Connect-seq to superimpose molecular on anatomical neural circuit maps
Contributed by Linda B. Buck, December 28, 2019 (sent for review July 16, 2019; reviewed by Liqun Luo and Hongkui Zeng)
Significance
Single-cell transcriptomics has emerged as a powerful means to define the molecular heterogeneity of brain neurons. However, which of the neurons with known transcriptomes interact with each other in specific neural circuits is largely unknown. Here, we devised a strategy, termed “Connect-seq,” which combines retrograde viral tracing and single-cell transcriptomics to determine the molecular identities of individual upstream neurons in a defined circuit. Using Connect-seq, we uncovered a large variety of signaling molecules expressed in neurons upstream of hypothalamic neurons that control physiological responses to stress. Information obtained by Connect-seq can be used to overlay molecular maps on anatomical neural circuit maps and generate molecular tools for probing the functions of individual circuit components.
Abstract
The mouse brain contains about 75 million neurons interconnected in a vast array of neural circuits. The identities and functions of individual neuronal components of most circuits are undefined. Here we describe a method, termed “Connect-seq,” which combines retrograde viral tracing and single-cell transcriptomics to uncover the molecular identities of upstream neurons in a specific circuit and the signaling molecules they use to communicate. Connect-seq can generate a molecular map that can be superimposed on a neuroanatomical map to permit molecular and genetic interrogation of how the neuronal components of a circuit control its function. Application of this method to hypothalamic neurons controlling physiological responses to fear and stress reveals subsets of upstream neurons that express diverse constellations of signaling molecules and can be distinguished by their anatomical locations.
Data Availability
Data deposition: Raw sequencing data related to this study have been archived in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database https://www.ncbi.nlm.nih.gov/geo (accession no. GSE139923).
Acknowledgments
We thank J. Delrow, A. Marty, A. Dawson, and R. Meredith at the Fred Hutchinson Cancer Research Center (FHCRC) Genomics Facility for their assistance with RNA-seq and A. Berger, S. Dozono, and B. Raden, at the FHCRC Flow Cytometry Facility for their assistance with flow sorting. We thank X. Ye, A. Spray, and E. Albrecht for technical assistance and members of the Buck Laboratory for helpful discussions. This work was supported by the Millen Literary Trust (E.J.L.), the Howard Hughes Medical Institute (L.B.B.), NIH Grants R01 DC015032 (L.B.B.), R01 DC016442 (L.B.B.), and DP2 HD088158 (C.T.), the Paul G. Allen Frontiers Foundation (through the Allen Discovery Center for Cell Lineage Tracing), and an Alfred P. Sloan Fellowship (C.T.). L.B.B. is on the Board of Directors of International Flavors & Fragrances.
Supporting Information
Appendix (PDF)
- Download
- 14.27 MB
References
1
L. Luo, E. M. Callaway, K. Svoboda, Genetic dissection of neural circuits: A decade of progress. Neuron 98, 865 (2018).
2
H. Zeng, Mesoscale connectomics. Curr. Opin. Neurobiol. 50, 154–162 (2018).
3
H. Zeng, J. R. Sanes, Neuronal cell-type classification: Challenges, opportunities and the path forward. Nat. Rev. Neurosci. 18, 530–546 (2017).
4
A. Zeisel et al., Molecular architecture of the mouse nervous system. Cell 174, 999–1014.e22 (2018).
5
R. A. Romanov et al., Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes. Nat. Neurosci. 20, 176–188 (2017).
6
J.-F. Poulin, B. Tasic, J. Hjerling-Leffler, J. M. Trimarchi, R. Awatramani, Disentangling neural cell diversity using single-cell transcriptomics. Nat. Neurosci. 19, 1131–1141 (2016).
7
Y. M. Ulrich-Lai, J. P. Herman, Neural regulation of endocrine and autonomic stress responses. Nat. Rev. Neurosci. 10, 397–409 (2009).
