Perineuronal nets decrease membrane capacitance of peritumoral fast spiking interneurons in a model of epilepsy

May combine two claims.

GBM14 implantation nullifies changes in firing threshold and Vm of PTC neurons, with no apparent difference to sham or contralateral brains.

“This indeed nullified the changes in firing threshold and Vm with no apparent difference to sham or contralateral brains (Fig. 6e, Supplementary Fig. 4b, c).”

May combine two claims.

The membrane capacitance (Cm) of FSNs in GM6001-treated PTC was lower than in nontreated PTC but similar to sham.

“Lower Cm of FSNs in the PTC of GM6001-treated (28.27 ± 1.36, n = 20(5)) than nontreated (43.84 ± 3.74, n = 13(8)), but similar to sham (30.11 ± 1.99, n = 15(6)).”

• Also stated in: § Biophysical changes in peritumoral neurons · boosted

  “Most importantly, FSNs in GM6001-treated mice brain slices showed profound differences in Cm and input−output curves, and both were essentially identical to the sham (Fig. 7b, e, f).”

May combine two claims.

In GBM14, WFA staining shows largely intact PNNs even in close proximity to the tumor (0.2-0.4 mm), whereas in GBM22, PNNs are largely degraded.

“In GBM14, WFA staining showed largely intact PNNs even in close proximity to the tumor (0.2–0.4 mm), whereas in GBM22, PNNs were largely degraded (Fig. 3a, c).”

May combine two claims.

The Rin of excitatory neurons in GM6001-treated PTC was not significantly different from nontreated PTC but was different from sham.

“Input resistance (Rin) of excitatory neurons in GM6001-treated PTC (244.64 ± 9.24, n = 27(6)) was not significantly different than in nontreated PTC (288.18 ± 15.82, n = 23(10)), but than in sham (298.63 ± 14.47, n = 39(10)).”

May combine two claims.

GM6001-treated mice retain structural integrity of PNNs in the PTC, as evident by PNNs around cell body and proximal dendrites, more WFA peaks, and higher WFA intensity compared to sham-treated mice.

“Indeed, GM6001-treated mice retained structural integrity of PNNs in the PTC (Fig. 5f−i) as evident by the presence of PNNs around cell body and proximal dendrites (Fig. 5f, middle panels), more WFA peaks (Fig. 5g, h), and higher WFA intensity (Fig. 5i) compared to sham-treated mice.”

May combine two claims.

GM6001 treatment reduces gelatinase activity within the glioma and in the PTC.

“Confirming our prediction, GM6001 treatment reduced gelatinase activity within the glioma (Fig. 5a, middle panels) and in the PTC (Fig. 5d).”

May combine two claims.

The PTC in close proximity to the tumor (<0.3 mm) shows disintegrated PNNs surrounded by highly reactive astrocytes, while essentially intact PNNs are observed around equally high GFAP-expressing astrocytes in more distal (>0.6 mm) PTC.

“while the PTC in close proximity to the tumor (<0.3 mm) showed disintegrated PNNs surrounded by highly reactive astrocytes (Fig. 4a (bottom square), Fig. 4b, c), we observed essentially intact PNNs around equally high GFAP (glial fibrillary acidic protein) expressing astrocytes in more distal (>0.6 mm) PTC (Fig. 4a (top square), 4b, c).”

May combine two claims.

The action potential (AP) frequency of FSNs in GM6001-treated PTC was higher than in nontreated PTC but not higher than in sham.

“Higher AP frequency of FSNs in GM6001-treated PTC (n = 12(5)) than in nontreated PTC (n = 18(11)) but not than in sham (n = 10(7)).”

May combine two claims.

WFA intensity peaks are significantly reduced in the line profile of peritumoral PNNs, indicating large holes in the PNN.

“We confirmed the same by observing significant reduction in the WFA intensity peaks (see methods) in the line profile of peritumoral PNNs, an indicative of large holes in the PNN (Fig. 2c−e).”

May combine two claims.

Membrane capacitance and input resistance of PTC neurons remain unaffected.

“however, their membrane capacitance (Cm) and input resistance (Rin) remained unaffected (Fig. 6b, c).”

May combine two claims.

The resting membrane potential (Vm) of FSNs in GM6001-treated PTC was not significantly different from nontreated GBM22 PTC but was significantly different from sham.

