Necessity of Hippocampal Neurogenesis for the
Therapeutic Action of Antidepressants in Adult
Tarique D. Perera1*, Andrew J. Dwork1,5, Kathryn A. Keegan1, Lakshmi Thirumangalakudi1, Cecilia M.
Lipira1, Niamh Joyce1, Christopher Lange2, J. Dee Higley4, Gorazd Rosoklija1,5,6, Rene Hen1, Harold A.
Sackeim1, Jeremy D. Coplan3
1Department of Psychiatry, College of Physicians and Surgeons, Columbia University Medical Center and New York State Psychiatric Institute, New York, New York, United
States of America, 2Department of Radiology, State University of New York at Brooklyn, Brooklyn, New York, United States of America, 3Department of Psychiatry, State
University of New York at Brooklyn, Brooklyn, New York, United States of America, 4Brigham Young University, Provo, Utah, United States of America, 5Department of
Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University Medical Center and New York State Psychiatric Institute, New York, New York, United
States of America, 6Macedonian Academy of Sciences and Arts, Skopje, Macedonia
Background: Rodent studies show that neurogenesis is necessary for mediating the salutary effects of antidepressants.
Nonhuman primate (NHP) studies may bridge important rodent findings to the clinical realm since NHP-depression shares
significant homology with human depression and kinetics of primate neurogenesis differ from those in rodents. After
demonstrating that antidepressants can stimulate neurogenesis in NHPs, our present study examines whether neurogenesis
is required for antidepressant efficacy in NHPs.
Materials/Methodology: Adult female bonnets were randomized to three social pens (N=6 each). Pen-1 subjects were
exposed to control-conditions for 15 weeks with half receiving the antidepressant fluoxetine and the rest receiving saline-
placebo. Pen-2 subjects were exposed to 15 weeks of separation-stress with half receiving fluoxetine and half receiving
placebo. Pen-3 subjects 2 weeks of irradiation (N=4) or sham-irradiation (N=2) and then exposed to 15 weeks of stress and
fluoxetine. Dependent measures were weekly behavioral observations and postmortem neurogenesis levels.
Results: Exposing NHPs to repeated separation stress resulted in depression-like behaviors (anhedonia and subordinance)
accompanied by reduced hippocampal neurogenesis. Treatment with fluoxetine stimulated neurogenesis and prevented
the emergence of depression-like behaviors. Ablation of neurogenesis with irradiation abolished the therapeutic effects of
fluoxetine. Non-stressed controls had normative behaviors although the fluoxetine-treated controls had higher
neurogenesis rates. Across all groups, depression-like behaviors were associated with decreased rates of neurogenesis
but this inverse correlation was only significant for new neurons in the anterior dentate gyrus that were at the threshold of
Conclusion: We provide evidence that induction of neurogenesis is integral to the therapeutic effects of fluoxetine in NHPs.
Given the similarity between monkeys and humans, hippocampal neurogenesis likely plays a similar role in the treatment of
clinical depression. Future studies will examine several outstanding questions such as whether neuro-suppression is
sufficient for producing depression and whether therapeutic neuroplastic effects of fluoxetine are specific to
Citation: Perera TD, Dwork AJ, Keegan KA, Thirumangalakudi L, Lipira CM, et al. (2011) Necessity of Hippocampal Neurogenesis for the Therapeutic Action of
Antidepressants in Adult Nonhuman Primates. PLoS ONE 6(4): e17600. doi:10.1371/journal.pone.0017600
Editor: Tadafumi Kato, RIKEN Brain Science Institution, Japan
Received November 11, 2010; Accepted February 1, 2011; Published April 15, 2011
Copyright: ? 2011 Perera et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided to Dr. Perera from the National Institute of Mental Health (NIMH) (KO8 MH70954) and NARSAD (Young Investigator Award) and
to Dr. Coplan from NIMH (RO1MH59990). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Major depression is consistently associated with decreased
hippocampal volumes and deficits in hippocampus-dependent
cognition , . Some of these deficits may reflect structural
changes in the hippocampal dentate gyrus. In preclinical studies,
factors that predispose to depression, such as social stress , ,
maternal neglect , and drug abuse  decrease rates of new
neuron formation (neurogenesis) in the dentate gyrus and cause
cell atrophy and death in the CA1/CA3 region of the adult rodent
hippocampus. Interventions that ameliorate major depression,
including antidepressant medications, electroconvulsive therapy
(ECT) , exercise, and environmental enrichment  stimulate
dentate gyrus neurogenesis. These findings led to the hypotheses
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that suppression of neurogenesis leads to depression, and that
stimulation of neurogenesis is required for treating depression ,
. Despite generating widespread interest, this hypothesis is
based mainly on indirect evidence derived mostly from rodents. A
major limitation of rodent studies is that the phenomenological
complexities of major depression are not evident in lower
mammals. In addition, the speed and extent of neuronal
maturation and the proliferation of neuronal precursor cells in
the primate hippocampus is almost 10-fold less than in rodents
. Since there are no established methods of non-invasively
detecting neurogenesis in humans, terminal studies of nonhuman
primates are the best available options to examine the clinical
relevance of these findings. Macaque monkeys are available for
research in larger numbers than apes, and they display a richer
repertoire of affective behaviors than New World monkeys.
Bonnet macaques, in particular, form strong peer attachments
that can be disrupted to produce plausible, core symptoms of
depression , .
In the two previous studies related to neurogenesis in primates,
hippocampal neurogenesis was suppressed in both adult marmoset
monkeys exposed to acute intruder stress  and in juvenile
rhesus macaques exposed to acute prenatal stress . We
reported that treatment with electroconvulsive stimulation (ECS),
the pre-clinical equivalent of antidepressant electroconvulsive
therapy (ECT), stimulated hippocampal cell proliferation and
neurogenesis in adult bonnet macaques . In the current study,
we examined whether the therapeutic behavioral effects of
antidepressant treatment required the induction of neurogenesis
in adult bonnet macaques.
