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Oxytocin-receptor-expressing neurons in the parabrachial nucleus regulate fluid intake

Nature Neurosciencevolume 20pages17221733 (2017) | Download Citation

Abstract

Brain regions that regulate fluid satiation are not well characterized, yet are essential for understanding fluid homeostasis. We found that oxytocin-receptor-expressing neurons in the parabrachial nucleus of mice (OxtrPBN neurons) are key regulators of fluid satiation. Chemogenetic activation of OxtrPBN neurons robustly suppressed noncaloric fluid intake, but did not decrease food intake after fasting or salt intake following salt depletion; inactivation increased saline intake after dehydration and hypertonic saline injection. Under physiological conditions, OxtrPBN neurons were activated by fluid satiation and hypertonic saline injection. OxtrPBN neurons were directly innervated by oxytocin neurons in the paraventricular hypothalamus (OxtPVH neurons), which mildly attenuated fluid intake. Activation of neurons in the nucleus of the solitary tract substantially suppressed fluid intake and activated OxtrPBN neurons. Our results suggest that OxtrPBN neurons act as a key node in the fluid satiation neurocircuitry, which acts to decrease water and/or saline intake to prevent or attenuate hypervolemia and hypernatremia.

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Acknowledgements

We thank B. Roth and K. Deisseroth for AAV plasmid constructs. M. Chiang assisted with animal husbandry. S. Tsang and K. Kafer helped to generate the Oxtr Cre mice. We thank J. Chen for help with surgery for Calca Cre/+ experiments and C. Roman for help with surgery for Cck Cre/+ experiments. M. McKinley, B. Jarvie, C. Roman and members of the Palmiter lab provided helpful discussion and feedback. P.J.R. was supported by an Australian American Fellowship and a National Health and Medical Council of Australia CJ Martin Fellowship. C.A.C. was supported by a fellowship from Hope Funds for Cancer Research. R.D.P. was supported by a US National Institutes of Health grant (R01-DA24908). Inscopix provided the calcium-imaging equipment and supplies via their DECODE grant program.

Author information

Affiliations

  1. Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA

    • Philip J. Ryan
    • , Silvano I. Ross
    • , Carlos A. Campos
    • , Victor A. Derkach
    •  & Richard D. Palmiter
  2. Department of Biochemistry, University of Washington, Seattle, Washington, USA

    • Philip J. Ryan
    • , Silvano I. Ross
    • , Carlos A. Campos
    • , Victor A. Derkach
    •  & Richard D. Palmiter
  3. The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia

    • Philip J. Ryan

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Contributions

P.J.R. and R.D.P. conceived and designed the study. P.J.R. performed and analyzed the experiments. S.I.R and P.J.R. performed the immunohistochemistry and counting of cells. V.A.D. performed electrophysiological experiments. C.A.C. performed GCaMP6 studies. R.D.P. generated Oxtr Cre mice and provided equipment and reagents. P.J.R. wrote the manuscript with input from R.D.P and other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Philip J. Ryan or Richard D. Palmiter.

Integrated supplementary information

  1. Supplementary Figure 1 Generation of Oxtr Cre:GFP mice

    Diagram showing: top, insertion of ires-mnCre:GFP construct just 3’ of the termination codon of the Oxtr gene; bottom, the targeting vector. Some key restriction enzymes sites used for cloning are shown. See Methods for details

