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.
Highlights
The molecular features of synapses in the hippocampus underpin current models of learning and cognition
We report a study of the nano-architecture of synapses in the hippocampal formation by combining the technology of genetically modified mice, where endogenous Post Synaptic Density 95 (PSD95) was labelled with fluorescent proteins, with two orthogonal forms of super-resolution microscopy
Tandem affinity purification (TAP) tags were fused in-frame to the carboxyl terminus of endogenous PSD95 using gene targeting and we have used the same strategy with eGFP and mEos[2] tags, expressing the fusion proteins under the control of PSD95’s regulatory elements
Summary
The molecular features of synapses in the hippocampus underpin current models of learning and cognition. We used mice that were genetically engineered to express PSD95 fused to either eGFP or mEos[2] (PSD95-eGFP, PSD95-mEos2), which are suitable for use with two orthogonal super-resolution methods: gated stimulated emission depletion (g-STED) microscopy and photoactivated localisation microscopy (PALM) Using these cross-validating approaches and highly quantitative methods, we performed the first systematic large-scale study of PSD95 nanoarchitecture across the hippocampal formation in brain sections from mature mice. We found characteristic anatomical distributions of PSD95 NCs in distinct hippocampal regions, cell-types and locations within the dendritic tree Large, complex synapses such as the thorny excresences of CA3 pyramidal neurons were found to contain multiple NCs. This study demonstrates the power of combining investigations of synapse nano-architecture with large-scale anatomical approaches to define basic mechanisms of synapse structure. The tools and approaches described here have wide application in vitro and in vivo
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