Differential Effects of Social and Physical Environmental Enrichment On Brain Plasticity, Cognition, and Ultrasonic Communication in Rats

R E S EA R C H A R T I C L E
Differential Effects of Social and Physical

Environmental Enrichment on Brain Plasticity,
Cognition, and Ultrasonic Communication in Rats
Juan C. Brenes,1,2,3 Martin Lackinger,4 G€unter U. H€oglinger,5 Gerhard Schratt,4 Rainer K.W. Schwarting,1*
and Markus W€ohr1*
1
Behavioral Neuroscience, Experimental and Biological Psychology, Philipps-University of Marburg, 35032 Marburg, Germany
2
Institute for Psychological Research, University of Costa Rica, Rodrigo Facio Campus, 2060 San Pedro, Costa Rica
3
Neuroscience Research Center, University of Costa Rica, Rodrigo Facio Campus, 2060 San Pedro, Costa Rica
4
Biochemical and Pharmacological Center, Institute of Physiological Chemistry, Philipps-University of Marburg, 35032 Marburg,
Germany
5
Technical University M€unchen & German Center for Neurodegenerative Diseases (DZNE) M€unchen, Department for Translational
Neurodegeneration, 81377 M€unchen, Germany
ABSTRACT in the emission of prosocial 50-kHz ultrasonic vocaliza-

Environmental enrichment (EE) exerts beneficial effects tions (USV) paralleled by a lack of social approach in
on brain plasticity, cognition, and anxiety/depression, response to them, consistent with the intense world
leading to a brain that can counteract deficits underly- syndrome/theory of autism. In contrast, social EE had
ing various brain disorders. Because the complexity of only minor effects on brain plasticity and cognition, but
the EE commonly used makes it difficult to identify led to increased prosocial 50-kHz USV emission rates
causal aspects, we examined possible factors using a and enhanced social approach behavior. Importantly,
2 3 2 design with social EE (two vs. six rats) and phys- social deficits following physical EE were prevented by
ical EE (physically enriched vs. nonenriched). For the additional social EE. The finding that social EE has posi-
first time, we demonstrate that social and physical EE tive whereas physical EE has negative effects on social
have differential effects on brain plasticity, cognition, behavior indicates that preclinical studies focusing on
and ultrasonic communication. Expectedly, physical EE EE as a potential treatment in models for neuropsychi-
promoted neurogenesis in the dentate gyrus of the hip- atric disorders characterized by social deficits, such as
pocampal formation, but not in the subventricular zone, autism, should include social EE in addition to physical
and, as a novel finding, affected microRNA expression EE, because its lack might worsen social deficits.
levels, with the activity-dependent miR-124 and miR- J. Comp. Neurol. 524:1586–1607, 2016.
132 being upregulated. Concomitant improvements in
cognition were observed, yet social deficits were seen C 2015 Wiley Periodicals, Inc.
V
INDEXING TERMS: enriched environment; neurogenesis; microRNA; learning and memory; ultrasonic vocalization;
social behavior; autism; RRID: AB_10013660; AB_2160651; nif-0000-3092; nif-0000-30176; nlx_152478;
nlx_144442
Environmental enrichment (EE) is a combination of

“inanimate and social stimulation” (Rosenzweig et al.,
1978), which is widely used to study experience- Grant sponsor: The Deutsche Forschungsgemeinschaft; Grant num-
bers: HO 2402/6-1; SCHR 1136/3-1; SCHW 559/10-1; WO 1732/4-1.
dependent changes in rodent brain and behavior. In the
*CORRESPONDENCE TO: Rainer Schwarting, Behavioral Neuroscience,
laboratory, EE is typically composed of groups of Experimental and Biological Psychology, Philipps-University of Marburg,
rodents living in large housing cages with objects that Gutenbergstr. 18, 35032 Marburg, Germany. E-mail: schwarti@staff.uni-
marburg.de; and Markus W€ ohr, Behavioral Neuroscience, Experimental and
are periodically changed to stimulate curiosity and Biological Psychology, Philipps-University of Marburg, Gutenbergstr. 18,
exploration, often in combination with running wheels. 35032 Marburg, Germany. E-mail: markus.woehr@staff.uni-marburg.de
EE is thought to improve well-being by helping animals Received December 18, 2014; Revised June 23, 2015;
Accepted June 23, 2015.
to fulfill some of their ethological needs, while reducing DOI 10.1002/cne.23842
Published online August 10, 2015 in Wiley Online Library
C 2015 Wiley Periodicals, Inc.
V (wileyonlinelibrary.com)
1586 The Journal of Comparative Neurology | Research in Systems Neuroscience 524:1586–1607 (2016)
Environmental enrichment: Brain and behavior
the stress of captivity, and is probably the best transla- In contrast, relatively few studies have examined the
tional model to investigate positive life experiences act- effects of EE on social behavior. This is surprising
ing either as protective or curative factors in because brain plasticity processes, particularly hippo-
neurological and psychiatric disorders (for reviews, see campal neurogenesis, have been repeatedly linked to
Nithianantharajah and Hannan, 2006; van Praag et al., social behaviors. Thus, social experiences strongly
2000). affect adult neurogenesis, with social defeat (Becker
Early studies showed that EE leads to increased brain et al., 2008; Czeh et al., 2007) and isolation (Lu et al.,
weight and cortical thickness (Bennett et al. 1969; Dia- 2003; Stranahan et al., 2006) having negative effects,
mond et al., 1976; Renner and Rosenzweig, 1986; while mimicking rough-and-tumble play through tickling
Rosenzweig et al., 1978), possibly due to dendritic and (W€ohr et al., 2009) and mating (Leuner et al., 2010;
synaptic growth (Faherty et al., 2003; Leggio et al., Spritzer et al., 2009) can lead to increased adult neuro-
2005; Rampon et al., 2000b). Furthermore, EE pro- genesis. The few available EE studies mostly found
motes adult hippocampal neurogenesis (Kempermann increased social behavior following EE (Green et al.,
et al., 1997) and the integration of newborn cells into 2010; Laviola et al., 2004; Morley-Fletcher et al., 2003;
functional circuits (for review, see Kempermann et al., Neugebauer et al., 2004; Schneider et al., 2006), yet
2010). Such cellular changes are associated with others reported no effects or inconsistent findings
altered expressions of genes involved in synaptic func- (Pe~na et al., 2006; Renner and Rosenzweig, 1986).
tion and cellular plasticity, including increased levels of One type of social behavior, namely rodent communi-
brain-derived neurotrophic factor (BDNF) and postsy- cation, has not yet been investigated in EE research,
naptic density protein 95 (PSD95) (Kuzumaki et al., but can be studied by means of ultrasonic vocalizations
2010; Rampon et al., 2000a), consistent with enhanced (USV), which serve as socially relevant and situation-
glutamatergic signaling and synaptic strength, as dependent affective signals in rats (for review, see
revealed by long-term potentiation (Foster and Dumas, Brudzynski, 2013; W€ohr and Schwarting, 2013). The so-
2001; Green and Greenough, 1986). In addition to glu- called 50-kHz USV are typical for appetitive juvenile
tamate, EE affects various other neurotransmitter sys- social interactions, especially rough-and-tumble play
tems (for review, see Solinas et al., 2010), particularly and tickling, or adult mating (Burgdorf and Panksepp,
mesocorticolimbic dopamine (DA; Bezard et al., 2003; 2001; Burgdorf et al., 2008; Knutson et al., 1998; Pan-
Neugebauer et al., 2004), resulting, for instance, in ksepp and Burgdorf, 2000; Sales, 1972; Schwarting
altered psychomotor responses to amphetamine (Bardo et al., 2007; W€ohr et al., 2009). Rats also emit 50-kHz
et al., 1995; Bowling and Bardo, 1994; Bowling et al., USV when being separated from conspecifics, suggest-
1993; Cain et al., 2012; Gill et al., 2012). ing that they serve a communicative function, namely,
Regarding cognition, EE leads to improved learning to maintain or (re)establish social contact (Schwarting
and memory as assessed by spatial mazes (Bennett et al., 2007; W€ohr et al., 2008). Such an affiliative func-
et al., 2006; Kempermann et al., 1997; Leggio et al., tion was confirmed by means of playback experiments,
2005; Nilsson et al., 1999) and novel object recognition showing that 50-kHz USV elicit social approach behav-
(Bruel-Jungerman et al., 2005; Rampon et al., 2000b). ior in recipients, in both males (W€ohr and Schwarting,
EE also affects exploratory behavior and habituation 2007, 2009, 2012) and females (Willadsen et al.,
learning (Brenes et al., 2009; Elliott and Grunberg, 2014). Importantly, social approach behavior in
2005; Neugebauer et al., 2004; Zimmermann et al., response to 50-kHz USV is paralleled by phasic dopa-
2001), whereas olfactory social discrimination learning mine (DA) release in the nucleus accumbens (Willuhn
is unchanged (Pe~na et al., 2006; Rampon et al., et al., 2014), supporting the idea of a perception-and-
2000b). In addition to learning and memory, EE is action-loop because 50-kHz USV can be triggered by
known to reduce anxiety- and depression-related behav- electrical stimulation of the mesolimbic DA system
ior (Brenes et al., 2008, 2009; Roy et al., 2001; Schnei- (Burgdorf et al., 2000) and by DAergic psychostimu-
der et al., 2006). Furthermore, several studies showed lants, such as amphetamine (for review, see Rippberger
that EE can reverse deficits in models of brain dysfunc- et al., 2015).
tion, including brain damage, dementia, and aging The present study was designed to determine the dif-
(Bezard et al., 2003; Speisman et al., 2013; van Dellen ferential effects of social and physical EE during adoles-
et al., 2000; Will et al., 1976; Wolf et al., 2006). It has cence on brain plasticity, cognition, and ultrasonic
thus been hypothesized that EE leads to a brain that communication in rats. In general, the complexity of EE
can counteract or compensate for deficits underlying commonly used in the laboratory makes it difficult to
various brain dysfunctions (for review, see Nithianan- identify specific factors causing such changes. There-
tharajah and Hannan, 2009; van Praag et al., 2000). fore, the exact causes of the EE effects are the subject
The Journal of Comparative Neurology | Research in Systems Neuroscience 1587

