Sucking patterns and behavioral state in 1- and 2-day-old full-term infants.

OBJECTIVE: To provide a comprehensive analysis of the temporal structure of sucking in full-term neonates. DESIGN: Descriptive study. SETTING: Newborn nursery in a city teaching institution. PATIENTS/PARTICIPANTS: Fifty-six full-term infants with a mean birth weight of 3,128±370 g completed sucking assessments on the first and second day of life. METHODS: A 5-minute sucking assessment was completed on the first and second day of life. Instruments included an Infant Nutritive Sucking Apparatus and the Anderson Behavioral Assessment Scale. RESULTS: The number of sucks (p<.001), intersuck width (p=.008) and interburst width (p<.05) were significantly different between the first and second day of life. On the second day of life the infants generated significantly more sucks, a decrease in interburst width and a decrease in intersuck width. There was a significant increase in the presence of an alert behavioral state from the first to second sucking assessment (p<.01). In addition, with a more alert infant state there was an increased time spent bursting (p<.001). CONCLUSION: Our results show that sucking analysis is sensitive to infant status and suggest that the development of sucking methodology can be considered as a useful clinical tool to assess the normal developmental course of sucking patterns.

Caudal brainstem delivery of ghrelin induces fos expression in the nucleus of the solitary tract, but not in the arcuate or paraventricular nuclei of the hypothalamus.

Ghrelin increases food intake when injected into either the forebrain or hindbrain ventricles. Brain areas activated by ghrelin after forebrain delivery have been examined using Fos immunohistochemistry and include the hypothalamic arcuate (Arc) and paraventricular (PVN) nuclei, and the nucleus of the solitary tract (NTS) in the medulla. It is not clear, however, if ghrelin applied directly to the hindbrain activates forebrain structures. Therefore, we examined Fos expression in the Arc, PVN, and NTS after injecting ghrelin into the fourth ventricle. Animals treated with a hyperphagic dose of ghrelin had greater levels of Fos expression in the NTS at the level of the area postrema than animals injected with vehicle. Ghrelin did not, however, increase Fos expression in the Arc or PVN in rats with open or occluded cerebral aqueducts. Given the importance of caudal brainstem (CBS) catecholamine pathways in the control of food intake, we performed double-labeling experiments to evaluate the potential overlap between tyrosine hydroxylase TH and ghrelin-induced Fos expression. Ghrelin did not increase Fos in TH-positive neurons in the NTS, suggesting that ghrelin delivered to the fourth ventricle does not act through catecholaminergic pathways. Nevertheless, the local (NTS), but not distal (Arc and PVN), induction of Fos suggests the presence of partially independent forebrain and hindbrain circuits that respond to ghrelin. These data support the NTS as a target of ghrelin action by building upon prior findings of increases in food intake in response to third- and fourth-ventricle ghrelin delivery.

Distinct forebrain and caudal brainstem contributions to the neuropeptide Y mediation of ghrelin hyperphagia.

Neuropeptide Y (NPY) has been implicated in the downstream mediation of ghrelin hyperphagia, with the site of action for both peptides considered to be intrinsic to the hypothalamus. Here, however, we observed robust hyperphagia with caudal brainstem (CBS) (fourth intracerebroventricular) ghrelin delivery and, moreover, that this response was reversed with coadministration of either of two NPY receptor antagonists (1229U91 and D-Tyr27,36, D-Thr32 NPY27-36) with contrasting NPY receptor subtype-binding properties. The same results were obtained after forebrain (third intracerebroventricular) administration, but the sites for both ghrelin and antagonist action were open to question, given the caudal flow of cerebrospinal fluid (CSF) through the ventricular system. To control for this, we occluded the cerebral aqueduct to restrict CSF flow between the forebrain and CBS ventricles and tested all combinations (same and cross ventricle) of ghrelin (150 pmol/1 microl) and NPY receptor antagonist delivery. With fourth intracerebroventricular ghrelin delivery after aqueduct occlusion, preadministration of either of the two antagonists through the same cannula reversed the hyperphagic response but neither was effective when delivered to the third ventricle. With third intracerebroventricular ghrelin administration, however, 1229U91 reversed the ingestive response only when delivered to the fourth ventricle, whereas D-Tyr27,36) D-Thr32 NPY27-36 was effective only when delivered to the forebrain. These results demonstrate distinct mediating pathways (due to location and subtypes of relevant NPY receptor) for the hyperphagic response driven separately by forebrain and CBS ghrelin administration.

Role of the duodenum and macronutrient type in ghrelin regulation.

