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

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.

Presynaptic receptors exert a powerful influence on evoked release of neurotransmitters. This project aims to determine the molecular mechanisms and organising principles that govern presynaptic receptor function at individual central synapses. We will achieve this by combining state-of-the-art methods developed by the applicants and collaborators. We will complement an existing multi-photon imaging system (coupled with single-cell electrophysiology) with a two-photon uncaging facility. This will allow us to probe presynaptic receptor function at sub-micron resolution in acute brain slices. We will focus on several distinct synapses in the hippocampus (mossy fibre synapses in CA3 and in the hilus, s. lacunosum-moleculare interneuron -interneuron and s.oriens - s. lacunosum-moleculare inetrneuron connected pairs) and address the identity and location of different types of presynaptic glutamate receptors and Ca2+ channels. We will also use two-photon photobleaching to study how activity and neurotransmitter receptors can affect chemical compartmentalisation and structural changes in axonal varicosities. The anticipated results will provide important insights into the extent, consequences and heterogeneity of presynaptic receptor actions in the brain.