INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CENTRAL INFUSION of either synthetic oxytocin (OT) or glucagon-like peptide-1 (GLP-1) inhibits deprivation-induced food intake in rats (1, 2, 13, 28, 30, 31). Hypothalamic OT- and hindbrain GLP-1-containing neurons are activated in rats after various treatments that promote anorexia; such treatments include systemic administration of lithium chloride, lipopolysaccharide, or cholecystokinin octapeptide (16, 17, 19, 32). Furthermore, pharmacological studies provided evidence that central OT and GLP-1 receptor signaling pathways contribute to the anorexigenic effects of several such treatments (5, 14, 15, 25).

OT neurons that project to sites within the brain are located in parvocellular subnuclei of the paraventricular nucleus of the hypothalamus (PVN) (27). GLP-1 neurons are located in the hindbrain, within and near caudal levels of the dorsal vagal complex (DVC) (8, 9, 11). Direct, reciprocal neural connections exist between the hypothalamic and hindbrain regions where OT and GLP-1 neurons are located (19, 20, 24, 26). Hypothalamic OT neurons express GLP-1 receptors (12, 33), and central administration of GLP-1 activates c-Fos expression in OT neurons (10). These findings suggest that the anorexigenic effect of centrally administered GLP-1 might result from activation of OT neurons and signaling pathways originating in the PVN. The present study was initiated to test this hypothesis. However, we also considered the possibility that the anorexigenic effect of centrally administered OT may be due to activation of hindbrain GLP-1 neurons and signaling pathways, because OT neurons are known to project to regions of the caudal brain stem where GLP-1 neurons are located. Thus we determined the effects on feeding of centrally administered GLP-1 in rats with and without prior blockade of OT receptors, and we also determined the effects on feeding of centrally administered OT in rats with and without prior blockade of GLP-1 receptors. In addition, immunocytochemical methods were used to determine whether OT-positive nerve fibers are associated with GLP-1-positive neurons in the caudal medulla and whether central administration of OT activates c-Fos expression by these neurons. Some of the results have been presented in abstract form (23).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Male Sprague-Dawley rats (Zivic Miller, Zelionople, PA or Harlan Laboratories, Indianapolis, IN) were initially housed individually in hanging wire cages in a controlled environment (24°C, lights on from 0700 to 1900). In this environment, rats had ad libitum access to water and pelleted rat chow (Purina). All experimental procedures were reviewed and approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

Cannulation procedures. Rats weighing 225-250 g were anesthetized with halothane (1.5-2% in oxygen) and placed into a stereotaxic frame with the incisor bar positioned 3.3 mm below horizontal zero. Rats were fitted with chronic indwelling 26-gauge stainless steel guide cannulas (Plastics One) aimed at the lateral ventricle. Guide cannulas were positioned 1.5 mm lateral to bregma on the coronal suture with the tip protruding 4.5 mm below the surface of the skull. Cannulas were fixed to the skull with anchor screws and dental acrylic and fitted with removable obturators that extended 0.5 mm beyond the tip of the guide cannula.

Automated food intake measurements. At least 1 wk after surgery, rats with ventricular cannulas were moved in groups of 6-10 to a new environment (24°C, lights on from 0300 to 1500). Rats were housed individually in clear Plexiglas boxes (25 × 30-cm floor, 22-cm height) with stainless steel rod floors, each equipped with a computer-driven pellet delivery and monitoring system (Med Associates). Drinking water was available ad libitum from sipper tubes within each box, but food access was restricted to a daily 4-h period beginning at lights out (1500). At lights out, a single 45-mg chow pellet (Precision Dustless Pellets, Bio-Serv, Frenchtown, NJ) was delivered automatically to a shallow feeding trough in each cage, thereby breaking a photobeam crossing the base of the trough. A new pellet was delivered automatically each time the rat removed the preceding pellet. The cumulative number of pellet deliveries (photobeam breaks) was recorded automatically every 30 min for the 4-h feeding period. Cage trays beneath the open rod floors were routinely inspected to ensure that delivered pellets were consumed. Data were collected and stored using Med PC software (Med Associates). Rats were acclimated to the new environment and feeding schedule for at least 1 wk before experimental manipulations, by which time stable 4-h daily food intakes of ~7-8% body wt were achieved. Rats were handled every 2 days for a brief period between 0100 and 0200 to record body weights, which remained steady or increased throughout each study. Because the rats' body weights ranged from 261 to 293 g on testing days in these studies, food intake is expressed as percent body weight in each experiment.

