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Chemosensory modulation of neural circuits for sodium appetite

Abstract

Sodium is the main cation in the extracellular fluid and it regulates various physiological functions. Depletion of sodium in the body increases the hedonic value of sodium taste, which drives animals towards sodium consumption1,2. By contrast, oral sodium detection rapidly quenches sodium appetite3,4, suggesting that taste signals have a central role in sodium appetite and its satiation. Nevertheless, the neural mechanisms of chemosensory-based appetite regulation remain poorly understood. Here we identify genetically defined neural circuits in mice that control sodium intake by integrating chemosensory and internal depletion signals. We show that a subset of excitatory neurons in the pre-locus coeruleus express prodynorphin, and that these neurons are a critical neural substrate for sodium-intake behaviour. Acute stimulation of this population triggered robust ingestion of sodium even from rock salt, while evoking aversive signals. Inhibition of the same neurons reduced sodium consumption selectively. We further demonstrate that the oral detection of sodium rapidly suppresses these sodium-appetite neurons. Simultaneous in vivo optical recording and gastric infusion revealed that sodium taste—but not sodium ingestion per se—is required for the acute modulation of neurons in the pre-locus coeruleus that express prodynorphin, and for satiation of sodium appetite. Moreover, retrograde-virus tracing showed that sensory modulation is in part mediated by specific GABA (γ-aminobutyric acid)-producing neurons in the bed nucleus of the stria terminalis. This inhibitory neural population is activated by sodium ingestion, and sends rapid inhibitory signals to sodium-appetite neurons. Together, this study reveals a neural architecture that integrates chemosensory signals and the internal need to maintain sodium balance.

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Acknowledgements

We thank the members of the Oka laboratory and D. J. Anderson for discussion and comments; B. Lowell and M. Krashes for providing PDYN–Cre mice; A. Fejes-Toth for HSD2–Cre mice; and Y. Peng for real-time mouse tracking software. This work was supported by Startup funds from California Institute of Technology. Y.O. is supported by the Searle Scholars Program, the Mallinckrodt Foundation, the McKnight Foundation, the Klingenstein-Simons Foundation, and the National Institutes of Health (NIH) (R56MH113030, R01NS109997). D.K. is supported by the NIH (R01 DK108797 and R01 NS107315). H.E. is supported by the Japan Society for the Promotion of Science.

Reviewer information

Nature thanks Charles Bourque, Ivan de Araujo and Michael McKinley for their contribution to the peer review of this work.

Author information

S.L. and Y.O. conceived the research programme and designed the experiments. S.L. performed the experiments and analysed the data, with help from V.A. and Y.O. H.E. and B.H. performed intragastric surgery. Y.Z. performed all slice patch-clamp recordings. D.K. generated and maintained the PDYN–GFP animals. S.L. and Y.O. wrote the paper. Y.O. supervised the work.

Competing interests

The authors declare no competing interests.

Correspondence to Yuki Oka.

Extended data figures and tables

Extended Data Fig. 1 Behavioural paradigms for sodium-appetite induction and histological analysis of the pre-LC.

a, Experimental protocols for inducing thirst and sodium appetite. Intraperitoneal injection of furosemide (FURO) (50 mg per kg body weight) was used to induce sodium appetite. b, Sodium-depleted animals showed a strong preference for sodium whereas water-deprived animals preferred water over sodium (n = 9). c, Water-deprivation for 48 h induced robust c-FOS expression in the subfornical organ. However, it did not activate the pre-LC (representative image for one out of four mice that were tested). d, FISH showing that PDYN–Cre expression (visualized in the Ai3 transgenic line, green) overlaps with endogenous PDYN transcripts in the pre-LC (red, one out of two mice). e, Pre-LCPDYN neurons also overlap with FOXP2 expression, which is a known marker in the pre-LC43,44 (93.8 ± 1.1% (mean ±s.e.m.); n = 3). Scale bars, 50 μm. **P < 0.01 by two-tailed Wilcoxon test. Data are mean ± s.e.m.

