JNM J Neurogastroenterol Motil, Vol. 29 No. 1 January, 2023 pISSN: 2093-0879 eISSN: 2093-0887 https://doi.org/10.5056/jnm22009 Journal of Neurogastroenterology and Motility Original Article The Stressed Gut: Region-specific Immune and Neuroplasticity Changes in Response to Chronic Psychosocial Stress Beatriz Lobo,1,5,6,7* Mónica Tramullas,1,8 Beate-C Finger,1 Kevin W Lomasney,1,3 Caroll Beltran,1,9 Gerard Clarke,1 Javier Santos,5,6,7,10 Niall P Hyland,1,2 Timothy G Dinan,1,4 and John F Cryan1,3* 1 APC Microbiome Ireland, Departments of 2Physiology, 3Anatomy and Neuroscience, 4Psychiatry and Neurobehavioural Science, University College Cork, Ireland; 5Digestive System Research Unit, Laboratory of Neuro-Immuno-Gastroenterology, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Hospital Universitari, Barcelona, Spain; 6Department of Gastroenterology, Vall d’Hebron Hospital Universitari, Passeig Vall d’Hebron Barcelona, Spain; 7Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain; 8Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad de Cantabria, Santander, Spain (Current address); 9Laboratory of Immunogastroenterology, Gastroenterology Unit, Hospital Clinico Universidad de Chile, Faculty of Medicine Universidad de Chile, Santiago, Chile; and 10Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain Background/Aims Chronic psychological stress affects gastrointestinal physiology which may underpin alterations in the immune response and epithelial transport, both functions are partly regulated by enteric nervous system. However, its effects on enteric neuroplasticity are still unclear. This study aims to investigate the effects of chronic unpredictable psychological stress on intestinal motility and prominent markers of enteric function. Methods Adult male C57BL/6J mice were exposed to 19 day of unpredictable stress protocol schedule of social defeat and overcrowding. We investigated the effects on plasma corticosterone, food intake, and body weight. In vivo gastrointestinal motility was assessed by fecal pellet output and by whole-gastrointestinal transit (using the carmine red method). Tissue monoamine level, neural and glial markers, neurotrophic factors, monoamine signaling, and Toll-like receptor expression in the proximal and distal colon, and terminal ileum were also assessed. Results Following chronic unpredictable psychological stress, stressed mice showed increased food intake and body weight gain (P < 0.001), and reduced corticosterone levels (P < 0.05) compared to control mice. Stressed mice had reduced stool output without differences in water content, and showed a delayed gastrointestinal transit compared to control mice (P < 0.05). Stressed mice exhibited decreased mRNA expression of tyrosine hydroxylase (Th ), brain-derived neurotrophic factor (Bdnf ) and glial cell-derived neurotrophic factor (Gdnf ), as well as Toll-like receptor 2 (Tlr2 ) compared to control (P < 0.05), only proximal colon. These molecular changes in proximal colon were associated with higher levels of monoamines in tissue. Conclusion Unpredictable psychological chronic stress induces region-specific impairment in monoamine levels and neuroplasticity markers that may relate to delayed intestinal transit. (J Neurogastroenterol Motil 2023;29:72-84) Key Words Enteric nervous system; Gastrointestinal motility; Nerve tissue proteins; Neuronal plasticity; Stress, psychological ⓒ 2023 The Korean Society of Neurogastroenterology and Motility 72 J Neurogastroenterol Motil, Vol. 29 No. 1 January, 2023 www.jnmjournal.org Gut Neuroplasticity in Chronic Stress Received: January 24, 2022 Revised: May 5, 2022 Accepted: June 3, 2022 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. *Correspondence: Beatriz Lobo and John F Cryan are equally responsible for this study. Beatriz Lobo, PhD, MD Laboratory of Neuro-Immuno-Gastroenterology, Digestive Diseases Research Unit. Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain Tel: +34-93-489-4035, E-mail: beatriz.lobo@vhir.org John F Cryan, PhD Department of Anatomy and Neuroscience, University College Cork, room 3.86 Western Gateway Building, Ireland Fax: +353-0214205497, E-mail: j.cryan@ucc.ie chronic psychological stress affects intestinal functions (motility and secretion) and to examine neuroplasticity and immune-related Introduction changes that may underpin such effects. Chronic exposure to stress can negatively affect gastrointestinal (GI) functions1-3 and plays an important role in the development exacerbation of GI disorders as irritable bowel syndrome (IBS).4-6 Materials and Methods IBS is defined by recurrent abdominal pain associated with altered bowel habits,7 and it is one of the most common functional GI Animals disorders in Western countries with a significant socioeconomic Adult male C57BL/6J mice (n = 60; 8 weeks of age) were impact.8 Diverse pathological factors, including physical and psy- used as experimental mice, and adult male CD-1 mice (n = 20; chological stressors, have been proposed to disturb gut-brain bi- 9-10 weeks of age) were used as resident aggressors, and additional directional communication.3-7,9 These factors lead to gut dysmotility C57BL/6J mice (n = 8; 8-9 weeks of age) were used to train the and enhanced visceral pain,10-12 and contribute to the development aggressor CD-1 mice, all animals were purchased from Envigo of IBS. Brain-gut axis communication involves coordinated re- (Huntingdon, UK). All mice were singly housed in standard sponses of the enteric nervous system (ENS), the autonomic ner- mouse