CRF in the paraventricular nucleus mediates gastric

and colonic motor response to restraint stress

H. MijNNIKES, B. G. SCHMIDT, H. E. RAYBOULD, AND Y. TACHI?
Center for Ulcer Research and Education, Veterans Affairs Wadsworth Medical Center, Department
of Medicine and Brain Research Institute, University of California, Los Angeles, California 90024

H. Miinnikes, B. G. Schmidt, H. E. Raybould, and Y. as well as CRF receptors have been located in several
Tach6. CRF in the paraventricular nucleus mediates gastric brain nuclei known to play a role in the regulation of
and colonic motor response to restraint stress. Am. J. PhysioZ. visceral function (3, 26). In particular, the paraventric-
262 (Gastrointest. Liver Physiol. 25): GP37-G143, 1992.-The ular nucleus of the hypothalamus (PVN) contains nu-
role of corticotropin-releasing factor (CRF) in the paraventric-
merous CRF-like immunoreactive neurons and fibers as
ular nucleus of the hypothalamus (PVN) in the control of
gastric emptying of a nonnutrient meal and colonic transit was well as CRF receptors (3, 24, 25). Subpopulations of
investigated in conscious fasted rats chronically implanted with neurons in the PVN send direct projections to medullary
hypothalamic cannulas and catheters in both the stomach and and spinal preganglionic neurons controlling autonomic
proximal colon. CRF (0.06-0.6 nmol) microinfused unilaterally nervous system activity (14, 25). There is also evidence
into the PVN resulted in a dose-dependent inhibition of gastric in rats that CRF microinjected into the PVN inhibits
emptying (O-51%) and stimulation of colonic transit (O-93%). gastric acid secretion (28) and that electrical stimulation
CRF (0.6 nmol) -induced delay in gastric emptying was inhibited of this nucleus influences gastric motility (19). Therefore,
by 50% by subdiaphragmatic vagotomy or atropine methyl the aim of the present study was to determine whether
nitrate (1 mg/kg ip), whereas the stimulation of colonic transit CRF in the PVN may play a role in mediating the effect
was completely abolished by atropine methyl nitrate and re-
duced by 19% by vagotomy. Microinfusion of CRF (0.6 nmol)
of restraint stress on gastric and colonic transit in con-
into the lateral hypothalamus or central amygdala had no scious rats. Part of this work has been reported in ab-
effect. Restraint exposure for 1 h delayed gastric emptying and stract form (15).
stimulated colonic transit by 28 and 78%, respectively. Bilateral
microinfusion of the CRF antagonist a-helical CRF-(9-41) MATERIALS AND METHODS
(13 nmol) into the PVN before restraint abolished stress-
induced alterations of gastric and colonic transit. The CRF Animals
antagonist did not alter basal gastric and colonic transit under Male Sprague-Dawley rats (Harlan, Indianapolis, IN) weigh-
basal conditions. These data indicate that the PVN is a specific ing 290-370 g were housed under conditions of controlled
responsive site for central CRF-induced alterations of gastric illumination (12:12-h light-dark cycle), humidity, and temper-
and colonic transit and suggest that endogenous CRF in the ature (22 t 2OC) for at least 7 days before the surgical procedure.
PVN plays a role in mediating restraint stress-related altera- They were fed a standard rat diet (Purina Laboratory Chow)
tions of gastrointestinal transit. and maintained in colony cages. After surgery, rats were housed
individually and fed a liquid diet (Isocal tube feeding formula-
lateral hypothalamus; central amygdala; central nervous system
control of gastrointestinal transit; gastrointestinal transit; tion, Mead Johnson Nutritional Division, Evansville, IN). An-
imals had ad libitum access to the liquid diet and tap water and
brain; restraint; corticotropin-releasing factor antagonist
gained weight under these conditions.
