enteric glial cells are associated with stress‐induced...

Enteric glial cells are associated with stress-induced

colonic hyper-contraction in maternally separated rats

Y. FUJIKAWA, K. TOMINAGA, F. TANAKA, T. TANIGAWA, T. WATANABE, Y. FUJIWARA & T. ARAKAWA

Department of Gastroenterology, Osaka City University Graduate School of Medicine, Osaka, Japan

Key Messages

• Combination of infantile maternal separation with additional acute stress in adulthood is a model frequently

used to investigate the pathogenesis of stress-associated irritable bowel syndrome (IBS).

• Enteric glial cells (EGCs) play important roles in the regulation of gastrointestinal function, but manifestations

of colonic EGCs in IBS patients remain unknown.

• We evaluated the changes in colonic EGCs and their association with contraction in maternally separated rats

with additional acute stress.

• EGCs exhibited hyperplasia of their processes that apparently overlapped with the neurons and changes in

bulbous swelling of terminals, and were also associated with stress-induced hyper colonic contraction.

• Such changes in EGCs may underlie the pathogenesis of IBS.

Abstract

Background Enteric glial cells (EGCs) play important

roles in enteric integrity and regulation of gastrointes-

tinal function. However, whether EGCs undergo

pathophysiological changes in stress-associated gastro-

intestinal disorders is unknown.We investigated struc-

tural and functional alterations in colonic EGCs and

their roles in colonic contraction in an irritable bowel

syndrome (IBS) model. Methods As a chronic stress,

male Wistar rats underwent 3-h maternal separation

during postnatal days 2–14. As an acute stress, we used

water-immersion stress (4 h) in adulthood (at 8 weeks).

We quantitatively and morphologically evaluated

enteric neurons and EGCs using whole-mount longitu-

dinal muscle-myenteric plexus preparations. Colonic

contraction was analyzed with electrical field stimula-

tion (EFS). Key Results Glial fibrillary acidic protein

(GFAP) expression and thenumber of total, cholinergic,

and nitrergic neurons were unchanged in maternally

separatedratswithacutestress (combinedstress:anIBS

model) comparedwithcontrols.However, thedensityof

GFAP-positive EGC processes that apparently over-

lapped with the neurons and the extent of bulbous

swelling of terminals increased according to the stress

intensity:control,acutestress,maternalseparation,and

combined stress. EFS-induced colonic contractions

were significantly greater in the combined stress rats

than in controls.Higher dose of fluorocitrate, a selective

inhibitor of EGC metabolism, was required to inhibit

both EFS-induced contraction and EGCs activation in

thecombinedstressratsthanincontrols.Conclusions&

Inferences Colonic EGCs exhibited structural altera-

tions according to the stress intensity. EGCs were

associated with stress-induced colonic hyper-contrac-

tion in thecombinedstress rats,whichmayunderlie the

pathogenesis of IBS.

Keywords functional gastrointestinal disorders, glial

cells,irritablebowelsyndrome,maternalseparation,stress.

INTRODUCTION

Gastrointestinal physiological functions, such as

motility, visceral sensitivity, and mucosal barrier

function, are controlled by the enteric nervous system

Address for Correspondence

Kazunari Tominaga, Department of Gastroenterology, OsakaCity University Graduate School of Medicine, 1-4-3,Asahimachi, Abeno-ku, Osaka 545-8585, Japan.Tel: +81-6-6645-3811; fax: +81-6-6645-3813;e-mail: [email protected]: 25 September 2014Accepted for publication: 5 April 2015

© 2015 John Wiley & Sons Ltd1010

Neurogastroenterol Motil (2015) 27, 1010–1023 doi: 10.1111/nmo.12577

Neurogastroenterology & Motility

(ENS), which coordinates with the epithelium and

smooth muscle.1 Such functions are influenced by

environmental or psychological/physical stressors.2

The pathogenesis of irritable bowel syndrome (IBS)

involves both the central nervous system (CNS) and

colon. Central nervous system changes alter percep-

tion, and postinfectious mucosal inflammation is

associated with abdominal symptoms of IBS.3–8 Irrita-

ble bowel syndrome patients often experienced phys-

ical abuse as children (infantile stressor), and IBS

develops following additional stress in adulthood.9

Considering the brain–gut interaction, colonic dys-

functions influenced by psychological/physical stres-

sors may cause bowel dysfunction including

abdominal pain, discomfort, diarrhea, and constipation

in IBS patients.10 However, macroscopic changes are

absent in the colon of IBS patients. Therefore, inves-

tigation of microscopic changes in the colon in a model

of chronic infantile stress with adult acute stress,

which is important for the pathogenesis of IBS, is

required.

Enteric glial cells (EGCs), major components of the

ENS, are present in two major regions, the submucosal

and myenteric plexuses, together with enteric neu-

rons.11 Enteric glial cells regulate epithelial cell prolif-

eration, increase tight junction-associated protein

expression via the release of 15-deoxy-D12,14-prostaglan-

din J212 and s-nitrosoglutathione,13 and interact with

enteric neurons through purinergic signaling and

changes in intracellular Ca2+ levels.14,15 Thus, EGCs

contribute to mucosal barrier function, enteric neuro-

transmission, neuronal protection, and neurogenesis.11

Conversely, disruption of EGC function causes fulmin-

ant intestinal inflammation with degeneration of my-

enteric neurons,16 impairment of gastrointestinal

motility, and phenotypic alterations in enteric neu-

rons.17,18 Thus, EGCs play important roles in enteric

integrity and the regulation of gastrointestinal function

via theENS rather than by simply providing a supportive

framework for neurons. However, whether any changes

in EGCs occur in stressful conditions and how stress-

induced changes in EGCs affect colonic contraction are

unknown.

