Site selective activation of lateral hypothalamic mGluR1 and R5 receptors elicits feeding in rats.

Recent findings from our lab indicate that metabotropic glutamate receptor (mGluR) activation elicits eating, and the goal of the current study was to specify whether the lateral hypothalamus (LH) is the actual brain site mediating this effect. To examine this issue we injected the selective mGluR group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) unilaterally into the LH and surrounding regions (n=5-8 subjects/brain site) of satiated adult male Sprague-Dawley rats and measured elicited feeding. We determined that 1.0 nmol elicited food intake only within the LH. Increasing the dose to 10 or 25 nmol produced a more sustained effect in the LH, and also elicited eating in several other brain sites. These results, demonstrating that the LH mediates the eating elicited by low doses of DHPG, suggest that the LH may contain mGluR whose activation can produce eating behavior.

Activation of lateral hypothalamic mGlu1 and mGlu5 receptors elicits feeding in rats.

Metabotropic glutamate receptors (mGluRs) have been popular drug targets for a variety of central nervous system (CNS) disease models, ranging from seizures to schizophrenia. The current study aimed to determine whether mGluRs participate in lateral hypothalamic (LH) stimulation of feeding. To this end, we used satiated adult male Sprague-Dawley rats stereotaxically implanted with indwelling bilateral LH guide cannulas to determine if injection of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), a broad mGluR group I and II agonist, would elicit feeding. Administration of 100 nmol ACPD induced feeding with a short latency. Similarly, unilateral LH injection of the selective mGluR group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) elicited significant feeding beginning 60 min postinjection and continuing until 4 h postinjection. Administration of the mGluR5 agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) produced a smaller delayed feeding response. These delayed but prolonged eating responses suggest that activation of LH mGluR1 and/or mGluR5 might be sufficient to elicit feeding. To determine which subtypes were involved, LH DHPG injections were preceded by LH injection of either the group I antagonist n-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC), the mGluR1 antagonist 6-amino-n-cyclohexyl-n,3-dimethylthiazolo[3,2-a]benzimi dazole-2-carboxamide hydrochloride (YM-298198) or the mGluR5 antagonist 3-((2-methyl-4-thiazolyl)ethynyl)pyridine (MTEP), and food intake was measured. PHCCC blocked DHPG-elicited feeding, and each of the other antagonists produced significant feeding suppression. These findings suggest roles for mGluR1 and/or mGluR5 in lateral hypothalamic circuits capable of stimulating feeding behavior.

N-methyl-D-aspartate receptor subunit NR2B is widely expressed throughout the rat diencephalon: an immunohistochemical study.

Glutamate (Glu), a major excitatory neurotransmitter within the hypothalamus and thalamus, acts upon many receptors, including the N-methyl-D-aspartate (NMDA) subtype. Abundant evidence suggests that variations in the subunit composition of NMDA receptors (NMDA-Rs) contribute to differences in Glu's immediate electrophysiological effects as well as to the patterns of signal transduction cascades it triggers to mediate long-term changes in neuronal function. We have previously shown that hypothalamic NMDA-Rs containing the NR2B subunit may be involved in the control of eating as well as in the mediation of physiological responses to osmotic stimuli. To broaden our understanding of diencephalic NMDA-R participation in other functions, we localized the NR2B subunit in the diencephalon of the adult male rat using immunoperoxidase, immunogold, and immunofluorescence techniques and an affinity-purified polyclonal antibody specific for the NR2B subunit of the NMDA-R. In addition, we used a monoclonal NR2B antibody with immunoperoxidase detection to confirm the NR2B distribution seen with the polyclonal antibody. In the hypothalamus, the highest levels of NR2B immunoreactivity (-ir) were found in the magnocellular neurosecretory system, including the paraventricular and supraoptic nuclei. A new finding was that intense NR2B-ir was present within perivascular "accessory" magnocellular groups of this system, including the nucleus circularis, anterior fornical nucleus, and scattered clusters of lateral hypothalamic cells apposed to blood vessels. Robust NR2B-ir was also present within the arcuate nucleus, the median eminence, and the tuberal nucleus, and light immunostaining was found in all other hypothalamic nuclei examined. In the thalamus, the highest NR2B-ir was observed in the medial habenula and the anterodorsal, paraventricular, rhomboid, reticular, and dorsal lateral geniculate nuclei. As in the hypothalamus, all thalamic nuclei examined displayed at least light immunostaining for this subunit. Control sections, including those incubated with the polyclonal NR2B antibody preadsorbed with its fusion protein, were virtually devoid of immunostaining. This demonstration that the NR2B subunit of the NMDA-R is widely distributed in the diencephalon, implicates it in a wide variety of functions, and provides a useful anatomical framework for establishing a comprehensive map of Glu receptor populations within this major subdivision of the brain.

