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Connect-Seq to Superimpose Molecular on Anatomical Neural Circuit Maps

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Connect-Seq to Superimpose Molecular on Anatomical Neural Circuit Maps Connect-seq to superimpose molecular on anatomical neural circuit maps Naresh K. Hanchatea,1, Eun Jeong Leea,1, Andria Ellisb,2, Kunio Kondoha,2,3, Donghui Kuanga, Ryan Basomc, Cole Trapnellb,d, and Linda B. Bucka,d,4 aBasic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; bDepartment of Genome Sciences, University of Washington, Seattle, WA 98115; cGenomics and Bioinformatics Shared Resource, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and dThe Brotman Baty Institute for Precision Medicine, Seattle, WA 98195 Contributed by Linda B. Buck, December 28, 2019 (sent for review July 16, 2019; reviewed by Liqun Luo and Hongkui Zeng) The mouse brain contains about 75 million neurons interconnected odors (8). The viral tracing studies provide an anatomical map of in a vast array of neural circuits. The identities and functions of neurons directly presynaptic to CRHNs, but the molecular individual neuronal components of most circuits are undefined. identities of those neurons are unknown. Here we describe a method, termed “Connect-seq,” which combines If one could identify genes whose expression defines subsets of retrograde viral tracing and single-cell transcriptomics to uncover neurons upstream of CRHNs in different brain areas, one would the molecular identities of upstream neurons in a specific circuit have molecular tools to explore which subsets mediate responses and the signaling molecules they use to communicate. Connect- to different stressors and to manipulate those subsets to gain seq can generate a molecular map that can be superimposed on a insight into how they function. Information regarding neuro- neuroanatomical map to permit molecular and genetic interrogation transmitters and neuromodulators used by the upstream neurons of how the neuronal components of a circuit control its function. to communicate with CRHNs would, in addition, provide in- Application of this method to hypothalamic neurons controlling formation relevant not only to an understanding of neural circuit physiological responses to fear and stress reveals subsets of up- functions, but also potential insights into pharmacological inter- stream neurons that express diverse constellations of signaling mol- ventions to modify the functions of specific circuit components. ecules and can be distinguished by their anatomical locations. To test the Connect-seq strategy, we infected Cre recombinase- expressing CRHNs with a Cre-dependent Pseudorabies virus single-cell RNA-seq | neural circuits | stress (PRV) that travels retrogradely across synapses. Using flow cytom- NEUROSCIENCE etry,weisolatedsinglevirus-infectedneuronsandthenusedsingle- he brain contains a multitude of neural circuits that control cell RNA sequencing (RNA-seq) to define the transcriptomes of Tdiverse functions, but the specific neuronal components of individual upstream neurons. These experiments revealed a most circuits and their respective roles are largely a mystery (1–3). The application of single-cell RNA sequencing techniques has Significance allowed rapid advances in defining the transcriptomes of brain neurons (1–6). However, much less is known about which of the Single-cell transcriptomics has emerged as a powerful means to vast numbers of individual neurons with known transcriptomes are define the molecular heterogeneity of brain neurons. How- interconnected to control specific functions. Here we devised a ever, which of the neurons with known transcriptomes interact strategy, “Connect-seq,” to obtain additional information about with each other in specific neural circuits is largely unknown. the neuronal components of specified neural circuits. The strategy Here, we devised a strategy, termed “Connect-seq,” which couples the use of conditional neurotropic viruses that cross syn- combines retrograde viral tracing and single-cell transcriptomics apses with transcriptome analysis of single upstream neurons in- to determine the molecular identities of individual upstream fected with virus to obtain the molecular identities of individual neurons in a defined circuit. Using Connect-seq, we uncovered a neurons connected in a circuit. large variety of signaling molecules expressed in neurons up- To test the strategy, we focused on neural circuits that transmit stream of hypothalamic neurons that control physiological re- signals to corticotropin-releasing hormone neurons (CRHNs) in sponses to stress. Information obtained by Connect-seq can be the paraventricular nucleus of the hypothalamus (PVN) (7). In used to overlay molecular maps on anatomical neural circuit response to danger, CRHNs induce increases in blood stress maps and generate molecular tools for