Evaluation of antagonist efficacy and selection of peptide doses. Lyophilized peptides were dissolved in sterile 0.15 M NaCl no more than 30 min before infusion and were delivered intracerebroventricularly in a volume of 5 µl. The efficacy of the antagonists used to block OT receptors {[d(CH₂)₅, Tyr(Me)², Orn⁸]vasotocin; Peninsula Laboratories} and GLP-1 receptors (des His¹ Glu⁹-exendin 4; American Peptides) has been reported (14, 15, 25, 29). The potency of each batch of antagonist used in the present study was also tested in pilot experiments associated with the present work. In the first pilot study (n = 6 rats), we determined that intracerebroventricular infusion of 10 µg of the OT receptor blocker by itself did not significantly affect cumulative 4-h food intake, but it did block the feeding-suppressive effect of synthetic OT (1 µg; Peninsula Laboratories) (Fig. 1A). Interestingly, the OT receptor antagonist did not attenuate the grooming behavior that was consistently elicited within 2-3 min after intracerebroventricular infusion of OT. In this first pilot experiment, we also determined that intracerebroventricular infusion of vehicle (0.15 M NaCl) did not affect cumulative 4-h food intake compared with the noninfused control condition (Fig. 1A). In the second pilot study (n = 5 rats), a similar approach was used to determine that 10 µg of GLP-1 receptor blocker infused alone did not significantly affect food intake, but it did block the feeding-suppressive effect of synthetic GLP-1 (7-36) amide (1 µg; Peninsula Laboratories) (Fig. 1B).

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Results of 2 pilot experiments to verify the efficacy of synthetic peptides and receptor antagonists used in these studies. Cumulative 4-h food intake (displayed as group means ± SE) was recorded in rats deprived of food for the preceding 20 h and given access to food at lights out. A: in the first experiment, rats received no infusion or intracerebroventricular infusion of oxytocin (OT; 1 µg) (n = 6) or OT receptor blocker (10 µg) followed 10 min later by OT (1 µg) (n = 6) or vehicle (0.15 M NaCl) (n = 5) or OT receptor blocker alone (10 µg) (n = 5). All rats were tested as a group in each condition in the order indicated, with 3 or 4 baseline recovery days between each test. One rat was removed from the study because of a loose cannula on the vehicle infusion day, and so data for the vehicle and OT blocker alone conditions were collected from 5 rats rather than 6. Food intake was significantly inhibited only after intracerebroventricular infusion of OT (* P < 0.05 compared with each other treatment). B: in the second experiment, a separate group of 5 rats received no infusion or intracerebroventricular infusion of glucagon-like peptide-1 (GLP-1; 1 µg) or GLP-1 receptor blocker (10 µg) followed 10 min later by GLP-1 (1 µg) or GLP-1 receptor blocker alone (10 µg). All 5 rats were tested in each condition in the order indicated, with 3 or 4 recovery days between each test. Food intake was significantly inhibited only after intracerebroventricular infusion of GLP-1 (* P < 0.05 compared with each other treatment). BW, body weight.

Feeding experiment 1. Rats (n = 11) were infused intracerebroventricularly with either 0.15 M NaCl vehicle or GLP-1 receptor antagonist (10 µg) followed 15 min later by synthetic OT (1 µg). Food access began 20 min after OT infusion, at lights out (1500). Cumulative pellet intake values were collected every 30 min for 4 h. Rats were tested twice, so that all 11 rats were included in both treatment conditions over 2 testing days, with 6 baseline days intervening. Baseline 4-h intakes were recorded on the day before each of the 2 testing days.

