Chapter 75
Enteric Neurobiology of Stress
Sumei Liu and Jackie D. Wood
75.1  STRESS: CASE EXAMPLES
A routine history revealed that Ms. A, a 44-yr-old attor-
ney, had experienced up to six episodes of cramping lower
abdominal pain associated with watery diarrhea per month
over a period of 15 years. These symptoms were worsened
by stress associated with anticipation of a courtroom trial
or the stress of presentation of the defense of a client dur-
ing the actual trial.
Barbara, a 22-year-old second year medical student,
walked to the front of the lecture auditorium with a ques-
tion for the professor at the end of the final lecture in a
series on gastrointestinal physiology. The question: Can
you explain to me why I routinely experienced lower
abdominal distress and diarrhea as I prepared for and
anticipated the examinations in the medicine course mod-
ules throughout the year?
A male guinea pig was held in a supine position by
placing a tie around each ankle and gently extending and
tying each of the four extremities to metal hooks at the
corners of a rectangular flat platform. Thus restrained, the
guinea pig was placed in a 4°C cold room for 2 hours. A
second male guinea pig of the same weight and age and
from the same holding rack in the vivarium was caged at
room temperature and allowed to feed and drink H2O ad
libitum during the 2 hour period. Figure 75.1 shows fecal
output for both guinea pigs during the period of cold-
restraint stress in the 2 hour period.
The three examples illustrate an expanding body of
converging clinical and experimental evidence that impli-
cates stress as a factor in disordered gastrointestinal func-
tion in both humans and animals. Stressful life events in
humans can be associated with the onset, symptom exac-
erbation, and reactivation of gastrointestinal disorders,
which include irritable bowel syndrome (IBS), ulcerative
colitis, Crohn’s disease, gastroesophageal reflux disease,
and peptic ulcer disease.1–6 Exposing rats or guinea pigs
to stress inhibits gastric contractions and gastric empty-
ing while stimulating contractile and secretory activity
in the colon.7–12 Stress, in rats, exacerbates colitis3 and
Physiology of the Gastrointestinal Tract, Two Volume Set. DOI: 10.1016/B978-0-12-382026-6.00075-0
© 2012 Elsevier Inc. All rights reserved.
FIGURE 75.1  Effects of cold-restraint stress on fecal pellet out-
put in guinea pig.  (A) The non-stressed control remained caged in its
normal environment in the vivarium. (B) Guinea pig was restrained in a
supine position by placing a tie around each ankle and loosely extending
and tying each of the four extremities to metal hooks at the corners of
a wooden rectangular flat platform and placed in a 4°C cold room for 2
hours.
initiates colitis in the cotton-top tamarin model for ulcera-
tive colitis and colon cancer.6 Acutely applied forms of
stress — among which are handling, water avoidance,
abdominal surgery, acoustic stimulation, wrap restraint at
room temperature, swimming, and ether anesthesia — all
suppress gastric emptying and simultaneously increase
secretion and propulsive motility in the large intestine of
mice, rats, guinea pigs, and dogs. Anger, fear, pre-surgical
anxiety, and intense exercise are forms of stress that exac-
erbate the symptoms of IBS.9,13,14 Cold exposure, painful
stimuli, dichotomous listening tests, fear, anxiety, anger, or
stressful interviews all stimulate colonic motor activity in
healthy human volunteers.14,15
75.2  STRESS AND THE SYMPATHETIC
NERVOUS SYSTEM
Earlier physiological explanation for the above exam-
ples of stress-associated gastrointestinal symptoms would
likely have been that effects of stress reflect a brain–gut
interaction mediated by activation of the sympathetic
division of the autonomic nervous system. The sympa-
thetic nervous hypothesis had credibility because the
2001
2002
neuroendocrine components in the stress response were
known to include increased secretion of epinephrine and
norepinephrine by the sympathetic nervous system and
adrenal glands.16 Nevertheless, sympathetic activation
is no longer a plausible explanation for stress-related gut
symptoms. Descending spinal activation of the sympa-
thetic nervous system cannot explain the stress-associ-
ated symptoms of cramping lower abdominal pain, fecal
urgency, and watery diarrhea in humans or the similar
effects of stress in animals.
Advances in what is known about the sympathetic
interface with the enteric nervous system (ENS) rule out
sympathetic involvement.17,18 The majority of sympathetic
noradrenergic fibers entering the small and large intestine
innervate ganglion cells in the ENS. Sympathetic activa-
tion and the release of norepinephrine inside the ENS
inhibits firing of secretomotor neurons in the submucosal
plexus, thereby suppressing the neurogenic secretion
responsible for generation of loose watery stools.17,19 Once
released in the ENS, norepinephrine acts at presynaptic
alpha2a receptors to suppress transmission at both slow and
fast excitatory synapses and at excitatory neuromuscular
junctions.20,21 Sympathetic inhibition of neurotransmis-
sion in the synaptic microcircuitry makes improbable any
suggestion that sympathetic activation is responsible for
initiation of the powerful propulsive motility in the colon
that accounts for cramping lower abdominal pain in dogs
and humans.22 Noradrenergic inhibitory synapses on
secretomotor neurons “shut down” secretion in the face
of sympathetically reduced intestinal blood flow making
it unlikely that stress-associated diarrhea involves sympa-
thetic activation (see Chapter 21). The evidence against
sympathetic activation directs attention to involvement of
other enteric neurophysiological mechanisms that come
into play in stress and will be dealt with in this chapter.
75.3  PSYCHOGENIC STRESS AND
FUNCTIONAL GASTROINTESTINAL
DISORDERS
The symptoms in the “stressed-out” attorney and medi-
cal student are characteristic of functional gastrointestinal
disorders (FGIDs). FGIDs are those disorders in which no
abnormal metabolic or physical processes, which account
for the symptoms, can be identified.23 IBS is an example
of a significant FGID, which compromises the quality of
life for 10–20% of humans worldwide.24–27 Predominant
symptoms of IBS are abnormal defecation associated with
abdominal pain, both of which are exacerbated by psy-
chogenic stress.28 Psychological stress and negative life
experiences are widely recognized as exacerbating psycho-
social factors in IBS.24,29–32
SECTION  |  VI  Consequences of Disregulated Physiology
75.4  PSYCHOGENIC STRESS AND ENTERIC
MAST CELLS
Figure 75.2 is a heuristic model for the physiology of
brain–gut interactions in the stressed animal or human.
A unifying concept emerges from consideration that a
major impact of stress on the intestinal tract is reflected by
symptoms reminiscent of the diarrhea-predominant form
of IBS. Cramping abdominal pain, acute fecal urgency,
and explosive watery diarrhea are hallmarks not only
of diarrhea-predominant IBS, but also infectious enteri-
tis, radiation-induced enteritis, food allergy, and noxious
mucosal irritation (e.g., senna laxatives). The concept
starts with the evidence that enteric mast cells receive
brain-derived input in the stressed individual. The same
mast cells intercept and become sensitized to microor-
ganisms and other sensitizing antigens that find their way
across the intestinal mucosal barrier. Mast cells detect
threats of this nature to the integrity of the whole animal
and signal their presence to the ENS, which responds by
calling up from its program library a program of special-
ized integrated secretion and motility that removes the
threat from the lumen.33–36 Threat removal starts with
stimulation of mucosal secretion, which flushes the threat-
ening agent out of the mucosa into the lumen and holds it
in suspension. The secretory response then links to pow-
erful propulsive motility, which propels the secretions
together with the offending agent quickly in the anal direc-
tion. Cramping abdominal pain accompanies the strong
propulsive contractions.22,37 Urgency is experienced when
arrival of the liquid bolus distends the rectosigmoid region
and reflexively opens the internal anal sphincter with con-
tinence protection now provided only by spinal reflexes
that contract the puborectalis and external anal sphincter.
Sensory information arriving in the brain from receptors in
the rapidly distending rectosigmoid accounts for the con-
scious sensation of urgency and fear of incontinence might
further exacerbate the individual’s emotional stress. The
symptom of acute explosive watery diarrhea becomes self-
explanatory in this scenario.
Enteric mast cells are implicated as a key step in the
initiation of stress-evoked symptoms (Figures 75.2 and
75.3). Enteric mast cells are packed with granules that are
sites of storage for a broad mix of preformed chemical
mediators (Figure 75.3). Antigens and neural input stimu-
late the mast cells to release mediators, which diffuse into
the extracellular milieu as paracrine signals to other kinds
of cells including the neurons and glia of the ENS. Mast
cells express high-affinity receptors for IgE antibodies or
other immunoglobulins on their surfaces. When antibod-
ies to a sensitizing antigen occupy the receptors and cross-
linking occurs by interaction of the sensitizing antigen
with the bound antibody, a flood of different mediators is
released from the mast cells (Figure 75.3).
Chapter  |  75  Enteric Neurobiology of Stress 2003

