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

Pre and Post Synaptic NMDA Effects Targeting Purkinje Cells in the Mouse Cerebellar Cortex

N-methyl-D-aspartate (NMDA) receptors are associated with many forms of synaptic plasticity. Their expression level and subunit composition undergo developmental changes in several brain regions. In the mouse cerebellum, beside a developmental switch between NR2B and NR2A/C subunits in granule cells, functional postsynaptic NMDA receptors are seen in Purkinje cells of neonate and adult but not juvenile rat and mice. A presynaptic effect of NMDA on GABA release by cerebellar interneurons was identified recently. Nevertheless whereas NMDA receptor subunits are detected on parallel fiber terminals, a presynaptic effect of NMDA on spontaneous release of glutamate has not been demonstrated. Using mouse cerebellar cultures and patch-clamp recordings we show that NMDA facilitates glutamate release onto Purkinje cells in young cultures via a presynaptic mechanism, whereas NMDA activates extrasynaptic receptors in Purkinje cells recorded in old cultures. The presynaptic effect of NMDA on glutamate release is also observed in Purkinje cells recorded in acute slices prepared from juvenile but not from adult mice and requires a specific protocol of NMDA application

Mécanisme d'action de la toxine epsilon de Clostridium perfringens dans le système nerveux central

La toxine epsilon ( ) produite par les bactéries Clostridium perfringens de type B et D est responsable de graves entéro-toxémies chez les ovins et bovins. Elle fait partie des 5 agents létaux les plus puissants. Facile à produire sous forme de protéine recombinante elle a été identifiée comme un agent potentiellement utilisable dans des actions bio-terroristes. créée des lésions principalement dans les reins et le cerveau des animaux exposés. Dans le rein, agit en se liant et en s insérant dans la membrane plasmique des cellules pour y former des pores perméables aux cations, qui seraient responsables de la mort cellulaire. Les dommages cérébraux provoqués par sont localisés, symétriques et bilatéraux. Ceci suggère que la toxine a une action spécifique directement sur certains types cellulaires du tissu nerveux.L objectif de mon doctorat a été de déterminer si a une action neuronale spécifique et d en comprendre les mécanismes. Mon travail a porté sur la caractérisation de l action de T sur les neurones et les cellules gliales du cervelet. La première étape fut de compléter la caractérisation de ce modèle dans le cadre des études électrophysiologiques. Nous avons notamment étudié le rôle des canaux calcium dépendants du potentiel de type N et P/Q dans le contrôle de la neurotransmission aux synapses excitatrices et inhibitrices dans les tranches organotypiques.L étude de la liaison de ET au tissu cérébral a révélé un très fort marquage de l hippocampe, du cervelet, et de la substance blanche. Dans le cervelet, nos travaux ont permis de démontrer que ET lie spécifiquement les cellules des grains et les oligodendrocytes. A l inverse les neurones inhibiteurs et les astrocytes ne fixent pas ET.L application de ET sur des tranches organotypiques de cervelet provoque une augmentation massive de l activité synaptique spontanée reçue par la cellule de Purkinje. Des approches pharmacologiques nous ont permis de conclure que cette augmentation d activité était exclusivement due à une action de ET propagée depuis le corps cellulaire des cellules des grains. Dans les cellules des grains, ET induit une dépolarisation très rapide des cellules suffisante pour déclencher des bouffées de potentiels d actions. Des approches biochimiques nous ont également permis de vérifier que les cellules des grains libèrent de grandes quantités de glutamate après application de ET.La question qui découle de ces résultats était de savoir si la dépolarisation des cellules des grains est la conséquence de la formation du pore de ET, ou s il s agit plutôt de l activation de voies de signalisations intracellulaires. Pour aborder cette problématique, nous avons mesuré directement le courant transmembranaire des cellules des grains lors d une application de toxine. Cette approche nous a permis d observer l apparition très rapide d une conductance transmembranaire dans ces cellules, confortant l hypothèse de la formation d un pore. Ces résultats n excluent cependant pas complètement l implication de voies de signalisation intracellulaires. En effet, des expériences d imagerie calcique nous ont permis d observer une augmentation de calcium après l application de ET sur des cellules des grains en culture bien que le pore formé par ET ne soit pas perméable au calcium. De plus, cette entrée calcique a pu être diminuée par l utilisation d antagonistes des récepteurs NMDA. Ces résultats suggèrent qu il existe plusieurs modes d action pour ET qui peuvent agir en parallèle dans les cellules.L identification des neurones et des oligodendrocytes comme étant des cibles de ET apporte une nouvelle vision des mécanismes d action de la toxine. La spécificité de ET pour certains types cellulaires et son activité très puissante sur les neurones permet d expliquer le pouvoir létal de cette toxine, qui est cent fois supérieur à celui des autres toxines du même type.Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin ranking among the 10 most potent poisonous substances. ET-intoxicated animals express very severe neurologic disorders associated with marked increase in neurotransmitter (including glutamate) release and neuronal cell death. These effects are generally proposed to result from vasogenic brain edemas. However the possibility that ET directly attacks the neurons has been postulated, albeit debated. We have analyzed the effect of ET on cerebellar preparations. Using immune-straining techniques we found that ET binds to granule layer and markers revealed that the toxin binds to the cell body of the granule neurons and to oligodendrocytes, but not astroglial cells or nerve ending. When applied onto granule cell primary culture, ET induced intracellular Ca2+ rise and fast cell fragmentation, which appear related to its pore-forming activity. Electrophysiological investigations and pharmacological analysis of the ET effects on neurotransmission using cultured cerebellar slices revealed that ET depolarizes directly granule cells of glutamate release. ET had no direct action on nerve terminals and no membrane effects induced by ET in granule cells maintained in the nerve tissue, suggest that the membrane depolarization induced by ET may be unrelated to its poreforming activity. These data shed another light on the etiology of the neurologic disorders caused by ET.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Repetitive applications of NMDA promote the facilitatory effect on mEPSC frequency.