J.C. Brenes et al.
of considerable speculation (for review, see Kemper- and kept in a climate-controlled room with a 12:12-
mann et al., 2010). Typically, EE consists of a combina- hour light–dark schedule (lights on at 07:00 hours). On
tion of both social and physical factors, hindering the PND22–25, they were handled for 4 consecutive days.
assessment of their single contributions, particularly Because emission of 50-kHz ultrasonic vocalizations
when animals raised under EE conditions are housed in (USV) is characterized by considerable interindividual
groups, whereas controls are housed individually, i.e., variability (Schwarting et al., 2007; W€ohr et al., 2009),
exposed to social isolation, which is known to have experimental groups were counterbalanced according
adverse effects on brain and behavior (Fone and Pork- to interindividual 50-kHz USV emission rates by means
ess, 2008). Only recently have studies trying to sepa- of the cage test, a simple and reliable test for assess-
rate out these components been conducted, mainly ing 50-kHz USV (Schwarting et al., 2007; Natusch and
focusing on social isolation effects on locomotor activ- Schwarting, 2010; W€ohr et al., 2008). The cage test
ity, anxiety-related behavior, and spatial cognition, or was conducted on 2 consecutive days (PND26–27) for
body weight gain and feeding (Elliott and Grunberg, 5 minutes each. Based on the average number of 50-
2005; Schrijver et al., 2002; Zaias et al., 2008). kHz USV, rats were split into the four experimental
Here, we used a 2 3 2 experimental design with the housing conditions, resulting in groups that did not dif-
factors social (two vs. six rats per cage) and physical fer in spontaneous emission of 50-kHz USV (P >
(enriched vs. nonenriched cages) EE, resulting in four 0.050). All animal work was conducted according to the
experimental housing conditions: standard control (CO: relevant national and international guidelines.
two rats in a nonenriched cage), social enrichment (SE:
six rats in a nonenriched cage), physical enrichment (PE: Experimental housing conditions
two rats in an enriched cage), and physical plus social A modified version of our routine EE protocol without
enrichment (PESE: six rats in an enriched cage). This running wheels was implemented (Brenes et al., 2009).
allowed us to separate out the contribution of individual Rats (n 5 12 per condition) were kept for 5 weeks
EE components, namely, social EE and physical EE. Impor- (PND33–69) in one of the following conditions: standard
tantly, we employed an EE protocol without the often control (CO: two rats in a nonenriched cage), social
enrichment (SE: six rats in a nonenriched cage), physical
used running wheels, which allowed us to exclude the
enrichment (PE: two rats in an enriched cage), and physi-
possibility that potential EE effects might simply be attrib-
cal plus social enrichment (PESE: six rats in an enriched
uted to running exercise. As measures for experience-
cage). Briefly, PESE rats were housed in commercial pet
dependent brain plasticity we assessed hippocampal neu-
cages (85 cm length 3 46 cm width 3 75 cm height),
rogenesis, i.e., cell proliferation (proliferating cell nuclear
which consisted of two rectangular wooden platforms
antigen [PCNA]) and survival (5-bromodeoxyuridine [BrdU])
(46 3 23 cm each) placed at 30 cm and 51 cm above
in the dentate gyrus (DG), the immediate early gene and
the cage floor, respectively, two wooden stairs, three
transcription factor c-fos, the cAMP response binding pro-
metal food dispensers, and a stainless steel grid with a
tein1 (CREB1), and changes in microRNA (miRNA) expres-
bottle of water placed over it. All other groups were
sion, focusing on miRNAs that are activity-regulated and
housed in normal polycarbonate Macrolon type IV cages
involved in postnatal neuronal development and plasticity
(size: 380 3 200 3 590 mm, plus high stainless steel
(miR-124, -132, and -137; for review, see Schratt, 2009;
covers). The PE/PESE cages contained the following
McNeill and Van Vactor, 2012). At the behavioral level,
items: bedding, pieces of towel paper, roll papers, grass
we studied cognition by means of a habituation learning nests (three for the PESE cage and one for each PE
paradigm and the place object recognition test (PORT), cage), wood sticks, feeding balls filled with towel paper
both strongly associated with the brain plasticity proc- and food pellets, cardboard boxes, and nonchewable
esses assessed. Finally, we determined whether EE leads plastic, glass and metal objects. Except for grass nests,
to changes in social behavior and ultrasonic communica- bedding, and towel papers, enriching items were only
tion, including both sender and receiver, by means of our temporarily available in the cages. Two or three times a
established 50-kHz USV radial maze playback paradigm week, half of these objects were either relocated or
(for review, see Seffer et al., 2014). replaced by a new set of similar objects. As a nutritional
stimulus, 10 g of sweetened puffed wheat cereals
MATERIALS AND METHODS (Smacks, Kellogg’s) were randomly hidden at different
Animals places inside the PE/PESE cages once or twice a week.
Forty-eight juvenile male Wistar rats (HsdCpb:WU; In all groups, food (Altromin, Lage, Germany) and water
Harlan-Winkelmann, Horst, The Netherlands) were (0.0004% HCL-solution) were available ad libitum. Except
group-housed (six per cage) on postnatal day (PND) 21 for behavioral testing and cage cleaning, animals
1588 The Journal of Comparative Neurology | Research in Systems Neuroscience

remained undisturbed. Cage cleaning, including changes during exploration of a cage containing scents from a cage
of bedding material, was carried out at least once a mate (Fig. 5A); and 2) time- and amplitude-matched white
week. noise, which served as a control for novelty-induced
changes in behavior (Fig. 5B). Both stimulus types were
Behavioral characterization presented at 69 dB (measured from a distance of 40 cm)
Once a week during 4 consecutive weeks, rats were for 1 minute with a sampling rate of 192 kHz in a 16-bit for-
tested in the cage test and open field paradigms mat. Playback of acoustic stimuli was monitored by two
(PND41, PND48, PND56, and PND63). During the fourth ultrasonic microphones. Each rat was exposed to both
week, playback of prosocial 50-kHz USV (PND64-65) stimulus types in a counterbalanced manner with an inter-
and place object recognition test (PORT; PND66-67) stimulus interval of 10 minutes after being habituated to
were conducted. In the fifth week, the rats were treated the elevated radial arm maze for 15 minutes. Testing was
with 2.5 mg/kg (intraperitoneally [i.p.]) amphetamine performed under dim red light (10 lx) in a testing room
(PND68–69; the results of this experiment are reported with no other rats present.
elsewhere). At about 45 minutes after amphetamine
injection, rats were transcardially perfused. In all behav- Place object recognition test (PORT)
ioral tests, equipment was thoroughly cleaned with The test apparatuses were two open field chambers (60
0.1% acetic acid solution between subjects. Test order 3 60 3 60 cm; Eckart et al., 2012) illuminated with
was counterbalanced within and across testing days. red light (10 lx) and equipped with a white plastic
disc (5 cm diameter each) attached to the middle of
Cage test one of the walls at 40 cm above the box floor to facili-
Our routine cage test protocol was used (Schwarting tate spatial orientation. The test consisted of three
et al., 2007; Natusch and Schwarting, 2010; W€ohr parts, i.e., habituation, sample trial, and test trial. On
et al., 2008). Briefly, a given rat was separated from the first day (habituation trial), animals were allowed to
conspecifics and individualized in a clean cage (type III) habituate to the empty open fields for 5 minutes.
with fresh bedding (Tapvei) for a 5-minute 50-kHz USV Twenty-four hours later they were exposed to two iden-
recording session. For USV detection, an ultrasonic tical objects for 5 minutes (sample trial), either two sil-
microphone was mounted centrally at 35 cm above the ver iron cylinders (5 cm in diameter, 8 cm high) or two
floor of the cage, which was illuminated by dim red solid glass pillars (6 cm in diameter, 8 cm high).
light (7 lx). Locomotor activity (the number of cage- Objects and spatial location were counterbalanced
halves crossed with at least three paws) and rearing across groups. In the sample trial, objects were placed
behavior were scored off-line from videotapes. in the back corners of the box, with the objects situ-
ated 15 cm away from the walls. During the intertrial
Open field interval animals were returned to their home cages for
As previously described (Schwarting et al., 2007; Wo€hr 30 minutes. Afterwards, rats were again allowed to
and Schwarting, 2008), open field activity was automati- explore the objects (test trial), but now one of them
cally monitored (TruScan, Photo beam Sensor-E63-22, was displaced to one of the front corners of the open
Coulbourn Instruments, Allentown, PA) for 10 minutes in field. Object exploration was scored whenever the rat’s
two acrylic boxes (40 3 40 3 40 cm) equipped with nose touched the object or when it was directed
two grids of infrared sensor beams mounted horizontally toward it within a distance of 2 cm. Climbing onto an
2.5 cm and 14.5 cm above the floor that measured loco- object was not recorded as exploration unless the
motion (distance traveled in m) and rearing (n), respec- snout of the rat was directed towards it by less than 2
tively. For USV detection, an ultrasonic microphone was cm. The relative amount of time spent exploring the dis-
mounted centrally at 45 cm above the floor of the box. placed object as compared with the nondisplaced one
Open field testing was conducted immediately after the was taken as a memory index [exploration time dis-
cage test under dim red light (7 lx). placed object/(exploration time displaced object 1
exploration time stationary object) 3 100]. Object
Playback of prosocial 50-kHz USV exploration, locomotor activity (the number of lines
Testing for social approach behavior in response to play- crossed with at least three paws), and rearing behavior
back of prosocial 50-kHz USV was performed by using our were manually scored off-line from videotapes.
established 50-kHz USV radial maze playback paradigm
(W€
ohr and Schwarting, 2007, 2009, 2012; for review, see Ultrasonic recording and analysis
Seffer et al., 2014). Acoustic stimuli presented were: 1) USV were monitored with UltraSoundGate Condenser
natural 50-kHz USV recorded from an adult male Wistar rat Microphones (CM16; Avisoft Bioacoustics, Berlin,

J.C. Brenes et al.
Germany) and recorded with Avisoft Recorder 2.7 soft- mined by the isolation of RNA from whole-brain slices.
ware (sampling rate: 214,285 Hz; format: 16 bit). High- The expression of c-Fos and CREB1 mRNA was used
resolution spectrograms (frequency resolution: 0.488 as an indirect marker of neural activity, whereas
kHz, time resolution: 0.512 ms) were obtained after a changes in precursor (pre-) and mature miR-124 and
fast Fourier transformation (512 FFT-length, 100% frame, miR-132 were taken as indicative of brain plasticity
Hamming window, 75% time window overlap), by using processes induced by EE. As a control, pre-miR-137
the Avisoft SASLabPro 4.38 software. Experienced was used, which was not expected to vary with
observers manually counted the numbers of USV, with treatments.
USV emitted within a frequency range of 20–32 kHz
being considered as 22-kHz USV and USV between 32 Tissue preparation and immunohistochemistry
and 96 kHz as 50-kHz USV (Schwarting et al., 2007). If Rats were sacrificed 45 minutes after amphetamine
two 50-kHz USV elements were at least 0.048 seconds administration (PND69) with 100 mg/kg pentobarbital
apart, two independent 50-kHz USV were counted. Any i.p. (Merial, Hallbergmoos, Germany) and perfused
change in peak frequency higher than 5 kHz either transcardially using 0.9% saline followed by 4% (wt/vol)
within a single 50-kHz USV element, as the zigzag shape paraformaldehyde in 0.1 mol/L phosphate-buffered
in TRILL calls, or between two or more overlapped 50- saline (PBS; pH 7.4; W€ohr et al., 2009). Postfixed (4%
kHz USV elements, as in STEP calls, was considered as paraformaldehyde, 24 hours, 48C), cryoprotected (20%
a modulation in peak frequency (frequency-modulation sucrose, 48 hours, 48C), and snap-frozen (methylbutane,
[FM]). Therefore, a FLAT call was scored when peak fre- 2308C, 2 minutes) brains were cut on a freezing micro-
quency changes within a single call element were equal tome (CM33050S, Leica, Wetzlar, Germany) in 40-lm
to or lower than 5 kHz. However, the difference coronal sections. Sections were collected in 10 regu-
between the start and the end peaks could be higher larly spaced series, and stored in 0.1 mol/L PBS con-
than 5 kHz, i.e., in 50-kHz USV with a flat shape in taining 0.02% (wt/vol) sodium azide at 48C. For
either an upward or downward direction. When a funda- immunohistochemistry, free-floating sections were incu-
mental flat 50-kHz USV had at least one short flat ele- bated successively for 30 minutes with 0.1% H2O2 in
ment overlapping at the start and/or at the end of the 0.1 mol/L PBS to block endogenous peroxidase activ-
50-kHz USV, a STEP was counted. At least one of these ity, and for 30 minutes with 3% vol/vol donkey serum
short steps had to be 5 kHz higher than the fundamen- in 0.1 mol/L PBS to inhibit nonspecific binding. Sam-
tal call. TRILL calls were considered when at least one ples were pretreated with 2 N HCl (378C, 20 minutes)
frequency-modulation occurred or when at least two and neutralized with borate buffer (pH 8.5, 10 minutes,
frequency peaks in opposed directions were 5 kHz room temperature) for BrdU detection or with 70% etha-
apart within a single call. nol (2208C, 30 minutes) for PCNA detection prior to
incubation with the respective primary antibodies, i.e.,
Neuronal characterization anti-BrdU or anti-PCNA antibody (both from DAKO,
In addition to the behavioral tests, the rate of new- Glostrup, Denmark; Cat# M0744, RRID:AB_10013660
born cells in the subventricular zone (SVZ) and the DG and Cat# M0879, RRID:AB_2160651, respectively;
of the hippocampal formation was investigated. To this 1:500 and 1:1,000, 48C, 12 hours). All antibodies were
aim, animals were treated with BrdU (Sigma, St. Louis, diluted in 0.1 mol/L PBS with 2.5% donkey serum and
MO), which allows the identification of cells that 0.15% Triton X-100 (Sigma). Sections were then incu-
undergo division when BrdU is systemically available, bated for 1 hour at room temperature with the appro-
i.e., during the injection period (H€oglinger et al., 2004). priate biotinylated secondary antibody (donkey anti-rat
BrdU was dissolved at 5 mg/ml in 0.9% NaCl and IgG# 712-005-150 or donkey anti-mouse IgG# 715-
injected i.p. at doses of 100 mg/kg from PND48–52 005-150, Dianova, Hamburg, Germany) in 0.1 mol/L
(see Fig. 1A; 5 consecutive days according to W€ohr PBS with 3% donkey serum and 0.15% Triton X-100
et al., 2009). Solutions were prepared freshly each day. (Sigma). The avidin–biotin method was used to amplify
The last BrdU dose was at least 15 days prior to the the signal (ABC kit, Vector, Burlingame, CA) and 3,3’-
day of perfusion (PND69). This interval is thought to diaminobenzidine tetrachloride/NiCl2 was used to visu-
give labeled cell sufficient time to differentiate and alize bound antibodies. To exclude nonspecific labeling,
mature. In addition, we used an endogenous marker of the primary antibodies were omitted.
cell division, PCNA, as a punctual measure of ongoing
cell proliferation occurring only at the time of perfusion Image analysis
(H€oglinger et al., 2004). Finally, the expression of c-Fos Immunolabeled cells were counted stereologically on
and CREB1 mRNA and different miRNAs was deter- every 10th section of regularly spaced, 40-lm-thick

