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Distinct structural plasticity in the hippocampus and amygdala of the middle-agedcommon marmoset (Callithrix jacchus)

Michael W. Marlatt a, Ingrid Philippens b, Erik Manders c, Boldizsár Czéh d,1, Marian Joels a,e,Harm Krugers a, Paul J. Lucassen a,⁎a Swammerdam Institute for Life Science — Center for Neuroscience, University of Amsterdam, Amsterdam, The Netherlandsb Biomedical Primate Research Centre (BPRC), Rijswijk, The Netherlandsc Center for Advanced Microscopy, University of Amsterdam, Amsterdam, The Netherlandsd Clinical Neurobiology Laboratory, German Primate Center, Leibniz Institute for Primate Research, Göttingen, Germanye Department Neuroscience & Pharmacology, University Medical Center Utrecht, The Netherlands

a b s t r a c ta r t i c l e i n f o

Article history:Received 16 December 2010Revised 25 March 2011Accepted 6 May 2011Available online 14 May 2011

Keywords:Adult neurogenesisStressNonhuman primateDoublecortinPSA-NCAMAging

Adult neurogenesis in the primate brain is generally accepted to occur primarily in two specific areas; thesubgranular zone (SGZ) of the hippocampal dentate gyrus (DG) and the subventricular zone (SVZ) of thelateral ventricles. Hippocampal neurogenesis is well known to be downregulated by stress and aging inrodents, however there is less evidence documenting the sensitivity of neuroblasts generated in the SVZ. Inprimates, migrating cells generated in the SVZ travel via a unique temporal stream (TS) to the amygdala andentorhinal cortex. Using adult common marmoset monkeys (Callithrix jacchus), we examined whether i)adult-generated cells in themarmoset amygdala differentiate into doublecortin-positive (DCX+) neuroblasts,and ii) whether lasting changes occur in DCX-expressing cells in the DG or amygdala when animals areexposed to 2 weeks of psychosocial stress.A surprisingly large population of DCX+ immature neurons was found in the amygdala of these 4-year-oldmonkeys with an average density of 163,000 DCX+ cells per mm3. Co-labeling of these highly clustered cellswith PSA-NCAM supports that a subpopulation of these cells are migratory and participate in chain-migrationfrom the SVZ to the amygdala in middle-aged marmosets. Exposure to 2 weeks of isolation and social defeatstress failed to alter the numbers of BrdU+, or DCX+ cells in the hippocampus or amygdala when evaluated2 weeks after psychosocial stress, indicating that the current stress paradigm has no long-term consequenceson neurogenesis in this primate.

© 2011 Elsevier Inc. All rights reserved.

Introduction

Adult neurogenesis occurs in at least two specific locations in thebrains of adult mammals: the subventricular (SVZ) and subgranularzones (SGZ) located in the lateral ventricles and hippocampal dentategyrus respectively. These zones are unique, specialized micro-environments that support the production of new cells. They contain

neural stem cells (NSCs) with the capacity to proliferate, migrate, anddifferentiate into adult, functional neurons. Neuronal differentiationof adult-generated cells represents a novel form of structural plasticitywith considerable relevance for olfaction and cognition (Dupret et al.,2008; Lledo et al., 2006). This process can be identified throughmarkers such as Doublecortin (DCX), amicrotubule associated proteinfound selectively in immature, migratory neurons. DCX is required forthe proper migration of immature neurons (Bai et al., 2003; Francis etal., 1999) and is considered an endogenous marker for adultneurogenesis (Brown et al., 2003; Rao and Shetty, 2004).

In the hippocampus, immature neurons migrate from the SGZ intothe granule cell layer (GCL) of the dentate gyrus (DG) where theymature into granule neurons. Similarly, neuroblasts born in the SVZmigrate tangentially towards the olfactory bulb along the rostralmigratory stream (RMS)(Kornack and Rakic, 2001; Lois and Alvarez-Buylla, 1994), an area that has recently also been identified in humanbrain (Curtis et al., 2007). Migratory, DCX-positive (+) neuronspresent in the primate SVZ were found to co-express polysialylatedneural cell adhesion molecule (PSA-NCAM), identifying a migratory

Experimental Neurology 230 (2011) 291–301

Abbreviations: SVZ, subventricular zone; SGZ, subgranular zone; NSCs, neural stemcells; DG, dentate gyrus; GCL, granule cell layer; RMS, rostral migratory stream; BrdU,bromodeoxyuridine; tLV, temporal horn of lateral ventricles; TS, temporal stream; DCX,doublecortin; PSA-NCAM, polysialylated neural cell adhesion molecule; BM, basalmedial; BLVM, basolateral ventral medial amygdala nucleus; BLI, basolateral interme-diate amygdala nucleus; EC, entorhinal cortex; VACo, ventral cortical amygdaloidnucleus; Me, medial; La, lateral amygdala.⁎ Corresponding author at: SILS-CNS, University of Amsterdam, Science Park 904,

