Gene expression and protein distribution of orexins and orexin receptors in rat retina

doi:10.1016/j.neuroscience.2011.04.011

Neuroscience

Volume 189, 25 August 2011, Pages 146-155

https://doi.org/10.1016/j.neuroscience.2011.04.011 Get rights and content

Abstract

Orexins, composed of orexin A and orexin B, are identified as endogenous ligands of two orphan G-protein-coupled receptors: orexin 1 and orexin 2 receptors (OX₁R and OX₂R). Orexins are implicated in regulating wake/sleep states, feeding behaviors, etc. Using reverse transcription-polymerase chain reactive (RT-PCR) analysis and immunofluorescence double labeling, we investigated the distributions of orexin A, orexin B, OX₁R and OX₂R in rat retina. RT-PCR analysis revealed the presence of mRNAs of prepro-orexin, OX₁R and OX₂R in rat retina. Immunostaining for orexin A and orexin B was observed in many cells in the inner nuclear layer and the ganglion cell layer. In the outer retina, horizontal cells, labeled by calbindin, and bipolar cells, labeled by homeobox protein Chx10, were orexin A- and orexin B-positive. In the inner retina, two orexins were both found in GABAergic amacrine cells (ACs), including dopaminergic and cholinergic ones, stained by tyrosine hydroxylase and choline acetyltransferase respectively. Glycinergic ACs, including AII ACs, also expressed orexins. Weak to moderate labeling for orexin A and orexin B was diffusely distributed in the inner plexiform layer. Additionally, orexins were expressed in almost all ganglion cells (GCs) retrogradely labeled by cholera toxin B subunit. Specifically, double-labeling experiments demonstrated that melanopsin-positive GCs (intrinsically photosensitive retinal GCs, ipRGCs) were labeled by two orexins. Morever, OX₁R immunoreactivity was observed in most of GCs and all dopaminergic ACs, as well as in both outer and inner plexiform layers. In contrast, no obvious OX₂R immunostaining was detectable in the rat retina. These results suggest that orexins may modulate the function of neurons, especially in the inner retina. We further hypothesize that the orexin signaling via ipRGCs may be involved in setting the suprachiasmatic nucleus (SCN) circadian clock.

Highlights

▶mRNAs of prepro-orexin and OXRs are present in rat retina. ▶Orexin A and orexin B are expressed in HCs, BCs, ACs and GCs. ▶OX1R-IR is observed in dopaminergic ACs, GCs and in both plexiform layers. ▶No obvious OX₂R-IR is detectable throughout the retina.

Section snippets

Experimental procedures

A total of 34 male albino rats (Sprague–Dawley, 42–49 days of age) were used in this study. The experiments were carried out in accordance with the NIH guidelines for the Care and Use of Laboratory Animals and the regulations of Fudan University on the ethical use of animals. All efforts were made to minimize the number of animals used and their suffering.

Gene expression of prepro-orexin and OXRs in rat retina

Both orexin A and orexin B are produced from a common precursor polypeptide, prepro-orexin by proteolytic processing (de Lecea et al., 1998, Sakurai et al., 1998). After extracting total RNA from rat retinas, RT-PCR analysis was conducted to detect the mRNA transcripts of prepro-orexin, OX₁R and OX₂R. As shown in Fig. 1, the expected RT-PCR products of approximately ∼486 bp, ∼462 bp and ∼526 bp were amplified for prepro-orexin, OX₁R and OX₂R, respectively. No signal was observed when the

Discussion

While Savaskan et al. (2004) reported the expression of orexin A and orexin B in cells resembling ACs and GCs in aged human retina, these authors did not definitely identify the orexin-positive cells by double labeling experiments. In the present work, we first showed the presence of prepro-orexin mRNA in rat retina. By immunocytochemistry we further demonstrated the expression of orexin A and B in different types of retinal neurons. A schematic diagram, summarizing these results, is shown in

Acknowledgments

We would like to thank Dr. D. Pow for providing the rat anti-glycine antibody as a gift. This work was supported by grants from the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2006CB500805, 2007CB512205, 2011CB504602), the National Natural Science Foundation of China (30770698, 30930034, 31070967, 30821002), and the 211 Project sponsored by the Ministry of Education of China.

