Single channel recording from small mammalian presynaptic nerve terminals.

The human brain is composed of nerve cells that communicate with each other by sending and receiving chemical signals, called neurotransmitters. This form of communication is tightly controlled by a set of proteins called ion channels. The activity of these ion channel proteins determines when chemical signals are sent. Thus, if we are to understand chemical signalling in the human brain we must understand how ion channels control that signalling. Although there are technologies that allow us to measure the activity of ion channel proteins, in most cases they cannot be applied directly at the most important area for chemical signalling: the synapse. The synapse is a tiny area (about one millionth of a metre across) which has the special function of sending and receiving chemical signals between nerve cells. The reason we cannot measure ion channel activity at most synapses is simply because they are too small. However, we have made a major breakthrough in this area by applying methods used in nanotechnology (the science concerned with things that are about one thousand millionth of a metre in size). By applying methods adopted from nanotechnology we have been able to show that for the first time it is possible to record the activity of ion channel proteins at synapses and thus to radically improve our understanding of chemical signalling in the brain. This technology will eventually have extensive implications for researchers studying the brain in both health and disease. This proposal thus seeks funding to refine our methods for general use and to begin the enormous task of measuring the array of ion channel activity that controls chemical signalling in the brain.

The ion channels that control neurotransmitter release at presynaptic terminals play a vital role in human and animal physiology, in both health and disease. Unfortunately, technical limitations associated with the small size of most CNS synapses prevent the most informative approach towards characterizing these channels; direct single channel recording. As a consequence of this, presynaptic channels at small terminals are largely uncharacterised at the single channel level. We are proposing the use and further development of an approach that will radically improve our understanding by allowing us to make single channel recordings from small, intact presynaptic boutons. Our method is based on a combination of patch clamp recording, high resolution topographical imaging (using scanning ion conductance microscopy; SICM), and confocal microscopy. By combining these techniques we will be able to locate and patch clamp small presynaptic terminals. We thus plan to characterise both voltage-gated and ligand-gated ion channels in synaptic terminals from two key brain regions; the cerebellum and the hippocampus. We have provided proof-of-principle for our approach in cultured neurons but we also plan to extend this technology so that it can also be used in brain slice preparations.