- Email: [email protected]
Cold exposure elevates thyrotropin-releasing hormone gene expression in medullary raphe nuclei: Relationship with vagally mediated gastric erosions
Neuroscience Vol. 61, No. 3, pp. 655~63, 1994
Pergamon
0306-4522(94)E0110-P
Elsevier ScienceLtd Copyright © 1994 IBRO Printed in Great Britain. All rights reserved 0306-4522/94 $7.00 + 0.00
COLD EXPOSURE ELEVATES THYROTROPIN-RELEASING HORMONE GENE EXPRESSION IN M E D U L L A R Y RAPHE NUCLEI: RELATIONSHIP WITH VAGALLY MEDIATED GASTRIC EROSIONS H. YANG,*~ S. V. W u , t T. ISHIKAWA~ and Y. TACHI~t tCURE/Gastroenteric Biology Center, VA Wadsworth Medical Center, Department of Medicine and Brain Research Institute, UCLA, Los Angeles, CA 90073, U.S.A. *Division of Psychosomatic Research, NIMH, Chiba, Japan Abstract--The stimulation of thyrotropin release by cold is associated with an increase in thyrotropin-releasing hormone gene expression in the paraventricular nucleus of the hypothalamus. Cold exposure also stimulates autonomic outflow to viscera. There is evidence that caudal raphe nuclei are involved in autonomic regulation through thyrotropin-releasing hormone projections to the dorsal vagal complex and spinal cord. To determine whether cold modulates thyrotropin-releasing hormone gene expression in the caudal raphe nuclei, the effect of cold exposure on thyrotropin-releasing hormone messenger RNA levels in the rat lower brainstem was examined by quantitative Northern blot analysis and thyrotropin-releasing hormone messenger RNA was localized by in situ hybridization. The gastric responses to cold exposure were also assessed in sham or vagotomized rats with pylorus ligation. Thyrotropin-releasing hormone messenger RNA signal was detected in the RNA extracted from the medulla and hypothalamus but not from the amygdala, periaqueductal gray or cerebellum. Cold exposure (4°C) for 1 or 3 h increased thyrotropin-releasing hormone messenger RNA levels in the medulla by 77 + 37 and 142 + 39% respectively. In situ hybridization histochemistry showed that the increase in silver grain density occurred exclusively in the raphe pallidus and raphe obscurus. Exposure to cold stress for 2 h stimulated gastric acid secretion and resulted in gastric lesion formation in sham but not vagotomized rats. There are established thyrotropin-releasing hormone projections from the raphe pallidus and obscurus to the dorsal vagal complex. Thyrotropin-releasing hormone is known to exert an excitatory effect on neurons of the dorsal motor nucleus of the vagus, leading to vagally mediated stimulation of gastric function and erosions. The increased thyrotropin-releasing hormone gene expression in the raphe pallidus and obscurus induced by cold supports an important role of endogenous thyrotropin-releasing hormone contained in caudal raphe nuclei-dorsal vagal complex pathways in vagally mediated changes in gastric function induced by cold.
Cold exposure is known to induce a rapid release of thyrotropin-releasing hormone ( T R H ) in the median eminence, which leads to the pituitary secretion of thyrotropin) The portal release of hypothalamic T R H is associated with a parallel increase in neuronal activity and T R H m R N A in the paraventricular nucleus o f the hypothalamus (PVN) as assessed by immunohistochemistry for Fos protein and in situ hybridization for T R H m R N A . s,62,63 Cold exposure also stimulates both the parasympathetic and sympathetic nervous systems. 22m In particular, hypothermia induces a vagal-dependent and atropine-sensitive stimulation of gastric secretory and m o t o r functions as well as the formation of gastric lesions within 3 h. 2''~'32'56Convergent functional and anatomical evidence indicates that endogenous T R H in the dorsal vagal complex (DVC) is involved in the vagally *To whom correspondence should be addressed. Abbreviations: DVC, dorsal vagal complex; PVN, paraven-
trieular nucleus of the hypothalamus; SSC, standard saline citrate; TRH, thyrotropin-releasing hormone. 655
mediated gastric response to cold exposure) ° T R H containing fibers and terminals in the D V C originate exclusively from neurons in the raphe pallidus and obscurus and to a lesser extent in the parapyramidal region, where T R H m R N A , p r o - T R H and T R H have been localized. 23,25,37,47 Chemical or electrical activation of the raphe pallidus or obscurus neurons increase gastric acid secretion, motility and lesion formation through vagal pathways in rats and c a t s . 9'16'29'35'55'57'58In addition, we recently showed that microinjection of T R H antibody into the D V C blocked the acid and motility responses to chemical activation of raphe pallidus neurons. 9'5s These data indicate that endogenous T R H is released from nerve terminals in the D V C upon stimulation of caudal raphe nuclei. Very little is known about the regulation of T R H gene expression in lower brainstem nuclei s,42,53 compared with in the PVN. s,21,62,63 The purpose of the present study was to investigate whether exposure to cold for 1-3 h increases levels of T R H gene expression in the medulla in conscious rats. Northern blot
656
H. YANG et al.
analysis was used to m e a s u r e q u a n t i t a t i v e c h a n g e s in T R H m R N A a n d in situ h y b r i d i z a t i o n to locate the signals. T h e a l t e r a t i o n s in gastric acid secretion a n d m u c o s a l integrity i n d u c e d by cold were also m o n i t o r e d in s h a m a n d v a g o t o m i z e d rats with p y l o r u s ligation.
at 60°C with 0.2 M NazCO 3 and 0.2 M NaHCO 3 for 15 min in order to obtain fragments about 150 bases in length. Northern blot analysis
In a separate experiment, in rats under light ether anesthesia a laparotomy was performed and the pylorus was ligated. Ten minutes after recovery from anesthesia, conscious rats were placed in semi-restraining cages either at room temperature or at 4°C. An additional group of rats was maintained singly in home cages at room temperature. Two other groups received bilateral cervical vagotomy or sham operation, respectively, before ligating the pylorus, then 10 min later both groups were placed singly in semirestraining cages at 4°C. Two hours later, rats were killed by decapitation. The stomachs were removed and gastric secretion collected. The measurements of gastric secretion volume, acid concentration and mucosal erosions were performed as described previously, t8 Gastric acid output was calculated by multiplying the values of acid concentration by the volume of secretion for each rat.
Groups of rats were exposed for 60, 90, 120 or 180 min period at 4°C or room temperature under identical conditions of light illumination (n = 5~/group). Cold exposed rats were decapitated at the end of each period, and rats maintained at room temperature were decapitated in between cold exposed rats. Brains were rapidly removed, then the hypothalamus, amygdala, periaqueductal gray, cerebellum and medulla were dissected, immediately placed in powdered dry ice and kept frozen at - 8 0 ° C until RNA extraction. The landmarks for dissection of the various regions of the brain were as follows. Hypothalamus: rostral to caudal, from the optic chiasm to the mammillary body; lateral section from the midline, c. 2 mm and dorsal section from the ventral surface, c. 2.5 ram. Amygdala: rostral to caudal was the same as the hypothalamus, from the lateral border of the optic tract laterally and dorsally to the rhinal fissure. Periaqueductal gray: rostral to caudal, from the posterior commissure to the caudal part of the inferior colliculus, the area around the aqueduct with a radial of c. 1.5 mm. Cerebellum whole: Medulla: rostral to caudal, from the level of the appearance of the facial nerve to the level of obex; lateral, c. 1.5 mm; dorsal section from the ventral surface, c. 2.5 mm. In addition, in the group of rats exposed to cold for 180min, the stomach was also removed for measurements of gastric lesions. Northern blot analysis was performed as described previously. 6° Total RNA was extracted from the hypothalamus, amygdala, periaqueductal gray, cerebellum and medulla by a rapid method developed by Chomczynski and Sacchi. 7 Denatured RNA samples (20 #g/sample) were separated on a 1.2% formaldehyde gel with buffer circulation and were then transferred by electroblotting to a nylon membrane (Hybridization, 0.45 pm, 10x 15cm, catalog no. RPN.1510N; Amersham Corp., Arlington Heights, IL). RNA was immobilized to the membranes by UV cross-linking followed by baking in a vacuum oven for l h at 80°C. RNA blots were prehybridized with the hybridization buffer for 6-10 h and then incubated with fresh hybridization buffer in the presence of radiolabeled TRH probe for 24-48 h at 55°C. Blots were washed and exposed to X A R film (Eastman Kodak Co., Rochester, NY) for one to three days at -80°C. The relative densities of the m R N A signals were measured quantitatively by using a Bio-Rad Densitometer (Model 620). The consistency of RNA loading and transferring was assessed by rehybridizing with an 18S ribosomal RNA oligo-deoxynucleotide probe (5'CGG CAT GTA TTA GCT CTA GAA TTA CCA CAG 3') labeled by a standard 5' end-labeling technique. 2s
Hybridization probe The 1322 bp EcoRI pro-TRH DNA fragment cloned in plasmid pUC12 (kindly provided by Dr R. Goodman, Vollum Institute, OR) was resubcloned into pGEM-3. The antisense RNA was synthesised by T 7 RNA polymerase after linearizing the plasmid with SmaI. The cRNA probe was synthesized using 1 #g SmaI-linearized plasmid DNA with the transcription buffer (40mM Tris-HCl, 6 m M MgC12, 2 mM spermidine and 10 mM NaC1, pH 7.5) in the presence of 10 mM dithiothreitol, 0.5 mM of each of ATP, GTP and CTP, 12 # M UTP and 50 #Ci [a_32p] UTP (for the probe used in Northern blot analysis) or 100 pCi [~-ssS]UTP (for the probe used in in situ hybridization). The reaction was initiated by adding 10 U of T 7 RNA polymerase and incubated at 37°C for 60 min. After the transcription, the plasmid D N A was removed by digesting with ribonucleasefree deoxyribonuclease I (RQI DNase). All the reagents used in the transcription were from Promega (Madison, WI). Radiolabeled R N A was purified by phenol--chloroform extraction and alcohol precipitation. For in situ hybridization, the probe was hydrolysed by incubating
In situ hybridization Rats maintained in semi-restraining cages either at 4°C (n = 3) or at room temperature (n = 2) for 2 h were decapitated. Brains were rapidly removed and immediately immersed in 4% paraformaldehyde at 4°C for 15 h and soaked in 25% sucrose solution. The frozen tissues were sectioned coronally on a microtome cryostat at 20 # m at the level of the hypothalamus (interaural 6.88-7.20 mm from Paxinos and Watson's plates 38) and medullar (interaural - 3 . 7 2 mm to - 4 . 6 8 mm) and mounted onto ribonucleasefree, gelatin-coated glass slides. The tissues were allowed to dry completely, then fixed in 4% paraformaldehydephosphate-buffered saline for 30 min, washed three times in phosphate-buffered saline for 10 min each time, and dehydrated for 5 min in 70% and 100% ethanol, respectively. After air drying, slides were stored at - 2 0 ° C until hybridization. Hybridization was performed using a commercial in situ hybridization kit (Oncor, Gaithersberg, MD). In brief, the slides were washed in 2 x standard saline citrate (SSC) to remove the ethanol and incubated in pretreatment 1 and
EXPERIMENTAL PROCEDURES
Animals
Male Sprague-Dawley rats weighing 230-290 g (Bantin and Kingman, CA) were maintained on rat Purina chow and tap water ad libitum and housed under conditions of controlled temperature (20 + I°C) and illumination (light on 6.00 a.m. to 6.00 p.m.). Experiments were performed on animals deprived of food for 18 h but allowed free access to water until the beginning of the experiments. All animals were killed between 1.00 p.m. and 5.00 p.m. A previous study indicated that there were only small circadian variations in TRH m R N A in the PVN between 2.00 and 6.00 p.m. 62 Cold exposure
Conscious rats were placed individually in stainless steel cylindrical cages with flat bottoms ( 16 cm x 5.5 cm × 5.5 cm) and with perforations to allow ventilation. Such semi-restraining cages were shown previously by Robert et al. 45 not to induce stress. Rats housed in semi-restraining cages were maintained either at room temperature (23 + 2°C) or at 4°C for various time periods. Measurement o f gastric secretion and lesions
657
Cold activates TRH gene expression in caudal raphe 2 solutions, then washed again and the tissues dehydrated with 70%-100% ethanol. The hybridization was carried out at 52°C. The [~t-3sS] UTP-labeled TRH probe was dissolved in 20 mM dithiothreitol and diluted with 9 vols of hybridization mix solution to reach approximately 3-5 x 105 c.p.m./10/~1. After the slides and the probe were preheated to 52°C, 10/~1 of denatured probe/hybridization mix solution were added to each slide and the tissues were covered with a coverslip. The hybridization was carried out in a humidified atmosphere for 3-5 h, then the coverslips were removed. The non-specific binding was washed out by 50% formamide/2 x SSC at 52°C and the formamide was removed by washing in 2 x SSC at room temperature to prevent the inhibition of the ribonuclease. Slides were incubated at 37°C for 30 min in a post-treatment solution which contained ribonuclease to remove tingle-stranded RNA without degrading the complex probe/target. Then the slides were dehydrated in graded concentrations of ethanol and air dried. Tissue sections were coated with radiographic emulsion, air dried for 4-5 h, transferred to the Black Slide Boxes and kept at 4°C for three to eight weeks. Autoradiograms were developed using the Developer and the Fixer in the Kit. The slides were stained with Toluidine Blue and observed under a microscope with light or dark field. Specificity of the in situ hybridization was assessed by adding a 50-fold excess of cold probe synthesized with UTP instead of [35S]-UTP to compete with the radiolabeled probe during the hybridization.