Brain regions where cholecystokinin suppresses feeding in rats.

The gut-brain peptide, cholecystokinin (CCK), inhibits food intake when injected either systemically or within the brain. To determine whether CCK's effect in the brain is anatomically specific, CCK-8 (0. 8, 4, 20, 100, 500 pmol) was microinjected into one of 14 different brain sites of rats, and its impact on subsequent food intake was measured. CCK-8 at 500 pmol significantly suppressed intake during the first hour post-injection following administration into six hypothalamic sites (anterior hypothalamus, dorsomedial hypothalamus, lateral hypothalamus, paraventricular nucleus, supraoptic nucleus, ventromedial hypothalamus) and two hindbrain sites (nucleus tractus solitarius, fourth ventricle). Although lower doses were sometimes effective (anterior hypothalamus, dorsomedial hypothalamus, nucleus tractus solitarius), there appeared to be no significant difference in potency among sites. Injections into the medial amygdala, nucleus accumbens, posterior hypothalamus, dorsal raphe, and ventral tegmental area were either ineffective or produced a delayed response. The higher doses required for most sites, as well as the widespread effectiveness of CCK-8 within the hypothalamus, suggest that spread of CCK-8 to adjacent brain sites, and (or) to the periphery, may have been required for anorexia to occur. Findings reported in an accompanying paper provide strong evidence that paraventricular nucleus injection of CCK-8 (500 pmol) did not increase plasma CCK-levels sufficiently to suppress feeding by a peripheral mechanism. Together, these results suggest that CCK may be acting as a neurotransmitter or neuromodulator within two different brain regions to produce satiety - one region which includes the nucleus tractus solitarius in the hindbrain, and another more distributed region within the medial-basal hypothalamus.

Eating induced by perifornical cAMP is behaviorally selective and involves protein kinase activity.

It has previously been shown that agents that increase endogenous cAMP elicit robust eating when injected into the perifornical hypothalamus (PFH) but not when injected into surrounding brain sites, suggesting that PFH cAMP may play a role in eating control. We report here that bilateral microinjection of the adenylyl cyclase activator 7-deacetyl-7-O-(N-methylpiperazino)-gamma-butyryl-forskolin dihydrochloride (MPB forskolin; 300 nmol/0.3 microl) into the PFH is sufficient to elicit intense eating (up to 15.7 +/- 2.3 g in 2 h) in satiated rats, without concomitant effects on other behaviors, including gnawing and drinking. In contrast, the inactive analog 1, 9-dideoxyforskolin is ineffective, suggesting that the effects of MPB forskolin are behaviorally selective and pharmacologically specific. We also show that injection of the protein kinase A inhibitor H-89 (100 nmol) into the PFH reduced MPB forskolin-induced eating by up to 50%. Collectively, these results suggest that increased cAMP production in a single brain area may be sufficient to selectively generate a patterned, goal-oriented behavior by activating cAMP-dependent protein kinase.

Similar feeding patterns are induced by perifornical neuropeptide Y injection and by food deprivation.