Figure 1. Effects of social and physical environmental enrichment (EE) on cell proliferation and survival in the dentate gyrus (DG) of the
hippocampal formation. A: 1) Experimental design showing time-points of i.p. BrdU administion (PND, postnatal days). 2) Illustration of rat
brain and hippocampal formation; 3) Schematic overview on the information provided by each cellular marker. B: Effect of housing condi-
tions on the number of BrdU1 cells in the DG. C: Effect of housing conditions on the number of PCNA1 cells in the DG. D–G: Representa-
tive coronal hippocampal sections immunostained for BrdU. H–K: Representative coronal hippocampal sections immunostained for PCNA.
Data are shown as mean 1 SEM. P values: P P 0.050 main effect physical enrichment;* P 0.050 versus CO; # P 0.050 versus SE.
CO, standard control (two rats in a nonenriched cage); SE, social enrichment (six rats in a nonenriched cage); PE, physical enrichment
(two rats in an enriched cage); PESE, physical plus social enrichment (six rats in an enriched cage).
sections using a semiautomatic stereology system Quantitative real-time PCR

(Imager.M2 Axio microscope, Zeiss, Oberkochen, Ger- As previously reported (Siegel et al., 2009; Fiore et al.,
many and Stereoinvestigator, MBF Bioscience, Williston, 2009), quantitative real-time polymerase chain reaction
VT) in the SVZ (2.2 to 20.4 mm in relation to bregma) (qRT-PCR) was performed with a 7300 Real Time PCR
and the subgranular zone of the DG (24.1 to 25.5 System (Applied Biosystems, Foster City, CA;
mm; Paxinos and Watson, 1998), covering its entire RRID:nlx_144442) using iTaq SybrGreen Supermix with
dorsoventral extension. The rate of cell proliferation ROX (Bio-Rad, Hercules, CA; Cat# 1725852, RRID:nif-
was expressed as BrdU1 or PCNA1 cells per lm3. 0000-30176) for mRNAs and pre-miRNAs (primer
sequences are available on request) and TaqMan Micro-
RNA Assays (U6 snRNA and has-mir132; Applied Bio-
RNA isolation systems; Cat# 4440887 and Cat# 4427975,
The remaining slices of the immunohistochemistry anal- RRID:nlx_144442, respectively) for the detection of
ysis were stored in antifreeze solution at 2208C. mature miRNAs. U6 was used as qRT-PCR normalization
For each animal, four whole-brain slices including the control and the average of triplicate CT values from
hippocampual formation (24.1 to 25.5 mm) were taken each sample was used to calculate the relative RNA
for RNA isolation. The total RNA was isolated using amount (2-DCT). Brain samples were taken from two
Recover-All-Total-Nucleic-Acid-Isolation-Kit (Ambion, Carls- independent cohorts of rats (n 5 24 per cohort), with
bad, CA; Cat# AM1975, RRID:nif-0000-3092) as described each cohort including subjects of all experimental
in the manual. TurboDNase (Ambion; Cat# AM2238, groups. Consequently, qRT-PCR analysis was performed
RRID:nif-0000-3092) was used to remove DNA. RNA con- in two independent runs. Detection of c-fos, CREB1,
centrations were determined by measuring absorbance pre-miR-124, pre-miR-137, and mature miR-132 varied
(A260) on the NanoDrop 2000c Spectrophotometer between both cohorts (c-fos: F1,48 5 8.818, P 5 0.005;
(Thermo Scientific, Wilmington, DE; RRID:nlx_152478). CREB1: F1,48 5 7.106, P 5 0.011; pre-miR-124: F1,48

J.C. Brenes et al.
5 68.575, P < 0.001; pre-miR-137: F1,36 5 18.688, P Fig. 1C). As compared with CO rats, the number of
< 0.001; mature miR-132: F1,48 5 108.638, P < BrdU1 cells in the DG was enhanced by 65% in PE rats
0.001). Importantly, however, variability between and by 77% in PESE rats, whereas no change was
cohorts did not affect the effects of social and physical observed in SE rats. When comparing individual housing
EE, because no interactions between cohort and experi- conditions, PESE rats had significantly more BrdU1 cells
mental housing conditions, i.e., social and physical EE, than CO and SE rats, but not PE rats (LSD: all P <
were detected (all P > 0.050). 0.050; all other P > 0.050; for exemplary hippocampal
slices see Fig. 1D–G). Similar results were obtained for
Data analysis and statistics PCNA. The number of PCNA1 cells in the DG was
The particular contribution of either social or physical enhanced by 29% in PE rats and by 61% in PESE rats, as
EE was determined by comparing groups of two versus compared with CO rats, whereas only a minor increase
six rats irrespective of physical EE (effect of social EE) (16%) was observed in SE rats. Group comparison revealed
and groups of rats with or without physical EE irrespec- that PESE rats had significantly more PCNA1 cells than
tive of social EE (effect of physical EE), respectively, by CO rats, but not SE and PE rats (LSD: P < 0.050; all
two-way analyses of variance (ANOVAs; social EE 3 other P > 0.050; for exemplary hippocampal slices see
physical EE). For all cage test and open field parame- Fig. 1H–K). Importantly, EE effects were specific to the
ters, three-way ANOVAs for repeated measures (4 PND DG, because no differences in the level of either BrdU1 or
3 social EE 3 physical EE) were conducted. One-way PCNA1 cells were detected in the subventricular zone
ANOVA analysis followed by protected least significant (SVZ; all P > 0.050).
difference (LSD) Fisher post hoc test was used for sin- Together, the data show that physical EE without run-
gle group comparisons when appropriate. ANOVAs or ning wheels led to enhanced cell proliferation and/or
paired t-tests were used to compare 50-kHz USV sub- survival in the DG, whereas social EE had no net effects
types and behavior in the playback experiment. Cohort on the levels of BrdU1 and PCNA1 cells by its own.
was used as an additional factor in the statistical analy- However, social EE appeared to have a minor additive
ses of c-fos, CREB1, and miRNA expression levels. For effect to physical EE, because PCNA1 cells in particular
all statistical tests the level of significance was defined tended to be higher in PESE than in PE rats.
as P 0.050. Data are expressed as mean 6 standard
error of the mean (SEM). Physical but not social EE increased c-fos,
CREB1, and activity-dependent miRNA
RESULTS expression
Physical but not social EE promoted adult Upregulation of immediate early genes, such as c-fos
hippocampal neurogenesis or zif-268, has been repeatedly detected in brains of
EE is known to induce several neuromorphological rats exposed to EE (Rampon et al., 2000a; Solinas
and neurochemical changes underlying improved learn- et al., 2009), whereas miRNAs have been rarely studied
ing and memory, with increased hippocampal neurogen- (Kuzumaki et al., 2011). Complementing the analysis of
esis being one of the most prominent (for review, see cell proliferation and survival, c-fos and CREB1 mRNA
Kempermann et al., 2010). The first aim of the present expression levels were measured as markers of func-
study was to better understand the factors underlying tional activity in brain slices containing the hippocampal
EE effects on hippocampal neurogenesis. After two formation. In addition, miRNA levels were determined in
weeks of exposure to one of our four experimental the same samples. The short noncoding miRNAs act as
housing conditions, rats received daily i.p. injections of post-transcriptional regulators of gene expression by
BrdU for 5 consecutive days to label newly generated complementary binding to the 30 untranslated region of
cells. Two weeks after the last injection, the number of target mRNAs and mediate experience-dependent
surviving cells was examined. In addition, the number changes in brain plasticity (for review, see Schratt,
of cells expressing PCNA protein was quantified to 2009). We measured expression of the activity-
assess the level of cell proliferation occurring at the dependent precursor and/or mature miR-124 and miR-
time of perfusion (H€oglinger et al., 2004; Fig. 1A). 132, both of which are known to positively regulate
EE significantly affected cell proliferation and/or sur- dendritic morphogenesis, synaptic plasticity, and/or
vival in the DG of the hippocampal formation, as neurogenesis (Magill et al., 2010; Luikart et al., 2011,
assessed by BrdU (physical EE: F1,46 5 8.497, P 5 2012; Remenyi et al., 2010; Vo et al., 2005; Wayman
0.006; all other P > 0.050; Fig. 1B) and PCNA (physical et al., 2008). Thus, we anticipated higher expression
EE: F1,44 5 4.075, P 5 0.050; all other P > 0.050; levels in enriched rats, as EE is known to enhance

dendritic spine number and density (Faherty et al., particularly the mature miR-132 tended to be higher in
2003; Leggio et al., 2005; Rampon et al., 2000b). As a PESE than in PE rats. Increased expression levels of
control, pre-miR-137 was selected, because its overex- activity-dependent miRNAs in rats exposed to physical
pression inhibits dendritic morphogenesis rather than EE are in line with enhanced hippocampal cell prolifera-
increasing it (Silber et al., 2008; Szulwach et al., 2010; tion, survival, and activation following physical EE, sug-
Smrt et al., 2010; Fig. 2A). gesting that experience-dependent changes induced by
EE significantly affected c-fos expression levels (phys- EE could involve miRNA regulation.
ical EE: F1,48 5 5.097, P 5 0.029; all other P > 0.050;
Fig. 2B). In comparison with CO rats, c-fos expression Physical but not social EE improved learning
was enhanced by 21% in PE rats and by 30% in PESE and memory
rats, whereas only a minor increase of 3% was observed Besides brain plasticity, EE is known to affect behav-
in SE rats. Likwise, CREB1 was significantly affected by ior, with improved learning and memory being the most
EE (physical EE: F1,48 5 11.201, P 5 0.002 and social consistent outcome (for reviews see: Nithianantharajah
3 physical EE: F1,48 5 4.418, P 5 0.042; all other P > and Hannan, 2006; van Praag et al., 2000). To test
0.050; Fig. 2C). As compared with CO rats, CREB1 lev- whether the changes in brain plasticity following physi-
els were strongly enhanced by 52% in PE rats, whereas cal EE are associated with changes in cognitive func-
relatively moderate increases of 13% and 25% were tions, we first assessed habituation learning. To this
observed in SE and PESE rats, respectively. Individual aim, a given rat was individually exposed to a cage
group comparisons showed that PE rats had signifi- with clean bedding, referred to as cage test. Immedi-
cantly more CREB1 expression than CO and SE rats,
ately after the cage tests the rat was individually tested
but not PESE rats (LSD: all P < 0.050; all other P >
in an adjacent open field. Both tests were conducted
0.050). At the miRNA level, the activity-dependent pre-
once per week for four weeks whereas rats
miR-124 and pre-miR-132 were affected by EE (physical
were exposed to the experimental housing conditions
EE: F1,48 5 5.297, P 5 0.027 and F1,48 5 5.271, P 5
(Fig. 3A).
0.027, respectively; all other P > 0.050; Fig. 2D,E). As
EE significantly affected locomotor activity in both
compared with CO rats, levels of pre-miR-124 and pre-
tests, i.e., cage test and open field. In the open field,
miR-132 were enhanced by 25% and 26%, respectively,
physically enriched rats, i.e., PE and PESE, displayed
in PE rats, and by 20% and 27%, respectively, in PESE
significantly lower levels of locomotor activity than non-
rats, whereas only minor increases of 3% and 7%,
physically enriched rats, i.e., CO and SE (physical EE:
respectively, were observed in SE rats. Levels of pre-
F1,44 5 18.867, P < 0.001), but also social EE led to
miR-137 were not affected by EE (all P > 0.050; Fig.
reduced locomotor activity (social EE: F1,44 5 8.619, P
2F). In addition to pre-miR, mature levels of miR-132
5 0.005; all other P > 0.050), probably reflecting
were assessed, which were strongly affected by EE
(physical EE: F1,48 5 6.580; P 5 0.014; all other P > reduced psychomotor activation following social and
0.050; Fig. 2G). As compared with CO rats, miR-132 physical EE (Fig. 3B). A comparison of housing condi-
levels were enhanced by 17% in PE rats, and by 38% in tions over the four open field tests showed that PESE
PESE rats, whereas only a minor increase of 7% was rats consistently displayed significantly less locomotor
observed in SE rats, again indicating strong effects of activity than CO, SE, and PE rats, with PE rats also dis-
physical but not social EE. A comparison of individual playing significantly reduced locomotor activity during
housing conditions revealed that PESE rats had signifi- the last open field test, as compared with CO but not
cantly higher miR-132-levels than CO and SE rats, but SE rats (LSD: all P < 0.050; all other P > 0.050). In
not PE rats (LSD: all P < 0.050; all other P > 0.050). support of habituation learning, locomotor activity in
Together, these data show that physical EE without the open field decreased during testing (time: F3,176 5
running wheels led to enhanced neuronal activity levels, 308.410, P < 0.001; Fig. 3C) and with repeated testing
as assessed by the immediate early gene c-fos and over weeks (week: F3,176 5 18.858, P < 0.001; Fig.
CREB1, and changes in miRNA expression levels, with 3D). Within-session habituation was enhanced by physi-
the activity-dependent precursor and/or mature miR- cal EE (time 3 physical EE: F3,176 5 2.477, P 5
124 and miR-132 being upregulated, whereas pre-miR- 0.009), but not social EE (all P > 0.050; Fig. 3C).
137, the negative regulator of neuronal maturation, was Between-session habituation, in contrast, was not
not affected. As for cell proliferation and survival, social affected seperately by the individual experimental hous-
EE had no net effects on c-fos, CREB1, and miRNA ing conditions, namley social and physical EE (all P >
expression levels by its own. However, social EE 0.050; Fig. 3D). Despite an overall similar tendency
appears to have a minor additive effect to physical EE: toward reduced locomotor activity with repeated

J.C. Brenes et al.
Figure 2. Effects of social and physical environmental enrichment (EE) on c-fos mRNA, CREB1 mRNA, and microRNA (miRNA) expression.
A: 1) Experimental design showing when brain samples were obtained (PND, postnatal days). 2) Simplified schematic overview on miRNA
biogenesis. B: Expression of c-Fos mRNA. C: Expression of CREB1 mRNA. D: Expression of pre-miR-124. E: Expression of pre-miR-132. F:
Expression of pre-miR-137. G: Expression of mature miR-132. Data are shown as mean 1 SEM. P values: P P 0.050 main effect physi-
cal enrichment; * P 0.050 versus CO; # P 0.050 versus SE. For group abbreviations see Figure 1.

