Skip to main content
NCBI home page
VA Author Manuscripts logoLink to VA Author Manuscripts
. Author manuscript; available in PMC: 2021 Feb 25.
Published in final edited form as: Neurogastroenterol Motil. 2020 Feb 21;32(6):e13826. doi: 10.1111/nmo.13826

Environmental Enrichment Prevents Chronic Stress-Induced Brain-Gut Axis Dysfunction Through a GR-Mediated Mechanism in the Central Nucleus of the Amygdala

A Orock 1, T Louwies 1, T Yuan 1, B Greenwood-Van Meerveld 1,2,3
PMCID: PMC7906280  NIHMSID: NIHMS1671623  PMID: 32084303

Abstract

Introduction:

Cognitive behavioral therapy (CBT) improves quality of life of patients with irritable bowel syndrome (IBS), a disorder characterized by chronic visceral pain and abnormal bowel habits. Whether CBT can actually improve visceral pain in IBS patients is still unknown. The aim of this study is to evaluate whether environment enrichment (EE), the animal analog of CBT, can prevent stress-induced viscero-somatic hypersensitivity through changes in glucocorticoid receptor (GR) signaling within the central nucleus of the amygdala (CeA).

Methods:

Rats were housed in either standard housing (SH) or EE for 7 days before and during daily water avoidance stress (WAS) exposure (1-h/day for 7 days). In the first cohort, visceral and somatic sensitivity were assessed via visceromotor response to colorectal distention and von Frey Anesthesiometer 24h and 21 days after WAS. In another cohort, the CeA was isolated for GR mRNA quantification.

Results:

EE for 7 days before and during the 7 days of WAS persistently attenuated visceral and somatic hypersensitivity when compared to rats placed in SH. EE exposure also prevented the WAS-induced decrease in GR expression in the CeA.

Conclusions:

Pre-exposure to short-term EE prevents the stress-induced downregulation of GR, and inhibits visceral and somatic hypersensitivity induced by chronic stress. These results suggest that a positive environment can ameliorate stress-induced pathology and provide a non-pharmacological therapeutic option for disorders such as IBS.

Keywords: Environmental Enrichment, pain, rat, visceral hypersensitivity

INTRODUCTION

The usefulness of psychotherapy, such as cognitive behavioral therapy (CBT) that encompasses cognitive and behavioral strategies that aim to improve the underlying thinking or behavioral patterns, extends far beyond the treatment of stress-related mood and psychological disorders such as anxiety and depression [1–3]. Nowadays, CBT is also being used to improve the quality of life in patients suffering from chronic pain, such as musculoskeletal pain [4–6], pelvic pain [7], and arthritic pain [8].

Clinical evidence suggests that CBT also improves in the overall symptomatology of patients with irritable bowel syndrome (IBS) [9]. IBS a common gastrointestinal disorders characterized by chronic abdominal pain, due to increased visceral sensitivity, and abnormal bowel habits. These symptoms severely affect the quality of life of IBS patients, which make up 10–20% of the general population [10]. The high prevalence of IBS and severely reduced of quality of life of IBS-patients translates into significant losses of work productivity and high health care costs for the general society [11–13]. As common comorbidities of IBS often include stress-related disorders such as depression and anxiety [14–16], IBS is regarded as a functional disorder of the brain-gut axis. In support of this hypothesis, brain imaging studies of IBS patients have revealed higher activity in the limbic brain during colorectal distention and abnormal connectivity between regions involved in the pain matrix [17, 18]. Interestingly, some of these brain regions that display higher activity, such as the amygdala, are also involved in the hypothalamic-pituitary-adrenal (HPA) axis, which is responsible for the body’s neuroendocrine response to stress. The amygdala exerts a positive feedback control on HPA activity, and hyper reactivity of this brain region may contribute to the HPA axis dysregulation that is often observed in IBS patients. Moreover, increased amygdala activity may be responsible for the reported exaggeration of symptomatology during or after stressful periods [19].

The importance of HPA axis dysregulation has been shown in animal models where IBS-like symptomatology can be easily recapitulated with several stress models. We have previously shown that repetitive daily exposure to a water avoidance stress (WAS) is sufficient to induce persistent visceral and somatic hypersensitivity through amygdala-mediated mechanisms [20–23]. Furthermore, we have shown that WAS-induced decreases in glucocorticoid receptor (GR) in the central nucleus of the amygdala (CeA) are responsible for the onset of visceral and somatic hypersensitivity after WAS [24].

The etiology of visceral pain IBS is poorly understood and the mechanisms through which CBT improves pain perception in IBS patients also remains to be elucidated. The animal-equivalent of CBT, environment enrichment (EE), could be a potential model to study the beneficial effects of CBT in animal models recapitulating IBS-like visceral pain. EE exposes animals to an environment that is both physically and socially more stimulating than standard rodent housing condition. This enriched environment leads to enhanced cognitive and physiological wellbeing [25–29], and has been successfully used to decrease recovery time following chronic inflammatory pain [30], and reverses neuropathic pain in rodents [31, 32]. Moreover, pre-exposure to EE attenuated the negative effects of social defeat stress [33].

