The electrophysiological analysis of orexin neurons

Orexins/hypocretins are a recently described pair of neuropeptides. Orexin-A is a 33 amino-acid residue polypeptide with an N-terminal pyroglutamyl residue and C-terminal amidation. It has two intrachain disulfide bridges, while orexin-B is a 28 amino-acid linear polypeptide with C-terminal amidation. The actions of orexins are mediated via two G protein-coupled receptors named the orexin-l (OX1R) and orexin-2 receptor (OX2R). Orexins were implicated in energy homeostasis and arousal, however, recent reports suggest that mice with a targeted deletion of the prepro-orexin gene display a phenotype strikingly similar to human narcolepsy. We have previously reported transgenic studies in which the 3.2-kb fragment of the 5-upstream region of the human prepro-orexin gene is sufficient to express the Escherichia coli beta-galactosidase (lacZ) gene in orexin neurons. In the present study, we used the same human prepro-orexin gene fragment as the promoter to specifically express EGFP in orexin neurons to visualize and identify from other neurons. Immunohistochemical experiments using anti-orexin antibody revealed that 80% of orexin neurons expressed EGFP in two-lines of transgenic mice. Orexin neurons were dissociated enzymaticaly from lateral hypothalamus and were adhered to slide glass for electrophysiological experiments. Spontaneous action potential was recorded by means of whole-cell patch clamp technique in the current-clamp mode.