Figure 3. Effects of social and physical environmental enrichment (EE) on habituation learning and declarative memory. A: 1) Experimental
design showing when open field and cage tests were performed (PND, postnatal days). 2) Picture illustrating the open field test. B: Cumu-
lative locomotion. C: Locomotion per minute over the 10-minute session (all weeks averaged). D: Locomotion over weeks. E: 1) Experimen-
tal design showing when the object recognition trials took place. 2) Pictures illustrating the testing arena during the different trials. F:
Percentage of time spent exploring the displaced object. G: Time spent exploring the two objects in the sample trial expressed as percen-
tages of total exploration time. H: Locomotion on habituation trial. I: Locomotion on sample trial. J: Locomotion on test trial. Data are
shown as mean 1 SEM. S P 0.050 main effect social enrichment; P P 0.050 main effect physical enrichment; * P 0.050 versus
CO; # P 0.050 versus SE; 1 P 0.050 versus PE; X P 0.050 versus week 1. For group abbreviations see Figure 1.

J.C. Brenes et al.
testing, however, reductions in locomotor activity from namely, increased hippocampal cell proliferation, sur-
the first to the fourth open field tests were weak in CO vival, and activation, along with enhanced levels of
rats and did not reach statistical significance (t11 5 activity-dependent miRNAs.
1.176, P 5 0.264), but were pronounced in SE (t11 5
4.268, P 5 0.001), PE (t11 5 3.758, P 5 0.003), and Physical EE reduced, whereas social EE
PESE (t11 5 3.983, P 5 0.002) rats. Similar findings enhanced, the relative number of prosocial
were obtained in the cage test (not shown). Faster 50-kHz USV
within- and between-session habituation in cage test To test whether social and physical EE differentially
and open field in PE and PESE rats probably reflects affect rat social ultrasonic communication, rats were sep-
enhanced habituation learning in rats exposed to physi- arated from conspecifics and singly exposed to a cage
cal EE during adolescence. once per week for 4 weeks; then separation-induced 50-
We also assessed place object recognition by means kHz USV emission rates were assessed (Fig. 4A). Impor-
of the PORT, a test for hippocampus-dependent declar- tantly, we differentiated between call subtypes, namely,
ative-like episodic memory (for review, see Dere et al., FLAT and FM calls. For example, 50-kHz USV occur when
2007) known to be improved by EE (Bennett et al., rats are separated from conspecifics while being isolated
2006; Kempermann et al., 1997; Leggio et al., 2005; in an empty cage, with FLAT calls, a subtype character-
Nilsson et al., 1999). Rats were first exposed to two
ized by a nearly constant frequency, being most promi-
identical objects, termed the sample trial. After a delay
nent (Schwarting et al., 2007; W€ohr et al., 2008). In
of 30 minutes, they were again allowed to explore the
contrast, FM 50-kHz USV, such as STEP and TRILL, are
objects, but in this trial, one of two objects was dis-
less prominent there, but occur at high rates in appetitive
placed to a different location, termed the test trial (Fig.
situations, such as rough-and-tumble play in juveniles
3E). The relative amount of time spent exploring the
(Burgdorf et al., 2008; Knutson et al., 1998).
displaced object, as compared with the nondisplaced
EE significantly affected the emission of 50-kHz USV
one, was taken as the memory index.
in the cage test. Physically enriched rats, i.e., PE and
EE significantly affected performance in the PORT.
PESE, displayed significantly lower 50-kHz USV emis-
Physically enriched rats, i.e., PE and PESE, spent signifi-
sion rates than nonphysically enriched rats, i.e., CO and
cantly more time exploring the displaced object than
SE (physical EE: F1,44 5 9.362, P < 0.004; all other P
nonphysically enriched rats, i.e., CO and SE, indicating
> 0.050; Fig. 4B). When comparing housing conditions
enhanced place object learning in rats exposed to phys-
in the first, second, third, and fourth cage tests, we
ical but not social EE during adolescence (physical EE:
F1,44 5 8.215, P 5 0.006; all other P > 0.050; Fig. found PE and PESE rats to emit significantly fewer 50-
3F). Comparing housing conditions showed that PESE kHz USV than CO but not SE rats, with the exception
rats spend significantly more time exploring the dis- of the first test, in which only a trend was observed
placed object than CO and SE, but not PE rats (LSD: all (LSD: all P < 0.050; all other P > 0.050). In contrast
P < 0.050; all other P > 0.050). Importantly, enhanced to locomotor activity, there was no general decrease in
performance in the PORT was not due to differences in 50-kHz USV emission with repeated testing over weeks
the motivation to explore objects, because rats did not (week: F3,132 5 1.903, P 5 0.132; Fig. 4C). In fact,
differ in object exploration and preference during the changes in 50-kHz USV emission were dependent on
sample trial (all P > 0.050; Fig. 3G). This also shows housing conditions (week 3 physical EE: F3,176 5
that differences in locomotor activity between experi- 2.913, P 5 0.037; all other P > 0.050) and even
mental housing conditions, as observed in cage test increased over weeks from the first to the fourth test in
and open field, but also in the PORT (not shown in CO rats (t11 5 2.520, P 5 0.028), but were unchanged
detail; Fig. 3H–J), are unlikely to be due to a general in SE (t11 5 1.411, P 5 0.186), PE (t11 5 0.045, P 5
decrease in the motivation to explore. 0.965), and PESE (t11 5 1.670, P 5 0.123) rats.
Together, these data show that physical EE without EE also significantly affected the 50-kHz USV profile
running wheels led to improved learning and memory, in the cage test. FLAT calls were the most prominent
as assessed by habituation learning and place object type in all experimental housing conditions and
recognition, whereas social EE had no net effects by its occurred with an overall occurrence rate of 78% as
own. However, social EE appeared to have a minor compared with STEP (15%) and TRILL (7%). Socially
additive effect to physical EE, because in both the enriched rats, i.e., SE and PESE, emitted significantly
habituation learning and the PORT, PESE rats tended to more FLAT and less FM calls, particularly STEP but not
outperform PE rats. This pattern is consistent with the TRILL, than the nonsocially enriched counterparts, i.e.,
changes in brain plasticity following physical EE, CO and PE (social EE: F1,44 5 10.451, P 5 0.003; F1,44

Figure 4. Effects of social and physical environmental enrichment (EE) on spontaneous 50-kHz ultrasonic vocalizations (USV) in the cage
test. A: 1) Experimental design showing when open field and cage tests were performed (PND, postnatal days). 2) Picture illustrating the
cage test. B: Cumulative spontaneous 50-kHz USV. C: Spontaneous 50-kHz USV over weeks. D: Spontaneous 50-kHz USV profiles. Each
area represents the group mean of a given 50-kHz USV subtype, expressed as the percentage of all 50-kHz USV. For significant differen-
ces in the 50-kHz USV profile charts see main text. Data are shown as mean 1 SEM. P P 0.050 main effect physical enrichment; * P
0.050 versus CO; X P 0.050 versus week 1. For group abbreviations see Figure 1.
5 12.472, P 5 0.001; F1,44 5 2.301, P 5 0.136; Together, these data show that physical EE without
respectively; Fig. 4D). In contrast, physical EE had no running wheels led to reduced emission of 50-kHz USV
effect (all P > 0.050). PESE rats emitted significantly in cage test and open field, without changing the 50-
more FLAT but fewer STEP calls than CO and PE but kHz USV profile. In contrast, social EE had no net
not SE rats (LSD: all P < 0.050; all other P > 0.050). effects on emission rates by its own, but led to an
Aversive 22-kHz USV were only rarely observed (aver- increase in the relative amount of FLAT calls. This indi-
age call number per rat/cage test: <0.1 22-kHz USV) cates that 50-kHz USV emission rate and qualitative
and occurred significantly less often than 50-kHz USV features of 50-kHz USV can be independently modu-
in all experimental housing conditions (all P < 0.050), lated by social and physical EE.
with experimental housing conditions not differing from
each other (all P > 0.050). Similar findings were
obtained in the open field (not shown), despite the fact Physical EE reduced, whereas social EE
that USV emission was relatively low, which is probably enhanced, social approach behavior in
due to the short intertest interval between cage test response to playback of prosocial 50-kHz
and open field and the fact that 50-kHz USV emission USV
is typically inhibited when animals are confronted with Despite the increasing interest in understanding
a mild stress context, such as open fields without bed- ultrasonic communication in rodents, most USV studies
ding material (W€ohr et al., 2008; Natusch and Schwart- focus on the emission of USV by the sender, whereas
ing, 2010). the behavioral responses elicited in the receiver in