Brain STRESS

ENTERIC EFFECTOR
NERVOUS SYSTEM SYSTEMS

Interneurons Muscle
Information
Brain to Mast Cell Connection

Sensory Motor
Processing Glands
Neurons Neurons
Reflexes
Program Blood Vessels
Library

INTESTINAL
BEHAVIOR
Mast Cell
Mediators (Hyper-Secretion)

Inflammatory
Surveillance
Antibodies (Power Propulsion)

Antigen
SYMPTOMS
MAST CELL
(Diarrhea)
(Pain)

FIGURE 75.2  Heuristic model for the physiology of brain–gut interactions in stressed animals or humans.  The ENS is a minibrain with
sensory neurons, interneurons, and motor neurons. Enteric mast cells are in position to detect foreign antigens and signal their presence to the ENS.
Stimulated mast cells release multiple paracrine mediators (e.g., histamine and proteases) simultaneously. Some of the mediators signal the ENS while
others are attractant factors for polymorphonuclear leukocytes responsible for acute inflammatory responses. The ENS responds to the mast cell signal
by initiating a program of coordinated secretion and propulsive motility that expels the source of antigenic stimulation from the bowel. Symptoms of
cramping abdominal pain and acute urgency and diarrhea result from operation of the neural program. Neural inputs to mast cells from the brain stimu-
lates simultaneous release of chemoattractant factors for inflammatory cells, which establish inflammatory surveillance at a dangerous interface and
chemical signals to the ENS with effects that mimic the symptoms of antigenic stimulation. Stress acts through the brain–mast cell connection.

that manage access across the mucosal barrier.38,39 Results

FIGURE 75.3  Multiple mast cell mediators, which become signals
to other systems in the gut, are stored in granules inside enteric mast
cells or are synthesized from membrane lipids. Close spatial asso-
ciation of inflammatory/immune cells with the mast cell reflects release
of chemoattractant factors from the mast cell in the inflamed mucosa/
submucosa.
Experimental infections with nematode intestinal para-
sites (e.g., Trichinella spiralis) in animals stimulate prolif-
eration of enteric mast cells and are experimental models
for mast cell functions in detection and signaling of the
presence of sensitizing antigens and infectious invaders
from nematode-infected animals and food-allergic models
have expanded our understanding of mast cell involvement
in enteric immunoneural communication. This work shows
that a second exposure to antigens isolated from the infec-
tious agent (e.g., intestinal nematodes in rats, guinea pigs,
or dogs) or food products (e.g., β-lactoglobulin in milk-
sensitized guinea pigs) results in predictable integration of
intestinal motility and secretory behavior.35,37,39,40–42
Recognition of sensitizing antigens by antibodies
bound to Fc receptors on the sensitized mast cells triggers
degranulation and release of multiple mediators. Once in
the interstitial compartment, the mediators become para-
crine messages to the ENS, which responds by suspending
operation of other programs in its library and “running”
a defense program that integrates secretion and special-
ized propulsive motility to eliminate the antigen from the
lumen. Copious neurogenic secretion and elevated intesti-
nal blood flow followed by orthograde power propulsion
(see Chapter 21) of the luminal contents are the behavioral
components of the program.22,37 Mast cells are equipped
and strategically placed to recognize foreign agents that
2004
threaten whole-body integrity and to signal the ENS to
program a defensive response to eliminate the threat. A
similar form of ENS-programmed defense takes place in
the upper half of the jejunum, duodenum, and stomach in
the form of emesis.
Mast cell function in immunoneural communication in
the gut is an immune counterpart of sensory detection and
information coding in sensory neurophysiology. In sensory
physiology, sensory neurons are genetically programmed
to express detection mechanisms for specific stimuli (e.g.,
receptors for touch, temperature, or pH) that remain fixed
throughout the life of the individual. Mast cells, on the
other hand, acquire specific detection capabilities through
the flexibility of recognition functions inherent in synthe-
sis by the immune system of new specific antibodies that
become attached to Fc receptors (e.g., FceRI receptors in
allergies) on the mast cells.43 Detection specificity for for-
eign antigens is acquired and reinforced throughout life
due to formation of new antibodies that bind to and remain
bound to the mast cell immunoglobulin receptors. Output
signals from mast cells, which are triggered by cross-link-
ing of antigens with the attached antibodies, are chemical
in nature and analogous to chemical output signals (i.e.,
neurotransmitters) from sensory neurons to second-order
neurons in the brain and spinal cord. Both mast cells and
sensory neurons ultimately code information on the sensed
parameter by releasing a chemical message that is decoded
by information processing circuits in the nervous system.
75.4.1  Mast Cells and the Brain–Gut
Connection in Stress
Enteric mast cells, in addition to their sensing functions,
are relay points for transmission of information on emo-
tional status from the brain to the ENS (see Figure 75.2).
This is a brain–gut connection that links central psycho-
logical status (e.g., psychogenic or physical stress) to a
specific state of the digestive tract via mast cell signal-
ing. Release of mast cell mediators, evoked in this man-
ner, activates an “alarm program” in the ENS program
library, which when “running,” produces the same symp-
toms of diarrhea and abdominal distress as antigen-evoked
degranulation in food allergies and infectious enteritis.
The brain-to-enteric mast cell connection can be demon-
strated experimentally as a release of mast cell proteases
in a Pavlovian-like conditioned response. Assays for the
release of mast cell proteases into the systemic circulation
or into the intestinal lumen are useful tools for measure-
ment and timing of degranulation of enteric mast cells.44
Release of mast cell proteases into the systemic circula-
tion can be evoked as a Pavlovian-conditioned response
in laboratory rats by light or auditory stimuli after several
trials of pairing these stimuli with injection of a sensitiz-
ing antigen, such as ovalbumin.45 In humans, release of
SECTION  |  VI  Consequences of Disregulated Physiology
mast cell proteases and histamine into the lumen of the
small intestine takes place as a response to stress of cold-
induced pain and mimics responses to sensitizing antigens
in food allergies (Figure 75.4). Santos et al.46 instructed
subjects to dip one hand into a container of ice-cold H2O
for 1 minute at 15 second intervals repeatedly for 15
minutes. Continuous aspiration and analysis of the con-
tents from subjects’ small intestine show appearance of
mast cell proteases and histamine statistically correlated
with the cold-H2O stressor (Figure 75.4). The speed of
the stress response most likely reflects neurotransmission
over a neural pathway to the enteric mast cells and makes
it unlikely that the responses resulted from any form of
endocrine signaling from the brain.
A neural highway for central nervous signaling to
enteric mast cells is suggested by close histological prox-
imity between vagal and spinal sensory afferent nerves
and enteric mast cells.47 Elevated expression of histamine
in intestinal mast cells occurs in response to vagal nerve
stimulation and also suggests existence of a brain–mast
cell connection.48 Stimulation of neurons in the brainstem
by intracisternal injection of thyrotropin-releasing hor-
mone (TRH) evokes degranulation of enteric mast cells
and release of mast cell protease II into the lumen of rat
small intestine and adds to the evidence for a brain–mast
cell connection.49 Vagotomy or administration of the mus-
carinic receptor antagonist, atropine, suppresses the action
of intracisternal TRH on release of mast cell mediators,
including release of mast cell protease II and prostaglandin
D2 in the small intestine. The evidence for a brain–mast
cell connection takes on significance for understanding
stress-associated symptoms of gut origin, because the
symptoms associated with release of mast cell mediators
are the same irrespective of whether mast cells are activated
by antigen-antibody cross-linking in allergies and infec-
tions or by input down the brain–gut axis during stress.
75.5  NEUROBIOLOGY OF STRESS
Symptomatic manifestations of stress in upper and lower
regions of the digestive tract are different. Cold-restraint
stress in rats evokes inflammation and erosions in the
stomach. Intracerebral injection of TRH mimics the gas-
tric responses to cold-restraint stress. In the large intestine,
restraint stress alters motility, induces a diarrheal state,
and exacerbates nociceptive pain responses to distension
in association with increased release of histamine from
enteric mast cells.50 Like the effects of centrally acting
TRH on gastric mucosal pathology, intracerebroventricular
injection of corticotropin-releasing factor (CRF) mimics
large intestinal responses to stress.8,51 CRF receptor antag-
onists or pretreatment with mast cell-stabilizing drugs sup-
presses the stress-evoked responses in the large bowel.9,10
Neuropharmacology suggests that CRF is working as a
Chapter  |  75  Enteric Neurobiology of Stress 2005

