Alterations in Prefrontal-Limbic Functional Activation
and Connectivity in Chronic Stress-Induced Visceral
Zhuo Wang1, Marco A. Ocampo1, Raina D. Pang2, Mihail Bota3, Sylvie Bradesi5,6, Emeran A. Mayer5,6,7,
Daniel P. Holschneider1,2,4,6*
1Department of Psychiatry and Behavioral Sciences, University of Southern California, Los Angeles, California, United States of America, 2Program in Neuroscience,
University of Southern California, Los Angeles, California, United States of America, 3Department of Biological Sciences, University of Southern California, Los Angeles,
California, United States of America, 4Departments of Neurology, Biomedical Engineering, Cell & Neurobiology, University of Southern California, Los Angeles, California,
United States of America, 5Veterans Administration Greater Los Angeles Healthcare System, Los Angeles, California, United States of America, 6Center for Neurobiology
of Stress, Department of Medicine, University of California Los Angeles, Los Angeles, California, United States of America, 7Departments of Physiology, Psychiatry and
Biobehavioral Sciences, University of California Los Angeles, Los Angeles, California, United States of America
Repeated water avoidance stress (WAS) induces sustained visceral hyperalgesia (VH) in rats measured as enhanced
visceromotor response to colorectal distension (CRD). This model incorporates two characteristic features of human irritable
bowel syndrome (IBS), VH and a prominent role of stress in the onset and exacerbation of IBS symptoms. Little is known
regarding central mechanisms underlying the stress-induced VH. Here, we applied an autoradiographic perfusion method
to map regional and network-level neural correlates of VH. Adult male rats were exposed to WAS or sham treatment for
1 hour/day for 10 days. The visceromotor response was measured before and after the treatment. Cerebral blood flow (CBF)
mapping was performed by intravenous injection of radiotracer ([14C]-iodoantipyrine) while the rat was receiving a 60-
mmHg CRD or no distension. Regional CBF-related tissue radioactivity was quantified in autoradiographic images of brain
slices and analyzed in 3-dimensionally reconstructed brains with statistical parametric mapping. Compared to sham rats,
stressed rats showed VH in association with greater CRD-evoked activation in the insular cortex, amygdala, and
hypothalamus, but reduced activation in the prelimbic area (PrL) of prefrontal cortex. We constrained results of seed
correlation analysis by known structural connectivity of the PrL to generate structurally linked functional connectivity (SLFC)
of the PrL. Dramatic differences in the SLFC of PrL were noted between stressed and sham rats under distension. In
particular, sham rats showed negative correlation between the PrL and amygdala, which was absent in stressed rats. The
altered pattern of functional brain activation is in general agreement with that observed in IBS patients in human brain
imaging studies, providing further support for the face and construct validity of the WAS model for IBS. The absence of
prefrontal cortex-amygdala anticorrelation in stressed rats is consistent with the notion that impaired corticolimbic
modulation acts as a central mechanism underlying stress-induced VH.
Citation: Wang Z, Ocampo MA, Pang RD, Bota M, Bradesi S, et al. (2013) Alterations in Prefrontal-Limbic Functional Activation and Connectivity in Chronic Stress-
Induced Visceral Hyperalgesia. PLoS ONE 8(3): e59138. doi:10.1371/journal.pone.0059138
Editor: Thomas Boraud, Centre national de la recherche scientifique, France
Received December 23, 2012; Accepted February 12, 2013; Published March 1 , 2013
Copyright: ? 2013 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The research was supported by United States National Institutes of Health grants P50DK064539 (Mayer) and R24AT002681 (Mayer). The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Considerable evidence links stress with the onset and symptom
exacerbation in irritable bowel syndrome (IBS) [1–3]. To better
understand the underlying mechanisms underlying this stress
sensitivity, and to identify novel targets for drug development,
stress-based animal models for IBS have been established and
extensively studied, using as stressors electric foot shock ,
maternal separation , social defeat and overcrowding , as
well as repeated water avoidance stress (WAS) . Visceromotor
responses measured as abdominal electromyographic signals
evoked by colorectal distension (CRD), are most commonly used
to assess stress-induced visceral hyperalgesia, modeling a cardinal
symptom of IBS. However, given the multidimensional nature of
pain, the visceromotor response in rodents likely reflects only a
portion of the complex human visceral pain experience. In recent
years, functional brain imaging technology has emerged as a
powerful tool to bridge the measurement gap between preclinical
and clinical pain research, providing an objective measurement of
pain in humans and laboratory animals alike. Comparing
alterations in CRD-evoked brain responses in stress-induced
visceral hyperalgesic rodents and that reported in IBS patients
by human brain imaging studies can provide important validation
for the stress-based animal models for human IBS. A better
understanding of such stress-induced alterations in brain nocicep-
tive responses is critical to delineating the underlying mechanisms.
There are few published reports on functional brain mapping
studies in stress-induced visceral hyperalgesia animal models. Stam
et al.  examined in rats the effect of foot shock on CRD-evoked
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expression of c-Fos, a gene marker of neuronal activity. In the
central amygdala, as well as prelimbic, infralimbic, insular, and
cingulate cortices, previously shocked rats showed reduced c-Fos
expression following CRD compared with no-shock controls.
Wouters et al.  used H215O microPET to map CRD-evoked
functional brain activation in maternal-separated rats before and 1
day after 1 hour of WAS. Following WAS, rats showed CRD-
evoked activation in new areas, including the somatosensory
cortex and hippocampus, and greater deactivation in the frontal
cortex. While these studies provided important evidence that
stress-induced visceral hyperalgesia is associated with alterations in
brain responses to CRD, due to the use of anesthesia in both
studies, it is difficult to compare the results directly with human
brain imaging findings .
We have recently adapted an autoradiographic cerebral blood
flow (CBF) perfusion mapping method to the rat CRD model .