The CNS consists of neurons and glial cells (astro-

cytes, microglia, and oligodendrocytes). The number

and structure of astrocytes are influenced by psycho-

social stressors.19,20 Pharmacological glial ablation in

cortical regions induces depressive-like behaviors sim-

ilar to those observed after chronic unpredictable

stress.21 Furthermore, chronic stress, including

repeated water avoidance or maternal separation,

causes visceral hypersensitivity due to a decrease

in glutamate transporters in spinal glia.22,23 Thus,

astrocytes are important for the pathogenesis of stress-

associated disorders including mood disorders24 and

IBS. On the basis of evidence regarding glial fibrillary

acidic protein (GFAP) expression25 and electrophysio-

logical function26 in these glial cells, we hypothesize

that EGCs may be structurally and functionally mod-

ulated during the pathogenesis of stress-associated

gastrointestinal disorders like IBS. Maternal separa-

tion, a well-established experimental model of early

life stress, with additional acute stress in adulthood

causes colonic hyper-motility, barrier dysfunction, and

visceral hypersensitivity via various neurotransmit-

ters.27–29 Therefore, we investigated quantitative and

structural changes in EGCs and their role in colonic

motility using electrical field stimulation (EFS) and

fluorocitrate as a selective inhibitor of EGC metabo-

lism in maternally separated rats with additional acute

stress as an IBS model.

MATERIALS AND METHODS

Animals

Primiparous timed-pregnant female Wistar rats (Charles RiverJapan, Inc., Yokohama, Japan, gestational day 14) were allowedfree access to water and food, and were maintained at 22–24 °Cwith controlled lighting (light from 08:00 to 20:00 h daily). Theanimal ethics committee of Osaka City University approved allexperiments.

Drugs

Acetylcholine (ACh; Nihon Yakkyoku Hou, Tokyo, Japan), atro-pine,amuscarinicAChreceptorantagonist (NihonYakkyokuHou),ondansetron, a selective 5-hydroxytryptamine (5-HT) 3 receptorantagonist (Sigma, St. Louis,MO,USA), SB-204070hydrochloride, aselective 5-HT 4 receptor antagonist (Sigma), D-(�)-2-amino-5-phosphonopentanoic acid (AP5), a selective N-methyl-D-aspartate(NMDA) antagonist (Sigma), and tetrodotoxin (Wako Pure Chemi-cal, Osaka, Japan) were diluted with distilled water. DL-fluorocitricacid barium salt (Sigma), a selective inhibitor for glial cells, wasdissolvedindistilledwater,andbeforeuse,sodiumsulfatewasaddedto precipitate barium from the solution.18,30

Maternal separation

Maternalseparationwasperformeddailyforthreeconsecutivehours(09:00–12:00) on postnatal days 2–14.28 Briefly, pupswere separatedfrom their dams and placed in individual cages on heated pads (28–30 °C) in another room. Control pupswere left with their dams. Onpostnatal day 22, all pups were weaned. Only male pups wereanalyzed to avoid hormonal cycle-induced variations.

Acute stress

We used an established rat model of fatigue with continuousstressful restraint as an acute stress.31 Briefly, rats were housed for4 h (08:00–12:00) in cages filled with water (23 � 1 °C) to a height

© 2015 John Wiley & Sons Ltd 1011

Volume 27, Number 7, July 2015 Enteric glia and stress-induced colonic contraction

of 2.0 cm at 8 weeks of age, which increases plasma adrenocor-ticotropic hormone levels and delays gastric emptying withoutmacroscopic damage.32

Experimental protocol

Maternal separation was considered chronic infantile stress, andacute stress was considered additional adult stress in adulthood.We designated four groups to investigate the effect of thesestresses on EGCs: (i) control (no stress); (ii) acute stress alone inadulthood; (iii) maternal separation alone prior to weaning; and(iv) maternal separation prior to weaning and acute stress inadulthood (combined stress). All rats were killed by cervicaldislocation at 8 weeks of age or 24 h after acute stress, and distalcolonic tissue samples were obtained.

Western blotting

Proteins (30 lg) were extracted from distal colonic tissue in buffer(50 mM Tris–HCl, 150 mM NaCl, 0.5% sodium deoxycholate,0.1% sodium dodecyl sulfate [SDS], 1% nonidet P-40, 10 mMethylenediaminetetraacetic acid, protease inhibitor cocktail[Roche, Mannheim, Germany]), separated on a 10% SDS gel, andtransferred to polyvinylidene difluoride membranes. After block-ing with 5% skim milk, membranes were incubated overnight at4 °C with an anti-GFAP (2 lg/mL, #ab48059; Abcam, Cambridge,UK) primary antibody. After washing, membranes were incubatedwith a secondary antibody (anti-rabbit IgG-horseradish peroxidase,1:1000, #NA934; Amersham, Buckinghamshire, UK) followed byenhanced chemiluminescence reagents (GE Healthcare Life Sci-ence, Tokyo, Japan). Western blot bands were detected using aLAS-3000 imaging system (Fujifilm,Tokyo, Japan). Membraneswere stripped using Restore Western Blotting Stripping Buffer(Thermo Scientific, Yokohama, Japan) and reprobed with b-actin(0.6 lg/mL, #A5441; Sigma). Immunoblots were quantified usingNIH Image-J software, version 1.44 (National Institutes of Health,Bethesda, MD, USA). Results were expressed as a ratio of GFAP tob-actin.