(A) Aspiration (B) Aspiration

Cold Cannula
Antigen Cannula
0.6 Stress Balloons 0.5

Mast Cell Tryptase Release

Mast Cell Tryptase Release
Challenge Balloons
(Egg Albumin)

(Units/15 min)
(Units/15 min)
0.4 0.3

0.2 0.2

0 0
15 0 15 30 45 60 15 0 15 30 45 60 75 90
Time (Minutes) Time (Minutes)
FIGURE 75.4  Cold-pain stress or antigen challenge degranulates enteric mast cells in humans.  A group of patients with confirmed allergy to
egg albumin and a group of normal subjects were asked to immerse one hand into ice-cold H2O for 1 minute at 15 second intervals in 2 successive 15
minute trials. (A) Degranulation of mast cells measured as amounts of tryptase in aspirates from the small intestine. Cold-pain stress evoked release of
mast cell tryptase in both food-allergic patients (O) and normal subjects (•). (B) Egg albumin, infused into the small intestinal lumen, evoked mast cell
degranulation manifested as elevated mast cell tryptase in aspirates from food-allergic patients, but not in normal subjects. (Adapted from 46).

neurotransmitter in the neural networks in the brain and

“Puff” Pipette
ENS to generate stress-induced large intestinal symptoms (CRF, Urocortin, etc.)
of abdominal pain, fecal urgency, and diarrhea. Microelectrode