J.C. Brenes et al.
response to it are rarely studied. By using a playback 50-kHz USV despite intact acoustic information
paradigm, we have repeatedly shown that 50-kHz USV processing.
elicit social approach behavior in the recipients (W€ohr Deficits in social exploration and approach induced
and Schwarting, 2007, 2009, 2012). Because EE by exposure to physical EE were prevented when rats
affected the emission of 50-kHz USV serving prosocial were exposed to social EE in addition to physical EE,
functions, we asked whether social approach elicited by i.e., PESE rats. With the exception of a lack of locomo-
playback of such 50-kHz USV (W€ohr and Schwarting, tor inhibition in response to time- and-amplitude white
2007, 2009, 2012) is also affected by social and physi- noise (t11 5 1.095; P 5 0.297; Fig. 5C), PESE rats dis-
cal EE. To this aim, rats were tested for behavioral played a behavioral response pattern very similar to
changes in response to playback of prosocial 50-kHz that of CO and SE rats and showed social exploration
USV (Fig. 5A) or time- and amplitude-matched white when exposed to 50-kHz USV (t11 5 3.530; P 5 0.005;
noise (Fig. 5B). The latter stimulus served as a control Fig. 5D). As in CO and SE rats, social exploration was
for novelty-induced changes in behavior. directed toward the ultrasonic speaker producing 50-
EE significantly affected social approach behavior eli- kHz USV, as reflected in a significant preference toward
cited by playback of 50-kHz USV. In line with previous proximal arms in comparison with distal arms (t11 5
studies (W€ ohr and Schwarting 2007, 2009, 2012), rats 5.413; P < 0.001; Fig. 5F) and an increased time spent
not exposed to physical EE, i.e., CO and SE rats, dis- on proximal arms during playback when compared with
played locomotor inhibition in response to time- and the 5 minutes before (t11 5 4.162; P 5 0.031; Fig. 5F).
amplitude-matched white noise (CO: t11 5 3.306; P 5 The prosocial effects of social EE were also reflected in
0.007; SE: t11 5 5.099; P < 0.001; Fig. 5C), whereas a significant difference in the time spent proximal dur-
ing playback of 50-kHz USV between rats exposed to
playback of 50-kHz USV led to an increase in locomotor
social EE, i.e., SE and PESE rats, and rats not exposed
activity, reflecting the induction of social exploration
to social EE, i.e., CO and PE rats (social EE: F1,48 5
(CO: t11 5 2.524; P 5 0.028; SE: t11 5 2.681; P 5
9.683; P 5 0.003; Fig. 5F), in the absence of an effect
0.021; Fig. 5D). When the direction of the 50-kHz USV-
of physical EE (all P < 0.050). Such an effect was not
induced social exploration seen in CO and SE rats was
seen for locomotor activity in response to time- and
analyzed by measuring the time spent on proximal and
amplitude-matched white noise (all P > 0.050) and 50-
distal arms (Fig. 5E), significant preferences toward the
kHz USV (all P > 0.050). Preferences toward the ultra-
arms proximal to the speaker producing 50-kHz USV
sonic speaker producing time- and amplitude-matched
were found in both groups (CO: t11 5 3.373; P 5
white noise were not detected (all P > 0.050), and the
0.003; SE: t11 5 5.105; P < 0.001; Fig. 5F). In addi-
groups did not differ in their behavioral profile before
tion, when the time spent on proximal arms in the 5
playback of time- and amplitude-matched white noise
minutes before and during playback of 50-kHz USV was
and 50-kHz USV (all P > 0.050). Two exemplary track
compared, significant increases in the time spent on profiles during playback of prosocial 50-kHz USV are
proximal arms during playback were found in both shown for each experimental housing condition in Fig-
groups (CO: t11 5 2.470; P 5 0.031; SE: t11 5 3.787; ure 5G.
P 5 0.003; Fig. 5F). Together, these data show that social EE led to
In rats exposed to physical EE only, i.e., PE rats, enhanced social exploration and approach in response
time- and amplitude-matched white noise led to a simi- to 50-kHz USV, whereas physical EE led to social defi-
lar reduction in locomotor activity as seen in rats not cits, as indicated by a lack of social exploration and
exposed to physical EE, i.e., CO and SE rats (t11 5 approach in PE rats when exposed to 50-kHz USV.
8.612; P 5 0.001; Fig. 5C). However, in contrast to the Importantly, however, deficits in social exploration and
latter, PE rats did not show social exploration when approach due to physical EE were rescued by exposing
exposed to 50-kHz USV (t11 5 1.421; P 5 0.182; Fig. rats to social EE in addition, i.e., in PESE rats. This indi-
5D). In line with the lack of an induction of social cates that enhanced affiliative behavior provided by
exploratory behavior, no preference toward proximal rearing rats in larger groups during adolescence has
arms was found when the time spent on proximal and positive effects on the rats’ social development that
distal arms was compared (t11 5 1.578; P 5 0.143; counteract or compensate for deficits induced by physi-
Fig. 5F). Also, the time spent on proximal arms before cal EE. This view is supported by the fact that rearing
and during playback of 50-kHz USV did not differ (t11 rats in larger groups also favored the emission of FLAT
5 0.713; P 5 0.490; Fig. 5F). This behavioral response calls with presumably prosocial functions. Observed
pattern indicates deficits in social exploratory and effects were specific to playback of prosocial 50-kHz
approach behavior in PE rats when exposed to prosocial USV because they were not observed in response to

Figure 5. Effects of social and physical environmental enrichment (EE) on approach in response to playback of prosocial 50-kHz ultrasonic
vocalizations (USV). Exemplary spectrograms of acoustic stimuli used for playback. A: Natural 50-kHz USV. B: Time- and amplitude-matched
white noise. C: Locomotor activity before and during playback of time- and amplitude-matched white noise. D: Locomotor activity before and
during playback of prosocial 50-kHz USV. E: 1) Experimental design showing when the playback experiment was performed (PND, postnatal
days); 2) Illustration of the radial maze used for playback. F: Time spent on proximal and distal arms in response to prosocial 50-kHz USV.
The time spent on proximal arms before playback of prosocial 50-kHz USV is shown as dashed (mean) and dotted lines (SEM). G: Two exem-
plary track profiles during playback of prosocial 50-kHz USV are shown for each experimental group. Data are shown as mean 1 SEM. S P
0.050 main effect social enrichment; * P 0.050 versus before; # P 0.050 versus distal. For group abbreviations see Figure 1.

J.C. Brenes et al.
our acoustic control, time- and amplitude-matched rats. This indicates that enhanced affiliative behavior
white noise. provided by rearing rats in larger groups during adoles-
cence has positive effects on the rats’ social develop-
DISCUSSION ment that counteract or compensate for deficits
EE is known to exert a variety of beneficial effects on induced by physical EE. Together, our data show that
brain and behavior, including enhanced brain plasticity, social and physical EE have differential effects on brain
improved learning and memory, and reduced anxiety- plasticity, cognition, and ultrasonic communication in
and depression-like behavior, and hence it was hypothe- rats.
sized that EE leads to a brain that can counteract or
compensate for deficits underlying various neurological Effects of social and physical EE on brain
and psychiatric disorders (for reviews, see Nithianan- plasticity and cognition
tharajah and Hannan, 2006; van Praag et al., 2000). In Adult hippocampal neurogenesis
general, however, the complexity of EE commonly used Among the many effects of EE on brain plasticity proc-
in the laboratory makes it difficult to identify specific esses, increased hippocampal neurogenesis is probably
factors causing the observed changes. The aim of the one of the most prominent (for review, see Kemper-
present study was to better understand these factors mann et al., 2010). Whereas many studies attributed
by using a 2 3 2 design with the factors social EE (two enhanced hippocampal neurogenesis following EE to a
vs. six rats per cage) and physical EE (enriched vs. non- combination of several factors, others see exercise as
enriched cages), all without running wheels. We showed the main contributor. For instance, it has been sug-
that physical EE but not social EE leads to enhanced gested that the effects of EE on hippocampal neurogen-
cell proliferation and/or survival in the DG of the hippo- esis are exclusively due to exercise, because cell
campal formation, increased neuronal activity levels, as proliferation and survival were found to be enhanced
assessed by the immediate early gene c-fos and only when running wheels were accessible (Kobilo
CREB1, and changes in miRNA expression levels, with et al., 2011; Mustroph et al., 2012). Our data suggest,
the activity-dependent precursor and/or mature miR- however, that wheel running exercise might be suffi-
124 and miR-132 being upregulated, whereas pre-miR- cient but not necessary for the induction of hippocam-
137 was not affected. Consistent with enhanced plas- pal neurogenesis, because enhanced cell proliferation
ticity, we found that physical EE leads to improved and/or survival were observed under physical EE condi-
learning and memory, as assessed by habituation learn- tions without running wheels, in agreement with recent
ing and place object recognition. Social EE, in contrast, reports in mice and rats (Freund et al., 2013; Speisman
had no net effect on brain plasticity, and no evidence et al., 2013).
for changes in learning and memory was obtained, yet Enhanced neurogenesis in physical EE without run-
PESE rats often outperformed PE rats, indicating an ning wheels suggests that other factors play important
additive effect of social EE. Furthermore, the numbers roles as well, such as the social environment (Freund
of 50-kHz USV emitted in cage test and open field et al., 2013; Speisman et al., 2013). It is well known
were not affected by social EE, whereas physical EE that social isolation has adverse effects on cognition
consistently led to reduced prosocial 50-kHz USV emis- (Fone and Porkess, 2008) and it has thus been
sion. In contrast, however, social EE, but not physical hypothesized that the social component of EE may play
EE, increased the relative amount of FLAT calls, indicat- a role in hippocampal neurogenesis, yet specific studies
ing that 50-kHz USV emission rate and qualitative fea- have been missing (for review, see Kempermann et al.,
tures of 50-kHz USV can be independently modulated 2010). In support of this hypothesis, however, it was
by the two EE factors. Whereas social EE, i.e., rearing shown that adult hippocampal neurogenesis correlates
rats in larger groups, favored the emission of FLAT calls with individual differences in establishing territories in
with presumably prosocial functions, physical EE large social groups housed in enriched environments
reduced the propensity to emit them. Furthermore, (Freund et al., 2013). Our present data show that social
social EE enhanced social exploration and approach in EE alone did not lead to significant net enhancement of
response to 50-kHz USV, whereas physical EE led to cell proliferation and/or survival when compared with
social deficits, as indicated by a lack of social explora- non-socially enriched animals, yet the effects of physi-
tion and approach in PE rats when exposed to such 50- cal EE varied according to the number of animals per
kHz USV. Importantly, deficits in social behavior cage, with animals raised in larger groups displaying
induced by physical EE were rescued by exposing rats higher cell proliferation and/or survival rate. In fact,
to social EE in addition to physical EE, i.e., in PESE when housing conditions were compared, only PESE

rats differed significantly from CO rats. This is possibly endured upon the expression of the mature miRNA.
due to higher social activity in cages with more animals, Pre-miR-132 has been considered as a rapid response
in line with a study by Fabel et al. (2009) showing that gene because its induction phase parallels that of c-fos
exercise mainly enhances cell proliferation, whereas EE (Nudelman et al., 2010; Vo et al., 2005). In line with
leads to a increase in cell survival (for review, see Kem- this idea, we found that physical EE induced both c-fos
permann et al., 2010). Thus, one potential reason for and pre-miR-132 expression to a similar degree. The
the enhanced cell proliferation despite the lack of run- fact that miR-132 can be rapidly induced but with a
ning wheels is social EE and perhaps the exercise asso- persistent expression, suggests that miR-132 may act
ciated with it, for instance in the form of rough-and- as a signal-dependent switch that regulates neuronal
tumble play behavior (Burgdorf et al., 2008; Knutson homeostasis over the long term (Vo et al., 2005). This
et al., 1998). In fact, we recently found that mimicking can account for the differences found between precur-
rough-and-tumble play enhances hippocampal cell pro- sor and mature miRNA levels, with social stimulation
liferation (W€
ohr et al., 2009), whereas social isolation again having a modulatory effect on brain plasticity
has negative effects (Lu et al., 2003; Stranahan et al., changes induced by physical EE. As for hippocampal
2006). cell proliferation and survival, social EE had no net
effects on miRNA expression levels by its own. How-
Expression of miRNAs ever, social EE appeared to have a minor additive effect
Consistent with the EE effects on neurogenesis, we fur- to physical EE, with only PESE rats differing significantly
ther showed for the first time distinct contributions of from CO rats when housing conditions were compared.
social and physical EE to miRNA expression levels posi- Importantly, our observation that environmental factors,
tively associated with dendritic spine morphogenesis, namely, EE, exert prominent effects on miRNA expres-
synaptic plasticity, and/or neurogenesis (Magill et al., sion levels is in line with our recent finding that social
2010; Luikart et al., 2011, 2012; Remenyi et al., 2010; isolation induces alterations in miRNA expression func-
Vo et al., 2005; Wayman et al., 2008). Specifically, the tionally linked to cognitive impairment (Valluy et al.,
activity-dependent precursor and/or mature miR-124 2015). However, enhanced miR-132 expression follow-
and miR-132 were enhanced by EE, in contrast to pre- ing EE contrasts with a recent study by Kuzumaki et al.
miR-137 which was selected as a control miRNA, (2011) in which no effects of EE on miR-132 expression
because its overexpression inhibits dendritic morpho- levels were found. Besides the fact that we used rats
genesis rather than increasing it (Silber et al., 2008; as compared with mice, conflicting results are possibly
Szulwach et al., 2010; Smrt et al., 2010). due to the fact that we used juvenile animals, whereas
The enhancement of miR-132 expression following EE Kuzumaki et al. (2011) studied older animals, as it is
in brain samples including the hippocampal formation is known that miR-132 expression levels peak around ado-
in accordance with findings showing that acute lescence (Miller et al., 2012; Nudelman et al., 2010).
experience-dependent neural activation induces a tran-
sient increase in pre-miR-132 expression (Nudelman Cognition
et al., 2010). The activity-dependent miR-132, known to Physical EE without running wheels led to improved
be regulated by BDNF and CREB1, has been shown to habituation learning and place object recognition,
promote mature spine morphology by targeting the whereas social EE had no net effects by its own. How-
spine inhibitor GTPase p250GAP (Remenyi et al., 2010; ever, social EE appeared to have a minor additive effect
Vo et al., 2005; Wayman et al., 2008). Also, the neuro- to physical EE in both paradigms, because only PESE
trophic action of BDNF is thought to be directly medi- rats differed from controls in the single-group compari-
ated by miR-132 (Luikart et al., 2011, 2012; Numakawa sons. Importantly, the pattern of results is consistent
et al., 2011). In addition, dendritic branching and syn- with the changes in brain plasticity following physical
aptic integration of newborn hippocampal neurons is EE, namely, increased hippocampal cell proliferation,
modulated by miR-132 (Magill et al., 2010; Luikart survival, and activation, along with enhanced levels of
et al., 2011, 2012). Therefore, it is tempting to specu- activity-dependent miRNAs. Our findings are in line with
late that EE effects on cell proliferation and/or survival enhanced hippocampus-dependent learning following
were at least partly mediated by miR-132 through EE, as assessed in the Morris water maze, radial arm
BDNF and CREB1 downstream activation in the hippo- maze, and novel object recognition (Bennett et al.,
campal formation. This view is supported by the fact 2006; Bruel-Jungerman et al., 2005; Kempermann et al.,
that also CREB1 was upregulated by physical EE. The 1997; Leggio et al., 2005; Nilsson et al., 1999; Rampon
upregulation of miR-132 induced by physical EE et al., 2000b). Also, the result of enhanced habituation
occurred at the level of the precursor and its effect learning following EE is consistent with the literature