1098 XH Amsterdam. Fax: +31 20 525 7709.E-mail address: P.J.Lucassen@uva.nl (P.J. Lucassen).

1 Present address: Molecular Neurobiology, Max Planck Institute of Psychiatry,Munich, Germany.

0014-4886/$ – see front matter © 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.expneurol.2011.05.008

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pathway to the striatum (Bedard et al., 2006). In parallel with othermarkers of neurogenesis, DCX expression in the rodent hippocampusdecreases strongly with age and is reduced after stress as well (Heineet al., 2004a, b; Lucassen et al., 2010; Oomen et al., 2007; Rao et al., etal., 2006).

A distinct, less well characterized migratory stream exists inprimates allowing neuroblasts to migrate directly to the amygdala, anarea associated with emotional memories (Roozendaal et al., 2009).The temporal stream (TS) has only been described in rhesus andsquirrel monkeys, however due to structural homology it is suspectedto exist in other primates. A cell fate study combined with migrationof lipophilic dye identified a stream of cells extending from thetemporal horn of the lateral ventricles (tLV) to the amygdala andpiriform cortex (Bernier et al., 2002). The contribution of cells foundin the TS to the amygdala has yet to be quantified.

Here, we studied neurogenesis in the hippocampus, amygdala andassociated cortex of four-year-old, middle-aged marmoset monkeysusing BrdU and DCX immunocytochemistry and further establishedthe migratory nature of the DCX+ immature neurons using co-labeling for PSA-NCAM. Secondly, although stress and stress hormoneexposure reduces neurogenesis in the DG of young rodents andmonkeys (Czeh et al., 2002; Gould et al., 1998; Oomen et al., 2007), itwas not known whether psychosocial stress affects the DG andamygdala to the same extent in this primate species. In rodents,reductions in neurogenesis after psychosocial stress are long lastingand can persist despite a normalization of cortisol levels (Buwalda etal., 2005; van Bokhoven et al., 2011). Stress has also been shown toreduce neurogenesis in adult primates (Gould et al., 1998; Pencea etal., 2001). We thus studied whether isolation and social defeat stressaltered neurogenesis (DCX+) and newborn cell survival (BrdU+) in alasting manner in the marmoset DG and amygdala and studied their

brains following an additional 2 week recovery and resocializationperiod in order to mitigate acute effects and allow a normalization ofthe initial cortisol rises.

Materials and methods

Stress exposure in adult marmosets

All experimental procedures were approved by the AnimalExperimentation Committee of the Dutch Institute of AppliedScientific Research (TNO-DEC # 2148). All aspects of animal care aredescribed in Standard Operating Procedures in agreement with thecurrent guidelines of the European Community. Ten 4-year-old adultmale (n=5) and female (n=5) common marmosets (Callithrixjacchus) were subjected to a 6-week experiment. All monkeys werebred and raised at the Biomedical Primate Research Centre (BPRC),Rijswijk, The Netherlands, and randomized to stress (n=5, 3:2 male:female) and control groups (n=5, 3:2 male: female) prior to the startof the study. The stress protocol (Fig. 1), a 6-week paradigm, wascomposed of three segments; normal housing for behavioralobservation (days 0–12), isolation stress and social defeat (stresscohort only) (days 12 to 27), followed by a re-socialization period of2 weeks (days 27–40). This design was employed in order to studywhether social stress had lasting consequences on neurogenesis andstructural plasticity.

During the second week of isolation, animals from the stresscohort were placed in the territory of a dominant monkey of the samesex for 10 min to additionally expose them to a social-defeatparadigm. Animals were observed and tested during these periodsusing separate read-outs including behavioral observation and bodyweight. Cortisol concentrations were determined in 24-hour urinesamples collected in metabolic cages. Samples were collected twiceduring the normal housing, once in the second week of the isolationperiod and once at the end of the experimental period directly out ofthe bladder during euthanasia. 25 μl of the urine sample was appliedin antibody-coated tubes in duplicate using commercial radioimmu-noassays (Corti-Cote) according to standard procedures (MP Bio-medicals Europe, Illkirch France). Subsequently, 0.5 ml of “cortisoltracer solution” with 125I-cortisol was applied, which binds incompetition with the urine cortisol. After incubation of 45 min at37 °C the liquid was removed. This was followed by separation ofbound and unbound radioactivity and counting of bound radioactivityto determine cortisol concentration.