Based on the aforementioned studies, (pre-) exposure to EE could also be a potential treatment for visceral and somatic pain. Preclinical data on whether EE could reverse stress-induced visceral and somatic hypersensitivity is lacking. Therefore, we set out to test the efficacy of EE in an animal model that recapitulates IBS-like symptomatology. We hypothesized that (pre-) exposure to EE would protect against the decrease in GR expression in the CeA and the consequential development of visceral and somatic hypersensitivity, induced by repetitive daily exposure to a psychological stressor. In addition, we tested the hypothesis that the potential beneficial effects of EE on stress-induced visceral and somatic hypersensitivity persist after EE has ended. Our data will provide important insights into the efficacy of EE in stress-induced visceral and somatic pain and whether this model is useful to study the underlying mechanisms of CBT-induced pain relief in IBS patients.

METHODS:

Ethical statement and assurances

All procedures were approved by the Veterans Affairs (VA) Health Care System (1604–001, 1809–001) and the University of Oklahoma Health Sciences Center (OUHSC) (15–070-SSHIL, 18–061-SSHIL) institutional animal care and use committees (IACUCs). Experiments were designed to minimize pain and distress to the animals per the Guide for Care and Use of Laboratory Animals (8th edition, 2011).

Animals:

The experiments were performed on adult male (250–300g; 74–84 days old) Fischer-344 (F344) rats (Charles Rivers Laboratory, Wilmington MA, USA). All the animals were maintained on a 12-h light/dark cycle at 21°C with ad libitum access to food and water. The animals were acclimated to the laboratory and experimenter one week before experimentation.

Environmental Enrichment and Standard Housing procedures:

The enriched environment (EE) consisted of larger cages (78cm L × 52cm W × 100cm H) with ample bedding, toys, food enrichment (sweetened cereal, seed/nut mix), and burrowing tunnels in groups of 4 rats/enrichment cage. The remaining half of the animals remained in standard double-housed shoe-box cages (SH) with filter tops (43cm L × 20cm W × 21.5cm H). Both cohorts were kept in the same room of the animal facility with the same light-dark cycle and temperature. The animals were initially left to acclimate to the facility for 7 days before being moved to their designated housing conditions to habituate for another 7 days.

Water Avoidance Stress (WAS):

The WAS procedure was performed daily for 1-h between 9:00 am and noon. Rats in the WAS group were placed on a platform (8 × 8 × 8 cm) mounted in the center of a white semi-transparent plastic container (50 × 35 × 33 cm) filled with fresh, room temperature water to 1 cm below the surface of the platform. Animals in the sham stress group (SHAM) were placed in similar containers but without water. Animals were weighed every day before WAS/SHAM procedure. All animals were left undisturbed in the containers for 60 min before being returned to their home cages. The number of fecal pellets produced during the WAS or SHAM stress (fecal pellet output, FPO) was recorded to evaluate stress-induced colonic motility and autonomic output.

Assessment of Visceral Sensitivity

Visceral sensitivity was assessed as previously described [34]. Briefly, visceral sensitivity thresholds were determined by recording visceromotor responses (VMR) to colorectal distension (CRD) in rats that were unrestrained and freely moving in a standard cage. Rats were fasted overnight in preparation for visceral sensitivity assessment. Rats were then placed under anesthesia (2–2.5% isoflurane) and a 5-cm latex balloon catheter was inserted approximately 8-cm into the colon via the anal cavity and fixed in place with surgical tape around the tail. Rats were placed into a clean cage with fresh bedding and allowed 30 min to recover. After recovery, CRD was induced by inflating the balloon and maintaining an isobaric pressure using a constant-pressure barostat (G&J Electronics, Toronto, ON, Canada). CRD was performed at graded pressures of 0 (baseline), 20, 40, and 60 mmHg for 10 min each in a randomized order with a 10 min recovery period between distension periods. VMR was measured by quantifying the number of abdominal contractions during each period of CRD.

Assessment of Somatic Sensitivity:

Somatic sensitivity was measured as previously described [24, 35]. Briefly, rats were placed in Plexiglas enclosure units with elevated wire mesh floor and allowed to acclimate for 30 minutes. The IITC 2390 series Electronic Von Frey Anesthesiometer (IITC Life Science, Woodland Hills, CA) was used to assess somatic sensitivity. Following acclimatization, somatic sensitivity was measured by placing the rigid tip of the wand through the wire mesh bottom of the cage onto the plantar area of the hind paw and slowly pressing upward until a withdrawal reflex was observed and the minimum amount of force required to elicit a withdrawal reflex was recorded. The procedure was then repeated an additional three times using the same point on either hind paw with 5-min intervals between each testing session and an average was used as the final withdrawal force for each animal.