In contrast to the requirement of sedation or restraint in fMRI and
microPET studies, the perfusion method allows functional brain
mapping in awake and nonrestrained rats. This is particularly
important when studying brain mechanisms related to stress and
affect related pain modulation, as brain networks involved in
nociception, stress and affect significantly overlap, and are subject
to influences by anesthetics [9,11]. Using this method, we have
shown that patterns of brain activation in response to acute CRD
and in expectation of CRD in the rat are in general agreement
with that reported in the human brain imaging literature
[10,12,13]. Here, we applied perfusion mapping to characterize
the effect of repeated WAS, which we have previously shown to
induce long-lasting visceral hyperalgesia , on CRD-evoked
functional brain activation.
Repeated, daily WAS (7–10 days) induces a chronic visceral
hyperalgesia in the rodent model. This hyperalgesia persists for
periods as long as one month after cessation of the stress [7,14],
something not seen after single day stress exposure . These
observations suggest that chronic/subchronic stress results in a
functional reorganization of the nociceptive response. It has been
proposed that an important brain mechanism underlying stress-
induced visceral hyperalgesia involves chronic stress induced
impairment of prefrontal-cortico-limbic pain modulation .
The prefrontal cortex (PFC) has been implicated in this
corticolimbic regulatory circuit. To examine this hypothesis, we
applied seed-region correlational analysis to assess changes in
CRD-evoked functional connectivity (FC) of the PFC in the rat
following stress. The prelimbic cortex (PrL) of the PFC was chosen
as the seed based on our previously reported findings of robust
activation of this region both during acute CRD  and in
expectation of CRD , and the observation that activity of PrL
and amygdala were anticorrelated (e.g. showed a negative
correlation) during expectation of CRD .
One major limitation of FC analysis is that correlation based
analysis does not address causality. Furthermore, due to multiple
comparisons, false positive findings are inevitable when a simple
significance threshold is applied. Constraining FC analysis with
structural connectivity (SC) information can reduce the number of
false positive reports, as well as provide directionality for the
otherwise non-directional FC networks. The concept of anatom-
ically constrained FC analysis has been implemented in effective
connectivity analysis of human and animal brain imaging data
[17,18], but has been largely limited to small-scale networks.
Recent studies have suggested a direct association between FC and
SC in the human brain by combining resting-state fMRI with
structural diffusion tensor imaging (DTI) measurement . With
the recent surge in efforts to construct connectome databases for
human and rodent brain, it has become possible to combine and
compare SC and FC at the whole brain level. Here, we
constrained FC analysis with complete SC information of the PrL
based on reports from tract tracing experiments manually collated
in the Brain Architecture Management System (BAMS, http://
brancusi.usc.edu/) [20,21]. The resulting structurally linked functional
connectivity (SL-) network keeps only functional connections over
direct structural projections. An SLFC network inherits direction-
ality information from the SC network and the sign of functional
interaction (positive or negative) from the FC network, constituting
a substantive step toward understanding the causality in brain
Materials and Methods
Adult male Wistar rats (2–2.5 month old) were purchased from
Harlan Sprague Dawley (Indianapolis, IN, USA) and were
individually housed in the vivarium on a 12-hour light/12-hour
dark cycle with free access to water and rodent chow. All
experiments were conducted under a protocol approved by the
Institutional Animal Care and Use Committee of the University of
Southern California, an institution accredited by the Association
for Assessment and Accreditation of Laboratory Animal Care,
International. All work was in accordance with the guidelines of
the Committee for Research and Ethical Issues of the Interna-
tional Association or the Study of Pain. The numbers of animals in
each group were as follows: Sham stress/0-mmHg CRD, n=9;
Sham stress/60-mmHg CRD, n=10; WAS/0-mmHg CRD,
n=10; WAS/60-mmHg CRD, n=10.
One week before the start of WAS treatment (Fig. 1), animals
were anesthetized (isoflurane 2% in 70% oxygen and 30% nitrous
oxide). The right external jugular vein was cannulated with a 5
French silastic catheter (Dow Corning Corp., Midland, MI, USA),
advanced into the superior vena cava. The port at the distal end of
the catheter was tunneled subcutaneously and externalized
dorsally in the region rostral to the scapula. Subsequently, a
telemetry transmitter was implanted to measure abdominal EMG.
Such implants can be turned on and off with an external magnet
and send a radiofrequency signal of EMG acitvity to a receiver
platform placed underneath the rat’s cage. The body of the
transmitter (TA11-CTA-F40, Data Sciences Intl., St. Paul, MN,
USA) was implanted subcutaneously on the dorsum of the animal
caudal to the scapula. A skin incision was made on the abdomen
and electrodes of the transmitter were tunneled subutaneously to
the abdominal incision. Tips of the eletrodes were bared, placed in
parallel (0.5 cm apart), and stitched into the left external oblique
musculature, just superior to the inguinal ligament. The receiver
platform was linked via a data exchange matrix to a PC computer.
All animals were allowed to recover for seven days. The catheter
was flushed every other day postoperatively to ensure patency
(0.3 mL of sterile 0.9% saline, followed by 0.1 mL taurolidine-
citrate catheter lock solution, Access Technologies, Skokie, IL,
Assessment and quantification of the VMR to CRD
VMR to CRD was assessed as described before . Briefly,
under light isoflurance anesthesia (1.5% isoflurance 6 3 min), a
flexible latex balloon (length=6 cm) was inserted intra-anally such
that its caudal end was 1 cm proximal to the anus. The silicon
tubing connecting the balloon and the barostat (Distender Series
II, G&J Eletronics Inc., Toronto, Canada) was fixed to the base of
the tail with adhesive tape and covered by a stainless steel spring
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for protection against animal biting. Animals were allowed to
recover for 30 min in the experiment cage, the floor of which was
covered with bedding from the animal’s home cage. The CRD
procedure consisted of two series of phasic distension to constant
pressure of 10, 20, 40, and 60 mmHg with 20-s duration and 4-
min interstimulus intervals. The visceromotor response was
quantified by measuring EMG activity in the external oblique
musculature. EMG signals were recorded telemetrically at a
sampling rate of 1 kHz, digitized and stored on a PC computer
with the Dataquest ART 3.0 software (Data Sciences Intl., St.