Bromodeoxyuridine injection and immunohistochemistry

To analyze whether stress exposure had affected the survival rateof newborn cells, 5-bromo-2′-deoxyuridine (10 mg/ml dissolved in0.007 MNaOH/0.9% NaCl, at a dose of 200 mg/kg., Sigma)was injectedsubcutaneously before onset of isolation, i.e. at day 12. Twenty-eight

Fig. 1. Timeline for BrdU injection during a 6-week study. Marmosets were observed for2 weeks. Prior to beginning the stress component all animals were injected with 5-bromo-2′-deoxyuridine (BrdU). Stress cohort animals were housed in isolation for1 week followed by 1 week of isolation and daily social-defeat in the territory of adominant male animal. Stress animals were then given 2 weeks of re-socializationbefore euthanization.

Table 1Primary antibodies.

Target protein Immunogen Species Dilution Source/Cat. no.

Anti-BrdU 5-bromo-2′-deoxyuridine Rat 1:1000 Accurate ChemicalWestbury NY USAClone BU1/75 (ICR1)

Anti-DCX Peptide corresponding to aa 385–402 of human DCX Goat 1:800 Santa Cruz BiotechnologySanta Cruz CA, USAsc-8066

Anti-PSA-NCAM Viable Meningococcus group B (strain 355) Mouse 1:400 MilliporeBillerica MA, USAMAB5324

Anti-GFAP GFAP isolated from cow spinal cord Rabbit 1:1000 DakoGlostrup, DenmarkZ0334

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days later, i.e. at day 40, which would allow sufficient time fornewborn cells to undergo neuronal differentiation, animals wereeuthanized by decapitation under isoflurane/NO2 inhalation anesthe-sia and blood was collected from each marmoset after decapitation.The brains were then removed immediately. The right hemispherewas washed and placed in 4% paraformaldehyde overnight forimmunohistochemistry while the left hemispheres were kept innitrogen and used for proteomics analysis in a separate study.

Before sectioning from a dry-ice cooled tissue block on a slidingmicrotome, the brains were transferred to 30% sucrose in 0.1 M PBS,pH 7.4, until they sank. Brains were then cut in the coronal plane in40 μm sections; free floating sections were washed thoroughly in0.05 M PBS, pre-incubated with 0.3% H2O2, and incubated with theprimary antibodies (Table 1). Antibodies were diluted in 1% BSA —

0.05 M PBS, 0.5% Triton for 1 h at room temperature and thenovernight at 4°C: rat anti-BrdU (Accurate Chemical Westbury NY),rabbit polyclonal anti-GFAP (DakoCytomation, Denmark), goat anti-DCX (Santa Cruz Biotechnology), mouse PSA-NCAM (Millipore,Billerica, Massachusetts), corresponding secondary antibodies were

incubated at 1:200 for 2 h at room temperature. ABC reagent(Vestastain Elite; Vector Laboratories, Burlingame, CA) was appliedfor 2 h at room temperature. Sections were double stained for BrdUand DCX with Diaminobenzidine+0.08% Nickel (DAB-N; SigmaGermany) and DAB respectively leaving a black precipitate on BrdU+nuclei and a brown precipitate on DCX+ cells.

Quantification

The sampling volume of the DG and amygdala was determined bydrawing contours around these regions of interest using theStereoInvestigator system (Microbrightfield, Williston, VT). In thehippocampus, DCX+ and BrdU+ cells were present in small numbersand all positive cells (on average 14 markers per section) weretherefore quantified manually. Markers in the DG were counted using20× and 40× objectives in 3 mid-level hippocampal sections. BrdU+nuclei in the amygdala were also hand-counted individually aftermorphological assessment again using 3 selected sections matched tothe same anterior–posterior level. The amygdala ranges from

Fig. 2. DCX expression in the marmoset hippocampus. Low numbers of DCX+ cell bodies are found mainly in the first and second inner third of the granular cell layer (GCL), some ofwhich are bipolar (left arrow in A). Their dendrites are short and their dendritic extent generally small (right arrow in A). DCX+ cells are often oriented in a radial manner and seemto undergo secondary division (arrow in B). Some DCX+dendrites can be followed through the GCL (arrowheads in C). Double labeling for DCX and BrdU identified clear examples ofnewborn BrdU-positive cells and doublets (arrows in D), that rarely co-express DCX (arrowheads in D). Figure E shows an example of a BrdU+ newborn cell (left arrow) in thesubgranular zone next to the hilus (H), that weakly co-expresses DCX. In the same subregion, a single labeled DCX+ cell is present with its dendrite running parallel to the GCL(arrowheads in E). Inset shows another example of a BrdU/DCX+ cell within the GCL. Fig. F shows 2 DCX+ cells with well developed dendrites (arrowhead) close to a BrdU+newborn cell (arrow) located in the entorhinal cortex.

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approximately +5.50 through+3.64 mm Bregma as published in themarmoset brain atlas (Palazzi and Bordier, 2008), also available online(Tokuno et al., 2009). Sections for quantification were identified at thelevel of approximately +4.28 mm Bregma. To quantify DCX+ cells inthe amygdala a different strategy was employed due to the presenceof a very large number of cells. Quantification was completed usingthe StereoInvestigator system by systematically sampling regions ofinterest at regular intervals. Overcounting bias is avoided by the use ofupper and lower guard zones, i.e. excluding cells found within 1.5 μmof the top and bottom of the dehydrated section according to standardstereological procedures.