Statistical analysis Results are presented as mean _+ S.E.M. Multiple group comparisons were calculated by ANOVA followed by Duncan's contrast. Statistical significance was assigned when P < 0.05. RESULTS
Influence of coM exposure on gastric acid secretion and lesion formation R a t s m a i n t a i n e d at a m b i e n t r o o m t e m p e r a t u r e in semi-restraining cages h a d n o change in gastric acid c o n c e n t r a t i o n , o u t p u t a n d mucosal integrity comp a r e d with those kept in s t a n d a r d cages as measured
2 h after pylorus ligation (Table 1). In rats placed in semi-restraining cages at 4°C for 2 h, gastric acid c o n c e n t r a t i o n a n d o u t p u t increased by 8 6 % a n d 216% respectively, c o m p a r e d with rats m a i n t a i n e d at r o o m temperature. In addition, gastric h e m o r rhagic lesions were f o u n d in the glandular p a r t o f the s t o m a c h (Table 1). Bilateral cervical v a g o t o m y abolished cold-induced stimulation o f gastric acid secretion a n d lesion f o r m a t i o n (Table 2).
Influence of cold exposure on medullary thyrotropinreleasing hormone mRNA expression Northern blot analysis Northern blot analysis of total RNA ( 2 0 # g / s a m p l e ) isolated from the h y p o t h a l a m u s , medulla, amygdala, periaqueductal gray a n d cerebellum was performed using a labeled T R H c R N A probe. A single b a n d o f 1 . 6 k b representing the p r o p e r size of published T R H m R N A 24 was detected in the h y p o t h a l a m u s a n d the medulla, b u t n o t in the amygdala, periaqueductal gray a n d cerebellum (Fig. 1). Equal a m o u n t s o f R N A (20/~g/sample) from the medulla of rats m a i n t a i n e d at r o o m t e m p e r a t u r e or exposed to cold for 60, 90, 120 a n d 180rain were further analysed by the T R H probe. T R H m R N A levels increased after cold exposure c o m p a r e d with rats m a i n t a i n e d at r o o m t e m p e r a t u r e u n d e r conditions t h a t were otherwise the same (Fig. 2). Q u a n titative analysis of the T R H m R N A signal showed that the density o f the T R H m R N A signal was significantly increased by 77 + 37% after 60 min o f cold exposure a n d was m a i n t a i n e d elevated by 110 + 27, 110 _+ 20 a n d 142 _+ 39% w h e n cold exposure extended to 90, 120 a n d 180rain periods, respectively (Table 3). Gastric lesions were also observed in each rat exposed to cold for 1 8 0 m i n (3.3 _+ 0.9 m m 2, n = 6).
Table 1. Effect of cold exposure on gastric acid secretion and lesion formation in pylorus-ligated rats n
Acid cone.§ (mmol/l)
Volume~ (ml/2 h)
6 6 10
51 _+8 58 + 9 109 -+ 4*t
4.0_+0.3 2.7 _+ 0.4* 4.8 + 0.3t
Treatments~: Freely moving at 23°C Restraint at 23°C Restraint at 4°C
Acid output§ (~mol/2 h)
Erosions§ (mm 2)
199+32 165 _+40 519 + 39 *t
0.02_+0.02 0.00 + 0.00 0.89 -+ 0.35
:[:The pylorus was ligated under light ether anesthesia and 10 min later, rats were returned to home cages or placed in semi-restraining cages either at room temperature or at 4°C for 2 h, then killed for the measurement of gastric secretion and erosions. §Mean _+ S.E.M. of the group. *P < 0.05 compared with freely moving rats at 23°C. t £ < 0.05 compared with rats restrained at 23°C. Table 2. Effect of vagotomy on gastric acid secretion in conscious pylorus ligated rats exposed to cold Treatmentst Sham Vagotomy
Acid conc.~ (mmol/ml)
Volume:~ (ml/2 h)
Acid output~ (~umol/2 h)
Erosions:[: (mm 2)
103_+8 32 _+ 9*
4.0_+0.6 0.3 _+0. ! *
411+63 9 _+ 3*
0.53_+ 0.17 0.0 _ 0.0"
t i n rats under light ether anesthesia, sham operation or bilateral cervical vagotomy was performed and the pylorus was ligated. After regaining the righting reflex, rats were placed in semi-restraining cages at 4°C for 2 h then killed for the measurement of gastric secretion and erosions. :~Mean + S.E.M. of four rats per group; *P < 0.01, Student t-test. NSC 61/~-1
658
H. YANG et al.
- 28S
~-
H
M
A
P
18S 1.6kb
C
Fig. 1. Northern blot analysis o f T R H mRNA in rat brain. Total RNA (20 #g/sample) was extracted from the hypothalamus (H), medulla (M), amygdala (A), periaqueductal gray (P) and cerebellum (C). Samples were hybridized with the TRH probe. TRH mRNA signals are located in the position of 1.6 kb. Only the hypothalamus and medulla show the TRH mRNA signal.
In situ hybridization Brainstem sections from rats m a i n t a i n e d at r o o m t e m p e r a t u r e or exposed to cold were m o u n t e d on the same slide a n d hybridized with a T R H c R N A probe.
Over 9 0 % of the T R H m R N A signal observed in the medulla was located in the raphe pallidus a n d raphe obscurus, including the area s u r r o u n d i n g the raphe obscurus. A few T R H m R N A signals could also be
Time after cold exposure
0
60
90
120
(min)
180
- 28S
TRH - 1.6kb
- 28S
18S - 18S
Fig. 2. Northern blot analysis of the effect of cold exposure on medullary TRH gene expression. The pictures show the membrane loaded with medullary total RNA extracted from rats maintained either at room temperature (0 min) or exposed to cold for 60, 90, 120 or 180 rain, respectively, and hybridized separately with TRH or 18S ribosomal RNA probes. The TRH mRNA signals are located in the 1.6 kb position.
659
Cold activates TRH gene expression in caudal raphe Table 3. Effect of cold exposure on medullary thyrotropin-releasing hormone mRNA levels in rats Time of cold exposure (min)
01"
60
90
120
180
Relative densitometric unit:l:
2.3 + 0.2
4.1 __+0.9*
4.9 + 0.6*
4.9 + 0.5*
5.6 -I-0.9*
*P < 0.05 compared with control rats. tRats maintained in semi-restraining cages at room temperature served as control. ~Mean_ S.E.M. of five to six rats in each group. Data from Northern blot analysis were quantitatively analysed using a Bio-Rad Densitometer.
