Invasive behaviour and depolarization effect of Pseudomonas fluorescens on rat cerebellar granule neurons

Previous studies have shown that Pseudomonas fluorescens exerts cytotoxic effects on neurons and glial cells. In the present work, we investigated the time course effect of Pseudomonas fluorescens MF37 and of its lipopolysaccharide (LPS) on cultured rat cerebellar granule neurons. The kinetics of binding of P. fluorescens to cerebellar granule neurons is identical to that of cortical neurons but the binding index is lower, suggesting the presence of a reduced number of binding sites. As demonstrated by measurement of the concentration of nitrites in the culture medium, P. fluorescens induces a rapid stimulation (3 h) of the nitric oxide synthase (NOS) activity of the cells. In contrast, LPS extracted from P. fluorescens requires a long lag phase (24 h) before observation of an activation of NOS. Measurement of the resting membrane potential of granule neurons showed that within 3 h of incubation, there was no difference of effect between the action of P. fluorescens and that of its LPS endotoxin. Two complementary approaches allowed us to demonstrate that P. fluorescens MF37 presents a rapid invasive behaviour, suggesting a mobilisation of calcium in its early steps of action. The present study reveals that P. fluorescens induces the sequential activation of a constitutive calcium dependent NOS and that of an inducible NOS activated by LPS. Ours results also suggest that P. fluorescens cytotoxicity and invasion are not mutually exclusive events. Key Words: Cytotoxicity, Lipopolysaccharide, Patch-clamp, Invasion, Pseudomonas fluorescens Afr. J. Clin. Exper. Microbiol. Vol.6(1) 2005: 1-13