Opioid-induced feeding: Localization of sensitive brain sites

These experiments were designed to identify brain sites at which opioids might act to influence ingestive behavior and to determine which opioid receptor types are involved. After food deprivation, rats were given microinjections of naloxone into several brain regions and food intake was measured. Injections into or near the paraventricular (PVN) or ventromedial (VMH) hypothalamic nuclei or the globus pallidus (GP) reduced food intake; injections into the striatum or lateral hypothalamus (LH) were ineffective. A second study examined the ingestive effects of roughly equimolar doses (1.43-1.75 nmol) of dynorphin A (DYN), beta-endorphin (beta-END), and D-Ala2,D-Leu5-enkephalin (DADLE) when injected into 4 different brain regions. Only DYN significantly increased food intake, and this effect was seen only with injections into the PVN and VMH. Beta-END stimulated water intake when injected into the PVN, VMH and GP but not the LH. Further studies indicated that with PVN injections, DYN was effective at a dose as low as 0.47 nmol, and that a higher dose of DADLE (4.39 nmol) did stimulate food intake. These studies support an important role for dynorphin and the kappa opioid receptor in the regulation of feeding and suggest that the opioid regulation of food and water intake can be differentiated both by sites of action and by effective agonists.     

Phaseolus vulgaris leuco-agglutinin tracing of intrahypothalamic connections of the lateral, ventromedial, dorsomedial and paraventricular hypothalamic nuclei in the rat

Intrahypothalamic connections of the lateral (LHA), ventromedial (VMH), dorsomedial (DMH) and paraventricular (PVN) hypothalamic nuclei were studied with anterograde transport of iontophoretically injected Phaseolus vulgaris leuco-agglutinin and the immunocytochemical detection of labeled structures. The LHA was found to give rise to a minor projection in the VMH, whereas the VMH in reverse maintains few connections with the ventromedial part of the tuberal LHA. Tracer deposits in both the LHA and VMH resulted in anterograde terminal labeling in the DMH. The DMH, in turn, donates a small number of projections to the LHA and VMH. The main projection of the DMH is aimed at the parvocellular paraventricular nucleus. Direct outflow pathways from the VMH to the PVN were not found, but lectin injections in the LHA on the other hand gave rise to terminal labeling in both the parvocellular and magnocellular divisions of the PVN. The PVN in turn was found to give only minor reciprocal projections to the LHA, DMH and VMH. These findings indicate that the main stream of connections in the hypothalamus runs from the LHA and VMH to the DMH, and from the DMH to the PVN. The identified circuitry patterns were discussed with respect to the role of the hypothalamus in the control of homeostasis and metabolic regulation, and more specifically in relation to the modulation of the hormone release from the pancreas and adrenal glands.     

Hypothalamic sites of neuronal histamine action on food intake by rats

To identify sites of histaminergic modulation of food intake, histamine H₁-receptor antagonist was microinfused into the rat hypothalamus, the ventromedial hypothalamus (VMH), the lateral hypothalamus (LHA), the paraventricular nucleus (PVN), the dorsomedial hypothalamus (DMH), or the preoptic anterior hypothalamus (POAH), during the early light period. Feeding, but not drinking, was elicited in 100% of the rats (P<0.01) that were bilaterally microinfused with 26 nmol chlorpheniramine into the VMH. Unilateral infusion into the VMH did not affect food intake at doses of 26 or 52 nmol. Feeding was also induced by bilateral microinfusion into the PVN, but only the 52 nmol dose was effective. Bilateral infusions into the LHA, the DMH or the POAH did not affect ingestive behavior. Feeding induced by an H₁-antagonist was completely abolished in all 7 rats tested when endogenous neuronal histamine was decreased by pretreatment with α-fluoromethylhistidine (100 mg/kg). The findings suggest that H₁-receptors in the VMH and the PVN, but not in the LHA, the DMH or the POAH, may be involved in histaminergic suppression of foof intake.     

GABA-related feeding control in genetically obese rats

Feeding in response to glucoprivation induced by 2-deoxy-D-glucose (2-DG) is impaired in genetically obese (Zucker) rats. Muscimol, a GABAA-agonist (0.5 nmol/0.5 microliter in each area) increased food intake in lean rats over 3 h but in fatty rats only at 30 min after infusion into the VMH. Injection of muscimol into the DMH and PVN increased feeding of both phenotypes. Picrotoxin, a non-competitive GABAA-antagonist (0.1 nmol/0.5 microliter) increased food intake after infusion into the LH of both phenotypes and decreased food intake over a 3 h period when infused into the VMH. DMH and PVN of fatty rats. In the lean littermates, picrotoxin was only effective in reducing food intake at 30 min after infusion into the VMH and PVN but not the DMH. The present results suggest that the fatty Zucker rat has a disturbance in the GABA-related regulatory mechanism of feeding behavior in the ventromedial hypothalamus, which may be responsible for the impaired response to glucoprivation found in these rats.     