75.5.1  CRF CRF or CRF Receptor

CRF in the “big brain” is expressed predominantly in the
hypothalamic paraventricular nucleus. Nevertheless, it is
distributed in neural elements throughout the brain.52 The
majority of the endocrine, neurochemical, electrophysi-
ological, and behavioral effects, which are reported to
occur after injection of CRF into the brain, are essentially
the same as those associated with experimentally induced
stress. Virtually all of the effects of intracerebral applica-
tions of CRF can be reduced or abolished by administra-
tion of CRF receptor antagonists, and this reinforces belief
in a predominant central role for CRF neurotransmission
in stress.53–56ab Chronic stress is known to exert an inhibi-
tory effect on hair growth in animals and humans. CRF
and adrenocorticotropic hormone (ACTH), aside from
being key mediators in the endocrine and neuroimmune
responses to stress, inhibit the growth cycles of cutane-
ous hair follicles.56b Intraperitoneal administration of the
non-selective CRF1/CRF2 receptor antagonist, astressin-
B, in nude mice with a transgene that overexpressed CRF,
induces pigmentation and hair regrowth.56b
Application of experimental methods for recording
electrical and synaptic behavior in single neurons in con-
cert with immunohistochemistry has greatly advanced
insight into the role of CRF in the brain and in the ENS
(Figure 75.5). Application of electrophysiological methods
generally finds that exposure to CRF stimulates firing of
single neurons in the hypothalamus,57 cerebral cortex,58
cerebellar Purkinje cells in culture,59 hippocampus,60,61
locus coeruleus,62 periaqueductal gray,63 and motor neu-
rons in the lumbar spinal cord.58 CRF-evoked neuronal
ENTERIC
NEURON
Fluorescent
Tags
Primary
Antibody
Secondary
Antibodies
FIGURE 75.5  Indirect immunofluorescence and intraneuronal elec-
trophysiological recording with “sharp” microelectrodes are used to
study the neurobiology of CRF and its receptors in the ENS. CRF or
other members of the CRF family of peptides are applied to the neurons
by nitrogen pressure ejection (“puffs”) from fine-tipped micropipettes. In
immunohistochemistry allied with the electrophysiology, primary anti-
bodies recognize and bind to CRF or its receptor types and fluorescently
tagged second antibodies bind to and mark the location of bound primary
antibodies.
excitation in hippocampal slice preparations is associated
with reduction of the hyperpolarizing responses that follow
bursts of spikes,60 and this is reminiscent of suppression of
hyperpolarizing after potentials by certain neurotransmit-
ters/modulators in ENS neurons (Chapter 21).64,65
CRF, when functioning as a neurotransmitter or neu-
romodulator in the CNS, alters the plasticity of synaptic
transmission in forms that may be observed as long-term
potentiation or long-term depression depending on the
studied region of the brain or spinal cord.66–68 This is rele-
vant in terms of ramifications of prolonged stress, because
long-term changes in synaptic strength are associated
with memory functions in the CNS. Exposure to CRF
2006
produces long-lasting enhancement of synaptic efficacy
in the hypothalamus.69,70 Antagonists at both CRF1R
and CRF2R receptors depress the induction of long-term
potentiation in the CA1 region of the hippocampus.71
In the cerebellum, CRF receptor antagonists inhibit the
induction of long-term depression of synaptic transmis-
sion.72 In the spinal cord, CRF converts long-term synaptic
suppression to facilitation.66
75.5.2  CRF in Stress
Acutely applied forms of stress (e.g., water avoidance,
handling, acoustic stimulation, abdominal surgery, move-
ment restraint, swimming, or ether anesthesia) each inhibit
gastric emptying in mice, rats, guinea pigs, and dogs.73
Similarly in human subjects, anger, fear, preoperative anxi-
ety, and intense exercise are forms of stress that result in
slowing of gastric emptying and exacerbation of IBS
symptoms.13,14 Acute stress, intracerebral injection of CRF,
or urocortin 1 suppresses propulsive motility and transit in
the small intestine of animals.74–78 Moreover, acute psy-
chological stress suppresses the small intestinal migrating
motor complex in humans.79 Unlike the inhibitory effects
on gastric and small intestinal motility, stress stimulates
contractile activity of the colonic musculature, accelerates
colon transit, and stimulates defecation in animal mod-
els, human volunteers, and IBS patients.14,75,80,81 Central
administration of CRF or urocortin 1 mimics effects of
stress by stimulating colonic motility, increasing colonic
transit, and accelerating fecal pellet output in rats.82
Urocortin 1 is included in the CRF family of mammalian
messenger peptides,83 which also includes urocortin 284 and
urocortin 385 and two non-mammalian CRF-like peptides,
sauvagine86 and urotensin 1.87 Intracerebral administra-
tion of non-selective or selective CRF1R receptor antago-
nists suppresses the effects of both stress and administration
of CRF or urocortin 1 on colonic motor activity.75,80,82,88
Although urocortins binds with high affinity to CRF2R
receptors in the brain, CRF2R receptor antagonists do not
attenuate stress-induced acceleration of colonic transit or the
actions of CRF and urocortin-1 on colonic motility.82
75.5.3  CRF Putative Neurotransmission in
the ENS
Downstream from the brain, CRF signaling inside the
ENS is implicated in stress-evoked alterations in motil-
ity and mucosal secretory and barrier functions. An early
suggestion was that CRF, which does not enter the brain
from the blood, is actively transported out of the brain into
the systemic circulation.89 This implies that CRF, after
release inside the brain during stress, might enter and be
carried by the blood, in hormonal fashion, to the gastroin-
testinal tract where it produces its actions by binding to
SECTION  |  VI  Consequences of Disregulated Physiology
CRF receptors expressed by ENS neurons. Nevertheless,
other lines of early evidence suggested that CRF might be
expressed in organs outside the brain and be released to act
locally. Outside the brain, expression of CRF immunore-
activity has been reported for the digestive system, entero-
chromaffin cells, and by immune/inflammatory cells.90–94
Current evidence suggests that intestinal manifesta-
tions of stress reflect the release of CRF as a neurotrans-
mitter/neuromodulator in the integrative microcircuits of
the ENS well outside the hypothalamic-pituitary-adrenal
(HPA) axis. Earlier evidence supporting this was find-
ing that intraperitoneal administration of CRF mimics
stress-evoked changes that are mediated by a local action
inside the intestine. As early as 1992, Hanani and Wood95
found that applications of CRF to ENS neurons evoked
firing in the guinea pig small intestinal myenteric plexus,
raising the possibility of an ENS signal function for CRF
reminiscent of its signaling function in the brain (Figure
75.6). The stress-like effects of peripheral administration
of CRF cannot be attributed to action in the brain and are
consistent with a signaling function for CRF in the ENS
analogous to its neurotransmitter function in the CNS.96
Intraperitoneal administration of CRF evokes delayed
gastric emptying that is mediated by stimulation of CRF2R
receptors and stimulates colonic transit mediated by the
CRF1R receptor.97 Microelectrode recording in concert
with immunohistochemistry in ENS neurons identifies the
CRF1R receptor as the predominant subtype expressed by
the ganglion cell bodies in the guinea pig intestine (see
Figure 75.5).98
Aside from effects on gastrointestinal motility, stress
strongly influences intestinal glandular secretion and
mucosal barrier function in both animal models and human
subjects (see Chapter 74). For example, intravenous or
intraperitoneal injection of CRF evokes acute watery
diarrhea in rats.11 In a related manner, psychological stress
in human subjects reduces H2O absorption and converts
absorption of Na and Cl to secretion of NaCl and H2O
in the jejunum.99,100 Elevated secretion in these cases
is associated with increased blood flow in support of the
secretory response, which is attributed to elevated firing of
secretomotor/vasodilator neurons in the individuals’ sub-
mucosal plexus. The result of elevated secretomotor neuro-
nal activity is increased liquidity of the intestinal contents
and a predisposition to neurogenic secretory diarrhea
(Figure 75.7). In rats, exposure to restraint or water avoid-
ance stress or intraperitoneal administration of CRF
induces rapid onset watery diarrhea associated with stimu-
lation of mucosal secretion and increased epithelial barrier
permeability in the colon.11,12,101,102
The connection between CRF and stress-evoked
mucosal changes in animals has pathophysiological, clini-
cal, and therapeutic implications. It suggests that CRF sig-
naling in the ENS might have direct involvement in the
Chapter  |  75  Enteric Neurobiology of Stress
(A) AH-Neuron
Post-spike AH
Post-spike AH
CRF (30 nM) 20mV
40s
(B) S-Neuron
1st “Puff” CRF (25µm)
40mV
10s
2nd “Puff” CRF (25µm)
FIGURE 75.6  CRF-evoked excitatory responses are different for
AH- and S-type enteric neurons when recorded intraneuronally with
“sharp” microelectrodes. (A) Application of 30 nM CRF in the bath-
ing medium evoked a slow excitatory postsynaptic potential-like response
consisting of slowly activating membrane depolarization, increased input
resistance, action potential discharge, and suppression of the hyperpo-
larizing response (AH) at the end of each action potential. Downward
deflections on the trace are electrotonic potentials evoked by repetitive
injection of hyperpolarizing current pulses through the electrode. Increased
amplitude of the electrotonic potentials during the depolarizing response
reflects increase in input resistance due to closure of potassium channels.
(B) Two continuous traces showing application of 25 µM CRF by micro
pressure ejection (100 ms “puffs”; see Figure 75.5). CRF evoked mem-
brane depolarization accompanied by intense discharge of action potentials.
Application of a second puff 2 minutes later, apparent on the bottom trace,
evoked a much weaker response indicative of tachyphylaxis. S-type neu-
rons, but not AH-neurons show tachyphylaxis to CRF.
stress-induced symptoms of watery diarrhea, urgency, and
cramping lower abdominal pain that are present in FGIDs
and mucosal inflammation in humans. CRF function as a
neurotransmitter/neuromodulator in the microcircuitry
that controls activity of secretomotor/vasodilator neurons
is likely to be the underlying factor in effects of stress to
open the mucosal barrier and allow sensitizing antigens to
cross the barrier and evoke or exacerbate mucosal inflam-
matory responses in the colon. This is supported by evi-
dence that CRF acts to increase mucosal permeability to
2007
large organic molecules in mucosal biopsies from human
colon and that pretreatment with the CRF receptor antago-
nist, α-helical CRH (9-41), blocks this action of CRF.103
Cold temperature stress and the presence of the microflora
in the lumen are the only requirements for development of
inflammatory bowel disease in a primate model for ulcera-
tive colitis and colon cancer.6 Moreover, psychological
stress exacerbates ulcerative colitis and leads to relapse in
human patients is common knowledge.5,104,105
75.5.4  CRF in Mucosal Secretion
Application of CRF to the serosal side of flat-sheet prepa-
rations of rat or guinea pig colon in Ussing flux chambers
stimulates electrogenic chloride secretion, which can be
measured as elevation of baseline short circuit current across
the mucosa. Increases in mucosal permeability to large mol-
ecules, such as horseradish peroxidase and mannitol, occur
in parallel with the CRF-evoked increases in chloride secre-
tion.101 CRF-evoked increases in chloride secretion are
suppressed or abolished by application of nerve-blocking
agents (e.g., tetrodotoxin) or CRF receptor antagonists (e.g.,
α-helical CRH (9-41).101 Blockade of CRF-evoked mucosal
secretion by nerve-blocking agents is reliable evidence that
this action of CRF results from stimulating the firing of
secretomotor neurons in the submucosal plexus.
75.5.5  CRF in the ENS
Aside from stimulation of neurogenic secretion, multiple
forms of evidence, analogous to the evidence for the “big
brain,” point to the neural networks of the ENS “minibrain” as
the site where peripheral CRF-related mechanisms contribute
to stress-induced changes in intestinal motility and mucosal
function. The evidence can be summarized as follows:
1. Application of CRF to segments of guinea pig small
intestine in vitro evokes muscle contractions that are
blocked by tetrodotoxin, indicating that the CRF-
evoked contractions are mediated by stimulation of
excitatory musculomotor neurons.106
2. Exposure of rat isolated duodenal and colonic segments
to CRF enhances neurally mediated phasic contrac-
tions and neurally mediated peristaltic propulsion, and
this action is prevented by the peptidergic CRF recep-
tor antagonist α-helical CRF (9-41) or the non-peptide
receptor antagonist, CP-154,526.107,108
3. Application of CRF to the myenteric or submucosal
plexus of guinea pig small and large intestine evokes exci-
tatory responses, which are blocked by selective CRF1R,
but not CRF2R antagonists, in single neurons recorded
with intracellular microelectrodes (see Figure 75.6).95,98
4. H2O-avoidance stress or intraperitoneal injection of
CRF induces elevated expression of c-fos, a marker of
2008 SECTION  |  VI  Consequences of Disregulated Physiology

