrenergic Denervation Lesion..............................................................................107
Experiment V - Vascular endothelial growth factor induces neurogenesis in the adult brain ..............107
Experiment VI - Transient expression of doublecortin during adult neurogenesis ...............................108
HISTOLOGICAL PROTOCOLS............................................................................................................................109
BrdU-DAB Protocol..................................................................................................................................109
BrdU Triple Immunofluoroscence Protocol.............................................................................................110
Immunofluoroscence Protocol.................................................................................................................110
Standard TUNEL Protocol.......................................................................................................................111
TUNEL + Immunofluorescence Protocol ................................................................................................111
TUNEL + BrdU Protocol...........................................................................................................................112
Nissl Staining............................................................................................................................................112
DAPI/Propidium iodide Staining ..............................................................................................................113
ANTIBODIES ....................................................................................................................................................114
DNA PURIFICATION ........................................................................................................................................115
PCR...............................................................................................................................................................115
STEREOLOGY..................................................................................................................................................115
Adaptations for specific experiments ......................................................................................................118
MICROSCOPY..................................................................................................................................................119
NEURONAL DIFFERENTIATION..........................................................................................................................119
STATISTICAL ANALYSIS....................................................................................................................................119
CHEMICALS.....................................................................................................................................................121
APPENDIX ........................................................................................................................................................125
TABLE I...........................................................................................................................................................125
Interventions influencing Neurogenesis in Dentate gyrus......................................................................126
CURRICULUM VITAE ........................................................................................................................................128
BIBLIOGRAPHY...............................................................................................................................................132
ACKNOWLEDGEMENTS ................................................................................................................................161
7LIST OF ABBREVIATIONS
5,7-DHT 5,7-dihydroxytryptamine
5-HT serotonin
5-HTP 5-hydroxytryptophan
AMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
BDNF brain-derived neurotrophic factor
BrdU bromodeoxyuridine
CA1/CA3 Ammon’s horn or cornu ammonis of the hippocampus
cAMP cyclic adenosine monophosphate
CBF cholinergic basal forebrain
ChAT choline acetyltransferase
CNS central nervous system
CPS cryoprotectant solution
CY5 Indodicarbocyanine
DAPI 4,6-diamidino-2-phenylindole
DBH dopamine beta-hydroxylase
DCX doublecortin
DG dentate gyrus
DNA deoxyribonucleic acid
EGF epidermal growth factor
FGF-2 basic fibroblast growth factor
FITC fluorescein isothiocyanate
Flk-1 fetal liver kinase-1
GABA gamma-amino butyric acid
GCL granule cell layer
hr hour(s)
HVC higher vocal center
ICV intracerebroventricular
IGF-1 insulin-like growth factor-1
KA kainate
LC locus coeruleus
LV lateral ventricle
MCL mitral cell layer
min minute
ML molecular layer
NET norepinephrine transporter
NeuN neuronal nuclear antigen
NMDA N-methyl-D-aspartate
8NSE neuronal specific enolase
NT-3 neurotrophin-3
OB olfactory bulb
ON over night
PB phosphate buffer
PBS phosphate buffered saline
PCNA proliferating cell nuclear antigen
PCPA p-chlorophenylalanine
PFA paraformaldehyde
PGL periglomerular layer
PI propidium iodide
PSA-NCAM polysialylated neural cell adhesion molecule
Rb retinoblastoma gene
RECA rat endothelial cell antigen
RHOX rhodamineX
RMS rostral migratory stream
RT room temperature
SGZ subgranular zone
SVZ subventricular zone
TBS tris buffered saline
TGF-ß tumor growth factor-ß
TH tyrosine hydroxylase
TUC-4 TOAD (turned on after division)/Ulip/CRMP
TUNEL terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling
TX100 triton-X100
VEGF vascular endothelial growth factor
9INTRODUCTION
Generation of new neurons in the adult mammalian brain
The discovery that stem, or progenitor, cell populations produce neurons in the
adult brain highlights the dynamics of a previously assumed “static” organ. During
development, all organs are created from groups of proliferating cells, which have the
potential to differentiate into those cell types that are needed for a functional organ. In
adult tissues, these organ-specific stem cells are thought to play a major role in
1-3replacement of cells lost to physiological wear and injury . The existence of neural
progenitor cells in the adult central nervous system (CNS) is therefore seen as a
paradox, since the brain has classically been thought of as a structure with very limited
regenerative capacity. Nevertheless, in recent years, it became evident that the adult
brain is generating certain neuronal populations through a process termed “adult
neurogenesis”.
As early as 1912, scientists have noted mitotic activity in the cells of the lateral
4ventricle of the adult rat . And although Altman proposed the concept of persistent
5neurogenesis in the adult rodent brain as early as 1965 , the neuroscience community
was hesitant to recognize the importance of this observation until 1977 when Kaplan
3and Hinds were able to confirm, through the use of electron microscopy and H-
thymidine labeling, the neuronal fate of newly generated cells in the dentate gyrus (DG)
6and subventricular zone (SVZ)/olfactory bulb (OB) . Through the use of better labeling
methods for newborn cells, it is now generally accepted that the DG of the hippocampus
and the OB are the two main areas of adult neurogenesis in the rodent.
The fact that new neurons are generated in the adult mammalian CNS has major
implications for the study of the brain’s regenerative capacity. (1) Neural plasticity is not
restricted to axonal sprouting and synaptic remodeling. The possibility of addition or
replacement of neurons could provide a fundamentally new approach to compensate for
neuronal loss during aging and after injury or disease. (2) The existence of multipotent
progenitor cells in the adult brain provides the possibility to study their functional
behavior either in vivo or after isolation in vitro. This allows new insights into important
concepts of progenitor cell biology, such as self-renewal, multipotentiality, lineage-
10