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Activation of c-fos expression in the rat inferior olivary nucleus by ghrelin
Neuroscience Letters 353 (2003) 157–160 www.elsevier.com/locate/neulet
Activation of c-fos expression in the rat inferior olivary nucleus by ghrelin Weizhen Zhang, Theodore R. Lin, Yuexian Hu, Yongyi Fan, Lili Zhao, Michael W. Mulholland* Department of Surgery, Michigan Gastrointestinal Peptide Center, University of Michigan, 2101 Taubman, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0346, USA Received 7 April 2003; received in revised form 20 August 2003; accepted 22 August 2003
Abstract Ghrelin, a novel 28-amino-acid hormone secreted by gastric oxyntic glands, stimulates food intake and induces adiposity. We examined whether ghrelin activates the inferior olivary nucleus. Systemic administration of ghrelin (37 nmol/kg) induced the expression of c-fos immunoreactivity in inferior olive neurons (n ¼ 6 rats). The number of neurons containing c-fos staining was significantly increased in the ghrelin-treated rats (65 ^ 14 vs.11 ^ 6 positive neurons, n ¼ 5). No significant difference in c-fos-positive neurons was observed between left (32 ^ 5) and right (33 ^ 6) inferior olivary nuclei. The number of c-fos-positive neurons in rats with bilateral vagotomy was not significantly different from those with intact vagal nerves. The present study demonstrates that ghrelin induces c-fos expression in inferior olivary nucleus via a central mechanism. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Ghrelin; c-fos; Immediate-early gene; Inferior olive; Brain stem; Rat
Ghrelin, a novel 28-amino-acid peptide, is produced primarily in endocrine cells of the stomach fundus [5]. Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) and has been reported to stimulate growth hormone release in human and rat, following either peripheral or central administration [1,12, 17]. Whereas ghrelin was originally reported to stimulate growth hormone release, the wide distribution of its receptor, GHS-R, has suggested a potentially broader array of actions. Expression of ghrelin and its related receptor in the kidney indicates that ghrelin may play an important role in the regulation of renal function [7]. In rats, ghrelin has been found to stimulate food intake [9,17], to induce adiposity [14], and to increase body weight [9,14, 17], suggesting a possible role in regulation of feeding behavior and energy metabolism. The ghrelin-induced adiposity is centrally mediated by reducing fat utilization [14]. This result suggests that ghrelin reduces sympathetic nervous system activity. In humans, ghrelin has been demonstrated to reduce cardiac afterload and increase cardiac output [8]. In the gastrointestinal system, ghrelin has been recently reported to regulate secretion of gastric acid [2,6], gastric motility [6,13], and pancreatic protein * Corresponding author. Tel.: þ 1-734-936-3236; fax: þ1-734-763-5615. E-mail address: [email protected] (M.W. Mulholland).
output [18]. These results suggest that ghrelin coordinates multiple functions in the gastrointestinal system, although the mechanisms involved remain largely unknown. Stimulation of gastric secretion has been observed in rats with central or peripheral administration of ghrelin [2,6]. The inferior olivary nucleus is the largest nuclear group in the brain stem. It consists of a convoluted band of cells that lie dorsal to the pyramid. This nucleus is the most characteristic and striking feature of the medulla. The inferior olive has long been considered as a major afferent of the cerebellum and plays an important role in the control of movement. Recent studies have defined new roles for the inferior olivary nucleus. Activation of c-fos in the inferior olivary neurons has been reported following hemorrhagic stress [3], in response to aortic depressor nerve stimulation and after restraint or swim stress [1,11], suggesting that inferior olive is involved in stress-related cardiovascular regulation. In rats, inferior olivary neurons have been demonstrated to modulate metabolic heat production by relaying central thermal signals to peripheral thermoregulatory effectors such as brown adipose tissue and skeletal muscle [15]. This effect is mediated by the tonic inhibition of the sympathetic nervous system, suggesting that inferior olivary neurons may play an important role in functional interactions between the motor and thermoregulatory systems. In addition to integration of central and somatic
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.08.083
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W. Zhang et al. / Neuroscience Letters 353 (2003) 157–160
