modification of hippocampal neurogenesis and neuroplasticity by social environments

Modification of hippocampal neurogenesis and neuroplasticityby social environments

Lin Lu,a,c Guobin Bao,b Hai Chen,a Peng Xia,b Xueliang Fan,a Jisheng Zhang,a Gang Pei,b

and Lan Maa,*a National Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, 138 Yi Xue Yuan Road,

Shanghai 200032, Chinab Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road,

Shanghai 200031, Chinac Behavioral Neuroscience Branch, IRP/NIDA, National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, MD 21224, USA

Received 9 December 2002; revised 20 March 2003; accepted 24 April 2003

Abstract

Synaptic plasticity and neurogenesis in the brain are affected by environmental stimuli. The present study was designed to investigatethe effects of social environments on learning and memory, neurogenesis, and neuroplasticity. Twenty-two-day-old rats were housed inisolation or in groups for 4 or 8 weeks and injected intraperitoneally with bromodeoxyuridine to detect proliferation among progenitor cells.The animals were also tested for learning in a water maze and for hippocampal CA1 long-term potentiation in vivo and in vitro. The resultsshow that the number of newborn neurons in the dentate gyrus and the learning in a water maze decreased significantly in rats reared inisolation for 4 or 8 weeks, as compared with grouped controls. Induction of long-term potentiation in the CA1 area of rat hippocampus invivo and in vitro was also significantly reduced by isolation. Furthermore, the effects of isolation rearing on spatial learning, hippocampalneurogenesis, and long-term potentiation could be reversed by subsequent group rearing. These findings demonstrated that socialenvironments can modify neurogenesis and synaptic plasticity in adult hippocampal regions, which is associated with alterations in spatiallearning and memory.© 2003 Elsevier Science (USA). All rights reserved.

Keywords: Social environments; Neurogenesis; Long-term potentiation; Synaptic plasticity; Hippocampus; Isolation; Learning and memory

Introduction

Events experienced in early life may contribute to theexpression or exacerbation of a variety of physical andpsychological disorders. Rearing animals in isolation is arelevant paradigm for studying early life stress and forunderstanding the genesis of certain neurological and psy-chiatric diseases (Myhrer, 1998; Whitaker-Azmitia et al.,2000). The so-called isolation syndrome has been well char-acterized and consists of spontaneous and conditioned lo-comotor hyperactivity (Heidbreder et al., 2000), enhancedresponses to novel environments, greater tendency toward

preservation, deficits in prepulse inhibition, and altered re-sponse to the behavioral effects of drugs such as opioids andamphetamine-like psychostimulants (Morutto and Phillips,1997; Smith et al., 1997). Accumulating evidence demon-strates that social environments in early life significantlyinfluence not only the organization of behavior but also thedevelopment of the brain. In particular, social isolationimpairs memory processes but the mechanism for this effectis not known (Myhrer, 1998; Nilsson et al., 1999).

Unlike cells in most tissues, which undergo generationand replacement throughout life, most neurons of the mam-malian brain are generated entirely during early develop-ment, either before birth or shortly thereafter, and are notreplaced if lost (Rakic, 1985). However, recent evidence hasshown that in certain brain areas such as hippocampus,

* Corresponding author. Fax: �86-21-64174579.E-mail address: [email protected] (L. Ma).

R

Available online at www.sciencedirect.com

Experimental Neurology 183 (2003) 600–609 www.elsevier.com/locate/yexnr

0014-4886/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved.doi:10.1016/S0014-4886(03)00248-6

olfactory bulb, and neocortex, new neurons are also gener-ated during adulthood (Elizabeth and Charles, 2002; Gouldet al., 1997, 1999, 2001; Rakic, 2002; Rocht et al., 2002).The newly generated cells with neuronal features have beendetected recently in the dentate gyrus of humans, in autopsymaterial of patients exposed to the thymidine analog 5-bro-modeoxyuridine (BrdU) at advanced age (Elizabeth andCharles, 2002). The production and survival of these new-born neurons may contribute to neuroplasticity and a varietyof hippocampus related functions, including learning andmemory (Snyder et al., 2001; Van Praag et al., 1999; Vartyet al., 1999).

Hippocampal neurogenesis is dependent on both genetic(Kempermann et al., 1997b) and environmental factors(Bengzon et al., 1997; Jones et al., 1992), but the mecha-nisms underlying the effect of social interaction in early andadult life on hippocampal neurogenesis are largely un-known. Therefore, this study investigated the influence ofearly social isolation on neurogenesis and neuroplasticity inhippocampus and their association with the alterations inspatial learning by early social isolation. For this purposewe used a water maze task for spatial learning and deter-mined hippocampal long-term potentiation (LTP), a modelof synaptic plasticity thought to underlie memory and learn-ing processes (Bliss and Collingridge, 1993; Malenka andNicoll, 1993).

Materials and methods

Animals and housing conditions

Male Sprague–Dawley rats (Shanghai Center of Exper-imental Animals, Chinese Academy of Sciences) were used.Rats were reared at weaning under two different conditions:isolation rearing (one per cage) and group rearing (five percage). Thus, in the first animal model, rats randomly re-ceived 4 weeks of isolation rearing (S4w) or group rearing(G4w) from Postnatal Days 22 to 49. In the second animalmodel, rats randomly received 8 weeks of isolation rearing(S4w/S4w), 8 weeks of group rearing (G4w/G4w), of 4weeks of isolation and 4 weeks of group (S4w/G4w) rearingfrom Postnatal Days 22 to 77. Rats were given free accessto food and water and were maintained on a constant light/dark cycle with constant temperature and humidity. Allanimal treatments were strictly in accordance with the Na-tional Institutes of Health Guide for the Care and Use ofLaboratory Animals.

