Hierarchical neural architecture underlying thirst regulation

ABSTRACT 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. Access through

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DOPAMINE SUBSYSTEMS THAT TRACK INTERNAL STATES Article Open access 13 July 2022 PARALLEL GUT-TO-BRAIN PATHWAYS ORCHESTRATE FEEDING BEHAVIORS Article 03 December 2024 THE PARASUBTHALAMIC

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_The Mouse Brain in Stereotaxic Coordinates_ 2nd edn (Academic, 2001) Download references ACKNOWLEDGEMENTS 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). AUTHOR INFORMATION Author notes * Sertan Kutal Gokce and Sangjun Lee: These authors contributed equally to this work.

AUTHORS AND AFFILIATIONS * Computation and Neural Systems, California Institute of Technology, Pasadena, California, USA Vineet Augustine & Yuki Oka * Division of Biology and Biological

Engineering, California Institute of Technology, Pasadena, California, USA Vineet Augustine, Sertan Kutal Gokce, Sangjun Lee, Bo Wang, Carlos Lois & Yuki Oka * Department of Physiology

and Kavli Institute for Fundamental Neuroscience, University of California, San Francisco, California, USA Thomas J. Davidson * Department of Clinical Biochemistry, University of Cambridge,

Cambridge, UK Frank Reimann & Fiona Gribble * Howard Hughes Medical Institute, Stanford University, Stanford, California, USA Karl Deisseroth * Department of Bioengineering, Stanford

University, Stanford, California, USA Karl Deisseroth Authors * Vineet Augustine View author publications You can also search for this author inPubMed Google Scholar * Sertan Kutal Gokce

View author publications You can also search for this author inPubMed Google Scholar * Sangjun Lee View author publications You can also search for this author inPubMed Google Scholar * Bo

Wang View author publications You can also search for this author inPubMed Google Scholar * Thomas J. Davidson View author publications You can also search for this author inPubMed Google

Scholar * Frank Reimann View author publications You can also search for this author inPubMed Google Scholar * Fiona Gribble View author publications You can also search for this author

inPubMed Google Scholar * Karl Deisseroth View author publications You can also search for this author inPubMed Google Scholar * Carlos Lois View author publications You can also search for

this author inPubMed Google Scholar * Yuki Oka View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS V.A. and Y.O. conceived the research program

and designed experiments. V.A., with assistance from S.K.G., S.L. and Y.O., carried out the experiments and analysed data. B.W. and C.L. performed all slice patch-clamp recordings. T.J.D.

and K.D. provided technical advice on setting up fibre photometry. F.R. and F.G. generated and provided _Glp1r-cre_ mice. V.A. and S.K.G. together with Y.O. wrote the paper. Y.O. supervised

the entire work. CORRESPONDING AUTHOR Correspondence to Yuki Oka. ETHICS DECLARATIONS COMPETING INTERESTS 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. ADDITIONAL INFORMATION 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. EXTENDED DATA FIGURE 3 THE SFO RECEIVES SPARSE MONOSYNAPTIC INPUT FROM MNPONNOS NEURONS. 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. EXTENDED DATA FIGURE 4 NEURAL DYNAMICS OF SFONNOS

AND MNPONNOS NEURONS. 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. EXTENDED DATA FIGURE 5 MAPPING OF INHIBITORY INPUTS TO THE SFO. 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. EXTENDED DATA FIGURE 6

THE MNPOGLP1R POPULATION DOES NOT OVERLAP WITH NNOS-EXPRESSING NEURONS. 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. EXTENDED DATA FIGURE 7 _IN VIVO_ ACTIVATION PATTERNS OF MNPOGLP1R AND SFONNOS NEURONS UPON INGESTION. 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, Δ_F_ change) were quantified for SFOnNOS neurons (GCaMP6s, dark blue bars; control, light blue bars). A significant shift in the baseline activity (Δ_F_ 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.

EXTENDED DATA FIGURE 9 NEURAL PROJECTIONS FROM NNOS+ AND GLP1R+ MNPO NEURONS. 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. SUPPLEMENTARY INFORMATION LIFE

SCIENCES REPORTING SUMMARY (PDF 80 KB) SUPPLEMENTARY INFORMATION This file contains Supplementary Table 1. (PDF 132 kb) SUPPLEMENTARY DATA This file contains RNA sequencing data. (XLSX 399

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Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 03 August 2017 * Accepted: 03 January 2018 * Published: 28 February 2018 * Issue Date: 08 March 2018 * DOI:

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