The effect of age on prepro-orexin gene expression and contents of orexin A and B in the rat brain

Orexin A and B (hypocretin 1 and 2) are hypothalamic peptides, which are synthesized in the lateral hypothalamus. Orexins participate in the regulation energy balance, food intake, vigilance and several endocrine and autonomic functions. The widespread projections of the orexin neurons suggest that they may have a role in coordination of different brain activities. The effects of ageing on the orexin system have not been studied previously. Prepro-orexin gene expression in the lateral hypothalamus, and the contents of orexin A and B peptides in the lateral hypothalamus and hypothalamus were measured in young, middle-aged and old (3, 12 and 24 months) rats. In the course of ageing, the expression of the prepro-orexin gene and the levels of orexin A and B decreased; the main decrease occurred by 12 months. Sleep deprivation for 6h increased slightly the expression of prepro-orexin gene in young rats. Deterioration of the orexin system may play a role in the phenomenon associated with aging, e.g. decreased consolidation of vigilance states, endocrine changes and dysfunctions of autonomic nervous system.     

Onset of feeding at birth—Perinatal development of the hypothalamic mechanisms that induce appetite and feeding in the newborn

Orexin-A and -B are hypothalamic peptides which, in the adult brain, are associated with arousal, increased vigilance, and the seeking and ingestion of food. Because the fetus is mostly asleep, and hunger is a physiological state unlikely to arise until birth, we hypothesized that orexigenic neurons in the lateral and dorso-medial hypothalamic areas (LHA, DMH) and their projections to the locus coeruleus (LC) would develop only near the time of birth. We therefore determined orexin expression in fetal sheep, where birth occurs over a tightly regulated interval of 146–148 days gestation. Immunohistochemistry was used to determine the presence and distribution of orexin-A positive fibres and cells at the level of the hypothalamus and LC in fetal (125–137 and 145+ days gestation age) and newborn sheep brains. Orexin was measured by radioimmunoassay in plasma samples taken from chronically catheterised fetal and newborn sheep, and in CSF taken from fetuses and lambs at postmortem. Orexin-A positive cells bodies were observed in the hypothalamus, and orexin-A fibres were found throughout all hypothalamic, thalamic, and brain stem regions of all the fetal and newborn brains examined. Orexin-A was present in plasma and CSF at similar concentrations in fetal and newborn sheep. The presence of orexin in hypothalamic neurons and CSF throughout late gestation suggests that orexinergic regulation of hunger, appetite and the sleep/wake cycle is inhibited, by mechanisms yet to be identified, until the time of parturition.     

Morphological study of orexin neurons in the hypothalamus of the Long-Evans rat,with special reference to co-expression of orexin and NADPH-diaphorase or nitric oxide synthase activities

Orexins, novel neuropeptides, are exclusively localized in the hypothalamus and implicated in the regulation of a variety of activities, including food intake and energy balance. Nitric oxide (NO), an unconventional neurotransmitter, is widely present in numerous brain regions including the hypothalamus, and has similar physiological roles to those of the orexins. The present study was undertaken to examine the distribution of orexin neurons and the presence of neuronal nitric oxide synthase (nNOS) in the orexin neurons to clarify whether NO interacts with the orexins in the neuronal regulation activities in the Long-Evans rat. We used two double-labeling methods: nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry in combination with orexin immunohistochemistry, and double-labeling fluorescent immunohistochemistry for orexin and nNOS. The majority of the orexin immunoreactive neurons were localized mainly in the areas of the dorsomedial hypothalamic nucleus (DMN), the dorsal part of the perifornical nucleus (PEF) and lateral hypothalamic area. The orexin immunoreactive cell bodies were medium in size, and triangular, round, elliptic, and fusiform in shape. The sizes and shapes of orexin neurons in the different parts were similar. Cell bodies coexpressing the orexin and nNOS or NADPH-d were present in the areas of the DMN and the PEF, and the nerve fibers containing orexin and nNOS were distributed in the DMN and PEF, arcuate nucleus (ARN) and ventromedial hypothalamic nucleus (VMH). These results provide morphological evidence that there exists a population of nNOS- or NADPH-d-/orexin-coexpressing neurons in the orexinergic cell group in the hypothalamus, and taken together with previous findings, suggest that NO may play a role in the mechanisms by which orexin neurons regulate food intake and energy balance.     

