Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior.

The dentate gyrus of the mammalian hippocampus continuously generates new neurons during adulthood. These adult-born neurons become functionally active and are thought to contribute to learning and memory, especially during their maturation phase, when they have extraordinary plasticity. In this Review, we discuss the molecular machinery involved in the generation of new neurons from a pool of adult neural stem cells and their integration into functional hippocampal circuits. We also summarize the potential functions of these newborn neurons in the adult brain, their contribution to behavior, and their relevance to disease.

Seizure-induced strengthening of a recurrent excitatory circuit in the dentate gyrus is proconvulsant.

Epilepsy is a devastating brain disorder for which effective treatments are very limited. There is growing interest in early intervention, which requires a better mechanistic understanding of the early stages of this disorder. While diverse brain insults can lead to epileptic activity, a common cellular mechanism relies on uncontrolled recurrent excitatory activity. In the dentate gyrus, excitatory mossy cells (MCs) project extensively onto granule cells (GCs) throughout the hippocampus, thus establishing a recurrent MC-GC-MC excitatory loop. MCs are implicated in temporal lobe epilepsy, a common form of epilepsy, but their role during initial seizures (i.e., before the characteristic MC loss that occurs in late stages) is unclear. Here, we show that initial seizures acutely induced with an intraperitoneal kainic acid (KA) injection in adult mice, a well-established model that leads to experimental epilepsy, not only increased MC and GC activity in vivo but also triggered a brain-derived neurotrophic factor (BDNF)-dependent long-term potentiation (LTP) at MC-GC excitatory synapses. Moreover, in vivo induction of MC-GC LTP using MC-selective optogenetic stimulation worsened KA-induced seizures. Conversely, Bdnf genetic removal from GCs, which abolishes LTP, and selective MC silencing were both anticonvulsant. Thus, initial seizures are associated with MC-GC synaptic strengthening, which may promote later epileptic activity. Our findings reveal a potential mechanism of epileptogenesis that may help in developing therapeutic strategies for early intervention.

Enriched Environment Promotes Adult Hippocampal Neurogenesis through FGFRs.

The addition of new neurons to existing neural circuits in the adult brain remains of great interest to neurobiology because of its therapeutic implications. The premier model for studying this process has been the hippocampal dentate gyrus in mice, where new neurons are added to mature circuits during adulthood. Notably, external factors such as an enriched environment (EE) and exercise markedly increase hippocampal neurogenesis. Here, we demonstrate that EE acts by increasing fibroblast growth factor receptor (FGFR) function autonomously within neurogenic cells to expand their numbers in adult male and female mice. FGFRs activated by EE signal through their mediators, FGFR substrate (FRS), to induce stem cell proliferation, and through FRS and phospholipase Cγ to increase the number of adult-born neurons, providing a mechanism for how EE promotes adult neurogenesis.SIGNIFICANCE STATEMENT How the environment we live in affects cognition remains poorly understood. In the current study, we explore the mechanism underlying the effects of an enriched environment on the production of new neurons in the adult hippocampal dentate gyrus, a brain area integral in forming new memories. A mechanism is provided for how neural precursor cells in the adult mammalian dentate gyrus respond to an enriched environment to increase their neurogenic output. Namely, an enriched environment acts on stem and progenitor cells by activating fibroblast growth factor receptor signaling through phospholipase Cγ and FGF receptor substrate proteins to expand the pool of precursor cells.

The Population Tracking Model: A Simple, Scalable Statistical Model for Neural Population Data.

Our understanding of neural population coding has been limited by a lack of analysis methods to characterize spiking data from large populations. The biggest challenge comes from the fact that the number of possible network activity patterns scales exponentially with the number of neurons recorded ([Formula: see text]). Here we introduce a new statistical method for characterizing neural population activity that requires semi-independent fitting of only as many parameters as the square of the number of neurons, requiring drastically smaller data sets and minimal computation time. The model works by matching the population rate (the number of neurons synchronously active) and the probability that each individual neuron fires given the population rate. We found that this model can accurately fit synthetic data from up to 1000 neurons. We also found that the model could rapidly decode visual stimuli from neural population data from macaque primary visual cortex about 65 ms after stimulus onset. Finally, we used the model to estimate the entropy of neural population activity in developing mouse somatosensory cortex and, surprisingly, found that it first increases, and then decreases during development. This statistical model opens new options for interrogating neural population data and can bolster the use of modern large-scale in vivo Ca[Formula: see text] and voltage imaging tools.

What is memory? The present state of the engram.

The mechanism of memory remains one of the great unsolved problems of biology. Grappling with the question more than a hundred years ago, the German zoologist Richard Semon formulated the concept of the engram, lasting connections in the brain that result from simultaneous "excitations", whose precise physical nature and consequences were out of reach of the biology of his day. Neuroscientists now have the knowledge and tools to tackle this question, however, and this Forum brings together leading contemporary views on the mechanisms of memory and what the engram means today.

