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Cited by 133Neurogenesis and neuroplasticity mechanismsNeuroscience and Neuropharmacology ResearchMemory and Neural Mechanisms

Differential Expression and Cell-Type Specificity of Perineuronal Nets in Hippocampus, Medial Entorhinal Cortex, and Visual Cortex Examined in the Rat and Mouse

Open access
Lensjø, Kristian Kinden, Christensen, Ane Charlotte, Tennøe, Simen, Fyhn, Marianne, Hafting, Torkel·2017·eneuro
DOI 10.1523/eneuro.0379-16.2017PMC PMC5461557
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Abstract

Perineuronal nets (PNNs) are specialized extracellular matrix (ECM) structures that condense around the soma and proximal dendrites of subpopulations of neurons. Emerging evidence suggests that they are involved in regulating brain plasticity. However, the expression of PNNs varies between and within brain areas. A lack of quantitative studies describing the distribution and cell-specificity of PNNs makes it difficult to reveal the functional roles of PNNs. In the current study, we examine the distribution of PNNs and the identity of PNN-enwrapped neurons in three brain areas with different cognitive functions: the dorsal hippocampus, medial entorhinal cortex (mEC) and primary visual cortex (V1). We compared rats and mice as knowledge from these species are often intermingled. The most abundant expression of PNNs was found in the mEC and V1, while dorsal hippocampus showed strikingly low levels of PNNs, apart from dense expression in the CA2 region. In hippocampus we also found apparent species differences in expression of PNNs. While we confirm that the PNNs enwrap parvalbumin-expressing (PV+) neurons in V1, we found that they mainly colocalize with excitatory CamKII-expressing neurons in CA2. In mEC, we demonstrate that in addition to PV+ cells, the PNNs colocalize with reelin-expressing stellate cells. We also show that the maturation of PNNs in mEC coincides with the formation of grid cell pattern, while PV+ cells, unlike in other cortical areas, are present from early postnatal development. Finally, we demonstrate considerable effects on the number of PSD-95-gephyrin puncta after enzymatic removal of PNNs.

AI snapshot

Written from the paper’s full text.

TL;DR

Perineuronal nets (PNNs) differ markedly in density, laminar distribution, morphology, and cell-type specificity across rat and mouse hippocampus, medial entorhinal cortex (mEC), and primary visual cortex (V1). Enzymatic PNN removal with chondroitinase ABC altered excitatory and inhibitory puncta differently in mEC and V1, suggesting region-specific roles in plasticity and synaptic organization.

Key findings

  • PNN-enwrapped neuron fractions were similar in rat V1 and mEC (8.4% vs 7.9%, 5997 neurons counted), but laminar distributions differed: V1 had highest expression in Layers IV/V, while mEC was dense in superficial Layers II/III.
  • The dorsal hippocampus was largely PNN-sparse except CA2, where PNN-like structures overlapped with PCP4 and predominantly enwrapped CaMKII-positive excitatory neurons (~80%); in rat CA2, <20% of PNNs colocalized with PV+ neurons, whereas mouse CA2 showed 75.4 ± 5.9% WFA overlap with PV.
  • In mEC, more PNNs colocalized with PV+ cells in mice (87%) than rats (74%); in rats, ~25% of mEC PNNs colocalized with other cell types, including reelin-positive cells (28.6 ± 2.5% in dorsal mEC) and only ~5% with calbindin.
  • Unilateral chABC in rats reduced PSD-95 puncta in mEC (p < 0.002) and increased gephyrin puncta in V1 (p < 0.03), with no significant changes in puncta clustering in either area.

Methods

  • Used 14 adult and 7 juvenile male Long Evans rats and 5 adult male C57BL/6 mice; brain sections were stained with WFA for PNNs plus markers including PV, CaMKII, PCP4, reelin, calbindin, GFAP, PSD-95, and gephyrin.
  • PNN expression, colocalization, and development were assessed by immunohistochemistry, confocal microscopy, and manual counting; hippocampal cell counting was excluded due to dense packing.
  • Adult rats received unilateral stereotaxic injections of chABC or aCSF into V1 and mEC, with retrograde tracer injections in some experiments; puncta were quantified with Imaris spot detection and clustered using a friends-of-friends algorithm, with statistics by t test or Mann–Whitney U test.

