CURE: Digestive Diseases Research Center, Department of Veterans Affairs Greater Los Angeles Healthcare System, and Digestive Diseases Division, Department of Medicine and Brain Research Institute, University of California, Los Angeles, California 90073
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ABSTRACT |
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Acute cold exposure-induced activation of gastric myenteric neurons in conscious rats was examined on longitudinal muscle-myenteric plexus whole mount preparations. Few Fos-immunoreactive (IR) cells (<1/ganglion) were observed in 24-h fasted rats semirestrained at room temperature. Cold exposure (4°C) for 1-3 h induced a time-related increase of Fos-IR cells in corpus and antral myenteric ganglia with a maximal plateau response (17 ± 3 and 18 ± 3 cells/ganglion, respectively) occurring at 2 h. Gastric vagotomy partly prevented, whereas bilateral cervical vagotomy completely abolished, Fos expression in the myenteric cells induced by cold exposure (2 h). Hexamethonium (20 mg/kg) also prevented 3-h cold exposure-induced myenteric Fos expression by 76-80%, whereas atropine or bretylium had no effect. Double labeling revealed that cold (3 h)-induced Fos-IR myenteric cells were mainly neurons, including a substantial number of choline acetyltransferase-containing neurons and most NADPH-diaphorase-positive neurons. These results indicate that acute cold exposure activates cholinergic as well as nitrergic neurons in the gastric myenteric ganglia through vagal nicotinic pathways in conscious rats.
bretylium; nicotamide adenine dinucleotide phosphate-diaphorase; hexamethonium; atropine
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INTRODUCTION |
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THE GASTRIC MYENTERIC PLEXUS innervates smooth muscle and mucosal layers (15, 40) and receives a dense and intricate network of vagal efferent axons (2, 20). Recent studies reported that electrical stimulation of the rat cervical vagus nerve induces widespread Fos expression in the gastric myenteric plexus in anesthetized rats (57, 58). Similarly, intracisternal (i.c.) injection of thyrotropin-releasing hormone (TRH) analog, known to activate vagal preganglionic neurons in the dorsal motor nucleus (DMN) (6, 47) and increase gastric vagal efferent discharges (39), activates gastric myenteric neurons and glia in rats (31). However, the gastric myenteric response to environmental stimuli, which activate the gastric vagal pathways, has not been studied.
Acute exposure of fasted rats to cold induces vagal cholinergic-mediated stimulation of gastric secretion, contractility, and erosion formation as the consequence of the activation of medullary TRH pathways (10, 17, 19, 28, 37, 52). Cold exposure stimulates Fos expression in the DMN and specific medullary TRH-synthesizing neurons located in the raphe pallidus, raphe obscurus, and the parapyramidal regions (7, 53). Activation of the raphe nuclei and parapyramidal regions induces vagal stimulation of gastric secretory and motility functions through projections from these nuclei to the DMN (26, 44, 48, 53). TRH or its analog, injected i.c., induces vagally mediated gastric responses similar to those induced by cold exposure (47). Besides the vagus, cold exposure also increases the activity of the sympathetic nervous system (1). Collectively, these data indicate that acute cold exposure is a relevant environmental stimulus for examination of the neuronal response of the gastric myenteric plexus to autonomic nervous system-mediated adaptive visceral changes (1).
In the present study, we assessed the activation of gastric myenteric cells, especially neurons, in response to acute cold exposure in conscious rats. Immunohistochemical detection of the nuclear protein Fos was used as a marker of cellular activation (33) in gastric longitudinal muscle-myenteric plexus (LMMP) whole mount preparations (30). Double staining of Fos and cuprolinic blue, an established marker for enteric neurons (21), was used to assess the neuronal identity of Fos-immunoreactive (IR) myenteric cells. The role of extrinsic autonomic innervation in cold exposure-induced change in the gastric myenteric activity was investigated by surgical (vagotomy) and pharmacological (pretreatments with antagonists of nicotinic or muscarinic receptors and adrenergic blocking agent) approaches. We also examined the biochemical coding of myenteric Fos-IR cells in cold-exposed rats by using double labeling of Fos with choline acetyltransferase (ChAT) (32) or with nicotamide adenine dinucleotide phosphate-diaphorase (NADPH-d), an established marker for nitric oxide (NO)-synthesizing neurons (12, 51, 56).
