Title Page
Contents
LIST OF ABBREVIATIONS 15
ABSTRACT 19
INTRODUCTION 21
A. Cannabis: A new kind of medicine 21
B. THC vs. synthetic cannabinoids 22
C. Issues of evaluating cannabinoid addiction 24
1. Cannabinoid self-administration 24
2. Cannabinoid-conditioned place preference 26
3. Other cannabinoid-induced behaviors 27
D. Purpose of the study 29
CHAPTER 1. POTENTIAL ANIMAL MODEL FOR CANNABINOID ABUSE SUSCEPTIBILITY 30
Ⅰ. INTRODUCTION 31
A. Transgenic mice models for addiction sensitivity 31
B. Aim of the chapter 35
Ⅱ. MATERIALS AND METHODS 36
A. Animals 36
B. Drugs 37
C. Behavioral tests 40
D. Statistical analyses 50
Ⅲ. RESULTS 51
A. Validation of the Cryab KO mice 51
B. General behavior of Cryab KO mice 52
C. Addiction-associated behaviors of Cryab KO and WT mice 57
Ⅳ. DISCUSSION 64
Ⅴ. SUMMARY 68
CHAPTER 2. MECHANISM UNDERLYING CANNABINOID ABUSE SUSCEPTIBILITY 69
Section A: Dopaminergic Pathway and Addiction 70
Ⅰ. INTRODUCTION 70
Ⅱ. MATERIALS AND METHODS 77
Ⅲ. RESULTS 82
Ⅳ. DISCUSSION 86
Ⅴ. SUMMARY 89
Section B: Neuroinflammation and Addiction 90
Ⅰ. INTRODUCTION 90
Ⅱ. MATERIALS AND METHODS 99
Ⅲ. RESULTS 108
Ⅳ. DISCUSSION 116
Ⅴ. SUMMARY 120
CONCLUSION OF THE STUDY 121
REFERENCES 122
국문초록 140
Table 1. The PCR primers and conditions for genotyping 37
Table 2. Sequences for endocannabinoid- and dopamine-related oligonucleotides 79
Table 3. Sequences for inflammation-related oligonucleotides 102
Table 4. Primary antibody information 104
Figure 1. Re-emergence of synthetic cannabinoids 24
Figure 2. Cryab alteration following methamphetamine and cocaine treatment 34
Figure 3. Effects of cocaine in Cryab KO mice. 35
Figure 4. Increased response of Cryab KO mice to cannabinoids 35
Figure 5. Representative synthetic cannabinoids 38
Figure 6. Addiction-related behavioral experiment outline 39
Figure 7. The open-field test 41
Figure 8. The Y-maze test 42
Figure 9. The rota-rod test 43
Figure 10. The cliff avoidance test 44
Figure 11. The elevated plus-maze 45
Figure 12. The electroencephalographic test 46
Figure 13. The intravenous SA paradigm 48
Figure 14. The CPP assay 50
Figure 15. Absence of Cryab/CRYAB expression in Cryab KO mice 51
Figure 16. Locomotor activity of Cryab KO and WT mice. 53
Figure 17. Short-term memory assessment in Cryab KO and WT mice. 54
Figure 18. Motor coordination and balance of Cryab KO and WT mice. 55
Figure 19. Impulsivity assessment of Cryab KO and WT mice. 55
Figure 20. Anxiety levels in Cryab KO and WT mice. 56
Figure 21. EEG of Cryab KO and WT mice. 57
Figure 22. Responses of Cryab KO mice in cannabinoid SA. 59
Figure 23. Behavior of Cryab KO mice in cannabinoid CPP test. 60
Figure 24. Response of Cryab KO mice to the locomotor-reducing effect of cannabinoids. 61
Figure 25. EEG power changes after acute and repeated JWH-018 treatment. 63
Figure 26. Unidentified mechanism behind cannabinoid abuse susceptibility 68
Figure 27. Cannabinoid mediation of dopaminergic activity 73
Figure 28. Dopamine-based hypothesis for the response of Cryab KO mice to cannabinoids 76
Figure 29. The qRT-PCR workflow 80
Figure 30. The ELISA workflow 81
Figure 31. Endocannabinoid system-related mRNA levels. 83
Figure 32. Dopamine system-related mRNA levels. 84
Figure 33. Dopamine concentration in mice NAC. 85
Figure 34. Desensitized dopamine system in the behavior of Cryab KO mice to cannabinoids 89
Figure 35. Drugs of abuse and inflammation. (A) Drugs of abuse activate NF-κB in microglia, releasing inflammatory cytokines. (B) Cytokines activate NF-κB in astrocytes, which... 92
Figure 36. Anti-inflammatory pathways involving CRYAB 97
Figure 37. Inflammation-based hypothesis for the behavior of Cryab KO mice 98
Figure 38. The Western blotting workflow 105
Figure 39. The Immunofluorescence workflow 106
Figure 40. Neuroinflammation-related mRNA expressions in mice. 109
Figure 41. Protein expressions along the PI3K-AKT-GSK3 pathway. 110
Figure 42. Inflammation-related protein expressions in mice. 111
Figure 43. Glutamate transporter mRNA levels in mice. 112
Figure 44. Plasticity-related protein expressions in mice. 113
Figure 45. NF-κB immunoreactivity in mice from JWH-018 SA. 114
Figure 46. Effect of LPS pre-treatment on JWH-018 CPP in WT mice. 115
Figure 47. Probable effect of Cryab on synaptic plasticity-associated addiction development 120
Figure 48. Potential mechanism for Cryab-mediated cannabinoid abuse susceptibility 121