Pre- and post-developmental neurogenesis in primates

Pre- and post-developmental neurogenesis in primates

Pasko Rakic*

Department of Neurobiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA

Abstract

The [3H]TdR or bromodeoxyuridine (BrdU) methods for marking DNA replication were used to determine time of origin of over 125

classes of neurons in the 34 structures of the macaque monkey central nervous system. The analysis revealed that each neuronal class is

generated in a precisely defined developmental period, the duration of which did not depend on the size and final number of neurons in a

given structure. An exception was the granule cell class of the olfactory bulb and dentate gyrus that continues genesis at a low rate even after

sexual maturity. Thus, most neurons of the primate brain, including cerebral cortex last the entire life span and are normally not renewable.

Overcoming resistance of the adult primate’s brain to acquisition of functionally competent neurons as a form of therapy would benefit from

insight into why neurogenesis normally ceases at a specific developmental period as well as why there are species-specific and regional

differences in the capacity for neurogenesis. q 2002 Elsevier Science B.V. All rights reserved.

Keywords: Neuronal stem cell; Neuronal regeneration; Cerebral cortex

1. Introduction

The enormous interest in research on the embryonic and

neural stem cells is to a large degree inspired by the hope

that they can be used to substitute for lost or damaged

neurons that are normally not being replaced or replaceable.

It is well established that many organs in the human body,

such as the skin or liver are adept at regenerating them-

selves, while other tissues such as the brain and heart are

not [1]. The apparent resistance of the human brain to

renewal of its neuronal constituents stands in contrast to

the higher level of neurogenesis observed in non-mamma-

lian vertebrates such as fish, amphibians, reptiles and birds

(e.g. [2–6]). Adult neurogenesis in these species has been

associated with ether continuous growth and/or turnover of

neurons, and is usually, but not always correlated with the

power for regeneration. Thus, species with a higher capacity

for cell turnover are indispensable model systems for unra-

veling the basic mechanisms essential for restoration of

tissues in an adult organism with lesser or no capacity [7].

Most of what we know about the time of neuron origin in

mammals has been learned from autoradiographic studies

done in mice and rats using radioactive thymidine ([3H]dT)

(e.g. [8–10]). Studies in rodents have provided the basis for

our present understanding of neuronal production in the

mammalian brain in general. Because adult neurogenesis

in mammals is more restricted, it has received renewed

attention only after the introduction of bromodeoxyuridine

(BrdU) labeling as a method for detection of possible neuro-

nal production [11]. In terms of adult neurogenesis in adult

rodents, so far unambiguous evidence has been obtained

only for the interneurons of the olfactory bulb and dentate

gyrus (e.g. [12,13]). Since considerable difference in the

magnitude of neurogenesis has been observed within the

same rodent species depending on their genetic background

[14,15], it is important to examine the time of neuronal

origin in an Old World nonhuman primate that is phylogen-

etically, genetically and anatomically closer to human.

2. Prenatal neurogenesis

In order to determine the timing and sequence of neuron

origin in nonhuman primates, we have initiated a compre-

hensive research program designed to examine the brain of

developing and adult macaque monkeys exposed to [3H]dT,

and more recently to BrdU. Pregnancy in macaque monkey

species lasts 165 days (determined by the time of conception

at the middle of the 28-day menstrual cycle); and, the post-

natal periods of infancy, adolescence and puberty are also

well established [16]. Since [3H]dT is a DNA-specific

nucleotide that is incorporated into nuclear DNA during

the S phase of the cell cycle, the amount of its incorporation

in a given cell is directly proportional to the number of

reduced silver grains in the photosensitive emulsion over-

laying its nucleus. Although the total duration of the cell

cycle in the macaque is 3–4 times longer than in mice, the

phase of DNA synthesis lasts about 8 h in both species [17].

Clinical Neuroscience Research 2 (2002) 29–39

1566-2772/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved.

PII: S1566-2772(02)00005-1

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* Tel.: 11-203-785-4330; fax: 11-203-785-5263.

E-mail address: [email protected] (P. Rakic).