Feeding experiment 2. A separate group of rats (n = 9) was infused centrally with either 0.15 M NaCl vehicle or OT receptor antagonist (10 µg) followed 15 min later by infusion of GLP-1 (7-36) amide (1 µg). Food access began at lights out (1500), 20 min after GLP-1 infusion. Cumulative pellet intake values were collected every 30 min for 4 h. Rats were tested twice, so that all nine rats were included in both treatment conditions over 2 testing days, with 6 baseline days intervening. Baseline 4-h intakes were recorded on the day before each of the 2 testing days.

Food intake data analysis. Pellet delivery data for each rat were converted to grams (number of pellets × 45 mg), expressed as percent body weight (% BW), and then combined by experimental treatment group. Values are expressed as group means ± SE. Treatment-related differences in food intake within each experiment were tested for statistical significance by using one-way ANOVA, with the first infusion substance [vehicle, receptor blocker, or no infusion (baseline day)] as the independent variable. When F values indicated significant overall main treatment effects, ANOVA was followed up with post hoc t-tests using Dunn's (Bonferroni) correction procedure to control for multiple comparisons. Differences were considered significant when P  0.05.

Verification of intraventricular cannula placement. At the conclusion of each feeding study, rats were anesthetized (83 mg ketamine and 17 mg xylazine ip; Fort Dodge Labs), infused centrally with 5 µl of India ink (50% in distilled water), and perfused transcardially with 0.15 M NaCl (50 ml) followed by 4% paraformaldehyde (300 ml). Brains were removed from the skull and inspected visually for evidence of ventricular diffusion of ink. Ink was observed in the lateral, third, and fourth ventricles in each animal included in this report.

Immunocytochemical study. A separate group of rats with chronic lateral ventricular guide cannulas was infused centrally with 5 µl of vehicle (0.15 M NaCl; n = 5) or vehicle containing 1 µg OT (n = 5) between 1000 and 1100. Three additional rats were handled and had their guide cannula obturators loosened and retightened, but they received no central infusions. Four hours later, rats were anesthetized, infused centrally with 5 µl of 50% India ink, and perfused transcardially with 0.15 M NaCl (50 ml) followed by fixative (2% paraformaldehyde, 1% acrolein; 250 ml). Fixed brains were removed from the skull, bisected coronally at the level of the pineal gland, and inspected visually for evidence of ventricular diffusion of ink. Ink was observed in the lateral, third, and fourth ventricles in each animal included in this report.

Immunocytochemical data analysis. General patterns of treatment-related c-Fos expression were evaluated qualitatively by microscopic inspection of immunolabeled brain stem and forebrain sections. In addition, a quantitative analysis of c-Fos expression was conducted in brain stem tissue sections reacted for c-Fos and GLP-1. For this purpose, GLP-1-positive neurons in each rat were counted in six to eight hindbrain tissue sections (at 150-µm intervals) from the level of the pyramidal decussation caudally through the mid-area postrema rostrally. GLP-1-positive cells were identified by the presence of brown cytoplasmic immunoreactivity and were counted only when they displayed a visible nucleus. Cells were considered c-Fos positive when their nucleus contained blue-black nuclear immunoreactivity, regardless of intensity, and they were considered c-Fos negative when they displayed a visible nucleus lacking blue-black immunoreactivity. The number of GLP-1-positive neurons and the proportion that expressed c-Fos were determined for each rat; percentage activation values were then combined by treatment group and presented as means ± SE. Group differences in GLP-1 neuronal activation were tested for statistical significance by using one-way ANOVA, with treatment condition (no central infusion, vehicle infusion, or OT infusion) as the independent variable. When F values indicated significant overall main treatment effects, ANOVA was followed up with post hoc comparisons using Dunn's (Bonferroni) correction procedure to control for multiple comparisons. Differences were considered significant when P  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Feeding experiment 1. Average cumulative baseline intakes on the days preceding each of the 2 experimental treatment days did not differ significantly at any time point; thus these were combined to generate a single set of baseline values. As shown in Fig. 2, central administration of saline vehicle followed by OT (1 µg) significantly suppressed food intake compared with baseline at each 30-min time point during the 4-h monitoring period (P  0.05 for each comparison). Central administration of GLP-1 receptor antagonist (10 µg) blocked the anorexigenic effect of OT, such that intake values in rats receiving the GLP-1 receptor blocker followed by OT (1 µg) were not significantly different from baseline at any time point.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect on food intake of centrally infused OT (Saline; OT) compared with the effect of central OT preceded by central GLP-1 receptor antagonist (GLP-1 rec X; OT). Cumulative food intake was measured every 30 min for 4 h, beginning 20 min after the second intracerebroventricular infusion. Baseline intakes were recorded on the day before each experimental treatment day (see text). Intakes are expressed as % BW ± SE. OT dose = 1 µg in 5 µl saline vehicle; GLP-1 rec X dose = 10 µg in 5 µl saline vehicle. Cumulative intake values after central infusion of saline followed by OT are significantly smaller at each time point compared with baseline (P  0.05 for each comparison). Conversely, cumulative intakes are similar to baseline when central OT infusion is preceded by GLP-1 receptor blocker.

