Title Page
Contents
Part I. Decreased Expression of Glycine Receptors (GlyRs) in the Cerebellum of Ataxic mutant, Pogo Mouse 5
Introduction 6
Materials and Methods 10
1. Animals 10
2. Tissue preparation 10
3. Antibodies 11
4. Immunohistocemistry 11
5. Western blotting analysis 12
6. Image capture and photography 14
7. Densitometry and Statistical analysis 15
Results 16
1. Glycine Receptor α1 (GlyRα1) expression in the ataxic Pogo cerebellum 16
2. Glycine Receptor α2 (GlyRα2) expression in the ataxic Pogo cerebellum 22
3. Glycine Receptor α3 (GlyRα3) expression in the ataxic Pogo cerebellum 24
Discussion 31
Reference 37
Abstract 46
국문요약 48
Part II. Expression of Glycine Transporter (GlyT2) in the Cerebellum of Ataxic mutant, Pogo Mouse 50
Introduction 51
Materials and Methods 55
1. Animals 55
2. Tissue preparation 55
3. Immunohistocemistry 56
4. Western blotting analysis 57
5. Image capture and photography 59
6. Densitometry and statistical analysis 60
Results 61
1. Decreased expression of glycine transporter 2 (GlyT2) in cerebellar cortex. 61
2. Expression of glycine transporter 2 (GlyT2) in deep cerebellar nuclei (DCN) 64
3. Quantitative analysis of glycine transporter 2 (GlyT2) by western blotting 68
Discussion 70
Reference 75
Abstract 83
국문요약 85
Table1. Used antibodies and their sources in this experiments 11
Fig. I-1. Immunohistochemical distribution of GlyRα1 in the control (A, B, C, D, E, F) and Pogo (G, H, I, J, K) mice cerebellum. GlyRα1 was expressed by Purkinje cells, granule cells, basket and stellate cells in... 19
Fig. I-2. Bar graph shows the quantification of GlyRα1 immunoreactivity of relative optical density values in the cerebellum of control and ataxic Pogo mice. Immunoreactivity of GlyRα1 is decreased in four transverse... 20
Fig. I-3. GlyRα1-immunoreacted sections (30.㎛) from a control and Pogo at the level of the deep cerebellar nuclei (DCN). The intensity of immunostaining in the somata, as well as density of GlyRα1 were... 21
Fig. I-4. Photograph of GlyRα2 immunoreactivity in sagttal sections of control mice cerebellum and Pogo mice cerebellum. We can not detected of anti- GlyRα2 in the cerebellar cortex and deep cerebellar nuclei of control and Pogo mice Scale Bar = 400 ㎛ 23
Fig. I-5. Microphotographs taken from GlyRα3 immunoperoxidase stained, frontal sections though the vermis of the cerebellum of control (Left panels: A, B, C, D, E) and Pogo mice (Right panels: F, G, H, I, J).... 26
Fig. I-6. The bar chart indicates the relative optical density of GlyRα3 immunoreactive in the cerebellar cortex. GlyRα3 immunoreactivity was significant decreased in III lobule, VI lobule, V lobule and VI lobule.... 27
Fig. I-7. Photomicrographs showed the spatial distribution of immnoreactivity for GlyRα3 in the DCN of control and Pogo mice. GlyRα3 positive neurons in medial, interposed and lateral nuclei.... 28
Fig. I-8. The bar graph shows relative optical density values of the respective immunohistochemistry in the DCN. GlyRα1 and GlyRα3 immunoreactivity in Pogo mice is significant decreased in all three... 29
Fig. I-9. Western blot of expression GlyRα-subunit (GlyRα1, GlyRα2, GlyRα3) from cerebellum of control and Pogo mice. (A) anti- GlyRα subunits recognizes a single polypeptide, apparent around... 30
Fig. II-1. Light microphotographs taken from the coronal section of the control mice (A, B, C, D) and ataxic Pogo mice (E, F, G, H) cerebellum. GlyT2 immunoreactivity was showed stellate, basket cells in the... 62
Fig. II-2. The bar chart indicates the relative optical density of GlyT2 immunoreactivity in the cerebellar cortex. GlyT2 immunoreactivity is significant decreased in III lobule and V loblue in anterior zone, VI lobule... 63
Fig. II-3. Distribution of GlyT2 immunoreactivity in the deep cerebellar nuclei (DCN) of control mice and ataxia mice. GlyT2 positive neurons in medial, interposed and lateral nuclei. GlyT2 immunopositive somata, are... 66
Fig. II-4. Bar charts represent relative optical density values of the respective immunohistochemistry in the DCN. GlyT2 immunoreactivity in ataxic Pogo mice is significant decreased in all three subdivisions of the... 67
Fig. II-5. Western blot analysis of GlyT2 protein levels in the cerebellum of control mice and ataxia mice. (A) Immunoblot stained by GlyT2 antibody demonstrating a single band at 87.6KDa of molecular weight : control... 69