Characterization of the neurogenic niche in the aging dentate gyrus using iterative immunofluorescence imaging.

Advancing age causes reduced hippocampal neurogenesis, associated with age-related cognitive decline. The spatial relationship of age-induced alterations in neural stem cells (NSCs) and surrounding cells within the hippocampal niche remains poorly understood due to limitations of antibody-based cellular phenotyping. We established iterative indirect immunofluorescence imaging (4i) in tissue sections, allowing for simultaneous detection of 18 proteins to characterize NSCs and surrounding cells in 2-, 6-, and 12-month-old mice. We show that reorganization of the dentate gyrus (DG) niche already occurs in middle-aged mice, paralleling the decline in neurogenesis. 4i-based tissue analysis of the DG identifies changes in cell-type contributions to the blood-brain barrier and microenvironments surrounding NSCs to play a pivotal role to preserve neurogenic permissiveness. The data provided represent a resource to characterize the principles causing alterations of stem cell-associated plasticity within the aging DG and provide a blueprint to analyze somatic stem cell niches across lifespan in complex tissues.

Development of the mammalian cortical hem and its derivatives: the choroid plexus, Cajal-Retzius cells and hippocampus.

The dorsal medial region of the developing mammalian telencephalon plays a central role in the patterning of the adjacent brain regions. This review describes the development of this specialized region of the vertebrate brain, called the cortical hem, and the formation of the various cells and structures it gives rise to, including the choroid plexus, Cajal-Retzius cells and the hippocampus. We highlight the ontogenic processes that create these different forebrain derivatives from their shared embryonic origin and discuss the key signalling pathways and molecules that influence the patterning of the cortical hem. These include BMP, Wnt, FGF and Shh signalling pathways acting with Homeobox factors to carve the medial telencephalon into district progenitor regions, which in turn give rise to the choroid plexus, dentate gyrus and hippocampus. We then link the formation of the lateral ventricle choroid plexus with embryonic and postnatal neurogenesis in the hippocampus.

An early Sox2-dependent gene expression programme required for hippocampal dentate gyrus development.

The hippocampus is a brain area central for cognition. Mutations in the human SOX2 transcription factor cause neurodevelopmental defects, leading to intellectual disability and seizures, together with hippocampal dysplasia. We generated an allelic series of Sox2 conditional mutations in mouse, deleting Sox2 at different developmental stages. Late Sox2 deletion (from E11.5, via Nestin-Cre) affects only postnatal hippocampal development; earlier deletion (from E10.5, Emx1-Cre) significantly reduces the dentate gyrus (DG), and the earliest deletion (from E9.5, FoxG1-Cre) causes drastic abnormalities, with almost complete absence of the DG. We identify a set of functionally interconnected genes (Gli3, Wnt3a, Cxcr4, p73 and Tbr2), known to play essential roles in hippocampal embryogenesis, which are downregulated in early Sox2 mutants, and (Gli3 and Cxcr4) directly controlled by SOX2; their downregulation provides plausible molecular mechanisms contributing to the defect. Electrophysiological studies of the Emx1-Cre mouse model reveal altered excitatory transmission in CA1 and CA3 regions.

Intermediate progenitors support migration of neural stem cells into dentate gyrus outer neurogenic niches.

The hippocampal dentate gyrus (DG) is a unique brain region maintaining neural stem cells (NCSs) and neurogenesis into adulthood. We used multiphoton imaging to visualize genetically defined progenitor subpopulations in live slices across key stages of mouse DG development, testing decades old static models of DG formation with molecular identification, genetic-lineage tracing, and mutant analyses. We found novel progenitor migrations, timings, dynamic cell-cell interactions, signaling activities, and routes underlie mosaic DG formation. Intermediate progenitors (IPs, Tbr2+) pioneered migrations, supporting and guiding later emigrating NSCs (Sox9+) through multiple transient zones prior to converging at the nascent outer adult niche in a dynamic settling process, generating all prenatal and postnatal granule neurons in defined spatiotemporal order. IPs (Dll1+) extensively targeted contacts to mitotic NSCs (Notch active), revealing a substrate for cell-cell contact support during migrations, a developmental feature maintained in adults. Mouse DG formation shares conserved features of human neocortical expansion.