DCX/PSA-NCAM immunofluorescence

To establish whether DCX+ neurons were indeed migratory, co-expression of DCX and PSA-NCAM was examined using doubleimmunofluorescence and confocal imaging. Sections were co-incu-bated with antibodies for DCX and PSA-NCAM and then incubatedindependently with Alexa-488 donkey anti-goat and Alexa-647donkey anti-mouse (1:200 Molecular Probes, Carlsbad, California).Fluorescent signals were imaged with a Zeiss LSM 510 confocal laser-scanningmicroscope; emission signals from the Alexa-488 and Alexa-647 probes were assigned to green and red channels, respectively.Images obtained by immunofluorescence were not digitally manip-ulated in any way but were cropped to provide clearly framed images.

Antibody characterization

The monoclonal mouse anti-PSA-NCAM detects a single band onWestern blot according to manufacturer's technical information;recognizing the 180-kDa, highly sialiated form of NCAM foundpredominantly in embryonic brain (Rougon et al., 1986). The stainingpattern we observed is identical to that described in previous reports(Palmer et al., 2000; Pieribone et al., 2002).

The goat polyclonal anti-DCX detects a single band at 40 kDa onWestern blot of adult rat OB (Brown et al., 2003; Suzuki et al., 2007).

Staining morphology for DCX+ cells was identical to a priorpublication (Oomen et al., 2010).

Rat monoclonal anti-BrdU reacts with BrdU in single-strandedDNA (manufacturer's technical information).We observed no labelingwhen anti-BrdU was incubated with non-BrdU-injected rats used as acontrol. The morphological pattern of staining we observed wasidentical to that described in previous reports (Cameron and McKay,2001; Czeh et al., 2007).

The rabbit polyclonal anti-GFAP antibody precipitates one distinctGFAP protein from cow brain extract (manufacturer's technicalinformation). The antibody identifies a 50-kd band in western blotanalysis of brain tissue (Knabe and Kuhn, 2000). The distribution andmorphology of GFAP+ astrocytes matched previous descriptions(Yang et al., 2007).

Results

Hippocampal neurogenesis

In the dentate gyrus, BrdU+ cells were confined to the granule celllayer, SGZ, and hilus. The black immunostained BrdU+ nuclei had around morphology making them easy to identify; only cells in the SGZand GCL were used for quantitative analysis. DCX+ cells appeared asimmature neurons or neuroblasts within the granule cell layer with arobust cytoplasmic staining. Many labeled cells had secondary andtertiary processes that were relatively short and lacked furtherbifurcations. These processes typically extending in a radial mannerthrough the granule cell layer (Figs. 2A–C); generally, the morphologywas similar to other published images e.g. DG of rodents (Brown et al.,2003; Oomen et al., 2010). In the DG, approximately 3-foldmore DCX+cells were observed than BrdU+ cells. BrdU: control 9910±3120(mean±SE) cells/mm3, stress cohort 6220±820 (mean±SE) cells/mm3, DCX: control 32000±3640 (mean±SE) cells/mm3, stress 24300±3670 (mean±SE) cells/mm3. Rare double-positive BrdU/DCX cellswere observed in the DG (Fig. 2D); the two markers were never co-localized in the hilus. Populations of BrdU- and DCX-positive cells were

Fig. 3. Photomicrographs illustrating PSA-NCAM expression in thewall of the lateral ventricle and caudal amygdala. A–B) Immunostaining for PSA-NCAM identified expression in thewall of the lateral ventricle (LV). C–D) Immunostaining can be observed where hippocampus and temporal horn of the lateral ventricle (tLV) end near the ventral amygdala.

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not significantly correlated (Pearson 2-tailed test, R=0.32, p=0.08) inthe marmoset DG. This corresponds with a higher number of immatureneuroblasts transiently expressing DCX and a lower number of BrdU+surviving cells.

Neurogenic markers in the amygdala

As neural stem cells located in the lateral ventricle are known todeliver new astrocytes and neurons to the amygdala and neocortex,we analyzed coronal sections directly rostral of the hippocampus.Immunostaining for PSA-NCAM identified expression in the wall ofthe lateral ventricle (Fig. 3A). The lateral ventricle ends adjacent to thecaudal amygdala and PSA-NCAMwas observed in amanner consistentwith lateral migration from the SVZ (Fig. 3B). In the caudal amygdala,DCX+ cells were first observed in the ventral basolateral nucleus

where it borders the temporal horn of the lateral ventricle (tLV).Adjacent to the ventricle, DCX+ cells line the amygdala where itborders the hippocampus and entorhinal cortex (EC). The highestdensity of cells was observed closest to the ventricle and slightly moredispersed at locations further from the ventricle. In these immediateareas, no BrdU+ cells were observed.

