Physiol Rev 94: 991–1026, 2014
doi:10.1152/physrev.00004.2014
REGULATION AND FUNCTION OF ADULT
NEUROGENESIS: FROM GENES TO COGNITION
James B. Aimone, Yan Li, Star W. Lee, Gregory D. Clemenson, Wei Deng, and Fred H. Gage
Cognitive Modeling Group, Sandia National Laboratories, Albuquerque, New Mexico; and Laboratory of Genetics,
Salk Institute for Biological Studies, La Jolla, California
Aimone JB, Li Y, Lee SW, Clemenson GD, Deng W, Gage FH. Regulation and
Function of Adult Neurogenesis: From Genes to Cognition. Physiol Rev 94: 991–1026,
2014; doi:10.1152/physrev.00004.2014.—Adult neurogenesis in the hippocampus
is a notable process due not only to its uniqueness and potential impact on cognition but
also to its localized vertical integration of different scales of neuroscience, ranging from
molecular and cellular biology to behavior. This review summarizes the recent research regarding
the process of adult neurogenesis from these different perspectives, with particular emphasis on
the differentiation and development of new neurons, the regulation of the process by extrinsic and
intrinsic factors, and their ultimate function in the hippocampus circuit. Arising from a local neural
stem cell population, new neurons progress through several stages of maturation, ultimately
integrating into the adult dentate gyrus network. The increased appreciation of the full neurogenesis process, from genes and cells to behavior and cognition, makes neurogenesis both a unique
case study for how scales in neuroscience can link together and suggests neurogenesis as a
potential target for therapeutic intervention for a number of disorders.
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NSCS AND THE NEUROGENIC NICHE
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I. INTRODUCTION
The neurogenesis field has made remarkable progress in the
past two decades (82, 111, 124, 370). Less than 20 years
ago, the neuroscience community’s appreciation of adult
neurogenesis was quite limited. Despite having been characterized using a range of techniques, the fact that young
neurons continue to be incorporated into the adult brain
was not widely accepted until the mid 1990s (128, 150).
Several factors precipitated this shift. First, immunohistological techniques for labeling dividing cells with nucleotide
analogs [e.g., bromodeoxyuridine (BrdU)] and protein
markers that were specific to neurons (e.g., NeuN), coupled
with confocal imaging, allowed the confident identification
of adult-born neurons. Second, these tracing techniques revealed that the incorporation of young neurons did not
simply occur at a persistent low rate of residual development; rather, it was heavily regulated by behavioral factors
such as stress, age, exercise, and enrichment. Finally, the
positive identification of adult-born neurons in the human
hippocampus, and even in aged individuals, demonstrated
that this phenomenon was potentially relevant to human
cognition (95).
Neurogenesis has been described in several brain regions of
multiple species. It was identified early on in songbirds
(253) and has since been observed in other bird species,
reptiles, and fish (372). In mammals, neurogenesis appears
to be considerably more limited, with robust levels limited
to the dentate gyrus (DG) region of the hippocampus and
the olfactory bulb (OB). New neurons have also been reported in other areas, with the neocortex (52, 120, 126) and
hypothalamus (168) attracting the most attention, although
the extent of neurogenesis in these areas remains controversial (273). It is worth noting that the existence of OB neurogenesis [in which maturing neurons migrate from a stem
cell population in the subventricular zone (SVZ)] in humans
is itself controversial, with differing reports having been
presented in recent years (35, 75, 288). Human neurogenesis in the DG is generally accepted, although the levels of
new neuron production have only been characterized in
limited cases (95, 315).
Both a microcosm of neural development and a never-before-appreciated form of neural circuit plasticity, neurogenesis has recently attracted investigations from perspectives
ranging from molecular neuroscience studies of the genetic
regulation of young neurons to studies of the computational
and behavioral implications of neurogenesis at circuit
scales. In this review, we focus primarily on the process of
DG neurogenesis (FIGURE 1), summarizing the full process
from the origins of new neurons as neural stem cells (NSCs)
residing in the subgranular zone (SGZ) through their maturation into fully functional granule cells (GCs). We describe the regulation of the neurogenesis process, which is
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>4 wk
3 wk
2 wk
1 wk
Basket
cell
type 2
3 days
type 1
Astrocyte
to CA3
Proliferation
Differentiation
Survival
FIGURE 1. Illustration of the development of dentate gyrus granule cells from stem cells to fully mature
neurons. New neurons arise from two populations of primitive cells, the slowly dividing type 1 cells, also known
as radial glial cells, and the more rapidly amplifying type 2 neural progenitor cells. Over the next few weeks, cells
differentiate into neurons, slowly developing dendritic arborizations and axonal projections. Between 2 and 3
wk of age, new neurons begin to receive excitatory input from cortical perforant path axons, and by 4 – 8 wk,
their physiology and anatomy begin to approach those of fully mature neurons.
influenced by molecular, network, and behavioral sources.
We then review the current view of the role of neurogenesis
in the DG’s function in learning and memory, ending with a
description of current and future techniques for studying
new neurons.
A. Characterization of Neurogenesis
It is important to briefly summarize the methods for tracing
new neurons, as the techniques used to label a dividing and
maturing cell often influence the nature and interpretation
of the experiments described below. The most common
method for labeling dividing cells involves the incorporation of a traceable molecule into DNA. Because DNA synthesis is generally limited to mitosis, at least at measurable
levels, it has been used as a marker of neurogenesis. The first
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neurogenesis studies utilized tritiated (3H) thymidine (10),
permitting the radiographic tracing of cells that had been
born at the time of injection. In the 1990s, another thymidine analog, BrdU, was developed because this molecule
could be detected using immunohistology. This development was a vital advance since immunolabeling could permit fate identification of dividing cells through the colabeling of other markers, such as the neuronal marker NeuN or
the glial marker glial fibrillary acidic protein (GFAP). BrdU
labeling remains widely used, as are its sister molecules IdU
and CldU (for its iodide and chloride equivalents, respectively), in large part because it can measure both proliferation rates and survival. It is worth noting that BrdU, despite
its usefulness, is not an ideal marker for several reasons,
including potential toxicity, nonspecificity (damaged cells
may incorporate it), and histological limitations (172).
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Other immunohistological markers can be used to identify
proliferating cells; for neurogenesis work, the most widely
used is Ki67 (245). Similarly, there are several markers for
specific stem cell phases, such as Sox-2 and nestin, which
will be briefly described below. It is important to note that,
while these protein markers are specific and do not require
preceding injections, they are also transient and not particularly suitable for long-term labeling or quantification. Genetic tracing studies, such as cre-lox systems in which a
transiently activated promoter permanently labels a neuron
with a marker such as lacZ or green fluorescent protein
(GFP), are increasingly used for long-term quantification
(140, 250). Finally, the most common approach is retroviral labeling of dividing cells with a marker such as GFP.
Retroviral labeling is fairly sparse, which limits quantification but can provide highly accurate birthdating, can show
full morphologies of new neurons (371), and is suitable for
imaging in live slices for electrophysiology studies (343).
Several of these methods will be described at greater length
later in the text.
B. Neurogenesis in Humans
For obvious reasons, measuring neurogenesis in humans is
considerably more difficult. While human neurogenesis was
originally confirmed using BrdU (95), the sample sizes were
too low for thorough quantification, and subsequent studies using histological markers left some doubt regarding the
overall levels of neurogenesis in humans (299, 300). Recently, Frisen and colleagues (315) described a technique
whereby rates of new neuron birth in humans could be
estimated by taking advantage of increased radioactive carbon in the atmosphere due to above-ground nuclear testing
(315). For roughly 15 years, levels of 14C increased steadily
above historic levels; thereafter, levels decayed steadily due
to international agreements to limit above-ground testing.
Upon entering food supplies, 14C incorporates into DNA in
a many similar to that seen in the original [3H]thymidine
studies. This technique enables estimates of overall levels of
neurogenesis, showing a turnover rate of GCs at ⬃1.75% a
year. Notably, subsequent studies with this technique did
not observe new neuron turnover in the OB; however, it
may be possible that new neuron incorporation rates there
were not well suited for this technique. Interestingly, Frisen
and colleagues (96) did observe new neurons in the human
striatum, which has not been widely observed in rodents but
has in other mammals such as rabbits (44, 206).
II. NSCS AND THE NEUROGENIC NICHE
The ability of NSCs to self-renew, proliferate, and differentiate into all cell types of the nervous system makes understanding these cells and the factors that regulate this process
critical to developing potential therapies for a number of
neurodegenerative diseases. In the adult brain, neurogenesis
is well documented to continue throughout life in only two
regions: the SVZ of the lateral ventricles and the SGZ of the
DG. Although additional areas have been reported to support adult neurogenesis, we do not focus on those studies in
this review. In both regions, NSCs give rise to neural progenitor cells (NPCs), which are limited in proliferation and
differentiate into neurons or glia (111). NPCs from the SVZ
migrate along the rostral migratory stream and supply newborn neurons for the OB; those in the SGZ migrate a short
distance into the GC layer of the DG and integrate into the
existing circuitry of the hippocampus.
There has been ongoing debate regarding which cell population within the SGZ is the “true” NSC population, if such
a thing can be appropriately defined (41). Considerable evidence exists from experiments in mice leveraging promoters from genes such as Gfap, Nestin, Sox2, and Mash to
perform lineage analysis (93, 314, 321). These promoters
each appear to label slightly different, though overlapping,
populations of cells, including both radial glia cells (RGCs),
often referred to as type 1 cells, and nonradial type 2 cells.
Several studies have demonstrated that RGCs can give rise
to type 2 cells through asymmetric division; however, it is
debated whether RGCs can replicate through symmetric
division (41). It is also not known whether type 2 cells can
revert to an RGC state. However, it is known that from type
2, cells generate what appears to be a more fate-committed
intermediate progenitor cell (IPC) population, sometimes
referred to type 3 cells, which has a distinct expression
profile, including transcription factors such as Tbr2, and
appears constrained to a future neuronal fate (137). It is
unclear when and at what rates astrocytes arise from this
process as well, with competing hypotheses existing about
whether glia are an end product of neuronal differentiation
(93) or a separation differentiation path for multipotent
cells (42), although it appears that the gliogenesis process
may be equivalently complex (46). Notably, it does not
appear that oligodendrocytes are a product of this process
in vivo (42), although they may arise from a separate pool
of progenitors (149).
Since which neurons in the above process are indeed selfrenewing and multipotent is still contested, in this review
we broadly define NSCs as those cells that are capable of
self-renewal, albeit slowly, and broadly multipotent. Likewise, we refer to NPCs as those cells that proliferate quickly
and remain capable of becoming glia and neurons but are
limited in the number of progeny they can produce. In addition, with regard to neurogenesis and its regulation, it is
often useful to consider three higher level processes: cell
proliferation, neuronal differentiation, and cell survival
(FIGURE 1). Each aspect is critical to the overall levels of
neurogenesis. For example, NPC proliferation occurs in
other regions of the adult brain, but the cells do not differentiate into neurons, becoming glia instead. However,
NPCs isolated from these nonneurogenic regions, such as
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cortex and optic nerve, in the adult brain, retain the potential to become neurons in vitro following treatment with
fibroblast growth factor fibroblast growth factor-2 (FGF2), indicating that extrinsic factors play a major role in
stimulating NPCs to differentiate into neurons (262). Further supporting the idea of a neurogenic microenvironment
is the finding that the adoption of neuronal cell fates by
NPCs under normal physiological conditions is limited in
the adult brain to a few regions such as the SVZ and SGZ,
but transplantation of NPCs from the SVZ into ectopic
regions of the adult brain results in gliogenesis, in which
NPCs become oligodendrocytes and astrocytes (298). In
contrast, NPCs from a nonneurogenic region, such as the
spinal cord, can differentiate into neurons when transplanted into the DG, supporting the idea that external cues
from the local microenvironment promote neuronal differentiation of NPCs (306).
The SVZ and SGZ represent neurogenic niches or local
microenvironments that permit and support neurogenesis.
Studies have identified key components and factors of the
neurogenic niche in a number of ways, including in vitro
coculture experiments and in vivo manipulations of neurogenesis. While there are a number of underlying similarities
between the two environments, in this review we focus on
the critical elements relevant to the hippocampal neurogenic niche.
A. Microglia
The neurogenic niche supports and promotes neurogenesis
through both secreted factors and cell-cell contact. Microglia are the resident macrophages and primary immune cells
of the brain, and they have a multitude of functions, ranging
from phagocytosis to neuroprotection. In the adult brain,
microglia in the resting state continuously survey the local
environment, with their dynamic processes interacting with
a number of cell types, including astrocytes, neurons, and
endothelial cells (249). In the adult DG, microglia are found
distributed evenly in the hilus and along the border of the
GC layer (360). In the adult hippocampus, resting microglia
are associated with GC death (277); more specifically, interactions between microglia and NPCs regulate neurogenesis via phagocytosis (310). Most newborn cells in the adult
DG fail to survive past the first week (159, 310); eventually,
the number of surviving newborn cells stabilizes after 3– 4
wk (159, 310, 326). The decline in newborn cells within the
first week is largely due to apoptotic cell death, which is
mediated by microglia, as their processes are localized and
engulf apoptotic BrdU-positive cells (310). These studies
investigating the behavior of resting and unchallenged microglia demonstrate the active role these cells play in monitoring and maintaining the local environment of an unperturbed adult brain.
The contribution of microglia to the neurogenic niche appears to depend largely on their secretion of cytokines and
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chemokines. Additionally, it is the balance of proinflammatory and anti-inflammatory signaling that is critical to
whether or not the environment supports or prohibits neurogenesis (28, 49, 61). Classically activated microglia secrete the cardinal proinflammatory cytokines, including tumor necrosis factor-␣ (TNF-␣) and interleukin-6 (IL-6),
that can inhibit NSCs from differentiating into neurons in
favor of astrocytes (61). However, depending on the environment, individual cytokines can exhibit proneurogenic or
antineurogenic properties. For example, transforming
growth factor-␤ (TGF-␤) is a cytokine secreted by microglia
that is known to activate both proinflammatory and antiinflammatory pathways, and its expression has been shown
to both induce (28) and inhibit neurogenesis (48), illustrating its ability to positively and negatively regulate this process.
