Research ArticleEpilepsy

Pannexin-1 channels contribute to seizure generation in human epileptic brain tissue and in a mouse model of epilepsy

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Science Translational Medicine  30 May 2018:
Vol. 10, Issue 443, eaar3796
DOI: 10.1126/scitranslmed.aar3796
  • Fig. 1 Cortical slices from epileptic patients display spontaneous interictal and induced ictal epileptic activities ex vivo.

    (A) Example of a preoperative axial fluid attenuation inversion recovery (FLAIR) magnetic resonance image showing a focal cortical dysplasia type IIb embedded in the right frontal lobe (arrow), with cortical thickening and hypersignal, gray/white matter blurring, and transmantle sign. (B) Representative immunohistochemical labeling of a human cortical slice with neuronal (NeuN) and astrocytic [glial fibrillary acidic protein (GFAP)] markers. Scale bar, 25 μm. (C) Picture of a slice placed in a MEA chamber for recording (left) and zoom of the recording area (right). Scale bars, 4 mm (left) and 200 μm (right). (D) Representative MEA recording of spontaneous IIDs in Ct artificial cerebrospinal fluid (ACSF) (left). The expanded trace of a single IID is shown on the right. The correspondent time-frequency plot of IID activity is shown under the traces. Scale bars, 2 s (left), 100 ms (right), and 20 μV. (E) Quantification of IID (white) and ID (gray) frequency and duration. (F) Representative MEA recordings of LVF (left) or HS (right) IDs in proepileptic ACSF. The expanded traces of a single ID are shown on the right. The correspondent time-frequency plots are shown under the traces. IDs, black dashed line; PIDs, black dots. Scale bars, 50 s (left) and 20 s (right) for LVF and HS IDs; 500 μV (LVF IDs) and 100 μV (HS IDs). (G) Quantification of ID average duration (red-filled dots) and ID type (histograms) during recordings of the first 10 IDs in each slice (n = 464 IDs and 61 slices from 35 patients). (H) Quantification of the percentage of active electrodes, maximum depth, and lateral extension (MEA maximum lateral extension of recording area, 3.11 mm) of IIDs (white) and IDs (gray) (IIDs, n = 42 slices from 26 patients; IDs, n = 61 slices from 35 patients; P < 0.001 by Mann-Whitney test). Asterisks indicate statistical significance performed on raw data (***P < 0.001).

  • Fig. 2 Properties of IID and ID-like events differ according to human brain tissue type.

    (A) Distribution of brain tissue specimens showing spontaneous IIDs in Ct ACSF (left) and IDs in proepileptic ACSF (right) from glioma (orange), MCD (green), and DNET (blue) patients (IIDs, P = 0.0210; n = 24, 15, and 8 tested glioma, MCD, and DNET patients, respectively; χ2 test; IDs, P = 0.1867; n = 32, 13, and 7 tested glioma, MCD, and DNET patients, respectively; χ2 test). (B) Quantification of IID frequency (P = 0.0232, Kruskall-Wallis test) and duration (P = 0.0495, Kruskall-Wallis test) in glioma (n = 28 slices from 18 patients), MCD (n = 9 slices from 5 patients), and DNET (n = 5 slices from 3 patients) brain samples. (C) Quantification of ID delay (P = 0.6799, Kruskall-Wallis test), frequency [P = 0.0188, one-way analysis of variance (ANOVA) with Tukey post hoc test], duration (P = 0.2675, one-way ANOVA with Tukey post hoc test), and type (P < 0.0001, χ2 test) in glioma (n = 30 slices from 17 patients), MCD (n = 15 slices from 9 patients), and DNET (n = 9 slices from 5 patients) brain samples. (D) Quantification of ID average duration (red-filled dots) and ID type (histograms) during recordings of the first 10 IDs in each slice from glioma (n = 218 IDs and 30 slices from 17 patients), MCD (n = 134 IDs and 15 slices from 9 patients), and DNET (n = 69 IDs and 9 slices from 5 patients) samples. (E) Quantification of the percentage of active electrodes, maximum depth, and lateral extension (MEA maximum lateral extension of recording area, 3.11 mm) of IIDs (empty columns) and IDs (striped columns) recorded from glioma (P < 0.0001 for all the parameters; IIDs, n = 28 slices from 18 patients; IDs, n = 30 slices from 17 patients), MCD (P < 0.0001 and P = 0.0059 for active electrodes and lateral extension, respectively; IIDs, n = 9 slices from 5 patients; IDs, n = 15 slices from 9 patients), and DNET (P < 0.0001 for active electrodes and lateral extension; IIDs, n = 5 slices from 3 patients; IDs, n = 9 slices from 5 patients) samples (two-way ANOVA with Tukey post hoc test). Asterisks indicate statistical significance performed on raw data (*P < 0.05, **P < 0.01, and ***P < 0.001).

  • Fig. 3 Panx1 channels in cortical slices from epileptic patients are strongly activated under ictal conditions.

