Pharmacology, Biochemistry and Behavior 76 (2003) 481 – 486

www.elsevier.com/locate/pharmbiochembeh

Environmental enrichment lowers stress-responsive hormones in

singly housed male and female rats
Emily E. Belz, Jamilyn S. Kennell, R. Kenneth Czambel, Robert T. Rubin, Michael E. Rhodes*
Center for Neurosciences Research, Allegheny-Singer Research Institute, Allegheny General Hospital, 8 S.T.,
320 East North Avenue, Pittsburgh, PA 15212, USA
Received 9 June 2003; received in revised form 26 August 2003; accepted 3 September 2003

Abstract

Structural and social aspects of an environmental system can influence the physiology and behavior of animals occupying that system.
This study examined the physiological effects of environmental enrichment (EE) with KongR ToysR and NestletsR on stress-responsive
hormones of the hypothalamic – pituitary – adrenal (HPA) axis under basal and mild stress conditions in singly housed, jugular vein-
cannulated, male and female rats.
Animals of both sexes housed with EE had significantly lower baseline adrenocorticotropic hormone (ACTH) and corticosterone (CORT)
concentrations compared to those housed without EE. ACTH responses to the mild stress of saline injection were significantly lower in
female rats housed with EE.
Interaction with the Kong Toys and Nestlets appears to have provided the rats with a diversion from monotonous cage life, resulting in
lower HPA axis activity before and after mild stress. These results are important because low, stable baselines are essential for accurately
discerning pharmacological and other influences on the HPA axis.
D 2003 Elsevier Inc. All rights reserved.

Keywords: ACTH; CORT; Environmental enrichment; HPA axis; Stress

1. Introduction instinctive tendencies. Environmental enrichment (EE) can

be defined as using objects to improve the quality of life of
Stress is a variable that often is overlooked in research on the caged animal, thus distracting the animal from an
laboratory animals. It is representative of a living condition otherwise monotonous environment. For example, rats tend
that is unfavorable and damaging to the organism’s homeo- to tear materials such as cellulose paper or straw into small
stasis or adaptive state (Martini et al., 2000). Slight changes pieces to create a nest (Scharmann, 1991). NestletsR, com-
in a laboratory animal’s environment can alter its behavior mercially available, 2
2-in. squares composed of sterilized,
during an experiment. Factors such as caging, social dis- pulped, virgin cotton fiber, provide an animal with a soft,
ruption, restraint, transport, noise, routine cleaners, and warm, nest-like environment. Animals housed with Nestlets
lighting can significantly impact the behavior of laboratory are less aggressive, gain more weight, and are easier to
animals (Barnard and Hou, 1988; Jain and Baldwin, 2003). handle—behaviors compatible with reduced stress (Hobbs et
Adaptive responses to these factors include adverse physi- al., 1997). Another object for EE is the KongR Toy, a 2- to 3-
ological and behavioral consequences, as well as alterations in.-long rubber toy originally designed in larger sizes for
in neuroendocrine and autonomic systems (Martini et al., dogs. Kong Toys augment an animal’s natural affinity for
2000). gnawing.
Stress reduction may be achieved by enriching an ani- The hypothalamic –pituitary –adrenal (HPA) axis is an
mal’s environment with devices that promote its normal important modulator of the stress response. HPA activity is
reflected peripherally by plasma concentrations of adreno-
* Corresponding author. Tel.: +1-412-359-6038; fax: +1-412-359- corticotropic hormone (ACTH) and corticosterone (CORT),
4499. released from the anterior pituitary and adrenal cortex,
E-mail address: mrhodes@wpahs.org (M.E. Rhodes). respectively. Conditions such as chronic cannula implanta-

0091-3057/$ – see front matter D 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.pbb.2003.09.005
482 E.E. Belz et al. / Pharmacology, Biochemistry and Behavior 76 (2003) 481–486