Although hypothalamic injections of neuropeptide Y (NPY) induce robust feeding, there is little information about the patterns of feeding elicited by this peptide. To reveal these patterns, NPY (0, 8, 24, 78, 235 pmol/10 nl) was injected into the perifornical hypothalamus (PFH) of satiated adult male rats and their subsequent food intake was monitored every minute for 24 h. For comparison, feeding patterns were similarly observed following fasts of 0, 3, 6, 9, 12, and 24 h. The results demonstrated that NPY and food deprivation both produced dose- or deprivation-dependent increases in food intake that were most evident in the first 6 h. The increased intakes induced by NPY were characterized by combinations of increased meal size and frequency, with the predominant effects being increases in the size of and decreased latency to eat the first meal. Similarly, fasting progressively increased food intake by combinations of increased meal size and frequency, with the predominant effects being increases in the size of and decreased latency to eat the first meal. These similarities between NPY-induced and food deprivation-induced feeding are consistent with a stimulatory role for endogenous NPY in deprivation-induced feeding. These findings also suggest that NPY may increase eating by acting on mechanisms of both meal initiation and of meal termination.

Stimulation of eating by the second messenger cAMP in the perifornical and lateral hypothalamus.

Despite intense study of neurotransmitters mediating hypothalamic controls of food intake, little is known about which second messengers are critical for these mechanisms. To determine whether adenosine 3',5'-cyclic monophosphate (cAMP) might participate in these mechanisms, we injected the membrane-permeant cAMP analog 8-bromo-cAMP (8-BrcAMP) hypothalamically in satiated rats. Injection of 8-BrcAMP (10-100 nmol) into the perifornical (PFH) and lateral hypothalamus (LH) dose dependently stimulated food intake of up to 15.7 g in 2 h. Significantly smaller responses were obtained with thalamic injections. In contrast to the strong stimulatory effects of PFH and LH 8-BrcAMP, cAMP and 8-bromo-guanosine 3',5'-cyclic monophosphate (100 nmol) were ineffective, suggesting a chemically specific, intracellular action. Consistent with this, combined PFH injection of 7-deacetyl-7-O-(N-methylpiperazino)-tau-butyryl-forskolin dihydrochloride and 3-isobutyl-1-methylxanthine, agents that increase endogeneous cAMP, stimulated eating of up to 9.9 g in 2 h. These results demonstrate that increases in PFH/LH cAMP can elicit complex, goal-oriented behavior, suggesting an important role for cAMP in hypothalamic mechanisms stimulating food intake.

Evidence that neuropeptide Y and dopamine in the perifornical hypothalamus interact antagonistically in the control of food intake.

Mapping studies have revealed that the perifornical hypothalamus (PFH) is a primary locus for both the feeding-stimulatory effect of neuropeptide Y (NPY) and the anorectic effect of catecholamines (CAs), suggesting that NPY and CAs may interact antagonistically there. To investigate this, the CA-releasing agent amphetamine (AMPH) was injected through indwelling guide cannulas into the PFH of satiated adult male rats 5 min prior to injection of NPY (78 pmol/0.3 microliters) and food intake was measured 1, 2, and 4 h later. Amphetamine (50-200 nmol) dose-dependently reduced NPY feeding, usually eliminating it at the higher doses. The receptors mediating this effect were investigated by sequential injection of various CA antagonists, AMPH, and NPY into the PFH. Neither the alpha- nor beta-adrenergic receptor antagonists phentolamine (100 nmol) or propranolol (200 nmol) significantly affected AMPH suppression of NPY feeding. In contrast, the dopamine receptor antagonist haloperidol (5 nmol) abolished AMPH suppression of NPY feeding, suggesting that dopamine (DA) mediates the AMPH effect. To examine this, epinephrine (EPI, 50-200 nmol) and DA (25-200 nmol) were tested for suppression of NPY-induced feeding. While EPI had no significant effect, DA at the maximally effective dose (50 nmol) reduced the NPY feeding response by 36% or more. These findings provide convergent evidence for antagonistic interactions between endogenous DA and NPY in the control of eating behavior.

Feeding responses to perifornical hypothalamic injection of neuropeptide Y in relation to circadian rhythms of eating behavior.