Extended Data Fig. 2 Sodium appetite induced by the photostimulation of pre-LCPDYN neurons.

a, Photostimulation of pre-LCPDYN neurons increased the intake of a lower concentration of NaCl (0.06 M and 0.15 M, n = 5 for eYFP, n = 4 for ChR2). b, Photostimulation triggered sodium appetite in both sexes (left, n = 7 female, n = 4 male), at any time of the day (right, n = 7). Data were partially reanalysed from Fig. 1e and g. c, Left, pre-LCPDYN-stimulated animals favoured NaCl over KCl (n = 9). Right, NaCl consumption was reduced in the presence of amiloride (n = 8). 0.5 M solutions were used for NaCl and KCl. d, Left, representative plots showing lick events during the 5 s of water or KCl access. Right, the effect of amiloride on water and KCl intake was quantified under water deprivation and sodium depletion. The total number of licks from five trials with amiloride was averaged and divided by that without amiloride (n = 9). *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Mann–Whitney U-test or Friedman test (with Dunn’s multiple comparison). Data are mean ± s.e.m.

Extended Data Fig. 3 Optogenetic and chemogenetic inhibition of pre-LCPDYN neurons.

a, Electrophysiological recording in fresh brain slices. Illumination with 473-nm light strongly suppressed firing of pre-LCPDYN neurons expressing iC++ (10 out of 10 neurons from two mice). b, Representative image of AAV-DIO-iC++-eYFP expression in the pre-LC of a PDYN–Cre animal (one out of seven mice). c, Suppression of pre-LCPDYN neurons did not affect water intake in water-deprived animals (n = 5). d, AAV-DIO-eYFP controls for optogenetic inhibition (n = 5). e, AAV-DIO-hM4Di(Gi)-mCherry was bilaterally injected into the pre-LC. Representative recording demonstrates chemogenetic inhibition of pre-LCPDYN neurons by CNO (13 out of 14 neurons from two mice). f, Representative image of AAV-DIO-hM4Di(Gi)-mCherry expression in the pre-LC (one out of nine mice). g, Chemogenetic inhibition of pre-LCPDYN neurons reduced sodium intake in sodium-depleted animals. The same manipulation did not affect thirst (n = 9). h, CNO administration did not affect thirst or sodium appetite in animals that were injected with AAV-DIO-mCherry (n = 7). Scale bars, 50 μm. **P < 0.01 by two-tailed Wilcoxon test. Data are mean ± s.e.m.

Extended Data Fig. 4 Training paradigm for negative reinforcement assay.

a, A diagram of the training paradigm using foot shock (FS). Each lever press pauses continuous foot shock for 20 s. b, Total number of lever presses in each condition during the 30-min session (n = 5 for eYFP and n = 6 for ChR2). c, Animals were conditioned to press the lever without foot-shock pre-training sessions (n = 6). *P < 0.05 by two-tailed Wilcoxon test. Data are mean ± s.e.m.

Extended Data Fig. 5 In vivo activity of pre-LCPDYN neurons after ingestive behaviours.

a, Placement of an implanted optic fibre and GCaMP6s expression in the pre-LC. Scale bar, 50 μm. b, A low concentration (0.06 M) of NaCl had inhibitory effects on pre-LCPDYN neurons (n = 7). c, Licking an empty spout had no inhibitory effect on pre-LCPDYN neurons (n = 4 for eYFP, n = 4 for GCaMP6s). d, Peristimulus time histogram of GCaMP signals around the start of sodium ingestion. Data were magnified from c and Fig. 3a. Fluorescence changes (ΔF/F) from −1 to 0 s were calculated. e, Activity change per lick was quantified for Fig. 3d and e. *P < 0.05, **P < 0.01 by two-tailed Wilcoxon or two-tailed Mann–Whitney U-test. Data are mean ± s.e.m.