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MATERIALS AND METHODS |
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Animals
Male Sprague-Dawley rats (Harlan Laboratory, San Diego, CA) weighing 280-320 g were housed under controlled conditions (22-24°C, light on from 6:00 AM to 6:00 PM). Animals had free access to Purina Rat Chow (Ralston Purina, St. Louis, MO) and tap water. Rats were deprived of food but not water for 16 h (gastric-vagotomized rats) or 24 h (all other rats) before acute cold exposure. Studies were conducted under the protocol of the Department of Veterans Affairs Animal Component of Research.Drugs
The following drugs were used: hexamethonium chloride (Sigma, St. Louis, MO), a nicotinic receptor antagonist (34), bretylium tosylate (Sigma), an adrenergic blocking agent (8), and atropine sulfate (Sigma), a muscarinic receptor antagonist. All drugs were dissolved in sterile saline immediately before use.Treatments
Acute cold exposure. Fasted conscious rats were placed individually in a flat-bottom cylindrical stainless steel cage (16 cm × 5.5 cm × 5.5 cm) with perforations to allow ventilation. The semirestrained rats were maintained either at room temperature (23 ± 2°C) or in a cold room (4°C) for a 60- to 180-min period as previously described (7, 52).
Gastric vagotomy. Gastric vagotomy was performed in 24-h fasted rats that were anesthetized by intraperitoneal (i.p.) injection of a 3:1 (vol/vol) mixture of ketamine (75 mg/kg; Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (5 mg/kg; Mobay, Shawnee, KS). After a midline abdominal incision, the lower esophagus was exposed and ~0.5 cm of the anterior and posterior gastric vagal branches were resected under a microscope using forceps. Sham-operated animals underwent the same procedure without resection of the gastric vagal branches. After surgery, rats were housed individually and fed with a liquid diet (Ensure; Abbott Laboratories, Columbus, OH), which was removed 16 h before cold exposure.
Acute bilateral cervical vagotomy. Acute bilateral cervical vagotomy was performed under short enflurane anesthesia (3-5 min, 5% vapor concentration in oxygen; Ethrane, Ohmeda Pharmaceutical Products Division, Liberty Corner, NJ) in 24-h fasted rats. After a cervical midline incision, bilateral cervical vagal trunks were cut. Atropine (8 mg/kg) was injected i.p. 10 min before the surgery to alleviate acute respiratory complications associated with bilateral cervical vagotomy (11). Sham-operated rats underwent the same procedure (including atropine administration) without sectioning of the vagus nerves.
Experimental Protocols
Effect of cold exposure on Fos expression in gastric myenteric cells. Conscious rats were semirestrained, maintained either at room temperature for 180 min or in cold (4°C) for 60, 90, 120, or 180 min, and then euthanized by an overdose of pentobarbital (90 mg/kg i.p.; Abbott Laboratories, North Chicago, IL). Stomachs were collected for Fos immunohistochemistry and double-staining procedures.
Effect of vagotomy on cold exposure-induced Fos expression in
gastric myenteric cells.
Rats that underwent gastric (48 h) or bilateral cervical (
30 min)
vagotomy, or the corresponding sham operations, were placed in
semirestraint cages and exposed to cold (4°C) for 2 h. One group
of sham gastric-vagotomized rats was semirestrained at room temperature. At the end of the 2-h cold exposure, rats were euthanized and stomachs were collected for Fos immunohistochemistry.
Effect of cholinergic and adrenergic blockade on cold
exposure-induced Fos expression in gastric myenteric cells.
Rats received a subcutaneous (s.c.) injection of one of the following
chemicals, saline (15 min), bretylium (25 mg/kg;
15 min),
hexamethonium (10, 20, or 40 mg/kg;
30 min), or atropine sulfate (1 mg/kg;
30 min) and were exposed to cold for a 2- or 3-h period. When
the exposure to cold lasted for 3 h, a second s.c. injection of
the same dose of bretylium or atropine was given 75 min after the
beginning of cold exposure. The regimen of drug administration was
based on previous functional studies showing pharmacological effects of
these drugs (14, 54). Rats were euthanized at the end of
cold exposure. The stomachs were collected and processed for Fos immunohistochemistry.
