Joshua P. Nixon

A comparative analysis of the distribution of immunoreactive orexin A and B in the brains of nocturnal and diurnal rodents

BackgroundThe orexins (hypocretins) are a family of peptides found primarily in neurons in the lateral hypothalamus. Although the orexinergic system is generally thought to be the same across species, the orexins are involved in behaviors which show considerable interspecific variability. There are few direct cross-species comparisons of the distributions of cells and fibers containing these peptides. Here, we addressed the possibility that there might be important species differences by systematically examining and directly comparing the distribution of orexinergic neurons and fibers within the forebrains of species with very different patterns of sleep-wake behavior.MethodsWe compared the distribution of orexin-immunoreactive cell bodies and fibers in two nocturnal species (the lab rat, Rattus norvegicus and the golden hamster, Mesocricetus auratus) and two diurnal species (the Nile grass rat, Arvicanthis niloticus and the degu, Octodon degus). For each species, tissue from the olfactory bulbs through the brainstem was processed for immunoreactivity for orexin A and orexin B (hypocretin-1 and -2). The distribution of orexin-positive cells was noted for each species. Orexin fiber distribution and density was recorded and analyzed using a principal components factor analysis to aid in evaluating potential species differences.ResultsOrexin-positive cells were observed in the lateral hypothalamic area of each species, though there were differences with respect to distribution within this region. In addition, cells positive for orexin A but not orexin B were observed in the paraventricular nucleus of the lab rat and grass rat, and in the supraoptic nucleus of the lab rat, grass rat and hamster. Although the overall distributions of orexin A and B fibers were similar in the four species, some striking differences were noted, especially in the lateral mammillary nucleus, ventromedial hypothalamic nucleus and flocculus.ConclusionThe orexin cell and fiber distributions observed in this study were largely consistent with those described in previous studies. However, the present study shows significant species differences in the distribution of orexin cell bodies and in the density of orexin-IR fibers in some regions. Finally, we note previously undescribed populations of orexin-positive neurons outside the lateral hypothalamus in three of the four species examined.

Evaluation of a quantitative magnetic resonance imaging system for whole body composition analysis in rodents

We evaluated the EchoMRI‐900 combination rat and mouse quantitative magnetic resonance (QMR) body composition method in comparison to traditional whole‐body chemical carcass composition analysis (CCA) for measurements of fat and fat‐free mass in rodents. Live and postmortem (PM) QMR fat and lean mass measurements were obtained for lean, obese and outbred strains of rats and mice, and compared with measurements obtained using CCA. A second group of rats was measured before and after 18 h food or water deprivation. Significant positive correlations between QMR and CCA fat and lean mass measurements were shown for rats and mice. Although all live QMR fat and lean measurements were more precise than CCA for rats, values obtained for mice significantly differed from CCA for lean mass only. QMR performed PM slightly overestimated fat and lean values relative to live QMR but did not show lower precision than live QMR. Food deprivation reduced values for both fat and lean mass; water deprivation reduced estimates of lean mass only. In summary, all measurements using this QMR system were comparable to those obtained by CCA, but with higher overall precision, similar to previous reports for the murine QMR system. However, PM QMR measurements slightly overestimated live QMR values, and lean and fat mass measurements in this QMR system are influenced by hydration status and animal size, respectively. Despite these caveats, we conclude that the EchoMRI QMR system offers a fast in vivo method of body composition analysis, well correlated to but with greater overall precision than CCA.

Brain orexin promotes obesity resistance

Resistance to obesity is becoming an exception rather than the norm, and understanding mechanisms that lead some to remain lean in spite of an obesigenic environment is critical if we are to find new ways to reverse this trend. Levels of energy intake and physical activity both contribute to body weight management, but it is challenging for most to adopt major long‐term changes in either factor. Physical activity outside of formal exercise, also referred to as activity of daily living, and in stricter form, spontaneous physical activity (SPA), may be an attractive modifiable variable for obesity prevention. In this review, we discuss individual variability in SPA and NEAT (nonexercise thermogenesis, or the energy expended by SPA) and its relationship to obesity resistance. The hypothalamic neuropeptide orexin (hypocretin) may play a key role in regulating SPA and NEAT. We discuss how elevated orexin signaling capacity, in the context of a brain network modulating SPA, may play a major role in defining individual variability in SPA and NEAT. Greater activation of this SPA network leads to a lower propensity for fat mass gain and therefore may be an attractive target for obesity prevention and therapy.