Extended Data Fig. 6 Functional analysis of the NTSHSD2→pre-LCPDYN connections.

a, Functional validation of PDYN–GFP transgenic animals. Similar to the PDYN–Cre line, GFP-positive neurons in the pre-LC were activated by sodium depletion in PDYN–GFP mice (one out of two mice). b, A diagram of optogenetic stimulation of NTSHSD2 neurons. FOXP2-positive pre-LC neurons express c-FOS after HSD2 stimulation (n = 6 hemispheres from 3 mice). c, Relationship between the number of NTSHSD2 neurons in the NTS and c-FOS-positive neurons in the pre-LC. More than 95% of c-FOS-positive neurons expressed FOXP2. d, The number of FOXP2-positive neurons was not affected by the ablation of NTSHSD2 neurons (n = 18 hemispheres from 9 mice for +CASP3, and n = 8 hemispheres from 4 mice for –CASP3). Scale bars, 50 μm. **P < 0.01 by two-tailed Wilcoxon test. Data are mean ± s.e.m.

Extended Data Fig. 7 Histological analysis of putative upstream brain structures of pre-LCPDYN neurons.

a, Control monosynaptic tracing experiments without rabies glycoprotein (one out of three mice). Scale bars, 100 μm. b, Most PDYN neurons in the dBNST (green) are inhibitory neurons (red, 77.3 ± 1.7% (mean ± s.e.m.); n = 3). c, CAV2-positive neurons in the dBNST, labelled in a retrograde manner from the pre-LC (red), are inhibitory neurons (one out of three mice). Scale bars, 50 μm.

Extended Data Fig. 8 Downstream projections of pre-LCPDYN neurons.

a, PDYN–Cre mice were injected with AAV-DIO-ChR2-mCherry into the pre-LC. Representative axonal projections are shown (one out of six mice). ARC, arcuate nucleus; DMH, dorsomedial hypothalamic nucleus; LH, lateral hypothalamus; PVT, paraventricular thalamic nucleus; vBNST, ventral bed nucleus of the stria terminalis; VTA, ventral tegmental area. Scale bars, 50 μm. b, A wiring diagram of upstream and downstream neural connections of pre-LCPDYN neurons. It is feasible that the ventral tegmental area and lateral hypothalamus process the reward aspect of sodium appetite45,46, whereas the BNST and paraventricular thalamic nucleus may regulate preference and valence towards sodium47,48. Besides the hindbrain, the BNST also receives interoceptive information from subfornical neurons that express angiotensin receptor (SFOAgtr1a)15.

Supplementary information

Supplementary Table 1

Reporting Summary

Supplementary Video 1

Robust licking behaviour toward rock salt induced by the photostimulation of pre-LCPDYN neurons. Light was delivered for 1 sec every 4 sec.

Supplementary Video 2

A control experiment of video 1 without photostimulation.

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Fig. 1: Genetic and functional identification of sodium-appetite neurons in the pre-LC.
Fig. 2: Activation of pre-LCPDYN neurons drives an aversive motivational signal.
Fig. 3: Sodium-appetite neurons are rapidly modulated by chemosensory signals of sodium.
Fig. 4: Oral sodium detection promotes satiation of sodium appetite by suppressing pre-LCPDYN neurons.
Fig. 5: Pre-LCPDYN neurons receive both homeostatic and sensory inputs.
Extended Data Fig. 1: Behavioural paradigms for sodium-appetite induction and histological analysis of the pre-LC.
Extended Data Fig. 2: Sodium appetite induced by the photostimulation of pre-LCPDYN neurons.
Extended Data Fig. 3: Optogenetic and chemogenetic inhibition of pre-LCPDYN neurons.
Extended Data Fig. 4: Training paradigm for negative reinforcement assay.
Extended Data Fig. 5: In vivo activity of pre-LCPDYN neurons after ingestive behaviours.
Extended Data Fig. 6: Functional analysis of the NTSHSD2→pre-LCPDYN connections.
Extended Data Fig. 7: Histological analysis of putative upstream brain structures of pre-LCPDYN neurons.
Extended Data Fig. 8: Downstream projections of pre-LCPDYN neurons.

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