Neural circuits for appetites are regulated by both homeostatic perturbations and ingestive behaviour. However, the circuit organization that integrates these internal and external stimuli is unclear. Here we show in mice that excitatory neural populations in the lamina terminalis form a hierarchical circuit architecture to regulate thirst. Among them, nitric oxide synthase-expressing neurons in the median preoptic nucleus (MnPO) are essential for the integration of signals from the thirst-driving neurons of the subfornical organ (SFO). Conversely, a distinct inhibitory circuit, involving MnPO GABAergic neurons that express glucagon-like peptide 1 receptor (GLP1R), is activated immediately upon drinking and monosynaptically inhibits SFO thirst neurons. These responses are induced by the ingestion of fluids but not solids, and are time-locked to the onset and offset of drinking. Furthermore, loss-of-function manipulations of GLP1R-expressing MnPO neurons lead to a polydipsic, overdrinking phenotype. These neurons therefore facilitate rapid satiety of thirst by monitoring real-time fluid ingestion. Our study reveals dynamic thirst circuits that integrate the homeostatic-instinctive requirement for fluids and the consequent drinking behaviour to maintain internal water balance.
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We thank B. Ho, A. Qin and M. Liu for technical assistance, D. J. Anderson for sharing Ai110 mice, members of the Oka laboratory, and J. R. Cho for comments. We also thank N. Shah for Casp3 viruses, N. F. Dalleska, and the Beckman Institute at Caltech for technical assistance. This work was supported by Startup funds from the President and Provost of 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 Okawa Foundation, the McKnight Foundation and the Klingenstein-Simons Foundation, and National Institutes of Health U01 (U01 NS099717).
F.G. is a consultant for Kallyope. Y.O. has disclosed these methods and findings to the Caltech Office of Technology Transfer, with provisional patent number CIT-7938-P. The other authors declare no competing financial interests.
Reviewer Information Nature thanks M. McKinley 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 Figure 1 Optogenetic activation MnPOnNOS and OVLTnNOS neurons induces robust water intake in satiated mice.
a, Water restriction (top) and SFOnNOS photostimulation (bottom) induces robust c-Fos expression in the SFO, MnPO and OVLT, compared to control conditions. A majority of c-Fos signals in these areas overlapped with nNOS-expressing neurons. The graph shows the quantification of the overlap between nNOS and c-Fos signals (n = 3 mice). c-Fos signals in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) overlapped with vasopressin (AVP)-expressing neurons. b, MnPO (top) and OVLT (bottom) excitatory neurons visualized in VGlut2/Ai110 transgenic mice co-stained with nNOS (red, antibody staining). MnPOnNOS and OVLTnNOS neurons co-express a glutamatergic marker. 92.2 ± 4.9% of nNOS-expressing neurons were excitatory, and 80.9 ± 2.6% of excitatory neurons are nNOS-expressing in the MnPO (n = 3 mice). Magnified images are shown on the right. c, Left, scheme of the control experiments for monosynaptic rabies tracing. Right, a representative image of the MnPO of an nNOS-cre mouse transduced with AAV-EF1a-FLEX-TVA-mCherry (red) followed by EnvA G-deleted Rabies-eGFP (bottom). No eGFP+ cells were present in the SFO (top, one of two mice) d, Photostimulation of ChR2-expressing MnPOnNOS and OVLTnNOS neurons (red bars, n = 8 and 4 mice for MnPO and OVLT respectively) triggered intense drinking; control mice infected with AAV-DIO-eYFP showed no such response (grey bars, n = 5 mice). Photostimulated mice showed a strong preference for water over a highly concentrated NaCl solution (500 mM, right panel). *P < 0.05, **P < 0.01; by two-tailed Mann–Whitney U test. All error bars show mean ± s.e.m. Scale bars, 50 μm.