Depending on the mode of activation, activated microglia
can release proinflammatory or anti-inflammatory cytokines, modulating the immune response supporting or suppressing neurogenesis (49, 233). In in vitro studies, microglia are most commonly activated with lipopolysaccharide
(LPS). LPS is an endotoxin from the gram-negative bacterial
cell wall that activates microglia through Toll-like receptor-4 to induce both morphological changes and release of
cytokines and nitric oxide (37, 243). Coculturing LPS-activated microglia or conditioned media (CM) with NPCs
decreases neuronal differentiation of NPCs (49, 233). In
contrast, both IL-4- and interferon (IFN)-␥-stimulated
microglia induce neuronal differentiation of NPCs, illustrating the potential proneurogenic effects of activated
microglia (49).
Interestingly, it is not only the mode of activation but also a
temporal component that determines whether activated microglia induce or inhibit neurogenesis. For instance, coculturing acutely activated microglia with LPS (after 24 h of
LPS treatment) or CM with NPCs decreases neuronal differentiation and increases glial differentiation (50, 233).
NPCs also exhibit increased TUNEL staining when cocultured with CM from LPS-activated microglia, illustrating
the potent effects of acutely activated microglia on NPC
activity (233). However, CM from chronically activated
microglia with LPS (after 72 h of LPS treatment) does not
significantly decrease neuronal differentiation of NPCs
(50), likely due to a difference in secreted cytokine profiles
from acutely and chronically activated microglia. While
acutely activated microglia release more proinflammatory
cytokines, including IL-1␣ and IL-6, chronically activated
microglia release more anti-inflammatory proteins, like
IL-10 and PGE2 (50).
Further studies have evaluated the activity of microglia by
manipulating levels of neurogenesis. Two of the most common ways to increase neurogenesis are through running and
exposure to an enriched environment (EE), which appear to
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increase neurogenesis via different mechanisms enhancing
cell proliferation and cell survival, respectively. The effect
of running on cell proliferation is robust, as depicted in the
increase in the numbers of BrdU cells in the DG with running (257, 350). However, exercise does not result in an
increase in the overall number of microglia in the DG or
increase the percentage of activated microglia, illustrating
that the overall microglia profile remains unaffected by running (257). Additionally, running does not modulate the
expression of MHCII or cytokines in the DG, suggesting
that the effect of running on neurogenesis is not mediated
by microglia (257). While running does not increase the
number of total microglia or level of gene expression, running does influence the interaction between microglia and
NPCs. Microglia isolated from runners induce more neurosphere formation of NPCs in vitro compared with microglia
isolated from nonrunner mice (350). Fractalkine, whose
receptor CX3CR1 is found on microglia (21), is critical in
maintaining microglia in the resting, inactivated state (60).
In vivo, CX3CR1-deficient mice, which have disrupted fractalkine signaling, exhibit decreased numbers of BrdU-positive cells as well as Dcx-positive cells, indicating a decrease
in overall neurogenesis (21, 350). Infusion of a blocking
antibody targeting CX3CR1 in running mice generated
fewer neurospheres compared with wild-type runner mice,
suggesting that the resting state of microglia is necessary for
normal NPC activity and is an integral component of the
neurogenic niche (350).
Studies have also used aged animals to investigate the role
of microglia in the neurogenic niche. While hippocampal
neurogenesis occurs throughout adulthood, there is a significant decline in the rate of neurogenesis with increasing
age. Aged animals have significantly less NPC proliferation,
neuronal differentiation, and newborn neuron survival
compared with younger animals (43, 134, 173). Age-related changes are also seen in microglia, and increased levels
of inflammation and oxidative stress, both of which
strongly inhibit neurogenesis, are observed in aged animals
(182). In addition, microglia profiles of aged animals include more activated microglia as well as higher levels of
TNF-␣ and IL-6 secretion upon activation, resulting in
elevated levels of proinflammatory cytokines in the brain
(203, 251).
Aged animals have lower levels of anti-inflammatory signaling, such as the chemokine fractalkine. Intracerebroventricular infusion of fractalkine into aged animals restores hippocampal cell proliferation, suggesting that the presence of
fractalkine helps to provide a noninflammatory environment supportive of neurogenesis. This phenomenon appears to be mediated by IL-1␤, as blocking CX3CR1 increases IL-1␤ protein levels in the hippocampus (21). These
data further highlight the sensitivity of neurogenesis to the
balance of pro- and anti-inflammatory cytokines and
chemokines.
In addition to inflammation, microglia play a key role in
modulating levels of oxidative stress by producing the antioxidant glutathione (136, 198). Microglia from aged mice
have significantly lower glutathione levels than microglia
from young mice (251), and oxidation products are found
in microglia in aged mice (133), suggesting that microglia
are less effective in attenuating oxidative stress in aged mice.
These data illustrate how changes in the activity of microglia with age potentially contribute to the age-related decline in neurogenesis by altering the neurogenic niche.
B. Astrocytes
In addition to microglia, NPCs interact with other cell types
present in the neurogenic niche (FIGURE 1). Astrocytes represent one of the major contributors to the neurogenic
niche, as coculturing with astrocytes, both directly and
without contact, induces neuronal differentiation of NPCs
(27, 256, 313). The relationship between astrocytes and
NPCs depends on a number of factors, including gene expression of the astrocytes. Coculturing astrocytes with neurospheres has revealed the importance of GFAP and vimentin in mediating cell-cell contact between these two cell
types (358). Wild-type neurospheres differentiated into
neurons at a greater rate when cocultured with astrocytes
without GFAP and vimentin expression, indicating that
these intermediate filament proteins negatively regulated
neurogenesis in vitro (358). This inhibitory effect may be
mediated by Jagged1 and Notch signaling pathways, as Jagged1 mRNA is significantly decreased in astrocytes without
GFAP and vimentin (358). Other proteins in hippocampal
astrocytes, such as ephrins, have also been identified to play
a role in regulating neurogenesis through cell-cell contact
(18). Specifically, ephrin-B2 expressed by hippocampal astrocytes induced neuronal differentiation of NPCs by activating ␤-catenin signaling, and shRNA-mediated knockdown of astrocytic ephrin-B2 resulted in reduced hippocampal neurogenesis (18).
The region of origin of astrocytes plays a significant role in
their ability to promote neurogenesis of NPCs. Astrocytes
isolated from the adult hippocampus but not spinal cord
promote neuronal differentiation of NPCs in coculture
through both soluble factors and cell-cell contact (313).
While astrocytes derived from the hippocampus and cortex are able to induce neuronal differentiation, those isolated from the hippocampus are more effective, suggesting hippocampal astrocytes release more potent neurogenic factors (256).
Analyzing gene expression from astrocytes isolated from
different regions of the central nervous system has identified
a number of key factors. Two of these factors released by
neurogenesis-promoting astrocytes are IL-1␤ and IL-6,
which are proinflammatory cytokines that, in combination
with other factors such as vascular cell adhesion molecule-1
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(VCAM-1) and interferon-induced protein 10 (IP-10), increase neuronal differentiation from NPCs (27). While proinflammatory cytokines generally inhibit neurogenesis, this
inhibition depends largely on the concentration of the cytokines. More importantly, it is the combination of factors
secreted by astrocytes into the local microenvironment that
produces the neurogenic niche (27). In contrast, astrocytes
isolated from the spinal cord express more insulin-like
growth factor binding protein-6 (IGFBP-6), decorin, and
enkephalin, which negatively regulate neurogenesis (27).
One factor that is continuously expressed in the hippocampus throughout adulthood is Wnt3a, representing a promising key factor of the neurogenic niche (197). Wnt3a is
produced by astrocytes, and ␤-catenin signaling is stimulated in NPCs, indicating a possible pathway by which astrocytes directly regulate NPC activity (197). The Wnt signaling pathway has been shown to regulate NPC proliferation and differentiation in adult neurogenesis in vitro and in
vivo (175, 197, 356). Wnt modulates adult neurogenesis by
upregulating NeuroD1 expression, which is an essential
transcription factor in the generation of GCs and neuronal
differentiation from NPCs (175). In response to Wnt, NPCs
increase expression of LINE-1, a retrotransposon that is
important in NPC survival, demonstrating that Wnt regulates multiple facets of adult neurogenesis in the hippocampus (175). Other components of the Wnt pathway, including Dickkopf-1 and secreted frizzled-related protein 3, have
been recently implicated in the regulation of neurogenesis
by aging and behavior (143, 297).
Glial metabolism appears to be a key modulator of adult
hippocampal neurogenesis. Selective inhibition of glial metabolism can be accomplished by treating cells with fluorocitrate, which inhibits glial aconitase, leading to reduced
levels of glutamine (106). While it is unclear if there are
direct effects on NPCs, fluorocitrate treatment results in a
decrease in NPC proliferation both in vivo and in vitro,
suggesting that a critical metabolite produced by astrocytes
may be necessary for NPC proliferation (58). ATP from
astrocytic metabolism serves as a signaling molecule to increase NPC proliferation in vitro and in animal models with
defective astrocytic ATP release (58). Behaviorally, animals
deficient in astrocytic ATP release exhibit depression-like
behavior, which can be reversed with the administration of
ATP (59), supporting the link between adult neurogenesis
and depression-like behavior (283).
Studies have further investigated the role of astrocytes in the
neurogenic niche using aged animals to evaluate how agerelated changes in astrocytes correlate with their low levels
of neurogenesis. The levels of hippocampal growth factors
that are known to promote neurogenesis, including insulin
growth factor-I (IGF-I) and vascular endothelial growth
factor (VEGF), are significantly reduced in aged rats compared with young rats (36, 305). While there are conflicting
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reports about whether levels of FGF-2 or its receptor are
decreased in the hippocampus of aged rats, it is clear that
this pathway is affected negatively with age (36, 305). The
overall density of hippocampal astrocytes, which are immunopositive for GFAP, remains the same throughout adulthood; however, the density of astrocytes that express FGF-2
declines with age in the DG as well as other regions of the
hippocampus, suggesting that the local environment for
NPCs progressively becomes less supportive of neurogenesis with age (305). The altered profile of astrocytes in the
aged hippocampus suggests that astrocytes are a major contributor to maintaining a neurogenic niche, and the decline
in their secreted, proneurogenic factors correlates with the
decline in hippocampal neurogenesis with age.
Interestingly, brain-derived neurotrophic factor (BDNF) is
a neurotrophin that regulates hippocampal neurogenesis
(194, 263). TrkB, a high-affinity receptor for BDNF, is expressed on NPCs (194), and levels of BDNF are highest in
the hippocampus and hypothalamus in rat brains (154).
Studies have demonstrated that hippocampal astrocytes
minimally produce BDNF (281, 282), and BDNF is highly
localized within the nucleus of GCs of the DG (154). While
BDNF protein levels in the hippocampus do not decline
with age, as do the previously discussed factors (38, 153,
178), its receptor TrkB decreases with increasing age in the
hippocampus of rats (74, 311). There is also evidence that
serum levels of BDNF in humans are correlated with hippocampal volume and memory, as illustrated by the lower
levels seen in older people with smaller hippocampi and
poor memory (94).
C. Vasculature
In addition to secreting factors to NPCs, astrocytes are
tightly linked physically with endothelial cells, wrapping
their endfeet around blood vessels (FIGURE 1). The vasculature represents an abundant source of extrinsic factors that
can modulate adult neurogenesis. Illustrating this possibility, NPCs can be found in clusters in close proximity to
blood vessels (264, 304). The physical location of NPCs in
relation to blood vessels suggests that NPCs may receive
critical factors from the vasculature to stimulate proliferation, neuronal differentiation, and survival (264). Endothelial cells secrete soluble factors that increase NPC proliferation, neurogenesis (303), maturation, and migration, in
part by secreting BDNF (191).
The intimate relationship between NPCs and the vasculature is much more evident in animals with access to a running wheel. As discussed earlier, running is a potent stimulator of adult hippocampal neurogenesis, which is accompanied by an increase in cerebral blood flow in the DG
(266). The number of blood vessels does not increase with
running, but the surface area covered by blood vessels is
significantly increased in the DG (344). The increase in the
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density of blood vessels of runners is specific to the DG and
not the entire hippocampus (66), a finding that is consistent
with the increased cerebral blood flow observed in running
mice (266). Although the number of blood vessels remains
stable in the DG with running, there is evidence of angiogenesis in the molecular layer of the hippocampus with
running, because newborn cells colabeled with an endothelial cell marker, rat endothelial cell antigen-1, are observed
in this region (92). Angiogenesis and neurogenesis rely
heavily on the circulating growth factors and nutrients supplied by the vasculature.
VEGF is a factor that has been shown to promote proliferation of not only endothelial cells but also NPCs (145).
Expression of VEGF and its receptors is not limited to endothelial cells but also expressed in NPCs, neurons, and
glial cells. VEGF mRNA and protein are expressed in the
hippocampus, colocalizing with GFAP and NeuN (57,
196). VEGF-induced neurogenesis is mediated predominantly through the Flk-1 receptor rather than Flt-1. Expression of Flk-1, but not Flt-1, is robust in the SGZ, and Flk-1
colocalizes with BrdU-positive cells in the SGZ (57, 145).
Adult rat NPCs express both VEGF and Flk-1, and VEGF
stimulates neurogenesis via Flk-1 in vitro (292). In addition
to local signaling, VEGF upregulation in the periphery is
essential to the increase in neurogenesis induced by running,
illustrating its broad range of influence in modulating neurogenesis (98).