Place navigation task

This test was performed as previously described (Morriset al., 1986; Pu et al., 2002). The apparatus consisted of acircular black-painted swimming pool (160 cm in diameter� 68 cm high), filled with water at 20°C and made opaqueby the addition of ink. The pool was located in a large

testing room containing many objects that the animals canuse as cues for spatial orientation. The position of theseenvironmental cues remained unchanged throughout the en-tire testing period. The pool was divided into four quadrantswith four starting locations called North (N), East (E), South(S), and West (W) at equal distances on the rim. Theinvisible black platform (diameter 10 cm and submerged 1.5cm below the water line) was placed in the center of thenortheast quadrant. On Day 1, the animals were habituatedto the swimming pool environment. They were placed in-dividually on the platform for 15 s and in the pool for 60 sand allowed to swim. They were then returned to the homecages and experienced another habituation trial after 10 min.During the training period of the task, rats were given threetrials per day to find the hidden platform for 7 consecutivedays (maximum trial duration 60 s, 15 s reinforcement onthe platform, 30 s recovery period between trials). Each ratwas gently placed into the water, with the nose pointingtoward the wall at one of the starting points designated S,W, and E. Rats that failed to find the hidden platform weregiven a score of 60 s and then physically placed on theplatform for 15 s. The time required for rats to climb ontothe platform was recorded as escape latency. The length ofswim path was recorded and analyzed by a computer con-nected to a video tracking system. The average of the threetrials was scored for the given day. The swim path andlatency for finding the platform during the water maze testwere analyzed with a one-way analysis of variance(ANOVA), which was followed by a Fisher PLSD post hoctest at each time interval.

BrdU injections

Rats were injected with BrdU (Sigma; 50 mg/kg, ip)dissolved in phosphate buffer. To evaluate the effect ofisolation rearing on cell proliferation, G4w (n � 8), S4w (n� 8), G4w/G4w (n � 7), S4w/S4w (n � 7), and S4w/G4w(n � 7) groups received BrdU twice daily on the last 3 days(Days 47–49 for 4-week rearing or Days 75–77 for 8-weekrearing) of their rearing treatments to label newborn braincells and these rats were sacrificed at the end of the 4- or8-week isolation or group rearing treatment (i.e., 24 h afterthe last BrdU injection). Alternatively, in some other exper-iments designed to evaluate the effect of isolation rearing onthe survival of BrdU-labeled newborn brain cells, rats weregiven BrdU injections twice daily for 3 days (PostnatalDays 47–49) to label newborn cells and then reared for 4weeks in groups (BrdU-G4w, n � 8) or in isolation (BrdU-S4w, n � 7); and these animals were sacrificed at the end ofthe 4-week isolation or group rearing treatment (i.e., 4weeks after the last BrdU injection).

Histological procedures

Rats were deeply anesthetized with chloral hydrate (400mg/kg, ip) and perfused with 200 ml of phosphate buffer

601L. Lu et al. / Experimental Neurology 183 (2003) 600–609

saline (PBS, pH 7.3) containing heparin (5 � 104 unit/ml),followed by 300 ml of 4% paraformaldehyde in 0.1 M ofphosphate buffer (pH 7.3). After a 24-h postfixation inparaformaldehyde, 40-�m frontal sections of the brain werecut on a vibratome and collected in 0.1 M PBS (pH 7.4).Free-floating sections were processed in a standard immu-nohistochemical procedure (Kornack and Rakic, 1999; Mal-berg et al., 2000). For BrdU labeling, sections were treatedwith 2 N HCl (30 min at 37°C) and then rinsed in 0.1 Mborate buffer (pH 8.5) for 5 min. Sections were extensivelywashed with PBS, preincubated for 45 min in PBS contain-ing 0.3% Triton X-100 and 3% normal goat serum (blockingsolution), and incubated under agitation for 72 h at 4°C withmouse monoclonal BrdU antibody (1/200, Sigma) diluted inPBS containing 0.3 Triton X-100 and 1% goat normalserum. The sections were then incubated under agitation for2 h with a biotin-labeled goat anti-mouse IgG antibody(1/200, Vector). Sections from all animals were processedin parallel and immunoreactivities were visualized by thebiotin–streptavidin technique (Elite ABC kit, Vector) with3,3�-diaminobenzidine as chromogen.

The phenotype of newly born cells was examined bydouble-labeled immunohistofluorescence according to apreviously described method (Gould et al., 1997; Van Praaget al., 1999). Briefly, sections were incubated with theprimary antibody. The antibodies used were as follows:mouse anti-BrdU (1: 500; Sigma), rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibodies (1:1,000,Sigma), a marker of astroglia, and a rabbit monoclonalanti-TOAD-64 (1/1,000, kindly provided by Dr. P. C. Liu,Fudan University), a marker of immature neurons. GFAPand TOAD-64 antibodies bound were visualized with aTexas red-labeled anti-rabbit IgG antibody (1/500, Sigma)and BrdU molecules were revealed by a FITC-labeled anti-mouse IgG antibody (1/400, Sigma).