All animal work has been conducted according to relevant
national and international guidelines. In accordance with the
recommendations of the Weatherall report, ‘‘The use of non-
human primates in research.’’ the following statement to this effect
has been included to document the details of animal welfare and
steps taken to ameliorate suffering in all work involving non-
human primates: This work was conducted at the Nonhuman
Primate Facility of the State University of New York Downstate
Medical Center with permission from its Institutional Animal Care
and Use Committee (IACUC) The protocol number is 01-217-04,
approved on 12/16/04. The welfare of the animals conformed to
the requirements of National Institutes of Mental Health (NIMH). All
animals were housed in pens exceeding the stipulated sizes
requirements. Animals were maintained in large group houses
under 12-hour dark and light cycles, and were given access to food
and water ad libitum. Animals were engaged with a variety of
psychologically enriching tasks. No animal was physically harmed
or knowingly exposed to potential infection.
Subjects and interventions: Adult female bonnet macaques were
matched based on age, weight, social rank, and timing of
menstruation, and randomized to a Control pen (n=6) or a
Stress pen (n=6). Using the chronic stress paradigm developed in
rhesus macaques , we exposed subjects in the Stress pen to
social isolation for two days followed by social reunion on the
remaining 5 days, repeated for a total duration of 15-weeks. The
monkeys in the Control pen remained in social housing for those
15-weeks. During this period, half the subjects in each pen
(Control-Drug and Stress-Drug groups) were treated with the
selective serotonin reuptake inhibitor (SSRI), fluoxetine. In order
to minimize the stress of administration, we used Prozac-weekly
preparation (Eli Lilly Corp.), at a dose of 13.5 mg/kg infused via
nasogastric tube (NGT) under sedation (ketamine 5 mg/kg and
xylazine 1 mg/kg), once per week for 15-weeks. This dose was
equivalent to a daily dose of 2 mg/kg of the drug. The remaining
half (Control-Placebo and Stress-Placebo) received the same
treatment with saline placebo via NGT. All groups were injected
with the thymidine analog bromodeoxyuridine (BrdU) (100 mg/
kg/day, I.V.) under ketamine/xylazine sedation for 5 days during
week-7 of interventions (Stress/Control+Drug/Placebo) and then
sacrificed by transcardiac perfusion with normal saline (500 ml/
kg) followed by 4% paraformaldehyde (500 mg/kg) under deep
anesthesia with pentobarbital (15 mg/kg, I.V.) on week-16 of
In a follow-up study, a social group of female bonnet macaques
(Radiation-Stress-Drug group) were anesthetized with ketamine
and xylazine, and given bilateral temporal lobe irradiation at a
dose of 20Gy (n=2) or 30Gy (n=2) fractionated into 10
treatments over 2-weeks. This regimen was based on rodent
studies, where irradiation effectively suppressed neurogenesis
without causing significant necrosis or behavioral side effects
, , . A control group received ‘sham’ irradiation
(anesthesia only) (n=2). A Philips RT250 X-ray machine provided
an X-irradiation beam (250 kVp, 15 mA, 0.4 cm Thoreau’s filter,
HVL=0.3 mm3, 50 cm FSD (Focal Spot to Skin Distance). A
customized shield made of cerabend (a lead alloy) was placed to
cover the head with a large exterior 3.7 cm61.8 cm rectangular
hole (with rounded edges) that was collimated into a small internal
field 2 cm in diameter. The X-ray beam was intended to target the
entire hippocampus while sparing adjacent temporal lobe
structures and the optic chiasm. Before starting irradiation, a
plain film X-ray image was taken to confirm the positioning of the
beam. After 2-weeks of irradiation or sham treatment, the subjects
rested for 3-weeks to allow the acute effects of irradiation to
dissipate as per previous rodent research . Next, these
monkeys were exposed to 15-weeks of repeated separation stress
and concurrent antidepressant treatment with fluoxetine, injected
with BrdU on week-7 and sacrificed on week-16, using the same
methods described above.
Note: Since high- and low-dose irradiation had equivalent
effects, the four irradiated animals were collapsed into one
Radiated-Stress-Drug group (n=4). Similarly, the sham-irradiated
animals were collapsed into the Stress-Drug group (n=5) because
they were exposed to similar experimental conditions. Table 1
presents a schematic diagram of the study design.
Home-cage behaviors were quantified through one-way mirrors
by trained raters (inter-rater correlation coefficient, ICC.0.96) for
3-days a week. The frequency of 40 behaviors typical to bonnet
Table 1. Study Design: Schematic illustrating study design
Time 2 wks 3 wks15 wks
Baseline Control & Placebo (n=3)
Control & Drug (n=3)
BaselineStress & Placebo (n=3)
Stress & Drug (n=5)
XRTRestStress & Drug (n=4)
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macaques  was scored for each animal at 30-second intervals
per session. These behaviors were then collapsed into seven
subscales determined a priori (Table 2). On the final week of
observations (week-15), all animals were subjected to a single (60-
seconds) exposure to a masked human intruder based on
previously established methods . Behavioral ratings were
acquired during 5-minutes of intruder exposure and for 30-
minutes post-exposure. The average behavioral scores for the
seven behavioral subscales were analyzed in 3-week blocks in
order to reduce fluctuations of individual sessions. Repeated
measures analysis of covariance were conducted for each
behavioral subscale, with baseline scores as covariates, and
repeated measures involving time in 3-week time blocks over a
total of 15-weeks. Effects within each behavioral domain were
separately tested using Bonferroni post-hoc testing (p,0.05)
The left hippocampus was cut into 40 mm sections and
immuno-stained to detect and quantify cell proliferation and
neurogenesis rates using our previous methods . Using
standard peroxidase methods we determined the following:
granule cells proliferating at the time of sacrifice (on week-15)
identified by the expression of the mitotic marker Ki67 (mouse
anti-Ki67 antibody, 1:200; Vector Laboratories, Burlingame, CA);
mitotic cells that took up BrdU at the time of injection (week-7)
and survived until sacrifice (week-15) identified by BrdU labeling
(mouse anti-BrdU, 1:200; Becton Dickinson, San Jose, CA); and
new neurons that were still immature, detected by the expression
of the microtubule-associated protein doublecortin (DCX) ,
 (goat anti-doublecortin, 1:200; Santa Cruz, CA). The
secondary antibody was biotinylated anti-mouse IgG (1:200;
Vector Laboratories, Burlingame, CA), visualized with avidin-
biotin complex solution (Vector Laboratories, Burlingame, CA),
and diaminobenzidine (DAB; Sigma-Aldrich, St Louis, MO).