  2. Supplementary Figure 2 Oxytocin receptor expression in the parabrachial nucleus

    (a) Coronal sections 90 µm apart from Oxtr Cre/+ ::Ai14 mouse demonstrating oxytocin receptor (Oxtr) expression in the parabrachial nucleus (PBN) from bregma −5.1 to −5.5; scale bar, 500 µm. (b) Selection of brain images demonstrating robust Oxtr expression; AD, anterodorsal thalamic nucleus; CeA, central nucleus of amygdala; EPd, dorsal endopiriform nucleus; DMV, dorsal motor nucleus of the vagus; DR, dorsal raphé nucleus; GP, globus pallidus; XII, hypoglossal nucleus; MD, mediodorsal thalamic nucleus; NAc, nucleus accumbens; PBN, parabrachial nucleus; PVT, paraventricular thalamic nucleus; pVH, periventricular nucleus of the hypothalamus; SFO, subfornical organ; V, trigeminal motor nucleus; VMH, ventromedial hypothalamic nucleus; scale bar, 500 µm (n = 3). (c) Representative RNAscope® image of PBN demonstrating coexpression of Oxtr mRNA in 80 ± 3% Oxtr:TdTomato-expressing neurons (n = 3). Scale bar, 100 μm; scp, superior cerebellar peduncle. (d) Oxtr agonist, TGOT, increased spiking frequency in OxtrPBN neurons by 3.7 ± 0.55-fold, which was inhibited by Oxtr antagonist, atosiban to 1.6 ± 0.14 fold (n = 4/4 OxtrPBN neurons). Data were normalized to spiking frequency prior to TGOT application

  3. Supplementary Figure 3 Chemogenetic activation of OxtrPBN neurons decreases fluid intake acutely

    (a) Overnight NaCl was not significantly different, but overnight water intake remained slightly decreased following CNO injection in hM3Dq-injected vs control mCherry-injected mice after 24-h dehydration (n = 7/group; unpaired 2-tailed Student’s t-test; NaCl: t(12) = 0.5656; p = 0.5821; water: t(12) = 2.311; p = 0.0394). (b) Control mCherry-injected mice consumed more NaCl and less water when food was absent; but there was no significant difference in fluid intake after CNO injection in hM3Dq-injected mice (n = 7/group in food present; 6/group in food absent; 2-way ANOVA; NaCl: interaction F(1,22) = 0.7401; p = 0.3989; main effect of viral genotype F(1,22) = 32.44; p < 0.0001; main effect of food availability F(1,22) = 11.77; p = 0.0024; water: interaction F(1,22) = 4.946; p = 0.0367). (c) Overnight baseline fluid intake was not significantly different following vehicle or CNO injection (n = 7/group; 2-way RM ANOVA; NaCl: interaction F(1,12) = 1.806; p = 0.2038; water: interaction F(1,12) = 0.1977; p = 0.6645). d,e, Acute OxtrPBN activation demonstrated no significant difference in Ensure or water intake when Ensure was limited to (d) ~1.6 mL (n = 6/group; 2-way RM ANOVA; Ensure: interaction F(8,80) = 1.168; p = 0.3285; water: interaction F(8,80) = 1.027; p = 0.4228) or (e) ~0.7 mL (n = 6/group; 2-way RM ANOVA; Ensure: interaction F(8,80) = 1.182; p = 0.3202; water: interaction F(8,80) = 1.313; p = 0.2489). (f) Overnight fluid intake was not significantly different after hypertonic saline ip injection in hM3Dq- or vs mCherry-injected mice (n = 7/group; unpaired 2-tailed Student’s t-test; NaCl: t(12) = 0.2868; p = 0.7791; water: t(12) = 1.354; p = 0.2008). (g) Daily intake of NaCl and water was not significantly different between the start and end of experimentation (n = 7/group; 2-way RM ANOVA; NaCl: interaction F(1,12) = 0.1273; p = 0.7275; water: interaction: F(1,12) = 4.243; p = 0.0618). (h) Percentage Fos in OxtrPBN neurons in hM3Dq-injected vs mCherry-injected Oxtr Cre/+ mice (n = 7/group; unpaired 2-tailed Student’s t-test; t(12) = 19.28; p < 0.0001). Data expressed as mean ± s.e.m. ****p < 0.0001; **p < 0.01; *p < 0.05. See Supplementary Information for statistical analyses

  4. Supplementary Figure 4 Chronic OxtrPBN neuron inactivation does not affect fluid intake at baseline or following various homeostatic challenges

