Fluid intake is an essential innate behaviour that is mainly caused by two distinct types of thirst1,2,3. Increased blood osmolality induces osmotic thirst that drives animals to consume pure water. Conversely, the loss of body fluid induces hypovolaemic thirst, in which animals seek both water and minerals (salts) to recover blood volume. Circumventricular organs in the lamina terminalis are critical sites for sensing both types of thirst-inducing stimulus4,5,6. However, how different thirst modalities are encoded in the brain remains unknown. Here we employed stimulus-to-cell-type mapping using single-cell RNA sequencing to identify the cellular substrates that underlie distinct types of thirst. These studies revealed diverse types of excitatory and inhibitory neuron in each circumventricular organ structure. We show that unique combinations of these neuron types are activated under osmotic and hypovolaemic stresses. These results elucidate the cellular logic that underlies distinct thirst modalities. Furthermore, optogenetic gain of function in thirst-modality-specific cell types recapitulated water-specific and non-specific fluid appetite caused by the two distinct dipsogenic stimuli. Together, these results show that thirst is a multimodal physiological state, and that different thirst states are mediated by specific neuron types in the mammalian brain.
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The behavioural and histological data that support the findings are available from the corresponding author on reasonable request. Raw and fully processed scRNA-seq data are available at the NCBI Gene Expression Omnibus (GEO accession no. GSE154048).
The R code used to perform the scRNA-seq analysis is available from the corresponding author on reasonable request.
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We thank the members of the Oka laboratory, D. J. Anderson, M. Thomson and S. Chen for helpful discussion and comments; B. Ho and A. Koranne for maintaining and genotyping animal lines; J. Park and the Single-Cell Profiling Center (SPEC) in the Beckman Institute at Caltech for technical assistance with scRNA-seq; B. Lowell and M. Krashes for generously sharing Pdyn-Cre mice; and L. Luo for a generous gift of TRAP2 mice. This work was supported by Startup funds from the President and Provost of the California Institute of Technology and the Biology and Biological Engineering Division of California Institute of Technology. Y.O. is also supported by the Searle Scholars Program, the Mallinckrodt Foundation, the McKnight Foundation, the Klingenstein-Simons Foundation, the New York Stem Cell Foundation and the NIH (R56MH113030 and R01NS109997). J.N. is supported by the NIH (U19MH114830).
The authors declare no competing interests.
Peer review information Nature thanks Benjamin R. Arenkiel, Charles W. Bourque and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Thirst-state-dependent drinking behaviour and genetic labelling of active neurons.
a, FOS expression in the SFO (left) and the OVLT (right) under the four thirst states (SFO: n = 6 sections from 4 mice for control, n = 6 sections from 6 mice for osmotic thirst, n = 5 sections from 4 mice for hypovolaemic thirst, and n = 6 sections from 6 mice for water deprivation; OVLT: n = 3 sections from 3 mice for control, n = 8 sections from 7 mice for osmotic thirst, n = 5 sections from 4 mice for hypovolaemic thirst, n = 7 sections from 7 mice for water deprivation). b, Water and 0.3M NaCl consumption in sated control animals. The number of total licks for water (grey) and 0.3M saline (red) were quantified during a one-hour session (n = 9 mice for each group). c, Water (grey) and 0.3M KCl intake (orange) under osmotic and hypovolaemic thirst states. The number of total licks was quantified during a one-hour session (n = 6 mice). d, Experimental diagram for TRAP2 activity-dependent genetic labelling. TRAP2/Ai14 double transgenic animals were challenged with osmotic stress by i.p. injection of NaCl solution in the presence of 4-OHT. Osmolality sensitive cells (upper) express Cre-ER under the promoter of Fos gene, which turns on tdTomato expression (red). In osmolality insensitive cells, the same stimulus does not induce tdTomato expression (bottom). e, Genetic labelling of thirst-sensitive neurons in the OVLT of TRAP2/Ai14 mice. Experimental design to label activated neurons under osmotic thirst and hypovolaemic thirst (top). Osmolality sensitive neurons (Osm-TRAP, red) in the OVLT (bottom) overlapped with NaCl-induced acute FOS expression (green). Individual labelling and merged images are shown. By contrast, a significantly smaller fraction of Osm-TRAP neurons was co-labelled with hypovolaemia-induced FOS. Scale bars, 50 μm. f, Quantification of OVLT TRAP2 experiments (n = 6 sections from 4 mice for Osm-Osm, n = 5 sections from 5 mice for Osm-Hvol). g, TRAP labelling in the SFO and the OVLT of sated control animals (n = 6 sections from 3 mice for SFO, n = 3 sections from 2 mice for OVLT). *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Wilcoxon matched-pairs signed rank test or Mann–Whitney test. Data are shown as mean ± s.e.m.