messages, the inferior olivary nucleus also coordinates visceral signals [4,10]. While ghrelin plays an important role in coordinating gastrointestinal function and the inferior olivary nucleus relays visceral signals to cerebellum to coordinate gastrointestinal movement, it is unknown whether ghrelin affects inferior olivary neurons. We hypothesized that ghrelin activates inferior olivary neurons to coordinate gastrointestinal motility. The objective of this study was to determine whether intravenous injection of ghrelin activates inferior olivary neurons. Activation of inferior olivary neurons was assessed using immunohistochemical detection of the nuclear protein marker c-fos. Bilateral vagotomy was performed to determine if loss of vagal afferents and efferents would prevent c-fos activation of inferior olivary neurons in response to ghrelin challenge. Male Sprague– Dawley rats (140 – 160 g, Harlan, Indianapolis, IN) were kept in individual cages and were given chow food and water ad libitum. All experiments were performed between 09:00 and 12:00 h in rats anesthetized with intramuscular injection of a mixture of urethane and ketamine (13 and 87 mg/kg body weight, respectively) and were approved by the University of Michigan Committee on Use and Care of Animals. Ghrelin was dissolved in 0.9% normal saline. Ghrelin (37 nmol/kg, a dose previously reported to stimulate food intake [14,17] and to activate hypothalamic neurons [16]) or 0.9% saline was injected intravenously into male rats 90 min before perfusion. Acute bilateral subdiaphragmatic vagotomy was performed immediately prior to ghrelin administration. Through a midline laparotomy, the esophagus was exposed. Subdiaphragmatic vagal trunks were exposed halfway between the diaphragm and the gastric cardia and both anterior and posterior trunks were transected. Acute bilateral cervical vagotomy was performed as follows. Through a midline cervical incision, bilateral cervical vagal trunks were exposed and sectioned. In some animals, sham operation was performed without sectioning vagus nerves. After experiments, rats were anesthetized by an intramuscular injection of sodium pentobarbital (75 mg/kg body weight) and perfused transcardially, first for 10 min with 100 ml of 0.1 M phosphate buffer (pH 7.4) and then for 15 min with 150 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. The brain stem was removed and postfixed for 2 days at 4 8C, then kept in 0.1 M phosphate buffer containing 20% sucrose for 24 h. The medulla oblongata containing the dorsal motor nucleus of the vagus was sectioned into 50-mm slices at the interaural level of 2 4.24 to 2 5.08 mm according to the atlas of Paxinos and Watson using a freezing microtome. All slices were incubated for 24 h at 4 8C with a polyclonal rabbit anti-fos antibody (1:800) ( fos AB-5, Oncogene Research Products, Cambridge, MA), then incubated at room temperature for 1 h with FITCconjugated goat anti-rabbit IgG (1:300) (Jackson Immunoresearch Laboratory Inc., West Grove, PA). c-fos-Positive cells were counted in three sections per animal at the level
of area postrema and means of cells per section were calculated. Data were expressed as mean ^ S.E.M. Statistical differences between control and treatment were assessed by unpaired Student’s t-test. Data were considered significant different when P , 0:05. Systemic administration of ghrelin induced c-fos expression in inferior olivary neurons in vagus-intact anesthetized rats (Fig. 1A), while there were a few or no positive cells in rats injected with normal saline (Fig. 1B). The majority of c-fos-immunoreactive nuclei were located in the cap of Kooy of the medial nucleus and the beta subnucleus of the inferior olive. Scattered cells were also observed in other subnuclei of the medial nucleus. The number of c-fos-positive cells per section in the inferior olivary nucleus was significantly increased to 65 ^ 14 (n ¼ 6) compared with 11 ^ 6 positive neurons (n ¼ 5) in rats injected with normal saline (P , 0:05). No significant difference in c-fos-positive neurons was observed between left (32 ^ 5) and right sides (33 ^ 6) of inferior olivary nuclei (P . 0:05). Rats with bilateral subdiaphragmatic vagotomy demonstrated a significant elevation in the numbers of c-foslabeled neurons in inferior olive in response to ghrelin (37 nmol/kg body weight). The number of c-fos-positive neurons in rats with bilateral subdiaphragmatic vagotomy was not significantly different from those animals with intact vagal nerves (59 ^ 8 versus 58 ^ 15, n ¼ 3, P . 