Decoding the development of the human hippocampus.

The hippocampus is an important part of the limbic system in the human brain that has essential roles in spatial navigation and the consolidation of information from short-term memory to long-term memory1,2. Here we use single-cell RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis to illustrate the cell types, cell linage, molecular features and transcriptional regulation of the developing human hippocampus. Using the transcriptomes of 30,416 cells from the human hippocampus at gestational weeks 16-27, we identify 47 cell subtypes and their developmental trajectories. We also identify the migrating paths and cell lineages of PAX6+ and HOPX+ hippocampal progenitors, and regional markers of CA1, CA3 and dentate gyrus neurons. Multiomic data have uncovered transcriptional regulatory networks of the dentate gyrus marker PROX1. We also illustrate spatially specific gene expression in the developing human prefrontal cortex and hippocampus. The molecular features of the human hippocampus at gestational weeks 16-20 are similar to those of the mouse at postnatal days 0-5 and reveal gene expression differences between the two species. Transient expression of the primate-specific gene NBPF1 leads to a marked increase in PROX1+ cells in the mouse hippocampus. These data provides a blueprint for understanding human hippocampal development and a tool for investigating related diseases.

Transcription Factor 4 Safeguards Hippocampal Dentate Gyrus Development by Regulating Neural Progenitor Migration.

The dentate gyrus (DG) of the hippocampal formation plays essential roles in learning and memory. Defective DG development is associated with neurological disorders. Here, we show that transcription factor 4 (Tcf4) is essential for DG development. Tcf4 expression is elevated in neural progenitors of the dentate neuroepithelium in the developing mouse brain. We demonstrate that conditional disruption of Tcf4 in the dentate neuroepithelium leads to abnormal neural progenitor migration guided by disorganized radial glial fibers, which further leads to hypoplasia in the DG. Moreover, we reveal that Wnt7b is a key downstream effector of Tcf4 in regulating neural progenitor migration. Behavioral analysis shows that disruption of integrity of the DG impairs the social memory highlighting the importance of proper development of the DG. These results reveal a critical role for Tcf4 in regulating DG development. As mutations in TCF4 cause Pitt-Hopkins syndrome (PTHS) characterized by severe intellectual disability, our data also potentially provide insights into the basis of neurological defects linked to TCF4 mutations.

The Microtubule Severing Protein Katanin Regulates Proliferation of Neuronal Progenitors in Embryonic and Adult Neurogenesis.

Microtubule severing regulates cytoskeletal rearrangement underlying various cellular functions. Katanin, a heterodimer, consisting of catalytic (p60) and regulatory (p80) subunits severs dynamic microtubules to modulate several stages of cell division. The role of p60 katanin in the mammalian brain with respect to embryonic and adult neurogenesis is poorly understood. Here, we generated a Katna1 knockout mouse and found that consistent with a critical role of katanin in mitosis, constitutive homozygous Katna1 depletion is lethal. Katanin p60 haploinsufficiency induced an accumulation of neuronal progenitors in the subventricular zone during corticogenesis, and impaired their proliferation in the adult hippocampus dentate gyrus (DG) subgranular zone. This did not compromise DG plasticity or spatial and contextual learning and memory tasks employed in our study, consistent with the interpretation that adult neurogenesis may be associated with selective forms of hippocampal-dependent cognitive processes. Our data identify a critical role for the microtubule-severing protein katanin p60 in regulating neuronal progenitor proliferation in vivo during embryonic development and adult neurogenesis.

Suppressor of fused controls perinatal expansion and quiescence of future dentate adult neural stem cells.

Adult hippocampal neurogenesis requires the quiescent neural stem cell (NSC) pool to persist lifelong. However, establishment and maintenance of quiescent NSC pools during development is not understood. Here, we show that Suppressor of Fused (Sufu) controls establishment of the quiescent NSC pool during mouse dentate gyrus (DG) development by regulating Sonic Hedgehog (Shh) signaling activity. Deletion of Sufu in NSCs early in DG development decreases Shh signaling activity leading to reduced proliferation of NSCs, resulting in a small quiescent NSC pool in adult mice. We found that putative adult NSCs proliferate and increase their numbers in the first postnatal week and subsequently enter a quiescent state towards the end of the first postnatal week. In the absence of Sufu, postnatal expansion of NSCs is compromised, and NSCs prematurely become quiescent. Thus, Sufu is required for Shh signaling activity ensuring expansion and proper transition of NSC pools to quiescent states during DG development.