probing the functions of hormones that act on multiple tissue systems to coordinate ap- individual circuit components. propriate physiological responses. Surges in blood stress hormones occur in response to a variety of external and internal dangers or Author contributions: N.K.H., K.K., and L.B.B. designed research; N.K.H. and E.J.L. per- “ ” formed research; N.K.H., K.K., and D.K. contributed new reagents/analytic tools; N.K.H., stressors, including predator odors, tissue injury, inflammation, E.J.L., A.E., R.B., C.T., and L.B.B. analyzed data; and N.K.H. and L.B.B. wrote the paper. and emotional and psychological stress (7, 8). Studies using clas- Reviewers: L.L., Stanford University; and H.Z., Allen Institute for Brain Science. sical neuroanatomical and neurophysiological approaches have The authors declare no competing interest. provided a large body of information on PVN inputs that may affect CRHNs and the effects of classical neurotransmitters on Published under the PNAS license. – Data deposition: Raw sequencing data related to this study have been archived in the CRHN activity and function (7, 9 12). However, the exact iden- National Center for Biotechnology Information Gene Expression Omnibus (GEO) database tities of upstream neurons that control CRHN functions and the https://www.ncbi.nlm.nih.gov/geo (accession no. GSE139923). mechanisms by which they do so have not been defined. 1N.K.H. and E.J.L. contributed equally to this work. Viral tracing studies indicate that CRHNs receive direct syn- 2A.E. and K.K. contributed equally to this work. aptic input from neurons in 31 different brain areas (8). These 3Present address: Department of Homeostatic Regulation, Division of Endocrinology and include 19 areas of the hypothalamus, a brain area that inte- Metabolism, National Institute for Physiological Sciences, 444-8585 Myodaiji, Okazaki, grates information from the body and external environment and Aichi, Japan. organizes fitting behavioral and physiological responses. Neu- 4To whom correspondence may be addressed. Email: [email protected]. rons two or more synapses upstream of CRHNs are seen in This article contains supporting information online at https://www.pnas.org/lookup/suppl/ additional areas, including one small area of the olfactory cortex doi:10.1073/pnas.1912176117/-/DCSupplemental. that has proved key to stress hormone responses to predator First published February 7, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1912176117 PNAS | February 25, 2020 | vol. 117 | no. 8 | 4375–4384 Downloaded by guest on October 4, 2021 large diversity of neurotransmitter and neuromodulator signaling to CRHNs, but the possibility that a minor fraction are two molecules in neurons directly upstream of CRHNs, including synapses upstream cannot be absolutely excluded. Microscopic more than 40 different neuropeptides. Many individual neurons examination of cells sorted into microtiter plates confirmed that coexpressed multiple different signaling molecules. These in- the methods used deposited only a single cell per well (Materials cluded neurons coexpressing neuropeptides with neurotransmit- and Methods and SI Appendix, Fig. S2). cDNAs were prepared ters or biogenic amines, as well as neurons coexpressing different from each sorted cell and tested for GFP expression by PCR. neuropeptides. Upstream neurons expressing specific signaling We then conducted RNA-seq (15, 16) on individual GFP- molecules mapped to selected brain areas, demonstrating that expressing cells by Illumina-sequencing cDNAs prepared from Connect-seq can provide molecular tools with which to dissect the those cells (SI Appendix,Fig.S3A). Gene expression in indi- functions of individual neuronal components of neural circuits. vidual cells (SI Appendix,Fig.S3B) was determined by align- ment to the mouse genome using standard methods (17, 18). Results Infection of cells with PRV was confirmed by aligning sequence Transcriptome Analysis of Neurons Upstream of CRHNs. We first data to PRV genome data (Materials and Methods and SI Ap- developed a Pseudorabies virus, PRVB180, which has Cre pendix,Fig.S3C and D). We then examined the transcriptome recombinase-dependent expression of thymidine kinase (TK) of each cell for expression of markers of neurons or glia (Ma- fused to green fluorescent protein (GFP) (Fig. 1A). After infecting terials and Methods) and proceeded to analyze 117 cells that neurons expressing Cre, this virus will travel retrogradely across expressed neuronal markers. synapses to infect upstream neurons. We injected PRVB180 into Neurons were sequenced at an average of ∼6.5 million reads the PVN of CRH-IRES-Cre (CRH-Cre) mice (Fig. 1B), which per cell. Comparison of sequenced reads and numbers of genes express Cre in CRHNs (13). Following infection with PRVB180, expressed in neurons did not indicate a correlation (Pearson’s
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