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Content archived on 2024-06-16

Development of flash photolysis for deep uncaging in vivo and high throughput characterisation of neurotransmitter gated ion channels in drug discovery

Final Report Summary - PHOTOLYSIS (Development of flash photolysis for deep uncaging in vivo and high throughput characterisation of neurotransmitter gated ion channels in drug discovery)

Signalling within and between cells is usually with small, diffusible molecular messengers that impart specificity, spatial range and direction to the signal propagation. Each messenger binds to and activates one or a few kinds of receptor on the target cells. There are many examples in cell-cell signalling: the neurotransmitters glutamate, Gamma amino butyric acid (GABA) and glycine mediate fast millisecond timescale synaptic transmission in the brain; there are slower 'neuromodulators' that affect neuronal excitability; local hormones mediate defence mechanisms in the immune system and vascular inflammatory responses; and there are diffusible morphogens in development and long term cell control. Intracellular signals include calcium ions which are involved in many secretory and contractile processes, small nucleotide second messengers such as cAMP and cGMP, and the inositol phosphates.

The speed and efficiency of transmission is achieved with close apposition, on a micron scale, of the release site and the target receptors. This presents the major technical problem in cell physiology and neuroscience of mimicking the spatiotemporal scale of physiological events in an experimental context. PHOTOLYSIS is an experimental tool that has been increasingly applied to cell physiology over three decades. The idea is straightforward, an inert photolabile precursor of the physiological ligand - the 'cage'- is equilibrated in the tissue and the ligand released by a pulse of light. When combined with laser microscopy, the spatiotemporal requirements to mimic physiological events can in principle be met and the implementation and development of this idea is fundamental to the PHOTOLYSIS programme.

Areas identified for improvement were the photochemistry of two-photon excitation and the availability of probes; the spatial and temporal modification of the excitation light; the application of the method in drug discovery. The PHOTOLYSIS consortium comprised of photochemists, photonics experts, high throughput specialists and neuroscientists to facilitate the development and application of photolysis in neuroscience and cell physiology.

A number of significant advances have been made in all aspects of the initial proposal, and it is clear that the deviations from the initial proposals have followed good scientific practice. Two patents have been awarded for novel photochemistry. 10 new probes are available to neuroscience and cell physiology research communities. Significant improvements have been made photonics applied to microscopy and photolysis, in the sources available for excitation and the spatial distribution of excitation in full field microscopy.

Through the PHOTOLYSIS research project, Flyion has now achieved the ability to implement uncaging capabilities with optical means. To this day this feature is completely unique in the area of automated patch clamp systems. The output of the results the study on the modification of neural network discharge by transplantation of cells over-expressing ion channels provided insight on the understanding of the mechanism of action of fibroblasts on neuronal network, and specifically on the behaviour of parkinsonian rats when transplanted to the GPi. The experimental results enabled Genegraft company to move forward with non-human primate study. Finally, significant results have been obtained in neuroscience research with a total of close to 20 papers published.