Immunohistochemistry
Tissue preparation. The stomach was opened along the greater curvature, pinned flat, and fixed overnight in 0.1 M sodium phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde and 14% picric acid. After being rinsed in PBS, the corpus and the antrum were separated and dissected under surgical microscope to obtain the LMMP whole mount preparations. The mucosa was scraped off, and the submucosa and the circle muscle were carefully removed using fine forceps. The LMMP whole mount preparation includes myenteric plexus adhered on the longitudinal muscle. To avoid regional differences, in each rat, ~0.5 cm2 of preparation dissected from the middle portion of the ventral corpus (~0.4 mm from boundary between corpus and antrum, 0.7 mm from lesser curvature, and 1.0 mm from greater curvature) and the entire antrum preparation were collected for morphological staining.
Fos immunohistochemistry. Fos staining was performed as described previously (29, 30). Briefly, the LMMP whole mount preparations were rinsed in PBS and incubated with 0.3% hydrogen peroxide to remove endogenous peroxidase activity. The tissues were incubated for 24 h at 4°C with a polyclonal rabbit anti-Fos serum (Fos Ab-5, 1:10,000; Oncogene Research Products, Cambridge, MA) diluted in PBS containing 0.1% sodium azide and 0.3% Triton X-100 (PBS-T, pH 7.4) and then rinsed in PBS and incubated for 1 h at room temperature with biotinylated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:1,000. Finally, preparations were processed using the standard biotin-avidin-horseradish peroxidase methodology (22). Fos immunoreactivity was detected as dark brown nuclear staining. Immunohistochemical controls were routinely performed following the same procedure except that the primary antibody was replaced by PBS-T.
Fos immunohistochemistry combined with neuronal staining using cuprolinic blue (quinolinic phthalocyanine). Slightly modified cuprolinic blue counterstaining (21) was used to assess the neuronal identity of myenteric cells expressing Fos. Briefly, LMMP whole mount preparations were incubated in PBS-T containing 0.3% H2O2 followed by washing with PBS and 0.05 M sodium acetate buffer (pH 5.6). The preparations were stained for 2 h at 42°C in cuprolinic blue (0.3% quinolinic phthalocyanine in 0.05 M sodium acetate-1.0 M magnesium chloride buffer, pH 4.9; Electron Microscopy Sciences, Fort Washington, PA). After being washed in PBS, tissues were processed for Fos immunohistochemistry as described above. Neurons were recognized by the turquoise cuprolinic blue staining in cytoplasm, and nuclear Fos immunoreactivity was revealed as brown staining.
Double immunolabeling of Fos and ChAT. After incubations with monoclonal mouse Fos antibody TF161 (1:500; gift of Dr. K. Riabowol, University of Calgary, Calgary, AB, Canada) overnight at 4°C and biotinylated goat anti-mouse IgG (1:100, Sigma) for 2 h at room temperature, the preparations were processed using avidin-biotin-peroxidase with diaminobenzidine enhanced with nickel ammonia sulfate as the first chromogen. After being washed, the preparations were incubated with a polyclonal goat anti-ChAT serum (1:500; no. AB144p, Chemicon International) overnight at 4°C followed by biotinylated donkey anti-goat IgG (1:1,000; Jackson ImmunoResearch) for 1 h at room temperature and then processed using avidin-biotin-peroxidase procedure with diaminobenzidine as the second chromogen. Fos immunoreactivity was detected as a dark blue nuclear reaction product, and ChAT immunoreactivity appeared as a brown staining in the cytoplasm.
Double labeling of Fos and NADPH-d.
Double labeling was performed using nitroblue tetrazolium formazan
histochemical staining followed by Fos immunohistochemistry. LMMP whole
mount preparations were rinsed in PB and incubated for 30-60 min
at 37°C in PB containing 0.3% Triton X-100, 1 mg/ml -NADPH, and
0.1 mg/ml nitroblue tetrazolium. After a further rinsing, tissues were
processed for Fos immunohistochemistry as described in Fos
immunohistochemistry. Fos immunoreactivity was detected as
brown nuclear staining, and NADPH-d appeared as a dark blue reaction
product in the cytoplasm.
