Potentiometric dyes are used to image spatial/temporal patterns of electrical activity in cells and tissues. This laboratory has been actively engaged in the development of this technology for the membrane potential imaging of both excitable and non-excitable systems. Sensing voltage in excitable cells or tissues is more challenging because it requires that the dye respond to voltage changes with
The first Aim i s to synthesize potentiometric indicators with improved photostability. This will, of course, be of general benefit to experimentalists, enabling them to extend the duration of optical recording measurements and minimize photodynamic damage to the biological preparation. More specifically, it will allow for the use of narrow band emission collection to maximize the sensitivity of the measurement during intense laser illumination.
The second Aim i s to determine the mechanism(s) by which the SHG signal from dye-stained membranes is sensitive to membrane potential. This will allow us to more rationally design new dyes that can produce large SHG responses with sufficient speed to measure action potentials. In the third Aim, we will apply these new technologies to the study of electrical signals in single spines in cerebellar Purkinje cells. In addition to providing a great test bed for the dye technologies, these studies will explore the fundamental question of whether a spine can compartmentalize electrical inputs. Depolarizations restricted to Purkinje spines would have important consequences for our understanding of synaptic plasticity in these neurons. In our fourth Aim, we will engage in a variety of collaborations with cardiologist and neuroscientists to develop customized dyes and methods for their experimental needs. We will also continue to supply dyes that are not commercially available to the optical recording community.
This project will develop new functional contrast agents that will permit the imaging of electrical activity in excitable tissue with sub-cellular resolution. This technology will be applied to the study of normal and diseased heart. It will also be used to understand information processing in the brain at the level of a single synapse.
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