    (a) Injection of AAV-DIO-GFP:TetTox in OxtrPBN neurons. (b) Representative images of GFP expression in OxtrPBN neurons in GFP:TetTox- vs control YFP-injected mice; scp, superior cerebellar peduncle; dl, dorsolateral; el, external lateral; scale bar 100 µm. (c) Chronic OxtrPBN inactivation does not significantly alter baseline saline preference at different concentrations (0.075, 0.3, 0.5 M) (n = 6 TetTox, 7 YFP; unpaired 2-tailed Student’s t-test; 0.075 M NaCl: t(11) = 0.4967; p  = 0.6292; 0.3 M NaCl: t(11) = 1.005; p = 0.3364; 0.5 M NaCl: t(11) = 0.09961; p = 0.9224), and does not significantly change fluid intake following normal saline ip, either (d) acutely (n = 6 TetTox, 7 YFP; 2-way RM ANOVA; NaCl: interaction F(8,88) = 0.7729; p = 0.6275; water: interaction F(8,88) = 0.4721; p = 0.8728) or (e) overnight (n = 6 TetTox, 7 YFP; unpaired 2-tailed Student’s t-test; NaCl: t(11) = 0.8014; p = 0.4398; water: t(11) = 0.6858; p = 0.5070). (f) Overnight NaCl intake remained significantly increased in TetTox-injected mice (n = 6 TetTox, 7 YFP; unpaired 2-tailed Student’s t-test; NaCl: t(11) = 2.760; p = 0.0186; water: t(11) = 0.8881; p = 0.3935). Chronic OxtrPBN inactivation does not significantly change fluid intake following (g) 1 M mannitol ip (n = 6 TetTox; 7 YFP; 2-way RM ANOVA; NaCl: interaction F(4,44) = 0.4638; p = 0.7619; water: interaction F(4,44) = 1.524; p = 0.2118) or (h) 30% PEG sc (n = 6 TetTox; 7 YFP; 2-way RM ANOVA; NaCl: interaction F(4,44) = 1.494; p = 0.2203; water: interaction F(4,44) = 0.3461; p = 0.8453). (i) There was no significant correlation between the level of GFP:TetTox expression and the amount of fluid intake after 24-h dehydration (n = 6; Pearson product-moment correlation; NaCl: r = 0.3167; p = 0.5408; water: r = 0.4335; p = 0.3905). Data are expressed as mean ± s.e.m. *p < 0.05. See Supplementary Information for statistical analyses

  5. Supplementary Figure 5 Acute OxtrPBN neuron inhibition increases fluid intake after dehydration, but does not affect baseline food or fluid intake

    (a) Injection of AAV-DIO-hM4Di:mCherry in OxtrPBN neurons. (b) Representative images of hM4Di and mCherry expression in OxtrPBN neurons; scp, superior cerebellar peduncle; dl, dorsolateral; el, external lateral; scale bar 100 µm. (c) Acute OxtrPBN inhibition increases NaCl and water intake following 24-h dehydration (n = 5 hM4Di, 6 mCherry; 2-way RM ANOVA; NaCl: interaction F(8,72) = 2.962; p = 0.0064; water: interaction F(8,72) = 2.289; p = 0.0304), but (d) does not significantly alter fluid intake at baseline during the light cycle (n = 5 hM4Di, 6 mCherry; 2-way RM ANOVA; NaCl: interaction F(8,72) = 0.1264; p = 0.9979; water: interaction F(8,72) = 0.6017; p = 0.7734). (e) OxtrPBN inhibition does not significantly alter food intake during the dark or light cycle (n = 5 hM4Di, 6 mCherry; 2-way RM ANOVA; baseline food: interaction F(8,72) = 0.2429; p = 0.9811; post 24-h fast: interaction F(8,72) = 1.040; p = 0.4510). (f) There was no significant correlation between the level of hM4Di:mCherry expression and the amount of fluid intake after 24-h dehydration (n = 5; Pearson product-moment correlation; NaCl: r = 0.6649; p = 0.2208; water: r = −0.6857; p = 0.2013). Data are expressed as mean ± s.e.m. **p < 0.01; *p < 0.05. See Supplementary Information for statistical analyses