III. DEVELOPMENT AND MATURATION
PROCESS OF ADULT-BORN NEURONS
In the adult SGZ, NSCs give rise to NPCs, which in turn
generate immature neurons. Immature neurons migrate a
short distance to the granule cell layer (GCL). Adult-born
neurons follow the same outside-in layering pattern as developmentally born neurons in DG, with adult-born neurons preferentially staying in the inner or middle of the GCL
and cells born in embryonic and postnatal stages of development found in the outer GCL (219). Extracellular matrix
molecules, such as the Reelin signaling pathway (45), tyrosine kinase receptors (70), and chemoattractive and repulsive signals (362), may play important roles in the migration of adult-born neurons. While the role of extracellular matrix molecules in neurodevelopment has been
extensively studied, a recent study investigated the effects of
manipulating Reelin and Disabled-1 (Dab1) on newborn
cell migration and dendritic maturation in the hippocampus
(327). In mice that overexpressed Reelin under the Ca2⫹/
calmodulin-dependent protein kinase II (CaMKII) promoter, 2-wk-old neurons displayed greater dendritic branching and arborization, indicative of faster maturation, compared with those found in wild-type littermates. Consistent
with this finding, conditional deletion of Dab1 in newborn
neurons resulted in aberrant dendritic extensions and shorter,
fewer processes. Disturbed migration and integration are often
identified in pathological conditions such as epilepsy, schizophrenia, and neurodegenerative diseases (63).
A. Morphological Maturation of Adult-Born
Neurons
It takes roughly 2 mo for newborn neurons to reach morphological maturity (FIGURE 1). Although no significant
structural differences are observed between fully mature
adult-born and perinatal-born neurons, the maturation
process is delayed in the adult (259, 371). It is likely that
differences in the local niche between the adult and neonatal
brain contribute to the different maturation rate. In the
adult hippocampus, by roughly 1 wk after differentiation,
newborn neurons can be seen extending their apical dendrite into the molecular layer, and spines are first detected in
the dendrite at 16 days post division (dpd). Spine density
significantly increases between 21 dpd and 28 dpd (371),
which corresponds to a critical period for neurogenesisrelated learning and memory (325). The number of spines
appears to reach a plateau by 56 dpd. Furthermore, during
these initial 2 mo, the spines remain highly plastic, dynamic,
and regulated by neural activity (371).
Axon mossy fibers reach to the CA3 region before the first
dendritic spine is detected (371) (FIGURE 2). The thin axon
fibers are first found in the hilus at 10 dpd and continue
growing until they reach to CA3 (371). Axonal boutons in
hilus are significantly smaller than those in the CA3 region
(330), consistent with what has been classically observed of
mossy fibers. Synapses arising from adult-born neurons can
be identified by electron microscopy (EM) as early as 17
dpd onto postsynaptic targets in the CA3 and the hilus
(330). These axon boutons form synapses on thorny excrescences and dendritic shafts of neurons in CA3 and hilus,
respectively. The overall size of these mossy terminals, the
number of presynaptic vesicles, and the number of activated
zones continue to increase until roughly 75 dpd, when they
are comparable to those of perinatal-born neurons. In addition, some thin axon fibers form en passant synapses onto
the dendrites of GABAergic interneurons. At a population
level, synapses from 3- to 4-mo-old neurons were found to
be functional by use of Channelrhodopsin-2 (ChR2) (330).
A subsequent study using a similar optogenetic technique
demonstrated that the maturation of mossy fiber axonal
contacts onto CA3 from young neurons had a roughly similar time course as dendritic maturation, with newborn
mossy terminals passing through several stages of increased
plasticity (129).
Notably, the spine formation process of adult-born neurons
appears to be different from that of perinatal-born neurons
in that adult-born neurons preferentially target preexisting
synapses, and little is known about the underlying mechanisms (332). One hypothesis is that glutamate spillover may
play a chemoattractive role and induce filopodia growth
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EC2
EC3
CA1
SUB
EC5
CA3
DG
EC2
DG
CA3
EC3
Pyramidal neuron
Hilar interneuron
MOPP cell
Granule cell - newborn
Garnule cell - adult
Basket cell
Mossy cell
EC2 neuron
EC3 neuron
EC5
SUB
CA1
FIGURE 2. Anatomy of hippocampal circuit into which new neurons integrate. Neurogenesis is localized to
the dentate gyrus (DG) region, where only excitatory granule cells are continually produced throughout life. The
DG has a complex local circuitry, with both inhibitory interneurons and excitatory feedback neurons (mossy
cells) participating in the network’s behavior. Granule cells in the DG project to the CA3 region, which in addition
to a robust recurrent connection then projects to the CA1 region. The CA1 then projects back to the entorhinal
cortex and subiculum regions, closing the “hippocampal loop.”
towards active synapses (331, 332). Local synaptic activity
may induce glutamate release and activate glutamate receptors in filopodia, which induce new filopodia to target the
existing synapse (331). Recent evidence suggests that local
astrocytes may also contribute to this targeting process
(171). Three-dimensional EM analysis shows that both synaptic outputs (axon boutons) and inputs (dendritic spines)
from adult-born neurons form intermediate structures on
which multiple synaptic partners are shared at a single synaptic structure (i.e., multisynapse boutons) (330, 332). As
neurons mature, the proportion of multisynapse bouton
structures decreases.
This structural difference parallels the differences between
the physiologies of immature and mature GCs. Newborn
neurons display a high input resistance (97), receive less
inhibition (193), and have been shown to exhibit considerably greater synaptic plasticity (115, 295). Because of these
unique properties, young neurons are likely to be more
excitable than mature neurons (97, 231, 232), and thus, in
response to presynaptic inputs, the synapses formed by
newborn neurons in the multisynapse boutons may be more
998
dynamic than the existing synapses. The increased dynamics of young synapses may allow new protrusions to enlarge
and subsequently lead to the eventual retraction of spines
from the mature neurons (331).
B. Physiological Maturation of Adult-Born
Neurons
For integration into the DG circuit, adult-born neurons follow the same process as perinatal neurons: silent synapse,
GABA excitatory response, glutamatergic inputs, and
GABA inhibitory response (97).
The amino acid GABA is the major inhibitory neurotransmitter in the adult brain (170). However, GABA plays very
different roles in NPCs and their progeny (114). The presence of functional GABA receptors is observed as early as in
type 2 progenitor cells (333). After neuronal fate is determined, yet prior to receiving synaptic inputs, adult-born
neurons are tonically activated by GABA released at extrasynaptic sites (81, 114). GABA’s tonic effects are able to
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REGULATION AND FUNCTION OF ADULT NEUROGENESIS
regulate the excitability threshold for neurons (117, 351),
including reducing its own tonic effect, enhancing longterm potentiation (LTP) and improving performance on
hippocampus-dependent spatial learning (19, 69). In mature cortical and hippocampal neurons, ␣5 or ␴ subunits
are primarily components of extrasynaptic GABAA receptors (117, 351). Little is known about the major components of the extrasynaptic GABAA receptors in adult-born
neurons and about how GABA’s tonic effects regulate their
maturation, integration, and function.
This gradual increase of inhibition, together with the
changes in the intrinsic physiology of young neurons and
the onset of glutamatergic synapse formation in adult-born
neurons, sets up a critical period that is a unique feature of
young neurons during their maturation (193). This critical
period is particularly important for the function of newborn
neurons in memory encoding (6, 82).
Functional GABA and glutamate receptors are detected in
3-day-old immature neurons (97), but GABAergic synaptic
events are not detected until neurons are a week old. Similar
to what is seen in type 2 progenitor cells and neurons in
developing brain, high [Cl⫺]i and expression of Na⫹-K⫹
transporter NKCC1 in immature neurons cause GABA to
depolarize instead of hyperpolarize cells. This initial depolarizing GABA phenotype has been shown to be critical for
the maturation of adult-born neurons (64, 114). Knocking
down NKCC1 in immature neurons leads to significant defects in both GABAergic and glutamatergic synapse formation as well as in dendritic development of new neurons
(114). Two possibilities may explain this phenotype: manipulation of NKCC1 expression may convert GABA from
excitatory to inhibitory, which causes the defect in synapse
formation and in morphology, or NKCC1 manipulation
changes the [Cl⫺]i inside of the cell. [Cl⫺]i is very critical for
cell function (144). [Cl⫺]i may act as an intracellular messenger to directly regulate expression of genes such as the
GABAA receptor subunit (320) and KCC2 (141). Changing
[Cl⫺]i itself may induce malformation of synapses and dendritic arborization. Different combinations of GABAA receptor subunits are involved in GABAergic tonic and phasic
response (117). Developmentally decreasing [Cl⫺]i in a cell
might contribute to the switch of GABA from its tonic to
phasic effects (261, 320) as well as the activation of the
glutamatergic synapse (352).
As described above, the maturation of adult-born GCs generally mimics the neuronal maturation process observed in
development, albeit at a slower time scale. The same holds
true for the dynamics and regulation of gene transcription
in maturing GCs. Many of the gene expression profiles observed in development are also evident in adult-born GCs,
and these genetic configurations are integral to the regulation of the maturation process. Details concerning the transcriptional and genetic regulation of neurogenesis have recently been examined in depth elsewhere (100, 139, 156,
207, 237). These processes fit into the “control” processes
underlying neurogenesis as described by Kempermann
(156), and mechanistically would be expected to be similar
between developmental and adult-born neurogenesis.
Rather than fully repeating that information here, we refer
the reader to those reviews and focus our discussion on
several examples in depth, emphasizing the circuit and behavioral factors that regulate the proliferation and differentiation of new neurons that will be specific to the regulation
and integration adult neurons.
Glutamatergic inputs are detected in 3-wk-old adult-born
neurons. Interestingly, glutamatergic currents in immature
neurons display a lower threshold for the induction of LTP
facing stimulation of the perforant pathway (PP) (115) in a
specific time window. Developmentally regulated synaptic
expression of NR2B containing NMDA receptors partially
contributes to the mechanisms for LTP induction in immature neurons (115).
Fast, perisomatic GABAergic inhibitory currents are recorded in neurons later than functional glutamatergic inputs. Adult-born neurons receive local inhibitory inputs
from all three subregions of DG: molecular layers, GCLs,
and hilus (193) (FIGURE 2). With maturation, inhibitory
inputs to adult-born neurons gradually increase. This developmental change is due to an increase in the number of
inhibitory synapses as well as their synaptic strength (193).
IV. REGULATION OF NEUROGENESIS
The regulation of neurogenesis can be targeted at several
steps of the overall process. As described above, stem cells
pass through several distinct morphologically and genetically identifiable stages, most notably a slowly dividing
RGC stage and a more rapidly proliferating neural progenitor stage. Although it is still somewhat a matter of debate
whether RGCs truly self-renew, it appears clear that cells of
the morphologically more compact progenitor population
are essentially fate-committed to become neurons after a
few rounds of amplification at most (88, 93, 321).
A. Network Level Regulation
The substantial regulatability of NSCs and NPCs suggests
that local circuit activity likely can affect proliferation and
differentiation while in the stem cell state (FIGURE 3). Recently, Song et al. (314) demonstrated that nestin-expressing RGCs in the SGZ were responsive to GABA through ␥2
receptors (314). This GABA response appeared to be tonically active and originate from nearby, but not synaptically
connected, parvalbumin (PV) basket cells. This GABA, presumably from spillover from GABAergic synapses, suppressed the symmetric proliferation of RGLs, instead biasing them to quiescence. Thus increased PV-basket cell ac-
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>4 wk
3 wk
2 wk
1 wk
Basket
cell
3 days
type 2
type 1
Astrocyte
to CA3
Proliferation
Differentiation
?
Survival
?
GABA
Glutamate
Serotonin (5HT)
Norepinephrine (NE)
Acetylcholine (ACh)
Dopamine (DA)
FIGURE 3. Regulation of neurogenesis by local circuit factors. Each of the three stages of neurogenesis–
proliferation, differentiation, and survival–is a target of regulation by network factors. Local GABA, the primary
inhibitory neurotransmitter in the brain, appears to suppress proliferation while inducing differentiation and
survival. Glutamate, the primary excitatory neurotransmitter, is necessary for proper survival as well. Modulatory neurotransmitters, which have been implicated in numerous mental health conditions, appear to
regulate different parts of the process; for instance, serotonin induces proliferation, whereas acetylcholine is
necessary for proper maturation and survival.
tivity, which likely corresponds to overall increased DG
activity, will lower proliferation of stem cells, whereas decreased DG activity will likely lower tonic GABA levels and
increase proliferation.
This direct effect of regulation of RGCs by GABA, and presumably inverse correlation of proliferation with overall network activity, is notable with respect to another recent study
by Dranovsky et al. (88) showing that exercise and EE increase
1000
the number of neurons that derive from the RGC population
(while preserving overall numbers of RGCs), whereas social
isolation significantly increases the density of RGCs. The
model proposed by the authors is that EE increases the proliferation of the intermediate NPC population and that social
isolation increases the RGC population directly.
As described by Song, the effect of GABA on RGCs is likely
due to diffusion from nearby synapses. Diffuse GABA is
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only an indirect measure of network activity, and it is not
long after progressing from the RGC stage that NPCs and
very young new neurons begin to receive more direct
GABAergic and glutamatergic inputs (through NMDA receptors) (80, 114, 215, 217, 260, 326, 333, 353). Responses to GABA progress from tonic to “phasic” GABA
roughly around the time of activation of DCX as a label
and, when tracked using retrovirus, within the first several
days post injection (97, 114). Notably, the response to
GABA is depolarizing in NPCs and young neurons due to
the distinctly high reversal potential of [Cl⫺] because of the
preferential expression of the NKCC1 ion pump as opposed
to the KCC2 ion pump (114). In NPCs, this depolarizing
effect of GABA is necessary for the influx of [Ca2⫹] through
voltage-gated calcium channels (333); in turn, calcium activity has been linked to the critical pro-neuronal differentiation gene NeuroD1 (80). Activation of local interneurons
in hilus elicits a GABAA receptor-sensitive synaptic response in type 2 progenitor cells. High [Cl⫺]i in type 2
progenitor cells makes the effects of GABA depolarizing as
opposed to hyperpolarizing. GABA mediates excitation in
NPCs and subsequently induces calcium influx via voltagegated calcium channels, which promote NeuroD expression
and lead differentiation towards a neuronal fate. Recently,
it was reported that GABA released from PV-positive interneurons in the hilus promoted radial glia-like quiescent
NSCs to actively divide (314). GABA can also provide a
trophic effect in early development, such as promoting
DNA synthesis in proliferating neurons (202) and cell motility of immature neurons (32, 216). Notably, the role of
GABA in neuronal development has also been well characterized in the SVZ, where it is essential to regulating proliferation of progenitors (201).