8
K. Kondoh et al., A specific area of olfactory cortex involved in stress hormone responses to predator odours. Nature 532, 103–106 (2016).
9
L. Senst, J. Bains, Neuromodulators, stress and plasticity: A role for endocannabinoid signalling. J. Exp. Biol. 217, 102–108 (2014).
10
B. Myers, J. R. Scheimann, A. Franco-Villanueva, J. P. Herman, Ascending mechanisms of stress integration: Implications for brainstem regulation of neuroendocrine and behavioral stress responses. Neurosci. Biobehav. Rev. 74, 366–375 (2017).
11
C. S. Johnson, J. S. Bains, A. G. Watts, Neurotransmitter diversity in pre-synaptic terminals located in the parvicellular neuroendocrine paraventricular nucleus of the rat and mouse hypothalamus. J. Comp. Neurol. 526, 1287–1306 (2018).
12
G. Aguilera, Y. Liu, The molecular physiology of CRH neurons. Front. Neuroendocrinol. 33, 67–84 (2012).
13
M. J. Krashes et al., An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger. Nature 507, 238–242 (2014).
14
B. Tasic et al., Adult mouse cortical cell taxonomy revealed by single cell transcriptomics. Nat. Neurosci. 19, 335–346 (2016).
15
S. Picelli et al., Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).
16
I. C. Macaulay et al., G&T-seq: Parallel sequencing of single-cell genomes and transcriptomes. Nat. Methods 12, 519–522 (2015).
17
C. Trapnell, L. Pachter, S. L. Salzberg, TopHat: Discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).
18
C. Trapnell et al., Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).
19
L. B. Buck, C. I. Bargmann, “Smell and taste: The chemical senses” in Principles of Neuroscience, E. Kandel, J. Schwartz, T. Jessell, S. Siegelbaum, A. J. Hudspeth, Eds. (McGraw-Hill, New York, 2012), pp. 712–742.
20
J. M. Aubry, V. Bartanusz, S. Pagliusi, P. Schulz, J. Z. Kiss, Expression of ionotropic glutamate receptor subunit mRNAs by paraventricular corticotropin-releasing factor (CRF) neurons. Neurosci. Lett. 205, 95–98 (1996).
21
W. E. Cullinan, GABA(A) receptor subunit expression within hypophysiotropic CRH neurons: A dual hybridization histochemical study. J. Comp. Neurol. 419, 344–351 (2000).
22
I. Mody, J. Maguire, The reciprocal regulation of stress hormones and GABA(A) receptors. Front. Cell. Neurosci. 6, 4 (2012).
23
J. I. Wamsteeker Cusulin, T. Füzesi, A. G. Watts, J. S. Bains, Characterization of corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus of Crh-IRES-Cre mutant mice. PLoS One 8, e64943 (2013).
24
J. S. Bains, J. I. Wamsteeker Cusulin, W. Inoue, Stress-related synaptic plasticity in the hypothalamus. Nat. Rev. Neurosci. 16, 377–388 (2015).
25
R. L. Cole, P. E. Sawchenko, Neurotransmitter regulation of cellular activation and neuropeptide gene expression in the paraventricular nucleus of the hypothalamus. J. Neurosci. 22, 959–969 (2002).
26
T. Murakami et al., Stress-related activities induced by predator odor may become indistinguishable by hinokitiol odor. Neuroreport 23, 1071–1076 (2012).
27
M. Matsukawa, M. Imada, T. Murakami, S. Aizawa, T. Sato, Rose odor can innately counteract predator odor. Brain Res. 1381, 117–123 (2011).
28
D. Wacker, R. C. Stevens, B. L. Roth, How ligands illuminate GPCR molecular pharmacology. Cell 170, 414–427 (2017).
29
A. E. Granstedt, M. L. Szpara, B. Kuhn, S. S. Wang, L. W. Enquist, Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus. PLoS One 4, e6923 (2009).