EMOTIONS AND EXPOSURE to stressful stimuli are well
known to alter gastrointestinal (GI) motor function in Surgery
experimental animals and humans (16, 27, 32). More- Rats were anesthetized using a mixture of ketamine (75 mg/
over, stress-related alteration of bowel function is a kg ip; Fort Dodge Laboratories, Fort Dodge, IA) and xylazine
prevalent clinical GI syndrome (22). Growing evidence (5 mg/kg ip; Mobay, Shawnee, KS) and implanted with silicone
indicates that corticotropin-releasing factor (CRF) is catheters (1.2 mm ID, 1.7 mm OD) into the gastric fundus and
involved in mediating the effects of various stressors on the proximal colon. In one group of rats, vagotomy was per-
formed immediately after catheter implantation by circular
GI motor function (1,29). CRF injected into the cerebro-
seromuscular myotomy of the esophagus at 2 cm from the
spinal fluid mimics the effects of certain forms of stress gastroesophageal junction and ligation of the vagal trunk trav-
on GI transit, most markedly inhibiting gastric emptying eling with the left gastric artery. Catheters were fixed at the GI
of a nonnutrient meal and increasing colonic transit in wall by a purse-string suture and subcutaneously exteriorized
rats and mice (12, 23, 31, 33). CRF action is central at the interscapular region, where they were secured at the
nervous system (CNS) -mediated through the autonomic animal’s skin. Additionally, a 22-gauge guide cannula was po-
nervous system independently from activation of the sitioned unilaterally 3 mm above the PVN, the lateral hypo-
pituitary-adrenal axis (12, 31). Furthermore, injection of thalamus (LH), or the central amygdaloid nucleus (CA) by
the selective CRF antagonist a-helical CRF-(9-41) (18) using a stereotaxic instrument (David Kopf Instruments, Tu-
junga, CA). Groups of rats to be exposed to restraint were
into the cerebrospinal fluid prevents the alterations of
implanted with 26-gauge bilateral guide cannulas positioned 3
gastric and colonic transit induced by exposure to wrap mm above the PVN. The stereotaxic coordinates used for guide
restraint, noise, cold, and surgery in rodents (2, 13, 30, cannula placement were derived from the atlas of Paxinos and
33). However, the specific brain nuclei at which CRF or Watson (17). The cannulas were anchored by dental cement
the CRF antagonist acts to influence GI motor function and stainless steel screws affixed to the skull. All experiments
are still unknown. CRF immunoreactive cells and fibers were performed 6-7 days after surgery in 18-h liquid food- but
0193~1857/92 $2.00 Copyright 0 1992 the American Physiological Society G137

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Copyright © 1992 American Physiological Society. All rights reserved.
G138 CRF IN PVN MEDIATES GI TRANSIT DURING RESTRAINT

not water-deprived awake rats chronically implanted with brain ‘Cr (0.4 &i) in 23.5 ml of 0.15 M NaCl. Gastric emptying was
cannulas as well as gastric and colonic catheters. calculated as previously described (33): 100 - (disintegrations
per minute in sample x lOO/disintegrations per minute in
Drugs standard). To assess the gastric emptying rate, the emission of
1 ml of the supernatant was determined for 1 min in a gamma-
The following drugs were used: synthetic rat CRF (kindly counter. The large bowel, separated from the cecum, was cut
provided by Dr. J. Rivier, Salk Institute) was dissolved imme- into 10 equal segments. To assess the movement of the marker,
diately before the experiment in 0.1% bovine serum albumin the emission of each segment was determined for 1 min, and
(BSA) in saline. a-Helical CRF-(9-41) (Bachem, Torrance, the geometric center was calculated as previously described
CA) was dissolved just before injection in sterile saline warmed (33). Small numbers indicate a slow transit, and high numbers
at 37°C. Alcian blue 8GX, atropine methyl nitrate, and xan- signify a fast transit.
thum gum were obtained from Sigma (St. Louis, MO) and ‘Cr
from New England Nuclear (Boston, MA). Alcian blue, atro-
Brain Histology
pine methyl nitrate, and xanthum gum were dissolved in 0.15
M NaCl. Brain sections of 42 ,um were examined after performing a
Nissl staining. Visualization of the tip of the injection needle
Study Design track was considered as microinfusion site and marked on
plates reproduced from the atlas of Paxinos and Watson (17).
Effect of CRF microinjection into the hypothalamic nuclei. When alcian blue dye was detected in the third ventricle,
CRF or vehicle (0.1% BSA in saline) was microinfused into animals were excluded from data analyses (:20% of all PVN
hypothalamic nuclei in conscious rats lightly restrained during microinjections with saline or CRF).
the microinjection procedure. A total volume of 100 nl was
delivered over 30 s through a 30G injection cannula (Plastic
Products, Roanoke, VA) lowered 3 mm below the end of the Statistical Analysis
guide cannula using a 2-~1 Hamilton syringe. Then, a semiliquid Results are expressed as means of: SE. Data were analyzed
nonabsorbable marker (250 mg of xanthum gum initially dis- by ANOVA, and differences between groups were evaluated by
solved in 10 ml of 0.15 M NaCl containing radioactive marker the Newman-Keuls multiple range test.