Extended Data Figure 2 MnPOnNOS neurons are necessary for the induction of drinking by SFOnNOS photostimulation.
a, Casp3-TEVp efficiently eliminates SFOnNOS neurons (right) without affecting MnPOnNOS neurons (left). c-Fos expression pattern is shown after water-restriction (red). b, Rastor plots representing licking events during the 5-s session with photostimulation. c, Ablation of MnPOnNOS (MnPOx) but not SFOnNOS (SFOx) neurons attenuated the drinking response to OVLTnNOS photostimulation (left, 10 min, blue box). Quantification of the number of licks during the 10-min light-on period (right, n = 9 mice for controls and MnPOx and n = 7 mice for SFOx). d, 5-s brief-access assays to examine the necessity of MnPOnNOS neurons. Acute inhibition of MnPOnNOS neurons by CNO injection severely reduced SFOnNOS-stimulated (left, n = 5 mice for CNO, n = 3 mice for vehicle, and n = 6 mice for no i.p.) and dehydration-induced water intake (middle, n = 7 mice for CNO, n = 5 mice for vehicle, and n = 3 mice for no i.p.). However, the same treatment did not suppress sucrose consumption (300 mM, right, n = 6 mice for CNO, n = 5 mice for vehicle, and n = 3 mice for no i.p.). Control mice transduced by AAV-DIO-mCherry in the MnPO showed no reduction after water or food-restriction (n = 3 mice). e, mCherry control for Fig. 1g. Cumulative water intake in nNOS-cre mice transduced with AAV-DIO-mCherry in the MnPO, AAV-DIO-ChR2-eYFP in the SFO under photostimulated (left, n = 5 mice) or water-restricted conditions (middle, n = 6 mice), and sucrose (300 mM) intake under food-restricted conditions (right, n = 5 mice). f, Intraperitoneal injection of mannitol robustly activated SFOnNOS neurons with (red trace) or without (black trace) CNO injection (left). CNO injection drastically suppressed drinking behaviour without changing the activity of SFOnNOS neurons (middle, n = 4 mice). Plasma osmolality was increased by the injection of mannitol (right, n = 5 mice). *P < 0.05, **P < 0.01, by paired two-tailed t-test or Kruskal–Wallis one-way ANOVA test with Dunn’s correction for multiple comparisons. All error bars and shaded areas show mean ± s.e.m. Scale bar, 50 μm.
a, Left, schematic for the assessment of the MnPOnNOS → SFO monosynaptic connection (left). Right, whole-cell patch-clamp recording from SFO neurons was performed with optogenetic stimulation of MnPOnNOS → SFO projections. Excitatory synaptic currents were measured in the presence (red trace) or absence (black trace) of CNQX (10 μM) + dl-APV (25 μM) after photostimulation (2 ms, blue arrowheads). Most SFOnNOS neurons (12 out of 16 cells, labelled with mCherry, middle panel) or SFOnon-nNOS neurons (14 out of 16 cells, right panel) did not receive monosynaptic input from MnPOnNOS neurons. b, Representative image (one out of three mice) of robust c-Fos expression (red) in the MnPO (top) but not in the SFO (bottom) by photostimulation of ChR2 expressing MnPOnNOS neurons. Scale bar, 50 μm.
a, Left, Schematic of fibre photometry experiments from SFOnNOS (top) and MnPOnNOS (bottom) neurons. nNOS-cre mice were injected with AAV-FLEX-GCaMP6s or eYFP into the SFO and MnPO. Right, representative traces showing the real-time activity of the SFOnNOS (blue trace) and MnPOnNOS (green trace) populations with water intake in water-restricted mice. Grey traces show the activity of eYFP control mice. Corresponding lick patterns are also shown (lower traces). SFOnNOS and MnPOnNOS neurons are rapidly and persistently inhibited by water drinking. b, SFOnNOS and MnPOnNOS neurons are sensitive to thirst-inducing stimuli. Intraperitoneal injection of NaCl (2 M, 300 μl) in a water-satiated animal robustly activated SFOnNOS (blue) and MnPOnNOS (green) neurons. c, Quantification of the neuronal responses. During liquid intake (black bars, n = 4 mice for SFO, n = 6 mice for MnPO) and sodium loading (grey bars, n = 5 mice), both SFOnNOS and MnPOnNOS neurons showed opposite activity changes. All error bars show mean ± s.e.m.