Fluorescent labeling was used to detect the maturational fate of
BrdU-labeled cells by co-labeling with markers of mature neurons,
i.e., NeuN (mouse monoclonal anti-NeuN, 1:200; Chemicon,
Temecula CA), astroglia i.e., GFAP (mouse anti-GFAP, 1:200;
Dako, Carpinteria, CA) , and microglia i.e., Iba-1 (rabbit-anti-
Iba-1, 1:500; Dako, Carpinteria, CA) . Additionally, we
identified dentate gyrus neurons that were synaptically active by
labeling for the immediate early gene c-Fos ,  (rabbit anti-
c-Fos, 1:20,000; Calbiochem, Gibbstown, NJ) and new neurons
that were at a hyperplastic stage of neuronal maturation by
labeling for the NMDA receptor subunit II  (mouse anti-
NMDA-subunit II, 1:25; Millipore, Temecula, CA). The second-
ary antibodies were a mixture of Alexa 568-conjugated goat anti-
rat IgG and Alexa 488-conjugated goat anti-mouse IgG (or anti-
rabbit IgG for Iba-1) (1:200; Invitrogen, Carlsbad CA). Since
doublecortin (DCX) had not been previously used to detect
neurogenesis in bonnet macaques, we confirmed its neuronal
specificity in these animals by demonstrating co-labeling between
DCX and the immature neuronal marker TUC-4 (rabbit anti-
TUC-4, 1:200; Millipore, Temecula, CA) and the absence of co-
labeling with the astroglial marker GFAP . Finally, we
carefully inspected irradiated hippocampal sections for evidence
of inflammation, apoptosis, or necrosis using hematoxylin and
eosin (H & E) and Hoechst stains.
The maturational stage of DCX-expressing cells was deter-
mined by comparing morphology with corresponding DCX-
expressing neurons in the rodent hippocampus . The post-
mitotic age of these cells was estimated by comparing morphology
to the appearance of BrdU-TUC-4 co-labeled hippocampal cells
in adult rhesus macaques (phylogenetically close to bonnets) that
were sacrificed at different time-intervals following BrdU injections
. Using this approach, DCX-expressing hippocampal neurons
were divided into the following three different maturational stages.
Stage 1: DCX-expressing neurons that lack dendrites or have
rudimentary dendrites were estimated to be approximately 1–3
weeks-old because they corresponded to Stage 1 and early Stage 2
according to Ngwenya et al.  (Fig. 2A). Stage 2: DCX-
expressing neurons with dendrites that had secondary branches
extending no further than the inner molecular layer were
estimated to be 3–5 weeks old because they corresponded to late
Stage 2 according to Ngwenya et al.  (Fig. 2A). Stage 3: DCX-
expressing neurons with mature dendrites that had tertiary
branches and extended into the outer molecular layer were
estimated at 6–7 weeks old because they corresponded to late
Stage 3 according to Ngwenya et al.  (thus .5-weeks old) and
because they did not co-label with BrdU (thus ,8-weeks old)
(Fig. 2A). Stage 4: BrdU-labeled cells that co-expressed NeuN
Table 2. Behavioral Categories: Set of 40 behaviors observed during home cage ratings were collapsed into eight subscales for
Domain Behavior Symptoms
1. AffiliationIn close physical contact or proximity of other animals, huddling together.
2. Hedonia Grooming others, toy play, sharing toys with others, playfully provoking dominants, embracing others, genital contact or sexual
3. Exploration Explores cages walls or objects, watches doors for windows or doors for investigators.
4. Self- directedBody scratching, shaking, grooming self.
5. Subordinance Being chased, grabbed, pushed, bitten, or non-sexually mounted by dominant. Displaying subordinance postures or expressions (i.e.,
grimacing, yawning, lip smacking, presenting rear, raised tail, curled feet, rigid posture) when threatened or approached by dominant.
6. Dominance Biting, chasing, growling, grabing, or non-sexual mounting another animal. Staring with canines, scowling, growling or taking
threatening posture directed at subordinate. Inducing subordinance behavior in other animal’s without over threat. Dominance displays
7. Vigilance Pacing or stereotypy ($3 repetitive moves). Anxious posture (i.e., piloerection, freezing/rigid posture, raised tail, curled feet startle
response, yawn) not induced by dominant animal.
8. Anhedonia Alone and immobile, slumped or collapsed body posture, lack of purposeful eye movements or responsiveness to environmental
stimuli, rejecting social advances.
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were deemed to be 8–10 weeks old because BrdU was injected 8–9
weeks prior to euthanasia (Fig. 2B). DCX-expressing cells were
further grouped based on anterior or posterior dentate gyrus
location, given that there appears to be an anterior-posterior
dichotomy in hippocampal function . The anterior dentate
gyrus was defined by coronal levels resembling Bregma-10.88 mm
Figure 1. Behavioral Results. a. Anhedonia scores. Anhedonia scores showed an effect of group (F4,65=5.6, p=0.0001), time (F5,65=17.1,
p,0.0001), group and time interaction (F20,65=3.4, p=0.0001). Bonferroni post-hoc tests showed that Stress-Placebo and Radiation-Stress-Drug
groups had greater anhedonia compared to Control-Drug, Control-Placebo, and Stress-Drug groups by 13–15 weeks (p,0.01). Error bars represent
standard error of mean. b. Hierarchy scores in stressed subjects. Hierarchy (defined as the difference between dominance and subordinance scores)
showed an interaction between group and time (F10, 35=2.1, p=0.04). Bonferroni post-hoc tests showed that the Stress-Drug group had higher
hierarchy scores compared to Stress-Placebo (p,0.01) and Radiation-Stress-Drug (p,0.05) by week 13–15. The controls showed no changes in
hierarchy over the 15 weeks (Fig.S1). c. Affiliation scores in homecage. Affiliation (physical contact and huddling) showed an effect of group (F4,
65=4.1, p=0.023) and time (F5, 65=2.6, p=0.033) and a group by time interaction (F20, 65=2.4, p=0.005). Bonferroni post-hoc tests showed that the
stressed subjects (Stress-Placebo, Stress-Drug, and Radiation-Stress-Drug) had greater affiliation compared to non-stressed subjects (Control-Drug
and Control-Placebo) (p,0.05) during most of weeks 1–15.