“Cr) was injected into the stomach (0.4 &i in 1.5 ml of
xanthum gum) and the colon (0.4 &i in 0.2 ml of xanthum
gum), and the catheters were flushed with 0.2 ml of 0.15 M RESULTS
NaCl. Rats were returned to their home cages and maintained
in a nonstressful environment. Sixty minutes after injection of Effects of CRF Microinjected Into the PVN
markers, animals were anesthetized with ketamine (75 mg/kg
ip) and xylazine (5 mg/kg ip), and 100 nl of 0.05% alcian blue Unilateral microinfusion of CRF into the PVN at 0.06,
8GX was microinfused into the brain under the same conditions 0.2, or 0.6 nmol dose dependently inhibited gastric emp-
as CRF. Stomach and colon were carefully excised to avoid any tying of a semisolid nonnutrient meal and stimulated
dislocation of the markers. After transcardial perfusion with colonic transit in conscious rats as measured 1 hr after
0.15 M NaCl and 10% formaldehyde the brain was removed peptide and GI administration of markers (Fig. 1). At 0.2
and conserved in 20% sucrose for 24 h. nmol CRF, gastric emptying was significantly reduced
In one group of rats, pretreatment with atropine methyl from 75 & 9 to 44 t 12% (P < 0.05) and colonic transit
nitrate (1 mg/kg ip in 1 ml/kg) was performed 15 min before
injection of the GI markers.
was increased from 3.2 t 0.4 to 4.9 t 0.5 (P < 0.01).
Effect of restraint stress and microinjection of CRF antagonist. Microinfusion at 0.6 nmol into the PVN further inhibited
One week before the experiment, rats chronically implanted gastric emptying to 35 t 8% (P < 0.005) and markedly
with bilateral cannulas (Plastic Products) into the PVN were increased geometric center to 6.2 t 0.8 (P < 0.005) (Fig.
microinjected bilaterally with CRF (0.6 nmol/site). Animals 1). One-third of these animals showed a profound diar-
that responded to CRF by increased fecal output and diarrhea rhea. Microinfusion of CRF at 0.6 nmol into sites just
were selected for study on the effect of restraint on gastric and adjacent to the boundaries of the PVN (Fig. 2) did not
colonic motor function. Animals were subjected for 60 min to alter significantly the rate of gastric emptying (60 t 14%,
a nonulcerogenic form of restraint (13). Each rat was placed in n = 6) or the average geometric center (3.4 t 0.3, n = 6).
a metal mesh tube allowing slight movements of head and legs
Pretreatment with atropine methyl nitrate (1 mg/kg
but not of the trunk. a-Helical CRF-(9-41) (13 nmol/site in
250 nl) or vehicle (sterile water) was bilaterally microinfused
ip) or truncal vagotomy did not modify basal gastric
into the PVN through a 33-gauge injection cannula 15 min emptying (72 t 7 and 69 t 5%, respectively) or colonic
before restraint exposure. Higher volume was used for the transit (geometric center, 3.5 t 0.5 and 2.7 t 0.3, respec-
microinfusion of CRF antagonist to ensure proper delivery of tively) in rats microinfused with vehicle into the PVN
the peptide in solution and not in suspension. Subsequent compared with control group (gastric emptying, 75 t 9%
procedures of injection of the markers into the stomach and geometric center, 3.2 t 0.4) (Fig. 1). Pretreatment with
colon were performed as described above. Histological evalua- atropine methyl nitrate and truncal vagotomy reduced
tion after the experiment confirmed a correct placement within by 50% the inhibitory effect of CRF (0.6 nmol) microin-
the PVN in -80% of the animals. The remaining 20% were fused into the PVN on gastric emptying (Fig. 1). The
mislocated into the third ventricle and thus excluded from the
stimulation of colonic transit induced by microinfusion
data analyses.
Measurement of gastric emptying and colonic transit. The of CRF (0.6 nmol) into the PVN was completely abol-
stomach was homogenized in 10 ml of 0.15 M NaCl and then ished by atropine methyl nitrate pretreatment and atten-
0.15 M NaCl was added to the homogenate to yield a total uated by truncal vagotomy from 6.2 t 0.8 (CRF) to 5.0
volume of 25 ml, which then was centrifuged at 5,000 g for 20 t 0.8 (CRF plus vagotomy), but values were still signif-
min. A standard solution was prepared by diluting 1.5 ml of icantly higher than the vehicle-treated group (Fig. 1).

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Copyright © 1992 American Physiological Society. All rights reserved.