Long-term alterations in body weight do not affect the expression of melanocortin receptor-3 and -4 mRNA in the ovine hypothalamus

The pro-opiomelanocortin-derived peptides and the melanocortin receptors are implicated in various functions within the CNS including the regulation of food intake. In the present study, we used in situ hybridization, with specific 35S-labelled ovine riboprobes to map the expression of melanocortin receptor-3 (MC3-R) and -4 (MC4-R) mRNA in the diencephalon and brainstem of normal female sheep. Furthermore, we examined the effect of long-term alterations in energy balance on the distribution and expression of MC3-R and MC4-R mRNA in food-restricted and ad libitum-fed ovariectomized female sheep. The distribution of melanocortin receptors generally resembled that of the rat. A high number of MC3-R-labelled cells were seen in the ventral division of the lateral septum and the medial preoptic area. In the hypothalamus, a moderate number of MC3-R-labelled cells was observed in the lateral hypothalamic area while other nuclear groups had low to intermediate numbers of MC3-R-labelled cells. The distribution of MC4-R mRNA was generally similar to that of MC3-R mRNA in the septal/preoptic and hypothalamic regions, with a high number of labelled cells present in the intermediate division of the lateral septum. Within the hypothalamus, no MC4-R mRNA expression was observed in the arcuate nucleus. There was more widespread distribution of moderate to low numbers of MC4-R mRNA-expressing cells in the brainstem compared to that of MC3-R mRNA. Unlike findings in the rat, only a low number of cells expressed melanocortin receptor mRNA in the ovine hypothalamic nuclei associated with feeding behavior. The number of melanocortin receptor-labelled cells and the level of expression (silver grains/cell) in the hypothalamic feeding centers was similar in food-restricted and ad libitum-fed animals.These findings suggest that long-term alterations in metabolic status do not change the melanocortin receptor mRNA distribution and/or expression in the sheep hypothalamus.     

The role of the endogenous opiates in zinc deficiency anorexia

Anorexia is a major symptom of zinc deficiency, but the mechanism(s) for this anorexia are poorly defined. Recent studies have suggested an integral role for endogenous opiate peptides in appetite regulation. Dynorphin, a leucine-enkephalin containing opiate peptide, is a potent inducer of spontaneous feeding. In this study we showed that zinc deficient animals were relatively resistant to dynorphin-induced feeding. Measurement of dynorphin levels using a highly sensitive radioimmunoassay showed that zinc deficient animals had lower levels of dynorphin in the hypothalamus than did ad lib fed animals, with weight restricted animals having intermediate values. [3H]-naloxone binding was significantly increased to isolated brain membranes from zinc deficient animals using 1 nM unlabeled naloxone when compared to ad lib fed controls with the weight restricted animals again having intermediate values. These data suggest that abnormalities in endogenous opiate regulation of appetite may well play a role in the anorexia of zinc deficiency. The effects of zinc deficiency on endogenous opiate action appear to include alterations in receptor affinity, a post-receptor defect and alterations in the synthesis and/or release of dynorphin.     

Dysregulation of dynorphins in Alzheimer disease

The opioid peptides dynorphins may be involved in pathogenesis of Alzheimer disease (AD) by inducing neurodegeneration or cognitive impairment. To test this hypothesis, the dynorphin system was analyzed in postmortem samples from AD and control subjects, and subjects with Parkinson or cerebro-vascular diseases for comparison. Dynorphin A, dynorphin B and related neuropeptide nociceptin were determined in the Brodmann area 7 by radioimmunoassay. The precursor protein prodynorphin, processing convertase PC2 and the neuroendocrine pro7B2 and 7B2 proteins required for PC2 maturation were analyzed by Western blot. AD subjects displayed robustly elevated levels of dynorphin A and no differences in dynorphin B and nociceptin compared to controls. Subjects with Parkinson or cerebro-vascular diseases did not differ from controls with respect to any of the three peptides. PC2 levels were also increased, whereas, those of prodynorphin and pro7B2/7B2 were not changed in AD. Dynorphin A levels correlated with the neuritic plaque density. These results along with the known non-opioid ability of dynorphin A to induce neurodegeneration suggest a role for this neuropeptide in AD neuropathology.     