“Unaltered resting membrane potential (Vm) of FSNs in the PTC of GM6001-treated (62.88 ± 1.40, n = 18(5)) than in nontreated (GBM22) PTC (61.67 ± 1.01, n = 12(8)), but significantly different than in sham (69.85 ± 0.67, n = 7(6)).”

May combine two claims.

The gradient of NeuN+ cell loss extends up to 0.6 mm from the tumor margin, with significantly lower cell density compared to sham and contralateral brains.

“The gradient of NeuN+ cell loss was observed up to 0.6 mm from the tumor margin, the furthest distance evaluated, with significantly lower cell density compared to sham and contralateral brains (Fig. 1a).”

May combine two claims.

Excitatory neurons in GM6001-treated PTC were depolarized relative to nontreated PTC and had Vm similar to sham.

“Resting membrane potential (Vm) of excitatory neurons in GM6001-treated PTC (66.62 ± 0.77, n = 27(8)) remained similar to sham (67.02 ± 0.38, n = 98(15)), but was significantly higher than the nontreated PTC (61.49 ± 0.86, n = 80(11)).”

• Also stated in: § Biophysical changes in peritumoral neurons · plain

  “Excitatory neurons in GM6001-treated PTC were depolarized (Fig. 7g);”

May combine two claims.

The input−output curve of excitatory neurons in GM6001-treated PTC showed lower excitability with small current injections, similar to sham, but somewhat increased excitability with larger current injections than sham.

“Moreover, the input−output curve showed a lower excitability with small current injections, similar to that of sham, yet still showed somewhat increased excitability with larger current injections than sham (Fig. 7k, l).”

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

Excitatory neurons gradually depolarized during ChABC superfusion.

“Excitatory neurons gradually depolarized during ChABC superfusion (Supplementary Fig. 8a, b (right)), a phenomenon we did not study further.”

May combine two claims.

In the Hodgkin−Huxley simulation, maximal firing rate increased nonlinearly with increasing stimulation and decreased progressively with increasing specific membrane capacitance.

“Maximal firing rate (z-axis) increased nonlinearly with increasing stimulation (y-axis), and decreased progressively with increasing specific membrane capacitance (x-axis).”

• AssumptionRests on an assumption: The first-order Hodgkin−Huxley approximation and Neuronify model with identical electrical properties adequately represent the biophysical behavior of FSNs.

May combine two claims.

There is a 2-5-fold decrease in PV interneuron density within 0.6 mm of glutamate-releasing tumor, suggesting excitotoxic loss.

“In agreement with prior studies, we observed a 2–5-fold decrease in the PV interneurons density within 0.6 mm of glutamate-releasing tumor suggesting their excitotoxic loss.”

May combine two claims.

ChABC pretreatment significantly shortened the latency of ictal-like events but not interictal-like events.

“ChABC pretreatment significantly shortened the latency of ictal-like events (Fig. 9e−g) but not interictal-like events (Fig. 9f).”

• Also stated in: § PNN degradation facilitates cortical epileptiform activity · plain

  “ictal-like activity (control, 73.67 ± 6.69 min, n = 12(6); ChABC-treated, 38.83 ± 3.40 min, n = 8(5); ***P < 0.001, Welch's t test).”

May combine two claims.

Surviving PV interneurons show ~50% reduction in firing frequency, a phenomenon independent of tumor-released Glu.

“Interestingly, the surviving PV interneurons show ~50% reduction in firing frequency, a phenomenon that was independent of the tumor-released Glu.”

May combine two claims.

Peritumoral excitatory neurons are significantly depolarized, have a reduced activation threshold and increased discharge frequency, but only when proximal to a tumor that expresses the Glu-releasing SXC transporter.

“Prior studies had already implicated the assiduous release of Glu from the tumor as instrumental, and we indeed find that peritumoral excitatory neurons are significantly depolarized, have a reduced activation threshold and increased discharge frequency but only when proximal to a tumor that expresses the Glu-releasing SXC transporter.”

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

Action potential amplitudes shortened as firing frequency increased.

“We observed that the amplitudes of APs shortened as the firing frequency increased; therefore, we set a minimum 15 mV deflection from the steady-state response as a qualifying criterion for a spike to be identified as an AP.”