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Orexins A and B (OXA and OXB) and their receptors are expressed in the retina of both human and rodents and play a vital role in regulating signal transmission circuits in the retina. There is an anatomical-physiological relationship between the retinal ganglion cells and suprachiasmatic nucleus (SCN) through glutamate as a neurotransmitter and retinal pituitary adenylate cyclase-activating polypeptide (PACAP) as a co-transmitter. SCN is the main brain center for regulating the circadian rhythm, which governs the reproductive axis. The impact of retinal orexin receptors on the hypothalamic-pituitary-gonadal axis has not been investigated. Retinal OX1R or/and OX2R in adult male rats by 3 µl of SB-334867 (1 µg) or/and 3 µl of JNJ-10397049 (2 µg) were antagonized via intravitreal injection (IVI). Four time-periods were considered (3, 6, 12, and 24 h) for the controls without any treatment, SB-334867, JNJ-10397049, and SB-334867 + JNJ-10397049 groups. Antagonizing retinal OX1R or/and OX2R resulted in a significant elevation of retinal PACAP expression compared to control animals. In addition, expression of GnRH increased non-significantly in the hypothalamus over the 6 h of the study, and the serum concentration of LH decreased significantly in the SB-334867 group after 3 h of injection. Furthermore, testosterone serum levels declined significantly, especially within 3 h of injection; serum levels of progesterone were also exposed to a significant rise at least within 3 h of injection. However, the retinal PACAP expression changes were mediated by OX1R more effectively than by OX2R. In this study, we report the retinal orexins and their receptors as light-independent factors by which the retina affects the hypothalamic-pituitary-gonadal axis.
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The peptide hormone cholecistokinin (CCK) plays a key role in the central and peripheral nervous system. It is known to be involved in the digestive physiology and in the regulation of food intake. Moreover, the CCK expression has also been detected in the retina of different vertebrates, including fish, although its biological activity in this tissue remains to be elucidated. In literature no data are yet available about the CCK-immunoreactivity in the zebrafish retina during development. Therefore, the aim of the study was to investigate the distribution of sulfated cholecystokinin octapeptide (CCK8-S) as a well preserved form during evolution in the zebrafish retina from 3 days post hatching (dph) until adult stage, using immunohistochemistry in order to elucidate the potential role of this protein in the development and maintenance of normal retinal homeostasis. The cellular distribution of CCK in the retina was similar from 3 dph to 40 days post fertilization (dpf) when immunoreactivity was found in the photoreceptors layer, in the outer plexiform layer, in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer (GCL). Immunohistochemical localization at 50 dpf as well as in the adult stage was observed in a subpopulation of amacrine cells in the proximal inner nuclear layer, in the inner plexiform layer, in displaced amacrine cells and in retinal ganglion cells in the GCL. Our results demonstrate for the first time the occurrence of CCK in the zebrafish retina from larval to adult stage with a different pattern of distribution, suggesting different roles of CCK during retinal cells maturation.
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In addition, RBCs also receive GABAergic feedback from amacrine cells (Chavez et al., 2010; Chavez et al., 2006; Dowling and Boycott, 1966; Grunert and Martin, 1991; Sterling and Lampson, 1986), mediated by both GABAA receptors (GABAARs) and GABAC receptors (GABACRs) expressed in axon terminals of RBCs (Du and Yang, 2000; Euler and Wassle, 1998; Fletcher et al., 1998; Yu et al., 2006). Immunostaining for orexin A and orexin B is observed in horizontal cells and BCs in the outer retina and in GABAergic and glycinergic amacrine cells in the inner retina in rat (Liu et al., 2011). Weak to moderate labeling for these peptides is also diffusely distributed in the inner plexiform layer (IPL).
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¹: These authors contributed equally to the work.

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Cellular and Molecular NeuroscienceResearch PaperGene expression and protein distribution of orexins and orexin receptors in rat retina

Abstract

Highlights

Section snippets

Experimental procedures

Gene expression of prepro-orexin and OXRs in rat retina

Discussion

Acknowledgments

Trends Neurosci

Neuron

Neurosci Lett

Neurosci Lett

Neuroscience

Neuroscience

Neuroscience

Neuron

Neuroscience

Brain Res

Cell

Neurosci Lett

Neuroscience

FEBS Lett

Neuroscience

Brain Res Bull

Orexin receptor-1 (OX-R1) immunoreactivity in chemically identified neurons of the hypothalamus: focus on orexin targets involved in control of food and water intake

Eur J Neurosci

Melanopsin and inner retinal photoreception

Cell Mol Life Sci

Phototransduction by retinal ganglion cells that set the circadian clock

Science

Orexin B/hypocretin 2 increases glutamatergic transmission to ventral tegmental area neurons

Eur J Neurosci

GABAergic synapses made by a retinal dopaminergic neuron

Proc Natl Acad Sci U S A

Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems

Proc Natl Acad Sci U S A

The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity

Proc Natl Acad Sci U S A

The hypocretins and sleep

FEBS J

Expression of the LIM-homeodomain protein Isl1 in the developing and mature mouse retina

J Comp Neurol

Cellular and Molecular Neuroscience
Research Paper
Gene expression and protein distribution of orexins and orexin receptors in rat retina