Novel localizations of TRPC5 channels suggest novel and unexplored roles: A study in the chick embryo brain.

Mammalian TRPC5 channels are predominantly expressed in the brain, where they increase intracellular Ca2+ and induce depolarization. Because they augment presynaptic vesicle release, cause persistent neural activity, and show constitutive activity, TRPC5s could play a functional role in late developmental brain events. We used immunohistochemistry to examine TRPC5 in the chick embryo brain between 8 and 20 days of incubation, and provide the first detailed description of their distribution in birds and in the whole brain of any animal species. Stained areas substantially increased between E8 and E16, and staining intensity in many areas peaked at E16, a time when chick brains first show organized patterns of whole-brain metabolic activation like what is seen consistently after hatching. Areas showing cell soma staining match areas showing Trpc5 mRNA or protein in adult rodents (cerebral cortex, hippocampus, amygdala, cerebellar Purkinje cells). Chick embryos show protein staining in the optic tectum, cerebellar nuclei, and several brainstem nuclei; equivalent areas in the Allen Institute mouse maps express Trpc5 mRNA. The strongest cell soma staining was found in a dorsal hypothalamic area (matching a group of parvicellular arginine vasotocin neurons and a pallial amygdalohypothalamic cell corridor) and the vagal motor complex. Purkinje cells showed strong dendritic staining at E20. Unexpectedly, we also describe neurite staining in the septum, several hypothalamic nuclei, and a paramedian raphe area; the strongest neurite staining was in the median eminence. These novel localizations suggest new unexplored TRPC5 functions, and possible roles in late embryonic brain development.

Melanin-concentrating hormone (MCH) neurons in the developing chick brain.

The present study was undertaken because no previous developmental studies exist on MCH neurons in any avian species. After validating a commercially-available antibody for use in chickens, immunohistochemical examinations first detected MCH neurons around embryonic day (E) 8 in the posterior hypothalamus. This population increased thereafter, reaching a numerical maximum by E20. MCH-positive cell bodies were found only in the posterior hypothalamus at all ages examined, restricted to a region showing very little overlap with the locations of hypocretin/orexin (H/O) neurons. Chickens had fewer MCH than H/O neurons, and MCH neurons also first appeared later in development than H/O neurons (the opposite of what has been found in rodents). MCH neurons appeared to originate from territories within the hypothalamic periventricular organ that partially overlap with the source of diencephalic serotonergic neurons. Chicken MCH fibers developed exuberantly during the second half of embryonic development, and they became abundant in the same brain areas as in rodents, including the hypothalamus (by E12), locus coeruleus (by E12), dorsal raphe nucleus (by E20) and septum (by E20). These observations suggest that MCH cells may play different roles during development in chickens and rodents; but once they have developed, MCH neurons exhibit similar phenotypes in birds and rodents.

Activation of state-regulating neurochemical systems in newborn and embryonic chicks.

Coordinated activity in different sets of widely-projecting neurochemical systems characterize waking (W) and sleep (S). How and when this coordination is achieved during development is not known. We used embryos and newborns of a precocial bird species (chickens) to assess developmental activation in different neurochemical systems using cFos expression, which has been extensively employed to examine cellular activation during S and W in adult mammals. Similarly to adult mammals, newborn awake chicks showed significantly higher cFos expression in W-active hypocretin/orexin (H/O), serotonergic Dorsal Raphe, noradrenergic Locus Coeruleus and cholinergic Laterodorsal and Pedunculopontine Tegmental (Ch-LDT/PT) neurons when compared to sleeping chicks. cFos expression was significantly correlated both between these systems, and with the amount of W. S-active melanin-concentrating hormone (MCH) neurons showed very low cFos expression with no difference between sleeping and awake chicks, possibly due to the very short duration of S episodes. In embryonic chicks, cFos expression was low or absent across all five systems at embryonic day (E) 12. Unexpectedly, a strong activation was seen at E16 in H/O neurons. The highest activation of Ch-LDT/PT (also S-active) and MCH neurons was seen at E20. These data suggest that maturation of arousal systems is achieved soon after hatching, while S-control networks are active in late chick embryos.