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

The saturating/maximum firing frequency supported by fast-spiking neurons (FSNs) in the experimental conditions was achieved by injecting 400-500 pA.

“The observed saturating/maximum firing frequency was found to be achieved by injecting 400−500 pA; therefore, we capped the highest injected current to 500 pA.”

May combine two claims.

Glutamate release is necessary but insufficient to drive peritumoral epilepsy; an additional loss of GABAergic inhibition is required.

“Glu release, while necessary, is insufficient to drive peritumoral epilepsy3. Rather an additional loss of GABAergic inhibition is required”

• cites [3] Campbell, S. L. et al. GABAergic disinhibition and impaired KCC2 cotransporter activity underlie tumor‐associated epilepsy. Glia 63, 23–36 (2015). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

PNNs act as an electrostatic insulator that reduces specific membrane capacitance, functionally akin to myelin sheaths around axons, thereby permitting FSNs to exceed physiological firing rates.

“These studies uncovered a hitherto unknown role of PNNs as an electrostatic insulator that reduces specific membrane capacitance, functionally akin to myelin sheaths around axons, thereby permitting FSNs to exceed physiological firing rates.”

May combine two claims.

Enhanced glutamatergic drive in tumor-associated epilepsy results from glutamate released from gliomas via the system Xc transporter (SXC) encoded by SLC7A11, which is upregulated in about 54% of glioma patients.

“The enhanced glutamatergic drive is the result of glutamate (Glu) being assiduously released from gliomas via system Xc transporter (SXC) encoded by the SLC7A11 gene that is upregulated in about 54% of glioma patients4.”

• cites [4] Robert, S. M. et al. SLC7A11 expression is associated with seizures and predicts poor survival in patients with malignant glioma. Sci. Transl. Med. 7, 289ra286 (2015). sourceNot checked yet

  Not checked in the free run.

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

The cellular and molecular changes underlying tumor-associated epilepsy collectively point to a decrease in GABAergic and increase in glutamatergic transmission as the major contributors.

“The cellular and molecular changes underlying tumor-associated epilepsy collectively point to a decrease in GABAergic and increase in glutamatergic transmission as the major contributors3–6.”

• cites [3] Campbell, S. L. et al. GABAergic disinhibition and impaired KCC2 cotransporter activity underlie tumor‐associated epilepsy. Glia 63, 23–36 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [4] Robert, S. M. et al. SLC7A11 expression is associated with seizures and predicts poor survival in patients with malignant glioma. Sci. Transl. Med. 7, 289ra286 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [5] Pallud, J. et al. Cortical GABAergic excitation contributes to epileptic activities around human glioma. Sci. Transl. Med. 6, 244ra289 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [6] Buckingham, S. C. et al. Glutamate release by primary brain tumors induces epileptic activity. Nat. Med. 17, 1269–1274 (2011). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Tumor-released proteolytic enzymes degrade PNNs, resulting in increased membrane capacitance, reduced firing, and hence decreased GABA release.

“Tumor-released proteolytic enzymes degrade PNNs, resulting in increased membrane capacitance, reduced firing, and hence decreased GABA release.”

May combine two claims.

PNNs decrease specific membrane capacitance of FSNs, permitting them to fire action potentials at supra-physiological frequencies.

“We show that PNNs decrease specific membrane capacitance of FSNs permitting them to fire action potentials at supra-physiological frequencies.”

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

About 80% of PV+ FSNs are surrounded by perineuronal nets (PNNs).

“About 80% of PV+ FSNs14 are surrounded by perineuronal nets (PNNs)”

• cites [14] Carulli, D. et al. Animals lacking link protein have attenuated perineuronal nets and persistent plasticity. Brain 133, 2331–2347 (2010). sourceNot checked yet

  Not checked in the free run.

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

PNNs may restrict local ionic concentration due to a high density of negatively charged glycosaminoglycans, thereby regulating the microenvironment of FSNs and influencing their intrinsic properties.

“PNNs may also restrict local ionic concentration due to a high density of their negatively charged constituent glycosaminoglycans (GAGs)18, thereby regulating the microenvironment of FSNs and influencing their intrinsic properties19.”