Once differentiation is complete and neuronal maturation
begins, the young neurons begin to develop considerably
more sophisticated connectivity to the local circuit, which
in response regulates the maturation process (97, 179, 215,
217). GABA remains depolarizing in young neurons until
roughly 2 wk of age (114), which roughly corresponds to
the time in which glutamatergic spines develop on immature dendrites (371). Glutamatergic inputs become more
and more important as immature neurons develop. Initially
the primary influence of glutamate is through NMDA receptors, which are notable for their permeability to Ca2⫹.
NMDA appears to be expressed in NPCs and has been
shown in vitro to have a direct effect on internal calcium
levels (80); however, physiology studies typically do not
show any glutamatergic signaling in NPCs or very young
neurons in slice (97, 333), which is not surprising given
their localization to the SGZ, where few synapses are glutamatergic. By roughly 2.5 wk of age, young neurons do
have dendrites with spines in the molecular layer and receive glutamatergic inputs. NMDA appears to be significant
in these early stages of excitatory synapse formation (115,
326, 367), with NMDA activation involving the NR1 sub-
unit being critical in the survival of young neurons passing
through the 2- to 3-wk-old “critical period” (326). Notably, the requirement of NMDA for neuronal survival appears to be relative to the surrounding circuit; global
blockade of NMDA in the circuit mitigates the effect of
the conditional NR1 knockout to some extent (326). The
NMDA-dependent survival of young neurons corresponds to the timing of EE’s effects on survival (325), as
well as a particular period of increased plasticity in maturing neurons (115).
1. Neuromodulators and neurogenesis regulation
In addition to the local circuit effects on neurogenesis, several of the numerous modulatory systems that have effects
on hippocampal function have been shown to affect the
proliferation and differentiation of NSCs and NPCs and the
maturation of adult-born neurons (FIGURE 3). Of these,
serotonin (5-HT) has been the most heavily studied, in part
due to its relationship with depression and stress (121,
283). Attention to serotonergic effects on the NSC population was stimulated by the observation that 5-HT-selective
reuptake inhibitor (SSRI) antidepressants such as fluoxetine
could directly induce increased levels of proliferation (209).
Subsequent studies showed that the putative 5-HT-increasing effects of SSRIs were consistent with a direct effect of
5-HT on proliferation that likely arises through the 5-HT1A
receptor, with possible contributions through the 5-HT1B
and 5-HT2A receptors (25, 271, 289). Serotoninergic fibers
project to the DG from two distinct regions, the median
raphe (MR) nucleus and the dorsal raphe (DR) nucleus
(188). MR projections are known to provide substantial
inputs to the varied interneuron populations in the hilus
and throughout the hippocampus, often onto the ionotropic (unique among the monoaminergic systems) 5-HT3
receptors (110, 345). In contrast, the DR projections to the
DG terminate preferentially in the SGZ region, with fairly
small axonal terminations that appear to be positioned for
diffuse release of 5-HT (169). As a result, it appears that the
DR projection is best positioned to impact the neurogenesis process directly; however, the MR may well regulate
local circuit activity through an indirect effect via local
interneurons.
The effects of the other major neuromodulatory systems
[norepinephrine (NE), dopamine (DA), acetylcholine
(ACh)] on neurogenesis are less well understood (346) (FIGURE 3). ACh is one of the more attractive candidates for a
potential relationship, as the cholinergic systems are known
to have a substantial relationship with the hippocampus
and the DG in particular. Depletions of ACh (through lesions of basal forebrain cholinergic regions) lead to a reduction in neurogenesis (71, 148); however, high doses of selfadministered nicotine also decrease neurogenesis (1). The
␣7-nAChR receptor was shown to be essential for the
proper maturation of adult-born GCs, with decreased survival of new neurons and reduced synaptic development in
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those young neurons that survive in ␣7KO mice (55). Like
ACh, NE has substantial impacts on the DG (130), and the
limited studies with reference to neurogenesis suggest that it
is required for normal neurogenesis; selective lesions of NEprojecting fibers decrease neurogenesis considerably (174)
and increases in NE through blockade of NE-axon autoreceptor inhibition lead to increased proliferation and survival (278). One recent study using an in vitro assay suggests that the effects of proliferation may be through ␤2
receptors (218). Finally, the dopaminergic system, which is
notoriously difficult to study in vivo, appears to have effects
on neurogenesis as well; however, these studies have generally investigated the effects of antipsychotic drugs and Parkinson’s disease models on neurogenesis, so the direct effects are difficult to elucidate (346). It does appear that D2
receptors are involved in in vivo DG neurogenesis (365).
Furthermore, it has been reported that DA can selectively
impact the LTP potential of maturing neurons (238), so it is
reasonable that DA could affect the maturation process of
new neurons as well. Notably, these systems are all tightly
coupled and known to be related to behaviors such as stress
and exercise that are associated with neurogenesis modification (see below), so the absence of clear links is more
indicative of the challenges in studying these systems in
isolation in vivo than of an indictment of their functional
importance.
B. Regulation by Local Signaling
In addition to the direct effects of neuronal activity on proliferation and differentiation, the DG network is capable of
regulating neurogenesis through local astrocytes. Wnt signaling has previously been shown to play a critical role in
the neuronal differentiation of NSCs (197). Wnt3a is secreted by astrocytes in the SGZ, whose factors have been
shown to be capable of permitting neuronal differentiation
(313). Through the canonical Wnt signaling pathway,
␤-catenin acts as a transcriptional activator of TCF/LEF
transcription factors that in turn regulate the expression of
Wnt target genes. Two such targets are the transcription
factors NeuroD1 and Prox1 (112, 151, 175, 183).
Originally identified for its ability to activate the insulin
gene promoter (246, 247), NeuroD1 has been shown to
play a crucial role in neuronal differentiation (112, 183).
NeuroD1 (also known as NeuroD and BETA2) is a basic
helix-loop-helix (bHLH) transcription factor required for
the survival and maturation of adult-born neurons. NeuroD1 null mice die from early-onset diabetes due to loss of
expression of the insulin gene (246), so to study the role of
NeuroD1 on adult neurogenesis, a transgenic mouse expressing a floxed NeuroD1 gene and ROSA26 YFP reporter
was crossed with a tamoxifen (TAM)-inducible CRE recombinase under the transcriptional control of nestin (118,
177). A dose-dependent injection of tamoxifen allowed the
conditional knockout of NeuroD1 in all nestin-expressing
1002
stem cells and their progeny. Compared with wild-type
mice, in the NeuroD1 knockout animals, deletion of NeuroD1 did not alter the number of YFP-expressing recombined cells 6 days post TAM injection; however, 40 days
after injection, the number of YFP-positive cells was significantly reduced (112). Morphological analysis revealed no
difference in proliferation (YFP/Ki67) at 6 days, further
supporting the idea that NeuroD1 is not required in early
NPCs. However, reductions in both YFP/Prox-1 and YFP/
DCX at 40 days post TAM injection indicate a critical role
for NeuroD1 during the later stages of differentiation/maturation. The dendritic length of YFP cells in NeuroD1
knockout mice was reduced as well. These data together
highlight NeuroD1 as a critical intrinsic transcription factor
that regulates survival and maturation of immature granule
neurons as they become mature granule neurons.
Another target of the Wnt signaling pathway is Prox1.
Prox1 is unique in that its expression is restricted to the DG
(180), whereas NeuroD1 is expressed in both neurogenic
regions of the brain. Prox1 has previously been demonstrated to be a target of TCF/LEF signaling (151). In vitro
analysis using ChIP as well as a luciferase reporter assay
revealed two functional TCF/LEF sites within the Prox1
enhancer region. This finding was further confirmed in vivo.
Knockdown of TCF/LEF transcription through a retrovirus
expressing a dominant negative form of LEF (dnLEF) led to
a reduction in colocalization with Prox1 compared with
cells expressing a control GFP retrovirus. In addition, overexpression of a dominant active form of ␤-catenin induced
significant ectopic expression of the Prox1 protein.
To determine the regulation of adult neurogenesis by
Prox1, viral methods were used to either overexpress or
inhibit Prox1 expression (151). In the knockdown studies, a
lentivirus expressing an shRNA against Prox1 was injected
into animals. Two weeks later, CldU was injected to label a
newborn cell population, and 3 wk after CldU administration, a dramatic reduction in the number of CldU/DCXpositive cells was observed. Interestingly, however, the researchers did not observe a reduction in the number of
NSCs expressing Sox2 and GFAP, indicating that Prox1
affected the number of newborn neurons (DCX) without
altering the proliferative NSC populations. Overexpression
of Prox1 into newborn cells, using a retrovirus, enhanced
neuronal differentiation 1 wk after injection, as demonstrated by colocalization with DCX. To determine whether
Prox1 played a role in the maintenance of mature GCs,
animals were injected with BrdU to label a newborn cell
population. Sixteen weeks later, when these neurons fully
matured, the lentivirus shRNA Prox1 was injected to knock
down Prox1 levels. Interestingly, there was no difference in
the number of BrdU cells compared with the control animals. These data suggest that Prox1 is required for the
neuronal differentiation of newborn neurons but not nec-
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REGULATION AND FUNCTION OF ADULT NEUROGENESIS
essarily for the maintenance and survival of these neurons
once they have reached maturity.
C. Regulation by Extrinsic Factors
Neurogenesis is a highly dynamic process and is regulated by
many extrinsic factors as well as intrinsic factors (FIGURE 4).
These external manipulations are known to both positively
and negatively impact levels of neurogenesis throughout the
life of mammals. Some of the most prominent positive regulators of hippocampal neurogenesis include EE, voluntary
exercise, and diet (118, 234, 256, 257). In contrast, factors
such as aging and stress have been shown to dramatically
reduce levels of neurogenesis (126, 220). In either case,
manipulations of these common elements of life play a significant part in the way that adult hippocampal neurogenesis is regulated.
One important observation is that in contrast to the molecular and circuit level regulatory factors described above, the
extrinsic regulation of neurogenesis has been challenging to
quantify with the same level of precision. While running
>4 wk
3 wk
2 wk
1 wk
Basket
cell
3 days
type 2
type 1
Astrocyte
to CA3
Proliferation
Differentiation
Survival
Learning
Running
Enrichment
Stress
FIGURE 4. Regulation of neurogenesis by behaviors. Neurogenesis is regulated by many behavioral factors
as well. Running is one of the most potent inducers of neurogenesis, targeting the proliferation of neural
progenitor cells. Enrichment has a complementary effect, increasing the survival of neurons at a critical stage
of their maturation. In contrast, stress is a severe negative regulator of new neuron birth, suppressing
proliferation. The effects of learning are more complex, suppressing the neurogenesis process at some stages
while increasing it at other stages.
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and stress were among the earliest known neurogenesis
modulators and are among the most potent regulatory factors, studies focused on extrinsic regulation, particularly
with regard to enrichment and diet, have been somewhat
more challenging to interpret.
One reason is that the variability between individual animals and their responses to the manipulation can be quite
substantial. For example, 40 mice were housed together in
one large EE and their exploratory activity was recorded
with radiofrequency identification transponders over 3 mo.
Despite the similar genetic background of all 40 mice, there
was huge variability in their exploration of the environment. Furthermore, levels of neurogenesis correlated with
the amount of exploratory activity of each individual mouse
(108). This variability in exploration can be extrapolated to
other manipulations, including running, diet, and stress. An
additional complication is the variability in experimental
design, which makes cross-laboratory comparisons challenging. The following sections describe how the continuous process of neurogenesis is actively regulated by experiences we encounter on a daily basis, with the caveat that
most of these findings remain phenomenological with only
limited insight into the underlying mechanisms.
1. EE and Running
An EE can be defined as an environment that provides sensory, social, and motor stimulation. This environment may
consist of an enlarged enclosure along with tunnels, huts,
toys, running wheels, and other animals to provide more
social interactions. One of the first studies to demonstrate
the impact of EE on adult hippocampal neurogenesis labeled newborn cells with BrdU in mice that lived in an EE
for 40 days. The researchers discovered that there was a
small but significant increase in the number of surviving
BrdU cells when animals were killed 4 wk later (160). Although there was no effect on cell proliferation in these
animals, the mice that were exposed to the EE showed significantly higher numbers of total granule neurons in the
hippocampus. In an effort to determine the long-term effects of EE, 10-mo-old mice were housed in an EE for
roughly half of their life (10 mo) (158). The number of
proliferating cells, as determined by BrdU 1 day prior to EE
exposure, increased (although not significant), as did the
neuronal differentiation of newborn cells. Cells labeled 4
wk prior to death were more than fivefold more likely to
differentiate into a neuron in mice that lived in an EE compared with mice that were housed in a standard cage. In
multiple instances, these enrichment-induced increases in
neurogenesis levels were correlated with improved performance on hippocampus-dependent behavior such as in the
Morris water maze (MWM).
Living in an EE for 4 wk is associated with increases in
neurotrophins and growth factors, including VEGF and
BDNF, in the rodent hippocampus. In rats, there is a signif-
1004
icant increase in VEGF mRNA expression after 4 wk of
living in an EE (57). The increase in hippocampal neurogenesis with exposure to an EE appears to be dependent on
VEGF expression, as shRNA-mediated knockdown of
VEGF in hippocampal neurons inhibits EE-induced neurogenesis (57). In mice, expression of BDNF mRNA is upregulated, but VEGF, nerve growth factor (NGF), and epidermal growth factor (EGF) are not observed after exposure to
an EE for 4 wk (176). Upregulation of BDNF is due to
histone modifications of the BDNF promoter (increased histone H3K4 trimethylation of BDNF P3 and P6 promoters,
and decreased histone H3K9 trimethylation of BDNF P4
promoter and histone H3K27 trimethylation of BDNF P3
and P4 promoters).
