Opposing effects of hypoxia on catecholaminergic locus coeruleus and hypocretin/orexin neurons in chick embryos

Abstract

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.

Optimized CUBIC protocol for 3D imaging of chicken embryos at single-cell resolution

“…To confirm that the optimized CUBIC-clearing and immunostaining protocol provides comparable results to standard histology and immunohistochemistry, we compared our results with those of Godden et al (2014), Landry et al (2014Landry et al ( , 2016 and Chan et al (2016). The stained cells obtained with our optimized protocol showed identical spatial distributions to those obtained in these previous studies at both E16 ( Fig.…”

“…These staining combinations were previously used by Landry et al (2014Landry et al ( , 2016 and Chan et al (2016) in chicken embryos and post-hatched chicks. Light-sheet microscopy of the CUBIC-cleared brains produced images with sufficient resolution to clearly recognize single labelled cells in the hypothalamus (Fig.…”

Chick embryos have the same pattern of hypoxic lower‐brain activation as fetal mammals

“…The intensity of cFos staining and the overall morphology of serotonergic and catecholaminergic neurons did not differ among the hypoxic and normoxic groups (Fig. in Landry et al, ). Normal instantaneous heart rate changes have been recorded in E18–19 chick embryos (10% O 2 for 2 h, returned to normoxia; Khandoker et al, ), suggesting the proper functioning of brain circuitry.…”

“…At younger ages, more severe hypoxia than used here leads to brain apoptosis, detectable after 6 h of normoxic recovery (Pozo Devoto et al, ). The modest (15% O 2 ) and medium hypoxia (10% O 2 ) are known to be nonlethal at E18 for the exposure times used here (Ferner and Mortola, ; Mortola JP, personal communication; Landry et al, ). We also did not see any of the morphological changes previously described in apoptotic serotonergic neurons (Rahim et al, ), nor any differences in the shape or size of cell nuclei (revealed by cFos and DAPI staining) between control and hypoxic embryos.…”

“…Sections spanning the brainstem and hypothalamus (1 section every 240 μm) were stained in the present experiment. cFos expression was evaluated in these brain areas using the same colorimetric protocol described previously for the study of the pallium (Landry et al, ). All procedures were performed at room temperature, and washes were done in phosphate‐buffered saline (PBS; 140 m M NaCl, 8 m M Na 2 HPO 4 , 2.7 m M KH 2 PO 4 , 1.5 m M KCl, pH 7.4).…”