tion for serial blood sampling necessitate that animals 2.2. Environmental enrichment
usually housed in a group be housed individually after
surgery. Both current housing conditions and previous Two environments were used. One group of animals was
housing experience can influence ACTH and CORT in male singly housed under standard laboratory conditions. A
and female rats: Female rats housed in a group were shown second group of animals had both Kong Toys (Bio-Serv,
to have lower CORT baselines than female rats housed Frenchtown, NJ, USA) and Nestlets (Ancare, Bellmore, NY,
individually, whereas male rats housed in a group had higher USA) in their cages. Kong Toys remained in the cage for the
CORT baselines than male rats housed individually (Brown duration of housing. A new Nestlet was presented when the
and Grunberg, 1995). In a follow-up study from the same bedding from each cage was changed every 3 days.
laboratory, individually housed male rats that had been
previously housed in a group had higher CORT baselines 2.3. Blood sampling
compared to male rats that had not been previously housed
in a group (housed individually) (Brown and Grunberg, The two-person procedure for blood sampling from
1996). cannulated animals was used: One person gently contained
Because our experiments incorporate singly housed, the animals that typically remained calm after daily handling
jugular vein-cannulated male and female rats into pharma- for 3 –4 days before experimentation, while the other person
cological dosing studies (e.g., Rhodes et al., 2001a,b, 2002), collected the blood sample. Sampling was completed in less
alleviating potential stress from single housing is impera- than 1 min. During experimentation, the stainless-steel
tive. The present study therefore attempted to reduce the cannula plug was removed, the heparin –polyvinylpyrroli-
stress of individual housing and resulting basal HPA axis done (PVP; 100 IU/ml) lock solution (Sigma, St. Louis,
activity by providing EE with Nestlets and Kong Toys. We MO, USA) was aspirated, and 300 – 350 Al blood was
also examined HPA responses to the mild stress of saline withdrawn into a 1-ml tuberculin syringe, immediately
injection. transferred into microcollection tubes, and stored on ice.
A replacement solution of warm, buffered physiological
SAL equal to the amount of blood withdrawn was infused
2. Materials and methods through the cannula, the cannula was injected with 0.02 ml
lock solution, and the stainless-steel plug was reinserted.
2.1. Animals

Eight-week-old, jugular vein-cannulated, male and fe-

male Sprague – Dawley rats weighing 200– 225 g (Taconic
Farms, Germantown, NY, USA) were housed singly in a
standard, shoebox-style polycarbonate cage (24
46
20
cm) with woodchip bedding, a wire-top lid and filter-top
plastic cap in a well-ventilated, temperature- and humidity-
controlled environment (22 – 25 jC, 50 – 75% humidity)
under a standard 12-h light:dark cycle (lights on at 0700
h). Male animals were housed within sight, hearing, and
smell of other male animals. Likewise, female animals
were housed within sight, hearing, and smell of other
female animals. Intermingling of cages of animals of each
sex housed with or without enrichment insured that tem-
perature and humidity were random variables. Laboratory
rat chow and water were available ad libitum. Animals
were allowed 3 – 4 days to acclimate to the housing
conditions; during this time they were handled daily for
2 –3 min and, on the day before blood sampling experi-
ments, administered an intraperitoneal injection of physio-
logical saline (SAL, 1 ml/kg) to acclimate the animals to
the procedure of intraperitoneal injection. Experiments
were performed between 0900 and 1300 h to minimize
the influence of circadian variations in plasma hormone
concentrations. All experiments were approved by the
Institutional Animal Care and Use Committee and were Fig. 1. Environmental enrichment. Examples of Kong Toys and Nestlets
conducted in accordance with National Institutes of Health before (A, B) and after (C) presentation to singly housed male and female
guidelines. rats.
 E.E. Belz et al. / Pharmacology, Biochemistry and Behavior 76 (2003) 481–486 483

The plasma was separated by centrifugation, quickly 2.5. Statistical analysis

frozen at  80 jC, and stored until hormone analyses.
Baseline blood samples were collected at  25 and  15 Multiple experiments were performed under the same
min before SAL injection. The two samples were averaged conditions to attain N = 138 animals for the baseline analy-
to yield a mean baseline at  20 min. SAL (1 ml/kg) was sis. Of these 138 animals, 42 animals were administered
administered intraperitoneally to animals after the baseline SAL at 0 min. All animals from the SAL response groups
blood samples were collected. Additional samples were are included in the baseline analysis. The overall N was
collected 10, 20, 40, and 60 min after SAL administration. necessary to compensate for nonpatent cannula occurrences.
Group Ns varied, because there was insufficient sample for
2.4. Hormone assays the analysis of both hormones from a few of the animals.
Statistically significant differences between groups were
Multiple assays were performed; each assay contained determined by two-way analysis of variance (ANOVA)
random samples from all experimental groups. Plasma (Sex
Environment) for baseline analysis and by three-
samples were analyzed in singlet for ACTH and in duplicate way ANOVA (Sex
Environment
Time) for SAL-re-
for CORT. ACTH was determined by a highly specific sponse analysis, with time as a repeated measure. Tukey–
immunoradiometric assay (Nichols Institute, San Juan Cap- Kramer and Fisher’s LSD tests were used for post hoc
istrano, CA, USA) (Raff and Findling, 1989). Inter- and comparisons. Significance was considered as P < .05.
intra-assay coefficients of variation were 6.2% and 3.8%,
respectively. The minimum detectable ACTH concentration
was 2.6 pg/ml. CORT was analyzed by RIA with materials 3. Results
obtained in kit form (ICN Pharmaceuticals, Costa Mesa,
CA, USA). Antibody specificities compared to CORT were 3.1. Behavioral observations
less than 0.5% for all other steroids. Inter- and intra-assay
coefficients of variation were less than 10%. The minimum Animals of both sexes consistently chewed their Kong
detectable CORT concentration was 2.3 ng/ml. Toys and shredded their Nestlets (Fig. 1). It appeared that