BDNF induces phosphorylation of mitogen/stress-activated
kinase 1 (MSK1) (272). MSK1 is also activated by the
ERK1/2 or MAPK signaling cascades and is involved in
CREB and H3 phosphorylation, activation of the NF␬B
pathway, and the regulation of immediate early genes (for
review, see Ref. 17). There is recent evidence that MSK1 is
involved in regulating hippocampal synaptic plasticity and
not only baseline but also EE-induced adult hippocampal
neurogenesis (72, 152). In a mouse model without functional MSK1, MSK1⫺/⫺ mice had less proliferation and
fewer immature neurons in the DG compared with wildtype mice (152). After exposure to EE for 40 days, wild-type
mice had increased cell proliferation and CREB phosphorylation, whereas these remained unchanged in MSK1⫺/⫺
mice (152). These data suggest that MSK1 is part of a critical signaling pathway involved in regulating EE-induced
hippocampal neurogenesis.
Running wheels were used in these particular EE paradigms, and it was later discovered that voluntary exercise,
i.e., running, was one of the most salient features of the EE
(340, 341). EEs are composed of multiple types of sensory
enrichment and, although exposure to these environments
has been shown to positively affect adult neurogenesis, it
was unknown which component(s) contributed most to this
effect. To answer that question, adult mice were assigned to
random groups based on the different types of enrichment
the animals experienced during exposure to an EE (341).
These groups were termed learners, swimmers, runners,
and enriched (without running wheel). Only the running
and enriched groups of animals exhibited increased levels of
neurogenesis. Consistent with previous findings, enrichment, even without a running wheel, had the biggest impact
on the survival of newborn neurons 4 wk after BrdU labeling. Runners, however, displayed a dramatic increase in the
number of proliferating cells, ultimately leading to an overall increase in the number of surviving newborn neurons at
4 wk post BrdU labeling. Both swimmers and learners
showed no differences in either cell proliferation or cell
survival compared with control animals. Interestingly, run-
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REGULATION AND FUNCTION OF ADULT NEUROGENESIS
ning also had an influence on synaptic plasticity, specifically
enhancing LTP in the DG of the hippocampus (340). Like
enrichment, the increase in neurogenesis and enhanced LTP
associated with running also correlated with improvement
in hippocampus-dependent behavior such as in the MWM.
More recently, several studies have suggested that the notable increase in neurogenesis observed with enrichment
can be attributed mainly to physical activity associated with
exercise (167, 239) (FIGURE 4). Animals were separated into
four groups: control, enriched only, enriched running, and
running. Both the enriched running and running groups
were allowed free access to running wheels. Groups that
were housed with running wheels (enriched running and
running) demonstrated significantly higher levels of neurogenesis than controls, and this enhancement was positively
correlated with an improvement in MWM performance
(121). Although the enrichment-only group did not show
enhanced levels of neurogenesis, they did demonstrate a
faster habituation to the open field test compared with controls, suggesting beneficial effects of enrichment independent of neurogenesis. Additionally, one study has suggested
that the beneficial effects of EE are independent of hippocampal neurogenesis (225). Following complete ablation
of hippocampal neurogenesis through x-focal irradiation,
irradiated mice exposed to an EE performed as well as sham
control enriched animals on the MWM, suggesting that the
previously observed enhancement of enriched mice in
MWM behavior was not attributable to hippocampal neurogenesis. Although the extent to which EE influences adult
neurogenesis is debatable, one recent study has discovered
that it affects neurogenesis on an individual basis (109).
Forty mice tagged with radiofrequency identification transponders were allowed to live in an EE for 3 mo. Interestingly, adult neurogenesis correlated positively with the exploration of individual mice (determined by “roaming entropy”). Mice that spent more time actively roaming the
environment showed a correlation with increased neurogenesis levels, suggesting that the impact of EE is ultimately
dependent on the mouse’s individual experiences within the
environment.
Although both enrichment and exercise have been shown to
enhance levels of neurogenesis, they may work through different mechanisms. Enrichment has a greater influence on
cell survival and neuronal differentiation, whereas running
appears to affect cell proliferation (341). Enrichment and
exercise increase neurogenesis to different extents (although this varies from study to study), but the application
of both of these manipulations together results in a synergistic effect on neurogenesis (99). Although the enhancements of neurogenesis associated with both running and
enrichment are consistent with increases in trophic factors
such as BDNF and NGF, the underlying differences between the two manipulations remain unclear. It is clear,
however, that the intense physical activity associated with
exercise leads to dramatic changes in the vasculature and
blood-brain barrier permeability (258). Due to this increase
in angiogenesis, circulating hormones and growth factors
are more readily delivered to the hippocampus through the
permeable blood-brain barrier and ultimately promote the
proliferation of new neurons in the DG. Circulating growth
factors that are induced through exercise include VEGF
(98), IGF-I (334), and FGF-2 (56), all of which have been
shown to influence hippocampal neurogenesis. Furthermore, the effects of running and enrichment may also involve neuromodulators such as ACh and 5-HT, which we
described above. More recently, in an animal model that
lacked brain 5-HT, basal levels of adult neurogenesis were
not affected by the permanent depletion of 5-HT, but it was
required for exercise-induced neurogenesis (166).
Many studies have revealed the underlying molecular mechanism of regulating neurogenesis by exercise and enrichment. Running has been shown to increase thickness (12)
and metabolic capacity (221) in specific regions of cortex.
Neural plasticity and electrical activity have also been associated with exercise. Running and EE-associated motor activities increase cerebral blood flow (364), blood-brain barrier permeability (301), and glucose metabolism (348).
These changes combined might increase hormone and
growth factor level, such as VEGF (145), GDNF, and
BDNF (286). BDNF in particular has received considerable
attention in neurogenesis regulation. Proliferation of NPCs
in vivo is boosted by exogenous BDNF injection into the
hippocampus (293). Together with NT3, BDNF promotes
the differentiation and maturation of adult NPCs in culture.
The expression of BDNF is regulated by neural activity and
plasticity. Increasing BDNF mRNA and protein has been
found to be associated with memory acquisition and consolidation (33, 184). Interestingly, the signaling of BDNF
promotes the survival and maturation of GABAergic inhibitory neurons. A recent study indicates that BDNF might
potentially link neural activity and GABA-mediated effects
on neurogenesis (354).
2. Aging
While there are clear benefits of manipulations such as exercise, enrichment, and diet, other external factors are consistent with a decline in hippocampal neurogenesis levels.
The most dramatic and well-studied of these negative regulators is aging. Aging is consistent with significant reductions in cell proliferation, survival, and neuronal differentiation. With the use of BrdU to label dividing cells at a given
time point, proliferation in the DG was significantly reduced in aged (21 mo) rats compared with middle-aged (6
mo) rats. Overall numbers of newborn neurons decreased
roughly eight- to ninefold from middle-aged to aged rats,
indicating the dramatic decrease in neurogenesis associated
with aging (173). Another study observing cell proliferation
in rats at broader time points indicated that this age-related
decrease in neurogenesis was observed as early as 6 wk of
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age (when compared with 2-wk-old rats) and persisted until
12 mo, where it began to level off. When DG volume was
quantified in these animals, the volume increased from 2 to
6 wk and then remained relatively stable through 24 mo of
age (134). When comparing cell proliferation and total DG
volume during aging, the initial decrease in proliferation
from 2 to 6 wk may be attributed to the fact that the brain
has not yet fully developed, suggesting a peak in cell proliferation at this time. Similar observations have been noted in
mice. Cell proliferation in mice decreases from 1 to 2 mo of
age and progressively declines until 18 mo, where the significant decreases in proliferation are no longer observed
(34, 43). Survival of these newborn labeled cells was dramatically reduced in 3- to 6-mo-old mice compared with
18-mo-old mice when BrdU was quantified 4 wk after labeling. Consistent with the decrease in cell survival, colocalization of BrdU and the neuronal marker NeuN was also
substantially reduced in both 3- and 6-mo-old mice compared with 18-mo-old animals, indicating a decrease in neuronal differentiation with age. Interestingly, this decrease in
neuronal differentiation was offset by an increase in glial
differentiation (173, 344).
The age-related decline in hippocampal neurogenesis is
likely a result of a reduction in NSC population and/or
activity. The sex-determining region Y-box 2 (Sox2) and
bHLH gene Hes5 have been identified as NSC markers
(204, 293). In aged rats, the Sox2⫹ population remains
unchanged but proliferation rates are decreased, suggesting
that a decrease in NSC activity is the major contributor to
the reduced hippocampal neurogenesis with age (131). In
contrast, examination of Hes5⫹ cells in aged mice revealed
an overall reduction in the total number of Hes5⫹ cells,
indicating that there is a significant decrease in the NSC
population with age (204). Specifically, the largest decrease
was found in the proliferative horizontal Hes5⫹ cell population in aged mice. While it is unclear from these two
studies whether the NSC population remains intact, there is
a consistent finding of decreased proliferative activity of
NSCs in aged animals. A combination of intrinsic and extrinsic age-related changes is likely to contribute to decreased hippocampal neurogenesis in aged animals (see Ref.
185 for review). A recent study identified that Wnt signaling
from astrocytes regulates the expression of survivin, which
is important for cell proliferation, in NPCs (229). Additionally, as discussed earlier in this review, the neurogenic niche
is altered with increasing age; microglia and astrocytes transition from anti-inflammatory and antioxidative to proinflammatory signaling pathways.
Exercise and enrichment are capable of increasing the number of newborn neurons in the DG fivefold, and aging is
associated with an eight- to ninefold decrease, demonstrating the extent to which external manipulations are capable
of affecting adult hippocampal neurogenesis. Given the
breadth with which changes in the external environment
1006
influence neurogenesis, it is interesting to see how these
external factors interact with one another. The most widely
studied interaction is how enrichment and exercise affect
aging in animals. As previously mentioned, when 10-moold mice (a time when decreased neurogenesis is already
present) lived in an EE for 10 more months, they exhibited
(at the age of 20 mo) a fivefold increase in the number of
newborn neurons compared with control animals (158).
The improvement in neurogenesis levels was consistent
with performance in the MWM task. Interestingly, even at
advanced stages of aging, when the reduction in neurogenesis and cognitive decline are most severe, voluntary exercise has been shown to mitigate many of these deficits. Animals, 18 mo of age, that were allowed access to a running
wheel for 45 days demonstrated a remarkable increase in
cell survival and neuronal differentiation, ultimately leading to an increase in the total number of new neurons (344).
Aged enriched animals exhibited improvements in the
MWM.
3. Stress
Along with aging, another extrinsic factor negatively regulating hippocampal neurogenesis is stress (FIGURE 4). In the
laboratory setting, there are various types of stressors used
to observe the effects of stress on adult neurogenesis. Acute
stressors are defined as a single stressful event, and chronic
stressors are multiple stressful events over a period of time;
however, both types of stressors vary based on their method
of application (physical stressor, social stressor, and odor
stressor) and duration (for a review, see Ref. 296). Despite
the variability in how stress is applied, the general consensus of these studies is that stress has a substantial effect on
DG cell proliferation. Tree shrews were subjected to acute
psychosocial stress, for 1 h, when two males were placed
together to establish a dominance/subordinance hierarchy
that caused stress for the subordinate animal (125). After
the stressful experience, animals received a BrdU injection
and were killed 2 h later to determine cell proliferation.
Tree shrews that were exposed to psychosocial stress exhibited significantly reduced numbers of proliferating BrdUpositive cells. Similarly, adult marmoset monkeys were subjected to a resident-intruder psychosocial stress model and,
from this one stressful experience, intruder monkeys demonstrated a significant decrease in cell proliferation (127).
There is one study, however, that did not observe an effect
of stress on cell proliferation. Rats placed in a similar acute
psychosocial situation did not exhibit a decrease in cell
proliferation but rather a decrease in neuronal survival
(328). Experiments utilizing a chronic psychosocial stressor
method have observed similar decreases in cell proliferation
in tree shrews, rats, and mice (76, 77, 104). Just like acute
stressors, one study has also identified an inverse effect of
chronic stress on cell survival (268). Interestingly, some
studies have found that when animals are put through behavioral tasks, such as the MWM, the stress associated with
either extended training paradigms (20) or even the novelty
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REGULATION AND FUNCTION OF ADULT NEUROGENESIS
of the task itself (91) is sufficient to reduce levels of neurogenesis. Animals that were exposed to multiple mild stressors such as cage tapping/tilting, auditory exposure to predators, cages with water or damp bedding, or housing with
temporary light fluctuations were assessed for levels of neurogenesis. Survival of BrdU cells was significantly decreased
in response to chronic exposure of two random mild stressors, across multiple mouse strains. This decrease in cell
survival was consistent with a decrease in neuronal differentiation as quantified by BrdU and NeuN colocalization
(228). Although slight differences in stress application and
species of animal may account for the variability observed,
the effects of stress on cell proliferation and cell survival
have been well documented.
Stress is associated with a release of glucocorticoids into the
bloodstream, which have been shown to modulate neurogenesis (53, 123) and potentially relate neurogenesis to its
hypothesized role in mood (186). Following performance of
an adrenalectomy to reduce circulating adrenal hormones
in the bloodstream of rats, an increase in the number of
GFAP-positive cells in the DG was observed (123). This
increase was reduced when corticosterone was introduced
through the drinking water, although not to the level of
sham treated animals. In a similar experiment, rats were
given subcutaneous injections of corticosterone and a significant decrease in the number of proliferating cells in the
DG was observed (53). An adrenalectomy of these animals
dramatically increased the number of proliferating cells.
These experiments suggest a role for circulating glucocorticoids in the bloodstream and their negative regulation of
adult neurogenesis, hinting at a possible mechanism by
which stress can affect adult neurogenesis. Interestingly,
one of the most potent positive regulators of adult neurogenesis, exercise, is also associated with an increase in glucocorticoids in the bloodstream; however, these seemingly
contradictory occurrences could be partially explained by
the massive upregulation of circulating growth factors accompanying exercise. This influx of circulating growth factors may overshadow the negative effects of stress-induced
glucocorticoids. Finally, expression of glucocorticoid receptor (GR) is robust in the GCL and colocalizes with both
NeuN and DCX (98). Additionally, glucorticoids may act
directly on NPCs, as treatment with a GR agonist reduces
cell proliferation in vitro. shRNA-mediated knockdown of
glucocorticoid receptors in the hippocampus disrupted migration and positioning of newborn cells in the GCL, suggesting a critical role for glucocorticoid receptors in mediating the integration of newborn cells into the existing circuitry (105).
