Byline: Ruo-Xu. Liu, Jie. Ma, Bin. Wang, Tian. Tian, Ning. Guo, Shao-Jun. Liu
Whether endogenous neurogenesis occurs in the adult cortex remains controversial. An increasing number of reports suggest that doublecortin (DCX)-positive neurogenesis persists in the adult primate cortex, attracting enormous attention worldwide. In this study, different DCX antibodies were used together with NeuN antibodies in immunohistochemistry and western blot assays using adjacent cortical sections from adult monkeys. Antibody adsorption, antigen binding, primary antibody omission and antibody-free experiments were used to assess specificity of the signals. We found either strong fluorescent signals, medium-weak intensity signals in some cells, weak signals in a few perikarya or near complete lack of labeling in adjacent cortical sections incubated with the various DCX antibodies. The putative DCX-positive cells in the cortex were also positive for NeuN, a specific marker of mature neurons. However, further experiments showed that most of these signals were either the result of antibody cross reactivity, the non-specificity of secondary antibodies or tissue autofluorescence. No confirmed DCX-positive cells were detected in the adult macaque cortex by immunofluorescence. Our findings show that DCX-positive neurogenesis does not occur in the cerebral cortex of adult primates, and that false-positive signals (artefacts) are caused by antibody cross reactivity and autofluorescence. The experimental protocols were approved by the Institutional Animal Care and Use Committee of the Institute of Neuroscience, Beijing, China (approval No. IACUC-AMMS-2014-501).
Because of the importance of the neocortex and its intricate connections, endogenous neurogenesis in the cerebral cortex of adult primates has attracted enormous attention worldwide for the treatment of neurological disease and aging. Previous studies using BrdU labeling reported endogenous neurogenesis in the cerebral cortex of adult primates (Gould et al., 1999; He et al., 2019). However, Kornack and Rakic showed that the so-called endogenous neurogenesis in the adult primate cortex was caused by the spatial overlap of BrdU-positive nuclei and the perikarya of NeuN-positive neurons, leading to the wrong conclusion (Kornack and Rakic, 2001; Rakic, 2002). Numerous reports have subsequently described the presence or absence of endogenous neurogenesis in the cerebral cortex of nonhuman primates by doublecortin (DCX) immunolabeling (Qin et al., 2000; Fung et al., 2011; Nacher and Bonfanti, 2015).
DCX is a microtubule and actin filament-associated protein (Dhaliwal et al., 2015; Moslehi et al., 2017; Han et al., 2018; Zhou et al., 2019). Because of its specific expression in neural precursors and newly-generated immature neurons in many brain regions of both developing and adult mammals, DCX has been used as a specific marker of neural precursors and immature neurons to evaluate potential endogenous neurogenesis in the adult brain (Francis et al., 1999; Gleeson et al., 1999; Brown et al., 2003; Schaar et al., 2004; Tsukada et al., 2005; Fournier et al., 2018; Shahsavani et al., 2018). Using DCX immunolabeling, a few studies reported that DCX-positive immature neurons are not detectable in the cerebral cortex of adult primates (Bonfanti and Nacher, 2012; Stanton et al., 2015). However, the overwhelming majority of studies have reported that DCX-positive cells persist in...