Feeding experiment 2. As in experiment 1, average baseline intakes on the days preceding each experimental treatment day did not differ significantly, and so these data were combined to generate a single set of baseline values. As shown in Fig. 3, central administration of saline vehicle followed by GLP-1 (7-36) amide (1 µg) significantly suppressed food intake relative to baseline at each time point examined during the 4-h monitoring period (P  0.05 for each comparison). Prior administration of OT receptor antagonist (10 µg) did not significantly alter the anorexigenic effect of GLP-1 (7-36) amide at any time point during the 4-h monitoring period, such that food intake remained significantly depressed relative to baseline (P  0.05 for each comparison).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect on food intake of centrally infused GLP-1 [(7-36) amide] (Saline; GLP-1) compared with the effect of central GLP-1 preceded by central OT receptor antagonist (OT rec X; GLP-1). Cumulative food intake was measured every 30 min for 4 h, beginning 20 min after the second intracerebroventricular infusion. Baseline intakes were recorded on the day before each experimental treatment day (see text). Intakes are expressed as % BW ± SE. GLP-1 dose = 1 µg in 5 µl saline vehicle; OT rec X dose = 10 µg in 5 µl saline vehicle. Cumulative intake values after central infusion of saline followed by GLP-1 are significantly smaller at each time point compared with baseline (P  0.05 for each comparison). Cumulative intakes remain significantly depressed compared with baseline when central GLP-1 infusion is preceded by OT receptor blocker (P  0.05 for each comparison).

Immunocytochemical study. As shown in Fig. 4, relatively few GLP-1-positive cells (4 ± 1%) expressed c-Fos in control rats that were handled but received no intracerebroventricular infusion. Conversely, central administration of synthetic OT (1 µg) activated c-Fos expression in the majority (72 ± 19%) of GLP-1-immunoreactive neurons (Figs. 4 and 5A). The proportion of GLP-1-positive neurons expressing c-Fos after intracerebroventricular infusion of 0.15 M NaCl vehicle (24 ± 11%; Fig. 5B) was significantly greater than the proportion activated in noninfused controls but was significantly less than the proportion activated in rats infused centrally with OT (P  0.05 for each comparison; Fig. 4). The hindbrain distribution of c-Fos- and/or GLP-1-positive neurons in two representative rats (one receiving vehicle intracerebroventricularly and one receiving OT intracerebroventricularly) is illustrated schematically in Fig. 6. Although central OT infusion activated the majority of GLP-1 neurons, these comprised but a subset of the total population of activated medullary neurons (Fig. 6, right). Determining the chemical phenotypes of these additional activated neurons was not a goal of the present study, but our initial observations indicate that they include catecholaminergic neurons of the A2 and A1 cell groups (23).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Percentage of GLP-1-immunoreactive neurons activated to express c-Fos immediate-early gene product. Relatively few GLP-1 neurons are activated in rats killed 4 h after handling and cannula manipulation but which received no intracerebroventricular (i.c.v.) infusion (No infusion). Moderate activation of GLP-1 neurons is observed in rats killed 4 h after i.c.v. infusion of 5 µl 0.15 M NaCl vehicle (Saline i.c.v.). Conversely, robust activation of GLP-1 neurons is observed 4 h after i.c.v. infusion of 5 µl of saline containing 1 µg OT (OT i.c.v.). Activation values for all 3 groups are significantly different from one another (P < 0.05 for each comparison).