a, Left, a schematic for retrograde tracing of inhibitory inputs to the SFO by HSV-mCherry. Shown are the major inhibitory inputs to the SFO. Right, quantification of HSV-positive neurons (n = 4 mice). LS, lateral septum; MS, medial septum; BNST, bed nucleus of the stria terminalis; MPA, medial preoptic area. b, Monosynaptic retrograde rabies tracing of SFOnNOS neurons. Left, a representative image of the SFO of an nNOS-cre mouse transduced with AAV-CA-FLEX-RG and AAV-EF1a-FLEX-TVA-mCherry followed by EnvA G-deleted Rabies-eGFP. Right, almost no eGFP-positive neurons in the MnPO (green, 5.4 ± 1.3%, n = 4 mice) overlapped with excitatory nNOS-expressing neurons (blue). Maximum inputs to the SFOnNOS neurons are from the MnPO, followed by the MS, LS, MPA and OVLT (n = 4 mice). All error bars show mean ± s.e.m. Scale bars, 50 μm. The mouse brain in this figure has been reproduced from the mouse brain atlas45.
a, nNOS antibody staining (green) of the MnPO from a Glp1r-cre/Ai9 transgenic mouse expressing tdTomato in MnPOGLP1R neurons (red). No substantial overlap was observed between these populations (4.3 ± 0.9% of GLP1R-expressing neurons, n = 3 mice). b, Fluorescence in situ hybridization (FISH) shows that a majority of Ai9 expression (red, 91.9 ± 2.4%, n = 3 mice) closely overlaps with endogenous GLP1R expression (green). c, Left, a diagram showing optogenetic stimulation of MnPOGLP1R neurons transduced with AAV-DIO-ChR2-eYFP or AAV-DIO-eYFP. Right, stimulation of ChR2-expressing MnPOGLP1R neurons inhibited drinking after water restriction as compared to eYFP controls (n = 7 mice for ChR2, n = 6 mice for controls, blue box indicates the Light-ON period). For statistical analysis, we used the same dataset as for 0–10 min from Fig. 2e. d, GLP1 has minor effects on acute drinking behaviour. A diagram of whole-cell recording from MnPOGLP1R neurons is shown on the left. A GLP1 agonist, exendin-4 (Ex-4), had no effect on the firing frequency of MnPOGLP1R neurons in brain slice preparation (middle, n = 6 neurons). However, there was a small decrease in the resting membrane potential (right, n = 6 neurons). e, Enzyme-linked immunosorbent assay analysis of plasma GLP1 levels. Feeding behaviour induced robust plasma GLP1 secretion whereas water intake did not (n = 5 mice for WD + W and FD, n = 6 mice for control and WD, and n = 7 mice for FD + F). f, Left, intra-cranial injection of Ex-4 (red trace, n = 7 mice) into the MnPO had no effect on water intake after water deprivation as compared to vehicle injection (artificial cerebrospinal fluid, black trace, n = 7 mice). Right, a representative injection pattern visualized with fluorescent Ex-4 FAM. *P < 0.05, **P < 0.01, two-tailed Mann–Whitney U test or paired t-test or Kruskal–Wallis one-way ANOVA test with Dunn’s correction for multiple comparisons. All error bars and shaded areas show mean ± s.e.m. Scale bars, 50 μm.