cages (33X15X13 cm) with only white bedding without vous system, the hypothalamic-pituitary-adrenal axis, and neuro- additional enrichment, and had ad libitum access to food and tap immuno endocrine mediators.13,14 Chronic psychological stress water under a 12-hour light/dark schedule (light on at 5 AM) with increases HPA axis and autonomic nervous system activity which standard controlled conditions (21 ± 1℃, 55 ± 10% humidity) promotes modulation in monoaminergic systems signaling includ- throughout the study. None of the procedures required a fasting ing the dopaminergic (DA), noradrenergic (NA), and serotonergic period. Once weekly, home cages were cleaned and equipped with (5-HT) circuitries involved in the regulation of gut physiology and new white bedding before stool collection by the same researcher. brain-gut axis function.1,3-6,11,14,15 Much of our knowledge about All animal care and experimental procedures were carried out fol- chronic stress-induced dysfunction of GI physiology and brain- lowing the European Community Council Directive (86/609/ gut axis comes from rodent models,9 though the specific molecular EEC) and approved by the Animal Experimentation and Ethics mechanisms underlying stress-induced enteric neuroplasticity re- Committee. main elusive.16-18 Clinical data show neuroplastic changes (increased neuronal outgrowth as well as features of enteric glial cells activa- Experimental Design tion) in the colonic mucosa of IBS patients associated with visceral hypersensitivity.19,20 Different studies support the role of neuroim- Chronic psychosocial stress procedure mune activation in psychological stress-induced visceral hyperal- One day before starting the chronic unpredictable stress (CUS) gesia.18,21 In line with this, we have demonstrated that chronic psy- protocol, mice were balanced based on body weight and randomly chological stress in mice results in increased visceral hypersensitivity assigned to either the CUS or control group (n = 10 each). After- associated with up-regulation of Toll-like receptor (TLR) 4 and wards, experimental mice were randomized into 3 separate cohorts (n microglia activation within CNS, as well as increased social anxiety- = 20 each) for GI motility and secretomotory function, molecular like behaviour.22,23 Therefore, this study aims to investigate whether markers, and behavior analysis (Fig. 1). CUS procedure was car- Vol. 29, No. 1 January, 2023 (72-84) 73 Beatriz Lobo, et al Whole GI transit Cohort 1 Corticosterone in plasma Measurement of daily food intake and body weight Day Body composition analysis 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Cohort 2 Social interaction test Sacrifice Ussing chambers Cohort 3 Intestinal gene and protein expression of ENS-related markers Corticosterone in plasma Randomization Social defeat Intestinal tissue HPLC neurotransmitters Overcrowding Spleen cytokines assay Faecal pellet output Figure 1. Timeline of the experimental procedure. Mice (n = 60) were exposed to 19 days of unpredictable chronic social stress paradigm. Fecal pellet were collected at day 5, 12, and 19 (indicated by arrow heads). Mice were split into 3 separated cohorts (n = 20): a sample, gastrointestinal (GI) motility and behavioral testing group. In the sample group plasma corticosterone, tissue neurotransmitters (high-performance liquid chroma- tography [HPLC]), mRNA and protein expression were assessed as well as ex vivo cecum ion transport (Ussing chambers). In the motility testing group red carmine dye was administered 2 hours following last stress session to assess the whole GI transit time and plasma corticosterone was de- termined. In the behavioral testing group, the social interaction test and body composition analysis were carried out. ENS, enteric nervous system. ried out as described previously.22 Briefly, mice were exposed to an ously in the social interaction test (Supplementary Methods).22 unpredictable mixed model that comprised social defeat and over- crowding sessions throughout 19 days (Supplementary Methods). Neurotransmitters determination Tissue concentration of NA, DA, 5-HT, and their metabolites Corticosterone assay were quantified in the full-thickness terminal ileum, proximal and At the end of the experimental paradigm and procedures, mice distal colon tissue from both experimental groups by high-perfor- were sacrificed just at the beginning of the dark phase (between mance liquid chromatography (HPLC) coupled to electrochemical 4:30 PM and 6 PM) based on our previous findings for this CUS detection, using a modification of a previously described procedure protocol,22,23 and on the circadian pattern of plasma corticosterone (Supplementary Methods).26 concentrations, which are higher during the early dark phase. Trunk blood was collected into EDTA-coated tubes. Plasma corti- Quantitative real-time polymerase chain reaction costerone concentration was quantified with commercially available To determine the differential transcriptional profile associated enzyme immunoassay kits, namely Enzo ADI-901-097 (Enzo Life with the regulation of the ENS functions, total RNA was extracted Sciences, Exeter, UK, LTD) according to the manufacturer’s in- from full thickness terminal ileum, proximal and distal colon with structions (Supplementary Methods). the Qiagen RNeasy Lipid Mini Kit (Qiagen, Valencia, CA,USA). Complementary DNA was synthesized with 1 mg total RNA with Body composition analysis random primers. Quantitative changes in mRNA levels were esti- The following day of the last stress session (between 9 AM and mated by quantitative real-time polymerase chain reaction (Supple- 1 PM), fat and lean body mass were determined. Measurements mentary Methods). were carried out with conscious mice in a 3-minute trial using the Minispec mq benchtop NMR spectrometer (Bruker Instruments, Protein isolation and Western blotting Rheinstetten, Germany).24,25 Western blot was performed to determine the protein levels of glial fibrillary acidic protein (GFAP), S100 calcium-binding protein Social interaction test B (S100B), and protein gene product 9.5 (PGP 9.5) from homog- The following day of the last stress session (between 9 AM and enized tissues from the proximal colon (Supplementary Methods). 