Epub – bioRxiv
Characterization of the three-dimensional synaptic and mitochondrial nanoarchitecture within glutamatergic synaptic complexes in postmortem human brain via focused ion beam-scanning electron microscopy

Glausier JR, Bouchet-Marquis C, Maier M, Banks-Tibbs T, Wu K, Ning J, Melchitzky D, Lewis DA, Freyberg Z

Glutamatergic synapses are the primary site of excitatory synaptic signaling and neural communication in the cerebral cortex. Electron microscopy (EM) studies in non-human model organisms have demonstrated that glutamate synaptic activity and functioning are directly reflected in quantifiable ultrastructural features. Thus, quantitative EM analysis of glutamate synapses in ex vivo preserved human brain tissue has the potential to provide novel insight into in vivo synaptic functioning. However, factors associated with the acquisition and preservation of human brain tissue have resulted in persistent concerns regarding the potential confounding effects of antemortem and postmortem biological processes on synaptic and sub-synaptic ultrastructural features. Thus, we sought to determine how well glutamate synaptic relationships and nanoarchitecture are preserved in postmortem human dorsolateral prefrontal cortex (DLPFC), a region that substantially differs in size and architecture from model systems. Focused ion beam-scanning electron microscopy (FIB-SEM), a powerful volume EM (VEM) approach, was employed to generate high-fidelity, fine-resolution, three-dimensional (3D) micrographic datasets appropriate for quantitative analyses. Using postmortem human DLPFC with a 6-hour postmortem interval, we optimized a tissue preservation and staining workflow that generated samples of excellent ultrastructural preservation and the high-contrast staining intensity required for FIB-SEM imaging. Quantitative analysis of sub-cellular, sub-synaptic and organelle components within glutamate axo-spinous synapses revealed that ultrastructural features of synaptic function and activity were well-preserved within and across individual synapses in postmortem human brain tissue. The synaptic, sub-synaptic and organelle measures were highly consistent with findings from experimental models that are free from antemortem or postmortem effects. Further, dense reconstruction of neuropil revealed a unique, ultrastructurally-complex, spiny dendritic shaft that exhibited features characteristic of neuronal processes with heightened synaptic communication, integration and plasticity. Altogether, our findings provide a critical proof-of-concept that ex vivo VEM analysis provides a valuable and informative means to infer in vivo functioning of human brain.

Characterization of the three-dimensional synaptic and mitochondrial nanoarchitecture within glutamatergic synaptic complexes in postmortem human brain via focused ion beam-scanning electron microscopy. Glausier JR, Bouchet-Marquis C, Maier M, Banks-Tibbs T, Wu K, Ning J, Melchitzky D, Lewis DA, Freyberg Z. bioRxiv. 2024 Feb 28:2024.02.26.582174. doi: 10.1101/2024.02.26.582174. [Epub ahead of print] PMID: 38463986 PMCID: PMC10925168.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10925168/