CURE: Digestive Diseases Research Center, Veterans Affairs Greater Los Angeles Healthcare System, Department of Medicine, Digestive Diseases Division, and Brain Research Institute, University of California, Los Angeles, California 90073
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ABSTRACT |
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Corticotropin-releasing factor (CRF) injected peripherally induces clustered spike-burst activity in the proximal colon through CRF1 receptors in rats. We investigated the effect of intraperitoneal CRF on proximal colon ganglionic myenteric cell activity in conscious rats using Fos immunohistochemistry on the colonic longitudinal muscle/myenteric plexus whole mount preparation. In vehicle-pretreated rats, there were only a few Fos immunoreactive (IR) cells per ganglion (1.2 ± 0.6). CRF (10 µg/kg ip) induced Fos expression in 19.6 ± 2.1 cells/ganglion. The CRF1/CRF2 antagonist astressin (33 µg/kg ip) and the selective CRF1 antagonist CP-154,526 (20 mg/kg sc) prevented intraperitoneal CRF-induced Fos expression in the proximal colon (number of Fos-IR cells/ganglion: 2.7 ± 1.2 and 1.0 ± 1.0, respectively), whereas atropine (1 mg/kg sc) had no effect. Double labeling of Fos with protein gene product 9.5 revealed the neuronal identity of activated cells that were encircled by varicose fibers immunoreactive to vesicular acetylcholine transporter. Fos immunoreactivity was mainly present in choline acetyltransferase-IR nerve cell bodies but not in the NADPH-diaphorase-positive cells. These results indicate that peripheral CRF activates myenteric cholinergic neurons in the proximal colon through CRF1 receptor.
acetylcholine; corticotropin-releasing factor; CP-154,526; enteric nervous system; Fos; NADPH-diaphorase
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INTRODUCTION |
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CORTICOTROPIN-RELEASING FACTOR (CRF) is a 41-amino acid peptide of hypothalamic origin, initially established to be the main regulator of stress-related activation of the pituitary adrenal axis (55). Subsequent studies established that the activation of CRF pathways in the brain also plays an important role in the behavioral, autonomic, visceral, and immune responses to stress (15, 50). In addition, increasing evidence indicates that CRF and CRF-related peptides administered peripherally exert actions on the cardiovascular, immune, gastrointestinal, vascular, and reproductive systems that may have pathophysiological relevance (3, 18, 29, 31, 39). In the lower gut, peripheral administration of CRF stimulates propulsive colonic motor function as shown by the increase in colonic motility, transit, and defecation in rats (7, 29, 56). Likewise, intravenous injection of CRF increases motility in the sigmoid colon in healthy subjects and more prominently in patients with irritable bowel syndrome (10).
The biological actions of CRF are mediated through activation of
CRF1 and CRF2 receptors that have been cloned
from two distinct genes (42). CRF receptors belong to the
seven transmembrane domain family positively coupled to adenylate
cyclase via G proteins (42). The development of peptide
antagonists nonselective for CRF1 and CRF2,
namely -helical CRF9-41 and, more recently, astressin (14), as well as selective CRF1
antagonists, CP-154,526 and antalarmin (11, 48), provides
tools to assess the biological role of CRF receptors. In rats, we
previously reported (29) that astressin or CP-154,526
injected peripherally antagonized intraperitoneal CRF-induced clustered
spike-burst activity in the proximal colon and defecation. CP-154,526
injected peripherally also alleviates water avoidance stress-induced
defecation and morphine withdrawal stress-induced diarrhea in rats
(28, 29). In monkeys, oral administration of antalarmin
reduced a broad range of anxiety-related biological responses to an
acute social stress including urination and defecation
(15). These observations support the implication of CRF
receptors, mainly CRF1, in the colonic motor response to
acute stress. However, the mechanism(s) through which peripheral CRF
stimulates colonic motility remain largely unknown (29,
30). At the cellular level, intracellular recording of enteric
neuronal activity has demonstrated that CRF applied directly on a
longitudinal muscle/myenteric plexus (LMMP) preparation from guinea pig
ileum excites myenteric neurons (16). These data suggest a
possible action of CRF at the level of the colonic myenteric nervous system.
The present study was undertaken to characterize the receptor-mediated action of rat/human CRF (r/hCRF) injected peripherally on the activity of myenteric cells in the rat proximal colon using c-Fos expression. Growing evidence indicates that immunohistochemical detection of Fos is a relevant approach to map enteric neuron activation in response to physiological and pathophysiological stimuli (32, 34, 43).
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MATERIALS AND METHODS |
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Animals
Male Sprague-Dawley rats (Harlan, San Diego, CA) weighing 250-300 g were maintained under controlled environmental conditions (12:12-h light-dark cycle, 22 ± 1°C) with free access to food (Purina Rat Chow) and tap water for at least 7 days before the experiments. Rats were accustomed to single housing and handled daily for 2 days before the experiments, which were carried out between 9:00 AM and 12:00 PM. Protocols (99-127-07) were approved by the Animal Research Committee of the Veteran Affairs Greater Los Angeles Healthcare System.Substances and Treatments
r/hCRF and astressin, cyclo(30-33)-[D-Phe12,Nle21,38,Glu30, Lys33]-r/hCRF12-41 (Salk Institute, Clayton Foundation Laboratories for Peptide Biology, La Jolla, CA) were synthesized as previously described (14) and kept atExperimental Protocols
Effects of intraperitoneal CRF on Fos expression in myenteric cells in vehicle- or astressin-pretreated rats. Rats (n = 4-6/group) were injected intraperitoneally with either vehicle (water) or astressin (33 µg/kg) 15 min before the injection of CRF (10 µg/kg ip) or saline. The regimen of CRF injection was selected based on previous studies showing the induction of clustered spike-burst activity in the proximal colon and inhibition of CRF action by astressin administered at the same dose (29).
