Many computations in the nervous system occur at the level of individual neurites within large extensively branched arbors (i.e., subcellular processing). Most neurites operate in dense neuropils, in which processes of diverse cell types are tightly packed and abundantly interconnected. To understand subcellular processing, we need to measure neurite responses to physiological stimuli and relate them to local patterns of synaptic inputs. To delineate the functional architecture of neuropils and reveal the logic of their connectivity, we need to characterize neurite responses and synapse patterns at high density. Neurite responses can be observed by two-photon imaging, and synaptic inputs can be reconstructed in serial-section electron microscopy (ssEM). A number of technical obstacles have precluded the combination of these techniques (i.e., functional connectomics) to study subcellular processing in dense neuropils. Here, we develop new tools and approaches to overcome these obstacles.
In Aim 1, we develop genetic, viral, and computational tools for multispectral two-photon calcium imaging and signal demixing to enable dense functional characterization of neuropils.
In Aim 2, we devise a novel strategy for combining two-photon imaging and ssEM (i.e., multimodal imaging), and establish a high-throughput ssEM method for analyzing local synaptic connectivity patterns in the context of larger-scale circuit wiring (i.e., multiresolution imaging). We use our advances to study amacrine cells (ACs), a diverse class of retinal interneurons. The neurites of more than 50 AC types extract salient visual information in a dense neuropil the inner retina. We will acquire a complete functional connectomic dataset of ACs. This dataset, which will be made publicly available, will form the basis of a future R01 application to study the mechanisms of subcellular processing in ACs, the functional architecture of the AC neuropil, and the logic of its connectivity.

Public Health Relevance

Because many important computations occur at the level of individual neurites (i.e., subcellular processing), a better understanding of nervous system function and dysfunction requires tools that can reveal subcellular processing in the dense neuropils that most neurites occupy. Here, we develop such tools and use them to analyze the function and connectivity of retinal interneurons (i.e., amacrine cells). Insights into the subcellular processing of amacrine cells are fundamental to understanding the visual function of the retina, may aid in the identification of circuit diseases, and can function as benchmarks for testing the performance of rescue strategies for retinal degenerations.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21EY030623-01
Application #
9809180
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Greenwell, Thomas
Project Start
2019-08-01
Project End
2021-07-31
Budget Start
2019-08-01
Budget End
2020-07-31
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Washington University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130