Limitations

  • The paper states that automated PNN/cell counting produced false positives or negatives, so colocalization was counted manually; hippocampal cell density prevented accurate cell-number analysis in that region.
  • The authors caution that chABC effects may partly reflect degradation of chondroitin sulfate proteoglycans generally, since PNN-embedded chondroitin sulfate proteoglycans make up only 2–5% of total chondroitin sulfate proteoglycans in rat brain.
  • The paper notes large variability between animals, especially for PSD-95 puncta, and that the clustering analysis did not account for the reduction in total puncta; additional repeated spine imaging was said to be needed.

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

Integrity signals

Registry

Crossref · 10.1523/eneuro.0379-16.2017

Cites

88 of 88 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 80 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    In V1, PNNs enwrapped the cell soma and a short section of the proximal dendrites.

    “In V1, the PNNs enwrapped the cell soma and a short section of the proximal dendrites.”
    Quote in the paperSubstantial claim
  • Findingown data

    The total fraction of PNN-enwrapped neurons was similar between V1 and mEC (8.4% in V1 and 7.9% in mEC; 5997 neurons counted in total).

    “The total fraction of PNN enwrapped neurons was, however, similar between V1 and mEC (8.4% in V1 and 7.9% in mEC, 5997 neurons counted in total).”
    Quote in the paperNot a substantial claim
  • Findingown data

    In rats, approximately 25% of PNNs in mEC colocalized with other cell types.

    “In rats, ∼25% of the PNNs in mEC colocalized with other cell types”
    Quote in the paperSubstantial claim
  • Findingown data

    More PNNs colocalized with PV+ cells in mice (87%) compared with rats (74%).

    “More PNNs colocalized with PV+ cells in mice (87%) compared with rats (74%).”
    Quote in the paperSubstantial claim
  • Findingown data

    Counterstaining with reelin revealed almost complete overlap between the tracer and reelin in mEC.

    “Counter-staining with reelin revealed an almost complete overlap between the tracer and reelin in mEC”
    Quote in the paperSubstantial claim
  • May combine two claims. 46
  • Classified as probably not a substantial claim. 35

  • May combine two claims.

    Findingown data

    In rat V1, PNNs were distributed across cell layers except Layer I and the upper part of Layer V, with highest expression in Layers IV and V.

    “In the rat, PNNs were distributed across cell Layers in V1, apart from Layer I which is largely void of cell bodies, and the upper part of Layer V. The highest expression was found in Layers IV and V”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The dorsal hippocampus had a sparse and area-specific PNN expression pattern, with only a few weak PNNs in CA1 stratum pyramidale and some in stratum oriens.

    “The PNN expression of the dorsal hippocampus stands out with a very sparse and area-specific expression pattern (Fig. 1A,C, right). Only a few weak PNNs were found in CA1 and they were located in the stratum pyramidale, and some in stratum oriens.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In mEC, PNNs were present around the cell soma but continued along long stretches of the dendritic segments.

    “In contrast, PNNs in the mEC were similarly present around the cell soma but continued along long stretches of the dendritic segments”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In CA2, WFA staining was intense and enwrapped all cell bodies in the stratum pyramidale; CA2 had the highest density of WFA-enwrapped cells of all areas investigated, but its PNN microstructure was more diffuse than in cortex.

    “In CA2, however, the WFA staining was intense, enwrapping all cell bodies in the stratum pyramidale. The CA2 showed the highest density of WFA-enwrapped cells of all areas investigated, but the microstructure of the PNNs were more diffuse compared with the cortex”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In CA2, PNNs located in the neuropil of the pyramidal cell layer enwrapped all cell somas but only a minor part of the processes.

    “In the CA2 of the hippocampus, on the other hand, PNNs located in the neuropil of the pyramidal cell layer enwrapped all cell somas but only a minor part of the processes”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PNN expression and laminar distribution differed greatly between V1, mEC, and the dorsal hippocampus CA regions.

    “We found that both the level of expression and laminar distribution of PNNs differed greatly between these areas (Fig. 1A,B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In rat mEC, superficial layers showed dense WFA labeling while few PNNs were located in deeper layers.