Quantification

All slides were coded prior to quantitative analysis, andthe code was not broken until the quantification was com-pleted. In general, coded slides were examined for BrdU-positive cells in the dentate gyrus, which were quantifiedusing the optical fractionator method in which every sixthsection (240 �m apart) through the rostral/caudal extent ofthe hippocampus (bregma 1.40 to 7.60 mm) was examined(Eisch et al., 2000; Van Praag et al., 1999; West et al.,1991). All BrdU-positive cells within the granule cell layerand hilus of the dentate gyrus were counted regardless ofsize or shape. To enable counting of cell clusters, cells wereexamined under �400 and �1000 magnification. BrdU-positive cells within the subgranular zone that were withintwo cell body widths of the granule cell layer were consid-ered part of the granule cell layer (Gould et al., 1997; Kuhnet al., 1996). The total numbers of BrdU-positive cells in thedentate gyrus were multiplied by 6 and are reported as totalnumber of cells per region (mean � SE). Raw data for cell

counts were subjected to one-way ANOVA followed byDunnett’s post hoc comparisons. For immunofluorescenceanalysis, BrdU-positive cells were first examined under afluorescence microscope for double labeling with TOAD-64or GFAP. Confirmation of double labeling was performedon a laser confocal microscope (Leica Microsystems) usinga �50 objective. Eighty to one hundred BrdU-positive cellsper rat were subjected to confocal analysis for verificationof co-localization within the granule cell layer, and co-localization with TOAD-64, GFAP, or neither was deter-mined from scans in single optical planes 1 �m thick. Thesections from the same rostral/caudal level were analyzedby using the image analysis system. The volume of thegranule cell layer was measured in each animal on the sameseries of sections by the Cavalieri method using a countinggrid with an area associated with the counting points of2500 �m2 at 10-fold magnification. Means were determinedfor each experimental group, and average volume was re-ported as cubic micrometers.

Electrophysiology

Recordings of field excitatory postsynaptic potentials(EPSPs) were made from the CA1 stratum radiatum of thehippocampus in response to ipsilateral stimulation of theSchaffer collateral/commissural pathway using techniquessimilar to those described previously (Xu et al., 1997,1998). Rats (n � 6) were anesthetized with urethane (1.1–1.5 g/kg, ip) and maintained at 37°C. The recording elec-trode was inserted 3.4 mm posterior to bregma and 2.5 mmright of the midline, and the stimulating electrode wasinserted 4.2 mm posterior to bregma and 3.8 mm right of themidline. The optimal placement of the electrodes in thestratum radiatum of the CA1 region of the dorsal hippocam-pus was determined using electrophysiological criteria andwas verified by postmortem examination. Test field EPSPswere evoked by stimulating with a square-wave constantcurrent pulse of 50-�s duration at a rate of 0.033 Hz. At thebeginning of each experiment, an input–output curve wasgenerated to determine the maximum EPSP amplitude. Dur-ing the experiments, the stimulus intensity was set at a levelthat evoked a field EPSP amplitude of 55–65% of themaximum. LTP was induced by high-frequency stimulation(HFS) as 20 pulses at 200 Hz, repeated three times at 30-sintervals. The EPSP slope was measured and averaged ev-ery 3 min.

In experiments on in vitro LTP recording, rats (n � 7 forS4w/S4w, n � 8 for S4w/G4w, and n � 6 for G4w/G4w)were anesthetized with urethane and decapitated, and thebrains were quickly removed into chilled artificial cerebro-spinal fluid (125 mM NaCl, 2.5 mM KCl, 1.5 mMNaH2PO4, 25 mM NaHCO3, 1.5 mM CaCl2, 1.3 mMMgCl2, 10 mM dextrose, 0.001 mM bicuculline methobro-mide, pH 7.4), and continuously bubbled with 95% O2/5%CO2. The brain was affixed to a vibratome and cut into400-�m slices. The slices were incubated at 32°C for 30

602 L. Lu et al. / Experimental Neurology 183 (2003) 600–609

min in a circulating perfusion chamber and then maintainedat room temperature. Individual slices were transferred tothe recording chamber as needed, and experiments wereperformed in artificial cerebrospinal fluid maintained at 32� 1°C. Sharpened tungsten, bipolar stimulating electrodesand 1-M recording electrodes filled with 150 mM NaClwere used for testing LTP. For CA1 responses, the record-ing electrode and the stimulating electrode (toward CA3)were positioned in the stratum radiatum of field CA1, aidedby a microscope (Olympus BX50wi) with a �40 objectivelens. Responses were evoked with single biphasic currentpulses (10–400 �A), adjusted to yield a response 30% ofmaximum. All evoked responses were initially tested withpaired-pulse stimuli. Individual synaptic responses wereelicited at 15-s intervals. After at least 10 min of stablebaseline responses, LTP was induced by a burst of 50 pulsesat 100 Hz; bursts were repeated four times at 30-s intervals.Recordings continued for 45 min after LTP induction.

Statistical Analysis

Statistical significance was determined either withANOVA followed by a post hoc analysis or with a two-tailed Student t test for independent samples. Data wereconsidered significant at P � 0.05. Statistical analysis wasperformed using cell number for immunohistochemistry,latencies (s) and swimming distance (m) for behavior, orEPSP amplitude for electrophysiological recording.