Individual differences in wheel-running rhythms are related to temporal and spatial patterns of activation of orexin A and B cells in A diurnal rodent (Arvicanthis niloticus)

This study investigated the relationship between the orexins and patterns of activity in the diurnal Nile grass rat, Arvicanthis niloticus. Some individuals of this species switch to a more nocturnal pattern when given access to a running wheel, while others continue to be most active during the day. In both day- and night-active grass rats, the percentages of orexin A (OXA) and orexin B (OXB) cells expressing Fos were highest when animals were actively running in wheels. In night-active animals, removal of the running wheel significantly decreased OXA and OXB cell Fos expression. Additionally, in night-active animals, clear regional differences were apparent. In these animals the presence of a wheel induced higher percentages of Fos in both OXA and OXB cells in medial regions of the lateral hypothalamus than in lateral regions. In night-active animals without access to wheels, this medial-lateral gradient was present only in OXA cells. No regional differences were observed in day-active animals. This study demonstrates that individual differences in the patterns of activation of OXA and OXB cell populations are related to differences in the temporal pattern of wheel running. We also present evidence that orexin cells have projections to the intergeniculate leaflet that appear to make contact with neuropeptide-Y cells. We discuss the possibility that these fibers may be involved in relaying feedback regarding the activity state of the animal to the circadian system through these projections.

Sleep disorders, obesity, and aging: the role of orexin.

The hypothalamic neuropeptides orexin A and B (hypocretin 1 and 2) are important homeostatic mediators of central control of energy metabolism and maintenance of sleep/wake states. Dysregulation or loss of orexin signaling has been linked to narcolepsy, obesity, and age-related disorders. In this review, we present an overview of our current understanding of orexin function, focusing on sleep disorders, energy balance, and aging, in both rodents and humans. We first discuss animal models used in studies of obesity and sleep, including loss of function using transgenic or viral-mediated approaches, gain of function models using exogenous delivery of orexin receptor agonist, and naturally-occurring models in which orexin responsiveness varies by individual. We next explore rodent models of orexin in aging, presenting evidence that orexin loss contributes to age-related changes in sleep and energy balance. In the next section, we focus on clinical importance of orexin in human obesity, sleep, and aging. We include discussion of orexin loss in narcolepsy and potential importance of orexin in insomnia, correlations between animal and human studies of age-related decline, and evidence for orexin involvement in age-related changes in cognitive performance. Finally, we present a summary of recent studies of orexin in neurodegenerative disease. We conclude that orexin acts as an integrative homeostatic signal influencing numerous brain regions, and that this pivotal role results in potential dysregulation of multiple physiological processes when orexin signaling is disrupted or lost.

Orexin A decreases lipid peroxidation and apoptosis in a novel hypothalamic cell model

Current data support the idea that hypothalamic neuropeptide orexin A (OxA; hypocretin 1) mediates resistance to high fat diet-induced obesity. We previously demonstrated that OxA elevates spontaneous physical activity (SPA), that rodents with high SPA have higher endogenous orexin sensitivity, and that OxA-induced SPA contributes to obesity resistance in rodents. Recent reports show that OxA can confer neuroprotection against ischemic damage, and may decrease lipid peroxidation. This is noteworthy as independent lines of evidence indicate that diets high in saturated fats can decrease SPA, increase hypothalamic apoptosis, and lead to obesity. Together data suggest OxA may protect against obesity both by inducing SPA and by modulation of anti-apoptotic mechanisms. While OxA effects on SPA are well characterized, little is known about the short- and long-term effects of hypothalamic OxA signaling on intracellular neuronal metabolic status, or the physiological relevance of such signaling to SPA. To address this issue, we evaluated the neuroprotective effects of OxA in a novel immortalized primary embryonic rat hypothalamic cell line. We demonstrate for the first time that OxA increases cell viability during hydrogen peroxide challenge, decreases hydrogen peroxide-induced lipid peroxidative stress, and decreases caspase 3/7 induced apoptosis in an in vitro hypothalamic model. Our data support the hypothesis that OxA may promote obesity resistance both by increasing SPA, and by influencing survival of OxA-responsive hypothalamic neurons. Further identification of the individual mediators of the anti-apoptotic and peroxidative effects of OxA on target neurons could lead to therapies designed to maintain elevated SPA and increase obesity resistance.

Neuropeptides controlling energy balance: orexins and neuromedins

In this chapter, we review the feeding and energy expenditure effects of orexin (also known as hypocretin) and neuromedin. Orexins are multifunctional neuropeptides that affect energy balance by participating in regulation of appetite, arousal, and spontaneous physical activity. Central orexin signaling for all functions originates in the lateral hypothalamus-perifornical area and is likely functionally differentiated based on site of action and on interacting neural influences. The effect of orexin on feeding is likely related to arousal in some ways but is nonetheless a separate neural process that depends on interactions with other feeding-related neuropeptides. In a pattern distinct from other neuropeptides, orexin stimulates both feeding and energy expenditure. Orexin increases in energy expenditure are mainly by increasing spontaneous physical activity, and this energy expenditure effect is more potent than the effect on feeding. Global orexin manipulations, such as in transgenic models, produce energy balance changes consistent with a dominant energy expenditure effect of orexin. Neuromedins are gut-brain peptides that reduce appetite. There are gut sources of neuromedin, but likely the key appetite-related neuromedin-producing neurons are in the hypothalamus and parallel other key anorectic neuropeptide expression in the arcuate to paraventricular hypothalamic projection. As with other hypothalamic feeding-related peptides, hindbrain sites are likely also important sources and targets of neuromedin anorectic action. Neuromedin increases physical activity in addition to reducing appetite, thus producing a consistent negative energy balance effect. Together with the other various neuropeptides, neurotransmitters, neuromodulators, and neurohormones, neuromedin and orexin act in the appetite network to produce changes in food intake and energy expenditure, which ultimately influences the regulation of body weight.