Orexin neuronal changes in the locus coeruleus of the aging rhesus macaque

Orexin neuropeptides regulate arousal state and excite the noradrenergic locus coeruleus (LC), so it is plausible that an age-related loss of orexin neurons and projections to the LC contributes to poor sleep quality in elderly humans and nonhuman primates. To test this hypothesis we examined orexin B-immunoreactivity in the lateral hypothalamic area (LHA) and the LC of male rhesus macaques (Macaca mulatta) throughout the life span. Orexin perikarya, localized predominantly in the LHA, showed identical distribution patterns irrespective of age. Similarly, orexin neuron number and serum orexin B concentrations did not differ with age. In contrast, orexin B-immunoreactive axon density in the LC of old animals was significantly lower than that observed in the young or adult animals. Furthermore, the age-related decline was associated with a significant decrease in tyrosine hydroxylase (TH) mRNA in the LC, despite no change in TH-immunoreactive neuron number. Taken together, these data suggest that age-related decreases in excitatory orexin innervation to the noradrenergic LC may contribute to the etiology of poor sleep quality in the elderly.     

Caspase-3 activity is reduced after spinal cord injury in mice lacking dynorphin: differential effects on glia and neurons

Dynorphins are endogenous opioid peptide products of the prodynorphin gene. An extensive literature suggests that dynorphins have deleterious effects on CNS injury outcome. We thus examined whether a deficiency of dynorphin would protect against tissue damage after spinal cord injury (SCI), and if individual cell types would be specifically affected. Wild-type and prodynorphin(-/-) mice received a moderate contusion injury at 10th thoracic vertebrae (T10). Caspase-3 activity at the injury site was significantly decreased in tissue homogenates from prodynorphin(-/-) mice after 4 h. We examined frozen sections at 4 h post-injury by immunostaining for active caspase-3. At 3-4 mm rostral or caudal to the injury, >90% of all neurons, astrocytes and oligodendrocytes expressed active caspase-3 in both wild-type and knockout mice. At 6-7 mm, there were fewer caspase-3(+) oligodendrocytes and astrocytes than at 3-4 mm. Importantly, caspase-3 activation was significantly lower in prodynorphin(-/-) oligodendrocytes and astrocytes, as compared with wild-type mice. In contrast, while caspase-3 expression in neurons also declined with further distance from the injury, there was no effect of genotype. Radioimmunoassay showed that dynorphin A(1-17) was regionally increased in wild-type injured versus sham-injured tissues, although levels of the prodynorphin processing product Arg(6)-Leu-enkephalin were unchanged. Our results indicate that dynorphin peptides affect the extent of post-injury caspase-3 activation, and that glia are especially sensitive to these effects. By promoting caspase-3 activation, dynorphin peptides likely increase the probability of glial apoptosis after SCI. While normally beneficial, our findings suggest that prodynorphin or its peptide products become maladaptive following SCI and contribute to secondary injury.     

Responses of hypothalamic orexin-containing neurons to cyclophosphamide, EHF-irradiation of the skin, and their combination in rats.

Orexins are neuromediators that participate in the regulation of feeding behavior, energy metabolism, circadian rhythms and perception of pain. The aim of the present study was to clarify the responses of the hypothalamic orexin-containing neurons to an intraperitoneal injection of cyclophosphamide (CPA), extremely high frequency (EHF)-electromagnetic stimulation of skin, which is used to modulate side effects of cytostatics and their combination. The activation of orexin-containing neurons was determined by recording of the intensity of c-Fos protein expression. Injection of cyclophosphamide (40mg/kg) or EHF-irradiation of the skin decreased the staining of orexin-containing neurons, which was most pronounced in the subfornical region of the lateral hypothalamic area (LHAs). A redistribution of orexin from the perinuclear space to the processes of these cells took place, which occurs after the activation and the expression of the c-fos-gene. c-Fos protein was expressed in most neurons with minimum content of orexin, i.e. activation of these neurons correlated with the redistribution of orexins caused by skin EHF-irradiation and injection of cyclophosphamide (CPA). EHF-irradiation of the skin before and after injection of CPA increased the staining of orexin-containing neurons, i.e. it prevented the redistribution of orexin.     