• cites [18] Morawski, M. et al. Ion exchanger in the brain: quantitative analysis of perineuronally fixed anionic binding sites suggests diffusion barriers with ion sorting properties. Sci. Rep. 5, 16471 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [19] Härtig, W. et al. Cortical neurons immunoreactive for the potassium channel Kv3. 1b subunit are predominantly surrounded by perineuronal nets presumed as a buffering system for cations. Brain Res. 842, 15–29 (1999). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Tumor-associated seizures are the consequence of impaired GABAergic inhibition due to an overall loss of peritumoral fast spiking interneurons (FSNs) and a significantly reduced firing rate of those that remain.

“We show that tumor-associated seizures are the consequence of impaired GABAergic inhibition due to an overall loss of peritumoral fast spiking interneurons (FSNs) concomitant with a significantly reduced firing rate of those that remain.”

May combine two claims.

The PTC exhibits a spatial gradient of higher to lower gelatinase activity from the glioma border to the distal PTC that is negatively correlated with WFA intensity, suggesting the tumor as the likely source of MMPs.

“PTC exhibited a spatial gradient of higher to lower gelatinase activity from the glioma border to the distal PTC (Fig. 5b) that appeared negatively correlated with the WFA intensity (Fig. 5c), suggesting the tumor as the likely source of MMPs.”

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

Approximately 10% of all NeuN+ neurons in cortex are PV+.

“In agreement with the literature24–26, ~10% of all NeuN+ neurons in cortex were PV+ (Fig. 1b, c)”

• cites [24] Ren, J., Aika, Y., Heizmann, C. & Kosaka, T. Quantitative analysis of neurons and glial cells in the rat somatosensory cortex, with special reference to GABAergic neurons and parvalbumin-containing neurons. Exp. Brain Res. 92, 1–14 (1992). sourceNot checked yet

  Not checked in the free run.

• cites [25] Le Magueresse, C. & Monyer, H. GABAergic interneurons shape the functional maturation of the cortex. Neuron 77, 388–405 (2013). sourceNot checked yet

  Not checked in the free run.

• cites [26] Beaulieu, C. Numerical data on neocortical neurons in adult rat, with special reference to the GABA population. Brain Res. 609, 284–292 (1993). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Seizures originate from the PTC, and previous studies show altered excitation−inhibition balance in the PTC due to changes in glutamatergic and GABAergic activity.

“Seizures originate from the PTC, and previous studies3,5,6 show altered excitation−inhibition balance in the PTC due to changes in glutamatergic and GABAergic activity.”

• cites [3] Campbell, S. L. et al. GABAergic disinhibition and impaired KCC2 cotransporter activity underlie tumor‐associated epilepsy. Glia 63, 23–36 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [5] Pallud, J. et al. Cortical GABAergic excitation contributes to epileptic activities around human glioma. Sci. Transl. Med. 6, 244ra289 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [6] Buckingham, S. C. et al. Glutamate release by primary brain tumors induces epileptic activity. Nat. Med. 17, 1269–1274 (2011). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Real-time digestion of PNNs suggests that PNNs act as an insulator that decreases specific membrane capacitance of the cell, thereby permitting an increase in the maximally achievable firing rate of FSNs.

“Importantly, real-time digestion of PNNs suggests that PNNs act as an insulator that decreases specific membrane capacitance of the cell thereby permitting an increase in the maximally achievable firing rate of FSNs.”

The quote only partly states this claim.

Experimental limitations including incubation duration and low enzymatic activity prevented sufficient PNN degradation with the MMP cocktail.

“Unfortunately, experimental limitations including incubation duration and low enzymatic activity prevented us to achieve sufficient PNN degradation.”

May combine two claims.

PNN degradation alone is sufficient to reduce the firing frequency of FSNs, thereby decreasing inhibitory tone.

“We show that PNN degradation alone is sufficient to reduce the firing frequency of FSNs thereby decreasing inhibitory tone”

May combine two claims.

Preventing PNN degradation significantly rescued the biophysical characteristics of inhibitory neurons but did not affect excitatory neurons, which were still under the influence of tumor-released glutamate.

“Preventing PNN degradation significantly rescued the biophysical characteristics of inhibitory neurons but without affecting excitatory neurons, which were still under the influence of tumor-released glutamate.”

• AssumptionRests on an assumption: The elevation in glioma-released glutamate persists in GM6001-treated animals and continues to drive heightened excitability of excitatory neurons.