Hypothalamic injection of neuropeptide Y (NPY) can elicit eating in satiated rats, and the perifornical hypothalamus (PFH) is the site where this effect is most pronounced (48). Additionally, there is a well-documented circadian rhythm of spontaneous eating behavior. Our objective was to determine whether there are daily rhythms of sensitivity to NPY in the PFH that might contribute to this behavioral rhythm. To accomplish this, the effectiveness in eliciting eating of PFH injection of NPY was examined at six different time points in the light-dark cycle. Neuropeptide Y (78 pmol/10 nl) or vehicle (10 nl) were injected through chronically implanted guide cannulas into the PFH of satiated adult male rats and food intake was measured 1, 2, and 4 h later. In animals on 12-12 h light-dark cycles, these injections were given 1 h before and after the onset of the light and dark phases, and in the middle of these phases. Additionally, dose-response effects of NPY were examined at two points: the first hour of both the dark and the light phases. The results show that NPY was effective at every time tested, and that the magnitude of the peptide-elicited intakes was primarily additive to the underlying patterns of spontaneous intake, with only a modest daily cycle of sensitivity to NPY. Consistent with this, NPY dose-dependently increased intake in the early light and in the early dark, and the magnitude of these effects across doses was similar at these times. This suggests that the sensitivity of the PFH system mediating NPY eating exhibits only a modest daily cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

The perifornical area: the major focus of (a) patchily distributed hypothalamic neuropeptide Y-sensitive feeding system(s).

Neuropeptide Y (NPY), a neurochemical found in high concentrations within hypothalamic neurons, is believed to participate in the control of eating behavior and body energy balance and elicits a powerful eating response when injected into the hypothalamus. To delineate precisely the locus of this effect, NPY (78 pmol) or its artificial cerebrospinal fluid vehicle was injected in the extremely small volume of 10 nl through chronic guide cannulae into an array of 47 different hypothalamic areas in satiated rats and the elicited food intake was measured. To determine the anatomical resolution of this technique, the spread and recovery of [125I]NPY injected in 10 nl was also assessed. Results indicate that as much as 95% of the injected label was recovered within the brain tissue and guide cannulae and that 100% of the tissue label was localized to within 0.8 mm of the injection site. Behavioral results show that the perifornical hypothalamus (PFH), at the level of the caudal paraventricular nucleus, is the most sensitive hypothalamic site for NPY-induced eating. NPY there elicited mean increases in food intake of 12.5 g over baseline at 1 h and 20.0 g at 4 h postinjection. Injections bracketing the PFH in all directions were substantially less effective. Additionally, significant effects were also observed in at least seven other sites that were distributed throughout the hypothalamus. These findings suggest both that the PFH may be the primary hypothalamic site containing feeding-related NPY-sensitive receptors and that other sites distributed within the hypothalamus also can mediate NPY's effects.

Evidence for neuropeptide Y mediation of eating produced by food deprivation and for a variant of the Y1 receptor mediating this peptide's effect.

Neuropeptide Y (NPY) elicits eating when injected directly into the paraventricular nucleus (PVN) or perifornical hypothalamus (PFH). To identify the essential regions of the NPY molecule and the relative contributions of Y1 and Y2 receptors, the eating stimulatory potency of NPY was compared to that of its fragments, analogues, and agonists when injected into the PVN or PFH of satiated rats. Additionally, antisera to NPY was injected into the cerebral ventricles (ICV) to determine whether passive immunization suppresses the eating produced by mild food deprivation. Tests with NPY fragments revealed that NPY(2-36) was surprisingly potent, nearly three times more so than intact NPY. In contrast, fragments with further N-terminal deletions were progressively less effective or ineffective, as was the free acid form of NPY. Collectively, this suggests that both N- and C-terminal regions of NPY participate in the stimulation of eating. Tests with agonists revealed that the putative Y1 agonist [Pro34]NPY elicited a strong dose-dependent feeding response, while the putative Y2 agonist, C2-NPY, had only a small effect at the highest doses. Although this suggests mediation by Y1 receptors, the uncharacteristically high potency of NPY(2-36) may additionally suggest that the receptor subtype underlying feeding is distinct from that mediating other responses. Additional results revealed that ICV injection of antisera to NPY, which should inactivate endogenous NPY, produced a concentration-dependent suppression of eating induced by mild food deprivation. This finding, along with published work demonstrating enhanced levels of hypothalamic NPY in food-deprived rats, suggests that endogenous NPY mediates the eating produced by deprivation.