View larger version (189K): [in this window] [in a new window]   Fig. 5.   Color photomicrographs illustrating c-Fos expression (black nuclear label) in GLP-1-positive neurons (brown cytoplasmic label) after central infusion of 1 µg OT (A) or after central infusion of 0.15 M NaCl vehicle (B). Large arrows in A and B point out some of the activated GLP-1-positive neurons. Small arrows in B point out GLP-1 neurons that do not express c-Fos. C: close appositions (arrows) between OT-immunolabeled fibers and terminals (black) and GLP-1-positive neurons (brown). C, inset: higher-magnification view of OT-positive fibers apposed to a GLP-1-positive cell. Scale bar in C = 100 µm, applies also to A and B; same bar = 50 µm in C, inset.

View larger version (42K): [in this window] [in a new window]   Fig. 6.   Schematic diagram indicating the distribution of GLP-1- and/or c-Fos-positive neurons in the caudal brain stem of 2 representative rats. The line down the middle divides the labeling of 1 control rat (left: vehicle i.c.v.) from the labeling observed in a rat after OT i.c.v. (right). Small dots are c-Fos-positive (activated) neurons. Open circles are GLP-1-positive neurons. Filled circles are GLP-1-positive, activated neurons. Labeling data were plotted manually (using Adobe Illustrator) onto closely matched rat brain stem image files (atlas levels 70, 71, and 72) downloaded from Swanson LW (1998/1999) Brain Maps: Structure of the Rat Brain (2nd edition), Elsevier, Amsterdam.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Results from this study indicate that the anorexigenic effect of centrally infused OT is significantly attenuated in rats after pharmacological antagonism of central GLP-1 receptors, whereas the anorexigenic effect of centrally infused GLP-1 is fully maintained after blockade of central OT receptors. Our immunocytochemical data demonstrate that the majority of hindbrain GLP-1 neurons are activated to express c-Fos after central infusion of an anorexigenic dose of OT. Furthermore, the close apposition of OT-immunoreactive fibers and terminals with GLP-1-positive perikarya suggests that GLP-1 neurons might respond directly to endogenous OT released at synapses in the caudal medulla (a possibility that must be confirmed by ultrastructural analysis). The new findings presented here support the hypothesis that central GLP-1 neurons and signaling pathways play a functionally important role in treatment-induced anorexia in rats, including the well-known anorexia that results from activation of central OT neural signaling pathways.

No abnormal motor effects were produced by the dose of OT used in the present report (1 µg). This dose significantly suppressed food intake relative to baseline, although the magnitude of this effect (~26% average suppression across all time points) was smaller than the 50-60% suppression reported after central infusion of a higher dose of OT (13). We elected to use the lower effective dose in the present work because we found that a higher dose of synthetic OT (10 µg) sometimes caused barrel-rolling and seizurelike activity lasting ~5-10 min.

The 4-h postinfusion survival time used in the c-Fos expression study was based on preliminary work in which lateral ventricular infusion of 0.15 M NaCl vehicle activated c-Fos expression in the majority of hindbrain GLP-1-immunopositive neurons in rats killed 60-90 min after the intracerebroventricular infusion (23). Subsequent experiments indicated that vehicle-induced c-Fos expression in GLP-1 neurons was significantly reduced when rats were perfused 4 h after the intracerebroventricular infusion. Because the anorexigenic effect of centrally administered OT persisted for at least 3-4 h (see Fig. 2), a 4-h postinfusion survival interval was considered valid for analysis of OT-induced c-Fos expression that was temporally related to OT-induced anorexia but separable from the nonspecific effects of the intracerebroventricular infusion itself. Even in rats killed 4 h after infusion, however, approximately six times as many GLP-1 neurons were activated in rats after intracerebroventricular infusion of saline vehicle compared with the proportion activated in control rats that were handled but received no infusion. Nevertheless, central infusion of OT activated significantly more GLP-1 neurons compared with either control condition (i.e., ~18 times more than in noninfused controls and 3 times more than in vehicle-infused controls; see Fig. 4).