0:05), suggesting that activation of c-fos expression by ghrelin in inferior olivary neurons does not require the afferent input from the gastrointestinal system. To determine whether ghrelin-induced c-fos expression in inferior olivary neurons requires vagal afferent input from a more rostral level, bilateral cervical vagotomy was performed and the effect of ghrelin on c-fos activation was assessed. As shown in Fig. 2, ghrelin (37 nmol/kg body weight) induced a significant increment in c-fos expression in inferior olivary neurons in rats with bilateral cervical vagotomy. The number of c-fos-positive neurons in rats with bilateral cervical vagotomy was not significantly different from those with intact vagal nerve (51 ^ 14 versus 39 ^ 8, n ¼ 4 and n ¼ 6, P . 0:05). Few or no c-fos-positive neurons were observed in rats with bilateral cervical vagotomy and saline injection. The present study demonstrates that in anesthetized rats, inferior olivary neurons respond to systemic ghrelin with a significant increase in c-fos immunoreactivity. This increase occurs regardless of the integrity of the vagal nerve, suggesting that ghrelin induces c-fos expression in inferior olivary neurons via a central mechanism. The inferior olivary nucleus is an important structure in the brain stem that integrates both somatic and visceral messages to cerebellum. Previous studies have demonstrated that there exists a direct projection from the abdominal region to the inferior olive via a vagal communicating branch [4]. Response in the inferior olive nucleus has also been reported following stimulation of
W. Zhang et al. / Neuroscience Letters 353 (2003) 157–160
159
Fig. 1. c-fos immunoreactivity in inferior olivary neurons. (A) Control rats injected with 0.9% normal saline demonstrated few or no c-fos staining. Scale bar: 100 mm. (B) Systemic ghrelin evoked a substantial, bilaterally symmetrical increase in c-fos immunoreactivity in anesthetized rats.
gastrointestinal and peritoneal receptors [10]. These results suggest that the inferior olivary nucleus may play a role in coordinating the gastrointestinal function. Our observation that ghrelin activates the inferior olivary nucleus suggests that ghrelin may be involved in the coordination of the pattern of the gastrointestinal motility. This finding is consistent with previous reports demonstrating that ghrelin stimulates gastric and intestinal motility and that this effect is independent of the vagal nerve [2,6]. The mechanism through which peripheral ghrelin activates inferior olivary neurons awaits further investi-
gation. Our results demonstrate that the ghrelin effect on inferior olivary neurons does not require the presence of intact vagal innervation. Two observations support this notion. First, bilateral subdiaphragmatic vagotomy, in which afferent signals from abdominal region were blocked, did not alter c-fos expression induced by ghrelin in inferior olivary neurons. Second, bilateral cervical vagotomy, in which afferent signals caudal to the nodose ganglia were abolished, also demonstrated no effect on ghrelin-induced cfos expression in inferior olivary neurons. These results demonstrate that neither afferent signals from gastrointestinal tract nor afferent signals from cardiovascular system contribute to the ghrelin-induced activation of inferior olivary nucleus. In conclusion, this study demonstrates that ghrelin induces c-fos expression in the inferior olivary nucleus, likely via a central mechanism.
References
Fig. 2. c-fos immunoreactivity in rats with bilateral cervical vagotomy. Inferior olivary neurons in rats with bilateral cervical vagotomy responded to ghrelin with an increase in c-fos expression. The number of c-fos-positive neurons was not significantly different from those in rats with sham bilateral cervical vagotomy (P , 0:05). Scale bar: 100 mm.
[1] W.E. Cullinan, J.P. Herman, D.F. Battaglia, H. Akil, S.J. Watson, Pattern and time course of immediate early gene expression in rat brain following acute stress, Neuroscience 64 (1995) 477–505. [2] Y. Date, M. Nakazato, M. Kojima, T. Kuroiwa, S. Matsukura, K. Kangawa, M. Nakazato, Ghrelin acts in the central nervous system to stimulate gastric acid secretion, Biochem. Biophys. Res. Commun. 280 (2001) 904 –907. [3] N.J. Dun, S.L. Dun, N.L. Chiaia, Hemorrhage induces fos immunoreactivity in rat medullary catecholaminergic neurons, Brain Res. 608 (1993) 223–232. [4] J.L. Fitzakerley, G.E. Lucier, Connections of a vagal communicating branch in the ferret. II. Central projections, Brain Res. Bull. 20 (1988) 479–486. [5] M. Kojima, H. Hosoda, Y. Date, M. Nakazato, H. Matsuo, K. Kangawa, Ghrelin is a growth-hormone-releasing acylated peptide from stomach, Nature 402 (1999) 656 –660. [6] Y. Masuda, T. Tanaka, N. Inomata, N. Ohnuma, S. Tanaka, Z. Itoh, H. Hosoda, M. Kojima, K. Kangawa, Ghrelin stimulates gastric acid secretion and motility in rats, Biochem. Biophys. Res. Commun. 276 (2000) 905–908. [7] K. Mori, A. Yoshimoto, K. Takaya, K. Hosoda, H. Ariyasu, K.
