Title Page
Contents
ABSTRACT 11
Chapter 1. Background 13
1.1. Introduction 13
1.2. The medial prefrontal cortex 15
1.2.1. Characteristics of the medial prefrontal cortex 15
1.2.2. The input and output system of the medial prefrontal cortex 16
1.2.3. Stress and the medial prefrontal cortex 16
1.2.4. The role of the medial prefrontal cortex in cognition and stress-related disorders 18
1.3. Src family kinase 21
1.3.1. Characteristics of Src family kinase 21
1.3.2. Characteristics of Fyn 23
1.3.3. Interaction between Fyn and a FYN binding protein 24
1.4. Effects of Fyn on other brain regions 25
1.4.1. Fyn and the hippocampus 25
1.4.2. Fyn and the amygdala 26
1.5. Aims of the present study 27
Chapter 2. Altered FYN expression in the medial prefrontal cortex reduces resistance to stress-induced memory impairments 31
2.1. Introduction 31
2.2. Materials and Methods 34
2.2.1. Subject 34
2.2.2. Cell lines and culture 34
2.2.3. RNA extraction 34
2.2.4. RT-qPCR detection 35
2.2.5. Viral vector production 35
2.2.6. Stereotaxic surgery 36
2.2.7. Acute stress paradigm 36
2.2.8. Apparatus and experimental environment 37
2.2.9. Behavioral procedure: object recognition task 39
2.2.10. Analysis of behavior 41
2.2.11. Brain preparation 43
2.2.12. Western blot 43
2.2.13. Immunofluorescent 44
2.2.14. Image acquisition and analysis 45
2.2.15. Statistical analysis 45
2.3. Results 46
2.3.1. Validation of Fyn siRNA candidates 46
2.3.2. Validation of Fyn knockdown 48
2.3.3. Down-regulation of FYN in the mPFC resulted in impaired recognition memory in response to the 20-min stress 51
2.3.4. Validation of Fyn overexpression 56
2.3.5. The overexpression of FYN in the mPFC led to a reduction in stress resistance, resulting in impairments of recognition memory 60
2.4. Discussion 65
Chapter 3. Decreased expression of FYB in the medial prefrontal cortex renders recognition memory susceptible to stress 71
3.1. Introduction 71
3.2. Materials and Methods 75
3.2.1. Subject 75
3.2.2. Cell lines and culture 75
3.2.3. RNA extraction 75
3.2.4. RT-qPCR detection 76
3.2.5. Viral vector production 76
3.2.6. Stereotaxic surgery 77
3.2.7. Acute stress paradigm 77
3.2.8. Apparatus and experimental environment 77
3.2.9. Behavioral procedure: object recognition task 78
3.2.10. Analysis of behavior 78
3.2.11. Brain preparation 79
3.2.12. Western blot 79
3.2.13. Immunofluorescent 80
3.2.14. Image acquisition and analysis 80
3.2.15. Statistical analysis 81
3.3. Results 82
3.3.1. Validation of Fyb siRNA candidates 82
3.3.2. Validation of Fyb knockdown virus 84
3.3.3. Reduced FYB in the mPFC increased stress susceptibility 87
3.3.4. Decreased FYB expression in Fyn knockdown samples from mPFC 92
3.4. Discussion 95
Chapter 4. Effects of reduced FYN level on stress-induced neuronal activity in the medial prefrontal cortex 98
4.1. Introduction 98
4.2. Materials and Methods 100
4.2.1. Subject 100
4.2.2. Viral vector production 100
4.2.3. Stereotaxic surgery 101
4.2.4. Acute stress paradigm 101
4.2.5. Brain preparation 101
4.2.6. Western blot 102
4.2.7. Immunofluorescent 102
4.2.8. Image acquisition and analysis 103
4.2.9. Statistical analysis 103
4.3. Results 104
4.3.1. Changes in the expression levels of FYN and FYB after stress exposure 104
4.3.2. Changes in the expression levels of c-Fos after stress exposure 106
4.3.3. Impact of the reduced FYN expression on stress-induced alterations of the neuronal activities in the mPFC 108
4.4. Discussion 112
Chapter 5. Conclusion 116
References 119
Abstract (in Korean) 146
Table 1. Mean total time (in s± SEM) in the arena for scrambled and Fyn knockdown groups to reach 30-s exploration criterion. 55
Table 2. Mean total time (in s± SEM) in the arena for Fyn overexpression and empty groups to reach 30-s exploration criterion. 64
Table 3. Mean total time (in s± SEM) in the arena for scrambled and Fyb knockdown groups to reach 30-s exploration criterion. 91
Figure 1. Structure of Src family tyrosine kinase 30
Figure 2. A photograph of two objects used in an object recognition task 38
Figure 3. A schematic diagram of the object recognition task 40
Figure 4. Image of the scoring program start screen 42
Figure 5. Validation of Fyn siRNA 47
Figure 6. Validation for AAV-mediated Fyn knockdown in the mPFC 49
Figure 7. Histological validation for AAV-mediated Fyn knockdown in the mPFC 50
Figure 8. Effect of 20-min stress upon Fyn knockdown in the mPFC 54
Figure 9. Vector map of WT Fyn AAV 57
Figure 10. Validation for AAV-mediated FYN overexpression in the mPFC 58
Figure 11. Histological validation for AAV-mediated FYN overexpression in the mPFC 59
Figure 12. Effect of 20-min stress upon Fyn overexpression in the mPFC 63
Figure 13. Validation of Fyb siRNA candidates by RT-qPCR 83
Figure 14. Validation of reduced FYB expression by western blot 85
Figure 15. Validation of reduced FYB expression in the mPFC 86
Figure 16. An ineffective brief 20-min stress impairs recognition memory in rats with Fyb knockdown virus infusion 90
Figure 17. Decreased tendency of FYB level in Fyn knockdown 93
Figure 18. Reduction of FYB level in Fyn knockdown 94
Figure 19. Alterations in the levels of FYN and FYB protein expression were measured at a time point 4 h post-exposure to stress 105
Figure 20. The level of c-Fos expression in the mPFC following exposure to stress 107
Figure 21. Quantifying the levels of c-Fos expression in the mPFC with either scramble or shFYN in the rats under each stress 111