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Phenylthiourea (PTU) is commonly used for inhibiting melanization of zebrafish embryos. In this study, the standard treatment with 0.2 mM PTU was demonstrated to specifically reduce eye size in larval fish starting at three days post-fertilization. This effect is likely the result of a reduction in retinal and lens size of PTU-treated eyes and is not related to melanization inhibition. This is because the eye size of tyr, a genetic mutant of tyrosinase whose activity is inhibited in PTU treatment, was not reduced. As PTU contains a thiocarbamide group which is presented in many goitrogens, suppressing thyroid hormone production is a possible mechanism by which PTU treatment may reduce eye size. Despite the fact that thyroxine level was found to be reduced in PTU-treated larvae, thyroid hormone supplements did not rescue the eye size reduction. Instead, treating embryos with six goitrogens, including inhibitors of thyroid peroxidase (TPO) and sodium-iodide symporter (NIS), suggested an alternative possibility. Specifically, three TPO inhibitors, including those that do not possess thiocarbamide, specifically reduced eye size; whereas none of the NIS inhibitors could elicit this effect. These observations indicate that TPO inhibition rather than a general suppression of thyroid hormone synthesis is likely the underlying cause of PTU-induced eye size reduction. Furthermore, the tissue-specific effect of PTU treatment might be mediated by an eye-specific TPO expression. Compared with treatment with other tyrosinase inhibitors or bleaching to remove melanization, PTU treatment remains the most effective approach. Thus, one should use caution when interpreting results that are obtained from PTU-treated embryos.

The present studies assessed the extent to which the adiposity signal leptin and the brain-gut hormone cholecystokinin (CCK), administered alone or in combination, give rise to interoceptive sensory cues like those that are produced by a low (1h) level of food deprivation. Rats were trained with cues arising from 1 to 24-h food deprivation as discriminative stimuli. For one group, 24-h food deprivation predicted the delivery of sucrose pellets, whereas 1-h food deprivation did not. Another group received the reversed deprivation level-sucrose contingency. After asymptotic performance was achieved, the effects of leptin and CCK on food intake and on discrimination performance were tested under 24-h food deprivation. In Experiment 1a, leptin administered into the third cerebroventricle (i3vt) at 3.5 or 7.0 microg doses had little effect, compared to saline on food intake or discriminative responding. In Experiment 1b, leptin (7.0 microg, i3vt) combined with CCK-8 (2 microg/kg, i.p.) reduced food intake significantly, but the findings indicated that CCK-8 alone produces interoceptive discriminative cues more like those produced by 1- than 24-h food deprivation. Experiment 2a tested rats with i.p. leptin (0.3 and 0.5mg/kg). Although neither dose suppressed intake, the 0.3mg/kg dose produced interoceptive cues like 1-h food deprivation. Experiment 2b tested two doses of CCK-8 (2 and 4 mg/kg, i.p.) and found significant intake suppression and generalization of discrimination with both doses of CCK-8. These findings suggest a role for both leptin and CCK in the production of sensory consequences that correspond to "satiety".

At a simple behavioral level, food intake and body weight regulation depend on one's ability to balance the tendency to seek out and consume food with the ability to suppress or inhibit those responses. Accordingly, any factor that augments the tendency to engage in food seeking and eating or that interferes with the suppression of these behaviors could produce (a) caloric intake in excess of caloric need; (b) increases in body weight leading to obesity. This paper starts with the idea that excess body weight and obesity stem from a failure or degradation of mechanisms that normally function to inhibit eating behavior. Unlike previous approaches, we focus not on failures of traditional physiological (e.g., neural, hormonal) regulatory control mechanisms, but on disruptions of inhibitory learning and memory processes that may help to regulate energy intake. This view of energy dysregulation as a type of "learning disorder" leads us to the hippocampus, a brain structure that has long been regarded as an important substrate for learning and memory and which we think may be critically involved with a specific type of memory inhibition function that could contribute to the suppression of food intake. With this focus, the search for environmental origins of the current obesity epidemic in Western populations is directed toward factors that alter hippocampal functioning. We conclude by offering a preliminary account of how consumption of foods high in saturated fats might lead to impaired hippocampal function, reduced ability to inhibit caloric intake and, ultimately, to increased body weight.