Since [3H]dT injected intravenously into the macaque is

present in the blood stream for about 10 min, dating the

time of last cell division in this species is particularly accu-

rate [18]. Since DNA doubles during cell division, a labeled

neuron can be considered with confidence as new only if the

number of silver grains over a given nucleus is at least 50%

of the number recorded over the maximally labeled nuclei in

the given specimen (Fig. 1 and Refs. [19,20]). This proce-

dure may underestimate by about 0.2% the number of divid-

ing cells, since cells that are at the vary beginning or the

very end of their 8-h long S phase, during the 10 min time

when [3H]dT is available, may be missed. Such a stringent

criterion is essential to avoid the much larger problem of

misinterpreting the background label, the possible non-

scheduled DNA synthesis, or other biological and technical

artifacts as a sign of the last cell division [21–23]. However,

the application of this high criterion is a small price to pay

for the high reliability and accuracy of the results obtained.

In order to obtain unambiguous data on the time of cell

origin, most animals in this project received a single injec-

tion of [3H]dT, which was sufficient to intensively label

dividing cells in both embryonic and adult brain. Cells

that were in their last division at the time of [3H]dT expo-

sure remain permanently labeled, as have been readily

observed even in animals sacrificed seven years after injec-

tion (half-life of the tritium (3H) is 12 years). The autoradio-

graphic material was prepared from over 120 monkeys

exposed at various pre and postnatal ages and sacrificed at

intervals ranging from 1 h to several years. The brains were

dissected into 7000 gapless 8-mm thick sections and,

depending on the size of the structure, every second to

every tenth section processed for fine-resolution autoradio-

graphy [9,20,24]. Hence, the prepared material in this

collection has been examined by over 30 researchers from

eight different countries (see Acknowledgement), who

focused their analysis on brain structures ranging from the

spinal cord and brain stem to the cerebellum, the various

forebrain subdivisions and the retina.

Because of prolonged childhood and long life span in

primates we expected neurogenesis to be much more

pronounced after birth than in rodents. To our surprise, we

found that analogous classes of neurons in the monkey are

actually generated at comparatively earlier embryonic

stages in relation to birth than in rodents. This is evident

when timing of the neurogenetic periods of the major struc-

tures of the monkey brain is compared to the time of concep-

tion and birth (Fig. 2). We have examined over one hundred

classes of neurons (e.g. [19,20,25–49]). Provided here will

only be a simple graphic representation of the time of a

‘cell’s birthday’, followed by a brief description of the

neurogenetic gradients in the cerebral cortex.

As presented in Fig. 2, most classes of neurons compris-

ing the monkey central nervous system were generated

during a specific and highly restricted gestational period.

The only exceptions are the granule cells of the olfactory

bulb, cerebellum and hippocampus, which continue their

P. Rakic / Clinical Neuroscience Research 2 (2002) 29–3930

Fig. 1. Incorporation of [3H]dT as marker of cell division. (a) Graphic

representation of the level of DNA during different phases of cell cycle

(G1 and G2, gap phases; D, mitotic division; S, synthesis phase). The

method of [3H]dT incorporation following a single injection of the isotope

is stoichiometric and thus allows distinction between doubling of DNA

content during the S phase, signifying mitotic division, and the light label

of less than 50% of the maximum grain counts that cannot ascertain that the

cell has divided. (b) The Purkinje cells in the middle and on the left can be

considered as heavily labeled while the five grains over the cell nucleus on

the right may be due to a variety of biological and technological factors,

some of which are discussed in the text. Importantly, if the animal were

injected with ten, instead of a single [3H]dT dose, this cell may be as

heavily labeled as the one in the middle and could be falsely interpreted

as divided. From Rakic [22].

genesis after birth. The duration of the neurogenetic period

of a given structure did not depend on its size and final

number of centers. This point can be illustrated by the

period of neurogenesis in the visual system (Fig. 3). For

example, 1.2 million ganglion cells in the retina are gener-

ated within 40 days (E30–70 [41]) whereas 1.5 million

neurons in their synaptic targets, the lateral geniculate

nucleus, are produced during a period five times shorter

(E36–43 [20]). Even after taking into account that these

two structures display a somewhat different rate of

P. Rakic / Clinical Neuroscience Research 2 (2002) 29–39 31

Fig. 2. Time of neuron origin of the major classes of neurons in the macaque central nervous system. The age of animals in post-conceptual (E ¼ embryonic)

days is represented on the horizontal line, ending at term (E165 ¼ birth) and thereafter in postnatal (P) days. The horizontal lines corresponding to each

neuronal class indicate the animal’s age when an injection of [3H]thymidine results in a heavy label in the neuronal nuclei when the monkeys are killed

postnatally. The dotted line corresponding to the dentate gyrus indicates continued neurogenesis based on the data obtained by BrdU/NeuN double labeling in

adult monkeys. See text for further explanation.