found along the ventral edge of the brainstem in the parapyramidal region (data not shown). In rats exposed to cold for 120 min, the TRH m R N A signal in the raphe pallidus and the raphe obscurus was markedly denser than in rats maintained at room temperature (Fig. 3A, B). Adding a 50-fold excess of cold probe almost completely abolished the hybridization signal in medullary raphe nuclei (Fig. 3C). The PVN used as a control for TRH m R N A signal also showed dense labeling in rats exposed to cold for 120 min (Fig. 3D). DISCUSSION
Pylorus-ligated rats exposed to cold for 2 h in semi-restraining cages had an increased gastric acid secretion and developed gastric hemorrhagic erosions compared with rats maintained at room temperature. The gastric changes were related to cold exposure and not to housing in semi-restraining cages since there was no increase in gastric acid secretion or change in gastric mucosal integrity between groups kept at room temperature, either in semi-restraining cages or in standard cages. These results are in agreement with previous observations that rats maintained in these semi-restraining cages were not stressed and have no observable c-fos expression in the brain. 5"45 More severe gastric erosions were also observed after 3 h exposure to cold in rats without pylorus ligation. The difference in severity of the gastric lesions between these two groups may be related to the different duration of cold exposure and the release of protective factors in the stomach, such as calcitonin gene-related peptide triggered by abdominal surgery and pylorus ligation. ~°,39,6~The stimulation of gastric acid secretion and the formation of gastric mucosal lesions involved vagal pathways, since the gastric response was no longer observed in vagotomized rats. Previous studies performed under different experimental conditions of cold exposure such as immersion in water at 20°C or lowering body temperature by refrigerant pack also demonstrated a vagally mediated stimulation of gastric acid secretion and erosion formation in fasted rats. 2,32,33,56 Functional and anatomical evidence support a role of medullary TRH in the vagal stimulation of gastric function and ulcer formation induced by cold) ° Immunoneutralization by TRH antibody injected into the cerebrospinal fluid inhibits cold water immersion and cold restraint-induced stimulation of gastric acid secretion and lesion formation. 4'13'~5'32'33 TRH
microinjected into the DVC is unique among other transmitters to induce a vagal-dependent stimulation of gastric secretory and motor function and the formation of gastric lesions in rats. 1°'14'19'34'46'49'59Electrophysiological studies showed that TRH causes a direct postsynaptic excitatory effect on dorsal motor nucleus neurons 3°'4~'52and increases efferent activity in the gastric branch of the vagus. 48 These observations were well correlated with the high density of TRH receptors and TRH receptor m R N A localized mainly on vagal preganglionic motoneurons. 6'27 Combined immunocytochemical and retrograde tracing techniques further demonstrated that TRH immunoreactive fibers and terminals make asymmetric synaptic contacts on dendrites of gastric vagal motoneurons? TM The dense TRH-immunoreactive nerve fibers and terminals in the DVC arise exclusively from perikarya located in the raphe pallidus, rapbe obscurus and to a small extent in the parapyramidal regions, where TRH m R N A and pro-TRH have been located.23,25,37,47
Results from the present study provide strong evidence that medullary TRH is involved in the vagally mediated gastric response to cold. Cold exposure increased TRH m R N A levels in the lower brainstem within the first hour. The changes in TRH m R N A persisted for the duration of cold exposure, as reflected by the 142% increase in signal density in Northern blot analysis observed at 180 min. The similarity of the signal density of 18S ribosomal R N A shows the consistency of the equal sample loading, transfer and illumination under these conditions. The specificity of the response was validated by several observations. First, as expected, a single band corresponding to the appropriate size of 1.6 kb for TRH m R N A 21'24was selectively increased after cold stress. Second, Northern blot analysis of total RNA extracted from different brain regions showed that TRH m R N A signal is present only in the hypothalamus and the medulla, where the densest localization of pro-TRH-containing perikarya have been reported. 23 No signal was observed in the periaqueductal gray, amygdala or cerebellum. These results are consistent with in situ hybridization histochemicai studies showing only a cluster of weakly hybridizing cells in the periaqueductal gray and amygdala and no positive cells in the cerebellum. 47 In the present study, in situ hybridization was used to localize the TRH m R N A signal in the medulla. Data revealed that TRH m R N A signal is confined exclusively in neurons of the caudal medullary raphe
Fig. 3. Light micrographs of the brainstem from rats maintained at room temperature (A) or in cold (4r'C; B) for 120 min. Coronal sections were exposed for eight weeks. Adding 50-fold excess of cold probe simultaneously with the radiolabeled probe into the hybridization mix solution almost completely prevented the hybridization in section from a non-stressed rat (C). TRH mRNA signal was also observed in the PVN in rats exposed to cold for 120 min (D). Tissues stained with Toluidine Blue. Rpa, raphe pallidus; Rob, raphe obscurus; PVN, paraventricular nucleus of the hypothalamus. Scale bar = 500 #m.
Cold activates TRH gene expression in caudal raphe complex (raphe obscurus and pallidus). After 2 h of cold exposure, a more intense signal was observed in the caudal raphe nuclei compared with rats maintained at room temperature. The specificity of the response was shown by the abolition of the hybridization signal when the radiolabeled probe was combined with a 50-fold excess of cold probe during the hybridization. In addition, dense TRH mRNA signal could be observed in the PVN of cold exposed rats as reported previously,62 giving further support for the specificity of the probe. Cold-induced changes in TRH gene expression in the forebrain were found to be specific to neurons localized in the PVN and preoptic area, whereas TRH mRNA levels in the thalamus were not modified by cold. 8'62'63 Enhanced TRH gene expression in PVN neurons by cold has been associated with an increase in TRH release in the median eminence, resulting in the stimulation of pituitary-thyroid hormone secretion. ~7'26 Although direct measurement of TRH release in the DVC in response to cold cannot be directly assessed as in the median eminence,26 the TRH antibody microinjected into the DVC or the cerebrospinal fluid blocked the vagally mediated stimulation of gastric function and ulcer formation induced by cold exposure or chemical activation of the raphe pallidus neurons.4,9,13,3z33,58 These latter findings suggest that changes in TRH mRNA in medullary raphe nuclei may be parallel with changes in TRH release from their terminal projections to the DVC, leading to vagaily mediated stimulation of gastric function. This is further supported by recent evidence that cold exposure for 3 h under the same semi-restraining conditions results in a marked activation of neurons located in the raphe pallidus, raphe obseurus and parapyramidal regions, as well as in the dorsal motor nucleus of the vagus, as shown by numerous Fos-positive neurons in these nuclei? The mechanisms through which cold exposure increases TRH mRNA expression in the raphe pallidus and obscurus are still to be investigated. Little is known about factors that influence the regulation of TRH gene expression in these nuclei? One study showed that changes in blood pressure are associated with alterations in TRH mRNA in the raphe pallidus, s However, it is unlikely that the observed increase in TRH gene expression is secondary to the elevation in blood pressure and the decrease in heart rate induced by cold exposure. 31 Cardiovascular changes induced for 1 h by peripheral epinephrine administration did not influence TRH mRNA in the raphe pallidus and obscurus while a decrease in mean
66t
arterial blood pressure induced by nitroprusside increased TRH mRNA selectively in the raphe pallidus and not the raphe obscurus) TRH neurons in the caudal raphe nuclei contribute projections to both the DVC and the spinal cord, where terminals have been identified apposing preganglionic sympathetic neurons. ~'4° Functional studies have shown that TRH acts in the medulla and intrathecally to increase blood pressure through activation of the sympathetic activity. 12,36,5~ Similarly, cold exposure increases blood pressure and sympathetic activity.31 The present study demonstrates that cold-induced increase in TRH mRNA in the raphe obscurus and paUidus may have implications not only to the understanding of the mechanism of vagally mediated alterations of gastric function but may also encompass the sympathetic responses to cold. CONCLUSIONS Our results demonstrate that cold exposure for 1-3 h elevated brainstem TRH mRNA selectively in the caudal medullary raphe nuclei in conscious rats. Cold exposure for 2-3 h also increased gastric acid secretion and induced lesion formation through vagal pathways in fasted rats. Previous studies indicate that the TRH projections from the raphe pallidus and obscurus to the DVC exert an excitatory effect on preganglionic vagal neurons, leading to vagally mediated stimulation of gastric secretory and motor function and erosions formation. Taken together, the present data support an important role of endogenous TRH contained in caudal raphe nuclei-DVC pathways 54 in the parasympathetic response to cold. Previous studies have shown that cold increases TRH mRNA in specific nuclei of the hypothalamus associated with pituitary secretion and thermoregulation. The increased cellular levels of TRH mRNA in response to cold exposure in both the PVN and caudal raphe nuclei may have a bearing on the integrated endocrine and autonomic responses to this stimulus. Acknowledgements--This work was supported by the National Institute of Mental Health, Grant MH-00663, and the National Institute of Diabetes and Digestive and Kidney Diseases, Grants DK-30110. We are indebted to Drs J. H. Walsh and A. Soil (CURE/DDC) and Dr Ma Kinodan (Geriatric Research Education and Clinical Center) for their support. The authors wish to thank Drs Monica Chen, Martin Martin and Mari Nogami for the help received during the performance of the studies. Paul Kirshbaum is also acknowledged for helping in the preparation of the manuscript.
REFERENCES
l. Appcl N. M., Wessendorf M. W. and Elde R. P. (1987) Thyrotropin-releasing hormone in spinal cord: coexistencewith serotonin and substance P in fibers and terminals apposing identified preganglionic sympathetic neurons. Brain Res. 415, 137-143. 2. Arai I., Muramatsu M. and Aihara H. (1986) Body temperature dependency of gastric regional blood flow, acid secretion and ulcer formation in restraint and water-immersion stressed rats. Jap. J. Pharmac. 40, 501-504.
