To understand hindbrain pathways involved in the control of food intake, we examined roles for calcitonin receptor (CALCR)-containing neurons in the NTS. Ablation of NTS Calcr abrogated the long-term suppression of food intake, but not aversive responses, by CALCR agonists. Similarly, activating Calcr NTS neurons decreased food intake and body weight but (unlike neighboring Cck NTS cells) failed to promote aversion, revealing that Calcr NTS neurons mediate a non-aversive suppression of food intake. While both Calcr NTS and Cck NTS neurons decreased feeding via projections to the PBN, Cck NTS cells activated aversive CGRP PBN cells while Calcr NTS cells activated distinct non-CGRP PBN cells. Hence, Calcr NTS cells suppress feeding via non-aversive, non-CGRP PBN targets. Additionally, silencing Calcr NTS cells blunted food intake suppression by gut peptides and nutrients, increasing food intake and promoting obesity. Hence, Calcr NTS neurons define a hindbrain system that participates in physiological energy balance and suppresses food intake without activating aversive systems.

The most primitive portion of the brain, known as the hindbrain, has circuits that are widely thought to mediate only the short-term suppression of feeding by gut signals under normal physiologic conditions. These hindbrain circuits have been generally thought to suppress feeding through nausea and other aversive responses when activated strongly, as by drugs that mimic gut peptides. However, researchers at the University of Michigan identified a hindbrain system that not only participates in the physiological control of long-term energy balance, but also strongly and durably suppresses food intake without activating aversive systems or symptoms. In addition to revising our concept of food intake control by the hindbrain, these findings identify a circuit that represents a potentially ideal target for the treatment of obesity.

While gut-peptide-responsive cells in the NTS (and in the adjacent area postrema [AP]) play prominent roles in meal termination and the control of meal size (), the gut peptide receptors on these cells only modestly (if at all) impact the long-term physiologic control of food intake and energy balance (). Hence, conventional wisdom holds that while NTS cells play important roles in the suppression of feeding in response to gut peptide receptor agonism and the control of meal termination, these NTS neurons do not participate in the physiologic regulation of long-term energy balance. This idea has not been tested directly by interfering with the overall function of NTS neurons, though. Thus, in addition to understanding the roles for NTS cells in the anorectic response to pharmacologic CALCR agonism and defining hindbrain systems that mediate the non-aversive suppression of food intake, it will be important to directly determine roles for NTS cells in the physiologic control of food intake and energy homeostasis. Here, we examined roles for Calcrcells, demonstrating their role in mediating the non-aversive suppression of food intake and revealing their importance for the long-term control of energy balance.

Normal food ingestion (or nutrient infusion into the gut) mediates positive reinforcement even while stimulating meal termination (). Therefore, the circuits that terminate normal feeding must differ at least in part from those that convey aversive signals in response to gut malaise, and it should be possible to identify populations of NTS neurons that suppress food intake without activating CGRPneurons or causing aversion.

One line of thinking holds that meal-terminating brainstem circuits must also mediate aversive responses, such that overfeeding (and other gut-malaise-associated stimuli) would promote aversive signals by more strongly activating these circuits than would a normal-sized meal. Indeed, previous work has demonstrated that calcitonin gene-related peptide (CGRP)-containing neurons of the parabrachial nucleus (PBN; CGRPcells) mediate aversion (as well as anorexia) in response to a variety of cues of gastrointestinal malaise (including that induced by intraperitoneal LiCl) and that silencing CGRPcells increases meal size (). For instance, stimulating PBN projections from Cck-expressing NTS (Cck) neurons activates CGRPcells and promotes aversion and anorexia ().

Calcr is widely distributed in the brain, including in several areas linked to food intake, including the hypothalamic arcuate nucleus (ARC), the paraventricular hypothalamic nucleus (PVH), and the amygdala (). The nucleus tractus solitarius (NTS; a CNS region crucial for integrating gut-derived prandial signals and promoting meal termination) () also contains Calcr cells (Calcrneurons). Importantly, while agonists for CALCR (and other gut peptide receptors) decrease food intake, they also produce aversive responses that mimic gut malaise (), potentially limiting their therapeutic utility.

Obesity affects over one-third of the adult population in developed countries, leading to diabetes, cardiovascular disease, and other conditions that cause substantial morbidity and mortality ( https://www.cdc.gov/obesity/data/adult.html ). Unfortunately, most current medical therapies are ineffective in treating obesity (). Agents that mimic the action of gut peptides (such as agonists [including calcitonin and amylin] for the calcitonin receptor [CALCR] and glucagon-like peptide-1 receptor [GLP1R]) suppress long-term food intake and promote significant weight loss, though (). Thus, it is important to understand the neural mechanisms by which such peptides mediate their anorexic effects.

Furthermore, exposure to HFD resulted in a 25%–30% increase in food intake by Calcr-TT mice compared to controls ( Figure 7 A) over the 6 weeks of HFD exposure, promoting an additional 8 g of weight gain compared to controls. Body composition analysis revealed that adipose mass represented the majority of the excess weight gain by Calcr-TT mice ( Figures 7 E and 7F). Thus, silencing Calcrcells increases long-term food intake, body weight, and adiposity (especially in mice exposed to a palatable diet), revealing that these NTS neurons play an important role in overall energy balance, not just in the short-term suppression of food intake in response to gut-derived signals.

