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Cited by 17Neuroscience and Neuropharmacology ResearchAdvanced Electron Microscopy Techniques and ApplicationsClassical Anatomy and Histology

The DOI matches its Crossref record; 79 of the 83 references it cites were found in a registry or an index; 19 of the 40 sources its claims cite were checked for support.

Unambiguous identification of asymmetric and symmetric synapses using volume electron microscopy

Open access
Cano-Astorga, Nicolás, Plaza-Alonso, Sergio, Turegano-Lopez, Marta, Rodrigo-Rodríguez, José, Merchan-Perez, Angel, DeFelipe, Javier·2024·Frontiers in Neuroanatomy
DOI 10.3389/fnana.2024.1348032PMC PMC11026665
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Abstract

The brain contains thousands of millions of synapses, exhibiting diverse structural, molecular, and functional characteristics. However, synapses can be classified into two primary morphological types: Gray’s type I and type II, corresponding to Colonnier’s asymmetric (AS) and symmetric (SS) synapses, respectively. AS and SS have a thick and thin postsynaptic density, respectively. In the cerebral cortex, since most AS are excitatory (glutamatergic), and SS are inhibitory (GABAergic), determining the distribution, size, density, and proportion of the two major cortical types of synapses is critical, not only to better understand synaptic organization in terms of connectivity, but also from a functional perspective. However, several technical challenges complicate the study of synapses. Potassium ferrocyanide has been utilized in recent volume electron microscope studies to enhance electron density in cellular membranes. However, identifying synaptic junctions, especially SS, becomes more challenging as the postsynaptic densities become thinner with increasing concentrations of potassium ferrocyanide. Here we describe a protocol employing Focused Ion Beam Milling and Scanning Electron Microscopy for studying brain tissue. The focus is on the unequivocal identification of AS and SS types. To validate SS observed using this protocol as GABAergic, experiments with immunocytochemistry for the vesicular GABA transporter were conducted on fixed mouse brain tissue sections. This material was processed with different concentrations of potassium ferrocyanide, aiming to determine its optimal concentration. We demonstrate that using a low concentration of potassium ferrocyanide (0.1%) improves membrane visualization while allowing unequivocal identification of synapses as AS or SS.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

This protocol paper shows that 0.1% potassium ferrocyanide in FIB-SEM brain tissue preparation preserves excellent membrane contrast while allowing unambiguous identification of asymmetric (AS) and symmetric (SS) synapses. VGAT immunolabeling confirmed that SS were formed by VGAT-positive terminals and AS by VGAT-negative terminals.

Key findings

  • With 1% potassium ferrocyanide, EM image quality was excellent but SS were difficult to identify because their thin PSD resembled surrounding non-synaptic membranes; AS remained visible but their PSD appeared thinner than surrounding membranes (201 slices, 361.68 μm³).
  • With 0.1% potassium ferrocyanide, EM quality remained excellent and AS and SS could be clearly distinguished through serial sections; in that stack, 88 SS and 836 AS were unambiguously identified (299 slices, 538.02 μm³).
  • In VGAT pre-embedding immunocytochemistry with 0.1% potassium ferrocyanide, analysis of 266 serial images (4,184 μm³) identified 265 AS and 23 SS; all SS (8% of total synapses) were formed by VGAT-positive terminals, while all AS (92%) were established by VGAT-negative terminals.
  • The authors recommend 0.1% potassium ferrocyanide, as it improves membrane visualization while still permitting PSD-based identification of SS.

Methods

  • Four adult female C57BL/6 mice (8 weeks old) were used: two for testing potassium ferrocyanide concentrations (one mouse per condition) and two for VGAT validation (one with 0.1% potassium ferrocyanide, one without). Brains were perfusion-fixed with 4% PFA, sectioned at 150 μm, postfixed, osmicated with 0%, 0.1%, or 1% potassium ferrocyanide, dehydrated, en bloc stained with uranyl acetate, and embedded in Araldite.
  • FIB-SEM imaging used a 20 nm milling step and 5 nm/pixel in X-Y, giving a voxel size of 5 nm × 5 nm × 20 nm; stacks were aligned in FIJI with rigid registration and visualized/segmented in EspINA. Synapses were identified by PSD, presynaptic vesicles, and cleft across serial sections.
  • VGAT pre-embedding immunocytochemistry included cryoprotection in 30% sucrose, liquid nitrogen permeabilization, anti-VGAT primary antibody (1:2000), biotinylated secondary antibody, ABC amplification, DAB detection, and EM processing with 0.1% potassium ferrocyanide plus 7% glucose in the osmium solutions.

