ET depolarizes granule cells in cultured slices.

<p>A, Schematic representation of the recording configuration (Whole Cell). B–E, typical membrane potential changes recorded in granule cells (using the Current Clamp mode) adjusted at −60 mV, after application of 10⁻⁷ M ET but without (B, <i>n</i> = 15) or after pre-treatment for 10 min with (C, <i>n</i> = 6) Bicuculline (Bicu, 10⁻⁵ M), (D, <i>n</i> = 7) CNQX (10⁻⁵ M) or, (E, <i>n</i> = 8) a cocktail of Bicuculline (10⁻⁵ M), CNQX (10⁻⁵ M) and TTX (10⁻⁶ M). F, quantification of the delay and amplitude of the depolarization induced by ET. For the corresponding <i>n</i>, see above. All comparisons <i>vs</i> ET alone are <i>n.s.</i> G, Changes in membrane resistance of the granule cells before (white bar) and 5 min after 10⁻⁷ M ET (black bar) (<i>n</i> = 15, <i>p<</i>0.001). Same scale for all voltage traces.</p

Abrupt current changes induced by ET in membrane patches.

<p>A, Schematic representation of the recording configuration (Cell attached). ET (10⁻⁷ M) was applied inside the patch-pipette. B–C, typical membrane current changes recorded after sealing the patch-pipette onto a granule cell membrane (membrane potential maintained at −45 mV using the Voltage Clamp mode), without pre-treatment (B_i, B_ii) or after pre-treatment (C) for 30 min with 1 mM of methyl-β-cyclodextrin (MβCD). The corresponding average delays (D) before current changes were detected, and average amplitude of the detected current changes (E), and distribution amplitude (F) of the observed current changes. Grey and black bars denote experiments performed using ET alone (mean from 34 recordings) or after pre-treatment with MβCD (mean from 16 recordings) respectively.</p

Membrane current induced by ET in granule cells.

<p>Granule cells were maintained under voltage clamp using the whole cell configuration. A, a recording taken from a series of 15 independent experiments (granule cells hold at −75 mV) during which slices were preincubated for 10 min with TTX (10⁻⁵ M), TEA (1 mM), 4-AP (2 mM), CNQX (10⁻⁵ M) and bicuculline (10⁻⁵ M) before application of ET (10⁻⁷ M, arrow). Note the abrupt large inward current step that manifests action of ET on the membrane characteristics. B, During the course of the same series of experiments, membrane holding potential was changed from −75 mV to −115 mV, followed by depolarizing ramps from −115 mV to −15 mV, before returning to −75 mV. This paradigm was performed before application of ET and after the toxin had induced an abrupt change in the whole cell current, as illustrated in A. Typical currents (before: grey; after ET: black) are shown. C) Currents traces were pooled under control (before ET) or after ET and averaged, to build the I =  f(V) relationship.</p

ET stains granule cells and oligodendrocytes but not astrocytes or nerve endings.

<p>A–D: column i: ET-staining (green), column ii: specific cell-marker immunoreactivity (red) and DRAQ5 DNA signal (cyan), column iii: merge of the ET and cell-marker immunoreactivities. In all experiments ET was applied for 5 min 10⁻⁷ M. A: ET and MAP-2, B: ET and synaptotagmin. C: ET and CNPase. D: ET and GFAP. Scale bars are 10 µm in A, B, D, and 50 µm in C.</p

ET staining in mouse cerebellum and hippocampus slices.

<p>A, Western blot of rabbit anti-epsilon toxin antibodies. Crude culture supernatant of <i>C. perfringens</i> type D was submitted to immunoblotting experiment using immunoaffinity purified rabbit anti-epsilon toxin (1∶2000). Molecular weight markers are indicated. The labeled band at 36 kDa corresponds to ET. ET-immuno staining obtained when tissue slices were incubated with (B, D–F) or without (C) ET (10⁻⁷ M) for 5 min before fixation. B, mouse cerebellum. Scale bar  = 500 µm. C, Immuno-staining obtained in absence of ET: cerebellar cortex was submitted to double immunostaining against ET and calbindin, which is expressed by the Purkinje cells in their dendrites (i.e. in the molecular layer), cell body, and axons (present in granule cells layer and white matter). Scale bar  = 40 µm. D, Magnification of a cerebellar lobule from (B); Scale bar  = 250 µm. C–D: WM: white matter, GL: granule cells layer, PCL: Purkinje cells layer, ML: molecular layer. E and F, ET-staining obtained in hippocampus: in the CA4 region (E) and in dentate gyrus (F); Scale bars are 50 and 100 µm, respectively. E–F: CA4 denotes presence of large pyramidal cells, MFL: mossy fiber layer, GCL: granule cell layer, MLH: molecular layer of hippocampus, *: capillary blood vessel.</p

