Understanding synaptic tranmission is essential in understanding the neuronal basis of health and disease. Development and function of synaptic connections profoundly depend on the mechanisms of vesicular targeting and transport. This includes transport of synaptic vesicles as well as trafficking of synaptic receptors. Vesicular transport is highly specific, yet the molecular mechanisms of its specificity are not well understood. In this project a method will be developed that allows tracking of individual molecules over extended periods of time. Vesicular transport and targeting has in the past been studied by overexpressing GFP-labeled proteins. One disadvantage of the method is that the transported species must be highly overexpressed. Another disadvantage is the bleaching of the fluorophores. More importantly a better understanding of the recycling would require tracking of single molecules over extended periods of time with high accuracy. Here an approach will be used using fluorescent nanoparticles with long lifetime. Fluorescent particles functionalized to bind specific proteins will be microinjected into retinal amacrine cells in the intact retina and will be followed over extended periods of time using multiphoton microscopy.