Immunofluorescent staining

Distal colon tissues were obtained by opening the distal colonalong the mesenteric border, pinned flat with the mucosal side up,and fixed with acetone or 4% paraformaldehyde (4 °C, 1 h).Whole-mount longitudinal muscle-myenteric plexus (LMMP)tissues were prepared by removing the mucosa and submucosa,and partly removing the circular muscle layer. After blockingwith 5% normal donkey serum in phosphate-buffered salinecontaining 0.3% Triton-X 100 (4 °C, 1 h), the LMMP preparationwas incubated overnight at 4 °C with primary antibodies. Rabbitanti-GFAP (a marker of glial cell cytoplasm, 2 lg/mL, #ab48050;Abcam), mouse anti-GFAP (1 lg/mL, #556328; BD Pharmingen,NJ, USA), mouse anti-HuC/D (a marker of enteric neurons,0.4 lg/mL, #A21271; Life Technologies, Carlsbad, CA, USA), goatanti-choline acetyltransferase (ChAT; a marker of cholinergicneurons, 10 lg/mL, #AB144P; Millipore, Temecula, CA, USA),goat anti-neuronal nitric oxide synthase (nNOS; a marker ofnitrergic neurons, 4 lg/mL, #ab72428; Abcam), and rabbit anti-phosphorylated extracellular signal-regulated kinase 1/2 (pERK 1/2; a marker of glial activation, diluted at 1:250, #4370; CellSignaling, Danvers MA, USA)33 were used as primary antibodies.Tissues were washed with phosphate-buffered saline and incu-bated with secondary antibodies (donkey anti-rabbit Alexa Fluor

488, 4 lg/mL, #A21206; goat anti-mouse or donkey anti-goatAlexa Fluor 633, 4 lg/mL, #A21050 or #A21082, respectively;Molecular Probes) for double staining for 2 h at room temperature.Preparations were visualized with confocal microscopy (LAS AF,TCS SP5; Leica Microsystems, Tokyo, Japan) with a 409 oil-immersion objective lens (numerical aperture 1.25) or a 639 oil-immersion objective lens (numerical aperture 1.4). Z-series ofscans (0.5 lm) and single 512 9 512 pixel images (0.76 lm/pixel)were captured.

Quantitative analyses of enteric neurons

To assess the number of enteric neurons per ganglion, LMMPpreparations from control and maternal separation with acutestress groups (n = 4 per group) were immunostained with anti-bodies for HuC/D, ChAT, and nNOS in separate tissues. Fiveganglia (in a 150- to 200-lm wide region of each ganglion) fromeach rat (n = 4, each group) were randomly selected throughoutthe tissue preparation, and all positive cells (for each antibody) ina ganglion were counted. Data were expressed as the meannumber of positive cells per ganglion in each group.

Quantitative analysis of areas with glialprocesses that apparently overlapped withneurons

We measured areas of GFAP-positive hyperplasia of glialprocesses that apparently overlapped with neurons after con-verting the original color images to binary images in adjustingto a similar image as reliable as possible using NIH Image-Jsoftware (version 1.44) shown in Fig. 3B. Later, a mean ratio ofthe total area of EGC processes (white) to the whole area of theHuC/D-positive neurons (black) was evaluated in the fixedthresholds condition. While blinded to the tissue source, we (YFand KT) also counted EGC processes that apparently overlappedwith each neuronal cell body per ganglion and examined thestructure of the EGC processes in detail. Image analyses wereperformed at least for 100–150 neurons in 10 ganglia that wererandomly selected from each rat in each group (n = 4). Afterconfirming characteristic and representative features of EGCs,particularly the terminal glial processes, the glial processes wereclassified according to their terminal structure as eitherfilamentous (no obvious neck or bulbous terminal swelling) orleaf-like (a neck region with bulbous terminal swelling). Weevaluated and calculated a mean ratio of leaf-like processes tototal processes per ganglion in the above four groups (at8 weeks), and in maternal separation group at different timepoints (4, 8, 12, and 48 weeks, respectively).

Colonic contraction

Segments (1.5 cm in length) of the distal colon were promptlyremoved. Colonic segments were then ligated at each end witha silk thread and suspended longitudinally in a 5-mL organ bathinside an electrode sleeve and held at a resting tension of 1 g.Colonic tissues were allowed to equilibrate for 90 min in anorgan bath filled with Tyrode’s solution (composition [mM]: 137NaCl, 2.68 KCl, 1.8 CaCl2�2 H2O, 0.41 NaH2PO4, 11.9NaHCO3, 5.6 glucose, 1.0 MgCl2�6 H2O) at 37 °C and gassedwith 95% O2 and 5% CO2. Colonic contractile responses weredetected using isotonic force transducers (TD-111T; NihonKohden, Tokyo, Japan) and amplified with a high-gain DCamplifier (AD-632J; Nihon Kohden). After equilibration, maxi-


Y. Fujikawa et al. Neurogastroenterology and Motility

mal contractions were measured by high K+ stimulation(75 mmol/L). After rinsing and stabilization, we performedcolonic contraction experiments using EFS with pulses of 0.5-ms duration and 30 V for 10 s (SEN-3401; Nihon Kohden) atintervals of 10 min.34

In experiment 1 Electrical field stimulation was applied at 1-, 3-,and 10-Hz frequencies, and colonic contraction at each frequencywas evaluated three times in each rat in maternal separation withacute stress rats and control rats (n = 6). Electrical field stimula-tion-induced colonic contractions were normalized as a percent-age of the maximal contraction stimulated by high K+.

In experiment 2 To investigate whether EGCs participate incolonic contraction in stress conditions, we used fluorocitrateduring EFS. Fluorocitrate, a metabolic toxin, is incorporated intothe glial cells and then inhibits the tricarboxylic acid cycle.Administration of fluorocitrate inhibits EGC function and intes-tinal motility in mice and is thus considered a selective toxin forEGCs.18,30 After basal contraction was confirmed three timeswith EFS (3 Hz), we added fluorocitrate (100, 300, and 1000 lM) tothe organ bath and recorded the amplitude of colonic contractionwith EFS at 10-min intervals for 100 min in the maternalseparation with acute stress rats and control rats (n = 5–7). Theeffects of fluorocitrate on EFS-induced contractions wereexpressed as a percentage relative to basal contraction.

In experiment 3 To investigate the association betweenenteric neurons and colonic contraction, ondansetron (10�9 M),SB-204070 (10�6 M), AP5 (10�5 M), atropine (10�6 M), or tetrodo-toxin (10�6 M) was added to the organ bath 10 min prior to EFS(3 Hz) in the maternal separation with acute stress rats andcontrol rats (n = 4).

