Life Inside a Thin Section: Tomography

Chen, Xiaobing; Winters, Christine A.; Reese, Thomas S.

doi:10.1523/jneurosci.2992-08.2008

Cited by 40 publications

(52 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The corresponding cytoplasmic sides of these two types of transmembrane structures in tomograms differed markedly in size and shape, consistent with the fact that C-terminal tails of NMDA receptors, especially GluN2 C tails, are much larger than those of any of the AMPAR subunits (3,4,37,67). The size of the NMDAR cytoplasmic domain is predicted to be about twice that of AMPARs (3).…”

Section: Analysis Of Ampar-and Nmdar-type Structures By Em Tomographymentioning

confidence: 72%

“…Reconstruction of the core PSD structures by EM tomography reveals a prominent class of membrane-associated vertical filaments that associate with transmembrane structures and with horizontal elements (3,4,37,67). The vertical filaments appear to be essential to the assembly of the PSD because their elimination can potentially lead to disassembly of entire segments of the core PSD structure (3,37).…”

Section: Discussionmentioning

confidence: 99%

“…The vertical filaments appear to be essential to the assembly of the PSD because their elimination can potentially lead to disassembly of entire segments of the core PSD structure (3,37). The suggestion that vertical filaments contain PSD-95 (3) is thus far supported by several lines of evidences: positive labeling of the vertical filament for PSD-95 (3,4,37,67); distinct orientation of N and C termini of PSD-95 relative to the postsynaptic membrane (37); close matching of length (16-20 nm) of the vertical filaments with expected length of an extended PSD-95 or SAP97 molecule (3,37); close matching of their density of 368/0.1 μm 2 to the copy number of PSD-95 family proteins in the PSD (51,52,75); and loss of vertical filaments upon knockdown of PSD-95 (37).…”

Section: Discussionmentioning

confidence: 99%

“…Dissociated rat hippocampal neurons (E20) were plated on confluent glia layers, either on 35-mm coverslips for conventional EM and immuno-EM or in 3-mm gold specimen chambers for high-pressure freezing (3,4,67). Cultures were maintained in 10% CO 2 at 35°C in custom MEM (Invitrogen) supplemented with 2 mM Glutamax 1 (Invitrogen), N3 (a growth factor mixture containing apotransferrin, putrescine, selenium, triiodothyronine, insulin, progesterone, and corticosterone), 2% FBS (Invitrogen), and 5% horse serum (HyClone) in a HeraCell incubator (4,84).…”

Section: Methodsmentioning

confidence: 99%

“…For STEM tomography data analysis, PSD diameters are derived from the segmented and rendered PSD areas measured in Amira (FEI) (for details, see SI Materials and Methods). For TEM tomography data analysis, all core structural elements are classified, rendered, and counted according to previously published criteria (3,4,67). The density of a particular structure is calculated by counting its number divided by the postsynaptic membrane area it occupied (details are in SI Materials and Methods).…”

Section: Methodsmentioning

confidence: 99%

See 4 more Smart Citations

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

Chen

Levy

Hou

et al. 2015

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

253

201

View full text Add to dashboard Cite

The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-D-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre-or postsynaptic membrane, and depletes the number of membraneassociated PSD-95-like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membraneassociated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.T he postsynaptic density (PSD) at excitatory glutamatergic synapses, appearing in electron micrographs as a prominent electron-dense thickening lining the postsynaptic membrane (1) is a complex macromolecular machine positioned across from synaptic vesicle release sites at the presynaptic active zone. The PSD clusters and organizes neurotransmitter receptors and signaling molecules at the postsynaptic membrane, transmits and processes synaptic signals, and can undergo structural changes to encode and store information (2-5). Two types of ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs), present at PSDs of excitatory synapses (6-10) mediate almost all synaptic transmission in the brain (11). Biochemistry and mass spectrometry of the detergent-extracted cellular fraction of PSDs have additionally identified many proteins associated with AMPAR and NMDAR complexes (12, 13).The membrane-associated guanylate kinases (MAGUKs), a class of abundant scaffold proteins consisting of PSD-95, PSD-93, synapse-associated protein (SAP)102, and SAP97, interact directly with NMDARs (14-18). These MAGUK proteins share conserved modular structures consisting of three...

show abstract

Section: Analysis Of Ampar-and Nmdar-type Structures By Em Tomographymentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

Chen

Levy

Hou

et al. 2015

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

253

201

View full text Add to dashboard Cite

show abstract

Electron tomographic analysis of synaptic ultrastructure

Burette

Lesperance

Crum

et al. 2012

J of Comparative Neurology

View full text Add to dashboard Cite

Synaptic function depends on interactions among sets of proteins that assemble into complex supramolecular machines. Molecular biology, electrophysiology, and live-cell imaging studies have provided tantalizing glimpses into the inner workings of the synapse, but fundamental questions remain regarding the functional organization of these “nano-machines.” Electron tomography reveals the internal structure of synapses in three dimensions with exceptional spatial resolution. Here we report results from an electron tomographic study of axospinous synapses in neocortex and hippocampus of the adult rat, based on aldehyde-fixed material stabilized with tannic acid in lieu of postfixation with osmium tetroxide. Our results provide a new window into the structural basis of excitatory synaptic processing in the mammalian brain.

show abstract

Electron tomography on γ‐aminobutyric acid‐ergic synapses reveals a discontinuous postsynaptic network of filaments

Linsalata

Chen

Winters

et al. 2014

J of Comparative Neurology

View full text Add to dashboard Cite

The regulation of synaptic strength at γ-aminobutyric acid (GABA)-ergic synapses is dependent on the dynamic capture, retention, and modulation of GABA A-type receptors by cytoplasmic proteins at GABAergic postsynaptic sites. How these proteins are oriented and organized in the postsynaptic cytoplasm is not yet established. In order to better understand these structures and gain further insight into the mechanisms by which they regulate receptor populations at postsynaptic sites, we utilized electron tomography to examine GABAergic synapses in dissociated rat hippocampal cultures. GABAergic synapses were identified and selected for tomography using a set of criteria derived from the structure of immunogold-labeled GABAergic synapses. Tomography revealed a complex postsynaptic network composed of filaments that extend ∼100 nm into the cytoplasm from the postsynaptic membrane. The distribution of these postsynaptic filaments was strikingly similar to that of the immunogold label for gephyrin. Filaments were interconnected through uniform patterns of contact, forming complexes composed of 2-12 filaments each. Complexes did not link to form an integrated, continuous scaffold, suggesting that GABAergic postsynaptic specializations are less rigidly organized than glutamatergic postsynaptic densities.

show abstract

Life Inside a Thin Section: Tomography

Abstract: Editor's Note: Toolboxes are intended to briefly highlight and evaluate an emerging approach or a resource that is becoming widely used in neuroscience. For more information, see http://www.jneurosci.org/misc/itoa.shtml.

Cited by 40 publications

References 52 publications

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

Electron tomographic analysis of synaptic ultrastructure

Electron tomography on γ‐aminobutyric acid‐ergic synapses reveals a discontinuous postsynaptic network of filaments

Contact Info

Product

Resources

About