May combine two claims.

Neither resting membrane potential nor input−output curve of FSNs can be attributed to tumor-released Glu, as they are identical in neurons with disintegrated PNNs recorded in PTCs of GBM14 and GBM22.

“In contrast to excitatory cells, neither resting membrane potential nor input−output curve (Fig. 6j−l) could be attributed to the tumor-released Glu as they were identical in neurons with disintegrated PNNs recorded in the PTCs of GBM14 and GBM22-implanted brains (Fig. 6k).”

May combine two claims.

Tumor-secreted molecules, likely Glu and proteolytic enzymes, cause a distance-dependent loss of interneurons and degradation of PNNs, respectively.

“Taken together, these data suggest that tumor-secreted molecules, likely Glu and proteolytic enzymes, cause a distance-dependent loss of interneurons and degradation of PNNs, respectively.”

May combine two claims.

Changes in the excitability of FSNs appear intrinsic and are associated with disintegration of PNNs and a ~25% increase in cell capacitance as the primary differences between PTC and control cortex.

“In contrast, changes in the excitability of FSNs appear intrinsic, and are associated with disintegration of PNNs and ~25% increase in cell capacitance as the primary differences between PTC and control cortex.”

May combine two claims.

Spike frequency of excitatory neurons in GBM22 PTC is significantly reduced on blocking glutamatergic neurotransmission, confirming they are driven by glioma-released Glu rather than intrinsic to the cell.

“In addition, the spike frequency of excitatory neurons in GBM22 PTC was significantly reduced on blocking the glutamatergic neurotransmission, further confirming them driven by glioma-released Glu rather than intrinsic to the cell (Supplementary Fig. 5b).”

May combine two claims.

Metalloproteinase inhibitors prevented seizures and MMP-9 knockout mice were less susceptible to pentylenetetrazole-induced seizures.

“Moreover, metalloproteinase inhibitors prevented seizures51 and MMP-9 knockout mice were less susceptible to pentylenetetrazole-induced seizures52.”

• cites [51] Pollock, E., Everest, M., Brown, A. & Poulter, M. O. Metalloproteinase inhibition prevents inhibitory synapse reorganization and seizure genesis. Neurobiol. Dis. 70, 21–31 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [52] Wilczynski, G. M. et al. Important role of matrix metalloproteinase 9 in epileptogenesis. J. Cell. Biol. 180, 1021–1035 (2008). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Experimental digestion of PNNs in normal brain phenocopied the change in capacitance and the observed excitability changes.

“Experimental digestion of PNNs in normal brain phenocopied the change in capacitance and the observed excitability changes.”

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

Heightened MMP activity has been reported in other models of epilepsy, with elevated MMP level associated with epileptiform activity.

“While we suggest a pivotal role for the MMP activity in glioma-associated epilepsy, heightened MMP activity has also been reported in other models of epilepsy46 with elevated MMP level being associated with epileptiform activity47–50.”

• cites [46] Dubey, D. et al. Increased metalloproteinase activity in the hippocampus following status epilepticus. Epilepsy Res. 132, 50–58 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [47] Jourquin, J. et al. Neuronal activity‐dependent increase of net matrix metalloproteinase activity is associated with MMP‐9 neurotoxicity after kainate. Eur. J. Neurosci. 18, 1507–1517 (2003). sourceNot checked yet

  Not checked in the free run.

• cites [48] Kim, G. W. et al. The role of MMP-9 in integrin-mediated hippocampal cell death after pilocarpine-induced status epilepticus. Neurobiol. Dis. 36, 169–180 (2009). sourceNot checked yet

  Not checked in the free run.

• cites [49] Szklarczyk, A., Lapinska, J., Rylski, M., McKay, R. D. & Kaczmarek, L. Matrix metalloproteinase-9 undergoes expression and activation during dendritic remodeling in adult hippocampus. J. Neurosci. 22, 920–930 (2002). sourceNot checked yet

  Not checked in the free run.

• cites [50] Takács, E. et al. Matrix metalloproteinase-9 activity increased by two different types of epileptic seizures that do not induce neuronal death: a possible role in homeostatic synaptic plasticity. Neurochem. Int. 56, 799–809 (2010). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The PNN that extends from the proximal dendrites across the cell body to the axon hillock increases membrane thickness, which in turn decreases specific membrane capacitance.