Paul, MN, USA). EMG waveforms were lowcut filtered at 20 Hz
to eliminate movement interference, and then full-wave rectified.
Area under the curve (AUC) was calculated for the 20-s distension
period normalized by the 20-s before-distension baseline. The
visceromotor response was assessed on the day before (day 0) and
the day after (day 11) WAS or sham treatment (Fig. 1). EMG AUC
was further normalized to response to 60-mmHg CRD on day 0
and expressed as a percentage of this baseline value. EMG signals
were not recorded in 1 sham-treated rat due to equipment failure
and not included in the visceromotor response analysis.
Water avoidance stress protocol
The protocol was as described before . Briefly, the test
apparatus consisted of a plexiglas tank (45 cm length 6 25 cm
width620 cm height) with a block (8 cm length68 cm width6
10 cm height) affixed to the center of the floor of the tank. The
tank was filled with fresh room temperature water (25uC) to within
1 cm of the the top of the block. The animals were placed on the
block for a period of 1 hour daily for 10 consecutive days. Sham
treatment consisted of placing the rats in the dry tank for 1 hour
On day 11, animals were allowed to rest for 15 min following
the last distension of the CRD series. A piece of silastic tubing was
filled with radiotracer [14C]-iodoantipyrine (125 mCi/kg in
300 mL of 0.9% saline, American Radiolabelled Chemicals, St.
Louis, MO, USA). The radiotracer-filled tubing was then
connected to the animal’s cannula on one end, and to a syringe
filled with euthanasia agent (pentobabital 50 mg/mL, 3 M
potassium chloride) on the other. The animal was allowed to rest
for another 5 min before receiving one episode of 60-mmHg.
Thirty-five seconds after the onset of the distension, radiotracer
was infused at 2.25 mL/min by a motorized pump, followed
immediately by 0.7 mL of euthanasia solution, which resulted in
cardiac arrest within ,10 s, a precipitous fall of arterial blood
pressure, termination of brain perfusion, and death . This 10-s
time window provided the temporal resolution during which the
distribution of regional CBF (rCBF)-related tissue radioactivity was
mapped. Half of each treatment group received no distension
(0 mmHg) during CBF mapping and served as controls.
Brain slicing and autoradiography
Brains were rapidly removed, flash frozen in methylbutane on
dry ice (,255uC) and embedded in optimal cutting temperature
compound (Sakura Finetek Inc., Torrance, CA,USA). Brains were
subsequently sectioned on a cryostat (HM550 series, Microm
International GmbH, Walldorf, Germany) at 218uC into 20-mm
thick coronal slices, with an inter-slice spacing of 300 mm. Slices
were heat-dried on glass slides and exposed to Kodak Biomax MR
films (Eastman Kodak, Rochester, NY, USA) for 3 days at room
temperature. Images of brain sections were then digitized on an 8-
bit gray scale using a voltage stabilized light box (Northern Lights
Illuminator, Interfocus Imaging Ltd., Cambridge, UK) and a
Retiga 4000R charge-coupled device monochrome camera
(Qimaging, Surrey, Canada). Autoradiographic CBF mapping in
rodents has a spatial resolution of 100-mm, and hence, can provide
information on sub-regional activation, such as in individual
Functional brain mapping data analysis
rCBF-related tissue radioactivity was quantified by autoradiog-
raphy and analyzed on a whole-brain basis using statistical
parametric mapping (SPM, version 5, Wellcome Centre for
Neuroimaging, University College London, London, UK). Re-
cently, we and others have developed and validated an adaptation
of SPM for use in rodent brain autoradiograph . In
preparation for the SPM analysis, a 3-dimensional reconstruction
of each animal’s brain was conducted using 57 serial coronal
sections (starting at , bregma +4.5 mm) with a voxel size of
40 mm 6300 mm 640 mm. Adjacent sections were aligned
manually in Photoshop (version 9.0, Adobe Systems Inc., San
Jose, CA, USA) and using TurboReg, an automated pixel-based
registration algorithm implemented in ImageJ (version 1.35,
http://rsbweb.nih.gov/ij/). This algorithm registered each section
sequentially to the previous section using a nonwarping geometric
model that included rotations and translations (rigid-body
transformation) and nearest-neighbor interpolation. One ‘‘artifact
free’’ brain was selected as reference. All brains were spatially
normalized to the reference brain in SPM. Spatial normalization
consisted of applying a 12-parameter affine transformation
followed by a nonlinear spatial normalization using 3D discrete
cosine transforms. All normalized brains were then averaged to
create a final rat brain template. Each original 3D-reconstructed
Figure 1. Experimental design. Visceral motor response (VMR) to colorectal distension (CRD) was measured before (day 0, baseline) and after (day
11) water avoidance stress (WAS) or sham treatment. Each time, CRDs of 10-, 20-, 40-, and 60-mmHg (duration=20 s, interstimulus interval=4 min)
were delivered twice for each pressure level. On day 11, following VMR measurement, cerebral blood flow (CBF) mapping was performed while the
animal was receiving a 60-mmHg CRD or no distension (0-mmHg control).
