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A neural basis for tonic suppression of sodium appetite

Nature Neuroscience volume 23, pages 423–432 (2020)Cite this article

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Abstract

Sodium appetite is a powerful form of motivation that can drive ingestion of high, yet aversive concentrations of sodium in animals that are depleted of sodium. However, in normal conditions, sodium appetite is suppressed to prevent homeostatic deviations. Although molecular and neural mechanisms underlying the stimulation of sodium appetite have received much attention recently, mechanisms that inhibit sodium appetite remain largely obscure. Here we report that serotonin 2c receptor (Htr2c)-expressing neurons in the lateral parabrachial nucleus (LPBNHtr2c neurons) inhibit sodium appetite. Activity of these neurons is regulated by bodily sodium content, and their activation can rapidly suppress sodium intake. Conversely, inhibition of these neurons specifically drives sodium appetite, even during euvolemic conditions. Notably, the physiological role of Htr2c expressed by LPBN neurons is to disinhibit sodium appetite. Our results suggest that LPBNHtr2c neurons act as a brake against sodium appetite and that their alleviation is required for the full manifestation of sodium appetite.

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Fig. 1: LPBNHtr2c neurons are regulated by bodily sodium content.
Fig. 2: Activation of LPBNHtr2c neurons suppresses sodium intake.
Fig. 3: Inhibition of LPBNHtr2c neurons increases sodium intake.
Fig. 4: Stimulation of the LPBNHtr2c → CeA projection suppresses sodium intake.
Fig. 5: Htr2c inhibits LPBN neurons via KATP channels.
Fig. 6: Htr2c in the LPBN is necessary to disinhibit sodium appetite during hypovolemia.

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The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Code availability

Custom code used in this study is available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank G.S.B. Suh (KAIST) for helpful comments on the manuscript, S.-H. Lee (KAIST) for technical help with viral tracing experiments and D. Kim (KAIST) for technical help with imaging. This work was supported by grants from the Samsung Science & Technology Foundation (SSTF-BA1901-11 to J.-W.S.), the KAIST Future Systems Health Care Project (to J.-W.S.), the KAIST Venture Research Program for Graduate and PhD students (to S.P.), the American Heart Association (16SDG27260001 to C.L.) and the National Institutes of Health (R01 DK114036 to C.L. and R01 DK100699, R01 DK119169, DK119130 5830 to K.W.W.). S.P. was supported in part by stipends from the National Research Foundation of Korea (NRF-2015M3A9E7029177 and NRF-2016R1C1B2006614 to J.-W.S.).

Author information

Authors and Affiliations

  1. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea

    Seahyung Park & Jong-Woo Sohn

  2. The Center for Hypothalamic Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Kevin W. Williams & Chen Liu

  3. Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Chen Liu

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Contributions

S.P. and J.-W.S. designed the experiments. S.P. conducted experiments and analyzed data. C.L. and K.W.W. provided reagents and expertise. C.L. generated and validated Htr2c-2A-iCre mice. S.P. and J.-W.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Chen Liu or Jong-Woo Sohn.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks Yuki Oka and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 LPBN neurons respond to hypertonic saline.

Hypertonic saline-loaded mice show more Fos in the LPBN than saline-loaded controls. Two-tailed Mann-Whitney test, Saline N = 5 mice, Hypertonic saline N =5 mice. P = 0.0317. All data represented as mean ± s.e.m. *P < 0.05.

Extended Data Fig. 2 Generation of an Htr2c-2A-iCre mouse line.

(a) Htr2c-2A-iCre knock-in mice in which the coding sequences of Htr2c and iCre are linked by a viral peptide bridge 2A sequence. (b) Detection of wildtype and targeted Htr2c alleles by PCR genotyping. Similar results were obtained with at least 3 independent replications. (c) Immunohistochemical analysis of Htr2c-2A-iCre activity using a tdTomato reporter. Note high levels of tdTomato expression in the choroid plexus (CP). CC = corpus callosum. Similar results were obtained with at least 3 mice. Scale bar = 100 μm Source data.

Source data

Extended Data Fig. 3 EYFP-expressing LPBNHtr2c neurons are inhibited during sodium depletion.