(A) (B) (C)

Cholinergic Secretomotor/ Cholinergic Secretomotor/ Non-cholinergic Secretomotor/

Vasodilator Neuron Vasodilator Neuron Vasodilator Neuron

Arteriole Arteriole Arteriole

Crypt Crypt Crypt

Secretion Secretion Secretion

FIGURE 75.7  Three kinds of secretomotor neurons in guinea pig small intestine.  (A) Cholinergic secretomotor/non-vasodilator neurons express
NPY and innervate the secretory glands (crypts of Lieberkühn), but not arterioles. (B) Cholinergic secretomotor/vasodilator neurons contain calretinin
and innervate the secretory glands and periglandular arterioles. (C) Non-cholinergic secretomotor/vasodilator neurons innervate the secretory glands and
periglandular arterioles. These neurons contain VIP in all species and are the only neurons in the submucosal plexus that express CRF.

neuronal activation, in colonic myenteric neurons, and (A) Myenteric (B) Submucosal
this is blocked by peripheral application of the CRF1/ Plexus Plexus
CRF2R antagonist, astressin, or the selective peptider-
gic CRF1R receptor antagonist, CP-154,526.109,110 Calbindin--------------------0% Calbindin--------------------0%
5. RT-PCR and Western blot analysis detects CRF1R Calretinin--------------------0% Calretinin--------------------0%
receptor mRNA transcripts, and protein and immu- ChAT ----------------------4.5% ChAT ------------------------0%
nohistochemical analysis reveals the expression of
CRF1 Receptor ------------0% CRF1 Receptor ------------0%
CRF1R protein by neurons in both myenteric and sub-
mucosal plexuses.108,111 NOS -----------------------5.0% NOS --------------------------0%

NPY --------------------------0% NPY --------------------------0%

75.5.6  CRF Localization in the ENS Serotonin -------------------0% Serotonin -------------------0%

Immunohistochemical localization of CRF in the ENS Somatostatin---------------0% Somatostatin---------------0%