Effects of ip CRF on Fos expression in colonic myenteric cells in vehicle- or CP 154,526-pretreated rats. Rats (n = 6-9/ group) were injected subcutaneously (0.5 ml) with either vehicle (DMSO + cremophor + saline) or CP 154,526 (20 mg/kg) 30 min before the intraperitoneal injection of CRF (10 µg/kg) or saline. The dose of CP 154,526 was previously reported to antagonize CRF (10 µg/kg ip)-induced stimulation of proximal colonic motility (29).
Effects of intraperitoneal CRF on Fos expression in colonic myenteric cells in vehicle- or atropine-pretreated rats. Rats were injected subcutaneously with vehicle (n = 4) or atropine (1 mg/kg; n = 3), and 30 min later, CRF (10 µg/kg) was injected intraperitoneally.
In all experiments, rats were euthanized by decapitation 1 h after the intraperitoneal injection of saline or CRF. The proximal colon was immediately removed and processed for immunohistochemistry.Tissue Preparation
Tissue preparation was performed as previously described (32, 34). The proximal colon was opened longitudinally along the mesenteric border in PBS (pH 7.4) containing nifedipine (10Immunohistochemistry
Antibodies and reagents. The following polyclonal and monoclonal primary antibodies were used: rabbit anti-Fos (fos Ab-5; Cat# PC38, Oncogene Research Products, Cambridge, MA); rabbit antivesicular acetylcholine transporter (VChAT, Model H-V006; Phoenix Pharmaceuticals, Mountain View, CA); goat anticholine acetyltransferase (ChAT, model AB144P; Chemicon International, Temecula, CA); rabbit anti-human protein gene product 9.5 (PGP 9.5, Model RA 95101; Ultraclone Limited, Wellow Isle of Wight, UK), and mouse anti-Fos (model TF161; gift from Dr. K. A. Sharkey, University of Calgary, Calgary, Alberta, Canada). The secondary antibodies used were biotinylated goat anti-rabbit IgG, goat anti-rabbit and donkey anti-mouse IgG conjugated to FITC, donkey anti-mouse IgG conjugated to tetramethylrhodamine (TRITC), and donkey anti-goat IgG conjugated TRITC (Jackson ImmunoResearch, WestGrove, PA).
The following chemicals were used: Triton X-100, hydrogen peroxide, andFos immunolabeling. The immunohistochemical procedure used was previously described (32, 34). Briefly, LMMP whole mount preparations from the proximal colon were rinsed (3 times for 10 min each) in PBS (pH 7.4) and incubated for 30 min at room temperature with PBS containing 0.3% hydrogen peroxide to remove endogenous peroxidase activity. After a further rinsing in PBS, tissues were incubated for 1 h at room temperature with normal goat serum (3%) in PBS containing 0.3% Triton X-100 to block nonspecific binding (blocking solution). Tissues were then incubated for 48 h at 4°C with a polyclonal rabbit anti-Fos (fos Ab-5, 1:10,000) diluted in the blocking solution mentioned above. After the primary antibody, tissues were rinsed (3 times for 10 min each) in PBS and incubated for 2 h at room temperature with biotinylated goat anti-rabbit secondary antibody (1:500). Finally, tissues were rinsed (3 times for 10 min each) in PBS (pH 7.4) and processed using the standard biotin-avidin-horseradish peroxidase methodology (21). Tissues were examined using a light microscope (Zeiss Axioskop; Carl Zeiss, Thornwood, NY) and photographed (Kodak Technical Pan film). Fos immunoreactivity was also examined using indirect immunofluorescence. LMMP whole mount preparations were rinsed (3 times for 10 min each) in PBS containing 0.1% Triton X-100 (PBS-T; pH 7.4) and incubated for 1 h at room temperature with a blocking solution (3% normal goat serum in PBS containing 0.1% Triton X-100). Tissues were then incubated for 48 h at 4°C with a polyclonal rabbit anti-Fos (fos Ab-5, 1:5,000). After the first incubation, tissues were rinsed (3 times for 10 min each) in PBS-T and incubated for 1 h at room temperature with goat anti-rabbit IgG conjugated to FITC (1:50) or TRITC (1:100). All antibodies were diluted in PBS-T containing 0.1% BSA (antibody diluent). Tissues were given a final wash (3 times for 10 min each) with PBS-T, mounted in bicarbonate-buffered glycerol (pH 8.6), and examined using Zeiss LSM 510 laser scanning microscope (Carl Zeiss). Immunohistochemical controls involved incubation of tissues in blocking solution (3% normal goat serum in PBS containing 0.3% Triton X-100) followed by antibody diluent in place of primary antibody and processed as above. No positive staining was observed under these conditions.
Double labeling. The double-labeling procedure was performed as previously described (34) and involved Fos immunolabeling with PGP 9.5, VChAT, ChAT, and NADPH diaphorase (NADPH-d) in the LMMP whole mount preparations from the proximal colon. PGP 9.5, a well established marker for neuronal cell bodies and axons in the central and peripheral nervous systems (23, 34), was used to ascertain the neuronal identity of colonic myenteric cells expressing Fos. ChAT, the enzyme for acetylcholine synthesis (27, 47), VChAT, a specific marker of cholinergic axons (2, 27), and NADPH-d activity for histochemical detection of nitric oxide synthase (NOS) (4, 53) were used to assess the biochemical coding of cells expressing Fos.
