# Tools and approaches for functional connectomics of dense neuropils

> **NIH NIH R21** · WASHINGTON UNIVERSITY · 2020 · $196,875

## Abstract

PROJECT SUMMARY
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.

## Key facts

- **NIH application ID:** 9980918
- **Project number:** 5R21EY030623-02
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Daniel Kerschensteiner
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $196,875
- **Award type:** 5
- **Project period:** 2019-08-01 → 2021-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9980918

## Citation

> US National Institutes of Health, RePORTER application 9980918, Tools and approaches for functional connectomics of dense neuropils (5R21EY030623-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9980918. Licensed CC0.

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