Experimental Protocol:

Fisher 344 (F344) male rats were used to validate our model for stress induced visceral sensitivity. These rats were placed in SH cages during the WAS/SHAM (1-h/day for 7 days) procedure (n=6/group) before assessing visceral and somatic hypersensitivity. A second cohort of F344 rats was randomly divided into SH or EE housing conditions (randomization.com) and animals were acclimated to their housing and laboratory environments for 7 days. After 7 days of acclimation, the rats in both housing conditions were further divided into 4 groups; SH+WAS, EE+WAS, SH+SH and EE+SH (n=8 per group). WAS (1-h/day) for 7 days was carried out on rats in the WAS group while the sham protocol was carried out on the SHAM group. The animals were continually housed in the EE or SH groups during the WAS/SHAM protocol. Somatic sensitivity was assessed on day 6 of WAS/SHAM, then animals were then fasted overnight on day 7 (12–18 hours) and visceral sensitivity was assessed on day 8 after the last WAS/SHAM. Following visceral sensitivity assessment, a subset of these animals (n=4) (both EE and SH animals) were placed in SH cages and left undisturbed for 3 additional weeks. At the end of this period, visceral and somatic sensitivity were measured again to determine whether a brief exposure to EE had persistent effects on stress-induced hypersensitivity.

RNAscope assay for GR expression:

Rats were deeply anesthetized with 5% isofluorane before being euthanized. Whole brains were collected and snap frozen for cryosectioning. Non-consecutive amygdalar slides (10 μm) were collected and stored in −80°C. GR mRNA expression was analyzed using RNAscope 2.5 Chromogenic Assay following the manufactory’s protocol. Briefly, sections were post-fixed in 4% PFA and dehydrated in a graded ethanol series (50%, 70% and 100%). The tissue was treated with hydrogen peroxide (REF #:322330, ACD, Newark, CA) and protease IV (REF #: 322340, ACD), followed by probe hybridization (Rn-Nr3c1, 466991, ACD) in 40°C for 2 hours. Signal was detected using RNAscope 2.5 HD Detection Reagent · BROWN (REF #: 322310, ACD). Images were captured with a Zeiss AxioVert 200m Inverted microscope in bright field with 20X objective. From each animal (n=4/group), three slides were imaged and the images were analyzed with ImageJ by an experimenter blinded to the animal groups.

Statistics

Data are represented as means ± SD and analyses were performed using PRISM 8. Cohort sizes were determined by power analysis based on a preliminary study involving a similar experimental design. A Grubbs outlier test was performed on all data and outliers were excluded from data analysis. Daily FPO and VMR to CRD data was evaluated using a two-way (stress, housing) analysis of variance (ANOVA) with repeated measures followed by Bonferroni’s post hoc test to determine if there were significant differences between the groups. 2-tailed t-tests or one-way ANOVA followed by Bonferroni’s post-hoc tests were utilized to investigate differences between somatic sensitivity thresholds between the two (SH or WAS) or four (SH+WAS, SH+SH, EE+WAS and EE+SH) cohorts respectively. Grubbs’ test was used to identify and exclude outliers.

RESULTS

Pre-exposure to an enriched environment inhibits WAS-induced colonic and somatic hypersensitivity

Previous studies showed that pre-exposure to EE induced resiliency against social defeat stress [33]. To test whether pre-exposure to EE would prevent the development of stress-induced visceral and somatic hypersensitivity, EE rats were exposed to 2 weeks of EE (1 week before and during the 7-day WAS), whereas SH control rats were housed under standard conditions (Figure 1A). WAS significantly increased colonic motility, quantified as the average daily FPO, of SH+WAS (n=8) and EE+WAS (n=8) animals, when compared to SH+SHAM (n=8) and EE+SHAM (n=8), suggesting that the animals were not habituated to the stressor (P<0.001, Figure 1B). A Two-Way ANOVA was used to analyze the differences in visceral sensitivity after WAS between the groups. We observed a main effect of housing condition (group factor (F(3, 28)=6.694 P=0.0015)), a main effect of pressure (F(3, 84)=180.1, p<0.0001) and a housing condition × pressure interaction (F(9, 84)=6.392, P<0.0001). In Figure 1C, after Bonferroni post-hoc corrections, we observed that SH+WAS animals displayed a significantly higher number of abdominal contractions at 40 mmHg (P<0.05) and 60 mmHg (P<0.0001), when compared to SH+SHAM controls. In contrast, EE+WAS animals displayed a significantly lower number of abdominal contractions at 40 mmHg (P<0.05) and 60 mmHg (P<0.0001), when compared to SH+WAS animals. Visceral sensitivity in EE+WAS animals was comparable to animals exposed to SH+SHAM and EE+SHAM (P>0.9999). Somatic sensitivity was assessed with a One-Way ANOVA, which showed significant differences in housing conditions (F(3, 28)=39.62; P<0001). A Bonferroni post-hoc test revealed that the withdrawal threshold of SH+WAS animals was significantly lower when compared to SH+SHAM animals (P<0.0001). In contrast the withdrawal threshold of EE+WAS animals was significantly higher than SH+WAS animals (P<0.0001), and comparable to SH+SHAM and EE+SHAM animals (Figure 1D).