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brain was then spatially normalized to the template. Normalized
brains were smoothed with a Gaussian kernel (FWHM =36voxel
dimension in the coronal plane). A nonbiased, voxel-by-voxel
analysis of regional brain activation was performed. Voxels for
each brain failing to reach a specified threshold in optical density
(70% of the mean voxel value) were masked out to eliminate the
background and ventricular spaces without masking gray or white
matter. We implemented a Student’s t-test at each voxel. For each
treatment (WAS or sham), a t-contrast was calculated comparing
the 60-mmHg CRD to the 0-mmHg control subgroup. Threshold
for significance was set at P,0.05 at the voxel level and an extent
threshold of 100 contiguous voxels. This combination reflected a
balanced approach to control both type I and type II errors. The
minimum cluster criterion was applied to avoid basing our results
on significance at a single or small number of suprathreshold
voxels. Brain regions were identified according to a rat brain atlas
. In addition, we ran a factorial analysis to identify rCBF
changes reflecting WAS x CRD interaction. Threshold for
significance was set at P,0.05 at the voxel level and an extent
threshold of 100 contiguous voxels. Data interpretation was
focused on gray matter.
Structurally linked functional connectivity of the
To test the hypothesis that WAS may result in altered
corticolimbic modulation during noxious visceral stimulation, we
applied seed-region correlation analysis to assess differences in the
CRD-evoked FC of the PrL of the PFC between treatment groups.
The seed region of interest (ROI) was hand drawn in MRIcro
(version 1.40, http://cnl.web.arizona.edu/mricro.htm) for the
right hemisphere over the template brain according the rat brain
atlas  and intersected with clusters defining regional functional
activation in the PrL area based on the SPM analysis. The result
was one unilateral seed ROI representing the PrL region for each
treatment type showing CRD-evoked functional activation. Mean
optical density of the seed ROI was extracted for each animal
using the MarsBaR toolbox for SPM (version 0.42, http://
marsbar.sourceforge.net/). Correlation analysis was performed in
SPM for each 60-mmHg CRD subgroup using the seed values as a
covariate. Threshold for significance was set at P,0.05 at the
voxel level and an extent threshold of 100 contiguous voxels.
Regions showing significant correlations (positive or negative) in
rCBF with the PrL are considered functionally connected with the
Anatomical (structural) connectivity of the PrL in the rat was
extracted from BAMS. BAMS includes a large set of rat structural
connections collated from the literature, or directly inserted by
neuroanatomists [20,21]. The collation methodology of neuroan-
atomical data employed in BAMS is fully described in Bota et al.
. Briefly, the connectivity patterns of gray matter regions are
collated as reported in the published references recorded in
BAMS, or as inferred by collators. The connectivity information is
collated from the textual descriptions, and from the maps
associated with references. Each connectivity report is associated
with a qualitative strength, and with supporting textual annota-
tions. Connectivity reports inferred by collators are associated with
textual annotations that describe the annotation process. Overall,
the rat PrL is associated with about 1600 connectivity reports in
BAMS, with 106 gray matter regions that receive inputs from it
and 177 regions that send outputs to it. The inputs of the rat PrL
are associated with 37 references, and its outputs 62 references.
The set of qualitative strengths of neuroanatomical connections
collated in BAMS includes 10 values. Here, this set was encoded
on a linear scale from 1 to 7, with 1 being very strong and 7 being
very weak. Regions with ‘very weak’ connection to or from PrL, as
well as connectivity reports with the strength ‘fibers of passage’,
were removed for simplification. Only ipsilateral connections were
included due to incomplete understanding of cross-hemispheric
Results of the SPM seed correlation analysis were only analyzed
for those regions structurally connected with the PrL. This is
equivalent to taking an intersection of the structural and functional
connectivity network of the PrL, resulting in an SLFC network
Water avoidance stress induced visceral hyperalgesia
Ten days of WAS induced significant increases in visceromotor
response to CRD on day 11 as compared to day 0 baseline
(Fig. 3A, n=20, main effect of ‘Day’ F(1, 19)=18.19, P,0.001,
two-way repeated measures ANOVA with ‘Day’ and ‘CRD’ as the
within-subjects factors). In comparison, the visceromotor response
was only moderately increased in the sham-treated animals
primarily due to an increase in response to 60-mmHg CRD
(Fig. 3B, n=18, main effect of ‘Day’, F(1,17)=4.67, P=0.045,
two-way repeated measures ANOVA). Compared to the sham
condition, stressed rats showed significantly greater increases in the
visceromotor response on day 11 (Fig. 3C, main effect of
‘Treatment’, F(1,36)=4.47, P=0.042, mixed model ANOVA
with CRD as the within-subjects factor and ‘Treatment’ the
Comparison of CRD-evoked functional brain activation in
WAS- and sham-treated rats
CRD-evoked functional brain activation was assessed for each
treatment type by contrasting the subgroup receiving 60-mmHg
CRD, and the one receiving no CRD (0-mmHg control) of the
same treatment type (Fig. 4). Sham-treated rats showed CRD-
evoked functional activation (increase in rCBF) in the PrL,
primary motor, frontal area 3, primary and secondary somato-
sensory, anterior and posterior insular, and temporal association
cortices, as well as in the dorsal caudate putamen, amygdala
(lateral amygdaloid n., central amygdaloid n.), and superior olive.
Sham rats also showed CRD-evoked deactivation (decrease in
rCBF) in the retrosplenial, entorhinal, piriform, and secondary
visual cortices, hippocampus, subiculum, thalamus (habenular n.,
mediodorsal n., posterior n. group, parafascicular n., ventral
posteromedial n., ventral posterolateral n.), cerebellum (cerebellar
lobule, cerebellar hemisphere), and areas of the brainstem
(superior colliculus, dorsomedial periaqueductal gray, red n.;
caudal linear n. of the raphe).
In contrast, WAS-treated rats showed CRD-evoked brain
activation in a drastically different pattern. Major differences
were noted in the magnitude and extent of regional activation.
Greater activation in the WAS rats was noted in the anterior and
posterior insula, and in the amygdala (central n., lateral n.,
basolateral n., basomedial n., medial n., bed nucleus of the stria
terminalis intraamygdaloid division). The WAS rats also showed
significant activation in the hypothalamus (medial preoptic area,
medial preoptic n., lateral preoptic area, ventromedial hypotha-
lamic n.), nucleus accumbens, and bed nucleus of the stria
terminalis (medial division), which was not seen in the sham rats.