160
[8]
[9]
[10]
[11]
[12]
W. Zhang et al. / Neuroscience Letters 353 (2003) 157–160 Yahata, M. Mukoyama, A. Sugawara, H. Hosoda, M. Kojima, K. Kangawa, K. Nakao, Kidney produces a novel acylated peptide, ghrelin, FEBS Lett. 486 (2000) 213–216. N. Nagaya, M. Kojima, M. Uematsu, M. Yamagishi, H. Hosoda, H. Oya, Y. Hayashi, K. Kangawa, Hemodynamic and hormonal effects of human ghrelin in healthy volunteers, Am. J. Physiol. 280 (2001) R1483–R1487. M. Nakazato, N. Murakami, Y. Date, M. Kojima, H. Matsuo, K. Kangawa, S. Matrukura, A role for ghrelin in the central regulation of feeding, Nature 409 (2001) 194 –198. J. Perrin, J. Crousillat, Response of single units in the inferior olive nucleus to stimulation of the splanchnic afferents in the cat, J. Auton. Nerv. Syst. 2 (1980) 15–22. S.D. Rutherfurd, R.E. Widdop, F. Sannajust, W.J. Louis, A.L. Gundlach, Expression of c-fos and NGFI-A messenger RNA in the medullar oblongata of the anesthetized rat following stimulation of vagal and cardiovascular afferents, Mol. Brain Res. 13 (1992) 310– 312. K. Takaya, H. Ariyasu, N. Kanamoto, H. Iwakura, A. Yoshimoto, M. Harada, K. Mori, Y. Komatsu, T. Usui, A. Shimatsu, Y. Ogawa, K. Hosoda, T. Akamizu, M. Kojima, K. Kangawa, K. Nakao, Ghrelin strongly stimulates growth hormone release in humans, J. Clin. Endocrinol. Metab. 85 (2000) 4908–4911.
[13] L. Trudel, C. Tomasetto, M.C. Rio, M. Bouin, V. Plourde, P. Eberling, P. Poitras, Ghrelin/motilin-related peptide is a potent prokinetic to reverse gastric postoperative ileus in rat, Am. J. Physiol. 282 (2002) G948–G952. [14] M. Tschop, D.L. Smiley, M.L. Heiman, Ghrelin induces adiposity in rodents, Nature 407 (2000) 908–913. [15] T. Uno, M. Shibata, Role of inferior olive and thoracic IML neurons in nonshivering thermogenesis in rats, Am. J. Physiol. 280 (2001) R536–R546. [16] L. Wang, D.H. Saint-Pierre, Y. Tache, Peripheral ghrelin selectively increases fos expression in neuropeptide Y-synthesizing neurons in mouse hypothalamic arcuate nucleus, Neurosci. Lett. 325 (2002) 47– 51. [17] A.M. Wren, C.J. Small, H.L. Ward, K.G. Murphy, C.L. Dakin, S. Taheri, A.R. Kennedy, G.H. Roberts, D.G. Morgan, M.A. Ghatei, S.R. Bloom, The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion, Endocrinology 141 (2000) 4325–4328. [18] W.Z. Zhang, M. Chen, X. Chen, B.J. Segura, M.W. Mulholland, Inhibition of pancreatic protein secretion by ghrelin in rat, J. Physiol. 537 (2001) 231–236.