Figure 1.

Figure 1.

Open in a new tab

Exposure to EE for 1 week before and during WAS inhibits stress-induced colonic and somatic hypersensitivity. (A) Experimental design. (B) WAS increased the FPO of rats compared to SHAM, irrespective of housing condition (p<0.01). (C) Housing rats in EE for 1 week before and during EE prevented the induction of visceral hypersensitivity at 40 mmHg (p<0.05) and 60 mmHg (p<0.001), and (D) also inhibited WAS-induced somatic hypersensitivity (p<0.001).

Pre-exposure to EE also attenuates the persistent effects of WAS on colonic and somatic sensitivity

To assess the persistent effects of EE, a subset of SH and EE animals (n=4/group) were housed in standard cages after the visceral sensitivity assessment. At day 28, visceral and somatic sensitivity were assessed again (Figure 2A). A Two-Way ANOVA was used to reanalyze visceral sensitivity and revealed a main effect of previous housing conditions (EE or SH) (F(3, 12)=3.805, p=0.04) and a main effect of pressure (F(3, 36)=113.4, p<0.0001), but the housing × pressure interaction effect was not significant (F(9, 36)=1.637, p=0.142). As shown in Figure 2B, after Bonferroni post-hoc corrections, the number of abdominal contractions of SH+WAS animals was still significantly higher when compared to SH+SHAM animals at 60 mmHg (P<0.01). The number of abdominal contractions of EE+WAS animals was still significantly lower than SH+WAS (P<0.01) and remained comparable to SH+SHAM and EE+SHAM animals (p>0.9999). A One-Way ANOVA once again revealed significant differences in somatic sensitivity between the groups (F(3, 11)=7.144; p=0.006). After Bonferonni post hoc corrections, the withdrawal threshold of SH+WAS animals was still significantly lower when compared to SH+SHAM animals (P<0.05) and the withdrawal threshold of EE+WAS animals was still significantly higher when compared to SH+WAS animals (p=0.0062). Moreover, the withdrawal threshold of EE+WAS animals remained comparable to SH+SHAM and EE+SHAM animals (p=0.42) (Figure 2C).

Figure 2.

Figure 2.

Open in a new tab

Pre-exposure to EE inhibits the persistent stress-induced colonic and somatic hypersensitivity. (A) Experimental design. At day 28, SH+WAS rats still displayed visceral hypersensitivity (p<0.01) (B) and somatic hypersensitivity (p<0.01) (C) when compared to SH+SHAM. EE+WAS animals still did not display either visceral hypersensitivity or somatic hypersensitivity.

Environmental Enrichment during WAS is not sufficient to prevent stress-induced visceral and somatic hypersensitivity

To assess whether pre-exposure to EE is necessary to prevent WAS-induced visceral and somatic hypersensitivity, another cohort of animals (n=4/group) was housed in EE only during the WAS procedure (Figure 3A). The WAS-induced increase in colonic motility and autonomic output, assessed via quantification of FPO, was similar between EE+WAS and SH+WAS animals (Figure 3B). A Two-Way ANOVA was used to assess visceral sensitivity and revealed a main effect of pressure (F(1.17, 5.884)=62.85, p=0.002)), but failed to reveal an effect of housing condition (F(1, 5)=0.855) or housing × stress interaction (F(3, 15) =0.23, p=0.86). The number of abdominal contractions of SH+WAS and EE+WAS animals were similar (p=0.99), and were significantly higher when compared to aforementioned SH+SHAM controls (Figure 3C). In addition, a One-Way ANOVA did not reveal any significant differences in withdrawal threshold between SH+WAS and EE+WAS animals. The withdrawal threshold of both groups was lower than aforementioned SH+SHAM control levels (P=0.75) (Figure 3D).

Figure 3:

Figure 3:

Open in a new tab

EE during WAS only was not sufficient to prevent stress induced colonic and somatic hypersensitivity. (A) Experimental design. (B) FPO was not significantly different between EE+WAS and SH+WAS groups (P=0.67). (C) SH+WAS and EE+WAS had higher number of contractions when compared to SHAM controls at 40mmHg and 60mmHg (p<0.05). There was no significant difference in the VMR response between SH+WAS and EE+WAS (p=0.99). (D) SH+WAS and EE+WAS animals had a significantly lower withdrawal threshold when compared to SHAM controls (p=0.016). There was no difference in withdrawal threshold between EE+WAS and SH+WAS animals (p=0.76).

