Regular articleChanges in orexin (hypocretin) neuronal expression with normal aging in the human hypothalamus
Introduction
In the central nervous system, orexin A and B (OxA and OxB), also called hypocretin 1 and 2, are endogenous neuropeptides generated by the cleavage of the precursor protein, prepro-orexin (PPO) (de Lecea et al., 1998, Sakurai et al., 1998, Sakurai et al., 1999). PPO is synthesized within orexin (Ox) secreting neurons in the hypothalamus (Sakurai et al., 1999). In the adult human hypothalamus, Ox neuropeptides are highly expressed in neurons in the perifornical and dorsal medial hypothalamus (PeF and DMH), with moderate expression in the lateral hypothalamus (LH) (Thannickal et al., 2003). The Ox neuropeptides, on synthesis, are packaged into synaptic vesicles, with dynorphin and glutamate, and transported down the axon to synaptic terminals (Zhang et al., 2001). Although Ox neurons are confined to the hypothalamus, immunoreactive synaptic terminals are observed throughout the brain except in the cerebellum (Nambu et al., 1999). OxA and OxB bind exclusively to 2 G-protein-coupled postsynaptic receptors, orexin receptor 1 and 2. OxA binds to orexin receptor 1 with greater affinity than OxB, whereas OxA and OxB bind to orexin receptor 2 with equal affinity (Sakurai et al., 1998). Both Ox and glutamate, modulate neuronal depolarization, although compared with glutamate, Ox promotes prolonged neuronal excitation (Schöne et al., 2014).
Loss of Ox immunoreactive (Ox-ir) neurons as seen in narcolepsy in humans reduces the ability to maintain a sleep or wake state and is accompanied by frequent and rapid transitions between wakefulness and rapid eye movement sleep, and excessive daytime sleepiness (Sakurai, 2007, Thannickal et al., 2000, Thannickal et al., 2009). Additional roles of Ox have been demonstrated using PPO knockout and Ox neuron ablation mice whereby they were found to develop features of narcolepsy, had decreased food intake and/or appetite, yet a tendency to develop type 2 diabetes, indicating that Ox also has a role in both intake and energy regulation (Hara et al., 2005). Comparing the 2 types of mice, mice with neuron ablation develop more severe metabolic dysfunction (Hara et al., 2001, Hara et al., 2005).
Multiple studies with conflicting result have examined changes in Ox expression during development and aging. Prenatal expression of Ox and its receptors have been reported in the rat brain (Van Den Pol et al., 2001, Yamamoto et al., 2000). Postnatally in the human, Aran et al. (2012), found that OxA concentration in the cerebral spinal fluid (CSF) of infants increased from birth until 2–4 postnatal months and then decreased throughout childhood and puberty. However, Kanbayashi et al. (2002) found no change in CSF OxA concentration between birth and 4 years of age. Fronczek et al. (2005) remains the only study to examine Ox-ir expression in the infant and child age range (1–18 years); however, this study only including 3 data points for the infant age range (6, 7, and 9 months; all 3 of those infants died of sudden infant death syndrome) and no data points in the childhood/adolescence age range (4–18 years). Therefore, there is little data available regarding Ox-ir expression in the human infant and child age ranges. In older humans, the number of Ox-ir neurons was found unchanged between 50 and 90 years (Fronczek et al., 2005, Fronczek et al., 2012).
This study therefore aimed to: (1) clarify if changes in Ox-ir neurons occur between infants, children, younger; and older adults by quantifying the relative number and density of Ox-ir neurons in the hypothalamus; (2) determine any regional (DMH, PeF, and LH) differences or effects of age; and (3) confirm that OxA and OxB are colocalized in the same hypothalamic neurons in the human across all ages.
Section snippets
Tissue collection
Tissue sections (7 μm) from the hypothalamus at the tuberal level were obtained from formalin fixed paraffin embedded tissue blocks and mounted on 3-aminopronopyltriethoxysilane coated slides. Tissue from four age groups was examined; infants (0–1 year; n = 7) and children (4–10 years; n = 8) from the Department of Forensic Medicine (Sydney, New South Wales, Australia), and young (22–32 years; n = 4) and older (48–60 years; n = 7) adults from the NSW Tissue Resource Centre, University of Sydney
Colocalization and morphology
OxA and OxB were colocalized in all Ox-ir neurons in the DMH, PeF, and LH (Fig. 2), and this was consistent across all ages. Furthermore, no staining was observed in negative control sections.
Ox-ir neurons were round, elliptical, or fusiform, and either unipolar or multipolar. Ox-ir neurons averaged 50 μm in size (range of 35–60 μm) (Fig. 3). The Ox-ir neurons tended to be smaller in the DMH and the PeF (40 ± 10 μm [mean ± SD]) than in the LH (50 ± 10 μm [mean ± SD]), although this was not
Discussion
The key finding from this study was an overall decrease (averaging 23%–25%) in the proportion and density of Ox-ir neurons from infancy to older age (0–60 years) in the human hypothalamus. Regionally, this was confined to the DMH and the PeF. Additionally, OxA and OxB were colocalized in all neurons in all age groups.
Conclusion
This study demonstrates that OxA and OxB co-localize in the human hypothalamus and that from infancy to late adulthood, there is a progressive 23% decrease in the proportion of Ox-ir neurons, and a 25% reduction in the density of Ox-ir neurons in the tuberal level of the hypothalamus, largely confined to the DMH and the PeF. Between younger and older adults a 10% decrease in expression is observed. Based on these findings we suggest that high levels of Ox expression in infants may have a role
Disclosure statement
The authors report no conflicts of interest.
