Hypocretin/Orexin (H/O) neuropeptides are released by a discrete group of neurons in the vertebrate hypothalamus which play a pivotal role in the maintenance of waking behavior and brain state control. Previous studies have indicated that the H/O neuronal development differs between mammals and fish; H/O peptide-expressing cells are detectable during the earliest stages of brain morphogenesis in fish, but only towards the end of brain morphogenesis (by ∼85% of embryonic development) in rats. The developmental emergence of H/O neurons has never been previously described in birds. With the goal of determining whether the chick developmental pattern was more similar to that of mammals or of fish, we investigated the emergence of H/O-expressing cells in the brain of chick embryos of different ages using immunohistochemistry. Post-natal chick brains were included in order to compare the spatial distribution of H/O cells with that of other vertebrates. We found that H/O-expressing cells appear to originate from two separate places in the region of the diencephalic proliferative zone. These developing cells express the H/O neuropeptide at a comparatively early age relative to rodents (already visible at 14% of the way through fetal development), thus bearing a closer resemblance to fish. The H/O-expressing cell population proliferates to a large number of cells by a relatively early embryonic age. As previously suggested, the distribution of H/O neurons is intermediate between that of mammalian and non-mammalian vertebrates. This work suggests that, in addition to its roles in developed brains, the H/O peptide may play an important role in the early embryonic development of non-mammalian vertebrates.

Terrestrial vertebrate embryos face a risk of low oxygen availability (hypoxia) that is especially great during their transition to air-breathing. To better understand how fetal brains respond to hypoxia, we examined the effects of low oxygen availability on brain activity in late-stage chick embryos (day 18 out of a 21-day incubation period). Using cFos protein expression as a marker for neuronal activity, we focused on two specific, immunohistochemically identified cell groups known to play an important role in regulating adult brain states (sleep and waking): the noradrenergic neurons of the Locus Coeruleus (NA-LC), and the Hypocretin/Orexin (H/O) neurons of the hypothalamus. cFos expression was also examined in the Pallium (the avian analog of the cerebral cortex). In adult mammalian brains, cFos expression changes in a coordinated way in these areas. In chick embryos, oxygen deprivation simultaneously activated NA-LC while deactivating H/O-producing neurons; it also increased cFos expression in the Pallium. Activity in one pallial primary sensory area was significantly related to NA-LC activity. These data reveal that at least some of the same neural systems involved in brain-state control in adults may play a central role in orchestrating prenatal hypoxic responses, and that these circuits may show different patterns of coordination than seen in adults.

cFos expression (indicating a particular kind of neuronal activation) was examined in embryonic day (E) 18 chick embryos after exposure to 4 h of either normoxia (21% O2), modest hypoxia (15% O2), or medium hypoxia (10% O2). Eight regions of the brainstem and hypothalamus were surveyed, including seven previously shown to respond to hypoxia in late-gestation mammalian fetuses (Breen et al., 1997; Nitsos and Walker, 1999b). Hypoxia-related changes in chick embryo brain activation mirrored those found in fetal mammals with the exception of the medullary Raphe, which showed decreased hypoxic activation, compared with no change in mammals. This difference may be explained by the greater anapyrexic responses of chick embryos relative to mammalian fetuses. Activation in the A1/C1 region was examined in more detail to ascertain whether an O2-sensitive subpopulation of these cells containing heme oxygenase 2 (HMOX2) may drive hypoxic brain responses before the maturation of peripheral O2-sensing. HMOX2-positive and -negative catecholaminergic cells and interdigitating noncatecholaminergic HMOX2-positive cells all showed significant changes in cFos expression to hypoxia, with larger population responses seen in the catecholaminergic cells. Hypoxia-induced activation of lower-brain regions studied here was significantly better correlated with activation of the nucleus of the solitary tract (NTS) than with that of HMOX2-containing A1/C1 neurons. Together, these observations suggest that (1) the functional circuitry controlling prenatal brain responses to hypoxia is strongly conserved between birds and mammals, and (2) NTS neurons are a more dominant driving force for prenatal hypoxic cFos brain responses than O2-sensing A1/C1 neurons.

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