En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics.

Autor:	Lu Z; Department of Psychology and Neuroscience, Life Sciences Centre, Dalhousie University, Halifax, NS, Canada.; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States., Xu CS; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States.; Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, United States., Hayworth KJ; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States., Pang S; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States.; Yale School of Medicine, New Haven, CT, United States., Shinomiya K; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States., Plaza SM; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States., Scheffer LK; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States., Rubin GM; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States., Hess HF; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States., Rivlin PK; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States.; Applied Physics Laboratory, Johns Hopkins University, Laurel, MD, United States., Meinertzhagen IA; Department of Psychology and Neuroscience, Life Sciences Centre, Dalhousie University, Halifax, NS, Canada.; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States.
Jazyk:	angličtina
Zdroj:	Frontiers in neural circuits [Front Neural Circuits] 2022 Dec 16; Vol. 16, pp. 917251. Date of Electronic Publication: 2022 Dec 16 (Print Publication: 2022).
DOI:	10.3389/fncir.2022.917251
Abstrakt:	Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly Drosophila , in particular, we need to dissect the brain, connectives, and ventral nerve cord as a single continuous unit, fix and stain it, and undertake automated segmentation of neuron membranes. To achieve this, we designed a protocol using progressive lowering of temperature dehydration (PLT), a technique routinely used to preserve cellular structure and antigenicity. We combined PLT with low temperature en bloc staining (LTS) and recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection. Here we report three different PLT-LTS methods designed to meet the requirements for FIB-SEM imaging of the Drosophila brain. These requirements include: good preservation of ultrastructural detail, high level of en bloc staining, artifact-free microdissection, and smooth hot-knife cutting to reduce the brain to dimensions suited to FIB-SEM. In addition to PLT-LTS, we designed a jig to microdissect and pre-fix the fly's delicate brain and central nervous system. Collectively these methods optimize morphological preservation, allow us to image the brain usually at 8 nm per voxel, and simultaneously speed the formerly slow rate of FIB-SEM imaging. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2022 Lu, Xu, Hayworth, Pang, Shinomiya, Plaza, Scheffer, Rubin, Hess, Rivlin and Meinertzhagen.)
Databáze:	MEDLINE
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