4. Diet
Lifestyle choices such as exercise and diet have a tremendous impact on many aspects of brain function, including
mood, energy metabolism, behavior, cognitive function,
and hippocampal neurogenesis. Many studies have investi-
gated which specific foods or compounds can provide a
cognitive advantage or disadvantage. Rodents on a diet that
lacks essential vitamins or minerals exhibit decreased hippocampal neurogenesis, which is accompanied by impaired
learning and memory (for review, see Ref. 316). In contrast,
rodents on a diet supplemented with polyphenols or omega
3 fatty acids show increased hippocampal neurogenesis as
well as improved performance on cognitive tests (for review, see Ref. 316).
While there is an overwhelming amount of literature on diet
and cognitive function, there are many factors that need to
be considered before drawing conclusions about the effect
of diet on cognitive function. Many of these studies do not
take into account which compounds are being absorbed,
how they are metabolized, and which specific metabolite
acts directly in the brain to improve cognition. Furthermore, it is unclear how studies in rodents can be applied to
humans, specifically with regards to dose and if humans can
consume a comparable amount of the compounds. For
these reasons, it is even more difficult to comment on in
vitro studies in which compounds are directly applied to
neurons to determine the effects of cell survival or function.
Given the complicated nature of these studies, here we review a handful of studies that focus on specific flavonoids or
flavonoid-rich diets in rodents and humans to provide a
preview of a burgeoning field of cognitive neuroscience research.
Flavonoids are a subset of polyphenols that are found in
many different foods such as berries, parsley, green tea, and
cocoa (for review, see Refs. 31, 210). Epicatechin, found in
high concentrations in tea and cocoa, has been shown to
improve spatial memory retention in adult mice (342).
When epicatechin is combined with running in adult mice,
the cognitive improvement is more robust, and this combination is associated with an increase in spine density, suggesting that a synergy exists between diet and exercise
(342).
Identifying individual compounds that are effective in
enhancing neurogenesis and cognitive function is of particular interest to developing dietary interventions, particularly with the elderly population. Cognitive functions
decline with increasing age, and a number of studies have
investigated the potential use of flavonoid consumption
to prevent this decline. In elderly humans (70 –74 yr old),
consumption of chocolate, wine, and tea is associated in
a dose-dependent manner with better test scores on a
battery of cognitive tests (254). A 10-yr study examined
the cognitive function of subjects aged 65 or older as
assessed with psychometric tests. Those with the greatest
amount of flavonoid intake performed better at both the
beginning and end of the study compared with those with
the lowest intake. While the lowest flavonoid group
scored 2.1 points lower compared with their baseline, the
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highest flavonoid group scored 1.2 points lower on the
Mini-mental status examination (MMSE), suggesting
that flavonoid intake positively correlated with cognitive
function (189). However, not all diets with high flavonoid content attenuate the cognitive decline associated
with increasing age. Over a 5-yr study, subjects aged 65
or older who adhered to a more Mediterranean diet high
in fruits and vegetable consumption exhibited a slower
decline as assessed by the MMSE but not by other cognitive measures (102). In human studies, there are countless variables that cannot be controlled from one individual to the next, which makes it extremely difficult to
determine the most influential factors in improved cognitive functions. As mentioned above, lifestyle choices,
including physical and mental exercises, have a tremendous impact on cognitive performance and can vary extensively in these subjects. Additionally, individuals in
these studies not only consumed different amounts of
flavonoids but also from a variety of sources. While flavonoid intake is self-reported from an individual’s diet,
the person is typically unaware of how the processing of
food alters the quantity of flavonoids in a given vegetable
or product, which makes it difficult to determine how
much of which flavonoids had the greatest impact on
cognition.
berry-supplemented aged rats compared with control aged
rats (62).
Enhanced cell proliferation may be mediated by the increase
in IGF-I signaling, which is a known regulator of neurogenesis, as a result of the blueberry diet (62). While this study
also used a blueberry supplement in lieu of a specific flavonoid, the findings were of particular interest because they
linked the effects of blueberries on cognition to hippocampal neurogenesis, identifying possible mechanisms such as
neurogenesis and growth factor signaling through which blueberries modulate cognitive function. IGF-I and other growth
factors activate signaling cascades such as MAPK and CREB,
which are involved fundamentally in learning and memory
processes. Hippocampal levels of CREB phosphorylation and
downstream target genes such as BDNF and Bcl-2 decreased
with age but could be rescued with administration of green tea
catechins in aged mice (192). Similar age-related declines in
hippocampal levels of phospho-ERK1/2 and phosphorylated
Akt were reversed in aged rats on a blueberry-supplemented
diet (359). These studies reveal potential signaling pathways
and mechanisms that may be crucial in enhancing cognitive
function and synaptic plasticity by the flavonoids and/or other
compounds found in the diets.
5. Learning
Human studies are complicated by nature and, while the
majority of studies suggest flavonoids provide positive benefits to cognitive functions, it remains unclear which particular aspect of cognition is most improved with flavonoid
consumption (208). Studies using rodents on specific flavonoid-rich diets report enhanced learning and memory.
Changes in cognitive function are apparent in aged mice
and their ability to complete hippocampus-dependent cognitive tasks efficiently. With age, hippocampal neurogenesis
declines dramatically, and this decline is accompanied by a
slower learning curve in the MWM task as the aged mice
take longer to swim to the hidden platform compared with
young mice (344). The effectiveness of supplementing the
diet with flavonoid-rich extracts from berries or tea is evident in a number of motor and cognitive tasks in aged
animals. For example, aged rats who received grape juice
(10 –50%) for 6 – 8 wk exhibited improved performance on
behavioral tests, including the hippocampus-dependent
MWM, rod walking, and accelerating rotarod, all tasks that
are normally impaired in aged rats (309). Consistent with
this finding, diets supplemented with blueberry (1.86%),
spinach (0.91%), or strawberry (1.48%) extract reversed
the cognitive and motor deficits associated with age, as
assessed by a working memory water maze paradigm and
rod walk in aged rats (146). Aged rats on a blueberrysupplemented (2%) diet for 8 wk made fewer errors on a
radial arm water maze task than those on a control diet.
This improved performance corresponded to an increase in
the number of newborn cells in the hippocampus of blue-
1008
Interestingly, what was initially one of the more controversial relationships between behavior and neurogenesis is the
effect of learning on new neurons (FIGURE 4). While there is
considerable research into the effects of new neurons on
hippocampus-dependent learning (see sect. V), studies of
the effects of learning itself on the proliferation and survival
of new neurons initially showed mixed results (122, 340).
This initial confusion likely stems from two things: 1) not
all learning paradigms and tasks are equivalent, and 2) the
effect of learning appears to be temporally complex, with
learning promoting the survival of some young neurons
while suppressing the survival of earlier-born neurons
(87). Notably, the training alone does not appear sufficient; in a study of aged rats, only the subset of animals
that learned the task showed the aforementioned effects
on survival (89).
Subsequent studies have illustrated that the effects of learning on neurogenesis are quite complex. Interestingly, the
negative effects of learning on survival noted above appear
to be necessary for performance on the task, suggesting the
relationship between learning and survival is acutely important (90). Learning affects not only the survival of cells but
also the maturation of newborn neurons, with training in
MWM increasing the complexity of dendritic arborization
and the spine density of adult-born GCs (187, 337). Indeed,
this learning effect appears to influence what the neurons
will eventually respond to (155, 319, 325).
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REGULATION AND FUNCTION OF ADULT NEUROGENESIS
V. FUNCTION OF NEUROGENESIS
Despite its extensive characterization, the functional importance of adult neurogenesis, particularly in humans, remains a big question for the broader community. Although
the existence of adult neurogenesis was generally accepted
by the mid 1990s, the appreciation that adult-born neurons
had a meaningful role, as opposed to being simply a functionless evolutionary artifact (a “neural appendix” of
sorts), has taken much longer. Presently, the question is not
whether neurogenesis is relevant for cognition in rodents;
rather, the debate has shifted towards the scope of its function (e.g., is neurogenesis function equivalent to DG function?) and its relevance in primates. This sharp transition
from skepticism about its relevance to ascribing it a key role
in memory formation has arisen due to several factors, including theoretical and computational examinations of
how new neurons would relate to the well-characterized
DG network, focused behavioral studies designed to test
clear hypotheses on function, and sophisticated knockdown approaches that can specifically and sometimes reversibly impair neurogenesis.
Previous reviews have exhaustively described the numerous
behavioral studies that have targeted neurogenesis (82).
Here, we will take a different approach by explaining the
major functional hypotheses that are currently being con-
A
sidered by the community, highlighting both the theoretical
and computational justification for the hypotheses as well
as the associated supporting behavioral evidence (5, 252)
prior to describing the broader approaches to characterizing neurogenesis knockdown behavior. Notably, the following hypotheses are not exclusive of one another, because
the unique time scales of neurogenesis maturation may allow new neurons to have different impacts on memory at
different stages of their development.
A. Hypothesis 1: Role for Adult
Neurogenesis in Pattern Separation
The primary function that has been most associated with
adult neurogenesis is pattern separation (4, 285) (FIGURE 5).
Broadly, pattern separation can be thought of as a network
process whose outputs are less similar, or less overlapping
to one another, than its inputs.
1. What is pattern separation?
The concept of “pattern separation” has been used somewhat differently in different domains (i.e., computational,
behavioral, and physiology), so it helps to define the concept more directly as it relates to the DG. Broadly speaking,
a computational pattern separation function can be attribHypothesized Mechanisms
B
New learning directed to new neurons
A
B
Higher DG activity without new neurons
A
Different
Increased
interference
Same
B
FIGURE 5. Pattern separation theory for the neurogenic dentate gyrus. Left: illustration of the cognitive
phenomenon of pattern separation. Two events, consisting of highly similar objects and configurations, can be
learned to be different if neurogenesis is present in the DG, whereas without neurogenesis the memories will
be the same. Right: potential mechanisms for how neurogenesis may improve pattern separation. Top right:
having new neurons available can permit the second event to utilize new neurons instead of the same old
neurons to encode the differences in contexts. Bottom right: an alternative mechanism is that reducing
neurogenesis increases the baseline activity of mature granule cells, leading to higher statistical overlap (and
thus interference) between representations.
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uted to almost any neural network, as individual neurons
respond in different ways to different possible inputs. Behaviorally, pattern separation is more typically linked to
discrimination tasks, which are straightforward to design
metrics for, but are often difficult to attribute to specific
regions. Electrophysiology studies of pattern separation are
often in between these definitions; they can investigate the
correlation of neuronal activity within a region but, because
it is remains difficult to measure large populations simultaneously, one cannot fully investigate the computational
mechanisms underlying the separation.
The computational pattern separation function that has
been typically associated with the DG is more specifically
related to the hippocampus’ function in memory formation.
Information provided by the cortex (which presumably is in
the form of high level event and spatial representations) to
the hippocampus is potentially highly correlated: two different contexts may be composed of many of the same objects and spatial features yet ideally should be stored as
distinct memories. This decorrelation of cortical inputs to
optimally form memories has been attributed to the DG for
several reasons (223, 255, 335, 336). First, the large number of GCs relative to the principal cell numbers of the
entorhinal cortex (EC) and CA3 is similar to the machine
learning technique of projecting information into a higher
dimensional space to better classify or separate it (e.g., support vector machines; Refs. 23, 82). Having more neurons
available overall allows fewer neurons to be used to represent the same information, and sparse codes are ideally
suited for reducing overlap (which can equate to interference) between DG outputs. Second, the high tonic inhibition arising from both feed-forward and feedback inhibitory populations and related low activity of GCs in vivo are
well suited to achieve this sparse coding, supporting the idea
that this form of separation is indeed used by the DG (147,
190). Finally, the sparsely projecting yet powerful mossy
fiber output synapses of GCs are potentially well suited for
utilizing a sparse, separated code during memory formation
(135). The hypothesis that the DG as a whole has a pattern
separation role has been further validated by behavioral
studies in mice and rats, in which DG lesions impair
spatial discrimination (116, 161, 222, 234), and in human imaging studies, in which the CA3/DG area appears
to be particularly involved in the discrimination of similar visual objects (24).
Not surprisingly, given this long-held view of the DG, there
are numerous computational models that have examined
the role of new neurons in pattern separation (29, 355,
361). It is worth noting that these models have typically not
examined the role of new neurons in the aforementioned
rationale for the DG in pattern separation (i.e., why a highly
divergent, sparsely active network needs new neurons).
Rather, in these models, neurogenesis reduces interference
by directing new learning towards new neurons (a common
1010
theme through almost all neurogenesis models; Ref. 5), thus
ensuring that new memories and old memories are encoded
distinctly. In some respect, while this function can be related
to the pattern separation theory, the mechanism somewhat
contrasts with the fairly passive, classic DG pattern separation motif. One possibility is that these two views of pattern
separation can be unified if the activation of “classic” separating mature neurons and directed learning on immature
neurons were distinct in the timing regime (i.e., when they
fire is as important as what they fire to) (276).
2. Evidence for a neurogenesis pattern separation
function
While the computational mechanisms explaining a neurogenesis/pattern separation link are not fully understood,
behavioral data using different neurogenesis knockdown
approaches have provided strong evidence of a connection
between neurogenesis and tasks involving spatial discrimination. Several spatial discrimination tasks have been used,
including the radial-arm maze (67, 291), fear context discrimination (244, 284), and the two-choice discrimination
task (67, 73, 224). This latter task has become one of the
more prominent tasks to examine spatial discrimination, as
it uses a touchscreen operant chamber that permits a relatively large number of observations and typically shows
that the discrimination of proximal (in space) choices is
selectively impaired without neurogenesis.