 CRF IN PVN MEDIATES GI TRANSIT DURING RESTRAINT G139
UVEHICLE (vehicle, 75 _+ 5%; CRF antagonist, 83 t 7%) and the
BKRF, 0.06 nmol colon (vehicle, 3.2 t 0.4; CRF antagonist, 3.7 t 0.3). In
BlSPCRF, 0.20 nmol
loo-
-CRF, 0.60 nmol rats microinjected bilaterally into the PVN with a-helical
CRF-(g-41) 15 min before stress exposure, restraint did
(3 80- not significantly alter gastric emptying (72 t 6%) and
Z- colonic transit (3.6 t 0.5) (Fig. 4).
c
II 60- p(O.01
Zn DISCUSSION
W#?
o-
E 409
CRF injected into the cerebrospinal fluid is well estab-
lished to decrease gastric emptying and to increase co-
5 ionic transit in rats (12, 31, 33). In the present study,
c3 zo- microinfusion of CRF into the PVN also resulted in a
pronounced and dose-dependent inhibition of gastric
5 5 5 6 emptying of a non-nutrient meal and stimulation of
NO PRETREATMENT ATROPINE VAGOTOMY colonic transit in conscious, unrestrained, and fasted
1 mg/kg I.P.
rats. The threshold dose to induce alterations of gastric
and colonic transit was the same, suggesting that both
the stomach and the colon are equally responsive to
central CRF. These data indicate that the PVN is a
pa.oos
responsive site of action for CRF-induced alterations of
gastric and colonic transit and provide the first demon-
stration that the PVN can influence colonic motor func-
tion in conscious rats. The PVN has been established
previously to influence gastric secretion and contractility
(6, 19, 21, 28) and as reported here, transit. These find-
ings along with previous data indicate that the PVN
might play an important integrative role in the control
of both upper and lower gut function.
O-
NO PRETREATMENT ATROPINE VAGOTOMY Several lines of evidence indicate that CRF action in
1 m9/k9 1.P. the PVN to alter gastric and colonic transit is site
Fig. 1. Dose-dependent inhibitory effect on gastric emptying and stim- specific. First, the effects of PVN microinjections could
ulatory effect on colonic transit of unilateral corticotropin-releasing not be due to leakage into the ventricular system, since
factor (CRF) microinfusion into the paraventricular nucleus of the microinjection of a dye at the end of each experiment
hypothalamus (PVN). Effects of atropine pretreatment or vagotomy allowed us to eliminate from the analysis microinjections
on these alterations. Bars represent means t SE of the number of rats
indicated on each bar.
that were leaking into the third ventricle. It is also
unlikely that CRF acts by diffusing to another site
Effect of CRF Microinjected Into the LH, Central through backtracking along the cannula, since CRF (0.6
Amygdala, and Outside the Boundary of the PVN nmol; Fig. 3) microinjected outside of the boundary of
the PVN into medial hypothalamic sites, including sites
Unilateral microinfusion of CRF into the lateral hy- ventral and dorsal to the PVN, had no effect. Second,
pothalamus (0.2 or 0.6 nmol) or into the central amygdala we investigated the lateral hypothalamus, since it con-
(0.6 nmol) did not significantly modify gastric emptying tains CRF-like immunoreactivity (CRF-LI) and CRF
or colonic transit compared with the vehicle-treated receptors (3, 25). The LH has also been shown to influ-
group (Fig. 3). ence GI motor function (4,11,20), and CRF microinfused
CRF (0.2 nmol) microinfused into other medial hy- into the LH reduced gastric acid secretion at 0.45 nmol
pothalamic sites (Fig. 2), such as the anterior and medial and inhibited acid secretion significantly at a dose of 0.9
preoptic hypothalamic area (n = 4), bed nucleus of stria nmol CRF (28). However, microinjection of CRF into
terminalis (n = 2), or anterior hypothalamic nucleus (n the LH at doses (0.2 and 0.6 nmol) found to be active in
= 2), did not significantly influence gastric emptying (70 the PVN had no effect on both monitored parameters.
t 7%) or colonic transit (3.8 t 0.2) in conscious rats. Although it cannot be excluded that CRF injected into
the LH at a higher dose may have altered GI transit, the
Effect of Restraint Stress and CRF-Antagonist present data show that the PVN is a more sensitive site
Microinjection Into the PVN of action. In addition, CRF (0.6 nmol) microinfused into
the central amygdala had no effect on gastric or colonic
In animals microinjected with vehicle into the PVN, transit. This forebrain area contains abundant CRF-LI
restraint exposure for 1 h markedly inhibited gastric positive cells and terminals and also CRF receptors (3,
emptying to 54 t 5% (P c 0.005) and stimulated colonic 5). Furthermore, the CA is thought to be involved in
motor function, increasing the geometric center to 5.7 t autonomic and neuroendocrine responses to stress as
0.4 (P < 0.01) (Fig. 4). In a nonstressful environment, CY- well as in induction of experimental gastric ulcer (5).