a, b, Violin plots of log-normalized expression of cell-type-defining genes for SFO (a) and OVLT (b) major cell classes with maximum counts per million (max CPM). Bar graph shows profiling resolution per cell type in median genes/cell. c, d, Heat maps of cell-type-specific gene expression in the major cell classes of SFO (c) and OVLT (d). Gene expression data are z-scored with warmer colours indicating higher gene expression. e, Transcriptomic neuron types in the OVLT region (n = 4109 cells) shown in a UMAP embedding (left). Based on in situ hybridization data from Allen Brain Atlas, cell types were annotated into three anatomic classes: OVLT internal (green), external(red), and regional (both inside and outside of the OVLT) (yellow). We excluded non-OVLT cell types (red) for further analyses (Fig. 2, Extended Data Fig. 2h). f, Violin plot of log-normalized gene expression for all neuron types in the OVLT area. Neuron types outside the OVLT are shown in grey. g, h, Heat map of neuron-type-specific gene expression in the SFO (g) and the OVLT (h).
Extended Data Fig. 3 Expression of putative osmoregulatory channels or hormone receptors and cellular comparison between the SFO and the OVLT.
a, Dotplot of cell-type-specific expression for putative osmosensory ion channels and receptor genes for osmoregulatory hormone systems in major cell classes in the SFO and the OVLT (dot size is proportional to % of cells with transcript count >0 expression, colour scale represents z-scored average gene expression, n = 7,950 and 6,161 cells for SFO and OVLT respectively). b, Dotplot of neuron-type-specific expression for putative osmosensory ion channels and receptor genes for osmoregulatory hormone systems in neuron types in the SFO and OVLT30,33,34,35,36,37,38,39,40,41,42,43,44. Although some of the putative genes are not enriched in the SFO or OVLT, they may function outside the LT to regulate thirst (n = 2,642 and 1,511 neurons for SFO and OVLT respectively). c, Evaluation of transcriptional homology between SFO and OVLT cell classes based on Spearman correlation between average expression of top 850 most variable genes from the SFO and OVLT, respectively (n = 1,224 genes total). Euclidean distance matrix between cell types was calculated based on the Spearman correlation coefficients between cell types, which were then hierarchically clustered using Ward agglomeration. d, Same analysis on transcriptional homology between SFO and OVLT neuron types based on top 200 most variable genes from the SFO and OVLT (n = 315 genes total).