programmed cell death [50,51], the duration of the neuro-

genetic period is significantly different for neuronal popula-

tions of a similar size. The final number of neurons in a

given structure is species-specific and depends on the initial

size of the founder cell pool in the proliferative zones that

becomes exhausted at a specific time [29,52]. The conclu-

sion, reached after completion of a series of these studies,

was that most identifiable neurons in a species are generated

during well-defined developmental periods prior to birth.

A detailed account of neurogenesis in various brain struc-

tures of the large macaque nervous system will not be

provided here, except for the cerebral neocortex, which has

recently received particular attention. This phylogenetically

new area of the brain is essential for long term memory and

representational knowledge [53] and is associated with

primate ascent [54]. We have conducted a series of studies

on the neocortex to determine the timing and sequence of

neurogenesis in the diverse cytoarchitectonic areas. The

results revealed that corticogenesis begins almost simulta-

neously, between E38-E40, in all areas examined (Fig. 4

and Refs. [25,42,47,55,56]). However, the time of cessation

of neurogenesis was found to be more variable and has

ranged between E70, in the limbic cortex [47] to E102 in

the visual cortex [25]. In each area, neurons settle into a

distinct inside-outside neurogenetic gradient, in which the

earlier generated neurons occupy the deep layers and the


Fig. 3. Diagrammatic representation of the time of origin of the retinal ganglion cells (RG), neurons in the dorsal lateral geniculate nucleus (LGNd) and primary

visual cortex (VC) of the macaque monkey. Neurogenetic gradients outlined schematically in the retina (B), lateral geniculate nucleus (C) and striate cortex 17

(D) are based on a large series of [3H]TdR-labeled animals processed for autoradiography and the more detailed quantitative data can be found in Refs.

[20,25,41].

later generated neurons become situated more superficially.

Thus, the typical inside-out gradient of neurogenesis, known

from initial studies in rodents (e.g. [57–59]) is clearly visible

in the monkey in which layer specificity is more pronounced

and the cohorts of isochronously generated neurons even-

tually occupy a relatively narrow strata within the cortex

(Fig. 4 and Ref. [25]). The sole exception to the inside-out

sequence of neurogenesis in primates was encountered in

layer I, contrary to rodents, whereby neurogenesis continues

throughout the entire period of corticogenesis [49].

Most cortical neurons in the macaque monkey are gener-

ated in the proliferative zones situated near the embryonic

cerebral ventricle, which then migrate radially to the

increasingly distant and more convoluted cortical plate

[60]. Due to the size of the cerebrum of the fetal monkey,

at the end of the neurogenetic period, the migratory neurons

assume a progressively longer time to reach their final desti-

nation in the cortical plate and increasingly rely more on

radial glial guidelines [61]. It has been recently shown that

GABAergic interneurons of the cerebral cortex in rodents

are generated in the ganglionic eminence (GE) of the ventral

telencephalon and then migrate tangentially to the develop-

ing cortex [62–66]; and, in the mouse, most if not all inter-

neurons originate in the embryonic GE [67]. In contrast, in

the human fetus only 35% of the GABAergic interneurons,

or about 10% of the total number of cortical neurons, arrive

to the cortex from this proliferative center; thus, about 90%

of the total cortical population originates in the VZ/SVZ

[68]. Since postmitotic neurons at later stages of develop-

ment require more than two weeks to settle into their final

positions, we estimate that about one month before birth

(between E125 and 135), all cortical neurons in the macaque

are settled in their final laminar and radial positions.

We have observed in all animals exposed to [3H]dT post-

natally, labeled astrocytes, oligodendrocytes and endothelial

cells; but cells with typical neuronal morphology were not

intensively radiolabeled [27–29]. It should be emphasized

that neuronal cells in the mature primate neocortex can be

easily identified in autoradiograms and only the heavily 3H-

dT labeled nuclei belonged to non-neuronal cells, indicating

that the doses and method for detecting new cells were appro-

priate. It is also important to recognize that in all other

currently examined mammalian species, genesis of cortical

neurons was also found to be confined to the prenatal and

neonatal developmental periods (e.g. [57–59,69–74]).