helical CRF- (9-41) (13 nmol/site) bilaterally microin- However, our data do not support an action of CRF at
fused into the PVN did not modifv transit in the stomach this site in influencing GI transit. Taken together. these

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Copyright © 1992 American Physiological Society. All rights reserved.
G140 CRF IN PVN MEDIATES
results emphasize the specificity of CRF action in the
PVN to regulate GI transit. However, a recent study
indicates that CRF can also influence gastric motor
function through an action at the brain stem level. Mi-
croinfusion of CRF into the dorsal vagal complex inhibits
gastric contractility stimulated by central vagal activa-
tion (9).
Our technique of microinjection at a volume of 100 nl
does not allow us to accurately discriminate between
macro- and parvocellular microinjection sites. Neverthe-
less, a further assessment of the efficacy of CRF microin-
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Copyright © 1992 American Physiological Society. All rights reserved.
GI TRANSIT DURING RESTRAINT
Fig. 2. Diagram of coronal sections of the
rat brain showing each microinfusion site
used in the data analyses for the dose of 0.6
nmol CRF microinjected into various brain
nuclei (PVN, l ; LH, A; CA, n ; other sites, *)
and outside the boundaries of these nuclei.
Injection sites of vehicle were similarly dis-
tributed as CRF sites and are not shown.
Numbers in each section give the distance
of the plate from the bregma. Plates are
adapted from Paxinos and Watson (17).
fusion into the PVN at a dose of 0.6 nmol indicates that
the most responsive sites for influencing both gastric and
colonic transit were located within or near anterior and
medial parvocellular parts of the nucleus. It should be
also mentioned that at a 0%nmol dose, CRF microin-
fused into the posterior areas of the nucleus did not
affect GI transit, whereas microinfusion of the peptide
into or near the medial and most pronounced anterior
parvocellular parts of the PVN induced the most signif-
icant response. These observations suggest that the ef-
fects of CRF on gastric and colonic transit are mainly
 CRF IN PVN MEDIATES GI TRANSIT DURING RESTRAINT G141
LATERAL HYPOTHALAMUS CENTRAL AMYGDALA
well correlated with neuroanatomical studies suggesting
that this hypothalamic nucleus can be divided into func-
tional zones on the basis of efferent projections to auto-
nomic centers and anterior or posterior pituitary (26).
The parvocellular division of the hypothalamic nucleus
contains most of the cell group projecting to the inter-
mediolateral column of the spinal cord and to the dorsal
vagal complex (14, 25).
Previous studies (12, 23, 31) have shown that altera-
tions of GI transit induced by injection of CRF into the
6
cerebrospinal fluid are mediated through the autonomic
nervous system in rats and mice. Increase in colonic
transit is prevented by vagotomy but not sympathetic
blockade (l2), whereas delay in gastric emptying was
found to be blocked (31) or not modified by vagotomy
but reserved by bretylium (12). In the present study, the
role of the parasympathetic nervous system in mediating
CRF in the PVN-induced alterations of GI transit was
investigated using truncal vagotomy and peripheral mus-
carinic blockade by atropine methyl nitrate, which does
not cross the blood-brain barrier. The inhibitory effect
6
1 of CRF (0.6 nmol) microinfused into the PVN on gastric
0.6 0.6
emptying was reduced by 50% either by pretreatment
CRF (nmol/rat) with atropine methyl nitrate (1 mg/kg ip) or vagotomy,
Fig. 3. CRF microinfusion unilaterally into the lateral hypothalamus whereas the stimulation of colonic transit was completely
or the central amygdala did not alter gastric or colonic transit. Bars blocked by atropine methyl nitrate and partially reduced
represent means & SE of the number of rats indicated on each bar. by truncal vagotomy. These results indicate that the
increase in colonic transit is primarily mediated by pe-
I VEHICLE
loo m a-HUICAL CRF (g-41), 13 nmol ripheral muscarinic receptors, probably through activa-
1
tion of sacral parasympathetic pathways, since the block-
ing effect of truncal vagotomy is only 36%. Peripheral
vagal-muscarinic pathways contribute also to the expres-
p(O.005 sion of peptide action on gastric emptying. However,
other mechanisms may involve activation of sympathetic
preganglionic neurons acting through postganglionic
neurons and a-adrenoreceptors along with presynaptic
inhibition of acetylcholine release. Populations of neu-
a 6 6 6
rons in the PVN have monosynaptic connections to the
dorsal vagal complex in the brain stem and to the inter-
CONTROL RESTRAINT (lh)
mediolateral column in the spinal cord innervating par-
asympathetic and sympathetic preganglionic cells, re-
spectively (14, 25). These projections provide the neu-
roanatomical basis for the influence of the PVN on vagal
p(O.01
and sympathetic as well as on sacral parasympathetic
outflow and thereby for the effects on the stomach and
T-l colon.
