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A resource from 3D electron microscopy of hippocampal neuropil for user training and tool development.

Harris KM, Spacek J, Bell ME, Parker PH, Lindsey LF, Baden AD, Vogelstein JT, Burns R - Sci Data (2015)

Bottom Line: All axons, dendrites, glia, and synapses were reconstructed in a cube (~10 μm(3)) surrounding a large dendritic spine, a cylinder (~43 μm(3)) surrounding an oblique dendritic segment (3.4 μm long), and a parallelepiped (~178 μm(3)) surrounding an apical dendritic segment (4.9 μm long).Representative synapses are quantified from varying section planes, and microtubules, polyribosomes, smooth endoplasmic reticulum, and endosomes are identified and reconstructed in a subset of dendrites.The original images, traces, and Reconstruct software and files are freely available and visualized at the Open Connectome Project (Data Citation 1).

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Center for Learning and Memory, Institute for Neuroscience, University of Texas at Austin, 1 University Station C7000 , Austin, Texas 78712, USA.

ABSTRACT
Resurgent interest in synaptic circuitry and plasticity has emphasized the importance of 3D reconstruction from serial section electron microscopy (3DEM). Three volumes of hippocampal CA1 neuropil from adult rat were imaged at X-Y resolution of ~2 nm on serial sections of ~50-60 nm thickness. These are the first densely reconstructed hippocampal volumes. All axons, dendrites, glia, and synapses were reconstructed in a cube (~10 μm(3)) surrounding a large dendritic spine, a cylinder (~43 μm(3)) surrounding an oblique dendritic segment (3.4 μm long), and a parallelepiped (~178 μm(3)) surrounding an apical dendritic segment (4.9 μm long). The data provide standards for identifying ultrastructural objects in 3DEM, realistic reconstructions for modeling biophysical properties of synaptic transmission, and a test bed for enhancing reconstruction tools. Representative synapses are quantified from varying section planes, and microtubules, polyribosomes, smooth endoplasmic reticulum, and endosomes are identified and reconstructed in a subset of dendrites. The original images, traces, and Reconstruct software and files are freely available and visualized at the Open Connectome Project (Data Citation 1).