In experiment 4 In addition to EFS experiments, we examinedcolonic contraction in response to ACh (10�11 to 10�4 M). Coloniccontractions were expressed as relative percentages of the con-traction induced by a maximum ACh dose (10�4 M) in thematernal separation with acute stress rats and control rats (n = 6).Effective dose 50% (ED50) of the maximal contraction induced byACh was analyzed using Prism5 (GraphPad Software, San Diego,CA, USA). To examine the effect of fluorocitrate on ACh-inducedsmooth muscle contraction, we also investigated the contractileresponse to ACh (10�6 M) before and after administration offluorocitrate for 60 min (n = 4).

Effect of fluorocitrate on pERK 1/2 expression inEGCs

To examine whether fluorocitrate affects pERK1/2 expression inEGCs, LMMP tissue was immunohistochemically analyzed withan anti-pERK1/2 antibody and anti-GFAP antibody. Briefly, distalcolonic tissues were incubated with fluorocitrate or vehicle for60 min in 5-mL tubes with Tyrode’s solution at 37 °C, gassedwith 95% O2 and 5% CO2, and then stimulated with high K+ for10 min. Because high K+ stimulates both the neurons and CNSglia/astrocytes,35 tissues were treated with tetrodotoxin (10�6 M)for 10 min before high K+ treatment to inhibit the neurons. Aftertreatment, tissues were washed, and immunohistochemicalanalyses were performed. For pERK1/2 activation analysis, weused the previously reported method.18 Briefly, the mean back-ground pixel intensity for each photomicrograph was determinedby measuring the background pixel intensity in three to foursquare areas surrounding the ganglion. Next, an area of interest

was drawn around the ganglion, and the pixel intensity within theganglion was measured. The previously determined backgroundpixel intensity was then subtracted from the immunofluorescentganglion pixel intensity. Seven to eight ganglia per animal (n = 4–6) for each tissue treatment were measured, and the data wereexpressed as mean pixel intensity per lm2.

Statistical analysis

Data were expressed as the means � SE. We statistically analyzedEGC protein expression, the number of enteric neurons, EFS-induced colonic contractions and its inhibition by some drugsincluding fluorocitrate, and pERK1/2 expression between the con-trol andstressedgroupsusinga t-test.Wealsoanalyzed thearea ratioof glial process overlappedneurons, the ratioof leaf-like processes tototal processes per ganglion, and intensity of pERK1/2 expressionamongmultiple groups using analysis of variancewith a Bonferronicorrection. Furthermore, we have performed the statistical analysisforED50valuesbetweenthetwoconditionsbyextrasum-of-squaresF-test using Prism5. The significance level was set at p < 0.05.

RESULTS

Quantitative analysis of EGC protein expressionin stressed and control rats

Western blotting analysis was used to examine GFAP

expression in distal colonic tissue obtained from

maternally separated rats with acute stress or control

rats. Notably, no changes in GFAP expression were

observed between the two groups (Fig. 1).

Quantitation of the number of enteric neurons instressed and control rats

We investigated enteric neuron populations in LMMP

preparations from stressed and control rats. The mean

number of HuC/D-positive neurons (28.4 � 2.9, stress;

21.8 � 2.6, control), ChAT-positive neurons

(18.0 � 1.3, stressed; 13.4 � 1.9, control), and nNOS

neurons (6.1 � 0.6, stressed; 4.5 � 1.2, control) per

ganglion was slightly but not significantly changed by

the combined stresses (Fig. 2).

Histological analysis of EGCs in various stressedconditions (acute stress, maternal separation, andmaternal separation with acute stress)

Immunostaining was performed to investigate the mor-

phology andquantity ofGFAP-positive glial processes in

LMMP preparations. Most EGC processes (GFAP posi-

tive) were observed around HuC/D-positive neurons in

the two-dimensional projections. In the control group, a

limited number of EGC processes that apparently over-

lapped with neurons were seen; however, the density of

these EGC processes hyperplasia that apparently over-



lapped with neurons progressively increased in the

stressed rats (Fig. 3A). The two-dimensional area with

apparent overlap of these EGC processes with the neu-

ronswassemi-quantitativelymeasuredasapercentageof

the area encompassing the HuC/D-positive neurons

(Fig. 3B).Thearea ratio significantly increasedaccording

tothestressintensity (13.5%,acutestress;20.5%,maternal

separation;45.8%,maternal separationwithacutestressvs

9.1%, control; Fig. 3C). Between acute stress and chronic

stress (maternal separation), there was also significant

difference in the ratio (p < 0.001). In addition, combination

ofmaternalseparationwithacutestressstrikinglyincreased

it compared with that in acute stress alone and maternal

separation alone, respectively (p < 0.001).

Terminal structure of EGC processes in variousstressed conditions

Characteristic and representative features of EGCs,

particularly terminal glial processes, were notable in

the stressed rats (Fig. 4A). These glial processes were

classified according to their terminal structure as

either filamentous (no obvious neck or bulbous

terminal swelling) or leaf-like (a neck region with a

bulbous terminal swelling; Fig. 4B). The ratio of leaf-

like processes to total processes per ganglion tended to

be high in the maternally separated rats (6.6%),

although the ratio in the control rats was 1.2%

(p = 0.08; Fig. 4C). Furthermore, the ratio was dramat-

ically increased in the maternal separation with acute

stress group (15.2%, p < 0.001 vs other groups;

Fig. 4C). In addition, a stress-free period (4 weeks)

GFAP

β-actin

Control MS+AS

GFA

P/β-

actin

0

0.5

1.0

1.5

Figure 1 Western blots of GFAP expression in maternal separation

with acute stress and control rats. Upper panel shows GFAP expression

in distal colonic tissue extracts. Densitometric analysis of GFAP

expression is shown in the lower panel. No differences were observed

between control rats and maternal separation with acute stress rats

(n = 10 per group) using a t-test analysis. Each column represents the

mean � SEM. MS, maternal separation; AS, acute stress.

