Two-photon imaging of neurons, glia and vascular cells in acute brain slices of adult mice.
In this project we will use recombinant adeno-associated virus (rAAV) mediated transfer of genetically encoded indicators (GECI) into the brain of living mice. With the rAAV mediated gene delivery robust expression of GECI can be seen in targeted brain cells 2 weeks after injection. We will use optimized green and red-shifted Ca2+ indicators (e.g. GCaMP6f, jRGECo1a, jRCaMP1a) for detecting Ca2+ responses in astrocytic processes, neurons and vascular cells of acute brain slices during electrical stimulation of axons. We will use various promoters for targeting the gene expression in select brain cells; GFAP for expression in glia, SYN for neurons, NG2 for pericytes and smooth muscle cells, and Tie2 for endothelial cells. We will also use fluorescent indicators for extracellular neurotransmitters (e.g. iGluSnFR for glutamate) and aim at performing simultaneous imaging of Ca2+ signaling and neurotransmitter release using dual channel imaging. Furthermore, we will also deliver opsins (e.g. ChR2, halorhodopsin) and monitor the effects of optogenetically-induced cell activity changes.
Using electrical stimulation, optogenetics and GECI we will test whether astrocytes are involved in neuroplasticity and neurovascular coupling, i.e. mediating signals from active synapses to the vasculature in order to change the blood flow. Moreover, we will characterize the down-stream signaling mechanisms involved. Specifically, we will test the hypothesis that AQP4 and its anchoring protein alpha-syntrophin are crucial for normal neuron-glial-vascular communication. We will also investigate the role of Kir4.1 (potassium channel), carbonic anhydrase 4 and IP3R2 (a Ca2+ release receptor) in separate gene knockout mice.
Using electrical stimulation, optogenetics and GECI we will test whether astrocytes are involved in neuroplasticity and neurovascular coupling, i.e. mediating signals from active synapses to the vasculature in order to change the blood flow. Moreover, we will characterize the down-stream signaling mechanisms involved. Specifically, we will test the hypothesis that AQP4 and its anchoring protein alpha-syntrophin are crucial for normal neuron-glial-vascular communication. We will also investigate the role of Kir4.1 (potassium channel), carbonic anhydrase 4 and IP3R2 (a Ca2+ release receptor) in separate gene knockout mice.