Mechanosensation of the heart and gut elicits hypometabolism and vigilance in mice

Abstract

Interoception broadly refers to awareness of one's internal milieu. Although the importance of the body-to-brain communication that underlies interoception is implicit, the vagal afferent signalling and corresponding brain circuits that shape perception of the viscera are not entirely clear. Here, we use mice to parse neural circuits subserving interoception of the heart and gut. We determine that vagal sensory neurons expressing the oxytocin receptor (Oxtr), referred to as NG^Oxtr, send projections to cardiovascular or gastrointestinal tissues and exhibit molecular and structural features indicative of mechanosensation. Chemogenetic excitation of NG^Oxtr decreases food and water consumption, and remarkably, produces a torpor-like phenotype characterized by reductions in cardiac output, body temperature and energy expenditure. Chemogenetic excitation of NG^Oxtr also creates patterns of brain activity associated with augmented hypothalamic-pituitary-adrenal axis activity and behavioural indices of vigilance. Recurrent excitation of NG^Oxtr suppresses food intake and lowers body mass, indicating that mechanosensation of the heart and gut can exert enduring effects on energy balance. These findings suggest that the sensation of vascular stretch and gastrointestinal distention may have profound effects on whole-body metabolism and, possibly, mental health.

Extended Data Fig. 1. Impact of bilateral NG injection of Oxtr^Cre mice with the Cre-inducible AAV_PHP.S-FLEX-tdTomato or AAV₅-hSyn-DIO-Gq-mCherry on fluorophore expression in the hindbrain.

(a) Representative confocal scans of coronal sections spanning the brainstem of Oxtr^Cre mice that received a bilateral NG injection with the Cre-inducible AAV_PHP.S-FLEX-tdTomato. (b) Corresponding atlas schematics of representative coronal sections highlighting the area postrema (AP), nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus (DMNV), and nucleus ambiguous (NA). (c) Representative coronal section depicting tdTomato-labeled fibers in the AP and NTS; however, the DMNV and NA are unlabeled. (d, left) Schematic of bilateral injection of AAV_PHP.S-FLEX-tdTomato into the NG. (middle) Representative higher magnification images of the AP, NTS, DMNV, and (right) the NA. (e, left) Schematic of bilateral injection of AAV₅-hSyn-DIO-mCherry into the NG. (middle) Representative higher magnification images of the AP, NTS, DMNV, (right) the and NA. CC; Central Canal, 4V; 4th Ventricle. Scale Bars; 1 mm (a, c), 100 μm (d, e). n= 6–8 sections per 4 mice (tdTom groups) and 2–3 sections per 5 mice (Gq-mCherry groups). Schematics (d) and (e) made with Biorender.com.

Extended Data Fig. 2. Acutely isolated Gq-NG^Oxtr neurons depolarize during CNO bath application.

(a) Gq-NG^Oxtr neurons expressing mCherry acutely dissociated from the mouse NG are visualized with DIC and epifluorescence microscopy. (b, non-glowing, top) Representative whole-cell current clamp recordings of control NG neurons or (red glowing, bottom) neurons expressing Gq-mCherry. After a 60-s baseline recording, CNO (10 μM) was bath applied for 5 min. (c) Average membrane potentials of neurons at baseline and CNO were significantly different between groups (2-Way ANOVA). Multiple comparisons demonstrate that control neurons exposed to CNO failed to depolarize (Tukey post hoc) while CNO significantly depolarized Gq-DG^Oxtr neurons (Tukey post hoc). (d) The same data as (c) expressed as the Δ membrane potential during CNO application. CNO significantly depolarized membrane potential in Gq-NG^Oxtr compared to control neurons (unpaired, two-tailed t-test). Center line is median, edges show quartiles, and error bars are min/max. Recordings acquired from a total of 6 Gq and 5 Control neurons; n= 2 mice.

Extended Data Fig. 3. Representative graphs of individual temperature traces following CNO administration.

CNO (0.3 mg/kg i.p.) was administered at ZT11. Data expressed in 10 min bins.

Extended Data Fig. 4. Acute chemogenetic activation of NG^Oxtr results in decreased (a) food intake and (b) core body temperature in female mice.

CNO (0.3 mg/kg i.p.) was administered at ZT11. n=9 mice. Data represented as mean ± SEM. Within subjects 2-way repeated measures ANOVA, Šídák’s multiple comparisons test, *p<0.05.

Extended Data Fig. 5. The effects of acute CNO administration are recapitulated in male Gq-NG^Oxtr mice maintained on high fat diet.

Impact of CNO (0.3 mg/kg i.p.) or saline administered at ZT11 on hourly (a) food and (b) water intake, (c) oxygen consumption (VO₂), (d) respiratory exchange ratio (RER), (e) body temperature, (f) systolic blood pressure (SBP), (g) heart rate (HR) and (h) change in body weight in Gq-NG^Oxtr mice (n=7). Data represented as mean ± SEM. (a-g) Within subjects two-way repeated measures ANOVA with Šídák’s multiple comparisons test, (h) Paired, two-tailed t-test, *p<0.05.

