Intersectional Strategies to Understand the Function of Diverse Neural Circuits

St. Jude Children'S Research Hospital, Memphis TN

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Abstract

Project Summary Next-generation sequencing has enabled a rapid expansion in cellular taxonomy across biological systems. However, to probe the function of these newly defined cell types, researchers require a molecular toolkit that can precisely target cells based on a combination of features. To address this need, this project proposes the development of novel intersectional approaches that will restrict transgene expression to defined cell populations based on multiple characteristics, such as combinatorial gene expression and activity state during defined behaviors. While a handful of intersectional strategies currently exist, their limitations, such as complicated design parameters and limited spatial or temporal resolution, preclude their widespread use. The intersectional strategies proposed here address these limitations to provide two new types of tools: 1) a Cre and Flp-recombinase dependent AAV vector that is easily modified to accommodate a variety of transgenes and promoters and 2) a transgenic mouse line that restricts Cre or Flp recombinase expression to neurons activated in a tightly-defined time window (~30 minutes). Using these tools, our goal is to address a major unanswered question in neuroscience: how do discrete populations of neurons in the brain generate diverse behaviors? Towards this, we focus on a small group of neurons in the brainstem nucleus locus coeruleus (LC) that are known to regulate different forms of arousal. Despite their small number, LC neurons are the brain?s main source of norepinephrine, a neurotransmitter that promotes a wide range of behaviors related to arousal. Traditionally, the LC has been classified as molecularly homogeneous, since all neurons within it express NE. Thus, it is not clear how the LC achieves its functional diversity. We propose that distinct subsets of LC neurons, which may differ in their connectivity and molecular identity beyond NE expression, are responsible for promoting different arousal responses. Using the intersectional tools described here, combined with single cell RNA sequencing and a novel behavioral paradigm, we will identify the molecular landscape and brain-wide connectivity of discrete LC neuron populations that promote opposing types of arousal (positive or aversive). Identifying functional diversity in the LC also has implications for human health, as perturbations in LC- related neural circuits are thought to significantly contribute to mood disorders such as depression and anxiety. Understanding how LC neurons accurately generate different forms of arousal may also illuminate how particular alterations in these neural circuits promote specific mood disorder phenotypes.