title: Visualization of Ca2+ Filling Mechanisms upon Synaptic Inputs in the Endoplasmic Reticulum of Cerebellar Purkinje Cells.
abstract: The endoplasmic reticulum (ER) plays crucial roles in intracellular Ca(2+) signaling, serving as both a source and sink of Ca(2+), and regulating a variety of physiological and pathophysiological events in neurons in the brain. However, spatiotemporal Ca(2+) dynamics within the ER in central neurons remain to be characterized. In this study, we visualized synaptic activity-dependent ER Ca(2+) dynamics in mouse cerebellar Purkinje cells (PCs) using an ER-targeted genetically encoded Ca(2+) indicator, G-CEPIA1er. We used brief parallel fiber stimulation to induce a local decrease in the ER luminal Ca(2+) concentration ([Ca(2+)]ER) in dendrites and spines. In this experimental system, the recovery of [Ca(2+)]ER takes several seconds, and recovery half-time depends on the extent of ER Ca(2+) depletion. By combining imaging analysis and numerical simulation, we show that the intraluminal diffusion of Ca(2+), rather than Ca(2+) reuptake, is the dominant mechanism for the replenishment of the local [Ca(2+)]ER depletion immediately following the stimulation. In spines, the ER filled almost simultaneously with parent dendrites, suggesting that the ER within the spine neck does not represent a significant barrier to Ca(2+) diffusion. Furthermore, we found that repetitive climbing fiber stimulation, which induces cytosolic Ca(2+) spikes in PCs, cumulatively increased [Ca(2+)]ER. These results indicate that the neuronal ER functions both as an intracellular tunnel to redistribute stored Ca(2+) within the neurons, and as a leaky integrator of Ca(2+) spike-inducing synaptic inputs.
authors.affiliation: Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo 133-0033, Japan, and.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Okubo
authors.affiliation: Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo 133-0033, Japan, and.
authors.forename: Junji
authors.initials: J
authors.lastname: Suzuki
authors.affiliation: Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo 133-0033, Japan, and.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Kanemaru
authors.affiliation: Laboratory for Physical Biology, RIKEN Quantitative Biology Center, Kobe 650-0047, Japan.
authors.forename: Naotoshi
authors.initials: N
authors.lastname: Nakamura
authors.affiliation: Laboratory for Physical Biology, RIKEN Quantitative Biology Center, Kobe 650-0047, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Shibata
authors.affiliation: Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo 133-0033, Japan, and iino@m.u-tokyo.ac.jp.
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Iino
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002461:Cell Line, Transformed; D002531:Cerebellum / Q000166:cytology*; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D066298:In Vitro Techniques; D049408:Luminescent Agents / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D018408:Patch-Clamp Techniques; D011689:Purkinje Cells / Q000187:drug effects / Q000648:ultrastructure*; D013569:Synapses / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D014161:Transduction, Genetic; D014162:Transfection
chemical_list: D018691:Excitatory Amino Acid Antagonists; D049408:Luminescent Agents; D002118:Calcium
keywords: Purkinje cell; calcium; endoplasmic reticulum; inositol 1,4,5-trisphosphate; metabotropic glutamate receptor