A number of recent studies implicate the gut-brain peptide ghrelin as a putative "hunger signal". Most of these studies, however, rely on either consummatory behavior (in humans or nonhuman animals) or self-report (in humans) to draw conclusions regarding the orexigenic properties of this peptide. The present study employs the deprivation intensity discrimination paradigm to assess the interoceptive sensory properties of ghrelin in rats. In this paradigm, one group of rats was placed in a training context and presented with sucrose pellets when 24 h food deprived, but not when 1 h food deprived (24+ group). A second group was trained using the opposite sucrose-deprivation level contingency (1+ group). Learning in this paradigm was demonstrated by animals approaching the food delivery location more frequently under their rewarded compared to their non-rewarded deprivation condition (prior to actual pellet delivery). After asymptotic performance of this discrimination was achieved, these animals (1 h food deprived) were administered ghrelin or saline, either i.p. (3 or 6 nmol) or i3vt (0.1 or 1 nmol), placed in the training context, and appetitive responses were measured. Testing was conducted in extinction, eliminating confounding effects of food consumption. Results of these tests showed that 6 nmol i.p. ghrelin and 0.1 and 1 nmol i3vt ghrelin all generalized to a state of 24 h food deprivation, indicating that exogenous ghrelin has sensory properties in common with the stimuli produced by 24 h food deprivation. These results support the notion that endogenous ghrelin contributes to an interoceptive hunger cue, and that this may be a mechanism by which ghrelin influences food intake and appetitive behavior.

Experiment 1 used Pavlovian conditioning procedures to show that rats formed distinct memorial representations of 2 (peanut oil and sucrose pellets) unconditioned stimuli (USs) that could be activated by 2 different conditioned stimuli (CSs). After training in Experiment 2, rats injected with the lipid antimetabolite Na-2-mercaptoacetate (MA) responded more to the CS for oil than to the CS for sucrose. This pattern was not shown by rats that received isotonic saline or systemic 2-deoxy-d-glucose (a glucose antimetabolite). By contrast, intracerebroventricular infusion of the glucose antimetabolite 5-thioglucose selectively promoted responding to the CS for sucrose (Experiment 4). Thus, lipoprivic and glucoprivic treatments selectively promoted the activation of the memories of fat and carbohydrate USs, respectively. In Experiment 3, the capacity of MA to augment responding to a CS for oil was abolished for rats that received subdiaphragmatic vagal deafferentation. This indicates that the capacity of lipoprivic signals to selectively activate the representations of fat USs may depend on vagal afferent fibers.

Experiments employing infusions of nutrients into the gastrointestinal tract commonly deliver large volumes of solutions without evaluating the possibility that reflux of the infusate to more orad sites may occur. To assess this possibility for one conventional paradigm, rats with gastric fistulas and intestinal catheters were infused intraduodenally (4.0 to 5.0 cm distal to pylorus) in association with a meal. Infusions of 0.0 ml to 15.0 ml of 3% glucose and a dye marker or 0.9% saline containing a dye marker were delivered at 1 ml/min, and stomach contents were assayed for the infusates. All three probes were detected in stomach contents. The glucose marker proved the most sensitive of the three and indicated that duodenogastric reflux occurred in a dose-dependent manner with infusions > 2.5 ml.

