Colocalization of orexin a and glutamate immunoreactivity in axon terminals in the tuberomammillary nucleus in rats
Section snippets
Experimental procedures
We used 10 male Sprague–Dawley rats weighing 250–300 g, following protocols approved by the Harvard Medical School and Beth Israel Deaconess Medical Center Animal Care and Use Committees. Protocols were designed to minimize the use of animals and to prevent suffering.
Animals were anesthetized with chloral hydrate (350 mg/kg i.p.) and perfused with a saline flush followed by 500 ml of a solution containing 4% paraformaldehyde and 0.3% glutaraldehyde in 0.1 M phosphate buffer (PB) at pH 7.4. The
Light microscopy
We examined the light microscopic appearance of the orexin-ir axons and terminals in the TMN and neighboring structures in the posterior hypothalamus. As the axons leave the lateral hypothalamic area (LHA), they tend to be concentrated in a medial stream that innervates the TMNd and adjacent hypothalamic sites, and in a lateral stream that runs along the pial surface of the hypothalamus to reach the TMNv. The axons were relatively thin and gave off round, smooth boutons with a diameter that was
Discussion
In the present study we confirmed the intense projection of orexin-ir axons to both the dorsomedial and the ventrolateral divisions of the TMN. We showed that these orexin axons make primarily asymmetric synaptic contacts with TMN neurons, and demonstrated that orexin immunoreactivity is confined to a subpopulation of large, dense core vesicles, whereas the same terminals contain small clear vesicles which in most cases can be shown to be glutamatergic.
Acknowledgements
Supported by USPHS grant HL60292, and by Fondecyt Lineas Complementarias grant 8980006. M.Y. is an Investigator of the Howard Hughes Medical Institute.
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