Results

Effect of social isolation on spatial learning

The results show that rats housed individually for 4weeks (S4w) demonstrated impaired performance on thespatial learning task as compared with those reared ingroups for 4 weeks (G4w). As shown in Fig. 1, the isolatedrats spent more time and swam greater distances to find andclimb onto the hidden platform (for latency to find theplatform, F (1, 15) � 13.51, P � 0.01, and for swimmingdistance, F (1, 15) � 9.54, P � 0.01), which is in agreementwith earlier studies (Pacteau et al., 1989; Wainwright et al.,1993). Similarly, there was a significant difference (F (2,22) � 10.89, P � 0.01) in the latency to find the platformbetween rats reared in isolation for 8 weeks (S4w/S4w) andthose that were group-housed during this period (G4w/G4w). However, rats reared in isolation for the first 4 weeksand subsequently group-housed for 4 weeks (S4w/G4w)showed a significant recovery in spatial memory (Fig. 1). Asshown in Figs. 1C and D, the swimming distance that thegroup-housed rats covered was significantly shorter thanthat of isolation-housed rats (for 4-week rearing, F (1, 15) �7.58, P � 0.01, and for 8-week rearing, F(2, 22) � 5.75, P� 0.05). However, no significant difference in swimmingspeed was observed between different housing conditions

(for 4-week rearing, F (1, 15) � 2.13, P � 0.05, and for8-week rearing, F (2, 22) � 1.89, P � 0.05).

Effects of social isolation on hippocampal neurogenesis

BrdU-positive cells were detected in the dentate gyrus ofthe hippocampal region in all groups of rats (Figs. 2A–C).In addition, BrdU-positive cells could be detected in thehilar area of the dentate gyrus (CA4). As shown in Fig. 3,the number of BrdU-positive cells in dentate gyrus of S4wrats (4256 � 389) was only 73% that of the G4w rats (5842� 456). Thus, 4-week isolation rearing resulted in a signif-icant decrease in cell proliferation in dentate gyrus as com-pared with group rearing (F (1, 13) � 12.51, P � 0.01).Similarly, there was a significant difference between thenumbers of BrdU-positive cells in the dentate gyrus ofS4w/S4w and G4w/G4w rats (F (1, 15) � 15.51, P � 0.01).Interestingly, in comparison with rats reared in isolation for8 consecutive weeks, rats isolated in the first 4 weeks andthen group-housed had more BrdU-positive cells (F (1, 15)� 6.45, P � 0.05) (Fig. 3). These results suggest that socialinteraction could increase the number of newborn cells indentate gyrus of rats that experienced isolation previously.

The effect of social environments on the survival of theprogeny of the dividing progenitor cells was assessed next.The rats were subjected to repeated injections of BrdU tolabel the newborn cells and then were reared for 4 weekseither in isolation (BrdU-S4w) or in groups (BrdU-G4w),

Fig. 1. Effects of housing environment on performance in the Morris watermaze. Rats were housed in groups for 4 weeks (G4w) or 8 weeks (G4w/G4w), housed singly for 4 weeks (S4w) or 8 weeks (S4w/S4w), or housedsingly for 4 weeks and then in groups for 4 weeks (S4w/G4w). Thehidden-platform version of the water maze task was used to assess spatiallearning. The data are presented as means � SEM of escape latency (s).Animals housed in groups performed significantly better in the spatiallearning task, and group rearing during the last 4 weeks of the 8-weekrearing period could reverse the worse spatial learning induced by isolationin the first 4 weeks. *P � 0.05; #P � 0.01 versus group rearing.


and the BrdU-positive cells in dentate gyrus of rat hip-pocampus were examined. As shown in Table 1 and Figs.2D and E, the number of BrdU-positive cells in BrdU-S4wrats (3012 � 385) significantly decreased as compared withthat in BrdU-G4w rats (4746 � 458), indicating that thesurvival of the newborn cells in rat hippocampus is affectedby social environment.

The phenotype of the newborn cells was determined by

examining their morphology and location in the dentategyrus and the co-localization of BrdU-positive cells withneuronal and glial markers. As demonstrated in Fig. 4,BrdU-positive cells in the isolated and grouped rats did notshow any significant difference in morphology, location, ordepth in the granule cell layer. The BrdU-positive stainingco-localized mainly with the neuronal phenotype markerTOAD-64 as indicated in Fig. 4D, and rarely with the

Fig. 2. Proliferation and survival of BrdU-labeled cells in dentate gyrus of hippocampus. To estimate ongoing proliferation, BrdU was injected during thelast 3 days of the 4-week or 8-week rearing period, and the BrdU-positive cells per dentate gyrus 24 h after the last BrdU injection were observed in theG4w/G4w (A), S4w/S4w (B), and S4w/G4w (C) groups of rats. To estimate survival of the labeled cells, the rats were injected with BrdU for 3 days andthen subjected to 4-week isolation rearing (BrdU–S4w, D) or 4-week group rearing (BrdU–G4w, E), and the BrdU-positive cells in dentate gyrus ofhippocampus 4 weeks after BrdU labeling were observed.


astroglial marker GFAP (Fig. 4C). The confocal analysis ofco-localization of BrdU-positive cells with TOAD-64 orGFAP signal indicate that the BrdU-positive newborn cellswe observed in dentate gyrus were mainly neurons. Asshown in Table 1, 58% of BrdU-labeled cells were TOAD-64-positive in group-reared rats, but only 35% of BrdU-labeled cells were TOAD-64-postive in isolation-reared rats(t (14) � 3.71, P � 0.01). For group-reared rats 8.9% ofBrdU-labeled cells and for isolated rats, 9.2% of BrdU-labeled cells were GFAP-positive. These results indicatethat most BrdU-labeled cells in the granule layer are neu-rons. In addition, no significant differences were observedbetween grouped and isolated rats with respect to the totalnumber of granule cells (t (14) � 1.23, P � 0.05) or thevolume of the granule cell layer (t (14) � 1.48, P � 0.05).In summary, these data demonstrate that isolation decreasesthe birth of new cells (primarily neurons) in the dentategyrus of rats.