  6. Supplementary Figure 6 OxtrPBN neurons in mid and rostral PBN show no significant difference in Fos expression

    a,c,e, Quantification of mid OxtrPBN co-expression of Fos and Oxtr in (a) salt depletion (n = 4 salt returned, 4 salt depleted, 3 control; 1-way ANOVA; Fos/Oxtr: interaction F(2,7) = 2.727; p = 0.1331; Oxtr/Fos: interaction F(2,7) = 0.3097; p = 0.7432), (c) fluid deprivation (n = 4 fluid returned, 3 fluid deprived; unpaired 2-tailed Student’s t-test; Fos/Oxtr: t(5) = 1.335; p = 0.2394; Oxtr/Fos: t(5) = 0.4049; p = 0.7023) and (e) and hypertonic saline experiments (n = 4 for 1 M saline, 3 normal saline; unpaired 2-tailed Student’s t-test; Fos/Oxtr: t(5) = 0.5084; p = 0.6328; Oxtr/Fos: t(5) = 1.319; p = 0.2444). b,d,f, Quantification of rostral OxtrPBN co-expression of Fos and Oxtr in (b) salt depletion (n = 4 salt returned, 4 salt depleted, 3 control; 1-way ANOVA; Fos/Oxtr: interaction F(2,7) = 2.008; p = 0.2046; Oxtr/Fos: interaction F(2,7) = 1.582; p = 0.2711), (d) fluid deprivation (n = 4 fluid returned, 3 fluid deprived; unpaired 2-tailed Student’s t-test; Fos/Oxtr: t(5) = 0.5977; p = 0.5761; Oxtr/Fos: t(5) = 0.3396; p = 0.7479) and (f) and hypertonic saline (n = 4 for 1 M saline, 3 normal saline; unpaired 2-tailed Student’s t-test; Fos/Oxtr: t(5) = 1.716; p = 0.1469; Oxtr/Fos: t(5) = 2.300; p = 0.0698). Data are expressed as mean ± s.e.m. See Supplementary Information for statistical analyses

  7. Supplementary Figure 7 Downstream and upstream projections of OxtrPBN neurons

    (a) Injection of AAV-DIO-synaptophysin:mCherry in OxtrPBN neurons demonstrates less prominent downstream projections to (b) the nucleus of the solitary tract (NTS), lateral posterior thalamus, periventricular hypothalamus, tuberal nucleus, paraventricular nucleus of the hypothalamus (PVH) and supraoptic nucleus (SON); (n = 2) scale bar, 200 µm. (c) Percentage Fos in caudal, mid and rostral OxtrPBN neurons in Oxt Cre/+ ::Oxtr Cre/+ mice injected with hM3Dq or mCherry in PVH (n = 3/group; unpaired 2-tailed Student’s t-test; caudal: t(4) = 4.385; p = 0.0118; mid: t(4) = 1.807; p = 0.1451; rostral: t(4) = 2.278; p = 0.0850). (d) Injection of AAV1-DIO-synaptophysin:mCherry in PVH of Oxtr Cre/+ mice demonstrates no visible projections in PBN (n = 3); 3V, third ventricle; thal, thalamic nuclei; scp, superior cerebellar peduncle; dl, dorsolateral PBN; el, external lateral PBN; scale bar, 100 µm. (e) Injection of AAV-DIO-hM3Dq into the PVH and AAV-DIO-YFP into the PBN of Oxtr Cre/+ mice. (f) Following CNO, Fos is robustly expressed in the PVH and thalamus, but (g) there was minimal expression in the PBN (n = 3). (h) Representative live image of the lateral PBN prior to electrophysiological recordings demonstrating OxtPVH fibers and OxtrPBN neurons; scale bar, 200 μm. (i) Acute OxtPVH stimulation decreases water intake following 1 M mannitol ip (n = 7 hM3Dq, 8 mCherry; 2-way RM ANOVA; NaCl: interaction F(4,52) = 0.6187; p = 0.6512; water: interaction F(4,52) = 4.120; p = 0.0057), but (j) does not significantly alter fluid intake following 48-h salt appetite (n = 7 hM3Dq, 8 mCherry; 2-way RM ANOVA; NaCl: interaction F(8,104) = 1.056; p = 0.3999; water: interaction F(8,104) = 0.6962; p = 0.6941) or (k) 30% PEG sc (n = 7 hM3Dq, 8 mCherry; 2-way RM ANOVA; NaCl: interaction F(4,52) = 0.3265; p = 0.8589; water: interaction F(4,52) = 0.5231; p = 0.7191), or (l) food intake at baseline or after a 24-h fast (n = 7 hM3Dq, 8 mCherry; 2-way RM ANOVA; baseline food: interaction F(8,104) = 0.3734; p = 0.9325; post 24-h fast: interaction F(8,104) = 0.2928; p = 0.9670). (m) Percentage Fos in OxtPVH neurons in hM3Dq-injected vs mCherry-injected Oxt Cre/+ mice (n = 7 hM3Dq, 8 mCherry; unpaired 2-tailed Student’s t-test; t(13) = 12.50; p < 0.0001). Data are expressed as mean ± s.e.m. ****p < 0.0001; *p < 0.05. See Supplementary Information for statistical analyses