Effects of Cholecystokinin in the Supraoptic Nucleus and Paraventricular Nucleus are Negatively Modulated by Leptin in 24‐h Fasted Lean Male Rats

Cholecystokinin (CCK) and leptin are two important satiety factors that are considered to act in synergy to reduce meal size. Peripheral injection of CCK activates neurones in several hypothalamic nuclei, including the supraoptic (SON) and paraventricular (PVN) nuclei and neurones in the brainstem of fed rats. We investigated whether peripheral leptin would modulate the effects of CCK on neuronal activity in the hypothalamus and brainstem of fasted rats by investigating Fos expression in the PVN, SON, arcuate nucleus, ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), area postrema (AP) and the nucleus tractus solitarii (NTS). Male rats, fasted for 24 h, received either one i.p. injection of vehicle, leptin or CCK‐8 alone, or received one injection of vehicle or leptin before an i.p. injection of CCK‐8. We found that CCK increased Fos expression in the PVN and SON as well as in the NTS and AP, but had no effect on Fos expression in the arcuate nucleus, VMH or DMH compared to vehicle. Leptin injected alone significantly increased Fos expression in the arcuate nucleus but had no effect on Fos expression in the VMH, DMH, SON, PVN, AP or NTS compared to vehicle. Fos expression was significantly increased in the AP in rats injected with both leptin and CCK compared to rats injected with vehicle and CCK. Unexpectedly, there was significantly less Fos expression in the PVN and SON of fasted rats injected with leptin and CCK than in rats injected with vehicle and CCK, suggesting that leptin attenuated CCK‐induced Fos expression in the SON and PVN. However, Fos expression in the NTS was similar in fasted rats injected with vehicle and CCK or with leptin and CCK. Taken together, these results suggest that leptin dampens the effects of CCK on Fos expression in the SON and PVN, independently from NTS pathways, and this may reflect a direct action on magnocellular neurones.     

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.     

Effects of hypothalamic lesion on pancreatic autonomic nerve activity in the rat

The effects of hypothalamic lesions and intravenous glucose infusion on the efferent activity of vagal and splanchnic nerves to the pancreas were studied in anesthetized rats. Lesions of the ventromedial hypothalamic (VMH), the dorsomedial hypothalamic (DMH) and the paraventricular (PVN) nuclei increased vagal and reduced splanchnic nerve activity. Lesion of the lateral hypothalamic area (LHA) decreased pancreatic vagal nerve activity, and produced either increased or decreased activity of pancreatic splanchnic nerve. Intravenous glucose infusion increased activity of the vagal nerve and reduced that of the splanchnic nerve. These glucose responses were influenced by hypothalamic lesions only slightly or not at all. The findings suggest that hypothalamic modulation of pancreatic hormone secretion involves both the parasympathetic and sympathetic nervous systems, and provide evidence that not only the VMH and the LHA but also the DMH and the PVN are involved in this mechanism.     

Interrelations between lateral, dorsomedial and ventromedial hypothalamic nuclei in the rat. An HRP study

Afferents of the lateral (LH) and ventromedial (VMH) hypothalamic nuclei were studied with the horseradish peroxidase method. The aim was to investigate relations between these two centers presumed to be involved in the regulation of food intake. Special attempts were made to produce HRP injections limited to intranuclear dimensions, which was achieved by iontophoretic delivery of the tracer. The results indicate that LH and VMH do not maintain direct interconnections. Both nuclei, however, appear to have numerous afferents from the dorsomedial hypothalamic nucleus (DMH) in common, which led us to extend our analysis to the DMH. DMH injections of HRP resulted in retrograde labeling of somata in both LH and VMH, suggesting a reciprocal relationship of DMH with these latter nuclei. The possible significance of such a LH-DMH-VMH relationship in the food intake control circuitry is discussed. The other labeling of afferents resulting from HRP injections localized to LH, DMH and VMH is described and discussed as regards their morphological significance. A number of these connections confirm studies using anterograde transport techniques, but others have not been described before, including an extensive projection to the VMH from the mesencephalic perpendicular nucleus.     