Limitations

  • The paper does not include a formal limitations section.
  • Evident from the text: the sample is small—four mice total, with only one mouse per potassium ferrocyanide condition and one mouse per VGAT condition.
  • Evident from the text: the unlabeled ferrocyanide comparison was restricted to mouse primary somatosensory cortex layers II and III, and VGAT validation was performed in the immunostaining penetration zone rather than across whole tissue; tested potassium ferrocyanide concentrations were limited to 0%, 0.1%, and 1%.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3389/fnana.2024.1348032

Cites

79 of 79 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

19 of 40 cited sources checked · 6 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Quantitative analyses of synapses in the neuropil have shown that most synapses on dendritic shafts are AS (~80%), with relatively few being SS (~20%).

    “In fact, quantitative analyses of synapses in the neuropil have shown that most synapses on dendritic shafts are AS (∼80%), with relatively few being SS (∼20%) (Beaulieu et al., 1992; Peters et al., 2008; Hsu et al., 2017; Calì et al., 2018; Santuy et al., 2018b; Domínguez-Álvaro et al., 2019, 2021a,b; Yakoubi et al., 2019b; Montero-Crespo et al., 2020, 2021; Cano-Astorga et al., 2021, 2023;”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Beaulieu et al., 1992 source
    • cites Peters et al., 2008 source

      Table 1 and the Results section on postsynaptic components give the percentage of each synapse type that contacts a shaft—not the percentage of shaft synapses that is each type. Combining the reported 87% asymmetric and 13% symmetric overall with the young layer 2/3 shaft-contact percentages (16.5% and 78.5%) gives approximately 58% asymmetric and 42% symmetric synapses on shafts, rather than the claimed 80% and 20%. (LLM, conf=0.87)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites Hsu et al., 2017 source

      The Results section (‘Postsynaptic targets, location and distribution of asymmetric synapses’; page unavailable) quantifies asymmetric synapses on dendritic shafts in mouse and monkey cortex. It does not report the proportion of all shaft synapses that are asymmetric versus symmetric, so the claimed ~80%/~20% split is unsupported by this paper. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • Premisecitation

    Focused Ion Beam Milling and Scanning Electron Microscopy (FIB-SEM) enables automated serial sectioning of large volumes of tissue, without any mechanical interaction with the sample.

    “This technology was chosen because it enables automated serial sectioning of large volumes of tissue, without any mechanical interaction with the sample (e.g., see Knott et al., 2008; Merchán-Pérez et al., 2009; Titze and Genoud, 2016; Kubota et al., 2018; Rollenhagen et al., 2020).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Knott et al., 2008 source

      The Introduction and Discussion support automated serial imaging by ion-beam milling rather than mechanical cutting. But the Discussion says the method was limited in milling large surfaces and needed further improvements for large-scale imaging, so this paper does not fully support the claim that it enables sectioning of large volumes. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites Merchán-Pérez et al., 2009 source
    • cites Titze and Genoud, 2016 source
  • Premisecitation

    In individual electron microscopy sections, the synaptic cleft and the densities in the pre- and postsynaptic membranes appear blurred in 40 to 60% of synaptic junctions.

    “For instance, in individual sections, the synaptic cleft and the densities in the pre-and postsynaptic membranes appear blurred in a significant proportion of synaptic junctions, ranging from 40 to 60%.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites DeFelipe et al., 1999 source
  • Premisereasoning

    The preference of glutamatergic axons for spines and GABAergic axons for shafts is often misinterpreted as implying that synapses on dendritic shafts are mostly SS.

    “However, this characteristic is often misinterpreted as implying that synapses on dendritic shafts are mostly SS (Motta et al., 2019; Karimi et al., 2020; Loomba et al., 2022).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Motta et al., 2019 source

      The Synaptic specificity section (page unavailable) reports that excitatory glutamatergic axons preferentially innervate spine heads and inhibitory axons preferentially innervate dendritic shafts. It does not say that this preference is often misinterpreted, or establish whether shaft synapses are mostly SS. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites Karimi et al., 2020 source

      The Results (‘Shaft vs. spine synapses’; page unavailable) describe spine-preferring axons as putatively excitatory and shaft-preferring axons as putatively inhibitory, but also test for excitatory synapses on shafts. The Results (‘More detailed pyramidal cell type classification’; page unavailable) report that 44% and 60% of shaft synapses onto L5 slender-tufted apical dendrites were likely excitatory in layers 1 and 2, respectively. The paper supports the warning that shaft location does not always establish inhibitory identity, but does not say that the inference is often misinterpreted or establish that shaft synapses generally are not mostly symmetric. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites Loomba et al., 2022 source
  • Interpretationreasoning

    The masking of membrane specializations is probably one of the reasons why many ultrastructural studies that used high concentrations of potassium ferrocyanide/ferricyanide (1.5-3.0%) provided no data on SS.

