Elsevier

Neurobiology of Aging

Volume 36, Issue 1, January 2015, Pages 292-300
Neurobiology of Aging

Regular article
Changes in orexin (hypocretin) neuronal expression with normal aging in the human hypothalamus

https://doi.org/10.1016/j.neurobiolaging.2014.08.010Get rights and content

Abstract

Animal studies have shown that decreased orexin expression changes sleep regulation with normal aging. This study examined orexin A and B expression in the tuberal hypothalamus in infants (0–1 year; n = 8), children (4–10 years; n = 7), young adults (22–32 years; n = 4), and older (48–60 years; n = 7) adults. Neuronal expression was defined by the percentage positive orexin immunoreactive (Ox-ir) neurons in the whole tuberal hypothalamus, and in the dorsal medial (DMH), perifornical, and lateral hypothalamus. In addition, the number of Ox-ir neurons/mm2, regional distribution, and co-localization were examined. Within the whole tuberal hypothalamic section, there was a 23% decrease in the percentage of Ox-ir neurons between infants and older adults (p < 0.001), and a 10% decrease in older compared with younger adults (p = 0.023). These changes were confined to the DMH and/or perifornical hypothalamus. There was a 9%–24% decrease in Ox neurons/mm2 in adults compared with infants and/or children (p ≤ 0.001). These results demonstrate a decrease in Ox expression with normal human maturation and aging. This may contribute to changes in sleep regulation during development and with aging.

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

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