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Abstract: In a previous study, we noted the presence of enlarged “spot-like” structures that are immunoreactive to hypocretin in specific regions of brainstem of aged cats; similar structures were not seen in the same regions of adult cats. In the present study, electron microscopy was combined with hypocretin immunohistochemistry to examine the ultrastructure of these enlarged “spot-like” structures, which were found to consist of enlarged axonal terminals. The terminals were comprised of a large pale core with a dark peripheral rim. Many granules were present within the core; most of these granular structures were not immunostained. On the other hand, the rim contained a high density of hypocretin immunoreactivity; in this peripheral region, large dense-core vesicles and small synaptic vesicles without dense cores were observed. In addition, mitochondria in the peripheral rim region exhibited high electron density, which is indicative of the presence of age-related degenerative changes. Synaptic contacts were observed between the enlarged terminals and adjacent dendrites. Most of these synapses were asymmetric (Gray type I), although a few of them were symmetric (Gray type II). These data suggest that hypocretin transmission is altered during aging, which would be expected to result in age-related changes in the functioning of the hypocretinergic system. [Copyright &y& Elsevier]

The distribution of hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) immunoreactivities in the cat brainstem was examined using immunohistochemical techniques. Hcrt-1- and hcrt-2-positive fibers with varicosities were detected in almost all brainstem regions. However, no hcrt-1- or hcrt-2-immunoreactive neuronal somata were observed in the cat brainstem. Both hcrt-1- and hcrt-2-labeled fibers exhibited different densities in distinct regions of the brainstem. In most brainstem regions, the intensity of hcrt-1 immunoreactivity was higher than that of hcrt-2 immunoreactivity. The highest densities of hcrt-1- and hcrt-2-positive fibers were found in the nucleus raphe dorsalis (RD), the laterodorsal tegmental nucleus (LDT) and the locus coeruleus (LC), suggesting an important role for these peptides in functions related to sleep–wake behavior. [Copyright &y& Elsevier]

Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) are two recently discovered hypothalamic neuropeptides. In the present study, using double immunofluorescent techniques, the co-localization of hcrt-1 and hcrt-2 was examined in neuronal soma and fibers/terminals located, respectively, in the cat hypothalamus and brainstem. In the hypothalamus, all hcrt-1 positive neuronal soma also displayed hcrt-2 immunoreactivity. In the brainstem, both hcrt-1 and hcrt-2 antibodies labeled the same fibers/terminals, indicating that hcrt-1 and hcrt-2 co-localize not only in the neuronal soma (hypothalamus) but also in their fibers/terminals (brainstem). If both peptides are released following neuronal activity, then the distinct effects of these peptides in the brain are likely to depend on the types of postsynaptic receptors that are activated. [Copyright &y& Elsevier]

Terminals of hypothalamic hypocretin-containing neurons are observed within brainstem nuclei involved in the control of sleep and wakefulness. Because aged humans, cats and other species exhibit changes in sleep and wakefulness in old age, we were interested in examining age-related changes in hypocretin/orexin projections to the following brainstem regions which are associated with the regulation of sleep and wakefulness: the dorsal raphe nucleus, the laterodorsal tegmental nucleus, the pedunculo-pontine tegmental nucleus and the locus coeruleus. Based upon the results of immunohistochemical determinations, in all the regions examined, round or oval ‘spot-like’ structures were observed in aged cats. Many of these ‘spot-like’ structures resembled enlarged varicosities of a nature that would be expected to disrupt hypocretin neurotransmission. In addition, a site-specific decrease in immunostaining was observed in the locus coeruleus in old cats compared with adult controls; this result likely reflects a decrease in the number of labeled fibers, which indicates that there occurs a degeneration of hypocretinergic function in conjunction with old age. The proceeding changes may account for some of sleep–wake disturbance which are observed in aged animals as well as elderly humans. [Copyright &y& Elsevier]