“Akin to the myelin sheaths surrounding axons, the PNN that extends from the proximal dendrites across the cell body to the axon hillock increases the membrane thickness, which in turn decreases the specific membrane capacitance.”

May combine two claims.

PNN damage is primarily attributed to the proteolytic activity of glioma-released MMPs and consequently caused the cell's membrane capacitance to increase.

“PNN damage was found to be primarily attributed to the proteolytic activity of glioma-released MMPs and consequently caused the cell’s membrane capacitance to increase.”

May combine two claims.

PNNs act as an electrostatic insulator that decreases the cell's effective membrane capacitance and concomitantly increases the cell's maximally attainable firing frequency, which is directly linked to GABA release and maintenance of inhibitory tone.

“Further biophysical analysis and modeling results revealed that the PNNs must act as an electrostatic insulator that decreases the cell’s effective membrane capacitance and concomitantly increase the cell’s maximally attainable firing frequency, which is directly linked to GABA release and maintenance of inhibitory tone.”

• AssumptionRests on an assumption: The first-order Hodgkin−Huxley approximation and Neuronify model with identical electrical properties adequately represent the biophysical behavior of FSNs.

May combine two claims.

An intact PNN reduces membrane capacitance and is essential to allow FSNs to sustain their maximum firing frequency.

“Taken together, these data suggest that an intact PNN reduces membrane capacitance and is essential to allow FSNs to sustain their maximum firing frequency.”

May combine two claims.

PNNs and CSPGs protect neurons from oxidative stress and glutamate excitotoxicity, and combined actions of glioma-released proteases and Glu might explain the selective vulnerability of PV neurons over principal neurons in the PTC.

“PNNs and CSPGs play crucial roles in protecting neurons from oxidative stress33 and glutamate excitotoxicity42 and combined actions of glioma-released proteases and Glu might explain the selective vulnerability of PV neurons over principle neurons in the PTC.”

• cites [33] Okamoto, M., Mori, S. & Endo, H. A protective action of chondroitin sulfate proteoglycans against neuronal cell death induced by glutamate. Brain Res. 637, 57–67 (1994). sourceNot checked yet

  Not checked in the free run.

• cites [42] Rempe, R. G. et al. Matrix metalloproteinase-mediated blood−brain barrier dysfunction in epilepsy. J. Neurosci. 38, 4301–4315 (2018). sourceNot checked yet

  Not checked in the free run.

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

PNN degradation may also alter synaptic activity and hence network excitability.

“However, it is important to recognize that in addition to changes in the excitability of inhibitory neurons as suggested here and by others55, PNN degradation may also alter synaptic activity56 and hence network excitability.”

• cites [55] Balmer, T. S. Perineuronal nets enhance the excitability of fast-spiking neurons. eNeuro 3, ENEURO.0112-0116.2016 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [56] McRae, P. A. & Porter, B. E. The perineuronal net component of the extracellular matrix in plasticity and epilepsy. Neurochem. Int. 61, 963–972 (2012). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S4 (based).

ChABC degrades CSPGs in the interstitial matrix and PNNs irrespective of their structural arrangement.

“ChABC degrades CSPGs in the interstitial matrix and PNNs irrespective of their structural arrangement.”

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

Staley and colleagues demonstrated that CSPG digestion changes the transmembrane Cl− gradient, which can alter GABAergic neurotransmission.

“Staley and colleagues demonstrated that CSPGs digestion changes the transmembrane Cl− gradient, which can alter GABAergic neurotransmission20.”

• cites [20] Glykys, J. et al. Local impermeant anions establish the neuronal chloride concentration. Science 343, 670–675 (2014). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Gliomas act as a localized source of seizure-provoking Glu and PNN-degrading metalloproteinases, establishing a gradient of spread that explains the observed gradient of excitotoxic cell loss and PNN destruction decreasing with distance from the tumor.

“Gliomas act as a localized source of seizure provoking Glu and PNN degrading metalloproteinases28 thereby establishing a gradient of spread, which explains the observed gradient of excitotoxic cell loss and PNN destruction that decreases with distance from the tumor.”

• cites [28] Rao, J. S. Molecular mechanisms of glioma invasiveness: the role of proteases. Nat. Rev. Cancer 3, 489–501 (2003). sourceNot checked yet

  Not checked in the free run.