3. Postdevelopmental neurogenesis

From the beginning of the study on primate neurogenesis,


Fig. 4. Diagrammatic representation of the positions of heavily labeled neurons in the four representative cytoarchitectonic cortical areas. Each monkey had

been injected with 10 mCi/kg of [3H]dT at a selected embryonic day and sacrificed postnatally. On the left is a diagrammatic representation of the approximate

position of layers I–VI and the white matter (WM). Embryonic days (E) are represented on the horizontal line, starting with the beginning of the second fetal

month (E34) and ending at term (E165). Positions of the vertical lines indicate the embryonic day on which an animal has received a pulse of [3H]dT. On each

vertical line, short horizontal markers indicate positions of the heavily labeled neurons encountered in one 2.5-mm long strip of cortex. Blue: brown, area 24 of

Brodmann; green, area 11; blue, area 46; red, area 17. Gray: layer I neurons in the primate are generated throughout the entire period of neurogenesis in each

area. From Rakic [22].

it was clear that granule cells of the hippocampus and cere-

bellum are generated to a considerable extent postnatally

[19,27–29,36]. However, we were even more impressed

that among over the hundreds of classes of neurons exam-

ined, only the class of small interneurons (granule cells) and

few rod photoreceptors at the edge of the retina are gener-

ated after birth in the macaque monkey. While production of

the rods halts a few days after birth [41], the production of

the cerebellar granule cells continues during infancy and the

juvenile period, but could not be observed in animals

injected with [3H]dT after the third postnatal month [27].

While we noticed that proliferation of cells in the subgra-

nular zone of the dentate gyrus continues after sexual matur-

ity (third year of life) the methods for determining the nature

of these small cells at the time did not allow conclusive

distinction between granule cells (neurons) and astrocytes

(glia). Application of autoradiography at the electron micro-

scopic level revealed heavily labeled cells with only the

ultrastructural characteristics of astrocytes and oligodendro-

cytes [39]. Thus, we concluded that “if neurogenesis has

been missed as a result of methodological limitations, the

number of newly generated neurons must be very small and

thus hardly comparable to massive levels of neurogenesis

reported in birds and rodents “ [39]. At the time, the olfac-

tory bulb had not been dissected and examined with the

[3H]dT autoradiographic method.

After introduction of immunohistochemistry for BrdU and

the immunomarkers for neurons, we re-examined this issue

and found double labeled cells in the dentate gyrus of the

adult monkey [75]. To prove that these were indeed new

cells, we followed their developmental history from their

last division in the subgranular zone, through the migration

phase of bipolar cells, and finally to their differentiation in the

dentate gyrus [75]. No new neurons were formed by division

of the pre-existing hippocampal neurons. Rather, they origi-

nated from a remnant of the pluripotential stem cells situated

in the subgranular zone of the dentate gyrus in the hippocam-

pus. We estimated that in the dentate gyrus of the adult

monkey (5.5–16.5 years of age) one new granule cell is

generated each day per 24 000 existing cells, or 0.004% of

the granule cell population [75], which is about one order of

magnitude less than in mice [15]. It is not known whether

these cells in the primate incorporate into appropriate synap-

tic circuits, or how long they survive. It they are destined to

replace existing neurons, it would take over 65 years or about

three average life spans of the macaque to turn over all gran-

ule cells in the dentate gyrus. However, since the majority of

newly generated neurons die shortly after their genesis [76],

in reality it would take much longer. Since there is a consid-

erable genetic difference in the rate of neurogenesis in the

dentate gyrus even among different strains of mice [14,15], it

is not surprising that the difference may exist between species

millions of years apart in the evolutionary scale. At this point

it is not known whether new granule cells in the primate

dentate gyrus become incorporated into proper synaptic

circuits and what is their function.

We have also found that the subependymal zone (SEZ) in

the adult monkey telencephalon, similar to other mammals,

generates glial cells as well as prospective neurons that

migrate tangentially, via the rostral migratory stream

(RMS) and olfactory nerve, to the olfactory bulb [77].

These SEZ/RMS cells in adult monkeys are morphologi-

cally and biochemically similar to migrating cells described

in adult rodents (e.g. [12,28]) and form chains that are

immunopositive for surface markers, TuJ1 and PSA-

NCAM that characterize this migratory stream [77,78].

