Dataset Information

2P-NucTag: on-demand phototagging for molecular analysis of functionally identified cortical neurons.

ABSTRACT: Neural circuits are characterized by genetically and functionally diverse cell types. A mechanistic understanding of circuit function is predicated on linking the genetic and physiological properties of individual neurons. However, it remains highly challenging to map the functional properties of transcriptionally heterogeneous neuronal subtypes in mammalian cortical circuits in vivo. Here, we introduce a high-throughput two-photon nuclear phototagging (2P-NucTag) approach optimized for on-demand and indelible labeling of single neurons via a photoactivatable red fluorescent protein following in vivo functional characterization in behaving mice. We demonstrate the utility of this function-forward pipeline by selectively labeling and transcriptionally profiling previously inaccessible 'place' and 'silent' cells in the mouse hippocampus. Our results reveal unexpected differences in gene expression between these hippocampal pyramidal neurons with distinct spatial coding properties. Thus, 2P-NucTag opens a new way to uncover the molecular principles that govern the functional organization of neural circuits.

SUBMITTER: Shi J

PROVIDER: S-EPMC10996538 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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2P-NucTag: on-demand phototagging for molecular analysis of functionally identified cortical neurons.

Shi Jingcheng J Nutkovich Boaz B Kushinsky Dahlia D Rao Bovey Y BY Herrlinger Stephanie A SA Tsivourakis Emmanouil E Mihaila Tiberiu S TS Conde Paredes Margaret E ME Malina Katayun Cohen-Kashi KC O'Toole Cliodhna K CK Yong Hyun Choong HC Sanner Brynn M BM Xie Angel A Varol Erdem E Losonczy Attila A Spiegel Ivo I

bioRxiv : the preprint server for biology 20250826

Neural circuits are characterized by genetically and functionally diverse cell types. A mechanistic understanding of circuit function is predicated on linking the genetic and physiological properties of individual neurons. However, it remains highly challenging to map the molecular properties onto functionally heterogeneous neuronal subtypes in mammalian cortical circuits <i>in vivo</i> . Here, we introduce a high-throughput two-photon nuclear phototagging (2P-NucTag) approach for on-demand and ...[more]

PMID: 38585980

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Project description:Acute regulation of CO2 and pH homeostasis requires sensory feedback from peripheral (carotid body) and central (central) CO2/pH sensitive cells - so called respiratory chemoreceptors. Subsets of brainstem serotonin (5-HT) neurons in the medullary raphe are CO2 sensitive or insensitive based on differences in embryonic origin, suggesting these functionally distinct subpopulations may have unique transcriptional profiles. Here, we used Patch-to-Seq to determine if the CO2 responses in brainstem 5-HT neurons could be correlated to unique transcriptional profiles and/or unique molecular markers and pathways. First, firing rate changes with hypercapnic acidosis were measured in fluorescently labeled 5-HT neurons in acute brainstem slices from transgenic, Dahl SS (SSMcwi) rats expressing T2/ePet-eGFP transgene in Pet-1 expressing (serotonin) neurons (SS ePet1-eGFP rats). Subsequently, the transcriptomic and pathway profiles of CO2 sensitive and insensitive 5-HT neurons were determined and compared by single cell RNA (scRNAseq) and bioinformatic analyses. Low baseline firing rates were a distinguishing feature of CO2 sensitive 5-HT neurons. scRNAseq of these recorded neurons revealed 166 differentially expressed genes among CO2 sensitive and insensitive 5-HT neurons. Pathway analyses yielded novel predicted upstream regulators, including the transcription factor Egr2 and Leptin. Additional bioinformatic analyses identified 6 candidate gene markers of CO2 sensitive 5-HT neurons, and 2 selected candidate genes (CD46 and Iba57) were both expressed in 5-HT neurons determined via in situ mRNA hybridization. Together, these data provide novel insights into the transcriptional control of cellular chemoreception and provide unbiased candidate gene markers of CO2 sensitive 5-HT neurons. Methodologically, these data highlight the utility of the patch-to-seq technique in enabling the linkage of gene expression to specific functions, like CO2 chemoreception, in a single cell to identify potential mechanisms underlying functional differences in otherwise similar cell types.

Dataset Information

2P-NucTag: on-demand phototagging for molecular analysis of functionally identified cortical neurons.

Publications

2P-NucTag: on-demand phototagging for molecular analysis of functionally identified cortical neurons.

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