Probing presynaptic receptor function with two-photon uncaging, Ca2+ imaging and photobleaching

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Synapses that transmit information among neurons are modulated in a highly diverse manner by an interplay of intrinsic and extrinsic factors. This contributes both to the richness of information processing by brain circuits and to their stability, which can however break down in disease states such as epilepsy. Several methods are available to study rather indirectly how chemical receptors interact with calcium signals within individual neurotransmitter-releasing nerve processes. However, they cannot be readily integrated with one another: some can only be used with living neurons, while others require the tissue to be frozen or chemically treated. We propose to achieve a much more detailed insight into the function of individual synapses by incorporating a novel method to manipulate the local concentration of chemically active substances with laser-evoked excitation of molecules. This method relies on hitting individual electrons with two photons in very rapid succession, thus evoking a highly localised and brief change in the state of the atoms. Such localised manipulations are necessary to study synapses, because they normally function on a space- and time-scale of microns and milliseconds. We will apply this method to several topical questions that are relevant to normal brain function and to epilepsy. Specifically, we will investigate how the spatial distribution of neurotransmitter receptors and different proteins mediating calcium movements into nerve processes interact with one another. We will also ask whether some changes that occur in epilepsy are caused by alterations in the local expression of these receptors. The work will shed light on how neuronal signalling underlies cognitive processes and how abnormal synaptic function contributes to seizures.