Quantitative analysis and statistics. Fos-IR cells, cuprolinic blue-, ChAT-IR-, and NADPH-d-positive neurons, and double-staining neurons were counted under microscopy in 25 ganglia from each corpus or antral preparation and expressed as a mean count per myenteric ganglion. Myenteric ganglia were recognized as clearly delineated groups of neurons separated by well-defined internodal fiber tracts (30). The mean from each animal was used to calculate the group mean. Data are expressed as means ± SE of the number of cells or neurons per ganglion. Images of gastric myenteric plexus from rats exposed to cold or maintained at room temperature were taken under identical conditions. Comparisons between group mean values were performed with one-way ANOVA followed by Duncan's contrast. A P value <0.05 was considered statistically significant.
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RESULTS |
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Cold Exposure Induces Fos Expression in Gastric Myenteric Plexus
Conscious rats fasted for 24 h and semirestrained at room temperature for 3 h showed few Fos-IR cells in the gastric myenteric plexus (<1/ganglion) (Figs. 1 and 2). In contrast, cold exposure for 60-180 min triggered a time-related induction of Fos expression within the myenteric ganglia in both the corpus and antrum (Figs. 1 and 2). The maximal response was reached at 120 min and was sustained to the end of cold exposure (180 min) (Fig. 2). The numbers of Fos-IR cells at 120 and 180 min were similar in the corpus and antrum and were twofold higher than the numbers at 60 and 90 min (Fig. 2).
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Vagotomy Prevents Cold Exposure-Induced Fos Expression in Gastric Myenteric Plexus
Compared with corresponding sham-operated/cold-exposed rats, acute bilateral cervical vagotomy completely abolished cold exposure (2 h)-induced Fos expression in the corpus and antral myenteric plexus, whereas gastric vagotomy significantly reduced the number of Fos-positive cells by 52% and 49%, respectively (Table 1). Sham gastric vagotomy (
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Hexamethonium, But Not Bretylium or Atropine, Prevents Cold Exposure-Induced Fos Expression in Gastric Myenteric Plexus
Hexamethonium pretreatment (10 mg/kg,
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Most Fos-IR Cells in Gastric Myenteric Plexus of Cold-Exposed Rats Are Ganglionic Neurons
Cuprolinic blue staining revealed individual myenteric neurons with clearly outlined cytoplasm (Fig. 5). The total numbers of cuprolinic blue-positive cells in corpus or antral myenteric ganglia were similar in rats semirestrained for 3 h at room temperature or in cold conditions (Table 3). About 90-93% of the cuprolinic blue-positive neurons expressed Fos after 3 h of cold exposure (Fig. 5, B and D), whereas the double-stained neurons were rare in rats maintained at room temperature (Table 3 and Fig. 5, A and C). These results indicate that the majority of Fos-IR myenteric cells observed in acute cold-exposed rats were neurons.
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ChAT immunoreactivity was detected in fibers surrounding myenteric
cells and barely detected in the cytoplasm in rats semirestrained at
room temperature (Fig. 6, A
and C). However, ChAT immunoreactivity was present in the
cytoplasm of a substantial number of ganglionic cells in 3-h
cold-exposed rats. Part of the ChAT-IR neurons expressed Fos (Fig. 6,
B and D).
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NADPH-d-Fos double staining yielded low background and revealed
well-defined NADPH-d-positive cells similar to those described for
nitric oxide synthase (NOS)-containing neurons (Ref. 23; Fig. 7). Fibers displaying NADPH-d
activity were most prominent in the internodal fiber tracts (Fig. 7).
About 8-10 NADPH-d-positive neurons per ganglion and rare Fos-IR
cells were observed in both corpus and antral myenteric plexus in rats
semirestrained at room temperature for 3 h (Fig. 7, A
and C). Cold exposure for 3 h did not change the number
of NADPH-d-positive neurons but did induce Fos expression in numerous
myenteric cells (Fig. 7, B and D). The double
staining revealed that 90-100% of NADPH-d-positive neurons
expressed Fos after a 3-h cold exposure (Fig. 7, B and D). The proportion of ganglionic NADPH-d-Fos-IR neurons
represented ~35-40% of the total number of Fos-IR cells in both
corpus and antrum after cold exposure.
