Michel Simonneau

Expression of voltage-dependent sodium and transient potassium currents in an identified sub-population of dorsal root ganglion cells acutely isolated from 12-day-old mouse embryos.

The electrophysiological properties of a subset of dorsal root ganglion (DRG) neurons microdissected from 12-day-old (E12) mouse embryos and acutely isolated were analyzed as soon as 3 after their isolation. Two classes of neurons were defined according to their mean diameter. The larger diameter class was examined in this study. They display uniform cytoskeletal properties with co-expression of vimentin and neurofilament triplet proteins. Patch-clamp methods also revealed a homogeneous and limited repertoire of ionic channels that included (1) a TTX-sensitive Na+ current whose properties are similar to that reported in mature mammalian neurons, and (2) two types of K+ currents that can be compared with the delayed rectifier (Ik) and the transient (Ia) potassium currents found in other mammalian preparations. It may be possible to use this in vitro model to examine the development of new types of currents, such as Ca2+ currents during neuronal growth and differentiation.

Dihydropyridines interact with calcium-independent potassium currents in embryonic mammalian sensory neurons.

Early embryonic sensory neurons have two K currents resembling delayed rectifier and transient K currents of mature neurons. However, in contrast to those of adult neurons, the embryonic currents can hardly be separated either by electrophysiological or pharmacological methods, limiting their characterisation at these developmental stages. Using the whole-cell recording technique, we found that dihydropyridines (DHPs) inhibit the non-inactivating component of the Ca-independent K currents of 13-day mouse embryo dorsal-root ganglion (DRG) cells. The inhibitory effect of nicardipine began around 0.5 μM and was nearly complete at 5 μM while Na currents were not altered. This effect was reversible and voltage-dependent. The same results were obtained using another DHP Ca antagonist, nimodipine, whereas Bay K 8644, a DHP Ca agonist, had no effect. Kinetic properties of the DHP-insensitive K current have been described and compared with those of transient K currents found in differentiated neurons. These results suggest that both Ca and K channels have DHP sites, possibly homologous, at this developmental stage. The DHP inhibition of Ca-independent K channels provides a new tool with which to study K channels both at a molecular level and during DRG development.

Storage and growth of neuroblastoma cells immobilized in calcium-alginate beads

SummaryMouse neuroblastoma cells (N18) were immobilized in calcium-alginate gel beads. Under standard culture conditions (37° C; 5% CO2), cell growth was observed inside the beads. The number of cells increased threefold during 7 days of culture with cell division and differentiation visualized by electron microscopy. Cell properties maintained after short-term storage (2–3 days at 4° C) included: (i) properties of voltage-dependent ionic channels tested by patch-clamp electrophysiological techniques; (ii) expression of cell-adhesion membrane proteins tested by immunohistochemistry (iii) morphological differentiation obtained by depletion of foetal calf serum in culture medium. The advantages of such an immobilization technique as applied to neurone cells are discussed.

Both upstream and intragenic sequences of the human neurofilament light gene direct expression oflacZ in neurons of transgenic mouse embryos

Initial expression of the neurofilament light gene coincides with the appearance of postmitotic neurons. To investigate the molecular mechanisms involved in neuron-specific gene expression during embryogenesis, we generated transgenic mice carrying various regions of the human neurofilament light gene (hNF-L) fused to thelacZ reporter gene. We found that 2.3 or 0.3 kb of the hNF-L promoter region directs expression oflacZ in neurons of transgenic embryos. Addition of 1.8 kb hNF-L intragenic sequences (IS) enlarges the neuronal pattern of transgene expression. The 2.3-kb hNF-L promotelacZ-IS construct contains all regulatory elements essential for both spatial and temporal expression of the hNF-L gene during embryogenesis and in the adult. The use of a heterologous promoter demonstrated that the 1.8-kb hNF-L intragenic sequences are sufficient to direct the expression oflacZ in a NF-L-specific manner both temporally and spatially during development and in the adult. We conclude that these hNF-L intragenic sequences containcis-acting DNA regulatory elements that specify neuronal expression. Taken together, these results show that the neurofilament light gene contains separate upstream and intragenic elements, each of which directslacZ expression in embryonic neurons.