1 PM), social avoidance behavior was assessed as described previ- 74 Journal of Neurogastroenterology and Motility Gut Neuroplasticity in Chronic Stress Ireland) was quantified using custom mouse Multi-Spot 96 Well In vivo assessment of gastrointestinal motility Plates (Meso Scale Discovery, Rockville, MD, USA) according to Fecal pellet output and water content. Fecal pellets manufacturer’s recommendations (Supplementary Methods). expelled were collected from each animal for 90 minutes during the dark phase on days 5, 12, and 19 just after the bedding change once Statistical Methods a week, as well, the water content in feces was assessed (Supplemen- Data are represented in the text and figures as mean values ± tary Methods). SEM. Statistical analysis was performed using repeated measures Whole gastrointestinal transit time. A cohort of experi- two-way ANOVA for overall effects in the analysis of body weights mental mice (n = 20) was administered carmine dye by oral ga- followed by Bonferroni post hoc test. The Student’s t test and the vaged 2 hours following the last stress, GI transit time was consid- non-parametric Mann-Witney’s U test were used to compare 2 ered as the time between gavage and first observance of a red fecal independent groups. Differences were considered significant at P pellet (Supplementary Methods). < 0.05. All analysis was carried out with GraphPad Prism software (GraphPad Software Inc, La Jolla, CA, USA). Ex vivo Ussing chamber experiments Experiments reported in this study used mouse caecum due to its optimal size and different segments of the colon were processed Results and used for subsequent analysis. Paired mucosa submucosa prepa- rations from each animal (n = 6) were mounted in Ussing cham- Chronic Psychological Stress Induces a Significant bers (Harvard Apparatus, Kent, UK) (Supplementary Methods). Increase in Body Weight, Food Intake, and Significant Changes in Body Composition Spleen cytokine assays Chronic psychological stress significantly induced an increase The cytokines amount such as IL-1β, 6, 10, 12, IFN-γ, mouse in body weight in mice compared with control mice, from day 10 keratinocyte-derived cytokine, and TNF-α on spleen cells cultured to the end of the CUS procedure (stress: F [1332] = 71.5, P < with lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate 0.001) (stress X day: F [8332] = 4.0, P < 0.001; Supplementary (PMA), and lipoteichoic acid (LTA) (Sigma-Aldrich, Wicklow, Fig. 1A), consistent with the previous report.17 Thus, at the end A B C D Control 100 140 220 300 Stress Interaction ratio [time interaction Plasma corticosterone (ng/mL) Time of whole gut transit (min) Time of whole gut transit (min) * zone (target/no target)]*100 120 80 100 200 60 165 ** 80 40 60 **** 100 40 110 20 100 20 0 0 0 Control Stress Control Stress Control Stress 0 50 100 150 Corticosterone (ng/mL) Figure 2. Stress had effects on behavior and induced changes in evening plasma corticosterone levels and total gastrointestinal (GI) motility. (A) Social avoidance was analyzed by social interaction test. Exposure to chronic stress caused a significant increase in social avoidance (n = 10/group). (B) Plasma corticosterone levels were measured by enzyme-linked immunosorbent assay. Exposure to chronic stress caused a significant decrease in evening plasma corticosterone levels (n = 20/group). (C) Total GI transit time was measured after gavage of red carmine in control and stress mice (n = 9-10/group); total GI transit time was significantly increased in stressed mice; (D) however, total GI transit time was not associated with plasma corticosterone levels (n = 8-9/group) (Spearman’s rank correlation). (E) Data represent mean ± SEM. *P < 0.05,**P < 0.01 ****P < 0.0001; *by t test (A, B), Mann Whitney test (C), and Spearman’s rank correlation (D) represent significant difference between stress and control groups. Vol. 29, No. 1 January, 2023 (72-84) 75 Beatriz Lobo, et al of the experimental paradigm, stressed mice showed a higher food values were comparable in the cecum of both experimental groups intake than control mice (3.7 ± 0.1 vs 4.3 ± 0.0 g, P < 0.001; (Supplementary Fig. 2A). Electrogenic Na+ absorption was present n = 20) (Supplementary Fig. 1A). In CUS mice, fat mass was in the cecum of both groups, as evidenced by the amiloride-induced significantly reduced (P = 0.003, n = 20) compared to controls, decrease of Isc without significant difference among them (Supple- while lean mass increased in stressed animals (P = 0.003, n = 20) mentary Fig. 2C-E). The addition of bethanechol and forskolin (Supplementary Fig. 1B), which replicates earlier findings.22,23 elicited a sustained increase in Isc that was similar between groups. To assess the CUS impact on the intestinal epithelial barrier integ- Chronic Psychosocial Stress Reduces