662
H. YANG et al.
3. Arancibia S., Tapia-Arancibia L., Astier H. and Assenmacher I. (1989) Physiological evidence for cq-adrenergic facilitatory control of the cold-induced TRH release in the rat, obtained by push-pull cannulation of the median eminence. Neurosci. Lett. 100, 169-174. 4. Basso N., Bagarani M., Pekary E., Genco A. and Materia A. (1988) Role of thyrotropin-releasing hormone in stress ulcer formation in the rat. Dig. Dis. Sci. 33, 819-823. 5. Bonaz B. and Tach6 Y. (1993) C-Fos expression in specific brain nuclei during cold restraint-stress-induced increase in colonic transit and gastric lesions in rats. Gastroenterology 104, A43. 6. Calza L., Giardino L., Ceccatelli S., Zanni M., Elde R. and Hokfelt T. (1992) Distribution of thyrotropin-releasing hormone receptor messenger RNA in the rat brain: an in situ hybridization study. Neuroscience 251, 891-909. 7. Chomczynski P. and Saechi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analyt. Biochem. 162, 156-159. 8. Dolan D. H., Nichols M. F., Fletcher D., Schadt J. and Zoeller R. T. (1992) Cold- and ethanol-induced hypothermia reduces cellular levels of mRNA-encoding thyrotropin-releasing hormone (TRH) in neurons of the preoptic area. Molec. Cell. Neurosci. 3, 425-432. 9. Garrick T., Prince M., Yang H., Ohning G. and Tach6 Y. (1994) Raphe pallidus stimulation increases gastric contractility via TRH projections to the dorsal vagal complex. Brain Res. 636, 343-347. 10. Garrick T., Stephens R., Ishikawa T., Sierra A., Avidan A., Weiner H. and Tach6 Y. (1989) Medullary sites for TRH analogue stimulation of gastric contractility in the rat. Am. J. Physiol. 256, GI011-GI015. 11. Hayase M. and Takeuchi K. (1986) Gastric acid secretion and lesion formation in rats under water-immersion stress. Dig. Dis. Sci. 31, 166-171. 12. Helke C. J. and Phillips E. T. (1988) Thyrotropin-releasing hormone receptor activation in the spinal cord increases blood pressure and sympathetic tone to the vasculature and the adrenals. J. Pharmac. exp. Ther. 245, 41-46. 13. Hernandez D. E., Arredondo M. E., Xue B. G. and Jennes L. (1990) Evidence for a role of brain thyrotropin-releasing hormone (TRH) on stress gastric lesion formation in rats. Brain Res. Bull. 24, 693-695. 14. Hernandez D. E. and Emerick S. G. (1988) Thyrotropin-releasing hormone: medullary site of action to induce gastric ulcers and stimulate acid secretion. Brain Res. 459, 148-152. 15. Hernandez D. E., Jennes L. and Emerick S. G. (1987) Inhibition of gastric acid secretion by immunoneutralization of endogenous brain thyrotropin-releasing hormone. Brain Res. 401, 381-384. 16. Hornby P. J., Rossiter C. D., White R. L., Norman W. P., Kuhn D. H. and Gillis R. A. (1990) Medullary raphe: a new site for vagally mediated stimulation of gastric motility in cats. Am. J. Physiol. 258, G637-G647. 17. Ishikawa K., Kakegawa T. and Susuki M. (1984) Role of hypothalamic paraventricular nucleus in the secretion of thyrotropin under adrenergic and cold-stimulated conditions in the rat. Endocrinology 114, 352-358. 18. Ishikawa T. and Tach6 Y. (1988) Intrahypothalamic microinjection of calcitonin prevents stress-induced gastric lesions in rats. Brain Res. Bull. 20, 415-419. 19. Ishikawa T., Yang H. and Tach6 Y. (1988) Medullary sites of action of the TRH analogue, RX 77368, to stimulate gastric acid secretion in the rat. Gastroenterology 95, 1470-1476. 20. Kirfily A., Silt6 G. and Tach6 Y. (1993) Role of nitric oxide in the gastric cytoprotection induced by central vagal stimulation. Eur. J. Pharmac. 240, 299-301. 21. Koller K. J., Wolff R. S., Warden M. K. and Zoeller R. T. (1987) Thyroid hormones regulate levels of thyrotropin-releasing hormone mRNA in the paraventricular nucleus. Proc. nam. Acad. Sci. U.S.A. 84, 7329-7333. 22. LeBlanc J. and Cot6 J. (1967) Increase vagal activity in cold-adapted animals. Can. J. Physiol. Pharmac. 45, 745-749. 23. Lechan R. M., Wu P. and Jackson I. M. D. (1987) Immunolocalization of the thyrotropin-releasing hormone prohormone in the rat central nervous system. Endocrinology 119, 1210-1216. 24. Lee S. L., Stewart K. and Goodman R. H. (1988) Structure of the gene encoding rat thyrotropin releasing hormone. J. biol. Chem. 263, 16604-16609. 25. Liao N., Bulant M., Nicolas P., Vaudry H. and Pelletier G. (1990) lmmunoelectron microscopic localization of thyrotropin-releasing hormone (TRH) precursor in the rat raphe nuclei. Peptides 11, 397-400. 26. Lin M. T., Wang P. S., Chuang J., Fan L. J. and Won S. J. (1989) Cold stress or a pyrogenic substance elevates thyrotropin-releasing hormone levels in the rat hypothalamus and induces thermogenic reactions. Neuroendocrinology 50, 177-181. 27. Manaker S. and Rizio G. (1989) Autoradiographic localization of thyrotropin-releasing hormone and substance P receptors in the rat dorsal vagal complex. J. comp. Neurol. 290, 516-526. 28. Martin M. G., Wu S. V. and Walsh J. H. (1993) Hormonal control of intestinal Fc receptor gene expression and immunoglobulin transport in suckling rats. J. clin. Invest. 91, 2844-2849. 29. McCann M., Hermann G. E. and Rogers R. C. (1989) Nucleus raphe obscurus (nRO) influences vagal control of gastric motility in rats. Brain Res. 486, 181-184. 30. McCann M. J., Hermann G. E. and Rogers R. C. (1989) Thyrotropin-releasing hormone: effects on identified neurons of the dorsal vagal complex. J. auton, nerv. Syst. 26, 107 112. 3 I. McCarty R. (1985) Sympathetic-adrenal medullary and cardiovascular response to acute cold stress in adult and aged rats. J. auton, nerv. Syst. 12, 15-22. 32. Niida H., Takeuchi K. and Okabe S. (1991) Role of thyrotropin-releasing hormone in acid secretory response induced by lowering of body temperature in the rat. Eur. J. Pharmac. 198, 137-142. 33. Niida H., Takeuchi K., Ueshima K. and Okabe S. (1991) Vagally mediated acid hypersecretion and lesion formation in anesthetized rat under hypothermic conditions. Dig. Dis. Sci. 36, 441-448. 34. Okuma Y., Osumi Y., Ishikawa T. and Mitsuma T. (1987) Enhancement of gastric acid output and mucosal blood flow by tripeptide thyrotropin releasing hormone microinjected into the dorsal motor nucleus of the vagus in rats. Jap. J. Pharmac. 43, 173-178. 35. Okumura T., Uehara A., Taniguchi Y., Watanabe Y., Tsuji K., Kitamori S. and Namiki M. (1993) Kainic acid injection into medullary raphe produces gastric lesions through the vagal system in rats. Am. J. Physiol. 264, G655-G658. 36. Paakkari I., Nurminen M. L. and Siren A. L. (1986) Cardioventilator effects of TRH in anaesthetized rats: role of the brain stem. Eur. J. Pharmac. 122, 131-134. 37. Palkovits M., Mezey E., Eskay R. L. and Brownstein M. J. (1986) Innervation of the nucleus of the solitary tract and the dorsal vagal nucleus by thyrotropin-releasing hormone-containing raphe neurons. Brain Res. 373, 246-251.
Cold activates TRH gene expression in caudal raphe
663
38. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. pp. 1-116. Academic Press, Orlando, FL. 39. Plourde V., Wong H. C., Walsh J. H., Raybould H. E. and Tach6 Y. (1993) CGRP antagonists and capsaicin on celiac ganglia partly prevent postoperative gastric ileus. Peptides 14, 1225-1229. 40. Poulat P., Sandillon F., Marlier L., Rajaofetra N., Olivier C. and Privat A. (1992) Distribution of thyrotropin-releasing hormone in the rat spinal cord with special reference to sympathetic nuclei: a light- and electron-microscopic immunocytochemical study. J. NeurocytoL 21, 157-170. 41. Raggenbass M., Vozzi C., Tribollet E., Dubois-Dauphin M. and Dreifuss J. J. (1990) Thyrotropin-releasing hormone causes direct excitation of dorsal vagal and solitary tract neurones in rat brainstem slices. Brain Res. 5311, 85-90. 42. Riley L. A., Jonakait G. M. and Hart R. P. (1993) Serotonin modulates the levels of mRNAs coding for thyrotropin-releasing hormone and preprotachykinin by different mechanisms in medullary raphe neurons. Molec. Brain Res. 17, 251-257. 43. Rinaman L. and Miselis R. R. (1990) Thyrotropin-releasing hormone-immunoreactive nerve terminals synapse on the dentrites of gastric vagal motoneurons in the rat. J. comp. Neurol. 294, 235-251. 44. Rinaman L., Miselis R. R. and Kreider M. S. (1989) Ultrastructural localization of thyrotropin-releasing hormone immunoreactivity in the dorsal vagal complex in rat. Neurosci. Lett. 104, 7-12. 45. Robert A., Nezamis J. E., Lancaster C. and Hanchar A. J. (1979) Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCI, NaOH, hypertonic NaCI, and thermal injury. Gastroenterology 77, 433-443. 46. Rogers R. C. and Hermann G. E. (1987) Oxytocin, oxytocin antagonist, TRH, and hypothalamic paraventricular nucleus stimulation effects on gastric motility. Peptides 8, 505-513. 47. Segerson T. P., Hoefler H., Childers H., Wolfe H. J., Wu P., Jackson I. M. D. and Lechan R. M. (1987) Localization of thyrotropin-releasing hormone prohormone messenger ribonucleic acid in rat brain by in situ hybridization. Endocrinology 121, 98-107. 48. Somiya H. and Tonoue T. (1984) Neuropeptides as central integrators of autonomic nerve activity: effects of TRH, SRIF, VIP and bombesin on gastric and adrenal nerves. Regul. Pept. 9, 47-52. 49. Stephens R. L., Ishikawa T., Weiner H., Novin D. and Tach6 Y. (1988) TRH analog, RX 77368, injected into the dorsal vagal complex stimulates gastric secretion in rats. Am. J. Physiol. 254, G63943643. 50. Tach6 Y., Yang H. and Yoneda M. (1993) Vagal regulation of gastric function involves thyrotropin-releasing hormone in the medullary raphe nuclei and dorsal vagal complex. Digestion 54, 65-72. 51. Tan D. P. and Tsou K. (1985) Differential motor and blood pressure effects of intrathecal oxytocin and TRH in the rat. Peptides 6, 1191-1193. 52. Travagli R. A., Gillis R. A. and Vicini S. (1992) Effects of thyrotropin-releasing hormone on neurons in the rat dorsal motor nucleus of the vagus, in vitro. Am.J. Physiol. 263, G508~3517. 53. Van den Bergh P., Octave J.-N. and Lechan R. M. (1991) Muscle denervation increases thyrotropin-releasing hormone (TRH) biosynthesis in the rat medullary raphe. Brain Res. 566, 219-224. 54. Wessendorf M. W., Appel N. M., Molitor T. W. and Elde R. P. (1990) A method for immunofluorescent demonstration of three coexisting neurotransmitters in rat brain and spinal cord, using the fluorophores fluorescin, lissamine rhodamine, and 7-amino-4-methylcoumarin-3-acetic acid. J. Histochem. Cytochem. 38, 1859-1877. 55. White R. L. Jr, Rossiter C. D., Hornby P. J., Harmon J. W., Kasbekar D. K. and Gillis R. A. (1991) Excitation of neurons in the medullary raphe increases gastric acid and pepsin production in cats. Am. J. Physiol. 260, G91-96. 56. Witty R. T. and Long J. F. (1970) Effect of ambient temperature on gastric secretion and food intake in the rat. Am. J. Physiol. 219, 1359-1362. 57. Yang H., Ishikawa T. and Tach6 Y. (1990) Microinjection of TRH analogs into the raphe pallidus stimulates gastric acid secretion in the rat. Brain Res. 531, 280-285. 58. Yang H., Ohning G. and Tach6 Y. (1993) TRH in dorsal vagal complex mediates acid response to excitation of raphe pallidus neurons in rats. Am. J. Physiol. 265, G88043886. 59. Yang H., Stephens R. L. and Tach6 Y. (1992) TRH analogue microinjected into specific medullary nuclei stimulates gastric serotonin secretion in rats. Am. J. Physiol. 262, G216~3222. 60. Yang H., Wong H., Wu V., Walsh J. H. and Tach6 Y. (1990) Somatostatin monoclonal antibody immunoneutralization increases gastrin and gastric acid secretion in urethane-anesthetized rats. Gastroenterology 99, 659-665. 61. Yoneda M. and Tach6 Y. (1992) Central thyrotropin-releasing factor analog prevents ethanol-induced gastric damage through prostaglandins in rats. Gastroenterology 102, 1568-1574. 62. Zoeller R. T., Kabeer N. and Albers H. E. (1990) Cold exposure elevates cellular levels of messenger ribonucleic acid encoding thyrotropin-releasing hormone in paraventricular nucleus despite elevated levels of thyroid hormones. Endocrinology 127, 2955-2962. 63. Zoeller R. T. and Rudeen P. K. (1992) Ethanol blocks the cold-induced increase in thyrotropin-releasing hormone mRNA in paraventricular nuclei but not the cold-induced increase in thyrotropin. Molec. Brain Res. 13, 321-330.