  8. Supplementary Figure 8 Chronic OxtPVH inactivation increases water intake following dehydration, salt depletion, mannitol and PEG

    (a) Injection of AAV-DIO-GFP:TetTox in OxtPVH neurons. (b), Representative images of GFP expression in OxtPVH neurons in GFP:TetTox- vs control YFP-injected mice; 3V, third ventricle; (n = 6 TetTox, 8 YFP) scale bar 100 µm. (c) Chronic OxtPVH inactivation does not significantly alter baseline saline preference at different concentrations (0.075, 0.3, 0.5 M) (n = 6 TetTox, 8 YFP; unpaired 2-tailed Student’s t-test; 0.075 M NaCl: interaction t(12) = 0.2346; p = 0.8184; 0.3 M NaCl: interaction t(12) = 0.2509; p = 0.8061; 0.5 M NaCl: interaction t(12) = 0.6848; p = 0.5065), but (d) but significantly increases water following 24-h dehydration (n = 6 TetTox, 8 YFP; 2-way RM ANOVA; NaCl: interaction F(8,96) = 1.098; p = 0.3712; water: interaction F(8,96) = 4.073; p = 0.0003), (e) 48-h salt depletion (n = 6 TetTox, 8 YFP; 2-way RM ANOVA; NaCl: interaction F(8,96) = 0.7341; p = 0.6612; water: interaction F(8,96) = 6.866; p < 0.0001), (f) demonstrates a trend to increase water following 0.5 M saline ip (n = 6 TetTox, 8 YFP; 2-way RM ANOVA; NaCl: interaction F(4,48) = 0.1292; p = 0.9711; water: interaction F(4,48) = 2.183; p = 0.0850), and (g) increases water following 1 M mannitol ip (n = 6 TetTox, 8 YFP; 2-way RM ANOVA; NaCl: interaction F(4,48) = 0.8378; p  = 0.5080; water: interaction F(4,48) = 3.628; p = 0.0116), and (h) and 30% PEG sc (n = 6 TetTox, 8 YFP; 2-way RM ANOVA; NaCl: interaction F(4,48) = 1.296; p = 0.2848; water: interaction F(4,48) = 3.617; p = 0.0118). Data are expressed as mean ± s.e.m. ****p < 0.0001; **p < 0.01; *p < 0.05; φ p = 0.085. See Supplementary Information for statistical analyses