Temporal Embryonic Origin Critically Determines Cellular Physiology in the Dentate Gyrus.

The dentate gyrus, the entry gate to the hippocampus, comprises 3 types of glutamatergic cells, the granule, the mossy and the semilunar granule cells. Whereas accumulating evidence indicates that specification of subclasses of neocortical neurons starts at the time of their final mitotic divisions, when cellular diversity is specified in the Dentate Gyrus remains largely unknown. Here we show that semilunar cells, like mossy cells, originate from the earliest stages of developmental neurogenesis and that early born neurons form age-matched circuits with each other. Besides morphology, adult semilunar cells display characteristic electrophysiological features that differ from most neurons but are shared among early born granule cells. Therefore, an early birthdate specifies adult granule cell physiology and connectivity whereas additional factors may combine to produce morphological identity.

Gestational stress induced differential expression of HDAC2 in male rat offspring hippocampus during development.

Accumulating evidence from preclinical and clinical studies indicates prenatal exposure to stress or excess glucocorticoids can affect offspring brain. HDAC2 is an important target of glucocorticoid. Here we detected HDAC2 expression in male offspring hippocampus from gestational restraint stressed rat during development and the relationship between HDAC2 expression and behaviors and neurogenesis in male offspring. Pregnant rats received restrained stress during the last week of pregnancy. Expressions of HDAC2 in offspring hippocampus were detected on postnatal 0 day (P0) and 60 days (P60). Neurogenesis was evaluated by Doublecortin (DCX) staining on P60. Anxiety-like behavior and cognition were detected in open field, elevated plus maze, novel object recognition test, and Barnes maze. We found that HDAC2 expression in the hippocampus of male prenatally stressed offspring (MPSO) was similar to the male control offspring on P0, but significantly lower on P60. Corresponding to the decreased expression of HDAC2 in MPSO hippocampus at P60, neurogenesis in the dentate gyrus of MPSO was significantly lower than the control male offspring. And MPSO also showed greater anxiety and poorer learning and memories abilities than control male offspring. These showed that HDAC2 could partly explain the effects of gestational stress on male offspring behaviors.

Dentate Granule Neurons Generated During Perinatal Life Display Distinct Morphological Features Compared With Later-Born Neurons in the Mouse Hippocampus.

In nonhuman mammals and in particular in rodents, most granule neurons of the dentate gyrus (DG) are generated during development and yet little is known about their properties compared with adult-born neurons. Although it is generally admitted that these populations are morphologically indistinguishable once mature, a detailed analysis of developmentally born neurons is lacking. Here, we used in vivo electroporation to label dentate granule cells (DGCs) generated in mouse embryos (E14.5) or in neonates (P0) and followed their morphological development up to 6 months after birth. By comparison with mature retrovirus-labeled DGCs born at weaning (P21) or young adult (P84) stages, we provide the evidence that perinatally born neurons, especially embryonically born cells, are morphologically distinct from later-born neurons and are thus easily distinguishable. In addition, our data indicate that semilunar and hilar GCs, 2 populations in ectopic location, are generated during the embryonic and the neonatal periods, respectively. Thus, our findings provide new insights into the development of the different populations of GCs in the DG and open new questions regarding their function in the brain.

Ectopic neurogenesis induced by prenatal antiepileptic drug exposure augments seizure susceptibility in adult mice.

