Chronic in vivo imaging defines age-dependent alterations of neurogenesis in the mouse hippocampus.

Neural stem cells (NSCs) generate new neurons throughout life in the mammalian hippocampus1. Advancing age leads to a decline in neurogenesis, which is associated with impaired cognition2,3. The cellular mechanisms causing reduced neurogenesis with advancing age remain largely unknown. We genetically labeled NSCs through conditional recombination driven by the regulatory elements of the stem-cell-expressed gene GLI family zinc finger 1 (Gli1) and used chronic intravital imaging to follow individual NSCs and their daughter cells over months within their hippocampal niche4,5. We show that aging affects multiple steps, from cell cycle entry of quiescent NSCs to determination of the number of surviving cells, ultimately causing reduced clonal output of individual NSCs. Thus, we here define the developmental stages that may be targeted to enhance neurogenesis with the aim of maintaining hippocampal plasticity with advancing age.

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.

Long-term self-renewing stem cells in the adult mouse hippocampus identified by intravital imaging.

Neural stem cells (NSCs) generate neurons throughout life in the mammalian hippocampus. However, the potential for long-term self-renewal of individual NSCs within the adult brain remains unclear. We used two-photon microscopy and followed NSCs that were genetically labeled through conditional recombination driven by the regulatory elements of the stem cell-expressed genes GLI family zinc finger 1 (Gli1) or achaete-scute homolog 1 (Ascl1). Through intravital imaging of NSCs and their progeny, we identify a population of Gli1-targeted NSCs showing long-term self-renewal in the adult hippocampus. In contrast, once activated, Ascl1-targeted NSCs undergo limited proliferative activity before they become exhausted. Using single-cell RNA sequencing, we show that Gli1- and Ascl1-targeted cells have highly similar yet distinct transcriptional profiles, supporting the existence of heterogeneous NSC populations with diverse behavioral properties. Thus, we here identify long-term self-renewing NSCs that contribute to the generation of new neurons in the adult hippocampus.

Adult-Born Hippocampal Neurons Undergo Extended Development and Are Morphologically Distinct from Neonatally-Born Neurons.

During immature stages, adult-born neurons pass through critical periods for survival and plasticity. It is generally assumed that by 2 months of age adult-born neurons are mature and equivalent to the broader neuronal population, raising questions of how they might contribute to hippocampal function in old age when neurogenesis has declined. However, few have examined adult-born neurons beyond the critical period or directly compared them to neurons born in infancy. Here, we used a retrovirus to visualize functionally relevant morphological features of 2- to 24-week-old adult-born neurons in male rats. From 2 to 7 weeks, neurons grew and attained a relatively mature phenotype. However, several features of 7-week-old neurons suggested a later wave of growth: these neurons had larger nuclei, thicker dendrites, and more dendritic filopodia than all other groups. Indeed, between 7 and 24 weeks, adult-born neurons gained additional dendritic branches, formed a second primary dendrite, acquired more mushroom spines, and had enlarged mossy fiber presynaptic terminals. Compared with neonatal-born neurons, old adult-born neurons had greater spine density, larger presynaptic terminals, and more putative efferent filopodial contacts onto inhibitory neurons. By integrating rates of cell birth and growth across the life span, we estimate that adult neurogenesis ultimately produces half of the cells and the majority of spines in the dentate gyrus. Critically, protracted development contributes to the plasticity of the hippocampus through to the end of life, even after cell production declines. Persistent differences from neonatal-born neurons may additionally endow adult-born neurons with unique functions even after they have matured.SIGNIFICANCE STATEMENT Neurogenesis occurs in the hippocampus throughout adult life and contributes to memory and emotion. It is generally assumed that new neurons have the greatest impact on behavior when they are immature and plastic. However, since neurogenesis declines dramatically with age, it is unclear how they might contribute to behavior later in life when cell proliferation has slowed. Here we find that newborn neurons mature over many months in rats and may end up with distinct morphological features compared with neurons born in infancy. Using a mathematical model, we estimate that a large fraction of neurons is added in adulthood. Moreover, their extended growth produces a reserve of plasticity that persists even after neurogenesis has declined to low rates.

A combination of running and memantine increases neurogenesis and reduces activation of developmentally-born dentate granule neurons in rats.

During hippocampal-dependent memory formation, sensory signals from the neocortex converge in the dentate gyrus. It is generally believed that the dentate gyrus decorrelates inputs in order to minimize interference between codes for similar experiences, often referred to as pattern separation. The proportion of dentate neurons that are activated by experience is therefore likely to impact how memories are stored and separated. Emerging evidence from mouse models suggests that adult-born neurons can both increase and decrease activity levels in the dentate gyrus. However, the conditions that determine the direction of this modulation, and whether it occurs in other species, remains unclear. Furthermore, since the dentate gyrus is composed of a heterogeneous population of cells that are born throughout life, newborn neurons may not modulate all cells equally. We aimed to investigate whether adult neurogenesis in rats regulates activity in dentate gyrus neurons that are born at the peak of early postnatal development. Adult neurogenesis was increased by subjecting rats to an alternating running and memantine treatment schedule, and it was decreased with a transgenic GFAP-TK rat model. Activity was measured by Fos expression in BrdU+ cells after rats explored a novel environment. Running+memantine treatment increased adult neurogenesis by only 17%, but completely blocked experience-dependent Fos expression. In contrast, GFAP-TK rats had a 68% reduction in adult neurogenesis but normal experience-dependent Fos expression. The inconsistent relationship between neurogenesis and Fos expression suggests that neurogenesis does not regulate DG activity during exploration of a novel environment. Nonetheless, running and memantine may benefit disorders where there is elevated activity in the dentate gyrus, such as anxiety and age-related memory impairments.

Differential Effects of Extended Exercise and Memantine Treatment on Adult Neurogenesis in Male and Female Rats.

Adult neurogenesis has potential to ameliorate a number of disorders that negatively impact the hippocampus, including age-related cognitive decline, depression, and schizophrenia. A number of treatments enhance adult neurogenesis including exercise, NMDA receptor antagonism, antidepressant drugs and environmental enrichment. Despite the chronic nature of many disorders, most animal studies have only examined the efficacy of neurogenic treatments over short timescales (≤1 month). Also, studies of neurogenesis typically include only 1 sex, even though many disorders differentially impact males and females. We tested whether two known neurogenic treatments, running and the NMDA receptor antagonist, memantine, could cause sustained increases in neurogenesis in male and female rats. We found that continuous access to a running wheel (cRUN) initially increased neurogenesis, but effects were minimal after 1 month and completely absent after 5 months. Similarly, a single injection of memantine (sMEM) transiently increased neurogenesis before returning to baseline at 1 month. To determine whether neurogenesis could be increased over a 2-month timeframe, we next subjected rats to interval running (iRUN), multiple memantine injections (mMEM), or alternating blocks of iRUN and mMEM. Two months of iRUN increased DCX+ cells in females and iRUN followed by mMEM increased DCX+ cells in males, indicating that neurogenesis was increased in the later stages of the treatments. However, thymidine analogs revealed that neurogenesis was minimally increased during the initial stages of the treatments. These findings highlight temporal limitations and sex differences in the efficacy of neurogenic manipulations, which may be relevant for designing plasticity-promoting treatments.