Effects of staurosporine on exocytosis and endocytosis at frog motor nerve terminals.

Observations of the dynamic staining and destaining of FM1-43 in frog motor nerve terminals (Henkel and Betz, 1995) suggested that staurosporine might shorten the interval between exocytosis and endocytosis, inducing a "kiss and run" mode of exocytosis and endocytosis. We tested this hypothesis by using FM1-43 imaging (to measure the time course of FM1-43 endocytosis), intracellular recording of evoked synaptic potentials (to measure acetylcholine release), and electron microscopy (to examine synaptic vesicle distribution). Staurosporine reduced FM1-43 uptake during but not after a tetanus, increased the speed of end plate potential (EPP) amplitude rundown, and greatly slowed the recovery from synaptic depression. Ultrastructural observations showed pronounced vesicle depletion near active zones after tetanic stimulation in staurosporine-treated preparations. These results suggest that staurosporine acted primarily to impair mobilization of synaptic vesicles during tetanic stimulation.

Resting potential of rat cerebellar granule cells during early maturation in vitro.

The survival of rat cerebellar granule cells maintained in vitro is enhanced by a KCl-enriched medium. This effect is classically interpreted as resulting from a higher cytosolic calcium concentration. This implies the presence of voltage-dependent Ca2+ channels and a membrane potential that can respond to changes in external K+. Since previous studies cast a doubt on these two conditions, we reinvestigated the resting membrane potential and Ca2+ influxes in rat cerebellar granule neurones during the first week in vitro using a fluorescence imaging approach. Membrane potential was assessed with the fluorescent dye bis-oxonol, and intracellular free calcium with Fura-2. Resting potential was shown to progressively decrease from -40 mV at the first day in vitro to -60 mV at day 7. At all times in culture, as early as day 0, cells were depolarized when external KCl concentration was increased from 5 to 30 mM. This depolarization resulted in an increased cytosolic calcium concentration due to Ca2+ influx through L-type and N-type voltage-activated Ca2+ channels, functional at day 0. Gross estimations of the permeabilities of Na+ and Cl- were obtained at various times in culture by measuring the changes in resting potential brought about by a reduction of their external concentration. A progressive increase of the relative permeability to K+ ions seems to underlie the evolution of the resting potential with time.

Forskolin's structural analogue 1,9-dideoxyforskolin has Ca2+ channel blocker-like action in rat cerebellar granule cells.

Forskolin, routinely used as a specific activator of the cAMP pathway, is also a blocker of various ionic channels in a cAMP-independent way. We investigated, in rat cerebellar granule cells in culture, the effects of forskolin and its structural analogue 1,9-dideoxyforskolin on Ca2+ entry. Changes in cytosolic free Ca2+ concentration ([Ca]i) were monitored using fura-2 microfluorimetry. The increase in [Ca]i observed in response to membrane depolarization by 30 mM KCI was reduced by 20% in the presence of 100 microM forskolin, and by 71% with the same concentration of 1,9-dideoxyforskolin. A dose-response curve for 1,9-dideoxyforskolin gave an estimated IC50 of 54 microM. Additional experiments using the patch-clamp technique showed that 100 microM 1,9-dideoxyforskolin inhibit voltage-activated Ca2+ currents by 63%, although forskolin had no significant effect in the same conditions. This blocking effect of 1,9-dideoxyforskolin is not specific of a given Ca2+ channel type.

Increased axonal regrowth of lesioned rat sciatic nerve by veratrylguanidine methane sulfonate.

Neurotrophic factors appear as essential factors for normal development and repair of the nervous tissue. Veratrylguanidine methane sulfonate, has been shown to induce important neurite outgrowth of cultured dorsal root ganglia isolated from newborn rats. Its action was similar to that of NGF and was found to be additive to that of NGF. In order to see if this compound was able to stimulate axonal growth in adult animals, we examined the effect of this substance on the regeneration of the lesioned sciatic nerve. Using histochemical, immunohistochemical and ultrastructural studies, it is shown that a single intraperitoneal injection of veratrylguanidine methane sulfonate significantly increases the axonal growth during repair of the adult rat sciatic nerve. The efficiency of this substance is explained by its good targeting and long life time in the sciatic nerve.

Neurite outgrowth of neurons of rat dorsal root ganglia induced by new neurotrophic substances with guanidine group.

In order to search for compounds mimicking the neurotrophic activities, a series of substances possessing the guanidine group of isaxonine (2-isopropylamino-pyrimidine), were synthesized and tested for their ability to induce neurite outgrowth in cultures of explants of rat dorsal root ganglia. It is reported that several of these compounds, at concentrations ranging from 10(-5) to 10(-7) M, showed important effects on neurite outgrowth in vitro, similar to that obtained with the nerve growth factor NGF. These effects were found to be additive to that of NGF.