Elsevier

Neuroscience

Volume 119, Issue 4, 16 July 2003, Pages 1033-1044
Neuroscience

Colocalization of orexin a and glutamate immunoreactivity in axon terminals in the tuberomammillary nucleus in rats

https://doi.org/10.1016/S0306-4522(03)00238-0Get rights and content

Abstract

The orexins (also known as hypocretins) are peptide neurotransmitters made by hypothalamic neurons that are thought to play an important role in regulating wake–sleep states. One terminal area for orexin neurons is the tuberomammillary nucleus, a histaminergic cell group that is wake-active, but the relationship of the orexinergic terminals to the tuberomammillary neurons has not been examined in detail. We studied the ultrastructure of orexin A-immunoreactive axons and terminals in the tuberomammillary nucleus using pre- and post-embedding electron microscopic protocols. We confirmed an abundant projection of orexin-immunoreactive boutons to both dorsal and ventral divisions of the tuberomammillary nucleus. These terminals made asymmetric synaptic contacts with proximal and intermediate dendrites of tuberomammillary neurons. They contained small, clear synaptic vesicles and up to 30–40 dense core vesicles were seen per terminal in a single section. Both pre- and post-embedding immunostaining revealed that orexin immunoreactivity was localized to the dense core vesicles, which were always at a distance from the synaptic specialization. We also found glutamate immunoreactivity in the small synaptic vesicles which were at the active zone of the synapses of many of the same terminals. Orexinergic afferents to the tuberomammillary neurons contain separate populations of orexinergic and glutamatergic vesicles, suggesting that the release of these neurotransmitters may be differentially regulated.

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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