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