Dual color Ca2+ imaging of neuron-astrocyte interaction

[Speaker] Eiji Shigetomi:1
[Co-author] Yukiho J Hirayama:1, Kazuhiro Ikenaka:2, Kenji F Tanaka:3, Haruhiko Bito:4, Schuichi Koizumi:1
1:Department of Neuropharmacology, University of Yamanashi, Japan, 2:Division of Neurobiology and Bioinfomatics, NIPS, Japan, 3:Department of Neuropsychiatry, Keio University School of Medicine, Japan, 4:Department of Neurochemistry, Graduate School of Medicine, University of Tokyo, Japan

Astrocytes support synapses structurally and nutritionally. In addition, emerging evidence suggest that astrocytes release neuroactive substances in Ca2+-dependent manner to regulate synaptic functions. P2Y1 receptor is one of Gq-protein coupled receptors that is thought to play a central role in Ca2+ signals in astrocytes. However, role of astrocytic P2Y1 receptor in neuronal circuits is still largely unknown. In order to solve this problem, we increased the expression of P2Y1 receptors selectively in astrocytes and imaged Ca2+ dynamics in both neurons and astrocytes simultaneously to investigate neuronal consequence of astrocytic P2Y1 receptors.

Methods: Using AAV-mediated gene delivery into mice brain, we introduced red fluorescent genetically-encoded Ca2+ indicator (GECI), R-CaMP2 and green fluorescent GECI, GCaMP6f, into neurons and astrocytes, respectively. To increase P2Y1 receptor specifically into astrocytes, we used Tet-off conditional transgenic mice line (astrocyte P2Y1 overexpression mice). AAVs were injected into the hippocampus and 3-4 week after injection, acute brain slices were cut to image Ca2+ dynamics in both neurons and astrocytes using two-photon microscopy.

Results: We successfully introduced both GECIs to visualize Ca2+ activates from both neurons and astrocytes. Dendritic Ca2+ in neurons and fine process Ca2+ in astrocytes were easily to be monitored with high magnification imaging. In astrocyte P2Y1 overexpression mice, astrocytes displayed enhanced Ca2+ activities which occurred spontaneously and by P2Y1 receptor agonist. When the Schaffer collateral was stimulated electrically, dendritic Ca2+ signals increased immediately after the stimulus but astrocytic Ca2+ increase was delayed about a few seconds, suggesting that Ca2+-dependent mechanism in astrocytes may be slower than neuronal one. Ca2+ increase in astrocytes was reduced by P2Y1 receptor antagonist. fEPSP recorded from CA1 region of the hippocampus by stimulation of the Schaffer collateral was reduced by P2Y receptor antagonist in astrocyte P2Y1 receptor overexpression mice but not in control mice, suggesting that astrocytic P2Y1 receptor facilitate excitatory synaptic transmission in CA1 region.

Conclusion: Dual color Ca2+ imaging using R-CaMP2 and GCaMP6f revealed spatiotemporal dynamics of dendritic Ca2+ and astrocytic process Ca2+. This approach will provide useful information to understand how astrocytes regulate synapses.

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