Astrocytes play well documented supportive roles in the brain. In addition, emerging roles for astrocytes include signaling to and from neurons, and regulation of local blood flow. Certain astrocyte functions are correlated with, or regulated by, cytosolic calcium transients, which are considered a physiological signal reflecting astrocyte excitability. Astrocytes interact with neurons and blood vessels primarily with their distal processes, but it is currently not possible to measure calcium signals non-invasively in astrocyte processes either in vitro in tissue slice preparations or in vivo. A method for measuring calcium in astrocyte processes in slices and in vivo would enable the rigorous testing of mechanistic hypotheses regarding astrocyte roles in brain function in the healthy CNS, and would open up new ways to study the impact of reactive astrogliosis, which occurs in response to all forms of injury and disease, on these same CNS functions. To develop such a method, we have modified a genetically encoded calcium sensor called GCaMP2 to carry a membrane tethering domain (called Lck) on its N terminus, thus generating Lck-GCaMP2. Our findings thus far show that Lck-GCaMP2 allows the non-invasive imaging of calcium levels in astrocytes near the membrane and in processes in cell cultures. This level of resolution is possible because the Lck-GCaMP2 is selectively and highly expressed in the plasma membrane, providing micrometer scale spatial information. This approach will help reveal when, where and how astrocytes are activated during physiological and pathophysiological processes. In this proposal we seek to exploit our new approach and provide important new resources for the astrocyte signaling community by generating and characterizing novel transgenic mice that will allow calcium imaging in astrocyte processes in tissue slices and in vivo. We have two specific aims.
In Aim 1 we will manufacture gene constructs that target Lck-GCaMP2 to astrocytes and use these constructs to establish founder lines of transgenic mice.
In Aim 2 we will characterize and use these Lck- GCaMP2 transgenic mice to study mechanisms and functions of calcium signaling in astrocyte processes in hippocampal slices. We will test two hypotheses: (i) that astrocytes display spatially compartmentalized calcium signaling and (ii) that TGF2 has direct, receptor mediated effects on calcium signaling in the processes of reactive astrocytes, thereby providing a mechanism through which reactive astrogliosis could influence neurons. The work proposed here will provide novel, well characterized optical reporter mice that will allow us and others to measure precisely localized calcium signals in astrocyte somata and processes within intact tissue structures such as brain slices and in vivo. These new reporter mice will be valuable general tools for the astrocyte community by allowing researchers to measure local astrocyte calcium signals in processes that are currently inaccessible to conventional imaging methods.
We will develop mouse models that will allow us and other researchers to monitor and track calcium signaling in astrocytes in vitro and in vivo. The availability of these mice would constitute exceptional tools with which to study the role of astrocytes in the normal healthy brain and in diseases of the nervous system, including epilepsy and neurodegeneration as well as during brain injury and repair.
|Shigetomi, Eiji; Jackson-Weaver, Olan; Huckstepp, Robert T et al. (2013) TRPA1 channels are regulators of astrocyte basal calcium levels and long-term potentiation via constitutive D-serine release. J Neurosci 33:10143-53|
|Shigetomi, Eiji; Bushong, Eric A; Haustein, Martin D et al. (2013) Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses. J Gen Physiol 141:633-47|
|Tong, Xiaoping; Shigetomi, Eiji; Looger, Loren L et al. (2013) Genetically encoded calcium indicators and astrocyte calcium microdomains. Neuroscientist 19:274-91|
|Hamby, Mary E; Coppola, Giovanni; Ao, Yan et al. (2012) Inflammatory mediators alter the astrocyte transcriptome and calcium signaling elicited by multiple G-protein-coupled receptors. J Neurosci 32:14489-510|
|Reeves, Alexander M B; Shigetomi, Eiji; Khakh, Baljit S (2011) Bulk loading of calcium indicator dyes to study astrocyte physiology: key limitations and improvements using morphological maps. J Neurosci 31:9353-8|
|Shigetomi, Eiji; Tong, Xiaoping; Kwan, Kelvin Y et al. (2011) TRPA1 channels regulate astrocyte resting calcium and inhibitory synapse efficacy through GAT-3. Nat Neurosci 15:70-80|
|Shigetomi, Eiji; Kracun, Sebastian; Khakh, Baljit S (2010) Monitoring astrocyte calcium microdomains with improved membrane targeted GCaMP reporters. Neuron Glia Biol 6:183-91|
|Shigetomi, Eiji; Kracun, Sebastian; Sofroniew, Michael V et al. (2010) A genetically targeted optical sensor to monitor calcium signals in astrocyte processes. Nat Neurosci 13:759-66|