PSD95 nanoclusters are postsynaptic building blocks in hippocampus circuits

Abstract

The molecular features of synapses in the hippocampus underpin current models of learning and cognition. Although synapse ultra-structural diversity has been described in the canonical hippocampal circuitry, our knowledge of sub-synaptic organisation of synaptic molecules remains largely unknown. To address this, mice were engineered to express Post Synaptic Density 95 protein (PSD95) fused to either eGFP or mEos2 and imaged with two orthogonal super-resolution methods: gated stimulated emission depletion (g-STED) microscopy and photoactivated localisation microscopy (PALM). Large-scale analysis of ~100,000 synapses in 7 hippocampal sub-regions revealed they comprised discrete PSD95 nanoclusters that were spatially organised into single and multi-nanocluster PSDs. Synapses in different sub-regions, cell-types and locations along the dendritic tree of CA1 pyramidal neurons, showed diversity characterised by the number of nanoclusters per synapse. Multi-nanocluster synapses were frequently found in the CA3 and dentate gyrus sub-regions, corresponding to large thorny excrescence synapses. Although the structure of individual nanoclusters remained relatively conserved across all sub-regions, PSD95 packing into nanoclusters also varied between sub-regions determined from nanocluster fluorescence intensity. These data identify PSD95 nanoclusters as a basic structural unit, or building block, of excitatory synapses and their number characterizes synapse size and structural diversity.

Architecture of the Mouse Brain Synaptome

“…Advanced computer vision and machine learning algorithms were developed to segment individual synaptic puncta, measure their spatial distribution, and classify them in an unsupervised manner. The validity of detection and quantification of synaptic punctum parameters by SDM was established by the high correlation of SDM data with published data (Broadhead et al., 2016) from laser scanning confocal microscopy (LSCM) and super-resolution gated stimulated emission depletion (g-STED) microscopy (Figure S7). Synapse diversity was evident in different neuronal types and brain regions; for example, hippocampal CA3 pyramidal neurons had large PSD95-eGFP puncta (characteristic of “thorny excrescence” synapses), in contrast to the small puncta in pyramidal neurons of the somatosensory cortex (Figure S3).…”

Glutamatergic synapses are structurally and biochemically complex because of multiple plasticity processes: long-term potentiation, long-term depression, short-term potentiation and scaling

“…Note that slot proteins are assumed be grouped into hyperslots; the addition of a hyperslot after LTP induction would account for the observed sudden large increases in AMPA-mediated currents (adapted from [69] The advent of super-resolution methods, photo activated localization microscopy (PALM) and stimulated emission depletion (STED), has suddenly begun to provide information about the molecular substructure of the synapse, providing direct evidence that it has a modular character. As shown in figure 3a, PSD-95 is concentrated within a small number (up to about five) of subclusters (nanodomains; 70-80 nm diameter) [75][76][77]. Moreover, the diameter of nanodomains has a relatively small variance (figure 3b), suggestive of a stereotyped modular subunit.…”

Gradation (approx. 10 size states) of synaptic strength by quantal addition of structural modules

“…The modular growth model that we propose would require that functional states have sizes that differ in a quantal rather than proportional manner, given the linear dependence of synapse size on AMPA nanoclusters [14,16,17] ( figure 1). Therefore, we investigated whether the data from Bartol et al [13] might also be consistent with quantally distributed states.…”

“…The above analysis of synapse structure was based on EM data, but recently super-resolution microscopy [14][15][16][17] has revealed other data relevant to understanding the gradation of synaptic size and strength. Various techniques, including stimulated emission depletion (STED) microscopy, singleparticle tracking photoactivation localization microscopy (sptPALM), universal point accumulation in nanoscale topography (uPAINT) and direct stochastic optical reconstruction microscopy (dSTORM), have been employed to explore the molecular organization of synapses under the diffraction limit.…”