B. Hypothesis 2: Role for Adult
Neurogenesis in Encoding Temporal
Context
While the pattern separation function above has a long
history in the DG, a related potential function for new neurons represents a new perspective on DG and hippocampal
memory formation. As mentioned previously, one observation of almost all computational models of adult neurogenesis is that new neurons are the primary recipients of learning (5). From one perspective, driving learning to young
neurons can improve pattern separation by ensuring that
new memories and old memories do not interfere with
one another (361). However, this direct pattern separation role of new neurons is most evident when the process
is considered from a temporal perspective. Specifically,
although memories formed at different times would be
separated because different populations of young neurons were involved, this separation would not apply for
memories for novel events experienced at or around the
same time (FIGURE 6).
Notably, unlike pattern separation, the role of new neurons
in encoding time is an added function of neurogenesis that is
distinct from prior hypotheses of DG function. This potential role was arrived at independently by two groups using
slightly different theoretical rationales and has been subse-
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REGULATION AND FUNCTION OF ADULT NEUROGENESIS
Hypothesized Mechanism
Time
Overlapping young
neurons form temporal
association
Different young neuron
population creates temporal
separation
FIGURE 6. Temporal coding theory for the neurogenic dentate gyrus. Left: illustration of the cognitive
phenomenon of temporal episodic coding. Two events with very little similarity occur within a few days of each
other (an unexpected “visitor” coming and buying a new truck). The temporal proximity of these events is
sufficient to lead to a relationship in their long-term episodic memories. In contrast, another event several
months later (a major snow storm) would not be associated due to the time elapsed. Right: mechanism for
temporal coding by new neurons. The two temporally close events would activate separate populations of
mature granule cells (pattern separation) but would use overlapping populations of immature neurons (pattern
integration). This overlap is time dependent; as time passes, the immature neuron population matures and
another set of young neurons appears to encode future events. This effectively increases separation for events
far apart in time.
quently observed in several computational studies (6, 7,
30). In both cases, young neurons have been shown to be
better suited to encode novel information than mature neurons, which have already been configured to encode familiar information.
1. Evidence for temporal separation and
neurogenesis
The big challenge for the temporal separation theory for
neurogenesis is the lack of clear behavioral evidence for
such a function. Perhaps due to its status as a novel type of
memory, there are no direct animal behavior tests for this
function similar to the spatial discrimination tasks that have
been used to argue for a general pattern separation role.
Nonetheless, there is some evidence that tasks that require
integration of events that occur close in time require neurogenesis. For example, the hippocampus has long been
known to be required for trace conditioning, a form of fear
conditioning that uses a time delay between the conditioned
stimulus (i.e., tone) and the unconditioned stimulus (i.e.,
eyepuff), and this task was one of the first behaviors for
which a neurogenesis dependence was observed (307, 308).
However, although a temporal component can be attributed to this and other tasks, it is important to note that these
studies were not designed to examine the relationship of
neurogenesis to temporal coding but rather to hippocampal
memory in general. Notably, Morris et al. (235) have recently reported a time-dependent task that was motivated
by this neurogenesis hypothesis. While they did not ablate
new neurons, they observed that the DG as a whole is necessary for temporal associations over longer time scales
(235). It remains to be seen if other specific tasks that investigate the role of time in behavior directly can be developed
and if these tasks depend on neurogenesis.
C. Hypothesis 3: Role for Adult
Neurogenesis in Memory Resolution
A somewhat different perspective on neurogenesis function
relates to what new neurons encode as opposed to how new
neurons encode (FIGURE 7). In addition to its pattern separation function, the other primary function attributed to the
DG is referred to as conjunctive encoding (161, 255). Basi-
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Hypothesized Mechanism
Mature neurons are sufficient
to encode familiar features
…but not novel features
Young neurons are capable of
encoding all inputs
High resolution
memory
Low resolution
memory
FIGURE 7. Memory resolution theory for the neurogenic dentate gyrus. Left: illustration of different resolution memories. All events that are remembered have a number of features that are not encoded. In a
“high-resolution” memory, more details about the event are remembered; while the memory may not be
perfect, it is much higher fidelity. In a “low-resolution” memory, less about the original event is encoded within
memory, perhaps focusing on a lower number of features or coarse representations (“tree” instead of “maple
tree”). Right: mechanism for increasing memory resolution by neurogenesis. Mature neurons and young
neurons encode different types of information; mature neurons are powerful at representing what they have
encoded in the past, whereas young neurons are capable of encoding novel events. The combination of both
populations facilitates the encoding of more features robustly within memories.
cally, conjunctive encoding simply refers to the DG’s (and
downstream hippocampus’s) integration of spatial and
nonspatial information into memories. Rather than simply
encoding place or objects, DG neurons likely encode a
higher representation that combines the what and where
(and potential when, see above) of experienced events for
encoding into memory.
The challenge of linking the varied forms of pattern separation described above, combined with the appreciation
that the DG may be involved in a more complex role in
memory formation, has led us to propose that the DG,
aided in large part through its neurogenesis process, has a
substantial role in dictating the “resolution” of memories
that are encoded by the hippocampus (4, 347). The DG
memory resolution hypothesis is as follows. First, the sparse
representation of the DG population and strong inhibition
suggest that mature GCs are highly selective to what they
respond to (i.e., “tightly tuned”). In addition to making
1012
them excellent pattern separators, this high selectivity
makes mature GCs very informative about those features
that they respond to. When a mature neuron is activated,
there is only a relatively sparse set of features that could
have been responsible. As a result, when an event is encoded, the set of mature neurons activated provides a very
“high-resolution” encoding of those familiar features that
drove their activity. There is a notable downside to this
high-resolution coding: if each mature GC only encodes a
finite number of features, it stands to reason that there
will be features that are not encoded well by any mature
GCs. While there are a substantial number of GCs (in
rodents the GC layer is one of the largest forebrain regions by number), it is unlikely that they can guarantee
that any event that may be encountered will have mature
GCs capable of encoding it.
In contrast, young GCs are theoretically far less selective in
response to the inputs. This lack of selectivity could be due
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REGULATION AND FUNCTION OF ADULT NEUROGENESIS
to several features, namely, their altered physiological properties (2, 97, 114, 232, 269) and differential excitatory and
inhibitory connectivity (193, 371). Individually, this lack of
selectivity reduces the information content of young neurons relative to their mature counterparts and, in an absolute sense, makes networks with young neurons less effective at pattern separation (6). However, as a population, the
broad tuning of young neurons can form a powerful distributed code that can uniquely represent features, and because
this code consists of low-information units, it is likely capable of representing any cortical input that may be provided.
Alone, this combination of sparse (mature GCs) and distributed (immature GCs) coding schemes is a powerful approach to episodic memory encoding. However, improving
the high-information sparse-coding representations over
time is probably beneficial. Thus the memory resolution
hypothesis predicts that low-information young neurons
eventually become high-information mature neurons by
virtue of their increased propensity for synaptic plasticity
(115, 295). By focusing plasticity towards young neurons,
the mature neurons can essentially fix their coding on familiar features indefinitely, with little interference over time.
Notably, this directing of learning towards immature neurons to preserve the representations of mature neurons has
been a key feature of numerous computational models of
neurogenesis (5, 6, 13, 14, 361).
1. Evidence for a memory resolution function for
neurogenesis
The principal prediction of the memory resolution hypothesis is that mature neurons will be highly selective to what
they respond to and that young neurons will be somewhat
less discriminating when young, but that they will mature to
represent events from their development. This prediction is
difficult to validate directly due to the challenges associated
with in vivo physiology in the DG and because there is no
clear method to determine which features are salient to an
animal at a given time. However, there have been numerous
studies using immediate early gene (IEG) assessments of DG
activity during experiences in EEs and behavioral tasks (9,
83, 155, 275, 319, 325) and of CA3 following neurogenesis
disruption (248). The results from these IEG studies have
been somewhat mixed and at times difficult to interpret for
a number of reasons (3). In summary, it appears that young
neurons do learn to encode what they are exposed to when
young; however, the overall activity of both the young and
mature populations is quite low and it is unclear how representative the IEG-labeled population is of the active population.
From a behavioral level, the memory resolution hypothesis
would suggest that animals without neurogenesis should
show little effects on memory tasks that rely principally on
familiar features; however, tasks that involve the flexible
use of novel information would be predicted to suffer.
There have not been many attempts to break down behavioral tasks in this manner while investigating neurogenesis;
however, one recent study used diptheria toxin (DT) to
ablate adult-born neurons that were thought of as previously specializing to the trained event, showing that the loss
of already trained adult-born neurons did impair subsequent performance on those tasks (16).
2. Effect of neurogenesis reduction on behavior
Given that the neurogenesis occurs in the hippocampus, a
critical brain region for declarative memory in humans, it
has long been thought that adult-born neurons should contribute to hippocampus-dependent learning and memory.
With the use of methylazoxymethanol acetate (MAM), a
DNA methylating agent, to ablate neurogenesis, it was first
shown that hippocampal neurogenesis was critical for the
formation of hippocampus-dependent association memories using a trace version of a fear conditioning protocol and
was dispensable for hippocampus-independent memory using a delayed version of a fear conditioning protocol (307).
However, later studies of the consequences of adult neurogenesis ablation using irradiation or other nonspecific
methods showed rather inconsistent data, possibly due to
the nonspecific nature of neurogenesis ablating approaches,
the behavioral assays used, and variations in experimental
conditions from study to study (82). The latest studies using
more specific methods for neurogenesis manipulation and
behavioral tasks aimed at DG-related functions have not
only established a role for adult-born neurons in hippocampus-dependent learning and memory but also revealed several key functional features of adult neurogenesis (82).
First, as described above, adult neurogenesis appears to be
required for pattern separation, which is currently described as a key function of the DG. This critical role for
adult neurogenesis was revealed by using tasks that tested
animals’ ability to discriminate between closely located spatial positions or similar contexts (67, 73, 284). Recent molecular and physiological studies demonstrated that the DG
can achieve pattern separation by using distinct population
codes and different firing patterns to represent different
environmental inputs (83, 190). It will be interesting to test
whether such an event-specific activity pattern is affected by
altering the rate of adult neurogenesis. In addition, tasks
related to pattern separation have been noted to improve in
response to genetic and behavioral manipulations that increase neurogenesis (73, 284).
Second, adult neurogenesis is more critically involved in
memory discrimination at the time of memory recall. Mice
with adult-born neurons ablated after the acquisition of
contextual or spatial memories showed an impaired behavioral performance upon recall of those memories (16, 129).
Using a contextual fear conditioning paradigm, Tonegawa
and colleagues (199, 274) recently reported that optoge-
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AIMONE ET AL.
netic activation of GCs responding to a contextual exposure
episode was sufficient to create an artificial fear memory to
the exposed context and that artificially activating those
GCs subsequent to the fear memory formation was sufficient to induce a fear response in a neutral environment.
Further studies are needed to determine whether adult-born
GCs are involved in forming false memories.
Third, adult neurogenesis is also required for the long-term
consolidation of contextual memory. After the formation of
the contextual fear memory, the recall of fear memory is
initially dependent on the hippocampus but becomes independent of the hippocampus after several weeks (11). Kitamura et al. (164) showed that ablation of neurogenesis by
irradiation prolonged the hippocampus dependency of contextual fear memory in mice, suggesting a role for neurogenesis in promoting system memory consolidation. Given
that the hippocampus is considered to be a temporary storage site for memory, neurogenesis may serve as a mechanism
for forgetting in the hippocampus through general retroactive
interference, regardless of memory content (107).
Fourth, adult-born neurons at different maturation stages
may make different contributions to learning and memory,
given the differences in their physiological characteristics.
By reducing the number of adult-born DGCs at different
maturation stages in Nestin-tk transgenic mice, it was
found that immature adult-born DGCs but not their mature
counterparts were required for learning and memory (85).
Furthermore, specifically silencing the adult-born DGCs at
4 wk of age but not their 2- or 8-wk-old counterparts by
optogenetic techniques after task acquisition resulted in impaired memory recall (129). Finally, blocking the output of
both developmentally born and adult-born mature DGCs
did not impair contextual discrimination in mice but caused
a deficit in the recall of previously formed spatial or contextual memories when presented with an incomplete set of
cues (244). These results led Tonegawa and colleagues
(244) to propose that immature GCs and mature GCs were
responsible for pattern separation and pattern completion,
respectively. However, this hypothesis is challenged by the
observations that ablation or inhibition of young adultborn GCs impaired spatial and contextual memory recall
and by the finding that optogenetically inhibiting GC
activity did not affect the recall of contextual fear memory (16, 129, 162). Future studies are needed to resolve
this disparity.
In addition to its role in learning and memory, the hippocampus has been implicated in emotional regulation. Instead of a unitary structure, the functional role of the hippocampus varies along its dorsoventral axis, with the dorsal
and ventral parts involved in cognitive and emotional functions, respectively (26, 101). This hypothesis was supported
by both differential connections between the hippocampus
and other brain regions and distinct lesion effects, as well as
1014
a recent optogenetic characterization along the dorsoventral axis (162, 236, 322). Early studies of hippocampal
neurogenesis also revealed a correlation between the rate of
neurogenesis and emotional status, with increased neurogenesis being observed in antidepressant-treated animals
and decreased neurogenesis in stressed animals, as discussed above. It has therefore been proposed that adult
hippocampal neurogenesis could also be an important affective regulator (142).
Although initial correlative studies provided experimental
evidence for this hypothesis, subsequent studies reported
data inconsistent with this hypothesis, suggesting that there
is a rather complicated relationship between hippocampal
neurogenesis and affective states in animal models. For
example, the increase in NPC proliferation observed with
antidepressants is dependent on the strain of mice studied: antidepressants only enhance NPC proliferation in
129SvEv and DBA mice but not in C56BL/6, balb/c, or A/J
mice (78, 138, 227, 289). Furthermore, stress does not always cause a reduction in neurogenesis. It was recently
reported that, instead of inhibiting neurogenesis, acute
stress actually increased proliferation of NPCs in the dorsal
hippocampus, leading to enhanced activity in newborn GCs
and an improvement in fear extinction (163).
