Ⅰ. Introduction 1.1 Synapses are key regulatory sites of neuronal networks 1 1.2 Synaptic cell adhesion molecules (CAMs) regulate the development and characteristics of synapses 1 1.3 SLITRK family proteins regulate excitatory and inhibitory synapses 2 1.3.1 SLITRK related brain disorders 3 1.3.2 Slitrk2 regulates excitatory synapses through interaction with PTPσ 4 1.4 Astrocytes play an important role through interactions with neurons 4 1.4.1 Astrocyte play roles in regulation of synapse formation and synaptic activity 5 1.5 Tripartite synapse 6 1.5.1 Dysfunction of astrocytes can lead to both neurodevelopmental and neurodegenerative disorders 8 1.5.2 Astrocytic cell adhesion molecules 11 1.5.3 Aquaporin-4 (AQP4) plays a crucial role during neurotransmission 13
Ⅱ. Material and Methods 2.1 Expression vectors 15 2.2 Antibodies 15 2.3 Cell culture 16 2.4 Production of recombinant viruses 16 2.5 In situ hybridization 18 2.6 Primary neuron and mixed glia culture 18 2.7 Immunocytochemistry, and image acquisition and analysis 19 2.8 Immunohistochemistry, and image acquisition and analysis 20 2.9 Quantitative RT-PCR in cultured cells 20 2.10 Cultured neuron electrophysiology 21 2.11 Hippocampal CA1 pyramidal neuron electrophysiology 22 2.12 Animals 23 2.13 Stereotactic surgery 23 2.14 Data analysis 24
Ⅲ. Results 3.1 Verification of astrocytic Slitrk2 expression in vitro and in vivo 25 3.2 Astrocytic Slitrk2 deficiency affects excitatory synaptic transmission in hippocampal cultured neurons 29 3.3 Hippocampal CA1-specific astrocytic Slitrk2 deletion affects excitatory synaptic transmission 35 3.4 Hippocampal CA1-specific astrocytic Slitrk2 deletion affects excitatory synaptic formation 39 3.5 Astrocytic Slitrk2-cKO phenotype is abolished in PTPσ-cKO hippocampal cultured neurons 40 3.6 Astrocytic Slitrk2-cKO affects AQP4 expression upregulation in hippocampal CA1 42
Ⅳ. Discussion 46 Ⅴ. References 50 Ⅵ. 요약문 55
Figures
Figure 1. Identification of Slitrk1 and Slitrk2 mRNA level in WT mouse. Figure 2. Identification of mRNA level in cultured neuron, astrocyte and microglia. Figure 3. Generation of conditional Slitrk2 knockout mice. Figure 4. Validation of astrocytic Slitrk2 KO in cultured astrocyte. Figure 5. Excitatory synaptic transmission is enhanced in neurons co-cultured in with Slitrk2-deleted astrocytes. Figure 6. Excitatory synaptic transmission is enhanced in neurons co-cultured with astrocytes derived from Aldh1l1-Slitrk2 mice. Figure 7. No significant difference is observed in synaptic transmission by treatment of both Slitrk2 WT and KO ACM. Figure 8. Verification of astrocyte-specific GfaABC1D promoter operation. Figure 9. Excitatory synaptic transmission is enhanced in hippocampal CA1-specific astrocytic Slitrk2-cKO mice. Figure 10. Evoked excitatory synaptic transmission is not affected in hippocampal CA1-specific astrocytic Slitrk2-cKO mice. Figure 11. Excitatory synaptic formation is enhanced in hippocampal CA1-specific astrocytic Slitrk2-cKO mice. Figure 12. The astrocytic Slitrk2-cKO phenotype is not observed in PTPσ-cKO co-cultured neurons. Figure 13. AQP4 expression level is upregulated in hippocampal CA1-specific astrocytic Slitrk2-cKO mice. Figure 14. Summary of astrocytic Slitrk2 functions in tripartite synapses.