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DISCUSSION |
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The present results show that acute cold exposure induced Fos expression in the gastric myenteric ganglia in conscious semirestrained rats. The maximal plateau response (16-20 Fos-IR cells/ganglion) was reached at 120 min. In contrast, rats semirestrained for 180 min at room temperature showed almost no or only rare Fos-positive cells in the gastric myenteric ganglia. Because our experiments were performed in rats fasted for 16 or 24 h, the confounding effect of feeding, known to induce Fos expression in gastric myenteric plexus (13), was avoided. Double staining of Fos and cuprolinic blue (21) revealed that the major proportion of Fos-IR cells were gastric myenteric neurons.
Converging evidence supports the idea that central vagal stimulation is involved in acute cold exposure-induced Fos expression in gastric myenteric neurons. First, we previously reported that cold exposure induces Fos expression in medullary DMN, which is indicative of vagal preganglionic neuronal activation (7, 50). Second, acute cold exposure increases the amplitude and frequency of vagal efferent activity in rats as measured by direct electrophysiological recording (10). Third, recent studies showed that electrical stimulation of vagal efferents (57) or central vagal activation by i.c. TRH analog (31) induces Fos expression in the gastric myenteric plexus. Fourth, in the present study, bilateral cervical vagotomy completely abolished the 2-h cold exposure-induced Fos expression in gastric myenteric ganglia. It is unlikely that the abolition by vagotomy resulted from a nonspecific effect of acute surgical stress, because Fos expression induced by cold exposure was not altered in acute sham-operated rats that underwent the same anesthesia and surgical procedures except for sectioning of the vagus. In addition, gastric vagotomy performed 48 h earlier also reduced cold-induced Fos expression in the gastric myenteric ganglia. Selective gastric vagotomy was less efficient compared with bilateral cervical vagotomy. This may be related to vagal inputs from branches rostral to the site of the gastric vagotomy, including the hepatic and celiac branches, which also contribute to the gastric vagal innervation, although to a lesser extent (4, 5). Finally, vagal mediation is supported by the fact that pretreatment with bretylium (25 mg/kg), an adrenergic blocking agent (8), did not alter Fos expression in gastric myenteric ganglia induced by cold exposure. Bretylium injected at similar or lower doses blocked central corticotropin-releasing factor-induced modulation of gastric motor function (25) but had no effect on cold-induced gastric erosion formation (9). Anterograde tracing studies showed that vagal efferent terminals form a dense network encircling or making putative contacts with nearly all myenteric neurons in the rat corpus and antrum (20). The profuse and widespread presence and similar magnitude of vagus-dependent Fos expression in both the corpus and antral myenteric ganglia after cold exposure is consistent with vagal efferent innervation of the stomach (20).
The present pharmacological studies indicate that the vagus-dependent Fos expression induced by cold exposure is mediated by nicotinic acetylcholine receptors. Pretreatment with hexamethonium (20 mg/kg), a nicotinic receptor antagonist, significantly reduced the numbers of Fos-IR cells in the corpus and antral myenteric plexus by 76% and 80%, respectively, in rats exposed to cold for 3 h. The remaining Fos expression did not result from use of a submaximal dose because hexamethonium at 40 mg/kg did not further decrease the number of Fos-IR cells. The remaining Fos expression in the presence of nicotinic blockage may represent the activation of neurons by other excitatory transmitters (ATP, serotonin, and/or glutamate), which have been demonstrated to participate in the noncholinergic excitatory transmission in the guinea pig enteric nervous system (16). In contrast to hexamethonium, the muscarinic receptor antagonist atropine did not affect the gastric myenteric Fos expression induced by acute cold exposure. Likewise, atropine had no effect on the gastric myenteric Fos expression induced by electrical stimulation of the vagus (57) or by i.c. injection of TRH analog (31). Atropine administered at similar or even lower doses completely blocked acute cold exposure-induced vagal stimulation of gastric motor and secretory functions as well as gastric lesion formation (24, 28, 38). Therefore, it is unlikely that the afferent inputs triggered from activated gastric mucosal and smooth muscle cells after cold exposure contribute to the gastric myenteric Fos expression. Consequently, the present findings indicate that cold-induced Fos expression in gastric myenteric ganglia is largely mediated by vagal preganglionic stimulatory input through nicotinic synapse, whereas vagal postganglionic activation through muscarinic receptors does not play a role.