Despite our findings that central infusion of saline vehicle activated c-Fos expression in GLP-1 neurons, vehicle infusion did not inhibit food intake in our pilot study (Fig. 1A). It is possible that intracerebroventricular saline produced a relatively transient anorexigenic effect that was not apparent in this study. Alternatively, the neuronal activation produced by intracerebroventricular saline infusion may simply be insufficient to suppress feeding in the absence of some other necessary signal. In this regard, vehicle-induced c-Fos expression in GLP-1 neurons is not accompanied by significant activation of either hindbrain catecholaminergic neurons or hypothalamic OT-positive neurons (23). In contrast, GLP-1-positive neurons, hindbrain catecholaminergic neurons, and OT-positive neurons are commonly activated by several treatments that inhibit food intake, such as systemic administration of lithium chloride (LiCl), cholecystokinin, or lipopolysaccharide (16, 17, 19, 22, 32).

Two previous reports indicated that endogenous central GLP-1 receptor signaling is an important factor underlying LiCl-induced anorexia (18, 25). However, the precise brain location(s) of receptors that mediate the presumed anorexigenic and/or nauseogenic effects of endogenous GLP-1 remain unclear. The present study was inspired by previous work suggesting a possible interaction of GLP-1 signaling pathways with central OT neurons. Hypothalamic regions that contain OT neurons are specifically targeted by GLP-1-immunoreactive axon terminals (9), and OT neurons are activated after central infusion of synthetic GLP-1 (10). Furthermore, pretreatment of rats with a central OT receptor antagonist reduces the anorectic effects of LiCl (14). Because the anorectic effects of LiCl are also attenuated after blockade of central GLP-1 receptors, we hypothesized that GLP-1 may inhibit food intake due to activation/recruitment of central OT signaling pathways. However, blockade of central OT receptors did not attenuate the anorexigenic effect of centrally infused GLP-1 in the present study. Instead, the converse was true: the anorexigenic effect of centrally infused OT was completely eliminated after pharmacological antagonism of central GLP-1 receptors.

These findings suggest that anorexigenic responses in which central OT neural pathways have been implicated may also include a functionally "downstream" role for GLP-1. In addition to its role in LiCl-induced anorexia, central OT receptor signaling contributes to anorexia in rats after systemic administration of hypertonic saline (14) and after central infusion of corticotropin-releasing factor (CRF) (15). Importantly, these anorexigenic treatments also inhibit vagally mediated gastric motility and emptying (4, 5), as does central infusion of GLP-1 (7). The present study confirmed that OT-immunopositive nerve terminals are closely apposed to GLP-1-positive neurons in the DVC and that these neurons are activated by central infusion of an anorexigenic dose of OT. The OT-containing projection from the PVN to the DVC provides a tonic inhibitory influence over vagally mediated gastric motility that is further amplified by anorexigenic treatments (3, 4). Thus experimental treatments that activate central OT signaling pathways may inhibit gastric motility and food intake, at least in part, due to OT-mediated recruitment/activation of hindbrain GLP-1 neurons. If so, then functional blockade of central GLP-1 signaling pathways should also attenuate the gastric inhibitory effects of centrally infused OT and other anorexigenic treatments, such as systemically administered LiCl or centrally infused CRF, parallel with attenuation of treatment-induced anorexia. This has not yet been examined. However, the ability of restraint stress to stimulate colonic motility and fecal output in rats is completely reversed after blockade of central GLP-1 receptors (6), implicating GLP-1 more generally as a candidate neurotransmitter in stress-induced changes in gut motility.

Perspectives

Among the many brain areas that potentially mediate treatment-induced anorexia, it is likely that the DVC (including its GLP-1 neural constituents) plays a central role. As the major brain stem relay center for visceral sensory information and descending projections from the hypothalamus and limbic forebrain, the DVC is well positioned to integrate interoceptive cues with cognitive/emotional states and thereby serve a "comparator function" for organizing appropriate behavioral and autonomic responses. Direct and relayed projections from the DVC to somatic and autonomic motor nuclei provide potential routes by which the DVC might exert context-dependent control over both the ingestion and digestion of food. The present findings should serve to focus greater attention on the DVC and particularly on its resident GLP-1 neurons as an integral component of the central neural circuits that underlie ingestive behavior.