To directly assess a potential role for Calcrneurons in energy homeostasis, we examined food intake and body weight in Calcr-TT mice for 7 weeks on a chow diet, followed by an additional 6 weeks on HFD ( Figure 7 ). We found that Calcr-TT mice exhibited a tendency toward increased body weight on a chow diet. Indeed, the continuous analysis of food intake over 24 h in these mice revealed increased nocturnal food intake, and we also observed increased cumulative food intake over 7 weeks of home cage chow feeding ( Figures 7 B and 7C).

(A–F) Pairs of Calcr cre mice were matched for initial body weight and injected with AAV Flex−TetTox−GFP (Calcr TT ) or AAV Flex−mCherry (Ctrl). Mice were subjected to chow diet for 7 weeks, followed by another 6 weeks on HFD. (A) Body weight (change from baseline) for the duration of the experiment is shown (n = 10 in each group). (B) Food intake was continuously monitored over 24 h in a TSE system during the seventh week after surgery (n = 5 per group). Cumulative food intake on chow (C, n = 10 per group) and HFD (D, n = 10 per group) are shown. Body composition was determined after 7 weeks on chow diet (E, n = 7 in control group, n = 5 in Calcr TT group) and 6 weeks on HFD (F, n = 5 per group). Mean ± SEM is shown. Two-way ANOVA, Sidak’s multiple comparisons test was performed in (A), (C), and (D). Two-way repeated-measures ANOVA was performed for (B). Unpaired t test was performed for (E) and (F). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. ns, not significant.

We also examined the potential role for Calcrcells in the strong appetite-suppressing effects of the amino acid Leu. Intra-NTS Leu (NTS-Leu) suppresses food intake () without producing a CTA ( Figure S7 E). Indeed, we found that NTS-Leu promotes the activation of Calcrcells ( Figures S7 C and S7D). Furthermore, NTS-Leu failed to suppress food intake in Calcr-TT mice, although NTS-Leu suppressed food intake normally in Cck-TT mice with silenced Cckneurons ( Figures 6 E–6G).

Because the innervation of Calcrcells by vagal afferents in the nodose ganglion suggested that Calcrcells receive input from the gut, we examined their activation by a variety of anorectic agonists for gut peptide receptors, including sCT, CCK, and exendin-4 (Ex4, a GLP1R agonist). Each of these peptides, as well as refeeding following a fast, increased FOS-IR in Calcrcells, as well as in other NTS cells ( Figures S7 A and S7B). To examine roles for Calcrcells in the anorectic response to these peptides, we bilaterally injected AVV(which mediates the cre-dependent expression of tetanus toxin [TetTox] to prevent synaptic neurotransmitter release []) into the NTS of Calcrmice ( Figure 6 A). These Calcr-TT mice exhibited an attenuated suppression of food intake in response to sCT, as expected ( Figure 6 B). Furthermore, Calcr-TT mice also exhibit blunted suppression of food intake by CCK and Ex4 ( Figures 6 C and 6D). Thus, Calcrneurons contribute to the anorectic response to a variety of food-intake-suppressing gut peptide receptor agonists.

(E–G) Suppression of food intake at the onset of the dark cycle in response to the intra-NTS injection of vehicle (ACSF) or Leu for control (E, GFP, n = 6), Calcr TT (F, n = 7), and CCK TT (G, n = 8) mice. Shown is mean ± SEM. Two-way ANOVA, Sidak’s multiple comparisons test was performed, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. ns, not significant.

(B–D) Food intake was measured over the first 4 h of treatment with (B) sCT (150 μg/kg, n = 18 in control group, n = 16 in Calcr TT group), (C) EX4 (150 μg/kg, n = 14 in control group, n = 10-12 in Calcr TT group), and (D) CCK (100 μg/kg; n = 5 in both groups). Mean ± SEM, unpaired t test was performed between Ctrl and Calcr TT groups. ∗ p < 0.05, ∗∗ p < 0.01.

To understand the potential roles for Calcrneurons in the control of food intake, we examined the afferent inputs to these cells by means of single-synapse retrograde tracing with defective rabies virus () ( Figure S6 ). This analysis revealed strong inputs from the nodose ganglion and a variety of forebrain sites, including the PVH, lateral hypothalamic area (LHA), and central amygdala.

To directly examine the ability of Calcrand Cckcells to activate CGRPcells, we bred Calcrand Cckonto the Calcabackground (), which permits the GFP-dependent detection of CGRPcells. We injected AAVinto the NTS of these mice and examined the distribution of innervation and FOS-IR within the PBN and its colocalization with GFP-labeled CGRPcells ( Figures 5 D, 5E, and S5 ). This analysis confirmed the more dorsal medial distribution of projections and FOS-IR within the PBN following the activation of Calcrneurons than for Cckcells. Furthermore, although Cckcells promoted strong FOS accumulation in CGRPcells, Calcrcells did not. Thus, Calcrneurons suppress food intake via the PBN but (unlike Cckcells) poorly activate aversive CGRPcells, consistent with their non-aversive suppression of food intake. These data thus reveal the existence of a population of non-CGRP PBN neurons that mediates the non-aversive suppression of food intake by Calcrcells.

Because CGRPcells contribute to the suppression of food intake during the activation of Cckneurons and mediate aversive responses (), we examined the potential innervation and regulation of CGRPcells by Calcrcells. In our initial experiments, we stained for hM3Dq-mCherry and CGRP (which identifies the region containing CGRPcells but poorly reveals CGRPsoma) in Calcr-Dq and Cck-Dq mice ( Figure S5 ). While Cck-Dq mice demonstrated substantial mCherry innervation of the CGRP-IR PBN field, Calcr-Dq mice exhibited little overlap between the main PBN projection field of Calcrcells and CGRPcells. Furthermore, sCT promoted FOS-IR in the CGRPfield in CalcrKO and control mice ( Figure S5 ). Thus, unlike Cckcells, it appears that Calcrcells neither innervate CGRPcells nor are required for the activation of CGRPcells by sCT.