<p>A. Current traces showing mEPSCs before (left) and after (right) a second application of NMDA. B (same cell as in A). Histogram representing the number of mEPSCs as a function of recording time (bin 10 s). During the short horizontal bars NMDA is applied at 10⁻⁵ M (bars 1 and 2) and at 5×10⁻⁶ M. (bar 3). C. Cumulative plots of inter-event intervals in control conditions (black curve) in the presence of NMDA after the first application (1, red curves) and second application (2, blue curve).</p

NMDA increases the frequency of mEPSCs in Purkinje cells recorded in a young (P18) slice culture.

<p>Upper record: currents traces obtained at −40 mV in the presence of TTX and Gabazine before (left) and after NMDA application (right). Transient inward currents represent mEPSCs. Lower graphs: (same cell as in A) cumulative probability plots of inter-event intervals (left) under control conditions (black trace) and in the presence of NMDA (red trace); amplitude histogram distribution under control conditions (middle) and in the presence of NMDA (right). In both cases the amplitude distribution was fitted using a gaussian function with a peak value indicated on each graph. Note that in the presence of NMDA the distribution of inter-event intervals is shifted towards shorter intervals whereas the amplitude distribution is almost unchanged.</p

A postsynaptic response to NMDA in old Purkinje cells.

<p>A. NMDA-induced inward current recorded in a 45 day old Purkinje cell in the presence of the AMPA receptor antagonist NBQX. B. (left) represents the number of Purkinje cells responding to NMDA with an inward current as a function of their age. B (right) mean amplitude of the NMDA inward current (10⁻⁵ M, for 30 s). C, single NMDA channel recordings in outside-out patches, left: current traces illustrating the NMDA channels, middle: I/V plots obtained with two outside-out giving a conductance close to 45 pS, right: voltage dependent blockade of the NMDA channels.</p

The effect of NMDA on mEPSC frequency is seen in juvenile but not adult mice.

<p>A. Histogram representing the number of mEPSCs recorded in a Purkinje cell from a slice prepared from a P40 mouse as a function of recording time (bin 10 s). During the horizontal bars NMDA is applied at 5×10⁻⁴ Note the absence of NMDA effect on mEPSC frequency. B. The effect of NMDA on mEPSC mean frequency recorded in Purkinje cell from acute slices prepared with juvenile (left, 7 cells) and adult mice (right, 5 cells). A representation with box plots indicating the mean values (thick line) and median values (thin line) has been adopted because of the non-normal distribution of frequency values. The bottom and the top of the box represent the 25^th and 75 th percentile respectively.</p

NMDA increases the frequency of mEPSCs in Purkinje cells recorded in acute slices prepared from P15–16 mice.

<p>A, B. Upper panels illustrate current traces recorded at −60 mV in the presence of TTX and Gabazine before (left) and after NMDA application (right). Lower panels show histograms representing the number of mEPSC as a function of recording time (bin 10 s). NMDA 10⁻⁴ M is applied as indicated by the horizontal bars. Note in A that the NMDA effect on mEPSC frequency is observed after the third application of NMDA, whereas in B the response is observed after a long-lasting (10 minute) application of NMDA.</p