Involvement of hypothalamic neuropeptide Y in pentazocine induced suppression of food intake in rats

The potent orexigenic peptide neuropeptide Y (NPY) has been considered as a possible endogenous ligand for a subpopulation of sigma receptors (SigR). However, their mutual interaction with reference to feeding behavior remains poorly understood. In the present study, we explored the possible interaction between sigma1 receptors (Sig1R) agonist, pentazocine, and NPY on food intake in satiated rats. While pentazocine dose-dependently reduced the food intake, NPY significantly increased it at 2, 4 and 6 h post injection time points. In combination studies, pretreatment with NPY (0.1 nmol/rat, intra-PVN) normalized the inhibitory effect of pentazocine (60 μg/rat, intra-PVN) on food intake. Similarly, pre-treatment with pentazocine (30 μg/rat, intra-PVN) significantly antagonized the orexigenic effect of NPY (0.5 and 1.0 nmol/rat, intra-PVN). Moreover, pentazocine treatment decreased NPY immunoreactivity in arcuate (ARC), paraventricular (PVN), dorsomedial (DMH) and ventromedial (VMH) nuclei of hypothalamus. However, no change was observed in lateral hypothalamus (LH). Study implicates the reduced NPY immunoreactivity for the anorectic effect observed following pentazocine injections. Therefore, the concomitant activation of the NPYergic system along with the Sig1R agonist treatment may serve a useful purpose in the management of the unwanted side effects related to energy homeostasis.     

Amygdala enterostatin induces c-Fos expression in regions of hypothalamus that innervate the PVN

Enterostatin selectively inhibits the intake of the dietary fat after both central and peripheral administration. Our previous studies have shown that a central site of action is the central nucleus of amygdala. Serotonergic agonists administered into the paraventricular nucleus (PVN) inhibit fat intake and serotonergic antagonists block the feeding suppression induced by amygdala enterostatin, suggesting that there are functional connections between the PVN and amygdala that affect the feeding response to enterostatin. Our purpose was to identify the anatomic and functional projections from the amygdala to the PVN and hypothalamic area that are responsive to enterostatin, by using a retrograde tracer fluorogold (FG) and c-Fos expression. Rats were injected with fluorogold unilaterally into the PVN and a chronic amygdala cannula was implanted ipsilaterally. After 10 days recovery, rats were injected with either enterostatin (0.1 nmol) or saline vehicle (0.1 microl) into the amygdala and sacrificed 2 h later by cardiac perfusion under anesthesia. The brains were subjected to dual immunohistochemistry to visualize both FG and c-Fos-positive cells. FG/c-Fos double-labeled cells were found in forebrain regions including the PVN, amygdala, lateral hypothalamus (LH), ventral medial hypothalamus (VMH) and arcuate nucleus (ARC). The data provides the first anatomical evidence that enterostatin activates amygdala neurons that have functional and anatomic projections directly to the PVN and also activates neurons in the arcuate, LH and VMH, which innervate the PVN.     

Neuronal histamine in the hypothalamus suppresses food intake in rats

Using probes to manipulate hypothalamic neuronal histamine, we report here that changes in neuronal histamine modulate physiological feeding behavior in rats. Infusion of α-fluoromethylhistidine (FMH), a “suicide” inhibitor of histidine decar☐ylase (HDC), into the third cerebroventricular induced feeding in the early light phase when the histamine synthesis was most accelerated. FMH at an optimum 2.24 μmol dose elicited feeding in 100% of rats. Treatment of FMH specifically and selectively decreased concentration of histamine without affecting concentrations of catecholamines in the hypothalamus. Immediately before the dark phase, when the histamine synthesis was normally lower, FMH infusion did not affect feeding-related parameters such as meal size, meal duration or latency to eat. Conversely, thioperamide, which facilitates both synthesis and release of neuronal histamine by blocking presynaptic autoinhibitory H₃ receptors, significantly decreased food intake after infusion of a 100-nmol dose into the third cerebroventricle. The effect of thioperamide was abolished with i.p. injection of 26 μmol/kg chlorpheniramine, an H₁ antagonist. FMH at 224 nmol was microinfused bilaterally into the feeding-related nuclei in the hypothalamus. The ventromedial nucleus (VMH) and the paraventricular nucleus (PVN), but not the lateral hypothalamus, the dorsomedial hypothalamus or the preoptic anterior hypothalamus were identified as the active sites for the modulation. Neuronal histamine may convey suppressive signals of food intake through H₁ receptors in the VMH and the PVN with diurnal fluctuation.     