J.C. Brenes et al.
(Brenes et al., 2009; Elliott and Grunberg, 2005; Neuge- extinction-like effect by accelerating learning that no
bauer et al., 2004; Zimmermann et al., 2001). social encounter follows the emission of separation-
induced 50-kHz USV. In support of the latter, we have
Effects of social and physical EE on recently shown that social approach normally occurs
separation-induced 50-kHz USV emission during first exposure to playback of 50-kHz USV, but
Rodent USV serve important communicative and not in response to a second exposure even 1 week
affective functions (for review, see Brudzynski, 2013; later, an effect that can be blocked by post-trial treat-
W€ohr and Schwarting, 2013), and our findings show for ment with the amnesic drug scopolamine (W€ohr and
the first time that the emission of 50-kHz USV depends Schwarting, 2012). This indicates that a particular type
on both social and physical EE. High rates of 50-kHz of learning and memory for social acoustic stimuli is
USV typically occur during social interactions, such as active during ultrasonic communication, which might
rough-and-tumble play in juveniles (Burgdorf et al., also encompass 50-kHz USV emission and not just the
2008; Knutson et al., 1998) or mating in adulthood behavioral response to them.
(Burgdorf et al., 2008; Sales, 1972). However, rats also
emit 50-kHz USV when being separated from conspe- Effects of social and physical EE on social
cifics, with FLAT calls being most prominent (Schwart- approach in response to 50-kHz USV
ing et al., 2007; W€ohr et al., 2008). Such 50-kHz USV As outlined above, it has repeatedly been shown that
serve communicative functions as social contact calls EE can lead to a number of beneficial effects, especially
to (re)establish or maintain social proximity, as revealed in case of models of brain dysfunctions, and our pres-
by means of playback experiments (Willadsen et al., ent data on adult hippocampal neurogenesis, miRNA
2014; W€ ohr and Schwarting, 2007, 2009, 2012). Here, expression levels, and learning and memory are consist-
we showed that physical EE led to an overall reduced ent with that view. However, our results obtained in the
emission of separation-induced 50-kHz USV in both USV playback paradigm seem to contrast with such an
tests, i.e., cage test and open field, whereas social EE auspicious view. On the one hand, social EE had posi-
had no net effects by its own. However, social but not tive effects, because it not only enhanced the relative
physical EE led to an increase in the relative amount of amount of prosocial 50-kHz USV, i.e., FLAT calls, but it
FLAT calls. This indicates that 50-kHz USV emission also led to enhanced social exploration and approach in
rate and qualitative call features can be independently response to such signals. Physical EE, on the other
modulated by social and physical EE. Whereas a combi- hand, caused social deficits, as indicated by a lack of
nation of motor, cognitive, and sensory stimulation social exploration and approach in PE rats when
reduced the propensity to call, rearing rats in larger exposed to 50-kHz USV. This finding is consistent with
groups favored the emission of FLAT calls with presum- the intense world syndrome/theory of autism of Mark-
ably prosocial functions. The latter is in line with ram and Markram (2010; see also Markram et al.,
enhanced social behavior in rodents reared under social 2007), who hypothesized that the core neurophysiologi-
EE conditions (Green et al., 2010; Laviola et al., 2004; cal pathology of autism is “excessive neuronal informa-
Morley-Fletcher et al., 2003; Neugebauer et al., 2004; tion processing and storage in local circuits of the
Schneider et al., 2006) and might reflect increased brain, which gives rise to hyper-functioning of brain
social competence, namely, the ability to perceive and regions most affected. Such hyper-functioning in differ-
process social information to optimize behavior in a ent bran regions is proposed to cause hyper-
given social context (Taborsky and Oliveira, 2012). perception, hyper-attention, and hyper-memory that
Emission of 50-kHz USV after separation from conspe- could potentially explain the full spectrum of symptoms
cifics may signal a positive affective state reflecting the in autism” (Markram et al., 2007). In line with this idea,
receptiveness to engage in social interactions, while physical EE might have led to “hyper-plasticity” and
reducing the likelihood of intraspecific aggression. “hyper-reactivity” (increased levels of c-fos, CREB1, and
The inhibitory effects of physical EE on USV emission miRNAs), together with “hyper-memory” (improved
may be explained by two nonmutually exclusive factors: learning and memory in open field and object recogni-
1) physical EE may reduce the salience of stimuli elicit- tion), but reduced social functioning in terms of both
ing 50-kHz USV associated with social exploration, in sender (reduced prosocial 50-kHz USV emission) and
agreement with current and previous data of enhanced receiver (lack of social approach behavior in response
habituation in enriched rats (Brenes et al., 2009; Elliott to 50-kHz USV playback). Of note, whereas the reduc-
and Grunberg, 2005; Neugebauer et al., 2004; Zimmer- tion in prosocial 50-kHz USV emission following physi-
mann et al., 2001); and 2) physical EE may induce an cal EE was seen in two independent behavioral test

paradigms, namely, cage test and open field, lack of adulthood, irrespective of whether the animals were
social approach behavior in response to 50-kHz USV treated with valporate acid before or not. This means
playback was seen in only one behavioral test para- that the combination of both factors, i.e., social and
digm; a replication appears to be needed. Also, it would physical EE, has positive effects on social behavior, and
be interesting to see how direct social interactions are this is exactly what was found in the present study, in
affected by physical EE. which the highest levels of social approach were seen
Although our findings are in line with the intense in PESE rats, i.e., rats exposed to both social and physi-
world syndrome/theory of autism, they might appear cal EE.
surprising because EE has successfully been used to As outlined above, increased social contact provided
reverse social deficits in animal models of autism by living in larger groups during adolescence appears to
(Schneider et al., 2006) and fragile X syndrome (Oddi enhance social competence (Taborsky and Oliveira,
et al., 2014; Restivo et al., 2005). It was also found to 2012). In fact, early social EE has been found to affect
ameliorate the negative effects of prenatal stress and social behavior in different domains. For example, it is
inflammation on social behavior (Connors et al., 2014; known to increase social grooming and other forms of
Laviola et al., 2004; Morley-Fletcher et al., 2003). social interaction (Green et al., 2010; Laviola et al.,
Schneider et al. (2006) therefore proposed EE as an 2004; Morley-Fletcher et al., 2003; Neugebauer et al.,
“important tool for the treatment of autism.” Our 2004; Schneider et al., 2006). Also, mice reared in a
results, however, suggest exactly the opposite, namely, communal nest, an early form of social EE, more
that exposure to physical EE results in social deficits. promptly achieve the social status of either the domi-
Importantly, the observed deficits were specific for nant or the subordinate, display a marked propensity to
behavioral changes in response to playback of prosocial interact socially, and show a pronounced emotional
50-kHz USV, because no effects were observed in response that is modulated by the social context (Bran-
response to our acoustic control, time- and amplitude- chi et al., 2006; for review, see Branchi, 2009). The
matched white noise. Although there are many potential emission of USV and appropriate responses to such
reasons for the observed discrepancy, it is particularly USV is an important part of the social repertoire of
relevant to highlight the fact that we used normal rodents. For instance, rats prefer conspecifics that emit
healthy rats in our study and not a model characterized abundant 50-kHz USV over those calling at lower rates
by brain dysfunction. This difference might actually be (Panksepp et al., 2002). Rough-and-tumble play, usually
of importance because reversal of deficits by EE were associated with high levels of 50-kHz USV (Burgdorf
mostly obtained in models with severe cognitive impair- et al., 2008; Knutson et al., 1998), is altered in deaf
ments (Oddi et al., 2014; Restivo et al., 2005, Speis- (Siviy and Panksepp, 1987) and devocalized (Kisko
man et al., 2013; Will et al., 1976; Wolf et al., 2006). It et al., 2015) rats. Similarly, devocalization of male rats
would therefore be of interest to see whether physical disrupts sexual behavior by reducing proceptive and
EE also exerts beneficial effects in a model displaying receptive behavior in females, yet such deficits are
social deficits but intact cognitive functions. Further- reversed by playback of male USV (Thomas et al.,
more, it is important to highlight that the deficits in 1982; White and Barfield, 1990). The present study is
social exploration and approach induced by physical EE in line with these findings and shows for the first time
were prevented when rats were exposed to social EE in that variation in the level of social stimulation provided
addition to physical enrichment, i.e., in PESE rats. This during adolescence alters social approach in response
indicates that enhanced affiliative behavior provided by to 50-kHz USV.
rearing rats in larger groups during adolescence has
positive effects on the rats’ social development that Methodological considerations
counteract or compensate for deficits induced by physi- The two physical EE conditions, namely, PE and
cal EE. In fact, Oddi et al. (2014) observed improved PESE, as used in the present study, differed in the size
social functioning in the fragile X mouse model follow- of the cage. This difference in cage size is explained by
ing social EE, and in the study by Schneider et al. the fact that the space available per rat and number of
(2006), reversal of autism-related behavioral deficits enrichment items were proportional to the number of
induced by valporate acid was obtained in rats exposed rats housed together to avoid crowding. It is unlikely
to social and physical EE, and not just physical EE. that the size of the cage had a prominent impact on
Moreover, Schneider et al. (2006) found that exposure the results obtained. For most significant physical EE
to social and physical EE during early development effects, these two groups did not differ from each
results in more rough-and-tumble play behavior in juve- other, indicating that differences in cage dimensions
niles and higher levels of social exploratory behavior in and number of enrichment items were largely irrelevant

J.C. Brenes et al.
for the measures determined, except for some minor ROLE OF AUTHORS
additive effects seen in PESE rats. The only experiment All authors had full access to all the data in the
in which the two physical EE conditions clearly differed study and take responsibility for the integrity of the
from each other was the prosocial 50-kHz USV play- data and the accuracy of the data analysis. J.B., R.S.,
back experiment. However, considering that PE rats dis- and M.W. conceived and designed the experiments;
played a lack of approach behavior in response to J.B., M.L., and M.W. performed the experiments; J.B.
prosocial 50-kHz USV, whereas PESE rats displayed and M.W. analyzed the data; G.H., G.S., R.S., and M.W.
enhanced social approach behavior, one can attribute contributed reagents/materials/analysis tools; J.B.,
these differences to the presence of additional conspe- R.S., and M.W. wrote the manuscript; J.B., M.L., G.H.,
cifics in the PESE condition and not to the size of the G.S., R.S., and M.W. gave final approval of the
cage, as opposite effects are unlikely to be explained manuscript.
by even more enrichment items in the PESE as com-
pared with the PE condition. CONFLICT OF INTEREST STATEMENT
The authors have no conflicts of interest.
CONCLUSIONS
In summary, we showed that social and physical EE LITERATURE CITED
have differential effects on brain plasticity, cognition, Bardo MT, Bowling SL, Rowlett JK, Manderscheid P, Buxton
and ultrasonic communication in rats. Physical EE led ST, Dwoskin LP. 1995. Environmental enrichment attenu-
to enhanced brain plasticity, as revealed by means of ates locomotor sensitization, but not in vitro dopamine
release, induced by amphetamine. Pharmacol Biochem
cell proliferation and survival in the DG of the hippo- Behav 51:397–405.
campal formation, as well as c-fos, CREB1, and miRNA Becker C, Zeau B, Rivat C, Blugeot A, Hamon M, Benoliel JJ.
expression levels. Concomitant improvements were 2008. Repeated social defeat-induced depression-like
behavioral and biological alterations in rats: involvement
observed in learning and memory paradigms, yet social
of cholecystokinin. Mol Psychiatry 13:1079–1092.
deficits were seen in the emission of prosocial 50-kHz Bennett EL, Rosenzweig MR, Diamond MC. 1969. Rat brain:
USV and paralleled by a lack of social approach in effects of environmental enrichment on wet and dry
response to them, consistent with the intense world weights. Science 163:825–826.
Bennett JC, McRae PA, Levy LJ, Frick KM. 2006. Long-term
syndrome/theory of autism. In contrast, social EE had continuous, but not daily, environmental enrichment
only minor effects on brain plasticity and cognition, but reduces spatial memory decline in aged male mice. Neu-
led to increased relative emission rates of prosocial 50- robiol Learn Mem 85:139–152.
Bezard E, Dovero S, Belin D, Duconger S, Jackson-Lewis V,
kHz USV and enhanced social approach behavior. Przedborski S, Piazza PV, Gross CE, Jaber M. 2003.
Importantly, social deficits following physical EE were Enriched environment confers resistance to 1-methyl-4-
prevented by additional social EE. This shows that phenyl-1,2,3,6-tetrahydropyridine and cocaine: involve-
ment of dopamine transporter and trophic factors.
social and physical EE can have independent, additive,
J Neurosci 23:10999–11007.
or even opposite effects, depending on the biological or Bowling SL, Bardo MT. 1994. Locomotor and rewarding
behavioral domain studied. The finding that social EE effects of amphetamine in enriched, social, and isolate
has positive whereas physical EE has negative effects reared rats. Pharmacol Biochem Behav 48:459–464.
Bowling SL, Rowlett JK, Bardo MT. 1993. The effect of envi-
on social behavior is highly relevant for enrichment ronmental enrichment on amphetamine-stimulated loco-
research. For example, it indicates that preclinical stud- motor activity, dopamine synthesis and dopamine
ies focusing on EE as a potential treatment in models release. Neuropharmacology 32:885–893.
Branchi I. 2009. The mouse communal nest: investigating the
for neuropsychiatric disorders characterized by social epigenetic influences of the early social environment on
deficits, such as autism, should include social EE in brain and behavior development. Neurosci Biobehav Rev
addition to physical EE, because its lack might worsen 33:551–559.
Branchi I, D’Andrea I, Fiore M, Di Fausto V, Aloe L, Alleva E.
social deficits. A better understanding of such differen- 2006. Early social enrichment shapes social behavior
tial effects may help to uncover specific factors under- and nerve growth factor and brain-derived neurotrophic
lying beneficial and adverse effects of EE in distinct factor levels in the adult mouse brain. Biol Psychiatry
60:690–696.
behavioral domains.
Brenes JC, Rodrıguez O, Fornaguera J. 2008. Differential effect
of environment enrichment and social isolation on
ACKNOWLEDGMENTS depressive-like behavior, spontaneous activity and sero-
The authors thank Marıa Fernanda Urquijo and Natalie tonin and norepinephrine concentration in prefrontal cor-
Heyse for their help in this project. The funders had no tex and ventral striatum. Pharmacol Biochem Behav 89:
85–93.
role in study design, data collection and analysis, decision Brenes JC, Padilla M, Fornaguera J. 2009. A detailed analysis
to publish, or preparation of the manuscript. of open-field habituation and behavioral and

