Differential induction of c-fos expression in brain nuclei by noxious and non-noxious colonic distension:

Brain Research 966 (2003) 253–264 www.elsevier.com / locate / brainres

Research report

Differential induction of c-fos expression in brain nuclei by noxious and non-noxious colonic distension: role of afferent C-fibers and 5-HT 3 receptors a, *, Jens Ruter ¨ ¨ a , Matthias Konig ¨ b , Christoph Grote b , Peter Kobelt a , Hubert Monnikes Burghard F. Klapp a , Rudolf Arnold b , Bertram Wiedenmann a , Johannes J. Tebbe b a

¨ , Berlin, Germany Department of Internal Medicine, Charite´ , Humboldt-Universitat b ¨ , Marburg, Germany Department of Internal Medicine, Philipps-Universitat Accepted 10 December 2002

Abstract Experimental animal models have been established to gain insight into the pathogenesis and the mechanisms of visceral hyperalgesia in the irritable bowel syndrome (IBS). However, data about the mechanisms and pathways involved in the induction of neuronal activity in forebrain and midbrain structures by a physiological GI stimulus, like colonic distension (CD), in the range from non-noxious to noxious intensities are scarce. Thus, the effect of proximal CD with non-noxious (10 mmHg) and noxious (40 and 70 mmHg) stimulus intensities on neuronal activity in brain nuclei, as assessed by c-fos expression, was established. In additional studies, the role of vagal and non-vagal afferent sensory C-fibers and 5-HT 3 receptors in the mediation of visceral nociception was investigated in this experimental model at noxious colonic distension (70 mmHg). At CD, the number of c-Fos like immunoreactivity (c-FLI)-positive neurons increased pressure-dependently in the nucleus of the solitary tract (NTS), rostral ventrolateral medulla (RVLM), nucleus cuneiformis (NC), periaqueductal gray (PAG), and the amygdala (AM). In the dorsomedial (DMH) and ventromedial nucleus (VMH) of the hypothalamus, as well as in the thalamus (TH), neuronal activity was also increased after CD, but independently of stimulus intensities. A decrease of the CD-induced c-fos expression after sensory vagal denervation by perivagal capsaicin treatment was only observed in brainstem nuclei (NTS and RVLM). In all other activated brain nuclei examined, the CD-related induction of c-fos expression was diminished only after systemic neonatal capsaicin treatment. In the NTS and RVLM, a trend of decrease of c-fos expression was also observed after systemic neonatal capsaicin treatment. In order to assess the role of the 5-HT 3 receptor in CD-induced neuronal activation of brain nuclei, animals were pretreated with the 5-HT 3 receptor antagonist granisetron (1250 mg / kg, i.p. within 18 h before CD). Pretreatment with granisetron significantly reduced the number of c-FLI-positive cells / section in the NTS by 40%, but had no significant effect on the CD-induced c-fos expression in other brain areas. The data suggest that distinct afferent pathways and transmitters are involved in the transmission of nociceptive information from the colon to the brain nuclei activated by proximal colonic distension. Activation of NTS neurons at such a condition seems to be partially mediated via capsaicin-sensitive vagal afferents and 5-HT 3 receptors. In contrast, activation of brain nuclei in the di- and telencephalon by nociceptive mechanical stimulation of the proximal colon, as assessed by c-fos expression, is partially mediated by capsaicin-sensitive, non-vagal afferents, and independent of neurotransmission via 5-HT 3 receptors. The modulation of CD-induced c-fos expression exclusively in the NTS by granisetron points to a role of 5-HT 3 receptor antagonists in the modulation of vago-vagal sensomotoric reflexes rather than an influence on forebrain nuclei involved in nociception.  2002 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Somatic and visceral afferents Keywords: Brain–gut axis; Irritable bowel syndrome; Rat; Vagal afferent; Visceral nociception; 5-HT 3 receptor Abbreviations: AM, Amygdala; AP, Area postrema; CD, Colonic distension; c-FLI, c-fos-like immunoreactivity; Dc-FLI-C, Increase in the average number of c-FLI-positive cells / section in comparison to controls; DMH, Dorsomedial nucleus of the hypothalamus; NC, Nucleus cuneiformis; NTS, Nucleus of the solitary tract; PAG, Periaqueductal gray; RVLM, Rostral ventrolateral medulla; TH, Thalamus; VMH, Ventromedial nucleus of the hypothalamus *Corresponding author. Department of Medicine, Division of Hepatology, Gastroenterology, Endocrinology and Metabolic Diseases, and Division of ´ Humboldt-Universitat ¨ zu Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Tel.: 149-30-450-553391; Psychosomatics and Psychotherapy Charite, fax: 149-30-450-553991. ¨ E-mail address: [email protected] (H. Monnikes). 0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(02)04197-5

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1. Introduction Several pathophysiological mechanisms have been suggested to play a role in the genesis of symptoms in patients with irritable bowel syndrome (IBS); among others visceral hypersensitivity [44,61], autonomic nervous system dysregulation [68], alterations of gastrointestinal (GI) motility [50], and abnormalities in neurotransmitter systems such as serotonin [3]. There is considerable evidence from several groups of investigators that these pathophysiological disturbances lead to imbalances in the communication along the brain–gut axis [42,50]. Patients with IBS present with a variety of symptoms, including chronic abdominal pain or discomfort, and disordered gastrointestinal function, i.e. diarrhea or constipation [69]. While the gastrointestinal symptoms of these patients vary, chronic or recurrent pain or discomfort is a conditio sine qua non for the diagnosis of IBS [67]. Furthermore, it has been shown that at least a subgroup of IBS patients shows a hyperalgesic response to visceral stimuli, and discomfort in response to colorectal balloon distension under experimental conditions [44,61]. Thus, assessments of drug effects on visceral sensation at colorectal balloon distension have been performed to determine if various compounds might be beneficial in the treatment of IBS patients [19,36,58]. Experimental animal models have been established to gain insight into the pathogenesis and the mechanisms of visceral hyperalgesia. Also, in pharmacological studies the role of various neurotransmitters and effects of several drugs on visceral sensitivity to colonic distension have been investigated [9]. Colonic distension (CD) is a non-invasive, reproducible visceral stimulus with intensities ranging from sublime to painful that can be used in humans as well as in animal models. It has been shown in experimental rats that noxious CD-pressures (40 mmHg or higher) induce a range of pseudoaffective responses, including vasomotor, visceromotor, and respiratory responses [16,54]. In previous studies it has been shown that noxious distension of hollow viscera (i.e. colorectum, esophagus, stomach) induces a specific pattern of c-fos expression in the rat spinal cord, nucleus of the solitary tract (NTS) and limbic brain structures [38,70–72]. Induction of c-fos expression is a well established marker of neuronal activation, and immunohistological detection of c-Fos-like immunoreactivity (c-FLI) allows a mapping of activated brain nuclei on a single cell level [49,63]. However, little is known about the effect of CDstimulus intensities in the range from non-noxious to noxious on c-fos expression in forebrain and midbrain structures. Therefore, in the present study, we established a rat model to identify brain nuclei involved in the processing of visceral nociceptive stimuli of different intensities due to distension of the proximal colon, as assessed by induction of c-fos expression.