Whereas sham rats showed activation in the anterior dorsal aspect
of caudate putamen, WAS rats showed activation in the posterior
and anterior ventral aspects of caudate putamen. Importantly,
reduced activation in the PrL was noted in the WAS rats compared
to sham. Deactivation in the cingulate cortex area 1 and 2 (Cg1,
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Cg2) was noted in the WAS but not the sham rats. WAS rats
showed a similar pattern of deactivation as that seen in the sham
condition, though to a lesser extent. Factorial analysis confirmed
significant WAS x CRD interaction in the PrL, posterior insula,
retrosplenial cortex, secondary visual cortex, anterior striatum,
accumbens nucleus, bed nucleus of the stria terminalis medial
division, medial preoptic nucleus, hippocampus, and central
nucleus of the amygdala.
Differences in the structurally linked functional
connectivity of the prelimbic cortex
Seed correlation-based FC analysis was constrained by SC
information of the PrL to generate SLFC of the PrL cortex (Fig. 5).
In sham rats, PrL/PFC SLFC during noxious visceral stimulation
was characterized by negative FC with the amygdala, whose nuclei
provide either afferent input (basomedial n., posterior part; medial
n., posteroventral part; amygdalohippocampal area, posteromedial
part; posterolateral cortical n.; amygdalopiriform transition area)
(PrL[-]rAmygdala) or bidirectional connections of the lateral
amygaloid nucleus to the PrL (PrL[-]rRLa). In addition, sham
treated rats demonstrated the following SLFC of PrL cortex
during noxious visceral stimulation:
1. Positive FC with a cluster of cortical regions over largely
bidirectional structural connections, including orbital (ventral),
secondary motor, cingulate, retrosplenial (dysgranular, granular),
anterior insular, and ectorhinal cortices (PrL[+]rCtx).
2. Positive FC over projections to the striatum (PrL[+]Striatum)
or from lateral orbital cortex (PrL[+]rLO).
3. Negative FC over afferent projections from the hippocampal
formation (CA1, dorsolateral entorhinal cortex) (PrL[-]rHippo-
4. Negative FC with the hypothalamus over efferent projections
to medial preoptic nucleus, the anterior hypothalamic area
(anterior part), and subventricular zone (PrL[-]Hypothalamus),
and afferent projections from anterior hypothalamic (central part)
and supramammillary nucleus (PrL[-]rHypothalamus).
5. Positive FC with the thalamus over efferent projections to the
ventral anterior, ventral lateral, anterior ventral, and reticular
nuclei, and medial and lateral habenular nuclei (PrL[+]Thalamus),
as well as bidirectional connections with medial dorsal and central
6. Negative FC over efferent projection to the dorsomedial and
dorsolateral periaqueductal gray in the midbrain (PrL[-]PAG).
WAS-induced changes in SLFC were most noticeable in
relation to a cluster of cortical regions and to the amygdala.
Whereas PrL in the sham animals showed significant, positive FC
with a cortical cluster consisting of secondary motor, dorsal
posterior cingulate (pCg1), ventral cingulate (Cg2), retrosplenial
(dysgranular, granular) cortices, this connectivity turned negative in
the stressed rats. In the sham rats, negative FC was noted between
the PrL and the amygdala whose nuclei provide afferent input
(basomedial n., posterior part; medial n., posterior ventral part;
amygdalohippocampal area, posterior medial part; posterolateral
cortical n., amygdalopiriform transition area) or bidirectional
connections (lateral n.) to the PrL. In contrast, in the stressed rats,
this negative functional connectivity was absent or turned positive
(lateral n., anterior cortical n., posterolateral cortical n.).
In addition, WAS-induced alterations in SLFC of the PrL/PFC
included the following:
1. Changes from positive to negative FC with the thalamus
(ventral anterior, ventral lateral, anterior ventral, mediodorsal,
centromedial, medial and lateral habenular nuclei) (PrL[-]R/r/
r RThalamus), subthalamic nucleus, and zona incerta.
2. Changes from negative to positive FC with the hypothalamus
(medial preoptic n.; anterior hypothalamic area, anterior and
central parts; subventricular zone) (PrL[+]R/rHypothalamus).
3. New (no FC in sham rats) positive FC with infralimbic,
posterior insular, and piriform cortices (PrL[+]R/r RCtx),
hypothalamus (medial mammillary n., ventromedial n., lateral
preoptic area)(PrL[+]r/r RHypothalamus), dorsal subiculum,
nucleus accumbens, claustrum, dorsal endopiriform nucleus,
ventral pallidum, and nucleus of the diagonal band.
4. New, negative FC with thalamus (posterior, reuinens,
rhomboid, centrolateral nuclei)(PrL[-]R/r/r RThalamus),
and brainstem areas (substantia nigra reticulata and compacta,
isthmic and mesencephalic reticular formation, lateral and
ventrolateral periaqueductal gray, dorsal raphe n., dorsal and
laterodorsal tegmental n., pontine n., central gray, subcoeruleus n.
Neurobiological sequelae of chronic stress have been the subject
of extensive research. For example, it has been well established
that chronic stress in rodents can induce both visceral [6,7], as well
as somatic hypersensitivity [25–27]. Few studies have examined
stress-induced changes in functional activation of brain nociceptive
Figure 2. Structurally linked functional connectivity. Structural (SC) and functional connectivity (FC) networks are combined to create a
structurally linked functional connectivity (SLFC) network such that final network contains all regions that the SC and FC networks have in common.
Note the SLFC network inherits directionality information (denoted with arrows) from the SC network and the sign of functional interaction (positive/
red or negative/blue correlation) from the FC network.
