THE ROLE OF THE ENDOPLASMIC RETICULUM IN SYNAPTIC PLASTICITY

In neurons the endoplasmic reticulum (ER) is a complex organelle, distributed throughout the soma, axon and dendritic arbor, and present in some dendritic spines. While well known for its role in protein synthesis, transport, and calcium storage, less is known about ER calcium release and refilling dynamics, and how this affects synaptic function. It has previously been shown that ER is stably present in 10% of dendritic spines while 70% are transiently visited (Perez-Alvarez et al., Nat Commun. 2020). ER-containing spines have strong synapses with a higher number of GluA2 receptors, which do not undergo structural long-term potentiation but readily undergo long-term depression.
Using two-photon microscopy, we investigate dendritic calcium dynamics in rat organotypic hippocampal slices. We transfect CA1 pyramidal neurons with genetically targeted, low affinity ER calcium sensors, which allow us to quantify ER calcium responses while manipulating calcium levels through pharmacology, electrophysiology and optogenetics.
Preliminary experiments with optogenetic stimulation revealed strong artifacts due to photoswitching of the commonly used ER-GCaMP-210 calcium sensor. Introduction of a point mutation close to the fluorophore reduced, but did not eliminate, the photoswitching at a wavelength of 500 nm. This led us to develop and test novel sensors suitable for all-optical experiments with optogenetic stimulation. To investigate the dynamic response and recovery of ER Ca2+ in apical dendrites, we performed simultaneous cytosolic and ER calcium imaging while electrically stimulating the basal dendrites. Collectively, these tools will allow us to disentangle the contributions of different calcium sources at dendritic spines during synaptic plasticity protocols.