neurochemical antidepressant-like effects in postweaning 2013. Emergence of individuality in genetically identical

enriched rats. Behav Brain Res 197:125–137. mice. Science 340:756–759.
Brudzynski SM. 2013. Ethotransmission: communication of Gill MJ, Arnold JC, Cain ME. 2012. Impact of mGluR5 during
emotional states through ultrasonic vocalization in rats. amphetamine-induced hyperactivity and conditioned
Curr Opin Neurobiol 23:310–317. hyperactivity in differentially reared rats. Psychopharma-
Bruel-Jungerman E, Laroche S, Rampon C. 2005. New neurons cology (Berl) 221:227–237.
in the dentate gyrus are involved in the expression of Green EJ, Greenough WT. 1986. Altered synaptic transmission
enhanced long-term memory following environmental in dentate gyrus of rats reared in complex environments:
enrichment. Eur J Neurosci 21:513–521. evidence from hippocampal slices maintained in vitro.
Burgdorf J, Panksepp J. 2001. Tickling induces reward in ado- J Neurophysiol 55:739–750.
lescent rats. Physiol Behav 72:167–173. Green TA, Alibhai IN, Roybal CN, Winstanley CA, Theobald DE,
Burgdorf J, Knutson B, Panksepp J. 2000. Anticipation of Birnbaum SG, Graham AR, Unterberg S, Graham DL,
rewarding electrical brain stimulation evokes ultrasonic Vialou V, Bass CE, Terwilliger EF, Bardo MT, Nestler EJ.
vocalization in rats. Behav Neurosci 114:320–327. 2010. Environmental enrichment produces a behavioral
Burgdorf J, Kroes RA, Moskal JR, Pfaus JG, Brudzynski SM, phenotype mediated by low cyclic adenosine monophos-
Panksepp J. 2008. Ultrasonic vocalizations of rats (Rattus phate response element binding (CREB) activity in the
norvegicus) during mating, play, and aggression: behav- nucleus accumbens. Biol Psychiatry 67:28–35.
ioral concomitants, relationship to reward, and self- H€oglinger GU, Rizk P, Muriel MP, Duyckaerts C, Oertel WH,
administration of playback. J Comp Psychol 122:357– Caille I, Hirsch EC. 2004. Dopamine depletion impairs
367. precursor cell proliferation in Parkinson disease. Nat
Cain ME, Mersmann MG, Gill MJ, Pittenger ST. 2012. Dose- Neurosci 7:726–735.
dependent effects of differential rearing on Hoffmann LC, Sch€utte SR, Koch M, Schwabe K. 2009. Effect
amphetamine-induced hyperactivity. Behav Pharmacol of “enriched environment” during development on adult
23:744–753. rat behavior and response to the dopamine receptor ago-
Connors EJ, Shaik AN, Migliore MM, Kentner AC. 2014. Envi- nist apomorphine. Neuroscience 158:1589–1598.
ronmental enrichment mitigates the sex-specific effects Kempermann G, Kuhn HG, Gage FH. 1997. More hippocampal
of gestational inflammation on social engagement and neurons in adult mice living in an enriched environment.
the hypothalamic pituitary adrenal axis-feedback system. Nature 386:493–495.
Brain Behav Immun 42:178–190. Kempermann G, Fabel K, Ehninger D, Babu H, Leal-Galicia P,
Czeh B, M€uller-Keuker JI, Rygula R, Abumaria N, Hiemke C, Garthe A, Wolf SA. 2010. Why and how physical activity
Domenici E, Fuchs E. 2007. Chronic social stress inhibits promotes experience-induced brain plasticity. Front Neu-
cell proliferation in the adult medial prefrontal cortex: rosci 4:189.
hemispheric asymmetry and reversal by fluoxetine treat- Kisko TM, Himmler BT, Himmler SM, Euston DR, Pellis SM.
ment. Neuropsychopharmacology 32:1490–1503. 2015. Are 50-kHz calls used as play signals in the play-
Dere E, Huston JP, De Souza Silva MA. 2007. The pharmacology, ful interactions of rats? II. Evidence from the effects of
neuroanatomy and neurogenetics of one-trial object recog- devocalization. Behav Processes 111:25–33.
nition in rodents. Neurosci Biobehav Rev 31:673–704. Knutson B, Burgdorf J, Panksepp J. 1998. Anticipation of play
Diamond MC, Ingham CA, Johnson RE, Bennett EL, elicits high-frequency ultrasonic vocalizations in young
Rosenzweig MR. 1976. Effects of environment on mor- rats. J Comp Psychol 112:65–73.
phology of rat cerebral cortex and hippocampus. Kobilo T, Liu QR, Gandhi K, Mughal M, Shaham Y, van Praag
J Neurobiol 7:75–85. H. 2011. Running is the neurogenic and neurotrophic
Eckart MT, Huelse-Matia MC, Schwarting RKW. 2012. Dorsal stimulus in environmental enrichment. Learn Mem 18:
hippocampal lesions boost performance in the rat sequen- 605–609.
tial reaction time task. Hippocampus 22:1202–1214. Kuzumaki N, Ikegami D, Tamura R, Hareyama N, Imai S,
Elliott BM, Grunberg NE. 2005. Effects of social and physical Narita M, Torigoe K, Niikura K, Takeshima H, Ando T,
enrichment on open field activity differ in male and female Igarashi K, Kanno J, Ushijima T, Suzuki T, Narita M.
Sprague-Dawley rats. Behav Brain Res 165:187–196. 2011. Hippocampal epigenetic modification at the brain-
Fabel K, Wolf SA, Ehninger D, Babu H, Leal-Galicia P, derived neurotrophic factor gene induced by an enriched
Kempermann G. 2009. Additive effects of physical exer- environment. Hippocampus 21:127–132.
cise and environmental enrichment on adult hippocampal Laviola G, Rea M, Morley-Fletcher S, Di Carlo S, Bacosi A, De
neurogenesis in mice. Front Neurosci 3:50. Simone R, Bertini M, Pacifici R. 2004. Beneficial effects
Faherty CJ, Kerley D, Smeyne RJ. 2003. A Golgi-Cox morpho- of enriched environment on adolescent rats from
logical analysis of neuronal changes induced by environ- stressed pregnancies. Eur J Neurosci 20:1655–1664.
mental enrichment. Brain Res Dev Brain Res 141:55–61. Leggio MG, Mandolesi L, Federico F, Spirito F, Ricci B, Gelfo
Fiore R, Khudayberdiev S, Christensen M, Siegel G, Flavell F, Petrosini L. 2005. Environmental enrichment promotes
SW, Kim TK, Greenberg ME, Schratt GM. 2009. Mef2- improved spatial abilities and enhanced dendritic growth
mediated transcription of the miR379–410 cluster regu- in the rat. Behav Brain Res 163:78–90.
lates activity-dependent dendritogenesis by fine-tuning Leuner B, Glasper ER, Gould E. 2010. Sexual experience pro-
Pumilio2 protein levels. EMBO J 28:697–710. motes adult neurogenesis in the hippocampus despite
Fone KC, Porkess MV. 2008. Behavioural and neurochemical an initial elevation in stress hormones. PLoS One 5:
effects of post-weaning social isolation in rodents- e11597
relevance to developmental neuropsychiatric disorders. Lu L, Bao G, Chen H, Xia P, Fan X, Zhang J, Pei G, Ma L. 2003.
Neurosci Biobehav Rev 32:1087–1102. Modification of hippocampal neurogenesis and neuroplas-
Foster TC, Dumas TC. 2001. Mechanism for increased hippo- ticity by social environments. Exp Neurol 183:600–609.
campal synaptic strength following differential experi- Luikart BW, Bensen AL, Washburn EK, Perederiy JV, Su KG, Li
ence. J Neurophysiol 85:1377–1383. Y, Kernie SG, Parada LF, Westbrook GL. 2011. miR-132
Freund J, Brandmaier AM, Lewejohann L, Kirste I, Kritzler M, mediates the integration of newborn neurons into the
Kr€uger A, Sachser N, Lindenberger U, Kempermann G. adult dentate gyrus. PLoS One 6:e19077.