(a) Cell-attached recordings taken from Htr2c-2A-iCre mice injected with AAV5-E1fa-DIO-EYFP. (b) Representative cell-attached recording of LPBNHtr2c neurons after 7 days of control diet, or 7 days of sodium-deficient diet. (c) Firing frequency of LPBNHtr2c neurons between control diet-fed mice (left) and sodium-deficient diet-fed mice (right). Two-tailed Mann-Whitney test. Control diet n = 21 cells, sodium-deficient diet n = 24 cells. Control diet 1.2 ± 0.3 Hz, Sodium-deficient diet 0.4 ± 0.2 Hz, P = 0.007. All data represented as mean ± s.e.m. **P < 0.01.

Extended Data Fig. 4 CNO injections in AAV-DIO-mCherry-injected controls do not affect food intake, water intake or 300 mM NaCl intake.

(a) Htr2c-2A-iCre mice injected with AAV2-hSyn-DIO-mCherry showed no changes in food intake, body weight or 300 mM NaCl intake. (b) No changes in water intake or 300 mM NaCl intake during sodium depletion. N = 5 mice. Water intake during sodium depletion: Two-Way Repeated Measures ANOVA: time (F(4, 16) = 20.52, P < 0.0001), treatment (F(1, 4) = 0.5421, P = 0.5024), interaction (F(4, 16) = 0.6448, P = 0.6385). 300 mM NaCl intake during sodium depletion: Two-Way Repeated Measures ANOVA: time (F(4,16) = 108.1, P < 0.0001), treatment (F(1, 4) = 0.1128, P = 0.7538), interaction (F(4, 16) = 0.3142, P = 0.8643). (c) No changes in food intake or body weight when using a control diet. N = 5 mice. Control diet food intake: Two-Way Repeated Measures ANOVA: time (F(3,12) = 223.9, P < 0.0001), treatment (F(1, 4) = 0.2222, P = 0.6619), interaction (F(3,12) = 2.689, P = 0.0924). All data represented as mean ± s.e.m.

Extended Data Fig. 5 LPBNHtr2c neuron activation can cause non-sodium related effects.

(a) Unilateral injection of AAV5-hSyn-DIO-hM3Dq into the LPBN of Htr2c-2A-iCre mice. (b) Activation of LPBNHtr2c neurons decreased water and 300 mM NaCl intake during dehydration in a two-bottle choice assay. N =11. Water intake during dehydration: Two-Way Repeated Measures ANOVA: time (F(4,40) = 67.06, P < 0.0001), treatment (F(1, 10) = 8.707, P = 0.0145), interaction (F(4, 40) = 6.329, P = 0.0005). Post hoc tests using the Bonferroni correction. 300 mM NaCl intake during dehydration: Two-Way Repeated Measures ANOVA: time (F(4,40) = 31.38, P < 0.0001), treatment (F(1, 10) = 17.76 P = 0.0018), interaction (F(4, 40) = 14.06 P = 0.0001). (c) Activation of LPBNHtr2c neurons decreased food during a fast refeeding regardless of sodium content. N = 11. Control diet food intake (left): Two-Way Repeated Measures ANOVA: time (F(3,30) = 111.0, P < 0.0001), treatment (F(1, 10) = 51.2, P < 0.0001), interaction (F(3, 30) = 32.00, P < 0.0001). Sodium-deficient diet food intake (right): Two-Way Repeated Measures ANOVA: time (F(3,30) = 50.59, P < 0.0001), treatment (F(1, 10) = 27.74, P = 0.0004), interaction (F(3, 30) = 18.81, P < 0.0001). All post hoc tests using the Bonferroni correction. All data represented as mean ± s.e.m. ****P < 0.0001.

Extended Data Fig. 6 LPBNHtr2c neurons colocalise with CGRP.

(a) Schematic of experiment. Unilateral injection of AAV5-EF1a-DIO -EYFP into the LPBN of Htr2c-2A-iCre mice to visualise LPBNHtr2c neurons (left). Colocalisation of EYFP labelled LPBNHtr2c neurons with CGRP (right). Cyan = EYFP, Magenta = CGRP, Blue = DAPI. Scale bar = 100 μm. (b) Estimation of colocalisation between LPBNHtr2c neurons and CGRP neurons. 3 slices taken from each mice. N = 7 mice.

