Clostridium perfringens Epsilon Toxin Targets Granule Cells in the Mouse Cerebellum and Stimulates Glutamate Release

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca2+ rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons

Caractérisation des mécanismes de régulation intracellulaires des récepteurs de l'acétylcholine de type nicotinique résistants à l'a-bungarotoxine exprimés sur les dorsal unpaired median (DUM) neurones de la blatte Periplaneta americana

Les récepteurs de l'acétylcholine de type nicotinique (nAChRs) résistant à l'alpha-bungarotoxine exprimés sur les corps cellulaires de cellules neurosécrétrices (les DUM neurones) isolées du dernier ganglion abdominal de la blatte Periplaneta americana ont été étudiés au moyen des techniques de patch-clamp (configuration cellule entière) et de mesure de la concentration en calcium intracellulaire. Ces résultats mettent en évidence des caractéristiques entièrement nouvelles des nAChRs des insectes. Deux sous-types de récepteurs distincts (nAChR1 et nAChR2) ont été caractérisés. Ces deux nAChRs peuvent être séparés par leurs caractéristiques pharmacologiques, électrophysiologiques et par différents mécanismes de régulation intracellulaires (phosphorylation/déphosphorylation). La concentration en AMPc intracellulaire contrôle l'activité d'une protéine kinase (PKA) et d'une protéine phosphatase (PP1/2A) qui ont des effets antagonistes sur nAChR1. Le complexe calcium/calmoduline (CaM) module l'activité d'une adénylate cyclase et active une enzyme de type CaM kinase II qui potentialise la réponse de nAChR1 en partie par l'inhibition de la PP1/2A. Deux enzymes de la famille des PKC (PKC1 et PKC2) modulent nAChR1 de manière opposée et sont apparentées aux PKC " classiques " (PKC1) et " atypiques ". L'utilisation d'une sonde sensible au calcium, le fura 2, démontre que l'activation d'un récepteur de type muscarinique de sous-type M1 permet de d'augmenter la concentration en calcium intracellulaire et contrôle l'activation de ces PKC. Les effats d'un insecticide : l'imidaclopride, ont également été étudiés. Dans les conditions physiologiques, cette molécule active uniquement nAChR1 et son efficacité peut être modulée par ces processus intracellulaires de phosphorylation/déphosphorylation. L'insensibilité de nAChR2 vis-à-vis de l'imidaclopride s'explique par les caractéristiques fonctionnelles de ce récepteur. Le canal ionique de nAChR2 est perméable aux ions potassium et ouvert dans les conditions physiologiques, il se ferme en présence d'agoniste. Ces caractéristiques de nAChR2 suggèrent ainsi l'existence d'un nouveau type de récepteur ionotrope dont le canal ionique, ouvert au repos, est fermé lors de son activation.The a-bungarotoxin-resistant nicotinic acetylcholine receptors (nAChRs) expressed on the cell bodies of neurosecretory cells (DUM neurones) isolated from the terminal abdominal ganglia of the cockroach Periplaneta americana were studied using the patch-clamp technique (whole-cell recording configuration) and intracellular calcium imaging. These results demonstrate new characteristics of insect nAChRs.Two distinct nAChRs subtypes (nAChR1 et nAChR2) were characterised.These two receptors can be separated according to their pharmacological and electrophysiological properties and subtype-specific intracellular modulations (phosphorylation/dephosphorylation). First, the intracellular cAMP concentration controls both the activity of a protein kinase (PKA) and a protein phosphatase (PP1/2A) that modulate in opposite directions nAChR1. Second, the calcium/calmodulin complex (CaM) modulates adenylate cyclase and activates a CaM kinase II. This latter enzyme potentiates nAChR1 function partly through the inhibition of the PP1/2A. Third, we identified two distinct PKC that differentialy "up- and down-" regulate nAChR1 function (PKC1 and PKC2). These enzymes are related to the "classical" (PKC1) and "atypical (PKC2) PKC subtypes. Using the calcium-sensitive probe fura 2 we shown that the activation of M1 muscarinic receptor leads to an increase in intracellular calcium concentration and modulates PKC1 and PKC2 activities. The effects of the neonicotinoid insecticide imidacloprid were also studied. In physiological conditions, this compound only activates nAChR1 and its efficiency is affected by intracellular phosphorylation/dephosphorylation processes. The functional characteristics of nAChR2 can explain its insensivity to imidacloprid. The ionic channel of nAChR2 is permeable to potassium ions and open in physiological conditions. This channel closes following agonist application. nAChR2 might therefore represent a new kind of ionotropic receptor with an open channel in resting conditions that is closed when the receptor is activated.ANGERS-BU Lettres et Sciences (490072106) / SudocSudocFranceF

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 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