In the midrostrocaudal level, the basolateral nucleus is subdividedinto the ventral medial (BLVM) and intermediate (BLI) subregions.The high density of DCX+ cells extended through these regionsbordering the EC (Figs. 4A–G). Interestingly, DCX+ neurons in theBLVM and BLI have projections extending through the ventral corticaland medial nuclei, located adjacent to the optic tract. In contrast toDCX+ cells in the GCL, the processes of these cells in the amygdalahad no appreciable orientation (Figs. 4A–H). As rare DCX-expressingastrocytes have been documented in the human cortex (Verwer et al.,

Fig. 4. A–D. Photomicrographs illustrating the distribution of DCX+ cells over different anatomical levels of the marmoset amygdala. A) In sections immediately adjacent to thehippocampus cells reside in the basolateral and lateral amygdala nuclei. B–D)DCX+cells are primarily found in the ventral nuclei but also in the lateral nucleus. DCX+cells are also foundin1st and2nd layers of the entorhinal cortex. The areabetween these twopopulations is largely devoidofDCX+positive cells. Numerousdensely clusteredDCX+cell bodies arepresent intheventromedial (BLVM)and intermediate parts (BLI) of thebasolateral amygdaloidnucleus, that are borderedby theentorhinal cortex (EC). DCX+cell bodies are further abundant in theventral anteriorcortical nucleus of the amygdala (VACo). Numerous fibers leave the BLVM and BLI and traverse the basomedial amygdaloid nucleus (BM) projecting towards the medialamygdaloid nucleus (Me) and anterior amygdaloid area (AA), whereDCX+fiber clusters are seen, likely representing fiber endings (Fig. 4G). Details of projectingDCX+fibers are shownin Figs. 3E and F. This projection is more extensive in the anterior regions of the basolateral nucleus (Figs. 4A, B) than in other regions (Figs. 4C–F).

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2007), we also investigated this possibility by immunostainingsections with anti-GFAP antibody. In these areas, mature GFAP-expressing astrocytes had a uniform distribution within nuclei of theamygdala and had a cellular morphology entirely distinct from theDCX-expressing cells (data not shown).

In the amygdala, the highest density of BrdU+ cells occurred in theBLVM and BLI subregions rostral of the tLV (Fig. 5). BrdU+nuclei weremore sporadically distributed throughout the other nuclei of theamygdala and were not highly clustered, or e.g. limited to theintersection between the amygdala and EC. BrdU+ and DCX+ wererarely co-localized in this region (Supplemental Fig. 1). Within theamygdala, a significant positive correlation existed for BrdU and DCX(Pearson 2-tailed test, R=0.59, p=0.002).

DCX+ cells were found in entorhinal cortical (EC) layers I and II(Fig. 5D), BrdU+ and DCX+ cells were co-imaged in this area(Fig. 5F). BrdU+ nuclei were also found in the inner layers of the EC, asubregion of the EC largely devoid of DCX+ cells. These BrdU+ cellswere most often observed as a single nucleus and only occasionallyseen in clusters or doublets. The DCX+ cells displayed a complexdendritic morphology distinct from the clustered amygdala popula-tion where they appeared less mature due to their close proximity to

each other. DCX+ cells in the ECwere present in clusters which is alsodistinct from the organization seen in the amygdala.

In the amygdala, BrdU+ cells were found at densities of 3560±530and 2780±206 cells/mm3 for control and stress cohorts respectively,DCX+ cells were found at averages of 163,000±56,900 and 164,000±7590 cells/mm3 cells in each amygdalar midlevel section. DCX+ cellsare more than 50 timesmore prevalent than BrdU+ cells. These figuresmay underestimate the population of cells found within specific nucleiof the amygdala due to difficulty in properly delineating these highlyspecific areas. The DCX+ cell population can be found across nuclei ofthe amygdala (Fig. 6); these nuclei lack histochemical markersnecessary to determine their borders (personal communication withXavier Palazzi, author of the marmoset brain atlas).