Cell type-specific expression of the mouse peripherin gene requires both upstream and intragenic sequences in transgenic mouse embryos

Peripherin is a neuron-specific type III intermediate filament protein expressed in well-defined populations of neurons projecting towards peripheral targets. To investigate the molecular mechanisms by which a gene is expressed in a specific subset of neurons, we used a transgenic approach in order to define peripherin gene sequences that are necessary for cell-type specific expression. Transgenic mice carrying different various genomic regions of the mouse peripherin gene fused to the Escherichia coli lacZ reporter gene were generated. We used three different peripherin/lacZ constructs containing either 5.8 kb upstream sequences, or both 5.8 kb upstream and 1.1 kb intragenic sequences, or 1.1 kb intragenic sequences associated with an heterologous promoter. Analysis of lacZ gene expression in transgenic mouse embryos showed that cell type-specific expression of the mouse peripherin gene requires both upstream and intragenic sequences. Analysis of transgenic mouse lines expressing the construct containing both upstream and intragenic sequences showed that this transgene contains all regulatory elements essential for both spatial and temporal expression of the mouse peripherin gene during embryogenesis. Furthermore, lacZ+ positive cells isolated from these transgenic lines by fluorescence-activated cell sorting (FACS) can be stained with a peripherin antibody, demonstrating that the transgene containing both upstream and intragenic sequences is expressed in peripherin neurons. These mouse peripherin upstream and intragenic sequences can now be used to identify cis-acting regulatory elements and transcription factors involved in peripherin gene regulation.

Scorpion venom inhibits selectively Ca2+-activated K+ channels in situ

Using patch‐clamp techniques, a study was made of the component of Leiurus quinquestriatus scorpion venom which caused a blockade of one class of membrane potassium channels, the calcium activated potassium (BK) channels. This blockade was obtained on channels in their native lipidic environment and was specific for this class of channels as other types of potassium channels were not affected by this venom.

Calcium alginate immobilization of mammalian neurons: A potential tool to purify ligands of neuronal membrane proteins

We report here improved immobilization conditions which permitted (i) to immobilize mouse neuroblastoma cells in calcium alginate beads, (ii) to test the functions of using patch clamp techniques and (iii) to quantitatively analyze ligand interactions with voltage-dependent sodium channels in neurons immobilized inside alginate beads. These results qualify this immobilization technique as a biotechnological tool to isolate and/or purify ligands of neuronal membrane proteins.

Large unit conductance voltage chloride channels are expressed in mouse neural crest cells and embryonic dorsal root ganglion cells

The early expression of voltage-activated chloride channels of large unitary conductance (450 pS in symmetrical 140 mM KCl) was demonstrated using patch-clamp techniques in two preparations: (i) neural crest cells isolated from 9-day-old (E9) mouse embryos and (ii) acutely isolated dorsal root ganglion cells isolated from E12 mouse embryos. Properties of these ionic channels have been analyzed using single channel recordings and the group mean of these single channels.

Alginate immobilized mammalian neurons: a potential tool to isolate new neuronal ligands.

We developed improved immobilization conditions which permitted (i) to immobilize neuroblastoma cells (N18) in calcium-alginate gel beads, (ii) to test the function of ionic channels using patch-clamp electrophysiological techniques and (iii) to quantitatively analyze ligand interactions with voltage-dependent sodium channels in neurons inside the beads. These results qualify this immobilization technique for the isolation and/or purification of ligands specific for neuronal cells.

Environmental signals and neural crest cells.

Cell lineage analysis in both the central and peripheral nervous system of vertebrates has revealed that many neural progenitor cells are multipotent. These observations have raised the general issue of when and how such multipotent progenitors generate their various differentiated progeny. The environment of these progenitors controls the cell lineage decisions in the neural crest. This review considers the roles of the environmental signals in the context of the development of several different neural crest-derived lineages.