adds to the lines of evidence suggesting that it is a key
neurotransmitter in the integrative microcircuitry that
mediates stress-evoked changes in intestinal behavior. It
is expressed in neuronal cell bodies and nerve fibers in
the myenteric and submucosal plexuses of the small and
large intestine and the myenteric plexus of the stomach in
guinea pigs (Figure 75.8).112 Most, if not all, of the CRF
immunoreactive nerve fibers, are presumed to be intrin-
sic because they remain in organotypic culture after time-
dependent degeneration of extrinsic sympathetic and spinal
afferent fibers. The numbers of ganglia and ganglion cells
with CRF-IR are low in the stomach and increase progres-
sively in the aboral direction with the largest numbers
appearing in the distal colon.112
Ninety-five percent of CRF immunoreactive neurons in
the myenteric plexus have Dogiel type I morphology (i.e.,
an oval or round cell body with a single long process and
Substance P-------------4.2% Substance P----------------0%
VIP----------------------------0% VIP--------------------------77%
FIGURE 75.8  Proportions of neurons that express definitive chemi-
cal codes in combination with CRF immunoreactivity in guinea pig
small bowel. (A) In the myenteric plexus, only cholinergic (ChAT)
neurons and nitrergic (NOS) and substance Pergic neurons coexpress
CRF. (B) In the submucosal plexus only VIPergic neurons express CRF.
(Adapted from Liu et al. 112.)
several short lamellar dendrites). This kind of morphology
is characteristic for secretomotor/vasodilator, musculomo-
tor, and interneurons in the ENS.112 Less than 5% of CRF
immuno-positive myenteric neurons have Dogiel type II
morphology (i.e., a relatively large cell body with two or
more long processes; Figure 75.8). Neurons with Dogiel
Chapter  |  75  Enteric Neurobiology of Stress
type II morphology belong to a special class of ENS
interneurons, called AH-type, based on electrophysiologi-
cal properties that include long-lasting after-hyperpolariza-
tion associated with discharge of action potentials (Figure
75.6; see Chapter 21).65 About half of the CRF immuno-
reactive neurons with Dogiel type I morphology in the
myenteric plexus project their axons in the oral direction
along the intestine and the other half project in the aboral
direction. Divisions of projections in this manner would be
expected if the oral projections were from excitatory mus-
culomotor neurons and those going in the aboral direction
were inhibitory musculomotor neurons.
CRF is expressed most strongly in the submucosal
plexus. The proportion of the total number of ganglion
cells expressing CRF in the submucosal plexus is about
14-fold greater than the proportion in the myenteric
plexus. Virtually all of the submucosal ganglia contained at
least one CRF-immunoreactive neuronal cell body. When
evaluated as a percent of the total submucosal neuronal
population, similar percentages of CRF-immunoreactive
neurons are found in the duodenum, jejunum, ileum, and
proximal and distal colon in guinea pigs.112 All CRF-
immunoreactive submucosal neurons have Dogiel type I
uniaxonal morphology. Dogiel type II submucosal neurons
do not express CRF.
75.5.7  Distribution of CRF in Relation to
ENS Chemical Codes
ENS neurons are classified by their shapes, function, and
histochemical/immunohistochemical staining properties
and other characteristic features including the structures
they innervate (e.g., longitudinal or circular muscle coats
of the intestinal muscularis externa or intestinal secre-
tory glands), the transmitters they release, and the synap-
tic inputs they receive. Impressive progress has occurred
in the last 25 years in terms of success in combining the
various neuronal biomarkers into individual profiles that
distinguish each functionally identified neuronal class
in guinea pig small intestinal ENS (see Chapter 19).113
Double labeling immunohistochemistry provides an effec-
tive means for identification of chemical biomarker codes
for subpopulations of CRFergic neuronal profiles (Figure
75.5). The distribution of immunoreactive chemical codes
that are expressed with CRF in the myenteric and submu-
cosal plexuses of guinea pig ileum appear in Figure 75.8.
75.5.7.1  Myenteric Plexus
Immunoreactivity for choline acetyltransferase (ChAT)
is a code for neurons that express and probably release
acetylcholine as a neurotransmitter. It is a biomarker for
excitatory musculomotor neurons and two subsets of
secretomotor neurons. CRF is expressed by 4.5%% of
2009
the cholinergic neurons and 57% of CRF immunoreactive
neurons that express the cholinergic code, suggesting that
CRF might be released with acetylcholine when the neu-
rons fire in the myenteric plexus in guinea pigs. Substance
P is a marker for a subpopulation of excitatory musculo-
motor neurons. CRF is expressed by 4% of substance P
immunoreactive neurons and 55% of CRF immunoreac-
tive neurons express the substance P code, suggesting that
CRF might be released with substance P when these exci-
tatory musculomotor neurons fire. Immunoreactivity for
nitric oxide synthase (NOS) is a marker code for inhibi-
tory musculomotor neurons in the myenteric plexus. CRF
is expressed by 5% of the neurons with NOS and 38% of
CRF immunoreactive neurons express the (NO) code, sug-
gesting that CRF might be co-released with the inhibitory
musculomotor transmitter, NO, when the neurons fire.
In terms of chemical codes, morphology, and direc-
tionality of axonal projections, a majority of the myenteric
neurons, which express CRF, fit the profile for excitatory
or inhibitory musculomotor neurons. The neurons with
ChAT and substance P coexpression correspond to a sub-
group of cholinergic/substance Pergic neurons, with single
axonal morphology and orally projecting axons, identified
as excitatory musculomotor neurons to the circular muscle
coat.114,115 These neurons may be responsible for some of
the CRF-positive nerve fibers that can be seen running par-
allel to the muscle fibers in the circular muscle layer of the
small and large intestine where they release their excitatory
neurotransmitters at neuromuscular junctions.98 The neu-
rons with NOS immunoreactivity correspond to a subset of
inhibitory musculomotor neurons with aboral projections to
the circular muscle and descending interneurons involved
in local motility reflexes.114,115 It might be significant that
the CRF percentages are similar for motor neurons that
release excitatory or inhibitory neurotransmitters at intesti-
nal neuromuscular junctions. This suggests that CRF might
be co-released with acetylcholine, substance P, or NO from
subgroups of excitatory or inhibitory musculomotor neu-
rons. Evidence that this could be involved in generation
of motility patterns comes from studies in which periph-
eral administration of CRF stimulates propulsive motility
in the colon as reflected by elevated contractile activity,
accelerated transit, and ejection of fecal pellets in rats.116
Nevertheless, how this might relate to association of CRF
and stress-induced changes in motility remains unresolved.
Aside from acetylcholine, substance P, and NO, addi-
tional established chemical codes in the myenteric plexus
include somatostatin, serotonin, calbindin, and calretinin.
Small diameter uniaxonal neurons that express calretinin
make up about one-fourth of the neurons in the myenteric
plexus of guinea pig small intestine.117,118 These neurons
are also cholinergic and identified as excitatory motor neu-
rons that innervate and contract the longitudinal muscle
coat when they fire. Calbindin immunoreactivity identifies
2010
ENS neurons with multipolar Dogiel type II morphology
and AH-type electrophysiological behavior (see Chapters
19, 21, 22, 81). Somatostatin immunoreactivity marks a
group of descending interneurons in the guinea pig mye-
nteric plexus, and serotonin is expressed by a different
population of descending interneurons.113 None of these
four subpopulations express CRF.
75.5.7.2  Submucosal Plexus
CRF is expressed exclusively by neuronal cell bodies
that coexpress vasoactive intestinal peptide (VIP) in the
submucosal plexus (Figures 75.7 and 75.8). VIP is an
established neurotransmitter released by non-cholinergic
secretomotor neurons at neuroepithelial junctions.119 CRF
immunoreactivity is not found in submucosal neurons
that express ChAT, a neurochemical code for cholinergic
secretomotor innervation of the mucosal epithelium. VIP
is a chemical code for secretomotor/vasodilator neurons
(Figure 75.7).112,119 Colocalization with VIP suggests that
CRF is released together with VIP when secretomotor
neurons fire. Like the implications for coexpression with
excitatory and inhibitory neurotransmitters in musculomo-
tor neurons to circular muscle, coincident release at neu-
roepithelial and neurovascular junctions might function to
modulate the responses of the secretory glands and blood
vessels individually in response to input from a single
population of secretomotor/vasodilator neurons. In view of
established significance of CRF in stress-related opening
of the mucosal barrier, it might be questioned if VIP/CRF
co-release is significant in terms of secretomotor influence
on mucosal permeability.
Cholinergic secretomotor neurons express immuno-
reactivity for neuropeptide Y (NPY) in the submucosal
plexus, while cholinergic secretomotor/vasodilator neurons
express calretinin.113,115 Substance P and NeuN are marker
codes for submucosal neurons with Dogiel type II mor-