Plasma rity, transepithelial resistance of cecal explants among groups was Corticosterone Levels analyzed, demonstrating no significant difference between stressed CUS protocol induced a reduction in evening corticosterone mice and controls (Supplementary Fig. 2B). levels in plasma at day 19 compared to controls (Fig. 2A). Chronic Psychosocial Stress Induces Changes in Chronic Psychosocial Stress Induces Social Enteric Neurotransmitters in Proximal Colon Avoidance Behavior Based on our observation that chronic stress alters GI motility, Similar to our previous data,23 the animals exposed to 19 days of we examined along different intestinal full-thickness sections the CUS exhibited pronounced social avoidance behaviour as reflected content of diverse neurotransmitters as well as the expression pat- by an alteration in the social interaction ratio compared with the tern of genes involved in their regulation. Differences in noradrena- control group (Fig. 2B). lin and dopamine (catecholamines) levels between both groups were found only in the proximal colon, where CUS mice displayed Chronic Psychosocial Stress Delays Gastrointestinal higher catecholamine content than controls (Fig. 3A). While cat- Transit echolamines levels in the proximal colon were increased, we did ob- Following 5 days of CUS, the fecal water content was increased serve reduced expression of tyrosine hydroxylase (Th ), the enzyme in stressed mice compared with control mice, while no differences that controls catecholamine synthesis, which was inversely related to were observed in fecal pellet counts (Table 1). However, at the end catecholamines content in that segment (Fig. 3B), indicating a pos- of the procedure, water content was comparable to control mice sible adaptive compensatory mechanism for biosynthesis regulation. whereas stool output was significantly reduced (Table 1), indicat- On the other hand, CUS mice showed lower levels of the specific ing that CUS protocol appears to delay GI motility. Consistently, metabolite of DA, 4-hydroxyvanillic acid (HVA) in both colonic during the dark cycle, CUS mice displayed significant delay in GI segments, especially in the proximal colon, where it was undetect- transit compared to control animals (Fig. 2C) that was not associ- able. Notably, CUS mice showed lower total content of DA and its ated with decreased plasma corticosterone levels (Fig. 2D). metabolites, hydroxyvanillic acid, and DOPAC, in the distal colon. 5-HT and 5-hydroxyindoleacetic acid levels were higher only in Stress Had No Impact on Ex Vivo Cecum Ion Transport the proximal colon of CUS-exposed animals (Fig. 3A), indicating Under basal conditions, Cl--secretory short-circuit current (Isc) increasing 5-HT turnover. Again, differential changes in the ex- pression of key components of 5-HT system evoked by stress along Table 1. Comparison of Number of Fecal Pellets and Fecal Water the GI tract were observed. P11 was significantly up-regulated in Content Between Groups (n = 16-20 group) Over Chronic Unpre- the distal colon, whereas expression of Htr4 was up-regulated in dictable Stress Procedure the proximal colon. No significant change in the expression of the Control Stress solute carrier family 6, Slc6a4 (Sert ), was observed in any segment Day analysed (Fig. 4C). Overall, these findings point to a differential Fecal pellet Fecal water Fecal pellet Fecal water output content (%) output content (%) neurotransmission pattern in stress response along the GI tract, lim- ited to proximal colon which may explain the motility impairment. 5 2.5 ± 0.4 61.6 ± 1.4 2.6 ± 0.4 77.4 ± 3.7** 12 2.9 ± 0.4 65.8 ± 2.0 2.2 ± 0.4 68.8 ± 2.5 19 3.1 ± 0.4 60.7 ± 2.1 1.8 ± 0.3* 63.5 ± 2.0 *P < 0.05, **P < 0.01 by Mann Whitney test. Data represent mean ± SEM. 76 Journal of Neurogastroenterology and Motility Gut Neuroplasticity in Chronic Stress A Control 175 Control 5400 Control 750 Stress 150 Stress 3900 Stress 2400 600 ** 125 NA (ng/g) DA (ng/g) HVA (ng/g) 900 450 100 * 75 500 300 50 250 150 25 0 0 0 Ileum Proximal Distal Ileum Proximal Distal Ileum Proximal Distal 4500 Control 6000 Control 80 Control 3500 Stress DA + DOPAC + HVA Stress Stress 4500 2500 60 DOPAC (ng/g) 5-HT (g/g) 2000 2500 *** (ng/g) 1500 ** 2000 40 1000 ** 1000 20 500 0 0 0 Ileum Proximal Distal Ileum Proximal Distal Ileum Proximal Distal 800 Control 70 000 Control 5-HT + 5-HIAA (ng/g) Stress Stress 600 ** 5-HIAA (ng/g) 45 000 400 ** 20 000 ** 200 7500 5000 2500 0 0 Ileum Proximal Distal Ileum Proximal Distal B 2.0 Control 800 r = 0.68; P = 0.006 150 r = 0.63; P = 0.022 Stress Th/-actin (mRNA) NA ng/g tissue DA ng/g tissue 1.5 600 100 1.0 400 * 50 0.5 200 Control Control Stress Stress 0 Ileum Proximal Distal 0 0.5 1.0 1.5 0 0.5 1.0 1.5 Fold-change TH Fold-change TH Figure 3. Chronic stress induces changes in enteric neurotransmitters and down regulated the expression of tyrosine hydroxylase (Th ) mRNA in proximal colon. Levels of noradrenaline (NA), dopamine (DA), serotonin (5-HT), and metabolites from dopamine and 5-HT were measured by high-performance liquid chromatography (HPLC) in ileum and colon in control and stress mice (n = 7-8/group). (A) Levels of NA, DAc, and 5-HT were significantly greater than those in control mice only in proximal colon of stressed mice. No HVA levels were detected in proximal colon of stressed mice. The total levels of Dopamine plus its metabolites (homovanillic acid [HVA] and 3,4‑dihydroxyphenylacetic acid [DOPAC]) were significantly lower in distal colon of stressed mice than control. Total levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) were signifi- cantly greater in proximal colon of stressed mice than controls. Quantitative real-time polymerase chain reaction was used to quantify to measure transcripts encoding Th in small intestine and colon of control and stressed mice (n = 10/ group). (B) In proximal colon transcript levels of Th negatively correlated with the concentration of NA and DA. Data represent mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 by t test (A, B) and Pearson analysis (B). Vol. 29, No. 1 January, 2023 (72-84) 77 Beatriz Lobo, et al A Control 1.5 Control 1.5 Control Gdnf/-actin (mRNA) Bdnf/-actin (mRNA) Gfap/-actin (mRNA) Stress Stress Stress 1.5 * 1.0 1.0 P = 0.089 P = 0.084 1.0 * * * 0.5 0.5 0.5 0 0 0 Ileum Proximal Distal Ileum Proximal Distal Ileum Proximal Distal B 2.0 r = 0.74; P < 0.001 2.0 r = 0.38; P = 0.120 Gdnf/-actin (mRNA) Bdnf/-actin (mRNA) 1.5 1.5 1.0 1.0 0.5 0.5 Control Control Stress Stress 0 0.5 1.0 1.5 0 0.5 1.0 1.5 C 1.5 1.5 2.0 Control Control Control P = 0.052 Htr4/-actin (mRNA) Stress Stress Stress Sert/-actin (mRNA) p11/-actin (mRNA) * 1.5 1.0 1.0 * 1.0 0.5 0.5 0.5 0 0 0 Ileum Proximal Distal Ileum Proximal Distal Ileum Proximal Distal Figure 4. Chronic stress down-regulate transcripts encoding enteric glia, neurotrophins in proximal colon. Quantitative real-time polymerase chain reaction was used to analyze glial fibrillary acidic protein (Gfap ), glial cell-derived neurotrophic factor (Gdnf ), and brain-derived neurotrophic fac- tor (Bdnf ) expression levels in small intestine and colon of control and stressed mice (n = 10/group). (A) Transcripts encoding Gfap, Gdnf , and Bdnf were significantly decreased in proximal colon after chronic stress when compared with control mice. (B) In proximal colon also a strong cor- relation between Gfap and Gdnf expression levels was observed. (C) Expression levels of serotonin transporter, solute carrier family 6 Slc6a4 (Sert ), was not different between stressed and control mice, expression levels of S100 calcium binding protein A10 (p11 ) were significantly increased in distal colon of stress mice and the increase of serotonin receptor 4 (Htr4 ) receptor mRNA levels were nearly significant in proximal colon of stress mice. Data represent mean ± SEM, *P < 0.05 by t test. derived neurotrophic factor (Gdnf ) mRNA and brain-derived Chronic Stress Induces Segmental-specific Differential neurotrophic factor (Bdnf ) mRNA in the proximal colon of mice Expression Patterns in Genes Associated With the exposed to CUS. Whereas the distal colon of mice exposed to CUS Regulation and Integrity of the Enteric Nervous revealed an up regulation of Gfap mRNA and down-regulation of System Gdnf and Bdnf mRNA. Interestingly, only in the proximal colon Given that CUS-induced GI motility and neurotransmitter mRNA levels of Gfap were related to the mRNA levels of Gdnf (P dysfunction, we next examined if CUS could differentially modu- < 0.001, Fig. 4B). No significant changes in mRNA levels of these late the expression of ENS markers involved in regulating intestinal transcripts were observed in the ileum of mice exposed to CUS. plasticity and motility along the different intestinal segments. As Based on our observations, we next measured the protein expres- shown in Figure 4A, there was a down-regulation of Gfap mRNA sion of 2 glia markers (GFAP and S100B) and neuron (PGP 9.5) (glial cell marker), and neurotrophic factors (NTFs) as glial cell- in the proximal colon. Mice exposed to CUS showed a significant 78 Journal of Neurogastroenterology and Motility Gut Neuroplasticity in Chronic Stress PGP 9.5/-actin (fold change) S100B/-actin (fold change) GFAP/-actin (fold change) 1.0 1.0 1.0 * * 0.5 0.5 0.5 0 0 0 Control Stress Control Stress Control Stress GFAP S 100B PGP 9.5 -actin -actin -actin Figure 5. Effects of chronic stress on enteric glia population and neurons in proximal colon. Western blot analysis of specific glia markers, glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein B (S-100B), and neurons (protein gene product 9.5 [PGP 9.5]) in proximal colon of stressed and control mice (n = 7-8/group). Protein fold-change was calculated for each sample with reference to the average of the target protein to β-actin ratio of the control group. The protein expression of enteric glia markers were significantly decreased in proximal colon after chronic stress when compared with control mice. Data represent mean ± SEM, *P < 0.05 by Mann Whitney test. 1.5 Control 2.5 Control P = 0.065 Stress Stress Tlr2/-actin (mRNA) Tlr4/-actin (mRNA) P = 0.088 2.0 1.0 1.5 ** 1.0 0.5 0.5 0 0 Ileum Proximal Distal Ileum Proximal Distal Figure 6. Transcripts encoding Toll-like receptor 2 (Tlr2 ) are downregulated in the proximal colon of stressed mice and correlated with levels of transcripts encoding genes involved in enteric nervous system (ENS) function. Quantitative real-time polymerase chain reaction was used to analyze Tlr2 and Tlr4 expression levels in small intestine and colon of control and stressed mice (n = 10/group). Transcripts encoding Tlr2 were downregulated in the proximal colon of stressed mice. The increase expression levels of Tlr4 in proximal and distal colon of stressed mice did not reach statistical significance. Data represent mean ± SEM.