Paradox of pattern separation and adult neurogenesis: A dual role for new neurons balancing memory resolution and robustness.

Hippocampal adult neurogenesis is thought to subserve pattern separation, the process by which similar patterns of neuronal inputs are transformed into distinct neuronal representations, permitting the discrimination of highly similar stimuli in hippocampus-dependent tasks. However, the mechanism by which immature adult-born dentate granule neurons cells (abDGCs) perform this function remains unknown. Two theories of abDGC function, one by which abDGCs modulate and sparsify activity in the dentate gyrus and one by which abDGCs act as autonomous coding units, are generally suggested to be mutually exclusive. This review suggests that these two mechanisms work in tandem to dynamically regulate memory resolution while avoiding memory interference and maintaining memory robustness.

Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing.

In vivo two-photon calcium imaging would benefit from the use of multiple excitation beams to increase scanning speed, signal-to-noise ratio and field of view or to image different axial planes simultaneously. Using spatiotemporal multiplexing we circumvented light-scattering ambiguity inherent to deep-tissue multifocal two-photon microscopy. We demonstrate calcium imaging at multiple axial planes in the intact mouse brain to monitor network activity of ensembles of cortical neurons in three spatial dimensions.

Delayed formation of neural representations of space in aged mice.

Aging is associated with cognitive deficits, with spatial memory being very susceptible to decline. The hippocampal dentate gyrus (DG) is important for processing spatial information in the brain and is particularly vulnerable to aging, yet its sparse activity has led to difficulties in assessing changes in this area. Using in vivo two-photon calcium imaging, we compared DG neuronal activity and representations of space in young and aged mice walking on an unfamiliar treadmill. We found that calcium activity was significantly higher and less tuned to location in aged mice, resulting in decreased spatial information encoded in the DG. However, with repeated exposure to the same treadmill, both spatial tuning and information levels in aged mice became similar to young mice, while activity remained elevated. Our results show that spatial representations of novel environments are impaired in the aged hippocampus and gradually improve with increased familiarity. Moreover, while the aged DG is hyperexcitable, this does not disrupt neural representations of familiar environments.

Delayed formation of neural representations of space in aged mice.

Aging is associated with cognitive deficits, with spatial memory being very susceptible to decline. The hippocampal dentate gyrus (DG) is important for processing spatial information in the brain and is particularly vulnerable to aging, yet its sparse activity has led to difficulties in assessing changes in this area. Using in vivo two-photon calcium imaging, we compared DG neuronal activity and representations of space in young and aged mice walking on an unfamiliar treadmill. We found that calcium activity was significantly higher and less tuned to location in aged mice, resulting in decreased spatial information encoded in the DG. However, with repeated exposure to the same treadmill, both spatial tuning and information levels in aged mice became similar to young mice, while activity remained elevated. Our results show that spatial representations of novel environments are impaired in the aged hippocampus and gradually improve with increased familiarity. Moreover, while the aged DG is hyperexcitable, this does not disrupt neural representations of familiar environments.

Transcriptional regulation of neural stem cell expansion in the adult hippocampus.

Experience governs neurogenesis from radial-glial neural stem cells (RGLs) in the adult hippocampus to support memory. Transcription factors (TFs) in RGLs integrate physiological signals to dictate self-renewal division mode. Whereas asymmetric RGL divisions drive neurogenesis during favorable conditions, symmetric divisions prevent premature neurogenesis while amplifying RGLs to anticipate future neurogenic demands. The identities of TFs regulating RGL symmetric self-renewal, unlike those that regulate RGL asymmetric self-renewal, are not known. Here, we show in mice that the TF Kruppel-like factor 9 (Klf9) is elevated in quiescent RGLs and inducible, deletion of Klf9 promotes RGL activation state. Clonal analysis and longitudinal intravital two-photon imaging directly demonstrate that Klf9 functions as a brake on RGL symmetric self-renewal. In vivo translational profiling of RGLs lacking Klf9 generated a molecular blueprint for RGL symmetric self-renewal that was characterized by upregulation of genetic programs underlying Notch and mitogen signaling, cell cycle, fatty acid oxidation, and lipogenesis. Together, these observations identify Klf9 as a transcriptional regulator of neural stem cell expansion in the adult hippocampus.