It has been suggested that vagal afferents, in particular sensory vagal C-fibers, modulate the sensitivity to visceral stimuli by affecting the activity of cerebral pain control centers like the descending bulbospinal pain inhibitory system [9]. Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is widely used to selectively excite and eventually destroy primary sensory C-fibers. At local administration capsaicin depolarizes vagal fibers and induces an increased permeability to calcium, which has neurotoxic effects on sensory neurons [40]. Systemic application of capsaicin at high doses to neonatal animals inhibits the development of C-fibers [12,41]. Thus, in additional experiments we used perivagal and systemic neonatal capsaicin treatment to study the role of vagal or spinal C-fibers in the afferent neuronal transmission from the colon to the brain at CD. Serotonin (5-HT 3 ) is thought to play a role in visceral nociceptive mechanisms [9]. There is also considerable evidence that serotonin is involved in the regulation of motility and sensation in the gut [18,24]. Several studies involving antagonists at the 5-HT 3 receptor have implicated an important role of serotonergic pathways in the pathogenesis of IBS and have led to the use of 5-HT 3 receptor antagonists in the treatment of IBS patients, e.g. alosetron has been reported to significantly reduce pain scores at colorectal distension [19]. Interestingly, recent studies suggest that 5-HT 3 receptors in the gut are predominantly of vagal afferent origin [22]. Therefore, in additional studies, animals were pretreated with the 5-HT 3 receptor antagonist granisetron to investigate the role of the 5-HT 3 receptor in mediating activation of forebrain and hindbrain structures at CD.

2. Materials and methods

2.1. Animals Male Sprague–Dawley rats (Winkelmann Co., Borchen, Germany) weighing 250–300 g were housed under conditions of controlled illumination (12:12 h light / dark cycle), humidity, and temperature (2262 8C) for at least 7 days prior to the surgical procedure. They were fed a standard rat diet (Altromin, Lage, Germany) and tap water ad libitum and maintained in colony cages. The animals were deprived of food but not water 18 h before each experiment. Each experimental group included six rats. Animal care and experimental procedures followed institutional ethics guidelines and conformed to the requirements of the state authority for animal research conduct.

2.2. Drugs Capsaicin (Sigma, St Louis, USA) was sonicated with 0.1 ml Tween 80 (Sigma) for 10 min, made up to 1 ml with olive oil (Sigma), and mixed thoroughly.

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Granisetron was dissolved in sterile, isotonic NaCl solution immediately before the injection.

2.3. Perivagal capsaicin treatment The perivagal treatment with capsaicin was performed as described before [49,59]. Rats were anesthetized with xylazine (10 mg / kg, i.p.; Bayer, Leverkusen, Germany) and ketamine (100 mg / kg, i.p.; Parke–Davis, Freiburg, Germany). Both cervical vagi were exposed by a midline neck incision and the vagal trunk was carefully freed from the right and left carotid arteries and exposed for a distance of 3–4 mm. To avoid a diffusion of capsaicin into the surrounding tissue, strips of parafilm were placed around the exposed vagus nerve. Then a small pledge of cotton wool soaked in capsaicin solution was applied to the nerve for 30 min. Further drops of capsaicin solution were applied every 10 min to a maximum of 0.05 ml. Finally, the area was thoroughly rinsed with saline and dried with swabs and the neck incision was closed. The distension experiments were performed 3 weeks after the capsaicin treatment. The animals of the control groups were treated with vehicle solution.

2.4. Neonatal capsaicin treatment Pregnant female Sprague–Dawley rats were obtained from Winkelmann Co. Neonatal rats were treated with capsaicin solution (50 mg / kg, i.p.) or with vehicle solution (olive oil) at days 2 and 3 after birth as described before [41]. The animals were weaned at day 21 of age and grown to final weights of 250–300 g. Distension experiments were started between postnatal days 100–120.

2.5. Granisetron treatment ¨ The granisetron (GlaxoSmithKline, Munchen, Germany) treatment was performed in adjustment to a rationale described before [52]. Thus, granisetron was injected intraperitoneally at a dose of 500 mg / kg 18 h before the distension experiment. The animals were treated with additional doses of granisetron (375 mg / kg, i.p.) at 30 and 15 min before the start of the experiment.

2.6. Colonic distension A catheter made of tygon tube (o.d.,1.5 mm) and provided with a flexible latex balloon (2 cm in length) at the tip was carefully inserted into the proximal colon via the anal canal and fixed at a distance of 8 cm with adhesive tape at the tail of the awake animal. A computercontrolled Barostat-apparatus connected with a 60 ml syringe was used to inflate the balloon until reaching the scheduled pressures (10, 40 or 70 mmHg above basal balloon pressure at the respective volumes) in the freely moving animal. At the stimulation experiments six disten-

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sion cycles with a duration of 10 min were each followed by intervals without balloon distension (0 mmHg) of 10 min. The total time of the distension procedure was 120 min.

2.7. c-Fos immunohistology This method was performed as described before [49]. At the end of the distension procedure, the animals were anesthetized with xylazine (10 mg / kg, i.p.) and ketamine (100 mg / kg, i.p.), and transcardially perfused with phosphate-buffered saline (PBS) buffer (0.1 M) followed by Zamboni’s fixative (2% formaldehyde and 2% picric acid in 0.1 M PBS buffer). The brains were removed, postfixed in Zamboni’s fixative and cryoprotected in 25% sucrose. Then, free-floating brain sections (30 mm) were stained for c-Fos-like immunoreactivity (c-FLI) using the avidin– biotin–peroxidase method. The primary antibody (rabbit anti c-fos protein polyclonal antiserum, Calbiochem, USA) was applied at a dilution of 1:1000 with 0.5% Triton X, 1% goat serum, and 1% bovine serum albumin (BSA) for 48 h at 4 8C. The biotinylated secondary antibody (goat anti-rabbit immunoglobulin G) at a dilution of 1:200 and 0.5% Triton X was applied for 90 min. Diaminobenzidine was used as the chromogen. The stained sections were mounted on chromium–aluminium–gelatine-coated slides, air dried, and placed under a coverslip with DePeX. Brain sections were examined using bright-field microscopy. The same lot of antibody was used for each study outlined below. The primary c-fos antibody was omitted in one well of each immunohistological reaction as a negative control. In each study, every staining process included free-floating sections of all groups using the same buffers and solutions.

2.8. Experimental design 2.8.1. Study 1: effects of colonic balloon distension on c-fos expression in brain nuclei The first study was performed to investigate the effects of proximal colonic distension with non-noxious (10 mmHg) or noxious (40, 70 mmHg) intensities on neuronal activity in brain nuclei, as assessed by induction of c-fos expression. Animals with the balloon inserted into the colon but not distended (0 mmHg), and animals without balloon placement into the colon served as controls. Induction of c-fos expression was determined by changes in the number of c-FLI-positive cells in brain nuclei. 2.8.2. Study 2: effects of perivagal capsaicin on colonic balloon distension-induced stimulation of c-fos expression in brain nuclei The second study was performed to determine the

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effects of afferent vagal C-fiber denervation on proximal CD-induced nociceptive stimulation of neuronal activity in brain nuclei. At 3 weeks before CD experiments with a noxious distension pressure of 70 mmHg, perivagal capsaicin treatment was performed as described above. The average number of c-FLI-positive cells / section was compared to the results of control experiments in animals perivagally treated with vehicle solution 3 weeks before CD experiments.

2.8.3. Study 3: effects of neonatal capsaicin on colonic balloon distension-induced stimulation of c-fos expression in brain nuclei The third study was performed to investigate the effects of vagal plus spinal sensory C-fiber denervation on altered neuronal activity in brain nuclei (as assessed by c-FLI) due to noxious (70 mmHg) colonic balloon distension of the proximal colon. Neonatal rats were pretreated sytemically with capsaicin as described above. Experiments with a colonic distension pressure of 70 mmHg were performed when the animals had gained a weight of 250–300 g.

2.8.4. Study 4: effects of granisetron on colonic balloon distension-induced stimulation of c-fos expression in brain nuclei The fourth study was performed to determine the role of 5-HT 3 receptor-mediated neurotransmission on noxious proximal CD-induced stimulation of neuronal activity in brain nuclei (distension pressure 70 mmHg). Thus, animals were pretreated with an i.p. injection of the 5-HT 3 receptor antagonist granisetron as described above. The control group was pretreated with the vehicle solution, accordingly.

2.9. Data and statistical analysis Semiquantitative assessment of c-fos expression was performed as described before [51]. Briefly, the number of c-FLI-positive cells were counted in brain areas where colonic balloon distension induced c-fos expression, i.e. AM, AP, DMH, NC, NTS, PAG, RVLM, TH, and VMH. Cells with dark brown or black nuclear c-FLI staining above the generally low background at bright-field microscopy were identified as c-FLI-positive cells. Quantification of c-FLI-positive cells in the brain areas mentioned above was performed in every third of all coronal sections throughout the rostrocaudal extent of these brain areas. Anatomic correlations were made according to landmarks given in a stereotaxic atlas [57]. c-FLI-positive cells were counted in identical numbers of sections per rat separately for each side of the brain for the various brain nuclei investigated. In every immunohistological reaction identical numbers of brains were processed for all groups

investigated to accomplish maximal consistency of the results. This proceeding was strictly performed in a singleblind method. No differences in the number of c-FLIpositive cells between the two sides of the brain were observed. Thus, the average number of c-FLI-positive cells per section was calculated in each rat for the brain nuclei mentioned above. Data are expressed as mean6S.E.M. of the average number of cells / section of the respective brain are of all rats. The data were analyzed by analysis of variance, and differences between groups were evaluated by the Student Newman–Keul’s test; P,0.05 was considered significant.

3. Results

3.1. Effects of colonic balloon distension on c-fos expression in brain nuclei Systematic screening of coronal brain sections of the whole brain revealed CD-induced changes in neuronal activity in all treated animals in the nucleus of the solitary tract (NTS), rostral ventrolateral medulla (RVLM), periaqueductal gray (PAG), nucleus cuneiformis (NC), amygdala (AM), thalamus (TH), dorsomedial hypothalamus (DMH) and ventromedial hypothalamus (VMH), as determined by increased density of c-FLI-positive cells (Fig. 1). In the NTS, the increase in the average number of c-FLI-positive cells / section was dependent on the pressure intensity (Table 1). There was a significant 19-fold and 50-fold increase in the average number of c-FLI-positive cells / section in comparison to control conditions (Dc-FLIC) in animals treated with a colonic distension pressure of 40 and 70 mmHg, respectively. In the RVLM, the Dc-FLI-C was increased pressuredependently from 10-fold at 10 mmHg to 44-fold at 40 mmHg, and 64-fold at 70 mmHg, respectively (Table 1). A pressure-related effect of colonic distension on c-fos expression was also observed in the PAG and the NC. In the PAG, the Dc-FLI-C was significant at 10 mmHg (9-fold), 40 mmHg (14-fold), and 70 mmHg (19-fold), respectively (Table 1). In the NC, the increase in Dc-FLI-C was 18-fold at 10 mmHg, 32-fold at 40 mmHg, and 44-fold at 70 mmHg, respectively (Table 1). Colonic balloon distension also resulted in increased neuronal activity in hypothalamic nuclei. In the DMH, there was a 3-fold increase in the average number of c-FLI-positive cells at 10 mmHg, and a 9-fold increase at 40 mmHg and 70 mmHg in comparison to the control condition (Table 1). In the VMH, the Dc-FLI-C was 3-fold and 12-fold at these distension pressures (Table 1). Induction of c-fos expression at colonic balloon distension was also observed in the TH. In comparison to the control condition, there was a 4-fold increase in the

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Fig. 1. Sample photomicrographs of Fos immunohistochemistry showing (a) a caudal section of the NTS of a rat treated with colonic distension at a distension pressure of 70 mmHg and (b) a higher magnification of the same section depicting c-fos-positive neurons counted as positive (full arrow) and negative (dotted arrow).

average number of c-FLI-positive cells / section in all groups treated with colonic balloon distension irrespective of the various (10–70 mmHg) pressure intensities (Table 1). In the AM, Dc-FLI-C was also dependent on the stimulus intensity. A 6-fold, 12-fold, and 16-fold increase was observed at distension pressures of 10, 40, and 70 mmHg, respectively (Table 1).

In the RVLM, PAG, NC, AM, VMH, DMH, and TH, a significant increase in the average number of c-FLI-positive neurons in comparison to the control group was already found at non-noxious colonic stimulation by catheter placement or colonic distension at a pressure of 10 mmHg. In all of these brain areas except the TH, c-fos expression increased even further at colonic distension pressures of 40 or 70 mmHg. A significant difference

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Table 1 Effects of colonic balloon distension with different pressure intensities on c-fos expression in brain nuclei, as determined by the average number of c-Fos-LI-positive neurons / section Control

n56 NTS RVLM PAG NC AM VMH DMH TH

1.160.5 0.360.1 4.161.2 1.560.7 9.962.7 3.461.0 13.563.9 18.061.9

Balloon placement (0 mmHg) n56 3.060.7 1.561.6 23.065.5 a 13.462.7 a 70.2613.7 a 15.962.9 a 33.765.4 a 25.767.8

10 mmHg

40 mmHg

n56

n56

1.760.6 3.461.6 a 35.563.2 a 26.767.0 a 62.767.7 a 10.262.4 44.968.1 a 65.568.9 a,b

70 mmHg

n56 a,b

20.562.9 14.963.1 a,b 58.6610.9 a,b 47.767.0 a,b 127.664.8 a,b 40.964.1 a,b 118.614.9 a,b 71.267.4 a,b

55.564.4 a,b 21.661.0 a,b 76.567.2 a,b 66.464.9 a,b 161.0616.8 a,b 44.662.2 a,b 116.267.0 a,b 76.262.7 a,b

Data are expressed as Mean6S.E.M. b P,0.05 vs. balloon placement (0 mmHg). a P,0.05 vs. control.

between CD-induced c-fos expression at balloon placement and a colonic distension pressure of 10 mmHg was only found in the TH.

3.3. Effects of systemic neonatal capsaicin treatment on colonic distension-induced stimulation of c-fos expression in brain nuclei

3.2. Effects of perivagal capsaicin treatment on colonic balloon distension-induced stimulation of c-fos expression in brain nuclei In animals treated with perivagal capsaicin, CD-induced stimulation of neuronal activity was diminished in the NTS and the RVLM. Capsaicin pretreatment reduced the increase in the number of c-FLI-positive cells induced by colonic balloon distension with 70 mmHg in the NTS by 28% and in the RVLM by 17%, respectively (Table 2). In contrast, no significant changes of CD-related induction of c-fos expression was observed in the other brain nuclei, as assessed by c-FLI (Table 2).

Systemic neonatal treatment with capsaicin resulted in a considerable, but statistically non-significant reduction of the CD-induced increase in the average number of c-FLIpositive cells in the NTS by 32% and the RVLM by 45% (Table 3). In the PAG and the NC a significant reduction of the CD-induced c-fos expression by 48% and 40%, respectively, was observed, as determined by the reduction in the average number of c-FLI-positive cells / section. Neonatal capsaicin treatment also significantly diminished the CD-induced increase in the number of c-FLI-positive cells in the AM, TH, and VMH by 43%, 65%, and 27%, respectively (Table 3). In contrast, pretreatment with capsaicin had no effect on neuronal activity in the DMH.

Table 2 Effects of perivagal capsaicin treatment on noxious (70 mmHg) colonic balloon distension-induced stimulation of c-fos expression in brain nuclei, as determined by the average number of c-Fos-LI-positive neurons / section

Table 3 Effects of systemic, neonatal treatment with capsaicin (100 mg / kg, i.p.) on noxious (70 mmHg) colonic balloon distension-induced stimulation of c-fos expression in brain nuclei, as determined by the average number of c-Fos-LI-positive neurons / section

NTS RVLM PAG NC AM VMH DMH TH

Vehicle (n56)

Capsaicin (n56)

41.162.6 21.360.7 68.863.2 64.966.0 132.6622.2 17.462.0 117.5610.2 75.666.0

29.463.2 a 17.760.8 a 67.360.4 88.465.6 151.1620.2 18.662.4 111.168.7 73.167.6

Data are expressed as Mean6S.E.M. a P,0.05 vs. perivagal vehicle treatment.

NTS RVLM PAG NC AM VMH DMH TH

Vehicle (n56)

Capsaicin (n56)

124.5638.4 91.7626.9 198.4622.1 164.6619.1 527.16177.7 177.166.1 234.7616.3 413.96182.3

84.4619.1 50.668.9 102.1613.4 a 98.168.5 a 302.4632.2 a 112.9612.6 a 200.0631.3 146.1624.1 a

Data are expressed as Mean6S.E.M. a P,0.05 vs. neonatal treatment with vehicle.

¨ et al. / Brain Research 966 (2003) 253–264 H. Monnikes Table 4 Effect of granisetron treatment (1.25 mg / kg, i.p.) on noxious (70 mmHg) colonic balloon distension-induced stimulation of c-fos expression in brain nuclei, as determined by the average number of c-Fos-LI-positive neurons / section

NTS RVLM PAG NC AM TH VMH DMH

Vehicle (n56)

Granisetron (n56)

48.9610.1 22.662.5 74.7613.2 71.565.4 160.3616.7 84.3617.9 20.264.4 123.667.8

30.366.2 a 15.564.0 74.1615.2 74.8611.4 174.267.2 85.366.6 16.362.4 125.465.1

Data are expressed as Mean6S.E.M. a P,0.05 vs. treatment with vehicle i.p.

3.4. Effects of treatment with granisetron on colonic balloon distension-induced stimulation of c-fos expression in brain nuclei Pretreatment with the 5-HT 3 receptor antagonist granisetron significantly reduced the CD-induced stimulation of neuronal activity in the NTS. The increase in the average number of c-FLI-positive cells / section in this brain area was reduced by 38% (Table 4). A statistically non-significant reduction in the number of c-FLI-positive cells by 31% was observed in the RVLM. In contrast, granisetron had no effect on the colonic balloon distension-induced stimulation of c-fos expression in the other brain nuclei (Table 4).

4. Discussion In the first study of the present work we established an animal model to investigate pathways and mechanisms mediating the afferent transmission of nociceptive information from the gut to brain areas involved in the processing of physiological noxious and non-noxious mechanical stimuli. Thus, we examined the effects of colonic balloon distension with a pressure of 10 mmHg (non-noxious), and 40 or 70 mmHg (noxious) on neuronal activity in brain nuclei, as assessed by changes in c-Foslike immunoreactivity (c-FLI). Induction of c-fos expression is a well established marker of neuronal activation and assessment of changes in c-FLI allows a semiquantitative mapping of activated brain areas in response to peripheral stimuli [20,63]. The present data provide evidence that noxious and non-noxious visceral stimulation by distension of the proximal colon with different pressure intensities result in distinct responses of neuronal activity in different brain

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nuclei. We observed an increased c-fos expression in forebrain and hindbrain areas, which among other functions have been proposed to be involved in gut–brain interaction (NTS, RVLM, VMH, DMH) [48], nociception (PAG, NC, TH) [32,37,38,79] or emotional responses (AM) [11]. Interestingly, the brain nuclei activated by CD match the localization of Phaseolus vulgaris leucoagglutinin (PHAL)-labeled terminal projections arising from regions around the central canal that have been proposed to be part of the dorsal column pathway involved in visceral nociception [75,77]. This visceral pathway has been shown to be more effective than the spinothalamic tract in activating thalamic areas, triggering increases in regional blood flow and eliciting behavioral responses to visceral nociceptive stimuli. Thus, the pattern of activated brain nuclei suggests that the dorsal column pathway is involved in the afferent transmission of nociceptive information from the colon to the brain inducing neuronal activation in the c-fos-positive brain areas as observed in the present study. The response patterns to noxious and non-noxious colonic distension showed differences between these brain areas. In several brain nuclei, balloon placement or nonnoxious CD resulted in a similar increase of c-FLI, and noxious stimulation further increased c-fos expression in these areas dependent on stimulus intensity (i.e. RVLM, PAG, NC, AM). In other nuclei, the response to the noxious stimuli of CD with 40 and 70 mmHg, as assessed by changes in c-FLI, was of similar extent (i.e. VMH, DMH). In the NTS, only distension pressures of 40 mmHg or higher induced c-fos expression. In the thalamus, a significant response of c-FLI was observed at CD but not at balloon placement. Taken together, the brain nuclei, which are activated by a graded mechanical, visceral stimulus in the colon, show different response characteristics to the various intensities of CD. In the present study, we were able to show that proximal CD only induces c-FLI in the NTS at pressures with noxious intensity, but not at non-noxious distension pressures. Colonic distension with 10 mmHg did not alter c-fos immunoreactivity. However, at CD with a pressure of 40 mmHg and 70 mmHg the number of c-FLI-positive cells increased 20-fold and 50-fold, respectively. The 40 and 70 mmHg distension pressures have been proposed to be stimuli with noxious intensity [72]. On the other hand, distension pressures below 10 mmHg have been demonstrated to be below the threshold to induce cardiovascular and visceromotor responses [54] and do not represent an aversive stimulus as determined in the passive avoidance test [55]. Despite the fact that the present study does not define the exact threshold at which CD induces c-fos in the NTS, the data suggest that the NTS only responds to colonic distension of substantial intensity. This observation may reflect the fact that distension of the colon by stool also occurs under physiological conditions. Thus, CD with non-noxious pressures may not affect neuronal activity in

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the NTS, at least as determined by c-fos expression, despite the fact that the NTS plays an important role in the regulation of gastrointestinal function. With respect to the NTS, our results are in accordance with previous findings, demonstrating an activation of neurons in the NTS by colonic distension as an indicator of enhanced neuronal activity of the NTS induced by visceral noxious stimuli [25,32,37,71]. It has been proposed that the NTS has a specific viscerotopic organisation with vagal afferent terminal fields of the colon being localized primarily in the commissural subnucleus inside the NTS [2]. In the present study, we also observed c-FLI at CD predominantly in these subnuclei of the NTS. The RVLM, PAG, and NC showed a similar pattern of neuronal activation, with a stepwise increase in the number of c-FLI-positive cells / section throughout the full range of stimulation intensities from balloon placement to 70 mmHg. This is noteworthy since all of these brain areas have been proposed to be involved in the processing of viscerosensory information and in descending pain inhibitory systems [4,5,8,53,76,79]. The present observations are in accordance with previous studies which reported that painful visceral and somatic stimuli enhance c-fos expression in RVLM and PAG neurons [15,34,37,62]. In addition, it has been demonstrated that c-fos is expressed in the NC after direct electric stimulation of the PAG [64]. Furthermore, it has been shown before that catecholaminergic neurons in the NTS which express c-fos after noxious stimulation of the stomach are connected to PAG neurons [15]. These experiments suggest that information regarding visceral stimuli is transmitted directly from the NTS to the PAG. However, the present data are only partially in accordance with this concept, since in contrast to the PAG, no effect of non-noxious CD at 10 mmHg on c-fos expression was observed in the NTS. The distension-induced increase in the number of c-FLIpositive neurons counted in the ventromedial nucleus and dorsomedial nucleus of the hypothalamus showed a similar response pattern. The non-noxious stimuli of balloon placement and CD with 10 mmHg induced similar increases of c-FLI. The noxious distension with 40 and 70 mmHg further increased c-fos expression, whereby no significant differences were observed between the two noxious stimuli. These two areas of the hypothalamus have been proposed to be involved in the modulation of pain processing. The dorsomedial hypothalamus receives afferent input from the NTS [66] and has efferent connections to the nucleus cuneiformis [5], and to the amygdala [73]. The ventromedial hypothalamus has likewise connections to the nucleus cuneiformis [5]. The present data support the idea that these hypothalamic areas, which are part of the limbic system, are involved in the processing of noxious and non-noxious mechanical visceral stimuli from the distal GI tract. In this context it is of interest that the amygdala, which

is also part of the limbic system, showed a similar response to the experimental paradigm used in this study. Visceral stimulation by CD caused an intensity-dependent increase of c-FLI in the amygdala complex. However, similar to the results obtained in the hypothalamic areas mentioned above, no significant difference between the effects of the low (10 mmHg), non-noxious stimulus and catheter placement was observed. The amygdala has also been implicated in descending pain control mechanisms [10,43], and previous studies also found that noxious visceral stimuli cause enhanced c-fos expression in this area [71]. The present study provides evidence that non-noxious colonic stimuli also induce an activation of neurons in limbic structures, particularly in the central amygdala. Several previous studies support the involvement of the thalamus in visceral nociception. It has been shown that colonic distension enhances the neuronal activity and c-fos expression of thalamic neurons [1,71]. Electrophysiological studies performed on single cell neurons have demonstrated a close correlation between the electrophysiological responses of thalamic neurons and the intensity of the distension pressure [33,78]. In the present study, CD but not balloon placement induced a pronounced increase in the number of c-FLI-positive neurons / section in the thalamus, especially in the mediodorsal thalamic nucleus, median centrum and submedial nucleus. However, no significant differences in the number of c-FLI-positive cells were detected between the different stimulus intensities of 10, 40 and 70 mmHg. These results suggest a rather uniform activation of thalamus neurons at CD, at least as assessed by c-fos expression, once a mechanical stimulus even of low intensity is applied to the colon. Induction of c-fos expression in hypothalamic and thalamic areas of the limbic system due to colonic distension has been reported in a previous study [71]. However, the present study demonstrates for the first time distinct response patterns of these brain nuclei which are dependent on the stimulus intensity. Taken together, the first study of the present experimental work demonstrates that in the processing of mechanical colonic stimuli a distinct response pattern to various stimulus intensities, as assessed by c-FLI, can be observed in brain structures of the diencephalon and telencephalon, which are involved in nociception and are thought to facilitate higher levels of integration in aversive behavior. Thus, in the PAG, NC, and AM balloon placement or non-noxious CD resulted in a similar increase of c-FLI, and noxious stimulation further increased c-fos expression in these areas dependent on stimulus intensity, while the response to the noxious stimuli of CD with 40 and 70 mmHg was similar in the VMH and DMH. In the thalamus, significant and graded responses of c-FLI were observed at CD but not at balloon placement. In the second and third studies, capsaicin treatment was performed to study the involvement of C-fiber sensory nerves in the mediation of proximal colonic distension-

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induced neuronal activation in brain nuclei. Local application of this neurotoxin to the vagus nerve allows to determine the role of capsaicin-sensitive, sensory vagal fibers in the afferent neurotransmission from the gut to the brain [28]. Systemic, neonatal treatment with capsaicin, which selectively destroys unmyelinated afferent fibers [14] and causes a general loss of C-fiber sensory innervation of the GI tract, was performed to additionally assess the role of non-vagal C-fibers in the activation of brain areas at mechanical stimulation of the colon with noxious intensity. As the primary c-fos antibody used in these experiments has shown great variability in staining from lot to lot, the observed discrepancy in number of c-fos-positive neurons between the control group of the perivagal capsaicin experiments and of the neonatal capsaicin experiments is most likely due to the fact that different antibody lots were used for each set of experiments. Our results showed a significant reduction of c-fos expression after colonic distension with 70 mmHg by perivagal treatment with capsaicin in the NTS and a less pronounced, yet significant reduction of c-fos expression in the RVLM. The NTS represents the primary relay station of vagal afferent neurotransmission from the GI tract to midbrain and forebrain areas [60], and the RVLM may be activated via projections from the NTS [15], but also from other brain areas. This could be the reason for the more pronounced effect of perivagal capsaicin treatment on the NTS in comparison to the RVLM in this experimental model. Additionally, a trend of a decrease in c-fos expression was observed in the NTS and the RVLM after systemic, neonatal application of capsaicin. In all other brain areas activated by the mechanical colonic stimulus, except the DMH, a significant attenuation of c-fos expression was observed exclusively after systemic, neonatal capsaicin treatment. These data suggest an involvement of both capsaicinsensitive vagal afferents and capsaicin-sensitive, nonvagal, presumably spinal afferent fibers in the transmission of sensoric information from the proximal colon to the CNS. The question of how sensoric information is transmitted from the colon to the brain is discussed controversially in the literature. Thus, evidence for vagal afferent innervation of the colon is provided by studies showing vagal intraganglionic laminar endings (IGLE’s) in the cecum and colon [7] and by anterograde tracing studies showing connections between the colon and the dorsal vagal complex [6]. Conversely, tracing studies have shown that the NTS receives afferent input from a region around the central canal in the lumbosacral spinal cord [75], which has been described as a visceral processing region, suggesting that spinal in addition to vagal afferents are involved in transmitting nociceptive information from the colon to the NTS at CD. We did not examine c-fos expression in the spinal cord; yet, other studies have shown that noxious colorectal distension induces c-fos expression

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in the spinal cord [37]. Given the fact that only systemic capsaicin treatment diminished c-fos expression in the examined midbrain areas, a picture emerges in which nociceptive stimuli from the colon modulate neuronal activity in brainstem areas, such as the NTS, via spinal and vagal pathways. On the other hand, information on nociceptive stimuli seems to be transmitted exclusively by spinal pathways to midbrain areas in the hypothalamus and the thalamus. Furthermore, it has been proposed that afferent vagal pathways modulate the neuronal activity in brain areas activated via spinal pathways by nociceptive stimuli. The present data do not support this idea with respect to sensory vagal C-fibers and noxious colonic distension, since perivagal capsaicin treatment had no significant effect on CD-induced activation of the brain nuclei, where systemic capsaicin significantly attenuated the CD-induced c-fos expression, as mentioned above. The remaining c-fos expression seen in the NTS after colonic distension and concomitant systemic, neonatal capsaicin treatment is possibly due to the increased cardiovascular response associated with colonic distension of noxious intensity. Since we did not monitor cardiovascular function in our experiments, we cannot deduce such an effect from our data. However, it has been shown that colorectal distension of 70 mmHg is associated with an increase in cardiovascular function [54], and it is also known that neurons in the RVLM [47] and the NTS [45,46] are involved in the CNS control of cardiovascular function. However, it has been shown that cardiovascular reflexes are also mediated by capsaicin-sensitive afferent neurons [29,65], which makes it unlikely that the remaining c-fos expression in the NTS and the RVLM after CD and neonatal capsaicin treatment is mainly due to afferent input from the cardiovascular system. Most likely, these c-fos-positive neurons are an expression of incomplete destruction of afferent C-fibers by systemic capsaicin treatment, since it has been shown before that neonatal capsaicin only destroys 30% of the number of vagal neurons in the left and right nodose ganglia [12] and 41–75% of unmyelinated fibers in spinal dorsal roots [27]. In the fourth study, an antagonist of the 5-HT 3 receptor, granisetron, was given peripherally before colonic distension in order to determine an involvement of the 5-HT 3 receptor in the afferent neurotransmission of information about nociceptive stimuli from the gut to the activated brain nuclei. In the present study, the distension-induced c-fos expression in the NTS was reduced by peripheral pretreatment with granisetron. There is some evidence that 5-HT 3 receptors are involved in the mediation of visceral noxious stimuli induced by colonic distension [17,52]. The NTS is the primary relay station for vagal afferents and has one of the highest densities of 5-HT 3 receptors in the brain [21,39]. 5-HT 3 receptors mediate pre- and postsynaptic neuronal responses in the NTS [23,56], and it has been shown that NTS neurons receiving vagal sensoric input can

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be activated by 5-HT 3 receptors [74]. However, peripheral 5-HT 3 receptors have also been detected on the vagus nerve in high density [30,35]. The present data are only partially in agreement with previous studies which suggest an involvement of 5-HT 3 receptors in the afferent transmission of nociceptive stimuli from the gut to the central nervous system via spinal pathways. In these studies, it was found that alosetron, another 5-HT 3 receptor-antagonist, reduces the number of c-FLI-positive neurons in the spinal cord following repeated CD [36]. In the present study, we did not investigate the effects of granisetron on CD-induced c-fos expression in the spinal cord. However, in contrast to the effects of systemic capsaicin treatment, which attenuated the CD-induced stimulation of neuronal activity in forebrain and hindbrain nuclei, presumably by spinal pathways, we did not observe any effect on the CD-induced c-fos expression in these brain nuclei by pretreatment with the 5-HT 3 receptor antagonist. Thus, the present data do not suggest that 5-HT 3 receptors are involved in the afferent neurotransmission of nociceptive information from the distal gut to the activated brain nuclei via spinal pathways. How granisetron reduces c-fos expression in the NTS, as observed here, cannot be proven by our study. Since granisetron has been shown to cross the blood–brain barrier in rats [31] and 5-HT 3 receptors have been demonstrated in the NTS in high density [21] it is possible that the diminished c-fos expression after peripheral granisetron treatment is due to a direct action of granisetron in the NTS. On the other hand, since granisetron treatment diminished c-fos expression only in the same brain nuclei as the perivagal capsaicin treatment our data provide at least indirect evidence for an involvement of afferent vagal C-fibers in the transmission of nociceptive information at mechanical stimulation of the colon from the gut to brainstem nuclei via 5-HT 3 receptor-associated mechanisms. This concept is in agreement with electrophysiological data, which showed an antagonistic effect of the 5-HT 3 receptor antagonist cilansetron on vagal mucosal afferent terminals in the jejunum [26]. Yet, further studies would have to be performed to prove this concept, e.g. morphological studies to show the coexpression of 5-HT 3 receptors and the receptor for capsaicin, the vanilloid receptor subtype 1 (VR-1) on the same vagal afferent fibers projecting from the ascending colon to the NTS, or functional studies combining sensory vagal denervation and 5-HT 3 receptor blockade. Taken together, the effect of granisetron observed in the present study on c-fos expression in brainstem areas is due to a blockade of 5-HT 3 receptors located either directly on neurons in the NTS or on vagal afferents. The latter mechanism has been suggested before with respect to an altered afferent neurotransmission at digestive disturbances after intestinal anaphylaxis [13]. In summary, the results of the present study point out a distinct response pattern to mechanical noxious and non-

noxious colonic distension in brain areas involved in the central nervous system processing of painful visceral stimuli and gut–brain interaction. Neurons in the NTS seem to be stimulated partially via capsaicin-sensitive afferents and 5-HT 3 receptors at colonic distension. In this brain area, a discrimination between noxious and nonnoxious stimuli as well as between noxious stimuli of different intensities occurs. A less distinct differentiation between various levels of colonic stimulation can be observed in structures of the diencephalon and telencephalon, which are involved in a higher level of integrating aversive behavior in response to noxious visceral stimuli. In contrast to the areas of the brainstem, these brain nuclei seem to be activated mostly by capsaicin-sensitive, non-vagal afferents. However, the 5-HT 3 receptor does not seem to be involved in the afferent neurotransmission of information about noxious distension of the proximal colon to these brain areas. These results from an experimental animal model may help to further illuminate the complex mechanisms involved in the central processing and afferent transmission of information about nociceptive events in the gut. The modulation of c-fos expression only in the NTS by granisetron rather suggests a role of 5-HT 3 receptor antagonists in the modulation of vago-vagal sensomotoric reflexes than an influence on primary pain centers of the forebrain. This points to the potential use of 5-HT 3 receptor antagonists in modulating motility and secretion rather than visceral hypersensitivity associated with the irritable bowel syndrome.

Acknowledgements This work was supported by grants from the German Research Foundation (DFG) to H.M. (DFG: Mo¨ 458 / 4-1) and J.J.T. (DFG: Te 307 / 1-1).

References [1] E.D. al-Chaer, K.N. Westlund, W.D. Willis, Potentiation of thalamic responses to colorectal distension by visceral inflammation, NeuroReport 7 (1996) 1635–1639. [2] S.M. Altschuler, J. Escardo, R.B. Lynn, R.R. Miselis, The central organisation of the vagus nerve innervating the colon of the rat, Gastroenterology 104 (1993) 502–509. [3] C.P. Bearcroft, D. Perrett, M.J. Farthing, Postprandial plasma 5hydroxytryptamine in diarrhoea predominant irritable bowel syndrome: a pilot study, Gut 42 (1998) 42–46. [4] M.M. Behbehani, Functional characteristics of the midbrain periaqueductal gray, Prog. Neurobiol. 46 (1995) 575–605. [5] J.F. Bernard, M. Peschanski, J.M. Besson, Afferents and efferents of the rat cuneiformis nucleus: an anatomical study with reference to pain transmission, Brain Res 490 (1989) 181–185. [6] H.R. Berthoud, A. Jedrzejewska, T.L. Powley, Simultaneous labeling of vagal innervation of the gut and afferent projections from the

¨ et al. / Brain Research 966 (2003) 253–264 H. Monnikes

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18] [19]

[20] [21]

[22]

[23]

[24]

[25]

[26]

visceral forebrain with dil injected into the dorsal vagal complex in the rat, J. Comp. Neurol. 301 (1990) 65–79. H.R. Berthoud, L.M. Patterson, F. Neumann, W.L. Neuhuber, Distribution and structure of vagal afferent intraganglionic laminar endings (IGLEs) in the rat gastrointestinal tract, Anat. Embryol. (Berl) 195 (1997) 183–191. D. Bouhassira, L. Villanueva, D. Le Bars, Effects of systemic morphine on diffuse noxious inhibitory controls: role of the periaqueductal grey, Eur. J. Pharmacol. 216 (1992) 149–156. L. Bueno, J. Fioramonti, M. Delvaux, J. Frexinos, Mediators and pharmacology of visceral sensitivity: from basic to clinical investigations, Gastroenterology 112 (1997) 1714–1743. R. Burstein, S. Potrebic, Retrograde labeling of neurons in the spinal cord that project directly to the amygdala or the orbital cortex in the rat, J. Comp. Neurol. 335 (1993) 469–485. R.N. Cardinal, J.A. Parkinson, J. Hall, B.J. Everitt, Emotion and motivation: the role of the amygdala, ventral striatum, and prefrontal cortex, Neurosci. Biobehav. Rev. 26 (2002) 321–352. C. Carobi, A quantitative investigation of the effects of neonatal capsaicin treatment on vagal afferent neurons in the rat, Cell Tissue Res. 283 (1996) 305–311. N. Castex, J. Fioramonti, M.J. Fargeas, L. Bueno, C-fos expression in specific rat brain nuclei after intestinal anaphylaxis: involvement of 5-HT3 receptors and vagal afferent fibers, Brain Res. 688 (1995) 149–160. F. Cervero, H.A. McRitchie, Neonatal capsaicin does not affect unmyelinated efferent fibers of the autonomic nervous system: functional evidence, Brain Res. 239 (1982) 283–288. L.W. Chen, Z.R. Rao, J.W. Shi, Catecholaminergic neurons in the nucleus tractus solitarii which send their axons to the midbrain periaqueductal gray express Fos protein after noxious stimulation of the stomach: a triple labeling study in the rat, Neurosci. Lett. 189 (1995) 179–181. R.W. Colburn, D.W. Coombs, C.C. Degnan, L.L. Rogers, Mechanical visceral pain model: chronic intermittent intestinal distention in the rat, Physiol. Behav. 45 (1989) 191–197. R.M. Danzebrink, G.F. Gebhart, Evidence that spinal 5-HT1, 5-HT2 and 5-HT3 receptor subtypes modulate responses to noxious colorectal distension in the rat, Brain Res. 538 (1991) 64–75. F. De Ponti, M. Tonini, Irritable bowel syndrome: new agents targeting serotonin receptor subtypes, Drugs 61 (2001) 317–332. M. Delvaux, D. Louvel, J.P. Mamet, R. Campos-Oriola, J. Frexinos, Effect of alosetron on responses to colonic distension in patients with irritable bowel syndrome, Aliment. Pharmacol. Ther. 12 (1998) 849–855. M. Dragunow, R. Faull, The use of c-fos as a metabolic marker in neuronal pathway tracing, J. Neurosci. Methods 29 (1989) 261–265. D.R. Gehlert, S.L. Gackenheimer, D.T. Wong, D.W. Robertson, Localization of 5-HT3 receptors in the rat brain using [3H]LY278584, Brain Res. 553 (1991) 149–154. J. Glatzle, C. Robin, T.T. Zittel, C. Sternini, H. Raybould, 5-HT3 receptor (5-HT3R) immunoreactive fibers in the mucosa and myenteric plexus of the rat duodenum are predominately of vagal afferent origin, in: XI. European Symposium on Neurogastroenterology and ¨ Motility 2002, Tubingen website, 2002. S.R. Glaum, P.A. Brooks, K.M. Spyer, R.J. Miller, 5-Hydroxytryptamine-3 receptors modulate synaptic activity in the rat nucleus tractus solitarius in vitro, Brain Res. 589 (1992) 62–68. J.R. Grider, A.E. Foxx-Orenstein, J.G. Jin, 5-Hydroxytryptamine 4 receptor agonists initiate the peristaltic reflex in human, rat, and guinea pig intestine, Gastroenterology 115 (1998) 370–380. D.L. Hammond, R. Presley, K.R. Gogas, A.I. Basbaum, Morphine or U-50,488 suppresses Fos protein-like immunoreactivity in the spinal cord and nucleus tractus solitarii evoked by a noxious visceral stimulus in the rat, J. Comp. Neurol. 315 (1992) 244–253. K. Hillsley, C. Eeckhout, D. Grundy, Cilansetron acts at its site of absorption to antagonize the sensitivity of mesenteric afferent fibres

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42] [43] [44]

[45]

[46]

263

to 5-hydroxytryptamine in the rat jejunum, Neurosci. Lett. 278 (2000) 137–140. A. Hiura, Y. Sakamoto, Quantitative estimation of the effects of capsaicin on the mouse primary sensory neurons, Neurosci. Lett. 76 (1987) 101–106. P. Holzer, Capsaicin: cellular targets, mechanisms of action and selectivity for thin sensory neurons, Pharmacol. Rev. 43 (1991) 143–201. M.W. Hougland, K.H. Durkee, A.E. Hougland, Innervation of guinea pig heart by neurons sensitive to capsaicin, J. Auton. Nerv. Syst. 15 (1986) 217–225. D. Hoyer, C. Waeber, A. Karpf, H. Neijt, J.M. Palacios, [3H]ICS 205-930 labels 5-HT3 recognition sites in membranes of cat and rabbit vagus nerve and superior cervical ganglion, NaunynSchmiedeberg’s Arch. Pharmacol. 340 (1989) 396–402. C.T. Huang, C.F. Chen, T.H. Tsai, Pharmacokinetics of granisetron in rat blood and brain by microdialysis, Life Sci. 64 (1999) 1921– 1931. C.H. Hubscher, K.J. Berkley, Responses of neurons in caudal solitary nucleus of female rats to stimulation of vagina, cervix, uterine horn and colon, Brain Res. 664 (1994) 1–8. K. Kawakita, E. Sumiya, K. Murase, K. Okada, Response characteristics of nucleus submedius neurons to colo-rectal distension in the rat, Neurosci. Res. 28 (1997) 59–66. K.A. Keay, C.I. Clement, B. Owler, A. Depaulis, R. Bandler, Convergence of deep somatic and visceral nociceptive information onto a discrete ventrolateral midbrain periaqueductal gray region, Neuroscience 61 (1994) 727–732. G.J. Kilpatrick, B.J. Jones, M.B. Tyers, Binding of the 5-HT3 ligand, [3H]GR65630, to rat area postrema, vagus nerve and the brains of several species, Eur. J. Pharmacol. 159 (1989) 157–164. C.M. Kozlowski, A. Green, D. Grundy, F.M. Boissonade, C. Bountra, The 5-HT(3) receptor antagonist alosetron inhibits the colorectal distention induced depressor response and spinal c-fos expression in the anaesthetised rat, Gut 46 (2000) 474–480. M. Lanteri-Minet, P. Isnardon, J. de Pommery, D. Menetrey, Spinal and hindbrain structures involved in visceroception and visceronociception as revealed by the expression of Fos, Jun and Krox-24 proteins, Neuroscience 55 (1993) 737–753. M. Lanteri-Minet, J. Weil-Fugazza, P.J. de Pommery, D. Menetrey, Hindbrain structures involved in pain processing as revealed by the expression of c-Fos and other immediate early gene proteins, Neuroscience 58 (1994) 287–298. A.M. Laporte, T. Koscielniak, M. Ponchant, D. Verge, M. Hamon, H. Gozlan, Quantitative autoradiographic mapping of 5-HT3 receptors in the rat CNS using [125I]iodo-zacopride and [3H]zacopride as radioligands, Synapse 10 (1992) 271–281. S.J. Marsh, C.E. Stansfeld, D.A. Brown, R. Davey, D. McCarthy, The mechanism of action of capsaicin on sensory C-type neurons and their axons in vitro, Neuroscience 23 (1987) 275–289. G.P. McGregor, J.M. Conlon, Regulatory peptide and serotonin content and brush-border enzyme activity in the rat gastrointestinal tract following neonatal treatment with capsaicin; lack of effect on epithelial markers, Regul. Pept. 32 (1991) 109–119. R. Meier, M. Meyer, L. Degen, Irritable colon, Ther. Umsch. 54 (1997) 654–660. N.B. Mena, R. Mathur, U. Nayar, Amygdalar involvement in pain, Indian J. Physiol. Pharmacol 39 (1995) 339–346. H. Mertz, B. Naliboff, J. Munakata, N. Niazi, E.A. Mayer, Altered rectal perception is a biological marker of patients with irritable bowel syndrome, Gastroenterology 109 (1995) 40–52. S.W. Mifflin, R.B. Felder, An intracellular study of time-dependent cardiovascular afferent interactions in nucleus tractus solitarius, J. Neurophysiol. 59 (1988) 1798–1813. S.W. Mifflin, R.B. Felder, Synaptic mechanisms regulating cardiovascular afferent inputs to solitary tract nucleus, Am. J. Physiol. 259 (1990) H653–H661.

264

¨ et al. / Brain Research 966 (2003) 253–264 H. Monnikes

[47] T.A. Milner, D.J. Reis, V.M. Pickel, S.A. Aicher, R. Giuliano, Ultrastructural localization and afferent sources of corticotropinreleasing factor in the rat rostral ventrolateral medulla: implications for central cardiovascular regulation, J. Comp. Neurol. 333 (1993) 151–167. [48] H. Monnikes, G. Lauer, R. Arnold, CCK induces c-fos expression in the locus coeruleus, the nucleus of the solitary tract, and the paraventricular nucleus of the hypothalamus via capsaicin-sensitive pathways and CCK-A receptors, Gut 37 (1995) A93. [49] H. Monnikes, G. Lauer, R. Arnold, Peripheral administration of cholecystokinin activates c-fos expression in the locus coeruleus / subcoeruleus nucleus, dorsal vagal complex and paraventricular nucleus via capsaicin-sensitive vagal afferents and CCK-A receptors, Brain Res. 770 (1997) 277–288. [50] H. Monnikes, J.J. Tebbe, M. Hildebrandt, P. Arck, E. Osmanoglou, M. Rose, B.F. Klapp, B. Wiedenmann, I. Heymann-Monnikes, Role of stress in functional gastrointestinal disorders. Evidence for stressinduced alterations in gastrointestinal motility and sensitivity, Dig. Dis. 19 (2001) 201–211. [51] H. Monnikes, G. Lauer, C. Bauer, J. Tebbe, T.T. Zittel, R. Arnold, Pathways of Fos expression in locus coeruleus, dorsal vagal complex and PVN in response to intestinal lipid, Am. J. Physiol. 273 (1997) R2059–R2071. [52] H.E. Moss, G.J. Sanger, The effects of granisetron, ICS 205-930 and ondansetron on the visceral pain reflex induced by duodenal distension, Br. J. Pharmacol. 100 (1990) 497–501. [53] T.J. Ness, G.F. Gebhart, Quantitative comparison of inhibition of visceral and cutaneous spinal nociceptive transmission from the midbrain and medulla in the rat, J. Neurophysiol. 58 (1987) 850– 865. [54] T.J. Ness, G.F. Gebhart, Colorectal distension as a noxious visceral stimulus: physiologic and pharmacologic characterization of pseudaffective reflexes in the rat, Brain Res. 450 (1988) 153–169. [55] T.J. Ness, A. Randich, G.F. Gebhart, Further behavioral evidence that colorectal distension is a ‘noxious’ visceral stimulus in rats, Neurosci. Lett. 131 (1991) 113–116. [56] A. Nosjean, M. Hamon, R. Laguzzi, c-Fos induction in the rostroventrolateral medulla by 5-HT3 receptor activation in the nucleus tractus solitarius, NeuroReport 9 (1998) 373–378. [57] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1997. [58] A. Prior, N.W. Read, Reduction of rectal sensitivity and postprandial motility by granisetron, a 5HT 3 receptor antagonist, in patients with irritable bowel syndrome (IBS), Aliment. Pharmacol. Ther. 7 (1993) 175–180. [59] H. Raybould, Y. Tache, Cholecystokinin inhibits gastric motility and emptying via a capsaicin-sensitive vagal pathway in rats, Am. J. Physiol. 255 (1988) G242–G246. [60] H.E. Raybould, K.C. Lloyd, Integration of postprandial function in the proximal gastrointestinal tract. Role of CCK and sensory pathways, Ann. N.Y. Acad. Sci. 713 (1994) 143–156. [61] J. Ritchie, Pain from distention of the pelvic colon by inflating a balloon in the irritable bowel syndrome, Gut 14 (1973) 125–132. [62] L. Rodella, R. Rezzani, M. Gioia, G. Tredici, R. Bianchi, Expression of Fos immunoreactivity in the rat supraspinal regions following noxious visceral stimulation, Brain Res. Bull. 47 (1998) 357–366. [63] S.M. Sagar, F.R. Sharp, T. Curran, Expression of c-fos protein in

[64]

[65]

[66]

[67]

[68]

[69] [70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

brain: metabolic mapping at the cellular level, Science 240 (1988) 1328–1331. G. Sandner, G. Di Scala, B. Rocha, M.J. Angst, C-fos immunoreactivity in the brain following unilateral electrical stimulation of the dorsal periaqueductal gray in freely moving rats, Brain Res. 573 (1992) 276–283. J. Staszewska-Woolley, D.E. Luk, P.N. Nolan, Cardiovascular reflexes mediated by capsaicin sensitive cardiac afferent neurones in the dog, Cardiovasc. Res. 20 (1986) 897–906. G. ter Horst, P. de Boer, P.G. Luiten, J. Van Willigen, Ascending projections from the solitary tract nucleus to the hypothalamus. A Phaseolus vulgaris lectin tracing study in the rat, Neuroscience 31 (1989) 785–797. W.G. Thompson, G.F. Longstreth, D.A. Drossman, K.W. Heaton, E.J. Irvine, S.A. Muller-Lissner, Functional bowel disorders and functional abdominal pain, Gut 45 (Suppl. 2) (1999) II43–II47. G. Tougas, Irritable bowel syndrome: new approaches to its pharmacological management, Can. J Gastroenterol 15 (Suppl. B) (2001) 12B–13B. G. Tougas, The autonomic nervous system in functional bowel disorders, Gut 47 (Suppl. 4) (2000) iv78–iv80. R.J. Traub, T. Herdegen, G.F. Gebhart, Differential expression of c-fos and c-jun in two regions of the rat spinal cord following noxious colorectal distention, Neurosci. Lett. 160 (1993) 121–125. R.J. Traub, E. Silva, G.F. Gebhart, A. Solodkin, Noxious colorectal distention induced-c-Fos protein in limbic brain structures in the rat, Neurosci. Lett. 215 (1996) 165–168. R.J. Traub, P. Pechman, M.J. Iadarola, G.F. Gebhart, Fos-like proteins in the lumbosacral spinal cord following noxious and non-noxious colorectal distention in the rat, Pain 49 (1992) 393– 403. H.P. Volz, G. Rehbein, J. Triepel, M.M. Knuepfer, H. Stumpf, G. Stock, Afferent connections of the nucleus centralis amygdalae. A horseradish peroxidase study and literature survey, Anat. Embryol. (Berl.) 181 (1990) 177–194. Y. Wang, A.G. Ramage, D. Jordan, In vivo effects of 5-hydroxytryptamine receptor activation on rat nucleus tractus solitarius neurones excited by vagal C-fibre afferents, Neuropharmacology 36 (1997) 489–498. C.C. Wang, W.D. Willis, K.N. Westlund, Ascending projections from the area around the spinal cord central canal: a Phaseolus vulgaris leucoagglutinin study in rats, J. Comp. Neurol. 415 (1999) 341–367. M. Wiberg, Reciprocal connections between the periaqueductal gray matter and other somatosensory regions of the cat midbrain: a possible mechanism of pain inhibition, Ups. J. Med. Sci 97 (1992) 37–47. W.D. Willis Jr., K.N. Westlund, The role of the dorsal column pathway in visceral nociception, Curr. Pain Headache Rep. 5 (2001) 20–26. S.W. Yang, K.A. Follett, J.G. Piper, T.J. Ness, The effect of morphine on responses of mediodorsal thalamic nuclei and nucleus submedius neurons to colorectal distension in the rat, Brain Res. 779 (1998) 41–52. F.P. Zemlan, M.M. Behbehani, Nucleus cuneiformis and pain modulation: anatomy and behavioral pharmacology, Brain Res. 453 (1988) 89–102.