It has been demonstrated that the alterations of GI
transit induced by CRF injection into the cerebrospi .nal
fluid are independent of the hypothalamic-hypophyseal-
OJ '
10 6 6 6 adrenal axis as shown by experiments in hypophysecto-
CONTROL RESTRAINT (1 h) mized or adrenalectomized rats or with naloxone pre-
treatment (12, 23, 31). The complete blockade of the
Fig. 4. Pretreatment with CRF antagonist a-helical CRF-(9-41) mi-
croinfused bilaterally into the PVN blocks restraint of stress-induced
colonic response to CRF into the PVN by atropine
alterations of gastric emptying and colonic transit. Bars represent suggests that a PVN-pituitary contribution is unlikely.
means & SE of the number of rats indicated on each bar. Further studies in hypophysectomized animals wi ll bring
conclusive evidence ruling out such a possibility.
mediated by medial and anterior areas corresponding to Restraint has been shown to inhibit gastric emptying
parvocellular cell groups. These findings are parallel to and to increase colonic transit of nonnutrient marker in
previous results in which a similar subset distribution of fasted, conscious rats (13, 33, present observations).
responsive sites in the PVN was observed for the inhib- There is convincing evidence from several groups of
itory effect of bombesin on gastric acid secretion in rats investigators that endogenous CRF may mediate these
(6). These neuropharmacological observations are also effects (13, 33) through brain pathways yet to be eluci-

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Copyright © 1992 American Physiological Society. All rights reserved.
G142 CRF IN PVN MEDIATES GI TRANSIT DURING RESTRAINT

dated. The present neuropharmacological observations Received 20 August 1990; accepted in final form 12 August 1991.
along with existing neuronatomical data support a role
of CRF in the PVN in mediating restraint-induced alter- REFERENCES
ations of gastric and colonic transit. First, unilateral
microinfusion of CRF into the PVN resembles the effects 1. Buena, L. Role of corticotropin-releasing factor in the genesis of
of restraint stress on gastric and colonic transit. Second, gastrointestinal motor disturbances induced by stress: an overview.
bilateral microinfusion of the CRF antagonist a-helical In: Stress and Digestive MotiZity, edited by L. B&no, S. Collins,
and J. L. Junien. Montrouge: Libbey Eurotext, 1989, p. 141-149.
CRF-(9-41) into the PVN abolished restraint stress- 2. Buena, L., and M. Gu& Evidence for the involvement of corti-
induced alterations of gastric and colonic transit. Third, cotropin-releasing factor in the gastrointestinal disturbances in-
various physical and psychological stressors, including duced by acoustic and cold stress in mice. Brain Res. 441: l-4,
restraint, activate CRF-containing neurons in the PVN, 1988.
resulting in an increase of CRF mRNA in this nucleus 3. De Souza, E. B., T. R. Insel, M. H. Perrin, J. Rivier, W. W.
Vale, and M. J. Kuhar. Corticotropin-releasing factor receptors
(7, 8). Moreover, ultrastructural studies revealed that are widely distributed within the rat central nervous system: an
CRF terminals can synapse on CRF neurons and den- autoradiographic study. J. Neurosci. 5: 3189-3203, 1985.
drites, indicating that CRF itself could modulate the 4. Feng, H. S., R. J. Brobeck, and F. P. Brooks. Lateral hy-
secretory activity of the CRF neuronal network in the pothalamic sites in cats for stimulation of gastric antral contrac-
hypothalamic paraventricular nucleus (24). Our obser- tions. CZin. Inuest. Med. 10: 140-144, 1987.