Activation of c-fos expression in the rat inferior olivary nucleus by ghrelin Weizhen Zhang, Theodore R. Lin, Yuexian Hu, Yongyi Fan, Lili Zhao, Michael W. Mulholland* Department of Surgery, Michigan Gastrointestinal Peptide Center, University of Michigan, 2101 Taubman, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0346, USA Received 7 April 2003; received in revised form 20 August 2003; accepted 22 August 2003
Abstract Ghrelin, a novel 28-amino-acid hormone secreted by gastric oxyntic glands, stimulates food intake and induces adiposity. We examined whether ghrelin activates the inferior olivary nucleus. Systemic administration of ghrelin (37 nmol/kg) induced the expression of c-fos immunoreactivity in inferior olive neurons (n ¼ 6 rats). The number of neurons containing c-fos staining was significantly increased in the ghrelin-treated rats (65 ^ 14 vs.11 ^ 6 positive neurons, n ¼ 5). No significant difference in c-fos-positive neurons was observed between left (32 ^ 5) and right (33 ^ 6) inferior olivary nuclei. The number of c-fos-positive neurons in rats with bilateral vagotomy was not significantly different from those with intact vagal nerves. The present study demonstrates that ghrelin induces c-fos expression in inferior olivary nucleus via a central mechanism. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Ghrelin; c-fos; Immediate-early gene; Inferior olive; Brain stem; Rat
Ghrelin, a novel 28-amino-acid peptide, is produced primarily in endocrine cells of the stomach fundus [5]. Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) and has been reported to stimulate growth hormone release in human and rat, following either peripheral or central administration [1,12, 17]. Whereas ghrelin was originally reported to stimulate growth hormone release, the wide distribution of its receptor, GHS-R, has suggested a potentially broader array of actions. Expression of ghrelin and its related receptor in the kidney indicates that ghrelin may play an important role in the regulation of renal function [7]. In rats, ghrelin has been found to stimulate food intake [9,17], to induce adiposity [14], and to increase body weight [9,14, 17], suggesting a possible role in regulation of feeding behavior and energy metabolism. The ghrelin-induced adiposity is centrally mediated by reducing fat utilization [14]. This result suggests that ghrelin reduces sympathetic nervous system activity. In humans, ghrelin has been demonstrated to reduce cardiac afterload and increase cardiac output [8]. In the gastrointestinal system, ghrelin has been recently reported to regulate secretion of gastric acid [2,6], gastric motility [6,13], and pancreatic protein * Corresponding author. Tel.: þ 1-734-936-3236; fax: þ1-734-763-5615. E-mail address: [email protected] (M.W. Mulholland).
output [18]. These results suggest that ghrelin coordinates multiple functions in the gastrointestinal system, although the mechanisms involved remain largely unknown. Stimulation of gastric secretion has been observed in rats with central or peripheral administration of ghrelin [2,6]. The inferior olivary nucleus is the largest nuclear group in the brain stem. It consists of a convoluted band of cells that lie dorsal to the pyramid. This nucleus is the most characteristic and striking feature of the medulla. The inferior olive has long been considered as a major afferent of the cerebellum and plays an important role in the control of movement. Recent studies have defined new roles for the inferior olivary nucleus. Activation of c-fos in the inferior olivary neurons has been reported following hemorrhagic stress [3], in response to aortic depressor nerve stimulation and after restraint or swim stress [1,11], suggesting that inferior olive is involved in stress-related cardiovascular regulation. In rats, inferior olivary neurons have been demonstrated to modulate metabolic heat production by relaying central thermal signals to peripheral thermoregulatory effectors such as brown adipose tissue and skeletal muscle [15]. This effect is mediated by the tonic inhibition of the sympathetic nervous system, suggesting that inferior olivary neurons may play an important role in functional interactions between the motor and thermoregulatory systems. In addition to integration of central and somatic
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.08.083
158
W. Zhang et al. / Neuroscience Letters 353 (2003) 157–160
messages, the inferior olivary nucleus also coordinates visceral signals [4,10]. While ghrelin plays an important role in coordinating gastrointestinal function and the inferior olivary nucleus relays visceral signals to cerebellum to coordinate gastrointestinal movement, it is unknown whether ghrelin affects inferior olivary neurons. We hypothesized that ghrelin activates inferior olivary neurons to coordinate gastrointestinal motility. The objective of this study was to determine whether intravenous injection of ghrelin activates inferior olivary neurons. Activation of inferior olivary neurons was assessed using immunohistochemical detection of the nuclear protein marker c-fos. Bilateral vagotomy was performed to determine if loss of vagal afferents and efferents would prevent c-fos activation of inferior olivary neurons in response to ghrelin challenge. Male Sprague– Dawley rats (140 – 160 g, Harlan, Indianapolis, IN) were kept in individual cages and were given chow food and water ad libitum. All experiments were performed