30
K. M. McCarthy, D. W. Tank, L. W. Enquist, Pseudorabies virus infection alters neuronal activity and connectivity in vitro. PLoS Pathog. 5, e1000640 (2009).
31
T. Hökfelt et al., Neuropeptides–An overview. Neuropharmacology 39, 1337–1356 (2000).
32
M. P. Nusbaum, D. M. Blitz, E. Marder, Functional consequences of neuropeptide and small-molecule co-transmission. Nat. Rev. Neurosci. 18, 389–403 (2017).
33
M. S. Dicken, R. E. Tooker, S. T. Hentges, Regulation of GABA and glutamate release from proopiomelanocortin neuron terminals in intact hypothalamic networks. J. Neurosci. 32, 4042–4048 (2012).
34
S. T. Hentges, V. Otero-Corchon, R. L. Pennock, C. M. King, M. J. Low, Proopiomelanocortin expression in both GABA and glutamate neurons. 29, 13684–13690 (2009).
35
R. Chen, X. Wu, L. Jiang, Y. Zhang, Single-cell RNA-Seq reveals hypothalamic cell diversity. Cell Rep. 18, 3227–3241 (2017).
36
S. El Mestikawy, A. Wallén-Mackenzie, G. M. Fortin, L. Descarries, L. E. Trudeau, From glutamate co-release to vesicular synergy: Vesicular glutamate transporters. Nat. Rev. Neurosci. 12, 204–216 (2011).
37
N. X. Tritsch, A. J. Granger, B. L. Sabatini, Mechanisms and functions of GABA co-release. Nat. Rev. Neurosci. 17, 139–145 (2016).
38
T. M. Hahn, J. F. Breininger, D. G. Baskin, M. W. Schwartz, Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons. Nat. Neurosci. 1, 271–272 (1998).
39
C. F. Elias et al., Leptin activates hypothalamic CART neurons projecting to the spinal cord. Neuron 21, 1375–1385 (1998).
40
M. Zelikowsky et al., The neuropeptide Tac2 controls a distributed brain state induced by chronic social isolation stress. Cell 173, 1265–1279.e19 (2018).
41
E. S. Lein et al., Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176 (2007).
42
J. N. Campbell et al., A molecular census of arcuate hypothalamus and median eminence cell types. Nat. Neurosci. 20, 484–496 (2017).
43
L. E. Pomeranz et al., Gene expression profiling with cre-conditional pseudorabies virus reveals a subset of midbrain neurons that participate in reward circuitry. J. Neurosci. 37, 4128–4144 (2017).
44
W. E. Allen et al., Thirst-associated preoptic neurons encode an aversive motivational drive. Science 357, 1149–1155 (2017).
45
K. Sakurai et al., Capturing and manipulating activated neuronal ensembles with CANE delineates a hypothalamic social-fear circuit. Neuron 92, 739–753 (2016).
46
M. I. Ekstrand et al., Molecular profiling of neurons based on connectivity. Cell 157, 1230–1242 (2014).
47
C. J. Guenthner, K. Miyamichi, H. H. Yang, H. C. Heller, L. Luo, Permanent genetic access to transiently active neurons via TRAP: Targeted recombination in active populations. Neuron 78, 773–784 (2013).
48
Z. A. Knight et al., Molecular profiling of activated neurons by phosphorylated ribosome capture. Cell 151, 1126–1137 (2012).
49
B. Tasic et al., Shared and distinct transcriptomic cell types across neocortical areas. Nature 563, 72–78 (2018).
50
J. DeFalco et al., Virus-assisted mapping of neural inputs to a feeding center in the hypothalamus. Science 291, 2608–2613 (2001).
51
F. Schnütgen et al., A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat. Biotechnol. 21, 562–565 (2003).
52
B. W. Banfield, G. A. Bird, Construction and analysis of alphaherpesviruses expressing green fluorescent protein. Methods Mol. Biol. 515, 227–238 (2009).