Doublecortin and PSA-NCAM in the amygdala and entorhinal cortex

Since DCX is typically expressed in migratory neurons, we nextimmunostained sections from the amygdala for PSA-NCAM. Celladhesion is prevented when a carbohydrate such as polysialic acidbinds extracellular domains of neural cell adhesion molecule,allowing cells to migrate. Our results indicated that PSA-NCAM had

Fig. 5. Photomicrographs illustrating the dense clusters of DCX+ cells and fibers in the amygdala. The highest density of BrdU-labeled cells occurred in the BLVM and BLI subregions(Figs. A and B, resp.) while lower densities are found in the VACo (C). DCX+ fibers in the VACo appeared highly interconnected and have different orientations. This is in contrast tothe DCX+ fibers in the BLI (arrow in 2B) or the DCX+ fibers leaving the BLVM and BLI, most of which were directed towards Me and AA (A and arrow in B; see also Fig. 4A). Inaddition, considerable numbers of DCX+ cells were present in themore basolateral parts of the entorhinal cortex (EC)where DCX expression wasmainly confined to the outer layersI/II (Figure D). Higher magnifications show this group of cells, similar to the VACo, to be strongly interconnected with many DCX+ dendrites touching other DCX+ cells or dendrites(Figs. D–G, arrows in E). Double labeling for BrdU (black) and DCX (brown) identified the presence of newborn cells in the EC (arrows in Fig. F). In the lower layers III/IV, isolated cellsare occasionally found. Their dendrites often have a radial orientation (Fig. G, H) and frequent bifurcations (arrow in H).

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a very similar distribution to DCX in the amygdala. This providesevidence that DCX+ amygdala bound neuroblasts have a migratorycapacity (Figs. 7A–L). The BLVM and BLI have large populations ofdensely packed DCX+ cells, most commonly found dispersed andwithout a distinct orientation. Co-imaging of DCX expression and PSA-NCAM at highmagnification revealed that many of the DCX+ cells areindeed tangentially contacting PSA-NCAM sheathed axons. However,autonomous cell bundles were also observed. At lower magnification,PSA-NCAM was expressed on highly clustered DCX+ cells. While itwas clear that most of the DCX+ cells expressed PSA-NCAM, somecells in the amygdala were observed without a strong overlap withPSA-NCAM.

In the entorhinal cortex DCX and PSA-NCAMwere also co-imaged,indicating that these cells may have an immature and/or migratorycapacity (Figs. 7M–O). These cells were morphologically differentfrom the amygdala bound cells, and typically had branchedextensions. However, these processes also express PSA-NCAMperhaps indicating structural plasticity in the EC. None of the observedimages of PSA-NCAM expression suggested radial migration from theamygdala to the EC.

Delayed influence of psychosocial stress

The stress protocol was designed to evaluate lasting rather thanacute consequences of psychosocial stress on structural plasticity. RIAurine analysis indicated that the monkeys had indeed experiencedstress; mean cortisol concentrations were significantly elevatedduring the 2 weeks of isolation and social defeat in the stress cohort(Fig. 8A; pb0.01 student's one-tailed t-test) as compared to thecontrol group for the same 6 weeks period. During the re-socializationperiod, there was a significant normalization in measured cortisollevels (pb0.05 student's one-tailed t-test). When the groups wereevaluated by sex, no significant differences were observed (Fig. 8B).

Semi-quantitative analysis of the hippocampal DG for the presenceof BrdU and DCX positive cells did not reveal significant differences inthe distribution or number between stress and control cohorts(Fig. 8C) (BrdU; pb0.14, DCX; pb0.16; student's t-test). Similarly, inthe amygdala, stress effect on BrdU+ and DCX+ cell populationswere not significantly different, nor were obvious differences incellular morphology observed in animals in the stress compared tocontrol cohort (BrdU; pb0.19, DCX; pb0.49 student's t-test).

Discussion

The main finding of this study is the presence of a surprisinglylarge population of DCX-expressing newborn cells in the amygdala of4-year-old (middle-aged) common marmosets. Moreover, low num-bers of adult generated BrdU+ cells and immature DCX+ neuronswere found in the hippocampus, entorhinal and adjoining temporalcortex, as also seen in other primate species and as was expected foranimals this age. Exposure to a 2-week period of social stress followedby a 2-week recovery period revealed no lasting effects of stress onstructural plasticity in the marmoset.

Some recent studies have now documented structural plasticityand/or DCX expression not only in the DG but also in cortical and/oramygdalar regions in various species including primates, butgenerally not to such an extent as found here (Bernier et al., 2002;Cai et al., 2009; Gomez-Climent et al., 2008; Gould, 2007; Guo et al.,2010; Jabes et al., 2010; Tonchev et al., 2003; Xiong et al., 2008; Zhanget al., 2009). Indeed, our data from the DG and amygdala providesquantitative evidence of a large population of neuroblasts and/orimmature neurons in the amygdala when compared to a knownneurogenic zone. The low numbers of newborn neurons found in thehippocampal DG are consistent with the low rates reported earlier inmiddle aged and aging marmosets (Leuner et al., 2007), however thelarge population of DCX+ cells present in the amygdala of middle-aged animals was unpredicted.

