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.

+)-evoked phospholipase D1 (PLD1) activation and blocks the formation of phosphatidic acid at the plasma membrane. We identify βPix as the guanine nucleotideexchange factor integrating Rac1 activation to PLD1 and the exocytotic process. Finally, we show that the presence of the scaffolding protein Scrib at the plasma membrane is essential for βPix/Rac1-mediated PLD1 activation and exocytosis. As PLD1 has recently emerged as a promoter of membrane fusion in various exocytotic events, our results define a novel molecular pathway linking a Rho GTPase, Rac1, to the final stages of Ca 2+ -regulated exocytosis in neuroendocrine cells. Supplementary material available online at

Deletion of both alleles of the P ⁄ Q-type Ca 2+ -channel Ca v 2.1(a 1A ) subunit gene in mouse leads to severe ataxia and early death. Using cerebellar slices obtained from 10 to 15 postnatal days mice and cultured for at least 3 weeks in vitro, we have analysed the synaptic alterations produced by genetically ablating the P ⁄ Q-type Ca 2+ -channels, and compared them with the effect of pharmacological inhibition of the P ⁄ Q-or N-type channels on wild-type littermate mice. Analysis of spontaneous synaptic currents recorded in Purkinje cells (PCs) indicated that the P ⁄ Q-type channels play a prominent role at the inhibitory synapses afferent onto the PCs, with the effect of deleting Ca v 2.1(a 1A ) partially compensated. At the granule cell (GC) to PC synapses, both N-and P ⁄ Qtype Ca 2+ -channels were found playing a role in glutamate exocytosis, but with no significant phenotypic compensation of the Ca v 2.1(a 1A ) deletion. We also found that the P ⁄ Q-but not N-type Ca 2+ -channel is indispensable at the autaptic contacts between PCs. Tuning of the GC activity implicates both synaptic and sustained extrasynaptic c-aminobutyric acid (GABA) release, only the former was greatly impaired in the absence of P ⁄ Q-type Ca 2+ -channels. Overall, our data demonstrate that both P ⁄ Q-and N-type Ca 2+ -channels play a role in glutamate release, while the P ⁄ Q-type is essential in GABA exocytosis in the cerebellum. Contrary to the other regions of the CNS, the effect of deleting the Ca v 2.1(a 1A ) subunit is partially or not compensated at the inhibitory synapses. This may explain why cerebellar ataxia is observed at the mice lacking functional P ⁄ Q-type channels.