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circuits, which can bring new insights into the underlying
mechanisms. Using autoradiographic, perfusion-based functional
brain mapping, we studied regional and network-level neural
correlates of WAS-induced visceral hyperalgesia in awake,
nonrestrained rats. Our main findings were that stressed rats
showed greater CRD-evoked activation than sham-treated rats in
the amygdala, insular cortex, and hypothalamus, but reduced
activation in the prelimbic area of PFC (PrL/PFC). Profound
differences between the stressed and sham rats were noted in the
structurally linked functional connectivity of the PrL/PFC with
cortical, limbic and brainstem areas. In particular, while negative
functional connections were noted between the PrL/PFC and
amygdala in the sham condition, these were absent in the stressed
rats. These findings, in association with an exaggerated viscero-
motor response to CRD in the stressed animals, provide direct
evidence that stress amplifies sensory and affective responses to
nociceptive stimuli, with impairment of PFC-mediated pain
modulation as a candidate central mechanism for stress-induced
visceral hyperalgesia. We focus the discussion on stress-induced
Stress-induced changes in activation of amygdala and
PFC evoked by noxious CRD
In the current study, stressed compared to sham treated rats
showed greater CRD-evoked activation broadly across the amyg-
dala, with the central amygdaloid nucleus showing the most
significant differences. These findings are consistent with an
extensive literature on stress induced sensitization of the amygdala.
For example, the amygdala has been implicated in chronic stress-
induced sensitization of anxiety- and fear-related responses to an
pain can increase excitability and responsiveness of subsets of
neurons of the central n., a primary output of the amygdala to brain
regions involved in autonomic regulation [31,32]. Chronic restraint
stress has also been shown to increase excitability of pyramidal
neuronsinthe lateral amygdaloidnucleus , aswell as toincrease
the number of dendritic spines in the amygdala . Such stress-
induced neural plasticity may mediate enhanced responses to
noxious visceral stimulus. Further, chronic local application of the
stress hormone corticosterone to the amygdala leads to visceral
hyperalgesia, suggesting the amygdala as a site where stress
hormone can modulate visceral nociception .
Activation of the PrL during acute visceral pain in nonstressed
rats has previously been reported [10,36]. In the current study,
stressed compared to sham treated rats showed a striking
hypoactivation in the PrL in response to CRD. The PrL is a part
of the medial PFC in rodents. This brain region is thought to have
features of dorsolateral PFC and anterior cingulate cortex of
primates [37–41]. The PFC is a brain area vulnerable to chronic
stress. For example, chronic restraint stress [42–44] and corticoste-
roneadministration  have both been shown to induce retraction
of dendrites and loss of synaptic connections in the PFC, in line with
reduced functional activation as observed in the current study.
Chronic psychosocial stress in human subjects impairs PFC-
dependent attentional control and disrupts FC within a frontopa-
rietal network, including the PFC . In contrast to results of the
current study, Gibney et al.  reported exaggerated c-fos
expression in the PrL in visceral hypersensitive Wistar-Kyoto rats
compared to control Sprague-Dawley rats, albeit in the absence of a
chronic stressor. These differences in PrL changes associated with
visceral hyperalgesia may be attributable to different animal models
of visceral hyperalgesia used, different experimental protocols
(anesthetized vs. awake rats, no stress vs. WAS), and different
modalities of measurement (c-fos vs. cerebral blood flow).
Structurally linked functional connectivity analysis and
Functional interaction between brain regions can be analyzed
through FC analysis of brain imaging data. Here, we focused FC
Figure 3. Effect of repeated water avoidance stress (WAS) on
visceromotor response (VMR) to colorectal distension (CRD).
(A) VMR measured as electromyographic (EMG) area under the curve
(AUC) and expressed as % of control (baseline VMR to 60-mmHg CRD)
was significantly increased following WAS on day 11 compared to day 0
baseline (n=20, F(1, 19)=18.19, P,0.001, two-way repeated measures
ANOVA). (B) Repeated sham procedure caused a moderate increase in
VMR, primarily to 60-mmHg CRD (n=18, F(1,17)=4.67, P=0.045, two-
way repeated measures ANOVA). (C) Compared to sham-treated rats,
stressed rats showed significantly greater increases in VMR after
treatment (F(1,36)=4.47, P=0.042, mixed model ANOVA). Data are
expressed as means 6 SEM.
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Figure 4. Comparison of colorectal distension (CRD)-induced functional brain activation in stressed and sham rats. Statistically
significant increases (red scale) and decreases (blue scale) in regional cerebral blood flow (rCBF) contrasting the subgroup receiving 60-mmHg CRD
and the one receiving no distension (0-mmHg) are shown for the sham (left column) and water avoidance stress (WAS)-treated rats (middle column)
over representative coronal slices of the brain template with anterior-posterior coordinates given relative to the bregma (n=9 or 10/subgroup).
Results of factorial analysis are also colorcoded showing regions with significant WAS x CRD interaction (right column). Abbreviations: aINS (anterior
insular cortex), BL (basolateral amygdaloid n.), BM (basomedial amygdaloid n.), Cb (cerebellar lobule), Ce (central amygdaloid n.), Cg1/Cg2 (cingulate
ctx. area 1/area 2), CPu (caudate putamen), fi (fimbria), Fr3 (frontal ctx. area 3), GP (globus pallidus), Hb (habenular n.), HPC (hippocampus), La (lateral
amygdaloid n.), LD (laterodorsal thalamic n.), LPO (lateral preoptic area), M1 (primary motor ctx.), MD (mediodorsal thalamic n.), Me (medial
amygdaloid n.), MPA (medial preoptic area), MPO (medial preoptic n., medial part), NAcc (n. accumbens), RS (retrosplenial ctx.), p1PAG(p1
periaqueductal gray), PF (parafascicular thalamic n.), pINS (posterior insular ctx.), PLH (peduncular part of lateral hypothalamus), Pir (piriform ctx.), Po
(posterior thalamic n.), PrL (prelimbic ctx.), S1 (primary somatosensory ctx.), S2 (secondary somatosensory ctx.), SFi (septofimbrial n.), SO (superior
olive), STIA (bed nucleus of the stria terminalis, intraamygdaloid division), STM (bed nucleus of the stria terminalis, medial division), V2 (secondary
visual ctx.), VMH (ventromedial hypothalamic n.), VPL/VPM (ventral posteromedial/posterolateral thalamic n.). The left side of each coronal image
represents the left side of the rodent brain.