In humans and other mammals, a region of the brain known as the hippocampus plays important roles in memory. New experiences guide cells in the hippocampus known as radial-glial neural stem cells (RGLs) to divide to make new neurons and other types of cells involved in forming memories. Each time an RGL divides, it can choose to divide asymmetrically to maintain a copy of itself and make a new cell of another type, or divide symmetrically (a process known as symmetric self-renewal) to produce two RGLs. Symmetric self-renewal helps to restore and replenish the pool of stem cells in the hippocampus that are lost due to injury or age, allowing us to continue making new neurons. Proteins known as transcription factors are believed to control how RGLs divide. Previous studies have identified several transcription factors that regulate the RGLs splitting asymmetrically to make neurons and other cells. But the identities of the transcription factors that regulate symmetric self-renewal in the adult hippocampus have remained elusive. Here, Guo et al. searched for transcription factors that regulate symmetric self-renewal of RGLs in mice. The experiments found that RGLs that are resting and not dividing (referred to as 'quiescent') have higher levels of a transcription factor called Klf9 than RGLs that are actively dividing. Loss of the gene encoding Klf9 triggered quiescent RGLs to start dividing, and further experiments showed that Klf9 directly inhibited symmetric self-renewal. Guo et al. then used an approach called in vivo translational profiling to generate a blueprint that revealed new insights into the molecular processes involved in this symmetric division. These findings pave the way for researchers to develop strategies that may expand the numbers of stem cells in the hippocampus. This could eventually be used to help replenish brain circuits with neurons and improve the memory of individuals with Alzheimer's disease or other conditions that cause memory loss.

Donor-derived vasculature is required to support neocortical cell grafts after stroke.

Neural precursor cells (NPCs) transplanted into the adult neocortex generate neurons that synaptically integrate with host neurons, supporting the possibility of achieving functional tissue repair. However, poor survival and functional neuronal recovery of transplanted NPCs greatly limits engraftment. Here, we test the hypothesis that combining blood vessel-forming vascular cells with neuronal precursors improves engraftment. By transplanting mixed embryonic neocortical cells into adult mice with neocortical strokes, we show that transplant-derived neurons synapse with appropriate targets while donor vascular cells form vessels that fuse with the host vasculature to perfuse blood within the graft. Although all grafts became vascularized, larger grafts had greater contributions of donor-derived vessels that increased as a function of their distance from the host-graft border. Moreover, excluding vascular cells from the donor cell population strictly limited graft size. Thus, inclusion of vessel-forming vascular cells with NPCs is required for more efficient engraftment and ultimately for tissue repair.

Treadmill-based task for assessing spatial memory in head-fixed mice.

Several mouse in vivo neuronal recording techniques require head fixation. Head-fixed treadmill walking can be used to design tasks that enable the study of neural activity in the context of behavior. Here, we provide a detailed protocol for constructing a treadmill with tactile spatial cues, training mice on a rewarded behavioral task, and analyzing behavioral data. We discuss common problems and solutions we have developed to optimize training. Finally, we demonstrate how to test spatial memory performance using this task.

AAV ablates neurogenesis in the adult murine hippocampus.

Recombinant adeno-associated virus (rAAV) has been widely used as a viral vector across mammalian biology and has been shown to be safe and effective in human gene therapy. We demonstrate that neural progenitor cells (NPCs) and immature dentate granule cells (DGCs) within the adult murine hippocampus are particularly sensitive to rAAV-induced cell death. Cell loss is dose dependent and nearly complete at experimentally relevant viral titers. rAAV-induced cell death is rapid and persistent, with loss of BrdU-labeled cells within 18 hr post-injection and no evidence of recovery of adult neurogenesis at 3 months post-injection. The remaining mature DGCs appear hyperactive 4 weeks post-injection based on immediate early gene expression, consistent with previous studies investigating the effects of attenuating adult neurogenesis. In vitro application of AAV or electroporation of AAV2 inverted terminal repeats (ITRs) is sufficient to induce cell death. Efficient transduction of the dentategyrus (DG)- without ablating adult neurogenesis- can be achieved by injection of rAAV2-retro serotyped virus into CA3. rAAV2-retro results in efficient retrograde labeling of mature DGCs and permits in vivo two-photon calcium imaging of dentate activity while leaving adult neurogenesis intact. These findings expand on recent reports implicating rAAV-linked toxicity in stem cells and other cell types and suggest that future work using rAAV as an experimental tool in the DG and as a gene therapy for diseases of the central nervous system should be carefully evaluated.

Internally mediated developmental desynchronization of neocortical network activity.

During neocortical development, neurons exhibit highly synchronized patterns of spontaneous activity, with correlated bursts of action potential firing dominating network activity. This early activity is eventually replaced by more sparse and decorrelated firing of cortical neurons, which modeling studies predict is a network state that is better suited for efficient neural coding. The precise time course and mechanisms of this crucial transition in cortical network activity have not been characterized in vivo. We used in vivo two-photon calcium imaging in combination with whole-cell recordings in both unanesthetized and anesthetized mice to monitor how spontaneous activity patterns in ensembles of layer 2/3 neurons of barrel cortex mature during postnatal development. We find that, as early as postnatal day 4, activity is highly synchronous within local clusters of neurons. At the end of the second postnatal week, neocortical networks undergo a transition to a much more desynchronized state that lacks a clear spatial structure. Strikingly, deprivation of sensory input from the periphery had no effect on the time course of this transition. Therefore, developmental desynchronization of spontaneous neuronal activity is a fundamental network transition in the neocortex that appears to be intrinsically generated.