The arrival of nutrients in the gastrointestinal tract suppresses intake. To specify the neural pathways and receptor locations of this feedback, we examined the effects of intraduodenal infusions of 10 nutrients plus saline on short-term food intake of rats with selective deafferentations of vagal celiac branches. Three response profiles were observed: 1) isotonic saline, 5.6% glycerol, and 3% fructose did not inhibit intake of controls or selectively deafferented animals; 2) 3% glucose, 3% maltose, 3% L-phenylalanine, 12% Isocal, and 1.4% oleic acid suppressed intake of controls, but this inhibition was eliminated by vagal celiac deafferentation; and 3) 3% casein hydrolysate and 24% Isocal suppressed intake of controls and rats with selective vagotomies, although the latter exhibited significantly less suppression. In addition, elimination of celiac afferents chronically reduced meal size (i.e., first 30-min intake) without reducing daily food intake or body weight. Furthermore, D-phenylalanine infusions produced a delayed suppression of food intake in controls (possibly from intraluminal irritation); however, this reduction was eliminated with celiac deafferentation. Overall, this experiment indicates that vagal celiac afferents are critical for preabsorptive detection of some energy-yielding molecules or properties of nutrient solutions (as well as, perhaps, intraluminal inflammation), but not others, which are still detected, although only partially.

We have developed a dorsal intracranial surgery that is minimally invasive and gives excellent access to either afferent or efferent vagal rootlets to produce selective deafferentations or deefferentations in the rat. We have combined this new unilateral afferent rhizotomy with a contralateral celiac branch cut (to completely deafferent the intestines) and a duodenal catheter placement 4 cm distal to the pylorus. Animals were maintained with 17 h/day access to a nutritionally complete liquid diet. Measures of first meal size, daily intake, and body weight before and after both surgeries indicated that animals with unilateral vagal deafferentiations recovered as fast and completely as sham-operated controls. Intraduodenal oleate (1.2 kcal) infusions reduced the size of the first meal in surgical controls (by 64%; P < 0.01) but not in the deafferented rats. A dual wheat germ agglutinin-horseradish peroxidase/Fluorogold protocol provides verification of sensory and motor lesions. The selective vagal deafferentation provided by the new surgery offers a useful model for determining gastrointestinal sites of nutrient detection and separating pre- and postabsorptive consequences of a meal.

To test whether the route of insulin delivery has a major effect on the increase in daily food intake associated with chronic insulin treatment, insulin was continuously infused into either the vena cava (VC) or the hepatic portal (HP) vein of 23 diabetic Lewis rats. Increasing insulin doses in both the VC (2 to 6 U/day) and HP (1.5 to 3.5 U/day) groups significantly increased daily food intake (p < .05). Intake was higher in the VC group at 3 U/day but not at 2U/day. When insulin was delivered at a low fixed dose, daily food intake of both the VC and HP groups only increased after urinary glucose losses increased. The rate of weight gain increased significantly in the VC varied group (p < .05). Insulin administration also increased energy expenditure (p < .01). These results suggest that the extent of the increase in daily food intake and body weight that occurs with peripheral exogenous insulin administration is dependent on the route of infusion.

The effects of insulin dose and diet composition on daily food intake were investigated by IV infusion of insulin in doses of 2 to 5 U/day into diabetic rats consuming either a high CHO or high fat diet. The daily food intake of the diabetic rats on both diets increased significantly over baseline levels (p < .01) at the low insulin doses and was maintained at these elevated levels through the 5 U/day dose. Insulin increased the rate of weight gain from Ig/day during baseline to 2 and 2.5 g/day in high CHO and high fat fed diabetics (p < .01). These results show that treatment of diabetic rats with continuous low doses of IV insulin results in a 40% increase in daily food intake regardless of the diet consumed and this increase is accompanied by an increase in rate of body weight gain. While the high fat fed diabetics were relatively hypoglycemic, these increases in intake are not the result of insulin-induced hypoglycemia, since blood glucose concentrations are significantly elevated when the increases occur at the lower insulin doses (p < .01). Thus, peripheralinsulin infused at physiological levels stimulates rather than inhibits daily food intake.