Fig. 2. Baseline ACTH and CORT in male and female rats housed singly with or without environmental enrichment (EE). Each bar represents the
mean F S.E.M. Solid bars = control groups; Striped bars = EE groups. Ns per group for ACTH and CORT, respectively: male control = 48, 44; male EE = 25,
20; female control = 41, 37; female EE = 24, 20. * P < .05, difference between EE and control groups; yP < .05, difference between males and females.
484 E.E. Belz et al. / Pharmacology, Biochemistry and Behavior 76 (2003) 481–486

the females were more active in shredding the Nestlets 3.3. Responses to SAL injection
and sleeping on or next to them. It also was our obser-
vation that after the animals experienced living in an Fig. 3 shows the effects of housing on ACTH and CORT
enriched environment, they were easier to handle during responses to SAL injection.
acclimation periods and experiments. Vocalizations and For ACTH, there were significant main effects for
observable behaviors (e.g., exploratory behavior, rearing) environment [ F(1,61) = 46.8, P < .0001] and time
also did not appear to differ between groups, suggesting [ F(6,61) = 4.7, P=.0001]. Both male and female rats
that these behaviors were not influenced by housing housed with EE had significantly lower absolute ACTH
conditions. responses to the mild stress of SAL injection ( P < .05)
compared to control rats. Both male and female EE groups
3.2. Baseline hormone concentrations had significantly lower ACTH responses compared to the
control groups ( P < .01), because of their lower baseline
Fig. 2 shows the effects of housing on baseline ACTH and hormone concentrations. In the female control group,
CORT. Both ACTH [ F(1,137) = 24.8, P < .0001] and CORT ACTH responses to SAL at + 40 min were significantly
[ F(1,137) = 18.8, P < .0001] concentrations were significant- higher, by 52%, and at + 60 min, by 95%, compared to
ly lower in the groups housed with EE. For baseline ACTH baseline ( Ps < .05). In contrast, in the female EE group,
concentrations, there were no significant sex differences in ACTH responses to SAL were not significantly different at
either the control or the EE groups. For baseline CORT either + 40 or + 60 min (15% and 48% increases, respec-
concentrations, there was a significant main effect of sex tively) compared to baseline.
[ F(1,137) = 34.7, P < .0001], reflected in the significantly For CORT, there were significant main effects for envi-
higher CORT baselines in females in both the control and the ronment [ F(1,60) = 10.6, P=.0019], and sex [ F(1,60) = 24.0,
EE groups ( Ps < .05). P < .0001], as well as for time [ F(6,60) = 21.9, P < .0001].

Fig. 3. ACTH and CORT responses to saline (SAL) injection in male and female rats housed singly with or without environmental enrichment (EE). Baseline
blood samples collected at  25 and  15 min prior to intraperitoneal injection of SAL (1 ml/kg) were averaged to yield the  20 min baseline value.
Postinjection samples were collected at + 10, + 20, + 40, and + 60 min. Arrows (z) indicate time of intraperitoneal SAL injection. Each square represents the
mean F S.E.M. Open squares = control groups; solid squares = EE groups. Ns per group for ACTH and CORT, respectively: Male control = 13, 13; male EE = 8,
7; female control = 14, 14; female EE = 7, 7.
 E.E. Belz et al. / Pharmacology, Biochemistry and Behavior 76 (2003) 481–486 485