**P < 0.01 by Mann Whitney test. concomitant reduction in the protein expression of GFAP and compared with the control group (Supplementary Table). In con- S100B yet protein expression of PGP 9.5 tended to be reduced, trast, similar amounts of IL-6, TNF-α, IL-10, and IL-12 were though not significantly (Fig. 5). produced by splenocytes upon stimulation with lipoteichoic acid and PMA in both groups (data not shown). Then, based on the poten- Chronic Psychosocial Stress Increases tial role of TLRs signaling in the development and maintenance of Lipopolysaccharide-stimulated Release of a proper ENS, we analyzed the differential expression of transcripts Spleen Inflammatory Mediators encoding Tlr2 and Tlr4 between both experimental groups in the In order to examine if disruption of the ENS and intestinal different intestinal segments. The proximal colon of CUS mice dysfunction can reflect a systemic inflammatory process through revealed an up regulation of Tlr2 mRNA (P < 0.01). Whereas TLRs activation, we evaluated the differential cytokine produc- both colonic segments of mice exposed to CUS showed a slight in- tion in TLR-ligand-mediated in the periphery. Spleens from both crease of Tlr4 mRNA (Fig. 6A). Together with the transcriptional groups were stimulated with a selective TLR2 and TLR4 agonist. changes induced by CUS exposure in the proximal colon of genes In agreement with our previous results,24 splenocytes of CUS mice involved in ENS homeostasis, we wanted to examine whether an displayed increased levels of IL-12, IL-6, TNF-α, and mouse association with changes in Tlr2 expression in this segment could keratinocyte-derived cytokine in response to LPS, a TLR4 agonist, be observed. Interestingly, significant positive correlations were ob- Vol. 29, No. 1 January, 2023 (72-84) 79 Beatriz Lobo, et al Table 2. Pearson’s Correlation Analysis Between Tlr2 and Tlr4 Table 3. Correlation Between Neurotransmitters Concentration Expression and Levels of Transcripts Encoding Enteric Nervous (Noradrenaline, Dopamine, and Serotonin) and Expression Levels of System-relevant Genes Involved in Motility in the Proximal Colon of Transcripts Encoding Genes Involved in the Function of ENS in the Control and Stressed Mice Proximal Colon of Control and Stress Mice Tlr2 mRNA Tlr4 mRNA Gene NA (ng/g) DA (ng/g) 5-HT (µg/g) Gene mRNA r P -value r P -value mRNA r P -value r P -value r P -value Gfap 0.55 0.002 a –0.15 0.564 Gfap –0.54 0.048 a –0.41 0.143 –0.48 0.065 Th 0.77 0.010a 0.03 0.893 Gdnf –0.45 0.092 –0.31 0.253 –0.53 0.034a Gdnf 0.48 0.004a 0.24 0.332 Bdnf –0.57 0.026a –0.49 0.067 –0.70 0.003a Bdnf 0.67 0.003a 0.08 0.745 Htr4 0.16 0.576 0.43 0.105 0.31 0.245 Htr4 –0.52 0.024a 0.61 0.008a Tlr2 –0.57 0.028a –0.55 0.033a –0.73 0.001a Tlr , Toll-like receptor; Gfap , glial fibrillary acidic protein; Th , tyrosine hy- Tlr4 –0.06 0.843 0.47 0.088 0.14 0.614 droxylase; Gdnf , glial cell-derived neurotrophic factor; Bdnf , brain-derived NA, noradrenaline; DA, dopamine; 5-HT, serotonin; Gfap , glial fibrillary neurotrophic factor; Htr4 , serotonin receptor 4. acidic protein; Gdnf , glial cell-derived neurotrophic factor; Bdnf , brain- a P < 0.05 was considered statistically significant. derived neurotrophic factor; Htr4 , serotonin receptor 4; Tlr , Toll-like receptor. a P < 0.05 was considered statistically significant. served between all of them Table 2. rate compared with controls was observed. In addition, the increase Stress-induced Changes in Neurotransmitter Levels of NA and DA levels is associated with a reduction in the expres- Correlate With the Expression of Selective Genes sion of Th , the rate-limiting enzyme in catecholamines biosynthesis. Involved in Enteric Nervous System Homeostasis However, despite the descriptive nature of this work, it could be Growing evidence highlights the critical role of neurotrans- considered as a potential constipation-predominant IBS model. mitters in the regulation of GI physiology and homeostasis main- Overall, our data reveal that CUS causes intestinal dysmotility tenance. To support this idea, we analyzed the relationship in the related to transcriptional changes associated with the regulation of proximal colon between amine content and the expression levels of the ENS activity in an intestinal-region-specific manner. It is worth all transcripts measured. Catecholamine content in the proximal co- noting that, intestinal motility tests were done in both experimental lon of CUS inversely correlated with Gfap, Bdnf , and Tlr2 expres- groups without fasting and during the dark (active) cycle. sion. In this colonic segment, we also found a significant negative Emerging evidence shows that mood disorders are comorbid correlation between 5-HT levels and Gdnf, Bdnf , and Tlr2 expres- with functional constipation and constipation-predominant IBS. sion Table 3. Moreover, neurological disorders are often associated with GI dysmotility,27,28 explained by the structural and neurochemical simi- larities that share ENS with CNS. It requires a proper network of Discussion enteric neuronal and glial cells with balanced neurotransmission for In the present study, we provide novel molecular insights into GI physiological function regulation.29,30 stress-induced mechanisms evoking constipation and enteric neu- Chronic constipation is a multifactorial disorder with a complex roplasticity. First, in our chronic stress model, a slow gut transit is pathogenesis that includes abnormalities within the ENS.31,32 In observed and accompanied by a decrease in fecal output without terms of animal models, different approaches have been used to changes in fecal water content. Second, along the intestinal seg- provide a better understanding of the pathophysiology of constipa- ments evaluated, chronic psychosocial stress