Fos with PGP9.5, VChAT, or ChAT. Indirect immunofluorescence was used for the double labeling. LMMP whole mount preparations from the proximal colon were rinsed (3 times for 10 min each) with PBS-T (pH 7.4). After being rinsed, tissues were incubated separately for 48 h at 4°C with a monoclonal Fos antibody (TF161; 1:500) alone or combined with the following polyclonal antibodies: 1) rabbit anti-human PGP 9.5 (1:500), 2) rabbit anti-VChAT (1:500), or 3) goat anti-ChAT (1:1,000). Primary as well as secondary antibodies were mixed before use. After the first incubation, tissues were washed with PBS-T and incubated for 1 h at room temperature with donkey anti-mouse IgG conjugated to FITC (1:50) or TRITC (1:100) alone or combined to goat anti-rabbit IgG conjugated to TRITC (1:100) or FITC (1:50), or donkey anti-goat IgG conjugated to TRITC (1:100). All antibodies were diluted in antibody diluent. Tissues were given a final wash (3 times for 10 min each) with PBS-T and mounted in bicarbonate-buffered glycerol (pH 8.6). The antibodies raised against Fos (fos Ab-5 and TF161), VChAT, and ChAT have been previously used in the gut tissues (32-34, 44, 49, 57). Controls were performed by incubating a few LMMP whole mount preparations in antibody diluent in place of the primary antibodies and processed as above. Tissues were examined using the Zeiss LSM 510 laser scanning microscope (Carl Zeiss), whose fluorescent optics with both FITC/TRITC filters allowed the dual-labeling analysis by overlapping the labeling from different primary antibodies. For each examined colonic LMMP whole mount preparation, single and/or double stainings were detected using TRITC, FITC, or FITC/TRITC filters. Images were viewed and captured using the computerized image-analysis system coupled with the Zeiss LSM laser scanning microscope. No positive staining was seen in controls.
Fos with NADPH-d.
The double-labeling NADPH-d and Fos was performed using nitroblue
tetrazolium formazan histochemical staining followed by Fos
immunohistochemistry in the colonic LMMP whole mount preparation as
previously described (57). Briefly, LMMP whole mount
preparations were rinsed (3 times for 10 min each) in phosphate buffer
(PB; pH 7.4) and incubated for 30-60 min at 37°C in PB
containing 0.3% Triton X-100, 0.5 mg/ml of -NADPH, and 0.1 mg/ml of
nitroblue tetrazolium. After a further rinsing (6 times for 20 min
each) in PBS containing 0.3% Triton X-100 (pH 7.4), tissues were
incubated for 48 h at 4°C with a polyclonal rabbit anti-Fos (fos
Ab-5; 1:10,000) and processed using the standard
biotin-avidin-horseradish peroxidase methodology (20).
Nickel was excluded from the chromogen (diaminobenzine) to discriminate
NADPH-d staining from Fos immunoreactivity. NADPH-d activity was
detected as dark blue formazan products into the nerve cell bodies,
whereas Fos immunoreactivity was detected as brown nuclear staining.
Tissues were examined using a light microscope (Nikon Labophot-2
Microscope, Melville, NY) and photographed (Kodak Ektachrome 64 T film).
Quantitative analysis. Monoclonal (TF161) and polyclonal (fos Ab-5) antibodies detect Fos protein in the myenteric ganglia (32-34, 49). To avoid a possible discrepancy in the magnitude of Fos expression detected with TF161 and fos Ab-5, we blindly counted the number of Fos-positive nuclei stained only with the polyclonal antibody (fos Ab-5). In each rat, the number of Fos-IR cells as well as NADPH-d-positive neurons was counted in 25 myenteric ganglia randomly selected in each examined piece (0.25 cm2) of the LMMP whole mount preparation and expressed as a mean number per myenteric ganglion. The mean number of Fos-immunoreactive (IR) cells per myenteric ganglion from each animal was used to generate a mean number for each experimental group.
Statistical analysis. Data are expressed as means ± SE and analyzed by one-way ANOVA followed by Dunn's test to identify differences between individual treatment groups. The confidence limit for significance was set at P < 0.05.
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RESULTS |
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Effects of Intraperitoneal CRF and CRF-Receptor Antagonists
There was a low number of Fos-IR cells (1.2 ± 0.6 cells/ganglion) in the myenteric plexus of the proximal colon 1 h after the intraperitoneal injection of saline in conscious rats pretreated with water (Figs. 1A and 2). CRF (10 µg/kg ip) significantly increased Fos expression in the proximal colon myenteric ganglia to 19.6 ± 2.1 Fos-IR cells/ganglion in water-pretreated rats (Figs. 1C and 2). Astressin (33 µg/kg ip) prevented intraperitoneal CRF-induced Fos expression in the myenteric ganglia of proximal colon (2.7 ± 1.2 Fos-IR cells/ganglion; Figs. 1E and 2). Astressin followed by intraperitoneal saline did not influence the low basal Fos expression in the colonic myenteric ganglia (0.8 ± 0.4 Fos-IR cells/ganglion; Fig. 2).
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Likewise, the number of Fos-IR cells in the myenteric plexus of
proximal colon was low in rats injected subcutaneously with vehicle or
CP-154,526 (20 mg/kg) 30 min before the intraperitoneal injection of
saline (1.3 ± 0.9 and 0 ± 0 Fos positive cells/ganglion, respectively; Figs. 1B and 3).
In vehicle (subcutaneous) pretreated rats, CRF (10 µg/kg ip)
significantly increased the number of Fos-IR cells in the myenteric
ganglia of the proximal colon (Figs. 1D and 3). CRF action
was antagonized by CP-154,526, and the number of Fos-IR cells per
ganglion (1.0 ± 1.0) was no longer significantly different from
that of control groups (Figs. 1F and 3).
