Effect of calcium on nicotine-induced current expressed by an atypical alpha-bungarotoxin-insensitive nAChR2

Two distinct native alpha-bungarotoxin (alpha-Bgt)-insensitive nicotinic acetylcholine receptors (nAChRs), named nAChR1 and nAChR2, were identified in the cockroach Periplaneta americana dorsal unpaired median (DUM) neurons. They differed in their electrophysiological, pharmacological properties and intracellular regulation pathways. nAChR2 being an atypical nicotinic receptor closed upon agonist application and its current-voltage relationship resulted from a reduction in potassium conductance. In this study, using whole-cell patch-clamp technique, we demonstrated that calcium modulated nAChR2-mediated nicotine response. Under 0.5 microM alpha-Bgt and 20 mM d-tubocurarine, the nicotine-induced inward current amplitude was strongly reduced in the presence of intracellularly applied BAPTA or bath application of calcium-free solution. In addition, using cadmium chloride, we showed that nicotine response was modulated by extracellular calcium through plasma membrane calcium channels. Moreover, extracellular application of caffeine and thapsigargin reduced nAChR2-mediated response. Together these experiments revealed a complex calcium-dependent regulation of nAChR2

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