|  Until
recently, it was assumed that neurogenesis, or the production of new neurons,
occurs only during development and never in the adult organism. The famous
neuroanatomist Cajal stated that “Once development was ended, the
fonts of growth and regeneration of the axons and dendrites dried up irrevocably.
In adult centers, the nerve paths are something fixed and immutable: everything
may die, nothing may be regenerated.” This statement holds true
for most of the regions of the adult brain. However, there are two adult
brain areas in which neurogenesis is observed: the subventricular zone
of the anterior lateral ventricles gives rise to cells that become neurons
in the olfactory bulb, and the subgranular zone in the dentate gyrus generates
new granule cell neurons in the hippocampus, a brain region that is important
for learning and memory.
The
initial studies that suggested that the adult brain could generate new
neurons were largely ignored. In the 1960s Joseph Altman and coworkers
published a series of papers reporting that some dividing cells in the
adult brain survived and differentiated into cells with morphology similar
to neurons. They used tritiated thymidine autoradiography to label the
cells. Tritiated thymidine is incorporated into the DNA of dividing cells.
They found that the highest density of labeling was in the subventricular
zone and in the dentate gyrus of the hippocampus. It was known that the
dentate gyrus of the hippocampus is essentially devoid of glia. Therefore,
Altman attributed the labeling in this region to the uptake of thymidine
by dentate granule cells. However, he could not prove that the adult-generated
cells were neurons rather than glia, since no phenotypic markers were
available that could be used in conjunction with thymidine autoradiography.
The absence of specific markers for neurons and glia and continued skepticism
surrounding the novel concept of adult neurogenesis limited further development
of the research.
In
the mid 1970s and the early 1980s, Michael Kaplan and his colleagues reexamined
the initial observations using the electron microscope and added substantial
confidence that neurogenesis could occur in the adult brain. Combining
electron microscopy and tritiated thymidine labeling, they showed that
labeled cells in the rat dentate gyrus have ultrastructural characteristics
of neurons, such as dendrites and synapses. However, most researchers
did not consider this to be evidence of significant neurogenesis in adult
mammals. It was still not possible to prove that the new cells were neurons.
In addition, the concept that there may be brain stem cells that could
proliferate, migrate, and then differentiate into new neurons had not
yet been introduced. It was therefore thought that mature neurons would
have to replicate, an idea that most researchers found incredible. Furthermore,
the possible relevance of the findings for humans was underestimated because
there was no evidence of neurogenesis in primates.
In
the mid 1980s, astonishing findings in adult canaries by Fernando Nottebohm
and his student Steve Goldman stimulated this fledgling field. They discovered
that neurogenesis occurs in brain areas that mediate song learning. Using
a combination of tritiated thymidine with ultrastructural and electrophysiological
techniques, they provided evidence that the new cells were neurons. In
addition, they showed that there is a peak in the production of new neurons
at the time of year birds acquire songs. Nottebohm and his coworkers also
showed that neurogenesis in the hippocampal complex of adult chickadees
is correlated with seed-storing behavior. They focused on the hippocampus
because this structure in relatively larger in seed-storing than nonstoring
birds and because it plays an important role in spatial learning. Chickadees
store seeds in the fall and then retrieve them after days or weeks. In
one study, the birds were captured at different times of the year, injected
with tritiated thymidine, released, and recaptured 6 weeks later. It was
found that there was significant seasonality in the number of hippocampal
cells labeled with tritiated thymidine. Birds that had received the label
in October had more labeled cells than chickadees that had received the
label at other times during the year. Taken together, these results raised
the possibility that new neurons play a functional role in the mature
brain and led to a revived interest in possible neurogenesis in adult
mammals.
Despite
the observations of neurogenesis in the adult avian brain, confusion over
the mechanism of cell genesis in the adult brain persisted. In the early
1990s, a series of experiments in the adult mouse by Bartlett in Australia;
Reynolds, Weiss, and coworkers in Canada; and Temple in the United States,
and in the adult rat by Ray, Gage, and colleagues, revealed that cells
with stem cell properties could be isolated and expanded in culture. Under
a variety of culture conditions with different factors, these isolated
cells can be induced to proliferate and differentiate into glia or neurons.
Specifically, fibroblast growth factor and to a lesser extent epidermal
growth factor stimulate proliferation of progenitor cells in culture.
Factors that have been found to be important for neuronal differentiation
in cultured progenitor cells are retinoic acid and cyclic AMP. In addition,
neurotrophins such as NGF, BDNF, and NT-3 have been found to influence
neuronal differentiation and transmitter phenotype, whereas CNTF can regulate
glial differentiation of precursor cells. These observations in vitro
provided a mechanism for the neurogenesis in the adult brain in vivo.
Mature committed neurons were not dividing. A population of immature stem-like
cells exists in the brain. It is likely that the proliferation and differentiation
of this population results in neurogenesis.
It
is interesting that immature or stem cells that can divide and give rise
to neurons in culture can be isolated from many areas of the adult brain
and spinal cord, not just from the subventricular zone and hippocampus.