To define potential differences in the food-intake-suppressing systems engaged by Calcrand Cckneurons, we identified their downstream projections by the cre-dependent expression of a synaptophysin-mCherry (Syn-mCherry) fusion protein in these cells following the unilateral injection of AAVinto the NTS of Calcrand Cckanimals. This analysis revealed projections to the PBN, PVH, and dorsomedial hypothalamic nucleus (DMH) from both Calcrand Cckneurons ( Figures S4 A–S4H). Similarly, activation of Calcrand Cckcells each increased FOS-IR in the NTS, PBN, PVH, and DMH, although the Calcrcells appeared to promote FOS-IR in the PVH and DMH more strongly than did Cckcells ( Figures S4 I–S4T). Interestingly, while the distribution of projections and Dq-stimulated FOS-IR within the target regions was similar in most cases, Calcrcells targeted a PBN region slightly more dorsal and medial to the region targeted by Cckcells ( Figures S4 B, S4F, S4J, S4N, and S4R). Despite this difference in PBN projection targets, the optogenetic activation of PBN terminals from either Calcror Cckneurons suppressed food intake at the onset of the dark cycle and following an overnight fast ( Figures 5 A–5C ).

(E) Quantification of FOS-positive CGRP PBN neurons from the groups in (D). Mean ± SEM is shown; n = 3 per group. One-way ANOVA, Tukey’s multiple comparisons was performed, different letters indicate differences, p < 0.05.

(D) Representative images of FOS (purple) and CGRP cells (GFP, green) in the PBN of mice on the Calca cre−GFP background 2 h after treatment with vehicle (Veh), sCT (150 μg/kg, IP), LiCl (126 mg/kg, IP), or CNO (1 mg/kg, IP) in Calcr NTS -Dq (Calcr Dq ) and Cck NTS -Dq (CCK Dq ) mice. Scale bar, 150 μm; scp, superior cerebellar peduncle.

(B and C) Food intake over the first 2 h of the dark cycle (B) or during the first hour following the return of food to overnight-fasted mice (C) under control (Ctrl) or blue-light-stimulated (Light) conditions. Mean ± SEM is shown, paired t test was performed, ∗ p < 0.05, ∗∗ p < 0.01. Dark cycle: Calcr ChR2 n = 12, CCK ChR2 n = 9; refeeding: Calcr ChR2 n = 12, CCK ChR2 n = 11.

Because Cckcells promote CTA formation while Calcrcells play no role in this process, these NTS cell types must act via different downstream circuits, at least in part. Nutritional signals from the gut suppress the activity of ARC NAG neurons (), presumably via a brainstem circuit. Because we found that the chemogenetic activation of Calcrcells suppressed ghrelin-simulated food intake (which is largely mediated by the activation of NAG neurons) ( Figure 4 A), we tested the potential role for Calcrcells in the inhibition of NAG cells. The fasting-induced activation of NAG cells can be monitored by the accumulation of FOS-IR in the medial basal ARC (MB-ARC; Figures 4 B–4D) (); this fasting-induced MB-ARC activation can be decreased by refeeding. Similarly, the chemogenetic activation of Calcrcells suppressed fasting-induced MB-ARC FOS-IR ( Figures 4 C and 4D). Thus, Calcrcells inhibit fasting-induced MB-ARC FOS-IR cells, suggesting that hindbrain neurons control hypothalamic circuits crucial for the regulation of feeding. While the inhibition of MB-ARC cells may contribute to the suppression of food intake by Calcrneurons, the activation of Cckneurons also blunts fasting-induced MB-ARC FOS ( Figures 4 C and 4D). Hence, the difference in downstream signaling between Calcrand Cckcells must lie elsewhere.

(D) Quantification of ARC FOS for the groups shown in (C); n = 5, 6, 4, 9, and 5, respectively. Mean ± SEM is shown. Two-way ANOVA, Sidak’s multiple comparisons test for (A). One-way ANOVA, Tukey’s multiple comparisons was performed in (D). Different letters indicate difference between conditions, p < 0.05.

(C) Representative images showing ARC FOS (black) 3 h after the onset of the light cycle in ad libitum-fed (Fed), overnight fasted, overnight fasted and refed (Fast-refed), and overnight fasted with CNO (1 mg/kg, IP, 2 h prior to perfusion) in Calcr Dq and CCK Dq mice. Scale bar, 150 μm; 3V, third cerebral ventricle. Dashed line shows the limits of the MB-ARC region used for quantification.

(A) Calcr NTS -Dq mice were treated with vehicle (Veh), Ghrelin (Gh, 400 μg/kg, IP), or Gh plus CNO (IP, 1 mg/kg) 3 h after the onset of the light cycle (9 AM) and food intake was monitored over the subsequent 6 h. Veh: n = 14, Gh and Gh+CNO: n = 12.