    “In contrast, the superficial layers of mEC showed dense WFA labeling, while few PNNs were located in the deeper layers.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Similar diffuse staining was observed in CA3 and the granular cell layer of the DG, but intensity was much lower than in CA2.

    “While a similar diffuse staining was observed in CA3 and in the granular cell layer of the DG, the intensity was much lower than in the CA2.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Sections stained with WFA revealed overlapping cells with the tracer and WFA, particularly in Layer II, but only few such colocalized cells were found.

    “The sections from the tracer experiments were also stained with WFA, revealing overlapping cells with the tracer and WFA, in particular in Layer II”
    • Also stated in: § Results · plain
      “However, only few such colocalized cells were found”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In rat hippocampus, very few discernable PNNs were found apart from CA2 and to some extent CA3, both of which showed a dense but diffuse matrix.

    “In the rat hippocampus, however, we found very few discernable PNNs apart from that of the CA2 and to some extent in the CA3, which both showed a dense but diffuse matrix”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    When injecting retrograde tracer in DG, projecting neurons were strongly labeled in Layer II of mEC, with some labeling throughout Layer III.

    “when injecting the tracer in DG, we found projecting neurons strongly labeled with the tracer in Layer II of mEC, and some labeling throughout Layer III”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A large fraction of PNNs in mouse hippocampus colocalized with PV+ neurons, but this was not the case in rats, except for the few weak PNNs in CA1.

    “Furthermore, while a large fraction of PNNs in mouse hippocampus colocalized with PV+ neurons, this was not the case in rats, with the exception of the few weak PNNs in the CA1”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PCP4 and WFA markers overlapped in CA2, supporting that PNN-positive neurons in CA2 differ from those in other hippocampal subregions.

    “By counterstaining sections with WFA, we found that these markers overlapped, supporting that the PNN-positive neurons in CA2 differ from those in other sub-regions of the hippocampus”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In mouse, PNNs were present in all subregions of the hippocampus, with a structure similar to those in cortical areas.

    “In the mouse, PNNs were present in all sub regions of the hippocampus, with a structure similar to those in the cortical areas”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In mEC, there were significant species differences in the fraction of PNNs colocalizing with PV+ cells and in the fraction of PV+ cells without PNNs.

    “In mEC, however, we found significant species differences, both in the fraction of PNNs colocalizing with PV+ cells and also the fraction of PV+ cells without PNNs”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The CA2 of mice was similar in PNN structure but not as densely packed as in the rat, and as much as 75% of PNNs enwrapped PV+ cells.

    “The CA2 of mice was similar in terms of PNN structure, but not as densely packed as in the rat, and as much as 75% of PNNs enwrapped PV+ cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There was a tendency toward reduced clustering after chABC treatment in mEC, but overall high variation between data sets, particularly for excitatory puncta (PSD-95).

    “There was a tendency toward reduced clustering after chABC treatment in mEC, but overall, high variation between the data sets, in particular for excitatory puncta (PSD-95).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The most notable difference occurred between P17 and P20 when PNNs became more clearly defined, after which WFA intensity gradually increased and PNNs formed along longer stretches of dendrites.

    “The most notable difference occurred between P17 and P20 when the PNNs became more clearly defined, after which we observed a gradual increase in WFA intensity and PNNs forming along longer stretches of the dendrites”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    chABC treatment caused a significant reduction of PSD-95 puncta in mEC (p = 0.002) and no significant effect in V1 (p = 0.7).

    “The chABC treatment caused a significant reduction of PSD-95 puncta (red) in mEC (p = 0.002, Mann–Whitney U test), and no significant effect in V1 (p = 0.7, Student’s t test).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Based on tracer experiments, there appear to be different populations of stellate cells, with a subset of hippocampus projecting stellate cells that colocalized with WFA.

    “Based on our tracer experiments it appears as if there are different populations of stellate cells, with a subset of hippocampus projecting stellate cells that colocalized with WFA (Fig. 4C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    In V1, PNNs have a uniform distribution, usually packing around the soma and proximal dendrite.

    “The PNNs in V1 had a uniform distribution with a structure usually described packing around the soma and proximal dendrite (Wegner et al., 2003).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Wegner et al., 2003

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Among few PNNs in rat hippocampus, only a relatively small fraction colocalized with PV+ neurons, while a high fraction colocalized in most areas of mouse hippocampus.