caused a marked tion where changes in density and/or altered morphology of the decrease in the expression of glial-specific molecular markers, Gfap ENS, as well as altered neurotransmission, have been found.33,34 To and S100B , at the transcriptional and protein level in the proximal date, less is known about the mechanism by which chronic stress colon. Importantly, stressed mice exhibit also in the proximal colon impacts normal enteric neurotransmission and both motor and a significant down-regulation of 2 neurotrophic factors; further- sensory functions. Here, chronic stress-induced imbalance in neu- more, the Gdnf expression reduction is in parallel with changes in rotransmitters levels within the proximal colon with accompanying transcription levels of Gfap . Again, only in the proximal colon of molecular changes that might compromise the ENS function. mice exposed to CUS, a significant increase in monoamines content Interestingly, our data show again that those mice exposed to 80 Journal of Neurogastroenterology and Motility Gut Neuroplasticity in Chronic Stress CUS exhibited social anxiety-like behavior.23 Two animal models tential role of TLR-mediated signalling.46-48 It is known that differ- that resemble IBS-like features exhibited increased stress-induced ent TLRs are expressed in both CNS and ENS, regulating enteric neurochemical and ultrastructural changes in ENS in both the neuronal plasticity and neurotransmission.49 Indeed, the absence of small intestine17 and distal colon,18 and an association between en- TLR2 and TLR4 signaling alters GI motility may, in part, results teric glial cells (EGCs) activity and higher colonic contractility.18 from their role in modulating the magnitude of motor response elic- Advances in enteric neurobiology support EGCs as important ited by 5-HT in the proximal colon,50 enteric neurotransmission,47,48 players in enteric neuroplasticity, neurotransmission,35 and mucosal and glial phenotype by regulating NTFs production. Furthermore, immune response balance.28,36,37 a significant reduction in Tlr2 expression is identified in the proxi- Another interesting feature is their contribution to the regula- mal colon of stressed mice concomitant with a reduction of Gfap, tion of motility patterns and their outstanding plasticity, although Gdnf , and Th expression, besides being associated with the tissue today remains unclear under which specific conditions they switch.38 monoamine content. Numerous reports show EGCs loss in colon biopsy specimens from Taken together, all these findings suggest a potential association patients with altered GI motility.20,28,39 In the present study, stressed between changes in neuroplasticity through Tlr2 and stress-induced mice exhibit different expression patterns of glial-specific molecular slow intestinal transit. markers between the proximal colon and distal colon compared Indeed, our laboratory has shown different TLRs expres- with controls, and changes in Gfap expression in the proximal colon sion profiles in colonic mucosa in 2 distinct stress animal models,51 are inversely correlated with the NA levels. Notably, ion transport besides in this CUS paradigm we have exhibited the contribution in the caecum was not affected by chronic stress-related molecular of TLR4-dependent mechanism to stress-induced visceral hyper- changes in the ENS of the proximal colon. sensitivity through activation of microglia within the CNS.23 As Indeed, this result could explain that mice exposed to CUS well, other IBS stress-based rodent model advocated for the role exhibited reduced stool output without changes in water content. of TLR4 signaling coupled with increased colonic glial activation Although not shown in this study, in the presence of impaired intes- in visceral pain.52 In this line, our study shows an increased of both tinal transit, dysbiosis is the rule, what could determine the balance Gfap and Tlr4 expression in the distal colon of stressed mice. between absorption and secretion, but also cecal permeability, by On the other hand, an enhanced expression of TLR2 and multiple mechanisms, being one of the most frequent the increased TLR4 on splenic macrophages has been shown in a model of re- levels of 5-HT and tryptamine that mediates colonic secretion peated social defeat.53 Therefore, our group has also shown periph- through serotonin 4 receptor. Despite that we cannot associate the eral enhanced TLR4 activity associated with a visceral hyperalgesic motility readouts with neurotransmitter concentration, increased phenotype in DIO mice.54 Our current results indicate heightened levels of DA levels in the proximal colon might be the main factor, TLR4 activity of peripheral immune cells following CUS results which explains CUS-induced intestinal dysmotility. Thus, it has in increased LPS-induced release of several pro-inflammatory cy- been previously shown the inhibitory effect of dopamine on intesti- tokines. Finally, it is important to note that various animal models nal motility through its receptor D2.40 have proved how acute or repetitive exposure to homotypic stress, Of interest, preclinical studies have previously reported altered mediates intestinal pathology and psychiatric disorders.55-57 intestinal transit, whereas fecal water content41 or ion transport were However, the predictive validity of those models remains dis- unchanged after pharmacological disruption of EGCs function.42 puted because in socially organized mammals, dominant stressors NTFs as well as EGCs are important players in