Environmental Enrichment prevents the WAS-induced decrease of GR expression in the CeA

We next aimed to study a potential molecular mechanism, through which EE might confer its protective effects, in the EE paradigm that prevented visceral and somatic hypersensitivity. As we have previously shown that WAS induces visceral and somatic hypersensitivity through a GR-mediated mechanism, this mechanism was a likely candidate for the protective effects of EE. In order to study the molecular mechanism, another cohort of animals underwent 2 weeks of EE (1 week before and during the 7-day WAS), while SH control rats remained in standard housing conditions (Figure 4A). Using an RNAScope assay, we measured the levels of GR immunoreactive cells in the CeA (Figure 4B). A One-way ANOVA revealed a significant difference between the groups (F(3, 43)=6.632; p=0.0009). A Bonferroni’s post hoc tests revealed that SH+WAS rats (n=11 slides) had significantly reduced GR expression when compared to SH+SHAM (p=0.02; n=12 slides)(Figure 4C). In contrast, GR expression in the CeA of EE+WAS animals significantly higher (p=0.003; n=12 slides), when compared to SH+WAS animals, and comparable to SH+SHAM (Figure 4C).

Figure 4:

Figure 4:

Open in a new tab

EE prevents WAS-induced decrease of GR expression in the CeA. (A) Experimental design. (B) Representative pictures of CeA showing GR immunoreactive cells. (C) SH+WAS rats had significantly lower GR expression levels when compared to either SH+SHAM controls (p=0.02) or EE+WAS (p=0.003). GR expression was not significantly different between SH+SHAM and EE+WAS rats.

DISCUSSION:

In the current study, we sought to evaluate the efficacy of two EE paradigms to ameliorate stress-induced visceral and somatic hypersensitivity, which was induced using an experimental model of psychological stress. Our results clearly show that the combination of pre-exposure to EE and exposure to EE during stress is critical to prevent the stress-induced decrease in GR expression as well as visceral and somatic hypersensitivity. In contrast, when EE exposure only overlapped with the stress paradigm, the protective effects of EE exposure did not outweigh the deleterious effects of chronic stress. As a result, EE was unable to prevent visceral and somatic hypersensitivity. Our results also clearly indicate that the beneficial effects of EE extended long after stress exposure had ended, as these animals did not develop any form of hypersensitivity after they were housed in SH conditions.

CBT is a known therapy to treat neurological disorders such as mood disorders, anxiety and depression [1–3]. As IBS patients often report similar neurological disorders, the efficacy of CBT to improve the quality of life of IBS patients may not be surprising [9]. Moreover, anecdotal clinical evidence suggest that CBT is an efficient intervention to treat visceral pain in IBS patients. However, the exact mechanisms of how and when CBT improves visceral pain perception in IBS patients remain unknown. Our experimental data adds support to the notion that CBT could become a valuable alternative to treat IBS-like visceral pain. Our results concur with previous reports which suggest that pre-exposure to EE is pivotal to prevent the negative effects of social defeat stress [33]. We have yet to address the question of whether exposure to EE after stress could ameliorate visceral and somatic hypersensitivity. Parent-Vachon et al. and Vachon et al., who studied the effects of EE on neuropathic and inflammatory pain, showed that EE after the insult occurred, ameliorated pain behaviors [36, 37]. Therefore, it seems plausible that EE after stress exposure might also be beneficial to ameliorate stress-induced hypersensitivity.

An important caveat of our current findings is that only the EE paradigm that included pre-exposure to EE attenuated the development of stress-induced visceral pain. When animals were only housed in an enriched environment in the same week that WAS occurred, EE failed to protect against the development of visceral and somatic hypersensitivity. Changing the animals to the EE housing condition at the beginning of the WAS regime may have caused additional stress, which ultimately masked the beneficial effects of EE. By allowing the animals to adapt to the EE conditions before the initiation of the WAS procedure, the potential confounds of a novel environment stressor were removed. Moreover, multiple CBT sessions are typically required before patients begin seeing significant improvements in pain sensitivity [38], suggesting that increasing the duration of exposure to EE may be required to prevent stress-induced pain behavior. Several reports showed that housing animals in SH cages, after being exposed to EE, induces a depressive-like phenotype [39, 40]. In this regard, our animals did not develop visceral or somatic hypersensitivity after being housed in SH at the end of the WAS period. Differences in EE conditions, exposure time (2 weeks vs 4 weeks), and housing conditions (double housing vs. single housing after EE) between our study and the work of Smith et al. may account for these conflicting results.