Patterns of Wheel Running Are Related to Fos Expression in Neuropeptide-Y-Containing Neurons in the Intergeniculate Leaflet of Arvicanthis niloticus

Avariety of nonphotic influences on circadian rhythms have been documented in mammals. In hamsters, one such influence, running in a novel wheel, is mediated in part by the pathway extending from neuropeptide-Y (NPY)-containing cells within the intergeniculate leaflet (IGL) of the thalamus to the hypothalamic suprachiasmatic nucleus (SCN). Arvicanthis niloticus is a species in which all individuals are diurnal with respect to general activity and body temperature when they are housed without a running wheel, but access to a running wheel induces a subset of individuals to become nocturnal. In the first study, the authors evaluated the possibility that nocturnal and diurnal patterns of wheel running in Arvicanthis are correlated with differences in IGL function. Adult male Arvicanthis housed in a 12:12 light-dark (LD) cycle were monitored in wheels, classified as nocturnal or diurnal, and then perfused either 4 h after lights-on or 4 h after lights-off. Sections through the intergeniculate leaflet were processed for immunohistochemical labeling of Fos and NPY. The percentage of NPY cells that expressed Fos was significantly influenced by an interaction between time of day and phenotype such that it rose from night to day in diurnal animals, and from day to night in nocturnal animals. In the second experiment, the authors established that running in a wheel actually induces Fos in the IGLof Arvicanthis. Specifically, the proportion of NPY cells expressing Fos was increased by access to wheels in nocturnal animals at night and in diurnal animals during the day. In the third experiment, the authors established that lesions of the IGL eliminate NPY fibers within the SCN, suggesting that these IGL cells project to the SCN in this species as has been established in other rodents. Together, these data demonstrate a clear difference in NPY cell function in nocturnal and diurnal Arvicanthis that appears to be caused, at least in part, by the differences in their wheel-running patterns, and that NPY cells within the IGL project to the SCN in Arvicanthis.

NEURAL ACTIVATION IN AROUSAL AND REWARD AREAS OF THE BRAIN IN DAY-ACTIVE AND NIGHT-ACTIVE GRASS RATS

In the diurnal unstriped Nile grass rat (Arvicanthis niloticus) access to a running wheel can trigger a shift in active phase preference, with some individuals becoming night-active (NA), while others continue to be day-active (DA). To investigate the contributions of different neural systems to the support of this shift in locomotor activity, we investigated the association between chronotype and Fos expression during the day and night in three major nuclei in the basal forebrain (BF) cholinergic (ACh) arousal system - medial septum (MS), vertical and horizontal diagonal band of Broca (VDB and HDB respectively) -, and whether neural activation in these areas was related to neural activity in the orexinergic system. We also measured Fos expression in dopaminergic and non-dopaminergic cells of two components of the reward system that also participate in arousal - the ventral tegmental area (VTA) and supramammillary nucleus (SUM). NAs and DAs were compared to animals with no wheels. NAs had elevated Fos expression at night in ACh cells, but only in the HDB. In the non-cholinergic cells of the BF of NAs, enhanced nocturnal Fos expression was almost universally seen, but only associated with activation of the orexinergic system for the MS/VDB region. For some of the areas and cell types of the BF, the patterns of Fos expression of DAs appeared similar to those of NAs, but were never associated with activation of the orexinergic system. Also common to DAs and NAs was a general increase in Fos expression in non-dopaminergic cells of the SUM and anterior VTA. Thus, in this diurnal species, voluntary exercise and a shift to a nocturnal chronotype changes neural activity in arousal and reward areas of the brain known to regulate a broad range of neural functions and behaviors, which may be also affected in human shift workers.

Orexin fibers form appositions with Fos expressing neuropeptide-Y cells in the grass rat intergeniculate leaflet.

Neuropeptide-Y (NPY) cells in the intergeniculate leaflet (IGL) are known to modulate effects of arousal on the mammalian circadian system. However, the route through which this information reaches the IGL has not been established. Here, we provide evidence that the orexins (hypocretins) are uniquely positioned as a potential source of activity state feedback to the IGL in the grass rat, Arvicanthis niloticus. Specifically, many NPY cells in the grass rat IGL exhibit orexin-A (OXA) fiber appositions. Furthermore, NPY cells contacted by OXA fibers are significantly more likely to express Fos during nocturnal wheel running than are NPY cells without such contacts (P < 0.001).