    “Among the few PNNs found in the rat hippocampus, only a relatively small fraction were colocalized with PV+ neurons, while a high fraction colocalized in most areas of mouse hippocampus.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Maturation of PNNs in mEC closely follows the timing reported for development of grid cells, and is very similar to PNN maturation seen in primary sensory cortices.

    “We show that the maturation of PNNs in this region closely follows the timing reported for development of grid cells, and is very similar to the PNN maturation seen in primary sensory cortices (Ye and Miao, 2013; Fig. 6).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Ye and Miao, 2013

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    In mEC, PNNs pack around the soma but extend along long stretches of dendrites.

    “The nets in mEC also packed around the soma but extended along long stretches of the dendrites.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    We found large effects on the number of putative inhibitory and excitatory puncta, and the ratio between these after chABC treatment.

    “We found large effects on the number of putative inhibitory and excitatory puncta, and the ratio between these after chABC treatment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Colocalization between PV+ cells and PNNs increases dramatically during P14-P20 and stable spatial representations appear.

    “i.e., during the same period that the colocalization between PV+ cells and PNNs increases dramatically and stable spatial representations appear (Fig. 7B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both stellate and pyramidal grid cells partly colocalized with PNNs.

    “both of which partly colocalized with PNNs.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationown data

    Differences between PNN expression in rats and mice emphasize the importance of taking into account species differences when dissecting the functional roles of PNNs.

    “Moreover, the differences between PNN expression in rats and mice emphasize the importance of taking into account species differences when dissecting the functional roles of PNNs.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Rats and mice have been used interchangeably in functional studies of PNNs.

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    For analysis of PSD-95 and gephyrin puncta, treated and untreated hemispheres from the same rat were compared and values normalized to the mean of the same area in the control hemisphere.

    “For analysis of both number and distribution of PSD-95 and gephyrin puncta, we always compared the treated and untreated hemispheres from the same rat and normalized values to the mean of the same area in the control hemisphere.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    PNNs mainly enwrap a subpopulation of inhibitory neurons, the fast-spiking PV+ inhibitory neurons, but the overlap is not exclusive.

    “The PNNs mainly enwrap a subpopulation of inhibitory neurons, the fast-spiking parvalbumin-expressing (PV+) inhibitory neurons, but the overlap is not exclusive”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Kosaka and Heizmann, 1989

      Not checked in the free run.

    • cites Härtig et al., 1992

      Not checked in the free run.

    • cites Wegner et al., 2003

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    As the objective was to compare chABC-treated and control datasets, the exact choice of linking length is not important as long as it is kept constant.

    “As the objective was to perform a comparison between the two datasets (chABC treated vs control), the exact choice of linking length is not important as long as it is kept constant.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In neocortex, PV+ cells mature in parallel with the assembly of PNNs late in postnatal development as the critical period comes to an end.

    “In the neocortex, the PV+ cells mature in parallel with the assembly of the PNNs late in postnatal development as the critical period comes to an end”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Hockfield et al., 1990

      Not checked in the free run.

    • cites Pizzorusso et al., 2002

      Not checked in the free run.

    • cites Ye and Miao, 2013

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PNNs play a role in regulating plasticity during development, learning, and memory processing.

    “Recent work indicates that PNNs play a role in regulating plasticity during development, learning, and memory processing”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Bartus et al., 2012

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Removal of PNNs, both by enzymatic degradation and by partial genetic knock-out, has been shown to increase plasticity comparable to juvenile levels in several areas of the brain.

    “removal of PNNs, both by enzymatic degradation and by partial genetic knock-out, has been shown to increase plasticity comparable to juvenile levels in several areas of the brain”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Pizzorusso et al., 2002

      Not checked in the free run.

    • cites Gogolla et al., 2009

      Not checked in the free run.

    • cites Carulli et al., 2010

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    PNNs are specialized structures of extracellular matrix that condense around the cell soma and proximal dendrites of subpopulations of neurons.

    “Perineuronal nets (PNNs) are specialized structures of extracellular matrix (ECM) that condense around the cell soma and proximal dendrites of subpopulations of neurons”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Hockfield and McKay, 1983

      Not checked in the free run.