(Accepted 12 January 1994)
Pergamon
0306-4522(94)E0110-P
Elsevier ScienceLtd Copyright © 1994 IBRO Printed in Great Britain. All rights reserved 0306-4522/94 $7.00 + 0.00
COLD EXPOSURE ELEVATES THYROTROPIN-RELEASING HORMONE GENE EXPRESSION IN M E D U L L A R Y RAPHE NUCLEI: RELATIONSHIP WITH VAGALLY MEDIATED GASTRIC EROSIONS H. YANG,*~ S. V. W u , t T. ISHIKAWA~ and Y. TACHI~t tCURE/Gastroenteric Biology Center, VA Wadsworth Medical Center, Department of Medicine and Brain Research Institute, UCLA, Los Angeles, CA 90073, U.S.A. *Division of Psychosomatic Research, NIMH, Chiba, Japan Abstract--The stimulation of thyrotropin release by cold is associated with an increase in thyrotropin-releasing hormone gene expression in the paraventricular nucleus of the hypothalamus. Cold exposure also stimulates autonomic outflow to viscera. There is evidence that caudal raphe nuclei are involved in autonomic regulation through thyrotropin-releasing hormone projections to the dorsal vagal complex and spinal cord. To determine whether cold modulates thyrotropin-releasing hormone gene expression in the caudal raphe nuclei, the effect of cold exposure on thyrotropin-releasing hormone messenger RNA levels in the rat lower brainstem was examined by quantitative Northern blot analysis and thyrotropin-releasing hormone messenger RNA was localized by in situ hybridization. The gastric responses to cold exposure were also assessed in sham or vagotomized rats with pylorus ligation. Thyrotropin-releasing hormone messenger RNA signal was detected in the RNA extracted from the medulla and hypothalamus but not from the amygdala, periaqueductal gray or cerebellum. Cold exposure (4°C) for 1 or 3 h increased thyrotropin-releasing hormone messenger RNA levels in the medulla by 77 + 37 and 142 + 39% respectively. In situ hybridization histochemistry showed that the increase in silver grain density occurred exclusively in the raphe pallidus and raphe obscurus. Exposure to cold stress for 2 h stimulated gastric acid secretion and resulted in gastric lesion formation in sham but not vagotomized rats. There are established thyrotropin-releasing hormone projections from the raphe pallidus and obscurus to the dorsal vagal complex. Thyrotropin-releasing hormone is known to exert an excitatory effect on neurons of the dorsal motor nucleus of the vagus, leading to vagally mediated stimulation of gastric function and erosions. The increased thyrotropin-releasing hormone gene expression in the raphe pallidus and obscurus induced by cold supports an important role of endogenous thyrotropin-releasing hormone contained in caudal raphe nuclei-dorsal vagal complex pathways in vagally mediated changes in gastric function induced by cold.
Cold exposure is known to induce a rapid release of thyrotropin-releasing hormone ( T R H ) in the median eminence, which leads to the pituitary secretion of thyrotropin) The portal release of hypothalamic T R H is associated with a parallel increase in neuronal activity and T R H m R N A in the paraventricular nucleus o f the hypothalamus (PVN) as assessed by immunohistochemistry for Fos protein and in situ hybridization for T R H m R N A . s,62,63 Cold exposure also stimulates both the parasympathetic and sympathetic nervous systems. 22m In particular, hypothermia induces a vagal-dependent and atropine-sensitive stimulation of gastric secretory and m o t o r functions as well as the formation of gastric lesions within 3 h. 2''~'32'56Convergent functional and anatomical evidence indicates that endogenous T R H in the dorsal vagal complex (DVC) is involved in the vagally *To whom correspondence should be addressed. Abbreviations: DVC, dorsal vagal complex; PVN, paraven-
trieular nucleus of the hypothalamus; SSC, standard saline citrate; TRH, thyrotropin-releasing hormone. 655
mediated gastric response to cold exposure) ° T R H containing fibers and terminals in the D V C originate exclusively from neurons in the raphe pallidus and obscurus and to a lesser extent in the parapyramidal region, where T R H m R N A , p r o - T R H and T R H have been localized. 23,25,37,47 Chemical or electrical activation of the raphe pallidus or obscurus neurons increase gastric acid secretion, motility and lesion formation through vagal pathways in rats and c a t s . 9'16'29'35'55'57'58In addition, we recently showed that microinjection of T R H antibody into the D V C blocked the acid and motility responses to chemical activation of raphe pallidus neurons. 9'5s These data indicate that endogenous T R H is released from nerve terminals in the D V C upon stimulation of caudal raphe nuclei. Very little is known about the regulation of T R H gene expression in lower brainstem nuclei s,42,53 compared with in the PVN. s,21,62,63 The purpose of the present study was to investigate whether exposure to cold for 1-3 h increases levels of T R H gene expression in the medulla in conscious rats. Northern blot
656
H. YANG et al.
analysis was used to m e a s u r e q u a n t i t a t i v e c h a n g e s in T R H m R N A a n d in situ h y b r i d i z a t i o n to locate the signals. T h e a l t e r a t i o n s in gastric acid secretion a n d m u c o s a l integrity i n d u c e d by cold were also m o n i t o r e d in s h a m a n d v a g o t o m i z e d rats with p y l o r u s ligation.
at 60°C with 0.2 M NazCO 3 and 0.2 M NaHCO 3 for 15 min in order to obtain fragments about 150 bases in length. Northern blot analysis
In a separate experiment, in rats under light ether anesthesia a laparotomy was performed and the pylorus was ligated. Ten minutes after recovery from anesthesia, conscious rats were placed in semi-restraining cages either at room temperature or at 4°C. An additional group of rats was maintained singly in home cages at room temperature. Two other groups received bilateral cervical vagotomy or sham operation, respectively, before ligating the pylorus, then 10 min later both groups were placed singly in semirestraining cages at 4°C. Two hours later, rats were killed by decapitation. The stomachs were removed and gastric secretion collected. The measurements of gastric secretion volume, acid concentration and mucosal erosions were performed as described previously, t8 Gastric acid output was calculated by multiplying the values of acid concentration by the volume of secretion for each rat.
Groups of rats were exposed for 60, 90, 120 or 180 min period at 4°C or room temperature under identical conditions of light illumination (n = 5~/group). Cold exposed rats were decapitated at the end of each period, and rats maintained at room temperature were decapitated in between cold exposed rats. Brains were rapidly removed, then the hypothalamus, amygdala, periaqueductal gray, cerebellum and medulla were dissected, immediately placed in powdered dry ice and kept frozen at - 8 0 ° C until RNA extraction. The landmarks for dissection of the various regions of the brain were as follows. Hypothalamus: rostral to caudal, from the optic chiasm to the mammillary body; lateral section from the midline, c. 2 mm and dorsal section from the ventral surface, c. 2.5 ram. Amygdala: rostral to caudal was the same as the hypothalamus, from the lateral border of the optic tract laterally and dorsally to the rhinal fissure. Periaqueductal gray: rostral to caudal, from the posterior commissure to the caudal part of the inferior colliculus, the area around the aqueduct with a radial of c. 1.5 mm. Cerebellum whole: Medulla: rostral to caudal, from the level of the appearance of the facial nerve to the level of obex; lateral, c. 1.5 mm; dorsal section from the ventral surface, c. 2.5 mm. In addition, in the group of rats exposed to cold for 180min, the stomach was also removed for measurements of gastric lesions. Northern blot analysis was performed as described previously. 6° Total RNA was extracted from the hypothalamus, amygdala, periaqueductal gray, cerebellum and medulla by a rapid method developed by Chomczynski and Sacchi. 7 Denatured RNA samples (20 #g/sample) were separated on a 1.2% formaldehyde gel with buffer circulation and were then transferred by electroblotting to a nylon membrane (Hybridization, 0.45 pm, 10x 15cm, catalog no. RPN.1510N; Amersham Corp., Arlington Heights, IL). RNA was immobilized to the membranes by UV cross-linking followed by baking in a vacuum oven for l h at 80°C. RNA blots were prehybridized with the hybridization buffer for 6-10 h and then incubated with fresh hybridization buffer in the presence of radiolabeled TRH probe for 24-48 h at 55°C. Blots were washed and exposed to X A R film (Eastman Kodak Co., Rochester, NY) for one to three days at -80°C. The relative densities of the m R N A signals were measured quantitatively by using a Bio-Rad Densitometer (Model 620). The consistency of RNA loading and transferring was assessed by rehybridizing with an 18S ribosomal RNA oligo-deoxynucleotide probe (5'CGG CAT GTA TTA GCT CTA GAA TTA CCA CAG 3') labeled by a standard 5' end-labeling technique. 2s
Hybridization probe The 1322 bp EcoRI pro-TRH DNA fragment cloned in plasmid pUC12 (kindly provided by Dr R. Goodman, Vollum Institute, OR) was resubcloned into pGEM-3. The antisense RNA was synthesised by T 7 RNA polymerase after linearizing the plasmid with SmaI. The cRNA probe was synthesized using 1 #g SmaI-linearized plasmid DNA with the transcription buffer (40mM Tris-HCl, 6 m M MgC12, 2 mM spermidine and 10 mM NaC1, pH 7.5) in the presence of 10 mM dithiothreitol, 0.5 mM of each of ATP, GTP and CTP, 12 # M UTP and 50 #Ci [a_32p] UTP (for the probe used in Northern blot analysis) or 100 pCi [~-ssS]UTP (for the probe used in in situ hybridization). The reaction was initiated by adding 10 U of T 7 RNA polymerase and incubated at 37°C for 60 min. After the transcription, the plasmid D N A was removed by digesting with ribonucleasefree deoxyribonuclease I (RQI DNase). All the reagents used in the transcription were from Promega (Madison, WI). Radiolabeled R N A was purified by phenol--chloroform extraction and alcohol precipitation. For in situ hybridization, the probe was hydrolysed by incubating
In situ hybridization Rats maintained in semi-restraining cages either at 4°C (n = 3) or at room temperature (n = 2) for 2 h were decapitated. Brains were rapidly removed and immediately immersed in 4% paraformaldehyde at 4°C for 15 h and soaked in 25% sucrose solution. The frozen tissues were sectioned coronally on a microtome cryostat at 20 # m at the level of the hypothalamus (interaural 6.88-7.20 mm from Paxinos and Watson's plates 38) and medullar (interaural - 3 . 7 2 mm to - 4 . 6 8 mm) and mounted onto ribonucleasefree, gelatin-coated glass slides. The tissues were allowed to dry completely, then fixed in 4% paraformaldehydephosphate-buffered saline for 30 min, washed three times in phosphate-buffered saline for 10 min each time, and dehydrated for 5 min in 70% and 100% ethanol, respectively. After air drying, slides were stored at - 2 0 ° C until hybridization. Hybridization was performed using a commercial in situ hybridization kit (Oncor, Gaithersberg, MD). In brief, the slides were washed in 2 x standard saline citrate (SSC) to remove the ethanol and incubated in pretreatment 1 and
EXPERIMENTAL PROCEDURES
Animals
Male Sprague-Dawley rats weighing 230-290 g (Bantin and Kingman, CA) were maintained on rat Purina chow and tap water ad libitum and housed under conditions of controlled temperature (20 + I°C) and illumination (light on 6.00 a.m. to 6.00 p.m.). Experiments were performed on animals deprived of food for 18 h but allowed free access to water until the beginning of the experiments. All animals were killed between 1.00 p.m. and 5.00 p.m. A previous study indicated that there were only small circadian variations in TRH m R N A in the PVN between 2.00 and 6.00 p.m. 62 Cold exposure
Conscious rats were placed individually in stainless steel cylindrical cages with flat bottoms ( 16 cm x 5.5 cm × 5.5 cm) and with perforations to allow ventilation. Such semi-restraining cages were shown previously by Robert et al. 45 not to induce stress. Rats housed in semi-restraining cages were maintained either at room temperature (23 + 2°C) or at 4°C for various time periods. Measurement o f gastric secretion and lesions
657
Cold activates TRH gene expression in caudal raphe 2 solutions, then washed again and the tissues dehydrated with 70%-100% ethanol. The hybridization was carried out at 52°C. The [~t-3sS] UTP-labeled TRH probe was dissolved in 20 mM dithiothreitol and diluted with 9 vols of hybridization mix solution to reach approximately 3-5 x 105 c.p.m./10/~1. After the slides and the probe were preheated to 52°C, 10/~1 of denatured probe/hybridization mix solution were added to each slide and the tissues were covered with a coverslip. The hybridization was carried out in a humidified atmosphere for 3-5 h, then the coverslips were removed. The non-specific binding was washed out by 50% formamide/2 x SSC at 52°C and the formamide was removed by washing in 2 x SSC at room temperature to prevent the inhibition of the ribonuclease. Slides were incubated at 37°C for 30 min in a post-treatment solution which contained ribonuclease to remove tingle-stranded RNA without degrading the complex probe/target. Then the slides were dehydrated in graded concentrations of ethanol and air dried. Tissue sections were coated with radiographic emulsion, air dried for 4-5 h, transferred to the Black Slide Boxes and kept at 4°C for three to eight weeks. Autoradiograms were developed using the Developer and the Fixer in the Kit. The slides were stained with Toluidine Blue and observed under a microscope with light or dark field. Specificity of the in situ hybridization was assessed by adding a 50-fold excess of cold probe synthesized with UTP instead of [35S]-UTP to compete with the radiolabeled probe during the hybridization.