53
J. P. Card, L. W. Enquist, Transneuronal circuit analysis with pseudorabies viruses. Curr. Protoc. Neurosci. 9, 1.5.1–1.5.28 (2001).
54
K. Franklin, G. Paxinos, The Mouse Brain in Stereotaxic Coordinates (Elsevier Inc., ed. 3, 2008).
55
N. K. Hanchate et al., Single-cell transcriptomics reveals receptor transformations during olfactory neurogenesis. Science 350, 1251–1255 (2015).
56
bcl2fastq conversion software (version 1.8.4) (Illumina, Inc., San Diego, CA).
57
F. Krueger, Trim Galore, Version 0.4.4. http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/. Accessed 14 March 2017.
58
R Core Team, R: A language and environment for statistical computing (R Foundation for Statistical Computing, Vienna, Austria, 2019). https://www.R-project.org/. Accessed 22 March 2017.
59
RStudio Team (2015). RStudio: Integrated development for R. Version 1.2.1206. http://www.rstudio.com/. Accessed 12 December 2018.
60
H. Wickham, ggplot2: Elegant graphics for data analysis (Springer-Verlag, New York, NY, 2016).
61
M. I. Ekstrand, L. W. Enquist, L. E. Pomeranz, The alpha-herpesviruses: Molecular pathfinders in nervous system circuits. Trends Mol. Med. 14, 134–140 (2008).
62
G. Ugolini, Advances in viral transneuronal tracing. J. Neurosci. Methods 194, 2–20 (2010).
63
L. Rinaman, J. P. Card, L. W. Enquist, Spatiotemporal responses of astrocytes, ramified microglia, and brain macrophages to central neuronal infection with pseudorabies virus. J. Neurosci. 13, 685–702 (1993).
64
J. P. Card et al., Pseudorabies virus infection of the rat central nervous system: Ultrastructural characterization of viral replication, transport, and pathogenesis. J. Neurosci. 13, 2515–2539 (1993).
Information & Authors
Information
Published in
Classifications
Copyright
© 2020. Published under the PNAS license.
Data Availability
Data deposition: Raw sequencing data related to this study have been archived in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database https://www.ncbi.nlm.nih.gov/geo (accession no. GSE139923).
Submission history
Published online: February 7, 2020
Published in issue: February 25, 2020
Keywords
Acknowledgments
We thank J. Delrow, A. Marty, A. Dawson, and R. Meredith at the Fred Hutchinson Cancer Research Center (FHCRC) Genomics Facility for their assistance with RNA-seq and A. Berger, S. Dozono, and B. Raden, at the FHCRC Flow Cytometry Facility for their assistance with flow sorting. We thank X. Ye, A. Spray, and E. Albrecht for technical assistance and members of the Buck Laboratory for helpful discussions. This work was supported by the Millen Literary Trust (E.J.L.), the Howard Hughes Medical Institute (L.B.B.), NIH Grants R01 DC015032 (L.B.B.), R01 DC016442 (L.B.B.), and DP2 HD088158 (C.T.), the Paul G. Allen Frontiers Foundation (through the Allen Discovery Center for Cell Lineage Tracing), and an Alfred P. Sloan Fellowship (C.T.). L.B.B. is on the Board of Directors of International Flavors & Fragrances.
Authors
Competing Interests
The authors declare no competing interest.
Metrics & Citations
Metrics
Altmetrics
Citations
Cite this article
Connect-seq to superimpose molecular on anatomical neural circuit maps, Proc. Natl. Acad. Sci. U.S.A.
117 (8) 4375-4384,
https://doi.org/10.1073/pnas.1912176117
(2020).
Copied!
Copying failed.
Export the article citation data by selecting a format from the list below and clicking Export.
Cited by
Loading...
View Options
View options
PDF format
Download this article as a PDF file
DOWNLOAD PDFLogin options
Check if you have access through your login credentials or your institution to get full access on this article.
Personal login Institutional LoginRecommend to a librarian
Recommend PNAS to a LibrarianPurchase options
Purchase this article to access the full text.