HuC/D ChAT nNOS

Num

ber o

f ent

eric

neu

rons

/gan

glio

n

35

30

25

20

15

10

5

0

Control

MS+AS

A HuC/D ChAT nNOS

Control

MS+AS

B

Figure 2 Total number of enteric, cholinergic, and nitrergic neurons in maternal separation with acute stress rats and control rats. (A) Mean number

of HuC/D-positive neurons, ChAT-positive neurons, and nNOS-positive neurons per ganglion in LMMP tissue obtained from maternal separation

with acute stress and control rats (n = 4 per group, five ganglia per animal). No differences were observed between control rats and maternal

separation with acute stress rats using a t-test analysis. Each column represents the mean � SEM. (B) Representative images of HuC/D-positive

neurons, ChAT-positive neurons, and nNOS-positive neurons; scale bar = 50 lm. MS, maternal separation; AS, acute stress.



after acute stress discontinuation reversed the struc-

tural changes to close to the ratio of maternal separa-

tion alone (8.8%, p < 0.05 vs maternal separation with

acute stress; Fig. 4C). Regarding the time course in the

maternal separation group, leaf-like changes in the

terminal glial processes also appeared at 4 weeks

(7.1%) and lasted until at least 12 weeks. At 48 weeks,

the ratio decreased to 2.5%, although the ratio in the

age-matched control rats was 0.9% (Fig. 4D).

Evaluation of EFS-induced colonic contractionsin stressed and control rats

Electrical field stimulation induced longitudinal colo-

nic contractions in a frequency-dependent manner (1–10 Hz) in maternal separation with acute stress and

control rats (Fig. 5A). Electrical field stimulation-

induced colonic contractions were inhibited by pre-

treatment with atropine or tetrodotoxin (data not

shown). Notably, EFS-induced contractions were sig-

nificantly greater in the maternal separation with

acute stress rats than the control rats at 1 and 3 Hz

frequency (1 Hz; 47.6% control and 68.3% stressed;

p < 0.05; 3 Hz; 69.3% control and 87.3% stressed;

p < 0.05; Fig. 5B), while, EFS-induced contractions at

10 Hz frequency were 77.6% (control) and 87.6%

(stressed; p = 0.092; Fig. 5B).

Effect of fluorocitrate on EFS-induced coloniccontraction in stressed and control rats

Fluorocitrate (100–1000 lM) decreased EFS-induced

colonic contractions in both groups in a time- and

dose-dependent manner (Fig. 6A). However, the

inhibitory effect of fluorocitrate on EFS-induced

colonic contraction differed between maternal sepa-

ration with acute stress and control rats. The

percentage of fluorocitrate (100 lM) inhibition after

60 min were 6.6% in the maternal separation with

acute stress rats and 37.1% in control (p = 0.06;

A

B C

Control AS MS MS+ASGFAP

HuC/D

GFAP

HuC/D

GFAP

HuC/D

GFAP

HuC/D

45

40

35

30

25

20

15

10

5

0Control AS MS MS+AS

Ove

rlapp

ed a

rea

by E

GC

s pr

oces

ses

to th

e ne

uron

s/ne

ural

bod

y (%

)

#

*

§

Overlapped area by EGCs processes (white area)

Area of HuC/D-positive neuron(black area)

Figure 3 Quantitative analysis of area with apparent overlap of EGCs processes with the neurons. LMMP tissue was obtained from experimental rats

and immunostained as described in the Materials and Methods. (A) GFAP-positive EGCs (green) were found around HuC/D-positive enteric neurons

(red) in the colonic ganglion. The HuC/D-positive enteric neurons were gradually overlapped by the GFAP-positive EGCs in response to stress; scale

bars = 25 lm. (B) GFAP-positive regions were converted to white images. The red line surrounds a single HuC/D-positive neuron. (C) The ratio of

white area to black area gradually increased with stress (n = 4 per group, 10 ganglia, and 100–150 neurons per group) using variance with a Bonferroni

correction. Each column represents the mean � SEM. *p < 0.05 vs the control group; #p < 0.001 vs the control and acute stress groups; §p < 0.001 vs

the control, acute stress, and maternal separation groups. MS, maternal separation; AS, acute stress.



Fig. 6B). Furthermore, the percentage of fluorocitrate

(300 lM) was significantly weaker in the maternal

separation with acute stress rats than in the control

rats (37.4% stressed and 75.7% control; p < 0.01;

Fig. 6B). However, we found no differences in the

percentage of fluorocitrate inhibition at 1000 lMbetween the two groups (72.5% stressed and 78.5%

control; p = 0.62; Fig. 6B).

Effect of ACh on colonic contraction in thestressed and control rats

Acetylcholine enhanced colonic contraction in the

maternal separation with acute stress rats in a dose-

dependentmanner (Fig. 6C). The ED50was significantly

lower in the maternal separation with acute stress rats

(6.4 � 0.12 9 10�8 M) than in the control rats

(2.2 � 0.07 9 10�7 M; n = 6 per group, p = 0.0047).

The inhibition ratesoffluorocitrate (100 and300 lM)after

60 min on ACh-induced colonic contraction were small

(100 lM: 21.5% control and 10.3% stressed; 300 lM:

24.5%controland17.2%stressed).Wefoundnodifferences

in inhibition rates between the two groups (Fig. 6D).

Effects of serotonin receptor antagonists and anNMDA receptor antagonist on EFS-inducedcolonic contraction in stressed and control rats

Ondansetron, SB-204070, and AP5 partially decreased

EFS-induced colonic contraction in both groups. How-

ever, we found no differences in the inhibitory effect of

these drugs between maternal separation with acute

stress and control rats (Ondansetron; 5.3% control and

8.5% stressed; SB-204070, 21.3% control and 29.5%

stressed; AP5 58.6% control and 43.8% stressed; Fig. 7).