    • cites Celio and Blumcke, 1994

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductreasoning

    All colocalization analysis was performed by manual counting, with all images counted by two investigators independently.

    “We therefore chose to perform all colocalization analysis by manual counting, by overlaying the sections with a grid and using the counter tool. All images were counted by two investigators independently.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Most reelin-expressing WFA-positive cells are intrinsic to the entorhinal network, and only a subpopulation project to the dorsal hippocampus.

    “indicating that most of the reelin-expressing WFA-positive cells are intrinsic to the entorhinal network, and only a subpopulation project to the dorsal hippocampus.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Sections from each rat were treated identically, and stained and imaged in parallel.

    “The sections from each rat were treated in an identical fashion, and stained and imaged in parallel.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PNNs seem to have an important function in restricting adult brain plasticity.

    “In more general terms, PNNs seem to have an important function in restricting adult brain plasticity.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Pizzorusso et al., 2002

      Not checked in the free run.

    • cites Gogolla et al., 2009

      Not checked in the free run.

    • cites Carulli et al., 2010

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    While PNNs mainly colocalize with PV+ neurons in visual cortex, they predominantly colocalize with CaMKII and PCP4-expressing neurons in CA2 of the hippocampus.

    “In particular, while PNNs mainly colocalize with PV+ neurons in visual cortex, they predominantly colocalize with CaMKII and PCP4-expressing neurons in CA2 of the hippocampus.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Measurements of the distance between selected puncta showed that 2 μm was a good choice of linking length for FOF clustering.

    “Measurements of the distance between a selected set of puncta showed that 2 μm was a good choice of linking length, as a shorter or longer distance would yield clusters of very few puncta or only a few clusters with almost all puncta clustered, respectively.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All data were tested for normality by Wilks-Shapiro test, and further analysis was performed accordingly.

    “All data were tested for normality by Wilks-Shapiro test, and further analysis was performed accordingly (Student’s t test or Mann–Whitney U test).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    PNNs are found throughout the brain with profound variations in expression pattern and morphology between brain regions.

    “The PNNs are found throughout the brain with profound variations in expression pattern and morphology between brain regions”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Seeger et al., 1994

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    These changes may be indicative of a network in a high plasticity state, as similar effects have been observed after environmental enrichment.

    “These changes may be indicative of a network in a high plasticity state, as similar effects have been observed after a period of environmental enrichment, a paradigm known to cause increased plasticity (Donato et al., 2013).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Donato et al., 2013

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The mEC is believed to be the hub in a distributed neural network coding for spatial representation that is fed into the hippocampus and used as a critical component of episodic memories.

    “The mEC is believed to be the hub in a distributed neural network coding for spatial representation that is fed into the hippocampus and used as a critical component of episodic memories”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Buzsaki and Moser, 2013

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    chABC treatment causes changes to the number of synapses and spine motility in hippocampus and V1, respectively.

    “Previous work has shown that chABC treatment causes changes to the number of synapses and spine motility in hippocampus and V1, respectively (de Vivo et al., 2013; Donato et al., 2013).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites de Vivo et al., 2013

      Not checked in the free run.

    • cites Donato et al., 2013

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    chABC treatment effectively collapses PNNs and abolishes WFA staining.

    “This treatment effectively collapses the PNNs and abolishes WFA staining (Brückner et al., 1998).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Brückner et al., 1998

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    V1 has been a canonical system to study sensory processing and critical period plasticity for decades.

    “The V1 has been a canonical system to study sensory processing and critical period plasticity for decades”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Hensch, 2005

      Not checked in the free run.

    • cites Wandell and Smirnakis, 2009

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Our results demonstrate that PNNs are heterogeneous in expression, cell-type specificity and morphology.

    “Our results demonstrate that PNNs are heterogeneous in expression, cell-type specificity and morphology.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PNNs enwrapping other cell types have been found in several brain areas.

    “However, PNNs enwrapping other cell types have been found in several brain areas”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Alpar et al., 2006

      Not checked in the free run.

    • cites Carstens et al., 2016

      Not checked in the free run.

    • cites Morikawa et al., 2017

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PV+ cells have been proposed as key regulators of plasticity, both during development and in adulthood.

    “The PV+ cells have been proposed as key regulators of plasticity, both during development and in adulthood”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Hensch, 2005

      Not checked in the free run.

