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Immunohistochemical and genetic approaches for visualization of glutamatergic neurons in the mouse CNS [Elektronische Ressource] / vorgelegt von Neven Ashoor

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120 pages
Immunohistochemical and Genetic Approaches for Visualization of Glutamatergic Neurons in the Mouse CNS Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Neven Ashoor aus Giza, Ägypten Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 17. November 2005 Vorsitzender der Promotionskomission: Prof. Dr. D.-P. Häder Erstberichterstatter: Prof. Dr. W. Kreis Zweitberichterstatter: Prof. Dr. H.U. Zeilhofer Contents Abbreviations 1 Summary 3 Zusammenfassung 5 1. Introduction 7 1. 1. Glutamate 7 1. 2. Glutamate receptors 7 1. 2. 1. Ionotropic glutamate receptors 7 1. 2. 2. Metabotropic glutamate receptors 9 1. 3. Glutamate transporters 9 1. 3. 1. Plasma membrane excitatory amino acid transporters (EAAT) 9 1. 3. 2. Vesicular glutamate transporter (VGLUT) 10 1. 4. VGLUTs : Histological markers for glutamatergic neurons 13 1. 4. 1. Biochemical characterization of the VGLUTs 14 1. 4. 2. Distribution of the VGLUT isoforms inside and outside the rat CNS 14 1. 5. Bacterial Artificial Chromosome 15 1. 5. 1. Factors affecting the The BAC stability 16 1. 5. 2. BAC versus classic gene targeting 17 1. 5. 3.
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Immunohistochemical and Genetic Approaches for Visualization
of Glutamatergic Neurons in the Mouse CNS




Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades
















vorgelegt von
Neven Ashoor
aus
Giza, Ägypten




Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der
Universität Erlangen-Nürnberg


































Tag der mündlichen Prüfung: 17. November 2005
Vorsitzender der Promotionskomission: Prof. Dr. D.-P. Häder
Erstberichterstatter: Prof. Dr. W. Kreis
Zweitberichterstatter: Prof. Dr. H.U. Zeilhofer

Contents
Abbreviations 1
Summary 3
Zusammenfassung 5

1. Introduction 7
1. 1. Glutamate 7
1. 2. Glutamate receptors 7
1. 2. 1. Ionotropic glutamate receptors 7
1. 2. 2. Metabotropic glutamate receptors 9
1. 3. Glutamate transporters 9
1. 3. 1. Plasma membrane excitatory amino acid transporters (EAAT) 9
1. 3. 2. Vesicular glutamate transporter (VGLUT) 10
1. 4. VGLUTs : Histological markers for glutamatergic neurons 13
1. 4. 1. Biochemical characterization of the VGLUTs 14
1. 4. 2. Distribution of the VGLUT isoforms inside and outside the rat CNS 14
1. 5. Bacterial Artificial Chromosome 15
1. 5. 1. Factors affecting the The BAC stability 16
1. 5. 2. BAC versus classic gene targeting 17
1. 5. 3. Mammalian CNS analysis using BAC mediated transgenesis 18
1. 6. Fluorescent proteins as markers 19
1. 7. Objectives and approaches 20

2. Materials and methods 21
2. 1. Immunohistochemistry 21
2. 1. 1. Tissue preparation 21
2. 1. 2. Immunoperoxidase staining 21
2. 1. 3. Immunofluorescence staining 22
2. 1. 4. Antibodies 23
2. 1. 5. Data analysis 24
2. 2. Molecular Biology 26

2. 2. 1. Solutions and buffers 26
2. 2. 2. Plasmids 28
2. 2. 3. Nutrient media 29
2. 2. 4. DNA extraction 29
2. 2. 4. 1. Preparation of plasmid DNA 29
2. 2. 4. 2. Preparation of BAC DNA 30
2. 2. 4. 3. Preparation of purified BAC DNA for pronuclear injection 30
2. 2. 4. 4. Preparation of genomic DNA from mouse tail biopsies 32
2. 2. 5. Determination of DNA concentration 32
2. 2. 6. Preparation of competent E. coli cells 33
2. 2. 7. Transformation of competent cells 34
2. 2. 8. Polymerase Chain Reaction 34
2. 2. 9. DNA digestion with restriction endonucleases 37
2. 2. 10. Gel electrophoresis techniques 37
2. 2. 11. Ligation of DNA fragments 38
2. 2. 12. Homologous recombination for targeted modification of the BAC 39
2. 2. 13. Southern blot analysis 40
2. 3. Cell Culture 42
2. 3. 1. Cultivation and growth conditions of HEK-293 cells and
SH-SY5Y neuroblastoma cells 42
2. 3. 2. Transfection and visualization of the cells 42