Monoaminergic activity at the level of the hypothalamus and striatum: relationship to anticipated feeding and pancreatic insulin responses

The present study examined monoaminergic activity at the level of the lateral hypothalamus (LH), ventromedial hypothalamus (VMH) and ventral striatum associated with the conditioned cephalic phase insulin release. Partially food-deprived male Wistar rats were divided into 3 groups. Two of the groups were conditioned to drink a 50% glucose solution each morning for 3 weeks; the control group received an amount of glucose equal to the amount drunk by the experimental animals with their afternoon meal. On test day, conditioned animals were sacrificed either just prior to glucose presentation or 2 min following consumption of the solution; control animals were sacrificed at the same time. Gas chromatography/mass spectrometry was used to determine the levels of the monoamines noradrenaline (NA), dopamine (DA) and serotonin (5-HT), as well as their principal metabolites dihydroxyphenylethyleneglycol, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in the LH and VMH. DA and DOPAC levels were assayed in the striatum. Although serum glucose levels were unchanged, animals conditioned to drink glucose had significantly higher serum insulin levels. This increased insulin was associated with increased content of 5-HT and 5-HIAA at the level of the LH and VMH, increased NA content in the LH, increased DOPAC levels in the VMH as well as increases in the ratio of DOPAC to DA in the striatum. A regression analysis showed that 5-HIAA at the level of the LH related closely to serum insulin levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neural connectivity between the hypothalamic supramammillary nucleus and appetite‐ and motivation‐related regions of the rat brain

The supramammillary nucleus (SuM) has an emerging role in appetite control. We have shown that the rat SuM is activated during hunger or food anticipation, or by ghrelin administration. In the present study, we characterised the connectivity between the SuM and key appetite‐ and motivation‐related nuclei in the rat. In adult wild‐type rats, or rats expressing Cre recombinase under the control of the tyrosine hydroxylase (TH) promoter (TH‐Cre rats), we used c‐Fos immunohistochemistry to visualise and correlate the activation of medial SuM (SuMM) with activation in the lateral hypothalamic area (LH), the dorsomedial hypothalamus (DMH) or the ventral tegmental area (VTA) after voluntary consumption of a high‐sugar, high‐fat food. To determine neuroanatomical connectivity, we used retrograde and anterograde tracing methods to specifically investigate the neuronal inputs and outputs of the SuMM. After consumption of the food there were positive correlations between c‐Fos expression in the SuMM and the LH, DMH and VTA (P = 0.0001, 0.01 and 0.004). Using Fluoro‐Ruby as a retrograde tracer, we demonstrate the existence of inputs from the LH, DMH, VTA and ventromedial hypothalamus (VMH) to the SuMM. The SuMM showed reciprocal inputs to the LH and DMH, and we identified a TH‐positive output from SuMM to DMH. We co‐labelled retrogradely‐labelled sections for TH in the VMH, or for TH, orexin and melanin‐concentrating hormone in the LH and DMH. However, we did not observe any colocalisation of immunoreactivity with any retrogradely‐labelled cells. Viral mapping in TH‐Cre rats confirms the existence of a reciprocal SuMM‐DMH connection and shows that TH‐positive cells project from the SuMM and VTA to the lateral septal area and cingulate cortex, respectively. These data provide evidence for the connectivity of the SuMM to brain regions involved in appetite control, and form the foundation for functional and behavioural studies aiming to further characterise the brain circuitry controlling eating behaviours.     

Role of the ventromedial hypothalamus in the regulation of adenohypophyseal immunoreactive dynorphin in the rat

In this study, we have examined the role of the dorsomedial (DMH), ventromedial (VMH) and arcuate (ARH) nuclei of the hypothalamus in the control of hypothalamic and pituitary immunoreactive (ir) dynorphin (Dyn) A and ir-Dyn B in the rat, by evaluating the effect of discrete, bilateral radiofrequency lesions in these structures. Lesions limited to the VMH reduced the content of ir-Dyn in the anterior pituitary but not in the neurointermediate lobe or in the hypothalamus. Gel chromatographic analysis of anterior pituitary extracts confirmed that ir-Dyn is mainly associated with high molecular weight forms containing Dyn A and Dyn B in their sequence. Anterior pituitary extracts of VMH-lesioned rats displayed a clearly lower proportion of these forms. Destruction of the DMH affected only the hypothalamic content of ir-Dyn; ablation of the ARH did not cause any significant change. Our results suggest that ablation of the VMH may disrupt critical neuronal connections to the median eminence originating in this nucleus or crossing it and participating in control of the adenohypophyseal pool of ir-Dyn.     