Figure 2. Images of Doublecortin-expressing new neurons at different stages of maturation and BrdU-NeuN co-labeled cells. a.
Doublecortin-expressing cells in the SGZ at different stages of maturation. Stage 1. Immature neurons that lack dendrites, or have short dendrites
that lack branches. Stage 2. Differentiating neurons with dendrites that have secondary branches and extend no further than the inner molecular
layer. Stage 3. Neurons with dendrites that have tertiary branches and extend into the outer molecular layer. b. Confocal image of BrdU-NeuN co-
labeled cells. Stage 4 BrdU-labeled cells (green) that also express the mature neuronal marker NeuN (red) with the overlayed images of NeuN and
BrdU labeling (yellow).
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through Bregma-13.50 mm in the rhesus macaque brain atlas by
Paxinos et al. . The rest of the dentate gyrus was designated as
the posterior segment.
All unambiguously labeled (e.g. BrdU, Ki67, DCX, Iba-1, c-Fos
etc.) cells in every 20thsection (average of 15 per animal) of the left
subgranular zone (SGZ) were conducted by two independent
raters (ICC .0.90) who were blinded to animal identity. The SGZ
was defined as a 50 mm band at the border of the granule cell layer
(GCL) and hilus. The total number of counted cells was divided by
the volume of the SGZ (length of SGZ in each section X 50 mm
width X 40 mm thickness before post-mounting shrinkage of 50%–
70%). Rates of labeled cells were expressed as a density per mm3
of the subgranular zone (SGZ). The volume of the granule cell
layer (GCL) of left hippocampal sections stained with 49, 6-
diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO)
was estimated according to the Cavalieri Principle  using an
Olympus BS 52 research microscope fitted with the Olympus
DP72 camera. For all data, repeated measures ANOVA was
conducted using irradiation, stress, and drug treatment as
between-subject variables and counts of new neurons as the
dependent measure with 2 within-subject factors: maturity, with 4
levels (Stages 1–4), and region, with 2 levels (anterior and posterior
dentate gyrus regions).
Exposure of monkeys that received placebo (Stress-Placebo
group, n=3) to repeated social separation stress led to gradual
increases in behavioral scores for anhedonia (a behavioral
composite of collapsed postures, inactivity, and blank stares)
(Fig. 1A) and decreases in scores for hierarchy (total subordinate
behaviors subtracted from total dominant behaviors) (Fig. 1B).
Antidepressant treated cage-mates in the Stress pen (Stress-Drug,
n=3) did not show increases in anhedonia (Fig. 1A) or decreases
in hierarchy (Fig. 1B). Affiliation scores (reflecting enhanced
between-subject contact) were increased in stressed animals,
irrespective of whether they were treated (Stress-Drug) or not
(Stress-Placebo) compared to non-stressed controls (Control-Drug
and Control-Placebo) (F4,65=4.1, p=0.023) (Fig. 1C) throughout
the 15-weeks of repeated separation-stress, with the greatest
increases noted on days of reunion. Affiliation scores were also
increased in the Stress groups compared to Controls in response to
acute human intruder stress on week-15 (F4,65=4.1, p,0.05) (Fig.
S1C). Non-stressed controls that received treatment (Control-
Drug, n=3) did not behaviorally differ from cage-mates that did
not receive fluoxetine (Control-Placebo, n=3) in terms of
anhedonia (Fig. 1A) and affiliation scores (Fig. 1C).
The monkeys that underwent temporal lobe irradiation and
were then exposed to separation stress and antidepressant
treatment (Radiation-Stress-Drug group, n=4) showed progres-
sive increased anhedonia and decreased hierarchy scores com-
pared to sham irradiated (anesthesia only) cage-mates (Stress-
Drug, n=2) (Fig. S1B). These behavioral changes in the irradiated
animals closely paralleled changes in the Stress-Placebo group in
terms of anhedonia (Fig. 1A) and hierarchy (Fig. 1B). By week 13–
15 of stress, anhedonia scores showed an effect of group
(F4,65=5.6, p=0.0001) that resulted from increased scores in
Stress-Placebo and Radiation-Stress-Drug subjects compared to
Control-Drug, Control-Placebo, and Stress-Drug groups (p,0.01)
per Bonferroni post-hoc tests (Fig. 1A). Hierarchy scores also
showed an interaction between group and time (F10,35=2.1,
p=0.04) by week 13–15 with decreases in Stress-Placebo (p,0.01)
and Radiation-Stress-Drug (p,0.05) groups compared to Control-
Placebo subjects per Bonferroni post-hoc tests (Fig. 1B). The only
exception was a transient increase in anhedonia scores in the
Radiation-Stress-Drug group immediately after the completion of
irradiation (XRT) in the absence of stress (Fig. 1A). The non-
stressed Controls showed no increases in anhedonia (Fig. 1A) or
changes in hierarchy over 15-weeks (Fig. 1B and Fig. S1A). There
were no significant group differences in any of the other behavioral
categories, i.e., self-stimulation, environmental exploration, and
vigilance (data not shown), and there were no manifestations of
physical distress or weight change.
Total neurogenesis rates, represented by the density of DCX-
expressing cells in the subgranular zone, were increased in the
Stress-Drug and Control-Drug groups and decreased in the Stress-
Placebo and Radiation-Stress-Drug group, compared to rates in
the Control-Placebo group (p,0.05) (Fig. 3A). Repeated measures
ANOVA showed within-subjects interactions of neuronal maturity
(stages 1–3) and region (anterior or posterior) that were of
borderline significance (F2,4=3.3, p=0.05). Between-subject
effects were significant for drug (F13,91=26.6, p,0.001) and
irradiation (F13,91=19.4, p=0.001), but not for stress (F13,91=0.6,
p=0.45). When examining between-subject effects of maturity
and region, there were significant interactions of region, maturity,
and stress (p=0.006) and region, maturity, and drug (p=0.04).
