Rapid Ultrastructural Changes in the PSD and Surrounding Membrane after Induction of Structural LTP in Single Dendritic Spines

Abstract

The structural plasticity of dendritic spines is considered to be an important basis of synaptic plasticity, learning, and memory. Here, we induced input-specific structural LTP (sLTP) in single dendritic spines in organotypic hippocampal slices from mice of either sex and performed ultrastructural analyses of the spines using efficient correlative light and electron microscopy. We observed reorganization of the PSD nanostructure, such as perforation and segmentation, at 2-3, 20, and 120 min after sLTP induction. In addition, PSD and nonsynaptic axon-spine interface (nsASI) membrane expanded unevenly during sLTP. Specifically, the PSD area showed a transient increase at 2-3 min after sLTP induction. The PSD growth was to a degree less than spine volume growth at 2-3 min and 20 min after sLTP induction but became similar at 120 min. On the other hand, the nsASI area showed a profound and lasting expansion, to a degree similar to spine volume growth throughout the process. These rapid ultrastructural changes in PSD and surrounding membrane may contribute to rapid electrophysiological plasticity during sLTP.

Cryo-Confocal Imaging for CLEM Mapping in Brain Tissues

“…CLEM workflow, especially when functional LM imaging and EM examination are combined, can be powerful tools for imaging cells or tissues to correlate structure and function. We previously studied structural plasticity of dendritic spines using a CLEM workflow with ssSEM [6][7], however, a technical question remained in terms of the temporal resolution of the fixation process. To capture the morphological change of spine plasticity, we ideally want to fix the tissue within 2-3 minutes of glutamate uncaging to preserve the initial stage of the phenomena.…”

“…A pyramidal neuron expressing GFP fluorescence was imaged with 2-photon light microscopy using a 60× water immersion lens (LUMPlan FLN 60× 1.00, OLYM-PUS) with 1×-30× digital zoom to capture the target cell body, dendrite, and spine. Glutamate uncaging was performed on one single spine as previously described [7] to induce structural plasticity. Subsequent spine growth was monitored for approximately 1 minute (Figure 1A).…”

“…This structural plasticity of dendritic spines is thought to be the basis of learning and memory and has been extensively characterized [3][4][5]. Following excitation, the tissue can be fixed and observed using CLEM to characterize ultrastructural changes induced in the spine [6][7]. Since initial structural changes in the dendritic spine start on a short time scale (∼several seconds), capturing the process of structural plasticity requires rapid fixation of the tissue.…”

Regulation of the Stability and Localization of Post-synaptic Membrane Proteins by Liquid-Liquid Phase Separation

“…First, PSD in synapses shows the exchange of component proteins between the inside and outside of PSD (Kuriu et al, 2006 ; Sharma et al, 2006 ). PSD also shows the incorporation of cytosolic proteins (Bosch et al, 2014 ) and the rapid rearrangement of its structure (Blanpied et al, 2008 ; Kerr et al, 2011 ; Sun et al, 2021 ) upon stimulation and Ca 2+ influx. In addition, although PSD is defined as a highly dense area under electron microscopy, it exhibits multiplexed shapes, such as fenestrated, horseshoe, and segmented (Toni et al, 2001 ; Borczyk et al, 2019 ).…”

“…It is known that the size of PSD is correlated with the number of AMPAR nanodomains, the size of dendritic spines, and synaptic strength (Harris et al, 1992 ; Noguchi et al, 2005 ; Nair et al, 2013 ; Meyer et al, 2014 ). The Ca 2+ influx during LTP mediates an increase in the size and complexity of PSD (Sun et al, 2021 ), and the growth of PSD (Harris, 2020 ). On the contrary, LTD induction results in a loss of PSD components, such as PSD-95 (Bingol and Shang, 2011 ; Compans et al, 2021 ).…”