Amperometric Measurement of Vesicular Exocytosis of Neurotransmitters: Deciphering its Biological and Physicochemical Meaning

Christian AMATORE

Ecole Normale Supérieure. Département de Chimie, Paris. http://helene.ens.fr/w3amatore/

Living cells exchange information through the emission of chemical messengers. The importance of such messengers has been widely recognized by biologists. However what is less understood is how these chemical messengers are released by the cell in its outer-cytoplasmic fluids. This difficulty is easily understood when one becomes aware that most of these releases occur in the atto- or femtomole ranges which prevents the use of classical analytical methods. We wish to show here that ultramicroelectrodes may prove extremely useful for monitoring such events.

In this lecture we will be concerned by exocytosis of neurotransmitters although similar method can be applied to investigate oxidative stress cellular bursts. The target cells are chromaffin cells which are located above kidneys. These cells produce the initial adrenaline burst which induces fast body reactions; they are used in neurosciences as standard models for synaptic exocytosis by cathecolaminergic neurons. Prior to exocytosis, adrenaline is contained in highly concentrated solutions into a gel matrix packed into small vesicles dispersed in the cytoplasm near the cell membrane. Stimulation of the cell by divalent ions induces the fusion of the vesicles membranes with that of the cell and hence the release of the intravesicular content into the outer-cytoplasmic region.

Electrochemical data permit to describe the whole process of exocytosis with a precision that has never been achieved before by patch-clamp techniques. This enables to investigate kinetically these events and conclude upon the physicochemical origin of the individual factors which govern vesicular release. Based on this analysis, one may propose a first-time explanation for the biologically known relationship between size of an exocytotic vesicle and type of release : mostly through pore-release for vesicles less than 25-30 nm radius and containing less than 10,000-20,000 neurotransmitter molecules ; mostly through full-fusion release for larger vesicles.