Studies examining the causal relationship between hippocampal neurogenesis and affective regulation also suggest that hippocampal neurogenesis is not a master regulator of emotional status in rodents; instead, neurogenesis
may influence the emotional circuitry indirectly. In the first
studies addressing the causality of neurogenesis and affective regulation, Santarelli et al. (289) reported that hippocampal neurogenesis was required for the effectiveness of
fluoxetine (an antidepressant of the SSRI class) in certain
tests for anxiety- and depression-like behaviors, such as the
novelty suppressed feeding task and the splash test. However, subsequent studies showed that the dependence of
antidepressant efficacy on hippocampal neurogenesis was
not found in other tests for anxiety- and depression-like
behaviors, such as the open field test or forced swimming
test (78). In addition, hippocampal neurogenesis was only
involved in the efficacy of limited classes of antidepressants
(267), probably because anxiety and depression have complex etiologies and different classes of antidepressants may
have different therapeutic targets. A recent study showed
that hippocampal neurogenesis also played a role in stress
adaptation, which in turn could influence anxiety- and depression-like behavior in a distinctive way in different, commonly used behavioral tasks (312).
In summary, the relationship between hippocampal neurogenesis and emotional regulation is rather complicated. Because of the heterogeneous nature of depression and poorly
understood neural networks underlying the disease, it is
difficult to pinpoint the exact mechanism for adult-born
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REGULATION AND FUNCTION OF ADULT NEUROGENESIS
GCs in affective regulation. It is possible that adult-born
GCs are part of brain networks that underlie certain aspects
of emotional disorders and are affected by certain antidepressants, making hippocampal neurogenesis indirectly involved in emotional regulation. For example, as discussed
above, hippocampal neurogenesis is thought to be an important factor in pattern separation, so impaired neurogenesis may result in overgeneralization to emotional stimuli,
which are found in certain types of anxiety disorders. Indeed, a recent study showed that there was a negative correlation between performance in a pattern separation task
and the score received on the Depression Anxiety Stress
Scales (302). Hence, it could be the hippocampal neurogenesis-mediated cognitive function that plays a role in affective regulation.
3. Relationship of neurogenesis and cognitive deficits
in humans
Given the challenges associated with directly measuring
neurogenesis in humans, the role of new neurons in human
cognitive disorders remains unknown. Nonetheless, the
lower neurogenesis levels typically seen in aged or diseased
mice suggest that human neurogenesis may be potentially
lower in psychiatric and neurological conditions and, in
turn, linked to impaired cognition. For instance, both stress
and aging, which have dramatic effects on neurogenesis
levels in mice, are well known to be associated with memory
impairments in clinical human populations (40, 205). Likewise, patients who have undergone radiation therapy for
brain tumors are known to experience cognitive and memory deficits. While radiation clearly has broad effects on
neural tissue, it is notable that radiation remains one of the
most potent techniques for experimentally ablating neurogenesis and that the observed cognitive deficits are consistent with functions linked to neurogenesis (270, 279).
Despite the lack of direct evidence, there are several studies
that have sought to link cognitive functions related to neurogenesis in rodents to human clinical conditions. In particular, the pattern separation function described above has
been measured in humans and observed to be impaired in
aged individuals. Furthermore, this age-dependent decline
of function can be correlated with a regional fMRI effect
specific to the DG/CA3 area (317, 366). In all, the observations that human cognition correlates with neurogenesisrelated behaviors support the contention that activities and
interventions that improve neurogenesis may be useful
strategies to alleviate cognitive deficits in humans.
VI. TECHNOLOGY FOR STUDYING THE
FUNCTION OF ADULT NEUROGENESIS
As adult neurogenesis has become an increasingly well-accepted concept over the past couple of decades, focus has
been directed toward developing new technologies to inves-
tigate both the NSC development processes and the function of adult neurogenesis. There is also increasing interest
in developing techniques to manipulate the levels of neurogenesis for both scientific and clinical purposes. Below we
discuss the development of these new techniques designed
to study adult neurogenesis.
A. Regulating the Number of Adult-Born
Neurons
Adult hippocampal neurogenesis can be influenced by many
factors, such as the genetic background of the animal, the
age of the animal (185), mood (283), and housing environment (341). The number of adult-born neurons can be regulated at the levels of cell proliferation, differentiation, and
survival.
Recently, various strategies have been developed to disrupt
or augment neurogenesis specifically in the hippocampus.
These methods provide spatial and cellular specificity to
manipulate neurogenesis in the adult brain. The Herpes
simplex virus type 1 thymidine kinase (HSV-1 TK) is a
phosphotransferase that phosphorylates a broad range of
pyrimidine nucleosides and purine nucleosides, including
thymidine, deoxycytidine, and ganciclovir (GCV), which is
a synthetic analog of 2’-deoxy-guanosine (65). Phosphorylated GCV is further metabolized to its triphosphate, GCVTP, and incorporates into the nascent DNA, which induces
chromosome breaks and sister chromatid exchanges (329)
and thus causes cell apoptosis (280, 329). Combining GCV
with a genetically expressed HSV-1 TK in adult-born neurons by use of a specific promoter such as GFAP and nestin
(226, 363) has been used to specifically ablate adult-born
neurons (85, 290).
DT is another toxin that is widely used for lineage-specific
ablation (265). DT is synthesized by Corynebacterium
dipheriae (220). It has been adapted for expression in mammalian cells (220), and a single molecule might be sufficient
to kill a cell. DT is a secretory precursor polypeptide and
can be cleaved into two fragments, A (DTA) and B (DTB)
chains, joined by a disulfide bridge. DTB binds to the receptor on the cell surface and is absorbed into the cells via
endocytosis. Once DTA is transported into the cytoplasm, it
catalyzes the transfer of ADP-ribose moiety of nicotinamide
adenine dinucleotide (NAD⫹) to a modified histidine residue on elongation factor 2, resulting in termination of protein synthesis (68) and subsequent cell death. Ideally, the
delivery of DT into NPCs by retrovirus could specifically
ablate newborn neurons in the adult brain. Since mice and
rats are naturally resistant to DT, the genetic expression of
an identified monkey DT receptor (a membrane-anchored
form of the heparin-binding EGF-like growth factor) (241) in
NPCs in these species provides another approach that ablates
newborn neurons in the adult brain induced by DT (47, 287).
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Only a fraction of the cells generated in the adult brain
survives more than several months because the majority
(⬃60 – 80% in standard housing) undergoes apoptosis (54,
79). Therefore, antiapoptotic techniques can potentially be
used to increase neurogenesis. A recent example of this
approach is the use of a mouse line with a genetic knockout
of the proapoptotic gene Bax in NPCs to selectivity increase
adult neurogenesis (284).
B. Mapping the Circuit of Adult-Born
Neurons
To date, most of the anatomical characterization of how
new neurons physically connect to the existing circuitry has
relied on circumstantial evidence from electrophysiology
and the gross morphology of the neurons (97, 179, 214,
371). Developing more advanced techniques for dissecting
the circuit of adult-born neurons is potentially very critical
for fully understanding their integration and ultimately
their function.
A genetically modified rabies virus was developed recently
to trace the monosynaptic input of particular cell types
(357). It has been successfully used to map the circuits of the
piriform cortical neurons (132, 230), amygdala (132), retinal ganglion cells (318), and motor neurons (368). Unlike
traditional tracers, this genetically modified rabies virus is
able to target specific cell types and is subsequently retrogradely transported to presynaptic neurons only across one
synapse, thereby labeling direct connections onto the initially infected cells (51).
Two modifications are made to generate this genetically
modified rabies virus. 1) Rabies glycoprotein is necessary
for a virus to retrogradely transport from target neurons to
their presynaptic partners. Replacing rabies glycoprotein
with fluorescent protein in a virus genome allows visualization of the presynaptic neurons and prevents the rabies virus
from jumping across multiple synapses. 2) Pseudotyping
rabies virus with EnvA then allows the rabies virus to target
a specific cell type. EnvA is an avian sarcoma and leukosis
viral envelope protein and can be used to guide virus infection specifically into cells that express the TVA viral receptor. Because TVA is only found in birds and not in mammals,
introducing TVA into host cells in mice restricts EnvA pseudotyped rabies virus infection to only the particular cell type.
The use of this technique to map the circuit of newborn
neurons in adult hippocampus requires both the genetically
modified rabies virus and help from the aforementioned
techniques to target young neurons. To get rabies glycoprotein and TVA to be specifically expressed in newborn neurons, the Moloney murine leukemia retrovirus can be used
to specifically target genes to nonquiescent NSCs, NPCs,
and their progeny. Therefore, combining a retrovirus and a
1016
genetically modified rabies virus is an ideal tool to study the
monosynaptic circuit of adult-born neurons (86, 195, 349).
C. Imaging the Activity of Adult-Born
Neurons
Ultimately, fully understanding the function of immature
neurons will likely require an ability to observe the activity
of young neurons in the context of the functioning DG
circuit during behavior. A recent technique gaining widespread use in rodents is large-scale calcium imaging of the
neural circuits. Combining optical microscopy with a fluorescent [Ca2⫹] indicator can be used to record activity of
population of neurons. Because action potentials trigger
Ca2⫹ influx through voltage-gated calcium channels,
changes of free [Ca2⫹] can be used as a reliable readout for
neural activity. The most popular techniques to qualitatively measure [Ca2⫹] have been the use of either synthetic
dyes or proteins that change their intensity of fluorescence
upon calcium binding (212).
Chemical indicators are often a derivative of BAPTA, an
EGTA homolog (338). They have high selectivity for calcium, and their fluorescence significantly increases in response to binding calcium (339). Membrane-permeable indicators (369) or dextran-conjugated indicators (240) such
as Oregon Green BAPTA-1 (OGB-1) and fluo 4 have been
widely used in functional imaging in vivo. This technique
has been used in slice studies of the DG (213). In one study,
the DG was bathed with a calcium indicator and retroviruslabeled, adult-born, and embryonically born neurons could
then be measured in the same experimental condition in
vitro (213).
Genetically encoded calcium indicators (GECIs) overcome
several of the primary limitations of chemical indicators,
such as cell type specificity. GECIs can be targeted to specific neuronal populations and are able to be stably expressed in cells over months. GECIs are based on fusions of
fluorescent proteins and calcium buffers, such as calmodulin (CaM) (242) or troponin C (TnC) (211). When calcium
binds to CaM/TnC, conformational changes induce
changes in fluorescence intensity (242). Long-term expression of GECIs in adult-born neurons by a retrovirus or
progenitor-specific promoter would allow the imaging of
the activity of newborn neurons in behaving animals in vivo
over long periods of time and would greatly facilitate the
demonstration of what adult-born neurons encode.
D. Controlling the Activity of Adult-Born
Neurons
Manipulating the activity of newborn neurons in the adult
brain is required to further understand the function of adult
neurogenesis. Inactivating or activating newborn neurons
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REGULATION AND FUNCTION OF ADULT NEUROGENESIS
by genetics or viral vector using optogenetic strategies (103)
has the potential to directly investigate the impact of adultborn neurons on the local circuit. The most common optogenetic tools are Chlamydomonas reinhardtii channelrhodopsin-2 (ChR2) and Natronomonas pharaonis halorhodopsin (NpHR), which are microbial light-sensitive
proteins. ChR2 is a cation channel that mainly allows Na⫹
to enter the cell and activate cells following exposure to blue
light (479 nm). NpHR is a chloride pump that activates
upon illumination with 580 nm yellow light and causes
inhibition of neurons. The millisecond temporal scale of
optogenetics allows control of neuronal activity in real
time. The introduction of ChR2 to adult-born neurons by
retrovirus has shown that newborn neurons generated in
the adult brain are able to form functional synapses with
their postsynaptic target CA3 pyramidal neurons (330). Direct activation using ChR2 of a group of neurons in the DG
that are activated during fear conditioning has been shown
to be sufficient to mediate the fear memory recall (200). It
will be interesting to directly activate or inhibit newborn
neurons by optogenetics during learning.
In addition to optogenetics, other methods can be used to
control neuronal activity. Tetanus toxin (TeTx) is a neurotoxin produced by the bacteria Clostridium tetani (165).
TeTx suppresses neurotransmitter release by cleaving the
synaptic vesicle-associated membrane protein VAMP2/synaptobrevin2 (294). Therefore, expressing TeTx in neurons
will inhibit their output and inactivate their function. A
similar method involves the use of the Drosophila allatostatin (AL) receptor (AlstR), which is a G protein-coupled
inward rectifier K⫹ channel (39). Applying AL to AlstRexpressing neurons blocks generation of action potentials
(181). Inducible and rapidly reversible AL-dependent silencing of a cell has been demonstrated in cortex and spinal
cord in the anesthetized rat, mouse, and monkey (119, 323,
324). Finally, an engineered muscarinic receptor, hM3Dq,
is specifically activated by a synthetic ligand, clozapine-Noxide (CNO), but not by its endogenous ligand, acetylcholine (15). CNO activates Gq signaling pathway in hippocampal neurons and induces burst firing (8, 113).
E. Future of Neurogenesis Technology and
Research
While current technology has provided us many insights
into how these new neurons arise and what they do, there is
still much that we need to learn about these new cells.
Ultimately, understanding the cellular nature of the neurogenesis process from NSC to GC as well the functions of
adult-born neurons is going to require a combination of
many of these approaches. For instance, externally regulatable genetic approaches manipulating how progenitors respond to their local environment and external cues will
reveal much about the proliferation and differentiation processes. In short, while the technological advances of the last
couple of decades have revealed most of what we know
about adult neurogenesis, we may yet learn that we still
know very little.
ACKNOWLEDGMENTS
We thank Mary Lynn Gage for editorial comments and
assistance.
Current address of G. D. Clemenson: Dept. of Neurobiology and Behavior, Univ. of California Irvine, Irvine, CA
92697.
Address for reprint requests and other correspondence:
F. H. Gage, Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, CA 92037 (e-mail: gage@salk.
edu).
GRANTS
This work was supported by the James S. McDonnell Foundation, JPB Foundation, Mather’s Foundation, Glenn Center of Aging, and National Institutes of Health Grant R01MH090258-04. J. B. Aimone is supported by Sandia National Laboratories’ Laboratory Directed Research and
Development (LDRD) program. Sandia National Laboratories is a multiprogram laboratory managed and operated
by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the United States Department
of Energy’s National Nuclear Security Administration under Contract DE-AC04-94AL85000.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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