phology and AH-type electrical and synaptic behavior.120
None of these neuronal populations in the submucosal
plexus express CRF.
There are far fewer CRF-positive nerve fibers in sub-
mucosal ganglia than in ganglia in the myenteric plexus.
There are no CRF fibers in over half of the ganglia in the
submucosal plexus.112 On the other hand, CRF nerve fib-
ers are abundant in the interganglionic fiber tracts. It
seems that rather than forming synapses within the gan-
glia, most of the CRF neurons appear to send their axons
outside their home ganglia to release neurotransmitters at
synapses with neurons and at neuroeffector junctions. The
microanatomy of VIP immunoreactive neurons in the sub-
mucosal plexus is essentially the same as for the neurons
that contain CRF. VIP and CRF neurons have few axonal
projections to synapses with other neurons inside their
ganglia of residence. Instead, their axons leave the ganglia
SECTION  |  VI  Consequences of Disregulated Physiology
and continue on through several ganglia without forming
contacts with other ganglion cells.121 Immunoreactive CRF
fibers in the interganglionic fiber tracts are most likely pro-
jections from CRF/VIP secretomotor/vasodilator neurons,
because they appear alongside submucosal arterioles and
both VIP and CRF persist in isolated submucosal prepa-
rations after degeneration of extrinsic innervation in orga-
notypic culture.112 Persistence of CRF fibers in isolated
submucosal preparations after several days in culture indi-
cates that few, if any, of the CRF neurons in the myenteric
plexus project their axons to the submucosal plexus.
75.6  CRF RECEPTORS
CRF and CRF-like peptides exert their signaling functions
through specific G-protein-coupled receptors. Two types of
CRF receptors have been cloned, characterized, and named
CRF1R and CRF2R.122 Each receptor is associated with
distinctly different physiological events that reflect unique
distributions in various tissues and differing profiles of
pharmacological specificity. The mRNA transcript for the
CRF1R is widely expressed in the mammalian brain and
pituitary gland and reduced levels are found in peripheral
organs, including testis, ovaries, and adrenal glands.123–125
CRF2R is expressed in the brain and peripheral tissues,
including heart, lung, gastrointestinal tract, skeletal mus-
cle, testis, and ovaries.126–128 CRF1R and CRF2R are
expressed in the intestinal tract where mRNA transcripts
and Western blot protein expression can be found for crude
extracts from rat colon.110,129 CRF1R and CRF2R immu-
noreactivity is colocalized with neuron specific enolase
and neurofilament protein in the rat colon. This was a
seminal indication (seen in 2004) that CRF receptors are
expressed by neurons in the ENS.111a
CRF binds with high affinity to CRF1R and with lower
affinity to CRF2R. Urocortin 1 binds with high affinity to
CRF1R and CRF2R.130 Urocortin 2 and urocortin 3 are
putative endogenous ligands for CRF2R based on exclusive
binding to CRF2R and absence of CRF1R binding.84,85,131
Currently available, selective CRF receptor antagonists
now enable quantitative investigational identification of
CRF receptor types involved in specific actions of CRF
at the tissue, organ, and whole animal levels of biologi-
cal organization. Earlier receptor antagonists were CRF
family peptides with modified amino acid sequences that
were eventually followed by development of non-peptide
antagonists that avoided many of the pitfalls associated
with peptides, especially in human therapeutics. Among
the currently available antagonists for research are the pep-
tides, astressin, and α-helical CRF (9-41), which are non-
selective CRF antagonists that have high affinity for both
CRF1R and CRF2R.132 Non-peptide CRF antagonists,
CP154526, NBI27914, NBI35965, and antalarmin, each
have strong selectivity for CRF1R; whereas the peptidergic
Chapter  |  75  Enteric Neurobiology of Stress
antagonists antisauvagine-30 and the long-acting analog
astressin2-B have high selectivity for CRF2R.10,132
75.6.1  CRF Receptor Expression in Relation
to ENS Codes
Early results from work on CRF receptor gene expres-
sion suggested that CRF2R is the predominant receptor
subtype expressed in the gastrointestinal tract and that the
CRF1R transcript is expressed mainly in the brain and pitu-
itary gland.123,124,126,127 Immunoreactivity for CRF1R and
CRF2R was reported, subsequently, for the upper gastroin-
testinal tract.111a CRF1R is found in a narrow zone in the
upper oxyntic gland region of the gastric corpus. CRF2R
is localized to gastric parietal cells and to enteroendocrine
cells in the stomach and duodenal mucosa and to the lumi-
nal surfaces of the crypts and around blood vessels in rat
colon. Double staining finds CRF1R coexpressed with the
neuronal markers, neuron-specific enolase and neurofila-
ment protein, in the myenteric and submucosal plexuses.111a
Immunofluorescence, RT-PCR, and Western blot evidence
now suggests that CRFR1 is the predominant stress-asso-
ciated receptor type expressed by neurons in guinea pig
and rat ENS. CRFR2 stimulation, in rat colon, offsets to
a degree stress-evoked changes mediated by CRFR1.111b
Moreover, RT-PCR reveals mRNA transcripts for CRF1R
gene expression in both myenteric and submucosal plex-
uses.98,111,129 Immunoreactivity for CRF1R is reported to be
expressed also by mucosal epithelial and enteroendocrine
cells in rat,110,129 but not guinea pig.98 In general, immuno-
histochemical localization of CRFR1 protein is consistent
with RT-PCR and Western blot evidence for expression of
CRF1R by ENS neurons. Moreover, pharmacological data
obtained with selective CRF receptor antagonists in electro-
physiological studies (see the following section) show the
excitatory actions of CRF and urocortin 1 on ENS neurons
to be mediated exclusively by CRF1R.98
75.6.2  Double-labeling Immunofluorescence
Double-labeling immunohistochemical methods are pro-
ductive for identification of the classes of ENS neurons
that express CRF1R (Figure 75.5). Figure 75.9 shows
data for the distribution of CRF1R in relation to CRF and
some of the known chemical codes for myenteric and sub-
mucosal plexuses in guinea pig small intestine. CRF1R is
found in neurons that are neighbors to neurons that express
CRF in both the myenteric and submucosal plexuses.
However, immunoreactivity for CRF is never found in neu-
rons that express CRF1R, which implies that specific sub-
sets of neurons express CRF as a neurotransmitter, while
other specific subsets express the postsynaptic receptor for
CRF neurotransmission (Figure 75.10).
2011
(A) Myenteric (B) Submucosal
Plexus Plexus
Calbindin--------------------86% Calbindin------------------100%
Calretinin ---------------------0% Calretinin---------------------0%
ChAT ------------------------74% ChAT -----------------------60%
NOS ---------------------------0% NPY -------------------------34%
NPY ---------------------------0% Somatostatin --------------29%
Somatostatin ----------------0% Substance P -------------100%
VIP -----------------------------0% VIP ----------------------------0%
FIGURE 75.9  Proportions of each neurochemical neuronal type
that express immunoreactivity for CRF1R in guinea pig small
bowel.  (A) In the myenteric plexus, only calbindin-positive neurons
(i.e., AH-type) and cholinergic (ChAT-positive) neurons express CRF1R.
(B) In the submucosal plexus, all calbindin- and substance P-positive
neurons express immunoreactivity for CRF1R. VIP-positive neurons do
not express CRF1R. (Adapted from Liu et al. 112.)
75.6.2.1  Myenteric Plexus
Immunoreactivity for the calcium-binding protein, calbi-
ndin, is a marker for neurons with AH-type electrophysi-
ological behavior and multipolar Dogiel II morphology
in the ENS.113,114,133 Most calbindin neurons (86%) in
the myenteric plexus express CRF1R (Figures 75.9 and
75.10). This is consistent with electrophysiological results
showing that CRF evokes slowly activating membrane
depolarization and action potential discharge in AH-type
neurons with Dogiel II morphology (Figure 75.6). CRF1R
is expressed by 74% of all cholinergic ENS neurons in
the myenteric plexus, as revealed by colocalization with
ChAT. Based on ChAT expression, at least ten differ-
ent functionally identified subpopulations of guinea pig
myenteric neurons can release acetylcholine at neuron-to-
neuron synapses or at neuroeffector junctions; included are
ascending and descending interneurons, calbindin-positive
AH-type neurons, and excitatory motor neurons to the
musculature and mucosal secretory glands.113
Intestinal smooth muscle, in the guinea pig, does not
express CRF1R, which suggests that CRF1R does not
mediate any direct myogenic effect of musculomotor CRF
release on contractility and therefore propulsive or mixing
motility.105 Moreover, finding of a lack of expression of
CRF1R by interstitial cells of Cajal implies that alteration
of intestinal motor responses seen during exogenous appli-
cations of CRF or during exposure of an animal to stress
does not involve any action of CRF at CRF1R receptors on
functions of interstitial cells of Cajal.105 On the other hand,
exposure of intestinal preparations to CRF in vitro evokes
excitatory junction potentials in the circular muscle of
guinea pig small intestine that reflect input from excitatory
musculomotor neurons.134 This probably reflects activation
2012 SECTION  |  VI  Consequences of Disregulated Physiology

