Visualization of Ca2+ Filling Mechanisms upon Synaptic Inputs in the Endoplasmic Reticulum of Cerebellar Purkinje Cells

• Published in: The Journal of neuroscience 2015-12, Vol.35 (48), p.15837-15846
• Main Authors: Okubo, Yohei, Suzuki, Junji, Kanemaru, Kazunori, Nakamura, Naotoshi, Shibata, Tatsuo, Iino, Masamitsu
• Format: Article
• Language: English
• Subjects: Animals; Calcium - metabolism; Calcium Signaling - physiology; Cell Line, Transformed; Cerebellum - cytology; Cricetinae; Endoplasmic Reticulum - drug effects; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum - ultrastructure; Excitatory Amino Acid Antagonists - pharmacology; In Vitro Techniques; Luminescent Agents - metabolism; Male; Membrane Potentials - drug effects; Mice; Mice, Inbred C57BL; Patch-Clamp Techniques; Purkinje Cells - drug effects; Purkinje Cells - ultrastructure; Synapses - drug effects; Synapses - physiology; Synapses - ultrastructure; Transduction, Genetic; Transfection
• ISSN: 0270-6474; 1529-2401
• DOI: 10.1523/JNEUROSCI.3487-15.2015

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.