Role of serotonin in the regulation of the dynorphinergic system by a kappa-opioid agonist and cocaine treatment in rat CNS

It has been shown that chronic cocaine increases prodynorphin mRNA in the caudate putamen and decreases it in the hypothalamus. In addition, treatment with a kappa-opioid receptor agonist produced the opposite effect on prodynorphin gene expression in these brain regions and also evoked a decrease in the hippocampus. It is already known that kappa-opioid receptor agonists decrease the development of sensitization to some of the behavioral effects of cocaine. The serotonin system has also been shown to regulate dynorphin gene expression and a continuous infusion of fluoxetine induced prodynorphin gene expression in the same pattern as the kappa-opioid agonist (+)(5a,7a,8b)-N-methyl-N-[7-(1-pyrrolidinyl)-1 oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U-69593) in the brain regions investigated. It is interesting to note that treatment with a continuous infusion of cocaine produced different effects on this parameter. To determine whether serotonin plays a role in the regulation of prodynorphin mRNA by kappa-opioid agonists or cocaine, rats were treated with the serotonin depleter parachloroamphetamine (PCA). Beginning 24 h later, rats were treated with the selective kappa-opioid agonist U-69593 for 5 days or continuously with cocaine for 7 days and prodynorphin mRNA was measured. Prodynorphin mRNA was decreased significantly in the hypothalamus, caudate putamen, and hippocampus of rats treated with a single injection of PCA. Subsequent to PCA administration the effects of U-69593 or cocaine on prodynorphin mRNA were differentially affected across brain regions. Prodynorphin gene expression was still increased by U-69593 treatment in the hypothalamus and decreased in the caudate putamen. Cocaine treatment still produced a decrease in this parameter in the hypothalamus and an increase in the caudate putamen. In contrast, in the hippocampus, the decrease in prodynorphin mRNA produced by U-69593 was no longer evident after PCA and cocaine, which previously had no effect, now increased it in the serotonin-depleted group. These findings suggest that serotonin is necessary to maintain normal levels of dynorphin mRNA in all of the investigated brain areas and that the regulation of prodynorphin mRNA expression by chronic treatment with a kappa-opioid receptor agonist or cocaine requires serotonin in the hippocampus, but not in the hypothalamus or caudate putamen.     

Hypothalamic neuronal networks and feeding-related peptides involved in the regulation of feeding

The hypothalamus is a region of the brain that plays a critical role in feeding regulation. It has been revealed by various physiological experiments that the feeding-regulating center is confined to the ventromedial hypothalamus, lateral hypothalamus (LH) and arcuate nucleus (ARC). Many kinds of neurons in these areas of the hypothalamus express factors such as melanin-concentrating hormone (MCH), neuropeptide Y (NPY), proopiomelanocortin (POMC), orexin (OX) and ghrelin, which have been implicated in feeding regulation. In tissues of the periphery, two critical factors involved in feeding regulation, leptin and ghrelin, have been identified. Both hormone peptides are secreted mainly from adipose and stomach tissue, respectively, and are considered to function via their receptors mainly through several hypothalamic nuclei that play important roles in the regulation of appetite. The present review looks mainly at the functional significance of feeding-regulation factors, such as those described above, and the humoral and neuronal interactions among these compounds in the hypothalamus by drawing on published reports of morphological and physiological analyses. Immunohistochemical and in situ hybridization experiments indicate that both leptin and ghrelin receptors are distributed in the hypothalamus and that there are reciprocal interactions between MCH and OX neurons in the LH. Morphological and physiological studies on single living cells isolated from fresh rat hypothalamus or with receptor agonist and antagonist combined with immunohistochemisry clearly demonstrate that both leptin and OX reciprocally regulate NPY- and POMC-containing neurons in the ARC and that ghrelin may regulate feeding status independently through direct OX and NPY pathways. In this way, cross-talking systems in the hypothalamus play a role in determining feeding states.     

Expression of the M3 Muscarinic Receptor on Orexin Neurons that Project to the Rostral Ventrolateral Medulla

Activation of central cholinergic receptors causes a pressor response in rats, and the hypothalamus is important for this response. Projections from hypothalamic orexin neurons to the rostral ventrolateral medulla (RVLM) are involved in sympatho‐excitation of the cardiovascular system. A small population of orexin neurons is regulated by cholinergic inputs through M₃ muscarinic acetylcholine receptor (M₃R). To elucidate whether the M₃R on orexin neurons is involved in cardiosympathetic regulation through the RVLM, we examined the presence of the M₃R on retrograde‐labeled RVLM‐projecting orexin neurons. The retrograde tracer was unilaterally injected into the RVLM. Within the hypothalamus, retrograde‐labeled neurons were located predominantly ipsilateral to the injection side. In the anterior hypothalamus (?1.5 to ?2.3 mm to the bregma), retrograde‐labeled neurons were densely distributed in the paraventricular nuclei and scattered in the retrochiasmatic area. At ?2.3 to ?3.5 mm from the bregma, labeled neurons were located in the regions where orexin neurons were situated, that is, the tuberal lateral hypothalamic area, perifornical area, and dorsomedial nuclei. Very few retrograde‐labeled neurons were observed in the hypothalamus at ?3.5 to ?4.5 mm from the bregma. About 19.5% ± 1.6% of RVLM‐projecting neurons in the tuberal hypothalamus were orexinergic. The M₃R was present on 18.7% ± 3.0% of RVLM‐projecting orexin neurons. Injection of a muscarinic agonist, oxotremorine, in the perifornical area resulted in a pressor response, which was attenuated by a pretreatment of atropine. We conclude that cholinergic inputs to orexin neurons may be involved in cardiosympathetic regulation through the M₃R on the orexin neurons that directly project to the RVLM. Anat Rec, 299:660–668, 2016. © 2016 Wiley Periodicals, Inc.     