  9. Supplementary Figure 9 Characterization of upstream projections to OxtrPBN neurons

    (a) Injection of AAV-DIO-hM3Dq:mCherry in OxtPVH neurons of Oxt Cre/+ mice and bilateral cannula into PBN. (b) Acute OxtPVH activation by CNO ip decreases 2-h water consumption after 24-h dehydration during the light cycle (n = 6/group; unpaired 2-tailed Student’s t-test; NaCl: interaction t(10) = 0.3017; p = 0.7690; water: interaction t(10) = 2.484; p = 0.0323), but (c) demonstrates no significant change in 2-h NaCl or water consumption at baseline (n = 6/group; 2-way RM ANOVA; NaCl: interaction F(1,10) = 0.9098; p = 0.3627; water: interaction F(1,10) = 2.118; p = 0.1763). (d) Representative histological image demonstrating cannula placement in PBN (n = 6/group). (e) Injection of AAV-DIO-hM3Dq:mCherry in OxtPVH neurons and AAV-DIO-YFP in OxtrPBN neurons of Oxt Cre/+ ::Oxtr Cre/+ mice and bilateral cannula into PBN. (f) Following intra-PBN CNO, we saw some Fos expression in OxtPVH neurons, (g) in OxtrPBN neurons, and (h) some scattered Fos in the NTS (n = 3). (i) Injection of AAV-hM3Dq:mCherry in NTS and AAV-DIO-YFP in OxtrPBN neurons of Oxtr Cre/+ mice. (j) Following CNO ip, we observed robust Fos expression in hM3Dq-expressing NTS neurons (n = 6) and (k) minimal Fos expression in control DsRed-expressing NTS neurons (n = 5). (l) Injection of AAV-DIO-hM3Dq:mCherry in CCKNTS neurons of Cck Cre/+ mice. m,n, Following CNO ip, we observed Fos in the (m) NTS and (n) PBN of hM3Dq-injected mice (n = 5 hM3Dq, 4 mCherry). Data are expressed as mean ± s.e.m. dl, dorsolateral PBN; el, external lateral PBN; scp, superior cerebellar peduncle. *p < 0.05. Scale bars, 100 μm. See Supplementary Information for statistical analyses

  10. Supplementary Figure 10 Model of neural circuits that suppress fluid intake

    (a) Model of OxtrPBN neural circuitry illustrating OxtPVH projections to OxtrPBN neurons, likely projections from CCKNTS neurons to OxtrPBN neurons, and major OxtrPBN projections to CeA, BNST, OVLT, AVPV and MnPO. (b) OxtrPBN neurons are proposed to decrease or prevent hypernatremia ± hypervolemia by decreasing NaCl ± water intake. Inputs to OxtrPBN neurons arise from OxtPVH and NTS neurons. NTS neurons receive signals about volume and osmolarity status from peripheral baroceptors and visceral osmoreceptors (and likely from oropharyngeal and upper gastrointestinal receptors); while OxtPVH neurons receive signals from forebrain osmoreceptors and angiotensin II (ANG II). OxtPVH neurons project to PBN and can also release oxytocin peripherally to increase renal NaCl excretion. NTS neurons likely project to both OxtrPBN and CGRPPBN neurons, which can decrease NaCl, water and food intake

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–10 and Supplementary Tables 1 and 2

  2. Life Sciences Reporting Summary

  3. Supplementary Video 1

    Calcium fluorescence in OxtrPBN neurons during rehydration (2× speed). Upper panel: video of mouse behavior during water rehydration following 24-h dehydration; lower panel: corresponding calcium fluorescence activity in OxtrPBN neurons demonstrating low activity during dehydration and initial presentation of water, followed by increasing activity during drinking bouts, with decreasing activity between bouts (n = 94 neurons from 3 mice)

  4. Supplementary Video 2

    Calcium fluorescence in OxtrPBN neurons during a prolonged drinking bout (2× speed). Upper panel: video of mouse behavior during drinking bout; lower panel: corresponding calcium fluorescence activity in OxtrPBN neurons demonstrating activity during drinking bout

  5. Supplementary Video 3

    Calcium fluorescence in hM3Dq-injected OxtrPBN neurons following intraperitoneal CNO (10× speed).Calcium fluorescence in OxtrPBN neurons following injection of CNO ip, demonstrating increasing activity over ~15 min, which lasted at least 2 h (n = 83 neurons from 2 mice)

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https://doi.org/10.1038/s41593-017-0014-z