    • cites Donato et al., 2013

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    PNNs may support the high activity of PV+ cells through ion buffering, protection against reactive oxygen radicals, and a role in plasticity through capture of Otx2.

    “The PNNs may support the high activity of PV+ cells through ion buffering, protection against reactive oxygen radicals and their role in plasticity through capture of the transcription factor Otx2”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Beurdeley et al., 2012

      Not checked in the free run.

    • cites Cabungcal et al., 2013

      Not checked in the free run.

    • cites Liu et al., 2013

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The dense expression of PNNs in mEC Layers II and III and high colocalization with PV+ cells indicates a role for PNNs in the circuitry for spatial navigation.

    “The dense expression of PNNs in Layers II and III and high colocalization with PV+ cells that are known to be a key player in the grid cell network, indicates a role for the PNNs in the circuitry for spatial navigation (Fig. 4).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The mEC is a hub in a distributed network for navigation in the brain.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The FOF algorithm directly returns which puncta belong to a given cluster.

    “Compared with other clustering algorithms, the FOF algorithm has the advantage of directly returning which puncta belong to a given cluster.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    The different levels of expression, cell-type specificity and appearance of PNNs between brain regions and species may indicate that PNNs may have different functions depending on network architecture and function.

    “The different levels of expression, cell-type specificity and appearance of the PNNs between brain regions and species may indicate that the PNNs may have different functions depending of the network architecture and function.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Grid cells are likely to be both stellate (reelin-positive) and pyramidal (calbindin-positive) cells.

    “Emerging evidence suggests that the grid cells are likely to be both stellate (reelin-positive) and pyramidal (calbindin-positive) cells (Sun et al., 2015)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Sun et al., 2015

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Grid cells are likely interconnected through a network of mono-synaptically coupled PV+ cells, which is believed to contribute to the rigid activity pattern of the grid cell network.

    “The grid cells are likely interconnected through a network of mono-synaptically coupled PV+ cells, which is believed to contribute to the rigid activity pattern of the grid cell network (Couey et al., 2013, Buetfering et al., 2014).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Couey et al., 2013

      Not checked in the free run.

    • cites Buetfering et al., 2014

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Perturbations of PNNs in V1 by chABC or deletion of link protein Crtl-1 reopens for ocular dominance plasticity in V1.

    “Perturbations of PNNs in V1 by chABC or deletion of the link protein Crtl-1 reopens for ocular dominance plasticity in V1 (Pizzorusso et al., 2002; Carulli et al., 2010).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Pizzorusso et al., 2002

      Not checked in the free run.

    • cites Carulli et al., 2010

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    This process differs from critical period plasticity in that it is largely dependent on other cellular mechanisms, and on the action of matrix metalloproteinases (MMPs).

    “This process differs from critical period plasticity in that it is largely dependent on other cellular mechanisms, and on the action of matrix metalloproteinases (MMPs) (Ranson et al., 2012; Pielecka-Fortuna et al., 2015).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Ranson et al., 2012

      Not checked in the free run.

    • cites Pielecka-Fortuna et al., 2015

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The FOF algorithm is reasonably fast with a time complexity of O = (n 2).

    “and is reasonably fast with a time complexity of O = (n 2), where n = total number of puncta.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    CA2 is an area resistant to synaptic change under normal conditions, but a state switch such as induced by PNN removal or caffeine opens for synaptic plasticity in the circuitry.

    “Thus, it appears that while the CA2 is an area resistant to synaptic change under normal conditions, it may be that a state switch such as that induced by PNN removal or caffeine opens for synaptic plasticity in the circuitry.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Study design and conductnone given

    To the best of our knowledge, this is a novel tool in the study of synaptic organization.

    “To the best of our knowledge, this is a novel tool in the study of synaptic organization.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Principal neurons in CA2 differ from surrounding areas in morphology, gene expression, physiologic properties, and connections to other areas.

    “The principal neurons found in this area differ from the areas around in terms of morphology, gene expression, physiologic properties, and connections to other areas (Caruana et al., 2012; Hitti and Siegelbaum, 2014; Mankin et al., 2015)”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Caruana et al., 2012

      Not checked in the free run.

    • cites Hitti and Siegelbaum, 2014

      Not checked in the free run.