5. Gray, T. S. The organization and possible function of amygdaloid
vations indirectly support this concept. There is some corticotropin-releasing factor pathways. In: dorticotropin-Releas-
evidence that most CRF-LI neurons in the PVN are of ing Factor: Basic and Clinical Studies of a Neuropeptide, edited by
intranuclear origin, but is is uncertain whether all CRF E. B. De Souza and C. B. Nemeroff. Boca Raton, FL: CRC, 1989,
terminals originate from within that nucleus (24). The p. 53-68.
PVN receives neuronal input from several brain areas, 6. Gunion, M. W., and Y. Tache. Bombesin microinfusion into the
paraventricular nucleus suppresses gastric acid secretion in the rat.
e.g., the dorsal vagal complex, locus coeruleus, parabrach- Brain Res. 422: 118-128, 1987.
ial nucleus, the subfornical organ, and the hypothalamus 7. Haas, D. A., and S. R. George. Single or repeated mild stress
itself (26), and neural input to CRF neurons influences increases synthesis and release of hypothalamic corticotropin-
the CRF release from neurosecretory cells in the PVN releasing factor. Brain Res. 461: 230-237, 1988.
(25, 26). In particular, abundant neuropeptide Y (NPY) 8. Harbuz, M. S., and S. L. Lightman. Responses of hypothalamic
and pituitary mRNA to physical and psychological stress in the
input to CRF neurons in that nucleus has been demon- rat. J. EndocrinoZ. 122: 705-711, 1989.
strated (26). NPY injected into the lateral ventricle 9. Heymann-Monnikes, I., Y. Tache, M. Trauner, H. Weiner,
blocks intracerebroventricular CRF- and stress-induced and T. Garrick. CRF microinjected into the dorsal vagal complex
increase in cecal motor motility in rats (lo), suggesting inhibits TRH analog- and kainic acid-stimulated gastric contrac-
tility in rats. Brain Res. 554: 139-144, 1991.
a possible interaction between CRF and NPY in the
10. Jimenez, M., and L. Buena. Inhibitory effects of neuropeptide
PVN in stress-related alterations of GI motility. Y (NPY) on CRF and stress-induced cecal motor response in rats.
In summary, our results demonstrate that the PVN is Life Sci. 47: 205-211, 1990.
responsive to CRF to alter propulsive motor function of 11. Lee, Z. L. Effects of stimulation of the satiety and feeding centers
the stomach and colon in a similar fashion as restraint- on gastric, cecal and rectal motility in the rat. Acta Med. Okayama
36:213-222,1982.
stress does in conscious rats. CRF action is site specific 12. Lenz, H. J., M. Burlage, A. Raedler, and H. Greten. Central
and cannot be duplicated in other lateral or medial nervous system effects of corticotropin-releasing factor on gas-
hypothalamic sites and the central amygdaloid nucleus. trointestinal transit in the rat. Gastroenterology 94: 598-602, 1988.
The effect of exogenous CRF in the PVN on gastric 13. Lenz, H. J., A. Raedler, H. Greten, W. W. Vale, and J. E.
Rivier. Stress-induced gastrointestinal secretory and motor re-
emptying is partially mediated by vagal-cholinergic path-
sponses in rats are mediated by endogenous corticotropin-releasing
ways. The stimulation of colonic transit by CRF microin- factor. Gastroenterology 95: 1510-1517, 1988.
jetted into the PVN is cholinergic dependent; the ana- 14. Luiten, P. G. M., G. J. Ter Horst, H. Karst, and A. B.
tomical substrate of this pathway still remains to be Steffens. The course of paraventricular hypothalamic efferents to
determined. Our results further indicate that endogenous autonomic structures in medulla and spinal cord. Brain Res. 329:
374-378,1985.
CRF in the PVN is involved in mediating restraint 15. Monnikes, H., H. E. Raybould, and Y. Tache. CRF microin-
stress-induced alterations of gastric and colonic motor fused into the paraventricular nucleus (PVN) inhibits gastric emp-
activity. Our findings provide insight in the comprehen- tying and stimulates colonic transit in the conscious rat (Abstract).
sion of hypothalamic nuclei regulating GI transit. This Gastroenterology 98: A512, 1990.
information may have implications for the understand- 16. Narducci, F., W. J. Snape, W. M. Battle, Jr., R. L. London,
and S. Cohen. Increased colonic motility during exposure to a
ing of underlying mechanisms through which stress al- stressful situation. Dig. Dis. Sci. 30: 40-44, 1985.
ters GI motor function and may affect functional GI 17. Paxinos, G., and C. Watson. In: The Rat Brain in Stereotaxic
disorders such as the irritable bowel syndrome. Coordinates. Sydney: Academic, 1982, p. l-82.