a, A diagram of scRNA-seq-based stimulus-to-cell-type mapping protocol. As previously reported, regular scRNA-seq results in artificial induction of IEGs in all neuron types stemming from tissue dissociation21,22. Performing scRNA-seq with a transcriptional blocker during tissue dissociation suppresses artificial induction of IEGs revealing the stimulus or behaviour induced IEG expression pattern. b, Regular scRNA-seq induces high levels of Fos expression in all SFO and OVLT neuron types. Data are shown as a violin plot of log-normalized Fos transcript count data. c, In the presence of actinomycin D, artificial induction of IEGs in non-stimulated SFO and OVLT neurons is abolished. 10x Chromium Controller image was provided by 10x Genomics. d, Expression of Fos in SFO and OVLT major cell classes under distinct thirst states (SFO excitatory neurons n = 931, 689, 775, 706; SFO inhibitory neurons n = 935, 714, 997, 793; SFO LT astrocytes n = 2,085, 1,907, 2,544, 3,177; SFO astrocytes n = 110, 138, 97, 265; OVLT area excitatory neurons n = 2,623, 3,027, 2,115, 2,489; OVLT area inhibitory neurons n = 853, 831, 661, 773; OVLT LT astrocytes n = 1,229, 1,087, 1,133, 1,238; OVLT astrocytes n = 1,736, 1,225, 1,384, 1,353). Data are shown as mean ± s.e.m. e, Expression of other IEGs (Nr4a1 and Fosl2) in SFO and OVLT neuron types under distinct thirst states. All data were analysed with two-tailed Kruskal–Wallis test with Dunn’s post-test. P -values are shown on a log10(p) scale.
Extended Data Fig. 5 Canonical correlation analysis based alignment of transcriptomic neuron types under different physiological conditions.
a, A diagram illustrating the misalignment of cell types under distinct physiological states with regular graph-based clustering analysis. b, The canonical correlation analysis (CCA) workflow for realigning cell types for joint analysis of transcriptomic data sets. c, UMAP embedded scRNA-seq data from SFO and OVLT neurons under distinct thirst states without alignment (left panel), with CCA alignment (middle panel) and cell-type identification on CCA aligned data (right panel). d, e, Violin plots of cell-type defining marker genes in CCA aligned stimulus-to-cell-type mapping data sets for SFO (e) and OVLT (d) respectively.
Extended Data Fig. 6 Multi-colour in situ hybridization for anatomical validation of transcriptomic cell types.
a, Quantification of SFO Htr7- and Rxfp1-positive cells and their overlap in the SFO (n = 17 sections from 6 animals). Scale bar, 20 μm. Nuclei are visualized by DAPI staining (white). b, Quantification of Bmp3- and Rxfp1-positive cells and their overlap in the OVLT (n = 15 sections from 8 animals). Scale bar, 20 μm. c, Rxfp1-and Pdyn-positive cells co-express Fos under water deprived conditions. Representative images from 8, 3, 8 and 3 sections from 2 independent experiments for SFO RXFP1/FOS, OVLT RXFP1/FOS, SFO PDYN/FOS and OVLT PDYN/FOS stains respectively. Scale bar, 10 μm. d, Cell types labelled by Rxfp3 (SFO) and Cpne4 (OVLT) express Fos under osmotic thirst conditions (left). Cell types labelled by Htr7 (SFO) and Bmp3 (OVLT) express Fos under hypovolaemic thirst (right). Representative images from 2, 2, 3 and 2 sections from 2 independent experiments for SFO RXFP3/FOS, OVLT CPNE4/FOS, SFO HTR7/FOS and OVLT BMP3/FOS stains respectively. Scale bar, 10 μm.
Extended Data Fig. 7 Genetic targeting of osmotic and hypovolaemic thirst-activated cell populations in the SFO and the OVLT.