    (A) Top: Representative basal EtBr uptake (red) in neurons (blue) and astrocytes (green) in cortical tissue from epileptic patients under Ct condition. Bottom: EtBr uptake (red) under proepileptic conditions [6 mM K+, 0 mM Mg2+(6K+ 0Mg2+); left] and in the presence of 10Panx (400 μM; right). Scale bar, 25 μm. (B) Quantification of neuronal (left) and astrocytic (right) EtBr uptake (normalized to Ct ACSF) in cortical slices from epileptic patients treated with CBX (200 μM) or 10Panx (400 μM) in 6K+ 0Mg2+ ACSF (n = 8; P < 0.0001, repeated-measures one-way ANOVA with Tukey post hoc test). (C) EtBr uptake (red) in control (Ct) and under proepileptic conditions (6K+ 0Mg2+) in control cortical tissue from nonepileptic subjects. (D) Quantification of neuronal (left) and astrocytic (right) EtBr uptake (normalized to Ct ACSF) in cortical slices from nonepileptic subjects treated with CBX (200 μM) or 10Panx (400 μM) in 6K+ 0Mg2+ ACSF (n = 3; P = 0.7223 and P = 0.1963 for neurons and astrocytes, respectively, repeated-measures one-way ANOVA with Tukey post hoc test). Asterisks indicate statistical significance performed on raw data (***P < 0.001).

  • Fig. 4 Panx1 channels contribute to the induction and maintenance of ictal-like events.

    (A) Example of ID activity recorded from a human epileptic cortical slice before (left) and after (right) the treatment with 10Panx (400 μM; 30 min). IDs, black dashed line; IIDs, white dots; PIDs, black dots. Scale bars, 10 s and 100 μV. (B) Quantification of 10Panx effect on ID frequency and duration (P = 0.0049; n = 11 slices from four glioma patients and one MCD patient; Wilcoxon matched-pair test). Glioma and MCD slice values are indicated by orange- and green-filled circles, respectively. (C) Example of ID activity that is successfully induced once (left) but that fails to subsequently occur when the second induction is performed in the presence of 10Panx (right). IDs, black dashed line; IIDs, white dots; PIDs, black dots. Scale bars, 10 s and 200 μV. (D) Quantification of 10Panx effect on ID frequency and duration (P = 0.0039; n = 9 slices from three glioma patients, two MCD patients, and one DNET patient; Wilcoxon matched-pair test). Glioma, MCD, and DNET slice values are indicated by orange-, green-, and blue-filled circles, respectively. Asterisks indicate statistical significance (**P < 0.01).

  • Fig. 5 Panx1 channels do not alter basal activity of human cortical slices and are not activated under basal conditions.

    (A) Schematic representation of human cortical slice position on MEA (left); the orange-filled squares indicate the electrodes whose traces are shown in middle and right panels. Representative traces of unsynchronized sparse activity recorded under basal conditions in the absence (black traces) and in the presence (gray traces) of 10Panx. Single isolated events are indicated by red asterisks. Scale bars, 10 s and 10 μV. (B) Quantification of event frequency (P = 0.2843; n = 6 slices from two glioma patients and one control patient; paired t test). (C) EtBr uptake (red) in epileptic patients cortical tissue under basal conditions (Ct ACSF) and in the presence of 10Panx (400 μM). Scale bar, 25 μm. (D) Quantification of neuronal (left) and astrocytic (right) EtBr uptake (normalized to Ct ACSF) in slices treated with CBX (200 μM) or 10Panx (400 μM) in Ct ACSF (n = 6; P = 0.2097, repeated-measures two-way ANOVA with Tukey post hoc test).

  • Fig. 6 ATP signaling plays a prominent role in Panx1 channel–mediated effects on ID activity via P2 receptor activation.

    (A) Schematic representation of the experimental design to measure ATP release from human cortical slices. (B) Quantification of ATP release measured by luminescence detection in 500 μl of ACSF containing a single cortical slice under basal (white) and proepileptic conditions (dark gray) and in the presence of 10Panx (striped dark gray) (n = 6; repeated-measures one-way ANOVA with Tukey post hoc test). (C) Example of ID activity recorded from human epileptic cortical tissue before (left trace) and after the treatment with PPADS and RB-2 (30 μM; middle trace), and after the additional blockage of Panx1 channels by 10Panx (400 μM; right trace). Scale bars, 10 s and 500 μV. (D) Quantification of ATP receptor inhibition effect and of Panx1 channel inhibition effect after ATP receptor blockade on ID frequency and duration (P = 0.0151; n = 7 slices from three glioma patients and one DNET patient; Friedman test with Dunn post hoc test) and on PID frequency (P = 0.0064; n = 7 slices from three glioma patients and one DNET patient; repeated-measures one-way ANOVA with Tukey post hoc test). Glioma and DNET slice values are indicated by orange- and blue-filled circles, respectively. (E) Example of ID activity recorded from human epileptic tissues in the absence (top trace) and in the presence of SCH58261 (100 nM; bottom trace). Scale bars, 10 s and 100 μV. (F) Quantification of A2 receptor inhibition effect on ID frequency and duration (P = 0.0349 and 0.1412, respectively; n = 5 slices from one glioma patient and one MCD patient; paired t test) and on PID frequency (P = 0.2252; n = 5 slices from one glioma and one MCD patient; paired t test). Glioma and MCD slice values are indicated by orange- and green-filled circles, respectively. Asterisks indicate statistical significance (*P < 0.05, **P < 0.01, and ***P < 0.001).