Extended Data Fig. 6. Acute activation of NG^Oxtr promotes vigilance and reduces locomotor activity.

Schematics of the (a) light dark box (LDB), (d) elevated plus maze (EPM) and (g) open field (OF) arena. Gq-NG^Oxtr made (b) fewer entries into the light and (c) spent less time in the light side of the LDB. Gq-NG^Oxtr mice (e) made fewer entries and (f) travelled less distance in the EPM. Gq-NG^Oxtr (h) made fewer center entries and (i) travelled less in the OF. (b,c,e,f) n= 10 CON-NG^Oxtr, 12 Gq-NG^Oxtr; (h) n= 14 CON-NG^Oxtr, 13 Gq-NG^Oxtr; (i) n= 14 CON-NG^Oxtr, 12 Gq-NG^Oxtr. Unpaired, two-tailed t-tests. Schematics (a, d, g) made with Biorender.com.

Extended Data Fig. 7. Cardiometabolic effects of chronic CNO administration in CON-NG^Oxtr mice.

CNO (0.3 mg/kg, i.p.) was administered (left) every other day for 11 days at ZT11 (1h before lights off) or (right) ZT23 (1h before lights on). Impact of chronic CNO administration on (a,c) food intake, (b, d) water intake, (e, g) body temperature, (f, h) VO₂, (i, k) RER, (j, l) SBP or (m, n) HR. CON-NG^Oxtr data are averaged between baseline (2 days), no injection (5 days) and CNO injection days (6 days). (a,b,f,i) n=15; (c, d, e, g, h, m, n) n=7; (j,l) n= 6. Within-subject two-way repeated measures ANOVA with Tukey’s multiple comparisons test. *p<0.05 BASELINE vs CNO INJ, #p<0.05 NO INJ vs CNO INJ, &p<0.05 BASELINE vs NO INJ.

Extended Data Fig. 8. Chronic administration of CNO (0.3 mg/kg i.p.) to male Gq-NG^Oxtr mice results in (a) weightloss attributed to (b) loss in fat mass but not (c) lean mass.

n=8 mice. Within subject two-way repeated measures ANOVA and Šídák’s multiple comparisons test.

Extended Data Fig. 9. Chronic activation of NG^Oxtr does not induce anxiety-like behavior or reduce social preference.

Schematics of the (a) light dark box (LDB), (d) elevated plus maze (EPM) and (g) three-chamber social interaction tests. (b) Entries into and (c) duration of time spent in the light side of the LDB did not differ between groups. (e) Open arm entries and (f) distance travelled in the EPM did not differ between groups. Chronic NG^Oxtr activation did not affect (h) preference for investigating a novel mouse or (i) distance travelled in the three-chamber social interaction test. n= 8 CON-NG^Oxtr, 7 Gq-NG^Oxtr. Unpaired, two-tailed t-tests. Schematics (a, d, g) made with Biorender.com.

Fig. 1.. Subpopulations of neurons within the NG contain Oxtr(s) and form structural endings in the stomach, duodenum and aortic arch that are likely to monitor tension and stretch.

(a) Schematic illustrating application of (b-e) AAV₅-hSyn-DIO-mCherry or (f-q) AAV_PHP.S-FLEX-tdTomato bilaterally into the NG. (b-e) Images and quantification of co-localization of mCherry and (b, c) Oxtr mRNAs or (d, e) NeuN in NG^Oxtr. (f-k) tdTomato fibers arising from NG^Oxtr are found in the (f-i) stomach and (j) duodenum and form structural endings [IGLEs (h, j_inset) and IMAs (i)] that are suggestive of their involvement in mechanosensation. Note that distance between sections in panels h and i is 100 μm. (k) Comparison of IGLE density across the length of the duodenum in males and females (n= 3 males, 3 females). (l-m) tdTomato fibers arising from NG^Oxtr are also found in the aortic arch (n= 3 male mice). (n-p) Co-localization of Piezo1, Piezo2 and Trpa1 mRNAs in tdTomato-labeled NG^Oxtr(n,o) n= 3 mice, (p) n= 4 mice (q) Similar innervation patterns in the aortic arch were also observed in Oxtr^2A-Cre mice delivered AAV_PHP.S-FLEX-tdTomato into the NG (n= 4 mice). (r) Representative 40x images of the NG of mice trapped with either saline (left) or PE+MC combo (right). Red arrows point to example TRAP+ neurons, Green arrows to Oxtr+ neurons, and yellow arrows exemplify TRAP+Oxtr+. (s) Number of Oxtr+ neurons that were trapped with either saline or PE+MC combo (n= 5 mice). Scale = 10 μm (b, d, j_inset, n_inset-p_inset); 20 μm (r); 50 μm (n-p); 100 μm (h-j, m); 500 μm (l, q); 1 mm (f, g). Data presented as mean ± SEM. (k) 2-way ANOVA with Šídák’s multiple comparisons test. (s) Unpaired 2-tailed t-test. Schematics made with Biorender.com.