    “This is probably one of the reasons why many ultrastructural studies that utilized high concentrations of potassium ferrocyanide/ferricyanide (1.5–3.0%) provided no data on SS (e.g., Harris and Stevens, 1989; Hayworth et al., 2014; Yakoubi et al., 2019a; Yin et al., 2020; Gour et al., 2021; Phelps et al., 2021; Peddie et al., 2022; Turner et al., 2022).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Harris and Stevens, 1989 source

      The Serial electron microscopy methods report that tissue from two animals was treated with 1.5% potassium ferrocyanide, supporting the citation as an example of a study using the stated concentration range. The paper does not say that this treatment masked membrane specializations or caused an absence of SS data; it instead discusses possible staining or fixation limitations in visualizing smooth endoplasmic reticulum. (LLM, conf=0.80)

      Experimental — accuracy not yet measured
    • cites Hayworth et al., 2014 source

      In Sample preparation > Tissue processing (page not provided), Hayworth et al. say they often use (R)OTO staining and that its thickening and darkening of membranes can make synapses harder to identify in single sections. Our copy does not specify 1.5–3.0% potassium ferrocyanide/ferricyanide or say that masking membrane specializations caused the absence of SS data. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites Yakoubi et al., 2019a

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim. 14
  • It may state two separate claims as one. 17

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Volume electron microscopy studies have commonly employed potassium ferrocyanide or potassium ferricyanide.

    “However, volume electron microscopy studies have commonly employed potassium ferrocyanide (Harris and Stevens, 1988, 1989; Harris et al., 1992, 2015; Medalla et al., 2007; Hua et al., 2015) or potassium ferricyanide (Tapia et al., 2012).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Harris and Stevens, 1988 source
    • cites Medalla et al., 2007 source
    • cites Tapia et al., 2012 source

  • Probably not a substantial claim. It may state two separate claims as one.

    Premisecitation

    Potassium ferrocyanide and potassium ferricyanide facilitate the reconstruction of cellular processes and the automatic segmentation of electron microscope images.

    “These compounds facilitate the reconstruction of cellular processes and the automatic segmentation of electron microscope images.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Harris and Stevens, 1988 source

      The tissue-preparation passage reports using potassium ferrocyanide, and the Three-dimensional reconstructions section describes reconstructing the resulting electron micrographs. The paper does not say ferrocyanide facilitated reconstruction, does not report using potassium ferricyanide, and describes tracing membrane boundaries rather than automatic image segmentation. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites Medalla et al., 2007 source
    • cites Tapia et al., 2012 source

      The Membrane Contrasting Agents section says either potassium ferricyanide or ferrocyanide improves osmium staining of membranes. The Introduction links the staining method to segmentation of neuronal membranes and 3D reconstruction, but the copy does not establish that these compounds facilitate automatic image segmentation specifically. Page numbers are unavailable in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim. It may state two separate claims as one.

    Premisecitation

    Automated volume electron microscopy methods are a valuable and efficient approach for identifying synapses in three dimensions and have become the gold standard technique for this purpose.

    “Fortunately, the recent introduction of automated volume electron microscopy methods has proven to be a valuable and efficient approach for identifying synapses in three dimensions, becoming the gold standard technique for this purpose for this task (Merchán-Pérez et al., 2009).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Merchán-Pérez et al., 2009 source

      The Introduction and Results (pages not available in our copy) describe automated FIB/SEM imaging that enables efficient three-dimensional identification and counting of synapses. The paper does not call it the gold standard, and its findings about FIB/SEM do not establish that status for automated volume electron microscopy methods generally. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • It may state two separate claims as one.

    Interpretationcitation

    The use of a lower concentration of potassium ferrocyanide (0.1%) shows an improvement in membrane visualization while still allowing the PSD of the SS to be identified.