Bonferroni post-hoc tests showed that between-subjects effects
were only significant for Stage 3 of maturity (in DCX-expressing
cells) and the anterior region of the dentate gyrus (p,0.01)
(horizontal bar in Fig. 3A). Compared to the Control-Placebo
group, fluoxetine treatment increased Stage 3 anterior dentate
gyrus neurons in the Stress-Drug and Control-Drug groups by
250% and 300%, respectively, while these rates were suppressed in
the Stress-Placebo and Radiation-Stress-Drug groups by 84% and
90%, respectively (Fig. 3A). Further characterization showed that
DCX-expressing granule cells at Stage 3 of maturity did not co-
label with BrdU, NeuN, c-Fos, or the NR2B subunit of the NMDA
receptor (not shown).
The rates of cell proliferation at the time of sacrifice (Ki67-
expressing cells) was decreased intheStress-Placebogroup (p,0.01)
when compared to Control-Placebo, while subjects treated with
fluoxetine (Stress-Drug, Control-Drug) showed modest increases
There were no significant group differences in precursor survival
(BrdU-labeled cells) across all groups (Fig. 4A). The maturational
speed ([number of DCX-expressing neurons at Stage 3 of
maturation]/[total DCX-expressing neurons]) of Drug-treated
groups (Control-Drug and Stress-Drug) was significantly increased
when compared to Placebo groups (Control-Placebo and Stress-
Placebo), and to the Irradiation-Stress-Drug group (p,0.001)
(Fig. S2A). In terms of maturational fate of proliferating cells in
the non-irradiated subjects (Control-Drug, Control-Placebo, Stress-
Drug, Stress-Placebo), 52.9%614.9% of BrdU-labeled cells
co-labeled fortheneuronal markerNeuN, compared toa significant
decrease of 21.2%611.6% in irradiated subjects (Radiation-Stress-
Drug) (p,0.01) (Fig. 4B). By contrast, 64.0%68.9% of BrdU-
labeled cells in the irradiated subjects expressed Iba-1 and displayed
microglial morphology compared to just 8.9%67.5% in the non-
irradiated subjects (Fig. 4B), which were shown to be significantly
different (p,0.0001). There was no increase in astrocytosis,
apoptosis, or necrosis per H & E staining and Hoechst staining
(not shown) nor higher rates of microglial cells (Iba-1 positive,
BrdU-negative) cells in the Radiation-Stress-Drug group compared
to the Stress-Drug subjects (Fig. S2B).
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Correlation between behavior and histology
We conducted linear regression analysis of correlations between
anhedonia ratings and neurogenesis rates including all five groups
(Fig. 3B). Anhedonia scores at each of the five different
observational periods served as the dependent variable and the
six subdivisions of neurogenesis rates served as independent
variables. The analyses revealed that anhedonia scores at the final
assessment block (weeks 13–15) inversely correlated with the
density of new neurons that were in the anterior dentate gyrus and
at Stage 3 of development (r2=0.67, p,0.0001) (Fig. 3B). By
contrast, the number of immature Stage 1 and 2 neurons in the
anterior dentate gyrus and the number of neurons at any
maturational stage in the posterior dentate gyrus did not correlate
with anhedonia scores at any of the behavioral assessment periods.
Anhedonia scores at weeks 10–12 inversely correlated with the
density of BrdU-NeuN co-labeled cells at Stage 4 of development
in the anterior dentate gyrus (r2=0.28, p=0.02) (Fig. 3B). The
only other histological measure associated with behavioral scores
was the rates of c-Fos expression in the anterior dentate gyrus,
shown by the density of c-Fos-BrdU co-labeled cells (Fig. S3A),
which positively correlated with anhedonia scores at the final time
point during weeks 13–15 (r2=0.31, p=0.008) (Fig. S3B). In
terms of GCL volume, there were no overall group differences, but
Bonferroni post hoc comparisons indicated significant volume
reductions in the Stress-Placebo and Radiation-Stress-Drug
(p,0.01) groups when compared with Control-Placebo monkeys
Our results show that bonnet macaques exposed to repeated
social separation stress display depression-like features associated
with suppression of hippocampal neurogenesis. Treatment with
the antidepressant fluoxetine blocked emergence of depressive
behaviors and stimulated neurogenesis. Ablating neurogenesis
with temporal lobe irradiation abolished the salutary effects of the
antidepressant and led to depression-like behavior in response to
chronic stress. Together, these results provide the first evidence
that hippocampal neurogenesis may play a role in the treatment of
depression in NHPs similar to previous findings in rodents .
By using a nonhuman primate paradigm, we had the
opportunity to more effectively replicate depression-related
behaviors. The bonnet macaques exposed to repeated social
separation stress displayed increases in anhedonia that involved a
cluster of symptoms typically seen in depressive monkeys ,
including macaques . Critically, this behavioral profile
possesses significant face validity as an analog of clinical
anhedonia, a core symptom of major depression . The
increases in the anhedonia scores were accompanied by decreases
in hierarchy scores. Social subordinance is a hallmark of both
chronic anxiety and depression in monkeys , . This
behavior of the NHPs in the setting of repeated social separation
stress parallels clinical depression, as interpersonal loss is the
predominant trigger of depression in humans  and chronic
stress is a major epidemiological risk factor for major depression
and chronic anxiety disorders .
The animals treated with fluoxetine did not demonstrate
anhedonia-related behaviors. This is consistent with its therapeutic
effect and with previous findings that sertraline, another SSRI,
ameliorated anxiety-related behaviors and alcohol abuse in rhesus
macaques that were exposed to repeated separation stress .