Although the known role for the NTS in the control of food intake is consistent with the notion that Calcrand Cckcells suppress food intake directly, it is also possible that the activation of these cells could promote other effects (e.g., pain) that indirectly blunt feeding. We thus bilaterally injected AAVto express the Gi-coupled inhibitory hM4Di DREADD in Calcrand Cckcells (Calcr-Di and Cck-Di mice, respectively) ( Figure 3 A), permitting their acute inhibition by CNO. We found that CNO increased food intake following an overnight fast similarly in Calcr-Di and Cck-Di mice ( Figure 3 B). Furthermore, while the hM4Di-mediated inhibition of Calcrcells did not alter CTA formation in response to LiCl administration, CNO administration blocked CTA formation in Cck-Di mice ( Figures 3 C and 3D). Thus, while Cckcells provoke aversive responses while suppressing food intake and are required for CTA formation in response to LiCl, Calcrcells similarly suppress food intake but are neither necessary nor sufficient to mediate a CTA.

(D) Quantification of CTA to HFD produced by LiCl injection paired with silencing Calcr NTS or CCK NTS neurons (n = 11 in vehicle and LiCl groups, n = 8 for in Calcr Di and CCK Di groups). Mean ± SEM is shown. Paired t test was performed for each time point in (B), ∗∗ p < 0.01. One-way ANOVA, Tukey’s multiple comparisons was performed in (D), different letters indicate difference between conditions, p < 0.05.

(B) Food intake for the first 6 h of refeeding following an overnight fast in the indicated groups of mice following treatment with vehicle (Veh) or CNO (IP, 1 mg/kg). Assay was performed using a crossover design with 1 week between conditions. Control: n = 5; Calcr Di and CCK Di : n = 8 each.

Because of the overlap between Calcrand Thcells, we also examined the response to activating Thcells ( Figures S3 H–S3L). While (like Calcrcells) we found that Thactivation failed to promote a CTA, activating Thcells only weakly suppressed food intake compared to Calcrcells; thus, we continued to focus on Calcrneurons to understand NTS cells that mediate the non-aversive suppression of food intake. The more robust suppression of food intake by Calcrthan Thcells might reflect some property of the non-Th Calcrcells that mediate a stronger anorectic response than Thcells.

Interestingly, despite the rapid and complete suppression of short-term food intake by both Calcrand Cckcells, only the activation of Cckcells promoted a CTA ( Figures 2 E–2G). Furthermore, in the two-flavor choice paradigm, animals consumed more of the flavor that was paired with the activation of Calcrcells ( Figure S3 G); the lack of significant change in the preference ratio likely reflects the lower sensitivity of this measure due to normalization for total volume consumed. Thus, not only is Calcr in NTS neurons crucial for the anorectic (but not aversive) response to sCT treatment, but the activation of Calcrcells causes the acute and chronic suppression of food intake while promoting reinforcement rather than aversion.

CNO treatment promoted FOS-IR in mCherry-expressing cells in the NTS of Calcr-Dq and Cck-Dq mice, consistent with the DREADD-mediated activation of Calcrand Cckcells, respectively, in these animals ( Figure 2 A). The activation of Calcror Cckcells at the onset of the dark cycle or following an overnight fast completely abrogated the intake of normal chow for 2 h and continued to dramatically reduce food intake for several hours ( Figures S3 E and S3F), although food intake suppression was less robust in Cck-Dq mice than in Calcr-Dq animals by 4 h after refeeding in fasted animals ( Figure 2 B). Furthermore, the twice-daily injection of CNO reduced food intake by 70% for the first 24 h and then by approximately 50% thereafter in Calcr-Dq mice ( Figure 2 C). CNO reduced long-term food intake less dramatically in Cck-Dq mice, but both mouse lines lost approximately 10% of their body weight over the 4-day treatment ( Figures 2 C and 2D). Thus, both Calcrand Cckcells suppress food intake and body weight, although Calcrcells mediate a greater long-term suppression of feeding than Cckcells.

Because the chemogenetic activation of Cckcells has been shown to promote the aversive suppression of food intake (), we utilized these cells as comparators for Calcrneurons ( Figure 2 ). We bilaterally injected AAVinto the NTS of Calcror Cckmice to cre-dependently express the Gq-coupled (activating; hM3Dq) designer receptor exclusively activated by designer drugs (DREADD) in Calcrand Cckcells, permitting their activation by the injection of clozapine-N-oxide (CNO) (). We used the post hoc detection of mCherry (which is fused to hM3Dq in AAV) in these Calcr-Dq and Cck-Dq mice to ensure that we analyzed only mice with NTS-restricted hM3Dq expression ( Figure 2 A).

All graphs: Mean ± SEM is shown. Two-way ANOVA, Sidak’s multiple comparisons test for (B), (C), and (D). One-way ANOVA, Tukey’s multiple comparisons was performed in (E), (F), and (G). Different letters indicate difference between groups, p < 0.05.

(E–G) CTA was determined for control, Calcr hm3dq , and CCK hm3dq groups in which stimulus was paired with HFD (E, n = 10, 10, 11, and 9 for control, LiCl, CCK Dq , and Calcr Dq groups, respectively), Saccharin (F, n = 13, 11, 8, and 12 for control, LiCl, CCK Dq , and Calcr Dq groups, respectively) or one of two flavors (G, n = 11, 12, and 8 for control, LiCl, and Calcr Dq groups, respectively).

(C and D) Daily food intake (C) and body weight (D) were measured during 3 days of vehicle, 4 days of CNO (IP, 1 mg/kg, BID), and 2 additional days of vehicle injection. Food intake and body weight were normalized to baseline. Control: n = 12; Calcr Dq : n = 12; and CCK Dq : n = 7.

