Transform-based backprojection for volume reconstruction of large format electron microscope tilt series

Lawrence, Albert F.; Bouwer, James C.; Perkins, Guy; Ellisman, Mark H.

doi:10.1016/j.jsb.2005.12.012

Cited by 102 publications

(116 citation statements)

References 50 publications

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“…The 4-to 5-m-thick sections were sent to Osaka, Japan, for tomographic imaging by using the 3 MeV Hitachi H3000U ultra-high voltage EM. Dendritic shafts and spines in the digital slices from the tomograms were segmented, 3D-reconstructed, and measured by using IMOD and NCMIR's in-house reconstruction algorithm called TxBR (43). Spine head volumes and spine lengths were analyzed for protrusions that are entirely included in the tomograms.…”

Section: Methodsmentioning

confidence: 99%

Regulation of spine morphology and spine density by NMDA receptor signaling in vivo

Ultanir

Kim²,

Hall³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

155

136

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Dendritic spines are the major sites of excitatory synaptic transmission in the CNS, and their size and density influence the functioning of neuronal circuits. Here we report that NMDA receptor signaling plays a critical role in regulating spine size and density in the developing cortex. Genetic deletion of the NR1 subunit of the NMDA receptor in the cortex leads to a decrease in spine density and an increase in spine head size in cortical layer 2/3 pyramidal neurons. This process is accompanied by an increase in the presynaptic axon bouton volume and the postsynaptic density area, as well as an increase in the miniature excitatory postsynaptic current amplitude and frequency. These observations indicate that NMDA receptors regulate synapse structure and function in the developing cortex.cortex ͉ development ͉ synapse D endritic spines are bulbous membrane protrusions that form the postsynaptic specializations of the vast majority of excitatory synapses in the CNS (1-3). During the first postnatal week, highly motile and short-lived dendritic filopodia are abundant on cortical pyramidal neurons (4). These actin-rich protrusions make immature synapses along their length or tip (5). The dendritic spines that are present during the first two postnatal weeks display an immature morphology (5), and the spine head is highly motile, with protrusions extending and retracting from the head (6). After the second postnatal week, the spine density increases, and spines attain a mature morphology with bulbous heads similar to that seen in the adult (7). Dendritic spines are sites of excitatory synaptic transmission, and their structure and density are important measures of synaptic function.An important feature of dendritic spines is that their volume and density can be dynamically regulated. Stimuli that induce long-term potentiation (LTP) and long-term depression in hippocampal slices lead to rapid changes in spine volume by activation of NMDA receptors (8-10). Several lines of evidence suggest that NMDA receptor signaling might influence spine volume by recruitment of AMPA receptors (AMPARs). Immature synapses in the CNS have a low AMPAR:NMDA receptor (NMDAR) ratio and gradually acquire AMPARs during development (11,12). The increase in the AMPAR:NMDAR ratio at synapses during development is thought to be mediated by NMDAR-induced recruitment of AMPARs (13-16). This increase in surface AMPARs could influence spine volume because it has been reported that overexpression of the GluR2 AMPAR subunit in hippocampal cultures leads to an increase in spine size (17). NMDAR signaling also has been implicated in regulating spine density. In hippocampal slices, LTP-inducing stimuli or glutamate application leads to the rapid induction of new spines/filopodia, which is blocked by NMDAR antagonists (18,19).These observations suggest a model in which NMDARs recruit AMPARs to developing synapses, which drives spine growth and maturation. This model predicts that loss of NMDARs should lead to smaller AMPA currents and smaller spines. Her...

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Section: Methodsmentioning

confidence: 99%

Regulation of spine morphology and spine density by NMDA receptor signaling in vivo

Ultanir

Kim²,

Hall³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

155

136

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“…Back projection was performed using the TxBr package (Lawrence et al, 2006). Surface renderings were visualized in Amira (Mercury/TGS, San Diego, CA).…”

Section: 33mentioning

confidence: 99%

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

Sosinsky

Crum

Jones

et al. 2008

Journal of Structural Biology

Self Cite

112

114

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The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were 1) high pressure frozen without fixation, 2) high pressure frozen following fixation, and 3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.

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“…The final pixel size in our reconstructions was 1.41 nm. The IMOD package (Kremer et al, 1996) was used for rough alignment with the fine alignment and reconstruction performed using the TxBR package (Lawrence et al, 2006). One of the HPF/FS single-tilt series reconstructions (310 nm thickness) had a (global) resolution estimated by using the even/odd Fourier shell correlation (Cardone et al, 2005) and software provided by Alasdair Steven (NIH) to be 8.4 nm at a threshold of 0.3; the other two single-tilt reconstructions analyzed likely had similar resolution estimates because they had similar thicknesses and angular increment and range.…”

Section: Electron Tomographymentioning

confidence: 99%

Electron tomographic analysis of cytoskeletal cross-bridges in the paranodal region of the node of Ranvier in peripheral nerves

Perkins

Sosinsky

Ghassemzadeh

et al. 2008

Journal of Structural Biology

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The node of Ranvier is a site for ionic conductances along myelinated nerves and governs the saltatory transmission of action potentials. Defects in the cross-bridging and spacing of the cytoskeleton is a prominent pathologic feature in diseases of the peripheral nerve. Electron tomography was used to examine cytoskeletal-cytoskeletal, membrane-cytoskeletal, and heterologous cell connections in the paranodal region of the node of Ranvier in peripheral nerves. Focal attachment of cytoskeletal filaments to each other and to the axolemma and paranodal membranes of the Schwann cell via narrow cross-bridges was visualized in both neuronal and glial cytoplasms. A subset of intermediate filaments associates with the cytoplasmic surfaces of supramolecular complexes of transmembrane structures that are presumed to include known and unknown junctional proteins. Mitochondria were linked to both microtubules and neurofilaments in the axoplasm and to neighboring smooth endoplasmic reticulum by narrow cross-bridges. Tubular cisternae in the glial cytoplasm were also linked to the paranodal glial cytoplasmic loop juxtanodal membrane by short cross-bridges. In the extracellular matrix between axon and Schwann cell, junctional bridges formed long cylinders inking the two membranes. Interactions between cytoskeleton, membranes, and extracellular matrix associations in the paranodal region is likely critical not only for scaffolding, but also for intracellular and extracellular communication.

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Transform-based backprojection for volume reconstruction of large format electron microscope tilt series

Cited by 102 publications

References 50 publications

Regulation of spine morphology and spine density by NMDA receptor signaling in vivo

Regulation of spine morphology and spine density by NMDA receptor signaling in vivo

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

Electron tomographic analysis of cytoskeletal cross-bridges in the paranodal region of the node of Ranvier in peripheral nerves

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