WAS is known to elevate circulating CORT, and repeated exposure to elevated CORT causes persistent HPA axis dysregulation [20, 41, 42]. Although we did not measure CORT levels in our animals, based on the increased WAS-induced colonic motility in the SH and the EE group, we can conclude that both groups are stressed. In other words, exposure to EE did not prevent the mounting of the stress-response. Various studies have shown that alterations in HPA axis and/or its associated regions reactivity, due to chronic psychological stress, underlie visceral and somatic hypersensitivity [20, 43]. From our previous studies, we have shown that WAS causes visceral and somatic hypersensitivity through the downregulation of GR expression in the CeA [44]. In addition, stereotaxic targeting of the CeA with CORT or knocking down GR expression in the CeA were sufficient to induce visceral and somatic sensitivity [45, 46]. Taken together, these studies highlight the importance of GR signaling in the CeA for the development of visceral and somatic hypersensitivity. Therefore, GR signaling in the CeA was a likely candidate mechanism through which two weeks of EE exposure confers its protective effects. Using RNAscope assays, we first confirmed that WAS significantly reduces GR expression in the CeA. As discussed above, this WAS-induced decrease in GR is responsible for the development of visceral hypersensitivity [47, 48]. Our results clearly demonstrate that exposure to EE before and during WAS prevented the WAS-induced downregulation of GR in the CeA. As a result, the EE-mediated prevention of GR decrease translated into the prevention of WAS-induced visceral hypersensitivity. Our results concur with others who demonstrated that WAS-triggered GR translocation in the basolateral amygdala (BLA) or WAS-induced increase in c-Fos expression (a marker for neuronal activity) in the central nucleus of the amygdala [49, 50]. As the BLA sends signals to the CeA, EE might have reduced input from the BLA to the CeA, which prevented the development of visceral and somatic hypersensitivity. Future studies are needed to elucidate the molecular mechanisms that prevent the WAS-induced decrease in GR expression. For instance, our group demonstrated that chronic stress activates epigenetic mechanisms in the CeA that underlie WAS-induced changes in GR expression. Whether EE impacts enzymes responsible for histone acetylation or DNA methylation in the CeA is currently unknown. As these epigenetic mechanisms require time to be embedded into the CeA, they might have been established during the pre-exposure which might explain why EE concurrent with WAS did not protect against stress-induced visceral and somatic hypersensitivity. Although we did not verify the mechanism, based on our prior knowledge, we assume that EE concurrent with WAS did not prevent the WAS-induced downregulation of GR in the CeA, which lead to the development of visceral and somatic hypersensitivity in this paradigm.

In conclusion, our results demonstrate that pre-exposure to an enriched environment can prevent the decrease of GR in the CeA induced by chronic psychological stress, which results in the prevention of long-term visceral and somatic hypersensitivity. We hereby demonstrate the efficacy of the EE paradigm, which now can be used in future studies to investigate the underlying mechanisms of EE-mediated protective/preventative effects. Moreover, this study highlights the beneficial effects of CBT as a potential new therapy for individuals with a high risk for developing stress-induced visceral hypersensitivity.

ACKNOWLEDGEMENTS

Preliminary results of this study have been presented at the 2019 American Neurogastroenterology and Motility society meeting. Dr. Greenwood-Van Meerveld would like to acknowledge the funding provided by her Department of Veterans Affairs Merit Grant - I01BX002188-03. Dr. Greenwood-Van Meerveld is the recipient of a Senior Research Career Scientist award (1IK6BX003610-01) from the Department of Veterans Affairs. We would also like to acknowledge Dawn Prusator PhD, and Jay Love for their help in collecting some of the enrichment data.

Footnotes

Disclosures

BGVM and AO, designed the research study. AO performed the behavioral procedures and analyzed the behavioral experiments. TY performed and analyzed the molecular experiments. AO performed the statistical analysis. AO, TL, TY and BGVM each contributed to the preparation of the paper.

Conflict of Interest: None of the authors have any conflicts of interest.