Effects of isolation on long-term potentiation

To determine effects of social environments on synapticplasticity, LTP was induced in hippocampus of isolation-and group-reared rats in vivo and in vitro. As shown in Fig.5, high-frequency stimulation effectively induced a normalEPSP in the CA1 region of Gw4/Gw4, Sw4/Sw4, or Sw4/Gw4 animals. The induced EPSPs lasted at least 150 min (at180–210 % of the pre-HFS baseline) in G4w/G4w rats(Figs. 5A and B), which is in 2agreement with a previouslystudy (Xu et al., 1998). In contrast, in S4w (data not shown)and S4w/S4w rats, the EPSP amplitudes were significantlylower than those of G4w/G4w rats after high-frequencystimulation (Figs. 5A and B). The estimated half-time of theEPSP decline was 40–50 min. However, the EPSP ampli-

tudes in S4w/G4w rats were comparable to those of G4w/G4w rats.

The LTP was also recorded in the hippocampal CA1 areain vitro on brain slices. As shown in Figs. 5C and D, nodifference was found among the three groups in the initialexcitatory postsynaptic potential amplitudes, suggestingthat isolation does not affect basal synaptic efficacy. Therecordings in the CA1 area show that EPSP amplitude wassignificantly increased after administration of high-fre-quency stimuli in G4w/G4w rats (Figs. 5C and D). Similarto what we have observed in the in vivo recording experi-ments, isolation treatment decreased CA1 LTP. However,there was no difference in the magnitude of CA1 LTPbetween control rats and rats that were returned to the groupconditions during the last 4 weeks of rearing. Thus, ourresult demonstrates that isolation rearing significantly re-duces LTP in the CA1 area and this effect can be reversedby subsequent group rearing.

Discussion

Rats reared under conditions of isolation from weaningexpress a number of behavioral changes relative to theirsocially reared counterparts. For instance, isolated rats arehyperactive, exhibit deficits in prepulse inhibition of thestartle response, and also demonstrate learning and memoryimpairments on certain tasks (Jones et al., 1992; Paulus etal., 2000; Varty et al., 1999). Some of these behavioralchanges resemble clinical aspects of schizophrenia and Alz-heimer’s disease. Thus, early isolation in rat is regarded asa useful animal model for studying the neurobiological basisof these diseases, and other psychiatric or neurological dis-orders involving neurodevelopment deficits. In the presentexperiment, rats reared under conditions of social isolationdemonstrated impairments of learning and memory, which

Table 1Stereological data from hippocampusa

Group rearing Isolation rearing

Number of surviving BrdU-labeledcells

4746 � 458 3012 � 385*

% TOAD-64-positive 58.2 � 2.3 35.2 � 3.1*% GFAP-positive 8.96 � 1.6 9.28 � 3.2

Number of granule cells (106) 2.81 � 0.21 2.98 � 0.18Volume of granule cell layer

(mm3)2.95 � 0.11 2.81 � 0.14

a To estimate the effect of social environments on survival of newbornneurons, rats were administered BrdU to label newborn cells and then werereared for an additional 4 weeks either individually (BrdU-S4w) or ingroups (BrdU-G4w). The total number of BrdU-positive cells per dentategyrus 4 weeks after BrdU labeling was determined. Data are means�SEM. There were no significant differences in the number of granulecells and the volume of the granule cell layer between the two rearingconditions. However, there were significant increases in the number ofsurviving BrdU-labeled cells and percentage of TOAD-64-positive cells.

* Significantly different from group-reared rats.

Fig. 3. Effects of rearing condition on proliferation of BrdU-labeled cellsin dentate gyrus of hippocampus. To estimate ongoing proliferation, thetotal number of BrdU-positive cells per dentate gyrus 24 h after the lastBrdU injection was observed in the G4w, S4w, G4w/G4w, S4w/G4w, andS4w/S4w groups of animals. More BrdU-labeled cells were observed in thedentate gyrus of the G4w and G4w/G4w groups than in the S4w orS4w/S4w groups. Subsequent group rearing during the last 4 weeks of the8-week rearing period reversed the decreases in BrdU-positive cells in-duced by rearing in isolation in the first 4 weeks.


is in agreement with previous reports (Nilsson et al., 1999;Varty et al., 1999). Interestingly, when the isolated rats wereregrouped during subsequent rearing, they showed a recov-ery of learning and memory. These findings demonstratethat early social environments modify hippocampus-depen-dent functions and, somewhat surprisingly, these effects canbe reversed by subsequent rearing in group housing.