The molecular mechanisms that channel newly generated

neurons to the olfactory bulb via the RMS and repels

them from entering into other neighboring structures has

been elucidated in the mouse [79]. However, in the monkey

it takes about 3 months for cells to complete their migration

via the RMS to become mature neurons, probably reflecting

a lower rate of genesis as well as migration and a protracted

tempo of differentiation. The relatively smaller daily rate of

neurogenesis in the olfactory system in the adult primate

compared to that observed in rodents can be explained by

the reduced size of the olfactory bulb in relation to the large

expansion of the primate cerebral cortex and other structures

engaged in cognitive functions [80,81].

Although all classes of neurons in the large macaque

nervous system have not been examined with the new meth-

ods, the overall assessment of the presently available data is

that neurogenesis in adult primates is more limited than in

rodents [22,75,77,82]. A similar limit appears to exist in the

human forebrain (e.g. [83]). Thus, the report of a large daily

influx of new neurons to the association neocortex in maca-

que monkey, particularly the principal sulcus of the prefron-

tal area [84,85] came as a surprise and has necessitated re-

examination using the same method. Thus we performed a

study on additional 10 macaque monkeys of mixed gender,

some of which were raised in a group and exposed to enriched

environment [82]. Examination of the cerebral cortex follow-

ing BrdU injections to these animals revealed labeled cells in

the subependymal zone of the lateral cerebral ventricle [82]

as previously observed by other investigators (e.g. [86,87]).

However, we also found that cells doubly-stained with TuJ1

(a marker for immature neurons) were entering the rostral

migratory stream on way to the olfactory bulb, where they

differentiate into granule cells [77]. Although BrdU-labeled

nuclei were also observed in the neocortex, examination of

several thousand cells using triple-labeled immunofluores-

cent histochemistry for BrdU, NeuN (a marker for mature

neurons) and GFAP (a marker for astrocytes) yielded none

that met the basic criteria for a new neuron [77]. Likewise, the

massive migratory stream to the principal sulcus that was

suggested to be a conduit for new neurons to the prefrontal

cortex [84] could not be identified.

A more detailed evaluation of the available evidence about

adult neurogenesis and the problems of the methods used for

detecting new cells can be found elsewhere [21–23,82,88]. In

short, the review of the caveats of the present state of the art

shows that BrdU incorporation alone can not be taken as a


definitive marker for cell division, but rather a marker for

DNA synthesis, which can also occur in nondividing cells

and can actually be enhanced by a variety of factors and

conditions including the process of programmed cell death

(e.g. [89]). It is often overlooked that BrdU is toxic and may

act as a mutagen or stimulator of DNA synthesis if given in

high and repeated doses (e.g. [90]). Finally, one has to

exclude the possibility that BrdU labeling or polyploidy is

not due to the fusion of the newly generated blood born cells

with the nuclei of existing, damaged or aged neurons [91,92].

Thus, BrdU incorporation in the cell’s nucleus is essential,

but not a sufficient indication that a given cell has actually

divided [21,22,23]. Before concluding that the occasional

BrdU labeled cell found in the adult or aging brain tissue is

a newly generated neuron, it is essential to exclude BrdU

labeling as not resulting from DNA synthesis in response to

cell damage, abortive mitoses, polyploidy, or the initial steps

in naturally occurring cell death.