Distribution of alpha-neoendorphin immunoreactivity in the diencephalon and the brainstem of the dog

Alpha-neoendorphin (-NE) is an opiate decapeptide derived from the prodynorphin protein. Its anatomical distribution in the brain of mammals other than the rat, particularly in carnivores, is less well known than for other opiate peptides. In the present work, we have charted the distribution of -NE immunoreactive fibers and perikarya in the diencephalon and the brainstem of the dog. The highest densities of labeled fibers were found in the substantia nigra and in patches within the nucleus of the solitary tract. Moderate densities appeared in the arcuate nucleus (Ar), median eminence, entopeduncular nucleus, ventral tegmental area, retrorubral area, periaqueductal central gray, interpeduncular nucleus and lateral parabrachial nucleus. Groups of numerous labeled perikarya were localized in the magnocellular hypothalamic nuclei, Ar and in the central superior and incertus nuclei in the metencephalon. Moreover, less densely packed fibers and cells appeared widely distributed throughout many nuclei in the region studied. These results are discussed with regard to the pattern described in other species. In addition, the present results were compared with the distribution of met-enkephalin immunoreactivity in the diencephalon and the brainstem of the dog that we have recently described. Although the distributions of these two peptides overlap in many areas, the existence of numerous differences suggest that they form separate opiate systems in the dog.     

Distribution of alpha-neoendorphin immunoreactivity in the diencephalon and the brainstem of the dog.

Alpha-neoendorphin (alpha-NE) is an opiate decapeptide derived from the prodynorphin protein. Its anatomical distribution in the brain of mammals other than the rat, particularly in carnivores, is less well known than for other opiate peptides. In the present work, we have charted the distribution of alpha-NE immunoreactive fibers and perikarya in the diencephalon and the brainstem of the dog. The highest densities of labeled fibers were found in the substantia nigra and in patches within the nucleus of the solitary tract. Moderate densities appeared in the arcuate nucleus (Ar), median eminence, entopeduncular nucleus, ventral tegmental area, retrorubral area, periaqueductal central gray, interpeduncular nucleus and lateral parabrachial nucleus. Groups of numerous labeled perikarya were localized in the magnocellular hypothalamic nuclei, Ar and in the central superior and incertus nuclei in the metencephalon. Moreover, less densely packed fibers and cells appeared widely distributed throughout many nuclei in the region studied. These results are discussed with regard to the pattern described in other species. In addition, the present results were compared with the distribution of met-enkephalin immunoreactivity in the diencephalon and the brainstem of the dog that we have recently described. Although the distributions of these two peptides overlap in many areas, the existence of numerous differences suggest that they form separate opiate systems in the dog.     

Stimulation of hypothalamic opioid peptide release by lithium is mediated by opioid autoreceptors: evidence from a combined in vitro, ex vivo study