18. Rivier, J., C. Rivier, and W. Vale. Synthetic competitive
The authors are grateful to Dr. Mark Gunion for helping set up the antagonists of corticotropin-releasing factor: effect of ACTH se-
experimental model; Dr. Jean Rivier, Salk Institute, La Jolla, CA, for cretion in the rat. Science Wash. DC 224: 889-891, 1984.
his generous donation of rat CRF; and Paul Kirshbaum for editorial 19. Rogers, R. C., and G. E. Hermann. Oxytocin, oxytocin antag-
assistance. onist, TRH, and hypothalamic paraventricular nucleus stimulation
This work was supported by the National Institutes of Health Grants effects on gastric motility. Peptides 8: 505-513, 1987.
DK-33061 and MH-00663. H. Monnikes is the recipient of a fellowship 20. Rostad, H. Colonic motility in the cat. 3. Influence of hypothal-
from the Deutsche Forschungsgemeinschaft. amic and mesencephalic stimulation. Acta Physiol. Stand. 89: 104-
Address for reprint requests: Y. Tache, Center for Ulcer Research 115,1973.
and Education, VA Wadsworth Medical Ctr., Bldg. 115, Rm. 203, 21. Sakaguchi, T., and M. Ohtake. Inhibition of gastric motility
Wilshire and Sawtelle Blvd., Los Angeles, CA 90073. induced by activation of the hypothalamic paraventricular nucleus.

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 CRF IN PVN MEDIATES GI TRANSIT DURING RESTRAINT G143

Brain Res. 335: 365-367, 1985. 29. Tache, Y., M. W. Gunion, and R. Stephens. CRF: central
22. Sandler, R. S. Epidemiology of irritable bowel syndrome in the nervous system action to influence gastrointestinal function and
United States. Gastroenterology 99: 409-415, 1990. role in the gastrointestinal response to stress. In: Corticotropin-
23. Sheldon, R. J., Q. Jiang, F. Porreca, and L. A. Fisher. Releasing Factor: Basic and Clinical Studies of a Neuropeptide,
Gastrointestinal motor effects of corticotropin-releasing factor in edited by E. B. DeSouza and C. B. Nemeroff. Boca Raton, FL:
mice. Regul. Peptides 28: 137-151, 1990. CRC, 1989, p. 300-307.
24. Silverman, A. J., A. HOU-Yu, and W. P. Chen. Corticotropin- 30. Tache, Y., E. Barquist, R. L. Stephens, and J. Rivier. Ab-
releasing factor synapses within the paraventricular nucleus of the dominal surgery- and trephination-induced delay in gastric emp-
hypothalamus. Neuroendocrinology 49: 291-299, 1989. tying is prevented by intracisternal injection of CRF antagonist in
25. Swanson, L. W., and P. E. Sawchenko. Hypothalamic integra- the rat. J. Gastrointest. Motility 3: 19-25, 1991.
tion: organization of the paraventricular and supraoptic nuclei. 31. Tache, Y., M. Maeda-Hagiwara, and C. M. Turkelson. Cen-
Annu. Rev. Neurosci. 6: 269-324, 1983. tral nervous system action of corticotropin-releasing factor to
26. Swanson, L. W., P. E. Sawchenko, and R. W. Lind. Regula-
inhibit gastric emptying in rats. Am. J. Physiol. 253 (Gastrointest.
tion of multiple peptides in CRF parvocellular neurosecretory
Liver Physiol. 16): G241-G245, 1987.
neurons: implications for the stress response. Prog. Brain Res. 68:
169-190,1986. 32. Williams, C. L., and T. F. Burks. Stress, opioids, and gastroin-
27. Tache, Y. Stress-induced alterations of gastric emptying. In: testinal transit. In: Neuropeptides and Stress, edited by Y. Tache,
Stress and Digestive Motility, edited by E. B&no, S. Collins, and J. E. Morley, and M. R. Brown. New York: Springer-Verlag, 1988.
J. L. Junien. Montrouge: Libbey Eurotext, 1989, p. 123-132. 33. Williams, C. L., J. M. Peterson, R. G. Villar, and T. F.
28. Tache, Y., and M. Gunion. Corticotropin-releasing factor: cen- Burks. Corticotropin-releasing factor directly mediates colonic
tral action to influence gastric secretion. Federation Proc. 44: 255- responses to stress. Am. J. Physiol. 253 (Gastrointest. Liver Physiol.
258,1985. 16): G582-G586,1987.

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Copyright © 1992 American Physiological Society. All rights reserved.