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Effects of atropine on CRF action.
CRF (10 µg/kg ip)-induced Fos expression in myenteric ganglia of the
proximal colon (15.8 ± 1.2 Fos-IR cells/ganglion,
n = 4) in subcutaneous saline-pretreated rats was not
modified by atropine (1 mg/kg sc; 15.0 ± 1.9 Fos-IR
cells/ganglion, n = 3). Photomicrographs illustrate the
similar occurrence of Fos-IR induced by intraperitoneal CRF in the
myenteric ganglia of rats pretreated subcutaneously either with vehicle
(Fig. 4A) or atropine (Fig. 4B).
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Neuronal Identity of Fos-IR Cells in the Myenteric Ganglia
Double labeling of Fos with PGP 9.5.
In rats injected intraperitoneally with water followed by saline, the
proximal colon myenteric plexus displayed abundant ganglionic cells
labeled with PGP 9.5, whereas a small number of Fos-IR nuclei was
localized in PGP 9.5-IR cell bodies (Fig.
5A). In rats pretreated with
water, CRF (10 µg/kg ip) induced Fos expression in the
glanglionic myenteric neurons as illustrated by the double labeling of
Fos/PGP 9.5 in all Fos-positive cells (Fig. 5B).
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Double labeling of Fos with VChAT or ChAT. The monoclonal Fos (TF161) was used for the double labeling. Proximal colonic tissues from rats injected intraperitoneally with saline showed abundant myenteric ganglionic nerve and varicose fibers immunoreactive to VChAT, whereas Fos immunoreactivity was rare or absent (Fig. 5C). In contrast, proximal colonic tissues from rats injected with CRF (10 µg/kg ip) exhibited ganglionic Fos-IR cells encircled by varicose fibers immunoreactive to VChAT (Fig. 5D).
Colonic tissues from control rats injected intraperitoneally with saline and double labeled with Fos/ChAT did not show Fos-IR cells in the myenteric ganglia (Fig. 6A). However, varicose fibers and a few nerve cell bodies immunoreactive to ChAT were visible in the myenteric ganglia where Fos IR was absent (Fig. 6, C and D). After intraperitoneal injection of CRF, there was abundant Fos-IR nuclei (Fig. 6B), which were found in the majority of ChAT-IR nerve cell bodies surrounded by ChAT-IR varicose fibers (Fig. 6, D and F).
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Double labeling of Fos with NADPH-d.
In the colonic tissues from control rats pretreated with vehicle
(intraperitoneal) and 15 min later with saline, the myenteric plexus
displayed 9.2 ± 0.4 NADPH-d-positive neurons/ganglion
(n = 3), whereas Fos-IR cells were absent (Fig.
7A). A network of ganglionic
NADPH-d-positive varicose fibers was also clearly visible. Occasionally
NADPH-d-positive fibers arising from positive nerve cell bodies and
traveling into the internodal strands were also visible in the
myenteric plexus (Fig. 7A). CRF injected intraperitoneally did not modify the number of NADPH-d-positive neurons in the proximal colon myenteric ganglia (9.3 ± 0.5 neurons/ganglion;
n = 5) in vehicle-pretreated rats. Double labeling of
NADPH-d and Fos showed abundant ganglionic Fos-IR cells, which were not
localized in NADPH-d-positive nerve cell bodies (Fig. 7B).
Pretreatment with astressin (33 µg/kg ip) prevented Fos expression
but did not affect the number of NADPH-d-positive neurons (Fig.
7C). Similar results were observed with CP-154,526 (data not
shown).
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DISCUSSION |
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The present study demonstrates that CRF injected intraperitoneally in conscious rats induces Fos expression detected by immunohistochemistry in the myenteric plexus of the proximal colon 60 min after peptide administration. This response is specific to CRF, as shown by the rare Fos IR in the colonic myenteric plexus after the intraperitoneal injection of saline, whereas ~19 Fos-IR cells/ganglion were observed after intraperitoneal CRF. The double labeling of Fos with the neuronal marker PGP 9.5 (24) revealed the neuronal identity of activated myenteric cells. These data provide the first evidence of Fos expression in myenteric neurons of the proximal colon in response to the activation of CRF-signaling pathways established to be involved in colonic motor alterations induced by acute stress (7, 29, 56). Fos expression has been extensively used to map, at the cellular level, stimuli-related activation of neuronal circuits in the central nervous system (25, 35). This approach has also been successfully applied to the enteric nervous system embedded in the wall of stomach (5, 9, 34, 57) and small intestine (33, 40, 43, 45). However, Fos induction in colonic myenteric ganglia has received less attention, and investigations so far have been limited to the response of the distal colonic myenteric cells to acute experimental colitis (32, 49).
The present findings indicate that intraperitoneal CRF-induced Fos expression in the proximal colonic myenteric neurons is CRF-receptor mediated. First, the specific CRF-receptor antagonist astressin, which displays equal high binding affinity to CRF1, and either splice variants of the CRF2 receptors (41) injected peripherally abolished intraperitoneal CRF-induced Fos expression. Second, similar inhibition of CRF-induced ganglionic Fos expression was induced by the specific CRF1 antagonist CP-154,526 (48). We previously reported (29) that CRF (10 µg/kg) injected intraperitoneally induced a new pattern of clustered spike-burst activity in the proximal colon and defecation, which were both antagonized by astressin and CP-154,526 at similar doses used in the present study. Together, these data support an important role of the CRF1 receptor in the activation of myenteric neurons and propulsive motor activity in the proximal colon after peripheral administration of CRF.