Extended Data Fig. 7 Downstream circuitry of LPBNHtr2c neurons.

(a) Axonal projections visualised by unilateral injection of AAV5-EF1a-DIO-EYFP into the LPBN of Htr2c-2A-iCre mice. aca = anterior commissure (anterior part), acp = anterior commissure (posterior part), dBNST = bed nucleus of the stria terminalis (lateral division), fr = fasciculus retroflexus, ml = medial lemniscus, MnPO = median preoptic nucleus, opt = optic tract, PVH = paraventricular nucleus of the hypothalamus, scp = superior cerebellar peduncle, SPF = subparafascicular thalamus, st = stria terminalis, vBNST = bed nucleus of the stria terminalis (ventral part), VPPC = ventral posterior thalamus (parvicellular part). Scale bar = 400 μm. N = 7 mice. (b) Schematic of experiment. Bilateral injection of AAV2-DIO-hChR2 (H134R)-p2A-mCherry into the LPBN of Htr2c-2A-iCre mice (left). Voltage-clamp recordings taken at downstream sites (traces on the right). Blue rectangles indicate times of photostimulation.

Extended Data Fig. 8 Illustration showing the approximate spatial distribution of mCPP-responsive neurons within the LPBN.

C = central LPBN, D = dorsal LPBN, E = external LPBN, I = internal LPBN, KF = Kolliker’s Fuse, V = ventral LPBN.

Extended Data Fig. 9 Hyperpolarisation of LPBNHtr2c neurons is not dependent on Htr1b and is postsynaptic.

(a) Representative current clamp recording of an LPBNHtr2c neuron pre-treated with SB216641 (200 nM). Hyperpolarisation in response to mCPP persists in presence of SB216641. Arrows indicate time at which current steps were applied. (b) Voltage deflections in response to hyperpolarising currents from the same neuron, showing decreased input resistance in response to mCPP. Current steps in trace made in 10 pA increments from -50 pA to 0 pA. (c) Representative current clamp recording of an LPBNHtr2c neuron pre-treated with TTX (500 nM), kynurenic acid (KA, 1 mM) and picrotoxin (PTX, 50 μM), showing hyperpolarisation in response to mCPP. Arrows indicate time at which current steps were applied. (d) Voltage deflections in response to hyperpolarising currents from the same neuron, showing decreased input resistance in response to mCPP. Current steps in trace made in 10 pA increments from -50 pA to 0 pA. (e) Representative current clamp recording of an LPBNHtr2c neuron from a fasted mouse (18 hours), showing hyperpolarisation in response to mCPP. Arrows indicate time at which current steps were applied. (f) Voltage deflections in response to hyperpolarising currents from the same neuron, showing decreased input resistance in response to mCPP. Current steps in trace made in 10 pA increments from -50 pA to 0 pA. (g) Graph summarising change in membrane potential in response to mCPP under conditions tested. Neither pre-treatment with SB216641, Synaptic blockers nor recordings from fasted mice changed the magnitude of response to mCPP. mCPP n = 10 cells, SB216641 n = 5 cells, Synaptic block n = 3 cells, Fasted n = 8 cells. Data represented as mean ± s.e.m.

Extended Data Fig. 10 Serotoninergic projections to the LPBN.

(a) Schematic of experiment. Unilateral injection of Lumafluor retrobeads into the LPBN of wildtype mice to visualise upstream neurons. (b) Arrows indicate colocalised cells. Aq = aqueduct, DR = dorsal raphe. Scale bar = 200 μm. N = 6 mice (upper). MnR = median raphe. Scale bar = 100 μm. N = 6 mice (lower).

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Supplementary Table 1: Adjusted P values for post hoc comparisons.

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Park, S., Williams, K.W., Liu, C. et al. A neural basis for tonic suppression of sodium appetite. Nat Neurosci 23, 423–432 (2020). https://doi.org/10.1038/s41593-019-0573-2

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