Fig. 2.. Separate populations of NG^Oxtr innervate the stomach or aortic arch.

(a) Schematic illustrating the application of AAV_rg-Flex-tdTomato and -EGFP to the aortic arch and stomach (of Oxtr^Cre mice), respectively. (b) Confocal image of the whole NG collected from one such mouse (n=6 mice). (c) Images of the distribution of Aortic Arch-tdTomato and Stomach-EGFP fibers throughout the rostrocaudal extent of the NTS; rostrocaudal distance from bregma is denoted in the lower left-hand corner (n=6 mice). (d) Schematic illustrating the application of AAV_rg-Flex-tdTomato applied to the stomach. RNAscope in situ hybridization for (e) Piezo1- and (f) Piezo2- mRNA(s) in NG^Oxtr that innervate the stomach (i.e., Stomach-tdTomato) n= 3 mice. Scale = 100 μm (b and c_insets), 10 μm (e, f). Schematics made with Biorender.com.

Fig. 3.. Acute stimulation of NG^Oxtr affects cardiometabolic parameters and patterns of neuronal activation within brain regions implicated in conditioned taste avoidance and stress responses.

(a) Schematic of treatment groups. Impact of CNO (0.3 mg/kg i.p.) or saline administered at ZT11 on (b) food intake, (c) oxygen consumption (VO₂), (d) respiratory exchange ratio (RER), (e) core body temperature, (f) systolic blood pressure (SBP), (g) heart rate (HR) and (h) water intake in Gq-NG^Oxtr or CON-NG^Oxtr mice. Fos immunoreactivity was assessed 90 min following CNO administration. (i) Representative images and (j) quantification of colocalization of Fos, GIPR, and VGAT within the AP and NTS. (k) Representative images and quantification of (l, top) Fos and (l, bottom) Fos, Calca colocalization within the PBN of Gq-NG^Oxtr mice. (m) Change in flavor preference following conditioned taste avoidance paradigm. (n) Representative images and (o) quantification of Fos expression and colocalization with CRH-expressing neurons in the PVN. (p, top) Plasma corticosterone 60 min after saline or CNO administration and (bottom) time course of plasma corticosterone response to CNO. Data shown as mean ± SEM, (b) n= 12 CON, 19 Gq; (c,d) n=12 CON, 20 Gq; (e,f,g) n= 4 CON, 5 Gq; (h) n= 11 CON, 20 Gq; (j) n= 4 CON, 4 Gq; (l, top panel) n= 5 CON, 5 Gq; (l, bottom panel) n= 5 CON, 4 Gq; (m) n= 6 CON, 7 Gq; (o) n= 4 CON, 4 Gq; (p, top panel) n= 8 CON, 8 Gq; (p, bottom panel) n= 5 CON, 5 Gq. Scale = 200 μm (i_top); 10 μm (i_bottom); 1 mm (k_top); 50 μm (k_bottom); 100 μm (n). Two-way repeated measures ANOVA with Šídák’s multiple comparisons test; (c,d) mixed effects analysis with Šídák’s multiple comparisons test; (j, l, o) unpaired 2-tailed t-tests, (m,p) Two-way repeated measures ANOVA with Fisher’s LSD tests. Figures b-h: *p<0.05 Gq-Saline vs Gq-CNO. Schematic (a) made with Biorender.com.

Fig. 4.. Chronic activation of NG^Oxtr affects cardiometabolic parameters, resulting in persistent reductions in body weight.

CNO (0.3 mg/kg, i.p.) was administered every other day for 11 days at ZT11 (left, 1h before lights off) or ZT23 (right, 1h before lights on). Impact of chronic administration on (a,c) food intake, (b,d) water intake, (e,g) body temperature, (f,h,i,k) metabolic and (j,l,m,o) cardiovascular parameters in Gq-NG^Oxtr mice. Data are averaged between baseline (2 days), no injection (5 days) and CNO injection (6 days). See Supp. Fig. 7 for corresponding data in CON-NG^Oxtr mice. (n,p) Body weight in Gq-NG^Oxtr and CON-NG^Oxtr mice. (a,b,f,i) n= 13 Gq; (c,d,e,g,h,j,k,l,m,o) n= 8 Gq; (n) n= 17 CON, 13 Gq; (p) n= 11 CON, 8 Gq. Data expressed as mean ± SEM. (a-m,o) Within subjects two-way repeated measures ANOVA with Tukey’s posthoc tests; or (n) 2-way repeated measures ANOVA with Šídák’s multiple comparisons test or (p) mixed effects model with Šídák’s multiple comparisons test. For figures a-m, o: *p<0.05 BASELINE vs CNO INJ, #p<0.05 NO INJ vs CNO INJ, &p<0.05 BASELINE vs NO INJ. For figures n and p, *p<0.05.