Distribution of orexin-1 receptor-green fluorescent protein- (OX1-GFP) expressing neurons in the mouse brain stem and pons: Co-localization with tyrosine hydroxylase and neuronal nitric oxide synthase
Introduction
The neuropeptides orexin-A and orexin-B are produced via the proteolytic cleavage of a 130 amino acid long prepro-orexin protein, coded by a single gene transcript into 33 and 28 amino acid peptides, respectively (Sakurai et al., 1998). The prepro-orexin gene and protein are expressed by subsets of neurons located around the fornix, lateral hypothalamic area and dorsomedial hypothalamus. While the distribution of these neurons is localized within the hypothalamus, they have widespread projections that extend throughout the central nervous system (Peyron et al., 1998, Date et al., 1999). Unsurprisingly, the orexinergic system has been implicated in a range of diverse functions including the control of the sleep–wake cycle (Inutsuka and Yamanaka, 2013, Xu et al., 2013), thermoregulation (Tupone et al., 2011), feeding (Kay et al., 2014), reward (Lawrence, 2010, Xu et al., 2013), and neuroendocrine (Inutsuka and Yamanaka, 2013) and cardiovascular regulation (Antunes et al., 2001, Carrive, 2013, Ciriello et al., 2013).
The actions of both orexin-A and orexin-B are mediated by two different G-protein-coupled receptors: orexin-1 (OX1) and orexin-2 (OX2) (Sakurai et al., 1998). Pharmacological studies demonstrate that both orexin-A and orexin-B have similar affinities for OX2 receptors, whereas orexin-A is 10-fold more selective for OX1 receptors than orexin-B (Sakurai et al., 1998). As such, orexin-A is widely regarded as being selective for OX1 receptors. Activation of OX1 receptors within the ventrolateral periaqueductal gray has been shown to induce anti-nociception (Ho et al., 2011), while in the hindbrain, OX1 receptors modulate meal size (Parise et al., 2011). In the rostral ventrolateral medulla (RVLM), OX1 receptors have been suggested to be expressed on sympathoexcitatory bulbospinal neurons as exogenous application of orexin-A has been shown to elicit increases in blood pressure, sympathoexcitation and increases baroreflex sensitivity (Shahid et al., 2012).
There have been few studies that have mapped the distribution of the OX1 and OX2 receptor subtypes within the mammalian brain. There have, however, been comprehensive studies describing the distribution of the OX1 receptor messenger RNA (Marcus et al., 2001, Hervieu et al., 2001). This is most likely due to the fact that producing specific antibodies to G-protein-coupled receptors has been challenging. Many of the available antibodies appear to lack specificity and produce high levels of background staining. To circumvent this issue, we used an enhanced green fluorescent protein (GFP) reporter mouse to characterize the distribution of OX1 receptors within the mouse brain stem and pons. Additionally, given the evidence for interactions between the orexinergic, nitrergic (Shih and Chuang, 2007, Xiao et al., 2013) and catecholaminergic (Shahid et al., 2012, Soya et al., 2013) systems, we also performed a systematic analysis of OX1-GFP co-localization with the neuronal isoform of nitric oxide synthase (NOS1) and a synthetic enzyme for catecholamines, tyrosine hydroxylase (TH).
Section snippets
Experimental procedures
All experimental protocols used in this study were performed in accordance with the Prevention of Cruelty to Animals Act, Australia 1986, conformed with the guidelines set out by the National Health and Medical Research Council of Australia (2007), and were approved by the Florey Institute of Neuroscience and Mental Health Animal Ethics Committee.
To determine the distribution of OX1 receptors in the brainstem and pons, adult male transgenic OX1-GFP (bacterial artificial chromosome (BAC))
Validation of staining specificity
Incubation of mouse brainstem sections in the GFP antisera revealed robust staining that co-localized in exactly the same cells as those expressing intrinsic GFP fluorescence. No staining was observed in control brainstem sections from wildtype mice that do not express GFP. Incubation of mouse brain stem sections with the NOS1- and TH-antisera revealed the same patterns of distribution for nNOS (Gotti et al., 2005) and TH (Ginovart et al., 1996, Puskás et al., 2010) staining previously reported
Discussion
In the present study we systematically mapped the anatomical distribution of OX1-GFP expression throughout the brain stem and pons of OX1-GFP transgenic reporter mice. We observed discrete and specific expression in a number of nuclei distributed throughout the medulla oblongata and pons. Although our results are highly consistent with the distribution of OX1 mRNA, we failed to detect OX1 receptor expression in areas of the medulla where OX1 receptor protein expression has previously been
Conclusion
Using an OX1-GFP transgenic reporter mouse, we extensively mapped the anatomical distribution of OX1 receptors and their co-localization with NOS1 and TH throughout the medulla oblongata and pons. Our results are in agreement with the reported expression of OX1 receptor mRNA in the rat brainstem and pons (Marcus et al., 2001). However, to the best of our knowledge, no other study has systematically mapped the OX1 receptor protein in the mouse brain. Based on the distribution of OX1-GFP, our
Acknowledgments
We thank Dr Danny Winder (Vanderbilt) and Dr Paul Kenny (Mount Sinai School of Medicine) for the OX1R-eGFP founder mice. This work was supported by the National Health and Medical Research Council (NH&MRC; Australia) and the Victorian Government through the Operational Infrastructure Scheme. LCB is supported by an NH&MRC Early Career Fellowship. CNM and AJL are supported by NH&MRC Research Fellowships (566819 and 1020737 respectively).
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