In order to determine whether insulin receptors in the brain or in the periphery are more important for the control of food intake, four doses of insulin (0.1, 0.5, 1.0 and 2.0 U/day) were each infused continuously over 24 h into rats with chronic catheters in the common carotid artery (C) or the superior vena cava (VC). Daily food intake was unchanged from baseline levels by insulin doses of 0.1 to 1.0U. However, intake increased by 8.1 +/- 1.3 kcal for the C rats and by 10.2 +/- 1.8 kcal for the VC rats during the 2.0U infusion and was also increased on the subsequent day, by 10.9 +/- 1.9 kcal and 15.4 +/- 3.0 kcal for C and VC groups respectively. Rats were fed half an hour after insulin infusions began and measures of short-term intake made over the next 30 min were unaffected by any of the insulin doses. When injected into C catheters prior to sacrifice greater than 90% of 57Co labelled microspheres were recovered in arterioles of the brain, skull and facial regions. As brain blood flow is 1.8% of cardiac output, insulin levels in the brain blood supply would have been substantially greater for C rats. The similar increases in food intake for C and VC rats clearly show that insulin fails to produce greater effects when infused directly into the brain. Instead, these findings suggest that the effect of insulin on daily food intake is predominantly a function of insulin receptor activation in the periphery.

Three types of nutrient were infused intravenously for 30 min before and during the 17 h when rats were fed to assess the effects of these nutrients on short-term and daily food intake. Infusions of glucose (34 kcal/day for 4 days) reduced food intake from saline baseline levels by 18.8 +/- 1.9 kcal/day. This represents an oral intake reduction equivalent to 55% of each calorie infused. When amino acids were delivered intravenously at 10 and then 20 kcal/day for 4 days, food intake was reduced by 11.9 +/- 1.4 and 20.4 +/- 2.4 kcal/day, which represent oral intake reductions of 112 and 103% of infused calories, respectively. Food intake was reduced 8.2 +/- 0.5 and 16.6 +/- 1.4 kcal/day (or 41 and 42% of calories infused), when a lipid emulsion was delivered at 20 or 40 kcal/day for 6 days, respectively. A combination of all three nutrients (i.v. diet composed of 50% glucose, 13% amino acids, and 37% lipid by calories) identical to the nutrient composition of the oral diet was infused at 20 and then 40 kcal/day for 6 days. Food intakes were reduced by 14.8 +/- 2.0 and 32.3 +/- 3.3 kcal/day, representing 74 and 80% of infused calories. Recovery of food intake to previous saline baseline levels was rapid and complete after the glucose and amino acid infusions but was delayed and incomplete after the lipid and intravenous diet conditions. These results indicate that infused amino acids or a balanced intravenous diet is more effective than glucose or fats in inhibiting daily food intake.

Studies on two different types of one-way crossed-intestines rats have shown that daily food intake is controlled by either endogenous gut signals or absorbed nutrients and their metabolic consequences, or both. If the amount of incoming ingested food is metered somewhere in the body, this could only occur in the gut or liver. The capacity of the liver to determine the amount of water-soluble nutrient absorbed was assessed by portacaval shunt and found to be inadequate. Infusion of nutrients directly into the bloodstream show that plasma nutrients provide part of the signal that inhibits daily food intake, but that endogenous gut signals must play some role. Insulin, an important hormone in the movement of plasma nutrients into cells, was found to stimulate food intake at low infusion doses. IV nutrients raise the level of plasma nutrients and lower daily food intake, while insulin, which inhibits the release of endogenous fuels and moves exogenous fuels into cells, lowers plasma nutrients and stimulates daily intake. Thus, the integrated level of all plasma nutrients may be a major controller of daily food intake.

To determine whether feeding-generated gut signals act in conjunction with intravenous nutrients to suppress food intake, nutrients were infused intravenously to dark-fed rats during different portions of the 12:12 h light-dark cycle. During 4-day test periods rats received 35.5-37 kcal/day of a solution containing 25% glucose-4.25% amino acids. Food intakes were reduced by 40.5 +/- 1.1 and 30.7 +/- 1.8 kcal or 110 and 87% of the calories infused in the dark (fed) phase over 8 and 12 h, respectively. When administered in the light phase while rats were fasted, 8- and 12-h infusions reduced food intake significantly less than in the dark: 21.3 +/- 1.1 and 19.6 +/- 0.7 kcal/day or only 57 and 55% of the infused calories, respectively. With 24-h infusions, as in previous studies, food intake was reduced by 24.5 +/- 2.1 kcal/day or 68% of the infused calories. These results suggest that gut signals generated by concurrent feeding act with intravenous glucose and amino acids to produce a more compensatory reduction in daily food intake.

