Nervous Conduction of Excitation Without Action Potentials
3 Oct, 2007 12:08 pm
The functioning of neuronal networks involves the conduction of excitation along the axon which is the wire-like part of the neuron. This excitation was hitherto held to rely only on an electric phenomenon, the action potential, triggered by transient modifications in the concentration of ions from each side of the membrane. However, a recent study in the peripheral nervous system has established that in contradiction of one of the central dogmas in Neurosciences, excitation can be conducted by molecules without electrical charges. This discovery opens up new perspectives for research in neuronal functioning from a fundamental and clinical viewpoint.
This advance was made possible by the use of an integrated physiological model in the mammal. It is an in vitro preparation consisting of the stomach and duodenum connected by nervous fibres to a peripheral nervous centre: the coeliac plexus. When the gastric mechanoreceptors are activated, the excitation is conducted to the coeliac plexus where it activates neurons which inhibit the duodenal motility. At the beginning of the nineties, using neuropharmacological techniques, we made a surprising discovery: the afferent and efferent messages organizing this gastroduodenal inhibitory (GIR) reflex along the nervous fibres are independent of action potentials (Mazet et al, 1993). We then showed that the neurotransmitter released in the coeliac plexus to activate the neurons inhibiting the duodenum was gaseous, nitric oxide (NO), (Quinson et al, 1999). At this stage of our research, the nature of the mechanism conducting the excitation was unknown. The only parameter we had was the velocity of this atypical conduction of excitation, about 1cm/min. This is intermediate between the lowest speed for action potentials (0.1m/sec) and the highest speed for axonal flows (40 cm/day). So we proposed the hypothesis of the activation of a second messenger sequence. It quickly appeared that the demonstration of this hypothesis required the use of complementary techniques and we started a collaborative study with 2 biochemical teams and a technical workshop, thus regrouping researchers from several French universities (Paul Cezanne and Paul Sabatier) and research institutions (CNRS, INSERM and INRA).
In the course of our research, we first showed that during the GIR there is an increase along the nerve fibres in the concentration of a particular membrane lipid, ceramide. This lipid belongs to the sphingolipid family, so named because at their discovery at the end of the nineteen century they were as enigmatic as the sphinx. So we focused our research on this molecule which was of great interest to us. We know today that ceramide is not only a membrane constituent but also a second messenger (which can activate within the cell cytoplasm other molecules involved in various cell functions). We then showed that superfusion of the nerve fibres with an agonist of ceramide crossing the membrane triggers an inhibition of duodenal motility mimicking that observed during GIR. This result was obtained even when the action potentials were blocked by a toxin (tetrodotoxin). GIR and the increase in ceramide concentration were blocked by superfusion of the nerve fibres with a specific inhibitor of the sphingomyelinase, the enzyme which products ceramide from sphingomyeline.
Conversely, superfusion of the nerve fibres with bacterial sphingomyelinase triggers an inhibition of duodenal motility and an increase in the ceramide concentration.
In fact, sphingolipids are mainly located in specialized areas of the membrane called microdomains or lipid rafts. These areas are involved in many key events of cell functioning and behave as signalling platforms. We fist provided evidence for the presence of rafts in the nerve fibres organizing GIR. They represent low density fractions extracted from membranes; they are also enriched in molecules such as cholesterol, specific proteins (annexin II, tubulin) and sphingolipids (GM1 ganglioside). We then established that raft integrity was necessary to the conduction of excitation without action potentials. Indeed, superfusion of the nerve fibres with a drug disrupting the rafts (β-Methyl-Cyclodextrin) inhibited GIR and prevented the increase in ceramide concentration.
We then looked for the second messengers activated in the cytoplasm by ceramide production. We identified calcium released from the internal stores, NO and cyclic guanosine monophosphate (cGMP). Lastly, we showed that the sequence, ceramide, calcium, NO and cGMP is activated in cascade along the nerve fibre which explains the conduction of this atypical excitation.
These findings were published online on July 18 2007 in the journal PLoS ONE.
So the neurons appear to be be fitted with two modes of conduction of excitation, fast and slow involving respectively action potentials and second messengers. The first mode is suited for rapid functioning and the other for slower regulations. Our study opens up new perspectives for research in the fundamental and clinical fields of neuronal functioning.
Our research is funded by Paul Cezanne University, Paul Sabatier University, CNRS, INSERM and INRA.
Fasano C., et al, Neuronal Conduction of Excitation without Action Potentials Based on Ceramide Production. PLoS ONE 2007; 2(7): e612. doi:10.1371/journal.pone.0000612