RESUMO
The hippocampus hosts the continuous addition of new neurons throughout life-a phenomenon named adult hippocampal neurogenesis (AHN). Here we revisit the occurrence of AHN in more than 110 mammalian species, including humans, and discuss the further validation of these data by single-cell RNAseq and other alternative techniques. In this regard, our recent studies have addressed the long-standing controversy in the field, namely whether cells positive for AHN markers are present in the adult human dentate gyrus (DG). Here we review how we developed a tightly controlled methodology, based on the use of high-quality brain samples (characterized by short postmortem delays and ≤24 h of fixation in freshly prepared 4% paraformaldehyde), to address human AHN. We review that the detection of AHN markers in samples fixed for 24 h required mild antigen retrieval and chemical elimination of autofluorescence. However, these steps were not necessary for samples subjected to shorter fixation periods. Moreover, the detection of labile epitopes (such as Nestin) in the human hippocampus required the use of mild detergents. The application of this strictly controlled methodology allowed reconstruction of the entire AHN process, thus revealing the presence of neural stem cells, proliferative progenitors, neuroblasts, and immature neurons at distinct stages of differentiation in the human DG. The data reviewed here demonstrate that methodology is of utmost importance when studying AHN by means of distinct techniques across the phylogenetic scale. In this regard, we summarize the major findings made by our group that emphasize that overlooking fundamental technical principles might have consequences for any given research field.