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3Ds and representative EMs of subcellular structures.(a–d) Dendritic segment d008 from the apical dendrite volume. (a) 3D reconstruction of the dendrite (yellow), excitatory synapses (red), and a subset of organelles that were traced and reconstructed on sections 100–153. (b) Enlarged view of microtubules (mt, royal blue lines) and EM where they appear circular and regularly spaced throughout the shaft of the dendrite on section 148 (royal blue arrowheads). A circular stamp shape was placed manually in the center of each microtubule. Since stamp shapes are closed objects they can represent objects in 3D. The jagged appearance is due to subtle misalignments of the image sections and manual placement of the stamps. 3D of a multivesicular body (teal ovoid) and EM as it appears (teal arrowhead) on section 117. (c) 3D of SER (orange) and EM from section 137, where the SER was tubular and distributed throughout the dendritic shaft. A spine apparatus (sa, arrow), consisting of folds of SER interspersed with dense plates, is illustrated from section 102 in a spine head where part of the PSD was also present (psd, red arrowhead). (d) 3DEM of the mitochondrion (mito, fuchsia) where it appears compact on sections 67 and 111, but distended between where it might be dividing or in a different functional state as visualized on section 88. Scale cube in (d) is 200 nm per side and scale bar is 200 nm for all 3Ds and EMs, respectively, in (b–d). (e–h) 3Ds and representative EMs of subcellular structures in the branched central dendrite from the apical dendrite volume. (e) 3D reconstruction of the dendrite (yellow), excitatory synapses (red), and a subset of organelles that were traced and reconstructed on sections 100–152. EM from section 139 shows d000B branching off from d000A. (f) 3DEM of 3 mitochondria (green, pink, fuchsia) and polyribosomes (black spheres). An EM from section 114 shows the branch point of the green mitochondrion (mito, arrowheads) as it enters d000B and also continues along the length of d000A. (g) 3D of a portion of the dendrite where spine contains a polyribosome. Section 83 illustrates an EM from section 83 of a polyribosome in a spine neck protruding from dendritic segment d000A (pr, arrowhead, named d000Arn20 in the trace files for Reconstruct). (h) 3DEM of SER encircling a vacuole (teal blue) of an autophagosome complex. EM from section 123 shows the vacuole (vac) encircled by SER (ser, arrowhead) in close proximity to an autophagosome (auto, arrowhead) and a mitochondrion (mito, arrowhead). Scale cube 1 μm3 for 3DEM in (e) and 0.125 μm3 (500 nm per side) for 3DEMs in (f–h) and scale bars are 200 nm in (f–h).
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f7: 3Ds and representative EMs of subcellular structures.(a–d) Dendritic segment d008 from the apical dendrite volume. (a) 3D reconstruction of the dendrite (yellow), excitatory synapses (red), and a subset of organelles that were traced and reconstructed on sections 100–153. (b) Enlarged view of microtubules (mt, royal blue lines) and EM where they appear circular and regularly spaced throughout the shaft of the dendrite on section 148 (royal blue arrowheads). A circular stamp shape was placed manually in the center of each microtubule. Since stamp shapes are closed objects they can represent objects in 3D. The jagged appearance is due to subtle misalignments of the image sections and manual placement of the stamps. 3D of a multivesicular body (teal ovoid) and EM as it appears (teal arrowhead) on section 117. (c) 3D of SER (orange) and EM from section 137, where the SER was tubular and distributed throughout the dendritic shaft. A spine apparatus (sa, arrow), consisting of folds of SER interspersed with dense plates, is illustrated from section 102 in a spine head where part of the PSD was also present (psd, red arrowhead). (d) 3DEM of the mitochondrion (mito, fuchsia) where it appears compact on sections 67 and 111, but distended between where it might be dividing or in a different functional state as visualized on section 88. Scale cube in (d) is 200 nm per side and scale bar is 200 nm for all 3Ds and EMs, respectively, in (b–d). (e–h) 3Ds and representative EMs of subcellular structures in the branched central dendrite from the apical dendrite volume. (e) 3D reconstruction of the dendrite (yellow), excitatory synapses (red), and a subset of organelles that were traced and reconstructed on sections 100–152. EM from section 139 shows d000B branching off from d000A. (f) 3DEM of 3 mitochondria (green, pink, fuchsia) and polyribosomes (black spheres). An EM from section 114 shows the branch point of the green mitochondrion (mito, arrowheads) as it enters d000B and also continues along the length of d000A. (g) 3D of a portion of the dendrite where spine contains a polyribosome. Section 83 illustrates an EM from section 83 of a polyribosome in a spine neck protruding from dendritic segment d000A (pr, arrowhead, named d000Arn20 in the trace files for Reconstruct). (h) 3DEM of SER encircling a vacuole (teal blue) of an autophagosome complex. EM from section 123 shows the vacuole (vac) encircled by SER (ser, arrowhead) in close proximity to an autophagosome (auto, arrowhead) and a mitochondrion (mito, arrowhead). Scale cube 1 μm3 for 3DEM in (e) and 0.125 μm3 (500 nm per side) for 3DEMs in (f–h) and scale bars are 200 nm in (f–h).

Mentions: Also for training and identification purposes, subcellular structures were named (Table 3 (available online only)) and traced in dendrites D008 and D000A-B from the apical dendrite volume (Fig. 7). The short reconstructed dendritic segments (Fig. 7a) contained microtubules that typically spanned their entire length (Fig. 7b), as would be expected from our prior work showing them to be about 80 μm long in the CA1 dendrites67. SER formed a continuous network of tubules, branches, and lakes in the dendritic shaft (Fig. 7c)52 and occasionally expanded into dendritic spines to form a spine apparatus (Figs 2a, 7c). Mitochondria spanned the full lengths of these dendritic segments. One appears to have been in multiple functional states, or dividing, with both compact and distended regions (Fig. 7d). The apical dendrite (Fig. 7e) had a mitochondrion that branched into a side dendritic branch (Fig. 7f). Polyribosomes occurred in the dendritic shaft and some spines (Fig. 7g). Endosomal compartments (Table 3 (available online only)), including autophagosomes (Fig. 7h), were also identified and traced in these two dendrites. Multivesicular bodies (Fig. 7b) were included in the endosomal compartments, although some might in fact be exosomes68. Standard names for all of the traced subcellular components in Reconstruct are summarized in Table 3 (available online only) along with the total number of objects for each component. The OCP sliders are described next.


