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

Abstract

Interoception broadly refers to awareness of one's internal milieu. Vagal sensory afferents monitor the internal milieu and maintain homeostasis by engaging brain circuits that alter physiology and behavior. While the importance of the body-to-brain communication that underlies interoception is implicit, the vagal afferents and corresponding brain circuits that shape perception of the viscera are largely unknown. Here, we use mice to parse neural circuits subserving interoception of the heart and gut. We determine vagal sensory afferents expressing the oxytocin receptor, hereafter referred to as NDG^Oxtr, send projections to the aortic arch or stomach and duodenum with molecular and structural features indicative of mechanosensation. Chemogenetic excitation of NDG^Oxtr significantly 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 NDG^Oxtr also creates patterns of brain activity associated with augmented hypothalamic-pituitary-adrenal axis activity and behavioral indices of vigilance. Recurrent excitation of NDG^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 mental health.

Fig. 1.. Subpopulations of neurons within the NDG ganglia 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 AAV₅-hSyn-DIO-mCherry (b-e) or AAV_PHP.S-FLEX-tdTomato (f-q) bilaterally into the NDG. (b-e) Images and quantification of co-localization of mCherry and (b, c) Oxtr mRNAs or (d, e) NeuN in NDG^Oxtr. (f-k) tdTomato fibers arising from NDG^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 NDG^Oxtr are also found in the aortic arch. (n-p) Co-localization of Piezo1, Piezo2 and Trpa1 mRNAs in tdTomato-labeled NDG^Oxtr. (q) Similar innervation patterns in the aortic arch were observed in Oxtr^2A-cre knock-in mice (used in Bai et al.) delivered AAV_PHP.S-FLEX-tdTomato, in NDG. Scale = 10 μm (b, d, j_inset, n_inset-p_inset); 50 μm (n-p); 100 μm (h-j, m); 500 μm (l, q); 1 mm (f, g). Bars represent SEM. *, P <0.05. Schematics made with Biorender.com.

Fig. 2.. Separate populations of NDG^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, respectively (of Oxtr-Cre mice). (b) Confocal image of the whole NDG collected from one such mouse. (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. (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 NDG^Oxtr that innervate the stomach (i.e., Stomach-tdTomato). Anatomical data are representative of 6 mice. Scale = 100 μm (b and c_insets), 10 μm (e, f). Schematics made with Biorender.com.

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

(a) schematic of bilateral nodose injection treatment groups. (b) hourly food intake, (c) oxygen consumption (VO₂) (d) respiratory exchange ratio (RER) (e) core body temperature, (f) systolic blood pressure (g) heart rate (h) hourly water intake (i) representative image of neuronal activation within the area postrema (AP) and nucleus of the solitary tract (NTS) 90 min following CNO administration (j) patterns of neuronal activation and colocalization of c-fos, GIPR, and VGAT within the AP (left panel) and NTS (right panel) following 90 min following CNO administration (k) representative image of neuronal activation and Calca colocalization within the parabrachial nucleus (PBN) 90 min following CNO administration (l) patterns of c-fos expression and colocalization with Calca-expressing neurons in the PBN 90 min following CNO administration (m). Change in flavor preference following conditioned taste avoidance paradigm (n) representative image of c-fos expression (left panel) and colocalization with CRH-expressing neurons in the paraventricular nucleus (PVN) of the hypothalamus 90 min following CNO administration (o) c-fos expression within the PVN (top panel) and colocalization of c-fos with CRH-expressing neurons within the PVN (bottom panel) (p) plasma corticosterone response to CNO (0.3 mg/kg i.p.) administration Data shown as mean ± s.e.m., n= 12 CON, 22 Gq (b, c, d, h); n=4 CON, 5 Gq (e, f, g); n= 6 CON, 7 Gq (m); n= 5 CON, 5 Gq (p). Statistical analysis performed by two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test (a, b, e, f, g, h) or mixed effects analysis with Dunnett’s multiple comparisons testd (c, d), two-way or repeated measures ANOVA with Fisher’s LSD (p). Schematic (a) made with Biorender.com.

Fig. 4.. Chronic activation of NDG^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 ZT 11 (1h before lights off, left panel) or ZT23 (1h before lights on, right panel). Chronic administration reduced food intake (a,c), temperature (b,d) metabolic (e-h) and cardiovascular parameters (i-l), and body weight (m,n). Gq-NDG^Oxtr data are averaged between baseline (2 days), no injection (5 days) and CNO injection days (6 days). Data shown as mean ± s.e.m., (a,e,f) n= 13 Gq; (b,I,j) n=8 Gq (m) n=17 CON, 13 Gq (n) n=11 CON, 8 Gq. 2 way RM ANOVA or mixed effects model with Dunnett’s multiple comparisons tests (a-l) or mixed effects model with Šídák’s multiple comparisons test (m,n). *, p<0.05 between BASELINE and CNO INJ; #, p<0.05 between NO INJ and CNO INJ.