(B) Food (chow) intake during CNO (1 mg/kg, IP) treatment of control (n = 23–25), Calcr Dq (n = 7–20), and CCK Dq (n = 5–9) mice during the first 4 h of the dark cycle (left panel) and the first 6 h following refeeding in overnight fasted mice (right panel).

To understand the relationship of Calcrneurons to previously studied populations of NTS neurons, we examined the potential colocalization of Calcrcells with NTS neurons that express LepRb, cholecystokinin (Cck), or tyrosine hydroxylase (Th) ( Figures S3 A–S3D). While LepRband Cckwere distinct from Calcrcells, a significant proportion of Thcells contain Calcr (and vice versa); Thcells do not overlap significantly with Cckcells.

To understand the potential role for Calcrneurons in the aversive response to sCT, we utilized the conditioned taste aversion (CTA) assay, in which pairing of a stimulus with exposure to a novel tastant (e.g., grape flavor or 0.15% saccharine in drinking water, or high-fat diet [HFD]) inhibits the subsequent consumption of aversive stimulus-paired tastants. Like LiCl, sCT promotes a CTA in normal mice () ( Figure 1 K). Interestingly, although deletion of NTS Calcr abrogated the ability of sCT to reduce food intake ( Figures 1 E–1J), sCT treatment promoted a CTA in CalcrKO mice similarly to control animals ( Figure 1 L), revealing that NTS Calcr is not required for the formation of a CTA to sCT, thus suggesting that Calcrcells might suppress food intake without participating in aversive responses.

In contrast to the reduced efficacy of sCT and davalintide (a co-agonist for the CALCR and amylin receptor []) on food intake suppression in CalcrKO mice ( Figures 1 E–1J), CalcrKO and control mice responded similarly to the NPY2R agonist, PYY3-36, and lipopolysaccharide (LPS) ( Figures S2 P–S2R), consistent with the specificity of NTS Calcr for the anorexic response to CALCR agonists. Overall, these data suggest that while NTS Calcr is not required for the control of energy balance at baseline, Calcr in the NTS mediates the long-term effects of CALCR agonists on food intake and body weight.

While we found that sCT suppressed food intake normally in CalcrKO mice for the first 2 h at the onset of the dark cycle, the anorectic effect of sCT in CalcrKO mice attenuated by 4 h, and sCT failed to suppress 24 h food intake and reduce body weight during 3 days of sCT administration in these animals ( Figures 1 E–1G). Indeed, 3-day treatment with sCT promoted body weight gain (p = 0.047) in the CalcrKO mice. Thus, not only are NTS Calcr (Calcr) neurons crucial for the anorectic response to sCT, but an unidentified set of Calcr neurons elsewhere in the brain may mediate an orexigenic signal that is normally overcome by the anorexic signal from the Calcrcells.

To determine the anorectic response of these KO animals to CALCR agonists, we examined feeding following sCT administration. We found that sCT decreased food intake by >40% during the first 4 h of the dark cycle and that 3 days of twice-daily sCT administration suppressed food intake and lowered body weight similarly in CalcrKO and CalcrKO mice and their controls ( Figures S2 J–S2O). Thus, Calcr expression in PVH Sim1 neurons and hypothalamic LepRb neurons is not required for the suppression of food intake by sCT.

Our analysis revealed no alteration in food intake, body weight, or body composition in CalcrKO, CalcrKO, and CalcrKO mice ( Figures S2 A–S2I). Hence, Calcr expression in the PVH, the NTS, and in ARC LepRb neurons does not meaningfully contribute to long-term energy balance under normal conditions.

As expected, CalcrKO mice exhibited reduced salmon calcitonin (sCT)-stimulated NTS FOS-immunoreactivity (IR) ( Figures 1 C, 1D, and S1 F). In contrast, CalcrKO mice exhibited unchanged sCT-stimulated FOS-IR in the ARC ( Figures S1 B and S1C), and we detected increased sCT-stimulated FOS in the PVH of CalcrKO mice despite decreased PVH Calcr expression in CalcrKO mice ( Figures S1 D and S1E). These findings suggest that most sCT-stimulated FOS in the ARC and PVH is mediated indirectly (by other CALCR cells) and that PVH CALCR may suppress the sCT-dependent activation of PVH neurons by other CALCR-expressing cells.

We sought to understand the roles for specific Calcr-expressing neurons for the control of food intake. Based upon Calcr expression patterns in the hypothalamus, we crossed Calcr) onto the Sim1) or Lepr) backgrounds (CalcrKO [knockout] and CalcrKO mice, respectively) ( Figure S1 A) to ablate Calcr in neurons of the PVH and in leptin receptor (LepRb) neurons (primarily neuropeptide Y-, agouti-related peptide-, and gamma-aminobutyric-acid-containing (NAG) cells of the ARC) (). We also injected AAVor AAV(control) into the NTS of Calcrmice to ablate Calcr expression in this brain region (CalcrKO mice) ( Figure 1 A). We examined AAV reporter expression in the brains of all injected animals following the completion of experiments to ensure correct targeting ( Figure 1 B).

All graphs: Mean ± SEM is shown. One-way ANOVA, Tukey’s multiple comparisons was performed in (D), (H), (K), and (L), different letters indicate differences between groups, p < 0.05. The same test was performed for each time point in (E); ns, not significant. Two-way ANOVA, Sidak’s multiple comparisons test was performed for (F, G, I, and J). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

(K and L) Conditioned taste aversion (CTA) was determined following the IP injection of Veh, LiCl (126 mg/kg), or sCT (150 μg/kg) in control (K) or control and Calcr NTS KO mice (L); n = 7–10 per group.