A resource from 3D electron microscopy of hippocampal neuropil for user training and tool development.

Harris KM, Spacek J, Bell ME, Parker PH, Lindsey LF, Baden AD, Vogelstein JT, Burns R - Sci Data (2015)

3Ds and representative EMs of subcellular structures.(a–d) Dendritic segment d008 from the apical dendrite volume. (a) 3D reconstruction of the dendrite (yellow), excitatory synapses (red), and a subset of organelles that were traced and reconstructed on sections 100–153. (b) Enlarged view of microtubules (mt, royal blue lines) and EM where they appear circular and regularly spaced throughout the shaft of the dendrite on section 148 (royal blue arrowheads). A circular stamp shape was placed manually in the center of each microtubule. Since stamp shapes are closed objects they can represent objects in 3D. The jagged appearance is due to subtle misalignments of the image sections and manual placement of the stamps. 3D of a multivesicular body (teal ovoid) and EM as it appears (teal arrowhead) on section 117. (c) 3D of SER (orange) and EM from section 137, where the SER was tubular and distributed throughout the dendritic shaft. A spine apparatus (sa, arrow), consisting of folds of SER interspersed with dense plates, is illustrated from section 102 in a spine head where part of the PSD was also present (psd, red arrowhead). (d) 3DEM of the mitochondrion (mito, fuchsia) where it appears compact on sections 67 and 111, but distended between where it might be dividing or in a different functional state as visualized on section 88. Scale cube in (d) is 200 nm per side and scale bar is 200 nm for all 3Ds and EMs, respectively, in (b–d). (e–h) 3Ds and representative EMs of subcellular structures in the branched central dendrite from the apical dendrite volume. (e) 3D reconstruction of the dendrite (yellow), excitatory synapses (red), and a subset of organelles that were traced and reconstructed on sections 100–152. EM from section 139 shows d000B branching off from d000A. (f) 3DEM of 3 mitochondria (green, pink, fuchsia) and polyribosomes (black spheres). An EM from section 114 shows the branch point of the green mitochondrion (mito, arrowheads) as it enters d000B and also continues along the length of d000A. (g) 3D of a portion of the dendrite where spine contains a polyribosome. Section 83 illustrates an EM from section 83 of a polyribosome in a spine neck protruding from dendritic segment d000A (pr, arrowhead, named d000Arn20 in the trace files for Reconstruct). (h) 3DEM of SER encircling a vacuole (teal blue) of an autophagosome complex. EM from section 123 shows the vacuole (vac) encircled by SER (ser, arrowhead) in close proximity to an autophagosome (auto, arrowhead) and a mitochondrion (mito, arrowhead). Scale cube 1 μm3 for 3DEM in (e) and 0.125 μm3 (500 nm per side) for 3DEMs in (f–h) and scale bars are 200 nm in (f–h).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4555877&req=5

