Near-isotropic, reconstructed volume electron microscopy (FIB-SEM) of mouse dorsal striatum (jrc_mus-dorsal-striatum)

<b>Sample</b>: Wild-type, 4 month old mouse, strain: C57BL/6;129 from Jackson Lab<b>Sample Description</b>: Close appositions between the membrane of the endoplasmic reticulum (ER) and other intracellular membranes have important functions in cell physiology. These include lipid homeostasis, regulation of Ca2+ dynamics, and control of organelle biogenesis and dynamics. Although these membrane contacts have previously been observed in neurons, their distribution and abundance have not been systematically analyzed. Here, we have used focused ion beam-scanning electron microscopy to generate 3D reconstructions of intracellular organelles and their membrane appositions involving the ER (distance ≤30 nm) in different neuronal compartments. ER–plasma membrane (PM) contacts were particularly abundant in cell bodies, with large, flat ER cisternae apposed to the PM, sometimes with a notably narrow lumen (thin ER). Smaller ER–PM contacts occurred throughout dendrites, axons, and in axon terminals. ER contacts with mitochondria were abundant in all compartments, with the ER often forming a network that embraced mitochondria. Small focal contacts were also observed with tubulovesicular structures, likely to be endosomes, and with sparse multivesicular bodies and lysosomes found in our reconstructions. Our study provides an anatomical reference for interpreting information about interorganelle communication in neurons emerging from functional and biochemical studies.<b>Protocol</b>: Four month old female mice C57BL/6;129 were anesthetized with a ketamine/xylazine anesthetic mixture before perfusion with 2% depolymerized paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, postfixed in 2% OsO4, 1.5% K4Fe(CN)6, 0.1 M sodium cacodylate buffer 1 h. Subsequently, specimens were stained in block with 2% aqueous uranyl acetate (1 h), dehydrated in increasing concentrations of ethanol, and embedded in Epon then re-embedded in durcupan.<b>Contributions</b>: Sample provided by Yumei Wu, Christina Whites, and Pietro De Camilli (Yale), prepared for imaging by Yumei Wu and Christina Whites (Yale), with imaging and post-processing by C. Shan Xu (Yale), Kenneth Hayworth (HHMI/Janelia), Gleb Shtengel, and Harald Hess(HHMI/Janelia).<b>Acquisition ID</b>: jrc_mus-dorsal-striatum<b>Voxel size (nm)</b>: 4 x 4 x 4.44 (X, Y, Z)<b>Data dimensions (µm)</b>:<b> </b>10.2 x 10.3 x 6.0 (X, Y, Z)<b>Imaging start date</b>: 2016-09-09<b>Imaging duration (days)</b>: 3<b>Landing energy (eV)</b>: 700<b>Imaging current (nA)</b>: 0.20<b>Scanning speed (MHz)</b>: 0.400<b>Dataset URL</b>: s3://janelia-cosem-datasets/jrc_mus-dorsal-striatum/jrc_mus-dorsal-striatum.zarr/recon-2/em/<b>Visualization Website</b>: https://openorganelle.janelia.org/datasets/jrc_mus-dorsal-striatum<b>Publication</b>:<b> </b>Wu et al., 2017; Xu et al., 2017; Xu et al., 2021