It was recently found that the rate of neurogenesis in therat dentate gyrus decreases with age (Kuhn et al., 1996), andthe newborn neurons in this brain area are inhibited byprenatal stress (Lemaire et al., 2000). The present study wasundertaken to investigate whether neurogenesis in the adultcentral nervous system could be affected by early socialenvironments. The dentate gyrus in the hippocampal regionwas studied because of its documented potential for neuro-genesis in young animals, its circuitry, and its important rolein learning and memory (Gould et al., 2000; Kesner et al.,

2000). We found that living in an isolated environmentdecreases the number of newborn progenitor-derived cellsin the dentate gyrus, and that subsequent social interactioncan increase the rate of neurogenesis that has been attenu-ated by isolation. The exogenous cues responsible for themodification of progenitor progeny and the spatial memoryremain unidentified, as do the signal pathway mediating thiseffect at the cellular level. Recent evidence has shown thatthe rate of neurogenesis in the adult dentate gyrus is affectedby stimulation of NMDA receptor (Cameron et al., 1995).Activation of NMDA receptor decreases the rate of adulthippocampal neurogenesis, while glutamatergic deafferen-tation and treatment with NMDA receptor antagonists haveopposite effects (Nacher et al., 2001). Hormonal status alsoinfluences adult neurogenesis. It was demonstrated recentlythat adrenal steroids negatively regulate adult hippocampalneurogenesis, resulting in increased neurogenesis (Cameron

Fig. 4. BrdU-labeled cells in dentate gyrus of adult hippocampus. BrdU-positive cells (green) were observed in the dentate gyrus in G4w/G4w (A) orS4w/S4w (B) rats. Confocal images of BrdU-positive cells (green) overlaid (yellow) with neuronal phenotype TOAD-64 (red, D) or astroglial marker GFAP(red, A–C) showing that newborn cells were mainly neurons. The BrdU-positive cells cells co-localized primarily with TOAD-64 as indicated by the yellownuclear staining (D), and rarely stained for GFAP (C).


and McKay, 1999; Cameron et al., 1998). Furthermore,prenatal stress and adult stress have been shown to reduceneurogenesis in young and adult dentate gyrus (Gould andTanapat, 1999; Lemaire et al., 2000). Thus, the effects ofstress and adrenal steroids on neurogenesis should not beoverlooked. However, isolation rearing does not alter adre-nal steroid levels (Nilsson et al., 1999), which suggests thatmodification of neurogenesis by environmental stimuli maynot be stress and adrenal steroid dependent.

The characteristics of LTP, including rapid formation,stability, synapse specificity, and reversibility, make it anattractive model for certain forms of learning and memory(Bliss et al., 1983). In the present study, the basal levels ofEPSP amplitude appear unchanged in the isolated rats.However, both in vivo and in vitro EPSP recordings showthat induction of LTP was significantly attenuated in theCA1 area of the isolated rat. In addition, subsequent grouprearing reversed this effect. Induction of a significant de-crease in CA1 LTP by isolation, concurrent with reducedneurogenesis and impairment of water maze performance,raises the possibility that newborn granule cells play a rolein the formation of CA1 LTP. Newborn cells in the adultbrain, similar to those generated during development, may

affect hippocampus physiology more than mature cells do(Gould and Tanapat, 1999; Snyder et al., 2001), althoughthe newborn cells are a small percentage of the total cells inthe granule cell layer.

The decrease in synaptic plasticity and impairment oflearning and memory may be a result of the reduced numberof newborn cells. However, several other variables may beimportant as well. Isolation may result in reduced trophicfactor production (Pham et al., 1999), and dopamine andserotonin levels (Heidbreder et al., 2000). Some of thesevariables may exert multiple influences on learning andmemory, long-term potentiation, and neurogenesis. Indeed,there is evidence that increased serotonin levels enhancecell proliferation and reduced serotonin levels, caused bydihydroxytryptamine lesions, diminish dentate LTP (Bliss,et al., 1983). In addition, mutation of the 5-HT2C receptorimpairs dentate gyrus LTP and learning (Tecott et al., 1998).However, It remains to be determined whether such factorsinduced isolation-reduced synaptic plasticity and learningindependently or act indirectly.

Taken together, our findings demonstrate that isolatedrearing results in decreased neurogenesis and reduced LTPin hippocampus and impairment of performance in a water

Fig. 5. Effects of rearing condition on induction of LTP in the CA1 area. LTP was significantly suppressed in both S4w/S4w and S4w (data not shown) ratsas compared with group-reared (G4w/G4w) rats, and the suppressed LTP could recover after social interaction among rats (S4w/G4w) was allowed.Extracellular recording at hippocampal CA1 synapses was performed by stimulation of the Schaffer collateral/commissural inputs to CA1 pyramidal cells.(A, B) Results from in vivo recording of anesthetized rats. (C, D) Results from in vitro recording in hippocampal slices. Arrows indicate high-frequencystimulation. n � 5–8 in each group. Test field excitatory postsynaptic potential (fEPSP) was evoked by stimulating with a square-wave constant current pulseof 50-�s duration at a rate of 0.033 Hz. The slope of fEPSP was measured and averaged every 3 min. At the beginning of each experiment, an input–outputcurve was generated to determine the maximal fEPSP slope, and then the intensity of the stimulus was set at a level that evoked a fEPSP slope of 55–65%of the maximum. Statistical comparisons between baseline and poststimulation values were made using Student’s t test.


maze in rat. Furthermore, these effects induced by isolationcan be reversed by subsequent group rearing, suggestingthat environmental stimuli modify neurogenesis and synap-tic plasticity. However, the neurobiological mechanismsunderlying these effects modified by social environmentsare to be determined.

Acknowledgments

We thank Dr. Y. Shaham for critical reading of themanuscript and helpful suggestions and Y. J. Lu for clericalwork. This work was supported in part by grants from theNational Natural Science Foundation of China (30230130,39825110, 30000050, and 20021003), the Ministry of Sci-ence and Technology (G1999054003 and G1999053907),the National Foundation for Postdoctoral Research, theMinistry of Education, and Shanghai Municipal Commis-sions for Education and Science and Technology.