The orexigenic hormone ghrelin is implicated in preprandial hunger and meal initiation in part because circulating levels increase before meals and decrease after food intake. The mechanisms underlying postprandial ghrelin suppression are unknown. Although most ghrelin is produced by the stomach, we have shown that neither gastric nutrients nor gastric distension affect ghrelin levels. We hypothesized that the nutrient-sensing mechanism regulating ghrelin is in the duodenum, the second richest source of ghrelin. To test whether duodenal nutrient exposure is required for ghrelin suppression, we infused nutrients into either the proximal duodenum or proximal jejunum in rats bearing chronic intestinal cannulas. At 0, 30, 60, 90, 120, 180, 240, and 300 min after infusions, blood was sampled via jugular-vein catheters for ghrelin, insulin, and glucose measurements. To elucidate further the mechanisms governing nutrient-related ghrelin suppression, we also assessed the ghrelin responses to isocaloric (3 kcal) infusions of glucose, amino acids, or lipids delivered into the stomach or small intestine of chronically catheterized rats. Regardless of macronutrient type, the depth and duration of ghrelin suppression were equivalent after gastric, duodenal, and jejunal infusions. Glucose and amino acids suppressed ghrelin more rapidly and strongly (by approximately 70%) than did lipids (by approximately 50%). Because jejunal nutrient infusions suppressed ghrelin levels as well as either gastric or duodenal infusions, we conclude that the inhibitory signals mediating postprandial ghrelin suppression are not derived discretely from either the stomach or duodenum. The relatively weak suppression of ghrelin by lipids compared with glucose or amino acids could represent one mechanism promoting high-fat dietary weight gain.

Central structures necessary and sufficient for ingestive and glycemic responses to Urocortin I administration.

CNS delivery of Urocortin I (UcnI), a member of the corticotropin-releasing factor family, suppresses feeding behavior and increases plasma glucose. The sites of action necessary and sufficient for these responses remain unclear. The contribution of the caudal brainstem was explored using chronically maintained decerebrate (CD) and neurologically intact control rats given fourth-ventricle injections of UcnI. Ingestive and glycemic responses were evaluated, and Fos immunoreactivity was measured in the paraventricular nucleus of the hypothalamus (PVN), the parabrachical nucleus (PBN), the rostral ventrolateral medulla (RVLM), and the nucleus of the solitary tract (NTS). CD rats, like the neurologically intact controls, decreased intraoral food intake and had elevated plasma glucose in response to Unc1 injections, indicating that forebrain structures are not required for these behavioral and physiological actions of UcnI. Fos immunohistochemistry, however, revealed notable differences in the pattern of UcnI-induced activation between intact and CD rats. UcnI-related activation was observed in each of the four aforementioned brain areas of neurologically intact rats but only in the NTS of CD rats. The intact behavioral and physiological responses to UcnI in the absence of neural activation in the PVN, PBN, and RVLM help limit the list of structures necessary for the stimulation and mediation of these responses to UcnI and suggest that the NTS may serve as a primary site of UcnI action.

Attenuation of lipopolysaccharide anorexia by antagonism of caudal brain stem but not forebrain GLP-1-R.

The central glucagon-like peptide-1 (GLP-1) system has been implicated in the control of feeding behavior. Here we explore GLP-1 mediation of the anorexic response to administration of systemic LPS and address the relative importance of caudal brain stem and forebrain GLP-1 receptor (GLP-1-R) for the mediation of the response. Fourth-intracerebroventricular delivery of the GLP-1-R antagonist exendin-(9-39) (10 microg) did not itself affect food intake in the 24 h after injection but significantly attenuated the otherwise robust (approximately 60%) reduction in food intake obtained after LPS (100 microg/kg) treatment. This result highlights a role for caudal brain stem GLP-1-R in the mediation of LPS anorexia but does not rule out the possibility that forebrain receptors also contribute to the response. Forebrain contribution was addressed by delivery of the GLP-1-R antagonist to the third ventricle with the caudal flow of cerebrospinal fluid blocked by occlusion of the cerebral aqueduct. Exendin-(9-39) delivery thus limited to forebrain did not attenuate the anorexic response to LPS. These data suggest that LPS anorexia is mediated, in part, by release of the native peptide acting on GLP-1-R within the caudal brain stem.

Food deprivation after treatment blocks the multiple-day hyperphagic response to SHU9119 administration.

The multiple-day hyperphagic effect of the melanocortin 3/4 receptor antagonist, SHU9119, is apparently abolished when rats are food-deprived for 24 h after central (4th-ventricular) injection. Here, we affirmed this indication, and addressed the possibility that the orexigenic potency of SHU9119 is simply masked by the refeeding hyperphagia that follows food deprivation. This explanation is discounted by our finding that the drug response in ad libitum-fed rats and the deprivation response are expressed at different, and non-overlapping, times of day. We then asked whether food consumption during a hypothesized critical period in the hours after treatment is necessary for expression of the hyperphagic response to SHU9119, or alternatively, whether blood-borne signals that emerge only after an extended period of food restriction underlie the drug-state interaction. Evidence favoring the latter interpretation was derived from a series of four experiments over which the timing and duration of food access after drug administration was varied. The results indicate an interaction between melanocortin receptor activity and the metabolic state of the animal, and constrain our thinking about the peripheral signals and central mechanisms that underlie this interaction.