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

Others have suggested altered lateral mobility of AMPAR or changes in local diffusion of Ca2+ in brain tissue following PNN degradation.

“Others have suggested altered lateral mobility of AMPAR53 or changes in the local diffusion of Ca2+ in brain tissue54 following PNN degradation.”

• cites [53] Frischknecht, R. et al. Brain extracellular matrix affects AMPA receptor lateral mobility and short-term synaptic plasticity. Nat. Neurosci. 12, 897–904 (2009). sourceNot checked yet

  Not checked in the free run.

• cites [54] Hrabětová, S., Masri, D., Tao, L., Xiao, F. & Nicholson, C. Calcium diffusion enhanced after cleavage of negatively charged components of brain extracellular matrix by chondroitinase ABC. J. Physiol. 587, 4029–4049 (2009). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

GM6001 increased the number of FSNs, most likely due to the known neuroprotective effect of PNNs shielding FSNs from glutamate excitotoxicity.

“In addition, GM6001 increased the number of FSNs most likely due to the known neuroprotective effect of the PNNs33 shielding FSNs from glutamate excitotoxicity33.”

• cites [33] Okamoto, M., Mori, S. & Endo, H. A protective action of chondroitin sulfate proteoglycans against neuronal cell death induced by glutamate. Brain Res. 637, 57–67 (1994). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The presence of PNN increases maximal sustainable firing frequency and greatly enhances evoked activity even at lower frequencies.

“Hence, these data suggest that not only does the presence of PNN increases maximal sustainable firing frequency, but also the evoked activity is indeed greatly enhanced even at lower frequencies.”

• AssumptionRests on an assumption: The first-order Hodgkin−Huxley approximation and Neuronify model with identical electrical properties adequately represent the biophysical behavior of FSNs.

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Other populations of FSNs in brain may be similarly modulated by PNNs and similarly vulnerable to enzymatic damage of PNNs following injury and disease.

“Other populations of FSNs in brain may be similarly modulated by PNNs, and similarly vulnerable to enzymatic damage of PNNs following injury and disease.”

May combine two claims.

PNN tunes the cell's membrane capacitance thereby modulating its firing frequency.

“As illustrated in summary Supplementary Fig. 10, our data suggest that PNN tunes cell’s membrane capacitance thereby modulating its firing frequency.”

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

Membrane capacitance and series resistance were not compensated unless otherwise stated.

“The membrane capacitance (Cm) and series resistance were not compensated unless otherwise stated.”

Classified as probably not a substantial claim.

A line profile intensity graph showing an almost flat line with only a few low intensity peaks indicates a disintegrated PNN by glioma.

“A line profile intensity graph showing almost flat line with only few low intensity peaks indicates disintegrated PNN by glioma as exemplified in Fig. 2d.”

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

All animal procedures were approved and performed in accordance with ethical guidelines set by Virginia Tech Institutional Animal Care and Use Committee (IACUC).

“All animal procedures were approved and performed in accordance with the ethical guidelines set by Virginia Tech Institutional Animal Care and Use Committee (IACUC).”

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

Glu may cause an increase in MMP expression and activity level.

“Note that Glu may also cause an increase in MMP expression and activity level as reported by a recent study41.”

• cites [41] Lukes, A., Mun-Bryce, S., Lukes, M. & Rosenberg, G. A. Extracellular matrix degradation by metalloproteinases and central nervous system diseases. Mol. Neurobiol. 19, 267–284 (1999). sourceNot checked yet

  Not checked in the free run.

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

The model methodology, including model interactions and parameter values, has been used in recent studies.

“This similar methodology, both model interactions and parameter values, has been used in recent studies61–63.”

• cites [61] Guerrier, C. & Holcman, D. Hybrid Markov-mass action law model for cell activation by rare binding events: application to calcium induced vesicular release at neuronal synapses. Sci. Rep. 6, 35506 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [62] Howell, B., Medina, L. E. & Grill, W. M. Effects of frequency-dependent membrane capacitance on neural excitability. J. Neural Eng. 12, 056015 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [63] Weinberg, S. H. Membrane capacitive memory alters spiking in neurons described by the fractional-order Hodgkin−Huxley model. PLoS ONE 10, e0126629 (2015). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

The data are sufficiently normally distributed and the variance within groups is sufficiently similar to be used for parametric tests.