support- in natural conditions are frequently heterotypic and complex, and ing neurotransmission and neuroplasticity,43 indeed colonic Bdnf best represented by social experiences.58 The combination of social modulates both intestinal motor and sensory functions.44,45 Our data defeat and overcrowding that we used in this study reflects better suggest their role in the regulation of neurotransmission processes the stress experienced by humans on a daily basis. in the proximal colon as reflected by an inverse association between In addition, our study has several limitations: we did not con- NTFs transcripts and total tissue levels of 5-HT and NA, although duct a metagenomics analysis of the intestinal microbiome to assess we do not find statistical differences in protein expression of pan- the impact of CUS in gut microbiota and its metabolic activity, so neuronal marker PGP 9.5 between groups. we cannot rule out the influence of microbiota on gut motility, ENS The cellular and molecular pathways underlying the ENS or- disruption, and secretion/absorption process through Tlr signaling; ganization and activity are unclear, yet recent studies suggest the po- second, in our model, only male mice have been employed, and Vol. 29, No. 1 January, 2023 (72-84) 81 Beatriz Lobo, et al therefore, we recognize the potential sex-bias in our conclusions and Beca Estada l’estranger (Societat Catalana Digestología) 2011 on the impact of chronic stress on colonic neuroplasticity and neu- and PI19/01643 (Beatriz Lobo); PI17/01902 (Javier Santos) rotransmission. Therefore, studies comparing responses between CIBEREHD (Javier Santos); Vall d’Hebron Institut de Recerca. sexes are certainly warranted; third, gut motility and secretion/ FONDECYT #1181699 and #11121527; MECESUP 0608 absorption are certainly regulated by multiple signaling pathways Universidad de Chile (Caroll Beltran). The work described herein not analyzed in this study. Thus, further work is necessary to clarify was supported by APC Microbiome Ireland, funded by Sci- whether other pathways participate in the regulation of gut motility ence Foundation Ireland (SFI), through the Irish Government’s in constipation. National Development Plan. The authors and their work were Unfortunately, the present design was limited in exploring the supported by SFI (Grant No. 02/CE/B124, 07/CE/B1368, and relationship between stress anxiety-like behavior and the delay in GI SFI/12/RC/2273). transit time in the same cohort, as well the lack of functional charac- terizations analyses focused on EGCs and neurons are required in Conflicts of interest: The authors disclose the following: Javier Santos serves as a consultant for Noventure and discloses present order to provide further insight into regional functional changes in or past recent scientific collaborations with Salvat, Norgine, Alfa- motility. Sigma, Cosmo, Adare, Devintec Pharma, Pileje, and Danone. Finally, further mechanistic studies are needed to determine John F Cryan has been an invited speaker at meetings organized by how these transcriptional changes contribute to stress-induced Precision Biotics, Alkermes, and Janssen and has received research visceral hypersensitivity and motility dysfunction. While our work funding from Cremo, Nutricia, DuPont (IFF), and is a consultant does not provide a specific underlying mechanism, it offers an over- for Alkermes and Nestlé. Timothy G Dinan has been an invited view of how the effects of chronic stress could vary across GI tract speaker at meetings organized by Servier, Lundbeck, Janssen, and segments. These findings might reflect the complex nature of the AstraZeneca and has received research funding from Cremo, Nu- chronic psychological stress impact on intestinal motility. Thus, ad- tricia. The remaining authors declare no competing interests. The vancing our understanding of the mechanism(s) by which the ENS study was carried out in APC Microbiome Institute Biosciences responds to stress serves to elucidate the biology supporting stress- Building, University College Cork, Western Road, Cork, Ireland. related GI disorders, and requires further evaluation as therapeutic targets to counteract the consequences of chronic stress. This study Author contributions: Beatriz Lobo conceived of the experi- would give new insights into stress-induced constipation, and fur- ments; Beatriz Lobo, Mónica Tramullas, Beate-C Finger, Niall P ther studies in constipation-predominant IBS are needed to investi- Hyland, and John F Cryan supervised the experiments; Beatriz gate molecular changes related to the ENS integrity function. Lobo, Mónica Tramullas, Beate-C Finger, Kevin W Lomasney, and Caroll Beltran conducted the experiments; Beatriz Lobo wrote the initial draft of the manuscript; and Beatriz Lobo, Mónica Supplementary Materials Tramullas, Gerard Clarke, Javier Santos, John F Cryan, and Timo- Note: To access the supplementary table, figures, and methods thy G Dinan wrote subsequent drafts of the manuscript. mentioned in this article, visit the online version of Journal of Neu- rogastroenterology and Motility at http://www.jnmjournal.org/, and at https://doi.org/10.5056/jnm22009. References Acknowledgements: We would like to thank Rachel D Mo- 1. Mayer EA. The neurobiology of stress and gastrointestinal disease. Gut loney and Mr Patrick Fitzgerald for their technical assistance and 2000;47:861-869. Dr Bruno M Godinho for help with spleen stimulation studies. 2. Vicario M, Guilarte M, Alonso C, et al. 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