a, Spearman correlation between Fos expression under distinct thirst states and cell-type-specific and thirst-state-specific marker genes. Thirst-state-specific marker genes (Rxfp1 and Pdyn) show higher correlation with Fos expression compared to cell-type-specific genes. b, Two-colour in situ hybridization of Pdyn and Rxfp1. These gene expression patterns are mostly distinct with minor overlap (arrowhead). Representative images from 8 and 2 slices from 2 independent experiments for SFO (left) and OVLT (right) respectively. Scale bar, 10 μm. c, Validation of Cre expression in Pdyn-Cre and Rxfp1-Cre lines. 95.5% of Pdyn-Cre and 100% of Rxfp1-Cre expression matched endogenous gene expression. Representative images from 4 and 2 slices from 2 independent experiments for PDYN/CRE and RXFP1/CRE labelling, respectively. Scale bar, 10 μm. d, Immunostaining of the SFO (top) and OVLT (bottom). Shown are PDYN-positive neurons in Pdyn-Cre/Ai3 animals (representative images out of 8 slices from 4 mice for SFO and out of 2 slices from 2 mice for OVLT, left) and RXFP1-positive neurons in Rxfp1-Cre/Ai14 animals (representative images out of 6 slices from 3 mice for both SFO and OVLT, right). PDYN- and RXFP1-positive neurons (red) are a partial population of ETV1-positive excitatory neurons (green). Almost all (>90%) PDYN- and RXFP1-positive neurons expressed Etv1. RXFP1 and PDYN data are from Fig. 4d. Scale bar, 10 μm.
Extended Data Fig. 8 Characterization of Rxfp1-Cre and Pdyn-Cre activation-derived consumption phenotypes.
a, Photostimulation of RXFP1 neurons in the SFO triggered robust drinking preference to pure water (n = 9 mice), while photostimulation of SFOPdyn neurons induced indiscriminate intake of both water and 0.5 M KCl (n = 6 mice). We observed similar preference in OVLT neurons (n = 6 mice for Rxfp1-Cre, and n = 4 mice for Pdyn-Cre). b, Drinking patterns of Rxfp1-Cre and Pdyn-Cre animals to different concentrations and various salts. Photoactivation of SFORxfp1 induced robust pure water drinking, while the same animal avoided NaCl (0.3 M, n = 4 mice), KCl (0.3 M, n = 5 mice), MgCl2 (0.05 M, n = 5) and CaCl2 (0.05M, n = 5). Animals that receive stimulation in SFOPdyn neurons accepted all of the above solutions (n = 7 mice for NaCl and KCl, 5 mice for MgCl2 and CaCl2). c, Photostimulation of SFOPdyn and SFORxfp1 neurons triggered comparable total fluid intake (n = 7 mice for SFOPdyn, n = 5 mice for SFORxfp1). The total lick number over 20 trials was quantified. d, Photostimulation of SFOPdyn neurons did not drive sodium-licking behaviour (n = 6 animals). Schematic of rock salt behaviour test (left). Representative salt licking raster plots under sodium deprivation (-Sodium), sated (- Light) and photostimulation (+ Light) are presented (middle). Triangle marks the start time of recording. The total bout duration is quantified (right). e, Hypovolaemic stress failed to activate sodium appetite neurons in Pre-LC. Representative images of FOS (red) and FOXP2 expression (a genetic marker for sodium appetite neurons, green) under sated (Control), hypovolaemic thirst (Furosemide) and sodium deprived conditions (Sodium deprivation). Quantification shows percentage of activated sodium appetite neurons (double positive / FOXP2 positive neurons, right, n = 4 mice per group). Scale bar, 50 μm. *P < 0.05, **P < 0.01, by two-tailed Wilcoxon matched-pairs signed-rank test, Mann–Whitney test, Friedman test or Kruskal–Wallis test followed by a Dunn’s post-test. Data are shown as mean ± s.e.m.
| Statistical summary table.
Optogenetic stimulation of SFORxfp1 neurons. Optogenetic activation of Rxfp1 neurons in SFO induced robust pure water consumption (left) but the same animals avoided hyperosmotic saline (0.5M NaCl, right). Light was delivered for 10 sec.
Optogenetic stimulation of SFOPdyn neurons. Optogenetic stimulation of Pdyn neurons in SFO triggered consumption of both water (left) and hyperosmotic salt solution (0.5M NaCl, right). Light was delivered for 10 sec.
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Pool, AH., Wang, T., Stafford, D. et al. The cellular basis of distinct thirst modalities. Nature 588, 112–117 (2020). https://doi.org/10.1038/s41586-020-2821-8
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