(H–J) Control (Ctrl, blue) and Calcr NTS KO (KO, red) mice were treated with Davalintide (560 μg/kg, IP, twice daily) and food intake was measured for the first 4 h of the dark cycle (H) or food intake (I) and body weight (J) were measured daily over 3 days of injections (dashed lines); n = 18 per group.

(F and G) Daily food intake (F) and body weight (G) were measured during 2 days of vehicle, 3 days of sCT (IP, 150 μg/kg, BID), and 3 additional days of vehicle injection. Food intake and body weight were normalized to baseline. n = 18 in control group, n = 8–10 in Calcr NTS KO group.

(E) Control and Calcr NTS KO mice were treated with Veh or sCT (150 μg/kg, IP) and food intake was measured for the subsequent 4 h. n = 8 in 1-h and 2-h groups, n = 26 in 4-h groups.

(D) Quantification of FOS-IR cells in the NTS of mice treated as in (C). Shown is mean ± SEM; n = 4–10 per group.

Discussion

NTS neurons mediate the long-term suppression of food intake by gut peptide mimetics and are required for the normal physiologic regulation of feeding and energy balance. Moreover, the activation of CalcrNTS neurons is neither necessary nor sufficient for the acquisition of a CTA. In the context of the CTA-associated food intake suppression that results from the activation of CckNTS cells ( Roman et al., 2017 Roman C.W.

Sloat S.R.

Palmiter R.D. A tale of two circuits: CCKNTS neuron stimulation controls appetite and induces opposing motivational states by projections to distinct brain regions. NTS neurons also demonstrate that the NTS systems that control the aversive and non-aversive suppression of feeding can be distinguished. Indeed, we show that while CalcrNTS and CckNTS cells both inhibit MB-ARC neurons, CalcrNTS cells do not activate aversive CGRPPBN cells (as do CckNTS cells), but rather suppress food intake via distinct PBN targets. Our findings also reveal that CalcrNTS cells not only participate in the short-term response to nutritional and gut peptide signals, but also play important roles in the long-term control of food intake and body weight, demonstrating a role for brainstem circuits in the control of overall energy balance, especially during exposure to HFD. Our findings demonstrate that Calcrneurons mediate the long-term suppression of food intake by gut peptide mimetics and are required for the normal physiologic regulation of feeding and energy balance. Moreover, the activation of Calcrneurons is neither necessary nor sufficient for the acquisition of a CTA. In the context of the CTA-associated food intake suppression that results from the activation of Cckcells (), our findings with Calcrneurons also demonstrate that the NTS systems that control the aversive and non-aversive suppression of feeding can be distinguished. Indeed, we show that while Calcrand Cckcells both inhibit MB-ARC neurons, Calcrcells do not activate aversive CGRPcells (as do Cckcells), but rather suppress food intake via distinct PBN targets. Our findings also reveal that Calcrcells not only participate in the short-term response to nutritional and gut peptide signals, but also play important roles in the long-term control of food intake and body weight, demonstrating a role for brainstem circuits in the control of overall energy balance, especially during exposure to HFD.

Gianini et al., 2013 Gianini L.M.

White M.A.

Masheb R.M. Eating pathology, emotion regulation, and emotional overeating in obese adults with Binge Eating Disorder. NTS cells, which contribute to the long-term suppression of food intake without provoking a CTA, represent a potentially useful target for the control of food intake. While the activation of Gcg-expressing NTS neurons in mice also suppresses food intake without activating a CTA, the decrease in food intake mediated by these cells is relatively small and transient ( Gaykema et al., 2017 Gaykema R.P.

Newmyer B.A.

Ottolini M.

Raje V.

Warthen D.M.

Lambeth P.S.

Niccum M.

Yao T.

Huang Y.

Schulman I.G.

et al. Activation of murine pre-proglucagon-producing neurons reduces food intake and body weight. Because the activation of aversive symptoms can limit the utility of treatments designed to decrease food intake and body weight (), agents that activate neurons that suppress food intake without simultaneously stimulating aversive systems would represent ideal therapeutic agents. Hence Calcrcells, which contribute to the long-term suppression of food intake without provoking a CTA, represent a potentially useful target for the control of food intake. While the activation of Gcg-expressing NTS neurons in mice also suppresses food intake without activating a CTA, the decrease in food intake mediated by these cells is relatively small and transient (), and their downstream targets have not been identified.

NTS cells in CALCR agonist-mediated anorexia, Calcr in Sim1 or LepRb neurons is not required for this response to CALCR agonists. Furthermore, the undiminished PVH and ARC FOS responses to sCT in CalcrSim1KO and CalcrLepRbKO mice, respectively, suggest that much sCT-induced hypothalamic FOS may be mediated indirectly, via Calcr neurons that project into the hypothalamus (potentially including CalcrNTS cells). Furthermore, CalcrLepRb neurons mainly represent orexigenic ARC NAG neurons ( Pan et al., 2018 Pan W.

Adams J.M.

Allison M.B.

Patterson C.

Flak J.N.

Jones J.

Strohbehn G.

Trevaskis J.

Rhodes C.J.

Olson D.P.