    “In conclusion, the use of a lower concentration of potassium ferrocyanide (0.1%), as we propose here, shows an improvement in membrane visualization, while still allowing the PSD of the SS to be identified (Domínguez-Álvaro et al., 2018, 2019, 2021a,b; Montero-Crespo et al., 2020, 2021; Cano-Astorga et al., 2021, 2023).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Domínguez-Álvaro et al., 2018 source

      The Electron microscopy methods (page not available) specify 0.1% potassium ferrocyanide, and the Synaptic three-dimensional analysis methods and Synaptic density results say that symmetric synapses could be identified by their thin postsynaptic density. The paper does not report a comparison showing that this concentration improved membrane visualization. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites Montero-Crespo et al., 2020 source

      The Materials and methods (Tissue processing for EM; page unavailable) report using 0.1% potassium ferrocyanide. The 3D analysis of synapses section says symmetric synapses could be identified by their thin postsynaptic densities. The paper does not report a comparison showing that this concentration improved membrane visualization. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Cano-Astorga et al., 2021 source

      Materials and Methods (Electron Microscopy; page unavailable) reports using 0.1% potassium ferrocyanide. Materials and Methods (Three-Dimensional Analysis of Synapses; page unavailable) says the researchers identified symmetric synapses by their thin postsynaptic densities. The paper does not report a comparison showing that the lower concentration improved membrane visualization. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • It may state two separate claims as one.

    Premisecitation

    Glutaraldehyde fixation is associated with increased background fluorescence and limited antibody penetration into tissues.

    “Firstly, this fixative is associated with increased background fluorescence and limited antibody penetration into tissues, as demonstrated, for example, by Stradleigh (2015).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Stradleigh (2015) source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    A clear preference of glutamatergic axons (forming AS) for dendritic spines and of GABAergic axons (forming SS) for dendritic shafts is reported in the literature.

    “A clear preference of glutamatergic axons (forming AS) for dendritic spines and GABAergic axons (forming SS) for dendritic shafts is reported in the literature (reviewed in DeFelipe et al., 2002; for a recent study, see Cano-Astorga et al., 2023).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites DeFelipe et al., 2002 source
    • cites Cano-Astorga et al., 2023 source

  • Probably not a substantial claim.

    Findingnone given

    Increasing concentrations of potassium ferrocyanide make postsynaptic densities thinner.

    “However, identifying synaptic junctions, especially SS, becomes more challenging as the postsynaptic densities become thinner with increasing concentrations of potassium ferrocyanide.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    VGAT-positive axon terminals established symmetric synapses, while VGAT-negative axon terminals established asymmetric synapses.

    “These VGAT-positive terminals established SS, while VGAT-negative axon terminals established AS (Figure 4).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    At 1% potassium ferrocyanide, asymmetric synapses remained visible, but their postsynaptic density appeared thinner compared to the thicker surrounding membranes.

    “In the case of AS, they remained visible, but their PSD appeared thinner compared to the thicker surrounding membranes (Figure 1; Supplementary Figures S2.1A, S2.2A, S2.3A, S2.4A).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    VGAT immunoreactivity was distributed across all layers of the mouse primary somatosensory cortex, with numerous stained puncta scattered in the neuropil.

    “VGAT immunoreactivity was distributed across all layers of the mouse primary somatosensory cortex, where numerous stained puncta were scattered in the neuropil (Supplementary Figure S1A).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    In the FIB/SEM material, VGAT-positive axon terminals established symmetric synapses (SS), while VGAT-negative axon terminals established asymmetric synapses (AS).

    “These VGAT-positive terminals established SS, while VGAT-negative axon terminals established AS (Figure 4).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    In the neuropil within the penetration zone of immunostaining, 266 serial images corresponding to 4,184 μm3 were analyzed, and 265 AS and 23 SS were identified.

    “We analyzed 266 serial images in the neuropil within the penetration zone of immunostaining, corresponding to 4,184 μm3, and identified 265 AS and 23 SS.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    All SS (8% of total synapses) were formed by VGAT-positive terminals, while all AS (92%) were established by VGAT-negative terminals.

    “All SS (8% of total synapses) were formed by VGAT-positive terminals, while all AS (92%) were established by VGAT-negative terminals (Figure 6).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    The use of potassium ferrocyanide or ferricyanide is not necessary for the morphological identification of synapses.

    “However, it should be noted that the use of potassium ferrocyanide or ferricyanide is not necessary for the morphological identification of synapses (see, for example, Peters et al., 1991).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Peters et al., 1991

  • It may state two separate claims as one.