Figure 5. Stress-induced changes in the structurally linked functional connectivity (SLFC) of the prelimbic cortex (PrL) during
colorectal distension (CRD). The SLFC network contains both directionality information of the underlying structural connections (denoted with
arrows) and the sign of functional interaction (positive/red or negative/blue statistically significant correlation).
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analysis on the PrL and constrained FC results with SC
information of the PrL. The integration of whole brain-level FC
and SC information reflects a recent trend in human brain
imaging field to take advantage of rapid advances in the Human
Connectome Project . With the Mouse Connectome Project
(http://www.mouseconnectome.org/), BAMS , and other
rodent structural connectome projects well underway, a similar
approach has become feasible for animal research. Based on the
complete information of the SC of the PrL collated in BAMS, we
were able to thoroughly investigate stress-induced changes in
SLFC of the PrL. The first advantage of SLFC over regular FC is
the simplification of the connectivity network by the removal of
FC connections not substantiated by structural projections. FC
connections without SC may be false positive findings, or indirect
FC connections. The second advantage of SLFC is that it
combines SC directionality with the sign of FC. The resulting
SLFC can help generate new hypotheses about the causality in the
brain circuits. While both SC and FC data were binarized for
simplification in this study, the strength of each SC and FC
connection can also be integrated into SLFC analysis. The current
study represents one of the first rodent studies to present a detailed
integration of structural information into FC analyses.
Structurally linked functional connectivity of the PFC-
amygdala circuit and the effects of stress
In rodents, the PrL is reciprocally connected with the lateral and
basolateral nuclei of the amgydala, and sends efferent projections
to the central nucleus and anterior part of basomedial nucleus, and
receives afferent projections from medial, posterior, and cortical
nuclei and posterior part of the basomedial nucleus of the
amygdala. In sham rats, PrL/PFC SLFC during CRD was
characterized in our study by negative FC with the amygdala
nuclei over mostly afferent projections from the amygdala (PrL[-]
rAmygdala), except bidirectional connection with the lateral
nucleus (PrL[-]r RLa). These SLFC results suggest that in sham
animals, PrL/PFC inhibits the amygdala through its projection to
the lateral amygdalar nucleus, whereas the amygdala may in
return inhibit PrL/PFC activity though its lateral, basomedial,
medial, and cortical nuclei.
Bidirectional PFC-amygdala interactions have been extensively
studied in humans and in laboratory animals, and changes in these
interactions have been implicated in the regulation of negative
emotion, mood and pain [48–56]. The inhibitory interaction
between the PrL/PFC and amygdala is likely bidirectional. On the
one hand, it has been well documented that PFC regulates
amygdala-mediated responses to aversive stimulus . On the
other hand, ample evidence exists for amygdala-mediated
modulation of PFC , particularly under aversive condition
[59,60]. We have recently applied correlation-based FC and graph
theoretical analysis to characterize brain activation at the network
level in expectation of visceral pain . Animals previously
trained with a step-down passive avoidance paradigm using
noxious CRD as the aversive stimulus demonstrated negative FC
between the amygdala and areas of the PFC (including PrL and
dorsal and ventral cingulate cortex, Cg1, Cg2) when reexposed to
the conditioned context—a finding that was interpreted as
evidence for inhibitory corticolimbic modulation.
In the current study, stressed rats compared to sham rats
showed substantial changes in the SLFC of PrL/PFC. The
negative FC with the amygdala seen in the sham disappeared, and
in its place appeared a few positive connections (with the lateral
and corticoamygdaloid nuclei). Our results are consistent with
those of Correll et al.  who using in-vivo extracellular neural
recordings reported that chronic cold stress enhanced the acute
footshock-induced response of the central amygdaloid nucleus,
and that chronic stress weakened prefrontal inhibitory regulation
of this response. Our data suggest similar chronic stress-induced
disinhibition of the amygdala by the PFC.
Stress-induced changes in activation of other brain
regions during noxious CRD
Previous imaging studies in animal and human subjects
[reviewed by 61] have also implicated the anterior cingulate
cortex in visceral pain processing and regulation, with the majority
of visceral distension studies reporting enhanced activation of mid-
cingulate subregions. Here, the observed deactivation in the
cingulate area in the stressed rats and no activation in the sham
rats was unexpected. Previously, we have reported cingulate
activation in male, naı ¨ve rats (with no prior experience of CRD)
receiving acute, noxious CRD . In the current study, rats
received a series of CRD eleven days prior, as well as 20 minutes
prior to the CBF perfusion procedure. This difference in protocol
may have contributed to this different pattern of cingulate
activation. For example, CBF level in the cingulate areas in the
0-mmHg control rats may have been elevated from baseline due to
prior exposure to repeated, noxious CRD. The exact cause and
implication of cingulate deactivation in the stressed rats remains to
be further investigated.
Whereas both sham and stressed rats showed CRD-evoked
activation in the anterior and posterior insula, activation in the
stressed rats was much greater in amplitude and extent. We have
reported CRD-evoked activation of the anterior and posterior
insula in normal (nonstressed), male rats , as well as activation
of anterior insula in expectation of CRD . Activation of the
insula in response to acute rectal distension is the most consistently
reported finding in human brain imaging studies , and
alterations of insular functional activation have been reported in
IBS patients. The posterior insula in its role as primary
interoceptive cortex mediates sensory processing of pain and is
part of a sensorimotor network, whereas the anterior insula is
involved in a salience network closely linked to emotional arousal
. In the rat, anterior insular cortex may modulate pain
processing through its projection to the amygdala and periaque-
ductal gray in the rat [63,64].
