| Abstract [eng] |
During development, synaptic pruning removes excess synapses and contributes to the formation of mature neuronal network. Synaptic pruning is performed by microglia that phagocytose synaptic material. It is essential to achieve optimal neural network and healthy nervous system. The impairments of synaptic pruning were linked to a variety of pathological conditions, including schizophrenia and autism spectrum disorders. Emerging data suggests that neuronal activity regulates synaptic pruning in competition-based mechanism. To investigate the role of neuronal activity in synaptic pruning we developed experimental approaches for the inhibition of neuronal activity and their visualization. First, we established organotypic hippocampal slice culture using Thy1::EGFP mice to provide robust labeling of excitatory pyramidal neurons of hippocampus in maturing brain tissue. Second, we used Thy1::EGFP, RC::LSL-tdTomato, Cx3cr1::Cre mice to visualize direct interactions between neurons and microglia. Third, we optimized conditions to use AAV8/2-CaMKIIα::hM4Di(Gi)-mCherry-WPRE-hgHp(A) designer receptor exclusively activated by designer drugs (DREADD) to inhibit neuronal activity and visualize inhibited neurons. We determined that this DREADD construct is not suitable to perform morphological analysis of axonal boutons and dendritic spines but could be used with other neuron visualization tools. Finally, we proposed a strategy to analyze morphology of axonal boutons and dendritic spines of neurons inhibited during peak of synaptic pruning utilizing co-localization of EGFP and mCherry signals. |