Vagotomy dissociates short- and long-term controls of circulating ghrelin.

Plasma ghrelin levels are responsive to short- and long-term nutrient fluctuation, rapidly decreasing with food consumption and increasing with food deprivation or weight loss. We hypothesized a vagal contribution to both responses. Nutrient-related ghrelin suppression may be mediated by gastrointestinal load-related vagal afferent activity, or depend upon vagal efferent input to the foregut, where most ghrelin is produced. Similarly, the deprivation-induced ghrelin rise could require state-related vagal afferent or efferent activity. Here, we examined the role of the vagus nerve in the regulation of plasma ghrelin by sampling blood from rats with subdiaphragmatic vagotomy and from sham-operated controls over 48 h of food deprivation, and before and after gastric gavage of liquid diet. Vagotomy affected neither baseline ghrelin levels nor the suppression of ghrelin by a nutrient load. The food deprivation-induced elevation of plasma ghrelin levels ( approximately 160% of baseline), however, was completely prevented by subdiaphragmatic vagotomy. In a separate experiment, the deprivation-related rise in plasma ghrelin was substantially reduced by atropine methyl nitrate treatment, indicating that the response to fasting is driven by increased vagal efferent tone. The dissociation between nutrient load- and deprivation-related ghrelin responses indicates that the regulation of circulating ghrelin levels involves separate mechanisms operating through anatomically distinct pathways.

Hyperphagic effects of brainstem ghrelin administration.

The role of ghrelin in feeding control has been addressed from a largely hypothalamic perspective, with little attention directed at ingestive consequences of stimulation of the peptide's receptor, the growth hormone secretagogue receptor (GHS-R), in the caudal brainstem. Here, we demonstrate a hyperphagic response to stimulation of GHS-R in the caudal brainstem. Ghrelin (150 pmol) delivered to the third and fourth ventricles significantly and comparably increased cumulative food intake, with maximal response approximately 3 h after injection. The meal patterning effects underlying this hyperphagia were also similar for the two placements (i.e., significant reduction in the time between injection and first-meal onset, an increase in the number of meals taken shortly after the injection, and a trend toward an increase in the average size of the first meals that approached but did not achieve statistical significance). In a separate experiment, ghrelin microinjected unilaterally into the dorsal vagal complex (DVC) significantly increased food intake measured 1.5 and 3 h after treatment. The response was obtained with a 10-pmol dose, establishing the DVC as a site of action with at least comparable sensitivity to that reported for the arcuate nucleus. Taken together, the results affirm a caudal brainstem site of action and recommend further investigation into multisite interactions underlying the modulation of ingestive behavior by ghrelin.

Meal-related ghrelin suppression requires postgastric feedback.

Plasma ghrelin levels are rapidly suppressed by ingestion or gastric delivery of nutrients. Given that the majority of circulating ghrelin appears to be of gastric origin, we addressed the contribution of gastric distention or nutrient sensitivity to this response. Awake, unrestrained rats received intragastric infusions of glucose or water (1 ml/min for 12 min) with gastric emptying either proceeding normally or prevented by inflation of a pyloric cuff. When emptying was permitted, glucose infusion reduced ghrelin level by approximately 50%, and, in agreement with previous data, water infusions were without effect. Ghrelin level was not affected by either infusate when gastric emptying was prevented, thereby discounting a role for gastric distention in the meal-related ghrelin response. That glucose and water infusions were similarly ineffective when the pylorus was occluded shows, further, that gastric chemosensation is not a sufficient trigger for the ghrelin response. We conclude that the meal-related suppression of plasma ghrelin requires postgastric (pre- or postabsorptive) stimulation.

Behavioral processes underlying the intake suppressive effects of melanocortin 3/4 receptor activation in the rat.