Stressed rats also showed significant CRD-evoked activation in
the hypothalamus, bed nucleus of stria terminalis, accumbens
nucleus and ventral striatum, but deactivation in the cingulate
cortex, which were all absent in the sham rats. The hypothalamus
is believed to be modulated by PFC, and in turn regulates activity
of descending inhibitory and facilitatory pathways through
periaqueductal gray and pontomedullary nuclei [16,65–67]. The
ventromedial hypothalamus has also been implicated in the
generation of the affective dimension of pain . Increased
hypothalamic activation to rectal distension has been observed in
IBS patients as compared to healthy controls . The bed
nucleus of the stria terminalis, considered extended amygdala,
receives heavy projection from the basolateral amygdala and
projects to the hypothalamus and brainstem areas, and partici-
pates in anxiety and stress responses . Chronic immobilization
stress has been shown to induce dendritic remodeling of neurons of
the bed nucleus of the stria terminalis . Collectively,
augmented activation of the hypothalamus, bed nucleus of the
stria terminalis, and amygdala may underlie increased pain
responses in the affective dimension, as well as increased
descending facilitatory pain modulation.
The nucleus accumbens and ventral striatum participate in
reward responses and positive emotional states. The accumbens
nucleus and ventral striatum are also considered part of the
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emotional motor system , serving perhaps as a gateway
between the limbic system and the motor system . Human
brain imaging studies have reported activation of these structures
by noxious thermal stimuli , as well as in expectation of
aversive somatic stimuli . Interestingly, in a treatment study of
IBS patients, Berman et al.  showed that Alosetron, a
serotonin receptor antagonist, elicited decreases in rCBF in the
amygdala, ventral striatum, and dorsal pons, in significant
correlation with symptom (abdominal pain) reduction. Their
findings suggest a possible role of the ventral striatum in central
pain sensitization in IBS.
Structurally linked functional connectivity of the PFC to
other brain regions and the effects of chronic stress
During visceral noxious stimulation, sham treated rats demon-
strated a positive SLFC of the PrL cortex with the thalamus,
anterior insula and other cortical areas. This is consistent with an
integrative role of PrL/PFC in the processing of visceral input. In
addition, the negative connectivity of PrL/PFC with limbic areas
(amygdala, hypothalamus) and periaqueductal gray in the
midbrain is consistent with PFC-limbic-periaqueductal gray
inhibitory modulation [77,78]. Water avoidance stress induced
substantial changes in the SLFC of the PrL with the thalamus,
hypothalamus, and brainstem. Connections with the thalamus
which demonstrated significant positive FC in the sham, appeared
significantly negative in the WAS rats, whereas FC showed the
reverse pattern with the hypothalamus (excluding the subthalamic
n., and zona incerta). In the stressed rats, but not the sham, PrL
demonstrated significant negative FC with areas of the brainstem,
including the substantia nigra, reticular formation, periaqueductal
gray, dorsal raphe nucleus, tegmental nucleus, pontine nucleus,
central gray, and subcoeruleus nucleus alpha. This complex
pattern of alterations in SLFC suggests profound changes in how
the PrL/PFC interacts with other cortical and subcortical areas
during visceral pain processing as a result of chronic stress.
Translational implications of rodent brain imaging
findings in the study of pain
Human functional brain imaging has been extensively applied
to investigate central processing and modulation of pain, including
visceral pain, and to characterize alterations in central pain
responses in functional pain disorders, including IBS [16,61]. A
recent quantitative meta-analysis of imaging data from 19
published studies reported that compared to healthy controls,
IBS patients have greater engagement of regions associated with
emotional arousal (perigenual anterior cingulate cortex and
amygdala) and homeostatic afferent processing (anterior mid-
cingulate cortex, medial thalamic regions, mid-insula, areas of the
midbrain). In contrast, controls show greater reliable activation in
cortical regions involved in modulation of pain and emotion
(lateral and medial prefrontal cortex, Brodmann Area 49), which is
largely absent in IBS patients . In striking agreement with
these human brain imaging findings, the current study showed
that stressed rats, compared to sham, had much greater activation
in the insula and amygdala, but reduced activation in PrL, a PFC
region with features of the dorsolateral PFC [37,38] and the
anterior cingulate cortex [40,41] of primates. This adds to our
previous reports of remarkable homology in functional brain
activation between the rat and human in response to acute noxious
CRD in both males and females, and in expectation of CRD in
In conclusion, chronic stress induced marked alterations in
CRD-evoked functional brain activation characterized by hypoacti-
vation of the prelimbic area of PFC and hyperactivation of the insular
cortex, amygdala, and hypothalamus. Structurally linked func-
tional connectivity analysis further revealed stress-induced disrup-
tion of PFC-limbic (amygdala and hypothalamus) inhibitory
interaction during CRD. Dysfunction of the PFC, including
impairment of the PFC-limbic regulatory circuit, is strongly
implicated as a central mechanism contributing to stress-induced
visceral hyperalgesia. The findings of hypoactivation in PFC and
hyperactivation in limbic/paralimbic structures in the homeostatic
afferent processing network and emotional arousal network are in
general agreement with human brain imaging findings on altered
brain responses to noxious visceral stimulus in IBS patients. These
findings provide further support for the face and construct validity
of the WAS animal model for human IBS. Functional brain
mapping in awake, nonrestrained rodents can be a powerful tool
for bridging animal and human visceral pain research, for gaining
new mechanistic insights, and for preclinical drug evaluation with
presumably greater predictive power . Future work will need
to evaluate if activation/deactivation patterns reported in our
study would be different and allow for animal-to-human transla-
tion if another painful stressor were used (e.g. electric shock;
Conceived and designed the experiments: ZW DPH EAM SB. Performed
the experiments: ZW MAO RDP DPH. Analyzed the data: ZW MB DPH.
Contributed reagents/materials/analysis tools: ZW MB DPH. Wrote the
paper: ZW DPH EAM MB.
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