  • Fig. 7 PBN and MFQ inhibit IDs in human cortical slices and chronic seizures in KA mice.

    (A) Top: Schematic representation of the experimental protocol. Human cortical slices placed on MEA are incubated under proepileptic conditions to induce IDs; after ID induction, PBN or MFQ is applied. Bottom: Example of ID activity recorded from a human epileptic cortical slice before (left; black traces) and after (right) the treatment with PBN (1 mM; 30 min; top; dark gray trace) or MFQ (100 nM; 30 min; bottom; light gray trace). IDs, black dashed line; IIDs, white dots; PIDs, black dots. Scale bars, 10 s and 200 μV. (B) Quantification of PBN (top) and MFQ (bottom) effect on ID frequency and duration (ID frequency: PBN, P = 0.0313; n = 6 slices from two glioma and two MCD patients; Wilcoxon matched-pair test; MFQ, P = 0.0367; n = 6 slices from five glioma patients; paired t test). Glioma and MCD slice values are indicated in orange- and green-filled circles, respectively. (C) Left: Schematic representation of the experimental protocol. Mice are injected unilaterally with KA in the CA1 area of the right hippocampus; they are then left for 3 weeks for settling of the chronic epileptic phenotype before performing 24- to 48-hour EEG recordings. Right: Examples of EEG recordings in wild-type mice (+/+) before (black; top trace) and 0 to 4 hours after a single intraperitoneal injection of PBN (200 mg/kg; gray) or MFQ (20 mg/kg; light gray), and in +/+ and Panx1−/− mice (black; bottom trace). Scale bars, 30 min and 250 μV. The seizure indicated by a red asterisk (top) is zoomed in the red rectangle. Scale bar, 5 s. (D) Quantification of seizure frequency and total time spent in seizures (normalized to +/+ mice) 0 to 4 hours after PBN or MFQ treatment and in Panx1−/− mice (seizure frequency: PBN and MFQ, P = 0.0156; n = 7; MFQ, P = 0.0476; n = 6; Wilcoxon matched-pair test; +/+, n = 10; Panx1−/−, n = 5; P = 0.0329, unpaired t test with Welch correction; total time spent in seizures: PBN, P = 0.0307; n = 7; MFQ, P = 0.0453; n = 6; Wilcoxon matched-pair test; +/+, n = 10; Panx1−/−, n = 5; P = 0.0395, unpaired t test with Welch correction). Asterisks indicate statistical significance (*P < 0.05). p.t., post-treatment.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/443/eaar3796/DC1

    Materials and Methods

    Fig. S1. Absence of spontaneous IIDs and induced IDs in control tissues from epileptic patients.

    Fig. S2. Unaltered ID induction and maintenance in the presence of scPanx.

    Fig. S3. Generation of Panx1−/− mice.

    Fig. S4. Epileptic activity recorded in a patient cortical area in vivo and ex vivo after surgical resection.

    Fig. S5. MEA recordings of epileptic activities in human cortical slices.

    Table S1. Clinical, imaging, and neuropathological characteristics of patients involved in the study.

    Table S2. Properties of IIDs and IDs recorded from the different types of epileptic tissues.

    Table S3. Raw data (provided as an Excel file).

  • Supplementary Material for:

    Pannexin-1 channels contribute to seizure generation in human epileptic brain tissue and in a mouse model of epilepsy

    Elena Dossi, Thomas Blauwblomme, Julien Moulard, Oana Chever, Flora Vasile, Eleonore Guinard, Marc Le Bert, Isabelle Couillin, Johan Pallud, Laurent Capelle, Gilles Huberfeld, Nathalie Rouach*

    *Corresponding author. Email: nathalie.rouach{at}college-de-france.fr

    Published 30 May 2018, Sci. Transl. Med. 10, eaar3796 (2018)
    DOI: 10.1126/scitranslmed.aar3796

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Absence of spontaneous IIDs and induced IDs in control tissues from epileptic patients.
    • Fig. S2. Unaltered ID induction and maintenance in the presence of scPanx.
    • Fig. S3. Generation of Panx1−/− mice.
    • Fig. S4. Epileptic activity recorded in a patient cortical area in vivo and ex vivo after surgical resection.
    • Fig. S5. MEA recordings of epileptic activities in human cortical slices.
    • Table S1. Clinical, imaging, and neuropathological characteristics of patients involved in the study.
    • Table S2. Properties of IIDs and IDs recorded from the different types of epileptic tissues.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3. Raw data (provided as an Excel file).

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