J.C. Brenes et al.
Luikart BW, Perederiy JV, Westbrook GL. 2012. Dentate gyrus Panksepp J, Gordon N, Burgdorf J. 2002. Empathy and the
neurogenesis, integration and microRNAs. Behav Brain action-perception resonances of basic socio-emotional
Res 227:348–355. systems of the brain. Behav Brain Sci 25:43–4.
Magill ST, Cambronne XA, Luikart BW, Lioy DT, Leighton BH, Paxinos GT, Watson C. 1998. The rat brain in stereotaxic coor-
Westbrook GL, Mandel G, Goodman RH. 2010. micro- dinates. Academic Press, London.
RNA-132 regulates dendritic growth and arborization of Rampon C, Jiang CH, Dong H, Tang YP, Lockhart DJ, Schultz
newborn neurons in the adult hippocampus. Proc Natl PG, Tsien JZ, Hu Y. 2000a. Effects of environmental
Acad Sci U S A 107:20382–20387. enrichment on gene expression in the brain. Proc Natl
Markram K, Markram H. 2010. The intense world theory—a Acad Sci U S A 97:12880–12884.
unifying theory of the neurobiology of autism. Front Hum Rampon C, Tang YP, Goodhouse J, Shimizu E, Kyin M, Tsien
Neurosci 4:224. JZ. 2000b. Enrichment induces structural changes and
Markram H, Rinaldi T, Markram K. 2007. The intense world recovery from nonspatial memory deficits in CA1
syndrome—an alternative hypothesis for autism. Front NMDAR1-knockout mice. Nat Neurosci 3:238–244.
Neurosci 1:77–96. Remenyi J, Hunter CJ, Cole C, Ando H, Impey S, Monk CE,
McNeill E, Van Vactor D. 2012. MicroRNAs shape the neuro- Martin KJ, Barton GJ, Hutvagner G, Arthur JS. 2010. Reg-
nal landscape. Neuron 75:363–379. ulation of the miR-212/132 locus by MSK1 and CREB in
Miller BH, Zeier Z, Xi L, Lanz TA, Deng S, Strathmann J, response to neurotrophins. Biochem J 428:281–291.
Willoughby D, Kenny PJ, Elsworth JD, Lawrence MS, Roth Renner MJ, Rosenzweig MR. 1986. Social interactions among
RH, Edbauer D, Kleiman RJ, Wahlestedt C. 2012. Micro- rats housed in grouped and enriched conditions. Dev
RNA-132 dysregulation in schizophrenia has implications Psychobiol 19:303–313.
for both neurodevelopment and adult brain function. Restivo L, Ferrari F, Passino E, Sgobio C, Bock J, Oostra BA,
Proc Natl Acad Sci U S A 109:3125–3130. Bagni C, Ammassari-Teule M. 2005. Enriched environ-
Morley-Fletcher S, Rea M, Maccari S, Laviola G. 2003. Envi- ment promotes behavioral and morphological recovery in
ronmental enrichment during adolescence reverses the a mouse model for the fragile X syndrome. Proc Natl
effects of prenatal stress on play behaviour and HPA Acad Sci U S A 102:11557–11562.
axis reactivity in rats. Eur J Neurosci 18:3367–3374. Rippberger H, van Gaalen M, Schwarting RKW, W€ohr M. 2015.
Mustroph ML, Chen S, Desai SC, Cay EB, DeYoung EK, Environmental and pharmacological modulation of
Rhodes JS. 2012. Aerobic exercise is the critical variable amphetamine-induced 50-kHz ultrasonic vocalizations in
in an enriched environment that increases hippocampal rats. Curr Neuropharmacol 12:220–232.
neurogenesis and water maze learning in male C57BL/6J Rosenzweig MR, Bennett EL, Hebert M, Morimoto H. 1978.
mice. Neuroscience 219:62–71. Social grouping cannot account for cerebral effects of
Natusch C, Schwarting RKW. 2010. Using bedding in a test enriched environments. Brain Res 29;153:563–576.
environment critically affects 50-kHz ultrasonic vocaliza- Roy V, Belzung C, Delarue C, Chapillon P. 2001. Environmen-
tions in laboratory rats. Pharmacol Biochem Behav 96: tal enrichment in BALB/c mice: effects in classical tests
251–259. of anxiety and exposure to a predatory odor. Physiol
Neugebauer NM, Cunningham ST, Zhu J, Bryant RI, Middleton Behav 74:313–320.
LS, Dwoskin LP. 2004. Effects of environmental enrich- Sales GD. 1972. Ultrasound and mating behaviour in rodents
ment on behavior and dopamine transporter function in with some observations on other behavioural situations.
medial prefrontal cortex in adult rats prenatally treated J Zool 168:149–164.
with cocaine. Brain Res Dev Brain Res 153:213–223. Schneider T, Turczak J, Przewłocki R. 2006. Environmental
Nilsson M, Perfilieva E, Johansson U, Orwar O, Eriksson PS. enrichment reverses behavioral alterations in rats prena-
1999. Enriched environment increases neurogenesis in tally exposed to valproic acid: issues for a therapeutic
the adult rat dentate gyrus and improves spatial mem- approach in autism. Neuropsychopharmacology 31:36–46.
ory. J Neurobiol 39:569–578. Schratt G. 2009. microRNAs at the synapse. Nat Rev Neuro-
Nithianantharajah J, Hannan AJ. 2006. Enriched environments, sci 10:842–849.
experience-dependent plasticity and disorders of the Schrijver NC, Bahr NI, Weiss IC, W€urbel H. 2002. Dissociable
nervous system. Nat Rev Neurosci 7:697–709. effects of isolation rearing and environmental enrichment
Nudelman AS, DiRocco DP, Lambert TJ, Garelick MG, Le J, on exploration, spatial learning and HPA activity in adult
Nathanson NM, Storm DR. 2010. Neuronal activity rap- rats. Pharmacol Biochem Behav 73:209–224.
idly induces transcription of the CREB-regulated micro- Schwarting RK, Jegan N, W€ohr M. 2007. Situational factors,
RNA-132, in vivo. Hippocampus 20:492–498. conditions and individual variables which can determine
Numakawa T, Richards M, Adachi N, Kishi S, Kunugi H, ultrasonic vocalizations in male adult Wistar rats. Behav
Hashido K. 2011. MicroRNA function and neurotrophin Brain Res 182:208–222.
BDNF. Neurochem Int 59:551–558. Seffer D, Schwarting RK, W€ohr M. 2014. Pro-social ultrasonic
Oddi D, Subashi E, Middei S, Bellocchio L, Lemaire-Mayo communication in rats: insights from playback studies.
V, Guzman M, Crusio WE, D’Amato FR, Pietropaolo S. J Neurosci Methods 234:73–81.
2014. Early social enrichment rescues adult behav- Siegel G, Obernosterer G, Fiore R, Oehmen M, Bicker S,
ioral and brain abnormalities in a mouse model of Christensen M, Khudayberdiev S, Leuschner PF, Busch
fragile X syndrome. Neuropsychopharmacology 40: CJ, Kane C, H€ubel K, Dekker F, Hedberg C, Rengarajan
1113–1122. B, Drepper C, Waldmann H, Kauppinen S, Greenberg ME,
Pe~na Y, Prunell M, Dimitsantos V, Nadal R, Escorihuela RM. Draguhn A, Rehmsmeier M, Martinez J, Schratt GM.
2006. Environmental enrichment effects in social investi- 2009. A functional screen implicates microRNA-138-
gation in rats are gender dependent. Behav Brain Res dependent regulation of the depalmitoylation enzyme
174:181–187. APT1 in dendritic spine morphogenesis. Nat Cell Biol 11:
Panksepp J, Burgdorf J. 2000. 50-kHz chirping (laughter?) in 705–716.
response to conditioned and unconditioned tickle- Silber J, Lim DA, Petritsch C, Persson AI, Maunakea AK, Yu M,
induced reward in rats: effects of social housing and Vandenberg SR, Ginzinger DG, James CD, Costello JF,
genetic variables. Behav Brain Res 115:25–38. Bergers G, Weiss WA, Alvarez-Buylla A, Hodgson JG.

2008. miR-124 and miR-137 inhibit proliferation of glio- RH, Impey S. 2008. An activity-regulated microRNA con-
blastoma multiforme cells and induce differentiation of trols dendritic plasticity by down-regulating p250GAP.
brain tumor stem cells. BMC Med 6:14. Proc Natl Acad Sci U S A 105:9093–9098.
Siviy SM, Panksepp J. 1987. Sensory modulation of juvenile White NR, Barfield RJ. 1990. Effects of male pre-ejaculatory
play in rats. Dev Psychobiol 20:39–55. vocalizations on female receptive behavior in the rat
Smrt RD, Szulwach KE, Pfeiffer RL, Li X, Guo W, Pathania M, (Rattus norvegicus). J Comp Psychol 104:140–146.
Teng ZQ, Luo Y, Peng J, Bordey A, Jin P, Zhao X. 2010. Will BE, Rosenzweig MR, Bennett EL. 1976. Effects of differen-
MicroRNA miR-137 regulates neuronal maturation by tar- tial environments on recovery from neonatal brain
geting ubiquitin ligase mind bomb-1. Stem Cells 28: lesions, measured by problem-solving scores and brain
1060–1070. dimensions. Physiol Behav 16:603–611.
Solinas M, Thiriet N, El Rawas R, Lardeux V, Jaber M. 2009. Willadsen M, Seffer D, Schwarting RK, W€ohr M. 2014. Rodent
Environmental enrichment during early stages of life ultrasonic communication: male prosocial 50-kHz ultra-
reduces the behavioral, neurochemical, and molecular sonic vocalizations elicit social approach behavior in
effects of cocaine. Neuropsychopharmacology 34:1102– female rats (Rattus norvegicus). J Comp Psychol 128:56–
1111. 64.
Solinas M, Thiriet N, Chauvet C, Jaber M. 2010. Prevention Willuhn I, Tose A, Wanat MJ, Hart AS, Hollon NG, Phillips PE,
and treatment of drug addiction by environmental enrich- Schwarting RK, W€ohr M. 2014. Phasic dopamine release
ment. Prog Neurobiol 92:572–592. in the nucleus accumbens in response to pro-social 50
Speisman RB, Kumar A, Rani A, Pastoriza JM, Severance JE, kHz ultrasonic vocalizations in rats. J Neurosci 34:
Foster TC, Ormerod BK. 2013. Environmental enrichment 10616–10623.
restores neurogenesis and rapid acquisition in aged rats. W€ohr M, Houx B, Schwarting RKW, Spruijt B. 2008. Effects of
Neurobiol Aging 34:263–274. experience and context on 50-kHz vocalizations in rats.
Spritzer MD, Weinberg A, Viau V, Galea LA. 2009. Prior sexual Physiol Behav 93:766–776.
experience increases hippocampal cell proliferation and W€ohr M, Kehl M, Borta A, Sch€anzer A, Schwarting RK,
decreases risk assessment behavior in response to acute H€oglinger GU. 2009. New insights into the relationship
predator odor stress in the male rat. Behav Brain Res of neurogenesis and affect: tickling induces hippocampal
200:106–112. cell proliferation in rats emitting appetitive 50-kHz ultra-
Stranahan AM, Khalil D, Gould E. 2006. Social isolation delays sonic vocalizations. Neuroscience 163:1024–1030.
the positive effects of running on adult neurogenesis. W€ohr M, Schwarting RKW. 2007. Ultrasonic communication in
Nat Neurosci 9:526–533. rats: can playback of 50-kHz calls induce approach
Szulwach KE, Li X, Smrt RD, Li Y, Luo Y, Lin L, Santistevan behavior? PLoS One 2:e1365.
NJ, Li W, Zhao X, Jin P. 2010. Cross talk between micro- W€ohr M, Schwarting RKW. 2009. Ultrasonic communication in
RNA and epigenetic regulation in adult neurogenesis. rats: effects of morphine and naloxone on vocal and
J Cell Biol 189, 27–41. behavioral responses to playback of 50-kHz vocaliza-
Taborsky B, Oliveira RF. 2012. Social competence: an evolu- tions. Pharmacol Biochem Behav 94:285–295.
tionary approach. Trends Ecol Evol 27:679–688. W€ohr M, Schwarting RKW. 2012. Testing social acoustic mem-
Thomas DA, Howard SB, Barfield RJ. 1982. Male produced ory in rats: effects of stimulus configuration and long-
ultrasonic vocalizations and mating patterns in female term memory on the induction of social approach behav-
rats J Comp Psychol. 96:807–815. ior by appetitive 50-kHz ultrasonic vocalizations. Neuro-
Valluy J, Bicker S, Aksoy-Aksel A, Lackinger M, Sumer S, Fiore biol Learn Mem 98:154–164.
R, W€ust T, Seffer D, Metge F, Dieterich C, W€ohr M, W€ohr M, Schwarting RKW. 2013. Affective communication in
Schwarting R, Schratt G. 2015. A coding-independent rodents: ultrasonic vocalizations as a tool for research
function of an alternative Ube3a transcript during neuro- on emotion and motivation. Cell Tissue Res 354:81–97.
nal development. Nat Neurosci 18:666–673. Wolf SA, Kronenberg G, Lehmann K, Blankenship A, Overall R,
van Dellen A, Blakemore C, Deacon R, York D, Hannan AJ. Staufenbiel M, Kempermann G. 2006. Cognitive and
2000. Delaying the onset of Huntington’s in mice. Nature physical activity differently modulate disease progression
404:721–722. in the amyloid precursor protein (APP)-23 model of Alz-
van Praag H, Kempermann G, Gage FH. 2000. Neural conse- heimer’s disease. Biol Psychiatry 60:1314–1323.
quences of environmental enrichment. Nat Rev Neurosci Zaias J, Queeney TJ, Kelley JB, Zakharova ES, Izenwasser S.
1:191–198. 2008. Social and physical environmental enrichment dif-
Vo N, Klein ME, Varlamova O, Keller DM, Yamamoto T, ferentially affect growth and activity of preadolescent
Goodman RH, Impey S. 2005. A cAMP-response element and adolescent male rats. J Am Assoc Lab Anim Sci 47:
binding protein-induced microRNA regulates neuronal 30–34.
morphogenesis. Proc Natl Acad Sci U S A 102:16426– Zimmermann A, Stauffacher M, Langhans W, W€urbel H. 2001.
16431. Enrichment-dependent differences in novelty exploration
Wayman GA, Davare M, Ando H, Fortin D, Varlamova O, in rats can be explained by habituation. Behav Brain Res
Cheng HY, Marks D, Obrietan K, Soderling TR, Goodman 121:11–20.

Differential Effects of Social and Physical Environmental Enrichment On Brain Plasticity, Cognition, and Ultrasonic Communication in Rats

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Differential Effects of Social and Physical

ABSTRACT in the emission of prosocial 50-kHz ultrasonic vocaliza-

Environmental enrichment (EE) is a combination of

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sections using a semiautomatic stereology system Quantitative real-time PCR

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neurochemical antidepressant-like effects in postweaning 2013. Emergence of individuality in genetically identical

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