A Molecular Logic of Sensory Coding Revealed by Optical Tagging of Physiologically-Defined Neuronal Types
Date of Award
Doctor of Philosophy (PhD)
Neural circuit analysis relies on having molecular markers for specific cell types. However, for a cell type identified only by its circuit function, the process of identifying markers remains a laborious task. Here, we report an approach called photoactivated intersectional physiology sequencing (PIPseq), a technique for tagging cells based on their functional properties and then harvesting them for RNA sequencing. To achieve this, we exploited two coexpressed fluorescent proteins, GCaMP and photoactivatable mCherry (PAmCherry): GCaMP permitted recording neuronal activity by large-scale calcium imaging, and PAmCherry enabled labeling selected neurons by two-photon photoactivation (PA). We also developed infrastructure for accurate and sensitive real-time physiological subtyping, quantitative expression profiling, and efficient methods for post-hoc validation.
We leveraged PIPseq to the challenge of mapping receptor-ligand pairings among vomeronasal sensory neurons (VSNs). Among 20 functional cell types revealed by exhaustive light sheet calcium imaging, PIPseq was capable of selecting even rare functional types (∼0.2%) and enriching them by nearly one hundred-fold. It also demonstrated that, just like rare functional types, prevalent types were composed of a single receptor types. Together with in-vivo ectopic expression of vomeronasal receptors, PIPseq identified the complete combinatorial molecular code for a specific set of ligands, and revealed that the primary sequence of a vomeronasal receptor was an unexpectedly strong predictor of functional similarity.
As a preliminary further application of PIPseq, we considered the mechanism of sexually-dimorphic neuronal responses in the vomeronasal sensory population. From light sheet calcium imaging, a particular functional cell type was observed only in male mice, but not in female mice. This cell type was strongly responsive to female specific ligands. PIPseq enabled the identification of a vomeronasal receptor gene expressed in this cell type. Interestingly, the expression level of the vomeronasal receptor gene was indifferent between male and female, indicating that the functional dimorphism depends on factors other than total gene expression.
To summarize, PIPseq is an efficient and exhaustive approach to discovering the expression profile of cells responsible for particular circuit functions. Using this approach, we shed light on molecular logic of vomeronasal chemosensation and sexually-dimorphic sensory responses.
Chair and Committee
Timothy E. Holy
Yehuda Ben-Shahar, Joseph C. Corbo, Harrison W. Gabel, Paul H. Taghert,
Lee, Donghoon, "A Molecular Logic of Sensory Coding Revealed by Optical Tagging of Physiologically-Defined Neuronal Types" (2019). Arts & Sciences Electronic Theses and Dissertations. 1763.
Available for download on Tuesday, August 15, 2119
Permanent URL: https://doi.org/10.7936/q9hx-as12