Myers Jr., M.G. Essential Role for Hypothalamic Calcitonin Receptor‒Expressing Neurons in the Control of Food Intake by Leptin. NTSKO mice, consistent with the notion that sCT activates a set of orexigenic neurons (such as CalcrLepRb/NAG cells) but that this effect is normally masked by the anorexigenic action of CalcrNTS cells. Consistently, we find that CalcrNTS neuron activation overcomes feeding driven by ghrelin. In contrast to the important role for Calcr in Calcrcells in CALCR agonist-mediated anorexia, Calcr in Sim1 or LepRb neurons is not required for this response to CALCR agonists. Furthermore, the undiminished PVH and ARC FOS responses to sCT in CalcrKO and CalcrKO mice, respectively, suggest that much sCT-induced hypothalamic FOS may be mediated indirectly, via Calcr neurons that project into the hypothalamus (potentially including Calcrcells). Furthermore, Calcrneurons mainly represent orexigenic ARC NAG neurons (), suggesting that the sCT-dependent direct activation of these cells might increase (rather than decrease) feeding. Indeed, we found that sCT slightly increases long-term food intake in CalcrKO mice, consistent with the notion that sCT activates a set of orexigenic neurons (such as Calcr/NAG cells) but that this effect is normally masked by the anorexigenic action of Calcrcells. Consistently, we find that Calcrneuron activation overcomes feeding driven by ghrelin.

Sim1 and CalcrLepRb neurons are not required for the suppression of food intake by sCT, non-NTS Calcr neurons must participate in the acute anorectic response to sCT (during the first 1–2 h of treatment), even though CalcrNTS cells mediate the long-term suppression of food intake. Indeed, AP lesions can attenuate the sCT-mediated suppression of food intake over 1–2 h in rats ( Braegger et al., 2014 Braegger F.E.

Asarian L.

Dahl K.

Lutz T.A.

Boyle C.N. The role of the area postrema in the anorectic effects of amylin and salmon calcitonin: behavioral and neuronal phenotyping. NTS cells do not contribute to the aversive effects of sCT, mediate aversion, or contribute to the activation of CGRPPBN cells, non-NTS Calcr cells must mediate the CGRPPBN-activating and aversive responses to sCT. Since the AP contributes to aversive signaling, including nausea ( Wang et al., 2013 Wang T.J.C.

Fontenla S.

McCann P.

Young R.J.

McNamara S.

Rao S.

Mechalakos J.G.

Lee N.Y. Correlation of Planned Dose to Area Postrema and Dorsal Vagal Complex with Clinical Symptoms of Nausea and Vomiting in Oropharyngeal Cancer (OPC) patients treated with radiation alone using IMRT. AP cells mediate the CTA-producing effects of sCT. CalcrAP cells could also mediate the CalcrNTS-independent short-term suppression of food intake by sCT. Unfortunately, we and others have been unable to specifically target the AP by stereotaxic injection in mice, preventing us from directly testing this possibility. While Calcrand Calcrneurons are not required for the suppression of food intake by sCT, non-NTS Calcr neurons must participate in the acute anorectic response to sCT (during the first 1–2 h of treatment), even though Calcrcells mediate the long-term suppression of food intake. Indeed, AP lesions can attenuate the sCT-mediated suppression of food intake over 1–2 h in rats (), suggesting that sCT action via AP Calcr neurons might mediate the short-term attenuation of food intake in response to CALCR agonists. Similarly, because Calcrcells do not contribute to the aversive effects of sCT, mediate aversion, or contribute to the activation of CGRPcells, non-NTS Calcr cells must mediate the CGRP-activating and aversive responses to sCT. Since the AP contributes to aversive signaling, including nausea (), it is possible that Calcrcells mediate the CTA-producing effects of sCT. Calcrcells could also mediate the Calcr-independent short-term suppression of food intake by sCT. Unfortunately, we and others have been unable to specifically target the AP by stereotaxic injection in mice, preventing us from directly testing this possibility.

NTS and CalcrNTS cells, respectively, we first used MB-ARC FOS-IR as a surrogate to examine the control of NAG cells by CalcrNTS neurons, since gut signals (that are presumably mediated by the hindbrain) inhibit these cells ( Beutler et al., 2017 Beutler L.R.

Chen Y.

Ahn J.S.

Lin Y.C.

Essner R.A.

Knight Z.A. Dynamics of Gut-Brain Communication Underlying Hunger. NTS neuron activation blunts ghrelin-induced hyperphagia (which is mediated by NAG cells). Indeed, we found that the activation of CalcrNTS neurons inhibited MB-ARC FOS-IR. While the ultimate circuit underlying this effect will require further studies, it is possible that CalcrNTS cells project to and activate the GABAergic DMH cells that directly inhibit NAG cells ( Garfield et al., 2016 Garfield A.S.

Shah B.P.

Burgess C.R.

Li M.M.

Li C.

Steger J.S.

Madara J.C.

Campbell J.N.

Kroeger D.

Scammell T.E.

et al. Dynamic GABAergic afferent modulation of AgRP neurons. To understand the potential circuit differences that underlie the aversive versus non-aversive suppression of food intake by Cckand Calcrcells, respectively, we first used MB-ARC FOS-IR as a surrogate to examine the control of NAG cells by Calcrneurons, since gut signals (that are presumably mediated by the hindbrain) inhibit these cells () and because we found Calcrneuron activation blunts ghrelin-induced hyperphagia (which is mediated by NAG cells). Indeed, we found that the activation of Calcrneurons inhibited MB-ARC FOS-IR. While the ultimate circuit underlying this effect will require further studies, it is possible that Calcrcells project to and activate the GABAergic DMH cells that directly inhibit NAG cells ().