Our quantitative analysis of the DG and amygdala demonstratedthat structural plasticity persists in the amygdala of marmosetmonkeys, consistent with a previous study in squirrel monkeys(Saimiri sciureus) that reported approximately 14 BrdU+ nuclei peramygdala section (Bernier et al., 2002); this was the result of a50 mg/kg intravenous injection of BrdU and coronal sectioning at40 μm. Using sections of identical thickness, we here identifiedapproximately 30 BrdU+ nuclei and 1600 DCX+ cells in eachcoronal section in the amygdala of middle aged commonmarmosets. Importantly, the earlier study established that migra-tory cells from the SVZ differentiated into mature neurons in theamygdala (41% BrdU+/NeuN+). A small population of BrdU+ cells(7%) was found to be MAP-2+ in the temporal stream andamygdala 28 days after BrdU injection. Our findings are inagreement with these results demonstrating structural plasticityin the amygdala.

Fig. 6. Anatomical representation of DCX+ cells present in the marmoset amygdala. Rostral of the hippocampus, DCX+ cells appear closely associated to the temporal horn of theLateral Ventricle (tLV) where the caudal amygdala begins. In midlevel sections, DCX+ cells are found as a dense band in multiple nuclei of the amygdala where it borders theentorhinal cortex (Er). This includes the BLVM, BLI, VACo, and lateral (LA) nuclei. In the rostral amygdala this band of cells occupies a smaller volume of the nuclei. The present areasof interest illustrated here are found from approximately Bregma +5.50 through +3.64 mm.

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The staining patternwe see for DCX is consistentwith earlier evidenceof neurogenesis in the amygdala of New World monkeys. Previously,newborn neurons, observed as a thick band of Bcl-2 expressing cells, andchains of PSA-NCAM+ cells were observed to invade the amygdala(Bernier et al., 2002). Our double immunofluorescence analysis confirmsthe presence of DCX+cell bodies and projecting axons that co-expressPSA-NCAM in the amygdala, thereby verifying the migratory capacity ofcells in this structure. We also documented DCX+/PSA-NCAM+cells inthe first and second cortical layers of the entorhinal cortex withimmunoreactivity absent in the area separating these cortical layersfrom the amygdala. This is consistent with findings that oligodendroglialprogenitors reside in piriform cortex (Guo et al., 2010).

Our results from the DG are consistent with the known kinetics ofthese two markers: BrdU labels newborn cells while DCX, at least inrodents, is expressed for approximately 10 days. (Brown et al., 2003;Couillard-Despres et al., 2005; Francis et al., 1999; Friocourt et al.,2003).We observed rare colocalization of BrdU and DCX in the DG andamygdala, a finding that demonstrates that at least some of these cellsare newly generated. In the amygdala, the DCX+ population wassignificantly higher, approximately 50 times more prevalent thanBrdU+ cells. This represents a distinctly different relationship fromthat found in the DG, perhaps indicating that DCX is not transientlyexpressed.

At this time we have no evidence that DCX+ cells in the amygdalaare functionally integrated. However, based on protein expression weexpect they participate in mechanisms of structural or synapticplasticity. DCX is typically associated with cell migration due todevelopmental regulation necessary for cell migration (Bai et al.,2003). However, prenatally born cells can also maintain DCXexpression and an immature neuronal phenotype (Gomez-Climentet al., 2008). DCX is additionally expressed by a subset of matureneurons. In adult rat brain, differentiated neurons in select areas,including the piriform cortex, co-express DCX and PSA-NCAM(Nacher et al., 2001), reviewed by Gomez-Climent (Gomez-Climentet al., 2010). These finding agree with observations that DCXexpression is not limited to migratory neuroblasts alone and may beinvolved in other aspects of cellular orchestration of synapticplasticity. DCX coprecipitates with adapter proteins involved withprotein sorting and vesicular trafficking (Friocourt et al., 2001). Theformation of new neurites implies the participation of microtubules.Indeed, DCX participation in microtubule reorganization has beenlinked to axonal outgrowth or synaptogenesis (Ribak et al., 2004). Inthis respect, it is interesting to note that in a rodent model dendriticarborization was increased in neurons of the basolateral amygdalafollowing chronic stress (Vyas et al., 2004).

Amygdala-bound DCX+ cells express PSA-NCAM which stronglysuggests that these cells participate in chain migration, an unusualtype of cell displacement where large bulks of cells are arranged incontact with axon bundles. This is in agreement with previousfindings in primates (Kornack and Rakic, 2001; Pencea et al., 2001)where migrating chains of cells have been observed along the lengthof the entire lateral ventricle system, both in humans and nonhumanprimates (Bernier et al., 2000; Curtis et al., 2007; Doetsch and Alvarez-Buylla, 1996; Pencea et al., 2001). Our stainings are consistent withearlier evidence of the temporal stream however verification is ideallycompleted with stainings in the sagittal plane.