An in vivo model of functional and vascularized human brain organoids.

Differentiation of human pluripotent stem cells to small brain-like structures known as brain organoids offers an unprecedented opportunity to model human brain development and disease. To provide a vascularized and functional in vivo model of brain organoids, we established a method for transplanting human brain organoids into the adult mouse brain. Organoid grafts showed progressive neuronal differentiation and maturation, gliogenesis, integration of microglia, and growth of axons to multiple regions of the host brain. In vivo two-photon imaging demonstrated functional neuronal networks and blood vessels in the grafts. Finally, in vivo extracellular recording combined with optogenetics revealed intragraft neuronal activity and suggested graft-to-host functional synaptic connectivity. This combination of human neural organoids and an in vivo physiological environment in the animal brain may facilitate disease modeling under physiological conditions.

Beyond excitation/inhibition imbalance in multidimensional models of neural circuit changes in brain disorders.

A leading theory holds that neurodevelopmental brain disorders arise from imbalances in excitatory and inhibitory (E/I) brain circuitry. However, it is unclear whether this one-dimensional model is rich enough to capture the multiple neural circuit alterations underlying brain disorders. Here, we combined computational simulations with analysis of in vivo two-photon Ca2+ imaging data from somatosensory cortex of Fmr1 knock-out (KO) mice, a model of Fragile-X Syndrome, to test the E/I imbalance theory. We found that: (1) The E/I imbalance model cannot account for joint alterations in the observed neural firing rates and correlations; (2) Neural circuit function is vastly more sensitive to changes in some cellular components over others; (3) The direction of circuit alterations in Fmr1 KO mice changes across development. These findings suggest that the basic E/I imbalance model should be updated to higher dimensional models that can better capture the multidimensional computational functions of neural circuits.

In vivo imaging of dendritic pruning in dentate granule cells.

We longitudinally imaged the developing dendrites of adult-born mouse dentate granule cells (DGCs) in vivo and found that they underwent over-branching and pruning. Exposure to an enriched environment and constraint of dendritic growth by disrupting Wnt signaling led to increased branch addition and accelerated growth, which were, however, counteracted by earlier and more extensive pruning. Our results indicate that pruning is regulated in a homeostatic fashion to oppose excessive branching and promote a similar dendrite structure in DGCs.

Directly Reprogrammed Human Neurons Retain Aging-Associated Transcriptomic Signatures and Reveal Age-Related Nucleocytoplasmic Defects.

Aging is a major risk factor for many human diseases, and in vitro generation of human neurons is an attractive approach for modeling aging-related brain disorders. However, modeling aging in differentiated human neurons has proved challenging. We generated neurons from human donors across a broad range of ages, either by iPSC-based reprogramming and differentiation or by direct conversion into induced neurons (iNs). While iPSCs and derived neurons did not retain aging-associated gene signatures, iNs displayed age-specific transcriptional profiles and revealed age-associated decreases in the nuclear transport receptor RanBP17. We detected an age-dependent loss of nucleocytoplasmic compartmentalization (NCC) in donor fibroblasts and corresponding iNs and found that reduced RanBP17 impaired NCC in young cells, while iPSC rejuvenation restored NCC in aged cells. These results show that iNs retain important aging-related signatures, thus allowing modeling of the aging process in vitro, and they identify impaired NCC as an important factor in human aging.

Circuit level defects in the developing neocortex of Fragile X mice.

Subtle alterations in how cortical network dynamics are modulated by different behavioral states could disrupt normal brain function and underlie symptoms of neuropsychiatric disorders, including Fragile X syndrome (FXS). Using two-photon calcium imaging and electrophysiology, we recorded spontaneous neuronal ensemble activity in mouse somatosensory cortex. Unanesthetized Fmr1(-/-) mice exhibited abnormally high synchrony of neocortical network activity, especially during the first two postnatal weeks. Neuronal firing rates were threefold higher in Fmr1(-/-) mice than in wild-type mice during whole-cell recordings manifesting Up/Down states (slow-wave sleep, quiet wakefulness), probably as a result of a higher firing probability during Up states. Combined electroencephalography and calcium imaging experiments confirmed that neurons in mutant mice had abnormally high firing and synchrony during sleep. We conclude that cortical networks in FXS are hyperexcitable in a brain state-dependent manner during a critical period for experience-dependent plasticity. These state-dependent network defects could explain the intellectual, sleep and sensory integration dysfunctions associated with FXS.