Extrasynaptic transmission: investigating synaptic vesicle fusion at non-conventional release sites in hippocampal neurons

Overview: The brain of a mammal is arguably the most sophisticated of all biological structures and scientists are very motivated to understand, in detail, how it works. When we talk about brain activity, the essence of this is the communication of information between individual brain cells, or 'neurons'. Although a great deal is known about this process, new experimental findings which add to our understanding of brain function are emerging all the time. This proposal will examine an important and only recently identified aspect of neuron-to-neuron communication which impacts on our knowledge of brain operation. To investigate this phenomenon, state-of-the-art methods will be employed which allow movements of molecules in living brain cells to be directly observed. Research like this adds greatly to our knowledge of how the brain works at the most fundamental level and could have important implications for understanding forms of brain disease or even inspiring the design of new and more sophisticated computer technology. Detail: Most neuron-to-neuron information transfer relies on release of a chemical 'transmitter' from a source neuron to a target neuron. Transmitter release is a complex process requiring lots of different types of specific molecules. These are concentrated at specialized sites in neurons known as presynaptic terminals, and the established view is that these terminals are therefore the only location within a brain cell where transmitter release can occur. Recently, however, researchers have found this idea to be inaccurate: in certain types of neuron, transmitter can be released at other 'non-specialized' sites within a source neuron. This is called 'extrasynaptic' release, meaning literally 'release away from the presynaptic terminal'. The mechanism allowing this process to occur, however, is not understood. The most likely explanation for how extrasynaptic release is achieved is that molecular machines needed for transmitter release can be readily moved from normal presynaptic terminals to new parts of the neuron. Confirmation of this idea, the manner in which it is controlled and what types of machinery are actually necessary, though, remains to be established. A knowledge of this would greatly expand our understanding of the necessary and sufficient components of chemical-neurotransmission, and will be investigated in this proposal. Experiments will rely on an important system for studying transmitter release: neurons grown on glass to form miniature brain circuits. With this system, presynaptic molecules can be visualized in living cells with microscopes and sensitive cameras. A second type of experimental approach will even allow neurons to be viewed at a resolution beyond the limits of a light microscope so that detailed information about the structure of extrasynaptic release sites can be obtained. Using these methods, experiments will focus on investigating how release sites are constructed, characterizing their properties, and comparing them to normal presynaptic terminals. The idea of extrasynaptic release is an emerging theme in neurobiology and has important potential roles in contributing to the communication between brain cells. This makes it a very worthwhile area of study. Moreover, incorrect control of transmitter release machinery is thought to be associated with some brain-related diseases; therefore, understanding this fundamental process may even offer clues about the treatment of forms of brain-disorders.

The classical view of fast neurotransmitter release at central neurons is that exocytosis occurs exclusively at specialized ultrastructurally-defined active zones in presynaptic terminals. However, recent work has challenged this idea suggesting that at some central connections, vesicle fusion can also take place at extrasynaptic or ectopic release sites with important functional consequences for neuronal signalling. In hippocampal cultured neurons, an established system for studying neurotransmission, synaptic vesicles arising from stable synapses can readily aggregate at extrasynaptic sites and undergo Ca2+-triggered fusion with normal release kinetics. Moreover, preliminary experiments suggest that vesicles can even exocytose as they transit along the axon. How the highly-regulated and coordinated process of vesicle fusion can occur at such non-specialized sites remains unclear, however. To address this issue, fluorescence tagging and time-lapse imaging methods will be employed here to examine the composition of extrasynaptic release sites and the dynamics of their formation and operation. Experiments will also utilize immunocytochemistry, newly-developed optical glutamate sensors and Ca2+-imaging methods to explore the functional consequences of extrasynaptic transmitter release. Using acute hippocampal slices and ultrastructural investigation, the facility for extrasynaptic release in native tissue will also be examined. The capability of neurons to undergo extrasynaptic vesicle fusion undermines the conventional view of highly compartmentalized neurotransmission, suggesting that release machinery can be readily available across presynaptic structures. Understanding how this is achieved will shed new light on what constitutes the minimal complement of active zone machinery necessary for vesicle exocytosis, and further our knowledge of the fundamental mechanisms underlying information transfer.