The role of NCAM is well established as assembly of the CNSarchitecture depends on this transmembrane protein (Hoffman et al.,1982). The extracellular domains, when bound by the carbohydratepolysialic acid (PSA), prevent cell–cell adhesion, and permit structuralplasticity in the brain (Bonfanti, 2006; Finne, 1982). PSA is attached

Fig. 8. Stress induction during a 2-week period is not associated with reducedneurogenesis in the dentate gyrus or amygdala. A) Urinary corticosterone levels aresignificantly elevated in middle aged marmosets exposed to a combined isolation andsocial defeat stress (student's t-test p=0.004). B) Although reductions were present inDCX, in neither the hippocampus nor the amygdala were significant differences foundin the number of BrdU+ or DCX+ cells after stress exposure (DG: BrdU (p=0.14), DCX(p=0.16), amygdala: BrdU (p=0.18), DCX (p=0.48).

Fig. 7. DCX+ cells in the adult marmoset express PSA-NCAM. A–C, D–F) A dense band of DCX+ cells co-express PSA-NCAM on extensions that run laterally in the plane in thebasolateral intermediate nucleus. G–I, J–L) A diffuse band of DCX+ cells co-express PSA-NCAM on extensions that run tangentially. M–O) DCX+ cells in the piriform cortex display amore mature morphology with branched processes that express PSA-NCAM. P–R) Two immature neurons in the DG express PSA-NCAM confirming that these molecular markers ofneurogenesis are found in a known neurogenic zone. Scale bars=50 μm (A and G), 20 μm (D, J, M, and P).

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exclusively toNCAMin the brain; PSA-NCAM immunoreactivity therebyidentifies migratory cells exclusively. Coordinated expression of PSA-NCAM and DCX is conserved in populations of cells migrating from theSVZ (Luzzati et al., 2003;Nacher et al., 2002; Pencea et al., 2001; Ponti etal., 2006; Yang et al., 2004). These findings have not been extended tohumans yet. Migratory progenitor cells are present in the human RMS(Kam et al., 2009) but evaluation of the same molecular markers failedto identifymigration through the TS to the amygdala (Sanai et al., 2004).

Exposure to isolation and social-defeat stress is known to represent asevere stressor for these social animals. Indeed, a significant increase inurinary cortisol was induced during stress conditions and normalized atthe time of sacrifice. Depressive-like behaviors were commonlyobserved until the last day of sacrifice in the stress group (not quantifiedsystematically), however the current stress paradigm failed to signif-icantly affect BrdU+ or DCX+ cell numbers. Despite the rise in cortisol,the nature and duration of these stressors during a 2-week exposureperiod may have been too mild to significantly reduce structuralplasticity in a lastingmanner. In rodent studies of stress onhippocampalneurogenesis or amygdalar plasticity (Vyas et al., 2004), stress durationis often prolonged, i.e. applied for 21 days prior to immediate sacrifice,and frequently contains a physical component, like restraint, that wasnot present here. If we assume transient expression of DCX, lasting fromapproximately 4 to 14 days in new neuroblasts, a putative initialreduction in DCX+ cells might have normalized during the 2 weeks ofre-socialization. In the current paradigm animals were sacrificed28 days after BrdU injection. In a previous paper on marmosets, asignificant reduction in neural stem cell proliferation was reportedwhen sacrifice occurred shortly after stress exposure (Gould et al.,1998). In rodents, changes in neurogenesis after chronic stress (Czeh etal., 2002; Oomen et al., 2007) were indeed shown to be reversiblefollowing a recovery period (Heine et al., 2004a, b).

In conclusion, neurogenesis in the DG and amygdala of adultmarmosets was found to be insensitive to a psychosocial stressparadigm that included a recovery period. Quantification of twomarkers of neurogenesis provide evidence there were no long-lastingchanges in the population of surviving BrdU-labeled cells and DCX+neuroblasts in each anatomical region. Whether the population ofcells in the amygdala is entirely dependent on SVZ migration remainsto be determined, but a large pool of migratory neuroblasts is presentin the amygdala that is unlikely to be supported fully by new cellmigration from the lateral ventricle. Given the similarities indistribution in other primate species, this suggests evolutionaryconservation of structural plasticity outside the classic SVZ and SGZzones. The exact functional role of these DCX+ cells in the amygdalaremains to be further discovered.

Supplementarymaterials related to this article can be found onlineat doi:10.1016/j.expneurol.2011.05.008.

Acknowledgments

MWM and PJL are supported by the European Union (MEST-CT-2005-020013). PJL is additionally supported by the Dutch BrainFoundation and Internationale Stichting Alzheimer Onderzoek (ISAO).We wish to thank and acknowledge technical assistance from: Ms.Jose Wouda (UvA, SILS-CNS, Amsterdam, The Netherlands) for herassistance with processing tissue and optimizing staining techniques,Ms. Danielle Straub (University Medical Center, Utrecht, The Nether-lands) for executing the animal behavior work and biotechnicalhandling, and Ms. Shizuka Aoki (Johns Hopkins University, Baltimore,MD, USA) for her assistance preparing anatomical drawings.

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