The previously reported vagally dependent, atropine-sensitive stimulation of gastric function induced by acute cold exposure (45, 46, 55) is indicative of cholinergic myenteric neuronal activation. ChAT immunoreactivity has been successfully used to visualize cholinergic neurons and their processes in the central nervous system but less successfully applied to the peripheral cholinergic system, especially in rats (41). In the present study, we used a commercial ChAT antibody that has been reported to stain myenteric neurons in the guinea pig stomach (dilution 1:100) treated with colchicine (32) and in the rat ileum (dilution 1:50) without colchicine treatment (27). At 1:500 dilution, we observed dense ChAT-IR fibers surrounding the gastric ganglionic cells, whereas no ChAT immunoreactivity was found in the cytoplasm in rats maintained at room temperature. The ChAT-IR fibers may represent the vagal preganglionic fibers, which are of central origin. After a 3-h cold exposure, a substantial number of myenteric Fos-positive cells contained ChAT immunoreactivity in their cytoplasm. These findings are indicative of activation of cholinergic neurons as well as increased intracellular ChAT content under these conditions. However, the number of ChAT-IR neurons in cold-exposed rats observed in the present study was still lower than that reported by Nakajima et al. (35) in rat stomach (~60%) detected by a specific antiserum against peripheral ChAT. This may be a result of the limitation of the antibody used in the present study to react with the peripheral ChAT, which is encoded by an alternative splice variant of ChAT mRNA and expressed preferentially in the peripheral nervous system (49).
Gastric circular and longitudinal muscle layers are innervated by excitatory cholinergic as well as inhibitory nitrergic neurons located in the myenteric plexus (32). ChAT- and NADPH-d-containing neurons are not colocalized in rat enteric nervous system (35), and these two separate populations were reported to constitute >95% of gastric myenteric neurons in guinea pigs (43). Neuroanatomic studies also revealed that a large population of NADPH-d-positive gastric myenteric neurons receives vagal efferent contacts (2, 3). In addition, NOS gene expression in rat gastric myenteric plexus is stimulated by vagal activation through nicotinic receptors (36). In the present study, there were ~8-10 NADPH-d-positive neurons per gastric myenteric ganglion, which is in agreement with a previous report (2). Nearly all myenteric NADPH-d-positive neurons expressed Fos in rats exposed to cold for 3 h. The Fos-NADPH-d-positive neurons represented ~35-40% of the whole population of myenteric Fos-IR cells after cold exposure. NO in the myenteric nervous system plays an important role in the nonadrenergic, noncholinergic relaxation of smooth muscles (36). In rats, the axonal projections of NADPH-d- or NOS-positive gastric myenteric neurons provide an extensive network of fibers running within the circular smooth muscle layer (2, 23). In addition, in the guinea pig, the gastric circular and longitudinal muscle layers receive ascending excitatory cholinergic innervation and descending inhibitory nitrergic innervation (32, 42). The coactivation of cholinergic and nitrergic pathways in the gastric myenteric plexus may have functional relevance to the stimulation of gastric propulsive contractility observed during acute cold exposure (17, 18, 28).
In summary, acute cold exposure for 1-3 h induces a time-related Fos expression in corpus and antral myenteric neurons in conscious fasted rats. The activation of gastric myenteric neurons is mediated by vagal nicotinic pathways and includes cholinergic and NOS-synthesizing neurons, suggesting a central regulation of both excitatory and inhibitory myenteric pathways during acute cold exposure. These results provide neuroanatomic and neurochemical evidence at the gastric myenteric level for the vagal cholinergic-dependent stimulation of gastric function induced by acute cold exposure (18, 28, 38, 52, 53).
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ACKNOWLEDGEMENTS |
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We thank Paul Kirsch for assistance in the preparation of the manuscript.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-50255 (H. Yang), DK-30110 (Y. Taché), and DK-41301 (CURE Animal Core).
Address for reprint requests and other correspondence: H. Yang, CURE: DDRC, VA GLAHS, Bldg. 115, Rm. 203, 11301 Wilshire Blvd., Los Angeles, CA 90073 (E-mail: hoyang{at}ucla.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 November 2000; accepted in final form 23 April 2001.
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