Epilepsy is a neurological disorder often associated with seizure that affects ∼0.7% of pregnant women. During pregnancy, most epileptic patients are prescribed antiepileptic drugs (AEDs) such as valproic acid (VPA) to control seizure activity. Here, we show that prenatal exposure to VPA in mice increases seizure susceptibility in adult offspring through mislocalization of newborn neurons in the hippocampus. We confirmed that neurons newly generated from neural stem/progenitor cells (NS/PCs) are integrated into the granular cell layer in the adult hippocampus; however, prenatal VPA treatment altered the expression in NS/PCs of genes associated with cell migration, including CXC motif chemokine receptor 4 (Cxcr4), consequently increasing the ectopic localization of newborn neurons in the hilus. We also found that voluntary exercise in a running wheel suppressed this ectopic neurogenesis and countered the enhanced seizure susceptibility caused by prenatal VPA exposure, probably by normalizing the VPA-disrupted expression of multiple genes including Cxcr4 in adult NS/PCs. Replenishing Cxcr4 expression alone in NS/PCs was sufficient to overcome the aberrant migration of newborn neurons and increased seizure susceptibility in VPA-exposed mice. Thus, prenatal exposure to an AED, VPA, has a long-term effect on the behavior of NS/PCs in offspring, but this effect can be counteracted by a simple physical activity. Our findings offer a step to developing strategies for managing detrimental effects in offspring exposed to VPA in utero.

Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults.

New neurons continue to be generated in the subgranular zone of the dentate gyrus of the adult mammalian hippocampus. This process has been linked to learning and memory, stress and exercise, and is thought to be altered in neurological disease. In humans, some studies have suggested that hundreds of new neurons are added to the adult dentate gyrus every day, whereas other studies find many fewer putative new neurons. Despite these discrepancies, it is generally believed that the adult human hippocampus continues to generate new neurons. Here we show that a defined population of progenitor cells does not coalesce in the subgranular zone during human fetal or postnatal development. We also find that the number of proliferating progenitors and young neurons in the dentate gyrus declines sharply during the first year of life and only a few isolated young neurons are observed by 7 and 13 years of age. In adult patients with epilepsy and healthy adults (18-77 years; n = 17 post-mortem samples from controls; n = 12 surgical resection samples from patients with epilepsy), young neurons were not detected in the dentate gyrus. In the monkey (Macaca mulatta) hippocampus, proliferation of neurons in the subgranular zone was found in early postnatal life, but this diminished during juvenile development as neurogenesis decreased. We conclude that recruitment of young neurons to the primate hippocampus decreases rapidly during the first years of life, and that neurogenesis in the dentate gyrus does not continue, or is extremely rare, in adult humans. The early decline in hippocampal neurogenesis raises questions about how the function of the dentate gyrus differs between humans and other species in which adult hippocampal neurogenesis is preserved.

Aberrant Epigenetic Gene Regulation in GABAergic Interneuron Subpopulations in the Hippocampal Dentate Gyrus of Mouse Offspring Following Developmental Exposure to Hexachlorophene.

Maternal hexachlorophene (HCP) exposure causes transient disruption of hippocampal neurogenesis in mouse offspring. We examined epigenetically hypermethylated and downregulated genes related to this HCP-induced disrupted neurogenesis. Mated female mice were dietary exposed to 0 or 100 ppm HCP from gestational day 6 to postnatal day (PND) 21 on weaning. The hippocampal dentate gyrus of male offspring was subjected to methyl-capture sequencing and real-time reverse transcription-polymerase chain reaction analyses on PND 21. Validation analyses on methylation identified three genes, Dlx4, Dmrt1, and Plcb4, showing promoter-region hypermethylation. Immunohistochemically, DLX4+, DMRT1+, and PLCB4+ cells in the dentate hilus co-expressed GAD67, a γ-aminobutyric acid (GABA)ergic neuron marker. HCP decreased all of three subpopulations as well as GAD67+ cells on PND 21. PLCB4+ cells also co-expressed the metabotropic glutamate receptor, GRM1. HCP also decreased transcript level of synaptic plasticity-related genes in the dentate gyrus and immunoreactive granule cells for synaptic plasticity-related ARC. On PND 77, all immunohistochemical cellular density changes were reversed, whereas the transcript expression of the synaptic plasticity-related genes fluctuated. Thus, HCP-exposed offspring transiently reduced the number of GABAergic interneurons. Among them, subpopulations expressing DLX4, DMRT1, or PLCB4 were transiently reduced in number through an epigenetic mechanism. Considering the role of the Dlx gene family in GABAergic interneuron migration and differentiation, the decreased number of DLX4+ cells may be responsible for reducing those GABAergic interneurons regulating neurogenesis. The effect on granule cell synaptic plasticity was sustained until the adult stage, and reduced GABAergic interneurons active in GRM1-PLCB4 signaling may be responsible for the suppression on weaning.