REFERENCES

  • 1.Hofmann SG, Wu JQ, and Boettcher H, Effect of cognitive-behavioral therapy for anxiety disorders on quality of life: a meta-analysis. J Consult Clin Psychol, 2014. 82(3): p. 375–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Hans E and Hiller W, A meta-analysis of nonrandomized effectiveness studies on outpatient cognitive behavioral therapy for adult anxiety disorders. Clin Psychol Rev, 2013. 33(8): p. 954–64. [DOI] [PubMed] [Google Scholar]
  • 3.Chambless DL and Ollendick TH, Empirically supported psychological interventions: controversies and evidence. Annu Rev Psychol, 2001. 52: p. 685–716. [DOI] [PubMed] [Google Scholar]
  • 4.Castro MM, et al. , The cognitive behavioral therapy causes an improvement in quality of life in patients with chronic musculoskeletal pain. Arq Neuropsiquiatr, 2012. 70(11): p. 864–8. [DOI] [PubMed] [Google Scholar]
  • 5.Crofford LJ, Psychological aspects of chronic musculoskeletal pain. Best practice & research. Clinical rheumatology, 2015. 29(1): p. 147–155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Bernardy K, et al. , Efficacy of cognitive-behavioral therapies in fibromyalgia syndrome - a systematic review and metaanalysis of randomized controlled trials. J Rheumatol, 2010. 37(10): p. 1991–2005. [DOI] [PubMed] [Google Scholar]
  • 7.Haugstad GK, et al. , Continuing improvement of chronic pelvic pain in women after short-term Mensendieck somatocognitive therapy: results of a 1-year follow-up study. Am J Obstet Gynecol, 2008. 199(6): p. 615.e1–8. [DOI] [PubMed] [Google Scholar]
  • 8.Ismail A, et al. , Cognitive behavioural therapy and pain coping skills training for osteoarthritis knee pain management: a systematic review. Journal of physical therapy science, 2017. 29(12): p. 2228–2235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Lackner JM, et al. , Improvement in Gastrointestinal Symptoms After Cognitive Behavior Therapy for Refractory Irritable Bowel Syndrome. Gastroenterology, 2018. 155(1): p. 47–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Peery AF, et al. , Burden of gastrointestinal disease in the United States: 2012 update. Gastroenterology, 2012. 143(5): p. 1179–1187.e3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Tuteka A, et al. , Bowel Disorders in Gulf War Veterans. Gastroenterology 2008, 2008(134(4, Suppl 1): A-31): p. 182. [Google Scholar]
  • 12.Lovell RM and Ford AC, Global prevalence of and risk factors for irritable bowel syndrome: a meta-analysis. Clin Gastroenterol Hepatol, 2012. 10(7): p. 712–721.e4. [DOI] [PubMed] [Google Scholar]
  • 13.Dunphy RC, et al. , Visceral and cutaneous hypersensitivity in Persian Gulf war veterans with chronic gastrointestinal symptoms. Pain, 2003. 102(1–2): p. 79–85. [DOI] [PubMed] [Google Scholar]
  • 14.Riedl A, et al. , Somatic comorbidities of irritable bowel syndrome: a systematic analysis. J Psychosom Res, 2008. 64(6): p. 573–82. [DOI] [PubMed] [Google Scholar]
  • 15.Popa S-L and Dumitrascu DL, Anxiety and IBS revisited: ten years later. Clujul medical (1957), 2015. 88(3): p. 253–257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Banerjee A, et al. , Anxiety and Depression in Irritable Bowel Syndrome. Indian journal of psychological medicine, 2017. 39(6): p. 741–745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Bonaz B, et al. , Central processing of rectal pain in patients with irritable bowel syndrome: an fMRI study. Am J Gastroenterol, 2002. 97(3): p. 654–61. [DOI] [PubMed] [Google Scholar]
  • 18.Naliboff BD, et al. , Sex-related differences in IBS patients: central processing of visceral stimuli. Gastroenterology, 2003. 124(7): p. 1738–47. [DOI] [PubMed] [Google Scholar]
  • 19.Chang L, et al. , Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in irritable bowel syndrome. Neurogastroenterol Motil, 2009. 21(2): p. 149–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Myers B and Greenwood-Van Meerveld B, Differential involvement of amygdala corticosteroid receptors in visceral hyperalgesia following acute or repeated stress. Am J Physiol Gastrointest Liver Physiol, 2012. 302(2): p. G260–6. [DOI] [PubMed] [Google Scholar]
  • 21.Myers B, McKlveen JM, and Herman JP, Neural Regulation of the Stress Response: The Many Faces of Feedback. Cellular and molecular neurobiology, 2012: p. 10.1007/s10571-012-9801-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Beaulieu S, et al. , Participation of the central amygdaloid nucleus in the response of adrenocorticotropin secretion to immobilization stress: opposing roles of the noradrenergic and dopaminergic systems. Neuroendocrinology, 1987. 45(1): p. 37–46. [DOI] [PubMed] [Google Scholar]
  • 23.Myers B, Dittmeyer K, and Greenwood-Van Meerveld B, Involvement of amygdaloid corticosterone in altered visceral and somatic sensation. Behav Brain Res, 2007. 181(1): p. 163–7. [DOI] [PubMed] [Google Scholar]
  • 24.Johnson AC and Greenwood-Van Meerveld B, Knockdown of steroid receptors in the central nucleus of the amygdala induces heightened pain behaviors in the rat. Neuropharmacology, 2015. 93: p. 116–123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Baumans V and Van Loo PL, How to improve housing conditions of laboratory animals: the possibilities of environmental refinement. Vet J, 2013. 195(1): p. 24–32. [DOI] [PubMed] [Google Scholar]
  • 26.Simpson J and Kelly JP, The impact of environmental enrichment in laboratory rats--behavioural and neurochemical aspects. Behav Brain Res, 2011. 222(1): p. 246–64. [DOI] [PubMed] [Google Scholar]