between 09:00 and 12:00 h in rats anesthetized with intramuscular injection of a mixture of urethane and ketamine (13 and 87 mg/kg body weight, respectively) and were approved by the University of Michigan Committee on Use and Care of Animals. Ghrelin was dissolved in 0.9% normal saline. Ghrelin (37 nmol/kg, a dose previously reported to stimulate food intake [14,17] and to activate hypothalamic neurons [16]) or 0.9% saline was injected intravenously into male rats 90 min before perfusion. Acute bilateral subdiaphragmatic vagotomy was performed immediately prior to ghrelin administration. Through a midline laparotomy, the esophagus was exposed. Subdiaphragmatic vagal trunks were exposed halfway between the diaphragm and the gastric cardia and both anterior and posterior trunks were transected. Acute bilateral cervical vagotomy was performed as follows. Through a midline cervical incision, bilateral cervical vagal trunks were exposed and sectioned. In some animals, sham operation was performed without sectioning vagus nerves. After experiments, rats were anesthetized by an intramuscular injection of sodium pentobarbital (75 mg/kg body weight) and perfused transcardially, first for 10 min with 100 ml of 0.1 M phosphate buffer (pH 7.4) and then for 15 min with 150 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. The brain stem was removed and postfixed for 2 days at 4 8C, then kept in 0.1 M phosphate buffer containing 20% sucrose for 24 h. The medulla oblongata containing the dorsal motor nucleus of the vagus was sectioned into 50-mm slices at the interaural level of 2 4.24 to 2 5.08 mm according to the atlas of Paxinos and Watson using a freezing microtome. All slices were incubated for 24 h at 4 8C with a polyclonal rabbit anti-fos antibody (1:800) ( fos AB-5, Oncogene Research Products, Cambridge, MA), then incubated at room temperature for 1 h with FITCconjugated goat anti-rabbit IgG (1:300) (Jackson Immunoresearch Laboratory Inc., West Grove, PA). c-fos-Positive cells were counted in three sections per animal at the level
of area postrema and means of cells per section were calculated. Data were expressed as mean ^ S.E.M. Statistical differences between control and treatment were assessed by unpaired Student’s t-test. Data were considered significant different when P , 0:05. Systemic administration of ghrelin induced c-fos expression in inferior olivary neurons in vagus-intact anesthetized rats (Fig. 1A), while there were a few or no positive cells in rats injected with normal saline (Fig. 1B). The majority of c-fos-immunoreactive nuclei were located in the cap of Kooy of the medial nucleus and the beta subnucleus of the inferior olive. Scattered cells were also observed in other subnuclei of the medial nucleus. The number of c-fos-positive cells per section in the inferior olivary nucleus was significantly increased to 65 ^ 14 (n ¼ 6) compared with 11 ^ 6 positive neurons (n ¼ 5) in rats injected with normal saline (P , 0:05). No significant difference in c-fos-positive neurons was observed between left (32 ^ 5) and right sides (33 ^ 6) of inferior olivary nuclei (P . 0:05). Rats with bilateral subdiaphragmatic vagotomy demonstrated a significant elevation in the numbers of c-foslabeled neurons in inferior olive in response to ghrelin (37 nmol/kg body weight). The number of c-fos-positive neurons in rats with bilateral subdiaphragmatic vagotomy was not significantly different from those animals with intact vagal nerves (59 ^ 8 versus 58 ^ 15, n ¼ 3, P . 0:05), suggesting that activation of c-fos expression by ghrelin in inferior olivary neurons does not require the afferent input from the gastrointestinal system. To determine whether ghrelin-induced c-fos expression in inferior olivary neurons requires vagal afferent input from a more rostral level, bilateral cervical vagotomy was performed and the effect of ghrelin on c-fos activation was assessed. As shown in Fig. 2, ghrelin (37 nmol/kg body weight) induced a significant increment in c-fos expression in inferior olivary neurons in rats with bilateral cervical vagotomy. The number of c-fos-positive neurons in rats with bilateral cervical vagotomy was not significantly different from those with intact vagal nerve (51 ^ 14 versus 39 ^ 8, n ¼ 4 and n ¼ 6, P . 0:05). Few or no c-fos-positive neurons were observed in rats with bilateral cervical vagotomy and saline injection. The present study demonstrates that in anesthetized rats, inferior olivary neurons respond to systemic ghrelin with a significant increase in c-fos immunoreactivity. This increase occurs regardless of the integrity of the vagal nerve, suggesting that ghrelin induces c-fos expression in inferior olivary neurons via a central mechanism. The inferior olivary nucleus is an important structure in the brain stem that integrates both somatic and visceral messages to cerebellum. Previous studies have demonstrated that there exists a direct projection from the abdominal region to the inferior olive via a vagal communicating branch [4]. Response in the inferior olive nucleus has also been reported following stimulation of
W. Zhang et al. / Neuroscience Letters 353 (2003) 157–160
159
Fig. 1. c-fos immunoreactivity in inferior olivary neurons. (A) Control rats injected with 0.9% normal saline demonstrated few or no c-fos staining. Scale bar: 100 mm. (B) Systemic ghrelin evoked a substantial, bilaterally symmetrical increase in c-fos immunoreactivity in anesthetized rats.