a, SFOnNOS neurons are negatively and chronically regulated by water drinking. Representative responses of SFOnNOS (blue traces) to different types of liquids under water-restricted conditions: a control empty bottle, isotonic saline, silicone oil and water. Each stimulus was presented for 30 s (shaded box). Quantification of the responses is shown in the bottom panel. Activity change (left, area under curve) and baseline activity shift (right, ΔF change) were quantified for SFOnNOS neurons (GCaMP6s, dark blue bars; control, light blue bars). A significant shift in the baseline activity (ΔF change) was observed only in response to water ingestion (n = 6 mice for saline, n = 7 mice for empty, silicone oil and water, n = 5 mice for eYFP). b, Shown are representative responses of SFOnNOS neurons (blue traces) to an empty bottle, peanut butter, and 300 mM sucrose solution under food-restricted conditions (n = 7 mice for empty and peanut butter, n = 5 mice for sucrose, n = 5 mice for all eYFP recordings). c, Activity change per lick was quantified for MnPOGLP1R neurons (GCaMP6s, red bars; eYFP, grey bars) under water-restricted conditions (left, n = 6 mice for saline and silicone oil, n = 7 mice for empty and water, n = 6 mice for all eYFP controls) and food-restricted conditions (right, n = 6 mice for empty and peanut butter, n = 7 mice for sucrose, n = 6 mice for all eYFP controls). All data were reanalysed from Fig. 3b, c. d, Normalized fluorescence change of SFOnNOS (top) and MnPOGLP1R (bottom) neurons from individual mice during licking an empty bottle and water under water-restricted, or sucrose under food-restricted conditions. e, MnPOGLP1R activation is independent of instinctive need. Left, fibre photometry recording of MnPOGLP1R neurons while activating the SFOnNOS neurons. GCaMP6s was virally expressed in MnPOGLP1R neurons for recording calcium dynamics while activating SFOnNOS neurons by hM3Dq-mCherry under the CamKII promoter. Middle, intraperitoneal CNO injection and water deprivation induce water drinking, which robustly activates MnPOGLP1R neurons (red and blue traces respectively). Right, activity change (area under the curve) and licks were quantified for natural thirst and CNO activation (n = 5 mice). *P < 0.05, **P < 0.01, ***P < 0.001, paired two-tailed t-test or Kruskal–Wallis one-way ANOVA test with Dunn’s correction for multiple comparisons. All error bars show mean ± s.e.m.
Extended Data Figure 8 Acute inhibition or chronic ablation of MnPOGLP1R neurons causes overdrinking.
a, b, Acute inhibition of hM4Di-expressing MnPOGLP1R neurons by CNO modestly increases water consumption at the onset of drinking. Drinking behaviour was monitored for 30 min after the injection of CNO (a); magnified data (0–1 min) is shown in b (n = 8 mice). c, d, mCherry controls for acute inhibition of MnPOGLP1R neurons. Drinking behaviour was monitored for 30 min after the injection of CNO or vehicle under water-deprived conditions with free access to saline (c) or water (d). No significant difference was found between mice injected with CNO and vehicle (n = 6 mice). e, Schematic for the genetic ablation of MnPOGLP1R neurons with AAV-flex-Casp3-TEVp (left) in Glp1r-cre/Ai9 mice. Compared to a control animal (right), a Casp3-injected animal displayed almost no GLP1R-expressing neurons in the MnPO (middle, representative image from one out of four mice). In both cases, GLP1R-expressing neurons were labelled using Glp1r-cre/Ai9 transgenic mice. f, Genetic ablation of MnPOGLP1R neurons (red trace, n = 4 mice) recapitulates the overdrinking phenotype similar to the acute inhibition by hM4Di (Fig. 5b), compared to control eYFP group (black trace, n = 6 mice). **P < 0.01, by two-tailed Mann–Whitney U test. All error bars and shaded areas show mean ± s.e.m. Scale bar, 50 μm. The mouse brain in this figure has been reproduced from the mouse brain atlas45.
a, b, Left, schematics for mapping downstream targets of MnPO neurons using AAV-DIO-mCherry (a) or AAV-DIO-eYFP (b). Right, the major outputs from MnPO neurons. nNOS-cre (a) and Glp1r-cre (b) mice were injected with AAV-DIO-mCherry and AAV-DIO-eYFP in the MnPO respectively, and the axon projections were examined using reporter expression. Shown are the injection sites and main representative downstream targets (one out of three mice). Arc, Arcuate Nucleus; DMH, dorsomedial hypothalamic nucleus; DRN, dorsal raphe nucleus; LH, lateral hypothalamus; MRN, median raphe nucleus; PAG, periaqueductal gray; PVH, paraventricular hypothalamic nucleus; PVT, paraventricular thalamic nucleus; SON, supraoptic nucleus. Scale bars, 50 μm. The mouse brain in this figure has been reproduced from the mouse brain atlas45.
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Augustine, V., Gokce, S., Lee, S. et al. Hierarchical neural architecture underlying thirst regulation. Nature 555, 204–209 (2018). https://doi.org/10.1038/nature25488
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