It is noteworthy that, although cortical neurons in the

monkey are produced prenatally, the volume of the cerebral

cortex continues to grow at a considerable degree postna-

tally. Detailed examination of the cytological maturation of

the primate neocortex reveals that its postnatal expansion is

due to multiple aspects of cellular growth and maturation

and a large addition of non-neuronal cells. First, the size of

neurons in the primate cortex doubles during the postnatal

period in both monkey and human [93–95]. Second, after

cessation of neurogenesis and completion of neuronal

migration to the cortical plate, the formation of axonal

connections and synaptogenesis continues at an exponential

pace during the postnatal period in both monkeys and

human [96–98]. Third, postnatal development of the

primate cerebrum is also associated with a massive invasion

of various types of glial and endothelial cells. Although

formation of astrocytes in the macaque monkey begins

before birth [99–101], the major wave of gliogenesis that

includes oligodendrocytes occurs during the neonatal and

postnatal period when these cells are produced in both the

SEZ as well as in the GE [86,87,95]. The BrdU labeled

oligodendrocytes can be seen throughout the white matter

of the forebrain including the corpus callosum. Small ellip-

soid nuclei and bipolar shape resemble migrating neurons,

but their size, distributions along myelinated tracts, their

tinctorial properties and absence of reactivity to neuronal

markers indicate their glial nature [22,23,28]. The majority

of oligodendroglial cells, which in the primate cerebrum

outnumber neurons by severalfold, are generated until

puberty. However, gliogenesis continues during adulthood

in both the SEZ, as well as in situ within the white matter

and cerebral cortex itself throughout the prolonged primate

life span in both monkeys and human [28,82,95,102,103].

4. Evolutionary and medical perspective

With the notable exception of the granule cells in the

dentate gyrus and olfactory bulb, most neurons in the

primate brain including our own, appear to be generated

before birth or the early neonatal period and then persist

during many decades of the primate life span. In contrast,

cells in many other organs of the body are being recycled

every few months [104]. In a more primitive organism such

as hydra and even in lower vertebrates such as amphibians

and reptiles, the capacity for self renewal is much higher, as

their individual organs or appendages can be restored

following amputation [105]. Newts and salamanders are,

for example, capable of a spectacular degree of entire

limb regeneration [106]. This capacity, also called

epimorphic restoration, is not possible in most birds and

mammals. The general decline in capacity for cell renewal

and epimorphic restoration during the course of evolution

has puzzled evolutionary biologists for years. Why should

such a useful ability be lost? One explanation offered was

that, in warm-blooded animals, it has lost survival value.

While cold-blooded animals can survive in their ecological

niche or a sufficient length of time without movement and

food to allow re-grow of a new limb, warm-blooded animals

die from hunger or are killed by predators before their limbs

or organs can be restored (e.g. [105,107]).

The gradual diminishing capacity of neuronal turnover in

the adult brain during vertebrate evolution is equally

puzzling. The heart is the only other organ that is not

capable of renewing its contractile cells under ether normal

or pathological conditions. [108]. Thus, the argument of

‘loss of survival value’ made for the other organs (see

above and Ref. [107]) could also apply to the heart or

brain. In the latter case, the process of neuronal prolifera-

tion, migration and the formation of elongated axons that

form precise synaptic connections takes much more time

than most mammals, in their natural environment, survive.

In spite of these negative evolutionary pressures, it seems

reasonable to explore whether there may be some positive

survival value for keeping the same set of neurons for an entire

life span. The need for a permanent population of neurons may

be a biological necessity for storage of learned behavior in all

species. Even in organisms which have unusual evolutionary

adaptation, like the male canary, that undergoes seasonal turn-

over of particular neuronal types, have a subpopulation of

neurons that are nonrenewable and apparently important in

memory function (e.g. [109]). Could increasing the proportion

of the nondividing neurons that last during the entire life span

in the brains of the mammals help their survival? Could such

neurons have a particular functional significance in primates

including human? I have hypothesized that during vertebrate

evolution the ability for neuronal renewal and regeneration

may have been traded for the ability to retain them throughout

the entire life span in order to enhance retention of long-term

memory and use the learned behavior for their survival [28].

Preservation of acquired information within a permanent

population of neurons in these species may be more valuable

for the survival of an organism than the introduction of ‘naive’

neurons. Is the active population of stem cells found in the SEZ


and subgranular zone which produce granule cells for the

olfactory bulb or dentate gyrus in the adult primate, a rudiment

left from our predecessors, similar to the appendix in the diges-

tive system, which has lost survival value? Or alternatively, is

it a small but important aspect of the adult function in this

particular structure? Irrespective of what the final answer on

these questions may eventually be, it is essential to continue

research on these systems in order to learn how newly formed

neurons are incorporated into the circuitry and what may be

their function in species where this is a more common event.

Understanding the basis of the difference between species may

help to elucidate the significance of the newly generated gran-

ule cells as well as to design a method on how to enhance this

process if deemed functionally useful.

