Elsevier

Neuroscience

Volume 103, Issue 3, 21 March 2001, Pages 777-797
Neuroscience

Gene expression and protein distribution of the orexin-1 receptor in the rat brain and spinal cord

https://doi.org/10.1016/S0306-4522(01)00033-1Get rights and content

Abstract

Orexins-A and -B are neuropeptides derived from a single precursor prepro-orexin. The mature peptides are mainly expressed in the lateral hypothalamic and perifornical areas. The orexins have been implicated in the control of arousal and appear to be important messengers in the regulation of food intake. Two receptors for orexins have been characterised so far: orexin-1 and -2 receptors. To gain a further understanding of the biology of orexins, we studied the distribution of the orexin-1 receptor messenger RNA and protein in the rat nervous system. We first assessed the expression profile of the orexin-1 receptor gene (ox-r1) in different regions by using quantitative reverse transcription followed by polymerase chain reaction. Using immunohistochemical techniques, we investigated the distribution of orexin-1 receptor protein in the rat brain using a rabbit affinity-purified polyclonal antiserum raised against an N-terminal peptide. The orexin-1 receptor was widely and strongly expressed in the brain. Thus, immunosignals were observed in the cerebral cortex, basal ganglia, hippocampal formation, and various other subcortical nuclei in the hypothalamus, thalamus, midbrain and reticular formation. In particular, robust immunosignals were present in many hypothalamic and thalamic nuclei, as well as in the locus coeruleus. The distribution of the receptor protein was generally in agreement with the distribution of the receptor messenger RNA in the brain as reported previously by others and confirmed in the present study. In addition, we present in situ hybridisation and immunohistochemical data showing the presence of orexin-1 receptor messenger RNA and protein in the spinal cord and the dorsal root ganglia. Finally, due to the shared anatomical and functional similarities between orexins and melanin-concentrating hormone, we present a comparison between the neuroanatomical distribution of the orexin-1 receptor and melanin-concentrating hormone receptor protein-like immunoreactivities in the rat central nervous system, and discuss some functional implications.

In conclusion, our neuroanatomical data are consistent with the biological effects of orexins on food intake and regulation of arousal. In addition, the data suggest other physiological roles for orexins mediated through the orexin-1 receptor.

Section snippets

Animals

Adult male Sprague–Dawley rats (200–250 g; Charles River, UK) were kept in a fixed 12-h light–dark cycle with food and water provided ad libitum. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act (86609EEC) and all efforts were made to minimise the number of animals used and their suffering.

Peptides

All peptides were synthesised using solid-phase methodology on a model 432A Applied Biosystem Synthethiser. Peptide purity was estimated by chromatography

Taqman reverse transciption–polymerase chain reaction analysis of orexin-1 receptor mRNA expression in rat nervous system

The primary data generated by Taqman RT–PCR consist of a threshold cycle value, indicating the PCR cycle at which the PCR product is detectable above an arbitrary threshold. The system was calibrated using known numbers of copies of genomic DNA. When the threshold cycle generated by these standards was plotted against the genomic DNA copy number on a logarithmic scale, the data points lay on a straight line (not shown). With threshold cycle values resulting from the cDNA samples from various

Discussion

In the present work, we have reported the distribution of OX-R1 protein in the rat CNS and its mRNA in the spinal cord and dorsal root ganglia by using quantitative RT–PCR, in situ hybridisation and immunohistochemistry. Specificity of the antiserum raised against an N-terminal peptide of the OX-R1 rat orthologue sequence40 was demonstrated using OX-R1-transfected HEK293 cells. We could not detect a specific immunosignal in western blot experiments (after denaturing sodium dodecyl

Conclusion

Using a selective antiserum raised against OX-R1, we have characterised the brain and spinal cord distribution of OX-R1 in the rat. Gene expression experiments (in situ hybridisation and quantitative RT–PCR) broadly confirmed our immunohistochemical findings: the receptor gene is expressed widely throughout the rat CNS, as also reported by others.51 Our study would support the involvement of orexins in regulating feeding and drinking behaviours, neuroendocrine control and arousal, through the

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