RATIONALE: Central application of MTII, a melanocortin 3/4 receptor agonist, reduces food intake. The behavioral mechanisms underlying the anorexia, however, have not been evaluated. OBJECTIVES: We examined the ingestive behavioral effects of MTII at the microstructural level using two complementary approaches. METHODS: Rats were given daily 2-h sessions during which they drank 12.5% glucose solution; the time of occurrence of each lick event was recorded. We compared rats' glucose intake 30 min after the fourth ICV injection of 0.1, 0.33, and 1.0 nmol MTII or vehicle. The licking patterns were examined to discern effects on parameters related to taste processes and others related to post-ingestive inhibitory feedback. A second experiment directly analyzed the effect of MTII on motor performance by examining whether drug treated rats would, like controls, adjust licking output to maintain meal size when lick volume was shifted from 8 to 4 microl. RESULTS: Meal size was reduced by MTII in a dose-dependent manner (20-50%) in both experiments. Rats treated with MTII compensated for decreased lick volume by substantially increasing the number of licks emitted. Licking parameters associated with taste evaluation were not significantly affected by MTII, whereas parameters associated with post-ingestive inhibition varied as a function of treatment. CONCLUSIONS: Results suggest that MTII reduces intake by amplifying post-ingestive feedback inhibition. That MTII-treated rats increase the number of licks emitted in response to the lick volume reduction discounts the suggestion that intake inhibition is secondary to disruption of motor performance.

The neuroanatomical axis for control of energy balance.

The hypothalamic feeding-center model, articulated in the 1950s, held that the hypothalamus contains the interoceptors sensitive to blood-borne correlates of available or stored fuels as well as the integrative substrates that process metabolic and visceral afferent signals and issue commands to brainstem mechanisms for the production of ingestive behavior. A number of findings reviewed here, however, indicate that sensory and integrative functions are distributed across a central control axis that includes critical substrates in the basal forebrain as well as in the caudal brainstem. First, the interoceptors relevant to energy balance are distributed more widely than had been previously thought, with a prominent brainstem complement of leptin and insulin receptors, glucose-sensing mechanisms, and neuropeptide mediators. The physiological relevance of this multiple representation is suggested by the demonstration that similar behavioral effects can be obtained independently by stimulation of respective forebrain and brainstem subpopulations of the same receptor types (e.g., leptin, CRH, and melanocortin). The classical hypothalamic model is also challenged by the integrative achievements of the chronically maintained, supracollicular decerebrate rat. Decerebrate and neurologically intact rats show similar discriminative responses to taste stimuli and are similarly sensitive to intake-inhibitory feedback from the gut. Thus, the caudal brainstem, in neural isolation from forebrain influence, is sufficient to mediate ingestive responses to a range of visceral afferent signals. The decerebrate rat, however, does not show a hyperphagic response to food deprivation, suggesting that interactions between forebrain and brainstem are necessary for the behavioral response to systemic/ metabolic correlates of deprivation in the neurologically intact rat. At the same time, however, there is evidence suggesting that hypothalamic-neuroendocrine responses to fasting depend on pathways ascending from brainstem. Results reviewed are consistent with a distributionist (as opposed to hierarchical) model for the control of energy balance that emphasizes: (i) control mechanisms endemic to hypothalamus and brainstem that drive their unique effector systems on the basis of local interoceptive, and in the brainstem case, visceral, afferent inputs and (ii) a set of uni- and bidirectional interactions that coordinate adaptive neuroendocrine, autonomic, and behavioral responses to changes in metabolic status.

Evidence that the caudal brainstem is a target for the inhibitory effect of leptin on food intake.

Three experiments were performed to investigate the hypothesis that leptin action within the caudal brain stem (CBS) contributes to its intake inhibitory effects. The first experiment evaluated the anatomical distribution of leptin receptor mRNA in rat CBS using a sensitive fluorescence in situ hybridization method with a riboprobe specific for the long form of the leptin receptor (Ob-Rb). An Ob-Rb mRNA hybridization signal was detected in neurons of several CBS nuclei involved in the control of food intake, including the dorsal vagal complex and parabrachial nucleus. A strong hybridization signal was also obtained from neuronal cell bodies of a number of other structures including the hypoglossal, trigeminal, lateral reticular, and cochlear nuclei; locus ceruleus; and inferior olive. The anatomical profile revealed by fluorescence in situ hybridization was in good agreement with immunocytochemical analysis with an antibody specific to Ob-Rb. In a second experiment, exploring the relevance of CBS Ob-Rb to feeding behavior, rats were given a fourth intracerebroventricular (i.c.v.) injection of leptin (0.1, 0.83, or 5.0 microg; n = 9-11/group) or vehicle 30 min before lights-out on three consecutive days. The two higher doses reduced food intake significantly at 2, 4, and 24 h after injection and caused significant reductions of body weight. The dose-response profiles for fourth i.c.v. administration were indistinguishable from those obtained from separate groups of rats that received leptin via a lateral i.c.v. cannula. In the last experiment, a ventricle-subthreshold dose of leptin (0.1 microg) microinjected unilaterally into the dorsal vagal complex suppressed food intake at 2, 4, and 24 h. The results indicate that the CBS contains neurons that are potentially direct targets for the action of leptin in the control of energy homeostasis.