The biochemical coding of CRF1-mediated Fos expression in myenteric neurons was investigated by immunohistochemical double-labeling techniques. With the use of a commercially available ChAT antibody (44), we visualized a few cell bodies immunoreactive to ChAT in the rat proximal colon, as reported in mice large intestine (44). After intraperitoneal injection of CRF, all ganglionic ChAT-IR cells were Fos positive. We observed only a small proportion of ganglionic nerve cell bodies immunoreactive to ChAT, as previously described (44). Recent studies (54) have reported the existence of ChAT mRNA splice variant that has been identified to be preferentially expressed in the peripheral nervous system (pChAT). With the use of an antibody raised against pChAT, 69% of total neuronal somata of myenteric ganglion in the rat colon were pChAT positive (37, 54). The findings obtained with pChAT antibody indicate that cholinergic neurons comprise a major subpopulation of proximal and distal colonic myenteric neurons. This could also explain the small population of Fos-positive ganglionic cholinergic neurons observed in the present study using a ChAT antibody not directed to pChAT.
The detection of NADPH-d activity is a well-established cytochemical maker for the presence of NO synthase activity in myenteric neurons of the rat gut (4). We counted ~8-9 NADPH-d-positive neurons per myenteric ganglion in the LMMP whole mount preparations of rat proximal colon. These data are consistent with Takahashi and Owyang's previous study (53) reporting 9.8 ± 1.1 NADPH-d-positive cell/ganglion in the rat proximal colon. Interestingly, CRF did not induce Fos expression in NADPH-d-positive neurons. It is unlikely that the lack of Fos in NADPH-d neurons reflects the inability of these myenteric cells to express Fos. Indeed, we previously demonstrated (57) Fos expression in NADPH-d-positive gastric myenteric neurons in response to vagal cholinergic stimulation by acute cold exposure. Previous reports (1, 37) indicate that NADPH-d-positive neurons in the rat colonic myenteric ganglia are not immunoreactive to antibodies raised against ChAT or pChAT. The present observation that intraperitoneal CRF induced Fos expression in ChAT-IR cells but not in the labeled subpopulation of nitrinergic myenteric neurons of the proximal colon provides evidence that these cholinergic and nitrinergic neurons are differentially influenced by peripheral CRF.
The exact site(s) and mechanism(s) of intraperitoneal CRF action to induce CRF1-mediated Fos expression in the colonic myenteric neurons cannot be inferred from the present studies. Fos expression is induced in the guinea pig myenteric neurons as a consequence of activation of motor function (43). Studies (19) performed in LMMP strips of guinea pig ileum established that the preferential CRF1 agonist ovine CRF (51)-induced contractile response is blocked by atropine. In the present study, atropine injected peripherally at a dose (1 mg/kg) that prevented the CRF receptor-mediated colonic propulsive response to restraint stress (7) and basal contractile activity in the proximal colon (52) did not alter Fos expression induced by intraperitoneal CRF. Even a higher dose of atropine (2 mg/kg) resulted only in a small decrease in the number of Fos-positive cells (unpublished observations). Collectively these observations indicate that intraperitoneal CRF-induced Fos expression in the myenteric ganglia is unlikely to be secondary to muscarinic activation of colonic motor function.
Indirect evidence supports a possible site of action of CRF directly on myenteric neurons, although the distribution of CRF1 receptors in rat enteric neurons of the colon is yet to be established. Indeed, an in vitro intracellular microelectrode recording study indicates that the preferential CRF1 agonist ovine CRF (51) acts directly on both AH/type-2 and S/type-1 neurons in the LMMP from guinea pig ileum to evoke a prolonged excitatory response (16), suggesting the presence of CRF1 receptor on the enteric neurons. Alternatively, CRF may also influence preganglionic cholinergic input to myenteric neurons. Cholinergic nicotinic activation induces marked Fos expression in gastric myenteric neurons (34, 57) and excitation of myenteric neurons in the colon assessed electrophysiologically (6). With the use of an antibody raised against VChAT to label terminal fields of the cholinergic nervous system (46), we found abundant VChAT-IR fiber bundles in the myenteric ganglia but rarely in cell bodies of rat proximal colon as previously reported (44) in mice. Abundant varicose fibers positive for ChAT were also detected in strands and around somata, suggesting a possible implication of the nicotinic cholinergic pathway(s). Fos-IR neurons induced by intraperitoneal CRF were encircled by varicose fibers immunoreactive to VChAT or ChAT, providing anatomical support for possible action of CRF to modulate cholinergic input to myenteric neurons expressing Fos. This will need to be further examined with the use of nicotinic blockade. However, any possible action of CRF presynaptically to increase cholinergic excitation of myenteric neurons would not be consistent with the lack of Fos induction in the NADPH-d-containing neurons. Indeed, nicotinic/muscarinic-receptor activation is a major stimulus for gene transcription of NOS and NO release in the enteric nervous system (22, 38). Other studies also showed that endogenous acetylcholine stimulates NO-containing myenteric neurons in the gut, including the proximal colon (22, 57). However, we could not exclude the possibility that CRF recruits enteric circuitry that inhibits cholinergic input to nitrinergic neurons.