NTS and CalcrNTS cells similarly suppress MB-ARC neuron activity. Thus, the difference between the effects of CckNTS and CalcrNTS neurons on aversion must lie in a distinct circuit. The DMH and/or PVH, both of which are innervated and activated by CckNTS and CalcrNTS cells, could also contribute to the suppression of food intake by these NTS cells types. Indeed, activation of the CckNTS→PVH circuit suppresses food intake ( D’Agostino et al., 2016 D’Agostino G.

Lyons D.J.

Cristiano C.

Burke L.K.

Madara J.C.

Campbell J.N.

Garcia A.P.

Land B.B.

Lowell B.B.

Dileone R.J.

Heisler L.K. Appetite controlled by a cholecystokinin nucleus of the solitary tract to hypothalamus neurocircuit. NTS cells is non-aversive ( D’Agostino et al., 2016 D’Agostino G.

Lyons D.J.

Cristiano C.

Burke L.K.

Madara J.C.

Campbell J.N.

Garcia A.P.

Land B.B.

Lowell B.B.

Dileone R.J.

Heisler L.K. Appetite controlled by a cholecystokinin nucleus of the solitary tract to hypothalamus neurocircuit. NTS and CalcrNTS cells are unlikely to underlie the difference in aversive signaling by the NTS cell types. While the inhibition of MB-ARC neurons may contribute to the suppression of food intake by NTS neurons, Cckand Calcrcells similarly suppress MB-ARC neuron activity. Thus, the difference between the effects of Cckand Calcrneurons on aversion must lie in a distinct circuit. The DMH and/or PVH, both of which are innervated and activated by Cckand Calcrcells, could also contribute to the suppression of food intake by these NTS cells types. Indeed, activation of the Cck→PVH circuit suppresses food intake (). Given that this projection from Cckcells is non-aversive (), however, these differences in PVH projections between Cckand Calcrcells are unlikely to underlie the difference in aversive signaling by the NTS cell types.

PBN cells in the aversive suppression of food intake ( Campos et al., 2016 Campos C.A.

Bowen A.J.

Schwartz M.W.

Palmiter R.D. Parabrachial CGRP Neurons Control Meal Termination. NTS neurons to suppress feeding via the PBN. While PBN projections from CalcrNTS cells suppress feeding, these neurons innervate a distinct PBN region compared to CckNTS cells and poorly activate CGRPPBN neurons compared to CckNTS cells. Hence, non-aversive, non-CGRP PBN neurons contribute to the anorectic effects of CalcrNTS cells. Consistently, the PBN region activated by CalcrNTS cells appears similar to that which relays a positive-valence vagal response to nutrient ingestion ( Han et al., 2018 Han W.

Tellez L.A.

Perkins M.H.

Perez I.O.

Qu T.

Ferreira J.

Ferreira T.L.

Quinn D.

Liu Z.W.

Gao X.B.

et al. A Neural Circuit for Gut-Induced Reward. Because of the important roles played by CGRPcells in the aversive suppression of food intake (), we examined the ability of Calcrneurons to suppress feeding via the PBN. While PBN projections from Calcrcells suppress feeding, these neurons innervate a distinct PBN region compared to Cckcells and poorly activate CGRPneurons compared to Cckcells. Hence, non-aversive, non-CGRP PBN neurons contribute to the anorectic effects of Calcrcells. Consistently, the PBN region activated by Calcrcells appears similar to that which relays a positive-valence vagal response to nutrient ingestion ().

NTS neurons contribute to the suppression of food intake by a variety of gut peptide-mimetic stimuli (e.g., CCK and Ex4), as well as CALCR agonists. CalcrNTS cells, but not CckNTS cells, also mediate the suppression of food intake by amino acids in the hindbrain (which do not produce a CTA). Although Calcr in CalcrNTS neurons is not required for normal energy balance, our data reveal the requirement for signaling by CalcrNTS neurons in the maintenance of long-term food intake control and energy homeostasis, especially in animals exposed to HFD. The importance of non-aversive CalcrNTS cells to limit the consumption of HFD is not only consistent with the failure of silencing CGRPPBN cells to alter long-term energy balance ( Campos et al., 2016 Campos C.A.

Bowen A.J.

Schwartz M.W.

Palmiter R.D. Parabrachial CGRP Neurons Control Meal Termination. NTS cells and their downstream circuits represent potentially useful therapeutic targets for the treatment of obesity. Calcrneurons contribute to the suppression of food intake by a variety of gut peptide-mimetic stimuli (e.g., CCK and Ex4), as well as CALCR agonists. Calcrcells, but not Cckcells, also mediate the suppression of food intake by amino acids in the hindbrain (which do not produce a CTA). Although Calcr in Calcrneurons is not required for normal energy balance, our data reveal the requirement for signaling by Calcrneurons in the maintenance of long-term food intake control and energy homeostasis, especially in animals exposed to HFD. The importance of non-aversive Calcrcells to limit the consumption of HFD is not only consistent with the failure of silencing CGRPcells to alter long-term energy balance (), but also suggests an important role for these cells in controlling the incentive value of palatable food. Thus, Calcrcells and their downstream circuits represent potentially useful therapeutic targets for the treatment of obesity.

Overall, CalcrNTS neurons define a hindbrain system that suppresses food intake without activating aversive systems and participates in physiological energy balance (as well as in the anorectic response to pharmacologic activation). These results also demonstrate the separability of NTS circuits that mediate aversive anorexia from those that mediate the non-aversive suppression of food intake.