Effect of fluorocitrate on high K+-induced pERK1/2 expression in stressed and control rats

As a measure of EGC activation, pERK1/2 expression

was measured in LMMP tissue. At the upper panels

(Fig. 8A), weak pERK1/2 expression and the co-local-

ization with GFAP was observed in the colonic tissues

treated with no treatment. High K+ stimulation

induced pERK1/2 expression in EGCs (Fig. 8A). Mean

intensity of pERK1/2 expression by high K+ was

significantly higher in the maternal separation with

Control AS MS MS+AS

Filamentous

Leaf-like

BulgingHead

Neck

20

15

10

5

0Control AS MS MS+AS MS+AS

4 weeks afterAS discontinuation

*

#

Leaf

-like

pro

cess

es /t

otal

pro

cess

es (%

)

Control■MS

10

8

6

4

2

04 8 12 48 48

Age (weeks)

Leaf

-like

pro

cess

es /t

otal

pro

cess

es (%

)

A

B C D

Figure 4 Quantitative analysis of terminal structural changes in EGCs. (A) The images in the upper panels are magnified images of the respective

squares with dotted lines in Fig. 3A; scale bars = 10 lm. (B) Representative image and illustration of the terminal glial structure for filamentous and

leaf-like processes. (C) The ratio of leaf-like processes to the total processes per ganglion is higher in the maternal separation alone and maternal

separation with acute stress rats than in the control rats using variance with a Bonferroni correction. The increase in the ratio of leaf-like processes by

acute stress in the maternally separated rats decreased to levels similar to before acute stress by 4 weeks after acute stress discontinuation (n = 4 per

group, 10 ganglia, and about 700 processes per group). Each column represents the mean � SEM. *p < 0.001 vs the control, acute stress, and maternal

separation rats; #p < 0.05 vs maternal separation with acute stress. (D) Time course of leaf-like processes in the maternal separation rats. Appearance

of leaf-like processes was already increased at 4 weeks of age and changes in leaf-like processes were observed even at 48 weeks. MS, maternal

separation; AS, acute stress.



acute stress rats than in the control rats (6.1 � 0.4

pixel intensity per lm2 vs 3.2 � 0.2 pixel intensity per

lm2; p < 0.01; Fig. 8A and C). Pretreatment with

fluorocitrate (300 lM) prior to addition of the high K+

did not inhibit EGC pERK1/2 expression in the

stressed rats (5.8 � 0.5 pixel intensity per lm2; Fig. 8B

and C). However, a higher dose of fluorocitrate

(1000 lM) was needed to inhibit EGC pERK1/2 expres-

sion in the stressed rats (2.0 � 0.3 pixel intensity per

lm2; Fig. 8B and C).

DISCUSSION

Our results demonstrate that structural changes (elon-

gation and/or bulbous terminal swelling of processes) in

colonic EGCs were more notable in the maternal

separation with acute stress rats than in controls.

Enteric glial cellsmay be associatedwith stress-induced

colonic hyper-contraction in maternally separated rats.

Enteric glial cells, an ENS component, undergo

several rounds of postnatal division.36 After differenti-

ation, EGCs are generally static and are present around

neurons and neuronal processes in normal conditions in

adulthood.11 Various in vivo and in vitro studies have

demonstrated increased EGC proliferation resulting

from inflammation or stimulation with proinflamma-

tory cytokines.16,37,38 However, no previous reports

have investigated how the ENS including EGCs is

affected by stress. Firstly, we evaluated quantitative

changes in GFAP and various populations of enteric

neurons including HuC/D-positive neurons, nNOS-

positive neurons, and ChAT-positive neurons. Stress

had no effect onGFAP expression in colonic tissue or on

the number and distribution of the above different

populations of enteric neurons in LMMP preparations.

However, in contrast to the lack of quantitative changes

observed with GFAP expression, we observed marked

differences in histology in stressed rats. In response to

stress, hyperplasia of EGCs (especially bulbous terminal

swelling of processes) was present as processes that

apparently overlapped (GFAP positive) with neurons in

the two-dimensional projections. Accordingly, the

observed histological changes in EGCs mainly resulted

from structural changes in each type of EGC process in

A1 Hz 3 Hz 10 Hz 1 Hz 3 Hz 10 Hz

Control MS+AS

B 100

80

60

40

20

01 3 10

EFS (Hz)

Col

onic

con

trac

tion

(% o

f res

pons

e to

hig

h K

+ )

**

○●

●

●

MS+AS (n = 6)Control (n = 6)

●

Figure 5 Representative traces of EFS-

induced colonic contraction in distal colon

segments from maternal separation with

acute stress rats and control rats. (A) EFS-

induced colonic contraction was recorded as

the amplitude of each trace. The bar

indicates the period of EFS (10 s). Colonic

contractile traces induced by EFS were

recorded at frequencies of 1, 3, and 10 Hz in

the control and maternal separation with

acute stress rats. (B) Quantitative data for

EFS-induced contraction in the control (open

circle) and maternal separation with acute

stress rats (closed circle) were analyzed at

each frequency using a t-test. Each data

point represents the mean � SEM. *p < 0.05

vs control at each frequency (n = 6 per

group). MS, maternal separation; AS, acute

stress.



response to stress. In addition, we observed differences

in such structural changes in EGC processes according

to the stress intensity: control, acute stress alone,

maternal separation alone, and combined stress. In

addition, the effect of the combined stress on structural

changes in EGC processes was remarkable but not

simply additive. Glial fibrillary acidic protein, an

intermediate filament protein in mature CNS glia/

astrocytes, regulates cell migration and morphology by

providing structural stability to CNS glia/astrocyte

processes.39 Chronic stress affects the length, branching,

and volume of CNS glia/astrocyte processes.20 In addi-

tion, environmental interventions early in life such as

neonatal handling or physical exercise increase the den-

sity and degree of branching and length of CNS glia/

astrocyte processes in adulthood.40,41 Considering the

above evidence of the association between CNS glia/

astrocytes and stress, the observed histological changes in

EGCsmaymainly result from structural changes in EGC

processes (elongation and bulbous terminal swelling of

A

100

80

120

60

40

20

00 10 20 30 40 50 60 70 80 90 100

Time after fluorocitrate treatment (min)

Col

onic

con

trac

tion

(% o

f bas

al c

ontr

actio

n)□○△

Control 100 μM fluorocitrate (n = 6)Control 300 μM fluorocitrate (n = 7)Control 1000 μM fluorocitrate (n = 5)