The biochemical coding of activated myenteric neuron by intraperitoneal CRF may have a bearing with the increase in proximal colonic motility (29). Cholinergic myenteric neurons play a major role in stimulating colonic motility in the proximal colon in various animal species, including the rat through muscarinic excitatory input to the muscles (12, 17). The observed activation of cholinergic neurons, as evidenced by Fos expression, may contribute to the increased colonic contractile response to CRF, as observed in conscious rats (29) and in vitro colonic preparation (19, 29, 30). The NO released from enteric neurons is well established to restrain basal and inhibit nerve-stimulated mechanical contractile activity in the proximal colon, whereby relaxation for mixing, storage of liquid feces, absorption, and propulsion are facilitated (26, 36, 38, 53). The absence of simultaneous cholinergic and nitrinergic myenteric neuron activation observed in the present study may leave unopposed the excitatory cholinergic input to the proximal colonic smooth muscles induced by CRF.
In summary, data obtained in the present study show that CRF injected intraperitoneally induces Fos expression in proximal colon myenteric neurons through the activation of the CRF1 receptor in conscious rats. CRF induced Fos expression in cholinergic myenteric neurons but not in a subpopulation of inhibitory neurons exhibiting NADPH-d activity. CRF action was not altered in the presence of atropine, indicating that ganglionic myenteric Fos induction is not secondary to the activation of muscarinic receptors either on the myenteric ganglia or colonic muscles (8, 12). The CRF1-mediated activation of ganglionic neurons in the rat proximal colon by intraperitoneal CRF may have relevance to the neural mechanisms through which stress stimulates colonic propulsive motor function, as recently demonstrated in functional studies (29).
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ACKNOWLEDGEMENTS |
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The authors thank Dr. J. Rivier (Clayton Foundation Laboratories, La Jolla, CA) for the generous supply of peptides, Dr. E. D. Pagani (Central Research Division, Pfizer, Croton, CT) for the supply of CP-154,526, and P. Kirsch for assistance in the preparation of the manuscript.
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FOOTNOTES |
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This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-57238 (to Y. Taché), DK-57238-01A1S1 (to M. Million), and DK-41301 (Animal Core, Y. Taché; Imaging Core, G. Sachs).
Dr. C. Maillot was a fellow granted by the French Foreign Office, The "Conseil Regional of Normandie," University Hospital of Rouen and GlaxoSKB.
Address for reprint requests and other correspondence: M. Miampamba, CURE: DDRC, Veterans Affairs Greater Los Angeles Healthcare System, Bldg. 115, Rm. 203, 11301 Wilshire Blvd., Los Angeles, CA 90073 (Email: mmiampam{at}ucla.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 9, 2002;10.1152/ajpgi.00434.2001
Received 10 October 2001; accepted in final form 7 January 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aimi, Y,
Kimura H,
Kinoshita T,
Minami Y,
Fujimura M,
and
Vincent SR.
Histochemical localization of nitric oxide synthase in rat enteric nervous system.
Neuroscience
53:
553-560,
1993[ISI][Medline].
2.
Arvidsson, U,
Riedl M,
Elde R,
and
Meister B.
Vesicular acetylcholine transporter (VAChT) protein: a novel and unique marker for cholinergic neurons in the central and peripheral nervous systems.
J Comp Neurol
378:
454-467,
1997[ISI][Medline].
3.
Baigent, SM.
Peripheral corticotropin-releasing hormone and urocortin in the control of the immune response.
Peptides
22:
809-820,
2001[ISI][Medline].
4.
Belai, A,
Schmidt HH,
Hoyle CH,
Hassall CJ,
Saffrey MJ,
Moss J,
Forstermann U,
Murad F,
and
Burnstock G.
Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut.
Neurosci Lett
143:
60-64,
1992[ISI][Medline].
5.
Berthoud, HR,
Patterson LM,
and
Zheng H.
Vagal-enteric interface: vagal activation-induced expression of c-Fos and p-CREB in neurons of the upper gastrointestinal tract and pancreas.
Anat Rec
262:
29-40,
2001[ISI][Medline].
6.
Browning, KN,
and
Lees GM.
Myenteric neurons of the rat descending colon: electrophysiological and correlated morphological properties.
Neuroscience
73:
1029-1047,
1996[ISI][Medline].
7.
Castagliuolo, I,
Lamont JT,
Qiu B,
Fleming SM,
Bhaskar KR,
Nikulasson ST,
Kornetsky C,
and
Pothoulakis C.
Acute stress causes mucin release from rat colon: role of corticotropin releasing factor and mast cells.
Am J Physiol Gastrointest Liver Physiol
271:
G884-G892,
1996
8.
Christofi, FL,
Palmer JM,
and
Wood JD.
Neuropharmacology of the muscarinic antagonist telenzepine in myenteric ganglia of the guinea-pig small intestine.
Eur J Pharmacol
195:
333-339,
1991[ISI][Medline].
9.
Dimaline, R,
Miller SM,
Evans D,
Noble PJ,
Brown P,
and
Poat JA.
Expression of immediate early genes in rat gastric myenteric neurones: a physiological response to feeding.
J Physiol (Lond)
488:
493-499,
1995[Abstract].
10.
Fukudo, S,
Nomura T,
and
Hongo M.
Impact of corticotropin-releasing hormone on gastrointestinal motility and adrenocorticotropic hormone in normal controls and patients with irritable bowel syndrome.
Gut
42:
845-849,
1998
11.
Gilligan, PJ,
Robertson DW,
and
Zaczek R.
Corticotropin releasing factor (CRF) receptor modulators: progress and opportunities for new therapeutic agents.
J Med Chem
43:
1641-1660,
2000[ISI][Medline].
12.
Gomez, A,
Martos F,
Bellido I,
Marquez E,
Garcia AJ,
Pavia J,
and
Sanchez de la Cuesta
Muscarinic receptor subtypes in human and rat colon smooth muscle.
Biochem Pharmacol
43:
2413-2419,
1992[ISI][Medline].
14.