While longevity of neurons may be useful for storage of

long-term memory and life-long experience in the cerebral

neocortex, the decline in capacity for neurogenesis may be

an impediment for replacement of lost neurons in the motor

and sensory systems following trauma, in neurodegenera-

tive disorders or natural aging [110]. With the notable

exception of the granule cells of the dentate gyrus [111],

the neurons in the human central nervous system shows no

sign of natural turnover [83]. The lack of frank regeneration

of neural tissues of the adult human brain is due partly to its

inability to generate new neurons, a limitation that has

severely hindered the development of therapies for neuro-

logical injury or degeneration (e.g. [112]). Although stem

cells in the SEZ of the human brain appears to cease to

produce cortical neurons under normal condition in vivo,

its cells can be induced to produce neurons in vitro indicat-

ing that the suppression may be exerted by local cellular

milieu [114]. Resistance of differentiated neurons to divi-

sion in vivo seems to be so powerful that no malignancy

derived from mature neurons has yet been recorded in the

adult human cerebrum, whereas gliomas derived from glial

cells are very frequent [113]. Thus, identification of the

inhibitory mechanism preventing neuronal proliferation

could be equally as important for cancer research as it is

for restorative neuroscience. The powerful molecular

mechanisms that prevent neurons in adult mammalian

brain to divide are a complete mystery. Therefore, in an

effort to enhance neurogenesis as a form of therapy in the

brain, we must also to increase our knowledge of how and

why capacity for cell division has declined during evolution.

It is our hope that modern biomedical science can overcome

this naturally evolving impediment while simultaneously

preventing uncontrolled neurogenesis formation of tumors

and preservation of an individual’s identity.

Lack of neuronal turnover in the major structures of the

human brain is not a reason for a decline in our efforts to find

a way to replace cells lost due to disease and aging. To the

contrary, it calls for more intense, sophisticated research

aimed at finding how to overcome the natural resistance

of the human brain to spontaneous regeneration than

hitherto has been possible [1]. Indeed, due to development

of ever more sophisticated methods, there are some good

reasons for hope. For example, there is a growing body of

evidence that embryonic and even adult neuronal stem cells

in the SEZ are highly plastic [115–117]. However, to gener-

ate functionally meaningful substitution therapy it is essen-

tial to reproduce sequentially restriction steps that occur

normally during formation of specific neuronal phenotypes

and their point-to-point connectivity. At present, our ability

to direct either endogenous or exogenous stem cells to

migrate, acquire desired neuronal phenotype and finally

incorporate appropriately into an already established adult

neuro- and synaptoarchitecture in order to restore lost func-

tion in a predictable manner is still rather limited [118,119].

While cells in organs such as the liver (where function does

not depend on positional information of the cells) can be

successfully replaced, the projection neurons in large and

stereotypically organized structures such as the cerebral

cortex, acquire their final site and form long distance synap-

tic connections through precise developmental sequences

[120]. So far, these neurons have been refractory to natural

replacement and regeneration in response to injury.

However, if we discover the genes and factors that inhibit

neurogenesis in the adult human brain, we might eventually

find a way to induce neurons in individuals affected with

specific neurodegenerative disorders or following stroke

and trauma, either by stimulation or blocking inhibition of

mitotic activity or by transplanting neuronal stem cells. As a

first step, we need to learn more how to regulate stem cell

differentiation in an unresponsive environment along speci-

fic neuronal and glial cell fates, and also how to make

appropriate synaptic connections [110,112,121–123]. To

generate such connections in the large adult human brain

may not be a trivial task but, as I have argued elsewhere, the

word impossible is not in the vocabulary of contemporary

neuroscience [110].

Acknowledgements

This work was supported in part by the National Institute of

Heath of the U.S. Public Health Service. I am grateful to my

colleagues J.-P. Bourgeois, S. Brand, T. Bump, M. Cooper, B.

Csillik, J. Dekker, M. Eckenhoff, P. Goldman-Rakic, B. Gran-

ger, B. Gould, N. Halfon, E. Knyhar-Csilik, J. Kordower, D.

Kornack, I. Kostovic, M. LaVail, N. Lenn, P. Levitt, M.

Lidow, R. Nowakowski, M. Ogren, P. Piecinski, D. Rapaport,

D. Schmechel, M. Schwartz, R. Sidman, F. Van Eereburg, G,

Wikmark, N. Zecevic and D. Yasamura who, over the years,

have used the collection of autoradiographs in my laboratory

to make crucial observations as well as engender insightful

discussions about the role of timing in brain development. I

also thank Dr. E. Markakis and J. Breunig for helpful

comments on the manuscript.

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Pre- and post-developmental neurogenesis in primates