Statistical analysis Results are presented as mean _+ S.E.M. Multiple group comparisons were calculated by ANOVA followed by Duncan's contrast. Statistical significance was assigned when P < 0.05. RESULTS
Influence of coM exposure on gastric acid secretion and lesion formation R a t s m a i n t a i n e d at a m b i e n t r o o m t e m p e r a t u r e in semi-restraining cages h a d n o change in gastric acid c o n c e n t r a t i o n , o u t p u t a n d mucosal integrity comp a r e d with those kept in s t a n d a r d cages as measured
2 h after pylorus ligation (Table 1). In rats placed in semi-restraining cages at 4°C for 2 h, gastric acid c o n c e n t r a t i o n a n d o u t p u t increased by 8 6 % a n d 216% respectively, c o m p a r e d with rats m a i n t a i n e d at r o o m temperature. In addition, gastric h e m o r rhagic lesions were f o u n d in the glandular p a r t o f the s t o m a c h (Table 1). Bilateral cervical v a g o t o m y abolished cold-induced stimulation o f gastric acid secretion a n d lesion f o r m a t i o n (Table 2).
Influence of cold exposure on medullary thyrotropinreleasing hormone mRNA expression Northern blot analysis Northern blot analysis of total RNA ( 2 0 # g / s a m p l e ) isolated from the h y p o t h a l a m u s , medulla, amygdala, periaqueductal gray a n d cerebellum was performed using a labeled T R H c R N A probe. A single b a n d o f 1 . 6 k b representing the p r o p e r size of published T R H m R N A 24 was detected in the h y p o t h a l a m u s a n d the medulla, b u t n o t in the amygdala, periaqueductal gray a n d cerebellum (Fig. 1). Equal a m o u n t s o f R N A (20/~g/sample) from the medulla of rats m a i n t a i n e d at r o o m t e m p e r a t u r e or exposed to cold for 60, 90, 120 a n d 180rain were further analysed by the T R H probe. T R H m R N A levels increased after cold exposure c o m p a r e d with rats m a i n t a i n e d at r o o m t e m p e r a t u r e u n d e r conditions t h a t were otherwise the same (Fig. 2). Q u a n titative analysis of the T R H m R N A signal showed that the density o f the T R H m R N A signal was significantly increased by 77 + 37% after 60 min o f cold exposure a n d was m a i n t a i n e d elevated by 110 + 27, 110 _+ 20 a n d 142 _+ 39% w h e n cold exposure extended to 90, 120 a n d 180rain periods, respectively (Table 3). Gastric lesions were also observed in each rat exposed to cold for 1 8 0 m i n (3.3 _+ 0.9 m m 2, n = 6).
Table 1. Effect of cold exposure on gastric acid secretion and lesion formation in pylorus-ligated rats n
Acid cone.§ (mmol/l)
Volume~ (ml/2 h)
6 6 10
51 _+8 58 + 9 109 -+ 4*t
4.0_+0.3 2.7 _+ 0.4* 4.8 + 0.3t
Treatments~: Freely moving at 23°C Restraint at 23°C Restraint at 4°C
Acid output§ (~mol/2 h)
Erosions§ (mm 2)
199+32 165 _+40 519 + 39 *t
0.02_+0.02 0.00 + 0.00 0.89 -+ 0.35
:[:The pylorus was ligated under light ether anesthesia and 10 min later, rats were returned to home cages or placed in semi-restraining cages either at room temperature or at 4°C for 2 h, then killed for the measurement of gastric secretion and erosions. §Mean _+ S.E.M. of the group. *P < 0.05 compared with freely moving rats at 23°C. t £ < 0.05 compared with rats restrained at 23°C. Table 2. Effect of vagotomy on gastric acid secretion in conscious pylorus ligated rats exposed to cold Treatmentst Sham Vagotomy
Acid conc.~ (mmol/ml)
Volume:~ (ml/2 h)
Acid output~ (~umol/2 h)
Erosions:[: (mm 2)
103_+8 32 _+ 9*
4.0_+0.6 0.3 _+0. ! *
411+63 9 _+ 3*
0.53_+ 0.17 0.0 _ 0.0"
t i n rats under light ether anesthesia, sham operation or bilateral cervical vagotomy was performed and the pylorus was ligated. After regaining the righting reflex, rats were placed in semi-restraining cages at 4°C for 2 h then killed for the measurement of gastric secretion and erosions. :~Mean + S.E.M. of four rats per group; *P < 0.01, Student t-test. NSC 61/~-1
658
H. YANG et al.
- 28S
~-
H
M
A
P
18S 1.6kb
C
Fig. 1. Northern blot analysis o f T R H mRNA in rat brain. Total RNA (20 #g/sample) was extracted from the hypothalamus (H), medulla (M), amygdala (A), periaqueductal gray (P) and cerebellum (C). Samples were hybridized with the TRH probe. TRH mRNA signals are located in the position of 1.6 kb. Only the hypothalamus and medulla show the TRH mRNA signal.
In situ hybridization Brainstem sections from rats m a i n t a i n e d at r o o m t e m p e r a t u r e or exposed to cold were m o u n t e d on the same slide a n d hybridized with a T R H c R N A probe.
Over 9 0 % of the T R H m R N A signal observed in the medulla was located in the raphe pallidus a n d raphe obscurus, including the area s u r r o u n d i n g the raphe obscurus. A few T R H m R N A signals could also be
Time after cold exposure
0
60
90
120
(min)
180
- 28S
TRH - 1.6kb
- 28S
18S - 18S
Fig. 2. Northern blot analysis of the effect of cold exposure on medullary TRH gene expression. The pictures show the membrane loaded with medullary total RNA extracted from rats maintained either at room temperature (0 min) or exposed to cold for 60, 90, 120 or 180 rain, respectively, and hybridized separately with TRH or 18S ribosomal RNA probes. The TRH mRNA signals are located in the 1.6 kb position.
659
Cold activates TRH gene expression in caudal raphe Table 3. Effect of cold exposure on medullary thyrotropin-releasing hormone mRNA levels in rats Time of cold exposure (min)
01"
60
90
120
180
Relative densitometric unit:l:
2.3 + 0.2
4.1 __+0.9*
4.9 + 0.6*
4.9 + 0.5*
5.6 -I-0.9*
*P < 0.05 compared with control rats. tRats maintained in semi-restraining cages at room temperature served as control. ~Mean_ S.E.M. of five to six rats in each group. Data from Northern blot analysis were quantitatively analysed using a Bio-Rad Densitometer.