    Premisecitation

    In the cerebral cortex, most asymmetric synapses are excitatory (glutamatergic), while symmetric synapses are inhibitory (GABAergic).

    “In the cerebral cortex, since most AS are excitatory (glutamatergic), and SS are inhibitory (GABAergic)”
    • Also stated in: § Introduction · boosted
      “Importantly, it has been demonstrated that, in the cerebral cortex, most AS are excitatory (glutamatergic), while SS are inhibitory (GABAergic) (Colonnier, 1968; Gray, 1969; Peters and Kaiserman-Abramof, 1969; Houser et al., 1984; Peters and Palay, 1996; Ascoli et al., 2008).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Colonnier, 1968 source
    • cites Gray, 1969 source
    • cites Peters and Kaiserman-Abramof, 1969 source

  • It may state two separate claims as one.

    Premisecitation

    Asymmetric (AS) synapses have a thick postsynaptic density, while symmetric (SS) synapses have a thin postsynaptic density.

    “AS and SS have a thick and thin postsynaptic density, respectively.”
    • Also stated in: § Introduction · plain
      “The most noticeable distinction lies in the postsynaptic density: AS have a thick postsynaptic density, while SS have a thin postsynaptic density.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Gray, 1959

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Colonnier, 1968 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Potassium ferrocyanide in combination with osmium tetroxide enhances the visualization of cellular membranes.

    “Later, the introduction of potassium ferrocyanide (potassium hexacyanoferrate (II); K4[Fe(CN)6]; Karnovsky, 1971; White et al., 1979; McDonald, 1984) or potassium ferricyanide (potassium ferricyanide (III); K3[Fe(CN)6]; Rivlin and Raymond, 1987) in combination with osmium tetroxide was used to enhance the visualization of cellular membranes, as well as certain aspects of cell morphology.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Karnovsky, 1971

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites White et al., 1979 source
    • cites McDonald, 1984 source

  • It may state two separate claims as one.

    Premisereasoning

    Symmetric synapses are the most challenging type to identify, because asymmetric synapses may resemble symmetric synapses in certain planes of section.

    “Furthermore, SS are the most challenging type to identify, as AS may resemble SS in certain planes of section.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductreasoning

    The perspective deformation resulting from the 36° angle of incidence of the SEM is automatically corrected by the microscope software during acquisition, so no distortion is present in the final images.

    “The resulting perspective deformation is automatically corrected by the microscope software during acquisition (SmartSEM 6.02; Carl Zeiss Microscopy Ltd.), so no distortion is present in the final images.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim.

    Study design and conductnone given

    The identification of synapses relies on examining all serial sections where each individual synapse is visible.

    “Moreover, the identification of synapses relies on examining all serial sections where each individual synapse is visible.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim.

    Premisecitation

    In the perisomatic innervation of pyramidal cells, only symmetric synapses (SS) are established by VGAT-immunoreactive axon terminals.

    “Further verification of the morphology of synaptic contacts made by VGAT-positive boutons in the neuropil was obtained by examining the perisomatic innervation of pyramidal cells by VGAT-immunoreactive axon terminals, where it is known that only SS are established (DeFelipe and Fariñas, 1992).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites DeFelipe and Fariñas, 1992 source

  • Probably not a substantial claim.

    Study design and conductnone given

    Utilizing EspINA software, the 3D course of axons can be followed within stacks of sections to confirm the nature (asymmetric or symmetric) of synapses established in all their synaptic contacts.

    “Additionally, utilizing EspINA software, the 3D course of the axons can be followed within stacks of sections to confirm the nature (AS or SS) of the synapses established in all their synaptic contacts.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    The finding that only VGAT-positive boutons establish SS corroborates the correspondence between SS and inhibitory synapses, and between AS and excitatory synapses, in the cerebral cortex.

    “The fact that only VGAT-positive boutons establish SS corroborates the widely accepted correspondence between SS and inhibitory synapses, as well as between AS and excitatory synapses in the cerebral cortex, as reported in previous studies.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: VGAT immunoreactivity reliably identifies inhibitory (GABAergic) presynaptic terminals, so that synapses formed by VGAT-positive boutons are inhibitory.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Potassium ferrocyanide and potassium ferricyanide in combination with osmium tetroxide work equally well, increasing the quality of electron microscopy images.