■●▲

MS+AS 100 μM fluorocitrate (n = 6)MS+AS 300 μM fluorocitrate (n = 5)MS+AS 1000 μM fluorocitrate (n = 5)

C

Col

onic

con

trac

tion

(% re

spon

se to

AC

h 10

–4)

ACh (M)

○● MS+AS (n = 6)

Control (n = 6) D □ Control

■ MS+AS

100

80

60

40

20

0

Inhi

bitio

n ra

te o

f EFS

indu

ced

con

trac

tion

(% o

f bas

al c

ontr

actio

n)

100 300Fluorocitrate (μM)

B100

80

60

40

20

0100 300 1000

Fluorocitrate (μM)

Inhi

bitio

n ra

te o

f EFS

indu

ced

con

trac

tion

(% o

f bas

al c

ontr

actio

n)

*

□ Control

■ MS+AS

Figure 6 Effect of fluorocitrate on EFS-induced colonic contraction and ACh-induced colonic contraction in distal colon segments from maternal

separation with acute stress rats and control rats. (A) Quantitative analyses of EFS-induced contractions (3 Hz) at 10-min intervals for 100 min after

treatment with 100 lM (squares), 300 lM (circles), and 1000 lM fluorocitrate (triangles) in control (dotted line) and maternal separation with acute

stress rats (solid line). Each data point represents the mean � SEM (n = 5–7 per group). (B) Inhibition rate of colonic contraction 60 min after

treatment in the control and maternal separation with acute stress rats was analyzed using a t-test. Each column represents the mean � SEM.

*p < 0.01 vs control (n = 5–7 per group). (C) ACh-induced colonic contraction was evaluated at different doses of ACh in maternal separation with

acute stress (closed circle) and control (open circle) rats. The ED50 of ACh-induced colonic contraction was calculated in both groups from these

contraction curve (n = 6 per group). (D) Inhibition rate of the ACh-induced contraction 60 min after fluorocitrate (100, 300 lM) treatment in the

control and maternal separation with acute stress rats was analyzed using a t-test. Each column represents the mean � SEM (n = 4 per group). MS,

maternal separation; AS, acute stress.



processes) in response to stress intensity. These results are

the first report of alterations in EGC processes (from

filamentous to leaf-like) in a model of stress-associated

IBS. In addition, we also found that leaf-like changes in

terminal glial processes in maternally separated rats

appeared at 4 weeks and lasted at least until 12 weeks.

Furthermore, the leaf-like changes were observed even at

48 weeks. In contrast, the leaf-like change in terminal

glial processes caused by acute adult stress was reversed

during a stress-free period (4 weeks) after discontinuation

of acute stress. Together, these findings suggested that the

effects of chronic stress prior to weaning were difficult to

attenuate, whereas the effects of acute stress in adulthood

may be relatively easy to be attenuate. Both the effects of

acute and chronic stress on such structural changes and

the differences in attenuation of structural changes

between acute adult and chronic infantile stress may

indicate that synergistic effects on EGC structuresmay be

due to irreversible effects of the chronic infantile stress

(maternal separation). These findings may indicate clin-

ical importance of infantile stress regarding IBS. However,

the physiological significance of these stress-induced

changes in EGCmorphology remains unknown. A previ-

ous report showed that L-type calciumchannel expression

is up-regulated in the colonic smooth muscle cells of

maternally separated rats, resulting in increased colonic

motility.42 We also confirmed that colonic contractions

caused by ACh were significantly increased in the stress

group compared with the control. Thus, among several

gastrointestinal physiological functions, maternal separa-

tion with or without acute stress in adulthood may affect

colonic motility via not only hypersensitivity of smooth

muscle but also neuronal activity including EGC.

The structure of dendritic spine heads is reported to

change to a stubby, thin, or mushroom-like morphol-

ogy following exposure to several types of stress. Head

size is associated with neuronal excitability in CNS

regions (amygdala and hippocampus) that are highly

sensitive to stress.43 Accordingly, the leaf-like changes

in EGC processes may reflect changes in glial activity.

In the present study, EFS-induced contraction was

increased in a frequency-dependent manner and was

greater in the maternal separation with acute stress

rats than in the control rats. This enhancement may be

due to an association of the extent of hyperplasia of

EGC processes with colonic contraction. Therefore, we

examined the role of EGCs in colonic contraction

using fluorocitrate because it is also a selective inhib-

itor of EGC metabolism.18 Fluorocitrate inhibited EFS-

induced colonic contraction in a time- and dose-

dependent manner in the maternal separation with

acute stress rats and in the control rats. Fluorocitrate

inhibition was less effective in IBS model rats than in

controls for both colonic contraction and pERK1/2

expression after high K+ stimulation. Considering the

pharmacological properties of fluorocitrate (a selective

metabolic toxin for EGCs), these results may be due to

differences in EGC morphology. In other words, the

difference in the effects of fluorocitrate on EFS-induced

colonic contraction between stressed and control rats

may indicate increased metabolic activation of EGCs,

particularly the leaf-like type of EGCs. However,

enteric neurons, rather than EGCs, may be directly

involved in stress-induced colonic hyper-contraction

because ACh-induced contraction was also enhanced

in the stress rats. Furthermore, psychological stress

activates colonic cholinergic neurons as shown by Fos

expression.44 To evaluate the above possibility, selec-

tive inhibition of cholinergic neurons is required

during EFS-induced contractions. However, no easy

method is available to directly and selectively inhibit

colonic cholinergic neurons. Therefore, we evaluated

the effects of serotonin and glutamate on EFS-induced

contraction in this model using respective receptor

100

80

60

40

20

0

Inhi

bitio

n ra

te o

f EFS

indu

ced

cont

ract

ion

(% o

f bas

al c

ontr

actio

n)

Control MS+ASOndansetron

100

80

60

40

20

0

Inhi

bitio

n ra

te o

f EFS

indu

ced

cont

ract

ion

(% o

f bas

al c

ontr

actio

n)

Control MS+ASSB-204070

100

80

60

40

20

0

Inhi

bitio

n ra

te o

f EFS

indu

ced

cont

ract

ion

(% o

f bas

al c

ontr

actio

n)

Control MS+ASAP5

Figure 7 Involvement of serotonergic and glutamatergic neurons in EFS-induced contraction. Inhibition rate of the EFS-induced contraction 10 min

after ondansetron, SB-204070, or AP5 treatment in the control and maternal separation with acute stress rats was analyzed using a t-test. Each

column represents the mean � SEM (n = 4 per group). MS, maternal separation; AS, acute stress.



antagonist, because these mediators can affect a release

of ACh from cholinergic neurons resulting in colonic

contraction.45,46 We found no significant difference in

inhibition rates by these antagonists in EFS-induced

contraction between both groups. Therefore, colonic

hyper-contraction caused by the above chronic and

acute stresses may be dominantly due to EGCs hyper-

plasia.