Repeated hypothalamic stimulation with neuropeptide Y increases daily carbohydrate and fat intake and body weight gain in female rats.

Neuropeptide Y (NPY), repeatedly injected in the hypothalamic paraventricular nucleus (PVN), produces dramatic obesity and overeating in female rats maintained on a single nutritionally complete diet. In the present study, we investigated whether these effects could also be obtained in animals with a choice of three pure macronutrients: protein, carbohydrate, and fat. Female rats with indwelling PVN cannulas were injected with NPY (235 pmol) or its saline vehicle every 8 hr for 6 days. A third group was left undisturbed. Consumption of each macronutrient and body weight were measured every 24 hr for 6 days preinjection, 6 days during injections, and 21 days after the injections were terminated. Relative to vehicle or preinjection rates of body weight gain (approximately 1.5 g/day), NPY dramatically enhanced weight gain to a rate of 9.3 g/day and more than doubled total daily food intake. This augmentation was accounted for by increases in carbohydrate intake (+26.4 kcal/day) and fat intake (+48.5 kcal/day), with no significant potentiation of protein consumption. When the NPY injections were terminated, body weight and macronutrient intake returned to control levels within 1 or 2 weeks. These findings are consistent with a role for NPY in hypothalamic mechanisms of macronutrient intake and body weight regulation and suggest that disturbances in brain NPY may contribute to the development of eating and weight disorders.

Patterns of extracellular 5-hydroxyindoleacetic acid (5-HIAA) in the paraventricular hypothalamus (PVN): relation to circadian rhythm and deprivation-induced eating behavior.

Daily rhythms in extracellular levels of the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were examined in the region of the paraventricular nucleus (PVN), using intracerebral microdialysis combined with high performance liquid chromatography and electrochemical detection. Samples of PVN dialysate, from 11 rats on a 12/12 hr light/dark cycle, were collected and assayed for 5-HIAA every 2 hr for 3 days. During the first 2 days the rats were given free access to food. During the 3rd day they were deprived of food for a 24-hr period and then given food for 4 hr. The results showed that in freely-feeding rats, there was a 24-hr rhythm in the levels of 5-HIAA, with a marked transient peak just after the beginning of the dark portion of the light/dark cycle and stable levels at all other times. When the animals were food-deprived, PVN levels of this metabolite remained stable, and the early dark peak was abolished, suggesting that it might have been consequent to the eating behavior which normally occurred at this time. In the 4-hr refeeding period, there were no changes in 5-HIAA levels, despite the intense eating behavior which occurred during this time. These patterns of 5-HIAA in the PVN region, taken together with previous evidence, suggest that PVN serotonin metabolism may increase in association with feeding specifically in the early portion of the nocturnal eating period, when it may play a role in controlling food intake and macronutrient selection.

Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity.

Neuropeptide Y (NPY), a putative neurotransmitter abundant in the brain, has recently been shown to act within the hypothalamus, inducing a powerful eating response and a specific appetite for carbohydrates. In the present study, NPY (235 pmol) injected bilaterally in the paraventricular nucleus three times a day for 10 days caused approximately a two-fold increase in daily food intake, a six-fold increase in the rate of body weight gain and a three-fold increase in the body fat of female rats. Subsequently, the food intake and body weight of these subjects decreased precipitously, reaching control levels 20 days postinjection. These findings, demonstrating that exogenous NPY is capable of overriding mechanisms of satiety and body weight control, suggest that disturbances in NPY function may play a role in some disorders of eating behavior and body weight regulation.

Feeding and drinking elicited by central injection of neuropeptide Y: evidence for a hypothalamic site(s) of action.