  • 27.Bennett JC, et al. , Long-term continuous, but not daily, environmental enrichment reduces spatial memory decline in aged male mice. Neurobiol Learn Mem, 2006. 85(2): p. 139–52. [DOI] [PubMed] [Google Scholar]
  • 28.Harburger LL, Lambert TJ, and Frick KM, Age-dependent effects of environmental enrichment on spatial reference memory in male mice. Behav Brain Res, 2007. 185(1): p. 43–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Sampedro-Piquero P and Begega A, Environmental Enrichment as a Positive Behavioral Intervention Across the Lifespan. Curr Neuropharmacol, 2017. 15(4): p. 459–470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Gabriel AF, et al. , Enriched environment and the recovery from inflammatory pain: Social versus physical aspects and their interaction. Behav Brain Res, 2010. 208(1): p. 90–5. [DOI] [PubMed] [Google Scholar]
  • 31.Vachon P, et al. , Alleviation of chronic neuropathic pain by environmental enrichment in mice well after the establishment of chronic pain. Behav Brain Funct, 2013. 9: p. 22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Tai LW, Yeung SC, and Cheung CW, Enriched Environment and Effects on Neuropathic Pain: Experimental Findings and Mechanisms. Pain Pract, 2018. 18(8): p. 1068–1082. [DOI] [PubMed] [Google Scholar]
  • 33.Lehmann ML and Herkenham M, Environmental enrichment confers stress resiliency to social defeat through an infralimbic cortex-dependent neuroanatomical pathway. J Neurosci, 2011. 31(16): p. 6159–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Myers B and Greenwood-Van Meerveld B, Corticosteroid receptor-mediated mechanisms in the amygdala regulate anxiety and colonic sensitivity. Am J Physiol Gastrointest Liver Physiol, 2007. 292(6): p. G1622–9. [DOI] [PubMed] [Google Scholar]
  • 35.Prusator DK and Greenwood-Van Meerveld B, Sex-related differences in pain behaviors following three early life stress paradigms. Biology of sex differences, 2016. 7: p. 29–29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Parent-Vachon M and Vachon P, Environmental enrichment alleviates chronic pain in rats following a spared nerve injury to induce neuropathic pain. A preliminary study. Veterinary medicine (Auckland, N.Z.), 2018. 9: p. 69–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Vachon P, et al. , Alleviation of chronic neuropathic pain by environmental enrichment in mice well after the establishment of chronic pain. Behavioral and brain functions : BBF, 2013. 9: p. 22–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Beehler GP, et al. , Brief Cognitive Behavioral Therapy For Chronic Pain: Results From a Clinical Demonstration Project in Primary Care Behavioral Health. The Clinical Journal of Pain, 2019. 35(10): p. 809–817. [DOI] [PubMed] [Google Scholar]
  • 39.Morano R, et al. , Loss of Environmental Enrichment Elicits Behavioral and Physiological Dysregulation in Female Rats. Frontiers in behavioral neuroscience, 2019. 12: p. 287–287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Smith BL, et al. , Behavioral and physiological consequences of enrichment loss in rats. Psychoneuroendocrinology, 2017. 77: p. 37–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Besheer J, et al. , The effects of repeated corticosterone exposure on the interoceptive effects of alcohol in rats. Psychopharmacology, 2012. 220(4): p. 809–822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.McEwen BS, Physiology and neurobiology of stress and adaptation: central role of the brain. Physiol Rev, 2007. 87(3): p. 873–904. [DOI] [PubMed] [Google Scholar]
  • 43.Meerveld BG-V and Johnson AC, Mechanisms of Stress-induced Visceral Pain. Journal of neurogastroenterology and motility, 2018. 24(1): p. 7–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Tran L, et al. , Importance of epigenetic mechanisms in visceral pain induced by chronic water avoidance stress. Psychoneuroendocrinology, 2013. 38(6): p. 898–906. [DOI] [PubMed] [Google Scholar]
  • 45.Greenwood-Van Meerveld B, et al. , Stereotaxic delivery of corticosterone to the amygdala modulates colonic sensitivity in rats. Brain Res, 2001. 893(1–2): p. 135–42. [DOI] [PubMed] [Google Scholar]
  • 46.Johnson AC and Greenwood-Van Meerveld B, Knockdown of steroid receptors in the central nucleus of the amygdala induces heightened pain behaviors in the rat. Neuropharmacology, 2015. 93: p. 116–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Arnett MG, et al. , The role of glucocorticoid receptor-dependent activity in the amygdala central nucleus and reversibility of early-life stress programmed behavior. Translational psychiatry, 2015. 5(4): p. e542–e542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Kolber BJ, et al. , Central amygdala glucocorticoid receptor action promotes fear-associated CRH activation and conditioning. Proceedings of the National Academy of Sciences, 2008. 105(33): p. 12004–12009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Novaes LS, et al. , Environmental enrichment protects against stress-induced anxiety: Role of glucocorticoid receptor, ERK, and CREB signaling in the basolateral amygdala. Neuropharmacology, 2017. 113(Pt A): p. 457–466. [DOI] [PubMed] [Google Scholar]
  • 50.Reichmann F, Painsipp E, and Holzer P, Environmental enrichment and gut inflammation modify stress-induced c-Fos expression in the mouse corticolimbic system. PloS one, 2013. 8(1): p. e54811–e54811. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES