Neuronal activity and CRF receptor gene transcription in the brains of rats with colitis

Christophe Porcher,1 Valérie Sinniger,1,2 Aurélie Juhem,1 Patrick Mouchet,1 and Bruno Bonaz1,2

1Groupe d'Etude du Stress et des Interactions Neuro-Digestives (EA 3744), and 2Département d'Hépato-Gastroenterologie, Hôpital Albert Michallon, Centre Hospitalier Universitaire, Cedex 09, France

Submitted 29 March 2004 ; accepted in final form 1 June 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We aimed to characterize neuronal and corticotropin-releasing factor (CRF) pathways at the acute phase of a model of colitis in rats. Male rats received an intracolonic injection of either vehicle (controls) or trinitrobenzenesulfonic acid (TNBS) and were killed 1, 2, 3, 4, 6, 12, or 24 h later. Coronal frozen sections of the brain were cut and mRNAs encoding the rat c-fos, CRF1 receptor, and CRF2{alpha},{beta} receptors were assayed by in situ hybridization histochemistry. Localization of these transcripts within CRF-immunoreactive (CRF-ir) neurons of the paraventricular nucleus (PVN) of the hypothalamus was also determined. Intracolonic TNBS induced c-fos mRNA expression in brain nuclei involved in the autonomic, behavioral, and neuroendocrine response to a stimulus (PVN, amygdala, locus coeruleus, parabrachial nucleus, nucleus of the solitary tract) and in circumventricular organs (lamina terminalis, subfornical organ, area postrema). CRF pathways, particularly in the PVN, were activated in this model as represented by a robust signal of c-fos and CRF1 receptor transcripts in the PVN and numerous CRF-ir neurons expressed c-fos or CRF1 receptor transcripts in the PVN of TNBS-treated animals. No expression of CRF2 receptor transcripts was observed in the PVN, either in basal conditions or after TNBS. These neuroanatomical data argue for an involvement of CRF pathways, through CRF1 receptor, within the PVN in TNBS-induced colitis.

c-fos; colon; hypothalamus; inflammation; in situ hybridization; stress


IDIOPATHIC INFLAMMATORY BOWEL diseases, including Crohn's disease and ulcerative colitis, are complex disorders with multiple pathogenic factors including cellular and humoral inflammatory cascades, genetic susceptibility, and environmental influences (28). Instillation of trinitrobenzenesulfonic acid (TNBS) into the distal colon of the rat has been developed by Morris et al. (20) as a simple and reproducible model of chronic intestinal inflammation in rat. This model is useful for the study of the etiopathogenesis of chronic intestinal inflammation and is suitable for assessing potential treatments.

There is now a large body of evidence that the brain is informed during neurogenic and systemic immunogenic stimuli, controlling numerous responses necessary to restore the homeostasis during psychological stress, infection, and inflammatory processes (33). The hypothalamic-pituitary-adrenal (HPA) axis has a pivotal role in this response. Corticotropin-releasing factor (CRF) is a 41-amino acid peptide recognized as a major regulator of pituitary adrenocorticotropin (34). This neuropeptide coordinates a wide variety of physiological responses caused by sustained stresses (10). The paraventricular nucleus (PVN) of the hypothalamus is the main source for CRF in the brain (29). The action of CRF is mediated by its receptors. Two G protein-coupled receptor subtypes, CRF1 receptor (23) and CRF2{alpha}/CRF2{beta} receptors (16), were recently cloned. CRF1 receptor mRNA is predominantly expressed in the brain (24). CRF2{alpha} receptor is the predominant neuronal receptor subtype within the brain, whereas the CRF2{beta} receptor form is localized in nonneuronal elements like the choroid plexus and cerebral blood vessels (16). In the periphery, CRF2{beta} receptor mRNA is expressed in both cardiac and skeletal muscle with lower levels observed in both lung and intestine (16). This heterogeneous distribution of CRF1 receptor and CRF2 receptor mRNAs suggests distinctive functional roles for each receptor in CRF-related brain and systemic systems. We have observed a robust transcriptional activation of the gene encoding the CRF1 receptor in the rat PVN after an acute immobilization stress, whereas a downregulation of the expression of the transcript was observed in chronic conditions (6).

A marker of neuronal activation, c-fos, is rapidly and transiently expressed in neurons of the central nervous system in response to somatocutaneous or visceral sensory stimuli or to central injection of CRF (5, 7, 9). Expression of the gene is typically measured by in situ hybridization, whereas the protein is usually visualized by using immunocytochemical techniques. Consequently, this marker appears as a useful tool to study gut-brain interactions.

Previous studies have looked at the influence of colitis on c-fos expression in the brain and changes in CRF expression. Expression of c-fos has been observed in the nucleus of the solitary tract (NTS), area postrema (AP), parabrachial nucleus (PB), and locus coeruleus (LC) in a model of colonic inflammation induced by mustard oil (17) or formalin (18). Kresse et al. (12) have shown that TNBS colitis increased CRF heterogeneous nuclear RNA (hnRNA) (primary transcript) and mRNA signals in the magnocellular part of the PVN and supraoptic nucleus (SON) of the hypothalamus but did not modify CRF hnRNA signal in the parvocellular part of the PVN.

In the present study, we first investigated the kinetic and distribution of c-fos mRNA in the rostrocaudal extent of the brain during the acute-phase response of an experimental colitis induced by instillation of TNBS in rats chronically fitted with a colonic catheter. Second, we examined the involvement of CRF pathways in this model through the changes in CRF1 receptor and CRF2 receptor mRNAs in the brain and the localization of these transcripts in CRF perikarya in the PVN.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Adult male Sprague-Dawley rats (250–300 g; Charles River) were individually housed in a temperature-controlled environment (22 ± 1°C) with food and water available ad libitum and a 12:12-h light-dark cycle. Animals were allowed a minimum of 7 days to adapt to housing conditions before any manipulation. Because of the diurnal variations in corticosteroid secretion and c-fos expression in specific brain areas (11), experiments were performed at the same time of day (between 10:00 AM and 1:00 PM). Animals were deprived of food but not water for at least 18 h before the experiments. Animal care and experiments were conducted in accordance with the "Guide for the Care and Use of Laboratory Animals" [DHEW Publication No. (NIH) 85-23, Revised 1978, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205] and the legal French Ministry of Agriculture (authorization no. 38-R-1001).

Colitis induction. Induction of colitis was adapted from Morris et al. (20) with some modifications. Rats were anesthetized with a mixture of pentobarbital sodium and chloralhydrate (4 ml/kg ip; Sanofi, Libourne, France). A silicone catheter (ID, 1.2 mm; OD, 1.7 mm) was chronically implanted into the distal colon, 7–8 cm from the anus. The catheter was fixed at the colonic wall by a purse-string suture, tunneled subcutaneously, and externalized at the back of the neck where it was secured at the animal's skin. After surgery, animals were housed separately for at least 7 days before testing. Experiments were performed in conscious, freely moving rats equipped with this chronic catheter to avoid the possible confounding effects of stress-induced manipulation of the animals on c-fos expression. Inflammation of the colon was induced through the catheter with a single intracolonic administration of 0.25 ml of 50% ethanol containing 30 mg of TNBS (Fluka; St. Quentin, Fallavier, France). The instillation procedure required ~5 s to complete. The catheter was then slowly flushed with 0.2 ml of air to expel any vehicle or TNBS agent remaining in the catheter. Control animals received 0.25 ml of 0.9% saline after the same procedure. Animals administered vehicle or TNBS then had free access to food and water.

Assessment of colonic damage was performed at the time of in situ hybridization processing. When animals were killed (overdose of pentobarbital sodium; 200 mg/kg ip), the distal colon was isolated just before initiation of the perfusion, opened by a longitudinal incision, rinsed with saline, and then pinned out on a wax block. The colon was immediately examined under microscope, and any visible damage was scored on a 0–5 scale according to Morris et al. (20). A 1- to 3-cm segment of the distal colon at the site of instillation was then resected, and coronal frozen sections (7 µm) were routinely stained with hematoxylin and eosin. Macroscopic and histological assessment by light microscopy were performed in a blinded fashion on coded slides.

In situ hybridization histochemistry. To follow the time course of c-fos and CRF receptor transcripts, rats were killed (overdose of pentobarbital sodium; 200 mg/kg ip) 1, 2, 3, 4, 6, 12, or 24 h after administration of TNBS or saline. Protocol was performed as previously described (6). Animals were killed and then transcardially perfused with saline followed by 4% paraformaldehyde in 0.1 M borax buffer (pH 9.5 at 4°C). Brains were rapidly removed from the skull, postfixed for 4 to 8 days in the same fixative at 4°C, and then subsequently cryoprotected overnight at 4°C in the same fixative containing 10% sucrose. Coronal frozen sections (30 µm) were cryostat cut (Microm) from the olfactory bulb to the end of the medulla. Sections were collected in a cold cryoprotectant solution (0.05 M sodium phosphate buffer, 30% ethylene glycol, 20% glycerol) and then stored at –20°C for further processing. Hybridization histochemical localization of each transcript (c-fos mRNA, CRF1 receptor, CRF2{alpha} and CRF2{beta} receptors mRNAs) was carried out in one in six series (every 6th section) of brain sections using 35S-labeled cRNA probes. Protocols for riboprobe synthesis, hybridization, and autoradiographic localization of the mRNA signals were performed as previously described (6). All solutions were treated with diethyl pyrocarbonate (DEPC) and sterilized to prevent RNA degradation. Tissue sections were mounted onto gelatin- and poly-L-lysine-coated slides, vacuum-dried, fixed in 4% paraformaldehyde for 20 min, and digested by proteinase K [10 µg/ml in 100 mM Tris·HCl (pH 8.0), and 50 mM EDTA (pH 8.0), at 37°C for 25 min]. Brain sections were then rinsed in sterile DEPC-treated water followed by a solution of 0.1 M triethanolamine (TEA; pH 8.0), acethylated in 0.25% acetic anhydride in 0.1 M TEA, and dehydrated through graded concentrations of alcohol (50, 70, 95, and 100%). After vacuum drying for a minimum of 2 h, 90 µl hybridization mixture (107 counts·minute–1/ml–1) was spotted on each slide, sealed under a coverslip, and incubated at 60°C for 15–20 h in a slide warmer. Coverslips were then removed, and the slides were rinsed four times in 4 x SSC (150 mM NaCl and 15 mM Tris·NaCl citrate buffer, pH 7.0) at room temperature. Sections were digested by RNase A (20 µg/ml in a solution of 500 mM NaCl, 10 mM Tris·HCl, pH 8.0, and 1 mM EDTA, pH 8.0) at 37°C for 30 min, rinsed in descending concentrations of SSC (2, 1, and 0.5 times), washed in 0.1 x SSC for 30 min at 60°C, and dehydrated through graded concentrations of alcohol. After a 2-h period of drying in a vacuum, sections were exposed at 4°C to X-ray films (Kodak) for 17 h (c-fos mRNA), 24 h (CRF1 receptor mRNA), or 68 h (CRF2{alpha}/{beta} receptor mRNAs). After development of the X-ray film, sections were defatted in xylene and dipped into NTB2 nuclear emulsion (Kodak; diluted 1:1 with distilled water). Slides were exposed for 7 days (c-fos mRNA), 14 days (CRF1 receptor mRNA), or 21 days (CRF2{alpha}/{beta} receptor mRNAs), developed in D19 developer (Kodak) for 3.5 min at 14–16°C, washed for 15 s in water, and fixed in rapid fixer (Kodak) for 5 min. Thereafter, sections were rinsed in running distilled water for 1 h, counterstained with thionin (0.25%), dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with DPX mountant.

cRNA probe synthesis and preparation. The rat c-fos probe (2.0 kb) was generated from the EcoR1 fragment of rat c-fos cDNA (Dr. I. Verma, The Salk Institute, La Jolla, CA), subcloned into pBluescript SK-1 (Stratagene, La Jolla, CA), and linearized with SmaI and HindIII (Pharmacia) for antisense and sense probes, respectively. The rat CRF1 receptor probe (1.3 kb) was generated from the PstI-PstI fragment of the rat prCRF PP1.3-BS cDNA (Dr. W. Vale, Peptide Biology Laboratory, The Salk Institute), subcloned into pBluescript II SK (Stratagene), and linearized with BamHI and HindIII (Pharmacia) for antisense and sense probes, respectively. The pBluescript SK+ plasmids containing either a 275-bp insert of the rat CRF2{alpha} receptor cDNA or a 200-bp insert of a rat CRF2{beta} receptor cDNA (Dr. T. Lovenberg, Neurocrine Biosciences, La Jolla, CA) were linearized with HindIII and BamHI to generate antisense and sense probes, respectively. These two probes (CRF2{alpha} receptor and CRF2{beta} receptor) have no overlap with one another and have no similarity to the CRF1 receptor probe. Radioactive cRNA copies were synthesized by incubating 250 ng (CRF1 receptor and CRF{beta} receptor mRNAs) or 1 µg (CRF2{alpha} receptor mRNAs) of linearized plasmid in (in mM) 6 MgCl2, 40 Tris (pH 7.9), 2 spermidine, 10 NaCl, 10 dithiothreitol, 0.2 ATP/GTP/CTP, and 200 µCi of [{alpha}-35S]UTP (cat. no. NEG039H; Dupont New England Nuclear), 40 U RNAsin (Promega, Madison, WI), and 20 U of T7 (c-fos and CRF1 receptor antisense probes; CRF2{alpha} receptor and CRF2{beta} receptor sense probes) or T3 (c-fos and CRF1 receptor sense probes; CRF2{alpha} receptor and CRF2{beta} receptor antisense probes) RNA polymerase for 60 min at 37°C. Unincorporated nucleotides were removed by using the amonium-acetate method; 100 µl of DNAse solution (1 µl DNAse, 5 µl of 5 mg/ml tRNA, 94 µl of 10 mM Tris/10 mM MgCl2) was added, and 10 min later, an extraction was accomplished by using a phenol-chloroform solution. The probes were precipitated with 80 µl of 5 M ammonium acetate and 500 µl of 100% ethanol for 20 min on dry ice. After centrifugation (14,000 revolutions/min) for 15 min, the supernatant was removed, and the pellet was dried and then resuspended in 100 µl of 10 mM Tris/1 mM EDTA. A concentration of 107 cpm probe was mixed into 1 ml of hybridization solution [(in µl) 500 formamide, 60 5 M NaCl, 10 1 M Tris, pH 8.0, 2 0.5 M EDTA, pH 8.0, 50 20x Denhardt's solution, 200 50% dextran sulfate, 50 10 mg/ml tRNA, and 10 1 M dithiothreitol (118 DEPC water-treated water per volume of probe used)]. This solution was mixed and heated for 5 min at 65°C before being spotted on slides.

Combination of immunocytochemistry with in situ hybridization. Immunocytochemistry (CRF-immunoreactive neurons) was combined with the in situ hybridization histochemistry protocol (c-fos, CRF1 receptor and CRF2{alpha}/{beta} receptor mRNAs) to determine whether CRF neurons located mainly in the hypothalamic PVN express the gene encoding c-fos, CRF1 receptor, or CRF2 receptor after TNBS-induced colitis. Protocol was performed as previously described (6). Every sixth tissue slice was processed by means of the avidin-biotin peroxidase method. Briefly, slices were washed in sterile DEPC-treated 0.05 M potassium phosphate-buffered saline (KPBS) and incubated at 4°C with CRF antibody mixed in sterile KPBS, 0.4% Triton X-100, 0.25% heparin sodium salt USP (ICN Biomedicals, Aurora, Ohio), and 1% bovine serum albumin (fraction V; Sigma, St. Louis, MO). Sections were then incubated overnight with a rabbit anti-human/rat CRF serum (code no. PBL rc 70, 8/9/83 bleed), a generous gift of Dr. Wylie Vale (Peptide Biology Laboratory, The Salk Institute), used at a concentration of 1:10,000. Brain slices were rinsed in sterile KPBS and incubated for 60 min with a secondary biotinylated goat anti-rabbit IgG (1:1,500 dilution in KPBS containing heparin; Vector Laboratories). Sections were then rinsed with sterile KPBS and incubated with an avidin-biotin complex (Vectastain ABC elite kit, Vector Laboratories) for 60 min at room temperature. Thereafter, tissues were rinsed in sterile KPBS and reacted in a mixture containing sterile KPBS, the chromogen 3,3'-diaminobenzidine tetrahydrochloride (0.04%), and 1% hydrogen peroxide (H2O2). Sections were then rinsed in sterile KPBS, mounted onto poly-L-lysine-coated slides, desiccated under vacuum overnight, and then processed for in situ hybridization as described in In situ hybridization histochemistry with the difference of dehydration (alcohol 50, 70, 95, 100%), which was shortened to avoid decoloration of CRF cells (brown staining), a 24- to 68-h exposure to X-ray film, a l0–15 days exposure to NTB2 nuclear emulsion, and the absence of counterstaining with thionin. The presence of c-fos, CRF1 receptor or CRF2 receptor transcripts was evidenced as agglomeration of silver grains in perikarya, whereas CRF immunoreactivity within the cell cytoplasm was stained in brown.

Qualitative and quantitative analysis. Analysis was performed as previously described (6). Anatomical identification of brain structures was essentially on the basis of the atlas of Paxinos and Watson (22). For qualitative analysis, the relative intensity of c-fos, CRF1 receptor, and CRF2{alpha}/{beta} receptor mRNA signals throughout the brain of each animal was assessed on X-ray film images and graded according to the scale of undetectable (0), low (+), moderate (++), strong (+++), or very strong signal (++++). A semiquantitative analysis of hybridization signals for c-fos and CRF1 receptor mRNAs in the PVN was carried out in nuclear emulsion-dipped slides over the confines of cells of the hypothalamic PVN using a Zeiss Optical System (Axioscop) coupled to a multimedia PC computer (ASC computer) and Image software (Alcatel TITN Answare). The ratio luminosity of the signal/surface of the PVN (expressed in pixels) was analyzed on matched sections for all animals and expressed in arbitrary units. Because of the lack of basal expression of c-fos and CRF1 receptor mRNAs in the PVN, each unilateral medial nucleus was digitized under brightfield illumination and then subjected to densitometric analysis under darkfield at a magnification of x10, yielding measurements of mean refraction density in arbitrary units (RDAU). The RDAU was corrected for the average background in subtracting the signal measured immediately outside the PVN.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macroscopic and microscopic evaluation of colitis. Intracolonic administration of 0.9% saline alone did not produce any behavioral modification of the animals as well as any macroscopically or histologically detectable damage of the rectocolon.

All animals receiving TNBS/ethanol showed reduced fluid and food intake, diarrhea, piloerection, lack of preening, and a reduced level of activity after induction of colitis. Administration of TNBS was not painful as observed by the absence of any abdominal contractions consisting in the contraction of the flank muscles associated with inward movements of the hindlimb or with whole body stretching (7). Macroscopic damages of the colon were observed at the site of TNBS instillation between 1 and 24 h. The site of inflammation generally involved the rectum until the splenic flexure. Damages were rarely observed proximal to the splenic flexure. No macroscopic abnormality of the liver, spleen, kidney, ileum, jejunum, or duodenum was observed. According to Morris et al. (20), the macroscopic damage score was between 2 and 5 for all rats. Linear ulcers with no significant inflammation were observed at 1 h post-TNBS (score: 2). Linear ulcers with inflammation at one, two, or more sites were observed at 2–6 h post-TNBS (score: 3–4). Extensive damage was observed at 12–24 h (score: 5). Histological examination showed broad-based mucosal ulcers with a surface layer of necrotic ulcer slough. The inflammatory infiltrate associated with the ulcers consisted of neutrophils, eosinophils, lymphocytes, and plasma cells extended through the full thickness of the bowel wall. Infiltration of polymorphonuclear leukocytes in the mucosa and submucosa started 1 h after induction of colitis. Leucocytes were observed in the serosa at 6 h, and at 12 h, all of the wall was infiltrated with polymorphonuclear leukocytes. Destruction of the glands in the mucosa started 4 h after colitis and was almost completed at 24 h. Edema of the mucosa and submucosa was observed at 1 h and then regularly increased.

Expression of the gene encoding c-fos in the brain of rats with colitis. With sense probe, brain-hybridized tissues did not exhibit detectable signal in any of the regions that showed positive signal with antisense probe (data not shown).

In animals receiving intracolonic injection of saline, the pattern of c-fos expression in the brain was comparable with untreated animals. As we previously described (6), basal levels of c-fos mRNA were observed in numerous brain regions (Fig. 1, Table 1) predominantly in the dorsal endopiriform nucleus, cerebral cortex, hippocampus, thalamus (anterodorsal nucleus), pontine gray, spinal trigeminal nucleus, and cerebellum.



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Fig. 1. Representative example of the distribution of the mRNA encoding the immediate-early gene c-fos in the rat brain 2 h after intracolonic administration of vehicle (control) or trinitrobenzensulfonic acid (TNBS; colitis). These rostrocaudal coronal sections (30 µm) of TNBS-treated rats exhibited a positive signal on X-ray film (Kodak XAR 5) for c-fos transcript in the bed nucleus of the stria terminalis (BST), piriform cortex (Pir), subfornical organ (SFO), supraoptic nucleus (SON) of the hypothalamus, suprachiasmatic nucleus (SCh), cerebral cortex (Cx), paraventricular nucleus of the thalamus (PV), paraventricular nucleus (PVN; parvo- and magnocellular part) of the hypothalamus, central nucleus of the amygdala (CeA), locus coeruleus (LC), posterodorsal tegmental nucleus (PDTg), lateral parabrachial nucleus (LPB), external cuneate nucleus (ECu), area postrema (AP), A1/C1 cathecolaminergic nuclei, and nucleus of the solitary tract (NTS).

 

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Table 1. Relative expression of c-fos mRNA across the rostrocaudal extent of the brain of controls and trinitrobenzenesulfonic acid (TNBS)-induced colitis rats

 
In animals receiving 30 mg TNBS/ethanol, a strong signal for c-fos mRNA was observed in several nuclei and areas of the brain, including the medial preoptic area (MPOA)/organum vasculosum of the lamina terminalis (OVLT); the SON; the parvocellular and magnocellular subdivision of the PVN; the arcuate nucleus/mediane eminence; the LC; the NTS, mainly in its medial subdivision, and the AP. A significant transcription of c-fos was also observed in the lateral preoptic area, intermediate part of the lateral septal nucleus, bed nucleus of the stria terminalis, anterior hypothalamic nucleus, subfornical organ (SFO), suprachiasmatic nucleus, dorsomedial hypothalamic area, PVN of the thalamus, nucleus reuniens, central nucleus of the amygdala (CeA), ventral part of the tuberomammillary nucleus, laterodorsal tegmental nucleus (LDT), external lateral part of the PB, dorsal division of the ambiguus nucleus, and the lateral reticular nucleus. In the medulla, c-fos transcript was also observed in the cuneate and gracile nuclei (Fig. 1, Table 1). The c-fos mRNA signal appeared at 1 h, peaked at 2 h, and then regularly decreased to completely vanish at 6–12 h. In the PVN, the c-fos signal appeared 1 h after colitis in the parvocellular subdivision and then increased to peak at 2 h to be present not only in the parvo- but also in the magnocellular part. The signal decreased at 3 h and was only present in the parvocellular part. The signal then regularly decreased at 4 h in the parvocellular part with some expression at the periphery of the magnocellular part. At 6–12 h, the signal was barely detectable in the all nucleus with only a few expressions in the dorsal and ventral part of the parvocellular subdivision. At 24 h, the signal had almost completely vanished with only a little expression in the dorsal parvocellular part (Figs. 2 and 3).



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Fig. 2. Time-related influence of intracolonic (ic) administration of vehicle (control; A) or TNBS (BH) on the expression of c-fos mRNA in the PVN of the rat hypothalamus. These photographs depict darkfield photomicrographs of dipped autoradiographs of hybridized 30-µm sections with c-fos riboprobe through identical areas of the right PVN 1 (B), 2 (C), 3 (D), 4 (E), 6 (F), 12 (G), and 24 (H) h after ic injections. Note the positive hybridization signal in the parvo (p)- and magno (m)-cellular divisions of the PVN 2 h after TNBS, whereas the PVN of control rats did not display detectable c-fos transcript. 3V, third ventricle. Magnification, x110.

 


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Fig. 3. Effect of intracolonic administration of vehicle (controls) vs. TNBS on the refraction density (RD) for c-fos and coricotropin-releasing factor 1 (CRF1) receptor mRNA signals in the PVN of the rat hypothalamus. The RD in arbitrary units (RDAU) was quantified in both sides of the PVN using a Zeiss Optical System (Axioscop) coupled to a PC computer (ASC computer) and Image software (Alcatel TITN Answare). Results represent means ± SE, and statistical analysis was performed by using a 3-way ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control animals.

 
Expression of the gene encoding the CRF1 receptor in the brain of rats with colitis. Brain-hybridized tissues with the sense probe did not exhibit a detectable signal in any of the regions that showed a positive signal with the antisense probe (data not shown).

We (6) and others (25) have observed basal levels of CRF1 receptor mRNA in control rats in numerous regions of the brain (Fig. 4, Table 2), such as the frontal cortex, cingulate and piriform cortices, medial and basolateral nuclei of the amygdala, caudal division of the zona incerta, red nucleus, LDT, pontine gray, cerebellum, nucleus incertus, spinal nucleus of the trigeminal nerve, principal sensory nucleus of the trigeminal nerve, suprageniculate nucleus, and external cuneate nucleus. A low-to-moderate signal was also observed in the medial septal nucleus, nucleus of the diagonal band, bed nucleus of the stria terminalis, hippocampus, SON, dorsomedial nucleus of the hypothalamus, arcuate nucleus of the hypothalamus, parafascicular nucleus, the interpeduncular nucleus, nucleus prepositus, medial vestibular nucleus, and spinal nucleus of the trigeminal nerve. An undetectable signal of CRF1 receptor mRNA was observed in the PVN as previously demonstrated (Figs. 35).



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Fig. 4. Representative example of the distribution of the mRNA encoding the CRF1 receptor in the rat brain 3 h after intracolonic administration of vehicle (control) or TNBS (colitis). These rostrocaudal coronal sections (30 µm) of TNBS-treated rats exhibited a positive signal on X-ray film (Kodak XAR 5) for the CRF1 receptor transcript in various layers of the Cx, Pir, parvocellular part of the PVN of the hypothalamus, medial nucleus of the amygdala (MeA), dorsomedial hypothalamus (DM), red nucleus (RN), laterodorsal tegmental nucleus (LDT), pontine nuclei (Pn), external cuneate nucleus (ECu), and spinal nucleus of the trigeminal nerve interpolar part (Sp5I). Note the selective induction of the CRF1 receptor mRNA in the PVN of TNBS-treated animals.

 

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Table 2. Qualitative analysis of the corticotropin-releasing factor (CRF1) receptor mRNA signal throughout the brain of controls or TNBS-induced colitis rats.

 


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Fig. 5. Time-related influence of ic administration of vehicle (control; A) or TNBS (BH) on the expression of CRF1 receptor mRNA in the PVN of the rat hypothalamus. These darkfield photomicrographs of dipped autoradiographs of hybridized 30-µm coronal sections with CRF1 receptor cRNA probe throughout identical areas of the right PVN were taken 1 (B), 2 (C), 3 (D), 4 (E), 6 (F), 12 (G), and 24 (H) h after ic injections. No CRF1 receptor transcript was observed in controls, whereas animals receiving TNBS exhibited a dense and selective signal in the parvocellular subdivision of the PVN 2, 3, and 4 h after TNBS. Magnification, x110.

 
In animals exposed to colonic injection of TNBS/ethanol, CRF1 receptor mRNA signal selectively appeared in the parvo-PVN (dorsomedial part) at 1 h, was more detectable at 2 h, peaked at 3 h, and then rapidly decreased to completely vanished at 12 h (Figs. 3 5). Moreover, no significant increase in the intensity of the CRF1 receptor signal was observed in other regions and nuclei of the brain (Fig. 4, Table 2).

Expression of gene encoding the CRF2 receptors in the brain of rats with colitis. In contrast to the CRF1 receptor, which is widely distributed throughout the rat brain, mRNA encoding the CRF2{alpha} receptor is highly localized in very few regions of the brain in basal conditions. Indeed, a positive hybridization signal was detected in the lateral septal nucleus, principal nucleus of the bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, corticoamygdaloid nucleus, enthorinal cortex, and interpeduncular nucleus (Fig. 6). TNBS-induced colitis did not alter the endogenous expression of CRF2{alpha} receptor transcript in these spontaneously expressing structures (Fig. 6). We did not see any convincing evidence of positive signal for the CRF2{alpha} receptor transcript within the hypothalamic PVN and the SON on brain sections either exposed on X-ray film or dipped in NTB-2 nuclear emulsion (data not shown). A positive signal was hybridized with CRF2{beta} antisense only in the choroid plexus and not in any other region of controls and TNBS-treated animals (data not shown).



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Fig. 6. Distribution of the mRNA encoding the CRF2{alpha} receptor in the rat brain 3 h after intracolonic administration of vehicle (control) or TNBS. These rostrocaudal coronal sections (30 µm) of TNBS- and vehicle-treated rat brains exhibit a highly localized positive signal on X-ray film for the CRF2{alpha} receptor transcript in the lateral septal nucleus (LS). VMH, ventromedial hypothalamic nucleus.

 
Induction of c-fos and CRF1 receptor transcripts in CRF-immunoreactive neurons. In control rats, none of the CRF-immunoreactive neurons exhibited a positive signal for either c-fos, CRF1 receptor, or CRF2 receptor transcripts in the parvocellular neurosecretory zone of the PVN, whereas numerous CRF-immunoreactive neurons were positive for c-fos or CRF1 receptor transcripts in TNBS-induced colitis (Fig. 7). No expression of CRF2 receptor transcript was observed in CRF-immunoreactive neurons in the PVN after colitis (data not shown).



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Fig. 7. Expression of c-fos (A and B) and CRF1 receptor (C and D) mRNAs in CRF-immunoreactive (CRF-ir) perikarya located in the hypothalamic PVN of vehicle (A and C)- and TNBS (B and D)-treated animals. Animals were fixed with 4% paraformaldehyde borax 2 h after intracolonic injection of vehicle or TNBS (CRF-ir/c-fos mRNA) or 3 h thereafter (CRF-ir/CRF1 receptor mRNA). Immunocytochemistry (CRF protein, neurons) was performed on the same brain sections (30 µm) before in situ hybridization histochemistry (CRF1 receptor and c-fos mRNAs, small grains). Arrowheads show CRF neurons expressing the gene encoding c-fos (B) or CRF1 receptor (D) mRNAs in TNBS-treated animals, whereas very few CRF-containing neurons were positive for either transcripts in the PVN of vehicle-treated animals (A and B). Magnification, x625.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study shows that a validated model of colonic inflammation, produced by an intracolonic injection of a mixture of ethanol and TNBS, induces at the acute-phase response c-fos transcript in brain nuclei involved in the autonomic, behavioral, and neuroendocrine response to inflammation, as well as in circumventricular organs (CVOs) and that CRF pathways, in particular in the PVN, are activated in this model.

The present study provides evidence that colitis induces strong expression of c-fos in multiple structures of the brain, including most of the CVOs (i.e., the AP-SFO-OVLT), but also the MPOA, SON, PVN (parvo- and magnocellular part), arcuate nucleus, LC, NTS, CeA, LDT, and LPB. The brain distribution of c-fos was comparable with other studies using transsynaptic retrograde tracing from the colon to the brain (36). The pattern of c-fos transcript observed in the present study is most likely induced through a dual pathway. The first pathway is most likely represented by the activation of visceral afferents through the aggression of the mucosa by the mixture of ethanol/TNBS, whereas the second pathway is most likely activated by interleukins. Nociceptive pathways may be involved in the present study, because some of the brain nuclei activated by colitis are also activated after intraperitoneal injection of acetic acid (7), a well-known model of noxious somatovisceral stimulus. However, the present study does not provide evidence nor does it provide adequate reference in support of the fact that nociceptive pathways are one of the pathways involved in the present neuronal activation pattern. In addition, no abdominal contraction was observed after intracolonic instillation of TNBS as observed after intraperitoneal acetic acid (7). The spinal cord was not studied in the present study. However, we have already observed Fos protein in the lumbosacral spinal cord (L6-S1) of rats with TNBS-induced colitis and, to a lesser extent, at the thoracolumbar level (T10-L2) in laminae known to receive splanchnic visceral afferents as represented by laminae I-IIo, V, VII, and X and in the sacral parasympathetic nucleus (30). Among brain nuclei activated in the present study, the cuneate nucleus and the gracile nucleus are known to receive axons from neurons near the central canal at sacral (L6-S1) and thoracic levels of the spinal cord; these neurons are part of the postsynaptic dorsal column, a pathway that conveys visceral signals, in particular from the rectocolon (1). The pattern of c-fos transcript observed after colitis is also close to that observed after an immune challenge, as represented by intraperitoneal injection of lipopolysaccharides (24) in which a strong expression of c-fos transcript is observed in the CVOs. Immune systems might use several pathways and sites of entry to communicate with the brain and to activate the neurons responsible for the activation of the HPA axis (26). The noradrenergic (A1, A2, A6) and adrenergic (C1-C3) pathways from the brain stem could mediate the influence of different cytokines of systemic origin produced during the acute-phase response to stimulate the parvo- and magnocellular neurons of the hypothalamic PVN (26). Data argue for an effect of IL on vagal afferents, especially IL-1{beta}. In TNBS-induced colitis, it has already been made clear that ethanol acts primarily as a "barrier breaker" allowing TNBS access to subepithelial space, thus activating a variety of intestinal cells to release inflammatory cytokines (14). We did not assess tissue and/or plasma levels of interleukins in the present study. However, Tateishi et al. (32) have looked at interleukin levels in the early and chronic phase of colitis; i.e., at 5, 15, 30, 45 min; 1, 2, 6, and 12 h; 1 and 3 days; and 1, 2, and 4 wk after TNBS instillation in the rat colon. They observed an early increase in plasma and/or tissue levels of TNF-{alpha} and IL-6. However, the release of IL-1{beta} did not occur before 6 h, whereas the peak of c-fos induction is 2 h, which does not correspond. TNF-{alpha}, known to act through the vagus nerve (13), may be another candidate. Finally, both bulbospinal and vagal pathways are most likely involved, because spinal cord transsection decreases transsynpatic retrograde tracing in the NTS after injection into the colon (36). However, c-fos mRNA is expressed not only in the NTS and in the A1 to C1 regions, but also in the LC, LPB, and LDT. These nuclei could either relay the information from the NTS to the PVN or modulate themselves some of the effects of cytokines on neuroendocrine functions. The NTS also projects massively to the PB nucleus, which in turn projects to the parvocellular subdivision of the PVN (27). The cholinergic cell group of the LDT can also directly modulate parvocellular PVN neurons. The LC has few projections to the PVN; it is thus possible that the LC interacts indirectly with PVN neurons through other brain stem nuclei, such as the LDT and PB, which are part of the nuclei at the origin of the CRF innervation of the PVN (8). A strong signal for c-fos was also detected in several layers of the median eminence, indicating the possibility that systemic immune factors alter the release of neuropeptides directly within the infundibulum. In our study, the c-fos transcript was also observed in the magno-PVN and the SON, known to contain arginine vasopressin (AVP). This neuromediator is released into the hypophyseal-portal circulation by an immune challenge (3). Most of this AVP is believed to derive from magnocellular neurosecretory neurons and thus participates in the control of the HPA axis during various stressors. One can wonder that fasting could have an effect on the neuronal activation observed in our study. Although the animals administered with vehicle or TNBS had free access to food after the fasting period, we did not look at eating pattern at the time (2 or 3 h) at which most of the data are presented. Barbier et al. (2) have shown that elevated plasma leptin concentration, correlated with the degree of inflammation and associated with anorexia, were induced in rats during the early stages of experimental colitis but proved transient. Plasma leptin concentration increased fourfold 8 h after colitis, whereas 4 h after colitis, there was no significant change in plasma leptin concentration in comparison with saline-treated animals. In this study, the authors showed that changes in food intake after colitis were paralleled by changes in body weight. Rats lost weight within 24 h and experienced the greatest drop in body weight on days 3 and 4, which is presently far away from the times at which we looked for c-fos and CRF receptor expression in our study. Consequently, at this early time (2–3 h post-TNBS) we think that the anorectic effect of colitis at that time is not able to interfere with our results.

In this study, we did not perform myeloperoxidase activity as a marker of inflammation. We looked only at macroscopic and histological damage of the colon after TNBS. Yamada et al. (37), in comparing two models of colitis in rats (acetic acid vs. TNBS intracolonic), have shown that colonic myeloperoxidase activity was significantly increased compared with control at 48 h but not at 6 h post-TNBS as observed by Tateishi et al. (32). In the same study, the authors observed a significant increase of mucosal permeability and wet-dry ratios of the colon at 2 and 6 h after TNBS, and they found an acute microscopic inflammation at 2 h post-TNBS.

Central action of IL-6 includes stimulation of the HPA axis through a neuroendocrine CRF-mediated mechanism (21). This cytokine may thus be considered an important mediator in the neuroimmune interface. After systemic IL-6, Vallières et al. (35), observed c-fos mRNA signal, 1 h after intravenous injection, in the same regions of the ones observed in our study. These data provide clear evidence that systemic administration of IL-6 triggers expression of c-fos within all the sensorial CVOs, suggesting that OVLT, SFO, median eminence, and AP play an important role in the interface between circulating IL-6 and neuronal activity. The sensorial CVOs seem therefore to be located in a privileged position to mediate the acute-phase response of an immune challenge (26).

In agreement with these data, we show that CRF pathways are activated by colitis as represented by 1) the topographical distribution of the c-fos mRNA signal in the parvocellular PVN, an area rich in CRF perikarya (29); 2) the combination of in situ hybridization with immunohistochemistry, which showed that numerous CRF perikarya expressed the immediate-early gene; and 3) the selective expression of CRF1 receptor mRNA in the PVN within CRF immunoreactive cells. Activation of CRF pathways could play a determinant role in the integration of the information received from the periphery to restore homeostasis via the HPA axis. We (6) previously observed a selective expression of CRF1 receptor transcript in the parvocellular PVN after an acute immobilization stress, whereas a downregulation of this transcript was observed in chronic conditions. Although the gene encoding CRF1 receptor is widely distributed in the brain in basal conditions, neither immune challenge nor immobilization stress seemed able to modulate the transcription of this receptor in most of these spontaneously expressing regions (Refs. 6, 25, and present study). In contrast, the CRF1 receptor transcript is induced quite selectively within the population of PVN cells in response to stressful conditions (6, 25). Such specific induction may indicate that 1) neuroendocrine CRF is the only CRF that can be regulated by its own receptor during stress, 2) induction of the CRF1 receptor in the parvocellular PVN is a determinant mechanism involved in the restoration of the peptide depletion after the stress period, and 3) the constitutive expression of the gene encoding the CRF1 receptor is not regulated in a stress-dependent manner. In our study, the rapid expression of c-fos in PVN-containing neurons and the temporal changes are consistent with the hypothesis that the transcription factor encoded by this gene may be acting as an early intracellular signal for direct or indirect activation of CRF1 receptor gene.

Apparently, CRF2 receptors are not involved in the present model because of the absence of any modification of this transcript in the brain. Confusing data exist regarding the presence or absence of the CRF2{alpha} receptor within the PVN. Although Lovenberg et al. (16) have described its presence, we (present study) and others (4) have failed to show any signal for the CRF2{alpha} receptor mRNA in the PVN of neither control nor challenged rats. The discrepancy between the original description of the CRF2 receptor distribution and more recent publications may be related to the low transcript levels and the CRF2 receptor probe that exhibits nonspecific labeling when overexposed.

In conclusion, peripheral colitis triggers the activation of the neuroendocrine system via hypothalamic CRF. The aim of this activation is to provide a counterregulatory mechanism that critically modulates inflammatory events through the hypersecretion of glucocorticoids. Indeed, it is well established that central CRF has a protective effect on experimental colitis (19). Direct evidence of the anti-inflammatory role of hypothalamic CRF has been shown by an enhanced susceptibility of genetically CRF-hyporesponsive Lewis rats to inflammatory injury (31). A blunting of this anti-inflammatory limb thus contributes to the stress-related worsening of colitis by leaving unchecked the proinflammatory functions triggered by acute stress as observed in experimental conditions (12) and in humans (15).


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. Bonaz, Département d'Hépato-Gastroenterologie, Hôpital Albert Michallon, Centre Hospitalier Universitaire, 38043 Grenoble Cedex 09, France (E-mail: Bruno.Bonaz{at}ujf-grenoble.fr)

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.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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