The therapeutic effects of fluoxetine in the stressed animals could
not be explained by medication side effects because behavioral
ratings in drug- and placebo-treated non-stressed controls did not
differ. These behavioral changes did not result from emotional
indifference, a potential side effect of fluoxetine treatment ,
because the drug-treated animals showed the same level of distress,
in the form of affiliation, as placebo-treated subjects at the time of
social reunion. Affiliation, an indicator of acute anxiety in bonnet
macaques , increased not only during reunions but also during
acute intruder stress. In summary, fluoxetine treatment specifically
prevented stress-induced depressive behavior (anhedonia) and
chronic anxiety behavior (subordinance), but had no impact on
acute anxiety (affiliation). These behavioral effects of the
medication are consistent with its therapeutic profile in humans
Fluoxetine treatment had a profound effect on neurogenesis:
non-irradiated fluoxetine-treated animals showed high rates of
newly formed neurons at all stages of maturation in both the
anterior and posterior dentate gyrus compared to their placebo-
treated and irradiated counterparts. Fluoxetine treatment seems to
have induced neurogenesis by increasing the speed of neuronal
maturation, similar to its effects in mice , without increasing
precursor cell survival (BrdU-labeled cells) or proliferation (Ki67-
expressing cells). This is in contrast to the significant increase in
proliferation and 600% increase in neurogenesis seen after ECT
, and parallels the more robust clinical efficacy of ECT in
treating depression compared to pharmacological agents such as
fluoxetine . Moreover, in the present study, chronic stress
suppressed neurogenesis and cell proliferation in general, a pattern
previously reported in adult rodents and tree shrews exposed to
chronic stress , . This is in contrast with recent evidence in
rodents that chronic stress reduces the level of neuronal
differentiation without decreasing proliferation rates .
Despite dramatic increases in new neurons following fluoxetine
treatment, only a subpopulation of these, the Stage 3 DCX-
expressing cells located in the anterior dentate gyrus, correlated
with behavioral changes (anhedonia and hierarchy). The anatomic
location of these new neurons in the anterior dentate gyrus is
intriguing in light of the evidence that the anterior (ventral or
temporal in rodents) hippocampus specifically mediates limbic
behavior  via anatomic connections with the amygdala and
prefrontal subregions  and supports the hypothesis that the
antidepressant effect is linked with neurogenesis only in the
Figure 3. Neurogenesis rates. a. Neurogenesis rates based on maturational stage and regional distribution Neurogenesis rates at the three
maturational stages were divided based on location in the anterior (left panel) or posterior (right panel) dentate gyrus. Multivariate analysis
conducted on these six subdivisions showed an overall effect of experimental group (F4,5=90.01, p,0.0001). Univariate analysis of experimental
group effects for each of the six sub-divisions of neurogenesis rates revealed significant effects only for Stage 3 neurons in the anterior dentate gyrus
(F4,5=22.25, p,0.0001). Bonferroni post-hoc tests showed that only Stage 3 anterior dentate gyrus neurons were reduced in both depressive groups
(Stress-Placebo and Stress-Drug-Radiation) compared to non-depressed groups (p,0.05). Data were Log10transformed for statistical analysis to
correct for uneven variance. b. Correlation between anhedonia scores and neurogenesis rates. Multiple regression analysis was conducted between
anhedonia scores obtained at 5 different observation periods and the 6 subdivisions of neurogenesis rates. Anhedonia rates at the final observation
period (weeks 13–15) inversely correlated only with Stage 3 anterior dentate gyrus neurons (r2=0.52, p,0.001). Since the regression curve appeared
non-linear, a second degree polynomial fit was found to be the best (r2=0.67, p,0.0001). Anhedonia rates at weeks 10–12 inversely correlated with
Stage 4 anterior dentate gyrus neurons (r2=0.28, p=0.02). Likewise, a second-degree polynomial fit was found to be the best (r2=0.34, p,0.01).
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Figure 4. a. Left panel: Precursor cell survival rates. BrdU-labeling represented proliferating hippocampal precursors that took up
BrdU on week-7 and survived until sacrifice on week-15. Log10transformed rates of BrdU-labeled cells in the SGZ did not show an effect of
experimental group (F4,13=1.7, p=0.46). b. Right panel: Precursor cell proliferation rates. Hippocampal cell proliferation rates at the time of sacrifice
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anterior region of the dentate gyrus . Likewise, neurogenesis
rates in the posterior dentate gyrus failed to correlate with
depressive behavior, consistent with the notion that the posterior
hippocampus (dorsal or septal in rodents) mainly mediates spatial
The specific association between depressive behavior and Stage
3 neurons was surprising, however. Although these cells had
mature dendrites, they expressed immature neuronal markers
(DCX and TUC-4), but not NeuN. They also failed to express c-
Fos, suggesting that they did not participate in activation of
synaptic circuitry, as well as the NR2B subunit of the NMDA
receptor. These findings suggest that the Stage 3 granule cells had
not entered the hyperplastic stage of neuronal maturation
described in the adult mouse hippocampus. During this stage, at
4–6 weeks of age, differentiating granule cells enter a period of
enhanced synaptic plasticity associated with NR2B expression and
a lower threshold for LTP . External stimuli preferentially
activate these hyperplastic new neurons over less excitable mature
granule cells . Although presumed to be 6–7 weeks old, the
Stage 3 neurons in the bonnet macaque seemed be less mature
than the hyperplastic cells described in the mouse , and
resembled younger, 3-week-old, mouse granule cells ,
reflecting the fact that duration of neuronal maturation in
monkeys is 2–3 fold longer than in rodents . Nonetheless,
the fact that granule cells that were presumably 6–7 weeks old
displayed an immature histological profile is surprising because in
our previous study approximately 50% of 4-week old granule cells
expressed NeuN following ECS treatment , and this rate of
maturation is consistent with several other studies in nonhuman
primates , , , . It is possible that primate neuronal
maturation takes many months because TUC-4-BrdU co-labeling
has been detected ,100 days post-BrdU injection . On the
other hand, it has been shown that TUC-4 can occasionally persist
in mature neurons in the monkey brain .
