Arizono, M., Bannai, H., Nakamura, K., Niwa, F., Enomoto, M., Matsu-Ura, T., … Mikoshiba, K.
Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Division of Neuronal Network, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Calcium Oscillation Project, ICORP-SORST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan. Department of Physiology, Juntendo University, Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate School of Frontier Science, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Metabotropic glutamate receptor (mGluR)-dependent calcium ion (Ca²+) signalling in astrocytic processes regulates synaptic transmission and local blood flow essential for brain function. However, because of difficulties in imaging astrocytic processes, the subcellular spatial organization of mGluR-dependent Ca²+ signalling is not well characterized and its regulatory mechanism remains unclear. Using genetically encoded Ca²+ indicators, we showed that despite global stimulation by an mGluR agonist, astrocyte processes intrinsically exhibited a marked enrichment of Ca²+ responses. Immunocytochemistry indicated that these polarized Ca²+ responses could be attributed to increased density of surface mGluR5 on processes relative to the soma. Single-particle tracking of surface mGluR5 dynamics revealed a membrane barrier that blocked the movement of mGluR5 between the processes and the soma. Overexpression of mGluR or expression of its carboxyl terminus enabled diffusion of mGluR5 between the soma and the processes, disrupting the polarization of mGluR5 and of mGluR-dependent Ca²+ signalling. Together, our results demonstrate an mGluR5-selective diffusion barrier between processes and soma that compartmentalized mGluR Ca²+ signalling in astrocytes and may allow control of synaptic and vascular activity in specific subcellular domains.
… “All other Ca2+ imaging experiments in cultured astrocytes, including measurements of spontaneous Ca2+ transients, were recorded with a 490-nm light-emitting diode (LED) illumination system (450 to 550 nm, precisExcite, CoolLED) as a light source.”…
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