ET stimulates excitatory and inhibitory synaptic transmission onto the Purkinje cells.

<p>A, right: spontaneous PSC detected in voltage-clamped Purkinje cells maintained at −60 mV, in absence (Cont) or 5 min after ET (10⁻⁷ M) application. The relative mean frequencies (Freq) and amplitudes (Ampl) of spontaneous EPSC (upper graph) or IPSC (lower graph), before (white bar) or 5–7 min after 10⁻⁷ M ET was added (black bar), <i>n</i> = 15 distinct experiments. B–D, same kind of measurements but after pre-treatment (B) with TTX (10⁻⁶ M for 10 min, <i>n</i> = 18, (C) with bicuculline (10⁻⁵ M for 5 min) to block the IPSC (<i>n</i> = 17), or (D) CNQX (10⁻⁵ M for 5 min) to block the EPSC (<i>n</i> = 18). The frequencies and amplitudes are presented as percent of control condition (i.e. without any treatment, white bars) or after pre-treatment (grey bars), and after subsequent application of ET (black bars). **: <i>p</i><0.01, *: <i>p</i><0.05, otherwise <i>n.s.</i> Same scale for all current traces.</p

Effect of ET on primary cultures of cerebellum highly enriched in granule cells.

<p>Ai, Aii and Aiii, primary cultures of cerebellar cells. Ai and Aii, 10⁻⁷ M ET was applied in PBS buffer (i.e. containing no Ca²⁺ ions) for 5 min after fixation of the cells, then the ET- (green) and GFAP-(red) immunoreactivities were revealed. Cell nuclei were labeled using DRAQ5 (cyan). GFAP-positive cells (astrocytes) are not labeled by ET. Aiii, same kind of experiment except that the culture was pretreated with methyl-β-cyclodextrin (MβCD, 1 mM for 30 min at 37°C) before fixation. B, the glutamate concentration (µM) in extracellular medium was determined from cerebellar primary cultures using the Amplex Red Assay. ET was applied for 10 min at the indicated final concentrations, under the following conditions: ET alone (red) ET after pretreatment with methyl-β-cyclodextrin (blue), ET in physiological medium containing no Ca²⁺ (10 mM EGTA; 0 mM CaCl₂; cyan). Data points are mean ± SEM determined from triplicate determinations. C, extracellular lactate-deshydrogenase (LDH) levels (using the Cytotoxicity Detection Kitplus assay). Data points are mean ± SEM (<i>n</i> = 4). In B and C, lower black horizontal lines denote basal levels (i.e. without ET), and upper dashed red lines denote the maximal values determined after granule cell lysis was induced by hypo-osmotic shock. D, averaged Fura-2 measurements of intracellular [Ca²⁺] from 25 granule cells, in absence (black, denoted as Basal)) or after addition of 10⁻⁷ M ET (red), with ET after preincubation of the cells with methyl-β-cyclodextrin (1 mM for 30 min, blue), or with medium containing 10 mM EGTA-0 mM Ca²⁺ (cyan). The green curve shows control cells preincubated only with methyl-β-cyclodextrin. Inner graph: relative [Ca²⁺] changes (% of control) determined 10 min after addition of ET in absence (red, n = 15) or presence of methyl-β-cyclodextrin (blue, n = 5) or in EGTA-0 Ca²⁺ medium (cyan, n = 14). ET <i>vs</i> control: <i>p</i><0.001, ET + MβCD <i>vs</i> control or MβCD alone: <i>n.s.</i></p