gastrointestinal and peritoneal receptors [10]. These results suggest that the inferior olivary nucleus may play a role in coordinating the gastrointestinal function. Our observation that ghrelin activates the inferior olivary nucleus suggests that ghrelin may be involved in the coordination of the pattern of the gastrointestinal motility. This finding is consistent with previous reports demonstrating that ghrelin stimulates gastric and intestinal motility and that this effect is independent of the vagal nerve [2,6]. The mechanism through which peripheral ghrelin activates inferior olivary neurons awaits further investi-
gation. Our results demonstrate that the ghrelin effect on inferior olivary neurons does not require the presence of intact vagal innervation. Two observations support this notion. First, bilateral subdiaphragmatic vagotomy, in which afferent signals from abdominal region were blocked, did not alter c-fos expression induced by ghrelin in inferior olivary neurons. Second, bilateral cervical vagotomy, in which afferent signals caudal to the nodose ganglia were abolished, also demonstrated no effect on ghrelin-induced cfos expression in inferior olivary neurons. These results demonstrate that neither afferent signals from gastrointestinal tract nor afferent signals from cardiovascular system contribute to the ghrelin-induced activation of inferior olivary nucleus. In conclusion, this study demonstrates that ghrelin induces c-fos expression in the inferior olivary nucleus, likely via a central mechanism.
References
Fig. 2. c-fos immunoreactivity in rats with bilateral cervical vagotomy. Inferior olivary neurons in rats with bilateral cervical vagotomy responded to ghrelin with an increase in c-fos expression. The number of c-fos-positive neurons was not significantly different from those in rats with sham bilateral cervical vagotomy (P , 0:05). Scale bar: 100 mm.
[1] W.E. Cullinan, J.P. Herman, D.F. Battaglia, H. Akil, S.J. Watson, Pattern and time course of immediate early gene expression in rat brain following acute stress, Neuroscience 64 (1995) 477–505. [2] Y. Date, M. Nakazato, M. Kojima, T. Kuroiwa, S. Matsukura, K. Kangawa, M. Nakazato, Ghrelin acts in the central nervous system to stimulate gastric acid secretion, Biochem. Biophys. Res. Commun. 280 (2001) 904 –907. [3] N.J. Dun, S.L. Dun, N.L. Chiaia, Hemorrhage induces fos immunoreactivity in rat medullary catecholaminergic neurons, Brain Res. 608 (1993) 223–232. [4] J.L. Fitzakerley, G.E. Lucier, Connections of a vagal communicating branch in the ferret. II. Central projections, Brain Res. Bull. 20 (1988) 479–486. [5] M. Kojima, H. Hosoda, Y. Date, M. Nakazato, H. Matsuo, K. Kangawa, Ghrelin is a growth-hormone-releasing acylated peptide from stomach, Nature 402 (1999) 656 –660. [6] Y. Masuda, T. Tanaka, N. Inomata, N. Ohnuma, S. Tanaka, Z. Itoh, H. Hosoda, M. Kojima, K. Kangawa, Ghrelin stimulates gastric acid secretion and motility in rats, Biochem. Biophys. Res. Commun. 276 (2000) 905–908. [7] K. Mori, A. Yoshimoto, K. Takaya, K. Hosoda, H. Ariyasu, K.
160
[8]
[9]
[10]
[11]
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