New Methods for High-Speed Monitoring of Sensory-Driven Synaptic Activity Across All Inputs to Single Living Neurons in the Context of the Intact Cerebral Cortex

The goal of this proposal is to develop new methods for high speed monitoring of sensory-driven synaptic activity across all inputs to single living neurons in the context of the intact cerebral cortex. Although our focus is on understanding how synaptic inputs are integrated across a single neuron embedded in an intact circuit, the next generation random access imaging technology we propose is more broadly applicable for monitoring multi-cellular activity representing large intra-and inter areal neuronal networks. The approach improves on the speed and sensitivity of current random-access technology by nearly 2 orders of magnitude, enabling high- throughput interrogation of up to 104 independent locations within a fraction of a millisecond and compatible with imaging using next generation voltage sensitive indicators. In Aim 1 we propose to generate a comprehensive structural map that will allow random access scanning of all excitatory and inhibitory synapses on functionally defined pyramidal cell types expressing a genetically encoded Ca+2 indicator. The data generated in this Aim will be used to develop image segmentation algorithms to quickly convert structural images of the dendritic tree and the associated synapses into a 3D location map with grid coordinates for sparse sampling of activity patterns at known locations using a fast random access imaging approach described in Aim 2. In Aim 2 we will construct and develop an imaging system allowing high throughput, random addressing within 10-100 ms of approximately 10,000 locations corresponding to all excitatory synapses and other functionally relevant dendritic and somal sites on a single neuron. In Aim 3 we will test and validate the utility of our approach.

PUBLIC HEALTH RELEVANCE: We propose the development of next generation in vivo, random access imaging technology. While our proof of concept study addresses how calcium signals from synaptic inputs are integrated across a single neuron embedded in an intact circuit, the technology we propose is more broadly applicable, and would significantly impact the ability to monitor multi-cellular activity representing large intra-and inter areal communication networks within the intact brain in a behaving animal. Moreover, since the time constant of voltage signals is several orders of magnitude shorter than for calcium, the increased efficiency over previous approaches will be critical for future monitoring of voltage signals in large networks when more sensitive voltage indicators become available.