Stem cells also have been isolated from areas that are non-neurogenic
such as the septum, striatum, spinal cord, cerebral cortex, corpus callosum,
optic nerve, and eye. In culture, these cells are multipotent and can
give rise to neurons and glia. This suggests that the potential for neurogenesis
exists throughout the nervous system but that the signals necessary for
neurogenesis have been lost or that inhibitory mechanisms may prevent
its occurrence. Understanding these processes may have important implications
for repair and recovery from injury in the adult brain.
In
the 1990s, research pertaining to neurogenesis in vivo made great conceptual
and technical progress. Researchers Stanfield and Trice showed that tritiated
thymidine–labeled cells in the dentate gyrus project axons to hippocampal
area CA3, the target for mature granule cell neurons. In their experiment,
they injected the retrograde tracer fluorogold into area CA3 of tritiated
thymidine–treated animals. Subsequently, cells that were double-labeled
for tritiated thymidine and fluorogold were observed. Another important
step forward was the use of the thymidine analog 5-bromo-3'-deoxyuridine
(BrdU), a traceable analog of uridine, which is incorporated into the
genome of cells undergoing cell division. The advantage of BrdU over thymidine
autoradiography is that the cells can be visualized by using immunocytochemistry.
This method allows for a more accurate estimate of the number of new cells
with stereological techniques. In addition, BrdU immunocytochemistry can
be used in combination with now available specific markers for neurons,
such as NeuN and calbindin, and for glia, such as s100b
and glial fibrillary acidic protein (GFAP). Double-labeling for BrdU and
NeuN can be used to demonstrate convincingly whether a newborn cell has
become a neuron.
Kuhn
and coworkers in the Gage laboratory were the first to use BrdU labeling
in combination with neuronal and glial markers, demonstrating that neurogenesis
occurs throughout the lifespan of the adult rodent. They also showed that
there is a time course over which neurogenesis occurs. When BrdU-labeled
cells were examined a few days after the last BrdU injection, the majority
of the cells did not co-label for any of the mature neuronal or glial
markers; this finding suggests that these are immature, proliferating
cells. At 4 weeks after the last BrdU injection, about 60% of the BrdU-positive
cells co-labeled with the neuronal marker NeuN.
Until
the 1990s, studies that provided evidence for neurogenesis were considered
irrelevant to the primate or human brain. It was assumed that neurogenesis
has become restricted throughout evolution, as the brain became more intricate.
Thus lizards and other reptiles can regenerate and replace neurons after
being damaged, whereas in the complex human brain, the addition of new
neurons could conceivably disturb the intricate wiring of neuronal connections.
A few years ago, however, Gould, McEwen, Fuchs, and colleagues provided
evidence for neurogenesis in the hippocampus of the primate-like tree
shrew and also in the marmoset monkey. Studies using rhesus monkeys, which
are evolutionarily closer to humans than marmosets, by these investigators
as well as by Rakic and Kornack confirmed that neurogenesis occurs in
adult nonhuman primates.
At
about the same time, the possible occurrence of neurogenesis in humans
was studied. Administering BrdU and then examining cell proliferation
in tumor biopsies is occasionally used to monitor tumor progression in
patients with cancer. Because BrdU is a small soluble molecule, it is
distributed throughout the body, including the brain, and thus can be
a marker for cell division and neurogenesis in humans. In 1998, Eriksson,
Gage, and coworkers reported that five cancer patients who had received
BrdU between 15 days and more than 2 years earlier showed neurogenesis
as revealed by co-labeling of BrdU with markers of mature neurons in the
dentate gyrus. These studies clearly demonstrate that neurogenesis, at
least in the dentate gyrus, is a process that persists throughout life
in mammalian species, including humans.
Although
the genesis of new neurons in the dentate gyrus of the hippocampus of
adult mammals is now a generally accepted phenomenon, the functional role
of the new neurons remains unclear. A variety of environmental, behavioral,
genetic, neuroendocrine, and neurochemical factors can influence the proliferation
and survival of newborn cells. For example, researchers in the Gage laboratory
found that mice housed in an enriched environment (a larger cage with
toys, tunnels, and more opportunity for physical activity, learning, and
social interaction than in standard housing) have an increased number
of new neurons in the dentate gyrus. To determine which element of the
enriched environment is critical for the enhanced neurogenesis, mice were
assigned to groups with a learning task, wheel running, enrichment, or
standard housing. Similar to environmental enrichment, voluntary exercise
in a running wheel enhanced the survival of newborn neurons in the dentate
gyrus, whereas the other conditions had no effect on cell genesis (Fig.
1). In addition, both enrichment and wheel running led to improved
spatial memory function. Other investigators showed that new neurons are
generated in the hippocampus after stroke and seizures. These findings
have led to the assumption that newborn neurons may be involved in cognition
as well as brain repair. However, the conclusions from these studies are
based on correlation. The functional role of newborn neurons in the adult
brain remains to be determined.
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