Multiple pathways regulate the expression of genes encoding sodium channel subunits in developing neurons.

In primary cultures of fetal neurons, activation of sodium channels with either alpha-scorpion toxin or veratridine caused a rapid and persistent decrease of mRNAs encoding beta2 and different sodium channel alpha mRNAs. In contrast, beta1 subunit mRNA was up-regulated by sodium channel activation. This phenomenon was calcium-independent. The effects of activating toxins on mRNAs of different sodium channel subunits were mimicked by membrane depolarization. An important aspect of this study was the demonstration that cAMP also caused rapid reduction of alphaI, alphaII and alphaIII mRNA levels whereas beta1 subunit mRNA was up regulated and beta2 subunit mRNA was not affected. Sodium channel activation by veratridine was shown to increase cAMP immunoreactivity in cultured neurons, but alphaII mRNA down-regulation induced by activating toxins was not reversed by protein kinase A antagonists, indicating that this phenomenon is not protein kinase A dependent. The effects of cAMP and membrane depolarisation were antagonized by the PKA inhibitor H89. These results are indicative of the existence of multiple and independent regulatory pathways modulating the expression of sodium channel genes in the developing central nervous system.

Progress of the ITER equatorial vis/IR wide angle viewing system optical design.

The equatorial vis/IR wide angle viewing system is present in four ITER diagnostic equatorial ports. This instrument will cover a large field of view with high spatial and temporal resolutions, to provide real time temperature measurements of plasma facing components, spectral data in the visible range, information on runaway electrons, and pellet tracking. This diagnostic needs to be reliable, precise, and long lasting. Its design is driven by both the tokamak severe environment and the high performances required for machine protection. The preliminary design phase is ongoing. Paramount issues are being tackled, relative to wide spectral band optical design, material choice, and optomechanical difficulties due to the limited space available for this instrument in the ports, since many other diagnostics and services are also present. Recent progress of the diagnostic optical design and status of associated R&D are presented.

Internalization of voltage-dependent sodium channels in fetal rat brain neurons: a study of the regulation of endocytosis.

In fetal rat brain neurons, activation of voltage-dependent Na+ channels induced their own internalization, probably triggered by an increase in intracellular Na+ level. To investigate the role of phosphorylation in internalization, neurons were exposed to either activators or inhibitors of cyclic AMP- and cyclic GMP-dependent protein kinases, protein kinase C, and tyrosine kinase. None of the tested compounds mimicked or inhibited the effect of Na+ channel activation. An increase in intracellular Ca2+ concentration induced either by thapsigargin, a Ca(2+)-ATPase blocker, or by A23187, a Ca2+ ionophore, was unable to provoke Na+ channel internalization. However, Ca2+ seems to be necessary because both neurotoxin- and amphotericin B-induced Na+ channel internalizations were partially inhibited by BAPTA-AM. The selective inhibitor of Ca2+/calmodulin-dependent protein kinase II, KN-62, caused a dose-dependent inhibition of neurotoxin-induced internalization due to a blockade of channel activity but did not prevent amphotericin B-induced internalization. The rate of increase in Na+ channel density at the neuronal cell surface was similar before and after channel internalization, suggesting that recycling of internalized Na+ channels back to the cell surface was almost negligible. Pretreatment of the cells with an acidotropic agent such as chloroquine prevented Na+ channel internalization, indicating that an acidic endosomal/lysosomal compartment is involved in Na+ channel internalization in neurons.

Direct regenerationin vitro and transient GUS expression inMentha xpiperita.

Genetic transformation of peppermint is known to be very difficult essentially because of low efficiency regeneration. A regeneration protocol allowing 51% shooting frequency is proposed. Transient ß-glucuronidase expression and adjustment of selection pressure with kanamycin are also reported. The final retained method to attempt peppermint transformation is:Agrobacterium inoculation or biolistic treatment of the first apical leaves ofin vitro clones, regeneration in the dark with kanamycin (1 mg l(-1)) and 6-benzylaminopurine (2 mg l(-1)), followed by selection of regenerated shoots with 200 mg 1(-1) kanamycin.

Identification of an axonal determinant in the C-terminus of the sodium channel Na(v)1.2.

To obtain a better understanding of how hippocampal neurons selectively target proteins to axons, we assessed whether any of the large cytoplasmic regions of neuronal sodium channel Na(v)1.2 contain sufficient information for axonal compartmentalization. We show that addition of the cytoplasmic C-terminal region of Na(v)1.2 restricted the distribution of a dendritic-axonal reporter protein to axons. The analysis of mutants revealed that a critical segment of nine amino acids encompassing a di-leucine-based motif mediates axonal compartmentalization of chimera. In addition, the Na(v)1.2 C-terminus is recognized by the clathrin endocytic pathway both in non-neuronal cells and the somatodendritic domain of hippocampal neurons. The mutation of the di-leucine motif located within the nine amino acid sequence to alanines resulted in the loss of chimera compartmentalization in axons and of internalization. These data suggest that selective elimination by endocytosis in dendrites may account for the compartmentalized distribution of some proteins in axons.