3. Results 43
3. 1. Regional distribution of VGLUT2-IR throughout the mouse CNS 43
3. 1. 1. Telencephalon 49
3. 1. 2. Diencephalon 52
3. 1. 3. Mesencephalon 54
3. 1. 4. Cerebellum 54
3. 1. 5. Medullary regions and brain stem 55
3. 1. 6. Spinal cord 55
3. 2. Generation of VGLUT2-DsRed2 BAC transgenic mice 56
3. 2. 1. Screening of a mouse BAC library 56

3. 2. 2. Trageted modification of the BAC 58
3. 2. 2. 1. Construction of the recombination cassette 58
3. 2. 2. 2. Subcloning the recombination cassette into the shuttle vector 64
3. 2. 2. 3. Homologous recombination 65
3. 2. 3. Confirmation of the BAC modification 65
3. 2. 3. 1. Southern blot analysis 65
3. 2. 3. 2. Determination of integration site DsRed2 in the BAC 67
3. 2. 3. 3. Confirmation of absence of gross rearrangement 69
3. 2. 3. 4. Expression of VGLUT2-DsRed2 transgene construct in cell culture 70
3. 2. 4. Pronuclear injection 72
3. 2. 5. Breeding and genotyping of transgenic mice 73
3. 2. 6. Imaging of DsRed2 in BAC transgenic mice 77

4. Discussion 81
4. 1. VGLUT2 distribution and function throughout the mouse CNS 81
4. 2. Utility of BAC transgenic mice encoding a fluorescent marker 83
4. 3. Electrophysiology using genetically modified mice 83
4. 4. Role of BAC transgenic mice in the CNS analysis 84
4. 5. Considerations for the generation of BAC transgenic mice 85
4. 6. Analysis of transgenic mice and attempted controls 87
4. 7. Possible factors contributing in these results 88
4. 8. DsRed as a reporter protein 89
4. 9. Perspectives of a monomeric red fluorescent protein 91
4. 10. Suggestions for successful VGLUT2-FP BAC transgenic mice 91

5. Appendix 93
5.1. Construction and maps of the cloning vectors 93
5. 1. 1. pDsRed2-N1 93
5. 1. 2. pGEM-T Easy building vector 94
5. 1. 3. pBACe3.6 BAC vector 95
5. 1. 4. pSV1.RecA shuttle vector 96


6. References 98

7. Acknowledgement 112

8. Curriculum vitae 113



Abbreviations

Amp ampicillin
ATP adenosine triphosphate
ADP adenosine diphosphate
BAC bacterial artificial chromosome
BES N,N-Bis (2-hydroxyethyl)-2-aminoethanesulfonic Acid
bp base pair
BSA bovine serum albumin
LacZ beta galactosidase gene
CAP chloramphenicol
cDNA complementary deoxyribonucleic acid
CIAP calf intestinal alkaline phosphatase
CNS central nervous system
DAB 3`,3`-Diaminobenzidine
DMEM Dulbecco`s modified Eagle medium
DMSO dimethylsulfoxide
DNA deoxyribonucleic acid
DNTP deoxynucleoside triphosphate
dsDNA double stranded DNA
DsRed Discosoma red fluorescent protein
E.coli Escherichia coli
Et OH ethanol
Et Br ethidium bromide
FA fusaric acid
FCS fetal calf serum
GFP green fluorescent protein
GTBE glycine tris borate EDTA
HEK human embryonic kidney cells
h hour
IF immunofluorescence
IGLEs intraganglionic laminar endings
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IPTG isopropyl-ß-D-thiogalactoside
IR immunoreactivity
IU international unit
LB Luria-Bertani medium
MeOH methanol
mRNA messenger ribonucleic acid
NaAc sodium acetate
NMDA N-methyl-D-aspartate receptor
OD optical density
PCR polymerase chain reaction
PFA paraformaldehyde
PFEG pulsed field electrophoresis gel
Poly A polyadenylation signal
rpm round per minute
RT room temperature
SDS sodium dodecyl sulfate solution
TAE tris acetate EDTA
Taq Thermus aquaticus DNA polymerase
TB tryptone broth
TBE tris borate EDTA
TBS tris buffered saline
Tet tetracycline
TE tris HCl EDTA
UV ultraviolet
VGLUT (1, 2, 3) vesicular glutamate transporter (1, 2, 3)
VGLUT2-IR VGLUT2-Immunoreactivity
X-gal 5-bromo-4-chloro-3-indolyl-ß-D-galactoside
YAC yeast artificial chromosome
ZNS Zentral-Nerven-System