References

Bengzon, J., Kokaia, Z., Elmer, E., Nanobashvili, A., Kokaia, M., Lindvall,O., 1997. Apoptosis and proliferation of dentate gyrus neurons aftersingle and intermittent limbic seizures. Proc. Natl. Acad. Sci. USA 94,10432–10437.

Bliss, T.V., Collingridge, G.L., 1993. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31–39.

Bliss, T.V., Goddard, G.V., Riives, M., 1983. Reduction of long-termpotentiation in the dentate gyrus of the rat following selective depletionof monoamines. J. Physiol. 334, 475–491.

Cameron, H.A., McKay, R.D., 1999. Restoring production of hippocampalneurons in old age. Nat. Neurosci. 2, 894–897.

Cameron, H.A., Tanapat, P., Gould, E., 1998. Adrenal steroids and N-methyl-D-aspartate receptor activation regulate neurogenesis in thedentate gyrus of adult rats through a common pathway. Neuroscience82, 349–354.

Cameron, H.A., McEwen, B.S., Gould, E., 1995. Regulation of adultneurogenesis by excitatory input and NMDA receptor activation in thedentate gyrus. J. Neurosci. 15, 4687–4692.

Eisch, A.J., Barrot, M., Schad, C.A., Self, D.W., Nestler, E.J., 2000.Opiates inhibit neurogenesis in the adult rat hippocampus. Proc. Natl.Acad. Sci. USA 97, 7579–7584.

Elizabeth, G., Charles, G., 2002. Gross neurogenesis in adult mammals:some progress and problems. J. Neurosci. 22, 619–623.

Eriksson, P.S., Perfilieva, E., Bjork-Eriksson, T., Alborn, A., Nordborg, C.,Peterson, D.A., Gage, F.H., 1998. Neurogenesis in the adult humanhippocampus. Nat. Med. 4, 1313–1317.

Gould, E., Beylin, A., Tanapat, P., Reeves, A., Shors, T.J., 1999. Learningenhances adult neurogenesis in the hippocampal formation. Nat. Neu-rosci. 2, 260–265.

Gould, E., Vail, N., Wagers, M., Gross, C.G., 2001. Adult-generatedhippocampal and neocortical neurons in macaques have a transientexistence. Proc. Natl. Acad. Sci. USA 98, 10910–10917.

Gould, E., Tanapat, P., 1999. Stress and hippocampal neurogenesis. Biol.Psychiatry 46, 1472–1479.

Gould, E., McEwen, B.S., Tanapat, P., Galea, L.A.M., Fuchs, E., 1997.Neurogenesis in the dentate gyrus of the adult tree shrew is regulatedby psychosocial stress and NMDA receptor activation. J. Neurosci. 17,2492–2498.

Gould, E., Tanapat, P., Hastings, N.B., Shors, T.J., 1999. Neurogenesis inadulthood: a possible role in learning. Trends Cogn. Sci. 3, 186–192.

Gould, E., Tanapat, P., Rydel, T., Hastings, N., 2000. Regulation ofhippocampal neurogenesis in adulthood. Biol. Psychiatry 48, 715–720.

Heidbreder, C.A., Weiss, I.C., Domeney, A.M., Pryce, C., Homberg, J.,Hedou, G., Feldon, J., Moran, M.C., Nelson, P., 2000. Behavioral,neurochemical and endocrinological characterization of the early socialisolation syndrome. Neuroscience 100, 749–768.

Jones, G.H., Hernandez, T.D., Kendall, D.A., Marsden, C.A., Robbins,T.W., 1992. Dopaminergic and serotonergic function following isola-tion rearing in rats: study of behavioural responses and postmortem andin vivo neurochemistry. Pharmacol. Biochem. Behav. 43, 17–35.

Kempermann, G., Kuhn, H.G., Gage, F.H., 1997a. More hippocampalneurons in adult mice living in an enriched environment. Nature 386,493–495.

Kempermann, G., Kuhn, H.G., Gage, F.H., 1997b. Genetic influence onneurogenesis in the dentate gyrus of adult mice. Proc. Natl. Acad. Sci.USA 94, 10409–10414.

Kesner, R.P., Gilbert, P.E., Wallenstein, G.V., 2000. Testing neural net-work models of memory with behavioral experiments. Curr. Opin.Neurobiol. 10, 260–265.

Kornack, D.R., Rakic, P., 1999. Continuation of neurogenesis in the hip-pocampus of the adult macaque monkey. Proc. Natl. Acad. Sci. USA96, 5768–5773.

Kuhn, H.G., Dickinson-Anson, H., Gage, F.H., 1996. Neurogenesis in thedentate gyrus of the adult rat: age-related decrease of neuronal progen-itor proliferation. J. Neurosci. 16, 2027–2033.

Lemaire, V., Koehl, M., Le Moal, M., Abrous, D.N., 2000. Prenatal stressproduces learning deficits associated with an inhibition of neurogenesisin the hippocampus. Proc. Natl. Acad. Sci. USA 97, 11032–11037.

Malberg, J.E., Eisch, A.J., Nestler, E.J., Duman, R.S., 2000. Chronicantidepressant treatment increases neurogenesis in adult rat hippocam-pus. J. Neurosci. 20, 9104–9110.

