Date Published: January 31, 2017
Publisher: Public Library of Science
Author(s): Olesya T. Shevchouk, Gregory F. Ball, Charlotte A. Cornil, Jacques Balthazart, Paul A. Bartell.
In songbirds, neurogenesis in the song control nucleus HVC is sensitive to the hormonal and social environment but the dynamics of this process is difficult to assess with a single exogenous marker of new neurons. We simultaneously used three independent markers to investigate HVC neurogenesis in male and female canaries. Males were castrated, implanted with testosterone and housed either alone (M), with a female (M-F) or with another male (M-M) while females were implanted with 17β-estradiol and housed with a male (F-M). All subjects received injections of the two thymidine analogues, BrdU and of EdU, respectively 21 and 10 days before brain collection. Cells containing BrdU or EdU or expressing doublecortin (DCX), which labels newborn neurons, were quantified. Social context and sex differentially affected total BrdU+, EdU+, BrdU+EdU- and DCX+ populations. M-M males had a higher density of BrdU+ cells in the ventricular zone adjacent to HVC and of EdU+ in HVC than M-F males. M birds had a higher ratio of BrdU+EdU- to EdU+ cells than M-F subjects suggesting higher survival of newer neurons in the former group. Total number of HVC DCX+ cells was lower in M-F than in M-M males. Sex differences were also dependent of the type of marker used. Several technical limitations associated with the use of these multiple markers were also identified. These results indicate that proliferation, recruitment and survival of new neurons can be independently affected by environmental conditions and effects can only be fully discerned through the use of multiple neurogenesis markers.
Adult neurogenesis was first discovered in the rat hippocampus , however, it was a series of experiments in songbirds that conclusively demonstrated the production, functional integration and electrophysiological activity of newborn neurons in the adult brain , triggering a new wave of interest in the phenomenon. Songbirds continue to be a useful model for the study of adult neurogenesis due to some unique features of the phenomenon in this taxon such as widespread migration of newborn neurons throughout the telencephalon, higher rates of proliferation than in mammals and the establishment of long-distance projections made by the newborn neurons in certain cases [3,4]. One specific neurogenic region, the song control nucleus HVC (used as a proper name), is of particular interest due to its important and specific role in the regulation of song behavior. By investigating the regulation of HVC neurogenesis, we can not only gain insight into the molecular and cellular aspects of this process, but also probe for the function of adult neurogenesis.
Most studies on adult songbird neurogenesis to have date employed a single proliferation marker. However due to the limitations of each marker, these investigations could be missing valuable information about the dynamics and regulation of neurogenesis. BrdU and other exogenous markers label small populations of cells born at specific times immediately after injections, doublecortin labels a broad population of neurons born over a large period before brain collection, although some disagreement exists concerning how long this labeling will last (see [14,69,70]). Exogenous markers are non-specific as regards the cell type that they label (in the brain they label both new neurons and glial/endothelial cells even if the former are more numerous, >70% of the total than the latter based on the co-localization with DCX; see  for discussion concerning the HVC of canaries) but largely label in a specific manner newly born cells. On the other hand doublecortin is neuron-specific but may also label neurons undergoing other types of plasticity. To exploit the advantages of both approaches, we combined here doublecortin and two exogenous markers, EdU and BrdU, to investigate effects of social context on HVC neurogenesis in male and female canaries. Each approach revealed substantially divergent patterns of neurogenesis as a function of the social condition or the sex of the birds.
These results, despite their limited power related to small final sample sizes, demonstrate that the use of multiple markers is a very useful tool to understand the complexities of environmental influences on HVC neurogenesis. A limited number of endogenous markers have been validated for use in songbirds, including doublecortin that is particularly useful because it is neuron-specific and discriminates two different stages of neuroblast development, especially when combined with different analogues of thymidine which enable us to follow the trajectories of newborn neuron populations born at a specific time relative to the treatments administrated. Technical problems are however associated with the simultaneous use of multiple thymidine analogs including cross-reactivity in their detection and potential toxicity of EdU that should only be used as a marker of proliferation and injected less than 24 hours before brain collection. Even with these limitations, the present data suggest that proliferation, recruitment and survival of new neurons can be independently affected by environmental conditions with DCX providing cumulative information not necessarily reflected in measures of single new populations (BrdU+ or EdU+).