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Summary

Glutamate is the main excitatory neurotransmitter in the mammalian central
nervous system. The specific roles of various glutamatergic subpopulations,
however, have remained elusive. This is in part due to the difficulty of identifying
them in live brain or spinal cord slices, where the neuronal subtypes are not
readily distinguishable. Another critical point was to define a specific marker for
glutamatergic neurons. Glutamate (being a general metabolic precursor),
glutaminase and plasma membrane glutamate transporters are not suitable for this
purpose as they have been also detected in non-glutamatergic neurons.
Three isoforms of vesicular glutamate transporters (VGLUT1, VGLUT2, VGLUT3)
with complementary distribution throughout the CNS have been recently cloned
and are considered to represent the most specific markers so far for glutamatergic
neurons. The regional distribution of the three VGLUT isoforms has been studied
in detail in the rat CNS.
This study examined the distribution of the VGLUT2 immunoreactivity in the CNS
of the adult mouse. Results showed that the VGLUT2 protein was most intense in
the spinal cord, thalamus and hypothalamus (diencephalon) and brain stem.
Certain layers of the cortex, cerebellum and hippocampus showed dense VGLUT2
innervation. VGLUT2 puncta were enriched in pain processing areas of the spinal
cord, the thalamocortical pathway and the periaqueductal gray (PAG).
In order to provide a useful tool for the characterization of glutamatergic pathways,
an attempt was made to generate BAC (Bacterial Artificial Chromosome)
transgenic mice expressing red fluorescent protein (DsRed2) under the
transcriptional control of the VGLUT2 gene 17 amino acids upstream of the
translational start of VGLUT2. DsRed2 coding cDNA was precisely integrated
inframe into exon 2 of the VGLUT2 gene in a BAC clone by two consecutive
rounds of homologous recombination (Yang et al., 1997).
Under the control of the human cytomegalovirus (CMV) promoter, proper
transcription of the construct, splicing of the transcript and the fluorescence of the
resulting protein were obtained in transfected HEK-293 cells and SH-SY5Y
neuroblastoma cells.
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Following pronuclear injection of CsCl gradient purified BAC DNA in 505 oocytes,
109 ainmals were born and 10 transgenic founders could be identified. All the
founders exhibited germline transmission and gave rise to independent lines with
varying copy numbers. The expression of the VGLUT2-DsRed2 construct was
analyzed in live and fixed brain and spinal cord slices of the transgenic offspring.
Surprisingly, no fluorescence could be detected in the expected regions where
VGLUT2 immunoreactivity was defined in this study. A weak fluorescence could
be detected in the ganglion nodosum, where the presence of VGLUT2 was
reported by Tong et al. (2001).
The reasons for this are not clear. Some suggestions for these unexpected
phenomena could be that the DsRed2 RNA or protein might be less stable than
the products of the replaced VGLUT2 gene. The N-terminal overhang (acting as a
targeting sequence) might have participated in protein-protein interactions with
other endogenous proteins or other proximity-related phenomena in-vivo.
Furthermore this might have led to irreversible binding affecting the tetramer
formation necessary for the chromophore maturation of DsRed2. Similar problems
have arisen in other groups, who have used DsRed2 as an in-vivo marker.
To solve this, Campbell et al. (2002) developed the first true monomeric red
fluorescent protein and further attempts were performed by Shaner et al., 2004 to
introduce more reliable red shifted markers. A further step in this project would be
the generation of BAC transgenic mice in which the VGLUT2 gene is replaced with
one of the monomeric forms of the red shifted proteins or the EGFP, which proved
to be a reliable marker in transgenic studies. Such a transgenic mouse will be
useful in detailed investigation of the anatomical, physiological and molecular
analysis of subpopulations of glutamatergic neurons, thus provide important tools
for the anatomical and functional characterization of glutamatergic pathways.






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