Malenka, R.C., Nicoll, R.A., 1993. NMDA-receptor-dependent synapticplasticity: multiple forms and mechanisms. Trends Neurosci. 16, 521–527.

Morris, R.G., Anderson, E., Lynch, G.S., Baudry, M., 1986. Selectiveimpairment of learning and blockade of long-term potentiation by anN-methyl-D-aspartate receptor antagonist, AP5. Nature 319, 774–776.

Morutto, S.L., Phillips, G.D., 1997. Isolation rearing enhances the loco-motor stimulant properties of intra-perifornical sulpiride, but impairsthe acquisition of a conditioned place preference. Psychopharmacology33, 224–232.

Myhrer, T., 1998. Adverse psychological impact, glutamatergic dysfunc-tion, and risk factors for Alzheimer’s disease. Neurosci. Biobehav. Rev.23, 131–139.

Nacher, J., Rosell, D.R., Alonso-Llosa, G., McEwen, B.S., 2001. NMDAreceptor antagonist treatment induces a long-lasting increase in thenumber of proliferating cells, PSA-NCAM-immunoreactive granuleneurons and radial glia in the adult rat dentate gyrus. Eur. J. Neurosci.13, 512–520.

Nilsson, M., Perfilieva, E., Johansson, U., Orwar, O., Eriksson, P.S., 1999.Enriched environment increases neurogenesis in the adult rat dentategyrus and improves spatial memory. J. Neurobiol. 39, 569–578.

Pacteau, C., Einonm, D., Sinden, J., 1989. Early rearing environment anddorsal hippocampal ibotenic acid lesions: long-term influences on spa-tial learning and alternation in the rat. Behav. Brain Res. 34, 79–96.

Paulus, M.P., Varty, G.B., Geyer, M.A., 2000. The genetic liability tostress and ostweaning isolation have a competitive influence on behav-ioral organization in rats. Physiol. Behav. 68, 389–394.

Pham, T.M., Ickes, B., Albeck, D., Soderstrom, S., Granholm, A.C., Mo-hammed, A.H., 1999. Changes in brain nerve growth factor levels andnerve growth factor receptors in rats exposed to environmental enrich-ment for one year. Neuroscience 94, 279–286.

Pu, L, Bao, G., Xu, N., Ma, L., Pei, G., 2002. Hippocampal long-termpotentiation is reduced by chronic opiate treatment and can be restoredby re-exposure to opiates. J. Neurosci. 22, 1914–1921.

Rakic, P., 1985. Limits of neurogenesis in primates. Science 227, 1054–1056.


Rakic, P., 2002. Neurogenesis in adult primate neocortex: an evaluation ofthe evidence. Nat. Rev. Neurosci. 3, 65–71.

Rocht, C., Gheusi, G., Vincent, J.D., Lledo, P.M., 2002. Enriched odorexposure increases the number of newborn neurons in the adult olfac-tory bulb and improves odor memory. J. Neurosci. 22, 2679–2689.

Smith, J.K., Neill, J.C., Costall, B., 1997. Bidirectional effects of dopamineD2 receptor antagonists on responding for a conditioned reinforcer.Psychopharmacology 131, 23–33.

Snyder, J.S., Kee, N., Wojtowicz, J.M., 2001. Effects of adult neurogenesison synaptic plasticity in the rat dentate gyrus. J. Neurophysiol. 85,2423–2431.

Tecott, L.H., Logue, S.F., Wehner, J.M., Kauer, J.A., 1998. Perturbeddentate gyrus function in serotonin 5-HT2C receptor mutant mice.Proc. Natl. Acad. Sci. USA 95, 15026–15031.

Van Praag, H., Christie, B.R., Sejnowski, T.J., Gage, F.H., 1999. Runningenhances neurogenesis, learning, and long-term potentiation in mice.Proc. Natl. Acad. Sci. USA 96, 13427–13431.

Van Praag, H., Kempermann, G., Gage, F.H., 1999. Running increases cellproliferation and neurogenesis in the adult mouse dentate gyrus. Nat.Neurosci. 2, 266–270.

Varty, G.B., Marsden, C.A., Higgins, G.A., 1999. Reduced synaptophysinimmunoreactivity in the dentate gyrus of prepulse inhibition-impairedisolation-reared rats. Brain Res. 824, 197–203.

Wainwright, P.E, Levesque, S., Krempulec, L., Bulman-Fleming, B., Mc-Cutcheon, D., 1993. Effects of environmental enrichment on corticaldepth and Morris-maze performance in B6D2F2 mice exposed prena-tally to ethanol. Neurotoxicol. Teratol. 15, 11–20.

West, M.J., Slomianka, L., Gundersen, H.J.G., 1991. Unbiased stereologi-cal estimation of the total number of neurons in the subdivisions of therat hippocampus using the optical fractionator. Anat. Rev. 231, 482–497.

Whitaker-Azmitia, P., Zhou, F., Hobin, J., Borella, A., 2000. Isolation-rearing of rats produces deficits as adults in the serotonergic innerva-tion of hippocampus. Peptides 21, 1755–1759.

Xu, L., Anwyl, R., Rowan, M.J., 1997. Behavioural stress facilitates theinduction of long-term depression in the hippocampus. Nature 387,497–500.

Xu, L., Anwyl, R., Rowan, M.J., 1998. Spatial exploration induces apersistent reversal of long-term potentiation in rat hippocampus. Nature394, 891–894.


modification of hippocampal neurogenesis and neuroplasticity by social environments

Documents