MOTIVATION

Understand how synaptic terminals release neurotransmitter and how synaptic vesicles are recycled

Hippocampal neurons n culture making synaptic contacts among themselves. The brighter spots are synaptic buttons stained with synaptopHluorin, a synaptic vesicle marker

APPROACH

To monitor exocytosis and endocytosis with maximal time and spatial resolution. We try to monitor single synaptic vesicles with a miliseconds time resolution, spatial of nanometers and a sensitivity in cell membrane capacaitance measurements of attofarads.

METHODS

High resolution cell membrane capacitance measurements

1. Single cell amperometry and cell membrane capacitance measurements

2. Total Internal Reflection Fluorescence Microscopy (TIRFM)

3. Stable transfection of cultured cells with fluorescently labelled proteins

BACKGROUND

We study single vesicle exocytosis and endocytosis with high resolution electrophysiological methods. Millisecond time resolution is achieved by measuring the cell membrane capacitance with the patch clamp technique and amperometry. The combination of these two methods have been very useful to determine the fine details of vesicle fusion at the level of single vesicles; from observing how the cell membrane surface area increases as the vesicle membrane collapses into the plasma membrane to monitor the time course of release of secretory products to the extracellular space..

The data obtained in our lab in peritoneal mast cells cells several years ago revealed that during transient vesicle fusion there is release of the vesicle contents to the extracellular space. These results showed that the flicker observed in 1984 by Fernandez, Neher and Gomperts were actually functional, in contrast to what was believed by the time. We demonstrated a new mechanism of release during granule exocytosis, i.e. a secretory vesicle could fuse to the plasma membrane by the opening of a fusion pore and through that small opening release part or all of the vesicle contents to the extracellular space. Our results revived a debated held for three decades about the cellular mechanism underlying Quantal Transmitter Release. In the late 70s and early 80s two different hypothesis were propose

Classical hypothesis of transmitter release by John Heuser and Thomas Reese

The classical and widely accepted mecanism for reuse of synatic vesicles is that upon arrival of an action potential to the presynaptic terminal and entry of calcium via voltage gated channels, a single synaptic vesicle fuses with the presynaptic membrane releasing its quantum of neurotransmitter to the synaptic cleft. After fusion the vesicle membrane collapses into the presynaptic membranes. The excess of presynaptic membrane is restored by internalization of membrane in a lateral position to the active zone by clathrin-mediated endocytosis.

Alternative hypothesis by Bruno Ceccarelli et al.

The alternative hypothesis of Ceccarelli et al. proposes that most of transmitter relese occurs by a transient fusion of the synaptic vesicle with the presynaptic membrane. Full collapse of vesicle membrane into the presynaptic membrane only occurs after the snaptic vesicle has been reused and reloaded several times of neurotransmitter.