Positive Feed-forward
Synaptic Network
Directions of Transmission

(CRF)

Calbindin-Positive
AH-Dogiel II Neurons

(CRF)

Non-Cholinergic Secretomotor/
Vasodilator Neurons
(VIP-Positive Neurons)

Secretomotor
Neurons

Secretory Glands

FIGURE 75.10  Calbindin-positive AH-Dogiel morphological type II interneurons are networked into positive feed-forward synaptic cir-
cuits.  Feed-forward synaptic excitation is a mechanism for rapid initiation of synchronous discharge in multiple neural elements of the circuit. Output
from the feed-forward circuit drives populations of secretomotor neurons, which in turn drive the behavior of the secretory epithelium and vasculature.
Positive feed-forward synaptic networks synchronize simultaneous firing of large numbers of motor neurons to ensure coordination of secretion around
and along an extended length of bowel. VIP-positive, non-cholinergic secretomotor vasodilator neurons are the only neurons that express CRF in the
submucosal plexus. When they fire, these neurons are postulated to release CRF as an excitatory neurotransmitter at postsynaptic CRF1R receptors
expressed on calbindin-positive interneurons in positive feed-forward synaptic networks that drive mucosal secretion.

of CRF1R on cholinergic and/or substance Pergic muscu-
lomotor neurons.
75.6.2.2  Submucosal Plexus
Nearly all of the neurons in the submucosal plexus of
guinea pig small intestine fit into one or the other of four
neurochemical classes (Figure 75.7).112,113 These include:
(1) neurons with multipolar Dogiel II morphology and
coexpressed immunoreactivity for calbindin, ChAT, and
SP; (2) neurons with a single axon and immunoreactiv-
ity for calretinin and ChAT; (3) neurons with single axon
morphology that coexpress NPY and ChAT; and (4) single
axonal neurons immunoreactive for VIP. Three of these
subclasses are motor neurons that stimulate mucosal secre-
tion when they fire. The first are VIP-expressing, non-
cholinergic secretomotor/vasodilator neurons that innervate
the mucosal epithelium and small arterioles. Second are
cholinergic secretomotor/vasodilator neurons that contain
calretinin, and the third subclass includes cholinergic secre-
tomotor neurons that contain NPY and do not innervate the
vasculature (Figure 75.7).
Calbindin-positive neurons with Dogiel II multipolar
morphology and AH-type electrophysiological behavior
innervate and activate subsets of secretomotor neurons.
A single nicotinic synapse connects AH-type axons with
secretomotor neurons.121,135–137
All calbindin-containing submucosal neurons with
Dogiel II morphology, like their counterparts in the mye-
nteric plexus, express CRF1R (Figure 75.9). CRF1R
appears also to be expressed by cholinergic/NPY secreto-
motor neurons that do not innervate the vasculature. On
the other hand, the receptor is not expressed by VIPergic,
non-cholinergic secretomotor/vasodilator neurons nor is it
expressed by cholinergic/calretinin-containing secretomo-
tor/vasodilator neurons.
Calbindin-positive AH-type neurons are interconnected
to form positive feed-forward circuits in which firing of
one member of the circuit evokes simultaneous firing in
all neighboring members (Figure 75.10). This is a form of
synaptic connectivity in which the neurons of the circuit
make recurrent excitatory synaptic connections with each
other.138–140 Feed-forward connectivity in the circuit causes
excitation to build rapidly to firing threshold in each of the
Chapter  |  75  Enteric Neurobiology of Stress
neurons that make up the circuit. Rapid buildup of firing in
the individually interconnected neurons in the circuit ensures
simultaneous activation of the whole network around the
circumference and along the length of a segment of bowel.
Neurons in the circuit have excitatory synaptic connections
with adjacent motor neurons. In this arrangement, output of
the circuit functions to simultaneously activate large pools
of motor neurons around the circumference and up and
down an intestinal segment. When submucosal secretomo-
tor neurons are activated in this manner, the effect is to dis-
tribute secretion of electrolytes, H2O, and mucin uniformly
around the circumference and along a length of bowel.141
The observation that calbindin-containing neurons (AH-type
neurons) express receptors for CRF, but no CRF, while VIP-
expressing, non-cholinergic secretomotor/vasodilator neu-
rons express CRF, but no receptors for CRF, suggests that
these neurons might be the source of CRF synaptic input to
AH-type neurons that make up the feed-forward circuit that
drives the cholinergic secretomotor/vasodilator neurons that
contain calretinin and the cholinergic secretomotor neurons
that contain NPY and do not innervate the vasculature.
75.7  SOURCES FOR CRF
VIP and acetylcholine are firmly established as submu-
cosal neurotransmitters at neuroepithelial and neurovas-
cular junctions that increase blood flow in concert with
stimulation of mucosal secretion.119,142 The CRF hypoth-
esis for stress-evoked stimulation of mucosal secretion and
increased barrier permeability is supported also by firm
evidence. Localization of CRF1R in the submucosal ENS
suggests that the neuronal site of action for CRF-evoked
effects of stress-induced diarrhea and opening of the
mucosal barrier to large molecules is restricted to cholin-
ergic/NPY secretomotor neurons that do not innervate
the vasculature. That this might be the case is suggested
by reports that pretreatment with the muscarinic receptor
antagonist, atropine, suppresses stress-induced stimula-
tion of mucosal secretion and increased permeability of the
mucosal barrier in animals12,101,102,143 and humans.99
The expression of receptors for CRF by AH-Dogiel
type II neurons, putative cholinergic secretomotor neu-
rons, and enteric mast cells raises a question of the ori-
gin of the CRF that could act to stimulate the receptors.
Based on the immunohistochemical localization, cholin-
ergic and substance P excitatory musculomotor neurons
or nitrergic inhibitory musculomotor neurons could be a
source of CRF release. Calbindin-positive AH-Dogiel type
II neurons, which comprise about 20% of the neurons in
the myenteric plexus,112 do not express CRF and therefore
are not a likely source. In the submucosal plexus, the only
apparent neuronal source for CRF release is the population
of VIPergic, non-cholinergic secretomotor/vasodilator neu-
rons (Figures 75.8 and 75.10).
2013
Outside the ENS, CRF is expressed near the base of
crypts in human colon and coexpressed with serotonin in
mucosal enterochromaffin cells.93 A large proportion of
calbindin-positive, AH-Dogiel II neurons in the myenteric
plexus express CRF1R, which might become a target for
CRF release from enterochromaffin cells (Figure 75.9).
Neurites from AH-Dogiel II neurons project downward
through the circular muscle coat to end in submucosal/
mucosal regions.144 The terminals in the submucosa
express serotonergic 5-HT3 receptors. Stimulation by
5-HT, released from enterochromaffin cells, fires the ter-
minals.147 Once initiated, the action potentials propagate
along the neurites in a retrograde manner to the cell body
and may fire the cell body depending on its synaptic/para-
crine input and level of excitability when they arrive (see
Chapter 19). Based on its immunohistochemical locali-
zation, CRF1R is expected to be operative alongside the
5-HT3 receptors at the AH-Dogiel II terminals.
CRF also is released locally in sites of inflammation in
animal models and inflamed joints of patients with rheuma-
toid arthritis, which suggests that CRF might have proin-
flammatory actions in the gut.145–148 Immunoreactivity
for CRF expression is significantly increased in mucosal
inflammatory cells in ulcerative colitis and is correlated
with the intensity of the inflammation.149 Expression is
restricted to inflammatory cells and is not increased in ente-
rochromaffin cells or mucosal epithelial cells in ulcerative
colitis. The association of CRF with inflammatory bowel
disease is reminiscent of evidence that subepithelial mast
cells in mucosal biopsies from human colon express immu-
noreactivity for CRF1R and CRF2R, and our preliminary
results show coexpression of CRF1R and CRF2R recep-
tors by mast cells in guinea pig colon and human jejunum
and CRF-evoked release of histamine from mast cells in
guinea pig colon.102,150 Release of chemoattractant factors
for inflammatory/immune cells occurs in concert with the
collection of other mast cell mediators in response to CRF
input. This might well be a significant factor in the associa-
tion of mucosal inflammation with stress (see Figure 75.2).
75.7.1  CRF Action on ENS Neurons
Intracellular recording with “sharp” microelectrodes is
used to study CRF1R receptor pharmacology in neurons
in the ENS (Figure 75.5).98 Electrophysiological actions
of CRF on ENS neurons are essentially the same for the
guinea pig small and large intestine. Application of CRF
in vitro evokes, with an EC50 of 25 nM, slowly activat-
ing slow EPSP-like depolarizing responses associated
with increased excitability in myenteric neurons (Figure
75.6). CRF stimulates excitability in this manner for both
neurons with single axons and S-type electrophysiologi-
cal behavior and Dogiel type II multipolar neurons with
AH-type electrophysiological behavior. An increase in
2014
membrane input resistance attributed to closure of K
channels is associated with the depolarizing responses in
AH-type neurons.151 Changes in input resistance in S-type
neurons are inconsistent, with input resistance decreased
or unchanged. The action of CRF reflects direct activa-
tion of receptors on the neuron, rather than indirect syn-
aptic input from CRF-sensitive neurons elsewhere in the
preparation, because blockade of synaptic transmission by
tetrodotoxin or lowering of Ca2 and elevation of Mg2
in the bathing medium does not change the CRF-evoked
depolarizing responses.98 Tachyphylaxis to CRF occurs
rapidly in S-type neurons (Figure 75.6) and does not occur
in AH-type neurons. This may be significant in terms of
the putative neurotransmitter role for CRF in the model in
Figure 75.10.
The excitatory responses to CRF in ENS neurons are
mediated exclusively by CRF1R. Astressin, a non-selective
CRF1R/CRF2R receptor antagonist, or the selective
CRF1R receptor antagonist, NBI 27914, both block the
excitatory responses to CRF. The CRF2R antagonist, anti-
sauvagine-30, has been tested and does not suppress or
otherwise change depolarizing responses to CRF, which is
consistent with other evidence that CRF1R mediates exci-
tatory responses to CRF in ENS neurons in guinea pig.
75.7.2  CRF–Mast Cell Connection
Evidence is accumulating that enteric mast cells are inner-
vated by CRFergic ENS neurons. Firing of the neurons
and release of CRF at postsynaptic receptors on the mast
cells is expected to release a deluge of mast cell-signaling
molecules. These may be mediators stored in granules
(e.g., histamine, proteases, and bradykinin) or obtained by
synthesis from membrane lipids (e.g., prostaglandins and
platelet-activating factor). This expectation is reinforced
by findings of immunoreactivity for CRF1R and CRF2R
receptor subtypes expressed by subepithelial mast cells in
mucosal biopsies from human colon102 and by mast cells
in guinea pig colon and human jejunum.150 Application of
CRF to segments of guinea pig and human small intestine
in vitro evokes release of mast cell histamine and protease
II into the bathing medium as determined by ELISA.150
These findings are an incentive for adding innervation of
enteric mast cells by CRF secretomotor neurons to a pos-
tulated circuit diagram for CRF neurotransmission in the
submucosal plexus in a scenario for stress, ENS, and mast
cell interaction (see Figure 75.2). The outcome of which
results in stimulation of neurogenic secretion, compromise
of the mucosal barrier, and enhanced propulsive motility.
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