found along the ventral edge of the brainstem in the parapyramidal region (data not shown). In rats exposed to cold for 120 min, the TRH m R N A signal in the raphe pallidus and the raphe obscurus was markedly denser than in rats maintained at room temperature (Fig. 3A, B). Adding a 50-fold excess of cold probe almost completely abolished the hybridization signal in medullary raphe nuclei (Fig. 3C). The PVN used as a control for TRH m R N A signal also showed dense labeling in rats exposed to cold for 120 min (Fig. 3D). DISCUSSION
Pylorus-ligated rats exposed to cold for 2 h in semi-restraining cages had an increased gastric acid secretion and developed gastric hemorrhagic erosions compared with rats maintained at room temperature. The gastric changes were related to cold exposure and not to housing in semi-restraining cages since there was no increase in gastric acid secretion or change in gastric mucosal integrity between groups kept at room temperature, either in semi-restraining cages or in standard cages. These results are in agreement with previous observations that rats maintained in these semi-restraining cages were not stressed and have no observable c-fos expression in the brain. 5"45 More severe gastric erosions were also observed after 3 h exposure to cold in rats without pylorus ligation. The difference in severity of the gastric lesions between these two groups may be related to the different duration of cold exposure and the release of protective factors in the stomach, such as calcitonin gene-related peptide triggered by abdominal surgery and pylorus ligation. ~°,39,6~The stimulation of gastric acid secretion and the formation of gastric mucosal lesions involved vagal pathways, since the gastric response was no longer observed in vagotomized rats. Previous studies performed under different experimental conditions of cold exposure such as immersion in water at 20°C or lowering body temperature by refrigerant pack also demonstrated a vagally mediated stimulation of gastric acid secretion and erosion formation in fasted rats. 2,32,33,56 Functional and anatomical evidence support a role of medullary TRH in the vagal stimulation of gastric function and ulcer formation induced by cold) ° Immunoneutralization by TRH antibody injected into the cerebrospinal fluid inhibits cold water immersion and cold restraint-induced stimulation of gastric acid secretion and lesion formation. 4'13'~5'32'33 TRH
microinjected into the DVC is unique among other transmitters to induce a vagal-dependent stimulation of gastric secretory and motor function and the formation of gastric lesions in rats. 1°'14'19'34'46'49'59Electrophysiological studies showed that TRH causes a direct postsynaptic excitatory effect on dorsal motor nucleus neurons 3°'4~'52and increases efferent activity in the gastric branch of the vagus. 48 These observations were well correlated with the high density of TRH receptors and TRH receptor m R N A localized mainly on vagal preganglionic motoneurons. 6'27 Combined immunocytochemical and retrograde tracing techniques further demonstrated that TRH immunoreactive fibers and terminals make asymmetric synaptic contacts on dendrites of gastric vagal motoneurons? TM The dense TRH-immunoreactive nerve fibers and terminals in the DVC arise exclusively from perikarya located in the raphe pallidus, rapbe obscurus and to a small extent in the parapyramidal regions, where TRH m R N A and pro-TRH have been located.23,25,37,47
Results from the present study provide strong evidence that medullary TRH is involved in the vagally mediated gastric response to cold. Cold exposure increased TRH m R N A levels in the lower brainstem within the first hour. The changes in TRH m R N A persisted for the duration of cold exposure, as reflected by the 142% increase in signal density in Northern blot analysis observed at 180 min. The similarity of the signal density of 18S ribosomal R N A shows the consistency of the equal sample loading, transfer and illumination under these conditions. The specificity of the response was validated by several observations. First, as expected, a single band corresponding to the appropriate size of 1.6 kb for TRH m R N A 21'24was selectively increased after cold stress. Second, Northern blot analysis of total RNA extracted from different brain regions showed that TRH m R N A signal is present only in the hypothalamus and the medulla, where the densest localization of pro-TRH-containing perikarya have been reported. 23 No signal was observed in the periaqueductal gray, amygdala or cerebellum. These results are consistent with in situ hybridization histochemicai studies showing only a cluster of weakly hybridizing cells in the periaqueductal gray and amygdala and no positive cells in the cerebellum. 47 In the present study, in situ hybridization was used to localize the TRH m R N A signal in the medulla. Data revealed that TRH m R N A signal is confined exclusively in neurons of the caudal medullary raphe
Fig. 3. Light micrographs of the brainstem from rats maintained at room temperature (A) or in cold (4r'C; B) for 120 min. Coronal sections were exposed for eight weeks. Adding 50-fold excess of cold probe simultaneously with the radiolabeled probe into the hybridization mix solution almost completely prevented the hybridization in section from a non-stressed rat (C). TRH mRNA signal was also observed in the PVN in rats exposed to cold for 120 min (D). Tissues stained with Toluidine Blue. Rpa, raphe pallidus; Rob, raphe obscurus; PVN, paraventricular nucleus of the hypothalamus. Scale bar = 500 #m.
Cold activates TRH gene expression in caudal raphe complex (raphe obscurus and pallidus). After 2 h of cold exposure, a more intense signal was observed in the caudal raphe nuclei compared with rats maintained at room temperature. The specificity of the response was shown by the abolition of the hybridization signal when the radiolabeled probe was combined with a 50-fold excess of cold probe during the hybridization. In addition, dense TRH mRNA signal could be observed in the PVN of cold exposed rats as reported previously,62 giving further support for the specificity of the probe. Cold-induced changes in TRH gene expression in the forebrain were found to be specific to neurons localized in the PVN and preoptic area, whereas TRH mRNA levels in the thalamus were not modified by cold. 8'62'63 Enhanced TRH gene expression in PVN neurons by cold has been associated with an increase in TRH release in the median eminence, resulting in the stimulation of pituitary-thyroid hormone secretion. ~7'26 Although direct measurement of TRH release in the DVC in response to cold cannot be directly assessed as in the median eminence,26 the TRH antibody microinjected into the DVC or the cerebrospinal fluid blocked the vagally mediated stimulation of gastric function and ulcer formation induced by cold exposure or chemical activation of the raphe pallidus neurons.4,9,13,3z33,58 These latter findings suggest that changes in TRH mRNA in medullary raphe nuclei may be parallel with changes in TRH release from their terminal projections to the DVC, leading to vagaily mediated stimulation of gastric function. This is further supported by recent evidence that cold exposure for 3 h under the same semi-restraining conditions results in a marked activation of neurons located in the raphe pallidus, raphe obseurus and parapyramidal regions, as well as in the dorsal motor nucleus of the vagus, as shown by numerous Fos-positive neurons in these nuclei? The mechanisms through which cold exposure increases TRH mRNA expression in the raphe pallidus and obscurus are still to be investigated. Little is known about factors that influence the regulation of TRH gene expression in these nuclei? One study showed that changes in blood pressure are associated with alterations in TRH mRNA in the raphe pallidus, s However, it is unlikely that the observed increase in TRH gene expression is secondary to the elevation in blood pressure and the decrease in heart rate induced by cold exposure. 31 Cardiovascular changes induced for 1 h by peripheral epinephrine administration did not influence TRH mRNA in the raphe pallidus and obscurus while a decrease in mean
66t
arterial blood pressure induced by nitroprusside increased TRH mRNA selectively in the raphe pallidus and not the raphe obscurus) TRH neurons in the caudal raphe nuclei contribute projections to both the DVC and the spinal cord, where terminals have been identified apposing preganglionic sympathetic neurons. ~'4° Functional studies have shown that TRH acts in the medulla and intrathecally to increase blood pressure through activation of the sympathetic activity. 12,36,5~ Similarly, cold exposure increases blood pressure and sympathetic activity.31 The present study demonstrates that cold-induced increase in TRH mRNA in the raphe obscurus and paUidus may have implications not only to the understanding of the mechanism of vagally mediated alterations of gastric function but may also encompass the sympathetic responses to cold. CONCLUSIONS Our results demonstrate that cold exposure for 1-3 h elevated brainstem TRH mRNA selectively in the caudal medullary raphe nuclei in conscious rats. Cold exposure for 2-3 h also increased gastric acid secretion and induced lesion formation through vagal pathways in fasted rats. Previous studies indicate that the TRH projections from the raphe pallidus and obscurus to the DVC exert an excitatory effect on preganglionic vagal neurons, leading to vagally mediated stimulation of gastric secretory and motor function and erosions formation. Taken together, the present data support an important role of endogenous TRH contained in caudal raphe nuclei-DVC pathways 54 in the parasympathetic response to cold. Previous studies have shown that cold increases TRH mRNA in specific nuclei of the hypothalamus associated with pituitary secretion and thermoregulation. The increased cellular levels of TRH mRNA in response to cold exposure in both the PVN and caudal raphe nuclei may have a bearing on the integrated endocrine and autonomic responses to this stimulus. Acknowledgements--This work was supported by the National Institute of Mental Health, Grant MH-00663, and the National Institute of Diabetes and Digestive and Kidney Diseases, Grants DK-30110. We are indebted to Drs J. H. Walsh and A. Soil (CURE/DDC) and Dr Ma Kinodan (Geriatric Research Education and Clinical Center) for their support. The authors wish to thank Drs Monica Chen, Martin Martin and Mari Nogami for the help received during the performance of the studies. Paul Kirshbaum is also acknowledged for helping in the preparation of the manuscript.
REFERENCES
l. Appcl N. M., Wessendorf M. W. and Elde R. P. (1987) Thyrotropin-releasing hormone in spinal cord: coexistencewith serotonin and substance P in fibers and terminals apposing identified preganglionic sympathetic neurons. Brain Res. 415, 137-143. 2. Arai I., Muramatsu M. and Aihara H. (1986) Body temperature dependency of gastric regional blood flow, acid secretion and ulcer formation in restraint and water-immersion stressed rats. Jap. J. Pharmac. 40, 501-504.