Vascular pattern of the dentate gyrus is regulated by neural progenitors.

Neurogenesis is a vital process that begins during early embryonic development and continues until adulthood, though in the latter case, it is restricted to the subventricular zone and the subgranular zone of the dentate gyrus (DG). In particular, the DG's neurogenic properties are structurally and functionally unique, which may be related to its singular vascular pattern. Neurogenesis and angiogenesis share molecular signals and act synergistically, supporting the concept of a neurogenic niche as a functional unit between neural precursors cells and their environment, in which the blood vessels play an important role. Whereas it is well known that vascular development controls neural proliferation in the embryonary and in the adult brain, by releasing neurotrophic factors; the potential influence of neural cells on vascular components during angiogenesis is largely unknown. We have demonstrated that the reduction of neural progenitors leads to a significant impairment of vascular development. Since VEGF is a potential regulator in the neurogenesis-angiogenesis crosstalk, we were interested in assessing the possible role of this molecule in the hippocampal neurovascular development. Our results showed that VEGF is the molecule involved in the regulation of vascular development by neural progenitor cells in the DG.

NMDA Receptor GluN2 Subtypes Control Epileptiform Events in the Hippocampus.

NMDA receptors (NMDARs) play a key role in synaptic plasticity and excitotoxicity. Subtype-specific role of NMDAR in neural disorders is an emerging area. Recent studies have revealed that mutations in NMDARs are a cause for epilepsy. Hippocampus is a known focal point for epilepsy. In hippocampus, expression of the NMDAR subtypes GluN1/GluN2A and GluN1/GluN2B is temporally regulated. However, the pharmacological significance of these subtypes is not well understood in epileptic context/models. To investigate this, epilepsy was induced in hippocampal slices by the application of artificial cerebrospinal fluid that contained high potassium but no magnesium. Epileptiform events (EFEs) were recorded from the CA1 and DG areas of hippocampus with or without subtype-specific antagonists. Irrespective of the age group, CA1 and DG showed epileptiform activity. The NMDAR antagonist AP5 was found to reduce the number of EFEs significantly. However, the application of subtype-specific antagonists (TCN 201 for GluN1/GluN2A and Ro 25-69811 for GluN1/GluN2B) revealed that EFEs had area-specific and temporal components. In slices from neonates, EFEs in CA1 were effectively reduced by Ro 25-69811, but were largely insensitive to TCN 201. In contrast, EFEs in DG were equally sensitive to both of the subtype-specific antagonists. However, the differential sensitivity for the antagonists observed in neonates was absent in later developmental stages. The study provides a functional insight into the NMDAR subtype-dependent contribution of EFEs in hippocampus of young rats, which may have implications in treating childhood epilepsy and avoiding unnecessary side effects of broad spectrum antagonists.

Dentate granule progenitor cell properties are rapidly altered soon after birth.

Neurogenesis occurs during the embryonic period and ceases soon after birth in the neocortex, but continues to occur in the hippocampus even in the adult. The embryonic neocortex has radial glia or progenitor cells expressing brain lipid-binding protein (BLBP), whereas the adult hippocampus has radial granule progenitor cells expressing BLBP and glial fibrillary acidic protein (GFAP) in the subgranular zone. We previously found that embryonic hippocampal granule progenitor cells express GFAP, but not BLBP, indicating that these cells are different from both embryonic neocortical and adult granule progenitor cells. In the present study, as the first step towards understanding the mechanism of persistent hippocampal neurogenesis, we aimed to determine the stage at which embryonic-type granule progenitors become adult-type progenitors using mouse Gfap-GFP transgenic mice. During the embryonic stages, Gfap-GFP-positive (Gfap-GFP+) cells were distributed in the entire developing dentate gyrus (DG), whereas BLBP-positive (BLBP+) cells were mainly present in the fimbria and subpial region, and to some extent in the DG. Up to postnatal day 0 (P0), double-positive cells were scarcely detected. However, at P1, one-third of the Gfap-GFP+ cells in the DG suddenly began to weakly express BLBP. Thereafter, Gfap-GFP+/BLBP+ cells rapidly increased in number, and extended their radial processes in the inner granular cell layer. At P14 and in the adult, two-thirds of the Gfap-GFP+ cells in the subgranular zone showed BLBP immunoreactivity. These results suggest that the properties of hippocampal granule progenitor cells are rapidly altered from an embryonic to adult type soon after birth.