f7: 3Ds and representative EMs of subcellular structures.(a–d) Dendritic segment d008 from the apical dendrite volume. (a) 3D reconstruction of the dendrite (yellow), excitatory synapses (red), and a subset of organelles that were traced and reconstructed on sections 100–153. (b) Enlarged view of microtubules (mt, royal blue lines) and EM where they appear circular and regularly spaced throughout the shaft of the dendrite on section 148 (royal blue arrowheads). A circular stamp shape was placed manually in the center of each microtubule. Since stamp shapes are closed objects they can represent objects in 3D. The jagged appearance is due to subtle misalignments of the image sections and manual placement of the stamps. 3D of a multivesicular body (teal ovoid) and EM as it appears (teal arrowhead) on section 117. (c) 3D of SER (orange) and EM from section 137, where the SER was tubular and distributed throughout the dendritic shaft. A spine apparatus (sa, arrow), consisting of folds of SER interspersed with dense plates, is illustrated from section 102 in a spine head where part of the PSD was also present (psd, red arrowhead). (d) 3DEM of the mitochondrion (mito, fuchsia) where it appears compact on sections 67 and 111, but distended between where it might be dividing or in a different functional state as visualized on section 88. Scale cube in (d) is 200 nm per side and scale bar is 200 nm for all 3Ds and EMs, respectively, in (b–d). (e–h) 3Ds and representative EMs of subcellular structures in the branched central dendrite from the apical dendrite volume. (e) 3D reconstruction of the dendrite (yellow), excitatory synapses (red), and a subset of organelles that were traced and reconstructed on sections 100–152. EM from section 139 shows d000B branching off from d000A. (f) 3DEM of 3 mitochondria (green, pink, fuchsia) and polyribosomes (black spheres). An EM from section 114 shows the branch point of the green mitochondrion (mito, arrowheads) as it enters d000B and also continues along the length of d000A. (g) 3D of a portion of the dendrite where spine contains a polyribosome. Section 83 illustrates an EM from section 83 of a polyribosome in a spine neck protruding from dendritic segment d000A (pr, arrowhead, named d000Arn20 in the trace files for Reconstruct). (h) 3DEM of SER encircling a vacuole (teal blue) of an autophagosome complex. EM from section 123 shows the vacuole (vac) encircled by SER (ser, arrowhead) in close proximity to an autophagosome (auto, arrowhead) and a mitochondrion (mito, arrowhead). Scale cube 1 μm3 for 3DEM in (e) and 0.125 μm3 (500 nm per side) for 3DEMs in (f–h) and scale bars are 200 nm in (f–h).
Mentions: Also for training and identification purposes, subcellular structures were named (Table 3 (available online only)) and traced in dendrites D008 and D000A-B from the apical dendrite volume (Fig. 7). The short reconstructed dendritic segments (Fig. 7a) contained microtubules that typically spanned their entire length (Fig. 7b), as would be expected from our prior work showing them to be about 80 μm long in the CA1 dendrites67. SER formed a continuous network of tubules, branches, and lakes in the dendritic shaft (Fig. 7c)52 and occasionally expanded into dendritic spines to form a spine apparatus (Figs 2a, 7c). Mitochondria spanned the full lengths of these dendritic segments. One appears to have been in multiple functional states, or dividing, with both compact and distended regions (Fig. 7d). The apical dendrite (Fig. 7e) had a mitochondrion that branched into a side dendritic branch (Fig. 7f). Polyribosomes occurred in the dendritic shaft and some spines (Fig. 7g). Endosomal compartments (Table 3 (available online only)), including autophagosomes (Fig. 7h), were also identified and traced in these two dendrites. Multivesicular bodies (Fig. 7b) were included in the endosomal compartments, although some might in fact be exosomes68. Standard names for all of the traced subcellular components in Reconstruct are summarized in Table 3 (available online only) along with the total number of objects for each component. The OCP sliders are described next.

Bottom Line: All axons, dendrites, glia, and synapses were reconstructed in a cube (~10 μm(3)) surrounding a large dendritic spine, a cylinder (~43 μm(3)) surrounding an oblique dendritic segment (3.4 μm long), and a parallelepiped (~178 μm(3)) surrounding an apical dendritic segment (4.9 μm long).Representative synapses are quantified from varying section planes, and microtubules, polyribosomes, smooth endoplasmic reticulum, and endosomes are identified and reconstructed in a subset of dendrites.The original images, traces, and Reconstruct software and files are freely available and visualized at the Open Connectome Project (Data Citation 1).

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Center for Learning and Memory, Institute for Neuroscience, University of Texas at Austin, 1 University Station C7000 , Austin, Texas 78712, USA.

ABSTRACT
Resurgent interest in synaptic circuitry and plasticity has emphasized the importance of 3D reconstruction from serial section electron microscopy (3DEM). Three volumes of hippocampal CA1 neuropil from adult rat were imaged at X-Y resolution of ~2 nm on serial sections of ~50-60 nm thickness. These are the first densely reconstructed hippocampal volumes. All axons, dendrites, glia, and synapses were reconstructed in a cube (~10 μm(3)) surrounding a large dendritic spine, a cylinder (~43 μm(3)) surrounding an oblique dendritic segment (3.4 μm long), and a parallelepiped (~178 μm(3)) surrounding an apical dendritic segment (4.9 μm long). The data provide standards for identifying ultrastructural objects in 3DEM, realistic reconstructions for modeling biophysical properties of synaptic transmission, and a test bed for enhancing reconstruction tools. Representative synapses are quantified from varying section planes, and microtubules, polyribosomes, smooth endoplasmic reticulum, and endosomes are identified and reconstructed in a subset of dendrites. The original images, traces, and Reconstruct software and files are freely available and visualized at the Open Connectome Project (Data Citation 1).

Show MeSH
Related in: MedlinePlus