    • cites Mankin et al., 2015

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    This can be further improved by using an already existing algorithm that compares puncta on a grid, giving a time complexity of O = Nlog(n).

    “This can be further improved by using an already existing algorithm that compares puncta on a grid, giving a time complexity of O = Nlog(n).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PNNs have been reported in the mEC of humans.

    “PNNs have been reported in the mEC of humans (Pantazopoulos et al., 2010; Lendvai et al., 2013).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Pantazopoulos et al., 2010

      Not checked in the free run.

    • cites Lendvai et al., 2013

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The FOF algorithm is purely geometric and does not impose any assumptions on the data.

    “Furthermore, it is purely geometric and does not impose any assumptions on the data in the calculations”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    It may be that lack of PNNs allows the hippocampus to retain the unique plasticity necessary for learning and memory processing throughout life.

    “It may be that it is the lack of PNNs that allows for the hippocampus to retain the unique plasticity necessary for learning and memory processing throughout life.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Prolonged sensory deprivation causes a shift in ocular dominance in adult V1.

    “While the adult V1 network shows limited degree of experience-dependent plasticity in adulthood, prolonged sensory deprivation does cause a shift in ocular dominance (Sawtell et al., 2003; Tagawa et al., 2005; Sato and Stryker, 2008).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Sawtell et al., 2003

      Not checked in the free run.

    • cites Tagawa et al., 2005

      Not checked in the free run.

    • cites Sato and Stryker, 2008

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CA2 is more resistant to ischemia-induced cell death.

    “and CA2 is more resistant to ischemia-induced cell death (Sadowski et al., 1999; Zao et al., 2007).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Sadowski et al., 1999

      Not checked in the free run.

    • cites Zao et al., 2007

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Our analysis does not take into account the reduction in total number of puncta.

    “our analysis does not take into account the reduction in total number of puncta.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Dense expression of PNNs in mEC and the cell types being enwrapped in PNNs point toward a role in stabilizing the network.

    “their dense expression in mEC and the cell types being enwrapped in PNNs point toward a role in stabilizing the network.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The grid map is stable over time and ensembles of grid cells operate in synchrony and retain their temporal relationship, indicating a hard-wired neural network.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationreasoning

    This may be a highly useful tool for the study of structural network organization and stability.

    “This may be a highly useful tool for the study of structural network organization and stability.”
    Quote in the paperNot a substantial claim
72 of 72

  • The paper relies on this without support.

    Rats and mice have been used interchangeably in functional studies of PNNs.

    “The two species have nonetheless been used interchangeably in functional studies of PNNs.”

  • The paper relies on this without support.

    It remains unknown how the distribution and cell-type specificity of PNNs varies between mice and rats.

    “it remains unknown how the distribution and cell-type specificity varies between the widely used laboratory models mice and rats.”

  • The paper relies on this without support.

    It remains unclear whether the level of PNN expression in a brain area is correlated with the level of plasticity in that area.

    “Furthermore, it remains unclear if the level of PNN expression in a brain area is correlated with the level of plasticity in that area.”

  • The paper relies on this without support.

    It remains elusive how findings from V1 correspond to the development and expression pattern in mEC.

    “it remains elusive how the findings from this primary sensory cortex correspond to the development and expression pattern in mEC.”

  • The paper relies on this without support.

    It remains unknown whether PNN assembly in mEC follows a similar postnatal development as reported for V1.

    “If the PNN assembly in the mEC follow a similar postnatal development as reported for V1 (Ye and Miao, 2013) remains unknown.”

  • The paper relies on this without support.

    It remains unclear how removal of PNNs affects the stability and organization of synapses.

    “it remains unclear how removal of PNNs affects the stability and organization of synapses.”

  • The paper relies on this without support.

    The hippocampus is critical for encoding new memories and is reciprocally connected with mEC.

    “The hippocampus is critical for encoding new memories and is reciprocally connected with mEC.”

  • The paper relies on this without support.

    Grid cells are most abundant in Layers II and III of mEC.

    “with, e.g., grid cells being most abundant in Layers II and III.”

  • The paper relies on this without support.

    The grid map is stable over time and ensembles of grid cells operate in synchrony and retain their temporal relationship, indicating a hard-wired neural network.