Acknowledgements
Research funded by the SIDS Stampede, Australia, and the Miranda Bradshaw Foundation. The tissue used in this study was provided by the NSW Brain Bank Network and NSW Forensic and Analytical Science Service. MAB763 was a kind gift from Associate Professor Pascal Carrive, School of Medical Sciences, University of New South Wales. The authors acknowledge the facilities, and scientific and technical assistance of the Australian Microscopy and Microanalysis Research Faculty at the Australian Centre
References (76)
- et al.
Age-and gender-specific changes of hypocretin immunopositive neurons in C57Bl/6 mice
Neurosci. Lett.
(2010) - et al.
Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation
Cell
(1999) - et al.
Sleep, circadian rhythms, and delayed phase in adolescence
Sleep Med. Rev.
(2007) - et al.
Orexin neuronal changes in the locus coeruleus of the aging rhesus macaque
Neurobiol. Aging
(2007) Aging-related sleep changes
Clin. Geriatr. Med.
(2008)- et al.
Hypocretin (orexin) loss in Alzheimer's disease
Neurobiol. Aging
(2012) - et al.
Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity
Neuron
(2001) - et al.
Difference in obesity phenotype between orexin-knockout mice and orexin neuron-deficient mice with same genetic background and environmental conditions
Neurosci. Lett.
(2005) - et al.
Effects of single and chronic intracerebroventricular administration of the orexins on feeding in the rat
Peptides
(1999) - et al.
Orexin receptors in the developing piglet hypothalamus, and effects of nicotine and intermittent hypercapnic hypoxia exposures
Brain Res.
(2013)
The hypothalamic clock and its control of glucose homeostasis
Trends Endocrinol. Metabol.
Hypothalamic contribution to sleep–wake cycle development
J. Neurosci.
Excitotoxic degeneration of hypothalamic orexin neurons in slice culture
Neurobiol. Dis.
Entrainment of temperature and activity rhythms to restricted feeding in orexin knock out mice
Brain Res.
Age-related loss of orexin/hypocretin neurons
J. Neurosci.
Measurement of hypocretin/orexin content in the mouse brain using an enzyme immunoassay: the effect of circadian time, age and genetic background
Peptides
Effects of changes in energy homeostasis and exposure of noxious insults on the expression of orexin (hypocretin) and its receptors in the brain
Brain Res.
Age-related changes in plasma orexin-A concentrations
Exp. Gerontol.
Calcium signaling in the ER: its role in neuronal plasticity and neurodegenerative disorders
Trends Neurosci.
Adolescent development, hypothalamic-pituitary-adrenal function, and programming of adult learning and memory
Prog. Neuropsychopharmacol. Biol. Psychiatry
Changes in circadian rhythms and sleep quality with aging: mechanisms and interventions
Neurosci. Biobehav. Rev.
Distribution of orexin neurons in the adult rat brain
Brain Res.
Activation of orexin neurons in dorsomedial/perifornical hypothalamus and antidepressant reversal in a rodent model of depression
Neuropharmacology
The effect of age on prepro-orexin gene expression and contents of orexin A and B in the rat brain
Neurobiol. Aging
Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior
Cell
Structure and function of human prepro-orexin gene
J. Biol. Chem.
Developmental and aging change of orexin-A and-B immunoreactive neurons in the male rat hypothalamus
Neurosci. Lett.
Coreleased orexin and glutamate evoke nonredundant spike outputs and computations in histamine neurons
Cell Rep.
Calcium, cellular aging, and selective neuronal vulnerability in Parkinson's disease
Cell Calcium
Reduced number of hypocretin neurons in human narcolepsy
Neuron
Adrenergic nerve smooth endoplasmic reticulum calcium buffering declines with age
Neurobiol. Aging
Chronobiology of aging: temperature, sleep-wake rhythms and entrainment
Neurobiol. Aging
Postnatal development of orexin/hypocretin in rats
Mol. Brain Res.
Orexin (hypocretin)-like immunoreactivity in the cat hypothalamus: a light and electron microscopic study
Sleep
CSF levels of hypocretin-1 (orexin-A) peak during early infancy in humans
Sleep
Developmental divergence of sleep-wake patterns in orexin knockout and wild-type mice
Eur. J. Neurosci.
Muscarinic-2 and orexin-2 receptors on GABAergic and other neurons in the rat mesopontine tegmentum and their potential role in sleep–wake state control
J. Comp. Neurol.
Dynamic changes of orexin A and leptin in obese children during body weight reduction
Physiol. Res.
Cited by (64)
Targeting the orexin/hypocretin system for the treatment of neuropsychiatric and neurodegenerative diseases: From animal to clinical studies
2023, Frontiers in NeuroendocrinologyUnderstanding the aging hypothalamus, one cell at a time
2022, Trends in NeurosciencesDepression and stress levels increase risk of liver cancer through epigenetic downregulation of hypocretin
2022, Genes and DiseasesCitation Excerpt :At the molecular level, epigenetic regulation influences gene expression in susceptible individuals, and which may be associated with the interactions among environmental stress exposure, endocrine modulation and immune dysfunction.12 Hypocretin (HCRT, also known as Orexin)13 is an excitatory neuropeptide secreted by neurons on the lateral side of the hypothalamus and around the fornix,14 which acts via two G protein-coupled receptors, hypocretin-receptor-1 and -2.15 HCRT and its receptors are found in many brain regions including the prefrontal cortex (PFC), hippocampus, almond nucleus and ventral tegmental area.16–19
Intranasal insulin and orexins to treat age-related cognitive decline
2021, Physiology and BehaviorThe orexin/hypocretin system in neuropsychiatric disorders: Relation to signs and symptoms
2021, Handbook of Clinical Neurology