The fact that behavioral changes correlated with the density of
newly-formed Stage 3 neurons that had not yet entered the
were identified by the expression of Ki67. Log10transformed rates of Ki67 expressing cells showed an overall effect of group (F4,13=6.8, p,0.001)
stemming from decreased counts in Stress-Placebo compared to all other groups per Bonferroni post-hoc tests (p,0.01). b Left panel: Maturational
fate of BrdU-labeled cells on week 7. The percentage of BrdU-labeled cells that co-labeled with NeuN was designated as new neurons and BrdU-
labeled cells that co-labeled with Iba-1 were designated as microglia. Two-way ANOVA showed an overall interaction between group and
maturational stage (P,0.0001), as well as an effect of group (p=0.036), and maturational fate (p,0.0001). Bonferroni post-hoc tests showed greater
levels of BrdU-NeuN co-labeling (p,0.01) and lower levels of BrdU-Iba-1 co-labeling (P,0.001) in the 4 non-irradiated subjects (Control-Drug,
Control-Placebo, Stress-Drug, and Stress-Placebo) compared to irradiated subjects (Radiation-Stress-Drug group).
Figure 5. Volume of granule cell layer. Two-way ANOVA with Bonferroni post-hoc comparisons showed a significant decrease in granule cell
layer volumes of Stress-Placebo and Radiation-Stress-Drug groups when compared with control-placebo (p,0.01).
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hyperplastic stage suggest that any role in mood regulation would
have to involve indirect mechanisms. This role may involve
reducing nonspecific activation in the dentate gyrus, since the rate
of biochemical synaptic activation (c-Fos expression) of mature
granule cells in the anterior dentate gyrus was reduced in bonnet
macaques with high rates of neurogenesis and positively correlated
with increases in depressive behavior (anhedonia ratings). This
potential role is supported by a study where depressive behavior
produced by bulbectomy in rats was associated with increased
hippocampal c-Fos expression, while the reduction of depression
with fluoxetine treatment decreased hippocampal c-Fos expression
. Neurogenesis may also play a role in reducing interference of
older memories, thereby facilitating the acquisition of novel
experiences, as it has been proposed to occur in rodents . The
reduction of synaptic excitability of mature granule cells by
immature neurons provides a putative mechanism by which this
reduction of interference may occur.
In the irradiation group, the animals showed increases in
anhedonia and decreases in hierarchical behaviors despite antide-
pressant treatment, similar to the Stress-Placebo group. Although
irradiation was administered at the beginning of the study, these
animals did not display sustained increases in depressive behaviors
until they were exposed to several weeks of stress. There was,
however, a transient increase in anhedonia scores immediately
following irradiation prior to the initiation of separation stress.
These symptoms appear to have resulted from sedation or side
effects of irradiation because the symptoms increased acutely (not
gradually), were unaccompanied by hierarchical changes, and
dissipated within 3-weeks, which is the typical duration of acute
behavioral side effects of irradiation in mice .
The depressive behavior seen in the irradiated animals was
associated with depressed levels of neurogenesis and altered
maturational fate (the majority of the BrdU-labeled cells
differentiated into microglia rather than neurons). The neurosup-
pressive effect of irradiation was subtler in the monkeys than in
mice, where irradiation abolished all possibility of neurogenesis for
months , . In our subjects, cell proliferation resumed by
weeks 13–16. Cell proliferation rates in the irradiated monkeys
were normal by the time of sacrifice, with a majority of newly
formed cells expressing Iba-1 and displaying microglial morphol-
ogy. The total number of microglia, however, remained
unchanged. Cell proliferation probably resumed over the 19
weeks between irradiation and sacrifice, likely potentiated by
fluoxetine treatment in the interim. Irradiation may have reduced
neuronal differentiation of mitotic stem cells, as shown by Steele
and Lange , possibly by delaying the commitment of precursor
cells to neuronal fate, as recently reported by our collaborators
(Dranovsky, Hen et al., Science, in press). The predominance of
microglia in this population raises the question of whether these
mitotic cells represented an inflammatory reaction to irradiation,
as has been reported in rodents . Reduced neurogenesis
accompanied by microglial proliferation has also been reported in
mice  and rhesus macaques  exposed to brain injury. That
these cells represent an inflammatory reaction is unlikely since
both H & E staining and Hoechst staining failed to show evidence
of increased astrocytosis, apoptosis, or necrosis in the irradiated
hippocampi, and the total number of microglia was unaffected.
This suggests that even if inflammatory macrophagic extravasation
took place, and gave way to proliferation and differentiation into
new microglial cells as late as weeks 7–8 (time of BrdU injection),
then the stimulating inflammation response must have resolved by
the time of sacrifice (week 15).
Because the method of ablation used in this study was relatively
nonspecific, it was important to ascertain that the depressive
behavior detected in the Radiated-Stress-Drug group did not
result from delayed complications of irradiation such as inflam-
mation and necrosis , . To this end, we verified that the
salient behavioral differences in the bonnet macaques occurred
immediately prior to the time of sacrifice (week 13–15), at which
point there was no evidence of inflammation or necrosis in the
irradiated hippocampi despite extensive histopathological inspec-
tion. Therefore, we conclude that the loss of antidepressant
efficacy in the irradiated monkeys most likely resulted from the
reduction of neurogenesis.
In addition to reducing neurogenesis rates, chronic stress also
reduced hippocampal granule cell layer (GCL) volume by 22% in
the Stress-Placebo group and by 25% in the Radiation-Stress-
Drug group, as compared with the Control-Placebo group. This is
in agreement with previous research in rats , , and tree
shrews . Only one primate study has examined GCL volume
after prenatal stress and found significant reductions in juvenile
rhesus macaques . Fluoxetine, on the other hand, may have
prevented stress-induced reduction in GCL volume, as GCL
dimensions in the Stress-Drug group did not differ from those of
Control-Placebo subjects. This finding is in agreement with the
prevention of stress-related shrinkage in GCL volume in tree
shrews treated with the antidepressant tianeptine . Temporal
lobe irradiation abolished any protective effect of fluoxetine and
decreased GCL volume in the Radiation-Stress-Drug group. This
was not surprising because irradiation has been shown to decrease
brain volume in fetal rhesus monkeys .
The interpretation of these data needs to be tempered by the
small sample size. This is an unavoidable problem intrinsic to
nonhuman primate research because of the limited availability of
subjects. Nevertheless, conducting this experiment in monkeys
revealed subtleties in the association between hippocampal
neurogenesis and limbic behavior that not could not be detected
in rodents , , for instance, that the regulation of
neurogenesis was associated with changes only in depressive
(anhedonia) and chronic-anxiety (subordinance) symptoms and
was unrelated to changes in acute anxiety phenomena (affiliation
and vigilance). Using NHPs also allowed us to localize and stage
the population of new neurons correlated to behavioral changes in
the primate brain, which differs in both anatomy and cellular
kinetics to that of the rodent.