Gulyas, J,
Rivier C,
Perrin M,
Koerber SC,
Sutton S,
Corrigan A,
Lahrichi SL,
Graig AG,
Vale W,
and
Rivier J.
Potent, structurally constrained agonists and competitive antagonists of corticotropin-releasing factor.
Proc Natl Acad Sci USA
92:
10575-10579,
1995
15.
Habib, KE,
Weld KP,
Rice KC,
Pushkas J,
Champoux M,
Listwak S,
Webster EL,
Atkinson AJ,
Schulkin J,
Contoreggi C,
Chrousos GP,
McCann SM,
Suomi SJ,
Higley JD,
and
Gold PW.
Oral administration of a corticotropin-releasing hormone receptor antagonist significantly attenuates behavioral, neuroendocrine, and autonomic responses to stress in primates.
Proc Natl Acad Sci USA
97:
6079-6084,
2000
16.
Hanani, M,
and
Wood JD.
Corticotropin-releasing hormone excites myenteric neurons in the guinea-pig small intestin.
Eur J Pharmacol
211:
23-27,
1992[ISI][Medline].
17.
Hasler, WL,
Kurosawa S,
and
Chung OY.
Regional cholinergic differences between distal and proximal colonic myenteric plexus.
Am J Physiol Gastrointest Liver Physiol
258:
G404-G410,
1990
18.
Heinrichs, SC,
and
Taché Y.
Therapeutic potential of CRF receptor antagonists: a gut-brain perspective.
Expert Opin Investig Drugs
10:
647-659,
2001[ISI][Medline].
19.
Hollt, V,
Garzon J,
Schulz R,
and
Herz A.
Corticotropin-releasing factor is excitatory in the guinea-pig ileum and activates an opioid mechanism in this tissue.
Eur J Pharmacol
101:
165-166,
1984[ISI][Medline].
20.
Hsu, SM,
and
Raine L.
Protein A, avidin, and biotin in immunohistochemistry.
J Histochem Cytochem
29:
1349-1353,
1981[Abstract].
21.
Hsu, SM,
Raine L,
and
Fanger H.
Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures.
J Histochem Cytochem
29:
577-580,
1981[Abstract].
22.
Iversen, HH,
Wiklund NP,
Olgart C,
and
Gustafsson LE.
Nerve stimulation-induced nitric oxide release as a consequence of muscarinic M1 receptor activation.
Eur J Pharmacol
331:
213-219,
1997[ISI][Medline].
23.
Krammer, HJ,
Karahan ST,
Rumpel E,
Klinger M,
and
Kuhnel W.
Immunohistochemical visualization of the enteric nervous system using antibodies against protein gene product (PGP) 9.5.
Anat Anz
175:
321-325,
1993[Medline].
24.
Krammer, HJ,
Karahan ST,
Sigge W,
and
Kuhnel W.
Immunohistochemistry of markers of the enteric nervous system in whole- mount preparations of the human colon.
Eur J Pediatr Surg
4:
274-278,
1994[ISI][Medline].
25.
Krukoff, TL.
Expression of c-fos in studies of central autonomic and sensory systems.
Mol Neurobiol
7:
247-263,
1993[ISI][Medline].
26.
Kumano, K,
Fujimura M,
Oshima S,
Yamamoto H,
Hayashi N,
Nakamura T,
and
Fujimiya M.
Effects of VIP and NO on the motor activity of vascularly perfused rat proximal colon.
Peptides
22:
91-98,
2001[ISI][Medline].
27.
Li, ZS,
and
Furness JB.
Immunohistochemical localisation of cholinergic markers in putative intrinsic primary afferent neurons of the guinea-pig small intestine.
Cell Tissue Res
294:
35-43,
1998[ISI][Medline].
28.
Lu, L,
Liu D,
Ceng X,
and
Ma L.
Differential roles of corticotropin-releasing factor receptor subtypes 1 and 2 in opiate withdrawal and in relapse to opiate dependence.
Eur J Neurosci
12:
4398-4404,
2000[ISI][Medline].
29.
Maillot, C,
Million M,
Wei JY,
Gauthier A,
and
Taché Y.
Peripheral corticotropin-releasing factor and stress-stimulated colonic motor activity involve type 1 receptor in rats.
Gastroenterology
119:
1569-1579,
2000[ISI][Medline].
30.
Mancinelli, R,
Azzena GB,
Diana M,
Forgione A,
and
Fratta W.
In vitro excitatory actions of corticotropin-releasing factor on rat colonic motility.
J Auton Pharmacol
18:
319-324,
1998[ISI][Medline].
31.
McLean, M,
and
Smith R.
Corticotropin-releasing hormone in human pregnancy and parturition.
Trends Endocrinol Metab
10:
174-178,
1999[ISI][Medline].
32.
Miampamba, M,
and
Sharkey KA.
c-Fos expression in the myenteric plexus, spinal cord and brainstem following injection of formalin in the rat colonic wall.
J Auton Nerv Syst
77:
140-151,
1999[ISI].
33.
Miampamba, M,
Tan DT,
Oliver MR,
Sharkey KA,
and
Scott RB.
Intestinal anaphylaxis induces Fos immunoreactivity in myenteric plexus of rat small intestine.
Am J Physiol Gastrointest Liver Physiol
272:
G181-G189,
1997
34.
Miampamba, M,
Yang H,
Sharkey KA,
and
Taché Y.
Intracisternal TRH analog induces Fos expression in gastric myenteric neurons and glia in conscious rats.
Am J Physiol Gastrointest Liver Physiol
280:
G979-G991,
2001
35.
Morgan, JI,
and
Curran T.
Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun.
Annu Rev Neurosci
14:
421-451,
1991[ISI][Medline].