“Data are sufficiently normal distributed and variance within groups is sufficiently similar to be used for parametric tests.”

May combine two claims.

Real-time ChABC superfusion at 1 U/ml for 50 min at 32-33 °C reliably degraded PNNs but left identifiable traces around FSNs to confirm the presence of PNNs prior to digestion.

“This protocol also reliably degraded PNNs but left identifiable traces around the FSNs to confirm the presence of PNNs around patched cells prior to the digestion (Supplementary Fig. 7).”

May combine two claims.

ChABC treatment at 0.5 U/ml for 45 min at 33 °C reliably degraded PNNs but spared traces around FSNs, allowing identification of PNN-positive cells.

“These parameters of PNNs digestion by ChABC (enzyme concentration—0.5 U/ml, incubation time—45 min, incubation temperature—33 °C) reliably degraded PNNs but spared traces around the FSNs thereby allowing the identification of PNN-positive cells (Supplementary Fig. 6a, b).”

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

Glutamatergic neurotransmission was blocked by superfusing D-AP-5 and CNQX, and the blockage was confirmed by the disappearance of spontaneous EPSCs.

“For few experiments, we superfused ACSF containing 50 µM D-2-amino-5-phosphonovalerate (D-AP-5) and 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to block glutamatergic neurotransmission and confirmed the blockage by observing disappearance of spontaneous EPSCs.”

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

Staining was performed in a large batch of duplicate sections from 5 to 7 mice of each treatment group to minimize procedure-related variability.

“To minimize procedure-related variability, we preformed staining in a large batch of duplicate sections from 5 to 7 mice of each treatment group.”

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

The Hodgkin-Huxley model was first described by Hodgkin and Huxley and modified by Abbott and Kepler.

“This model was first described in the seminal work by Hodgkin and Huxley, and modified by Abbot and Kepler59,60.”

• cites [59] Abbott, L. & Kepler, T. B. Model neurons: from Hodgkin−Huxley to Hopfield. In Statistical Mechanics of Neural Networks (ed. Garrido, I.) (Springer, Berlin, 1990).Not checked yet

  Not checked in the free run.

• cites [60] Hodgkin, A. L. & Huxley, A. F. A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. 117, 500–544 (1952). sourceNot checked yet

  Not checked in the free run.

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

PNNs are stable structures known to lock synapses in place upon closure of the developmental window of plasticity, and several CNS pathologies present with upregulated ECM and PNN remodeling enzymes including MMPs.

“Although PNNs are stable structures and known to lock synapses in place upon closure of the developmental window of plasticity17,38,39, several CNS pathologies40, including glioma and epilepsy, present with upregulated ECM and PNN remodeling enzymes including MMPs.”

• cites [17] Tsien, R. Y. Very long-term memories may be stored in the pattern of holes in the perineuronal net. Proc. Natl. Acad. Sci. USA 110, 12456–12461 (2013). sourceNot checked yet

  Not checked in the free run.

• cites [38] Hu, H., Gan, J. & Jonas, P. Fast-spiking, parvalbumin + GABAergic interneurons: From cellular design to microcircuit function. Science 345, 1255263 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [39] Pizzorusso, T. et al. Reactivation of ocular dominance plasticity in the adult visual cortex. Science 298, 1248–1251 (2002). sourceNot checked yet

  Not checked in the free run.

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

Different patterns of PV and NeuN intensity peaks flanked by WFA peaks indicate whether a cell is present inside the PNN and the status of the PNN.

“Different patterns of PV and NeuN intensity peaks flanked by WFA peaks indicate whether a cell is present inside the PNN and the status of PNN.”

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

Potential damage to PNNs during patching was minimized by applying minimum positive pressure while approaching cells.

“A potential damage to the PNNs was minimized during the patching procedure by applying minimum positive pressure while approaching cells.”

May combine two claims.

The aforementioned mechanisms could alter the excitation−inhibition balance in favor of seizures and may contribute to a loss of inhibition in tumor-associated cortex.

“The aforementioned mechanisms could alter the excitation−inhibition balance in favor of seizures and may well contribute to a loss of inhibition in tumor-associated cortex.”