We also understand the possibility that fluorocitrate,

an inhibitor of the tricarboxylic acid cycle (TCA), may

affect neurons, interstitial cells of Cajal (ICC), and

smooth muscle cells. However, examining the separate

role of these components on EGCs in the above

contraction is very difficult. Tetrodotoxin, a represen-

tative neurotoxin, completely inhibited EFS-induced

contraction within 10 min. In contrast, during a long-

term period after fluorocitrate incubation, both EFS-

induced contraction and spontaneous colonic contrac-

tion also occurred. If fluorocitrate is a neurotoxic

agent, the agent would have inhibited the EFS-induced

contraction and exhibited similar effects on EFS-

induced contraction in both groups with different

Control(no treatment)

Control(vehicle)

MS+AS(vehicle)

pERK 1/2 GFAP Merge pERK 1/2 GFAP Merge

Controlfluorocitrate(300 μM)

MS+ASfluorocitrate(300 μM)

MS+ASfluorocitrate(1000 μM)

Vehicle Fluorocitrate (μM)300 300 1000

Pixe

l int

ensi

ty/μ

m2

* * □ Control

■ MS+AS

A

C

B

Figure 8 Effect of fluorocitrate on high K+-induced pERK1/2 expression in EGCs in maternal separation with acute stress rats and control rats. (A) At

the upper panels, pERK1/2 expression (green), GFAP expression (red), and their co-localization in the colonic tissues with no treatment. Stimulation

with high K+ induced pERK1/2 expression. Co-localization with GFAP expression (red; EGCs) was observed in LMMP tissue obtained from control

rats and maternal separation with acute stress rats. (B) Fluorocitrate (300 lM) inhibited high K+-induced pERK1/2 expression in the control rats. In the

maternal separation with acute stress rats, high K+-induced pERK1/2 expression was inhibited by high doses of fluorocitrate (1000 lM) but not by

300 lM fluorocitrate (n = 4 per group); scale bars = 100 lm. (C) Intensity of pERK1/2 expression in control rats (vehicle and fluorocitrate: 300 lM)

was analyzed using a t-test and intensity of pERK1/2 expression in maternal separation with acute stress rats (vehicle and fluorocitrate: 300 and

1000 lM) was analyzed using variance with a Bonferroni correction. Each column represents the mean � SEM (7–8 per ganglia per each rat, n = 4 per

each group). *p < 0.001 vs control (vehicle and 300 lM fluorocitrate, respectively) and maternal separation with acute stress rats (1000 lMfluorocitrate). MS, maternal separation; AS, acute stress.



extent of EGC processes. Furthermore, incubation of

fluorocitrate faintly decreased ACh-induced contrac-

tion in both groups. Together with all these findings,

we believe that fluorocitrate must mainly inhibit

EGCs as introduced in the previous papers,18,30

although we could not absolutely exclude the possibil-

ity that fluorocitrate may affect neurons and smooth

muscle cells.

Our study has some limitations. We could not

elucidate the mechanism(s) relevant to morphological

changes in EGCs after stress. Our preliminary data

suggested that administration of an a-helical cortico-

tropin-releasing factor antagonist prior to acute stress

prevented the stress-associated morphological changes

in EGC processes. Thus, in acute stress conditions, the

hypothalamic–pituitary–adrenal axis including various

humoral factors may affect the observed morphological

changes in EGC processes, at least in part. Accordingly,

the mechanism may be associated with stress-related

mediators including glucocorticoids, glutamate, sero-

tonin, gamma-aminobutyric acid, and brain-derived

neurotrophic factor. Therefore, these humoral factors

may be associated with EGC process proliferation in

this model. Further examinations are required to

investigate these possibilities.

CONCLUSION

Structural changes (elongation and/or bulbous terminal

swelling of processes) in colonic EGCs were noted in

the maternal separation with acute stress rats. Enteric

glial cell were also associated with stress-induced

colonic hyper-contraction in maternally separated rats.

Such microscopic changes in the ENS may underlie the

pathogenesis of stress-associated diseases such as IBS.

ACKNOWLEDGMENTS

We are indebted to Prof. Miyako Takaki of Nara MedicalUniversity School of Medicine and to Associate Prof. YasutakaAzuma and Prof. Masayoshi Takeuchi of Osaka PrefectureUniversity Graduate School of Life and Environmental Sciencefor their expert technical assistance and supervision.

FUNDING

This study was supported in part by a Grant-in-Aid for ScientificResearch from the Ministry of Education, Culture, Sports, Scienceand Technology in Japan.

DISCLOSURE

Tetsuo Arakawa has participated in advisory committees forOtsuka Pharmaceutical Co. Ltd. and Eisai Co. Ltd. YasuhiroFujiwara has participated in advisory committees for Eisai Co.Ltd. All other authors declare that they have no conflicts ofinterest.

AUTHOR CONTRIBUTION

YF & KT designed the research study and performed histolog-ical and functional analyses; YF performed statistical analysesand wrote the paper; FT, TT, TW, and YF were experimentaladvisers; KT is an overall director; TA is a supervisor of overallstudy.

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enteric glial cells are associated with stress‐induced...

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