Feeding response to central orexins

Orexin A and orexin B were microinjected into the perifornical hypothalamus (PFH), lateral hypothalamus (LH), hypothalamic paraventricular nucleus (PVN), and ventral tegmental area (VTA) of male Sprague-Dawley rats. Orexin B (15 nmol) was also injected into the lateral cerebral ventricle (i.c.v.). Orexin A (>/=500 pmol) stimulated feeding in the PFH and LH, but not in the VTA or PVN. Orexin B stimulated feeding only when injected i.c.v.     

Hypothalamic neuronal histamine modulates ad libitum feeding by rats

Manipulating histamine endogenously, its effects on brain functions were assessed in rats. alpha-Fluoromethylhistidine (FMH), an inhibitor of histamine synthesis, elicited feeding (P less than 0.01) after intra-third cerebroventricular infusion at the early light phase when hypothalamic histamine was normally highest. No periprandial drinking was observed. The effect of FMH was attenuated, and thioperamide, an antagonist of auto-inhibitory effects on both histamine synthesis and release at presynaptic H3-receptor, conversely suppressed food intake (P less than 0.05), when these probes were carried out during the minimum histamine level early in the dark period. Bilateral microinfusion of FMH into the ventromedial hypothalamus (VMH) and the paraventricular nucleus (PVN) selectively induced feeding, but the infusion into the remaining sites of the hypothalamus had no effect. These data show that neuronal histamine plays a physiological role in feeding suppression through the VMH and the PVN in the rat.     

Mapping of hypothalamic sites involved in the effects of NPY on body temperature and food intake

The objective of the present study was to identify hypothalamic sites that might be implicated in the effects of neuropeptide Y (NPY) on both body temperature and food intake. For this purpose, the effects of direct microinjections of NPY in several doses (0.156–20 μg) into discrete hypothalamic nuclei on body temperature were examined in rats. To examine specificity of effects, food consumption of animals following injections was also measured. Results indicate that the influence of NPY on body temperature varies with the hypothalamic region where the peptide is administered. NPY had no effect on temperature after administration into the ventromedial (VMH) and the perifornical hypothalamus (PeF). However, a significant hypothermia was seen following administration into the preoptic (POA) and arcuate nucleus (Arc), and hyperthermia was seen after injection into the paraventricular nucleus (PVN). Finally, a biphasic effect was observed after injection into the lateral hypothalamus (LH): hyperthermia with relatively small doses and hypothermia with higher doses. Similar effects were obtained when administred into the third ventricle (3V) but in an inverted dose-related fashion: hypothermia at low and hyperthermia at higher doses. For feeding, NPY consistently increased food intake in all regions examined, with the strongest effect obtained after administration into the PeF. The present results clearly dissociate the effects of NPY on food intake and body temperature, and demonstrate that these effects are related to specific hypothalamic nuclei.     

l-DOPA produces regional changes in glucose utilization which form discrete anatomic patterns in motor nuclei and the hypothalamus of rats

Systemic injections of l-DOPA (200 mg/kg) combined with a peripheral decarboxylase inhibitor (Carbidopa) produced increased motor activity and changes in regional cerebral glucose utilization similar to and different from changes seen with other dopaminergic drugs in previous studies. Similarities were an increased glucose utilization (relative optical density measurement) in the subthalamic and entopeduncular nuclei and the substantia nigra. A major difference was a suggested decreased glucose utilization in the striatum with l-DOPA treatment rather than the increases observed with apomorphine in other studies. Also, glucose utilization in the paraventricular nucleus of the hypothalamus was decreased. Finally, specific, discrete patterns were visible in motor nuclei of the l-DOPA-treated rats which were not visible with other dopaminergic drugs: the ventral posterior striatum was increased compared with the dorsal striatum and the compacta region of the substantia nigra contained unusual round spots and patterns of high-normal density and low-normal density, especially in the lateral compacta region. In one case, the density pattern in the substantia nigra was similar to the field of dopamine cell bodies and their dendrites which extend into the reticulata region. The results confirm the importance of activity in the subthalamic nucleus, globus pallidus, and entopeduncular nucleus, as well as the substantia nigra for dopaminergic drug effects either through striatal dopamine receptors or through dopamine receptors in each of these nuclei. In addition, new “functional units” in the substantia nigra were revealed.