The lysosome or lysosome-related organelle may serve as a Ca 2+ store in the boutons of hippocampal pyramidal cells

Abstract

Boutons are specialised presynaptic compartments that lie along the axons of central neurons. Release of neurotransmitter from boutons is tightly regulated by the level of intracellular calcium [Ca 2+] i. A rise in Ca 2+ level may be generated in several ways; entry of extracellular Ca 2+ via voltage gated calcium channels (VGCCs), entry via ligand-operated channels (LOCs) or the release of Ca 2+ from intracellular Ca 2+ stores. The role of Ca 2+ stores in boutons remains poorly understood, despite recent work indicating that the release of Ca 2+ from the endoplasmic reticulum (ER) may contribute to transmitter release. In this study we assess whether the lysosome or a closely related organelle functions as a Ca 2+ store in the boutons of hippocampal pyramidal neurones. Lysosomes are small acidic organelles more commonly known for their role in degrading redundant cellular constituents. Using a fluorescent lysosomal marker, we show that lysosomes are located in the axons of hippocampal CA3 neurones. Selective pharmacological lysis of the lysosomes with glycyl-phenylalanine 2-napthylamide (GPN) generates rapid, highly focal Ca 2+ transients within the axon and increases the frequency of spontaneous miniature excitatory post-synaptic currents (mEPSCs), revealing that the organelle contains Ca 2+ at a concentration sufficient to evoke transmitter release. Confocal laser scanning microscopy, combined with electrophysiology is used to monitor the action potential evoked increases in [Ca 2+] i in boutons. We show that disruption of lysosomes compromises action potential evoked [Ca 2+] i but this effect is occluded if the ER is discharged. Conversely, disruption of the lysosome does not appear to impact on the capacity of the ER to release Ca 2+. These results suggest that the lysosome may serve as a Ca 2+ store within hippocampal boutons, with a Ca 2+ signalling role that is unique from that of the ER.