Autism spectrum disorder-associated genes and the development of dentate granule cells.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by severe clinical symptoms such as the deficiency of the social communication, repetitive and stereotyped behaviors, and restricted interests. Although complex genetic and environmental factors are thought to contribute to the development of ASD, the precise etiologies are largely unknown. Neuroanatomical observations have been made of developmental abnormalities in different brain regions, including dentate gyrus of hippocampus, which is widely accepted as the center for learning and memory. However, little is known about what roles ASD-associated genes play in the development of hippocampal dentate granule cells. In this article, we summarized functions and pathophysiological significance of 6 representative ASD-associated genes, SEMA5A, PTEN, NLGN, EN-2, FMR1, and MECP2, by focusing on the development of dentate gyrus. We then introduced a recently developed gene transfer method directed to neonatal dentate granule cells. This new method will be useful for elucidating physiological as well as pathophysiological significance of ASD-associated genes in the development of hippocampal formation.

COUP-TFI mitotically regulates production and migration of dentate granule cells and modulates hippocampal Cxcr4 expression.

Development of the dentate gyrus (DG), the primary gateway for hippocampal inputs, spans embryonic and postnatal stages, and involves complex morphogenetic events. We have previously identified the nuclear receptor COUP-TFI as a novel transcriptional regulator in the postnatal organization and function of the hippocampus. Here, we dissect its role in DG morphogenesis by inactivating it in either granule cell progenitors or granule neurons. Loss of COUP-TFI function in progenitors leads to decreased granule cell proliferative activity, precocious differentiation and increased apoptosis, resulting in a severe DG growth defect in adult mice. COUP-TFI-deficient cells express high levels of the chemokine receptor Cxcr4 and migrate abnormally, forming heterotopic clusters of differentiated granule cells along their paths. Conversely, high COUP-TFI expression levels downregulate Cxcr4 expression, whereas increased Cxcr4 expression in wild-type hippocampal cells affects cell migration. Finally, loss of COUP-TFI in postmitotic cells leads to only minor and transient abnormalities, and to normal Cxcr4 expression. Together, our results indicate that COUP-TFI is required predominantly in DG progenitors for modulating expression of the Cxcr4 receptor during granule cell neurogenesis and migration.

Late Effect of Developmental Exposure to 3,3'-Iminodipropionitrile on Neurogenesis in the Hippocampal Dentate Gyrus of Mice.

The effects of developmental exposure to 3,3'-iminodipropionitrile (IDPN), a neurotoxicant that causes proximal axonopathy, on mouse hippocampal neurogenesis was examined. Pregnant mice were exposed to IDPN at 0, 600, or 1200 ppm in their drinking water from gestational day 6 to postnatal day (PND) 21. On PND 21, male offspring showed increased postmitotic neuron-specific NeuN-immunoreactive(+) granule cell numbers in the dentate subgranular zone (SGZ) and granule cell layer (GCL) and decreased glutamate receptor gene Grin2d levels in the dentate gyrus at 1200 ppm. On PND 77, decreased numbers were observed for TBR2+ progenitor cells in the SGZ at ≥600 ppm and GFAP+ stem cells, DCX+ progenitor cells and immature granule cells, NeuN+ immature and mature granule cells, PCNA+ proliferating cells in the SGZ and/or GCL, and immunoreactive cells for ARC or FOS, immediate-early gene products related to neuronal and synaptic plasticity, in the GCL at 1200 ppm. Additionally, at 1200 ppm of IDPN, downregulation of Kit, the gene encoding the stem cell factor (SCF) receptor, and upregulation of Kitl, encoding SCF, were observed in the dentate gyrus. Therefore, maternal IDPN exposure in mice affects neurogenesis involving glutamatergic signals at the end of developmental exposure, with late effects suppressing SGZ cell proliferation, reducing the broad range of granule cell lineage population, which may be responsible for SCF receptor downregulation. The upregulated SCF was likely a feedback response to the decreased receptor level. These results suggest that reduced SCF signaling may cause suppressed neuronal and synaptic plasticity.