All-optical investigation of the role of CaMKII in long-term plasticity in the hippocampus and the development of a method for ultrastructural analysis of synapses

Abstract

Dynamic changes in synaptic efficacy and structure, termed synaptic plasticity, are a major mechanism of information storage in the brain. Calcium–calmodulin-dependent protein kinase II (CaMKII) is one of the most important memory molecules that, through its autophosphorylation feature, transforms transient activation due to synaptic activity-related increases in calcium into longer-lasting changes in synaptic strength. Whether CaMKII is essential to induce and/or to maintain synaptic plasticity remains controversial. I took advantage of optogenetic tools to investigate the role of CaMKII in synaptic plasticity by inducing plasticity and manipulating relevant signaling pathways at the same time. Specifically, I induced spike-timing-dependent plasticity (STDP) at Schaffer collateral synapses in rat hippocampal slice cultures by optogenetic stimulation of two neuronal populations expressing spectrally separated channelrhodopsins. The all-optical protocol induced timing-dependent long-term potentiation (tLTP), increasing synaptic strength both acutely and more interestingly, chronically. Optical inhibition of CaMKII during tLTP induction complete blocked acute tLTP, but interestingly, chronic tLTP did still emerge. Optical activation of CaMKII, on the other hand, accomplished by photoactivatable CaMKII, induced acute functional, structural and ultrastructural alterations at synapses. Structural plasticity often developed in a spatially clustered manner. However, these CaMKII-activation-induced synaptic alterations did not last for more than one day. Together, these data suggest that activity-dependent potentiation of synaptic inputs has two phases: CaMKII is necessary and sufficient for the induction of early LTP. A second, CaMKII-independent mechanism, possibly through the persistent activity of protein kinase Mζ (PKMζ), is responsible for the selective strengthening of inputs days later. Although significant progress has been made in understanding the mechanism of information storage at the synaptic level, our knowledge of the mechanisms of long-term synaptic information storage and the ultrastructural anatomical features of synaptic plasticity is still rudimentary. For this reason, I developed a genetic labeling method that allows the visualization of synapses of interest under electron microscopy (EM) in a robust and versatile manner. I use two organelle-targeted peroxidases, dAPEX2-NES and SYP-HRP, and the Cre/LoxP system, which together allow specific labeling of optogenetically manipulated synapses at the EM level. Optimizing transfection, stimulation, fixation and image analysis, I have established a pipeline for the ultrastructural analysis of synapses after optogenetic stimulation.