Neuropeptide Y (NPY), which exists in very high concentrations in the brain, has been shown to elicit a powerful feeding response and a small drinking response in satiated rats. In order to delineate the brain sites sensitive to these effects, NPY was injected through chronic guide cannulas into seven different brain regions, and the food and water intake of satiated rats was measured one hr postinjection. Injection of NPY (78 pmoles) into hypothalamic areas, namely the paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), and lateral hypothalamus (LH), elicited a strong feeding response; in contrast, injections into extra-hypothalamic areas, namely the amygdala, thalamus, and periaqueductal gray, were completely ineffective. Administration of NPY into the PVN and VMH also elicited a small drinking response; however, all other areas, including the LH, were insensitive to this effect. The findings that NPY was effective in the hypothalamus, as opposed to sites anterior, posterior, lateral or dorsal to this structure, suggest a hypothalamic site(s) of action for this neuropeptide.

Disproportionate increases in locomotor activity in response to hormonal and photic stimuli following regional neurochemical depletions of serotonin.

The role of forebrain serotonin in behavior-related energy output was assessed in two locomotor activity tests conducted 3 and 6 months after bilateral, intrahypothalamic microinfusion of the serotonin neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT). The serotonin-depleted animals exhibited a long-lasting and stable increase in energy expenditure as locomotor activity. This increased activity was investigated at the behavioral level by relating the hyperactivity to estrous cycle, photoperiod and body weight. Although the serotonin depletion-induced hyperactivity occurred in all photoperiod and estrous cycle stages, its magnitude was disproportionately increased during light and estrus. This hyperactivity could not be related to decreases in body weight because the serotonin-depleted animals weighed significantly more than the control animals. These animals responded to the weight loss that normally accompanies wheel running by increasing their activity to the same proportion as the other groups. The neuroanatomical and neurochemical substrate of the increased locomotor activity was investigated with a regional neurochemical assay for serotonin, dopamine and norepinephrine. This assay revealed that the toxin had no effect on dopamine or norepinephrine in any structure analyzed; however, serotonin was depleted in the hippocampus, septum and, to a lesser degree, in the hypothalamus. Serotonin levels were negatively correlated with overall activity. The magnitude of the disproportionate increase in activity during light and estrus was negatively correlated with hippocampal serotonin level. These results indicate that forebrain depletions of serotonin differentially affect the control of activity exerted by the phases of the photoperiod and estrous cycle. However, the modulation of activity levels by decreases in body weight remains intact.

Increased food intake, body weight, and adiposity in rats after regional neurochemical depletion of serotonin.

Long-term changes in food and water intake, body weight, and skeletal growth were assessed following bilateral hypothalamic microinfusions of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). The analysis of ingestive behavior included assessment of photoperiodic effects and responsivity to alterations in dietary composition. The neuroanatomical and neurochemical selectivity of the serotonergic depletion was documented by regional spectrofluorometric analysis for serotonin, dopamine, and norepinephrine. The results indicated that norepinephrine and dopamine were not depleted in the brain areas examined. However, substantial depletion of serotonin occurred in the septum, hippocampus, and hypothalamus of the 5,7-DHT (12 microgram) group. During feeding of a high-fat diet, the serotonin-depleted group exhibited a long-lasting and stable hyperphagia which led to an increase in body weight. This hyperphagia occurred during both phases of the photoperiod but was disproportionately distributed, occurring to a greater extent in the light phase. Because body weight increased in the absence of increased skeletal growth or intestinal weight, it is concluded that the increased body weight reflected increased adiposity. The serotonin-depleted group also exhibited an essential light-phase hyperdipsia, increasing water intake above that which would be accounted for by the light-phase hyperphagia. Because the light-phase hyperdipsia was counterbalanced by a dark-phase hypodipsia, there was no change in the 24-hour water intake. The hyperphagia exhibited by the 5,7-DHT (12 microgram) group was associated with both hippocampal and hypothalamic serotonergic depletion. The results of this investigation provide the first clear evidence of an inhibitory role of serotonin in food intake and point to the importance of maximizing both neurochemical and neuroanatomical selectivity in such investigations.