Two-photon imaging of neurons, glia and vascular cells in acute brain slices of adult mice.
Formål: This project is a continuation of FOTS application 11255. In this project we seek to gain new insight into the role of astrocytes in health and disease (e.g. learning and memory, epilepsy or Alzheimers disease). 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 to 4 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 in acute brain slices during electrical stimulation of axons or "puffing" of various agonists. We will use various promoters for targeting the gene expression in select brain cells; e.g. 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 various neuromodulators (e.g. dopamin, ATP, NA) and aim at performing simultaneous imaging of Ca2+ signalling and neurotransmitter/-modulator 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. Finally, we will use designer receptors exclusively activated by designer drugs (DREADDs) to activate selected cells or pathways.
Skadevirkninger: The mice will be subjected to minor surgery for the injection of the rAAV encoding GECIs. Subsequently, after the viral vectors are expressed, the mice will be sacrificed and acute brain slices will be used for electrophysiology and imaging experiments. The burden on the mice in relation to surgery is considered mildly severe.
Nytteverdi: Using electrical stimulation, direct agonist application, optogenetics, GECI and DREADDs we will investigate the role of astrocytes 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 signalling mechanisms involved. Hopefully, the obtained knowledge from these experiments will lead to new and deeper understanding of the astrocytes role in health and disease.
Antall dyr og art: The total number of mice is estimated to be 590 (310 wild type mice and 280 mutant mice from twelve different mutant lines).
3R: Unfortunately, there are no alternative to the use of animals in these experiments, since
it require viable and intact nervous tissue. The applicant and co-workers have extensive experience with the experimental procedures involved and the number of mice used will be kept to a minimum. The mice will be treated with well-established routines for anesthesia, analgesia and postoperative care, and will be housed in an enriched environment.
Skadevirkninger: The mice will be subjected to minor surgery for the injection of the rAAV encoding GECIs. Subsequently, after the viral vectors are expressed, the mice will be sacrificed and acute brain slices will be used for electrophysiology and imaging experiments. The burden on the mice in relation to surgery is considered mildly severe.
Nytteverdi: Using electrical stimulation, direct agonist application, optogenetics, GECI and DREADDs we will investigate the role of astrocytes 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 signalling mechanisms involved. Hopefully, the obtained knowledge from these experiments will lead to new and deeper understanding of the astrocytes role in health and disease.
Antall dyr og art: The total number of mice is estimated to be 590 (310 wild type mice and 280 mutant mice from twelve different mutant lines).
3R: Unfortunately, there are no alternative to the use of animals in these experiments, since
it require viable and intact nervous tissue. The applicant and co-workers have extensive experience with the experimental procedures involved and the number of mice used will be kept to a minimum. The mice will be treated with well-established routines for anesthesia, analgesia and postoperative care, and will be housed in an enriched environment.