662
H. YANG et al.
3. Arancibia S., Tapia-Arancibia L., Astier H. and Assenmacher I. (1989) Physiological evidence for cq-adrenergic facilitatory control of the cold-induced TRH release in the rat, obtained by push-pull cannulation of the median eminence. Neurosci. Lett. 100, 169-174. 4. Basso N., Bagarani M., Pekary E., Genco A. and Materia A. (1988) Role of thyrotropin-releasing hormone in stress ulcer formation in the rat. Dig. Dis. Sci. 33, 819-823. 5. Bonaz B. and Tach6 Y. (1993) C-Fos expression in specific brain nuclei during cold restraint-stress-induced increase in colonic transit and gastric lesions in rats. Gastroenterology 104, A43. 6. Calza L., Giardino L., Ceccatelli S., Zanni M., Elde R. and Hokfelt T. (1992) Distribution of thyrotropin-releasing hormone receptor messenger RNA in the rat brain: an in situ hybridization study. Neuroscience 251, 891-909. 7. Chomczynski P. and Saechi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analyt. Biochem. 162, 156-159. 8. Dolan D. H., Nichols M. F., Fletcher D., Schadt J. and Zoeller R. T. (1992) Cold- and ethanol-induced hypothermia reduces cellular levels of mRNA-encoding thyrotropin-releasing hormone (TRH) in neurons of the preoptic area. Molec. Cell. Neurosci. 3, 425-432. 9. Garrick T., Prince M., Yang H., Ohning G. and Tach6 Y. (1994) Raphe pallidus stimulation increases gastric contractility via TRH projections to the dorsal vagal complex. Brain Res. 636, 343-347. 10. Garrick T., Stephens R., Ishikawa T., Sierra A., Avidan A., Weiner H. and Tach6 Y. (1989) Medullary sites for TRH analogue stimulation of gastric contractility in the rat. Am. J. Physiol. 256, GI011-GI015. 11. Hayase M. and Takeuchi K. (1986) Gastric acid secretion and lesion formation in rats under water-immersion stress. Dig. Dis. Sci. 31, 166-171. 12. Helke C. J. and Phillips E. T. (1988) Thyrotropin-releasing hormone receptor activation in the spinal cord increases blood pressure and sympathetic tone to the vasculature and the adrenals. J. Pharmac. exp. Ther. 245, 41-46. 13. Hernandez D. E., Arredondo M. E., Xue B. G. and Jennes L. (1990) Evidence for a role of brain thyrotropin-releasing hormone (TRH) on stress gastric lesion formation in rats. Brain Res. Bull. 24, 693-695. 14. Hernandez D. E. and Emerick S. G. (1988) Thyrotropin-releasing hormone: medullary site of action to induce gastric ulcers and stimulate acid secretion. Brain Res. 459, 148-152. 15. Hernandez D. E., Jennes L. and Emerick S. G. (1987) Inhibition of gastric acid secretion by immunoneutralization of endogenous brain thyrotropin-releasing hormone. Brain Res. 401, 381-384. 16. Hornby P. J., Rossiter C. D., White R. L., Norman W. P., Kuhn D. H. and Gillis R. A. (1990) Medullary raphe: a new site for vagally mediated stimulation of gastric motility in cats. Am. J. Physiol. 258, G637-G647. 17. Ishikawa K., Kakegawa T. and Susuki M. (1984) Role of hypothalamic paraventricular nucleus in the secretion of thyrotropin under adrenergic and cold-stimulated conditions in the rat. Endocrinology 114, 352-358. 18. Ishikawa T. and Tach6 Y. (1988) Intrahypothalamic microinjection of calcitonin prevents stress-induced gastric lesions in rats. Brain Res. Bull. 20, 415-419. 19. Ishikawa T., Yang H. and Tach6 Y. (1988) Medullary sites of action of the TRH analogue, RX 77368, to stimulate gastric acid secretion in the rat. Gastroenterology 95, 1470-1476. 20. Kirfily A., Silt6 G. and Tach6 Y. (1993) Role of nitric oxide in the gastric cytoprotection induced by central vagal stimulation. Eur. J. Pharmac. 240, 299-301. 21. Koller K. J., Wolff R. S., Warden M. K. and Zoeller R. T. (1987) Thyroid hormones regulate levels of thyrotropin-releasing hormone mRNA in the paraventricular nucleus. Proc. nam. Acad. Sci. U.S.A. 84, 7329-7333. 22. LeBlanc J. and Cot6 J. (1967) Increase vagal activity in cold-adapted animals. Can. J. Physiol. Pharmac. 45, 745-749. 23. Lechan R. M., Wu P. and Jackson I. M. D. (1987) Immunolocalization of the thyrotropin-releasing hormone prohormone in the rat central nervous system. Endocrinology 119, 1210-1216. 24. Lee S. L., Stewart K. and Goodman R. H. (1988) Structure of the gene encoding rat thyrotropin releasing hormone. J. biol. Chem. 263, 16604-16609. 25. Liao N., Bulant M., Nicolas P., Vaudry H. and Pelletier G. (1990) lmmunoelectron microscopic localization of thyrotropin-releasing hormone (TRH) precursor in the rat raphe nuclei. Peptides 11, 397-400. 26. Lin M. T., Wang P. S., Chuang J., Fan L. J. and Won S. J. (1989) Cold stress or a pyrogenic substance elevates thyrotropin-releasing hormone levels in the rat hypothalamus and induces thermogenic reactions. Neuroendocrinology 50, 177-181. 27. Manaker S. and Rizio G. (1989) Autoradiographic localization of thyrotropin-releasing hormone and substance P receptors in the rat dorsal vagal complex. J. comp. Neurol. 290, 516-526. 28. Martin M. G., Wu S. V. and Walsh J. H. (1993) Hormonal control of intestinal Fc receptor gene expression and immunoglobulin transport in suckling rats. J. clin. Invest. 91, 2844-2849. 29. McCann M., Hermann G. E. and Rogers R. C. (1989) Nucleus raphe obscurus (nRO) influences vagal control of gastric motility in rats. Brain Res. 486, 181-184. 30. McCann M. J., Hermann G. E. and Rogers R. C. (1989) Thyrotropin-releasing hormone: effects on identified neurons of the dorsal vagal complex. J. auton, nerv. Syst. 26, 107 112. 3 I. McCarty R. (1985) Sympathetic-adrenal medullary and cardiovascular response to acute cold stress in adult and aged rats. J. auton, nerv. Syst. 12, 15-22. 32. Niida H., Takeuchi K. and Okabe S. (1991) Role of thyrotropin-releasing hormone in acid secretory response induced by lowering of body temperature in the rat. Eur. J. Pharmac. 198, 137-142. 33. Niida H., Takeuchi K., Ueshima K. and Okabe S. (1991) Vagally mediated acid hypersecretion and lesion formation in anesthetized rat under hypothermic conditions. Dig. Dis. Sci. 36, 441-448. 34. Okuma Y., Osumi Y., Ishikawa T. and Mitsuma T. (1987) Enhancement of gastric acid output and mucosal blood flow by tripeptide thyrotropin releasing hormone microinjected into the dorsal motor nucleus of the vagus in rats. Jap. J. Pharmac. 43, 173-178. 35. Okumura T., Uehara A., Taniguchi Y., Watanabe Y., Tsuji K., Kitamori S. and Namiki M. (1993) Kainic acid injection into medullary raphe produces gastric lesions through the vagal system in rats. Am. J. Physiol. 264, G655-G658. 36. Paakkari I., Nurminen M. L. and Siren A. L. (1986) Cardioventilator effects of TRH in anaesthetized rats: role of the brain stem. Eur. J. Pharmac. 122, 131-134. 37. Palkovits M., Mezey E., Eskay R. L. and Brownstein M. J. (1986) Innervation of the nucleus of the solitary tract and the dorsal vagal nucleus by thyrotropin-releasing hormone-containing raphe neurons. Brain Res. 373, 246-251.
Cold activates TRH gene expression in caudal raphe
663
38. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. pp. 1-116. Academic Press, Orlando, FL. 39. Plourde V., Wong H. C., Walsh J. H., Raybould H. E. and Tach6 Y. (1993) CGRP antagonists and capsaicin on celiac ganglia partly prevent postoperative gastric ileus. Peptides 14, 1225-1229. 40. Poulat P., Sandillon F., Marlier L., Rajaofetra N., Olivier C. and Privat A. (1992) Distribution of thyrotropin-releasing hormone in the rat spinal cord with special reference to sympathetic nuclei: a light- and electron-microscopic immunocytochemical study. J. NeurocytoL 21, 157-170. 41. Raggenbass M., Vozzi C., Tribollet E., Dubois-Dauphin M. and Dreifuss J. J. (1990) Thyrotropin-releasing hormone causes direct excitation of dorsal vagal and solitary tract neurones in rat brainstem slices. Brain Res. 5311, 85-90. 42. Riley L. A., Jonakait G. M. and Hart R. P. (1993) Serotonin modulates the levels of mRNAs coding for thyrotropin-releasing hormone and preprotachykinin by different mechanisms in medullary raphe neurons. Molec. Brain Res. 17, 251-257. 43. Rinaman L. and Miselis R. R. (1990) Thyrotropin-releasing hormone-immunoreactive nerve terminals synapse on the dentrites of gastric vagal motoneurons in the rat. J. comp. Neurol. 294, 235-251. 44. Rinaman L., Miselis R. R. and Kreider M. S. (1989) Ultrastructural localization of thyrotropin-releasing hormone immunoreactivity in the dorsal vagal complex in rat. Neurosci. Lett. 104, 7-12. 45. Robert A., Nezamis J. E., Lancaster C. and Hanchar A. J. (1979) Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCI, NaOH, hypertonic NaCI, and thermal injury. Gastroenterology 77, 433-443. 46. Rogers R. C. and Hermann G. E. (1987) Oxytocin, oxytocin antagonist, TRH, and hypothalamic paraventricular nucleus stimulation effects on gastric motility. Peptides 8, 505-513. 47. Segerson T. P., Hoefler H., Childers H., Wolfe H. J., Wu P., Jackson I. M. D. and Lechan R. M. (1987) Localization of thyrotropin-releasing hormone prohormone messenger ribonucleic acid in rat brain by in situ hybridization. Endocrinology 121, 98-107. 48. Somiya H. and Tonoue T. (1984) Neuropeptides as central integrators of autonomic nerve activity: effects of TRH, SRIF, VIP and bombesin on gastric and adrenal nerves. Regul. Pept. 9, 47-52. 49. Stephens R. L., Ishikawa T., Weiner H., Novin D. and Tach6 Y. (1988) TRH analog, RX 77368, injected into the dorsal vagal complex stimulates gastric secretion in rats. Am. J. Physiol. 254, G63943643. 50. Tach6 Y., Yang H. and Yoneda M. (1993) Vagal regulation of gastric function involves thyrotropin-releasing hormone in the medullary raphe nuclei and dorsal vagal complex. Digestion 54, 65-72. 51. Tan D. P. and Tsou K. (1985) Differential motor and blood pressure effects of intrathecal oxytocin and TRH in the rat. Peptides 6, 1191-1193. 52. Travagli R. A., Gillis R. A. and Vicini S. (1992) Effects of thyrotropin-releasing hormone on neurons in the rat dorsal motor nucleus of the vagus, in vitro. Am.J. Physiol. 263, G508~3517. 53. Van den Bergh P., Octave J.-N. and Lechan R. M. (1991) Muscle denervation increases thyrotropin-releasing hormone (TRH) biosynthesis in the rat medullary raphe. Brain Res. 566, 219-224. 54. Wessendorf M. W., Appel N. M., Molitor T. W. and Elde R. P. (1990) A method for immunofluorescent demonstration of three coexisting neurotransmitters in rat brain and spinal cord, using the fluorophores fluorescin, lissamine rhodamine, and 7-amino-4-methylcoumarin-3-acetic acid. J. Histochem. Cytochem. 38, 1859-1877. 55. White R. L. Jr, Rossiter C. D., Hornby P. J., Harmon J. W., Kasbekar D. K. and Gillis R. A. (1991) Excitation of neurons in the medullary raphe increases gastric acid and pepsin production in cats. Am. J. Physiol. 260, G91-96. 56. Witty R. T. and Long J. F. (1970) Effect of ambient temperature on gastric secretion and food intake in the rat. Am. J. Physiol. 219, 1359-1362. 57. Yang H., Ishikawa T. and Tach6 Y. (1990) Microinjection of TRH analogs into the raphe pallidus stimulates gastric acid secretion in the rat. Brain Res. 531, 280-285. 58. Yang H., Ohning G. and Tach6 Y. (1993) TRH in dorsal vagal complex mediates acid response to excitation of raphe pallidus neurons in rats. Am. J. Physiol. 265, G88043886. 59. Yang H., Stephens R. L. and Tach6 Y. (1992) TRH analogue microinjected into specific medullary nuclei stimulates gastric serotonin secretion in rats. Am. J. Physiol. 262, G216~3222. 60. Yang H., Wong H., Wu V., Walsh J. H. and Tach6 Y. (1990) Somatostatin monoclonal antibody immunoneutralization increases gastrin and gastric acid secretion in urethane-anesthetized rats. Gastroenterology 99, 659-665. 61. Yoneda M. and Tach6 Y. (1992) Central thyrotropin-releasing factor analog prevents ethanol-induced gastric damage through prostaglandins in rats. Gastroenterology 102, 1568-1574. 62. Zoeller R. T., Kabeer N. and Albers H. E. (1990) Cold exposure elevates cellular levels of messenger ribonucleic acid encoding thyrotropin-releasing hormone in paraventricular nucleus despite elevated levels of thyroid hormones. Endocrinology 127, 2955-2962. 63. Zoeller R. T. and Rudeen P. K. (1992) Ethanol blocks the cold-induced increase in thyrotropin-releasing hormone mRNA in paraventricular nuclei but not the cold-induced increase in thyrotropin. Molec. Brain Res. 13, 321-330.
(Accepted 12 January 1994)