36.
Mule, F,
D'Angelo S,
Amato A,
Contino I,
and
Serio R.
Modulation by nitric oxide of spontaneous mechanical activity in rat proximal colon.
J Auton Pharmacol
19:
1-6,
1999[ISI][Medline].
37.
Nakajima, K,
Tooyama I,
Yasuhara O,
Aimi Y,
and
Kimura H.
Immunohistochemical demonstration of choline acetyltransferase of a peripheral type (pChAT) in the enteric nervous system of rats.
J Chem Neuroanat
18:
31-40,
2000[ISI][Medline].
38.
Nakamura, K,
Takahashi T,
Taniuchi M,
Hsu CX,
and
Owyang C.
Nicotinic receptor mediates nitric oxide synthase expression in the rat gastric myenteric plexus.
J Clin Invest
101:
1479-1489,
1998
39.
Parkes, DG,
Weisinger RS,
and
May CN.
Cardiovascular actions of CRH and urocortin: an update.
Peptides
22:
821-827,
2001[ISI][Medline].
40.
Parr, EJ,
and
Sharkey KA.
The use of constitutive nuclear oncoproteins to count neurons in the enteric nervous system of the guinea pig.
Cell Tissue Res
277:
325-331,
1994[ISI][Medline].
41.
Perrin, MH,
Sutton SW,
Cervini LA,
Rivier JE,
and
Vale WW.
Comparison of an agonist, urocortin, and an antagonist, astressin, as radioligands for characterization of CRF receptors.
J Pharmacol Exp Ther
288:
729-734,
1999
42.
Perrin, MH,
and
Vale WW.
Corticotropin releasing factor receptors and their ligand family.
Ann NY Acad Sci
885:
312-328,
1999
43.
Ritter, RC,
Costa M,
and
Brookes SH.
Nuclear Fos immunoreactivity in guinea pig myenteric neurons following activation of motor activity.
Am J Physiol Gastrointest Liver Physiol
273:
G498-G507,
1997
44.
Sang, Q,
and
Young HM.
The identification and chemical coding of cholinergic neurons in the small and large intestine of the mouse.
Anat Rec
251:
185-199,
1998[ISI][Medline].
45.
Sayegh, AI,
and
Ritter RC.
CCK-A receptor activation induces fos expression in myenteric neurons of rat small intestine.
Regul Pept
88:
75-81,
2000[ISI][Medline].
46.
Schafer, MK,
Eiden LE,
and
Weihe E.
Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. II. The peripheral nervous system.
Neuroscience
84:
361-376,
1998[ISI][Medline].
47.
Schemann, M,
Sann H,
Schaaf C,
and
Mader M.
Identification of cholinergic neurons in enteric nervous system by antibodies against choline acetyltransferase.
Am J Physiol Gastrointest Liver Physiol
265:
G1005-G1009,
1993
48.
Schulz, DW,
Mansbach RS,
Sprouse J,
Braselton JP,
Collins J,
Corman M,
Dunaiskis A,
Faraci S,
Schmidt AW,
Seeger T,
Seymour P,
Tingley FD, III,
Winston EN,
Chen YL,
and
Heym J.
CP-154,526: a potent and selective nonpeptide antagonist of corticotropin releasing factor receptors.
Proc Natl Acad Sci USA
93:
10477-10482,
1996
49.
Sharkey, KA,
Parr EJ,
and
Keenan CM.
Immediate-early gene expression in the inferior mesenteric ganglion and colonic myenteric plexus of the guinea pig.
J Neurosci
19:
2755-2764,
1999
50.
Steckler, T,
and
Holsboer F.
Corticotropin-releasing hormone receptor subtypes and emotion.
Biol Psychiatry
46:
1480-1508,
1999[ISI][Medline].
51.
Suman-Chauhan, N,
Carnell P,
Franks R,
Webdale L,
Gee NS,
McNulty S,
Rossant CJ,
Van Leeuwen D,
MacKenzie R,
and
Hall MD.
Expression and characterisation of human and rat CRF2- receptors.
Eur J Pharmacol
379:
219-227,
1999[ISI][Medline].
52.
Sun, Y,
Fihn BM,
Jodal M,
and
Sjovall H.
Effects of neural blocking agents on motor activity and secretion in the proximal and distal rat colon: evidence of marked segmental differences in nicotinic receptor activity.
Scand J Gastroenterol
35:
380-388,
2000[ISI][Medline].
53.
Takahashi, T,
and
Owyang C.
Regional differences in the nitrergic innervation between the proximal and the distal colon in rats.
Gastroenterology
115:
1504-1512,
1998[ISI][Medline].
54.
Tooyama, I,
and
Kimura H.
A protein encoded by an alternative splice variant of choline acetyltransferase mRNA is localized preferentially in peripheral nerve cells and fibers.
J Chem Neuroanat
17:
217-226,
2000[ISI][Medline].
55.
Turnbull, AV,
and
Rivier C.
Corticotropin-releasing factor (CRF) and endocrine response to stress: CRF receptors, binding protein, and related peptides.
Proc Soc Exp Biol Med
215:
1-10,
1997[Abstract].
56.
Williams, CL,
Peterson JM,
Villar RG,
and
Burks TF.
Corticotropin-releasing factor directly mediates colonic responses to stress.
Am J Physiol Gastrointest Liver Physiol
253:
G582-G586,
1987
57.
Yuan, PQ,
Taché Y,
Miampamba M,
and
Yang H.
Acute cold exposure induces vagally mediated Fos expression in gastric myenteric neurons in rats.
Am J Physiol Gastrointest Liver Physiol
281:
G560-G568,
2001