In both the control and the EE groups, male and female intriguing to manipulate by the animals (e.g., golf balls,
CORT responses to SAL were similar. wood dowels, caged peach pit). In another study, Nyla-
bones, comparable to Kong Toys, were actively chewed by
many rats because they augmented the gnawing instinct
4. Discussion (Watson, 1993). Animals that did not chew the bone
occupied their time by moving the bone around the cage.
Social aspects of an environmental system may play a Therefore, providing laboratory rats with EE for chewing
significant role in influencing the physiology and behavior will, at the very least, occupy their time as well as allow
of the animals occupying that system. Because rodents are them to exercise a fundamental instinctive behavior.
social animals, isolated housing can produce stress (Barnard EE may provide multiple benefits to animals (Würbel,
and Hou, 1988). Many current studies house rats or mice in 2001). An environment to which an animal cannot easily
grouped environments, which is seemingly beneficial to the adapt may cause stress (Beaver, 1989; van de Weerd and
animals. Conditions such as cannula implantation for serial Baumans, 1995). Male mice housed with EE have lower
blood sampling or drug administration necessitate that CORT responses to the stress of cat odor compared to male
animals be singly housed. This prevents group-housed mice housed under standard conditions (Roy et al., 2001),
animals from scratching, biting, or gnawing, and thus suggesting that EE can reduce the HPA response to an
destroying the cannulae of other animals. environmental signal such as cat odor. Other benefits of EE
The results of our study support the concept that the in rodents include larger forebrains and a better-developed
structural aspects of an environmental system can influence brain structure (Beaver, 1989), increased granule cell num-
the physiology and behavior of the animals occupying that ber and hippocampal volume (Kempermann et al., 1997),
system. This is indicated by the significantly lower baseline enhanced problem-solving and exploratory behaviors (Stu-
ACTH and CORT concentrations in the male and female delska and Kemble, 1979; Scharmann, 1991), and delayed
rats housed with EE. Compared to baseline, ACTH decline of these physiological and behavioral measures with
responses to SAL injection were significantly lower in advancing age (e.g., Fernández-Teruel et al., 2002).
female rats housed with EE, but not in males. This may Interactions between animals and the researcher are cru-
have been related to the mild stress of the SAL injection; in cial to maintaining low stress-responsive hormone baselines.
other work, male rats reared with EE (bedding material, Postnatal handling of rat pups produces significantly lower
shelves, plastic tunnels, hay, ropes, and a hut) had lower plasma ACTH responses to a variety of stressors compared to
ACTH responses to a more severe, 20-min restraint stress nonhandled pups (Viau et al., 1992). In our animals, whatever
than did male rats reared in isolation (Schrijver et al., 2002). stress-reducing benefits the acclimation and restraint process
The lower ACTH responses to restraint stress may have provided were significantly augmented by EE. The effects of
derived from the greater complexity of the enriched envi- handling before and after mild stress in the present study are
ronment. Future studies that determine ACTH and CORT difficult to determine; future studies using cannula extensions
responses to moderate stressors (e.g., footshock or restraint) whereby blood sampling can occur with minimal contact to
in singly housed male and female rats with and without the animal (Thrivikraman et al., 2002) would clarify the
Kong Toys and Nestlets may further elucidate the sex potential interaction of stress and handling.
differences observed in the present study. In summary, the hormone effects observed in our study
In all our studies with singly housed, jugular vein- are likely due to interaction of the rats with Kong Toys and
cannulated rats, the females had consistently greater CORT Nestlets, providing the animals with a diversion from
baselines and greater absolute CORT responses to SAL and monotonous cage life and resulting in both lower basal
other pharmacological challenges compared to male rats HPA hormones and, in the females, reduced ACTH
(Rhodes et al., 2001a,b, 2002). Because females typically responses to the mild stress of SAL injection. These results
have higher baseline and stress hormone responses (Rhodes are important because low and stable baselines, which can
and Rubin, 1999), EE appears to be useful in lowering result from an enriched environment, are essential for
baseline ACTH and CORT concentrations, as well as ACTH accurately discerning HPA responses to pharmacological
responses to stress, particularly in females. Lower baseline and other challenges.
hormone concentrations should produce a greater sensitivity
of the animal to the effects of experimental manipulation.
Rats show a distinct preference for chewable objects, Acknowledgements
allowing them to exhibit their natural tendency to gnaw and
tear (Chmiel and Noonan, 1995). Out of numerous forms of The technical assistance of Steven J. Walsh is gratefully
enrichment, a block of wood with predrilled holes appeared acknowledged. Supported by a 2002 American Society of
to stimulate the greatest interest in rats (Chmiel and Noonan, Pharmacology and Experimental Therapeutics (ASPET)
1995). Little interest was shown in pipes or partitions that Summer Undergraduate Research Fellowship (SURF)
would appeal to a wall-hugging tendency, and similarly, no Award (Glenn E. Ullyot Fund) to EEB and by NIH grant
interest was shown for objects that were reasoned to be MH28380-24 to RTR.
486 E.E. Belz et al. / Pharmacology, Biochemistry and Behavior 76 (2003) 481–486

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