The effect of both chronic and acute lithium treatment on hypothalamic opioid peptides was investigated. Acute treatment with lithium was found to stimulate the release of beta-endorphin, dynorphin and Met-enkephalin from perfused rat hypothalamic slices. Application of tetrodotoxin was found to have no effect upon the stimulation indicating it to be mediated at the nerve terminal level. The release of hypothalamic opioid peptides is known to be under the chronic control of a system of inhibitory autoreceptors. Blockade of these autoreceptors with, for example, the opioid receptor antagonist naloxone causes a release of all three opioid peptides. Simultaneous addition of naloxone and lithium was found to have no additive effect on the release of any opioid, suggesting lithium acts via an inhibition of the inhibitory autoreceptor. Preincubation with pertussis toxin prevented the lithium stimulation of dynorphin and Met-enkephalin, but not beta-endorphin, release, indicating lithium interacts with a G-protein to affect the autoreceptor controlling the release of dynorphin and Met-enkephalin. Chronic treatment with lithium in vivo (10 days) had no effect on the basal release or hypothalamic content of any of the opioids, but prevented the naloxone-stimulated release of dynorphin and Met-enkephalin in vitro. Long-term treatment with lithium would thus appear to inactivate the autoreceptor(s) controlling their release. These data demonstrate a lithium-stimulated release of hypothalamic beta-endorphin, Met-enkephalin and dynorphin, apparently mediated via an inhibition of the autoreceptors controlling their release. Chronic treatment with lithium permanently inactivated the autoreceptor(s) controlling the release of dynorphin and Met-enkephalin but not beta-endorphin. Lithium would appear to mediate its effects upon Met-enkephalin and dynorphin release via an interaction with a pertussis toxin-sensitive G-protein. The mechanisms underlying its release of beta-endorphin are at present uncertain.     

Effects of leptin and orexin-A on food intake and feeding related hypothalamic neurons

The lateral hypothalamic area (LHA) and the ventromedial hypothalamic nucleus (VMH) have historically been implicated in ingestive behavior, energy balance and body mass regulation. The LHA is more closely associated with the initiation of eating; whereas the VMH mediates the cessation of eating. The parvocellular part of the paraventricular nucleus (pPVN) is also included in the suppressing mechanism. Recently, two hypothalamic peptides, orexin-A and orexin-B, localized in the posterior and lateral hypothalamic perifornical region were discovered in the rat brain and they increase food intake. Leptin, a protein encoded by an obesity gene, expressed in adipose tissue and released into the blood also affects food intake. Orexin and leptin receptors have been localized in the LHA, pPVN, and VMH. The purpose of this study was to measure food intake in the rat in response to leptin and orexin-A; and to determine their electrophysiological effects on feeding related hypothalamic neurons. Results clearly show that leptin suppresses food intake whereas orexin-A increases food intake. These differences are associated with leptin and orexin-A modulatory effects on LHA, pPVN, and VMH glucose responding neurons. In the LHA, leptin inhibits a larger proportion of both glucose-sensitive neurons (GSNs) and non-GSNs. In the pPVN, leptin increases more GSNs in comparison to non-GSNs. Whereas in the VMH, leptin increases the activity of glucoreceptor neurons (GRNs) in comparison to non-GRNs. Orexin-A had opposite effects: increases activity of GSNs more than the non-GSNs in the LHA and significantly suppresses GRNs in the VMH. In the pPVN, orexin-A had no observable effects on neurons that have a low density of orexin 2 receptors. Results are discussed in terms of hypothalamic neural circuits that are sensitive to endogenous food intake inducing and reducing substances.     

Enhanced release of adenosine in rat hind paw following spinal nerve ligation: involvement of capsaicin-sensitive sensory afferents

Dopaminergic projections to the forebrain arising from the mesencephalic ventral tegmentum modulate information processing in cortical and limbic sites. The lateral hypothalamus is crucial for the coordination of behavioral responses to interoceptive cues. The presence of a hypothalamic input to the ventral tegmental area has been known for some time, but the organization of this pathway has received little attention. Among the neuropeptides found in the hypothalamus are the orexins, which are selectively expressed in the lateral hypothalamus and adjacent perifornical area and are critically involved in homeostatic regulatory processes, including arousal and feeding. We examined the anatomical relationships between orexin and dopamine neurons in rats, with particular attention to characterizing the lateral hypothalamic projection to midbrain dopamine neurons.Iontophoretic deposits of the retrograde tracer FluoroGold into the ventral tegmental area revealed a large number of retrogradely-labeled cells that formed a band extending from the medial perifornical area arching dorsally over the fornix and then ventrolaterally into the lateral hypothalamus; approximately 20% of these cells expressed orexin A-like immunoreactivity. Moreover, axons that were anterogradely labeled from the lateral hypothalamus were seen throughout the ventral tegmental area, and were often in close proximity to the dendrites and somata of dopamine neurons. Dopamine and orexin fibers were found to codistribute in the medial prefrontal cortex; orexin fibers were present in lower density in the medial shell of the nucleus accumbens, and the central and posterior basolateral nuclei of the amygdala.We conclude that the lateral hypothalamic/perifornical projection represents an anatomical substrate by which interoceptive-related signals may influence forebrain dopamine function.