    “Furthermore, it has been reported that the use of either reagent in combination with osmium tetroxide work equally well (Rivlin and Raymond, 1987), increasing the quality of electron microscopy images.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Rivlin and Raymond, 1987 source

  • Probably not a substantial claim. The paper may give no reason why it holds.

    Premisenone given

    The high concentrations of glutaraldehyde commonly employed for electron microscopy are incompatible with other methods currently used to examine the microanatomy of the brain, such as intracellular injections in fixed tissue.

    “Furthermore, the high concentrations of glutaraldehyde commonly employed for electron microscopy are incompatible with other methods currently utilized to examine the microanatomy of the brain, such as intracellular injections in fixed tissue.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Brain tissue fixed with glutaraldehyde allows differentiation between AS and SS based on the shape of synaptic vesicles: AS typically exhibit round vesicles, whereas some SS display pleomorphic vesicles, including both round and elongated forms.

    “It is worth noting that brain tissue fixed with glutaraldehyde allows for the differentiation between AS and SS based on the shape of synaptic vesicles. Specifically, AS typically exhibit round vesicles, whereas some SS display pleomorphic vesicles, including both round and elongated forms (e.g., Peters and Palay, 1996).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Peters and Palay, 1996 source
26 of 26

  • The paper relies on this without support.

    The primary fixative used does not override the main feature utilized for distinguishing between asymmetric and symmetric synapses, which is the thickness of electron-dense labeling in the postsynaptic density.

    “Importantly, the primary fixative used does not override the main feature utilized for distinguishing between AS and SS, which is the thickness of electron-dense labeling in the postsynaptic density (PSD).”

  • The paper relies on this without support.

    VGAT immunoreactivity specifically identifies GABAergic axon terminals.

    “To validate SS observed using this protocol as GABAergic, experiments with immunocytochemistry for the vesicular GABA transporter were conducted on fixed mouse brain tissue sections.”
    “These VGAT-positive terminals established SS, while VGAT-negative axon terminals established AS (Figure 4).”

  • The paper relies on this without support.

    The effect of potassium ferrocyanide concentration on membrane visualization and synapse identification is not confounded by inter-animal variability, despite using one mouse per concentration.

    “Two of these mice were utilized to assess various concentrations of potassium ferrocyanide, with one mouse assigned to each condition.”

  • The paper relies on this without support.

    VGAT immunoreactivity reliably identifies inhibitory (GABAergic) presynaptic terminals, so that synapses formed by VGAT-positive boutons are inhibitory.

    “The fact that only VGAT-positive boutons establish SS corroborates the widely accepted correspondence between SS and inhibitory synapses, as well as between AS and excitatory synapses in the cerebral cortex, as reported in previous studies.”
4 of 4

Citations

Cites

Flagged first, then by citations

4 of the 83 references couldn’t be matched to a known paper.

Research works printed but not linked: 1

  1. Karnovsky, 1971, Use of ferrocyanide-reduced osmium tetroxide in electron microscopy

Cited by

Sorted by citations

Related

Metadata

Authors
Cano-Astorga, N., Plaza-Alonso, S., Turegano-Lopez, M., Rodrigo-Rodríguez, J., Merchan-Perez, A., & DeFelipe, J.
Affiliations
Consejo Superior de Investigaciones Científicas (ES)
Corresponding author
DeFelipe, Javier
ORCID
DeFelipe, Javier
Venue
Frontiers in Neuroanatomy
Publisher
Frontiers Media SA
Published
2024-04-05
Volume
18
Pages
1348032-1348032
ISSN
1662-5129 (ISSN-L)
DOI
10.3389/fnana.2024.1348032
PMC
PMC11026665
OpenAlex
W4393987059
Semantic Scholar
565faa100592396d6f7cfb751bb87a7b357e872b
Cited by
17
References
83
Citations by year
2026: 4 · 2025: 8 · 2024: 5
FWCI
2.70
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Neuroanatomy (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
  • Health Sciences › Medicine › Anatomy › Classical Anatomy and Histology
Keywords
asymmetric synapses, symmetric synapses, postsynaptic density, potassium ferrocyanide, membrane staining, vesicular GABA transporter, GABAergic synapses, glutamatergic synapses, cerebral cortex, synaptic morphology, focused ion beam milling

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

A language model drew the claims from the paper on 2026-10-12, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).

A second model labelled each claim on 2026-10-12 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).

Cited sources are checked with a language model’s help (gpt-6-sol).

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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