This research plan proposes a major technical advancement in fluorescent imaging of ion dynamics by designing modular, tunable nanosensors with narrow emission spectra. Under two-photon microscopy, these nanosensors will quantitatively image the spatio-temporal dynamics of sodium fluxes in dendritic spines, which has never been done before. Dendritic spines are tiny, semi-autonomously neuronal compartments that exhibit a fascinating relationship between their structure and their function in processing synaptic signals. This dynamic structure-function relationship provides extraordinarily high input specificity while also allowing for rapid modifications that give rise t plasticity in neural circuits. Understanding spinal physiology may vastly enhance our causal knowledge for a broad range of diseases from Alzheimer's to neuropathic pain as well as basic processes in learning and memory. In order to gain be able to image sodium fluxes, we need to design fluorescent nanosensors that can enter into spines and rapidly measure local sodium concentrations. For this reason, we will use a novel design we call PUNQs for Photostable, Ultra-fast, Nano-optode, Quantum dots. These new PUNQs are modular, tunable, and have narrow emission spectra. Thus, with existing chemistry, they can be easily modifiable to study new ion and small-molecule targets, their dynamic ranges can be adjusted for the target analyte's physiologic concentration, and they can be multiplexed together. This research will produce new insight into the elusive dynamics of sodium in dendritic spines, and the PUNQ platform will be applicable to any research involving ion dynamics.
The specific aims of this research are: 1) Designing PUNQs with optimal size and chemical properties to achieve physiologically-relevant analyte sensitivity and selectivity 2) Delivering PUNQs into small cellular subcompartments at effective concentrations. 3) Measuring dendritic and somatic Na+ fluxes in response to glutamatergic excitation at dendritic spines. This interdisciplinary research merges the Clark laboratory's experience with nanosensor development, the Bhatia laboratory's expertise in multifunctional nanoparticle development, and the Sabatini laboratory's expertise in neurobiology. The proposed research and training plan will elucidate the role of sodium in dendritic spines, and provide a high-value, flexible tool to study ion dynamics within individual cells. Finally, research will provide me with valuable experience that will prepare me for a future as an independent investigator in biomedical research.
This research plan proposes a major technical advancement in fluorescent imaging of ion dynamics by designing modular, tunable nanosensors with narrow emission spectra. These nanosensors will be used to image sodium dynamics in dendritic spines, and they will be expanded to imaging multiple ions simultaneously in the future.
|Ruckh, Timothy T; Clark, Heather A (2014) Implantable nanosensors: toward continuous physiologic monitoring. Anal Chem 86:1314-23|
|Ruckh, Timothy T; Mehta, Ankeeta A; Dubach, J Matthew et al. (2013) Polymer-free optode nanosensors for dynamic, reversible, and ratiometric sodium imaging in the physiological range. Sci Rep 3:3366|