    “The grid map is stable over time and ensembles of grid cells operate in synchrony and retain its temporal relationship indicating a hard-wired neural network.”

  • The paper relies on this without support.

    The mEC is a hub in a distributed network for navigation in the brain.

    “The mEC has been proposed to be a hub in a distributed network for navigation in the brain.”
10 of 10

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Both hippocampus and perirhinal cortex are rich in PV interneurons enwrapped with PNNs, and PNNs are also present on some hippocampal pyramidal neurons.

    “Both hippocampus and perirhinal cortex are rich in PV interneurons enwrapped with PNNs, and PNNs are also present on some hippocampal pyramidal neurons [46, 101, 102].”

    From The extracellular matrix and perineuronal nets in memory (2022) · cites this paper as [101]

    The Results and the Discussion section on hippocampal PNNs report nets on CA2 pyramidal neurons and PV-positive neurons enwrapped by nets in parts of the mouse hippocampus. They do not support describing the hippocampus generally as rich in enwrapped PV interneurons: hippocampal PNNs were sparse outside CA2 in rats, and their association with PV neurons varied by species. The paper examines medial entorhinal cortex, not perirhinal cortex, so it does not establish the perirhinal part of the claim. Page numbers were not available in our copy. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • PNNs predominantly enwrap PV inhibitory neurons in most brain areas, but they surround excitatory neurons in the CA2.

    “While PNNs predominantly enwrap PV inhibitory neurons in most brain areas, they surround excitatory neurons in the CA2 [66, 71].”

    From The extracellular matrix and perineuronal nets in memory (2022) · cites this paper as [71]

  • The WFA signal in CA2 is PNN-specific.

    “This agrees with recent studies that emphasize that the WFA signal in CA2 is PNN-specific37,38.”

    From Elimination of the four extracellular matrix molecules tenascin-C, tenascin-R, brevican and neurocan alters the ratio of excitatory and inhibitory synapses (2019) · cites this paper as [37]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Two different references have the same DOI

    Minor

    The references describe different articles published in different years but assign both the identical DOI, 10.1111/ejn.12792.

  • Two different reagents have the same RRID

    Minor

    The GFAP antibody and the Neurotrace Nissl reagent are listed as different reagents with different targets, but both are assigned RRID AB_10013382.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. 10.1111/ejn.12792 · DOI · Paper page

Cited by

Sorted by citations

Authors
Lensjø, K. K., Christensen, A. C., Tennøe, S., Fyhn, M., & Hafting, T.
Affiliations
University of Oslo (NO)
Corresponding author
Hafting, Torkel
ORCID
Venue
eneuro
Publisher
Society for Neuroscience
Published
2017-05
Volume
4
Issue
3
Pages
ENEURO.0379-16.2017
ISSN
2373-2822 (ISSN-L)
DOI
10.1523/eneuro.0379-16.2017
PMC
PMC5461557
OpenAlex
W2618096481
Semantic Scholar
7a87921e0d623bb8110dab7e94406ea922ffb1d4
Cited by
133
References
88
Citations by year
2026: 12 · 2025: 22 · 2024: 18 · 2023: 8 · 2022: 13 · 2021: 16 · 2020: 12 · 2019: 19 · 2018: 9 · 2017: 4
FWCI
5.09
Type
journal article
Language
en
Open access
Licence
Locations
  • eNeuro (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • NORA - Norwegian Open Research Archives (submittedVersion, cc-by)
Funders
Research Council of Norway (204939, 217920, 250259, 231248)
Topics
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Memory and Neural Mechanisms
Keywords
perineuronal nets, extracellular matrix, hippocampus, medial entorhinal cortex, primary visual cortex, parvalbumin interneurons, grid cells, neuroplasticity, species differences, CA2, postnatal development
MeSH terms
Disks Large Homolog 4 Protein, Reelin Protein, Animals, Carrier Proteins, Chondroitin Sulfates, Extracellular Matrix, Hippocampus, Male, Membrane Proteins, Mice, Inbred C57BL, Nerve Net, Nerve Tissue Proteins, Neurons, Parvalbumins, Visual Cortex, Statistics, Nonparametric, Entorhinal Cortex, Rats, Long-Evans, Mice, Rats, Calbindins

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.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

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.

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