Current theories state that physiological levels of insulin inhibit daily food intake and reduce body weight. To test whether insulin induces satiety, systematically increasing doses of insulin from 2.0 to 5.0 U/day were infused intravenously into streptozotocin-induced diabetic rats. Food intake increased significantly from 70.0 +/- 1.4 kcal/day during the saline baseline up to 102.2 +/- 1.9 kcal/day in the 3.5 U/day insulin infusion and then stabilized at 95.9 +/- 0.5 kcal/day for the remaining doses (p less than 0.05). Retained energy values (kcal of food intake minus kcal of urinary glucose loss) also increased from 69.9 +/- 1.4 kcal/day to stabilize at 95 kcal/day (p less than 0.001). Food intake and retained energy of normal controls remained unchanged at 75.4 +/- 1.6 kcal/day for the duration of the study. With elevated food intake and retained energy values after the 3.5 U/day insulin dose, the diabetic rats gained more weight than the normal controls (p less than 0.01). Contrary to expectations, increasing the amount of insulin infused through the physiological range results in a 40% increase in daily food intake and a rapid gain in body weight.

This is a report of the concentrations of DA, DOPAC, HVA, 5-HT, and 5-HIAA in the brain of spontaneously diabetic male Wistar rats. These rats showed a marked increase in blood and urine glucose, polydipsia, polyuria, and weight loss that had an onset 11-23 days earlier. Controls were litter mates with no hyperglycemia, glucosuria or weight reduction. The spontaneously diabetic rats showed a significant reduction of DOPAC in the striatum, and DOPAC, HVA, and 5HIAA in the olfactory tubercles (to 69, 61, 62, and 65% of their respective controls). No changes were found in the concentrations of DA or 5-HT. Thus the spontaneously diabetic rats showed a marked reduction in striatal and mesolimbic DA and mesolimbic 5-HT metabolism. This reduction in metabolism could be the consequence of a reduction in the formation of DA and 5-HT.

Rats received large, bilateral lesions of the ventromedial hypothalamus. Water or saline intakes, urine outputs and body temperatures were observed for up to 24 hr after surgery. Fifty percent of the operated animals drank excessively and died within 4-6 hr when permitted access to water. Urine outputs were low and symptoms of water intoxication were evident. When allowed access to saline, outputs rose and the number of animals which survived increased as the saline concentration increased. Body temperatures approached 40 degrees C during drinking, but did not differ from operated animals which refused to drink. It was concluded that the deposition of metallic ions strongly stimulates a hypothalamic drinking system which results in overhydration and water intoxication death.

To assess the effect of gut signals on food intake two types of nutrients were infused intravenously for 17.5 hours in 17 hour fed rats. In the first experiment a solution of 25% d-glucose and 4.25% amino acids (Travasol) was infused at levels of 26 and 52 kcal/day for two consecutive four-day periods. During infusion periods, food intake was reduced from saline baseline levels by 18.9 +/- 1.7 and 34.8 +/- 1.8 kcal/day, respectively. This represents an oral intake reduction of approximately 70% of the infused calories. In contrast, food intake was reduced 17.4 +/- 1.7 kcal/day below saline baseline levels when 40 kcal of Nutralipid were infused. The reduction in food intake was only 43% of the lipid calories infused. These results indicate that infusions of glucose and amino acids are more effective than infusion of fats in inhibiting daily food intake, that gut signals associated with absorption of fat provide important satiety signals and that removal of fat from the bloodstream has relatively little effect on daily food intake.

A method for chronic or acute cannulation of the third ventricle of the rat is described. This technique, in which successful cannulation is verified by the aspiration of a minute quantity of cerebrospinal fluid, is simple, reliable and produces minimal tissue distortion. The results of an experiment employing the technique and which compared the effects of direct intrahypothalamic and intraventricular administration of phentolamine on stimulus-bound feeding are briefly described.