Evaluating the predictive value of doublecortin as a marker for adult neurogenesis in canaries (Serinus canaria)

Abstract

Doublecortin (DCX) is an important microtubule-associated protein involved in the migration of young neurons into the cortical layers of the brain during early human development. The continued expression of DCX in brain areas with protracted neuron recruitment has promoted this endogenous protein as a popular indirect tool to monitor adult neurogenesis in a variety of species. However, little is known about its possible involvement in other cellular processes and a thorough validation of DCX as a quantitative measure for neurogenesis is generally lacking. Here we investigated the relationship between DCX expression and neuron recruitment in the brains of adult canaries (Serinus canaria), a species well-known for its adult neurogenesis. We examined the age and functional state of DCX-labeled cells by using mitotic and neuron-specific markers, retrograde tracings, and immediate early gene colocalizations. Although DCX expression was high in brain areas implicated in adult neurogenesis, DCX-expressing neurons were also abundant in regions that do not recruit new neurons. Moreover, DCX expression was observed in adult, active neurons, differentiated projection neurons, and birth-dated neurons of up to 1 year of age. Season and testosterone treatment affected DCX expression in two song control nuclei, HVC and Area X, but did not correlate with known patterns of neuron recruitment. Together, these results demonstrate that DCX expression is not exclusive to young migrating neurons, and does not predict neuron recruitment equally throughout the canary brain. Therefore, DCX labeling needs careful validation for each brain region separately in each species analyzed when used to quantify adult neurogenesis.

What Makes a Marker a Good Marker?

“…This is not the case, however, as we observed clear regional differences in the relationship between the two labeling methods in neurogenic and nonneurogenic brain areas in our study [Vellema et al, 2014: fig. 7B].…”

“…Similarly, we also observed DCX-labeling in mature neurons throughout the adult canary brain, including regions in the midbrain and hindbrain that have not previously been documented to recruit new neurons [Vellema et al, 2014]. The suggestion of Balthazart and Ball [2014] that these DCX-expressing mid-and hindbrain regions may constitute 'undiscovered' neurogenic brain areas seems unlikely, and over 30 years of neurogenesis research in the canary brain do not support it.…”

“…The suggestion of Balthazart and Ball [2014] that these DCX-expressing mid-and hindbrain regions may constitute 'undiscovered' neurogenic brain areas seems unlikely, and over 30 years of neurogenesis research in the canary brain do not support it. It is possible to detect small numbers of BrdU-labeled cells in the songbird mid-and hindbrain after BrdU injection, but we have demonstrated before that these BrdU-labeled cells are not associated with neuron recruitment and are not immunoreactive for DCX or other neuronal markers [Vellema et al, 2010[Vellema et al, , 2014.…”

“…For an antibody to be useful as a molecular marker, it needs to specifically and selectively identify the object of interest. The validation process to determine if a marker is specific and selective is often laborious but is highly essential for establishing whether a new marker qualifies as a reliable tool.In a critical review of our work, Balthazart and Ball [2014] argue that the microtubule-associated protein doublecortin (DCX) qualifies as a reliable marker for quantifying adult neurogenesis in canaries, despite observations that DCX is expressed in mature neurons in both neurogenic and nonneurogenic brain regions in canaries [Vellema et al, 2014] and mammals [e.g. …”

A Method for Exploring Adult Neurogenesis in the Songbird Brain

“…Recently, doublecortin (DCX) has been shown to be expressed transiently in young neurons (Balthazart et al 2008), providing a method for measuring relatively long-term changes in neuron addition without the need for prior, in vivo exposure to cell division markers-an attribute attractive for studies of wild birds where capture-recapture is needed when measuring conventional cell birth markers. However, recent work indicates that in some brain regions, DCX continues to be expressed at cell ages of 4-12 mo, long after they have differentiated and established projections (Vellema et al 2014), raising concerns about the utility of DCX as a marker of recently formed neurons. Moreover, in many cases it is desirable to follow a cohort of cells formed in discrete time windows.…”