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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.

Adult hippocampal neurogenesis (AHN) gives rise to new neurons throughout life. This phenomenon takes place in more than 120 mammalian species, including humans, yet its occurrence in the latter was questioned after one study proposed the putative absence of neurogenesis markers in the adult human hippocampus. In this regard, we showed that prolonged fixation impedes the visualization of Doublecortin immature neurons in this structure, whereas other authors have suggested that a dilated post-mortem delay (PMD) underlies these discrepancies. Nevertheless, the individual and/or additive contribution of fixation and the PMD to the detection (or lack thereof) of other AHN markers has not been studied to date. To address this pivotal question, we used a tightly controlled experimental design in mice, which allowed the dissection of the relative contribution of the aforementioned factors to the visualization of markers of individual AHN stages. Fixation time emerged as the most prominent factor globally impeding the study of this process in mice. Moreover, the visualization of other particularly sensitive epitopes was further prevented by prolonged PMD. These results are crucial to disambiguate current controversies related to the occurrence of AHN not only in humans but also in other mammalian species.

The rodent hippocampus generates new neurons throughout life. This process, named adult hippocampal neurogenesis (AHN), is a striking form of neural plasticity that occurs in the brains of numerous mammalian species. Direct evidence of adult neurogenesis in humans has remained elusive, although the occurrence of this phenomenon in the human dentate gyrus has been demonstrated in seminal studies and recent research that have applied distinct approaches to birthdate newly generated neurons and to validate markers of adult-born neurons. Our data point to the persistence of AHN until the 10th decade of human life, as well as to marked impairments in this process in patients with Alzheimer's disease. Moreover, our work demonstrates that the methods used to process and analyze postmortem human brain samples can limit the detection of various markers of AHN to the point of making them undetectable. In this Dual Perspectives article, we highlight the critical methodological aspects that should be strictly controlled in human studies and the robust evidence that supports the occurrence of AHN in humans. We also put forward reasons that may account for current discrepancies on this topic. Finally, the unresolved questions and future challenges awaiting the field are highlighted.

Adult neurogenesis occurs in a few selected regions of the mammalian brain. One such region is the hippocampus, the so-called gateway to memory, where adult hippocampal neurogenesis (AHN) occurs. Here, we provide a comprehensive description of the methods used in our laboratory to unambiguously detect a population of immature neurons in the human hippocampus until the 10th decade of life. The criteria used to refine and develop the current protocol include obtaining post-mortem human samples of remarkable quality and under tightly controlled conditions for immunohistochemistry (IHC) studies, optimizing tissue processing and histological procedures, establishing criteria to reliably validate antibody signal and performing unbiased stereological cell counts. Moreover, we provide a detailed description of the parameters that, in our view, should be reported in human AHN studies. The opposing results obtained by introducing slight variations in the methodological conditions should be considered by future studies that seek to increase our knowledge of this fascinating process. By applying simple and inexpensive tissue pre-treatments, this protocol, which can be completed in 7 days, might be applicable to a variety of IHC studies performed on other tissues of human (or animal) origin.

Frontotemporal dementia (FTD) is characterized by neuronal loss in the frontal and temporal lobes of the brain. Here, we provide the first evidence of striking morphological alterations in dentate granule cells (DGCs) of FTD patients and in a mouse model of the disease, TauVLW mice. Taking advantage of the fact that the hippocampal dentate gyrus (DG) gives rise to newborn DGCs throughout the lifetime in rodents, we used RGB retroviruses to study the temporary course of these alterations in newborn DGCs of female TauVLW mice. In addition, retroviruses that encode either PSD95:GFP or Syn:GFP revealed striking alterations in the afferent and efferent connectivity of newborn TauVLW DGCs, and monosynaptic retrograde rabies virus tracing showed that these cells are disconnected from distal brain regions and local sources of excitatory innervation. However, the same cells exhibited a predominance of local inhibitory innervation. Accordingly, the expression of presynaptic and postsynaptic markers of inhibitory synapses was markedly increased in the DG of TauVLW mice and FTD patients. Moreover, an increased number of neuropeptide Y-positive interneurons in the DG correlated with a reduced number of activated egr-1+ DGCs in TauVLW mice. Finally, we tested the therapeutic potential of environmental enrichment and chemoactivation to reverse these alterations in mice. Both strategies reversed the morphological alterations of newborn DGCs and partially restored their connectivity in a mouse model of the disease. Moreover, our data point to remarkable morphological similarities between the DGCs of TauVLW mice and FTD patients.SIGNIFICANCE STATEMENT We show, for the first time to our knowledge, that the population of dentate granule cells is disconnected from other regions of the brain in the neurodegenerative disease frontotemporal dementia (FTD). These alterations were observed in FTD patients and in a mouse model of this disease. Moreover, we tested the therapeutic potential of

The hippocampus is one of the most affected areas in Alzheimer’s disease (AD) . Moreover, this structure hosts one of the most unique phenomena of the adult mammalian brain, namely, the addition of new neurons throughout life . This process, called adult hippocampal neurogenesis (AHN), confers an unparalleled degree of plasticity to the entire hippocampal circuitry . Nonetheless, direct evidence of AHN in humans has remained elusive. Thus, determining whether new neurons are continuously incorporated into the human dentate gyrus (DG) during physiological and pathological aging is a crucial question with outstanding therapeutic potential. By combining human brain samples obtained under tightly controlled conditions and state-of-the-art tissue processing methods, we identified thousands of immature neurons in the DG of neurologically healthy human subjects up to the ninth decade of life. These neurons exhibited variable degrees of maturation along differentiation stages of AHN. In sharp contrast, the number and maturation of these neurons progressively declined as AD advanced. These results demonstrate the persistence of AHN during both physiological and pathological aging in humans and provide evidence for impaired neurogenesis as a potentially relevant mechanism underlying memory deficits in AD that might be amenable to novel therapeutic strategies.