An outstanding question that warrants investigation is whether
suppression of neurogenesis was sufficient to produce depressive
behavior in the absence of stress. Although neurogenesis was
acutely suppressed in the irradiated group, appearance of
depressive behavior was delayed by several weeks. This suggests
that neuro-suppression per se was not sufficient for producing
depression in the irradiated monkeys and that exposure to several
weeks of chronic stress was also needed. This view is supported by
studies in which chronic uncontrollable stress consistently
suppresses neurogenesis in rats , but only a fraction of these
animals developed learned helplessness . Although suppression
of neurogenesis seemed insufficient to produce acute depression-
like behavior in the irradiated animals, the later appearance of
these behaviors despite fluoxetine treatment demonstrate that
stimulating neurogenesis was necessary for mediating antidepres-
sant efficacy. This has also been reported in mouse studies, in
which ablating hippocampal neurogenesis did not produce
behavioral abnormalities at baseline (in the absence of treatment),
but abolished the therapeutic effects of antidepressant treatment in
the setting of stressful conditions , .
Another outstanding issue, highlighted previously by Wang et al.
 that future studies must address is whether neurogenesis plays
a similar role in the treatment of preexisting depressive symptoms
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(similar to its clinical application), as it does in the prevention of
depression during chronic stress. While the mechanisms needed to
prevent depression are likely similar to those critical for reversing
depression, it will be necessary to confirm this with a specifically-
tailored study design. Future studies should also examine whether
the timing of antidepressant response correlates with induction of
neurogenesis. From our data, we assume that Stage 3 neurons take
6–7 weeks to develop in monkeys and maybe longer (perhaps 8–9
weeks) in humans. This implies that antidepressants would need at
least 2-months to up-regulate this population in depressed patients.
This speed of induction is too long to explain initial response to
antidepressants, but is more consistent with the 8–10 weeks needed
for complete remission of clinical depression . There is some
evidence that clinical remission is signaled by cognitive improve-
ments that appear weeks after the initial response .
Interestingly, blocking hippocampal neurogenesis disrupts analo-
gous cognitive function in rodents  several weeks after ablation
. Therefore, it is conceivable that hippocampal neurogenesis
mediates delayed cognitive improvements associated with remis-
sion of depression but has no role in initial affective response to
treatment (see review by Perera et al. ).
This study provides strong support for a link between the
treatment of depression and the regulation of a specific population
of new hippocampal neurons. The fact that this relationship was
demonstrated in a plausible model of depression in anthropoid
monkeys lends strong support for a similar role for neurogenesis in
humans. Future studies are needed to confirm and expand these
findings. If they do, new hippocampal neurons may serve as a
marker of risk for depression and provide a tangible target for
developing more effective and tolerable antidepressants.
of stress, the hierarchy scores and rank did not differ throughout
the 15-week period of testing. b. Anhedonia scores in irradiation
(XRT) pen. The XRT pen housed subjects that were irradiated
(Radiation-Stress-Drug group, n=4) and matched cage-mates that
received sham irradiation (anesthesia only) (Stress-Drug group,
n=2). Compared to the control-placebo group, the irradiated
subjects showed increases in anhedonia ratings at two time points:
during the 3-week baseline period (immediately following
irradiation, prior to stress/drug exposure) and during weeks 10–
15 of stress/drug exposure (p,0.05). c. Affiliation scores during
acute intruder stress on week 15. Intruder stress at week-15
a. Hierarchy scores in control subjects. In the absence
increased affiliative behavior in the Stress-Placebo and Stress-Drug
groups compared to Control-Placebo groups (p,0.05). There was
no effect of drug treatment.
maturational speed was calculated as the percentage of DCX-
expressing neurons with mature dendrites (Stage 3) among all
DCX-expressing cells (Stages 1–3). One-way ANOVA showed an
overall effect of group (p=0.001) and Bonferroni’s multiple
comparison post-hoc test showed that the fraction of DCX Stage 3
cells was significantly higher in the drug-treated groups (Control-
Drug and Stress-Drug) compared to Placebo-treated groups
(Control-Placebo and Stress-Placebo), and to irradiated subjects
(Radiation-Stress-Drug) (p,0.05). b. Total number of microglia.
The total number of microglia did not differ between irradiated
subjects and non-irradiated subjects (p=0.8127). c. Fluorescent
images of microglial cells. Images of newly-generated microglia
where fluorescent images Iba-1-expressing cells (red), BrdU-
expressing cells (green), and DAPI-expressing cells (blue) were
a. Maturational speed: Drug Vs. Placebo.The
dentate gyrus. The density of c-Fos-BrdU co-labeled neurons
(mm3) in the anterior SGZ did not differ across all groups
(p=0.16).b. Correlation between Anhedonia scores and c-Fos
expression in the anterior dentate gyrus. Anhedonia scores on
weeks 13–15 correlated with increases in c-Fos expression in the
anterior dentate gyrus (r2=0.31, p=0.008), but not with c-Fos
expression in the posterior dentate gyrus (not shown).
a. c-Fos-expressing granule cells in the anterior
We wish to thank the following: Dr. Elizabeth Gould for immunohistology
protocols and assistance with confocal microscopy. We acknowledge Dr.
Bruce Scharf, Dr. Leonard Rosenblum, Ms. Yelena Nemirovskaya, Ms.
Natasa Draskovic, Ms. Laura Anderson, and Mr. Mohammed Anwar for
Conceived and designed the experiments: TP HS AD JC RH. Performed
the experiments: TP KK NJ CL LT DH GR. Analyzed the data: TP JC
AD. Contributed reagents/materials/analysis tools: TP AD JC. Wrote the
paper: TP CL LT JC KK.
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Neurogenesis Necessity for Antidepressants Action
PLoS ONE | www.plosone.org13 April 2011 | Volume 6 | Issue 4 | e17600